本文選題：豬丹毒桿菌 + 生物學(xué)特性��； 參考：《安徽農(nóng)業(yè)大學(xué)》2015年碩士論文

【摘要】：豬丹毒(Swine erysipelas)是由豬丹毒桿菌(Erysipelothrix rhusiopathiae)引起的一種人畜共患傳染病,已有二十多年極少見到,但近幾年,無論國內(nèi)、國外豬丹毒的發(fā)生都有明顯的增多。SpaA蛋白是豬丹毒桿菌的主要免疫保護(hù)性抗原,而且該抗原幾乎存在于所有毒力較強(qiáng)的豬丹毒桿菌中。本研究對分離自安徽省8個不同地區(qū)豬場的豬丹毒桿菌進(jìn)行生物學(xué)特性研究,鑒定SpaA蛋白的免疫原性,確定其對小鼠的免疫保護(hù)性,為選擇良好的亞單位疫苗候選單位提供參考,為豬丹毒的防控及豬丹毒新型疫苗的研究提供理論依據(jù)。通過形態(tài)學(xué)及培養(yǎng)特性觀察、生化試驗(yàn)、PCR方法對菌株進(jìn)行鑒定,并進(jìn)行藥物敏感性試驗(yàn)及商品化豬丹毒G4T10株弱毒疫苗免疫保護(hù)實(shí)驗(yàn)。利用PCR擴(kuò)增豬丹毒桿菌SpaA基因,連接pGEX-6P-1載體,將鑒定為陽性的重組載體轉(zhuǎn)化至大腸桿菌中,加入誘導(dǎo)劑常溫誘導(dǎo)表達(dá),獲得的蛋白經(jīng)過聚丙烯酰氨凝膠電泳和Western-blotting分析鑒定;重組蛋白免疫小鼠,檢測血清中IgG抗體和Th1/Th2細(xì)胞因子含量;臨床分離自敗血型病例及疹塊型病例豬丹毒桿菌攻擊免疫后小鼠,測定其免疫保護(hù)性,采集小鼠臟器制作病理組織切片,觀察病理組織變化。結(jié)果顯示:共分離到29株豬丹毒桿菌,源自8個地區(qū)的豬丹毒桿菌分離菌具有較一致的形態(tài)特征和相似的生化特性。29株豬丹毒桿菌對氨芐西林、頭孢曲松敏感率均達(dá)100%,其次是青霉素93%、紅霉素89.7%和頭孢噻肟75.9%,對其他13種藥物則表現(xiàn)不同程度的耐藥性。8株不同地區(qū)豬丹毒桿菌分離菌的LD50在14.3-2.36×10~2 CFU/mL之間,顯示分離菌對小鼠均具有較強(qiáng)的致病力。商品化豬丹毒G4T10株弱毒疫苗2次頸部皮下免疫小鼠后,分別用劑量為100LD50的8株豬丹毒桿菌分離菌腹腔攻毒小鼠,免疫保護(hù)率為100%。豬丹毒桿菌SpaA基因在大腸桿菌中實(shí)現(xiàn)表達(dá),獲得大小為75 kD的目的蛋白,能夠與豬丹毒桿菌陽性血清反應(yīng);重組蛋白能誘導(dǎo)小鼠產(chǎn)生較高水平的特異性抗體,同時誘導(dǎo)Th1、Th2細(xì)胞因子水平顯著升高,SpaA重組蛋白對臨床分離自敗血型病例及疹塊型病例豬丹毒桿菌的免疫保護(hù)率為100%,蛋白免疫組與攻毒對照組相比,病理組織變化差異明顯。結(jié)果表明:安徽地區(qū)豬丹毒發(fā)生有上升趨勢,不同地區(qū)的豬丹毒桿菌分離菌具有較為一致的生物學(xué)特性,青霉素類和頭孢類抗菌藥物有顯著療效,使用豬丹毒G4T10株弱毒疫苗可產(chǎn)生有效的免疫保護(hù)力。成功表達(dá)的SpaA重組蛋白具有良好的免疫原性,能誘導(dǎo)機(jī)體產(chǎn)生較高水平的體液免疫應(yīng)答及細(xì)胞免疫應(yīng)答,且對臨床分離自敗血型病例及疹塊型病例豬丹毒桿菌攻擊小鼠均具有較好的免疫保護(hù)性,可以作為良好的亞單位疫苗的候選單位,為豬丹毒新型疫苗的研究及豬丹毒的防控提供理論依據(jù)。
[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.
【學(xué)位授予單位】：安徽農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S852.61

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