CN115747170B - Cowpea chlorotic mottle virus-polypeptide complex and application thereof in osteoporosis treatment - Google Patents
Cowpea chlorotic mottle virus-polypeptide complex and application thereof in osteoporosis treatment Download PDFInfo
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Abstract
The invention provides a cowpea chlorotic mottle virus-polypeptide compound and application thereof in osteoporosis treatment, and belongs to the technical field of biological medicines. The compound is obtained by grafting polypeptide on the surface of cowpea chlorotic mottle virus; the polypeptide is an amino acid sequence shown as SEQ ID NO. 1. The compound can effectively inhibit the formation and differentiation of osteoclasts, can effectively improve the bone mass and the bone mass of osteoporosis, and is used for preventing and/or treating the osteoporosis. In addition, the compound has strong safety, no cytotoxicity, can reduce side effects caused by completely blocking the RANKL-RANK signal channel, and has no obvious side effects on organs such as heart, liver, spleen, lung, kidney and the like. The compound has the advantages of safety, no toxicity, stability, good effect and the like, and has good application prospect in the aspect of treating osteoporosis.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a cowpea chlorotic mottle virus-polypeptide compound and application thereof in osteoporosis treatment.
Background
Osteoporosis (osteoporosis) is a systemic metabolic disorder characterized by reduced bone density and bone mass and by destruction of bone tissue microstructure, most commonly seen in postmenopausal women. The nationwide osteoporosis patient in 2020 is estimated to rise to 5.333 million in 2050. Along with the acceleration of the aging process of population, osteoporosis, fracture and other complications become serious public health problems faced by China. The main approaches for treating osteoporosis currently include reducing bone resorption, increasing bone formation, and the like. The anti-bone resorption drugs include bisphosphate drugs, osteoclast differentiation factor inhibitors, etc. The biphosphate type medicine enters mature osteoclasts when the osteoclasts perform bone resorption function through hydroxyapatite combined in bone tissues, prevents the osteoclasts from further functioning and induces apoptosis of the osteoclasts, so that bone resorption is effectively inhibited. However, there are also limitations to bisphosphates, including: is deposited in bone tissue so that the metabolic rate in the body is slow, affects only the function of mature osteoclasts without affecting the differentiation and formation of osteoclasts, causes the accumulation of a large number of osteoclasts with loss of function, etc.
The monoclonal antibody of the receptor activator ligand RANKL of the nuclear factor ĸ B is a specific monoclonal antibody of the receptor activator ligand RANKL of the osteoclast differentiation factor inhibitor, and can block the binding of the RANKL and the receptor RANK thereof by binding with the RANKL and inhibit the activation of the RANKL-RANK signal pathway, thereby effectively reducing the differentiation formation of the osteoclast and weakening the bone resorption function. However, since the RANKL-RANK signaling pathway plays an important role in the immune, inflammatory, infectious and other processes, blocking the RANKL-RANK signaling pathway may increase side effects such as risk of infection while reducing osteoclast formation and function.
It can be seen that the existing medicines for treating osteoporosis have certain problems. The study of a drug that can inhibit osteoclast differentiation and formation without increasing the risk of infection is of great importance for the treatment of osteoporosis.
Disclosure of Invention
The invention aims to provide a cowpea chlorotic mottle virus-polypeptide compound and application thereof in osteoporosis treatment.
The invention provides a cowpea chlorotic mottle virus-polypeptide compound, which is a compound obtained by grafting polypeptide on the surface of cowpea chlorotic mottle virus; the polypeptide is an amino acid sequence shown as SEQ ID NO. 1. The grafting means that the polypeptide is connected to the surface of cowpea chlorotic mottle virus through an amide bond.
Further, when preparing the cowpea chlorotic mottle virus-polypeptide compound, the mass ratio of the cowpea chlorotic mottle virus to the polypeptide is 1: (0.1 to 50).
Further, the mass ratio of the cowpea chlorotic mottle virus to the polypeptide is 1:50.
the invention also provides a preparation method of the cowpea chlorotic mottle virus-polypeptide compound, which comprises the following steps:
and (3) activating carboxyl on the polypeptide, mixing with cowpea chlorotic mottle virus, performing condensation reaction on the activated carboxyl and amino on the cowpea chlorotic mottle virus in a solvent, eluting the reaction mixture, and dialyzing to obtain the cowpea chlorotic mottle virus.
Further, the method comprises the steps of,
the activation is activation of carboxyl groups on the polypeptide using EDC and NHS;
and/or, the solvent is PBS buffer solution or Tris buffer solution;
and/or the condensation reaction is to incubate for 1-12 hours at 4-25 ℃;
and/or, the eluting is eluting through a desalting column;
and/or, the dialysis is dialysis in PBS buffer.
Further, the method comprises the steps of,
the molar ratio of the polypeptide to EDC to NHS is 1:0.9:1;
and/or, the eluted eluent is PBS buffer solution; and/or, the elution speed is 0.5 mL/min; and/or the elution volume at elution is 24 mL;
and/or, the molecular cutoff at dialysis is 14 kDa; and/or, the dialysis time is 12 hours; and/or, changing the dialysis fluid every 3 hours during the dialysis.
The invention also provides application of the cowpea chlorotic mottle virus-polypeptide compound in preparing a medicament for preventing and/or treating osteoporosis.
Further, the drug is a drug that inhibits osteoclast formation.
Further, the drug is a drug that inhibits the expression of c-Fos, CTSK and/or Acp5 genes.
The invention also provides a medicine which is prepared by taking the cowpea chlorotic mottle virus-polypeptide compound as an active ingredient and adding pharmaceutically acceptable auxiliary materials or auxiliary ingredients.
Compared with the prior art, the invention has the beneficial effects that:
the cowpea chlorotic mottle virus-polypeptide compound provided by the invention can effectively inhibit the formation and differentiation of osteoclasts, can effectively improve the bone mass and the bone mass of osteoporosis, and is used for preventing and/or treating the osteoporosis. In addition, the compound has strong safety, no cytotoxicity, can reduce side effects caused by completely blocking the RANKL-RANK signal channel, and has no obvious side effects on organs such as heart, liver, spleen, lung, kidney and the like. The compound has the advantages of safety, no toxicity, stability, good effect and the like, and has good application prospect in the aspect of treating osteoporosis.
It should be apparent that, in light of the foregoing, various modifications, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.
The above-described aspects of the present invention will be described in further detail below with reference to specific embodiments in the form of examples. It should not be understood that the scope of the above subject matter of the present invention is limited to the following examples only. All techniques implemented based on the above description of the invention are within the scope of the invention.
Drawings
FIG. 1 is a characterization result of RM-CCMVs prepared in example 2: a is the result of a volume exclusion chromatography (FPLC) elution; b is an ultraviolet absorption spectrum of the ultraviolet spectrophotometer for representing RM-CCMVs; c is the dynamic mechanical particle size test result of CCMVs and RM-CCMVs; d is a Zeta potential test result of CCMVs and RM-CCMVs; e is a TEM detection result of CCMVs; f is TEM detection result of RM-CCMVs; bar=50 nm in E and F.
FIG. 2 is a CCK-8 assay for the effect of RM-CCMVs prepared in example 2 on bone marrow macrophage (BMDM) growth: a is the detection result of different concentrations of RM-CCMVs processed for 1 day; b is the detection result of the RM-CCMVs with different concentrations processed for 2 days; c is the detection result of the RM-CCMVs with different concentrations for 3 days.
FIG. 3 shows the results of flow cytometry to examine the cell entry rate and stability of RM and RM-CCMVs prepared in example 2: a is a flow cytometry detection result graph of 1 day, 3 days and 5 days of treating cells; b is a cell entering rate statistical chart.
FIG. 4 shows the inhibitory effect of qPCR on the osteoclast-associated gene of RW-CCMVs prepared in example 2; a is the detection of the expression inhibition effect of c-Fos genes; b is the detection of the expression inhibition effect of CTSK genes; c is the detection of the expression inhibition effect of Acp5 gene.
FIG. 5 shows the effect of TRAP staining to detect the inhibition of osteoclast formation by RW-CCMVs prepared in example 2: a is TRAP staining pattern; b is a statistical graph of the number of osteoclast formation.
FIG. 6 shows the improvement of bone mass in osteoporotic mice by RW-CCMVs prepared in example 2, as measured by Micro-CT and TRAP staining: a is a Micro-CT image; b is TRAP dyeing picture; c is a statistical chart of BV/TV, tb. Th, tb. Sp, tb. N and other parameters; d is a statistical graph of the number of osteoclasts in each group.
FIG. 7 shows the effect of HE staining on the tissue structure of the organs of RW-CCMVs prepared in example 2.
Detailed Description
The materials and equipment used in the embodiments of the present invention are all known products and are obtained by purchasing commercially available products.
The experiment is carried out by (1) Sichuan natural science foundation 2022NSFSC1511; (2) The university of Sichuan Hua Xi oral hospital explores and develops project basis and application basis research project RD-02-202207 support.
EXAMPLE 1 preparation of cowpea chlorotic mottle virus-polypeptide complexes of the invention
The preparation of the cowpea chlorotic mottle virus-polypeptide compound mainly comprises the following steps:
1. preparation of RANK domain related polypeptides
Firstly, according to the amino acid sequence of the RANK key domain Motif 2/3, the polypeptide with the length of 10 amino acids is prepared and named RM. The polypeptide sequence of 10 amino acids in length is: SRPVQEQGGA (SEQ ID NO. 1). The polypeptide is prepared by adopting a conventional polypeptide solid-phase synthesis method.
2. Amplification of cowpea chlorotic mottle virus nanoparticles
Cowpea chlorotic mottle virus used in this experiment was from ATCC and its platform number was Bio-46614.
The CCMVstock was an aqueous solution with a pH of 5, a NaOAc concentration of 1M and a DTT concentration of 5 mM.
Cowpea Chlorotic Mottle Virus (CCMV) is a virus nanoparticle (virus nanoparticles, VNPs) that can be amplified by artificially infecting cowpea leaves with CCMV and purifying it through a series of separations, in particular as follows:
sowing cowpea seeds in a plant cultivation room, growing young leaves 7-10 days later, and infecting CCMVvirus solution (the solvent is CCMVstorage solution, and the concentration of the CCMVvirus is 1 mg/mL) on young leaves by artificial friction, specifically: picking up 6-7 young cowpea leaves, grinding the young cowpea leaves in a mortar, adding 100 mu L of CCMVV virus solution and 100 mL distilled water, fully and uniformly mixing, dipping a little turbid liquid by using a finger belly, repeatedly rubbing on the surfaces of the young cowpea leaves until slight scars appear on the surfaces, culturing for 8-10 days, picking up all cowpea leaves, breaking walls by a wall breaking machine, filtering by gauze, centrifuging to obtain a supernatant, obtaining a crude product containing a large amount of protein, and then mixing the centrifuged supernatant with PEG6000 for 10:1 (volume mass ratio, mL/g), precipitating the protein, centrifuging to obtain a protein precipitate, suspending the precipitate with a CCMV stock solution to obtain a CCMV crude product, and subjecting the CCMV crude product to gradient centrifugation (40000 rpm,17 h,4 ℃) by means of an ultracentrifuge to obtain CCMV nanoparticles, and dialyzing the CCMV nanoparticles into the CCMV stock solution (molecular retention of 14 kDa, dialysis time of 7 days, changing the solution once a day).
3. Preparation of cowpea chlorotic mottle virus-polypeptide complex
The compound is prepared by modifying the surface of the CCMVs by condensation reaction according to the mass ratio of 5:1 of the CCMVs prepared in the step 2 to the polypeptides (RMs) prepared in the step 1. The preparation method comprises the following steps:
firstly, mixing RM and EDC/NHS in a molar ratio of 1:0.9:1, taking PBS buffer solution as a solvent, reacting for 10-15min at room temperature, then adding the obtained mixed solution into CCMV solution (the solvent is PBS buffer solution), and reacting RM activated carboxyl with amino on the surface of CCMV to form an amide bond, wherein the mass ratio of CCMV to RM is 5:1, and incubating at 4 ℃ for 2 hours. The reaction mixture was then eluted through a desalting column with PBS buffer (ph=7.2) at a rate of 0.5 mL/min and a rinse volume of 24 mL. After elution, the eluent is placed into a dialysis bag with the molecular cutoff of 14 kDa, dialyzed in PBS dialysate for 12 hours, the dialysate is changed every 3 hours, cowpea chlorotic mottle virus-polypeptide complex (RM-CCMVV) is obtained after dialysis, and the obtained solution is stored at the temperature of minus 20 ℃.
EXAMPLE 2 preparation of cowpea chlorotic mottle virus-polypeptide complexes of the invention
According to the method described in example 1, only in step 3, the mass ratio of CCMVs to RMs was changed to 1:50 to prepare RM-CCMVs.
EXAMPLE 3 preparation of cowpea chlorotic mottle virus-polypeptide complexes of the invention
According to the method described in example 1, only in step 3, the mass ratio of CCMVs to RMs was changed to 1:1 to prepare RM-CCMVs.
EXAMPLE 4 preparation of cowpea chlorotic mottle virus-polypeptide complexes of the invention
According to the method described in example 1, only in step 3, the mass ratio of CCMVs to RMs was changed to 1:5 to prepare RM-CCMVs.
EXAMPLE 5 preparation of cowpea chlorotic mottle virus-polypeptide complexes of the invention
According to the method described in example 1, only in step 3, the mass ratio of CCMVs to RMs was changed to 1:10 to prepare RM-CCMVs.
EXAMPLE 6 preparation of cowpea chlorotic mottle virus-polypeptide complexes of the invention
According to the method described in example 1, only in step 3, the mass ratio of CCMVs to RMs was changed to 1:20 to prepare RM-CCMVs.
The beneficial effects of the present invention are demonstrated by specific test examples below.
Test example 1 characterization of cowpea chlorotic mottle virus-polypeptide complex (RM-CCMVs)
1. Experimental method
The RM-CCMV and CCMV prepared in example 2 were characterized using a volume chromatography exclusion column (FPLC), an ultraviolet spectrophotometer, zeta potential, transmission electron microscopy, etc.
Wherein the mobile phase of the volume chromatography exclusion column (FPLC) is PBS buffer (ph=7.2), the elution rate of the buffer is 0.5 mL/min, the elution volume is 24 mL, the detection samples are all dispersed in the PBS buffer, and the samples are filtered (filter pore size is 200 μm) before loading, so that large impurities are removed to avoid blocking the column.
The detection wavelength of the ultraviolet spectrophotometer is 220-700 nm, the scanning speed is 100 nm/min, and the detection sample is RM-CCMVV solution after FPLC separation and purification.
The sample cell used for Zeta potential detection is a graphite electrode, the required sample volume is 1 mL, and the detection sample is RM-CCMVA solution after FPLC separation and purification.
And (3) the detection voltage of the transmission electron microscope is 400V, 5 mu L of RM-CCMVV solution separated and purified by FPLC is dripped on a copper mesh, the excessive liquid is gently sucked by filter paper, then 5 mu L of uranyl acetate staining solution is dripped, the room temperature is stained for 45-60 s, the excessive liquid is sucked by filter paper again, and the room temperature is dried and waits for detection.
2. Experimental results
The characterization result of RM-CCMVs is shown in FIG. 1. FIG. 1A shows the separation and purification of the prepared RM-CCMVs by FPLC, and the experimental results show that the RM-CCMVs are eluted at a elution volume of about 11.5 and ml, and the fractions are collected and further characterized. FIG. 1B is a graph showing the UV absorption spectrum of the RM-CCMVs by a UV spectrophotometer, indicating that the RM-modified CCMVs do not significantly change in the UV absorption spectrum. Fig. 1C shows the results of dynamic mechanical particle size testing (DLS), which shows that the modified RM-CCMV particle size is not significantly changed from the CCMV before modification, and the size is about 30 nm. The Zeta potential test results of FIG. 1D show that both the modified RM-CCMVs are slightly negatively charged and have no significant difference. FIG. 1E is a TEM test result of CCMVs, showing that the size is consistent with the DLS test result; FIG. 1F shows TEM examination of RM-CCMVs, showing that the size and morphology are similar to those of CCMVs, consistent with the DLS test results.
The above experimental results demonstrate that RM-CCMVs do not significantly change in the physical properties.
Test example 2 cell experiments of RM-CCMVs
1. CCK-8 detection of the cytotoxicity of RM-CCMVs
1. Experimental method
Bone marrow macrophages (BMDM) in the logarithmic growth phase were taken and prepared as cell suspensions (cell density: 1.5 ten thousand), and then RM-CCMVs (medium preparation) prepared in example 2 were added to 200. Mu.L of the cell suspensions at concentrations of 5. Mu.M, 1. Mu.M, 0.2. Mu.M, and 0.04. Mu.M, respectively, and the bone marrow macrophages (BMDM) were incubated at 37℃for 1 day, 2 days, and 3 days, and the cell activities were examined using CCK8, respectively, after incubation. Bone marrow macrophages without RM-CCMVs were used as control (Ctrl).
2. Experimental results
CCK8 detection results after bone marrow macrophages (BMDM) were treated for 1 day, 2 days and 3 days at different concentrations of RM-CCMV are shown in fig. 2. Compared with a control group (Ctrl), the CCK8 detection results of BMDM after 1 day, 2 days and 3 days of RM-CCMVs with all concentrations have no obvious statistical difference, which indicates that the RM-CCMVs prepared by the invention do not influence the growth of bone marrow macrophages and have no cytotoxicity.
2. Flow cytometry for detecting cell entry rate and stability of RM-CCMVs
1. Experimental method
RM-CCMVs were prepared as described in example 2 using a Rodamine B-labeled RM, and the following experiments were performed:
bone marrow macrophages (BMDM) in the logarithmic growth phase were taken and configured as a cell suspension (cell density 10 ten thousand), then a 1. Mu.M concentration of Rodamine B-labeled RM or RM-CCMVs (all formulated with medium) was added to the 1.5 mL cell suspension, and bone marrow macrophages (BMDM) were incubated at 37℃for 1, 3 and 5 days, after which the efficiency of RM or RM-CCMVs entry into the cells and the time of presence in the cells were examined by flow cytometry. Untreated bone marrow macrophages were used as control (Ctrl).
2. Experimental results
The experimental results are shown in fig. 3: the uptake rate of RM is obviously lower than RM-CCMV, and RM-CCMV can be efficiently taken up by cells and can exist stably in the cells, so that the cell entry efficiency and stability of RM-CCMV are improved compared with pure polypeptide. Meanwhile, the flow cytometry detection is based on a fluorescence signal, and only after RM is successfully grafted on CCMVs, the fluorescence signal can be detected, so that the preparation success of the RM-CCMVs is further proved.
3. qPCR detection of inhibiting effect of RM-CCMVs on osteoclast-related genes
1. Experimental method
qPCR detects the inhibiting effect of RW-CCMVs on the osteoclast-related genes. Bone marrow macrophages (BMDM) in the logarithmic growth phase were prepared as a cell suspension (cell density: 100 ten thousand), and RM-CCMVs (medium preparation) prepared in example 2 were added to the cell suspension of 3 ml at concentrations of 5. Mu.M, 1. Mu.M, 0.2. Mu.M, and 0.04. Mu.M, respectively, and the bone marrow macrophages (BMDM) were incubated at 37℃for 1 day, and then ranKL was added thereto for in vitro osteoclast induction at a concentration of 100 ng/ml for 4 days. After induction, the expression levels of the c-Fos, CTSK and Acp5 genes in the cells were examined. Only RANKL-induced cells were used as control (RANKL) without RM-CCMVs.
2. Experimental results
The inhibitory effect of RM-CCMVs on the expression of c-Fos, CTSK and Acp5 genes is shown in FIG. 4. As can be seen from fig. 4: 5. mu M and 1 mu M RM-CCMVs have remarkable inhibition effect on the expression of the osteoclast related genes such as c-Fos, CTSK, acp5 and the like.
4. TRAP staining to detect inhibiting effect of RM-CCMVs on osteoclast formation
1. Experimental method
The RM-CCMVs used in this experimental procedure were those prepared in example 2.
Cells were TRAP stained 4 days after treatment of BMDM cells with control solvents PBS, RANKL, RANKL +RM, RANKL+CCMVs, RANKL+RM-CCMVs, respectively. The specific method comprises the following steps:
bone marrow macrophages (BMDM) in the logarithmic growth phase were prepared as cell suspensions (cell density: 10 ten thousand), RM-CCMVs at a concentration of 1. Mu.M, RM or CCMVs at a concentration of 1. Mu.M (the concentration of CCMVs is equivalent according to the loading rate) were added to the 1 mL cell suspensions, the bone marrow macrophages (BMDM) were incubated at 37℃for 1 day, then 100 ng/ml of RANKL was added to each group, and the control solvent group was added with an equivalent amount of PBS, followed by incubation at 37℃for 4 days. After the cell culture was completed, the cells were fixed with 4% paraformaldehyde for 30 minutes, and then stained with TRAP.
2. Experimental results
The experimental results are shown in fig. 5: neither RM nor CCMVs alone inhibit osteoclast formation, whereas RM-CCMVs of 1. Mu.M are effective in inhibiting osteoclast formation.
Test example 3 animal experiment with RM-CCMVs
1. Micro-CT and TRAP staining detection for improving bone mass of osteoporosis mice by RM-CCMVs
1. Experimental method
A mouse osteoporosis model (OVX) was established by resecting the bilateral ovaries of C57/BL6 mice. The next day after model establishment, RM, CCMV, RM-CCMVs (prepared in example 2) were injected into the osteoporotic mice via the tail vein, each at a concentration of 1. Mu.M (100. Mu.L in PBS as vehicle) and 8 times (2 injections per week). After injection, micro-CT scan was performed to reconstruct three-dimensional femur trabeculae in mice and analyzed for differences between groups of BV/TV (bone tissue volume/total volume), tb. Th (bone trabecula thickness), tb. Sp (bone trabecula gap), tb. N (bone trabecula number), etc. The improvement of the bone mass of the mice with osteoporosis by RM-CCMVs was observed.
2. Experimental results
The experimental results are shown in fig. 6: RM-CCMVs significantly improve the values of parameters such as BV/TV, tb. Th, tb. Sp, etc. of the femur of the osteoporosis mice. There was no significant statistical difference between RM or CCMV groups and osteoporosis mice. The RM-CCMVs can obviously improve the bone quantity and bone microstructure of the femoral bone trabecula of the osteoporosis mouse; meanwhile, the RM-CCMVs can obviously reduce the number of osteoclasts in the femur of the osteoporosis mouse.
2. HE staining to observe influence of RM-CCMV on organ tissue structure
1. Experimental method
A mouse osteoporosis model (OVX) was established by resecting the bilateral ovaries of C57/BL6 mice. The next day after model establishment, RM, CCMV, RM-CCMVs (prepared in example 2) were injected into the osteoporotic mice via the tail vein, each at a concentration of 1. Mu.M (100. Mu.L in PBS as vehicle) and 8 times (2 injections per week). After the injection, various organs of the mice were dissected and separated, HE staining was performed, and the influence of RM-CCMVs on the organ tissue structure was observed.
2. Experimental results
The experimental results are shown in fig. 7: the microstructure of each organ was not significantly different from group to group. Experimental results illustrate: RM-CCMVs have good biocompatibility, and do not cause obvious damage to other organs such as heart, liver, spleen, lung, kidney and the like while improving bone mass and bone microstructure of an osteoporosis mouse.
In summary, the invention provides a cowpea chlorotic mottle virus-polypeptide compound which can effectively inhibit the formation and differentiation of osteoclasts, can effectively improve the bone mass and the bone mass of osteoporosis, and is used for preventing and/or treating the osteoporosis. In addition, the compound has strong safety, no cytotoxicity, can reduce side effects caused by completely blocking the RANKL-RANK signal channel, and has no obvious side effects on organs such as heart, liver, spleen, lung, kidney and the like. The compound has the advantages of safety, no toxicity, stability, good effect and the like, and has good application prospect in the aspect of treating osteoporosis.
Claims (8)
1. A cowpea chlorotic mottle virus-polypeptide complex, characterized in that: a compound obtained by grafting polypeptide on the surface of cowpea chlorotic mottle virus; the polypeptide is an amino acid sequence shown in SEQ ID NO. 1; the carboxyl grafted into polypeptide reacts with amino on the surface of cowpea chlorotic mottle virus to form an amide bond;
when the cowpea chlorotic mottle virus-polypeptide compound is prepared, the mass ratio of the cowpea chlorotic mottle virus to the polypeptide is 1:50.
2. the method for preparing cowpea chlorotic mottle virus-polypeptide complex according to claim 1, characterized in that: it comprises the following steps:
mixing polypeptide with EDC and NHS, reacting, mixing with cowpea chlorotic mottle virus, condensing carboxyl activated by the polypeptide with amino on the surface of cowpea chlorotic mottle virus in a solvent to form an amide bond, eluting a reactant, and dialyzing to obtain the product;
the condensation reaction is to incubate at 4 ℃ for 2 hours;
the elution is through a desalting column;
the eluted eluent is PBS buffer solution;
the molar ratio of the polypeptide to EDC to NHS is 1:0.9:1;
the molecular cut-off during dialysis was 14 kDa.
3. The preparation method according to claim 2, characterized in that:
the solvent is PBS buffer solution or Tris buffer solution;
and/or, the dialysis is dialysis in PBS buffer.
4. The preparation method according to claim 2, characterized in that:
the eluting speed is 0.5 mL/min during the elution; and/or the elution volume at elution is 24 mL;
and/or, the dialysis time is 12 hours; and/or, changing the dialysis fluid every 3 hours during the dialysis.
5. Use of the cowpea chlorotic mottle virus-polypeptide complex according to claim 1 for the preparation of a medicament for the treatment of osteoporosis.
6. Use according to claim 5, characterized in that: the medicine is a medicine for inhibiting the formation of osteoclast.
7. Use according to claim 6, characterized in that: the medicine is used for inhibiting the expression of c-Fos, CTSK and/or Acp5 genes.
8. A medicament for treating osteoporosis, which is characterized in that: the cowpea chlorotic mottle virus-polypeptide complex according to claim 1 is used as the only active ingredient, and pharmaceutically acceptable auxiliary materials are added.
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