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目的研究中国黄热疫苗生产用减毒株与世界卫生组织(WHO)黄热疫苗标准株的基因序列,了解其核苷酸序列变异程度,和与其相关的氨基酸是否发生突变。方法根据基因库(GenBank)中收录的黄热病毒(Yellow Fever Virus,YFV)17D序列设计引物,提取该两株YFV的核酸,逆转录后进行聚合酶链反应(Polymerase Chain Reaction,PCR)扩增病毒各部分基因片段,PCR产物纯化后直接测序,5’和3’端TA克隆后进行测序,然后进行遗传进化分析。经核苷酸序列测定进行序列比对分析。结果经序列测定后,进行两株YFV的序列比对,并与GenBank中收录的其它相关YFV17D疫苗株和野毒株进行序列比对,发现中国黄热疫苗生产株与WHO黄热疫苗标准株间的核苷酸序列并未发生较大变异,核苷酸和氨基酸的同源性分别约为99%和99%。其重要病毒抗原E蛋白也未发生较大突变,但发现E区与毒力密切相关的173位氨基酸发生回复突变,进一步通过系统发生分析说明两者亲缘关系十分接近。结论传代过程中,中国黄热疫苗株和WHO黄热疫苗标准株在基因水平上除个别外未发生较大改变,其基因组具有一定的稳定性。
Objective To study the gene sequence of attenuated strain of yellow fever vaccine in China and the standard strain of Yellow fever vaccine of World Health Organization (WHO) to find out whether the variation of nucleotide sequence and the amino acids related to it are mutated. Methods Based on the sequence of 17F of Yellow Fever Virus (YFV) contained in GenBank, the two YFV nucleic acids were extracted and reverse transcribed for polymerase chain reaction (PCR) amplification The virus fragments of various parts of the PCR products were purified directly sequenced, 5 ’and 3’ TA cloned after sequencing, and then genetic analysis. Sequence alignment analysis was performed by nucleotide sequencing. Results After sequence determination, the sequence alignment of two strains of YFV was carried out. The sequence alignment of other YFV17D vaccine strains and wild-type strains in GenBank revealed that the Chinese yellow fever vaccine strains and WHO yellow fever vaccine standard strains There was no significant variation in the nucleotide sequence, nucleotide and amino acid homologies were about 99% and 99%, respectively. The major virus antigen E protein also did not occur a major mutation, but found in E region and virulence closely related to the amino acid mutation occurs back 173, further phylogenetic analysis shows that the two are very close genetic relationship. Conclusion During the process of passage, the standard strains of yellow fever vaccine and yellow fever vaccine of China did not change much at the gene level except for a few cases, and the genome of the yellow fever vaccine and the WHO standard vaccine had a certain stability.