【作者】 刘雅婷；

【导师】 曹刚强； 毕利军；

【作者基本信息】 郑州大学 ， 遗传学， 2012， 硕士

【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。更多还原

【Abstract】 Recently, in the genomes of the most bacteria (40%) and almost all of the archaea (90%), it was discovered that a group of clustered regularly interspaced short palindromic repeats (CRISPR). For bacteria, CRISPR is an adaptive immune mechanism, which is used to defense against bacteriophages and viruses so that bacteria could survive in the harsh natural environmental. It is because the CRISPR system provides the bacteria with phage-specific immunity, so it will have a broad prospect in both biomedicine and bioindustry.Currently, tuberculosis is one of the highest date rate of single pathogenic bacterium infectious diseases which induced by the single pathogenic bacterium. China is one of the high tuberculosis burden countries and the population of tuberculosis patients ranks second in the world, second only to India. In China, the population of dying from tuberculosis is about250,000every year, which is more than the twice of the sum of all kinds of infectious diseases. However, in recent years, the high drug-resistance strains of Mycobacterium tuberculosis increased gradually in all over the world, which is because Mycobacterium tuberculosis prone to generate drug resistance, and even caused the tuberculosis outbreak. At the same time, it made that the current drug has been insufficient to restrain Mycobacterium tuberculosis growth. So, it is greatly important to investigate deeply to the pathogenic mechanism and the immune mechanism of Mycobacterium tuberculosis, the pathogenic bacterium of tuberculosis. The CRISPR system is one of the important immune mechanisms for Mycobacterium tuberculosis. Therefore, this study does a preliminary exploration on the CRISPR system of Mycobacterium tuberculosis.From some previous studies on certain bacteria, it was reasonable to deduce the presence of CRISPR system in Mycobacterium tuberculosis.The process of CRISPR system contains two steps. First, the phage invaded into the bacteria. The Cas protein recognized it, and integrate its DNA into the host genome. Then, in the second phage invasion, CRISPR system could identify the exogenous phage, and end the immune process by using the specific ribose endonuclease to cut the invaded phage. In this process, Casl and Cas2involved in obtaining the phage information, and Cas6protein, a ribose endonuclease which could cut CRISPR repeat RNA, plays an important role in the defense process.In this study, three recombinant expression plasmids (MBP-Rv2816, MBP-Rv2817and MBP-Rv2824) were successfully constructed, and highly expressed in the pronucleus. The target genes Rv2816, Rv2817, Rv2824corresponded to three target proteins Casl-mbp, Cas2-mbp and Cas6-mbp, respectively. Vector pPDB-His-MBP was selected as the expression plasmid, and the size of protein marker MBP was45KDa. The MBP marker in the vector could enhance the solubility of the fusion proteins, particularly the eukaryotic protein, which were over-expressed in the bacteria. The purification of protein was carried out by employing Ni2+-affinity chromatography (IMAC). The His-carrying fusion protein specifically binded to the gel matrix (i.e. the chromatography column) of IMAC. The column-binding fusion protein could be separated by the elution using a highly concentrated (400mM) imidazole buffer solution.With reference to the expression of these three target proteins, the fusion proteins Casl-mbp and Cas6-mbp depicted excellent solubility after16℃induced expression; while Cas2-mbp was severely degraded after purification. The fusion protein Cas6-mbp was used for the follow-up study, i.e. used to establish in vitro systems of Cas6-mbp severing CRISPR-repeat RNA and severing CRISPR-spacer RNA. The experimental samples were divided into two groups, which used CRISPR-repeat RNA and CRISPR-spacer RNA as the reaction substrate, respectively. While the amount of RNA maintained the same, the amounts of Cas6-mbp were designed to increase according to a concentration gradient for both groups. In this experiment, another RNA-negative group was set up as the control group. From the results, Protein Cas6appeared to be able to sever CRISPR-repeat RNA, but not able to sever CRISPR-spacer RNA.In this paper, the presence of CRISPR system in Mycobacterium tuberculosis was preliminarily interpreted. In addition, the existence of Cas1, Cas2and Cas6genes in the genome of mycobacterium tuberculosis, as well as the validity of expression purification on these genes, were examined. Among them, protein Cas6could serve as an endoribonuclease. The findings of this study may probably facilitate a further understanding on the immunological mechanism of Mycobacterium tuberculosis, so as to constitute an important reference for the biomedicine studies in the future.更多还原