結(jié)核分枝桿菌毒力基因mce1a侵襲性機(jī)制研究及微生物16S rDNAs快速分類鑒定
發(fā)布時(shí)間：2018-09-14 15:01
【摘要】：近年,結(jié)核分枝桿菌(Mycobacterium tuberculosis,Mtb)感染所致結(jié)核病(tuberculosis,TB)的發(fā)病率和死亡率一直高居不下。在全球,Mtb感染者幾占總?cè)丝诘娜种?每年TB新發(fā)和死亡患者分別在900萬(wàn)和200萬(wàn)人左右。在中國(guó),TB(主要是肺結(jié)核)現(xiàn)有患者約500萬(wàn)人(80%在農(nóng)村),數(shù)量居世界第二位,其發(fā)病數(shù)與死亡數(shù)一直高居法定報(bào)告甲乙類傳染病的首位。目前,傳統(tǒng)卡介苗的預(yù)防效果己不甚理想,并且耐藥Mtb感染正逐漸增多。然而,Mtb的具體致病機(jī)制仍不十分清楚,這為研發(fā)新型疫苗和更加敏感有效的抗TB藥物造成了很大障礙。本研究立足于Mtb侵襲相關(guān)基因mcela,首先,探討了該基因在Mtb潛伏生存與活性增殖狀態(tài)下的表達(dá)特性,并對(duì)其全長(zhǎng)和截短序列分別進(jìn)行了克隆、原核表達(dá)及純化。其次,設(shè)計(jì)、轉(zhuǎn)錄合成了兩條mcela特異性小干擾RNAs(small interfering RNAs,siRNAs),并通過(guò)HEK 293-T細(xì)胞,對(duì)siRNAs的抑制活性與抑制效率進(jìn)行了鑒定。在上述工作基礎(chǔ)上,進(jìn)一步通過(guò)小鼠巨噬細(xì)胞RAW 264.7和人結(jié)腸癌上皮細(xì)胞CT 26.CL25,進(jìn)行了T-McelA蛋白的細(xì)胞刺激實(shí)驗(yàn)、mcela特異性siRNA的體外干擾實(shí)驗(yàn)以及重組E.coli(表達(dá)完整McelA蛋白)的體外侵襲實(shí)驗(yàn),著重探討了McelA蛋白介導(dǎo)細(xì)菌侵襲的作用機(jī)制。此外,本研究還通過(guò)對(duì)多種微生物16S rDNAs的序列比對(duì)與進(jìn)化樹(shù)分析,分別設(shè)計(jì)、合成了細(xì)菌通用及特異性引物,建立了實(shí)時(shí)熒光定量PCR(real-time fluorescence quantitative PCR,FQ-PCR)快速分類與檢測(cè)方法。 一、毒力基因mcela在Mtb潛伏生存與活性增殖狀態(tài)下的表達(dá)特性 Mtb屬專性需氧細(xì)胞內(nèi)寄生菌,感染后能在宿主體內(nèi)以休眠狀態(tài)長(zhǎng)期潛伏生存,并在一定條件下復(fù)蘇、大量增殖從而引發(fā)疾病。鑒于Mtb在低于25℃時(shí)停止生長(zhǎng),故低溫條件下長(zhǎng)時(shí)間存活的Mtb菌株可以作為一種非增殖狀態(tài)的體外模型。本研究選取兩種標(biāo)準(zhǔn)株(強(qiáng)毒株H37Rv和弱毒株H37Ra)及八種臨床分離株(非耐藥四株,耐利福平或異煙肼各兩株),分別以4℃保存3 m與37℃培養(yǎng)2 w～5 w為其潛伏生存和活性增殖狀態(tài)。通過(guò)溶菌酶、蛋白酶K及1%SDS裂解Mtb,提取、純化總RNA,并以不含RNase的DNase消化去除DNA。設(shè)計(jì)、合成mcela特異性引物,以看家基因16S rRNA為平行參照,通過(guò)RT-PCR分別檢測(cè)上述樣品mcela的表達(dá)。結(jié)果顯示,處于非增殖期的十種Mtb菌株,mcela基因均未見(jiàn)表達(dá);而處于活性增殖期的上述菌株,mcela基因均有穩(wěn)定表達(dá),且在不同菌株的表達(dá)水平相似。上述結(jié)果初步提示,mcela基因的表達(dá)可能與Mtb侵襲力或潛伏生存能力有關(guān)。 二、McelA蛋白介導(dǎo)細(xì)菌侵襲的作用機(jī)制 首先,進(jìn)行了全長(zhǎng)與截短mcela基因的克隆及原核表達(dá)。設(shè)計(jì)Exo-primer、In-orimer及In-primer2共三對(duì)引物,通過(guò)巢式PCR,分別擴(kuò)增Mtb(H37Rv)全長(zhǎng)與截短的mcela基因——mcela和T-mcela,并將其克隆至6xHis原核表達(dá)載體pROEX-HTb和熱誘導(dǎo)表達(dá)載體pBV220,分別通過(guò)IPTG或42℃加熱誘導(dǎo)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，
本文編號(hào)：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，
本文編號(hào)：2243051
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