贝氏柯克斯体的免疫蛋白质组学研究

发布时间：2018-06-12 03:38

本文选题：贝氏柯克斯体 + Q热　；参考：《中国人民解放军军事医学科学院》2011年博士论文

【摘要】：贝氏柯克斯体(Coxiella burnetii)是Q热的病原菌,对人和动物有极强的感染力,可通过气溶胶扩散经呼吸道进入体内导致感染。人类感染贝氏柯克斯体可引发肺炎、肝炎、心内膜炎等严重疾病。贝氏柯克斯体的高致病性和对环境、理化因素的高抵抗力使其成为重要的生物战剂/生物恐怖剂。 Q热最常用的特异性诊断方法为血清学诊断,包括间接免疫荧光(IFA)技术,补体结合(CF)实验,酶联免疫吸附(ELISA)技术等。这些检测方法都需要纯化的贝氏柯克斯体全菌作为抗原,由于提取纯化贝氏柯克斯体的防护要求高、工艺复杂,造成了这些检测方法难以大规模推广和使用。疫苗接种为预防贝氏柯克斯体感染的有效手段。虽然灭活贝氏柯克斯体全菌疫苗具有良好的免疫保护效果,但是其难以制备和较强的副作用限制了它的推广应用。因此,许多国家和地区都致力于研制安全、可靠、副作用小的新型Q热疫苗。本研究通过高通量的免疫蛋白质组学来筛选贝氏柯克斯体菌体蛋白抗原,并制备相应的重组蛋白,以及评价重组蛋白抗原的免疫原性和免疫保护效能,为Q热分子疫苗和分子诊断试剂的研发提供候选蛋白抗原。本研究采用泛影葡胺线性密度梯度法从鸡胚培养物中分离出贝氏柯克斯体新桥株。用纯化的贝氏柯克斯体新桥株腹腔接种感染小鼠,于感染后1、2、3、4周分别处死小鼠,收集不同感染时期的抗血清,并采用间接免疫荧光法检测感染小鼠血清特异性抗体水平。再用贝氏柯克斯体特异的实时荧光定量PCR检测感染小鼠组织中的贝氏柯克斯体载量,以分析新桥株对BALB/c小鼠的感染特征。结果显示感染后4周内,小鼠肝、脾、肺组织中均检出大量贝氏柯克斯体,第1周检出量最高,脾的含量显著高于肝和肺,肝的含量显著高于肺。随着感染时间延长,贝氏柯克斯体检出量呈下降趋势,但血清特异性抗体水平则继续升高。随后利用双向电泳技术将纯化贝氏柯克斯体全菌蛋白进行分离,用贝氏柯克斯体实验感染小鼠血清与转移到PVDF膜上的贝氏柯克斯体蛋白进行免疫印迹反应,并通过蛋白质谱技术完成了对这些蛋白的鉴定。结果显示1、2、3、4周感染小鼠血清鉴定的印迹反应阳性蛋白分别为0、4、9、14个,其中印迹反应最强烈的4个蛋白为GroEL、Com1、Mip、OmpH。用Q热患者血清与贝氏柯克斯体蛋白进行免疫印迹反应,鉴定出了15个印迹反应阳性蛋白,其中有9个蛋白为Q热患者血清与贝氏柯克斯体感染小鼠血清共同识别抗原。采用分子克隆技术,将鉴定的免疫印迹反应阳性蛋白的基因克隆和重组表达。除了rspB基因,其他19个印迹反应阳性蛋白基因均被高效表达。采用镍离子亲和层析方法(Ni-NTA)分别从IPTG诱导的转化菌中纯化19个重组蛋白。将纯化的重组蛋白点制成一张蛋白芯片,以此芯片与贝氏柯克斯体感染的鼠血清反应。结果与贝氏柯克斯体感染1、2、3、4周小鼠血清反应的蛋白为0、16、19、18个,其中反应荧光强度最强的4个重组蛋白依次为GroEL、Mip、OmpH、Com1。将蛋白芯片与56份Q热病人血清和25份正常人血清进行反应,当单份病人血清与单个蛋白点的反应荧光信号值大于正常人血清与对应蛋白点反应荧光信号平均值加2个标准差时结果为阳性。结果GroEl、YbgF、RplL、Mip、OmpH、Com1、Dnak共7个重组蛋白被急性晚期Q热病人血清识别的阳性率大于40%。因此,这些蛋白有望成为构建Q热分子诊断试剂的侯选蛋白。将重组蛋白抗原GroEL、Com1、Mip、YbgF分别刺激体外培养的小鼠骨髓来源的树突状细胞,以贝氏柯克斯体全菌抗原(WCA)和大肠杆菌脂多糖(LPS)作阳性对照,以pET-32a(+)表达的标签蛋白(TrxA)作阴性对照,并以蛋白洗脱缓冲液(elution buffer)作为模拟刺激。24h后收集抗原刺激的树突状细胞,用流式细胞仪分析树突状细胞表面分子,结果发现不同抗原刺激树突状细胞的表面分子表达显著高于模拟刺激组。再将不同抗原激活的树突状细胞分别腹腔转移至正常小鼠,转移后1天、7天、14天分别用贝氏柯克斯体毒株攻击受体小鼠,并采用定量PCR检测小鼠脾脏贝氏柯克斯体载量,结果显示接受WCA、Com1或Mip激活树突状细胞的小鼠柯克斯体载量显著低于未接受任何刺激的阴性对照组,而接受其它抗原(GroEL/YbgF/TrxA/LPS)激活树突状细胞的小鼠贝氏柯克斯体载量与阴性对照组相比无显著性差异。说明重组蛋白抗原Com1和Mip能够诱导特异性免疫保护,为贝氏柯克斯体保护性抗原。将重组蛋白GroEL、Com1、Mip刺激的树突状细胞分别与纯化的CD4+和CD8+T细胞共培养,以全菌抗原(WCA)、标签蛋白TrxA、elution buffer刺激的树突状细胞与T细胞共培养分别作为阳性和阴性对照。24h后收集抗原刺激的树突状细胞,用流式细胞技术分析T细胞表面分子,结果发现全菌抗原(WCA)和重组蛋白GroEL、Com1、Mip刺激树突状细胞的表面分子CD69的表达水平显著高于对照刺激组。再用流式细胞技术分析T细胞细胞因子表达情况,结果显示与WCA、Com1或Mip激活树突细胞相互作用的T细胞的IFN-γ表达水平均显著高于与其它组的T细胞,提示保护性抗原刺激的树突状细胞介导的特异性免疫保护与该抗原刺激T细胞表达高水平的IFN-γ以及随后激活巨噬细胞的杀菌活性密切相关。
[Abstract]:Coxiella burnetii, a pathogen of Q heat, is a pathogen of Q fever and is highly infectious to humans and animals. It can spread through the respiratory tract by aerosol diffusion into the body and lead to infection. Human infection of the bainite body can cause severe diseases such as pneumonia, hepatitis, endocarditis and other serious diseases. The high pathogenicity and environmental and physicochemical factors of the bainite Kirk body High resistance makes it an important biological agent / bioterrorism agent.
The most commonly used specific diagnostic method for Q fever is serological diagnosis, including indirect immunofluorescence (IFA), complement binding (CF), and enzyme linked immunosorbent (ELISA). All these methods need to be purified by purified bainite Kirk body as antigen, because the extraction and purification of bainite body is high, and the process is complicated. These methods are difficult to be popularized and used in a large scale. Vaccination is an effective means to prevent bainite Kirk infection. Although the inactivation of the bainite whole strain vaccine has a good immune protection effect, its difficult preparation and strong side effects restrict its application. Therefore, many countries and regions have committed to the application. The development of a new Q heat vaccine with safe, reliable and small side effects. This study screened the bainitic Kirk body protein antigen by high throughput Immunoproteomics, prepared the corresponding recombinant protein, and evaluated the immunogenicity and immune protection efficacy of the recombinant protein antigen, which provided the research and development of Q hot molecular vaccine and molecular diagnostic reagent. Candidate protein antigen.
In this study, the new bainite new bridge strain was isolated from chicken embryo culture from chicken embryo culture by linear density gradient method. The mice were inoculated with the purified bainite new bridge strain, the mice were killed at 1,2,3,4 weeks after 1,2,3,4 infection, the antiserum of different infection period was collected, and the indirect immunofluorescence method was used to detect the infected mice blood. The level of specific antibody was clear. The bainite somatic load in the infected mice tissues was detected by the bainite specific real time fluorescence quantitative PCR PCR to analyze the infection characteristics of the new bridge strain on BALB/c mice. The results showed that a large number of bainite Kirk bodies were detected in the liver, spleen and lung tissues of the mice within 4 weeks after the infection, and the first weeks was the highest, the spleen was the highest. The content of the liver and the lung was significantly higher than that of the lung. As the time of infection was prolonged, the amount of bainite Kirk showed a downward trend, but the serum specific antibody level continued to rise.
Then the purified bainite whole bacteria protein was purified by two-dimensional electrophoresis, and the bainite body experiment was used to infect the serum of the mice and the bainite Kirk body protein transferred to the PVDF membrane to be immunoblotting, and the egg white was identified by the protein mass spectrometry technology. The results showed that the 1,2,3,4 week was infected with the blood of the mice. The results showed that the 1,2,3,4 week was infected with the blood of mice. The positive proteins of the Western blot identification were 0,4,9,14, respectively, of which 4 of the most intense imprinting proteins were GroEL, Com1, Mip, and OmpH. used Q fever patients' serum and bainite Kirk body protein for Western blotting, and 15 positive proteins were identified, of which 9 proteins were sera from Q fever patients and the infection of bainite Kirk body. The mouse sera co identified the antigen.
Cloning and recombinant expression of the identified immunoblotting positive proteins were cloned with molecular cloning technology. Except for rspB gene, 19 other positive protein genes were highly expressed. 19 recombinant proteins were purified from IPTG induced transformed bacteria by nickel ion affinity chromatography (Ni-NTA). The recombinant protein was purified. A protein chip was made to react with the mouse sera infected by bainite Kirk. The results were 0,16,19,18 in the serum of 1,2,3,4 week mice infected with Bainite body infection, of which 4 recombinant proteins with the strongest fluorescent intensity were GroEL, Mip, OmpH, Com1., and 56 sera and 25 portions of Q fever patients. In the normal human serum, the results were positive when the fluorescence signal of the single patient's sera and the single protein point was greater than the average value of the normal human serum and the corresponding protein point response fluorescence signal. Results GroEl, YbgF, RplL, Mip, OmpH, Com1, Dnak were 7 recombinant proteins in the patients with acute advanced Q fever. The rate is greater than 40%., so these proteins are expected to become candidate proteins for constructing Q thermal molecular diagnostic reagents.
Recombinant protein antigen GroEL, Com1, Mip, YbgF were used to stimulate dendritic cells derived from bone marrow of mice in vitro, respectively, with the positive control of bainitic Kirk body antigen (WCA) and Escherichia coli lipopolysaccharide (LPS), and the negative control of the labeled protein (TrxA) expressed by pET-32a (+), and the simulation of the protein elution buffer (elution buffer) as a simulation. Dendritic cells stimulated by.24h were collected and the surface molecules of dendritic cells were analyzed by flow cytometry. The results showed that the surface molecular expression of dendritic cells with different antigens was significantly higher than that in the simulated stimulus group. Then the dendritic cells activated by different antigens were transferred to normal mice, respectively, for 1 days, 7 days, and 14 days after metastasis. Do not use the bainite Kirk strain to attack the recipient mice, and use quantitative PCR to detect the load of the bainite Kirk body in the spleen of the mice. The results showed that the Kirk somatic load of mice receiving WCA, Com1 or Mip activated dendritic cells was significantly lower than that of the negative control group that did not receive any stimulation, while other antigens (GroEL/YbgF/TrxA/LPS) activated the dendritic cells. There was no significant difference in the load of the bainite Kirk body in the cells of the mice compared with the negative control group. It indicated that the recombinant protein antigen Com1 and Mip could induce specific immune protection, which was a protective antigen of bainite Kirk.
The recombinant protein GroEL, Com1, and Mip stimulated dendritic cells were co cultured with purified CD4+ and CD8+T cells respectively. The total bacterial antigen (WCA), tagged protein TrxA, elution buffer stimulated dendritic cells and T cells were co cultured as positive and negative control dendritic cells that collected antigen stimulated by the negative control, and the T cells were analyzed by flow cytometry. The cell surface molecules, the results showed that the expression level of the surface molecule CD69 of the dendritic cells stimulated by whole bacteria antigen (WCA) and recombinant protein GroEL, Com1, and Mip was significantly higher than that of the control stimulus group. The expression of cytokine in T cells was analyzed by flow cytometry, and the IFN- of T cells interacting with WCA, Com1 or Mip activated dendritic cells The level of gamma expression is significantly higher than that of T cells in other groups, suggesting that the specific immune protection mediated by dendritic cells stimulated by protective antigen is closely related to the antigen stimulated T cells to express the high level of IFN- gamma and the subsequent activation of the bactericidal activity of macrophages.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R392.1

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