【作者】 黄勇；

【导师】 张德纯；

【作者基本信息】 重庆医科大学 ， 临床检验诊断学， 2003， 硕士

【摘要】 目的：克隆大肠杆菌锰超氧化物歧化酶基因（sodA）并在保加利亚乳杆菌（L6032）中表达，为下一步SOD发酵奶的研制奠定基础。同时，观察SOD在保加利亚乳杆菌中的活性表达对该菌氧耐受性的影响。方法：1).根据PubMed基因库中报道的大肠杆菌MG1655全基因组DNA序列中SOD的编码基因序列设计引物，PCR扩增大肠杆菌锰超氧化物歧化酶基因，并将其克隆入乳酸菌表达载体pMG36e中组成重组质粒pMG36e-sod。2).对影响L6032电转化效率的多种因素进行初步探讨，优化该菌的电转化条件，并在此条件下将重组质粒pMG36e-sod电转入L6032中。用黄嘌呤氧化酶法对SOD的表达活性进行测定。同时，对保加利亚乳杆菌及其转化子在有氧条件下的存活情况及其在MRS平板上的生长情况进行比较，初步探讨SOD在L6032中的活性表达对其氧耐受性的影响。<WP=8>结果：1).构建了重组质粒pMG36e-sod，经酶切鉴定和PCR鉴定与预期大小完全吻合。核酸序列测定显示插入的sodA片段能正确编码SOD氨基酸序列。2).建立了较优化的电转化条件。在电场强度9kV/cm、电阻100Ω、细菌生长浓度OD600为0.7的条件下，采用pH6.0的柠檬酸盐缓冲液能获得103个转化子/ugDNA的转化效率。3).利用电转化成功地将重组质粒pMG36e-sod导入L6032中。用黄嘌呤氧化酶法测得SOD的表达活性约590.5 NU/mg prot。SOD的活性表达，能增强保加利亚乳杆菌对氧的耐受性，在有氧条件下培养相同时间，重组子在MRS平板上的生长优于原菌，形成的菌落也较大；在有氧条件下存放7-10天，重组子的存活率与原菌相比得到明显提高。结论：SodA在保加利亚乳杆菌中获得了成功表达，增强了该菌对氧的耐受性,有利于该菌的开发应用。同时也为下一步SOD发酵奶的研制奠定了基础。更多还原

【Abstract】 Objective: To enhance its tolerance to oxygen, the superoxide dismutase gene encoding the antioxidant enzyme Mn-SOD was cloned and expressed in Lactobacillus bulgaricus that lacks SOD. And this work would lay a foundation for developing SOD fermented milk.Methods and Results: 1).According to the sodA encoding sequence of E.coli MG1655,a pair of primers was designed and the sodA gene was amplified by PCR techniques. After purification, the sodA gene fragment was digested with restriction endonucleases XbaI and HindIII, and then linked with the expression vector pMG36e digested with the same enzymes to construct the recombinant plasmid pMG36e-sod. Restriction enzyme digestion, PCR identification and DNA sequencing analysis confirmed that the construction of pMG36e-sod was successful. 2).In order to optimize the electroporation procedures for Lactobacillus bulgaricus, several parameters were tested including the cell growth phase, voltage, resistance, and the composition of the wash and electroporation<WP=10>buffers. In our results the maxium transformation efficiency was about 103 transformants per μg of DNA under such conditions.3).The recombinant plasmid pMG36e-sod was transformed into Lactobacillus bulgaricus by electroportion. And the expression of sodA gene in Lactobacillus bulgaricus was verified by SOD activity assay. It was about 590.5NU/mgprotein. In the experiments we also found that the expression of sodA gene is beneficial to aerobic growth of Lactobacillus bulgaricus. Conclusions: 1)The recombinant plasmid pMG36e-sod was successfully constructed.2)Developing a effective electroporation procedure for Lactobacillus bulgaricus. 3)The sodA gene was expressed in Lactobacillus bulgaricus successfully. And expression of sodA gene in Lactobacillus bulgaricus can enhance its tolerance to oxygen.更多还原