人白细胞介素-6基因的克隆表达和纯化的研究

目的：研究人ＩＬ ６基因在大肠杆菌中的高效表达及表达产物的纯化。方法：采用ＲＴ－ＰＣＲ方法克隆了人ＩＬ ６ｃＤＮＡ，用ｐＢＶ２２０载体对ＩＬ ６基因进行了表达调控研究，用阴离子交换柱和凝胶柱对表达产物进行纯化，用ＭＴＴ法测定ｒｈＩＬ ６的活性。结果：表达调控研究发现，当ＳＤ序列到ＡＴＧ之间的距离为６ｂｐ和１０ｂｐ时在ＳＤＳ－ＰＡＧＥ胶上未见明显表达，而当距离为５ｂｐ、７ｂｐ、８ｂｐ、９ｂｐ时均可获高效表达，最高表达量占菌体总蛋白的２６％。表达产物经变性复性及纯化后产品纯度达９８％，比活性为１１×１０８Ｕ／ｍｇ。结论：本研究为ｒｈＩＬ ６的中试生产奠定了坚实的基础

Cloning expression and purification of human interleukin-6

Objective:To achieve high level expression of rhIL 6 cDNA in E.coli;To find out a simple and efficient purification scheme of the product.Methods:rhIL 6 cDNA was cloned by RT-PCR and inserted into pBV220 for expression.The distance between the SD sequence and ATG was optimized.The product was purified by anion exchange chromatography and gel filtration.Specific activity of the product was measured by MTT method.Results:It was found when the distance between the SD and ATG was 6 bp or 10 bp,there was no obvious expression detected by SDS PAGE,when the distance was 5 bp,7 bp,8 bp,9 bp,the expression level was high.The highest expression level accounted for 26% of the total cellular protein.After the two step purification,the product purity reached 98% and the specific activity reached 1.1×10 8 U/mg.Conclusion:This research has laid solid foundation for pilot scale production of rhIL 6.