犬新孢子虫MAG1与IFN-γ融合基因重组质粒的构建及原核共表达  

Prokaryotic co-expression of MAG1 of Neospora caninum and IFN-γ gene

作　　者：焦石[1] 贾立军[1] 张立霞[1] 钱年超[1] 刘明明[1] 黄国明[1] 张守发[1] 

机构地区：[1]延边大学农学院,吉林延吉133002

出　　处：《中国兽医学报》2013年第3期386-388,408,共4页Chinese Journal of Veterinary Science

基　　金：吉林省自然科学基金资助项目(201115230)

摘　　要：根据发表的犬新孢子虫MAG1基因序列(EF580924.1)和Bov IFN-γ基因序列(M29867.1)设计2对引物。分别以犬新孢子虫基因组DNA和pET28α-IFN-γ重组质粒为模板,通过重叠延伸拼接聚合酶链式反应(SOE-PCR)将犬新孢子虫MAG1基因与IFN-γ基因拼接,构建pMD18-T-MAG1-IFN-γ克隆质粒和pGEX-4T-MAG1-IFN-γ重组表达质粒,将鉴定正确的pGEX-4T-MAG1-IFN-γ重组表达质粒转化大肠杆菌BL21感受态细胞,IPTG诱导表达,SDS-PAGE和Western-blotting分析表达产物。结果表明,SOE-PCR扩增获得双基因融合片段大小为1 491bp,与GenBank中发表的MAG1(EF580924.1)和IFN-γ(M29867.1)核苷酸序列的同源性为99%;MAG1-IFN-γ融合基因表达蛋白大小为82 000,具有较好的反应原性。Two pairs of primers were designed according to the Neospora caninurn MAG1 gene （EF580924.1） and Bov IFN-γ, gene （M29867.1）sequence. MAG1 gene and IFN-y gene were con- nected by SOE-PCR. The recombinant plasmid pMD18-T-MAG1-IFN-γ, and pGEX-4T-MAG1- IFN-）, were constructed. The pGEX-4T-MAGI-IFN-γ plasmid was transformed into E. coli BL21. The expression products were analyzed by SDS-PAGE and Western-blotting. The results show that the size of two-gene fusion fragment is 1 491 bp. The gene shared 99% nucleotide sequence homology with MAG1 （EF580924.1） and IFN-γ, （M29867.1） in GenBank. The molecular weight of MAGI-IFN-γ fusion protein is 82 000. The protein can be recognized by positive serum of Neospora caninurn and it has strong reactiity.

关 键 词：犬新孢子虫 MAG1 IFN-Γ 原核共表达 

分 类 号：S852.723[农业科学—基础兽医学]