人Flt3配体基因的克隆及在Hela细胞中的表达

Clone of human Flt3 Ligand gene and its expression in Hela cells

目的:克隆人Flt3配体(FL)基因,检测其在Hela细胞中转录及表达蛋白的活性。方法:Trizol法提取K562细胞总RNA,经RT-巢式PCR得到FL的cDNA,PCR产物亚克隆入pGEM-T载体测序,构建pcDNA3/hFL。经Lipofectamine2000介导转入Hela细胞,RT-PCR法检测转染细胞中hFL基因的转录,ELISA法检测培养上清中hFL的含量,增殖和促生存实验鉴定hFL的活性。结果:所得FL基因序列正确。转染后的Hela细胞能转录FL基因,上清中hFL蛋白含量为56.8ng/ml/1,hFL能促Raji细胞生存及抑制凋亡(P<0.01),促进HL-60细胞增殖(P<0.05)。结论:构建的pcDNA3/hFL质粒在Hela细胞表达系统中能够有效转录,表达的hFL蛋白具有生物学活性。

To clone hFL gene, detect its transcription in Hela cells and the biological activity of hFL protein. Methods： The total RNA was extracted from K562 cells by Trizol, and hFL cDNA was cloned by RT-nested PCR, the PCR products were subcloned into pGEM-T for sequencing Target sequence was inserted into pcDNA3 to construct pcDNA3/hFL. Hela cells were transfected with the recombinant by Lipofectamine 2000. The transcription of hFL in the transfected cells was assayed by RT-PCR. The content of hFL protein in the culture supernatant was assayed by ELISA, and the biological activity was assayed by proliferation experiment and survival promoting experiment. Results： The hFL gene was transcripted effectively in transfected cells. The content of hFL protein in supernatant was 56.8ng/ml/l in 72h after transfected. The hFL protein stimulated the survival of the Raji cells and the proliferation of HL-60 （P〈0.05）, inhibited apoptosis of the Raji cells （P〈0.01）. Conclusion： hFL gene was transcripted effectively in Hela cells, the hFL protein translated by the transfectants showed obvious biological activity.