本文關(guān)鍵詞： 痤瘡丙酸桿菌抗體 胸膜肺炎放線桿菌 預(yù)防 感染 共同抗原表位　出處：《吉林大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：豬胸膜肺炎放線桿菌（Actinobacillus pleuropneumoniae，APP）是一種革蘭氏陰性小球桿菌，能夠造成豬傳染性胸膜肺炎，對世界養(yǎng)豬業(yè)造成巨大的經(jīng)濟損失。APP分為15個血清型，由于主要的血清型之間缺乏交叉免疫保護(hù)導(dǎo)致疫苗研究進(jìn)展緩慢。痤瘡丙酸桿菌（Propionibacterium acnes，PA）是一種廣泛存在于人或動物體表、體腔及病灶中的革蘭氏陽性短棒狀桿菌，具有佐劑作用，能夠刺激機體單核巨噬細(xì)胞系統(tǒng)及炎性因子的釋放，對抗多種病原菌的感染。 本課題組在前期系統(tǒng)篩選APP主要血清型之間的差異基因過程中，意外地發(fā)現(xiàn)PA與APP之間存在很強的交叉免疫反應(yīng)，PA可以預(yù)防APP感染。PA對APP感染的特異性預(yù)防作用是以體液免疫為主的，PA免疫后刺激B細(xì)胞產(chǎn)生的IgG抗體能夠顯著提高小鼠的存活率，抗體水平越高，保護(hù)效果越好。但是PA的抗體通過何種方式發(fā)揮作用，以及究竟是哪些表位誘導(dǎo)產(chǎn)生的抗體來預(yù)防APP感染尚不清楚。 為闡明PA抗體在預(yù)防APP感染過程中的作用方式，尋找PA與APP之間具有交叉免疫保護(hù)作用的共同抗原表位，促進(jìn)豬傳染性胸膜肺炎新型疫苗的研究，本研究首先分別構(gòu)建中性粒細(xì)胞、巨噬細(xì)胞損耗小鼠模型，給予足量PA抗體后用APP攻毒，測定小鼠保護(hù)率、肺部細(xì)菌定植量以及肺臟病理變化，評價PA抗體在預(yù)防APP感染過程中對中性粒細(xì)胞和巨噬細(xì)胞的依賴性，闡明PA抗體在預(yù)防APP感染過程中的作用方式。隨后構(gòu)建PA全基因組噬菌體隨機展示文庫，以APP1高免血清做為固相靶分子對構(gòu)建的文庫進(jìn)行篩選；對篩選到的蛋白進(jìn)行B細(xì)胞表位預(yù)測，并與APP1進(jìn)行比對，尋找與APP1同源率高的表位進(jìn)行合成；以PA高免血清及APP1高免血清對合成的多肽進(jìn)行篩選，篩選得到的多肽與KLH偶聯(lián)后免疫小鼠，ELISA檢測免疫后IgG水平、各抗血清與PA及APP的血清學(xué)反應(yīng)以及IL-4、IFN-γ水平，流式分析脾臟中CD4+/CD8+T細(xì)胞的比例，并測定APP1攻毒后各免疫組小鼠存活情況，綜合分析表位肽的免疫效果以及預(yù)防APP感染的能力；之后將篩選出的具有保護(hù)效果的共同抗原表位肽聯(lián)合免疫小鼠，APP1及APP5分別攻毒后，評價共同抗原表位肽保護(hù)小鼠對抗不同型APP感染的能力。 實驗表明抗PA的IgG能夠顯著促進(jìn)巨噬細(xì)胞對APP的吞噬作用，吞噬效果與抗APP的IgG相似；巨噬細(xì)胞損耗的小鼠，無論是否給予PA抗體，均不能有效對抗APP感染，PA抗體預(yù)防APP感染過程中必須依賴巨噬細(xì)胞；而中性粒細(xì)胞無論損耗與否，PA抗體均能夠保護(hù)小鼠對抗APP的感染，PA抗體在預(yù)防APP過程中不依賴于中性粒細(xì)胞。構(gòu)建PA全基因組噬菌體展示文庫，庫容量為105，應(yīng)用該文庫篩選得到10個PA與APP共同抗原蛋白；對篩選到的抗原蛋白進(jìn)行B細(xì)胞表位預(yù)測并與APP1進(jìn)行比對后，獲得6個與APP1同源率高的表位，分別為A2、Ba1、Ba2、Bb4、Bb5及C1；除A2外，其余五個表位均與抗PA高免血清及抗APP1高免血清有特異性反應(yīng)。五個多肽免疫小鼠后抗體水平均有所增加，多肽Ba1、Bb5及C1抗血清與PA及APP抗原具有特異性反應(yīng)，而多肽Ba2、Bb4沒有特異性反應(yīng)；免疫熒光顯示，Ba1、Bb5抗血清能夠較好地識別APP1。多肽Ba1、Bb5、C1免疫后小鼠的IL-4水平上升，而僅Ba5免疫的小鼠IFN-γ水平上調(diào)，表明Bb5誘導(dǎo)Th1/Th2混合免疫應(yīng)答，Ba1、C1傾向于Th2免疫應(yīng)答；多肽Ba1、Bb5免疫后CD4+/CD8+T細(xì)胞的比值高于對照組，CD4+T細(xì)胞占優(yōu)勢，體液免疫起主導(dǎo)作用；APP1攻毒后，多肽Ba1、Bb5對小鼠的保護(hù)率均為80%。多肽Ba1、Bb5聯(lián)合免疫小鼠后，對APP5型攻毒小鼠的保護(hù)率為90%，對APP1型攻毒小鼠的保護(hù)率為80%。 本研究初步闡明了PA抗體在預(yù)防APP感染中的作用方式，獲得并鑒定2個具有保護(hù)作用的PA與APP的共同抗原B細(xì)胞表位，為研究APP新型疫苗奠定基礎(chǔ)，同時也為異源疫苗研究提供有益的理論基礎(chǔ)。
[Abstract]:Actinobacillus pleuropneumoniae (Actinobacillus pleuropneumoniae APP) is a gram negative bacilli ball, to Porcine Contagious Pleuropneumonia caused the world pig industry caused enormous economic losses of.APP divided into 15 serotypes, because the main serotype lack of cross immune protection led to the slow pace of vaccine research. P.acne (Propionibacterium acnes PA) is a kind of widely exists in the human or animal body, body cavity and lesions of gram positive Corynebacterium parvum, with adjuvant effect, it can induce the mononuclear macrophage system and inflammatory factor release, against a variety of pathogenic bacteria.
The research group in the pre screening system between APP main serotype gene process, accidentally found a strong cross immune reaction between PA and APP, PA can prevent APP infection.PA specificity for the prevention of APP infection is mainly to humoral immune, IgG antibody after immunization with PA stimulated B cells can significantly improve the survival rate of mice, the antibody level is higher, the better protection. But the PA antibody by the way in which play a role, and what is what the antibody induced epitope to prevent APP infection is not clear.
In order to elucidate the PA antibody in the prevention of APP infection effect in the process of looking for a cross protection between PA and APP common epitope, promote the study of porcine contagious pleuropneumonia vaccine, this study firstly constructed neutrophils, macrophage loss model in mice, with plenty of PA antibody by APP after inoculation. Determination of mice protection rate, pulmonary bacterial colonization and lung pathological changes, evaluation of PA antibody in the prevention of APP infection in the process of neutrophil and macrophage dependent, PA antibody in the prevention of APP infection to clarify the mode of action in the process. Then construct PA whole genome phage display library, using APP1 hyperimmune serum to do on the construction of the solid target molecule library screening; on the screening of the protein of B cell epitope prediction, and compared with that of APP1 and APP1, looking for the high rate of homologous epitope synthesis PA; hyperimmune serum and APP1 on the synthesis of polypeptides were screened hyperimmune serum of mice immunized with KLH peptide, were obtained by coupling, ELISA detected the level of IgG, PA and APP in the serum and serological reaction and IL-4, IFN- levels, CD4+/CD8+T cells in the spleen type analysis and determination of proportion. After infection of APP1 all immunized mice survival, comprehensive analysis of epitope peptide immune effect and the ability to prevent APP infection; then screened the protective effect of common epitope peptide of mice immunized with APP1 and APP5, respectively, after challenge, evaluation of common epitope peptide protection ability of different type APP mice against the infection.
Experiments show that the anti PA IgG could significantly promote the phagocytosis of macrophage phagocytosis of APP, similar effect and anti APP IgG; macrophage depletion in mice, regardless of whether the given PA antibodies are not effective against APP infection, PA antibody dependent macrophage APP cells must prevent infection process; and whether or not the loss of neutrophils, PA antibody could protect mice against APP infection, PA antibody in the prevention of APP in the process is not dependent on neutrophils. Construct PA genome phage display library, a library with a capacity of 105, the application of the library screened 10 PA and APP common antigen protein; B cell epitope prediction and compared with that of APP1 the screening of the antigen protein was obtained after 6 APP1 and the high rate of homologous epitopes, respectively A2, Ba1, Ba2, Bb4, Bb5 and C1; except A2, the remaining five epitopes with hyperimmune serum anti PA and anti APP1 hyperimmune serum. Specific reaction. Antibody level increased, five peptides in mice immunized with peptide Ba1, Bb5 and C1 with PA antiserum and APP antigen has a specific reaction, and peptide Ba2, Bb4 no specific reaction; immunofluorescence showed that Ba1, Bb5 was better able to identify APP1. peptide Ba1, Bb5, IL-4 increased in mice the level of C1 and IFN- after immunization, mice immunized with Ba5 gamma levels increased only, show that the Th1/Th2 hybrid immune response induced by Bb5, Ba1, C1 tend to Th2 immune response; polypeptide Ba1, Bb5 immune CD4+/CD8+T cell ratio is higher than that of control group, CD4+T cells predominate, humoral immunity plays a leading role; after infection of APP1, polypeptide Ba1, Bb5 protected mice against the rate of 80%. peptide Ba1, Bb5 after immunization with the protection of APP5 challenged mice was 90%, the protection of APP1 against the virus in mice was 80%.
This study preliminarily clarified the way of PA antibody in the prevention of APP infection, and identified 2 protective B epitopes of PA and APP common antigen, which laid the foundation for the research of APP new vaccine, and also provided a useful theoretical basis for the research of heterologous vaccine.

【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S852.61

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