军事医学2016，Vol.40Issue(2)：142-146,5.DOI:10.7644/j.issn.1674-9960.2016.02.016
E6AP蛋白结构域突变真核表达载体的构建及其生物学功能检测
Construction and characterization of E6 AP deletion mutant expression vectors
摘要
Abstract
Objective To construct deletion mutant eukaryotic expression vectors of E 6AP labeled with myc tag and de-tect its activity in cancer cells .Methods E6AP deletion mutants were amplified from myc-E6AP plasmid by PCR and in-serted into the eukaryotic expression vector of pXJ-40-myc.The recombinant plasmids pXJ-40-myc-E6AP(1-494) and (495-852) were identified by enzyme digestion and sequencing , transfected into HEK293T cells and detected by Western blotting .CCK8 assay was performed to investigate the effect of the deletion mutants on breast cancer and gastric cancer cell proliferation.Results The E6AP deletion mutants of about 1479 and 1071 bp were successfully cloned and expressed .In contrast to E6AP(1-494), E6AP(495-852) could inhibit TP53 expression and promote proliferation of ZR75-1 and BGC-823 cells.Conclusion The eukaryotic expression vector of myc-E6AP(1-494)and myc-E6AP(495-852) is suc-cessfully constructed and expressed , which can facilitate the study of E6AP in related cancer development and progression .关键词
E6AP/p53/结构域突变/泛素化/肿瘤Key words
E6AP/p53/deletion mutants/ubiquitination/neoplasms分类
生物科学引用本文复制引用
熊加秀,丁丽华,营孙阳,陈志达,张亚楠,贾小萌,陈滢洁,刘文忠,叶棋浓..E6AP蛋白结构域突变真核表达载体的构建及其生物学功能检测[J].军事医学,2016,40(2):142-146,5.基金项目
国家自然科学基金资助项目(81101883,81272231, 31470773) （81101883,81272231, 31470773）
北京市科技新星计划资助项目(2009A38,2011112) （2009A38,2011112）
