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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2009, Vol. 29 Issue (03): 25-29    
研究报告     
组氨酸激酶(YycG)在筛选肺炎链球菌抑制剂中的应用
李南1,王非2,牛司强1,王虹1,李有强1,尹楠林1,张雪梅1,朱维良2,尹一兵1,胥文春1
1. 重庆医科大学医学检验系临床生物化学教研室,临床检验诊断学省部共建教育部重点实验室
2. 中国科学院上海药物研究所
Application of Histidine kinase (YycG) in screening inhibitor of Streptococcus pneumoniae
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摘要：

为了获得具有体外活性的肺炎链球菌组氨酸激酶YycG并利用其筛选寻找新的抑制剂。原核表达组氨酸激酶YycG的激酶功能域，经SDS-PAGE，Western blot鉴定及镍层析柱纯化后，采用激酶试剂盒检测其激酶活性；利用对其激酶活性的抑制作用从105种候选化合物中筛选有效的抑制剂，并通过实验验证抑制剂的抗菌作用。原核表达得到约35 kDa的目的蛋白激酶域片段YycG′，其纯度达 95 %，并具有体外水解ATP的激酶活性；利用其活性筛选得到数种不同抑制效果的小分子化合物，且体外验证具有较好的抑菌效果。通过肺炎链球菌组氨酸激酶YycG活性筛选找到的小分子抑制剂可为进一步研发与该菌相关的药物或消毒剂提供基础。
关键词： 组氨酸激酶;蛋白纯化;酶活性;体外筛选    
Abstract:

Objective: To obtain a purified protein fragment with ATP-dependent kinase activity of the kinase domain of histidine kinase YycG in Streptococcus pneumoniae by a prokaryotic expression system and make use of its activity to search for new bacterial inhibitors. Methods: The protein of YycG kinase domain was expressed by a prokaryotic expression, identified by SDS-PAGE and Western Blot, and then purified by His-tagged Ni affinity column, and finally the activity of this purified protein was determined by a Kinase-Glo Luminescent Kinase Assay; Effective inhibitors were screened out from 105 candidate compounds based on their inhibitory action on the kinase activity of YycG protein, and were testified for their antibacterial effects in vitro. Results: The interest protein expressed in protocaryon was about 35KDa, reaching a purity of 95%, with obvious kinase activity of hydrolyzing ATP. Several effective kinase inhibitors were filtered out, and a selective one was verified overt bactericidal effects in vitro assay. Conclusion: The kinase inhibitors screened out based on their action on YycG may be important cues for developing antibacterial drugs or disinfectant.
Key words: histidine kinase;purified protein;enzymatic activity;screening in vitro
收稿日期: 2008-10-27 出版日期: 2009-03-31
ZTFLH:  Q591  
基金资助:

国家自然科学基金:30671868;重庆市自然科学基金资助项目，编号：CSTC;2007BB5283;其它项目基金
通讯作者: 胥文春   
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李南 王非 牛司强 王虹 李有强 尹楠林 张雪梅 朱维良 尹一兵 胥文春

引用本文:

李南,王非,牛司强,王虹,李有强,尹楠林,张雪梅,朱维良,尹一兵,胥文春. 组氨酸激酶(YycG)在筛选肺炎链球菌抑制剂中的应用[J]. 中国生物工程杂志, 2009, 29(03): 25-29.

LI Na- Wang-Fei- Niu-Ci-Jiang- Wang-Gong- Li-Wei-Jiang- Yin-Nan-Lin- Zhang-Xue-Mei- Shu-Wei-Liang- Yin-Yi-Bing- Xu-Wen-Chun. Application of Histidine kinase (YycG) in screening inhibitor of Streptococcus pneumoniae. China Biotechnology, 2009, 29(03): 25-29.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/    或    https://manu60.magtech.com.cn/biotech/CN/Y2009/V29/I03/25

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