Modeling method for bonding affinity difference between annexin A5 and fluorescent protein FPs and phosphatidylserine PS caused by fusion of annexin A5 and fluorescent protein FPs and application of modeling method

Abstract

The invention discloses a modeling method for the binding affinity difference between annexin A5AnxA5 and fluorescent protein FPs and phosphatidylserine PS caused by fusion of annexin A5AnxA5 and fluorescent protein FPs and application of the modeling method. 18 kinds of binding conditions of AnxA5 based on fluorescent protein and apoptotic cells are detected through flow cytometry, and the binding affinity between AnxA5-FPs fusion protein and phosphatidylserine PS is obtained through data analysis; the PS affinity of the fusion protein is different, and the affinity constant Kd of the fusion protein with the highest PS affinity and the affinity constant Kd of the fusion protein with the lowest PS affinity are different by more than two orders of magnitude. The aggregation characteristic of the fluorescent protein is a factor influencing the PS affinity of the fusion protein; different fluorescent proteins and AnxA5 are subjected to fusion expression to generate different conformations, and the difference on the conformations causes the fusion proteins to have different PS binding modes, so that the difference of PS binding affinity is shown.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a modeling method for the difference in the binding affinity of annexin A5 (AnxA5) fused with fluorescent proteins (FPs) to phosphatidylserine (PS) and its application. Background Art

[0002] When cells undergo apoptosis, phosphatidylserine (PS) flips from the inner side of the cell membrane to the outer side of the cell membrane. This PS exposed on the cell surface can be used as a marker for apoptotic cells (M.A. O’Brien, et al., J Vet Emerg Crit Car 2008, 18(6):572 - 585; S.E. Logue, et al., Nat Protoc 2009, 4(9):1383 - 1395). Annexin A5 (AnxA5) is a phospholipid - binding protein that can bind to PS in a calcium - dependent manner (A.Q. Abbaady, et al., Front Physiol 2017, 8:317). Based on its high affinity for PS, at the nanomolar level, fluorescently labeled AnxA5 is widely used as an apoptosis detection probe (M. Stocker, et al., Protein Expr Purif 2008, 58(2):325 - 331). However, so far, most available and commercial AnxA5 probes are AnxA5 conjugated with fluorescent dyes. The preparation process of this type of probe involves complex operations and multiple purification steps, such as protein expression and purification, chemical conjugation reactions, and further removal of free fluorescein (A.Q. Abbaady, et al., Front Physiol 2017, 8:317; J. Wang, et al., Eur Biophys J 2015, 44(5):325 - 336). The whole process takes 3 days, including overnight dialysis. In addition, chemical conjugation always produces a heterogeneous mixture, and the labeled AnxA5 molecules differ in both the number and position of bound fluorescein. In addition, amine - directed chemical modification of AnxA5 may reduce its binding activity to the membrane (Tait JF, et al., J Nucl Med 2006, 47(9):1546 - 1553). Moreover, the complex preparation process means high production costs, which also increases the market price of the probe. More importantly, chemical dyes are prone to quenching and require strict light - avoidance treatment. The preparation process of this type of probe is complex and cumbersome, and its final product is usually a mixture with different labeling degrees, which is heterogeneous and has poor photostability, thus limiting their applications. Therefore, there is an urgent need to develop AnxA5 fluorescent probes with a simple preparation process, homogeneous end - products, and good photostability.

[0003] Since the green fluorescent protein (GFP) was isolated, it has been optimized to obtain the variant enhanced GFP (EGFP), which has extremely wide applications and thus occupies an important position (T.D. Craggs, Chem Soc Rev 2009, 38(10):2865 - 2875; W. Tao, et al., Stem Cells 2007, 25(3):670 - 678). By introducing different mutations into the GFP gene, many useful variants have been developed, such as blue, cyan, and yellow fluorescent proteins, etc. (R. Heim, et al., Curr Biol 1996, 6:178 - 182; H. Imamura, et al., PNAS 2023, 120(45):e2307687120). The emergence of these variants has greatly enriched the types of fluorescent proteins and broadened their application scope. In addition, fluorescent proteins are easy to use and inexpensive (Y.T. Kim, et al., Molecules 2022, 27(16):5248), and their genes can be mutated to improve various properties, such as excitation and emission wavelengths, brightness, pKa, maturation time, lifetime, and photostability. Previous work of the present invention confirmed that AnxA5 - EGFP has a higher affinity for membranes than AnxA5 - FITC (J. Wang, et al., Eur Biophys J 2015, 44(5):325 - 336), it labels uniformly, has higher brightness, and stronger photostability. In a study, sfGFP - AnxA5 was superior to fluorescein - conjugated AnxA5 in binding to phospholipids, making it more sensitive to label early apoptotic HeLa cells (A.Q. Abbaady, et al., Front Physiol 2017, 8:317). These studies have all demonstrated that AnxA5 based on fluorescent proteins is a promising apoptosis detection probe. Currently, the commercially available AnxA5 probes based on fluorescent proteins are only AnxA5 - EGFP (green fluorescence) and AnxA5 - mCherry (red fluorescence). Therefore, developing more AnxA5 probes based on fluorescent proteins with emission light ranges covering blue, cyan, and yellow is of great significance for expanding their application scope.

[0004] Fluorescent proteins have been used as tags for intracellular organelles and proteins, as well as components of fluorescent biosensors in gene encoding (L.M. Costantini, et al., Traffic 2012, 13(5):643 - 649; X.Y. Chen, et al., Adv Drug Deliv Rev 2013, 65(10):1357 - 1369; E.L. Snapp, Trends Cell Biol 2009, 19(11):649 - 655). The fluorescent protein tag is located at the C - terminus or N - terminus of the target protein and is generally considered to be functionally harmless. However, a recent study has shown that the choice of specific fluorescent protein is related to the function of the target protein (Y.W. Jiang, et al., Traffic 2017, 18(1):58 - 70). In addition, many fluorescent proteins tend to oligomerize (E.L. Snapp, Trends Cell Biol 2009, 19(11):649 - 655), and this property may affect the function of the target protein (L.M. Costantini, et al., Traffic 2012, 13(5):643 - 649; X.Y. Chen, et al., Adv Drug Deliv Rev 2013, 65(10):1357 - 1369). Therefore, when designing a strategy for generating fluorescent protein - tagged proteins, it is crucial to select the type of fluorescent protein to minimize interference with the function of the native protein. This ensures the optimization of experimental conditions and accurately reflects the function and characteristics of the native protein under undisturbed conditions. There are a wide variety of fluorescent proteins with different properties, and it is still unclear whether fusing with AnxA5 will affect its ability to label apoptosis and how it affects.

[0005] In summary, the present invention believes that fluorescent protein - labeled AnxA5 can be developed into a promising probe. However, whether the type of fluorescent protein will affect the function and characteristics of the AnxA5 protein and how it affects the function of AnxA5 have not had a clear answer so far. Therefore, analyzing the conformation of the fusion protein and its binding mode with PS from the perspective of structural biology helps to better understand the impact of fluorescent protein fusion on the function of AnxA5, especially on its affinity for PS. Methodological verification also provides strong support for structural insights, and there is no relevant report at present. These structural insights can provide guidance for the development of highly sensitive, fluorescent protein - based AnxA5 probes. Summary of the Invention

[0006] The object of the present invention is to provide a modeling method for the difference in the binding affinity of annexin A5 (AnxA5) fused with fluorescent proteins (FPs) to phosphatidylserine (PS) and its application.

[0007] To solve the problems of the prior art, the present invention provides the following technical solutions: In the first aspect of the present application, a method for modeling the difference in the binding affinity of annexin A5 (AnxA5) and fluorescent proteins (FPs) fused with phosphatidylserine (PS) is provided, characterized by comprising the following steps:

[0008] (1) Construct fusion proteins AnxA5-FPs of AnxA5 and different fluorescent proteins, and connect the C-terminus of AnxA5 and the N-terminus of the fluorescent protein through a linker peptide Linker by genetic engineering;

[0009] (2) Use a protein structure prediction tool to perform three-dimensional structure modeling on the AnxA5-FPs fusion protein to obtain a prediction model;

[0010] (3) Through molecular docking technology, perform binding simulation on the prediction model and the PS ligand, and analyze the binding energy and interaction mode;

[0011] (4) Based on the binding energy and interaction mode, screen out AnxA5-FPs fusion proteins with high affinity for PS.

[0012] Further, in step (1), the C-terminus of AnxA5 and the N-terminus of 18 fluorescent proteins are fused through a Gly-Ser4 linker peptide by genetic engineering;

[0013] In step (2), the protein structure prediction tool is AlphaFold2, and the following verification method is used to evaluate the credibility of the predicted structure:

[0014] The structural modeling used is performed by AlphaFold2, and the obtained prediction model is the rank0 model. The specific screening method is as follows: Use the alignment tool in PyMOL to calculate the root mean square deviation (RMSD) of Cα of each of the 5 prediction models of each fusion protein generated by AlphaFold2 (rank0-rank4), and perform inter-group comparison on all 5 sorted models. By sorting the models according to the average Cα RMSD, it is observed that the rank0 model always occupies the central position among the five prediction models of these six fusion proteins, that is, it is closest to the average Cα RMSD, which means that the rank0 model can represent the average conformation and can be selected for further analysis; then use Amber to relax the predicted structure to further optimize the structure.

[0015] (i) Calculate the predicted local distance difference test (pLDDT) and the predicted template modeling score (pTM);

[0016] (ii) Analyze the dihedral angle distribution of amino acid residues through Ramachandran plot; verify through the Structure Analysis and Verification Server SAVES v6.0, and evaluate using functions such as Ramachandran plot, ERRAT score, and Verify3D score; evaluate the reliability of the results through pLDDT and pTM scores. The pLDDT of the six modeled structures all exceed 90%, and the pTM score is approximately 0.6, proving that the models of the six fusion proteins are highly credible; the Ramachandran plot is also used to determine whether the protein conformation is reasonable by visualizing the dihedral angles of the main amino acid residues in the protein structure. For the modeling results of AnxA5-mCerulean3, AnxA5-TagRFP, AnxA5-mCherry, AnxA5-Venus, AnxA5-mVenus, and AnxA5-cpCitrine174, 92.70%, 92.90%, 92.40%, 92.30%, 91.90%, and 92.00% of the residues are located in the most favorable regions (red and yellow regions in the figure); ERRAT analyzes the statistics of non-bonded interactions between different atomic types, and the values of the six protein structures are approximately 95%, indicating that the six predicted structures are accurate and reliable.

[0017] (iii) Use ERRAT and Verify3D tools to verify the rationality of the structure.

[0018] Furthermore, in step (3), the molecular docking technology uses AutoDock Vina, specifically including:

[0019] (i) Dock the PS ligand with the predicted structure of AnxA5-FPs to simulate the binding conformation;

[0020] (ii) Calculate the binding energy (kcal / mol), and screen the conformation with the lowest binding energy as the optimal binding mode;

[0021] (iii) Analyze the hydrogen bonds, ionic interactions, and hydrophobic forces between AnxA5-FPs and PS.

[0022] Molecular docking was performed using the AutoDock vina server. Six predicted structures were defined as receptors, and PS was used as the ligand. The PS ligand is P5S, a phosphatidylserine analogue that exists in the complex crystal (PDB ID: 6SP2). Nine Ca2+ ions were added to the receptor by rewriting the PDB file according to the sample protein structure (PDB ID: 1A8A) because Annexins family proteins bind to PS based on calcium ions. Then, the docking complex was visualized using Pymol. Subsequently, 2D graphs were analyzed and deduced using Ligplot+v.2.2; the best docking pose was selected for all analyses; the binding energy was used to represent the affinity between the fusion protein and PS.

[0023] Furthermore, (5) according to the molecular docking results, site-directed mutagenesis was performed on specific amino acid residues involved in PS binding in the fluorescent protein to construct AnxA5-FPs mutants;

[0024] (6) The PS binding ability of the mutants was verified through experiments to confirm the accuracy of the structural model's prediction of affinity differences and to detect changes in their Kd values with PS.

[0025] Furthermore, in step (5), the site-directed mutagenesis was to mutate hydrophobic amino acids to hydrophilic amino acids, specifically including:

[0026] (i) Select residues in the fluorescent protein that form hydrophobic interactions with the fatty acid side chain of PS;

[0027] (ii) Mutate the residues to glycine Gly or serine Ser through gene editing technology to weaken the hydrophobic interaction;

[0028] In step (6), the Kd value of the AnxA5-mCerulean3 mutant was about two orders of magnitude higher than that of the wild type, indicating a significant reduction in its PS binding ability.

[0029] Furthermore, in step (6), based on the Kd values measured in claim 5, 5 AnxA5-FPs probes with the highest PS affinity were screened; the probes include AnxA5-TagBFP, AnxA5-mCerulean3, AnxA5-EGFP, AnxA5-Ypet, and AnxA5-TagRFP.

[0030] Furthermore, in step (6), when the fluorescent protein is mCerulean3, the mutation sites are Leu-338, Phe-339, Val-342, Val-343, Leu-346, Phe-445, and Val-451;

[0031] The fluorescent protein is selected from any one of 18 types including Venus, mVenus, Citrine, mCitrine, cpVenus173, cpCitrine174, EYFP, Ypet, mCherry, DsRed2, TagRFP, TagBFP, EBFP2, Cerulean, mCerulean, mCerulean3, EGFP, or ECFP;

[0032] The fluorescent proteins include fusion proteins AnxA5-Venus, AnxA5-mVenus, AnxA5-Citrine, AnxA5-mCitrine, AnxA5-cpVenus173, AnxA5-cpCitrine174, AnxA5-EYFP, AnxA5-Ypet, AnxA5-mCherry, AnxA5-Ypet, AnxA5-mCherry, AnxA5-DsRed2, AnxA5-TagRFP, AnxA5-TagBFP, AnxA5-EBFP2, AnxA5-Cerulean, AnxA5-mCerulean, AnxA5-mCerulean3, AnxA5-EGFP, AnxA5-ECFP, and AnxA5-Venus;

[0033] The nucleotide sequence of the fusion protein AnxA5-Venus is as shown in SEQ ID NO:1 in sequence;

[0034] The nucleotide sequence of the fusion protein AnxA5-mVenus is as shown in SEQ ID NO:2 in sequence;

[0035] The nucleotide sequence of the fusion protein AnxA5-Citrine is as shown in SEQ ID NO:3 in sequence;

[0036] The nucleotide sequence of the fusion protein AnxA5-mCitrine is as shown in SEQ ID NO:4 in sequence;

[0037] The nucleotide sequence of the fusion protein AnxA5-cpVenus173 is as shown in SEQ ID NO:5 in sequence;

[0038] The nucleotide sequence of the fusion protein AnxA5-cpCitrine174 is as shown in SEQ ID NO:6 in sequence;

[0039] The nucleotide sequence of the fusion protein AnxA5-EYFP is shown in SEQ ID NO:7 in sequence;

[0040] The nucleotide sequence of the fusion protein AnxA5-Ypet is shown in SEQ ID NO:8 in sequence;

[0041] The nucleotide sequence of the fusion protein AnxA5-mCherry is shown in SEQ ID NO:9 in sequence; The nucleotide sequence of the fusion protein AnxA5-Ypet is shown in SEQ ID NO:8 in sequence; The nucleotide sequence of the fusion protein AnxA5-mCherry is shown in SEQ ID NO:9 in sequence; The nucleotide sequence of the fusion protein AnxA5-DsRed2 is shown in SEQ ID NO:10 in sequence; The nucleotide sequence of the fusion protein AnxA5-TagRFP is shown in SEQ ID NO:11 in sequence; The nucleotide sequence of the fusion protein AnxA5-TagBFP is shown in SEQ ID NO:12 in sequence; The nucleotide sequence of the fusion protein AnxA5-EBFP2 is shown in SEQ ID NO:13 in sequence; The nucleotide sequence of the fusion protein AnxA5-Cerulean is shown in SEQ ID NO:14 in sequence; The nucleotide sequence of the fusion protein AnxA5-mCerulean is shown in SEQ ID NO:15 in sequence; The nucleotide sequence of the fusion protein AnxA5-mCerulean3 is shown in SEQ ID NO:16 in sequence; The nucleotide sequence of the fusion protein AnxA5-EGFP is shown in SEQ ID NO:17 in sequence; The nucleotide sequence of the fusion protein AnxA5-ECFP is shown in SEQ ID NO:18 in sequence; The nucleotide sequence of the fusion protein AnxA5-Venus is shown in SEQ ID NO:19 in sequence;

[0042] The amino acid sequences of the fusion proteins AnxA5-mVenus are shown successively as SEQ ID NO:20; the amino acid sequences of the fusion proteins AnxA5-Citrine are shown successively as SEQ ID NO:21; the amino acid sequences of the fusion proteins AnxA5-mCitrine are shown successively as SEQ ID NO:22; the amino acid sequences of the fusion proteins AnxA5-cpVenus173 are shown successively as SEQ ID NO:23; the amino acid sequences of the fusion proteins AnxA5-cpCitrine174 are shown successively as SEQ ID NO:24; the amino acid sequences of the fusion proteins AnxA5-EYFP are shown successively as SEQ ID NO:25; the amino acid sequences of the fusion proteins AnxA5-Ypet are shown successively as SEQ ID NO:26; the amino acid sequences of the fusion proteins AnxA5-mCherry are shown successively as SEQ ID NO:27; the nucleotide sequences of the fusion proteins AnxA5-DsRed2 are shown successively as SEQ ID NO:28; the nucleotide sequences of the fusion proteins AnxA5-TagRFP are shown successively as SEQ ID NO:29; the nucleotide sequences of the fusion proteins AnxA5-TagBFP are shown successively as SEQ ID NO:30; the nucleotide sequences of the fusion proteins AnxA5-EBFP2 are shown successively as SEQ ID NO:31; the nucleotide sequences of the fusion proteins AnxA5-Cerulean are shown successively as SEQ ID NO:32; the nucleotide sequences of the fusion proteins AnxA5-mCerulean are shown successively as SEQ ID NO:33; the nucleotide sequences of the fusion proteins AnxA5-mCerulean3 are shown successively as SEQ ID NO:34; the nucleotide sequences of the fusion proteins AnxA5-EGFP are shown successively as SEQ ID NO:35; the nucleotide sequences of the fusion proteins AnxA5-ECFP are shown successively as SEQ ID NO:36.

[0043] Eighteen fluorescent proteins were classified into three categories according to their sources, namely 14 fluorescent proteins derived from avGFP, 2 fluorescent proteins derived from eqFP578, and 2 fluorescent proteins derived from DsRed. These fluorescent proteins were all mutated and modified from the original progenitor proteins. By aligning the amino acid sequences of all fluorescent proteins in each category, it was found that the amino acid sequences were highly conserved among fluorescent proteins of the same source; using TBtools to perform amino acid sequence alignment and phylogenetic analysis of the 18 fluorescent proteins, a phylogenetic tree was generated, indicating that among the 18 fluorescent proteins, mVenus, TagBFP, TagRFP, DsRed2, and mCherry showed the highest similarity, while mCerulean, mCerulean3, mCitrine, and cpCitrine174 showed a greater distance compared to these fluorescent proteins;

[0044] In the present invention, the aggregation characteristics of the fusion protein were detected by dynamic light scattering experiment (DLS) and native polyacrylamide gel electrophoresis (Native-PAGE), indicating that the aggregation characteristics of the fluorescent protein is a factor affecting the PS affinity of AnxA5. Secondly, through homology modeling by AlphaFold2, the conformation of the fusion protein was obtained, indicating that different fluorescent proteins fused with AnxA5 would produce different conformations. Then, through molecular docking by AutoDock Vina, it was shown that this conformational difference would lead to different PS binding modes of the AnxA5-FPs fusion protein, and the aggregation characteristics of the fluorescent protein is a reason for the difference in PS affinity of AnxA5.

[0045] In the second aspect of the present application, a high-affinity AnxA5-FPs probe screened based on a modeling method is provided. The Kd value of the probe is 10 -7 M to 10 -9 M, and the detection sensitivity for early apoptotic cells is significantly higher than that of chemically labeled AnxA5-FITC; the probe is AnxA5-TagBFP, AnxA5-Cerulean, AnxA5-EGFP, AnxA5-Ypet, or AnxA5-TagRFP.

[0046] In the third aspect of the present application, an apoptosis detection kit is provided, which contains the high-affinity AnxA5-FPs probe described above and instructions for flow cytometry or fluorescence microscopy detection.

[0047] In the fourth aspect of the present application, an application of a cell apoptosis detection probe in flow cytometry, fluorescence microscopy detection, or in vivo PS externalization tracing is provided.

[0048] Beneficial effects: The present invention provides structural insights into the hundred-fold difference in the binding affinity to phosphatidylserine caused by the fusion of annexin A5 and fluorescent proteins, and for the first time, analyzes the potential effects of different fluorescent proteins on the function of AnxA5 from the perspective of structural biology. The structural insights of the present invention can more efficiently screen suitable fluorescent proteins for the preparation of high-affinity AnxA5 apoptosis detection probes for applications in the field of biotechnology.

[0049] Compared with the prior art, the present invention has the following advantages:

[0050] (1) The present invention discovers that the fusion of different fluorescent proteins can lead to a hundred-fold difference in the PS affinity of AnxA5, confirming the necessity of screening for high-affinity AnxA5 probes.

[0051] (2) The present invention, for the first time, analyzes from the perspective of structural biology the effects of different fluorescent proteins on the PS affinity of AnxA5, and the proposed structural insights provide a mechanistic explanation for the different PS affinities of AnxA5-FPs fusion proteins.

[0052] (3) The structural insights proposed by the present invention provide a reference for predicting the effects of more fluorescent proteins on the PS affinity of AnxA5. First, find suitable fluorescent proteins through predicted structures and then conduct experimental verification, so that high-PS-affinity AnxA5 probes can be more efficiently screened out, which is of great significance for the research in the field of cell apoptosis.

[0053] (4) The present invention comprehensively considers the types and sources of fluorescent proteins, the results of amino acid sequence alignment, and the PS affinity, and six AnxA5-FPs fusion proteins are selected for homology modeling and molecular docking. The fusion proteins are AnxA5-mCerulean3, AnxA5-TagRFP, AnxA5-mCherry, AnxA5-Venus, AnxA5-mVenus, and AnxA5-cpCitrine174. The present invention dissects and designs AnxA5-FPs fusion proteins with different PS affinity differences from the perspective of structural biology.

[0054] (5) The present invention can more efficiently screen suitable fluorescent proteins to prepare high-affinity, fluorescent protein-based AnxA5 apoptosis detection probes to meet the needs of biotechnology applications. Description of the Drawings

[0055] To more clearly illustrate the technical solutions in the embodiments of the present application, the following will briefly introduce the accompanying drawings required for use in the embodiments or the description of the prior art. Obviously, the accompanying drawings in the following description are only some embodiments of the present application. For those of ordinary skill in the art, without creative efforts, other accompanying drawings can be obtained based on these drawings.

[0056] Figure 1 Construction of the AnxA5-FPs expression plasmid of the present invention. (GS)4 serves as the fusion tag of AnxA5 and the fluorescent protein.

[0057] Figure 2 Excitation and emission spectra of the AnxA5-FPs fusion protein of the present invention

[0058] Among them, (A) Fusion protein of AnxA5 and green fluorescent protein; (2) Fusion protein of AnxA5 and 3 red fluorescent proteins; (C) Fusion protein of AnxA5 and 8 yellow fluorescent proteins.

[0059] Figure 3 Evaluation of the binding ability of AnxA5-FPs to apoptotic cell membranes by flow cytometry (taking AnxA5-EGFP as an example) of the present invention.

[0060] Among them, (A) The main gate R1 clears small debris. The R1 gate is selected from the dot plot of the flow cytometry results detected from apoptotic Jurkat cells for further analysis. Scatter plot of Jurkat cells in the R1 gate after being labeled with AnxA5-FPs. (B) Dot plot of flow cytometry for detecting apoptosis of Jurkat cells. The R2 event includes AnxA5-FPs positive cells, which may include apoptotic cells and necrotic cells. The R2 gate is used for further analysis of the mean fluorescence intensity. (C) Analysis of the mean fluorescence intensity of different concentrations of AnxA5-FPs binding to apoptotic cell membranes. Relationship between the PBI value and the protein concentration. Data are presented as mean ± SD.

[0061] Figure 4 Analysis of the affinity of AnxA5-FPs of the present invention for PS exposed on the cell surface (Jurkat cells are induced by etoposide).

[0062] Among them, (A)-(E) Fitting curves of the fluorescence intensity of AnxA5-FPs binding to PS exposed on the cell surface with different concentrations of AnxA5-FPs; (F) Kd value analysis based on the fitting curve of AnxA5-FPs binding to PS exposed on the cell surface. PBI: Protein binding ability index, representing the normalized fluorescence intensity of AnxA5-FPs binding to apoptotic cells. Data are shown as mean ± SD.

[0063] Figure 5Analysis of the affinity of AnxA5-FPs of the present invention for PS exposed on the cell surface (Jurkat cells were induced by camptothecin).

[0064] Among them, (A)-(E) Fitting curves of the fluorescence intensity of AnxA5-FPs binding to PS exposed on the cell surface with different concentrations of AnxA5-FPs; (F) Kd value analysis based on the fitting curves of AnxA5-FPs binding to PS exposed on the cell surface. PBI: Protein binding ability index, representing the normalized fluorescence intensity of AnxA5-FPs binding to apoptotic cells. Data are shown as mean ± SD.

[0065] Figure 6 Particle size distribution of the AnxA5-FPs fusion protein of the present invention.

[0066] Among them, (A) AnxA5 is fused with green fluorescent protein; (2) AnxA5 is fused with 8 yellow fluorescent proteins; (C) AnxA5 is fused with 3 red fluorescent proteins.

[0067] Figure 7 Native-PAGE analysis results of the AnxA5-FPs fusion protein of the present invention. The Kd value (×10-7, mol / L) of PS exposed on the cell surface binding to AnxA5-FPs is shown in red below the corresponding protein.

[0068] Figure 8 Sources and modification situations of 18 fluorescent proteins (FPs) of the present invention. Origins and engineering processes of 18 fluorescent proteins. Blue fluorescent protein (EBFP2), cyan fluorescent protein, green fluorescent protein (EGFP) and yellow fluorescent protein are modified from avGFP. Red fluorescent protein (TagRFP) and blue fluorescent protein (TagBFP) are derived from eqFP578. Red fluorescent proteins (DsRed2 and mCherry) are both derived from DsRed. The colors in the circles correspond to the emission wavelengths of the proteins. The Kd values of AnxA5-FPs binding to PS are shown in purple below the corresponding proteins.

[0069] Figure 9 Multiple sequence alignment of fluorescent proteins modified from avGFP of the present invention. The amino acid sequence of AnxA5 is not shown.

[0070] Figure 10 Multiple sequence alignment of fluorescent proteins modified from eqFP578 of the present invention. The amino acid sequence of AnxA5 is not shown.

[0071] Figure 11 Multiple sequence alignment of fluorescent proteins modified from DsRed of the present invention. The amino acid sequence of AnxA5 is not shown.

[0072] Figure 12 Phylogenetic tree of 18 fluorescent proteins (FPs) of the present invention, relative affinity constants (Kd) of 18 AnxA5-FPs, and homology models of six representative AnxA5-FPs generated by AlphaFold2.

[0073] Among them, (A) Ramachandran plot: Red, yellow, and white represent residues located in the favorable, allowed, and disallowed regions, respectively; (B) Surface plot shows the overall structure. AnxA5 protein is shown in gray, and calcium ions are shown in pink. The schematic diagram shows the size of six AnxA5-FPs, the angle, and the centroid distance between AnxA5 and the fluorescent protein.

[0074] Figure 13 Overall structures of the remaining 12 AnxA5-FPs fusion proteins generated by AlphaFold2 of the present invention. The cartoon shows the overall structure of the fusion protein. AnxA5 protein is shown in sky blue. Fluorescent proteins are shown in different colors and exhibit a typical β-barrel structure.

[0075] Figure 14 Binding surface and polar contacts of the AnxA5-FPs fusion protein of the present invention with PS. PS: Shown in samarium color. Calcium ions are shown in pink. The darker the color, the stronger the hydrophobicity.

[0076] Figure 15 Two-dimensional (2D) display results of the interaction forces between six AnxA5-FPs fusion proteins of the present invention and PS.

[0077] Figure 16 Expression of the AnxA5-mCerulean3 mutant of the present invention and its binding affinity to apoptotic cells.

[0078] Among them, (A) Amino acid sequences of mCerulean3 WT and mCerulean3 mutants; (B) SDS-PAGE analysis of purified AnxA5-mCerulean3 mutants; (C) Binding curves of AnxA5-mCerulean3 WT and AnxA5-mCerulean3 mutants to etoposide-induced apoptotic cells; (D) Binding curves of AnxA5-mCerulean3 WT and AnxA5-mCerulean3 mutants to camptothecin-induced apoptotic cells; (E) Comparison of the binding abilities of AnxA5-mCerulean3 WT and AnxA5-mCerulean3 mutants. Data are shown as mean ± SD. ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05.

[0079] Figure 17 To analyze the apoptosis detection ability of five AnxA5-FPs with high PS affinity by fluorescence microscopy.

[0080] Among them, (A) Five AnxA5-FPs were selected according to their PS affinity (the binding affinity of AnxA5-FPs to etoposide-induced apoptotic cells). Fluorescence microscopy analysis of apoptotic cells labeled with different concentrations of (B) AnxA5-TagBFP, (C) AnxA5-mCerulean3, (D) AnxA5-EGFP, (E) AnxA5-Ypet, and (F) AnxA5-TagRFP. Concentration note: The concentrations of the working solutions of the five AnxA5-FPs. These working solutions were diluted 20-fold to their final working concentrations.

[0081] Figure 18 To analyze the apoptosis detection ability of five AnxA5-FPs with high PS affinity by flow cytometry.

[0082] Among them, (A) Flow cytometry detection results of apoptotic cells labeled with different concentrations of AnxA5-TagBFP; (B) The proportion of AnxA5-TagBFP-positive cells after apoptotic cells were labeled with different concentrations of AnxA5-TagBFP; (C) Flow cytometry detection results of apoptotic cells labeled with different concentrations of AnxA5-mCerulean3; (D) The proportion of AnxA5-mCerulean3-positive cells after apoptotic cells were labeled with different concentrations of AnxA5-mCerulean3; (E) Flow cytometry detection results of apoptotic cells labeled with different concentrations of AnxA5-EGFP; (F) The proportion of AnxA5-EGFP-positive cells after apoptotic cells were labeled with different concentrations of AnxA5-EGFP; (G) Flow cytometry detection results of apoptotic cells labeled with different concentrations of AnxA5-Ypet; (H) The proportion of AnxA5-Ypet-positive cells after apoptotic cells were labeled with different concentrations of AnxA5-Ypet; (I) Flow cytometry detection results of apoptotic cells labeled with different concentrations of AnxA5-TagRFP; (J) The proportion of AnxA5-TagRFP-positive cells after apoptotic cells were labeled with different concentrations of AnxA5-TagRFP. Concentration note: The concentrations of the working solutions of the five AnxA5-FPs. These working solutions were diluted 20-fold to their final working concentrations. Data are expressed as mean ± standard deviation. Detailed implementation manner

[0083] In order to make the technical problems to be solved, technical solutions and beneficial effects of this application more clearly understood, the following further details this application in combination with embodiments. It should be understood that the specific embodiments described herein are only used to explain this application and are not used to limit this application.

[0084] In this application, the term "and / or" describes the association relationship of associated objects and indicates that three relationships may exist. For example, A and / or B may represent: A exists alone, A and B exist simultaneously, or B exists alone. Here, A and B may be singular or plural. The character " / " generally represents an "or" relationship between the associated objects before and after.

[0085] In this application, "at least one" means one or more, and "a plurality" means two or more. "At least one of the following" or its similar expressions refer to any combination of these items, including any combination of single item(s) or plural item(s). For example, "at least one of a, b, or c", or "at least one of a, b, and c" can both represent: a, b, c, a - b (i.e., a and b), a - c, b - c, or a - b - c, where a, b, and c can be single or plural respectively.

[0086] It should be understood that in various embodiments of this application, the magnitudes of the sequence numbers of the above processes do not mean the order of execution. Some or all of the steps can be executed in parallel or sequentially. The execution order of each process should be determined according to its function and internal logic, and should not constitute any limitation to the implementation process of the embodiments of this application.

[0087] The terms used in the embodiments of this application are only for the purpose of describing specific embodiments and are not intended to limit this application. The singular forms "a", "the", and "said" used in the embodiments of this application and the appended claims are also intended to include the plural forms unless the context clearly indicates otherwise.

[0088] The weights of the relevant components mentioned in the specification of the embodiments of this application not only can refer to the specific contents of each component, but also can represent the proportional relationship of the weights between each component. Therefore, as long as the contents of the relevant components in the specification of the embodiments of this application are scaled up or down in proportion, they are within the scope disclosed in the specification of the embodiments of this application. Specifically, the mass mentioned in the specification of the embodiments of this application can be mass units well-known in the chemical industry such as μg, mg, g, kg, etc.

[0089] The terms "first" and "second" are used only for descriptive purposes to distinguish objects such as substances from each other, and should not be construed as indicating or implying relative importance or implicitly specifying the quantity of the indicated technical features. For example, without departing from the scope of the embodiments of the present application, the first XX may also be referred to as the second XX, and similarly, the second XX may also be referred to as the first XX. Thus, the features defined with "first" and "second" may explicitly or implicitly include one or more of such features.

[0090] The following embodiments are helpful for further understanding the present invention, but the present invention is not limited thereto.

[0091] The first aspect of the embodiments of the present application provides a modeling method for the difference in the binding affinity of annexin A5 (AnxA5) fused with fluorescent proteins (FPs) to phosphatidylserine (PS), which is characterized by including the following steps:

[0092] (1) Construct fusion proteins AnxA5-FPs of AnxA5 and different fluorescent proteins, and connect the C-terminus of AnxA5 and the N-terminus of the fluorescent protein through a linker peptide Linker by genetic engineering;

[0093] (2) Use a protein structure prediction tool to perform three-dimensional structure modeling on the AnxA5-FPs fusion protein to obtain a prediction model;

[0094] (3) Perform binding simulation of the prediction model with the PS ligand through molecular docking technology, and analyze the binding energy and interaction mode;

[0095] (4) Based on the binding energy and interaction mode, screen out the AnxA5-FPs fusion protein with high affinity for PS.

[0096] In some embodiments, in step (1), the C-terminus of AnxA5 and the N-terminus of 18 fluorescent proteins are fused through a Gly-Ser4 linker peptide by genetic engineering;

[0097] In step (2), the protein structure prediction tool is AlphaFold2, and the following verification method is used to evaluate the credibility of the predicted structure:

[0098] The structural modeling used was performed by AlphaFold2, and the obtained prediction model was the rank0 model. The specific screening method is as follows: Calculate the root mean square deviation (RMSD) of the Cα atoms of the 5 prediction models of each fusion protein generated by AlphaFold2 (rank0 - rank4) using the alignment tool in PyMOL, and perform an inter-group comparison of all 5 ranked models. By ranking the models according to the average Cα RMSD, it was observed that the rank0 model always occupied the central position among the five prediction models of these six fusion proteins, that is, it was closest to the average Cα RMSD, which means that the rank0 model can represent the average conformation and can be selected for further analysis; then use Amber to relax the predicted structure to further optimize the structure.

[0099] (i) Calculate the predicted local distance difference test pLDDT and the predicted template modeling score pTM;

[0100] (ii) Analyze the dihedral angle distribution of amino acid residues through a Ramachandran plot; verify through the Structure Analysis and Verification Server SAVES v6.0, and evaluate using functions such as the Ramachandran plot, ERRAT score, and Verify3D score; evaluate the credibility of the results through the pLDDT and pTM scores. The pLDDT of the six modeled structures all exceeded 90%, and the pTM score was approximately 0.6, proving that the models of the six fusion proteins are highly credible; the Ramachandran plot was also used to determine whether the protein conformation is reasonable by visualizing the dihedral angles of the main amino acid residues in the protein structure. For the modeling results of AnxA5-mCerulean3, AnxA5-TagRFP, AnxA5-mCherry, AnxA5-Venus, AnxA5-mVenus, and AnxA5-cpCitrine174, 92.70%, 92.90%, 92.40%, 92.30%, 91.90%, and 92.00% of the residues were located in the most favorable regions (the red and yellow regions in the figure); ERRAT analyzed the statistics of non-bonded interactions between different atom types, and the values of the six protein structures were approximately 95%, indicating that the six predicted structures are accurate and reliable.

[0101] (iii) Use the ERRAT and Verify3D tools to verify the rationality of the structure.

[0102] In some embodiments, in step (3), the molecular docking technology uses AutoDock Vina, specifically including:

[0103] (i) Dock the PS ligand with the predicted structure of AnxA5-FPs to simulate the binding conformation;

[0104] (ii) Calculate the binding energy (kcal / mol) and select the conformation with the lowest binding energy as the optimal binding mode;

[0105] (iii) Analyze the hydrogen bonds, ionic interactions, and hydrophobic forces between AnxA5-FPs and PS.

[0106] Molecular docking was performed using the AutoDock vina server. Six predicted structures were defined as the receptors, and PS was used as the ligand. The PS ligand was P5S, a phosphatidylserine analogue present in the complex crystal (PDB ID: 6SP2). Nine Ca2+ were added to the receptor by rewriting the PDB file according to the sample protein structure (PDB ID: 1A8A) because Annexins family proteins bind to PS based on calcium ions. Then, the docking complex was visualized using Pymol. Subsequently, 2D maps were analyzed and deduced using Ligplot+v.2.2; the best docking pose was selected for all analyses; the binding energy was used to represent the affinity between the fusion protein and PS.

[0107] In some embodiments, (5) Based on the molecular docking results, site-directed mutagenesis was performed on specific amino acid residues involved in PS binding in the fluorescent protein to construct AnxA5-FPs mutants;

[0108] (6) The PS binding ability of the mutants was verified by experiments to confirm the prediction accuracy of the structural model for the affinity difference and to detect the change in its Kd value with PS.

[0109] In some embodiments, in step (5), the site-directed mutagenesis was to mutate hydrophobic amino acids to hydrophilic amino acids, specifically including:

[0110] (i) Select the residues in the fluorescent protein that form hydrophobic interactions with the fatty acid side chain of PS;

[0111] (ii) Mutate the residues to glycine (Gly) or serine (Ser) through gene editing technology to weaken the hydrophobic interaction;

[0112] In step (6), the Kd value of the AnxA5-mCerulean3 mutant was about two orders of magnitude higher than that of the wild type, indicating a significant reduction in its PS binding ability.

[0113] In some embodiments, in step (6), (a) Based on the Kd values measured in claim 5, the 5 AnxA5-FPs probes with the highest PS affinity were screened;

[0114] (b) The probes include AnxA5-TagBFP, AnxA5-mCerulean3, AnxA5-EGFP, AnxA5-Ypet, and AnxA5-TagRFP.

[0115] Further, in step (6), when the fluorescent protein is mCerulean3, the mutation sites are Leu-338, Phe-339, Val-342, Val-343, Leu-346, Phe-445, and Val-451;

[0116] The fluorescent protein is selected from any one of 18 types including Venus, mVenus, Citrine, mCitrine, cpVenus173, cpCitrine174, EYFP, Ypet, mCherry, DsRed2, TagRFP, TagBFP, EBFP2, Cerulean, mCerulean, mCerulean3, EGFP, or ECFP;

[0117] The fluorescent proteins include the fusion protein AnxA5-Venus, the fusion protein AnxA5-mVenus, the fusion protein AnxA5-Citrine, the fusion protein AnxA5-mCitrine, the fusion protein AnxA5-cpVenus173, the fusion protein AnxA5-cpCitrine174, the fusion protein AnxA5-EYFP, the fusion protein AnxA5-Ypet, the fusion protein AnxA5-mCherry, the fusion protein AnxA5-Ypet, the fusion protein AnxA5-mCherry, the fusion protein AnxA5-DsRed2, the fusion protein AnxA5-TagRFP, the fusion protein AnxA5-TagBFP, the fusion protein AnxA5-EBFP2, the fusion protein AnxA5-Cerulean, the fusion protein AnxA5-mCerulean, the fusion protein AnxA5-mCerulean3, the fusion protein AnxA5-EGFP, the fusion protein AnxA5-ECFP, and the fusion protein AnxA5-Venus;

[0118] The nucleotide sequence of the fusion protein AnxA5-Venus is shown in SEQ ID NO:1 in sequence;

[0119] The nucleotide sequence of the fusion protein AnxA5-mVenus is shown in SEQ ID NO:2 in sequence;

[0120] The nucleotide sequence of the fusion protein AnxA5-Citrine is shown in SEQ ID NO:3 in sequence;

[0121] The nucleotide sequence of the fusion protein AnxA5-mCitrine is shown in SEQ ID NO: 4;

[0122] The nucleotide sequence of the fusion protein AnxA5-cpVenus173 is shown in SEQ ID NO: 5;

[0123] The nucleotide sequence of the fusion protein AnxA5-cpCitrine174 is shown in SEQ ID NO: 6;

[0124] The nucleotide sequence of the fusion protein AnxA5-EYFP is shown in SEQ ID NO: 7;

[0125] The nucleotide sequence of the fusion protein AnxA5-Ypet is shown in SEQ ID NO: 8;

[0126] The nucleotide sequence of the fusion protein AnxA5-mCherry is shown in SEQ ID NO: 9; the nucleotide sequence of the fusion protein AnxA5-Ypet is shown in SEQ ID NO: 8; the nucleotide sequence of the fusion protein AnxA5-mCherry is shown in SEQ ID NO: 9; the nucleotide sequence of the fusion protein AnxA5-DsRed2 is shown in SEQ ID NO: 10; the nucleotide sequence of the fusion protein AnxA5-TagRFP is shown in SEQ ID NO: 11; the nucleotide sequence of the fusion protein AnxA5-TagBFP is shown in SEQ ID NO: 12; the nucleotide sequence of the fusion protein AnxA5-EBFP2 is shown in SEQ ID NO: 13; the nucleotide sequence of the fusion protein AnxA5-Cerulean is shown in SEQ ID NO: 14; the nucleotide sequence of the fusion protein AnxA5-mCerulean is shown in SEQ ID NO: 15; the nucleotide sequence of the fusion protein AnxA5-mCerulean3 is shown in SEQ ID NO: The nucleotide sequence of the fusion protein AnxA5-EGFP is shown in SEQ ID NO: 16; the nucleotide sequence of the fusion protein AnxA5-EGFP is shown in SEQ ID NO: 17; the nucleotide sequence of the fusion protein AnxA5-ECFP is shown in SEQ ID NO: 18; the nucleotide sequence of the fusion protein AnxA5-Venus is shown in SEQ ID NO: 19;

[0127] The amino acid sequence of the fusion protein AnxA5-mVenus is shown in SEQ ID NO: 20; the amino acid sequence of the fusion protein AnxA5-Citrine is shown in SEQ ID NO: 21; the amino acid sequence of the fusion protein AnxA5-mCitrine is shown in SEQ ID NO: 22; the amino acid sequence of the fusion protein AnxA5-cpVenus173 is shown in SEQ ID NO: 23; the amino acid sequence of the fusion protein AnxA5-cpCitrine174 is shown in SEQ ID NO: 24; the amino acid sequence of the fusion protein AnxA5-EYFP is shown in SEQ ID NO: 25; the amino acid sequence of the fusion protein AnxA5-Ypet is shown in SEQ ID NO: 26; the amino acid sequence of the fusion protein AnxA5-mCherry is shown in SEQ ID NO: 27; the nucleotide sequence of the fusion protein AnxA5-DsRed2 is shown in SEQ ID NO: 28; the nucleotide sequence of the fusion protein AnxA5-TagRFP is shown in SEQ ID NO: NO:29; the nucleotide sequence of the fusion protein AnxA5-TagBFP is shown in SEQ ID NO:30; the nucleotide sequence of the fusion protein AnxA5-EBFP2 is shown in SEQ ID NO:31; the nucleotide sequence of the fusion protein AnxA5-Cerulean is shown in SEQ ID NO:32; the nucleotide sequence of the fusion protein AnxA5-mCerulean is shown in SEQ ID NO:33; the nucleotide sequence of the fusion protein AnxA5-mCerulean3 is shown in SEQ ID NO:34; the nucleotide sequence of the fusion protein AnxA5-EGFP is shown in SEQ ID NO:35; and the nucleotide sequence of the fusion protein AnxA5-ECFP is shown in SEQ ID NO:36.

[0128] Eighteen fluorescent proteins were classified into three categories according to their sources, namely 14 fluorescent proteins derived from avGFP, 2 fluorescent proteins derived from eqFP578, and 2 fluorescent proteins derived from DsRed. These fluorescent proteins were all mutated and modified from the original progenitor proteins. By aligning the amino acid sequences of all fluorescent proteins in each category, it was found that the amino acid sequences were highly conserved among fluorescent proteins of the same source; TBtools was used to align the amino acid sequences of the 18 fluorescent proteins and perform phylogenetic analysis to generate a phylogenetic tree, indicating that among the 18 fluorescent proteins, mVenus, TagBFP, TagRFP, DsRed2, and mCherry showed the highest similarity, while mCerulean, mCerulean3, mCitrine, and cpCitrine174 showed a greater distance compared to these fluorescent proteins;

[0129] In this invention, the aggregation characteristics of the fusion proteins were detected by dynamic light scattering experiments (DLS) and native polyacrylamide gel electrophoresis (Native-PAGE), indicating that the aggregation characteristics of fluorescent proteins are a factor affecting the PS affinity of AnxA5. Secondly, through homology modeling by AlphaFold2, the conformation of the fusion proteins was obtained, indicating that different fluorescent proteins fused with AnxA5 would produce different conformations. Then, molecular docking was performed by AutoDock Vina, indicating that such conformational differences would lead to different PS binding modes of the AnxA5-FPs fusion proteins, and the aggregation characteristics of fluorescent proteins are a reason for the differences in PS affinity of AnxA5.

[0130] In the second aspect of the embodiments of this application, a high-affinity AnxA5-FPs probe screened based on a modeling method is provided. The Kd value of the probe is 10 -7 M to 10 -9 M, and the detection sensitivity for early apoptotic cells is significantly higher than that of chemically labeled AnxA5-FITC; the probe is AnxA5-TagBFP, AnxA5-Cerulean, AnxA5-EGFP, AnxA5-Ypet, or AnxA5-TagRFP.

[0131] In the third aspect of the embodiments of this application, an apoptosis detection kit is provided, which includes the above-mentioned high-affinity AnxA5-FPs probe and instructions for flow cytometry or fluorescence microscopy detection.

[0132] In the fourth aspect of the embodiments of this application, an application of a cell apoptosis detection probe in flow cytometry, fluorescence microscopy detection, or in vivo PS externalization tracing is provided.

[0133] Example 1

[0134] A modeling method for the fusion of annexin A5 (AnxA5) and fluorescent proteins (FPs) in the present invention, which results in differences in binding affinity to phosphatidylserine (PS), is characterized by the following steps:

[0135] (1) Construct fusion proteins AnxA5-FPs of AnxA5 and different fluorescent proteins, and connect the C-terminus of AnxA5 and the N-terminus of the fluorescent protein through a linker peptide Linker by genetic engineering; fuse the C-terminus of AnxA5 and the N-terminus of 18 fluorescent proteins through a Gly-Ser4 linker peptide by genetic engineering.

[0136] (2) Use a protein structure prediction tool to perform three-dimensional structure modeling on the AnxA5-FPs fusion protein to obtain a prediction model; the protein structure prediction tool is AlphaFold2, and the reliability of the predicted structure is evaluated through the following verification methods:

[0137] The structural modeling used is carried out by AlphaFold2, and the obtained prediction model is the rank0 model. The specific screening method is as follows: Calculate the root mean square deviation (RMSD) of Cα of the 5 prediction models of each fusion protein generated by AlphaFold2 (rank0-rank4) using the alignment tool in PyMOL, and perform an inter-group comparison of all 5 sorted models. By sorting the models according to the average Cα RMSD, it is observed that the rank0 model always occupies the central position among the five prediction models of these six fusion proteins, that is, it is closest to the average Cα RMSD, which means that the rank0 model can represent the average conformation and can be selected for further analysis; then use Amber to relax the predicted structure to further optimize the structure.

[0138] (i) Calculate the predicted local distance difference test pLDDT and the predicted template modeling score pTM.

[0139] (ii) Analyze the dihedral angle distribution of amino acid residues through a Ramachandran plot; verify through the Structure Analysis and Verification Server SAVES v6.0, and evaluate using functions such as the Ramachandran plot, ERRAT score, and Verify3D score; evaluate the reliability of the results through pLDDT and pTM scores. The pLDDT of the six modeled structures all exceeded 90%, and the pTM score was approximately 0.6, proving that the models of the six fusion proteins are highly credible; the Ramachandran plot is also used to determine whether the protein conformation is reasonable by visualizing the dihedral angles of the main amino acid residues in the protein structure. For the modeling results of AnxA5-mCerulean3, AnxA5-TagRFP, AnxA5-mCherry, AnxA5-Venus, AnxA5-mVenus, and AnxA5-cpCitrine174, 92.70%, 92.90%, 92.40%, 92.30%, 91.90%, and 92.00% of the residues are located in the most favorable regions (red and yellow regions in the figure); ERRAT analyzes the statistics of non-bonded interactions between different atom types, and the values of the six protein structures are approximately 95%, indicating that the six predicted structures are accurate and reliable.

[0140] (iii) Use the ERRAT and Verify3D tools to verify the rationality of the structure.

[0141] (3) Combine and simulate the predicted model with the PS ligand through molecular docking technology, and analyze the binding energy and interaction mode; the molecular docking technology uses AutoDock Vina, and performs molecular docking using the AutoDock vina server. Define the six predicted structures as receptors and PS as the ligand. The PS ligand is P5S, a phosphatidylserine analogue, which exists in the composite crystal (PDB ID: 6SP2). According to the sample protein structure (PDB ID: 1A8A), nine Ca2+ ions were added to the receptor by rewriting the PDB file because Annexins family proteins bind to PS based on calcium ions. Then, use Pymol to visualize the docking complex, and then use Ligplot+v.2.2 to analyze and deduce the 2D graph; the best docking pose is selected for all the analyses; the binding energy is used to represent the affinity between the fusion protein and PS.

[0142] (i) Dock the PS ligand with the predicted structure of AnxA5-FPs to simulate the binding conformation;

[0143] (ii) Calculate the binding energy (kcal / mol), and screen the conformation with the lowest binding energy as the optimal binding mode;

[0144] (iii) Analysis of hydrogen bonding, ionic interactions, and hydrophobic forces between AnxA5-FPs and PS.

[0145] (4) Based on the binding energy and interaction pattern, AnxA5-FPs fusion protein with high affinity to PS was screened.

[0146] (5) Based on the molecular docking results, site-directed mutagenesis was performed on the specific amino acid residues involved in PS binding in the fluorescent protein to construct an AnxA5-FPs mutant; the site-directed mutagenesis was to mutate the hydrophobic amino acids into hydrophilic amino acids, specifically including:

[0147] (i) selecting residues in the fluorescent protein that form hydrophobic interactions with the fatty acid side chains of PS;

[0148] (ii) mutating the residue to glycine (Gly) or serine (Ser) by gene editing technology to weaken the hydrophobic interaction;

[0149] (6) The PS binding ability of the mutant was verified experimentally, the accuracy of the structural model's prediction of affinity differences was confirmed, and the changes in its Kd value with PS were detected.

[0150] The Kd value of the AnxA5-mCerulean3 mutant was approximately two orders of magnitude higher than that of the wild type, indicating that its PS binding ability was significantly reduced.

[0151] In some embodiments, in step (6), based on the measured Kd values, five AnxA5-FPs probes with the highest PS affinity are screened; the probes include AnxA5-TagBFP, AnxA5-mCerulean3, AnxA5-EGFP, AnxA5-Ypet and AnxA5-TagRFP.

[0152] Furthermore, in step (6), when the fluorescent protein is mCerulean3, the mutation sites are Leu-338, Phe-339, Val-342, Val-343, Leu-346, Phe-445, and Val-451;

[0153] The fluorescent protein is selected from any one of 18 types: Venus, mVenus, Citrine, mCitrine, cpVenus173, cpCitrine174, EYFP, Ypet, mCherry, DsRed2, TagRFP, TagBFP, EBFP2, Cerulean, mCerulean, mCerulean3, EGFP, or ECFP;

[0154] The fluorescent proteins include the fusion proteins AnxA5-Venus, AnxA5-mVenus, AnxA5-Citrine, AnxA5-mCitrine, AnxA5-cpVenus173, AnxA5-cpCitrine174, AnxA5-EYFP, AnxA5-Ypet, AnxA5-mCherry, AnxA5-Ypet, AnxA5-mCherry, AnxA5-DsRed2, AnxA5-TagRFP, AnxA5-TagBFP, AnxA5-EBFP2, AnxA5-Cerulean, AnxA5-mCerulean, AnxA5-mCerulean3, AnxA5-EGFP, AnxA5-ECFP, and AnxA5-Venus;

[0155] The nucleotide sequence of the fusion protein AnxA5-Venus is shown in SEQ ID NO:1 in sequence;

[0156] The nucleotide sequence of the fusion protein AnxA5-mVenus is shown in SEQ ID NO:2 in sequence;

[0157] The nucleotide sequence of the fusion protein AnxA5-Citrine is shown in SEQ ID NO:3 in sequence;

[0158] The nucleotide sequence of the fusion protein AnxA5-mCitrine is shown in SEQ ID NO:4 in sequence;

[0159] The nucleotide sequence of the fusion protein AnxA5-cpVenus173 is shown in SEQ ID NO:5 in sequence;

[0160] The nucleotide sequence of the fusion protein AnxA5-cpCitrine174 is shown in SEQ ID NO:6 in sequence;

[0161] The nucleotide sequence of the fusion protein AnxA5-EYFP is shown in SEQ ID NO:7 in sequence;

[0162] The nucleotide sequence of the fusion protein AnxA5-Ypet is shown in SEQ ID NO:8 in sequence;

[0163] The nucleotide sequences of the fusion proteins AnxA5-mCherry are shown in SEQ ID NO:9 in sequence; the nucleotide sequences of the fusion proteins AnxA5-Ypet are shown in SEQ ID NO:8 in sequence; the nucleotide sequences of the fusion proteins AnxA5-mCherry are shown in SEQ ID NO:9 in sequence; the nucleotide sequences of the fusion proteins AnxA5-DsRed2 are shown in SEQ ID NO:10 in sequence; the nucleotide sequences of the fusion proteins AnxA5-TagRFP are shown in SEQ ID NO:11 in sequence; the nucleotide sequences of the fusion proteins AnxA5-TagBFP are shown in SEQ ID NO:12 in sequence; the nucleotide sequences of the fusion proteins AnxA5-EBFP2 are shown in SEQ ID NO:13 in sequence; the nucleotide sequences of the fusion proteins AnxA5-Cerulean are shown in SEQ ID NO:14 in sequence; the nucleotide sequences of the fusion proteins AnxA5-mCerulean are shown in SEQ ID NO:15 in sequence; the nucleotide sequences of the fusion proteins AnxA5-mCerulean3 are shown in SEQ ID NO:16 in sequence; the nucleotide sequences of the fusion proteins AnxA5-EGFP are shown in SEQ ID NO:17 in sequence; the nucleotide sequences of the fusion proteins AnxA5-ECFP are shown in SEQ ID NO:18 in sequence; the nucleotide sequences of the fusion proteins AnxA5-Venus are shown in SEQ ID NO:19 in sequence;

[0164] The amino acid sequence of the fusion protein AnxA5-mVenus is shown in SEQ ID NO: 20; the amino acid sequence of the fusion protein AnxA5-Citrine is shown in SEQ ID NO: 21; the amino acid sequence of the fusion protein AnxA5-mCitrine is shown in SEQ ID NO: 22; the amino acid sequence of the fusion protein AnxA5-cpVenus173 is shown in SEQ ID NO: 23; the amino acid sequence of the fusion protein AnxA5-cpCitrine174 is shown in SEQ ID NO: 24; the amino acid sequence of the fusion protein AnxA5-EYFP is shown in SEQ ID NO: 25; the amino acid sequence of the fusion protein AnxA5-Ypet is shown in SEQ ID NO: 26; the amino acid sequence of the fusion protein AnxA5-mCherry is shown in SEQ ID NO: 27; the nucleotide sequence of the fusion protein AnxA5-DsRed2 is shown in SEQ ID NO: 28; the nucleotide sequence of the fusion protein AnxA5-TagRFP is shown in SEQ ID NO: NO:29; the nucleotide sequence of the fusion protein AnxA5-TagBFP is shown in SEQ ID NO:30; the nucleotide sequence of the fusion protein AnxA5-EBFP2 is shown in SEQ ID NO:31; the nucleotide sequence of the fusion protein AnxA5-Cerulean is shown in SEQ ID NO:32; the nucleotide sequence of the fusion protein AnxA5-mCerulean is shown in SEQ ID NO:33; the nucleotide sequence of the fusion protein AnxA5-mCerulean3 is shown in SEQ ID NO:34; the nucleotide sequence of the fusion protein AnxA5-EGFP is shown in SEQ ID NO:35; and the nucleotide sequence of the fusion protein AnxA5-ECFP is shown in SEQ ID NO:36.

[0165] Eighteen fluorescent proteins were classified into three categories according to their sources, namely 14 fluorescent proteins derived from avGFP, 2 fluorescent proteins derived from eqFP578, and 2 fluorescent proteins derived from DsRed. These fluorescent proteins were all mutated and modified from the original progenitor proteins. By aligning the amino acid sequences of all fluorescent proteins in each category, it was found that the amino acid sequences were highly conserved among fluorescent proteins of the same source; using TBtools for amino acid sequence alignment and phylogenetic analysis of the 18 fluorescent proteins to generate a phylogenetic tree, indicating that among the 18 fluorescent proteins, mVenus, TagBFP, TagRFP, DsRed2, and mCherry showed the highest similarity, while mCerulean, mCerulean3, mCitrine, and cpCitrine174 showed a greater distance compared with these fluorescent proteins;

[0166] In the present invention, the aggregation characteristics of the fusion protein were detected by dynamic light scattering experiment (DLS) and native polyacrylamide gel electrophoresis (Native-PAGE), indicating that the aggregation characteristics of the fluorescent protein were a factor affecting the PS affinity of AnxA5. Secondly, through homology modeling by AlphaFold2, the conformation of the fusion protein was obtained, indicating that different fluorescent proteins fused with AnxA5 would produce different conformations. Then, through molecular docking by AutoDock Vina, it was shown that such conformational differences would lead to different PS binding modes of the AnxA5-FPs fusion protein, and the aggregation characteristics of the fluorescent protein were a reason for the difference in PS affinity of AnxA5.

[0167] Example 2

[0168] A high-affinity AnxA5-FPs probe screened by a modeling method of the present invention has a Kd value of 10 -7 M to 10 -9 M, and the detection sensitivity for early apoptotic cells is significantly higher than that of chemically labeled AnxA5-FITC; the probe is AnxA5-TagBFP, AnxA5-Cerulean, AnxA5-EGFP, AnxA5-Ypet, or AnxA5-TagRFP.

[0169] Example 3

[0170] An apoptosis detection kit of the present invention contains the high-affinity AnxA5-FPs probe described above and instructions for flow cytometry or fluorescence microscopy detection.

[0171] Example 4

[0172] Use of an apoptosis detection probe of the present invention in flow cytometry, fluorescence microscopy detection or in vivo PS externalization tracing.

[0173] Example 5

[0174] Cloning, construction, expression and purification of the fusion protein of Annexin A5 and fluorescent protein (AnxA5-FPs)

[0175] In previous work, the present invention has constructed the plasmid pET28a(+)-AnxA5-EGFP-his6 (as Figure 1 shown). The construction of other pET28a(+)-AnxA5-FPs-his6 plasmids was based on the pET28a(+)-AnxA5-EGFP-his6 plasmid as a template. The present invention replaced the gene sequence of EGFP with the gene sequences of other fluorescent proteins, including Venus, mVenus, cpVenus173, Citrine, mCitrine, cpCitrine174, EYFP, Ypet, mCherry, DsRed2, TagRFP, TagBFP, EBFP2, Cerulean, mCerulean, mCerulean3 ( Figure 1 shown). The correctly sequenced pET28a(+)-AnxA5-FPs-his6 plasmid was transformed into Escherichia coli BL21(DE3) cells for expression. Protein expression was induced with 1 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG) at 20 °C for 16 hours. 18 AnxA5-FPs fusion proteins were purified by Ni-NTA affinity column. After dialysis for 24 hours, they were stored in Tris buffer containing 20 mM Tris, 30 mM NaCl, pH 8.5.

[0176] Example 6

[0177] Detection of the excitation and emission spectra of the fusion protein of Annexin A5 and fluorescent protein (AnxA5-FPs)

[0178] To investigate whether the fusion of Annexin A5 affects the chromophore environment of the fluorescent protein, the present invention measured the fluorescence spectra of AnxA5-FPs fusion proteins. The specific operation is as follows: On an F-4500 fluorescence spectrophotometer (Hitachi, Japan), the present invention performed excitation and emission wavelength scans on the AnxA5-FPs (50 μg / mL) fusion protein, and recorded the excitation and emission spectra, and then analyzed the spectral data using GraphPad Prism 8.0. The results showed that there were no significant changes in the excitation and emission spectra of AnxA5-FPs (as Figure 2As shown, its excitation maximum and emission maximum (as shown in Table 1) are basically consistent with the excitation and emission maxima of the corresponding FPs (as shown in Table 2). This indicates that the fusion of AnxA5 does not significantly change the chromophore environment of the fluorescent protein, which is very important for fluorescence quantitative analysis.

[0179] Table 1

[0180]

[0181] Table 2

[0182]

[0183]

[0184] Example 7

[0185] Detection of the affinity of AnxA5-FPs fusion protein for PS exposed on the surface of apoptotic cells

[0186] To explore the PS-binding ability of AnxA5-FPs, the present invention attempted to stain apoptotic cells with AnxA5-FPs. Apoptosis of Jurkat cells was induced by adding etoposide at a final concentration of 25 μM or camptothecin at a final concentration of 1 μM. Then, the cells were collected by centrifugation at 2,500 rpm for 5 minutes, washed twice, and resuspended in binding buffer (10 mM Hepes, 140 mM NaCl, 2.5 mM CaCl2, pH 7.4) to a cell density of 1×106 cells / mL. Before that, the AnxA5-FPs probes were diluted to different concentrations, and then 200 μL of the protein dilution was added to 200 μL of the cell suspension to make the final concentrations 2 nM, 5 nM, 10 nM, 25 nM, 50 nM, 100 nM, 250 nM, 500 nM, 1000 nM, 2500 nM. After gentle vortexing, the mixture was incubated on ice for 30 minutes. Finally, after adding 1 μL of propidium iodide (PI) and mixing well, the cell suspension was immediately detected using a FACSCalibur system. AnxA5-EBFP2, AnxA5-TagBFP, AnxA5-Cerulean, AnxA5-mCerulean, AnxA5-mCeruelan3, and AnxA5-ECFP were diluted to different concentrations, and then 100 μL of the protein dilution was added to 100 μL of the cell suspension to make the final concentrations 5 nM, 10 nM, 25 nM, 50 nM, 100 nM, 250 nM. After gentle vortexing, the mixture was incubated on ice for 30 minutes. The present invention detected the fluorescence intensity of the binding of six AnxA5-FPs to PS exposed on the cell surface using an M200PRO multimode microplate reader (TECAN, Switzerland). The raw data was corrected by subtracting the average background signal of the untreated wells (blanks).

[0187] After the flow cytometry (FCM) analysis was completed, the R2-gated events in the FCM data were analyzed using FlowJo V10 software to determine the mean fluorescence intensity (MFI) of 12 AnxA5-FPs (as Figure 3 shown). For fluorescent proteins of the same color, the normalized MFI value was calculated using the formula: MFI / maximum MFI * 100 (%). The normalized fluorescence intensities of AnxA5-EBFP2, AnxA5-TagBFP, AnxA5-Cerulean, AnxA5-mCerulean, AnxA5-mCerulean3, and AnxA5-ECFP were calculated using the formula: (fluorescence intensity of cells labeled with AnxA5-FPs - fluorescence intensity of unstained cells) / maximum fluorescence intensity within the same group × 100 (%).

[0188] The normalized fluorescence intensity was used as the protein binding ability index (PBI). After data collection and analysis, the fitting curves of the PBIs of 18 AnxA5-FPs fusion proteins against different concentrations of AnxA5-FPs were plotted (as shown in Figure 4 A - E). According to the fitting curves, the relative affinity constants (Kd) were obtained (as shown in Figure 4 F and Table 3), which represent the PS binding ability of AnxA5-FPs. Similarly, Jurkat cells were induced with camptothecin, and the affinity analysis of AnxA5-FPs with apoptotic cells was performed. The fitting curves were plotted (as shown in Figure 5 A - E), and the relative affinity constants (Kd) were evaluated (as shown in Figure 5 F and Table 3). These results all indicate that the 18 AnxA5-FPs have different binding abilities to PS. This shows that the fusion of FPs results in a difference in the affinity of AnxA5 for PS by up to a hundredfold. Therefore, the present invention attempts to explain the reasons for their affinity differences.

[0189] Table 3

[0190]

[0191] Note: a Apoptotic cells were induced by etoposide;

[0192] b Apoptotic cells were induced by camptothecin.

[0193] Example 8

[0194] Detection of the aggregation state of AnxA5-FPs fusion proteins

[0195] As shown in Table 1, mVenus, mCitrine, and mCherry are monomers, while several FPs, such as EGFP, Venus, and Citrine, are weak dimers, and DsRed2 is a tetramer. To determine whether the oligomerization of AnxA5-FPs is related to their PS binding ability, the present invention measured the aggregation state of AnxA5-FPs. The aggregation state of AnxA5-FPs fusion proteins was analyzed by dynamic light scattering (DLS) experiments. The particle size distribution of AnxA5-FPs fusion proteins at a concentration of 3.0 mg / mL was measured using a Nano-size-Zeta potential analyzer (Malvern Instruments, ZS90). The test temperature was 4°C. The data were analyzed using MalvernZetasizer Software 7.12. The dynamic light scattering (DLS) results showed that AnxA5-FPs were uniformly distributed (as shown in Figure 6The diameters of the proteins were similar (2.4 nm to 3.3 nm) except for AnxA5-DsRed2 (~5.85 nm) and AnxA5-mCherry (~1.81 nm) (Table 4). Their aggregation states were also analyzed by Native-PAGE. The results showed that AnxA5-DsRed2 and AnxA5-TagRFP were multimers and dimers, respectively, while the other AnxA5-FPs had similar molecular weights and were monomers (e.g. Figure 7 (As shown). Dimeric AnxA5-TagRFP and multimeric AnxA5-DsRed2 have relatively high PS affinity. Studies prior to the present invention have shown that AnxA5-TagBFP is a dimer and also has a high affinity for PS. In summary, AnxA5-FPs that readily aggregate tend to have higher PS affinity.

[0196] Table 4

[0197]

[0198] Example 9

[0199] Comparison of protein structure modeling

[0200] The present invention explored how the fusion of fluorescent proteins structurally affected the function of AnxA5-FPs. First, the present invention used FPbase (https: / / www.fpbase.org) to query the phylogeny of 18 fluorescent proteins that were derived from avGFP, eqFP578 or DsRed and modified so that they could be divided into three groups (e.g. Figure 8 Then, the present invention uses CLUSTALW (https: / / www.genome.jp / tools-bin / clustalw) to perform multiple sequence alignment of fluorescent proteins. The multiple sequence alignment results show that among fluorescent proteins with the same origin, the amino acid sequences are highly conserved (such as Figure 9, as shown in Figures 10 and 11). To represent the relationships among these fluorescent proteins, the amino acid sequences of 18 fluorescent proteins were compared by cluster analysis. Among these fluorescent proteins, mVenus, TagBFP, TagRFP, DsRed2, and mCherry showed the highest similarity, while mCerulean, mCerulean3, mCitrine, and cpCitrine174 showed a greater distance compared to these fluorescent proteins. Considering comprehensively the similarity of fluorescent proteins, the types and sources of fluorescent proteins, and the relative affinity constant (Kd) of AnxA5-FPs, AnxA5-mCerulean3, AnxA5-TagRFP, AnxA5-mCherry, AnxA5-Venus, AnxA5-mVenus, and AnxA5-cpCitrine174 were selected for further homology modeling (as shown in Figure 12 Figure A).

[0201] Through the homology modeling method of AlphaFold2 and further structural optimization by Amber, the present invention obtained the relaxed models of six fusion proteins. The reliability of the results was evaluated by pLDDT and pTM scores (as shown in Table 5). The results showed that the pLDDT of the six modeled structures exceeded 90%, and the pTM score was approximately 0.6, proving that the models of the six fusion proteins were highly credible. The Ramachandran plot was also used to determine whether the protein conformation was reasonable by visualizing the dihedral angles of the main amino acid residues in the protein structure. The modeling results of AnxA5-mCerulean3, AnxA5-TagRFP, AnxA5-mCherry, AnxA5-Venus, AnxA5-mVenus, and AnxA5-cpCitrine174 are as shown in Figure 12 Figure A, and 92.70%, 92.90%, 92.40%, 92.30%, 91.90%, and 92.00% of the residues were in the most favorable regions (the red and yellow regions in the figure) (as shown in Table 6). ERRAT analyzed the statistics of non-bonded interactions between different atom types. The values of the six protein structures were approximately 95% (as shown in Table 6). The 3D structures corresponding to the six AnxA5-FPs are as shown in Figure 12 Figure B. These results indicate that the six predicted structures are accurate and reliable. The overall structures of the other twelve AnxA5-FPs are also as shown in Figure 13 Figure, showing the spatial relationships between AnxA5 and the remaining 12 fluorescent proteins.

[0202] Table 5

[0203]

[0204]

[0205] Note: a :AlphaFold: Prediction local distance difference test.

[0206] b :AlphaFold: Predictive template modeling score.

[0207] Table 6

[0208]

[0209] Note: a Pull-out plot: residues in the most favorable region [A,B,L].

[0210] b Pull-out diagram: append residues [a,b,l,p] in the allowed region.

[0211] c Pull-out diagram: residues in the allowed region [~a, ~b, ~l, ~p].

[0212] d Pull-out diagram: residues in the disallowed region.

[0213] e A composite quality factor generated by the ERRAT server.

[0214] f The average 3D-1D score generated by the Verify3D server.

[0215] AnxA5 and fluorescent protein were used as two domains, and the dihedral angles and center-to-center distances (dcent) of the two domains were measured (e.g. Figure 12 B and Table 7). The results showed that the six AnxA5-FPs had different sizes, and the dihedral angles and center-of-mass distances between AnxA5 and fluorescent proteins were also different. Specifically, the smaller dihedral angle and closer center-of-mass distance between AnxA5 and cpCitrine174 resulted in the complete exposure of the "convex surface" of AnxA5, making the binding of AnxA5-cpCitrine174 to PS completely dependent on AnxA5. The larger dihedral angles and farther center-of-mass distances between AnxA5 and the other five FPs made the fluorescent protein play a key role in the binding of AnxA5-FPs to PS. In summary, the differences in the conformations of the six AnxA5-FPs are mainly attributed to their size, dihedral angles and center-of-mass distances. Therefore, the present invention strongly speculates that the fusion mode of AnxA5 and fluorescent protein in the six AnxA5-FPs may lead to their different PS binding modes.

[0216] Table 7

[0217]

[0218] Example 10

[0219] Molecular docking of six AnxA5-FPs fusion proteins with different PS affinities to PS

[0220] Molecular docking was performed using the AutoDock vina server. The six predicted structures were defined as the receptor and PS as the ligand. Molecular docking was performed using P5S (a phosphatidylserine analogue present in the complex crystal (PDB ID: 6SP2)) with the representative modeled structures of the six fusion proteins. The best docking pose was selected for analysis. The binding energy represents the degree of affinity between the six AnxA5-FPs and PS (as shown in Table 8). AnxA5-mCerulean3 had the highest binding energy to PS (-9.01 kcal / mol), while AnxA5-cpCitrine174 had the lowest binding energy to PS (-7.7 kcal / mol) (as shown in Table 8). These results indicate that AnxA5-mCerulean3 binds most strongly to PS, while AnxA5-cpCitrine174 binds weakest to PS, and the other four proteins showed similar degrees of affinity to PS. The ligand P5S was recruited to the calcium-binding region of AnxA5. The charged group (NH3-) of the hydrophilic head of P5S is negatively charged and can generate electrostatic force with the positive charge of calcium ions fixed on the surface of the AnxA5 protein. At the same time, some polar amino acids in the AnxA5 calcium-binding region can generate hydrogen bond interactions with the hydrophilic head of P5S. The different spatial conformations of AnxA5 fused with six fluorescent proteins led to changes in the hydrophilic and hydrophobic regions on the surface of the six fusion proteins. AnxA5-mCerulean3 formed a highly hydrophobic region near the interface of AnxA5 and mCerulean3 (as shown in Figure 14 Figure A, the darker the color, the stronger the hydrophobicity), which made the hydrophilic head of PS firmly fixed on the surface of AnxA5, thus promoting the stability of PS. In addition, the hydrophobic fatty acid side chain in the ligand was fixed in the hydrophilic region near the interface of AnxA5 and mCerulean3 (as shown in Figure 14 Figure A). This binding mode stabilized PS firmly on the surface of AnxA5-mCerulean3, showing the highest PS affinity. In the AnxA5-TagRFP, AnxA5-mCherry, AnxA5-Venus, and AnxA5-mVenus fusion proteins, the hydrophobic cavity formed near the interface of AnxA5 and the fluorescent protein decreased, which weakened the stability of the hydrophilic head of PS (as shown in Figure 14However, they form a hydrophilic region near the interface between AnxA5 and the fluorescent protein, which can still stabilize the binding of the hydrophobic fatty acid side chains in the ligand (as shown in Figures B, C, D, and E). Figure 14 B, C, D, and E). Therefore, compared with AnxA5-mCeruelan3, the PS affinity of these four fusion proteins is reduced. In AnxA5-cpCitrine174, the hydrophilic head and hydrophobic fatty acid side chains of phosphatidylserine (PS) are fixed to the surface of AnxA5 in a calcium-dependent manner, and cpCitrine174 has no additional contribution to the binding of AnxA5-cpCitrine174 to PS (as shown in Figures 2 and 3). Figure 14 (F) Therefore, among the six fusion proteins, AnxA5-cpCitrine174 has the lowest PS affinity.

[0221] Table 8

[0222]

[0223] Next, the present invention demonstrates the interaction between six AnxA5-FPs fusion proteins and PS (e.g. Figure 15 Specifically, for AnxA5-mCerulean3, the oxygen atoms of the ligand (OXT and O15) generate ionic interactions (-6.1 and -5.6 kcal / mol) with 410CA immobilized on the AnxA5 surface, stabilizing the PS head group (as shown in Figure 2). Figure 15 A, as shown in Table 9). The nitrogen atom (N) of the ligand is spatially separated from the oxygen atom (OE1) of 370Glu of AnxA5-TagRFP. The generation of hydrogen bonds and ionic interactions indicates that TagRFP plays a key role in the binding of AnxA5-TagRFP to PS (e.g. Figure 15 B, as shown in Table 9). For AnxA5-mCherry, the nitrogen atom (N) of the ligand is spatially separated from the oxygen atom (O) of 303Asp of AnxA5-mCherry. Generate hydrogen bond interactions (-3.4kcal / mol) (such as Figure 15 C, as shown in Table 9). The oxygen atom of the ligand generates hydrogen bonds and ionic interactions with the carbon atom or nitrogen atom of 334Lys of AnxA5-mCherry (as shown in Table 9). Figure 15 C, as shown in Table 9), indicating the important role of mCherry in promoting PS binding. Figure 15As shown in D (Table 9), the nitrogen atom (N) of the ligand forms hydrogen bonds and ionic interactions with the oxygen atom (OD1) of 303Asp, respectively. The oxygen atom (O) of the ligand forms an ionic interaction with 410CA (-5.6 kcal / mol). For AnxA5-mVenus (as Figure 15 shown in E, Table 9), the oxygen atom (O15) forms hydrogen bonds, metal, and ionic interactions with the nitrogen atom of 261Gly and 410CA, respectively. These results indicate that the lack of interaction between Venus or mVenus and PS weakens the stability of PS on the surface of the fusion protein. In AnxA5-cpCitrine174 (as Figure 15 shown in F, Table 9), the oxygen atoms (O and O13) of the ligand form metal and ionic interactions with 406CA. The distances between the carbon atom (C2) of 103Ala and the oxygen atom (O), and between the oxygen atom (O) of 105Thr and the nitrogen atom (N) are and to form hydrogen bond interactions. In AnxA5-cpCitrine174, the PS binding ability completely depends on the interaction between AnxA5 and PS.

[0224] Table 9

[0225]

[0226]

[0227] Note: There are 320 amino acids in the AnxA5 protein. Amino acids numbered above 320 are located on the fluorescent protein.

[0228] In summary, the fusion of fluorescent proteins (FPs) can change the binding mode of AnxA5-FPs to PS. The fusion mode of AnxA5 and the fluorescent protein, the hydrophilic and hydrophobic cavities formed due to the fusion of the fluorescent protein, and the interactions formed between the fluorescent protein and PS are the key factors affecting the change in the binding mode of AnxA5-FPs to PS. The structural influence of the fluorescent protein on the PS affinity of AnxA5 is the result of the combined action of these different factors. Therefore, the influence of the fluorescent protein on the function of AnxA5 cannot be ignored.

[0229] Example 11

[0230] Cloning, construction, expression, purification of AnxA5-mCerulean3 mutant and detection of PS affinity

[0231] Based on the docking results, the Leu-338, Phe-339, Val-342, Val-343, Leu-346, Phe-445, and Val-451 residues of mCerulean3 have hydrophobic interactions with the fatty acid side chains of PS ( Figure 15 A). Therefore, site-directed mutagenesis was performed on these residues to verify whether they are involved in the binding affinity of AnxA5-mCerulean3 to PS. The Leu-338, Phe-339, Val-342, Val-343, Leu-346, Phe-445, and Val-451 residues of mCerulean3 were mutated to hydrophilic amino acids (L338G, F339S, V342G, V343G, L346G, F445S, V451G) to remove the active groups that stabilize the binding of the hydrophobic fatty acid side chains in the PS ligand (such as Figure 16 shown in A). The AnxA5-mCerulean3 mutants were expressed and purified according to the procedure of AnxA5-mCerulean3 wild type (WT) (such as Figure 16 shown in B), and then used for PS binding experiments (such as Figure 16 shown in C, D). Apoptosis of Jurkat cells was induced by etoposide at a final concentration of 25 μM or camptothecin at a final concentration of 1 μM. Cells were collected by centrifugation at 2,500 rpm, washed twice, and resuspended in binding buffer (10 mM Hepes, 140 mM NaCl, 2.5 mM CaCl2, pH 7.4) to a cell density of 1 × 106 cells / mL. Then, the AnxA5-mCherry probe was added to a final concentration of 400 nM and incubated with apoptotic cells on ice for 30 minutes. After that, the cells were labeled with different concentrations of AnxA5-mCerulean3 mutants and AnxA5-mCerulean3 WT, respectively. After incubation on ice for 30 minutes, the cell suspension was immediately measured by FACSCanto II. The Kd value of the AnxA5-mCerulean3 mutants was about two orders of magnitude higher than that of AnxA5-mCerulean3 WT, indicating the loss of its binding affinity ( Figure 16 E).

[0232] Example 12

[0233] Screening of High-Affinity AnxA5 Probes and Their Applications

[0234] According to the results of the present invention, five AnxA5 probes with high PS affinity and different fluorescence spectra were additionally screened out (such as Figure 17As shown in Figure A). Cells stained with AnxA5 can be detected by flow cytometry. Etoposide was used to induce apoptosis in Jurkat cells. After treatment, the cells were washed with PBS and then incubated with different concentrations of AnxA5-FPs preparations on ice for 30 minutes. All incubation procedures were carried out in 100 μL cell suspensions. 5 μL of different concentrations of AnxA5-FPs were added for staining. AnxA5-TagBFP and AnxA5-mCerulean3 were prepared into working solutions at concentrations of 200 μg / mL, 300 μg / mL, 500 μg / mL, and 800 μg / mL. The working solution concentrations of AnxA5-EGFP, AnxA5-Ypet, and AnxA5-TagRFP were 100 μg / mL, 200 μg / mL, 300 μg / mL, and 500 μg / mL, respectively. After adding 1 μL of PI, the cells were diluted to 400 μL with binding buffer and immediately used for flow cytometry detection. Cells stained with AnxA5-TagRFP were counterstained with 7-AAD.

[0235] In addition, AnxA5-labeled cells can also be detected by fluorescence microscopy. Cells were treated with etoposide and stained with 5 kinds of AnxA5-FPs including AnxA5-TagBFP, AnxA5-Cerulean, AnxA5-EGFP, AnxA5-Ypet, and AnxA5-TagRFP after 24 hours. AnxA5-TagBFP and AnxA5-mCerulean3 were prepared into working solutions at concentrations of 200 μg / mL, 300 μg / mL, 500 μg / mL, and 800 μg / mL. The working solution of AnxA5-EGFP was prepared at concentrations of 50 μg / mL, 100 μg / mL, 200 μg / mL, and 300 μg / mL. The working solutions of AnxA5-Ypet and AnxA5-TagRFP were prepared at concentrations of 100 μg / mL, 200 μg / mL, 300 μg / mL, and 500 μg / mL. The cell nuclei were stained with PI. 5 μL of different concentrations of AnxA5-FPs and 5 μL of PI (100 μg / mL) were added to 100 μL of cell solution for staining. These mixtures were incubated at room temperature for 15 minutes after gentle vortexing. Then, the cells were collected by centrifugation at 2500 rpm for 5 minutes, washed twice, and resuspended with binding buffer. Finally, the cell suspension was placed on a glass slide, covered with a coverslip, and immediately photographed with a fluorescence microscope (Leica DM-2500 fluorescence microscope). Since the fluorescence spectra of AnxA5-TagRFP and PI overlap, cells were stained only with AnxA5-TagRFP. Fluorescence signals were displayed in false colors.

[0236] In the present invention, apoptotic cells were observed to be labeled by these 5 kinds of AnxA5-FPs fusion proteins both by flow cytometry and fluorescence microscopy (such asFigure 17 As shown in B-F, Figure 18 as shown). In addition, the present invention uses AnxA5-TagRFP to detect the externalized PS on colonic capillaries, demonstrating the utility of this probe in in vivo imaging. A better understanding of the effect of fluorescent proteins on the PS-binding ability of AnxA5 contributes to the development of highly efficient and qualified apoptosis detection probes.

[0237] In summary, the fusion proteins of AnxA5 with 18 different fluorescent proteins show a hundred-fold difference in binding affinity to PS. First, the oligomeric properties of fluorescent proteins are a factor affecting the PS affinity of AnxA5. Second, considering the similarity of fluorescent proteins, the type and source of fluorescent proteins, and the relative affinity constant (Kd) of AnxA5-FPs, the present invention selected 6 AnxA5-FPs fusion proteins with different PS affinities for homology modeling and molecular docking with PS. The structural insights suggest that there are differences in the fusion modes of different fluorescent proteins with AnxA5, resulting in different conformations of the fusion proteins. These conformational differences lead to differences in the binding modes of the fusion proteins to PS, and thus show differences in PS affinity. Based on the affinity results of 18 fusion proteins with PS, the present invention screened out 5 high-affinity AnxA5 apoptosis detection probes with superior performance. The present invention provides structural insights and their applications for the fusion of annexin A5 and fluorescent proteins, which result in a hundred-fold difference in binding affinity to phosphatidylserine. The structural insights provided by the present invention help to better understand the effect of different fluorescent protein fusions on the function of AnxA5, and can more efficiently screen suitable fluorescent proteins to prepare high-affinity, fluorescent protein-based AnxA5 apoptosis detection probes to meet the needs of biotechnological applications.

[0238] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments. What is described in the above embodiments and the specification only illustrates the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention will have various changes and improvements. The scope of protection required by the present invention is defined by the appended claims, the specification and their equivalents.

Claims

1. A modeling method for the difference in the binding affinity of annexin A5 (AnxA5) fused with fluorescent proteins (FPs) to phosphatidylserine (PS), characterized in that: It includes the following steps: (1) Construct fusion proteins AnxA5-FPs of AnxA5 and different fluorescent proteins, and connect the C-terminus of AnxA5 and the N-terminus of the fluorescent protein through a linker peptide Linker by genetic engineering; (2) Use a protein structure prediction tool to perform three-dimensional structure modeling on the AnxA5-FPs fusion protein to obtain a prediction model; (3) Through molecular docking technology, perform binding simulation of the prediction model with the PS ligand, and analyze the binding energy and interaction mode; (4) Based on the binding energy and interaction mode, screen out AnxA5-FPs fusion proteins with high affinity for PS.

2. The modeling method according to claim 1, wherein: In step (1), the C-terminus of AnxA5 and the N-terminus of 18 fluorescent proteins are fused through a Gly-Ser4 linker peptide by genetic engineering; In step (2), the protein structure prediction tool is AlphaFold2, and the following verification methods are used to evaluate the credibility of the predicted structure: (i) Calculate the predicted local distance difference test pLDDT and the predicted template modeling score pTM; (ii) Analyze the dihedral angle distribution of amino acid residues through a Ramachandran plot; (iii) Use ERRAT and Verify3D tools to verify the rationality of the structure.

3. The modeling method according to claim 2, wherein: In step (3), the molecular docking technology uses AutoDock Vina, which specifically includes: (i) Dock the PS ligand with the predicted structure of AnxA5-FPs to simulate the binding conformation; (ii) Calculate the binding energy kcal / mol, and screen out the conformation with the lowest binding energy as the optimal binding mode; (iii) Analyze the hydrogen bonds, ionic interactions and hydrophobic forces between AnxA5-FPs and PS.

4. The modeling method according to claim 1, wherein: (5) According to the molecular docking results, perform site-directed mutagenesis on specific amino acid residues involved in PS binding in the fluorescent protein to construct AnxA5-FPs mutants; (6) Experimentally verify the PS binding ability of the mutants, confirm the prediction accuracy of the structural model for affinity differences, and detect the change in its Kd value with PS.

5. The modeling method according to claim 4, characterized in that: In step (5), the site-directed mutagenesis is to mutate hydrophobic amino acids into hydrophilic amino acids, which specifically includes: (i) Select residues in the fluorescent protein that form hydrophobic interactions with the fatty acid side chain of PS; (ii) Mutate the residues into glycine Gly or serine Ser through gene editing technology to weaken the hydrophobic interaction; In step (6), the Kd value of the AnxA5-mCerulean3 mutant is about two orders of magnitude higher than that of the wild type, indicating that its PS binding ability is significantly reduced.

6. The modeling method according to claim 5, wherein: In step (6), based on the Kd values measured in claim 5, screen out 5 AnxA5-FPs probes with the highest PS affinity; the probes include AnxA5-TagBFP, AnxA5-mCerulean3, AnxA5-EGFP, AnxA5-Ypet and AnxA5-TagRFP.

7. The modeling method according to claim 5, wherein: In step (6), when the fluorescent protein is mCerulean3, the mutation sites are Leu-338, Phe-339, Val-342, Val-343, Leu-346, Phe-445, and Val-451; The fluorescent protein is selected from any one of 18 kinds including Venus, mVenus, Citrine, mCitrine, cpVenus173, cpCitrine174, EYFP, Ypet, mCherry, DsRed2, TagRFP, TagBFP, EBFP2, Cerulean, mCerulean, mCerulean3, EGFP, or ECFP; The fluorescent protein includes fusion proteins AnxA5-Venus, fusion protein AnxA5-mVenus, fusion protein AnxA5-Citrine, fusion protein AnxA5-mCitrine, fusion protein AnxA5-cpVenus173, fusion protein AnxA5-cpCitrine174, fusion protein AnxA5-EYFP, fusion protein AnxA5-Ypet, fusion protein AnxA5-mCherry, fusion protein AnxA5-Ypet, fusion protein AnxA5-mCherry, fusion protein AnxA5-DsRed2, fusion protein AnxA5-TagRFP, fusion protein AnxA5-TagBFP, fusion protein AnxA5-EBFP2, fusion protein AnxA5-Cerulean, fusion protein AnxA5-mCerulean, fusion protein AnxA5-mCerulean3, fusion protein AnxA5-EGFP, fusion protein AnxA5-ECFP, and fusion protein AnxA5-Venus; The nucleotide sequence of the fusion protein AnxA5-Venus is shown in SEQ ID NO:1 in sequence; The nucleotide sequence of the fusion protein AnxA5-mVenus is shown in SEQ ID NO:2 in sequence; The nucleotide sequence of the fusion protein AnxA5-Citrine is shown in SEQ ID NO:3 in sequence; The nucleotide sequence of the fusion protein AnxA5-mCitrine is shown in SEQ ID NO:4 in sequence; The nucleotide sequence of the fusion protein AnxA5-cpVenus173 is shown in SEQ ID NO:5 in sequence; The nucleotide sequence of the fusion protein AnxA5-cpCitrine174 is shown in SEQ ID NO:6 in sequence; The nucleotide sequence of the fusion protein AnxA5-EYFP is shown in SEQ ID NO:7 in sequence; The nucleotide sequence of the fusion protein AnxA5-Ypet is shown in SEQ ID NO:8 in sequence; The nucleotide sequence of the fusion protein AnxA5-mCherry is as shown in SEQ ID NO: 9 in sequence; the nucleotide sequence of the fusion protein AnxA5-Ypet is as shown in SEQ ID NO: 8 in sequence; the nucleotide sequence of the fusion protein AnxA5-mCherry is as shown in SEQ ID NO: 9 in sequence; the nucleotide sequence of the fusion protein AnxA5-DsRed2 is as shown in SEQ ID NO: 10; the nucleotide sequence of the fusion protein AnxA5-TagRFP is as shown in SEQ ID NO: 11 in sequence; the nucleotide sequence of the fusion protein AnxA5-TagBFP is as shown in SEQ ID NO: 12 in sequence; the nucleotide sequence of the fusion protein AnxA5-EBFP2 is as shown in SEQ ID NO: 13 in sequence; the nucleotide sequence of the fusion protein AnxA5-Cerulean is as shown in SEQ ID NO: 14 in sequence; the nucleotide sequence of the fusion protein AnxA5-mCerulean is as shown in SEQ ID NO: 15 in sequence; the nucleotide sequence of the fusion protein AnxA5-mCerulean3 is as shown in SEQ ID NO: 16 in sequence; the nucleotide sequence of the fusion protein AnxA5-EGFP is as shown in SEQ ID NO: 17 in sequence; the nucleotide sequence of the fusion protein AnxA5-ECFP is as shown in SEQ ID NO: 18 in sequence; the nucleotide sequence of the fusion protein AnxA5-Venus is as shown in SEQ ID NO: 19 in sequence; The amino acid sequence of the fusion protein AnxA5-mVenus is shown in SEQ ID NO:20 in sequence; the amino acid sequence of the fusion protein AnxA5-Citrine is shown in SEQ ID NO:21 in sequence; the amino acid sequence of the fusion protein AnxA5-mCitrine is shown in SEQ ID NO:22 in sequence; the amino acid sequence of the fusion protein AnxA5-cpVenus173 is shown in SEQ ID NO:23 in sequence; the amino acid sequence of the fusion protein AnxA5-cpCitrine174 is shown in SEQ ID NO:24 in sequence; the amino acid sequence of the fusion protein AnxA5-EYFP is shown in SEQ ID NO:25 in sequence; the amino acid sequence of the fusion protein AnxA5-Ypet is shown in SEQ ID NO:26 in sequence; the amino acid sequence of the fusion protein AnxA5-mCherry is shown in SEQ ID NO:27 in sequence; the nucleotide sequence of the fusion protein AnxA5-DsRed2 is shown in SEQ IDNO:28; the nucleotide sequence of the fusion protein AnxA5-TagRFP is shown in SEQ ID NO:29; the nucleotide sequence of the fusion protein AnxA5-TagBFP is shown in SEQ ID NO:30; the nucleotide sequence of the fusion protein AnxA5-EBFP2 is shown in SEQ ID NO:31; the nucleotide sequence of the fusion protein AnxA5-Cerulean is shown in SEQ ID NO:32; the nucleotide sequence of the fusion protein AnxA5-mCerulean is shown in SEQ ID NO:33; the nucleotide sequence of the fusion protein AnxA5-mCerulean3 is shown in SEQ ID NO:34; the nucleotide sequence of the fusion protein AnxA5-EGFP is shown in SEQ ID NO:35; the nucleotide sequence of the fusion protein AnxA5-ECFP is shown in SEQ ID NO:

36.

8. A high-affinity AnxA5-FPs probe screened by the modeling method according to claim 1, characterized in that: The Kd value of the probe is 10 -7 M to 10 -9 M, and the detection sensitivity for early apoptotic cells is significantly higher than that of chemically labeled AnxA5-FITC; the probe is AnxA5-TagBFP, AnxA5-Cerulean, AnxA5-EGFP, AnxA5-Ypet or AnxA5-TagRFP.

9. An apoptosis detection kit, characterized in that: Comprising the high-affinity AnxA5-FPs probe according to any one of claims 9-10, and instructions for flow cytometry or fluorescence microscopy detection.

10. Use of the apoptosis detection probe according to claim 8 in flow cytometry, fluorescence microscopy detection or in vivo PS externalization tracing.

Images