西方马脑炎病毒E2基因的克隆与原核表达

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。

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

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.