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目的:克隆人IL- 3基因cDNA ,构建真核表达质粒并转导脐带血造血干细胞(HSC) ,探讨IL -3在HSC中的表达情况,从而为脐血HSC扩增及移植奠定基础。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 -3cDNA ,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HSC ,并于1～7d检测IL- 3表达情况。结果:pcDNA3/IL -3重组质粒的插入基因与I-L 3基因序列相同;pcDNA3/IL- 3转导脐血HSC后,经检测能够有效表达。结论:成功克隆了hIL- 3基因cDNA并构建了重组质粒pcD -NA3/IL- 3,该质粒转导脐血HSC后,能在短期内有效表达。
OBJECTIVE: To clone human IL-3 gene cDNA, construct eukaryotic expression plasmid and transduce cord blood hematopoietic stem cells (HSC), and to explore the expression of IL-3 in HSC, which will lay the foundation for the expansion and transplantation of HSC in umbilical cord blood. METHODS: IL-3 mRNA was extracted from human peripheral blood mononuclear cells and IL-3 cDNA was amplified by reverse transcription-polymerase chain reaction (RT-PCR). The eukaryotic expression vector pcDNA3 / IL- 3. The pcDNA3 / IL-3 plasmid was transduced into cord blood HSC, and the expression of IL-3 was detected from 1 to 7 days. Results: The inserted gene of pcDNA3 / IL-3 recombinant plasmid was the same as that of I-L 3 gene. PcDNA3 / IL-3 could be expressed efficiently by HSC in cord blood. CONCLUSION: The cDNA of hIL-3 gene was successfully cloned and the recombinant plasmid pcD-NA3 / IL-3 was constructed. After transfected with cord blood HSC, the recombinant plasmid pcDNA3-IL3 could effectively express in a short time.