金黄色葡萄球菌wood46株eno基因的克隆及原核表达  

Cloning and Prokaryotic Expression of the eno Gene from Staphylococcus aureus wood46

作　　者：曹宏伟[1] 李冬野[1] 刘哲[1] 崔玉东[1] 宋佰芬[1] 肖翠红[1] 王桂华[1] 李闰婷[1] 

机构地区：[1]黑龙江八一农垦大学生命科学技术学院,大庆163319

出　　处：《黑龙江八一农垦大学学报》2014年第4期46-49,65,共5页journal of heilongjiang bayi agricultural university

基　　金：黑龙江省教育厅面上项目(11541251)

摘　　要：为获得Eno的重组蛋白,采用PCR法从金黄色葡萄球菌wood46株基因组扩增eno基因,并连接到pMD18-T载体上,转化至BL21感受态细胞中;重组质粒经PCR及酶切鉴定后,送上海生工测序。将鉴定正确的质粒酶切回收产物与原核表达载体pET-32a连接,然后转化至BL21感受态细胞,对平板筛选的阳性质粒进行PCR和酶切鉴定。对鉴定正确的重组菌进行诱导表达并纯化。实验结果表明成功构建了pET32a-eno表达载体,并获得了该纯化蛋白。该实验结果为S.aureus亚单位疫苗研究奠定了一定的基础。To construct eno express vector and obtain the purified recombinant protein, eno gene of S. aureus strain wood 46 was amplified using PCR method from genomic DNA extraction. PCR product was cloned into the pMD18-T vector. The positive plasmids after identification of PCR and restriction enzyme digestion was digested, inserted into the PET32a expression vector and transformed into BL21 competent cells. The recombinant expression strains were identified by using PCR and restriction enzyme digestion methods. The competent cells were induced by IPTG to express the fusion protein and eno protein was purified by using AKTA purifier 100. SDS-PAGE was used for identification of the fusion expression and purified protein. The results of the identification showed that the expression vector pET32-eno was constructed successfully and the vector expressed eno protein in BL21 correctly. Theses results laid the certain foundation for the further study of subunit vaccine,

关 键 词：金黄色葡萄球菌 eno基因 克隆 表达 

分 类 号：Q785[生物学—分子生物学]