乙型脑炎病毒SA14-14-2株NS1蛋白基因在原核细胞中的高效表达及其表达产物的抗原性分析  

High expression of the nonstructural 1 genes of Japanese encephalitis virus (JEV) strain SA14-14-2 in E.coli and the antigenicity analysis of its expressed products

作　　者：李波[1] 李伯安[2] 侯俊[3] 胡艳[3] 曲芬[2] 郭桐生[2] 马洪滨[2] 貌盼勇[3] 毛远丽[2] 

机构地区：[1]解放军军医进修学院学员队,北京100853 [2]解放军第302医院临床检验中心 [3]解放军第302医院病毒研究所

出　　处：《解放军医学杂志》2008年第4期391-393,共3页Medical Journal of Chinese People's Liberation Army

基　　金：首都医学发展科研基金资助项目(C0302050202)

摘　　要：目的将乙型脑炎病毒NS1蛋白基因克隆至pET28-a(+)表达载体,构建原核表达载体pET-NS1,并使该基因在E.coli中高效表达,为进一步研制乙脑早期诊断试剂奠定基础。方法根据GenBank中提供的乙型脑炎病毒SA14-14-2株全基因序列设计引物,通过反转录及巢式PCR方法扩增目的片段,测序后连接pET28-a(+)载体,构建重组表达载体pET-NS1,转化大肠杆菌BL21(DE3)后,利用IPTG诱导获得高效表达。结果扩增出了1300bp的基因片段,与预期大小一致,测序后经Blast验证与已发表乙型脑炎病毒SA14-14-2株NS1蛋白基因序列同源性为100%,成功克隆至表达载体pET28-a(+)并获得高效表达,表达产物分子量约为45kD,Western blot分析表明该表达产物具有良好抗原性。结论该表达产物的稳定高效表达及其抗原特异性为乙脑的诊断试剂开发提供了依据。Objective To construct prokaryotic expression vector carrying Japanese encephalitis virus （JEV） NS1 gene, and to express the vector in E. coli, so to lay a foundation for the further development of JEV early diagnosis kit. Methods The NS1 gene was amplified by RT-PCR, the target gene and prokaryotic expression plasmid pET28a（＋） were digested by BamH I and Hind Ⅲ respectively. The target gene was then purified by DNA extraction kit. A 1∶3 molar ratio of vector： insert DNA ligated with T4 DNA ligase to construct the recombinant plasmid pET28a（＋）-NS1. The ligated products were transformed into E. coli BL21 （DE3） and the colonies were selected on LB medium with karamycin. After cultivation, positive colonies were picked out and the recombinant plasmid were identified by endonuclease digestions, PCR rand sequencing. The target protein was expressed with induction of IPTG. The expressed proteins mentioned above were then identified and analyzed by SDS-PAGE and Western-blotting respectively. Results The sequencing results of amplified products showed that JEV-NS1 RNA fragments were about 1 300bp in length which were similar as respected. Compared with the published sequence of SA14-14-2 with Blast, the homology of the nucleotide sequence was 100%. The molecular weight of expressed protein was about 45kD, the result of Western blotting proved the specific antigenicity of the protein. Conclusion The specific JEV nonstructural protein 1 is expressed in E. coli successfully and shows high specificity to the antibody. The stable expression of the protein and the analysis of its antigenic specificity provide foundation to develop the early stage diagnosis kit.

关 键 词：病毒非结构蛋白质类 克隆 分子 基因表达 抗原性 

分 类 号：R373-33[医药卫生—病原生物学] R373.21[医药卫生—基础医学]