小鼠PICK1与Orai1蛋白质间的相互作用研究

Research on Interaction between PICK1 and Orai1 of Mouse

从小鼠脑组织中提取总RNA,RT-PCR扩增PICK1及Orai1的CDS序列,构建原核表达载体GST-PICK1及真核表达载体myc-Orai1,重组质粒经SalⅠ和NotⅠ双酶切鉴定后测序.正确的质粒myc-Orai1在HEK293细胞中表达收获过表达蛋白,GST-PICK1进行原核表达并获得融合蛋白.将纯化后的GST-PICK1蛋白条带切下,进行蛋白质液相质谱分析.后将myc-Orai1蛋白与GST-PICK1蛋白混合,进行GST-pulldown实验,western-blot检测到myc-Orai1的条带.结果表明PICK1与Orai1在体外有相互作用.

Total RNA of mice brain was extracted, CDS fragment of PICK1 and Orail was obtained by RT-PCR, CDS were respectively sub-cloned into pGEX-4T-2 with GST tag to generate GST-PICK1 and sub-cloned into pRK5 with myc tag to get myc-Orail, and recombinants were sequenced after confirma- tion by digested with Sal I and Not I ~ Correct myc-Orail vector was expressed in HEK293 cells, GST- PICK1 vector was expressed in BL21 competent cells. GST-PICKi fusion proteins were analyzed with protein liquid mass spectrometry after being purified and cut SDS-PAGE gel of the target band. And then GST-PICK1 fusion protein and myc-Orail fusion protein were mixed to apply GST-pulldown exper- iment, and myc-Orail band was detected by western-blot. Result indicates PICK1 and Orail interacted in Vitro.