超氧化物歧化酶在酿酒酵母中的分泌表达

发布时间：2018-05-29 12:25

本文选题：人铜锌超氧化物歧化酶 + 酿酒酵母　；参考：《郑州大学》2005年硕士论文

【摘要】：背景和目的超氧化物歧化酶(SOD)是一种广泛存在于动物、植物、微生物中的金属酶,按其结合的金属离子可分为Cu/Zn-SOD、Mn-SOD、Fe-SOD三种,它们主要催化超氧化物阴离子自由基发生歧化反应,生成氧和过氧化氢,因而具有防御氧毒性,增强机体抗辐射损伤能力,还可预防衰老,在一些疾病如肿瘤、炎症及自身免疫疾病等的治疗中有良好的疗效。通过研究寄生虫及宿主体内SOD的含量、结构及功能有助于确立寄生虫免疫诊断指标、制备疫苗和寻找特异性抑制虫体相关酶的药物。其中,Cu/Zn-SOD(SOD1)分布最广,占总SOD的90%以上。它是一种普遍存在的酶,在家族性肌萎缩性侧索硬化症(FALS)、帕金森病(PD)、登革热、癌症、Down’s综合症、白内障和多种神经紊乱综合症中具有重要的生理学意义和治疗潜力。天然SOD来源有限,且具有异体蛋白免疫原性,外源SOD不易被人体接受等,使之在应用方面受到很大限制,SOD基因工程是广开酶源、降低成本和获得无抗原性的人源SOD的有效途径。目前国外对SOD进行了一系列的基础研究、应用开发和临床试验。国内主要集中在与应用有关的基础理论的研究上,如分离纯化及其性质等方面的研究,SOD基因克隆和表达方面的研究报道较少。利用大肠杆菌表达系统来生产SOD的生物技术研究已比较成熟,并已达到医学临床试验阶段,而用酿酒酵母等真核生物表达系统的研究和开发则较少。本文首次利用酿酒酵母交配因子(MF)α的分泌信号肽序列与hCu/Zn-SOD cDNA融合,构建了分泌表达载体,实现其在酿酒酵母中的克隆和分泌表达,其表达产物有可能不经过传统的纯化工艺而直接应用于化妆品、医疗保健食品等领域,发挥其抗氧化,抗衰老等功能。该酵母工程菌株的成功构建为开发hCu/Zn-SOD的基因工程产品奠定了良好的基础。
[Abstract]:Background and purpose Superoxide dismutase (SOD) is a kind of metallozyme widely found in animals, plants and microbes. According to the metal ions combined with it, it can be divided into three kinds: Cu- / Zn-SOD- Mn-SOD- Fe-SOD. They mainly catalyze the disproportionation of superoxide anion free radicals. Oxygen and hydrogen peroxide can be used to protect against oxygen toxicity, enhance the ability of body to resist radiation damage, and prevent aging. It has a good effect in the treatment of some diseases such as tumor, inflammation and autoimmune diseases. By studying the content, structure and function of SOD in parasite and host, it is helpful to establish the diagnostic index of parasite immunity, to prepare vaccine and to search for drugs that can specifically inhibit the enzyme related to parasite. Among them, CuZn-SOD1) is the most widely distributed, accounting for more than 90% of the total SOD. It is a ubiquitous enzyme that has important physiological and therapeutic potential in familial ALS, PD, dengue, Downs syndrome, cataract and multiple neurological disorders. The source of natural SOD is limited, and it has the immunogenicity of alloprotein, and the exogenous SOD is not easily accepted by human body, so its application is restricted greatly. It is an effective way to open the enzyme source, reduce the cost and obtain the human SOD without antigenicity. At present, a series of basic research, application development and clinical trials on SOD have been carried out abroad. There are few reports on the cloning and expression of superoxide dismutase (SOD) gene in China, such as isolation, purification and properties of superoxide dismutase (SOD) gene. The biotechnological research on the production of SOD by E. coli expression system has been mature and has reached the stage of medical clinical trial, but the research and development of eukaryotic expression system using Saccharomyces cerevisiae and other eukaryotes is less. In this paper, the secretory expression vector of Saccharomyces cerevisiae was constructed by fusion of the secreting signal peptide sequence of yeast mating factor MF- 伪 with hCu/Zn-SOD cDNA for the first time, and its cloning and secretory expression in Saccharomyces cerevisiae were realized. The expressed products may be directly used in cosmetics, health food and other fields without the traditional purification process, and play their functions of anti-oxidation and anti-aging. The successful construction of the yeast engineering strain laid a good foundation for the development of genetic engineering products of hCu/Zn-SOD.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R341

【引证文献】