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針對(duì)hiv-1的免疫方法和組合物的制作方法

文檔序號(hào):400075閱讀:577來(lái)源:國(guó)知局
專利名稱:針對(duì)hiv-1的免疫方法和組合物的制作方法
技術(shù)領(lǐng)域
本發(fā)明涉及用于預(yù)防HIV-1感染的DNA疫苗,以及對(duì)有患HIV感染危險(xiǎn)的受試者進(jìn)行接種的方法。
背景技術(shù)
盡管針對(duì)HIV-1進(jìn)行二十多的努力,但其全球流行一直困擾著人類。至今,大約2千2百萬(wàn)人死于AIDS,目前超過(guò)3千6百萬(wàn)人感染HIV-1。對(duì)發(fā)展中世界的影響不成比例,占總病例的95%。在5%接受抗逆轉(zhuǎn)錄病毒治療的人當(dāng)中,絕大部分由于副作用不能耐受現(xiàn)有藥物,另一部分具有藥物抗性病毒變體。盡管公共衛(wèi)生事業(yè)的發(fā)展可有助于延緩HIV-1在某些地區(qū)的傳播速度,但是很明顯,防護(hù)性的疫苗將是解決全球問(wèn)題的最令人滿意的方法。
目前,世界上50%以上的新HIV-1感染是由HIV-1病毒的亞型C引起的。亞型C從印度和緬甸傳到了中國(guó),估計(jì)是通過(guò)在國(guó)家西南部的靜脈注射吸毒者中間傳播的。云南省特別嚴(yán)重,約占全中國(guó)HIV-1病例中的一半以上。根據(jù)云南省衛(wèi)生局的資料,2000年該省IVDU中HIV-1感染流行率是29%,預(yù)計(jì)到2005年將達(dá)到40.7%。云南省的5個(gè)縣(文山、紅河、德洪、陵長(zhǎng)和大理)的流行率最高,估計(jì)為50-70%。HIV-1亞型C也已傳播到鄰省,例如四川和廣西,并引起很遠(yuǎn)的新疆的許多感染。
DNA接種,或遺傳免疫是疫苗學(xué)上有希望的新策略。先前設(shè)計(jì)針對(duì)HIV-1的疫苗的嘗試表明常規(guī)方法如蛋白質(zhì)/亞基或失活病毒對(duì)逆轉(zhuǎn)錄病毒的感染不起作用。另外,廣泛認(rèn)為活的減毒疫苗在用于對(duì)抗HIV-1時(shí)有不能接受的危險(xiǎn)。因此,用核酸免疫是新的、及時(shí)的想法。
發(fā)明簡(jiǎn)述本發(fā)明提供了針對(duì)HIV-1的DNA疫苗,以及最大實(shí)用性和效率的接種方法學(xué)策略。本發(fā)明的預(yù)防疫苗方案包括用包含兩種新DNA載體的接種物初始免疫,然后用表達(dá)相應(yīng)HIV-1蛋白質(zhì)的修飾的牛痘Ankara(Modified Vaccinia Ankara)(MVA)重組核酸加強(qiáng)免疫。
第一方面,本發(fā)明的特征在于核酸載體,其是從商品化的質(zhì)粒(pVAX1)(InvitrogenTM)開(kāi)發(fā)而來(lái)的(

圖1),并通過(guò)插入另外的啟動(dòng)子修飾。在一個(gè)實(shí)施方案中,該核酸載體包含人延伸因子1α(hEF1α)啟動(dòng)子(pADVAX)(圖2)第二方面,本發(fā)明的特征在于包含兩種HIV-1基因的核酸載體,其中每種HIV-1基因由不同的啟動(dòng)子控制。在更具體的實(shí)施方案中,本發(fā)明的核酸載體包含pCMV啟動(dòng)子和人延伸因子1α(hEF1α)啟動(dòng)子。在另一個(gè)實(shí)施方案中,HIV-1基因包含env和gag。在更具體的實(shí)施方案中,對(duì)核酸載體進(jìn)行密碼子優(yōu)化以在人類受試者中表達(dá)。在另一更具體的實(shí)施方案中,該核酸載體進(jìn)一步包含tPA前導(dǎo)序列(MDAMKRGLCCVLLLCGAVFVSAR)(SEQ ID NO1)。在更具體的實(shí)施方案中,核酸載體包含密碼子優(yōu)化的HIV-1 env基因和tPA前導(dǎo)序列,其包含SEQID NO7的序列和編碼SEQ ID NO8的氨基酸序列。在另一更具體的實(shí)施方案中,核酸載體包含密碼子優(yōu)化的HIV-1 gag基因和tPA前導(dǎo)序列,其包含SEQ ID NO9的核酸序列,編碼SEQ ID NO10的氨基酸序列。在另一實(shí)施方案中,核酸載體包含密碼子優(yōu)化的HIV-1 tPA-env和tPA-gag基因,其具有SEQ ID NO15的序列(ADVAXI)。
在另一實(shí)施方案中,HIV-1基因包含pol、nef和tat。在更具體的實(shí)施方案中,對(duì)核酸載體進(jìn)行密碼子優(yōu)化以在人類受試者中表達(dá)。在一個(gè)實(shí)施方案中,核酸載體包含密碼子優(yōu)化的HIV-1 pol和tPA序列,其包含SEQ ID NO11的序列(其編碼SEQ ID NO12的氨基酸序列)。在另一具體的實(shí)施方案中,核酸載體包含密碼子優(yōu)化的HIV-1 nef-tat融合基因序列和tPA前導(dǎo)序列,其包含SEQ ID NO13的序列(編碼SEQ ID NO14的氨基酸序列)。在另一實(shí)施方案中,核酸載體包含密碼子優(yōu)化的HIV-1tPA-pol和tPA-nef-tat基因,其具有SEQ ID NO16的序列(ADVAXII)。
第三方面,本發(fā)明的特征在于包含兩種核酸載體的接種物,其中每種載體包含兩個(gè)啟動(dòng)子,每個(gè)啟動(dòng)子控制一種或多種HIV-1基因的表達(dá)。在更具體的實(shí)施方案中,一種核酸載體包含pCMV啟動(dòng)子和hEF1α啟動(dòng)子,以及HIV-1基因gag和nev,另一種核酸載體包含pCMV啟動(dòng)子和hEF1α啟動(dòng)子,以及HIV-1基因pol、nef和tat。在另一個(gè)更具體的實(shí)施方案中,一種核酸載體包含pCMV啟動(dòng)子和hEF1α啟動(dòng)子,以及HIV-1基因gag-pol和nev,另一種核酸載體包含pCMV啟動(dòng)子和hEF1α啟動(dòng)子,以及HIV-1基因nef和tat。在更具體的實(shí)施方案中,接種物包含一種核酸載體(其包含密碼子優(yōu)化的HIV-1 tPA-env和tPAa-gag基因,具有SEQ IDNO15的序列(ADVAXI))和另一種核酸載體(其包含密碼子優(yōu)化的HIV-1tPA-pol和tPA-nef-tat基因,具有SEQ ID NO16的序列(ADVAXII))。
第四方面,本發(fā)明的特征在于包含兩條插入到MVA DelIII區(qū)的核酸序列的修飾的牛痘Ankara(MVA)載體,其中每條核酸序列受單獨(dú)的啟動(dòng)子控制。在更具體的實(shí)施方案中,第一種修飾的載體包含表達(dá)三種HIV-1序列,env和gal-pol融合序列的修飾的MVA載體,其中env序列是編碼SEQ ID NO18氨基酸序列的的tPA-δV2 env(SEQ ID NO17);融合基因序列tPA-gag-pol包含編碼SEQ ID NO20的氨基酸序列(ADMVA1)的SEQID NO19的序列。
第五方面,本發(fā)明的特征在于另一種修飾的載體,其包含表達(dá)五種HIV-1融合基因序列env、gal-pol和nef-tat的修飾的DNA。更具體而言,該修飾的MVA載體包含編碼氨基酸序列SEQ ID NO18的tPA-δV2 env(SEQ ID NO17);編碼SEQ ID NO20的氨基酸序列的tPA-gag-pol(SEQID NO19),以及編碼SEQ ID NO22的氨基酸序列(ADMVA2)的tPA-nef-tatSEQ ID NO21。
第六方面,本發(fā)明的特征在于針對(duì)HIV-1對(duì)人類受試者進(jìn)行接種的方法,其包含給予DNA疫苗,該疫苗包含一種含有ADVAXI(SEQ ID NO15)的核酸及另一種含有ADVAXII(SEQ ID NO16)的核酸。在一個(gè)實(shí)施方案中,用ADVAXI和II初步免疫后,用表達(dá)相應(yīng)HVI-1蛋白質(zhì)的修飾的牛痘Ankara(ADMVA2)載體進(jìn)行加強(qiáng)免疫。
第七方面,本發(fā)明的特征在于用于給予本發(fā)明的一種或多種核酸的試劑盒,其用于治療有患HIV相關(guān)疾病危險(xiǎn)或患有HIV相關(guān)疾病的人類受試者,其包含初始DNA疫苗ADVAXI和ADVAXII中的一種或兩種(單獨(dú)或聯(lián)合使用),以及用于制備或傳遞疫苗的試劑、緩沖液或溶液。該試劑盒還包括使用試劑盒中組分進(jìn)行接種或治療HIV相關(guān)疾病的用法說(shuō)明。該試劑盒還可包括進(jìn)行對(duì)本發(fā)明的核酸給藥的儀器和裝置,例如注射器和針。在本發(fā)明的一個(gè)實(shí)施方案中,該試劑盒包括兩種初始DNA疫苗ADVAXI和ADVAXII,單獨(dú)或聯(lián)合使用,以及加強(qiáng)疫苗ADMVA2,和用于制備或傳遞該疫苗的試劑、緩沖液或溶液,及使用該試劑盒的說(shuō)明。
本發(fā)明的其他特征和好處在發(fā)明詳述、附圖和實(shí)施例中很明顯。
附圖簡(jiǎn)述圖1是pVAX1的簡(jiǎn)圖。
圖2是pADVAX(包括人延伸因子1α(hEF1α)作為另一啟動(dòng)子的修飾的pVAX1)的簡(jiǎn)圖。
圖3是為ADVAXII而進(jìn)行的對(duì)pol基因的修飾的簡(jiǎn)要示意圖。PR=蛋白酶,RT=逆轉(zhuǎn)錄酶,IN=整合酶。蛋白酶中的缺失(DTGA)包含野生型基因的25-28位氨基酸。在逆轉(zhuǎn)錄酶中的點(diǎn)實(shí)變(M到G)是在野生型基因的184位。對(duì)用pVAX1-tPA-實(shí)變的pol(A)和單獨(dú)的pVAX1(B)轉(zhuǎn)染的293T細(xì)胞的上清液進(jìn)行Western印跡。未斷裂的tPA-pol是110KD。
圖4是對(duì)用nef構(gòu)建體單獨(dú)的載體、nef、tPA-nef和tpa-nef-tat轉(zhuǎn)染的293T細(xì)胞上MHC-I類的表達(dá)的流式細(xì)胞術(shù)分析結(jié)果。
圖5是ADVAXI和ADVAXII的簡(jiǎn)圖。
圖6是對(duì)來(lái)源于肽混合物的Env和Gag的IFN-γELISpol反應(yīng)。肽是有10處重疊的20聚體。每種混合物包括12種肽,除GagA-I是以前鑒定的BALB/c小鼠上CTL表位的特異的9聚體(AMGMLKDTI)(SEQ ID NO2)。Env P1包含24-144位的氨基酸,Env P4包含403-573位的氨基酸,GagP3包含251-380位的氨基酸,Gag A-1包含217-225位的氨基酸。
圖7顯示了用不同劑量的ADVAXI接種的小鼠中Env-和Gag-特異的IFN-γELESpot反應(yīng)。
圖8是從pLW7構(gòu)建pZC1和pZC3的簡(jiǎn)圖。
圖9是從pLW22構(gòu)建pZC22的簡(jiǎn)圖。
圖10.用不同的DNA疫苗接種的小鼠中血清抗Gag抗體。各組小鼠在0周、3周和6周用不同DNA疫苗IM(肌內(nèi)注射)免疫。在第8周時(shí)收集每只小鼠的血清,每組混合在一起,并用ELISA分析抗Gag抗體反應(yīng)。
圖11.對(duì)來(lái)源于肽混合物的pol、Tat和Nef的IFN-γELISpot反應(yīng)。肽是有11處重疊的15聚體,每種混合物包含25-30種肽。在我們修飾的基因中,Pol P2包含92-226位的氨基酸,Pol P4包含343-477位的氨基酸,Pol P7包含711-845位的氨基酸。Pol P8包含839-969位氨基酸,Nef P1和P2包括全部Nef蛋白。
圖12.用不同劑量的ADVAXII接種的小鼠中Pol-和Nef-Tat-特異性IFN-γELISpot反應(yīng)。如前所述,結(jié)果反映了對(duì)選擇的肽混合物的免疫應(yīng)答。
發(fā)明詳述在描述本發(fā)明的方法學(xué)、載體和組合物之前,應(yīng)當(dāng)理解本發(fā)明不限于特定的方法,或?qū)嶒?yàn)化合物以及所述的實(shí)驗(yàn)條件,因?yàn)樵摲椒ê蜅l件可能改變。也應(yīng)當(dāng)理解的是此處所用的術(shù)語(yǔ)只是為了描述特定的實(shí)施方案,不是為了限制,因?yàn)楸景l(fā)明的范圍只由所附的權(quán)利要求來(lái)限制。
在本說(shuō)明書和所附的權(quán)利要求中所用的單數(shù)形式除非上下文另有明確規(guī)定,包括復(fù)數(shù)范圍。因此例如,涉及到“方法”時(shí)包括一種或多種方法,和/或此處所述的和/或在閱讀本公開(kāi)內(nèi)容后對(duì)本領(lǐng)域技術(shù)人員很明顯的方法等。
除非另有規(guī)定,此處所用的技術(shù)和科學(xué)術(shù)語(yǔ)與本發(fā)明所屬的技術(shù)領(lǐng)域的普通技術(shù)人員通常理解的意義相同。盡管任何與此處所述的方法和材料類似或等價(jià)的方法和材料都可用于本發(fā)明的實(shí)踐或檢驗(yàn),但此處描述的是優(yōu)選的方法和材料。此處提到的所有出版物此處引用用來(lái)參考,以公開(kāi)和描述與出版物中涉及的方法和/或材料相關(guān)的方法和/或材料。
定義短語(yǔ)“與HIV感染相關(guān)的疾病”或“HIV-1相關(guān)疾病”等,此處是指此HIV感染為特征的疾病狀態(tài)。此類與HIV感染相關(guān)的疾病包括但不限于AIDS、卡波西肉瘤(Kaposi’s Sarcoma)、機(jī)會(huì)性感染例如由卡氏肺囊蟲(chóng)(Pneumocystis carinii)和結(jié)核分枝桿菌(Mycobacteriumtuberculosis)引起的感染;包括鵝口瘡、毛狀白斑和口腔潰瘍的口腔損傷;全身淋巴結(jié)??;帶狀皰疹;血小板減少癥;無(wú)菌性腦膜炎;神經(jīng)系統(tǒng)疾病如弓形體病、隱球菌病(cyrptoco ccosis)、CMV感染、原發(fā)性CNS淋巴瘤和HIV相關(guān)的疾??;末梢神經(jīng)?。话d癇發(fā)作和肌病。
有患HIV感染或HIV相關(guān)疾病“危險(xiǎn)”的人是指其HIV感染的危險(xiǎn)比群體的危險(xiǎn)高的人。
術(shù)語(yǔ)“治療上有效的劑量”是指因所給劑量而產(chǎn)生目的效果的劑量。確切的劑量取決于治療的目的,并可由本領(lǐng)域技術(shù)人員利用已知的技術(shù)確定(例如參見(jiàn)Lieberman(1992)Pharmaceutical Dosage FormsVOl.1-3;Lolyd(1999)The Art,Science and Technology ofpharmaceutical Compounding;and Pickar(1999)Dosage Calculations)。對(duì)于本發(fā)明的DNA疫苗的治療上有效的劑量是對(duì)個(gè)體HIV感染或AIDS治療中獲得成功的必需量,包括任何客觀的和主觀的標(biāo)準(zhǔn),如HIV病毒抑制、與HIV感染和AIDS有關(guān)的病狀的減少,或改善了病人的生理或心理健康。
發(fā)明概述本發(fā)明涉及將編碼病毒抗原的DNA直接注射入皮膚或肌肉的策略。然后局部細(xì)胞吸收該質(zhì)粒并表達(dá)外源蛋白本身,基本上在原位產(chǎn)生疫苗免疫原。該方法經(jīng)濟(jì)實(shí)用。但更重要的是,該方法可在體內(nèi)非常有效。
已知細(xì)胞介導(dǎo)的免疫(CMI)對(duì)控制HIV-1復(fù)制非常重要(Ogg等人,(1998)Science 2792103-6;Schmitz等人,(1999)Science283857-60;Jin等人,(1999)J.Exp.Med.189991-8;McMichael等人,(2001)Nature 410980-7)。也許由于涉及從頭表達(dá),看起來(lái)DNA接種可以針對(duì)CMI的生成產(chǎn)生更好的抗原呈遞作用。在一項(xiàng)研究中,DNA接種可至少部分保護(hù)恒河猴免受致病性SHIV的實(shí)驗(yàn)性攻擊(Barouch等,(2000)Science 290486-92)。但是,在重組載體聯(lián)合作為初始-加強(qiáng)免疫方案中,DNA接種甚至對(duì)刺激CMI和封閉SHIV的感染更有效(Robinson等,(1999)Nat.Med.5526-34;Hanke等,(1999)Vaccine17589-96;Hanke等,(1999)J.Virol.737524-32;Allen等,(2000)J.Immunol.1644968-78;Amara等,(2001)Science 29269-74;Barouch等,(2001)J.Virol.755151-8)。
在下述實(shí)驗(yàn)中,構(gòu)建了兩種獨(dú)特的初始DNA疫苗,其包括兩種雙啟動(dòng)子載體表達(dá)env和gag的ADVAXI,以及表達(dá)pol和nef-tat的ADVAXII(圖5)。這兩種疫苗所攜帶的全部五種HIV-1基因都在體外進(jìn)行了全面的評(píng)估,以確保在獨(dú)特的質(zhì)粒載體中的表達(dá)能力和安全性。該實(shí)驗(yàn)還描述了表達(dá)相應(yīng)HIV-1蛋白質(zhì)(ADMVA2)并用于接種ADVAXI和II后加強(qiáng)免疫應(yīng)答的修飾的Modified Vaccinia AnKara(修飾的牛痘Ankara)(MVA)載體。
給藥藥物可接受的載體部分地由預(yù)給藥的特定的組合物以及化合物給藥的特定方法所決定。本發(fā)明包括包含核酸的藥物組合物的傳遞。因此,存在許多該核酸藥物組合物的合適制劑(例如參見(jiàn)RemingtonsPharmaceutical Science,17thed.1989)??梢匀魏纬R?guī)的方式給藥,例如,注射、口服、吸入法、經(jīng)皮的應(yīng)用或直腸給藥。
適于非腸道給藥(例如肌內(nèi)的、皮內(nèi)的和皮下途徑)的制劑包括水性的和非水性等滲的無(wú)菌注射液,其可包括抗氧化劑、緩沖液、制菌劑,以及使制劑與受者血液等滲的溶質(zhì)、水性和非水性的無(wú)菌懸液(其可包括懸浮劑、增溶劑、加厚劑、穩(wěn)定劑和防腐劑)。在本發(fā)明的實(shí)踐中,組合物可通過(guò)例如靜脈注射、口腔、局部、腹膜內(nèi)、膀胱內(nèi)或鞘內(nèi)給藥。非腸道給藥是優(yōu)選的給藥方法。制劑可以單位劑量或多劑量密封在容器(如安瓿和小瓶)內(nèi)存在。
利用本領(lǐng)域已知的方法制備用于給藥的純化的疫苗溶液,包括過(guò)濾溶液來(lái)除菌、稀釋溶液、添加佐劑和穩(wěn)定溶液??梢詫⒃撘呙鐑龈?,制備干燥形式的針對(duì)HIV的疫苗,以便于運(yùn)輸和貯存。進(jìn)一步地可將該疫苗制成初始疫苗ADVAXI和ADVAXII單獨(dú)成聯(lián)合形式、單獨(dú)加強(qiáng)疫苗ADMVA2形式或可包括至少一種其它抗原的形式,只要所加的抗原不干擾初始或加強(qiáng)疫苗的效力、不另外或協(xié)同增加副作用和有害的反應(yīng)即可。
試劑盒本發(fā)明包括用于接種需要接種的對(duì)象的試劑盒。在一個(gè)實(shí)施方案中,本發(fā)明的試劑盒通常包括本發(fā)明的初始DNA疫苗,包括ADVAXI和ADVAXII(單獨(dú)地或聯(lián)合地)。在本發(fā)明的另一個(gè)實(shí)施方案中,試劑盒包括本發(fā)明的加強(qiáng)疫苗ADMVA2。在另一個(gè)實(shí)施方案中,試劑盒既包括單獨(dú)或聯(lián)合的初始疫苗ADVAXI和ADVAXII,也包括加強(qiáng)疫苗ADMVA2。試劑盒還包括給藥本發(fā)明疫苗必需的試劑、溶液或緩沖液,以及醫(yī)療設(shè)備,如注射器和針,還有如何使用試劑盒中組分的用法說(shuō)明。
DNA疫苗ADVAXI和II的構(gòu)建本發(fā)明的預(yù)防性疫苗方案包括用包含兩種新DNA載體的接種物初始免疫,然后用表達(dá)相應(yīng)HIV-1蛋白質(zhì)的修飾的牛痘Ankara(MVA)重組體加強(qiáng)免疫。用于瞬時(shí)疫苗的基因衍生自分化體C(clade C)菌株Circulating Recombinant Form007(循環(huán)重組型007)或HIVCHN.AD,其還包括分化體B(clade B)(在云南省內(nèi)的優(yōu)勢(shì)亞型)中的小片段。
大多數(shù)針對(duì)HIV-1的疫苗研究涉及病毒的結(jié)構(gòu)基因產(chǎn)物Env、Gag和Pol,其為包含潛在重要的免疫原性表位的蛋白質(zhì)。但是,越來(lái)越多的證據(jù)表明HIV-1調(diào)節(jié)蛋白質(zhì)如Nef和Tat的免疫原性重要性(Allen等,(2000)Nature 407386-90;Addo等,(2001)Pros.Natl.Acad.Sci.USA981781-6;Cafaro等,(1999)Nat.Med.5643-50;Pauza等,(2000)Proc,Natl.Acad.Sci.USA 973515-9;Cafaro等,(2001)Vaccine192862-77),特別是針對(duì)CMI誘導(dǎo)的重要性。因?yàn)樵摶虍a(chǎn)物在病毒生活周期的早期表達(dá),因此它們可能是免疫控制HIV-1感染的關(guān)鍵物質(zhì)。因此,結(jié)構(gòu)基因和調(diào)節(jié)基因都包括在本發(fā)明策略中,并被設(shè)計(jì)為包含最多的免疫原性表位。
本發(fā)明的DNA疫苗以Invitrogen的商品化質(zhì)粒pVAX1為基礎(chǔ)(圖1)。對(duì)該載體進(jìn)行特別的設(shè)計(jì)以用于開(kāi)發(fā)DNA疫苗,并構(gòu)建該載體以符合美國(guó)食品與藥品管理(FDA)的規(guī)定(Center for Biologics Evaluation andResearch,F(xiàn)DA,22 Dec 1996,Docket NO.96N-0400)。但是,通過(guò)插入另外的啟動(dòng)子對(duì)原始載體進(jìn)行修飾。從商品化的載體pBudCE4.1(Invitrogen)中利用PCR擴(kuò)增人延伸因子1α(hEF1α)啟動(dòng)子。將該啟動(dòng)子克隆到pVAX1的EcoR1/Not1位點(diǎn),并用測(cè)序驗(yàn)證新構(gòu)建體。其它人已對(duì)hEF1α啟動(dòng)子進(jìn)行了完善的性質(zhì)鑒定(Najjar等,(1999)Gene23041-5;Nishimura等,(1999)Vaccine 18675-80;Wallich等,(2001)Infect.Inmun.692130-6)。發(fā)現(xiàn)經(jīng)過(guò)對(duì)pVAX1的這種改變產(chǎn)生的pADVAX(圖2)允許另外基因插入片段的獨(dú)立的高水平表達(dá)。pADVAX的雙順?lè)醋幽芰Ρ壤脙?nèi)部核糖體進(jìn)入位點(diǎn)或IRES獲得的更有效(10-20倍)(Martinez-Salas(1999)Curr.Opin.Biotechnol.10458-64)。Western印跡表明在pADVAX的雙啟動(dòng)子的控制下的每一基因的蛋白質(zhì)表達(dá)水平可以達(dá)到pVAX1中由CMV啟動(dòng)子單獨(dú)驅(qū)動(dòng)的水平。
在構(gòu)建pADVAX載體后,準(zhǔn)備用于插入的病毒基因。合成包含最適于哺乳動(dòng)物表達(dá)的密碼子的HIV-1 env和gag基因。密碼子優(yōu)化可以促進(jìn)Rev/RRE-獨(dú)立的核輸出(Schneider等,(1997)J.Virol.714892-903;Kotsopoulou等,(2000)J.Virol.744839-52),并且一直發(fā)現(xiàn)其可加強(qiáng)病毒基因的表達(dá)。用重疊PCR將具有反映該理想密碼子選擇的序列的寡核苷酸(80-960聚體,有16-18處重復(fù))聯(lián)結(jié)起來(lái)。由此,基因表達(dá)提高100-1000倍(由ELISA或Western印跡測(cè)量)。
還通過(guò)摻入組織纖溶酶原激活劑(tPA)前導(dǎo)序列(MDAMKRGLCCVLLLCGAVFVSAR)(SEQ ID NO1)、置換env的天然序列和補(bǔ)充gag基因?qū)υ摶蜻M(jìn)行進(jìn)一步修飾。認(rèn)為該序列通過(guò)協(xié)助蛋白質(zhì)從內(nèi)質(zhì)網(wǎng)(ER)到高爾基體的運(yùn)輸而部分地增加表達(dá)(Haddad等,(1997)FEMS Immunol.Med.Microbiol.18193-202;Li等(1999)Infect.Immun.674780-6;Weiss等,(1999)Vaccine 18815-24;Qiu等,(2000)Virol.745997-6005)。通過(guò)該修飾,基因表達(dá)進(jìn)一步被增加3-5倍。在利用針對(duì)env基因產(chǎn)物的多克隆抗體進(jìn)行的Western印跡測(cè)量中,對(duì)通過(guò)密碼子優(yōu)化和加入tPA前導(dǎo)序列而得到的增加的env表達(dá)獲得類似的結(jié)果(結(jié)果未列出)。該結(jié)果確定了利用基因修飾保存了Env功能。在涉及帶有CD4/CCR5(HIV-1受體/共-受體)的Hela細(xì)胞的融合試驗(yàn)中,用tPA-優(yōu)化的env載體轉(zhuǎn)染的293T細(xì)胞能夠融合形成合胞體(結(jié)果未列出)。
通過(guò)適當(dāng)?shù)乃璧幕蛐揎?,將兩種HIV-1基因克隆到pADVAX中產(chǎn)生第一種疫苗ADVAXI。用Western印跡確證雙順子表達(dá)(結(jié)果未列出)。
再如上所述構(gòu)建第二種疫苗,用重疊PCR聯(lián)結(jié)密碼子優(yōu)化的寡核苷酸以合成pol、nef和tat。但是,采取另外的措施以確保體內(nèi)應(yīng)用的安全性。首先,在pol基因中包含蛋白酶(PR)活性位點(diǎn)的缺失。這可防止多肽的加工(Loeb等(1989)Nature 340307-400),結(jié)果由Western印跡確證(圖3)。采取了另外的預(yù)防措施,還在pol基因中的逆轉(zhuǎn)錄酶(RT)活性位點(diǎn)摻入了點(diǎn)突變(Wakefield等,(1992)J.Virol666806-12;Chao等,(1995)Nucleic Acid Res.23803-10)。
為了能將全部3種基因摻入到一個(gè)基于pADVAX的載體中,利用重疊PCR產(chǎn)生nef-tat融合基因。結(jié)果兩種基因序列都保持完整,因而在所得的融合蛋白質(zhì)中保留了所有免疫原性表位。同前一樣,將tPA前導(dǎo)序列加入到pol和nef-tat中。通過(guò)對(duì)相關(guān)載體轉(zhuǎn)染的293T細(xì)胞裂解物和上清液進(jìn)行Western印跡證實(shí)獲得的表達(dá)和分泌效率的增加。所用的抗體是多克隆兔抗-Nef抗體(由Cecilia Cheny-Mayer博士提供)。
重要的是,同Pol一樣,必須確保Nef-Tat融合蛋白質(zhì)體內(nèi)應(yīng)用的安全性。已知Nef下調(diào)CD4和I類MHC的表面表達(dá)(Collins等,(1998)Nature 391397-401;AiKen等,(1994)Cell 76853-64;Collins等,(1999)Immunol.Rev.16865-74),Tat具有免疫抑制作用(可能通過(guò)作為一般的反式激活蛋白起作用)(Goldstein,(1996)Nat.Med.2960-4;Garber等,(1999)Curr.Opin.Immunol.11460-5)。但是通過(guò)流式細(xì)胞術(shù)分析,我們證明tPA前導(dǎo)序列使Nef對(duì)I類MHC的表達(dá)的作用失效(圖4)。
類似地,在Nef-Tat融合蛋白質(zhì)中,Tat失去其反式激活的能力。這種現(xiàn)象在“MAGI”實(shí)驗(yàn)中很明顯,該實(shí)驗(yàn)中用到經(jīng)改造在功能性HIV-1 Tat存在下表達(dá)β-半乳糖苷酶基因的Hela細(xì)胞(Kimpton等,(1992)J.Virol.662232-9)。因此,在加入X-gal底物后,只有在Tat有活性時(shí),細(xì)胞才轉(zhuǎn)為藍(lán)色(結(jié)果未列出)。因此可以推出,該疫苗產(chǎn)生的Nef-Tat融合蛋白質(zhì)在體內(nèi)不會(huì)有免疫抑制作用。實(shí)際上,既使忽略了通常的反式激活的危險(xiǎn),也已發(fā)現(xiàn)編碼野生型HIV-1 tat的DNA在無(wú)免疫應(yīng)答的個(gè)體中用作疫苗也是安全的(Calarota等,(1999)J.Immunol.1632330-8)。
在ADVAXI和ADVAXII的構(gòu)建和體外性質(zhì)鑒定之后(如上所述),用ELISpot方法評(píng)估該載體的體內(nèi)免疫原性,因?yàn)樵摲椒焖?、可重?fù)、并對(duì)檢測(cè)CD8+和CD4+T細(xì)胞活性靈敏。用5種不同的疫苗pVAX1-env、pVAX1-gag、pVAX1-env、pVAX1-gag、pVAX1(對(duì)照)和ADVAXI免疫BALB/c小鼠。將各組處死的小鼠的脾細(xì)胞合并,并測(cè)定用Env和Gag抗原特異性肽混合物體外再刺激過(guò)程中干擾素-γ(IFN-γ)。針對(duì)兩種單啟動(dòng)子驅(qū)動(dòng)的載體(pVAX1-env和pAVX1-gag)都產(chǎn)生強(qiáng)的免疫應(yīng)答,每種在每一百萬(wàn)脾細(xì)胞中產(chǎn)生大約700個(gè)點(diǎn)形成細(xì)胞(spot-forming Cell,SFC)。由ADVAX1誘導(dǎo)的免疫應(yīng)答相對(duì)明顯,針對(duì)所檢測(cè)的Env和Gag肽混合物產(chǎn)生大約600SFC/百萬(wàn)脾細(xì)胞(圖6)。
總之,該細(xì)胞介導(dǎo)的免疫應(yīng)答定向針對(duì)至少九種不同的表位,包括一種被發(fā)現(xiàn)對(duì)CD8+或CD4+T細(xì)胞特異的表位(數(shù)據(jù)未列出)。沒(méi)有檢測(cè)到在ADVAXI的兩種基因產(chǎn)物間有免疫原性協(xié)同作用或干擾作用。劑量放大實(shí)驗(yàn)揭示了明顯的劑量-反應(yīng)效果(圖7)。
作為HIV-1疫苗的修飾的牛痘Ankara
目前痘苗病毒是在哺乳動(dòng)物細(xì)胞的細(xì)胞質(zhì)中表達(dá)基因的唯一的活載體。重組痘苗病毒已經(jīng)作為科學(xué)工具用于研究防護(hù)包括AIDS的特異性感染性疾病所需的免疫應(yīng)答的類型(Girard(1990)CaccerDetect.Prev.14411-3;Haynes(1996)The Lancet 348933-7;Moss(1996)Pros.Natl.Acad.Sci.USA 9311341-8)。由于痘苗病毒對(duì)人有感染性,因此使用活痘苗病毒時(shí)所關(guān)心的主要問(wèn)題是它們的安全性。不能在無(wú)免疫應(yīng)答的病人(例如帶HIV的患者、血癌患者或正接受化療的患者)中使用常規(guī)的痘苗病毒。為此,已經(jīng)開(kāi)發(fā)了幾種高度減毒的痘苗病毒菌株用作天花疫苗(Paoletti(1996)Proc.Natl.Acad.Sci.USA9311349-53)。盡管不再需要這些減毒的病毒進(jìn)行針對(duì)天花的免疫,但是它們?cè)谌祟愔械脑缙趹?yīng)用為指導(dǎo)篩選合適的AIDS疫苗的菌株提供了重要的安全信息。
三種高度減毒的和感染性的基于痘病毒的載體,包括NYVAC、金絲雀痘(Canarypox)(ALVAC)和修飾的牛痘Ankara(MVA),可用于作為人和獸醫(yī)藥物中重組疫苗的定向應(yīng)用(例如參見(jiàn),Moss等,(1996)Adv.Exp.Med.Biol.3977-13)。MVA菌株已應(yīng)用于大量的疫苗試驗(yàn)和超過(guò)120000人的初始免疫的臨床實(shí)踐。甚至在高?;颊呓邮艹跏济庖邥r(shí),也沒(méi)有發(fā)現(xiàn)與其使用相關(guān)的副作用(Mayr等,(1978)ZBL Bakt Hyg.1Abt.Orig.B 167375-90)。
MVA是宿主范圍限制的和高度減毒的痘苗病毒菌株。該MVA菌株已經(jīng)在雞胚胎成纖維細(xì)胞(CEF)中傳代超過(guò)570次,由于其基因組中包含六個(gè)相對(duì)于牛痘WR菌株的重大缺失,MVA菌株已經(jīng)失去了在大多數(shù)哺乳動(dòng)物細(xì)胞系中繁殖的能力(Altenburger等,(1989)Arch.Virol.10515-27;Meyer等,(1991)J.Gen.Virol.711031-8)。該缺失位于病毒基因組兩末端的附近,其中的一個(gè)缺失影響55K及32K的人類宿主范圍基因。進(jìn)一步分析表明病毒減毒部分由于大約三分之二的宿主范圍基因的缺失引起。MVA菌株在禽類細(xì)胞中生長(zhǎng)良好,但在測(cè)試的人類和大多數(shù)其它哺乳動(dòng)物細(xì)胞中不能增殖。然而,MVA DNA的復(fù)制看起來(lái)正常,并在人類細(xì)胞中合成早期和晚期的病毒蛋白(Sutter等,(1992)Proc.Natl.Acad.Sci.USA 8910847-51)。
已經(jīng)證明高度減毒和復(fù)制缺陷的重組MVA的免疫原性和保護(hù)效力比許多常規(guī)痘苗病毒(VV)強(qiáng)。利用感染復(fù)數(shù)(Multiplicity ofinfection,MOI)為1,該高度減毒株MVA唯一代表能誘導(dǎo)大量IFNα/β(其具有抗病毒作用)的VV。用五種熟知的常規(guī)VV菌株以及中國(guó)VV菌株TienTan(VVTT)作為重組疫苗不能誘導(dǎo)白細(xì)胞IFN(Buttner等,(1995)Vet,Immunol.46237-50)。在小動(dòng)物中,表達(dá)HA-NP基因的重組MVA不但誘導(dǎo)血清IgG抗體、粘膜IgA抗體和強(qiáng)CTL反應(yīng),也保護(hù)免疫的小鼠的肺免受流感病毒的侵襲,甚至在口服免疫之后也能如此(Bender等,(1996)J.Virol.706418-24)。最重要的是,用SIV/SHIV重組MVA免疫的恒河猴比用SIV重組NYCBH-VV免疫的恒河猴更有可能成為長(zhǎng)期不發(fā)展者(nonprogressor)(Hirsch等,(1996)703741-52;Amara等,(2001)Science 29269)。這些恒河猴,就象HIV-1感染的人類長(zhǎng)期不發(fā)展者一樣,具有低水平的、伴隨持續(xù)病毒復(fù)制限制的初始血漿病毒血(癥),其與維持正常淋巴細(xì)胞亞群和完整的淋巴樣結(jié)構(gòu)相關(guān)。該結(jié)果和先前的有關(guān)MVA在人體中的安全性的數(shù)據(jù)一起暗示了重組MVA在人類AIDS的預(yù)防性接種中的重要用途。目前,還沒(méi)有HIV重組MVA被構(gòu)建或用于人類HIV-1接種。
原始的MVA插入載體pLW22和pLW7分別靶向MVA基因組的Del II區(qū)和Del III區(qū),其從NIH的B.Moss處獲得?;谠搩煞N惠贈(zèng)的載體,產(chǎn)生兩種插入載體pZC22和pZC1,其同樣靶向Del II區(qū)和Del III區(qū)。載體pZC1經(jīng)pLW7修飾而來(lái)(圖8)。該新插入載體pZC1被構(gòu)建遞送兩種外源基因到MVA基因組的Del III區(qū),而不是遞送一種外源基因。由于pZC1帶有兩種不同的啟動(dòng)子,此處不存在潛在啟動(dòng)子競(jìng)爭(zhēng)的問(wèn)題。因此,pZC1可以遞送env和gag-pol到MVA的Del III區(qū),每種在單獨(dú)的不同牛痘啟動(dòng)子控制之下,但在同一插入盒中。由于已經(jīng)證實(shí)包膜免疫染色是靈敏的和可靠的,所以可以用env作為替代標(biāo)志物去篩選gag-pol的存在,其本身很難檢測(cè)。通過(guò)這種方法,對(duì)包膜染色呈陽(yáng)性的細(xì)胞也具有整合到基因組上的gag-pol基因。經(jīng)過(guò)多輪富集之后,可以通過(guò)western印記確證gag-pol的表達(dá),其比原位免疫染色更靈敏。
如下所述,將HIV-1 env和gag-pol都插入到pZC1中單獨(dú)的啟動(dòng)子下。通過(guò)同源重組將env-gag-pol pZC1插入到野生型MVA的Del III區(qū)。通過(guò)利用抗Env抗體的免疫染色鑒定重組的env-gag-pol MVA,并通過(guò)利用Western印記分析檢測(cè)Gag-pol的表達(dá)進(jìn)一步確證。因此,兩種基因都在雙啟動(dòng)子的構(gòu)建體中進(jìn)行表達(dá)。已經(jīng)通過(guò)利用抗Env抗體的富集/選擇進(jìn)一步增殖了重組的env-gag-pol MVA菌株(“ADMVA1”)。
另一種插入載體(pLW22)經(jīng)修飾產(chǎn)生pZC22(圖9),其遞送外源基因至MVA基因組的Del II區(qū)。修飾去除了報(bào)告基因。Del II區(qū)在DelIII區(qū)上游超過(guò)120kbp處。理論上,利用pZC1和pZC22載體都可以將多HIV-1基因重組進(jìn)單一的MVA基因組。
為了將5種HIV-1基因用于DNA接種,構(gòu)建了稱為ADMVA2的第2代ADMVA。將HIV-1 nef-tat基因插入pZC22,并經(jīng)同源重組將該nef-tat pZC22引入噬斑純化的ADMVA1的Del II區(qū)。通過(guò)利用抗Env和抗Nef抗體的雙重免疫染色鑒定該重組的ADMVA2。已經(jīng)通過(guò)利用抗Env和抗Nef抗體的選擇噬斑純化了該重組的ADMVA2。
用富集的ADMVA2評(píng)估感染后的細(xì)胞中五種HIV-1基因產(chǎn)物的表達(dá)。已經(jīng)通過(guò)western印記分析確證了全部5種基因的有效表達(dá)。另外,已經(jīng)確定全部5種基因都可從ADMVA2基因組DNA中擴(kuò)增。序列分析已經(jīng)確證了插入基因的身份。
ADMVA2的感染力可以達(dá)到108-109TCID50/mL,并且病毒很容易地以1∶10的比例擴(kuò)增。ADMVA2在體外傳代6次仍然穩(wěn)定。除雞胚胎成纖維細(xì)胞外,ADMVA2還可感染人細(xì)胞。
實(shí)施例提供下列實(shí)施例,為本領(lǐng)域普通技術(shù)人員提供如何制造和使用本發(fā)明的測(cè)定、篩選和治療方法的完整的公開(kāi)和描述,并不想限制本發(fā)明人認(rèn)為的其發(fā)明的范圍。已經(jīng)努力確保所用數(shù)據(jù)(例如量、溫度等)的準(zhǔn)確性,但是,應(yīng)考慮到某些實(shí)驗(yàn)錯(cuò)誤和誤差。除非另有說(shuō)明,份數(shù)是指重量份數(shù),分子量是指平均分子量,溫度是攝氏度,壓力是或接近大氣壓。
實(shí)施例1.方案直接ELISA測(cè)量免疫動(dòng)物中抗-HIV-1gag抗體Gag蛋白質(zhì)-樣品-抗小鼠IgG.ALP。用100μl溶于0.1M NaHCO3(pH9.6)溶液中的Gag蛋白質(zhì)(0.5μg/孔)4℃過(guò)夜包被培養(yǎng)板(Immulon-2,Dynex Technologies,Chantilly,VA或Costar EIA/RIA高結(jié)合的96孔板9018,Corning Inc.,Corning,NY)。用200μl的PBS洗一次板,加入含5%脫脂奶粉、0.5%6BSA的PBS室溫封閉1-2小時(shí)。將在封閉緩沖液中的無(wú)菌稀釋的動(dòng)物血清或?qū)φ占尤氲脚囵B(yǎng)板中,并在室溫溫育1小時(shí)。用含有0.05%Tween-20的PBS將板洗滌4次。加入堿性磷酸酶標(biāo)記的山羊抗小鼠IgG(Pharmingen BD,AKP多克隆山羊抗小鼠Ig,Cat#554000,lotM061790),1∶10000稀釋10ml封閉緩沖液中加入1μl的綴合物,并在室溫溫育板30分鐘。用AmpliQ洗滌緩沖液洗4次板。AmpliQ的使用方法如下在每孔中加入100μl的新配制的底物(50μl溶液A+50μl溶液B),室溫作用15分鐘。用AmpliQ終止溶液終止反應(yīng),并在15分鐘內(nèi)在490nm處讀板(AmpliQDAKO Diagnostics Ltd.)。
實(shí)施例2.競(jìng)爭(zhēng)性gp120-sCD4 ELISA方案抗gp120Abs-gp120-sCD4.IgG-抗人IgG.ALP。用溶于0.1M NaHCO3(pH8.6)溶液中的抗gp120抗體室溫過(guò)夜包被培養(yǎng)板。用PBS將板洗滌2次,加入含有4%脫脂奶粉、0.5%BSA的PBS室溫封閉1小時(shí)?;旌戏忾]液,并加入gp120上清,室溫作用1小時(shí)。用含有Tween-20的PBS將板洗滌4次。加入用封閉緩沖液無(wú)菌稀釋的sCD4或?qū)φ眨⒃谑覝販赜?小時(shí)。用含有Tween-20的PBS將板洗滌4次。加入固定濃度的sCD4-人IgG,并在室溫溫育板1小時(shí)。用含有Tween-20的PBS將板洗滌4次。加入堿性磷酸酶標(biāo)記的抗人IgG,并在室溫溫育板30分鐘。用AMPAK洗滌緩沖液將板洗滌4次。按如下的AMPAK操作方法進(jìn)行加入100μl的新配制的底物作用20分鐘,然后加入100μl的擴(kuò)增液作用10分鐘。用50μl 0.5M的H2SO4終止反應(yīng),在492nm處讀板。
實(shí)施例3.小鼠IFN-γELISpot試驗(yàn)第1天。將ELISpot過(guò)濾板進(jìn)行如下預(yù)包被加入用包被緩沖液1∶50稀釋的捕捉抗體(如小鼠IFN-γ)(例如在5ml包被緩沖液中加入125μl抗體)。在每孔中放置100μl的捕捉抗體/包被緩沖液,加蓋,然后放在4℃溫育過(guò)夜。
第2天。收獲細(xì)胞,用PBST接種4塊板。每孔用R10(200μl/孔)37℃封閉2小時(shí)。根據(jù)特定板的計(jì)劃加入細(xì)胞。加入肽,并在CO2培養(yǎng)箱中37℃溫育過(guò)夜。
第3天。用PBST洗板5次,然后,每孔加入100μl在1%BSA中1∶60稀釋的檢測(cè)抗體。將板4℃溫育過(guò)夜。
第4天。用PBST洗板4次,然后在每孔加入100μl在1%BSA中1∶60稀釋的SAP。將板室溫溫育2小時(shí),用PBST洗滌4次,然后用ddH2O洗滌1次。每孔中加入100μl底物,然后室溫避光溫育大約15分鐘或直到完全形成。用自來(lái)水洗板,徹底干燥,數(shù)點(diǎn)。
實(shí)施例4.體內(nèi)免疫原性評(píng)估用ELISpot試驗(yàn)如上所述評(píng)估針對(duì)ADVAXI和II的CMI反應(yīng)(也參見(jiàn)Hanke等,(1998)J.Gen.Virol 7983-90;Carvalho等,(2001)J.Immunol.Methods 252207-18;Tobery等,(2001)J.Immunol.Methods25459-66;Novitsky等,(2001)J.Virol.759210-28,該出版物此處全文引用以供參考)。
開(kāi)始用ADVAXI的GLP級(jí)貯存物(Aldevron,F(xiàn)argo,ND)免疫6-8周齡的雌性BALB/c小鼠。在0周、3周和6周IM給藥200μg的疫苗??偣?組、每組6只小鼠,每組進(jìn)行如下接種p VAX1-nev,p VAX1-gag,p VAX1-nev+p VAX1-gag,p VAX1(對(duì)照)和ADVAXI。肽代表如下的特異表位已知誘發(fā)CD4+細(xì)胞介導(dǎo)的反應(yīng)的Env34(VPVWKEAKTTLFCASDAKAY)(SEQ ID NO3),誘發(fā)CD8+細(xì)胞介導(dǎo)的反應(yīng)的Env43(RNVSSDGTYNETYNEIKNCS)(SEQ ID NO4),誘發(fā)CD4+細(xì)胞介導(dǎo)的反應(yīng)的Gag26(TSNPPIPVGDIYKRWIILGL)(SEQ ID NO5)和誘發(fā)CD8+細(xì)胞介導(dǎo)的反應(yīng)的Gag A-I(AMQMLKDTI)(SEQ ID NO6或2)。
第3次注射兩周后處死小鼠。合并每組的脾細(xì)胞,用ELISpot測(cè)定在用Env和Gag抗原特異性肽混合物(NIH AIDS研究和參考試劑)體外再刺激過(guò)程中其分泌干擾素-γ(IFN-γ)的能力。此時(shí),只有來(lái)自異源株(HIV96ZM65.8,目錄號(hào)3993)的Gag肽。類似地,在ELISpot測(cè)定時(shí)我們沒(méi)有整套的同源Env肽(HIVCHN.AD,目錄號(hào)4974,80%完整)。但是,結(jié)果表明針對(duì)兩種單啟動(dòng)子驅(qū)動(dòng)的載體(p VAX1-nev和p VAX1-gag)都有強(qiáng)的免疫應(yīng)答,每種產(chǎn)生大約700個(gè)點(diǎn)形成細(xì)胞(spot-formingcells,SFC)/百萬(wàn)脾細(xì)胞。由ADVAXI誘導(dǎo)的免疫應(yīng)答也很明顯,大約產(chǎn)生特異性針對(duì)所檢測(cè)的Env和Gag肽混合物的600SFC/百萬(wàn)脾細(xì)胞??蓴喽ǖ?,針對(duì)p VAX1對(duì)照的反應(yīng)為零,由于脾細(xì)胞混合物中缺失CD8+細(xì)胞,沒(méi)有檢測(cè)到針對(duì)GAG A-I的ELISpot反應(yīng)(圖6)。
總之,該細(xì)胞介導(dǎo)的免疫應(yīng)答定向針對(duì)至少9種不同的表位,包括被發(fā)現(xiàn)特異性針對(duì)CD8+或CD4+T細(xì)胞的表位(數(shù)據(jù)未列出)。沒(méi)有檢測(cè)到在ADVAXI的兩種基因產(chǎn)物之間有免疫原性協(xié)同作用和干擾作用的證據(jù)。劑量放大試驗(yàn)表明了明顯的劑量-反應(yīng)效果(圖7)。對(duì)于至少一種表位(Env34),發(fā)現(xiàn)150μg時(shí)的ELISpot反應(yīng)量是5μg時(shí)的大約7倍。但是,該劑量-反應(yīng)趨勢(shì)對(duì)所有檢測(cè)的表位都是正確的,不管是特異性針對(duì)CD4+或CD8+細(xì)胞介導(dǎo)的反應(yīng)。
實(shí)施例5.臨床前體內(nèi)免疫原性評(píng)估下列數(shù)據(jù)表明ADVAXI的體內(nèi)體液免疫原性。在小鼠試驗(yàn)中,在最后(第三次)免疫后兩周收集血清樣品,用ELISA測(cè)試抗-Gag抗體。盡管在用p VAX1-gag接種的小鼠中的滴度最高,在用ADVAXI免疫組中也有較大的滴度,其與接受p VAX1-env+p VAX1-gag的動(dòng)物中顯示的反應(yīng)相當(dāng)(圖10)。通過(guò)western印記也表明在ADVAXI組中收集的血清樣本中有針對(duì)Env的抗體反應(yīng)(數(shù)據(jù)未列或定量)。
用ADVAXII進(jìn)行了類似的體內(nèi)研究。具體地,用ADVAXII的GLP-級(jí)貯存物(Aldevron,F(xiàn)argo,ND)免疫6-8周齡的雌性BALB/c小鼠。在0周、3周和6周IM注射200μg疫苗。每組5只、共5組小鼠,每組進(jìn)行如下接種p VAX1-pol、p VAX1-nef-tat、p VAX1-pol+pVAX1-nef-tat、p VAX1(對(duì)照)和ADVAXII(只有雙啟動(dòng)子載體)。第三次注射后兩周處死小鼠。然后合并每組的脾細(xì)胞,并用ELISpot分析其在用Pol、Tat和Nef衍生的肽混合物體外再刺激過(guò)程中分泌干擾素-γ的能力。注意的是在分析中沒(méi)有分化體C。此處的抗原特異性肽是基于分化體B的共有序列的15聚體(NIH AIDS研究和參考試劑計(jì)劃Tat目錄號(hào)5138,Nef目錄號(hào)5189,和Pol目錄號(hào)6208)。但是,和ADVAXI試驗(yàn)一樣,我們發(fā)現(xiàn)單基因載體和雙啟動(dòng)子疫苗都有很好的反應(yīng)。例如,針對(duì)Pol混合物的反應(yīng)最好的是單獨(dú)的p VAX1-pol(300-800SFC/百萬(wàn)脾細(xì)胞,取決于混合物)。對(duì)于p VAX1-pol+p VAX1-nef-tat,結(jié)果為180-500SFC/百萬(wàn)脾細(xì)胞,對(duì)于ADVAXII,其反應(yīng)為180-600SFC/百萬(wàn)脾細(xì)胞。針對(duì)Tat混合物,對(duì)于p VAX1-nef-tat,其反應(yīng)大約為180SFC,對(duì)于p VAX1-pol+p VAX1-nef-tat和ADVAXII都是大約100SFC。利用Nef混合物,對(duì)于pVAX1-nef-tat,其反應(yīng)為30-200SFC,對(duì)于p VAX1-pol+p VAX1-nef-tat和ADVAXII都是20-150SFC(圖11)。
我們又用ADVAXII進(jìn)行了劑量放大研究,表明了明顯的劑量-反應(yīng)效果(圖12)。在0周、3周和6周給小鼠IM注射5μg、10μg、50μg、100μg或150μg的DNA。在第8周處死小鼠,合并脾細(xì)胞,用衍生自分化體B共有序列的肽體外再刺激(再次重申,沒(méi)有分化體C試劑)。Pol的反應(yīng)隨劑量的升高而升高,從10μg的250-450SFC到100μg的500-700SFC。(150μg時(shí)的反應(yīng)與100μg時(shí)的類似)。Nef的反應(yīng)范圍為大約20SFC至大約200SFC,Tat的反應(yīng)在大約25至大約100SFC之間。
進(jìn)行初始-加強(qiáng)疫苗方案的全部計(jì)劃如下將ADVAXI和ADVAXII聯(lián)合作為初始免疫組分,重組的MVA載體組成加強(qiáng)免疫的組分。因此將ADVAXI+II一起給藥作為聯(lián)合接種物進(jìn)行體外試驗(yàn)。在0周、3周和6周對(duì)各組小鼠IM注射5μg、10μg、50μg、100μg或150μg的ADVAXI+II。對(duì)照組注射pVAX1-gag、pVAX1-env、pVAX1-pol和pVAX1-nef-tat各50μg的混合物。在最后的免疫之后兩周,處死小鼠,將每組的脾細(xì)胞合并,用ELISpot測(cè)定干擾素-γ的釋放。用包含CD4+和CD8+T細(xì)胞表位的Env、Gag和Pol特異性肽進(jìn)行體外再刺激,同樣用自體亞型CTat和Nef序列進(jìn)行體外再刺激。如在單獨(dú)的ADVAXI和ADVAXII試驗(yàn)中看到的結(jié)果一樣,聯(lián)合接種物也對(duì)兩種載體顯示比較好的反應(yīng)。用ADVAXI+II接種的小鼠針對(duì)所有試驗(yàn)的肽(肽混合物)具有抗原特異性反應(yīng),并且該反應(yīng)是以劑量依賴的方式誘導(dǎo)的(表1)。引人注意的是,在該試驗(yàn)中發(fā)現(xiàn)用亞型C衍生的Nef和Tat肽混合物體外再刺激的脾細(xì)胞上的反應(yīng)特別強(qiáng)烈,相反,在ADVAXII試驗(yàn)中發(fā)現(xiàn)的反應(yīng)較輕微。這種差異可能是由于異體和自體蛋白質(zhì)之間的差異造成的。因此,我們相信,在ADVAXII中的nef-tat融合基因?qū)嶋H上可以誘導(dǎo)非常有效的免疫應(yīng)答。另外,聯(lián)合接種試驗(yàn)表明用干擾素-γELISpot測(cè)量在不同的抗原特異性反應(yīng)中沒(méi)有檢測(cè)到干擾。因此我們可得到結(jié)論可以認(rèn)為ADVAXI+II可以進(jìn)一步臨床開(kāi)發(fā)為針對(duì)HIV-1的初步-加強(qiáng)疫苗策略中的初步免疫的組分。
表1.針對(duì)聯(lián)合接種物和對(duì)照的抗原特異性干擾素-γELISpot反應(yīng)疫苗 IFN-γ點(diǎn)形成細(xì)胞(SFC)/106脾細(xì)胞(劑量)Gag 26 Gag A-I Env 34 Env T-I Pol 223 Pol YLI Pol VGI Tat pool Nef pool1 Nef pool2pVAX1-gag+pVAX1-env+pVAX1-pol+pVAX1-nef-tat 120 150 350 700 70 500 500 110400 300(每種50μg)ADVAXI+II 112 252 400 700 117 700 830 120225 370(150μg+150μg)ADVAXI+II 70 210 400 600 100 550 500 70 200 330(100μg+100μg)ADVAXI+II 30 100 220 500 84 400 420 50 140 160(50μg+50μg)ADVAXI+II 30 60 180 250 70 275 375 30 110 140(10μg+10μg)ADVAXI+II 10 30 110 180 60 250 370 20 70140(5μg+5μg)注針對(duì)兩種細(xì)胞輸入水平歸一化每百萬(wàn)脾細(xì)胞的點(diǎn)數(shù),并根據(jù)雙孔將每種樣品和抗原進(jìn)行平均。
Gag 26、Env 34和Pol 223包含CD4表位,而Gag A-I、Env T-I、Pol YLI和Pol VGI包含CD8表位。用于Tat和Nef的肽混合物衍生自自體亞型C序列。
序列表<110>Huang,YaoxingChen,ZhiweiHo,David<120>針對(duì)HIV-1的免疫方法和組合物<130>2570-1-003P<160>22<170>FastSEQ for Windows Version 4.0<210>1<211>23<212>PRT<213>人工序列<220><223>片段<400>1Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly1 5 10 15Ala Val Phe Val Ser Ala Arg20<210>2<211>9<212>PRT<213>人工序列<220><223>片段<400>2Ala Met Gly Met Leu Lys Asp Thr Ile1 5<210>3<211>20<212>PRT<213>人工序列<220><223>片段<400>3Val Pro Val Trp Lys Glu Ala Lys Thr Thr Leu Phe Cys Ala Ser Asp1 5 10 15Ala Lys Ala Tyr20<210>4<211>20<212>PRT<213>人工序列<220><223>片段<400>4Arg Asn Val Ser Ser Asp Gly Thr Tyr Asn Glu Thr Tyr Asn Glu Ile1 5 10 15Lys Asn Cys Ser20<210>5<211>20<212>PRT<213>人工序列<220><223>片段<400>5Thr Ser Asn Pro Pro Ile Pro Val Gly Asp Ile Tyr Lys Arg Trp lle1 5 10 15Ile Leu Gly Leu20<210>6<211>9<212>PRT<213>人工序列<220><223>片段<400>6Ala Met Gln Met Leu Lys Asp Thr Ile1 5<210>7<211>2520<212>DNA<213>人(Homo sapiens)<400>7atggatgcaa tgaagagagg gctctgctgt gtgctgctgc tgtgtggagc agtcttcgtt 60agcgccgccg agaacttgtg ggtgaccgtg tactacggcg tgcccgtgtg gaaggaggcc120aagaccaccc tgttctgcgc ctccgacgcc aaggcctacg agaaggaggt gcacaacgtg180tgggccaccc acgcctgcgt gcccaccgac cccaaccccc aggagatggt gttggagaac240gtgaccgaga acttcaacat gtggaagaac gacatggtga accagatgca cgaggacgtc300atcagcttgt gggaccagag cctgaagccc tgcgtgaagt tgacccccct gtgcgtgacc360ttggagtgca ggaacgtgag cagcaacggc acctacaacg agacctacaa cgagatcaag420aactgctcct tcaacgccac caccgtgttg agggacagga agcagaccgt gtacgccctg480ttctacaggc tggacatcgt gcccctgaac aagaagaact ccagcgagaa ctccagcgag540tactacaggt tgatcaactg caacacctcc gccatcaccc aggcctgccc caaggtgacc600ttcgacccca tccccatcca ctactgcacc cccgccggct acgccatcct gaagtgcaac660gacaagacct tcaacggcac cggcccctgc cacaacgtga gcaccgtgca gtgcacccac720ggcatcaagc ccgtggtgtc cacccagctg ctgttgaacg gcagcctggc cgagagggag780atcatcatca ggtccgagaa cctgaccaac aacgtgaaga ccatcatcgt gcacctgaac840cagtccgtgg agatcgtgtg caccaggccc aacaacaaca ccaggaagag catcaggatc900ggccccggcc agaccttcta cgccaccggc gacatcatcg gcgacatcag gcaggcccac960tgcaacatca gcaaggacaa gtggaaggag accttgcaga gggtgggcaa gaagttggcc 1020gagcacttcc ccaacaagac catcgagttc gcctcctcct ccggcggcga cctggagatc 1080accacccaca gcttcaactg caggggcgag ttcttctact gcaacacctc cagcctgttc 1140aacggcacct acatgcccaa cggcaccgag ggcaactcca gctccatcat caccatcccc 1200tgcaggatca agcagatcat caacatgtgg caggaggtgg gccgcgccat gtacgccccc 1260cccatcgagg gcaacatcac ctgcaagtcc aacatcaccg gcctgctgtt ggtgcgcgac 1320ggcggcaagg agaccaacga caccgagacc ttcaggcccg gcggcggcga catgagggac 1380aactggagga gcgagttgta caagtacaag gtggtggaga tcaagccctt gggcatcgcc 1440cccaccgccg ccaagaggag ggtggtggag agggagaaga gggccgtggg catcggcgcc 1500gtgttcctgg gcttcctggg cgccgccggc agcaccatgg gcgccgccag catcaccctg 1560accgtgcagg cccgccagct gctgagcggc atcgtgcagc agcagagcaa cctgctgcgc 1620gccatcgagg cccagcagca cctgctgcag ctgaccgtgt ggggcatcaa gcagctgcag 1680acccgcgttc tggccatcga gcgctacctg aaggaccagc agctgctggg catctggggc 1740tgcagcggca agctgatctg caccaccgcc gtgcactgga acagcagctg gagcaaccgc 1800agccaggagg agatctggaa caacatgacc tggatgcagt gggaccgcga gatcagcaac 1860tacaccaaca ccatctaccg cctgctggag gacagccaga accagcagga gcgcaacgag 1920aaggacctgc tggccctgga caactggaag aacctgtgga gctggttcga catcaccaac 1980tggctgtggt acatccgcat cttcatcatg atcgtgggcg gcctgatcgg cctgcgcatc 2040atcttcgccg tgctgagcat cgtgaaccgc gtgcgccagg gctacagccc cctgagcttc 2100cagaccctga cccccaaccc cggcggcccc gaccgcctgg gccgcatcga ggaggagggc 2160ggcgagcagg acaagaaccg cagcatccgc ctggtgaacg gcttcctggc cctggcctgg 2220gacgacctgc gcaacctgtg ccgcttcagc taccacctgc tgcgcgacct gctgctgatc 2280gtggcccgca tcgtggagct gctgggccgc cgcggctggg aggccctgcg ctactggtgg 2340aacctgctga agtactgggt gcaggagctg aagaacagcg ccgtgagcct gctgaacgcc 2400accgccatcg ccgtggccga gggcaccgac cgcgtgatcg aggtggtgca gggcgcctac 2460cgcgccatcc tgcacatccc ccgccgcatc cgccagggct tcgaggccgc cctgcagtaa 2520<210>8<211>839<212>PRT<213>人<400>8Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly1 5 10 15Ala Val Phe Val Ser Ala Ala Glu Asn Leu Trp Val Thr Val Tyr Tyr20 25 30Gly Val Pro Val Trp Lys Glu Ala Lys Thr Thr Leu Phe Cys Ala Ser35 40 45Asp Ala Lys Ala Tyr Glu Lys Glu Val His Asn Val Trp Ala Thr His50 55 60Ala Cys Val Pro Thr Asp Pro Asn Pro Gln Glu Met Val Leu Glu Asn65 70 75 80Val Thr Glu Asn Phe Asn Met Trp Lys Asn Asp Met Val Asn Gln Met85 90 95His Glu Asp Val Ile Ser Leu Trp Asp Gln Ser Leu Lys Pro Cys Val100 105 110Lys Leu Thr Pro Leu Cys Val Thr Leu Glu Cys Arg Asn Val Ser Ser115 120 125Asn Gly Thr Tyr Asn Glu Thr Tyr Asn Glu Ile Lys Asn Cys Ser Phe130 135 140Asn Ala Thr Thr Val Leu Arg Asp Arg Lys Gln Thr Val Tyr Ala Leu145 150 155 160Phe Tyr Arg Leu Asp Ile Val Pro Leu Asn Lys Lys Asn Ser Ser Glu165 170 175Asn Ser Ser Glu Tyr Tyr Arg Leu Ile Asn Cys Asn Thr Ser Ala Ile180 185 190Thr Gln Ala Cys Pro Lys Val Thr Phe Asp Pro Ile Pro Ile His Tyr195 200 205Cys Thr Pro Ala Gly Tyr Ala Ile Leu Lys Cys Asn Asp Lys Thr Phe210 215 220Asn Gly Thr Gly Pro Cys His Asn Val Ser Thr Val Gln Cys Thr His225 230 235 240Gly Ile Lys Pro Val Val Ser Thr Gln Leu Leu Leu Asn Gly Ser Leu245 250 255Ala Glu Arg Glu Ile Ile Ile Arg Ser Glu Asn Leu Thr Asn Asn Val260 265 270Lys Thr Ile Ile Val His Leu Asn Gln Ser Val Glu Ile Val Cys Thr275 280 285Arg Pro Asn Asn Asn Thr Arg Lys Ser Ile Arg Ile Gly Pro Gly Gln290 295 300Thr Phe Tyr Ala Thr Gly Asp Ile Ile Gly Asp Ile Arg Gln Ala His305 310 315 320Cys Asn Ile Ser Lys Asp Lys Trp Lys Glu Thr Leu Gln Arg Val Gly325 330 335Lys Lys Leu Ala Glu His Phe Pro Asn Lys Thr Ile Glu Phe Ala Ser340 345 350Ser Ser Gly Gly Asp Leu Glu Ile Thr Thr His Ser Phe Asn Cys Arg355 360 365Gly Glu Phe Phe Tyr Cys Asn Thr Ser Ser Leu Phe Asn Gly Thr Tyr370 375 380Met Pro Asn Gly Thr Glu Gly Asn Ser Ser Ser Ile Ile Thr Ile Pro385 390 395 400Cys Arg Ile Lys Gln Ile Ile Asn Met Trp Gln Glu Val Gly Arg Ala405 410 415Met Tyr Ala Pro Pro Ile Glu Gly Asn Ile Thr Cys Lys Ser Asn Ile420 425 430Thr Gly Leu Leu Leu Val Arg Asp Gly Gly Lys Glu Thr Asn Asp Thr435 440 445Glu Thr Phe Arg Pro Gly Gly Gly Asp Met Arg Asp Asn Trp Arg Ser450 455 460Glu Leu Tyr Lys Tyr Lys Val Val Glu Ile Lys Pro Leu Gly Ile Ala465 470 475 480Pro Thr Ala Ala Lys Arg Arg Val Val Glu Arg Glu Lys Arg Ala Val485 490 495Gly Ile Gly Ala Val Phe Leu Gly Phe Leu Gly Ala Ala Gly Ser Thr500 505 510Met Gly Ala Ala Ser Ile Thr Leu Thr Val Gln Ala Arg Gln Leu Leu515 520 525Ser Gly Ile Val Gln Gln Gln Ser Asn Leu Leu Arg Ala Ile Glu Ala530 535 540Gln Gln His Leu Leu Gln Leu Thr Val Trp Gly Ile Lys Gln Leu Gln545 550 555 560Thr Arg Val Leu Ala Ile Glu Arg Tyr Leu Lys Asp Gln Gln Leu Leu565 570 575Gly Ile Trp Gly Cys Ser Gly Lys Leu Ile Cys Thr Thr Ala Val His580 585 590Trp Asn Ser Ser Trp Ser Asn Arg Ser Gln Glu Glu Ile Trp Asn Asn595 600 605Met Thr Trp Met Gln Trp Asp Arg Glu Ile Ser Asn Tyr Thr Asn Thr610 615 620Ile Tyr Arg Leu Leu Glu Asp Ser Gln Asn Gln Gln Glu Arg Asn Glu625 630 635 640Lys Asp Leu Leu Ala Leu Asp Asn Trp Lys Asn Leu Trp Ser Trp Phe645 650 655Asp Ile Thr Asn Trp Leu Trp Tyr Ile Arg Ile Phe Ile Met Ile Val660 665 670Gly Gly Leu Ile Gly Leu Arg Ile Ile Phe Ala Val Leu Ser Ile Val675 680 685Asn Arg Val Arg Gln Gly Tyr Ser Pro Leu Ser Phe Gln Thr Leu Thr690 695 700Pro Asn Pro Gly Gly Pro Asp Arg Leu Gly Arg Ile Glu Glu Glu Gly705 710 715 720Gly Glu Gln Asp Lys Asn Arg Ser Ile Arg Leu Val Asn Gly Phe Leu725 730 735Ala Leu Ala Trp Asp Asp Leu Arg Asn Leu Cys Arg Phe Ser Tyr His740 745 750Leu Leu Arg Asp Leu Leu Leu Ile Val Ala Arg Ile Val Glu Leu Leu755 760 765Gly Arg Arg Gly Trp Glu Ala Leu Arg Tyr Trp Trp Asn Leu Leu Lys770 775 780Tyr Trp Val Gln Glu Leu Lys Asn Ser Ala Val Ser Leu Leu Asn Ala785 790 795 800Thr Ala Ile Ala Val Ala Glu Gly Thr Asp Arg Val Ile Glu Val Val805 810 815Gln Gly Ala Tyr Arg Ala Ile Leu His Ile Pro Arg Arg Ile Arg Gln820 825 830Gly Phe Glu Ala Ala Leu Gln835<210>9<211>1545<212>DNA<213>人<400>9atggacgcca tgaagcgcgg cctgtgctgc gtgctgctgc tgtgcggcgc cgtgttcgtg 60agcgcccgca tgggcgcccg cgccagcatc ctgcgcggcg gcaagctgga caagtgggag120aagatccgcc tgcgccccgg cggcaagaag cactacatgc tgaagcacct ggtgtgggcc180agccgcgagc tggagcgctt cgccctgaac cccggcctgc tggagaccag cgagggctgc240aagcagatca tcaagcagct gcagcccgcc ctgcagaccg gcaccgagga gctgcgcagc300ctgttcaaca ccgtggccac cctgtactgc gtgcacgagg gcatcgagat ccgcgacacc360aaggaggccc tggacaagat cgaggaggag cagaacaaga tccagcagaa gacccagcag420gccaagaagg ccgacgagaa ggtgagccag aactacccca tcgtgcagaa cctgcagggc480cagatggtgc accaggccat ctcccccagg accttgaacg cctgggtgaa ggtgatcgag540gagaaggcct tcagccccga ggtgatcccc atgttcaccg ccttgtccga gggcgccacc600ccccaggact tgaacaccat gttgaacacc gtgggcggcc accaggccgc catgcagatg660ttgaaggaca ccatcaacga ggaggccgcc gagtgggaca gggtgcaccc cgtgcacgcc720ggccccatcg cccccggcca gatgagggag cccaggggca gcgacatcgc cggcaccacc780agcaccctgc agggccagat cgcctggatg accagcaacc cccccgtgcc cgtgggcgag840atctacaaga ggtggatcat cctgggcttg aacaagatcg tgaggatgta cagccccgtg900agcatcttgg acatcaagca gggccccaag gagcccttca gggactacgt ggaccgcttc960ttcaagacct tgagggccga gcaggccacc caggacgtga agaactggat gaccgacacc 1020ttgttggtgc agaacgccaa ccccgactgc aagaccatct tgagggcctt gggccccggc 1080gcctccttgg aggagatgat gaccgcctgc cagggcgtgg gcggccccag ccacaaggcc 1140agggtgttgg ccgaggccat gagccaggcc aacggcacca tcctgatgca gaggagcaac 1200ttcaagggct ccaagaggat cgtgaagtgc ttcgactgcg gcaaggaggg ccacatcgcc 1260aggaactgca gggcccccag gaagaagggc tgctggaagt gcggcaagga gggccaccag 1320atgaaggact gcaccgagag gcaggccaac ttcttgggca agatctggcc ctcccacaag 1380ggcaggcccg gcaacttcct gcagagcagg cccgagccca ccgccccccc cgccgagagc 1440ttcaggttcg aggagaccac ccccgccccc aagcaggagc ccaaggacag ggagcccttg 1500acctccctga agtccctgtt cggcagcgac cccttgtccc agtaa 1545<210>10<211>514<2L2>PRT<213>人<400>10Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly1 5 10 15Ala Val Phe Val Ser Ala Arg Met Gly Ala Arg Ala Ser Ile Leu Arg
20 25 30Gly Gly Lys Leu Asp Lys Trp Glu Lys Ile Arg Leu Arg Pro Gly Gly35 40 45Lys Lys His Tyr Met Leu Lys His Leu Val Trp Ala Ser Arg Glu Leu50 55 60Glu Arg Phe Ala Leu Asn Pro Gly Leu Leu Glu Thr Ser Glu Gly Cys65 70 75 80Lys Gln Ile Ile Lys Gln Leu Gln Pro Ala Leu Gln Thr Gly Thr Glu85 90 95Glu Leu Arg Ser Leu Phe Asn Thr Val Ala Thr Leu Tyr Cys Val His100 105 110Glu Gly Ile Glu Ile Arg Asp Thr Lys Glu Ala Leu Asp Lys Ile Glu115 120 125Glu Glu Gln Asn Lys Ile Gln Gln Lys Thr Gln Gln Ala Lys Lys Ala130 135 140Asp Glu Lys Val Ser Gln Asn Tyr Pro Ile Val Gln Asn Leu Gln Gly145 150 155 160Gln Met Val His Gln Ala Ile Ser Pro Arg Thr Leu Asn Ala Trp Val165 170 175Lys Val Ile Glu Glu Lys Ala Phe Ser Pro Glu Val Ile Pro Met Phe180 185 190Thr Ala Leu Ser Glu Gly Ala Thr Pro Gln Asp Leu Asn Thr Met Leu195 200 205Asn Thr Val Gly Gly His Gln Ala Ala Met Gln Met Leu Lys Asp Thr210 215 220Ile Asn Glu Glu Ala Ala Glu Trp Asp Arg Val His Pro Val His Ala225 230 235 240Gly Pro Ile Ala Pro Gly Gln Met Arg Glu Pro Arg Gly Ser Asp Ile245 250 255Ala Gly Thr Thr Ser Thr Leu Gln Gly Gln Ile Ala Trp Met Thr Ser260 265 270Asn Pro Pro Val Pro Val Gly Glu Ile Tyr Lys Arg Trp Ile Ile Leu275 280 285Gly Leu Asn Lys Ile Val Arg Met Tyr Ser Pro Val Ser Ile Leu Asp290 295 300Ile Lys Gln Gly Pro Lys Glu Pro Phe Arg Asp Tyr Val Asp Arg Phe305 310 315 320Phe Lys Thr Leu Arg Ala Glu Gln Ala Thr Gln Asp Val Lys Asn Trp325 330 335Met Thr Asp Thr Leu Leu Val Gln Asn Ala Asn Pro Asp Cys Lys Thr340 345 350Ile Leu Arg Ala Leu Gly Pro Gly Ala SerLeu Glu Glu Met Met Thr355 360365Ala Cys Gln Gly Val Gly Gly Pro Ser His Lys Ala Arg Val Leu Ala370 375 380Glu Ala Met Ser Gln Ala Asn Gly Thr Ile Leu Met Gln Arg Ser Asn385 390 395 400Phe Lys Gly Ser Lys Arg Ile Val Lys Cys Phe Asn Cys Gly Lys Glu405 410 415Gly His Ile Ala Arg Asn Cys Arg Ala Pro Arg Lys Lys Gly Cys Trp420 425 430Lys Cys Gly Lys Glu Gly His Gln Met Lys Asp Cys Thr Glu Arg Gln435 440 445Ala Asn Phe Leu Gly Lys Ile Trp Pro Ser His Lys Gly Arg Pro Gly450 455 460Asn Phe Leu Gln Ser Arg Pro Glu Pro Thr Ala Pro Pro Ala Glu Ser465 470 475 480Phe Arg Phe Glu Glu Thr Thr Pro Ala Pro Lys Gln Glu Pro Lys Asp485 490 495Arg Glu Pro Leu Thr Ser Leu Lys Ser Leu Phe Gly Ser Asp Pro Leu500 505 510Ser Gln<210>11<211>2901<212>DNA<213>人<400>11atggacgcca tgaagcgcgg cctgtgctgc gtgctgctgc tgtgcggcgc cgtgttcgtg 60agcgcccgcc cccagatcac cctgtggcag cgccccctgg tgtccatccg cgtgggcggc120cagatcaagg aggccctgct ggacgacacc gtgttggagg aggtgaactt gcccggcaag180tggaagccca agatgatcgg cggcatcggc ggcttcatca aggtgaggca gtacgaccag240atccccatcg agatctgcgg caagaaggcc atcggcaccg tgttggtggg ccccaccccc300gtgaacatca tcggcaggaa catgttgacc cagctgggct gcaccctgaa cttccccatc 360agccccatcg agaccatccc cgtgaagctg aagcccggca tggacggccc ccgcgtgaag 420cagtggcccc tgaccgagga gaagatcaag gccctgaccg ccatctgcga cgagatggag 480aaggagggca agatcaccaa gatcggcccc gagaacccct acaacacccc cgtgttcgcc 540atcaagaaga aggacagcac caagtggcgc aagctggtgg acttccgcga gctgaacaag 600cgcacccagg acttctggga ggtgcagctg ggcatccccc accccgccgg cctgaagaag 660aagaagtccg tgaccgtgct ggacgtgggc gacgcctact tctccgtgcc cctgtacgag 720gacttccgca agtacaccgc cttcaccatc cccagcatca acaacgagac ccccggcatc 780cgctaccagt acaacgtgct gccccagggc tggaagggct cccccgccat cttccagtgc 840agcatgacca agatcctgga gcccttccgg gcccagaacc ccgagatcgt gatctaccag 900tacggcgacg acctgtacgt gggctccgac ctggagatcg gccagcaccg cgccaagatc 960gaggagttgc gcgagcacct gctgaagtgg ggcttcacca cccccgacaa gaagcaccag1020aaggagcccc ccttcctgtg gatgggctac gagctgcacc ccgacaagtg gaccgtgcag1080cccatccagc tgcccgagaa ggacagctgg accgtgaacg acatccagaa gctggtgggc1140aagctgaact gggccagcca gatctacccc ggcatcaagg tgcgccagct gtgcaagctg1200ctgcgcggcg ccaaggccct gaccgacatc atccccctga ccgaggaggc cgagctggag1260ctggccgaga accgcgagat cctgaaggag cccgtgcacg gcgcctacta cgacccctcc1320aaggacctga tcgccgagat ccagaagcag ggccaggacc agtggaccta ccagatctac1380caggagccct tcaagaacct gaagaccggc aagtacgcca agatgcgcac cgcccacacc1440aacgacgtga agcagctgac cgaggccgtg cagaagatct ccatggagag catcgtgatc1500tggggcaaga tccccaagtt ccgcctgccc atccagaagg agacctggga gacccgctgg1560accgcctact ggcaggccac ctggatcccc gagtgggagt tcgtgaacac cccccccctg1620gtgaagctgt ggtaccagct ggagaaggac cccatcgccg gcgtggagac cttctacgtg1680gacggcgccg ccaaccgcga gaccaagatg ggcaaggccg gctacgtgac cgaccgcggc1740cgccagaaga tcgtgtccct gaccgagacc accaaccaga agaccgagct gcaggccatc 1800tgcctggcct tgcaggactc cggctccgag gtgaacatcg tgaccgactc ccagtacgcc1860ctgggcatca tccaggccca gcccgacaag agcgagtccg agctggtgaa ccagatcatc1920gagcagctga tcaagaagga gcgcgtgtac ctgtcctggg tgcccgccca caagggcatc1980ggcggcaacg agcaggtgga caagctggtg agcaacggca tccgcaaggt gctgttcctg2040gacggcatcg acaaggccca ggaggagcac gagaagtacc acagcaactg gcgcgccatg2100gccagcgact tcaacctgcc ccccatcgtg gccaaggaga tcgtggccag ctgcgaccag2160tgccagctga agggcgaggc catgcacggc caggtggact gcagccccgg catctggcag2220ctggactgca cccacctgga gggcaagatc atcctggtgg ccgtgcacgt ggccagcggc2280tacatcgagg ccgaggtgat ccccgccgag accggccagg agaccgccta cttcatcctg2340aagctggccg gccgctggcc cgtgaagatc atccacaccg acaacggcag caacttcacc2400agcgccgccg tgaaggccgc ctgctggtgg gccggcatcc agcaggagtt cggcatcccc2460tacaaccccc agagccaggg cgtggtggag tccatgaaca aggagctgaa gaagatcatc2520ggccaggtgc gcgaccaggc cgagcacctg aagaccgccg tgcagatggc cgtgttcatc2580cacaacttca agcgcaaggg cggcatcggc ggctacagcg ccggcgagcg catcatcgac2640atcatcgcca ccgacatcca gaccaaggag ctgcagaagc agatcatcaa gatccagaac2700ttccgcgtgt actaccgcga cagccgcgac cccatctgga agggccccgc caagctgctg2760tggaagggcg agggcgccgt ggtgatccag gacaacagcg acatcaaggt ggtgccccgc2820cgcaaggcca agatcatcaa ggactacggc aagcagatgg ccggcgccga ctgcgtggcc2880agccgccagg acgaggacta g 2901<210>12<211>966<212>PRT<213>人<400>12Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly1 5 10 15Ala Val Phe Val Ser Ala Arg Pro Gln Ile Thr Leu Trp Gln Arg Pro20 25 30Leu Val Ser Ile Arg Val Gly Gly Gln Ile Lys Glu Ala Leu Leu Asp35 40 45Asp Thr Val Leu Glu Glu Val Asn Leu Pro Gly Lys Trp Lys Pro Lys50 55 60Met Ile Gly Gly Ile Gly Gly Phe Ile Lys Val Arg Gln Tyr Asp Gln65 70 75 80Ile Pro Ile Glu Ile Cys Gly Lys Lys Ala Ile Gly Thr Val Leu Val85 90 95Gly Pro Thr Pro Val Asn Ile Ile Gly Arg Asn Met Leu Thr Gln Leu100 105 110Gly Cys Thr Leu Asn Phe Pro Ile Ser Pro Ile Glu Thr Ile Pro Val115 120 125Lys Leu Lys Pro Gly Met Asp Gly Pro Arg Val Lys Gln Trp Pro Leu130 135 140Thr Glu Glu Lys Ile Lys Ala Leu Thr Ala Ile Cys Asp Glu Met Glu145 150 155 160Lys Glu Gly Lys Ile Thr Lys Ile Gly Pro Glu Asn Pro Tyr Asn Thr165 170 175Pro Val Phe Ala Ile Lys Lys Lys Asp Ser Thr Lys Trp Arg Lys Leu180 185 190Val Asp Phe Arg Glu Leu Asn Lys Arg Thr Gln Asp Phe Trp Glu Val195 200 205Gln Leu Gly Ile Pro His Pro Ala Gly Leu Lys Lys Lys Lys Ser Val210 215 220Thr Val Leu Asp Val Gly Asp Ala Tyr Phe Ser Val Pro Leu Tyr Glu225 230 235 240Asp Phe Arg Lys Tyr Thr Ala Phe Thr Ile Pro Ser Ile Asn Asn Glu245 250 255Thr Pro Gly Ile Arg Tyr Gln Tyr Asn Val Leu Pro Gln Gly Trp Lys260 265 270Gly Ser Pro Ala Ile Phe Gln Cys Ser Met Thr Lys Ile Leu Glu Pro275 280 285Phe Arg Ala Gln Asn Pro Glu Ile Val Ile Tyr Gln Tyr Gly Asp Asp290 295 300Leu Tyr Val Gly Ser Asp Leu Glu Ile Gly Gln His Arg Ala Lys Ile305 310 315 320Glu Glu Leu Arg Glu His Leu Leu Lys Trp Gly Phe Thr Thr Pro Asp325 330 335Lys Lys His Gln Lys Glu Pro Pro Phe Leu Trp Met Gly Tyr Glu Leu340 345 350His Pro Asp Lys Trp Thr Val Gln Pro Ile Gln Leu Pro Glu Lys Asp355 360 365Ser Trp Thr Val Asn Asp Ile Gln Lys Leu Val Gly Lys Leu Asn Trp370 375 380Ala Ser Gln Ile Tyr Pro Gly Ile Lys Val Arg Gln Leu Cys Lys Leu385 390 395 400Leu Arg Gly Ala Lys Ala Leu Thr Asp Ile Ile Pro Leu Thr Glu Glu405 410 415Ala Glu Leu Glu Leu Ala Glu Asn Arg Glu Ile Leu Lys Glu Pro Val420 425 430His Gly Ala Tyr Tyr Asp Pro Ser Lys Asp Leu Ile Ala Glu Ile Gln435 440 445Lys Gln Gly Gln Asp Gln Trp Thr Tyr Gln Ile Tyr Gln Glu Pro Phe450 455 460Lys Asn Leu Lys Thr Gly Lys Tyr Ala Lys Met Arg Thr Ala His Thr465 470 475 480Asn Asp Val Lys Gln Leu Thr Glu Ala Val Gln Lys Ile Ser Met Glu485 490 495Ser Ile Val Ile Trp Gly Lys Ile Pro Lys Phe Arg Leu Pro Ile Gln500 505 510Lys Glu Thr Trp Glu Thr Arg Trp Thr Ala Tyr Trp Gln Ala Thr Trp515 520 525Ile Pro Glu Trp Glu Phe Val Asn Thr Pro Pro Leu Val Lys Leu Trp530 535 540Tyr Gln Leu Glu Lys Asp Pro Ile Ala Gly Val Glu Thr Phe Tyr Val545 550 555 560Asp Gly Ala Ala Asn Arg Glu Thr Lys Met Gly Lys Ala Gly Tyr Val565 570 575Thr Asp Arg Gly Arg Gln Lys Ile Val Ser Leu Thr Glu Thr Thr Asn580 585 590Gln Lys Thr Glu Leu Gln Ala Ile Cys Leu Ala Leu Gln Asp Ser Gly595 600 605Ser Glu Val Asn Ile Val Thr Asp Ser Gln Tyr Ala Leu Gly Ile Ile610 615 620Gln Ala Gln Pro Asp Lys Ser Glu Ser Glu Leu Val Asn Gln Ile Ile625 630 635 640Glu Gln Leu Ile Lys Lys Glu Arg Val Tyr Leu Ser Trp Val Pro Ala645 650 655His Lys Gly Ile Gly Gly Asn Glu Gln Val Asp Lys Leu Val Ser Asn660 665 670Gly Ile Arg Lys Val Leu Phe Leu Asp Gly Ile Asp Lys Ala Gln Glu675 680 685Glu His Glu Lys Tyr His Ser Asn Trp Arg Ala Met Ala Ser Asp Phe690 695 700Asn Leu Pro Pro Ile Val Ala Lys Glu Ile Val Ala Ser Cys Asp Gln705 710 715 720Cys Gln Leu Lys Gly Glu Ala Met His Gly Gln Val Asp Cys Ser Pro725 730 735Gly Ile Trp Gln Leu Asp Cys Thr His Leu Glu Gly Lys Ile Ile Leu740 745 750Val Ala Val His Val Ala Ser Gly Tyr Ile Glu Ala Glu Val Ile Pro755 760 765Ala Glu Thr Gly Gln Glu Thr Ala Tyr Phe Ile Leu Lys Leu Ala Gly770 775 780Arg Trp Pro Val Lys Ile Ile His Thr Asp Asn Gly Ser Asn Phe Thr785 790 795 800Ser Ala Ala Val Lys Ala Ala Cys Trp Trp Ala Gly Ile Gln Gln Glu805 810 815Phe Gly Ile Pro Tyr Asn Pro Gln Ser Gln Gly Val Val Glu Ser Met820 825 830Asn Lys Glu Leu Lys Lys Ile Ile Gly Gln Val Arg Asp Gln Ala Glu835 840 845His Leu Lys Thr Ala Val Gln Met Ala Val Phe Ile His Asn Phe Lys850 855 860Arg Lys Gly Gly Ile Gly Gly Tyr Ser Ala Gly Glu Arg Ile Ile Asp865 870 875 880Ile Ile Ala Thr Asp Ile Gln Thr Lys Glu Leu Gln Lys Gln Ile Ile885 890 895Lys Ile Gln Asn Phe Arg Val Tyr Tyr Arg Asp Ser Arg Asp Pro Ile900 905 910Trp Lys Gly Pro Ala Lys Leu Leu Trp Lys Gly Glu Gly Ala Val Val915 920925Ile Gln Asp Asn Ser Asp Ile Lys Val Val Pro Arg Arg Lys Ala Lys930 935 940Ile Ile Lys Asp Tyr Gly Lys Gln Met Ala Gly Ala Asp Cys Val Ala945 950 955 960Ser Arg Gln Asp Glu Asp965<210>13<211>993<212>DNA<213>人<400>13atggacgcca tgaagcgcgg cctgtgctgc gtgctgctgc tgtgcggcgc cgtgttcgtg 60agcgcccgca tgggcggcaa gtggtccaag agcagcatcg tgggctggcc cgccatccgc120gagcgcattc gccgcaccga gcccgccgcc gacggcgtgg gcgccgtgtc tcgcgacctg180gagaagcatg gcgccatcac cagtaacaac accgccgaca ccaacgccga ctgcgcctgg240ctggagaccc aggaggagga ggaggtgggc ttccccgtcc gcccccaggt gcccttgcgc300cccatgacct tcaagggcgc cttggacctc agcttcttcc tgaaggagaa gggcggcctg360gaggggttga tctacagcaa gaagaggcag gagatcctgg acttgtgggt ctaccacacc420cagggctact tccccgactg gcacaactac acccccggcc ccggcgtccg cttccccctg480accttcggct ggtgcttcaa gctggtgccc gtggaccccg gcgaggtgga ggaggccaac540gagggcgaga acaactgctt gctgcacccc gtctgccagc acggcatgga cgacgagcac600cgcgaggtgc tgaagtggaa gttcgacagc cagctggccc accgccacag ggcccgcgag660ctgcacccgg agttctacaa ggactgcatg gagcccgtgg accccaacct ggagccctgg720aaccaccccg gcagccagcc cgagaccgcc tgcaacaact gctactgcaa gcgctgcagc780taccactgcc tggtgtgctt ccagaagaag ggcctgggca tcagctacgg ccgcaagaag840cgccgccagc gccgcagcgc cccccccagc agcgaggacc accagaaccc catcagcaag900cagcccctgc cccgcaccca gggcgacccc accggcagcg aggagagcaa gaagaaggtg960gagagcaaga ccaagaccga ccccttcgac tag 993<210>14<211>330<212>PRT<213>人<400>14Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly1 5 10 15Ala Val Phe Val Ser Ala Arg Met Gly Gly Lys Trp Ser Lys Ser Ser20 25 30Ile Val Gly Trp Pro Ala Ile Arg Glu Arg Ile Arg Arg Thr Glu Pro35 40 45Ala Ala Asp Gly Val Gly Ala Val Ser Arg Asp Leu Glu Lys His Gly50 55 60Ala Ile Thr Ser Asn Asn Thr Ala Asp Thr Asn Ala Asp Cys Ala Trp65 70 75 80Leu Glu Thr Gln Glu Glu Glu Glu Val Gly Phe Pro Val Arg Pro Gln85 90 95Val Pro Leu Arg Pro Met Thr Phe Lys Gly Ala Leu Asp Leu Ser Phe100 105 110Phe Leu Lys Glu Lys Gly Gly Leu Glu Gly Leu Ile Tyr Ser Lys Lys115 120 125Arg Gln Glu Ile Leu Asp Leu Trp Val Tyr His Thr Gln Gly Tyr Phe130 135 140Pro Asp Trp His Asn Tyr Thr Pro Gly Pro Gly Val Arg Phe Pro Leu145 150 155 160Thr Phe Gly Trp Cys Phe Lys Leu Val Pro Val Asp Pro Gly Glu Val
165 170 175Glu Glu Ala Asn Glu Gly Glu Asn Asn Cys Leu Leu His Pro Val Cys180 185 190Gln His Gly Met Asp Asp Glu His Arg Glu Val Leu Lys Trp Lys Phe195 200 205Asp Ser Gln Leu Ala His Arg His Arg Ala Arg Glu Leu His Pro Glu210 215 220Phe Tyr Lys Asp Cys Met Glu Pro Val Asp Pro Asn Leu Glu Pro Trp225 230 235 240Asn His Pro Gly Ser Gln Pro Glu Thr Ala Cys Asn Asn Cys Tyr Cys245 250 255Lys Arg Cys Ser Tyr His Cys Leu Val Cys Phe Gln Lys Lys Gly Leu260 265 270Gly Ile Ser Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Ser Ala Pro275 280 285Pro Ser Ser Glu Asp His Gln Asn Pro Ile Ser Lys Gln Pro Leu Pro290 295 300Arg Thr Gln Gly Asp Pro Thr Gly Ser Glu Glu Ser Lys Lys Lys Val305 310 315 320Glu Ser Lys Thr Lys Thr Asp Pro Phe Asp325 330<210>15<211>8186<212>DNA<213>人<400>15gctgcttcgc gatgtacggg ccagatatac gcgttgacat tgattattga ctagttatta 60atagtaatca attacggggt cattagttca tagcccatat atggagttcc gcgttacata 120acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat 180aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga 240gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc 300ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt 360atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat 420gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag 480tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc 540aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga 600ggtctatata agcagagctc tctggctaac tagagaaccc actgcttact ggcttatcga 660aattaatacg actcactata gggagaccca agctggctag ccgccaccat ggatgcaatg 720aagagggggc tctgctgtgt gctgctgctg tgtggagcag tcttcgttag cgccgccgag 780aacttgtggg tgaccgtgta ctacggcgtg cccgtgtgga aggaggccaa gaccaccctg 840ttctgcgcct ccgacgccaa ggcctacgag aaggaggtgc acaacgtgtg ggccacccac 900gcctgcgtgc ccaccgaccc caacccccag gagatggtgt tggagaacgt gaccgagaac 960ttcaacatgt ggaagaacga catggtgaac cagatgcacg aggacgtcat cagcttgtgg1020gaccagagcc tgaagccctg cgtgaagttg acccccctgt gcgtgacctt ggagtgcagg1080aacgtgagca gcaacggcac ctacaacgag acctacaacg agatcaagaa ctgctccttc1140aacgccacca ccgtgttgag ggacaggaag cagaccgtgt acgccctgtt ctacaggctg1200gacatcgtgc ccctgaacaa gaagaactcc agcgagaact ccagcgagta ctacaggttg1260atcaactgca acacctccgc catcacccag gcctgcccca aggtgacctt cgaccccatc1320cccatccact actgcacccc cgccggctac gccatcctga agtgcaacga caagaccttc1380aacggcaccg gcccctgcca caacgtgagc accgtgcagt gcacccacgg catcaagccc1440gtggtgtcca cccagctgct gttgaacggc agcctggccg agagggagat catcatcagg1500tccgagaacc tgaccaacaa cgtgaagacc atcatcgtgc acctgaacca gtccgtggag1560atcgtgtgca ccaggcccaa caacaacacc aggaagagca tcaggatcgg ccccggccag1620accttctacg ccaccggcga catcatcggc gacatcaggc aggcccactg caacatcagc1680aaggacaagt ggaaggagac cttgcagagg gtgggcaaga agttggccga gcacttcccc1740aacaagacca tcgagttcgc ctcctcctcc ggcggcgacc tggagatcac cacccacagc1800ttcaactgca ggggcgagtt cttctactgc aacacctcca gcctgttcaa cggcacctac1860atgcccaacg gcaccgaggg caactccagc tccatcatca ccatcccctg caggatcaag1920cagatcatca acatgtggca ggaggtgggc cgcgccatgt acgccccccc catcgagggc1980aacatcacct gcaagtccaa catcaccggc ctgctgttgg tgcgcgacgg cggcaaggag2040accaacgaca ccgagacctt caggcccggc ggcggcgaca tgagggacaa ctggaggagc2100gagttgtaca agtacaaggt ggtggagatc aagcccttgg gcatcgcccc caccgccgcc2160aagaggaggg tggtggagag ggagaagagg gccgtgggca tcggcgccgt gttcctgggc2220ttcctgggcg ccgccggcag caccatgggc gccgccagca tcaccctgac cgtgcaggcc2280cgccagctgc tgagcggcat cgtgcagcag cagagcaacc tgctgcgcgc catcgaggcc2340cagcagcacc tgctgcagct gaccgtgtgg ggcatcaagc agctgcagac ccgcgttctg2400gccatcgagc gctacctgaa ggaccagcag ctgctgggca tctggggctg cagcggcaag2460ctgatctgca ccaccgccgt gcactggaac agcagctgga gcaaccgcag ccaggaggag2520atctggaaca acatgacctg gatgcagtgg gaccgcgaga tcagcaacta caccaacacc2580atctaccgcc tgctggagga cagccagaac cagcaggagc gcaacgagaa ggacctgctg2640gccctggaca actggaagaa cctgtggagc tggttcgaca tcaccaactg gctgtggtac2700atccgcatct tcatcatgat cgtgggcggc ctgatcggcc tgcgcatcat cttcgccgtg2760ctgagcatcg tgaaccgcgt gcgccagggc tacagccccc tgagcttcca gaccctgacc2820cccaaccccg gcggccccga ccgcctgggc cgcatcgagg aggagggcgg cgagcaggac2880aagaaccgca gcatccgcct ggtgaacggc ttcctggccc tggcctggga cgacctgcgc2940aacctgtgcc gcttcagcta ccacctgctg cgcgacctgc tgctgatcgt ggcccgcatc3000gtggagctgc tgggccgccg cggctgggag gccctgcgct actggtggaa cctgctgaag3060tactgggtgc aggagctgaa gaacagcgcc gtgagcctgc tgaacgccac cgccatcgcc3120gtggccgagg gcaccgaccg cgtgatcgag gtggtgcagg gcgcctaccg cgccatcctg3180cacatccccc gccgcatccg ccagggcttc gaggccgccc tgcagtaaga attccgtgag3240gctccggtgc ccgtcagtgg gcagagcgca catcgcccac agtccccgag aagttggggg3300gaggggtcgg cgattgaacc ggtgcctaga gaaggtggcg cggggtaaac tgggaaagtg3360atgtcgtgta ctggctccgc ctttttcccg agggtggggg agaaccgtat ataagtgcag3420tagtcgccgt gaacgttctt tttcgcaacg ggtttgccgc cagaacacag gtaagtgccg3480tgtgtggttc ccgcgggcct ggcctcttta cgggttatgg cccttgcgtg ccttgaatta3540cttccacctg gctgcagtac gtgattcttg atcccgagct tcgggttgga agtgggtggg3600agagttcgag gccttgcgct taaggagccc cttcgcctcg tgcttgagtt gaggcctggc3660ctgggcgctg gggccgccgc gtgcgaatct ggtggcacct tcgcgcctgt ctcgctgctt3720tcgataagtc tctagccatt taaaattttt gatgacctgc tgcgacgctt tttttctggc3780aagatagtct tgtaaatgcg ggccaagatc tgcacactgg tatttcggtt tttggggccg3840cgggcggcga cggggcccgt gcgtcccagc gcacatgttc ggcgaggcgg ggcctgcgag3900cgcggccacc gagaatcgga cgggggtagt ctcaagctgg ccggcctgct ctggtgcctg3960gcctcgcgcc gccgtgtatc gccccgccct gggcggcaag gctggcccgg tcggcaccag4020ttgcgtgagc ggaaagatgg ccgcttcccg gccctgctgc agggagctca aaatggagga4080cgcggcgctc gggagagcgg gcgggtgagt cacccacaca aaggaaaagg gcctttccgt4140cctcagccgt cgcttcatgt gactccacgg agtaccgggc gccgtccagg cacctcgatt4200agttctcgag cttttggagt acgtcgtctt taggttgggg ggaggggttt tatgcgatgg4260agtttcccca cactgagtgg gtggagactg aagttaggcc agcttggcac ttgatgtaat4320tctccttgga atttgccctt tttgagtttg gatcttggtt cattctcaag cctcagacag4380tggttcaaag tttttttctt ccatttcagg tgtcgtgaag cggccgccgc caccatggac4440gccatgaagc gcggcctgtg ctgcgtgctg ctgctgtgcg gcgccgtgtt cgtgagcgcc4500cgcatgggcg cccgcgccag catcctgcgc ggcggcaagc tgggcaagtg ggagaagatc4560cgcctgcgcc ccggcgacaa gaagcactac atgctgaagc acctggtgtg ggccagccgc4620gagctggagc gcttcgccct gaaccccggc ctgctggaga ccagcgaggg ctgcaagcag4680atcatcaagc agctgcagcc cgccctgcag accggcaccg aggagctgcg cagcctgttc4740aacaccgtgg ccaccctgta ctgcgtgcac gagggcatcg agatccgcga caccaaggag4800gccctggaca agatcgagga ggagcagaac aagatccagc agaagaccca gcaggccaag4860aaggccgacg agaaggtgag ccagaactac cccatcgtgc agaacctgca gggccagatg4920gtgcaccagg ccatctcccc caggaccttg aacgcctggg tgaaggtgat cgaggagaag4980gccttcagcc ccgaggtgat ccccatgttc accgccttgt ccgagggcgc caccccccag5040gacttgaaca ccatgttgaa caccgtgggc ggccaccagg ccgccatgca gatgttgaag5100gacaccatca acgaggaggc cgccgagtgg gacagggtgc accccgtgca cgccggcccc5160attgcccccg gccagatgag ggagcccagg ggcagcgaca tcgccggcac caccagcacc5220ctgcagggcc agatcgcctg gatgaccagc aacccccccg tgcccgtggg cgagatctac5280aagaggtgga tcatcctggg cttgaacaag atcgtgagga tgtacagccc cgtgagcatc5340ttggacatca agcagggccc caaggagccc ttcagggact acgtggaccg cttcttcaag5400accttgaggg ccgagcaggc cacccaggac gtgaagaact ggatgaccga caccttgttg5460gtgcagaacg ccaaccccga ctgcaagacc atcttgaggg ccttgggccc cggcgcctcc5520ttggaggaga tgatgaccgc ctgccagggc gtgggcggcc ccagccacaa ggccagggtg5580ttggccgagg ccatgagcca ggccaacggc accatcctga tgcagaggag caacttcaag5640ggctccaaga ggatcgtgaa gtgcttcaac tgcggcaagg agggccacat cgccaggaac5700tgcagggccc ccaggaagaa gggctgctgg aagtgcggca aggagggcca ccagatgaag5760gactgcaccg agaggcaggc caacttcttg ggcaagatct ggccctccca caagggcagg5820cccggcaact tcctgcagag caggcccgag cccaccgccc cccccgccga gagcttcagg5880ttcgaggaga ccacccccgc ccccaagcag gagcccaagg acagggagcc cttgacctcc5940ctgaagtccc tgttcggcag cgaccccttg tcccagtaat ctagagggcc cgtttaaacc6000cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc6060gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa6120attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac6180agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg6240gcttctactg ggcggtttta tggacagcaa gcgaaccgga attgccagct ggggcgccct6300ctggtaaggt tgggaagccc tgcaaagtaa actggatggc tttcttgccg ccaaggatct6360gatggcgcag gggatcaagc tctgatcaag agacaggatg aggatcgttt cgcatgattg6420aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg6480actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg6540ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaagacg6600aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg6660ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc6720tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc6780tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc6840gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc6900aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gagcatgccc gacggcgagg6960atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct7020tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt7080tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc7140tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt7200tcttctgaat tattaacgct tacaatttcc tgatgcggta ttttctcctt acgcatctgt7260gcggtatttc acaccgcatc aggtggcact tttcggggaa atgtgcgcgg aacccctatt7320tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa7380atgcttcaat aatagcacgt gctaaaactt catttttaat ttaaaaggat ctaggtgaag7440atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg7500tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc7560tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag7620ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt7680cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac7740ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc7800gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt7860tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt7920gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc7980ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt8040tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca8100ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt8160tgctggcctt ttgctcacat gttctt 8186<210>16<211>8017<212>DNA<213>人<400>16gctgcttcgc gatgtacggg ccagatatac gcgttgacat tgattattga ctagttatta 60atagtaatca attacggggt cattagttca tagcccatat atggagttcc gcgttacata 120acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat 180aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga 240gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc 300ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt 360atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat 420gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag 480tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc 540aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga 600ggtctatata agcagagctc tctggctaac tagagaaccc actgcttact ggcttatcga 660aattaatacg actcactata gggagaccca agctggctag cgccgccacc atggacgcca 720tgaagcgcgg cctgtgctgc gtgctgctgc tgtgcggcgc cgtgttcgtg agcgcccgcc 780cccagatcac cctgtggcag cgccccctgg tgtccatccg cgtggggggc cagatcaagg 840aggccctgct ggacgacacc gtgttggagg aggtgaactt gcccggcaag tggaagccca 900agatgatcgg cggcatcggc ggcttcatca aggtgaggca gtacgaccag atccccatcg 960agatctgcgg caagaaggcc atcggcaccg tgttggtggg ccccaccccc gtgaacatca1020tcggcaggaa catgttgacc cagctgggct gcaccctgaa cttccccatc agccccatcg1080agaccatccc cgtgaagctg aagcccggca tggacggccc ccgcgtgaag cagtggcccc1140tgaccgagga gaagatcaag gccctgaccg ccatctgcga cgagatggag aaggagggca1200agatcaccaa gatcggcccc gagaacccct acaacacccc cgtgttcgcc atcaagaaga1260aggacagcac caagtggcgc aagctggtgg acttccgcga gctgaacaag cgcacccagg1320acttctggga ggtgcagctg ggcatccccc accccgccgg cctgaagaag aagaagtccg1380tgaccgtgct ggacgtgggc gacgcctact tctccgtgcc cctgtacgag gacttccgca1440agtacaccgc cttcaccatc cccagcatca acaacgagac ccccggcatc cgctaccagt1500acaacgtgct gccccagggc tggaagggct cccccgccat cttccagtgc agcatgacca1560agatcctgga gcccttccgg gcccagaacc ccgagatcgt gatctaccag tacggcgacg1620acctgtacgt gggctccgac ctggagatcg gccagcaccg cgccaagatc gaggagttgc1680gcgagcacct gctgaagtgg ggcttcacca cccccgacaa gaagcaccag aaggagcccc1740ccttcctgtg gatgggctac gagctgcacc ccgacaagtg gaccgtgcag cccatccagc1800tgcccgagaa ggacagctgg accgtgaacg acatccagaa gctggtgggc aagctgaact1860gggccagcca gatctacccc ggcatcaagg tgcgccagct gtgcaagctg ctgcgcggcg1920ccaaggccct gaccgacatc atccccctga ccgaggaggc cgagctggag ctggccgaga1980accgcgagat cctgaaggag cccgtgcacg gcgcctacta cgacccctcc aaggacctga2040tcgccgagat ccagaagcag ggccaggacc agtggaccta ccagatctac caggagccct2100tcaagaacct gaagaccggc aagtacgcca agatgcgcac cgcccacacc aacgacgtga2160agcagctgac cgaggccgtg cagaagatct ccatggagag catcgtgatc tggggcaaga2220tccccaagtt ccgcctgccc atccagaagg agacctggga gacccgctgg accgcctact2280ggcaggccac ctggatcccc gagtgggagt tcgtgaacac cccccccctg gtgaagctgt2340ggtaccagct ggagaaggac cccatcgccg gcgtggagac cttctacgtg gacggcgccg2400ccaaccgcga gaccaagatg ggcaaggccg gctacgtgac cgaccgcggc cgccagaaga2460tcgtgtccct gaccgagacc accaaccaga agaccgagct gcaggccatc tgcctggcct2520tgcaggactc cggctccgag gtgaacatcg tgaccgactc ccagtacgcc ctgggcatca2580tccaggccca gcccgacaag agcgagtccg agctggtgaa ccagatcatc gagcagctga2640tcaagaagga gcgcgtgtac ctgtcctggg tgcccgccca caagggcatc ggcggcaacg2700agcaggtgga caagctggtg agcaacggca tccgcaaggt gctgttcctg gacggcatcg2760acaaggccca ggaggagcac gagaagtacc acagcaactg gcgcgccatg gccagcgact2820tcaacctgcc ccccatcgtg gccaaggaga tcgtggccag ctgcgaccag tgccagctga2880agggcgaggc catgcacggc caggtggact gcagccccgg catctggcag ctggactgca2940cccacctgga gggcaagatc atcctggtgg ccgtgcacgt ggccagcggc tacatcgagg3000ccgaggtgat ccccgccgag accggccagg agaccgccta cttcatcctg aagctggccg3060gccgctggcc cgtgaagatc atccacaccg acaacggcag caacttcacc agcgccgccg3120tgaaggccgc ctgctggtgg gccggcatcc agcaggagtt cggcatcccc tacaaccccc3180agagccaggg cgtggtggag tccatgaaca aggagctgaa gaagatcatc ggccaggtgc3240gcgaccaggc cgagcacctg aagaccgccg tgcagatggc cgtgttcatc cacaacttca3300agcgcaaggg cggcatcggc ggctacagcg ccggcgagcg catcatcgac atcatcgcca3360ccgacatcca gaccaaggag ctgcagaagc agatcatcaa gatccagaac ttccgcgtgt3420actaccgcga cagccgcgac cccatctgga agggccccgc caagctgctg tggaagggcg3480agggcgccgt ggtgatccag gacaacagcg acatcaaggt ggtgccccgc cgcaaggcca3540agatcatcaa ggactacggc aagcagatgg ccggcgccga ctgcgtggcc agccgccagg3600acgaggacta ggaattccgt gaggctccgg tgcccgtcag tgggcagagc gcacatcgcc3660cacagtcccc gagaagttgg ggggaggggt cggcaattga accggtgcct agagaaggtg3720gcgcggggta aactgggaaa gtgatgtcgt gtactggctc cgcctttttc ccgagggtgg3780gggagaaccg tatataagtg cagtagtcgc cgtgaacgtt ctttttcgca acgggtttgc3840cgccagaaca caggtaagtg ccgtgtgtgg ttcccgcggg cctggcctct ttacgggtta3900tggcccttgc gtgccttgaa ttacttccac ctggctgcag tacgtgattc ttgatcccga3960gcttcgggtt ggaagtgggt gggagagttc gaggccttgc gcttaaggag ccccttcgcc4020tcgtgcttga gttgaggcct ggcctgggcg ctggggccgc cgcgtgcgaa tctggtggca4080ccttcgcgcc tgtctcgctg ctttcgataa gtctctagcc atttaaaatt tttgatgacc4140tgctgcgacg ctttttttct ggcaagatag tcttgtaaat gcgggccaag atctgcacac4200tggtatttcg gtttttgggg ccgcgggcgg cgacggggcc cgtgcgtccc agcgcacatg4260ttcggcgagg cggggcctgc gagcgcggcc accgagaatc ggacgggggt agtctcaagc4320tggccggcct gctctggtgc ctggcctcgc gccgccgtgt atcgccccgc cctgggcggc4380aaggctggcc cggtcggcac cagttgcgtg agcggaaaga tggccgcttc ccggccctgc4440tgcagggagc tcaaaatgga ggacgcggcg ctcgggagag cgggcgggtg agtcacccac4500acaaaggaaa agggcctttc cgtcctcagc cgtcgcttca tgtgactcca cggagtaccg4560ggcgccgtcc aggcacctcg attagttctc gagcttttgg agtacgtcgt ctttaggttg4620gggggagggg ttttatgcga tggagtttcc ccacactgag tgggtggaga ctgaagttag4680gccagcttgg cacttgatgt aattctcctt ggaatttgcc ctttttgagt ttggatcttg4740gttcattctc aagcctcaga cagtggttca aagttttttt cttccatttc aggtgtcgtg4800aagcggccgc cgccaccatg gacgccatga agcgcggcct gtgctgcgtg ctgctgctgt4860gcggcgccgt gttcgtgagc gcccgcatgg gcggcaagtg gtccaagagc agcatcgtag4920gctggcccgc catccgcgag cgcatccgcc gcaccgagcc cgccgccgac ggcgtgggcg4980ccgtgtctcg cgacctggag aagcatggcg ccatcaccag taacaacacc gccgacacca5040acgccgactg cgcctggctg gagacccagg aggaggagga ggtgggcttc cccgtccgcc5100cccaggtgcc cttgcgcccc atgaccttca agggcgcctt ggacctcagc ttcttcctga5160aggagaaggg cggcctggag gggttgatct acagccagaa gaggcaggag atcctggact5220tgtgggtcta ccacacccag ggctacttcc ccgactggca caactacacc cccggccccg5280gcgtccgctt ccccctgacc ttcggctggt gcttcaagct ggtgcccgtg gaccccggcg5340aggtggagga ggccaacgag ggcgagaaca actgcttgct gcaccccgtc tgccagcacg5400gcatggacga cgagcaccgc gaggtgctga agtggaagtt cgacagccag ctggcccacc5460gccacagggc ccgcgagctg cacccggagt tctacaagga ctgcatggag cccgtggacc5520ccaacctgga gccctggaac caccccggca gccagcccga gaccgcctgc aacaactgct5580actacaagcg ctgcagctac cactgcctgg tgtgcttcca gaagaagggc ctgggcatca5640gctacggccg caagaagcgc cgccagcgcc gtagcgcccc ccccagcagc gaggaccacc5700agaaccccat cagcaagcag cccctgcccc gcacccaggg cgaccccacc ggcagcgagg5760agagcaagaa gaaggtggag agcaagacca agaccgaccc cttcgactag tctagagggc5820ccgtttaaac ccgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt5880gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat5940aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg6000tggggcagga cagcaagggg gaggattggg aagacaatag caggcatgct ggggatgcgg6060tgggctctat ggcttctact gggcggtttt atggacagca agcgaaccgg aattgccagc6120tggggcgccc tctggtaagg ttgggaagcc ctgcaaagta aactggatgg ctttcttgcc6180gccaaggatc tgatggcgca ggggatcaag ctctgatcaa gagacaggat gaggatcgtt6240tcgcatgatt gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct6300attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct6360gtcagcgcag gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga6420actgcaagac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc6480tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg6540gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc6600aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca6660tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga6720cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg cgagcatgcc6780cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga6840aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca6900ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg6960cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct7020tcttgacgag ttcttctgaa ttattaacgc ttacaatttc ctgatgcggt attttctcct7080tacgcatctg tgcggtattt cacaccgcat caggtggcac ttttcgggga aatgtgcgcg7140gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat7200aaccctgata aatgcttcaa taatagcacg tgctaaaact tcatttttaa tttaaaagga7260tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt7320tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc7380tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc7440cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac7500caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac7560cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt7620cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct7680gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat7740acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt7800atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg7860cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt7920gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt7980tcctggcctt ttgctggcct tttgctcaca tgttctt 8017<210>17<211>2442<212>DNA<213>人<400>17atgagagtga cggggatcag gaagaactat cagcatttat ggagatgggg caccatgctc 60cttgggatgt tgatgatctg tagtgctgca gaaaacttgt gggtcacagt ctattatggg 120gtacctgtgt ggaaagaagc caaaactact ctattctgtg cgtcagatgc taaagcatat 180gagaaagaag tgcataatgt ctgggctaca catgcctgtg tacccacaga ccccaaccca 240caagaaatgg ttttggaaaa tgtaacagaa aattttaaca tgtggaaaaa tgacatggtg 300aatcagatgc atgaggatgt aatcagttta tgggatcaaa gcctaaagcc atgtgtaaag 360ttgaccccac tctgtgtcac tttagaatgt agaaatgtta gcagtaatgg tacctacaat 420gagacctaca atgagatcaa aaattgctct ttcggggccg ggtattatag attaataaat 480tgtaatacct cagccataac acaagcctgt ccaaaggtca cttttgatcc aattcctata 540cactattgca ctccagctgg ttatgcgatt ctaaagtgta atgataagac attcaatgga 600acaggaccat gccataatgt tagtacagta caatgtacac atggaattaa gccagtggta 660tcaactcaac tactgttaaa tggtagccta gcagaaagag agataataat tagatctgaa 720aatctgacaa acaatgtcaa aacaataata gtacatctta atcaatctgt agaaattgta 780tgtacaagac ccaacaataa tacaagaaaa agtataagga taggaccagg acaaacattc 840tatgcaacag gagacataat aggagatata agacaagcac attgtaacat tagtaaagat 900aaatggaatg aaactttaca aagggtaggt aaaaaattag cagaacactt ccctaataaa 960acaatagaat ttgcatcatc ctcaggaggg gacctagaaa ttacaacaca tagctttaat1020tgtagaggag aatttttcta ttgtaataca tcaatcctgt ttaatggtac atacatgcct1080aatggtacag aaggtaattc aagctcaatc atcacaatcc catgcagaat aaagcaaatt1140ataaacatgt ggcaggaggt aggacgagca atgtatgccc ctcccattga gggaaacata1200acatgtaaat caaatatcac aggactacta ttggtacgtg atggaggaaa agagacaaat1260gatacagaga cattcagacc tggaggagga gatatgaggg acaattggag aagtgaatta1320tataaatata aagtggtaga aattaagcca ttgggaatag cacccactgc agcaaaaagg1380agagtggtgg agagagaaaa aagagcagtg ggaataggag ctgtgttcct tgggttcttg1440ggagcagcag gaagcactat gggcgcggcg tcaataacgc tgacggtaca gtccagacaa1500ttgttgtctg gtatagtgca acagcaaagc aatttgctga gggctataga ggcgcaacag1560catctgttgc aactcacggt ctggggcatt aagcagctcc agacaagagt cctggctata1620gaaagatacc taaaggatca acagctccta gggatttggg gctgctctgg aaaactcatc1680tgcactactg ctgtacattg gaactccagt tggagtaaca gatctcaaga agagatttgg1740aataacatga cttggatgca gtgggataga gaaattagta attacacaaa cacaatatac1800aggttgcttg aagactcgca aaaccagcag gaaagaaatg aaaaagattt actagcattg1860gacaattgga aaaatctatg gagttggttt gacataacaa attggctgtg gtatataaga1920atattcataa tgatagtagg aggcttgata ggtttaagaa taatttttgc tgtgctctct1980atagtgaata gagttaggca gggatactca cctttgtcgt ttcagaccct taccccgaac2040ccagggggac ccgacaggct cggaagaatc gaagaagaag gtggagagca agacaaaaac2100agatccattc gattagtgaa cggattctta gcacttgcct gggacgacct gcggaacctg2160tgccgcttca gctaccacct cttgagagac ttactcttga ttgtagcaag gattgtggaa2220cttctgggac gcagggggtg ggaagccctc agatattggt ggaatctcct gaagtattgg2280gttcaggaac taaagaatag tgctgttagt ttgctcaatg ccacagctat agcagtagct2340gaggggacag atagggttat agaagtagta caaggagctt atagagctat tctccacata2400cctagaagaa taagacaggg ctttgaagca gctttgcaat aa 2442<210>18<211>813<212>PRT<213>人<400>18Met Arg Val Thr Gly Ile Arg Lys Asn Tyr Gln His Leu Trp Arg Trp1 5 10 15Gly Thr Met Leu Leu Gly Met Leu Met Ile Cys Ser Ala Ala Glu Asn20 25 30Leu Trp Val Thr Val Tyr Tyr Gly Val Pro Val Trp Lys Glu Ala Lys35 40 45Thr Thr Leu Phe Cys Ala Ser Asp Ala Lys Ala Tyr Glu Lys Glu Val50 55 60His Asn Val Trp Ala Thr His Ala Cys Val Pro Thr Asp Pro Asn Pro65 70 75 80Gln Glu Met Val Leu Glu Asn Val Thr Glu Asn Phe Asn Met Trp Lys85 90 95Asn Asp Met Val Asn Gln Met His Glu Asp Val Ile Ser Leu Trp Asp100 105 110Gln Ser Leu Lys Pro Cys Val Lys Leu Thr Pro Leu Cys Val Thr Leu115 120 125Glu Cys Arg Asn Val Ser Ser Asn Gly Thr Tyr Asn Glu Thr Tyr Asn130 135 140Glu Ile Lys Asn Cys Ser Phe Gly Ala Gly Tyr Tyr Arg Leu Ile Asn145 150 155 160Cys Asn Thr Ser Ala Ile Thr Gln Ala Cys Pro Lys Val Thr Phe Asp165 170 175Pro Ile Pro Ile His Tyr Cys Thr Pro Ala Gly Tyr Ala Ile Leu Lys180 185 190Cys Asn Asp Lys Thr Phe Asn Gly Thr Gly Pro Cys His Asn Val Ser195 200 205Thr Val Gln Cys Thr His Gly Ile Lys Pro Val Val Ser Thr Gln Leu210 215 220Leu Leu Asn Gly Ser Leu Ala Glu Arg Glu Ile Ile Ile Arg Ser Glu225 230 235 240Asn Leu Thr Asn Asn Val Lys Thr Ile Ile Val His Leu Asn Gln Ser245 250 255Val Glu Ile Val Cys Thr Arg Pro Asn Asn Asn Thr Arg Lys Ser Ile260 265 270Arg Ile Gly Pro GlyGln Thr Phe Tyr Ala Thr Gly Asp Ile Ile Gly275 280 285Asp Ile Arg Gln Ala His Cys Asn Ile Ser Lys Asp Lys Trp Asn Glu290 295 300Thr Leu Gln Arg Val Gly Lys Lys Leu Ala Glu His Phe Pro Asn Lys305 310 315 320Thr Ile Glu Phe Ala Ser Ser Ser Gly Gly Asp Leu Glu Ile Thr Thr325 330 335His Ser Phe Asn Cys Arg Gly Glu Phe Phe Tyr Cys Asn Thr Ser Ile340 345 350Leu Phe Asn Gly Thr Tyr Met Pro Asn Gly Thr Glu Gly Asn Ser Ser355 360 365Ser Ile Ile Thr Ile Pro Cys Arg Ile Lys Gln Ile Ile Asn Met Trp370 375 380Gln Glu Val Gly Arg Ala Met Tyr Ala Pro Pro Ile Glu Gly Asn Ile385 390 395 400Thr Cys Lys Ser Asn Ile Thr Gly Leu Leu Leu Val Arg Asp Gly Gly405 4l0 415Lys Glu Thr Asn Asp Thr Glu Thr Phe Arg Pro Gly Gly Gly Asp Met420 425 430Arg Asp Asn Trp Arg Ser Glu Leu Tyr Lys Tyr Lys Val Val Glu Ile435 440 445Lys Pro Leu Gly Ile Ala Pro Thr Ala Ala Lys Arg Arg Val Val Glu450 455 460Arg Glu Lys Arg Ala Val Gly Ile Gly Ala Val Phe Leu Gly Phe Leu465 470 475 480Gly Ala Ala Gly Ser Thr Met Gly Ala Ala Ser Ile Thr Leu Thr Val485 490 495Gln Ser Arg Gln Leu Leu Ser Gly Ile Val Gln Gln Gln Ser Asn Leu500 505 510Leu Arg Ala Ile Glu Ala Gln Gln His Leu Leu Gln Leu Thr Val Trp515 520 525Gly Ile Lys Gln Leu Gln Thr Arg Val Leu Ala Ile Glu Arg Tyr Leu530 535 540Lys Asp Gln Gln Leu Leu Gly Ile Trp Gly Cys Ser Gly Lys Leu Ile545 550 555 560Cys Thr Thr Ala Val His Trp Asn Ser Ser Trp Ser Asn Arg Ser Gln565 570 575Glu Glu Ile Trp Asn Asn Met Thr Trp Met Gln Trp Asp Arg Glu Ile580 585 590Ser Asn Tyr Thr Asn Thr Ile Tyr Arg Leu Leu Glu Asp Ser Gln Asn595 600 605Gln Gln Glu Arg Asn Glu Lys Asp Leu Leu Ala Leu Asp Asn Trp Lys610 615 620Asn Leu Trp Ser Trp Phe Asp Ile Thr Asn Trp Leu Trp Tyr Ile Arg625 630 635 640Ile Phe Ile Met Ile Val Gly Gly Leu Ile Gly Leu Arg Ile Ile Phe645 650 655Ala Val Leu Ser Ile Val Asn Arg Val Arg Gln Gly Tyr Ser Pro Leu660 665 670Ser Phe Gln Thr Leu Thr Pro Asn Pro Gly Gly Pro Asp Arg Leu Gly675 680 685Arg Ile Glu Glu Glu Gly Gly Glu Gln Asp Lys Asn Arg Ser Ile Arg690 695 700Leu Val Asn Gly Phe Leu Ala Leu Ala Trp Asp Asp Leu Arg Asn Leu705 710 715 720Cys Arg Phe Ser Tyr His Leu Leu Arg Asp Leu Leu Leu Ile Val Ala725 730 735Arg Ile Val Glu Leu Leu Gly Arg Arg Gly Trp Glu Ala Leu Arg Tyr740 745 750Trp Trp Asn Leu Leu Lys Tyr Trp Val Gln Glu Leu Lys Asn Ser Ala755 760 765Val Ser Leu Leu Asn Ala Thr Ala Ile Ala Val Ala Glu Gly Thr Asp770 775 780Arg Val Ile Glu Val Val Gln Gly Ala Tyr Arg Ala Ile Leu His Ile785 790 795 800Pro Arg Arg Ile Arg Gln Gly Phe Glu Ala Ala Leu Gln805 810<210>19<211>4404<212>DNA<213>人<400>19gccgccacca tggacgccat gaagcgcggc ctgtgctgcg tgctgctgct gtgcggcgcc 60gtgttcgtga gcgcccgcat gggtgcgaga gcgtcaatat taagaggggg aaaattagat 120aaatgggaaa aaattaggtt aaggccaggg ggaaagaaac actatatgct aaaacacata 180gtatgggcaa gcagagagct ggaaagattt gcacttaacc ctggcctttt agagacatca 240gaaggctgta aacaaataat aaaacagcta caaccagctc ttcagacagg aacagaggaa 300cttagatcat tattcaacac agtagcaact ctctattgtg tacatgcagg gatagaagta 360cgagacacca aagaagcctt agacaagata gaggaagaac aaaacaaaat gcagcaaaaa 420acacagcagg caaaagaggc tgacgggaag gtcagtcaaa attatcctat agtgcagaat 480ctccaagggc aaatggtgca ccaggccata tcacctagaa ctttgaatgc atgggtaaaa 540gtaatagagg aaaaggcttt tagcccagag gtaataccca tgtttacagc attatcagaa 600ggagccaccc cacaagattt aaacaccatg ttaaatacag tagggggaca tcaggcagcc 660atgcaaatgt taaaagatac tatcaatgaa gaggctgcag aatgggatag agtacatcca 720gtacatgcag ggcctattgc accaggccaa atgagagaac caaggggaag tgacatagca 780ggaactacta gtacccttca gggacaaata gcatggatga cgagtaaccc acctgttcca 840gtgggagaaa tctataaaag atggataatt ctggggttaa ataaaatagt aagaatgtat 900agccctgtga gcattttgga cataaaacaa gggccaaagg aaccctttag agactatgta 960gaccggttct ttaaaacttt aagagctgaa caagctacac aagatgtaaa aaattggatg1020acagacacct tgttggtcca aaatgcgaac ccagattgta agaccatttt aagagcatta1080ggaccagggg cttcattaga agagatgatg acagcatgtc agggagtggg aggacctaac1140cacaaagcaa gagtgttggc tgaggcaatg agccaagcaa acggtaccat actgatgcag1200agaagcaatt ttaaaggctc taaaagaatt gttaaatgtt tcaactgtgg caaggaaggg1260cacatagcca gaaattgcag ggcccctagg aaaaaaggct gttggaaatg tggaaaggaa1320ggacaccaaa tgaaagactg cactgaaagg caggctaatt ttttagggaa aatttggcct1380ccccacaagg ggaggccagg gaatttcctc cagagcagac cagagccaac agccccacca1440gcagagagct tcgggttcga ggagacaacc ccagctccga agcaggagcc gaaagacagg1500gaacccttaa cttccctcaa atcactcttt ggcagcgacc ccttgtctca acctcaaatc1560actctttggc agcgacccct tgtctcaata agagtagggg gccagataaa agaggctctc1620ttagatgata cagtattaga agaagtaaat ttgccaggaa aatggaaacc aaaaatgata1680ggaggaattg gaggttttat caaagtaaga caatatgatc aaatacctat agaaatttgt1740ggaaaaaagg ctataggtac agtattagtg ggacccacac ctgtcaacat aattggaaga1800aatatgttga ctcaacttgg atgcacacta aattttccaa tcagtcccat tgaaactata1860ccagtaaaat taaagccagg aatggatggc ccaagggtta aacaatggcc attgacagaa1920gagaaaataa aagcattaac agcaatttgt gatgaaatgg agaaggaagg aaaaattaca1980aaaattgggc ctgaaaatcc atataacact ccagtatttg ccataaaaaa gaaggacagt2040actaagtgga gaaaattagt agatttcagg gaactcaata aaagaactca agatttttgg2100gaagttcaat taggaatacc acacccagca gggttaaaaa agaaaaaatc agtgacagta2160ctggatgtgg gggatgcata tttttcagtt cctttatatg aagacttcag aaaatatact2220gcattcacca tacctagtat aaacaatgaa acaccaggga tcaggtatca atataatgtg2280cttccacagg gatggaaggg atcaccagca atattccagt gtagcatggc aaaaatctta2340gagcccttta gggcacaaaa tccagaaata gtcatctatc aatatggcga tgacttgtat2400gtaggatctg acttagagat agggcaacat agagcaaaaa tagaggagtt aagagaacat2460ctgttaaagt ggggatttac cacaccagac aagaaacatc agaaagaacc tccatttctt2520tggatggggt atgaactcca tcctgacaaa tggacagtac agcctataca gctgccagaa2580aaggatagct ggactgtcaa tgatatacag aagttagtgg gaaaattaaa ctgggcaagt2640cagatttacc caggaattaa agtaaggcaa ctttgtaaac tccttagggg ggccaaagca2700ctaacagaca taataccact aactgaagaa gcagaattgg agttggcaga aaacagggaa2760attctaaaag aaccagtaca tggagcatat tatgacccat caaaagactt gatagctgaa2820atacagaaac aggggcagga ccaatggaca tatcaaattt accaagaacc attcaaaaat2880ctgaaaacag ggaaatatgc aaaaatgagg actgcccaca ctaatgatgt aaaacagtta2940acagaggctg tgcagaaaat atccatggaa agcatagtaa tatggggaaa aattcctaaa3000tttaggttac ccatcccaaa agaaacctgg gagacacggt ggacagccta ttggcaagcc3060acctggattc ctgagtggga atttgttaat acccctccct tagtaaaatt atggtaccag3120ctggagaaag atcccatagc aggagtagaa actttctatg tagatggagc agctaatagg3180gaaactaaaa tgggaaaagc agggtatgtt actgacagag gaaggcagaa aattgtgtct3240ctaactgaaa caacaaatca gaagactgaa ttgcaagcaa tttgtctagc tttgcaagat3300tcaggatcag aagtaaatat agtaacagat tcacagtatg cattaggaat cattcaagca3360caaccagata agagtgagtc agagttagtt aaccaaataa tagaacaatt aataaaaaag3420gaaagggtct acctgtcgtg ggtaccagca cataaaggaa ttggaggaaa tgaacaagta3480gataaattag taagtaatgg aatcaggaaa gtgctatttc tagatggaat agataaagct3540caagaagagc atgaaaagta tcacagcaat tggagagcaa tggctagtga ctttaatctg3600ccacccatag tagcaaaaga aatagtagct agctgtgatc aatgtcagct aaaaggggaa3660gccatgcatg gacaagtaga ctgtagtcca gggatatggc aattagattg tacacattta3720gaaggaaaaa tcatcctggt agcagtccat gtagccagtg gctacataga agcagaagtt3780atcccagcag aaacaggaca agaaacagca tactttatac taaaattagc aggaagatgg3840ccagtcaaaa taatacatac agacaatggt agcaatttca ccagtgctgc agttaaggca3900gcctgttggt gggcaggtat ccaacaggaa tttggaattc cctacaatcc ccaaagtcag3960ggagtagtag aatccatgaa taaagaatta aagaaaatta tagggcaggt aagagatcaa4020gctgagcacc ttaagacagc agtacaaatg gcagtattca ttcacaattt taaaagaaaa4080ggggggattg gggggtacag tgcaggggaa agaataatag acataatagc aacagacata4140caaactaaag aattacaaaa acaaattata aaaattcaaa attttcgggt ttattacaga4200gacagcagag accccatttg gaaaggacca gccaaactac tctggaaagg tgaaggggca4260gtagtaatac aagataatag tgacataaag gtagtaccaa ggaggaaagc aaaaatcatt4320aagggctatg gaaaacagat ggcaggtgct gattgtgtgg caagtagaca ggatgaagat4380tagtaatttt ttatgcggcc gcta 4404<210>20<211>1457<212>PRT<213>人<400>20Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly1 5 10 15Ala Val Phe Val Ser Ala Arg Met Gly Ala Arg Ala Ser Ile Leu Arg20 25 30Gly Gly Lys Leu Asp Lys Trp Glu Lys Ile Arg Leu Arg Pro Gly Gly35 40 45Lys Lys His Tyr Met Leu Lys His Ile Val Trp Ala Ser Arg Glu Leu50 55 60Glu Arg Phe Ala Leu Asn Pro Gly Leu Leu Glu Thr Set Glu Gly Cys65 70 75 80Lys Gln Ile Ile Lys Gln Leu Gln Pro Ala Leu Gln Thr Gly Thr Glu85 90 95Glu Leu Arg Ser Leu Phe Asn Thr Val Ala Thr Leu Tyr Cys Val His100 105 110Ala Gly Ile Glu Val Arg Asp Thr Lys Glu Ala Leu Asp Lys Ile Glu115 120 125Glu Glu Gln Asn Lys Met Gln Gln Lys Thr Gln Gln Ala Lys Glu Ala130 135 140Asp Gly Lys Val Ser Gln Asn Tyr Pro Ile Val Gln Asn Leu Gln Gly145 150 155 160Gln Met Val His Gln Ala Ile Ser Pro Arg Thr Leu Asn Ala Trp Val165 170 175Lys Val Ile Glu Glu Lys Ala Phe Ser Pro Glu Val Ile Pro Met Phe180 185 190Thr Ala Leu Ser Glu Gly Ala Thr Pro Gln Asp Leu Asn Thr Met Leu195 200 205Asn Thr Val Gly Gly His Gln Ala Ala Met Gln Met Leu Lys Asp Thr210 215 220Ile Asn Glu Glu Ala Ala Glu Trp Asp Arg Val His Pro Val His Ala225 230 235 240Gly Pro Ile Ala Pro Gly Gln Met Arg Glu Pro Arg Gly Ser Asp Ile245 250 255Ala Gly Thr Thr Ser Thr Leu Gln Gly Gln Ile Ala Trp Met Thr Ser260 265 270Asn Pro Pro Val Pro Val Gly Glu Ile Tyr Lys Arg Trp Ile Ile Leu275 280 285Gly Leu Asn Lys Ile Val Arg Met Tyr Ser Pro Val Ser Ile Leu Asp290 295 300Ile Lys Gln Gly Pro Lys Glu Pro Phe Arg Asp Tyr Val Asp Arg Phe305 310 315 320Phe Lys Thr Leu Arg Ala Glu Gln Ala Thr Gln Asp Val Lys Asn Trp325 330 335Met Thr Asp Thr Leu Leu Val Gln Asn Ala Asn Pro Asp Cys Lys Thr340 345 350Ile Leu Arg Ala Leu Gly Pro Gly Ala Ser Leu Glu Glu Met Met Thr355 360 365Ala Cys Gln Gly Val Gly Gly Pro Asn His Lys Ala Arg Val Leu Ala370 375 380Glu Ala Met Ser Gln Ala Asn Gly Thr Ile Leu Met Gln Arg Ser Asn385 390 395 400Phe Lys Gly Ser Lys Arg Ile Val Lys Cys Phe Asn Cys Gly Lys Glu405 410 415Gly His Ile Ala Arg Asn Cys Arg Ala Pro Arg Lys Lys Gly Cys Trp420 425 430Lys Cys Gly Lys Glu Gly His Gln Met Lys Asp Cys Thr Glu Arg Gln435 440 445Ala Asn Phe Leu Gly Lys Ile Trp Pro Pro His Lys Gly Arg Pro Gly450 455 460Asn Phe Leu Gln Ser Arg Pro Glu Pro Thr Ala Pro Pro Ala Glu Ser465 470 475 480Phe Gly Phe Glu Glu Thr Thr Pro Ala Pro Lys Gln Glu Pro Lys Asp485 490 495Arg Glu Pro Leu Thr Ser Leu Lys Ser Leu Phe Gly Ser Asp Pr0 Leu500 505 510Ser Gln Pro Gln Ile Thr Leu Trp Gln Arg Pro Leu Val Ser Ile Arg515 520 525Val Gly Gly Gln Ile Lys Glu Ala Leu Leu Asp Asp Thr Val Leu Glu530 535 540Glu Val Asn Leu Pro Gly Lys Trp Lys Pro Lys Met Ile Gly Gly Ile545 550 555 560Gly Gly Phe Ile Lys Val Arg Gln Tyr Asp Gln Ile Pro Ile Glu Ile565 570 575Cys Gly Lys Lys Ala Ile Gly Thr Val Leu Val Gly Pro Thr Pro Val580 585 590Asn Ile Ile Gly Arg Asn Met Leu Thr Gln Leu Gly Cys Thr Leu Asn595 600 605Phe Pro Ile Ser Pro Ile Glu Thr Ile Pro Val Lys Leu Lys Pro Gly610 615 620Met Asp Gly Pro Arg Val Lys Gln Trp Pro Leu Thr Glu Glu Lys Ile625 630 635 640Lys Ala Leu Thr Ala Ile Cys Asp Glu Met Glu Lys Glu Gly Lys Ile645 650 655Thr Lys Ile Gly Pro Glu Asn Pro Tyr Asn Thr Pro Val Phe Ala Ile660 665 670Lys Lys Lys Asp Ser Thr Lys Trp Arg Lys Leu Val Asp Phe Arg Glu675 680 685Leu Asn Lys Arg Thr Gln Asp Phe Trp Glu Val Gln Leu Gly Ile Pro690 695 700His Pro Ala Gly Leu Lys Lys Lys Lys Ser Val Thr Val Leu Asp Val705 710 715 720Gly Asp Ala Tyr Phe Ser Val Pro Leu Tyr Glu Asp Phe Arg Lys Tyr725 730 735Thr Ala Phe Thr Ile Pro Ser Ile Asn Asn Glu Thr Pro Gly Ile Arg740 745 750Tyr Gln Tyr Asn Val Leu Pro Gln Gly Trp Lys Gly Ser Pro Ala Ile755 760 765Phe Gln Cys Ser Met Ala Lys Ile Leu Glu Pro Phe Arg Ala Gln Asn770 775 780Pro Glu Ile Val Ile Tyr Gln Tyr Gly Asp Asp Leu Tyr Val Gly Ser785 790 795 800Asp Leu Glu Ile Gly Gln His Arg Ala Lys Ile Glu Glu Leu Arg Glu805 810 815His Leu Leu Lys Trp Gly Phe Thr Thr Pro Asp Lys Lys His Gln Lys820 825 830Glu Pro Pro Phe Leu Trp Met Gly Tyr Glu Leu His Pro Asp Lys Trp835 840 845Thr Val Gln Pro Ile Gln Leu Pro Glu Lys Asp Ser Trp Thr Val Asn850 855 860Asp Ile Gln Lys Leu Val Gly Lys Leu Asn Trp Ala Ser Gln Ile Tyr865 870 875 880Pro Gly Ile Lys Val Arg Gln Leu Cys Lys Leu Leu Arg Gly Ala Lys885 890 895Ala Leu Thr Asp Ile Ile Pro Leu Thr Glu Glu Ala Glu Leu Glu Leu900 905 910Ala Glu Asn Arg Glu Ile Leu Lys Glu Pro Val His Gly Ala Tyr Tyr915 920 925Asp Pro Ser Lys Asp Leu Ile Ala Glu Ile Gln Lys Gln Gly Gln Asp930 935 940Gln Trp Thr Tyr Gln Ile Tyr Gln Glu Pro Phe Lys Asn Leu Lys Thr945 950 955 960Gly Lys Tyr Ala Lys Met Arg Thr Ala His Thr Asn Asp Val Lys Gln965 970 975Leu Thr Glu Ala Val Gln Lys Ile Ser Met Glu Ser Ile Val Ile Trp980 985 990Gly Lys Ile Pro Lys Phe Arg Leu Pro Ile Pro Lys Glu Thr Trp Glu995 10001005Thr Arg Trp Thr Ala Tyr Trp Gln Ala Thr Trp Ile Pro Glu Trp Glu101010151020Phe Val Asn Thr Pro Pro Leu Val Lys Leu Trp Tyr Gln Leu Glu Lys1025103010351040Asp Pro Ile Ala Gly Val Glu Thr Phe Tyr Val Asp Gly Ala Ala Asn104510501055Arg Glu Thr Lys Met Gly Lys Ala Gly Tyr Val Thr Asp Arg Gly Arg106010651070Gln Lys Ile Val Ser Leu Thr Glu Thr Thr Asn Gln Lys Thr Glu Leu107510801085Gln Ala Ile Cys Leu Ala Leu Gln Asp Ser Gly Ser Glu Val Asn Ile109010951100Val Thr Asp Ser Gln Tyr Ala Leu Gly Ile Ile Gln Ala Gln Pro Asp1105111011151120Lys Ser Glu Ser Glu Leu Val Asn Gln Ile Ile Glu Gln Leu Ile Lys112511301135Lys Glu Arg Val Tyr Leu Ser Trp Val Pro Ala His Lys Gly Ile Gly114011451150Gly Asn Glu Gln Val Asp Lys Leu Val Ser Asn Gly Ile Arg Lys Val115511601165Leu Phe Leu Asp Gly Ile Asp Lys Ala Gln Glu Glu His Glu Lys Tyr117011751180His Ser Asn Trp Arg Ala Met Ala Ser Asp Phe Asn Leu Pro Pro Ile1185119011951200Val Ala Lys Glu Ile Val Ala Ser Cys Asp Gln Cys Gln Leu Lys Gly120512101215Glu Ala Met His Gly Gln Val Asp Cys Ser Pro Gly Ile Trp Gln Leu122012251230Asp Cys Thr His Leu Glu Gly Lys Ile Ile Leu Val Ala Val His Val123512401245Ala Ser Gly Tyr Ile Glu Ala Glu Val Ile Pro Ala Glu Thr Gly Gln125012551260Glu Thr Ala Tyr Phe Ile Leu Lys Leu Ala Gly Arg Trp Pro Val Lys1265127012751280Ile Ile His Thr Asp Asn Gly Ser Asn Phe Thr Ser Ala Ala Val Lys128512901295Ala Ala Cys Trp Trp Ala Gly Ile Gln Gln Glu Phe Gly Ile Pro Tyr130013051310Asn Pro Gln Ser Gln Gly Val Val Glu Ser Met Asn Lys Glu Leu Lys131513201325Lys Ile Ile Gly Gln Val Arg Asp Gln Ala Glu His Leu Lys Thr Ala133013351340Val Gln Met Ala Val Phe Ile His Asn Phe Lys Arg Lys Gly Gly Ile1345135013551360Gly Gly Tyr Ser Ala Gly Glu Arg Ile Ile Asp Ile Ile Ala Thr Asp136513701375Ile Gln Thr Lys Glu Leu Gln Lys Gln Ile Ile Lys Ile Gln Asn Phe138013851390Arg Val Tyr Tyr Arg Asp Ser Arg Asp Pro Ile Trp Lys Gly Pro Ala139514001405Lys Leu Leu Trp Lys Gly Glu Gly Ala Val Val Ile Gln Asp Asn Ser141014151420Asp Ile Lys Val Val Pro Arg Arg Lys Ala Lys Ile Ile Lys Gly Tyr1425143014351440Gly Lys Gln Met Ala Gly Ala Asp Cys Val Ala Ser Arg Gln Asp Glu144514501455Asp<210>21<211>1011<212>DNA<213>人<400>21gccgccacca tggacgccat gaagcgcggc ctgtgctgcg tgctgctgct gtgcggcgcc 60gtgttcgtga gcgcccgcat ggggggcaag tggtcaaaaa gtagcatagt tggatggcct120gctataagag aaagaataag acgaactgaa ccagcagcag atggggtggg agcagtatct180cgagacctgg aaaaacatgg agcaatcaca agtaacaaca cagcagatac taatgctgat240tgtgcctggc tagaaacaca agaggaggag gaggtgggtt ttccagtcag acctcaggta300cccttaagac caatgacttt taagggagca ttggatctca gcttcttttt aaaagaaaag360gggggactgg aagggttaat ttactctaag aaaaggcaag agatccttga tttgtgggtc420tatcacacac aaggctactt ccctgactgg cacaactaca caccaggacc aggggtcaga480ttcccactga cttttgggtg gtgcttcaag ctagtaccag ttgacccagg ggaagtggaa540gaggccaatg aaggagaaaa caactgtttg ctacaccctg tctgccagca tggaatggat600gatgaacaca gagaagtatt aaagtggaag tttgacagtc aactagcaca cagacacagg660gcccgcgagc tacatccgga gttttacaaa gactgcatgg agccagtaga tcctaaccta720gagccctgga accatccagg aagtcagcct gaaactgctt gcaataactg ttattgtaaa780cgctgtagct accattgtct agtttgcttt cagaaaaaag gcttaggcat ttcctatggc840aggaagaagc ggagacagcg acgaagcgct cctccaagca gtgaggatca tcaaaatcct900atatcaaagc agcccttacc ccgaacccag ggggacccga caggctcgga agaatcgaag960aagaaggtgg agagcaagac aaaaacagat ccattcgatt agtaattttt t1011<210>22<211>330<212>PRT<213>人<400>22Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly1 5 10 15Ala Val Phe Val Ser Ala Arg Met Gly Gly Lys Trp Ser Lys Ser Ser20 25 30Ile Val Gly Trp Pro Ala Ile Arg Glu Arg Ile Arg Arg Thr Glu Pro35 40 45Ala Ala Asp Gly Val Gly Ala Val Ser Arg Asp Leu Glu Lys His Gly50 55 60Ala Ile Thr Ser Asn Asn Thr Ala Asp Thr Asn Ala Asp Cys Ala Trp65 70 75 80Leu Glu Thr Gln Glu Glu Glu Glu Val Gly Phe Pro Val Arg Pro Gln85 90 95Val Pro Leu Arg Pro Met Thr Phe Lys Gly Ala Leu Asp Leu Ser Phe100 105 110Phe Leu Lys Glu Lys Gly Gly Leu Glu Gly Leu Ile Tyr Ser Lys Lys115 120 125Arg Gln Glu Ile Leu Asp Leu Trp Val Tyr His Thr Gln Gly Tyr Phe130 135 140Pro Asp Trp His Asn Tyr Thr Pro Gly Pro Gly Val Arg Phe Pro Leu145 150 155 160Thr Phe Gly Trp Cys Phe Lys Leu Val Pro Val Asp Pro Gly Glu Val165 170 175Glu Glu Ala Asn Glu Gly Glu Asn Asn Cys Leu Leu His Pro Val Cys180 185 190Gln His Gly Met Asp Asp Glu His Arg Glu Val Leu Lys Trp Lys Phe195 200 205Asp Ser Gln Leu Ala His Arg His Arg Ala Arg Glu Leu His Pro Glu210 215 220Phe Tyr Lys Asp Cys Met Glu Pro Val Asp Pro Asn Leu Glu Pro Trp225 230 235 240Asn His Pro Gly Ser Gln Pro Glu Thr Ala Cys Asn Asn Cys Tyr Cys245 250 255Lys Arg Cys Ser Tyr His Cys Leu Val Cys Phe Gln Lys Lys Gly Leu260 265 270Gly Ile Ser Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Ser Ala Pro275 280 285Pro Ser Ser Glu Asp His Gln Asn Pro Ile Ser Lys Gln Pro Leu Pro290 295 300Arg Thr Gln Gly Asp Pro Thr Gly Ser Glu Glu Ser Lys Lys Lys Val305 310 315 320Glu Ser Lys Thr Lys Thr Asp Pro Phe Asp325 330
權(quán)利要求
1.包含兩個(gè)啟動(dòng)子的核酸載體。
2.權(quán)利要求1的核酸載體,其中啟動(dòng)子是pCMV和人延伸因子1α(hEF1α)。
3.權(quán)利要求2的核酸載體,其包含至少兩種核酸序列,其中每種在不同啟動(dòng)子的控制之下。
4.權(quán)利要求3的核酸載體,其中核酸序列是HIV-1核酸序列。
5.權(quán)利要求4的核酸載體,其中HIV-1核酸序列是env和gag。
6.權(quán)利要求5的核酸載體,其中HIV-1核酸序列進(jìn)一步包含異源前導(dǎo)序列。
7.權(quán)利要求6的核酸載體,其中前導(dǎo)序列是tPA(SEQ ID NO1)。
8.權(quán)利要求5的核酸載體,其中HIV-1序列是tPA-env(SEQ ID NO7)。
9.權(quán)利要求5的核酸載體,其中HIV-1序列是tPA-gag(SEQ ID NO9)。
10.權(quán)利要求4的核酸載體,其包含SEQ ID NO15的核酸序列。
11.包含權(quán)利要求10的核酸載體的DNA疫苗。
12.權(quán)利要求4的核酸載體,其中HIV-1核酸序列是pol、nef和tat或其融合序列。
13.權(quán)利要求12的核酸載體,其中HIV-1核酸序列進(jìn)一步包含異源前導(dǎo)序列。
14.權(quán)利要求13的核酸載體,其中前導(dǎo)序列是tPA(SEQ ID NO1)。
15.權(quán)利要求12的核酸載體,其中HIV-1 pol序列是SEQ ID NO11的序列。
16.權(quán)利要求5的核酸載體,其中nef和tat是包含SEQ ID NO13序列的融合nef-tat序列。
17.權(quán)利要求12的核酸載體,其包含SEQ ID NO16的核酸序列。
18.包含權(quán)利要求17的核酸載體的DNA疫苗。
19.包含兩種核酸載體的組合物,其中每種載體包含兩個(gè)啟動(dòng)子,每一個(gè)啟動(dòng)子都控制一種HIV-1基因的表達(dá)。
20.權(quán)利要求19的組合物,其中一種核酸載體包含pCMV啟動(dòng)子和hEF1α啟動(dòng)子,以及HIV-1基因gag和env,另一種核酸載體包含pCMV啟動(dòng)子和hEF1α啟動(dòng)子,以及HIV基因pol、nef和tat。
21.權(quán)利要求20的組合物,其包含SEQ ID NO15和16。
22.修飾的牛痘Ankara(MVA)載體,其包含至少三種插入到MVADelIII區(qū)的核酸序列,其中每一種核酸序列都在單獨(dú)的啟動(dòng)子控制之下。
23.權(quán)利要求22的修飾的MVA載體,其中核酸序列是HIV-1序列env、gag和pol或其融合序列。
24.權(quán)利要求23的修飾的MVA載體,其中核酸序列是SEQ ID NO17和SEQ ID NO19。
25.權(quán)利要求24的修飾的MVA載體,其進(jìn)一步包含HIV-1 nef-tat融合序列。
26.權(quán)利要求25的修飾的MVA載體,其中HIV nef-tat序列是SEQ IDNO21。
27.治療有患HIV相關(guān)疾病危險(xiǎn)的人類受試者的方法,其包含給予包含含SEQ ID NO15的一種核酸和含SEQ ID NO16的另一種核酸的核酸。
28.權(quán)利要求27的方法,其進(jìn)一步包含給予包含SEQ ID NO17、19和21的修飾的牛痘Ankara載體。
29.權(quán)利要求27的方法,其進(jìn)一步包含給予權(quán)利要求26的修飾的牛痘Ankara載體。
30.防護(hù)有患HIV-1相關(guān)疾病危險(xiǎn)的人類受試者的方法,其包含給予包含含SEQ ID NO15的一種核酸和含SEQ ID NO16的另一種核酸的DNA疫苗。
31.權(quán)利要求30的方法,其進(jìn)一步包含給予包含SEQ ID NO17、19和21的修飾的牛痘Ankara載體。
32.權(quán)利要求30的方法,其進(jìn)一步包含給予權(quán)利要求26的修飾的牛痘Ankara載體。
33.改善患HIV-1相關(guān)疾病的受試者中HIV-1相關(guān)疾病的方法,其包含給予包含含SEQ ID NO15的一種核酸和含SEQ ID NO16的另一種核酸的DNA疫苗。
34.權(quán)利要求33的方法,其進(jìn)一步包含給予包含SEQ ID NO17、19和21的修飾的牛痘Ankara載體(ADMVA2)。
35.權(quán)利要求33的方法,其進(jìn)一步包含給予權(quán)利要求26的修飾的牛痘Ankara載體。
36.用于對(duì)人類受試者提供針對(duì)HIV-1感染的防護(hù)的試劑盒,其包含包括SEQ ID NO15的序列的一種核酸載體、包括SEQ ID NO16的序列的另一種核酸載體和包括SEQ ID NO17、19和21的加強(qiáng)牛痘疫苗以及進(jìn)一步包含使用說(shuō)明。
全文摘要
本發(fā)明提供了用于治療有患HIV相關(guān)疾病危險(xiǎn)或正患有HIV相關(guān)疾病的受試者的核酸分子。本發(fā)明包括兩條初始的核酸分子ADVAXI和ADVAXII,以及加強(qiáng)牛痘核酸分子ADMVA2。本發(fā)明還包括用于治療和/或防護(hù)HIV相關(guān)疾病的方法,對(duì)患有HIV相關(guān)疾病或有患HIV相關(guān)疾病危險(xiǎn)的受試者接種的方法,以及包含本發(fā)明的核酸疫苗的試劑盒。
文檔編號(hào)C12N5/00GK1490056SQ0215489
公開(kāi)日2004年4月21日 申請(qǐng)日期2002年12月3日 優(yōu)先權(quán)日2002年10月18日
發(fā)明者Z·陳, Y·黃, D·D·霍, Z 陳, 霍 申請(qǐng)人:阿隆戴蒙德艾滋病研究中心
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