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Construction of the Tetramerizing Single Chain Fv Antibody Gene Specific for Human Prostate Specific Antigen and Its Expression in HeLa Cells
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Abstract:
Objective: To construct anti-human prostate specific antigen (PSA) single chain Fv antibody (scFv)/human p53 tetramerization domain fusion gene and express fusion protein in HeLa cells.Methods:The human IgG3 upper hinge/human p53 tetramerization domain fusion gene was obtained by recursive polymerase chain reaction (PCR), and was inserted into pUC19 to construct cloning plasmid pUC19/IgG3/p53. The anti-PSA scFv was then cloned into pUC19/IgG3/p53 to construct anti-PSA scFv /human p53 tetramerization domain fusion gene which was then subcloned into the pSecTag2-B expression plasmid. Then the pSecTag2-B plasmids concluding the fusion gene were transfected HeLa cells. The expression products were analyzed by both SDS-PAGE and Western blot, then were purified with Ni2+-NTA superflow affinity chromatography. The binding affinity for PC-3 cells was measured by flow cytometry.Results:The anti-PSA scFv/human p53 tetramerization domain fusion gene consisted of 891bp encoding 297 amino acid residues, and was the same as that reported before. The expression products of the tetrameric anti-PSA scFv, which relative molecular mass (Mr) was about 35 000, were confirmed by SDS-PAGE and Western blot. After purified with Ni2+-NTA superflow affinity chromatography, the tetrameric anti-PSA scFv showed significantly stronger binding to PC-3 cells than scFv.Conclusion: The tetrameric anti-PSA scFv which could bind to PC-3 cells has been successfully gained for the potential use in clinical studies.
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Clc Number:
R737.31
