HPV16 L1-E7重组腺病毒rAd5 HPV16 L1-E7的构建及克隆表达

CONSTRUCTION AND EXPRESSION OF AN ATTENUATED RECOMBINANT ADENOVIRUS OF HUMAN PAPILLOMAVIRUS TYPE 16 L1-E7

目的 :研制人乳头瘤病毒 16型 (HPV16)L1 E7重组腺病毒 ,以期获得防治宫颈癌的重组腺病毒减毒活疫苗和嵌合病毒样颗粒疫苗。方法 :以HPV16型野毒株HPV16 114/K为模板 ,利用PCR克隆技术构建可表达HPV16主要衣壳蛋白L1( 1 30 1aa)和转化蛋白E7( 1 60aa)融合基因的重组质粒 pUC19L1 E7,HPV16L1 E7DNA片段经质粒 pBSL1 E7转入腺病毒Ad5穿梭质粒 pCA14L1 E7,与腺病毒质粒pBHG10 共转染 2 93细胞 ,制备重组腺病毒rAd5HPV16L1 E7,密度梯度超速离心纯化HPV16嵌合L1 E7VLP。结果 :成功构建了HPV16L1 E7重组质粒 pUC19L1 E7,制备HPV16L1 E7重组腺病毒rAd5HPV 16L1 E7,HPV16L1 E7融合蛋白可在 2 93细胞中高效表达 ,可达细胞总蛋白的 10 %以上 ,并装配成嵌合病毒样颗粒 (cVLP)。结论 :成功制备了可高效表达HPV16L1 E7嵌合L1 E7VLP的重组腺病毒rAd5HPV 16L1 E7载体疫苗 ,为HPV重组腺病毒疫苗用于宫颈癌的防治打下了基础。构建的 pUC19L1载体可以比较方便的插入外源基因 ,用来构建多价疫苗 。

Objective:To develop an attenuated recombinant adenoviral vaccine against HPV16 infection and human cervical cancer,the recombinant adenoviral vaccine of HPV was constructedMethods:The plasmid pUC19L1E7 contained the major capsid protein L1 ORF and E7 ORF was constructed using polymerase chain reaction.The L1E7 DNA fragment was digested with HindIII and SmaI and inserted into pBluescript,named pBSL1E7,and then the L1E7 DNA fragment was cut with HindIII and XbaI and inserted into adenoviral shuttle plasmid pCA14,and cotransfected 293 cells with the adenovirus pBHG10 plasmid,harvested the recombinant adenovirus of HPV16.The expression level of the chimeric viruslike paricle was detected using ELISA, immunoblot assay and the electron microscopy.Results:The fusion L1E7 protein was expressed at the high level(10% of total cellular protein,TCR)and can self assembled into the chimeric viruslike particle(cVLP),which is morphologically indistinguishable from the naitive virions and display the conformational epitopes.Conclusion:These data indicated that we constructed the recombinant abenovirus of human papillomavirus type 16 L1E7,the L1E7 fusion protein can be expressed at high level,and can self assemble into the chimeric viruslike particles in human cells.The recombinant pUC19L1 plasmid was an ideal vector for constructing other chimeric vaccine carried the epitopes derived from virus and tumor antigen.