牛源多杀性巴氏杆菌粘附相关基因的体内表达及FimA蛋白功能的研究

发布时间：2018-09-08 11:43

【摘要】：多杀性巴氏杆菌是一种非运动性的、兼性球状杆菌,属于革兰氏阴性菌,巴氏杆菌科巴氏杆菌属。该菌能够引起多种动物感染,导致急性、败血性传染病,包括禽霍乱、牛出血性败血症、猪肺疫和猪萎缩性鼻炎、兔出血性败血症,甚至受到其感染的犬猫抓伤人类后可引起患者伤口脓肿和脑脊膜炎。通过荚膜抗原和脂多糖区分血清型,可分为5个血清群(A、B、D、E、F)和16个血清型(1、2、3、4、5、……、16),该病呈世界性分布,且可对畜牧业造成严重的经济损失。在我国,以往在牛群中流行的主要是以引起出血性败血症的荚膜血清B型为主,但近年来的研究发现以引起肺部感染的荚膜血清A型日益增多,且在全国多个省份均有发生多杀性巴氏杆菌包含多种毒力因子,包括荚膜、脂多糖、菌毛、粘附素、毒素、铁调蛋白、唾液酸代谢酶、透明质酸和外膜蛋白。这些毒力因子在多杀性巴氏杆菌致病机理中占有重要作用。研究发现,血清B型菌株能够紧密粘附于宿主细胞并进入胞内,而A型菌株仅能疏松的粘附于宿主细胞表面,且不能进行细胞内。目前,两种血清型菌株之间的粘附机制尚不清楚。其次,fim A基因可能编码菌毛亚基,是一种重要的毒力因子,但几乎没有对其功能的报道。本研究首先对血清A型和B型菌株粘附相关基因在体内的表达进行了分析,试图从转录水平探讨其粘附机制；然后对fim A 基因进行了克隆表达,制备其抗血清,并对其功能进行了初步研究；最后尝试构建A型多杀性巴氏杆菌△fim A株,为本实验室构建突变株提供一定的实践经验。主要研究内容及结果如下：1.3株多杀性巴氏杆菌粘附相关基因在小鼠体内表达的研究以本室保存的3株不同血清型、不同毒力的牛源多杀性巴氏杆菌PmCQ2 PmCQ6和PmB为对象,从转录水平研究ptfA、fimApfh A等5个粘附相关基因在小鼠肺脏的相对表达差异。结果表明PmCQ6中ptfA表达量与PmB几乎完全一致,我们推测ptfA并非A、B型多杀性巴氏杆菌粘附能力差异的关键因子,但可作为多杀性巴氏杆菌体内增殖的重要标志之一；同时,PmCQ6作为弱毒株,除ptfA基因各时间段表达量均高外,其余粘附相关基因表达量均低,而强毒株PmCQ2在24h粘附相关基因表达量明显增高,我们推测fimA,hsf-1,hsf-2和pfhA可能是致病过程中强弱毒株的重要因子。研究结果为牛源荚膜血清A型致病机制的深入探讨奠定了基础。2.PmCQ2基因fim A的原核表达及其功能的初步分析通过PCR克隆PmCQ2株基因fim A,并插入pET-30 a(+)质粒中进行测序分析,IPTG诱导表达重组蛋白FimA (rFimA), western blot分析其抗原性。结果表明：PmCQ2株的fim A基因与已报道的其他多杀性巴氏杆菌菌株znuA基因的同源性均高于97%,与其他种属的细菌的znuA基因的同源性在68%-74%之间；成功表达了CQ2菌株rFimA,SDS-PAGE显示其分子量为38kDa,与预期大小一致；Western blot试验证明,rFimA具有良好的抗原性；将PmCQ2全菌蛋白和纯化的rFimA蛋白分别免疫小鼠(剂量为50μg/只),三免后经致死性剂量的PmCQ2攻毒,保护率分别为100%和20%；粘附抑制试验结果显示,rFimA对多杀性巴氏杆菌粘附细胞具有一定的抑制抑制作用,推测该蛋白具有一定的粘附能力；同时,检测蛋白FimA在细菌内的定位,结果显示：FimA蛋白主要定位于细菌的细胞壁中。3.PmCQ2 fimA自杀载体的构建PCR扩增PmCQ2株fimA的多对上、下游同源臂,将其分别克隆至质粒pWSK-29.pBC-SK相应的酶切位点,然后将卡那霉素或四环素抗性基因盒分别插入至fimA的上、下游同源臂之间,共构建六个重组质粒：pWSK-fimA-Tet①、 pWSK-fimA-Tet②、pWSK-fimA-Tet③.pWSK-fimA-Tet④、pBC-fimA-Km及pBC-fimA-Tet;分别以电转、化转和原生质体三种方法进行转化,并尝试多个电击条件。但经多次试验发现：转化后菌落在Km平板上较多,而在四环素平板上生长较少,进一步研究发现,PmCQ2株基因组中可能含有Km抗性基因,但是以四环素进行筛选仍未筛选到正确的突变株。本研究对A型多杀性巴氏杆菌突变株构建条件进行了摸索,为进一步构建牛源多杀性巴氏杆菌基因敲除突变株奠定了基础。
[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.
【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.61

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