【作者】 尹晓敏；

【导师】 赵德明；

【作者基本信息】 中国农业大学 ， 基础兽医学， 2013， 博士

【摘要】 牛结核病(Bovine tuberculosis)是由牛分枝杆菌(Mycobacterium bovis, Mb)引起的一种慢性人兽共患传染病,给畜牧业发展和公共卫生带来了严重危害。Mb成功侵入巨噬细胞并在其胞内存活是其致病机制中的重要环节。因此,解析与细胞入侵相关的蛋白的功能将有助于控制牛结核病。牛分枝杆菌Mb1514基因编码一种推定侵入蛋白(本研究将其命名MbINV蛋白),但其确切功能尚不明确。本研究利用基因重组技术对该基因进行了克隆、原核表达、纯化和复性,首次成功获得了重组蛋白,并利用鼠巨噬细胞模型RAW264.7初步解析了该蛋白的生物功能。为更深入地研究该基因的功能和在机体免疫调节中的作用,成功构建了该基因的绿色荧光融合蛋白穿梭表达载体,并将其电转化至耻垢分枝杆菌后获得了重组菌株。具体研究内容如下：1.以Mb的DNA为模板,PCR扩增Mb1514基因并将其克隆至pEASY-T1载体。测序结果表明,克隆所得基因与GenBank上所公布的M. bovis AF2122/97的Mb1514基因序列100%同源。将该基因插入到原核表达载体pET-28a(+)中构建重组表达质粒,将其转化至大肠杆菌BL21(DE3),通过IPTG诱导蛋白表达。SDS-PAGE分析和Western blotting鉴定结果表明,MbINV蛋白以包涵体的形式在大肠杆菌中成功表达,分子量约为28kDa。利用镍柱对重组蛋白进行纯化,成功获得了纯化蛋白,将其复性后用于功能分析。2. Western blotting结果显示,重组MbINV蛋白质能够被兔抗牛分枝杆菌阳性血清特异性识别,说明该蛋白具有抗原性,提示该蛋白在诊断抗原的制备和亚单位疫苗的研发上具有潜在应用价值。3.用不同浓度的重组MbINV蛋白刺激RAW264.7,在不同时间点用CCK-8试剂盒检测细胞活性。结果显示,该蛋白对RAW264.7的活性有显著的抑制作用(P<0.05),且具剂量依赖性。4.用重组蛋白(终浓度10μg/mL)刺激RAW264.7细胞24h后,用细胞凋亡与坏死检测试剂盒检测细胞死亡方式。结果显示,该蛋白可导致RAW264.7坏死,说明该蛋白具有毒性。5.分别用MbINV蛋白和MbPPD以终浓度10μg/mL的剂量刺激RAW264.7细胞,同时设立PBS阴性对照。在刺激后不同时间点提取细胞的总RNA后反转录成cDNA。以β-actin为内参基因,采用相对实时荧光定量PCR方法检测促炎细胞因子’TNF-α、NOS2和IL-1β的mRNA表达水平。结果显示,相对于PBS阴性对照组,MbINV蛋白能够非常显著地上调RAW264.7细胞中3种促炎细胞因子的mRNA水平(P<0.01),且呈时间效应关系,在24h时达到最大值。MbPPD也能使3种促炎细胞因子的表达上调,但在刺激24h后显著低于MbINV处理组(P<0.05)。6.通过细胞侵入抑制试验测定了MbINV的侵入功能。结果显示,MbINV能够抑制牛分枝杆菌的侵入,且呈剂量依赖关系,证明该蛋白自身具有细胞侵入功能。7.成功构建了Mb1514-EGFP-pLAM12重组穿梭表达质粒,并获得其重组耻垢分枝杆菌菌株,为深入开展研究奠定了基础。更多还原

【Abstract】 Bovine tuberculosis (BTB) is a chronic zoonosis caused by Mycobacterium bovis (Mb), posing a great risk to public health besides a major economic problem. The ability of M. bovis to invade and survive in the macrophages is the key component in the pathogenesis of BTB. Therefore, analysis of the proteins responsible for cell invasion will help us to combat BTB. The Mb1514gene in Mb encodes a hypothetical invasion protein (designated here as MbINV protein), whose function has not yet been directly identified. In this study, the Mb1514gene from Mb was cloned, expressed in E. coli and purified. The prokaryotic recombinant MbINV protein was successfully obtained for the first time and its function was initially analized using a murine macrophage cell line, RAW264.7. For further study of the Mb1514gene, a Mb1514-EGFP fusion protein expressing shuttle vector was constructed and successfully transformed into Mycobacterium smegmatis by electroporation. The detailed research contents were described as follows:1. The Mb1514gene was amplified from the DNA template of M. bovis Beijing strain by PCR and cloned into pEASY-T1simple vector. Sequencing result showed that the gene cloned in this study is100%identical with that in M. bovis AF2122/97published in GenBank. The Mb1514gene was then sub-cloned into the prokaryotic expression vector pET-28a(+).The recombinant plasmids were transformed into E. coli BL21(DE3) to express the recombinant MbINV protein by IPTG induction. SDS-PAGE analysis and Western blotting assay showed that the protein was successfully expressed as an inclusion body in the cell lysis pellets, with a molecular weight of approximately28kDa. Recombinant proteins were purified by Ni Sepharose column and renatured for function analysis.2. The specific recognition of the recombinant MbINV proteins by the rabbit-anti-Mb polyclonal antibody demonstrated that the recombinant protein is immunogenic, which sμggests that MbINV is a potential candidate for a diagnostic biomarker or a subunit vaccine.3. To investigate the virulence of Mb1514, the RAW264.7cells were stimulated with the recombinant MbINV protein at various concentrations and the cell viabilities were detected at different time points with CCK-8Kit. The results demonstrated that recombinant MbINV protein significantly inhibited the viability of RAW264.7macrophages in a dose-dependent manner (P<0.05).4. Cell death type assay was carried out using Apoptosis and Necrosis Assay Kit at24h post stimulation of the RAW264.7cells with the recombinant MbINV protein at the final concentration of lOμg/mL. The result showed that MbINV caused cell necrosis, compared with the PBS control, indicating that this protein is toxic.5.To investigate the effect of MbINV protein on TNF-a, NOS2, and IL-1β mRNA expression, the RAW264.7cells were stimulated with recombinant MbINV (10μg/mL) or MbPPD (10μg/mL). As a control, cells were incubated with an equal volume of PBS. Total RNA was extracted at each time point after stimulation and the inflammatory cytokine mRNA expression was detected by quantitative RT-PCR. The results showed that the mRNA levels of the three factors were all up-regulated significantly (P<0.01) in a time-dependent manner, with the most pronounced effect observed at24h post-stimulation, compared with the PBS control (P<0.01). MbPPD stimulation also resulted in up-regulation of the mRNA levels of the three factors to various degrees, but is significantly lower than that induced by MbINV after24h post stimulation(P<0.05).6. A cell invasion inhibition assay was performed to investigate the invasion capability of MbINV. The result demonstrated that the cell invasion rates by Mb is markedly inhibited by MbINV in a concentration-dependant manner at24-h post infection, indicating that MbINV is cell invasive.7. For further study of the Mb1514gene, Mb1514-EGFP-pLAM12shuttle expression vectors were successfully constructed and transformed into the competent cells of Mycobacterium smegmatis Mc2155by electroporation. This provides a basis tool for further characterization of this gene.更多还原