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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 ,研究其在防治宫颈癌中的作用
Objective To prepare human papilloma virus (HPV) 16L1 E7 recombinant adenovirus in order to obtain a live attenuated recombinant adenovirus vaccine against cervical cancer. Methods The coding sequence of HPV16L1 E7 fusion gene pUC19L1 E7, 1 ~ 30 1 amino acids (L1) and 1 ~ 6 0AA of E7 were prepared by PCR cloning technique using HPV16 114 / K as a template. The recombinant adenoviruses were transfected into adenoviral shuttle plasmid pCA14L1 E7 and cotransfected into 293 cells with adenovirus plasmid pBHG10 to screen recombinant adenovirus rAd5HPV16L1 E7. The recombinant virus and its expression of L1 E7 protein were identified by PCR, ELISA and Western blotting. HPV16 chimeric L1 E7VLP was purified by density gradient ultracentrifugation and the morphology of VLP was observed by electron microscopy. Results PCR DNA sequence analysis showed that HPV16L1 E7 recombinant plasmid pUC19L1 E7 was successfully constructed and HPV16 recombinant adenovirus rAd5HPV16L1 E7 was obtained. L1 E7 protein was highly expressed in 293 cells and chimeric virus-like particles (cVLP) were formed. Conclusion The constructed recombinant adenovirus rAd5HPV16L1 E7 can effectively express HPV16L1 E7cVLP and can be further used in animal experiments to study its role in the prevention and treatment of cervical cancer