泰山松花粉多糖对水貂肺炎克雷伯氏菌荚膜多糖亚单位疫苗的免疫增强效果

发布时间：2018-06-10 15:25

  本文选题：肺炎克雷伯氏菌 + 分离与鉴定　； 参考：《山东农业大学》2015年硕士论文

【摘要】：肺炎克雷伯氏菌(Klebsiella pneumoniae,K.pneumoniae)是一种能够导致多种动物患病的条件性致病菌,属于产超广谱β-内酰胺酶(ESBLs)的肠杆菌属,β-内酰胺酶可以水解含氧亚氨基侧链的广谱头孢菌素及单胺类抗生素药物,并能介导细菌对这些抗生素产生耐药。该菌主要定植在患病动物及人的呼吸道和肠道,可引起呼吸道、消化道、泌尿道等多个部位感染。产β-内酰胺酶的细菌具有多重耐药的特性,使得临床的治疗成为一个棘手的问题。过去人们一直认为肺炎克雷伯氏菌对畜禽等多种动物的危害不大,因此一直未受到重视。但是,近些年来有关肺炎克雷伯氏菌引起毛皮动物疾病的报道迅速增加,加上其本身耐药性的产生,作为一种人畜共患传染病的病原,应该受到广泛的重视。荚膜多糖(caps ular polysaccharide,CPS)作为带荚膜细菌的保护性抗原及毒力因子,是细菌疫苗最重要的靶抗原之一。具有被开发利用为亚单位疫苗的潜力。因此,本研究对肺炎克雷伯氏菌荚膜多糖进行提取,以泰山松花粉多糖、蜂胶等作为免疫佐剂制成亚单位疫苗,为肺炎克雷伯氏菌的免疫防控提供新的技术支撑及理论依据。取病死水貂的病变组织肝、肺、气管组织等材料分离纯化细菌,对分离出的细菌进行形态特征、生化特性检查、药敏试验和16S rRNA基因序列的分子鉴定。经试验得知,从病死水貂的肺、肝、气管组织等同时检出一种细菌。综合分析得知分离出的菌株为肺炎克雷伯氏菌。采用十六烷基三甲基溴化铵(CTAB)沉淀多糖、乙醇除核酸、苯酚除蛋白的方法提取肺炎克雷伯氏菌荚膜多糖(CPS-K)。并将其分别加入泰山松花粉多糖、蜂胶和弗氏佐剂等体积配比制成亚单位疫苗。将40只雄性昆明小白鼠随机分为4组,分别为松花粉组(I)、蜂胶组(II)、弗氏佐剂组(III)和荚膜多糖(CPS)对照组(Ⅳ),分别于3 d、7 d、14 d对小鼠进行三次免疫,在首免后每隔7d检测一次淋巴细胞增值率、外周血抗体效价、外周血白细胞介素-2(IL-2)含量及CD4+、CD8+T淋巴细胞百分比含量,共检测8次。试验结果表明,仅注射荚膜多糖不能刺激小白鼠产生足够的保护性抗体,泰山松花粉多糖、蜂胶和弗氏佐剂三种免疫佐剂均可显著提高机体的细胞免疫和体液免疫水平,其中松花粉多糖组(I)细胞免疫和体液免疫水平与其他两组差异显著(p0.05),因此,松花粉多糖作为疫苗佐剂效果最好。本试验为肺炎克雷伯氏菌荚膜多糖亚单位疫苗的开发奠定了理论基础。
[Abstract]:Klebsiella pneumoniae K. pneumoniae is a conditional pathogen that can cause disease in many animals. ESBLsproducing extended-spectrum 尾 -lactamases (ESBLs), 尾 -lactamases can hydrolyze broad-spectrum cephalosporins and monoamine antibiotics containing oxygen iminodium side chain, and can mediate bacterial resistance to these antibiotics. The bacteria is mainly colonized in the respiratory and intestinal tract of infected animals and human beings, which can cause respiratory tract, digestive tract, urinary tract infection and so on. 尾-lactamases-producing bacteria are multidrug resistant, making clinical treatment a thorny problem. Klebsiella pneumoniae has always been regarded as not harmful to many animals, such as livestock and poultry, so it has not been paid much attention to. However, in recent years, the reports of fur animal diseases caused by Klebsiella pneumoniae have increased rapidly, and their own drug resistance, as a pathogen of zoonotic diseases, should be paid more and more attention. As the protective antigen and virulence factor of bacteria with capsule, Caps ular polysaccharide is one of the most important target antigens of bacterial vaccine. It has the potential to be exploited as a subunit vaccine. Therefore, in this study, the capsule polysaccharides of Klebsiella pneumoniae were extracted, the pine pollen polysaccharides and propolis were used as immune adjuvants to prepare subunit vaccine, which provided new technical support and theoretical basis for the prevention and control of Klebsiella pneumoniae. The diseased tissue liver, lung and trachea of the dead mink were isolated and purified. The isolated bacteria were examined for their morphological and biochemical characteristics, drug sensitivity test and molecular identification of 16s rRNA gene sequence. The results showed that a bacterium was detected from lung, liver and trachea of the dead mink at the same time. Comprehensive analysis showed that the isolated strain was Klebsiella pneumoniae. The polysaccharide of Klebsiella pneumoniae was extracted by cetyltrimethylammonium bromide (CTAB) precipitation, ethanol denucleic acid and phenol deproteinization. The subunit vaccine was prepared by adding Taishanpine pollen polysaccharide, propolis and Freund's adjuvant respectively. Forty male Kunming mice were randomly divided into 4 groups: pine pollen group (Ig), propolis group (鈪，

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