發(fā)布時間：2018-06-10 15:25

  本文選題：肺炎克雷伯氏菌 + 分離與鑒定　； 參考：《山東農(nóng)業(yè)大學(xué)》2015年碩士論文

【摘要】：肺炎克雷伯氏菌(Klebsiella pneumoniae,K.pneumoniae)是一種能夠?qū)е露喾N動物患病的條件性致病菌,屬于產(chǎn)超廣譜β-內(nèi)酰胺酶(ESBLs)的腸桿菌屬,β-內(nèi)酰胺酶可以水解含氧亞氨基側(cè)鏈的廣譜頭孢菌素及單胺類抗生素藥物,并能介導(dǎo)細菌對這些抗生素產(chǎn)生耐藥。該菌主要定植在患病動物及人的呼吸道和腸道,可引起呼吸道、消化道、泌尿道等多個部位感染。產(chǎn)β-內(nèi)酰胺酶的細菌具有多重耐藥的特性,使得臨床的治療成為一個棘手的問題。過去人們一直認為肺炎克雷伯氏菌對畜禽等多種動物的危害不大,因此一直未受到重視。但是,近些年來有關(guān)肺炎克雷伯氏菌引起毛皮動物疾病的報道迅速增加,加上其本身耐藥性的產(chǎn)生,作為一種人畜共患傳染病的病原,應(yīng)該受到廣泛的重視。莢膜多糖(caps ular polysaccharide,CPS)作為帶莢膜細菌的保護性抗原及毒力因子,是細菌疫苗最重要的靶抗原之一。具有被開發(fā)利用為亞單位疫苗的潛力。因此,本研究對肺炎克雷伯氏菌莢膜多糖進行提取,以泰山松花粉多糖、蜂膠等作為免疫佐劑制成亞單位疫苗,為肺炎克雷伯氏菌的免疫防控提供新的技術(shù)支撐及理論依據(jù)。取病死水貂的病變組織肝、肺、氣管組織等材料分離純化細菌,對分離出的細菌進行形態(tài)特征、生化特性檢查、藥敏試驗和16S rRNA基因序列的分子鑒定。經(jīng)試驗得知,從病死水貂的肺、肝、氣管組織等同時檢出一種細菌。綜合分析得知分離出的菌株為肺炎克雷伯氏菌。采用十六烷基三甲基溴化銨(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莢膜多糖(CPS-K)。并將其分別加入泰山松花粉多糖、蜂膠和弗氏佐劑等體積配比制成亞單位疫苗。將40只雄性昆明小白鼠隨機分為4組,分別為松花粉組(I)、蜂膠組(II)、弗氏佐劑組(III)和莢膜多糖(CPS)對照組(Ⅳ),分別于3 d、7 d、14 d對小鼠進行三次免疫,在首免后每隔7d檢測一次淋巴細胞增值率、外周血抗體效價、外周血白細胞介素-2(IL-2)含量及CD4+、CD8+T淋巴細胞百分比含量,共檢測8次。試驗結(jié)果表明,僅注射莢膜多糖不能刺激小白鼠產(chǎn)生足夠的保護性抗體,泰山松花粉多糖、蜂膠和弗氏佐劑三種免疫佐劑均可顯著提高機體的細胞免疫和體液免疫水平,其中松花粉多糖組(I)細胞免疫和體液免疫水平與其他兩組差異顯著(p0.05),因此,松花粉多糖作為疫苗佐劑效果最好。本試驗為肺炎克雷伯氏菌莢膜多糖亞單位疫苗的開發(fā)奠定了理論基礎(chǔ)。
[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，

本文編號：2003690