犬新孢子虫AMA1基因重组猫疱疹病毒1型转移载体的构建及真核表达

Construction and eukaryotic expression of recombinant feline herpesvirus type 1 transfer vector with Neospora caninum AMA1 gene

为构建犬新孢子虫AMA1基因重组猫疱疹病毒1型(FHV1)转移质粒pBS-TK-NcAMA1,并在真核细胞中表达,以重组质粒pVAX1-NcAMA1为模板,扩增犬新孢子虫AMA1基因,并将其亚克隆至重组质粒pBS-TK。通过转染试剂PEI将pBS-TK-NcAMA1转移载体转染至293T细胞,以制备的小鼠抗犬新孢子虫AMA1蛋白的特异性抗体为一抗,应用间接免疫荧光试验和Western-blot对基因的表达情况进行鉴定。结果显示,构建的病毒转移载体pBS-TK-NcAMA1可以在293T细胞内获得表达,表达蛋白的分子质量为68ku。这一研究成果为犬新孢子虫AMA1基因的重组猫疱疹病毒1型载体疫苗的研制奠定了基础。

To construct a recombinant feline herpesvirus type 1 transfer vector with Neospora caninum AMA1 gene,pBS-TK-NcAMA1,and to express AMA1 gene in eukaryotic cells,an AMA1 gene was amplified and subcloned into the recombinant plasmid pBS-TK vector.The recombinant feline herpesvirus type 1 transfer vector pBS-TK-NcAMA1 was then transfected into 293T cells by PEI.The expressed product in 293T cells was analyzed by indirect fluorescence assay and Western-blot.In result,the recombinant plasmid pBSTK-NcAMA1 expressed AMA1protein in 293T cells and the protein molecular weight was about 68ku.This study laid the foundation for construction of recombinant feline herpesvirus type 1vaccines using N.caninum AMA1.