KL2型鲍曼不动杆菌噬菌体解聚酶克隆及抗菌活性分析

Cloning and antibacterial activity analysis of KL2 type Acinetobacter baumannii bacteriophage depolymerase

摘要:

[背景] 噬菌体解聚酶是噬菌体在裂解细菌过程中产生的一种抗菌蛋白，关于鲍曼不动杆菌荚膜分型及常见型别噬菌体解聚酶的研究报道较少。[目的] 以KL2型鲍曼不动杆菌为研究对象，从噬菌体IME-AB2中克隆解聚酶，在大肠杆菌中进行可溶性表达并研究其体外抗菌活性。[方法] 应用二代测序及生物信息学方法鉴定鲍曼不动杆菌荚膜型，分析IME-AB2全基因组。应用分子克隆技术克隆ORF76假定的尾丝蛋白（Putative Tail Fiber）基因，构建重组表达载体pEASY-Blunt-E1-gp76，在大肠杆菌BL21（DE3）中诱导表达，通过Ni-NTA亲和层析纯化解聚酶，研究解聚酶体外抗菌活性。[结果] 构建了pEASY-Blunt-E1-gp76解聚酶重组表达质粒，该重组质粒在大肠杆菌中得到可溶性表达；体外活性分析显示，该重组蛋白在体外能够对所有的KL2型鲍曼不动杆菌具有较好的抗菌活性，解聚酶联合人和狗的血清具有很好的杀菌活性。[结论] 鉴定解聚酶并提高其抗菌谱具有重要意义，也是噬菌体及解聚酶用于治疗耐药菌研究领域急需解决的重要问题之一。

Abstract:

[Background] Bacteriophage depolymerase is a kind of antibacterial protein produced by bacteriophage in the process of bacterial lysis. There are few reports on the capsule typing and common types of Acinetobacter baumannii bacteriophage depolymerases. [Objective] The KL2 type A. baumannii was selected as the research object. The depolymerase was cloned from the phage IME-AB2, and soluble expression was carried out in Escherichia coli, and its activity in vitro was studied. [Methods] A. baumannii capsular typing was identified through next generation sequencing and bioinformatics analysis. The IME-AB2 whole genome was analyzed. The putative tail fiber gene of ORF76 was cloned. The recombinant expression vector pEASY-Blunt-E1-gp76 was constructed and induced to express in E. coli BL21(DE3). The depolymerase was purified by Ni-NTA affinity chromatography and the gp76 antibacterial activity was studied in vitro. [Results] The recombinant plasmid of pEASY-Blunt-E1-gp76 was successfully constructed and expressed in E. coli. In vitro activity analysis was showed that the recombinant protein had good antibacterial activity against all KL2 type A. baumannii in vitro, and the depolymerase combined with human or dog serum had good bactericidal activity. [Conclusion] It is of great significance to identify depolymerase and improve its antibacterial spectrum, and it is also one of the important problems to be solved urgently in the field of bacteriophage and depolymerase used in the treatment of drug-resistant bacteria.