坏死梭杆菌的分离鉴定及其白细胞毒素的原核表达与免疫原性分析

发布时间：2018-04-15 08:19

本文选题：腐蹄病 + 坏死梭杆菌　；参考：《甘肃农业大学》2017年硕士论文

【摘要】：坏死梭杆菌(Fusobacterium necrophorum)是引起绵羊腐蹄病和肝脓肿等疾病的常见病原菌之一,所致疾病给养羊业造成很大的经济损失。该病原产生的白细胞毒素是分泌毒素最强的毒力因子之一,能抑制中性粒细胞的吞噬作用,为细菌侵入宿主体内提供协助作用,从而导致疾病的发生。由于白细胞毒素的抗原表位区较分散,其中相对较集中的2407~2695之间的氨基酸序列还未见报道。因此,本研究拟从绵羊腐蹄病病料中分离坏死梭杆菌并开展白细胞毒素免疫原性的研究,以期为今后制备坏死梭杆菌所致疾病的有效疫苗奠定基础。(1)严格遵循厌氧条件下从病料中分离培养细菌,通过形态学观察、PCR扩增和系统发育树分析对分离菌株进行检测。结果显示:从感染腐蹄病的绵羊蹄部分离出长丝串珠状结构的菌体,经PCR扩增可知该分离株为坏死梭杆菌阳性,从系统发育树可知该分离株上传所得序列(KY808973)与澳大利亚分离株A39A8(JX678850.1)的亲缘关系最近,同源性达99%。(2)应用DNA star软件,依据坏死梭杆菌白细胞毒素全长基因的抗原表位区和开放性阅读框筛选出一段基因,依此设计引物来扩增目的基因,并将其克隆到pET-32a(+)表达载体上获得重组质粒,转化到表达菌BL21(DE3)中,经IPTG诱导获得蛋白表达,纯化出的蛋白通过Western-blot检测其反应原性。结果表明:重组蛋白主要以可溶性的形式表达,分子量大小约为35 ku,将其命名为lkt A35;Western-blot检测显示,重组蛋白可以与全菌灭活苗制备的高免血清结合,具有较好的反应原性。(3)用获得的重组蛋白与弗氏佐剂乳化后,免疫小鼠3次,制备多克隆抗体,用间接ELISA检测其抗体效价。结果显示:二免和三免的抗体效价分别达到1:64 000和1:128 000,表明本试验获得的多克隆抗体具有较高的抗体效价。
[Abstract]:Fusobacterium necrophorum (Fusobacterium necrophorum) is one of the common pathogens of sheep hoof rot and liver abscess.The leucocyte toxin produced by the pathogen is one of the most virulent factors secreting toxins, which can inhibit the phagocytosis of neutrophils and provide assistance for bacteria to invade the host body, thus leading to the occurrence of diseases.Due to the dispersion of antigenic epitopes of leukocyte toxins, the amino acid sequences between 2407 and 2695, which are relatively concentrated, have not been reported.Therefore, the aim of this study was to isolate Clostridium Necrosis from sheep hoof rot and to study the immunogenicity of leukocyte toxin.In order to lay the foundation for the preparation of the effective vaccine caused by Clostridium Necrosis in the future, the bacteria were isolated and cultured from the diseased materials under anaerobic condition strictly, and the isolated strains were detected by morphological observation PCR amplification and phylogenetic tree analysis.The results showed that filamentous beads were isolated from the hoof of sheep infected with hoof rot, and the strain was positive for Clostridium necrotizing by PCR amplification.The phylogenetic tree showed that the sequence KY808973) was most closely related to the Australian strain A39A8 (JX678850.1), and the homology was 99%. (2) DNA star software was used.According to the antigen epitope region and open reading frame of the full-length gene of Clostridium necrosin, a gene was screened out, and the target gene was amplified by using this primer and cloned into the expression vector pET-32a () to obtain the recombinant plasmid.The protein was induced by IPTG and the purified protein was detected by Western-blot.The results showed that the recombinant protein was mainly expressed in soluble form, and its molecular weight was about 35 ku. the recombinant protein was named lkt A35 Western-blot. The recombinant protein could bind to the high immune serum prepared by the whole bacterium inactivated vaccine.After emulsified with Freund's adjuvant, mice were immunized for 3 times to prepare polyclonal antibodies. The titers of polyclonal antibodies were detected by indirect ELISA.The results showed that the titers of the second and third immunizations reached 1:64 000 and 1: 128 000 respectively, which indicated that the polyclonal antibodies obtained in this experiment had higher titers.
【学位授予单位】：甘肃农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.61

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