弓形虫致密颗粒蛋白基因的表达及其重组蛋白免疫反应性的评价  

Expression of gene encoding the dense granule protein of Toxoplasma gondii and evaluation on the immuno-reactivity of its recombinant protein

作　　者：郑斌[1] 余南[1] 李卓雅[1] 郑焕钦[1] 何蔼[1] 詹希美[1] 

机构地区：[1]中山大学中山医学院寄生虫学教研室,广州510080

出　　处：《中国人兽共患病杂志》2005年第2期119-121,114,共4页Chinese Journal of Zoonoses

基　　金：国家自然科学基金资助(批准号:301708377);中山医学院抗SARS专项基金资助

摘　　要：目的将克隆入pGEX-4T-1的致密颗粒蛋白基因进行表达并对表达产物的免疫反应性进行评价。方法将重组表达质粒pGEX-4T-1/GRA7转入大肠埃希菌BL21,经IPTG诱导进行SDS变性蛋白质电泳,分别以Anti-GSTAntibody、免抗弓形虫阳性血清和人抗弓形虫阳性血清为一抗进行WesternBlot分析。用GSTrapFFHiTrapaffinitycolumns纯化重组蛋白,以此蛋白作为包被抗原,BLISA法检测抗弓形虫阴性、阳性血清。结果SDS变性蛋白质电泳显示在43KDa～66KDa蛋白条带之间有特异蛋白的表达,蛋白分子量大小与理论值相符。WesternBlot分析表明该重组蛋白为GST融合蛋白,且该蛋白能被人抗弓形虫阳性血清、兔抗弓形虫阳性血清所识别。ELISA结果表明该蛋白能与人抗弓形虫阳性血清、兔抗弓形虫阳性血清特异结合,而与抗弓形虫阴性血清无反应。结论弓形虫致密颗粒蛋白基因在大肠埃希菌中得到表达;该重组蛋白具有良好的免疫反应性。The aim of the present study is to express the gene encoding the dense granule protein-7 (GRA7) that has been cloned into the vector pGEX-4T-1 in Escherchia coli and to evaluate the immuno-reactivity of its recombinant protein.The recombinant plasmid pGEX-4T-1/GRA7. was transformed into E.coli BL21 and induced by IPTG for its expression. The expressed protein was analyzed by SDS-PAGE and Western blotting with positive sera,and purified by using the GSTrap FF HiTrap affinity columns. This purified protein was used as the coating antigen in the ELISA assay to test for the positive and negative sera. The experimental results showed that there was a special protein band observed between 43 kDa to 66 kDa as demonstrated in SDS-PAGE gel, and this protein was proved to be GST fusion protein that could react with human and rabbit positive sera as analyzed by Western blotting. It concludes that the gene encoding the dense granule protein 7 (GRA7) of Toxoplasma gondii can be expressed as a recombinant protein with excellent immuno-reactivity in E.coli.

关 键 词：刚地弓形虫 致密颗粒蛋白7 基因表达 免疫反应性 酶联免疫吸附试验 

分 类 号：R382.5[医药卫生—医学寄生虫学]