猪丹毒杆菌安徽分离株的生物学特性及SpaA蛋白免疫原性研究

发布时间：2018-04-26 23:32

本文选题：猪丹毒杆菌 + 生物学特性　；参考：《安徽农业大学》2015年硕士论文

【摘要】：猪丹毒(Swine erysipelas)是由猪丹毒杆菌(Erysipelothrix rhusiopathiae)引起的一种人畜共患传染病,已有二十多年极少见到,但近几年,无论国内、国外猪丹毒的发生都有明显的增多。SpaA蛋白是猪丹毒杆菌的主要免疫保护性抗原,而且该抗原几乎存在于所有毒力较强的猪丹毒杆菌中。本研究对分离自安徽省8个不同地区猪场的猪丹毒杆菌进行生物学特性研究,鉴定SpaA蛋白的免疫原性,确定其对小鼠的免疫保护性,为选择良好的亚单位疫苗候选单位提供参考,为猪丹毒的防控及猪丹毒新型疫苗的研究提供理论依据。通过形态学及培养特性观察、生化试验、PCR方法对菌株进行鉴定,并进行药物敏感性试验及商品化猪丹毒G4T10株弱毒疫苗免疫保护实验。利用PCR扩增猪丹毒杆菌SpaA基因,连接pGEX-6P-1载体,将鉴定为阳性的重组载体转化至大肠杆菌中,加入诱导剂常温诱导表达,获得的蛋白经过聚丙烯酰氨凝胶电泳和Western-blotting分析鉴定;重组蛋白免疫小鼠,检测血清中IgG抗体和Th1/Th2细胞因子含量;临床分离自败血型病例及疹块型病例猪丹毒杆菌攻击免疫后小鼠,测定其免疫保护性,采集小鼠脏器制作病理组织切片,观察病理组织变化。结果显示:共分离到29株猪丹毒杆菌,源自8个地区的猪丹毒杆菌分离菌具有较一致的形态特征和相似的生化特性。29株猪丹毒杆菌对氨苄西林、头孢曲松敏感率均达100%,其次是青霉素93%、红霉素89.7%和头孢噻肟75.9%,对其他13种药物则表现不同程度的耐药性。8株不同地区猪丹毒杆菌分离菌的LD50在14.3-2.36×10~2 CFU/mL之间,显示分离菌对小鼠均具有较强的致病力。商品化猪丹毒G4T10株弱毒疫苗2次颈部皮下免疫小鼠后,分别用剂量为100LD50的8株猪丹毒杆菌分离菌腹腔攻毒小鼠,免疫保护率为100%。猪丹毒杆菌SpaA基因在大肠杆菌中实现表达,获得大小为75 kD的目的蛋白,能够与猪丹毒杆菌阳性血清反应;重组蛋白能诱导小鼠产生较高水平的特异性抗体,同时诱导Th1、Th2细胞因子水平显著升高,SpaA重组蛋白对临床分离自败血型病例及疹块型病例猪丹毒杆菌的免疫保护率为100%,蛋白免疫组与攻毒对照组相比,病理组织变化差异明显。结果表明:安徽地区猪丹毒发生有上升趋势,不同地区的猪丹毒杆菌分离菌具有较为一致的生物学特性,青霉素类和头孢类抗菌药物有显著疗效,使用猪丹毒G4T10株弱毒疫苗可产生有效的免疫保护力。成功表达的SpaA重组蛋白具有良好的免疫原性,能诱导机体产生较高水平的体液免疫应答及细胞免疫应答,且对临床分离自败血型病例及疹块型病例猪丹毒杆菌攻击小鼠均具有较好的免疫保护性,可以作为良好的亚单位疫苗的候选单位,为猪丹毒新型疫苗的研究及猪丹毒的防控提供理论依据。
[Abstract]:Swine erysipelas is a zoonotic disease caused by Erysipelothrix rhusiopathiae. It has been rarely seen for more than 20 years, but in recent years, no matter in China, SpaA protein is the main immune protective antigen of erysipelas in foreign countries, and it exists in almost all virulent porcine erysipelas. The biological characteristics of porcine erysipelas isolated from 8 different pig farms in Anhui Province were studied in this study. The immunogenicity of SpaA protein was identified and its immunological protection to mice was determined. It provides a reference for the selection of good subunit vaccine candidate units and a theoretical basis for the prevention and control of porcine erysipelas and the study of new porcine erysipelas vaccine. According to the observation of morphology and culture characteristics, the strain was identified by biochemical test and PCR, and the drug sensitivity test and immune protection test of commercial porcine erysipelas G4T10 strain attenuated vaccine were carried out. The SpaA gene of erysipelas porcine was amplified by PCR and ligated with pGEX-6P-1 vector. The recombinant vector was transformed into Escherichia coli and induced by inducer at room temperature. The obtained protein was identified by polyacrylamide gel electrophoresis and Western-blotting analysis. The mice were immunized with recombinant protein to detect the levels of IgG antibody and Th1/Th2 cytokines, and the mice were immunized with erysipelas from septic type and rashes. Mouse viscera were collected to make pathological sections and the pathological changes were observed. The results showed that 29 strains of porcine erysipelas were isolated. The isolates from 8 regions had the same morphological characteristics and similar biochemical characteristics. 29 strains of erysipelas suis had similar biochemical characteristics to ampicillin. The sensitivity rate of ceftriaxone was 100%, followed by penicillin 933%, erythromycin 89.7% and cefotaxime 75.9%. The LD50 of the other 13 drugs was between 14.3-2.36 脳 10 ~ 2 CFU/mL. The results showed that the isolated bacteria had strong pathogenicity to mice. After subcutaneously immunized mice with commercial porcine erysipelas G4T10 strain attenuated vaccine twice, the mice were inoculated with 8 strains of porcine erysipelas isolated bacteria with dose of 100LD50, and the immune protection rate was 100%. The SpaA gene of erysipelas was expressed in Escherichia coli, and the target protein of 75 KD was obtained, which could react with the positive serum of erysipelas, and the recombinant protein could induce mice to produce a higher level of specific antibody. At the same time, the level of Th1 Th 2 cytokines induced by SpaA was significantly increased. The protective rate of SpaA recombinant protein against erysipelas was 100. The pathological changes in the protein immunized group were significantly different from those in the control group. The results showed that the incidence of porcine erysipelas in Anhui Province was on the rise, the isolates of erysipelas from different areas had the same biological characteristics, and penicillin and ceftaxime antimicrobial agents had remarkable curative effects. The use of porcine erysipelas G4T10 strain attenuated vaccine can produce effective immune protection. The recombinant SpaA protein successfully expressed has good immunogenicity and can induce a higher level of humoral and cellular immune responses. And it has good immune protection to the mice which were isolated from the clinical cases of septic type and rashes. It can be used as a good candidate for subunit vaccine. It provides theoretical basis for the study of new vaccine and prevention and control of porcine erysipelas.
【学位授予单位】：安徽农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.61

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