BiFC fluorescence complementation pair and application thereof

Abstract

The application provides a new BiFC fluorescence complementation pair or recombinant fluorescent protein and application thereof.The fluorescence complementation pair or recombinant fluorescent protein has lower self-assembly capability than the VN173 / VC155 fluorescence complementation pair, has significantly reduced background fluorescence signal, improves specificity of BiFC analysis, and has stronger fluorescence signal than the VN210 / VC210 fluorescence complementation pair, has significantly increased fluorescence signal, and improves sensitivity of BiFC analysis.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of protein detection, and particularly relates to a BiFC fluorescence complementation pair and application thereof. BACKGROUND

[0002] The network of signal pathways based on protein-protein interactions (PPIs) plays a very important role in many 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. In order to solve this problem, several visualization techniques for protein-protein interactions in living cells based on different principles have been developed and applied, of which the most representative two are fluorescence resonance energy transfer (FRET) technology and bimolecule fluorescence complementation (BiFC) technology.

[0003] BiFC is a technology based on protein complementation assay (PCA) for qualitatively or quantitatively analyzing the interaction and subcellular localization of proteins or polypeptide fragments in living cells or extracellular solution by using fluorescence microscope and fluorescence spectrometer, etc. It has the advantages of high flexibility, rapid and intuitive, wide application range, etc. This technology ingeniously breaks the fluorescent protein molecule at the appropriate position to produce two complementary fragments that do not emit fluorescence, and then the two complementary fragments are fused with the target protein for expression. 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 will be close to each other in space, and the complete fluorescent protein molecule with activity will be restored, thereby producing fluorescence under excitation. The fluorescence signal generated in this process can be detected by conventional instruments such as fluorescence microscope and fluorescence spectrometer, and therefore it is widely used in the research of protein interaction and drug screening.

[0004] At present, the commonly used BiFC fluorescence complementary pair at home and abroad is based on VN173 (encoding the 1-173th amino acid) and VC155 (encoding the 156-239th amino acid) of Venus fluorescent protein from Aequorea Victoria, but the co-expression of the complementary pair in cells produces a strong fluorescence signal, reduces the specificity of protein interaction analysis, and easily leads to false positive results.

[0005] Although the non-specificity of VN210 (encoding the 1-211th amino acid) and VC210 (encoding the 211-238th amino acid) fluorescence complementary pair in analyzing protein interaction is significantly reduced in 2012 by Mizuno et al., the fluorescence signal of the interaction polypeptide fragments after fusion is also weak, which affects the sensitivity of the analysis.

[0006] Therefore, how to optimize the design of the BiFC fluorescence complementary pair, both reducing the false positive produced by them in detecting protein interaction and improving the sensitivity in detection, is the focus of the present application. SUMMARY

[0007] The purpose of the present application is to provide a BiFC fluorescence complementary pair and its application, which can both reduce the false positive produced by them in detecting protein interaction and improve the sensitivity in detection.

[0008] The present application obtains a new VN198 fragment (encoding the 1-198th amino acid) by shortening the length of the VN210 fragment from the C-terminal of the Venus fluorescent protein, and at the same time, lengthening the length of the VC210 fragment to the N-terminal, so that the fragment includes the 10th and 11th beta folds of the Venus fluorescent protein, obtaining a VC198 fragment (encoding the 198-239th amino acid), and further obtaining a new BiFC fluorescence complementary pair VN198 / VC198. On this basis, the present application proves the superiority of the fluorescence complementary pair VN198 / VC198 in detecting the interaction between proteins by using the fluorescence complementary pair VN198 / VC198 to detect the interaction between proteins.

[0009] Therefore, the present application provides a BiFC fluorescence complementary pair or a recombinant fluorescent protein, which is produced by breaking the loop between the 9th and 10th beta folds of the fluorescent protein or inserting a Caspase-3 recognition sequence into the loop between the 9th and 10th beta folds of the fluorescent protein.

[0010] Preferably, the fluorescent protein is selected from the yellow fluorescent protein Venus, the cyan fluorescent protein mTurquiose2, the green fluorescent protein mNeonGreen and the 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 application, the BiFC fluorescence complementation pair or the recombinant fluorescent protein is selected from the group consisting of:

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

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

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

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

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

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

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

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

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

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

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

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

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

[0027] In another aspect, the present application provides an expression vector comprising the BiFC fluorescence complementation pair or recombinant fluorescent protein of the present application.

[0028] Preferably, the expression vector is selected from the group consisting of 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] In another aspect, the present application provides a cell comprising the BiFC fluorescence complementation pair or the recombinant fluorescent protein according to the present application, or the expression vector according to the present application.

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

[0031] In another aspect, the present application provides a system comprising the BiFC fluorescence complementation pair or the recombinant fluorescent protein according to the present application, the expression vector according to the present application, or the cell according to the present application.

[0032] In another aspect, the present application provides a kit comprising the BiFC fluorescence complementation pair or the recombinant fluorescent protein according to the present application, the expression vector according to the present application, the cell according to the present application, or the system according to the present application.

[0033] In another aspect, the present application provides a method for detecting the interaction between proteins, between polypeptide fragments, or between a protein and a polypeptide fragment, wherein the BiFC fluorescence complementation pair or the recombinant fluorescent protein according to the present application, the expression vector according to the present application, the cell according to the present application, the system according to the present application, or the kit according to the present application is used in the detection process.

[0034] Preferably, the protein or polypeptide fragment is selected from the group consisting of mTOR and FKBPs, programmed cell death protein PD-1 and programmed cell death protein 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 the recombinant fluorescent protein comprising the recognition sequence thereof,

[0035] More preferably, the FKBPs are selected from the group consisting of FKBP12 and FKBP52.

[0036] In another aspect, the present application provides a method for screening the promoter or inhibitor for promoting or inhibiting the interaction between proteins, between polypeptide fragments, or between a protein and a polypeptide fragment, wherein the BiFC fluorescence complementation pair or the recombinant fluorescent protein according to the present application, the expression vector according to the present application, the cell according to the present application, the system according to the present application, or the kit according to the present application is used in 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 recombinant fluorescent protein containing its recognition sequence.

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

[0039] Another aspect of the present application provides a method for detecting Caspase-3 activity, which uses the BiFC fluorescence complementation pair or recombinant fluorescent protein of the present application, the expression vector of the present application, the cell of the present application, the system of the present application, or the kit of the present application in the detection process.

[0040] Another aspect of the present application provides a method for screening candidate proteins capable of interacting with target proteins from a candidate protein library, which uses the BiFC fluorescence complementation pair or recombinant fluorescent protein of the present application, the expression vector of the present application, the cell of the present application, the system of the present application, or the kit of the present application in 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 recombinant fluorescent protein containing its recognition sequence.

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

[0043] Another aspect of the present application provides the BiFC fluorescence complementation pair or recombinant fluorescent protein of the present application, the expression vector of the present application, the cell of the present application, the system of the present application, or the kit of the present application for use in detecting the interaction between proteins, polypeptide fragments, or proteins and polypeptide fragments, or in analyzing Caspase-3 activity.

[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 recombinant fluorescent protein containing its recognition sequence.

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

[0046] In another aspect, the present application provides use of the BiFC fluorescence complementation pair or the recombinant fluorescent protein, the expression vector, the cell, the system or the kit of the present application in screening a promoter or an inhibitor of interaction between proteins, between polypeptide fragments, or between a protein and a polypeptide fragment.

[0047] Preferably, the proteins or polypeptide fragments are 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 comprising a recognition sequence thereof.

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

[0049] In another aspect, the present application provides use of the BiFC fluorescence complementation pair or the recombinant fluorescent protein, the expression vector, the cell, the system or the kit of the present application in detecting Caspase-3 activity.

[0050] In another aspect, the present application provides use of the BiFC fluorescence complementation pair or the recombinant fluorescent protein, the expression vector, the cell, the system or the kit of the present application in screening a candidate protein capable of interacting with a target protein from a candidate protein library.

[0051] Preferably, the candidate protein library or the target protein comprises 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 comprising a recognition sequence thereof.

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

[0053] In another aspect, the present application provides use of the BiFC fluorescence complementation pair or the recombinant fluorescent protein, the expression vector, the cell, the system or the kit of the present application in drug screening or drug evaluation, wherein the drug screening or drug evaluation involves interaction between proteins, between 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 proteins GSDMD-N and GSDMD-C, Tau protein, or Caspase-3 and 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 application has the following beneficial effects:

[0057] The fluorescent complementation pair or recombinant fluorescent protein screened by the present application, especially the fluorescent complementation pair VN198 / VC198, has lower self-assembly ability than the fluorescent complementation pair VN173 / VC155, has significantly reduced background fluorescence signal, and improves the specificity of BiFC analysis. At the same time, the fluorescent signal of the fusion interaction protein or fragment thereof is stronger than that of the fusion VN210 / VC210 fluorescent complementation pair, has significantly increased fluorescence signal, and improves the sensitivity of BiFC analysis. BRIEF DESCRIPTION OF DRAWINGS

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

[0059] Figure 1B is a loop structure diagram between the 9th and 10th beta folds and between the 9th and 10th beta folds of yellow fluorescent protein Venus;

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

[0061] Figure 1D is a loop structure diagram between the 9th and 10th beta folds and between the 9th and 10th beta folds of cyan fluorescent protein mTurquiose2;

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

[0063] Figure 1F is a loop structure diagram between the 9th and 10th beta folds and between the 9th and 10th beta folds of green fluorescent protein mNeonGreen;

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

[0065] Figure 1HStructure diagram of the 9th to 11th beta sheet of red fluorescent protein mCherry and the loop structure between the 9th and 10th beta sheet.

[0066] Figure 2A Structure diagram of pBiFC-VN173 and pBiFC-VC155 carrier;

[0067] Figure 2B Brief structure diagram of VN173 and VC155.

[0068] Figure 2C Structure diagram of pBiFC-VN210 and pBiFC-VC210 carrier;

[0069] Figure 2D Brief structure diagram of VN210 and VC210.

[0070] Figure 3A Structure diagram of pBiFC-VN198 and pBiFC-VC198 carrier;

[0071] Figure 3B Screening site diagram of new and old bimolecular fluorescence complementation pairs;

[0072] Figure 3C Brief structure diagram of VN198 and VC198.

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

[0074] Figure 4B Figure 4A After collecting the lysate of MCF7 cells after fluorescence imaging, the proteins in the cell lysate were separated by SDS-PAGE, and the Western blotting diagram of the target protein was detected by Flag antibody and HA antibody with β-actin as an internal reference, and the arrow indicates the target protein in each lane.

[0075] Figure 5A After about 24 hours, the results were observed using an inverted fluorescence microscope (the picture scale is 50 μm) after transfecting each plasmid or plasmid combination shown in the figure in HeLa, MCF7 and U2OS cells at a transfection dose of 3.0 μg / mL of each plasmid.

[0076] Figure 5B After collecting the lysate of MCF7 cells after fluorescence imaging, the proteins in the cell lysate were separated by SDS-PAGE, and the Western blotting diagram of the target protein was detected by Flag antibody and HA antibody with β-actin as an internal reference, and the arrow indicates the target protein in each lane. Figure 5A ​Cells were observed under fluorescence and lysed, total protein was extracted, Western Blotting was used to detect the expression of target protein in each transfected cell, with β-actin as internal reference, and the Western Blotting diagram was drawn;

[0077] Figure 5C HeLa cells were seeded in black transparent bottom 96-well cell culture plates, transfected with the plasmid combinations shown in the figure (the transfection dose of mCherry was 0.02 μg / mL, and the transfection dose of each other plasmid was 0.5 μg / mL), and the result map was observed using an inverted fluorescence microscope about 24 hours later (the picture scale was 50 μm);

[0078] Figure 5D To measure the Venus and mCherry fluorescence intensity of HeLa cell lysate using a multifunctional microwell plate detector, a column chart was drawn, and the ratio of the fluorescence intensity of each group interaction to the corresponding non-interaction group was calculated, where J represents bJun and F represents bFos. Figure 5C

[0079] To transfect the corresponding plasmid combinations into MCF7, HeLa and U2OS cells at a dose of 0.5 μg / mL per plasmid, and the result map was observed using an inverted fluorescence microscope about 24 hours later (the picture scale was 50 μm); Figure 6A

[0080] To transfect the plasmid combinations shown in the figure into HeLa cells at a transfection dose of 0.5 μg / mL per plasmid, and about 9 hours after adding 20 μM MG132 or the same volume of DMSO according to the figure, the result map was observed using an inverted fluorescence microscope (the picture scale was 50 μm); Figure 6B

[0081] To collect Figure 6C Cells were observed under fluorescence and lysed, total protein was extracted, Western Blotting was used to detect the expression of target protein in each transfected cell, with β-actin as internal reference, and the Western Blotting diagram was drawn; Figure 6B

[0082] To transfect the corresponding plasmid combinations into HeLa cells at a gradient dose (the figure shows the individual transfection dose of each plasmid), and the result map was observed using an inverted fluorescence microscope about 24 hours later (the picture scale was 50 μm). Figure 6D

[0083] Structure diagram of VN198-FKBP12, VN198-FKBP52 and mTOR-VC198 expression vectors; Figure 7A

[0084] Structure diagram of VN198-FKBP12, VN198-FKBP52 and mTOR-VC198 expression vectors;Figure 7B HeLa, MCF7 and U2OS cells were transfected with each plasmid at a dose of 0.5 μg / mL, respectively, and about 24 hours later, 50 μM FK506 or an equal volume of DMSO was added to the transfected cells as shown in the figure, about 1 hour later, 1 nM Rapamycin (RAP) or an equal volume of DMSO was added for treatment, and about 24 hours later, the results were observed using an inverted fluorescence microscope (the picture scale is 50 μm);

[0085] Figure 7C To collect Figure 7B After observing the fluorescence of HeLa cells, total cell protein was extracted, β-actin was used as an internal reference, and Western Blotting was used to detect the protein expression of the target protein in each transfected group.

[0086] Figure 8A For PD-L1-VN198 and PD-1-VC198 expression vector structure diagram;

[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 expression vectors for VN198 / VC198 and PD-L1-VN198 / PD-1-VC198, respectively, 8 hours after transfection, 20 μM of PD-1 antibody (Pembrolizumab) or an equal dose of control antibody was added as shown, and about 16 hours after treatment, the results were observed using an inverted fluorescence microscope (the picture scale is 25 μm);

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

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

[0092] Figure 9A For VN198-GSDMD-N and GSDMD-C-VC198 expression vector structure diagram;

[0093] Figure 9BThe results of the inverted fluorescence microscope observation after about 24 hours of transfection of the VN198 and VC198 expression vector pair, or the VN198-GSDMD-N and GSDMD-C-VC198 expression vector pair, in HEK293T and HeLa cells are shown in the figure (the picture scale is 25 μm);

[0094] Figure 9C The Western Blotting detection of the expression of the target proteins in each transfection group by Western Blotting using β-actin as an internal reference after 24 hours of transfection of different combinations of VN198-GSDMD-N and GSDMD-C-VC198 expression plasmids in HEK293T cells is shown in the figure.

[0095] Figure 10A The structural diagram of the BiFC-Tau expression vector (VN173-Tau, Tau-VC155, VN198-Tau and Tau-VC198) is shown in the figure.

[0096] Figure 10B The results of the fluorescence microscope imaging observation after 8 hours of transfection of the BiFC-Tau expression vector pair or its corresponding BiFC empty vector pair in HeLa cells are shown in the figure (the picture scale is 50 μm), wherein the control solvent, Forskolin (30 μM), or Forskolin (30 μM) and LMTX (5 μM) were added to the transfected cells for 36 hours of co-treatment according to the figure.

[0097] Figure 10C The schematic diagram of collecting the cell lysate in Figure 10B and performing protein quantification to calculate the relative fluorescence intensity of each group (fluorescence intensity of the BiFC-Tau complementary pair group / the corresponding BiFC empty vector pair group) is shown in the figure.

[0098] Figure 10B The schematic diagram of repeating the Figure 10C experiment in HEK293T cells is shown in the figure.

[0099] RFU is Relative Fluorescence Units;

[0100] The error bar represents the standard deviation measured from three repeated holes;

[0101] * represents p≤0.01;

[0102] Figure 10D The Western Blotting detection of the expression of the target proteins in each transfection group by Western Blotting using β-actin as an internal reference after 24 hours of transfection of various Tau fusion protein expression plasmids in HEK293T cells is shown in the figure.

[0103] Figure 10E Figure 4 is a Coomassie blue staining result of four purified proteins, VN198-Tau, Tau-VC198, VN198, and GST-VC198;

[0104] Figure 10F Figure 5 is a plot of the fluorescence intensity of the purified protein pairs (VN198 / GST-VC198, VN198-Tau / Tau-VC198) incubated at room temperature, and measured every 3 hours;

[0105] * represents p<0.0001;

[0106] Figure 10G Figure 6 is a plot of the fluorescence intensity of the purified protein complementary pair VN198-Tau / Tau-VC198 incubated with different concentrations of LMTX (0, 10, 25 μM) at room temperature, and measured at different time points.

[0107] Figure 10H Figure 7 is a schematic diagram of the detection of Caspase-3 activity by VN198-DEVD-VC198;

[0108] Figure 11A Figure 8 is a plot of the fluorescence intensity of the purified substrate recombinant protein VN198-DEVD-VC198 (1.0 ng / μl) incubated at 37°C for 18 hours, and then heated at 68°C for 5 minutes, and then measured again. The protein was divided into three groups: no Caspase-3 treatment, Caspase-3 (0.2 ng / μl) treatment, and Caspase-3 (0.2 ng / μl) and Caspase-3 inhibitor (Ac-DEVD-CHO, 20.0 μM) co-treatment.

[0109] Figure 11B Figure 9 is a Western blotting plot of the detection of the expression level of the substrate protein by Caspase-3 using Western blotting;

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

[0111] Inh. represents 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 11C HeLa cells were treated with different density of untreated, or DMSO, Etoposide, Caspase-3 inhibitor, Etoposide combined with Caspase-3 inhibitor, respectively. After 48 hours, HeLa cells were collected and lysed by cell lysis buffer. Cell lysates and in vitro purified substrate VN198-DEVD-VC198 (500 ng / mL) were incubated in vitro at 37 °C for 2 hours. Then, the mixture was heated at 68 °C for 5 minutes. The distribution of RFU reduction before and after heating with different cell numbers was obtained.

[0116] Figure 11D HeLa cells were transfected with each plasmid at a dose of 0.5 μg / mL. After 8 hours, cells transfected with VN198 / VC198 empty vector were not treated, while cells transfected with VN198-DEVD-VC198 were treated with Etoposide (30 μM) or equal volume of DMSO for 26 hours. The imaging results under fluorescence microscope were shown in the figure (the scale bar is 50 μm).

[0117] Figure 11E The results of flow cytometry analysis of HeLa cells in the 2nd and 3rd rows of Figure 11F

[0118] Figure 11G The results of Western blotting analysis of cells in the 2nd and 3rd rows of Figure 11H

[0119] HeLa cells were transfected with 0.5 μg / mL of GFP or VN198-DEVD-VC198 plasmid. After 8 hours, cells were treated with equal volume of control solvent, Etoposide (25 μM), or Etoposide (25 μM) combined with pan-Caspase inhibitor (Z-VAD-FMK, 100 μM). Z-VAD-FMK was added 4 hours after transfection. The decrease in RFU was detected with the same total protein amount in each group, and the results were presented in the form of a bar graph. Figure 11J Eto. represents Etoposide; Casp. 3 represents Caspase-3.

[0120]

[0121] The results of the experiment in Figure 11K HEK293T cells were repeated, in which Etoposide was replaced by Camp (Camptothecin, Camptothecin). Figure 11L

[0122] ​​Figure 11M Figure 2. The VN198-DEVD-VC198 substrate concentration-relative fluorescence intensity curve;

[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 different concentrations of substrate was detected in a black transparent 96-well cell culture plate using a multifunctional enzyme label meter.

[0124] Figure 11N Figure 3. The substrate-enzyme reaction curve;

[0125] Caspase-3, or 200 ng / mL of Caspase-3 and different concentrations of substrate VN198-DEVD-VC198 (0.25, 0.5, 1, 2.5, 5, 10 μg / mL) were reacted at 37°C for 2 hours, and the degree of RFU reduction before and after heating at 68°C was calculated for each group.

[0126] Figure 12A Figure 4. The enzymatic reaction kinetics curve;

[0127] The substrate VN198-DEVD-VC198 (500 ng / mL) and Caspase-3 enzyme (50 ng / mL) were reacted at 37°C for different times (0-5.5 hours), and the RFU reduction value at each time point was measured. The time point was taken as the horizontal axis, and the RFU reduction value was taken as the vertical axis to draw a curve.

[0128] Figure 12B Figure 5. The enzyme dose-substrate reaction curve;

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

[0130] Figure 12C Figure 6. The Caspase-3 activity of Etoposide-treated cells and DMSO-treated cells was calculated according to the enzyme dose-substrate reaction curve described above, and presented in the form of a column chart.

[0131] Figure 12D Figure 7. HEK293T cells were transfected with each pair of BiFC plasmids based on the structure of mCherry fluorescent protein at a dose of 1 μg / mL, and 0.2 μg / mL of mCerulean plasmid was transfected as an internal reference. The imaging results under fluorescence microscopy 48 hours after transfection are shown in the figure (the scale bar is 25 μm).

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

[0133] Figure 12E Figure 6 is a graph of the experimental results in HeLa cells repeated in Figure 5 (the picture scale is 50 pm) ; Figure 12F

[0134] Figure 12G Figure 8 is a graph of the experimental results in HeLa cells repeated in Figure 7 (the picture scale is 50 pm) ;

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

[0136] Figure 12A Figure 10 is a graph of the experimental results in HeLa cells repeated in Figure 9 (the picture scale is 50 pm) ; Figure 1A

[0137] Figure 1B Figure 12 is a graph of the experimental results in HeLa cells repeated in Figure 11 (the picture scale is 50 pm) ;

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

[0139] Figure 1C Figure 14 is a graph of the experimental results in HeLa cells repeated in Figure 13 (the picture scale is 50 pm) ; Figure 1D

[0140] Figure 1E Figure 16 is a graph of the fluorescence intensity of the cell lysates of each group of 10E, 11E and 12E, and the relative fluorescence intensity of different BiFC pairs in each group after correction with the internal reference fluorescent protein. The ratio of the fluorescence intensity of the interacting BiFC complementary pairs in each group to the fluorescence intensity of the empty vector control or non-interacting complementary pairs is further calculated. Figure 1F DETAILED DESCRIPTION 12C

[0141] ​​​​​The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention, and should not be construed as limiting the scope of protection 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 stated, the raw materials and reagents used in the following examples are commercially available products or can be prepared by known methods.

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

[0144] The term "Venus" as used in this article refers to a yellow fluorescent protein derived from the green fluorescent protein avGFP of the bioluminescent jellyfish Aequorea Victoria, which was artificially modified through site-directed mutagenesis to produce a yellow fluorescent protein. Its specific sequence is shown in GenBank: AAZ65844. The spatial structure of this protein is attached. Figure 1G As shown, the loop structure between the 9th and 11th β-folds and between the 9th and 10th β-folds is as follows. Figure 1H As shown.

[0145] The term "cyan fluorescent protein mTurquiose2" as used in this article refers to a cyan-fluorescent fluorescent protein derived from the green fluorescent protein avGFP of the bioluminescent jellyfish Aequorea Victoria, obtained through artificial site-directed mutagenesis. It is produced by mutating SCFP3A (GenBank: AAZ65848) to T65S / I146F. The spatial structure of this protein is shown in the attached figure. Figure 2A As shown, the loop structure between the 9th and 11th β-folds and between the 9th and 10th β-folds is as follows. Figure 2C As shown.

[0146] The term "mNeonGreen" as used in this article refers to a green fluorescent protein obtained by site-directed mutagenesis of LanYFP, a yellow-green fluorescent protein derived from the amphioxus branchiostoma lanceolatum. Its specific sequence is shown in GenBank: AGG56535. The spatial structure of this protein is attached. Figure 3A As shown, the loop structure between the 9th and 11th β-folds and between the 9th and 10th β-folds is as follows. Figure 2A As shown.

[0147] The term "red fluorescent protein mCherry" used in this article refers to a red fluorescent protein obtained by artificial site-directed mutagenesis of the shiitake mushroom coral *Discosoma* sp., specifically the DsRFP protein, and its sequence is shown in GenBank:AAV52164. The spatial structure of this protein is attached. Figure 2B As shown, the loop structure between the 9th and 11th β-folds and between the 9th and 10th β-folds is as follows. Figure 4A As shown.

[0148] The term “fluorescent complementary pair VN198 / VC198” as used in this article refers to the BiFC fluorescent complementary pair composed of the polypeptide fragment of the yellow fluorescent protein Venus consisting of amino acid residues 1-198 (abbreviated as VN198) and the polypeptide fragment of Venus consisting of amino acid residues 198-239 (abbreviated as VC198).

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

[0150] The term "FKBPs protein" as used in this article refers to the collective term for members of the FK506-binding protein family, which belongs to the immunophilin protein family. It binds to the immunosuppressants FK506 and rapamycin and has cis-trans-prolyl isomerase activity. Here, it refers to two members, FKBP12 and FKBP52.

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

[0152] The term "PD-L1 protein" used in this article refers to programmed death-ligand 1, a ligand for PD-1, encoded by the CD274 gene, and expressed on the surface of T cells, B cells, and various tumor cells. Its interaction with PD-1 inhibits T cell activation and cytokine production. In the course of infection or inflammation in normal tissues, this interaction is crucial for maintaining immune homeostasis and preventing autoimmunity. In the tumor microenvironment, this interaction inactivates the function of cytotoxic T cells, facilitating immune escape by tumor cells.

[0153] The term "GSDMD protein" used in this article refers to Gasdermin protein family member D, which is cleaved by Caspase 1 / 4 / 5 / 11, and the released N-terminal fragment oligomerizes in the cell membrane to form pores, inducing pyroptosis and inflammatory responses.

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

[0155] The term "Caspase-3 protein" as used in this article refers to cysteine-aspartic protease 3, which is encoded by the CASP3 gene and matured by cysteine-aspartic proteases Caspase-8 / 9 / 10. It cleaves substrates with DEVD amino acid residue sequences and induces apoptosis.

[0156] The following describes some of the materials and methods used in the embodiments of the present invention. In the following embodiments of the present invention, if the aforementioned materials and methods are required, they are used in the following manner.

[0157] (I) Carrier

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

[0159] The detailed spectra of the pBiFC-VN173 and pBiFC-VC155 vectors are attached. Figure 2C As shown, the aforementioned carriers can be obtained through purchase.

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

[0161] The specific spectra of the pBiFC-VN210 and pBiFC-VC210 vectors are attached. Figure 4A As shown, the former is constructed by inserting Flag-linker-VN210(1-211)-linker between NheI and HindIII restriction endonuclease in the pcDNA3.1(+) vector, and the latter is constructed by inserting Linker-VC210(211-239)-linker-HA between NotI and ApaI restriction endonuclease in the pcDNA3.1(+) vector. The above vector pairs are the starting vectors of this invention.

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

[0163] The detailed spectra of the pBiFC-VN198 and pBiFC-VC198 vectors are attached. Figure 4AThe pBiFC-VN210 and pBiFC-VC210 vectors are constructed on the basis of the pBiFC-VN198 and pBiFC-VC198 vectors, respectively.

[0164] The other vectors used in the present application are mainly constructed on the basis of the pBiFC-VN210, pBiFC-VC210, pBiFC-VN198 and pBiFC-VC198 vectors.

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

[0166] The present application involves 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 are purchased from the Cell Resource Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences (IBMS & CAMS). HeLa cells are 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, the cells were seeded and grown for 14-18 hours, then the plasmids were transfected into the cells with polyacrylamide (PEI) at a ratio of 1:2 (μg: μl). Unless otherwise specified, the final concentration of each BiFC plasmid in the culture medium was 1.0 μg / mL, and the final concentration of the internal reference fluorescent protein (mCherry, mCerulean, mTagBFP2) was 0.1 μg / mL.

[0169] (iii) Western blotting

[0170] The 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 inhibitors) was used to lyse the cells on ice for 1 hour, then centrifuged at 12,000 rpm for 15 minutes at 4°C, the proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the target protein level was detected by the corresponding antibody.

[0171] (iv) In vivo BiFC analysis

[0172] Live cell imaging was observed by inverted fluorescence microscope EVOS FL Auto 2 or Leica DMI8 (DFC9000 GT VSC-11009). When using the latter, the fluorescence proteins and the corresponding excitation wavelength (λ ex ) and emission wavelength (λem ) 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 lysate was detected by FlexStation 3 Multifunctional Microplate Reader (Molecular Devices) or Synergy H1 Multifunctional Microplate Reader (Biotek, USA). The λ ex and λ em of Synergy H1 were 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 were as follows: mCerulean and mTurquoise 2, 433 / 538; Venus and mNeonGreen, 488 / 538; mCherry, 580 / 620.

[0174] (V) Flow cytometry

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

[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 pET vectors, and the following plasmids were successfully constructed: 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 Ni-NTA purification system (Qiagen). The purified protein concentration was determined by BCA method, and the protein purity was evaluated by 10% SDS-PAGE and Coomassie blue staining. The protein solution was stored in the following buffer: 150 mM NaCl, 25 mM Tris-HCl, pH 7.2, 5% glycerol, 1 mM phenylmethylsulfonyl fluoride.

[0178] (vii) Analysis of Tau protein aggregation in vitro

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

[0180] (viii) Detection of Caspase-3 activity in vitro

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

[0182] Measurement plate and system: black transparent bottom 96-well cell culture plate was used, and the determination volume of each well was 100 μl.

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

[0184] When detecting the Caspase-3 activity in the apoptosis-induced and non-induced cells in vitro, the cell sample preparation process is as follows: the cells are induced by 30 μM etoposide for 20 hours, and the cells are treated with DMSO as a negative control. The cells are collected and lysed in pre-cooled lysis buffer for 1 hour, 500 μl of cell lysis buffer is used for incubation per 1×10 7 6 cells, and centrifuged at 4°C and 12,000 rpm for 15 minutes to collect the supernatant for standby.

[0185] Example 1: Obtaining of VN198 / VC198 BiFC fluorescence complementation pair

[0186] I. Currently commonly used BiFC fluorescence complementation pair based on the structure of Venus protein

[0187] 1. VN173 / VC155 fluorescence complementation pair

[0188] The pBiFC-VN173 / pBiFC-VC155 used for BiFC analysis of protein interaction based on the Venus fluorescent protein is a commonly used fluorescence complementation pair at home and abroad, and the plasmid and fluorescence complementation pair structure are shown in Figs. 1 and 2, respectively. Figure 4B and Fig. 2. Figure 5A

[0189] The present application finds that the above-mentioned VN173 and VC155 expression plasmids are co-transfected into HeLa, MCF7 and U2OS cells. Under the same conditions, a similar or even stronger fluorescence signal to Venus can be observed in the three kinds of transfected cells, as shown in the left two columns of Fig. 3. It is shown that there is a strong self-assembly ability between the VN173 / VC155 complementation pair in the cells, and false positive results are easy to occur in the detection of protein interaction. Figure 5B

[0190] 2. VN210 / VC210 fluorescence complementation pair

[0191] The VN210 / VC210 complementation pair is a BiFC complementation pair screened by Mizuno et al. based on the structure of Venus fluorescent protein in 2012, as shown in Figs. 4 and 5. Figure 3B and Fig. 5. 2D ​​As shown in the attached document. Figure 5C As shown in the middle column, no obvious fluorescence signal was detected in cells transfected with the empty VN210 / VC210 vector pair alone, indicating that the VN210 / VC210 pair has a weak self-assembly ability in cells.

[0192] VN210 was fused with bJun (encoding amino acid residues 257-334, NP_068607), and VC210 was fused with bFos (encoding amino acid residues 118-211, NP-071533) or bFosΔzip (encoding amino acid residues 118-211, with residues 179-193 deleted, denoted as Δzip) to construct VN210-bJun, bFos-VC210, and bFosΔzip-VC210 expression vectors. Interactions between bJun and bFos have been reported, but no interaction exists between bJun and bFosΔzip. (See attached...) Figure 5D The results showed that the fluorescence signal of cells co-expressing VN210-bJun / bFos-VC210 was significantly stronger than that of cells expressing the empty VN210 / VC210 vector pair, but significantly weaker than that of cells expressing VN173 / VC155, and the results were consistent across all three cell types. Furthermore, no significant fluorescence signal was detected in cells co-transfected with VN210-bJun / bFosΔzip-VC210. ​​Therefore, the VN210 / VC210 complementary pair can be used to analyze protein-protein interactions, exhibiting significantly improved specificity compared to the VN173 / VC155 complementary pair. However, the weak fluorescence signal of VN210-bJun / bFos-VC210 in detecting protein interactions reduces the sensitivity of this complementary pair, and false negative results are easily obtained for weakly interacting proteins.

[0193] The expression levels of all target proteins were detected by Western blotting. Results are attached. Figure 6A The results showed that the expression level of bFosΔzip-VC210 protein in cells co-transfected with VN210-bJun / bFosΔzip-VC210 was significantly lower than that in cells co-transfected with VN210-bJun / bFos-VC210.

[0194] To rule out the possibility that low expression of bFosΔzip-VC210 protein might lead to reduced fluorescence signal, this invention co-transfected MCF7 cells with VN210-bJun / bFos-VC210 or VN210-bJun / bFosΔzip-VC210, and further treated the VN210-bJun / bFosΔzip-VC210 transfected group with DMSO and MG132, respectively. (See attached...) Figure 6BAs shown, obvious fluorescent signals can be observed in VN210-bJun / bFos-VC210 group, however, no obvious fluorescent signals can be observed in VN210-bJun / bFosΔzip-VC210 group, no matter treated with MG132 or DMSO. Although the Western blotting results show that the expression level of bFosΔzip-VC210 is obviously increased after treated with MG132, as shown in Figure 6B, we believe that the undetectable fluorescent signals in VN210-bJun / bFosΔzip-VC210 transfected group may not be caused by the low expression level of bFosΔzip-VC210. Instead, the reason for the latter may be that bFosΔzip-VC210 loses the interaction with VN210-bJun and becomes unstable, and is degraded by ubiquitin-proteasome system. Figure 6C As shown, the Western blotting results show that the expression level of bFosΔzip-VC210 is obviously increased after treated with MG132. Therefore, we believe that the undetectable fluorescent signals in VN210-bJun / bFosΔzip-VC210 transfected group may not be caused by the low expression level of bFosΔzip-VC210. Instead, the reason for the latter may be that bFosΔzip-VC210 loses the interaction with VN210-bJun and becomes unstable, and is degraded by ubiquitin-proteasome system.

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

[0196] Based on the cleavage sites of VN173 / VC155 complementation pair and VN210 / VC210 complementation pair on Venus, the present application looks for new cleavage sites between the 173th and 210th positions of Venus, in order to obtain a site with lower self-assembly ability than VN173 / VC155 complementation pair, but with stronger fluorescent signal when the proteins specifically interact than VN210 / VC210 complementation pair.

[0197] Mizuno et al. conducted a systematic study on the complementation pairs designed based on Venus fluorescent protein in 2012, and the results showed that the VN210 / VC189 combination self-assembled to produce fluorescence and could not distinguish protein interaction from non-interaction, while the VN210 / VC210 complementation pair could distinguish interaction from non-interaction but the fluorescent signal was weak. Therefore, the present application shortens the length of VN210 fragment from C terminal to obtain a new VN198 fragment including the first to ninth β folds of Venus, and constructs a VN198-bJun fusion expression vector. The length of VC210 fragment is extended to N terminal, so that the new fragment includes the tenth and eleventh β folds of Venus, as shown in Figure 5B, and according to the different positions in the loop between the ninth and tenth β folds, VC198, VC194 and VC189 fragments are obtained, VN (VN210 or VN198) is fused with bJun fragment, and VC198, VC194 and VC189 are fused with bFos or bFosΔzip fragment, and the expression vectors constructed are transfected into HeLa cells and the fluorescent signals are observed, and mCherry is co-transfected as an internal control. Figure 6D

[0198] As shown in Figure 6A, obvious fluorescent signals can be observed in VN198-bJun / bFos-VC198 group, while no obvious fluorescent signals can be observed in VN198-bJun / bFosΔzip-VC198 group, no matter treated with MG132 or DMSO. As shown in Figure 6B, the Western blotting results show that the expression level of bFosΔzip-VC198 is obviously increased after treated with MG132. Therefore, we believe that the undetectable fluorescent signals in VN198-bJun / bFosΔzip-VC198 transfected group may not be caused by the low expression level of bFosΔzip-VC198. Instead, the reason for the latter may be that bFosΔzip-VC198 loses the interaction with VN198-bJun and becomes unstable, and is degraded by ubiquitin-proteasome system. Figure 7A ​As shown, the fluorescence signals of VN210-bJun or VN198-bJun co-expressed with bFos-VCs were significantly stronger than those co-expressed with bFosΔzip-VCs. Under the same transfection and fluorescence imaging conditions, regardless of whether interactions occurred between the fusion proteins, combinations containing the VN210 fragment produced stronger fluorescence signals than the corresponding combinations containing the VN198 fragment. Notably, among all complementary pairs, the VN210-bJun / bFos-VC210 pair had the weakest fluorescence intensity. The relative fluorescence intensity ratio (fluorescence intensity of interacting pairs / corresponding non-interacting fluorescence intensity) for each transfection pair was calculated. (See attached image.) Figure 7B The results showed that the fluorescence intensity ratio of the VN210 / VC198 and VN198 / VC198 complementary pairs was the largest. Meanwhile, this invention observed that the fluorescence intensity of the non-interacting group of the VN198 / VC198 complementary pair was lower than that of the VN210 / VC198 pair, and even lower than that of VN210 / VC210.

[0199] Therefore, this 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, this invention transfected MCF7, HeLa, and U2OS cells with the plasmid pairs VN210-bJun / bFos-VC210 and VN198-bJun / bFos-VC198, as well as the corresponding non-interacting plasmid pairs VN210-bJun / bFosΔzip-VC210 and VN198-bJun / bFosΔzip-VC198, respectively. Figure 7C As shown, under the same treatment conditions, for each cell type, the fluorescence intensity of the VN198-bJun / bFos-VC198 transfection group was significantly higher than that of the VN210-bJun / bFos-VC210 transfection group. However, the fluorescence signal of the corresponding non-interacting transfection group was very weak or even undetectable in all three cell types, regardless of whether it was the VN198 / VC198 combination or the VN210 / VC210 combination. This indicates that the VN198 / VC198 combination has higher sensitivity than the VN210 / VC210 combination in analyzing bJun-bFos interactions.

[0201] Furthermore, in the VN198 / VC198 combination, to investigate the relationship between the protein expression level of bFosΔzip and the fluorescence signal intensity of this group, according to the attached... Figure 8AThe HeLa cells were transfected with different combinations and treated with DMSO or MG132 for a certain time. The fluorescence microscope imaging results showed that the VN198-bJun / bFos-VC198 expression group had obvious fluorescence signals, while the VN198-bJun / bFosΔzip-VC198 group had no fluorescence signals whether treated with MG132 or not. After observing the fluorescence of each group, the cells were collected, and the expression levels of the target proteins of each group were analyzed. The Western Blotting results showed that the protein expression level of bFosΔzip-VC198 in the non-interaction group treated with MG132 was significantly higher than that in the non-interaction group treated with DMSO, as shown in the following figure: Figure 8B According to the above results, it is inferred that the results of the VN210 / VC210 combination are consistent, that is, the fluorescence signal is not detected in the VN198-bJun / 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 suggest that the transfection dose of the plasmid pair affects the self-assembly ability of the fluorescence complementation fragment. In order to exclude the interference of the plasmid dose on the self-assembly analysis, HeLa cells were transfected with different doses of N173 / VC155 complementary pairs, VN210 / VC210 complementary pairs, or VN198 / VC198 complementary pairs. As shown in the following figure: Figure 8C The proportion of yellow fluorescent cells transfected with the VN173 / VC155 pair increased with the increase of the plasmid transfection dose, but even if the lowest dose of plasmid reported in the literature was transfected, the VN173 / VC155 pair could still observe obvious fluorescence signals, but for VN210 / VC210 and VN198 / VC198 plasmid pairs, even if the highest plasmid dose was transfected, no obvious fluorescence signal was observed. This result suggests that the different self-assembly abilities of VN173 / VC15, VN210 / VC210 and VN198 / VC198 complementary pairs are determined by the differences in fragments.

[0203] In summary, the fluorescence complementary pair VN198 / VC198 screened by the present application has lower self-assembly ability than the VN173 / VC155 fluorescence complementary pair, has significantly reduced background fluorescence signals, and improves 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, which improves the sensitivity of BiFC analysis. Therefore, the fluorescence complementary pair VN198 / VC198 may be a new and more ideal BiFC fluorescence complementary pair.

[0204] Example 2: Application of VN198 / VC198 fluorescent complementary pairs in detecting interactions between proteins with important physiological functions

[0205] 1. Detecting the interaction between mTOR and FKBPs

[0206] Crystal structure analysis has confirmed that mTOR interacts with FKBPs (FKBP12 and FKBP52) through its FRB domain after rapamycin (RAP) induction. To further demonstrate the feasibility of the BiFC fluorescence complementary pair VN198 / VC198 screened in this invention for detecting mTOR / FKBP interactions, this invention constructed pBiFC-VN198-FKBP12, pBiFC-VN198-FKBP52, and pBiFC-mTOR-VC198 expression vectors, the structures of which are attached. Figure 9A As shown, FK506 and RAP are two novel immunosuppressants widely used in clinical organ transplantation to treat immune rejection and autoimmune diseases. The main intracellular targets of both FK506 and RAP are FKBPs. FK506 binds to FKBP12, specifically inhibiting calcineurin and suppressing T cell-mediated immune responses. RAP's binding to FKBPs promotes its binding to mTOR, inhibiting mTORC1 activity and reducing T cell and B cell activation. 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, follow the instructions in the appendix. Figure 9B The plasmids were constructed by co-transfection with the combination shown. After a certain period of time, the cells were treated with DMSO solvent, RAP alone, or FK506 and RAP co-treated. The fluorescence intensity of the cells in each transfection group was observed and recorded.

[0208] The results showed that, in the three cell lines derived from different tissues, the addition of RAP significantly induced fluorescence signals in FKBP12-mTOR and FKBP52-mTOR transfected cells compared to solvent-treated cells. If transfected cells were treated with both RAP and FK506, virtually no fluorescence signal or only a very weak fluorescence signal was observed in the transfected cells. The expression levels of the target proteins in each treatment group were detected by Western blotting of the collected HeLa cells, as shown in the attached figure. Figure 8CThe results of Western Blotting showed that the expression levels of FKBP12 and FKBP52 fusion proteins were relatively close in each group of cells treated with RAP and FK506, but for mTOR fusion protein, RAP treatment significantly up-regulated its expression level, but after the addition of FK506 treatment, the level of mTOR fusion protein was reduced to a certain extent compared with RAP treatment alone, but close to or still significantly higher than the DMSO treatment group.

[0209] In summary, when mTOR-VC198 and the expression plasmids of VN198-FKBP12 or VN198-FKBP52 were co-transfected into cells, no obvious fluorescence signal was observed, but after the addition of RAP to promote their interaction, obvious fluorescence signal was observed under fluorescence microscope. After the addition of FK506 reagent, the effect of RAP-induced fluorescence signal was significantly weakened or not observed.

[0210] 2, detecting the interaction between PD-1 and PD-L1

[0211] The crystal structure of human PD-1 / PD-L1 interaction in 2015 revealed that PD-1 and PD-L1 mainly interact through the extracellular region. In order to clarify whether the VN198 / VC198 fluorescence complementation pair can be used to visualize the interaction between PD-1 and PD-L1 in cells, the extracellular region of PD-1 and PD-L1 was fused to the N-terminus of VC198 and VN198, respectively. In order to enhance the membrane localization ability of PD-1 and PD-L1, the signal peptide sequence of IgK and the transmembrane region sequence of PDGFRβ were used to replace the corresponding sequences of themselves, and the corresponding expression vectors were successfully constructed, and the structure of the inserted fragments is shown in the following figure. Figure 10A

[0212] The above constructed vectors were transfected into HeLa and HEK293T cells, and the immune checkpoint inhibitor Pembrolizumab targeting the interaction of 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 control. As shown in the following figure, the fluorescence signal of the transfected cells was observed under fluorescence microscope. Figure 10B ​As 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 fluorescent signals, while the membrane-localized fluorescent signals of the cells treated with Pembrolizumab were weakened, but no fluorescent signals were detected in the cells transfected with the pair of empty vectors expressing VN198 / VC198. The HEK293T cells were co-transfected with the PD-L1-VN198 and PD-1-VC198 expression vectors, respectively, or separately, and the expression of the target proteins was detected by Western Blotting, although the level of the target proteins in the co-transfected cells was slightly lower than that in the separately transfected cells, as shown in FIG. 2. Figure 10B As shown.

[0213] The above results show 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 fluorescent signals, but the treatment of the transfected cells with Pembrolizumab, which inhibits the interaction of the fusion proteins, resulted in a significant weakening of the membrane-localized fluorescent signals compared with the treatment with the control antibody.

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

[0216] To analyze whether the VN198 / VC198 complementary pair can be used to analyze the interaction between GSDMD-N and GSDMD-C in living cells, the inventors inactivated the cell puncture activity of GSDMD-N by deletion mutation, constructed the expression vector pBiFC-VN198-GSDMD1-250Δ96-116Δ174-204 (referred to as pBiFC-VN198-GSDMD-N), and constructed the expression vector pBiFC-GSDMD251-484-VC198 (referred to as pBiFC-GSDMD-C-VC198), and the structure of the inserted fragments is shown in FIG. 3. Figure 10B As shown.

[0217] The above-mentioned plasmid pairs or their corresponding empty vector pairs were co-transfected into HeLa cells and HEK293T cells, and the expression of the target proteins was detected by Western Blotting, as shown in FIG. 4. Figure 10CThe results show that there is obvious fluorescence signal in the cytoplasm of cells transfected with VN198-GSDMD-N / GSDMD-C-VC198, but no fluorescence signal is detected in cells co-transfected with VN198 / VC198 pair. The HEK293T cells were transfected with VN198-GSDMD-N and GSDMD-C-VC198 expression vectors respectively or co-transfected, and the expression of the target protein was detected by Western Blotting. Although the level of the target protein of GSDMD-C-VC198 transfected alone was slightly lower than that of co-transfected, the expression of the target protein was detected, as shown in the following figure. Figure 10D

[0218] The above results show 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 level of living cells.

[0219] From the above experiments of the first to third parts, the cell model is used to analyze the interaction between 4 pairs of proteins or polypeptide fragments with important biological functions, including mTOR (2019-2114 aa, NP_001373429) and FKBP12 (1-108 aa, NP_463460) and FKBP52 (1-459 aa, NP_002005), programmed cell death protein 1 (PD1, 24-170 aa, NP_005009) and programmed cell death protein ligand 1 (PD-L1, 19-238 aa, NP_054862), N-terminal fragment (1-250Δ96-116Δ174-204, NP_079012) and C-terminal fragment (251-484) of cell pyroptosis related protein GSDMD, and the specific fluorescence between each pair is detected, and for the first three pairs, if the interaction between them is prevented, the fluorescence signal is weakened or not detected.

[0220] 4. Detection of aggregation between Tau proteins

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

[0222] The present application first constructs pBiFC-VN173-Tau, pBiFC-Tau-VC155, pBiFC-VN198-Tau, pBiFC-Tau-VC198 expression vectors, and the vector insertion sequences are as follows: Figure 10E ​As shown in the attached figure, HeLa cells were transfected with the VN173-Tau / Tau-VC155 and VN198-Tau / Tau-VC198 expression vector pairs and their corresponding empty vector pairs. The results observed under a fluorescence microscope are as follows. Figure 10F As shown.

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

[0224] Forskolin (FK) and LMTX are a tau protein aggregation inducer and inhibitor, respectively. After transfecting cells with the above vectors for 8 hours, DMSO or Forskolin alone, or Forskolin and LMTX co-treated the cells, were observed and recorded for fluorescence signals in each treatment. (See attached image) Figure 10G As shown, the VN173 / VC155 empty vector showed obvious whole-cell fluorescence signals in transfected cells. However, under the same imaging conditions, the VN198 / VC198 empty vector showed almost no obvious fluorescence signal in transfected cells, although both showed obvious cytoplasmic fluorescence signals after fusion with Tau, and the former's fluorescence signal was generally higher than the latter's, but the latter's increase was greater compared to the corresponding empty vector pair. After further treatment with Forskolin, both the VN173-Tau / Tau-VC155 and VN198-Tau / Tau-VC198 groups showed similar fluorescence intensities. After co-treatment with LMTX and Forskolin, compared with the FK-treated groups, the reduction in cell fluorescence intensity of the 173 / 155 and 198 / 198 pairs of Tau protein fusion cells was similar. (Collection attached...) Figure 10H After protein quantification, the relative fluorescence intensity of the cell lysates from each group was measured using a multi-functional microplate reader under the same total protein content. The fluorescence intensity of the Tau fusion protein transfected group was then calculated and divided by the fluorescence intensity of the corresponding empty vector transfected group. (See attached image) Figure 11AAs shown, the fluorescence intensity of cells transfected with Tau fusion vector pairs was significantly enhanced compared with the respective empty vector pairs, but the fluorescence intensity of VN198 / VC198 pair was more than 10 times that of VN173 / VC155 pair. After treatment with Forskolin, only the VN198-Tau / Tau-VC198 group detected a significantly increased fluorescence intensity, and after further co-treatment with LMTX and Forskolin, although the fluorescence intensity of cells of VN173 / VC155 pair and VN198 / VC198 pair fused with Tau protein was significantly reduced, the latter was reduced to a greater extent. Overall, 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 VN173-Tau / Tau-VC155 group. The experiment was repeated in HEK293T cells, and similar experimental results were obtained, as shown in FIG. 6. Figure 11B As shown, * represents p<0.01. Different combinations of expression plasmids were transfected in HEK293T cells, combined with FK (Forskolin) and LMTX drug treatment, as shown in FIG. 5. Figure 11C The results of Western Blotting suggest that the expression levels of each target protein in different treatment groups are close.

[0225] In addition, the present application expresses and purifies VN198-Tau, Tau-VC198 and their empty vector proteins VN198 and VC198 in vitro, and the protein purification results are shown in FIG. 6. Figure 11D

[0226] Firstly, the present application studies the feasibility of VN198-Tau / Tau-VC198 in detecting Tau protein aggregation and disaggregation in vitro. As shown in FIG. 4, under in vitro conditions, the fluorescence intensity of VN198-Tau / Tau-VC198 co-incubation solution was significantly higher than that of VN198 / GST-VC198 pair, and the fluorescence intensity of the former showed time-dependent increase within 24 hours of observation, so it can be concluded that VN198-Tau / Tau-VC198 fluorescence protein pair can be used to analyze Tau protein aggregation in vitro. Figure 11E

[0227] On the basis of the above experiments, the present application also studies whether VN198-Tau and Tau-VC198 can be used to detect the ability of LMTX to inhibit Tau protein aggregation in vitro. VN198-Tau and Tau-VC198 recombinant fusion proteins were respectively 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 more, as shown in FIG. 7. Figure 11F ​​The results can be seen that the fluorescence intensity of VN198-Tau / Tau-VC198 protein pair at each detection time point gradually decreases with the gradual increase of LMTX concentration.

[0228] Therefore, the present application concludes that VN198-Tau / Tau-VC198 can be used for detecting Tau protein aggregation in vivo and in vitro, wherein the specificity and sensitivity of VN198-Tau / Tau-VC198 in detecting Tau protein aggregation in living cells are significantly higher than those of VN173-Tau / Tau-VC155 pair.

[0229] In summary, compared with VN198 and VC198 complementary pair, the co-expression of VN198-Tau and Tau-VC198 pair in cells or the co-incubation of recombinant protein pair in solution can observe obvious fluorescence signal, Forskolin promotes fluorescence signal, and LMTX inhibits fluorescence signal.

[0230] 5. Detecting the activity of Caspase-3

[0231] The sequence Asp-Glu-Val-Asp (DEVD) recognized and cleaved by Caspase-3 is inserted into the 198 site of Venus fluorescent protein to construct a recombinant fluorescent protein substrate VN198-DEVD-VC198 which can be used for detecting the activity of Caspase-3, and the principle of detecting the activity of Caspase-3 is shown in the attached Figure 11G Without the presence of Caspase-3, VN198 and VC198 structure complement each other 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, VN198 and VC198 complementary pair are separated by heating at 68°C for 5 minutes, and the Caspase-3 activity in the cells or sample solution to be detected is analyzed by analyzing the fluorescence intensity change before and after heating.

[0232] Firstly, the VN198-DEVD-VC198 fusion protein is purified by in vitro prokaryotic expression and used for studying the feasibility of detecting the activity of Caspase-3 in vitro.

[0233] VN198-DEVD-VC198 is 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 attached Figure 11HThe fluorescence intensity results show that at 37°C, there was no significant change in fluorescence intensity among the treatment groups. However, after heating at 68°C for 5 minutes, the fluorescence intensity of the Caspase-3 treatment group was significantly reduced compared to the first and third groups mentioned above. (See attached image.) Figure 11J The Western blotting results shown indicate that Caspase-3 can recognize and cleave the VN198-DEVD-VC198 substrate, and Caspase-3 inhibitors significantly inhibit the cleavage effect. These results demonstrate that VN198-DEVD-VC198 is feasible for in vitro analysis of Caspase-3 enzyme activity.

[0234] To demonstrate that VN198-DEVD-VC198 can be used to detect Caspase-3 activity in live cells, this invention transfected HeLa cells with the VN198-DEVD-VC198 expression plasmid. The relative fluorescence intensity reduction was then measured in untreated (Blank), DMSO, Eto. (etoposide), Inh. (Ac-DEVD-CHO), and co-treated (Eto. and Inh.) cells to evaluate the feasibility of using VN198-DEVD-VC198 to analyze Caspase-3 activity. Figure 11K The results showed that, compared with other treatment groups, the etoposide-only treatment group had a higher cell count (8 × 10⁻⁶). 4 and 10×10 4 The decrease in relative fluorescence intensity was significantly increased in the first treatment group, while the decrease in relative fluorescence intensity was less in the other treatment groups and there were no significant differences among them. HeLa cells were transfected with VN198 / VC198 and VN198-DEVD-VC198 expression plasmids, and the latter-transfected cells were further treated with DMSO or Eto. Fluorescence signals of the cells after different transfections and treatments were observed using a fluorescence microscope. Figure 11L The results show that, compared with the DMSO group, the fluorescence signal of VN198-DEVD-VC198 cells transfected with etoposide was significantly reduced, but no obvious fluorescence signal was detected in cells transfected with the VN198 / VC198 empty vector pair. Quantitative analysis of the fluorescence intensity of the VN198-DEVD-VC198 expression plasmid transfection groups by flow cytometry revealed that the fluorescence signal of VN198-DEVD-VC198 cells treated with etoposide was significantly lower than that treated with DMSO, as shown in the attached figure. Figure 11M As shown. Similarly, the levels of the VN198-DEVD-VC198 fusion protein in the remaining cells were detected by flow cytometry, as shown in the attached figure. Figure 11NAs shown, Western Blotting results showed that the level of VN198-DEVD-VC198 fusion protein in Etoposide treated group was significantly lower than that in DMSO treated group. Therefore, it can be determined that VN198-DEVD-VC198 can be used to detect Caspase-3 activity in living cells. Likewise, the present application treated HeLa and HEK293T cells transfected with GFP or VN198-DEVD-VC198 with DMSO, Eto., Eto.+Inh., respectively, and further detected the fluorescence intensity changes of the cell lysates of these different treatments. As shown in Fig. 2A and Fig. 2B, only in VN198-DEVD-VC198 transfected cells, the fluorescence intensity of Eto. treated cells was significantly reduced compared with DMSO treatment, but the fluorescence intensity of Eto.+Inh. co-treated cells recovered to different degrees. Figure 12A (HeLa) and 11I (HEK293T), the fluorescence intensity of Eto. treated cells was significantly reduced compared with DMSO treatment, but the fluorescence intensity of Eto.+Inh. co-treated cells recovered to different degrees.

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

[0236] Firstly, the present application determined the linear range of fusion protein VN198-DEVD-VC198. The results showed that the substrate concentration in the range of 0-15 μg / mL showed a good linear relationship with the relative fluorescence intensity, as shown in Fig. 3A and Fig. 3B. Figure 12B To verify that VN198-DEVD-VC198 is a better substrate for detecting Caspase-3 activity in vitro, we incubated different concentrations of VN198-DEVD-VC198 recombinant protein with recombinant Caspase-3 at 37°C for 2 hours, while using the substrate without adding Caspase-3 enzyme as a control. After incubation, the fluorescence intensity of each solution was detected, and after heating at 68°C for 5 minutes, the fluorescence intensity of each solution was detected again, and the reduction value of the fluorescence intensity of each solution before and after heating was calculated. The results showed that whether or not Caspase-3 was added, the reduction value of the fluorescence intensity before and after heating had a good linear correlation with the substrate concentration, but the slope of the line of the enzyme added group was significantly higher than that of the no enzyme added group, suggesting that the substrate can be used to distinguish different Caspase-3 enzyme activity levels, as shown in Fig. 4A and Fig. 4B. Figure 12C

[0237] ​In addition, the present application analyzes the kinetic curve of Caspase-3 cleavage of VN198-DEVD-VC198. A certain concentration of Caspase-3 (50 ng / mL) is reacted with VN198-DEVD-VC198 (500 ng / mL) at 37°C for different time (0-5.5 hours), and the results show that the decrease of fluorescence intensity of the sample increases continuously with the extension of the detection time, and reaches the enzymatic reaction platform after 4 hours, as shown in the following figure: Figure 12D which indicates that the cleavage of the current concentration of Caspase-3 to the current concentration of substrate VN198-DEVD-VC198 takes about 4 hours to complete.

[0238] In order to study the dose-response relationship between the substrate VN198-DEVD-VC198 and the amount of Caspase-3 enzyme, the present application uses a fixed concentration of substrate VN198-DEVD-VC198 and different concentrations of Caspase-3 to analyze the enzyme dose-fluorescence intensity relationship. As shown in the following figure: Figure 12E , under the condition of low concentration of Caspase-3 (5-150 ng / mL), the decrease of fluorescence intensity and the dose of Caspase-3 show a significant correlation. On this basis, the present application further analyzes the Caspase-3 activity in different Eto. treated cell samples, and the results are shown in the following figure: Figure 12F , which suggests that the VN198-DEVD-VC198 substrate can be used to analyze the Caspase-3 enzyme activity level in vitro.

[0239] In summary, based on the VN198 / VC198 fluorescence complementary pair constructed, the substrate VN198-DEVD-VC198 which can be recognized and cleaved by Caspase-3 can be used to analyze the Caspase 3 enzyme activity in and outside the cell.

[0240] Example 3 Screening of other fluorescent protein fluorescence complementary pairs

[0241] Referring to the cleavage site of VN198 and VC198 in the Venus fluorescent protein, the present application further studies the feasibility of using the fluorescence complementary pairs with similar cleavage sites in other fluorescent proteins with similar barrel-shaped structures for BiFC analysis, including 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, this invention constructs a vector expressing the N-terminal amino acid fragment 1-196 of mCherry (abbreviated as CN196) and a vector expressing CN196-bJun. It also constructs a vector expressing the C-terminal amino acid fragment 189-236 of mCherry (abbreviated as CC189). Similarly, it constructs vectors expressing CC192 and CC196, as well as corresponding expression vectors constructed by fusing the N-terminus with bFos or bFosΔzip fragments, respectively.

[0243] Subsequently, the present invention will attach the vectors expressing CN196-bJun and bFos-CCs or bFosΔzip-CCs (including CC189, CC192 or CC196) or their corresponding empty vectors to the attached vectors. Figure 12G and attached ​ The combination of the two methods was co-transfected into HEK293T and HeLa cells, and the fluorescence signal was observed by fluorescence microscopy.

[0244] Using the same strategy, this invention is also based on mNeonGreen (with appendix) ​ and attached ​ ) and mTurquoise2 (attached) ​ and attached ​ The crystal structure of the corresponding complementary pair was constructed and transfected into HEK293T and HeLa cells.

[0245] The results showed that the fluorescence signals of the three fluorescent proteins exhibited a relatively consistent trend; that is, no obvious fluorescence signal was observed under a fluorescence microscope after transfection of cells with empty vectors without fusion interaction proteins. The fluorescence signals of cells transfected with expression vectors carrying the bJun and bFos fusion fragments were significantly stronger than those carrying the bJun and bFosΔzip fusion fragments. (See attached image) ​ As shown, cell lysates were collected and total protein was quantified. The feasibility of detecting protein interactions using complementary pairs of these fluorescent proteins was further evaluated by measuring and calculating the ratios of fluorescence intensity between the bJun / bFos interacting group and the empty vector group, and between the bJun / bFos interacting group and the bJun / bFosΔzip non-interacting group, under the same total protein content and internal control fluorescent proteins (mCherry, mNeonGreen, and mTurquoise 2) calibration conditions.

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

[0247] In summary, the complementary pairs of VN198 and VC198 were used to generate BiFC pairs in the Loop between the 9th and 10th beta strands of fluorescent proteins of the same species (mTurquoise2, derived from Aequorea victoria) and different species (mNeonGreen, derived from Branchiostoma lanceolatum; mCherry, derived from Discosoma sp.). The complementary pairs generated by cleavage in the Loop between the 9th and 10th beta strands of fluorescent proteins of the same species and different species all have good specificity and sensitivity when used in BiFC analysis, but the fluorescence signal of the mNeonGreen and mCherry complementary pairs is significantly weaker than that of the Venus and mTurquoise2 pairs.

[0248] Finally, it should be pointed out that the above examples are only used to illustrate the technical solutions of the present application and are not limiting. Although the present application 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 application can be modified or replaced equivalently without departing from the spirit and scope of the technical solutions of the present application, and all should be encompassed in the scope of the claims of the present application.

Claims

1. A BiFC fluorescence complementary pair, characterized in that: It is produced by the breakage of fluorescent protein within the loop between the 9th and 10th β-sheets; The fluorescent protein is a yellow fluorescent protein mVenus, the sequence of which is shown in GenBank: AAZ65844. The BiFC fluorescence complementary pair is VN198 / VC198, wherein, VN198 encodes amino acids 1-198 of the yellow fluorescent protein mVenus, while VC198 encodes amino acids 198-239 of the yellow fluorescent protein mVenus.

2. A recombinant fluorescent protein, characterized in that: It is generated by inserting a Caspase-3 recognition sequence into the loop between the 9th and 10th β-sheets of the fluorescent protein; The fluorescent protein is a yellow fluorescent protein mVenus, the sequence of which is shown in GenBank: AAZ65844. The recombinant fluorescent protein is VN198-DEVD-VC198, which is generated by inserting the Caspase-3 recognition sequence DEVD at the 198th amino acid position of the yellow fluorescent protein mVenus.

3. An expression carrier, characterized in that, It comprises the BiFC fluorescent complementary pair or recombinant fluorescent protein as described in claim 1 or 2.

4. The expression vector according to claim 3, characterized in that, The expression vectors are selected from pBiFC-VN198, pBiFC-VC198, pBiFC-VN198-bJun, pBiFC-bFos-VC198, pBiFC-bFosΔzip-VC198, pBiFC-VN198-FKBP12, pBiFC-VN198-FKBP52, pBiFC-mTOR-VC198, pBiFC-PD-L1-VN198, pBiFC-PD-1-VC198, and pBiFC-VN198-G. SDMD1-250Δ96-116Δ174-204, pBiFC-GSDMD251-484-VC198, pBiFC-VN198-Tau, pBiFC-Tau-VC198, pET-24b(+)-VN 198-Tau, pET-24b(+)-Tau-VC198, pET-24b(+)-VN198, pET-42a(+)-GST-VC198, pET-24b(+)-VN198-DEVD-VC198.

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

6. The cell according to claim 5, characterized in that, The cells were selected from human embryonic kidney cells HEK293T, human breast cancer cells MCF7, human osteosarcoma cells U2OS, and human cervical cancer cells HeLa.

7. A reagent kit, characterized in that, It comprises the BiFC fluorescent complementary pair or recombinant fluorescent protein as described in claim 1 or 2, the expression vector as described in claim 3 or 4, or the cell as described in claim 5 or 6.

8. A method for detecting interactions between proteins, between polypeptide fragments, or between proteins and polypeptide fragments, characterized in that, The detection process utilizes the BiFC fluorescence complementary pair as described in claim 1, the expression vector as described in claim 3 or 4, the cells as described in claim 5 or 6, or the kit as described in claim 7. Wherein, the expression vector of claim 3 or 4, the cell of claim 5 or 6, and the kit of claim 7 are all expression vectors, cells, and kits containing the BiFC fluorescent complementary pair of claim 1.

9. The method according to claim 8, characterized in that, 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, fragments of pyroptosis-associated proteins GSDMD-N and GSDMD-C, or Tau protein.

10. The method according to claim 9, characterized in that, The FKBPs are selected from FKBP12 or FKBP52.

11. A method for detecting Caspase-3 activity, characterized in that, The detection process uses the recombinant fluorescent protein of claim 2, the expression vector of claim 3 or 4, the cells of claim 5 or 6, or the kit of claim 7, wherein the method is not a method for diagnosing or treating the disease; Wherein, the expression vector of claim 3 or 4, the cell of claim 5 or 6, and the kit of claim 7 are all expression vectors, cells, and kits containing the recombinant fluorescent protein of claim 2.

12. A method for screening candidate proteins that can interact with a target protein from a candidate protein library, characterized in that, The screening process employed the BiFC fluorescent complementary pair as described in claim 1, the expression vector as described in claim 3 or 4, the cells as described in claim 5 or 6, or the kit as described in claim 7. Wherein, the expression vector of claim 3 or 4, the cell of claim 5 or 6, and the kit of claim 7 are all expression vectors, cells, and kits containing the BiFC fluorescent complementary pair of claim 1.

13. The method according to claim 12, characterized in that, The candidate protein library or target protein includes mTOR and FKBPs, programmed cell death protein PD-1 and programmed cell death protein ligand PD-L1, pyroptosis-associated proteins GSDMD-N and GSDMD-C, or Tau protein.

14. The method according to claim 13, characterized in that, The FKBPs are selected from FKBP12 or FKBP52.

15. The use of the BiFC fluorescent complementary pair of claim 1, the expression vector of claim 3 or 4, the cell of claim 5 or 6, or the kit of claim 7 in detecting interactions between proteins, between peptide fragments, or between proteins and peptide fragments; in, The expression vector of claim 3 or 4, the cell of claim 5 or 6, and the kit of claim 7 all contain the expression vector, cell, and kit of the BiFC fluorescence complementary pair of claim 1.

16. The application according to claim 15, characterized in that, 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-associated proteins GSDMD-N and GSDMD-C, or Tau protein.

17. The application according to claim 16, characterized in that, The FKBPs are selected from FKBP12 or FKBP52.

18. The use of the recombinant fluorescent protein of claim 2, the expression vector of claim 3 or 4, the cell of claim 5 or 6, or the kit of claim 7 in detecting or screening Caspase-3 activity, wherein the use is not a method for diagnosing or treating a disease; in, The expression vector of claim 3 or 4, the cell of claim 5 or 6, and the kit of claim 7 are all expression vectors, cells, and kits containing the recombinant fluorescent protein of claim 2.

19. The use of the BiFC fluorescent complementary pair of claim 1, the expression vector of claim 3 or 4, the cell of claim 5 or 6, or the kit of claim 7 in screening candidate proteins that can interact with the target protein from a candidate protein library; in, The expression vector of claim 3 or 4, the cell of claim 5 or 6, and the kit of claim 7 all contain the expression vector, cell, and kit of the BiFC fluorescence complementary pair of claim 1.

20. The application according to claim 19, characterized in that, The candidate protein library or target protein includes mTOR and FKBPs, programmed cell death protein PD-1 and programmed cell death protein ligand PD-L1, N-terminal and C-terminal fragments of pyroptosis-associated protein GSDMD, or Tau protein.

21. The application according to claim 20, characterized in that, The FKBPs are selected from FKBP12 or FKBP52.

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