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目的 :对高效表达的重组IL 6D2 4 PE4 0KDEL外毒素融合蛋白进行包涵体的分离、变性与复性以及纯化。方法 :对表达菌体进行超声破碎、洗涤、氧化还原法变性及复性 ,经离子交换层析。结果 :工程菌HB10 1/ pE0 1T的表达产物是以包涵体形式存在 ,菌体的破碎以高渗蔗糖溶液进行预处理后 ,再超声破碎效果最好 ;包涵体的洗涤则先用Triton X 10 0洗 2次 ,再用 8mol/L尿素洗涤一次效果更优 ;最佳变性条件为在 7mol/L盐酸胍中加入0 .0 6 5mmol/L的DTT和 0 .2mol/L的盐酸精氨酸 ;最佳复性条件为在 10℃复性保持 2 4h ,蛋白浓度保持在 30～80 μg/ml,并需加入 0 .2mol/L盐酸精氨酸 ;利用谷胱甘肽的氧化还原法 ,当氧化型谷胱甘肽与还原型谷胱甘肽的浓度比在 2∶1时所获得的活性成分得率最高 ;利用强弱离子交换结合的方法能获得纯度为 95 %的目的蛋白 ,所得融合蛋白可与IL 6及PEA抗体相结合并能特异性地杀伤高表达IL 6R的人多发性骨髓瘤细胞。结论 :本研究获得重组IL 6D2 4 PE4 0KDEL外毒素融合蛋白包涵体的分离、变性与复性条件和一条能获得大量高纯度该融合蛋白的纯化路线。
OBJECTIVE: To isolate, degenerate, renature and purify the highly expressed recombinant IL 6D2 4 PE4 0KDEL exotoxin fusion protein. Methods: The bacterial cells were sonicated, washed, denatured and refolded by redox method and analyzed by ion exchange chromatography. Results: The expression product of the engineered bacteria HB10 1 / pE0 1T existed in the form of inclusion bodies. The cell disruption was pretreated with hypertonic sucrose solution and then sonicated by ultrasound. The inclusion bodies were washed with Triton X 10 0 wash 2 times, and then 8mol / L urea once more effective; the best conditions for the denaturation of 7mol / L guanidine hydrochloride added 0. 06mmol / L of DTT and 0.2mol / L of arginine hydrochloride ; The best refolding conditions were refolding at 10 ℃ for 24 h and protein concentration at 30 ~ 80 μg / ml, and 0.2 mol / L arginine hydrochloride was added. Using glutathione redox method, When the concentration ratio of oxidized glutathione to reduced glutathione is 2: 1, the yield of the active ingredient is the highest; the target protein with a purity of 95% can be obtained by the method of strong and weak ion exchange combination The fusion protein binds IL6 and PEA antibodies and specifically targets human multiple myeloma cells that are highly IL6R-expressing. Conclusion: The isolation, denaturation and refolding conditions of inclusion body of recombinant IL 6D2 4 PE4 0KDEL exotoxin fusion protein were obtained in this study and a purification route for obtaining a large number of high purity fusion proteins was obtained.