水貂铜绿假单胞菌-肺炎克雷伯菌二联亚单位疫苗的研制及其应用

发布时间：2018-03-26 00:53

本文选题：铜绿假单胞菌　切入点：肺炎克雷伯菌　出处：《吉林大学》2017年硕士论文

【摘要】：目前水貂养殖以散养户为主,养殖户的防疫管理意识淡薄,高密度养殖、粪便污染和抗生素滥用导致携毒力因子的铜绿假单胞菌和肺炎克雷伯菌等环境常在菌出现并大量繁殖,病原菌引起的耐药性问题日益突出。近年来,铜绿假单胞菌和肺炎克雷伯菌引起的水貂肺炎常于水貂的换毛期暴发流行,给养殖户带来重大经济损失。鉴于目前市场应用的疫苗均为临床分离的完整菌体抗原存在携带耐药基因和毒力基因的风险,以及由于血清型的复杂致使免疫效果不确实的问题,本研究从临床分离的貂出血性肺炎强毒株-铜绿假单胞菌精制蛋白组份和肺炎克雷伯菌提取荚膜多糖作为抗原,制备了针对两病原的水貂铜绿假单胞菌-肺炎克雷伯菌二联亚单位疫苗,并应用于水貂养殖场新生仔貂的免疫,验证该疫苗对水貂肺炎的防控效果。本研究分别以常规方法提取并精制的肺炎克雷伯菌荚膜多糖和离子交换层析法提取临床貂出血性肺炎强毒分离株铜绿假单胞菌培养物中抗原成分,将各抗原以氢氧化铝为佐剂进行配苗,设置不同抗原组别,免疫小鼠后进行攻毒保护性实验,实验结果表明:铜绿假单胞菌菌体裂解上清液氢氧化铝佐剂抗原与铜绿假单胞菌灭活菌体抗原免疫小鼠后均能诱导小鼠产生特异性抗体,两种抗原诱导产生抗体水平无显著差异,即两种抗原针对铜绿假单胞菌临床分离强毒株的攻击的水貂具有相当水平的保护作用;肺炎克雷伯菌荚膜多糖具有良好的抗原性,诱导小鼠产生特异性抗体水平高于肺炎克雷伯菌菌体抗原,且差异显著(P㩳0.05)。为验证各抗原对原宿主动物水貂的免疫保护作用,将优化各抗原组份,按灭活疫苗免疫程序免疫水貂,攻毒保护性实验结果表明:铜绿假单胞菌菌体裂解上清液精制的多组份蛋白抗原诱导水貂产生的特异性抗体,抗体水平与铜绿假单胞菌灭活菌体抗原相比差异不显著。将铜绿假单胞菌菌体裂解上清液精制的多组份蛋白和肺炎克雷伯菌荚膜多糖抗原以氢氧化铝为佐剂进行配苗,制备出铜绿假单胞菌-肺炎克雷伯菌二联亚单位疫苗中荚膜多糖具有良好的抗原性,对水貂铜绿假单胞菌和肺炎克雷伯菌引起的肺炎具有100%的保护作用。批量制备铜绿假单胞菌-肺炎克雷伯菌二联亚单位疫苗,在水貂养殖场推广应用研究结果显示:在水貂生产中,铜绿假单胞菌-肺炎克雷伯菌二联亚单位疫苗免疫当年生产的仔貂对铜绿假单胞菌和肺炎克雷伯菌引起的水貂肺炎具有良好的保护作用。综上,肺炎克雷伯菌荚膜多糖与铜绿假单胞菌精制多蛋白组份抗原制备二联亚单位疫苗能够克服两病原的毒力增强及耐药性问题,对水貂具有良好的保护效果,可以作为一种用于水貂肺炎防控的安全、高效、无生物风险的亚单位候选疫苗。
[Abstract]:At present, mink farming is dominated by scattered farmers, whose awareness of epidemic prevention management is weak, high density culture, fecal pollution and abuse of antibiotics lead to the presence of Pseudomonas aeruginosa and Klebsiella pneumoniae, which carry virulence factors, and so on. In recent years, mink pneumonia caused by Pseudomonas aeruginosa and Klebsiella pneumoniae has become more prevalent in mink than in mink. Since the vaccines currently used in the market are all clinical isolates of intact bacterial antigens, there is a risk of carrying drug-resistant and virulent genes, and because of the complexity of serotypes, the immune effect is uncertain. In this study, capsule polysaccharides were extracted from purified protein components of Pseudomonas aeruginosa strain and Klebsiella pneumoniae strain as antigen. A binomial vaccine against Pseudomonas aeruginosa and Klebsiella pneumoniae was prepared and used to immunize minks in mink farms. To verify the efficacy of the vaccine in the prevention and control of mink pneumonia. In this study, we used routine extraction and purification of Klebsiella pneumoniae capsule polysaccharides and ion exchange chromatography to extract pseudomonas aeruginosa from mink hemorrhagic pneumonia isolates. Antigenic components in bacterial cultures, Each antigen was inoculated with aluminum hydroxide as adjuvant, and different antigen groups were set up. The results showed that both aluminum hydroxide adjuvant antigen and inactivated Pseudomonas aeruginosa antigen could induce the production of specific antibodies in mice after immunization with the supernatant of Pseudomonas aeruginosa. There was no significant difference between the two antigen-induced antibody levels, that is, the minks attacked by the two antigens against the clinical isolates of Pseudomonas aeruginosa had a significant protective effect, and Klebsiella pneumoniae capsule polysaccharides had good antigenicity. The specific antibody level induced in mice was higher than that of Klebsiella pneumoniae antigen, and the difference was significant (P < 0.05). In order to verify the immune protective effect of each antigen on mink, the antigen components will be optimized and the mink will be immunized according to the program of inactivated vaccine immunization. The results showed that the purified multicomponent protein antigen of Pseudomonas aeruginosa induced the specific antibody of mink. There was no significant difference between the antibody level and the inactivated bacterial antigen of Pseudomonas aeruginosa. The multicomponent protein purified from the supernatant of Pseudomonas aeruginosa and the capsule polysaccharide antigen of Klebsiella pneumoniae were mixed with aluminum hydroxide as adjuvant. The capsule polysaccharides of Pseudomonas aeruginosa and Klebsiella pneumoniae double subunit vaccine were prepared. Pseudomonas aeruginosa and Klebsiella pneumoniae have a 100% protective effect on pneumonia caused by Pseudomonas aeruginosa and Klebsiella pneumoniae. The results of the research on popularization and application in mink farms show that: in mink production, Pseudomonas aeruginosa-Klebsiella pneumoniae duplex subunit vaccine has a good protective effect on mink pneumonia caused by Pseudomonas aeruginosa and Klebsiella pneumoniae. The preparation of biphasic subunit vaccine with Klebsiella pneumoniae capsule polysaccharide and purified polyprotein component antigen of Pseudomonas aeruginosa can overcome the problems of virulence enhancement and drug resistance of the two pathogens and have a good protective effect on mink. It can be used as a safe, efficient and biohazard subunit candidate vaccine for mink pneumonia prevention and control.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S858.92

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