弓形虫缓殖子期特异性抗原1基因的克隆、表达及对重组抗原的免疫反应性分析  

Cloning and Expression of a Bradyzoite-Specific Gene of Toxoplasma gondii and Immunoreactive Analysis on the Recombinant Antigen

作　　者：王琼[1] 吴焜[1] 陈晓光[1] 郝丽[1] 程璐[1] 

机构地区：[1]南方医科大学病原生物学系,广州510515

出　　处：《中国寄生虫学与寄生虫病杂志》2007年第4期295-299,共5页Chinese Journal of Parasitology and Parasitic Diseases

基　　金：国家自然科学基金(No.30671837)~~

摘　　要：目的克隆与表达弓形虫缓殖子期特异性抗原1(BAG1)的基因,并分析重组抗原的免疫反应性。方法诱导体外培养的弓形虫RH株速殖子向缓殖子转化,用RT-PCR法从缓殖子期弓形虫扩增BAG1基因片段,进行序列分析;构建表达重组质粒pET32a(+)-BAG1,转入大肠埃希菌BL21(DE3)中诱导表达;表达蛋白经次氨基三乙酸镍(Ni-NTA)琼脂糖亲和层析纯化后,蛋白质印迹(Western blotting)分析与ELISA分析其免疫反应性。结果从缓殖子期弓形虫克隆的BAG1基因长690bp,构建的重组质粒pET32a(+)-BAG1经异丙基-β-D-硫代半乳糖苷(IPTG)诱导后,可高效表达重组BAG1。Western blotting分析显示纯化的重组BAG1(SAG1)能被弓形虫慢性感染血清识别。ELISA结果表明重组BAG1抗原检测350份人血清弓形虫IgG抗体的阳性率为17.4%,显著高于重组SAG1抗原的阳性率12.6%(P<0.05)。结论原核表达的重组BAG1抗原具有特异的免疫反应性。　Objective To clone and express bradyzoite antigen 1（BAG1） gene of T. gondii,and analyze the immunoreactivity of the recombinant product. Methods The differentiation of T. gondii RH strain tachyzoites into bradyzoites was induced in vitro,and the coding sequence of BAG1 was amplified from bradyzoites by RT-PCR. The PCR product was analyzed by sequencing. The BAG1 coding sequence was further subcloned into the plasmid pET32a（＋）. The plasmid pET32a（＋）-BAG1 was then transformed into BL21（DE3） to express after IPTG induction. The expression product was purified with Ni-NTA agarose and the purified BAG1 was further analyzed by Western blotting and ELISA. Results BAG1 cDNA was amplified from bradyzoites. After IPTG induction,BAG1 was expressed in a fusional form in E. coli. Western blotting showed that the purified recombinant protein could be specifically recognized by sera from mice chronically infected by T. gondii B36 strain. ELISA showed that the positive rate of T. gondii IgG antibodies of 350 human sera detected by the recombinant BAG1（17.4%） was higher than by recombinant SAG1 （12.6%）（P〈0.05）. Conclusion The expressed recombinant BAG1 shows a specific immunoreactivity.

关 键 词：弓形虫 缓殖子期抗原 基因表达 免疫反应性 

分 类 号：R384.11[医药卫生—医学寄生虫学]