本文選題：結核分枝桿菌　切入點：CRISPR系統(tǒng)　出處：《鄭州大學》2012年碩士論文　論文類型：學位論文

【摘要】：近來在大多數的細菌(40%)和幾乎所有的古細菌(90%)的基因組中,人們發(fā)現一類成簇的、有規(guī)律間隔的短回文重復結構家族(clusteredregularl y interspaced short palindromic repeats, CRISPR)。 CRISPR主要用于細菌防御外來噬菌體、病毒的侵襲,從而使細菌能夠在殘酷的自然環(huán)境中生存下來,是一種適應性的免疫機制。CRISPR系統(tǒng)的作用是提供對噬菌體特異性免疫,在生物醫(yī)學,生物工業(yè)上都有廣闊的前景。 結核病是目前單一致病菌中致死率最高的傳染病之一,我國是全球結核病高負擔國家之一,結核病患者人數位居世界第二。結核病的病原體是結核分枝桿菌,結核分枝桿菌易發(fā)生耐藥性,近年來世界各地結核分枝桿菌的多耐藥菌株和泛耐藥菌株逐漸增多,甚至引起暴發(fā)流行,使得目前的藥物已不足以抑制。因此,對于結核病的致病菌——結核分枝桿菌的致病機制和免疫機制的深入研究顯得極為重要。CRISPR系統(tǒng)是結核分枝桿菌重要的免疫機制之一,本研究著重于對結核分枝桿菌的CRISPR系統(tǒng)中的Cas蛋白進行初步探索。 根據目前在一些細菌中的研究成果,預測結核分枝桿菌也存在CRISPR系統(tǒng)。CRISPR系統(tǒng)包括一系列Cas蛋白,這些蛋白協助CRISPR系統(tǒng)發(fā)揮免疫作用。CRISPR系統(tǒng)發(fā)揮免疫作用分為兩個步驟。首先,噬菌體入侵細菌,Cas蛋白將其識別,并整合到宿主的基因組上。然后,噬菌體第二次入侵,CRISPR系統(tǒng)能夠識別外源噬菌體,用具有特異性核糖核酸內切酶功能的Cas蛋白將其切割,外源噬菌體失去毒力,免疫過程結束。在這個過程中,Cas1和Cas2蛋白參與獲取外源噬菌體基因的過程,Cas6蛋白是核糖核酸內切酶,能夠切割CRISPR repeat RNA,在宿主細菌防御外源噬菌體的過程中扮演了重要的角色。 本研究首先成功構建了3個重組表達質粒MBP-Rv2816、MBP-Rv2817以及MBP-Rv2824,并在原核中得到高效表達。目的基因Rv2816、Rv2817以及Rv2824對應的目的蛋白分別是Cas1-mbp、Cas2-mbp以及Cas6-mbp。我們選擇的表達質粒是PDB-His-MBP載體,MBP標簽大小為45KDa。載體上的標簽MBP可增加在細菌中過量表達的融合蛋白的溶解性,尤其是真核蛋白。純化蛋白使用的是鎳離子親和層析的方法。帶有His標簽的融合蛋白特異性結合在鎳離子親和層析膠體(即柱材料)上。結合在柱材料上的融合蛋白可用400mM高濃度咪唑的生理緩沖液進行洗脫。 就三個目的蛋白的表達情況來看,16℃誘導表達的融合蛋白Cas1-mbp和Cas6-mbp有較好的溶解性,Cas2-mbp在純化得到之后,降解程度嚴重。將融合蛋白Cas6-mbp用于后續(xù)功能的研究,在體外構建了Cas6-mbp蛋白切割CRISPR-repeat RNA和切割CRISPR-spacer RNA的體系。實驗樣品分為兩組,反應底物分別是CRISPR-repeat RNA和CRISPR-spacer RNA。在RNA的量不變的條件下,Cas6-mbp蛋白的量沿梯度濃度增高。實驗另外設置有RNA陰性對照。實驗結果發(fā)現Cas6蛋白能夠切割CRISPR-repeat RNA,但不能夠切割CRISPR-spacer RNA。 本研究初步闡釋了結核分枝桿菌中存在CRISPR系統(tǒng),驗證了在結核分枝桿菌基因組中確實存在Cas1、Cas2、Cas6蛋白基因,并且能夠進行表達純化,其中Cas6蛋白具有核糖核酸內切酶的功能。這一研究結果可能有助于對結核分枝桿菌免疫機制的深入了解,對以后的生物醫(yī)學發(fā)展有比較重要的參考價值。
[Abstract]:In recent years most of the bacteria and archaea (40%) almost all (90%) of the genome, it was found that a class of clustering, short palindromic repeat structure family regular intervals (clusteredregularl y interspaced short palindromic repeats, CRISPR). CRISPR is mainly used in bacterial defense against bacteriophages, viruses, bacteria and to survive in the harsh natural environment, is an adaptive immune mechanism of.CRISPR system's role is to provide the phage specific immunity, in biomedical, biological industry has broad prospects.
TB is currently one of the most infectious diseases caused by the single highest death rate of bacteria in China is one of the global TB burden countries, TB patients ranks second in the world. The causative agent of tuberculosis mycobacterium tuberculosis, Mycobacterium tuberculosis prone to drug resistance of Mycobacterium tuberculosis in recent years around the world of multi drug resistant strains and pan resistant strains gradually increased, and even caused the outbreak, the current drug has been insufficient to restrain. Therefore, further study on pathogenicity of Mycobacterium tuberculosis strains of tuberculosis pathogenic and immunological mechanisms is very important for the.CRISPR system is one of the important immunological mechanism of Mycobacterium tuberculosis, were investigated in this study focuses on the CRISPR system of Cas protein Mycobacterium tuberculosis in.
According to the current studies on certain bacteria, prediction of Mycobacterium tuberculosis has included a series of Cas protein CRISPR.CRISPR system, CRISPR system to assist these proteins playing a role in the immune system.CRISPR immune function is divided into two steps. Firstly, the bacteria phage Cas protein, its identification, and integrated into the host genome. Then, second phage invasion, CRISPR system can recognize exogenous phage, with specific Endoribonucleases functional Cas protein cut, exogenous phage lose virulence, immune processes. In this process, Cas1 and Cas2 proteins involved in the process of obtaining exogenous phage gene, Cas6 protein is Endoribonucleases can CRISPR repeat cut RNA, played an important role in the process of host bacteria phage in defense.
鏈爺絀墮鍏堟垚鍔熸瀯寤轟簡3涓噸緇勮〃杈捐川綺扢BP-Rv2816,MBP-Rv2817浠ュ強MBP-Rv2824,騫跺湪鍘熸牳涓緱鍒伴珮鏁堣〃杈，

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