Expression and identification of recombinant soluble single-chain variable fragment of monoclonal antibody MC3

World Journal of Gastroenterology : WJG
Feng-Tian HeYan Zhang

Abstract

To generate soluble single chain variable fragments (ScFv) of monoclonal antibody MC3 recognizing colorectal and gastric carcinomas. mRNA was isolated from the hybridoma cell line producing MC3 and the DNAs encoding variable domains of heavy and light chains (VH and VL) of the antibody were amplified separately by RT-PCR and assembled into ScFv DNA with a linker DNA. The ScFv DNA was ligated into the phagemid vector pCANTAB5E and the ligated sample was transformed into E.coli TG1. The transformed cells were infected with M13KO7 helper phage to yield recombinant phages. After two rounds of panning with gastric carcinoma cell line AGS highly expressing MC3-binding antigen, the phage clones displaying ScFv fragments of the antibody were selected by ELISA. 4 phage clones showing strong signal in ELISA were used to infect E.coli HB2151 to express soluble ScFvs. The soluble ScFvs were identified by Dot blot and Western blot, and their antigen-binding activity was assayed by ELISA. The VH and VL DNAs of the ScFv DNA derived from phage clone 19 were sequenced. The VH,VL and ScFv DNAs were about 340 bp, 320 bp and 750 bp respectively. After two rounds of panning to the recombinant phages, 18 antigen-positive phage clones were selected f...Continue Reading

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Citations

Jun 16, 2015·Japanese Journal of Infectious Diseases·Xiao ZhangYi Zeng
Apr 8, 2003·World Journal of Gastroenterology : WJG·Jin FangJin-Dan Song
Mar 3, 2005·Journal of Gastroenterology and Hepatology·Daiming FanKaichun Wu

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