免疫共沉淀-蛋白测序技术验证Periostin和SFRP2之间的相互作用  

Interaction between periostin and SFRP2 by co-immunoprecipitation and protein sequencing

作　　者：陶熹[1] 王珍祥[1] 袁顺忠[2] 李世荣[1] 吴军[2] 

机构地区：[1]第三军医大学西南医院整形外科,重庆400038 [2]第三军医大学西南医院全军烧伤研究所,创伤,烧伤与复合伤国家重点实验室,重庆400038

出　　处：《第三军医大学学报》2009年第21期2078-2080,共3页Journal of Third Military Medical University

基　　金：国家自然科学基金(39700185)~~

摘　　要：目的验证凋亡相关蛋白基因SFRP2(secreted frizzled-related protein2)和骨母细胞特异性因子-2Periostin(osteoblast-specific factor2)间的相互作用。方法分别构建重组载体pCMV-Myc-SFRP2和pCMV-HA-Periostin经双酶切鉴定正确,共转染HEK293细胞,抗Myc单克隆抗体沉淀Myc-SFRP2相互作用蛋白复合物后,进行SDS-PAGE、考马斯亮蓝染色,切下目的条带,利用HPLC-Chip/MS纳流液质联用技术进行肽序列分析,经SpectrumMill搜索Swissport数据库后鉴定蛋白质。结果经HPLC-Chip/MS纳流液质联用技术分析,免疫沉淀复合物内可检测出Periostin的表达。结论Peri-ostin与SFRP2间存在相互作用。Objective To confirm the interaction between periostin （ osteoblast-specific factor 2） and secreted frizzled-related protein 2 （ SFRP2） by co-immunoprecipitation and HPLC-Chip/MS techniques. Methods Expression vector encoding HA-tagged fusion protein HA-Periostin was constructed,identified and transfected into human embryo kidney 293 （ HEK293） cells alone or with the expression vector pMCV-Myc-SFRP2 containing Myc-tagged fusion protein Myc-SFRP2. The interaction between SFRP2 and periostin was detected by co-immunoprecipitation with the aid of anti-Myc antibody first. Then SDS-PAGE was perform,and the blots of the protein complexes were analyzed by HPLC-Chip/MS techniques. Spectrum Mill was employed to identify the protein complexes by searching Swissport database. Results From the Myc-SFRP2 immunoprecipitation complex,periostin was identified by protein sequencing. Conclusion There is an interaction between SFRP2 and periostin.

关 键 词：免疫共沉淀 相互作用 SFRP2 PERIOSTIN 瘢痕疙瘩 

分 类 号：R341[医药卫生—基础医学] R394-33