【摘要】：多殺性巴氏桿菌是一種非運(yùn)動(dòng)性的、兼性球狀桿菌,屬于革蘭氏陰性菌,巴氏桿菌科巴氏桿菌屬。該菌能夠引起多種動(dòng)物感染,導(dǎo)致急性、敗血性傳染病,包括禽霍亂、牛出血性敗血癥、豬肺疫和豬萎縮性鼻炎、兔出血性敗血癥,甚至受到其感染的犬貓抓傷人類(lèi)后可引起患者傷口膿腫和腦脊膜炎。通過(guò)莢膜抗原和脂多糖區(qū)分血清型,可分為5個(gè)血清群(A、B、D、E、F)和16個(gè)血清型(1、2、3、4、5、……、16),該病呈世界性分布,且可對(duì)畜牧業(yè)造成嚴(yán)重的經(jīng)濟(jì)損失。在我國(guó),以往在牛群中流行的主要是以引起出血性敗血癥的莢膜血清B型為主,但近年來(lái)的研究發(fā)現(xiàn)以引起肺部感染的莢膜血清A型日益增多,且在全國(guó)多個(gè)省份均有發(fā)生多殺性巴氏桿菌包含多種毒力因子,包括莢膜、脂多糖、菌毛、粘附素、毒素、鐵調(diào)蛋白、唾液酸代謝酶、透明質(zhì)酸和外膜蛋白。這些毒力因子在多殺性巴氏桿菌致病機(jī)理中占有重要作用。研究發(fā)現(xiàn),血清B型菌株能夠緊密粘附于宿主細(xì)胞并進(jìn)入胞內(nèi),而A型菌株僅能疏松的粘附于宿主細(xì)胞表面,且不能進(jìn)行細(xì)胞內(nèi)。目前,兩種血清型菌株之間的粘附機(jī)制尚不清楚。其次,fim A基因可能編碼菌毛亞基,是一種重要的毒力因子,但幾乎沒(méi)有對(duì)其功能的報(bào)道。本研究首先對(duì)血清A型和B型菌株粘附相關(guān)基因在體內(nèi)的表達(dá)進(jìn)行了分析,試圖從轉(zhuǎn)錄水平探討其粘附機(jī)制；然后對(duì)fim A 基因進(jìn)行了克隆表達(dá),制備其抗血清,并對(duì)其功能進(jìn)行了初步研究；最后嘗試構(gòu)建A型多殺性巴氏桿菌△fim A株,為本實(shí)驗(yàn)室構(gòu)建突變株提供一定的實(shí)踐經(jīng)驗(yàn)。主要研究?jī)?nèi)容及結(jié)果如下：1.3株多殺性巴氏桿菌粘附相關(guān)基因在小鼠體內(nèi)表達(dá)的研究以本室保存的3株不同血清型、不同毒力的牛源多殺性巴氏桿菌PmCQ2 PmCQ6和PmB為對(duì)象,從轉(zhuǎn)錄水平研究ptfA、fimApfh A等5個(gè)粘附相關(guān)基因在小鼠肺臟的相對(duì)表達(dá)差異。結(jié)果表明PmCQ6中ptfA表達(dá)量與PmB幾乎完全一致,我們推測(cè)ptfA并非A、B型多殺性巴氏桿菌粘附能力差異的關(guān)鍵因子,但可作為多殺性巴氏桿菌體內(nèi)增殖的重要標(biāo)志之一；同時(shí),PmCQ6作為弱毒株,除ptfA基因各時(shí)間段表達(dá)量均高外,其余粘附相關(guān)基因表達(dá)量均低,而強(qiáng)毒株P(guān)mCQ2在24h粘附相關(guān)基因表達(dá)量明顯增高,我們推測(cè)fimA,hsf-1,hsf-2和pfhA可能是致病過(guò)程中強(qiáng)弱毒株的重要因子。研究結(jié)果為牛源莢膜血清A型致病機(jī)制的深入探討奠定了基礎(chǔ)。2.PmCQ2基因fim A的原核表達(dá)及其功能的初步分析通過(guò)PCR克隆PmCQ2株基因fim A,并插入pET-30 a(+)質(zhì)粒中進(jìn)行測(cè)序分析,IPTG誘導(dǎo)表達(dá)重組蛋白FimA (rFimA), western blot分析其抗原性。結(jié)果表明：PmCQ2株的fim A基因與已報(bào)道的其他多殺性巴氏桿菌菌株znuA基因的同源性均高于97%,與其他種屬的細(xì)菌的znuA基因的同源性在68%-74%之間；成功表達(dá)了CQ2菌株rFimA,SDS-PAGE顯示其分子量為38kDa,與預(yù)期大小一致；Western blot試驗(yàn)證明,rFimA具有良好的抗原性；將PmCQ2全菌蛋白和純化的rFimA蛋白分別免疫小鼠(劑量為50μg/只),三免后經(jīng)致死性劑量的PmCQ2攻毒,保護(hù)率分別為100%和20%；粘附抑制試驗(yàn)結(jié)果顯示,rFimA對(duì)多殺性巴氏桿菌粘附細(xì)胞具有一定的抑制抑制作用,推測(cè)該蛋白具有一定的粘附能力；同時(shí),檢測(cè)蛋白FimA在細(xì)菌內(nèi)的定位,結(jié)果顯示：FimA蛋白主要定位于細(xì)菌的細(xì)胞壁中。3.PmCQ2 fimA自殺載體的構(gòu)建PCR擴(kuò)增PmCQ2株fimA的多對(duì)上、下游同源臂,將其分別克隆至質(zhì)粒pWSK-29.pBC-SK相應(yīng)的酶切位點(diǎn),然后將卡那霉素或四環(huán)素抗性基因盒分別插入至fimA的上、下游同源臂之間,共構(gòu)建六個(gè)重組質(zhì)粒：pWSK-fimA-Tet①、 pWSK-fimA-Tet②、pWSK-fimA-Tet③.pWSK-fimA-Tet④、pBC-fimA-Km及pBC-fimA-Tet;分別以電轉(zhuǎn)、化轉(zhuǎn)和原生質(zhì)體三種方法進(jìn)行轉(zhuǎn)化,并嘗試多個(gè)電擊條件。但經(jīng)多次試驗(yàn)發(fā)現(xiàn)：轉(zhuǎn)化后菌落在Km平板上較多,而在四環(huán)素平板上生長(zhǎng)較少,進(jìn)一步研究發(fā)現(xiàn),PmCQ2株基因組中可能含有Km抗性基因,但是以四環(huán)素進(jìn)行篩選仍未篩選到正確的突變株。本研究對(duì)A型多殺性巴氏桿菌突變株構(gòu)建條件進(jìn)行了摸索,為進(jìn)一步構(gòu)建牛源多殺性巴氏桿菌基因敲除突變株奠定了基礎(chǔ)。
[Abstract]:Pasteurella multocida is a non sports, facultative Bacillus globulus, belonging to Gram-negative bacteria, Pasteurella Pasteurella. It can cause a variety of animal infections, leading to acute and septic infectious diseases, including avian cholera, hemorrhagic septicemia, * swine pneumonia and atrophic rhinitis, hemorrhagic septicemia in rabbits, and even feel it. It can be divided into 5 serotypes (A, B, D, E, F) and 16 serotypes (1, 2, 3, 4, 5, 16) by capsular antigen and lipopolysaccharide. The disease is worldwide distributed and can cause serious economic losses to animal husbandry. Pasteurella multocida contains many virulence factors, including capsule, lipopolysaccharide, pili, adhesin, toxin, ferritin, sialic acid metabolizing enzyme. Hyaluronic acid and envelope proteins. These virulence factors play an important role in the pathogenesis of Pasteurella multocida. It has been found that serotype B can adhere tightly to the host cell and enter the cell, while type A can only adhere loosely to the surface of the host cell and can not carry out the cell. Secondly, FIM A gene may encode fimbriae subunit, which is an important virulence factor, but its function has not been reported. The cloning, expression, preparation of antiserum and preliminary study on its function were carried out. Finally, a mutant strain of Pasteurella multocida type A FIM A was constructed to provide some practical experience for the construction of the mutant strain in our laboratory. The main contents and results were as follows: 1.3 strains of Pasteurella multocida adhesion-related genes were expressed in mice. The relative expression of ptfA, fimApfh A and other five adhesion-related genes in the lungs of mice was studied by using three different serotypes of Pasteurella multocida PmCQ2 PmCQ6 and PMB with different virulence. PmCQ6 is one of the important markers for the proliferation of Pasteurella multocida in vivo, and the expression of other adhesion-related genes is low except for the high expression of ptfA gene at all time intervals, while the expression of adhesion-related genes in PmCQ2 is significantly higher than that in other virulent strains. We speculated that fimA, hsf-1, hsf-2 and pfhA might be important factors in the pathogenesis of virulent and virulent strains. The results laid a foundation for further study on the pathogenesis of bovine capsular serum type A. 2. Prokaryotic expression of fimA of PmCQ2 gene and preliminary analysis of its function were cloned by PCR and inserted into pET-30a (+) plasmid for detection. The results showed that the homology of FIM A gene of PmCQ2 strain with other reported Pasteurella multocida strains was higher than 97%, and the homology of znuA gene of other strains was between 68% and 74%. A, SDS-PAGE showed that its molecular weight was 38 kDa, consistent with the expected size; Western blot test showed that rFimA had good antigenicity; PmCQ2 whole bacterial protein and purified rFimA protein were immunized to mice (dose 50 ug/mouse) respectively; after three immunizations, the lethal dose of PmCQ2 was used to attack the virus, and the protective rate was 100% and 20% respectively; adhesion inhibition test showed that rFimA had good antigenicity. The results showed that rFimA could inhibit the adhesion of Pasteurella multocida cells to a certain extent, which suggested that rFimA had a certain adhesion ability. At the same time, the localization of FimA protein in bacteria showed that FimA protein was mainly localized in the cell wall of bacteria. 3. PmCQ2 fimA suicide vector construction PCR amplification of PmCQ2 strain fimA more pairs. Upper and downstream homologous arms were cloned into the corresponding digestion sites of plasmid pWSK-29.pBC-SK, and then kanamycin or tetracycline resistance gene cassettes were inserted into the upper and lower homologous arms of fimA to construct six recombinant plasmids: pWSK-fimA-Tet 1, pWSK-fimA-Tet 2, pWSK-fimA-Tet 3.pWSK-fimA-Tet 4, pBC-KM and pBC-fim-Tet 4. A-Tet; transformed by electroporation, transformation and protoplast respectively, and tried several electroporation conditions. However, many experiments showed that the transformed colonies grew more on Km plate and less on tetracycline plate. Further studies showed that the genome of PmCQ2 strain may contain Km resistance genes, but tetracycline screening was still possible. In this study, the construction conditions of mutant strain of Pasteurella multocida type A were explored, which laid a foundation for further construction of gene knockout mutant strain of Pasteurella multocida from cattle.
【學(xué)位授予單位】：西南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：S852.61

【參考文獻(xiàn)】

相關(guān)期刊論文 前9條

1 高鈺雙;馮甜;邱景富;楊瑞馥;周冬生;李迎麗;;肺炎克雷伯菌基因無(wú)痕敲除方法的建立[J];軍事醫(yī)學(xué);2013年06期

2 鄭艷;劉喜朋;劉建華;;外源載體高效轉(zhuǎn)化肺炎克雷伯菌的新途徑[J];微生物學(xué)報(bào);2007年04期

3 恩特馬克·布拉提白;彭清忠;嚴(yán)芳;何翠;吾魯木汗·那孜爾別克;;禽多殺性巴氏桿菌粘附蛋白Cp39的交叉免疫保護(hù)作用[J];微生物學(xué)報(bào);2010年03期

4 曾靜雯,吳彤,余旭平;多殺性巴氏桿菌鑒定與特性分析的分子生物學(xué)方法[J];畜牧獸醫(yī)科技信息;2005年09期

5 全國(guó)華;;禽慢性呼吸道病繼發(fā)巴氏桿菌病及脂肪肝綜合癥[J];畜牧獸醫(yī)雜志;2006年05期

6 馬文戈;于力;;牛源莢膜血清A型多殺性巴氏桿菌的分離鑒定[J];中國(guó)預(yù)防獸醫(yī)學(xué)報(bào);2008年10期

7 王帥濤;宮強(qiáng);彭永剛;程茗;秦翠麗;張敏;牛明福;;禽多殺性巴氏桿菌外膜蛋白和脂多糖的免疫保護(hù)效果[J];中國(guó)預(yù)防獸醫(yī)學(xué)報(bào);2011年08期

8 吾魯木汗·那孜爾別克;張宇鳳;龔鳳娟;澤田拓士;恩特馬克·布拉提白;;禽多殺性巴氏桿菌C48-3株ompH基因敲除突變株的構(gòu)建及其生物學(xué)功能[J];微生物學(xué)報(bào);2013年01期

9 倪欣濤;盧鳳英;苗雙;朱寅玉;邢林林;俞慧;祁晶晶;王桂軍;胡青海;;鴨疫里氏桿菌轉(zhuǎn)座子隨機(jī)突變庫(kù)的構(gòu)建[J];中國(guó)獸醫(yī)科學(xué);2014年04期

相關(guān)碩士學(xué)位論文 前1條

1 盧順;多殺性巴氏桿菌毒素單克隆抗體的制備及初步應(yīng)用[D];華中農(nóng)業(yè)大學(xué);2008年

，

本文編號(hào)：2230416