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。
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.
出处
《中国肿瘤临床》
CAS
CSCD
北大核心
2006年第16期923-926,共4页
Chinese Journal of Clinical Oncology
基金
吉林省科技发展计划项目基金资助(编号:20050703-6
20050402-3)