论文部分内容阅读
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 ,因此在制备HPV16多价疫苗方面具有广泛的应用价值
Objective: To develop human papillomavirus type 16 (HPV16) L1 E7 recombinant adenovirus in order to obtain live attenuated recombinant adenovirus vaccine against cervical cancer and chimeric virus-like particle vaccine. Methods: The recombinant plasmid pUC19L1 E7 expressing HPV16 major capsid protein L1 (130a1a) and transforming protein E7 (160a) fusion gene was constructed by PCR cloning technique using HPV16 114 / K as template. HPV16 L1 E7DNA fragment was transfected into adenoviral Ad5 shuttle plasmid pCA14L1 E7 by plasmid pBSL1 E7, and cotransfected 293 cells with adenovirus plasmid pBHG10 to prepare recombinant adenovirus rAd5HPV16L1 E7. The HPV16 chimeric L1 E7VLP was purified by density gradient ultracentrifugation. Results: The recombinant plasmid pUC19L1 E7 of HPV16L1 E7 was successfully constructed and the recombinant adenovirus rAd5HPV 16L1 E7 of HPV16L1 E7 was successfully constructed. The HPV16L1 E7 fusion protein was highly expressed in 293 cells up to 10% of the total cellular protein and assembled into a chimeric Virus-like particles (cVLP). Conclusion: The recombinant adenovirus rAd5HPV 16L1 E7 vector vaccine expressing HPV16L1 E7 chimeric L1 E7VLP was successfully prepared, which laid the foundation for the prevention and treatment of cervical cancer with HPV recombinant adenovirus vaccine. The constructed pUC19L1 vector can insert foreign gene conveniently and construct multivalent vaccine, so it has a wide range of application value in preparing multivalent HPV16 vaccine