【摘要】 为提高抗对硫磷基因工程四价抗体在大肠杆菌中可溶性表达量,本研究利用克隆技术得到四价抗体基因sc-sa,将该融合基因连接至表达载体pTO-T7的Ω序列和T7启动子下游,构建成功的表达质粒导入大肠杆菌OrigamiB(DE3),经IPTG诱导表达,SDS-PAGE和Western Blot鉴定表达产物,Ni亲和层析纯化蛋白,间接非竞争ELISA法测定该四价抗体的亲和力。结果表明在大肠杆菌OrigamiB(DE3)中表达分子量约为46kDa的四价抗体,0.1mmol/L的IPTG在30℃条件下诱导原核表达,外源蛋白占菌体总蛋白含量近50%,纯化后可溶性蛋白纯度在90%以上,ELISA结果显示该抗体与对硫磷结合呈阳性,抗体效价在1∶1×106以上,亲和常数为6.84×108L/mol。与母源抗体和相应单链抗体相比,利用pTO-T7载体四价抗体在大肠杆菌OrigamiB(DE3)中可实现高效表达,且抗体效价和亲和力均得到进一步提高。更多还原

【Abstract】 To improve expression of genetically engineered tetravalent antibodies against parathion in E.coli using optimal combination of expression plasmid and host,after being cloned,gene of tetravalent antibody,named sc-sa,was subcloned into downstream promoter of the vector pTO-T7 containing an enhancer from tabacoo mosauc virus(TMV),Ω sequence.Then the expression vector was used to transformed E.coli OrigamiB(DE3) for prokaryotic expression.The product was identified by SDS-PAGE and Western Blot.Activity of target protein was measured with ELISA after purification using Ni-NTA chelating affinity chromatography.The results showed that SDS-PAGE and Western Blot analysis demonstrated that the tetravalent antibody was highly in the form of both inclusion body and soluble protein with a purity up to 90%.The results of ELISA showed that antibody liter was about 1∶106 and affinity constant was 6.84×108L /mol.Compared to maternal antibody and ScFv(single chain Fv antibody),expression level and bioactivity of tetravalent antibody had improved significantlly with the application of expression vextor pTO-T7 in E.coli OrigamiB(DE3).更多还原