Construction, Expression And Characterization Of A Bivalent Single Chain Variable Fragment Specifically Binding To Hepatocellular Carcinoma Cell Line HepG2

Objectives To produce and characterize a bivalent single chain variable fragment (BsFv) specifically binding to HepG2 cell line, and campare the binding activity and advidity of BsFv and that of the parent scFv SLH-10.Methods Two pairs of PCR primers were designed to introduce an interlinker G4S and the restriction endonuclease AscI, and two fragments were amplified (SfiI-VH-VL-linker-AscI and AscI-VH-VL-NotI) from the parent scFv SLH-10 by PCR. Then the two fragments were cloned into the plasmid pAB1 to construct the expression vector pAB1-BsFv. Also the parent scFv was subcloned by SfiI/NotI digestion to generate the expression vector pAB1-scFv. After transformed into the bacterial TG1, the currect expression clone were identified. In order to increase the yield of functional antibodies in the culture supernatant or the bacterial periplasm, the expression in the shake-tube cultures with different conditions were tried, such as changing the temperature, the concentration of IPTG, or giving the addition into the culture. With the optimized expression condition, we performed the large-scale expression. And the products were purified by IMAC. SDS-PAGE and western blot analysis were performed. Then the cell-binding activity of the bivalent single-chain Fv(BsFv)and scFv were analyzed by FACS.Results Two different fragments(SfiI-VH-VL-linker -AscI and AscI-VH-VL-NotI) were obtained and identified. The bivalent single-chain Fv(BsFv)was then generated by linking the two scFv together and was identified by sequencing. After expression, the products were identified by SDS-PAGE and western blot, and found that the relative molecular mass of scFv is about 30kDa and BsFv is about 60kDa. They are consistent with what we expected. Through FACS, we found that the BsFv bind to selected cell lines more specifically than parent scFv.Conclusions We successfully constructed a bivalent single chain Fv. It has the advantage in both specificity and avidity by comparing to the parent scFv. The bivalent scFv is potential in the future research for application in tumor targeting.