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将人免疫缺陷病毒(HIV)gag p20基因插入表达质粒pBV220,得到重组表达质粒pCY7.在大肠杆菌中高效表达了p20蛋白.Western blot结果显示,大肠杆菌表达的p20蛋白能被抗p17单克隆抗体识别.用脱氧胆酸裂解重组表达菌,得到上清和沉淀两部分,SDS-聚丙烯酰胺凝胶电泳结果表明,p20蛋白主要存在于沉淀中.经用盐溶液洗涤2次后,沉淀中p20蛋白的纯度达到27.4％.再用1mol/L脲溶液洗2次沉淀,p20蛋白纯度提高到58.1％.用6 mol/L脲溶液溶解沉淀,上肝素亲和层析柱,用6 mol/L脲洗脱,得到4个蛋白峰.p20蛋白主要存在于第3峰,凝胶电泳和扫描分析表明,p20蛋白纯度为84.2％。
The human immunodeficiency virus (HIV) gag p20 gene was inserted into the expression plasmid pBV220 to obtain the recombinant expression plasmid pCY7.The p20 protein was highly expressed in E. coli.Western blot results showed that p20 protein expressed by E. coli can be detected by anti-p17 monoclonal antibody The recombinant protein was purified by deoxycholic acid to obtain the supernatant and the precipitate.The results of SDS-polyacrylamide gel electrophoresis showed that p20 protein was mainly present in the precipitate.After washing twice with salt solution, the content of p20 protein The purity of p20 protein was increased to 58.1%. The precipitate was dissolved in 6 mol / L urea solution and heparin affinity chromatography was carried out on a 6 mol / L urea solution The results showed that p20 protein mainly existed in the third peak, and gel electrophoresis and scanning analysis showed that the purity of p20 protein was 84.2%.