鸡柔嫩艾美耳球虫ZJ株TA4基因的克隆及原核表达  

Cloning and expression of TA4 gene of Eimeria tenella ZJ strain

作　　者：高翔[1] 杜爱芳[1] 张娟敏[2] 侯玉慧[1] 庞林海[1] 

机构地区：[1]浙江大学动物预防医学研究所,浙江杭州310029 [2]嘉兴市畜牧兽医站,浙江嘉兴314000

出　　处：《中国预防兽医学报》2008年第5期366-370,共5页Chinese Journal of Preventive Veterinary Medicine

基　　金：嘉兴市科技局(20043015);浙江银冠兽药饲料有限公司(H20041704)项目资助

摘　　要：本试验对E. tenella ZJ株的TA4基因进行了克隆和表达。根据已报道的柔嫩艾美尔球虫TA4基因序列设计引物,以孢子化卵囊总RNA为模板,用RT-PCR的方法扩增得到一特异片段,将扩增产物克隆至pMD18-T,转化DH5-α感受态,经酶切鉴定获得阳性重组质粒并对其进行测序。测序结果与参考序列比较,核苷酸同源性为99.1%。然后将重组质粒和表达载体pGEX-4T2分别以EcoRⅠ、XhoⅠ酶切后构建重组表达载体pGEX-TA4,并将其转化入大肠杆菌BL21中,提取质粒经酶切和PCR鉴定正确后,用IPTG诱导表达。表达产物经SDS-PAGE和Westernblot检测显示,TA4基因在大肠杆菌中获得表达;融合蛋白的分子量约为43ku,以包涵体形式存在;诱导6h的蛋白表达量可达到35.9%,采用抗GST抗体进行Westernblot,成功检测到了该特异性条带。In this experiment, the TA4 gene of E.tenelta ZJ Strain was amplified by RT-PCR from total RNA extracted from sporulated oocysts and cloned into pMD18-T vector. The sequencing results showed that the gene was 99.1% identical to the published nucleotide sequence of TA4. The gene was further subcloned into expression vector pGEX-4T2 for expressing in E.coli BL21. SDS-PAGE analysis indicated the GST-TA4 fusion protein is about 43 ku, about 35.9 % in total bacteria proteins, mainly in form of inclusion body. The recombinant protein was verified by Western blot with antibody against GST.

关 键 词：柔嫩艾美尔球虫 TA4基因 RT—PCR 原核表达 

分 类 号：S852.723[农业科学—基础兽医学]