瑞替普酶的分子克隆及其在大肠杆菌中使用 tac 启动子的表达。
Molecular cloning of Reteplase and its expression in E. coli using tac promoter.
作者信息
Aghaabdollahian Safieh, Rabbani Mohammad, Ghaedi Kamran, Sadeghi Hamid Mir Mohammad
机构信息
Department of Pharmaceutical Biotechnology, Isfahan Pharmaceutical Science Research Center, School of Pharmacy and Pharmaceutical Science, Isfahan University of Medical Sciences, Isfahan, I R Iran.
Department of Biology, Faculty of Sciences, University of Isfahan, Isfahan, I R Iran.
出版信息
Adv Biomed Res. 2014 Sep 12;3:190. doi: 10.4103/2277-9175.140622. eCollection 2014.
BACKGROUND AND AIMS
This study aimed to clone and express the reteplase cDNA, a thrombolytic agent used for the treatment of acute myocardial infarction and stroke, in E. coli, utilizing tac promoter for its expression.
MATERIALS AND METHODS
Reteplase cDNA was amplified by polymerase chain reaction (PCR) with designed primers. The product was then cloned into pTZ57R plasmid. The cloned cDNA was digested out and ligated into pGEX-5x-1 expression vector. The presence of the insert was confirmed by restriction digestion. By using 0.2, 0.5 and 1 mM isopropyl beta-D thiogalactopyranoside (IPTG), expression of reteplase was induced in E. coli TOP10 cells and analyzed by SDS-PAGE.
RESULTS
Electrophoresis of PCR product and also double digested recombinant pTZ57R plasmid, also, pGEX-5x-1 vector, showed a 1068bp band of reteplase. SDS-PAGE analysis showed a 60 KDa band of protein product induced with different concentrations of IPTG.
CONCLUSION
In the present study, reteplase cDNA was successfully cloned and expressed using tac promoter. This vector will be used for the optimization of the expression of reteplase in E. coli.
背景与目的
本研究旨在克隆瑞替普酶cDNA，并在大肠杆菌中利用tac启动子进行表达，瑞替普酶是一种用于治疗急性心肌梗死和中风的溶栓剂。
材料与方法
使用设计好的引物通过聚合酶链反应（PCR）扩增瑞替普酶cDNA。然后将产物克隆到pTZ57R质粒中。将克隆的cDNA切出并连接到pGEX-5x-1表达载体中。通过限制性酶切确认插入片段的存在。使用0.2、0.5和1 mM异丙基-β-D-硫代半乳糖苷（IPTG）在大肠杆菌TOP10细胞中诱导瑞替普酶的表达，并通过SDS-PAGE进行分析。
结果
PCR产物以及双酶切的重组pTZ57R质粒和pGEX-5x-1载体的电泳显示出一条1068bp的瑞替普酶条带。SDS-PAGE分析显示，不同浓度IPTG诱导产生的蛋白质产物有一条60 kDa的条带。
结论
在本研究中，利用tac启动子成功克隆并表达了瑞替普酶cDNA。该载体将用于优化瑞替普酶在大肠杆菌中的表达。
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