Construction Of ENO1 Single-Chain Variable Fragment And Human-Mouse Chimeric Antibody

Objective This study aims to construct prokaryotic and eukaryotic vectors of ENO1 single-chain variable fragment and eukaryotic vector of human-mouse chimeric antibody,they were expressed in BL21 and Expi293F^TMcells to obtain specific ENO1 single-chain antibody and human-mouse chimeric antibody.Methods 1.The pFast Bac-Dual-ENO1 single-chain variable fragment plasmid was stored in the laboratory as a template,the ENO1 single-chain variable fragment sequences were cloned on the pcDNA3.1（+）plasmids and p ET30a（+）plasmids to obtain recombination plasmids of the single-chain variable fragment.The p ET30a-ENO1 and pcDNA3.1-NEO1 single-chain variable fragments recombinant plasmid were verified by PCR and sequencing,and the recombinant plasmid with correct sequencing was transformed into competent cells DH5αto amplify and save plasmids;2.Using the ENO1 antibody plasmid stored in the laboratory as a template,through sequence optimization,the variable region sequence of the ENO1monoclonal antibody H1 was cloned into the plasmid pcDNA3.1（+）to obtain the chimeric antibody recombinant plasmid pcDNA3.1-VH and pcDNA3.1-VL,they were verified by PCR and sequencing,and the recombinant plasmid with correct sequencing was transformed into DH5αcompetent cells in order to amplify and rescue plasmids;3.Through molecular cloning to use the ENO1 antibody plasmid stored in the laboratory as a template,the variable region sequences of the ENO1 monoclonal antibody H1 were cloned into the plasmid p TRIOZ-h Ig G1 to obtain the VL-h Ig G1-VH recombinant plasmid;4.The p ET30a-ENO1single-chain variable fragment prokaryotic recombinant plasmid were transformed into BL21competent cells and induced expression with IPTG;5.Co-transfect pcDNA3.1-VH and pcDNA3.1-VL to express ENO1 chimeric antibody in Expi293F^TMcells.Results 1.The prokaryotic vector of pET30a-ENO1 single-chain variable fragment and the eukaryotic vector of pcDNA3.1-ENO1 single-chain variable fragment recombinant plasmid were successfully constructed;2.The prokaryotic vector of p ET30a-ENO1 single-chain variable fragment was successfully expressed in E.coli BL21,and the results of SDS-PAGE showed that the molecular weight was 32KDa,and the results was in line with the prediction;3.Recombinant plasmids pcDNA3.1-VH,pcDNA3.1-VL and p TRIOZ-ENO1 were successfully constructed;4.pcDNA3.1-VH and pcDNA3.1-VL recombinant plasmids were co-transfected into Expi293F^TMCells,SDS-PAGE results showed no specific expression.Conclusion 1.ENO1 single-chain antibody prokaryotic and eukaryotic expression vectors were successfully constructed;2.ENO1 single-chain antibody prokaryotic vectors were successfully expressed;3.ENO1 chimeric antibody eukaryotic vectors were successfully constructed,and no specific bands were seen in plasmid co-transfection.