肺炎链球菌自溶素基因的克隆、表达及蛋白纯化  

Cloning,expression of pneumococcal LytA gene and purification of its product

作　　者：徐江红[1] 陈兵[1] 章德广[1] 郭建[2] 顾凌澜[1] 

机构地区：[1]复旦大学附属眼耳鼻喉科医院耳鼻咽喉科,上海200031 [2]南昌大学生命科学学院,江西南昌330031

出　　处：《复旦学报（医学版）》2009年第2期191-194,200,共5页Fudan University Journal of Medical Sciences

基　　金：国家自然科学基金项目(30471872);上海市科委科研计划项目(06JC14014)

摘　　要：目的克隆肺炎链球菌自溶素(LytA)基因并在大肠埃希菌中表达及蛋白纯化。方法用PCR方法从肺炎链球菌基因组中克隆肺炎链球菌自溶素基因,然后将其插入原核表达载体pET32a(+),构建pET32a(+)-LytA重组质粒,经IPTG诱导使该基因在大肠埃希菌BL21(DE3)中表达,亲和层析法纯化目的蛋白,将获得的蛋白用Western blot鉴定。结果扩增出的DNA序列与GenBank中的LytA基因序列一致,成功构建了pET32a(+)-LytA重组质粒并在大肠埃希菌中高效表达,所表达的蛋白经Western blot证实为肺炎链球菌自溶素融合蛋白。结论成功克隆了肺炎链球菌自溶素基因,并将其在大肠埃希菌中表达、纯化,为新型肺炎链球菌疫苗的研制奠定了基础。Objective To clone the major autolysin（LytA） gene of Streptococcus pneumoniae and express it in E. coli BL21 （DE3） ,and to purify the recombinant LytA fusion protein. Methods The LytA gene was amplified from the total DNA of Streptococcus pneumoniae by PCR and was inserted into prokaryotic expressing vector pET32a（ ＋ ） to construct pET32a（ ＋ ） - LytA recombinant plasmid . The expression of LytA in BL21 （DE3） was induced by IPTG. The expressed protein was purified by affinity chromatography and was identified by Western blot assay. Results The DNA sequence of the cloned LytA gene was consistent with GenBank data. The recombinant plasmid pET 32a（ ＋ ） - LytA was constructed and expressed 56 000 fusion protein successfully. The specificity of autolysin fusion protein was verified by Western blot. Conclusions The LytA gene of Streptococcus pneumoniae has been successfully cloned and expressed in E. coli BL21 （DE3）,which paves a way for further studies on new generation pneumococcal vaccines.

关 键 词：肺炎链球菌 自溶素基因 克隆和表达 

分 类 号：R764.2[医药卫生—耳鼻咽喉科]