应用pCMV3-MdmX-RFP表达载体建立H1299^(p53R^(2)13X/MdmX)稳转细胞系

Establishment of the stable H1299^(p53R^(2)13X/MdmX)cell line using pCMV3-MdmX-RFP vector

[目的]探索应用pCMV3-MdmX-RFP表达载体构建H1299^(p53R^(2)13X/MdmX)稳转细胞系。[方法]用KpnⅠ和NotⅠ双酶切后连接,构建pCMV3-MdmX-RFP表达载体并测序。重组质粒转染H1299p53R^(2)13X细胞,用100μg/mL的潮霉素B筛选2 w。将筛选出的细胞无限稀释后分到96孔板中,每孔一个单细胞,并扩增成单细胞群,选出荧光较强的3个细胞克隆(C1、C2和B4)采用Western Blotting和基因组PCR验证。将建立的稳定细胞系命名为H1299^(p53R^(2)13X/MdmX)细胞系,用荧光显微镜观察稳转细胞系的第10代细胞,并用Western Blotting检测MdmX-RFP蛋白的表达情况。G418处理H1299^(p53R^(2)13X/MdmX)细胞后,Western Blotting检测全长p53(full-length p53,FL-p53)和截断的p53(Truncated p53,TR-p53)蛋白表达水平。[结果]测序结果显示,重组质粒pCMV3-MdmX-RFP与数据库中的基因序列相同。稳定细胞系中MdmX-RFP蛋白的高表达,在第10代仍然如此。与对照组相比,用G418处理H1299^(p53R^(2)13X/MdmX)细胞后,FL-p53蛋白水平升高1.6±0.12倍(P<0.05),TR-p53蛋白水平升高3±0.17倍(P<0.01)。[结论]构建的H1299^(p53R^(2)13X/MdmX)稳转细胞系过表达效率高,可直接用于后续实验。

[Objective]To construct a stable H1299~(p53R^(2)13X/MdmX)cell line using pCMV3-MdmX-RFP vector.[Method]Constructed and sequenced the expression vector for pCMV3-MdmX-RFP by double digestion with KpnⅠand NotⅠ,and then linked.The recombinant plasmid pCMV3-MdmX-RFP was transfected into H1299~(p53R^(2)13X)cells,and screened with 100μg/mL of hygromycin B for a fortnight.The screened cells were divided by infinite dilution into 96-well plates,one single cell per well,and expanded into single-cell colonies,and the three cell clones with stronger fluoresce(C1,C2 and B4)was selected for validation by Western Blotting and genome PCR.The established stable cell line was named H1299~(p53R^(2)13X/MdmX)cell line,and the 10th generation cells of the stable cell line was visualized with fluorescent microscope,and the expression of MdmX-RFP protein was confirmed by Western Blotting.The inducible expression level of full-length p53(FL-p53)and Truncated p53(TR-p53)protein was confirmed by Western Blotting after the H1299~(p53R^(2)13X/MdmX)cells were treated with G418.[Result]The sequencing results showed that the recombinant plasmid pCMV3-MdmX-RFP was identical to the gene sequence in the database.The high expression of MdmX-RFP protein in the stable cell line remained so in the tenth generation.Compared with control group,the FL-p53 was elevated 1.6±0.12 times(P<0.05)and the TR-p53 was elevated 3.0±0.17 times(P<0.01)protein expression was increased after treatment of H1299~(p53R^(2)13X/MdmX)cells with G418.[Conclusion]High overexpression efficiency of the constructed H1299~(p53R^(2)13X/MdmX)stable-transformation cell line,which can be used in subsequent experiments.