【作者】 林雪；

【导师】 徐丽；

【作者基本信息】 大连理工大学 ， 生物化工， 2010， 硕士

【摘要】 金黄色葡萄球菌蛋白A,简称蛋白A(Staphylococcus aureus protein A, SPA)能够特异性地与多种哺乳动物抗体结合,在抗体分析、分离以及医学领域有广泛的应用。但金黄色葡萄球菌为致病菌,蛋白A为细胞壁结合蛋白,因此,通过规模化培养金黄色葡萄球菌的方法获取蛋白A不仅有一定风险,且分离纯化也有一定难度。本论文的工作目标是研究利用基因工程技术生产重组蛋白A的方法。研究内容包括表达蛋白A的基因工程菌的构建以及培养条件(包括摇瓶和发酵罐培养)的优化。论文首先选择原核表达系统进行研究。选用大肠杆菌作为表达宿主菌,pET系列质粒作为表达载体,选择含有抗体结合域的不同长度的蛋白A编码基因进行基因构建。分别获得了四种不同分子量蛋白A的胞内表达,发现spa4的表达量最大,且与IgG结合活性良好,因此,选择该表达菌株进行后续研究。分别在摇瓶和发酵罐中进行了培养条件和诱导条件的优化。在摇瓶培养中,对诱导剂加入时间和加入量进行了初步优化,发现在菌体培养1～6小时时间段内,随着诱导剂加入时间的延后,工程菌的生长密度逐渐增加,但单位菌体蛋白A的表达量相差不大。诱导剂IPTG和乳糖均能有效诱导蛋白A表达,但乳糖对菌体生长的抑制作用较弱。在5 L发酵罐中,利用指数流加的补料方式,对补料速率进行优化。获得优化的培养模式为：以300 g/L的葡萄糖、50g/L的酵母提取物作为补料培养基,以0.1 h-1为理论比生长速率计算补料速率,在菌体OD600nm达到60时加入终浓度为0.5 mmol/L的IPTG。在优化的条件下,最终发酵液的菌体密度OD60nm达70以上,重组蛋白A表达量占菌体胞内总蛋白的比例约为22%,每升菌液重组蛋白A表达量约为2.6 g。为了简化重组蛋白A分离纯化的难度和成本,论文进一步对能够实现蛋白A外泌表达的真核表达系统进行了研究。利用毕赤酵母作为表达宿主、pPIC9K作为表达载体、选择仅含抗体结合功能区的蛋白A编码基因成功构建了外泌表达蛋白A的基因工程菌。在摇瓶培养中,考察了培养基组成,诱导剂甲醇加入量及诱导时间对重组蛋白A表达的影响。实验结果表明,利用BMGY/BMMY培养基,诱导阶段每24 h加入甲醇至终浓度0.5%,诱导84 h,目标蛋白的表达量最大。所表达的重组蛋白A与人IgG结合活性良好。通过本论文的研究,建立了重组蛋白A的原核和真核表达体系,对其培养和诱导条件进行了系统优化,研究结果为规模化生产重组蛋白A奠定了基础。更多还原

【Abstract】 Staphylococcus aureus protein A (SPA) which can specifically bind a variety of mammalian antibodies has a wide range of applications in antibody analysis, bioseparation and medicine. As protein A is a cell wall binding protein of pathogenic staphylococcus aureus, producing protein A by large-scale cultivation of staphylococcus aureus is risk and the purification process is also sophisticated.The research aims to produce recombinant protein A by genetic engineering technology in a safe and simple way, covering the construction of genetical engineering strains for expressing protein A and the optimization of culture conditions in shake flask and fermentor.First, prokaryotic expression system was selected. E.coli, pET series plasmid, and different lengths of protein A coding genes containing antibody binding domain were used as expression host, expression vector and target genes, respectively. Four different lengths of intracellular protein A with molecular weights were obtained. The results showed that spa4 had the maximum expression quantity and a good IgG binding activity. This strain was selected for further optimization of the culture and induction conditions in shake flask and fermentor. The effects of inducer concentration and adding time on bacteria growth and protein A expression were preliminarily studied in shake flask. The results showed that the engineering bacteria gradually increased with the delay of inducer added in 1 h to 6 h cell culture period, and the expression quantity per mass of bacteria did not change significantly. Besides, isopropyl (3-D-thiogalactopyranoside (IPTG) and lactose could effectively induce the expression of protein A, and lactose had the weaker inhibitory effect on bacteria growth. In 5 L fermentor, feeding rate was optimized. The optimal conditions for fermentation were obtained:the feeding rate of medium contained 300 g/L glucose,50 g/L yeast extract was calculated as theoretical specific growth rate of 0.1 h-1. IPTG was added to the concentration of 0.5 mmol/L when OD600nm was up to 60. The cell density of OD600nm in the final fermentation broth could reach more than 70, and the yield of recombinant protein A could be up to 2.6 g/L broth which accounted for about 22% of the total intracellular protein.In order to simplify the purification process of recombinant protein A and reduce its cost, eukaryotic expression system was studied further to realize the excretion of recombinant protein A. Genetic engineering yeast that could excrete recombinant protein A was successfully constructed by using Pichia pastoris GS115, pPIC9K and protein A coding genes containing only antibody binding domain as expression host, vector and target gene, respectively. In shake flask, effects of medium composition, concentration of methanol as the inducer and induction time on the expression of protein A were investigated. The results turned out that the optimal conditions were using BMGY/BMMY as medium and adding methanol to 0.5% every 24 h during the induction time of 84 h. The recombinant protein A had a good IgG binding activity.In this paper prokaryotic and eukaryotic expression system of recombinant protein A was established and culture and induction conditions were optimized. The results laid the foundation for large-scale production of recombinant protein A.更多还原