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  本文關(guān)鍵詞：結(jié)核分枝桿菌Rv0901基因功能研究　出處：《四川大學(xué)》2007年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 結(jié)核分枝桿菌 Rv0901 脂質(zhì)體轉(zhuǎn)染 重組恥垢分枝桿菌 凋亡

【摘要】： 結(jié)核病是由結(jié)核分枝桿菌引起的一種世界性傳染病。世界衛(wèi)生組織2004年的世界衛(wèi)生報告指出結(jié)核病已經(jīng)成為可治療的感染性疾病當(dāng)中的頭號致死性疾病。WHO感染性疾病的死亡登記和監(jiān)管記錄表明：2004年全球新增結(jié)核病人890萬，其中390萬例痰涂片陽性，而2004年全球結(jié)核病死亡人數(shù)則達(dá)到了170萬。隨著人口流動增加、HIV與結(jié)核分枝桿菌伴發(fā)感染以及結(jié)核分枝桿菌多重耐藥性菌株的出現(xiàn)等原因，結(jié)核分枝桿菌的傳播呈逐年穩(wěn)步增長趨勢。近年來研究發(fā)現(xiàn)，，結(jié)核病與多種慢性疾病有密切相關(guān)，如糖尿病，營養(yǎng)不良和由吸煙及大氣污染導(dǎo)致的呼吸系統(tǒng)疾病等等。因此，探索結(jié)核分枝桿菌的致病機(jī)制和新的防治策略顯得十分重要。而結(jié)核分枝桿菌H37Rv基因組測序的完成和更新，為結(jié)核分枝桿菌基因組學(xué)和蛋白組學(xué)的研究提供了重要而更加精確的資料。 初步研究表明，一組尚未確定生物學(xué)特征和功能的結(jié)核分枝桿菌假想蛋白基因，可能與其毒力致病機(jī)制有關(guān)。這些蛋白基因皆位于細(xì)胞壁和細(xì)胞活動基因及其調(diào)節(jié)蛋白編碼基因的下游，受同一操縱子控制，其編碼產(chǎn)物是構(gòu)成結(jié)核分枝桿菌細(xì)胞壁的重要構(gòu)件，其中尤以Rv0901基因最為引人注目。該基因在1998年基因組測序完成時被劃分為編碼未知蛋白類及保守的假想蛋白類基因，于2002年基因組更新時被重新劃入了細(xì)胞壁和細(xì)胞突(cell一wall andcell processes category)類基因。結(jié)核分枝桿菌細(xì)胞壁的特殊結(jié)構(gòu)和成分在其與宿主細(xì)胞的相互作用及其致病中發(fā)揮著重要的作用。為研究Rv0901基因的功能及其與結(jié)核分枝桿菌毒力、致病機(jī)制的關(guān)系，我們首先將該基因528bp片段從基因組中擴(kuò)增出，在原核融合表達(dá)載體pET32a(+)中成功表達(dá)了pET—Rv0901融合蛋白并制備了蛋白的多克隆抗體，為研究該基因功能奠定了基礎(chǔ)。繼而將Rv0901基因片段構(gòu)建進(jìn)入真核表達(dá)載體PcDNA3．1，在真核細(xì)胞中得以成功表達(dá)，再將該基因片段轉(zhuǎn)入小鼠腹腔巨噬細(xì)胞，研究該基因?qū)π∈蟾骨痪奘杉?xì)胞活性的影響。進(jìn)一步通過穿梭表達(dá)載體pMV261經(jīng)電轉(zhuǎn)化將Rv0901基因片段重組入不含該基因序列的無毒恥垢分枝桿菌(mc~2 155)，并在重組恥垢分枝桿菌(Rmc~2 155)中成功表達(dá)了19kDa的Rv0901編碼蛋白。將經(jīng)誘導(dǎo)表達(dá)的重組恥垢分枝桿菌和未重組的恥垢分枝桿菌同時以相同復(fù)數(shù)感染人單核巨噬細(xì)胞THP-1和BALB／c小鼠，比較重組株和未重組株的細(xì)胞學(xué)和動物學(xué)作用。結(jié)果發(fā)現(xiàn)，Rv0901基因片段轉(zhuǎn)入小鼠腹腔巨噬細(xì)胞后，小鼠腹腔巨噬細(xì)胞的凋亡增加，其培養(yǎng)液上清中的NO(一氧化氮)和IFN—γ(γ干擾素)釋放量增加。Rmc~2 155誘導(dǎo)THP-1細(xì)胞的凋亡率高于mc~2 155，其感染THP—1細(xì)胞后細(xì)胞的存活率低于mc~2 155的感染，且細(xì)胞培養(yǎng)液中NO的產(chǎn)生高于mc~2 155的感染。Rmc~2 155感染BALB／c小鼠后小鼠脾淋巴細(xì)胞增殖水平高于mc~2 155的感染，小鼠血清中IFN—γ和NO的釋放量均增加，但是較mc~2 155沒有顯著差異。以上結(jié)果提示Rv0901可能對于結(jié)核分枝桿菌在宿主細(xì)胞的存活以及調(diào)控宿主細(xì)胞凋亡以維持其生存環(huán)境的穩(wěn)定性中有一定作用。
[Abstract]:Tuberculosis is a worldwide infectious disease caused by Mycobacterium tuberculosis. The WHO in 2004 World Health report pointed out that TB has become infectious diseases can be treated in the most deadly disease.WHO infectious disease death registration and supervision records show that in 2004 the global new 8 million 900 thousand TB patients, including 3 million 900 thousand cases of sputum smear positive, and in 2004 the global TB deaths reached 1 million 700 thousand. With the increase of population mobility, HIV and Mycobacterium tuberculosis infection with Mycobacterium tuberculosis and multidrug-resistant strains have other reasons, the transmission of Mycobacterium tuberculosis showed an increasing trend. In recent years, the study found that there are closely related, tuberculosis and many chronic diseases such as diabetes, nutrition bad and caused by smoking and air pollution in the respiratory system diseases and so on. Therefore, exploring Mycobacterium tuberculosis Pathogenic mechanisms and new strategies for prevention and treatment are very important. The completion and update of H37Rv sequencing of Mycobacterium tuberculosis provide important and accurate data for the study of Mycobacterium tuberculosis genomics and proteomics.
The preliminary study shows that a group of Mycobacterium tuberculosis has not yet determined the biological characteristics and function of the hypothetical protein gene, which may be related to its virulence mechanism. These genes are located in the cell wall and cell activity gene and regulation protein encoding gene downstream by the same operating control, its encoding product is an important component of Mycobacterium tuberculosis the cell wall, especially in the Rv0901 gene is most conspicuous. The gene in 1998 to complete genome sequencing is divided into encoding unknown protein and conserved hypothetical protein gene, gene group in 2002 when the update was re assigned to the cell wall and cell process (cell wall andcell processes category) gene with special structure. And the components of the mycobacterial cell wall plays an important role in its interaction with the host cell and its pathogenicity. In order to study the Rv0901 gene The function and virulence of Mycobacterium tuberculosis and the pathogenic mechanism of the relationship, we will first, the 528bp gene fragment was amplified from the genome, in the prokaryotic fusion expression vector pET32a (+) successfully expressed pET Rv0901 fusion protein and polyclonal antibody protein was prepared, which laid the foundation for the research of the gene function and then Rv0901 gene were constructed into eukaryotic expression vector PcDNA3.1 was successfully expressed in eukaryotic cells, then the gene fragment into mouse peritoneal macrophages, study the genes that influence the activity of mouse peritoneal macrophages. The shuttle expression vector pMV261 by avirulent Mycobacterium smegmatis transformed Rv0901 gene fragment into plasmid does not contain the gene sequence (mc~2 155), and the recombinant Mycobacterium smegmatis (Rmc~2 155) in the expression of Rv0901 encoding protein 19kDa. The expression of recombinant. Mycobacterium smegmatis and recombinant Mycobacterium smegmatis infection of human monocyte macrophage THP-1 and BALB / c mice with the same number of recombinant strains and non recombinant strains of cytology and animal studies. Results showed that the Rv0901 gene fragment into mouse peritoneal macrophages, apoptosis of murine peritoneal macrophages increased in the culture medium in the supernatant of NO (nitric oxide) and IFN (interferon gamma) increased release of.Rmc~2 155 induced apoptosis of THP-1 cells was higher than that of mc~2 155, the infection of THP 1 cells after the cell survival rate was lower than 155 mc~2 infection, and NO in the cell culture medium of.Rmc~2 infection is higher than that of mc~2 155 BALB 155 infection / c mice after proliferation of spleen lymphocytes in mice was higher than that of mc~2 155 infection, release IFN gamma and NO in serum were increased, but no significant difference compared with mc~2 155. The above results suggest that Rv0901 may be on It has some effect on the survival of the host cell and the regulation of the host cell apoptosis in order to maintain the stability of its living environment.

【學(xué)位授予單位】：四川大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2007
【分類號】：R378

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