上海交通大学学报(医学版) ›› 2017, Vol. 37 ›› Issue (7): 930-.doi: 10.3969/j.issn.1674-8115.2017.07.008

• 论著(基础研究) • 上一篇    下一篇

分枝杆菌噬菌体重组体 TM4RpfE#br# 分枝杆菌噬菌体重组体 TM4RpfE的构建#br#

杜丽娟 1,杨婷 1,徐莉 1,邢爱英 2,刘忠泉 2,张宗德 2#,郭述良 1#   

  1. 1. 重庆医科大学附属第一医院呼吸与危重症医学科,重庆 400016;2. 北京市结核病胸部肿瘤研究所,北京 101100
  • 出版日期:2017-07-28 发布日期:2017-08-25
  • 通讯作者: 郭述良,电子信箱:guosl999@sina.com。张宗德,电子信箱:zzd417@163.com。# 为共同通信作者
  • 作者简介:杜丽娟(1990—),女,土家族,硕士生;电子信箱:13883721437@163.com
  • 基金资助:
    国家自然科学基金(81570010)

Construction of recombinant mycobacteriophage TM4RpfE#br#

DU Li-juan1, YANG Ting1, XU Li1, XING Ai-ying2, LIU Zhong-quan2, ZHANG Zong-de2#, GUO Shu-liang1#   

  1. 1. Department of Respiratory and Critical Care Medicine, the First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China; 2. Beijing Institute of Tuberculosis and Chest Cancer, Beijing 101100, China
  • Online:2017-07-28 Published:2017-08-25
  • Supported by:
    National Nature Science Foundation of China, 81570010

摘要: 目的 · 构建分枝杆菌噬菌体(TM4)与复苏促进因子(Rpf)的重组体(TM4-RpfE),为联合抗结核药物实现杀灭结核休眠菌、 缩短结核化疗疗程奠定实验基础。方法 · 电转化法将 pJV53 质粒转入耻垢分枝杆菌,制备重组工程菌;提取 TM4 基因组 DNA;重叠 PCR 扩增目的融合基因 hsp60-RpfE,多步重叠 PCR 扩增插入长片段 HHRH(homologous + hsp60-RpfE + homologous);电转化法将噬 菌体 DNA 与插入长片段 HHRH 共同转入重组工程菌,挑取单噬菌斑进行 PCR 和测序验证;SDS-PAGE 分析重组噬菌体的蛋白表达。 结果 · 重叠 PCR 扩增出全长 901 bp 大小的目的融合基因 hsp60-RpfE 和 1 873 bp 大小的插入长片段 HHRH;PCR 验证重组后的噬菌斑, 分别在 955 bp 和 301 bp 处出现特异性条带;SDS-PAGE 分析显示重组型噬菌体在 21 000 处出现特异性蛋白条带。结论 · 分枝杆菌噬 菌体重组体 TM4-RpfE 构建成功,初步验证目的基因 RpfE 得到表达。

关键词: 分枝杆菌噬菌体, 噬菌体重组, RpfE, pJV53 质粒, 潜伏结核感染

Abstract: Objective · To construct recombinant mycobacteriophage TM4-RpfE to lay a foundation for experimental research about how to eradicate Mycobacterium tuberculosis in combination with anti-tuberculosis drugs, and how to shorten treatment for tuberculosis ultimately.  Methods · Electrotransformation was used to introduce pJV53 plasmid into Mycobacterium smegmatis to prepare recombinant engineering bacteria. After amplification of hsp60-RpfE fusion gene by overlap PCR, a long gene fragment (homologous +hsp60-RpfE+homologous, HHRH) was amplified by multi-step overlap PCR. The DNA of mycobacteriophage TM4 and HHRH fragment were cotransfected  into the recombinant engineering bacteria by electrotransformation, then the recombinant phage from the single primary plaques were confirmed by PCR and sequencing. SDS-PAGE was used to analyze the protein expression in recombinant phage.  Results · The hsp60-RpfE fusion gene at the length of 901 bp and HHRH fragment at the length of 1 873 bp were identified by overlap PCR. The PCR product produced 955 bp and 301 bp DNA bands in the first generation plaques colony. SDS-PAGE analysis showed a specific protein band at 21 000 in the recombinant phages.  Conclusion · The recombinant mycobacterium phage TM4-RpfE was successfully constructed and the expression of target gene RpfE was initially verified.

Key words: mycobacteriophage, phage recombination, RpfE, pJV53 plasmid, latent tuberculosis infection