CN117327148A - Preparation and application of polypeptide targeting brain glioma and radionuclide labeled molecular probe - Google Patents
Preparation and application of polypeptide targeting brain glioma and radionuclide labeled molecular probe Download PDFInfo
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
- A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
- A61K51/04—Organic compounds
- A61K51/0474—Organic compounds complexes or complex-forming compounds, i.e. wherein a radioactive metal (e.g. 111In3+) is complexed or chelated by, e.g. a N2S2, N3S, NS3, N4 chelating group
- A61K51/0478—Organic compounds complexes or complex-forming compounds, i.e. wherein a radioactive metal (e.g. 111In3+) is complexed or chelated by, e.g. a N2S2, N3S, NS3, N4 chelating group complexes from non-cyclic ligands, e.g. EDTA, MAG3
- A61K51/048—DTPA (diethylenetriamine tetraacetic acid)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
- A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
- A61K51/04—Organic compounds
- A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/534—Production of labelled immunochemicals with radioactive label
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
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- Urology & Nephrology (AREA)
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- Proteomics, Peptides & Aminoacids (AREA)
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Abstract
The invention discloses preparation and application of a polypeptide targeting brain glioma and a radionuclide labeled molecular probe. The amino acid sequence of the polypeptide targeting the AGNPT2 protein is as follows: HSVPRHEV. The radionuclide-labeled molecular probe consists of the following components: polypeptide HSV targeting AGNPT2 protein, chelator DTPA and radionuclide 99m TcO, wherein an AGNPT2 protein targeting polypeptide HSV is covalently coupled to a chelator DTPA, a radionuclide 99m The TcO is sequestered to the chelating agent DTPA. The polypeptide targeting the ANGPT2 can be specifically combined with the ANGPT 2; the radionuclide marked molecular probe prepared by the invention is used as an imaging drug for brain glioma, and has the characteristics of long imaging time, easy preparation and the like.
Description
Technical Field
The invention belongs to the field of application of novel biomedical technology, and in particular relates to preparation and application of a polypeptide targeting brain glioma and a radionuclide labeled molecular probe.
Background
Glioma is a malignant primary brain tumor, which is classified by the World Health Organization (WHO) into grade I to IV by histology, with glioblastoma multiforme (Glioblastoma multiforme, GBM) with the highest proportion in grade IV being the most severe, with GBM patients having a median survival of only 12 months, extremely high recurrence and mortality, and extremely poor prognosis. Currently, the treatment modes of GBM mainly comprise surgical excision and radiotherapy and chemotherapy, however, the methods have certain limitations. Because GBM belongs to diffuse brain glioma, the operation is difficult to completely cut off, and the traditional radiotherapy and chemotherapy methods have larger toxic and side effects, so more effective treatment means are needed. Currently, the tumor targeted therapy technology is focused, has huge clinical application potential, but the number of targeted drugs applied to clinical glioma is limited. Therefore, the development of targeted diagnostic and therapeutic drugs for GBM is of great importance in providing survival for GMB patients, and is a hotspot and difficulty of current research in the field.
The role of angiogenin 2 (angptl 2) in tumor-induced angiogenesis has been gradually revealed. Inhibiting ANGPT2 expression can reduce tumor volume and inhibit tumor cell metastasis. Therefore, the inhibition of the expression of ANGPT2 has great clinical application prospect in tumor treatment. Based on TCGA data set and immunohistochemical analysis, the ANGPT2 is highly expressed in brain glioma tissues, is positively correlated with the stage and progress of brain glioma, is an oncogene of brain glioma, and has application potential in targeted therapy of brain glioma. The polypeptide is an important target ligand molecule, has the characteristics of easy transformation and optimization, stable chemical property and the like, and is an important ligand molecule for researching and developing brain glioma diagnosis and treatment medicines. At present, targeting polypeptides such as RGD and the like are applied to brain glioma diagnosis, so that the targeting polypeptides are developed aiming at ANGPT2 and have important clinical practical values in diagnosis and treatment of brain glioma.
Disclosure of Invention
Based on the above background, it is an object of the present invention to design and synthesize a polypeptide ligand with optimal binding pattern and affinity to the AGNPT2 protein, and finally obtain a polypeptide sequence targeting the AGNPT2 protein.
The amino acid sequence of the polypeptide of the target AGNPT2 protein provided by the invention is as follows: HSVPRHEV (histidine-serine-valine-proline-arginine-histidine-glutamic acid-valine, his-Ser-Val-Pro-Arg-His-Glu-Val), abbreviated HSV.
The present invention designs polypeptide ligands with optimal binding patterns and affinities to the AGNPT2 protein, and identifies the affinities of the polypeptides to the AGNPT2 protein.
In the embodiment of the invention, when the affinity of the polypeptide HSVPRHEV and the AGNPT2 protein is identified, the identification method of the affinity is a biological film interference technology, namely, 20 mu g/mL of the AGNPT2 protein is solidified on an NTA chip, 200 mu L of the polypeptide HSVPRHEV with the concentration of 100 mu M is added, and the identification result of the affinity of the HSV and the AGNPT2 is obtained through biological film interference analysis.
The application of the polypeptide HSV in preparing medicaments for diagnosing and treating brain glioma also belongs to the protection scope of the invention.
The second object of the invention is to provide a radionuclide-labeled molecular probe based on the polypeptide HSV and specifically targeting brain glioma.
The glioma may specifically be glioblastoma multiforme.
The invention designs and synthesizes a radionuclide-labeled molecular probe with specific targeting to human brain glioma cell transplantation tumor, and explores cell experiments and radionuclides 99m The targeting effect of the TcO marked HSV on brain glioma cell U87-MG cell transplantation tumor shows that the HSV has targeting effect on brain glioma and potential application value of developing brain glioma.
The radionuclide labeled molecular probe provided by the invention consists of the following components: polypeptide HSV targeting AGNPT2 protein, chelator DTPA and radionuclide 99m TcO, wherein an AGNPT2 protein targeting polypeptide HSV is covalently coupled to a chelator DTPA, a radionuclide 99m Chelating TcO to said chelationOn the agent DTPA.
The structural formula of the chelating agent DTPA is shown as follows:
the radionuclide-labeled molecular probe is expressed as: 99m TcO-DTPA-HSV, wherein, 99m TcO is radionuclide, DTPA is chelating agent, HSV is polypeptide targeting ANGPT2 protein.
It is still another object of the present invention to provide a method for preparing the above radionuclide-labeled molecular probe.
The radionuclide marked molecular probe is prepared by a method comprising the following steps:
1) Preparation of chelator-polypeptides
Reacting polypeptide HSV with DTPA-tetra (t-Bu ester) to obtain chelator-polypeptide DTPA-HSV;
2) Preparation of radionuclide-labeled chelator-polypeptide
Adding radionuclide into mixed solution of chelating agent-polypeptide DTPA-HSV, stannous chloride and buffer solution 99m TcO, reacting to obtain radionuclide labeled chelator-polypeptide, namely radionuclide labeled molecular probe 99m TcO-DTPA-HSV。
In the above method step 1), the molar ratio of the polypeptide HSV to DTPA-tetra (t-Bu ester) may be 1:2-3, which may be 1:3,
in the above method step 2), the buffer solution is 0.1M PBS buffer solution or physiological saline, the pH is 7.4,
the chelating agent-polypeptide DTPA-HSV, stannous chloride and radionuclide 99m The proportion of TcO can be: 0.1-0.3mg:0.1mg:2.5mCi;
the reaction temperature may be room temperature, and the reaction time may be 15-30min, specifically 30min.
Specifically, the operation of the above method step 2) is:
dissolving chelating agent-polypeptide DTPA-HSV in ultrapure water at a final concentration of 1mg/mL, mixing 300 μl of DTPA-HSV solution with 1mu.L of 1mg/mL stannous chloride solution was mixed in 500. Mu.L of PBS, and 2.5mCi was added 99m TcO ， The radionuclide-labeled molecular probe is prepared by reacting for 30min at room temperature 99m Tc-DTPA-HSV。
The application of the radionuclide marked molecular probe in preparing the diagnostic reagent for brain glioma also belongs to the protection scope of the invention.
In the application, the brain glioma is specifically glioblastoma multiforme.
The diagnostic reagent is used for imaging human brain glioma cells and in-vivo brain glioma cell transplantation tumors.
According to an embodiment of the invention, the selected human brain glioma cells are U87-MG cells and the selected in vivo brain glioma cell transplantation tumor is a BABL/c nude mouse subcutaneous transplantation tumor.
The radionuclide-labeled molecular probe (in particular 99m TcO-DTPA-HSV) has the following beneficial effects compared with the prior art:
1) The invention designs and screens out the targeting polypeptide specifically combined with the ANGPT2 by utilizing a molecular docking technology, and fills the blank of the ANGPT2 high affinity polypeptide ligand;
2) The radionuclide marked molecular probe prepared by the invention is used as an imaging drug for brain glioma, and has the characteristics of long imaging time, easy preparation and the like.
Drawings
FIG. 1 shows the structural formula of the polypeptide HSV in example 1 of the present invention.
FIG. 2 shows the HPLC and MS identification results of the polypeptide HSV produced in example 1 of the present invention.
FIG. 3 is a three-dimensional docking model of the polypeptide HSV and the ANGPT2 protein in example 1 of the present invention.
FIG. 4 shows the results of an affinity test for HSV and ANGPT2 protein according to example 2 of the present invention.
FIG. 5 shows the structural formula of FITC-HSV in example 3 of the present invention.
FIG. 6 shows the HPLC and MS identification of FITC-HSV prepared in example 3 of the present invention.
FIG. 7 shows the uptake of HSV by flow cytometer control U87-MG cells in example 3 of the present invention (co-culture).
FIG. 8 is a structural formula of DTPA-HSV prepared in example 4 of the present invention.
FIG. 9 shows the HPLC and MS identification results of DTPA-HSV prepared in example 4 of the present invention.
FIG. 10 is a photograph of an example 4 of the present invention 99m TLC detection of TcO-DTPA-HSV.
FIG. 11 is a photograph of an example 4 of the present invention 99m In vitro stability test results of TcO-DTPA-HSV.
FIG. 12 is a U87-MG cell pair of example 4 of the present invention 99m Ingestion results of TcO-DTPA-HSV. * p < 0.05, ×p < 0.01.
FIG. 13 shows a BABL/c nude mouse inoculated with U87-MG cells in example 5 of the present invention by tumor injection 99m TcO-DTPA-HSV 99m SPECT/CT imaging results of TcO.
Detailed Description
The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.
The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
EXAMPLE 1 Synthesis of HSV Polypeptides
Artificially synthesizing HSV, wherein the sequence of the HSV is histidine-serine-valine-proline-arginine-histidine-glutamic acid-valine, his-Ser-Val-Pro-Arg-His-Glu-Val, abbreviated as HSVPRHEV, and the structural formula is shown in figure 1;
Fmoc-His (Boc) -Wang Resin with substitution degree of 0.35mmol/g was selected, and Fmoc protecting group was removed after swelling. Coupling is sequentially carried out from the C end to the N end according to the polypeptide sequence until His is reached, and small samples are taken for cutting to judge the correctness of the polypeptide, wherein the side chain protecting groups of Ser, his, arg, glu are tBu, boc, pbf, otBu respectively; all amino acids were Fmoc protected with amino group alpha. The cleavage liquid (volume ratio is trifluoroacetic acid: ethanedithiol: phenol: triisopropylsilane: water=90:4:2:2:2) reacts with the linear peptide resin to obtain the linear peptide with all side chain protecting groups removed. Dissolving the linear peptide in water, purifying by semi-preparative chromatography, separating out qualified liquid, and collecting, rotary evaporating and freeze-drying to obtain the target polypeptide. HPLC and MS identification results of HSV are shown in FIG. 2;
the results of the interaction site of the two-way valve with the ANGPT2 are shown in figure 3. Arg at position 5 of the polypeptide forms a hydrogen bond with Cys at position 433 of ANGPT2, val at position 3 of the polypeptide forms a hydrogen bond with Cys at position 450 of ANGPT2, his at position 1 of the polypeptide forms a hydrophobic binding effect with Tyr at position 475 of ANGPT2, and His at position 1 of the polypeptide forms a hydrogen bond with Tyr at position 476 of ANGPT 2.
Example 2 affinity identification of HSV with ANGPT2 protein
ANGPT2 protein was diluted to 20. Mu.g/mL with PBS for chip immobilization and polypeptide was diluted to 100. Mu.M with PBST (pH 7.4). ANGPT2 protein solution was added dropwise to NTA chip, 200 μl of PBST (pH 7.4) was added as a control to different wells of the cured NTA chip, and 200 μl of 100 μm polypeptide was added to the other wells. The sensor was injected with 250 μl of PBS buffer (pH 7.4), the buffer was run at maximum flow rate (150 μl/min) to reach the signal baseline, and the flow rate of buffer was reduced to 20 μl/min to obtain a more stable baseline. The signals of HSV binding to the ANGPT2 protein are shown in FIG. 4. The result shows that the combination effect of HSV and ANGPT2 protein is strong, and the equilibrium dissociation constant KD of the interaction between the HSV and the ANGPT2 protein is 50.2nM.
EXAMPLE 3 analysis of uptake of HSV by U87-MG cells
1) FITC-HSV preparation
The synthesis steps are as follows: fmoc-His (Boc) -Wang Resin with substitution degree of 0.35mmol/g was selected, and Fmoc protecting group was removed after swelling. Coupling is sequentially carried out from the C end to the N end according to the polypeptide sequence until His is reached, and small samples are taken for cutting to judge the correctness of the polypeptide; wherein the side chain protecting groups of Ser, his, arg, glu are tBu, boc, pbf, otBu, respectively; all amino acids were protected with Fmoc at the alpha position. After determining that the Fmoc-HSVPRHEV mass spectrum of the fragment polypeptide is correct, removing Fmoc protecting group, adding FITC with 3 times of molar multiple, reacting, and ending the reaction after ninhydrin detection is negative. The cleavage liquid (volume ratio is trifluoroacetic acid: ethanedithiol: phenol: triisopropylsilane: water=90:4:2:2:2) reacts with the linear peptide resin to obtain the linear peptide with all side chain protecting groups removed. Dissolving the linear peptide in water, purifying by using semi-preparative chromatography, separating out qualified liquid, collecting, evaporating and freeze-drying to obtain FITC-HSV. The structural formula of FITC-HSV is shown in FIG. 5, and the HPLC and MS identification results are shown in FIG. 6.
2) Quantitative and qualitative analysis of uptake of HSV by U87-MG cells
The bEnd.3 and HUVEC and U87-MG cells were plated in the upper and lower Transwell chambers at a ratio of 1:5, respectively, cultured for 48 hours to simulate the blood brain barrier and the blood brain tumor barrier, the bEnd.3 and HUVEC cell culture broth in the upper Transwell chamber was aspirated, FITC-HSV and FITC with a final concentration of 20. Mu.g/mL containing 10% FBS were added, incubated at 37℃and the U87-MG cells in the lower Transwell chamber were digested with 0.25% trypsin at 4 and 8 hours, respectively, and washed 3 times with cold PBS and then resuspended in 300. Mu.L of cold PBS for flow-measuring the fluorescence intensity of the cells, and FITC without polypeptide as a control. The results showed that the FITC fluorescence intensity of the U87-MG cells at 8 hours was greater than that of the 4 hour and FITC groups, indicating that HSV was able to be taken up by the U87-MG cells (FIG. 7).
EXAMPLE 4, 99m Preparation of TcO-DTPA-HSV molecular probe
1) DTPA-HSV preparation
Fmoc-His (Boc) -Wang Resin with substitution degree of 0.35mmol/g was selected, and Fmoc protecting group was removed after swelling. Coupling is sequentially carried out from the C end to the N end according to the polypeptide sequence until His is reached, and small samples are taken for cutting to judge the correctness of the polypeptide; wherein the side chain protecting groups of Ser, his, arg, glu are tBu, boc, pbf, otBu, respectively; all amino acids were protected with Fmoc at the alpha position. After determining that the Fmoc-HSVPRHEV mass spectrum of the fragment polypeptide is correct, removing Fmoc protecting group, adding DTPA-tetra (t-Bu ester) with 3 times of molar multiple, reacting, and ending the reaction after ninhydrin detection is negative. The cleavage liquid (volume ratio is trifluoroacetic acid: ethanedithiol: phenol: triisopropylsilane: water=90:4:2:2:2) reacts with the linear peptide resin to obtain the linear peptide with all side chain protecting groups removed. Dissolving the linear peptide in water, purifying by using semi-preparative chromatography, separating out liquid with qualified purity, and collecting, evaporating and freeze-drying to obtain the DTPA-HSV. The structural formula of DTPA-HSV is shown in figure 8, and the HPLC and MS identification results are shown in figure 9.
2) 99m TcO marked DTPA-HSV
99m TcO-DTPA-HSV radioactive molecular probes include DTPA-HSV and radionuclides 99m TcO, the HSV and 99m the preparation method of the TcO is connected by DTPA, and comprises the following steps:
(1) 99m rinsing of TcO
5mL of physiological saline is extracted by a 5mL syringe, the molybdenum technetium generator is leached, the leaching solution is collected into a centrifuge tube, radioactivity is detected by a medical activity meter, and the activity is 5mCi.
(2) 99m TcO marked DTPA-HSV
DTPA-HSV was dissolved in ultrapure water at a final concentration of 1mg/mL, 300. Mu.L of LDTPA-HSV solution was mixed with 100. Mu.L of 1mg/mL stannous chloride solution in 500. Mu.L of PBS, and 2.5mCi was added 99m TcO, reacting for 30min at room temperature to obtain 99m Tc-DTPA-HSV radioactive molecular probe. The radiochemical purity of the radioactive molecular probe was checked by TLC, and as shown in fig. 10, the radiochemical purity was 100%.
3) 99m TcO-DTPA-HSV in vitro stability experiment
The prepared radioactive molecular probe tracer (100. Mu.L, 100. Mu. Ci) was placed in 1mL of 10% fetal bovine serum, incubated at 37℃for 0.5, 1, 2, 4, 6 and 8 hours, and then the radiochemical purity of the radioactive molecular probe was detected by TLC. As shown in FIG. 11, the stability of the radioactive molecular probe in 10% fetal bovine serum was high, and the radiochemical purity was 100% 6 hours after labeling.
4) 99m Cell uptake assay for TcO-DTPA-HSV
Inoculating U87-MG cells into a 6-well plate, incubating for 24 hours, and preparing in the step 2) 99m TcO-DTPA-HSV (about 25. Mu. Ci) was added separately to 6-well plates and the control group was added free 99m TcO into 6-well plates. The cells were incubated for 1, 2, 4 hours, washed 3 times with cold PBS, then lysed with 0.5mL of 1M NaOH, and counted with a gamma counter Wizard 2470. Uptake = gamma count of cells/25 μci 99m Gamma count of TcO x 100%. The results showed that free 99m Cell uptake was not significantly increased over time in the TcO control group 99m The TcO-DTPA-HSV group had progressively increased cellular uptake over time, indicating 99m TcO-DTPA-HSV has a targeted binding effect on U87-MG (FIG. 12).
EXAMPLE 5 in vivo imaging of U87-MG cell transplantation tumor
1) Establishment of U87-MG cell subcutaneous tumor animal model
Taking U87-MG-luc cells in logarithmic growth phase, digesting with 0.25% pancreatin, and adjusting cell density to 1×10 5 mu.L of the cell suspension was pipetted into the right forelimb axilla of the mice.
2) SPET/CT imaging of nude mice
U87-MG cell subcutaneous transplantation tumor nude mice are divided into 99m TcO group 99m The TcO-DTPA-HSV group, 99m TcO group in situ injection of 200. Mu. Ci via tumor 99m TcO， 99m Tumor in situ injection of 200 μCi into TcO-DTPA-HSV group 99m TcO-DTPA-HSV, images were acquired 5min, 0.5h, 1h, 2h and 3h post-dose using SPET/CT. The results show that the data obtained from the above-mentioned method, 99m 5min after TcO group administration, tumors were visualized, but at 1h, 2h and 3h, over time, 99m TcO gradually diffused throughout the body, indicating free form 99m The lack of targeting by TcO results in systemic circulation with blood; 99m the TcO-DTPA-HSV group was developed at 5min, 0.5h, 1h, 2h and 3h, only at the tumor site, and over time, 99m few diffusion of TcO-DTPA-HSV occurred, indicating that the radioactive molecular probe was able to continuously visualize brain glioma due to the targeted binding of HSV to brain glioma (FIG. 13).
The present invention is described in detail above. It will be apparent to those skilled in the art that the present invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with respect to specific embodiments, it will be appreciated that the invention may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains.
Claims (10)
1. A polypeptide targeting AGNPT2 protein, the amino acid sequence of which is as follows: HSVPRHEV, histidine-serine-valine-proline-arginine-histidine-glutamic acid-valine, his-Ser-Val-Pro-Arg-His-Glu-Val, abbreviated HSV.
2. Use of the AGNPT2 protein-targeting polypeptide of claim 1 for the preparation of a medicament for diagnosing and treating brain glioma.
3. The radionuclide labeled molecular probe consists of the following components: the polypeptide HSV targeting the AGNPT2 protein, chelator DTPA and radionuclide as claimed in claim 1 99m TcO, wherein an AGNPT2 protein targeting polypeptide HSV is covalently coupled to a chelator DTPA, a radionuclide 99m The TcO is sequestered to the chelating agent DTPA.
4. A radionuclide-labeled molecular probe according to claim 3, characterized in that: the structural formula of the chelating agent DTPA is shown as follows:
5. a method of preparing the radionuclide-labeled molecular probe of claim 3 or 4, comprising the steps of:
1) Preparation of chelator-polypeptides
Reacting polypeptide HSV with DTPA-tetra (t-Bu ester) to obtain chelator-polypeptide DTPA-HSV;
2) Preparation of radionuclide-labeled chelator-polypeptide
Adding radionuclide into mixed solution of chelating agent-polypeptide DTPA-HSV, stannous chloride and buffer solution 99m TcO, reacting to obtain radionuclide labeled chelator-polypeptide, namely radionuclide labeled molecular probe 99m TcO-DTPA-HSV。
6. The method according to claim 5, wherein: in step 1), the molar ratio of polypeptide HSV to DTPA-tetra (t-Bu ester) is 1:2-3.
7. The method according to claim 5 or 6, characterized in that: in the step 2), the buffer solution is 0.1M PBS buffer solution or physiological saline, the pH value is 7.4,
the chelating agent-polypeptide DTPA-HSV, stannous chloride and radionuclide 99m The proportion of TcO is as follows: 0.1mg-0.3mg:0.1mg:2.5mCi;
the reaction temperature is room temperature and the reaction time is 15-30min.
8. Use of the radionuclide-labeled molecular probe according to claim 3 or 4 for preparing a diagnostic reagent for brain glioma.
9. The use according to claim 8, characterized in that: in the application, the glioma glioblastoma multiforme.
10. The use according to claim 8, characterized in that: the diagnostic reagent is used for imaging human brain glioma cells and in-vivo brain glioma cell transplantation tumors.
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