摘要
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 domain deletion mutants of UHRF2 and express in HEK293 cells.Methods Five domain deletion mutants of UHRF2 were designed according to the location of domains.The genes encoding △UBL,△RING and △YDG ＋ △RING were amplified directly by PCR using recombinant plasmid pCMV-3xFlag-UHRF2 as a template,while those encoding △PHD and △YDG by overlapping PCR.The amplified genes were cloned into eukaryotic expression vector pCMV-3xFlag.The constructed recombinant plasmids were transfected to HEK293 cells in which the expression of recombinant protein was identified by Western blot.Results The length of PCR product of △UBL was 2 018 bp,while those of upstream,downstream and upstream ＋ downstream △PHD were 987,1 152 and 2 272 bp,those of upstream,downstream and upstream ＋ downstream △YDG were 1 232,692 and 1 827 bp,and those of △RING and △YDG ＋ △RING 2 163 and 1 287 bp,respectively.Restriction analysis and sequencing proved that all the expression vectors for domain deletion mutants were constructed correctly.All the relative molecular masses of expressed proteins in HEK293 cells transfected with the recombinant plasmids were consistent with those in theory.Conclusion Domain deletion mutants of UHRF2 were successfully expressed in HEK293 cells,which laid a foundation of further study on functions of various domains of UHRF2 as well as their interaction sites with other proteins.
出处
《中国生物制品学杂志》
CAS
CSCD
2013年第1期44-47,共4页
Chinese Journal of Biologicals
基金
国家自然科学基金资助项目(30872248)
重庆市科技委员会资助(CSTC
2008BB5400)
重庆市教育委员会资助(KJ080326)
