人EGFR显性负性突变体真核表达载体的构建、蛋白表达及亚细胞结构定位

Construction of the eukaryotic vector carrying human dominant negative epidermal growth factor receptor,its expression and sub-cellular localization in COS-7 cells

目的:构建人EGFR显性负性突变体真核表达载体pEGFPN1-DNEGFR,转染COS-7细胞,检测DNEGFR-EGFP的表达并进行亚细胞结构定位.方法:将RT-PCR(reverse tran-scription-polymerase chain reaction)方法扩增得到编码EGFR信号肽段、胞外区和跨膜区的cDNA,定向克隆至空载体pEG-FP-N1中,构建真核表达载体pEGFPN1-DNEGFR.经PCR扩增鉴定、双酶切鉴定、核苷酸序列测定以及生物信息学分析证明pEGFPN1-DNEGFR构建成功后,脂质体法转染体外培养的COS-7细胞,Western Blot检测DNEGFR-EGFP蛋白的表达,应用光谱激光扫描共聚焦显微镜对DNEGFR-EGFP亚细胞结构定位检测.结果:PCR扩增鉴定、双酶切鉴定、核苷酸序列测定以及生物信息学分析证实pEGFPN1-DNEGFR构建成功,并且Western Blot检测DNEGFR-EGFP蛋白的表达,光谱激光扫描共聚焦显微镜观察显示DNEGFR-EGFP蛋白主要定位于细胞膜.结论:成功构建人EGFR显性负性突变体真核表达载体,并在COS-7细胞胞膜上表达,为靶向EGFR显性负性策略在肿瘤基因治疗中的进一步研究打下基础.

AIM：To construct the eukaryotic expression vector（pEGFPN1-DNEGFR） carrying human dominant negative epidermal growth factor receptor（DNEGFR）,detect the expression and the sub-cellular localization of dominant negative epidermal growth factor receptor-enhanced green fluorescence protein （DNEGFR-EGFP） in COS-7 cells transfected with pEGFPN1-DNEGFR. METHODS：The cDNA coding signal peptide,extracellular ligand-binding domain and membrane-spanning region of epidermal growth factor receptor（EGFR） precursor was obtained by reverse transcription-polymerase chain reaction （ RT-PCR ）, then directionally cloned into the empty vector （pEGFP-N1） to con- struct pEGFPN1-DNEGFR. After confirmed by PCR amplification assay, double enzyme digestion, DNA sequencing and bioinfot^na- tics analysis of nucleotide sequence, pEGFPN1-DNEGFR was transfected into COS-7 cells, mediated by Lipofectamine 2000. The expression of and the sub-cellular localization of DNEGFR- EGFP in COS-7 cells were detected by Western Blot and Laser Scanning Spectral Confocal Microscope respectively. RESULTS： pEGFPN1-DNEGFR was constructed successfully, which was confirmed by PCR amplification assay, double enzyme digestion,DNA sequencing and bioinformatics analysis of nucleotide sequence. The expression of DNEGFR-EGFP was verified by Western Blot, and its predominant localization on the cell membrane of COS-7 cells was identified by Laser Scanning Spectral Confocal Microscope. CONCLUSION：The successful construction of pEGFPN1-DNEGFR, the expression and the sub-cellular localization of DNEGFR-EGFP in COS-7 cells, laid a solid foundation for further research on EGFR-targeted dominant negative strategy in cancer gene therapy.