狂犬病病毒aG株G基因的克隆与生物信息学分析

为了研究狂犬病病毒aG株的进化情况,分析狂犬病病毒的遗传变异和基因功能,采用BHK-21细胞培养繁殖狂犬病病毒aG株,运用RT-PCR扩增G基因,将其与pMD18-T simple vector连接后转化JM109感受态细胞,经PCR扩增和测序分析进行鉴定,用生物信息学软件预测其功能,并构建系统进化树。结果显示,狂犬病病毒G蛋白基因全长1 575bp,包含1个阅读框(ORF),编码524个氨基酸。生物信息学分析表明,G蛋白的分子质量为58 377.22u,理论等电点为7.080,半衰期为30h(体外哺乳类网状细胞),不稳定系数为42.99,脂肪指数为88.45,总平均亲水性为-0.106,最大疏水性为3.489,最小疏水性为-2.600,信号肽序列为1～19位氨基酸残基,跨膜区结构分析显示该蛋白为跨膜蛋白,预测可能包含有4个N-糖基化作用位点,2处基于cAMP和cGMP的蛋白激酶磷酸化位点,13处蛋白激酶C磷酸化位点,12处酪蛋白Ⅱ磷酸化位点;1处酪氨酸激酶磷酸化位点;9处N豆蒄酰化位点;1个酰胺化位点。系统进化分析表明,aG株G蛋白与DRV株进化距离最近。

Cloning and bioinformatics analysis of G gene of rabies virus aG strain

In order to monitor the evolutions of rabies virus aG strain,and to analyze the genetic vari-ation and function of G gene of aG strain,the total RNA was extracted from the rabies virus aG strain cultured in BHK-21.The G gene was amplified by RT-PCR and directly cloned into pMD18-T simple vector.The plasmid was transformed into Escherichia coli JM109,and then identified by PCR amplification and sequencing.Then the function of G gene was predicted by bioinformatics software and the phylogenetic tree of G gene was constructed by MEGA 5 software.The results indicated the complete G gene was 1 575 bp in length,encoding 524 aa.Bioinformatics analysis showed that molecular weight of G protein was 58 377.22 u,and the theoretical pI,estimated half-life,instability index and fatty index of G protein was 7.080,30 hours(mammalian reticulocytes in vitro),42.99 and 88.45,respectively.Grand average of hydropathicity of G protein was-0.106.The maximum and the minimum hydropathicity were 3.489 and-2.600 respectively.The signal peptide of the protein was 1-19 amino acid residues.Motif searching showed that G protein might have 4 N-glycosylation sites,2 cAMP and cGMP protein kinase phosphorylation sites,13 protein kinase C phosphorylation sites,12 casein kinaseⅡ phosphorylation sites,1 tyrosine protein kinase phosphorylation site,9 N myristoylation sites,and 1 amidation site.Analysis of phylogenetic tree indicated that the aG strain shared the closest evolutionary distance with rabies virus DRV strain.