噬菌体AB3裂解酶的抗菌谱及其抗菌机制

Antibacterial Spectrum and Antibacterial Mechanism of Bacteriophage AB3 Endolysin

目的明确噬茵体AB3裂解酶LysAB3的抗茵谱及其抗菌机制。方法利用扩散法检测裂解酶LysAB3的抗茵谱;扩增裂解酶LysAB3基因序列前端的354 bp以pET28a(+)为载体构建重组质粒,在大肠杆菌BL21(DE3)中表达,Ni^(2+)-NTA亲和层析柱纯化重组蛋白,得到删除了两性多肽结构的裂解酶LysAB3-D,并测定其抗茵率。结果裂解酶LysAB3可以裂解全部40株鲍曼不动杆菌菌株;删除了两性多肽结构后,裂解酶LysAB3-D的抗菌率由95.8%下降至33.3%。结论裂解酶LysAB3的抗茵谱较宽;其杀菌机制可能是通过其两性多肽结构增加细菌外膜通透性,辅助酶解催化域进入其中降解肽聚糖。

Objective To investigate the antibacterial spectrum and antibacterial mechanism of LysAB3. Method The gel dif- fusion assay is utilized to determine the antibacterial spectrum of LysAB3. The recombinant plasmid is constructed by inser- ting 354 bp in front of LysAB3 gene into the vector pET28a（ ＋ ）. After expression in E. coli BL21 （ DE3 ） and purification with Ni2 ＋ -NTA sepharose, LysAB3-D is acquired with lack of amphiphilic peptides structure, with the antibacterial activity determined. Results The gel diffusion assay showed that LysAB3 had bactericidal effects against 40 strains of Acinetobacter Baumannii. After the amphiphilic peptides structure was deleted, the antibacterial activity of LysAB3-D dropped from 95. 8% to 33.3%. Conclusion These results indicate that the antibacterial spectrum of LysAB3 is abroad relatively and the am- phiphilic peptides structure of LysAB3 increase permeability of bacterial outermembrane,which helps the enzymatic catalytic domain degrade the peptidoglycan.