食品科学 ›› 2005, Vol. 26 ›› Issue (5): 92-95.

• 基础研究 • 上一篇    下一篇

锰超氧化物歧化酶基因的克隆和在保加利亚乳杆菌中的表达

 黄勇, 张德纯   

  1. 重庆医科大学医学检验系
  • 出版日期:2005-05-15 发布日期:2011-09-19

Cloning and Expression of the Manganese Superoxide Dismutase Gene of E. coli in Lactobacillus bulgaricus

 HUANG  Yong, ZHANG  De-Chun   

  1. Department of Clinical Microbiology, Chongqing University of Medical Science
  • Online:2005-05-15 Published:2011-09-19

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,以提高该菌对氧的耐受性,同时为SOD发酵奶的研制奠定实验室基础。方法:PCR扩增大肠杆菌sodA,将其克隆入乳酸菌表达载体pMG36e中,并将该重组质粒pMG36e-sod电转入L6032中表达。用SOD测试盒测定SOD的表达活性,同时对L6032及其重组子在MRS平板上的生长情况及其在有氧条件下的存活情况进行比较,初步探讨SOD在L6032中的活性表达对其氧耐受性的影响。结果:构建了重组质粒pMG36e-sod,经酶切和PCR鉴定与预期完全吻合。核酸测序显示插入的sodA片段能正确编码SOD氨基酸序列。利用电转化成功地将重组质粒导入L6032中,SOD的表达活性约590.5NU/mgprot。SOD的活性表达,能增强保加利亚乳杆菌对氧的耐受性,在有氧条件下重组子的生长和存活率都优于原菌。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实验室基础。

关键词: 保加利亚乳杆菌, 超氧化物歧化酶, 基因克隆与表达

Abstract: Objective: Cloning manganese superoxide dismutase gene (sodA) to make it express in Lactobacillus bulgaricus (L6032) to enhance its tolerance to oxygen was studied. Moreover, a foundation for developing SOD fermented milk was established. Methods: Using PCR to amplify sodA and cloning it into the Lactic acid bacteria expression vector pMG36e and then transforming this recombinant plasmid pMG36e-sod into L6032 by electroporation were studied; SOD reagent was used to detect its activity and to compare the growing situation and living condition as aerobes in MRS culture medium between L6032 and L6032-SOD. A preliminary inquiry into the effect of anti-oxygen of L6032 with SOD expression was made. Results: Construct- ing the recombinant plasmid pMG36e-sod successfully has been identified with the prospect by means of enzyme digestion and PCR. The result of sequencing showed that the insert gene could encode SOD correctly; After successfully transforming pMG36e-sod into L6032 by electroporation the activity of SOD was about 590.5NU∕mg prot. Furthermore, enhancing L. bulgaricus’s tolerance to oxygen has not only made L6032-SOD grow better than L6032 under the same condition, but also improved the living rate of bacteria obviously. Conclusion: sodA has been successfully for expressed in L. bulgaricus to have enhanced its tolerance to oxygen. It was beneficial to develop and apply Lactobacillus. At the same time, this study has laid a foundation for further developing SOD fermented milk.

Key words: lactobacillus, superoxid dismutase, gene cloning and expression