泛素特异性蛋白酶53稳定表达细胞株的构建

Construction of human ubiquitin specific peptidase 53 overexpressing cell line

目的构建稳定过表达泛素特异性蛋白酶53(USP53)的食管癌Eca109细胞株。方法以GE公司购买的含USP53全长序列的质粒作为模板,使用聚合酶链反应(PCR)的方法扩增出USP53全长基因,将构建好的USP53慢病毒质粒和包装质粒共转染293T细胞获得浓缩慢病毒颗粒,再将慢病毒颗粒感染Eca109细胞,最后通过含有嘌呤霉素的培养基筛选稳定转染细胞株。结果过表达细胞株中的USP53的mRNA表达量(45.070±0.942)分别与对照组(1.091±0.075)及正常组(1.000±0.023)比较,差异均有统计学意义(P<0.05),Western blot实验显示过表达细胞株中标签蛋白HA明显高表达,对照组及正常组未见标签蛋白表达。结论成功构建人去泛素化酶USP53稳定高表达细胞株。

Objective To construct an Esophageal cancer Eca109 cell line which can stably overexpressing the ubiquitin specific peptidase 53 (USP53). Methods The USP53 full-length gene was amplified by Polymerase Chain Reaction (PCR) using a plasmid containing the full-length sequence of USP53 purchased by GE. The constructed USP53 lentiviral plasmid and packaging plasmid were co-transfected into 293T cells to obtain concentrated lentiviral particles, and then the lentiviral particles were infected with Eca109 cells. The medium containing puromycin was selected for stable transfected cell lines. Results The mRNA expression of USP53 in overexpressing cell lines (45.070±0.942) was significantly higher than that in the control group (1.091±0.075) and the normal group (1.000±0.023). The difference was statistically significant (P<0.05), Western blotting showed that the expression of the tagged protein HA was significant expressed in the overexpressed cell line, and no tagged protein expression was observed in the control group and the normal group. Conclusion Human ubiquitin specific peptidase 53 overexpressing cell line was successfully constructed.