猪源沙门氏菌菌蜕的制备及其免疫原性的研究
发布时间：2018-11-24 20:56
【摘要】：沙门氏菌(Salmonella)是重要的人畜共患病病原之一,对牲畜的繁殖和幼畜的健康带来严重威胁,临床上多表现为败血症和肠炎,也可使怀孕母畜发生流产,猪源沙门氏菌一直是危害养猪业发展和动物食品卫生上的重要病原体。细菌菌蜕是指缺少细胞浆、核酸、死的细菌菌体,它是经生理生化方法裂解后形成的完整细菌空壳,其内容物在裂解过程流失,因此具有良好的安全性,且制备和贮存方法简便,可以用来制备多价联合疫苗。与传统使用的变性灭活疫苗相比,菌蜕疫苗没有免疫原性的改变,具有佐剂的性质,且能够载入外源抗原、DNA疫苗、药物等物质,因此是一种优化的候选灭活型疫苗。通过生理生化特性、16SrDNA序列分析、种属特异性PCR鉴定等方法证明实验室保存的细菌为沙门氏菌,通过小鼠半数致死量和药敏试验,筛选毒性强且无耐氨苄抗性的沙门氏菌,为后续实验奠定基础。本实验室构建了温控裂解质粒PBVE220,将其导入沙门氏菌感受态细胞中,筛选得到质粒型猪源沙门氏菌菌蜕。对质粒型猪源沙门氏菌菌蜕进行溶菌动力学试验分析,经优化后的诱导条件,将质粒型猪源沙门氏菌菌蜕培养至OD600值约为0.4时,诱导6h后,得到裂解率为99.46%的沙门氏菌菌蜕,菌蜕数约为7.3×107个/mL,扫描电镜观察菌蜕表面结构完整,可见有内容物由溶菌孔道排出。最后,对质粒型猪源沙门氏菌菌蜕、沙门氏菌灭活苗、沙门氏菌灭活佐剂苗免疫原性比较分析,沙门氏菌菌蜕免疫小鼠后,随着免疫次数的增加,各免疫组血清IgG含量升高,且菌蜕BG组血清IgG含量明显高于灭活组和灭佐组,说明菌蜕疫苗能有效地刺激特异性体液免疫,小鼠脾淋巴细胞亚群检测分析表明质粒型猪源沙门氏菌菌蜕能诱导机体产生Th1型细胞免疫应答,菌蜕BG组细胞因子检测结果显示IFN-γ和IL-12分泌水平明显升高,且显著高于对照组,但IL-4分泌水平与对照组相比差异不显著,说明菌蜕BG组能诱导以Th1型细胞为主的保护性免疫应答。免疫保护效果说明,菌蜕BG组提供的保护率可达70%,且小鼠攻毒后各组织病理学变化可见,菌蜕组的临床症状轻于其他免疫组。综上所述,质粒型猪源沙门氏菌菌蜕能提供较好的安全性和免疫原性,为新型疫苗的研究提供依据,具有广阔的应用前景。
[Abstract]:Salmonella (Salmonella) is one of the most important zoonotic pathogens, which poses a serious threat to the reproduction of livestock and the health of young animals. The clinical manifestations are septicemia and enteritis, as well as miscarriage in pregnant females. Porcine salmonella is an important pathogen that endangers the development of pig industry and animal food hygiene. Bacterial slough refers to the lack of cytoplasm, nucleic acid and dead bacteria. It is a complete bacterial shell formed by physiological and biochemical methods, and its contents are lost in the process of lysis, so it has good safety. The method of preparation and storage is simple and can be used to prepare multivalent combined vaccine. Compared with the traditional denatured inactivated vaccine, the slough vaccine has no change in immunogenicity, has the property of adjuvant, and can load foreign antigen, DNA vaccine, drug and so on, so it is a kind of optimized candidate inactivated vaccine. By physiological and biochemical characteristics, 16SrDNA sequence analysis and species-specific PCR identification, it was proved that the bacteria kept in the laboratory were salmonella. The strains with strong toxicity and no resistance to ampicillin were screened by half lethal dose and drug sensitivity test in mice. It lays the foundation for the following experiment. The plasmids PBVE220, were constructed and introduced into the competent cells of Salmonella. The plasmids of salmonella porcine shedding were screened. The bacteriolytic kinetics of plasmid type porcine salmonella slough was analyzed. After the optimization of inducing conditions, the plasmid type porcine salmonella slough was cultured to a OD600 value of about 0. 4 and induced for 6 hours. Salmonella slough with a cleavage rate of 99.46% was obtained. The number of slough was about 7.3 脳 107 / mL,. The surface structure of the slough was observed by scanning electron microscope (SEM). Finally, the immunogenicity of salmonella slough, salmonella inactivated vaccine and salmonella adjuvant vaccine were compared and analyzed. After immunization with salmonella, the serum IgG content increased with the increase of immunization times. The serum IgG content in BG group was significantly higher than that in inactivated group and inactivated group, which indicated that the vaccine could stimulate specific humoral immunity effectively. The analysis of spleen lymphocyte subsets in mice showed that plasmid type of Salmonella spp. could induce Th1 type cellular immune response. Cytokines in BG group showed that the levels of IFN- 纬 and IL-12 were significantly increased. The level of IL-4 secretion was significantly higher than that of the control group, but the level of IL-4 secretion was not significantly different from that of the control group, which indicated that the mycelia BG group could induce the protective immune response of Th1 type cells. The protective effect of immune protection showed that the protective rate of BG group could reach 70%, and the histopathological changes of mice after toxin attack were obvious, and the clinical symptoms of the group were lighter than those of other immune groups. To sum up, plasmid type salmonella can provide better safety and immunogenicity, which provides basis for the study of new vaccine and has a broad application prospect.
【学位授予单位】：吉林农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S855.1
本文编号：2355020
[Abstract]:Salmonella (Salmonella) is one of the most important zoonotic pathogens, which poses a serious threat to the reproduction of livestock and the health of young animals. The clinical manifestations are septicemia and enteritis, as well as miscarriage in pregnant females. Porcine salmonella is an important pathogen that endangers the development of pig industry and animal food hygiene. Bacterial slough refers to the lack of cytoplasm, nucleic acid and dead bacteria. It is a complete bacterial shell formed by physiological and biochemical methods, and its contents are lost in the process of lysis, so it has good safety. The method of preparation and storage is simple and can be used to prepare multivalent combined vaccine. Compared with the traditional denatured inactivated vaccine, the slough vaccine has no change in immunogenicity, has the property of adjuvant, and can load foreign antigen, DNA vaccine, drug and so on, so it is a kind of optimized candidate inactivated vaccine. By physiological and biochemical characteristics, 16SrDNA sequence analysis and species-specific PCR identification, it was proved that the bacteria kept in the laboratory were salmonella. The strains with strong toxicity and no resistance to ampicillin were screened by half lethal dose and drug sensitivity test in mice. It lays the foundation for the following experiment. The plasmids PBVE220, were constructed and introduced into the competent cells of Salmonella. The plasmids of salmonella porcine shedding were screened. The bacteriolytic kinetics of plasmid type porcine salmonella slough was analyzed. After the optimization of inducing conditions, the plasmid type porcine salmonella slough was cultured to a OD600 value of about 0. 4 and induced for 6 hours. Salmonella slough with a cleavage rate of 99.46% was obtained. The number of slough was about 7.3 脳 107 / mL,. The surface structure of the slough was observed by scanning electron microscope (SEM). Finally, the immunogenicity of salmonella slough, salmonella inactivated vaccine and salmonella adjuvant vaccine were compared and analyzed. After immunization with salmonella, the serum IgG content increased with the increase of immunization times. The serum IgG content in BG group was significantly higher than that in inactivated group and inactivated group, which indicated that the vaccine could stimulate specific humoral immunity effectively. The analysis of spleen lymphocyte subsets in mice showed that plasmid type of Salmonella spp. could induce Th1 type cellular immune response. Cytokines in BG group showed that the levels of IFN- 纬 and IL-12 were significantly increased. The level of IL-4 secretion was significantly higher than that of the control group, but the level of IL-4 secretion was not significantly different from that of the control group, which indicated that the mycelia BG group could induce the protective immune response of Th1 type cells. The protective effect of immune protection showed that the protective rate of BG group could reach 70%, and the histopathological changes of mice after toxin attack were obvious, and the clinical symptoms of the group were lighter than those of other immune groups. To sum up, plasmid type salmonella can provide better safety and immunogenicity, which provides basis for the study of new vaccine and has a broad application prospect.
【学位授予单位】：吉林农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S855.1
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，本文编号：2355020
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