CN113514433B - Bimolecular fluorescence complementation technology capable of effectively identifying false positive signals - Google Patents
Bimolecular fluorescence complementation technology capable of effectively identifying false positive signals Download PDFInfo
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Abstract
The invention discloses a bimolecular fluorescence complementation (BiFC) technology capable of effectively identifying false positive signals, and the existing BiFC imaging technology is improved by adding a reference fluorescent protein. The invention connects a reference fluorescent protein and an N-terminal or C-terminal fragment of a labeled fluorescent protein to form a fusion protein, represents the expression level of a plasmid vector through the fluorescent signal intensity of the reference fluorescent protein, normalizes the fluorescent signal intensity of the labeled fluorescent protein for representing a BiFC signal, and takes the ratio of the fluorescent signal intensity of the labeled fluorescent protein to the fluorescent signal intensity of the reference fluorescent protein as a parameter for identifying a false positive signal. The method can be simply and efficiently applied to various BiFC (binary FC) marking systems for researching protein-protein interaction, can identify false positive signals on the premise of not influencing BiFC imaging effect, and solves the problem that real signals and false positive signals cannot be distinguished in the existing tool.
Description
Technical Field
The invention relates to a fluorescence imaging method for researching protein-protein interaction in living cells, in particular to a bimolecular fluorescence complementation technology capable of effectively identifying false positive signals.
Background
Signal pathway networks based on Protein-Protein Interactions (PPIs) play a very important role in numerous biological processes. Although some conventional experimental methods, such as Co-Immunoprecipitation (Co-Immunoprecipitation) and Yeast Two-Hybrid Assay (Yeast Two-Hybrid Assay), can identify protein-protein interactions relatively accurately, these techniques have certain limitations, for example, they do not allow direct observation and study of intracellular protein-protein interactions under physiological conditions of living cells. To solve this problem, several techniques for visualizing protein-protein interactions of living cells based on different principles are developed and applied one after another, and the two most representative techniques are Fluorescence Resonance Energy Transfer (FRET) technique and Bimolecular Fluorescence Complementation (BiFC) technique, respectively. The FRET technique is based on the energy transfer phenomenon of two fluorescent proteins with certain overlapping excitation and emission spectra when the two fluorescent proteins are close enough in space distance, can detect the instantaneously-occurring protein-protein interaction in real time, but has higher requirements on the distance and arrangement mode of the interaction of target proteins, and has limited detection capability for weaker interaction. The BiFC technology utilizes the structural complementarity between two non-fluorescent fragments, the N-terminal and the C-terminal of a fluorescent protein, to detect protein-protein interactions. The method fuses the two non-fluorescent fragments with two target proteins which can interact with each other respectively, if the two target proteins do interact with each other, the non-fluorescent fragments can be tightly combined and reformed into complete fluorescent protein due to the close spatial distance, and then the imaging and quantitative calculation can be carried out by using a fluorescence microscope. Compared with FRET technology, biFC technology has the advantages of easy realization, high sensitivity and the like, and can detect weak protein-protein interaction. In recent decades, experimental approaches based on BiFC technology have been widely used for the study of various types of protein-protein interactions.
Despite the above advantages, biFC technology still suffers from a high false positive signal when used to visualize protein-protein interactions. This is because even if the fusion protein is not linked to the N-and C-termini of the two non-fluorescent fragments, they are spatially close due to random collisions, resulting in spontaneous assembly and hence a large number of false positive signals. Current protocols do not effectively distinguish between true BiFC signals resulting from specific binding of the target protein and false positive signals resulting from random collisions. In addition, the intensity of BiFC fluorescence signal depends heavily on the transfection level of its expression plasmid in cells, and the difference of plasmid transfection level among cells can also influence the judgment of protein-protein interaction strength of researchers.
Disclosure of Invention
Aiming at the defects of the BiFC technology, the invention improves the existing BiFC imaging technology by adding a reference fluorescent protein method, aims to develop a fluorescence labeling method capable of effectively identifying false positive signals, and solves the problem that the existing tool can not distinguish real signals from false positive signals.
The technical scheme provided by the invention has the following strategies:
transforming a BiFC expression plasmid: the scheme of the invention uses a yellow fluorescent protein Venus widely applied to BiFC labeling as a report fluorescent protein for BiFC imaging. In addition, biFC systems may utilize otherFluorescent proteins are subjected to labeling imaging, including yellow fluorescent proteins YFP and Citrine, cyan fluorescent proteins CFP and Cerulean, red fluorescent proteins mCheerry, light conversion fluorescent proteins mEos3.2 and the like, and a BiFC system based on the fluorescent proteins can be theoretically modified by using the method disclosed by the invention. The realization of BiFC fluorescent labeling requires the application of molecular cloning technology to cut fluorescent protein into non-fluorescent fragment N-terminal and C-terminal at specific sites. The Venus fluorescent protein contains 238 amino acids, and a BiFC labeling system based on Venus can select various cleavage sites including 155 th amino acid, 173 th amino acid, 210 th amino acid and the like. The present protocol dissects the Venus protein into an N-terminal Venus-N (amino acids 1-172, VN) and a C-terminal Venus-C (amino acids 155-238, VC). In order to identify false positive signals, a fluorescent protein which does not interfere with the optical imaging of the report fluorescent protein is used as a reference fluorescent protein, and the reference fluorescent protein is connected with the N-terminal or C-terminal of the non-fluorescent fragment of the report fluorescent protein to form a fusion protein. The scheme of the invention uses a red fluorescent protein mCherry as a reference fluorescent protein, and the reference fluorescent protein is connected with VN to form a VN-mCherry mark fragment. In addition, blue fluorescent protein BFP, green fluorescent EGFP, red fluorescent protein mirFP670, mirFP670nano and mirFP703 and the like can also be used as reference fluorescent proteins. The complete mCherry protein and the cut Venus protein form a co-expression system, the intensity of the mCherry fluorescent signal can be used for representing the expression level of the plasmid, and the Venus fluorescent signal intensity for representing the BiFC signal is normalized-the ratio of the two fluorescent signal intensities (Venus fluorescent signal intensity/mCherry fluorescent signal intensity, F) Venus /F mCherry ) Can be used as a parameter for identifying false positive signals. As mentioned in the background art, biFC signal is easily affected by the difference of plasmid expression level between cells, therefore, the effective judgment of false positive signal can not be made only by using BiFC signal (for example, for the same pair of target proteins fused with non-fluorescent fragments, the stronger BiFC signal under a certain transfection condition is probably caused by the higher plasmid expression level under the condition), while the invention uses the intensity ratio of BiFC signal and reference fluorescent signal as evaluation parameter to eliminate the effect of plasmid expression level difference,in this new system, the ratio of fluorescence signal intensities F Venus /F mCherry The higher the level, the false positive signal can be reflected in the transfection condition, thereby realizing the effective identification of the false positive signal.
The strategy can be simply and efficiently applied to various BiFC (bipolar FC) marker systems for researching protein-protein interaction, can identify false positive signals on the premise of not influencing BiFC imaging effect, and can be used as an exogenous expression system to obtain the intracellular protein-protein interaction condition closest to the real state.
In a first aspect of the present invention, a BiFC labeling system capable of effectively identifying false positive signals is provided, which comprises a first vector for fusion expression of a protein a to be detected and an N-terminal fragment of a labeled fluorescent protein, and a second vector for fusion expression of a protein B to be detected and a C-terminal fragment of a labeled fluorescent protein, wherein the first vector or the second vector simultaneously expresses a reference fluorescent protein.
In the BiFC labeling system, the labeled fluorescent protein serving as a report fluorescent protein for BiFC imaging can be yellow fluorescent protein Venus widely applied to BiFC labeling, and can also be other fluorescent proteins, such as yellow fluorescent protein YFP and Citrine, cyan fluorescent protein CFP and Cerulean, red fluorescent protein mCheerry, light conversion fluorescent protein mEos3.2 and the like. Taking yellow fluorescent protein Venus as an example, various cleavage sites can be selected to divide the Venus fluorescent protein into a non-fluorescent N-terminal fragment and a non-fluorescent C-terminal fragment, and the cleavage sites can be 155 th amino acid, 173 th amino acid, 210 th amino acid and the like from the N terminal. In one embodiment of the invention, the Venus protein is cleaved into an N-terminal Venus-N fragment (peptide consisting of amino acid residues 1-172, VN) and a C-terminal Venus-C fragment (peptide consisting of amino acid residues 155-238, VC).
The reference fluorescent protein and the marked fluorescent protein do not interfere with each other on optical imaging. On the first carrier or the second carrier, the reference fluorescent protein is connected with the N-terminal fragment or the C-terminal fragment of the labeled fluorescent protein to form a fusion protein. The red fluorescent protein mCherry, the blue fluorescent protein BFP, the green fluorescent EGFP, the red fluorescent protein mirFP670, mirFP670nano and mirFP703 and the like can be used as reference fluorescent proteins.
In a second aspect of the present invention, based on the above BiFC labeling system, there is provided a fluorescent labeling method capable of effectively identifying false positive signals, comprising the steps of:
1) Constructing an N-terminal fragment and a C-terminal fragment of a labeled fluorescent protein based on a BiFC labeling system, and respectively naming the N-terminal fragment and the C-terminal fragment as FP _ N and FP _ C;
2) Selecting another fluorescent protein which does not interfere with the labeled fluorescent protein in optical imaging as a reference fluorescent protein and is named as REF _ FP;
3) Constructing a first vector of fusion expression of a protein A-FP _ N-REF _ FP to be detected and a second vector of fusion expression of a protein B-FP _ C to be detected; or constructing a first vector for fusion expression of the protein A-FP _ N to be detected and a second vector for fusion expression of the protein B-FP _ C-REF _ FP to be detected;
4) Transfecting living cells with the first vector and the second vector constructed in the step 3);
5) And (3) performing fluorescence microscope imaging on the transfected living cells, calculating the fluorescence signal intensity ratio of the labeled fluorescent protein and the reference fluorescent protein, and detecting the strength of a false positive signal.
In the step 1), the labeled fluorescent protein can be selected from yellow fluorescent proteins Venus, YFP and Citrine, cyan fluorescent proteins CFP and Cerulean, red fluorescent protein mCherry, light conversion fluorescent protein mEos3.2 and the like. Preferably, the yellow fluorescent protein Venus is used as a marked fluorescent protein, and the 155 th amino acid, the 173 th amino acid or the 210 th amino acid of the marked fluorescent protein is used as a segmentation site to segment the Venus protein into an N-terminal segment Venus-N (VN) and a C-terminal segment Venus-C (VC).
In the step 2), the reference fluorescent protein may be selected from red fluorescent protein mCherry, blue fluorescent protein BFP, green fluorescent EGFP, red fluorescent protein miRFP670, miRFP670nano, miRFP703, and the like. Preferably, the red fluorescent protein mCherry is used as the reference fluorescent protein.
In the step 3), the reference fluorescent protein and the N-terminal fragment or the C-terminal fragment of the labeled fluorescent protein are connected to form a fusion protein, and the intensity of the reference fluorescent signal can be used for representing the expression level of the vector.
In the step 5), the intensity of the labeled fluorescent signal for representing the BiFC signal is normalized by F FP Represents the intensity of the fluorescence signal of the labeled fluorescent protein, denoted by F REF_FP Representing the intensity of the fluorescence signal of the reference fluorescent protein, the ratio F of the two FP /F REF_FP Can be used as a parameter for identifying false positive signals, and the higher the ratio, the more false positive signals are generated. Therefore, the fluorescent labeling method can not only research the interaction between the protein A to be detected and the protein B to be detected, but also effectively identify false positive signals.
The invention has the following beneficial effects: false positive signals existing in the conventional BiFC imaging method are effectively identified, and the method is favorable for more accurately researching protein-protein interaction in living cells.
Drawings
FIG. 1 is a diagram of the structure of the plasmids used to express Gag-VN-mCherry and Gag-VC.
Figure 2. Results of fluorescence imaging and signal analysis of bifc labeled Gag under different plasmid transfection conditions, where: a is a wide-field microscope fluorescence imaging effect picture, and the scale bar =10 μm; b is the ratio F of the intensities of the Venus and mCherry fluorescence signals Venus /F mCherry Values in the graph represent mean ± sem, representing significant differences (independent samples T-test, P<0.001)。
Detailed Description
The following examples illustrate the improved BiFC technology of the present invention using the structural protein Gag of the HIV-1 virus as a model protein.
The assembly of HIV-1 virus on cell membranes to form virus particles requires the dependence on the polymerization of Gag proteins, so researchers can use BiFC technology to image the specific interaction between Gag proteins and further study the assembly process of HIV-1. In this example, two BiFC expression plasmids Gag-VN-mCherry and Gag-VC were constructed and used to express Gag proteins fused with VN-mCherry marker fragment and VC marker fragment, respectively. In order to verify that the invention can effectively identify false positive signals, the present example sets two different transfection conditions of high concentration and low concentration, respectively transfects 200ng (high concentration group) and 10ng (low concentration group) of BiFC expression plasmids (the mass ratio of Gag-VN-mCherry and Gag-VC always remains 1, and other details are shown in experimental method 2.2), and the specific implementation mode is as follows.
1 reagents and instruments
1.1 Primary reagents and materials
1) Plasmid HIV-1Gag-VN (for constructing a plasmid expressing Gag-VN-mCherry, see Experimental method 2.1) and plasmid HIV-1 Gag-VC (for expressing Gag-VC, structure shown in FIG. 1) (both from Ronald C.Montelo, original literature: distinglnternal transduction of organic infection Virus and Human Immunodeficiency Virus Type 1Gag dug Viral infection and Budding modified by Bimolecular fluorine compensation Assays (J virol.2007Oct;81 (20): 26-35.ub 2007 g 8.); the empty vector pBapo-CMV Neo (for keeping the total plasmid transfection amount constant, see Experimental method 2.2; commercially available from Takara Bio Inc.).
2) Related restriction enzyme, T4 ligase (available from New England Biolabs).
3) Competent E.coli cells.
4) Plasmid extraction kit (available from Omega Bio-tek).
5) Human cervical cancer cell lines (HeLa cells).
6) Containing 10% (vol/vol) fetal bovine serum (available from PAN) TM Biotech Co.) and 1 XGlutaMAX TM DMEM medium (available from CORNING corporation) (available from Thermo Fisher corporation); 10 XPBS (available from CORNING); trypsin (available from Thermo Fisher).
7) Transfection reagent
6 (available from Promega).
1.2 Main Instrument
1) PCR instrument, gel electrophoresis instrument, biochemical incubator, shaking table, constant temperature incubator.
2) Cell culture case, biological safety cabinet.
3) Fluorescence microscopy.
2 method of experiment
2.1 plasmid construction
An HIV-1 Gag-VN-mCherry plasmid (used for expressing Gag-VN-mChery, the structure is shown in figure 1) is constructed. The construction method comprises four steps. Firstly, using HIV-1Gag-VN plasmid as a template, carrying out PCR amplification by using a primer pair P1/P2 (primer sequences are shown in Table 1) to obtain a SpeI-EcoRI fragment (containing a partial coding region of Gag protein and a complete coding region of VN protein fragment) of the HIV-1Gag-VN, and then inserting an amplification product into a pEGFP-C1 plasmid which is cut by NheI and EcoRI (used for removing EGFP on the original plasmid) to obtain pGag PARTIAL VN-C1 plasmid. Secondly, taking pmCherry-C1 plasmid as a template, carrying out PCR amplification by using a primer pair P3/P4 (the primer sequence is shown in Table 1) to obtain a complete coding region of the mCherry protein, and then inserting the amplification product into pGag which is cut by NotI and AgeI (used for removing VN on the original plasmid) PARTIAL In the VN-C1 plasmid, pGag is obtained PARTIAL -mCherry-C1 plasmid. Thirdly, HIV-1Gag-VN plasmid is used as a template, a primer pair P5/P6 (the primer sequence is shown in table 1) is utilized for PCR amplification to obtain the complete coding region of the VN protein fragment, and then the amplification product is inserted into pGag which is digested by NotI and XhoI PARTIAL pGag was obtained from the-mCherry-C1 plasmid PARTIAL VN-mCherry-C1 plasmid. Fourth step, the pGag is digested with SpeI and AgeI PARTIAL Obtaining Gag from-VN-mCherry-C1 plasmid PARTIAL VN-mCherry fragment, and its insertion was cut with SpeI and AgeI (for removal of Gag from the plasmid) PARTIAL The HIV-1Gag-VN plasmid after VN) is used for obtaining the target plasmid HIV-1 Gag-VN-mCherry.
2.2 transfection of cells
Transfection reagents were used when cells were grown to 50% -70% coverage
6 Co-transfection of HIV-1 Gag-VN-mCherry and HIV-1 Gag-VC into HeLa cells. For the high concentration group, 100ng each of the HIV-1 Gag-VN-mCherry and HIV-1 Gag-VC transfection amount (i.e., 200ng total BiFC expression plasmid); for the low concentration group, HIV-1 Gag-VN-mCherry and HIV-1Gag-VC transfection amount is 5ng each (namely, the total amount of BiFC expression plasmid is 10 ng), and 190ng of empty vector pBapo-CMV Neo is added to keep the total transfection amount to be 200 ng.
2.3 fluorescence imaging and results analysis
1) After 24 hours of cell transfection, fluorescence imaging was performed using an OLYMPUS inverted fluorescence microscope (OLYMPUS IX 83), and cell images were acquired using the imaging software CellSens Dimension (parameter settings: the exposure time was set to 100ms and the electron multiplication gain (EM gain) was set to 30).
2) The fluorescence imaging results are shown as a in fig. 2. In both the high and low concentration groups, the Venus signal co-localized well with the mCherry signal on the cell membrane. In contrast, in the high concentration group, mCherry and Venus signals on the cell membrane appear as morphologically abnormal fluorescent plaques; whereas the mCherry and Venus signals on the cell membrane of the low concentration group of cells appear as smaller fluorescent spots, closer to the morphological structure of a typical viral assembly platform. Fluorescence imaging results suggest that more BiFC false positive signals were produced in the high concentration group of cells.
3) The fluorescence signal of the cell image was quantitatively analyzed. Opening a cell image by using image processing software ImageJ, drawing a cell outline by using a Freehand selection tool, respectively measuring the intracellular fluorescence intensity of a Venus channel and an mCherry channel by using a Measure command, drawing an extracellular region outline by using the same method, and measuring the background fluorescence intensity of each channel, wherein the signal intensity of the fluorescent protein corresponding to the channel is obtained by subtracting the background fluorescence intensity from the intracellular fluorescence intensity of each channel. By the method, the Venus fluorescence intensity F can be calculated Venus With mCherry fluorescence intensity F mCherry Ratio F of Venus /F mCherry The size of (a) can reflect the level of interaction between Gag-VN-mCherry and Gag-VC. Analysis and statistics of more than 20 cells gave F under the experimental conditions Venus /F mCherry Mean and standard error of ratio (analytical data are shown in tables 2 and 3, statistical results are shown in B of FIG. 2). In this example, F in the high concentration group Venus /F mCherry F ratio significantly higher than that of the low concentration group Venus /F mCherry The ratio confirms that the high concentration group cells generate more BiFC false positive signals, and further indicates that the morphologically abnormal fluorescent plaques observed on the cell membranes of the high concentration group cells (as shown in FIG. 2A) are caused by non-specific self-assembly of VN and VC (rather than specific interaction between Gag proteins).
TABLE 1 primer sequences used in this example
TABLE 2 results of analysis of fluorescence signals of the high concentration group
TABLE 3 fluorescent Signal analysis results of the Low concentration group
SEQUENCE LISTING
<110> Beijing university
<120> bimolecular fluorescence complementation technology capable of effectively identifying false positive signals
<130> WX2021-03-092
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Claims (8)
1. A bimolecular fluorescence complementary labeling system capable of effectively identifying false positive signals comprises a first carrier for fusion expression of a protein A to be detected and an N-terminal fragment of a labeled fluorescent protein and a second carrier for fusion expression of a protein B to be detected and a C-terminal fragment of the labeled fluorescent protein, and is characterized in that a reference fluorescent protein is fused and expressed on the N-terminal fragment or the C-terminal fragment of the labeled fluorescent protein on the first carrier or the second carrier, and the reference fluorescent protein and the labeled fluorescent protein are not interfered with each other in optical imaging.
2. The bimolecular fluorescent complementary labeling system of claim 1, wherein the labeled fluorescent protein is selected from, but not limited to, one of the following fluorescent proteins: yellow fluorescent proteins Venus, YFP and Citrine, cyan fluorescent proteins CFP and Cerulean, red fluorescent protein mCherry and light-converting fluorescent protein meeos 3.2.
3. The bimolecular fluorescent complementary labeling system of claim 1, wherein the reference fluorescent protein is selected from, but not limited to, one of the following fluorescent proteins: red fluorescent protein mCherry, blue fluorescent protein BFP, green fluorescent EGFP, red fluorescent protein miRFP670, miRFP670nano and miRFP703.
4. The bimolecular fluorescent complementary labeling system of claim 1, wherein the labeled fluorescent protein is yellow fluorescent protein Venus, the N-terminal fragment thereof is a peptide fragment consisting of amino acid residues 1 to 172 of Venus protein, and the C-terminal fragment thereof is a peptide fragment consisting of amino acid residues 155 to 238 of Venus protein.
5. A fluorescence labeling method capable of effectively identifying false positive signals, comprising the steps of:
1) Constructing an N-terminal fragment and a C-terminal fragment for marking the fluorescent protein based on a bimolecular fluorescent complementary marking system, and respectively naming the N-terminal fragment and the C-terminal fragment as FP _ N and FP _ C;
2) Selecting another fluorescent protein which does not interfere with the labeled fluorescent protein in optical imaging as a reference fluorescent protein and is named as REF _ FP;
3) Constructing a first vector of fusion expression of a protein A-FP _ N-REF _ FP to be detected and a second vector of fusion expression of a protein B-FP _ C to be detected; or constructing a first vector of the fusion expression of the protein A-FP _ N to be detected and a second vector of the fusion expression of the protein B-FP _ C-REF _ FP to be detected;
4) Transfecting living cells with the first vector and the second vector constructed in the step 3);
5) And (3) performing fluorescence microscope imaging on the transfected living cells, calculating the fluorescence signal intensity ratio of the labeled fluorescent protein and the reference fluorescent protein, and detecting the strength of a false positive signal.
6. The fluorescence labeling method of claim 5, wherein the labeled fluorescent protein in step 1) is selected from but not limited to one of the following fluorescent proteins: yellow fluorescent proteins Venus, YFP and Citrine, blue fluorescent proteins CFP and Cerulean, red fluorescent protein mCherry and light-converting fluorescent protein meeos 3.2; the reference fluorescent protein in step 2) is selected from but not limited to one of the following fluorescent proteins: red fluorescent protein mCherry, blue fluorescent protein BFP, green fluorescent EGFP, red fluorescent protein miRFP670, miRFP670nano and miRFP703.
7. The fluorescence labeling method of claim 5, wherein step 1) is carried out by constructing an N-terminal fragment consisting of amino acid residues 1 to 172 and a C-terminal fragment consisting of amino acid residues 155 to 238 of Venus yellow fluorescent protein; step 2) taking the red fluorescent protein mCherry as a reference fluorescent protein; and 3) connecting the reference fluorescent protein with the N-terminal fragment or the C-terminal fragment of the labeled fluorescent protein to form the fusion protein.
8. The fluorescent labeling method of claim 5, wherein in step 5), F is used FP Represents the intensity of the fluorescence signal of the labeled fluorescent protein, denoted by F REF_FP Representing the intensity of the fluorescence signal of the reference fluorescent protein, the ratio F of the two fluorescence signal intensities FP /F REF_FP As a parameter for identifying false positive signals.
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