PRL-3基因C104S位点突变体和CAAX缺失体的构建及表达  

Construction and expression of PRL-3 plasmid with C104S point mutation and CAAX deletion

作　　者：李伟军[1] 邢晓芳[1] 曲立科[1] 孟麟[1] 寿成超[1] 

机构地区：[1]北京大学临床肿瘤学院,北京肿瘤医院暨北京市肿瘤防治研究所生物化学与分子生物学研究室,恶性肿瘤发病机制及转化研究教育部重点实验室,北京100142

出　　处：《北京大学学报（医学版）》2009年第5期516-520,共5页Journal of Peking University:Health Sciences

基　　金：国家自然科学基金(30671024);国家重点基础研究发展计划项目基金(2009CB521805)资助~~

摘　　要：目的:分别构建肝再生磷酸酶-3(phosphatase of regenerating liver 3,PRL-3)基因C104S位点突变和C端CAAX缺失的myc标签和GFP荧光标签重组质粒pcDNA3-myc-PRL-3(C104S)、pEGFP-PRL-3(C104S)、pcDNA3-myc-PRL-3(△CAAX)和pEGFP-PRL-3(△CAAX),并进行真核表达,为PRL-3的后续功能研究提供有效的工具。方法:设计PRL-3基因C104S点突变、C端CAAX缺失的特异性引物,以pcDNA3-myc-PRL-3质粒为模板,通过基因克隆、一步法点突变的方法构建各重组质粒,通过酶切和测序鉴定后转染真核细胞,检测其在细胞中的表达并观察定位情况。结果:成功构建了4种重组质粒,并能在结肠癌细胞LoVo中表达。用激光共聚焦扫描显微镜对细胞中绿色荧光融合蛋白的定位观察发现,当PRL-3的CAAX位点缺失后,PRL-3由内膜系统大量入核,与理论预期相符。结论:获得了PRL-3基因C104S位点突变体和CAAX缺失体重组真核表达质粒并能在细胞中表达,为进一步研究PRL-3的功能提供了条件。Objective： To construct PRL-3 gene C104S point mutation and CAAX deletion mutants： pcDNA3-myc-PRL-3 （C104S）, pEGFP-PRL-3 （C104S）, pcDNA3-myc-PRL-3 （ACAAX） and pEGFP-PRL-3 （ACAAX）, and express these plasmids in eukaryotic cells. Methods： Recombinant plasmids were mutated with pcDNA3-myc-PRL-3 plasmid as template and specific primers. Mutants were identified by restriction enzyme digestion and DNA sequencing. Then these recombinant plasmids were transfected into LoVo cells. The expression of fusion proteins were detected by western blotting and the localization of fusion proteins were examined by GPF fluorescence labelling. Results： The mutants were successfully constructed and expressed in eukaryotic cells. PRL-3 （ /kCAAX ） relocates from plasma membrane/early endosome to the cytoplasm and/or nucleus, which provides a structural insight of PRL-3 protein. Conclusion： Construction of eukaryotic expression recombinant plasmids of PRL-3 gene C104S point mutation and CAAX deletion mutants provide a useful tool for the study of PRL-3＇ s role in cancer.

关 键 词：蛋白质酪氨酸磷酸酶 肿瘤 点突变 基因缺失 

分 类 号：R73-37[医药卫生—肿瘤]