[1]李东霞,杨振,潘少坤,等.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(03):74-80.
　Li Dongxia,Yang Zhen,Pan Shaokun,et al.Construction and Expression of Anti-CD20 Minibody in pET28a(+) of Prokaryotic Expression System[J].Journal of Nanjing Normal University(Natural Science Edition),2012,35(03):74-80.
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原核表达系统pET28a(+)中抗CD20微抗体的构建和表达()
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《南京师大学报（自然科学版）》[ISSN:1001-4616/CN:32-1239/N]

卷:
第35卷
期数:
2012年03期
页码:
74-80
栏目:
生命科学
出版日期:
2012-09-20

文章信息/Info

Title:
Construction and Expression of Anti-CD20 Minibody in pET28a(+) of Prokaryotic Expression System
作者:
李东霞;杨振;潘少坤;周楠楠;宋菲;曹祥荣;
南京师范大学生命科学学院，江苏省分子医学生物技术重点实验室，江苏南京210046
Author(s):
Li Dongxia; Yang Zhen; Pan Shaokun; Zhou Nannan; Song Fei; Cao Xiangrong
Jiangsu Key Laboratory of Molecular and Medical Biotechnology,School of Life Sciences, Nanjing Normal University,Nanjing 210046,China
关键词:
CD20; minibody; 原核表达; 大肠杆菌
Keywords:
CD20; minibody; prokaryotic expression; E． coli
分类号:
R733.1
摘要:
目的: 改造Rituximab，构建包含VL、VH 和CH3 的抗CD20 微抗体minibody，并探索诱导其可溶性表达的最佳方法． 方法: 首先通过overlap PCR 将Rituximab 的VL 和VH 通过Linker 连接在一起，成为单链抗体ScFv，再将ScFv 和人源IgG1 CH3 通过人源IgG1 铰链区连成minibody 的cDNA． 将其克隆至表达载体pET28a( + ) 中，并在大肠杆菌中用IPTG 诱导表达． 结果: SDS-PAGE 和Western blot 结果表明，抗CD20 的微抗体基因表达产物分子量约41. 88 kDa，在20℃、1. 0 mmol /L IPTG 诱导24 h 时minibody 的可溶性表达量最大，同时还有包涵体形式的蛋白． 可溶性抗体蛋白通过镍柱纯化，纯度达到90. 25%，包涵体经过变、复性获得了高纯度的抗体蛋白． 重组蛋白minibody 可特异性地被兔抗人IgG1 单克隆抗体识别，并且可特异性结合靶抗原CD20． 结论: 抗CD20 微抗体minibody 基因在此原核表达系统中成功表达，为其生物学功能和更好地针对B 淋巴系统恶性肿瘤的靶向治疗奠定了基础．
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 IgG1 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 chromatography， 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 immunological 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．

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备注/Memo

备注/Memo:
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) ．
通讯联系人: 曹祥荣，教授，博士生导师，研究方向: 肿瘤发生与基因治疗分子机制、动物染色体进化与基因组学． E-mail: caoxiangrong@ ninu． edu． cn
更新日期/Last Update: 2013-03-11