【摘要】 目的原核表达、纯化大肠杆菌不耐热肠毒素B亚单位(Heat-labile enterotoxin B subunit,LTB),并评价其黏膜免疫的佐剂作用。方法从产毒素大肠杆菌44815菌株中扩增出LTB基因,克隆至质粒pET-32a(+)中,构建重组原核表达质粒pET-32a(+)-LTB,转化大肠杆菌BL21(DE3)pLysS,IPTG诱导表达,表达的LTB经纯化后进行神经节苷脂GM1结合活性及黏膜免疫佐剂活性鉴定。结果重组表达质粒经双酶切及测序鉴定证明构建正确;重组LTB主要为可溶性表达,表达量约占菌体总蛋白的15%;纯化的LTB纯度可达95%以上,可与兔抗CTB血清发生特异性反应,且具有GM1结合活性及良好的黏膜免疫佐剂活性。结论在大肠杆菌中成功表达了重组LTB蛋白,纯化的LTB具有良好的黏膜免疫佐剂活性。更多还原

【Abstract】 Objective To express E.coli heat-labile enterotoxin B subunit(LTB) in prokaryotic cells and evaluate its mucosal immunoadjuvantivity.Methods LTB gene was amplified from toxinogenic E.coli 44815 strain and cloned into plasmid pET-32a(+).The constructed recombinant plasmid pET-32a(+)-LTB was transformed to E.coli BL21(DE3) pLysS and induced with IPTG.The expressed LTB was purified then determined for ganglioside(GM1) binding activity and mucosal immunoadjuvantivity.Results Restriction analysis and sequencing proved that recombinant plasmid pET-32a(+)-LTB was constructed correctly.The expressed LTB mainly existed in a soluble form,contained about 15% of total somatic protein and showed specific reaction with rabbit serum against CTB,binding activity to GM1 as well as high mucosal immunoadjuvantivity.Conclusion Recombinant LTB protein was successfully expressed in E.coli,which showed high mucosal immunoadjuvantivity after purification.更多还原