Prokaryotic expression, purification and biological activity determination of recombinant human fibroblast growth factor-20 and preparation of its polyclonal antisera
- Author:
Zhi-Guo JIANG
1
Author Information
1. Department of Molecular Oncology
- Publication Type:Journal Article
- From:
Academic Journal of Second Military Medical University
2006;27(9):950-952
- CountryChina
- Language:Chinese
-
Abstract:
Objective: To express and purify bioactive recombinant human fibroblast growth factor-20 (rhFGF-20) protein and to prepare its polyclonal antisera. Methods: FGF-20 cDNA was amplified from human prostate tissue by RT-PCR and was subcloned into expression vector pET-24a, which was then transformed into the E. coli DE3. Expression of rhFGF-20 protein was induced in E. coli DE3 and the protein was purified by Ni2+-NTA His-Bind Resins. The purity and biological activity of rh-FGF-20 protein were determined by SDS-PAGE and cell-proliferation assay, respectively. Two New Zealand white rabbits were immunized with rhFGF-20 protein to prepare polyclonal antisera and the titer of antibody was determined by double diffusion test. Results: rhFGF-20 protein was efficiently expressed in E. coli DE3 in the form of inclusion body and homogeneous protein was obtained after purification with Ni2-NTA His-Bind Resins. Cell proliferation assay indicated that rhFGF-20 dose-dependently (50-5 000 ng/ml) promoted fibroblast cells proliferation. The prepared polyclonal antisera, with a titer of 1:32, had immunoreation with hFGF-20. Conclusion: The expressed rhFGF-20 protein can stimulate the proliferation of fibroblast cells and the prepared antisera are antigen specific.
