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  

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

作　　者：陆路[1,2] 熊文[1,2] 张钰琨 王丽娜[3] 权春善[1,2] 范圣第[1,2] 

机构地区：[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1166003 [3]中国科学院大连化学物理研究所,大连116023

出　　处：《中国生物工程杂志》2014年第10期55-60,共6页China Biotechnology

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。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.

关 键 词：金黄色葡萄球菌 AgrC 无限制克隆法 巢式PCR 

分 类 号：Q786[生物学—分子生物学]