我国北方地区犊牛腹泻大肠杆菌HPI分子结构组成及其菌蜕的研究

发布时间：2018-01-09 05:11

本文关键词：我国北方地区犊牛腹泻大肠杆菌HPI分子结构组成及其菌蜕的研究　出处：《吉林农业大学》2016年博士论文　论文类型：学位论文

【摘要】：犊牛腹泻大肠杆菌病又称牛白痢,其病原为致病性大肠杆菌(Escherichia coli),临床症状多以急性腹泻、脱水等为主,本病全年均可发生、当气温骤变时更易多发,2-3周的犊牛易感染此病,本病死亡率较高,多呈地方性流行,一旦爆发,将给养牛业带来巨大的损失。耶尔森强毒力岛(HPI),其含有摄铁毒力基因簇,因此具有铁摄取功能,其在耶尔森菌感染机体过程中,发挥着重要作用。HPI可以通过质粒在不同菌株之间水平传播,因此,这些毒力相关基因可能在不同种属的菌株间、动物与人类间相互传播,近年来,HPI在致病性大肠杆菌、克雷伯菌、沙门菌等其他肠杆菌科的菌属中已被发现。菌蜕是近年来的一种新型疫苗,其多数由噬菌体Phix174的蛋白E裂解革兰氏阴性细菌表面而形成一孔状结构,其细菌内容物则由此排除,形成一个无活性、空壳状的“细菌”。菌蜕保留了细菌自身的天然表面抗原、且具有佐剂性能,因此可以加强免疫应答;而且,菌蜕具有制备简单、自身安全、运输方便、还可作多联疫苗等诸多优点,现已逐渐被人们所认识并研究。本研究以北方六省市的犊牛腹泻为研究材料,从中分离出致病性大肠杆菌,并对其O血清型进行鉴定,同时,检测大肠杆菌分离株HPI携带情况,并对其HPI相关毒力基因进行克隆与序列分析。在此基础上,筛选出一株疫苗候选株,制备成大肠杆菌菌蜕,并对其部分生物学特性以及免疫原性进行初步分析。结果显示:在北方六省市的420份犊牛腹泻样品中,共分离到大肠杆菌140株,分离率为33.33%(140/420);其犊牛腹泻大肠杆菌分属15种血清型,其中O14、O15、O101、O78、O36为优势血清型。140株犊牛腹泻大肠杆菌分离株中,共有59株HPI阳性、阳性率为42.14%(59/140);59株犊牛腹泻大肠杆菌HPI阳性菌株中,6种主要毒力因子的阳性率各不同:fyuA为49.15%(29/59)、asn_tRNA_intB为32.20%(19/59)、irp3为42.37%(25/59)、irp5为23.73%(14/59)、irp8为44.07%(26/59)、ybtA各为44.07%(26/59)。不同菌株的7种毒力基因序列具有较高的同源性,部分菌株毒力基因序列同源性与样品采集地点、时间有一定相关性;菌株携带HPI毒力基因的种类、数目与采集地点关联不密切,但携带irp2+fyuA+irp3+irp8+ybtA基因组合的菌株数量最多,占总数的11.86%(7/59)、且流行分布较广。本研究筛选出一株对氨苄青霉素敏感、致病性较强、生长速度较快、血清型为优势血清型的犊牛腹泻大肠杆菌菌蜕制备候选株HBTN1311222,血清型为O15型;并成功构建质粒型犊牛腹泻大肠杆菌菌蜕,在诱导6h时,裂解率为99.877%。犊牛腹泻大肠杆菌菌蜕可以诱导小鼠机体产生体液免疫反应与细胞免疫反应,可以诱导机体以Th1型细胞为主的保护性免疫应答;且可以对小鼠产生良好地免疫保护力、其保护率为80%;在小鼠攻毒后,菌蜕免疫的小鼠病理变化要略轻于灭活疫苗、灭活佐剂疫苗免疫小鼠的病理变化。研究结果将有助于明确我国北方地区犊牛腹泻大肠杆菌的分布情况、优势血清型;并为研究犊牛腹泻大肠杆菌HPI各毒力基因的功能提供数据支持;本研究成功制备的犊牛腹泻大肠杆菌菌蜕,将为犊牛腹泻的防治、尤其是新型疫苗的研制,奠定基础。
[Abstract]:Calf diarrhea Escherichia coli disease is also called bovine diarrhea, the pathogen of pathogenic Escherichia coli (Escherichia coli), the clinical symptoms of acute diarrhea, dehydration, the disease can occur throughout the year, when the temperature changes more multiple, 2-3 weeks of calves susceptible to the disease, the mortality of the disease is high, mostly local once the outbreak of epidemics, will bring huge losses to the cattle industry. Jerson high pathogenicity island (HPI), which contains iron uptake virulence gene cluster, therefore has the iron uptake function, the bacteria infection in Jerson body process, plays an important role in the level of.HPI can be spread between different strains by plasmid so. These possible virulence related genes in different species between strains, animal and human mutual communication, in recent years, HPI in the pathogenic Escherichia coli, Klebsiella, Salmonella and other Enterobacteriaceae bacteria have been found in bacterial ghosts in recent years is. A new type of vaccine, the most by phage Phix174 protein E cleavage of gram negative bacterial surface and form a porous structure, the bacterial content is thus excluded, forming a non active shell shaped bacteria. Bacterial ghosts retain natural bacterial surface antigen itself, and has adjuvant properties, so it can be to strengthen the immune response; moreover, bacterial ghost has the advantages of simple preparation, safety, convenient transportation, also can be used for many advantages of vaccine, has gradually been recognized and studied. This study in six provinces and cities in the north of the calf diarrhea as the research material, isolated from pathogenic Escherichia coli, and was identified. The O serotype and detection of Escherichia coli isolates carrying HPI, and the HPI cloning and sequence analysis of virulence related genes. On this basis, screening a strain of vaccine candidate strain, preparation of Escherichia coli bacterial ghosts, and the A preliminary analysis of the biological characteristics and immunogenicity. The results showed that: in 420 samples of calf diarrhea in six provinces and cities in the north, 140 strains of Escherichia coli were isolated, the isolation rate was 33.33% (140/420); the calf diarrhea Escherichia coli belonging to 15 serotypes, including O14, O15, O101, O78, O36 the dominant serotype.140 strains of Escherichia coli isolated from calf diarrhea, a total of 59 strains of HPI positive, the positive rate was 42.14% (59/140); 59 strains of calf diarrhea Escherichia coli HPI positive strains, the positive rate of the 6 main virulence factors of different fyuA 49.15% (29/59), asn_tRNA_intB 32.20% (19/59) irp3, 42.37% (25/59), irp5 23.73% (14/59), irp8 44.07% (26/59), ybtA 44.07% (26/59). 7 kinds of virulence gene sequences of different strains had high homology, partial sequence of virulence gene homology and location of sample collection, there is a certain correlation between the time of bacteria; Strains carrying HPI virulence genes, and the number of collection sites is not closely associated, but the number of strains carrying irp2+fyuA+irp3+irp8+ybtA gene combinations of the most, accounting for 11.86% of the total (7/59), popular and widely distributed. This study screened a strain sensitive to ampicillin, strong pathogenicity, growth speed, serotype advantage in serum the type of calf diarrhea Escherichia coli bacterial ghost preparation candidate strains HBTN1311222, serotype O15; plasmid and calf diarrhea Escherichia coli ghosts successfully constructed in the induction of 6h, cleavage rate of 99.877%. calf diarrhea Escherichia coli bacterial ghosts could induce humoral immune response and cellular immune response and protective immune response can induce to type Th1 cells in mice; and can produce good immune protection, the protection rate was 80%; in mice after infection of mice, the pathological changes of bacterial ghost immunity Slightly lighter than the inactivated vaccine, inactivated vaccine live pathological changes in mice. The results of the study will help to understand the distribution of calf diarrhea Escherichia coli in the northern area of China, and to study the dominant serotype; calf diarrhea Escherichia coli HPI virulence gene function can provide data support for the study of Escherichia coli successfully; bacterial ghost calf diarrhea preparation, will be for the prevention and treatment of diarrhea in calves, especially the development of new vaccines, lay the foundation.

【学位授予单位】：吉林农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S858.23

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