副猪嗜血杆菌细胞致死膨胀毒素对细胞和仔猪致病作用研究

发布时间：2018-02-23 07:25

本文关键词： 副猪嗜血杆菌细胞膨胀致死毒素细胞毒性抗吞噬致病作用　出处：《华中农业大学》2015年硕士论文　论文类型：学位论文

【摘要】：副猪嗜血杆菌(Haemophilus parasuis)是一种定植在猪上呼吸道的革兰氏阴性条件致病菌,是导致保育猪死亡的重要病原菌之一。由于研究起步较晚,人们对其毒力因子及致病机理仍不清楚,这在一定程度制约着对该病的防控。细胞致死膨胀毒素(Cytolethal distending toxin,CDT)是发现的唯一具有DNase活性的AB型细菌毒素,它能引起DSB(DNA double-strand break)反应,导致真核细胞周期的不可逆阻滞和靶细胞凋亡,在一些病原菌致病过程中发挥着重要作用。由于编码该毒素的基因簇具有相当的保守性,cdt基因很可能是H.parasuis重要的毒力因子,很有必要对其致病作用进行深入的研究。本课题以副猪嗜血杆菌中的cdt为研究对象,通过细胞实验和宿主动物实验从体外和体内两方面水平探究CDT的致病作用。取得的主要研究结果如下:1.自然转化获得cdt基因缺失株通过PCR的方法扩增得到cdt上下游同源臂,gm或kan抗性盒;将上游同源臂、抗性盒和下游同源臂用重叠PCR的方法重叠拼接后,连接到自杀性质粒p K18mobsac B上得到重组质粒p K18-cdt1-UGD和p K18-cdt2-UKD;通过两次自然转化获得JS0135?cdt1::gm?cdt2::kan,简称JS0135?cdt1?cdt2。通过PCR、Southern blot、DNA测序等鉴定方法,证明cdt双拷贝的基因缺失株构建成功。2.H.parasuis中的CDT对PK-15细胞具有毒性作用将不同剂量的野生菌株和cdt缺失菌株分泌蛋白与PK-15细胞互作,3h后继续培养24h、48h、72h,发现野生菌株分泌蛋白组可以使PK-15细胞稀疏,细胞膨胀,核浓染增大,出现空泡,甚至崩解,并且这种毒性作用具有时间和剂量的依赖性,定性地说明了CDT对PK-15细胞具有毒性作用。将适当菌量的野生菌株和cdt缺失菌株分别与PK-15细胞互作4h、6h,然后测细胞的LDH活性,发现野生菌株组细胞毒性强于缺失菌株组的细胞毒性,且差异极显著,定量地说明了CDT对PK-15细胞具有毒性作用。3.H.parasuis中的CDT与细菌的抗吞噬能力相关将野生菌株和cdt缺失菌株与猪肺泡巨噬细胞3D4/2互作,发现野生菌株抵抗3D4/2细胞的能力比缺失株强,说明H.parasuis中的CDT在细菌抗吞噬方面具有重要作用。4.H.parasuis中的CDT对仔猪具有一定程度的致病作用采用三个剂量组的野生菌株和cdt缺失菌株对H.parasuis阴性猪进行胸腔感染,通过临床症状、剖检病变、组织分菌、病理切片等指标比较野生株和缺失株的毒力差异及CDT引起仔猪的典型性病理变化。结果发现cdt缺失菌株较野生菌株毒力有所下降,但没有预期明显。
[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.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.28

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