本文關(guān)鍵詞：我國(guó)北方地區(qū)犢牛腹瀉大腸桿菌HPI分子結(jié)構(gòu)組成及其菌蛻的研究　出處：《吉林農(nóng)業(yè)大學(xué)》2016年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 犢牛腹瀉 大腸桿菌 耶爾森強(qiáng)毒力島 菌蛻 免疫原性

【摘要】：犢牛腹瀉大腸桿菌病又稱牛白痢,其病原為致病性大腸桿菌(Escherichia coli),臨床癥狀多以急性腹瀉、脫水等為主,本病全年均可發(fā)生、當(dāng)氣溫驟變時(shí)更易多發(fā),2-3周的犢牛易感染此病,本病死亡率較高,多呈地方性流行,一旦爆發(fā),將給養(yǎng)牛業(yè)帶來(lái)巨大的損失。耶爾森強(qiáng)毒力島(HPI),其含有攝鐵毒力基因簇,因此具有鐵攝取功能,其在耶爾森菌感染機(jī)體過(guò)程中,發(fā)揮著重要作用。HPI可以通過(guò)質(zhì)粒在不同菌株之間水平傳播,因此,這些毒力相關(guān)基因可能在不同種屬的菌株間、動(dòng)物與人類間相互傳播,近年來(lái),HPI在致病性大腸桿菌、克雷伯菌、沙門菌等其他腸桿菌科的菌屬中已被發(fā)現(xiàn)。菌蛻是近年來(lái)的一種新型疫苗,其多數(shù)由噬菌體Phix174的蛋白E裂解革蘭氏陰性細(xì)菌表面而形成一孔狀結(jié)構(gòu),其細(xì)菌內(nèi)容物則由此排除,形成一個(gè)無(wú)活性、空殼狀的“細(xì)菌”。菌蛻保留了細(xì)菌自身的天然表面抗原、且具有佐劑性能,因此可以加強(qiáng)免疫應(yīng)答;而且,菌蛻具有制備簡(jiǎn)單、自身安全、運(yùn)輸方便、還可作多聯(lián)疫苗等諸多優(yōu)點(diǎn),現(xiàn)已逐漸被人們所認(rèn)識(shí)并研究。本研究以北方六省市的犢牛腹瀉為研究材料,從中分離出致病性大腸桿菌,并對(duì)其O血清型進(jìn)行鑒定,同時(shí),檢測(cè)大腸桿菌分離株HPI攜帶情況,并對(duì)其HPI相關(guān)毒力基因進(jìn)行克隆與序列分析。在此基礎(chǔ)上,篩選出一株疫苗候選株,制備成大腸桿菌菌蛻,并對(duì)其部分生物學(xué)特性以及免疫原性進(jìn)行初步分析。結(jié)果顯示:在北方六省市的420份犢牛腹瀉樣品中,共分離到大腸桿菌140株,分離率為33.33%(140/420);其犢牛腹瀉大腸桿菌分屬15種血清型,其中O14、O15、O101、O78、O36為優(yōu)勢(shì)血清型。140株?duì)倥８篂a大腸桿菌分離株中,共有59株HPI陽(yáng)性、陽(yáng)性率為42.14%(59/140);59株?duì)倥８篂a大腸桿菌HPI陽(yáng)性菌株中,6種主要毒力因子的陽(yáng)性率各不同:fyuA為49.15%(29/59)、asn_tRNA_intB為32.20%(19/59)、irp3為42.37%(25/59)、irp5為23.73%(14/59)、irp8為44.07%(26/59)、ybtA各為44.07%(26/59)。不同菌株的7種毒力基因序列具有較高的同源性,部分菌株毒力基因序列同源性與樣品采集地點(diǎn)、時(shí)間有一定相關(guān)性;菌株攜帶HPI毒力基因的種類、數(shù)目與采集地點(diǎn)關(guān)聯(lián)不密切,但攜帶irp2+fyuA+irp3+irp8+ybtA基因組合的菌株數(shù)量最多,占總數(shù)的11.86%(7/59)、且流行分布較廣。本研究篩選出一株對(duì)氨芐青霉素敏感、致病性較強(qiáng)、生長(zhǎng)速度較快、血清型為優(yōu)勢(shì)血清型的犢牛腹瀉大腸桿菌菌蛻制備候選株HBTN1311222,血清型為O15型;并成功構(gòu)建質(zhì)粒型犢牛腹瀉大腸桿菌菌蛻,在誘導(dǎo)6h時(shí),裂解率為99.877%。犢牛腹瀉大腸桿菌菌蛻可以誘導(dǎo)小鼠機(jī)體產(chǎn)生體液免疫反應(yīng)與細(xì)胞免疫反應(yīng),可以誘導(dǎo)機(jī)體以Th1型細(xì)胞為主的保護(hù)性免疫應(yīng)答;且可以對(duì)小鼠產(chǎn)生良好地免疫保護(hù)力、其保護(hù)率為80%;在小鼠攻毒后,菌蛻免疫的小鼠病理變化要略輕于滅活疫苗、滅活佐劑疫苗免疫小鼠的病理變化。研究結(jié)果將有助于明確我國(guó)北方地區(qū)犢牛腹瀉大腸桿菌的分布情況、優(yōu)勢(shì)血清型;并為研究犢牛腹瀉大腸桿菌HPI各毒力基因的功能提供數(shù)據(jù)支持;本研究成功制備的犢牛腹瀉大腸桿菌菌蛻,將為犢牛腹瀉的防治、尤其是新型疫苗的研制,奠定基礎(chǔ)。
[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.

【學(xué)位授予單位】：吉林農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：S858.23

【參考文獻(xiàn)】

相關(guān)期刊論文 前5條

1 喻華英;底麗娜;;新疆犢牛腹瀉病病原菌的分離鑒定和藥敏試驗(yàn)[J];中國(guó)獸醫(yī)雜志;2012年05期

2 方光遠(yuǎn);張姝;張玉紅;顧亞鳳;陳鐘鳴;張志成;何小明;李志源;袁冬斌;;腹瀉犢牛大腸桿菌的分離鑒定和藥敏試驗(yàn)[J];中國(guó)獸醫(yī)雜志;2008年10期

3 方光遠(yuǎn);范紅結(jié);張玉紅;張姝;陳鐘鳴;顧亞鳳;何小明;;犢牛腹瀉大腸桿菌LEE和HPI毒力島相關(guān)基因的檢測(cè)[J];金陵科技學(xué)院學(xué)報(bào);2008年02期

4 儲(chǔ)衛(wèi)華;莊禧懿;陸承平;;嗜水氣單胞菌菌蛻的制備及其對(duì)銀鯽的口服免疫[J];微生物學(xué)報(bào);2008年02期

5 金文杰;鄭志明;秦愛(ài)建;劉岳龍;邵紅霞;張永志;黃訓(xùn)良;;禽致病性大腸桿菌中耶爾森菌強(qiáng)毒力島的分子流行病學(xué)調(diào)查[J];中國(guó)獸醫(yī)科學(xué);2006年10期

相關(guān)博士學(xué)位論文 前1條

1 羊云飛;牦牛、牧民源大腸桿菌分離鑒定、耐藥基因檢測(cè)、PFGE分析及耐藥性傳遞的研究[D];四川農(nóng)業(yè)大學(xué);2011年

，

本文編號(hào)：1400108