导　　师： 赵春芳;郝秀华

学科专业： 081703

授予学位： 硕士

作　　者： ;

机构地区： 吉林大学

摘　　要： 重组牛碱性成纤维细胞生长因子是一种新型的重组基因工程制品。其生物学作用极其广泛,它在血管形成、促进创伤愈合与组织修复、促进组织再生和神经组织生长发育过程中起着十分重要的作用。 对于采用含有具氨苄抗性、Lac启动子和编码牛碱性成纤维细胞生长因子重组质粒pUC18-bFGF基因质粒的大肠杆菌,首先在LB培养基中进行摇瓶培养,然后采用富含碳源和氮源的发酵培养基进行罐内高密度分批发酵培养,分别采用IPTG和乳糖进行诱导,均可获得了高效表达的菌体,并且采用相同的纯化方法对菌体进行纯化,获得所需的目的蛋白,经过检测完全符合质量要求。具体工作结果如下: 1、以IPTG诱导表达为主的传统发酵工艺的确立与优化 通过采用传统的摇瓶培养、高密度发酵培养工艺,确立了加入诱导剂的最佳时间和最佳浓度的选择。从而得到了以IPTG诱导表达为主的传统发酵最优工艺参数。 2、以乳糖代替IPTG诱导发酵工艺的改进 对采用IPTG诱导所存在的弊端进行了改进,确立以高效、低毒、安全的新型诱导表达方式,即用乳糖代替IPTG作为诱导剂,进行摇瓶培养、发酵罐培养,确定了采用乳糖诱导的最佳条件和发酵工艺的优化。 3、重组牛碱性成纤维细胞生长因子的纯化和检定 对获得的菌体进行纯化,通过初步纯化、CM离子交换层析、肝素亲和层析后,最终可以获得目的蛋白bFGF,对于所获得的目的蛋白进行蛋白含量、纯度、生物活性、等电点、外源性DNA残留量、紫外光谱、肽图检测后,可以得出,尽管采用的诱导方式不同,在菌体的收率方面有所差异,但所获得目的蛋白无论从数量还是质量上都无区别。 As same as other cell factor, basic fibroblast growth factor is a kind of normal trace substance of mammals and human body, but physiological function is very extensive and important in clinical. Recombinant basic Fibroblast Growth Factor of Bovine is a new type of recombinant products of genetic engineering. With the development of biotechnology, a lot of genetic engineering recombinant proteins were expressed in E. coli expression system for production. Lac promoter and its derivatives are widely used in this field. Usually this type of promoter is induced by isopropyl-β-D-galactoside /(IPTG/) to express the exogenous protein. In the production and research, the fermentation conditions of E. coli with gene plasmid having ampicillin resistance, Lac promoter and cow basic fibroblast growth factor /(bFGF/) were studied. In order to obtain the optimal expressing conditions of bFGF, shake-flask test was used. Under the induction of the inductive agent, bFGF can be secreted to cell periplasm. Using high density fed-batch fermentation technology with stepwise addition of glucose, eventually high efficiency, high density bacteria was obtained. After purification process, high purity protein bFGF was obtained. The physical and chemical and biological characteristics were confirmed by Western blot, the sequence analysis and biological activity detection experiment. IPTG is not suitable for large-scale industrial production due to high cost and toxicity. In view of the above questions, fermentation technology using lactose instead of IPTG was developed. IPTG or lactose was used as an inducer to acquire the fermentation products. Using the same technology, the fermentation products were purified to obtain the target protein bFGF, which was verified by SDS-PAGE verification method, biological activity determination method and high performance liquid chromatography. As shown in the data, the purity, biological activity and all the verification results of the target protein are homogeneous, and the target protein accord with the production requirements. Therefore, lactose can be used as inducer instead of IPTG to obtain cattle basic fibroblast growth factor in the fermentation process. The reseach was carried out in these studies as follows: 1、IPTG was used as the inducer in the fermentation process Using Shake-flask experiments, the best time of the inducer joined, optimal concentration and the influence on engineering bacterium growth of inducer were established. Using high density fed-batch fermentation technology with stepwise addition of glucose, eventually high density, high expression bacteria was obtained. Process parameters for the fermentation of IPTG revulsant were established, and the optimal fermentation technology was achieved. Experimental results showed that IPTG was added to 1mmol·L-1 concentration for 6 hours, After controlling the pH of fermentation fluid with stepwise adding glucose, and the addition time was 5 hours after fermentation cultivation. When bacteria light density values /(OD600/) reach about 35, stop fermenting. Bacterium body wet weighed was about 40 g.L -1, and the protein expression value of bacteria was above 20/%. 2、In order to improve the fermentation process using lactose as an inducer instead of IPTG In the Shake-flask experiments, lactose was used as an inducer instead of IPTG. By comparing the experimental results, the best time of the inducer joined, optimal concentration was established. After high density fermentation cultivation in fermentor, the optimal fermentation technology of lactose instead of IPTG. was achieved. Results showed that the best induction period was at the end of bacteria growth stages. When the final concentration of lactose was 20 g·L-1, the best production and expression of fermentation technology was achieved. In high density fermentation process, OD600 was between 20~ 25 after incubation about five hours, stop adding with glucose, but add with lactose with the concentration of 20/%, until the final concentration of lactose was 20 g·L-1. Fermentation terminates, until OD600 reaches 30~32 about fermentation 6 hours later. Compared with IPTG induction fermentation experiments, the different production of bacteria was collected by centrifugal separation. IPTG average yields for production was 63.67 g?L-1, and expression level was 21.55/%. Lactose average yields for production was 59.87 g·L-1, and expression level was 20.7/%. 3、Purification and characterization of recombinant bovine basic fibroblast growth factor The bacteria were qualified by electrophoresis. After a preliminary purification, the pure protein was exchanged through CM ion exchange chromatography and heparin affinity chromatography. Finally, we got the desired proteins bFGF. According to the biological manufacturing procedures and China pharmacopoeia of the People's Republic /(2005 edition/), bFGF was detected. Results showed that we could get 0.96 mg target proteins in 1 g bacteria after purification, and the protein recovery rate was 4.6/%. Biological activity reached 5.31×106 IU·ml-1. The purity of bFGF was above 98/% through SDS-PAGE gel electrophoresis and high performance liquid chromatographic detection. Peptide map and isoelectric point were in accordance with bFGF standard, and residual DNA detection also accord with the relevant provisions of China pharmacopoeia. For two inducer fermentation technology, such as the lactose and IPTG, the bacteria were purified through the same process to gain the target protein. According to the relevant regulations of the People's Republic of China pharmacopoeia /(2005 edition/), through activity detection /( MTT method/), active protein content detection /(Lowry method/), SDS-PAGE gel electrophoresis and high performance liquid chromatographic detection, the target proteins were in accord with the relevant standards, and deviation of the data between them is not significant. In Conclusion , our studies indicate that It was more complicated and troublesome using lactose as revulsant compared with IPTG.. Just because of this, there are few research reports about lactose used as revulsant to induce the expression of reorganization products. In the study on high density fermentation technology, research report about lactose is very rare. Nonetheless, lactose is of low prices and non-toxic, making use of lactose as the research revulsant for restructuring industrial production of gene engineering drugs has very important significance.

关 键 词： 碱性成纤维细胞生长因子 大肠杆菌 高密度发酵 乳糖 纯化 活性检测

分 类 号： [Q813]

领　　域： [生物学]