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目的测定我国人用狂犬病疫苗株4aGV、CTN-1V和PV2061株病毒糖蛋白G基因序列,并对其进行生物信息学分析。方法 RT-PCR扩增3株病毒糖蛋白G基因,分别克隆至pGEM-T载体中,转化大肠杆菌DH5α,筛选阳性克隆,进行测序。应用DNAstar MegAlign软件分析3株病毒糖蛋白的同源性,AntheProt 5.0及DNAstar Protean软件分析3株病毒糖蛋白的结构及潜在B细胞抗原表位的差异。结果 4aGV、CTN-1V和PV2061株病毒均属基因Ⅰ型狂犬病病毒;其糖蛋白G区基因同源性为76.0%～88.4%,G区ORF基因同源性为84.4%～91.5%,氨基酸序列同源性为90.5%～92.2%;3株病毒糖蛋白前19位氨基酸中均存在潜在信号肽结构,其中第19位氨基酸为三者潜在信号肽断裂位点,第17位氨基酸为4aGV株病毒糖蛋白潜在信号肽断裂位点;3株病毒糖蛋白分别在第463～476、464～475及463～475位氨基酸存在潜在可折叠螺旋的疏水性区域,为明显跨膜区域;4aGV、CTN-1V和PV2061株病毒糖蛋白氨基酸残基均富含潜在α螺旋、β折叠及卷曲结构,其比例分别为30%、31%、39%,28%、30%、41%和29%、30%、41%,易发生扭曲、折叠,形成丰富的二级结构;3株病毒糖蛋白潜在B细胞抗原表位位置与数量相似。结论已对3株人用狂犬病疫苗株G基因进行了全面生物信息学分析,为完善我国人用狂犬病疫苗生产用毒种的质控标准提供了支持。
Objective To determine the sequence of virus glycoprotein G gene of rabies vaccine strains 4aGV, CTN-1V and PV2061 in China and analyze its bioinformatics. Methods Three viral glycoprotein G genes were amplified by RT-PCR and cloned into pGEM-T vector respectively. The recombinant plasmid was transformed into E. coli DH5α. The positive clones were screened and sequenced. DNAstar MegAlign software was used to analyze the homology of the three viral glycoproteins. AntheProt 5.0 and DNAstar Protean software were used to analyze the structure of the three viral glycoproteins and the differences of potential B cell epitopes. Results The sequences of 4aGV, CTN-1V and PV2061 belonged to genotype I rabies virus. The homology of glycoprotein G region was 76.0% -88.4%, the homology of ORF gene in G region was 84.4% -91.5%, and the amino acid sequence The homology was 90.5% -92.2%. Potential signal peptides existed in the first 19 amino acids of the three virus glycoproteins, of which the 19th amino acid was the potential signal peptide cleavage site and the 17th amino acid was 4aGV strain Glycoprotein potential signal peptide cleavage site; three strains of viral glycoproteins in the 463 ~ 476,464 ~ 475 and 463 ~ 475 amino acids exist potential collapsible helical hydrophobic region, the obvious transmembrane region; 4aGV, CTN- The amino acid residues of 1V and PV2061 virus glycoproteins were all rich in potential α-helix, β-sheet and curly structure with the proportion of 30%, 31%, 39%, 28%, 30%, 41% and 29%, 30% , 41% respectively. It is easy to twist and fold to form abundant secondary structure. The positions and numbers of potential B cell epitopes of three virus glycoproteins are similar. Conclusions The bioinformatics analysis of G gene of three human rabies vaccine strains has been carried out, which provides support for improving the quality control standards for human virulent rabies vaccine production.