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

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.