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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  2014, Vol. 34 Issue (10): 55-60    DOI: 10.13523/j.cb.20141009
研究报告     
RF克隆结合巢式PCR构建金黄色葡萄球菌组氨酸蛋白激酶AgrC表达载体
陆路1,2, 熊文1,2, 张钰琨1,2, 王丽娜3, 权春善1,2, 范圣第1,2
1. 大连民族学院生物技术与资源利用国家民委-教育部重点实验室 大连 116600;
2. 大连民族学院生命科学学院 大连 116600;
3. 中国科学院大连化学物理研究所 大连 116023
Expression Vector Construction of Staphylococcus aureus Histidine Kinase AgrC Based on RF Cloning Combined with Nested PCR
LU Lu1,2, XIONG Wen1,2, ZHANG Yu-kun1,2, WANG Li-na3, QUAN Chun-shan1,2, FAN Sheng-di1,2
1. SEAC-ME Key Laboratory, Dalian 116600, China;
2. Department of Life Science, Dalian Nationalities University, Dalian 116600, China;
3. Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China
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摘要：

AgrC蛋白是位于金黄色葡萄球菌细胞膜上的组氨酸激酶,能够感受细胞外的化学信号并将信号传递到细胞内,进而调控细胞内与致病性相关的一系列基因的表达.利用无限制克隆法(RF克隆),并结合巢式PCR成功构建了AgrC表达载体pET-28a-AgrC.将表达载体电转入大肠杆菌中,并利用IPTG进行诱导表达AgrC蛋白.再经过低温超高压破碎、超高速离心、金属螯合层析与尺寸排阻层析等过程,分离纯化得到AgrC蛋白.
关键词： 金黄色葡萄球菌;  AgrC;  无限制克隆法;  巢式PCR    
Abstract:

AgrC is a membrane-embedded histidine kinase of Staphylococcus aureus that is thought to act as a sensor for the recognition of environmental signals and the transduction of signals into the cytoplasm so as to regulate and control a series of related pathogenic gene expression. Restriction-Free cloning and Nested PCR were used to construct an expression vector of pET-28a-AgrC successfully. Then, expression vector pET-28a-AgrC was transformed into E.coli C43 (DE3), and then, IPTG was added to induce expression. The expressed AgrC protein with a C-terminal His-tagged was purified using immobilized metal affinity chromatography (IMAC) and size exclusion chromatography (SEC). SDS-PAGE test showed that AgrC proteins were successfully separated and purified.
Key words: Staphylococcus aureus    AgrC    Restriction-Free cloning    Nested PCR
收稿日期: 2014-06-25 出版日期: 2014-10-25
ZTFLH:  Q786  
通讯作者: 权春善     E-mail: mikyeken@dlnu.edu.cn
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引用本文:

陆路, 熊文, 张钰琨, 王丽娜, 权春善, 范圣第. RF克隆结合巢式PCR构建金黄色葡萄球菌组氨酸蛋白激酶AgrC表达载体[J]. 中国生物工程杂志, 2014, 34(10): 55-60.

LU Lu, XIONG Wen, ZHANG Yu-kun, WANG Li-na, QUAN Chun-shan, FAN Sheng-di. Expression Vector Construction of Staphylococcus aureus Histidine Kinase AgrC Based on RF Cloning Combined with Nested PCR. China Biotechnology, 2014, 34(10): 55-60.

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https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20141009    或    https://manu60.magtech.com.cn/biotech/CN/Y2014/V34/I10/55


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