Protein purification and bacterial inhibition analysis of ORF693 of phage fkm18p

• Main Authors: Jen-Yi Wang, 王貞懿
• Other Authors: Chih-Hsin Hung
• Format: Others
• Language: zh-TW
• Published: 2014
• Online Access: http://ndltd.ncl.edu.tw/handle/y389sv

碩士 === 義守大學 === 化學工程學系暨生物技術與化學工程研究所 === 102 === Acinetobacter baumannii is an acidiphilic, obligate, aerobic and gram-negative bacterium. It is an opportunistic pathogen and people will fall to ill when they were under weak immunity. We have isolated several lytic bacteriophages from the environment to lysis A. baumannii and the km18p has the highest lytic efficiency. Phage km18p provided 100% protection and survival rate of mice that were inoculated with A. baumannii KM18. So this study will focus on clonong the lysin gene and detecting the bacterial inhibition efficiency of the expressed proteins. On this study, the lysin gene orf693 were cloned to pET21b to from the expression plasmid pEorf693. Plasmid pEorf693 was transferred to E. coli C43 (DE3) and induced by IPTG to over-expression. The induced protein showed the molecular weight about 70 kDa in SDS-PAGE, but it did not has lytic activity. If the purified proteins didn''t denatured and protein patterns with lysis activity were identified with zymogram method, we found that the protein pattern with 210 kDa molecular weight showed the lysis clear zone. We speculated that the translated lysin proteins were assembly as trimers structure in bacterium. When bacterium lysis activity was analysis with the paper disc, 1 μg/ml purified protein would present the obvious lysis activity and the diameter of inhibition zone would also increase during 120 hr. In the minimum inhibitory concentration (MIC) assay, purified proteins with minimum concentration of 0.5 mg/ml would inhibit the growth of A. baumannii KM18.