西方马脑炎病毒E2基因的克隆与原核表达  

Cloning and prokaryotic expression of E2 gene of Western equine encephalomyelitis virus

作　　者：马金柱[1,2,3] 王化磊[2] 郑学星[2] 赵永坤[2] 高玉伟[2] 王铁成[2] 黄耕[2] 杨松涛[2] 夏咸柱[2,3] 

机构地区：[1]黑龙江八一农垦大学生命科学技术学院,黑龙江大庆163319 [2]军事医学科学院军事兽医研究所,吉林长春130122 [3]东北农业大学动物医学学院,黑龙江哈尔滨150030

出　　处：《中国生物制品学杂志》2013年第6期776-779,共4页Chinese Journal of Biologicals

基　　金：公益性行业(农业)科研专项(201103032);"十一五"国家科技支撑计划重点项目(2010BAD04B03)

摘　　要：目的克隆并原核表达西方马脑炎病毒(Western equine encephalomyelitis virus,WEEV)E2基因。方法采用PCR法从重组质粒pVL1393-C-E中扩增E2基因,插入原核表达载体pET-30a(+)中,构建重组表达质粒pET-30a(+)-E2,转化E.coli BL21(DE3),IPTG诱导表达。表达产物经SDS-PAGE和Western blot进行鉴定。结果重组表达质粒pET-30a(+)-E2经PCR、双酶切及测序证明构建正确;表达的重组蛋白相对分子质量约为52 900,诱导6 h目的蛋白的表达量较高,约占菌体总蛋白的23.5%,重组蛋白主要以包涵体形式存在,可与鼠源His单克隆抗体特异性结合。结论克隆并原核表达了WEEV E2基因,为E2蛋白免疫原性及WEEV亚单位疫苗的研究奠定了基础。Objective To construct the E2 gene of Western equine encephalomyelitis virus（WEEV） and express in prokaryotic cells.Methods E2 gene was amplified from recombinant plasmid pVL1393-C-E by PCR and inserted into prokaryotic expression vector pET-30a（＋）.The constructed recombinant plasmid pET-30a（＋）-E2 was transformed to E.coli BL21（DE3） for expression under induction of IPTG.The expressed product was identified by SDS-PAGE and Western blot.Results PCR restriction analysis and sequencing proved that recombinant plasmid pET-30a（＋）-E2 was constructed correctly.The relative molecular mass of expressed product was about 52 900.The expression level reached a peak value of 23.5% of total somatic protein 6 h after induction.The recombinant E2 protein mainly existed in a form of inclusion body and showed specific binding to mouse monoclonal antibody against His.Conclusion The E2 gene of WEEV was cloned and expressed in prokaryotic cells,which laid a foundation of study on immunogenicity of E2 protein and development of WEEV subunit vaccine.

关 键 词：西方马脑炎病毒 E2基因 克隆 原核细胞 基因表达 

分 类 号：Q786[生物学—分子生物学]