人KRAS真核表达载体的构建及生物学功能鉴定

Construction of human KRAS eukaryotic expression vector and characterization of its biological function

目的构建带FLAG标签的Kirsten鼠肉瘤病毒癌基因(Kirsten rat sarcoma viral oncogene,KRAS)真核表达载体,获得其表达产物并鉴定同源二聚体。方法应用PCR技术从人乳腺文库中扩增出KRAS全长编码区基因,将其克隆到pCMV-FLAG2B载体中,BamHⅠ、XhoⅠ双酶切及测序得到阳性克隆;利用重组质粒转染人胚肾293T细胞,以SDS-PAGE和免疫印迹鉴定表达情况;免疫共沉淀检测FLAG2B-KRAS与Myc-KRAS的相互作用。结果双酶切和基因测序显示,FLAG2B-KRAS真核表达载体构建成功;SDS-PAGE和免疫印迹结果表明,FLAG2B-KRAS转染人胚肾293T细胞后成功表达;免疫共沉淀结果显示,FLAG2B-KRAS与Myc-KRAS在蛋白水平上具有相互作用,能形成同源二聚体,证明其具有生物学活性。结论成功构建FLAG2B-KRAS真核表达载体,为进一步探讨KRAS在肿瘤分子生物学中的作用及靶向治疗奠定了实验基础。

Objective To construct the eukaryotic expression vector of Kirsten rat sarcoma viral oncogene(KRAS)labeled with FLAG tag,obtain the expressed product and identify its interaction with Myc-KRAS at the protein level,namely KRAS homodimer.Methods A human KRAS coding region amplified from the mammary cDNA library by PCR was inserted into pCMV-FLAG2B,positive clones of which were identified using double enzyme digestion,namely BamHⅠ,XhoⅠand gene sequencing.The recombinant plasmid FLAG2B-KRAS transfected into human 293T cell lines was investigated and examined by SDS-PAGE and Western blotting.In addition,co-immunoprecipitation(Co-IP)was applied to determine the interaction between Myc-KRAS and FLAG2B-KRAS.Results The coding region of KRAS was successfully amplified by PCR and cloned into pCMV-FLAG2B vector,which was identified by double enzyme digestion and gene sequencing.FLAG2B-KRAS was successfully expressed in human 293T cell lines according to SDS-PAGE and Western blotting.Co-IP indicated that Myc-KRAS could interact with FLAG2B-KRAS,which verified its biological function,namely KRAS homodimer.Conclusion The eukaryotic expression vector FLAG2B-KRAS is successfully obtained,which can experimentally contribute to the further study of the role of KRAS in tumor molecular biology and targeted therapy.