人乳头瘤病毒16型野毒株L1-E7融合基因重组腺病毒载体构建及在293细胞中表达嵌合病毒样颗粒的研究  

Construction of an attenuated recombinant adenoviral vaccine of human papillomavirus type 16 L1-E7 and expression of the chimeric virus like particles in 293 cells

作　　者：卞继峰[1] 于修平[1] 赵蔚明[1] 杨海宁[1] 王芸[1] 胡海燕[1] 周亚滨[1] 贾继辉[1] 栾怡[1] 齐眉[1] 耿昭[1] 

机构地区：[1]山东大学医学院分子生物学实验室,济南250012

出　　处：《中华微生物学和免疫学杂志》2001年第4期368-372,共5页Chinese Journal of Microbiology and Immunology

基　　金：国家自然科学基金 (396 70 0 38);山东省自然科学基金(Y96C10 0 42 )资助项目

摘　　要：目的　制备人乳头瘤病毒HPV16L1 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。方法　以HPV16型的野毒株HPV16 114/K为模板 ,利用PCR克隆技术制备HPV16L1 E7融合基因pUC19L1 E7,含L1的 1～ 30 1氨基酸 (AA)和E7的 1～ 6 0AA的编码DNA序列 ;并转入腺病毒穿梭质粒pCA14L1 E7,与腺病毒质粒pBHG10共转染 2 93细胞 ,筛选重组腺病毒rAd5HPV16L1 E7。以PCR、ELISA和Westernblot方法鉴定重组病毒及其表达的L1 E7蛋白 ,密度梯度超速离心纯化HPV16嵌合L1 E7VLP ,电镜观察VLP的形态结构。结果　PCR DNA序列分析表明成功构建了HPV16L1 E7重组质粒pUC19L1 E7;并获得HPV16重组腺病毒rAd5HPV16L1 E7,在 2 93细胞中可高效表达L1 E7蛋白 ,并形成嵌合病毒样颗粒 (cVLP)。结论　构建的重组腺病毒rAd5HPV16L1 E7可有效表达HPV16L1 E7cVLP ,可进一步用于动物实验 。Objective Human papillomaviruses can not be cultured in vitro now, and have tumorgenicity. These properties of HPV severely hampered the vaccine development of human papillomavirus. In order to develop an attenuated recombinant adenovirus vaccine against HPV infection and human cervical cancer, the recombinant adenoviral of HPV16 was constructed. Methods The plasmid pUC19L1 E7 contained L1 ORF and E7 ORF was constructed using polymerase chain reaction. The L1 E7 DNA fragment was digested with HindⅢand SmaⅠ and inserted into pBluescript, to prepare pBSL1 E7. The L1 E7 DNA fragment was cut with HindⅢ and XbaⅠ and inserted into adenoviral shuttle plasmid pCA14, and then cotransfected 293 cells with the adenovirus pBHG10 plasmid, harvested the recombinant adenovirus of HPV16. The expression of the chimeric virus like particle was detected using ELISA, immunoblot assay and the electron microscopy. Results The fusion L1 E7 protein was expressed at high level and able to self assemble into chimeric virus like particle (cVLP), which is morphologically indistinguishable from the native virions and display the conformational epitopes. Conclusion We constructed the recombinant adenovirus of human papillomavirus type 16L1 E7, the L1 E7 fusion protein can be expressed in high level, and can self assemble into the chimeric virus like particles in human cells. The recombinant pUC19L1 plasmid was an potential vector for constructing other chimeric vaccines with the epitopes derived from virus and tumor antigen.

关 键 词：乳头状瘤病毒 病毒样颗粒 腺病毒载体 疫苗 宫颈肿瘤 宫颈癌 

分 类 号：R346[医药卫生—基础医学]