摘要
目的:在耻垢分枝杆菌中表达预测的噬菌体D29裂解酶基因片段,鉴定其细胞壁裂解活性。方法:在分析分枝杆菌噬菌体D29开放阅读框序列的基础上,用PCR法从噬菌体D29基因组DNA中扩增出与裂解酶同源性较高的序列gene10,将其克隆入大肠杆菌-分枝杆菌穿梭质粒后,在耻垢分枝杆菌中进行诱导表达。对表达产物进行SDS-PAGE检测,对诱导培养重组菌进行混浊度测定、透射电镜观察以鉴定其裂解活性。结果及结论:成功构建了重组穿梭质粒pUV-gene10并使gene10在耻垢分枝杆菌中获得表达,表达产物能有效降低宿主菌培养液混浊度,降解宿主菌细胞壁,证实gene10表达产物为噬菌体裂解酶,并推测其为非经典分泌蛋白。
Objective：To express mycobacteriophage D29 putative lysis gene in Mycobacterium smegmatis and identify its muralytic activity. Methods： Homological sequences of 89 ORFs encoded by mycobacteriophage D29 were blasted against DNA sequences in GenBank for possible functional identification, and ORF gene10 was found to be highly homological to lysins. This gene was cloned into E. coli-Mycobacterium shuttle plasmid pUV15tetORm and electroporated into M. smegmatis mc^2155. The expressed product was examined by SDS-PAGE. The lytic activity was identified by culture turbidity measurement and transmission electron microscopy （TEM）. Results and Conclusion： A recombinant shuttle plasmid pUV-gene10 was successfully constructed and gene10 was successfully expressed in Mycobacterium. The expressed product can effectively reduce the turbidity of host strain culture by hydrolyzing its cell wall. It is proved that gene 10 is a phage lysin, which is hypothesized to be a non-classical secretion protein.
出处
《军事医学科学院院刊》
CSCD
北大核心
2009年第2期113-116,共4页
Bulletin of the Academy of Military Medical Sciences
基金
国家科技重大专项(2008ZX100/03-016)
