新型肿瘤坏死因子免疫抑制剂的克隆和表达

Construction of immune inhibitor of new rhTNF-α

目的 :用鸡卵清溶菌酶 (henegg whitelysozyme ,HEL)T辅助细胞表位序列替换新型rhTNF α3’末端序列 ,构建和表达新型rhTNF α免疫抑制剂。方法 :克隆、表达、纯化和鉴定新型rhTNF α免疫抑制剂。结果 :DNA测序表明 ,重组新型hTNF αHEL的 3’末端序列与设计序列完全相同。将其转移到pBV2 2 0表达载体中 ,重组菌经 42℃诱导后做SDS PAGE分析 ,结果显示该重组体获得了表达 ,其表达量为菌体总蛋白量的 30 %,表达形式为包涵体 ,对该包涵体进行溶解和复性 ,再经阴离子交换柱纯化 ,获得的重组蛋白的纯度可达 90 %以上。免疫印迹鉴定结果证实 ,该表达蛋白可与抗TNF α抗体产生免疫反应。体外杀伤活性检测显示 ,该蛋白已完全丧失了新型TNF α的体外杀伤活性。结论 :上述实验证实 ,新型rhTNF α免疫抑制剂构建成功 ,该免疫抑制剂既保持了与TNF α抗体结合的能力又失去了TNF α的杀伤活性 ,为其下一步治疗作用的研究打下了基础。

Objective:To construct an immune inhibitor of new TNF-α,C-terminal sequence of new rhTNF-α was replaced with sequence of T-help cell of hen egg-white lysozyme(HEL).Methods:The rhTNF-α mutant was cloned 、expressed and purified.Results:The DNA sequencing analysis showed that the C-terminal sequence of new rhTNF-α mutant was correct.The mutant was inserted into pBV220 expression vector .After the recombinant bacteria was incubated at 42℃ for 4 h,a new band of the protein with relative molecular weight of 1.7 kD was shown on the gel.The band amounted to 30 % of total bacteria protein.Western blot showed that the mutant protein could associated with anti-TNF-α antibody.After the protein was purified by through a column of Q-Sepharose Fast Flow, the purity of the protein was above 90%.The biological activity of the protein was measured with L929 cells.The result showed that biological activity of the protein was totally lost.Conclusion:The experimental evidence demonstrated that the construction of new rhTNF-α mutant was successful.The mutant not only can be associated with anti-TNF-α antibody, but also lose the biological activity of original TNF-α. [