BiFC fluorescent complementary pair and application thereof

Abstract

The invention provides a novel BiFC fluorescent complementary pair or recombinant fluorescent protein and application thereof. The self-assembly capacity of the fluorescence complementary pair or recombinant fluorescent protein is lower than that of a VN173 / VC155 fluorescence complementary pair, background fluorescence signals are remarkably reduced compared with that of the VN173 / VC155 fluorescence complementary pair, the specificity of BiFC analysis is improved, meanwhile, fluorescence signals are stronger than that of a VN210 / VC210 fluorescence complementary pair, fluorescence signals are remarkably increased compared with that of the VN210 / VC210 fluorescence complementary pair, and the sensitivity of BiFC analysis is improved.

Description

Technical Field

[0001] The present invention relates to the field of protein detection, and particularly to a BiFC fluorescence complementation pair and its application. Background Art

[0002] Signal pathway networks based on Protein-Protein Interactions (PPIs) play very important roles in numerous biological processes. Although some traditional experimental methods, such as Co-Immunoprecipitation and Yeast Two-Hybrid Assay, can accurately identify protein-protein interactions, these techniques still have certain limitations. For example, they cannot directly observe and study protein-protein interactions in living cells under physiological conditions. To solve this problem, several visualization techniques for protein-protein interactions in living cells based on different principles have been successively developed and applied. The two most representative ones are Fluorescence Resonance Energy Transfer (FRET) technology and Bimolecule fluorescence complimentation (BiFC) technology.

[0003] BiFC is a technology developed based on protein complementation assay (PCA) for qualitatively or quantitatively analyzing the interactions and subcellular localization between proteins or polypeptide fragments in living cells or extracellular solutions using fluorescence microscopes and fluorescence spectrometers, etc. It has the advantages of high flexibility, fast and intuitive, and wide application range. This technology cleverly disconnects the fluorescent protein molecule at a suitable position to generate two non-fluorescent complementary fragments, and then fuses the two complementary fragments with the target proteins for expression respectively. When the target proteins are co-expressed in cells or co-incubated extracellularly, if there is an interaction between the target proteins, the two complementary fragments of the fluorescent protein approach each other spatially, restoring to form a complete and active fluorescent protein molecule, thus being stimulated to produce fluorescence. The fluorescent signal generated during this process can be detected by conventional instruments such as fluorescence microscopes and fluorescence spectrometers, so it is widely used in the study of protein interactions and drug screening.

[0004] Currently, the bimolecular fluorescence complementation pairs most commonly used at home and abroad are VN173 (encoding amino acids 1 - 173) and VC155 (encoding amino acids 156 - 239) based on the Venus fluorescent protein from Aequorea Victoria. However, the co-expression of this complementation pair in cells produces a strong fluorescence signal, reducing the specificity of protein interaction analysis and easily leading to false positive results.

[0005] Although in 2012, Mizuno et al. reported that the non-specificity of the VN210 (encoding amino acids 1 - 211) and VC210 (encoding amino acids 211 - 238) fluorescence complementation pair for analyzing protein interactions was significantly reduced, the fluorescence signals after they were respectively fused with interacting polypeptide fragments were also weak, affecting the sensitivity of the analysis.

[0006] Therefore, how to optimize the design of bimolecular fluorescence complementation pairs, which can not only reduce the false positives generated when detecting protein interactions but also improve the sensitivity during detection, is the main content elaborated in the present invention. Summary of the Invention

[0007] The object of the present invention is to provide a BiFC fluorescence complementation pair and its application, which can not only reduce the false positives generated when detecting protein interactions but also improve the sensitivity during detection.

[0008] In the present invention, by shortening the length of the VN210 fragment from the C-terminus of the Venus fluorescent protein, a new VN198 fragment (encoding amino acids 1 - 198) was obtained. At the same time, by extending the length of the VC210 fragment towards the N-terminus to make the fragment include the 10th and 11th β-strands of the Venus fluorescent protein, a VC198 fragment (encoding amino acids 198 - 239) was obtained, and thus a new BiFC fluorescence complementation pair VN198 / VC198 was obtained. On this basis, by using the fluorescence complementation pair VN198 / VC198 to detect the interaction between proteins, its superiority in detecting the interaction between proteins was confirmed.

[0009] For this reason, on the one hand, the present invention provides a BiFC fluorescence complementation pair or recombinant fluorescent protein, which is generated by cleavage within the loop between the 9th and 10th β-strands of the fluorescent protein, or by inserting a Caspase-3 recognition sequence within the loop between the 9th and 10th β-strands of the fluorescent protein.

[0010] Preferably, the fluorescent protein is selected from yellow fluorescent protein Venus, cyan fluorescent protein mTurquiose2, green fluorescent protein mNeonGreen, and red fluorescent protein mCherry.

[0011] More preferably, the yellow fluorescent protein Venus and the cyan fluorescent protein mTurquiose2 are derived from Aequorea victoria, the green fluorescent protein mNeonGreen is derived from Branchiostoma lanceolatum, and the red fluorescent protein mCherry is derived from Discosoma sp.

[0012] In a preferred embodiment of the present invention, the BiFC fluorescence complementation pair or the recombinant fluorescent protein is selected from:

[0013] VN198 / VC198, VN198 / VC189, VN198 / VC194, CN196 / CC196, CN196 / CC189, CN196 / CC192, NN200 / NC201, NN200 / NC188, NN200 / NC192, TN199 / TC200, TN199 / TC189, TN199 / TC194, VN198-DEVD-VC198, wherein,

[0014] VN198 encodes amino acids 1-198 of the yellow fluorescent protein Venus, and VC198 encodes amino acids 198-239 of the yellow fluorescent protein Venus.

[0015] VN198 encodes amino acids 1-198 of the yellow fluorescent protein Venus, and VC189 encodes amino acids 189-239 of the yellow fluorescent protein Venus.

[0016] VN198 encodes amino acids 1-198 of the yellow fluorescent protein Venus, and VC194 encodes amino acids 194-239 of the yellow fluorescent protein Venus.

[0017] CN196 encodes amino acids 1-196 of the red fluorescent protein mCherry, and CC196 encodes amino acids 196-236 of the red fluorescent protein mCherry.

[0018] CN196 encodes amino acids 1-196 of the red fluorescent protein mCherry, and CC189 encodes amino acids 189-236 of the red fluorescent protein mCherry.

[0019] CN196 encodes amino acids 1-196 of the red fluorescent protein mCherry, and CC192 encodes amino acids 192-236 of the red fluorescent protein mCherry.

[0020] NN200 encodes amino acids 1 - 200 of the green fluorescent protein mNeonGreen, and NC201 encodes amino acids 201 - 236 of the green fluorescent protein mNeonGreen.

[0021] NN200 encodes amino acids 1 - 200 of the green fluorescent protein mNeonGreen, and NC188 encodes amino acids 188 - 236 of the green fluorescent protein mNeonGreen.

[0022] NN200 encodes amino acids 1 - 200 of the green fluorescent protein mNeonGreen, and NC192 encodes amino acids 192 - 236 of the green fluorescent protein mNeonGreen.

[0023] TN199 encodes amino acids 1 - 199 of the cyan fluorescent protein mTurquiose2, and TC200 encodes amino acids 200 - 239 of the cyan fluorescent protein mTurquiose2.

[0024] TN199 encodes amino acids 1 - 199 of the cyan fluorescent protein mTurquiose2, and TC189 encodes amino acids 189 - 239 of the cyan fluorescent protein mTurquiose2.

[0025] TN199 encodes amino acids 1 - 199 of the cyan fluorescent protein mTurquiose2, and TC194 encodes amino acids 194 - 239 of the cyan fluorescent protein mTurquiose2.

[0026] VN198 - DEVD - VC198 is generated by inserting the Caspase - 3 recognition sequence DEVD at the 198th amino acid of the yellow fluorescent protein Venus.

[0027] On the other hand, the present invention provides an expression vector comprising the BiFC fluorescence complementary pair or recombinant fluorescent protein described in the present invention.

[0028] Preferably, the expression vector is selected from pBiFC-VN198, pBiFC-VC198, BiFC-VN198-bJun, pBiFC-VC198, pBiFC-bFos-VC198, pBiFC-bFosΔzip-VC198, pBiFC-bFos-VC189, pBiFC-bFosΔzip-VC189, pBiFC-bFos-VC194, pBiFC-bFosΔzip-VC194, pBiFC-VN198-FKBP12, pBiFC-VN198-FKBP52, pBiFC-mTOR-VC198, pBiFC-PD-L1-VN198, pBiFC-PD-1-VC198, pBiFC-VN198-GSDMD-N, pBiFC-GSDMD-C-VC198, pBiFC-VN198-Tau, pBiFC-Tau-VC198, pBiFC-CN196, pBiFC-CC189, pBiFC-CC192, pBiFC-CC196, pBiFC-CN196-bJun, pBiFC-bFos-CC189, pBiFC-bFos-CC192, pBiFC-bFos-CC196, pBiFC-bFosΔzip-CC189, pBiFC-bFosΔzip-CC192, pBiFC-bFosΔzip-CC196, pBiFC-NN200, pBiFC-NC188, pBiFC-NC192, pBiFC-NC201, pBiFC-NN200-bJun, pBiFC-bFos-NC188, pBiFC-bFos-NC192, pBiFC-bFos-NC201, pBiFC-bFosΔzip-NC188, pBiFC-bFosΔzip-NC192, pBiFC-bFosΔzip-NC201, pBiFC-TN199, pBiFC-TC189, pBiFC-TC194, pBiFC-TC200, pBiFC-TN199-bJun, pBiFC-bFos-TC189, pBiFC-bFos-TC194, pBiFC-bFos-TC200, pBiFC-bFosΔzip-TC189, pBiFC-bFosΔzip-TC194, pBiFC-bFosΔzip-TC200, pET-24b(+)-VN198-Tau, pET-24b(+)-Tau-VC198, pET-24b(+)-VN198, pET-42a(+)-GST-VC198, pcDNA3.1-VN198-DEVD-VC198, pET-24b(+)-VN198-DEVD-VC198.

[0029] On the other hand, the present invention provides a cell comprising the BiFC fluorescence complementary pair or recombinant fluorescent protein described in the present invention, or the expression vector described in the present invention.

[0030] Preferably, the cell is selected from human embryonic kidney cell HEK293T, human breast cancer cell MCF7, human osteosarcoma cell U2OS, and human cervical cancer cell HeLa.

[0031] On the other hand, the present invention provides a system comprising the BiFC fluorescence complementary pair or recombinant fluorescent protein described in the present invention, the expression vector described in the present invention, or the cell described in the present invention.

[0032] On the other hand, the present invention provides a kit comprising the BiFC fluorescence complementary pair or recombinant fluorescent protein described in the present invention, the expression vector described in the present invention, the cell described in the present invention, or the system described in the present invention.

[0033] On the other hand, the present invention provides a method for detecting the interaction between proteins, polypeptide fragments, or between a protein and a polypeptide fragment, which uses the BiFC fluorescence complementary pair or recombinant fluorescent protein described in the present invention, the expression vector described in the present invention, the cell described in the present invention, the system described in the present invention, or the kit described in the present invention during the detection process.

[0034] Preferably, the protein or polypeptide fragment is selected from mTOR and FKBPs, programmed cell death protein PD-1 and programmed cell death ligand PD-L1, pyroptosis-related protein GSDMD-N (N-terminal fragment of GSDMD) and GSDMD-C (C-terminal fragment of GSDMD), Tau protein, or Caspase-3 and a recombinant fluorescent protein containing its recognition sequence.

[0035] More preferably, FKBPs are selected from FKBP12 or FKBP52.

[0036] On the other hand, the present invention provides a method for screening promoters or inhibitors that promote or inhibit the interaction between proteins, polypeptide fragments, or between a protein and a polypeptide fragment, which uses the BiFC fluorescence complementary pair or recombinant fluorescent protein described in the present invention, the expression vector described in the present invention, the cell described in the present invention, the system described in the present invention, or the kit described in the present invention during the screening process.

[0037] Preferably, the protein or polypeptide fragment is selected from mTOR and FKBPs, programmed cell death protein PD-1 and programmed cell death protein ligand PD-L1, pyroptosis-related proteins GSDMD-N and GSDMD-C, Tau protein, or Caspase-3 and a recombinant fluorescent protein containing its recognition sequence,

[0038] More preferably, FKBPs are selected from FKBP12 or FKBP52.

[0039] On the other hand, the present invention provides a method for detecting Caspase-3 activity, which uses the BiFC fluorescence complementation pair or recombinant fluorescent protein, the expression vector, the cell, the system, or the kit of the present invention during the detection process.

[0040] On the other hand, the present invention provides a method for screening candidate proteins capable of interacting with a target protein from a candidate protein library, which uses the BiFC fluorescence complementation pair or recombinant fluorescent protein, the expression vector, the cell, the system, or the kit of the present invention during the screening process.

[0041] Preferably, the candidate protein library or target protein contains mTOR and FKBPs, programmed cell death protein PD-1 and programmed cell death protein ligand PD-L1, pyroptosis-related proteins GSDMD-N and GSDMD-C, Tau protein, or Caspase-3 and a recombinant fluorescent protein containing its recognition sequence,

[0042] More preferably, FKBPs are selected from FKBP12 or FKBP52.

[0043] On the other hand, the present invention provides the application of the BiFC fluorescence complementation pair or recombinant fluorescent protein, the expression vector, the cell, the system, or the kit of the present invention in detecting the interaction between proteins, polypeptide fragments, or between a protein and a polypeptide fragment, and Caspase-3 activity analysis.

[0044] Preferably, the protein or polypeptide fragment is selected from mTOR and FKBPs, programmed cell death protein PD-1 and programmed cell death protein ligand PD-L1, pyroptosis-related proteins GSDMD-N and GSDMD-C, Tau protein, or Caspase-3 and a recombinant fluorescent protein containing its recognition sequence,

[0045] More preferably, FKBPs are selected from FKBP12 or FKBP52.

[0046] On the other hand, the present invention provides the use of the BiFC fluorescence complementation pair or recombinant fluorescent protein, the expression vector, the cell, the system or the kit of the present invention in screening for promoters or inhibitors that promote or inhibit the interaction between proteins, polypeptide fragments, or between a protein and a polypeptide fragment.

[0047] Preferably, the protein or polypeptide fragment is selected from mTOR and FKBPs, programmed cell death protein PD-1 and programmed cell death protein ligand PD-L1, pyroptosis-related protein GSDMD-N and GSDMD-C, Tau protein, or Caspase-3 and a recombinant fluorescent protein containing its recognition sequence.

[0048] More preferably, the FKBPs are selected from FKBP12 or FKBP52.

[0049] On the other hand, the present invention provides the use of the BiFC fluorescence complementation pair or recombinant fluorescent protein, the expression vector, the cell, the system or the kit of the present invention in detecting the activity of Caspase-3.

[0050] On the other hand, the present invention provides the use of the BiFC fluorescence complementation pair or recombinant fluorescent protein, the expression vector, the cell, the system or the kit of the present invention in screening for candidate proteins capable of interacting with a target protein from a candidate protein library.

[0051] Preferably, the candidate protein library or the target protein contains mTOR and FKBPs, programmed cell death protein PD-1 and programmed cell death protein ligand PD-L1, pyroptosis-related protein GSDMD-N and GSDMD-C, Tau protein, or Caspase-3 and a recombinant fluorescent protein containing its recognition sequence.

[0052] More preferably, the FKBPs are selected from FKBP12 or FKBP52.

[0053] On yet another aspect, the present invention provides the use of the BiFC fluorescence complementation pair or recombinant fluorescent protein, the expression vector, the cell, the system or the kit of the present invention in drug screening or drug evaluation, wherein the drug screening or drug evaluation involves the interaction between proteins, polypeptide fragments, or between a protein and a polypeptide fragment.

[0054] Preferably, the protein or polypeptide fragment is selected from mTOR and FKBPs, programmed cell death protein PD-1 and programmed cell death protein ligand PD-L1, pyroptosis-related protein GSDMD-N and GSDMD-C, Tau protein, or Caspase-3 and a recombinant fluorescent protein containing its recognition sequence.

[0055] More preferably, the FKBPs are selected from FKBP12 or FKBP52.

[0056] Compared with the prior art, the present invention has the following beneficial effects:

[0057] The fluorescence complementation pair or recombinant fluorescent protein screened in the present invention, especially the fluorescence complementation pair VN198 / VC198, has a lower self-assembly ability than the VN173 / VC155 fluorescence complementation pair, and has a significantly reduced background fluorescence signal compared therewith, improving the specificity of BiFC analysis. At the same time, the fluorescence signal of its fusion interaction protein or fragment is stronger than that of the fusion VN210 / VC210 fluorescence complementation pair, and has a significantly increased fluorescence signal compared therewith, improving the sensitivity of BiFC analysis. Description of the Drawings

[0058] Figure 1A is the spatial structure diagram of the yellow fluorescent protein Venus;

[0059] Figure 1B is the structure diagram of the 9th to 11th β-sheets of the yellow fluorescent protein Venus and the loop between the 9th and 10th β-sheets;

[0060] Figure 1C is the spatial structure diagram of the cyan fluorescent protein mTurquiose2;

[0061] Figure 1D is the structure diagram of the 9th to 11th β-sheets of the cyan fluorescent protein mTurquiose2 and the loop between the 9th and 10th β-sheets;

[0062] Figure 1E is the spatial structure diagram of the green fluorescent protein mNeonGreen;

[0063] Figure 1F is the structure diagram of the 9th to 11th β-sheets of the green fluorescent protein mNeonGreen and the loop between the 9th and 10th β-sheets;

[0064] Figure 1G is the spatial structure diagram of the red fluorescent protein mCherry;

[0065] Figure 1HIt is a structural diagram of the 9th to 11th β-sheets of the red fluorescent protein mCherry and the loop between the 9th and 10th β-sheets.

[0066] Figure 2A It is a schematic diagram of the structures of the pBiFC-VN173 and pBiFC-VC155 vectors;

[0067] Figure 2B It is a brief schematic diagram of the structures of VN173 and VC155;

[0068] Figure 2C It is a schematic diagram of the structures of the pBiFC-VN210 and pBiFC-VC210 vectors;

[0069] Figure 2D It is a brief schematic diagram of the structures of VN210 and VC210.

[0070] Figure 3A It is a schematic diagram of the structures of the pBiFC-VN198 and pBiFC-VC198 vectors;

[0071] Figure 3B It is a schematic diagram of the screening sites for the new and old bimolecular fluorescence complementation pairs;

[0072] Figure 3C It is a brief schematic diagram of the structures of VN198 and VC198.

[0073] Figure 4A After transfecting each plasmid or plasmid combination shown in the figure at a transfection dose of 3.0 μg / mL for each plasmid in HeLa, MCF7, and U2OS cells, the results were observed using an inverted fluorescence microscope approximately 24 hours later (the scale bar of the picture is 50 μm);

[0074] Figure 4B It is Figure 4A After fluorescence imaging of MCF7 cells in [], the cells were collected and lysed, the proteins in the cell lysate were separated by SDS-PAGE, β-actin was used as an internal reference, and the western blot of the target protein was detected using Flag antibody and HA antibody. The target protein in each lane is indicated by an arrow.

[0075] Figure 5A After transfecting the plasmid combinations shown in the figure at a transfection dose of 0.5 μg / mL for each plasmid in HeLa cells, 20 μM MG132 or an equal volume of DMSO was added as shown in the figure approximately 24 hours later, and the results were observed using an inverted fluorescence microscope approximately 9 hours later (the scale bar of the picture is 50 μm);

[0076] Figure 5B It is for collecting Figure 5AObserve the cells after fluorescence and lyse them, extract the total cellular proteins, use β-actin as an internal reference, and detect the Western Blotting protein blot of the target protein expression in each transfected cell;

[0077] Figure 5C Seed HeLa cells into a black clear-bottom 96-well cell culture plate, transfect the cells according to the plasmid combinations shown in the figure (the transfection dose of mCherry is 0.02 μg / mL, and the transfection dose of each other plasmid is 0.5 μg / mL), and use an inverted fluorescence microscope to observe the result images approximately 24 hours later (the image scale bar is 50 μm);

[0078] Figure 5D Use a multi-functional microplate reader to measure Figure 5C the Venus and mCherry fluorescence intensities of the HeLa cell lysates in , plot the bar graph, and calculate the ratio of the fluorescence intensities of the interaction groups and the corresponding non-interaction groups, where J represents bJun and F represents bFos.

[0079] Figure 6A Transfect the corresponding plasmid combinations into MCF7, HeLa, and U2OS cells at a dose of 0.5 μg / mL for each plasmid, and use an inverted fluorescence microscope to observe the result images approximately 24 hours later (the image scale bar is 50 μm);

[0080] Figure 6B Transfect the plasmid combinations shown in the figure into HeLa cells at a transfection dose of 0.5 μg / mL for each plasmid, add 20 μM MG132 or an equal volume of DMSO according to the figure approximately 24 hours later, and use an inverted fluorescence microscope to observe the result images approximately 9 hours later (the image scale bar is 50 μm);

[0081] Figure 6C Collect Figure 6B Observe the cells after fluorescence and lyse them, extract the total cellular proteins, use β-actin as an internal reference, and detect the Western Blotting protein blot of the target protein expression in each transfected cell;

[0082] Figure 6D Transfect the corresponding plasmid combinations at gradient doses (the illustrated individual transfection dose for each plasmid) into HeLa cells, and use an inverted fluorescence microscope to observe the result images approximately 24 hours later (the image scale bar is 50 μm).

[0083] Figure 7A Schematic diagrams of the VN198-FKBP12, VN198-FKBP52, and mTOR-VC198 expression vectors;

[0084] Figure 7B HeLa, MCF7, and U2OS cells were transfected with each plasmid at a dose of 0.5 μg / mL. Approximately 24 hours later, 50 μM FK506 or an equal volume of DMSO was added to the transfected cells as shown in the figure. Approximately 1 hour later, 1 nM Rapamycin (RAP) or an equal volume of DMSO was added for treatment. Approximately 24 hours later, the results were observed using an inverted fluorescence microscope (the scale bar of the picture was 50 μm).

[0085] Figure 7C For harvesting Figure 7B After observing the fluorescence in HeLa cells, total cellular proteins were extracted. Using β-actin as an internal reference, Western Blotting was performed to detect the protein blotting images of the target protein expression in each transfection group.

[0086] Figure 8A Schematic diagrams of the PD-L1-VN198 and PD-1-VC198 expression vectors;

[0087] IgKsp represents the signal peptide sequence of IgK;

[0088] PDGFRβ513 - 561 is the transmembrane region sequence of PDGFR;

[0089] Figure 8B HeLa and HEK293T cells were transfected with the expression vectors for VN198 / VC198 and PD-L1-VN198 / PD-1-VC198 respectively. 8 hours after transfection, 20 μM of the PD-1 antibody (Pembrolizumab) or an equal dose of the control antibody was added as shown in the figure. Approximately 16 hours after treatment, the results were observed using an inverted fluorescence microscope (the scale bar of the picture was 25 μm).

[0090] BF stands for Bright Field (the same hereinafter); Ab stands for antibody;

[0091] Figure 8C HEK293T cells were transfected with the PD-L1-VN198 and PD-1-VC198 expression plasmids as shown in the figure for 24 hours. Total cellular proteins were extracted. Using β-actin as an internal reference, Western Blotting was performed to detect the protein blotting images of the target protein expression in each transfection group.

[0092] Figure 9A Schematic diagrams of the VN198-GSDMD-N and GSDMD-C-VC198 expression vectors;

[0093] Figure 9BIn HEK293T and HeLa cells, the expression vector pairs of VN198 and VC198, or the expression vector pairs of VN198-GSDMD-N and GSDMD-C-VC198 were transfected respectively. After about 24 hours, the results were observed using an inverted fluorescence microscope (the scale bar of the picture is 25 μm).

[0094] Figure 9C For HEK293T cells transfected with different combinations of VN198-GSDMD-N and GSDMD-C-VC198 expression plasmids as shown in the figure for 24 hours, the total cell proteins were extracted. Using β-actin as an internal reference, the Western Blotting was used to detect the protein blot of the target protein expression in each transfection group.

[0095] Figure 10A Schematic diagram of the BiFC-Tau expression vectors (VN173-Tau, Tau-VC155, VN198-Tau, and Tau-VC198);

[0096] Figure 10B In HeLa cells, the BiFC-Tau expression vector pairs or their corresponding BiFC empty vector pairs were transfected respectively. After 8 hours of transfection, according to the figure, the control solvent, Forskolin (30 μM), or Forskolin (30 μM) and LMTX (5 μM) were added to the transfected cells for a total treatment of 36 hours. The results were observed by imaging under a fluorescence microscope (the scale bar of the picture is 50 μm).

[0097] Figure 10C For collecting Figure 10B the cell lysates in and performing protein quantification, and calculating the relative fluorescence intensity of each group (the fluorescence intensity of the BiFC-Tau complementary pair group / the corresponding BiFC empty vector pair group);

[0098] Figure 10D For repeating Figure 10C the experiment in HEK293T cells;

[0099] RFU is Relative Fluorescence Units;

[0100] The error bars represent the standard deviation measured from three replicate wells;

[0101] * represents p ≤ 0.01;

[0102] Figure 10E For HEK293T cells transfected with various Tau fusion protein expression plasmids as shown in the figure for 24 hours, the total cell proteins were extracted. Using β-actin as an internal reference, the Western Blotting was used to detect the protein blot of the target protein expression in each transfection group;

[0103] Figure 10F Coomassie brilliant blue staining results of four purified proteins, namely VN198-Tau, Tau-VC198, VN198, and GST-VC198;

[0104] Figure 10G Curves showing the fluorescence intensity detected every 3 hours when in vitro purified protein pairs (VN198 / GST-VC198, VN198-Tau / Tau-VC198) were incubated at room temperature;

[0105] * represents p < 0.0001;

[0106] Figure 10H Curves showing the fluorescence intensity detected at different time points when the in vitro purified protein pair VN198-Tau / Tau-VC198 was incubated with different concentrations of LMTX (0, 10, 25 μM) at room temperature;

[0107] Figure 11A Schematic diagram of the principle for detecting Caspase-3 activity by VN198-DEVD-VC198;

[0108] Figure 11B For the in vitro expressed and purified substrate recombinant protein VN198-DEVD-VC198 (1.0 ng / μl), it was divided into 3 groups according to the treatment method: the group without Caspase-3 treatment, the group treated with Caspase-3 (0.2 ng / μl), and the group co-treated with Caspase-3 (0.2 ng / μl) and Caspase-3 inhibitor (Ac-DEVD-CHO, 20.0 μM). After reacting at 37°C for 18 hours to detect the fluorescence intensity, it was heated at 68°C for 5 minutes and the fluorescence intensity was detected again. The results are shown in the figure;

[0109] Figure 11C Western blotting protein blot for detecting the expression level of substrate protein by Caspase-3;

[0110] The green and red arrows respectively indicate the positions of the target proteins;

[0111] Inh. represents the Caspase-3 inhibitor Ac-DEVD-CHO;

[0112] VN-DEVD-VC is VN198-DEVD-VC198;

[0113] FL is the full length of VN198-DEVD-VC198;

[0114] VN is the VN198-DEV fragment;

[0115] Figure 11D HeLa cells with different densities were untreated or treated with DMSO, etoposide, Caspase-3 inhibitor, or etoposide combined with Caspase-3 inhibitor. After 48 hours, the HeLa cells were collected and lysed with cell lysis buffer. The cell lysates were co-incubated with the in vitro purified substrate VN198-DEVD-VC198 (500 ng / mL) at 37 °C for 2 hours, and then heated at 68 °C for 5 minutes to obtain a schematic diagram of the distribution of different cell numbers and the degree of RFU reduction before and after heating.

[0116] Figure 11E Each plasmid was transfected into HeLa cells at a dose of 0.5 μg / mL. After 8 hours, the cells transfected with the VN198 / VC198 empty vector were not treated, and the cells transfected with VN198-DEVD-VC198 were treated with etoposide (30 μM) or an equal volume of DMSO for 26 hours, and then imaged under a fluorescence microscope (the scale bar of the picture is 50 μm).

[0117] Figure 11F Quantitative analysis by flow cytometry Figure 11E Results of different fluorescence intensities of HeLa cells in the second and third rows in

[0118] Figure 11G Collection of Figure 11E Protein blot of the cells in the second and third rows in and detection of the corresponding protein expression levels by Western blotting

[0119] Figure 11H HeLa cells were transfected with 0.5 μg / mL of GFP or VN198-DEVD-VC198 plasmid. After 8 hours, the cells were treated with an equal volume of control solvent, etoposide (25 μM), or etoposide (25 μM) combined with a pan-Caspase inhibitor (Z-VAD-FMK, 100 μM) for 40 hours. Z-VAD-FMK was added 4 hours after transfection. The relative fluorescence intensity reduction value (Decrease in RFU) under the condition of detecting the same total protein amount in each group was presented in the form of a histogram.

[0120] Eto. represents etoposide; Casp.3 represents Caspase-3;

[0121] Figure 11I Repeating the Figure 11H experimental result histogram in HEK293T cells, where etoposide was replaced by Camp (Camptothecin);

[0122] Figure 11J It is a graph of substrate concentration - relative fluorescence intensity for VN198 - DEVD - VC198;

[0123] The substrate VN198 - DEVD - VC198 was diluted to 0, 25, 50, 100, and 150 μg / mL respectively by substrate dilution buffer, and the detection volume was made up to 100 μl with assay buffer. The relative fluorescence intensity of substrates at different concentrations was detected using a multimode microplate reader in a black clear - bottom 96 - well cell culture plate;

[0124] Figure 11K It is a graph of substrate - enzyme reaction;

[0125] Without Caspase - 3, or Caspase - 3 at 200 ng / mL was reacted with different concentrations of substrate VN198 - DEVD - VC198 (0.25, 0.5, 1, 2.5, 5, 10 μg / mL) at 37 °C for 2 hours, and the degree of RFU reduction before and after heating at 68 °C for each group was calculated;

[0126] Figure 11L It is a graph of enzyme - catalyzed reaction kinetics;

[0127] The substrate VN198 - DEVD - VC198 (500 ng / mL) was reacted with Caspase - 3 enzyme (50 ng / mL) at 37 °C for different times (0 - 5.5 hours), and the reduction values of RFU at each time point were measured. A graph was plotted with the time points as the horizontal axis and the reduction values of RFU as the vertical axis;

[0128] Figure 11M It is a graph of enzyme dosage - substrate reaction;

[0129] The substrate concentration was kept constant (500 ng / mL) and different amounts of Caspase - 3 enzyme (0 - 200 ng / mL) were incubated at 37 °C for 2 hours, and the reduction values of fluorescence intensity under different enzyme dosage conditions were calculated;

[0130] Figure 11N Based on the above enzyme dosage - substrate reaction curve, the Caspase - 3 activities of etoposide - treated cells and DMSO - treated cells were calculated and presented in the form of a bar graph.

[0131] Figure 12A For HEK293T cells transfected with each pair of BiFC plasmids designed based on the mCherry fluorescent protein structure at a dose of 1 μg / mL, and simultaneously transfected with 0.2 μg / mL mCerulean plasmid as an internal reference, it is a graph of the imaging results under a fluorescence microscope 48 hours after transfection (the scale bar of the picture is 25 μm);

[0132] CN is the N-terminal fragment of mCherry; CC is the C-terminal fragment of mCherry;

[0133] Figure 12B For repeating the Figure 12A experimental result graph in HeLa cells (the scale bar of the picture is 25 μm);

[0134] Figure 12C For HEK293T cells transfected with each pair of BiFC plasmids designed based on the mNeonGreen fluorescent protein structure at a dose of 1 μg / mL, and simultaneously transfected with 0.2 μg / mL mTagBFP2 plasmid as an internal reference, the imaging result graph under a fluorescence microscope after 48 hours of transfection (the scale bar of the picture is 50 μm);

[0135] NN is the N-terminal fragment of mNeonGreen; NC is the C-terminal fragment of mNeonGreen;

[0136] Figure 12D For repeating the Figure 12C experimental result graph in HeLa cells (the scale bar of the picture is 50 μm);

[0137] Figure 12E For HEK293T cells transfected with each pair of BiFC plasmids designed based on the mTurquoise2 fluorescent protein structure at a dose of 1 μg / mL, and simultaneously transfected with 0.2 μg / mL mTagBFP2 plasmid as an internal reference, the imaging result graph under a fluorescence microscope after 48 hours of transfection (the scale bar of the picture is 50 μm);

[0138] TN is the N-terminal fragment of mTurquoise2; TC is the C-terminal fragment of mTurquoise2;

[0139] Figure 12F For repeating the Figure 12E experimental result graph in HeLa cells (the scale bar of the picture is 50 μm);

[0140] Figure 12G For collecting Figure 12A 、 12C and the cell lysates of each group of 12E and detecting their fluorescence intensities. After correcting with the internal reference fluorescent protein, a bar graph of the relative fluorescence intensities of different BiFC pairs in each group is obtained, and further the ratio of the fluorescence intensity of the interacting BiFC complementary pairs in each group to that of the empty vector control or non-interacting complementary pairs is calculated. Specific implementation manners

[0141] The technical solution of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only for illustrative and explanatory purposes of the present invention, and should not be construed as limiting the protection scope of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0142] Unless otherwise specified, the raw materials and reagents used in the following embodiments are all commercially available products, or can be prepared by known methods.

[0143] In this specification and the claims, the words "comprising", "including" and "containing" mean "including but not limited to", and are not intended to exclude other parts, components, or steps.

[0144] As used herein, the term "yellow fluorescent protein Venus" refers to a yellow fluorescent protein obtained by artificially site-directed mutagenesis of the green fluorescent protein avGFP derived from Aequorea Victoria. Its specific sequence is shown in GenBank: AAZ65844. The spatial structure of this protein is as shown in the appendix Figure 1A shown, the 9th to 11th β-sheets and the loop structure between the 9th and 10th β-sheets are as shown in the appendix Figure 1B shown.

[0145] As used herein, the term "cyan fluorescent protein mTurquiose2" refers to a cyan fluorescent protein obtained by artificially site-directed mutagenesis of the green fluorescent protein avGFP derived from Aequorea Victoria. It is generated by mutating T65S / I146F from SCFP3A (GenBank: AAZ65848). The spatial structure of this protein is as shown in the appendix Figure 1C shown, the 9th to 11th β-sheets and the loop structure between the 9th and 10th β-sheets are as shown in the appendix Figure 1D shown.

[0146] As used herein, the term "green fluorescent protein mNeonGreen" refers to a green fluorescent protein obtained by artificially site-directed mutagenesis of the yellow-green fluorescent protein LanYFP derived from Branchiostoma lanceolatum. Its specific sequence is shown in GenBank: AGG56535. The spatial structure of this protein is as shown in the appendix Figure 1E shown, the 9th to 11th β-sheets and the loop structure between the 9th and 10th β-sheets are as shown in the appendix Figure 1F shown.

[0147] As used herein, the term "red fluorescent protein mCherry" refers to a red fluorescent protein obtained by artificial site-directed mutagenesis of the red fluorescent protein DsRFP derived from Discosoma sp. mushroom coral, and its specific sequence is shown in GenBank: AAV52164. The spatial structure of this protein is as shown in the appendix Figure 1G and shown in the appendix, the 9th to 11th β-sheets and the loop structure between the 9th and 10th β-sheets are as shown in the appendix Figure 1H and shown in the appendix.

[0148] As used herein, the term "fluorescent complementation pair VN198 / VC198" refers to a BiFC fluorescent complementation pair composed of a polypeptide fragment of the 1-198 amino acid residues of the yellow fluorescent protein Venus (abbreviated as VN198) and a polypeptide fragment of the 198-239 amino acid residues of Venus (abbreviated as VC198).

[0149] As used herein, the term "mTOR protein" refers to mammalian target of rapamycin, which is a highly conserved serine / threonine protein kinase and participates in the formation of two complexes, mTORC1 and mTORC2, and plays an important role in regulating cell autophagy, protein synthesis, and energy metabolism.

[0150] As used herein, the term "FKBPs protein" refers to the general term for members of FK506-Binding Protein, which belongs to the immunophilin protein family, binds the immunosuppressive agents FK506 and rapamycin, and has cis-trans prolyl isomerase activity. Here, it refers to two of its members, FKBP12 and FKBP52.

[0151] As used herein, the term "PD-1 protein" refers to programmed cell death protein 1, which is encoded by the PDCD1 gene, is expressed on the surface of activated T cells to regulate their functions, and is also expressed on various types of tumor cells and plays a role in anti-tumor immunity.

[0152] As used herein, the term "PD-L1 protein" refers to programmed death ligand 1, which is the ligand of PD-1, is encoded by the CD274 gene, is expressed on the surface of T cells, B cells, and various tumor cells, and the interaction with PD-1 inhibits the activation of T cells and the production of cytokines. During the infection or inflammation of normal tissues, this interaction is important for maintaining the homeostasis of the immune response and preventing the occurrence of autoimmunity. In the tumor microenvironment, this interaction inactivates the functions of cytotoxic T cells and is conducive to the immune escape of tumor cells.

[0153] As used herein, the term "GSDMD protein" refers to member D of the Gasdermin protein family, which is cleaved by Caspase1 / 4 / 5 / 11, etc. The released N-terminal fragment oligomerizes into pores on the cell membrane, inducing pyroptosis and inflammatory responses.

[0154] As used herein, the term "Tau protein" refers to microtubule-associated protein Tau, which is encoded by the MAPT gene. Its abnormal phosphorylation forms neurofibrillary tangles, which is one of the main pathological features of Alzheimer's disease.

[0155] As used herein, the term "Caspase-3 protein" refers to cysteine-aspartic protease 3, which is encoded by the CASP3 gene and is processed and matured by cysteine-aspartic proteases Caspase-8 / 9 / 10. It cleaves substrates with the DEVD amino acid residue sequence, inducing apoptosis.

[0156] The following describes the materials and methods used in the example part of the present invention. In the following examples of the present invention, if the said materials and methods are needed, they are carried out in the following manner respectively.

[0157] (I) Vectors

[0158] 1. pBiFC-VN173 and pBiFC-VC155 vectors

[0159] The specific maps of the pBiFC-VN173 and pBiFC-VC155 vectors are as shown in the appendix Figure 2A and the above vectors can be obtained by purchase.

[0160] 2. pBiFC-VN210 and pBiFC-VC210 vectors

[0161] The specific maps of the pBiFC-VN210 and pBiFC-VC210 vectors are as shown in the appendix Figure 2C The former is constructed by inserting Flag-linker-VN210(1-211)-linker between the NheI and HindIII restriction endonucleases of the pcDNA3.1(+) vector, and the latter is constructed by inserting Linker-VC210(211-239)-linker-HA between the NotI and ApaI restriction endonucleases of the pcDNA3.1(+) vector. The above vector pair is the starting vector of the present invention.

[0162] 3. pBiFC-VN198 and pBiFC-VC198 vectors

[0163] The specific maps of the pBiFC-VN198 and pBiFC-VC198 vectors are as shown in the appendix Figure 3AAs shown, it is constructed on the basis of the pBiFC-VN210 and pBiFC-VC210 vectors, and the above vector pair is the vector constructed for the present invention.

[0164] Other vectors used in the present invention are mainly constructed on the basis of the above pBiFC-VN210, pBiFC-VC210, pBiFC-VN198 and pBiFC-VC198 vectors.

[0165] (II) Cell lines, cell culture and transfection

[0166] The present invention relates to the following four cell lines: human embryonic kidney cells HEK293T, human breast cancer cells MCF7, human osteosarcoma cells U2OS and human cervical cancer cells HeLa.

[0167] Among them, HEK293T, MCF7 and U2OS cells were purchased from the Cell Resource Center of the Institute of Basic Medicine, Chinese Academy of Medical Sciences (IBMS&CAMS). HeLa cells were purchased from Hunan Fenghui Biotechnology Co., Ltd. (Changsha, China).

[0168] Cell culture conditions: Cultured in DMEM medium containing 10% fetal bovine serum. When transfecting cells, after inoculating and growing the cells for 14 - 18 hours, the plasmid was transfected into the cells at a ratio of 1:2 (μg:μl) with polyethylenimine (PEI). Unless otherwise specified, the final concentration of each BiFC plasmid in the medium was 1.0 μg / mL, and the final concentration of the internal reference fluorescent proteins (mCherry, mCerulean, mTagBFP2) was 0.1 μg / mL.

[0169] (III) Western blotting

[0170] Cells were lysed on ice for 1 hour with pre-cooled NP-40 lysis buffer (composition: 50 mM Tris-HCl pH 8.0, 1.0% Nonidet P-40, 150 mM NaCl, 1.0 mM EDTA, 10% glycerol, protease inhibitor), centrifuged at 12,000 rpm for 15 minutes at 4°C, and proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the target protein level was detected with the corresponding antibody.

[0171] (IV) In vivo BiFC analysis

[0172] Live cell imaging was observed by an inverted fluorescence microscope EVOS FL Auto 2 or Leica DMI8 (DFC9000 GT VSC-11009). When using the latter, the fluorescent proteins and the corresponding excitation wavelength (λ ex ) and emission wavelength (λem ) are as follows: mTagBFP2, 365 / 435 - 485; mCerulean and mTurquoise 2, 460 / 512 - 542; Venus and mNeonGreen, 490 / 512 - 542; mCherry, 580 / 595.

[0173] The fluorescence intensity of cell lysates was detected by a FlexStation 3 multimode microplate reader (Molecular Devices) or a Synergy H1 multimode microplate reader (Biotek, USA). The λ ex and λ em of each fluorescent protein for FlexStation 3 are as follows: mCerulean and mTurquoise 2, 433 / 538; Venus and mNeonGreen, 488 / 538; mCherry, 580 / 620. The λ ex and λ em of Synergy H1 are as follows: mCerulean and mTurquoise 2, 433 / 475; Venus and mNeonGreen, 495 / 530; mCherry, 580 / 620.

[0174] (V) Flow cytometry

[0175] HeLa cells were transfected with 0.5 μg / mL of VN198 - DEVD - VC198 expression plasmid. After 8 hours, DMSO or the apoptosis inducer etoposide (final concentration 30 μM) was added and the cells were incubated for 40 hours. The cells were collected and the proportion of Venus - positive cells in the samples was analyzed by the Accuri C6 Plus flow cytometer (BD Biosciences) through the FITC channel. Cells were collected for each sample under the same gating conditions.

[0176] (VI) Protein expression and purification

[0177] The coding sequences of VN198-DEVD-VC198, VN198-Tau, Tau-VC198, VN173-Tau, Tau-VC155, VN198, VN173, VC155 and VC198 were inserted into the pET vector to successfully construct the following plasmids: pET-24b(+)-VN198-DEVD-VC198, pET-24b(+)-VN198-Tau, pET-24b(+)-Tau-VC198, pET-24b(+)-VN198, pET-24b(+)-VN173, pET-42a(+)-GST-VC155 and pET-42a(+)-GST-VC198. Rosetta 2 cells were used for protein expression. The proteins were purified using the Ni-NTA purification system (Qiagen). The concentration of the purified proteins was determined by the BCA method, and the protein purity was evaluated by 10% SDS-PAGE and Coomassie blue staining. The protein solutions were stored in the following buffer: 150 mM NaCl, 25 mM Tris-HCl, pH 7.2, 5% glycerol, 1 mM phenylmethylsulfonyl fluoride.

[0178] (VII) In vitro aggregation analysis of Tau protein

[0179] VN198-Tau / Tau-VC198 was incubated in the reaction buffer (PBS solution containing 0.1 mM DTT) at room temperature with moderate shaking for 24 hours. VN198 / GST-VC198 was the corresponding negative control. The molar ratio of the VN fragment to the VC fragment in the reaction system was 1:3. The fluorescence intensity was detected every 2 hours using FlexStation 3 (λ ex = 488 nm, λ em = 538 nm). To evaluate the ability of LMTX to inhibit Tau protein aggregation, the recombinant protein fused with Tau was incubated with VN198-Tau / Tau-VC198 in the reaction buffer (PBS solution containing 0.1 mM DTT) and different concentrations of LMTX (0 μM, 10 μM and 25 μM) at room temperature with moderate shaking for 48 hours, and the fluorescence intensity of the solution was detected every 2 hours by a multi-functional microplate reader.

[0180] (VIII) In vitro detection of Caspase-3 activity

[0181] The cell lysis buffer composition: 50 mM HEPES pH 7.4, 5 mM CHAPS and 5 mM DTT. The analysis buffer composition: 20 mM HEPES pH 7.4, 2 mM EDTA, 0.1% CHAPS, 5 mM DTT.

[0182] Measurement orifice plate and system: A 96-well cell culture plate with a black transparent bottom was used, and the measured volume per well was 100 μl.

[0183] The specific operation steps are as follows: The reaction mixture was incubated at 37 °C in the dark for a certain period of time, and the reaction was terminated with an excessive amount of Caspase-3 inhibitor (Ac-DEVD-CHO), and then heated at 68 °C for 5 minutes. The fluorescence intensities before and after heating were detected using a FlexStation 3, and the excitation and emission wavelengths were 488 nm and 538 nm, respectively.

[0184] Regarding the preparation process of cell samples for in vitro detection of Caspase-3 activity in apoptotic and non-apoptotic cells, the following is the process: Cells were induced with 30 μM etoposide for 20 hours, and at the same time, cells were treated with DMSO as a negative control. The cells were collected and lysed in pre-cooled lysis buffer for 1 hour. Every 1 × 10 7 cells were incubated with 500 μl of cell lysis buffer for 1 hour, and the supernatant was collected by centrifugation at 4 °C and 12,000 rpm for 15 minutes for later use.

[0185] Example 1 Obtaining of the BiFC fluorescent complementary pair VN198 / VC198

[0186] I. Commonly used BiFC fluorescent complementary pairs designed based on the Venus protein structure currently

[0187] 1. VN173 / VC155 fluorescent complementary pair

[0188] pBiFC-VN173 / pBiFC-VC155 for BiFC analysis of protein-protein interactions based on the Venus fluorescent protein is a commonly used fluorescent complementary pair at home and abroad currently. Its plasmid and the structure of the fluorescent complementary pair are shown in Appendix Figure 2A and Appendix Figure 2B respectively.

[0189] The present invention found that the above-mentioned VN173 and VC155 expression plasmids were co-transfected into HeLa, MCF7, and U2OS cells. Under the same conditions, stronger fluorescent signals similar to or even stronger than Venus could be observed in the three transfected cell types, as shown in the left two columns of Appendix Figure 4A This indicates that there is a strong self-assembly ability between the VN173 / VC155 complementary pairs in cells, and false positive results are likely to occur when detecting protein-protein interactions.

[0190] 2. VN210 / VC210 fluorescent complementary pair

[0191] The VN210 / VC210 complementary pair is a BiFC complementary pair screened by Mizuno et al. in 2012 based on the Venus fluorescent protein structure, as shown in Appendix Figure 2C and 2DAs shown. As attached Figure 4A As shown in the middle column, no obvious fluorescence signal was detected in the cells transfected with the empty vector pair of VN210 / VC210 alone, indicating that the self-assembly ability of the VN210 / VC210 pair in cells is weak.

[0192] VN210 was fused with bJun (encoding amino acid residues 257 - 334, NP_068607), VC210 was fused with bFos (encoding amino acid residues 118 - 211, NP - 071533) or bFosΔzip (encoding amino acid residues 118 - 211, with a deletion of the 179 - 193 fragment, represented by Δzip) to construct VN210-bJun, bFos-VC210 and bFosΔzip-VC210 expression vectors. It has been reported that there is an interaction between bJun and bFos, and there is no interaction between bJun and bFosΔzip. As attached Figure 4A From the attached results, it can be seen that the fluorescence signal of the cells co-expressing VN210-bJun / bFos-VC210 was significantly stronger than that of the cells expressing the empty vector pair of VN210 / VC210, but significantly weaker than that of the cells expressing VN173 / VC155, and the results were consistent in the three types of cells. Moreover, no obvious fluorescence signal was detected in the cells co-transfected with VN210-bJun / bFosΔzip-VC210. Therefore, the VN210 / VC210 complementary pair can be used to analyze protein-protein interactions, and has significantly improved specificity compared with the VN173 / VC155 complementary pair. However, the fluorescence signal for detecting protein interactions of VN210-bJun / bFos-VC210 is weak, reducing the sensitivity of detection of this complementary pair, and false negative results are likely to be obtained for weakly interacting proteins.

[0193] The expression levels of all target proteins were detected by Western blotting. The results are as attached Figure 4B showed that the expression level of the bFosΔzip-VC210 protein in the cells co-transfected with VN210-bJun / bFosΔzip-VC210 was significantly lower than the expression level of the bFos-VC210 protein in the cells co-transfected with VN210-bJun / bFos-VC210.

[0194] To exclude the possibility that the low expression of the bFosΔzip-VC210 protein leads to a decrease in fluorescence signal, the present invention co-transfected VN210-bJun / bFos-VC210 or VN210-bJun / bFosΔzip-VC210 in MCF7 cells, and further treated the VN210-bJun / bFosΔzip-VC210 transfection group with DMSO and MG132 respectively. As attached Figure 5AAs shown, obvious fluorescence signals were observed in the VN210-bJun / bFos-VC210 group. However, for the VN210-bJun / bFosΔzip-VC210 group, no obvious fluorescence signals were observed whether treated with MG132 or DMSO. Although as attached Figure 5B As shown, the Western blotting results showed that treatment with MG132 did induce a significant increase in the expression level of bFosΔzip-VC210. Therefore, we believe that the undetectable fluorescence signals in the VN210-bJun / bFosΔzip-VC210 transfection group may not be due to the low expression level of bFosΔzip-VC210. Instead, the reason for the latter's decrease may be that bFosΔzip-VC210 loses its interaction with VN210-bJun, resulting in reduced stability and degradation by the ubiquitin-proteasome system.

[0195] 3. New BiFC fluorescence complementation pair VN198 / VC198

[0196] Referring to the cleavage sites of the VN173 / VC155 complementation pair and the VN210 / VC210 complementation pair on Venus, the present invention searched for new cleavage sites between the 173rd and 210th positions of Venus, with the expectation of obtaining a site with lower self-assembly ability than the VN173 / VC155 complementation pair but stronger fluorescence signals during protein-specific interactions than the VN210 / VC210 complementation pair.

[0197] Mizuno et al. conducted a systematic study on the complementation pairs designed based on the Venus fluorescent protein in 2012. The results showed that the VN210 / VC189 combination self-assembled to produce fluorescence and could not distinguish between protein interactions and non-interactions, while the VN210 / VC210 complementation pair could distinguish between interactions and non-interactions but had weak fluorescence signals. Therefore, the present invention shortened the length of the VN210 fragment from the C-terminus to obtain a new VN198 fragment including the 1st to 9th β-strands of Venus, and simultaneously constructed a VN198-bJun fusion expression vector. The length of the VC210 fragment was extended from the N-terminus so that the new fragment included the 10th and 11th β-strands of Venus, as attached Figure 3B As shown, and according to the different positions in the loop between the 9th and 10th β-strands, VC198, VC194, and VC189 fragments were obtained. VN (VN210 or VN198) was fused with the bJun fragment, and VC198, VC194, and VC189 were fused with the bFos or bFosΔzip fragment. The constructed expression vectors were transfected into HeLa cells and their fluorescence signals were observed, and mCherry was co-transfected as an internal reference control.

[0198] As attached Figure 5CAs shown, the fluorescence signals co-expressed by VN210-bJun or VN198-bJun and bFos-VCs are significantly stronger than the corresponding fluorescence signals co-expressed with bFosΔzip-VCs. Under the same transfection and fluorescence imaging conditions, regardless of whether there is an interaction between the fusion proteins, the combination containing the VN210 fragment produces a stronger fluorescence signal than the corresponding combination containing the VN198 fragment. Notably, among all the complementary pairs, the fluorescence intensity of the VN210-bJun / bFos-VC210 pair is the weakest. Calculate the relative fluorescence intensity ratio for each transfection pair (fluorescence intensity of interaction / fluorescence intensity of corresponding non-interaction). As attached Figure 5D The results show that the ratio of fluorescence intensities of the VN210 / VC198 and VN198 / VC198 complementary pairs is the largest. At the same time, the present invention observes that the fluorescence intensity of the non-interaction group of the VN198 / VC198 complementary pair is lower than that of the VN210 / VC198 pair, and even lower than that of the VN210 / VC210.

[0199] Therefore, the present invention preliminarily determines that the VN198 / VC198 complementary pair is more suitable as a fluorescence complementary pair for BiFC analysis.

[0200] To further verify whether the VN198 / VC198 combination is superior to the VN210 / VC210 combination, the present invention transfected MCF7, HeLa, and U2OS cells with the plasmid pairs of VN210-bJun / bFos-VC210 and VN198-bJun / bFos-VC198 and the corresponding non-interaction plasmid pairs VN210-bJun / bFosΔzip-VC210 and VN198-bJun / bFosΔzip-VC198, respectively. As Figure 6A shown, under the same treatment conditions, for each type of cell, the fluorescence intensity of the VN198-bJun / bFos-VC198 transfection group is significantly higher than that of the VN210-bJun / bFos-VC210 transfection group, while the fluorescence signals of the corresponding non-interaction transfection groups are very weak or even undetected in all three types of cells for both the VN198 / VC198 combination and the VN210 / VC210 combination, indicating that the VN198 / VC198 combination has higher sensitivity than the VN210 / VC210 combination in analyzing the bJun-bFos interaction.

[0201] In addition, in the VN198 / VC198 combination, to explore the relationship between the protein expression level of bFosΔzip and the fluorescence signal intensity of this group, according to the attached Figure 6BHeLa cells were transfected with the different combinations shown and treated with DMSO or MG132 for a certain period of time. The results of fluorescence microscopy imaging showed that obvious fluorescence signals could be observed in the VN198-bJun / bFos-VC198 expression group, while no fluorescence was detected in the VN198-bJun / bFosΔzip-VC198 group regardless of whether it was treated with MG132. Cells were harvested after observing fluorescence in each group above, and the expression levels of the target proteins in each group were analyzed. The results of Western Blotting showed that compared with the non-interacting group treated with DMSO, the protein expression level of bFosΔzip-VC198 in the non-interacting group treated with MG132 increased significantly, as shown in Figure 6C the appendix. It is speculated from this that it is consistent with the results in the VN210 / VC210 combination, that is, the lack of fluorescence signal detected in VN198-bJun / bFosΔzip-VC198 is not caused by the low protein expression level of bFosΔzip-VC198, and the lack of interaction between bJun and bFosΔzip may lead to the latter being easily degraded by the ubiquitin-proteasome system.

[0202] Some studies have suggested that the transfection dose of plasmid pairs affects the self-assembly ability of fluorescence complementary fragments. To exclude the interference of plasmid dose on self-assembly analysis, different doses of the N173 / VC155 complementary pair, VN210 / VC210 complementary pair, or VN198 / VC198 complementary pair were transfected into HeLa cells. As shown in Figure 6D the appendix, the proportion of yellow fluorescent cells transfected with the VN173 / VC155 pair increased with the increase of the plasmid transfection dose. However, even when the lowest dose of plasmid reported in the literature was transfected, obvious fluorescence signals could still be observed for the VN173 / VC155 pair. For the VN210 / VC210 and VN198 / VC198 plasmid pairs, no obvious fluorescence signals were observed even when transfected with the highest plasmid dose. This result suggests that the different self-assembly abilities of the VN173 / VC15, VN210 / VC210, and VN198 / VC198 complementary pairs are determined by the differences in the fragments.

[0203] In summary, the fluorescence complementary pair VN198 / VC198 screened in the present invention has a lower self-assembly ability than the VN173 / VC155 fluorescence complementary pair, and has a significantly reduced background fluorescence signal, improving the specificity of BiFC analysis. At the same time, the fluorescence signal of the VN198 / VC198 complementary pair is stronger than that of the VN210 / VC210 complementary pair, improving the sensitivity of BiFC analysis. Therefore, the fluorescence complementary pair VN198 / VC198 may be a new and more ideal BiFC fluorescence complementary pair.

[0204] Application of VN198 / VC198 Fluorescent Complementation Pair in Detecting the Interaction between Proteins with Important Physiological Functions

[0205] 1. Detection of the Interaction between mTOR and FKBPs

[0206] Crystal structures have confirmed that after induction by Rapamycin (RAP), mTOR interacts with FKBPs (FKBP12 and FKBP52) through its FRB domain. To further demonstrate the feasibility of the BiFC fluorescent complementation pair VN198 / VC198 screened by the present invention in detecting the mTOR / FKBP interaction, the present invention constructed pBiFC-VN198-FKBP12, pBiFC-VN198-FKBP52, and pBiFC-mTOR-VC198 expression vectors, the structures of which are as shown in the appendix Figure 7A FK506 and RAP are two new immunosuppressants widely used in the prevention of immune rejection in clinical organ transplantation and the treatment of autoimmune diseases. The main intracellular targets of FK506 and RAP are both FKBPs. FK506 binds to FKBP12 and specifically inhibits calcineurin, thereby inhibiting T cell-mediated immune responses. The binding of RAP to FKBPs promotes its binding to mTOR, inhibits the activity of mTORC1, and reduces the activation of T cells and B cells. The binding of FK506 to FKBPs competitively inhibits the binding of RAP to FKBPs and the formation of the RAP-FKBPs-mTOR complex

[0207] In HeLa, MCF7, and U2OS cells, the constructed plasmids were co-transfected according to the combinations shown in the appendix Figure 7B After transfection for a certain period of time, the cells were treated with the solvent DMSO, RAP alone, or a combination of FK506 and RAP, and the fluorescence intensity of each transfected group of cells was observed and recorded

[0208] The results showed that in the above three cell lines of different tissue origins, compared with the cells treated with the solvent, the addition of RAP significantly induced the production of fluorescent signals in the FKBP12-mTOR and FKBP52-mTOR transfected cells. If the transfected cells were treated with a combination of RAP and FK506, no or only very weak fluorescent signals were observed in the above transfected cells. The above HeLa cells were collected and the expression levels of the target proteins in each treatment group were detected by Western Blotting, as shown in the appendix Figure 7CAs shown in the figure. The results of Western Blotting showed that in the cells treated with different combinations of RAP and FK506, the expression levels of the FKBP12 and FKBP52 fusion proteins were relatively close. However, for the mTOR fusion protein, RAP treatment significantly up-regulated its expression level. But when FK506 was added simultaneously, the level of the mTOR fusion protein decreased to some extent compared with that treated with RAP alone, but was close to or still significantly higher than that of the DMSO treatment group.

[0209] In summary, when the expression plasmids of mTOR-VC198 and VN198-FKBP12 or VN198-FKBP52 were co-transfected into cells, no obvious fluorescence signal was observed. However, after adding RAP to promote their interaction, obvious fluorescence signals were observed under the fluorescence microscope. When the FK506 reagent was added simultaneously, the effect of RAP in inducing fluorescence signals was significantly weakened or not observed.

[0210] 2. Detection of the interaction between PD-1 and PD-L1

[0211] The crystal structure of the human PD-1 / PD-L1 interaction in 2015 revealed that PD-1 and PD-L1 mainly interact through their extracellular regions. To clarify whether the VN198 / VC198 fluorescence complementation pair can be used to visualize the interaction between PD-1 and PD-L1 in cells, in the present invention, the extracellular regions of PD-1 and PD-L1 were respectively fused to the N-terminus of VC198 and VN198. To promote the membrane localization ability of PD-1 and PD-L1, the corresponding sequences of PD-1 and PD-L1 were replaced with the signal peptide sequence of IgK and the transmembrane region sequence of PDGFRβ, thereby enhancing the membrane localization ability of PD-1 and PD-L1, and the corresponding expression vectors were successfully constructed. The structure of the inserted fragment is as shown in the appendix Figure 8A as shown.

[0212] The above constructed vectors were transfected into HeLa and HEK293T cells, and the immune checkpoint inhibitor Pembrolizumab targeting the interaction between PD-1 and PD-L1 and its isotype control antibody were added, and the fluorescence intensity and localization were observed. At the same time, cells transfected with only VN198 / VC198 were used as negative controls. As shown in the appendix Figure 8BAs shown, in the cells co-transfected with PD-L1-VN198 / PD-1-C198, the cells treated with the isotype control IgG antibody had obvious membrane-localized fluorescence signals. However, the membrane-localized fluorescence signals of the cells treated with Pembrolizumab were weakened, and no fluorescence signals were detected in the cells transfected with the empty vector pair of VN198 / VC198. When the PD-L1-VN198 and PD-1-VC198 expression vectors were transfected into HEK293T cells separately or co-transfected, the expression of the target proteins was detected by Western Blotting. Although the level of the target protein in the co-transfected cells was slightly lower than that in the cells transfected separately, as shown in the appendix Figure 8C shown.

[0213] The above results indicate that the PD-L1-VN198 / PD-1-VC198 complementary pair can be used to analyze the interaction between PD-1 and PD-L1 at the level of living cells.

[0214] In summary, the co-expression of the PD-L1-VN198 and PD-1-VC198 fusion proteins in cells detected obvious membrane-localized fluorescence signals. However, when the transfected cells were treated with the Pembrolizumab monoclonal antibody that inhibits their interaction, a significant decrease in the membrane-localized fluorescence signals was observed compared with the treatment with the control monoclonal antibody.

[0215] 3. Detection of the interaction between GSDMD-N and GSDMD-C

[0216] To analyze whether the VN198 / VC198 complementary pair can analyze the interaction between GSDMD-N and GSDMD-C in living cells, the present invention inactivated the cell perforation activity of GSDMD-N by deletion mutation, constructed the expression vector pBiFC-VN198-GSDMD1-250Δ96-116Δ174-204 (abbreviated as pBiFC-VN198-GSDMD-N), and simultaneously constructed the expression vector pBiFC-GSDMD251-484-VC198 (abbreviated as pBiFC-GSDMD-C-VC198). The structure of the inserted fragment is as shown in the appendix Figure 9A shown.

[0217] The above plasmid pairs or their corresponding empty vector pairs were co-transfected into HeLa cells and HEK293T cells. From the appendix Figure 9BAs shown by the results, obvious fluorescence signals were detected in the cytoplasm of cells transfected with VN198-GSDMD-N / GSDMD-C-VC198. However, no fluorescence signals were detected in cells co-transfected with the VN198 / VC198 pair. When the VN198-GSDMD-N and GSDMD-C-VC198 expression vectors were transfected into HEK293T cells separately or co-transfected, the expression of the target proteins was detected by Western Blotting. Although the level of the target protein transfected with GSDMD-C-VC198 alone was slightly lower than that in co-transfection, as shown in Appendix Figure 8C as follows.

[0218] The above results indicate that the VN198-GSDMD-N and GSDMD-C-VC198 vector pair can be used to analyze the interaction between GSDMD-N and GSDMD-C fragments at the live cell level.

[0219] As can be seen from the experiments in the above Sections 1-3, the fluorescence complementary pair of VN198 and VC198 was used to analyze the interactions between 4 pairs of protein or polypeptide fragments with important biological functions by using a cell model, including mTOR (2019-2114aa, NP_001373429) and FKBP12 (1-108aa, NP_463460) and FKBP52 (1-459aa, NP_002005), programmed cell death protein 1 (PD1, 24-170aa, NP_005009) and programmed cell death ligand 1 (PD-L1, 19-238aa, NP_054862), the N-terminal fragment (1-250Δ96-116Δ174-204, NP_079012) and C-terminal fragment (251-484) of the pyroptosis-related protein GSDMD. Specific fluorescence was detected for the interaction between each pair. For the first 3 pairs, if the interaction between them was blocked, the fluorescence signal weakened or could not be detected.

[0220] 4. Detection of aggregation between Tau proteins

[0221] The cell model co-transfected with the VN173-Tau and Tau-VC155 expression vector pair is called the Tau-BiFC cell model. It has currently been used to screen the FDA-approved Phase I clinical drug library.

[0222] The present invention first constructed pBiFC-VN173-Tau, pBiFC-Tau-VC155, pBiFC-VN198-Tau, and pBiFC-Tau-VC198 expression vectors, and the inserted sequences of the vectors are as shown in Appendix Figure 10AAs shown. The VN173-Tau / Tau-VC155, VN198-Tau / Tau-VC198 expression vector pairs and their corresponding empty vector pairs were transfected into HeLa cells respectively, and the results observed under a fluorescence microscope are as shown in the appendix Figure 10B shown.

[0223] The results showed that since VN173 / VC155 has strong self-assembly ability, there was no significant difference in the fluorescence intensity between the cells co-transfected with VN173-Tau / Tau-VC155 and those co-transfected with VN173 / VC155. Although the fluorescence signal of the former was mainly localized in the cytoplasm, while the fluorescence signal of the latter was distributed in both the cytoplasm and the nucleus. However, no obvious fluorescence signal was observed in the cells co-transfected with the VN198 / VC198 empty vector, but obvious fluorescence signals were observed in the cells co-transfected with VN198-Tau / Tau-VC198.

[0224] Forskolin (FK) and LMTX are a Tau protein aggregation inducer and inhibitor respectively. Eight hours after transfection of the above vector pairs into cells, DMSO or Forskolin was added alone to treat the cells, or the cells were co-treated with Forskolin and LMTX, and the fluorescence signals of each treated cell were observed and recorded. As shown in the appendix Figure 10B shown, obvious fluorescence signals with whole-cell localization were observed in the cells transfected with the VN173 / VC155 empty vector pair. However, under the same shooting conditions, almost no obvious fluorescence signals were observed in the cells transfected with the VN198 / VC198 empty vector pair. Although obvious fluorescence signals with cytoplasmic localization were observed after fusing Tau, and the fluorescence signal of the former was generally higher than that of the latter, the increase multiple of the latter was greater compared with the corresponding empty vector pair. After the cells were further treated with Forskolin, similar fluorescence signals were detected in both the VN173-Tau / Tau-VC155 and VN198-Tau / Tau-VC198 groups. After the cells were co-treated with LMTX and Forskolin, the degree of decrease in the fluorescence intensity of the cells with the 173 / 155 pair and 198 / 198 pair of Tau protein fusions was close compared with the group treated with FK alone. The lysates of each group of cells in the appendix were collected. After protein quantification, the relative fluorescence intensity of the lysates of each group of cells under the condition of the same total protein amount was measured by a multifunctional microplate reader, and the fluorescence intensity of the cells transfected with the Tau fusion protein group was divided by the fluorescence intensity of the cells transfected with the corresponding empty vector pair. As shown in the appendix Figure 10B Among them, the relative fluorescence intensity of each group of cell lysates was measured under the condition of the same total protein amount by a multifunctional microplate reader after protein quantification, and the fluorescence intensity of the cells transfected with the Tau fusion protein group was divided by the fluorescence intensity of the cells transfected with the corresponding empty vector pair. As shown in the appendix Figure 10CAs shown, compared with their respective empty vector pairs, the fluorescence intensity of transfected cells with Tau fusion vector pairs was significantly enhanced. However, the fluorescence intensity of the VN198 / VC198 pair increased by more than 10 times that of the VN173 / VC155 pair. After treatment with Forskolin, a significant increase in fluorescence intensity was detected only in the VN198-Tau / Tau-VC198 group. After further co-treatment with LMTX and Forskolin, although the fluorescence intensity of both the VN173 / VC155 pair and VN198 / VC198 pair fused with Tau protein decreased significantly, the latter decreased to a greater extent. Generally speaking, after removing the background fluorescence signal of the empty vector pair, the specificity and sensitivity of VN198-Tau / Tau-VC198 in detecting Tau protein aggregation and disaggregation were significantly higher than those of the VN173-Tau / Tau-VC155 group. Repeating this experiment in HEK293T cells also yielded similar experimental results, as shown in Attachment Figure 10D . * represents p<0.01. Different expression plasmid combinations as shown in Attachment Figure 10E were transfected into HEK293T cells, combined with drug treatments of FK (Forskolin) and LMTX. The results of Western Blotting indicated that the expression levels of each target protein were relatively close among different treatment groups.

[0225] In addition, the present invention expressed and purified VN198-Tau, Tau-VC198 and their empty vector proteins VN198 and VC198 in vitro, and the protein purification results are as shown in Attachment Figure 10F .

[0226] First, the present invention studied the feasibility of VN198-Tau / Tau-VC198 in detecting Tau protein aggregation and disaggregation in vitro. As shown in Attachment Figure 10G , under in vitro conditions, the fluorescence intensity of the co-incubation solution of VN198-Tau / Tau-VC198 was significantly higher than that of the VN198 / GST-VC198 pair, and the fluorescence intensity of the former showed a time-dependent increase during the 24-hour observation period. Therefore, it can be concluded that the VN198-Tau / Tau-VC198 fluorescent protein pair can analyze the Tau protein aggregation process in vitro.

[0227] Based on the above experiments, the present invention also studied whether VN198-Tau and Tau-VC198 can be used to detect the ability of LMTX to inhibit Tau protein aggregation in vitro. The recombinant fusion proteins of VN198-Tau and Tau-VC198 were co-incubated with different concentrations of LMTX (0 μM, 10 μM, 25 μM) in vitro for 0-48 hours, and the fluorescence intensity of the solution was measured every 4 hours or longer. From Attachment Figure 10HAs can be seen from the results, the fluorescence intensity of the VN198-Tau / Tau-VC198 protein pair at each detection time point gradually decreased as the LMTX concentration increased.

[0228] Therefore, the present invention draws the following conclusion: VN198-Tau / Tau-VC198 can be used to detect the Tau protein aggregation process in vitro and in vivo, and the specificity and sensitivity of VN198-Tau / Tau-VC198 in detecting Tau protein aggregation in living cells are significantly higher than those of the VN173-Tau / Tau-VC155 pair.

[0229] In summary, compared with the VN198 and VC198 complementary pair, obvious fluorescence signals can be observed when VN198-Tau and Tau-VC198 are co-expressed in cells or when the recombinant protein pair is co-incubated in solution. Forskolin promotes the fluorescence signal, and LMTX inhibits the fluorescence signal.

[0230] 5. Detection of Caspase-3 activity

[0231] The sequence Asp-Glu-Val-Asp (DEVD) recognized and cleaved by Caspase-3 was inserted into the 198th site of the Venus fluorescent protein to construct a recombinant fluorescent protein substrate VN198-DEVD-VC198 that may be used to detect Caspase-3 activity. The principle of this substrate for detecting Caspase-3 activity is as shown in the appendix Figure 11A As shown. In the absence of Caspase-3, VN198 and VC198 are structurally complementary to form a complete Venus fluorescent protein and can emit fluorescence. In the presence of Caspase-3, the DEVD sequence is recognized and cleaved by Caspase-3. The VN198 and VC198 complementary pair is separated by heating at 68°C for 5 minutes, and the Caspase-3 activity in the test cells or sample solution is analyzed by the change in fluorescence intensity before and after heating.

[0232] First, the VN198-DEVD-VC198 fusion protein was purified by in vitro prokaryotic expression and used to study the feasibility of detecting Caspase-3 activity in vitro.

[0233] VN198-DEVD-VC198 was divided into the following three groups: VN198-DEVD-VC198; VN198-DEVD-VC198 + Caspase-3; VN198-DEVD-VC198 + Caspase-3 + Ac-DEVD-CHO (Caspase-3 inhibitor), and incubated overnight at 37°C. From the appendix Figure 11BFrom the fluorescence intensity results shown, at 37 °C, there was no significant change in the fluorescence intensity among the treatment groups. However, after heating at 68 °C for 5 minutes, compared with the first and third groups above, the fluorescence intensity of the Caspase-3 treatment group was significantly reduced. As attached Figure 11C The Western blotting results shown indicate that Caspase-3 can recognize and cleave the VN198-DEVD-VC198 substrate, and the Caspase-3 inhibitor significantly inhibits the cleavage effect. These results suggest that VN198-DEVD-VC198 is feasible for in vitro analysis of Caspase-3 enzyme activity.

[0234] To prove that VN198-DEVD-VC198 can be used to detect Caspase-3 activity in living cells, the present invention transfected HeLa cells with the VN198-DEVD-VC198 expression plasmid, and then detected the relative fluorescence intensity reduction values in cells without treatment (Blank), DMSO, Eto. (etoposide), Inh. (Ac-DEVD-CHO), and co-treatment with Eto. and Inh. to evaluate the feasibility of VN198-DEVD-VC198 for analyzing Caspase-3 activity. Attached Figure 11D The results shown indicate that compared with other treatment groups, the etoposide alone treatment group significantly increased the reduction of intracellular relative fluorescence intensity at high cell numbers (8×10 4 and 10×10 4 ). The relative fluorescence intensity reduction of other treatment groups was less and there was no significant difference among them. VN198 / VC198 and VN198-DEVD-VC198 expression plasmids were transfected into HeLa cells respectively, and the latter transfected cells were further treated with DMSO or Eto. The fluorescence signals of different transfected and treated cells were observed by fluorescence microscopy. From the attached Figure 11E results shown, it can be seen that compared with the cells in the DMSO addition group, the fluorescence signal of VN198-DEVD-VC198 cells in the etoposide addition group was significantly reduced, but no obvious fluorescence signal was detected in the cells transfected with the VN198 / VC198 empty vector pair. By flow cytometry to quantitatively analyze the fluorescence intensity of the above VN198-DEVD-VC198 expression plasmid transfection group, it was found that the fluorescence signal of VN198-DEVD-VC198 cells treated with etoposide was significantly lower than that treated with DMSO, as attached Figure 11F shown. Similarly, the remaining cells used for flow analysis above were detected for the level of VN198-DEVD-VC198 fusion protein, as attached Figure 11GAs shown, the Western Blotting results indicate that compared with the DMSO-treated group, the level of VN198-DEVD-VC198 fusion protein in the etoposide-treated group is significantly reduced. Therefore, it can be determined that VN198-DEVD-VC198 can be used to detect Caspase-3 activity in living cells. Similarly, in HeLa and HEK293T cells transfected with GFP or VN198-DEVD-VC198, they were treated with DMSO, Eto., and Eto. + Inh. respectively, and the changes in fluorescence intensity of the cell lysates of these different treatments were further detected. As attached Figure 11H (HeLa) and 11I (HEK293T) shown, only in VN198-DEVD-VC198 transfected cells, it was observed that compared with the DMSO treatment, the fluorescence intensity of Eto.-treated cells was significantly reduced, but the fluorescence intensity of Eto. + Inh.-co-treated cells had a significant recovery to varying degrees.

[0235] Further study whether the purified VN198-DEVD-VC198 recombinant protein can be used to detect Caspase-3 activity in vitro.

[0236] First, the linear range of the fusion protein VN198-DEVD-VC198 was determined in this invention. The results show that there is a good linear relationship between the substrate concentration in the range of 0 - 15 μg / mL and the relative fluorescence intensity, as attached Figure 11J shown. To verify that VN198-DEVD-VC198 is a good substrate for detecting Caspase-3 activity in vitro, at 37 °C, different concentrations of VN198-DEVD-VC198 recombinant protein were co-incubated with recombinant Caspase-3 for 2 hours, and the substrate without adding Caspase-3 enzyme was used as a control. After the incubation, the fluorescence intensity of each solution was detected, and the fluorescence intensity of each solution was detected again after heating at 68 °C for 5 minutes, and the reduction value of the fluorescence intensity before and after heating of each solution was calculated. The results show that regardless of whether Caspase-3 is added, there is a good linear correlation between the reduction value of the fluorescence intensity before and after heating and the substrate concentration, but the slope of the straight line in the enzyme-added group is significantly higher than that in the non-enzyme-added group, indicating that this substrate can be used to distinguish different Caspase-3 enzyme activity levels. The specific results are as attached Figure 11K shown.

[0237] In addition, the present invention analyzed the kinetic curve of Caspase-3 cleaving VN198-DEVD-VC198. Caspase-3 (50 ng / mL) at a certain concentration was reacted with VN198-DEVD-VC198 (500 ng / mL) at 37 °C for different times (0 - 5.5 hours). The results showed that as the detection time extended, the decrease in the fluorescence intensity of the sample continuously increased and reached the enzyme reaction plateau after 4 hours. As shown in the appendix Figure 11L It indicates that the cleavage of the substrate VN198-DEVD-VC198 at the current concentration by Caspase-3 at the current concentration takes about 4 hours to complete.

[0238] To study the dose-response relationship between the substrate VN198-DEVD-VC198 and the amount of Caspase-3 enzyme, the present invention analyzed the relationship between enzyme dose and fluorescence intensity using a substrate VN198-DEVD-VC198 at a fixed concentration and different concentrations of Caspase-3. As shown in the appendix Figure 11M It indicates that under the condition of low concentration Caspase-3 (5 - 150 ng / mL), there was a significant correlation between the decrease in fluorescence intensity and the Caspase-3 dose. On this basis, the present invention further analyzed the Caspase-3 activity in cell samples treated with different Eto. The results are shown in the appendix Figure 11N It indicates that the substrate VN198-DEVD-VC198 can be used to analyze the Caspase-3 enzyme activity level in cell samples in vitro.

[0239] In summary, the substrate VN198-DEVD-VC198 constructed based on the VN198 / VC198 fluorescence complementation pair and recognizable and cleavable by Caspase-3 can better analyze the enzyme activity of Caspase 3 in samples inside and outside cells.

[0240] Example 3 Screening of Other Fluorescent Protein Fluorescence Complementation Pairs

[0241] Referring to the cleavage sites of VN198 and VC198 in the Venus fluorescent protein, the present invention further studied the feasibility of fluorescence complementation pairs with similar cleavage sites to the Venus fluorescent protein in other fluorescent proteins with similar barrel structures for BiFC analysis. These fluorescent proteins include the cyan fluorescent protein mTurquoise2 also derived from Aequorea Victoria, the green fluorescent protein mNeonGreen derived from Branchiostoma lanceolatum, and the red fluorescent protein mCherry derived from Discosoma sp.

[0242] First, the present invention constructed a vector expressing the amino acid fragment at positions 1-196 of the N-terminus of mCherry (abbreviated as CN196) and its vector expressing CN196-bJun. Meanwhile, a vector expressing the amino acid fragment at positions 189-236 of the C-terminus of mCherry (abbreviated as CC189) was also constructed. Similarly, vectors expressing CC192 and CC196 were constructed, as well as the corresponding expression vectors constructed by fusing the N-terminus with bFos or bFosΔzip fragments respectively.

[0243] Subsequently, the present invention co-transfected HEK293T and HeLa cells with the vectors expressing CN196-bJun and bFos-CCs or bFosΔzip-CCs (including CC189, CC192 or CC196) or their corresponding empty vector pairs in the combinations shown in Appendix Figure 12A and Appendix Figure 12B and observed their fluorescence signals through a fluorescence microscope.

[0244] Using the same strategy, the present invention also constructed corresponding complementary pairs based on the crystal structures of mNeonGreen (Appendix Figure 12C and Appendix Figure 12D ) and mTurquoise2 (Appendix Figure 12E and Appendix Figure 12F ) and co-transfected HEK293T and HeLa cells.

[0245] The results showed that the fluorescence signals of the above three fluorescent proteins all presented a relatively consistent trend, that is, no obvious fluorescence signal was observed under the fluorescence microscope after the empty vector pairs without fused interacting proteins were transfected into cells. The fluorescence signal of the cells transfected with the expression vector pairs carrying bJun and bFos fusion fragments respectively was significantly stronger than that carrying bJun and bFosΔzip fusion fragments. As shown in Appendix Figure 12G , cell lysates were collected and total protein quantification was performed. By measuring and calculating the ratios of the fluorescence intensities between the bJun / bFos interactome and the empty vector group, and between the bJun / bFos interactome and the non-interacting bJun / bFosΔzip group under the conditions of the same total protein amount and calibration with the internal reference fluorescent proteins (mCherry, mNeonGreen and mTurquoise 2), the feasibility of these fluorescent protein complementary pairs for detecting protein interactions was further evaluated.

[0246] The above results indicate that the BiFC fluorescent complementary pairs generated by cleavage within the Loop between the 9th and 10th β-strands of the barrel-shaped fluorescent proteins have good specificity and sensitivity and can be used to analyze the interactions between proteins or protein fragments.

[0247] In summary, referring to the cleavage sites of the VN198 and VC198 complementary pairs, among the fluorescent proteins with the same species origin (cyan fluorescent protein mTurquiose2, derived from Aequorea victoria) and different biological origins (green fluorescent protein mNeonGreen, derived from Branchiostoma lanceolatum; red fluorescent protein mCherry, derived from Discosoma sp.), the complementary pairs generated by cleavage within the Loop between the 9th and 10th β-strands all show good specificity and sensitivity when used for BiFC analysis. However, the fluorescence signals of the mNeonGreen and mCherry complementary pairs are significantly weaker than those of the Venus and mTurquoise2 pairs.

[0248] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention, and they should all be covered within the scope of the claims of the present invention.

Claims

1. A BiFC fluorescence complementation pair or recombinant fluorescent protein, characterized in that: It is generated by the cleavage of the fluorescent protein within the loop between the 9th and 10th β-strands, or by inserting a Caspase-3 recognition sequence within the loop between the 9th and 10th β-strands of the fluorescent protein; Preferably, the fluorescent protein is selected from yellow fluorescent protein Venus, cyan fluorescent protein mTurquiose2, green fluorescent protein mNeonGreen, and red fluorescent protein mCherry; More preferably, the yellow fluorescent protein Venus and cyan fluorescent protein mTurquiose2 are derived from Aequorea victoria, the green fluorescent protein mNeonGreen is derived from Branchiostoma lanceolatum, and the red fluorescent protein mCherry is derived from Discosoma sp.

2. The BiFC fluorescent complementary pair or recombinant fluorescent protein according to claim 1, which is selected from: VN198 / VC198, VN198 / VC189, VN198 / VC194, CN196 / CC196, CN196 / CC189, CN196 / CC192, NN200 / NC201, NN200 / NC188, NN200 / NC192, TN199 / TC200, TN199 / TC189, TN199 / TC194, VN198-DEVD-VC198, wherein, VN198 encodes amino acids 1-198 of green fluorescent protein Venus, and VC198 encodes amino acids 198-239 of green fluorescent protein Venus, VN198 encodes amino acids 1-198 of green fluorescent protein Venus, and VC189 encodes amino acids 189-239 of green fluorescent protein Venus, VN198 encodes amino acids 1-198 of green fluorescent protein Venus, and VC194 encodes amino acids 194-239 of green fluorescent protein Venus, CN196 encodes amino acids 1-196 of red fluorescent protein mCherry, and CC196 encodes amino acids 196-236 of red fluorescent protein mCherry, CN196 encodes amino acids 1-196 of red fluorescent protein mCherry, and CC189 encodes amino acids 189-236 of red fluorescent protein mCherry, CN196 encodes amino acids 1-196 of red fluorescent protein mCherry, and CC192 encodes amino acids 192-236 of red fluorescent protein mCherry, NN200 encodes amino acids 1-200 of green fluorescent protein mNeonGreen, and NC201 encodes amino acids 201-236 of green fluorescent protein mNeonGreen, NN200 encodes amino acids 1-200 of green fluorescent protein mNeonGreen, and NC188 encodes amino acids 188-236 of green fluorescent protein mNeonGreen, NN200 encodes the first to 200th amino acids of the green fluorescent protein mNeonGreen, and NC192 encodes the 192nd to 236th amino acids of the green fluorescent protein mNeonGreen. TN199 encodes the first to 199th amino acids of the cyan fluorescent protein mTurquiose2, and TC200 encodes the 200th to 239th amino acids of the cyan fluorescent protein mTurquiose2. TN199 encodes the first to 199th amino acids of the cyan fluorescent protein mTurquiose2, and TC189 encodes the 189th to 239th amino acids of the cyan fluorescent protein mTurquiose2. TN199 encodes the first to 199th amino acids of the cyan fluorescent protein mTurquiose2, and TC194 encodes the 194th to 239th amino acids of the cyan fluorescent protein mTurquiose2. VN198-DEVD-VC198 is generated by inserting the Caspase-3 recognition sequence DEVD at the 198th amino acid of the yellow fluorescent protein Venus.

3. An expression vector, characterized in that, It comprises the BiFC fluorescent complementation pair or recombinant fluorescent protein described in claim 1 or 2. Preferably, the expression vector is selected from pBiFC-VN198, pBiFC-VC198, BiFC-VN198-bJun, pBiFC-VC198, pBiFC-bFos-VC198, pBiFC-bFosΔzip-VC198, pBiFC-bFos-VC189, pBiFC-bFosΔzip-VC189, pBiFC-bFos-VC194, pBiFC-bFosΔzip-VC194, pBiFC-VN198-FKBP12, pBiFC-VN198-FKBP52, pBiFC-mTOR-VC198, pBiFC-PD-L1-VN198, pBiFC-PD-1-VC198, pBiFC-VN198-GSDMD-N, pBiFC-GSDMD-C-VC198, pBiFC-VN198-Tau, pBiFC-Tau-VC198, pBiFC-CN196, pBiFC-CC189, pBiFC-CC192, pBiFC-CC196, pBiFC-CN196-bJun, pBiFC-bFos-CC189, pBiFC-bFos-CC192, pBiFC-bFos-CC196, pBiFC-bFosΔzip-CC189, pBiFC-bFosΔzip-CC192, pBiFC-bFosΔzip-CC196, pBiFC-NN200, pBiFC-NC188, pBiFC-NC192, pBiFC-NC201, pBiFC-NN200-bJun, pBiFC-bFos-NC188, pBiFC-bFos-NC192, pBiFC-bFos-NC201, pBiFC-bFosΔzip-NC188, pBiFC-bFosΔzip-NC192, pBiFC-bFosΔzip-NC201, pBiFC-TN199, pBiFC-TC189, pBiFC-TC194, pBiFC-TC200, pBiFC-TN199-bJun, pBiFC-bFos-TC189, pBiFC-bFos-TC194, pBiFC-bFos-TC200, pBiFC-bFosΔzip-TC189, pBiFC-bFosΔzip-TC194, pBiFC-bFosΔzip-TC200, pET-24b(+)-VN198-Tau, pET-24b(+)-Tau-VC198, pET-24b(+)-VN198, pET-42a(+)-GST-VC198, pcDNA3.1-VN198-DEVD-VC198, pET-24b(+)-VN198-DEVD-VC198.

4. A cell, characterized in that, It comprises the BiFC fluorescent complementation pair or recombinant fluorescent protein described in claim 1 or 2, or the expression vector described in claim 3. Preferably, the cells are selected from human embryonic kidney cells HEK293T, human breast cancer cells MCF7, human osteosarcoma cells U2OS, and human cervical cancer cells HeLa.

5. A system, characterized in that, It comprises the BiFC fluorescent complementation pair or recombinant fluorescent protein described in claim 1 or 2, the expression vector described in claim 3, or the cells described in claim 4.

6. A kit, characterized in that, It comprises the BiFC fluorescent complementation pair or recombinant fluorescent protein described in claim 1 or 2, the expression vector described in claim 3, the cells described in claim 4, or the system described in claim 5.

7. A method for detecting the interaction between proteins, between polypeptide fragments, or between a protein and a polypeptide fragment, characterized in that, During the detection process, it uses the BiFC fluorescent complementation pair or recombinant fluorescent protein described in claim 1 or 2, the expression vector described in claim 3, the cells described in claim 4, the system described in claim 5, or the kit described in claim 6. Preferably, the protein or polypeptide fragment is selected from mTOR and FKBPs, programmed cell death protein PD-1 and programmed cell death ligand PD-L1, pyroptosis-related proteins GSDMD-N and GSDMD-C fragments, Tau protein, or Caspase-3 and the recombinant fluorescent protein containing its recognition sequence. More preferably, FKBPs are selected from FKBP12 or FKBP52.

8. A method for screening an enhancer or inhibitor that promotes or inhibits the interaction between proteins, between polypeptide fragments, or between a protein and a polypeptide fragment, characterized in that, During the screening process, it uses the BiFC fluorescent complementation pair or recombinant fluorescent protein described in claim 1 or 2, the expression vector described in claim 3, the cells described in claim 4, the system described in claim 5, or the kit described in claim 6. Preferably, the protein or polypeptide fragment is selected from mTOR and FKBPs, programmed cell death protein PD-1 and programmed cell death protein ligand PD-L1, pyroptosis-related proteins GSDMD-N and GSDMD-C, Tau protein, or Caspase-3 and a recombinant fluorescent protein containing its recognition sequence, More preferably, FKBPs are selected from FKBP12 or FKBP52.

9. A method for detecting Caspase-3 activity, characterized in that, During the detection process, the BiFC fluorescence complementation pair or recombinant fluorescent protein described in claim 1 or 2, the expression vector described in claim 3, the cell described in claim 4, the system described in claim 5, or the kit described in claim 6 is used.

10. A method for screening candidate proteins capable of interacting with a target protein from a candidate protein library, characterized in that, During the screening process, the BiFC fluorescence complementation pair or recombinant fluorescent protein described in claim 1 or 2, the expression vector described in claim 3, the cell described in claim 4, the system described in claim 5, or the kit described in claim 6 is used. Preferably, the candidate protein library or target protein contains mTOR and FKBPs, programmed cell death protein PD-1 and programmed cell death protein ligand PD-L1, pyroptosis-related proteins GSDMD-N and GSDMD-C, Tau protein, or Caspase-3 and a recombinant fluorescent protein containing its recognition sequence, More preferably, FKBPs are selected from FKBP12 or FKBP52.

11. Use of the BiFC fluorescence complementation pair or recombinant fluorescent protein described in claim 1 or 2, the expression vector described in claim 3, the cell described in claim 4, the system described in claim 5, or the kit described in claim 6 in detecting the interaction between proteins, polypeptide fragments, or between a protein and a polypeptide fragment, and in detecting Caspase-3 activity. Preferably, the protein or polypeptide fragment is selected from mTOR and FKBPs, programmed cell death protein PD-1 and programmed cell death protein ligand PD-L1, pyroptosis-related proteins GSDMD-N and GSDMD-C, Tau protein, or Caspase-3 and a recombinant fluorescent protein containing its recognition sequence, More preferably, FKBPs are selected from FKBP12 or FKBP52.

12. Use of the BiFC fluorescence complementation pair or recombinant fluorescent protein described in claim 1 or 2, the expression vector described in claim 3, the cell described in claim 4, the system described in claim 5, or the kit described in claim 6 in screening for promoters or inhibitors that promote or inhibit the interaction between proteins, polypeptide fragments, or between a protein and a polypeptide fragment. Preferably, the protein or polypeptide fragment is selected from mTOR and FKBPs, programmed cell death protein PD-1 and programmed cell death protein ligand PD-L1, pyroptosis-related proteins GSDMD-N and GSDMD-C, Tau protein, or Caspase-3 and a recombinant fluorescent protein containing its recognition sequence, More preferably, the FKBPs are selected from FKBP12 or FKBP52.

13. Use of the BiFC fluorescence complementary pair or fluorescent protein according to claim 1 or 2, the expression vector according to claim 3, the cell according to claim 4, the system according to claim 5, or the kit according to claim 6 in detecting or screening Caspase-3 activity.

14. Use of the BiFC fluorescence complementary pair or recombinant fluorescent protein according to claim 1 or 2, the expression vector according to claim 3, the cell according to claim 4, the system according to claim 5, or the kit according to claim 6 in screening candidate proteins capable of interacting with a target protein from a candidate protein library. Preferably, the candidate protein library or target protein comprises mTOR and FKBPs, programmed cell death protein PD-1 and programmed cell death ligand PD-L1, N-terminal and C-terminal fragments of pyroptosis-related protein GSDMD, Tau protein, or Caspase-3 and a recombinant fluorescent protein comprising its recognition sequence. More preferably, the FKBPs are selected from FKBP12 or FKBP52.

15. Use of the BiFC fluorescence complementation pair or recombinant fluorescent protein according to claim 1 or 2, the expression vector according to claim 3, the cell according to claim 4, the system according to claim 5 or the kit according to claim 6 in drug screening or drug evaluation, wherein, The drug screening or drug evaluation involves interactions between proteins, between polypeptide fragments, or between a protein and a polypeptide fragment. Preferably, the protein or polypeptide fragment is selected from mTOR and FKBPs, programmed cell death protein PD-1 and programmed cell death ligand PD-L1, pyroptosis-related protein GSDMD-N and GSDMD-C, Tau protein, or Caspase-3 and a recombinant fluorescent protein comprising its recognition sequence. More preferably, the FKBPs are selected from FKBP12 or FKBP52.

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