【摘要】：沙門氏菌(Salmonella)是重要的人畜共患病病原之一,對牲畜的繁殖和幼畜的健康帶來嚴重威脅,臨床上多表現(xiàn)為敗血癥和腸炎,也可使懷孕母畜發(fā)生流產(chǎn),豬源沙門氏菌一直是危害養(yǎng)豬業(yè)發(fā)展和動物食品衛(wèi)生上的重要病原體。細菌菌蛻是指缺少細胞漿、核酸、死的細菌菌體,它是經(jīng)生理生化方法裂解后形成的完整細菌空殼,其內(nèi)容物在裂解過程流失,因此具有良好的安全性,且制備和貯存方法簡便,可以用來制備多價聯(lián)合疫苗。與傳統(tǒng)使用的變性滅活疫苗相比,菌蛻疫苗沒有免疫原性的改變,具有佐劑的性質,且能夠載入外源抗原、DNA疫苗、藥物等物質,因此是一種優(yōu)化的候選滅活型疫苗。通過生理生化特性、16SrDNA序列分析、種屬特異性PCR鑒定等方法證明實驗室保存的細菌為沙門氏菌,通過小鼠半數(shù)致死量和藥敏試驗,篩選毒性強且無耐氨芐抗性的沙門氏菌,為后續(xù)實驗奠定基礎。本實驗室構建了溫控裂解質粒PBVE220,將其導入沙門氏菌感受態(tài)細胞中,篩選得到質粒型豬源沙門氏菌菌蛻。對質粒型豬源沙門氏菌菌蛻進行溶菌動力學試驗分析,經(jīng)優(yōu)化后的誘導條件,將質粒型豬源沙門氏菌菌蛻培養(yǎng)至OD600值約為0.4時,誘導6h后,得到裂解率為99.46%的沙門氏菌菌蛻,菌蛻數(shù)約為7.3×107個/mL,掃描電鏡觀察菌蛻表面結構完整,可見有內(nèi)容物由溶菌孔道排出。最后,對質粒型豬源沙門氏菌菌蛻、沙門氏菌滅活苗、沙門氏菌滅活佐劑苗免疫原性比較分析,沙門氏菌菌蛻免疫小鼠后,隨著免疫次數(shù)的增加,各免疫組血清IgG含量升高,且菌蛻B(tài)G組血清IgG含量明顯高于滅活組和滅佐組,說明菌蛻疫苗能有效地刺激特異性體液免疫,小鼠脾淋巴細胞亞群檢測分析表明質粒型豬源沙門氏菌菌蛻能誘導機體產(chǎn)生Th1型細胞免疫應答,菌蛻B(tài)G組細胞因子檢測結果顯示IFN-γ和IL-12分泌水平明顯升高,且顯著高于對照組,但IL-4分泌水平與對照組相比差異不顯著,說明菌蛻B(tài)G組能誘導以Th1型細胞為主的保護性免疫應答。免疫保護效果說明,菌蛻B(tài)G組提供的保護率可達70%,且小鼠攻毒后各組織病理學變化可見,菌蛻組的臨床癥狀輕于其他免疫組。綜上所述,質粒型豬源沙門氏菌菌蛻能提供較好的安全性和免疫原性,為新型疫苗的研究提供依據(jù),具有廣闊的應用前景。
[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- 緯 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.
【學位授予單位】：吉林農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S855.1

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本文編號：2355020