摘要
目的：研究人ＩＬ６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ＩＬ６ｃＤＮＡ，用ｐＢＶ２２０载体对ＩＬ６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ｒｈＩＬ６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１１×１０８Ｕ／ｍｇ。
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.
出处
《白求恩医科大学学报》
CSCD
1999年第1期14-17,共4页
Journal of Norman Bethune University of Medical Science
