中华皮肤科杂志 ›› 1999, Vol. 32 ›› Issue (5): 328-329.

• 论著 • 上一篇    下一篇

HIV-2 gag蛋白在大肠杆菌中的表达纯化

苏忠兰1, 金宁一2, 段正芳1, 王宏伟2, 李体远1, 李钟洙2, 王玉红2, 王洪军2, 殷震2   

  1. 1. 白求恩医科大学第二临床学院皮肤科 长春 130041;
    2. 长春农牧大学研究所基因工程实验室
  • 收稿日期:1998-10-14 修回日期:1999-06-03 出版日期:1999-10-15 发布日期:1999-10-15
  • 基金资助:
    国家自然科学基金!资助项目(39770661)

Expression and Purification of HIV-2 gag Protein in E.coli

SU Zhonglan1, JIN Ningyi2, DUAN Zhengfang1   

  1. Genetic Engineering Laboratory, Changchun University of Agriculture and Animal Sciences, Changchun 130062
  • Received:1998-10-14 Revised:1999-06-03 Online:1999-10-15 Published:1999-10-15

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摘要： 目的 在原核系统中高效表达及纯化人II型免疫缺陷病毒(HIV-2)gag蛋白,以期作为检测试剂的原料.方法 采用基因工程手段将编码II型人免疫缺陷病毒(HIV-2)核心蛋白的部分p55 gag1(G1)及全部p55 gag2(G2)基因片段分别克隆到原核高效表达载体 pET28a的T7噬菌体启动子下游,构建出重组表达质粒pET28aG1和 pET28aG2,将其转入不同的受体菌后,IPTG诱导下进行表达.经SDS-PAGE以及免疫印迹分析鉴定表达产物.在变性和非变性条件下,使用Ni-NTA亲和层析对gag蛋白进行了纯化.结果 构建出两个重组表达质粒pET28aG1和 pET28aG2,它们在受体菌BL21(DE3)plysS中有高的表达.pETY28aG1的表达产物相对分子质量为55000,表达的重组蛋白约占菌体总蛋白的10%,pET28aG2的表达产物相对分子质量为61000,表达的重组蛋白约占菌体总蛋白的6.8%,经免疫印迹分析表明,纯化前后的目的蛋白均可与HIV-2患者全血清发生特异性反应.结论 在大肠杆菌中表达及纯化了HIV-2Gag蛋白,具有良好的反应原性,认为可作为HIV-2检测试剂的原料.

关键词: HIV-2gag, 大肠杆菌, 表达, 纯化

Abstract: Objective To develop a test reagent with high level expression and purification of HIV-2 gag protein in prokaryotic system.Methods Expression with inserting HIV-2 p55gag2 (G2) and truncated p55gag1 (G1) into down stream of T7 promoter of prokaryotic expression vector pET28a,induced by IPTG and analyzed with SDS PAGE and Western blot.Purification with Ni NTA affinity chromatography under the conditions of both denaturation and undenaturation.Results Two recombinant plasmids,pET28aG1 and pET28aG2,were constructed,which were highly expressed in BL21(DE3)plysS.The products of pET28aG1 and pET28aG2 were 55kDa and 61kDa in weight,respectively The former is 10% and the later is 6.8% in the total protein.It was shown by Western blot that the expressed recombinant protein could specifically react with HIV-2 antiserum.Conclusion HIV-2 gag protein,expressed and purified in E.coli,with good immunogenicity,could be used for the test reagent.

Key words: HIV-2 gag, E.coli, Expression, Purification