表达HPV18E7E6的重组痘苗病毒构建及免疫效果的初步研究

摘要

目的 构建表达HPV18E7E6融合蛋白的重组痘苗病毒,并对E7E6蛋白的免疫原性进行研究.方法 将去除了转化活性的HPV18E6、E7基因融合,插入痘苗病毒重组质粒,通过同源重组构建表达HPV18E7E6的重组痘苗病毒,观察其免疫效果.结果 构建了表达E7E6融合蛋白的重组痘苗病毒,PCR鉴定及测序表明融合基因序列与设计相符,正确插入到痘苗病毒TK区域;Western-Blot检测表明该重组病毒能表达HPV18E7E6融合蛋白.免疫后的小鼠可产生E6、E7特异性抗体,但ELISPOT没检测到E7肽库刺激小鼠脾细胞产生分泌IFN-丫的阳性反应.结论 构建了一株表达HPV18E7E6融合蛋白的重组痘苗病毒,可以有效诱发小鼠产生针对E6、E7的体液免疫,但不能诱发产生相应的细胞免疫,为进一步研究不同动物模型中HPV18E6E7的细胞免疫特点提供了实验基础.%Objective To construct one recombinant vaccinia virus expressing the HPV18.E6 and E7fusion proteins as HPV18 therapeutic vaccine candidate, and test its immunogenicity. Methods The fusion E7E6genes were synthesized and mutated to inactivate their oncogenic potential, and inserted into a vaccine virus plasmid vector to construct one recombinant vaccinia virus. Finally its immunogenicity was characterized in immunized mice. Results One recombinant vaccinia virus expressing HPV18 E7E6 fusion proteins was constructed. Sequencing Results of PCR products and Western blot tests showed that the E7E6 fusion genes were correct and expressed in CEF cells infected with the recombinant vaccinia virus. The specific antibodies against E6and E7 proteins were elicited, however no positive responses were detected by ELISPOT in immunized mice. Conclusion s One recombinant vaccinia virus expressing HPVI8 E7E6 fusion proteins was generated and elicited specific antibodies against E6 and E7 proteins, but detected no positive cellular immune responses in immunized mice, which will provide the basis to develop the different animal model for examining the cellular immune responses of HPV18E6 and E7 proteins.