發(fā)布時(shí)間：2018-04-15 23:24

  本文選題：結(jié)核分枝桿菌 + SDS��； 參考：《西南大學(xué)》2012年碩士論文

【摘要】：結(jié)核病一直在全球廣泛流行,嚴(yán)重危害著人類健康,其病原菌——結(jié)核分枝桿菌(Mycobacterium tuberculosis, MTB)在1882年被德國(guó)科學(xué)家科赫首次鑒定。20世紀(jì)40年代以后,隨著抗結(jié)核藥物的問(wèn)世和卡介苗的預(yù)防接種,結(jié)核病的發(fā)病率和死亡率均明顯下降。但隨著耐藥菌株的出現(xiàn)、人口老齡化、人類免疫缺陷病毒感染者的增多和免疫抑制劑的應(yīng)用日益普遍,結(jié)核病又呈蔓延趨勢(shì)。結(jié)核分枝桿菌進(jìn)入人體后可能會(huì)引起原發(fā)性結(jié)核病,也有可能會(huì)在人體內(nèi)持續(xù)感染而不引起被感染者明顯的癥狀,當(dāng)宿主免疫力低下時(shí),潛伏性結(jié)核會(huì)被激活為活動(dòng)性結(jié)核。結(jié)核分枝桿菌的持續(xù)感染與其有效抵抗宿主體內(nèi)的各種脅迫因素有關(guān)。結(jié)核分枝桿菌經(jīng)呼吸道進(jìn)入人體肺部時(shí),將面臨多種不利因素的脅迫,例如氧化壓力、營(yíng)養(yǎng)缺乏、低氧、低pH值、肺部表面活性物質(zhì)等。 為了研究結(jié)核分枝桿菌抵抗肺部表面活性物質(zhì)脅迫相關(guān)基因,實(shí)驗(yàn)室前期工作利用大腸桿菌基因組文庫(kù)篩選到了抵抗肺部表面活性物質(zhì)類似物十二烷基硫酸鈉SDS的相關(guān)基因Rv0621,該基因編碼結(jié)核分枝桿菌的一個(gè)37kDa蛋白,經(jīng)疏水性分析,推測(cè)其為具有四個(gè)跨膜區(qū)的跨膜蛋白,并具有ATP/GTP結(jié)合位點(diǎn)的保守結(jié)構(gòu)域。我們?cè)诮Y(jié)核分枝桿菌的無(wú)毒模式菌株——恥垢分枝桿菌(Mycobacterium smegmatis)中進(jìn)行了Rv0621的性質(zhì)及功能研究,以期尋找Rv0621抵抗肺部表面活性物質(zhì)的機(jī)理。 本研究中,從GenBank數(shù)據(jù)庫(kù)中獲得MTB H37Rv Rv0621的核苷酸序列,設(shè)計(jì)引物,以MTB H37Rv全基因組為模板,體外擴(kuò)增獲得Rv0621基因,將PCR產(chǎn)物連接至pMD19-T,然后亞克隆至大腸桿菌表達(dá)質(zhì)粒pET32a(+)和大腸桿菌-分枝桿菌穿梭質(zhì)粒pNIT(myc),經(jīng)質(zhì)粒雙酶切及測(cè)序證明pET32a(+)-Rv0621和pNIT(myc)-Rv0621重組質(zhì)粒構(gòu)建成功。將重組質(zhì)粒分別轉(zhuǎn)化入大腸桿菌和恥垢分枝桿菌,分別用IPTG和己內(nèi)酰胺進(jìn)行誘導(dǎo)表達(dá),用SDS-PAGE和Western-Blot檢測(cè)了重組蛋白的表達(dá)。為了驗(yàn)證Rv0621編碼的蛋白是否會(huì)增加恥垢分枝桿菌在SDS下的存活率,用MTT法研究了重組菌在0.5%SDS下的存活能力。結(jié)核分枝桿菌作為一種重要的病原微生物,其細(xì)胞被膜有著獨(dú)特的組成和結(jié)構(gòu),可以通過(guò)屏蔽或外排藥物導(dǎo)致該菌的耐藥性,所以我們用藥敏法研究了Rv0621編碼的這一膜蛋白對(duì)恥垢分枝桿菌耐受結(jié)核藥物能力的影響。SDS能破壞結(jié)核分枝桿菌富含脂肪酸的細(xì)胞被結(jié)構(gòu),Rv0621可能會(huì)改變宿主的脂肪酸組分以應(yīng)對(duì)這一破壞,所以我們通過(guò)氣相色譜檢測(cè)了重組菌與空載對(duì)照的脂肪酸組成差異并檢測(cè)了導(dǎo)致該差異的基因轉(zhuǎn)錄水平。 實(shí)驗(yàn)結(jié)果顯示：Rv0621在大腸桿菌和恥垢分枝桿菌中成功異源表達(dá),Rv0621過(guò)表達(dá)對(duì)恥垢分枝桿菌的生長(zhǎng)速率影響不顯著,重組恥垢分枝桿菌在0.5%SDS下的存活率增加,對(duì)結(jié)核藥物尤其是利福平的耐受增加,重組恥垢分枝桿菌和空載對(duì)照菌的脂肪酸組分存在較大差異,重組恥垢分枝桿菌的十六烷酸和十八烷酸減少,相應(yīng)的不飽和脂肪酸十六碳烯-9-酸和十八碳烯-9-酸增加,經(jīng)RT-PCR驗(yàn)證,發(fā)現(xiàn)引起該脂肪酸變化的酶的編碼基因MSMEG2938和MSMEG5248的轉(zhuǎn)錄水平上調(diào)。肺泡表面活性物質(zhì)能破壞結(jié)核分枝桿菌富含脂肪酸的細(xì)胞被結(jié)構(gòu),而不飽和脂肪酸是微生物的重要組分,在微生物抵抗外界壓力中發(fā)揮著重要作用。我們推測(cè)Rv0621重組恥垢分枝桿菌不飽和脂肪酸的增加可能會(huì)改變其細(xì)胞膜的成分及功能,從而應(yīng)對(duì)SDS對(duì)恥垢分枝桿菌細(xì)胞膜產(chǎn)生的破壞,并對(duì)結(jié)核藥物產(chǎn)生屏蔽或外排,影響其進(jìn)入細(xì)胞發(fā)揮作用。從實(shí)驗(yàn)結(jié)果推測(cè),Rv0621編碼的蛋白是一個(gè)有意義的分子,對(duì)其繼續(xù)深入研究可以揭示結(jié)核分枝桿菌對(duì)肺部表面活性壓力耐受的機(jī)制,為開(kāi)發(fā)新的抗結(jié)核靶標(biāo)提供思路與基礎(chǔ)。
[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.

【學(xué)位授予單位】：西南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R378

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 ;Genes and regulatory networks involved in persistence of Mycobacterium tuberculosis[J];Science China(Life Sciences);2011年04期

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本文編號(hào)：1756295