藻蓝蛋白对Hela细胞CD59基因表达调控作用的研究

探讨了钝顶螺旋藻藻蓝蛋白(PC)对Hela细胞CD59基因表达的调控作用.以正常人CD59cDNA基因为模板,经PCR扩增后重组入真核表达质粒载体pALTER-MAX,然后利用阳离子脂质体(Lipfectamine-2000)将重组质粒和PcDNA共转染人子宫颈癌细胞(Hela)和对照用正常中国仓鼠卵巢细胞(Chinese hamster ovary,CHO)进行表达.用不同浓度的钝顶螺旋藻藻蓝蛋白作用于转染细胞,通过核酸分子杂交技术、免疫荧光标记法和ELISA法对细胞中CD59分子的表达进行检测.结果表明:成功构建了重组质粒pALTER-MAX-CD59,并将其导入真核细胞(Hela,CHO),经G418筛选获得了CD59分子高效表达的细胞克隆.用藻蓝蛋白作用于筛选出的转基因细胞,证实藻蓝蛋白可促进Hela细胞表面CD59蛋白的表达并抑制Hela细胞的增殖,而对于正常CHO细胞无明显作用.

Study of regulatory effect of phycocyanin on CD59 gene expression of Hela cells

The regulatory effect of phycocyanin from spirulina platensis on CD59 gene expression of Hela cells was studied. Human CD59cDNA was PCR-amplified and was cloned into the eukaryotic expression plasmid vector pALTER-MAX, and then the recombinant plasmid (pALTER-MAX-CD59) was transfected into Hela cells and normal CHO cells as control cells together with PcDNA by cation liposome(Lipfectamine-2000) . The two kinds of cells were disposed by different concentrations of phycocyanin in order to determine the expression of CD59 protein on the surface of cells by in situ hybridization, immunofluorescence and ELISA methods. Results illustrated the recombinant eukaryotic plasmid pALTER-MAX-CD59 was successfully constructed. The recombinant plasmid and PcDNA were cotransfected into Hela and CHO cells and the stable transfectants were sorted out by adding G418 into medium. The stable cell clones acted by phycocyanin were determined and the results showed phycocyanin can promote the expression of CD59 on the surface of Hela cells and hold back the reproduction of Hela cells, while it was no use to the CD59 gene expression and reproduction of normal control CHO cells.