牛源犬新孢子虫NcSRS2-NcGRA7融合基因的克隆与生物信息学分析

Cloning and bioinformatics analysis of NcSRS2-NcGRA7 fusion gene of bovine Neospora caninum

为了解牛源犬新孢子虫NcSRS2-NcGRA7融合基因的生物学特性,本试验提取牛源犬新孢子虫基因组DNA,应用PCR技术扩增犬新孢子虫表面蛋白基因NcSRS2和致密颗粒蛋白基因NcGRA7,SOE-PCR技术拼接NcSRS2和NcGRA7基因,构建NcSRS2-NcGRA7融合基因重组克隆质粒,并进行PCR鉴定、双酶切鉴定及生物信息学分析。结果,NcSRS2基因扩增片段大小为1 061 bp,NcGRA7基因扩增片段大小为364 bp,NcSRS2-NcGRA7融合基因扩增片段大小为1 482 bp;获得重组克隆质粒pMD18-NcSRS2-NcGRA7经PCR鉴定、双酶切鉴定正确;测序分析表明,与Gen-Bank中已发表的美国株犬新孢子虫(AF061249、AF176649)核苷酸序列同源性为99%;经DNAman等软件分析,预测NcSRS2-NcGRA7融合蛋白抗原指数较高,融合蛋白二级结构以α-螺旋和β-折叠为主,三级结构中2种蛋白独立折叠,并借助Linker互相连接,功能互不影响。本试验为牛源犬新孢子虫NcSRS2-NcGRA7融合蛋白的免疫学研究奠定了基础。

In order to understand the biological characteristic of NcSRS2-NcGRA7 fusion gene of bovine Neospora Caninum, genomic DNA of N. caninum was extracted. The surface protein gene NcSRS2 and granule protein gene NcGRA7 were amplified by PCR, respectively. Nc- SRS2-NcGRA7 fusion gene was synthesized by SOE-PCR. The NcSRS2-NcGRA7 cloning vector was constructed and identified by PCR am- plification, double digestion and bioinformatic analysis. The results showed that the sizes of NcSRS2, NcGRA7 and NcSRS2-NcGRA7 were 1061 bp, 364 bp and 1482 bp, respectively. The cloning vector was demonstrated to be correct by PCR and double digestion. Sequencing a- nalysis showed that the nucleotide sequences of NcSRS2 and NcGRA7 shared 99% homology with those published in GenBank （ AF061249, AF176649）. Prediction analysis by DNAman software indicated that the antigen index of NcSRS2-NcGRA7 was high. The main secondary structure of the fusion protein was mainly composed of α-helix and β-sheet. The tertiary structure analysis showed that the protein could fold into two independent regions connected with a linker. It indicated that the functions of NcSRS2 and NcGRA7 were not affected by each other. This study applies a foundation for the immunological functions of NcSRS2-NcGRA7 fusion protein of bovine N. caninum.