人类免疫缺陷病毒1型HXB2株Tat突变体的构建、原核表达及免疫原性分析  

Construction, prokaryotic expression and immunogenic analysis of HXB2 subtype Tat mutant of human immunodeficiency virus type-1

作　　者：李存枚[1] 邓松华[1] 曹洁[2] 王锦红[2] 陈璐[1] 黄德圣[1] 潘卫[2] 

机构地区：[1]安徽医科大学病理生理学教研室,合肥230032 [2]第二军医大学微生物学教研室

出　　处：《中华传染病杂志》2009年第9期517-521,共5页Chinese Journal of Infectious Diseases

基　　金：基金项目：国家自然科学基金资助项目（30872246、30872405）;国家“863”资助项目（2006AA02A238）;上海市基础研究重点项目（08JC1405200）

摘　　要：目的构建HIV一1HXB2株Tat基因半胱氨酸富集区3’末端移位突变体，经原核表达和纯化，分析该突变体蛋白（Tat—cct）的免疫原性。方法用PCR方法将HIV一1HXB2株Tat基因的半胱氨酸富集区（64～111位核苷酸）移位至Tat基因的3’末端，获得其突变体DNA序列，并构建其原核表达质粒pET32a—Tat—oct，转人大肠埃希菌E．coliBL21（DE3）中进行诱导表达及纯化。以Tat—cct融合表达蛋白免疫BALB／c小鼠，ELISA方法对该抗血清进行免疫原性检测分析。结果pET32aTat—cct重组质粒可在E．coliBI。21（DE3）中诱导表达，纯化后其融合蛋白相对分子质量约为31000。Tat—cct重组蛋白免疫小鼠诱导产生的抗体滴度为1：1600，该抗体与Tat—cot蛋白和Tat蛋白（1～101位氨基酸）均呈特异性结合反应。结论Tat突变体重组蛋白Tat—cct能在大肠埃希菌中有效表达，并较好保留其免疫原性，为HIV一1Tat疫苗的基础研究提供了有价值的实验结论。Objective To construct shifting mutant of cysteine rich region to 3＇ terminal of Tat gene of human immunodeficiency virus type-1 （HIV-1） HXB2 strain, and to analyze the immunogenicity of mutant protein （Tat-cct） after prokaryotically expressed and purified. Methods The cysteine-rich region （nucleotides 64--111） of Tat gene was shifted to 3＇ terminal of Tat of HIV1 HXB2 strain by polymerase chain reaction （PCR） and Tat mutant DNA sequence was obtained. Prokaryotic express plasmid pET32a-Tat cct was constructed and transformed into E. coli BL21 （DE3）, then Tat-cct protein was expressed and purified. BALB/c mice were immunized with the fusion protein Tat-cct, and immunogenicity of the immunized serum was detected by enzyme-linked immunosorbent assay （ELISA）. Results The recombinant plasmid pET32a-Tat-cct expressed in E. coli BL21 （DE3） and the relative molecular mass of the purified fusion protein was 31 000. The serum antibody titer of mice immunized with Tat cct recombinant protein was 1：1600, which binded specifically with both Tat-cct protein and Tat protein （amino acids 1--101）. Conclusions The recombinant protein Tat-cct of Tat mutant strain can be expressed efficiently in E. coli and well retains immunogenicity, which provides valuable information for basic research of HIV 1 Tat vaccine.

关 键 词：HIV-1 基因产物 TAT 重组蛋白质类 半胱氨酸 基因表达 免疫 大肠埃希菌 

分 类 号：R686[医药卫生—骨科学]