结核分枝杆菌毒力基因mce1a侵袭性机制研究及微生物16S rDNAs快速分类鉴定
发布时间：2018-09-14 15:01
【摘要】：近年,结核分枝杆菌(Mycobacterium tuberculosis,Mtb)感染所致结核病(tuberculosis,TB)的发病率和死亡率一直高居不下。在全球,Mtb感染者几占总人口的三分之一,每年TB新发和死亡患者分别在900万和200万人左右。在中国,TB(主要是肺结核)现有患者约500万人(80%在农村),数量居世界第二位,其发病数与死亡数一直高居法定报告甲乙类传染病的首位。目前,传统卡介苗的预防效果己不甚理想,并且耐药Mtb感染正逐渐增多。然而,Mtb的具体致病机制仍不十分清楚,这为研发新型疫苗和更加敏感有效的抗TB药物造成了很大障碍。本研究立足于Mtb侵袭相关基因mcela,首先,探讨了该基因在Mtb潜伏生存与活性增殖状态下的表达特性,并对其全长和截短序列分别进行了克隆、原核表达及纯化。其次,设计、转录合成了两条mcela特异性小干扰RNAs(small interfering RNAs,siRNAs),并通过HEK 293-T细胞,对siRNAs的抑制活性与抑制效率进行了鉴定。在上述工作基础上,进一步通过小鼠巨噬细胞RAW 264.7和人结肠癌上皮细胞CT 26.CL25,进行了T-McelA蛋白的细胞刺激实验、mcela特异性siRNA的体外干扰实验以及重组E.coli(表达完整McelA蛋白)的体外侵袭实验,着重探讨了McelA蛋白介导细菌侵袭的作用机制。此外,本研究还通过对多种微生物16S rDNAs的序列比对与进化树分析,分别设计、合成了细菌通用及特异性引物,建立了实时荧光定量PCR(real-time fluorescence quantitative PCR,FQ-PCR)快速分类与检测方法。 一、毒力基因mcela在Mtb潜伏生存与活性增殖状态下的表达特性 Mtb属专性需氧细胞内寄生菌,感染后能在宿主体内以休眠状态长期潜伏生存,并在一定条件下复苏、大量增殖从而引发疾病。鉴于Mtb在低于25℃时停止生长,故低温条件下长时间存活的Mtb菌株可以作为一种非增殖状态的体外模型。本研究选取两种标准株(强毒株H37Rv和弱毒株H37Ra)及八种临床分离株(非耐药四株,耐利福平或异烟肼各两株),分别以4℃保存3 m与37℃培养2 w～5 w为其潜伏生存和活性增殖状态。通过溶菌酶、蛋白酶K及1%SDS裂解Mtb,提取、纯化总RNA,并以不含RNase的DNase消化去除DNA。设计、合成mcela特异性引物,以看家基因16S rRNA为平行参照,通过RT-PCR分别检测上述样品mcela的表达。结果显示,处于非增殖期的十种Mtb菌株,mcela基因均未见表达;而处于活性增殖期的上述菌株,mcela基因均有稳定表达,且在不同菌株的表达水平相似。上述结果初步提示,mcela基因的表达可能与Mtb侵袭力或潜伏生存能力有关。 二、McelA蛋白介导细菌侵袭的作用机制 首先,进行了全长与截短mcela基因的克隆及原核表达。设计Exo-primer、In-orimer及In-primer2共三对引物,通过巢式PCR,分别扩增Mtb(H37Rv)全长与截短的mcela基因——mcela和T-mcela,并将其克隆至6xHis原核表达载体pROEX-HTb和热诱导表达载体pBV220,分别通过IPTG或42℃加热诱导目的基因
[Abstract]:In recent years, the incidence and mortality of tuberculosis (tuberculosis,TB) caused by Mycobacterium tuberculosis (Mycobacterium tuberculosis,Mtb) infection has been high. Almost 1/3 of the world's people are infected with TB, with 9 million new cases and 2 million deaths each year. In China, there are about 5 million patients (80% in rural areas) with TB (mainly pulmonary tuberculosis), ranking the second in the world. The number of morbidity and mortality has always been the highest in the statutory report of Class A and B infectious diseases. At present, the preventive effect of traditional BCG vaccine has not been satisfactory, and drug-resistant Mtb infection is increasing gradually. However, the specific pathogenesis of Mtb remains unclear, which poses a major obstacle to the development of new vaccines and more sensitive and effective anti- TB drugs. This study was based on the Mtb invasion-associated gene mcela,. Firstly, the expression characteristics of the gene were studied under the condition of latent survival and active proliferation of Mtb. The full length and truncated sequence of the gene were cloned, expressed and purified respectively. Secondly, two mcela specific small interfering RNAs (small interfering RNAs,siRNAs were designed and transcribed, and the inhibitory activity and inhibitory efficiency of siRNAs were identified by HEK 293-T cells. On the basis of the above work, In addition, murine macrophages (RAW 264.7) and human colon cancer epithelial cells (CT 26.CL25) were used to conduct cell stimulation assay of T-McelA protein and invasion of recombinant E.coli (expressing intact McelA protein) in vitro. The mechanism of bacterial invasion mediated by McelA protein was discussed. In addition, based on sequence alignment and phylogenetic tree analysis of 16s rDNAs of various microorganisms, general and specific primers of bacteria were designed and synthesized, and rapid classification and detection of real-time fluorescent quantitative PCR (real-time fluorescence quantitative PCR,FQ-PCR) were established. The expression of virulence Gene mcela in latent Survival and active Proliferation of Mtb belongs to specific aerobic cell parasitic bacteria. After infection, the host can remain dormant for a long time and be resuscitated under certain conditions. In view of the fact that Mtb stopped growing below 25 鈩，
本文编号：2243051
[Abstract]:In recent years, the incidence and mortality of tuberculosis (tuberculosis,TB) caused by Mycobacterium tuberculosis (Mycobacterium tuberculosis,Mtb) infection has been high. Almost 1/3 of the world's people are infected with TB, with 9 million new cases and 2 million deaths each year. In China, there are about 5 million patients (80% in rural areas) with TB (mainly pulmonary tuberculosis), ranking the second in the world. The number of morbidity and mortality has always been the highest in the statutory report of Class A and B infectious diseases. At present, the preventive effect of traditional BCG vaccine has not been satisfactory, and drug-resistant Mtb infection is increasing gradually. However, the specific pathogenesis of Mtb remains unclear, which poses a major obstacle to the development of new vaccines and more sensitive and effective anti- TB drugs. This study was based on the Mtb invasion-associated gene mcela,. Firstly, the expression characteristics of the gene were studied under the condition of latent survival and active proliferation of Mtb. The full length and truncated sequence of the gene were cloned, expressed and purified respectively. Secondly, two mcela specific small interfering RNAs (small interfering RNAs,siRNAs were designed and transcribed, and the inhibitory activity and inhibitory efficiency of siRNAs were identified by HEK 293-T cells. On the basis of the above work, In addition, murine macrophages (RAW 264.7) and human colon cancer epithelial cells (CT 26.CL25) were used to conduct cell stimulation assay of T-McelA protein and invasion of recombinant E.coli (expressing intact McelA protein) in vitro. The mechanism of bacterial invasion mediated by McelA protein was discussed. In addition, based on sequence alignment and phylogenetic tree analysis of 16s rDNAs of various microorganisms, general and specific primers of bacteria were designed and synthesized, and rapid classification and detection of real-time fluorescent quantitative PCR (real-time fluorescence quantitative PCR,FQ-PCR) were established. The expression of virulence Gene mcela in latent Survival and active Proliferation of Mtb belongs to specific aerobic cell parasitic bacteria. After infection, the host can remain dormant for a long time and be resuscitated under certain conditions. In view of the fact that Mtb stopped growing below 25 鈩，
本文编号：2243051
本文链接：https://www.wllwen.com/yixuelunwen/binglixuelunwen/2243051.html
最近更新
教材专著
