Gene Construction, Expression And Activity Identification Of Human Anti-HBsAg Single-chain Antibody

Objective: To construct the single-chain Fv gene of human anti-HBsAg and to analyse the expression of the constructed gene in eukaryotic cell and prokaryotic cell.Methods: A set of oligonucleotide primers were designed and used to amplify the VH and VL gene from anti-HBsAg Fab antibodies screened from phage antibody library. The products were cloned into pUC19 vector and their sequences were analysed. The VH and VL gene fragments were tethered by a peptide linker and a leader sequence coding region, with the leader sequence added at 5' terminus of each gene (L-VH-Linker-VL) and designated as L-ScFv. The L-ScFv genes were inserted into the eukaryotic fusion protein expression vector pEGFP-N3 and transfected transiently into COS-7 cells to express respectively. Jurkat cell lines were established to express ScFv fusion proteins stably. The products of stably transfect cells and their activities were analysed. The ScFv genes were then inserted into the prokaryotic fusion protein expressionvector pTAT-HA and expressed in E. coli BL21. After the expression products were purified by Ni-NTA colume, their activities were analysed. Results: The coding sequences of VH, VL, linker and leader sequence were correct. It was confirmed by restriction enzyme digestion analysis that EGFP fusion expression plasmids of ScFvs were successfully constructed. The expression of ScFvs fusion protein were detectable by fluorescence microscope directly and indirect immunofluorescence and immunohistochemistry analysis after transient expression in COS-7. After screening by G418, Jurkat cell lines were established to express ScFvs stably. The secretive expression of ScFv fusion proteins were confirmed by SDS-PAGE and Western Blot analysis in cell split products and condensed supernatant. And the binding specificity of the ScFv fusion protein with HBsAg was confirmed by indirect ELISA. After adding culture mediem of stably transfected Jurkat cells to hepatocarcinoma cells, the binding specificity of the ScFvs with HBsAg was further confirmed by observation by fluorescence microscope, indirect immunofluorescence and flow cytometer analysis. Prokaryotic expression plasmids pTAT-HA-ScFvs were successfully constructed. After one of them was expressed in E.coli BL21, the product was purified by Ni-NTA column. The activity of the purified product was confirmed by indirect ELISA analysis and was further confirmed by indirect immunofluorescence and immunohistochemistry after they were added to the culture medium of hepatocarcinoma cells.Conclusions: (1) Five human anti-HBsAg ScFv genes have been successfully constructed. (2 ) After transiently transfected in COS-7 cells, secreting expression were detect. Jurkat cell lines were construct to express ScFvs stably. The activities of the products were confirmed. (3) After one pTAT-HA-ScFv was expressed in E.coli BL21, the product was purified by Ni-NTA column and itsactivity was confirmed.