副猪嗜血杆菌nanH基因和at1基因的功能研究

发布时间：2018-03-23 15:37

本文选题：副猪嗜血杆菌　切入点：神经氨酸酶NanH　出处：《华中农业大学》2015年硕士论文

【摘要】：副猪嗜血杆菌(Haemophilus parasuis)属于革兰氏阴性菌,是猪群上呼吸道的条件性致病菌。本研究主要以副猪嗜血杆菌神经氨酸酶Nan H与自转运蛋白AT1为研究对象,探究其在副猪嗜血杆菌致病过程中发挥的主要功能。本课题取得的主要研究成果如下所示:1.副猪嗜血杆菌神经氨酸酶验证对菌株JS0135,缺失株JS0135Δnan H::kan(以下简称Δnan H)和互补菌株JS0135-Cnan H(以下简称Cnan H)进行神经氨酸酶活性检测,检测结果发现,互补菌株Cnan H的神经氨酸酶活性与缺失菌株相同,极显著低于野生菌株。后对其进行粘附实验,发现互补菌株Cnan H的粘附能力仍与缺失株相同,与野生株差异显著,因此该互补菌株并没有互补成功,缺失株构建成功。生长曲线测定发现,野生菌株与缺失株之间无显著性差异,且菌株在体外生长过程中不需要唾液酸提供营养。2.JS0135-Δnan H菌株的粘附能力、血清抗性和氧化耐受能力改变将菌株JS0135,Δnan H与猪肾上皮细胞(PK-15)与猪髋动脉内皮细胞(PIEC)互作,发现缺失株Δnan H的粘附能力比野生菌株JS0135强。与健康猪血清互作,发现缺失了nan H基因后,菌株的血清抗性降低。补体激活的经典途径在对菌株的杀伤中发挥一定作用。进行补体沉降实验则发现,在缺失株Δnan H表面沉积的补体要高于野生株JS0135。Western blot则发现菌株表面沉积的成分中存在Ig G蛋白。质谱分析则发现在菌株表面有C3分子沉积,并通过Western blot得到验证,且通过流式实验发现在缺失株表面沉积的补体C3要高于野生株。进行氧化耐受能力实验,发现缺失神经氨酸酶nan H基因后细菌的氧化耐受能力降低,从而发现nan H基因在菌株氧化耐受中发挥的重要作用。3.副猪嗜血杆菌自转蛋白at1基因缺失株的构建及其功能研究通过自然转化方法在JS0135中构建了at1基因缺失株JS0135Δat1::kan,以下简称(Δat1)。通过对PK-15细胞毒性实验,发现at1基因无细胞毒性作用。细菌的粘附实验表明at1基因缺失后,菌株对PK-15细胞的粘附能力增强;同时发现at1基因缺失后对细菌生物被膜形成以及自身凝集都没有影响。同时发现,缺失了at1基因后细菌的氧化耐受能力减弱,表明at1基因可能与细菌的氧化耐受能力有关。综合以上研究结果,唾液酸酶在副猪嗜血杆菌的致病机制中可能发挥重要作用,其通过减少细菌表面沉积的补体分子增强细菌对血清的抵抗作用。同时通过增强菌株的氧化耐受能力感染并侵入宿主。自转运蛋白AT1也具有增强细菌氧化耐受的功能。
[Abstract]:Haemophilus parasuis (Haemophilus parasuis) belongs to Gram-negative bacteria and is a conditional pathogen in the upper respiratory tract of swine population. This study focused on the neuraminidase Nan H and AT1 of Haemophilus parasuis. The main results of this study are as follows: 1. The neuraminidase test of Haemophilus parasuis strain JS0135, the missing strain JS0135 螖 nan H: kanand (螖 nan H) and the interaction between the two strains were investigated. The main results of this study are as follows: 1. The neuraminidase of Haemophilus parasuis strain JS0135, the missing strain JS0135 螖 nan H: K 谩 n (螖 nan H) and each other. The neuraminidase activity of JS0135-Cnan H (hereinafter referred to as Cnan H) was detected. The results showed that the neuraminidase activity of the complementary strain Cnan H was the same as that of the deficient strain, and was significantly lower than that of the wild strain. The adhesion of the complementary strain Cnan H was still the same as that of the missing strain. The difference between the strain and the wild strain was significant, so the complementary strain was not successful and the missing strain was successfully constructed. The growth curve showed that there was no significant difference between the wild strain and the missing strain. The strain did not need sialic acid to provide nutrition. 2. JS0135- 螖 nan H did not need to provide nutrition in vitro. Serum resistance and oxidative tolerance of strain JS0135, 螖 nan H interacted with porcine renal epithelial cells (PK-15) and porcine hip artery endothelial cells (PIECs). It was found that the adhesion ability of deletion strain 螖 nan H was stronger than that of wild strain JS0135. After interaction with healthy pig serum, the deletion of nan H gene was found. The classical pathway of complement activation played a certain role in killing the strain. The complement deposited on the surface of the missing strain 螖 nan H was higher than that of the wild strain JS0135.Western blot, and the IgG G protein was found in the component deposited on the surface of the strain, and the C3 molecule deposition was found on the surface of the strain by mass spectrometry, which was verified by Western blot. The results of flow test showed that the complement C3 deposited on the surface of the missing strain was higher than that of the wild strain, and the oxidative tolerance ability of bacteria was decreased after the deletion of nan H gene of neuraminidase. It was found that nan H gene plays an important role in the oxidative tolerance of the strain .3.The construction and function of the at1 gene deletion strain of Haemophilus parasuis autotropin were constructed in JS0135 by natural transformation method. The at1 gene deletion strain JS0135 螖 at 1: 1: kan. was constructed by natural transformation. Through the cytotoxicity test of PK-15 cells, It was found that at1 gene had no cytotoxicity. Bacterial adhesion test showed that the adhesion ability of the strain to PK-15 cells was enhanced after at1 gene deletion. It was also found that the absence of at1 gene had no effect on bacterial biofilm formation and self-agglutination. At the same time, the ability of bacterial oxidative tolerance was decreased after the deletion of at1 gene. These results suggest that sialidase may play an important role in the pathogenesis of Haemophilus parais. It enhances the bacterial resistance to serum by reducing the complement molecules deposited on the bacterial surface, and infects and invades the host by enhancing the ability of bacterial oxidation tolerance. The self-transporter AT1 also has the function of enhancing bacterial oxidative tolerance.
【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】：S852.61

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