表达EmIMP1-flagellin重组柔嫩艾美耳球虫激发鸡肠道粘膜免疫特性研究

Eimeria spp. are the most common protozoan pathogens of chickens. Infection by Eimeria causes coccidiosis which leads to significant economic losses in the poultry industry. Fortunately, Eimeria infection can be effectively controlled by vaccination with live Eimeria oocysts which can elicit high level antibody and cell mediated immune responses. But this kind of vaccine has not been widely used due to the difficult preparation, and high price. The establishment of transient and stable transfection systems in E. tenella now encourages the use of these parasites as novel vaccine delivery vehicles to express protective antigens from other eimeria species. As different eimeria species can parasitize in different gut of chickens, it is essential to study the intestinal mucosal immunity of chickens immunized with the recombinant E. tenella. In this study, firstly, we will construct the transgenic E. tenella expressing immune mapped protein 1 of E. maxima (EmIMP1) and flagellin ( ligand of TLR5) fusion protein; chickens will be immunized with the transgenic oocysts, and the intestinal mucosal humoral the cell-mediated immune responses of chickens will be studied respectively. Finally, immunized chickens will be challenged by E. maxima, and the clinical effects (intestinal lesion scores, body weights gain, and oocysts shedding) of chickens will be measured to evaluate the protection level of the recombinant E. tenella. Our research will provide some useful information for the development of coccidiosis vaccines.

鸡球虫病是危害养禽业最重要的疾病之一，疫苗免疫被认为是该病防治的重要途径。活卵囊疫苗已经研制成功，但该疫苗是多种虫种的混合体，存在着制备困难，剂量难以掌握，价格昂贵等缺点，因此，尚未得到广泛应用。球虫转染技术的发展使我们可以利用一种球虫表达其他球虫的保护性抗原，制备新型的重组球虫活卵囊疫苗，进而达到一苗多用，减少卵囊剂量的目的。但由于不同球虫寄生的部位不同，重组球虫活疫苗是否可以在鸡肠道的不同部位，激发同样的保护性免疫应答呢？为回答这一科学问题，本申请将构建表达EmIMP1-flagellin的重组柔嫩艾美耳球虫株，研究其在鸡肠道的不同部位激发的体液免疫应答和细胞免疫应答；并对免疫鸡用巨型艾美耳球虫攻虫，从鸡的体增重，肠道病变计分以及卵囊排出量方面对该重组球虫激发的免疫保护效果进行评价，以此证明鸡的肠道粘膜免疫具有共同粘膜免疫的特性，研究结果将有助于球虫病的免疫预防和疫苗开发。

疫苗递呈系统对于疫苗研究来说至关重要。本研究中，我们利用柔嫩艾美耳球虫表达巨型艾美耳球虫的免疫保护抗原EmIMP1，研究其激发的免疫反应以及免疫保护作用。为基于球虫的双价活疫苗提供基础。首先，构建球虫表达载体，转染后筛选稳定表达EmIMP1的柔嫩艾美耳球虫株，并对外源基因的表达进行鉴定。然后，进行免疫实验，检测免疫后的抗体反应以及细胞免疫应答；免疫后，分别用柔嫩艾美耳球虫和巨型艾美耳球虫进行攻虫试验，检测该疫苗对两种球虫的免疫保护作用。结果表明，该疫苗可以激发球虫特异性的体液和细胞免疫应答，并可以对球虫的感染提供部分保护。试验结果可以为开发基于球虫的新型活载体多价疫苗提供借鉴。