CAJ | 학술논문

　　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。
　　쌍분자형광호보（bimolecular fluorescence complementation，BiFC）시신근발전기래적연구단백질상호작용적기술，인기사용간편、검측령민、가재생리조건하직접관찰，이재생물각영역엄범응용。본시험장황색형광단백 EYFP 돌변위 Venus，병재제173위안기산후분할위량단실거활성적 VN 화 VC，경공전염표체불회산생형광；재삽입이피증명존재상호작용적저번식여호흡종합정병독（Porcine reproductive and respiratory syndrome virus ，PRRSV）적핵의각（nucleocapsid，N）단백경공전염표체시，가이관찰도형광；통과확증 MARC-145세포적세포골가단백α-tubulin 병삽입도 VN 화 VC，제여자신공표체시발출형광외，여 N 단백공표체시몰유형광산생，설명해 VN 화 VC 편단용우연구단백호작시가고적。본시험건립료 BiFC 계통，위이후응용도연구병독편마단백지간급기여숙주단백간적상호작용전정기출。
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.