狂犬病病毒4aG株G蛋白基因的克隆及其生物信息学分析

目的克隆狂犬病病毒(Rabies virus,RV)4aG株G蛋白基因,并与其他疫苗毒株G蛋白基因序列进行比较,为我国狂犬病疫苗的研制提供理论依据。方法从RV 4aG株中扩增G蛋白基因,并进行序列测定,利用生物信息学软件绘制系统进化树,预测G蛋白功能位点、二级结构和B细胞抗原表位,并与其他疫苗毒株进行比较。结果克隆的4aG株G蛋白基因序列与GenBank中登录的序列一致。进化树分析显示,4aG株与CVS株的进化关系较远;4aG、4aGV18、CVS、PV及RC-HL毒株跨膜区域在第460～479氨基酸之间,其他毒株在459～478氨基酸之间;PV株有5个糖基化位点,RV-97株有3个糖基化位点,其他毒株均有4个糖基化位点;G蛋白抗原表位位于氨基酸100～300及480～520之间;不同毒株G蛋白抗原位点区域有所不同,与CVS株比较,PV株抗原表位与其最为接近,4aG、4aGV18、CTN-1-31及Flury-HEP株与其抗原表位较为接近。结论4aG株G蛋白功能位点和抗原表位与CVS株相差较大,但其在Vero细胞上传代稳定,可用于制备Vero细胞纯化疫苗。

Cloning and bio-informatics analysis of G protein of RV aG strain

Objective The clone the gene encoding G protein of rabies virus(RV)4aG strain and compare its sequence with those of other vaccine strains so as to provide a theoretical basis for preparation of rabies vaccine in China.Methods G gene was amplified from RV 4aG strain and sequenced,based on which a phylogenetic tree was plotted by using bioinformatics software,and the functional site,secondary structure and B cell antigenic epitope of G protein were predicted and compared with those of other vaccine strains.Results The sequence of cloned G gene was identical to that reported in GenBank.Phylogenetic tree showed relatively remote relationship between 4aG and CVS strains.The transmembrane regions of 4aG,4aGV18,CVS,PV and RC-HL strains were located in amino acids 460 ～ 479,while those of other strains in amino acids 459 ～ 478.Five glycosylation sites were observed in PV strain,while 3 in RV-97 strain and 4 in other strains.The antigenic epitopes of G protein were located in amino acids 100 ～ 300 and 480 ～ 520,which were different in various strains.The antigenic epitopes of 4aG,4aGV18,CTN-1-31 and Flury-HEP strains showed high similarity,while that of PV strain shwoed the highest similarity,to that of CVS strain.Conclusion The G protein of 4aG strain showed significant difference with that of CVS strain in functional site and antigenic epitope.However,the strain was subcultured stably in Vero cells,and might be used for preparation of Vero cells-derived purified rabies vaccine.