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

doi:10.13523/j.cb.20141009

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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

作者信息 +

Expression Vector Construction of Staphylococcus aureus Histidine Kinase AgrC Based on RF Cloning Combined with Nested PCR

LU Lu^1,2, XIONG Wen^1,2, ZHANG Yu-kun^1,2, WANG Li-na³, QUAN Chun-shan^1,2, FAN Sheng-di^1,2

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文章历史 +

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.

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.

导出引用

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 https://doi.org/10.13523/j.cb.20141009

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[J]. China Biotechnology, 2014, 34(10): 55-60 https://doi.org/10.13523/j.cb.20141009

中图分类号： Q786

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