【作者】 孟玲；

【导师】 裴晓方；

【作者基本信息】 四川大学 ， 营养与食品卫生学， 2005， 硕士

【摘要】 目的: 扩增耐辐射奇球菌(Deinococcus radiodurans)锰超氧化物歧化酶(Mn-SOD)基因,构建pET-SOD重组子,实现Mn-SOD在大肠杆菌中的高效表达,通过金属离子亲和层析纯化表达蛋白,并对纯化蛋白的性质进行初步研究,为后续探讨耐辐射奇球菌的耐辐射机制以及对SOD的开发应用奠定基础。 方法: 本研究分为三部分: 1.耐辐射奇球菌锰超氧化物歧化酶基因原核表达载体的构建:用PCR方法自D.radiodurans基因组中扩增Mn-SOD目的片段。将该基因与原核表达质粒载体pET-30a(+)连接,构建重组质粒pET-SOD,并转化克隆宿主菌E.coli DH5α,利用卡那霉素抗性筛选阳性克隆,质粒小量提取后,双酶切和基因序列测定鉴定重组质粒。 2.超氧化物歧化酶融合蛋白的表达及纯化:重组质粒pET-SOD转化表达宿主菌E.coli BL21(DE3),IPTG诱导SOD融合蛋白表达,SDS-PAGE分析目的蛋白表达情况:通过镍金属离子亲和层析对SOD融合蛋白进行纯化,SDS-PAGE检测蛋白纯度和亚基分子量。 3.超氧化物歧化酶融合蛋白的性质研究:包括纯化产物总蛋白含量测定,SOD活性测定,SOD种类鉴定,SOD紫外吸收光谱测定,SOD的pH稳定性、温度稳定性研究。更多还原

【Abstract】 Objective:In this research, the gene of Mn-SOD of Deinococcus radiodurans was cloned into an expression plasmid and the recombinant SOD fusion protein was expressed in E.coli in high efficiency. Then the protein was purified by metal-chelating affinity chromatography and the purified SOD was studied. These results are to provide a foundation for further studies on the radioresistant mechanism of Deinococcus radiodurans and for further development and application of SOD.Methods:1. Cloning the gene of Mn-SOD of Deinococcus radiodurans and constructing the recombinant plasmid pET-SOD. SOD gene was amplified by PCR from genomic DNA of Deinococcus radiodurans and inserted into expression plasmid pET-30a(+) to construct pET-SOD. The recombinant plasmid was transformed into cloning host E.coli DH5α. The positive clones were selected by using Kanamycin resistance, and recombinant plasmid was identified by enzyme digestion and sequence analysis.2. Expression and purification of the recombinant SOD fusion protein. The recombinant plasmid pET-SOD was transformed into expression host E.coli BL21(DE3). The recombinant protein was expressed by the induction of isopropyl-β -D-thiogalactopyranoside (IPTG) and was analyzed by SDS-PAGE. Then the SOD fusion protein was purified by metal-chelating affinity chromatography and the purified product was assessed by SDS-PAGE.3. Study on the properties of the purified SOD fusion protein. The purified SOD was analyzed by determining total protein and enzyme activity, identifying its category, detecting its ultraviolet absorption spectrum, and testing activity stability under different conditions.Results:1. The recombinant expression plasmid pET-SOD was constructed. And after double enzyme digestion, the plasmid vector fragment (5.4Kb) and target gene fragment (0.6—0.7kb) were shown respectively. Sequence analysis indicated that the target SOD gene was 633bp and the coding sequence of SOD fusion protein is 780bp, coding 260 amino acid residues. There is a one-base difference between the obtained sequence and the previously published sequence in TIGR or GenBank. The homology was 99%.2. The expression protein of SOD was induced by IPTG at 37°C. The 29kD protein was found after SDS-PAGE, accordant with anticipation. After purified by affinity chromatography, the SOD fusion protein showed almost a single band after SDS-PAGE.3. The purified SOD was identified to be Mn-SOD. Its specific activity reaches 19344U/mg protein. In the ultraviolet absorption spectrum, it shows specific absorption at 280nm. It can maintain high activity in a wide pH range and it is also stable to high temperature.Conclusion:The recombinant expression plasmid pET-SOD was constructed successfully. The SOD fusion protein was expressed effectively and found by SDS-PAGE. By affinity chromatography, the purified product could be obtained conveniently and fast with a high purity, high enzyme activity, and good stability. These results will provide theoretical and experimental foundation for further studies on the radioresistant mechanism of Deinococcus radiodurans and on establishing a convenient and efficient way to product Mn-SOD. This will be helpful for the development and application of SOD.更多还原