结核分枝杆菌耐受表面活性物质SDS的基因Rv0621的性质及功能的深入研究

发布时间：2018-04-15 23:24

本文选题：结核分枝杆菌 + SDS　；参考：《西南大学》2012年硕士论文

【摘要】：结核病一直在全球广泛流行,严重危害着人类健康,其病原菌——结核分枝杆菌(Mycobacterium tuberculosis, MTB)在1882年被德国科学家科赫首次鉴定。20世纪40年代以后,随着抗结核药物的问世和卡介苗的预防接种,结核病的发病率和死亡率均明显下降。但随着耐药菌株的出现、人口老龄化、人类免疫缺陷病毒感染者的增多和免疫抑制剂的应用日益普遍,结核病又呈蔓延趋势。结核分枝杆菌进入人体后可能会引起原发性结核病,也有可能会在人体内持续感染而不引起被感染者明显的症状,当宿主免疫力低下时,潜伏性结核会被激活为活动性结核。结核分枝杆菌的持续感染与其有效抵抗宿主体内的各种胁迫因素有关。结核分枝杆菌经呼吸道进入人体肺部时,将面临多种不利因素的胁迫,例如氧化压力、营养缺乏、低氧、低pH值、肺部表面活性物质等。为了研究结核分枝杆菌抵抗肺部表面活性物质胁迫相关基因,实验室前期工作利用大肠杆菌基因组文库筛选到了抵抗肺部表面活性物质类似物十二烷基硫酸钠SDS的相关基因Rv0621,该基因编码结核分枝杆菌的一个37kDa蛋白,经疏水性分析,推测其为具有四个跨膜区的跨膜蛋白,并具有ATP/GTP结合位点的保守结构域。我们在结核分枝杆菌的无毒模式菌株——耻垢分枝杆菌(Mycobacterium smegmatis)中进行了Rv0621的性质及功能研究,以期寻找Rv0621抵抗肺部表面活性物质的机理。本研究中,从GenBank数据库中获得MTB H37Rv Rv0621的核苷酸序列,设计引物,以MTB H37Rv全基因组为模板,体外扩增获得Rv0621基因,将PCR产物连接至pMD19-T,然后亚克隆至大肠杆菌表达质粒pET32a(+)和大肠杆菌-分枝杆菌穿梭质粒pNIT(myc),经质粒双酶切及测序证明pET32a(+)-Rv0621和pNIT(myc)-Rv0621重组质粒构建成功。将重组质粒分别转化入大肠杆菌和耻垢分枝杆菌,分别用IPTG和己内酰胺进行诱导表达,用SDS-PAGE和Western-Blot检测了重组蛋白的表达。为了验证Rv0621编码的蛋白是否会增加耻垢分枝杆菌在SDS下的存活率,用MTT法研究了重组菌在0.5%SDS下的存活能力。结核分枝杆菌作为一种重要的病原微生物,其细胞被膜有着独特的组成和结构,可以通过屏蔽或外排药物导致该菌的耐药性,所以我们用药敏法研究了Rv0621编码的这一膜蛋白对耻垢分枝杆菌耐受结核药物能力的影响。SDS能破坏结核分枝杆菌富含脂肪酸的细胞被结构,Rv0621可能会改变宿主的脂肪酸组分以应对这一破坏,所以我们通过气相色谱检测了重组菌与空载对照的脂肪酸组成差异并检测了导致该差异的基因转录水平。实验结果显示：Rv0621在大肠杆菌和耻垢分枝杆菌中成功异源表达,Rv0621过表达对耻垢分枝杆菌的生长速率影响不显著,重组耻垢分枝杆菌在0.5%SDS下的存活率增加,对结核药物尤其是利福平的耐受增加,重组耻垢分枝杆菌和空载对照菌的脂肪酸组分存在较大差异,重组耻垢分枝杆菌的十六烷酸和十八烷酸减少,相应的不饱和脂肪酸十六碳烯-9-酸和十八碳烯-9-酸增加,经RT-PCR验证,发现引起该脂肪酸变化的酶的编码基因MSMEG2938和MSMEG5248的转录水平上调。肺泡表面活性物质能破坏结核分枝杆菌富含脂肪酸的细胞被结构,而不饱和脂肪酸是微生物的重要组分,在微生物抵抗外界压力中发挥着重要作用。我们推测Rv0621重组耻垢分枝杆菌不饱和脂肪酸的增加可能会改变其细胞膜的成分及功能,从而应对SDS对耻垢分枝杆菌细胞膜产生的破坏,并对结核药物产生屏蔽或外排,影响其进入细胞发挥作用。从实验结果推测,Rv0621编码的蛋白是一个有意义的分子,对其继续深入研究可以揭示结核分枝杆菌对肺部表面活性压力耐受的机制,为开发新的抗结核靶标提供思路与基础。
[Abstract]:Tuberculosis has been widely popular in the world, serious harm to human health, the pathogen Mycobacterium tuberculosis (Mycobacterium tuberculosis, MTB) in 1882 by German scientist Koch first identified.20 century in 40s, with the advent of anti tuberculosis drugs and BCG vaccination, TB incidence and mortality were significantly decreased. With the emergence of resistant strains, the aging of the population, the increasing application of human immunodeficiency virus infection and immune inhibitors have become more common, and this trend is spreading. Tuberculosis mycobacterium tuberculosis into the human body may cause primary tuberculosis, there may be persistent infection in the body but not caused by infection symptoms and when the host immunity, latent tuberculosis will be activated for active tuberculosis. Persistent infection of Mycobacterium tuberculosis and its effective against host All kinds of stress factors are involved. Mycobacterium tuberculosis will face various adverse factors such as oxidative stress, nutritional deficiency, hypoxia, low pH value, pulmonary surfactant and so on.
In order to study the resistance of Mycobacterium tuberculosis pulmonary surfactant stress related genes, the previous work using the library to screen the Escherichia coli genome Rv0621 gene related to resistance in pulmonary surfactant analogues twelve sodium dodecyl sulfate SDS, a gene encoding the 37kDa protein of Mycobacterium tuberculosis, by the analysis of hydrophobicity, the transmembrane protein with four the transmembrane region, and has a conserved ATP/GTP binding site. We are non-toxic pattern of Mycobacterium smegmatis strains of Mycobacterium tuberculosis (Mycobacterium smegmatis) in the nature and function of Rv0621, find the Rv0621 resistance mechanism of pulmonary surfactant in order.
In this study, MTB H37Rv Rv0621 acquired the nucleotide sequence from GenBank database to design primers, MTB H37Rv genome as template, Rv0621 gene was amplified by PCR, the PCR products were connected to the pMD19-T, and then cloned into Escherichia coli expression vector pET32a (+) and Escherichia coli Mycobacterium shuttle plasmid pNIT (myc) the plasmid, restriction enzyme digestion and sequencing confirmed that the pET32a (+) -Rv0621 and pNIT (myc) -Rv0621 recombinant plasmid was successfully constructed. The recombinant plasmids were transformed into Escherichia coli and Mycobacterium smegmatis, respectively using IPTG and caprolactam induced expression of recombinant protein was detected by SDS-PAGE and Western-Blot. In order to verify whether the survival rate Rv0621 encoding the protein will increase in Mycobacterium smegmatis SDS, recombinant strains in 0.5%SDS were investigated by MTT. The viability of Mycobacterium tuberculosis as an important pathogen, the Cell membrane has a unique composition and structure, can lead to drug resistance of the bacteria by shielding or efflux of drugs, so we use this method to study the sensitivity of membrane protein Rv0621 encoding effect on Mycobacterium smegmatis tolerance ability of.SDS tuberculosis drugs can destroy the Mycobacterium tuberculosis cells are rich in fatty acids, fat acid group Rv0621 may change the host to respond to this damage, so we detected by gas chromatography of fatty acid composition and the difference of recombinant vector control and detection the gene transcription level of the difference.
The experimental results showed that Rv0621 in Escherichia coli and Mycobacterium smegmatis successfully heterologous expression, effect of Rv0621 overexpression on the growth rate of Mycobacterium smegmatis was recombinant Mycobacterium smegmatis 0.5%SDS increased the survival rate of tuberculosis, especially rifampin resistance increased by recombinant Mycobacterium smegmatis and empty the control group were divided into fatty acid differences, sixteen alkyl acid of recombinant Mycobacterium smegmatis and eighteen acid reduced, corresponding unsaturated fatty acids increased sixteen carbon and eighteen carbon ene ene -9- acid, -9- acid, RT-PCR verification, found that the change of fatty acid enzyme encoding genes MSMEG2938 and MSMEG5248 the transcription level increased. The pulmonary surfactant can destroy Mycobacterium tuberculosis cells are rich in fatty acids, and unsaturated fatty acid is an important group of microorganisms, the microbial resistance to external pressure in Play an important role. We speculate that Rv0621 recombinant Mycobacterium smegmatis increased unsaturated composition and function may change the cell membrane fatty acids, which deal with SDS of Mycobacterium smegmatis cell membrane damage, and shielding or efflux of TB drugs affect their entry into cells. Inferred from experiments the Rv0621 encoding protein is a molecular, the further research can reveal the mechanism of Mycobacterium tuberculosis on the lung surface active pressure tolerance, provide ideas and basis for the development of new anti tuberculosis targets.

【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R378

【参考文献】