分枝杆菌噬菌体CJAUS5株holin基因的克隆与序列分析

Cloning and Sequence Analysis of Mycobacteriophage CJAUS5holin

关键词:  分枝杆菌  噬菌体  holin  基因克隆  序列分析

Key words:  mycobacterium  bacteriophage  holin  gene cloning  sequence analysis

摘要:

为了深入研究噬菌体裂解宿主菌的机制，开发噬菌体裂解相关蛋白的应用价值，本研究参照 GenBank 中登录的 holin基因序列设计特异性引物,以分枝杆菌噬菌体CJAUS5基因组为模板,经PCR 扩增出411bp的条带。构建重组质粒pMD19－T-holin,经双酶切鉴定呈阳性后进行测序分析。结果显示,克隆出的holin基因与已知分枝杆菌肌尾噬菌体holin序列同源性高达99.03%～99.51%；编码的氨基酸同源性高达99%～100%。对holin基因编码的蛋白进行分析表明,Holin蛋白具有亲水性C端和两个跨膜区域。本研究成功克隆了分枝杆菌噬菌体CJAUS5的holin基因并进行了氨基酸序列分析,为进一步研究 Holin蛋白的生物学特性及功能奠定了基础。

Abstract:

In order to further research the mechanisms of phage lysis host bacteria and develop the value of phage lysis related protein.Specific primers were designed based on the holin gene sequences of mycobacteriophage deposited in GenBank.Target segment (about 411 bp in length) was amplified by PCR with Mycobacteriophage CJAUS5 genome as a template.The segment was cloned into pMD19-T vector and sequencing the positive recombinant plasmid pMD19-T-holin after BamH I and EcoR I digestion. The results show that holin gene was successfully cloned and 99.03% to 99.51% sequence homology with other myoviridae mycobacteriophages’s holin gene and 99% to 100% amino acid homology.The analysis results of the protein encoded by holin gene show that it is a protein with a hydrophilic carboxy-terminal domain and two potential transmembrane domains.This research successfully cloned the holin gene of mycobacteriophage CJAUS5 and analyzed the amino acid sequence which laid the foundation for the futher study of Holin protein．