表达HPV16 E7基因的重组腺病毒质粒的构建及鉴定  

Construction and identification of a recombinant adenovirus expressing HPV16 E7 gene

作　　者：邵云亭 楼张蓉 符军 李山虎 黄芳 王芃 吴成君 Shao Yunting;Lou Zhangrong;Fu Jun;Li Shanhu;Huang Fang;Wang Peng;Wu Chengjun(School of Basic Medical Sciences,Faculty of Medicine,Dalian University of Technology,Dalian 116024,China;State Key Laboratory of Microbial Technology,Shandong University,Jinan 250100,China;Department of Cell Engineering,Beijing Institute of Biotechnology,Beijing 100850,China)

机构地区：[1]大连理工大学医学部基础医学院,116024 [2]山东大学微生物技术国家重点实验室,济南250100 [3]军事医学研究院生物工程研究所,北京100850

出　　处：《国际病毒学杂志》2024年第1期22-27,共6页International Journal of Virology

摘　　要：目的构建表达HPV16E7基因的重组腺病毒质粒.方法设计携带酶切位点和无酶切位点的两对引物以扩增ccdBKan目的片段.分别将ccdBKan与质粒pBR322-Ad4-eGFP共转至电转感受态GB08red-gyr462构建两种表达ccdBKan基因的重组Ad4质粒.利用ccdB正反筛选系统与ExoCET/Red重组结合将HPV16E7基因插入Ad4的E1A区,构建表达HPV16 E7基因的重组腺病毒质粒pBR322-Ad4-E 1 Amut-C16E7P,经酶切、测序验证.提取质粒pBR322-Ad4-EIAmut-C 16E7P释放基因组,转染293T细胞以包装和扩增病毒,用病毒感染293T细胞,检测HPV16E7基因在细胞内的转录水平和蛋白表达.重组病毒经肌肉接种BALB/C裸鼠,收集小鼠血清用于病毒中和实验.结果成功构建表达HPV16 E7基因的重组腺病毒质粒,并在293T细胞中成功包装出重组病毒.qRT-PCR和Western blotting验证了重组病毒HPV16 E7基因的成功表达,病毒中和实验结果提示经重组病毒免疫后的小鼠血清能够中和Ad4-HPV16E7.结论成功构建了表达HPV16E7基因的重组腺病毒株,为进一步研究HPV16 E7在癌症发病机制中的作用以及HPV感染相关癌症的治疗方法提供新的思路和依据,并且可能为HPV治疗性疫苗的研究探索提供了一种潜在策略.Objective To construct a recombinant adenovirus expressing the HPV16 E7 gene.Methods Two primer pairs,with and without respectively the insertion restriction site,were designed to amplify the target fragment of ccdBKan gene.Each of the ccdBKan products were transferred with the plasmid pBR322-Ad4-eGFP into electrocompetent strain of GB08red-gyr462 to construct two recombinant Ad4 plasmids expressing ccdBKan gene.The ccdB positive/negative selection method and ExoCET/Red recombination technology were used to insert the HPV16E7 gene into the E1A region of Ad4 and constructed a recombinant adenovirus plasmid pBR322-Ad4-E1Amut-C16E7P that can express HPV16 E7 gene.The recombinant adenovirus plasmid was confirmed by enzyme digestion and sequencing.The plasmid pBR322-Ad4-EIAmut-C16E7P was isolated to release the genome.The genome was used in the transfection of 293T cells for virus package and amplification.The viruses were used to infect 293T cells for the transcription level and protein expression of HPV16 E7 gene in the cells.The recombinant virus was inoculated into mice by intramuscular injection.The mouse serum was collected for virus neutralization assays.Results The recombinant adenovirus plasmid expressing the HPV16 E7 gene was constructed successfully and the recombinant virus Ad4-HPV16E7 was packaged in 293T cells.The qRT-PCR and western blotting results confirmed the successful expression of the HPV16 E7 gene of the recombinant adenovirus Ad4-HPV16E7 in 293T cells.Virus neutralization assay indicated that the serum from mice immunized with Ad4-HPV16E7 can neutralize the activity of Ad4-HPV16E7.Conclusions A recombinant adenovirus strain expressing the HPV16 E7 gene was successfully generated that provided new perspectives and a foundation for further investigating the role of HPV16 E7 in cancer pathogenesis and potential therapeutic strategies for HPV-induced cancers.Additionally,this work may also provide a potential strategy for exploring therapeutic HPV vaccines.

关 键 词：HPV16 E7基因 腺病毒 重组病毒 

分 类 号：R511[医药卫生—内科学]