黄河, 段秀梅, 谭岩, 刘力华, 方艳秋, 许淑芬, 姜艳芳, 刘小林. 人Flt3配体基因的克隆及在Hela细胞中的表达[J]. 中国肿瘤临床, 2006, 33(16): 923-926.
引用本文: 黄河, 段秀梅, 谭岩, 刘力华, 方艳秋, 许淑芬, 姜艳芳, 刘小林. 人Flt3配体基因的克隆及在Hela细胞中的表达[J]. 中国肿瘤临床, 2006, 33(16): 923-926.
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Huang He, Duan Xiumei, Tan Yan, . Clone of human Flt3 Ligand gene and its expression in Hela cells[J]. CHINESE JOURNAL OF CLINICAL ONCOLOGY, 2006, 33(16): 923-926.
Citation: Huang He, Duan Xiumei, Tan Yan, . Clone of human Flt3 Ligand gene and its expression in Hela cells[J]. CHINESE JOURNAL OF CLINICAL ONCOLOGY, 2006, 33(16): 923-926.
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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

    摘要
  • 摘要: 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。

     

    Abstract: Objective : 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/1 in 72h after transfected. The hFL protein stimulated the survival of the Raji cells and the proliferation of HL-60(P0.05), inhibited apoptosis of the Raji cells (P0.01). Conclusion : hFL gene was transcripted effectively in Hela cells, the hFL protein translated by the transfectants showed obvious biological activity.

     

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