小鼠GM-CSF基因的扩增及在HEK293T细胞中的分泌表达

Amplification of mouse GM-CSF gene and its expression in HEK293T cells

粒细胞-巨噬细胞集落刺激因子(GM-CSF)是机体免疫系统的重要细胞因子,具有生物佐剂作用,为研究GM-CSF的生物佐剂作用,本试验通过RT-PCR方法从小鼠脾脏细胞中扩增小鼠GM-CSF的cDNA,并将其插入到pcDNA3.1质粒中,构建成GM-CSF真核表达载体pcDNA3.1-GMCSF,并在真核细胞进行了瞬时表达。结果表明,本研究扩增的小鼠GM-CSF基因序列与GenBank序列完全一致,表达载体经脂质体介导转染HEK293T细胞,表达产物经western-blot检测,证明GM-CSF能够在HEK293T细胞中进行分泌表达。这为今后研究GM-CSF在动物疫苗,特别是DNA疫苗中的生物佐剂作用创造了必要的条件。

Granulocyte and macrophage colony-stimulating factor （GM-CSF） is an important cy- tokine in animal immune system and is also a biological adiuvant. To express GM-CSF in a se- cretory manner in eukaryotic cells, the mouse GM-CSF cDNA was amplified with RT-PCRfrom mouse spleen and was inserted into pcDNA3.1A expression vector to construct its eukary- otic expression vector, pcDNA3. 1-GMCSF. The transient expression of GM-CSF was per- formed in HEK293T cells transfected via liposome mediation. The results showed that the am-plified GM-CSF gene sequence was consistent with that published in GenBank. The recombi- nant mouse GM-CSF was detected by Western-blot in the culture medium of the transfected HEK293T cells, which indicates that the GM-CSF gene could be expressed and secreted in thecells. Those results provide the necessary conditions for further study on the GM-CSF adjuvant functions in animal vaccine, especially in animal DNA vaccine.