新孢子虫SAG1-SRS2融合基因真核表达质粒的构建及表达

Construction of a eukaryotic expression vector and its expression of NcSAG1-NcSRS2 fusion gene of Neospora caninum

根据犬新孢子虫NcSAG1-NcSRS2融合基因序列,设计了1对含有Kozak序列、终止密码子、BamHⅠ和XhoⅠ酶切位点的引物,以含有NcSAG1-NcSRS2融合基因的质粒pGEX-tNcP43-P36为模板,经PCR扩增获得NcSAG1-NcSRS2融合基因片段,用BamHⅠ和XhoⅠ双酶切该片段,回收得到含有以上2个酶切位点黏端的NcSAG1-NcSRS2融合基因,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pcNCSAG1-SRS2。经PCR鉴定、限制性内切酶酶切分析和克隆片段序列测定、比较,证实了重组质粒的正确性。将构建好的真核表达质粒转染到COS-7细胞中进行瞬时表达,经免疫荧光检测,证实了该载体能在细胞内进行蛋白表达。

The whole segment of NcSAG1-NcSRS2 fusion gene was amplified by PCR from plasmid pGEX-tNcP43-P36. The PCR product was digested with BamH I and Xho I , then inserted into pcDNA3.1 （＋） between BamH I and Xho I sites to generate a expression plasmid pcNCSAG1-SRS2, and transformed into Escherichia coli DH5a. The recombinant colonies were identified by the methods of restriction enzyme digestion, PCR and sequencing. The eukaryotic expression plasmid pcNCSAG1-SRS2 were successfully constructed. The expression of the NcSAG1-NcSRS2 fusion protein by COS-7 cells following transfection with the expression plasmid was detected by immunostaining with anti-Neospora caninum-bodies.