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目的构建表达中国流行株HIV-1 CRF_07 B/C亚型(CN54)膜蛋白gp140及其突变体,为HIV-1疫苗设计提供优秀的免疫原。方法构建gp140、gp140 ST质粒,将质粒瞬时转染293F细胞,120h后收获上清液,经纯化、浓缩、PBS置换后,利用SDS-PAGE电泳、Western-blot检测蛋白的表达,ELISA检测其与HIV-1阳性病人血清的反应性。结果 SDS-PAGE电泳、Western-blot试验证明成功表达了gp140、gp140 ST蛋白,并且gp140 ST是一个稳定的三聚体蛋白质。ELISA试验检测,发现表达的gp140 ST蛋白与HIV-1阳性病人血清的亲和力高于gp140蛋白。结论经过突变改造的gp140 ST具有稳定的三聚体构象,可作为HIV-1免疫原进行疫苗研究。
Objective To construct gp140 and its mutants expressing the HIV-1 CRF_07 B / C subtype (CN54) membrane protein of Chinese endemic strain and provide an excellent immunogen for HIV-1 vaccine design. Methods The plasmids were transiently transfected into 293F cells by gp140 and gp140 ST. The supernatants were harvested after 120 h. The supernatants were harvested and purified by SDS-PAGE and Western blotting. SDS-PAGE and Western blot were used to detect the expression of Serum reactivity of HIV-1-positive patients. Results SDS-PAGE and Western-blot showed that gp140 and gp140 ST proteins were successfully expressed and gp140 ST was a stable trimer protein. ELISA test showed that the expression of gp140 ST protein and HIV-1-positive patients serum affinity than gp140 protein. CONCLUSION: The mutated gp140 ST has a stable trimeric conformation and can be used as a vaccine for HIV-1 immunogens.