犬新孢子虫NcSRS2基因真核表达质粒的构建

Construction of an Eukaryotic Expression Plasmid for NcSRS2 Gene of Neospora caninum

根据犬新孢子虫NcSRS2基因序列,设计了1对含有Kozak序列、PstⅠ和XbaⅠ酶切位点的引物,以含有NcSRS2基因的质粒P43为模板,经PCR扩增获得NcSRS2ORF基因片段,用PstⅠ和XbaⅠ双酶切该片段,回收得到含有以上2个酶切位点黏端的NcSR2ORF基因,将此基因片段克隆至相同酶切回收后的pcDNA3.1(+)真核表达载体中,获得重组质粒pcNCSRS2。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, a pair of primers containing Kozak sequence, Pst Ⅰ and Xba Ⅰ enzyme digestion sites were designed . Using the plasmid P43 which contains NcSRS2 gene as template, the ORF region of NcSRS2 gene was amplified by polymerase chain reaction （PCR）, then the PCR product was digested with Pst Ⅰ and Xba Ⅰ ,and the eukaryotic expression vector pcDNA3.1 （＋） was also done , then the gene was cloned into the vector. Finally a recombinant plasmid named pcNcSRS2 was obtained, and was identified by PCR, restriction enzyme analysis and sequencing.