Abstract: Abstract: Objective To analyze the effect of signal peptide of chicken interleukin-10 ( chIL-10) on expressing in CHO-K1 cell line，and to establish a stable chIL-10 expressing cell line． Method Whole chIL-10F and signal peptide-deleting genetic fragment of chIL-10 were amplified with two pairs of primers and inserted into pEGFP-N1 vector． The obtained recombinant plasmids of N1-chIL-10F and N1-chIL-10M were transfected into CHO-K1 cells under G418 screening． Ｒesult Two stable cell lines of CHO-chIL-10F and CHO-chIL-10M were obtained， respectively，confirmed by ＲT-PCＲ and western blot assays． Confocal examination showed that the reporter protein GFP was expressed in the cytoplasm of CHO-chIL-10M and stronger than in the CHO-chIL-10F． Conclusion Ｒemoving signal peptide of chIL-10 improves expression of chIL-10 in the cytoplasm in vitro．

Key words: <p>Chicken interleukin-10, Signal peptide, Eukaryotic vector, CHO-KI cells</p>

摘要： 摘要: 目的 分析鸡白细胞介素 10( chicken interleukin-10，chIL-10) 的信号肽对体外表达的影响，以获得稳定表达 chIL-10 的细胞系。方法 采用 ＲT-PCＲ 方法从经脂多糖( LPS) 体外刺激的鸡外周血淋巴细胞中分别扩增含信号肽( euchIL-10F) 和去信号肽( euchIL-10M) 的 chIL-10 编码基因，克隆入含有报告基因 eGFP 和新霉素抗性基因 ( neo) 的真核表达载体 pEGFP-N1 中，获得重组质粒 N1-euchIL-10F 和 N1-euchIL-10M。分别转染中国仓鼠卵巢细胞系( CHO-K1) ，经 G418 加压筛选、纯化，获得稳定表达 chIL-10 蛋白的 CHO-K1 细胞系，利用激光共聚焦试验观察外源蛋白的细胞定位。结果 通过 ＲT-PCＲ、间接免疫荧光试验和免疫印迹试验，表明 euchIL-10F 和 euchIL-10M 均整合入细胞基因组中，且 N1-euchIL-10M 在胞浆大量表达较强的绿色荧光，而 N1-euchIL-10F 表达的荧光较弱，且胞浆内含量很低。结论 去除信号肽利于 chIL-10 基因在体外真核细胞胞浆的表达。

关键词: <, p>, 鸡白细胞介素-10, 信号肽, 真核表达, CHO-K1 细胞<, /p>