乙型脑炎减毒活疫苗生产株SA14-14-2全基因组序列测定及基因遗传稳定性

Seqencing and genetic stability of whole genome for prodution strain SA14-14-2 used in Japanese encephalitis vaccine(live)

目的对乙型脑炎减毒活疫苗生产株SA14-14-2株进行全基因组序列测定和分析,并观察该生产株在疫苗制备过程中的基因遗传稳定性。方法根据DNA序列数据库(Gen Bank)公布的SA14-14-2株的序列,设计合成7对引物,提取疫苗生产株SA14-14-2及其工作种子批生产的3批原液、3批成品疫苗的病毒RNA,通过RT-PCR方法扩增SA14-14-2株的cDNA片段,分别克隆到pGEM-T载体,转化至大肠埃希菌DH5α中,挑取阳性菌落克隆、鉴定后测定全序列并对序列进行比较分析,观察毒株在传代的过程中病毒滴度的稳定性。结果乙型脑炎减毒活疫苗生产株SA14-14-2株基因组全长10 976 bp,编码3 433个氨基酸。3批原液和3批成品疫苗的基因组全长为10 977 bp,比较分析发现,在3'端非编码区10 701处多一个G核苷酸的插入。与DNA序列数据库(Genbank)登录号为D90195的全长序列同源性分别是99.9%、99.9%、99.9%、99.9%、99.8%、99.9%、99.8%,其中E蛋白的同源性均为100%。SA14-14-2生产株的病毒滴度为7.22 lg PFU/mL,原液和成品疫苗的滴度分别为7.32、7.23、7.32、6.86、6.92、6.70 lg PFU/mL。结论乙型脑炎减毒活疫苗生产株SA14-14-2基因稳定,具有良好的一致性,为乙型脑炎减毒活疫苗的质量的稳定性提供了可靠依据。

Objective To sequence and analyze the whole genome of production strain SA14-14-2 used in Japanese encephalitis vaccine, live and to investigate it＇ s genetic stability in vaccine production process. Methods A total of 7 pairs of primers were synthesized according to the genome of SA14-14-2 strain published in GenBank. Viral RNAs were extracted from SA14-14-2 working strain and 3 bulk lots and 3 final products, from which cDNA fragments were amplified by RT- PCR. Then cDNA fragments were cloned into pGEM-T vector, and transformed into Escherichia coli DH5α. The positive colones were screened and sequenced. Sequence analysis and comparison were carried out, and the stability of virus titer was observed during the passaged process. Results The sequence analysis showed that whole genome of SA14-14-2 strain contains 10 976 nucleotides, encoding 3 433 amino acids, whereas there are 10 977 nucleotides in the bulk lots and the final products. This difference was due to an insertion of one G nucleotide at position 10 701 in the 3＇ non-coding region. The homologies of nucleotide were 99.9%, 99.9%, 99.9%, 99.9%, 99.8%, 99.9% and 99.8%, respectively, in comparison of the whole genomes, and all E proteins contained a complete homology （ 100% ） with that in SA14-14-2 strain published in GenBank with the accession number D90195. The virus titer was 7.22 lg PFU/mL for SA14-14-2 strain, while for the 3 bulk lots and the 3 final products were 7.32, 7.23,7.32, 6.86, 6.92, 6.70 lg PFU/mL, respectively. Conclusion The SA14-14-2 strain showed a high genetic stability and consistency in production of Japanese encephalitis vaccine, live, which provided a reliable basis for the quality stability of the vaccine.