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目的 克隆、测序人白细胞介素 10cDNA开放阅读框架 ,构建其真核表达载体。方法 刀豆蛋白A活化的人外周血单个核细胞培养后用于提取总RNA ,以随机引物行逆转录反应合成hIL 10cDNA第一链 ,并以hIL 10特异性引物行PCR扩增 ,将PCR产物克隆至pUC18中测序并构建真核表达载体pcDNA3.1hIL 10。结果 PCR扩出5 5 0bp特异性片段 ,经克隆至pUC18后测序表明序列同源性与GenBank报道完全一致 ,并克隆鉴定了真核表达载体pcDNA3.1hIL 10。结论 成功克隆了人白细胞介素 10cDNA开放阅读框架 ,序列分析证实与GenBank录入序列同源性达 10 0 % ,并成功构建了真核表达载体pcDNA3.1hIL 10。
Objective To clone and sequence human interleukin 10 cDNA open reading frame and construct its eukaryotic expression vector. Methods Concanavalin A-activated human peripheral blood mononuclear cells were used to extract total RNA. The first strand of hIL 10 cDNA was synthesized by random primer reverse transcription reaction. The PCR products were amplified by PCR with hIL 10 specific primers. Cloned into pUC18 and sequenced and constructed eukaryotic expression vector pcDNA3.1hIL 10. Results A total of 55bp fragment was amplified by PCR. The sequence was cloned into pUC18 and sequenced. The sequence homology was exactly the same as reported in GenBank. The eukaryotic expression vector pcDNA3.1hIL 10 was cloned and identified. Conclusion The open reading frame of human interleukin 10 cDNA was successfully cloned. The sequence analysis confirmed that the cDNA sequence shared 100% identity with GenBank. The eukaryotic expression vector pcDNA3.1hIL 10 was successfully constructed.