表达人乳头瘤病毒16型E6/E7融合蛋白的非复制型重组痘苗病毒的构建及免疫效果观察

采用基因工程方法将HPV16E6、E7基因融合后插入痘苗病毒载体,通过同源重组构建表达人乳头瘤病毒16型E6/E7融合蛋白的非复制型重组痘苗病毒疫苗,用C57BL/6小鼠观察其免疫原性和抗肿瘤移植情况。测序结果表明融合的HPV16E6、E7基因序列与设计相符;构建的非复制型重组痘苗病毒经Dot blot鉴定,显示有E6、E7融合基因的插入;Western blot检测表明该重组病毒在鸡胚成纤维细胞中能表达HPV16型E6/E7融合蛋白。动物免疫试验表明,该重组病毒在小鼠体内可诱发E6、E7特异性抗体;被免疫小鼠能抵抗TC-1肿瘤细胞的攻击。此结果为将来进一步研制HPV16、18型联合疫苗打下了基础。

Construction of Non-replicating Recombinant Vaccinia Virus Expressing Human Papillomavirus 16E6/E7 Fusion Protein and Study of Its Immunogenicity and Antitumor Response

The infection of human papillomavirus type 16 can cause neoplasia,that is closely associated with the development of cervical cancers.In this study, attempts were made to generate a HPV16 therapeutic vaccine candidate,which is capable of helping treatment of cervical cancer.The HPV16 E6/E7 fusion gene was constructed first and then inserted into a vaccinia virus vector.A strain of recombinant vaccinia virus was constructed through homologous recombination in chicken embryo fibroblast cells(CEF).Sequencing result showed that sequence of the mE6/E7 fusion gene was correct.Dot blot test confirmed that mE6/E7 fusion gene had been integrated in recombinant vaccinia virus DNA.Western blot test showed that the mE6/E7 fusion protein was expressed in CEF cells infected with the recombinant vaccinia virus.The ELISA result indicated that NTVJ16mE6/E7 could elicit specific antibodies against E6 and E7 proteins in immunized animals.In vivo tumor protection test indicated that tumor formation was retarded or prevented in C57BL/6 mice vaccinated with NTV16JmE6/E7,when challenged by syngenetic HPV16E6 and E7 transformed tumor cells.We had thus obtained a strain of replication-deficient recombinant vaccinia virus expressing HPV16 mE6/E7 fusion protein,it might have the potential to provide as a vaccine candidate for treatment of HPV-associated tumors and the prevention of precancerous transformations.