FGF21[Tyr^(20)]突变体的克隆、表达及纯化

Cloning,expression and purification of fibroblast growth factor 21 [Tyr^(20)]mutant

目的:将成纤维细胞生长因子21(FGF21)第20位氨基酸进行突变后,在大肠杆菌中表达,并进行纯化和生物活性检测。方法:以野生型的FGF21为模板,将FGF21第20位氨基酸Tyr进行PCR定点突变成Phe后与小分子泛素样修饰物(SUMO)融合,克隆至pET22b表达载体中,构建重组原核表达质粒pET22b-SUMO-FGF21[Tyr20],克隆至BL21(DE3),IPTG诱导表达,表达产物经SDS-PAGE电泳及Western blotting法进行鉴定,用Ni-NTA亲和层析柱纯化,SUMO蛋白酶将SUMO切除,SephadexG-50除盐。结果:通过PCR定点突变,成功地将FGF21第20位氨基酸进行了突变,重组质粒pET22b-SUMO-FGF21[Tyr20]经PCR和双酶切鉴定后测序与预期结果相符。经SDS-PAGE电泳分析,蛋白为可溶性,纯化后成功获得FGF21[Tyr20]突变体蛋白,Western blotting分析显示FGF21[Tyr20]突变体蛋白与FGF21抗体有特异性反应。结论:成功构建并高效可溶性表达SUMO-FGF21[Tyr20]的突变体基因,获得FGF21[Tyr20]蛋白。

Objective 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 FGF21as 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-PAGEelectrophoretic 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 FGF21antibody.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.