Fusion protein and fluorescence microplate reader method thereof for high-throughput cell apoptosis detection

Abstract

The invention discloses a fusion protein and a fluorescent microplate reader method for high-flux cell apoptosis detection, apoptotic cells are marked by fusion protein of annexin A5 (AnxA5) and fluorescent protein (FPs), and the fluorescence intensity of the fusion protein of AnxA5 and FPs is detected by a multifunctional microplate reader. And judging the exposure condition of the phosphatidylserine PS on the surface of the apoptotic cell according to the fluorescence signal. The method disclosed by the invention has the characteristics of simplicity and easiness in operation and rapidness in detection, and the method for detecting the fluorescence intensity by the microplate reader is applied to the detection of the cell apoptosis, so that the high-throughput detection of the cell apoptosis is realized. Compared with a flow cytometer and a fluorescence microscope, the multifunctional microplate reader has the advantages of low price, easiness in operation, simplicity in maintenance and easiness in popularization. The AnxA5 apoptosis detection probe is wide in selection range, is not limited by excitation wavelength and emission wavelength of a fluorescent marker, can simultaneously detect a plurality of samples at one time, and is convenient and fast.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a fusion protein and a fluorescence microplate reader method for high-throughput cell apoptosis detection. Background Art

[0002] Apoptosis is a form of programmed cell death, a process in which cells actively and physiologically die according to their own program under certain physiological or pathological conditions (M. Shamsipur, et al., Anal Chem 2016, 88(4): 2188-2197). It is crucial for maintaining homeostasis (M. Pierce, et al., J Fluoresc 2025, 35(2): 1111-1123; A. Pulfer, et al., eLife 2024, 12: RP90502) and regulating cell number by balancing cell proliferation (LG Ruan, et al., Langmuir 2018, 34(34): 10040-10047). In particular, it plays an indispensable role in organismal development and various diseases (H. Kim, et al., Adv Sci 2020, 7(24): 2002988). In addition, the detection of apoptosis levels can be used to evaluate drug efficacy and cytotoxicity (HYXue, et al., Sensor Actuat B-Chem 2024, 398: 134714; YYLi, et al., Biotechnol J 2024, 19(2): 2300443), as well as the effect of gene therapy (MNSoykan, et al., Biotechnol J 2024, 19(2): 2300496; P.Ladiwala, et al., Biotechnol J 2025, 20(3): e202400501). One of the recognized characteristics of early apoptosis is the externalization of phosphatidylserine (PS) to the outer leaflet of the cell membrane (HYXue, et al., Sensor Actuat B-Chem 2024, 398: 134714; G.Banfalvi, et al., Apoptosis 2017, 22(2): 306-323). Annexin A5 (AnxA5) can be used to analyze early apoptotic events by specifically detecting PS exposure (H.Kim, et al., Adv Sci 2020, 7(24): 2002988; MYGao, et al., Protein Sci 2025, 34(4): e70068). As a member of the membrane-bound protein (annexin family), AnxA5 binds to calcium ions (Ca 2+) exhibit high affinity for cell surface-exposed PS in a PS-dependent manner (W. Tang, et al., Acta Pharmacologica Sinica 2025, 46:852-866). Thus, PS egress on the cell surface can be targeted and visualized by fluorescently labeled AnxA5 (W. Tang, et al., Acta Pharmacologica Sinica 2025, 46:852-866; X. R. Zhang, et al., Signal Transduct Tar 2019, 6(1):235).

[0003] To date, fluorescent AnxA5 conjugates have been used to monitor apoptosis (LG Ruan, et al., Langmuir 2018, 34(34): 10040-10047; CM Worsley, et al., Plos One 2022, 17(6): e0270599), which can be detected by flow cytometry and fluorescence microscopy (J. Wang, et al., Eur Biophys J Biophy 2015, 44(5): 325-336). However, these techniques are often time-consuming and require expensive equipment, specialized skills, and lengthy preparation procedures (M. Pierce, et al., J Fluoresc 2025, 35(2): 1111-1123; G. Banfalvi, et al., Apoptosis 2017, 22(2): 306-323). Flow cytometers are typically equipped with only two common lasers, 488nm and 635nm, and fixed-band filters. The choice of fluorescent dyes is often limited by the laser and filter configurations. During detection, fluorescence overlap must also be considered, requiring adjustments for fluorescence compensation, which is complex and cumbersome. Furthermore, adding lasers and optical filters is expensive and infrequently used, leading to a waste of resources. Both of these instrument-based methods are limited by their laser configurations and the need for sample-by-sample testing, which restricts the ability to perform multiple tests simultaneously. Therefore, there is an urgent need to develop a more efficient, simpler, and high-throughput method for detecting apoptosis. Compared with flow cytometry and fluorescence microscopy, microplate readers have the advantages of being relatively inexpensive, compact, and easy to operate, while also offering a wider excitation wavelength range (K. Lewicki, et al., Talanta 2006, 70(4):876-882; F. Cheng, et al., Appl Microbiol Biot 2020, 104(7):2999-3009; J. Y. Choi, et al., J Biol Chem 2018, 293(5):1493-1503; J. Alley, et al., Assay Drug Dev Techn 2010, 8(1):73-84). They allow for rapid measurement of multiple samples in a microplate format (M. Mandalakis, et al., Anal Bioanal Chem 2017, 409(19):4539-4549). High throughput, ease of use, and low sample volume requirement are the key advantages of this instrument (M. Mandalakis, et al., Anal Bioanal Chem 2017, 409(19):4539-4549).

[0004] Currently, there is a lack of a fusion protein and its fluorescence microplate reader method for high-throughput cell apoptosis detection. Summary of the Invention

[0005] In order to solve the problems of the prior art, the present invention aims to provide a fusion protein and a fluorescence microplate reader method for high-throughput cell apoptosis detection.

[0006] To achieve the above objectives, the present invention provides the following technical solutions: First, the present application provides a fusion protein for high-throughput apoptosis detection. Second, the present application provides a fluorescence microplate reader method for high-throughput apoptosis detection. Third, the present application provides an application of a fusion protein of annexin A5 and a fluorescent protein or a fluorescence microplate reader method for detection.

[0007] In the first aspect of the present application, a fusion protein is provided for high-throughput cell apoptosis detection, which is a fusion protein of annexin A5AnxA5 and fluorescent protein FPs; the fluorescent protein is selected from any one of EBFP2, TagBFP, Cerulean, mCerulean, mCerulean3, ECFP, EGFP and TagRFP; the fusion protein includes AnxA5-EBFP2, AnxA5-TagBFP, AnxA5-Cerulean, AnxA5-mCerulean, AnxA5-mCerulean3, AnxA5-EGFP, AnxA5-ECFP and AnxA5-TagRFP.

[0008] Further, when the fluorescent protein is EBFP2, the nucleotide sequence of ANXA5-EBFP2 is shown in SEQ ID NO: 1, and the amino acid sequence of ANXA5-EBFP2 is shown in SEQ ID NO: 9; when the fluorescent protein is TagBFP, the nucleotide sequence of AnxA5-TagBFP is shown in SEQ ID NO: 2, and the amino acid sequence of AnxA5-TagBFP is shown in SEQ ID NO: 10; when the fluorescent protein is Cerulean, the nucleotide sequence of AnxA5-Cerulean is shown in SEQ ID NO: 3, and the amino acid sequence of AnxA5-Cerulean is shown in SEQ ID NO: 11; when the fluorescent protein is mCerulean, the nucleotide sequence of AnxA5-mCerulean is shown in SEQ ID NO: 4, and the amino acid sequence of AnxA5-mCerulean is shown in SEQ ID NO: 12; when the fluorescent protein is mCerulean3, the nucleotide sequence of AnxA5-mCerulean3 is shown in SEQ ID NO: NO:5, the amino acid sequence of the ANXA5-mCerulean3 is shown in SEQ ID NO:13; when the fluorescent protein is ECFP, the nucleotide sequence of ANXA5-ECFP is shown in SEQ ID NO:6, and the amino acid sequence of the ANXA5-ECFP is shown in SEQ ID NO:14; when the fluorescent protein is EGFP, the nucleotide sequence of ANXA5-EGFP is shown in SEQ ID NO:7, and the amino acid sequence of the ANXA5-EGFP is shown in SEQ ID NO:15; when the fluorescent protein is TagRFP, the nucleotide sequence of ANXA5-TagRFP is shown in SEQ ID NO:8, and the amino acid sequence of the ANXA5-TagRFP is shown in SEQ ID NO:16.

[0009] Furthermore, after the fusion protein was diluted 10-fold in the range of 0-0.15 μg / μL, when its concentration was lower than 0.015 μg / μL, there was no significant difference in fluorescence intensity from the background signal.

[0010] The second aspect of the present application provides a fluorescence microplate reader method for high-throughput cell apoptosis detection, comprising the following steps: (1) using a fusion protein of annexin A5 (AnxA5) and fluorescent protein FPs to label apoptotic cells; (2) detecting the fluorescence intensity of the fusion protein using a multifunctional microplate reader; and (3) determining the exposure of phosphatidylserine PS on the surface of apoptotic cells based on the fluorescence signal.

[0011] Furthermore, in step (1), about 8,000 cells are cultured in a black transparent bottom 96-well plate, and the cells are induced to undergo apoptosis using chemical drugs. The induced apoptotic cells are incubated with different concentrations of AnxA5 probes; the fusion protein of membrane-bound protein A5 (AnxA5) and fluorescent protein FPs specifically binds to the phosphatidylserine PS externalized on the surface of apoptotic cells.

[0012] Furthermore, in step (2), the fluorescence intensity of the fusion protein is detected by fluorescence using a multifunctional microplate reader, thereby reflecting the exposure degree of PS on the surface of apoptotic cells.

[0013] Furthermore, in step (2), after the fusion protein was diluted 10-fold in the range of 0-0.15 μg / μL, when the concentration was lower than 0.015 μg / μL, the fluorescence intensity had no significant difference from the background signal;

[0014] Fluorescent proteins emit fluorescence when excited by blue light, and this fluorescence can be detected by a microplate reader. The signal-to-noise ratio of AnxA5-EBFP2, AnxA5-TagBFP, AnxA5-Cerulean, AnxA5-mCerulean, AnxA5-mCerulean3, AnxA5-ECFP, and AnxA5-TagRFP excited by blue light is higher than that of AnxA5-EGFP;

[0015] As the final concentration of the fusion protein labeled cells increased, the fluorescence intensity of the apoptotic cells gradually increased and eventually reached a plateau. The microplate reader could monitor the changes in fluorescence intensity of apoptotic cells labeled with different concentrations of fusion protein, indicating that this method is feasible and reliable.

[0016] Furthermore, in step (3), the reliability of the microplate reader in detecting the fluorescence intensity of apoptotic cells labeled with the AnxA5-EGFP fusion protein was evaluated by comparing the measurement results of the microplate reader and flow cytometry; the correlation analysis results showed that there was a significant linear relationship between the two, but the Pearson r was only 0.9315 (p<0.0001), indicating that the fluorescence intensity measured by the microplate reader was higher than that by flow cytometry, suggesting that the high background signal of the AnxA5-EGFP fusion protein makes it unsuitable for microplate reader detection.

[0017] The third aspect of the present application provides an application of a fusion protein of annexin A5 and fluorescent protein or a fluorescence microplate reader method in detection.

[0018] Furthermore, the working concentration range of the fusion protein during detection is 0.015-0.15 μg / μL, within which a good signal-to-noise ratio and linear response can be obtained; the incubation time of the fusion protein in the microplate reader detection is 15-60 minutes and the temperature is 25-37°C to ensure sufficient binding and stable fluorescence signal.

[0019] Beneficial effects: The present invention solves the problems of existing apoptosis detection technologies, such as flow cytometry and fluorescence microscopy, through technological innovation, such as high cost, high technical requirements, and limited identification using AnxA5 labeled with fluorescent substances with specific excitation wavelengths. It provides an economical, efficient, easy-to-operate, high-throughput method for cell apoptosis detection, which can serve as an alternative to flow cytometry under certain conditions.

[0020] Compared with the existing technologies, the present invention has the following advantages: (1) Simple operation: The present invention uses a fusion protein of annexin A5 (AnxA5) and fluorescent proteins (FPs) to label apoptotic cells and detects the fluorescence intensity using a multifunctional microplate reader. Compared with flow cytometry and fluorescence microscopy, microplate readers are simple to operate and do not require complex instrument settings and professional skills, thus reducing the difficulty of operation and the dependence on professional technicians.

[0021] (2) High-throughput detection: Using a black transparent bottom 96-well plate for detection, multiple samples can be detected at the same time, greatly improving the detection efficiency. In the range of 0-0.15μg / μL, the fluorescence intensity of the fusion protein changes linearly with increasing concentration, and the correlation coefficient R 2 The results were greater than 0.98 (except AnxA5-EGFP), providing a reliable quantitative detection method for high-throughput screening.

[0022] (3) Low cost: The price of microplate reader is relatively cheap, the maintenance cost is low, and the preparation and use cost of fusion protein is also low. Compared with flow cytometry and fluorescence microscopy, the detection cost is greatly reduced, which is suitable for large-scale experiments and clinical applications.

[0023] (4) High detection sensitivity: At low concentrations (0.015-0.15 μg / μL), the fusion protein can specifically bind to the externalized phosphatidylserine (PS) on the surface of apoptotic cells and generate a fluorescent signal that can be detected by a microplate reader. The signal-to-noise ratio of fusion proteins such as AnxA5-EBFP2 and AnxA5-TagBFP excited by blue light is higher than that of AnxA5-EGFP, indicating that they can still provide high detection sensitivity at low concentrations.

[0024] (5) Good stability and repeatability: The fusion protein showed good stability and repeatability during the experiment, which provided a guarantee for obtaining reliable and consistent detection results. Not limited by instruments: The choice of fluorescent probes is no longer limited to common fluorescent dyes such as FITC and PE and AnxA5 labeled with common fluorescent proteins such as EGFP and TagRFP. The selection range of probes is wider and is not limited by the laser and filter configuration of the instrument. Experimenters can flexibly choose fluorescent markers according to their needs, which improves the flexibility and adaptability of the experiment.

[0025] (6) Fast detection speed: The microplate reader can detect multiple samples quickly, which can significantly improve the efficiency of the experiment and save time and resources. Low background signal: The fluorescence intensity of some fusion proteins (such as AnxA5-EBFP2, AnxA5-TagBFP, etc.) is not significantly different from the background signal at low concentrations, which helps to improve the accuracy and sensitivity of the detection and reduce the impact of background interference on the results.

[0026] (7) Wide range of applications: In addition to being used for apoptosis detection, the method can also be used to screen anticancer drugs, evaluate the apoptosis-inducing effect of drugs, and study the pathological mechanisms of apoptosis-related diseases. By detecting the level of apoptosis, the changes in apoptosis during the development of the disease can be explored, providing new ideas and methods for disease diagnosis and treatment. Given the popularity and ease of operation of the microplate reader, the method of the present invention is easy to promote and apply in different laboratories and medical institutions, and has good application prospects and market potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following briefly introduces the drawings required for use in the embodiments or descriptions of the prior art. 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 any creative work.

[0028] Figure 1 Figure 2 is a purification diagram of the AnxA5-blue / cyan FPs fusion protein of the present invention; wherein, ( Figure 1 A) SDS-PAGE analysis of purified AnxA5-FPs; ( Figure 1 B) Purity of AnxA5-FPs fusion protein.

[0029] Figure 2 is a basic characterization diagram of the AnxA5-FPs probe of the present invention; wherein, ( Figure 2 A) Particle size distribution of two AnxA5-blue FPs; ( Figure 2 B) Particle size distribution of four AnxA5-cyan FPs. ( Figure 2 C) Emission and excitation spectra of six AnxA5-FPs probes.

[0030] Figure 3 This is the non-denaturing gel electrophoresis analysis of the AnxA5-blue / cyan FPs fusion protein of the present invention.

[0031] Figure 4The figure shows the fitted straight line between the fluorescence signal ratio of AnxA5-FPs and its corresponding concentration obtained by the microplate reader detection method of the present invention (range: 0-0.15 μg / μL). The red dotted rectangle box: concentration range 0 to 0.015 μg / μL.

[0032] Figure 5 is a fitted straight line between the fluorescence intensity of AnxA5-FPs and its corresponding concentration obtained by the microplate reader detection method of the present invention; wherein, ( Figure 5 A) AnxA5-cyan FPs; ( Figure 5 B) AnxA5-blue FPs; ( Figure 5 C) AnxA5-EGFP; ( Figure 5 D) Fitted straight line between the fluorescence intensity of AnxA5-TagRFP and its corresponding concentration (range: 0-0.15 μg / μL). The red arrow shows the intercept of the straight line with the x-axis.

[0033] Figure 6 This is a diagram of etoposide inducing apoptosis in Jurkat cells according to the present invention; wherein, ( Figure 6 A) Flow cytometric analysis of apoptotic Jurkat cells induced by different concentrations of etoposide; ( Figure 6 B) Percentage of apoptotic cells in Jurkat cells induced by different concentrations of etoposide. The percentage of apoptotic cells in each group was compared with that in the control group. ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05.

[0034] Figure 7 The present invention is based on the fluorescence AnxA5 enzyme marker detection method to detect apoptotic cells; wherein, ( Figure 7 A) Flowchart of the microplate reader method for detecting cell apoptosis based on fluorescence AnxA5 (created by FigDraw 2.0); ( Figure 7 B) Fitting curve of the fluorescence intensity of AnxA5-FPs binding to PS exposed on the cell surface and different concentrations of AnxA5-FPs.

[0035] Figure 8 This is a correlation analysis diagram of the fluorescence intensity of AnxA5-TagRFP in apoptotic Jurkat cells detected by the microplate reader of the present invention and flow cytometry; wherein, ( Figure 8 A) Normalized AnxA5-TagRFP fluorescence intensity measured by microplate reader; ( Figure 8 B) Mean fluorescence intensity of AnxA5-TagRFP measured by flow cytometry; ( Figure 8C) Correlation line between the normalized fluorescence intensity of AnxA5-TagRFP measured by microplate reader (y-axis) and the average fluorescence intensity of AnxA5-TagRFP measured by flow cytometry (x-axis).

[0036] Figure 9 Correlation analysis of AnxA5-EGFP fluorescence intensity in apoptotic Jurkat cells detected by microplate reader and flow cytometry; ( Figure 9 A) Flow cytometric analysis of apoptotic cells labeled with AnxA5-EGFP and PI. Jurkat cells were induced with different concentrations of mitoxantrone hydrochloride (0, 0.5, 1.0, 1.5, 2.0, 2.5 μg / mL); ( Figure 9 B) The proportion of apoptotic cells induced by different concentrations of mitoxantrone hydrochloride was detected by flow cytometry; ( Figure 9 C) Average fluorescence intensity of AnxA5-EGFP positive cells measured by flow cytometry; ( Figure 9 D) Fluorescence intensity of apoptotic cells labeled with AnxA5-EGFP measured by microplate reader; ( Figure 9 E) Correlation line between the fluorescence intensity of apoptotic cells labeled with AnxA5-EGFP measured by microplate reader (y-axis) and the mean fluorescence intensity of AnxA5-EGFP-positive cells measured by flow cytometry (x-axis).

[0037] Figure 10 The fluorescence image of B16-F10 cells stained with the blue luminescent AnxA5 probe under continuous excitation conditions and its corresponding fluorescence intensity diagram are shown in the figure; wherein, ( Figure 10 A)AnxA5-mCerulean,AnxA5-mCerulean3,AnxA5-Cerulean,AnxA5-ECFP,( Figure 10 B)AnxA5-EBFP2,( Figure 10 C) Fluorescence microscopy images of apoptotic B16-F10 cells labeled with commercially available AnxA5-FITC within 25 minutes; ( Figure 10 D) Decrease in mean fluorescence intensity of apoptotic B16-F10 cells. The percentage of apoptotic cells in each group was compared with that in the control group. ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05. DETAILED DESCRIPTION

[0038] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0039] In this application, the term "and / or" describes the relationship between associated objects, indicating that three possible relationships exist. For example, A and / or B can represent: A exists alone, A and B exist simultaneously, 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.

[0040] In this application, "-one or more" means one or more, and "more than one" means two or more. "The following - one or more" or similar expressions refer to any combination of these items, including any combination of single items or plural items. For example, "a, b, or c - one or more", or "a, b, and c - one or more" can all mean: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, and c can be single or multiple.

[0041] 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. 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.

[0042] The terms used in the embodiments of the present application are for the purpose of describing specific embodiments only and are not intended to limit the present application. The singular forms "a", "an", "the" 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 otherwise.

[0043] The weights of the relevant components mentioned in the examples of this application may not only refer to the specific content of each component, but also represent the weight ratio between the components. Therefore, as long as the content of the relevant components is proportionally enlarged or reduced according to the examples of this application, it is within the scope disclosed in the examples of this application. Specifically, the mass described in the examples of this application may be a mass unit known in the chemical industry, such as μg, mg, g, kg, etc.

[0044] The terms "first" and "second" are used solely for descriptive purposes to distinguish objects, such as substances, from one another and should not be construed as indicating or implying relative importance or implicitly specifying the quantity of the technical features being referred to. For example, a first XX could also be referred to as a second XX, and similarly, a second XX could also be referred to as a first XX, without departing from the scope of the embodiments of this application. Thus, features defined as "first" or "second" may explicitly or implicitly include one or more of such features.

[0045] The first aspect of an embodiment of the present application provides a fusion protein for high-throughput cell apoptosis detection, which is a fusion protein of annexin A5 AnxA5 and fluorescent protein FPs; the fluorescent protein is selected from any one of EBFP2, TagBFP, Cerulean, mCerulean, mCerulean3, ECFP, EGFP and TagRFP; the fusion protein includes AnxA5-EBFP2, AnxA5-TagBFP, AnxA5-Cerulean, AnxA5-mCerulean, AnxA5-mCerulean3, AnxA5-EGFP, AnxA5-ECFP and AnxA5-TagRFP.

[0046] In some embodiments, when the fluorescent protein is EBFP2, the nucleotide sequence of ANXA5-EBFP2 is shown in SEQ ID NO: 1, and the amino acid sequence of ANXA5-EBFP2 is shown in SEQ ID NO: 9; when the fluorescent protein is TagBFP, the nucleotide sequence of AnxA5-TagBFP is shown in SEQ ID NO: 2, and the amino acid sequence of AnxA5-TagBFP is shown in SEQ ID NO: 10; when the fluorescent protein is Cerulean, the nucleotide sequence of AnxA5-Cerulean is shown in SEQ ID NO: 3, and the amino acid sequence of AnxA5-Cerulean is shown in SEQ ID NO: 11; when the fluorescent protein is mCerulean, the nucleotide sequence of AnxA5-mCerulean is shown in SEQ ID NO: 4, and the amino acid sequence of AnxA5-mCerulean is shown in SEQ ID NO: 12; when the fluorescent protein is mCerulean3, the nucleotide sequence of AnxA5-mCerulean3 is shown in SEQ ID NO: NO:5, the amino acid sequence of ANXA5-mCerulean3 is shown in SEQ ID NO:13; when the fluorescent protein is ECFP, the nucleotide sequence of ANXA5-ECFP is shown in SEQ ID NO:6, and the amino acid sequence of ANXA5-ECFP is shown in SEQ ID NO:14; when the fluorescent protein is EGFP, the nucleotide sequence of ANXA5-EGFP is shown in SEQ ID NO:7, and the amino acid sequence of ANXA5-EGFP is shown in SEQ ID NO:15; when the fluorescent protein is TagRFP, the nucleotide sequence of ANXA5-TagRFP is shown in SEQ ID NO:8, and the amino acid sequence of ANXA5-TagRFP is shown in SEQ ID NO:16.

[0047] In some embodiments, after the fusion protein is diluted 10-fold in the range of 0-0.15 μg / μL, when its concentration is lower than 0.015 μg / μL, the fluorescence intensity has no significant difference from the background signal.

[0048] The second aspect of the embodiment of the present application provides a fluorescence microplate reader method for high-throughput cell apoptosis detection, comprising the following steps: (1) using a fusion protein of annexin A5 (AnxA5) and fluorescent protein FPs to label apoptotic cells; culturing approximately 8,000 cells in a black transparent-bottom 96-well plate, inducing cell apoptosis with chemical drugs, and incubating the induced apoptotic cells with different concentrations of AnxA5 probes; and specifically binding the phosphatidylserine PS on the surface of apoptotic cells through the fusion protein of annexin A5 (AnxA5) and fluorescent protein FPs.

[0049] (2) Detecting the fluorescence intensity of the fusion protein using a multifunctional microplate reader; The fluorescence intensity of the fusion protein was detected by fluorescence using a multifunctional microplate reader, thereby reflecting the exposure degree of PS on the surface of apoptotic cells. After the fusion protein was diluted 10 times in the range of 0-0.15μg / μL, when the concentration was lower than 0.015μg / μL, the fluorescence intensity had no significant difference from the background signal; the fluorescent protein would emit fluorescence when excited by blue light, and this fluorescence could be detected by the microplate reader. The signal-to-noise ratio of AnxA5-EBFP2, AnxA5-TagBFP, AnxA5-Cerulean, AnxA5-mCerulean, AnxA5-mCerulean3, AnxA5-ECFP and AnxA5-TagRFP excited by blue light was higher than that of AnxA5-EGFP; as the final concentration of the fusion protein labeled cells increased, the fluorescence intensity of the apoptotic cells gradually increased and eventually reached a plateau. The microplate reader could monitor the changes in the fluorescence intensity of apoptotic cells labeled with different concentrations of fusion protein, indicating that this method was feasible and reliable.

[0050] (3) The exposure of phosphatidylserine (PS) on the surface of apoptotic cells was determined based on the fluorescence signal. The reliability of the microplate reader in detecting the fluorescence intensity of apoptotic cells labeled with AnxA5-EGFP fusion protein was evaluated by comparing the measurement results of the microplate reader and flow cytometry. The correlation analysis results showed that there was a significant linear relationship between the two, but the Pearson r was only 0.9315 (p < 0.0001), indicating that the fluorescence intensity measured by the microplate reader was higher than that by flow cytometry, suggesting that the high background signal of the AnxA5-EGFP fusion protein makes it unsuitable for microplate reader detection.

[0051] A third aspect of the embodiments of the present application provides an application of a fusion protein of annexin A5 and a fluorescent protein or a fluorescence microplate reader method in detection.

[0052] In some embodiments, the working concentration range of the fusion protein during detection is 0.015-0.15 μg / μL, within which a good signal-to-noise ratio and linear response can be obtained; the incubation time of the fusion protein in the microplate reader detection is 15-60 minutes at a temperature of 25-37°C to ensure sufficient binding and a stable fluorescent signal.

[0053] Example 1

[0054] The present invention discloses a fusion protein for high-throughput cell apoptosis detection, which is a fusion protein of annexin A5AnxA5 and fluorescent proteins FPs; the fluorescent protein is selected from any one of EBFP2, TagBFP, Cerulean, mCerulean, mCerulean3, ECFP, EGFP and TagRFP; and the fusion proteins include AnxA5-EBFP2, AnxA5-TagBFP, AnxA5-Cerulean, AnxA5-mCerulean, AnxA5-mCerulean3, AnxA5-EGFP, AnxA5-ECFP and AnxA5-TagRFP.

[0055] When the fluorescent protein is EBFP2, the nucleotide sequence of ANXA5-EBFP2 is shown in SEQ ID NO: 1, and the amino acid sequence of ANXA5-EBFP2 is shown in SEQ ID NO: 9; when the fluorescent protein is TagBFP, the nucleotide sequence of AnxA5-TagBFP is shown in SEQ ID NO: 2, and the amino acid sequence of AnxA5-TagBFP is shown in SEQ ID NO: 10; when the fluorescent protein is Cerulean, the nucleotide sequence of AnxA5-Cerulean is shown in SEQ ID NO: 3, and the amino acid sequence of AnxA5-Cerulean is shown in SEQ ID NO: 11; when the fluorescent protein is mCerulean, the nucleotide sequence of AnxA5-mCerulean is shown in SEQ ID NO: 4, and the amino acid sequence of AnxA5-mCerulean is shown in SEQ ID NO: 12; when the fluorescent protein is mCerulean3, the nucleotide sequence of AnxA5-mCerulean3 is shown in SEQ ID NO: When the fluorescent protein is ECFP, the nucleotide sequence of ANXA5-ECFP is shown in SEQ ID NO:6, and the amino acid sequence of ANXA5-ECFP is shown in SEQ ID NO:14. When the fluorescent protein is EGFP, the nucleotide sequence of ANXA5-EGFP is shown in SEQ ID NO:7, and the amino acid sequence of ANXA5-EGFP is shown in SEQ ID NO:15. When the fluorescent protein is TagRFP, the nucleotide sequence of ANXA5-TagRFP is shown in SEQ ID NO:8, and the amino acid sequence of ANXA5-TagRFP is shown in SEQ ID NO:16. After the fusion protein was diluted 10-fold in the range of 0-0.15 μg / μL, the fluorescence intensity was not significantly different from the background signal when its concentration was lower than 0.015 μg / μL.

[0056] Example 2

[0057] The present invention provides a fluorescence microplate reader method for high-throughput cell apoptosis detection, comprising the following steps: (1) labeling apoptotic cells with a fusion protein of annexin A5 (AnxA5) and fluorescent protein (FPs); culturing approximately 8,000 cells in a black transparent-bottom 96-well plate, inducing cell apoptosis with chemical drugs, and incubating the induced apoptotic cells with AnxA5 probes at different concentrations; and specifically binding the fusion protein of annexin A5 (AnxA5) and fluorescent protein (FPs) to the phosphatidylserine (PS) externalized on the surface of apoptotic cells.

[0058] (2) Detecting the fluorescence intensity of the fusion protein using a multifunctional microplate reader; The fluorescence intensity of the fusion protein was detected by fluorescence using a multifunctional microplate reader, thereby reflecting the exposure degree of PS on the surface of apoptotic cells. After the fusion protein was diluted 10 times in the range of 0-0.15μg / μL, when the concentration was lower than 0.015μg / μL, the fluorescence intensity had no significant difference from the background signal; the fluorescent protein would emit fluorescence when excited by blue light, and this fluorescence could be detected by the microplate reader. The signal-to-noise ratio of AnxA5-EBFP2, AnxA5-TagBFP, AnxA5-Cerulean, AnxA5-mCerulean, AnxA5-mCerulean3, AnxA5-ECFP and AnxA5-TagRFP excited by blue light was higher than that of AnxA5-EGFP; as the final concentration of the fusion protein labeled cells increased, the fluorescence intensity of the apoptotic cells gradually increased and eventually reached a plateau. The microplate reader could monitor the changes in the fluorescence intensity of apoptotic cells labeled with different concentrations of fusion protein, indicating that this method was feasible and reliable. (3) Determine the exposure of phosphatidylserine PS on the surface of apoptotic cells based on the fluorescence signal.

[0059] In step (3), the reliability of the microplate reader in detecting the fluorescence intensity of apoptotic cells labeled with the AnxA5-EGFP fusion protein was evaluated by comparing the measurement results of the microplate reader and flow cytometry. The correlation analysis results showed that there was a significant linear relationship between the two, but the Pearson r was only 0.9315 (p < 0.0001), indicating that the fluorescence intensity measured by the microplate reader was higher than that by flow cytometry, suggesting that the high background signal of the AnxA5-EGFP fusion protein makes it unsuitable for microplate reader detection.

[0060] Example 3

[0061] The invention discloses an application of a fusion protein of annexin A5 and fluorescent protein or a fluorescence enzyme marker method in detection.

[0062] The working concentration range of the fusion protein during detection is 0.015-0.15 μg / μL, within which a good signal-to-noise ratio and linear response can be obtained; the incubation time of the fusion protein in the microplate reader detection is 15-60 minutes and the temperature is 25-37°C to ensure sufficient binding and stable fluorescence signal.

[0063] Example 4

[0064] Preparation of blue light-excited AnxA5 probe

[0065] In the pET28a(+)-AnxA5-EGFP-his6 vector, blue / cyan fluorescent proteins, including EBFP2, TagBFP, Cerulean, mCerulean, mCerulean3 and ECFP, were used to replace EGFP, respectively, to construct the pET28a(+)-AnxA5-blue / cyan-FPs-his6 plasmid. The sequences of all primers are shown in Table 1. The pET28a(+)-AnxA5-blue / cyan-FPs-his6 plasmid was transformed into Escherichia coli BL21 (DE3) strain, induced with 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG), and then incubated at 20°C for 16 hours. The AnxA5-blue / cyan-FPs fusion protein was purified by Ni-NTA agarose affinity chromatography (as shown in Table 1). Figure 1 A), the purity of all fusion proteins reached more than 80% (as shown in Figure 1 The amino acid numbers and theoretical molecular weights (MW, kDa) of the AnxA5-blue / cyan-FPs fusion proteins are shown in Table 2. Overall, the results demonstrate that the AnxA5-blue / cyan-FPs fusion proteins were successfully expressed and purified. All six AnxA5-blue / cyan-FPs fusion proteins were stored in Tris buffer containing 20 mM Tris, 30 mM NaCl, pH 8.5.

[0066] Table 1

[0067]

[0068]

[0069] Table 2

[0070]

[0071] Example 5

[0072] Characterization of AnxA5-blue / cyan-FPs fusion protein

[0073] The oligomerization states of the six fluorescent proteins (EBFP2, TagBFP, Cerulean, mCerulean, mCerulean3 and ECFP) are different. As an apoptosis detection probe, the monomeric nature of AnxA5-FPs is crucial to the accuracy and reliability of apoptosis detection. Therefore, the present invention detects the particle size distribution of the AnxA5-blue / cyan FPs fusion protein. The particle size distribution of the fusion protein (3.0 mg / mL) was measured using a particle size analyzer (Malvern Instruments, ZS90). The DLS analysis results showed that the AnxA5-blue / cyan FPs fusion protein was homogeneous and highly monodispersed in the solution (e.g. Figure 2 A, B), hydrodynamic radius (R h ) varied between 2.8 nm and 3.8 nm, as shown in Table 3. In addition, the aggregation state of AnxA5-blue / cyan FPs fusion protein was analyzed by native-PAGE. The results showed that AnxA5-TagBFP was probably a multimer, while the other AnxA5-FPs were roughly at the same position on the gel and were probably monomers (e.g. Figure 3 R of AnxA5-TagBFP h There were no significant differences between the two AnxA5-FPs and the other AnxA5-FPs, with the exception of AnxA5-TagBFP, which migrated the slowest in native gel electrophoresis. This phenomenon may be due to the complex structure of the fusion protein and the inaccuracy of DLS detection of non-spherical particles. In summary, the purified AnxA5-blue / cyan FP fusion proteins were homogeneous and, with the exception of AnxA5-TagBFP, were monomeric.

[0074] Table 3

[0075]

[0076] The present invention also scanned the excitation and emission wavelengths of AnxA5-blue / cyan-FPs (50 μg / mL) fusion protein on a F-4500 fluorescence spectrophotometer (Hitachi, Japan). The results showed that the excitation and emission spectra of AnxA5-blue / cyan-FPs did not change significantly compared with the fluorescent protein alone (e.g. Figure 2 C), and its excitation maximum and emission maximum are shown in Table 4. 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.

[0077] Table 4

[0078]

[0079] Example 6

[0080] Concentration optimization of AnxA5-blue / cyan-FPs fusion protein for microplate reader detection

[0081] The AnxA5-blue / cyan-FPs fusion protein was diluted to different concentrations using 1× binding buffer (10mM Hepes, 140mM NaCl, 2.5mM CaCl2, pH 7.4). In a black transparent bottom 96-well microplate (Corning, New York, USA), the fluorescence intensity values ​​of 100μL of AnxA5-blue / cyan-FPs at different concentrations (0-1.5μg / μL and 0-0.15μg / μL) were detected using a TECAN Infinite M200PRO microplate reader (TECAN, Switzerland) at the optimal excitation and emission wavelengths. The AnxA5-blue / cyan-FPs fusion protein was diluted to different concentrations (0-0.15μg / μL) in a gradient manner, and the fluorescence intensity of each well was measured. The results showed that when the concentration of AnxA5-blue / cyan-FPs was lower than 0.015μg / μL, their fluorescence intensity was not significantly different from the background signal (such as Figure 4 The signal-to-noise ratios of AnxA5-FPs and AnxA5-TagRFP excited by blue light were higher than those of AnxA5-EGFP (as shown in Table 6), indicating that the microplate reader has higher sensitivity for detecting AnxA5-FPs and AnxA5-TagRFP excited by blue light. In addition, when the AnxA5-blue / cyan-FPs fusion protein was diluted in a 2-fold gradient, the fluorescence intensity increased with increasing AnxA5-blue / cyan-FPs concentration. When the concentration was lower than 0.15 μg / μL, the fitting curve was linear, with a correlation coefficient R 2 Greater than 0.98 (except AnxA5-EGFP) (such as Figure 5 The results show that within the concentration range designed for this experiment, the fluorescence intensity values ​​of AnxA5-blue FP, AnxA5-cyan FP, and AnxA5-TagRFP measured by the microplate reader have a good linear relationship with their content in the sample. In summary, blue-light-excited AnxA5-FPs and AnxA5-TagRFP are suitable probes for microplate reader detection.

[0082] Table 5

[0083]

[0084] Table 6

[0085]

[0086] Example 7

[0087] Detection of apoptotic samples and data analysis using a fluorescence-based microplate reader assay

[0088] The proposed microplate reader assay was applied to the detection of apoptotic samples. To perform a fluorescence-based microplate reader assay of AnxA5, the first step was to culture about 8,000 cells in a black-walled clear-bottom 96-well plate, followed by induction with chemical drugs. Jurkat cells were induced with 10 mM etoposide (as shown in Figure 6 The flowchart of this method is shown in Figure 7 A. The fluorescence intensity of AnxA5-Blue / Cyan-FPs fusion protein bound to apoptotic cells was analyzed using a M200 PRO multi-function microplate reader. The normalized fluorescence intensity was calculated according to the following formula: (fluorescence intensity of AnxA5-FPs labeled cells - fluorescence intensity of unstained cells) / maximum fluorescence intensity in the same group x 100 (%). The results showed that the fluorescence intensity of apoptotic cells gradually increased with the increase of the final concentration of AnxA5-Blue / Cyan-FPs used to label the cells, and finally reached a plateau (as shown in Figure 7 B). The microplate reader could effectively monitor the change of fluorescence intensity of apoptotic cells, which supported the feasibility and reliability of the proposed method. In summary, the microplate reader assay successfully monitored the change of fluorescence intensity of apoptotic cells labeled with different concentrations of AnxA5-Blue / Cyan-FPs, indicating the sensitivity of the method.

[0089] Test Example 1

[0090] Comparison of cell apoptosis detection using flow cytometry and microplate reader assay

[0091] To explore whether the AnxA5 probe can monitor the PS level in apoptotic samples to screen anticancer drugs, Jurkat cells were treated with different concentrations of etoposide, and then apoptotic cells were collected and incubated with AnxA5-TagRFP. The fluorescence intensity of AnxA5-TagRFP labeled apoptotic cells was detected using flow cytometry and microplate reader assay, respectively (as shown in Figure 8 A, Figure 8 B). The results of correlation analysis showed that there was a significant linear relationship between the fluorescence intensity of apoptotic cells measured using the microplate reader method and the results measured using flow cytometry (as shown in Figure 8C), Pearson r was 0.9744, p < 0.0001. This indicates that the results of the microplate reader assay are generally consistent with those of flow cytometry. However, the fluorescence intensity measured by the microplate reader is higher than that of the flow cytometry result. This may be because AnxA5-TagRFP is excited at its maximum excitation wavelength in the microplate reader, while the flow cytometer uses 488 nm excitation. In summary, the established microplate reader assay based on AnxA5-TagRFP can be used for apoptosis detection.

[0092] Similarly, the present invention also used different concentrations of mitoxantrone hydrochloride to induce Jurkat cells, then collected apoptotic cells and incubated with AnxA5-EGFP. Flow cytometry and microplate reader were used to detect the fluorescence intensity of AnxA5-EGFP-labeled apoptotic cells. The flow cytometry results showed that with the increase of mitoxantrone hydrochloride concentration, the proportion of apoptotic cells and the average fluorescence intensity of AnxA5-EGFP-positive cells increased significantly ( Figure 9 A- Figure 9 C). Similarly, when measured using a microplate reader, the fluorescence intensity of apoptotic cells labeled with AnxA5-EGFP also increased with increasing mitoxantrone hydrochloride concentrations ( Figure 9 D). Correlation analysis results showed that there was a linear relationship between the fluorescence intensity of apoptotic cells measured by the microplate reader and the results measured by flow cytometry ( Figure 9 E), but the Pearson r was only 0.9315 (p < 0.0001). This result indicates that the fluorescence intensity of apoptotic cells measured by the microplate reader is relatively poorly consistent with the results of flow cytometry, showing that the fluorescence intensity measured by the microplate reader is higher than that of the flow cytometry result. Therefore, AnxA5-EGFP, with its high background signal, may not be a suitable probe for detecting apoptosis using the microplate reader.

[0093] The higher Pearson's r value observed in the microplate reader-based assay results for AnxA5-TagRFP compared to AnxA5-EGFP indicates better concordance between the microplate reader and flow cytometric results, suggesting that AnxA5-TagRFP is more suitable for microplate reader detection. Therefore, microplate reader-based apoptosis detection is reliable, but requires an AnxA5 probe that is suitable for microplate reader detection. Comparison of the microplate reader-based properties of various AnxA5-FPs revealed that the blue-light-excited AnxA5 probe and AnxA5-TagRFP are more suitable for microplate reader detection than AnxA5-EGFP.

[0094] In summary, the present invention establishes a high-throughput, cost-effective method for apoptosis detection. Table 7 compares the characteristics of the microplate reader assay and flow cytometry assay in detail, highlighting the advantages of the microplate reader assay. In summary, the established microplate reader assay has the potential to serve as a reliable tool for apoptosis detection and, under certain conditions, as an alternative to flow cytometry.

[0095] Table 7

[0096]

[0097]

[0098] Test Example 2

[0099] Photostability of the blue-light-excited AnxA5 probe

[0100] As an important evaluation index for the application of fluorescent probes, photostability needs to be tested. Therefore, the photostability and long-term imaging capability of the AnxA5-blue / cyan FP probe were measured by continuous excitation under a fluorescence microscope. After incubation with AnxA5-FPs, apoptotic B16-F10 cells were continuously excited under a fluorescence microscope for 25 minutes (e.g. Figure 10 The results showed that compared with the commercially available AnxA5-FITC, the half-life (t 1 / 2 ) are more than 3 minutes (such as Figure 10 As the irradiation time increases, the average fluorescence intensity of AnxA5-mCerulean3 and AnxA5-FITC decreases more significantly (as shown in D). Figure 10 After 10 minutes of continuous excitation, the average fluorescence intensity of AnxA5-mCerulean3 and AnxA5-FITC remained less than 20%, while the average fluorescence intensity of AnxA5-EBFP2, AnxA5-Cerulean, and AnxA5-mCerulean probes still retained more than 30% (as shown in Figure 4A). Figure 10 Based on these results, the present invention concludes that blue-light-excited AnxA5-FPs, such as AnxA5-EBFP2, AnxA5-Cerulean, and AnxA5-mCerulean, have superior photostability compared to chemically labeled AnxA5-FITC and can meet the needs of apoptosis detection. For applications involving long-term imaging, such as several hours, AnxA5 probes fused with fluorescent proteins with improved photostability should be selected.

[0101] The basic principles, main features and advantages of the present invention are shown and described above. It should be understood by those skilled in the art that the present invention is not limited to the above embodiments. The above embodiments and descriptions are merely illustrative of the principles of the present invention. Various changes and improvements may be made to the present invention without departing from the spirit and scope of the present invention. The scope of protection claimed in the present invention is defined by the appended claims, the description and their equivalents.

Claims

1. A fusion protein for high-throughput apoptosis detection, characterized in that: The fusion protein is a fusion protein of annexin A5 AnxA5 and fluorescent protein FPs; the fluorescent protein is selected from any one of EBFP2, TagBFP, Cerulean, mCerulean, mCerulean3, ECFP, EGFP and TagRFP; the fusion protein includes AnxA5-EBFP2, AnxA5-TagBFP, AnxA5-Cerulean, AnxA5-mCerulean, AnxA5-mCerulean3, AnxA5-EGFP, AnxA5-ECFP and AnxA5-TagRFP.

2. The fusion protein for high-throughput apoptosis detection according to claim 1, characterized in that: When the fluorescent protein is EBFP2, the nucleotide sequence of ANXA5-EBFP2 is shown in SEQ ID NO: 1, and the amino acid sequence of ANXA5-EBFP2 is shown in SEQ ID NO: 9; When the fluorescent protein is TagBFP, the nucleotide sequence of the AnxA5-TagBFP is shown in SEQ ID NO: 2, and the amino acid sequence of the AnxA5-TagBFP is shown in SEQ ID NO: 10; When the fluorescent protein is Cerulean, the nucleotide sequence of AnxA5-Cerulean is shown in SEQ ID NO: 3, and the amino acid sequence of AnxA5-Cerulean is shown in SEQ ID NO: 11; When the fluorescent protein is mCerulean, the nucleotide sequence of AnxA5-mCerulean is shown in SEQ ID NO: 4, and the amino acid sequence of AnxA5-mCerulean is shown in SEQ ID NO: 12; When the fluorescent protein is mCerulean3, the nucleotide sequence of the AnxA5-mCerulean3 is shown in SEQ ID NO: 5, and the amino acid sequence of the ANXA5-mCerulean3 is shown in SEQ ID NO: 13; When the fluorescent protein is ECFP, the nucleotide sequence of the ANXA5-ECFP is shown in SEQ ID NO: 6, and the amino acid sequence of the ANXA5-ECFP is shown in SEQ ID NO: 14; When the fluorescent protein is EGFP, the nucleotide sequence of the ANXA5-EGFP is shown in SEQ ID NO: 7, and the amino acid sequence of the ANXA5-EGFP is shown in SEQ ID NO: 15; When the fluorescent protein is TagRFP, the nucleotide sequence of the ANXA5-TagRFP is shown as SEQ ID NO: 8, and the amino acid sequence of the ANXA5-TagRFP is shown as SEQ ID NO:

16.

3. The fusion protein for high-throughput apoptosis detection according to claim 1, characterized in that: After the fusion protein was diluted 10-fold in the range of 0-0.15 μg / μL, when its concentration was lower than 0.015 μg / μL, the fluorescence intensity had no significant difference from the background signal.

4. A fluorescence microplate reader method for high-throughput cell apoptosis detection, characterized in that The method comprises the following steps: (1) using the fusion protein of annexin A5 (AnxA5) and fluorescent protein FPs as claimed in claim 1 to mark apoptotic cells; (2) detecting the fluorescence intensity of the fusion protein using a multifunctional microplate reader; (3) Determine the exposure of phosphatidylserine PS on the surface of apoptotic cells based on the fluorescence signal.

5. The fluorescence microplate reader method for high-throughput cell apoptosis detection according to claim 4, characterized in that: In step (1), approximately 8,000 cells are cultured in a black transparent bottom 96-well plate, and apoptosis of the cells is induced by chemical drugs. The induced apoptotic cells are incubated with different concentrations of AnxA5 probes; the fusion protein of membrane-bound protein A5 (AnxA5) and fluorescent protein FPs specifically binds to the phosphatidylserine PS on the surface of apoptotic cells.

6. The fluorescence microplate reader method for high-throughput apoptosis detection according to claim 4, characterized in that: In step (2), the fluorescence intensity of the fusion protein is detected by fluorescence using a multifunctional microplate reader, thereby reflecting the exposure degree of PS on the surface of apoptotic cells.

7. The fluorescence microplate reader method for high-throughput apoptosis detection according to claim 4, characterized in that: In step (2), after the fusion protein is diluted 10-fold in the range of 0-0.15 μg / μL, when the concentration is lower than 0.015 μg / μL, the fluorescence intensity has no significant difference from the background signal; Fluorescent proteins emit fluorescence when excited by blue light, and this fluorescence can be detected by a microplate reader. The signal-to-noise ratio of AnxA5-EBFP2, AnxA5-TagBFP, AnxA5-Cerulean, AnxA5-mCerulean, AnxA5-mCerulean3, AnxA5-ECFP, and AnxA5-TagRFP excited by blue light is higher than that of AnxA5-EGFP; As the final concentration of the fusion protein labeled cells increased, the fluorescence intensity of the apoptotic cells gradually increased and eventually reached a plateau. The microplate reader could monitor the changes in fluorescence intensity of apoptotic cells labeled with different concentrations of fusion protein, indicating that this method is feasible and reliable.

8. The fluorescence microplate reader method for high-throughput apoptosis detection according to claim 6, characterized in that: In step (3), the reliability of the microplate reader in detecting the fluorescence intensity of apoptotic cells labeled with the AnxA5-EGFP fusion protein was evaluated by comparing the measurement results of the microplate reader and flow cytometry; the correlation analysis results showed that there was a significant linear relationship between the two, but the Pearson r was only 0.9315, p < 0.0001, indicating that the fluorescence intensity measured by the microplate reader was higher than that by flow cytometry, suggesting that the high background signal of the AnxA5-EGFP fusion protein makes it unsuitable for microplate reader detection.

9. Use of the fusion protein of Annexin A5 and fluorescent protein according to claim 1 or the fluorescence microplate reader method according to any one of claims 2 to 8 in detection.

10. The use according to claim 9, characterized in that: The working concentration range of the fusion protein during detection is 0.015-0.15 μg / μL, within which a good signal-to-noise ratio and linear response can be obtained; the incubation time of the fusion protein in the microplate reader detection is 15-60 minutes and the temperature is 25-37°C to ensure sufficient binding and stable fluorescence signal.

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