【作者】 孟庆玲；

【导师】 才学鹏； 吴润；

【作者基本信息】 甘肃农业大学 ， 预防兽医学， 2008， 博士

【摘要】 兔球虫病是由艾美耳科的球虫引起的一种寄生性原虫病,随着规模养兔的迅速发展,该病对我国养兔业的危害日益严重。目前,报道的感染家兔的艾美耳属球虫有15个种,其中斯氏艾美耳球虫(Eimeria stiedai,Es)的致病力最强、危害最大,它寄生于兔的肝胆管上皮细胞,能引起严重的肝球虫病。尽管化学药物是目前控制兔球虫病的常用手段,但随着耐药虫株的不断出现和化学药物残留问题的日益凸现,越来越多的学者寻求用免疫学方法来防控该病。本研究应用无球虫兔和单卵囊分离技术进行了Es LZ株的分离,运用RT-PCR技术成功扩增出Es LZ株微线蛋白5(MIC-5)基因,并进行了原核表达和纯化;克隆了兔IL-15基因;以含有双启动子的pBudCE4.1为载体,构建了真核表达质粒pBMIC-5-IL-15,通过脂质体转染BHK-21细胞,证实该真核表达质粒能正确表达重组蛋白Es MIC-5和IL-15。在上述研究的基础上,根据载体-宿主平衡致死系统的原理,将asd基因引入真核表达质粒pBMIC-5-IL-15中,相继电转化asd基因缺陷型减毒鼠伤寒沙门氏菌X3730和X4550,从而构建了pBMIC-5-IL-15-asd载体-沙门氏菌平衡致死系统。遗传稳定性试验表明,该载体能稳定存在于减毒鼠伤寒沙门氏菌X4550中。用携带重组质粒pBMIC-5-IL-15-asd的减毒鼠伤寒沙门氏菌X4550在无球虫兔上进行了免疫试验。结果表明,重组菌X4550/pBMIC-5-IL-15-asd能诱导机体产生较高水平的抗球虫细胞免疫应答和体液免疫应答,有较好的免疫保护作用。以上试验为兔肝球虫病口服疫苗的研制奠定了基础。更多还原

【Abstract】 Rabbit coccidiosis is a kind of sporozoan disease caused by Eimeriidae species, which jeopardizes rabbit breeding industry in our country. Among rabbit coccidian, Eimeria stiedai is the most virulent and hazardous coccidian, which parasitizes in the bile duct epithelial cells of rabbit, causing serious liver coccidiosis. Although chemotherapy is the conventional method to control rabbit coccidiosis, more and more scholars have sought immunological means to control the disease due to the increasing problems such as resistant strains and drug residue. In the study, E. stiedai LZ strain was isolated with coccidia-free rabbits by a single oocyst technique, then microneme protein 5 (Es MIC-5) gene was cloned from sporozoites of the E. stiedai LZ strain. Es MIC-5 was successfully expressed in E. coli by use of prokaryotic expression vector pETMIC-5. The expressed recombinant protein MIC-5 was purified for detection of specific antibody against E. stiedai. Rabbit IL-15 gene was amplified by RT-PCR, and MIC-5 and IL-15 were cloned into two multiple cloning sites (MCS) of eukaryotic expression vector pBudCE4.1 (the positive named as pBMIC-5-IL-15), respectively. The expression of MIC-5 and IL-15 in BHK-21 transfected by the recombinant plasmid pBMIC-5-IL-15 was confirmed by indirect immunofluorescent test. Moreover, asd gene from S. typhimtwium was introduced into DNA vaccine vector pBMIC-5-IL-15 and the Zeocin resistance gene was then fragmented and a new DNA vaccine vector pBMIC-5-IL-15-asd without an antibiotic resistance gene was constructed according to the principle of the balance-lethal system. Then pBMIC-5-IL-15-asd was transformed into ?asd S. typhimtmium X3730 and X4550 strains one after another. The results of genetic stability test showed DNA vaccine vector pBMIC-5-IL-15-asd stablely existed in the ?asd S. typhimtwium X4550 strain, which puts light on antibiotic resistance and instability of DNA vaccines in hosts. Based on the immunization of 45 coccidia-free rabbits, the results showed that the recombinant S. typhimtwium X4550 strain carrying DNA vaccine vector pBMIC-5-IL-15-asd could efficiently induce specific humoral and cellular immune responses to E. stiedai, supplying immune protection to some extent, which lays a solid foundation for further studies on oral vaccines to prevent liver coccidiosis in rabbits.更多还原