專(zhuān)利名稱(chēng)：流感A/Udorn/72(H3N2)基因組的核苷酸序列的制作方法
技術(shù)領(lǐng)域：
本發(fā)明涉及流感病毒菌株A/Udorn/72(H3N2)各節(jié)段的核苷酸序列。
背景技術(shù)：
流感病毒由命名為A、B和C的亞型構(gòu)成。流感A病毒具有8個(gè)節(jié)段的單負(fù)鏈RNA基因組，其編碼病毒生命周期所需的10種多肽(蛋白質(zhì))。這些蛋白質(zhì)的順序如下節(jié)段 12345678蛋白質(zhì)PB2 PB1 PA HA NP NA M1 NS1剪接產(chǎn)物 M2 NS2流感A亞型完整基因組的8個(gè)RNA節(jié)段各與核殼蛋白(NP)的多種亞基衣殼化，并與三聚聚合酶的一些分子(PB1、PB2和PA亞基)結(jié)合，從而形成核糖核蛋白復(fù)合物(RNP)(Lamb，R.A.，《流感病毒》(The Influenza Viruses)，第1-87頁(yè)，R.M.Krug編輯(Plenum Press，1989))。NP蛋白是結(jié)構(gòu)和轉(zhuǎn)錄/復(fù)制調(diào)節(jié)蛋白。這些結(jié)構(gòu)周?chē)鷩@著一層基質(zhì)蛋白(M1)，這種蛋白是病毒顆粒的主要結(jié)構(gòu)成分而且似乎在病毒核心和病毒包膜間起融合膜作用。這種宿主細(xì)胞衍生的包膜布滿(mǎn)兩種主要的病毒編碼的表面糖蛋白血凝素(HA)和神經(jīng)氨酸酶(NA)(這兩種表面糖蛋白是抗原性的決定簇)以及數(shù)量少很多的未糖基化的小蛋白M2(Lamb 1989；Lamb，R.A.等，Cell，40，627-633(1985))。M2蛋白是7#節(jié)段(還編碼M1蛋白)的剪接基因產(chǎn)物，其具有離子通道活性；剪接基因僅在感染的細(xì)胞中存在。類(lèi)似地，非結(jié)構(gòu)性NS2蛋白是8#節(jié)段(還編碼非結(jié)構(gòu)性NS1蛋白)的剪接基因產(chǎn)物；剪接基因僅在感染的細(xì)胞中存在。蛋白酶切割HA糖蛋白形成HA1和HA2。
流感病毒感染是由表面血凝素附著于含唾液酸的細(xì)胞受體引發(fā)的。這種病毒-細(xì)胞的初次相互反應(yīng)，通過(guò)受體介導(dǎo)的胞吞作用將病毒顆粒吸收到細(xì)胞內(nèi)。在核內(nèi)體的低pH條件下，HA經(jīng)歷了促進(jìn)HA2的疏水NH2末端區(qū)域和核內(nèi)體膜間相互作用的構(gòu)象變化，導(dǎo)致膜融合且隨后將核心RNP以及基質(zhì)蛋白(M1)釋放到細(xì)胞質(zhì)中。在RNP被轉(zhuǎn)運(yùn)到發(fā)生完整基因組的轉(zhuǎn)錄和復(fù)制的核心之前，在細(xì)胞質(zhì)中出現(xiàn)RNP和基質(zhì)蛋白的解離(Martin，K.，和Helenius，A.，Cell，67，117-130(1991)；Shapiro，G.I.，等，J.Virology，61，764-773(1987))。
在初次轉(zhuǎn)錄后，新合成的蛋白質(zhì)引發(fā)病毒基因組的復(fù)制，從而增加轉(zhuǎn)錄和蛋白合成。在病毒生命周期的這一階段，表面糖蛋白HA和NA開(kāi)始在質(zhì)膜的分散區(qū)域聚集，而新裝配的病毒則將從質(zhì)膜釋放。假設(shè)病毒的裝配是通過(guò)四種病毒編碼的結(jié)構(gòu)蛋白的細(xì)胞質(zhì)和/或跨膜區(qū)域間的某些相互作用起始的，這四種蛋白是膜錨著點(diǎn)蛋白(HA、NA和M2)和下面的基質(zhì)蛋白(M1)，基質(zhì)蛋白維持與RNP的緊密連接(Garoff，H.，等，“微生物學(xué)和分子生物學(xué)綜述”，62，1171-1190(1998)；Nayak，D.P.，ASM News，62，411-414(1996))。至今還未很好地定義基質(zhì)蛋白M1和RNP復(fù)合物間的接觸，以及8種RNP整套的摻入成熟病毒顆粒的機(jī)制。假定結(jié)構(gòu)成分間特定的分子接觸支配形態(tài)發(fā)生過(guò)程如何啟動(dòng)以及成熟病毒顆粒裝配的過(guò)程和從宿主細(xì)胞的表面出芽。
在病毒基因組復(fù)制的過(guò)程中，有大量流感A病毒亞型和抗原性變體通過(guò)RNA聚合酶的引入突變連續(xù)地析出。當(dāng)它們感染相同的細(xì)胞宿主時(shí)，兩種不同亞型的節(jié)段間，通過(guò)基因重配也發(fā)生突變。流感A病毒的這種生物學(xué)特性產(chǎn)生了新的病毒菌株，從而人們就必須周期性地更新抵抗流感的免疫原性組合物。因此，對(duì)新穎和革新的抵抗流感的免疫原性組合物的開(kāi)發(fā)而言，必須有流感A病毒某一特定菌株的完整核苷酸序列和全長(zhǎng)克隆的有效性。所以，需要確定流感A病毒的完整核苷酸序列并構(gòu)建含有該序列的全長(zhǎng)克隆。
具體說(shuō)，1972年首次分離了被命名為流感A/Udorn/72(H3N2)的流感A菌株。至今尚未測(cè)出聚合酶基因PB2、PB1和PA的核苷酸序列。已測(cè)序了編碼HA、NP、M1、M2、NS1和NS2的節(jié)段的核苷酸序列。然而，多年前采用儀器得到的那些不能提供精確的序列。因此，需要對(duì)這些節(jié)段重復(fù)測(cè)序以提供最新的精確序列。
發(fā)明概述本發(fā)明的目的是確定流感A/Udorn/72(H3N2)病毒菌株的完整核苷酸序列和7#和8# mRNA節(jié)段的剪接產(chǎn)物。本發(fā)明的另一目的是制備8種基因的各個(gè)克隆和剪接信息。
本發(fā)明的另一目的是確定這些基因和剪接信息的所有10種產(chǎn)物的演繹的氨基酸序列。
通過(guò)闡明分離的構(gòu)成流感A/Udorn/72(H3N2)菌株所有節(jié)段的核苷酸序列的核酸分子完成本發(fā)明的這些以及其它目的，這些核酸分子一起包含該菌株的完整核苷酸序列以及它們的剪接信息。所有這些核苷酸序列都是以正鏈、反基因組(antigenomic)信息鏈顯示的。
具體說(shuō)，由SEQ ID NO1、SEQ ID NO3、SEQ ID NO5、SEQ ID NO7、SEQID NO9、SEQ ID NO11、SEQ ID NO13和SEQ ID NO17及其生物學(xué)等效物構(gòu)成完整的核苷酸序列。
或者，完整的核苷酸序列包含HA序列的變體，命名為HA(P1)(SEQ IDNO21)，且所述的完整的核苷酸序列由SEQ ID NO1、SEQ ID NO3、SEQ ID NO5、SEQ ID NO9、SEQ ID NO11、SEQ ID NO13、SEQ ID NO17和SEQ ID NO21及其生物學(xué)等效物構(gòu)成。
在本發(fā)明的另一實(shí)施例中，這些分離的核苷酸分子定向于編碼各個(gè)蛋白質(zhì)的單獨(dú)分離的流感A病毒的核酸分子，且選自SEQ ID NO1、SEQ ID NO3、SEQ ID NO5、SEQ ID NO7、SEQ ID NO11和SEQ ID NO21(SEQ ID NO7的變體)及其生物學(xué)等效物。
在本發(fā)明的另一實(shí)施例中，這些單獨(dú)分離的核酸分子編碼具有選自SEQ IDNO2、SEQ ID NO4和SEQ ID NO6及其生物學(xué)等效物的氨基酸序列的蛋白質(zhì)。
本發(fā)明的另一實(shí)施例提供了單獨(dú)分離的流感A病毒的氨基酸序列，所述的序列選自SEQ ID NO2、SEQ ID NO4和SEQ ID NO6及其生物學(xué)等效物。
本發(fā)明的另一實(shí)施例提供了單獨(dú)的流感A病毒氨基酸序列，所述的序列具有SEQ ID NO8或SEQ ID NO12的序列。
在本發(fā)明的另一實(shí)施例中，使用具有流感A/Udorn/72(H3N2)菌株節(jié)段的核苷酸序列的分離的核酸分子(1)設(shè)計(jì)聚合酶鏈?zhǔn)椒磻?yīng)(PCR)的引物，用于PCR分析，以探測(cè)樣品中相應(yīng)的病毒節(jié)段的存在；或(2)設(shè)計(jì)和選擇用于ELISA的肽，以檢測(cè)樣品中由該節(jié)段產(chǎn)生的相應(yīng)的蛋白質(zhì)的存在。
發(fā)明詳述已出版了流感A/Udorn/72(H3N2)菌株一些節(jié)段的核苷酸序列及演繹的氨基酸序列。Yuferov等報(bào)道了第4節(jié)段的HA基因的序列(Yuferov，V.P.，等，Proceedings of the Academy of Sciences of the USSR，278，738-742(1984))。Buckler-White等報(bào)道了第5節(jié)段的NP基因的序列(Buckler-White，A.J.，和Murphy，B.R.，Virology，155，345-355(1986))。Markoff等報(bào)道了第6節(jié)段的NA基因的序列(Markoff，L.，和Lai，C.J.，Virology，119，288-297(1982))。Lamb等在1981年報(bào)道了第7節(jié)段的M1基因和M2基因剪接產(chǎn)物的序列(M1在Lamb，R.A.，和Lai，C.J.Virology，112，746-751(1981)(″Lamb Virology1981″)；M2在Lamb，R.A.等Proc.Natl.Acad.Sci.USA，78，4170-4174(1981)(″Lamb PNAS 1981″))。Lamb等在1980年報(bào)道了第8節(jié)段的NS1基因和NS2基因剪接產(chǎn)物的序列(Lamb，R.A.，和Lai，C.J.，Cell，21，475-485(1980))。
為了對(duì)這些節(jié)段進(jìn)行重測(cè)序并對(duì)尚未闡明過(guò)的那些節(jié)段進(jìn)行測(cè)序，在Madin-Darby犬腎(MDCK)細(xì)胞中培育流感A/Udorn/72(H3N2)菌株并從中純化。從純化的病毒顆粒提取病毒RNA，并用末端特異性引物進(jìn)行RT-PCR擴(kuò)增。將凝膠純化的基因克隆入pGemT載體(Promega)，并用多套引物測(cè)序。通過(guò)3’和5’連接和交換接頭的RT-PCR片段的測(cè)序，確定末端的序列(Galarza，J.M.等，J.Virol.，70，2360-2368(1996))。M2和NS2基因分別是第7和第8節(jié)段的mRNA的剪接產(chǎn)物。從用寡-dT和基因特異性引物進(jìn)行的RT-PCR得到的MDCK-流感A/Udorn感染的細(xì)胞純化的mRNA回收這些基因。RT-PCR產(chǎn)物是用凝膠純化的，并克隆入pGemT載體。用熒光染料終止子和AmpliTaq DNA聚合酶(Perkin-Elmer)并用Applied Biosystems ABI 377 DNA測(cè)序儀確定所有基因序列。重復(fù)這種步驟用于各片段的多個(gè)克??；得到的序列是一致的。
流感A/Udorn/72(H3N2)菌株的完整基因組總共13628個(gè)核苷酸。這是首次描述流感A/Udorn/72(H3N2)菌株的完整核苷酸序列。
在編碼區(qū)域、5’和3’非編碼區(qū)域(包括調(diào)節(jié)序列如啟動(dòng)子、增強(qiáng)子和聚腺苷酸化信號(hào))測(cè)定8個(gè)節(jié)段的序列。以下為這些節(jié)段、它們的核苷酸數(shù)量、它們分離的核酸分子序列(以正鏈、反基因組信息鏈顯示，即從5’到3’方向)、它們的編碼區(qū)域和氨基酸翻譯節(jié)段1#2341個(gè)核苷酸，SEQ ID NO1，編碼區(qū)域核苷酸28-2304，編碼PB2，759個(gè)氨基酸(SEQ ID NO2)。
節(jié)段2#2341個(gè)核苷酸，SEQ ID NO3，編碼區(qū)域核苷酸25-2295，編碼PB1，757個(gè)氨基酸(SEQ ID NO4)。
節(jié)段3#2233個(gè)核苷酸，SEQ ID NO5，編碼區(qū)域核苷酸25-2172，編碼PA，716個(gè)氨基酸(SEQ ID NO6)。
節(jié)段4#1765個(gè)核苷酸，SEQID NO7，編碼區(qū)域核苷酸30-1727，編碼HA，566個(gè)氨基酸(SEQ ID NO8)。
在一些方面，SEQ ID NO7和SEQ ID NO8與Yuferov等出版的HA的核苷酸和氨基酸序列不同。
Yuferov SEQ ID NO7或8密碼子#氨基酸#密碼子# 氨基酸#GACAspAAC Asn81-83 1881-83 18GGTGlyGGG Gly1101-1103358 1101-1103358TACTyrTTC Phe1485-1487486 1485-1487486GACAspAAC Asn1614-1616529 1614-1616529節(jié)段5#1565個(gè)核苷酸，SEQ ID NO9，編碼區(qū)域核苷酸46-1539，編碼NP，498個(gè)氨基酸(SEQ ID NO10)。
SEQ ID NO9和SEQ ID NO10與Buckler-White等出版的NP的核苷酸和氨基酸序列相同。
節(jié)段6#1466個(gè)核苷酸，SEQ ID NO11，編碼區(qū)域核苷酸20-1426，編碼NA，469個(gè)氨基酸(SEQ ID NO12)。
在一些方面，SEQ ID NO11和SEQ ID NO12與Markoff等出版的NA的核苷酸和氨基酸序列不同。
Markoff SEQ ID NO11或12密碼子# 氨基酸# 密碼子# 氨基酸#CAG Gln CTG Leu104-106 29104-106 29GGA Gly GCA Ala546-548177546-548177CAA Gln CAG Gln695-697226695-697226ACA Thr GCA Ala992-994325992-994325在氨基酸1中，甲硫氨酸的前導(dǎo)序列上游中也有一些不同。
節(jié)段7#1027個(gè)核苷酸，SEQ ID NO13，編碼區(qū)域核苷酸26-781，編碼M1，252個(gè)氨基酸(SEQ ID NO14)。
SEQ ID NO13和SEQ ID NO14與已出版的Lamb Virology 1981中的M1的核苷酸和氨基酸序列相同。
節(jié)段7#，剪接產(chǎn)物322個(gè)核苷酸，SEQ ID NO15，編碼區(qū)域核苷酸26-316，編碼M2，97個(gè)氨基酸(SEQ ID NO16)[第一剪接的氨基酸在殘基10，核苷酸53-55]。
SEQ ID NO15和SEQ ID NO16與已出版的Lamb PNAS 1981中的M2的核苷酸和氨基酸序列相同。
節(jié)段8#890個(gè)核苷酸，SEQ ID NO17，編碼區(qū)域核苷酸27-737，編碼NS1，237個(gè)氨基酸(SEQ ID NO18)。
SEQ ID NO17和SEQ ID NO18與Lamb等1980出版的NS1的核苷酸和氨基酸序列相同。
節(jié)段8#，剪接產(chǎn)物402個(gè)核苷酸，SEQ ID NO19，編碼區(qū)域核苷酸27-389，編碼NS2，121個(gè)氨基酸(SEQ ID NO20)[第一剪接的氨基酸在殘基11，核苷酸57-59]。
SEQ ID NO19和SEQ ID NO20與Lamb等1980出版的NS2的核苷酸和氨基酸序列相同。
如上所述，有另一種HA序列，命名為HA(P1)節(jié)段4#1764個(gè)核苷酸，SEQ ID NO21，編碼區(qū)域核苷酸30-1727，編碼HA，566個(gè)氨基酸(SEQ ID NO22)。
不希望被理論束縛，隨著時(shí)間的推移，HA(P1)序列中可能結(jié)合來(lái)自流感A/Udorn/72(H3N2)菌株的少量突變。HA(P1)序列(SEQ ID NO21)具有1764個(gè)核苷酸，比HA序列(SEQ ID NO7)少一個(gè)核苷酸，因?yàn)樵诠?jié)段4#的非編碼區(qū)域中有一個(gè)核苷酸缺失(1756位)。
本發(fā)明的兩種HA序列的核苷酸和氨基酸序列有如下差異核苷酸位點(diǎn) HAHA(P1) 氨基酸變化35 G A 沉默的81 A G Asn變?yōu)锳sp1103 G T 沉默的1486 T A Phe變?yōu)門(mén)yr1614 A G Asn變?yōu)锳sp1756 T 缺失 在編碼區(qū)域外部HA(P1)序列也與Yuferov等人的HA核苷酸序列有如下稍微的不同核苷酸位點(diǎn) HA(P1) Yuferov 氨基酸變化35 A G 沉默的1756缺失 T 編碼區(qū)域外部基因組中這些節(jié)段的mRNA的核酸序列、負(fù)鏈(即以3’到5’方向)是上述正鏈、反基因組信息鏈序列的互補(bǔ)。
除上述編碼流感A/Udorn/72(H3N2)病毒蛋白的核苷酸序列外，本發(fā)明還包括含各個(gè)流感A病毒核苷酸序列的分離的核酸分子，由于遺傳密碼的豐余在生物學(xué)上相當(dāng)于那些編碼病毒蛋白的序列，即其它核苷酸序列的特征與本文所述的核苷酸序列不同，但其編碼的蛋白質(zhì)的氨基酸序列與在SEQ ID NO1、SEQ IDNO3、SEQ ID NO5、SEQ ID NO7、SEQ ID NO11和SEQ ID NO21中任一編碼的氨基酸序列相同。
具體說(shuō)，本發(fā)明預(yù)計(jì)那些足以核苷酸重復(fù)任一SEQ ID NO1、SEQ ID NO3、SEQ ID NO5、SEQ ID NO7、SEQ ID NO11和SEQ ID NO21的序列，從而可以在標(biāo)準(zhǔn)高度嚴(yán)格Southern雜交條件下雜交，如由Sambrook等所述的(Sambrook，J.，等，《分子克隆實(shí)驗(yàn)室手冊(cè)》(Molecular CloningA LabortoryManul)，第2版，Cold Spring Harbor Laboratory Press，Cold Spring Harbor，N.Y.(1989))。
本發(fā)明還包括編碼與流感A/Udorn/72(H3N2)病毒蛋白的氨基酸序列不同的氨基酸序列的核苷酸序列，但其所編碼的氨基酸序列在生物學(xué)上與這些病毒蛋白(SEQ ID NO2、SEQ ID NO4和SEQ ID NO6)中的一種等效。如果這些氨基酸序列與病毒蛋白序列的差異僅在于少量的缺失、蛋白質(zhì)序列中的插入或替代，從而與病毒蛋白的序列相比這些序列的三級(jí)構(gòu)型基本未改變，就將這種氨基酸序列稱(chēng)為與任一病毒蛋白是生物學(xué)上等效的。
例如，氨基酸丙氨酸(一種疏水氨基酸)的密碼子可以被編碼另一種疏水性較弱的殘基(如甘氨酸)或疏水性更強(qiáng)的殘基(如纈氨酸、亮氨酸或異亮氨酸)替代。類(lèi)似地，將導(dǎo)致一種帶負(fù)電荷的(酸性)殘基替代另一種(如天冬氨酸替代谷氨酸)，或一種帶正電荷的(堿性)殘基替代另一種(如賴(lài)氨酸替代精氨酸或組氨酸)，以及基于親水性指數(shù)中殘基的相似性的替代，預(yù)計(jì)可產(chǎn)生生物學(xué)上等效的產(chǎn)物。預(yù)計(jì)將導(dǎo)致蛋白質(zhì)分子的N-端或C-端部分變化的核苷酸替代也不會(huì)改變蛋白質(zhì)的活性。
所建議的各種變化是本領(lǐng)域技術(shù)人員熟知的，且決定了所編碼的產(chǎn)物的結(jié)構(gòu)及生物活性的保留。
本發(fā)明還涉及分離的氨基酸序列，其包含選自SEQ ID NO2、SEQ ID NO4和SEQ ID NO6的各個(gè)流感A病毒氨基酸序列。
本發(fā)明還涉及單獨(dú)分離的SEQ ID NO8和SEQ ID NO12的氨基酸序列。
流感A/Udorn/72(H3N2)病毒的基本生物學(xué)特性取決于其由8個(gè)節(jié)段構(gòu)成的基因組。流感病毒的節(jié)段由負(fù)極性RNA的單鏈構(gòu)成，各病毒鏈具有其自身的特定核苷酸序列。
因此，病毒菌株間完整的差異鑒定就必須確定病毒的核苷酸序列。由于已清楚確定了完整的特異性核苷酸序列是由13,628個(gè)堿基構(gòu)成的(如本文所述)，就可在基因水平上鑒定病毒，因此這種鑒定方法提供了絕對(duì)的確定。
本文所述的序列可用于提供鑒定方法，該方法包括采用聚合酶鏈?zhǔn)椒磻?yīng)(PCR)方法檢測(cè)流感病毒反基因組DNA的核苷酸序列的一部分，以及制備肽用于ELISA檢測(cè)DNA產(chǎn)生的抗原。
具體一種流感病毒的完全測(cè)序以及克隆的基因組，對(duì)以該基因藍(lán)圖為基礎(chǔ)的新穎和革新的免疫原性組合物的研究和開(kāi)發(fā)是有利的。
將確定的突變引入上述序列的所需的一個(gè)或多個(gè)節(jié)段中。通過(guò)定向誘變引入一個(gè)或多個(gè)突變。然而，不能從病毒RNA直接產(chǎn)生突變體流感病毒，因?yàn)榛蚪M病毒RNA或反基因組cDNA都不能作為合成的直接模板。相反，在用NP衣殼化病毒后，必須通過(guò)病毒RNA聚合酶復(fù)合物將病毒RNA轉(zhuǎn)錄到正義mRNA中。
Pales等人(美國(guó)專(zhuān)利No.5,166,057，本文將其納入作為參考)描述了用于“拯救”流感A病毒節(jié)段的反向遺傳學(xué)輔助病毒依賴(lài)性系統(tǒng)。簡(jiǎn)單地說(shuō)，在三種聚合酶蛋白和NP存在下，通過(guò)體外合成制備核糖核蛋白(RNP)復(fù)合物。然后將該RNP復(fù)合物用于轉(zhuǎn)染真核細(xì)胞。隨后用流感A輔助病毒感染，產(chǎn)生具有衍生自克隆的cDNA節(jié)段的基因的病毒。然后用選擇方法從大量的輔助病毒中分離出所需的轉(zhuǎn)染子。
如果要拯救完整的突變體流感A菌株，則使用一種不同的系統(tǒng)。使用Neumann等(Neumann，G.，等，Proc.Natl.Acad.Sci.USA，96，9345-9350(1999)，本文將其納入作為參考)所述的系統(tǒng)，可以從克隆的cDNA制備修飾過(guò)的流感病毒菌株A/Udorn/72(H3N2)。這種以質(zhì)粒為基礎(chǔ)的系統(tǒng)不需要使用輔助病毒感染。簡(jiǎn)單地說(shuō)，用八種質(zhì)粒(各編碼菌株的病毒RNA節(jié)段且側(cè)翼于合適的RNA聚合酶啟動(dòng)子和終止子)以及另四種編碼病毒NP、PB2、PB1和PA蛋白的質(zhì)粒(用于在體內(nèi)細(xì)胞內(nèi)合成RNP)轉(zhuǎn)染細(xì)胞系，如人胚腎細(xì)胞系293T。添加另五種表達(dá)病毒結(jié)構(gòu)蛋白HA、NA、M1、M2和NS2的質(zhì)粒產(chǎn)量顯著增加。
在該系統(tǒng)的變更中，僅需要八種質(zhì)粒(Hoffmann，E.，等，Proc.Natl.Acad.Sci.USA，97，6108-6113(2000)，本文將其納入作為參考)。各質(zhì)粒包含兩種啟動(dòng)子、人RNA聚合酶I(pol I)啟動(dòng)子和人RNA聚合酶II(pol II)啟動(dòng)子。在用八種表達(dá)質(zhì)粒轉(zhuǎn)染真核細(xì)胞后，人pol I和II啟動(dòng)子分別轉(zhuǎn)錄質(zhì)粒模板。這導(dǎo)致了病毒mRNA和vRNA的合成，從而最終產(chǎn)生傳染性的流感A病毒。
由于8、12或17質(zhì)粒轉(zhuǎn)染系統(tǒng)的任何一種都不需要輔助病毒，因此無(wú)需噬斑純化就可回收轉(zhuǎn)染子病毒。這種系統(tǒng)促進(jìn)活的減毒流感A病毒的產(chǎn)生，用于抵抗包括新HA或NA亞型的流行病，其中使流感A/Udorn/72(H3N2)菌株的相應(yīng)節(jié)段突變以匹配新亞型的序列。
采用定向誘變將預(yù)定的突變引入流感A/Udorn/72(H3N2)菌株中編碼HA或NA的節(jié)段(其相應(yīng)于新循環(huán)亞型的序列)。通過(guò)標(biāo)準(zhǔn)重組DNA方法將這種突變引入病毒基因組的DNA拷貝中。
如果需要，用不同流感病毒菌株的相應(yīng)節(jié)段替代編碼流感A/Udorn/72(H3N2)菌株(或其突變體)的HA或NA的節(jié)段，產(chǎn)生重排列病毒。
在本發(fā)明的另一實(shí)施例中，用分離的具有流感A/Udorn/72(H3N2)菌株節(jié)段的核苷酸序列的核酸分子，制備寡核苷酸探針(從正鏈反基因組信息或從負(fù)鏈互補(bǔ)基因組信息)且表達(dá)這些肽(僅從正鏈反基因組信息)，可用于檢測(cè)體液或組織樣品中流感A菌株(或其突變體)的節(jié)段的存在。用核苷酸序列設(shè)計(jì)高度特異性和敏感性的診斷測(cè)試，以檢測(cè)樣品中病毒節(jié)段的存在。
以本文所述的病毒序列為基礎(chǔ)，用這些序列合成PCR引物。將測(cè)試樣品進(jìn)行RNA逆轉(zhuǎn)錄，隨后PCR擴(kuò)增選定的相應(yīng)于本文所述核苷酸序列(具有與該病毒菌株確定節(jié)段相同的核苷酸)的cDNA區(qū)域。在凝膠上鑒定擴(kuò)增的PCR產(chǎn)物，并用特異性核苷酸探針通過(guò)雜交驗(yàn)證它們的特異性。
用ELISA測(cè)試檢測(cè)病毒節(jié)段產(chǎn)生的蛋白質(zhì)的存在。設(shè)計(jì)并選擇出包含一個(gè)或多個(gè)不同的殘基(以本文所述的病毒序列為基礎(chǔ))的肽。然后將這些肽偶聯(lián)于半抗原(如匙孔 血藍(lán)蛋白(KLH))并用于疫苗動(dòng)物(如兔)，以產(chǎn)生單特異性多克隆抗體。然后這些多克隆抗體的選擇或多克隆和單克隆抗體的組合可用于“捕獲ELISA”以檢測(cè)該病毒節(jié)段產(chǎn)生的蛋白質(zhì)。
本文將所引用的參考文獻(xiàn)全部納入作為參考。
為了讓本發(fā)明更易理解，列出以下實(shí)施例。這些實(shí)施例僅用于說(shuō)明，對(duì)本發(fā)明的范圍無(wú)任何限制。
實(shí)施例實(shí)施例1流感A/Udorn/72(H3N2)菌株的測(cè)序如下進(jìn)行8個(gè)節(jié)段的分別測(cè)序RT-PCR方法用Madin-Darby犬腎(MDCK)細(xì)胞培育流感A/Udorn/72(H3N2)，并從澄清的培養(yǎng)物上清液中濃縮。用RNAeasy提取試劑盒(Qiagen)從100μl病毒原液制備基因組病毒RNA(vRNA)。用Oligotex mRNA試劑盒(Qiagen)，從流感A/Udorn感染的MDCK細(xì)胞的總mRNA中分離出節(jié)段7和8的mRNA的剪接產(chǎn)物(分別為M2和NS2)。將約1μg vRNA或者mRNA添加到裝有2pmol基因特異性引物和二乙基焦炭酸鹽處理的水(以抑制RNA酶)的Eppendorf管中，至12μl終體積。用寡-dT引物逆轉(zhuǎn)錄M2和NS2剪接信息。在70℃加熱RNA-引物混合物10分鐘，然后在冰上迅速冷凍。在該管中添加如下試劑4μl 5X第一條鏈的緩沖液(250mMTris-HCl pH 8.3，375mM KCl，15mM MgCl2)，2μl 0.1M DTT和1μl dNTP混合物(各為10mM dATP、dTTP、dGTP和dCTP)，并在42℃培育2分鐘。隨后，加入1μl SuperScript II(Gibco)逆轉(zhuǎn)錄酶，并在42℃再培育50分鐘。在70℃培育15分鐘終止RT反應(yīng)。將2μl上述RT添加到如下試劑的混合物中開(kāi)始PCR反應(yīng)50mM Tris-HCl(pH 8.0)、1.5mM MgCl2、50mM KCl、1μM各引物、0.2mM各dNTP和2單位Taq聚合酶。如下用Perkin-Elmer 9600 PCR儀擴(kuò)增DNA1輪，94℃變性2分鐘；30輪，94℃變性30秒；42℃退火30秒并在72℃延伸1分鐘。然后在72℃進(jìn)行一輪延伸若干分鐘。在1％瓊脂糖凝膠上分析PCR擴(kuò)增的DNA，并用Qiagen純化試劑盒純化。
測(cè)序方法將約500ng質(zhì)粒pGEM-T(含有相應(yīng)的由RT-PCR產(chǎn)生的DNA節(jié)段)添加到O.2ml裝有3.2pmol測(cè)序引物和8μl終止預(yù)備反應(yīng)混合液(Perkin-Elmer)的試管中，并將該試管置于Perkin-Elmer 9600 PCR儀中。通過(guò)25輪96℃10秒，50℃5秒和60℃ 4分鐘，擴(kuò)增反應(yīng)。然后在G50 Sepadex自旋柱上純化產(chǎn)物，并將其冷凍干燥，然后重懸浮于3μl 25mM EDTA(pH8.0)和50mg/ml葡聚糖藍(lán)色負(fù)載染料中。在ABI 377自動(dòng)測(cè)序儀上測(cè)試樣品，并用Sequencher(Gene CodesCorporation的序列分析程序)分析序列。
3’和5’端的核苷酸序列的確定在37℃，用10U煙草酸焦磷酸酶(Epicenter Technologies，Madison，Wis.)處理純化的基因組vRNA(2.5μg)(如上)30分鐘，以除去5’端的磷酸基團(tuán)。將處理過(guò)的RNA進(jìn)行苯酚-氯仿提取，并用乙醇沉淀。隨后，在37℃用50U T4 RNA-連接酶(Pharmaci Biotech)連接RNA的5’和3’端1小時(shí)，用苯酚-氯仿提取，用乙醇沉淀。逆轉(zhuǎn)錄連接的RNA，并用引物的正反對(duì)(如下)，用GeneAmp GoldRNA PCR試劑盒(PE Biosystems)在單次反應(yīng)中進(jìn)行PCR。如上所述，用各RNA節(jié)段的特異性引物進(jìn)行測(cè)序反應(yīng)(Galarza，J.M.，等，1996，J of Virol.702360-2368)。
用于流感A/Udron/72(H3N2)測(cè)序的引物以“F”標(biāo)記正向引物，且為反基因組加有義。反向引物用“R”標(biāo)記，且為基因組負(fù)有義。
PB2(節(jié)段1)引物引物F/R 引物序列 跨越的bp·RT引物1107 F5′-TATGGAAAGAATAAAAGAACTACGGAA-3′ 27-53(SEQ ID NO23)·PCR引物1108 R5′-TCGTTTTTAAACTATTCAACAT-3′ 2307-2328(SEQ ID NO24)1107 F5′-TATGGAAAGAATAAAAGAACTACGGAA-3′ 27-53(SEQ ID NO23)·測(cè)序引物1101 F5′-AGCAGGTCAATTATATTCAATATG-3′ 1-24(SEQ ID NO25)該引物序列與PB2基因序列有如下差異位點(diǎn) PB2基因1101 F5A G6A G7A T8G C9C A11 G T12 G T13 T A14 C T16 A T18 T C20 T A21 A T22 T A24 C G1102 R 5′-AACAAGGTCGTTTTTAAACTATTC-3′ 2312-2335(SEQ ID NO26)1103 F 5′-AGAACTCTATTCCAACAAATG-3′ 1816-1836(SEQ ID NO27)1104 R 5′-AATCGGATATTTCATTGCCAT-3′ 178-198(SEQ ID NO28；該引物序列在核苷酸195為C，而PB2基因序列為T(mén))1105 F 5′-AGAACTCTATTCCAACAAATGAGGGATGTAGTT-3′ 1816-1848(SEQ ID NO29；該引物序列在核苷酸1846為G，而PB2基因序列為C)1106 R 5′-AATCGGATATTTCATTGCCATCATCCATTTCAT-3′ 166-198(SEQ ID NO30；該引物序列在核苷酸195為C，而PB2基因序列為T(mén))1107 F 5′-TATGGAAAGAATAAAAGAACTACGGAA-3′27-53(SEQ ID NO23)1108 R 5′-TCGTTTTTAAACTATTCAACAT-3′ 2307-2328(SEQ ID NO24)1109 F 5′-AGACGTGGTGTTGGTAATGAA-3′ 2214-2234(SEQ ID NO31)1110 F 5′-CGAAGAGTTGACATAAACCCT-3′ 454-474(SEQ ID NO32)1111 R5′-TCATCCCTCAICCCCTCACAT-3′ 1943-1963(SEQ ID NO33；該引物序列在核苷酸1955為C；而PB2基因序列為G)1112 R5′-ATTTTCTGTTATCCTCTTGTCA-3′204-224(SEQ ID NO34；與實(shí)際PB2基因序列相比，該引物在核苷酸220后還具有一附加的核苷酸(T)1113 F5′-GTGGAGTCCGCTGTTTTGAG-3′ 2083-2102(SEQ ID NO35)1114 F5′-AGGATGGTGGACATTCTTAGG-3′ 904-924(SEQ ID NO36)1115 R5′-CCGATCAATGCTAACCACTAC-3′ 1507-1527(SEQ ID NO37)1116 F5′-AGCAAAAGCAGGTCAATTATATTCA-3′ 1-25(SEQ ID NO38)1117 R5′-AGTAGAAACAAGGTCGTTTTTAAAC-3′ 2317-2341(SEQ ID NO39)·用于確定末端序列的引物1104 R5′-AATCGGATATTTCATTGCCAT-3′ 178-198(SEQ ID NO28)1109 F5′-AGACGTGGTGTTGGTAATGAA-3′ 2214-2234(SEQ ID NO31)1112 R5′-ATTTTCTGTTATCCTCTTGTCA-3′204-224(SEQ ID NO34)1113 F5′-GTGGAGTCCGCTGTTTTGAG-3′ 2083-2102(SEQ ID NO35)PB1(節(jié)段2)引物引物F/R引物序列 跨越的bp·RT引物1215 F 5′-AGCAAAAGCAGGCAAACCAT-3′ 1-20(SEQ ID NO40；該引物序列在核苷酸4為A，而PB1基因序列為G)·PCR引物1216 R 5′-AGTAGAAACAAGGCATTTTTT-3′ 2321-2341(SEQ ID NO41)1215 F 5′-AGCAAAAGCAGGCAAACCAT-3′ 1-20(SEQ ID NO40)·測(cè)序引物1201 F 5′-TCGAGCTGAAGAAGCTATGG-3′ 1745-1764(SEQ ID NO42；該引物序列在核苷酸1752為G，而PB1基因序列為A，且該引物序列在核苷酸1761為A，而PB1基因序列為G)1202 R 5′-GTTCTGTTGACTGTGTCCAT-3′ 142-161(SEQ ID NO43)1203 F5′-TCGAGCTGAAGAAGCTATGGGAGCAGACCCGT-3′ 1745-1776(SEQ ID NO44)該引物序列與PB1基因序列有如下差異位點(diǎn)PB2基因1203 F1752 A G1761 G A1770 A G1776 C T1204 R 5′-GTTCTGTTGACTGTGTCCATGGTGTATCC-3′ 133-161(SEQ ID NO45)1205 F 5′-AGCGAAAGCAGGCAAACCATTTGAATGGATGTCAA-3′ 1-35(SEQ ID NO46)1206 R 5′-ATTAAAAACAAGGCATTTTTTCATGAAGGAC-3′2341-2311(SEQ ID NO47；該引物序列在核苷酸2337為A，而PB1基因序列為G，且該引物序列在核苷酸2340為T(mén)，而PB1基因序列為G)1207 F 5′-AATTTCCAGCATGGTGGAGGCCATGGTG-3′ 2154-2181(SEQ ID NO48)1208 F 5′-GGGATCTTTGAAAACTCGTG-3′ 325-344
(SEQ ID NO49)1209 R 5′-CAGCATTGTTTACAGACTC-3′ 1936-1954(SEQ ID NO50)1210 F 5′-ACCAAAGATGCAGAAAGAGG-3′706-725(SEQ ID NO51)1211 R 5′-CTCATATTGATTCCGACTAA-3′1438-1457(SEQ ID NO52)1212 R 5′-TGTCCACTTC CCTTTTTCTG A-3′ 172-192(SEQ ID NO53)1213 F 5′-ATTTTTCCCC AGTAGTTCAT AC-3′2118-2139(SEQ ID NO54)1214 F 5′-ACGCTGTTGC AACTACACAC TCCTG-3′ 1997-2021(SEQ ID NO55)1215 F 5′-AGCAAAAGCAGGCAAACCAT-3′1-20(SEQ ID NO40)1216 R 5′-AGTAGAAACAAGGCATTTTTT-3′ 2321-2341(SEQ ID NO41)·用于確定末端序列的引物1204 R 5′-GTTCTGTTGACTGTGTCCATGGTGTATCC-3′ 133-161(SEQ ID NO45)1212 R 5′-TGTCCACTTC CCTTTTTCTG A-3′ 172-192(SEQ ID NO53)1213 F 5′-ATTTTTCCCC AGTAGTTCAT AC-3′2118-2139(SEQ ID NO54)1214 F 5′-ACGCTGTTGC AACTACACAC TCCTG-3′ 1997-2021(SEQ ID NO55)PA(節(jié)段3)引物引物F/R 引物序列 跨越的bp·RT引物1315 F 5′-AGCAAAAGCA GGTACTGATT CGAGA-3′ 1-25(SEQ ID NO56)·PCR引物1315 F5′-AGCAAAAGCA GGTACTGATT CGAGA-3′1-25(SEQ ID NO56)1316 R5′-AGTAGAAACA AGGTACTTTT TTGGA-3′ 2209-2233(SEQ ID NO57)·測(cè)序引物1301 F5′-GAACCTGGAA CCTTTGATCT t-3′ 2053-2073(SEQ ID NO58)1302 R5′-ATTCACCACT GTCCAGGCCA T-3′ 280-300(SEQ ID NO59)該引物序列與PA基因序列有如下差異位點(diǎn) PA基因 1302 F294TC297TC300GA1303 F5′-GAACCTGGAA CCTTTGATCT TGAGGGGCTA-3′ 2053-2082(SEQ ID NO60；該引物序列在核苷酸2075為A，而PA基因序列為G)1304 R5′-ATTCACCACT GTCCAGGCCA TTGACGCGTC-3′ 271-300(SEQ ID NO61)該引物序列與PA基因序列有如下差異位點(diǎn) PA基因 1304 F274 G C275 C G276 G C277 T A294 T C297 T C300 G A1305 F5′-AGCAAAAGCA GGTAGTGATA G-3′ 1-21(SEQ ID NO62)
該引物序列與PA基因序列有如下差異位點(diǎn) PA基因 1302 F15 CG20 TA21 CG1306 R 5′-AGTAGAAACA AGGTA-3′ 2219-2233(SEQ ID NO63)1307 F 5′-AGCGAAAGCA GGTAGTGATT CGAGATGGA-3′ 1-29(SEQ ID NO64；該引物序列在核苷酸4為G，而PA基因序列為A)1308 R 5′-AGTAGAAACA AGGTACTTTT TTGGACAG-3′2206-2233(SEQ ID NO65)1309 R 5′-TCGATTTGTT GGAGTGACTG A-3′ 1782-1802(SEQ ID NO66)1310 F 5′-GCCGAACTTC TCCTGCCTTG A-3′684-704(SEQ ID NO67)1311 F5′-GTATTCAATA GCCTGTATG-3′ 1957-1975(SEQ ID NO68)1312 R5′-GACTTCTCTC CTTGTCACTC-3′ 386-405(SEQ ID NO69)1313 F 5′-ACGAGTCAGC TAAAGTGGGC A-3′ 1111-1131(SEQ ID NO70)1314 R 5′-GACACCTCTG CTGTGAAGTA A-3′ 1356-1376(SEQ ID NO71)1315 F 5′-AGCAAAAGCA GGTACTGATT CGAGA-3′1-25(SEQ ID NO56)1316 R 5′-AGTAGAAACA AGGTACTTTT TTGGA-3′ 2209-2233(SEQ ID NO57)1317 F 5′-GGAGCTGAGA AACCGAAGTT T-3′319-339(SEQ ID NO72)1318 R 5′-GACTTGGCCA ATAAAGTCCT A-3′1935-1955
(SEQ ID NO73)1319 R 5′-GCCTGCGCAT AGTTCCTGTG A-3′ 644-664(SEQ ID NO74)1320 R 5′-TCTACCACTA TTGACTCGCC-3′ 196-215(SEQ ID NO75)·用于確定末端序列的引物1301 F 5′-GAACCTGGAA CCTTTGATCT t-3′ 2053-2073(SEQ ID NO58)1303 F 5′-GAACCTGGAA CCTTTGATCT TGAGGGGCTA-3′ 2053-2082(SEQ ID NO60)1311 F5′-GTATTCAATA GCCTGTATG-3′ 1957-1975(SEQ ID NO68)1312 R 5′-GACTTCTCTC CTTGTCACTC-3′ 386-405(SEQ ID NO69)1320 R 5′-TCTACCACTA TTGACTCGCC-3′ 196-215(SEQ ID NO75)HA(節(jié)段4)引物引物F/R引物序列 跨越的bp·RT引物1401 F 5′-AGCAAAAGCA GGGGATAATT CTA-3′1-23(SEQ ID NO76)·PCR引物1401 F 5′-AGCAAAAGCA GGGGATAATT CTA-3′1-23(SEQ ID NO76)1402 R 5′-AGTAGAAACA AGGGTGTTTT TAA-3′ 1743-1765(SEQ ID NO77)·測(cè)序引物1401 F 5′-AGCAAAAGCA GGGGATAATT CTA-3′1-23(SEQ ID NO76)1402 R 5′-AGTAGAAACA AGGGTGTTTT TAA-3′ 1743-1765(SEQ ID NO77)1403 R 5′-ACAGTTTGTT CATTTCCGAG-3′ 1400-1419
(SEQ ID NO78)1404 R 5′-ACTTCAGGGT GTTTTGCTTA-3′ 1004-1023(SEQ ID NO79)1405 R 5′-GAACCCCCCA AATGTATAGT-3′ 605-624(SEQ ID NO80)1406 R 5′-TGAGGAACTC TGAACCAGCT-3′ 199-218(SEQ ID NO81)1407 F 5′-GTCACTAGTT GCCTCGTCAG-3′ 404-423(SEQ ID NO82)1408 F 5′-CCGGGAGACA TACTGGTAAT-3′ 792-811(SEQ ID NO83)1409 F 5′-CAAATCAATG GGAAACTGAA-3′ 1203-1222(SEQ ID NO84)1410 F 5′-TGAACTGAAG TCAGGATACA-3′ 1592-1611(SEQ ID NO85)·用于確定末端序列的引物1405 R 5′-GAACCCCCCA AATGTATAGT-3′ 605-624(SEQ ID NO80)1406 R 5′-TGAGGAACTC TGAACCAGCT-3′ 199-218(SEQ ID NO81)1409 F 5′-CAAATCAATG GGAAACTGAA-3′ 1203-1222(SEQ ID NO84)1410 F 5′-TGAACTGAAG TCAGGATACA-3′ 1592-1611(SEQ ID NO85)NP(節(jié)段5)引物引物F/R 引物序列 跨越的bp·RT引物1509 F 5′-AGCAAAAGCA GGGTTAATAA TCAC-3′ 1-24(SEQ ID NO86)·PCR引物1509 F 5′-AGCAAAAGCA GGGTTAATAA TCAC-3′ 1-24(SEQ ID NO86)1510 R 5′-AGTAGAAACA AGGGTATTTT TCCT-3′ 1542-1565(SEQ ID NO87)·測(cè)序引物1501 R 5′-TTGCACCTTC CATCATCCTT-3′ 1380-1399(SEQ ID NO88)1502 R 5′-GTCTATTCCC ACTAAAGAGT-3′ 932-951(SEQ ID NO89)1503 R 5′-TGCATCAGAG AGCACATCCT-3′ 529-548(SEQ ID NO90)1504 F 5′-GAACTCGTCC TTTATGACAA-3′ 364-383(SEQ ID NO91)1505 F 5′-AGAGCAATGG ATCAAGT-3′ 751-770(SEQ ID NO92；在核苷酸761-763上有三個(gè)堿基ATG的缺失)1506 F 5′-TACTATGGAA TCAAGTACTC-3′ 1161-1180(SEQ ID NO93)1507 R 5′-ATCAATCATC TTCCCGAC-3′ 130-147(SEQ ID NO94)1508 F 5′-AGTGTCCTTC CGTGGGCG-3′ 1410-1427(SEQ ID NO95)1509 F 5′-AGCAAAAGCA GGGTTAATAA TCAC-3′1-24(SEQ ID NO86)1510 R 5′-AGTAGAAACA AGGGTATTTT TCCT-3′ 1542-1565(SEQ ID NO87)·用于確定末端序列的引物1503 R 5′-TGCATCAGAG AGCACATCCT-3′ 529-548(SEQ ID NO90)1506 F 5′-TACTATGGAA TCAAGTACTC-3′1161-1180(SEQ ID NO93)NA(節(jié)段6)引物引物F/R 引物序列 跨越的bp·RT引物1601 F 5′-AGCAAAAGCA GGAGTGAAGA TGA-3′ 1-23(SEQ ID NO96)·PCR引物1601 F 5′-AGCAAAAGCA GGAGTGAAGA TGA-3′ 1-23(SEQ ID NO96)1602 R 5′-AGTAGAAACA AGGAGTTTTT TCTA-3′ 1443-1466(SEQ ID NO97)·測(cè)序引物1601 F 5′-AGCAAAAGCA GGAGTGAAGA TGA-3′ 1-23(SEQ ID NO96)1602 R 5′-AGTAGAAACA AGGAGTTTTT TCTA-3′ 1443-1466(SEQ ID NO97)1603 R 5’-TCGTTGTTTC TGGGTGTGTC-3′ 989-1008(SEQ ID NO98；該引物序列在核苷酸992為T(mén)，而NA基因序列為C)1604 R 5’-TTATCATACC CAGTGACACA-3′ 596-615(SEQ ID NO99)1605 R 5’-ATTATTATTG GTTCACACGG-3′ 173-192(SEQ ID NO100)1606 F 5’-TTATGTGTCA TGCGATCCTG-3′ 379-398(SEQ ID NO101)1607 F 5’-ATTGTTCATA TTAGCCCATT-3′ 803-822(SEQ ID NO102)1608 F 5’-ATAGGTCAGG TTATTCTGGT-3′1224-1243(SEQ ID NO103)·用于確定末端序列的引物1608 F 5’-ATAGGTCAGG TTATTCTGGT-3′1224-1243(SEQ ID NO103)1605 R 5’-ATTATTATTG GTTCACACG-3′ 173-192(SEQ ID NO100)M1(節(jié)段7)引物引物F/R 引物序列 跨越的bp·RT引物*1706 F 5’-AGCAAAAGCA GGTAG-3′1-15(SEQ ID NO104)·PCR引物1707 R 5’-AGTAGAAACA AGGTA-3′ 1013-1027(SEQ ID NO105)1706 F 5’-AGCAAAAGCA GGTAG-3′1-15(SEQ ID NO104)*1701R 5’-TTTACTCCAG CTCTATGCTG ACAA-3′ 985-1008(SEQ ID NO106)*這些引物將擴(kuò)增M1和M2基因·測(cè)序引物1701 R 5’-TTTACTCCAG CTCTATGCTG ACAA-3′ 985-1008(SEQ ID NO106)1702 R 5’-GATCCAGCCA TTTGCTCCAT-3′ 590-609(SEQ ID NO107)1703 R 5’-GAGGTGACAG GATTGGTCTT-3′ 169-188(SEQ ID NO108)1704 F 5’-CATGGACAGA GCAGTTAAAC-3′ 301-320(SEQ ID NO109)°1705F 5’-GCGAGTATCA TTGGGATCTT-3′ 801-820(SEQ ID NO110)°為M1和M2序列共有·用于確定末端序列的引物1705 F 5’-GCGAGTATCA TTGGGATCTT-3′ 801-820(SEQ ID NO110)1702 R 5’-GATCCAGCCA TTTGCTCCAT-3′ 590-609(SEQ ID NO107)1703 R 5’-GAGGTGACAG GATTGGTCTT-3′ 169-188(SEQ ID NO108)對(duì)序列M2而言，引物1701、1705和1706是足夠的。NS1(節(jié)段8)引物引物F/R引物序列跨越的bp·RT引物*1801F 5’-AGCAAAAGCA GGGTGACAAA GACA-3′ 1-24(SEQ ID NO111)·PCR引物1801 F 5’-AGCAAAAGCA GGGTGACAAA GACA-3′ 1-24(SEQ ID NO111)1802 R 5’-AGTAGAAACA AGGGTGTTTT TTAT-3′ 867-890(SEQ ID NO112)*1803R 5’-TTTTTTATCA TTAAATAAGC TGAA-3′ 851-874(SEQ ID NO113)*這些引物擴(kuò)增NS1和NS2基因?！y(cè)序引物°1801F 5’-AGCAAAAGCA GGGTGACAAA GACA-3′ 1-24(SEQ ID NO111)1802 R 5’-AGTAGAAACA AGGGTGTTTT TTAT-3′ 867-890(SEQ ID NO112)°1803R 5’-TTTTTTATCA TTAAATAAGC TGAA-3′ 851-874(SEQ ID NO113)1804 R 5’-AATTGCATTT TTGACATCCT-3′ 541-560(SEQ ID NO114)1805 R 5’-ATCTTCTCTA CTATCTGCTT-3′ 210-229(SEQ ID NO115)1806 F 5’-CAAGCAATCA TGGATAAGAA-3′ 387-406(SEQ ID NO116)°1807F 5’-GGCGAGAACA GCTAGGTCAA-3′ 692-711(SEQ ID NO117)°為NS1和NS2序列共有·用于確定末端序列的引物1807 F 5’-GGCGAGAACA GCTAGGTCAA-3′ 692-711(SEQ ID NO117)1804 R 5’-AATTGCATTT TTGACATCCT-3′ 541-560
(SEQ ID NO114)1806 F 5’-CAAGCAATCA TGGATAAGAA-3′387-406(SEQ ID NO116)1805 R 5’-ATCTTCTCTA CTATCTGCTT-3′210-229(SEQ ID NO115)對(duì)序列NS2而言，引物1801、1803和1807是足夠的。
實(shí)施例2PCR試驗(yàn)檢測(cè)流感A/Udorn/72(H3N2)菌株的節(jié)段用PCR試驗(yàn)檢測(cè)流感A/Udorn/72(H3N2)菌株的節(jié)段的存在。根據(jù)本文所述的病毒序列的同源性設(shè)計(jì)和選擇PCR引物。如下進(jìn)行該試驗(yàn)將樣品進(jìn)行RNA逆轉(zhuǎn)錄，PCR擴(kuò)增所選的相應(yīng)于本文所述的特異性核苷酸序列的cDNA區(qū)域。在凝膠上鑒定擴(kuò)增的PCR產(chǎn)物，并與特異性核苷酸探針雜交驗(yàn)證它們的特異性。
實(shí)施例3ELISA檢測(cè)由流感A/Udorn/72(H3N2)菌株的節(jié)段產(chǎn)生的抗原用ELISA檢測(cè)由流感A/Udorn/72(H3N2)菌株的節(jié)段產(chǎn)生的抗原的存在。根據(jù)本文所述的病毒序列的同源性設(shè)計(jì)和選擇肽。然后將這些肽偶聯(lián)于KLH，并用于免疫接種兔，以產(chǎn)生單特異性多克隆抗體。然后在“捕獲ELISA”中使用這些多克隆抗體的選擇或多克隆和單克隆抗體的組合，以檢測(cè)由該病毒節(jié)段產(chǎn)生的蛋白的存在。
序列表序列表<110>美國(guó)氰胺公司<120>流感A/Udorn/72(H3N2)基因組的核苷酸序列<130>AM100289PCT<140><141><150>60/213,650<151>2000-06-23<160>117<170>PatentIn Ver.2.1版本<210>1<211>2341<212>DNA<213>流感A病毒(Influenza A virus)<220><221>CDS<222>(28)..(2304)<400>1agcaaaagca ggtcaattat attcaat atg gaa aga ata aaa gaa cta cgg aat 54Met Glu Arg Ile Lys Glu Leu Arg Asn1 5ctg atg tcg cag tct cgc act cgc gag ata cta aca aaa acc aca gtg 102Leu Met Ser Gln Ser Arg Thr Arg Glu Ile Leu Thr Lys Thr Thr Val10 15 20 25gac cat atg gcc ata att aag aag tac aca tca ggg aga cag gaa aag 150Asp His Met Ala Ile Ile Lys Lys Tyr Thr Set Gly Arg Gln Glu Lys30 35 40aac ccg tca ctt agg atg aaa tgg atg atg gca atg aaa tat cca att 198Asn Pro Ser Leu Arg Met Lys Trp Met Met Ala Met Lys Tyr Pro Ile45 50 55aca gct gac aag agg ata aca gaa atg gtt cct gag aga aat gag caa 246Thr Ala Asp Lys Arg Ile Thr Glu Met Val Pro Glu Arg Asn Glu Gln60 65 70gga caa acc cta tgg agt aaa atg agt gat gcc ggg tca gat cga gtg 294Gly Gln Thr Leu Trp Ser Lys Met Ser Asp Ala Gly Ser Asp Arg Val75 80 85atg gta tca cct ttg gcg gtg aca tgg tgg aat aga aat gga cca gtg 342Met Val Ser Pro Leu Ala Val Thr Trp Trp Asn Arg Asn Gly Pro Val90 95 100 105aca agt acg gtt cat tat cca aaa gtc tac aag act tat ttt gat aaa 390Thr Ser Thr Val His Tyr Pro Lys Val Tyr Lys Thr Tyr Phe Asp Lys110 115 120gtc gaa agg tta aaa cat gga acc ttt ggc cct gtc cat ttt aga aac 438Val Glu Arg Leu Lys His Gly Thr Phe Gly Pro Val His Phe Arg Asn125 130 135caa gtc aaa ata cgc cga aga gtt gac ata aac cct ggt cat gca gac 486Gln Val Lys Ile Arg Arg Arg Val Asp Ile Asn Pro Gly His Ala Asp140 145 150ctc agt gcc aag gag gca caa gat gta atc atg gaa gtt gtt ttc ccc 534Leu Ser Ala Lys Glu Ala Gln Asp Val Ile Met Glu Val Val Phe Pro155 160 165aat gaa gtg ggg gcc agg ata cta acg tcg gaa tca caa tta aca ata 582Asn Glu Val Gly Ala Arg Ile Leu Thr Ser Glu Ser Gln Leu Thr Ile170 175 180 185acc aaa gag aaa aaa gaa gaa ctc caa gat tgc aaa att tct cct ttg 630Thr Lys Glu Lys Lys Glu Glu Leu Gln Asp Cys Lys Ile Ser Pro Leu190 195 200atg gtt gca tac atg tta gag aga gaa ctt gtc cga aaa acg aga ttt 678Met Val Ala Tyr Met Leu Glu Arg Glu Leu Val Arg Lys Thr Arg Phe205 210 215ctc cca gtt gct ggt gga aca agc agt gtg tac att gaa gtg tta cac 726Leu Pro Val Ala Gly Gly Thr Ser Ser Val Tyr Ile Glu Val Leu His220 225 230ttg act caa gga acg tgt tgg gaa cag atg tac act cca ggt gga gaa 774Leu Thr Gln Gly Thr Cys Trp Glu Gln Met Tyr Thr Pro Gly Gly Glu235 240 245gtg agg aat gac gat gtt gac caa agc cta att att gca gcc agg aac 822Val Arg Asn Asp Asp Val Asp Gln Ser Leu Ile Ile Ala Ala Arg Asn250 255 260 265ata gtg aga aga gca gca gta tca gca gat cca cta gca tct tta ttg 870Ile Val Arg Arg Ala Ala Val Ser Ala Asp Pro Leu Ala Ser Leu Leu270 275 280gag atg tgc cac agc aca ctg att ggc ggg aca agg atg gtg gac att 918Glu Met Cys His Ser Thr Leu Ile Gly Gly Thr Arg Met Val Asp Ile285 290 295ctt agg cag aac ccg acg gaa gaa caa gct gtg gat ata tgc aag gct 966Leu Arg Gln Asn Pro Thr Glu Glu Gln Ala Val Asp Ile Cys Lys Ala300 305 310gca atg gga ctg agg atc agc tca tcc ttc agt ttt ggt ggg ttc aca 1014Ala Met Gly Leu Arg Ile Ser Ser Ser Phe Ser Phe Gly Gly Phe Thr315 320 325ttt aag aga aca agc ggg tca tca atc aaa aga gag gaa gaa gtg ctt 1062Phe Lys Arg Thr Ser Gly Ser Ser Ile Lys Arg Glu Glu Glu Val Leu330 335 340 345acg ggc aat ctc caa aca ttg aaa ata agg gtg cat gag ggg tac gag 1110Thr Gly Asn Leu Gln Thr Leu Lys Ile Arg Val His Glu Gly Tyr Glu
350 355 360gag ttc aca atg gtg ggg aaa agg gca aca gct ata ctc aga aaa gca 1158Glu Phe Thr Met Val Gly Lys Arg Ala Thr Ala Ile Leu Arg Lys Ala365 370 375acc agg aga ttg gtt cag ctt ata gtg agt gga agg gac gag cag tca 1206Thr Arg Arg Leu Val Gln Leu Ile Val Ser Gly Arg Asp Glu Gln Ser380 385 390ata gcc gaa gcg ata att gta gcc atg gtg ttt tca caa gag gat tgc 1254Ile Ala Glu Ala Ile Ile Val Ala Met Val Phe Ser Gln Glu Asp Cys395 400 405atg ata aaa gca gtt aga ggt gac ctg aat ttc gtt aac agg gca aat 1302Met Ile Lys Ala Val Arg Gly Asp Leu Asn Phe Val Asn Arg Ala Asn410 415 420 425cag cgg ttg aat ccc atg cat caa ctt tta agg cat ttt cag aaa gat 1350Gln Arg Leu Asn Pro Met His Gln Leu Leu Arg His Phe Gln Lys Asp430 435 440gcg aaa gtg ctt ttt cag aat tgg gga att gaa cat atc gac aat gtg 1398Ala Lys Val Leu Phe Gln Asn Trp Gly Ile Glu His Ile Asp Asn Val445 450 455atg gga atg gtt gga gta tta cca gac atg act cca agc aca gag atg 1446Met Gly Met Val Gly Val Leu Pro Asp Met Thr Pro Ser Thr Glu Met460 465 470tca atg aga gga ata aga gtc agc aaa atg ggc gtg gat gaa tac tcc 1494Ser Met Arg Gly Ile Arg Val Ser Lys Met Gly Val Asp Glu Tyr Ser475 480 485agc aca gag agg gta gtg gtt agc att gat cgg ttt ttg aga gtt cga 1542Ser Thr Glu Arg Val Val Val Ser Ile Asp Arg Phe Leu Arg Val Arg490 495 500 505gac caa cgt ggg aat gta tta cta tct cct gag gag gtc agt gaa aca 1590Asp Gln Arg Gly Asn Val Leu Leu Ser Pro Glu Glu Val Ser Glu Thr510 515 520cag ggg aca gag aga ctg aca ata act tac tca tcg tca atg atg tgg 1638Gln Gly Thr Glu Arg Leu Thr Ile Thr Tyr Ser Ser Set Met Met Trp525 530 535gag att aat ggc cct gag tca gtg ttg gtc aat acc tat caa tgg atc 1686Glu Ile Asn Gly Pro Glu Ser Val Leu Val Asn Thr Tyr Gln Trp Ile540 545 550atc aga aac tgg gaa act gtt aaa att caa tgg tct cag aat cct aca 1734Ile Arg Asn Trp Glu Thr Val Lys Ile Gln Trp Ser Gln Asn Pro Thr555 560 565atg ttg tac aac aaa atg gaa ttt gag cca ttt cag tct tta gtt cct 1782Met Leu Tyr Asn Lys Met Glu Phe Glu Pro Phe Gln Ser Leu Val Pro570 575 580 585aag gcc att aga ggc caa tac agt gga ttt gtc aga act cta ttc caa 1830Lys Ala Ile Arg Gly Gln Tyr Ser Gly Phe Val Arg Thr Leu Phe Gln590 595 600caa atg agg gat gta ctt ggg aca ttt gat acc acc cag ata ata aaa 1878Gln Met Arg Asp Val Leu Gly Thr Phe Asp Thr Thr Gln Ile Ile Lys605 610 615ctt ctc ccc ttt gca gcc gcc cca cca aag caa agt aga atg cag ttc 1926Leu Leu Pro Phe Ala Ala Ala Pro Pro Lys Gln Ser Arg Met Gln Phe620 625 630tct tca ttg act gtg aat gtg agg gga tca ggg atg aga ata ctt gta 1974Ser Ser Leu Thr Val Asn Val Arg Gly Ser Gly Met Arg Ile Leu Val635 640 645agg ggc aat tct cct gta ttc aac tac aac aag acc act aaa aga cta 2022Arg Gly Asn Ser Pro Val Phe Asn Tyr Asn Lys Thr Thr Lys Arg Leu650 655 660 665aca att ctc gga aaa gat gct ggc act tta att gaa gac cca gat gaa 2070Thr Ile Leu Gly Lys Asp Ala Gly Thr Leu Ile Glu Asp Pro Asp Glu670 675 680agc aca tcc gga gtg gag tcc gct gtt ttg aga gga ttt ctc att cta 2118Ser Thr Ser Gly Val Glu Ser Ala Val Leu Arg Gly Phe Leu Ile Leu685 690 695ggt aag gaa gat aga aga tac gga cca gca tta agc atc aat gaa ctg 2166Gly Lys Glu Asp Arg Arg Tyr Gly Pro Ala Leu Ser Ile Asn Glu Leu700 705 710agt aac ctt gca aaa gga gaa aag gct aat gtg cta att ggg caa gga 2214Ser Asn Leu Ala Lys Gly Glu Lys Ala Asn Val Leu Ile Gly Gln Gly715 720 725gac gtg gtg ttg gta atg aaa cga aaa cgg gac tct agc ata ctt act 2262Asp Val Val Leu Val Met Lys Arg Lys Arg Asp Ser Ser Ile Leu Thr730 735 740 745gac agc cag aca gcg acc aaa aga att cgg atg gcc atc aat 2304Asp Ser Gln Thr Ala Thr Lys Arg Ile Arg Met Ala Ile Asn750 755taatgttgaa tagtttaaaa acgaccttgt ttctact 2341<210>2<211>759<212>PRT<213>流感A病毒<400>2Met Glu Arg Ile Lys Glu Leu Arg Asn Leu Met Ser Gln Sar Arg Thr1 5 10 15Arg Glu Ile Leu Thr Lys Thr Thr Val Asp His Met Ala Ile Ile Lys20 25 30Lys Tyr Thr Ser Gly Arg Gln Glu Lys Asn Pro Ser Leu Arg Met Lys
35 40 45Trp Met Met Ala Met Lys Tyr Pro Ile Thr Ala Asp Lys Arg Ile Thr50 55 60Glu Met Val Pro Glu Arg Asn Glu Gln Gly Gln Thr Leu Trp Ser Lys65 70 75 80Met Ser Asp Ala Gly Ser Asp Arg Val Met Val Ser Pro Leu Ala Val85 90 95Thr Trp Trp Asn Arg Asn Gly Pro Val Thr Ser Thr Val His Tyr Pro100 105 110Lys Val Tyr Lys Thr Tyr Phe Asp Lys Val Glu Arg Leu Lys His Gly115 120 125Thr Phe Gly Pro Val His Phe Arg Asn Gln Val Lys Ile Arg Arg Arg130 135 140Val Asp Ile Asn Pro Gly His Ala Asp Leu Ser Ala Lys Glu Ala Gln145 150 155 160Asp Val Ile Met Glu Val Val Phe Pro Asn Glu Val Gly Ala Arg Ile165 170 175Leu Thr Ser Glu Ser Gln Leu Thr Ile Thr Lys Glu Lys Lys Glu Glu180 185 190Leu Gln Asp Cys Lys Ile Ser Pro Leu Met Val Ala Tyr Met Leu Glu195 200 205Arg Glu Leu Val Arg Lys Thr Arg Phe Leu Pro Val Ala Gly Gly Thr210 215 220Ser Ser Val Tyr Ile Glu Val Leu His Leu Thr Gln Gly Thr Cys Trp225 230 235 240Glu Gln Met Tyr Thr Pro Gly Gly Glu Val Arg Asn Asp Asp Val Asp245 250 255Gln Ser Leu Ile Ile Ala Ala Arg Asn Ile Val Arg Arg Ala Ala Val260 265 270Ser Ala Asp Pro Leu Ala Ser Leu Leu Glu Met Cys His Ser Thr Leu275 280 285Ile Gly Gly Thr Arg Met Val Asp Ile Leu Arg Gln Asn Pro Thr Glu290 295 300Glu Gln Ala Val Asp Ile Cys Lys Ala Ala Met Gly Leu Arg Ile Ser305 310 315 320Ser Ser Phe Ser Phe Gly Gly Phe Thr Phe Lys Arg Thr Ser Gly Ser325 330 335Ser Ile Lys Arg Glu Glu Glu Val Leu Thr Gly Asn Leu Gln Thr Leu340 345 350Lys Ile Arg Val His Glu Gly Tyr Glu Glu Phe Thr Met Val Gly Lys355 360 365Arg Ala Thr Ala Ile Leu Arg Lys Ala Thr Arg Arg Leu Val Gln Leu370 375 380Ile Val Ser Gly Arg Asp Glu Gln Ser Ile Ala Glu Ala Ile Ile Val385 390 395 400Ala Met Val Phe Ser Gln Glu Asp Cys Met Ile Lys Ala Val Arg Gly405 410 415Asp Leu Asn Phe Val Asn Arg Ala Asn Gln Arg Leu Asn Pro Met His420 425 430Gln Leu Leu Arg His Phe Gln Lys Asp Ala Lys Val Leu Phe Gln Asn435 440 445Trp Gly Ile Glu His Ile Asp Asn Val Met Gly Met Val Gly Val Leu450 455 460Pro Asp Met Thr Pro Ser Thr Glu Met Ser Met Arg Gly Ile Arg Val465 470 475 480Ser Lys Met Gly Val Asp Glu Tyr Ser Ser Thr Glu Arg Val Val Val485 490 495Ser Ile Asp Arg Phe Leu Arg Val Arg Asp Gln Arg Gly Asn Val Leu500 505 510Leu Ser Pro Glu Glu Val Ser Glu Thr Gln Gly Thr Glu Arg Leu Thr515 520 525Ile Thr Tyr Ser Ser Ser Met Met Trp Glu Ile Asn Gly Pro Glu Ser530 535 540Val Leu Val Asn Thr Tyr Gln Trp Ile Ile Arg Asn Trp Glu Thr Val545 550 555 560Lys Ile Gln Trp Ser Gln Asn Pro Thr Met Leu Tyr Asn Lys Met Glu565 570 575Phe Glu Pro Phe Gln Ser Leu Val Pro Lys Ala Ile Arg Gly Gln Tyr580 585 590Ser Gly Phe Val Arg Thr Leu Phe Gln Gln Met Arg Asp Val Leu Gly595 600 605Thr Phe Asp Thr Thr Gln Ile Ile Lys Leu Leu Pro Phe Ala Ala Ala610 615 620Pro Pro Lys Gln Ser Arg Met Gln Phe Ser Ser Leu Thr Val Asn Val625 630 635 640Arg Gly Ser Gly Met Arg Ile Leu Val Arg Gly Asn Ser Pro Val Phe645 650 655Asn Tyr Asn Lys Thr Thr Lys Arg Leu Thr Ile Leu Gly Lys Asp Ala660 665 670Gly Thr Leu Ile Glu Asp Pro Asp Glu Ser Thr Ser Gly Val Glu Ser675 680 685Ala Val Leu Arg Gly Phe Leu Ile Leu Gly Lys Glu Asp Arg Arg Tyr690 695 700Gly Pro Ala Leu Ser Ile Asn Glu Leu Ser Asn Leu Ala Lys Gly Glu705 710 715 720Lys Ala Asn Val Leu Ile Gly Gln Gly Asp Val Val Leu Val Met Lys725 730 735Arg Lys Arg Asp Ser Ser Ile Leu Thr Asp Ser Gln Thr Ala Thr Lys740 745 750Arg Ile Arg Met Ala Ile Asn755<210>3<211>2341<212>DNA<213>流感A病毒<220><221>CDS<222>(25)..(2295)<400>3agcgaaagca ggcaaaccat ttga atg gat gtc aac ccg act tta ctt ttc51Met Asp Val Asn Pro Thr Leu Leu Phe1 5ttg aaa gtt cca gcg caa aat gcc ata agc acc aca ttc cct tat act 99Leu Lys Val Pro Ala Gln Asn Ala Ile Ser Thr Thr Phe Pro Tyr Thr10 15 20 25gga gat cct cca tac agc cat gga aca gga aca gga tac acc atg gac 147Gly Asp Pro Pro Tyr Ser His Gly Thr Gly Thr Gly Tyr Thr Met Asp30 35 40aca gtc aac aga aca cat caa tat tca gaa aaa ggg aag tgg aca aca 195Thr Val Asn Arg Thr His Gln Tyr Ser Glu Lys Gly Lys Trp Thr Thr45 50 55aac aca gaa act ggg gcg ccc caa ctt aac cca att gat gga cca cta 243Asn Thr Glu Thr Gly Ala Pro Gln Leu Asn Pro Ile Asp Gly Pro Leu60 65 70cct gag gat aat gag cca agt gga tat gca caa aca gac tgt gtc ctg 291Pro Glu Asp Asn Glu Pro Ser Gly Tyr Ala Gln Thr Asp Cys Val Leu75 80 85gaa gca atg gct ttc ctt gaa gaa tcc cac cca ggg atc ttt gaa aac 339Glu Ala Met Ala Phe Leu Glu Glu Ser His Pro Gly Ile Phe Glu Asn90 95 100 105tcg tgc ctt gaa acg atg gaa gtc gtt caa caa aca agg gtg gac aga 387Ser Cys Leu Glu Thr Met Glu Val Val Gln Gln Thr Arg Val Asp Arg110 115 120ctg acc caa ggt cgt cag acc tat gat tgg aca tta aac aga aat caa 435Leu Thr Gln Gly Arg Gln Thr Tyr Asp Trp Thr Leu Asn Arg Asn Gln125 130 135ccg gcc gca act gca tta gcc aac act ata gaa gtc ttc aga tcg aat 483Pro Ala Ala Thr Ala Leu Ala Asn Thr Ile Glu Val Phe Arg Ser Asn140 145 150ggt cta aca gct aat gag tcg gga agg cta ata gat ttc ctc aag gat 531Gly Leu Thr Ala Asn Glu Ser Gly Arg Leu Ile Asp Phe Leu Lys Asp155 160 165gtg atg gaa tca atg gat aaa gag gaa atg gag ata aca aca cac ttc 579Val Met Glu Ser Met Asp Lys Glu Glu Met Glu Ile Thr Thr His Phe170 175 180 185caa aga aaa aga aga gta aga gac aac atg acc aag aaa atg gtc aca 627Gln Arg Lys Arg Arg Val Arg Asp Asn Met Thr Lys Lys Met Val Thr190 195 200caa aga aca ata gga aag aag aag cag aga gtg aac aag aga agc tat 675Gln Arg Thr Ile Gly Lys Lys Lys Gln Arg Val Asn Lys Arg Ser Tyr205 210 215cta ata aga gca tta aca ttg aac aca atg acc aaa gat gca gaa aga 723Leu Ile Arg Ala Leu Thr Leu Asn Thr Met Thr Lys Asp Ala Glu Arg220 225 230ggt aaa tta aag aga aga gct att gca aca ccc ggg atg caa atc aga 771Gly Lys Leu Lys Arg Arg Ala Ile Ala Thr Pro Gly Met Gln Ile Arg235 240 245ggg ttc gtg tac ttt gtt gaa act cta gct agg agc att tgt gag aag 819Gly Phe Val Tyr Phe Val Glu Thr Leu Ala Arg Ser Ile Cys Glu Lys250 255 260 265ctt gaa cag tct gga ctt cca gtt gga ggt aat gaa aag aag gcc aaa 867Leu Glu Gln Ser Gly Leu Pro Val Gly Gly Asn Glu Lys Lys Ala Lys270 275 280ctg gca aat gtt gtg aga aag atg atg act aat tca caa gac aca gag 915Leu Ala Asn Val Val Arg Lys Met Met Thr Asn Ser Gln Asp Thr Glu285 290 295ctt tct ttc aca att act gga gac aat act aag tgg aat gaa aat caa 963Leu Ser Phe Thr Ile Thr Gly Asp Asn Thr Lys Trp Asn Glu Asn Gln300 305 310aat cct cga atg ttc ctg gcg atg att aca tat atc aca aaa aat caa 1011Asn Pro Arg Met Phe Leu Ala Met Ile Thr Tyr Ile Thr Lys Asn Gln315 320 325cct gaa tgg ttc aga aac att ctg agc atc gca ccc ata atg ttc tca 1059Pro Glu Trp Phe Arg Asn Ile Leu Ser Ile Ala Pro Ile Met Phe Ser330 335 340 345aac aaa atg gcg aga cta ggg aaa gga tac atg ttc gaa agt aag aga 1107Asn Lys Met Ala Arg Leu Gly Lys Gly Tyr Met Phe Glu Ser Lys Arg350 355 360atg aag ctc cga aca caa ata cca gca gaa atg cta gca agc att gac 1155Met Lys Leu Arg Thr Gln Ile Pro Ala Glu Met Leu Ala Ser Ile Asp365 370 375cta aag tat ttc aat gaa tca aca aga aag aaa att gag aaa ata aag 1203Leu Lys Tyr Phe Asn Glu Ser Thr Arg Lys Lys Ile Glu Lys Ile Lys380 385 390cct ctt cta ata gat ggc aca gcg tca ttg agt cct gga atg atg atg 1251Pro Leu Leu Ile Asp Gly Thr Ala Ser Leu Ser Pro Gly Met Met Met395 400 405ggc atg ttc aac atg cta agt acg gtt tta gga gtc tca atc ctg aat 1299Gly Met Phe Asn Met Leu Ser Thr Val Leu Gly Val Ser Ile Leu Asn410 415 420 425ctt ggg caa aag aaa tac acc aaa aca aca tac tgg tgg gat gga ctc 1347Leu Gly Gln Lys Lys Tyr Thr Lys Thr Thr Tyr Trp Trp Asp Gly Leu430 435 440caa tcc tct gat gat ttt gct ctc ata gtg aat gca cca aat cat gag 1395Gln Ser Ser Asp Asp Phe Ala Leu Ile Val Asn Ala Pro Asn His Glu445 450 455gga ata caa gca gga gtg gat aga ttc tac agg acc tgc aag tta gtc 1443Gly Ile Gln Ala Gly Val Asp Arg Phe Tyr Arg Thr Cys Lys Leu Val460 465 470gga atc aat atg agc aag aag aag tcc tat ata aat agg aca gga aca 1491Gly Ile Asn Met Ser Lys Lys Lys Ser Tyr Ile Asn Arg Thr Gly Thr475 480 485ttt gaa ttc aca agc ttt ttt tat cgc tat gga ttt gta gcc aat ttt 1539Phe Glu Phe Thr Ser Phe Phe Tyr Arg Tyr Gly Phe Val Ala Asn Phe490 495 500 505agc atg gag ctg ccc agt ttt gga gtg tct ggg att aat gag tca gct 1587Ser Met Glu Leu Pro Ser Phe Gly Val Ser Gly Ile Asn Glu Ser Ala510 515 520gat atg agc att gga gta aca gtg ata aag aac aac atg ata aac aat 1635Asp Met Ser Ile Gly Val Thr Val Ile Lys Asn Asn Met Ile Asn Asn525 530 535gac ctt gga cca gca aca gcc cag atg gct ctt caa ctg ttc atc aag 1683Asp Leu Gly Pro Ala Thr Ala Gln Met Ala Leu Gln Leu Phe Ile Lys540 545 550gac tac aga tat aca tat cgg tgc cac aga gga gac aca caa att cag 1731Asp Tyr Arg Tyr Thr Tyr Arg Cys His Arg Gly Asp Thr Gln Ile Gln555 560 565acg agg aga tca ttc gag cta aag aag ctg tgg gag caa acc cgc tca 1779Thr Arg Arg Ser Phe Glu Leu Lys Lys Leu Trp Glu Gln Thr Arg Ser570 575 580 585aag gca gga cta ttg gtt tca gat gga gga cca aac tta tac aat atc 1827Lys Ala Gly Leu Leu Val Ser Asp Gly Gly Pro Asn Leu Tyr Asn Ile590 595 600cgg aat ctt cac atc cct gaa gtc tgc tta aag tgg gag cta atg gat 1875Arg Asn Leu His Ile Pro Glu Val Cys Leu Lys Trp Glu Leu Met Asp605 610 615gag gac tat cag gga aga ctt tgt aat ccc ctg aat cca ttt gtc agc 1923Glu Asp Tyr Gln Gly Arg Leu Cys Asn Pro Leu Asn Pro Phe Val Ser620 625 630cat aag gag att gag tct gta aac aat gct gtg gta atg cca gct cat 1971His Lys Glu Ile Glu Ser Val Asn Asn Ala Val Val Met Pro Ala His635 640 645ggt cca gcc aag agc atg gaa tat gac gct gtt gca act aca cac tcc 2019Gly Pro Ala Lys Ser Met Glu Tyr Asp Ala Val Ala Thr Thr His Ser650 655 660 665tgg att ccc aag agg aac cgc tct att ctc aac aca agc caa agg gga 2067Trp Ile Pro Lys Arg Asn Arg Ser Ile Leu Asn Thr Ser Gln Arg Gly670 675 680att ctt gag gat gaa cag atg tat cag aag tgc tgc aac ctg ttc gag 2115Ile Leu Glu Asp Glu Gln Met Tyr Gln Lys Cys Cys Asn Leu Phe Glu685 690 695aaa ttt ttc ccc agt agt tca tac agg aga ccg gtt gga att tcc agc 2163Lys Phe Phe Pro Ser Ser Ser Tyr Arg Arg Pro Val Gly Ile Ser Ser700 705 710atg gtg gag gcc atg gtg tct agg gcc cgg att gat gcc aga att gac 2211Met Val Glu Ala Met Val Ser Arg Ala Arg Ile Asp Ala Arg Ile Asp715 720 725ttc gaa tct gga cgg att aag aaa gaa gag ttc gcc gag atc atg aag 2259Phe Glu Ser Gly Arg Ile Lys Lys Glu Glu Phe Ala Glu Ile Met Lys730 735 740 745atc tgt tcc acc att gaa gag ctc aga cgg caa aaa taatgaattt2305Ile Cys Ser Thr Ile Glu Glu Leu Arg Arg Gln Lys750 755agcttgtcct tcatgaaaaa atgccttgtt tctact 2341<210>4<211>757<212>PRT<213>流感A病毒<400>4Met Asp Val Asn Pro Thr Leu Leu Phe Leu Lys Val Pro Ala Gln Asn1 5 10 15Ala Ile Ser Thr Thr Phe Pro Tyr Thr Gly Asp Pro Pro Tyr Ser His
20 25 30Gly Thr Gly Thr Gly Tyr Thr Met Asp Thr Val Asn Arg Thr His Gln35 40 45Tyr Ser Glu Lys Gly Lys Trp Thr Thr Asn Thr Glu Thr Gly Ala Pro50 55 60Gln Leu Asn Pro Ile Asp Gly Pro Leu Pro Glu Asp Asn Glu Pro Ser65 70 75 80Gly Tyr Ala Gln Thr Asp Cys Val Leu Glu Ala Met Ala Phe Leu Glu85 90 95Glu Ser His Pro Gly Ile Phe Glu Asn Ser Cys Leu Glu Thr Met Glu100 105 110Val Val Gln Gln Thr Arg Val Asp Arg Leu Thr Gln Gly Arg Gln Thr115 120 125Tyr Asp Trp Thr Leu Asn Arg Asn Gln Pro Ala Ala Thr Ala Leu Ala130 135 140Asn Thr Ile Glu Val Phe Arg Ser Asn Gly Leu Thr Ala Asn Glu Ser145 150 155 160Gly Arg Leu Ile Asp Phe Leu Lys Asp Val Met Glu Ser Met Asp Lys165 170 175Glu Glu Met Glu Ile Thr Thr His Phe Gln Arg Lys Arg Arg Val Arg180 185 190Asp Asn Met Thr Lys Lys Met Val Thr Gln Arg Thr Ile Gly Lys Lys195 200 205Lys Gln Arg Val Asn Lys Arg Ser Tyr Leu Ile Arg Ala Leu Thr Leu210 215 220Asn Thr Met Thr Lys Asp Ala Glu Arg Gly Lys Leu Lys Arg Arg Ala225 230 235 240Ile Ala Thr Pro Gly Met Gln Ile Arg Gly Phe Val Tyr Phe Val Glu245 250 255Thr Leu Ala Arg Ser Ile Cys Glu Lys Leu Glu Gln Ser Gly Leu Pro260 265 270Val Gly Gly Asn Glu Lys Lys Ala Lys Leu Ala Asn Val Val Arg Lys275 280 285Met Met Thr Asn Ser Gln Asp Thr Glu Leu Ser Phe Thr Ile Thr Gly290 295 300Asp Asn Thr Lys Trp Asn Glu Asn Gln Asn Pro Arg Met Phe Leu Ala305 310 315 320Met Ile Thr Tyr Ile Thr Lys Asn Gln Pro Glu Trp Phe Arg Asn Ile325 330 335Leu Ser Ile Ala Pro Ile Met Phe Ser Asn Lys Met Ala Arg Leu Gly340 345 350Lys Gly Tyr Met Phe Glu Ser Lys Arg Met Lys Leu Arg Thr Gln Ile355 360 365Pro Ala Glu Met Leu Ala Ser Ile Asp Leu Lys Tyr Phe Asn Glu Ser370 375 380Thr Arg Lys Lys Ile Glu Lys Ile Lys Pro Leu Leu Ile Asp Gly Thr385 390 395 400Ala Ser Leu Ser Pro Gly Met Met Met Gly Met Phe Asn Met Leu Ser405 410 415Thr Val Leu Gly Val Ser Ile Leu Asn Leu Gly Gln Lys Lys Tyr Thr420 425 430Lys Thr Thr Tyr Trp Trp Asp Gly Leu Gln Ser Ser Asp Asp Phe Ala435 440 445Leu Ile Val Asn Ala Pro Asn His Glu Gly Ile Gln Ala Gly Val Asp450 455 460Arg Phe Tyr Arg Thr Cys Lys Leu Val Gly Ile Asn Met Ser Lys Lys465 470 475 480Lys Ser Tyr Ile Asn Arg Thr Gly Thr Phe Glu Phe Thr Ser Phe Phe485 490 495Tyr Arg Tyr Gly Phe Val Ala Asn Phe Ser Met Glu Leu Pro Ser Phe500 505 510Gly Val Ser Gly Ile Asn Glu Ser Ala Asp Met Ser Ile Gly Val Thr515 520 525Val Ile Lys Asn Asn Met Ile Asn Asn Asp Leu Gly Pro Ala Thr Ala530 535 540Gln Met Ala Leu Gln Leu Phe Ile Lys Asp Tyr Arg Tyr Thr Tyr Arg545 550 555 560Cys His Arg Gly Asp Thr Gln Ile Gln Thr Arg Arg Ser Phe Glu Leu565 570 575Lys Lys Leu Trp Glu Gln Thr Arg Ser Lys Ala Gly Leu Leu Val Ser580 585 590Asp Gly Gly Pro Asn Leu Tyr Asn Ile Arg Asn Leu His Ile Pro Glu595 600 605Val Cys Leu Lys Trp Glu Leu Met Asp Glu Asp Tyr Gln Gly Arg Leu610 615 620Cys Asn Pro Leu Asn Pro Phe Val Ser His Lys Glu Ile Glu Ser Val625 630 635 640Asn Asn Ala Val Val Met Pro Ala His Gly Pro Ala Lys Ser Met Glu645 650 655Tyr Asp Ala Val Ala Thr Thr His Ser Trp Ile Pro Lys Arg Asn Arg660 665 670Ser Ile Leu Asn Thr Ser Gln Arg Gly Ile Leu Glu Asp Glu Gln Met675 680 685Tyr Gln Lys Cys Cys Asn Leu Phe Glu Lys Phe Phe Pro Ser Ser Ser690 695 700Tyr Arg Arg Pro Val Gly Ile Ser Ser Met Val Glu Ala Met Val Ser705 710 715 720Arg Ala Arg Ile Asp Ala Arg Ile Asp Phe Glu Ser Gly Arg Ile Lys725 730 735Lys Glu Glu Phe Ala Glu Ile Met Lys Ile Cys Ser Thr Ile Glu Glu740 745 750Leu Arg Arg Gln Lys755<210>5<211>2233<212>DNA<213>流感A病毒<220><221>CDS<222>(25)..(2172)<400>5agcaaaagca ggtactgatt cgag atg gaa gat ttt gtg cga caa tgc ttc51Met Glu Asp Phe Val Arg Gln Cys Phe1 5aat ccg atg att gtc gag ctt gca gaa aag gca atg aaa gag tat gga 99Asn Pro Met Ile Val Glu Leu Ala Glu Lys Ala Met Lys Glu Tyr Gly10 15 20 25gag gat ctg aaa atc gaa aca aac aaa ttt gca gca ata tgc act cac 147Glu Asp Leu Lys Ile Glu Thr Asn Lys Phe Ala Ala Ile Cys Thr His30 35 40ttg gag ata tgt ttc atg tat tca gat ttt cat ttc atc aat gaa caa 195Leu Glu Ile Cys Phe Met Tyr Ser Asp Phe His Phe Ile Asn Glu Gln45 50 55ggc gag tca ata gtg gta gag ctt gat gat cca aat gca ctg tta aag 243Gly Glu Ser Ile Val Val Glu Leu Asp Asp Pro Asn Ala Leu Leu Lys60 65 70cac aga ttt gaa ata ata gag gga aga gac cgc aca atg gcc tgg aca 291His Arg Phe Glu Ile Ile Glu Gly Arg Asp Arg Thr Met Ala Trp Thr75 80 85gta gta aac agt att tgc aac act act gga gct gag aaa ccg aag ttt 339Val Val Asn Ser Ile Cys Asn Thr Thr Gly Ala Glu Lys Pro Lys Phe90 95 100 105ctg cca gat ttg tat gat tac aag gag aat aga ttc atc gag att gga 387Leu Pro Asp Leu Tyr Asp Tyr Lys Glu Asn Arg Phe Ile Glu Ile Gly110 115 120gtg aca agg aga gaa gtc cac ata tac tac ctt gaa aag gcc aat aaa 435Val Thr Arg Arg Glu Val His Ile Tyr Tyr Leu Glu Lys Ala Asn Lys125 130 135att aaa tct gag aat aca cac atc cac att ttc tca ttc act ggg gag 483Ile Lys Ser Glu Asn Thr His Ile His Ile Phe Ser Phe Thr Gly Glu140 145 150gaa atg gcc aca aag gcc gac tac act ctt gat gat gaa agc agg gct 531Glu Met Ala Thr Lys Ala Asp Tyr Thr Leu Asp Asp Glu Ser Arg Ala155 160 165agg atc aaa acc agg cta ttt acc ata aga caa gaa atg gcc aac aga 579Arg Ile Lys Thr Arg Leu Phe Thr Ile Arg Gln Glu Met Ala Asn Arg170 175 180 185ggc ctc tgg gat tcc ttt cgt cag tcc gaa aga ggc gaa gaa aca att 627Gly Leu Trp Asp Ser Phe Arg Gln Ser Glu Arg Gly Glu Glu Thr Ile190 195 200gaa gaa aga ttt gaa atc aca gga act atg cgc agg ctt gcc gac caa 675Glu Glu Arg Phe Glu Ile Thr Gly Thr Met Arg Arg Leu Ala Asp Gln205 210 215agt ctc ccg ccg aac ttc tcc tgc ctt gag aat ttt aga gcc tat gtg 723Ser Leu Pro Pro Asn Phe Ser Cys Leu Glu Asn Phe Arg Ala Tyr Val220 225 230gat gga ttc gaa ccg aac ggc tgc att gag ggc aag ctt tct caa atg 771Asp Gly Phe Glu Pro Asn Gly Cys Ile Glu Gly Lys Leu Ser Gln Met235 240 245tcc aaa gaa gtg aat gca aga att gaa cct ttt ctg aag aca aca cca 819Ser Lys Glu Val Asn Ala Arg Ile Glu Pro Phe Leu Lys Thr Thr Pro250 255 260 265aga cca atc aaa ctt ccg gat ggg cct cct tgt ttt cag cgg tcc aaa 867Arg Pro Ile Lys Leu Pro Asp Gly Pro Pro Cys Phe Gln Arg Ser Lys270 275 280ttc ctt ctg atg gat gct tta aaa tta agc att gaa gac cca agt cac 915Phe Leu Leu Met Asp Ala Leu Lys Leu Ser Ile Glu Asp Pro Ser His285 290 295gaa gga gag gga ata cca cta tat gat gcg atc aag tgc atg aga aca 963Glu Gly Glu Gly Ile Pro Leu Tyr Asp Ala Ile Lys Cys Met Arg Thr300 305 310ttc ttt gga tgg aaa gaa ccc tat atc gtc aaa cca cac gaa aag gga 1011Phe Phe Gly Trp Lys Glu Pro Tyr Ile Val Lys Pro His Glu Lys Gly315 320 325ata aat cca aat tat ctg ctg tca tgg aag caa gta ctg gca gaa cta 1059Ile Asn Pro Asn Tyr Leu Leu Set Trp Lys Gln Val Leu Ala Glu Leu330 335 340 345cag gac att gaa aat gag gag aag att cca aga act aaa aac atg aag 1107Gln Asp Ile Glu Asn Glu Glu Lys Ile Pro Arg Thr Lys Asn Met Lys350 355 360aaa acg agt cag cta aag tgg gca ctt ggt gag aac atg gca cct gag 1155Lys Thr Ser Gln Leu Lys Trp Ala Leu Gly Glu Asn Met Ala Pro Glu365 370 375aaa gta gac ttt gac aac tgt aga gac ata agc gat ttg aag caa tat 1203Lys Val Asp Phe Asp Asn Cys Arg Asp Ile Ser Asp Leu Lys Gln Tyr380 385 390gat agt gac gaa cct gaa tta agg tca ctt tca agc tgg atc cag aat 1251Asp Ser Asp Glu Pro Glu Leu Arg Ser Leu Ser Ser Trp Ile Gln Asn395 400 405gag ttc aac aag gca tgc gag ctg acc gat tca atc tgg ata gag ctc 1299Glu Phe Asn Lys Ala Cys Glu Leu Thr Asp Ser Ile Trp Ile Glu Leu410 415 420 425gat gag att gga gaa gac gtg gct cca att gaa tac att gca agc atg 1347Asp Glu Ile Gly Glu Asp Val Ala Pro Ile Glu Tyr Ile Ala Ser Met430 435 440agg agg aat tac ttc aca gca gag gtg tcc cat tgc aga gcc aca gaa 1395Arg Arg Asn Tyr Phe Thr Ala Glu Val Ser His Cys Arg Ala Thr Glu445 450 455tac ata atg aag ggg gta tac att aat act gcc ttg ctt aat gca tcc 1443Tyr Ile Met Lys Gly Val Tyr Ile Asn Thr Ala Leu Leu Asn Ala Ser460 465 470tgt gca gca atg gac gat ttc caa cta att ccc atg ata agc aag tgc 1491Cys Ala Ala Met Asp Asp Phe Gln Leu Ile Pro Met Ile Ser Lys Cys475 480 485aga act aaa gag gga agg cga aaa acc aat tta tat gga ttc atc ata 1539Arg Thr Lys Glu Gly Arg Arg Lys Thr Asn Leu Tyr Gly Phe Ile Ile490 495 500 505aaa gga aga tct cac tta agg aat gac acc gac gtg gta aac ttt gtg 1587Lys Gly Arg Ser His Leu Arg Asn Asp Thr Asp Val Val Asn Phe Val510 515 520agc atg gag ttt tct ctc act gac ccg aga ctt gag cca cat aaa tgg 1635Ser Met Glu Phe Ser Leu Thr Asp Pro Arg Leu Glu Pro His Lys Trp525 530 535gag aaa tac tgt gtc ctt gag ata gga gat atg cta cta aga agt gcc 1683Glu Lys Tyr Cys Val Leu Glu Ile Gly Asp Met Leu Leu Arg Ser Ala540 545 550ata ggc cag atg tca agg cct atg ttc ttg tat gtg agg aca aat gga 1731Ile Gly Gln Met Ser Arg Pro Met Phe Leu Tyr Val Arg Thr Asn Gly555 560 565aca tca aag att aaa atg aaa tgg gga atg gag atg aga cgt tgc ctc 1779Thr Ser Lys Ile Lys Met Lys Trp Gly Met Glu Met Arg Arg Cys Leu570 575 580 585ctt cag tca ctc caa caa atc gag agc atg att gaa gcc gag tct tct 1827Leu Gln Ser Leu Gln Gln Ile Glu Ser Met Ile Glu Ala Glu Ser Ser590 595 600gtc aaa gag aaa gac atg acc aaa gag ttt ttt gag aat aaa tca gaa 1875Val Lys Glu Lys Asp Met Thr Lys Glu Phe Phe Glu Asn Lys Ser Glu605 610 615aca tgg ccc att ggg gag tcc ccc aag gga gtg gaa gaa ggt tcc att 1923Thr Trp Pro Ile Gly Glu Ser Pro Lys Gly Val Glu Glu Gly Ser Ile620 625 630gga aag gtc tgt agg act tta ttg gcc aag tcg gta ttc aat agc ctg 1971Gly Lys Val Cys Arg Thr Leu Leu Ala Lys Ser Val Phe Asn Ser Leu635 640 645tat gca tcc cca caa ttg gaa gga ttt tea gcg gag tca aga aaa ctg 2019Tyr Ala Ser Pro Gln Leu Glu Gly Phe Ser Ala Glu Ser Arg Lys Leu650 655 660 665ctt ctt gtc gtt cag gct ctt agg gac aac ctt gaa cct gga acc ttt 2067Leu Leu Val Val Gln Ala Leu Arg Asp Asn Leu Glu Pro Gly Thr Phe670 675 680gat ctt ggg ggg cta tat gaa gca att gag gag tgc ctg att aat gat 2115Asp Leu Gly Gly Leu Tyr Glu Ala Ile Glu Glu Cys Leu Ile Asn Asp685 690 695ccc tgg gtt ttg ctt aat gcg tct tgg ttc aac tcc ttc cta aca cat 2163Pro Trp Val Leu Leu Asn Ala Ser Trp Phe Asn Ser Phe Leu Thr His700 705 710gca tta aga tagttgtggc aatgctacta tttgctatcc atactgtcca 2212Ala Leu Arg715aaaaagtacc ttgtttctac t 2233<210>6<211>716<212>PRT<213>流感A病毒<400>6Met Glu Asp Phe Val Arg Gln Cys Phe Asn Pro Met Ile Val Glu Leu1 5 10 15Ala Glu Lys Ala Met Lys Glu Tyr Gly Glu Asp Leu Lys Ile Glu Thr20 25 30Asn Lys Phe Ala Ala Ile Cys Thr His Leu Glu Ile Cys Phe Met Tyr35 40 45Ser Asp Phe His Phe Ile Asn Glu Gln Gly Glu Ser Ile Val Val Glu50 55 60Leu Asp Asp Pro Asn Ala Leu Leu Lys His Arg Phe Glu Ile Ile Glu65 70 75 80Gly Arg Asp Arg Thr Met Ala Trp Thr Val Val Asn Ser Ile Cys Asn85 90 95Thr Thr Gly Ala Glu Lys Pro Lys Phe Leu Pro Asp Leu Tyr Asp Tyr100 105 110Lys Glu Asn Arg Phe Ile Glu Ile Gly Val Thr Arg Arg Glu Val His115 120 125Ile Tyr Tyr Leu Glu Lys Ala Asn Lys Ile Lys Ser Glu Asn Thr His130 135 140Ile His Ile Phe Ser Phe Thr Gly Glu Glu Met Ala Thr Lys Ala Asp145 150 155 160Tyr Thr Leu Asp Asp Glu Ser Arg Ala Arg Ile Lys Thr Arg Leu Phe165 170 175Thr Ile Arg Gln Glu Met Ala Asn Arg Gly Leu Trp Asp Ser Phe Arg180 185 190Gln Ser Glu Arg Gly Glu Glu Thr Ile Glu Glu Arg Phe Glu Ile Thr195 200 205Gly Thr Met Arg Arg Leu Ala Asp Gln Ser Leu Pro Pro Asn Phe Ser210 215 220Cys Leu Glu Asn Phe Arg Ala Tyr Val Asp Gly Phe Glu Pro Asn Gly225 230 235 240Cys Ile Glu Gly Lys Leu Ser Gln Met Ser Lys Glu Val Asn Ala Arg245 250 255Ile Glu Pro Phe Leu Lys Thr Thr Pro Arg Pro Ile Lys Leu Pro Asp260 265 270Gly Pro Pro Cys Phe Gln Arg Ser Lys Phe Leu Leu Met Asp Ala Leu275280 285Lys Leu Ser Ile Glu Asp Pro Ser His Glu Gly Glu Gly Ile Pro Leu290 295 300Tyr Asp Ala Ile Lys Cys Met Arg Thr Phe Phe Gly Trp Lys Glu Pro305 310 315 320Tyr Ile Val Lys Pro His Glu Lys Gly Ile Asn Pro Asn Tyr Leu Leu325 330 335Ser Trp Lys Gln Val Leu Ala Glu Leu Gln Asp Ile Glu Asn Glu Glu340 345 350Lys Ile Pro Arg Thr Lys Asn Met Lys Lys Thr Ser Gln Leu Lys Trp355 360 365Ala Leu Gly Glu Asn Met Ala Pro Glu Lys Val Asp Phe Asp Asn Cys370 375 380Arg Asp Ile Ser Asp Leu Lys Gln Tyr Asp Ser Asp Glu Pro Glu Leu385 390 395 400Arg Ser Leu Ser Ser Trp Ile Gln Asn Glu Phe Asn Lys Ala Cys Glu405 410 415Leu Thr Asp Ser Ile Trp Ile Glu Leu Asp Glu Ile Gly Glu Asp Val420 425 430Ala Pro Ile Glu Tyr Ile Ala Ser Met Arg Arg Asn Tyr Phe Thr Ala435 440 445Glu Val Ser His Cys Arg Ala Thr Glu Tyr Ile Met Lys Gly Val Tyr450 455 460Ile Asn Thr Ala Leu Leu Asn Ala Ser Cys Ala Ala Met Asp Asp Phe465 470 475 480Gln Leu Ile Pro Met Ile Ser Lys Cys Arg Thr Lys Glu Gly Arg Arg485 490 495Lys Thr Asn Leu Tyr Gly Phe Ile Ile Lys Gly Arg Ser His Leu Arg500 505 510Asn Asp Thr Asp Val Val Asn Phe Val Ser Met Glu Phe Ser Leu Thr515 520 525Asp Pro Arg Leu Glu Pro His Lys Trp Glu Lys Tyr Cys Val Leu Glu530 535 540Ile Gly Asp Met Leu Leu Arg Ser Ala Ile Gly Gln Met Ser Arg Pro545 550 555 560Met Phe Leu Tyr Val Arg Thr Asn Gly Thr Ser Lys Ile Lys Met Lys565 570 575Trp Gly Met Glu Met Arg Arg Cys Leu Leu Gln Ser Leu Gln Gln Ile580 585 590Glu Ser Met Ile Glu Ala Glu Ser Ser Val Lys Glu Lys Asp Met Thr595 600 605Lys Glu Phe Phe Glu Asn Lys Ser Glu Thr Trp Pro Ile Gly Glu Ser610 615 620Pro Lys Gly Val Glu Glu Gly Ser Ile Gly Lys Val Cys Arg Thr Leu625 630 635 640Leu Ala Lys Ser Val Phe Asn Ser Leu Tyr Ala Ser Pro Gln Leu Glu645 650 655Gly Phe Ser Ala Glu Ser Arg Lys Leu Leu Leu Val Val Gln Ala Leu660 665 670Arg Asp Asn Leu Glu Pro Gly Thr Phe Asp Leu Gly Gly Leu Tyr Glu
675 680 685Ala Ile Glu Glu Cys Leu Ile Asn Asp Pro Trp Val Leu Leu Asn Ala690 695 700Ser Trp Phe Asn Ser Phe Leu Thr His Ala Leu Arg705 710 715<210>7<211>1765<212>DNA<213>流感A病毒<220><221>CDS<222>(30)..(1727)<400>7agcaaaagca ggggataatt ctattaacc atg aag act atc att gct ttg agc 53Met Lys Thr Ile Ile Ala Leu Ser1 5tac att ttc tgt ctg gtt ctc ggc caa aac ttt cca gga aat gac aac 101Tyr Ile Phe Cys Leu Val Leu Gly Gln Asn Phe Pro Gly Asn Asp Asn10 15 20agc aca gca acg ctg tgc ctg gga cat cat gcg gtg cca aac gga aca 149Ser Thr Ala Thr Leu Cys Leu Gly His His Ala Val Pro Asn Gly Thr25 30 35 40cta gtg aaa aca atc aca aat gat cag att gaa gtg act aat gct act 197Leu Val Lys Thr Ile Thr Asn Asp Gln Ile Glu Val Thr Asn Ala Thr45 50 55gag ctg gtt cag agt tcc tca acg ggg aaa ata tgc aac aat cct cat 245Glu Leu Val Gln Ser Ser Ser Thr Gly Lys Ile Cys Asn Asn Pro His60 65 70cga atc ctt gat gga ata gac tgc aca ctg ata gat gct cta ttg ggg 293Arg Ile Leu Asp Gly Ile Asp Cys Thr Leu Ile Asp Ala Leu Leu Gly75 80 85gac cct cat tgt gat ggc ttt caa aat gag aca tgg gac ctt ttc gtt 341Asp Pro His Cys Asp Gly Phe Gln Asn Glu Thr Trp Asp Leu Phe Val90 95 100gaa cgc agc aaa gct ttc agc aac tgt tac cct tat gat gtg cca gat 389Glu Arg Ser Lys Ala Phe Ser Asn Cys Tyr Pro Tyr Asp Val Pro Asp105 110 115 120tat gcc tcc ctt agg tca cta gtt gcc tcg tca ggc act ctg gag ttt 437Tyr Ala Ser Leu Arg Ser Leu Val Ala Ser Ser Gly Thr Leu Glu Phe125 130 135atc agt gaa ggc ttc act tgg act ggg gtc act cag aat ggg gga agc 485Ile Ser Glu Gly Phe Thr Trp Thr Gly Val Thr Gln Asn Gly Gly Ser140 145 150aat gct tgc aaa agg gga cct gat agc ggt ttt ttc agt aga ctg aac 533Asn Ala Cys Lys Arg Gly Pro Asp Ser Gly Phe Phe Ser Arg Leu Asn155 160 165tgg ttg tac aaa tca gga agc aca tat cca gtg ctg aac gtg act atg 581Trp Leu Tyr Lys Ser Gly Ser Thr Tyr Pro Val Leu Asn Val Thr Met170 175 180cca aac aat gac aat ttt gac aaa cta tac att tgg ggg gtt cac cac 629Pro Asn Asn Asp Asn Phe Asp Lys Leu Tyr Ile Trp Gly Val His His185 190 195 200ccg agc acg gac caa gaa caa acc agc cta tat gtt caa gca tca ggg 677Pro Ser Thr Asp Gln Glu Gln Thr Ser Leu Tyr Val Gln Ala Ser Gly205 210 215aga gtc aca gtc tct acc aag aga agc cag caa act ata atc ccg aat 725Arg Val Thr Val Ser Thr Lys Arg Ser Gln Gln Thr Ile Ile Pro Asn220 225 230atc ggg tct aga ccc tgg gta agg ggt ctg tct agt aga ata agc atc 773Ile Gly Ser Arg Pro Trp Val Arg Gly Leu Ser Ser Arg Ile Ser Ile235 240 245tat tgg aca ata gtt aaa ccg gga gac ata ctg gta att aat agt aat 821Tyr Trp Thr Ile Val Lys Pro Gly Asp Ile Leu Val Ile Asn Ser Asn250 255 260ggg aac cta att gct cct cgg ggt tat ttt aaa atg cgc act ggg aaa 869Gly Asn Leu Ile Ala Pro Arg Gly Tyr Phe Lys Met Arg Thr Gly Lys265 270 275 280agc tca ata atg agg tca gat gca cct att ggc acc tgc att tct gaa 917Ser Ser Ile Met Arg Ser Asp Ala Pro Ile Gly Thr Cys Ile Ser Glu285 290 295tgc atc act cca aat gga agc att ccc aat gac aag ccc ttt caa aac 965Cys Ile Thr Pro Asn Gly Ser Ile Pro Asn Asp Lys Pro Phe Gln Asn300 305 310gta aac aag atc aca tat ggg gca tgt ccc aag tat gtt aag caa aac 1013Val Asn Lys Ile Thr Tyr Gly Ala Cys Pro Lys Tyr Val Lys Gln Asn315 320 325acc ctg aag ttg gca aca ggg atg cgg aat gta cca gag aaa caa act 1061Thr Leu Lys Leu Ala Thr Gly Met Arg Asn Val Pro Glu Lys Gln Thr330 335 340aga ggc cta ttc agc gca ata gca ggt ttc ata gaa aat ggg tgg gag 1109Arg Gly Leu Phe Ser Ala Ile Ala Gly Phe Ile Glu Asn Gly Trp Glu345 350 355 360gga atg ata gac ggt tgg tac ggt ttc agg cat caa aat tct gag ggc 1157Gly Met Ile Asp Gly Trp Tyr Gly Phe Arg His Gln Asn Ser Glu Gly365 370 375aca gga caa gca gca gat ctt aaa agc act caa gca gcc atc gac caa 1205Thr Gly Gln Ala Ala Asp Leu Lys Ser Thr Gln Ala Ala Ile Asp Gln
380 385 390atc aat ggg aaa ctg aat agg gta atc gag aag acg aac gag aaa ttc 1253Ile Asn Gly Lys Leu Asn Arg Val Ile Glu Lys Thr Asn Glu Lys Phe395 400 405cat caa atc gaa aag gaa ttc tca gaa gta gaa ggg aga att cag gac 1301His Gln Ile Glu Lys Glu Phe Ser Glu Val Glu Gly Arg Ile Gln Asp410 415 420ctc gag aaa tac gtt gaa gac act aaa ata gat ctc tgg tct tac aat 1349Leu Glu Lys Tyr Val Glu Asp Thr Lys Ile Asp Leu Trp Ser Tyr Asn425 430 435 440gcg gag ctt ctt gtc gct ctg gag aac caa cat aca att gat ctg act 1397Ala G1u Leu Leu Val Ala Leu Glu Asn Gln His Thr Ile Asp Leu Thr445 450 455gac tcg gaa atg aac aaa ctg ttt gaa aaa aca agg agg caa ctg agg 1445Asp Ser Glu Met Asn Lys Leu Phe Glu Lys Thr Arg Arg Gln Leu Arg460 465 470gaa aat gct gag gac atg ggc aat ggt tgc ttc aaa ata ttc cac aaa 1493Glu Asn Ala Glu Asp Met Gly Asn Gly Cys Phe Lys Ile Phe His Lys475 480 485tgt gac aat gct tgc ata ggg tca atc aga aat ggg act tat gac cat 1541Cys Asp Asn Ala Cys Ile Gly Ser Ile Arg Asn Gly Thr Tyr Asp His490 495 500gat gta tac aga gac gaa gca tta aac aac cgg ttt cag atc aaa ggt 1589Asp Val Tyr Arg Asp Glu Ala Leu Asn Asn Arg Phe Gln Ile Lys Gly505 510 515 520gtt gaa ctg aag tca gga tac aaa aac tgg atc ctg tgg att tcc ttt 1637Val Glu Leu Lys Ser Gly Tyr Lys Asn Trp Ile Leu Trp Ile Ser Phe525 530 535gcc ata tca tgc ttt ttg ctt tgt gtt gtt ttg ctg ggg ttc atc atg 1685Ala Ile Ser Cys Phe Leu Leu Cys Val Val Leu Leu Gly Phe Ile Met540 545 550tgg gcc tgc cag aaa ggc aac att agg tgc aac att tgc att 1727Trp Ala Cys Gln Lys Gly Asn Ile Arg Cys Asn Ile Cys Ile555 560 565tgagtgtatt agtaattaaa aacacccttg tttctact 1765<210>8<211>566<212>PRT<213>流感A病毒<400>8Met Lys Thr Ile Ile Ala Leu Ser Tyr Ile Phe Cys Leu Val Leu Gly1 5 10 15Gln Asn Phe Pro Gly Asn Asp Asn Ser Thr Ala Thr Leu Cys Leu Gly
20 25 30His His Ala Val Pro Asn Gly Thr Leu Val Lys Thr Ile Thr Asn Asp35 40 45Gln Ile Glu Val Thr Asn Ala Thr Glu Leu Val Gln Ser Ser Ser Thr50 55 60Gly Lys Ile Cys Asn Asn Pro His Arg Ile Leu Asp Gly Ile Asp Cys65 70 75 80Thr Leu Ile Asp Ala Leu Leu Gly Asp Pro His Cys Asp Gly Phe Gln85 90 95Asn Glu Thr Trp Asp Leu Phe Val Glu Arg Ser Lys Ala Phe Ser Asn100 105 110Cys Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Ser Leu Arg Ser Leu Val115 120 125Ala Ser Ser Gly Thr Leu Glu Phe Ile Ser Glu Gly Phe Thr Trp Thr130 135 140Gly Val Thr Gln Asn Gly Gly Ser Asn Ala Cys Lys Arg Gly Pro Asp145 150 155 160Ser Gly Phe Phe Ser Arg Leu Asn Trp Leu Tyr Lys Ser Gly Ser Thr165 170 175Tyr Pro Val Leu Asn Val Thr Met Pro Asn Asn Asp Asn Phe Asp Lys180 185 190Leu Tyr Ile Trp Gly Val His His Pro Ser Thr Asp Gln Glu Gln Thr195 200 205Ser Leu Tyr Val Gln Ala Ser Gly Arg Val Thr Val Ser Thr Lys Arg210 215 220Ser Gln Gln Thr Ile Ile Pro Asn Ile Gly Ser Arg Pro Trp Val Arg225 230 235 240Gly Leu Ser Ser Arg Ile Ser Ile Tyr Trp Thr Ile Val Lys Pro Gly245 250 255Asp Ile Leu Val Ile Asn Ser Asn Gly Asn Leu Ile Ala Pro Arg Gly260 265 270Tyr Phe Lys Met Arg Thr Gly Lys Ser Ser Ile Met Arg Ser Asp Ala275 280 285Pro Ile Gly Thr Cys Ile Ser Glu Cys Ile Thr Pro Asn Gly Ser Ile290 295 300Pro Asn Asp Lys Pro Phe Gln Asn Val Asn Lys Ile Thr Tyr Gly Ala305 310 315 320Cys Pro Lys Tyr Val Lys Gln Asn Thr Leu Lys Leu Ala Thr Gly Met325 330 335Arg Asn Val Pro Glu Lys Gln Thr Arg Gly Leu Phe Ser Ala Ile Ala340 345 350Gly Phe Ile Glu Asn Gly Trp Glu Gly Met Ile Asp Gly Trp Tyr Gly355 360 365Phe Arg His Gln Asn Ser Glu Gly Thr Gly Gln Ala Ala Asp Leu Lys370 375 380Ser Thr Gln Ala Ala Ile Asp Gln Ile Asn Gly Lys Leu Asn Arg Val385 390 395 400Ile Glu Lys Thr Asn Glu Lys Phe His Gln Ile Glu Lys Glu Phe Ser405 410 415Glu Val Glu Gly Arg Ile Gln Asp Leu Glu Lys Tyr Val Glu Asp Thr420 425 430Lys Ile Asp Leu Trp Ser Tyr Asn Ala Glu Leu Leu Val Ala Leu Glu435 440 445Asn Gln His Thr Ile Asp Leu Thr Asp Ser Glu Met Asn Lys Leu Phe450 455 460Glu Lys Thr Arg Arg Gln Leu Arg Glu Asn Ala Glu Asp Met Gly Asn465 470 475 480Gly Cys Phe Lys Ile Phe His Lys Cys Asp Asn Ala Cys Ile Gly Ser485 490 495Ile Arg Asn Gly Thr Tyr Asp His Asp Val Tyr Arg Asp Glu Ala Leu500 505 510Asn Asn Arg Phe Gln Ile Lys Gly Val Glu Leu Lys Ser Gly Tyr Lys515 520 525Asn Trp Ile Leu Trp Ile Ser Phe Ala Ile Ser Cys Phe Leu Leu Cys530 535 540Val Val Leu Leu Gly Phe Ile Met Trp Ala Cys Gln Lys Gly Asn Ile545 550 555 560Arg Cys Asn Ile Cys Ile565<210>9<211>1565<212>DNA<213>流感A病毒<220><221>CDS<222>(46)..(1539)<400>9agcaaaagca gggttaataa tcactcactg agtgacatca aaatc atg gcg tcc caa 57Met Ala Ser Glnggc acc aaa cgg tct tat gaa cag atg gaa act gat ggg gaa cgc cag 105Gly Thr Lys Arg Ser Tyr Glu Gln Met Glu Thr Asp Gly Glu Arg Gln5 10 15 20aat gca act gag atc agg gca tcc gtc ggg aag atg att gat gga att 153Asn Ala Thr Glu Ile Arg Ala Ser Val Gly Lys Met Ile Asp Gly Ile25 30 35gga cga ttc tac atc caa atg tgc act gaa ctt aaa ctc agt gat tat 201Gly Arg Phe Tyr Ile Gln Met Cys Thr Glu Leu Lys Leu Ser Asp Tyr40 45 50gag ggg cga ttg atc cag aac agc tta aca ata gag aga atg gtg ctc 249Glu Gly Arg Leu Ile Gln Asn Ser Leu Thr Ile Glu Arg Met Val Leu55 60 65tct gct ttt gat gag aga agg aat aga tat ctg gaa gaa cat ccc agc 297Ser Ala Phe Asp Glu Arg Arg Asn Arg Tyr Leu Glu Glu His Pro Ser70 75 80gcg ggg aaa gat cct aag aaa act gga ggg ccc ata tac aag aga gta 345Ala Gly Lys Asp Pro Lys Lys Thr Gly Gly Pro Ile Tyr Lys Arg Val85 90 95 100gat aga aag tgg atg agg gaa ctc gtc ctt tat gac aaa gaa gaa ata 393Asp Arg Lys Trp Met Arg Glu Leu Val Leu Tyr Asp Lys Glu Glu Ile105 110 115agg cga atc tgg cgc caa gcc aat aat ggt gat gat gcg aca gct ggt 441Arg Arg Ile Trp Arg Gln Ala Asn Asn Gly Asp Asp Ala Thr Ala Gly120 125 130cta acc cac atg atg atc tgg cat tcc aat ttg aat gat aca aca tac 489Leu Thr His Met Met Ile Trp His Ser Asn Leu Asn Asp Thr Thr Tyr135 140 145cag agg aca aga gct ctt gtt cgc acc gga atg gat ccc agg atg tgc 537Gln Arg Thr Arg Ala Leu Val Arg Thr Gly Met Asp Pro Arg Met Cys150 155 160tct ctg atg cag ggt tcg act ctc cct agg agg tct gga gct gca ggc 585Ser Leu Met Gln Gly Ser Thr Leu Pro Arg Arg Ser Gly Ala Ala Gly165 170 175 180gct gca gtc aaa gga gtc ggg aca atg gtg atg gag ctg atc aga atg 633Ala Ala Val Lys Gly Val Gly Thr Met Val Met Glu Leu Ile Arg Met185 190 195atc aaa cgt ggg atc aac gat cgg aac ttc tgg aga ggt gag aat gga 681Ile Lys Arg Gly Ile Asn Asp Arg Asn Phe Trp Arg Gly Glu Asn Gly200 205 210cga aaa aca agg ggt gct tat gag aga atg tgc aac att ctc aaa gga 729Arg Lys Thr Arg Gly Ala Tyr Glu Arg Met Cys Asn Ile Leu Lys Gly215 220 225aaa ttt caa aca gct gca caa aga gca atg atg gat caa gtg aga gaa 777Lys Phe Gln Thr Ala Ala Gln Arg Ala Met Met Asp Gln Val Arg Glu230 235 240agc cgg aac cca gga aat gct gag atc gaa gat ctc ata ttt ttg gca 825Ser Arg Asn Pro Gly Asn Ala Glu Ile Glu Asp Leu Ile Phe Leu Ala245 250 255 260cgg tct gca cta ata ttg aga ggg tca gtt gct cac aaa tct tgt ctg 873Arg Ser Ala Leu Ile Leu Arg Gly Ser Val Ala His Lys Ser Cys Leu265 270 275cct gcc tgt gtg tat gga cct gcc gta gcc agt ggg tac gac ttc gaa 921Pro Ala Cys Val Tyr Gly Pro Ala Val Ala Ser Gly Tyr Asp Phe Glu280 285 290aaa gag gga tac tct tta gtg gga ata gac cct ttc aaa cta ctt caa 969Lys Glu Gly Tyr Ser Leu Val Gly Ile Asp Pro Phe Lys Leu Leu Gln295 300 305aac agc caa gta tac agc cta atc aga ccg aac gag aat cca gca cac 1017Asn Ser Gln Val Tyr Ser Leu Ile Arg Pro Asn Glu Asn Pro Ala His310 315 320aag agt cag ctg gtg tgg atg gca tgc aat tct gct gca ttt gaa gat 1065Lys Ser Gln Leu Val Trp Met Ala Cys Asn Ser Ala Ala Phe Glu Asp325 330 335 340cta aga tta tta agc ttc atc aga ggg acc aaa gta tct cca agg ggg 1113Leu Arg Leu Leu Ser Phe Ile Arg Gly Thr Lys Val Ser Pro Arg Gly345 350 355aaa ctt tca act aga gga gta caa att gct tca aat gaa aac atg gat 1161Lys Leu Ser Thr Arg Gly Val Gln Ile Ala Ser Asn Glu Asn Met Asp360 365 370act atg gaa tca agt act ctt gaa ctg aga agc agg tac tgg gcc ata 1209Thr Met Glu Ser Ser Thr Leu Glu Leu Arg Ser Arg Tyr Trp Ala Ile375 380 385agg acc aga agt gga gga aac act aat caa cag agg gcc tcc gca ggc 1257Arg Thr Arg Ser Gly Gly Asn Thr Asn Gln Gln Arg Ala Ser Ala Gly390 395 400caa atc agt gtg caa cct gca ttt tct gtg caa aga aac ctc cca ttt 1305Gln Ile Ser Val Gln Pro Ala Phe Ser Val Gln Arg Asn Leu Pro Phe405 410 415 420gac aaa tca acc atc atg gca gca ttc act ggg aat acg gag gga aga 1353Asp Lys Ser Thr Ile Met Ala Ala Phe Thr Gly Asn Thr Glu Gly Arg425 430 435acc tca gac atg agg gca gaa atc ata agg atg atg gaa ggt gca aaa 1401Thr Ser Asp Met Arg Ala Glu Ile Ile Arg Met Met Glu Gly Ala Lys440 445 450cca gaa gaa gtg tcc ttc cgt ggg cgg gga gtc ttc gag ctc tcg gac 1449Pro Glu Glu Val Ser Phe Arg Gly Arg Gly Val Phe Glu Leu Ser Asp455 460 465gaa aag gca acg aac ccg atc gtg ccc tct ttt gac atg agt aat gaa 1497Glu Lys Ala Thr Asn Pro Ile Val Pro Ser Phe Asp Met Ser Asn Glu470 475 480gga tct tat ttc ttc gga gac aat gca gag gag tac gac aat 1539Gly Ser Tyr Phe Phe Gly Asp Asn Ala Glu Glu Tyr Asp Asn485 490 495taaggaaaaa tacccttgtt tctact 1565<210>10<211>498<212>PRT<213>流感A病毒<400>10Met Ala Ser Gln Gly Thr Lys Arg Ser Tyr Glu Gln Met Glu Thr Asp1 5 10 15Gly Glu Arg Gln Asn Ala Thr Glu Ile Arg Ala Ser Val Gly Lys Met20 25 30Ile Asp Gly Ile Gly Arg Phe Tyr Ile Gln Met Cys Thr Glu Leu Lys35 40 45Leu Ser Asp Tyr Glu Gly Arg Leu Ile Gln Asn Ser Leu Thr Ile Glu50 55 60Arg Met Val Leu Ser Ala Phe Asp Glu Arg Arg Asn Arg Tyr Leu Glu65 70 75 80Glu His Pro Ser Ala Gly Lys Asp Pro Lys Lys Thr Gly Gly Pro Ile85 90 95Tyr Lys Arg Val Asp Arg Lys Trp Met Arg Glu Leu Val Leu Tyr Asp100 105 110Lys Glu Glu Ile Arg Arg Ile Trp Arg Gln Ala Asn Asn Gly Asp Asp115 120 125Ala Thr Ala Gly Leu Thr His Met Met Ile Trp His Ser Asn Leu Asn130 135 140Asp Thr Thr Tyr Gln Arg Thr Arg Ala Leu Val Arg Thr Gly Met Asp145 150 155 160Pro Arg Met Cys Ser Leu Met Gln Gly Ser Thr Leu Pro Arg Arg Ser165 170 175Gly Ala Ala Gly Ala Ala Val Lys Gly Val Gly Thr Met Val Met Glu180 185 190Leu Ile Arg Met Ile Lys Arg Gly Ile Asn Asp Arg Asn Phe Trp Arg195 200 205Gly Glu Asn Gly Arg Lys Thr Arg Gly Ala Tyr Glu Arg Met Cys Asn210 215 220Ile Leu Lys Gly Lys Phe Gln Thr Ala Ala Gln Arg Ala Met Met Asp225 230 235 240Gln Val Arg Glu Ser Arg Asn Pro Gly Asn Ala Glu Ile Glu Asp Leu245 250 255Ile Phe Leu Ala Arg Ser Ala Leu Ile Leu Arg Gly Ser Val Ala His260 265 270Lys Ser Cys Leu Pro Ala Cys Val Tyr Gly Pro Ala Val Ala Ser Gly275 280 285Tyr Asp Phe Glu Lys Glu Gly Tyr Ser Leu Val Gly Ile Asp Pro Phe290 295 300Lys Leu Leu Gln Asn Ser Gln Val Tyr Ser Leu Ile Arg Pro Asn Glu305 310 315 320Asn Pro Ala His Lys Ser Gln Leu Val Trp Met Ala Cys Asn Ser Ala325 330 335Ala Phe Glu Asp Leu Arg Leu Leu Ser Phe Ile Arg Gly Thr Lys Val340 345 350Ser Pro Arg Gly Lys Leu Ser Thr Arg Gly Val Gln Ile Ala Ser Asn355 360 365Glu Asn Met Asp Thr Met Glu Ser Ser Thr Leu Glu Leu Arg Ser Arg370 375 380Tyr Trp Ala Ile Arg Thr Arg Ser Gly Gly Asn Thr Asn Gln Gln Arg385 390 395 400Ala Ser Ala Gly Gln Ile Ser Val Gln Pro Ala Phe Ser Val Gln Arg405 410 415Asn Leu Pro Phe Asp Lys Ser Thr Ile Met Ala Ala Phe Thr Gly Asn420 425 430Thr Glu Gly Arg Thr Ser Asp Met Arg Ala Glu Ile Ile Arg Met Met435 440 445Glu Gly Ala Lys Pro Glu Glu Val Ser Phe Arg Gly Arg Gly Val Phe450 455 460Glu Leu Ser Asp Glu Lys Ala Thr Asn Pro Ile Val Pro Ser Phe Asp465 470 475 480Met Ser Asn Glu Gly Ser Tyr Phe Phe Gly Asp Asn Ala Glu Glu Tyr485 490 495Asp Asn<210>11<211>1466<212>DNA<213>流感A病毒<220><221>CDS<222>(20)..(1426)<400>11agcaaaagca ggagtgaag atg aat cca aat caa aag ata ata aca att ggc 52Met Asn Pro Asn Gln Lys Ile Ile Thr Ile Gly1 5 10tct gtc tct ctc acc att gca aca ata tgc ttc ctc atg cag att gcc 100Ser Val Ser Leu Thr Ile Ala Thr Ile Cys Phe Leu Met Gln Ile Ala15 20 25atc ctg gta act act gta aca ttg cat ttc aag caa tat gag tgc gac 148Ile Leu Val Thr Thr Val Thr Leu His Phe Lys Gln Tyr Glu Cys Asp30 35 40tcc ccc gcg aac aac caa gta atg ccg tgt gaa cca ata ata ata gaa 196Ser Pro Ala Asn Asn Gln Val Met Pro Cys Glu Pro Ile Ile Ile Glu45 50 55agg aac ata aca gag ata gtg tat ttg act aac acc acc ata gag aaa 244Arg Asn Ile Thr Glu Ile Val Tyr Leu Thr Asn Thr Thr Ile Glu Lys60 65 70 75gag ata tgc ccc aaa tta gtg gaa tac agg aat tgg tca aag ccg caa 292Glu Ile Cys Pro Lys Leu Val Glu Tyr Arg Asn Trp Ser Lys Pro Gln80 85 90tgt aaa att aca gga ttt gca cct ttt tct aag gac aat tca att cgg 340Cys Lys Ile Thr Gly Phe Ala Pro Phe Ser Lys Asp Asn Ser Ile Arg95 100 105ctt tct gct ggt ggg gac att tgg gtg acg aga gaa cct tat gtg tca 388Leu Ser Ala Gly Gly Asp Ile Trp Val Thr Arg Glu Pro Tyr Val Ser110 115 120tgc gat cct ggc aag tgt tat caa ttt gca ctc ggg cag ggg acc aca 436Cys Asp Pro Gly Lys Cys Tyr Gln Phe Ala Leu Gly Gln Gly Thr Thr125 130 135cta gac aac aaa cat tca aat gac aca ata cat gat aga acc cct cat 484Leu Asp Asn Lys His Ser Asn Asp Thr Ile His Asp Arg Thr Pro His140 145 150 155cga acc cta ttg atg aat gag ttg ggt gtt cca ttt cat ttg gga acc 532Arg Thr Leu Leu Met Asn Glu Leu Gly Val Pro Phe His Leu Gly Thr160 165 170agg caa gtg tgt ata gca tgg tcc agc tca agt tgt cac gat gga aaa 580Arg Gln Val Cys Ile Ala Trp Ser Ser Ser Ser Cys His Asp Gly Lys175 180 185gca tgg ctg cat gtt tgt gtc act ggg tat gat aaa aat gca act gct 628Ala Trp Leu His Val Cys Val Thr Gly Tyr Asp Lys Asn Ala Thr Ala190 195 200agc ttc att tac gat ggg agg ctt gta gac agt att ggt tca tgg tct 676Ser Phe Ile Tyr Asp Gly Arg Leu Val Asp Ser Ile Gly Ser Trp Ser205 210 215caa aat atc ctc agg acc cag gag tcg gaa tgc gtt tgt atc aat ggg 724Gln Asn Ile Leu Arg Thr Gln Glu Ser Glu Cys Val Cys Ile Asn Gly220 225 230 235act tgt aca gta gta atg act gat gga agt gct tca gga aga gct gat 772Thr Cys Thr Val Val Met Thr Asp Gly Ser Ala Ser Gly Arg Ala Asp240 245 250act aaa ata cta ttc att gaa gag ggg aaa att gtt cat att agc cca 820Thr Lys Ile Leu Phe Ile Glu Glu Gly Lys Ile Val His Ile Ser Pro255 260 265ttg tca gga agt gct cag cat gta gag gag tgt tcc tgt tat cct cga 868Leu Ser Gly Ser Ala Gln His Val Glu Glu Cys Ser Cys Tyr Pro Arg270 275 280tat cct ggt gtc aga tgt atc tgc aga gac aac tgg aaa ggc tct aat 916Tyr Pro Gly Val Arg Cys Ile Cys Arg Asp Asn Trp Lys Gly Ser Asn285 290 295agg ccc gtc gta gat ata aat gtg aaa gat tat agc att gat tcc agt 964Arg Pro Val Val Asp Ile Asn Val Lys Asp Tyr Ser Ile Asp Ser Ser300 305 310 315tat gtg tgc tca ggg ctt gtt ggc gac gca ccc aga aac aac gac aga 1012Tyr Val Cys Ser Gly Leu Val Gly Asp Ala Pro Arg Asn Asn Asp Arg320 325 330tct agc aat agc tat tgc cgg aat cct aac aat gag aaa ggg aat cac 1060Ser Ser Asn Ser Tyr Cys Arg Asn Pro Asn Asn Glu Lys Gly Asn His335 340 345gga gtg aaa ggc tgg gcc ttt gac gat gga aat gac gtg tgg atg gga 1108Gly Val Lys Gly Trp Ala Phe Asp Asp Gly Asn Asp Val Trp Met Gly350 355 360aga acg atc agc gag gat tca cgc tca ggt tat gaa acc ttc aaa gtc 1156Arg Thr Ile Ser Glu Asp Ser Arg Ser Gly Tyr Glu Thr Phe Lys Val365 370 375att ggt ggt tgg tcc aca cct aat tcc aaa ttg cag ata aat agg caa 1204Ile Gly Gly Trp Ser Thr Pro Asn Ser Lys Leu Gln Ile Asn Arg Gln380 385 390 395gtc ata gtt gac agc gat aat agg tca ggt tat tct ggt att ttc tct 1252Val Ile Val Asp Ser Asp Asn Arg Ser Gly Tyr Ser Gly Ile Phe Ser400 405 410gtt gag ggc aaa agc tgc atc aat agg tgc ttt tat gtg gag ttg ata 1300Val Glu Gly Lys Ser Cys Ile Asn Arg Cys Phe Tyr Val Glu Leu Ile415 420 425agg gga agg gaa cag gaa act aga gta tgg tgg acc tca aac agt att 1348Arg Gly Arg Glu Gln Glu Thr Arg Val Trp Trp Thr Ser Asn Ser Ile430 435 440gtt gtg ttt tgt ggc act tca ggt acc tat ggg aca ggc tca tgg cct 1396Val Val Phe Cys Gly Thr Ser Gly Thr Tyr Gly Thr Gly Ser Trp Pro445 450 455gat ggg gcg gac atc aat ctc atg cct ata taagctttcg caattttaga 1446Asp Gly Ala Asp Ile Asn Leu Met Pro Ile460 465aaaaactcct tgtttctact 1466<210>12<211>469<212>PRT<213>流感A病毒<400>12Met Asn Pro Asn Gln Lys Ile Ile Thr Ile Gly Ser Val Ser Leu Thr1 5 10 15Ile Ala Thr Ile Cys Phe Leu Met Gln Ile Ala Ile Leu Val Thr Thr20 25 30Val Thr Leu His Phe Lys Gln Tyr Glu Cys Asp Ser Pro Ala Asn Asn35 40 45Gln Val Met Pro Cys Glu Pro Ile Ile Ile Glu Arg Asn Ile Thr Glu50 55 60Ile Val Tyr Leu Thr Asn Thr Thr Ile Glu Lys Glu Ile Cys Pro Lys65 70 75 80Leu Val Glu Tyr Arg Asn Trp Ser Lys Pro Gln Cys Lys Ile Thr Gly85 90 95Phe Ala Pro Phe Ser Lys Asp Asn Ser Ile Arg Leu Ser Ala Gly Gly100 105 110Asp Ile Trp Val Thr Arg Glu Pro Tyr Val Ser Cys Asp Pro Gly Lys115 120 125Cys Tyr Gln Phe Ala Leu Gly Gln Gly Thr Thr Leu Asp Asn Lys His130 135 140Ser Asn Asp Thr Ile His Asp Arg Thr Pro His Arg Thr Leu Leu Met145 150 155 160Asn Glu Leu Gly Val Pro Phe His Leu Gly Thr Arg Gln Val Cys Ile165 170 175Ala Trp Ser Ser Ser Ser Cys His Asp Gly Lys Ala Trp Leu His Val180 185 190Cys Val Thr Gly Tyr Asp Lys Asn Ala Thr Ala Ser Phe Ile Tyr Asp195 200 205Gly Arg Leu Val Asp Ser Ile Gly Ser Trp Ser Gln Asn Ile Leu Arg210 215 220Thr Gln Glu Ser Glu Cys Val Cys Ile Asn Gly Thr Cys Thr Val Val225 230 235 240Met Thr Asp Gly Ser Ala Ser Gly Arg Ala Asp Thr Lys Ile Leu Phe245 250 255Ile Glu Glu Gly Lys Ile Val His Ile Ser Pro Leu Ser Gly Ser Ala260 265 270Gln His Val Glu Glu Cys Ser Cys Tyr Pro Arg Tyr Pro Gly Val Arg275 280 285Cys Ile Cys Arg Asp Asn Trp Lys Gly Ser Asn Arg Pro Val Val Asp290 295 300Ile Asn Val Lys Asp Tyr Ser Ile Asp Ser Ser Tyr Val Cys Ser Gly305 310 315 320Leu Val Gly Asp Ala Pro Arg Asn Asn Asp Arg Ser Ser Asn Set Tyr325 330 335Cys Arg Asn Pro Asn Asn Glu Lys Gly Asn His Gly Val Lys Gly Trp340 345 350Ala Phe Asp Asp Gly Asn Asp Val Trp Met Gly Arg Thr Ile Ser Glu355 360 365Asp Ser Arg Ser Gly Tyr Glu Thr Phe Lys Val Ile Gly Gly Trp Ser370 375 380Thr Pro Asn Ser Lys Leu Gln Ile Asn Arg Gln Val Ile Val Asp Ser385 390 395 400Asp Asn Arg Ser Gly Tyr Ser Gly Ile Phe Ser Val Glu Gly Lys Ser405 410 415Cys Ile Asn Arg Cys Phe Tyr Val Glu Leu Ile Arg Gly Arg Glu Gln420 425 430Glu Thr Arg Val Trp Trp Thr Ser Asn Ser Ile Val Val Phe Cys Gly435 440 445Thr Ser Gly Thr Tyr Gly Thr Gly Ser Trp Pro Asp Gly Ala Asp Ile450 455 460Asn Leu Met Pro Ile465<210>13<211>1027<212>DNA<213>流感A病毒<220><221>CDS<222>(26)..(781)<400>13agcaaaagca ggtagatatt gaaag atg agc ctt ctg acc gag gtc gaa acg 52Met Ser Leu Leu Thr Glu Val Glu Thr1 5tat gtt ctc tct atc gtt ccg tea ggc ccc ctc aaa gcc gag atc gcg 100Tyr Val Leu Ser Ile Val Pro Ser Gly Pro Leu Lys Ala Glu Ile Ala10 15 20 25cag aga ctt gaa gat gtc ttt gct gga aag aac aca gat ctt gag gct 148Gln Arg Leu Glu Asp Val Phe Ala Gly Lys Asn Thr Asp Leu Glu Ala30 35 40ctc atg gaa tgg cta aag aca aga cca atc ctg tca cct ctg act aaa 196Leu Met Glu Trp Leu Lys Thr Arg Pro Ile Leu Ser Pro Leu Thr Lys45 50 55ggg att ttg gga ttt gta ttc acg ctc acc gtg ccc agt gag cga gga 244Gly Ile Leu Gly Phe Val Phe Thr Leu Thr Val Pro Ser Glu Arg Gly60 65 70ctg cag cgt aga cgc ttt gtc caa aat gcc ctc aat ggg aat ggg gat 292Leu Gln Arg Arg Arg Phe Val Gln Asn Ala Leu Asn Gly Asn Gly Asp75 80 85cca aat aac atg gac aga gca gtt aaa ctg tat aga aaa ctt aag agg 340Pro Asn Asn Met Asp Arg Ala Val Lys Leu Tyr Arg Lys Leu Lys Arg90 95 100 105gag ata aca ttc cat ggg gcc aaa gaa ata gca ctc agt tat tct gct 388Glu Ile Thr Phe His Gly Ala Lys Glu Ile Ala Leu Ser Tyr Ser Ala110 115 120ggt gca ctt gcc agt tgc atg ggc ctc ata tac aac agg atg ggg gct 436Gly Ala Leu Ala Ser Cys Met Gly Leu Ile Tyr Asn Arg Met Gly Ala125 130 135gtg acc act gaa gtg gcc ttt ggc ctg gtt tgt gca acc tgt gaa cag 484Val Thr Thr Glu Val Ala Phe Gly Leu Val Cys Ala Thr Cys Glu Gln140 145 150att gct gac tcc cag cac agg tct cat agg caa atg gtg gca aca acc 532Ile Ala Asp Ser Gln His Arg Ser His Arg Gln Met Val Ala Thr Thr155 160 165aat cca cta ata aga cat gag aac aga atg gtt ctg gcc agc act aca 580Asn Pro Leu Ile Arg His Glu Asn Arg Met Val Leu Ala Ser Thr Thr170 175 180 185gct aag gct atg gag caa atg gct gga tca agt gag cag gca gca gag 628Ala Lys Ala Met Glu Gln Met Ala Gly Ser Ser Glu Gln Ala Ala Glu190 195 200gcc atg gag gtt gct agt cag gcc agg caa atg gtg cag gca atg aga 676Ala Met Glu Val Ala Ser Gln Ala Arg Gln Met Val Gln Ala Met Arg205 210 215gcc att ggg act cat cct agc tcc agt gct ggt cta aaa gat gat ctt 724Ala Ile Gly Thr His Pro Ser Ser Ser Ala Gly Leu Lys Asp Asp Leu220 225 230ctt gaa aat ttg cag gcc tat cag aaa cga atg ggg gtg cag atg caa 772Leu Glu Asn Leu Gln Ala Tyr Gln Lys Arg Met Gly Val Gln Met Gln235 240 245cga ttc aag tgaccctctt gttgttgctg cgagtatcat tgggatcttg 821Arg Phe Lys250cacttgatat tgtggattct tgatcgtctt tttttcaaat gcatctatcg attctttgaa 881cacggtctga aaagagggcc ttctacggaa ggagtacctg agtctatgag ggaagaatat 941cgaaaggaac agcagagtgc tgtggatgct gacgacagtc attttgtcag catagagctg 1001gagtaaaaaa ctaccttgtt tctact 1027<210>14<211>252<212>PRT<213>流感A病毒<400>14Met Ser Leu Leu Thr Glu Val Glu Thr Tyr Val Leu Ser Ile Val Pro1 5 l0 15Ser Gly Pro Leu Lys Ala Glu Ile Ala Gln Arg Leu Glu Asp Val Phe20 25 30Ala Gly Lys Asn Thr Asp Leu Glu Ala Leu Met Glu Trp Leu Lys Thr35 40 45Arg Pro Ile Leu Ser Pro Leu Thr Lys Gly Ile Leu Gly Phe Val Phe50 55 60Thr Leu Thr Val Pro Ser Glu Arg Gly Leu Gln Arg Arg Arg Phe Val65 70 75 80Gln Asn Ala Leu Asn Gly Asn Gly Asp Pro Asn Asn Met Asp Arg Ala85 90 95Val Lys Leu Tyr Arg Lys Leu Lys Arg Glu Ile Thr Phe His Gly Ala100 105 110Lys Glu Ile Ala Leu Ser Tyr Ser Ala Gly Ala Leu Ala Ser Cys Met115 120 125Gly Leu Ile Tyr Asn Arg Met Gly Ala Val Thr Thr Glu Val Ala Phe130 135 140Gly Leu Val Cys Ala Thr Cys Glu Gln Ile Ala Asp Ser Gln His Arg145 150 155 160Ser His Arg Gln Met Val Ala Thr Thr Asn Pro Leu Ile Arg His Glu165 170 175Asn Arg Met Val Leu Ala Ser Thr Thr Ala Lys Ala Met Glu Gln Met180 185 190Ala Gly Ser Ser Glu Gln Ala Ala Glu Ala Met Glu Val Ala Ser Gln195 200 205Ala Arg Gln Met Val Gln Ala Met Arg Ala Ile Gly Thr His Pro Ser2l0 215 220Ser Ser Ala Gly Leu Lys Asp Asp Leu Leu Glu Asn Leu Gln Ala Tyr225 230 235 240Gln Lys Arg Met Gly Val Gln Met Gln Arg Phe Lys245 250<210>15<211>322<212>DNA<213>流感A病毒<220><221>CDS<222>(26)..(316)<400>15agcaaaagca ggtagatatt gaaag atg agc ctt ctg acc gag gtc gaa acg 52Met Ser Leu Leu Thr Glu Val Glu Thr1 5cct atc aga aac gaa tgg ggg tgc aga tgc aac gat tca agt gac cct 100Pro Ile Arg Asn Glu Trp Gly Cys Arg Cys Asn Asp Ser Ser Asp Pro10 15 20 25ctt gtt gtt gct gcg agt atc att ggg atc ttg cac ttg ata ttg tgg 148Leu Val Val Ala Ala Ser Ile Ile Gly Ile Leu His Leu Ile Leu Trp30 35 40att ctt gat cgt ctt ttt ttc aaa tgc atc tat cga ttc ttt gaa cac 196Ile Leu Asp Arg Leu Phe Phe Lys Cys Ile Tyr Arg Phe Phe Glu His45 50 55ggt ctg aaa aga ggg cct tct acg gaa gga gta cct gag tct atg agg 244Gly Leu Lys Arg Gly Pro Ser Thr Glu Gly Val Pro Glu Ser Met Arg60 65 70gaa gaa tat cga aag gaa cag cag agt gct gtg gat gct gac gac agt 292Glu Glu Tyr Arg Lys Glu Gln Gln Ser Ala Val Asp Ala Asp Asp Ser75 80 85cat ttt gtc agc ata gag ctg gag taaaaa322His Phe Val Ser Ile Glu Leu Glu90 95<210>16<211>97<212>PRT<213>流感A病毒<400>16Met Ser Leu Leu Thr Glu Val Glu Thr Pro Ile Arg Asn Glu Trp Gly1 5 10 15Cys Arg Cys Asn Asp Ser Ser Asp Pro Leu Val Val Ala Ala Ser Ile20 25 30Ile Gly Ile Leu His Leu Ile Leu Trp Ile Leu Asp Arg Leu Phe Phe35 40 45Lys Cys Ile Tyr Arg Phe Phe Glu His Gly Leu Lys Arg Gly Pro Ser50 55 60Thr Glu Gly Val Pro Glu Ser Met Arg Glu Glu Tyr Arg Lys Glu Gln65 70 75 80Gln Ser Ala Val Asp Ala Asp Asp Ser His Phe Val Ser Ile Glu Leu85 90 95Glu<210>17<211>890<212>DNA<213>流感A病毒<220><221>CDS<222>(27)..(737)<400>17agcaaaagca gggtgacaaa gacata atg gat tcc aac act gtg tca agt ttt 53Met Asp Ser Asn Thr Val Ser Ser Phe1 5cag gta gac tgc ttc ctt tgg cat gtc cga aaa caa gtt gta gac caa 101Gln Val Asp Cys Phe Leu Trp His Val Arg Lys Gln Val Val Asp Gln10 15 20 25gaa cta ggt gat gcc cca ttc ctt gat cgg ctt cgc cga gat cag aag 149Glu Leu Gly Asp Ala Pro Phe Leu Asp Arg Leu Arg Arg Asp Gln Lys30 35 40tcc cta agg gga aga ggc agc act ctc ggt cta aac atc gaa gca gcc 197Ser Leu Arg Gly Arg Gly Ser Thr Leu Gly Leu Asn Ile Glu Ala Ala45 50 55acc cat gtt gga aag cag ata gta gag aag att ctg aag gaa gaa tct 245Thr His Val Gly Lys Gln Ile Val Glu Lys Ile Leu Lys Glu Glu Ser60 65 70gat gag gca ctt aaa atg acc atg gcc tcc aca cct gct tcg cga tac 293Asp Glu Ala Leu Lys Met Thr Met Ala Ser Thr Pro Ala Ser Arg Tyr75 80 85ata act gac atg act att gag gaa ttg tca agg gac tgg ttc atg cta 341Ile Thr Asp Met Thr Ile Glu Glu Leu Ser Arg Asp Trp Phe Met Leu90 95 100 105atg ccc aag cag aaa gtg gaa gga cct ctt tgc atc aga ata gac caa 389Met Pro Lys Gln Lys Val Glu Gly Pro Leu Cys Ile Arg Ile Asp Gln110 115 120gca atc atg gat aag aac atc atg ttg aaa gcg aat ttc agt gtg att 437Ala Ile Met Asp Lys Asn Ile Met Leu Lys Ala Asn Phe Ser Val Ile125 130 135ttt gac cgg cta gag acc cta ata tta cta agg gct ttc acc gaa gag 485Phe Asp Arg Leu Glu Thr Leu Ile Leu Leu Arg Ala Phe Thr Glu Glu140 145 150gga gca att gtt ggc gaa atc tca cca ttg cct tct ttt cca gga cat 533Gly Ala Ile Val Gly Glu Ile Ser Pro Leu Pro Ser Phe Pro Gly His155 160 165act att gag gat gtc aaa aat gca att ggg gtc ctc atc gga gga ctt 581Thr Ile Glu Asp Val Lys Asn Ala Ile Gly Val Leu Ile Gly Gly Leu170 175 180 185gaa tgg aat gat aac aca gtt cga gtc tct aaa act cta cag aga ttc 629Glu Trp Asn Asp Asn Thr Val Arg Val Ser Lys Thr Leu Gln Arg Phe190 195 200gct tgg gga agc agt aat gag aat ggg aga cct cca ctt act cca aaa 677Ala Trp Gly Ser Ser Asn Glu Asn Gly Arg Pro Pro Leu Thr Pro Lys205 210 215cag aaa cgg aaa atg gcg aga aca gct agg tca aaa gtt cga aga gat 725Gln Lys Arg Lys Met Ala Arg Thr Ala Arg Ser Lys Val Arg Arg Asp220 225 230aag atg gct gat tgaagaagtg agacacagac tgaagacaac agagaatagt 777Lys Met Ala Asp235tttgagcaaa taacattcat gcaagcctta cagctactat ttgaagtgga acaggagata 837agaactttct cgtttcagct tatttaatga taaaaaacac ccttgtttct act890<210>18<211>237<212>PRT<213>流感A病毒<400>18Met Asp Ser Asn Thr Val Ser Ser Phe Gln Val Asp Cys Phe Leu Trp1 5 10 15His Val Arg Lys Gln Val Val Asp Gln Glu Leu Gly Asp Ala Pro Phe20 25 30Leu Asp Arg Leu Arg Arg Asp Gln Lys Ser Leu Arg Gly Arg Gly Ser
35 40 45Thr Leu Gly Leu Asn Ile Glu Ala Ala Thr His Val Gly Lys Gln Ile50 55 60Val Glu Lys Ile Leu Lys Glu Glu Ser Asp Glu Ala Leu Lys Met Thr65 70 75 80Met Ala Ser Thr Pro Ala Ser Arg Tyr Ile Thr Asp Met Thr Ile Glu85 90 95Glu Leu Ser Arg Asp Trp Phe Met Leu Met Pro Lys Gln Lys Val Glu100 105 110Gly Pro Leu Cys Ile Arg Ile Asp Gln Ala Ile Met Asp Lys Asn Ile115 120 125Met Leu Lys Ala Asn Phe Ser Val Ile Phe Asp Arg Leu Glu Thr Leu130 135 140Ile Leu Leu Arg Ala Phe Thr Glu Glu Gly Ala Ile Val Gly Glu Ile145 150 155 160Ser Pro Leu Pro Ser Phe Pro Gly His Thr Ile Glu Asp Val Lys Asn165 170 175Ala Ile Gly Val Leu Ile Gly Gly Leu Glu Trp Asn Asp Asn Thr Val180 185 190Arg Val Ser Lys Thr Leu Gln Arg Phe Ala Trp Gly Ser Ser Asn Glu195 200 205Asn Gly Arg Pro Pro Leu Thr Pro Lys Gln Lys Arg Lys Met Ala Arg210 215 220Thr Ala Arg Ser Lys Val Arg Arg Asp Lys Met Ala Asp225 230 235<210>19<211>402<212>DNA<213>流感A病毒<220><221>CDS<222>(27)..(389)<400>19agcaaaagca gggtgacaaa gacata atg gat tcc aac act gtg tca agt ttt 53Met Asp Ser Asn Thr Val Ser Ser Phe1 5cag gac ata cta ttg agg atg tca aaa atg caa ttg ggg tcc tca tcg 101Gln Asp Ile Leu Leu Arg Met Ser Lys Met Gln Leu Gly Ser Ser Ser10 15 20 25gag gac ttg aat gga atg ata aca cag ttc gag tct cta aaa ctc tac 149Glu Asp Leu Asn Gly Met Ile Thr Gln Phe Glu Ser Leu Lys Leu Tyr30 35 40aga gat tcg ctt ggg gaa gca gta atg aga atg gga gac ctc cac tta 197Arg Asp Ser Leu Gly Glu Ala Val Met Arg Met Gly Asp Leu His Leu45 50 55ctc caa aac aga aac gga aaa tgg cga gaa cag cta ggt caa aag ttc 245Leu Gln Asn Arg Asn Gly Lys Trp Arg Glu Gln Leu Gly Gln Lys Phe60 65 70gaa gag ata aga tgg ctg att gaa gaa gtg aga cac aga ctg aag aca 293Glu Glu Ile Arg Trp Leu Ile Glu Glu Val Arg His Arg Leu Lys Thr75 80 85aca gag aat agt ttt gag caa ata aca ttc atg caa gcc tta cag cta 341Thr Glu Asn Ser Phe Glu Gln Ile Thr Phe Met Gln Ala Leu Gln Leu90 95 100 105cta ttt gaa gtg gaa cag gag ata aga act ttc tcg ttt cag ctt att 389Leu Phe Glu Val Glu Gln Glu Ile Arg Thr Phe Ser Phe Gln Leu Ile1l0 115 120taatgataaa aaa402<210>20<211>121<212>PRT<213>流感A病毒<400>20Met Asp Ser Asn Thr Val Ser Ser Phe Gln Asp Ile Leu Leu Arg Met1 5 10 15Ser Lys Met Gln Leu Gly Ser Ser Ser Glu Asp Leu Asn Gly Met Ile20 25 30Thr Gln Phe Glu Ser Leu Lys Leu Tyr Arg Asp Ser Leu Gly Glu Ala35 40 45Val Met Arg Met Gly Asp Leu His Leu Leu Gln Asn Arg Asn Gly Lys50 55 60Trp Arg Glu Gln Leu Gly Gln Lys Phe Glu Glu Ile Arg Trp Leu Ile65 70 75 80Glu Glu Val Arg His Arg Leu Lys Thr Thr Glu Asn Ser Phe Glu Gln85 90 95Ile Thr Phe Met Gln Ala Leu Gln Leu Leu Phe Glu Val Glu Gln Glu100 105 110Ile Arg Thr Phe Ser Phe Gln Leu Ile115 120<210>21<211>1764<212>DNA<213>流感A病毒<220><221>CDS<222>(30)..(1727)<400>21agcaaaagca ggggataatt ctattaacc atg aaa act atc att gct ttg agc 53Met Lys Thr Ile Ile Ala Leu Ser1 5tac att ttc tgt ctg gtt ctc ggc caa gac ttt cca gga aat gac aac 101Tyr Ile Phe Cys Leu Val Leu Gly Gln Asp Phe Pro Gly Asn Asp Asn10 15 20agc aca gca acg ctg tgc ctg gga cat cat gcg gtg cca aac gga aca 149Ser Thr Ala Thr Leu Cys Leu Gly His His Ala Val Pro Asn Gly Thr25 30 35 40cta gtg aaa aca atc aca aat gat cag att gaa gtg act aat gct act 197Leu Val Lys Thr Ile Thr Asn Asp Gln Ile Glu Val Thr Asn Ala Thr45 50 55gag ctg gtt cag agt tcc tca acg ggg aaa ata tgc aac aat cct cat 245Glu Leu Val Gln Ser Ser Ser Thr Gly Lys Ile Cys Asn Asn Pro His60 65 70cga atc ctt gat gga ata gac tgc aca ctg ata gat gct cta ttg ggg 293Arg Ile Leu Asp Gly Ile Asp Cys Thr Leu Ile Asp Ala Leu Leu Gly75 80 85gac cct cat tgt gat ggc ttt caa aat gag aca tgg gac ctt ttc gtt 341Asp Pro His Cys Asp Gly Phe Gln Asn Glu Thr Trp Asp Leu Phe Val90 95 100gaa cgc agc aaa gct ttc agc aac tgt tac cct tat gat gtg cca gat 389Glu Arg Ser Lys Ala Phe Ser Asn Cys Tyr Pro Tyr Asp Val Pro Asp105 110 115 120tat gcc tcc ctt agg tca cta gtt gcc tcg tca ggc act ctg gag ttt 437Tyr Ala Ser Leu Arg Ser Leu Val Ala Ser Ser Gly Thr Leu Glu Phe125 130 135atc agt gaa ggc ttc act tgg act ggg gtc act cag aat ggg gga agc 485Ile Ser Glu Gly Phe Thr Trp Thr Gly Val Thr Gln Asn Gly Gly Ser140 145 150aat gct tgc aaa agg gga cct gat agc ggt ttt ttc agt aga ctg aac 533Asn Ala Cys Lys Arg Gly Pro Asp Ser Gly Phe Phe Ser Arg Leu Asn155 160 165tgg ttg tac aaa tca gga agc aca tat cca gtg ctg aac gtg act atg 581Trp Leu Tyr Lys Ser Gly Ser Thr Tyr Pro Val Leu Asn Val Thr Met170 175 180cca aac aat gac aat ttt gac aaa cta tac att tgg ggg gtt cac cac 629Pro Asn Asn Asp Asn Phe Asp Lys Leu Tyr Ile Trp Gly Val His His185 190 195 200ccg agc acg gac caa gaa caa acc agc cta tat gtt caa gca tca ggg 677Pro Ser Thr Asp Gln Glu Gln Thr Ser Leu Tyr Val Gln Ala Ser Gly205 210 215aga gtc aca gtc tct acc aag aga agc cag caa act ata atc ccg aat 725Arg Val Thr Val Ser Thr Lys Arg Ser Gln Gln Thr Ile Ile Pro Asn220 225 230atc ggg tct aga ccc tgg gta agg ggt ctg tct agt aga ata agc atc 773Ile Gly Ser Arg Pro Trp Val Arg Gly Leu Ser Ser Arg Ile Ser Ile235 240 245tat tgg aca ata gtt aaa ccg gga gac ata ctg gta att aat agt aat 821Tyr Trp Thr Ile Val Lys Pro Gly Asp Ile Leu Val Ile Asn Ser Asn250 255 260ggg aac cta att gct cct cgg ggt tat ttt aaa atg cgc act ggg aaa 869Gly Asn Leu Ile Ala Pro Ara Gly Tyr Phe Lys Met Arg Thr Gly Lys265 270 275 280agc tca ata atg agg tca gat gca cct att ggc acc tgc att tct gaa 917Ser Ser Ile Met Arg Ser Asp Ala Pro Ile Gly Thr Cys Ile Ser Glu285 290 295tgc atc act cca aat gga agc att ccc aat gac aag ccc ttt caa aac 965Cys Ile Thr Pro Asn Gly Ser Ile Pro Asn Asp Lys Pro Phe Gln Asn300 305 310gta aac aag atc aca tat ggg gca tgt ccc aag tat gtt aag caa aac 1013Val Asn Lys Ile Thr Tyr Gly Ala Cys Pro Lys Tyr Val Lys Gln Asn315 320 325acc ctg aag ttg gca aca ggg atg cgg aat gta cca gag aaa caa act 1061Thr Leu Lys Leu Ala Thr Gly Met Arg Asn Val Pro Glu Lys Gln Thr330 335 340aga ggc cta ttc agc gca ata gca ggt ttc ata gaa aat ggt tgg gag 1109Arg Gly Leu Phe Ser Ala Ile Ala Gly Phe Ile Glu Asn Gly Trp Glu345 350 355 360gga atg ata gac ggt tgg tac ggt ttc agg cat caa aat tct gag ggc 1157Gly Met Ile Asp Gly Trp Tyr Gly Phe Arg His Gln Asn Ser Glu Gly365 370 375aca gga caa gca gca gat ctt aaa age act caa gca gcc atc gac caa 1205Thr Gly Gln Ala Ala Asp Leu Lys Ser Thr Gln Ala Ala Ile Asp Gln380 385 390atc aat ggg aaa ctg aat agg gta atc gag aag acg aac gag aaa ttc 1253Ile Asn Gly Lys Leu Asn Arg Val Ile Glu Lys Thr Asn Glu Lys Phe395 400 405cat caa atc gaa aag gaa ttc tca gaa gta gaa ggg aga att cag gac 1301His Gln Ile Glu Lys Glu Phe Ser Glu Val Glu Gly Arg Ile Gln Asp410 415 420ctc gag aaa tac gtt gaa gac act aaa ata gat ctc tgg tct tac aat 1349Leu Glu Lys Tyr Val Glu Asp Thr Lys Ile Asp Leu Trp Ser Tyr Asn425 430 435 440gcg gag ctt ctt gtc gct ctg gag aac caa cat aca att gat ctg act 1397Ala Glu Leu Leu Val Ala Leu Glu Asn Gln His Thr Ile Asp Leu Thr445 450 455gac tcg gaa atg aac aaa ctg ttt gaa aaa aca agg agg caa ctg agg 1445Asp Ser Glu Met Asn Lys Leu Phe Glu Lys Thr Arg Arg Gln Leu Arg460 465 470gaa aat gct gag gac atg ggc aat ggt tgc ttc aaa ata tac cac aaa 1493Glu Asn Ala Glu Asp Met Gly Asn Gly Cys Phe Lys Ile Tyr His Lys475 480 485tgt gac aat gct tgc ata ggg tca atc aga aat ggg act tat gac cat 1541Cys Asp Asn Ala Cys Ile Gly Ser Ile Arg Asn Gly Thr Tyr Asp His490 495 500gat gta tac aga gac gaa gca tta aac aac cgg ttt cag atc aaa ggt 1589Asp Val Tyr Arg Asp Glu Ala Leu Asn Asn Arg Phe Gln Ile Lys Gly505 510 515 520gtt gaa ctg aag tca gga tac aaa gac tgg atc ctg tgg att tcc ttt 1637Val Glu Leu Lys Ser Gly Tyr Lys Asp Trp Ile Leu Trp Ile Ser Phe525 530 535gcc ata tca tgc ttt ttg ctt tgt gtt gtt ttg ctg ggg ttc atc atg 1685Ala Ile Ser Cys Phe Leu Leu Cys Val Val Leu Leu Gly Phe Ile Met540 545 550tgg gcc tgc cag aaa ggc aac att agg tgc aac att tgc att 1727Trp Ala Cys Gln Lys Gly Asn Ile Arg Cys Asn Ile Cys Ile555 560 565tgagtgtatt agtaattaaa aacaccctgt ttctact 1764<210>22<211>566<212>PRT<213>流感A病毒<400>22Met Lys Thr Ile Ile Ala Leu Ser Tyr Ile Phe Cys Leu Val Leu Gly1 5 10 15Gln Asp Phe Pro Gly Asn Asp Asn Ser Thr Ala Thr Leu Cys Leu Gly20 25 30His His Ala Val Pro Asn Gly Thr Leu Val Lys Thr Ile Thr Asn Asp35 40 45Gln Ile Glu Val Thr Asn Ala Thr Glu Leu Val Gln Ser Ser Ser Thr50 55 60Gly Lys Ile Cys Asn Asn Pro His Arg Ile Leu Asp Gly Ile Asp Cys65 70 75 80Thr Leu Ile Asp Ala Leu Leu Gly Asp Pro His Cys Asp Gly Phe Gln85 90 95Asn Glu Thr Trp Asp Leu Phe Val Glu Arg Ser Lys Ala Phe Ser Asn100 105 110Cys Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Ser Leu Arg Ser Leu Val115 120 125Ala Ser Ser Gly Thr Leu Glu Phe Ile Ser Glu Gly Phe Thr Trp Thr130 135 140Gly Val Thr Gln Asn Gly Gly Ser Asn Ala Cys Lys Arg Gly Pro Asp145 150 155 160Ser Gly Phe Phe Ser Arg Leu Asn Trp Leu Tyr Lys Ser Gly Ser Thr165 170 175Tyr Pro Val Leu Asn Val Thr Met Pro Asn Asn Asp Asn Phe Asp Lys180 185 190Leu Tyr Ile Trp Gly Val His His Pro Ser Thr Asp Gln Glu Gln Thr195 200 205Ser Leu Tyr Val Gln Ala Ser Gly Arg Val Thr Val Ser Thr Lys Arg210 215 220Ser Gln Gln Thr Ile Ile Pro Asn Ile Gly Ser Arg Pro Trp Val Arg225 230 235 240Gly Leu Ser Ser Arg Ile Ser Ile Tyr Trp Thr Ile Val Lys Pro Gly245 250 255Asp Ile Leu Val Ile Asn Ser Asn Gly Asn Leu Ile Ala Pro Arg Gly260 265 270Tyr Phe Lys Met Arg Thr Gly Lys Ser Ser Ile Met Arg Ser Asp Ala275 280 285Pro Ile Gly Thr Cys Ile Ser Glu Cys Ile Thr Pro Asn Gly Ser Ile290 295 300Pro Asn Asp Lys Pro Phe Gln Asn Val Asn Lys Ile Thr Tyr Gly Ala305 310 315 320Cys Pro Lys Tyr Val Lys Gln Asn Thr Leu Lys Leu Ala Thr Gly Met325 330 335Arg Asn Val Pro Glu Lys Gln Thr Arg Gly Leu Phe Ser Ala Ile Ala340 345 350Gly Phe Ile Glu Asn Gly Trp Glu Gly Met Ile Asp Gly Trp Tyr Gly355 360 365Phe Arg His Gln Asn Ser Glu Gly Thr Gly Gln Ala Ala Asp Leu Lys370 375 380Ser Thr Gln Ala Ala Ile Asp Gln Ile Asn Gly Lys Leu Asn Arg Val385 390 395 400Ile Glu Lys Thr Asn Glu Lys Phe His Gln Ile Glu Lys Glu Phe Ser405 410 415Glu Val Glu Gly Arg Ile Gln Asp Leu Glu Lys Tyr Val Glu Asp Thr420 425 430Lys Ile Asp Leu Trp Ser Tyr Asn Ala Glu Leu Leu Val Ala Leu Glu435 440 445Asn Gln His Thr Ile Asp Leu Thr Asp Ser Glu Met Asn Lys Leu Phe450 455 460Glu Lys Thr Arg Arg Gln Leu Arg Glu Asn Ala Glu Asp Met Gly Asn465 470 475 480Gly Cys Phe Lys Ile Tyr His Lys Cys Asp Asn Ala Cys Ile Gly Ser485 490 495Ile Arg Asn Gly Thr Tyr Asp His Asp Val Tyr Arg Asp Glu Ala Leu500 505 510Asn Asn Arg Phe Gln Ile Lys Gly Val Glu Leu Lys Ser Gly Tyr Lys515 520 525Asp Trp Ile Leu Trp Ile Ser Phe Ala Ile Ser Cys Phe Leu Leu Cys530 535 540Val Val Leu Leu Gly Phe Ile Met Trp Ala Cys Gln Lys Gly Asn Ile545 550 555 560Arg Cys Asn Ile Cys Ile565<210>23<211>27<212>DNA<213>人工序列<220><223>人工序列的描述引物<400>23tatggaaaga ataaaagaac tacggaa 27<210>24<211>22<212>DNA<213>人工序列<220><223>人工序列的描述引物<400>24tcgtttttaa actattcaac at 22<210>25<211>24<212>DNA<213>人工序列<220><223>人工序列的描述引物<400>25agcaggtcaa ttatattcaa tatg24<210>26<211>24<212>DNA<213>人工序列<220><223>人工序列的描述引物<400>26aacaaggtcg tttttaaact attc24<210>27<211>21<212>DNA<213>人工序列<220><223>人工序列的描述引物<400>27agaactctat tccaacaaat g 21<210>28<211>21<212>DNA<213>人工序列<220><223>人工序列的描述引物<400>28aatcggatat ttcattgcca t 21<210>29<211>33<212>DNA<213>人工序列<220><223>人工序列的描述引物<400>29agaactctat tccaacaaat gagggatgta gtt 33<210>30<211>33<212>DNA<213>人工序列<220><223>人工序列的描述引物<400>30aatcggatat ttcattgcca tcatccattt cat 33<210>31<211>21<212>DNA<213>人工序列<220><223>人工序列的描述引物<400>31agacgtggtg ttggtaatga a 21<210>32<211>21<212>DNA<213>人工序列<220><223>人工序列的描述引物<400>32cgaagagttg acataaaccc t 21<210>33<211>21<212>DNA<213>人工序列<220><223>人工序列的描述引物<400>33tcatccctca tcccctcaca t21<210>34<211>22<212>DNA<213>人工序列<220><223>人工序列的描述引物<400>34attttctgtt atcctcttgt ca 22<210>35<211>20<212>DNA<213>人工序列<220><223>人工序列的描述引物<400>35gtggagtccg ctgttttgag 20<210>36<211>21<212>DNA<213>人工序列<220><223>人工序列的描述引物<400>36aggatggtgg acattcttag g 21<210>37<211>21<212>DNA<213>人工序列<220><223>人工序列的描述引物<400>37ccgatcaatg ctaaccacta c 21<210>38<211>25<212>DNA<213>人工序列<220><223>人工序列的描述引物<400>38agcaaaagca ggtcaattat attca 25<210>39<211>25<212>DNA<213>人工序列<220><223>人工序列的描述引物<400>39agtagaaaca aggtcgtttt taaac25<210>40<211>20<212>DNA<213>人工序列<220><223>人工序列的描述引物<400>40agcaaaagca ggcaaaccat 20<210>41<211>21<212>DNA<213>人工序列<220><223>人工序列的描述引物<400>41agtagaaaca aggcattttt t21<210>42<211>20<212>DNA<213>人工序列<220><223>人工序列的描述引物<400>42tcgagctgaa gaagctatgg 20<210>43<211>20<212>DNA<213>人工序列<220><223>人工序列的描述引物<400>43gttctgttga ctgtgtccat 20<210>44<211>32<212>DNA<213>人工序列<220><223>人工序列的描述引物<400>44tcgagctgaa gaagctatgg gagcagaccc gt 32<210>45<211>29<212>DNA<213>人工序列<220><223>人工序列的描述引物<400>45gttctgttga ctgtgtccat ggtgtatcc 29<210>46<211>35<212>DNA<213>人工序列<220><223>人工序列的描述引物<400>46agcgaaagca ggcaaaccat ttgaatggat gtcaa35<210>47<211>31<212>DNA<213>人工序列<220><223>人工序列的描述引物<400>47attaaaaaca aggcattttt tcatgaagga c31<210>48<211>28<212>DNA<213>人工序列<220><223>人工序列的描述引物<400>48aatttccagc atggtggagg ccatggtg28<210>49<211>20<212>DNA<213>人工序列<220><223>人工序列的描述引物<400>49gggatctttg aaaactcgtg 20<210>50<211>19<212>DNA<213>人工序列<220><223>人工序列的描述引物<400>50cagcattgtt tacagactc19<210>51<211>20<212>DNA<213>人工序列<220><223>人工序列的描述引物<400>51accaaagatg cagaaagagg 20<210>52<211>20<212>DNA<213>人工序列<220><223>人工序列的描述引物<400>52ctcatattga ttccgactaa 20<210>53<211>21<212>DNA<213>人工序列<220><223>人工序列的描述引物<400>53tgtccacttc cctttttctg a 21<210>54<211>22<212>DNA<213>人工序列<220><223>人工序列的描述引物<400>54atttttcccc agtagttcat ac 22<210>55<211>25<212>DNA<213>人工序列<220><223>人工序列的描述引物<400>55acgctgttgc aactacacac tcctg 25<210>56<211>25<212>DNA<213>人工序列<220><223>人工序列的描述引物<400>56agcaaaagca ggtactgatt cgaga 25<210>57<211>25<212>DNA<213>人工序列<220><223>人工序列的描述引物<400>57agtagaaaca aggtactttt ttgga 25<210>58<211>21<212>DNA<213>人工序列<220><223>人工序列的描述引物<400>58gaacctggaa cctttgatct t 21<210>59<211>21<212>DNA<213>人工序列<220><223>人工序列的描述引物<400>59attcaccact gtccaggcca t 21<210>60<211>30<212>DNA<213>人工序列<220><223>人工序列的描述引物<400>60gaacctggaa cctttgatct tgaggggcta 30<210>61<211>30<212>DNA<213>人工序列<220><223>人工序列的描述引物<400>61attcaccact gtccaggcca ttgacgcgtc 30<210>62<211>21<212>DNA<213>人工序列<220><223>人工序列的描述引物<400>62agcaaaagca ggtagtgata g 21<210>63<211>15<212>DNA<213>人工序列<220><223>人工序列的描述引物<400>63agtagaaaca aggta 15<210>64<211>29<212>DNA<213>人工序列<220><223>人工序列的描述引物<400>64agcgaaagca ggtagtgatt cgagatgga 29<210>65<211>28<212>DNA<213>人工序列<220><223>人工序列的描述引物<400>65agtagaaaca aggtactttt ttggacag28<210>66<211>21<212>DNA<213>人工序列<220><223>人工序列的描述引物<400>66tcgatttgtt ggagtgactg a 21<210>67<211>21<212>DNA<213>人工序列<220><223>人工序列的描述引物<400>67gccgaacttc tcctgccttg a 21<210>68<211>19<212>DNA<213>人工序列<220><223>人工序列的描述引物<400>68gtattcaata gcctgtatg 19<210>69<211>20<212>DNA<213>人工序列<220><223>人工序列的描述引物<400>69gacttctctc cttgtcactc 20<210>70<211>21<212>DNA<213>人工序列<220><223>人工序列的描述引物<400>70acgagtcagc taaagtgggc a 21<210>71<211>21<212>DNA<213>人工序列<220><223>人工序列的描述引物<400>71gacacctctg ctgtgaagta a 21<210>72<211>21<212>DNA<213>人工序列<220><223>人工序列的描述引物<400>72ggagctgaga aaccgaagtt t 21<210>73<211>21<212>DNA<213>人工序列<220><223>人工序列的描述引物<400>73gacttggcca ataaagtcct a21<210>74<211>21<212>DNA<213>人工序列<220><223>人工序列的描述引物<400>74gcctgcgcat agttcctgtg a 21<210>75<211>20<212>DNA<213>人工序列<220><223>人工序列的描述引物<400>75tctaccacta ttgactcgcc20<210>76<211>23<212>DNA<213>人工序列<220><223>人工序列的描述引物<400>76agcaaaagca ggggataatt cta 23<210>77<211>23<212>DNA<213>人工序列<220><223>人工序列的描述引物<400>77agtagaaaca agggtgtttt taa 23<210>78<211>20<212>DNA<213>人工序列<220><223>人工序列的描述引物<400>78acagtttgtt catttccgag 20<210>79<211>20<212>DNA<213>人工序列<220><223>人工序列的描述引物<400>79acttcagggt gttttgctta 20<210>80<211>20<212>DNA<213>人工序列<220><223>人工序列的描述引物<400>80gaacccccca aatgtatagt 20<210>81<211>20<212>DNA<213>人工序列<220><223>人工序列的描述引物<400>81tgaggaactc tgaaccagct20<210>82<211>20<212>DNA<213>人工序列<220><223>人工序列的描述引物<400>82gtcactagtt gcctcgtcag20<210>83<211>20<212>DNA<213>人工序列<220><223>人工序列的描述引物<400>83ccgggagaca tactggtaat20<210>84<211>20<212>DNA<213>人工序列<220><223>人工序列的描述引物<400>84caaatcaatg ggaaactgaa20<210>85<211>20<212>DNA<213>人工序列<220><223>人工序列的描述引物<400>85tgaactgaag tcaggataca20<210>86<211>24<212>DNA<213>人工序列<220><223>人工序列的描述引物<400>86agcaaaagca gggttaataa tcac 24<210>87<211>24<212>DNA<213>人工序列<220><223>人工序列的描述引物<400>87agtagaaaca agggtatttt tcct 24<210>88<211>20<212>DNA<213>人工序列<220><223>人工序列的描述引物<400>88ttgcaccttc catcatcctt20<210>89<211>20<212>DNA<213>人工序列<220><223>人工序列的描述引物<400>89gtctattccc actaaagagt 20<210>90<211>20<212>DNA<213>人工序列<220><223>人工序列的描述引物<400>90tgcatcagag agcacatcct 20<210>91<211>20<212>DNA<213>人工序列<220><223>人工序列的描述引物<400>91gaactcgtcc tttatgacaa 20<210>92<211>17<212>DNA<213>人工序列<220><223>人工序列的描述引物<400>92agagcaatgg atcaagt17<210>93<211>20<212>DNA<213>人工序列<220><223>人工序列的描述引物<400>93tactatggaa tcaagtactc 20<210>94<211>18<212>DNA<213>人工序列<220><223>人工序列的描述引物<400>94atcaatcatc ttcccgac18<210>95<211>18<212>DNA<213>人工序列<220><223>人工序列的描述引物<400>95agtgtccttc cgtgggcg18<210>96<211>23<212>DNA<213>人工序列<220><223>人工序列的描述引物<400>96agcaaaagca ggagtgaaga tga 23<210>97<211>24<212>DNA<213>人工序列<220><223>人工序列的描述引物<400>97agtagaaaca aggagttttt tcta 24<210>98<211>20<212>DNA<213>人工序列<220><223>人工序列的描述引物<400>98tcgttgtttc tgggtgtgtc20<210>99<211>20<212>DNA<213>人工序列<220><223>人工序列的描述引物<400>99ttatcatacc cagtgacaca20<210>100<211>20<212>DNA<213>人工序列<220><223>人工序列的描述引物<400>100attattattg gttcacacgg20<210>101<211>20<212>DNA<213>人工序列<220><223>人工序列的描述引物<400>101ttatgtgtca tgcgatcctg20<210>102<211>20<212>DNA<213>人工序列<220><223>人工序列的描述引物<400>102attgttcata ttagcccatt20<210>103<211>20<212>DNA<213>人工序列<220><223>人工序列的描述引物<400>103ataggtcagg ttattctggt20<210>104<211>15<212>DNA<213>人工序列<220><223>人工序列的描述引物<400>104agcaaaagca ggtag 15<210>105<211>15<212>DNA<213>人工序列<220><223>人工序列的描述引物<400>105agtagaaaca aggta 15<210>106<211>24<212>DNA<213>人工序列<220><223>人工序列的描述引物<400>106tttactccag ctctatgctg acaa 24<210>107<211>20<212>DNA<213>人工序列<220><223>人工序列的描述引物<400>107gatccagcca tttgctccat20<210>108<211>20<212>DNA<213>人工序列<220><223>人工序列的描述引物<400>108gaggtgacag gattggtctt20<210>109<211>20<212>DNA<213>人工序列<220><223>人工序列的描述引物<400>109catggacaga gcagttaaac20<210>110<211>20<212>DNA<213>人工序列<220><223>人工序列的描述引物<400>110gcgagtatca ttgggatctt20<210>111<211>24<212>DNA<213>人工序列<220><223>人工序列的描述引物<400>111agcaaaagca gggtgacaaa gaca 24<210>112<211>24<212>DNA<213>人工序列<220><223>人工序列的描述引物<400>112agtagaaaca agggtgtttt ttat 24<210>113<211>24<212>DNA<213>人工序列<220><223>人工序列的描述引物<400>113ttttttatca ttaaataagc tgaa 24<210>114<211>20<212>DNA<213>人工序列<220><223>人工序列的描述引物<400>114aattgcattt ttgacatcct20<210>115<211>20<212>DNA<213>人工序列<220><223>人工序列的描述引物<400>115atcttctcta ctatctgctt20<210>116<211>20<212>DNA<213>人工序列<220><223>人工序列的描述引物<400>116caagcaatca tggataagaa20<210>117<211>20<212>DNA<213>人工序列<220><223>人工序列的描述引物<400>117ggcgagaaca gctaggtcaa20
權(quán)利要求
1.一種分離的核酸分子，其特征在于，包含流感A/Udorn/72(H3N2)菌株的正鏈即反基因組有義鏈的完整核苷酸序列，，所述的完整核苷酸序列由以下序列構(gòu)成(a)SEQ ID NO1、SEQ ID NO3、SEQ ID NO5、SEQ ID NO7、SEQ9、SEQ ID NO11、SEQ ID NO13和SEQ ID NO17，或(b)SEQ ID NO1、SEQ ID NO3、SEQ ID NO5、SEQ ID NO9、SEQ ID NO11、SEQ ID NO13、SEQ ID NO17和SEQ ID NO21。
2.如權(quán)利要求1所述的分離的核酸分子，其特征在于，包含流感A/Udorn/72(H3N2)菌株的完整核苷酸序列，所述的完整序列由以下序列構(gòu)成SEQID NO1、SEQ ID NO3、SEQ ID NO5、SEQ ID NO7、SEQ ID NO9、SEQ ID NO11、SEQ ID NO13和SEQ ID NO17。
3.如權(quán)利要求1所述的分離的核酸分子，其特征在于，包含流感A/Udorn/72(H3N2)菌株的完整核苷酸序列，所述的完整序列由以下序列構(gòu)成SEQID NO1、SEQ ID NO3、SEQ ID NO5、SEQ ID NO9、SEQ ID NO11、SEQ IDNO13、SEQ ID NO17和SEQ ID NO21。。
4.一種分離的核酸分子，其特征在于，包含單個(gè)流感A病毒的正鏈即反基因組有義鏈的核苷酸序列，所述的序列選自SEQ ID NO1、SEQ ID NO3、SEQ IDNO5、SEQ ID NO7、SEQ ID NO11和SEQ ID NO21。
5.如權(quán)利要求4所述的分離的核酸分子，其特征在于，所述的流感A病毒核苷酸序列是SEQ ID NO1。
6.如權(quán)利要求4所述的分離的核酸分子，其特征在于，所述的流感A病毒核苷酸序列是SEQ ID NO3。
7.如權(quán)利要求4所述的分離的核酸分子，其特征在于，所述的流感A病毒核苷酸序列是SEQ ID NO5。
8.如權(quán)利要求4所述的分離的核酸分子，其特征在于，所述的流感A病毒核苷酸序列是SEQ ID NO7。
9.如權(quán)利要求4所述的分離的核酸分子，其特征在于，所述的流感A病毒核苷酸序列是SEQ ID NO11。
10.如權(quán)利要求4所述的分離的核酸分子，其特征在于，所述的流感A病毒核苷酸序列是SEQ ID NO21。
11.一種分離的核酸分子，其特征在于，包含單個(gè)流感A病毒的正鏈即反基因組有義鏈的核苷酸序列，所述的序列編碼蛋白質(zhì)，該蛋白質(zhì)具有選自以下的序列SEQ ID NO2、SEQ ID NO4和SEQ ID NO6。
12.如權(quán)利要求11所述的分離的核苷酸分子，其特征在于，所述的流感A病毒核苷酸序列編碼序列為SEQ ID NO2的蛋白質(zhì)。
13.如權(quán)利要求11所述的分離的核苷酸分子，其特征在于，所述的流感A病毒核苷酸序列編碼序列為SEQ ID NO4的蛋白質(zhì)。
14.如權(quán)利要求11所述的分離的核苷酸分子，其特征在于，所述的流感A病毒核苷酸序列編碼序列為SEQ ID NO6的蛋白質(zhì)。
15.一種分離的氨基酸序列，其特征在于，包含單個(gè)流感A病毒氨基酸序列，所述的序列選自SEQ ID NO2、SEQ ID NO4和SEQ ID NO6。
16.如權(quán)利要求15所述的分離的氨基酸序列，其特征在于，所述的流感A病毒的氨基酸序列是SEQ ID NO2。
17.如權(quán)利要求15所述的分離的氨基酸序列，其特征在于，所述的流感A病毒的氨基酸序列是SEQ ID NO4。
18.如權(quán)利要求15所述的分離的氨基酸序列，其特征在于，所述的流感A病毒的氨基酸序列是SEQ ID NO6。
19.一種分離的氨基酸序列，其包含單個(gè)流感A病毒的序列，其特征在于，所述的序列是SEQ ID NO8。
20.一種分離的氨基酸序列，其包含單個(gè)流感A病毒的序列，其特征在于，所述的序列是SEQ ID NO12。
全文摘要
本文公開(kāi)了負(fù)鏈流感A病毒菌株A/Udron/72(H3N2)的8個(gè)節(jié)段的完整核苷酸序列，以反基因組信息提供了完整的基因組序列。這些序列可用于診斷，且通過(guò)突變修飾可產(chǎn)生新的流感A變體菌株。
文檔編號(hào)C07K14/11GK1437612SQ01811649
公開(kāi)日2003年8月20日 申請(qǐng)日期2001年6月21日 優(yōu)先權(quán)日2000年6月23日
發(fā)明者J·M·加拉爾薩, T·E·萊瑟姆 申請(qǐng)人:美國(guó)氰胺公司