牛源犬新孢子虫NcGRA7基因的克隆及原核表达分析

Cloning and prokaryotic expression of bovine Neospora caninum NcGRA7 gene

为了解牛源犬新孢子虫NcGRA7基因的生物学特性,本实验采用PCR技术扩增牛源犬新孢子虫NcGRA7基因,并连接至pMD-18T载体中,构建重组质粒pMD18-NcGRA7,经PCR、酶切鉴定及测序分析,将NcGRA7基因连接到原核表达载体pGEX-4T-1中,构建重组表达质粒pGEX-NcGRA7,转化大肠杆菌BL21,筛选阳性克隆,将PCR和酶切鉴定正确的重组质粒进行IPTG诱导表达,SDS-PAGE和western blot分析表达产物。结果表明,扩增的NcGRA7基因片段大小为701 bp,编码233个氨基酸,与GenBank中登录的NcGRA7(AF176649)核苷酸序列同源性为98.7%;western blot分析表达蛋白的分子量为52 ku,具有较好的反应原性。本实验为犬新孢子虫NcGRA7基因疫苗研究奠定了基础。

To investigate the biological characteristics of bovine Neospora caninum NcGRA7 gene, the gene was amplified by PCR, sequenced and cloned into pGEX-4T-1 vector for expression in E.coli. The results showed that the length of NcGRA7 gene was 701 bp encoding 233 amino acids. The nucleotide sequence had 98.7% homology with that of AF176649 in GenBank. Western blot analysis showed that the expressed NcGRA7 recombinant protein was 52 ku and recognized by the positive serum against N.caninum. These results would facilitate future study of the N.caninum NcGRA7.