多重耐药鲍曼不动杆菌噬菌体Abp1毒力蛋白穿孔素的无细胞融合表达

Fusion expression of multidrug-resistant Acinetobacter baumannii phage Abp1 holin(holinAbp1)in a cell-free protein synthesis system

目的研究多重耐药鲍曼不动杆菌噬菌体Abp1毒力蛋白穿孔素holin(holinAbp1)的生物学特性及其对细菌的毒性作用,并通过无细胞蛋白表达系统对毒力蛋白穿孔素融合蛋白进行表达。方法利用生物信息学技术分析holinAbp1的蛋白结构特点,利用PCR技术从噬菌体Abp1基因组扩增holinAbp1基因,构建表达载体pET23bholinAbp1-gfp并转化至大肠杆菌BL21(DE3)中以及无细胞蛋白体系中表达,利用Western印迹鉴定蛋白表达情况。结果噬菌体Abp1的holin蛋白属于稳定疏水性蛋白,含有3个跨膜区域,属于Phage-holin-3-1家族,成功克隆了holinAbp1-gfp基因,holinAbp1-gfp蛋白在大肠杆菌内无法正确表达,对大肠杆菌表达宿主具有明显抑制作用,而在无细胞蛋白表达系统实现了蛋白表达。结论通过无细胞蛋白表达系统实现了holinAbp1-gfp融合蛋白表达,将为毒力蛋白的表达和噬菌体治疗药物的研发提供应用基础和技术支持。

Objective To study the biological characterizatics and antibacterial activity of the holin protein encoded by the holin gene from the Abp1 phage,a virulent phage targeting the multidrug-resistant Acinetobacter baumannii strain,AB1,and to express the fusion protein of holin by cell-free protein synthesis system(CFPS).Methods Characteristics of the holinAbp1 protein were predicted with bioinformatics,the holinAbp1 gene was amplified by PCR,recombined plasmid pET23b-holinAbp1-gfp was constructed and expressed in E.coli BL21(DE3)and CFPS system.The expression of holinAbp1-gfp was confirmed by Western blotting analysis.Results HolinAbp1 was a stable and hydrophobic protein with three transmembrane domains and belonged to phage-holin-3-1 family.Recombined plasmid pET23 b-holinAbp1-gfp was constructed.HolinAbp1-gfp could not be expressed in E.coli BL21(DE3)and the expression of holin protein could significantly inhibit the proliferation of E.coli BL21(DE3).However,holinAbp1-gfp was expressed in CFPS system.Conclusion The holin CFPS system is expressed by fluorescent protein fusion.These results can provide technical support for studies on virulence protein expression and development of bacteriophage therapeutic drugs.