專利名稱：：重組人乳頭瘤病毒l1衣殼蛋白的氨基酸序列及其應(yīng)用的制作方法
技術(shù)領(lǐng)域：
：本發(fā)明涉及人乳頭瘤病毒感染的預(yù)防和治療領(lǐng)域。具體地，本發(fā)明涉及重組人乳頭瘤病毒Ll衣殼蛋白的氨基酸序列，編碼該氨基酸序列的核苦酸序列，以及包含這些核苷酸序列的載體和宿主細(xì)胞，本發(fā)明還涉及由該氨基酸序列所組成的HPVL1蛋白五聚體在制備疫苗、藥物組合物、診斷抗原或抗體中的應(yīng)用。背條技術(shù)乳頭瘤病毒(P^z7/o/^Wn^)是一大類侵染人類和其他動(dòng)物的DNA病毒。其中人乳頭瘤病毒(/f"加cmp印/Z/o加av/nw;HPV)與人類的多種疾病相關(guān)，這些疾病包括良性疣和癌癥。乳頭瘤病毒含有兩個(gè)用于保護(hù)病毒染色體的結(jié)構(gòu)蛋白(或衣殼蛋白)，分別為Ll和L2。360個(gè)L1蛋白分子或72個(gè)L1五聚體形成病毒顆粒的外殼；L2位于L1外殼的內(nèi)部，又稱內(nèi)衣殼蛋白，通常每一個(gè)L2分子與一個(gè)L1五聚體相聯(lián)。因此，L1和L2是重要的候選免疫原。由于乳頭瘤病毒在長期進(jìn)化過程中所形成的寄主?；?，該病毒很難通過常規(guī)的細(xì)胞培養(yǎng)來繁殖擴(kuò)增，必須用基因重組的方法來生產(chǎn)該病毒衣殼蛋白。到目前為止，通過基因重組規(guī)模表達(dá)目標(biāo)基因有兩個(gè)基本選擇原核表達(dá)與真核表達(dá)。其中，原核表達(dá)相對(duì)于真核表達(dá)而言，具有產(chǎn)率高、成本低、系統(tǒng)相對(duì)簡單和技術(shù)相對(duì)成熟的優(yōu)點(diǎn)。但是，原核表達(dá)技術(shù)也存在著缺陷分子量大的生物大分子的活性表達(dá)相對(duì)困難，尤其是高等動(dòng)物及人類的很多基因的生物活性表達(dá)更為困難。困難的根本原因在于原核表達(dá)的宿主是低等的大腸桿菌等原核生物，基因表達(dá)時(shí)對(duì)表達(dá)產(chǎn)物多肽的加工、修飾元件及修飾過程與髙等生物有著重要區(qū)別，表達(dá)人類等髙等生物基因時(shí)，容易導(dǎo)致目標(biāo)蛋白分子的二級(jí)或髙級(jí)結(jié)構(gòu)發(fā)生錯(cuò)誤折疊，造成不正確的分子構(gòu)象，從而影響或喪失表達(dá)產(chǎn)物的生物活性。人乳頭瘤病毒是人類及其它高等哺乳動(dòng)物的專性寄生病毒，自身沒有基因表達(dá)系統(tǒng)，其全部基因的表達(dá)完全依賴其寄主一_髙等真核生物的蛋白表達(dá)、翻譯、加工和修飾體系。因此，在原核生物中表達(dá)象L1衣殼蛋白這樣的基因時(shí)，往往因?yàn)榉肿诱郫B及構(gòu)象發(fā)生錯(cuò)誤而喪失生物活性。例如，未加修飾的L1蛋白在大腸桿菌中表達(dá)時(shí)，表達(dá)產(chǎn)物常常形成不溶于水的包含體而沉淀。沉淀的L1蛋白目前無法使其回復(fù)生物活性，即無法恢復(fù)成分子構(gòu)象正確的可溶性蛋白。原核系統(tǒng)表達(dá)髙等生物基因的這些缺陷在一定程度上阻止了其巨大優(yōu)勢(shì)的發(fā)揮，是目前發(fā)酵工業(yè)還沒有全部使用原核發(fā)酵的主要原因之一。為了生產(chǎn)一些具有重要生物活性的多態(tài)類藥物或生物制劑，人們不得不釆用成本較高的、也較復(fù)雜的真核發(fā)酵方法。解決上述問題的辦法之一是在深入研究的基礎(chǔ)上，對(duì)目標(biāo)基因表達(dá)產(chǎn)物的序列進(jìn)行仔細(xì)分析，在不影響和改變蛋白分子三維結(jié)構(gòu)的基礎(chǔ)上，對(duì)目標(biāo)基因進(jìn)行一定的修飾，使其能夠在大腸桿菌等原核細(xì)胞中實(shí)現(xiàn)可溶性表達(dá)。在實(shí)現(xiàn)乳頭瘤病毒Ll蛋白原核活性表達(dá)的基礎(chǔ)上，表達(dá)的Ll蛋白分子能夠形成正確的分子構(gòu)象而可溶于水。可溶的L1分子由于三維構(gòu)象的緣故能夠自動(dòng)進(jìn)行分子聚合，形成Ll蛋白分子的多聚體。其中一種聚合方式是形成Ll蛋白分子的五聚體。在自然條件下，寄生于人體或其它哺乳動(dòng)物上皮細(xì)胞的乳頭瘤病毒在寄主細(xì)胞衰老及行將死亡前大量繁殖。繁殖時(shí)，病毒在啟動(dòng)自身基因組DNA復(fù)制的同時(shí)啟動(dòng)L1衣殼蛋白基因在寄主細(xì)胞中的表達(dá)，表達(dá)后的L1蛋白自動(dòng)形成五聚體結(jié)構(gòu)，然后這些五聚體自身進(jìn)一步自動(dòng)聚合，最后由72個(gè)五聚體形成一個(gè)野生病毒顆粒，每個(gè)病毒顆粒內(nèi)部儲(chǔ)藏一套病毒的DNA分子。人體對(duì)任何入侵病毒的識(shí)別主要是識(shí)別病毒表面的衣殼蛋白分子，沒有例外。人乳頭瘤病毒L1衣殼蛋白的五聚體分子表面構(gòu)成了人體的抗原識(shí)別表位(即抗原表位)。在病毒被識(shí)別后，人體產(chǎn)生特異抗體來中和病毒，對(duì)抗病毒的感染和危害。為了誘導(dǎo)人體產(chǎn)生對(duì)抗乳頭瘤病毒的特異抗體，我們通過生物工程的方法表達(dá)和純化病毒的衣殼蛋白，并以純化的Ll蛋白作為抗原來生產(chǎn)疫苗。重組L1蛋白五聚體本身就具備完整的抗原表位，因此就可以作為抗原用來制備疫苗。但在生產(chǎn)的過程中，部分L1五聚體可以自動(dòng)聚合形成與野生病毒顆粒大小完全一樣的類病毒顆粒(VLP，viruslikeparticle)。VLP的免疫原性及抗原性與Ll五聚體沒有實(shí)質(zhì)區(qū)別。為了降低生產(chǎn)成本，實(shí)現(xiàn)乳頭瘤病毒抗原的原核表達(dá)，有必要通過基因改造控制L1蛋白VLP的形成，這樣有利于使抗原分子的大小均勾一致，便于質(zhì)量控制，也有利于提高產(chǎn)率。
發(fā)明內(nèi)容(一)要解決的技術(shù)問題本發(fā)明的目的是提供一種重組人乳頭瘤病毒L1衣殼蛋白的氨基酸序列，使得在原核系統(tǒng)中表達(dá)的人乳頭瘤病毒Ll衣殼蛋白能溶于水，同時(shí)控制Ll五聚體的進(jìn)一步聚合；本發(fā)明的又一目的是提供上述重組人乳頭瘤病毒L1衣殼蛋白氨基酸序列的應(yīng)用。(二)技術(shù)方案本發(fā)明所述的人乳頭瘤病毒衣殼蛋白L1的重組氨基酸序列，是將野生型HPVLl蛋白的氨基酸序列N-末端的保守序列VYLPP或VYVPP上游的氨基酸序列替換成GSGGG，同時(shí)在其C-末端的保守序列LGRKFL下游第5個(gè)氨基酸處通過引入蛋白質(zhì)翻譯的終止密碼子形成相對(duì)于野生型的截短型序列，其中所述的HPV型選自HPV6、HPVll、HPV16、HPV18、HPV26、HPV31、HPV33、HPV35、HPV39、HPV42、HPV45、HPV51、HPV52、HPV53、HPV56、HPV58、HPV59、HPV66、HPV73、HPV82中的一種或多種。上述不同型HPV的野生型Ll蛋白的氨基酸序列對(duì)應(yīng)的Genebank登陸號(hào)分別為:HPV6-NC—000904,HPVll-NC一OO1525HPV26-NC—001583，HPV31-NC—001527，HPV39-NC一001535,HPV42-NC—00153,HPV52-NC001592,HPV53-NC001593,HPV16-AF402678,HPV18-AY262282HPV33-NC—001528,HPV35-NC—001529HPV45-NC—001590,HPV51-NC_001533HPV56-NC001594,HPV58-NC001443HPV59-NC—001635,HPV66-NC—001695，HPV73-X94165,HPV82-AF293961。采用上述策略進(jìn)行改造后，不同型HPV對(duì)應(yīng)的重組氨基酸序列分別為HPV6對(duì)應(yīng)的氨基酸序列如SEQIDN0:1所示，HPV11對(duì)應(yīng)的氨基酸序列如SEQIDN0:2所示，HPV16對(duì)應(yīng)的氨基酸序列如SEQIDNO:3所示，HPVl8對(duì)應(yīng)的氨基酸序列如SEQIDNO:4所示，HPV26對(duì)應(yīng)的氨基酸序列如SEQIDNO:5所示，HPV31對(duì)應(yīng)的氨基酸序列如SEQIDNO:6所示，HPV33對(duì)應(yīng)的氨基酸序列如SEQIDNO:7所示，HPV35對(duì)應(yīng)的氨基酸序列如SEQIDNO:8所示,HPV39對(duì)應(yīng)的氨基酸序列如SEQIDNO:9所示，HPV42對(duì)應(yīng)的氨基酸序列如SEQIDNO:10所示，HPV45對(duì)應(yīng)的氨基酸序列如SEQIDNO:11所示，HPV51對(duì)應(yīng)的氨基酸序列如SEQIDNO:12所示，HPV52對(duì)應(yīng)的氨基酸序列如SEQIDNO:13所示，HPV53對(duì)應(yīng)的氨基酸序列如SEQIDNO:14所示，HPV56對(duì)應(yīng)的氨基酸序列如SEQIDNO:15所示，HPV58對(duì)應(yīng)的氨基酸序列如SEQIDNO:16所示，HPV59對(duì)應(yīng)的氨基酸序列如SEQIDNO:17所示，HPV66對(duì)應(yīng)的氨基酸序列如SEQIDNO:18所示，HPV73對(duì)應(yīng)的氨基酸序列如SEQIDNO:19所示，HPV82對(duì)應(yīng)的氨基酸序列如SEQIDNO:20所示。采用上述策略進(jìn)行改造后，在原核系統(tǒng)中表達(dá)時(shí)得到的是HPVL1蛋白的五聚體，而且該五聚體能溶于水，即HPVL1蛋白實(shí)現(xiàn)了正確的空間折疊，具備了生物活性。由于編碼不同型HPVL1蛋白的DNA序列均具有較高的保守性，所以上述改造策略適用于所有的HPV型，得到的表達(dá)產(chǎn)物均為具備生物活性的HPVLl蛋白五聚體。本發(fā)明還涉及包含編碼上述氨基酸序列的核苷酸序列的核酸分子，編碼上述氨基酸序列的核苷酸序列的共同特征是表達(dá)得到的產(chǎn)物具有與野生型HPVLl蛋白相同的免疫特性。所述核酸分子包括但不限于以下形式編碼上述氨基酸序列的核苷酸序列與編碼HPV前期蛋白(如E6/E7)的核酸序列組成的融合序列。本發(fā)明也涉及包含上述核苷酸序列的表達(dá)載體，這些載體是通過將上述核酸序列克隆到包含合適啟動(dòng)子和其它合適轉(zhuǎn)錄表達(dá)調(diào)控原件的連接載體中得到的，其中用于連接的載體包括商業(yè)上可獲得的質(zhì)粒、細(xì)菌噬菌體和粘粒。優(yōu)選地，用于連接的載體選自pGEX-4T-l、pGEX-4T-2、pGEX-4T-3、pET-28a、pcDNA3.1或任何其它可用于原核表達(dá)的表達(dá)質(zhì)粒。更優(yōu)選地，用于連接的載體選自pGEX-4T-2。這里用到的分子克隆方法具體操作參見《分子克隆》(第三版，科學(xué)出版社，2002年8月出版)。本發(fā)明還涉及包含上述表達(dá)載體的宿主細(xì)胞，這些宿主細(xì)胞可選自大腸桿菌、昆蟲細(xì)胞、酵母細(xì)胞中的一種或幾種。優(yōu)選地，宿主細(xì)胞選自大腸桿菌。本發(fā)明進(jìn)一步涉及HPVL1蛋白五聚體，該五聚體蛋白的單體的一級(jí)結(jié)構(gòu)由上述重組氨基酸序列所組成，其二級(jí)結(jié)構(gòu)和高級(jí)結(jié)構(gòu)由其一級(jí)結(jié)構(gòu)所決定的。本發(fā)明還提供了生產(chǎn)上述HPVL1蛋白五聚體的方法，它包括如下步驟(1)將編碼上述重組氨基酸序列的核苷酸序列克隆到連接載體上，轉(zhuǎn)化大腸桿菌；(2)發(fā)酵培養(yǎng)上述大腸桿菌，并表達(dá)HPVL1蛋白；(3)分離、純化表達(dá)產(chǎn)物，得到HPVL1蛋白五聚體。本發(fā)明進(jìn)一步涉及包含一種或多種上述重組HPVL1蛋白五聚體的疫苗，該疫苗可應(yīng)用于制備預(yù)防HPV感染的疫苗。優(yōu)選地，本發(fā)明所述的疫苗包含HPV16的重組HPVLl蛋白五聚體，該五聚體蛋白的單體的一級(jí)結(jié)構(gòu)由SEQIDN0:3所示的氨基酸序列所組成。更優(yōu)選地，本發(fā)明所述的疫苗包含五種重組HPVLl蛋白五聚體，對(duì)應(yīng)的HPV型分別為HPV6、HPVll、HPV16、HPV18和HPV33,這五種HPVLl蛋白五聚體的單體的一級(jí)結(jié)構(gòu)分別由SEQIDNO:l、SEQIDNO:2、SEQIDNO:3、SEQIDNO:4、SEQIDNO:7所示的氨基酸序列所組成。本發(fā)明所述的疫苗的形式也可以是包含HPVLl蛋白五聚體+L2蛋白的組合蛋白。上述疫苗的免疫組分也包括一種生理上可接受的載體，包括但不限于能保持HPVL1蛋白五聚體完整性的簡單低濃度鹽溶液，例如10mMNaCl，O.lmMEDTA;或者更大范圍上講，載體包括代謝緩慢的大分子如蛋白質(zhì)、多糖、聚乳糖酸、聚甘油酸、復(fù)合氨基酸、以及失活病毒粒子等。藥理上適用的鹽也可在HPVL1蛋白五聚體的復(fù)合物中使用。例如，礦物鹽象鹽酸鹽，溴化鹽、磷酸鹽、硫酸鹽等；有機(jī)鹽類如乙酸鹽、甘露糖、苯甲酸等。免疫組分還包括液體，如水、生理鹽水、甘油、酒精，以及其他一些物質(zhì)如可濕劑、乳化劑、和pH緩沖劑。將上述重組HPVL1蛋白五聚體或HPVL1蛋白五聚體+L2蛋白的組合蛋白以免疫有效劑量接種給人體后，可引誘人體產(chǎn)生對(duì)這些重組蛋白的免疫反應(yīng)。人體的這種特異免疫反應(yīng)可幫助人體預(yù)防人乳頭瘤病毒的入侵或中和清除已經(jīng)入侵的人乳頭瘤病毒。由重組HPVLl蛋白五聚體或HPVLl蛋白五聚體+L2蛋白的組合蛋白制備的疫苗可采用多種方法作用于人體，包括靜脈、肌肉和皮下注射。本發(fā)明所述的疫苗在能引發(fā)對(duì)L1蛋白免疫反應(yīng)的有效劑量的范圍使用。用于增強(qiáng)免疫反應(yīng)的復(fù)合物的特定劑量依照Ll復(fù)合物不同而變化。一般而言，Ll五聚體的用量大約在l-500pg/Kg體重之間。上述劑量范圍并不排除更高或更低劑量的可能。例如，具體的劑量會(huì)根據(jù)是否環(huán)伴有其他藥物劑量而定，或取決于個(gè)人的藥代動(dòng)力學(xué)、藥物積累和代謝速率。本發(fā)明還涉及包含上述重組HPVL1蛋白五聚體的藥物組合物。該藥物組合物包括但不限于下列形式例如包含重組HPVL1蛋白五聚體、HPVE6/E7蛋白及其它藥物上可接受的輔料的藥物組合物。本發(fā)明還進(jìn)一步公開了上述重組HPVLl蛋白五聚體在制備HPV病毒免疫診斷抗原或抗體中的應(yīng)用。上述重組HPVLl蛋白五聚體可以用來產(chǎn)生對(duì)HPVLl蛋白有特異親和力的抗體。這些抗體可以被用來直接檢測生物樣品中是否存在或與特定病理階段相聯(lián)系的特定HPV型。HPVLl蛋白五聚體還能在免疫測定中用來檢測生物樣品中是否存在HPV病毒的抗體或抗原。包括特異結(jié)合到Ll蛋白的單鏈抗體在內(nèi)的多克隆或單克隆抗體能夠被本領(lǐng)域技術(shù)人員用成熟的技術(shù)生產(chǎn)出來。這些由特異抗原例如HPV不同亞型引誘產(chǎn)生的抗體具有識(shí)別特定的HPV亞型的特異性，例如HPV16和HPV18型。利用抗體反應(yīng)的免疫測定來檢測Ll蛋白包括ELISAs、Westernblot、放射免疫測定、免疫組化測定、免疫沉淀或其他方法。抗體和Ll五聚體都可作為檢測標(biāo)記，例如酶聯(lián)免疫、放射標(biāo)記、熒光或化學(xué)熒光?？贵w或L1五聚體可以固定在某個(gè)固體支持物上，例如玻璃或塑料波片，組織培養(yǎng)板、多孔板、試管、交換拄、交換柱填充物、蛋白；或者顆粒上如微球體，包括但不限于乳膠、聚苯乙?；虿Ａ颍换蛘弑∧ど先绱姿崂w維或尼龍膜。偶聯(lián)蛋白組分的方法為本領(lǐng)域技術(shù)人員所熟知。只要不防礙抗體原抗體特異結(jié)合的能力，任何偶聯(lián)方式都可使用.生物樣品可以是任何被懷疑含有HPV病毒的樣品，例如組織活檢、涂片、組織切樣如皮膚、子宮、生殖上皮細(xì)胞、喉、上呼吸道、結(jié)膜或口腔內(nèi)組織。(三)有益效果本發(fā)明通過對(duì)HPVL1野生型序列的改造，使得在原核系統(tǒng)中表達(dá)的重組HPVL1衣殼蛋白實(shí)現(xiàn)了正確的空間折疊，能溶于水，解決了目前利用原核系統(tǒng)中表達(dá)的HPVL1衣殼蛋白都是不溶于水的包含體(沒有生物活性)的難題，打破了用原核表達(dá)系統(tǒng)生產(chǎn)HPVLl衣殼蛋白的瓶頸。不僅如此，本發(fā)明還有效控制了HPVL1蛋白的聚合程度，表達(dá)得到的是與HPVL1的VLP具有相同免疫原性和抗原性的Ll五聚體，Ll五聚體相對(duì)于L1的VLP而言具有結(jié)構(gòu)更穩(wěn)定、大腸桿菌表達(dá)更容易實(shí)現(xiàn)的優(yōu)點(diǎn)。本發(fā)明使得利用原核表達(dá)系統(tǒng)工業(yè)生產(chǎn)HPVL1衣殼蛋白成為現(xiàn)實(shí)，相對(duì)于目前釆用的真核表達(dá)系統(tǒng)具有產(chǎn)品質(zhì)量更穩(wěn)定、產(chǎn)率更髙、成本低、質(zhì)量控制方便的優(yōu)點(diǎn)，具有重大的經(jīng)濟(jì)效益和社會(huì)效應(yīng)。圖1是重組改造后得到的HPV16Ll衣殼蛋白的SDS-聚丙烯酰胺凝膠電泳，圖中泳道l表示marker,泳道2和泳道3表示蛋白清液，泳道4和泳道5表示樹脂吸附的目的蛋白，泳道6、泳道7和泳道8表示酶切后的樹脂，泳道9表示目的蛋白洗脫液；圖2是重組改造后得到的HPV16L1衣充蛋白的液相色譜圖；圖3是HPV16L1蛋白的電鏡照片比較，其中圖3(a)表示HPV16Ll的VLP電鏡照片，圖3(b)表示重組改造后得到的HPV16L1五聚體電鏡照片。具體實(shí)施方式以下實(shí)施例用于說明本發(fā)明，但不用來限制本發(fā)明的范圍。實(shí)施例1以HPV16型為例說明對(duì)HPVLl衣殼蛋白氨基酸序列的重組改造1、人乳頭瘤病毒16型(HPV16)DNA的檢測和分離可能含有野生型HPV16病毒的臨床細(xì)胞樣本廢棄物(用于常規(guī)細(xì)胞形態(tài)學(xué)檢查的宮頸脫落細(xì)胞的剩余部分)購自北京市安貞醫(yī)院婦科門診。懸浮在1.0毫升水中的(約lxl()S個(gè))脫落細(xì)胞經(jīng)1000單位的蛋白酶K(購自華美生物制品有限公司)在攝氏55度處理300分鐘，經(jīng)10000g高速離心30分鐘，所得上清液可用于檢測是否存在HPV病毒。受感染的HPV16陽性細(xì)胞上清液可直接用于PCR擴(kuò)增HPV16病毒DNA序列的目標(biāo)基因片段。2、HPV16型L1蛋白DNA的分子克隆HPV16型L1衣殼蛋白基因由目標(biāo)基因特異寡核苷酸引物對(duì)經(jīng)DNA聚合酶鏈?zhǔn)椒磻?yīng)(PCR)獲得。經(jīng)典的PCR反應(yīng)體系包含20ugDNA模板、lxPCR緩沖液、正向和反向特異引物(濃度均為0,2^iM)、1.5mM鎂離子、1.0單位的TAQDNA聚合酶。反應(yīng)條件為攝氏95度變性5分鐘，經(jīng)36個(gè)PCR循環(huán)放大(每一循環(huán)為94度30秒，55度30秒，72度2分鐘)，反應(yīng)產(chǎn)物在72度下溫育10分鐘，然后停止反應(yīng)。PCR反應(yīng)的正向引物序列和反向引物序列見表l。PCR反應(yīng)擴(kuò)增放大的HPV16LlcDNA片段經(jīng)TA克隆(具體操作參見《分子克隆》第三版，科學(xué)出版社，2002年8月出版)克隆到一個(gè)T-Easyvector質(zhì)粒(購自美國Promega生物試劑公司)上。3、HPV16L1蛋白基因的修飾HPV16Ll蛋白的免疫源性及抗原性依賴于其正確的分子折疊所產(chǎn)生的正確三維構(gòu)象。對(duì)HPV16L1蛋白基因序列進(jìn)行修飾的目的是增加重組目標(biāo)基因原核表達(dá)時(shí)的可溶性，從而利用￡.c^/生產(chǎn)出具有生物活性的Ll蛋白。基因改造的基本步奏是設(shè)計(jì)特異引物，然后利用以特異引物為模板PCR擴(kuò)增放大L1目的基因片段。改造后的重組HPV16Ll蛋白基因DNA序列在其5，-端包含有5cwnHI限制性酶切位點(diǎn)，同時(shí)含有編碼GSGGG的DNA密碼子以取代編碼氨基酸序列N-末端的保守序列VYLPP上游氨基酸的密碼子。用于改造的正向引物序列和反向引物序列見表l。下游引物引入終止密碼子UAA，導(dǎo)致L1cDNA在3，-端減少約25個(gè)氨基酸。PCR反應(yīng)體系包含20ugDNA模板、lxPCR緩沖液、正向和反向特異引物(濃度均為0.2pM)、1.5mM鎂離子、1.0單位的TAQDNA聚合酶。反應(yīng)條件為攝氏95度變性5分鐘，經(jīng)36個(gè)PCR循環(huán)放大(每一循環(huán)為94度30秒，55度30秒，72度2分鐘)，反應(yīng)產(chǎn)物在72度下溫育IO分鐘，然后停止反應(yīng)。PCR反應(yīng)擴(kuò)增放大的HPV16LlcDNA產(chǎn)物經(jīng)SawHI和屈oI限制性酶酶切形成黏性末端，同時(shí)表達(dá)質(zhì)粒pGEX-4T-2(購自華美生物制品有限公司)DNA用Bamffl和XhoI酶切消化，再用T4連接酶(購自美國Promega生物試劑公司)連接PCR產(chǎn)物和表達(dá)質(zhì)粒。連接產(chǎn)物經(jīng)電轉(zhuǎn)化轉(zhuǎn)移到大腸桿菌BL21(購自華美生物制品有限公司)宿主細(xì)胞內(nèi)(電轉(zhuǎn)化的具體操作參見《分子克隆》第三版，科學(xué)出版社，2002年8月出版)。4、Ll蛋白的原核表達(dá)和檢測載有HPV16L1基因插入片段的表達(dá)質(zhì)粒導(dǎo)入大腸桿菌BL21細(xì)胞之后，使用LB培養(yǎng)基(配方見《分子克隆》第三版，科學(xué)出版社，2002年8月出版)進(jìn)行37度培養(yǎng)，菌種接入后生長12小時(shí)開始用表達(dá)誘導(dǎo)劑IPTG(購自美國Promega公司)誘導(dǎo)，9小時(shí)后收獲細(xì)胞，細(xì)胞用NiroSoaviNS2006型髙壓勻質(zhì)機(jī)在800bar下重復(fù)破碎細(xì)胞2次，顯微鏡檢查細(xì)胞破碎率達(dá)到90%以上。上清溶液中的L1蛋白用經(jīng)親和色譜純化蛋白預(yù)裝谷胱甘肽-瓊脂糖樹脂(Amersham公司生產(chǎn)的GlutathioneSepharose4B)色譜柱，取濃度為50%的GlutathioneSepharose4B勾漿放入色譜柱中(每200ml蛋白清液需要5-10ml勻漿)。用5-10倍的柱床體積的緩沖液A(組分為50mmol/LTric-HCl，200mmol/LNaCl，lmmol/LEDTA,pH8.0)洗滌樹脂，將蛋白清液加入色譜柱中，與樹脂混合均句并在室溫下作用20分鐘后放出濾過液，用10倍柱床體積的緩沖液A洗滌樹脂柱。檢測用SDS-聚丙烯酰胺凝膠電泳經(jīng)密度掃描(具體操作見《分子克隆》第三版，科學(xué)出版社，2002年8月出版)檢測目的蛋白的純度(凝膠電泳結(jié)果見附圖l),結(jié)果表達(dá)得到的HPV16Ll蛋白純度高達(dá)95%。再用液相分子排阻色譜法[用AmershamBiosciences生產(chǎn)的AKTAFPLC過濾系統(tǒng)進(jìn)行凝膠過濾色譜，凝膠過濾介質(zhì)為Amersham生產(chǎn)的HiloadSuperdex200,流動(dòng)相為pH7.4的磷酸鹽緩沖鹽溶液(成分見《分子克隆》第三版，科學(xué)出版社，2002年8月出版)]檢測，結(jié)果見附圖2，該圖表明表達(dá)得到的HPV16L1蛋白是可溶于水的，即具備了生物活性。然后用電鏡檢査純化的HPV16L1蛋白，結(jié)果見附圖3(b),對(duì)比附圖3(a)[HPV16L1的VLP電鏡照片]，可見得到的是HPV16L1五聚體。實(shí)施例2其余HPV型的L1衣殼蛋白氨基酸序列的重組改造其余HPV型的L1衣殼蛋白氨基酸序列的重組改造(包括HPV6、HPVll、HPV18、HPV26、HPV31、HPV33、HPV35、HPV39、HPV42、HPV45、HPV51、HPV52、HPV53、HPV56、HPV58、HPV59、HPV66、HPV73、HPV82)的操作步驟與檢測方法均與實(shí)施例1相同，不同之處見表l。表l<table>tableseeoriginaldocumentpage10</column></row><table><table>tableseeoriginaldocumentpage11</column></row><table>上述HPV型的L1衣殼蛋白氨基酸序列經(jīng)重組改造后，得到的結(jié)果均與實(shí)施例l中得到的HPV16Ll衣殼蛋白相同，即均得到了可溶于水的L1五聚體。序列表<110>馬潤林陳小江<120>重組人乳頭瘤病毒L1衣殼蛋白的氨基酸序列及其應(yīng)用<130>MA0702<160>20<170>Patentlnversion3.3<210>1<211>465<212>PRT<213>人工序列<220><223>HPV6Ll<400>1GlySerGlyGlyGlyValTyrValProProProAsnProValSerLys151015ValValAlaThrAspAlaTyrValThrAigThrAsnliePheTyrHis202530AlaSerSerSerArgLeuLeuAlaValGlyHisProTjtPheSerlie354045LysArgAlaAsnLysThrValValProLysValSerGlyTyrGinTyr505560ArgValPheLysValValLeuProAspProA幼LysPheAlaLeuPro65707580AspSerSerLeuPheAspProThrThrGinArgLeuValTrpAlaCys859095ThrGlyLeuGluValGlyA屯GlyGinProLeuGlyValGlyValSer100105110GlyHisProPheLeuAsnLysTyrAspAspValGluAsnSerGlySer115120125GlyGlyAsnProGlyGinAspAsnArgValAsnValGlyMetAspTyr130135140LysGinThrGinLeuCysMetValGlyCysAlaProProLeuGlyGlu145150155160HisTrpGlyLysGlyLysGinCysThrAsnThrProValGinAlaGly165170175AspCysProProLeuGluLeuHeThrSerVallieGinAspGlyAsp180185l卯MetValAspThrGlyPheGlyAlaMetAsnPheAlaAspLeuGinThr195200205<image>imageseeoriginaldocumentpage13</image>ValValAlaThrAspAlaTyrValLysArgThrAsnHePheTyrHis202530AlaSerSerSerAtgLeuLeuAlaValGlyHisProTyrTyrSerlie354045LysLysValAsnLysThrValValProLysValSerGlyTyrGinTyr505560ArgValPheLysValValLeuProAspProAsnLysPheAlaLeuPro65707580AspSerSerLeuPheAspProThrThrGinArgLeuValTrpAlaCys859095ThrGlyLeuGluValGlyA堪GlyGinProLeuGlyValGlyValSer100105110GlyHisProLeuLeuAsnLysTyrAspAspValGluAsnSerGlyGly115120125TyrGlyGlyAsnProGlyGinAspAsnArgValAsnValGlyMetAsp130135140TyrLysGinThrG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euPheAsnLysLeuAspAspThrGluAsnSerHisLeu115120125AlaThrValAsnAlaAspThrAspAsnArgAspAsnValSerValAsp130135140AsnLysGinThrGinLeuCyslielieGlyCysThrProProLeuGly145150155160GluHisTrpGlyHeGlyThrlieCysLysAsnThrGinThrGinA唯165170175GlyAspCysProProLeuGluLeulieSerSerHelieGluAspGly180185190AspMetHeAspThrGlyPheGlyAlaMetAspPheThrAlaLeuGin195200205AlaThrLysSerAspValProlieAsplieSerGinSerThrCysLys210215220TyrProAspTyrLeuLysMetSerAlaAspThrTyrGlyAsnSerMet225230235240PhePhePheLeuArgArgGluGinLeuPheAlaArgHisPheTyrAsn245250255LysAlaGlyAlaValGlyAspAlaHeProThrThrLeuTyrlieLys260265270GlyAlaGluSerGlyArgGluProProThrSerSerlieTyrSerAla275280285ThrProSerGlySerMetValThrSerAspAlaGinLeuPheAsnLys2卯295300ProTyrTrpLeuGinArgAlaGinGlyHisAsnAsnGlyHeCysTrp305310315320GlyAsnGinLeuPheValThrCysValAspThrThrArgSerThrAsn325330335LeuThrlieSerThrLeuSerAlaAlaSerAlaSerThrProPheLys340345350ProSerAspT>rLysGinPhelieArgHisGlyGluGluTyrGluLeu355360365GinPheliePheGinLeuCysLyslieThrLeuThrThrAspValMet370375380AlaTyrlieHisLeuMetAsnAlaSerlieLeuGluAspTrpAsnPhe385390395400GlyLeuThrLeuProProThrAlaSerLeuGluAspAlaTyrArgPhe405410415lieLysAsnSerAlaThrThrCysGinAtgAsnAlaProProValPro420425430LysGluAspProPheGinLysPheLysPheTrpAspValAspLeuLys435440445GluLysPheSerHeAspLeuAspGinPheProLeuGlyArgLysPhe450455460MetLeuGinAlaGly465<210>6<211>469<212>PRT<213>人工序列<220><223>HPV31LI<400>6GlySerGlyGlyGlyValTyrLeuProProValProValSerLysVal15'1015ValSerThrAspGluTyrValThrA@ThrAsnlieTyrTyrHisAla202530GlySerAlaArgLeuLeuThrValGlyHisProTyrTyrSerliePro354045LysSerAspAsnProLysLysHeValValProLysValSerGlyLeu505560GinTyrArgValPheArgValLeuProAspProAsnLysPheGly65707580PheProAspThrSerPheTyrAsnProGluThrGinArgLeuValTrp859095AlaCysValGlyLeuGluValGlyArgGlyGinProLeuGlyValGly100105110lieSerGlyHisProLeuLeuAsnLysPheAspAspThrGluAsnSer115120125AsnArgTyrAlaGlyGlyProGlyThrAspAsnArgGluCyslieSer130135140MetAspTyrLysGinThrGinLeuCysLeuLeuGlyCysLysProPro145150155160lieGlyGluHisTrpGlyLysGlySerProCysSerAsnAsnAlalie165170175ThrProGlyAspCysProProLeuGluLeuLysAsnSerVallieGin180185190AspGlyAspMetValAspThrGlyPheGlyAlaMetAspPheThrAla195200205LeuGinAspThrLysSerAsnValProLeuAsplieCysAsnSerlie210215220CysLysTyrProAspTyrLeuLysMetValAlaGluProTyrGlyAsp225230235240ThrLeuPhePheTyrLeuArgArgGluGinMetPheValArgHisPhe245250255PheAsnArgSerGlyThrValGlyGluSerValProThrAspLeuTyr260265270lieLysGlySerGlySerThrAlaThrLeuAlaAsnSerThrTyrPhe275280285ProThrProSerGlySefMetValThrSerAspAlaGinliePheAsn290295300LysProTyrTrpMetGinArgAlaGinGlyHisAsnAsnGlylieCys305310315320TrpGlyAsnGinLeuPheValThrValValAspThrThrArgSerThr325330335AsnMetSerValCysAlaAlalieAlaAsnSerAspThrThrPheLys340345350SerSerAsnPheLysGluTyrLeuArgHisGlyGluGluPheAspLeu355360365GinPheliePheGinLeuCysLyslieThrLeuSerAlaAsplieMet370375380ThrTyrHeHisSerMetAsnProAlalieLeuGluAspTrpAsnPhe385390395400GlyLeuThrThrProProSerGlySerLeuGluAspThrTyrArgPhe405410415ValThrSerGinAlalieThrCysGinLysThrAlaProGinLysPro420425430LysGluAspProPheLysAspTyrValPheTrpGluValAsnLeuLys435440445GluLysPheSerAlaAspLeuAspGinPheProLeuGlyArgLysPhe450455460LeuLeuGinAlaGly465<220><223>HPV33LI<400>7GlySerGlyGlyGlyValTyrLeuProProValProValSerLysVal151015ValSerThrAspGluTyrValSerArgThrSerlieTyrTyrTyrAla202530GlySerSerArgLeuLeuAlaValGlyHisProTyrPheSerHeLys354045<210><211><212><213>200710005099.4AsnProThrAsnAlaLysLysLeuLeuValProLysValSerGlyLeu505560GinTyrArgValPheA屯ValArgLeuProAspProAsnLysPheGly65707580PheProAspThrSerPheTyrAsnProAspThrGinArgLeuValTrp859095AlaCysValGlyLeuGlulieGlyArgGlyGinProLeuGlyValGly100105110HeSerGlyHisProLeuLeuAsnLysPheAspAspThrGluThrGly115120125AsnLysTyrProGlyGinProGlyAlaAspAsnArgGluCysLeuSer130135140MetAspTyrLysGinThrGinLeuCysLeuLeuGlyCysLysProPro145150155160ThrGlyGluHisTrpGlyLysGlyValAlaCysThrAsnAlaAlaPro165170175AlaAsnAspCysProProLeuGluLeuHeAsnThrlielieGluAsp180185190GlyAspMetValAspThrGlyPheGlyCysMetAspPheLysThrLeu195200205GinAlaAsnLysSerAspValProHeAsplieCysGlySerThrCys210215220LysTyrProAspTyrLeuLysMetThrSerGluProTyrGlyAspSer225230235240LeuPhePhePheLeuArgArgGluGinMetPheValArgHisPhePhe245250255AsnArgAlaGlyThrLeuGlyGluAlaValProAspAspLeuTyrlie260265270LysGlySerGlyThrThfAlaSerlieGinSerSerAlaPhePhePro275280285ThrProSerGlySerMetValThrSerGluSerGinUuPheAsnLys290295300ProTyrTrpLeuGinArgAlaGinGlyHisAsnAsnGlylieCysTrp305310315320GlyAsnGinValPheValThrValValAspThrThrArgSerThrAsn325330335M改ThrLeuCysThrGinValThrSerAspSerThrTyrLysAsnGlu340345350AsnPheLysGluTyrlieAfgHisValGluGluTyrAspLeuGinPhe355360365ValPheGinLeuCysLysValThrLeuThrAlaGluValMetThrTyr370375380lieHisAlaMetAsnProAspHeLeuGluAspTrpGinPheGlyLeu385390395400ThrProProProSerAlaSerLeuGinAspThrTyrArgPheValThr405410415SerGinAlalieThrCysGinLysThrValProProLysGluLysGlu420425430AspProLeuGlyLysTyrThrPheTipGluValAspLeuLysGluLys435440445PheSerAlaAspLeuAspGinPheProLeuGlyLysPheLeuLeu450455460GinAlaGly465<210>8<211>469<212>PRT<213>人工序列<220><223>HPV35LI<400>8GlySerGlyGlyGlyValTyrLeuProProValSerValSerLysVal151015ValSerThrAspGluTyrValThrArgThrAsnlieTyrTyrHisAla202530GlySerSerArgLeuLeuAlaValGlyHisProTyrTyrAlalieLys354045LysGinAspSerAsnLyslieAlaValProLysValSerGlyLeuGin505560TyrAigValPheArgValLysLeuProAspProAsnLysPh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rProValSerLysVal151015HeThrThrAspAlaTyrValLysArgThrThrliePheTyrHisAla202530GlySerSerArgLeuLeuThrValGlyHisProTyrTyrProHeSer354045LysSerGlyLysAlaAsplieProLysValSerAlaPheGinTyrArg505560ValPheArgValLeuProAspProAsnLysPheGlyLeuProAsp65707580ThrAsnHePheAsnProAspGinGluArgLeuValTrpAlaCysVal859095GlyLeuGlulieGlyArgGlyGinProLeuGlyValGlyValSerGly100105110HisProLeuPheAsnArgLeuAspAspThrGluSerSerSerlieAla115120125HeGinAspThrAlaProAspSerArgAspAsnValSerValAspPro130135140LysGinThrGinLeuCysHelieGlyCysAlaProAlalieGlyGlu145150155160HisTrpThrLysGlyThrAlaCysArgSerThrProThrThrAlaGly165170175AspCysProProLeuGluLeuHeAsnSerProlieGluAspGlyAsp180185l卯MetValAspThrGlyPheGlyAlaLeuAsnPheLysAlaLeuGinGlu195200205SerLysSerAspValPro'LeuAsplieValGinSerThrCysLysTyr210215220ProAspTyrLeuLysMetSerAlaAspAlaTyrGlyAspSerMetTrp225230235240PheTyrLeuArgArgGluGinLeuPheThrArgHisPhePheAsnArg245250255AlaGlyVallieGlyGluGluHeProAsnAspLeuTyrHeLysGly260265270SerAsnGlyArgAspProProProSerSerValTyrValAlaThrPro275280285SerGlySerMetlieThrSerGluAlaGinLeuPheAsnLysProTyr290295300TrpLeuGinArgAlaGinGlyHisAsnAsnGlylieCysTrpAsnAsn305310315320GinLeuPheValThrValValAspThrThrArgAsnThrAsnMetThr325330335UuSerAlaThrThrGinSerMetSerThrTyrAsnSerLysGinlie340345350LysGinTyrValArgHisAlaGluGluTyrGluLeuGinPheValPhe355360365GinLeuCysLyslieSerLeuSerAlaGluValMetAlaTyrLeuHis370375380ThrMetAsnSerThrLeuLeuGluAspTrpAsnlieGlyLeuSerPro385390395400ProValAlaThrSerLeuGluAspLysTyrArgTyrValLysSerAla405410415AlalieThrCysGinLysAspGinProProProGluLysGinAspPro420425430LeuSerLysTyrLysPheTrpGluValAsnLeuGinAsnSerPheSer435440445AlaAspLeuAspGinPheProLeuGlyArgLysPheLeuMetGinVal450455460Gly465<210>15<211>466<212>PRT<213>人工序列<220><223>HPV56U<400>15GlySerGlyGlyGlyVdTyrLeuProProThrProValSerLysVal151015ValAlaThrAspSerTyrValLysArgThrSerliePheTyrHisAla202530GlySerSerArgLeuLeuAlaValGlyHisProTyrTyrSerValThr354045LysAspAsnThrLysThrAsnHeProLysValSerAlaTyrGinTyr505560ArgValPheArgValArgLeuProAspProAsnLysPheGlyLeuPro65707580AspThrAsnlieTyrAsnProAspGinGluArgLeuValTrpAlaCys859095ValGlyLeuGluValGlyArgGlyGinProLeuGlyAlaGlyLeuSer100105110GlyHisProLeuPheAsnArgLeuAspAspThrGluSerSerAsnLeu115120125AlaAsnAsnAsnVallieGluAspSerAtgAspAsnlieSerValAsp130135140GlyLysGinThrGinLeuCyslieValGlyCysThrProAlaMetGly145150155160GluHisTrpThrLysGlyAlaValCysLysSerThrGinValThrThr165170175GlyAspCysProProLeuAlaLeulieAsnThrProlieGluAspGly180185190AspMetlieAspThrGlyPheGlyAlaMetAspPheLysValLeuGin195200205GluSerLysAlaGluValProLeuAsplieValGinSerThrCysLys210215220TyrProAspT>rLeuLysMetSerAlaAspAlaTyrGlyAspSerMet225230235240TrpPheTyrLeuArgAigGluGinLeuPheAlaArgHisTyrPheAsn245250255AigAlaGlyLysValGlyGluThrlieProAlaGluLeuTyrLeuLys260265270GlySerAsnGlyArgGluProProProSerSerValTyrValAlaThr275280285ProSerGlySerMetHeThrSerGluAlaGinLeuPheAsnLysPro2卯295300TyrTrpLeuGinArgAlaGinGlyHisAsnAsnGlylieCysTrpGly305310315320AsnGinLeuPheValThrValValAspTTirThrArgSerThrAsnMet325330335ThrlieSerThrAlaThrGluGinLeuSerLysTyrAspAlaArgLys340345350lieAsnGinTyrLeuArgHisValGluGluTyrGluLeuGinPheVal355360365PheGinLeuCysLysHeThrLeuSerAlaGluValMetAlaTyrLeu370375380HisAsnMetAsnAlaAsnLeuLeuGluAspTrpAsnlieGlyLeuSer385390395400ProProValAlaThrSerLeuGluAspLysTyrArgTyrValArgSer405410415ThrAlalieThrCysGinArgGluGinProProThrGluLysGinAsp420425430ProLeuAlaLysTyrLysPheTrpAspValAsnLeuGinAspSerPhe435440445SerThrAspLeuAspGinPheProLeuGlyArgLysPheLeuMetGin450455460LeuGly465<210>16<211>467<212>PRT<213>人工序列<220><223>HPV58Ll<400>16GlySerGlyGlyGlyValTyrLeuProProValProValSerLysVal151015ValSerThrAspGluTyrValSerArgThrSerHeTyrTyrTyrAla202530GlySerSerArgLeuLeuAlaValGlyAsnProTyrPheSerlieLys354045SerProAsnAsnAsnLysLysValLeuValProLysValSerGlyLeu505560GinTyrArgValPheArgValArgLeuProAspProAsnLysPheGly65707580PheProAspThrSerPheTyrAsnProAspThrGinArgLeuValTrp859095AlaCysValGlyLeuGlulieGlyArgGlyGinProLeuGlyValGly100105110ValSerGlyHisProTyrLeuAsnLysPheAspAspThrGluThrSer115120125AsnArgTyrProAlaGinProGlySerAspAsnArgGluCysLeuSer130135140MetAspTyrLysGinThrGinLeuCysLeulieGlyCysLysProPro145150155160ThrGlyGluHisTrpGlyLysGlyValA墓aCysAsnAsnAsnAlaAla165170175AlaThrAspCysProProLeuGluLeuPheAsnSerlielieGluAsp180185190GlyAspMetValAspThrGtyPheGlyCysMetAspPheGlyThrLeu195200205GinAlaAsnLysSerAspValProlieAsplieCysAsnSerThrCys210215220LysTyrProAspTyrLeuLysMetAlaSerGluProTyrGlyAspSer225230235240LeuPhePhePheLeuArgAigGluGinMetPheValArgHisPhePhe245250255AsnArgAlaGlyLysLeuGlyGluAlaValProAspAspLeuTyrlie260265270LysGlySerGlyAsnThrAlaValHeGinSerSerAlaPhePhePro275280285ThrProSerGlySerlieValThrSerGluSerGinLeuPheAsnLys290295300ProTyrTrpLeuGinArgAlaGinGlyHisAsnAsnGlylieCysTrp305310315320GlyAsnGinLeuPheValThrValValAspThrThrArgSerThrAsn325330335MetThrLeuCysThrGluValThrLysGluGlyThrTyrLysAsnAsp340345350AsnPheLysGluTyrValArgHisValGluGluTyrAspLeuGinPhe355360365ValPheGinLeuCysLyslieThrLeuThrAlaGluHeMetThrTyr370375380lieHisThrMetAspSerAsnlieLeuGluAspTrpGinPheGlyLeu385390395400ThrProProProSerAlaSerLeuGinAspThrTyrArgPheValThr405410415SerGinAlalieThrCysGinLysThrAlaProProLysGluLysGlu420425430AspProLeuAsnLysTyrThrPheTrpGluValAsnLeuLysGluLys435440445PheSerAlaAspLeuAspGinPheProLeuGlyArgLysPheLeuLeu450455460GinSerGly465<210>17<211>469<212>PRT<213>人工序列<220><223>HPV59L1<400>17GlySerGlyGlyGlyValTyrLeuProProProSerValAlaLysVal151015ValSerThrAspGluTyrValThrArgThrSerliePheTyrHisAla202530GlySerSerArgLeuLeuThrValGlyHisProTyrPheLysValPro354045LysGlyGlyAsnGlyArgGinAspValProLysValSerAlaTyrGin505560TyrArgValPheArgViedLysLeuProAspProAsnLysPheGlyLeu65707580ProAspAsnThrValT^rAspProAsnSerGinArgLeuValTrpAla859095CysValGlyValGlulieGlyAigGlyGinProLeuGlyValGlyLeul(W105110SerGlyHisProLeuTyrAsnLysLeuAspAspThrGluAsnSerHis115120125ValAlaSerAlaValAspThrLysAspThrA$AspAsnValSerVal130135140AspTyrLysGinThrGinLeuCyslieleGlyCysValProAlalie145150155160GlyGluHisTrpThrLysGlyThrAlaCysLysProThrThrValVal165170175GinGlyAspCysProProLeuGluLeulieAsnThrProlieGluAsp180185190GlyAspMetValAspThrGlyTyrGlyAlaMetAspPheLysLeuLeu195200205GinAspAsnLysSerGluValProLeuAsplieCysGinSerlieCys210215220LysTyrProAspTyrLeuGinMetSerAlaAspAlaTyrGlyAspSer225230235240MetPhePheCysLeuArgGluGinValPheAlaArgHisPheTrp245250255AsnArgSerGlyThrMetGlyAspGinLeuProGluSerLeuTyrlie260265270LysGlyThrAsplieAi"gAlaAsnProGlySerTyrLeuTyrSerPro275280285SerProSerGlySerValValThrSerAspSerGinLeuPheAsnLys2卯295300ProTyrTrpLeuHisLysAlaGinGlyLeuAsnAsnGlylieCysTrp305310315320HisAsnGinLeuPheLeuThrValValAspThrThrArgSerThrAsn325330335LeuSerValCysAlaSerThrThrSerSerlieProAsnValTyrThr340345350ProThrSerPheLysGluTyrAlaArgHisValGluGluPheAspLeu355360365GinPheliePheGinLeuCysLyslieThrLeuThrThrGluValMet370375380SerTyrlieHisAsnMetAsnThrThrlieLeuGluAspTrpAsnPhe385390395400GlyValThrProProProThrAlaSerLeuValAspThrTyrArgPhe405410415ValGinSerAlaAlaValThrCysGinLysAspThrAlaProProVal420425430LysGinAspProTyrAspLysLeuLysPheTipProValAspLeuLys435440445GluArgPheSerAlaAspLeuAspGinPheProLeuGlyArgLysPhe450455460<image>imageseeoriginaldocumentpage39</image>GlyGlyAsnGlyArgAspProProProSerSerValTyrValAlaThr275280285ProSerGlySerMetlieThrSerGluAlaGinLeuPheAsnLysPro290295300T>rTrpLeuGinArgAlaGinGlyHisAsnAsnGlyHeCysTrpGly305310315320AsnGinValPheValThrValValAspThrThrArgSerThrAsnMet325330335ThrlieAsnAlaAlaLysSerThrLeuThrLysTyrAspAlaArgGlu340345350lieAsnGinTyrLeuArgHisValGluGluTyrGluLeuGinPheVal355360365PheGinLeuCysLysHeThrLeuThrAlaGluValMetAlaTyrLeu370375380HisAsnMetAsnAsnThrLeuLeuAspAspTrpAsnlieGlyLeuSer385390395400ProProValAlaThrSerLeuGluAspLysTyrArgTyrlieLysSer405410415ThrAlalieThrCysGinArgGluGinProProAlaGluLysGinAsp420425430ProLeuAlaLysTyrLysPheTrpGluValAsnLeuGinAspSerPhe435440445SerAlaAspLeuAspGinPheProLeuGlyArgLysPheLeuMetGin450455460LeuGly465<210>19<211>470<212>PRT<213>人工序列<220><223>HPV73L1<400>19GlySerGlyGlyGlyValTyrLeuProProValSerValSerLysVal151015ValSerThrAspGluTyrValThrArgThrAsnlieTyrTyrTyrAla202530GlySerThrArgLeuLeuAlaValGlyHisProTyrPheProHeLys354045AspSerGinLysArgLysThrleValProLysValSerGlyLeuGin505560TyrArgValPheArgLeuArgLeuProAspProAsnLysPheGlyPhe65707580ProAspAlaSerPheTyrAsnProAspLysGluArgLeuValTrpAla859095CysSerGlyValGluValGlyArgGlyGinProLeuGlylieGlyThr100105110SerGlyAsnProPheMetAsnLysLeuAspAspThrGluAsnAlaPro115120125LysiyrlieAlaGlyGinAsnThrAspGlyArgGluCysMetSerVal130135140AspTyrLysGinThrGinLeuCyslieLeuGlyCysArgProProLeu145150155160GlyGluHisTrpGlyProGlyThrProCysThrSerGinThrValAsn165170175ThrGlyAspCysProProLeuGluLeuLysAsnThrProlieGinAsp180185190GlyAspMetlieAspValGlyPheGlyAlaMetAspPheLysAlaLeu195200205GinAlaAsnLysSerAspValProlieAsplieSerAsnThrThrCys210215220LysTyrProAspTyrLeuGlyMetAlaAlaAspProTyrGlyAspSer225230235240MetTrpPheTyrLeuArgArgGluGinMetPheValArgHisLeuPhe245250255AsnArgAlaGlyAspThrGlyAspLyslieProAspAspLeuMetlie260265270LysGlyThrGlyAsnThrAlaThrProSerSerCysValPheTyrPro275280285ThrProSerGlySerMetValSerSerAspAlaGinLeuPheAsnLys2卯295300ProTyrTrpLeuGinLysAlaGinGlyGinAsnAsnGlyHeCysTrp305310315320HisAsnGinLeuPheLeuThrValValAspThrThrArgSerThrAsn325330335PheSerValCysValGlyThrGinAlaSerSerSerThrThrThrTyr340345350AlaAsnSerAsnPheLysGluTyrLeuArgHisAlaGluGluPheAsp355360365LeuGinPheValPheGinLeuCysLysHeSerLeuThrThrGluVal370375380MetThrTyrlieHisSerMetAsnSerThrHeLeuGluGluTrpAsn385390395400PheGlyLeuThrProProProSerGlyThrLeuGluGluThrTyrArg405410415TyrValThrSerGinAlaHeSerCysGinArgProGinProProLys420425430GluThrGluAspProTyrAlaLysLeuSerPheTrpAspValAspLeu435440445LysGluLysPheSerAlaGluLeuAspGinPheProLeuGlyArgLys450455460PheLeuLeuGinLeuGly465470<210>20<211>469<212>PRT<213>人工序列<220><223>HPV83L1<400>20GlySerGlyGlyGlyValTyrLeuProProAlaProValSerArglie151015ValAsnThrGluGluTyrlieThrArgTiffGlyHeTyrTyrTyrAla202530GlySerSerArgLeuHeThrLeuGlyHisProTyrPheSerliePro354045LysThrAsnThrArgAlaGluHeProLysValSerAlaPheGinTyr505560AigValPheArgValGinLeuProAspProAsnLysPheGlyLeuPro65707580AspProAsnLeuPheAsnProAspThrAspArgLeuValTrpGlyCys859095ValGlyValGluValGlyArgGlyGinProLeuGlyValGlyLeuSer100105110GlyHisProLeuPheAsnLysTyrAspAspThrGluAsnSerArgPhe115120125AlaAsnGlyAsnAspGinGinAspValArgAspAsnlieSerValAsp130135140AsnLysGinThrGinLeuCyslielieGlyCysAlaProProlieGly145150155160GluHisTrpAlaThrGlyThrThrCysLysAsnValProValProGin165170175GlyAspCysProProLeuGluLeuValSerThrVallieGluAspGly180185190AspMetValAspThrGlyPheGlyAlaMetAspPheAlaAsnLeuGin195200205AlaThrLysSerAspValProLeuAsplieAlaGinSerValCysLys210215220TyrProAspTyrLeuLysMetSerAlaAspThrTyrGlyAsnSerMet225230235240PhePheHisLeuArgArgGluGinliePheAlaArgHisTyrTyrAsn245250255LysAlaGlyValValGlyAspAlaHeProAspLysAlaTyrlieLys260265270GlyThrGlyAlaGlyA堪AspProlieSerSerTyrlieTyrSerAla275280285ThrProSerGlySerMetlieThrSerAspSerGinliePheAsnLys2卯295300ProTyrTrpLeuHisArgAlaGinGlyHisAsnAsnGlyHeCysTrp305310315320AsnAsnGinLeuPhelieThrCysValAspThrThrLysSerThrAsn325330335LeuThrHeSerThrAlaValThrProSerValAlaGinThrPheThr340345350ProAlaAsnHieLysGinTyrlieArgHisGlyGluGluTyrGluLeu355360365GinPheliePheGinLeuCysLyslieThrLeuThrThrGlulieMet370375380AlaTyrLeuHisThrMetAspSerThrlieLeuGluGinTrpAsnPhe385390395400GlyLeuThrLeuProProSerAlaSerLeuGluAspAlaTyrArgPhe405410415ValLysAsnAlaAlaThrSerCysHisLysAspSerProProGinAla420425430LysGluAspProLeuAlaLysT^rLysPheTrpAsnValAspLeuLys435440445GluArgPheSerLeuAspLeuAspGinPheAlaLeuGlyArgLysPhe450455460LeuLeuGinlieGly46權(quán)利要求1.一種重組人乳頭瘤病毒L1衣殼蛋白的氨基酸序列，其特征在于將野生型HPVL1蛋白的氨基酸序列N-末端的保守序列VYLPP或VYVPP上游的氨基酸序列替換成GSGGG，同時(shí)在其C-末端的保守序列LGRKFL下游第5個(gè)氨基酸處通過引入蛋白質(zhì)翻譯的終止密碼子形成相對(duì)于野生型的截短型序列，其中所述的HPV型選自HPV6、HPV11、HPV16、HPV18、HPV26、HPV31、HPV33、HPV35、HPV39、HPV42、HPV45、HPV51、HPV52、HPV53、HPV56、HPV58、HPV59、HPV66、HPV73、HPV82中的一種或多種。2、根據(jù)權(quán)利要求1所述的氨基酸序列，其中HPV6對(duì)應(yīng)的氨基酸序列如SEQIDNO:l所示，HPV11對(duì)應(yīng)的氨基酸序列如SEQIDNO:2所示，HPV16對(duì)應(yīng)的氨基酸序列如SEQIDNO:3所示，HPV18對(duì)應(yīng)的氨基酸序列如SEQIDNO:4所示，HPV26對(duì)應(yīng)的氨基酸序列如SEQIDNO:5所示，HPV31對(duì)應(yīng)的氨基酸序列如SEQIDNO:6所示，HPV33對(duì)應(yīng)的氨基酸序列如SEQIDNO:7所示，HPV35對(duì)應(yīng)的氨基酸序列如SEQIDNO:8所示，HPV39對(duì)應(yīng)的氨基酸序列如SEQIDNO:9所示，HPV42對(duì)應(yīng)的氨基酸序列如SEQIDNO:10所示，HPV45對(duì)應(yīng)的氨基酸序列如SEQIDNO:ll所示，HPV51對(duì)應(yīng)的氨基酸序列如SEQIDNO:12所示，HPV52對(duì)應(yīng)的氨基酸序列如SEQIDNO:13所示，HPV53對(duì)應(yīng)的氨基酸序列如SEQIDNO:14所示，HPV56對(duì)應(yīng)的氨基酸序列如SEQIDNO:15所示，HPV58對(duì)應(yīng)的氨基酸序列如SEQIDNO:16所示，HPV59對(duì)應(yīng)的氨基酸序列如SEQIDNO:17所示，HPV66對(duì)應(yīng)的氨基酸序列如SEQIDNO:18所示，HPV73對(duì)應(yīng)的氨基酸序列如SEQIDNO:19所示，HPV82對(duì)應(yīng)的氨基酸序列如SEQIDNO:20所示。3、一種核酸分子，其特征在于它包含編碼權(quán)利要求1所述的氨基酸序列的核苷酸序列。4、一種表達(dá)載體，其特征在于它包含權(quán)利要求3所述的核苷酸序列。5、一種宿主細(xì)胞，其特征在于它包含權(quán)利要求4所述的載體。6、根據(jù)權(quán)利要求5所述的宿主細(xì)胞，其特征在于它為大腸桿菌細(xì)胞。7、一種HPVLl蛋白的五聚體，其特征在于它的一級(jí)結(jié)構(gòu)由權(quán)利要求l所述的氨基酸序列所組成，其二級(jí)結(jié)構(gòu)和高級(jí)結(jié)構(gòu)由上述一級(jí)結(jié)構(gòu)所決定。8、根據(jù)權(quán)利要求7所述的HPVL1蛋白的五聚體在制備預(yù)防HPV感染的疫苗中的應(yīng)用。9、根據(jù)權(quán)利要求8所述的應(yīng)用，其特征在于用于制備所述的預(yù)防HPV感染的疫苗的HPV型包括HPV16。10、根據(jù)權(quán)利要求8所述的應(yīng)用，其特征在于用于制備所述的預(yù)防HPV感染的疫苗的HPV型包括HPV6、HPVll、HPV16、HPV18和HPV33。11、根據(jù)權(quán)利要求7所述的HPVL1蛋白的五聚體在制備藥物組合物中的應(yīng)用.12、根據(jù)權(quán)利要求7所述的HPVL1蛋白的五聚體在制備免疫診斷抗原或抗體中的應(yīng)用。全文摘要本發(fā)明涉及重組人乳頭瘤病毒L1衣殼蛋白的氨基酸序列，編碼該氨基酸序列的核苷酸序列，和包含所述核苷酸序列的重組載體和表達(dá)宿主。本發(fā)明還涉及由該氨基酸序列所組成的HPVL1蛋白在制備疫苗、藥物組合物和診斷抗原或抗體中的應(yīng)用。本發(fā)明通過對(duì)HPVL1野生型序列的改造，使得在原核系統(tǒng)中表達(dá)的重組HPVL1衣殼蛋白能溶于水，而且表達(dá)得到的是與HPVL1的VLP具有相同免疫原性和抗原性的L1五聚體。本發(fā)明使得利用原核表達(dá)系統(tǒng)工業(yè)生產(chǎn)HPVL1衣殼蛋白成為現(xiàn)實(shí)，相對(duì)于目前采用的真核表達(dá)系統(tǒng)具有產(chǎn)品質(zhì)量更穩(wěn)定、產(chǎn)率更高、成本低、質(zhì)量控制方便的優(yōu)點(diǎn)，具有重大的經(jīng)濟(jì)效益和社會(huì)效應(yīng)。文檔編號(hào)C12N15/63GK101245099SQ200710005099公開日2008年8月20日申請(qǐng)日期2007年2月14日優(yōu)先權(quán)日2007年2月14日發(fā)明者陳小江,馬潤林申請(qǐng)人:馬潤林;陳小江