人抗地高辛单链抗体在大肠杆菌的表达

Studies on incolusion body expression of human scFv against digoxin in escherichia coli.

研究人抗地高辛(Dig)单链抗体(ADAscFv)在大肠杆菌(E.coli)的表达及包涵体变性和复性条件:方法:(1)用PCR扩增人ADAscFv基因,重组于克隆载体pGEM-T,NdeI+SaiI消化回收目的基因,插入pT7.7载体构建表达载体pT-ADAscFv,转化E.coliBL21(DE3),IPTG诱导表达,SDS-PAGE分析目的蛋白的表达情况,筛选高表达克隆;(2)提取纯化包涵体,SDS-PAGE分析其纯度,采用不同的方法及条件进行变性及复性,ELISA检测活性:结果:(1)SDS-PAGE显示,含有重组质粒pT-ADAscFv的F.coli菌株BL21(DE3)的表达产物中有一条约30kD的条带,其分子量与预期值相符,提取纯化的包涵体的纯度较高;(2)ELISA显示,透析复性法和稀释复性法均可使其复性。结论 成功地构建了人ADAscFv表达载体,实现了其在E.coli的表达并对其包涵体变性、复性条件进行了研究,为制备适于临床上诊断及治疗Dig中毒的人源性单抗奠定了基础。

Objective To investigate incolusion body expression of human scFv against digoxin in eseherichia coli（E, coli） and the conditions of denaturation and renaturation, and to increase its expression level. Methods （1）The ADAscFv gene was amplified from the experssion plamsid pHEN2 with PCR, cloned into the pGEM-T vector,digested with restriction enzyme NdeI and Sail, purified and cloned into pT7.7 vector to construct the expression plasmid pT-ADAscFv, transformed into E. coli strain BL21 （DE3） and induced by IPTG,SDS-PAGE was used to analyze the expression level and screen the positive clones; （2）incolusion body was extracted and purified, SDS-PAGE was used to analyze its purity, ELISA was used to analyze the activities of ADAscFv under various conditions of denaturation and renaturation. Results （ 1 ） SDS-PAGE showed that the E. con BL21 （DE3）carrying pT-ADAscFv could express a protein about 30kD, and the purity of extracted and purified incolusion body was high; （2）the protein could recover its activites by the methods of dialysis tenaturaion and dilution renaturation. Conclusions ADAscFv was successfully expressed in E. coli as incolusion bodles,and could recover its activities after denaturation and renaturation.