CN115991736B - CD137 targeting polypeptide and application thereof - Google Patents

Abstract

The invention relates to the technical fields of molecular biology and medicine, in particular to a CD137 targeting polypeptide and application thereof. The invention provides a CD137 targeting polypeptide, which is GGACIEEGQYCF. The polypeptide has higher affinity and specificity to CD137, can specifically and efficiently target CD137 positive cells, and can be used as a polypeptide molecular probe for detecting the condition of atherosclerosis vulnerable plaque so as to monitor the disease development in real time; the polypeptide can also be used as a targeting polypeptide for imaging of high-expression CD137 tissues, and has wide application prospect.

Description

CD137 targeting polypeptide and application thereof

Technical Field

The invention relates to the technical fields of molecular biology and medicine, in particular to a CD137 targeting polypeptide and application thereof.

Background

Acute coronary syndrome is the main cause of death of coronary heart disease patients, and the pathological basis is coronary artery atherosclerosis vulnerable plaque rupture or erosion secondary thrombosis to cause coronary artery complete or incomplete occlusion. Vulnerable plaque (Vulnerable plaque), unstable plaque, high-risk plaque, soft plaque) has not been well-defined, and the main characteristics include a large proportion of plaque lipid nuclei (more than 40%), a predominantly liquid cholesterol ester composition, thin fibrous cap (< 150 μm), internal inflammatory reactions and neovascularization. The risk of a "coronary heart disease event" is mainly dependent on the stability of the coronary plaque, rather than the degree of stenosis caused by the coronary plaque, so the imaging evaluation of vulnerable plaque and how to reverse vulnerable plaque is one of the hot spots of current coronary heart disease research. At present, imaging evaluation means for coronary atherosclerosis high-risk plaques are insufficient, and diagnostic means for vulnerable plaques at present are clinically insufficient, including intravascular ultrasound, optical coherence tomography, coronary enhanced CT, coronary enhanced nuclear magnetism and the like. The method comprises the following steps: intravascular ultrasound (IVUS): the current clinical gold standard for evaluating plaque is invasive examination, and the patient acceptance rate is low; there are limitations and security: the diameter of the catheter is 1mm, the pushing capability is poor, and the catheter cannot pass through the lesion under the condition that the lesion stenosis degree is serious or the blood vessel distortion is obvious; can produce halo artifacts, non-uniformity artifacts, guidewire artifacts, geometric distortions of images, blood echoes; thrombus cannot be reliably identified, rupture of a smaller plaque cannot be resolved, and the like. Optical Coherence Tomography (OCT): resolution is 10 times that of IVUS, up to 10 microns, and small plaque rupture can be detected, but the examination requires temporary blood flow interruption, exacerbates or induces myocardial ischemia, and is difficult to apply to open lesions. Coronary enhancement CT: sensitivity and specificity are relatively low, in particular as expressed in: (1) lesions of the far or tiny branches of the coronary artery cannot be found; (2) It is difficult to distinguish between lipid-rich plaques and plaques rich in fibrous tissue due to the overlapping CT density values; (3) The spatial resolution is not sufficient to date for the assessment of the fine tissue structure of coronary plaque, the assessment of the morphology and thickness of the fibrous cap of plaque is limited, and for smaller plaques the accuracy of CT density measurements is affected by partial volume effects. Coronary enhancement MRA: still in the preclinical research stage, the scanning time is long, the motion artifact is more, and the method is not widely applied to clinic. Most importantly, these several examinations are not early diagnoses nor reflect the pathophysiology of high risk plaques. The magnetic resonance soft salient has the advantages of high soft tissue and contrast resolution, and can clearly distinguish soft tissue structures; the method can carry out multiparameter and multiplane imaging, thus clearly showing the position and degree of the lesion and the relation between surrounding tissues and organs, accurately judging the lesion, and ensuring that the ferroferric oxide has been approved for clinical application. The fluorescence imaging sensitivity is high, and the ICG is already applied to clinical fundus fluorescence angiography and the like. Therefore, localization and quantification are diagnostically advantageous in the characterization of many lesions.

Most medical workers and researchers are well known for tumor immunity and targeted therapy, however cardiovascular immunity and targeted tracking and therapy are also emerging concepts and developments in the cardiovascular field in recent years. In recent years, the incidence and mortality of coronary heart disease in developed European and American countries have been reduced, but a part of special groups are found, and cardiovascular accidents still occur repeatedly despite various optimal intervention measures (including statin drugs), and Nilsson J points out that the cardiovascular accidents are determined by other mechanisms of atherosclerosis high-risk plaques, such as plaque blocking the lumen to cause hemodynamic changes, so that anti-inflammatory factors are secreted to be reduced, blood vessel inflammation caused by lipid such as cholesterol and autoimmune response of blood vessels to plaque components are caused, and points out that new methods for detecting and verifying pathophysiological mechanisms are needed. Antoniades C et al indicate that vascular wall inflammation is also a high risk factor for cardiovascular disease, that molecules that cause abnormal expression of vascular walls promote the progression of atherosclerosis, and that are very potential new therapeutic targets, but current methods for detecting coronary inflammation either lack specificity (e.g. plasma biomarkers) or are expensive and impractical (PET). Saba L et al clearly indicated that inflammatory markers are imaging tracer targets for detection of vulnerable plaques. Inflammation plays a key role in plaque destabilization, both innate immunity and adaptive immunity are involved, vascular endothelial cells, T cells are activated, and mononuclear/macrophages release a large number of pro-inflammatory factors, such as monocyte chemotactic protein (MCP-1), INF-gamma, TNF-alpha, etc., and released matrix degrading proteases degrade collagen fibers of fibrous caps; promoting plaque neovascularization. Olofsson PS et al demonstrate that CD137 is a costimulatory molecule expressed on the surface of vascular endothelial cells, activated T cells within plaque, and monocyte membranes with high specificity that when bound to a ligand promotes further activation and augmentation of leukocytes within plaque, secretion of inflammatory factors, and has potential as a target for high risk plaque tracking and treatment.

Disclosure of Invention

Based on this, it is necessary to provide a CD137 targeting polypeptide and its use.

In order to achieve the above purpose, the specific technical scheme of the invention is as follows:

first, the present invention provides a CD137 targeting polypeptide, the amino acid sequence of which is GGACIEEGQYCF.

Further, a nucleic acid encoding the polypeptide is provided.

Further, conjugates of the polypeptides are provided, the conjugates being obtained by covalent or non-covalent attachment or interaction of the polypeptides to a carrier.

Preferably, the carrier comprises any one or more of fluorescein, antibody, polymer, high molecular material, nano material, liposome, oily compound and inorganic material.

Further, there is provided the use of said polypeptide or said nucleic acid or said biological material or said conjugate in the manufacture of a medicament, diagnostic reagent, diagnostic kit or imaging formulation for the prevention or treatment of a CD137 mediated disease.

Preferably, the CD 137-mediated disease comprises atherosclerotic vulnerable plaque.

Further, there is provided the use of said polypeptide or said nucleic acid or said biological material or said conjugate in the preparation of a reagent for detecting the expression level of CD137 in a cell.

Further, a diagnostic reagent or kit for a CD137 mediated disease is provided, comprising the polypeptide or the conjugate.

Further, there is provided an imaging agent comprising said polypeptide or said conjugate.

Further, a preparation method of the fluorescence/magnetic resonance bimodal molecular probe is provided, which comprises the following steps:

(1) Hydrophobic Fe ₃ O ₄ Diluting the nano particles with tetrahydrofuran, adding DP-PEG-Mal, dissolving, heating the reaction solution, and carrying out oscillation reaction;

(2) Precipitating the nanoparticles with cyclohexane and washing, then removing the cyclohexane;

(3) Dissolving the dried nano particles in deionized water, performing centrifugal ultrafiltration, and washing with deionized water;

(4) DP-PEG-Mal modified Fe ₃ O ₄ The nano particles are dissolved in Tris-HCl buffer solution, added with polypeptide, stirred and mixed uniformly, and then added with ICG-SH, stirred and reacted.

(5) Centrifugal ultrafiltration was performed and washed with PBS buffer.

Based on the technical scheme, the invention has the following beneficial effects:

the method for dynamically monitoring the vulnerable plaque of the atherosclerosis is not strong in sensitivity and specificity, is noninvasive, and can dynamically monitor the vulnerable plaque, and on the basis of verifying that CD137 is expressed in a vulnerable plaque specimen of a patient and a vulnerable plaque model specimen of a mouse, the method is characterized in that polypeptide based on targeting CD137 protein is coupled with ferroferric oxide and ICG to identify in the vulnerable plaque model mouse of the atherosclerosis, so that the molecular probe which is high in safety, strong in sensitivity and specificity, noninvasive and capable of dynamically monitoring the vulnerable plaque is provided. The probe is a bimodal probe, can be used for fluorescence detection of the existence of CD137, and can be applied to magnetic resonance detection.

Drawings

FIG. 1 identifies an atherosclerosis vulnerable plaque model and CD137 immunohistochemical results;

FIG. 2 CD137@Fe ₃ O ₄ Schematic of the ICG probe preparation flow;

FIG. 3 CD137@Fe ₃ O ₄ The ICG probe in vitro represents the test result; a: a probe electron microscope image; b: the probe magnetic resonance T2 signal is in linear relation with the concentration; c: probe magnetic resonance T2 maps of different concentrations; d: ICG, fe ₃ O ₄ @ ICG fluorescence spectrum; e: fluorescent signals of probes with different concentrations; f: a probe cytotoxicity experiment;

FIG. 4 CD137@Fe ₃ O ₄ The ICG probe traces and images vulnerable plaque in the model mouse body; a: a magnetic resonance T2 sequence; b: a magnetic resonance fat-pressing sequence; c: signal condition 1 hour after contrast agent injection; d: t2 signal histogram of model mice 1 hour before contrast agent injection; e: fluorescence image of model mice 1 hour before and after probe injection; f: fluorescent signal histogram after probe injection for control and experimental groups; g: model rat isolated aortic arch and heart, lung, liver, double kidney, spleen, pancreas, gastrointestinal organ fluorescence signals;

FIG. 5H & E staining of normal carotid artery, carotid artery stable plaque, vulnerable plaque (100 μm), prussian blue staining (50 μm) and quantitative bar graph obtained 1 hour after probe injection in model and control mice.

Detailed Description

The following examples are illustrative of the invention and are not intended to limit the scope of the invention. The technical means used in the examples are conventional means well known to those skilled in the art unless otherwise indicated.

The experimental methods used in the examples below are conventional methods unless otherwise specified.

All materials, reagents, etc. in the examples described below are commercially available unless otherwise specified.

Example 1 modeling and characterization of vulnerable plaque atherosclerosis

1. Establishing an atherosclerosis vulnerable plaque model:

experimental group: 30 ApoE knockout c57/b6 background mice were purchased from carukang and fed a high fat diet for 6 months;

control group: 10 c57/b6 mice were fed the normal diet for 6 months.

2. Identification of vulnerable plaque model of atherosclerosis: performing ultrasonic examination on the mice in the experimental group and the mice in the control group in the step 1, and as shown in fig. 1A, finding that plaque forms and bulges in the tube cavities at the aortic arch and branches such as carotid arteries in the experimental group; as shown in fig. 1B, after sacrifice, the aorta and branches were removed for HE staining, and multiple plaques were found to form, with the plaques protruding into the lumen; as shown in fig. 1C, the sample was stained with oil red, and a large amount of lipid was found to accumulate under the intima, characteristic of vulnerable plaque, confirming successful molding.

3.CD137 immunohistochemistry is performed on vulnerable plaque, stable plaque and normal blood vessel after carotid endarterectomy of clinical patients, and the vulnerable plaque, stable plaque and normal blood vessel of the mouse aorta are respectively shown in fig. 1D and E, so that it can be confirmed that CD137 expression is obviously higher than that of the stable plaque and the normal blood vessel in the vulnerable plaque of human and mouse, and it is proved that CD137 is a potential target for tracing the vulnerable plaque with high sensitivity and specificity.

EXAMPLE 2 construction and screening of CD137 targeting polypeptide library

1. Experimental apparatus and materials

N-methylmorpholine (NMM), piperidine, trifluoroacetic acid (TFA), dichloromethane (DCM), ninhydrin, vitamin C, phenol, tetramethylurea Hexafluorophosphate (HBTU), piperidine, triisopropylsilane (TIS), ethylenediamine (EDT), N, N-Dimethylformamide (DMF), dehydrated ether, resins, methanol, various Fmoc-protected amino acids, alexa Fluor 647-anti-CD137 antibody, MB-strepitavidins (Streptavidin magnetic beads), strepitavidins-HRP (strepitavidins labeled horseradish peroxidase), ferric chloride hexahydrate (FeCl) ₃ ·6H ₂ O), sodium oleate, absolute ethyl alcohol, n-hexane, oleic acid, 1-octadecene, acetone, tetrahydrofuran, DP-PEG-Mal (molecular weight 2000 Da), cyclohexane, tris-HCl buffer (1 mM, pH 7.4), indocyanine green-mercapto (ICG-SH), ultrafiltration centrifuge tubes (molecular weight cut-off 100 kDa), PBS buffer (0.01M, pH 7.4), polypeptide synthesis tubes, shaking tables, vacuum water pumps, rotary evaporators, centrifuges, heating mantle, laser confocal microscopy (ZEISS LSM 710), all of which are commercially available.

Synthesis of CD137 "one bead one substance" polypeptide library

The Fmoc solid phase peptide synthesis method is adopted to synthesize the polypeptide library, and the specific method is to couple protected amino acids to solid phase resin one by one, then cleave peptide chains from the resin under strong acid and remove side chain protecting groups.

(1) 200mg of Tentagel-NH2 resin is weighed, circulated according to the solid-phase polypeptide synthesis program, and 200mg of Met and Gly are sequentially added for reaction;

(2) After the reaction is finished, the resin is divided into 3 parts, 80mg parts of Asp, asn, arg and equal amount of HBTU are respectively added into each tube for coupling, after the coupling is finished, the 3 tubes of resin are mixed after deprotection, the resin is divided into 3 parts, and 90mg of Glu, arg, lys is respectively added into each tube for coupling with the equal amount of HBTU;

(3) After the coupling is completed, the 3-tube resin is mixed after deprotection. The resin was then split equally into 5 parts, and 60mg of Ile, ser, thr, his, gln was added to each tube separately to couple with equal amounts of HBTU;

(4) After the coupling is completed, the 5-tube resin is mixed after deprotection. The resin was then split equally into 4 parts, and 80mg of Asp, asn, arg, ile and equal amount of HBTU were added to each tube for coupling;

(5) After the coupling is completed, the 5-tube resin is mixed after deprotection. Dividing the resin into 2 parts, and respectively adding Gly and Ala of 100 mg to each tube to couple with the same amount of HBTU;

(6) After the coupling is completed, the 2 tubes of resin are mixed after deprotection. The resin was then split equally into 4 parts, and 80mg of Glu, lys, thr, asp was added to each tube separately to couple with equal amounts of HBTU;

(7) After the coupling is completed, the 4-tube resin is mixed after deprotection. The resin was then split equally into 4 parts, and 80mg of His, tyr, thr, asn was added to each tube separately to couple with equal amounts of HBTU;

(8) After the coupling is completed, the 4-tube resin is mixed after deprotection. The resin was then split equally into 5 parts, and 80mg of Gln, lys, thr, tyr, arg was added to each tube separately to couple with equal amounts of HBTU;

(9) After the coupling is completed, the 5-tube resin is mixed after deprotection. The resin was then split equally into 3 parts, and 90mg of Glu, ser, arg was added to each tube separately to couple with equal amounts of HBTU;

(10) After the coupling is completed, the 3-tube resin is mixed after deprotection. The resin was then split equally into 5 parts, and 60mg of His, tyr, phe, asp, val was added to each tube separately to couple with equal amounts of HBTU;

(11) After the coupling is completed, the 5-tube resin is mixed after deprotection. The resin was then split equally into 4 parts, 80mg of Gln, asp, his, asn was added to each tube separately to couple with an equal amount of HBTU, and after coupling, the 4 tubes were deprotected and mixed. And (3) through methanol replacement and shrinkage steps, vacuum drying is carried out, and the dried resin loaded with the peptide library is obtained for standby.

Screening of CD137 Positive Polypeptides

(1) Washing the dried peptide library with 1 XPBS for 3 times, adding 5% skimmed milk, sealing the surface of peptide beads on a mixer at 37deg.C for 2h, and washing with 1 XPBS for 3 times;

(2) Mixing biotin (biotin) marked CD137 protein with a polypeptide library, incubating for 2 hours at 37 ℃, and washing 3 times with 1 XPBS;

(3) Then 100 mu L MB-strepitavidin and strepitavidin-HRP were added simultaneously to the peptide library and incubated for 2h at 37℃in a mixer under light-shielding conditions. The incubated polypeptide library EP-containing tube was placed on a magnetic rack. The positive polypeptides are magnetically attracted to the EP tube side wall, while the negative polypeptides settle to the EP tube bottom due to gravity.

After incubation of positive polypeptide beads with biotin-labeled receptor protein, positive peptide beads specifically recognize protein, and streptavidin labeled HRP and magnetic beads recognize positive peptide beads by recognizing biotin. The surface of the positive peptide beads is coated with a layer of magnetic beads which have magnetism, so that the positive peptide beads are captured by a magnetic field, and meanwhile, the positive peptide beads become blue due to the catalysis of horseradish peroxidase. Transferring positive peptide beads into a microchip array, dropwise adding hydrogen bromide for in-situ cleavage, identifying by MALDI-TOF-MS, and solving corresponding sequence information K through a Mascot database. The positive polypeptide fraction labeled fluorescence was resynthesized in sequence, MALDI-TOF identification and HPLC purification for subsequent testing. The 1 CD137 targeting polypeptide of the invention is prepared by chemical synthesis: GGACIEEGQYCF (SEQ ID NO: 1).

EXAMPLE 3 preparation of polypeptide conjugates

In this embodiment, the preparation of the polypeptide fluorescent/magnetic resonance bimodal conjugate is taken as an example, and the preparation method of the polypeptide conjugate is described as follows:

1. hydrophobic Fe ₃ O ₄ Synthesis of nanoparticles

10.8 g FeCl ₃ ·6H ₂ O and 36.5 sodium g oleate are dissolved in a mixed solvent consisting of 80 mL ethanol, 140 mL n-hexane and 60 mL deionized water. The reaction mixture was heated to 70 ℃ with stirring to react 4 h. Subsequently, the reaction solution was extracted through a separating funnel, leaving an upper organic phase, and the upper organic phase was washed three times with deionized water. After the washing was completed, n-hexane was removed by rotary evaporation, and dried under vacuum overnight to give a black waxy iron oleate complex. 3.6 g oleic acid iron complex and 3.39 g oleic acid are dissolved in 1-octadecene of 25 mL, heated to 310 ℃ at a heating rate of 3.3 ℃/min, and stirred under argon for 30 min. After the reaction is finishedThe reaction solution was cooled to room temperature under room temperature conditions, then precipitated by acetone, and subjected to magnetic separation. At the same time, the crude product of magnetic separation is hydrophobic Fe ₃ O ₄ Washing the nano particles with acetone for 3 times to obtain hydrophobic Fe ₃ O ₄ Nanoparticles, and stored in tetrahydrofuran.

2. Preparation of bimodal molecular probe of targeting CD137 protein

Hydrophobic Fe ₃ O ₄ The nanoparticles were diluted to 1 mg/mL with tetrahydrofuran. 10 mL of this solution was taken up in 150 mg of DP-PEG-Mal and dissolved. The reaction solution was heated to 60 ℃ by a metal bath and reacted with shaking 12 h. Subsequently, the nanoparticles were precipitated with cyclohexane and washed three times with cyclohexane, then dried under vacuum at room temperature to remove the cyclohexane. The dried nanoparticles were dissolved in deionized water, centrifuged and ultrafiltered with an ultrafiltration tube of 100 kDa cut-off and washed 3 times with deionized water to remove unmodified DP-PEG-Mal. DP-PEG-Mal modified Fe ₃ O ₄ The nanoparticles were dissolved in Tris-HCl buffer (1 mM, pH 7.4) at 3 mg/mL, followed by the addition of 360. Mu.g of the screened CD137 positive polypeptide, stirring and mixing at 37℃for 20 min, and further adding ICG-SH, stirring and reacting at 4℃for 48 h. Finally, probe CD137@Fe3O4/ICG was prepared by centrifugation ultrafiltration using an ultrafiltration tube of 100 kDa cut-off and washing 3 times with PBS buffer (0.01M, pH 7.4) to remove unconjugated CD137 positive polypeptide and ICG-SH, as shown in FIG. 2.

Example 4 functional validation of bimodal molecular probes targeting CD137 protein

1. In vitro characterization test of molecular probes CD137@Fe3O4/ICG

FIG. 3A is an electron microscope image thereof; as shown in FIG. 3B, the magnetic resonance T2 signal is linear with the probe concentration, R ² =0.997; as shown in fig. 3C, probe magnetic resonance T2 plots for different concentrations, with increasing iron concentration, the T2 signal increases; as shown in fig. 3D, ICG emits, fe3O4/ICG absorbs, and emits fluorescence spectra; as shown in FIG. 3E, the relationship between the fluorescence signal and the probe concentration is linear, R ² = 0.9809; as shown in FIG. 3F, the cytotoxicity test shows that the probe iron concentration is safe at 50. Mu.g or lessIs low in toxicity.

2. Molecular probe CD137@Fe3O4/ICG for tracing and imaging vulnerable plaque in vivo

To achieve targeted tracking of atherosclerotic vulnerable plaques, fluorescence and MRI imaging were performed after tail vein injection of cd137@fe3o4/ICG molecular probes in a mouse atherosclerosis model. In vivo MRI results: figure 4A shows a pre-injection magnetic resonance T2 sequence with higher signal shadows in the vessel lumen as indicated by the arrows; FIG. 4B shows a magnetic resonance fat liquoring sequence with higher signal shadow attenuation disappearing at the same level; FIG. 4C shows the disappearance of signal attenuation at the site shown after 1 hour of molecular probe injection; FIG. 4D also shows that the CD137@Fe3O4/ICG molecular probe has obvious T2 enhancement effect on carotid plaque, an image is acquired after 1h of probe injection, the T2 value is 10.69+ -1.91, the T2 value before probe injection is 14.03+ -1.02, and P < 0.05.

Fluorescence results: the fluorescence image of fig. 4E shows that the model mice had no fluorescence signal before probe injection and a significant fluorescence signal appeared 1 hour after injection; FIG. 4F shows that after injection of CD137@Fe3O4/ICG molecular probes, the signal intensity of plaques at aortic arch and carotid arteries in HFD fed group was much higher than in control group, especially at 1 hour (1 h: 1.14.+ -. 0.42X 10 ⁸ And 4.90+ -2.21×10 ⁶ ，p/sec/cm ² /sr，P <0.05). The results indicate that the molecular probe has specific response to vulnerable plaque; figure 4G shows that in vitro imaging of the aorta isolated from the HFD fed group injected with cd137@fe3o4/ICG further supports in vivo optical signals originating from aortic arch and carotid arteries upon active targeting.

The carotid row H & E staining and Prussian blue staining were separated after 1 hour of probe injection in model and control mice, respectively. As shown in FIG. 5, H & E staining shows that the probe can be positioned on vulnerable plaque, prussian blue quantitative analysis shows that vulnerable plaque has a significant difference from stable plaque, and normal artery Prussian blue staining is negative, further indicating that the CD137@Fe3O4/ICG molecular probe has specificity in targeting vulnerable plaque.

While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.

Claims (4)

1. A CD 137-targeting polypeptide, which has the amino acid sequence GGACIEEGQYCF.

2. Use of the polypeptide of claim 1 for the preparation of a reagent for detecting the expression level of CD137 in a cell.

3. The preparation method of the fluorescent/magnetic resonance bimodal molecular probe is characterized by comprising the following steps:

(1) Hydrophobic Fe ₃ O ₄ Diluting the nano particles with tetrahydrofuran, adding DP-PEG-Mal, dissolving, heating the reaction solution, and carrying out oscillation reaction;

(2) Precipitating the nanoparticles with cyclohexane and washing, then removing the cyclohexane;

(3) Dissolving the dried nano particles in deionized water, performing centrifugal ultrafiltration, and washing with deionized water;

(4) DP-PEG-Mal modified Fe ₃ O ₄ Dissolving the nano particles in Tris-HCl buffer solution, adding the polypeptide targeting CD137 in claim 1, stirring and uniformly mixing, adding ICG-SH, and stirring for reaction;

(5) Centrifugal ultrafiltration was performed and washed with PBS buffer.

4. A fluorescent/magnetic resonance bimodal molecular probe prepared by the method according to claim 3, comprising the polypeptide according to claim 1.