本文選題：肺炎克雷伯菌 + Ⅲ型菌毛　； 參考：《吉林大學(xué)》2010年碩士論文

【摘要】： 肺炎克雷伯菌(Klebsiella pneumoniae)是人與動物腸道和呼吸道常見的定植菌種,屬于腸桿菌科,是一種條件性致病菌。菌毛是其重要的致病因子之一,與細(xì)菌的粘附定植相關(guān),細(xì)菌可借助于菌毛尖端的粘附素(Adhesin)對宿主粘膜上皮細(xì)胞的粘附作用粘附到宿主組織器官,這是機體致病的首要條件。 本試驗以臨床分離的豬源肺炎克雷伯菌菌株Kpn4為研究對象,首先通過甘露糖敏感血凝與抵抗血凝試驗、PCR鑒定該菌生長表達的菌毛類型為Ⅲ型菌毛,并改良了Ⅲ型菌毛的體外表達條件,后經(jīng)SephsdexG—100凝膠過濾法、熱洗脫法及酸鹽沉淀法純化得到Ⅲ型菌毛蛋白。然后以Kpn4為抗原免疫4～6周齡BALB/C雌性小鼠,運用PEG融合法,經(jīng)兩次細(xì)胞融合,以Ⅲ型菌毛蛋白為一抗,通過間接ELISA法篩選,有限稀釋法克隆3次,得到3株可分泌Ⅲ型菌毛蛋白單克隆抗體的雜交瘤細(xì)胞株,分別命名為3A1、5D8、4F2,并對其效價、亞類、特異性、中和性、穩(wěn)定性和相對親和力進行了測定;結(jié)果表明制備出的三株穩(wěn)定分泌肺炎克雷伯菌Kpn4 McAb的雜交瘤細(xì)胞株3A1、5D8和4F2腹水效價依次為1∶3.2×106、1∶2.8×105和1∶1.8×105,培養(yǎng)上清效價依次為1∶3002、1∶2119、1∶1769。3A1為IgG2b亞型,5D8和4F2為IgM亞型。三株McAb的相對親和力為3A15D84F2;特異性測定結(jié)果表明,本試驗所制備的肺炎克雷伯Ⅲ型菌毛McAb特異性反應(yīng)顯示與大腸埃希菌,副傷寒沙門菌,銅綠假單胞菌,產(chǎn)單核細(xì)胞李斯特桿菌,胸膜肺炎放線桿菌均無交叉反應(yīng)性,具有較好的特異性。穩(wěn)定性試驗顯示三株雜交瘤細(xì)胞都能穩(wěn)定分泌Kpn4 McAb的能力。篩選效價高、中和活性好的1株單克隆抗體(3A1),建立雙抗夾心ELISA檢測方法,建立標(biāo)準(zhǔn)曲線,其標(biāo)準(zhǔn)曲線的回歸方程為y =0.4333x -0.2223,最低檢測限為62.5ng/mL,最適檢測范圍為62.5～4000 ng/mL。為該菌的快速診斷、噬菌體文庫的展示和阻斷菌毛粘附蛋白的作用機制及分子流行病學(xué)調(diào)查提供基礎(chǔ)。
[Abstract]:Klebsiella pneumoniae (Klebsiella pneumoniae) is a common colonized bacterium in intestinal tract and respiratory tract of human and animal. It belongs to Enterobacteriaceae and is a kind of conditional pathogenic bacteria. Bacterial pili is one of the important pathogenic factors, which is related to bacterial adhesion and colonization. Bacteria can adhere to host tissues and organs by means of adhesion to host mucosal epithelial cells by means of Adhesin, an adhesin at the tip of pili, which is the most important condition for organism pathogenicity. In this study, the clinical isolates of Klebsiella pneumoniae (Kpn4) were used as the research objects. Firstly, the growth and expression of Klebsiella pneumoniae was identified as type 鈪，

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