大肠杆菌生产药物蛋白的系统优化研究

【作者】 宋蕾；

【导师】 祁庆生； 王鹏；

【作者基本信息】 山东大学 ， 微生物及分子生物学， 2008， 硕士

【摘要】 大肠杆菌表达系统是基因工程中应用最广泛的也是最有效的表达重组蛋白的系统。大肠杆菌是目前遗传和生化背景最清晰的有机体,它良好的研究背景、易生长易操作的优点使其成为表达外源蛋白的首选系统。用传统方法难以制取的蛋白质、细胞因子等新型药物可以在大肠杆菌中大量表达生产。目前很多商品化的药用蛋白如胰岛素、干扰素、生长因子等都是用大肠杆菌系统生产的,占所有药用蛋白的三分之一。促红细胞生成素(EPO)是由肾脏和肝脏分泌的能促进红细胞生成的激素。利用基因工程研制成功的重组人红细胞生成素(rhEPO)价格相对较低,用于治疗肾性贫血取得了令人瞩目的疗效。干扰素-β(Interferon-β)是由成纤维细胞受病毒感染或IFN诱生所产生的一类具有生物活性的糖蛋白,能通过与靶细胞表面受体结合干扰人体内病毒复制和繁殖,是一种临床治疗病毒性疾病和肿瘤的有效生物反应调节剂。本实验对大肠杆菌表达系统中宿主菌、质粒和表达条件进行优化,选择EPO和IFN-β作为目的蛋白。在宿主菌的选择上,用大肠杆菌λDE3溶原菌BL21(DE3)和能编码表达少量T7溶菌酶的BL21(DE3)/pLys两种菌株。载体在pET系列表达载体上中筛选。同时为消除稀有密码子对表达的影响,目的蛋白EPO和IFN-β的基因用化学合成的方法得到。在表达条件的筛选上,用正交试验设计确定各种表达条件的影响。构建成功的表达载体在宿主菌中诱导后,经SDS-PAGE电泳检测,EPO和IFN-β表达量均在20%以上。正交试验结果分析后,选定0.8M IPTG、OD550为0.9、诱导时间4h作最佳诱导表达条件。试验结果表明,我们已经获得了大量表达EPO和IFN-β的菌株,优化得到最佳表达条件,为在大肠杆菌中表达各种外源药物蛋白打下基础。更多还原

【Abstract】 Genetic engineering pharmaceutical refers to the use of recombinant technology to overexpress physiological substances in bacteria,yeast and animal cells. These physiological substances include proteins,monoclonal antibodies,cytokines and other new types of drugs which are difficult to prepare using traditional methods. Genetic engineering pharmaceutical made a great impact on the pharmaceutical industry.It is playing an increasing important role in the prevention,diagnosis,control and eradication of diseases.The progress of genetic engineering pharmaceutical will make for the protection of human health and the protraction of the life course.Erythropoietin（EPO）is a model of successful development of the genetic engineering pharmaceutical.Formerly erythropoietin was obtained from the urea of anemia patients and the blood of sheep.The yield was very low and the production was extremely unstable.Its physico-chemical and biological properties were difficult of determine.These disadvantages block its large-scale application.The price of recombinant human erythropoietin（rhEPO）is much lower and curative effect is better in the therapy of renal anemia.Interferon-β（IFN-β）is a glycoprotein generated from fibroblast after affected by the virus or IFN.It is used for the treatment of viral diseases and tumor.In order to obtain a large number of cheap protein drugs,our experiment is designed to express these protein drugs in Escherichia coli expression system.We obtained the EPO and IFN-βgenes by chemical synthesis method for the elimination of the rare codons of EPO and IFN-βnative gene.The two genes were connected to expression vector pET series.Then the vectors were transformed into the host E.coli BL21（DE3）.Orthogonal experimental design was used to identify the influence of expression conditions.The yield of target protein is up to 30%after the induction of engineering strain. The result of orthogonal experimental design shows that the best induction condition is 0.8 M IPTG,OD₅₅₀=0.9,4h induction hours. The experimental results show that,we have succeed in constructing stable expression strains of erythropoietin and IFN-β,the strains can be used to produce vast and cheap proteins for the next phase of modifying the native proteins.更多还原