检验医学与临床Issue(18)：2703-2704,2707,3.DOI:10.3969/j.issn.1672-9455.2015.18.021
结核分枝杆菌去乙酰化酶2的克隆及酶学研究
Study on cloning and enzymology of deacetylase protein Sir2 from Mycobacterium tuberculosis H37Rv
林雅宁 1董剑 1徐忠玉1
作者信息
- 1. 中国人民解放军第一七五医院/厦门大学附属东南医院检验科，福建漳州 363000
- 折叠
摘要
Abstract
Objective To construct the deacetylase protein Sir2 prokaryotic expression vector of Mycobacteri‐um tuberculosis(M Tb) and to conduct the expression purification and enzymologic study .Methods Sir2 gene was amplified from M Tb H37Rv genomic DNA as the template by PCR and then the recombinant plasmid pET‐28a‐Sir2 was constructed .The expression protein was induced in E .coli BL2(DE3) and purified by Ni2 + ‐NTA afinity chroma‐tography .Finally the optimal reaction temperature and pH value of Sir2 ,and influence of pyrazinamide(PZA) on en‐zyme activity were detected by the acetylation enzyme test .Results Sir2 prokaryotic expression vector was success‐fully constructed and could be highly expressed in E .Coli BL21(DE3) .Then the purified Sir protein was conducted the NAD + dependent deacetylation activity research .The obtained optimal pH was 9 .0 and the optimal reaction tem‐perature was 25 ℃ .PZA had the inhibition effect to the Sir2 enzymatic activity under weakly acidic environment ,but without the inhibiting effect under the basic pH environment .Conclusion The highly purified protein is obtained by using themolecular cloning technique ,the optimal pH and optimal reaction temperature are also obtained ,PZA has the inhibiting effect on it ,which provides certain foundation for researching the antibacterial mechanism of ant‐tubercu‐lous drug PZA .关键词
结核分枝杆菌/去乙酰化酶/基因克隆/蛋白纯化/吡嗪酰胺Key words
mycobacterial/deacetylase/gene clone/expression and purification/PZA引用本文复制引用
林雅宁,董剑,徐忠玉..结核分枝杆菌去乙酰化酶2的克隆及酶学研究[J].检验医学与临床,2015,(18):2703-2704,2707,3.
