本文選題：貝氏柯克斯體 + Q熱　； 參考：《中國人民解放軍軍事醫(yī)學科學院》2011年博士論文

【摘要】：貝氏柯克斯體(Coxiella burnetii)是Q熱的病原菌,對人和動物有極強的感染力,可通過氣溶膠擴散經(jīng)呼吸道進入體內(nèi)導致感染。人類感染貝氏柯克斯體可引發(fā)肺炎、肝炎、心內(nèi)膜炎等嚴重疾病。貝氏柯克斯體的高致病性和對環(huán)境、理化因素的高抵抗力使其成為重要的生物戰(zhàn)劑/生物恐怖劑。 Q熱最常用的特異性診斷方法為血清學診斷,包括間接免疫熒光(IFA)技術(shù),補體結(jié)合(CF)實驗,酶聯(lián)免疫吸附(ELISA)技術(shù)等。這些檢測方法都需要純化的貝氏柯克斯體全菌作為抗原,由于提取純化貝氏柯克斯體的防護要求高、工藝復雜,造成了這些檢測方法難以大規(guī)模推廣和使用。疫苗接種為預防貝氏柯克斯體感染的有效手段。雖然滅活貝氏柯克斯體全菌疫苗具有良好的免疫保護效果,但是其難以制備和較強的副作用限制了它的推廣應用。因此,許多國家和地區(qū)都致力于研制安全、可靠、副作用小的新型Q熱疫苗。本研究通過高通量的免疫蛋白質(zhì)組學來篩選貝氏柯克斯體菌體蛋白抗原,并制備相應的重組蛋白,以及評價重組蛋白抗原的免疫原性和免疫保護效能,為Q熱分子疫苗和分子診斷試劑的研發(fā)提供候選蛋白抗原。 本研究采用泛影葡胺線性密度梯度法從雞胚培養(yǎng)物中分離出貝氏柯克斯體新橋株。用純化的貝氏柯克斯體新橋株腹腔接種感染小鼠,于感染后1、2、3、4周分別處死小鼠,收集不同感染時期的抗血清,并采用間接免疫熒光法檢測感染小鼠血清特異性抗體水平。再用貝氏柯克斯體特異的實時熒光定量PCR檢測感染小鼠組織中的貝氏柯克斯體載量,以分析新橋株對BALB/c小鼠的感染特征。結(jié)果顯示感染后4周內(nèi),小鼠肝、脾、肺組織中均檢出大量貝氏柯克斯體,第1周檢出量最高,脾的含量顯著高于肝和肺,肝的含量顯著高于肺。隨著感染時間延長,貝氏柯克斯體檢出量呈下降趨勢,但血清特異性抗體水平則繼續(xù)升高。 隨后利用雙向電泳技術(shù)將純化貝氏柯克斯體全菌蛋白進行分離,用貝氏柯克斯體實驗感染小鼠血清與轉(zhuǎn)移到PVDF膜上的貝氏柯克斯體蛋白進行免疫印跡反應,并通過蛋白質(zhì)譜技術(shù)完成了對這些蛋白的鑒定。結(jié)果顯示1、2、3、4周感染小鼠血清鑒定的印跡反應陽性蛋白分別為0、4、9、14個,其中印跡反應最強烈的4個蛋白為GroEL、Com1、Mip、OmpH。用Q熱患者血清與貝氏柯克斯體蛋白進行免疫印跡反應,鑒定出了15個印跡反應陽性蛋白,其中有9個蛋白為Q熱患者血清與貝氏柯克斯體感染小鼠血清共同識別抗原。 采用分子克隆技術(shù),將鑒定的免疫印跡反應陽性蛋白的基因克隆和重組表達。除了rspB基因,其他19個印跡反應陽性蛋白基因均被高效表達。采用鎳離子親和層析方法(Ni-NTA)分別從IPTG誘導的轉(zhuǎn)化菌中純化19個重組蛋白。將純化的重組蛋白點制成一張蛋白芯片,以此芯片與貝氏柯克斯體感染的鼠血清反應。結(jié)果與貝氏柯克斯體感染1、2、3、4周小鼠血清反應的蛋白為0、16、19、18個,其中反應熒光強度最強的4個重組蛋白依次為GroEL、Mip、OmpH、Com1。將蛋白芯片與56份Q熱病人血清和25份正常人血清進行反應,當單份病人血清與單個蛋白點的反應熒光信號值大于正常人血清與對應蛋白點反應熒光信號平均值加2個標準差時結(jié)果為陽性。結(jié)果GroEl、YbgF、RplL、Mip、OmpH、Com1、Dnak共7個重組蛋白被急性晚期Q熱病人血清識別的陽性率大于40%。因此,這些蛋白有望成為構(gòu)建Q熱分子診斷試劑的侯選蛋白。 將重組蛋白抗原GroEL、Com1、Mip、YbgF分別刺激體外培養(yǎng)的小鼠骨髓來源的樹突狀細胞,以貝氏柯克斯體全菌抗原(WCA)和大腸桿菌脂多糖(LPS)作陽性對照,以pET-32a(+)表達的標簽蛋白(TrxA)作陰性對照,并以蛋白洗脫緩沖液(elution buffer)作為模擬刺激。24h后收集抗原刺激的樹突狀細胞,用流式細胞儀分析樹突狀細胞表面分子,結(jié)果發(fā)現(xiàn)不同抗原刺激樹突狀細胞的表面分子表達顯著高于模擬刺激組。再將不同抗原激活的樹突狀細胞分別腹腔轉(zhuǎn)移至正常小鼠,轉(zhuǎn)移后1天、7天、14天分別用貝氏柯克斯體毒株攻擊受體小鼠,并采用定量PCR檢測小鼠脾臟貝氏柯克斯體載量,結(jié)果顯示接受WCA、Com1或Mip激活樹突狀細胞的小鼠柯克斯體載量顯著低于未接受任何刺激的陰性對照組,而接受其它抗原(GroEL/YbgF/TrxA/LPS)激活樹突狀細胞的小鼠貝氏柯克斯體載量與陰性對照組相比無顯著性差異。說明重組蛋白抗原Com1和Mip能夠誘導特異性免疫保護,為貝氏柯克斯體保護性抗原。 將重組蛋白GroEL、Com1、Mip刺激的樹突狀細胞分別與純化的CD4+和CD8+T細胞共培養(yǎng),以全菌抗原(WCA)、標簽蛋白TrxA、elution buffer刺激的樹突狀細胞與T細胞共培養(yǎng)分別作為陽性和陰性對照。24h后收集抗原刺激的樹突狀細胞,用流式細胞技術(shù)分析T細胞表面分子,結(jié)果發(fā)現(xiàn)全菌抗原(WCA)和重組蛋白GroEL、Com1、Mip刺激樹突狀細胞的表面分子CD69的表達水平顯著高于對照刺激組。再用流式細胞技術(shù)分析T細胞細胞因子表達情況,結(jié)果顯示與WCA、Com1或Mip激活樹突細胞相互作用的T細胞的IFN-γ表達水平均顯著高于與其它組的T細胞,提示保護性抗原刺激的樹突狀細胞介導的特異性免疫保護與該抗原刺激T細胞表達高水平的IFN-γ以及隨后激活巨噬細胞的殺菌活性密切相關(guān)。
[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.
【學位授予單位】：中國人民解放軍軍事醫(yī)學科學院
【學位級別】：博士
【學位授予年份】：2011
【分類號】：R392.1

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