本文關(guān)鍵詞： 副豬嗜血桿菌 細(xì)胞膨脹致死毒素 細(xì)胞毒性 抗吞噬 致病作用　出處：《華中農(nóng)業(yè)大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：副豬嗜血桿菌(Haemophilus parasuis)是一種定植在豬上呼吸道的革蘭氏陰性條件致病菌,是導(dǎo)致保育豬死亡的重要病原菌之一。由于研究起步較晚,人們對其毒力因子及致病機(jī)理仍不清楚,這在一定程度制約著對該病的防控。細(xì)胞致死膨脹毒素(Cytolethal distending toxin,CDT)是發(fā)現(xiàn)的唯一具有DNase活性的AB型細(xì)菌毒素,它能引起DSB(DNA double-strand break)反應(yīng),導(dǎo)致真核細(xì)胞周期的不可逆阻滯和靶細(xì)胞凋亡,在一些病原菌致病過程中發(fā)揮著重要作用。由于編碼該毒素的基因簇具有相當(dāng)?shù)谋Ｊ匦?cdt基因很可能是H.parasuis重要的毒力因子,很有必要對其致病作用進(jìn)行深入的研究。本課題以副豬嗜血桿菌中的cdt為研究對象,通過細(xì)胞實(shí)驗(yàn)和宿主動(dòng)物實(shí)驗(yàn)從體外和體內(nèi)兩方面水平探究CDT的致病作用。取得的主要研究結(jié)果如下:1.自然轉(zhuǎn)化獲得cdt基因缺失株通過PCR的方法擴(kuò)增得到cdt上下游同源臂,gm或kan抗性盒;將上游同源臂、抗性盒和下游同源臂用重疊PCR的方法重疊拼接后,連接到自殺性質(zhì)粒p K18mobsac B上得到重組質(zhì)粒p K18-cdt1-UGD和p K18-cdt2-UKD;通過兩次自然轉(zhuǎn)化獲得JS0135?cdt1::gm?cdt2::kan,簡稱JS0135?cdt1?cdt2。通過PCR、Southern blot、DNA測序等鑒定方法,證明cdt雙拷貝的基因缺失株構(gòu)建成功。2.H.parasuis中的CDT對PK-15細(xì)胞具有毒性作用將不同劑量的野生菌株和cdt缺失菌株分泌蛋白與PK-15細(xì)胞互作,3h后繼續(xù)培養(yǎng)24h、48h、72h,發(fā)現(xiàn)野生菌株分泌蛋白組可以使PK-15細(xì)胞稀疏,細(xì)胞膨脹,核濃染增大,出現(xiàn)空泡,甚至崩解,并且這種毒性作用具有時(shí)間和劑量的依賴性,定性地說明了CDT對PK-15細(xì)胞具有毒性作用。將適當(dāng)菌量的野生菌株和cdt缺失菌株分別與PK-15細(xì)胞互作4h、6h,然后測細(xì)胞的LDH活性,發(fā)現(xiàn)野生菌株組細(xì)胞毒性強(qiáng)于缺失菌株組的細(xì)胞毒性,且差異極顯著,定量地說明了CDT對PK-15細(xì)胞具有毒性作用。3.H.parasuis中的CDT與細(xì)菌的抗吞噬能力相關(guān)將野生菌株和cdt缺失菌株與豬肺泡巨噬細(xì)胞3D4/2互作,發(fā)現(xiàn)野生菌株抵抗3D4/2細(xì)胞的能力比缺失株強(qiáng),說明H.parasuis中的CDT在細(xì)菌抗吞噬方面具有重要作用。4.H.parasuis中的CDT對仔豬具有一定程度的致病作用采用三個(gè)劑量組的野生菌株和cdt缺失菌株對H.parasuis陰性豬進(jìn)行胸腔感染,通過臨床癥狀、剖檢病變、組織分菌、病理切片等指標(biāo)比較野生株和缺失株的毒力差異及CDT引起仔豬的典型性病理變化。結(jié)果發(fā)現(xiàn)cdt缺失菌株較野生菌株毒力有所下降,但沒有預(yù)期明顯。
[Abstract]:Haemophilus parasuis (Haemophilus parasuis) is a Gram-negative conditional pathogen in the upper respiratory tract of pigs, and is one of the most important pathogens that lead to the mortality of pigs. Because of the late research, the virulence factor and the pathogenic mechanism of Haemophilus parasuis are still unclear. This restricts the prevention and control of the disease to some extent. Cytolethal distending toxin CDT is the only type AB bacterial toxin found to have DNase activity, which can induce DSB(DNA double-strand breakup reaction and lead to irreversible arrest of eukaryotic cell cycle and apoptosis of target cells. It plays an important role in the pathogenicity of some pathogens. Because the gene cluster encoding the toxin is quite conserved, the CDT gene may be an important virulence factor in H. parasuis. It is very necessary to study its pathogenicity. In this study, cdt in Haemophilus parasuis was used as the research object. The pathogenicity of CDT was studied in vitro and in vivo by cell experiments and host animal experiments. The main results were as follows: 1.The cdt gene deletion strain was transformed naturally and cdt was obtained by PCR amplification. Homologous arm gm or kan resistance box; The upstream homologous arm, the resistance box and the downstream homologous arm were overlapped and spliced with overlapping PCR method. The recombinant plasmids pK18-cdt1-UGD and pK18-cdt2-UKDwere obtained from suicide plasmid pK18-cdt1-UGD and pK18-cdt2-UKDby two natural transformations. The recombinant plasmid pK18-cdt1-UGD and pK18-cdt2-UKDwas obtained by double natural transformation. Cdt1::gm? Cdt2: kan. for short JS0135? Cdt1? Cdt2.The method of DNA sequencing of PCRN Southern blotDNA was used. It is proved that the cdt double copy gene deletion strain was successfully constructed. 2. The CDT in H. parasuis has toxic effect on PK-15 cells. After 3 hours of interaction between the secreted proteins of different doses of wild strain and cdt deletion strain and PK-15 cell, we continue to culture for 24 h, 48 h and 72 h. Secreting proteins makes PK-15 cells sparse, Cells expand, nuclear staining increases, vacuoles appear, and even disintegrate, and the toxicity is time and dose dependent. It was proved qualitatively that CDT had toxic effect on PK-15 cells. The wild strains and cdt deficient strains were interacted with PK-15 cells for 4 h or 6 h, respectively. Then the cell LDH activity was measured. The results showed that the cytotoxicity of wild strain group was stronger than that of deletion strain group. The results showed that CDT in H. parasuis was related to the phagocytic ability of bacteria, which interacted with 3D 4 / 2 of porcine alveolar macrophages. It was found that wild strains were more resistant to 3D4 / 2 cells than the missing strains. The results show that CDT in H. parasuis plays an important role in bacteriological anti-phagocytosis. 4. CDT in H. parasuis has a certain degree of pathogenicity in piglets. H. parasuis negative pigs were infected by wild strains and cdt deficient strains in three dose groups. The virulence differences of wild and absent strains and the typical pathological changes of piglets induced by CDT were compared by dissecting pathological changes, tissue subcultures and pathological sections. The results showed that the virulence of cdt deficient strains was lower than that of wild strains, but the virulence of cdt deficient strains was less than that of wild strains.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S858.28

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本文編號：1526452