雙分子熒光互補技術(shù)是將熒光報告蛋白按照規(guī)則分成沒有熒光的兩段N-fragment及C-fragment，分別與誘餌蛋白和捕獲蛋白融合，如果誘餌蛋白和捕獲蛋白能發(fā)生相互作用，那么兩段不完整的熒光報告蛋白片段就會形成完整的熒光報告蛋白，在激發(fā)光的激發(fā)下發(fā)出熒光。該技術(shù)可以用于檢測蛋白-蛋白相互作用、藥物篩選等，可用熒光顯微鏡、激光共聚焦顯微鏡、流式細胞儀等進行熒光細胞的實時觀察、實時分選等。
（1）BiFC技術(shù)檢測優(yōu)勢：
活細胞分析，直觀、快速地判斷目標蛋白在活細胞中的定位和相互作用；
可以用于研究2種或2種以上的蛋白質(zhì)間相互作用；
具有很高的信噪比，弱蛋白相互作用也可以檢測到（>7nm）。
（2）可用于BiFC的熒光蛋白：
目前報道的用于BiFC檢測的熒光蛋白有GFP、BFP、CFP、YFP、Venus、citrine、cerulean、mCherry等。
常見的用于雙分子熒光互補技術(shù)的熒光蛋白及拆分位點
（3）BiFC技術(shù)的應(yīng)用：
A、BiFC檢測蛋白相互作用：
摘自Hox Proteins Display a Common and Ancestral Ability to Diversify Their Interaction Mode with the PBC Class Cofactors. Bruno Hudry, Sophie Remacle, Marie-Claire Delfini, René Rezsohazy, Yacine Graba, and Samir Merabet. PLoS Biol. 2012 Jun; 10(6): e1001351.
B、BiFC檢測細胞融合：
Validation of the BiFC approach to detect cell fusion.Two populations of COS-1 cells were transfected overnight with VN-Histone H3.1 and YC-Histone H3.1 respectively. The two populations of cells were then plated together one day prior to induction of fusion by PEG1500. (a–e) Typical BiFC fusion signals. Fusion signals (BiFC, green) were detected by fluorescence microscopy and images were obtained at high (a) and low magnification (d). Histone H3.1 was localized to the nucleus (b, Hoechst 33142 stain, blue; c, e, a merge with phase contrast). (f) Kinetic analysis of BiFC fusion signals. Two populations of COS-1 cells were transfected with plasmids encoding BiFC partners in the presence or absence of PEG1500. In addition, two populations of COS-1 cells were transfected with the same BiFC complex (i.e. VN-Histone H3.1+VN-Histone H3.1 or YC-Histone H3.1+YC-Histone H3.1). The number and location of cells per well with fusion signals were counted over a period of 24 h after fusion was induced. Three wells were counted for each condition. The mixture containing both BiFC partners induced to fuse via PEG showed the most dramatic increase in number of signals over time. In contrast, cell populations containing only one BiFC partner (i.e. VN- or YC-Histone H3.1) showed no formation of BiFC signals. (g) Time-lapse imaging of BiFC fusion signals. Cells were prepared as above and images were acquired after PEG-induced fusion at a frequency of 1/10 min over a period of 12 h. The upper panels show fusion signals and the lower panels show the same signals merged with a corresponding phase image. Scale bars in (a–g), 20 μm.
摘自Bimolecular fluorescence complementation analysis of eukaryotic fusion products. Ho-Pi Lin, Claudius Vincenz, Kevin W. Eliceiri, Tom K. Kerppola, and Brenda M. Ogle. Biol Cell. 2010 Aug 6; 102(Pt 9): 525–537.
C、BiFC檢測蛋白復(fù)合體在染色體上的定位：
Visualization of BiFC complexes on polytene chromosomes. (A) Diagram of BiFC complex formation on polytene chromosomes. The intensity of BiFC complex fluorescence at individual genomic loci is enhanced by the parallel alignment of hundreds of chromatids within the polytene chromosomes. (B) Examples of BiFC complex fluorescence (green) on polytene chromosomes stained using Hoechst (blue). The chromosomes were spread under acid-free conditions. The banding patterns of the polytene chromosomes stained using Hoechst are shown in the images to the right. Note the differences in chromosome spreading and extension that are necessary to map the localization of the BiFC complexes to individual genomic loci. Upper panel: an intact spread allowing mapping of a number of loci, including the two copies of the 55F locus, corresponding to the two homologues that were presumably separated during the squash procedure; Middle panel: a partial spread with broken arms, which are well extended and suitable for mapping a subset of the loci; Lower panel: a poorly extended spread on which the loci that are bound by BiFC complex cannot be easily identified. Scale bars: 10 μm. (C) Comparison of the localization of BiFC complexes and of the individual BiFC fusion proteins using acid-free (upper images) and conventional (middle and bottom images) squash protocols. Polytene chromosomes that prepared using the acid-free protocol have less sharp banding patters than those that are prepared using conventional squash protocols. Both dKeap1-CncC BiFC complexes and each of the fusion proteins bound the 55F locus. Both dKeap1 and CncC fusions bound many other loci, whereas the dKeap1-CncC BiFC complex bound the 55F locus with higher specificity. Scale bars: 5 μm.
摘自Visualization of the genomic loci that are bound by specific multiprotein complexes by bimolecular fluorescence complementation (BiFC) analysis on Drosophila polytene chromosomes. Huai Deng and Tom K Kerppola. Methods Enzymol. 2017; 589: 429–455.
D、BiFC檢測HIV病毒侵染宿主細胞：
Gag interactions with host proteins Staufen1 and IMP1 occur in the cytoplasm and at the plasma membrane of transfected HeLa and Jurkat T cells as determined by BiFC. (A) Top – schematic representation of BiFC method. Bottom – Rev-dependent Gag-VN and Gag-VC were co-transfected with pCMV-Rev in HeLa cells. At 24 hr post-transfection, cells were imaged by laser scanning confocal microscopy to detect BiFC. The white arrows indicate plasma membrane concentrated accumulations of Gag-Gag BiFC signals. (B) Gag-VN and Staufen1-VC (top panels) or Gag-VN and IMP1-VC (bottom panels) interactions identified by BiFC. BiFC signals for these interacting pairs were mainly detected in the cytoplasm (indicated by white arrows) and at or near the plasma membrane. (C) Interactions between Gag-VN with IMP1-KH(1-4)-VC (top) and with IMP1-RRM(1-2)-VC (bottom) as determined by BiFC analysis. Evidence for interaction is demonstrated by a green fluorescence signal. (D) The interaction between Gag-VN and Gag-VC (top) or Gag-VN and Staufen1-VC (bottom) was determined by BiFC in Jurkat T cells. Magnified sections demonstrate details on the shapes of BiFC signals/complexes. The size bars are equal to 10 μm.
摘自Live cell visualization of the interactions between HIV-1 Gag and the cellular RNA-binding protein Staufen1. Miroslav P Milev, Chris M Brown, and Andrew J Mouland. Retrovirology. 2010; 7: 41.
注：以上文章內(nèi)容僅供參考。
BiFC技術(shù)檢測蛋白-蛋白相互作用，廣泛應(yīng)用于植物、動物、微生物、病毒等的染色體、RNA、細胞融合等不同領(lǐng)域的研究。BiFC也可用于藥物開發(fā)，首先選取疾病相關(guān)的重要蛋白，基于BiFC技術(shù)開發(fā)BiFC穩(wěn)轉(zhuǎn)細胞株，不同的藥物處理后檢測熒光信號就可篩選潛在的治療藥物。
晶諾生物BiFC技術(shù)服務(wù)流程：
- 質(zhì)粒構(gòu)建：將目的基因連接至載體，構(gòu)建融合蛋白表達質(zhì)粒。目的基因需客戶提供，合成需另收費。
- 將質(zhì)粒轉(zhuǎn)染至細胞293或HepG2（或其他細胞，具體請咨詢我們，目前無法做植物細胞）。
- 熒光檢測。
- 交付檢測報告和融合質(zhì)粒。
注：本服務(wù)僅包含以上4個實驗內(nèi)容，如需增加實驗項目，請咨詢我們；生物實驗具有不確定性，不保證實驗結(jié)果與客戶預(yù)期完全一致，請知悉。
參考文獻
- Hox Proteins Display a Common and Ancestral Ability to Diversify Their Interaction Mode with the PBC Class Cofactors. Bruno Hudry, Sophie Remacle, Marie-Claire Delfini, René Rezsohazy, Yacine Graba, and Samir Merabet. PLoS Biol. 2012 Jun; 10(6): e1001351.
- Bimolecular fluorescence complementation analysis of eukaryotic fusion products. Ho-Pi Lin, Claudius Vincenz, Kevin W. Eliceiri, Tom K. Kerppola, and Brenda M. Ogle. Biol Cell. 2010 Aug 6; 102(Pt 9): 525–537.
- Visualization of the genomic loci that are bound by specific multiprotein complexes by bimolecular fluorescence complementation (BiFC) analysis on Drosophila polytene chromosomes. Huai Deng and Tom K Kerppola. Methods Enzymol. 2017; 589: 429–455.
- Live cell visualization of the interactions between HIV-1 Gag and the cellular RNA-binding protein Staufen1. Miroslav P Milev, Chris M Brown, and Andrew J Mouland. Retrovirology. 2010; 7: 41.
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