锦鲤疱疹病毒-CJ株ORF81基因的克隆及生物信息学分析

Cloning and bioinformatics analysis of ORF81 gene of koi herpesvirus-CJ strain

为了解锦鲤疱疹病毒中国吉林株(KHV-CJ)ORF81蛋白的结构特征和进化关系,用框镜鲤尾鳍原代细胞增殖KHV-CJ,提取其DNA,经PCR扩增,获得ORF81基因,将其克隆到pMD18-T载体中,构建重组质粒。应用生物信息学方法初步分析KHV-CJ ORF81基因的结构和功能,并与GenBank上已公布的3株KHV构建系统进化树。结果显示,获得了长771 bp的ORF81基因,编码256个氨基酸;预测ORF81基因的理论分子量为28 246.50 u,等电点为8.404;疏水性大于亲水性;信号肽切割部位最可能位于29位的S(丝氨酸);有4个跨膜区;抗原表位预测显示抗原性良好;结构预测显示,不存在N-糖基化位点、存在6个O-糖基化位点和11个磷酸化位点;系统进化树分析显示,与锦鲤疱疹病毒以色列株属同一分支。

To study its structure and function, the ORFS1 gene was amplified by PCR from koi herpesvirus China＇ s Jilin strains （KHV-CJ）infected. Mirror carp primary fin cells were then cloned into the vector pMD18-T to construct the recombinant plasmid. The structure and function of the gene were studied by means of bioinformatics software, including phylogenetic tree analysis compared with other three KHV strains published in GenBank. The results showed that the length of the ORFS1 gene, encoding 256 aa, was 771 bp. The molecular weight was 28 246. 50 u and the isoelectric point was 8. 404. The protein is hydrophobic. The most likely signal peptide cut was in 29 bit（Ser）. ORFS1 has four transmembrane regions. The antigenicity of ORP＇B1 was good. According to the protein structure analysis, there may be no N- glycosylation sites,6 O-glycosylation sites and 11 phosphorylation sites. Phylogenetic analysis showed that he ORFS1 gene of KHV-CJ was in the same branch with the Israel strain. Above all, ORF81 gene may have good immunogenicity. The study established the foundation for genetic background information, pathogenesis, molecular epidemiology and genetic engineering vaccine research of koi herpesvirus.