犬新孢子虫AMA1基因的生物信息学分析及原核表达载体构建

Bioinformatics analysis of AMA1 gene of Neospora caninum and construction of prokaryotic expression vector

为了解新孢子虫AMA1基因的生物信息学特性,并构建重组原核表达质粒p GEX-4T-1-Nc AMA1。本试验对牛源犬新孢子虫吉林株AMA1基因进行分子克隆,利用生物信息学软件对该基因进行编码蛋白等电点、信号肽、跨膜区、糖基化位点及疏水性分析,并将该基因片段亚克隆至原核表达载体p GEX-4T-1,构建了重组表达质粒p GEX-4T-1-Nc AMA1。结果显示,克隆的Nc AMA1基因片段长1 695 bp,与GenBank(AB265823.1)上发表的基因序列同源性99.9%;Nc AMA1基因编码蛋白等电点为5.43,在其N端及C端分别存在一个跨膜区,为不溶性蛋白,且Nc AMA1蛋白抗原指数较高,有潜在疫苗研究价值。

To understand the biological information of AMA1 gene of Neospora caninum （ NcAMA1 ） and construct the recombinant expres sion plasmid pGEX-4T- 1 -NcAMA1, the NcAMA1 gene of N. caninum Jilin strain was cloned, and the isoelectric point of the gene encoding protein, signal peptide, transmembrane region, glycosylation sites and hydrophobicity were analyzed using bioinformatics software. NcAMA1 gene was subcloned into the prokaryotic expression vector pGEX-4T-1 to construct the recombinant expression plasmid pGEX-4T-1-NcAMA1. The results showed that the length of cloned gene fragment NcAMA1 was 1 695 bp, and the homology with GenBank published gene sequence （AB265823.1） was 99. 9%. The isoelectric point of encoding protein is 5.43, and there is a transmembrane domain in its N-terminal and C-terminal, respectively. NcAMA1 is insoluble protein, and its antigens index is high, suggesting the potential in vaccine development.