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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 the predicted fragment of bacteriophage D29 lyase gene in Mycobacterium smegmatis and identify its cell wall lysis activity. Methods: Based on the analysis of open reading frame of mycobacterium phage D29, gene10 with higher homology to lytic enzyme was amplified by PCR from phage D29 genomic DNA and cloned into E.coli - Bacillus shuttle plasmid, induced expression in Mycobacterium smegmatis. The expression products were detected by SDS-PAGE, turbidity of induced recombinant strains were measured, and transmission electron microscopy was used to identify the lysis activity. RESULTS AND CONCLUSION: The recombinant shuttle plasmid pUV-gene10 was successfully constructed and gene10 was expressed in Mycobacterium smegmatis. The expression product can effectively reduce the turbidity of the host culture medium and degrade the cell wall of the host bacteria, and confirm that the gene10 product is phage lysis Enzymes, and presumably it is a non-canonical secreted protein.