犬新孢子虫NcSRS2基因原核表达质粒的构建

Construction of Prokaryotic Expression Plasmid of NcSRS2 Gene of Neospora caninum

根据已发表的犬新孢子虫NcSRS2基因序列,设计1对含有EcoRⅠ和NotⅠ酶切位点的引物。以提取犬新孢子虫虫体基因组DNA为模板,应用PCR扩增获得NcSRS2 ORF基因片段,将此基因片段克隆到pMD18-T Simple载体上,用EcoRⅠ和NotⅠ双酶切该片段,回收得到含有两个酶切位点黏端的Nc-SRS2 ORF基因,将此基因片段克隆至相同酶切回收后的PGEM-4T-2原核表达载体中,获得重组质粒pGEX-NcSRS2,经PCR鉴定,限制性内切酶分析和克隆片段序列测定比较,证实了重组质粒的正确性。

Based on the NcSRS2 gene sequence of Neospora caninum in the Genbank,a pair of primers containing EcoRⅠ and Not Ⅰ enyme digestion sites were designed. Using the Neospora caninum DNA,the ORF region of NcRSR2 gene was amplified by polymerase chain rection （PCR）and then inserted into PMD18-T Simple vector. Then the plasmid DNA was digested with Eco R Ⅰ and Not Ⅰ ,and the prokaryotic expression vector PGEM-4T-2 was prepared, the gene was cloned into the vector. Finally a recombinant plasmid named PGEX-NcRSR2 was obtained,and was identified by PCR, restriction enzyme analysis and sequencing. The accuracy of recombinant plasmid was confirmed.