分枝杆菌噬菌体D29 holin基因的克隆及其编码蛋白的生物信息学分析

Cloning and bioinformatic analysis of the gene encoding Holin protein from mycobacteriophage D29 holin

目的研究分枝杆菌噬菌体D29holin基因编码蛋白的理化特点及生物学特性,分析噬菌体抗结核菌的潜力。方法根据GenBank中登录的分枝杆菌噬菌体D29holin基因序列设计上、下游引物,以分枝杆菌噬菌体D29基因组为模板,PCR扩增holin基因,构建重组质粒pET32a-holin,进行双酶切鉴定、测序鉴定及生物信息学分析。结果 PCR扩增得到序列正确的holin基因产物并成功构建重组质粒pET32a-holin。进化分析显示分枝杆菌噬菌体D29holin基因与已知有尾分枝杆菌噬菌体Chy1、Chy4、Chy5holin基因亲缘关系较近。生物信息学分析显示,该基因编码Holin蛋白为稳定、疏水性蛋白,具有2个跨膜区域和亲水性C-端;二级结构中以α-螺旋和无规则卷曲为主,有信号肽,蛋白序列中存在2个丝氨酸磷酸化位点。结论分枝杆菌噬菌体D29 Holin蛋白的两个跨膜区及亲水性C-端构象发生变化,有助于阐明Holin"定时"打孔及启动噬菌体裂解细菌的机制,为研发抗结核Holin多肽药物奠定了基础。

Objectives To study the physicochemical characteristics and biological properties of the Holin protein encoded by the holin gene from mycobacteriophage D29 and to analyze the potential use of the phage in tuberculosis control.Methods Specific primers were designed for the mycobacteriophage D29 holin gene sequence registered in GenBank.The target segment was amplified with PCR using the mycobacteriophage D29 genome as a template.A recombinant plasmid pET32a-holin was successfully constructed and identified as correct according to PCR,restriction enzyme digestion,and sequencing.Bioinformatic data were analyzed with bioinformatics software.Results holin was cloned and amplified with PCR and its sequence was identified as correct,and the recombinant plasmid pET32a-holin was successfully constructed.Analysis of the phylogenetic tree of the mycobacteriophage indicated that the holin gene of mycobacteriophage D29had100% sequence similarity to the holin gene of mycobacteriophages Chy1,Chy4,and Chy5,indicating a close genetic relationship. Results of bioinformatics analysis indicated that the Holin protein encoded by holin is a stable and hydrophobic protein with two transmembrane domains and a hydrophilic C terminal.The protein sequence contains a signal peptide and two serine phosphorylation sites,and the secondary structure of the protein mainly consists of alpha helices and random coils. Conclusion The conformation of the two transmembrane regions and the hydrophilic C-terminal of the Holin protein is transformed.This finding will help clarify how Holin mediates ＂timed＂ membrane perforation and how Holin promotes the lysis of bacteria by mycobacteriophage D29.This work has laid the foundation for the development of antituberculosis drugs containing the polypeptide Holin.