抗对硫磷基因工程四价抗体在大肠杆菌中高效表达与鉴定

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。

HIGH LEVEL EXPRESSION OF GENETICALLY ENGINEERED TETRAVALENT ANTIBODIES AGAINST PARATHION IN E.coli AND ITS IDENTIFICATION

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).