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  

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

作　　者：张付凯[1] 乔亚奇[1] 王磊[1] 兰丽平[1] 潘家荣[1,2] 

机构地区：[1]中国农业科学院农产品加工研究所/农业部农产品加工与质量控制重点开放实验室,北京100193 [2]中国计量学院生命科学学院,浙江杭州310018

出　　处：《核农学报》2012年第1期118-122,175,共6页Journal of Nuclear Agricultural Sciences

基　　金：国家高技术研究发展计划课题(2006AA10Z449)

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。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 bioaetivity of tetravalent antibody had improved significantlly with the application of expression vextor pTO-T7 in E. coli OrigamiB （ DE3 ）.

关 键 词：Ω序列 四价抗体 原核表达 

分 类 号：Q78[生物学—分子生物学]