蛋白磷酸酶2A催化亚基β(PPP2Cβ)过表达慢病毒载体系统的构建及对K562红系分化的影响

Construction of Protein Phosphatase 2A Catalytic Subunit β(PPP2Cβ) Overexpression Lentiviral Vector and Its Effect on K562 Erythroid Differentiation

目的:构建蛋白磷酸酶2A的催化亚基PPP2Cβ慢病毒表达载体,获得较高滴度慢病毒颗粒,感染K562细胞建立稳定株,检测过表达PPP2Cβ对K562红系分化的影响。方法:将PPP2Cβ的CDS序列插入到第二代慢病毒表达载体FUGW,并与包装质粒p MD2G和ps PAX2共转染293T细胞,36、48h分两次收集慢病毒上清,应用流式细胞术检测病毒滴度。获得的病毒感染K562细胞,应用Western blot检测过表达效果。采用联苯胺染色以及实时定量PCR检测过表达PPP2Cβ对K562红系分化的影响。结果:成功构建了PPP2Cβ慢病毒表达载体,利用慢病毒系统包装PPP2Cβ的慢病毒,获得了高滴度的慢病毒颗粒,并成功建立了PPP2Cβ的K562细胞稳定株,促进了K562向红系分化。结论:过表达PPP2Cβ能使K562细胞自发的向红系分化。

Objective:To construct the ovexpression lentivirus vector of PPP2Cβ,the catalytic subunit of protein phosphatase 2A,so as to obtain high-titer packaged lentivirus particles,and to examine the effect of PPP2Cβ on the erythroid differentiation Methods:The CDS of PPP2Cβ was cloned into the second generation of lentivirus vector FUGW,which should be used to co-transfect HEK 293 T cells with the lentiviral expression vector and packaging vectors including pMD2 G and pSPAX2.Lentiviruses were harvested at 36 and 48 hours after transfection.Titers of viral stock were determined by using flow cytometric analysis.The Western blot was performed to detect the expression level of PPP2Cβ in K562 cells transinfected with the lentiviruses.Benzidine staining and real-time PCR analysis were used to assess the erythroid differentiation of K562 cells.Results:The PPP2Cβ overexpressing lentivirus vectors were constructed,the high-titer lentiviral particles were obtained,and then the PPP2Cβ overexpression K562 cell line was established and promote erythroid differentiation of K562 cells.Conclusion:This study suggests that overexpression PPP2Cβ can promote K562 cell erythroid differentiation.