重组小肿瘤坏死因子α拮抗剂的表达、纯化及其生物学活性

Expression,purification and biological activity of recombinant small tumor necrosis factor α antagonist

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 small TNFα antagonist （STNFtxA）, with a low relative molecular mass, binding to tumor necrosis factor receptor 1 （TNFR1）, and determine the biological activity of purified protein. Methods The STNFα A amino acid sequence with high binding ability to TNFR1 was designed via computational optimization, based on which target gene was designed and synthesized according to the codon bias of E. coli, than inserted into plasmid pET- 28a（＋ ）. The constructed recombinant plasmid pET28-STNFαA was transformed to competent E. coli BL21 （DE3） and for expression under induction of IFFG. The expressed product was identified by SDS-PAGE, purified by Ni Sepharose 6 Fast Flow chromatography, then analyzed by SDS-PAGE and HPLC, and determined for biological activity by MTF method. Results Restriction analysis showed that recombinant plasmid pET28-STNFαA was constructed correctly. The expressed STNFαA, with a relative molecular mass of about 17 000, contained about 25% of total somatic protein, mainly existed in a form of inclusion body, reached a purity of more than 95%, and showed dose-dependent inhibitory effect on TNFα. Conclusion STNFo^A was expressed successfully, which inhibited the cytotoxic biological activity mediated by TNFα. It laid a foundation of development of novel TNF antagonists.