犬新孢子虫SRS9基因真核重组质粒的构建及在Vero细胞中表达

Construction and expression of the recombinant eukaryotic expression plasmid NcSRS9 gene in Vero cells

为构建犬新孢子虫NcSRS9基因真核表达重组质粒,了解NcSRS9表面蛋白基因的生物学特性及其体外表达情况。本试验应用PCR技术获得目片段,构建克隆质粒pMD18-NcSRS9,经EcoRⅠ和BamHⅠ双酶切后构建真核表达重组质粒pVAX1-NcSRS9,利用脂质体介导法转染至Vero细胞,以间接免疫荧光方法(IFAT)检测NcSRS9基因在Vero细胞中的表达。结果显示,犬新孢子虫表面蛋白基因NcSRS9基因长度为1 191bp,与GenBank中(EF440644.1)发表的基因核苷酸序列同源性为99%,IFAT检测NcSRS9基因在Vero细胞中获得瞬时表达,为核酸疫苗研究奠定了基础。

In this research,Neospora Caninum surface protein NcSRS9 gene was amplified by PCR to understand NcSRS9 gene biological characteristic and expression in vitro.The target fragments were obtained by PCR amplification to construct pMD18-NcSRS9,and digested by EcoRⅠ and BamHⅠdouble enzyme to construct eukaryotic expression recombinant plasmid pVAX1-NcSRS9.The eukaryotic expression plasmid pVAX1-NcSRS9 was then transfected into Vero cells by liposome method.The expressed product in Vero cells was analyzed by IFAT.The results showed that the amplified fragment was 1 191 bp,and the homology with the corresponding gene sequence in Gen Bank(EF440644.1)was 99.9%.The expressed production of NcSRS9 was detected by indirect immunofluorescent assay.This research provided a foundation for the further studies on the DNA vaccine.