利用基因工程技术构建人WWP2基因的真核表达载体

Construction of Wwp2 Gene Eukaryotic Expression Vector via Genetic Engineering Technology

目的构建E3泛素连接酶WWP2基因的真核表达载体,并鉴定其表达。方法利用PCR扩增技术扩增人WWP2基因的编码序列,把WWP2基因插入酶切后的pc DNA3.1-Flag空载体,用PCR法、酶切法及基因测序的方法检测重组质粒的正确性,然后将所构建质粒pc DNA3.1-Flag-WWP2转染入人胚肾细胞HEK293T中,并利用免疫印记法检测其表达情况。结果 pc DNA3.1-Flag-WWP2真核表达载体构建成功,且能实现在HEK293T细胞中的表达。结论文章成功构建了带Flag标签的人WWP2真核表达载体,为后续的WWP2基因功能以及作为肺癌治疗新靶点的可行性研究奠定了基础。

Objective To construct a eukaryotic expression vector of E3 ubiquitin ligase WWP2 and to identify its expression.Method The coding sequence of human WWP2 gene was amplified by PCR,and then inserted into pcDNA3.1-Flag eukaryotic expression vector.After identification by PCR,restriction endonuclease analysis and sequencing,the recombinant plasmid pcDNA3.1-Flag-WWP2 was transfected into human HEK293T embryonic kidney cells,and western blotting was applied to detect the expression of WWP2 gene.Result The WWP2 gene expression sequence was successfully inserted into pcDNA3.1-Flag vector,and the recombinant vector could express in HEK293T cells detected by western blot.Conclusion Flag-tagged WWP2 gene eukaryotic expression vector was successfully constructed,laying a solid foundation for the further study of WWP2 function in cancer.