Anti-TNF inhibitors exert their therapeutic effect by inhibition of the excessive amounts of TNF-α within the body. Recombinant TNF-α should be produced in a soluble refolded form to investigate the effectiveness and efficiency of anti-TNF-α compounds. In this research, the designed cassette was subcloned in the pET28a expression vector and expressed in E. coli BL21 (DE3). The identity of the protein was confirmed through SDS-PAGE and Western blotting. After optimizing expression conditions, protein purification was performed using native Ni-NTA affinity chromatography. The biological activity of the soluble recombinant TNF-α was investigated using MTT assay. Also, the affinity of an anti-TNF-α agent, Altebrel, was investigated against the expressed protein through ELISA. Optimization of TNF-α expression conditions represented that the highest expression could be achieved at 37 °C using 0.5 mM IPTG 6 h post-induction. The recombinant protein represented an inhibitory effect on the L929 murine fibroblast cell line and was successfully detected by Altebrel in ELISA. Binding kinetics were also studied using Cimzia as an anti-TNF-α molecule and 7.2 E⁻¹³M was calculated as the equilibrium dissociation constant value (K_D). The significant expression level of the recombinant protein in the soluble form, its high purity, and assessment of its biological activity showed that the expressed protein could be used in tests of ELISA and MTT to assess the activity of anti-TNF-α agents.

抗 TNF 抑制剂通过抑制体内过量的 TNF-α 发挥其治疗作用。重组 TNF-α 应以可溶性重折叠形式生产，以研究抗 TNF-α 化合物的有效性和效率。在本研究中，设计的盒被亚克隆到 pET28a 表达载体中并在大肠杆菌中表达BL21 (DE3)。通过 SDS-PAGE 和蛋白质印迹确认蛋白质的身份。在优化表达条件后，使用天然 Ni-NTA 亲和层析进行蛋白质纯化。可溶性重组 TNF-α 的生物学活性用 MTT 法研究。此外，还通过 ELISA 研究了抗 TNF-α 试剂 Altebrel 对表达的蛋白质的亲和力。TNF-α 表达条件的优化表明，诱导后 6 小时使用 0.5 mM IPTG 可以在 37°C 下实现最高表达。该重组蛋白对 L929 鼠成纤维细胞系具有抑制作用，并在 ELISA 中被 Altebrel 成功检测到。还使用 Cimzia 作为抗 TNF-α 分子和 7.2 E ^-13研究了结合动力学M 计算为平衡解离常数值 (K _D )。可溶性重组蛋白的显着表达水平、高纯度和对其生物活性的评估表明，表达的蛋白可用于ELISA和MTT测试以评估抗TNF-α药剂的活性。