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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。
Objective To clone the G protein gene of Rabies virus (4a) strain and to compare with the G protein sequence of other vaccine strains to provide a theoretical basis for the development of rabies vaccine in China. Methods G protein gene was amplified from RV 4aG strain and sequenced. Bioinformatics software was used to draw the phylogenetic tree to predict the G protein functional site, secondary structure and B cell epitopes. The results were compared with other vaccine strains Compare. Results The sequence of the G protein gene of the cloned 4aG strain was consistent with the sequence registered in GenBank. The phylogenetic tree analysis showed that the evolutionary relationship between the strains 4aG and CVS was far. The strains 4aG, 4aGV18, CVS, PV and RC-HL had the transmembrane region between 460 and 479 amino acids and the other strains between 459 and 478 amino acids There were 5 glycosylation sites in PV strain, 3 glycosylation sites in RV-97 strain and 4 glycosylation sites in other strains. The G protein epitope was located between 100-300 amino acids and 480-420. The antigenic region of G protein of different strains was different. Compared with CVS strain, the epitope of PV strain was the closest to it. The sequences of 4aG, 4aGV18, CTN-1-31 and Flury-HEP strain and its antigenic epitope Bit closer. Conclusion The G protein function site and antigen epitope of 4aG strain are quite different from that of CVS strain. However, they are stable on Vero cells and can be used to prepare Vero cell vaccine.