犬新孢子虫新疆Nc-1株NcSRS2基因的克隆及原核表达  

Cloning and prokaryotic expression of the NcSRS2 gene from Xinjiang Nc-1 strain of Neospora caninum

作　　者：杨帆[1] 陈亮[1] 王莲芳[1] 巴音查汗[1] 

机构地区：[1]新疆农业大学动物医学学院,新疆乌鲁木齐830052

出　　处：《中国兽医科学》2010年第8期846-850,共5页Chinese Veterinary Science

基　　金：新疆维吾尔自治区高技术研究与发展计划项目(200811110)

摘　　要：根据GenBank中犬新孢子虫NcSRS2基因序列设计了1对引物,从新孢子虫病阳性牛血液中提取总DNA,经PCR扩增NcSRS2基因,将其插入原核表达质粒pGEX-4T-2中,重组质粒pGEX-4T-NcSRS2经测序确定后,转入表达菌株BL21,优化其表达条件,并探讨了GST-NcSRS2蛋白的生物活性。结果显示,在37℃条件下,经终浓度为0.5mmol/L的IPTG诱导表达4h后,获得融合蛋白。Western-blot分析表明该融合蛋白可与相应抗体发生特异性结合,rELISA检测与弓形虫和布鲁氏菌阳性血清无交叉反应。A pair of primers was designed according to NcSRS2 gene sequence of Neospora caninum available in GenBank.The NcSRS2 gene was amplified by PCR from genomic DNA extracted from the positive blood infected with N.caninum,then the purified PCR product was inserted into a prokaryotic expression plasmid pGEX-4T-2.The sequence of the fragment matched with the original sequence of NcSRS2.pGEX-4T-NcSRS2 was transformed into Escherichia coli BL21（DE3） competent cells for expression.The optimal expression of NcSRS2 was induced with 0.5mmol/L IPTG for 4 h at 37℃.Western-blot analysis showed that the expressed fusion protein could bind with the specific antibody,but the protein did not cross-react with positive sera with Toxoplasma gondii or Brucella in rELISA assay.

关 键 词：犬新孢子虫 新疆Nc-1株 NCSRS2基因 原核表达 

分 类 号：S852.723[农业科学—基础兽医学]