蛋白质硫辛酸修饰相关酶大肠杆菌研究模型的构建

doi:10.11843/j.issn.0366-6964.2019.05.018

畜牧兽医学报 ›› 2019, Vol. 50 ›› Issue (5): 1073-1081.doi: 10.11843/j.issn.0366-6964.2019.05.018

蛋白质硫辛酸修饰相关酶大肠杆菌研究模型的构建

朱可蒙, 谨瑾, 王宁, 辛九庆*, 刘恒贵*

中国农业科学院哈尔滨兽医研究所动物支原体病创新团队, 哈尔滨 150069

收稿日期:2018-11-19 出版日期:2019-05-23 发布日期:2019-05-23
通讯作者: 刘恒贵,主要从事动物疫苗与分子免疫学研究,E-mail:lhg7519@163.com;辛九庆,主要从事动物传染病及其病原分子流行病学研究,E-mail:Xinjiuqing2001@126.com
作者简介:朱可蒙(1993-),女,河南太康人,硕士生,主要从事猪肺炎支原体的研究,E-mail:zhukemeng0527@163.com;谨瑾(1993-),女,山西运城人,博士生,主要从事猪肺炎支原体的研究,E-mail:JIN109JIN@163.com
基金资助:
中央级公益性科研院所基本科研业务费专项（1610302017014）；国家重点研发计划（2016YFD0500908）

Establishment of E. coli Model for the Research of Lipoate-Protein Ligases

ZHU Kemeng, JIN Jin, WANG Ning, XIN Jiuqing*, LIU Henggui*

Animal Mycoplasma Innovation Team, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150069, China

Received:2018-11-19 Online:2019-05-23 Published:2019-05-23

摘要/Abstract

摘要：

为研究不同微生物硫辛酸修饰相关酶的功能，构建了大肠杆菌菌体内研究模型。利用Red同源重组系统对大肠杆菌的lplA、lipB基因依次进行敲除，获得大肠杆菌lplA及lipB双基因缺失株△lplA△lipB~DH5α。将lplA和lipB基因分别克隆到载体pBAD322G，构建基因回补株ClplA-△lplA△lipB~DH5α和ClipB-△lplA△lipB~DH5α。通过Western blot、质谱分析和补偿试验等方法检测缺失株和回补株对底物的硫辛酸修饰功能。结果显示：上述突变株构建成功并能稳定遗传，回补株构建成功并能发挥生物学功能。补偿试验结果显示，在甘油为碳源的M9基础培养基中，回补株ClipB-△lplA△lipB~DH5α能够生长，ClplA-△lplA△lipB~DH5α补偿硫辛酸后能够生长。以上结果表明，大肠杆菌DH5α硫辛酸修饰功能缺陷菌株构建成功，这有利于以大肠杆菌为生物模型进行其他微生物硫辛酸修饰相关酶的功能研究。

Abstract:

To investigate the function of lipoic acid metabolic enzymes in vivo, an Escherichia coli (E. coli) model was established.△lplA△lipB~DH5α mutant strain was obtained with deletion of lplA and lipB genes from DH5α strain using the Red homologous recombination system. The lplA and lipB genes were cloned into the expression vector of pBAD322G and transformed to the △lplA△lipB~DH5α mutant strain to construct the complementary strains of ClplA-△lplA△lipB~DH5α and ClipB-△lplA△lipB~DH5α. The lipoate modification on the substrates in the mutant and supplemented strains were analyzed with Western blot, mass spectrometric analysis and complementation experiment. The mutant strain △lplA△lipB~DH5α was very stable in the culture and displayed the phenotype of lplA and lipB deletion. Supplemented strain ClipB-△lplA△lipB~DH5α can grow in the M9 medium with glycerol as carbon source, ClplA-△lplA△lipB~DH5α can grow when LA is added to this medium. These results indicated that the E. coli model without lipoate modification function was successfully established. It will facilitate the functional study of enzymes associated with lipoic acid modification in other microorganism.

中图分类号:

S852.612

朱可蒙, 谨瑾, 王宁, 辛九庆, 刘恒贵. 蛋白质硫辛酸修饰相关酶大肠杆菌研究模型的构建[J]. 畜牧兽医学报, 2019, 50(5): 1073-1081.

ZHU Kemeng, JIN Jin, WANG Ning, XIN Jiuqing, LIU Henggui. Establishment of E. coli Model for the Research of Lipoate-Protein Ligases[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2019, 50(5): 1073-1081.

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蛋白质硫辛酸修饰相关酶大肠杆菌研究模型的构建

Establishment of E. coli Model for the Research of Lipoate-Protein Ligases

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