Construction and Expression of Single-Chain Antibody Derived from a New Clone of Monoclonal Antibody Against Human CD14 in CHO Cells

Abstract

Our aim was to construct the eukaryotic expressional system of single-chain antibody (ScFv2F9) derived from a new clone of monoclonal antibody named ZCH-7-2F9 against human CD14 antigen, and to obtain the ScFv2F9 protein with a high biological activity for further studies on ScFv2F9 immunotoxin and other kinds of genetically engineered antibodies. Primers were synthesized according to the gene sequence of ScFv2F9, 4 tandem glysines and 1 serine (Gly4Ser)3 linker and multiple cloning site(MCS) of pSectag2A vector, which included SfiI and EcoRI enzyme cleaving site, 6 × His and stop code TGA sequences. ScFv2F9 gene was amplified through splicing by overlap extension (SOE) using the high fidelity Taq polymerase. Then the gene was cloned to pGEM-T easy and pSectag2A vectors. Positive recombinants (pSectag2A/ScFv2F9) were identified through enzyme cleaving and sequenced before expression. The recombinant plasmids were transfected into Chinese hamster ovary (CHO) cells for expression. The relative molecular weight (MW) and the binding activity of the interesting protein were determined by SDS-PAGE, Western-blot and flow cytometry (FCM), respectively. The scFv2F9 VH is a member of the IgH gene family V1 subgroup and the scFv2F9 VL gene belongs to the Igκ gene family V10 subgroup. The recombined ScFv2F9 gene was identified to be functional by sequencing and expressing. The interesting protein could be detected in the culture supernatant of transformed CHO cells with a MW of 31 kDa. The blocking test showed that the positive cell percentages, the mean fluorescence intensity (MFI) and the peak of channel (peak Ch) were reduced by 90.02%, 63.3%and 63.38%, respectively after blockage of ScFv2F9. The ScFv2F9 gene was cloned and the interest protein against human CD14 antigen has been successfully expressed in eukaryotic cells with a high biological activity, which lays the foundation for further studies on its immunotoxin and other kinds of engineered antibodies.