摘要
将抗甲胎蛋白单链抗体(scFv-AFP)基因克隆至载体pET32a,转入大肠杆菌E.coli BL21(DE3),在不同的诱导剂浓度、诱导温度和时间下诱导pET32a-scFv-AFP表达,SDS-PAGE分析表明37℃培养4 h时加入0.4 mmol/L异丙基-β-D-硫代半乳糖苷(IPTG)诱导5 h为最适表达条件,目的蛋白表达量约为总蛋白的50%,其产物以包涵体形式存在。利用稀释法和透析法对包涵体复性,复性率为37.38%,活性蛋白产量达到200 mg/L。竞争抑制ELISA法初步鉴定活性,当scFv-AFP与亲本单抗浓度比为16∶1,竞争ELISA抑制率达到54.13%,说明scFv-AFP具有较好的抗原结合特异性。该研究为scFv-AFP的获得与抗体的进一步应用改造奠定了基础。
The single-chain variable fragment of alpha fetoprotein（scFv-AFP） gene was sub-cloned into vector pET32a and transformed into E.coli BL21（DE3）host.The expression of scFv-AFP was improved by optimizing the expressing conditions by changing different inducing temperature,time and concentration of IPTG.Then the inclusion body was denatured and renatured,the refolding yield of protein from inclusion body is about 37.38%.The competitive ELISA method was used to determine the immunologic activity of renatured protein,and the inhibition rate reached 54.13% while the concentration ratio of scFv and parent monoclonal antibodies was 16 ∶1.This work would be helpful for the further application and modification of scFv-AFP.
出处
《中国药科大学学报》
CAS
CSCD
北大核心
2012年第5期460-465,共6页
Journal of China Pharmaceutical University
基金
江苏省"青蓝工程"资助项目(2010)~~
