Preparation And Preliminary Research On Immunogenicity Of GST And FGF23C Termination Fusion Protein

Fibroblast growth factor23(FGF23) of the FGF19subfamily is one of the fibroblast growth factor familys and it had been successfully identified in2000. FGF23mainly expressed in bone, and then derived from the bone into the blood circulation to regulate the phosphate metabolism of kidney in the form of hormonal regulation. It is not only a major regulator to maintain phosphorus balance, but also an important regulation factor of vitamin D and parathyroid hormone. As a tune phosphorus factor, FGF23plays a key role in the pathogenesis of chronic kidney disease associated with secondary hyperparathyroidism. especially in the maintenance of early phosphorus steady of patients with chronic kidney disease (chronic kidney disease CKD). The metabolic level of FGF23is closely related to the prevalence of chronic kidney disease patients. In the early of CKD, FGF23is a kind of integrated indicators can help identify patients at risk and in need of treatment. Studies showed that the use of FGF23neutralizing antibodies can make the phosphate of hypophosphatemia in serum of rat and concentration of vitamin D to become normalization. Therefore, FGF23antibody especially the FGF23carbon end of71amino acid specific fragment antibody is expected to be used for developing as diagnostic reagents or therapeutic agents in chronic kidney disease, which shows a good application prospect. And FGF23is involved in many human diseases, thus FGF23antibody or FGF23C-terminal peptide analogs may eventually be used to treat low phosphate blood disease.Objective:The purpose of this study is genetically constructed and expressed engineered bacteria of fusion protein of glutathione-S-transferase (GST) with the activity fragment of clips-carbon end of the71amino acids (FGF23C-71AA) of the fibroblast growth factor23, and purified the activity target protein, and then the immunogenicity was detested for the fusion protein. All of these provide experimental data for the research of the preparation of the FGF23-specific monoclonal antibodies and also for the development of chronic kidney disease diagnostic reagents and the functions of FGF23physiological.Methods:According to the gene sequence of the specific fragments-the carbon end71amino acids of FGF23to design and synthesize a pair of primers. We utilized PCR technology to amplify FGF23c-71AA gene fragments, which were used the plasmid pET22b-fgf23as template. Then the gene was connected with the gene expression vector pGEX-4T-1, and transformed into BL21host cells, finally the expression of the gene was induced by IPTG. The successfully constructed expression bacteria were induced under different temperatures and IPTG concentration conditions. We took bacterial sample with a different time induced for screening the best expression conditions by SDS-PAGE method. Then we used Glutathione Sepharose4B affinity chromatography to purify GST-FGF23c-71AA fusion protein and Western blotting to identify the activity of target protein. The fusion protein was used to immunize Balb/C mice for the preparation of antiserum, and a sandwich ELISA to detect anti-serum titer indirectly. The same time, we constructed the GST protein expression vector using the same method to purife the GST protein. GST protein as control, the specificity of the antiserum was detected by ELISA testing.Results:Polyacrylamide gel electrophoresis results showed that the constructed expression bacterial was successfully expressed the target protein in about30KD, and almost all the soluble expression. The best expression condition is at25℃C,8h induced with0.4mM IPTG. After purification, the purity of the fusion protein is more than90%, and shows positive reaction with the commercialization of FGF23polyclonal antibody. ELISA method confirmed the fusion protein possess good specificity to the part immunogenicity of FGF23C-71AA fragment.Conclusion:We successfully constructed engineer bacteria of the fusion protein of GST-FGF23C-71AA. and purified the fusion protein with good immunogenicity. The preparation of the antigen can be used to obtain FGF23specificity of monoclonal antibodies and the study of biology function of FGF23.