弓形虫SAG1基因功能片段的克隆、表达与抗原性分析  

Cloning,expression and antigenicity analysis of gene encoding the major surface antigen 1 function of Toxoplasma gondii

作　　者：张建伟[1,2] 王海龙[1] 殷国荣[1] 刘转转[1] 刘晋平[2] 赵丰[3] 

机构地区：[1]山西医科大学医学寄生虫学研究所,寄生虫学教研室,太原030001 [2]山西农业大学动物科技学院 [3]山西医科大学第一临床医学院

出　　处：《山西医科大学学报》2009年第6期481-484,共4页Journal of Shanxi Medical University

基　　金：国家自然科学基金资助项目(30640057)

摘　　要：目的重组、表达与纯化弓形虫速殖子主要表面抗原1(SAG1)具有生物活性的功能多肽,分析其抗原性。方法根据SAG1的基因序列设计引物,截除其前端的信号肽和后端的疏水区,只扩增650bp的功能区域;将该片段重组入带有His标签的pET-30a(+)质粒,用异丙基-β-D-硫代半乳糖苷(IPTG)诱导其在E.coli中表达,调整诱导表达时间、IPTG浓度等条件,高效稳定地表达出目的蛋白;镍柱亲和层析法纯化目的蛋白,Western-blot检验纯化后蛋白的抗原性。结果成功构建pET-30a(+)/SAG1重组质粒,使其在E.coli中高效稳定地可溶性表达,并确定出在0.5mmol/LIPTG浓度下诱导12h为最佳表达条件,Western-blot显示纯化后的蛋白具有良好的抗原性。结论获得了可溶性的弓形虫SAG1基因功能片段表达的产物,其具有良好的抗原性。Objective To recombine, express and purify the truncated major surface protein 1 （ SAG1 ） gene of Toxoplasma gondii in E. coli,and to explore its antigenicity. Methods The gene encoding function peptide of SAG1 was amplified by PCR and inserted into expression vector pET30a（ ＋ ） ,then the recombinant plasmid was transformed into E. coli BL21 induced by IPTG. The soluble product was purified by Ni-NTA, and the purified recombinant protein was identified with Western-blotting. Results The recombinant plasmid was constructed successfully and high soluble expression was achived in E. coli. The induction of IPTG at the concentration of 0.5 mmo/L for 12 h was confirmed as optimum expression condition. Western-blotting results demonstrated that the purified product was specifically recognized by sera from rabbits immunized with the live Toxoplasma gondii tachyzoite. Conclusion Recombinant soluble SAG1 protein is successfully obtained with effective antigenicity. It will be used to carry out the further study in protein vaccine of toxoplasmosis.

关 键 词：弓形虫 SAG1 原核表达 蛋白纯化 抗原性 

分 类 号：R382.5[医药卫生—医学寄生虫学]