乙型脑炎病毒非结构4A蛋白编码基因重组子的构建及表达  

Construction and expression of a recombinant coding for nonstructural 4A protein derived from Japanese encephalitis virus

作　　者：翟永贞[1] 常彩芳[1] 冯国和[1] 

机构地区：[1]中国医科大学附属盛京医院感染科,辽宁沈阳110004

出　　处：《中国病原生物学杂志》2014年第7期599-602,612,共5页Journal of Pathogen Biology

基　　金：辽宁省教育厅基础研究计划资助项目(No.05L498);辽宁省博士科研启动基金项目计划(No.20131147)

摘　　要：目的构建流行性乙型脑炎病毒(JEV)非结构4A(NS4A)蛋白编码基因重组子并鉴定。方法以JEV SA14-14-2株Total RNA为模板,运用RT-PCR方法扩增JEV NS4A蛋白编码基因,克隆至pMD19-T Simple载体并测序。为便于分析JEV NS4A蛋白编码基因重组子在哺乳动物细胞中的表达,在JEV NS4A蛋白编码基因5'端附加FLAG序列,亚克隆至pcDNA3.1(+)载体中,构建重组子pJNS4A并作酶切及DNA测序分析;采用脂质体法将pJNS4A转染中华仓鼠卵巢(CHO)细胞,采用免疫荧光法检测转染的CHO细胞中JEV NS4A蛋白分布与表达。结果重组质粒pJNS4A经BamHⅠ/EcoRⅠ酶切释出的插入子在830bp左右,与JEV NS4A蛋白编码基因和FLAG基因序列之和(834bp)相一致。JEV NS4A蛋白编码基因重组质粒转染的CHO细胞可见绿色荧光标记,主要分布在胞膜。结论 pJNS4A构建成功,转染的CHO细胞可稳定表达JEV NS4A蛋白。Objectives To construction and identify a recombinant coding for NS4 Aprotein derived from Japanese encephalitis virus（JEV）. Methods Using total RNA of the SA14-14-2strain of JEV as a template,agene encoding JEV NS4 Awas amplified using RT-PCR and cloned into the pMD19-T simple vector.The pMD19-T simple vector containing the gene coding for JEV NS4 Awas designated pMD19-T simple-NS4 A.The pMD19-T simple-NS4 Awas subsequently identified with the restriction enzymes BamH I and EcoRI and sequenced,revealing it to be identical to sequences of JEV NS4 Adeposited in GenBank.In order to better analyze the expression of a recombinant coding for the JEV NS4 Agene in mammalian cells,a FLAG sequence was added to the 5＇end of the gene coding for JEV NS4 A.A 900-bp fragment of JEV NS4 Aand the FLAG sequence was digested from pMD19-T simple-NS4 Awith the restriction enzymes BamH I and EcoRI and then subcloned into the pcDNA3.1（＋）vector at the BamH I/EcoRI site.The result was designated pJNS4 A.The recombinant was confirmed using restriction enzyme analysis and DNA sequencing and it was then transfected into Chinese hamster ovary（CHO）cells using Lipofectamine 2000.Immunofluorescence was used to detect the distribution and expression of the NS4 Aprotein encoded by pJNS4 Ain transfected CHO cells. Results A DNA fragment（834bp）of the insert released from pJNS4 Awith BamH I/EcoR I restriction endonuclease agreed with the expected result.The recombinant fusion protein FLAG/NS4 Awas mainly located in the membrane of transfected CHO cells and seldom present in the cytoplasm of transfected CHO cells. Conclusion The recombinant pJNS4 Awas successfully constructed and transfected into CHO cells,and CHO cells transfected with pJNS4 Astably expressed the protein JEV NS4 A.

关 键 词：脑炎病毒 日本 病毒蛋白质类 NS4A蛋白 CHO细胞 

分 类 号：R373.31[医药卫生—病原生物学]