人IL-3基因cDNA的克隆及其在造血干细胞的表达

Clone of interleukin-3 cDNA and its expression in hematopoietic stem cell

目的 克隆人IL-3基因cDNA并转导人脐带血造血干细胞，然后研究这一细胞因子的表达情况，从而为脐血扩增及移植打下理论基础。方法 从人外周血单个核细胞中提取IL-3mRNA，用逆转录-聚合酶链反应（RT-PCR）扩增IL-3 cDNA，通过T／A克隆法，构建真核表达型载体pcDNA3／IL-3。将pcDNA3／IL-3质粒转导脐血造血干细胞，并于1—7d检测IL-3表达情况。结果 pcDNA3／IL-3重组质粒的目的基因与ID3基因序列相同；pcDNA3／IL-3转导脐血造血干细胞后，经检测能够有效表达。结论 成功克隆了hIL-3并构建重组质粒pcDNA3／IL-3，该质粒转导脐血造血干细胞后，能在短期内有效表达。

Objective To investgate the expression of IL-3 gene in hematopoietic stem cell （HSC） of umbilical cord blood. Methods Human peripheral blood mononuclear cells （PBMC） of healthy adult were purified by Ficoll gradient centrifugation and cultured in enriched Dulbecco＇ s Modified Eagle＇ s Medium （ DMEM）. Total RNA was yield by SV total RNA isolation system. IL-3 cDNA was synthesized by reverse transcription and polymerase chain reaction （RT-PCR） with Promega＇s Access RT-PCR System, and then cloned into pUCm-T vector which was verified by DNA sequencing. The pUCm-T/IL-3 was digested with BamH I and Xba I ;the IL-3 cDNA was subcloned into eukaryotic expression vector pcDNA3. The pcDNA3/IL-3 was transducted into umbilical cord blood HSC, and then IL-3 level was assayed by ELISA technique. Results Recombinant pUCm-T/IL-3 was constructed and transformed into DH5α. The objective fragment of pUCm-T/IL-3 was corresponded to genic sequence of IL-3. pcDNA3/IL-3 was significantly expressed in umbilical cord blood HSC. Conclusion The gene of hIL-3 is cloned and the secretory eukaryofic expression plasmid pcDNA3/IL-3 is constructed successfully. IL-3 level in supernatants of pcDNA3/IL-3- transducted HSC was higher than that of untransducted cell.