HIV-1 Gag蛋白酵母表达产物的纯化及免疫原性检测

Purification of HIV-1 Gag protein expressed in Pichia pastoris and analysis of its immunogenicity

目的 纯化HIV 1Gag蛋白 ,并检测其免疫原性。方法 将表达HIV 1Gag蛋白的酵母工程菌GS115 /pPICGAG接种到YPD培养基中多次传代后 ,用PCR检测其目的基因。该酵母工程菌在BMMY培养基中经甲醇诱导表达 ,上清液初步浓缩后上柱进行凝胶过滤层析 ,并将纯化蛋白免疫Balb/c小鼠 ,检测其血清抗体水平。结果 该酵母工程菌经多次传代后外源基因不丢失。Westernblot检测结果表明纯化蛋白具有很好的反应原性 ,其纯度可达 85 %。纯化蛋白免疫小鼠后可诱导特异性抗体产生。结论 表达HIV 1Gag蛋白的酵母工程菌具有很好的遗传稳定性 。

Objective To purify HIV-1 Gag protein and analyze its immunogenicity. Methods The engineering yeast strain GS115/pPICGAG expressing the HIV-1 Gag protein was inoculated into YPD medium and subcultured continuously, and the gene-of-interest was detected by PCR. After the engineering yeast strain expressed in BMMY medium under the induction of methanol, the supernatant of the culture was condensed and loaded into the chromatographic column for gel filtration chromatography. Balb/c mice were immunized with the purified protein and the serum antibody level was measured. Results The engineering yeast strain didn't lose the exogenous gene after the subculture. Western blot analysis showed that the purified protein had good reactogenicity with a purity of 85%. The purified protein could induce the specific antibodies in mice. Conclusion The engineering yeast strain expressing the HIV-1 Gag protein has good genetic stability and its expressed products can elicit the specific humoral immune response in mice.