【作者】 李俊；

【导师】 李祥瑞；

【作者基本信息】 南京农业大学 ， 预防兽医学， 2006， 硕士

【摘要】 鸡球虫病是一种病原寄生在细胞内的原虫疾病，给养禽业所带来的经济损失极为严重。目前，应用药物防治遇到了球虫耐药性和药物残留的问题，大大限制了养禽业发展，球虫防治需要寻求新的途径。实践证明，激发宿主细胞免疫反应来防治鸡球虫病是更有效的途径之一。作为第三代疫苗的DNA疫苗的一个显著优势是刺激机体产生广泛的细胞免疫反应和持久的免疫记忆，尤其应用特定的球虫保护性抗原联合一些细胞因子，构建调节型DNA疫苗，在球虫病防治中具有诱人的应用前景。为此我们选择球虫的抗原TA4、MZ5-7、SO7、pEtK2基因与鸡细胞因子IFN-γ基因，通过DNA重组技术将球虫的抗原基因分别和鸡细胞因子基因IFN-γ串联为一体，插入到相关真核表达载体当中，分别构建了pVAX1.0-TA4-IFN-γ、pVAX1.0-MZ5-7-IFN-γ、pVAX1.0-SO7-IFN-γ、pVAX1.0-pEtK2-IFN-γ四种免疫调节型核酸疫苗和pVAX1.0-TA4、pVAX1.0-MZ5-7、pVAX1.0-SO7、pVAX1.0-pEtK2四种基因疫苗，同时进行动物免疫保护性试验比较这些DNA疫苗的免疫保护效果。 设计特异性引物，利用本实验室克隆构建的pMD18-T-TA4、pMD18-T-MZ5-7、pMD18-T-SO7、pMD18-T-pETK2、pMD18-T-IFN-γ对E．tenella球虫基因TA4、MZ5-7、SO7、pEtK2、和鸡IFN-γ基因进行改造，以便于构建核酸疫苗。 用本实验室构建好的球虫E．tenella TA4、MZ5-7、SO7、pEtK2原核表达载体pET28b-TA4、pET28b-MZ5-7、pET32a(+)-SO7、pET-28a(+)-pEtK2进行诱导表达，并对表达蛋白进行纯化后注射大鼠，制备大鼠E．Tenella TA4、SO7、pEtK2蛋白抗血清。 将制备的TA4、SO7、pEtK2蛋白血清用于TA4、SO7、pEtK2基因疫苗在体内蛋白表达的检测；用大鼠子孢子抗血清检测MZ5-7基因疫苗在体内的表达。用特异性引物对TA4、MZ5-7、SO7、pEtK2基因疫苗在体内表达进行RT-PCR检测。结果表明构建的基因疫苗在体内能很好地转录表达，可用于动物保护性实验研究。 实验动物分为pVAX1.0-TA4-IFN-γ组、pVAX1.0-MZ5-7-IFN-γ组、pVAX1.0-SO7-IFN-γ组、pVAX1.0-pEtK2-IFN-γ组、pVAX1.0-TA4组、pVAX1.0-MZ5-7组、更多还原

【Abstract】 Avain coccocidiosis is a cytoplastic entozoic parasite disease that causes severe economic loss in the poultry industry . The emergence of drug-resistant parasites and the drugs residues problem restricted the development of poultry industry so that a new approach to prevent avain coccocidiosis should be found. It have proved that stimulating the cell immuno-reaction of the host is an effective approach to prevent avain coccocidiosis . A prominence advantage of DNA vaccine as a new generation of vaccine is that it can induce abroad cell immuno-reaction and form long perdured immunity memory, especially the immune regulative DNA vaccine which was constructed by special antigen together with some cytokine genes has more effect and prospect in the anti- coccocidiosis practice. In this investigation we selected the TA4、 MZ5-7、 SO7、 pEtK2 genes of Eimeria tenella antigen to construct DNA vaccine pVAX1.0- TA4、 pVAX1.0- MZ5-7, pVAX1.0-SO7, pVAX1.0- pEtK2 and combined with the chicken cytokine ChlFN-γ to construct immuno-regulative DNA vaccine pVAX1.0- TA4-IFN-γ pVAX1.0- MZ5-7-IFN-y, pVAX1.0- SO7-IFN-γ pVAX1.0- pEtK2-IFN-γ. The protective effects of these vaccines were compared in the experiment of chickens against the challenge of Eimeria tenella.For convenience to construct DNA vaccine, designed special primers were used to reconstruct Eimeria tenella antigen gene TA4, MZ5-7, SO7, pEtK2 and chicken cytokine ChlFN-γ by PCR method. And pMD18-T-TA4, pMD18-T-MZ5-7, pMD18-T-SO7, pMD18-T- pEtK2, pMD18-T-IFN-γ constructed by our laboratory were used as the PCR templates.IPTG was used to induce the expression of the E. tenella TA4, MZ5-7, SO7, pEtK2 proteins by vector pET28b-TA4, pET28b-MZ5-7, pET32a(+)-SO7, pET-28a(+)-pEtK2 constructed by our laboratory. The pET28b-MZ5-7 was not good expressed as expect. Purified TA4, SO7, pEtK2 proteins was used to immune rats for E. tenella TA4, SO7, pEtK2 anti-serums.These anti-serums were used to check the expression of TA4, SO7, pEtK2 DNA更多还原