用FRET方法研究与Mps1蛋白有相互作用的CENP-E蛋白结构域

目的：探索与Mps1蛋白有相互作用的CENP-E蛋白结构域。方法：将重组质粒pEGFP-CENPE2（674~1085位氨基酸）、pEGFP-CENPE3（1200~2134位氨基酸）转染人胚肾293(HEK293)细胞,采用受体漂白荧光共振能量转移方法（FRET方法）,检测EGFP-CENPE2、EGFP-CENPE3和Mps1间的能量转移率(Ef), 进一步用免疫共沉淀方法验证FRET的实验结果。结果：重组质粒转染HEK293细胞后经激光共聚焦显微镜观察重组质粒表达的融合蛋白与Mps1都存在着共定位；FRET检测结果显示EGFP-CENPE3和Mps1间的能量转移率为［(12.63±0.48)%, n=30］,pEGFP-CENPE2和Mps1间的能量转移率为［(3.07±0.21)%, n=30］，与对照组［(2.96±0.27)%, n=30］比较pEGFP-CENPE3和Mps1间的能量转移率差异存在显著性（p<0.05），免疫共沉淀实验结果显示EGFP-CENPE3与Mps1蛋白间存在相互作用。结论：FRET技术和免疫共沉淀实验证明了EGFP-CENPE3与Mps1间存在着相互作用。

Explore the Structural Domains of CENP-E Protein Interacting with Mps1 Protein by FRET Method

Objective: To explore the structural domains of the CENP-E protein that interact with Mps1 protein. Methods: Two recombinant vectors named pEGFP- CENPE2 (containing 674-1085 amino acids of CENP-E protein) and pEGFP-CENPE 3(containing 1200~2134 amino acids of CENP-E protein) were transfected into human embryo kidney 293 (HEK293) cells respectively. The respective energy transfer efficiency (Ef) between either EGFP-CENPE2 and Mps1, or EGFP-CENPE3 and Mps1 were detected by FRET through selective photobleaching of the acceptors. Results: Both recombinant proteins expressed in HEK293 cells transfected by the recombinant plasmids were found to co-localize with the Mps1 protein as confirmed by confocal microscopy. The Ef between EGFP-CENPE3 and Mps1 protein was ［(12.63±0.48)%, n=30］ and that between EGFP-CENPE3 and Mps1 protein was ［(3.17±0.21)%, n=30］ as revealed by the results from FRET，the result of FRET was confirmed by co Immunoprecipitate(CO-IP) method. When compared with that between the control and Mps1, the Ef between EGFP-CENPE3 and Mps1 was significantly higher（p<0.05）, The results of CO-I Pshowed that EGFP-CENPE3 could interact with Mps1 protein. Conclusion: There was interaction between EGFP-CENPE3 and Mps1 as was demonstrated by the methods of FRET and CO-IP.