| 摘要: |
| [目的]分离鉴定乙型脑炎病毒(JEV),确定分离毒株的基因分型及其E区段氨基酸的序列特征.[方法]采用BHK-21细胞培养法,从猪脑组织标本中分离出1株病毒,对其进行细胞和免疫荧光试验,同时采用RT-PCR扩增并克隆该毒株的PrM、E区段核苷酸序列,利用PrM(456～695位)区核苷酸序列与36株参考序列进行基因分型分析,并对E区核苷酸序列与减毒活疫苗株SA-14-14-2株的相应序列进行比对,分析分离株和疫苗株氨基酸的同源性及关键结构域的差异.[结果]分离出的病毒经细胞病变和直接免疫荧光试验证实为乙脑病毒,命名为SXBJ07;用该病毒感染BHK-21细胞,72 h后所有细胞均发生病变(CPE);用该病毒接种3日龄乳鼠,72 h之内即可导致乳鼠死亡;SXBJ07株PrM(456～695位)区基因分型证实,分离株属于基因Ⅰ型,E区段核苷酸和氨基酸与减毒活疫苗株SA-14-14-2株的同源性分别为87.7%和 97.0%,但在E蛋白的活性关键结构域有13个氨基酸存在差异.[结论]从陕西省境内首次成功分离到1株Ⅰ型乙脑病毒,初步确定了该分离株的分子特征,对乙型脑炎的流行病学研究及其防治具有重要意义. |
| 关键词: 猪 乙型脑炎乙脑病毒 分离鉴定 PrM基因分型 E基因 |
| DOI： |
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| 基金项目:国家自然科学基金 |
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| Isolation,identification and sequence analysis of the Japanese encephalitis virus SXBJ07 strain |
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| Abstract: |
| 【Objective】 The research isolated and identified Japanese encephalitis virus(JEV),and verified the genotype and amino acid sequence in E region of the new isolated strain. 【Method】 Virus strain was isolated from swine brain samples by cell culture preparation,the biological characteristics of the new isolate was preliminarily dstermined by inoculating BHK 21 cell and suckling mice,the PrM gene and E gene nucleotide sequences amplified and cloned from the isolated strain,the genotype analyzed according to PrM(456-695) sequences with the referenced sequences of 36 strains,and the E nucleotide sequences compared with the SA 14 14 2 live attenuated vaccine strain. 【Result】 Named SXBJ07 the new isolated strain was confirmed as Japanese encephalitis virus by cytopathic effect (CPE) test and immuno fluorescence assay (IFA).All BHK 21 cells were cytopathic after 72 h inoculating the isolated strain,inoculated suckling mice died within 72 h. The isolate was grouped into genotypeⅠof JEV,the homology of the nucleotide and amino acid sequence between the isolated strain and vaccine stain was 87.7% and 97.0% respectively,but there were amino acid substitutions in 13 sites of the key active domains. 【Conclusion】 A genotypeⅠJEV strain was isolated from Shaanxi province and the molecular characterization was preliminarily confirmed.This might be of great significance for the epidemiology and control researches of Japanese encephalitis. |
| Key words: swine Japanese encephalitis virus (JEV) isolation and identification PrM genotype E gene |
