干扰素-β1b的高效表达、纯化及抗病毒活性研究  

High Expression and Purification of IFN-β1b and Its Antiviral Activity

作　　者：李武平[1] 吕宏亮[1] 段招军[1] 张丽萍[1] 刘永清[1] 吴斌文[1] 陈勇[1] 张成海[1] 衣作安[1] 魏开坤[1] 侯云德[1] 

机构地区：[1]中国疾病预防控制中心病毒病预防控制所病毒基因工程国家重点实验室,北京100052

出　　处：《病毒学报》2002年第3期240-244,共5页Chinese Journal of Virology

摘　　要：IFN - β1b是大肠杆菌产生的 17位Cys被Ser替换的人IFN - β的类似物 ,为了获得高表达 ,使用了大肠杆菌的偏爱密码子 ,人工合成了IFN - β - 1b基因 ,插入质粒 pBV2 2 0中 ,转化大肠杆菌DH5α。IFN - β1b的制备过程 ,包括发酵和一系列的纯化步骤。经修饰 ,IFN - β1b基因在启动子PRPL 控制下发酵表达 ,合成的蛋白质以包涵体的形式存在。培养的细菌经收集、裂解后 ,将包涵体释放出来 ,包涵体经含SDS的溶液溶解 ,DTT还原。纯化过程包括有机溶剂抽提、分子筛层析、脱盐、氧化复性和反相层析 ,并用旋转蒸发除去有机溶剂。IFN - β -IFN-β1b,an analogue of human IFN-β, where serine was genetically engineered to substitute for cysteine at position 17,was produced in E.coli.In order to obtain high expression,we synthesized the gene of IFN-β-1b artificially using the preferred codons of E.coli,and inserted it into plasmid pBV220,and transformed to E.coli DH5α.The processes of IFN-β1b manufacturing consisted of fermentation and a series of purification steps.Briefly,a production strain of E.coli harboring the modified IFN-β gene was grown in fermenter.The synthesis of IFN-β was under the control of inducible promoter P RP L.The newly synthesized IFN-β was deposited in bacterial cells as inclusion bodies.The culture was harvested and bacterial cells were disruptd to release the intracellular IFN-β1b.IFN-β1b was then solubilized using a buffer solution containing sodium dodecyl sulphate(SDS)and reduced using dithiothreitol(DTT).Purification processes consisted of organic extraction,size exclusion chromatography,desalting,oxidation to form correct disulphide bond followed by reverse phase chromatography and rotary vaporization.The antiviral activity of IFN-β-1b on cell lines of different species was different.

关 键 词：干扰素-β1b 高效表达 纯化 抗病毒活性 反相高效层析 HPLC 

分 类 号：R978.7[医药卫生—药品] R394.8[医药卫生—药学]