人类免疫缺陷病毒1型Tat蛋白N末端缺失突变体融合蛋白的表达及其免疫原性分析  

Construction,Expression and Immunogenicity Analysis of a Tat N-terminusdeleted Mutant Fusion Protein of Human Immunodeficiency Virus Type 1

作　　者：张华群[1] 廖文婷[1] 陈秋莉[1] 葛宜兵[2] 杨界[1] 章萍萍[2] 祁培培[1] 刘超[1] 何婷[1] 王锦红[1] 潘卫[1] 曹洁[1] 

机构地区：[1]第二军医大学微生物学教研室上海市医学生物防护重点实验室,上海200433 [2]安徽医科大学病理生理学教研室,合肥230032

出　　处：《病毒学报》2011年第6期580-586,共7页Chinese Journal of Virology

基　　金：国家自然科学基金资助项目(编号:30872246;30972799);上海市基础研究重点项目(编号:08JC1405200)

摘　　要：本研究采用PCR方法从人类免疫缺陷病毒1型(Human immunodeficiency virus 1,HIV-1)HXB2株tat基因中扩增编码Tat蛋白N末端1-21位氨基酸缺失的突变体Tat22-101基因片段,构建其原核表达质粒pET32a-Tat22-101,经双酶切及测序验证后,转化大肠埃希菌BL21(DE3),进行IPTG诱导表达及Ni^(2+)-NTA柱亲和层析纯化。纯化后的突变体融合蛋白PET32a-Tat22-101经SDS-PAGE及Western blotting鉴定,其相对分子质量约为26.9kD。该融合蛋白免疫BALB/c小鼠,经ELISA检测结果表明,pET32a-Tat22-101融合蛋白不仅较好地保留其免疫原性,而且能诱导产生高滴度的针对Tat N末端区之外的Tat其他功能区表位的抗体,为进一步研究Tat生物学功能和研制新型HIV Tat疫苗奠定试验基础。In the study, a gene encoding Tat protein N terminal 1-21 amino acid residues-deleted mutant （Tat22-101） was amplified by PCR from a full length Tat gene of human immunodeficiency virus type 1, and the prokaryotic expression plasmid pET32a-Tat22-101 was constructed. After identification by digestion with endonucleases and sequencing, the recombinant plasmid pET32a-Tat22-101 was transformed into E. coli BL21（DE3） and expressed with IPTG induction. The mutant fusion protein with deleted Tat N terminal was purified by an affinity chromatography column Ni2^-NTA and subsequently identified by SDS-PAGE and Western blotting. The results showed that the molecular weight of the mutant protein was approximately 26.9kD. Furthermore, BALB/c mice were immunized with the mutant protein and the antisera were collected. ELISA results showed that the mutant protein preserved its immunogenicity, particularly it could improve the production of antibodies to other epitopes in addition to the N terminal epitope of Tat protein, which might provide some valuable information for the study of Tat functions as well as for development of potential novel HIV Tat vaccine.

关 键 词：人类免疫缺陷病毒1型 TAT蛋白 缺失突变 原核表达 免疫原性 

分 类 号：R373.9[医药卫生—病原生物学] Q78[医药卫生—基础医学]