Prokaryotic expression and polyclonal antibody preparation of camel FGF21 protein
The aim of this study was to provide protein and antibody materials for an in-depth study of the mode of action of camel fibroblast growth factor 21(FGF21)in regulating glycolipid metabolism.The camel FGF21 gene sequence(XM-010996368.2)was searched in the NCBI database,and the full-length FGF21 gene carrying His tag was synthesized after deleting the signal peptide segment,inserted into pET-28a(+)expression vector,and the recombinant expression plasmid pET-28a(+)-FGF21-His was obtained,and was transferred into E.coli Rosetta(DE3)for the expression of recombinant protein FGF21.The recombinant protein was purified by nickel ion affinity chromatography column,and the purified recombinant protein was identified by SDS-PAGE and Western-blot assay.The recombinant protein FGF21 was mixed with Freund's adjuvant and immunized New Zealand large white rabbits to prepare rabbit-derived polyclonal antibody serum.The polyclonal antibody serum was purified by using a protein a affinity chromatography purifi-cation column,and the purity of the purified polyclonal antibody was analyzed by using SDS-PAGE,and the reactivity of the purified polyclonal antibody was verified by using Western-blot and indirect ELSIA methods.The results showed that camel recombinant protein FGF21 was successfully expressed and purified,and the specific polyclonal antibody against FGF21 was prepared and purified after immunizing rabbits with it.This study provides materials for the detection of camel FGF21 protein by ELISA method,and lays the foundation for elucidating the functional mechanism of FGF21 in camel glycolipid metabolism.
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