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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 verify the interaction between the secreted frizzled-related protein 2 (SFRP2) and the osteoblast-specific factor 2 (SF-2). Methods The recombinant plasmids pCMV-Myc-SFRP2 and pCMV-HA-Periostin were identified by double enzyme digestion and co-transfected into HEK293 cells. Myc-SFRP2 interacting protein complex was precipitated with anti-Myc monoclonal antibody, Coomassie blue staining, the purpose of cutting the band, the use of HPLC-Chip / MS nanoflow LC / MS technology for peptide sequence analysis, the SpectrumMill search Swissport database identification of proteins. Results By HPLC-Chip / MS nanoflow LC / MS analysis, the expression of Periostin was detected in the immunoprecipitated complex. Conclusion There is interaction between Peri-ostin and SFRP2.