牛源犬新孢子虫NcSRS9基因的生物信息学分析及原核表达

Bioinformatics analysis and prokaryotic expression of NcSRS9 gene

为了解新孢子虫NcSRS9基因的生物信息学特性,本试验对牛源犬新孢子虫吉林株NcSRS9基因进行分子克隆,利用生物信息学软件对该基因进行编码蛋白等电点、信号肽、跨膜区、糖基化位点及疏水性分析,并将该基因片段亚克隆至原核表达载体pET-28α,构建了重组表达质粒pET-28α-NcSRS9,转化至大肠杆茵BL21中并诱导表达。结果显示,克隆的NcSRS9基因片段长969bp,与GenBank(EF440644.1)上发表的基因序列同源性100%。NcSRS9基因编码蛋白等电点为5.12,在其N末端存在一个跨膜区。SDS-PAGE和Western-blotting分析显示,NcSRS9基因在大肠杆菌内得到高效表达,表达的NcSRS9重组蛋白相对分子质量约为43 000(His约为7 000,NcSRS9约为36 000),可被牛抗新孢子虫阳性血清识别,说明表达的蛋白具有一定的反应原性。

To understand NcSRS9 gene-related biological information,N.caninumJilin strains NcSRS9 gene was cloned,the gene encoding protein isoelectric point,the signal peptide,transmembrane region,glycosylation sites and hydrophobicity were analyzed by using bioinformatics software.NcSRS9 gene was subcloned into the prokaryotic expression vector pET-28α,and the recombinant expression plasmid pET-28α-NcSRS9 was transformed into E.coli BL21 and induced the expression.The results showed that the length of cloned gene fragment NcSRS9 was 969bp,and the homology with GenBank（EF440644.1）published gene sequence was 100%.The isoelectric point of NcSRS9 gene encoding protein was 5.12,and there is a transmembrane domain in its Nterminal.SDS-PAGE and Western-blotting analysis showed that,NcSRS9 gene was highly expressed in E.coli BL21,the recombinant protein NcSRS9 molecular weight was about 43 000,which could be recogniced by anti-Neospora bovine sera,indicating that the protein has a certain reactogenicity.