结核分枝杆菌Cas蛋白表达纯化与功能研究

发布时间：2018-03-20 14:12

  本文选题：结核分枝杆菌　切入点：CRISPR系统　出处：《郑州大学》2012年硕士论文　论文类型：学位论文

【摘要】：近来在大多数的细菌(40%)和几乎所有的古细菌(90%)的基因组中,人们发现一类成簇的、有规律间隔的短回文重复结构家族(clusteredregularl y interspaced short palindromic repeats, CRISPR)。 CRISPR主要用于细菌防御外来噬菌体、病毒的侵袭,从而使细菌能够在残酷的自然环境中生存下来,是一种适应性的免疫机制。CRISPR系统的作用是提供对噬菌体特异性免疫,在生物医学,生物工业上都有广阔的前景。 结核病是目前单一致病菌中致死率最高的传染病之一,我国是全球结核病高负担国家之一,结核病患者人数位居世界第二。结核病的病原体是结核分枝杆菌,结核分枝杆菌易发生耐药性,近年来世界各地结核分枝杆菌的多耐药菌株和泛耐药菌株逐渐增多,甚至引起暴发流行,使得目前的药物已不足以抑制。因此,对于结核病的致病菌——结核分枝杆菌的致病机制和免疫机制的深入研究显得极为重要。CRISPR系统是结核分枝杆菌重要的免疫机制之一,本研究着重于对结核分枝杆菌的CRISPR系统中的Cas蛋白进行初步探索。 根据目前在一些细菌中的研究成果,预测结核分枝杆菌也存在CRISPR系统。CRISPR系统包括一系列Cas蛋白,这些蛋白协助CRISPR系统发挥免疫作用。CRISPR系统发挥免疫作用分为两个步骤。首先,噬菌体入侵细菌,Cas蛋白将其识别,并整合到宿主的基因组上。然后,噬菌体第二次入侵,CRISPR系统能够识别外源噬菌体,用具有特异性核糖核酸内切酶功能的Cas蛋白将其切割,外源噬菌体失去毒力,免疫过程结束。在这个过程中,Cas1和Cas2蛋白参与获取外源噬菌体基因的过程,Cas6蛋白是核糖核酸内切酶,能够切割CRISPR repeat RNA,在宿主细菌防御外源噬菌体的过程中扮演了重要的角色。 本研究首先成功构建了3个重组表达质粒MBP-Rv2816、MBP-Rv2817以及MBP-Rv2824,并在原核中得到高效表达。目的基因Rv2816、Rv2817以及Rv2824对应的目的蛋白分别是Cas1-mbp、Cas2-mbp以及Cas6-mbp。我们选择的表达质粒是PDB-His-MBP载体,MBP标签大小为45KDa。载体上的标签MBP可增加在细菌中过量表达的融合蛋白的溶解性,尤其是真核蛋白。纯化蛋白使用的是镍离子亲和层析的方法。带有His标签的融合蛋白特异性结合在镍离子亲和层析胶体(即柱材料)上。结合在柱材料上的融合蛋白可用400mM高浓度咪唑的生理缓冲液进行洗脱。 就三个目的蛋白的表达情况来看,16℃诱导表达的融合蛋白Cas1-mbp和Cas6-mbp有较好的溶解性,Cas2-mbp在纯化得到之后,降解程度严重。将融合蛋白Cas6-mbp用于后续功能的研究,在体外构建了Cas6-mbp蛋白切割CRISPR-repeat RNA和切割CRISPR-spacer RNA的体系。实验样品分为两组,反应底物分别是CRISPR-repeat RNA和CRISPR-spacer RNA。在RNA的量不变的条件下,Cas6-mbp蛋白的量沿梯度浓度增高。实验另外设置有RNA阴性对照。实验结果发现Cas6蛋白能够切割CRISPR-repeat RNA,但不能够切割CRISPR-spacer RNA。 本研究初步阐释了结核分枝杆菌中存在CRISPR系统,验证了在结核分枝杆菌基因组中确实存在Cas1、Cas2、Cas6蛋白基因,并且能够进行表达纯化,其中Cas6蛋白具有核糖核酸内切酶的功能。这一研究结果可能有助于对结核分枝杆菌免疫机制的深入了解,对以后的生物医学发展有比较重要的参考价值。
[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�鐮旂┒棣栧厛鎴愬姛鏋勫缓浜�3涓�閲嶇粍琛ㄨ揪璐ㄧ矑MBP-Rv2816,MBP-Rv2817浠ュ強MBP-Rv2824,骞跺湪鍘熸牳涓�寰楀埌楂樻晥琛ㄨ荆�

本文编号：1639433