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.
出处
《中国血吸虫病防治杂志》
CAS
CSCD
北大核心
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)
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
Toxoplasma gondii
Thioredoxin
Cloning
Prokaryotic expression
Identification
Polyclonal antibody
