荧光表达载体Podocin过表达对HEK293细胞中CD2AP分布的影响  

Construction of expression vector for podocin and its effect of transfection on CD2AP distribution in HEK293 cells

作　　者：沙玉根[1] 黄松明[1] 张爱华[1] 赵非[1] 陈荣华[2] 

机构地区：[1]南京医科大学附属南京市儿童医院肾脏科,江苏南京210008 [2]南京医科大学儿科研究所,江苏南京210029

出　　处：《南京医科大学学报（自然科学版）》2007年第6期527-529,533,F0002,共5页Journal of Nanjing Medical University(Natural Sciences)

基　　金：国家自然科学基金资助项目(30100081);南京市卫生局资助项目(ZKM06043)

摘　　要：目的:构建podocin荧光表达载体,并观察其转染HEK293后对CD2AP细胞内分布的影响。方法:PCR扩增NPHS2cDNA全长,通过T/A克隆连接至pGEMT-easy载体,应用XhoⅠ和BamHⅠ双酶切后,再将NPHS2全长亚克隆入pEGFP-C2。将构建好的荧光表达载体pEGFP-NPHS2转染HEK293细胞,通过免疫荧光的方法观察转染前后CD2AP细胞内的分布情况。结果:①成功构建pEGFP-NPHS2荧光表达载体;②Podocin表达载体转染HEK293细胞后,CD2AP由核周转为弥散的细胞质分布。结论:Podocin转染HEK293细胞可使CD2AP由核周转为弥散的细胞质分布。Objective：To construct the podocin immunofluorence expression vector and observe its effects of transfection on CD2AP distribution in HEK293 cells. Methods：The pGEMT-easy vector containing the full-length cDNA encoding the human podocin was cloned and digested with BamHI and XhoI. The digested full-length podocin was subcloned into pEGFP-C2. The constructed plasmids were transfected into HEK293 cells,and its effect on CD2AP distribution were observed by immunofluorescence. The interaction between podocin and CD2AP was detected by immunoprecipitation. Results： （1）The pEGFP-NPHS2 expression vector was successfully constructed and podocin exclusively located on HEK293 cell membrane. （2） After podocin transfection, CD2AP redistributed from perinucleus to cytoplasm in HEK293. Conclusion：Podocin can recruit CD2AP to redistribute from perinucleus to cytoplasm in HEK293 cells.

关 键 词：PODOCIN 表达载体 CD2AP 

分 类 号：Q74[生物学—分子生物学] R329.2[医药卫生—人体解剖和组织胚胎学]