Study on the construction and virulence observation of Rv2346c gene knockout strains of Mycobacterium tuberculosis mediated by bacteriophage

中华传染病杂志 Pub Date : 2018-08-15 DOI:10.3760/CMA.J.ISSN.1000-6680.2018.08.007

Xiaolin Chen, Sixia Chen, Jing Yao, Lei Shu, Kaili Deng

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引用次数: 1

Abstract

Objective To investigate the Rv2346c gene function through constructing Rv2346c gene knockout strains of Mycobacterium tuberculosis (M. tuberculosis) mediated by bacteriophage and observing its virulence after infecting mice lung tissue in vivo. Methods The affinal exchange sites (AES) of the target gene was built, and then integrated into the phage genomes of M. tuberculosis for harvesting the phagemids. The phagemids was imparted into Mycobacterium smegmatis to get recombinant phages with the same AES. A high titer of the recombinant phages was harvested through amplification in vitro. The M. tuberculosis was transfected and coated on solid medium with hygromycin resistance and cultured for 4 weeks at 37℃. Single clone was picked out and gene knock-out was confirmed by PCR. Then C57BL/6J mice were infected with either wild type strain (WT) or knockout strain (KO) of M. tuberculosis. Mice mortality, lung tissue inflammation and colony-forming units (CFU) counts in vitro were observed 6 to 8weeks post infection with different strains. Paired-samples t test was used for comparison between groups, chi-square test was used for comparison of rates. Results The products of PCR and inserted fragment sizes were consisted with the expectation and confirmed to be the target gene. The target fragment of Rv2346c was removed successfully and the mice were infected for 6-8 weeks. The mice infected with Rv2346c KO strain had reduced mortality (53% vs 20%, χ2=6.1112, P<0.05), lung tissue inflammation (1 040±89 vs 1 960±56, t=7.101 6, P<0.05) and CFU count in vitro (15.0±0.8 vs 90.0±1.5, t=23.036 1, P<0.05) compared with WT strain 6-8 weeks post infection. Conclusion Rv2346c gene knockout strains of M. tuberculosis mediated by bacteriophageis are successfully constructed, which establishes the foundation for the future gene function study of Rv2346c. Key words: Bacteriophages; Mycobacterium tuberculosis; Gene knockout strains; Rv2346c gene

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噬菌体介导结核分枝杆菌Rv2346c基因敲除菌株的构建及毒力观察

目的通过噬菌体介导构建结核分枝杆菌Rv2346c基因敲除株，并在体内观察其感染小鼠肺组织后的毒力，探讨其基因功能。方法构建靶基因的亲和交换位点（AES），并将其整合到结核分枝杆菌噬菌体基因组中以获得噬菌体。将噬菌体导入耻垢分枝杆菌中，得到具有相同AES的重组噬菌体。通过体外扩增获得了高滴度的重组噬菌体。将结核分枝杆菌转染并包被在具有潮霉素抗性的固体培养基上，在37℃下培养4周。筛选出单个克隆，并通过PCR确认基因敲除。然后用结核分枝杆菌的野生型菌株（WT）或敲除菌株（KO）感染C57BL/6J小鼠。在不同菌株感染后6至8周，观察小鼠死亡率、肺组织炎症和体外集落形成单位（CFU）计数。配对样本t检验用于组间比较，卡方检验用于比率比较。结果PCR产物和插入片段大小符合预期，确定为目标基因。Rv2346c的靶片段被成功去除，并且小鼠被感染6-8周。感染Rv2346c-KO株的小鼠在感染后6-8周的死亡率（53%vs 20%，χ2=6.1112，P<0.05）、肺组织炎症（1040±89 vs 1960±56，t=7.1016，P<0.05）和体外CFU计数（15.0±0.8 vs 90.0±1.5，t=23.036 1，P<0.05）均低于WT株。结论成功构建了噬菌体介导的结核分枝杆菌Rv2346c基因敲除菌株，为进一步研究Rv2346c基因功能奠定了基础。关键词：噬菌体；结核分枝杆菌；基因敲除菌株；Rv2346c基因

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来源期刊

中华传染病杂志

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5280

期刊介绍： The Chinese Journal of Infectious Diseases was founded in February 1983. It is an academic journal on infectious diseases supervised by the China Association for Science and Technology, sponsored by the Chinese Medical Association, and hosted by the Shanghai Medical Association. The journal targets infectious disease physicians as its main readers, taking into account physicians of other interdisciplinary disciplines, and timely reports on leading scientific research results and clinical diagnosis and treatment experience in the field of infectious diseases, as well as basic theoretical research that has a guiding role in the clinical practice of infectious diseases and is closely integrated with the actual clinical practice of infectious diseases. Columns include reviews (including editor-in-chief reviews), expert lectures, consensus and guidelines (including interpretations), monographs, short monographs, academic debates, epidemic news, international dynamics, case reports, reviews, lectures, meeting minutes, etc.