新孢子虫NcSRS2基因的亚克隆和表达  

Subcloning and expression of the NcSRS2 gene fragment of Neospora caninum

作　　者：余劲术[1] 刘群[1] 汪明[1] 

机构地区：[1]中国农业大学动物医学院

出　　处：《中国兽医杂志》2006年第4期3-6,共4页Chinese Journal of Veterinary Medicine

基　　金：国家自然科学基金(30371080);北京市自然科学基金(6042016)

摘　　要：本研究根据NcSRS2基因序列设计合成一对引物,将上、下游引物分别引入EcoRI,XhoI酶切位点,用PCR技术从pGEX-NcSRS2重组质粒扩增截去N端疏水氨基酸序列NcSRS2的基因片段(以下称dNcSRS2),插入到pGEX-6p-1质粒的多克隆位点,转化大肠杆菌BL21感受态细胞,于氨苄阳性LB培养平板上筛选阳性克隆,酶切及PCR鉴定;经IPTG诱导在E.coli中表达,用SDS-PAGE和免疫印迹分析表达产物并纯化。结果表明,新孢子虫dNcSRS2基因体外扩增产物与预期值相符,约1041bp;所构建pGEX-dNcSRS2重组质粒经双酶切与PCR鉴定,与预期结果一致;SDS-PAGE和免疫印迹显示,表达融合蛋白的分子量约为62.6kD,表达效率为32.3%,该蛋白具有特异的免疫反应性,为新孢子虫病诊断试剂盒的研制和疫苗的研制奠定了基础。One pair of primers were designed according to the sequence of NcSRS2. The fragment of dNcSRS2 gene was obtained by amplification from the recombinant pGEX-NcSRS2. By cloning target gene into a high level expression vector, pGEX-6p-1, a recombinant pGEX-dNcSRS2 was constructed. Then the recombinant plasmid, pGEX-dNcSRS2 was transformed into E. coli BL21（DE3）. The positive clones BL21 were identified by the methods of restriction endonucleace digestion, PCR and gene sequencing. The recombinant E. coli was induced by IPTG to express the fusion protein. The expressed and purified product was analyzed by SDS-PAGE. Results showed that the recombinant plasmid pGEX-dNcSRS2 was constructed and expressed in the E. coli BL21. The results of the SDS-PAGE showed that the molecular weight of expressed and purified aNcSRS2 protein was about 62.6 kD. The recombinant dNcSRS2 protein will be used for further studies in diagnosis and immunization of Neospora caninurn.

关 键 词：犬新孢子虫 NCSRS2基因 亚克隆 表达 

分 类 号：Q786[生物学—分子生物学]