表达APEC Ⅰ型菌毛的减毒鸡伤寒沙门菌重组疫苗的构建及免疫保护研究
发布时间：2018-10-04 21:04
【摘要】：鸡大肠杆菌病(Chicken Colibacillosis)是由禽致病性大肠杆菌(avian pathogenic Escherichia coli, APEC)引起的鸡体局部或全身性感染的传染病,是造成养禽业严重经济损失的主要传染病之一。目前,抗菌药物仍是防治鸡大肠杆菌病的主要措施,由此带来的生物安全问题受到广泛关注。针对鸡大肠杆菌病的防控,采取疫苗预防与合理用药相结合的综合措施是最为行之有效的方法。因此,开发强免疫效力且具有广谱保护性的新型APEC基因工程疫苗是解决该现状的一种有效途径。本研究选择减毒鸡伤寒沙门氏菌SG9R株为宿主菌,以asd基因为营养缺陷标志构建减毒鸡伤寒沙门氏菌染色体-质粒平衡致死系统表达禽源大肠杆菌Ⅰ型菌毛抗原,以探讨该重组口服疫苗对预防鸡大肠杆菌病的保护效果。1.禽源大肠杆菌Ⅰ型菌毛操纵子fim基因的克隆、表达及生物活性以禽源大肠杆菌分离株CE2基因组DNA为模板,采用长PCR技术扩增出编码Ⅰ型菌毛操纵子fim基因,克隆至表达质粒载体pBR322,构建和筛选出含fim完整基因并正确插入的pBR322-fim重组质粒,将该重组质粒转化至无菌毛的宿主大肠杆菌SE5000。该表达重组菌能分别与鸡红细胞、酵母细胞发生明显的凝集反应且均能被D-甘露糖所抑制；重组菌与鼠抗Ⅰ型菌毛亚单位蛋白FimA高免血清产生明显的凝集反应。电镜观察到重组菌表面长满菌毛,利用该重组菌免疫小鼠制备抗血清经交叉吸附纯化后获得Ⅰ型菌毛单因子血清,可有效用于禽源大肠杆菌Ⅰ型菌毛表达的检测。用重组菌pfim进行人肺腺上皮细胞A549体外粘附试验和粘附抑制试验,结果表明：重组菌pfim和野生株CE2均具有粘附肺腺上皮细胞的能力,且重组菌的粘附特性明显强于野生菌株,而D-甘露糖能有效地抑制上述重组菌或野生菌株对人肺腺上皮细胞的粘附结合。这为开发以Ⅰ型菌毛作为免疫原的基因工程疫苗的研制奠定了基础。2.禽伤寒沙门氏菌SG9R株asd缺失株平衡致死系统平台的构建禽伤寒沙门氏菌SG9R弱毒株在预防禽伤寒中发挥着较好作用,为将SG9R弱毒株开发为能携带外源基因的口服活疫苗载体且保持其原有免疫原性,利用Red同源重组系统对SG9R株基因组中编码天冬氨酸β-半醛脱氢酶的asd基因进行敲除,获得SG9RΔasd缺失突变株。该突变株在缺乏外源性DAP培养条件下发生溶菌死亡,而在添加DAP的培养基或导入携带asd基因的互补质粒后才能恢复生长,以此为基础建立以asd基因为营养缺陷标志的禽伤寒沙门氏菌染色体-质粒平衡致死系统。选择表达绿色荧光蛋白(GFP)基因作为报告基因,以携带链球菌asd基因的pYA3342质粒为表达载体,构建了表达绿色荧光蛋白的重组菌SG9R (pYA3342-GFP)。该重组菌在体外传至40代后,经荧光显微镜观察和流式细胞仪分析表明仍可稳定高效表达绿色荧光蛋白；同时,将该重组菌口服接种20日龄仔鸡,发现其广泛分布于鸡的脾脏、肝脏及肠道组织等,体内稳定性试验表明重组菌可在鸡体内停留2-3周。以上结果表明该缺失株可以用来作为宿主载体平衡致死系统来高效稳定表达外源基因,为弱毒禽伤寒沙门氏菌作为载体的基因工程活疫苗研制提供了优良的操作平台。3.禽源大肠杆菌Ⅰ型菌毛抗原在鸡伤寒沙门氏菌SG9RΔasd缺失株中的表达及免疫保护效果研究利用PCR技术扩增出禽源大肠杆菌表达Ⅰ型菌毛的fim操纵子基因并将其克隆至携带asd基因的组成型表达载体pYA3342,构建重组表达质粒pYA3342-fim,重组质粒经电转化至减毒鸡伤寒沙门氏菌SG9RΔasd缺失株,普通LB平板筛选阳性克隆,命名为SG9R (pYA3342-fim)。经甘露糖敏感性血凝试验与抗甘露糖血凝试验、酵母细胞凝集试验表明重组菌在普通培养条件下(37℃C,振荡培养)能够很好的表达Ⅰ型菌毛。同时,对重组疫苗的生长特性、遗传稳定性及口服安全性等生物学特性进行了评定。通过对3周龄非免疫鸡口服接种重组菌苗SG9R (pYA3342-fim)作为免疫组,SG9R (pYA3342)为空载体对照组,PBS为空白组,分别对各组试验鸡于不同时间段采集血清利用间接ELISA法测定抗Ⅰ型菌毛特异性IgG抗体动态水平,一次免疫14d后,免疫组血清抗体水平显著高于空载体对照组和空白组(P0.05),而空载体对照组与空白组之间无显著性差异(P0.05)；在整个免疫周期内(4周),免疫组鸡血清中特异性抗体水平均呈上升趋势。用禽致病性大肠杆菌强毒株QD2对各试验组鸡群经后胸气囊攻毒,实验观察10d内,免疫组、空载体对照组及空白对照组的死亡率分别为40.0%、73.3%、80.0%。试验结果表明,本实验构建重组活菌苗SG9R (pYA3342-fim)对禽大肠杆菌病的预防可以起到一定的保护效果。
[Abstract]:Chicken colibacis is an infectious disease caused by avian pathogenic Escherichia coli (APEC) or systemic infection, which is one of the major infectious diseases causing serious economic loss in poultry industry. At present, the anti-bacteria medicine is still the main measure to control the chicken colibacteremia, and the biological safety problem brought by the antibacterial medicine is widely concerned. According to the prevention and control of chicken colibacs, comprehensive measures combining vaccine prevention and rational drug administration are the most effective methods. Therefore, it is an effective way to develop a new APEC genetic engineering vaccine with strong immunopotency and broad-spectrum protection. In this study, the S. typhimurium SG9R strain was selected as host strain, and the asd gene was used as a nutrition defect marker to construct the Salmonella typhimurium chromosome-plasmid balanced lethal system to express avian source E. coli type I fimbriae antigen. To investigate the protective effect of the recombinant oral vaccine on the prevention of E. coli in chickens. The cloning, expression and biological activity of the Escherichia coli type I fimbrium gene of avian source are the template of CE2 genomic DNA of the avian source E. coli isolate, and the encoding type I fimbrium gene is amplified by using the long PCR technology, and cloned to the expression plasmid vector pBR322. A recombinant plasmid pBR322-fim containing fim complete gene was constructed and screened, and the recombinant plasmid was transformed into sterile wool host E. coli SE5000. The recombinant bacteria can react with chicken red blood cells and yeast cells respectively, and can be inhibited by D-mannose. Electron microscopy showed that the surface of recombinant bacteria was full of fimbriae, and the serum of type 鈪，
本文编号：2251872
[Abstract]:Chicken colibacis is an infectious disease caused by avian pathogenic Escherichia coli (APEC) or systemic infection, which is one of the major infectious diseases causing serious economic loss in poultry industry. At present, the anti-bacteria medicine is still the main measure to control the chicken colibacteremia, and the biological safety problem brought by the antibacterial medicine is widely concerned. According to the prevention and control of chicken colibacs, comprehensive measures combining vaccine prevention and rational drug administration are the most effective methods. Therefore, it is an effective way to develop a new APEC genetic engineering vaccine with strong immunopotency and broad-spectrum protection. In this study, the S. typhimurium SG9R strain was selected as host strain, and the asd gene was used as a nutrition defect marker to construct the Salmonella typhimurium chromosome-plasmid balanced lethal system to express avian source E. coli type I fimbriae antigen. To investigate the protective effect of the recombinant oral vaccine on the prevention of E. coli in chickens. The cloning, expression and biological activity of the Escherichia coli type I fimbrium gene of avian source are the template of CE2 genomic DNA of the avian source E. coli isolate, and the encoding type I fimbrium gene is amplified by using the long PCR technology, and cloned to the expression plasmid vector pBR322. A recombinant plasmid pBR322-fim containing fim complete gene was constructed and screened, and the recombinant plasmid was transformed into sterile wool host E. coli SE5000. The recombinant bacteria can react with chicken red blood cells and yeast cells respectively, and can be inhibited by D-mannose. Electron microscopy showed that the surface of recombinant bacteria was full of fimbriae, and the serum of type 鈪，
本文编号：2251872
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