Isolation and analysis of two endolysins derived from a Mycobacterium smegmatis phage BTCU-1

• Main Authors: Meng-Xuan Tu, 杜孟軒
• Other Authors: Kai-Chih Chang
• Format: Others
• Language: zh-TW
• Published: 2014
• Online Access: http://ndltd.ncl.edu.tw/handle/ccfu66

碩士 === 慈濟大學 === 醫學檢驗生物技術學系醫學生物技術碩士班 === 102 === Tuberculosis is an infectious disease which caused by Mycobacterium tuberculosis. Effective therapies existed are limited by the emergence of multidrug- resistant tuberculosis (MDR-TB) and extensively drug resistant tuberculosis (XDR-TB). Effective therapeutic regimens exist that are limited by the emergence of the inability of antibiotics to kill dormant organisms. Previous studies pointed out that endolysins produced from well assembled phage can hydrolyze the peptidoglycan of cell wall. We have discovered a Mycobacterium smegmatis bacteriophage which genome size of this phage is 46Kb by full genome sequencing and 72 open reading frames were predicted. We selected two suspected endolysin genes named lysA and lysB, cloned into E.coli and expressed, purified the protein to analyze their effect of bactericidal assay. The bactericidal test result showed that LysA and LysB could effectively restrain smegmatis. Under the Scanning Electron Microscope, it was observed that LysA and LysB could directly destroy smegmatis. The cytotoxicity effect of LysA and LysB was determined in HaCaT cell lines by WST-1 assay in vitro, there is no difference was found between negative control. Intracellular bactericidal activity assay showed that treatment of M. smegmatis–infected, RAW 264.7 macrophage, with LysA or LysB, resulted in a significant reduction in the number of viable intracellular bacilli. These results indicate that BTCU-1 and its cloned biologically active lytic modules have antimycobacterial activity, and suggest that they are good candidates for a therapeutic/disinfectant agent to control mycobacterial infections.