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结核分枝杆菌Rv3671c基因的克隆表达及免疫印迹检测 被引量:1
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作者 蒯守刚 崔振玲 +5 位作者 黄利华 刘忠华 麦广良 裴豪 刘君 何琳静 《临床检验杂志》 CAS CSCD 北大核心 2012年第11期906-908,共3页
目的构建结核分枝杆菌Rv3671c蛋白的重组质粒,在大肠埃希菌中表达并纯化重组蛋白Rv3671c,并评价结核分枝杆菌Rv3671c结构域多肽的免疫学特性。方法将编码结核分枝杆菌Rv3671c蛋白的基因克隆到pET-28a载体,并在大肠埃希菌中表达,镍亲和... 目的构建结核分枝杆菌Rv3671c蛋白的重组质粒,在大肠埃希菌中表达并纯化重组蛋白Rv3671c,并评价结核分枝杆菌Rv3671c结构域多肽的免疫学特性。方法将编码结核分枝杆菌Rv3671c蛋白的基因克隆到pET-28a载体,并在大肠埃希菌中表达,镍亲和层析和离子层析法纯化重组Rv3671c蛋白,透析复性和Lowry法测定蛋白质浓度。对蛋白质悬液进行SDS-PAGE分析并进行免疫印迹检测。结果结核分枝杆菌Rv3671c蛋白在大肠埃希菌中成功表达,层析纯化获得纯度为95.0%的重组Rv3671c蛋白,免疫印迹检测显示重组表达蛋白与结核病患者混合血清具有良好亲和性。结论 Rv3671c结构域多肽可作为新的结核病血清学检测方法的候选抗原肽组分。 展开更多
关键词 结核分枝杆菌 结核 Rv3671c蛋白 抗原 细菌蛋白质类
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基于结核分枝杆菌牛变种C68001随机突变体库筛选和鉴定成膜相关基因
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作者 时文健 徐磊 +5 位作者 张泽 杨蕊 辛凌翔 王楠 陈祥 鑫婷 《畜牧兽医学报》 北大核心 2025年第8期3992-4006,共15页
旨在利用结核分枝杆菌牛变种C68001菌株的突变体文库,高通量筛选鉴定影响生物膜形成的关键基因。本研究利用结核分枝杆菌牛变种C68001菌株的突变体文库,用苏通培养基进行培养,筛选生物膜表型差异明显的突变株,通过抗性标记挽救法及测序... 旨在利用结核分枝杆菌牛变种C68001菌株的突变体文库,高通量筛选鉴定影响生物膜形成的关键基因。本研究利用结核分枝杆菌牛变种C68001菌株的突变体文库,用苏通培养基进行培养,筛选生物膜表型差异明显的突变株,通过抗性标记挽救法及测序,确定突变基因位置;检测突变基因对菌株抗氧化性及抗SDS性能的影响;进一步以噬菌体系统构建靶标基因缺失、回补及过表达菌株,利用扫描电子显微镜检测靶基因对细菌菌丝影响,通过测定细菌的抗酸、抗氧化、抗SDS以及在小鼠巨噬细胞系J774A.1中的存活性能,检测靶标基因对细菌抗逆性和胞内存活的影响。利用突变体文库,筛选到8个生物膜形成缺陷的菌株,鉴定其中4个为单基因突变菌株,突变基因分别为Rv1096、Rv3425、Rv 3136和Rv 3671c;构建Rv 3671c基因敲除、回补和过表达菌株,通过PCR及Western blot验证构建成功,测试其生物膜形成能力、抗逆性和胞内存活性能,发现Rv 3671c基因可提高结核分枝杆菌牛变种抗SDS、生物膜形成及胞内存活等方面的能力。Rv 3671c基因在结核分枝杆菌牛变种C68001的生物膜形成和抗逆性能力中发挥重要作用。构建的结核分枝杆菌牛变种C68001菌株突变体文库可高效用于靶标基因的筛选和鉴定,为探索结核分枝杆菌牛变种的基因功能提供了可靠的工具。 展开更多
关键词 结核分枝杆菌牛变种 Himar 1转座子 随机插入突变库 生物膜 Rv 3671c
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The Strengths, Weaknesses, Opportunities and Threats (SWOT) Analysis of <i>Mycobacterium tuberculosis</i>: A Systematic Review 被引量:1
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作者 Michael Oluyemi Babalola 《Journal of Tuberculosis Research》 2015年第4期184-205,共22页
Background: Mycobacterium tuberculosis, the etiology of pulmonary and extra pulmonary tuberculosis is taunted to have predated the existence of mankind, and science has elucidated its presence in old Egyptian’ mummie... Background: Mycobacterium tuberculosis, the etiology of pulmonary and extra pulmonary tuberculosis is taunted to have predated the existence of mankind, and science has elucidated its presence in old Egyptian’ mummies, as it continues to evade current antibiotic treatments, wreck the havoc and decimate human populations. Presented here, are the Strengths, Weaknesses, Opportunities and Threats (SWOT) analysis of Mycobacterium tuberculosis, and the first proposal for the application of this innovative concept in the field of Tuberculosis research, to proffer holistic platform, focused knowledge, and strategies at undermining the prowess of the tubercle bacilli and overcoming its scourge. Materials and Methods: A systematic review was carried out to mine data on the strengths, the weaknesses, the opportunities and threats to M. tb, by review of several publications using meaningful theme and specific search phrases on the subject. Results: Strengths of Mycobacterium tuberculosis include: possession of abundant cell wall mycothiol;M. tb is highly contagious and requires low infectious dose (ID50) to establish infection;ability to specifically target and replicate in the host’ macrophages;ability to establish extrapulmonary multiorgan involvement;dual polymorphism i.e. existence in both an actively replicating form as well as or latent state;assumption of variable metabolic states;delayed seeding from the lungs of the replicating bacteria cells to the mediastinal lymph nodes;delayed macrophage apoptosis prior to bacterial growth and ultimate cellular necrosis;ability to shift to glyoxylate pathway during lipid metabolism in lieu of glucose during persistence phase in the host. Weaknesses of M. tuberculosis include: the requirement for growth of a membrane protein called Rv3671c during in vivo replication for survival in the acidic milieu of the macrophages and phagosome;M. tb is a fastidious slow growing bacterium with long generation time;establishment of productive infection in less than 10% of infected subjects;the bacterium is strictly an intracellular aerobic pathogen;and variable bacteria level of adenosine triphosphate. Opportunities harnessed by M. tb include: development and spread of resistant strains owing to inadequate and inappropriate drug treatment;limited efficacy and use of BCG Vaccine;MDR-TB is under-diagnosed in children;pathogenic synergy of coinfection of the Human Immunodeficiency Virus (HIV) and Mycobacterium tuberculosis (MTB);difficulty of TB screening in HIV-infected persons;immune status of the host;immigration;slow response of the cellular immune response to M. tbwhich enables constant endogenous reinfection of the host;anatomical vulnerability of the lungs;aerosols by inspired air is crucial for latent tuberculosis infection. Threats to M. tuberculosis include: the development and use of sensitive combination of microbiological tests as the gold-standard in HIV infected patients;annual TB test;selective isolation of TB patients by reintroduction of sanatoria;prioritizing genomic drug targets;sustenance of the global TB funds;development of potent vaccine;live imaging using computer tomography and positron electron tomography to characterize active TB in lesions;development and application of Infecton for imaging deep seated infections. 展开更多
关键词 SWOT ANALYSIS Mycobacterium tuberculosis Mycothiols GLYOXYLATE Pathway Rv3671c HIV COINFECTION Lungs Vulnerability Live Imaging Infecton Vaccine
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