结核分枝杆菌Lrp基因(Rv2779c)敲除菌株的构建与功能初探  

Construction and function of an Lrp gene(Rv2779c)deletion mutant of Mycobacterium tuberculosis

作　　者：郑继芳 尚园园 鲍生娟 黄海荣[1] 陈素婷[1] ZHENG Jifang;SHANG Yuanyuan;BAO Shengjuan;HUANG Hairong;CHEN Suting(National Clinical Laboratory on Tuberculosis,Beijing Key Laboratory for Drug-resistant Tuberculosis Research,Beijing Chest Hospital Affiliated to Capital Medical University,Beijing Tuberculosis and Thoracic Tumor Institute,Beijing 101149,China)

机构地区：[1]国家结核病临床实验室,耐药结核病研究北京市重点实验室,首都医科大学附属北京胸科医院/北京市结核病胸部肿瘤研究所,北京101149

出　　处：《实用医学杂志》2023年第19期2428-2433,共6页The Journal of Practical Medicine

基　　金：北京市自然科学基金(编号:5192006);北京市医院管理中心“青苗”计划(编号:QML20201601);通州区运河人才计划(编号:YHLD2018030)。

摘　　要：目的为深入研究结核分枝杆菌Rv2779c基因的功能,构建针对该基因的敲除突变菌株。方法以结核分枝杆菌H37Rv标准菌株基因组DNA为模板,PCR扩增Rv2779c基因上下游大约500 bp左右的片段作为同源重组的左右臂,并将同源重组左右臂构建到p0004s穿梭质粒上,将p0004s同源重组质粒构建到phAE159质粒中形成噬菌粒,包装形成的噬菌体侵染H37Rv后将同源重组元件整合到基因组中,通过重组元件中的潮霉素抗性标记对阳性克隆进行筛选,同时利用左右臂扩增及内部片段扩增的原理,对抗性筛选阳性克隆进行鉴定。比较Rv2779c基因敲除突变株与H37Rv标准菌株在正常7H9完全培养基及含高浓度利福平培养基中的生长差异。结果成功构建了包含Rv2779c上下游序列同源重组臂的p0004s-ΔRv2779c重组质粒,并将该质粒与含有分枝杆菌温度敏感型元件的phAE159质粒连接获得重组噬菌粒,最后将包装成功的噬菌体侵染H37Rv获得抗性筛选阳性的克隆菌株,PCR表明结核分枝杆菌Rv2779c基因敲除成功。Rv2779c基因敲除对结核分枝杆菌在正常培养基中生长几乎没有影响,但显著降低了结核分枝杆菌在高浓度利福平培养基中的存活率。结论首次成功构建了结核分枝杆菌Rv2779c敲除菌株,为后续进一步研究Rv2779c基因的功能奠定了重要基础。初步研究发现该基因影响结核分枝杆菌在含利福平培养基中的持留生长。Objective To explore the function of Mycobacterium tuberculosis(Mtb)Rv2779c gene,an Rv2779c deletion mutant was constructed.Methods Using the genomic DNA of the Mtb reference strain H37Rv as the template,a 500 bp upstream and downstream fragment of Rv2779c gene for homologous recombination was amplified by PCR,and then constructed into the shuttle plasmid p0004s.The homologous recombinant plasmid p0004s was constructed into temperature-sensitive phasmid phAE159 and then packaged as phage.After the phage infection,the homologous recombinant element was integrated into the H37Rv genome.The positive clones were screened by hygromycin resistance marker in the recombinant element.The resistance-positive clones were identified by PCR.The growth differences between Rv2779c deletion mutant and H37Rv WT strain in 7H9 complete medium and high concentration rifampicin medium were compared.Results The recombinant plasmid p0004s-ΔRv2779c containing the upstream and downstream sequence of Rv2779c homologous recombinant arm was successfully constructed,and the plasmid was ligated with phAE159 which contained mycobacterium temperature sensitive element to obtain the recombinant phasmid.Finally,the successfully packaged phage infected H37Rv and the resistance positive clone was selected.PCR results showed that the Rv2779c gene of Mtb was knocked out successfully.The knockout of Rv2779c had little effect on the growth of Mtb in normal medium,but significantly reduced the survival of Mtb in high concentrations of rifampin.Conclusion The Rv2779c deleted strain of Mtb was successfully constructed,which lays an important foundation for the functional study of Rv2779c gene.And preliminary studies revealed that this gene affected the growth of Mtb in rifampicin-containing medium.

关 键 词：结核分枝杆菌 基因敲除 Lrp/AsnC家族蛋白 Rv2779c基因 

分 类 号：S852.61[农业科学—基础兽医学]