荧光共振能量转移技术检测ITGB4BP与P311间的相互作用

目的 利用激光共聚焦显微镜中的荧光共振能量转移(FRET)技术来验证整合素4结合蛋白(ITGB4BP)和P311间的相互作用.方法 分别构建能在哺乳动物细胞中表达ITGB4BP的绿色荧光融合蛋白ITGB4BP-EGFP和表达P311的红色荧光融合蛋白P311-DsRed的重组载体pITGB4BP-EGFP和pP311-DsRed,经酶切与测序鉴定正确后,共转染人胚肾293(HEK293)细胞48 h后,采用受体漂白方法(P311-DsRed),检测ITGB4BP和P311间的能量转移率(E)和相互间的作用距离(R).结果 重组载体pITGB4BP-EGFP和pP311-DsRed经双酶切鉴定正确,转染293细胞后经激光共聚焦显微镜观察能分别表达融合蛋白ITGB4BP-EGFP和P311-DsRed,在细胞质和细胞核中存在着共定位,FRET检测结果显示其能量转移率为14%,两个分子间的作用距离为6.3 nm.结论 成功构建了ITGB4BP-EGFP和P311-DsRed融合蛋白真核表达重组载体,在哺乳动物细胞中进行了表达后,利用FRET技术证实了活体细胞内ITGB4BP和P311间存在着相互作用.

Interaction between ITGB4BP and P311 measured by fluorescence resonance energy transfer

Objective To detect the interaction between ITGB4BP and P311 by fluorescence resonance energy transfer(FRET).Methods Target fragments P311 and ITGB4BP were subcloned into the DsRed expression vector pDsRed1-N1 and pEGFP-N2,respectively.After the recombinants pP311-DsRed and pITGB4BP-EGFP identified by restriction enzyme digestion and sequencing analysis,the 2 constructs were co-transfected into human embryo kidney 293(HEK293)cells,then the energy transfer efficiency(E) and the molecular distances(R) between P311 and ITGB4BP were detected by FRET through selectively photobleaching the acceptors(P311-DsRed).Results Double restriction enzyme digestion showed that the 2 recombinant vectors,pP311-DsRed and pITGB4BP-EGFP were constructed correctly.Both vectors could express in HEK293 cells,and the fluorescence fusion proteins mainly distributed in the cytoplasm and nuclei and were co-localized.E was equal to 14% and R to 6.3 nm in our experiment.Conclusion The recombinant vectors pP311-DsRed and pITGB4BP-EGFP encoding P311-DsRed and ITGB4BP-EGFP distributing in the cytoplasm and nuclei in mammalian cells are constructed successfully,and the interaction between ITGB4BP and P311 can be detected by FRET.