人白细胞介素-10 cDNA的克隆、测序和真核表达载体的构建(英文)

Cloning,sequencing of human interleukin 10 cDNA and construction of its eukaryotic expression vector

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 open reading frame(ORF) of human interleukin 10 (hIL 10) cDNA for subsequent construction of eukaryotic expression vector.Methods Concanavalin A activated mononuclear cells from peripheral blood were cultured for total RNA extraction.With the use of random primers,cDNA was synthesized and then PCR was accomplished using the specific human interleukin 10 primers.Sequence analysis of pUC18hIL 10 was achieved with 377 type DNA sequencer;Eukaryotic expression vector pcDNA3.1hIL 10 was constructed with ligation.Results A 550bp DNA fragment was amplified with RT PCR.pUC18hIL 10 and pcDNA3.1hIL 10 were constructed and sequence analysis revealed 100% homology to published data in GenBank.Conclusion We successfully cloned human interleukin 10 cDNA ORF and constructed the eukaryotic expression vector pcDNA3.1hIL 10.