关键词:  成纤维细胞生长因子21［Tyr²⁰］突变体基因；克隆,分子；原核表达；纯化

Abstract:

To induce the mutation  of fibroblast growth factor 21 (FGF21)gene at the site of the 20th amino acid,express it in the E.coli BL21(DE3),purify the expressed product of FGF21［Tyr²⁰］ protein and detect the bioactivity.Methods  Using plasmid of wide FGF21 as a template,the site-specific mutagenesis of the 20th amino acid Tyr to Phe was performed by PCR,and it was fused with small ubiquitin-like modifier(SUMO) and cloned into vector pET22b. The constructed recombinant plasmid pET22b-SUMO-FGF21［Tyr²⁰］ was transformed to E.coli BL21(DE3) for expression under induction of IPTG. The expression product was identified with SDS-PAGE and Western blotting,purified with Ni-NTA affinity column,the SUMO was cut with SUMO enzyme and demineralizated with Sephadex G50.Results The site-specific mutagenesis of the 20th amino acid of FGF21 was made by PCR successfully.The  recombinant plasmid pET22b-SUMO- FGF21［Tyr²⁰］ after identification  by PCR,digestion and sequencing analysis  matched the expected result. Through SDS-PAGE electrophoretic analysis,the protein was soluble. After purification the  FGF21［Tyr²⁰］ mutant protein was successfully obtained.Western blotting analysis showed that the FGF21［Tyr²⁰］ mutant protein had specific reaction with FGF21 antibody.Conclusion The SUMO- FGF21［Tyr²⁰］ mutant gene is successfully constructed and expressed with high solubility,and the FGF21［Tyr²⁰］ protein is also purified and obtained.

Key words: FGF21［Tyr²⁰］ mutant gene;cloning,molecular;prokaryotic expression;purification