犬新孢子虫AMA1基因重组真核表达质粒的构建及其在Vero细胞中的表达

Construction of recombinant eukaryotic expression plasmid of AMA1 gene of Neospora caninumand its expression in Vero cells

为构建犬新孢子虫AMA1基因的重组真核表达质粒,了解其在Vero细胞中的表达情况,采用RT-PCR方法扩增牛源犬新孢子虫吉林株AMA1全长基因,构建了重组真核表达质粒pVAX1-NcAMA1;利用脂质体介导转染法将该重组质粒转染至Vero细胞,应用间接免疫荧光方法(IFA)和Western-blot技术检测AMA1基因在Vero细胞中的表达情况。结果显示,扩增的牛源犬新孢子虫吉林株AMA1基因的长度为1 695bp,与GenBank中相应基因序列(AB265823.1)的同源性为99.9%。成功构建了重组真核表达质粒pVAX1-NcAMA1,IFA检测发现AMA1基因在Vero细胞中获得了瞬时表达,表达产物经Western-blot鉴定,其分子质量约为68ku,且具有较好的反应原性。本试验为新孢子虫病核酸疫苗的进一步研究奠定了基础。

To construct recombinant eukaryotic expression plasmid of AMA1 gene of Neospora cani- hum and express it in Vero cells,the full-length fragment of NcAMA1 gene from bovine N. caninum Jilin strain was amplified by RT-PCR,and then subcloned into the expression vector pVAX-1 to construct a eu- karyotic expression plasmid pVAX1-AMA1. The recombinant was then transfected into the Vero cells by liposome method. The expressed product in Vero cells was analyzed by indirect immunofluorescence assay （IFA） and Western-blot. The results showed that the amplified fragment was 1 695 bp,and shared 99.9% homology with the corresponding gene sequence in GenBank （AB265823. 1）. The recombinant eukaryotic expression plasmid pVAX1-AMA1 was successfully constructed. IFA result showed that the AMA1 gene was transiently expressed,and Western-blotting result showed that expressed product was 68 ku in molecu- lar weight and had a positive reaction with anti-AMA1 serum. These results may provide a foundation for further studies on the development of nucleic acid vaccines against N. caninurn infections.