|Table of Contents|

Construction and Expression of Anti-CD20 Minibody in pET28a(+) of Prokaryotic Expression System(PDF)

《南京师大学报（自然科学版）》[ISSN:1001-4616/CN:32-1239/N]

Issue:
2012年03期
Page:
74-80
Research Field:
生命科学
Publishing date:

Info

Title:
Construction and Expression of Anti-CD20 Minibody in pET28a(+) of Prokaryotic Expression System
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
Keywords:
CD20; minibody; prokaryotic expression; E． coli
PACS:
R733.1
DOI:
-
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．

References:

［1］ Jemal A，Siegel R，Ward E，et al． Cancer statistics［J］． CA Cancer J Clin，2008，58( 2) : 71-96．
［2］ 师明磊，胡显文，陈惠鹏，等． 抗CD20 嵌合抗体的表达与活性鉴定［J］． 中国生物工程杂志，2005，25( 7) : 34-39．
［3］ Riley J K，Sliwkowski M X． CD20: a gene in search of a function［J］． Semin Oncol，2000， 27( 6 Suppl 12) : 17-24．
［4］ Du J，Wang H，Zhong C， et al． Structural basis for recognition of CD20 by therapeutic antibody Rituximab［J］． J Biol Chem， 2007，282( 20) : 15 073-15 080．［5］ 赵江宁，朱雅丽． 抗CD20 单克隆抗体-Rituximab［J］． 白血病． 淋巴瘤，2004， 13( 2) : 123-125．
［6］ John R G，Steven S H，Jeffrey T R，et al． An afucosylated anti-CD20 monoclonal antibody with greater antibody-dependent cellular cytotoxicity and B-cell depletion and lower complement-dependent cytotoxicity than rituximab［J］． Molecular Immunology， 2012，50( 3) : 134-141．
［7］ Hu S Z，Lousise S，Andrew R，et al． Minibody: A novel engineered anti-carcinoembryonic antigen antibody fragment ( single- chain Fv-CH3) which exhibits rapid，high-level targeting of xenografts［J］． Cancer Res，1996， 56( 13) : 3 055-3 061．
［8］ 温宁笑，袁红梅． 非霍奇金淋巴瘤单克隆抗体治疗的研究进展［J］． 现代诊断与治疗，2010，21( 4) : 221-223．
［9］ 陈森，饶青，王健祥，等． 抗人CD19 单链抗体基因的构建、表达及功能测定［J］． 生物工程学报，2005， 21( 5) : 686- 691．
［10］ Verma R，Boleti E，George A J． Antibody engineering: comparison of bacterial，yeast，insect and mammalian expression systems［J］． J Immunol Methods，1998，216( 1 /2) : 165-181．

Memo

Memo:
-
Last Update: 2013-03-11