摘要
目的通过谷胱甘肽巯基转移酶(GST)-促肾上腺皮质激素释放激素(CRH)原核表达条件的优化及纯化,获得可溶性好、纯度高的重组GST-CRH蛋白。方法通过对GST-CRH转化菌表达温度、菌液密度(OD600)、异丙基-β-D-硫代半乳糖苷(IPTG)浓度以及诱导时间的摸索,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)检测可溶性GST-CRH蛋白的表达情况,GST琼脂糖凝胶进行GST-CRH的纯化,Western Blot对表达的目的蛋白进一步鉴定。结果起始OD6000.8、IPTG工作浓度0.1 mmol/L、30℃诱导8 h为GST-CRH最优诱导条件;融合蛋白经Western Blot鉴定为表达的GST-CRH,纯化后纯度>95%。结论建立了GST-CRH的原核表达及纯化方法,获得了高纯度的GST-CRH可溶性蛋白。
Objective To obtain the recombinant corticotropin releasing hormone(CRH)protein with soluble,highpurity protein through optimizing prokaryotic expression condition and purifying glutathione thiol transferase(GST)-CRHprotein.Methods To detect the expression of soluble CRH protein through grope of the host strain GST-CRH temperatureof induction expression,the host strain concentration(OD600),IPTG concentration and induction time,the purification ofGST-CRH was performed by GST-CRH agarose gel.Western Blot assay was used for the expression identification of thetarget protein.Results The optimal conditions for the induction of CRH protein were determined:temperature of30℃,IPTG induced concentration0.1mmol/L,bacteria density(OD600)0.8,the induction time of8hours,purified GST-CRH>95%fusion protein was obtained.Conclusion The optimal expression conditions of GST-CRH are obtained,and thesoluble protein of high purity GST-CRH is also obtained.
作者
郁硕
陈锋
刘英富
霍景瑞
李光宗
张益
丁辉
樊毫军
YU Shuo;CHEN Feng;LIU Ying-fu;HUO Jing-rui;LI Guang-zong;ZHANG Yi;DING Hui;FAN Hao-jun(Respiratory Department, the Affiliated Hospital of Xinxiang Medical University, Henan 453000, China;Institute for Disaster and Emergency Rescue Medicine, the Affiliated Hospital of Logistics University of Chinese People’s Armed Police Force;Department of Cell Biology, Logistics University of Chinese People’s Armed Police Force)
出处
《天津医药》
CAS
2017年第2期146-150,共5页
Tianjin Medical Journal
基金
天津市科技计划项目(14ZCDZSY00033)
全军重点实验室开放基金(JY1402)
武警后勤学院附属医院科研平台开放基金(WYFKFM201602
WYKFZ201603)
