【摘要】 从ConA刺激的鸡脾细胞中扩增出鸡白细胞介素 2 (ChIL 2 )基因编码区。将该编码区cDNA序列和调控其转录的鸡痘病毒早晚期启动子 (PE L)的基因片段定向克隆到鸡痘病毒转移载体p1175中 ,获得重组转移载体p1175IL2 ,然后转染已感染鸡痘病毒 2 82E4疫苗株 (wt FPV)的鸡胚成纤维细胞 (CEF) ,质粒p1175IL2与wt FPV基因组DNA发生同源重组 ,产生了表达ChIL 2的重组鸡痘病毒rFPV IL2。通过在含X gal的营养琼脂上连续挑选蓝色病毒蚀斑 ,获得并纯化重组鸡痘病毒rFPV IL2。应用XTT PMS方法检测的rFPV IL2 (M .O .I 2 0 )感染CEF 72小时后细胞上清中表达的重组ChIL 2生物活性 ,效价为 3 6× 10 5u mL ,表明rFPV IL2能有效地表达ChIL 2。下一步将利用rFPV IL2在体内表达ChIL 2 ,研究ChIL 2的免疫增强作用及其作用机理。更多还原

【Abstract】 In order to determine the adjuvant effects of the chicken IL-2 (ChIL-2)on new generation vaccines, ChIL-2 gene was amplified from ConA-stimulated chicken spleen cells by RT-PCR and was directionally inserted into fowlpox virus (FPV) transferring vector p1175 under the control of FPV early/late promoter(PE/L), resulting in recombinant transferring vector p1175IL2. Then the p1175IL2 plasmid was transfected into chicken embryo fibroblasts (CEF) pre-infected with wild type FPV to generate recombinant fowlpox virus expressing ChIL-2 (rFPV-IL2). By selection of blue plaques on the CEF, overlaid with agar containing X-gal, rFPV-IL2 was obtained and purified. The supernatant from CEF monolayer infected with rFPV-IL2 (M.O.I 2.0) after 72 hours was detected for the production of ChIL-2 by XTT/PMS colorimetric assay. About 3.6×105u/mL of specific ChIL-2 activity was determined. The results show that rFPV-IL2 can express ChIL-2 effectively. rFPV-IL2 provides us with an effective tool for studying avian immunology as well as a potential vaccine-enhancing agent.更多还原