CN113295679A

CN113295679A - BRET living body imaging probe for detecting cell apoptosis

Info

Publication number: CN113295679A
Application number: CN202110632983.0A
Authority: CN
Inventors: 赵晟; 张越凌
Original assignee: Southeast University
Current assignee: Southeast University
Priority date: 2021-06-07
Filing date: 2021-06-07
Publication date: 2021-08-24
Anticipated expiration: 2041-06-07
Also published as: CN113295679B

Abstract

The invention discloses a BRET living body imaging probe for detecting apoptosis, which is used for evaluating the apoptosis at the living body level, and constructs an AnVPb apoptosis biosensor with high sensitivity and low background by combining Annex i n V and mNeon Green-teLuc fusion protein of bioluminescence resonance energy transfer BRET, thereby realizing the detection of fluorescence and bioluminescence for the apoptosis. The invention can be applied to conventional fluorescence-based in vitro apoptosis detection, and can also be applied to bioluminescence imaging of living deep tissues to track in vivo apoptotic cells. The in-vivo imaging probe of the invention proves that AnVPb has the function of tracking focus through a series of in-vivo imaging results, and particularly has good luminous performance in deep tissues, thereby realizing the evaluation of focus injury; the in-vivo imaging probe has high sensitivity, low price and easy operation, and is not easily limited by the type of a detected sample.

Description

BRET living body imaging probe for detecting cell apoptosis

Technical Field

The invention relates to the field of bioluminescence living body imaging, in particular to a BRET living body imaging probe for detecting apoptosis.

Background

Apoptosis, also known as programmed cell death, is an important biological process in normal tissue development and disease development. The development of new apoptosis detection technology is always one of active scientific research fields. Annexin V is a 36kDa phospholipid binding protein that is capable of binding at higher concentrations of Ca²⁺In the presence of a negatively charged phospholipid such as Phosphatidylserine (PS). In normal cells, PS is distributed onlyInside the lipid bilayer of the cell membrane. When apoptosis occurs, PS everts from the plasma membrane to the cell surface even in the early stages. Therefore, Annexin V is often used as a marker of an early apoptosis event in various apoptosis detection technologies.

In recent years, visual detection of apoptosis by non-invasive imaging methods has been widely used in basic research, and is expected to provide a rapid method for diagnosing human-related diseases mainly characterized by apoptosis. Annexin V-based specific imaging agents have been successfully applied in the study of various disease models and diagnosis at the clinical level, including cancer, myocardial infarction and ischemia, and atherosclerosis, among others. The Annexin V probe coupled with the far infrared fluorescent dye is also developed for detecting the living bodies of experimental animals, but the fluorescent dye needs exciting light, and the autofluorescence of the animals can increase the background signal of imaging. Therefore, it is important to develop better Annexin V probe molecules.

In recent years, studies on bioluminescent probes represented by luciferases have been increasingly conducted. Bioluminescence has the advantages of almost zero background signal, high signal-to-noise ratio, sensitivity higher than fluorescence, and the like. Therefore, the development of highly sensitive, strong-signal bioluminescent probes is a prerequisite for the application of bioluminescence in the field of in vivo imaging.

Currently, Bioluminescence systems have been widely used in the biomedical field, from hypersensitive bioassays, drug screening to high-sensitivity Bioluminescence imaging (BLI), involving bioluminescent proteins. The traditional bioluminescent protein systems are mainly Firefly Luciferase (FLuc) and Renilla Luciferase (RLuc). However, the applicability of these luciferases is often limited by their size, stability and luminescence efficiency. Therefore, the development of more penetrating biological reporter molecules is a necessary condition for solving the above problems.

Disclosure of Invention

The invention aims to provide a BRET living body imaging probe for detecting apoptosis, which is used for in vitro and in vivo experiments by constructing a probe plasmid AnVPb for detecting early apoptosis and a control probe mAnVPb, purifying the probe protein through a prokaryotic expression vector, and detecting the BRET performance of the probe; in an in vitro experiment, treating A172 cells with hydrogen peroxide to establish an oxidative stress induced apoptosis model, and staining the treated cells with probe protein to evaluate early apoptosis; in an in-vivo experiment, AnVPb is combined with a living body imaging technology to evaluate the apoptosis condition of cells in a focal region in an acute kidney injury model and a stroke model of a mouse so as to track the development of a focus, and the AnVPb is used for evaluating the intervention effect of Serpina3 protein with a neuroprotective effect in the stroke model.

The purpose of the invention can be realized by the following technical scheme:

according to the BRET principle, apoptosis probe AnVPb and control probe mAnVPb plasmids expressed in a prokaryotic expression system are constructed, expressed in escherichia coli respectively, and affinity purification is carried out by Ni-NTA to obtain apoptosis probe and control probe protein, and fluorescence excitation spectrum and emission spectrum determination are carried out on the purified apoptosis probe AnVPb and the control probe mAnVPb.

The AnVPb apoptosis probe plasmid is obtained by fusing Annexin V with green fluorescent protein mNeon Green and luciferase teLuc respectively and inserting the fusion into pET28Zs vector.

The mANVPb control probe plasmid is obtained by fusing mutant Annexin V with mutation sites with green fluorescent protein mNeonGreen and luciferase teLuc respectively and inserting the fusion into a pET28Zs vector.

Further, for the in vitro cell culture level, an oxidative stress induced apoptosis model is established by treating A172 cells with hydrogen peroxide, and the specific binding capacity of the treated apoptotic cells is specifically marked by probe proteins to evaluate early apoptosis.

On an in vivo level, an in vivo imaging probe AnVPb is combined with an in vivo imaging technology and used for in vivo evaluation of apoptosis of cells in a focal zone of a living animal and tracking of lesion development.

Further, the AnVPb constructs a control probe mAnVPb for experimental control, and the mutant Annexin V comprises four mutation sites, namely E72Q, D144N, E228Q and D303N.

Further, both AnVPb and mAnVPb coupled mNeonGreen to teLuc using the design concept of the NLuc variant.

Further, the construction and performance test of the living body imaging probe comprises the following steps:

s1, construction of Annexin V probe protein expression plasmids, and prokaryotic expression and purification of protein probes;

s2, performance determination and concentration-dependent activity detection of Annexin V probe BRET;

s3 and Annexin V probe protein are used for detecting early apoptosis in oxidative stress damage of in vitro cells;

evaluation of S4, Annexin V probe protein on apoptosis-related disease model at living level-model i: acute kidney injury models;

s5, evaluation of an apoptosis-related disease model by Annexin V probe protein at living body level-model II: a stroke model;

s6 and Annexin V probe protein track the evolution condition of the cerebral apoplexy focus at the living body level.

Further, the step S1 operates as follows:

design of prokaryotic expression vector of S11 and Annexin V probe protein

S111, transforming pET28a to obtain a high-efficiency expression vector pET28 Zs;

s112, coupling mNeon Green with teLuc;

s113, optimizing Annexin V;

s114, introducing an HA tag into the fusion protein, and connecting the proteins by Gly-Gly-Gly-Gly-Ser;

s115, inserting a pET28Zs vector to obtain an AnVPb probe and a control probe mAnVPb;

s12, and the Annexin V probe mAnVPb protein;

purifying S13 and Annexin V probe protein;

and S14, analyzing the concentration and purity of the protein.

Further, the operation of step S2 is as follows:

s21, measuring the fluorescence spectrum of the probe protein;

s22, measuring the bioluminescence spectrum of the probe protein;

s23, determination of the catalytic activity of the concentration-dependent probe protein.

Further, the operation of step S3 is as follows:

culturing S31 and A172 cells;

s32, establishing an oxidative stress model and detecting early apoptosis.

Further, the operation of step S4 is as follows:

s41 unilateral kidney injury model;

s42, injecting a probe;

s43, living body imaging.

The operation of step S5 is as follows:

s51 method for making cerebral surface light plug

Adopting cerebral cortex light embolism to make a model, comprising operation preparation, mouse anesthesia, target area irradiation, rose bengal injection and wound suture;

s52, injecting a probe;

s53, living body imaging;

s54, in vivo characterization of AnVPb bioluminescence signal decay.

Further, the operation of step S6 is as follows:

s61, molding the cerebral surface light plug;

s62, injecting a probe;

s63, living body imaging.

The invention has the beneficial effects that:

1. the in-vivo imaging probe of the invention proves that AnVPb has the function of tracking focus through a series of in-vivo imaging results, and particularly has good luminous performance in deep tissues, thereby realizing the evaluation of focus injury;

2. the in-vivo imaging method of the in-vivo imaging probe is low in price, easy to operate, wide in application range, not easy to be limited by the types of detected samples, capable of being applied to basic researches such as diagnosis and treatment of various diseases and capable of playing a greater role in drug screening and medical imaging;

3. the in-vivo imaging probe is used for evaluating early apoptosis; the AnVPb is combined with a living body imaging technology and is used for evaluating the apoptosis condition of cells in a focal area and tracking the development of a focus; can also be used to evaluate the intervention effect of anti-apoptotic drugs.

Drawings

The invention will be further described with reference to the accompanying drawings.

FIG. 1 is a diagram of the design and purification of Annexin V probe protein of the invention (a) chemical structure of DTZ (b) design scheme of luciferase teLuc and (c) SDS-PAGE identification of AnVPb and control probe mAnVPb protein purification;

FIG. 2 is a graph of in vitro characterization of the luminescent activity of Annexin V probes of the invention (a), (b) the emission spectra of AnVPb and mAnVPb under excitation light, (c) the emission spectra of AnVPb and mAnVPb catalytic substrates, and (d) the concentration-dependent activity test of AnVPb and mAnVPb;

FIG. 3 is a graph of the detection of apoptosis of the cells of the present invention (a) and (b) AnVPb, mAnVPb and Annexin V-Alexa Fluor 647/PI on H₂O₂Co-staining of apoptotic a172 cells;

FIG. 4 is a statistical plot of in vivo imaging and lesion evolution for the AKI model of the present invention;

FIG. 5 is a graph showing (a) modeling of a cortical plug, (b) in vivo imaging of a mouse after a lesion, and (c) attenuation of probe bioluminescent signal intensity according to the present invention;

fig. 6 is a living body imaging chart and a stroke focus evolution statistical chart of the invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1

Construction and purification of Annexin V probe protein

1) Design of Annexin V probe protein prokaryotic expression vector

The efficient expression vector pET28Zs is obtained by modifying a pET28a vector, the pET series vector is a system with most pronucleus expression proteins, the pET series vector is regarded as the 'gold standard' of pronucleus expression proteins in an escherichia coli expression system, and pET28a plasmid is partially optimized, and comprises an added protein tag (HA-tag, a protease cutting site, 6x His-tag and the like) and a plurality of suitable single enzyme cutting sites for molecular cloning, so that the modified efficient expression vector pET28Zs is obtained.

mNeonGreen was coupled to teLuc according to the design concept of the NLuc variant.

Human wild-type Annexin V (NM-001154.4) was obtained from GeneBank databases and was optimized using codon optimization software (GeneOptizer, Thermo) since the expressed protein was a human sequence.

An HA tag is introduced into the fusion protein, the proteins are connected by Gly-Gly-Gly-Gly-Ser and are provided with a 6 xHis tag, and the HA tag is synthesized by a general biological company after the sequence is optimized.

Annexin V and mutant Annexin V (E72Q, D144N, E228Q and D303N) with four mutation sites are respectively fused with teLuc and mNeonGreen and inserted into a pET28Zs vector to obtain an AnVPb apoptosis probe and an mAnVPb control probe, as shown in figure 1.

2) Inducible expression of Annexin V probe mAnVPb protein

Firstly, converting a constructed prokaryotic expression vector containing an AnVPb apoptosis probe and an mAnVPb control probe into Rosseta to obtain recombinant bacteria, selecting a monoclonal in a kanamycin (50 mu g/ml) liquid LB, and culturing at 37 ℃ and 200rpm overnight;

then, the ratio of 1: 100 is inoculated in a liquid LB containing kanamycin (50 mu g/ml), cultured for 1.5 to 2 hours at 37 ℃ and 200r, when the OD600 of the bacterial liquid is 0.6 to 0.8, IPTG (working concentration is 1mmol/L) is added, and induced expression is carried out for 4 hours at 37 ℃ and 200rpm in a constant temperature shaking table; centrifuging 3000g of bacterial liquid at 4 ℃ to obtain bacterial precipitates, wherein the bacterial precipitates can be seen to have obvious green color; the cells were resuspended in lysis buffer, sonicated in an ice-box (frequency 10s pulse-10s rest, 50% intensity, total duration 1h)14000g, 15min, 4 ℃ and the supernatant was collected by centrifugation.

Lysis buffer preparation ingredient table

3) Purification of Annexin V probe protein

Obtaining purified Annexin V probe protein by a Ni-IDA affinity chromatography method, firstly cleaning a chromatographic column by using a thallus buffer solution, adding Ni-IDA to fill the chromatographic column, adding the ultrasonic supernatant of the thallus into a nickel column, and controlling the flow rate to enable a flow-through liquid to flow out at the flow rate of 2 ml/min; washing off the contaminating proteins with at least 3 bed volumes of washing buffer (25mmol/L imidazole, cell buffer); the target protein was eluted with an equal volume of elution buffer (250mmol/L imidazole, cell buffer) and the eluate was collected.

The purity of AnVPb and mAnVPb probes was more than 90% analyzed by SDS-PAGE and Coomassie blue staining for protein concentration and purity, as shown in FIG. 1, at a concentration of about 20. mu.g/. mu.l, sufficient for subsequent in vivo level experiments.

Example 2

Performance determination and concentration-dependent activity detection of Annexin V probe BRET

1) Determination of fluorescent Spectroscopy of Probe proteins

Placing the diluted probe protein and the substrate on ice (keeping out of the sun), setting the program of the microplate reader as the range of excitation light of 400-520nm, and reading the value of the emitted light at 540nm (program 1); setting a microplate reader program as excitation light 470nm, reading the value of light emitted in the range of 490-650nm (program 2), and incubating the whole reaction system at 37 ℃; 50 μ l of the prepared AnVPb/mAnVPb was added to each well, the fluorescence intensity values were read under the conditions of procedure 1 and procedure 2, respectively, 3 multiple wells were set for each condition, and the data obtained were plotted as a spectrogram, as shown in FIG. 2.

2) Determination of bioluminescence spectra of probe proteins

Setting a microplate reader program to read the value of the emitted light in the range of 350-650nm, and incubating the whole reaction system at 37 ℃; 50 μ l of the prepared AnVPb/mAnVPb was added to each well, 50 μ l of the prepared substrate was added after the procedure was prepared, the fluorescence intensity value in the range of 350-650nm was immediately read, 3 multiple wells were set, and the obtained data was plotted as a spectrogram, as shown in FIG. 2.

3) Determination of the catalytic Activity of concentration-dependent Probe proteins

To the protein probes obtained in example 1, AnVPb and mAnVPb were added in different concentration gradients of 0.00078125 ng/. mu.l, 0.0015625 ng/. mu.l, 0.003125 ng/. mu.l, 0.00625 ng/. mu.l, 0.0125 ng/. mu.l, 0.025 ng/. mu.l, 0.05 ng/. mu.l, 0.1 ng/. mu.l, and 50. mu.l PBS as a control, respectively, and the whole reaction system was incubated at 37 ℃; 50 μ l of the prepared AnVPb/mAnVPb was added to each well, 50 μ l of the prepared substrate was added after the procedure was ready, the fluorescence intensity values in the range of 350-650nm were read immediately, 4 duplicate wells were set for each concentration, and the obtained data were expressed as a linear equation between the probe concentration and its bioluminescence intensity, as shown in FIG. 2.

Example 3

Detection of early apoptosis in oxidative stress injury of in vitro cells by Annexin V probe protein

1) A172 cell culture

A172 cell culture was performed using 10% fetal bovine serum, 100U/ml penicillin, antibiotic, DMEM medium. The culture conditions are as follows: 37 ℃ and 5% CO₂95% humidity, performing aseptic operation in a biological safety cabinet until the density of well-grown cells reaches about 90%, performing pancreatin digestion, counting cells, and performing cell counting in H₂O₂A172 cells were seeded into 24-well plates one day before induction at 50% of the total number of cells that could be cultured per well.

2) Establishment of oxidative stress model and detection of early apoptosis

With 200. mu.M H₂O₂Treating A172 cells, inducing in vitro oxidative stress and apoptosis for 12 h; then, the well-constructed AnVPb, the mAnVPb and the commercialized mitochondrial superoxide are respectively usedH co-staining with compound red fluorescent probe or Annexin V-Alexa Fluor 647-propidium iodide cell apoptosis detection kit₂O₂(ii) treated cells; annexin V-Alexa Fluor 647/PI, a superoxide red fluorescent probe (final concentration of 5nmol/ml) and AnVPb/mAnVPb (final concentration of 4. mu.g/ml) were added to 24 wells, the wells were incubated in an incubator for 30min after addition of various probe proteins, the culture medium was aspirated, rinsed once with medium, 500. mu.l fresh medium was added again, and the wells were examined under a fluorescent microscope as shown in FIG. 3.

Example 4

Evaluation of Annexin V probe protein on apoptosis-related disease model at in vivo level-model i: acute kidney injury model

1) Unilateral kidney injury model

Male ICR mice were randomly divided into two groups: control group (tail vein injection of mabvpb); experimental group (tail vein injection anspb); all mice were left with right pedicel occluded for 30min and left untreated; isoflurane is added into a small animal anesthesia machine, 0.5 percent chlorhexidine acetate is soaked in all surgical instruments for disinfection, and the surface of a surgical area is disinfected by 70 percent alcohol before starting surgery. Recording the weight of the mouse, observing the breath of the mouse in the anesthesia machine, and keeping the breath for 40-60 times per minute; placing the anesthetized mouse on a body temperature maintaining instrument with the back facing upwards, maintaining the anesthetized state of the mouse by using a general anesthesia machine, and coating eye cream.

The bilateral kidneys were positioned, and the skin on the 0.5% chlorhexidine acetate disinfected surface was swabbed with a cotton swab, from which the hair was removed; skin and muscle were cut from the lower edge of the back of about 0.5cm and the lower edge of the rib of about 0.5cm by a back-entry method, the renal pedicles on the left and right sides were exposed, the kidneys on the left side were gently pulled out and exposed on the body surface of the mouse (sham surgery), the right renal pedicle of the mouse was closed with a non-traumatic hemostatic clamp for 30min (the back of the mouse was gently covered with gauze wetted with 0.9% physiological saline during closing to maintain normal humidity), and then the perfusion was performed for 24 h.

The kidney is changed from cherry red to purple, which proves that the kidney is ischemic, after the clamping time is over, the hemostatic clamp is opened to recover the blood supply of the kidney, and if the color of the kidney is changed from purple black to gradually recover red, which proves that the reperfusion is successful; respectively returning the kidneys on the two sides to the original positions, and suturing the operation openings on the left side and the right side layer by using suture lines; the aseptic principle is paid attention to in the whole operation process, and important organs or large blood vessels are damaged in the molding process and are removed in time; closely paying attention to the vital signs of the mice in the operation process, simultaneously keeping the body temperature of the mice stable, and placing the mice after the operation on a heat preservation heating plate.

2) Probe injection

All mice were injected tail vein with purified control probe protein (mANVPb) and apoptosis detection probe protein (AnVPb) 4h and 20h before in vivo imaging, respectively; mu.l of probe protein (1mg/kg body weight, 1xPBS dilution) was injected intravenously to each mouse tail.

3) In vivo imaging

All mice are subjected to living body imaging after 4h and 24h of the unilateral renal ischemia model, the mice are placed into an anesthesia box and are anesthetized by isoflurane, 200 mul DTZ (final concentration of 30 muM) is injected into the abdominal cavity of all the mice after the mice are anesthetized, the mice are placed in the prone position in the imaging area of the animal imaging instrument one by one, and the bodies of the mice are positioned in the center of the visual field; mice are subjected to inhalation anesthesia during in-vivo imaging, the induction dose of isoflurane mixed with oxygen is 5%, the maintenance dose is 1%, the anesthetic dose is adjusted to keep the respiratory frequency of the mice at 25-35 times/min, all mice are subjected to DTZ substrate intraperitoneal injection before each imaging, and imaging is carried out after the injection, as shown in figure 4.

Example 5

Evaluation of Annexin V probe protein on apoptosis-related disease model at in vivo level-model ii: cerebral apoplexy model

1) Cerebral surface light plug molding

Because the survival rate of the cerebral cortex thrombus molding is high, the focus size is moderate, and the requirements required by experiments can be better met; the experiment was modeled using a cerebral cortex plug.

Operation preparation: 100mg/kg rose bengal reagent was prepared, all surgical instruments were soaked with 0.5% chlorhexidine acetate for sterilization, the surgical area was surface sterilized with 70% alcohol before starting surgery, the body weight of the mice was recorded, and the volume of injected rose bengal was calculated (i.e., intraperitoneal injection was performed according to the weight of 10. mu.l/g mouse, total injection volume was not more than 300ul each).

Anesthetizing the mice: the body weight of the mice is recorded, the volume of the 1% pentobarbital injection dose is calculated (i.e. the total injection volume is not more than 300ul according to the weight of the mice and intraperitoneal injection is carried out according to 10 mul/g), 5 minutes is timed after the anesthetic is injected, the anesthesia success is obtained by holding the feet of the mice without twitching, the respiration is carefully monitored in the whole operation process to avoid over-anesthesia, the respiration frequency is kept relatively constant (about 40-60 breaths per minute), and a small amount of eye-moistening ointment is applied to the eyes of the mice before the operation, so that the eyes are prevented from being dried and dehydrated to cause blindness during long-time anesthesia.

Target area irradiation: depilatory cream was used to remove the head tip, using a cotton swab to wipe the skin over a 0.5% chlorhexidine acetate sterile surface, using a scalpel to make an incision along the eye level to the midline of the neck, exposing the skull, and using a sterile cotton swab to dry the skull surface and remove the surface mucosa (using forceps or sterile cotton swab) completely; the mouse head was mounted on the device not too tightly, the head was held slightly to the left, covering an area of about 30mm2 at 2mm (left or right) from the anterior cranial face, and the fiber was brought into intimate contact with the cranial surface to avoid light scattering, but care was taken not to apply pressure to it.

Rose bengal injection: mice were given a dose of 10 μ l/g body weight by intraperitoneal rose injection, and after 5min, the light source was turned on to avoid any other light source from irradiating the animals, the other end of the optical fiber was connected to a 532nm laser source, and the laser intensity of the tip surface of the optical fiber inserted into the brain was 40mW for 10 minutes, as shown in fig. 5.

And (3) wound suturing: cleaning and disinfecting the wound surface with a cotton swab soaked with 0.5% chlorhexidine acetate to avoid dehydration, suturing the muscular layer of the scalp with a reverse cutting needle and absorbable suture, suturing the skin with a nylon suture, applying a small amount of antibiotic ointment to the wound surface, discontinuing the anesthetic gas, discontinuing the anesthetic, carefully removing the mouse from the fixture, placing it on a preheated heating pad until it is fully awake, and returning to the cage.

2) Probe injection, the procedure was as in example 4

3) In vivo imaging, the procedure was as in example 4, and the imaging results, as shown in FIG. 5,

4) in vivo characterization of AnVPb bioluminescence signal decay

Imaging after injecting substrates for 1min, 2min, 5min, 10min and 30min, respectively, carrying out statistics and calculation of bioluminescence intensity on a lesion area of a mouse injected with an AnVPb probe by using ImageJ software, removing the mouse with a failed model, selecting a graph of 4 mouse living imaging, representing bioluminescence attenuation conditions in a probe body, selecting a rectangular graph capable of completely covering all lesion areas of the mouse as a fixed area, checking bioluminescence pixels of the fixed lesion area in an imaging picture of each mouse at 1min, 2min, 5min, 10min and 30min after injecting DTZ, and calculating a photograph gray value of the lesion area under the same area, wherein the photograph gray value is shown in figure 5.

Example 6

Annexin V probe protein for tracking cerebral apoplexy focus evolution condition at living body level

1) Molding the surface light plug of the brain, which has the same operation steps as the surface light plug of the brain in the embodiment 5;

2) injecting a probe, wherein the operation steps are the same as those of the probe injection in the example 4;

3) living body imaging was performed in the same manner as in example 4.

18 ICR mice are 9 weeks old and have the weight of 18-20 g; mice were divided into two groups, control group: tail vein injection of mabpb; experimental group: injecting AnVPb into tail vein, injecting all probes into tail vein 4h before living body imaging, removing mice which are unsuccessful in molding or die in the middle and have abnormal weight, and finally injecting equal amount of probes into 6 mice in experimental group and 6 mice in control group 2 days after molding, 4 days after molding and 6 days after molding when stroke model is completed; the probe formulation and injection amount were the same as in example 4, as shown in FIG. 6.

In the description herein, references to the description of "one embodiment," "an example," "a specific example" or the like are intended to mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

The foregoing shows and describes the general principles, essential 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 embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed.

Claims

1. A BRET living body imaging probe for detecting apoptosis is characterized in that according to the BRET principle, apoptosis probe AnVPb and control probe mAnVPb plasmids expressed in a prokaryotic expression system are constructed, respectively expressed in escherichia coli, and affinity purification is carried out by Ni-NTA to obtain an apoptosis probe and control probe protein, and fluorescence excitation spectrum and emission spectrum determination are carried out on the purified apoptosis probe AnVPb and the control probe mAnVPb;

the AnVPb apoptosis probe plasmid is obtained by respectively fusing Annexin V with green fluorescent protein mNeon Green and luciferase teLuc and inserting the fusion into a pET28Zs vector;

2. The BRET in vivo imaging probe for detecting apoptosis according to claim 1, used for assessing apoptosis at in vitro and in vivo levels, wherein for in vitro cell culture levels, a model of oxidative stress-induced apoptosis is established by treating A172 cells with hydrogen peroxide, and early apoptosis is assessed by specifically labeling the specific binding capacity of apoptotic cells after treatment with a probe protein; on an in vivo level, an in vivo imaging probe AnVPb is combined with an in vivo imaging technology and used for in vivo evaluation of apoptosis of cells in a focal zone of a living animal and tracking of lesion development.

3. The BRET in-vivo imaging probe for detecting the apoptosis of claim 1, wherein the AnVPb constructs a control probe mAnVPb for experimental control, and the mutant Annexin V comprises four mutation sites, namely E72Q, D144N, E228Q and D303N.

4. The BRET in vivo imaging probe for detecting apoptosis according to claim 1, wherein both AnVPb and mAnVPb adopt the design concept of NLuc variant, and mNeonGreen is coupled with teLuc.

5. The BRET in vivo imaging probe for detecting apoptosis according to claim 1, wherein the construction and performance test of said in vivo imaging probe comprises the following steps:

6. The BRET in vivo imaging probe for detecting apoptosis according to claim 5, wherein said step S1 is operated as follows:

design of prokaryotic expression vector of S11 and Annexin V probe protein

s112, coupling mNeon Green with teLuc;

s113, optimizing Annexin V;

s12, and the Annexin V probe mAnVPb protein;

purifying S13 and Annexin V probe protein;

and S14, analyzing the concentration and purity of the protein.

7. The BRET in vivo imaging probe for detecting apoptosis according to claim 5, wherein the operation of step S2 is as follows:

s21, measuring the fluorescence spectrum of the probe protein;

s22, measuring the bioluminescence spectrum of the probe protein;

8. The BRET in vivo imaging probe for detecting apoptosis according to claim 5, wherein the operation of step S3 is as follows:

culturing S31 and A172 cells;

s32, establishing an oxidative stress model and detecting early apoptosis.

9. The BRET in vivo imaging probe for detecting apoptosis according to claim 5, wherein the operation of step S4 is as follows:

s41 unilateral kidney injury model;

s42, injecting a probe;

s43, living body imaging;

the operation of step S5 is as follows:

s51 method for making cerebral surface light plug

s52, injecting a probe;

s53, living body imaging;

s54, in vivo characterization of AnVPb bioluminescence signal decay.

10. The BRET in vivo imaging probe for detecting apoptosis according to claim 5, wherein the operation of step S6 is as follows:

s61, molding the cerebral surface light plug;

s62, injecting a probe;

s63, living body imaging.