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目的构建UHRF2不同结构域缺失突变体,并在HEK293细胞中表达。方法根据UHRF2不同结构域位置特征,构建5种不同结构域缺失突变体;以重组质粒pCMV-3xFlag-UHRF2为模板,PCR法直接扩增△UBL、△RING和△YDG+△RING编码基因,重叠PCR法扩增△PHD和△YDG编码基因,定向克隆至pCMV-3xFlag真核表达载体中,构建重组表达质粒,转染HEK293细胞,Western blot鉴定重组蛋白的表达。结果 UHRF2的结构域缺失体△UBL、△PHD的上游和下游及上下游合并、△YDG的上游和下游及上下游合并、△RING和△YDG+△RING的PCR产物分别可见2 018、987、1 152、2 272、1 232、629、1 827、2 163和1 287 bp的特异条带;UHRF2各结构域缺失体的重组表达质粒经双酶切和测序鉴定,证明构建正确;重组质粒转染HEK293细胞表达的重组蛋白大小均与理论值相符。结论成功在HEK293细胞中表达了UHRF2不同结构域缺失突变体,为进一步研究UHRF2各结构域的功能及其与其他蛋白质的相互作用位点奠定了基础。
Objective To construct the deletion mutants of different domains of UHRF2 and express them in HEK293 cells. Methods According to the location characteristics of different domains of UHRF2, five different domain deletion mutants were constructed. The genes encoding △ UBL, △ RING and △ YDG + △ RING were directly amplified by PCR using the recombinant plasmid pCMV-3xFlag-UHRF2 as template. The genes encoding △ PHD and △ YDG were amplified by PCR and cloned into pCMV-3xFlag eukaryotic expression vector. The recombinant plasmid was constructed and transfected into HEK293 cells. The recombinant protein was identified by Western blot. Results The deletion of the domain of UHRF2 △ UBL, △ PHD upstream and downstream and upstream and downstream integration, △ YDG upstream and downstream and upstream and downstream integration, △ RING and △ YDG + △ RING PCR products were seen 2 018,987,1 152,2 272,1 232,629,1 827,2 163 and 1 287 bp. The recombinant expression plasmids of each domain of UHRF2 were identified by double enzyme digestion and sequencing, which proved that the recombinant plasmid was constructed correctly. The size of recombinant protein expressed in HEK293 cells was consistent with the theoretical value. Conclusion The deletion mutants of different domains of UHRF2 were successfully expressed in HEK293 cells, which laid the foundation for further study on the function of each domain of UHRF2 and its interaction with other proteins.