乙型脑炎病毒减毒中间株SA14—12—1—7基因组全序列的测定

本研究通过对乙型脑炎活疫苗减毒过程中间株SA14 12 1 7株进行全序列测定和分析 ,进一步了解乙脑活疫苗减毒及其稳定性的分子机制。根据已发表的SA14 14 2株及SA14 株的序列 ,设计 6对重叠引物 ,涵括整个乙脑病毒的基因组 ,通过RT PCR扩增出SA14 12 1 7株的各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序。结果表明SA14 12 1 7株基因组全序列长 10 976个核苷酸 ,从 96到 10 394为一个长开放读码框 ,编码 3432个氨基酸。与野毒株SA14 和疫苗株SA14 14 2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 ,E区有 5个位点与疫苗株一致而与野毒株不同 ,3个位点与野毒株一致而与疫苗株不同 ,推测与其容易产生回复突变、恢复毒力有关。此外 ,NS3、NS5和 3′NTR的几个位点可能与病毒毒力稳定性相关。综上所述 ,乙脑病毒减毒中间株的基因组全序列基本类似于已发表的序列 ,若干突变位点影响病毒的弱毒性及毒力的稳定性。全序列的测定对于研究疫苗株的减毒机理具有重要意义

Complete Sequence Analysis of the Full-length Genome of the Japanese Encephalitis Virus Attenuated Strain SA_(14)-12-1-7

To sequence the full length genome of Japanese encephalitis virus attenuated strain SA\-\{14\} 12 1 7,direct information about the genomic structure and the possible relationship to the attenuated and stability mechanism has been provided in this study.Six pairs of primers were designed according to the published sequences of SA\-\{14\} 14 2 and SA\-\{14\} strains,covering full length genome of the JEV virus.Using RT PCR,cDNA fragments of SA\-\{14\} 12 1 7 strain were got and cloned into pGEM T vector,then transformed into competent TG1 hosts.The positive colones were screened and then the inserted fragments were sequenced.The results of sequence analysis showed that genome of SA\-\{14\} 12 1 7 strain consistes of 10976 nucleotides and containes a single open reading frame of 10299 nt which encodes 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.In E region,5 sites are the same as SA\-\{14\} 14 2 and other 3 sites are the same as SA\-\{14\} strain.Some sites in NS3,NS5 and 3'NTR may relate with the attenuated stability.So the sequence of SA\-\{14\} 12 1 7 strain genome is similar to the published information,several mutation sites are responsibe for the attenuation of the virus and the virulence stability.The sequence analysis would aid in understanding attenuated mechanism of the vaccine.