人乳头瘤病毒16型晚期蛋白L1基因重组MVA病毒的构建及鉴定

目的研制预防人乳头瘤病毒(HPV)感染的重组修饰的Ankara痘病毒(MVA)活载体疫苗。方法将HPV16L1基因插入MVA病毒转移载体psc11M2的P7·5启动子的下游,构建转移质粒psc11M2-L1,与MVA共转染鸡胚成纤维细胞(CEF),进行同源重组。通过9轮蓝斑筛选,以PCR和Westernblot鉴定重组病毒。结果获得的含目的片段的重组MVA病毒,表达产物与鼠抗人HPV16L1抗体发生阳性反应,目的蛋白相对分子质量约为55000。结论已成功地构建了可正确表达HPV16L1基因的重组MVA病毒株。

Construction and Identification of Recombinant Modified Vaccine Virus Ankara for Expression of Late Protein L1 Gene of Human Papillomavirus Type 16

Objective To prepare recombinant modified vaccine virus Ankara(MVA) for the prevention of infection with human papillomavirus(HPV).Methods Insert the late protein L1 gene of HPV16 downstream to the P_ 7.5 promoter of transferring vector psc11M2.Co-transfect chikc embryo fibroblast(CEF) with the constructed recombinant plasmid psc11M2-L1 and MVA for homologous recombination.After nine cycles of screening,identify the recombinant MVA by PCR and the expressed product by Western blot.Results The recombinant MVA containing the target gene fragment was obtained.The expressed product,with a relative molecular weight of 55 000,reacted with mouse anti-human HPV16 L1 antibody.Conclusion The recombinant MVA for expression of HPV16 L1 gene was successfully constructed.