用FRET方法研究与Mps1蛋白有相互作用的CENP-E蛋白结构域

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

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。

Objective： To explore the structural domains of the CENP-E protein that interact with Mpsl 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 Mpsl, or EGFP-CENPE3 and Mpsl were detected by FRET through selective photobleaching of the acceptors. Results ： Both recombinant proteins expressed in HEK293 ceils transfected by the recombinant plasmids were found to co-localize with the Mpsl protein as confirmed by confocal microscopy. The Ef between EGFP-CENPE3 and Mpsl protein was [ （ 12.63 ± 0.48 ） %, n = 30 ] and that between EGFP-CENPE3 and Mpsl 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 Mpsl, the Ef between EGFP-CENPE3 and Mpsl was significantly higher（p 〈 0.05）, The results of CO-I Pshowed that EGFP-CENPE3 could interact with Mpsl protein. Conclusion： There was interaction between EGFP-CENPE3 and Mpsl as was demonstrated by the methods of FRET and CO-IP.