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

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.

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

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.