SFRP2与Periostin的相互作用在瘢痕疙瘩形成机制中的研究

Experimental Study of Interaction between SERP 2 and Periostin in the Formation of Keloid

目的利用pull-down技术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。方法构建能在哺乳动物细胞中表达带HA标签的Periostin融合蛋白(HA-Periostin)的重组载体pCMV-HA-Periostin,经酶切鉴定正确后,和表达带Myc标签的融合蛋白(Myc-SFRP2)的重组真核表达载体pCMV-HA-SFRP2,单独或共转染人293细胞,利用pull-down技术验证Periostin与SFRP2间的相互作用。结果成功构建重组载体pCMV-HA-Periostin,与抗Myc单克隆抗体沉淀Myc-SFRP2相互作用蛋白复合物后,可以检测到HA-Periostin的表达。通过体外蛋白质结合实验证实了Periostin与SFRP2间的相互作用。结论成功构建带HA标签的Periostin融合蛋白(HA-Periostin)的重组载体,利用pull-down技术证实了Periostin与SFRP2间的存在相互作用。

Objective To investigate the interaction between Periostin （osteoblast-specifie factor 2） and SFRP 2 （Secreted frizzled-related protein 2） in the formation of keloid. Methods HA-tagged fusion protein （HA- Periostin） expression vector was constructed, identified and transfected into human embryo kidney 293 （HEK293） cells alone or with Myc-tagged fusion protein （Myc-SFRP 2） expression vector pMCV-Myc-SFRP 2. The interaction between SFRP 2 and Periostin was detected by pull-down technique. Results Double restriction enzyme digestion showed that pCMV-HA-Periostin was constructed successfully. When Myc-SFRP 2 was immunoprecipitanted, HA- Periostin was identified. And the interaction between SFRP 2 and Periostin was found by GST pull-down and His pull-down technique. Conclusion The recombinant vector pCMV-HA- Periostin was constructed successfully. The interaction between SFRP 2 and Periostin could be identified by GST pull-down and His pull-down.