摘要
构建牛瑟氏泰勒虫P23基因片段原核重组表达质粒,表达重组蛋白。采用PCR方法扩增牛瑟氏泰勒虫P23表面蛋白基因,将扩增产物与pMD18-T-Simple载体连接,测序,验证扩增产物。将P23基因片段克隆到载体pGEX-4T-3上,经酶切分析、PCR鉴定后,IPTG诱导表达,最后用SDS-PAGE和Western blotting分析鉴定表达产物。结果表明克隆的P23基因片段为684 bp,重组质粒pGEX-4T-3/P23构建成功;SDS-PAGE显示目的蛋白相对分子质量约为58 ku;Western blotting分析结果显示,与牛瑟氏泰勒虫阳性血清发生反应,而与牛瑟氏泰勒虫阴性血清无反应,表明牛瑟氏泰勒虫P23表面蛋白具有良好的抗原性和特异性,提示可以利用融合蛋白来建立检测抗体的间接ELISA诊断方法。
The experiment was conducted to construct prokaryotic recombinant expression plasmid of Theileria sergenti P23 gene and express the recombinant protein. P23 gene fragment was amplified by PCR, and ligated into pMD18-T-Simple vector. The recombinant plasmid was certified by PCR amplification, digestion with endonuclease and sequencing. A prokaryotic expression plasmid was constructed by inserting the P23 gene into pGEX-4T-3. After induced by IPTG, the expressed protein was analyzed by SDS-PAGE and Western-blotting. The resuits showed that, a fragment of 684 bp was obtained by PCR amplification and the expressed protein was 58 ku in molecular weight by SDS- PAGE analysis. The recombinant protein could react with the bovine sera against T. sergenti, but not with normal bovine sera in immuoblot assay. This study demonstrated that the recombinant protein had very good antigenicity and specificity.
出处
《畜牧与兽医》
北大核心
2009年第6期10-13,共4页
Animal Husbandry & Veterinary Medicine
基金
国家自然科学基金(30560112)
