致禽肾脏、肠道病变大肠杆菌菌影疫苗的制备及免疫效果的研究
发布时间：2018-08-09 09:38
【摘要】：禽类致病性大肠杆菌(Avain pathogenic Escherichia coli;APEC)的病原体会引起大多数的禽类感染大肠杆菌病。尽管对禽类致病性大肠杆菌病已经有一个多世纪的认识,但这种病仍然是影响全世界家禽业的主要地方性疾病之一。致禽肾脏、肠道病变大肠杆菌能使雏鸡肾脏肿大甚至出血,这在大肠杆菌的临床症状上是极其少见的,并且该菌对青、链霉素和庆大霉素有很强的耐药性,研发新型有效的疫苗对预防该病有着很重要的意义。随着耐药菌株和多重耐药株的产生,抗生素的效果也不如以前理想,灭活苗有较好的保护效果但存在血清型特异性的缺点,因此新型疫苗的研发逐渐成为科研工作者关注的热点。菌影(bacterial ghost,BG)是一种保留了细菌表面完整的形态结构和抗原决定簇,但却不含有核酸等物质的细菌空壳,具有很好的免疫原性和佐剂功能。本实验旨在制备高裂解效率的致禽肾脏、肠道病变大肠杆菌菌影,构建重组温控双裂解表达质粒,其方法为采用融合PCR的方法,通过15个柔性氨基酸linker将噬菌体中金黄色葡萄球菌基因组的核酸酶A基因(SN)和Φ174的裂解蛋白E基因串联,插入温控表达质粒pBV220。PCR扩增其温控双基因裂解表达系统后插入穿梭质粒pBA1100,构建重组穿梭质粒pBA1100-DLS-ES,并将构建好的重组穿梭质粒电转化致禽肾脏、肠道病变大肠杆菌中,低温集菌升温诱导制备菌影。结果表明,裂解效率可高达99.9999%,电镜检测显示细菌内容物被排出而保存了完整的细菌形态,成功制备了致禽肾脏、肠道病变大肠杆菌菌影,为新型疫苗的制备提供依据。通过动物试验对制备的致禽肾脏、肠道病变大肠杆菌菌影疫苗进行免疫保护效果研究。将健康的雏鸡,随机的分成4组,皮下分别接种用不同方式制备的疫苗进行免疫:甲醛灭活组,单裂解菌影苗免疫组和双裂解菌影苗免疫组,对照组。每组实验动物注射剂量都为0.2mL(含菌量约为1*109CFU/mL),对照组注射等体积(0.2mL)生理盐水,一免两周后加强免疫一次。二免后两周用致禽肾脏、肠道病变大肠杆菌7.3X107CFU/只的剂量进行肌肉注射攻毒,通过保护率和血清抗体动态检测等来进行免疫保护效果的研究。结果表明,菌影疫苗的免疫保护作用明显优于甲醛灭活苗组。本研究为耐药大肠杆菌病的预防和治疗提供了一种新技术和新工具。
[Abstract]:The pathogen of avian pathogenic Escherichia coli (Avain pathogenic Escherichia coli) causes most birds to be infected with E. coli. Although there has been more than a century of understanding of avian pathogenic Escherichia coli disease, it is still one of the major endemic diseases affecting poultry industry all over the world. E. coli, which causes kidney and intestinal lesions in poultry, can cause kidney enlargement or even bleeding in chickens, which is extremely rare in clinical symptoms of Escherichia coli, and the bacterium is highly resistant to cyanobactericin, streptomycin and gentamicin. It is very important to develop new and effective vaccine to prevent the disease. With the emergence of drug-resistant and multidrug resistant strains, the effect of antibiotics was not as good as before. Inactivated vaccine had better protective effect but had the disadvantage of serotype specificity. Therefore, the research and development of new vaccines has gradually become the focus of attention of researchers. Bacterial ghostBG is a kind of bacterial shell which retains the complete morphology and antigenic determinant of bacteria surface, but does not contain nucleic acid and other substances. It has good immunogenicity and adjuvant function. The purpose of this study was to prepare Escherichia coli (E. coli) with high lytic efficiency in poultry kidney and intestinal lesions, and to construct recombinant thermo-controlled double lytic expression plasmids. The method of fusion of PCR was used. The nuclease A gene (SN) of Staphylococcus aureus genome and the cleavage protein E gene of 桅 174 were linked by 15 flexible amino acid linker. The recombinant shuttle plasmid pBA1100-DLS-ESS was constructed by inserting the thermo-controlled double gene cleavage expression system into pBV220.PCR, and the recombinant shuttle plasmid pBA1100-DLS-ESS was constructed. The recombinant shuttle plasmid was electrotransformed into poultry kidney and intestinal lesion Escherichia coli. The bacteria shadow was prepared by low temperature collection and heating induction. The results showed that the cleavage efficiency could be as high as 99.99999.The electron microscopic examination showed that the bacterial contents were excreted and preserved the complete bacterial morphology, and successfully prepared the Escherichia coli shadow caused by poultry kidney and intestinal lesions, which provided the basis for the preparation of the new vaccine. The protective effect of the vaccine against Escherichia coli was studied by animal test. The healthy chicks were randomly divided into 4 groups, which were inoculated subcutaneously with vaccines prepared by different methods: formaldehyde inactivated group, single lytic bacteria vaccine immunization group and double lytic bacteria shadow vaccine immunization group, control group. Each group was injected with 0.2mL (containing about 1*109CFU/mL), while the control group was injected with normal saline of equal volume (0.2mL). Two weeks after the second immunization, the mice were injected intramuscularly with the dose of 7.3X107CFU/ in poultry kidney and intestinal lesions, and the protective effect was studied by dynamic detection of protective rate and serum antibody. The results showed that the immune protection of the vaccine was better than that of formaldehyde inactivated vaccine. This study provides a new technique and a new tool for the prevention and treatment of drug-resistant Escherichia coli disease.
【学位授予单位】：长江大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.4
本文编号：2173677
[Abstract]:The pathogen of avian pathogenic Escherichia coli (Avain pathogenic Escherichia coli) causes most birds to be infected with E. coli. Although there has been more than a century of understanding of avian pathogenic Escherichia coli disease, it is still one of the major endemic diseases affecting poultry industry all over the world. E. coli, which causes kidney and intestinal lesions in poultry, can cause kidney enlargement or even bleeding in chickens, which is extremely rare in clinical symptoms of Escherichia coli, and the bacterium is highly resistant to cyanobactericin, streptomycin and gentamicin. It is very important to develop new and effective vaccine to prevent the disease. With the emergence of drug-resistant and multidrug resistant strains, the effect of antibiotics was not as good as before. Inactivated vaccine had better protective effect but had the disadvantage of serotype specificity. Therefore, the research and development of new vaccines has gradually become the focus of attention of researchers. Bacterial ghostBG is a kind of bacterial shell which retains the complete morphology and antigenic determinant of bacteria surface, but does not contain nucleic acid and other substances. It has good immunogenicity and adjuvant function. The purpose of this study was to prepare Escherichia coli (E. coli) with high lytic efficiency in poultry kidney and intestinal lesions, and to construct recombinant thermo-controlled double lytic expression plasmids. The method of fusion of PCR was used. The nuclease A gene (SN) of Staphylococcus aureus genome and the cleavage protein E gene of 桅 174 were linked by 15 flexible amino acid linker. The recombinant shuttle plasmid pBA1100-DLS-ESS was constructed by inserting the thermo-controlled double gene cleavage expression system into pBV220.PCR, and the recombinant shuttle plasmid pBA1100-DLS-ESS was constructed. The recombinant shuttle plasmid was electrotransformed into poultry kidney and intestinal lesion Escherichia coli. The bacteria shadow was prepared by low temperature collection and heating induction. The results showed that the cleavage efficiency could be as high as 99.99999.The electron microscopic examination showed that the bacterial contents were excreted and preserved the complete bacterial morphology, and successfully prepared the Escherichia coli shadow caused by poultry kidney and intestinal lesions, which provided the basis for the preparation of the new vaccine. The protective effect of the vaccine against Escherichia coli was studied by animal test. The healthy chicks were randomly divided into 4 groups, which were inoculated subcutaneously with vaccines prepared by different methods: formaldehyde inactivated group, single lytic bacteria vaccine immunization group and double lytic bacteria shadow vaccine immunization group, control group. Each group was injected with 0.2mL (containing about 1*109CFU/mL), while the control group was injected with normal saline of equal volume (0.2mL). Two weeks after the second immunization, the mice were injected intramuscularly with the dose of 7.3X107CFU/ in poultry kidney and intestinal lesions, and the protective effect was studied by dynamic detection of protective rate and serum antibody. The results showed that the immune protection of the vaccine was better than that of formaldehyde inactivated vaccine. This study provides a new technique and a new tool for the prevention and treatment of drug-resistant Escherichia coli disease.
【学位授予单位】：长江大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.4
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