PcDNA3.1-rhGM-CSF重组质粒的构建及生物活性鉴定

Construction and biological identification of eukaryotic expression vector of PcDNA3.1-rhGM-CSF

目的构建PcDNA3.1-rhGM-CSF真核细胞表达载体,初步观察其对髓系白血病细胞K562细胞生长的影响。方法采用PCR、T-A克隆及基因定向克隆技术,将rhGM-CSF基因插入PcDNA3.1(+)空载体,构建了PcD-NA3.1-rhGM-CSF真核表达载体。经限制性内切酶酶切和DNA测序进行鉴定。转染人髓系白血病细胞K562细胞72h后,采用RT-PCR方法、NBT试验、MTT试验、免疫组化技术,观察和分析PcDNA3.1-rhGM-CSF在K562细胞中的表达及其对该细胞的影响。结论成功构建了PcDNA3.1-rhGM-CSF真核细胞表达载体;重组质粒PcDNA3.1-rh-GM-CSF能在K562细胞中表达;部分K562细胞向单核细胞系、巨噬细胞系分化。

Objective To construct eukaryotic expression vector of PeDNA3.1-hGM-CSF and investigate the effect of recombinant vector on suppressing K562 cell growth. Methods The eukaryotic expression vector PcDNA3.1-hGMCSF was constructed with PCR, T-A clone and directional cloning techniques, and transfected into K562. RT-PCR was used to affirm the expressing of the PcDNA3.1 -rhGM-CSF in K562. The cell proliferation assay and immunohistochemistry were used to observed the effect of PcDNA3.1 -rhGM-CSF on growth and differentiation of K562 cell. Results The recombinant eukaryotic expression vector PcDNA3.1-GM-CSF was constructed successfully. Recombinant vector PcDNA3.1-GM-CSF was expressed in transfeeted K562 cells. Transfected K562 cell could differentiate into monoeyte and macrophage cell. Conclusion The recombinant eukaryotic expression vector PcDNA3. 1-GM-CSF was successfully constructed..