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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。
Objective To study the physicochemical properties and biological characteristics of mycobacterium phage D29holin gene and to analyze the potential of bacteriophage against M. tuberculosis. Methods The upstream and downstream primers were designed according to the sequence of Mycobacterium phage D29holin registered in GenBank. The holin gene was amplified by PCR using Mycobacterium phage D29 as a template, and the recombinant plasmid pET32a-holin was constructed. And bioinformatics analysis. Results The correct holin gene was obtained by PCR and the recombinant plasmid pET32a-holin was successfully constructed. Phylogenetic analysis showed that the Mycobacterium phage D29holin gene was closely related to Chy1, Chy4 and Chy5holin genes. Bioinformatics analysis showed that the gene encoding Holin protein was a stable, hydrophobic protein with two transmembrane domains and hydrophilic C-terminal. The secondary structure was dominated by α-helix and random coil. The signal peptide There are two serine phosphorylation sites in the protein sequence. Conclusion The changes of the two transmembrane domains and the hydrophilic C-terminal conformation of Mycobacterium phage D29 Holin protein are helpful to elucidate the mechanism of Holin “timing ” to punch and start bacteriophage lytic bacteria. Peptide drugs laid the foundation.