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目的明确两性多肽结构对鲍曼不动杆菌(AB)外膜通透性的影响。方法 PCR扩增LysAB3基因及删除两性多肽结构的LysAB3-D基因,以pET28a(+)为载体构建重组质粒,在大肠杆菌BL21(DE3)中表达LysAB3及LysAB3-D,金属离子螯合亲和层析法纯化重组蛋白。将AB分别经LysAB3及LysAB3-D处理后,用扫描电子显微镜观察菌体形态,荧光显微镜观察菌体中是否有绿色荧光的聚集。结果经LysAB3处理后的AB,在扫描电子显微镜下可见菌体表面粗糙、皱缩,部分裂解为碎片;在荧光显微镜下可见菌体内有绿色荧光聚集。而删除两性多肽结构的LysAB3-D作用AB后,却没有上述现象的发生。结论两性多肽结构可增加AB外膜通透性,有助于裂解酶进入其中,达到抗菌目的。
Objective To determine the effect of amphipathic peptide structure on the outer membrane permeability of Acinetobacter baumannii (AB). Methods The LysAB3-D gene was amplified by PCR and deleted. The recombinant plasmid pET28a (+) was constructed. The LysAB3 and LysAB3-D were expressed in E. coli BL21 (DE3) Purification of Recombinant Proteins. After treating AB with LysAB3 and LysAB3-D respectively, the morphology of the cells was observed with a scanning electron microscope, and whether or not there was green fluorescence in the cells was observed under a fluorescence microscope. Results The LysAB3-treated AB showed that the surface of the cell was rough, collapsed and partially lysed into fragments under a scanning electron microscope. Green fluorescence was observed in the cells under a fluorescence microscope. The deletion of amphipathic peptide structure of LysAB3-D AB, but no such phenomenon. Conclusion The amphipathic peptide structure can increase the permeability of AB outer membrane and help the lyase enter into it, achieving the purpose of antibacterial.