[Construction of single-chain Fv Gene of Anti-HBsAg and its expression in COS-7 cells].
OBJECTIVE
To subclone the V(L) gene and V(H) genes of anti-HBsAg and construct the single-chain Fv gene and to analyse the expression of the constructed gene in COS-7 cells.


METHODS
A set of oligonucleotide primers were designed and used to amplify the V(H) and V(L) genes from Fab antibodies screened from phage antibody library. The products were cloned into pUC19 vector and their sequences were analysed. The V(H) and V(L) gene fragments were linked up by a peptide linker and a leader sequence added at 5' terminus of each gene (L-V(H)-Linker-V(L)) and designated as L-ScFv. The L-ScFv genes were inserted into the eukaryotic fusion protein expression vector pEGFP-N3 and transfected into COS-7 cells to express respectively.


RESULTS
The coding sequences of V(H),V(L), linker and leader in all constructs were all correct. The expression of ScFv fusion protein was detectable by fluorescence microscope after transient expression in COS-7. The secretive expression of L-ScFv was confirmed by SDS-PAGE and Western blot analysis. And the binding specificity of the ScFvs with HBsAg were proved by indirect ELISA.


CONCLUSIONS
Anti-HBsAg ScFv genes have been successfully constructed and expressed in COS-7 cells.
Wen
Wei-Hong
WH
Department of Biochemistry and Molecular Biology, Fourth Military Medical University, Xi'an 710032,China.
Zhao
Jing
J
Yu
Cui-Juan
CJ
Xu
Yan-Ming
YM
Jin
Ming
M
Wang
Cheng-Ji
CJ
Yang
An-Gang
AG
D000818:Animals; D019556:COS Cells; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006514:Hepatitis B Surface Antigens; D007135:Immunoglobulin Variable Region / Q000235:genetics; D057127:Single-Chain Antibodies
D006514:Hepatitis B Surface Antigens; D007135:Immunoglobulin Variable Region; D057127:Single-Chain Antibodies