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作 者:胡静平 姜羽婷 齐兴梅 HU Jing-ping;JIANG Yu-ting;QI Xing-mei(Institutes of Biology and Medical Sciences,Soochow University,Suzhou 215123,China)
出 处:《生物学杂志》2020年第2期101-103,共3页Journal of Biology
基 金:苏州大学大学生创新创业训练计划项目(No.2014xj048);国家自然科学基金项目(No.31771003,No.31400789);高等学校博士学科点专项科研基金新教师类项目(No.20133201120019)。
摘 要:结核分枝杆菌中的3个蛋白分别受intein调控,必须经过翻译后的蛋白剪接将intein去除才能成为有活性的蛋白,并且它们对结核杆菌的生长繁殖非常重要。这表明intein可以作为抗结核药物的一个新的作用靶点。因此,建立一个蛋白剪接抑制剂的高通量筛选系统对于大规模筛选抗结核新药尤为重要。将结核杆菌的RecA intein插入卡那霉素抗性蛋白基因中破坏其卡那霉素抗性,再通过intein剪接将其恢复。利用卡那霉素的LB平板筛选和Western Blot验证,结果表明依赖卡那霉素抗性的筛选系统可以精确地反映intein的剪接效率,因此可以作为intein剪接抑制剂的筛选系统。为了验证该系统的可行性,在含有卡那霉素的液体LB培养基中加入对intein剪接有抑制作用的顺铂,结果表明大肠杆菌的生长受到严重影响,从而直接反映顺铂对intein的剪接抑制作用。Mycobacterium tuberculosi s harbors three inteins in three critical proteins.Post-translational removal of the inteins is required for function of the proteins.Thus,inteins are potential targets for antimycobacterial agents.It is very important to develop a high throughput screening system for the isolation of antimycobacterial drugs that interfere with protein splicing.Therefore,the RecA intein coding sequence of Mycobacterium tuberculosis was inserted into kanamycin-resistance gene so that functional antibiotic resistance was restored upon protein splicing.Western Blot and kanamycin plate assay showed that the kanamycin resistance in LB plate was in accordance with the intein splicing efficiency,which was capable of screening system for intein inhibitors.To examine the utility of this screen system,the growth of E.coli transformed with plasmids containing kanamycin-intein fusions was significantly slowed by cisplatin,which showed that cisplatin toxicity could be suppress protein splicing by intein overexpression.
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