人肿瘤坏死因子α抑制肽-抗炎酸性尾巴融合蛋白的原核表达及纯化

Prokaryotic Expression and Purification of Fusion Protein of Human Tumor Necrosis Factor α Inhibitory Peptide and Anti-Inflammatory Acidic Tail

目的:表达和纯化人肿瘤坏死因子α抑制肽-抗炎酸性尾巴融合蛋白。方法:利用PCR搭接方法及基因合成方法获得目的基因,插入带有6×His标签的原核高效可溶性表达载体pET32a中,构建重组表达质粒pET32a-T9-ac-9,将重组表达质粒转化大肠杆菌BL21(DE3),经IPTG诱导目的基因表达;对融合蛋白进行Ni2+金属螯合柱纯化。结果:构建的重组表达质粒经PCR、内切酶鉴定及基因序列测定证实;目的蛋白在大肠杆菌中获得表达,SDS-PAGE显示相对分子质量为22.917×103;对表达产物进行了亲和层析纯化,从上清中获得了纯度较高的人肿瘤坏死因子α抑制肽-抗炎酸性尾巴融合蛋白。结论:获得了可溶性的人肿瘤坏死因子α抑制肽-抗炎酸性尾巴融合蛋白,为其生物学功能研究奠定了基础。

Objective： To prokaryotic express and purify the fusion protein of human tumor necrosis factor α inhibitory peptide with anti-inflammatory acidic tail in E.coli.Methods： The gene of interest was amplified by overlapping PCR and inserted into the prokaryotic expression vector pET32a with 6×His tag to build recombinant expression plasmid pET32a-T9-ac-9.The recombinant plasmid was transformed into E.coli BL21（DE3）,and the expression of fusion protein was induced by IPTG.Ni2＋ metal chelating column was utilized for high purification of the target protein.Results： Sequencing and restriction analysis revealed the recombinant plasmid was constructed successfully.The fusion protein was expressed in E.coli,SDS-PAGE showed a clear protein band with a relative molecular weight of 22.917 kD.The expression products were purified by affinity chromatography from ultrasonic supernatant of E.coli.Conclusion： The fusion protein of human tumor necrosis factor α inhibitory peptide with anti-inflammatory acidic tail was successfully expressed and purified,which makes the foundation of further study on its biological function.