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.
出处
《中国兽医科学》
CAS
CSCD
北大核心
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)
