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本文首次对我国现行狂犬病疫苗生产用毒株CTN-1进行全长基因组序列测定和分析,为CTN-1疫苗在我国实际应用中的优势提供理论基础。利用RT-PCR方法分段扩增CTN-1全基因组序列,随后将PCR产物克隆到T载体、测序、拼接,用MegAlign软件比较CTN-1全基因组序列与国内外狂犬病疫苗株和街毒株全基因组序列的同源性;再以糖蛋白(G)基因为模板,用ClustalX和MEGA4软件进行系统进化分析。测序结果表明CTN-1全基因组序列的长度为11925nt(GenBank登录号FJ959397),序列分析表明CTN-1为基因Ⅰ型;CTN-1株全基因组序列与国内外狂犬病疫苗株和街毒株全基因组之间的同源性是81.5%～93.4%;与美国蝙蝠分离株SHBRV18的同源性最低,仅为81.5%;与新近从中国国内分离的野毒株HN10株的同源性最高,达93.4%。系统进化分析结果表明,CTN-1与国内不同地区大多数分离的狂犬病街毒株聚类于同一组内;而我国另一疫苗株aG株与国外疫苗株如Flury、PM、PV、ERA、RC-HL和个别中国街毒株分在另一组内。G基因氨基酸对比也显示CTN-1株与国内大多数街毒株的同源性高于其他疫苗株,结果说明CTN-1株较其他疫苗株病毒更适合制备用于预防中国狂犬病的灭活疫苗。
This article for the first time on China’s current rabies vaccine production strain CTN-1 full-length genomic sequence analysis and analysis for the CTN-1 vaccine in our practical application of the advantages provide a theoretical basis. The whole genome sequence of CTN-1 was amplified by RT-PCR. The PCR products were cloned into T vector and sequenced. The complete genome sequence of CTN-1 was compared with that of rabies vaccine strains and street strains by MegAlign software Genomic sequence homology; and then use the glycoprotein (G) gene as a template, phylogenetic analysis using ClustalX and MEGA4 software. The sequence of CTN-1 was 11925nt (GenBank accession number FJ959397). The sequence analysis showed that CTN-1 was genotype Ⅰ. The complete genome sequence of CTN-1 was identical with the genomes of rabies vaccine strains and street strains at home and abroad The homology was 81.5% -93.4% with the lowest homology with that of SHBRV18 of American bat, which was only 81.5%. The homology was 93.4% with HN10 newly isolated from China %. Phylogenetic analysis showed that CTN-1 clustered together with most rabies street isolates in different regions of China. However, aG isolates from other vaccine strains in China such as Flury, PM, PV, ERA, RC -HL and individual Chinese street strains were grouped in another group. The amino acid comparison of G gene also showed that the CTN-1 strain had higher homology with most of the domestic street strains than other vaccine strains. The results indicated that CTN-1 strain was more suitable than other vaccine strains for preparing inactivated vaccine for rabies prevention in China .