柔嫩艾美耳球虫重组鸡痘病毒的构建与免疫保护研究

Construction of E. tenella Fowlpox Recombinant Virus and Immunity Protection

以鸡柔嫩艾美耳球虫(E.tenella)杂交株F2的RNA为模板,应用反转录聚合酶链式反应(RT-PCR)技术扩增了杂交株F2的Rhomboid蛋白家族相关基因,将PCR产物克隆至pMD18-T载体中,构建出克隆质粒pMD-Rhomboid.以KpnⅠ、PstⅠ双酶切重组质粒pMD-Rhomboid和鸡痘病毒载体质粒pUTA2,并将纯化的Rhomboid基因亚克隆至鸡痘病毒载体pUTA2复合启动子下游,构建出真核表达重组质粒pUTA-Rhomboid.采用脂质体转染技术,将该质粒转染FPV282E4株感染的鸡胚成纤维细胞(CEF)中,通过BrdU药物加压筛选,并通过RT-PCR和蛋白质印迹等方法检测,筛选出一株球虫鸡痘重组病毒rFPV-Rhomboid.进一步经CEF扩增病毒后,免疫雏鸡,监测免疫指标.结果表明:重组病毒接种鸡外周血中的CD4+、CD8+含量显著高于非免疫对照组(P<0.05);与对照组相比,重组病毒对鸡的增重效果差异显著(P<0.05),对E.tenella的攻击具有一定的保护作用,显示出较好的应用前景.

In order to construct an efficient and safe engineering vaccine against E. tenella, the relative gene of Rhomboid protein family of E. tenella F2 hybrid strain was amplified by RT-PCR technology and subcloned into the downstream of complex promoter of fowlpox virus vector pUTA2, recombinant fowlpox virus transfer vector was constructed successfully. Plasmid of fowlpox recombinant virus and fowlpox virus were co-transfected into chicken embryo fibroblast （CEF） and identified by BrdU drug, RT-PCR and Western blotting respectively. The result showed that the E. tenella fowlpox recombinant virus （rFPV-Rhomboid） was obtained. Furthermore, rFPV-Rhomboid was amplified in CEF, then chicken was immuned with rFPV-Rhomboid and the indication of immunity was detected. The result indicated that the contents of CD4^＋ and CD8^＋ in peripheral blood were significantly higher than that in control group （P〈0.05）. The weight gained of chicken vaccinated rFPV-Rhomboid was different from the chickens of control group significantly （P 〈 0.05）. The recombinant protein had immunogenicity and could induced strong immune response and immune protection ability.