刚地弓形虫硫氧还蛋白基因的克隆 表达及鉴定  

Cloning,fusion expression and identification of thioredoxin encoding gene from Toxoplasma gondii

作　　者：张梓岗[1] 陈小美[1] 苏丹华[1] 刘原[1] 付涛[1] 端木家苗 吴亮[1] 姜旭淦[1] 陈盛霞[1] 曹建平[2] 

机构地区：[1]江苏大学医学院,镇江212013 [2]中国疾病预防控制中心寄生虫病预防控制所

出　　处：《中国血吸虫病防治杂志》2016年第3期289-292,共4页Chinese Journal of Schistosomiasis Control

基　　金：国家自然科学基金(81301453);卫生部寄生虫病原与媒介生物学重点实验室开放课题(WSBKTKT201302);中国博士后科学基金特别资助(2015T80518);中国博士后科学基金(2014M561598);江苏省博士后科研计划(1402171C);江苏大学高级人才启动基金(13JDG023;13JDG127)

摘　　要：目的克隆刚地弓形虫硫氧还蛋白(Thioredoxin,Trx)基因,构建原核表达载体,通过诱导表达和纯化蛋白,免疫家兔制备多克隆抗体。方法采用PCR技术扩增刚地弓形虫Trx基因,克隆至原核表达载体p ET-28a(+)中,转化大肠埃希菌(E.coli)Rosetta,用IPTG诱导目的蛋白表达,采用镍亲和层析法获得纯化蛋白并免疫家兔制备多克隆抗体。利用Western blotting技术鉴定多克隆抗体的特异性。结果成功从刚地弓形虫c DNA中扩增出Trx目的基因,构建了Trx/p ET-28a(+)重组质粒,获得抗Trx重组蛋白的多克隆抗体。Western blotting技术检测出弓形虫Trx蛋白的特异性条带。结论用制备的兔抗Trx多克隆抗体能检测弓形虫Trx在速殖子内的表达,为进一步深入研究刚地弓形虫Trx功能奠定了基础。Objective To clone and express the thioredoxin （Trx） from RH strain tachyzoites of Toxoplasma gondii, establish the prokaryotic expression vector and purify the recombinant protein, then produce the polyclonal anti-Trx antibody in rabbits. Methods Trx fragment was amplified by PCR and cloned into the pET-28a（＋）veetor, and the recombinant protein was induced with IPTG and purified by Ni-NTA affinity chromatography. The polyclonal antibody specificity was detected by Western blotting. Results The trx gene was amplified from T. gondii eDNA by PCR. The recombinant plasmid trx/pET-28a（＋） was usefully constructed, and the recombinant TRX protein was expressed and purified. The TRX polyclonal antibody was also obtained. The specific band of TRX was detected by Western blotting. Conclusion Western blotting can detect the specificity of polyclonal anti-Trx antibody, which will facilitate the biological functions of Trx.

关 键 词：刚地弓形虫 硫氧还蛋白 克隆 原核表达 鉴定 多克隆抗体 

分 类 号：R382.5[医药卫生—医学寄生虫学]