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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 express and purify the human tumor necrosis factor alpha inhibitory peptide - anti-inflammatory acid tail fusion protein. Methods: The target gene was obtained by PCR overlap method and gene synthesis method, inserted into prokaryotic expression vector pET32a with 6 × His tag, and constructed recombinant expression plasmid pET32a-T9-ac-9. The recombinant plasmid was transformed into Escherichia coli BL21 (DE3), the target gene was induced by IPTG, and the fusion protein was purified by Ni2 + metal chelate column. Results: The constructed recombinant plasmid was confirmed by PCR, endonuclease identification and sequence analysis. The target protein was expressed in E. coli. SDS-PAGE showed that the relative molecular mass was 22.917 × 103. The expressed product was analyzed by affinity chromatography Purified, purified from the supernatant of human tumor necrosis factor α inhibitory peptide - anti-inflammatory acid tail fusion protein. CONCLUSION: Soluble human tumor necrosis factor-α inhibitor peptide-anti-inflammatory acid tail fusion protein was obtained, which laid the foundation for the study of its biological function.