Fusion protein of annexin A5 and fluorescent protein for apoptotic cell marking as well as preparation method and application of fusion protein

Abstract

The invention relates to the technical field of biology, and discloses an annexin A5 (AnxA5) and fluorescent protein (FPs) fusion protein for apoptotic cell marking and a preparation method and application thereof.The C-tail end of AnxA5 and N-tail ends of 18 fluorescent proteins are fused through (Gly-Ser) 4 connecting peptide through genetic engineering, an expression vector is constructed and transformed into escherichia coli BL21 (DE3), low-temperature induction of (1mM IPTG, 6mM IPTG) at the temperature of 20 DEG C is utilized, and the protein is obtained. Soluble expression is achieved through the fusion protein expression time (16 hours), and the AnxA5-FPs fusion protein high in homogeneity and light stability is obtained through Ni-NTA affinity purification. The complex process of chemical coupling is avoided, the product is uniform, the cost is low, the method can be widely applied to flow cytometry, fluorescence microscope detection and in-vivo apoptosis tracing, and an efficient tool is provided for cell apoptosis research.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a fusion protein of annexin A5 and fluorescent protein for marking apoptotic cells, and a preparation method and application thereof. Background Art

[0002] When apoptosis occurs in cells, phosphatidylserine (PS) will be turned from the inside of the cell membrane to the outside of the cell membrane. This PS exposed on the cell surface can be used as a marker for apoptotic cells (MAO'Brien, et al., J Vet Emerg CritCar 2008, 18 (6): 572-585; SELogue, 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 (AQ Abbaady, et al., Front Physiol 2017, 8: 317). Based on its high affinity for PS, which is 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, to date, most of the available, commercialized AnxA5 probes are fluorescent dye-coupled AnxA5. The preparation process of this type of probe involves complex operations and multiple purification steps, such as protein expression and purification, chemical coupling reaction, and further elimination of free fluorescein (AQ 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 coupling always produces a heterogeneous mixture, and the labeled AnxA5 molecules differ in 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). In addition, the complex preparation process also means high production costs, which will also increase the market price of the probe. More importantly, chemical dyes are easily quenched and need to be strictly protected from light. The preparation process of this type of probe is complicated and cumbersome, and its final product is usually a mixture of different labeling degrees, which is uneven and has poor photostability, thus limiting their application. Therefore, there is an urgent need to develop AnxA5 fluorescent probes with simple preparation process, uniform final product and good photostability.

[0003] Fluorescent proteins (FPs) are an indispensable part of modern biological research. As common fluorescent tags, they are often used for protein labeling and cell tracking (ZYWang, et al., Protein Sci 2021, 30 (11): 2298-2309; N.C. Shaner, et al., Nat Methods 2005, 2: 905-909; J.Zhang, et al., Nat Rev Mol Cell Biol 2002, 3: 906-918). Since the green fluorescent protein (GFP) was isolated, it has been optimized to obtain a variant enhanced GFP (EGFP), which is extremely widely used and thus occupies an important position (TD 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 (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 range. In addition, fluorescent proteins are easy to use and low in cost (YT 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, life span and photostability. The probes fused with fluorescent proteins are relatively uniform, with high fluorescence intensity and strong photostability. In addition, the fluorescent protein fusion probe does not involve the multi-step operation in the chemical coupling method, only the fusion protein needs to be expressed in prokaryotic cells and separated and purified, which is easy to operate and low in cost, and has great application value. Therefore, developing AnxA5 probes using a fluorescent protein-based strategy is an effective way to circumvent the limitations of chemical dyes.

[0004] AnxA5-GFP and AnxA5-EGFP probes have uniform labeling, higher brightness and stronger photostability (J. Wang, et al., Eur Biophys J 2015, 44 (5): 325-336; Stocker M, et al. Protein Expr Purif, 2008; 58 (2): 325-331). In previous work by our team, we found that AnxA5-FITC has a lower affinity for membranes than AnxA5-EGFP. In addition, in a study, sfGFP-AnxA5 was superior to fluorescein-coupled AnxA5 in binding to phospholipids, making it more sensitive to labeling of early apoptotic HeLa cells (AQ Abbaady, et al., Front Physiol 2017, 8: 317). These studies have demonstrated that fluorescent protein-based AnxA5 is a promising apoptosis detection probe. At present, the commercial fluorescent protein-based AnxA5 probes are only AnxA5-EGFP (green fluorescence) and AnxA5-mCherry (red fluorescence). Therefore, developing more fluorescent protein-based AnxA5 probes that emit light in the range of blue, cyan, and yellow is of great significance for expanding its application range, such as multicolor labeling and co-localization analysis with antibodies, dyes, etc. There are many types of fluorescent proteins with different properties. It is not clear whether fusion with AnxA5 will affect its ability to mark apoptosis. Therefore, it is of great application value to select suitable fluorescent proteins to fuse with AnxA5 and analyze their affinity with apoptotic cells, so as to screen out fusion proteins with higher affinity than AnxA5-EGFP and AnxA5-mCherry for early apoptosis detection.

[0005] In summary, we believe that fluorescent protein-tagged AnxA5 can be developed into a promising probe. However, whether the type of fluorescent protein affects the function and characteristics of AnxA5 protein and how it affects the function of AnxA5, there is no clear answer so far. Therefore, after AnxA5 is fused with multiple fluorescent proteins for expression, it is crucial to detect the ability of the fusion protein to mark apoptotic cells, so as to screen out high-affinity AnxA5 probes for cell apoptosis detection. Summary of the invention

[0006] The purpose of the present invention is to provide a fusion protein of annexin A5 and fluorescent protein for marking apoptotic cells, and a preparation method and application thereof.

[0007] In order to solve the problems of the prior art, the present invention provides the following technical solutions: In the first aspect, the present application provides a method for preparing a fusion protein of annexin A5 and a fluorescent protein for marking apoptotic cells;

[0008] In a second aspect, the present application provides a fusion protein of annexin A5 and a fluorescent protein prepared by a method;

[0009] In a third aspect, the present application provides a method for detecting apoptotic cells;

[0010] In a fourth aspect, the present application provides an application of a fusion protein in the preparation of an apoptotic cell detection reagent.

[0011] In a fifth aspect, the present application provides an apoptotic cell detection kit comprising the fusion protein.

[0012] The first aspect of the present application provides a method for preparing a fusion protein of annexin A5 and a fluorescent protein for marking apoptotic cells, comprising the following steps:

[0013] (1) Constructing a fusion gene expression vector in which the C-terminus of annexin A5 (AnxA5) and the N-terminus of fluorescent protein FPs are connected via a linker peptide;

[0014] (2) transforming the fusion gene expression vector into a host cell, and inducing expression with 1 mM isopropyl-β-D-1-thiogalactoside (IPTG) at 20° C. for 16 hours to achieve soluble expression of the fusion protein;

[0015] (3) The fusion protein was purified by Ni-NTA affinity chromatography.

[0016] Further, in step (1), the fluorescent protein is selected from any one of 18 kinds of Venus, mVenus, Citrine, mCitrine, cpVenus173, cpCitrine174, EYFP, Ypet, mCherry, DsRed2, TagRFP, TagBFP, EBFP2, Cerulean, mCerulean, mCerulean3, EGFP, or ECFP; and fusion proteins AnxA5-Venus, AnxA5-mVenus, AnxA5-Citrine, AnxA5-mCitrine, AnxA5-cpVenus173, AnxA5-cpCitrine174, AnxA5-EYFP, AnxA5-Ypet, AnxA5-mCherry, AnxA5-Ypet, are formed respectively. 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;

[0017] The nucleotide sequence of the fusion protein AnxA5-Venus is shown in SEQ ID NO: 1;

[0018] The nucleotide sequence of the fusion protein AnxA5-mVenus is shown in SEQ ID NO: 2;

[0019] The nucleotide sequence of the fusion protein AnxA5-Citrine is shown in SEQ ID NO: 3;

[0020] The nucleotide sequence of the fusion protein AnxA5-mCitrine is shown in SEQ ID NO: 4;

[0021] The nucleotide sequence of the fusion protein AnxA5-cpVenus173 is shown in SEQ ID NO: 5;

[0022] The nucleotide sequence of the fusion protein AnxA5-cpCitrine174 is shown in SEQ ID NO: 6;

[0023] The nucleotide sequence of the fusion protein AnxA5-EYFP is shown in SEQ ID NO: 7;

[0024] The nucleotide sequence of the fusion protein AnxA5-Ypet is shown in SEQ ID NO:8;

[0025] 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:16. 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;

[0026] 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:29. 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.

[0027] Through flow cytometry detection of 18 AnxA5-FPs fusion proteins and apoptotic cells and affinity analysis, 8 fusion proteins with higher affinity than AnxA5-EGFP and AnxA5-mCherry, 4 fusion proteins with equivalent affinity to AnxA5-EGFP and AnxA5-mCherry, and 4 fusion proteins with lower affinity than AnxA5-EGFP and AnxA5-mCherry were obtained; the emission light of these fusion proteins covers blue, cyan, yellow and red, supporting multi-color labeling and co-localization analysis.

[0028] After multiple experiments and optimization, the connecting peptide was determined to be (Gly-Ser) 4 Sequence; the host cell is Escherichia coli BL21 (DE3).

[0029] The second aspect of the present application provides a fusion protein of annexin A5 and fluorescent protein prepared by a method, the fusion protein is used to mark phosphatidylserine PS on the surface of apoptotic cells, and detect apoptotic cells by flow cytometry or fluorescence microscopy.

[0030] Further, in step (1), the affinity constant Kd value of the fusion protein and PS is 10 -5 M to 10 -8 M; 8 fusion proteins with higher affinity than AnxA5-EGFP and AnxA5-mCherry, namely, fusion protein AnxA5-TagBFP, fusion protein AnxA5-mCerulean3, fusion protein AnxA5-EBFP2, fusion protein AnxA5-Cerulean, fusion protein AnxA5-mCerulean, fusion protein AnxA5-ECFP, fusion protein AnxA5-Ypet and fusion protein AnxA5-TagRFP; 4 fusion proteins with comparable affinity to AnxA5-EGFP and AnxA5-mCherry, namely, fusion protein AnxA5-V enus, fusion protein AnxA5-EYFP, fusion protein AnxA5-cpVenus173, fusion protein AnxA5-DsRed2; 4 fusion proteins with lower affinity than AnxA5-EGFP and AnxA5-mCherry, namely fusion protein AnxA5-mVenus, fusion protein AnxA5-Citrine, fusion protein AnxA5-mCitrine, fusion protein AnxA5-cpCitrine174; the fusion protein with higher affinity than or equivalent to AnxA5-EGFP and AnxA5-mCherry, its emission light range covers blue, cyan, yellow and red.

[0031] The third aspect of the present application provides a method for detecting apoptotic cells, which is characterized by comprising the following steps:

[0032] (1) Use fusion proteins to mark apoptotic cells;

[0033] (2) The fluorescence intensity of the fusion protein is detected by flow cytometry, and the exposure degree of PS on the surface of apoptotic cells is determined according to the fluorescence signal.

[0034] Furthermore, in step (1), the labeling concentration of the fusion protein ranges from 2 nM to 2500 nM.

[0035] Furthermore, in step (1), the fusion protein has a higher detection sensitivity for early apoptotic cells than the chemically labeled AnxA5-FITC probe, and also has a higher detection sensitivity for early apoptotic cells than or equal to that of the AnxA5-EGFP fusion protein; the emission light of the fusion proteins AnxA5-TagBFP, AnxA5-mCerulean3, AnxA5-Ypet and AnxA5-TagRFP corresponds to blue, cyan, yellow and red, respectively; the labeling concentration range of the fusion protein is 25 nM to 500 nM.

[0036] The fourth aspect of the present application provides an application of a fusion protein in the preparation of an apoptotic cell detection reagent, the application including:

[0037] (1) The fusion protein is incubated with apoptotic cells to allow it to specifically bind to PS exposed on the cell surface;

[0038] (2) Detect apoptotic cells by fluorescence signals.

[0039] Furthermore, the apoptotic cell detection reagent is used for tracing early apoptotic cells in vitro or in vivo.

[0040] In a fifth aspect, the present application provides an apoptotic cell detection kit comprising a fusion protein, for detecting apoptotic cells by flow cytometry or fluorescence microscopy.

[0041] Beneficial effects: The present invention solves the problems of complex preparation and insufficient performance of existing probes through technical innovation, and provides a new tool for cell apoptosis detection with high efficiency, sensitivity and low cost. At the same time, the multiple fusion proteins obtained by the present invention with higher affinity than AnxA5-EGFP and AnxA5-mCherry can be used to more effectively detect early cell apoptosis.

[0042] Compared with the prior art, the present invention has the following advantages:

[0043] (1) The present invention directly expresses the fusion protein of AnxA5 and fluorescent protein through genetic engineering, avoiding the complicated coupling reaction, multi-step purification and free fluorescent removal steps in the traditional chemical labeling method. Experiments show that the preparation of the fusion protein only requires prokaryotic expression and Ni-NTA affinity purification (Example 3), which is simple and time-consuming (about 1 day), significantly reducing the production cost and process complexity.

[0044] (2) The chemically labeled AnxA5 probe of the present invention often leads to product heterogeneity due to differences in labeling sites and quantities, while the AnxA5-FPs probe expressed by gene fusion of the present invention has high uniformity (Example 3, Figure 3). In addition, fluorescent proteins have stronger photostability than chemical dyes (such as FITC) and do not require strict light protection (background technology comparison), which improves the practicality and storage convenience of the probe.

[0045] (3) The present invention systematically screened 18 fluorescent protein fusions and found that the affinity of different fusion proteins to phosphatidylserine (PS) differed by up to two orders of magnitude (Example 5, Table 3). The affinity was compared with that of AnxA5-EGFP and AnxA5-mCherry, and finally 8 fusion proteins with higher affinity than AnxA5-EGFP and AnxA5-mCherry were screened out, namely AnxA5-TagBFP, AnxA5-mCerulean3, AnxA5-EBFP2, AnxA5-Cerulean, AnxA5-mCerulean, AnxA5-ECFP, AnxA5-Ypet and AnxA5-TagRFP; fusion proteins with comparable affinity to AnxA5-EGFP and AnxA5-mCherry Four of them are AnxA5-Venus, AnxA5-EYFP, AnxA5-cpVenus173, and AnxA5-DsRed2; four of them are fusion proteins with lower affinity than AnxA5-EGFP and AnxA5-mCherry, namely AnxA5-mVenus, AnxA5-Citrine, AnxA5-mCitrine, and AnxA5-cpCitrine174. These fusion proteins have higher or equivalent affinity than AnxA5-EGFP and AnxA5-mCherry, and their emission light range covers blue, cyan, yellow, and red. Furthermore, the five high-affinity probes finally screened are blue AnxA5-TagBFP, cyan AnxA5-mCerulean3, yellow AnxA5-Ypet, and red AnxA5-TagRFP, and their Kd values ​​all reach 10 -7 M, showed excellent labeling efficiency in flow cytometry and fluorescence microscopy (Example 6, Figure 10-11 ), especially the detection sensitivity of early apoptotic cells is significantly higher than that of the traditional AnxA5-FITC probe ( Fig.11 D), and the sensitivity of detecting early apoptotic cells is higher than or equal to that of AnxA5-EGFP fusion protein ( Fig.11 D).

[0046] (4) The fluorescence spectrum of the fusion protein of the present invention covers multiple bands such as blue, cyan, green, yellow, and red (Example 4, Table 2), supporting multicolor labeling and co-localization analysis. Moreover, these probes have their own advantages, such as: when the fusion proteins of the blue and cyan spectra are combined with dyes such as propidium iodide (PI) for flow cytometry, there is no need to adjust the fluorescence compensation; the fluorescence intensity of the fusion protein of the yellow spectrum is higher ( Figure 4 ); The fusion protein of the red spectrum is not only suitable for in vitro apoptosis detection, but also suitable for in vivo apoptosis detection. In addition, the probe is suitable for flow cytometry and fluorescence microscopy detection (Examples 5-6), and can be extended to in vivo PS eversion tracing (Invention Content), meeting the diverse needs of basic research, drug screening and clinical diagnosis.

[0047] (5) The present invention realizes the large-scale preparation and screening of multiple AnxA5-fluorescent protein fusion probes for the first time (claims 1-4), filling the gap of the scarcity of AnxA5 probes based on fluorescent proteins. By revealing the influence of fluorescent protein types on AnxA5 function (Example 5), it provides an important theoretical basis for subsequent probe optimization, which has significant academic value and application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0049] Figure 1 The expression of the AnxA5-FPs fusion protein of the present invention after induction at 20°C and 37°C.

[0050] Among them, (A) SDS-PAGE analysis of protein expression in non-transformed (control) and transformed E. coli BL21 after induction at 20°C. (B) SDS-PAGE analysis of protein expression in non-transformed (control) and transformed E. coli BL21 after induction at 37°C. Lanes: labeled with the names of fluorescent proteins in AnxA5-FPs.

[0051] Figure 2 This is the soluble expression analysis of the AnxA5-FPs fusion protein of the present invention after induction at 37°C and 20°C.

[0052] Wherein, 'S' indicates the protein in the supernatant, and 'P' indicates the protein in the precipitate. (A) SDS-PAGE analysis of AnxA5-FPs fusion protein in the supernatant and precipitate of E. coli BL21 transformed after induction at 37°C. (B) SDS-PAGE analysis of AnxA5-FPs fusion protein in the supernatant and precipitate of E. coli BL21 transformed after induction at 20°C. (C) Ratio of AnxA5-FPs fusion protein present in the supernatant after induction at 37°C. (D) Ratio of AnxA5-FPs fusion protein present in the supernatant after induction at 20°C. Lanes: labeled with the name of the fluorescent protein in AnxA5-FPs. Data are presented as mean ± SD.

[0053] Figure 3 The invention relates to the expression and purification of the AnxA5-FPs fusion protein.

[0054] Among them, (A) SDS-PAGE analysis of purified AnxA5-FPs fusion protein. Lanes: labeled with the name of the fluorescent protein in AnxA5-FPs. (B) Purity of AnxA5-FPs fusion protein. Data are expressed as mean ± standard deviation.

[0055] Figure 4 It is the normalized fluorescence intensity of the AnxA5-FPs fusion protein of the present invention and the commercial AnxA5-FITC.

[0056] Data are presented as mean ± SD. The molar number of AnxA5-FPs and AnxA5-FITC is the same. The normalized fluorescence intensity of each group is compared with AnxA5-FITC. ns: no significant difference.

[0057] Figure 5 This is an experiment of binding of AnxA5-EGFP of the present invention to PS exposed on the cell membrane.

[0058] Among them, (A) Flow cytometry analysis of positive apoptotic cells labeled with AnxA5-EGFP at different concentrations. (B) Ratio of positive apoptotic cells labeled with AnxA5-EGFP at different concentrations. Data are expressed as mean ± standard deviation. The ratio of AnxA5-EGFP positive apoptotic cells in each group was compared with that in the 2nM group. The ratio of AnxA5-EGFP positive apoptotic cells in each group was compared with that in the 2nM group. ****p<0.0001,***p<0.001,**p<0.01,*p<0.05. ns: not significant.

[0059] Figure 6 This is an experiment of binding AnxA5-red fluorescent protein of the present invention to PS exposed on the cell membrane.

[0060] Among them, (A) Flow cytometry analysis of positive apoptotic cells labeled with AnxA5-mCherry at different concentrations. (B) The ratio of positive apoptotic cells labeled with AnxA5-mCherry at different concentrations. (C) Flow cytometry analysis of positive apoptotic cells labeled with AnxA5-DsRed2 at different concentrations. (D) The ratio of positive apoptotic cells labeled with AnxA5-DsRed2 at different concentrations. (E) Flow cytometry analysis of positive apoptotic cells labeled with AnxA5-TagRFP at different concentrations. (F) The ratio of positive apoptotic cells labeled with AnxA5-TagRFP at different concentrations. Data are expressed as mean ± standard deviation. The ratio of AnxA5 positive apoptotic cells in each group was compared with the 2nM group. ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05, ns: not significant.

[0061] Figure 7 This is an experiment of binding between AnxA5-yellow fluorescent protein of the present invention and PS exposed on the cell membrane.

[0062] Among them, (A)-(H) Flow cytometry analysis of positive apoptotic cells labeled with different concentrations of AnxA5-Venus, AnxA5-mVenus, AnxA5-Citrine, AnxA5-mCitrine, AnxA5-cpVenus173, AnxA5-cpCitrine174, AnxA5-Ypet and AnxA5-EYFP. (I)-(P) The proportion of positive apoptotic cells labeled with different concentrations of AnxA5-Venus, AnxA5-mVenus, AnxA5-Citrine, AnxA5-mCitrine, AnxA5-cpVenus173, AnxA5-cpCitrine174, AnxA5-Ypet and AnxA5-EYFP. The data are expressed as mean ± standard deviation. The proportion of AnxA5 positive apoptotic cells in each group was compared with the 2nM group. ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05, ns: not significant.

[0063] Figure 8 This is an experiment of binding between AnxA5-blue fluorescent protein of the present invention and PS exposed on the cell membrane.

[0064] Among them, (A) Flow cytometry analysis of positive apoptotic cells labeled with AnxA5-EBFP2 at different concentrations. (B) The ratio of positive apoptotic cells labeled with AnxA5-EBFP2 at different concentrations. (C) Flow cytometry analysis of positive apoptotic cells labeled with AnxA5-TagBFP at different concentrations. (D) The ratio of positive apoptotic cells labeled with AnxA5-TagBFP at different concentrations. Data are expressed as mean ± standard deviation. The ratio of AnxA5 positive apoptotic cells in each group was compared with the 2nM group. ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05, ns: not significant.

[0065] Fig. 9 This is an experiment of binding between AnxA5-cyan fluorescent protein of the present invention and PS exposed on the cell membrane.

[0066] Among them, (A) Flow cytometry analysis of positive apoptotic cells labeled with AnxA5-Cerulean at different concentrations. (B) The ratio of positive apoptotic cells labeled with AnxA5-Cerulean at different concentrations. (C) Flow cytometry analysis of positive apoptotic cells labeled with AnxA5-mCerulean at different concentrations. (D) The ratio of positive apoptotic cells labeled with AnxA5-mCerulean at different concentrations. (E) Flow cytometry analysis of positive apoptotic cells labeled with AnxA5-mCerulean3 at different concentrations. (F) The ratio of positive apoptotic cells labeled with AnxA5-mCerulean3 at different concentrations. (G) Flow cytometry analysis of positive apoptotic cells labeled with AnxA5-ECFP at different concentrations. (H) The ratio of positive apoptotic cells labeled with AnxA5-ECFP at different concentrations. Data are expressed as mean ± standard deviation. The ratio of AnxA5 positive apoptotic cells in each group was compared with the 2nM group. ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05, ns: not significant.

[0067] Fig.10 The ability of five AnxA5-FPs with high PS affinity to bind to apoptotic cells (induced by camptothecin) was evaluated by flow cytometry (chemically labeled AnxA5, AnxA5-FITC, was used as a control).

[0068] Fig.11 The figure shows the sensitivity analysis of the five AnxA5-FPs fusion proteins with high PS affinity for early apoptosis detection (compared with AnxA5-FITC).

[0069] Among them, (1) the percentage of living cells. (2) the percentage of AnxA5-FPs positive cells. (3) the percentage of early and late apoptotic cells. (D) the percentage of early apoptotic cells (bar graph). ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05. DETAILED DESCRIPTION

[0070] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present application more clearly understood, the present application is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.

[0071] In this application, the term "and / or" describes the association relationship of associated objects, indicating that there may be three relationships. For example, A and / or B can mean: A exists alone, A and B exist at the same time, and B exists alone. A and B can be singular or plural. The character " / " generally indicates that the associated objects are in an "or" relationship.

[0072] In this application, "at least one" means one or more, and "plurality" means two or more. "At least one of the following" or similar expressions refers to any combination of these items, including any combination of single items or plural items. For example, "at least one of a, b, or c", or "at least one of a, b, and c" can all mean: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, c can be single or multiple, respectively.

[0073] It should be understood that in the various embodiments of the present application, the size of the serial numbers of the above-mentioned processes does not mean the order of execution, some or all of the steps can be executed in parallel or sequentially, and the execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0074] The terms used in the embodiments of the present application are only for the purpose of describing specific embodiments, and are not intended to limit the present application. The singular forms "a", "said" and "the" used in the embodiments of the present application and the appended claims are also intended to include plural forms, unless the context clearly indicates other meanings.

[0075] The weight of the relevant components mentioned in the embodiment description of the present application can not only refer to the specific content of each component, but also represent the proportional relationship between the weights of the components. Therefore, as long as the content of the relevant components is proportionally enlarged or reduced according to the embodiment description of the present application, it is within the scope disclosed in the embodiment description of the present application. Specifically, the mass described in the embodiment description of the present application can be a mass unit known in the chemical industry such as μg, mg, g, kg, etc.

[0076] The terms "first" and "second" are used only for descriptive purposes to distinguish objects such as substances from each other, and should not be understood as indicating or implying relative importance or implicitly indicating the number of 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, features defined as "first" and "second" may explicitly or implicitly include one or more of the features.

[0077] The first aspect of the present application provides a method for preparing a fusion protein of annexin A5 and fluorescent protein for apoptotic cell marking, comprising the following steps:

[0078] (1) Constructing a fusion gene expression vector in which the C-terminus of annexin A5 (AnxA5) and the N-terminus of fluorescent protein FPs are connected via a linker peptide;

[0079] (2) transforming the fusion gene expression vector into a host cell, and inducing expression with 1 mM isopropyl-β-D-1-thiogalactoside (IPTG) at 20° C. for 16 hours to achieve soluble expression of the fusion protein;

[0080] (3) The fusion protein was purified by Ni-NTA affinity chromatography.

[0081] In some embodiments, in step (1), the fluorescent protein is selected from any one of 18 kinds of Venus, mVenus, Citrine, mCitrine, cpVenus173, cpCitrine174, EYFP, Ypet, mCherry, DsRed2, TagRFP, TagBFP, EBFP2, Cerulean, mCerulean, mCerulean3, EGFP, or ECFP; and the fusion proteins AnxA5-Venus, AnxA5-mVenus, AnxA5-Citrine, AnxA5-mCitrine, AnxA5-cpVenus173, AnxA5-cpCitrine174, AnxA5-EYFP, AnxA5-Ypet, AnxA5-mCherry, 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;

[0082] The nucleotide sequence of the fusion protein AnxA5-Venus is shown in SEQ ID NO: 1;

[0083] The nucleotide sequence of the fusion protein AnxA5-mVenus is shown in SEQ ID NO: 2;

[0084] The nucleotide sequence of the fusion protein AnxA5-Citrine is shown in SEQ ID NO: 3;

[0085] The nucleotide sequence of the fusion protein AnxA5-mCitrine is shown in SEQ ID NO: 4;

[0086] The nucleotide sequence of the fusion protein AnxA5-cpVenus173 is shown in SEQ ID NO: 5;

[0087] The nucleotide sequence of the fusion protein AnxA5-cpCitrine174 is shown in SEQ ID NO: 6;

[0088] The nucleotide sequence of the fusion protein AnxA5-EYFP is shown in SEQ ID NO: 7;

[0089] The nucleotide sequence of the fusion protein AnxA5-Ypet is shown in SEQ ID NO: 8;

[0090] 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:16. 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;

[0091] 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:29. 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.

[0092] In some embodiments, in step (2), the connecting peptide is determined to be a (Gly-Ser)4 sequence through multiple experiments and optimization; and the host cell is Escherichia coli BL21 (DE3).

[0093] A second aspect of an embodiment of the present application provides a fusion protein of annexin A5 and a fluorescent protein prepared by a method, wherein the fusion protein is used to label phosphatidylserine PS on the surface of apoptotic cells and detect apoptotic cells by flow cytometry or fluorescence microscopy.

[0094] In some embodiments, in step (1), the affinity constant Kd value of the fusion protein and PS is 10 -5M to 10 -8 M; the fusion proteins are 8 fusion proteins with higher affinity than AnxA5-EGFP and AnxA5-mCherry, namely, fusion protein AnxA5-TagBFP, fusion protein AnxA5-mCerulean3, fusion protein AnxA5-EBFP2, fusion protein AnxA5-Cerulean, fusion protein AnxA5-mCerulean, fusion protein AnxA5-ECFP, fusion protein AnxA5-Ypet and fusion protein AnxA5-TagRFP; 4 fusion proteins with comparable affinity to AnxA5-EGFP and AnxA5-mCherry, namely, fusion protein AnxA5- Venus, fusion protein AnxA5-EYFP, fusion protein AnxA5-cpVenus173, fusion protein AnxA5-DsRed2; 4 fusion proteins with lower affinity than AnxA5-EGFP and AnxA5-mCherry, namely fusion protein AnxA5-mVenus, fusion protein AnxA5-Citrine, fusion protein AnxA5-mCitrine, fusion protein AnxA5-cpCitrine174; the fusion protein with higher affinity than or equivalent to AnxA5-EGFP and AnxA5-mCherry, its emission light range covers blue, cyan, yellow and red.

[0095] A third aspect of the present application provides a method for detecting apoptotic cells, which is characterized by comprising the following steps:

[0096] (1) Use fusion proteins to mark apoptotic cells;

[0097] (2) The fluorescence intensity of the fusion protein is detected by flow cytometry, and the exposure degree of PS on the surface of apoptotic cells is determined according to the fluorescence signal.

[0098] In some embodiments, in step (1), the labeled concentration of the fusion protein ranges from 2 nM to 2500 nM.

[0099] In some embodiments, in step (1), the fusion protein has a higher detection sensitivity for early apoptotic cells than the chemically labeled AnxA5-FITC probe, and a higher detection sensitivity for early apoptotic cells than the AnxA5-EGFP fusion protein; the emission light of the fusion proteins AnxA5-TagBFP, AnxA5-mCerulean3, AnxA5-Ypet and AnxA5-TagRFP corresponds to blue, cyan, yellow and red, respectively; the labeling concentration range of the fusion protein is 25nM to 500nM.

[0100] The fourth aspect of the present application provides an application of a fusion protein in preparing an apoptotic cell detection reagent, and the application includes:

[0101] (1) The fusion protein is incubated with apoptotic cells to allow it to specifically bind to PS exposed on the cell surface;

[0102] (2) Detect apoptotic cells by fluorescence signals.

[0103] A fifth aspect of the embodiments of the present application provides an apoptotic cell detection kit comprising a fusion protein, for detecting apoptotic cells by flow cytometry or fluorescence microscopy.

[0104] In some embodiments, apoptotic cell detection reagents are used to track early apoptotic cells in vitro or in vivo.

[0105] Example 1

[0106] The method for preparing a fusion protein of annexin A5 and fluorescent protein for apoptotic cell marking of the present invention comprises the following steps:

[0107] (1) Constructing a fusion gene expression vector in which the C-terminus of annexin A5 (AnxA5) and the N-terminus of fluorescent protein FPs are connected via a linker peptide;

[0108] (2) transforming the fusion gene expression vector into a host cell, and inducing expression with 1 mM isopropyl-β-D-1-thiogalactoside (IPTG) at 20° C. for 16 hours to achieve soluble expression of the fusion protein;

[0109] (3) The fusion protein was purified by Ni-NTA affinity chromatography.

[0110] In step (1), the fluorescent protein is selected from any one of 18 kinds of Venus, mVenus, Citrine, mCitrine, cpVenus173, cpCitrine174, EYFP, Ypet, mCherry, DsRed2, TagRFP, TagBFP, EBFP2, Cerulean, mCerulean, mCerulean3, EGFP, or ECFP; respectively forming fusion protein 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;

[0111] The nucleotide sequence of the fusion protein AnxA5-Venus is shown in SEQ ID NO: 1;

[0112] The nucleotide sequence of the fusion protein AnxA5-mVenus is shown in SEQ ID NO: 2;

[0113] The nucleotide sequence of the fusion protein AnxA5-Citrine is shown in SEQ ID NO: 3;

[0114] The nucleotide sequence of the fusion protein AnxA5-mCitrine is shown in SEQ ID NO: 4;

[0115] The nucleotide sequence of the fusion protein AnxA5-cpVenus173 is shown in SEQ ID NO: 5;

[0116] The nucleotide sequence of the fusion protein AnxA5-cpCitrine174 is shown in SEQ ID NO: 6;

[0117] The nucleotide sequence of the fusion protein AnxA5-EYFP is shown in SEQ ID NO: 7;

[0118] The nucleotide sequence of the fusion protein AnxA5-Ypet is shown in SEQ ID NO:8;

[0119] 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:16. 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;

[0120] 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:29. 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.

[0121] In step (2), the connecting peptide was determined to be (Gly-Ser) through multiple experiments and optimization. 4 Sequence; the host cell is Escherichia coli BL21 (DE3).

[0122] Example 2

[0123] The invention discloses a fusion protein of annexin A5 and fluorescent protein prepared by a method. The fusion protein is used to mark phosphatidylserine PS on the surface of apoptotic cells and detect apoptotic cells by flow cytometry or fluorescence microscopy.

[0124] In step (1), the affinity constant Kd value of the fusion protein and PS is 10 -5 M to 10 -8M; the fusion proteins are 8 fusion proteins with higher affinity than AnxA5-EGFP and AnxA5-mCherry, namely, fusion protein AnxA5-TagBFP, fusion protein AnxA5-mCerulean3, fusion protein AnxA5-EBFP2, fusion protein AnxA5-Cerulean, fusion protein AnxA5-mCerulean, fusion protein AnxA5-ECFP, fusion protein AnxA5-Ypet and fusion protein AnxA5-TagRFP; 4 fusion proteins with comparable affinity to AnxA5-EGFP and AnxA5-mCherry, namely, fusion protein AnxA5- Venus, fusion protein AnxA5-EYFP, fusion protein AnxA5-cpVenus173, fusion protein AnxA5-DsRed2; 4 fusion proteins with lower affinity than AnxA5-EGFP and AnxA5-mCherry, namely fusion protein AnxA5-mVenus, fusion protein AnxA5-Citrine, fusion protein AnxA5-mCitrine, fusion protein AnxA5-cpCitrine174; the fusion protein with higher affinity than or equivalent to AnxA5-EGFP and AnxA5-mCherry, its emission light range covers blue, cyan, yellow and red.

[0125] Example 3

[0126] A method for detecting apoptotic cells of the present invention is characterized by comprising the following steps:

[0127] (1) Using fusion proteins to label apoptotic cells; the labeling concentration of the fusion proteins ranges from 2nM to 2500nM. The fusion proteins have a higher sensitivity to early apoptotic cells than the chemically labeled AnxA5-FITC probe, and also a higher sensitivity to early apoptotic cells than the AnxA5-EGFP fusion protein; the emission light of the fusion proteins AnxA5-TagBFP, AnxA5-mCerulean3, AnxA5-Ypet and AnxA5-TagRFP corresponds to blue, cyan, yellow and red, respectively; the labeling concentration of the fusion proteins ranges from 25nM to 500nM.

[0128] (2) The fluorescence intensity of the fusion protein is detected by flow cytometry, and the exposure degree of PS on the surface of apoptotic cells is determined according to the fluorescence signal.

[0129] Example 4

[0130] The application of a fusion protein of the present invention in preparing an apoptotic cell detection reagent includes:

[0131] (1) The fusion protein is incubated with apoptotic cells to allow it to specifically bind to PS exposed on the cell surface;

[0132] (2) Detect apoptotic cells by fluorescence signals.

[0133] A fifth aspect of the embodiments of the present application provides an apoptotic cell detection kit comprising a fusion protein, for detecting apoptotic cells by flow cytometry or fluorescence microscopy.

[0134] Apoptotic cell detection reagents are used to track early apoptotic cells in vitro or in vivo.

[0135] Example 5

[0136] Cloning and construction of fusion protein of AnxA5 and fluorescent protein (AnxA5-FPs)

[0137] In the past, we have constructed pET28a(+)-AnxA5-EGFP-his 6 The plasmid was constructed as follows: the EGFP coding sequence was amplified using primers NdeI-EGFP-F and XhoI-EGFP-R. After the PCR product was recovered, the primer BamHI-(GS) 4 -linker-F and XhoI-EGFP-R were amplified again, and (GS) was introduced after structure prediction and experimental screening 4 The final product was cloned into pET28a(+) to construct pET28a(+)-EGFP-his 6 Then, the coding sequence of human AnxA5 was amplified using primers NcoI-AnxA5-F and BamHI-AnxA5-R. The product was cloned into pET28a(+)-EGFP-his 6 The expression vector pET28a(+)-AnxA5-EGFP-his was constructed by fusion with the upstream of EGFP coding sequence. 6 . Restriction endonucleases and their recognition sites are indicated in italics. Other pET28a(+)-AnxA5-FPs-his 6 Plasmid construction: pET28a(+)-AnxA5-EGFP-his 6 Plasmid was used as template. We 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, and mCerulean3.

[0138] Example 6

[0139] The inducible expression of AnxA5 and fluorescent protein fusion protein (AnxA5-FPs) and the optimization of expression conditions were carried out. The correctly sequenced pET28a (+) -AnxA5-FPs-his 6 The plasmid was transformed into E. coli BL21 (DE3) cells for expression. 6 The various E. coli BL21 (DE3) were inoculated into 3 mL LB medium containing 50 μg / mL kanamycin and cultured at 37°C overnight. Then, the culture (30 μL) was inoculated into 3 mL LB medium containing 50 μg / mL kanamycin and cultured at 37°C for about 3 hours. When the bacteria grew to the mid-logarithmic phase (OD 600 =0.5-0.8), 1 mM isopropyl-β-D-1-thiogalactoside (IPTG) was added to induce gene expression, and the culture was continued at 20°C for about 16 hours, or 37°C for 12 hours. Afterwards, the bacterial pellet was collected by centrifugation, resuspended in 600 μL buffer (20 mM Tris, 250 mM NaCl, pH 7.4), and lysed by ultrasound. The expression of AnxA5-FPs at 20°C and 37°C was analyzed by SDS-polyacrylamide gel electrophoresis (PAGE). For solubility analysis, the bacterial pellet was lysed by ultrasound, and the suspension was centrifuged at 12,000 rpm for 20 minutes at 4°C. The supernatant and the precipitate were separated, and the expression of AnxA5-FPs in the supernatant and the precipitate at 20°C and 37°C was analyzed by SDS-PAGE. After induction of E. coli BL21(DE3) with IPTG at 20°C and 37°C, an additional protein band with a molecular weight (MW) of approximately 65 KDa was found in the cell lysate (e.g. Figure 1 This is consistent with the theoretical molecular weight of various AnxA5-FPs (as shown in Table 1). When the bacteria were induced at 20°C, compared with the induction at 37°C (as shown in Table 1), the Figure 2 A, C), the amount of AnxA5-FPs expressed in soluble form was greatly increased (as shown in Figure 2 B, D). The amino acid number and theoretical molecular weight (MW, KDa) of AnxA5-FPs fusion protein are shown in Table 1:

[0140] Table 1

[0141]

[0142]

[0143] Example 7

[0144] Purification of AnxA5-FPs fusion proteins

[0145] Protein expression was induced with 1 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG) at 20°C for 16 hours. The bacterial pellet was collected, resuspended in binding buffer (20 mM Tris, 250 mM NaCl, pH 7.4), and the cells were disrupted by ultrasound. The supernatant and the precipitate were then separated by centrifugation at 16,000 rpm for 20 minutes, and the supernatant was collected and filtered for protein with a 0.22 μm filter. The column was pre-equilibrated with binding buffer containing 40 mM imidazole, and imidazole was added to the supernatant to a final concentration of 40 mM. The supernatant was then loaded onto a Ni-NTA affinity column. After the sample loading was completed, the column was washed with 50 mM imidazole and then eluted with 250 mM imidazole. The eluate containing the AnxA5-FPs fraction was collected and dialyzed against Tris buffer (20 mM Tris, 30 mM NaCl, pH 8.5) for approximately 24 hours. After dialysis, the protein solution was collected and the protein concentration was quantified by the BCA method. 10 ng of protein was taken for SDS-PAGE analysis. AnxA5-EBFP2, AnxA5-TagBFP, AnxA5-Cerulean, AnxA5-mCerulean, AnxA5-mCerulean3, and AnxA5-ECFP were all stored in our laboratory. The AnxA5-FPs fusion protein was purified by Ni-NTA agarose affinity chromatography (e.g. Figure 3 The purity of all fusion proteins reached more than 80% (as shown in Figure 3 B). In summary, the results showed that AnxA5-FPs fusion proteins were successfully expressed and purified. All 18 AnxA5-FPs fusion proteins were stored in Tris buffer containing 20 mM Tris, 30 mM NaCl, pH 8.5.

[0146] Example 8

[0147] Characterization of AnxA5 and fluorescent protein fusion proteins (AnxA5-FPs)

[0148] On a F-4500 fluorescence spectrophotometer (Hitachi, Japan), we scanned the excitation and emission wavelengths of the AnxA5-FPs (50 μg / mL) fusion protein, recorded the excitation and emission spectra, and then analyzed the spectral data using GraphPad Prism 8.0. The results showed that the excitation and emission spectra of AnxA5-FPs did not change significantly compared with the individual fluorescent proteins, and its excitation maximum and emission maximum are shown in Table 2. This indicates that the fusion of AnxA5 did not significantly change the chromophore environment of the fluorescent protein, which is very important for quantitative fluorescence analysis. We also measured the fluorescence intensity of 18 AnxA5-FPs and AnxA5-FITC using a multifunctional microplate reader (BioTek, USA). Then, the concentrations of 18 AnxA5-FPs and AnxA5-FITC were determined by the BCA method. The normalized fluorescence intensity was calculated by the following formula: fluorescence intensity of AnxA5-FPs or AnxA5-FITC / nmol of protein. We compared the normalized fluorescence intensity of AnxA5-FP fusion protein and commercial AnxA5-FITC. The results showed that the normalized fluorescence intensity of AnxA5-EGFP, AnxA5-mCherry and AnxA5-DsRed2 was at the same order of magnitude as AnxA5-FITC, while the normalized fluorescence intensity of the other fifteen AnxA5-FPs was one order of magnitude higher than that of AnxA5-FITC (e.g. Figure 4 The fluorescence spectrum of AnxA5-FP fusion protein is shown in Table 2:

[0149] Table 2

[0150]

[0151]

[0152] Example 9

[0153] Affinity analysis of AnxA5-FPs fusion protein and PS exposed on the cell surface

[0154] To investigate the PS binding ability of AnxA5-FPs fusion protein, we attempted to use AnxA5-FPs to stain apoptotic cells. The specific method was as follows: Jurkat cells were induced to apoptosis 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 washed with binding buffer (10 mM Hepes, 140 mM NaCl, 2.5 mM CaCl 2 , pH 7.4) to a cell density of 1 × 10 6cells / mL. Prior to this, the AnxA5-FPs probe was diluted to different concentrations, and then 200 μL of protein dilution was added to 200 μL of cell suspension to make the final concentrations of 2nM, 5nM, 10nM, 25nM, 50nM, 100nM, 250nM, 500nM, 1000nM, and 2500nM. After gently vortexing, the mixture was incubated on ice for 30 minutes. Finally, after adding 1 μL of propidium iodide (PI) and mixing evenly, the cell suspension was immediately detected using a flow cytometer. The results showed that they can all bind to apoptotic cells, and their optimal labeling efficiency depends on their final concentration (such as Figure 5-9 That is, within a certain concentration range, AnxA5-negative and AnxA5-positive cells can be clearly distinguished.

[0155] After the flow cytometry (FCM) analysis was completed, the mean fluorescence intensity (MFI) of AnxA5-FPs positive cells was analyzed by FlowJo V10 software. For fluorescent proteins of the same color, the normalized MFI value was calculated using the formula: MFI / maximum MFI*100 (%). The normalized fluorescence intensity was used as the protein binding ability index (PBI). After data collection and analysis, fitting curves of the PBI of 18 AnxA5-FPs fusion proteins and different concentrations of AnxA5-FPs were drawn. According to the fitting curve, the relative affinity constant (Kd) (as shown in Table 3) was obtained, which represents the PS binding ability of AnxA5-FPs. These results all show that the 18 AnxA5-FPs have different binding abilities to PS. This shows that the fusion of FPs results in a hundred-fold difference in the affinity of AnxA5 for PS. The relative affinity constants (Kd) of the 18 AnxA5-FPs fusion proteins obtained according to the fitting curve are shown in Table 3:

[0156] Table 3

[0157]

[0158]

[0159] Note: a Apoptotic cells were induced by etoposide; b Apoptosis of cells is induced by Camptotheca acuminata.

[0160] Example 10

[0161] Sensitivity detection of early apoptosis by five AnxA5-FPs with high PS affinity

[0162] Based on the affinity analysis results of AnxA5-FPs fusion protein and PS, we screened out five proteins with high PS affinity (Kd value higher than 10 7Order of magnitude) and AnxA5 probes with different fluorescence spectra. Jurkat cells were induced with 0, 0.5, 1.0, 2.5, 5.0 and 10 μM camptothecin for 12 h and then labeled with five AnxA5-FPs at the same molar concentration, including AnxA5-TagBFP, AnxA5-mCerulean3, AnxA5-EGFP, AnxA5-Ypet, and AnxA5-TagRFP. After adding 1 μL PI, the cells were immediately used for flow cytometry analysis. AnxA5-TagRFP-stained cells were counterstained with 7-AAD. At the same time, apoptotic cells were labeled with a commercial apoptosis detection reagent AnxA5-FITC (YEASEN, Shanghai, China) as a control group. The percentages of cells labeled with these five AnxA5-FPs probes and AnxA5-FITC under control and camptothecin-treated conditions were almost the same (such as Fig.10 and Fig.11 Interestingly, when these labeled cells were divided into early and late apoptotic cells, we found that under all conditions, more early apoptotic cells were labeled with the five AnxA5-FPs probes than with AnxA5-FITC (e.g. Fig.10 and Fig.11 C and D). Meanwhile, the proportion of early apoptotic cells marked by probes such as AnxA5-TagBFP, AnxA5-mCerulean3, AnxA5-Ypet, and AnxA5-TagRFP was basically the same as that of early apoptotic cells marked by AnxA5-EGFP, and even more ( Fig.11 C and D). The above results indicate that the five AnxA5-FPs probes we screened are more sensitive in detecting early apoptotic cells.

[0163] In summary, the fusion protein of AnxA5 and 18 different fluorescent proteins is expressed in a soluble form when induced to express at 20°C, and can be purified by Ni-NTA affinity chromatography. These AnxA5-FPs fusion proteins can all bind to apoptotic cells, but there are differences in their optimal working concentrations. These fusion proteins show a hundred-fold difference in affinity in binding to PS. Based on the affinity results of 18 fusion proteins with PS, we screened out 5 high-affinity AnxA5 apoptosis detection probes with superior performance. Compared with the commercialized AnxA5-FITC probe, these 5 high-affinity AnxA5s are more sensitive in detecting early apoptotic cells. The fusion protein of membrane-attached protein A5 and fluorescent protein for apoptotic cell marking provided by the present invention, as well as its preparation method and application, can screen out suitable fluorescent proteins to prepare high-affinity, fluorescent protein-based AnxA5 apoptosis detection probes to meet the needs of biotechnology applications.

[0164] 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 to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, and the scope of protection of the present invention is defined by the attached claims, description and their equivalents.

Claims

1. A method for preparing a fusion protein of annexin A5 and fluorescent protein for apoptotic cell marking, characterized in that The steps include: (1) constructing a fusion gene expression vector in which the C-terminus of annexin A5 and the N-terminus of fluorescent protein FPs are connected via a linker peptide; (2) transforming the fusion gene expression vector into a host cell, and inducing expression with 1 mM isopropyl-β-D-1-thiogalactoside (IPTG) at 20° C. for 16 hours to achieve soluble expression of the fusion protein; (3) The fusion protein was purified by Ni-NTA affinity chromatography.

2. The preparation method according to claim 1, characterized in that: In step (1), the fluorescent protein is selected from any one of 18 kinds of Venus, mVenus, Citrine, mCitrine, cpVenus173, cpCitrine174, EYFP, Ypet, mCherry, DsRed2, TagRFP, TagBFP, EBFP2, Cerulean, mCerulean, mCerulean3, EGFP, or ECFP; respectively forming fusion protein 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; The nucleotide sequence of the fusion protein AnxA5-mVenus is shown in SEQ ID NO: 2; The nucleotide sequence of the fusion protein AnxA5-Citrine is shown in SEQ ID NO: 3; The nucleotide sequence of the fusion protein AnxA5-mCitrine is shown in SEQ ID NO: 4; The nucleotide sequence of the fusion protein AnxA5-cpVenus173 is shown in SEQ ID NO: 5; The nucleotide sequence of the fusion protein AnxA5-cpCitrine174 is shown in SEQ ID NO: 6; The nucleotide sequence of the fusion protein AnxA5-EYFP is shown in SEQ ID NO: 7; 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-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:

16. 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; 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:

29. 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; in step (1), the connecting peptide is a Gly-Ser4 sequence; and the host cell is Escherichia coli BL21 (DE3).

3. A fusion protein of annexin A5 and fluorescent protein prepared by the method of claim 1 or 2, characterized in that: The fusion protein is used to mark phosphatidylserine PS on the surface of apoptotic cells, and apoptotic cells are detected by flow cytometry or fluorescence microscopy.

4. The fusion protein according to claim 3, characterized in that: The affinity constant Kd value of the fusion protein and PS is 10 -5 M to 10 -8 M; the fusion proteins are 8 fusion proteins with higher affinity than AnxA5-EGFP and AnxA5-mCherry, namely, fusion protein AnxA5-TagBFP, fusion protein AnxA5-mCerulean3, fusion protein AnxA5-EBFP2, fusion protein AnxA5-Cerulean, fusion protein AnxA5-mCerulean, fusion protein AnxA5-ECFP, fusion protein AnxA5-Ypet and fusion protein AnxA5-TagRFP; 4 fusion proteins with comparable affinity to AnxA5-EGFP and AnxA5-mCherry, namely, fusion protein AnxA5- Venus, fusion protein AnxA5-EYFP, fusion protein AnxA5-cpVenus173, fusion protein AnxA5-DsRed2; 4 fusion proteins with lower affinity than AnxA5-EGFP and AnxA5-mCherry, namely fusion protein AnxA5-mVenus, fusion protein AnxA5-Citrine, fusion protein AnxA5-mCitrine, fusion protein AnxA5-cpCitrine174; the fusion protein with higher affinity than or equivalent to AnxA5-EGFP and AnxA5-mCherry, its emission light range covers blue, cyan, yellow and red.

5. A method for detecting apoptotic cells, characterized in that The steps include: (1) Using the fusion protein of claim 4 to mark apoptotic cells; (2) The fluorescence intensity of the fusion protein is detected by flow cytometry, and the exposure degree of PS on the surface of apoptotic cells is determined according to the fluorescence signal.

6. The detection method according to claim 5, characterized in that: In step (1), the labeling concentration of the fusion protein ranges from 2 nM to 2500 nM.

7. The detection method according to claim 6, characterized in that: In step (2), the fusion protein has a higher detection sensitivity for early apoptotic cells than the chemically labeled AnxA5-FITC probe, and also has a higher detection sensitivity for early apoptotic cells than or equal to that of the AnxA5-EGFP fusion protein; the emission lights of the fusion proteins AnxA5-TagBFP, AnxA5-mCerulean3, AnxA5-Ypet and AnxA5-TagRFP correspond to blue, cyan, yellow and red, respectively; the labeling concentration range of the fusion protein is 25 nM to 500 nM.

8. Use of the fusion protein according to claim 1 in preparing a reagent for detecting apoptotic cells, characterized in that: The applications include: (1) incubating the fusion protein with apoptotic cells to allow it to specifically bind to PS exposed on the cell surface; (2) Detect apoptotic cells by fluorescence signals.

9. The use according to claim 8, characterized in that: The apoptotic cell detection reagent is used for tracing early apoptotic cells in vitro or in vivo.

10. An apoptotic cell detection kit comprising the fusion protein according to claim 1.

Images