pEGFP-rhPLD_2融合基因在CHO细胞株中的稳定表达

目的建立可以稳定表达增强型绿色荧光蛋白-重组人磷脂酶D2(pEGFP-rhPLD2)的CHO细胞株,从而制备大量的重组PLD2(rhPLD2)蛋白,为rhPLD2的功能研究奠定基础。方法用脂质体介导pEGFP-rhPLD2重组质粒转染CHO细胞株,经G418加压筛选出阳性克隆,用RT-PCR、Western blot检测pEGFP-rhPLD2融合蛋白的表达,用倒置荧光显微镜观察增强型绿色荧光蛋白的表达。结果pEGFP-rhPLD2融合基因整合入细胞基因组DNA中,成功建立了pEGFP-rhPLD2在CHO细胞中的稳定表达体系。结论获得了稳定表达pEGFP-rhPLD2的CHO细胞株,为进一步研究rhPLD2的生物学功能及其在疾病预防治疗方面的作用奠定了实验基础。

The stable expression of the fusion gene pEGFP-rhPLD2 in CHO cell line

To establish a stable cell line expressing the EGFP recombinant human phospholipase D2 （PEGFP-rhPLD2） gene in CHO cells for the purpose to prepare large amount of rhPLD2, protein so as to provide the foundation for the study of the function of recombinant human PLD2 （rhPLD2）, the plasmid including the EGFP genes and recombinant human PLD2 was transfected into CHO cells by liposome-mediated gene transferred method. After screening with G418, the stable transfeetants were obtained and the fusion protein pEGFP-rhPLD2 was observed through the inverted fluorescence microscopy. The mRNA expression of gene pEGFP-rhPLD2 was identified by using RT-PCR assay and the protein expression was analyzed by Western blotting. In this way, the CHO cell line stably expressing pEGFP rhPLD2 protein was successively established which may lay a foundation for the further study on the biologic activities of recombinant human phospholipase D2 and its prophylactic function in diseases.