專利名稱：：一種抗病毒的融合蛋白及其編碼基因與應(yīng)用的制作方法
技術(shù)領(lǐng)域：
：本發(fā)明涉及一種抗病毒的融合蛋白及其編碼基因與應(yīng)用。
背景技術(shù)：
：核糖核酸(RNA)是生命活動中不可缺少的重要分子，具有傳遞遺傳信息、編碼蛋白質(zhì)等許多功能。有害RNA的產(chǎn)生導(dǎo)致多種疾病的發(fā)生。HIV病毒是一種RNA病毒，其基因組RNA是病毒遺傳信息的攜帶者，病毒RNA也是翻譯病毒蛋白的模板。HIV所引起的艾滋病是當(dāng)今世界上對人類健康威脅最大的流行性傳染病。在過去的十幾年中，各國投入了巨大的努力和巨額資金致力于研制開發(fā)抗艾滋病的藥物，目前世界上批準用于臨床的抗艾滋病藥物已有近二十種。但是，已有的任何一種藥物都不能完全清除人體內(nèi)的HIV病毒，幾種藥物組合使用的效果會優(yōu)于單一藥物治療，但也不能完全清除HIV病毒。HIV在復(fù)制過程中有很高的突變率，病人體內(nèi)的病毒呈異源性，其中一些病毒具有抗藥性，當(dāng)治療進行到一定階段時，這些抗藥病毒的數(shù)量會逐漸占據(jù)優(yōu)勢，使得現(xiàn)有的治療無效。而且這些抗病毒藥物的長期毒性已在臨床上顯示出來。開發(fā)一種無副作用而且不會產(chǎn)生抗藥性的抗艾滋病藥物是本領(lǐng)域每一個科學(xué)家的夢想。抗艾滋病藥物的副作用來源于藥物作用的不專一性；抗藥性的產(chǎn)生則是因為HIV病毒具有非常高的突變率，使作為藥物靶點的病毒蛋白可以通過少至一個氨基酸的改變而逃避藥物的抑制作用，但不影響病毒蛋白自身的功能。因此，理想的抗艾滋病藥物應(yīng)對HIV病毒具有極高的特異性，對作用靶點的突變又有一定的包容性。HIV是一種逆轉(zhuǎn)錄病毒，在感染過程中首先通過病毒表面包膜蛋白(envelope)與其受體的相互作用吸附到細胞表面，然后病毒質(zhì)膜與細胞質(zhì)膜融合，裸露的病毒顆粒進入到細胞中。在細胞質(zhì)中病毒的RNA被逆轉(zhuǎn)錄成雙鏈DNA，這段DNA存在于由病毒蛋白及宿主蛋白組成的整合前復(fù)合體(Pre-IntegrationComplex，簡稱PIC)中。PIC由細胞質(zhì)膜附近遷移到核膜附近并進入細胞核。在細胞核中病毒DNA由病毒的整合酶整合到宿主細胞的染色體中。上述過程度被稱為逆轉(zhuǎn)錄病毒生命周期的前期。整合的病毒DNA被轉(zhuǎn)錄成RNA，其中大部分經(jīng)過剪切后作為模板翻譯產(chǎn)生病毒蛋白，小部分全長RNA作為病毒遺傳物質(zhì)被病毒蛋白包裝成病毒顆粒出芽到細胞外成為未成熟病毒。在未成熟病毒中一些病毒蛋白以融合蛋白形式存在，經(jīng)過病毒蛋白酶剪切后病毒顆粒結(jié)構(gòu)重組成為有感染性的成熟病毒，此過程稱為逆轉(zhuǎn)錄病毒生命周期的后期。在HIV復(fù)制過程中，病毒自身編碼的Tat蛋白發(fā)揮著重要作用。Tat是一種RNA結(jié)合蛋白，能夠特異性結(jié)合到HIV病毒RNA上的TAR序列g(shù)ggtctctctggttagaccagatctgagcctgggagctctctggctaactagggaacccactgcttaagcctcaataaagct(ROBERTA.MARCINIAK，MARIANOA.GARCIA-BLANCO，ANDPHILLIPA.SHARP.(1990)IdentificationandcharacterizationofaHeLanuclearproteinthatspecificallybindstothetrans-activation-response(TAR)elementofhumanimmunodeficiencyvirus.PNAS，Vol.87，pp.3624-3628.)。Tat通過結(jié)合TAR序列實現(xiàn)HIV基因組的持續(xù)轉(zhuǎn)錄，是HIV生命周期中不可缺少的一環(huán)。ZAP是一個抗病毒因子，通過在胞質(zhì)中特異性降解病毒RNA從而抑制某些病毒的復(fù)制，如鼠白血病病毒(MLV)、辛德比斯病毒(SIN)。但是ZAP對HIV病毒作用并不明顯。ZAP作用機理的研究結(jié)果表明，ZAP本身不具有RNA酶的活性，但是能特異性結(jié)合到病毒RNA的特定序列上，并招募細胞內(nèi)具有降解RNA功能的蛋白復(fù)合物Exosome而特異性地降解所結(jié)合的RNA。
發(fā)明內(nèi)容本發(fā)明的目的是提供一種抗病毒的融合蛋白及其編碼基因與應(yīng)用。本發(fā)明所提供的抗病毒的融合蛋白，名稱為ZAP-Tat，是具有下述氨基酸殘基序列之一的蛋白質(zhì)1)序列表中的SEQID№1；2)將序列表中SEQID№1的氨基酸殘基序列經(jīng)過一個或幾個氨基酸殘基的取代和/或缺失和/或添加且可特異性降解HIV病毒RNA的蛋白質(zhì)。所述一個或幾個氨基酸殘基的取代和/或缺失和/或添加是指不多于十個氨基酸殘基的取代和/或缺失和/或添加。序列表中的SEQID№1由904個氨基酸殘基組成。自氨基端的第1位至776位氨基酸殘基為ZAP，自氨基端的第777位至805位氨基酸殘基為linker，自氨基端的第806位至881位氨基酸殘基為Tat，自氨基端的第884位至893位氨基酸殘基為myc，自氨基端的第899位至904位氨基酸殘基為6×HIS。上述抗病毒的融合蛋白(ZAP-Tat)的編碼基因也屬于本發(fā)明的保護范圍。抗病毒的融合蛋白(ZAP-Tat)的編碼基因，可具有下述核苷酸序列之一1)序列表中SEQID№2的核苷酸序列；2)編碼序列表中SEQID№1蛋白質(zhì)序列的DNA；3)在高嚴謹條件下可與序列表中的SEQID№2限定的DNA序列雜交的核苷酸序列。序列表中的SEQID№2由2712個核苷酸組成。自5′端的第1位至2328位核苷酸為ZAP編碼序列，自5′端的第2329位至2415位核苷酸為linker的編碼序列，自5′端的第2416位至2643位核苷酸為Tat的編碼序列，自5′端的第2650位至2679位核苷酸為myc的編碼序列，自5′端的第2695位至2712位核苷酸為myc的編碼序列。所述高嚴謹條件可為用0.1×SSPE(或0.1×SSC)，0.1％SDS的溶液，在65℃下雜交并洗膜。含有本發(fā)明基因的表達載體、細胞系及宿主菌均屬于本發(fā)明的保護范圍。本發(fā)明的融合蛋白包括可與病毒TAR序列特異結(jié)合的RNA結(jié)合蛋白亞基(Tat)和具有降解RNA功能的蛋白亞基(ZAP)，該融合蛋白通過RNA結(jié)合蛋白亞基結(jié)合到特定的靶RNA上，RNA降解亞基招募細胞內(nèi)的RNA酶，降解融合蛋白所結(jié)合的特定RNA。本發(fā)明的融合蛋白對HIV病毒具有明顯的抑制效果可用于制備抗病毒的藥物，如制備預(yù)防和/或治療艾滋病的藥物。圖1為ZAP-Tat等質(zhì)粒酶切電泳圖。圖2為利用Western免疫雜交方法檢測ZAP-Tat等在細胞內(nèi)的表達。圖3為分析ZAP-Tat對HIV載體表達的抑制效果。具體實施例方式下述實施例中的實驗方法，如無特別說明，均為常規(guī)方法。實施例1、融合蛋白ZAP-Tat的表達本實施例中，首先在ZAP的C-端接上聯(lián)接肽序列(命名為ZAP-linker)，并證明了該聯(lián)接肽序列不影響ZAP的功能。然后，將Tat通過聯(lián)接肽序列(linker)融合到ZAP的C-端，成為ZAP-Tat。使用聯(lián)接肽序列的目的是將ZAP和Tat兩個結(jié)構(gòu)域分開，使之不互相干擾彼此的活性。作為對照，還構(gòu)建了N-端含有69個來自ZAP的氨基酸殘基的Tat(命名為dTat)。1、linker的制備利用化學(xué)合成方法合成5′→3′鏈具有以下序列的一段雙鏈DNA5’-GGTGGAGGCTCTGGGGGAGGTACAGGAGGTGGCTCAGGTGGGGGCACTGGTGGAGGCAGTGGAGGTGGC-3’。該序列編碼一段聯(lián)接肽序列。然后對其用如下的引物進行PCR擴增，所用引物引入克隆所需的酶切位點上游引物linkersp5’-ATATAGGCGGCCGTGGTGGAGGCTCTGG-3’(下劃線部分核苷酸為NotI酶切識別位點，方框內(nèi)的核苷酸序列為XhoI酶切識別位點)下游引物linkerap5’-ATATAGGGGCCCTTGGCGCCGCCACCTCCACTGCCTC-3’(下劃線部分核苷酸為ApaI酶切識別位點，方框內(nèi)的核苷酸序列為EcoRV酶切識別位點，GGCGCC為KasI酶切識別位點)PCR的反應(yīng)體系為1μl模板DNA(5pM/μl)，1μl上游引物(50μM)，1μl下游引物(50μM)，1μlTag酶，4μldNTP混合物(2.5mM)，5μlPCR反應(yīng)緩沖液(10×)，37μldH2O。PCR的反應(yīng)程序為94℃退火2min，然后94℃，30sec；55℃，30sec；72℃，30sec共25個循環(huán)。得到的PCR產(chǎn)物進行凝膠電泳，結(jié)果表明得到124bp的條帶，結(jié)果如圖1中的泳道2所示，回收該條帶的DNA，對其測序表明所獲得的PCR片段即包含NotI-XhoI—聯(lián)接序列-EcoRV-KasI-ApaI的DNA片段。把該DNA片段命名為linker。圖1中，泳道1和9是DL2000plus的marker；泳道2是linker的PCR產(chǎn)物；泳道3是Tat的PCR產(chǎn)物；泳道4是pNZAP-Tat用XbaI和NheI雙酶切的片段；泳道5是pZAP-Tat用NheI和NotI雙酶切的片段；泳道6是pNZAPlinker用EcoRI和NotI雙酶切的片段；泳道7是pZAPlinker用NheI和NotI雙酶切的片段；泳道8是pdTat用XhoI和EcoRI雙酶切的片段。2、在質(zhì)粒pcDNA4/TO/myc-HisB中插入linkerpcDNA4/TO/myc-HisB(從Invitrogen購買)，用NotI和ApaI雙酶切pcDNA4/TO/myc-HisB，切膠回收得到5133bp的大片段，將步驟1所獲得的PCR片段也用NotI和ApaI雙酶切，切膠回收得到109bp片段，二者連接，轉(zhuǎn)化大腸桿菌TOP10，在含有50μg/ml氨芐青霉素的LB固體培養(yǎng)基上培養(yǎng)12小時，挑取單克隆進行培養(yǎng)。提取質(zhì)粒，用引物linkersp和linkerap進行PCR驗證，得到124bp片段，結(jié)果如圖1所示。對質(zhì)粒進行測序，結(jié)果表明該質(zhì)粒含有該段linker序列，將該質(zhì)粒命名為pcDNA4/TO/linker/myc-HisB。3、在質(zhì)粒pcDNA4/TO/linker/myc-HisB中插入NZAP序列將pcDNA4/TO/linker/myc-HisB用EcoRI和NotI雙酶切，切膠回收得到5215bp的大片段。質(zhì)粒pNZAP-myc(pNZAP-myc的構(gòu)建方法見GuangxiaGao，XueminGuo，StephenP.Goff1.(2002)InhibitionofRetroviralRNAProductionbyZAP，aCCCH-TypeZincFingerProtein.SCIENCEVOL297，1703-1706)，含有序列表中序列14(編碼序列表中序列13的NZAPmyc)的NZAPmyc編碼基因，也用EcoRI和NotI雙酶切，切膠回收得到1088的片段，二者連接，轉(zhuǎn)化大腸桿菌TOP10，在含有50μg/ml氨芐青霉素的LB固體培養(yǎng)基上培養(yǎng)12小時，挑取單克隆進行培養(yǎng)。提取質(zhì)粒，用EcoRI和NotI雙酶切得到了5215bp的pcDNA4/TO/linker/myc-HisB片段和1088bp的NZAP片段，結(jié)果如圖1中泳道6所示。對質(zhì)粒進行測序，結(jié)果表明該質(zhì)粒含有序列表中序列6的核苷酸序列，編碼序列表中序列5的NZAP-linker-myc，將該質(zhì)粒命名為pNZAPlinker。4、在載體pNZAPlinker上插入Tat蛋白編碼序列以質(zhì)粒pCMVTat(pCMVTat的構(gòu)建方法見文獻NARASIMHACHARSRINIVASAKUMAR，NATHALIECHAZAL，C.HELGA-MARIA，SUSANPRASAD，MARIE-LOUISEHAMMARSKJOLD，ANDDAVIDREKOSH.(1997)TheEffectofViralRegulatoryProteinExpressiononGeneDeliverybvHumanImmunodeficiencyVirusType1VectorsProducedinStablePackagingCellLines.JVI，Vol.71，No.8，5841-5848)作為模板，然后對其用如下的引物進行PCR擴增，所用引物引入克隆所需的酶切位點上游引物TATSP5’-GGCCGCGCTGCTGAGCC-3’下游引物TATAP5’-ATATAGTTCGAACCGCGGCTGCTTTGATAGA-3’(下劃線部分核苷酸為BstBI酶切識別位點)PCR的反應(yīng)體系為1μl模板(0.1μg/μl)，1μl上游引物(50μM)，1μl下游引物(50μM)，0.25μlTaq酶，4μldNTP混合物(2.5mM)，5μlPCRbuffer(10×)，37.75μldH20。PCR的反應(yīng)程序為94℃退火2min，然后94℃，30sec；55℃，30sec；72℃，30sec共25個循環(huán)。得到的PCR產(chǎn)物進行凝膠電泳，結(jié)果表明得到243bp的條帶，結(jié)果如圖1中的泳道3所示，回收該條帶的DNA，用BstBI酶切，切膠回收含有Tat基因的小片段233bp；載體pNZAPlinker用EcoRV和BstBI酶切，切膠回收含有NZAP和linker編碼基因的大片段6271bp，二者連接，轉(zhuǎn)化大腸桿菌TOP10，在含有50μg/ml氨芐青霉素的LB固體培養(yǎng)基上培養(yǎng)12小時，挑取單克隆進行培養(yǎng)，提取質(zhì)粒，用XbaI和NheI雙酶切，得到6027bp和477bp的片段，結(jié)果如圖1中泳道4所示。該質(zhì)粒命名為pNZAP-Tat，含有序列表中SEQID№10的核苷酸序列，編碼序列表中序列9的NZAP-Tat/myc。5、ZAP-Tat的表達載體pZAP-Tat的構(gòu)建將pNZAP-Tat用AflII和NotI雙酶切，凝膠電泳回收5372bp大片段作為載體，pZAP-myc(pZAP-myc的構(gòu)建方法見GuangxiaGao，XueminGuo，StephenP.Goff1.(2002)InhibitionofRetroviralRNAProductionbyZAP，aCCCH-TypeZincFingerProtein.SCIENCEVOL297，1703-1706)用AflII和NotI雙酶切，回收2701bp的ZAP片段，與上面制備的載體連接，轉(zhuǎn)化大腸桿菌TOP10，在含有50μg/ml氨芐青霉素的LB固體培養(yǎng)基上培養(yǎng)12小時，挑取單克隆進行培養(yǎng)，提取質(zhì)粒用NheI和NotI雙酶切，得到6141bp的載體片段和1932bp片段，結(jié)果如圖1中泳道5所示，該質(zhì)粒命名為pZAP-Tat，含有序列表中序列2的融合蛋白ZAP-Tat的編碼基因。6、ZAP、ZAP-linker、dTat的表達質(zhì)粒的構(gòu)建含有序列表中序列4(編碼序列表中序列3的ZAP-myc)的ZAP-myc編碼基因的質(zhì)粒pZAP-myc的構(gòu)建方法見文獻GuangxiaGao，XueminGuo，StephenP.Goff1.(2002)InhibitionofRetroviralRNAProductionbyZAP，aCCCH-TypeZincFingerProtein.SCIENCEVOL297.1703-1706。將質(zhì)粒pNZAPlinker用AflII和NotI雙酶切，凝膠電泳回收5162bp大片段作為載體，質(zhì)粒pZAP-myc也用AflII和NotI雙酶切，回收2701bp的ZAP片段，二者連接，轉(zhuǎn)化大腸桿菌TOP10，在含有50μg/ml氨芐青霉素的LB固體培養(yǎng)基上培養(yǎng)12小時，挑取單克隆進行培養(yǎng)，提取質(zhì)粒用NheI和NotI雙酶切，得到5931bp的載體片段和1932bp的片段，結(jié)果如圖1中泳道7所示。該質(zhì)粒命名為pZAPlinker，含有序列表中序列8的核苷酸序列，編碼序列表中序列7的ZAPlinker-myc。將質(zhì)粒pZAP-Tat用XhoI單酶切，回收5651bp的片段，用Klenow(NewEnglandBiolab)補平，自身連接，轉(zhuǎn)化大腸桿菌TOP10，在含有50μg/ml氨芐青霉素的LB固體培養(yǎng)基上培養(yǎng)12小時，挑取單克隆進行培養(yǎng)，提取質(zhì)粒，用XhoI和NotI雙酶切驗證，結(jié)果如圖1中泳道8所示。對用XhoI和EcoRI雙酶切為單一片段的質(zhì)粒測序，結(jié)果表明該質(zhì)粒含有該段dTat-myc序列，將該質(zhì)粒命名為pdTat，含有序列表中序列12的核苷酸序列，編碼序列表中序列11的dTat-myc。7、ZAP、ZAP-linker、dTat、ZAP-Tat的表達將pZAP-myc、pZAPlinker、pdTat、pZAP-Tat分別用磷酸鈣法轉(zhuǎn)染入293T細胞(購自ATCC)，質(zhì)粒用量為5μg/3.5cm培養(yǎng)皿，8小時后換液，用PBS緩沖液洗一次，加入新鮮培養(yǎng)基(DMEM∶FBS(胎牛血清)∶PS(青霉素和鏈霉素)＝100∶10∶1(體積比))，48小時后收集細胞，裂解后進行SDS-PAGE電泳，然后將蛋白轉(zhuǎn)到PVDF膜上，用myc抗體(9E10)(購自SantCruz)進行Western免疫檢測，檢測到ZAP、ZAP-linker、dTat、ZAP-Tat的蛋白表達，結(jié)果如圖2所示。實施例2、抗HIV功能檢測實施例1中，利用Western免疫雜交方法，首先證明了ZAP、ZAP-linker、dTat、ZAP-Tat在細胞內(nèi)的表達，本實施例分析了上述蛋白對HIVRNA的抑制情況，方法如下將表達ZAP、ZAPlinker、dTat、ZAP-Tat的質(zhì)粒與攜帶熒光素酶報告基因的HIV載體pHR’-CMV-luc共表達(pHR’-CMV-luc含有HIV的大部分骨架序列，包括5’和3’LTR，5’剪切位點，包裝信號，部分的gag基因編碼序列，3’剪切位點等，pHR’-CMV-luc的構(gòu)建方法見DavidDerse，ShawnA.Hill，PatriciaA.Lloyd，Hye-kyungChung，andBarryA.Morse.ExaminingHumanT-LymphotropicVirusType1InfectionandReplicationbyCell-FreeInfectionwithRecombinantVirusVectors.JVirol.2001September；75(18)8461-8468)，分析上述蛋白對HIV載體表達的抑制情況。為了檢測轉(zhuǎn)染效率，使用了表達海腎(Renilla)螢光素酶的質(zhì)粒pRL-TK(購自Promega)作為轉(zhuǎn)染效率的內(nèi)參。pHR’-CMV-luc和pRL-TK分別與下述四種質(zhì)粒中的一種用磷酸鈣法共轉(zhuǎn)染293A細胞(購自ATCC)pcDNA4/TO/myc-HisB，pZAP-myc，pZAPlinker和pZAP-Tat。所用質(zhì)粒的質(zhì)量比為pcDNA4/TO/myc-HisB或pZAP-myc或pZAP-linker或pZAP-Tat∶pHR’-CMV-luc∶pRL-TK＝100∶10∶1。先用胰蛋白酶消化以便收獲生長到對數(shù)期的293A細胞，以5×105個細胞/3.5cm培養(yǎng)皿的數(shù)量重新種入3.5cm培養(yǎng)皿，加入新鮮DMEM培養(yǎng)基于37℃、5％CO2的培養(yǎng)箱中培養(yǎng)16-24小時。然后用質(zhì)粒pcDNA4/TO/myc-HisB或pZAP或pZAP-linker或pZAP-Tat5μg，pHR’-CMV-luc0.5μg，pRL-TK0.05μg的量，磷酸鈣法分別轉(zhuǎn)染細胞。轉(zhuǎn)染后8小時，吸出培養(yǎng)皿中的培養(yǎng)基，用PBS緩沖液洗一次，重新加入新鮮培養(yǎng)基，培養(yǎng)48小時后，用Promega公司的Dual-luciferase試劑盒檢測熒光素酶活性，分別得到螢火蟲熒光素酶和海腎螢光素酶的熒光值。海腎螢光素酶的數(shù)值用來作為內(nèi)參，表明轉(zhuǎn)染效率的高低，螢火蟲熒光素酶的數(shù)值作為報告參數(shù)，表明在某一轉(zhuǎn)染效率下報告基因表達量的高低。螢火蟲熒光素酶和海腎(Renilla)螢光素酶的熒光值比值是將每組數(shù)值做歸一化處理，便于與某一標準值比較。以空質(zhì)粒pcDNA4/TO/myc-HisB共轉(zhuǎn)染組的螢火蟲熒光素酶和海腎螢光素酶的熒光值的比值作為對照，其它組的比值去除該值，所得比值即為抑制倍數(shù)，即抑制倍數(shù)＝空質(zhì)粒共轉(zhuǎn)染組比值/功能蛋白共轉(zhuǎn)染組比值。結(jié)果如圖3所示，表明pZAP-Tat表達的ZAP-Tat對HIV的抑制作用達到16倍以上，其它質(zhì)粒表達的蛋白對HIV的抑制作用不足4倍。圖3中，pcDNA4表示pcDNA4/TO/myc-HisB和pHR’-CMV-luc和pRL-TK共轉(zhuǎn)染組；ZAP表示pZAP-myc和pHR’-CMV-luc和pRL-TK共轉(zhuǎn)染組；ZAPlinker表示pZAPlinker和pHR’-CMV-luc和pRL-TK共轉(zhuǎn)染組；ZAP-Tat表示pZAP-Tat和pHR’-CMV-luc和pRL-TK共轉(zhuǎn)染組。序列表<160>14<210>1<211>904<212>PRT<213>人工序列<220><223><400>1MetAlaAspProGlyValCysCysPheIleThrLysIleLeuCysAla51015HisGlyGlyArgMetThrLeuGluGluLeuLeuGlyGluIleArgLeu202530ProGluAlaGlnLeuTyrGluLeuLeuGluThrAlaGlyProAspArg354045PheValLeuLeuGluThrGlyGlyGlnAlaGlyIleThrArgSerVal505560ValAlaThrThrArgAlaArgValCysArgArgLysTyrCysGlnArg65707580ProCysAspSerLeuHisLeuCysLysLeuAsnLeuLeuGlyArgCys859095HisTyrAlaGlnSerGlnArgAsnLeuCysLysTyrSerHisAspVal100105110LeuSerGluGlnAsnPheGlnIleLeuLysAsnHisGluLeuSerGly115120125LeuAsnGlnGluGluLeuAlaCysLeuLeuValGlnSerAspProPhe130135140PheLeuProGluIleCysLysSerTyrLysGlyGluGlyArgLysGln145150155160ThrCysGlyGlnProGlnProCysGluArgLeuHisIleCysGluHis165170175PheThrArgGlyAsnCysSerTyrLeuAsnCysLeuArgSerHisAsn180185190LeuMetAspArgLysValLeuThrIleMetArgGluHisGlyLeuSer195200205ProAspValValGlnAsnIleGlnAspIleCysAsnAsnLysHisAla210215220ArgArgAsnProProGlyThrArgAlaAlaHisProHisArgArgGly225230235240GlyAlaHisArgAspArgSerLysSerArgAspArgPheLeuHisAsn245250255SerLeuGluPheLeuSerProValValSerProLeuGlySerGlyPro260265270ProSerProAspValThrSerCysLysAspSerLeuGluAspValSer275280285ValAspValThrGlnLysPheLysTyrLeuGlyThrHisAspArgAla290295300GlnLeuSerProValSerSerLysAlaAlaGlyValGlnGlyProSer305310315320GlnMetArgAlaSerGlnGluPheSerGluAspGlyAsnLeuAspAsp325330335IlePheSerArgAsnArgSerAspSerSerSerSerArgAlaSerAla340345350AlaLysValAlaGlnArgAsnGluAlaValAlaMetLysMetGlyMet355360365GluValLysGlyLysLysGluAlaProAspIleAspArgValProPhe370375380LeuAsnSerTyrIleAspGlyValThrMetGluLysAlaSerValSer385390395400GlyIleProGlyLysLysPheThrAlaAsnAspLeuGluAsnLeuLeu405410415LeuLeuAsnAspThrTrpLysAsnValAlaLysProGlnAspLeuGln420425430ThrThrGlyArgIleThrAspSerGlyGlnAspLysAlaPheLeuGln435440445AsnLysTyrGlyGlyAsnProValTrpAlaSerAlaSerThrHisAsn450455460AlaProAsnGlySerSerGlnIleMetAspGluThrProAsnValSer465470475480LysSerSerThrSerGlyPheAlaIleLysProAlaIleAlaGlyGly485490495LysGluAlaValTyrSerGlyValGlnSerProArgSerGlnValLeu500505510AlaValProGlyGluAlaThrThrProValGlnSerAsnArgLeuPro515520525GlnSerProLeuSerSerSerSerHisArgAlaAlaAlaSerGlySer530535540ProGlyLysAsnSerThrHisThrSerValSerProAlaIleGluSer545550555560SerArgMetThrSerAspProAspGluTyrLeuLeuArgTyrIleLeu565570575AsnProLeuPheArgMetAspAsnHisGlyProLysGluIleCysGln580585590AspHisLeuTyrLysGlyCysGlnGlnSerHisCysAspArgSerHis595600605PheHisLeuProTyrArgTrpGlnMetPheValTyrThrThrTrpArg610615620AspPheGlnAspMetGluSerIleGluGlnAlaTyrCysAspProHis625630635640ValGluLeuIleLeuIleGluAsnHisGlnIleAsnPheGlnLysMet645650655ThrCysAspSerTyrProIleArgArgLeuSerThrProSerTyrGlu660665670GluLysProLeuSerAlaValPheAlaThrLysTrpIleTrpTyrTrp675680685LysAsnGluPheAsnGluTyrIleGlnTyrGlyAsnGluSerProGly690695700HisThrSerSerAspIleAsnSerAlaTyrLeuGluSerPhePheGln705710715720SerCysProArgGlyValLeuProPheGlnAlaGlySerGlnLysTyr725730735GluLeuSerPheGlnGlyMetIleGlnThrAsnIleAlaSerLysThr740745750GlnArgHisValValArgArgProValPheValSerSerAsnAspVal755760765GluGlnLysArgArgGlyProGluGlyGlyArgSerSerGlyGlyGly770775780SerGlyGlyGlyThrGlyGlyGlySerGlyGlyGlyThrGlyGlyGly785790795800SerGlyGlyGlyAspGlyArgAlaAlaGluProValAspProSerLeu805810815GluProTrpLysHisProGlySerGlnProArgThrAlaCysAsnAsn820825830CysTyrCysLysLysCysCysPheHisCysTyrAlaCysPheThrArg835840845LysGlyLeuGlyIleSerTyrGlyArgLysLysArgArgGlnArgArg850855860ArgAlaHisGlnAsnSerGlnThrHisGlnAlaSerLeuSerLysGln865870875880ProArgPheGluGlnLysLeuIleSerGluGluAspLeuAsnMetHis885890895ThrGlyHisHisHisHisHisHis900<210>2<211>2712<212>DNA<213>人工序列<220><223><400>2atggcagatcccggggtatgctgtttcatcaccaagatcctgtgcgcccacgggggccgt60atgaccctggaggaactgctgggtgagatcaggctccccgaggcgcagctctacgagctg120ctggagacggcggggcccgatcgcttcgtgctattggagactggaggccaggccgggatc180actcggtctgtagtggctactactcgagcccgcgtctgccgtcggaagtactgccagaga240ccctgcgacagcctgcacctctgcaagcttaatctgctcggccggtgccactatgcacag300tctcagcggaacctctgcaaatattctcacgatgttctctcggaacagaacttccagatc360ctgaagaatcatgagctctctgggcttaaccaagaggagctagcttgcctcctggtccaa420agcgacccttttttcctgcccgagatatgcaagagttacaaaggagagggccgaaaacag480acctgtgggcagccacagccatgcgagagactccacatctgtgagcacttcacccggggc540aactgcagttacctcaactgtctcaggtctcacaacctgatggacagaaaggtgttgacc600atcatgagggagcacgggctgagtcctgatgtggtccagaacatccaggacatctgcaac660aacaaacacgccaggaggaacccgcctggcacgagagctgcccatccacaccgcagaggc720ggcgcacacagagacagaagcaaaagcagagaccgcttccttcacaacagtctagaattt780ctctcacctgttgtctcacctctgggatctggtccgcctagcccagatgtcaccagctgt840aaagattccctggaggatgtgtctgtggatgtcacccagaagttcaagtacttggggacg900catgaccgtgcgcagctctccccagtctcatctaaggctgctggtgttcaaggacccagt960caaatgagagcaagccaagagttttcagaggatgggaatctagatgacatattttctagg1020aatcgttctgattcatcatcaagtcgagcctccgctgccaaggtggcacaaagaaatgaa1080gctgtggccatgaaaatgggcatggaggtcaagggcaagaaggaggctccagacatcgat1140cgggtcccatttttaaatagttatattgatggggtgaccatggaaaaagcatcggtctca1200ggaattccaggcaaaaagttcacagccaatgatctggaaaatttgctattacttaacgac1260acttggaagaatgtggctaagccccaggatctgcagaccacaggcagaatcactgacagt1320ggccaagacaaggcattcctgcagaataaatatggaggaaacccagtgtgggcaagtgca1380tccacccataatgccccaaatggctctagtcaaattatggatgaaactcctaatgtctct1440aaaagtagtaccagtggttttgccataaaaccagcaattgctggaggaaaagaagcagtc1500tattctggagttcagagtccgagaagccaggtcctagctgtgcctggggaggctactacc1560cctgtacagagcaacaggctgcctcagtcgcctctgtcttcctcaagccacagagctgca1620gcctctgggagccctggcaagaactccacccatacctctgtgagcccagccatcgagtct1680tcaaggatgacatcagaccccgatgagtatctcctacgctacatcctaaatcctttattt1740aggatggataatcatggcccgaaggaaatctgtcaggaccatctgtacaagggctgtcaa1800cagagccactgcgacaggagtcacttccatctgccctaccggtggcagatgttcgtatat1860accacttggagggacttccaggacatggagtctatcgaacaggcctattgtgatccccac1920gttgaactcattttgatagaaaaccatcagatcaatttccagaaaatgacctgtgactcc1980taccccatccgacgcctctccactccctcatatgaggaaaagccacttagtgctgtcttc2040gccaccaagtggatttggtattggaagaatgaatttaatgaatatatccagtatgggaat2100gagagcccaggccacaccagctctgacatcaactctgcgtacctggagtctttcttccag2160tcttgtcccaggggagttttgccattccaggctggttcacagaagtacgagttaagcttc2220caagggatgattcagacaaatatagcttccaagactcaaaggcatgttgtcagaaggcca2280gtatttgtttcttcgaacgatgtggagcagaagagaagaggtccagagggcggccgctcg2340agtggtggaggctctgggggaggtacaggaggtggctcaggtgggggcactggtggaggc2400agtggaggtggcgatggccgcgctgctgagccagtagatcctagtctagagccctggaag2460catccaggaagtcagcctaggactgcttgtaacaattgctattgtaaaaagtgttgcttt2520cattgctacgcgtgtttcacaagaaaaggcttaggcatctcctatggcaggaagaagcgg2580agacagcgacgaagagctcatcagaacagtcagactcatcaagcttctctatcaaagcag2640ccgcggttcgaacaaaaactcatctcagaagaggatctgaatatgcataccggtcatcat2700caccatcaccat2712<210>3<211>808<212>PRT<213>人工序列<220><223><400>3MetAlaAspProGlyValCysCysPheIleThrLysIleLeuCysAla51015HisGlyGlyArgMetThrLeuGluGluLeuLeuGlyGluIleArgLeu202530ProGluAlaGlnLeuTyrGluLeuLeuGluThrAlaGlyProAspArg354045PheValLeuLeuGluThrGlyGlyGlnAlaGlyIleThrArgSerVal505560ValAlaThrThrArgAlaArgValCysArgArgLysTyrCysGlnArg65707580ProCysAspSerLeuHisLeuCysLysLeuAsnLeuLeuGlyArgCys859095HisTyrAlaGlnSerGlnArgAsnLeuCysLysTyrSerHisAspVal100105110LeuSerGluGlnAsnPheGlnIleLeuLysAsnHisGluLeuSerGly115120125LeuAsnGlnGluGluLeuAlaCysLeuLeuValGlnSerAspProPhe130135140PheLeuProGluIleCysLysSerTyrLysGlyGluGlyArgLysGln145150155160ThrCysGlyGlnProGlnProCysGluArgLeuHisIleCysGluHis165170175PheThrArgGlyAsnCysSerTyrLeuAsnCysLeuArgSerHisAsn180185190LeuMetAspArgLysValLeuThrIleMetArgGluHisGlyLeuSer195200205ProAspValValGlnAsnIleGlnAspIleCysAsnAsnLysHisAla210215220ArgArgAsnProProGlyThrArgAlaAlaHisProHisArgArgGly225230235240GlyAlaHisArgAspArgSerLysSerArgAspArgPheLeuHisAsn245250255SerLeuGluPheLeuSerProValValSerProLeuGlySerGlyPro260265270ProSerProAspValThrSerCysLysAspSerLeuGluAspValSer275280285ValAspValThrGlnLysPheLysTyrLeuGlyThrHisAspArgAla290295300GlnLeuSerProValSerSerLysAlaAlaGlyValGlnGlyProSer305310315320GlnMetArgAlaSerGlnGluPheSerGluAspGlyAsnLeuAspAsp325330335IlePheSerArgAsnArgSerAspSerSerSerSerArgAlaSerAla340345350AlaLysValAlaGlnArgAsnGluAlaValAlaMetLysMetGlyMet355360365GluValLysGlyLysLysGluAlaProAspIleAspArgValProPhe370375380LeuAsnSerTyrIleAspGlyValThrMetGluLysAlaSerValSer385390395400GlyIleProGlyLysLysPheThrAlaAsnAspLeuGluAsnLeuLeu405410415LeuLeuAsnAspThrTrpLysAsnValAlaLysProGlnAspLeuGln420425430ThrThrGlyArgIleThrAspSerGlyGlnAspLysAlaPheLeuGln435440445AsnLysTyrGlyGlyAsnProValTrpAlaSerAlaSerThrHisAsn450455460AlaProAsnGlySerSerGlnIleMetAspGluThrProAsnValSer465470475480LysSerSerThrSerGlyPheAlaIleLysProAlaIleAlaGlyGly485490495LysGluAlaValTyrSerGlyValGlnSerProArgSerGlnValLeu500505510AlaValProGlyGluAlaThrThrProValGlnSerAsnArgLeuPro515520525GlnSerProLeuSerSerSerSerHisArgAlaAlaAlaSerGlySer530535540ProGlyLysAsnSerThrHisThrSerValSerProAlaIleGluSer545550555560SerArgMetThrSerAspProAspGluTyrLeuLeuArgTyrIleLeu565570575AsnProLeuPheArgMetAspAsnHisGlyProLysGluIleCysGln580585590AspHisLeuTyrLysGlyCysGlnGlnSerHisCysAspArgSerHis595600605PheHisLeuProTyrArgTrpGlnMetPheValTyrThrThrTrpArg610615620AspPheGlnAspMetGluSerIleGluGlnAlaTyrCysAspProHis625630635640ValGluLeuIleLeuIleGluAsnHisGlnIleAsnPheGlnLysMet645650655ThrCysAspSerTyrProIleArgArgLeuSerThrProSerTyrGlu660665670GluLysProLeuSerAlaValPheAlaThrLysTrpIleTrpTyrTrp675680685LysAsnGluPheAsnGluTyrIleGlnTyrGlyAsnGluSerProGly690695700HisThrSerSerAspIleAsnSerAlaTyrLeuGluSerPhePheGln705710715720SerCysProArgGlyValLeuProPheGlnAlaGlySerGlnLysTyr725730735GluLeuSerPheGlnGlyMetIleGlnThrAsnIleAlaSerLysThr740745750GlnArgHisValValArgArgProValPheValSerSerAsnAspVal755760765GluGlnLysArgArgGlyProGluGlyGlyArgSerSerLeuGluGly770775780ProArgPheGluGlnLysLeuIleSerGluGluAspLeuAsnMetHis785790795800ThrGlyHisHisHisHisHisHis805<210>4<211>2424<212>DNA<213>人工序列<220><223><400>4atggcagatcccggggtatgctgtttcatcaccaagatcctgtgcgcccacgggggccgt60atgaccctggaggaactgctgggtgagatcaggctccccgaggcgcagctctacgagctg120ctggagacggcggggcccgatcgcttcgtgctattggagactggaggccaggccgggatc180actcggtctgtagtggctactactcgagcccgcgtctgccgtcggaagtactgccagaga240ccctgcgacagcctgcacctctgcaagcttaatctgctcggccggtgccactatgcacag300tctcagcggaacctctgcaaatattctcacgatgttctctcggaacagaacttccagatc360ctgaagaatcatgagctctctgggcttaaccaagaggagctagcttgcctcctggtccaa420agcgacccttttttcctgcccgagatatgcaagagttacaaaggagagggccgaaaacag480acctgtgggcagccacagccatgcgagagactccacatctgtgagcacttcacccggggc540aactgcagttacctcaactgtctcaggtctcacaacctgatggacagaaaggtgttgacc600atcatgagggagcacgggctgagtcctgatgtggtccagaacatccaggacatctgcaac660aacaaacacgccaggaggaacccgcctggcacgagagctgcccatccacaccgcagaggc720ggcgcacacagagacagaagcaaaagcagagaccgcttccttcacaacagtctagaattt780ctctcacctgttgtctcacctctgggatctggtccgcctagcccagatgtcaccagctgt840aaagattccctggaggatgtgtctgtggatgtcacccagaagttcaagtacttggggacg900catgaccgtgcgcagctctccccagtctcatctaaggctgctggtgttcaaggacccagt960caaatgagagcaagccaagagttttcagaggatgggaatctagatgacatattttctagg1020aatcgttctgattcatcatcaagtcgagcctccgctgccaaggtggcacaaagaaatgaa1080gctgtggccatgaaaatgggcatggaggtcaagggcaagaaggaggctccagacatcgat1140cgggtcccatttttaaatagttatattgatggggtgaccatggaaaaagcatcggtctca1200ggaattccaggcaaaaagttcacagccaatgatctggaaaatttgctattacttaacgac1260acttggaagaatgtggctaagccccaggatctgcagaccacaggcagaatcactgacagt1320ggccaagacaaggcattcctgcagaataaatatggaggaaacccagtgtgggcaagtgca1380tccacccataatgccccaaatggctctagtcaaattatggatgaaactcctaatgtctct1440aaaagtagtaccagtggttttgccataaaaccagcaattgctggaggaaaagaagcagtc1500tattctggagttcagagtccgagaagccaggtcctagctgtgcctggggaggctactacc1560cctgtacagagcaacaggctgcctcagtcgcctctgtcttcctcaagccacagagctgca1620gcctctgggagccctggcaagaactccacccatacctctgtgagcccagccatcgagtct1680tcaaggatgacatcagaccccgatgagtatctcctacgctacatcctaaatcctttattt1740aggatggataatcatggcccgaaggaaatctgtcaggaccatctgtacaagggctgtcaa1800cagagccactgcgacaggagtcacttccatctgccctaccggtggcagatgttcgtatat1860accacttggagggacttccaggacatggagtctatcgaacaggcctattgtgatccccac1920gttgaactcattttgatagaaaaccatcagatcaatttccagaaaatgacctgtgactcc1980taccccatccgacgcctctccactccctcatatgaggaaaagccacttagtgctgtcttc2040gccaccaagtggatttggtattggaagaatgaatttaatgaatatatccagtatgggaat2100gagagcccaggccacaccagctctgacatcaactctgcgtacctggagtctttcttccag2160tcttgtcccaggggagttttgccattccaggctggttcacagaagtacgagttaagcttc2220caagggatgattcagacaaatatagcttccaagactcaaaggcatgttgtcagaaggcca2280gtatttgtttcttcgaacgatgtggagcagaagagaagaggtccagagggcggccgctcg2340agtctagagggcccgcggttcgaacaaaaactcatctcagaagaggatctgaatatgcat2400accggtcatcatcaccatcaccat2424<210>5<211>311<212>PRT<213>人工序列<220><223><400>5MetAlaAspProGlyValCysCysPheIleThrLysIleLeuCysAla151015HisGlyGlyArgMetThrLeuGluGluLeuLeuGlyGluIleArgLeu202530ProGluAlaGlnLeuTyrGluLeuLeuGluThrAlaGlyProAspArg354045PheValLeuLeuGluThrGlyGlyGlnAlaGlyIleThrArgSerVal505560ValAlaThrThrArgAlaArgValCysArgArgLysTyrCysGlnArg65707580ProCysAspSerLeuHisLeuCysLysLeuAsnLeuLeuGlyArgCys859095HisTyrAlaGlnSerGlnArgAsnLeuCysLysTyrSerHisAspVal100105110LeuSerGluGlnAsnPheGlnIleLeuLysAsnHisGluLeuSerGly115120125LeuAsnGlnGluGluLeuAlaCysLeuLeuValGlnSerAspProPhe130135140PheLeuProGluIleCysLysSerTyrLysGlyGluGlyArgLysGln145150155160ThrCysGlyGlnProGlnProCysGluArgLeuHisIleCysGluHis165170175PheThrArgGlyAsnCysSerTyrLeuAsnCysLeuArgSerHisAsn180185190LeuMetAspArgLysValLeuThrIleMetArgGluHisGlyLeuSer195200205ProAspValValGlnAsnIleGlnAspIleCysAsnAsnLysHisAla210215220ArgArgAsnProProGlyThrArgAlaAlaHisProHisArgArgGly225230235240GlyAlaHisArgAspArgSerLysSerArgAspArgSerCysGlyArg245250255SerSerGlyGlyGlySerGlyGlyGlyThrGlyGlyGlySerGlyGly260265270GlyThrGlyGlyGlySerGlyGlyGlyAspIleGlyAlaLysGlyPro275280285ArgPheGluGlnLysLeuIleSerGluGluAspLeuAsnMetHisThr290295300GlyHisHisHisHisHisHis305310<210>6<211>933<212>DNA<213>人工序列<220><223><400>6atggcagatcccggggtatgctgtttcatcaccaagatcctgtgcgcccacgggggccgt60atgaccctggaggaactgctgggtgagatcaggctccccgaggcgcagctctacgagctg120ctggagacggcggggcccgatcgcttcgtgctattggagactggaggccaggccgggatc180actcggtctgtagtggctactactcgagcccgcgtctgccgtcggaagtactgccagaga240ccctgcgacagcctgcacctctgcaagcttaatctgctcggccggtgccactatgcacag300tctcagcggaacctctgcaaatattctcacgatgttctctcggaacagaacttccagatc360ctgaagaatcatgagctctctgggcttaaccaagaggagctagcttgcctcctggtccaa420agcgacccttttttcctgcccgagatatgcaagagttacaaaggagagggccgaaaacag480acctgtgggcagccacagccatgcgagagactccacatctgtgagcacttcacccggggc540aactgcagttacctcaactgtctcaggtctcacaacctgatggacagaaaggtgttgacc600atcatgagggagcacgggctgagtcctgatgtggtccagaacatccaggacatctgcaac660aacaaacacgccaggaggaacccgcctggcacgagagctgcccatccacaccgcagaggc720ggcgcacacagagacagaagcaaaagcagagaccgttcctgcggccgctcgagtggtgga780ggctctgggggaggtacaggaggtggctcaggtgggggcactggtggaggcagtggaggt840ggcgatatcggcgccaagggcccgcggttcgaacaaaaactcatctcagaagaggatctg900aatatgcataccggtcatcatcaccatcaccat933<210>7<211>834<212>PRT<213>人工序列<220><223><400>7MetAlaAspProGlyValCysCysPheIleThrLysIleLeuCysAla151015HisGlyGlyArgMetThrLeuGluGluLeuLeuGlyGluIleArgLeu202530ProGluAlaGlnLeuTyrGluLeuLeuGluThrAlaGlyProAspArg354045PheValLeuLeuGluThrGlyGlyGlnAlaGlyIleThrArgSerVal505560ValAlaThrThrArgAlaArgValCysArgArgLysTyrCysGlnArg65707580ProCysAspSerLeuHisLeuCysLysLeuAsnLeuLeuGlyArgCys859095HisTyrAlaGlnSerGlnArgAsnLeuCysLysTyrSerHisAspVal100105110LeuSerGluGlnAsnPheGlnIleLeuLysAsnHisGluLeuSerGly115120125LeuAsnGlnGluGluLeuAlaCysLeuLeuValGlnSerAspProPhe130135140PheLeuProGluIleCysLysSerTyrLysGlyGluGlyArgLysGln145150155160ThrCysGlyGlnProGlnProCysGluArgLeuHisIleCysGluHis165170175PheThrArgGlyAsnCysSerTyrLeuAsnCysLeuArgSerHisAsn180185190LeuMetAspArgLysValLeuThrIleMetArgGluHisGlyLeuSer195200205ProAspValValGlnAsnIleGlnAspIleCysAsnAsnLysHisAla210215220ArgArgAsnProProGlyThrArgAlaAlaHisProHisArgArgGly225230235240GlyAlaHisArgAspArgSerLysSerArgAspArgPheLeuHisAsn245250255SerLeuGluPheLeuSerProValValSerProLeuGlySerGlyPro260265270ProSerProAspValThrSerCysLysAspSerLeuGluAspValSer275280285ValAspValThrGlnLysPheLysTyrLeuGlyThrHisAspArgAla290295300GlnLeuSerProValSerSerLysAlaAlaGlyValGlnGlyProSer305310315320GlnMetArgAlaSerGlnGluPheSerGluAspGlyAsnLeuAspAsp325330335IlePheSerArgAsnArgSerAspSerSerSerSerArgAlaSerAla340345350AlaLysValAlaGlnArgAsnGluAlaValAlaMetLysMetGlyMet355360365GluValLysGlyLysLysGluAlaProAspIleAspArgValProPhe370375380LeuAsnSerTyrIleAspGlyValThrMetGluLysAlaSerValSer385390395400GlyIleProGlyLysLysPheThrAlaAsnAspLeuGluAsnLeuLeu405410415LeuLeuAsnAspThrTrpLysAsnValAlaLysProGlnAspLeuGln420425430ThrThrGlyArgIleThrAspSerGlyGlnAspLysAlaPheLeuGln435440445AsnLysTyrGlyGlyAsnProValTrpAlaSerAlaSerThrHisAsn450455460AlaProAsnGlySerSerGlnIleMetAspGluThrProAsnValSer465470475480LysSerSerThrSerGlyPheAlaIleLysProAlaIleAlaGlyGly485490495LysGluAlaValTyrSerGlyValGlnSerProArgSerGlnValLeu500505510AlaValProGlyGluAlaThrThrProValGlnSerAsnArgLeuPro515520525GlnSerProLeuSerSerSerSerHisArgAlaAlaAlaSerGlySer530535540ProGlyLysAsnSerThrHisThrSerValSerProAlaIleGluSer545550555560SerArgMetThrSerAspProAspGluTyrLeuLeuArgTyrIleLeu565570575AsnProLeuPheArgMetAspAsnHisGlyProLysGluIleCysGln580585590AspHisLeuTyrLysGlyCysGlnGlnSerHisCysAspArgSerHis595600605PheHisLeuProTyrArgTrpGlnMetPheValTyrThrThrTrpArg610615620AspPheGlnAspMetGluSerIleGluGlnAlaTyrCysAspProHis625630635640ValGluLeuIleLeuIleGluAsnHisGlnIleAsnPheGlnLysMet645650655ThrCysAspSerTyrProIleArgArgLeuSerThrProSerTyrGlu660665670GluLysProLeuSerAlaValPheAlaThrLysTrpIleTrpTyrTrp675680685LysAsnGluPheAsnGluTyrIleGlnTyrGlyAsnGluSerProGly690695700HisThrSerSerAspIleAsnSerAlaTyrLeuGluSerPhePheGln705710715720SerCysProArgGlyValLeuProPheGlnAlaGlySerGlnLysTyr725730735GluLeuSerPheGlnGlyMetIleGlnThrAsnIleAlaSerLysThr740745750GlnArgHisValValArgArgProValPheValSerSerAsnAspVal755760765GluGlnLysArgArgGlyProGluGlyGlyArgSerSerGlyGlyGly770775780SerGlyGlyGlyThrGlyGlyGlySerGlyGlyGlyThrGlyGlyGly785790795800SerGlyGlyGlyAspIleGlyAlaLysGlyProArgPheGluGlnLys805810815LeuIleSerGluGluAspLeuAsnMetHisThrGlyHisHisHisHis820825830HisHis<210>8<211>2502<212>DNA<213>人工序列<220><223><400>8atggcagatcccggggtatgctgtttcatcaccaagatcctgtgcgcccacgggggccgt60atgaccctggaggaactgctgggtgagatcaggctccccgaggcgcagctctacgagctg120ctggagacggcggggcccgatcgcttcgtgctattggagactggaggccaggccgggatc180actcggtctgtagtggctactactcgagcccgcgtctgccgtcggaagtactgccagaga240ccctgcgacagcctgcacctctgcaagcttaatctgctcggccggtgccactatgcacag300tctcagcggaacctctgcaaatattctcacgatgttctctcggaacagaacttccagatc360ctgaagaatcatgagctctctgggcttaaccaagaggagctagcttgcctcctggtccaa420agcgacccttttttcctgcccgagatatgcaagagttacaaaggagagggccgaaaacag480acctgtgggcagccacagccatgcgagagactccacatctgtgagcacttcacccggggc540aactgcagttacctcaactgtctcaggtctcacaacctgatggacagaaaggtgttgacc600atcatgagggagcacgggctgagtcctgatgtggtccagaacatccaggacatctgcaac660aacaaacacgccaggaggaacccgcctggcacgagagctgcccatccacaccgcagaggc720ggcgcacacagagacagaagcaaaagcagagaccgcttccttcacaacagtctagaattt780ctctcacctgttgtctcacctctgggatctggtccgcctagcccagatgtcaccagctgt840aaagattccctggaggatgtgtctgtggatgtcacccagaagttcaagtacttggggacg900catgaccgtgcgcagctctccccagtctcatctaaggctgctggtgttcaaggacccagt960caaatgagagcaagccaagagttttcagaggatgggaatctagatgacatattttctagg1020aatcgttctgattcatcatcaagtcgagcctccgctgccaaggtggcacaaagaaatgaa1080gctgtggccatgaaaatgggcatggaggtcaagggcaagaaggaggctccagacatcgat1140cgggtcccatttttaaatagttatattgatggggtgaccatggaaaaagcatcggtctca1200ggaattccaggcaaaaagttcacagccaatgatctggaaaatttgctattacttaacgac1260acttggaagaatgtggctaagccccaggatctgcagaccacaggcagaatcactgacagt1320ggccaagacaaggcattcctgcagaataaatatggaggaaacccagtgtgggcaagtgca1380tccacccataatgccccaaatggctctagtcaaattatggatgaaactcctaatgtctct1440aaaagtagtaccagtggttttgccataaaaccagcaattgctggaggaaaagaagcagtc1500tattctggagttcagagtccgagaagccaggtcctagctgtgcctggggaggctactacc1560cctgtacagagcaacaggctgcctcagtcgcctctgtcttcctcaagccacagagctgca1620gcctctgggagccctggcaagaactccacccatacctctgtgagcccagccatcgagtct1680tcaaggatgacatcagaccccgatgagtatctcctacgctacatcctaaatcctttattt1740aggatggataatcatggcccgaaggaaatctgtcaggaccatctgtacaagggctgtcaa1800cagagccactgcgacaggagtcacttccatctgccctaccggtggcagatgttcgtatat1860accacttggagggacttccaggacatggagtctatcgaacaggcctattgtgatccccac1920gttgaactcattttgatagaaaaccatcagatcaatttccagaaaatgacctgtgactcc1980taccccatccgacgcctctccactccctcatatgaggaaaagccacttagtgctgtcttc2040gccaccaagtggatttggtattggaagaatgaatttaatgaatatatccagtatgggaat2100gagagcccaggccacaccagctctgacatcaactctgcgtacctggagtctttcttccag2160tcttgtcccaggggagttttgccattccaggctggttcacagaagtacgagttaagcttc2220caagggatgattcagacaaatatagcttccaagactcaaaggcatgttgtcagaaggcca2280gtatttgtttcttcgaacgatgtggagcagaagagaagaggtccagagggcggccgctcg2340agtggtggaggctctgggggaggtacaggaggtggctcaggtgggggcactggtggaggc2400agtggaggtggcgatatcggcgccaagggcccgcggttcgaacaaaaactcatctcagaa2460gaggatctgaatatgcataccggtcatcatcaccatcaccat2502<210>9<211>381<212>PRT<213>人工序列<220><223><400>9MetAlaAspProGlyValCysCysPheIleThrLysIleLeuCysAla151015HisGlyGlyArgMetThrLeuGluGluLeuLeuGlyGluIleArgLeu202530ProGluAlaGlnLeuTyrGluLeuLeuGluThrAlaGlyProAspArg354045PheValLeuLeuGluThrGlyGlyGlnAlaGlyIleThrArgSerVal505560ValAlaThrThrArgAlaArgValCysArgArgLysTyrCysGlnArg65707580ProCysAspSerLeuHisLeuCysLysLeuAsnLeuLeuGlyArgCys859095HisTyrAlaGlnSerGlnArgAsnLeuCysLysTyrSerHisAspVal100105110LeuSerGluGlnAsnPheGlnIleLeuLysAsnHisGluLeuSerGly115120125LeuAsnGlnGluGluLeuAlaCysLeuLeuValGlnSerAspProPhe130135140PheLeuProGluIleCysLysSerTyrLysGlyGluGlyArgLysGln145150155160ThrCysGlyGlnProGlnProCysGluArgLeuHisIleCysGluHis165170175PheThrArgGlyAsnCysSerTyrLeuAsnCysLeuArgSerHisAsn180185190LeuMetAspArgLysValLeuThrIleMetArgGluHisGlyLeuSer195200205ProAspValValGlnAsnIleGlnAspIleCysAsnAsnLysHisAla210215220ArgArgAsnProProGlyThrArgAlaAlaHisProHisArgArgGly225230235240GlyAlaHisArgAspArgSerLysSerArgAspArgSerCysGlyArg245250255SerSerGlyGlyGlySerGlyGlyGlyThrGlyGlyGlySerGlyGly260265270GlyThrGlyGlyGlySerGlyGlyGlyAspGlyArgAlaAlaGluPro275280285ValAspProSerLeuGluProTrpLysHisProGlySerGlnProArg290295300ThrAlaCysAsnAsnCysTyrCysLysLysCysCysPheHisCysTyr305310315320AlaCysPheThrArgLysGlyLeuGlyIleSerTyrGlyArgLysLys325330335ArgArgGlnArgArgArgAlaProGlnAspSerGlnThrHisGlnAla340345350SerLeuSerLysGlnProArgPheGluGlnLysLeuIleSerGluGlu355360365AspLeuAsnMetHisThrGlyHisHisHisHisHisHis370375380<210>10<211>1143<212>DNA<213>人工序列<220><223><400>10atggcagatcccggggtatgctgtttcatcaccaagatcctgtgcgcccacgggggccgt60atgaccctggaggaactgctgggtgagatcaggctccccgaggcgcagctctacgagctg120ctggagacggcggggcccgatcgcttcgtgctattggagactggaggccaggccgggatc180actcggtctgtagtggctactactcgagcccgcgtctgccgtcggaagtactgccagaga240ccctgcgacagcctgcacctctgcaagcttaatctgctcggccggtgccactatgcacag300tctcagcggaacctctgcaaatattctcacgatgttctctcggaacagaacttccagatc360ctgaagaatcatgagctctctgggcttaaccaagaggagctagcttgcctcctggtccaa420agcgacccttttttcctgcccgagatatgcaagagttacaaaggagagggccgaaaacag480acctgtgggcagccacagccatgcgagagactccacatctgtgagcacttcacccggggc540aactgcagttacctcaactgtctcaggtctcacaacctgatggacagaaaggtgttgacc600atcatgagggagcacgggctgagtcctgatgtggtccagaacatccaggacatctgcaac660aacaaacacgccaggaggaacccgcctggcacgagagctgcccatccacaccgcagaggc720ggcgcacacagagacagaagcaaaagcagagaccgttcctgcggccgctcgagtggtgga780ggctctgggggaggtacaggaggtggctcaggtgggggcactggtggaggcagtggaggt840ggcgatggccgcgctgctgagccagtagatcctagtctagagccctggaagcatccagga900agtcagcctaggactgcttgtaacaattgctattgtaaaaagtgttgctttcattgctac960gcgtgtttcacaagaaaaggcttaggcatctcctatggcaggaagaagcggagacagcga1020cgaagagctcctcaggacagtcagactcatcaagcttctctatcaaagcagccgcggttc1080gaacaaaaactcatctcagaagaggatctgaatatgcataccggtcatcatcaccatcac1140cat1143<210>11<211>194<212>PRT<213>人工序列<220><223><400>11MetAlaAspProGlyValCysCysPheIleThrLysIleLeuCysAla151015HisGlyGlyArgMetThrLeuGluGluLeuLeuGlyGluIleArgLeu202530ProGluAlaGlnLeuTyrGluLeuLeuGluThrAlaGlyProAspArg354045PheValLeuLeuGluThrGlyGlyGlnAlaGlyIleThrArgSerVal505560ValAlaThrThrArgSerSerGlyGlyGlySerGlyGlyGlyThrGly65707580GlyGlySerGlyGlyGlyThrGlyGlyGlySerGlyGlyGlyAspGly859095ArgAlaAlaGluProValAspProSerLeuGluProTrpLysHisPro100105110GlySerGlnProArgThrAlaCysAsnAsnCysTyrCysLysLysCys115120125CysPheHisCysTyrAlaCysPheThrArgLysGlyLeuGlyIleSer130135140TyrGlyArgLysLysArgArgGlnArgArgArgAlaProGlnAspSer145150155160GlnThrHisGlnAlaSerLeuSerLysGlnProArgPheGluGlnLys165170175LeuIleSerGluGluAspLeuAsnMetHisThrGlyHisHisHisHis180185190HisHis<210>12<211>582<212>DNA<213>人工序列<220><223><400>12atggcagatcccggggtatgctgtttcatcaccaagatcctgtgcgcccacgggggccgt60atgaccctggaggaactgctgggtgagatcaggctccccgaggcgcagctctacgagctg120ctggagacggcggggcccgatcgcttcgtgctattggagactggaggccaggccgggatc180actcggtctgtagtggctactactcgatcgagtggtggaggctctgggggaggtacagga240ggtggctcaggtgggggcactggtggaggcagtggaggtggcgatggccgcgctgctgag300ccagtagatcctagtctagagccctggaagcatccaggaagtcagcctaggactgcttgt360aacaattgctattgtaaaaagtgttgctttcattgctacgcgtgtttcacaagaaaaggc420ttaggcatctcctatggcaggaagaagcggagacagcgacgaagagctcctcaggacagt480cagactcatcaagcttctctatcaaagcagccgcggttcgaacaaaaactcatctcagaa540gaggatctgaatatgcataccggtcatcatcaccatcaccat582<210>13<211>285<212>PRT<213>人工序列<220><223><400>13MetAlaAspProGlyValCysCysPheIleThrLysIleLeuCysAla151015HisGlyGlyArgMetThrLeuGluGluLeuLeuGlyGluIleArgLeu202530ProGluAlaGlnLeuTyrGluLeuLeuGluThrAlaGlyProAspArg354045PheValLeuLeuGluThrGlyGlyGlnAlaGlyIleThrArgSerVal505560ValAlaThrThrArgAlaArgValCysArgArgLysTyrCysGlnArg65707580ProCysAspSerLeuHisLeuCysLysLeuAsnLeuLeuGlyArgCys859095HisTyrAlaGlnSerGlnArgAsnLeuCysLysTyrSerHisAspVal100105110LeuSerGluGlnAsnPheGlnIleLeuLysAsnHisGluLeuSerGly115120125LeuAsnGlnGluGluLeuAlaCysLeuLeuValGlnSerAspProPhe130135140PheLeuProGluIleCysLysSerTyrLysGlyGluGlyArgLysGln145150155160ThrCysGlyGlnProGlnProCysGluArgLeuHisIleCysGluHis165170175PheThrArgGlyAsnCysSerTyrLeuAsnCysLeuArgSerHisAsn180185190LeuMetAspArgLysValLeuThrIleMetArgGluHisGlyLeuSer195200205ProAspValValGlnAsnIleGlnAspIleCysAsnAsnLysHisAla210215220ArgArgAsnProProGlyThrArgAlaAlaHisProHisArgArgGly225230235240GlyAlaHisArgAspArgSerLysSerArgAspArgSerCysGlyArg245250255SerSerLeuGluGlyProArgPheGluGlnLysLeuIleSerGluGlu260265270AspLeuAsnMetHisThrGlyHisHisHisHisHisHis275280285<210>14<211>855<212>DNA<213>人工序列<220><223><400>14atggcagatcccggggtatgctgtttcatcaccaagatcctgtgcgcccacgggggccgt60atgaccctggaggaactgctgggtgagatcaggctccccgaggcgcagctctacgagctg120ctggagacggcggggcccgatcgcttcgtgctattggagactggaggccaggccgggatc180actcggtctgtagtggctactactcgagcccgcgtctgccgtcggaagtactgccagaga240ccctgcgacagcctgcacctctgcaagcttaatctgctcggccggtgccactatgcacag300tctcagcggaacctctgcaaatattctcacgatgttctctcggaacagaacttccagatc360ctgaagaatcatgagctctctgggcttaaccaagaggagctagcttgcctcctggtccaa420agcgacccttttttcctgcccgagatatgcaagagttacaaaggagagggccgaaaacag480acctgtgggcagccacagccatgcgagagactccacatctgtgagcacttcacccggggc540aactgcagttacctcaactgtctcaggtctcacaacctgatggacagaaaggtgttgacc600atcatgagggagcacgggctgagtcctgatgtggtccagaacatccaggacatctgcaac660aacaaacacgccaggaggaacccgcctggcacgagagctgcccatccacaccgcagaggc720ggcgcacacagagacagaagcaaaagcagagaccgttcctgcggccgctcgagtctagag780ggcccgcggttcgaacaaaaactcatctcagaagaggatctgaatatgcataccggtcat840catcaccatcaccat85權(quán)利要求1.一種抗病毒的融合蛋白，是具有下述氨基酸殘基序列之一的蛋白質(zhì)1)序列表中的SEQID№1；2)將序列表中SEQID№1的氨基酸殘基序列經(jīng)過一個或幾個氨基酸殘基的取代和/或缺失和/或添加且可特異性降解HIV病毒RNA的蛋白質(zhì)。2.根據(jù)權(quán)利要求1所述的抗病毒的融合蛋白，其特征在于所述病毒為HIV病毒。3.權(quán)利要求1或2所述的抗病毒的融合蛋白的編碼基因。4.根據(jù)權(quán)利要求3所述基因，其特征在于所述抗病毒的融合蛋白的編碼基因具有下述核苷酸序列之一1)序列表中SEQID№2的核苷酸序列；2)編碼序列表中SEQID№1蛋白質(zhì)序列的DNA；3)在高嚴謹條件下可與序列表中的SEQID№2限定的DNA序列雜交的核苷酸序列。5.含有權(quán)利要求3或4所述抗病毒的融合蛋白的編碼基因的表達載體。6.含有權(quán)利要求3或4所述抗病毒的融合蛋白的編碼基因的細胞系。7.含有權(quán)利要求3或4所述抗病毒的融合蛋白的編碼基因的宿主菌。8.權(quán)利要求1、2所述的抗病毒的融合蛋白及其編碼基因在制備抗病毒藥物中的應(yīng)用。9.根據(jù)權(quán)利要求8所述的應(yīng)用，其特征在于所述病毒為HIV病毒。10.根據(jù)權(quán)利要求9所述的應(yīng)用，其特征在于所述抗病毒藥物為預(yù)防和/或治療艾滋病的藥物。全文摘要本發(fā)明公開了一種抗病毒的融合蛋白及其編碼基因與應(yīng)用。本發(fā)明的抗病毒融合蛋白，是具有下述氨基酸殘基序列之一的蛋白質(zhì)1)序列表中的SEQID№1；2)將序列表中SEQID№1的氨基酸殘基序列經(jīng)過一個或幾個氨基酸殘基的取代和/或缺失和/或添加且可降解HIV病毒RNA的蛋白質(zhì)。該融合蛋白對HIV病毒感染具有明顯的抑制作用，可用于制備預(yù)防和/或治療艾滋病的藥物。文檔編號C12N15/62GK1763106SQ20051011423公開日2006年4月26日申請日期2005年10月21日優(yōu)先權(quán)日2005年10月21日發(fā)明者李志強,高光俠申請人:中國科學(xué)院生物物理研究所