Mobile website
Construction of the Tetramerizing Single Chain Fv Antibody Gene Specific for Human Prostate Specific Antigen and Its Expression in HeLa Cells
- Article
- Figures
- Metrics
- Preview PDF
- Reference
- Related
- Cited by
- Materials
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 Ni 2+ 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 Ni 2+ 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.
Keywords:
Project Supported:
Clc Number:
R392.11
