口腔医学研究 ›› 2018, Vol. 34 ›› Issue (6): 597-600.DOI: 10.13701/j.cnki.kqyxyj.2018.06.006

• 口腔微生物学研究 • 上一篇    下一篇

变异链球菌丙氨酸消旋酶基因表达纯化及活性测定

郭笑, 邱伟, 周学东, 程磊, 李明云*   

  1. 口腔疾病研究国家重点实验室 国家口腔疾病临床医学研究中心四川大学华西口腔医院 四川 成都 610041
  • 收稿日期:2017-12-05 出版日期:2018-06-20 发布日期:2018-06-21
  • 通讯作者: 李明云,E-mail: limingyun@scu.edu.cn
  • 作者简介:郭笑(1994~ ),女,河南林州市人,硕士在读,研究方向为龋病及其防治。
  • 基金资助:
    国家自然科学基金(编号:81400501、81430011);口腔疾病研究国家重点实验室专项经费(编号:SKLOD201525)

Gene Expression, Purification, and Activity Determination of Alanine Racemase from Streptococcus Mutans

GUO Xiao, QIU Wei, ZHOU Xue-dong, CHENG Lei, LI Ming-yun*   

  1. State Key Laboratory of Oral Diseases & National Clinical Research Center for Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu 610041, China
  • Received:2017-12-05 Online:2018-06-20 Published:2018-06-21

摘要: 变异链球菌是口腔主要的龋病病原菌。丙氨酸消旋酶通过合成右旋丙氨酸(D-alanine,D-ala)参与细菌细胞壁肽聚糖的生物合成过程,与细菌性疾病密切相关,近年来成为抗菌药物的又一理想靶位。D-环丝氨酸(D-cycloserine,DCS)是丙氨酸消旋酶不可逆性抑制剂。本研究通过聚合酶链反应克隆变异链球菌UA159的smu.1834c基因,经过酶切、连接,构建重组表达质粒pET-smu.1834c,诱导表达重组,纯化蛋白。测定DCS对丙氨酸消旋酶活性的抑制作用。成功诱导表达纯化smu.1834c基因编码蛋白,具有体外催化L-丙氨酸生成D-丙氨酸的活性。发现DCS能够抑制该蛋白的活性。本研究证实变异链球菌smu.1834c基因编码丙氨酸消旋酶。DCS能够抑制其活性,并且呈现一定的剂量相关性。

关键词: 变异链球菌, 丙氨酸消旋酶, 右旋丙氨酸, D-环丝氨酸

Abstract: Objective: To purify and measure activity of alanine racemase from Streptococcus mutans (S. mutans). Methods: In the study, expression of recombinant pET-smu.1834c was induced by IPTG, and the protein was purified using Ni-NTA resin with eluotropic buffers. Results: The protein encoded by smu.1834c was successfully purified and it was found that the protein could catalyze the conversion of L-Ala into D-Ala in vitro. Conclusion: D-cycloserine (DCS) was able to inhibit the activity of purified Alanine racemase (Alr) in a dose-dependent manner. This study confirmed that smu.1834c was encoded Alr in S. mutans. DCS can inhibit the activity of Alr.

Key words: Streptococcus mutans, Alanine racemase, D-alanine, D-cycloserine