痤疮丙酸杆菌（PA）抗体预防胸膜肺炎放线杆菌（APP）感染及PA与APP共同抗原表位的筛选与鉴定

发布时间：2018-02-22 01:46

本文关键词： 痤疮丙酸杆菌抗体胸膜肺炎放线杆菌预防感染共同抗原表位　出处：《吉林大学》2015年硕士论文　论文类型：学位论文

【摘要】：猪胸膜肺炎放线杆菌（Actinobacillus pleuropneumoniae，APP）是一种革兰氏阴性小球杆菌，能够造成猪传染性胸膜肺炎，对世界养猪业造成巨大的经济损失。APP分为15个血清型，由于主要的血清型之间缺乏交叉免疫保护导致疫苗研究进展缓慢。痤疮丙酸杆菌（Propionibacterium acnes，PA）是一种广泛存在于人或动物体表、体腔及病灶中的革兰氏阳性短棒状杆菌，具有佐剂作用，能够刺激机体单核巨噬细胞系统及炎性因子的释放，对抗多种病原菌的感染。本课题组在前期系统筛选APP主要血清型之间的差异基因过程中，意外地发现PA与APP之间存在很强的交叉免疫反应，PA可以预防APP感染。PA对APP感染的特异性预防作用是以体液免疫为主的，PA免疫后刺激B细胞产生的IgG抗体能够显著提高小鼠的存活率，抗体水平越高，保护效果越好。但是PA的抗体通过何种方式发挥作用，以及究竟是哪些表位诱导产生的抗体来预防APP感染尚不清楚。为阐明PA抗体在预防APP感染过程中的作用方式，寻找PA与APP之间具有交叉免疫保护作用的共同抗原表位，促进猪传染性胸膜肺炎新型疫苗的研究，本研究首先分别构建中性粒细胞、巨噬细胞损耗小鼠模型，给予足量PA抗体后用APP攻毒，测定小鼠保护率、肺部细菌定植量以及肺脏病理变化，评价PA抗体在预防APP感染过程中对中性粒细胞和巨噬细胞的依赖性，阐明PA抗体在预防APP感染过程中的作用方式。随后构建PA全基因组噬菌体随机展示文库，以APP1高免血清做为固相靶分子对构建的文库进行筛选；对筛选到的蛋白进行B细胞表位预测，并与APP1进行比对，寻找与APP1同源率高的表位进行合成；以PA高免血清及APP1高免血清对合成的多肽进行筛选，筛选得到的多肽与KLH偶联后免疫小鼠，ELISA检测免疫后IgG水平、各抗血清与PA及APP的血清学反应以及IL-4、IFN-γ水平，流式分析脾脏中CD4+/CD8+T细胞的比例，并测定APP1攻毒后各免疫组小鼠存活情况，综合分析表位肽的免疫效果以及预防APP感染的能力；之后将筛选出的具有保护效果的共同抗原表位肽联合免疫小鼠，APP1及APP5分别攻毒后，评价共同抗原表位肽保护小鼠对抗不同型APP感染的能力。实验表明抗PA的IgG能够显著促进巨噬细胞对APP的吞噬作用，吞噬效果与抗APP的IgG相似；巨噬细胞损耗的小鼠，无论是否给予PA抗体，均不能有效对抗APP感染，PA抗体预防APP感染过程中必须依赖巨噬细胞；而中性粒细胞无论损耗与否，PA抗体均能够保护小鼠对抗APP的感染，PA抗体在预防APP过程中不依赖于中性粒细胞。构建PA全基因组噬菌体展示文库，库容量为105，应用该文库筛选得到10个PA与APP共同抗原蛋白；对筛选到的抗原蛋白进行B细胞表位预测并与APP1进行比对后，获得6个与APP1同源率高的表位，分别为A2、Ba1、Ba2、Bb4、Bb5及C1；除A2外，其余五个表位均与抗PA高免血清及抗APP1高免血清有特异性反应。五个多肽免疫小鼠后抗体水平均有所增加，多肽Ba1、Bb5及C1抗血清与PA及APP抗原具有特异性反应，而多肽Ba2、Bb4没有特异性反应；免疫荧光显示，Ba1、Bb5抗血清能够较好地识别APP1。多肽Ba1、Bb5、C1免疫后小鼠的IL-4水平上升，而仅Ba5免疫的小鼠IFN-γ水平上调，表明Bb5诱导Th1/Th2混合免疫应答，Ba1、C1倾向于Th2免疫应答；多肽Ba1、Bb5免疫后CD4+/CD8+T细胞的比值高于对照组，CD4+T细胞占优势，体液免疫起主导作用；APP1攻毒后，多肽Ba1、Bb5对小鼠的保护率均为80%。多肽Ba1、Bb5联合免疫小鼠后，对APP5型攻毒小鼠的保护率为90%，对APP1型攻毒小鼠的保护率为80%。本研究初步阐明了PA抗体在预防APP感染中的作用方式，获得并鉴定2个具有保护作用的PA与APP的共同抗原B细胞表位，为研究APP新型疫苗奠定基础，同时也为异源疫苗研究提供有益的理论基础。
[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.

【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.61

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