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目的:构建基于中国HIV-1 CRF01_AE重组亚型包膜糖蛋白gp41 NHR结构域N51的亚单位疫苗,并进行免疫原性研究。方法:设计4条引物,运用重叠延伸PCR方法扩增出N51Fd基因,将其插入真核表达载体pFUSE-hIgG1-Fc2,构建重组质粒pFUSE/N51Fd并进行序列测定。Western blot法检测N51FdFc-AE重组蛋白的表达。用纯化蛋白免疫BALB/c小鼠后,ELISA法检测小鼠的抗体反应。结果:成功构建了pFUSE/N51Fd重组质粒,N51FdFc-AE重组蛋白在真核体系获得了高效表达,Western blot结果显示在相对分子质量(Mr)35 000处有目的蛋白条带。小鼠抗血清能特异性识别源于gp41 NHR的抗原,效价高达1∶102 400,平均效价为1∶51 200。结论:改造后的亚单位疫苗能有效激活机体的免疫响应,可用于HIV候选疫苗的研发。
OBJECTIVE: To construct a subunit vaccine based on the NHL domain N51 of the HIV-1 CRF01_AE recombinant subtype envelope glycoprotein and study its immunogenicity. Methods: Four primers were designed. The N51Fd gene was amplified by overlap extension PCR and inserted into eukaryotic expression vector pFUSE-hIgG1-Fc2. The recombinant plasmid pFUSE / N51Fd was constructed and sequenced. Western blot was used to detect the expression of N51FdFc-AE recombinant protein. BALB / c mice were immunized with the purified protein, and the antibody response in mice was detected by ELISA. Results: The recombinant plasmid pFUSE / N51Fd was successfully constructed. The recombinant protein N51FdFc-AE was highly expressed in eukaryotic system. The result of Western blot showed that the recombinant protein was expressed at the 35 000 molecular weight (Mr). Mouse antisera can specifically recognize antigens derived from gp41 NHR with titers as high as 1: 102 400 and an average titer of 1: 51 200. Conclusion: The modified subunit vaccine can effectively activate the immune response of the body and can be used in the research and development of HIV vaccine candidates.