BACKGROUND:In primary study, we obtained protein interacting with spinal cord injury and regeneration related gene No 10 (SCIRR10), which involving thyrotropin releasing hormone receptor 2 (TRH R2). However, the interaction between SCIRR10 and TRH R2 has been validated.OBJECTIVE:To explore whether SCIRR10 has effect on TRH R2 and to certify whether TRH R2 is special receptor of SCIRR10.METHODS:Recombinant plasmid expressing SCIRR10-GFP fusion protein and TRH R2-RFP fusion protein were constructed respectively using RT-PCR. The supernatant of these COS7 cells transfected with SCIRR10-GFP was collected and then added into those COS7 cells transfected with TRH R2-RFP, and later cells was cultu red for another 4 hours. Treated cells were washed,fixed with 40 g/L paraformaldehyde, mounted. The results were observed under a laser scanning confocal microscope.RESULTS AND CONCLUSION: RR10-GFP could be seen combined with TRH R2-RFP under fluorescence. No similar results could be found in the control group. The results of this experiment and primary studies demonstrated that, TRHR R2 is the receptor of SCIRR10. However, the interaction protein of SCIRR10 has its special ligand.%背景:前期研究中,通过酵母双杂交系统筛选出可能与脊髓损伤修复相关10 号基因(No 10,SCIRR10)编码蛋白相互作用的蛋白质,其中含有促甲状腺激素释放激素受体2(thyrotropin releasing hormone receptor 2,TRH R2),而SCIRR10 与TRHR2 是否存在相互作用尚未阐明.目的:验证SCIRR10 蛋白是否作用TRH R2 受体,进一步确认TRH R2 为SCIRR10 蛋白受体,为下一步研究提供可靠实验证据.方法:应用RT-PCR 方法扩增SCIRR10 和TRH R2 基因,构建带有绿色荧光蛋白和红色荧光蛋白标签的真核重组表达质粒GFP-SCIRR10 和RFP-TRH R2,分别转染真核细胞COS7,培养16 h 后,将表达SCIRR10-GFP 的细胞培养液加入到表达受体TRH R2-RFP 细胞中继续培养.4 h 后,用40 g/L 多聚甲醛固定细胞,于激光共聚焦显微镜下观察蛋白GFP-SCIRR10和蛋白RFP-TRH R2 两者相互结合情况.结果与结论:在荧光条件下仅能观察到SCIRR10-GFP 与TRH R2-RFP 相互结合,而其他对照没有观察到类似现象.通过实验以及前期研究结果证实TRHR R2 就是SCIRR10 作用受体.
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