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目的构建表达中国流行株HIV-1C亚型调控nef基因的重组质粒pVAX-nef,并免疫BALB/c小鼠,观察其免疫效果,为探索新型HIV DNA疫苗提供数据。方法利用分子生物学技术,将nef基因克隆到pVAX,并在体外进行表达与鉴定。以纯化的Nef蛋白作为包被抗原,用ELISA检测其体液免疫反应,用ELISPOT检测其细胞免疫反应。结果重组质粒pVAX-nef成功构建。接种小鼠后2周,ELISPOT结果显示产生了针对HIV特异的CD4和CD8细胞抗原表位的免疫应答,且与免疫剂量存在一定的正相关性。ELISA实验诱导产生了抗HIV-1特异性抗体,其中40μg免疫组诱导的抗体水平最高。结论重组质粒pVAX-nef免疫小鼠后可有效地诱导机体产生细胞免疫和体液免疫反应。
Objective To construct a recombinant plasmid pVAX-nef expressing the HIV-1 subtype of HIV-1 subtype of Chinese strain and immunize BALB / c mice to observe its immune effect and provide data for exploring novel HIV DNA vaccine. Methods Using molecular biology techniques, the nef gene was cloned into pVAX and expressed and identified in vitro. Purified Nef protein was used as coating antigen, humoral immune response was detected by ELISA, and cellular immune response was detected by ELISPOT. Results The recombinant plasmid pVAX-nef was successfully constructed. Two weeks after vaccination, the ELISPOT results showed that there was an immune response against HIV specific epitopes of CD4 and CD8 cells, and there was a certain positive correlation with the immunization dose. The anti-HIV-1 specific antibody was induced by ELISA experiment, and the highest level of antibody was induced by 40μg immune group. Conclusion The recombinant plasmid pVAX-nef immunized mice can effectively induce cellular immune response and humoral immune response.