穿透性Sox2蛋白的质粒构建及功能鉴定  

Construction of cell penetrated peptide 9R-Sox2 and identification of its function in mouse embryonic fibroblasts

作　　者：丁道芳[1] 王瑧[2] 李晓锋[1] 徐乐勤[1] 梁倩倩[1] 王拥军[1] 

机构地区：[1]上海中医药大学龙华医院脊柱病研究所,上海200032 [2]复旦大学肿瘤医院中心实验室,上海200433

出　　处：《中国药理学通报》2012年第12期1671-1674,共4页Chinese Pharmacological Bulletin

基　　金：国家基础研究计划(973计划)资助项目(No2010CB530400);国家自然科学基金重点项目(No30930111);长江学者计划(Teach people[2009]17);上海创新团队项目(No 08YZ56);曙光计划(No10GG20);上海高校创新团队项目(Shanghai Education Commission;Division 6[2009])

摘　　要：目的构建具有细胞性穿透性的9R-Sox2蛋白的重组质粒,使其在293T细胞中表达,并对所表达的9R-Sox2蛋白的功能进行鉴定。方法从小鼠胚胎干细胞中扩增Sox2基因,并且在其N端加上9个精氨酸短肽,此融合基因连接到pPYCAG载体上,pPYCAG-9R-Sox2和pPYCAG分别转染到293T细胞中。3 d后,对转染细胞用嘌呤霉素筛选,1周后筛选得到阳性克隆细胞株,对其进行细胞免疫荧光鉴定。表达9R-Sox2蛋白的细胞株进行裂解,取细胞裂解液加入至小鼠胚胎成纤维细胞的培养基中进行通透。48 h后,小鼠胚胎成纤维细胞进行细胞免疫荧光检测,并提取蛋白Western blot检测PCNA表达。结果用Sox2抗体进行细胞免疫荧光鉴定,结果均显示所有细胞表达9R-Sox2。在细胞裂解液处理的成纤维细胞,细胞免疫荧光结果显示9R-Sox2蛋白定位在细胞核,表明9R-Sox2构建和翻译正确。同时经含9R-Sox2的细胞裂解液处理的成纤维细胞,PCNA表达明显上调。结论成功构建表达9R-Sox2融合蛋白的pPYCAG-9R-Sox2重组质粒,并证明具有细胞通透及促细胞增殖功能。Aim To investigate the function of 9R-Sox2 in the mouse embryonic fibioblasts（MEFs） by constructing the recombinant plasmid pPYCAG-9R-Sox2 and expressing it in 293T cells.Methods Sox2 was amplified from mouse embryonic stem cells and modified by nine arginines to N-terminal,and this fused gene was cloned to pPYCAG vector.293T cells were transfected by recombinant plasmid pPYCAG-9R-Sox2 and pPYCAG respectively.These transfected cells were screened with puromycin three days later.The positive clones which expressed 9R-Sox2 would be obtained about one week later.The expression of 9R-Sox2 was qualified by cell immunofluorescence.The protein from 9R-Sox2 expressing cells was extracted and added into the medium for MEFs for 48 hours.The expression of PCNA and 9R-Sox2 wre detected in MEFs which had been treated with proteins containing 9R-Sox2.Results All 293T cells expressed 9R-Sox2 after being screened with puromycin,and protein extract from those positive clones penetrated the nuclei and detected by cell immunofluorescence,which indicated that 9R-Sox2 was coded and translated correctly.The expression level of PCNA was upregulated obviously in MEFs by treatment with protein extract containing 9R-Sox2.Conclusion Recombinant plasmid pPYCAG-9R-Sox2 is constructed successfully;9R-Sox2 is proved to enter the nuclei of the MEFs and promote its proliferation.

关 键 词：穿透性 9R-Sox2 细胞免疫荧光 WESTERN BLOT 增殖细胞核抗原 小鼠胚胎成纤维细胞 

分 类 号：R735.7[医药卫生—肿瘤]