CN120099052A

CN120099052A - A fusion protein of annexin A5 and fluorescent protein for apoptotic cell marking, and preparation method and application thereof

Info

Publication number: CN120099052A
Application number: CN202510310062.0A
Authority: CN
Inventors: 华子春; 高梦月
Original assignee: Targetpharma Laboratories Jiangsu Co ltd
Current assignee: Targetpharma Laboratories Jiangsu Co ltd
Priority date: 2025-02-28
Filing date: 2025-03-17
Publication date: 2025-06-06

Abstract

The present invention relates to the field of biotechnology, and discloses a fusion protein of annexin A5 (AnxA5) and fluorescent protein (FPs) for apoptotic cell labeling, and a preparation method and application thereof, wherein the C-terminus of AnxA5 is fused to the N-terminus of 18 fluorescent proteins via a (Gly-Ser) ₄ connecting peptide by genetic engineering, an expression vector is constructed and transformed into Escherichia coli BL21 (DE3), soluble expression is achieved by low-temperature induction at 20°C (1mM IPTG, 16 hours), and AnxA5-FPs fusion protein with high uniformity and strong photostability is obtained by Ni-NTA affinity purification. The present invention avoids the complex process of chemical coupling, has uniform products and low cost, can be widely used in flow cytometry, fluorescence microscopy detection and in vivo apoptosis tracing, and provides an efficient tool for apoptosis research.

Description

Fusion protein of annexin A5 and fluorescent protein for apoptotic cell labeling, and preparation method and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to fusion protein of annexin A5 and fluorescent protein for labeling apoptotic cells, and a preparation method and application thereof.

Background

When cells undergo apoptosis, phosphatidylserine (PS) everts from within the cell membrane to the outside of the cell membrane, and this cell surface-exposed PS can be used as a label (M.A.O'Brien,et al.,JVet Emerg Crit Car 2008,18(6):572-585;S.E.Logue,et al.,Nat Protoc 2009,4(9):1383-1395). for apoptotic cells, annexin A5 (AnxA 5) is a phospholipid-binding protein capable of binding to PS in a calcium-dependent manner (A.Q.Abbaady, et al, front Physiol 2017, 8:317). AnxA5, which is fluorescently labeled, is widely used as an apoptosis detection probe based on its high affinity for PS, at nanomolar levels (M.Stocker, et al Protein Expr Purif 2008,58 (2): 325-331). However, to date, most available, commercial AnxA5 probes are fluorochrome-conjugated AnxA5. The preparation of such probes involves complex procedures and multiple purification steps, such as protein expression and purification, chemical coupling reactions, and further exclusion of free fluorescein (a.q. Abband, et al, front physics 2017,8:317;J.Wang,et al, eur biophysis J2015,44 (5): 325-336). The whole procedure takes 3 days, including overnight dialysis. Furthermore, chemical coupling always results in heterogeneous mixtures, and the labeled AnxA5 molecules differ in both the number and position of bound luciferin. Furthermore, amine directed chemical modification of AnxA5 may decrease its binding activity to membranes (Tait JF, et al J Nucl Med 2006,47 (9): 1546-1553). Furthermore, the complicated manufacturing process also means high production costs, which also increases the market price of the probe. More importantly, the chemical dye is easy to quench and needs to be strictly protected from light. The preparation process of the probes is complex and cumbersome, the final products are usually mixtures with different labeling degrees, and the probes are nonuniform and have poor light stability, so that the application of the probes is limited. Therefore, development of an AnxA5 fluorescent probe which is simple in preparation process, uniform in final product and good in light stability is urgently needed.

Fluorescent Proteins (FPs) are an integral part of modern biological research. As a common fluorescent tag, they are often used for protein labeling and cell tracking (Z.Y.Wang,et al.,Protein Sci 2021,30(11):2298-2309;N.C.Shaner,et al.,Nat Methods 2005,2:905-909;J.Zhang,etal.,Nat Rev Mol Cell Biol 2002,3:906-918). since the isolation of Green Fluorescent Protein (GFP), it was optimized for variant-Enhanced GFP (EGFP), which is extremely widely used and thus occupies an important place (T.D.Craggs, chem Soc Rev 2009,38 (10): 2865-2875; W.tao, et al, STEM CELLS 2007,25 (3): 670-678). By introducing different mutations into the GFP gene, many useful variants have been developed, such as blue, cyan and yellow fluorescent proteins, etc. (R.Heim, et al, curr Biol 1996,6:178-182;H.Imamura,et al, PNAS2023,120 (45): e 2307687120). The appearance of the variants greatly enriches the variety of fluorescent proteins and widens the application range of the fluorescent proteins. In addition, fluorescent proteins are easy to use and inexpensive (Y.T.Kim, et al, molecules 2022,27 (16): 5248), the genes of which can be mutated to improve various properties such as excitation and emission wavelength, brightness, pKa, maturation time, lifetime and photostability. The probe fused with the fluorescent protein is relatively uniform, high in fluorescence intensity and high in light stability, and in addition, the fluorescent protein fusion probe does not involve multi-step operation in a chemical coupling method, and only prokaryotic expression, separation and purification of the fusion protein are needed, so that the operation is simple, the cost is low, and the application value is high. Therefore, the development of the AnxA5 probe using fluorescent protein based strategies is an effective approach to circumvent the chemical dye deficiency.

AnxA5-GFP and AnxA5-EGFP probes were labeled uniformly, with higher brightness and higher light stability (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 the work prior to this team, we found that AnxA5-FITC had a lower affinity for the membrane than AnxA5-EGFP. Furthermore, sfGFP-AnxA5 was superior to fluorescein-conjugated AnxA5 in binding to phospholipids in one study, making it more sensitive to labeling of early apoptotic HeLa cells (a.q. abband, et al, front Physiol 2017, 8:317). These studies all demonstrate that fluorescent protein-based AnxA5 is a promising apoptosis detection probe. Currently, only the fluorescent protein-based AnxA5 probes commercialized are AnxA5-EGFP (green fluorescence) and AnxA5-mCherry (red fluorescence). Therefore, the development of more AnxA5 probes which are based on fluorescent proteins and have the light emitting range covering blue, cyan and yellow has important significance for expanding the application range of the AnxA5 probes, such as multicolor labeling and co-localization analysis of antibodies, dyes and the like. Fluorescent proteins are various in variety and different in property, and whether the ability of labeling apoptosis is affected after the fluorescent proteins are fused with the AnxA5 is unclear, so that the fluorescent proteins are selected to be fused with the AnxA5 and the affinity of the fluorescent proteins with apoptotic cells is analyzed, and the screening of fusion proteins with affinity higher than that of the AnxA5-EGFP and the AnxA5-mCherry has important application value in early apoptosis detection.

In view of the above, we believe that fluorescent protein-labeled AnxA5 may be developed as a promising probe. However, whether the type of fluorescent protein affects the function and properties of the AnxA5 protein and how to affect the function of AnxA5 has not been a clear answer to date. Therefore, after the AnxA5 is fused and expressed with various fluorescent proteins, the labeling capacity of the fusion proteins on apoptotic cells is detected, so that it is important to screen out the high-affinity AnxA5 probe for apoptosis detection.

Disclosure of Invention

The invention aims to provide fusion protein of annexin A5 and fluorescent protein for labeling apoptotic cells, and a preparation method and application thereof.

In order to solve the problems of the prior art, the application provides the following technical scheme that in the first aspect, the application provides a preparation method of fusion protein of annexin A5 and fluorescent protein for apoptosis cell marking;

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

in a third aspect, the present application provides a method of detecting apoptotic cells;

in a fourth aspect, the application provides the use of a fusion protein in the preparation of an apoptotic cell detection reagent.

In a fifth aspect, the application provides an apoptotic cell detection kit comprising said fusion protein.

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

(1) Constructing a fusion gene expression vector in which the C-terminal of annexin A5 (AnxA 5) and the N-terminal of fluorescent protein FPs are connected through a connecting peptide (Linker);

(2) Transforming the fusion gene expression vector into a host cell, and carrying out induction expression by using 1mM isopropyl-beta-D-1-thiogalactoside IPTG for 16 hours at 20 ℃ to realize the soluble expression of the fusion protein;

(3) Purifying by Ni-NTA affinity chromatography to obtain fusion protein.

Further, in step (1), the fluorescent protein is selected from any one of Venus、mVenus、Citrine、mCitrine、cpVenus173、cpCitrine174、EYFP、Ypet、mCherry、DsRed2、TagRFP、TagBFP、EBFP2、Cerulean、mCerulean、mCerulean3、EGFP、 or ECFP 18, and forms a fusion protein AnxA5-Venus, a fusion protein AnxA5-mVenus, a fusion protein AnxA5-Citrine, a fusion protein AnxA5-mCitrine, a fusion protein AnxA5-cpVenus173, a fusion protein AnxA5-CPCITRINE174, a fusion protein AnxA5-EYFP, a fusion protein AnxA5-Ypet, a fusion protein AnxA5-mCherry, a fusion protein AnxA5-Ypet, a fusion protein AnxA5-mCherry, a fusion protein AnxA5-DsRed2, a fusion protein AnxA5-TagRFP, a fusion protein AnxA5-TagBFP, a fusion protein AnxA5-EBFP2, a fusion protein AnxA5-Cerulean, a fusion protein AnxA5-mCerulean, a fusion protein AnxA5-mCerulean, a fusion protein AnxA 5-XFP 5-mCherry, a fusion protein AnxA5-DsRed2, a fusion protein AnxA5-mCherry and an ECFP 5-ECFP;

The nucleotide sequence of the fusion protein AnxA5-Venus is shown as SEQ ID NO. 1 in sequence;

The nucleotide sequence of the fusion protein AnxA5-mVenus is shown as SEQ ID NO. 2 in sequence;

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-CPCITRINE is shown as SEQ ID NO. 6;

The nucleotide sequence of the fusion protein AnxA5-EYFP is shown in SEQ ID NO. 7 in sequence;

the nucleotide sequence of the fusion protein AnxA5-Ypet is shown as SEQ ID NO. 8 in sequence;

The nucleotide sequence of the fusion protein AnxA5-mCherry is shown in SEQ ID NO. 9 in sequence, the nucleotide sequence of the fusion protein AnxA5-Ypet is shown in SEQ ID NO. 8 in sequence, the nucleotide sequence of the fusion protein AnxA5-mCherry is shown in SEQ ID NO. 9 in sequence, the nucleotide sequence of the fusion protein AnxA5-DsRed2 is shown in SEQ ID NO. 10 in sequence, the nucleotide sequence of the fusion protein AnxA5-TagRFP is shown in SEQ ID NO. 11 in sequence, the nucleotide sequence of the fusion protein AnxA5-TagBFP is shown in SEQ ID NO. 12 in sequence, the nucleotide sequence of the fusion protein AnxA5-EBFP2 is shown in sequence in SEQ ID NO. 13, the nucleotide sequence of the fusion protein AnxA 5-Ceruean is shown in sequence in SEQ ID NO. 14 in sequence, the nucleotide sequence of the fusion protein AnxA5-mCerulean is shown in sequence in SEQ ID NO. 15, the nucleotide sequence of the fusion protein AnxA5-mCerulean is shown in sequence in SEQ ID NO. 16 in sequence, the nucleotide sequence of the fusion protein AnxA5-EGFP 2 is shown in sequence in SEQ ID NO. 18 in sequence;

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 in sequence, the amino acid sequence of the fusion protein AnxA 5-mcitrane is shown in SEQ ID NO. 22 in sequence, the amino acid sequence of the fusion protein AnxA5-cpVenus173 is shown in SEQ ID NO. 23 in sequence, the amino acid sequence of the fusion protein AnxA5-CPCITRINE174 is shown in SEQ ID NO. 24 in sequence, the amino acid sequence of the fusion protein AnxA5-EYFP is shown in SEQ ID NO. 25 in sequence, the amino acid sequence of the fusion protein AnxA5-Ypet is shown in SEQ ID NO. 26 in sequence, the amino acid sequence of the fusion protein AnxA5-mCherry is shown in SEQ ID NO. 27 in sequence, the nucleotide sequence of the fusion protein AnxA5-DsRed2 is shown in SEQ ID NO. 28 in sequence, the nucleotide sequence of the fusion protein AnxA5-TagRFP is shown in SEQ ID NO. 29, the nucleotide sequence of the fusion protein AnxA5-TagBFP is shown in sequence in SEQ ID NO. 30, the nucleotide sequence of the fusion protein AnxA 5-FP is shown in sequence shown in SEQ ID NO. 30, the nucleotide sequence of the fusion protein AnxA 5-35 is shown in sequence of the nucleotide sequence of the fusion protein AnxA 5-35 is shown in SEQ ID NO. 30, the nucleotide sequence of the fusion protein AnxA 5-35 is shown in sequence shown in SEQ ID NO. 35.

Through the flow detection of 18 AnxA5-FPs fusion proteins and apoptotic cells, and the affinity analysis, 8 fusion proteins with higher affinity than that of AnxA5-EGFP and AnxA5-mCherry are obtained, 4 fusion proteins with equal affinity to that of AnxA5-EGFP and AnxA5-mCherry are obtained, and 4 fusion proteins with lower affinity than that of AnxA5-EGFP and AnxA5-mCherry are obtained, wherein the emission light of the fusion proteins covers blue, cyan, yellow and red, and multicolor marking and co-localization analysis are supported.

The sequence of the connecting peptide (Gly-Ser) ₄ is determined through multiple experimental optimization, and the host cell is escherichia coli BL21 (DE 3).

In a second aspect, the present application provides a fusion protein of annexin A5 and a fluorescent protein prepared by the method, the fusion protein being used for labelling phosphatidylserine PS on the surface of apoptotic cells, and detecting apoptotic cells by flow cytometry or fluorescence microscopy.

Further, in step (1), the affinity constant Kd value of the fusion protein to PS is 10 ^-5 M to 10 ^-8 M, the fusion proteins are 8 fusion proteins having affinities higher than that of AnxA5-EGFP and AnxA5-mCherry, respectively fusion proteins AnxA5-TagBFP, fusion proteins AnxA5-mCerulean3, fusion proteins AnxA5-EBFP2, fusion proteins AnxA 5-Cerulan, fusion proteins AnxA5-mCerulean, fusion proteins AnxA5-ECFP, fusion proteins AnxA5-Ypet and fusion proteins AnxA5-TagRFP, the fusion proteins 4 having affinities equivalent to that of AnxA5-EGFP and AnxA5-mCherry are fusion proteins AnxA5-Venus, fusion proteins AnxA5-EYFP, fusion proteins AnxA5-cpVenus, fusion proteins AnxA 5-Dred 2, fusion proteins having affinities lower than that of AnxA5-EGFP and AnxA5-Ypet and fusion proteins AnxA5-TagRFP, respectively, and fusion proteins having affinities equivalent to AnxA5-mCherry, and fusion proteins having affinities equivalent to that of AnxA 5-35-blue, and fusion proteins having affinities equivalent to that of AnxA 5-blue, and that of the fusion proteins are equivalent to the dye, and the dye of the dye is 35.

The third aspect of the present application provides a method for detecting apoptotic cells, comprising the steps of:

(1) Labeling apoptotic cells with a fusion protein;

(2) Detecting the fluorescence intensity of the fusion protein by a flow cytometer, and judging the exposure degree of the PS on the surface of the apoptotic cell according to the fluorescence signal.

Further, in step (1), the concentration of the label of the fusion protein ranges from 2nM to 2500nM.

Further, in the step (1), the detection sensitivity of the fusion protein to early apoptosis cells is higher than that of a chemically-marked AnxA5-FITC probe, and the detection sensitivity of the fusion protein to early apoptosis cells is also higher than or equal to that of an AnxA5-EGFP fusion protein, wherein the emitted light of the fusion proteins AnxA5-TagBFP, anxA5-mCerulean, anxA5-Ypet and AnxA5-TagRFP respectively corresponds to blue, cyan, yellow and red, and the marker concentration range of the fusion protein is 25nM to 500nM.

In a fourth aspect, the present application provides an application of a fusion protein in preparing an apoptotic cell detection reagent, the application comprising:

(1) Incubating the fusion protein with apoptotic cells to bind specifically to the cell surface exposed PS;

(2) Apoptotic cells were detected by fluorescent signal.

Further, apoptotic cell detection reagents are used for in vitro or in vivo tracking of early apoptotic cells.

In a fifth aspect, the application provides a kit for detecting apoptotic cells comprising a fusion protein by flow cytometry or fluorescence microscopy.

The invention solves the problems of complex preparation, insufficient performance and the like of the existing probe through technical innovation, and provides a novel tool with high efficiency, sensitivity and low cost for apoptosis detection. Meanwhile, a plurality of fusion proteins with higher affinity than that of AnxA5-EGFP and AnxA5-mCherry can be used for more effectively detecting early apoptosis.

Compared with the prior art, the invention has the following advantages:

(1) The invention directly expresses the fusion protein of AnxA5 and fluorescent protein through genetic engineering, and avoids complex coupling reaction, multi-step purification and free fluorescein removal steps in the traditional chemical labeling method. Experiments show that the preparation of the fusion protein only needs prokaryotic expression and Ni-NTA affinity purification (example 3), the operation is simple and convenient, the time consumption is short (about 1 day), and the production cost and the process complexity are obviously reduced.

(2) The chemically labeled AnxA5 probes of the invention often resulted in product heterogeneity due to differences in the labeling sites and numbers, whereas the AnxA5-FPs probes of the invention expressed by gene fusion had a high degree of homogeneity (example 3, fig. 3). In addition, the light stability of the fluorescent protein is higher than that of a chemical dye (such as FITC), strict light-shielding treatment (contrast in the background technology) is not needed, and the practicability and storage convenience of the probe are improved.

(3) The present invention systematically screened 18 fluorescent protein fusions, and found that the affinity of the different fusion proteins to Phosphatidylserine (PS) was different by up to two orders of magnitude (example 5, table 3). And comparing with the affinities of AnxA5-EGFP and AnxA5-mCherry, 8 fusion proteins with affinities higher than that of AnxA5-EGFP and AnxA5-mCherry were finally selected, namely, 4 fusion proteins with affinities lower than that of AnxA5-TagBFP, anxA5-mCerulean3, anxA5-EBFP2, anxA5-Cerulean, anxA5-mCerulean, anxA5-ECFP, anxA5-Ypet and AnxA5-TagRFP, namely, 4 fusion proteins with affinities equivalent to that of AnxA5-EGFP and AnxA5-mCherry were respectively, namely, anxA5-Venus, anxA5-EYFP, anxA5-cpVenus173, anxA5-DsRed2, fusion proteins with affinities lower than that of AnxA5-EGFP and AnxA5-mCherry were respectively, namely, anxA5-mVenus, anxA 5-Citine, anxA5-mCitrine, anxA and AnxA5-mCherry were respectively, and the range of yellow color was higher than that of the blue color was emitted by the fusion proteins, or the blue color was equivalent to that of the blue color was increased by the fusion proteins. Further, the final 5 high affinity probes selected were blue AnxA5-TagBFP, cyan AnxA5-mCerulean3, yellow AnxA5-Ypet and red AnxA5-TagRFP, respectively, which all reached a Kd value of 10 ^-7 M, showed excellent labeling efficiency in flow cytometry and fluorescence microscopy (example 6, FIGS. 10-11), and in particular, the detection sensitivity for early apoptotic cells was significantly higher than that of the conventional AnxA5-FITC probe (FIG. 11D), while the detection sensitivity for early apoptotic cells was also higher than or equivalent to that of the AnxA5-EGFP fusion protein (FIG. 11D).

(4) The fluorescence spectrum of the fusion protein of the present invention covers multiple bands of blue, cyan, green, yellow, red, etc. (example 4, table 2), supporting multicolor labeling and co-localization analysis. The probes have the advantages of no need of adjusting fluorescence compensation when the fusion proteins with blue and cyan spectrums are combined with dyes such as Propidium Iodide (PI) and the like for flow detection, higher fluorescence intensity of the fusion protein with yellow spectrum (figure 4), and suitability for in-vitro apoptosis detection as well as in-vivo apoptosis detection. In addition, the probe is suitable for flow cytometry and fluorescence microscopy (examples 5-6), and can be expanded to in-vivo PS eversion tracing (summary of the invention) to meet the diversified requirements of basic research, drug screening and clinical diagnosis.

(5) The invention realizes the large-scale preparation and screening of a plurality of AnxA 5-fluorescent protein fusion probes for the first time (claims 1-4), and fills the gap that the types of AnxA5 probes based on fluorescent proteins are rare. By revealing the influence of fluorescent protein type on the function of AnxA5 (example 5), an important theoretical basis is provided for the subsequent probe optimization, and the method has remarkable academic value and application prospect.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings that are needed in the embodiments or the description of the prior art will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present application, and that other drawings can be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 shows the expression of the AnxA5-FPs fusion proteins of the invention after induction at 20℃and 37 ℃.

Wherein (A) SDS-PAGE analysis of non-transformed (control) and transformed E.coli BL21 protein expression after induction at 20 ℃. (B) SDS-PAGE analysis of non-transformed (control) and transformed E.coli BL21 protein expression after induction at 37 ℃. Lanes are labeled with the names of fluorescent proteins in AnxA 5-FPs.

FIG. 2 is a soluble expression analysis of the AnxA5-FPs fusion proteins of the invention after induction at 37℃and 20 ℃.

Where 'S' represents the protein in the supernatant and 'P' represents the protein in the precipitate. (A) SDS-PAGE analysis of the AnxA5-FPs fusion protein in supernatant and pellet in E.coli BL21 transformed after induction at 37 ℃. (B) SDS-PAGE analysis of the AnxA5-FPs fusion protein in supernatant and pellet in E.coli BL21 transformed after induction at 20 ℃. (C) The proportion of AnxA5-FPs fusion protein present in the supernatant after induction at 37 ℃. (D) Proportion of AnxA5-FPs fusion protein present in the supernatant after induction at 20 ℃. Lanes are labeled with the names of fluorescent proteins in AnxA 5-FPs. Data are expressed as mean ± standard deviation.

FIG. 3 shows the expression and purification of the AnxA5-FPs fusion proteins of the invention.

Wherein (A) SDS-PAGE analysis of purified AnxA5-FPs fusion proteins. Lanes are labeled with the names of fluorescent proteins in AnxA 5-FPs. (B) purity of AnxA5-FPs fusion protein. Data are expressed as mean ± standard deviation.

FIG. 4 shows normalized fluorescence intensities of AnxA5-FPs fusion proteins of the invention and commercial AnxA 5-FITC.

Wherein, data are expressed as mean ± standard deviation. The number of moles of AnxA5-FPs and AnxA5-FITC is the same. The normalized fluorescence intensity of each group was compared with AnxA 5-FITC. ns, no significant difference.

FIG. 5 is an experiment of binding of AnxA5-EGFP of the present invention to exposed PS on cell membranes.

Wherein (A) flow cytometry analysis charts of positive apoptotic cells labeled with different concentrations of AnxA 5-EGFP. (B) Proportion of positive apoptotic cells labeled with AnxA5-EGFP at different concentrations. Data are expressed as mean ± standard deviation. The proportion of AnxA5-EGFP positive apoptotic cells in each group was compared to the 2nM group. The proportion of AnxA5-EGFP positive apoptotic cells in each group was compared to the 2nM group. * P <0.0001, < p <0.001, < p <0.01, < p <0.05.ns, no significance.

FIG. 6 is an experiment showing binding of AnxA 5-red fluorescent protein of the present invention to exposed PS on cell membrane.

Wherein (A) flow cytometry analysis plots of positive apoptotic cells labeled with AnxA5-mCherry at different concentrations. (B) Proportion of AnxA5-mCherry labeled positive apoptotic cells at different concentrations. (C) Flow cytometry analysis of positive apoptotic cells labeled with AnxA5-DsRed2 at different concentrations. (D) Proportion 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) Proportion of positive apoptotic cells labeled with AnxA5-TagRFP at different concentrations. Data are expressed as mean ± standard deviation. The proportion of AnxA5 positive apoptotic cells in each group was compared to the 2nM group. * P <0.0001, p <0.001, p <0.01, p <0.05, ns are not significant.

FIG. 7 is an experiment showing binding of AnxA 5-yellow fluorescent protein of the present invention to exposed PS on cell membrane.

Wherein, (A) - (H) are different concentrations AnxA5-Venus、AnxA5-mVenus、AnxA5-Citrine、AnxA5-mCitrine、AnxA5-cpVenus173、AnxA5-cpCitrine174、AnxA5-Ypet and AnxA5-EYFP labeled positive apoptotic cells flow cytometry analysis chart. (I) - (P) ratio of AnxA5-Venus、AnxA5-mVenus、AnxA5-Citrine、AnxA5-mCitrine、AnxA5-cpVenus173、AnxA5-cpCitrine174、AnxA5-Ypet and AnxA5-EYFP labeled positive apoptotic cells at different concentrations. Data are expressed as mean ± standard deviation. The proportion of AnxA5 positive apoptotic cells in each group was compared to the 2nM group. * P <0.0001, p <0.001, p <0.01, p <0.05, ns are not significant.

FIG. 8 is an experiment showing binding of AnxA 5-blue fluorescent protein of the present invention to exposed PS on cell membrane.

Wherein (A) flow cytometry analysis plots of positive apoptotic cells labeled with AnxA5-EBFP2 at different concentrations. (B) Proportion 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) Proportion of positive apoptotic cells labeled with AnxA5-TagBFP at different concentrations. Data are expressed as mean ± standard deviation. The proportion of AnxA5 positive apoptotic cells in each group was compared to the 2nM group. * P <0.0001, p <0.001, p <0.01, p <0.05, ns are not significant.

FIG. 9 is an experiment of binding of AnxA 5-cyan fluorescent protein of the present invention to exposed PS on cell membrane.

Wherein (A) flow cytometry analysis of positive apoptotic cells labeled with AnxA5-Cerulean at different concentrations. (B) Proportion of AnxA5-Cerulean labeled positive apoptotic cells at different concentrations. (C) Flow cytometry analysis of positive apoptotic cells labeled with AnxA5-mCerulean at different concentrations. (D) Proportion of positive apoptotic cells labeled with AnxA5-mCerulean at different concentrations. (E) Flow cytometry analysis of positive apoptotic cells labeled with AnxA5-mCerulean at different concentrations. (F) Proportion of positive apoptotic cells labeled with AnxA5-mCerulean at different concentrations. (G) Flow cytometry analysis of positive apoptotic cells labeled with AnxA5-ECFP at different concentrations. (H) Proportion of AnxA5-ECFP labelled positive apoptotic cells at different concentrations. Data are expressed as mean ± standard deviation. The proportion of AnxA5 positive apoptotic cells in each group was compared to the 2nM group. * P <0.0001, p <0.001, p <0.01, p <0.05, ns are not significant.

FIG. 10 is a graph of the ability of five AnxA5-FPs of high PS affinity to bind to apoptotic cells (induced by camptothecins) assessed by flow cytometry in accordance with the present invention (with chemically labeled AnxA5, anxA5-FITC as control).

FIG. 11 is a sensitivity analysis of five AnxA5-FPs fusion proteins of the invention with high PS affinity for early apoptosis detection (compared to AnxA 5-FITC).

Wherein, (1) the percentage of living cells. (2) percentage of AnxA5-FPs positive cells. (3) percentage of early and late apoptotic cells. (D) percentage of early apoptotic cells (histogram). * P <0.0001, < p <0.001, < p <0.01, < p <0.05.

Detailed Description

In order to make the technical problems, technical schemes and beneficial effects to be solved more clear, the application is further described in detail below with reference to the embodiments. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the application.

In the present application, the term "and/or" describes an association relationship of an association object, which means that three relationships may exist, for example, a and/or B may mean that a exists alone, a and B exist together, and B exists alone. Wherein A, B may be singular or plural. The character "/" generally indicates that the context-dependent object is an "or" relationship.

In the present application, "at least one" means one or more, and "a plurality" means two or more. "at least one of" or the like means any combination of these items, including any combination of single item(s) or plural items(s). For example, "at least one (a), b, or c)", or "at least one (a, b, and c)", may each represent a, b, c, a-b (i.e., a and b), a-c, b-c, or a-b-c, wherein a, b, c may be single or multiple, respectively.

It should be understood that, in various embodiments of the present application, the sequence number of each process described above does not mean that the execution sequence of some or all of the steps may be executed in parallel or executed sequentially, and the execution sequence of each process should be determined by its functions and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

The terminology used in the embodiments of the application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. As used in this application and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

The weights of the relevant components mentioned in the description of the embodiments of the present application may refer not only to the specific contents of the components, but also to the proportional relationship between the weights of the components, so long as the contents of the relevant components in the description of the embodiments of the present application are scaled up or down within the scope of the disclosure of the embodiments of the present application. Specifically, the mass in the specification of the embodiment of the application can be a mass unit which is known in the chemical industry field such as mu g, mg, g, kg and the like.

The terms "first" and "second" are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or implicitly indicating the number of technical features indicated for distinguishing between objects such as substances from each other. For example, a first XX may also be referred to as a second XX, and similarly, a second XX may also be referred to as a first XX, without departing from the scope of embodiments of the application. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include one or more such feature.

An embodiment of the present application provides a method for preparing fusion protein of annexin A5 and fluorescent protein for apoptotic cell labeling, comprising the steps of:

(3) Purifying by Ni-NTA affinity chromatography to obtain fusion protein.

In some embodiments, in step (1), the fluorescent protein is selected from any of Venus、mVenus、Citrine、mCitrine、cpVenus173、cpCitrine174、EYFP、Ypet、mCherry、DsRed2、TagRFP、TagBFP、EBFP2、Cerulean、mCerulean、mCerulean3、EGFP、 or ECFP 18, forming fusion proteins AnxA5-Venus, fusion proteins AnxA5-mVenus, fusion proteins AnxA5-Citrine, fusion proteins AnxA5-mCitrine, fusion proteins AnxA5-cpVenus173, fusion proteins AnxA5-CPCITRINE174, fusion proteins AnxA5-EYFP, fusion proteins AnxA5-Ypet, fusion proteins AnxA5-mCherry, fusion proteins AnxA5-Ypet, fusion proteins AnxA5-mCherry, fusion proteins AnxA5-DsRed2, fusion proteins AnxA5-TagRFP, fusion proteins AnxA5-TagBFP, fusion proteins AnxA5-EBFP2, fusion proteins AnxA5-mCerulean, fusion proteins AnxA5-mCerulean, fusion proteins AnxA5-Ypet, fusion proteins AnxA 5-Cerulan, and ECFP 5-nus, respectively;

the nucleotide sequence of the fusion protein AnxA5-Venus is shown as SEQ ID NO.1 in sequence;

The nucleotide sequence of the fusion protein AnxA5-Citrine is shown as SEQ ID NO. 3 in sequence;

The nucleotide sequence of the fusion protein AnxA5-mCitrine is shown as SEQ ID NO. 4 in sequence;

The nucleotide sequence of the fusion protein AnxA5-cpVenus173 is shown as SEQ ID NO. 5 in sequence;

The nucleotide sequence of the fusion protein AnxA5-CPCITRINE174,174 is shown in SEQ ID NO. 6;

The nucleotide sequence of the fusion protein AnxA5-mCherry is shown as SEQ ID NO. 9 in sequence, the nucleotide sequence of the fusion protein AnxA5-Ypet is shown as SEQ ID NO. 8 in sequence, the nucleotide sequence of the fusion protein AnxA5-mCherry is shown as SEQ ID NO. 9 in sequence, the nucleotide sequence of the fusion protein AnxA5-DsRed2 is shown as SEQ ID NO. 10 in sequence, the nucleotide sequence of the fusion protein AnxA5-TagRFP is shown as SEQ ID NO. 11 in sequence, the nucleotide sequence of the fusion protein AnxA5-TagBFP is shown as SEQ ID NO. 12 in sequence, the nucleotide sequence of the fusion protein AnxA5-EBFP2 is shown as SEQ ID NO. 13 in sequence, the nucleotide sequence of the fusion protein AnxA5-Cerulean is shown as SEQ ID NO. 14 in sequence, the nucleotide sequence of the fusion protein AnxA5-mCerulean is shown as SEQ ID NO. 15 in sequence, the nucleotide sequence of the fusion protein AnxA5-TagRFP is shown as SEQ ID NO. 11 in sequence, the nucleotide sequence of the fusion protein AnxA5-EGFP 2 is shown as SEQ ID NO. 16 in sequence of the fusion protein AnxA5-EGFP 2 is shown as SEQ ID NO. 17 in sequence;

The amino acid sequence of the fusion protein AnxA5-mVenus is shown as SEQ ID NO. 20 in sequence, the amino acid sequence of the fusion protein AnxA5-Citrine is shown as SEQ ID NO. 21 in sequence, the amino acid sequence of the fusion protein AnxA5-mCitrine is shown as SEQ ID NO. 22 in sequence, the amino acid sequence of the fusion protein AnxA5-cpVenus is shown as SEQ ID NO. 23 in sequence, the amino acid sequence of the fusion protein AnxA5-CPCITRINE is shown as SEQ ID NO. 24 in sequence, the amino acid sequence of the fusion protein AnxA5-EYFP is shown as SEQ ID NO. 25 in sequence, the amino acid sequence of the fusion protein AnxA5-Ypet is shown as SEQ ID NO. 26 in sequence, the nucleotide sequence of the fusion protein AnxA5-mCherry is shown as SEQ ID NO. 27 in sequence, the nucleotide sequence of the fusion protein AnxA5-cpVenus is shown as SEQ ID NO. 28 in sequence, the nucleotide sequence of the fusion protein AnxA 5-49174 is shown as SEQ ID NO. 24 in sequence, the amino acid sequence of the fusion protein AnxA 5-XFP is shown as SEQ ID NO. 29, the fusion protein AnxA 5-XFP is shown as SEQ ID NO. 30, the fusion protein AnxA 5-XFP is shown as SEQ ID NO. 29, the amino acid sequence of the fusion protein AnxA 5-XFP is shown as SEQ ID NO. 29.

In some embodiments, in step (2), the connecting peptide is determined to be a (Gly-Ser) 4 sequence by multiple experimental optimization, and the host cell is E.coli BL21 (DE 3).

In a second aspect, the embodiment of the application provides a fusion protein of annexin A5 and fluorescent protein prepared by the method, wherein the fusion protein is used for marking phosphatidylserine PS on the surface of apoptotic cells, and apoptotic cells are detected by flow cytometry or a fluorescent microscope.

In some embodiments, in step (1), the affinity constant Kd of the fusion protein to PS is 10 ^-5 M to 10 ^-8 M, the fusion proteins are 8 fusion proteins having affinities higher than that of AnxA5-EGFP and AnxA5-mCherry, respectively fusion proteins AnxA5-TagBFP, fusion proteins AnxA5-mCerulean3, fusion proteins AnxA5-EBFP2, fusion proteins AnxA 5-Cerullan, fusion proteins AnxA5-mCerulean, fusion proteins AnxA5-ECFP, fusion proteins AnxA5-Ypet and fusion proteins AnxA5-TagRFP, fusion proteins equivalent to AnxA5-EGFP and AnxA5-mCherry affinity are fusion proteins AnxA5-Venus, fusion proteins AnxA5-EYFP, fusion proteins AnxA5-cpVenus, fusion proteins AnxA5-DsRed2, fusion proteins having affinities lower than that of AnxA5-ECFP and fusion proteins AnxA5-Ypet and fusion proteins AnxA5-TagRFP, respectively, and fusion proteins having affinities equivalent to AnxA5-mCherry of blue color, and fusion proteins having affinities higher than that of AnxA5-EGFP and AnxA5-mCherry are blue, respectively, and the fusion proteins having affinities of blue color of 35 and the fusion proteins are blue, the fusion proteins are the fusion proteins.

A third aspect of the embodiments of the present application provides a method for detecting apoptotic cells, comprising the steps of:

(1) Labeling apoptotic cells with a fusion protein;

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

In some embodiments, in step (1), the detection sensitivity of the fusion protein to early apoptotic cells is higher than that of a chemically labeled AnxA5-FITC probe, and the detection sensitivity of the fusion protein to early apoptotic cells is also higher than or equal to that of an AnxA5-EGFP fusion protein, wherein the emitted light of the fusion proteins AnxA5-TagBFP, anxA5-mCerulean3, anxA5-Ypet and AnxA5-TagRFP respectively corresponds to blue, cyan, yellow and red, and the labeling concentration of the fusion protein ranges from 25nM to 500nM.

In a fourth aspect, the present application provides an application of a fusion protein in preparing an apoptotic cell detection reagent, where the application includes:

(2) Apoptotic cells were detected by fluorescent signal.

In a fifth aspect, embodiments of the present application provide an apoptotic cell detection kit comprising a fusion protein for detecting apoptotic cells by flow cytometry or fluorescence microscopy.

In some embodiments, the apoptotic cell detection reagent is used for in vitro or in vivo tracking of early apoptotic cells.

Example 1

The invention relates to a preparation method of fusion protein of annexin A5 and fluorescent protein for apoptotic cell labeling, which comprises the following steps:

(3) Purifying by Ni-NTA affinity chromatography to obtain fusion protein.

In step (1), the fluorescent protein is selected from any one of Venus、mVenus、Citrine、mCitrine、cpVenus173、cpCitrine174、EYFP、Ypet、mCherry、DsRed2、TagRFP、TagBFP、EBFP2、Cerulean、mCerulean、mCerulean3、EGFP、 or ECFP 18, and forms a fusion protein AnxA5-Venus, a fusion protein AnxA5-mVenus, a fusion protein AnxA5-Citrine, a fusion protein AnxA5-mCitrine, a fusion protein AnxA5-cpVenus173, a fusion protein AnxA5-CPCITRINE174, a fusion protein AnxA5-EYFP, a fusion protein AnxA5-Ypet, a fusion protein AnxA5-mCherry, a fusion protein AnxA5-Ypet, a fusion protein AnxA5-mCherry, a fusion protein AnxA5-DsRed2, a fusion protein AnxA5-TagBFP, a fusion protein AnxA5-EBFP2, a fusion protein AnxA5-Cerulean, a fusion protein AnxA5-mCerulean, a fusion protein AnxA 5-353, a fusion protein AnxA 5-FP, an EGFP 5-ECFP and an ECFP 5-ECFP;

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 as SEQ ID NO. 21 in sequence; the amino acid sequence of the fusion protein AnxA5-mCitrine is shown as 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, 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 AnxA 5-Cerulan is shown in SEQ ID NO. 32, the nucleotide sequence of the fusion protein AnxA5-DsRed2 is shown in SEQ ID NO. 28, the nucleotide sequence of the fusion protein AnxA5-EGFP 5-nucleotide sequence is shown in SEQ ID NO. 35, and the nucleotide sequence of the fusion protein AnxA5-EGFP 5-nucleotide sequence is shown in SEQ ID NO. 35.

In the step (2), the sequence of the connecting peptide (Gly-Ser) ₄ is determined through multiple experimental optimization, and the host cell is escherichia coli BL21 (DE 3).

Example 2

The fusion protein of annexin A5 and fluorescent protein prepared by the method is used for marking phosphatidylserine PS on the surface of apoptotic cells, and apoptotic cells are detected by flow cytometry or a fluorescent microscope.

In step (1), the affinity constant Kd value of the fusion protein to PS is from 10 ^-5 M to 10 ^-8 M; the fusion proteins are 8 fusion proteins with higher affinity than the AnxA5-EGFP and the AnxA5-mCherry, namely fusion proteins AnxA5-TagBFP, fusion proteins AnxA5-mCerulean, fusion proteins AnxA5-EBFP2, fusion proteins AnxA5-Cerulean, fusion proteins AnxA5-mCerulean, fusion proteins AnxA5-ECFP, fusion proteins AnxA5-Ypet and fusion proteins AnxA5-TagRFP, fusion proteins 4 with the affinity equivalent to the AnxA5-EGFP and the AnxA5-mCherry, fusion proteins AnxA5-Venus, fusion proteins AnxA5-EYFP, fusion proteins AnxA5-cpVenus173, fusion proteins AnxA5-DsRed2, fusion proteins 4 with the affinity lower than the AnxA 5-Cerlean, fusion proteins AnxA5-mVenus, fusion proteins AnxA5-Ypet and fusion proteins AnxA5-TagRFP, fusion proteins with the affinity equivalent to the AnxA5-mCherry, fusion proteins with the affinity equivalent to the AnxA 5-Green, fusion proteins AnxA 5-blue color and the dye-35, or the fusion proteins with the affinity equivalent to the dye-blue color of the dye and the dye-blue dye of the dye.

Example 3

The invention relates to a method for detecting apoptotic cells, which is characterized by comprising the following steps:

(1) Apoptotic cells were labeled with fusion proteins ranging in concentration from 2nM to 2500nM. The detection sensitivity of the fusion protein to early apoptosis cells is higher than that of a chemically marked AnxA5-FITC probe, and the detection sensitivity of the fusion protein to early apoptosis cells is higher than or equal to that of an AnxA5-EGFP fusion protein, the emitted light of the fusion proteins AnxA5-tagBFP, anxA5-mCerulean3, anxA5-Ypet and AnxA5-TagRFP respectively corresponds to blue, cyan, yellow and red, and the marker concentration range of the fusion protein is 25nM to 500nM.

Example 4

The application of the fusion protein in preparing apoptotic cell detection reagent includes:

(2) Apoptotic cells were detected by fluorescent signal.

Apoptotic cell detection reagents are used for in vitro or in vivo tracking of early apoptotic cells.

Example 5

Cloning construction of fusion proteins of AnxA5 and fluorescent proteins (AnxA 5-FPs)

In past work, we have constructed pET28a (+) -AnxA5-EGFP-his ₆ plasmid by amplifying EGFP coding sequence using primers NdeI-EGFP-F and XhoI-EGFP-R. After recovery of the PCR products, the primers BamHI- (GS) ₄ -linker-F and XhoI-EGFP-R were used for re-amplification, introducing a (GS) ₄ linker fragment after structural prediction and experimental screening. The final product was cloned into pET28a (+) to construct the pET28a (+) -EGFP-his ₆ vector. Then, the coding sequence of human AnxA5 was amplified using primers NcoI-AnxA5-F and BamHI-AnxA 5-R. The product is cloned into pET28a (+) -EGFP-his ₆ and fused with the upstream of EGFP coding sequence to construct an expression vector pET28a (+) -AnxA5-EGFP-his ₆. Restriction enzymes and recognition sites therefor are shown in italics. The construction of other pET28a (+) -AnxA5-FPs-his ₆ plasmids takes pET28a (+) -AnxA5-EGFP-his ₆ plasmid as a template. We replaced the gene sequence of EGFP with that of other fluorescent proteins, including Venus、mVenus、cpVenus173、Citrine、mCitrine、cpCitrine174、EYFP、Ypet、mCherry、DsRed2、TagRFP、TagBFP、EBFP2、Cerulean、mCerulean、mCerulean3.

Example 6

Inducible expression of fusion protein of AnxA5 and fluorescent protein (AnxA 5-FPs) and optimization of expression conditions the plasmid pET28a (+) -AnxA5-FPs-his ₆ with correct sequence was transformed into E.coli BL21 (DE 3) cells for expression. Various E.coli BL21 (DE 3) transformed with pET28a (+) -AnxA5-FPs-his ₆ were inoculated into 3mL LB medium containing 50. Mu.g/mL kanamycin, and cultured overnight at 37 ℃. Then, the culture (30. Mu.L) was inoculated into 3mL of LB medium containing 50. Mu.g/mL of kanamycin, and cultured at 37℃for about 3 hours. When the bacteria grew to mid-log phase (OD ₆₀₀ = 0.5-0.8) in LB medium, gene expression was induced by the addition of 1mM isopropyl- β -D-1-thiogalactoside (IPTG), and cultivation was continued at 20 ℃ for about 16 hours, or 37 ℃ for 12 hours. After that, bacterial pellet was collected by centrifugation, resuspended in 600. Mu.L buffer (20mM Tris,250mM NaCl,pH7.4) and lysed by sonication. The expression of AnxA5-FPs at 20℃and 37℃was analyzed by SDS-polyacrylamide gel electrophoresis (PAGE). For the solubility analysis, after bacterial precipitation by sonication, the suspension was centrifuged at 12,000rpm for 20 minutes at 4 ℃. The supernatant and pellet were separated and analyzed for the expression of AnxA5-FPs in the supernatant and pellet at 20℃and 37℃by SDS-PAGE. After induction of E.coli BL21 (DE 3) with IPTG at 20℃and 37℃there was an additional protein band with a Molecular Weight (MW) of about 65kDa in the cell lysate (as shown in FIG. 1), which is consistent with the theoretical molecular weights of the various AnxA5-FPs (as shown in Table 1). When the bacteria were induced at 20 ℃, the amount of AnxA5-FPs expressed in soluble form was greatly increased (as shown in figures 2b, d) compared to 37 ℃ induction (as shown in figures 2a, C). The amino acid numbers and theoretical molecular weights (MW, kDa) of the AnxA5-FPs fusion proteins are shown in Table 1:

TABLE 1

Example 7

Purification of fusion proteins of AnxA5 and fluorescent proteins (AnxA 5-FPs)

Protein expression was induced with 1mM isopropyl- β -D-1-thiogalactoside (IPTG) at 20℃for 16 hours. Bacterial pellet was collected, resuspended in binding buffer (20mM Tris,250mM NaCl,pH7.4) and the cells disrupted by sonication. The supernatant and precipitate were then separated by centrifugation at 16,000rpm for 20 minutes, the supernatant was collected and the protein was filtered with a 0.22 μm filter. The column was pre-equilibrated with binding buffer containing 40mM imidazole and imidazole was added to the supernatant to a final concentration of 40mM. The supernatant was then applied to a Ni-NTA affinity column. After sample loading was completed, the column was washed with 50mM imidazole and then eluted with 250mM imidazole. The eluate containing the AnxA5-FPs fraction was collected and dialyzed against Tris buffer (20mM Tris,30mM NaCl,pH 8.5) for about 24 hours. After the dialysis was completed, protein solution was collected and protein concentration was determined by BCA, and 10ng of protein was taken for SDS-PAGE analysis. AnxA5-EBFP2, anxA5-TagBFP, anxA5-Cerulean, anxA-mCerulean, anxA5-mCerulean3 and AnxA5-ECFP were all kept in our laboratory. The AnxA5-FPs fusion proteins were purified by Ni-NTA agarose affinity chromatography (as shown in FIG. 3A), and all fusion proteins reached more than 80% purity (as shown in FIG. 3B). In summary, the results indicate successful expression and purification of the AnxA5-FPs fusion protein. The 18 AnxA5-FPs fusion proteins were all stored in Tris buffer containing 20mM Tris,30mM NaCl,pH 8.5.

Example 8

Characterization of fusion proteins of AnxA5 and fluorescent proteins (AnxA 5-FPs)

On an F-4500 fluorescence spectrophotometer (Hitachi, japan), we performed excitation and emission wavelength scans of AnxA5-FPs (50. Mu.g/mL) fusion proteins and recorded excitation and emission spectra, then analyzed the spectral data using GRAPHPAD PRISM 8.0.0. The results show that the excitation and emission spectra of AnxA5-FPs are not significantly changed compared to fluorescent protein alone, with the excitation and emission maxima shown in table 2. This suggests that the fusion of AnxA5 does not significantly alter the chromophore environment of the fluorescent protein, which is important for fluorescent quantitative analysis. We also measured the fluorescence intensities of 18 AnxA5-FPs and AnxA5-FITC using a multifunctional microplate reader (BioTek, USA). Then, the concentrations of 18 types of AnxA5-FPs and AnxA5-FITC were determined by the BCA method. Normalized fluorescence intensity was calculated by the following formula of the fluorescence intensity of AnxA5-FPs or AnxA5-FITC per nmol of protein. We compared the normalized fluorescence intensities of the AnxA5-FP fusion protein and the commercial AnxA 5-FITC. The results indicate that the normalized fluorescence intensities of AnxA5-EGFP, anxA5-mCherry and AnxA5-DsRed2 are on the same order of magnitude as AnxA5-FITC, while the normalized fluorescence intensities of the other fifteen AnxA5-FPs are all an order of magnitude higher than AnxA5-FITC (as shown in FIG. 4). The fluorescence spectra of the AnxA5-FP fusion protein are shown in Table 2:

TABLE 2

Example 9

Affinity analysis of AnxA5-FPs fusion proteins with cell surface exposed PS

To investigate the PS binding capacity of the AnxA5-FPs fusion proteins, we tried to stain apoptotic cells using AnxA 5-FPs. The specific method is that the apoptosis of Jurkat cells is induced by adding etoposide with a final concentration of 25 mu M or camptothecine with a final concentration of 1 mu M. Cells were then collected by centrifugation at 2,500rpm for 5 minutes, washed twice, and resuspended to a cell density of 1X 10 ⁶ cells/mL with binding buffer (10mM Hepes,140mM NaCl,2.5mM CaCl ₂, pH 7.4). Prior to this, the AnxA5-FPs probe was diluted to different concentrations, and then 200. Mu.L of the protein dilution was added to 200. Mu.L of the cell suspension to give final concentrations of 2nM, 5nM, 10nM, 25nM, 50nM, 100nM, 250nM, 500nM, 1000nM, 2500nM. After gentle vortexing, the mixture was incubated on ice for 30 minutes. Finally, after adding 1 μl of Propidium Iodide (PI) and mixing well, the cell suspension was immediately examined using a flow cytometer. The results indicate that they both bind to apoptotic cells and that their optimal labelling efficiency depends on their final concentration (as shown in FIGS. 5-9). That is, within a range of concentrations, anxA5 negative and AnxA5 positive cells can be clearly distinguished.

After completion of Flow Cytometry (FCM) analysis, the Mean Fluorescence Intensity (MFI) of AnxA5-FPs positive cells was analyzed by FlowJo V10 software. For fluorescent proteins of the same color, normalized MFI values MFI/maximum MFI 100 (%) were calculated using the formula. The normalized fluorescence intensity was used as a protein binding capacity index (PBI). After data collection and analysis, a fitted curve of PBI of 18 AnxA5-FPs fusion proteins with different concentrations of AnxA5-FPs was drawn. From the fitted curve, a relative affinity constant (Kd) was obtained (as shown in table 3), which represents PS binding capacity of AnxA 5-FPs. These results all indicate that 18 AnxA5-FPs have different binding capacities for PS. This suggests that fusion of FPs results in hundreds of fold differences in affinity of AnxA5 for PS. The relative affinity constants (Kd) of the 18 AnxA5-FPs fusion proteins obtained from the fitted curve are shown in Table 3:

TABLE 3 Table 3

Note that ^a apoptotic cells were induced by etoposide and ^b apoptotic cells were induced by camptotheca acuminata.

Example 10

Sensitivity detection of five AnxA5-FPs with high PS affinity for early apoptosis

According to the affinity analysis result of the AnxA5-FPs fusion protein and PS, five AnxA5 probes with high PS affinity (Kd value is higher than 10 ⁷ orders of magnitude) and different fluorescence spectra are screened. Jurkat cells were induced with 0, 0.5, 1.0, 2.5, 5.0 and 10. Mu.M camptothecins for 12 hours and then labeled with five AnxA5-FPs at the same molar concentrations, including AnxA5-TagBFP, anxA5-mCerulean, anxA5-EGFP, anxA5-Ypet, anxA5-TagRFP. After the addition of 1 μl PI, the cells were immediately used for flow cytometry analysis. AnxA5-TagRFP stained cells were counterstained with 7-AAD. Meanwhile, apoptotic cells were labeled with commercial apoptosis-detecting reagent AnxA5-FITC (YEASEN, shanghai, china) as a control group. The percentage of cells labeled with the five AnxA5-FPs probes and AnxA5-FITC under the control and camptothecin treatment conditions was almost the same (as shown in FIGS. 10 and 11A, B). Interestingly, when these labeled cells were divided into early and late apoptosis, we found that under all conditions, five types of AnxA5-FPs probe-labeled early apoptotic cells were more than AnxA5-FITC (as shown in FIGS. 10 and 11C, D), while the proportion of early apoptotic cells on AnxA5-TagBFP, anxA5-mCerulean, anxA5-Ypet, anxA5-TagRFP, etc. probe-labeled was substantially identical to, or even more than, the proportion of early apoptotic cells on AnxA5-EGFP (as shown in FIGS. 11C, D). The above results demonstrate that our 5 AnxA5-FPs probes screened are more sensitive in detecting early apoptotic cells.

In summary, fusion proteins of AnxA5 and 18 different fluorescent proteins, expressed in soluble form when induced at 20℃can be purified by Ni-NTA affinity chromatography. These AnxA5-FPs fusion proteins could bind to apoptotic cells, but there was a difference in their optimal working concentration. These fusion proteins exhibit a hundred-fold difference in affinity for binding to PS. Based on the affinity results of 18 fusion proteins and PS, 5 AnxA5 apoptosis detection probes with high affinity are screened, and the detection probes have excellent performance, and compared with commercial AnxA5-FITC probes, the 5 AnxA5 with high affinity are more sensitive in detecting early apoptotic cells. The fusion protein of annexin A5 and fluorescent protein for apoptotic cell labeling, and the preparation method and application thereof provided by the invention can screen out proper fluorescent protein to prepare an AnxA5 apoptosis detection probe with high affinity and based on fluorescent protein so as to meet the demands of biotechnology application.

The foregoing has shown and described the basic principles, principal features and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the foregoing embodiments, which have been described in the foregoing embodiments and description merely illustrates the principles of the invention, and various changes and modifications may be made therein without departing from the spirit and scope of the invention, the scope of which is defined in the appended claims, specification and their equivalents.

Claims

1. A method for preparing fusion protein of annexin A5 and fluorescent protein for labeling apoptotic cells, which is characterized by comprising the following steps:

(1) Constructing a fusion gene expression vector in which the C-terminal of annexin A5 and the N-terminal of fluorescent protein FPs are connected through a connecting peptide Linker;

(3) Purifying by Ni-NTA affinity chromatography to obtain fusion protein.

2. The method according to claim 1, wherein in the step (1), the fluorescent protein is selected from any one of Venus、mVenus、Citrine、mCitrine、cpVenus173、cpCitrine174、EYFP、Ypet、mCherry、DsRed2、TagRFP、TagBFP、EBFP2、Cerulean、mCerulean、mCerulean3、EGFP、 or ECFP 18, and forms a fusion protein AnxA5-Venus, a fusion protein AnxA5-mVenus, a fusion protein AnxA5-Citrine, a fusion protein AnxA5-mCitrine, a fusion protein AnxA5-cpVenus173, a fusion protein AnxA5-CPCITRINE174, a fusion protein AnxA5-EYFP, a fusion protein AnxA5-Ypet, a fusion protein AnxA5-mCherry, a fusion protein AnxA5-Ypet, a fusion protein AnxA5-mCherry, a fusion protein AnxA5-DsRed2, a fusion protein AnxA5-TagRFP, a fusion protein AnxA5-TagBFP, a fusion protein AnxA5-Cerulean, a fusion protein AnxA5-mCerulean, a fusion protein AnxA 5-mFP 3, an fusion protein AnxA 5-XFP 3, and an ECFP 5-ECFP;

The amino acid sequence of the fusion protein AnxA5-mVenus is shown as SEQ ID NO. 20 in sequence, the amino acid sequence of the fusion protein AnxA5-Citrine is shown as SEQ ID NO. 21 in sequence, the amino acid sequence of the fusion protein AnxA5-mCitrine is shown as SEQ ID NO. 22 in sequence, the amino acid sequence of the fusion protein AnxA5-cpVenus is shown as SEQ ID NO. 23 in sequence, the amino acid sequence of the fusion protein AnxA5-CPCITRINE is shown as SEQ ID NO. 24 in sequence, the amino acid sequence of the fusion protein AnxA5-EYFP is shown as SEQ ID NO. 25 in sequence, the amino acid sequence of the fusion protein AnxA5-Ypet is shown as SEQ ID NO. 26 in sequence, the amino acid sequence of the fusion protein AnxA5-mCherry is shown as SEQ ID NO. 27 in sequence, the nucleotide sequence of the fusion protein AnxA5-DsRed2 is shown as SEQ ID NO. 28 in sequence, the amino acid sequence of the fusion protein AnxA5-CPCITRINE is shown as SEQ ID NO. 24 in sequence, the amino acid sequence of the fusion protein AnxA 5-XFP is shown as SEQ ID NO. 29, the fusion protein AnxA 5-XFP is shown as SEQ ID NO. 30, the fusion protein AnxA 5-XFP is shown as SEQ ID NO. 29, the amino acid sequence of the fusion protein AnxA 5-XFP is shown as SEQ ID NO. 30 is shown as SEQ ID NO. 29, the connecting peptide is Gly-Ser4 sequence, and the host cell is Escherichia coli BL21 (DE 3).

3. A fusion protein of annexin A5 and fluorescent protein prepared by the method of claim 1 or 2, wherein the fusion protein is used for marking phosphatidylserine PS on the surface of apoptotic cells, and detecting the apoptotic cells by flow cytometry or a fluorescent microscope.

4. A fusion protein according to claim 3, wherein the fusion protein has an affinity constant Kd of 10 ^-5 M to 10 ^-8 M with PS, 8 fusion proteins having an affinity higher than that of AnxA5-EGFP and AnxA5-mCherry are respectively fusion proteins AnxA5-TagBFP, anxA5-mCerulean3, anxA5-EBFP2, anxA5-Cerulean, anxA5-mCerulean, anxA5-ECFP, anxA5-Ypet and AnxA5-TagRFP, 4 fusion proteins having an affinity equivalent to AnxA5-EGFP and AnxA5-mCherry are respectively fusion proteins AnxA5-Venus, anxA5-EYFP, anxA5-cpVenus, anxA5-DsRed2, lower affinity than AnxA5-ECFP and AnxA5-Ypet, and an fusion protein having an affinity equivalent to AnxA 5-XCherry 5-35, and a yellow color equivalent to AnxA 5-35, and a blue color equivalent to an affinity range of AnxA5-ECFP and an affinity equivalent to an affinity range of an affinity 5-mCherry 5-ECFP, an affinity equivalent to an affinity range of an affinity 5-ECfp and an affinity equivalent to an affinity range of an affinity-mCherry.

5. A method for detecting apoptotic cells, comprising the steps of:

(1) Labelling apoptotic cells with the fusion protein of claim 4;

6. The method according to claim 5, wherein in the step (1), the concentration of the label of the fusion protein is in the range of 2nM to 2500nM.

7. The method according to claim 6, wherein in the step (2), the detection sensitivity of the fusion protein to early apoptotic cells is higher than that of a chemically labeled AnxA5-FITC probe, and the detection sensitivity of the fusion protein to early apoptotic cells is also higher than or equal to that of an AnxA5-EGFP fusion protein, wherein the emitted light of the fusion proteins AnxA5-TagBFP, anxA5-mCerulean, anxA5-Ypet and AnxA5-TagRFP respectively corresponds to blue, cyan, yellow and red, and the concentration of the label of the fusion protein ranges from 25nM to 500nM.

8. The method for detecting apoptotic cells as claimed in claim 1 wherein said method comprises:

(1) Incubating the fusion protein with apoptotic cells to specifically bind to cell surface exposed PS;

(2) Apoptotic cells were detected by fluorescent signal.

9. The method according to claim 8, wherein the apoptotic cell detection reagent is used for in vitro or in vivo tracing of early apoptotic cells.

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