乙型脑炎病毒减毒活疫苗生产株SA_(14)-14-2基因组全序列的测定

Sequence analysis of the full-length genome of Japanese encephalitis virus vaccine SA_(14)-14-2 strain

目的 对乙型脑炎减毒活疫苗SA14 1 4 2生产株进行全序列测定和分析 ,为进一步了解其基因组结构与疫苗减毒机制的关系及研究疫苗生产的遗传学质控标准奠定基础。方法 根据已发表的SA14 1 4 2株及SA14株的序列 ,设计 6对相互重叠片段的引物 ,通过RT PCR扩增出SA14 1 4 2疫苗生产株的cDNA片段 ,分别克隆到pGEM T载体 ,转化至TG1受体菌中 ,挑取阳性克隆进行鉴定后测定全序列。结果 SA14 1 4 2生产株基因组全序列长 1 0 976个核苷酸 ,96到 1 0 3 94为一个长开放读码框 ,编码 3 43 2个氨基酸。与国外测定的SA14和SA14 1 4 2株的核苷酸序列和氨基酸序列相比 ,同源性均在 99%以上 ,突变位点分散于各个区域 ,以往推测的 7个可能与减毒相关的位点均未发生改变。1 2 96～ 1 50 6间有 4个突变位点 ,有可能作为疫苗质控的遗传学标记。结论 乙脑减毒活疫苗生产株的基因组全序列基本类似于已发表的序列 ,若干不同位点产生的原因可能在于病毒株的不同传代。全序列的测定对于研究疫苗株的减毒机理及疫苗的遗传学质控标准具有一定意义。

Objective To sequence the full length genome of Japanese encephalitis virus attenuated live vaccine SA 14 14 2 strain, provide direct information about the genomic structure and the possible relationship to the attenuated mechanism, and set up the basis for investigating the genetic quality control markers of the vaccine. Methods Six pairs of overlaping primers were designed according to the published sequences of SA 14 14 2 and SA 14 strains. Using RT PCR, cDNA fragments of SA 14 14 2 strain were got and cloned into pGEM T vector, then transformed into competent TG1 hosts. Positive clones were screened, identified and sequenced. Results Sequence analysis showed that genome of JEV vaccine SA 14 14 2 strain consisted of 10976 nucleotides and contained a single open reading frame of 10299 which encoded a polyprotein of 3432 amino acids. Compared with the published sequence of SA 14 14 2 and SA 14 strain, the homology of the nucleotide sequence and deduced amino acid sequence were all more than 99%, the mutation sites were located in different regions. Seven mutation sites responsible for the attenuation deduced were all consisted in. Among them, there were 4 mutated sites between 1296 and 1506 in the E region, this region may be used as genetic markers for the quality control of the vaccine. Conclusion Sequence of SA 14 14 2 vaccine genome is similar to the published data. Some mutations are among them probably due to passage of virus in difference cultures. The sequence analysis would be helpful to understanding attenuated mechanism of the vaccine and developing genetic quality control standard for vaccine production.