间日疟原虫乳酸脱氢酶的表达、纯化及免疫活性的鉴定  

Expression and Immunocompetence Characterization of Plasmodium vivax Lactate Dehydrogenase

作　　者：孙莉[1] 郝文波[1] 吴英松[1] 王穗海[1] 李明[1] 

机构地区：[1]南方医科大学生物技术学院,广州510515

出　　处：《热带医学杂志》2008年第3期198-200,共3页Journal of Tropical Medicine

基　　金：科技部973专项(No.2007CB513100);广州市科技计划项目(No.2002Z3-E4012);广东省医学科学技术研究基金(No.A2006365)

摘　　要：目的在大肠杆菌中表达间日疟原虫乳酸脱氢酶(PvLDH)与谷胱甘肽S-转移酶(GST)融合蛋白,对重组蛋白进行纯化并测定其免疫活性。方法将构建的LDH/pGEX-4T-1重组菌BL21接种于LB培养基中,异丙基硫代半乳糖苷(IPTG)诱导重组融合蛋白的表达,重组蛋白纯化后免疫小鼠制备特异性血清,ELISA检测血清效价,Western-blotting鉴定其免疫活性。结果重组质粒在大肠杆菌中表达PvLDH与谷胱甘肽S-转移酶(GST)的融合蛋白,表达产物主要以包涵体形式存在,经复性、纯化后免疫小鼠,能诱导小鼠产生特异性体液免疫应答,ELISA法测效价为1∶51200,Western-blotting结果显示该血清能特异性与间日疟和恶性疟患者全血发生反应,而不能与正常人起交叉反应。结论PvLDH在大肠杆菌中获得高效表达且表达产物具有良好的抗原性和免疫原性。Objective To express lactate dehydrogenase （LDH） gene of Plasmodium vivax in the E. coli BI221, and to purify and analyze immunocompetence of the recombinant protein. Methods The recombinant expression vector LDH/pGEX-4T-1 was transformed into E. coli BL21 and induced by IPTG （isopropyl-beta D-thiogalactoside）. Four mice （Kunming strain） were immunized with purified recombinant protein （antigen） and the polyclonal antibodies were collected. ELISA and Western-blot were carried out to identify the polyclonal antibodies. Results The LDH gene of P. vivax was successfully expressed in the E. coli BI221. By renaturation and chromatography, the expressed protein was purified. The purified induced the specific humoral responses in mice and the titer of the specific antibody was 1 ： 51200. Western-blot analysis indicates that there is a strong reaction between the antiserum and the blood of patient infected with P. vivax; however, there is no reaction between the antiserum and the normal human blood. Condusion The LDH gene of P. vivax has been successfully expressed in the E. coli BI221 and the expressed protein has high antigenicity.

关 键 词：恶性疟原虫 乳酸脱氢酶 表达 免疫活性 

分 类 号：R382.31[医药卫生—医学寄生虫学]