致禽腎臟、腸道病變大腸桿菌菌影疫苗的制備及免疫效果的研究
發(fā)布時間：2018-08-09 09:38
【摘要】：禽類致病性大腸桿菌(Avain pathogenic Escherichia coli;APEC)的病原體會引起大多數(shù)的禽類感染大腸桿菌病。盡管對禽類致病性大腸桿菌病已經(jīng)有一個多世紀的認識,但這種病仍然是影響全世界家禽業(yè)的主要地方性疾病之一。致禽腎臟、腸道病變大腸桿菌能使雛雞腎臟腫大甚至出血,這在大腸桿菌的臨床癥狀上是極其少見的,并且該菌對青、鏈霉素和慶大霉素有很強的耐藥性,研發(fā)新型有效的疫苗對預防該病有著很重要的意義。隨著耐藥菌株和多重耐藥株的產(chǎn)生,抗生素的效果也不如以前理想,滅活苗有較好的保護效果但存在血清型特異性的缺點,因此新型疫苗的研發(fā)逐漸成為科研工作者關注的熱點。菌影(bacterial ghost,BG)是一種保留了細菌表面完整的形態(tài)結(jié)構(gòu)和抗原決定簇,但卻不含有核酸等物質(zhì)的細菌空殼,具有很好的免疫原性和佐劑功能。本實驗旨在制備高裂解效率的致禽腎臟、腸道病變大腸桿菌菌影,構(gòu)建重組溫控雙裂解表達質(zhì)粒,其方法為采用融合PCR的方法,通過15個柔性氨基酸linker將噬菌體中金黃色葡萄球菌基因組的核酸酶A基因(SN)和Φ174的裂解蛋白E基因串聯(lián),插入溫控表達質(zhì)粒pBV220。PCR擴增其溫控雙基因裂解表達系統(tǒng)后插入穿梭質(zhì)粒pBA1100,構(gòu)建重組穿梭質(zhì)粒pBA1100-DLS-ES,并將構(gòu)建好的重組穿梭質(zhì)粒電轉(zhuǎn)化致禽腎臟、腸道病變大腸桿菌中,低溫集菌升溫誘導制備菌影。結(jié)果表明,裂解效率可高達99.9999%,電鏡檢測顯示細菌內(nèi)容物被排出而保存了完整的細菌形態(tài),成功制備了致禽腎臟、腸道病變大腸桿菌菌影,為新型疫苗的制備提供依據(jù)。通過動物試驗對制備的致禽腎臟、腸道病變大腸桿菌菌影疫苗進行免疫保護效果研究。將健康的雛雞,隨機的分成4組,皮下分別接種用不同方式制備的疫苗進行免疫:甲醛滅活組,單裂解菌影苗免疫組和雙裂解菌影苗免疫組,對照組。每組實驗動物注射劑量都為0.2mL(含菌量約為1*109CFU/mL),對照組注射等體積(0.2mL)生理鹽水,一免兩周后加強免疫一次。二免后兩周用致禽腎臟、腸道病變大腸桿菌7.3X107CFU/只的劑量進行肌肉注射攻毒,通過保護率和血清抗體動態(tài)檢測等來進行免疫保護效果的研究。結(jié)果表明,菌影疫苗的免疫保護作用明顯優(yōu)于甲醛滅活苗組。本研究為耐藥大腸桿菌病的預防和治療提供了一種新技術(shù)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.
【學位授予單位】：長江大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S852.4
本文編號：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.
【學位授予單位】：長江大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S852.4
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