南京师大学报（自然科学版）2012，Vol.35Issue(3)：74-80,7.
原核表达系统pET28a(+)中抗CD20微抗体的构建和表达
Construction and Expression of Anti-CD20 Minibody in pET28a( + ) of Prokaryotic Expression System
摘要
Abstract
Objective The research aimed to construct anti-CD20 minibody containing VL,VH and CH3,and to explore the best inducing condition of its soluble protein expression.Method The recombinant minibody cDNA was produced by overlap PCR.A short peptide linker was used to join VL and VH.The human IgGl hinge region was used to join VH and CH3.The recombinant minibody gene was subcloned into the expression vector of pET28a and expressed in E.coli BL21.Result SDS-PAGE and Western blot analysis showed that the recombinant protein was about 41.88 kDa and the optimized soluble protein expression condition of minibody was 1.0 mmol/L IPTG for 24 h at 20℃ ,the inclusion bodies were found in the precipitation after sonication.The soluble protein minibody was further purified Ni-NTA affinity chro- matography,the yield up to 90.25% ,the highly purity recombinant protein was obtained after a series of steps including inclusion bodies cell lysis,denaturation,purfication and renaturation.Western blot proved that the recombinant protein anti-CD20 minibody had immunologicai reactive ability against rabbit anti-human IgG,and can specific binding CD20 antigen.The successful expressed of minibody in the prokaryotic expression system was helpful for the future study of its biological function and target therapy to the B lymphoid leukemia and B lymphoma.关键词
CD20/minibody/原核表达/大肠杆菌Key words
CD20/minibody/prokaryotic expression/E.coli分类
医药卫生引用本文复制引用
李东霞,杨振,潘少坤,周楠楠,宋菲,曹祥荣..原核表达系统pET28a(+)中抗CD20微抗体的构建和表达[J].南京师大学报（自然科学版）,2012,35(3):74-80,7.基金项目
江苏舜唐生物工程有限公司资助项目,国家基础科学人才培养基金(J1103507). （J1103507）
