结核分枝杆菌蛋白酶体辅助因子PafC在氟喹诺酮类药物莫西沙星耐受中的作用

发布时间：2018-06-02 02:53

本文选题：蛋白酶体辅助因子 + 分枝杆菌　；参考：《西南大学》2014年硕士论文

【摘要】：结核病是由结核分枝杆菌(Mycobacterium tuberculosis,简称结核菌)引起的全球性感染性疾病。据世界卫生组织报告,全球三分之一的人感染过结核菌,而仅在2012年结核病患者就有860万,全球结核病死亡人数高达130万。结核病疫情如此严重,除了其致病菌结核菌的独特致病特性外,多重耐药结核(multi-drug resistant tuberculosis, MDR-TB)和广泛耐药结核(extensively drug resistant tuberculosis,XDR-TB)的出现以及和结核菌与人类免疫缺陷病毒(human immunodeficiency virus,HIV)共感染,使得结核病疫情更加难以控制。目前治疗结核病的一线药物包括异烟肼(Isoniazid, INH).利福平(Rifampicin, RIF)、吡嗪酰胺(Pyrazinamide, PZA)以及乙胺丁醇(Ethambutol, EMB)对MDR-TB和XDR-TB患者无效。因此二线抗结核药物氨基糖苷类抗生素、多肽类抗生素和氟喹诺酮类抗生素作为MDR-TB和XDR-TB患者的主要药物。其中氟喹诺酮药物因具备低毒副作用和大量的衍生物的特点而倍受重视。然而,针对氟喹诺酮药物的耐药菌的不断出现使得结核病防控雪上加霜。因此,我们试图寻找分枝杆菌耐氟喹诺酮药物的新机制,将相关基因作为潜在的氟喹诺酮药物增效剂靶点,为解决MDR-TB和XDR-TB的药物开发提供基础。本实验之前的工作初筛到了一株对莫西沙星超敏感的耻垢分枝杆菌(Mycobacteirum smegmatis)突变株M371,并鉴定了转座子插入的位点。本课题将进一步验证突变株M371超敏感的表型及其机制,并初步探索突变株在胁迫反应中的作用。首先,采用8倍野生型耻垢分枝杆菌最小抑菌浓度(minimum inhibitory concentration, MIC)的莫西沙星处理耻垢分枝杆菌野生型和突变株M371,记录处理不同时间后的菌落形成单位(colony-forming unit,CFU),计算存活率并作杀菌曲线,验证突变株M371的莫西沙星超敏感表型。然后,为了研究基因MSMEG_3888在耻垢分枝杆菌在天然耐受(intrinsic resistance)莫西沙星中的作用,构建突变株M371的回补菌株。而MSMEG_3888结核菌H37Rv中的对应基因为Rv2095c。将基因Rv2095c克隆并连接到穿梭质粒pALACE上,并将重组质粒pALACE-Rv2095c电转化入突变株M371,获得回补菌株。并采用Western-blot检测回补菌株中基因Rv2095c的表达和MIC实验判断对莫西沙星的敏感性。最后,探索突变株M371、野生株和回补菌株在表面性质方面的差异,并以过氧化氢(hydrogen peroxide,H2O2)及十二烷基磺酸钠(Sodium Dodecyl Sulfonate, SDS)模拟肺泡的环境来研究三株菌的胁迫反应。实验结果表明：耻垢分枝杆菌突变株M371的经莫西沙星处理存活率比野生型低,为莫西沙星超敏感表型。通过菌液PCR验证和基因测序,证明了成功构建了回补菌株M371-pALACE-Rv2095c。Western-blot验证了Rv2095c异源表达成功,并且回补菌株与野生型具有相同的耐受能力。由此证明了基因MSMEG_3888参与对莫西沙星的本底耐受。在生物表面性质方面,耻垢分枝杆菌野生型、突变株M371和回补菌株,三者在单菌落形态和滑动能力方面无明显差异；但在脂质系数方面差异明显,突变株较野生型明显下降。三者在SDS耐受水平无显著差异,突变株较野生株在双氧水耐受水平亦无明显差异。三株菌在胁迫应答中无明显差异。因此,本研究证明了基因MSMEG_3888的参与天然耐受,基因MSMEG_3888并不影响耻垢分枝杆菌的表面性质,亦不参与胁迫应答。
[Abstract]:Tuberculosis is a global infectious disease caused by Mycobacterium tuberculosis . According to the World Health Organization , one - third of the world ' s people have been infected with tuberculosis , but only in 2012 there were 8.6 million TB patients . The number of tuberculosis deaths in the world was 1.3 million . The presence of multi - drug resistant tuberculosis ( MDR - TB ) and broad - resistant tuberculosis ( XDR - TB ) , and tuberculosis and human immunodeficiency virus ( HIV ) , were more difficult to control , in addition to their unique pathogenic characteristics .

Currently , first - line drugs for tuberculosis include Isophthalazide ( INH ) . Rifampicin , rif , pyrazinamide ( PZA ) and ethambutol ( emb ) are ineffective for MDR - TB and XDR - TB patients . Therefore , two - line anti - tuberculosis drugs , such as aminoglycosides , polypeptide antibiotics and fluoroquinolone antibiotics , are important drugs for MDR - TB and XDR - TB patients .

Therefore , we try to find a new mechanism of fluoroquinolone drug for Mycobacterium tuberculosis , which will provide the basis for the drug development of MDR - TB and XDR - TB .

The mutant M371 was cloned and ligated into the shuttle plasmid pALACE . The results showed that the mutation strain M371 was cloned and ligated into the shuttle plasmid pALACE , and then the recombinant plasmid pALACE - Rv95c was transformed into the shuttle plasmid pALACE .

The results showed that the survival rate of M371 of Mycobacterium smegmatis mutant strain M371 was lower than that of wild type , which was more sensitive than wild type .
However , there was no significant difference between mutant strain and wild - type strain at SDS - tolerance level . There was no significant difference in the tolerance level between mutant strain and wild strain . There was no significant difference in the stress response .

Therefore , the study demonstrated that the gene MSV3888 was not involved in the surface properties of Mycobacterium smegmatis , nor was it involved in the stress response .
【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R52

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