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  

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

作　　者：田厚文[1] 任皎[1] 黄薇[1] 范江涛[1] 赵莉[1] 阮力[1] 

机构地区：[1]中国疾病预防控制中心病毒病预防控制所,北京100052

出　　处：《病毒学报》2006年第5期358-363,共6页Chinese Journal of Virology

基　　金：国家高技术研究发展计划(863)2002AA216041

摘　　要：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。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.

关 键 词：乳多空病毒科人乳头状瘤病毒16型 宫颈肿瘤 疫苗 早期蛋白 

分 类 号：R373[医药卫生—病原生物学]