双分子荧光互补系统的构建及其在病毒学中的初步应用

CONSTRUCTION OF THE BIMOLECULAR FLUORESCENCE COMPLEMENTATION ASSAY AND ITS APPLICATION FOR VIROLOGY

双分子荧光互补(bimolecular fluorescence complementation,BiFC)是新近发展起来的研究蛋白质相互作用的技术,因其使用简便、检测灵敏、可在生理条件下直接观察,已在生物各领域广泛应用。本试验将黄色荧光蛋白EYFP突变为Venus,并在第173位氨基酸后分割为两段失去活性的VN和VC,经共转染表达不会产生荧光;在插入已被证明存在相互作用的猪繁殖与呼吸综合征病毒(Porcine reproductive and respiratory syndrome virus,PRRSV)的核衣壳(nucleocapsid,N)蛋白经共转染表达时,可以观察到荧光;通过扩增MARC-145细胞的细胞骨架蛋白α-tubulin并插入到VN和VC,除与自身共表达时发出荧光外,与N蛋白共表达时没有荧光产生,说明该VN和VC片段用于研究蛋白互作是可靠的。本试验建立了BiFC系统,为以后应用到研究病毒编码蛋白之间及其与宿主蛋白间的相互作用奠定基础。

Bimolecular fluorescence complementation（BiFC） is a recently developed technique that has the capacity to detect weak or otherwise-transient protein-protein interactions in vivo with high specificity and sensitivity.Therefore,it has been widely employed in many areas of biological research.In the present study,the enhanced yellow fluorescent protein（EYFP） was mutated to Venus,which was then split into two fragments,VN（aa 1-173） and VC（aa 174-239）.PRRSV N protein,which has been reported to form dimers,was used to fuse with VN and VC.Co-transfection of these two plasmids produced strong fluorescent signal.In addition,alpha-tubulin gene from the MARC-145 cell line to be fused to the N-termini of VN and VC for use as negative controls.When the N protein and also fused to the N-termini of VN and VC for use as negative controls.When N protein and tubulin were co-transfected,no fluorescence was observed.These results demonstrated that BiFC assay was developed in the present study and could be used to study protein-protein interactions in the field of virology.