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目的表达能够与肿瘤坏死因子受体1(tumor necrosis factor receptor 1,TNFR1)结合的低相对分子质量重组小肿瘤坏死因子α拮抗剂(small TNFαantagonist,STNFαA),并对纯化后蛋白的生物学活性进行检测。方法利用计算机模拟优化设计出与TNFR1具有较高结合力的STNFαA氨基酸序列,根据大肠埃希菌“密码偏爱性”设计合成目的基因,插入质粒pET-28a(+)中,构建原核表达质粒pET28-STNFαA,转化感受态大肠埃希菌BL21(DE3),IPTG诱导表达,表达产物经SDS-PAGE鉴定后,采用Ni Sepharose 6 Fast Flow层析介质进行亲和纯化,纯化产物经SDSPAGE、HPLC分析;采用MTT法检测重组蛋白的生物学活性。结果重组表达质粒经双酶切鉴定证明构建正确;重组蛋白STNFαA相对分子质量约17 000,表达量约占菌体总蛋白的25%,以包涵体形式存在,纯度大于95%;重组蛋白STNFαA对TNFα的抑制作用呈浓度依赖性。结论已成功表达了STNFαA,该蛋白具有抑制TNFα介导的细胞毒生物活性作用,为全新的TNF拮抗剂的新药研究奠定了基础。
Objective To express low-level TNFα antagonist (STNFαA) that binds to tumor necrosis factor receptor 1 (TNFR1) and to study the biological activity of the purified protein Detection. Methods The amino acid sequence of STNFαA with high binding affinity to TNFR1 was designed by computer simulation. The gene of interest was designed and synthesized according to the Escherichia coli “password preference” and inserted into plasmid pET-28a (+) to construct prokaryotic expression plasmid pET28-STNFαA was transformed into competent E. coli BL21 (DE3) and induced by IPTG. The expressed product was identified by SDS-PAGE and affinity purified by Ni Sepharose 6 Fast Flow chromatography. The purified product was analyzed by SDSPAGE and HPLC The biological activity of the recombinant protein was detected by MTT assay. Results The recombinant plasmid STNFαA was confirmed by double enzyme digestion. The relative molecular mass of recombinant protein STNFαA was about 17 000, accounting for about 25% of the total bacterial proteins. The purity of recombinant protein STNFαA was more than 95% The inhibitory effect of TNFα was concentration-dependent. Conclusion STNFαA has been successfully expressed. This protein has the inhibitory effect on TNFα-mediated cytotoxicity and lays the foundation for new drug discovery of TNF antagonist.