HIV-1整合酶蛋白的可溶性表达及功能研究  

Prokaryotic Soluble Expression and Functional Study of HIV-1 Integrase Protein

作　　者：程绍辉[1] 马晓慧[1] 何红秋[1] 刘斌[1] 陈慰祖[1] 王存新[1] 

机构地区：[1]北京工业大学生命科学与生物工程学院,北京100022

出　　处：《中国生物工程杂志》2006年第1期22-26,共5页China Biotechnology

基　　金：国家自然科学基金资助项目(30500429);北京市教委重点基金资助项目(KZ200410005002)

摘　　要：HIV-1整合酶是HIV病毒复制中一个重要的酶,也是治疗艾滋病药物的一个重要靶点。为了开展以整合酶蛋白为靶点的抑制剂筛选,构建HIV-1整合酶重组质粒,在原核细胞中进行可溶性表达和功能研究。通过重叠PCR技术引入F185K和C280S突变于HIV-1B亚型标准株的整合酶cDNA片段中,PCR扩增片段克隆到pET-28a(+)表达载体中,构建重组质粒,在E.coli中进行整合酶基因表达,SDS-PAGE鉴定表达产物,亲和层析纯化蛋白,酶联免疫吸附实验方法测定整合酶的生物学活性。结果构建的重组质粒获得高效稳定的可溶性表达,ELISA实验证实该蛋白具有整合酶的3′切割DNA和5′链转移的活性。HIV-1整合酶蛋白的可溶性表达和活性研究为建立以整合酶为靶点的抗HIV药物筛选平台打下了基础。The pol gene of HIV-1 encodes mainly three enzymes： reverse transcriptase （ RT）, protease （PR） and integrase （IN）. Currently, FDA approved drugs targeting RT and PR are available and administered in various combinations, while no anti-IN drug was approved. HIV-1 integrase is an essential enzyme for the viral replication and an interesting target for the design of new pharmaceuticals for multi-drug therapy of AIDS. The 288 amino acids of IN （32kDa） recognizes specific sequences in the long terminal repeats （LTRs） of the retroviral DNA. The IN protein catalyzes the 3＇-processing step and the 5＇-strand transfer step reaction in vivo, which was called integration and this reaction could be analysed by ELISA Assay in vitro. It has been reported that F185K and C280S mutations of HIV-1 integrase would improve the enzyme solubility, and the catalytical activity of the enzyme was the same as that of the wild-type enzyme in vitro. In order to build the platform of screening inhibitor against integrase of HIV-1 virus, the IN enzyme was expressed and the function of integrase protein was assayed. The cDNA of clade B HIV-1 genome was used as a template, overlapped PCR was used to construct site mutagenesis of F185K/C280S and Nde I/Xho I restriction sites were brought in. The PCR product was cloned into the prokaryotic vector pET-28a（ ＋ ） to form a recombined plasmid, transferred into the host cell E. coli（BL21 DE3 ）. The recombined clones were identified by PCR and Nde I/Xho I digestion . The positive plasmid was sequenced, and the successfully recombined plasmid in the host cell was induced by IPTG. The expressed IN protein was purified by the Co ~ affinity chromatography column and SDS-PAGE was used to analyze the molecular weight and specificity. In addition, ELISA assay was used to analyze the function of the recombined IN protein. The recombinant protein was soluble, and molecular weight of the expression product was identical to the ex functional in 3＇ processing and 5＇strand tran

关 键 词：HIV-1整合酶 克隆 表达 ELISA 

分 类 号：Q786[生物学—分子生物学]