Soluble expression in Escherichia coli of functional ScFv of monoclonal antibody MC5 against human colorectal carcinoma

doi:10.3969/j.issn.1000-8861.2003.01.001

Abstract：Objective To prepare functional soluble single chain variable fragment(ScFv) of monoclonal antibody(McAb) MC5 against human colorectal carcinoma.Methods mRNA was isolated from the MC5-producing hybridoma cell line 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 human colorectal carci- noma cell line SW480 highly expressing MC5-binding antigen, the phage clones displaying ScFvs of MC5 were selected by ELISA. Two 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 activities were assayed by ELISA.Results VH,VL and ScFv DNAs were about 340, 320 and 750 bp respectively. 10 antigen-positive phage clones were selected from 25 preselected phage clones by ELISA. Both the soluble ScFvs derived from the 2 out of the 10 antigen-positive phage clones were about 32 000 u and concentrated in periplasmatic space, and could bind the antigen, and shared the same binding site with parental McAb MC5. Conclusion The functional soluble ScFv of MC5 is successfully produced, which may provide a possible novel targeting vehicle for in vitro and potential in vivo studies on human colorectal carcinoma.