HIV-1C亚型nef基因构建的DNA质粒诱导小鼠免疫反应的研究

Cellular and humoral immune responses induced by DNA plasmid constructed by HIV-1 clade C nef gene

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 DNA plasmid expressing HIV-1 clade C nef gene and to investigate its cellular and humoral immune responses. Methods The HIV-1 clade C nef gene was inserted into expression vector pVAX to construct the recombinant plasmid, pVAX-nef. The recombinant plasmid was transfected into 293T cell. Western blot was performed to examine the expression of nef gene. BALB/c mice were immunized with the recombinant plasmid and then the cellular immune response was analyzed by ELISPOT assay, the humoral immune response was tested by ELISA method. Results The pVAX-nef recombinant placid was successfully constructed and the Nef protein was successfully expressed in 293T cell. Two weeks after pVAX-nef recombinant plasmid inoculation, HIV-specific CD4^＋ and CD8^＋ immune responses were detected by ELISPOT assay, with a positive correlation between the dose of the recombinant plasmid and the immune response. ELISA method showed that anti-HIV-1 was successfully induced and the mice immunized with 40 μg pVAX-nef recombinant plasmid produced a highest level of anti-HIV-1. Conclusion The HIV-1 clade C DNA plasmid pVAX-nef could effectively induce cellular and humoral immune responses in BALB/c mice. These immunological effects showed positive correlation with immunization dose.