Application of trichina source RANKL targeting polypeptide TsPRTP21 in inhibition of osteoclast differentiation and prevention and treatment of bone destruction diseases

Abstract

The invention relates to an application of a trichina source RANKL targeting polypeptide TsPRTP21 in inhibiting osteoclast differentiation and preventing and treating bone destruction diseases. The polypeptide TsPRTP21 can inhibit downstream signal channels of osteoclast differentiation, reduce the generation of osteoclasts and relieve bone destructive diseases such as osteoporosis or rheumatoid arthritis, has no obvious cytotoxicity and hepatorenal toxicity, and provides a new scheme for treatment of bone destructive diseases.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and specifically to the application of a Trichinella - derived RANKL - targeting polypeptide TsPRTP21 in inhibiting osteoclast differentiation and preventing and treating bone destruction diseases. Background Art

[0002] Bone destruction diseases are a group of diseases that cause decreased bone density, altered bone structure and dysfunction due to local bone tissue loss, including various diseases such as osteoporosis, rheumatoid arthritis, etc. Common symptoms include local pain, pathological fractures, limited mobility, etc., and the harm is extremely extensive. Regarding the prevention and treatment of bone destruction diseases, in addition to treating the causes of bone destruction (such as inflammation, tumors, metabolic imbalance, etc.), inhibiting bone loss is an important strategy to reduce the disease burden. Currently, drugs for preventing and treating bone destruction are mainly divided into two categories according to their mechanisms of action: those that inhibit bone resorption and those that promote bone formation, and there are also drugs with dual functions of promoting bone formation and inhibiting bone resorption, such as romosozumab. Although there have been many advances in the field of drug research and development for preventing and treating bone destruction diseases, the efficacy of these new drugs is limited or there are certain side effects (such as inducing cardiovascular diseases, breast cancer, atypical femoral fractures, osteonecrosis of the jaw, etc.). Therefore, developing new drugs with different mechanisms of action and fewer side effects is of great significance for the prevention and treatment of bone destruction diseases.

[0003] The normal maintenance of the skeletal system is achieved by the dynamic balance between osteoclastic bone resorption and osteoblastic bone formation. Osteoclasts are tissue-specific multinucleated cells formed by the differentiation and fusion of monocytes / macrophages located on the bone surface. The receptor activator of nuclear factor-κB (RANK) on the surface of osteoclast precursor cells binds to the ligand RANKL of RANK produced by osteoblasts and other cells, initiating downstream signal cascades, differentiating into mature osteoclasts, and degrading and resorbing the bone matrix. Osteoprotegerin (OPG) secreted by osteoblasts is a decoy receptor for RANKL, which can competitively bind to RANKL with RANK, block the binding of RANKL to RANK on osteoclast precursor cells, and thus inhibit the formation, activation, and bone resorption function of osteoclasts. The OPG / RANKL / RANK signaling axis is a key link in regulating the formation, activation, and bone resorption of osteoclasts. Since the hyperfunction of bone resorption caused by the overactivation of osteoclasts is the main factor leading to bone destruction, the drug development targeting this signaling axis has become one of the important strategies for treating bone destructive diseases. Currently, the drugs targeting the RANKL / RANK targets in clinical practice are mainly monoclonal antibodies or targeting binding proteins. However, the long-term use of such drugs may have limitations such as immunogenicity. In recent years, with the structural analysis of the RANKL-RANK complex and the clarification of the key interaction sites, the targeting polypeptides or small molecule compounds designed for the binding site of RANKL and RANK can inhibit the binding of RANKL and RANK and the formation of osteoclasts by competitively binding to RANKL or RANK, reduce bone loss, and improve bone destructive diseases. Among them, polypeptide drugs have become a hot spot in drug development due to their high specificity, low immunogenicity, low toxicity, and easy synthesis.

[0004] Currently, there have also been certain progresses in the research of small molecule active polypeptides in bone tissue engineering. Among them, the research of polypeptide drugs targeting the RANKL / RANK / OPG axis for anti-osteoclast formation and activation is based on the design of OPG or RANK sequences, such as the polypeptide L3-3 mimicking RANK and the polypeptide OP3-4 mimicking OPG. However, since these polypeptides designed based on OPG and RANK are endogenous, the long-term use may induce the body to produce endogenous antibodies, neutralize the normal OPG or RANK in the body and affect their functions, and even may increase the risk of fractures. Therefore, it may be of great value to introduce new effector protein sequences for the design of polypeptide drugs. Summary of the Invention

[0005] Aiming at the defects existing in the prior art, the purpose of the present invention is to provide an application of a Trichinella - derived RANKL - targeting polypeptide TsPRTP21 in inhibiting osteoclast differentiation and preventing and treating bone destruction diseases. The polypeptide TsPRTP21 can inhibit the binding of RANKL to RANK by competitively binding to RANKL, reduce the formation and activation of osteoclasts, alleviate bone loss in bone destruction diseases such as osteoporosis or rheumatoid arthritis, and has no obvious cytotoxicity and liver - kidney toxicity, providing a new solution for the treatment of bone - destructive diseases.

[0006] To achieve the above - mentioned purpose, the technical solution adopted by the present invention is as follows:

[0007] A Trichinella - derived RANKL - targeting polypeptide TsPRTP21, characterized in that the amino acid sequence of the Trichinella - derived RANKL - targeting polypeptide TsPRTP21 is shown as SEQ ID NO.1.

[0008] An application of a Trichinella - derived RANKL - targeting polypeptide TsPRTP21, characterized in that the polypeptide TsPRTP21 can competitively bind to RANKL with RANK, inhibit the binding of RANKL to its receptor RANK, thereby inhibiting the downstream signaling pathway of osteoclast differentiation and reducing the formation and activation of osteoclasts.

[0009] The specific competitive binding of the polypeptide TsPRTP21 to RANKL with RANK is as follows: TsPRTP21 binds to receptor activator of nuclear factor - κB ligand (RANKL) and competitively inhibits the binding of RANKL to its receptor receptor activator of nuclear factor - κB (RANK).

[0010] On the basis of the above - mentioned scheme,

[0011] The polypeptide TsPRTP21 can inhibit the differentiation of murine RANKL - induced mouse bone marrow macrophages into osteoclasts.

[0012] On the basis of the above - mentioned scheme,

[0013] The polypeptide TsPRTP21 can inhibit the differentiation of human - derived RANKL - induced human peripheral blood monocytes into osteoclasts.

[0014] An application of a Trichinella - derived RANKL - targeting polypeptide TsPRTP21 in the research, development and manufacture of drugs for preventing and treating bone destruction diseases. The bone destruction diseases include: osteoporosis, rheumatoid arthritis and other diseases.

[0015] An application of a Trichinella polyubiquitin - like protein TsPULP in the specific binding sites with human - derived and murine RANKL, characterized in that:

[0016] The specific site can bind to both murine and human RANKL, thereby inhibiting the downstream pathway of osteoclast differentiation, reducing the generation of osteoclasts, and thus designing the RANKL-targeted polypeptide TsPRTP21;

[0017] The specific site is located at the 36th, 38th, 40th and 46th amino acids of the sequence shown in SEQ ID NO.4 (corresponding to the 7th, 9th, 11th and 17th amino acids of the sequence shown in SEQ ID NO.1).

[0018] Application of a specific site of Trichinella spiralis polyubiquitin-like protein TsPULP for binding to murine RANKL, characterized in that:

[0019] The specific amino acid site in TsPULP can bind to murine RANKL, thereby inhibiting the activation of the osteoclast signaling pathway and reducing the differentiation of murine RANKL-induced mouse bone marrow macrophages into osteoclasts;

[0020] The specific site is located at the 2nd, 5th, 6th, 7th, 36th, 38th, 40th, 44th and 46th amino acids of the sequence shown in SEQ ID NO.4.

[0021] Application of a specific site of Trichinella spiralis polyubiquitin-like protein TsPULP for binding to human RANKL, characterized in that:

[0022] The specific amino acid site in TsPULP can bind to human RANKL, thereby inhibiting the activation of the osteoclast signaling pathway and reducing the differentiation of human RANKL-induced human peripheral blood monocytes into osteoclasts;

[0023] The specific site is located at the 36th, 38th, 40th, 46th, 98th and 100th amino acids of the sequence shown in SEQ ID NO.4

[0024] Application of the Trichinella spiralis-derived RANKL-targeted polypeptide TsPRTP21 of the present invention in inhibiting osteoclast differentiation and preventing and treating bone destruction diseases, the beneficial effects are:

[0025] The polypeptide is used to bind to RANKL and competitively inhibit its binding to its receptor RANK, thereby inhibiting the downstream signaling pathway of osteoclast differentiation, reducing the generation of osteoclasts, inhibiting bone loss, alleviating bone destruction diseases such as osteoporosis or rheumatoid arthritis, and having no obvious cytotoxicity and hepatotoxicity and nephrotoxicity, providing a new solution for the treatment of bone destruction diseases. In addition, TsPRTP21 can be directly synthesized by chemical methods, has stable properties, large yield, low production cost, and can be optimized later to enhance its effect. Description of the Drawings

[0026] The present invention has the following attached drawings:

[0027] Figure 1 It is a molecular structure diagram of the complex of Trichinella spiralis polyubiquitin-like protein (TsPULP) predicted by AlphaFold2 and murine RANKL (mRANKL). The red amino acid residues in the figure are the amino acid residues on TsPULP analyzed by Chimera software that are at a distance from the amino acid residues;

[0028] Figure 2 It is the ability of wild-type and mutant TsPULP to inhibit the differentiation of mouse bone marrow macrophages (BMDMs) into osteoclasts; a is a typical TRAP staining diagram of wild-type and mutant TsPULP inhibiting the differentiation of mouse BMDMs into osteoclasts; b is a statistical chart of the number of TRAP-positive cells;

[0029] Figure 3 It is the SPR experiment to detect the binding affinity of wild-type and mutant TsPULP to mRANKL;

[0030] Figure 4 It is a molecular structure diagram of the complex of TsPULP predicted by AlphaFold2 and human RANKL (hRANKL). The red amino acid residues in the figure are the amino acid residues on TsPULP analyzed by Chimera software that are at a distance from the amino acid residues;

[0031] Figure 5 It is the ability of wild-type and mutant TsPULP to inhibit the differentiation of human peripheral blood mononuclear cells (PBMCs) into osteoclasts; a is a typical TRAP staining diagram of wild-type and mutant TsPULP inhibiting the differentiation of human PBMCs into osteoclasts; b is a statistical chart of the number of TRAP-positive cells;

[0032] Figure 6 It is the SPR experiment to detect the binding affinity of wild-type and mutant TsPULP to hRANKL;

[0033] Figure 7 It is the TsPRTP structure diagram, and the red-marked structure in the figure is TsPRTP21;

[0034] Figure 8 It is the binding of TsPRTP21 to RANKL; a is the SPR experiment result diagram of TsPRTP21 binding to mRANKL; b is the SPR experiment result diagram of TsPRTP21 binding to hRANKL;

[0035] Figure 9 TsPRTP21 inhibits the binding of RANKL to RANK; a is a competitive SPR experiment to verify that TsPRTP21 inhibits the binding of mRANKL to mRANK; b is a competitive SPR experiment to verify that TsPRTP21 inhibits the binding of hRANKL to hRANK.

[0036] Figure 10 TsPRTP21 inhibits the activation of the osteoclast signaling pathway induced by mRANKL in vitro; a is a typical Western blot image of the expression levels of proteins related to osteoclast differentiation in mouse BMDMs cells induced by mRANKL inhibited by TsPRTP21; b is a statistical chart of the Western blot gray values.

[0037] Figure 11 TsPRTP21 inhibits the differentiation of mouse BMDMs into osteoclasts induced by mRANKL in vitro; a is a typical TRAP staining image of the differentiation of mouse BMDMs into osteoclasts inhibited by TsPRTP21 induced by mRANKL; b is a statistical chart of the number of TRAP-stained positive cells of osteoclasts.

[0038] Figure 12 TsPRTP21 inhibits the activation of the osteoclast signaling pathway induced by hRANKL in vitro; a is a typical Western blot image of the expression levels of proteins related to osteoclast differentiation in human PBMCs induced by hRANKL inhibited by TsPRTP21; b is a statistical chart of the Western blot gray values.

[0039] Figure 13 TsPRTP21 inhibits the differentiation of human PBMCs into osteoclasts induced by hRANKL in vitro; a is a typical TRAP staining image of the differentiation of human PBMCs cells into osteoclasts inhibited by TsPRTP21 induced by hRANKL; b is a statistical chart of the number of TRAP-stained positive cells of osteoclasts.

[0040] Figure 14 TsPRTP21 alleviates bone loss in OVX mice; a is a typical image of micro-CT scanning of the femurs of OVX mice in each group; b is a statistical chart of the bone parameters of OVX mice.

[0041] Figure 15 TsPRTP21 inhibits osteoclastogenesis in the femurs of OVX mice; a is a typical TRAP staining image of the femurs of OVX mice in each group; b is a statistical chart of the number of TRAP-positive cells in the femurs.

[0042] Figure 16TsPRTP21 alleviates bone destruction in CIA mice; a is a typical micro-CT scan of the paws of CIA mice in each group; b is a statistical chart of the bone parameters of the paws of CIA mice.

[0043] Figure 17 TsPRTP21 inhibits the formation of osteoclasts in the paws of CIA mice; a is a typical TRAP staining picture of the paws of CIA mice in each group; b is a statistical chart of the number of TRAP-positive cells in the paws.

[0044] Figure 18 TsPRTP21 toxicity detection; a is the CCK8 experiment to detect the effect of TsPRTP21 on the cell viability of mouse BMDMs and human PBMCs; b is HE staining to detect the effect of TsPRTP21 on the liver tissue of OVX mice; c is HE staining to detect the effect of TsPRTP21 on the kidney tissue of OVX mice. Detailed implementation manners

[0045] The present invention will be further described in detail below with reference to the accompanying drawings.

[0046] 1. Determination of the binding site of TsPULP to RANKL

[0047] (1) Comparison of the binding sites of RANKL to TsPULP and RANK

[0048] The amino acid sequences of mRANKL (mRANKL, NP_035743, amino acid residues 162–316, shown in SEQ ID NO.2) and TsPULP (SEQ ID NO.4), and human RANKL (NP_003692, amino acid residues 162–317, shown in SEQ ID NO.3) and TsPULP were respectively input into the molecular docking algorithm AlphaFold2 (https: / / colab.research.google.com / github / sokrypton / ColabFold / blob / mai n / AlphaFold2.ipynbAlphaFold) to predict their protein structures and interaction sites. The model with the highest score was selected and imported into the UCSF Chimera tool to analyze the amino acids with an interaction distance less than between the two proteins, so as to predict the amino acid sites in TsPULP that can interact with RANKL. Mutants were designed according to the predicted amino acid sites for site-directed mutagenesis to further clarify the key interacting amino acids between the two. Figure 1 and Figure 4 The red amino acid residues in respectively show the possible binding sites of TsPULP predicted by AlphaFold2 to mRANKL and hRANKL.

[0049] (2) Expression and purification of TsPULP

[0050] Firstly, Trizol method was used to extract the mRNA of Trichinella spiralis. After reverse transcription, cDNA was obtained. The target gene TsPULP was amplified by PCR, and after double digestion, it was ligated to the PET-28a (containing His tag) vector. The recombinant vector was transformed into DH5α competent cells. After sequencing and comparison, the positive plasmid was obtained, and then it was transferred into DE3 competent cells and screened with kanamycin to complete the construction of the expression strain. The positive clone bacteria were inoculated into 1 L of LB medium. When the bacterial amount was amplified to OD600 = 0.6 - 0.8, IPTG was added and induced at 16 °C for 12 - 16 h. Then, it was centrifuged to collect the bacterial precipitate, resuspended and sonicated, and then centrifuged at high speed. Since TsPULP is an inclusion body, the supernatant was discarded, and the protein precipitate was dissolved with a binding buffer containing 6 M guanidine hydrochloride and sonicated with an output intensity of 30%, sonicated for 10 min, centrifuged at 12,000 rpm for 30 min, and the supernatant was collected and incubated with Ni2+ affinity chromatography medium for 2 h, washed with 50 mM imidazole to remove impurities, and eluted with 500 mM imidazole to obtain the target protein. Then, it was dialyzed overnight at 4 °C in 20 mM Tris-HCl, centrifuged at 12,000 rpm for 15 min, and the protein precipitate was collected. Then, a protein renaturation kit was used for denaturation and renaturation to obtain a soluble protein solution. The purity of the purified TsPULP was detected by SDS-PAGE gel electrophoresis and its concentration was determined by the BCA method.

[0051] (3) Purified expression of TsPULP mutants

[0052] All amino acid residues less than in the interaction surface between TsPULP and mRANKL were designed into 6 mutants (I2A / G5A, K6A / Q7A, H26A / R30A, Q36A / F38A / K40A, G44A / T46A, Q97A / E98A), and all amino acid residues less than in the interaction surface between hRANKL and TsPULP were designed into 4 mutants (V28A / R30A / L31A, Q36A / F38A, K40A / T46A, E98A / T100A), and site-directed mutagenesis was carried out with the FAST mutagenesis kit. Primers were designed before and after each mutation site, and PCR was used to mutate the target site. After nucleic acid electrophoresis detection, when positive bands appeared, DMT enzyme was added to the mutation system, and the unmutated products were digested at 37 °C for 4 h. Then, the digested system was transformed into DH5α competent cells, spread on LB solid medium, and single colonies were picked for gene sequencing to screen positive clones. The plasmids of positive clones were extracted, transformed into DE3 competent cells, expanded and induced for expression, and the target mutant proteins were obtained after purification according to the method in 1(3).

[0053] (4) Expression and purification of murine and human RANKL

[0054] Construct vectors according to the method described in 1(3) above. Construct murine RANKL (murine RANKL, mRANKL) and human RANKL (human RANKL, hRANKL) on the pEGX-6P-1 vector (expressing GST tag), screen positive clones, and induce protein expression. Induce expression for 12 - 16 h, centrifuge, collect bacterial precipitates, resuspend with binding buffer and then disrupt by sonication, centrifuge at high speed, collect the supernatant, incubate it with GST-binding resin for 2 h, and elute the target protein with elution buffer containing 10 mM reduced glutathione. Dialyze it into GST-tag protease cleavage buffer, add protease at a ratio of 2 U per 100 μg of protein, cleave at 4°C for 4 - 8 h, then add it to glutathione agarose resin for binding, centrifuge, and collect the supernatant (containing the target protein). Detect the purity of the purified target protein by SDS-PAGE gel electrophoresis and determine its concentration by the BCA method.

[0055] (5) Expression and purification of murine and human RANK

[0056] Construct murine RANK (murine RANK, mRANK) and human (human RANK, hRANK) onto the pET-28a vector according to the method in 1(3) for expression, purification, and renaturation, and determine the protein concentration by the BCA method.

[0057] (6) Determination of the key amino acid sites where TsPULP binds to mRANKL

[0058] ①Verify the ability of each mutant TsPULP to inhibit the differentiation of mouse bone marrow macrophages (BMDMs) into osteoclasts by TRAP staining of osteoclasts

[0059] After C57 mice were sacrificed by cervical dislocation, their femurs and tibias were taken, and the bone marrow cells were rinsed clean with PBS and placed in DMEM complete medium (containing 1% penicillin-streptomycin and 10% fetal bovine serum). They were cultured overnight in an incubator at 37°C with 5% CO2. The next day, they were centrifuged at 500 g for 5 min, and the adherent cells in the supernatant were collected and placed in DMEM complete medium containing 30 ng / ml M-CSF and cultured in the incubator. On the second day, 50 ng / ml mRANKL was added for induction, and wild-type and mutant TsPULP (I2A / G5A, K6A / Q7A, H26A / R30A, Q36A / F38A / K40A, G44A / T46A, Q97A / E98A) were added. The medium was changed every three days while keeping the concentrations of M-CSF, mRANKL, and the added proteins unchanged. After 5 - 7 days of induction, BMDMs were obtained. Mature osteoclasts can express a large amount of tartrate-resistant alkaline phosphatase (TRAP), which can be stained purple-red or wine-red by TRAP staining. Therefore, we used TRAP staining to analyze the effect of TsPULP on osteoclast differentiation in vitro. After cell fusion at 5 - 7 days, the supernatant was discarded, and the cells were fixed with 4% paraformaldehyde at room temperature for 10 min. Osteoclast staining was performed using a TRAP staining kit. After rinsing with double-distilled water, the nuclei were stained with hematoxylin, and after bluing with tap water, photographs were taken under an optical microscope. Cells containing more than three nuclei and with cytoplasm stained purple-red or wine-red were osteoclasts. The number of osteoclasts in each well was counted to observe the ability of each mutant TsPULP to inhibit the formation of mouse osteoclasts induced by mRANKL in vitro. Figure 2 The results showed that compared with the mRANKL group, the number of osteoclasts formed after adding wild-type TsPULP decreased significantly, while after adding mutants I2A / G5A, K6A / Q7A, Q36A / F38A / K40A, and G44A / T46A, there was no significant change in the number of osteoclasts compared with the mRANKL group, indicating that the ability of mutants I2A / G5A, K6A / Q7A, Q36A / F38A / K40A, and G44A / T46A to inhibit osteoclast differentiation decreased. Therefore, amino acids I2, G5, K6, Q7, Q36, F38, K40, G44, and T46 play important roles in TsPULP inhibiting osteoclast differentiation induced by mRANKL.

[0060] ② Verification of the change in the binding affinity between each mutant TsPULP and mRANKL by SPR experiment

[0061] The SPR protein-binding experiments were separately conducted on each mutant (I2A / G5A, K6A / Q7A, H26A / R30A, Q36A / F38A / K40A, G44A / T46A, Q97A / E98A) and the wild-type TsPULP protein with mRANKL. mRANKL was immobilized on a CM5 chip. Different concentrations of the wild-type and mutant TsPULP protein solutions were separately used as the mobile phase and flowed through the surface of the channel immobilized with mRANKL. The dissociation constant (KD) value was calculated based on the change data of the final refractive index of the protein surface, and the binding affinity between the TsPULP mutant and mRANKL was observed. Figure 3 The results showed that the KD values of the mutants I2A / G5A, K6A / Q7A, Q36A / F38A / K40A, and G44A / T46A were significantly decreased compared with the wild-type, indicating that the amino acids I2, G5, K6, Q7, Q36, F38, K40, G44, and T46 play important roles in the binding of TsPULP to mRANKL.

[0062] (7) Determination of the key amino acid sites for the binding of TsPULP to hRANKL

[0063] ① Verification of the ability of each mutant TsPULP to inhibit the differentiation of human peripheral blood mononuclear cells (PBMCs) into osteoclasts by osteoclast TRAP staining

[0064] Human peripheral blood was placed in a human peripheral blood mononuclear cell separation solution, and mononuclear cells (PBMCs) were extracted under the condition of differential centrifugation (3000 rpm, 30 min, with the acceleration and deceleration speed at gear 3). The human peripheral blood CD14 monocytes were extracted by immunomagnetic bead separation method using the CD14 MicroBead Kit monocyte sorting kit (Miltenyi Biotec GmbH, Germany) according to the kit instructions. + The monocytes were inoculated into a cell culture dish and added with αMEM complete medium containing 30 ng / ml M-CSF to induce them into macrophages. The obtained human macrophages were inoculated into a 48-well plate at 5×10 4 / well, and added with DMEM complete medium containing 30 ng / ml M-CSF and 50 ng / ml hRANKL. At the same time, the wild-type and mutant TsPULP (V28A / R30A / L31A, Q36A / F38A, K40A / T46A, E98A / T100A) were added. The cell fusion state was observed every day. After cell fusion on days 10 - 14, TRAP staining was performed according to the method in 1(6)①, and the number of osteoclasts was counted to observe the ability of each mutant TsPULP to inhibit the differentiation of human peripheral blood mononuclear cells induced by hRANKL into osteoclasts in vitro. Figure 5It was shown that compared with hRANKL, the number of osteoclasts formed decreased significantly after adding wild-type TsPULP, while there was no significant change in the number of osteoclasts after adding the mutants Q36A / F38A, K40A / T46A, and E98A / T100A compared with the hRANKL group. This indicates that the ability of these three mutants to inhibit osteoclast differentiation decreased significantly, suggesting that amino acids Q36, F38, K40, T46, E98, and T100 play important roles in TsPULP's inhibition of hRANKL-induced osteoclast differentiation.

[0065] ② Verification of the change in the binding affinity between each mutant TsPULP and hRANKL by SPR experiment

[0066] The SPR protein binding experiment of each mutant (V28A / R30A / L31A, Q36A / F38A, K40A / T46A, E98A / T100A) and wild-type TsPULP protein with hRANKL was carried out. hRANKL was immobilized on a CM5 chip, and different concentrations of wild-type and mutant TsPULP protein solutions were used as the mobile phase to flow through the surface of the channel immobilized with RANKL. According to the change data of the final refractive index of the protein surface, the dissociation constant (KD) value was calculated to observe the binding affinity between the TsPULP mutant and hRANKL. Figure 6 It was shown that the KD values of the mutants Q36A / F38A, K40A / T46A, and E98A / T100A decreased significantly compared with the wild-type, indicating that amino acids Q36, F38, K40, T46, E98, and T100 play important roles in the binding of TsPULP to hRANKL.

[0067] 2. TsPULP-derived RANKL-targeting polypeptide binds to RANKL and competitively inhibits the binding of RANKL to RANK

[0068] (1) Design of TsPULP-derived RANKL-targeting polypeptide

[0069] The ability of each TsPULP mutant to inhibit the binding of human or murine RANKL to RANK and to inhibit RANKL-induced osteoclast differentiation showed that the four amino-terminal sites Q36, F38, K40, and T46 play roles in binding to human and murine RANKL and inhibiting osteoclast differentiation. Therefore, we selected the amino acids 30-50 in TsPULP (RLRGGMQIFVKTLTGKTITLE) and synthesized a polypeptide (TsPRTP21) by solid-phase synthesis method to inhibit osteoclast formation and relieve bone loss. Figure 7 The red part in it shows the position of this peptide segment in the TsPULP protein.

[0070] (2) SPR experiment to verify the binding of TsPRTP21 to RANKL

[0071] Biotinylated TsPRTP21 was immobilized on an avidin-coupled chip. Different concentrations of mRANKL or hRANKL protein solutions were used as the mobile phase and flowed over the surface of the channel immobilized with TsPRTP21. The dissociation constant (KD) value was calculated based on the change data of the final protein surface refractive index, and the binding affinity of TsTsPRTP21 was observed. Figure 8 As shown in a, TsPRTP21 can bind to mRANKL, and the binding affinity between them is 4.71 nM. Figure 8 As shown in b, TsPRTP21 can bind to hRANKL, and the binding affinity between them is 7.83 nM. These results indicate that TsPRTP21 has strong binding affinity in the binding to both mRANKL and hRANKL.

[0072] (3) Competitive SPR experiment to verify the competitive binding of TsPRTP21 and RANK to RANKL

[0073] mRANK or hRANK was respectively immobilized on the CM5 chip. Different concentrations of TsPRTP21 protein solutions were incubated with 500 nM mRANKL or hRANKL at room temperature for 1 h. The mixture was used as the mobile phase and flowed over the surface of the channel immobilized with RANK. The ability of TsPRTP21 to competitively inhibit the binding of RANKL to RANK was observed through the change of the RU value of the binding of RANKL to RANK. Figure 9 As shown in a and b, with the increase of the concentration of TsPRTP21, the RU values of the binding of human and murine RANKL to RANK both decreased, indicating that TsPRTP21 can inhibit the binding of human and murine RANKL to RANK.

[0074] 3. TsPRTP21 inhibits the differentiation of mouse bone marrow macrophages into osteoclasts in vitro

[0075] (1) Detection of the activation level of osteoclast-related signaling pathways in mouse BMDMs by Western blot

[0076] Mouse BMDMs were obtained by the method of 1(6)① and seeded in 6-well plates. After the cells adhered, 50 ng / ml mRANKL was added, and TsPRTP21 was also added at the same time. The groups were as follows: ① PBS group; ② mRANKL group; ③ mRANKL + TsPRTP21 group. After stimulation for 60 min, the cells were collected, lysed with RIPA, and the expression of osteoclast-related signaling pathway proteins was detected by Western blot. Figure 10As shown in a and b, compared with the mRANKL group, after adding TsPRTP21, the expression levels of proteins related to osteoclast differentiation (p-p65, p-iκB, p-p38, p-ERK, p-JNK, NFAT) in mouse BMDMs decreased, indicating that TsPRTP21 can significantly inhibit the expression of osteoclast-related proteins in mouse BMDMs induced by mRANKL.

[0077] (2) Observation of the number of osteoclasts formed by TRAP staining

[0078] Mouse bone marrow BMDMs were seeded at 5×10 4 / well in a 48-well plate, and DMEM complete medium containing 30 ng / ml M-CSF and 50 ng / ml mRANKL was added. At the same time, TsPRTP21 was added, and the grouping was the same as above. The cell fusion state was observed every day. After cell fusion on days 5 - 7, the supernatant was discarded, and TRAP staining was performed according to the method in 1(6)①, and the number of osteoclasts in each well was counted to observe the effect of TsPRTP21 on the formation of mouse osteoclasts induced by mRANKL in vitro. Figure 11 As shown in a and b, compared with the mRANKL group, after adding TsPRTP21, the number of TRPA-positive osteoclasts decreased significantly, indicating that TsPRTP21 can significantly inhibit the formation of mouse osteoclasts in vitro.

[0079] 4. TsPRTP21 inhibits the differentiation of human peripheral blood monocytes into osteoclasts in vitro

[0080] (1) Verification by Western blot that TsPRTP21 inhibits the activation of the osteoclast signaling pathway in human PBMCs

[0081] The human PBMCs obtained in 1(7)① were seeded in a 6-well plate. After the cells adhered, 50 ng / ml hRANKL was added. At the same time, TsPRTP21 was added. The grouping was as follows: ① PBS group; ② hRANKL group; ③ hRANKL + TsPRTP21 group. After stimulation for 60 min, the cells were collected. After lysing the cells with RIPA, the expression of proteins in the osteoclast-related signaling pathway was detected by Western blot. Figure 12 As shown in a and b, compared with the hRANKL group, after adding TsPRTP21, the expression levels of proteins related to osteoclast differentiation (p-p65, p-iκB, p-p38, p-ERK, p-JNK, NFAT) in human PBMCs decreased, indicating that TsPRTP21 can significantly inhibit the expression of osteoclast-related proteins in human PBMCs induced by hRANKL.

[0082] (2) Observation of the number of osteoclasts formed by TRAP staining

[0083] The obtained human PBMCs were seeded at 5×10 4 cells per well in a 48-well plate, and DMEM complete medium containing 30 ng / ml M-CSF and 50 ng / ml hRANKL was added. At the same time, TsPRTP21 was added, and the grouping was the same as above. The cell fusion state was observed every day. After cell fusion on days 10 - 14, TRAP staining was performed according to the method of 1(6)①, and the number of osteoclasts was counted to observe the effect of TsPRTP21 on the differentiation of human PBMCs into osteoclasts induced by hRANKL in vitro. Figure 13 As shown in a and b, compared with the hRANKL group, after adding TsPRTP21, the number of TRPA-positive osteoclasts decreased significantly, indicating that TsPRTP21 can significantly inhibit the differentiation of human PBMCs into osteoclasts in vitro.

[0084] 5. TsPRTP21 alleviates bone loss in ovariectomized (OVX) mice

[0085] C57BL / 6 female mice were used to establish an animal model of osteoporosis in ovariectomized (OVX) mice by ovariectomy. They were randomly divided into 4 groups, with 8 mice in each group: ① sham operation group (Sham group); ② simple ovariectomy group (OVX group); ③ OVX + TsPRTP21 (2 mg / kg) group; ④ OVX + TsPRTP21 (4 mg / kg) group. Each group of mice was anesthetized with 3% sodium pentobarbital, and each mouse was intraperitoneally anesthetized at a dose of 0.1 ml / 100 g. The back side was shaved, disinfected with iodophor. In the OVX group, a longitudinal incision of about 1 cm was made on the back side, the skin and muscle were separated, ligated at the end of the uterus, and both ovaries were removed; in the sham operation group, only a part of the adipose tissue around the ovaries was removed, and after suturing, the mice were routinely raised for one week. The OVX + TsPRTP21 group was treated with TsPRTP21 by intraperitoneal injection one week later. The mice were sacrificed at the eighth week after modeling to observe the effect of TsPRTP21 on bone destruction in osteoporotic mice.

[0086] (1) Observation of the degree of bone loss in osteoporotic mice by Micro-CT and 3D modeling technology

[0087] Bilateral femurs of mice were taken and fixed in 4% paraformaldehyde. The fixed femurs were subjected to Micro-CT tomographic scanning using a Scanco Micro-CT 50 imaging system (SCANCO, Switzerland), and analyzed using three-dimensional analysis software (VGStudioMAX) to measure indices such as bone volume / tissue volume percentage (BV / TV), bone mineral density (BMD), trabecular bone number (Tb.N), and trabecular bone thickness (Tb.Th) of the femurs of each group of mice, and to observe the effect of TsPRTP21 on bone loss in osteoporotic mice. Figure 14 As shown in a and b, compared with the OVX group, after intraperitoneal injection of TsPRTP21, indices such as BV / TV, BMD, Tb.N, and Tb.Th were significantly increased, indicating that TsPRTP21 can significantly alleviate bone loss in OVX mice.

[0088] 2) Detection of osteoclast activation level in femurs of osteoporotic mice by TRAP staining

[0089] After decalcifying the fixed femurs, paraffin sections were made. After dewaxing, they were immersed in 0.1M CaCl 2 -MgCl 2 solution to activate tartrate-resistant acid phosphatase (TRAP). After rinsing with PBS, according to the instructions of the TRAP staining kit, the staining solution was prepared for TRAP staining. Staining was carried out at 37°C for 45 min. After rinsing with double-distilled water, the nuclei were stained with hematoxylin, and blued with tap water. After drying and mounting the slides, osteoclast counting was performed under a light microscope to observe the effect of TsPRTP21 on the number of osteoclasts in osteoporotic mice. Figure 15 As shown in a and b, compared with the OVX group, after intraperitoneal injection of TsPRTP21, the number of osteoclasts in the femurs of mice was significantly reduced, indicating that TsPRTP21 can inhibit the formation of osteoclasts in OVX mice.

[0090] 6. TsPRTP21 alleviates bone erosion in collagen-induced arthritis (CIA) mice

[0091] DBA / 1 male mice were randomly divided into four groups, including: ① normal control group (Control group); ② model group (CIA group); ③ CIA + TsPRTP21 group (1 mg / kg); ④ CIA + TsPRTP21 group (2 mg / kg). Bovine type II collagen (collagen II, CII) and complete Freund's adjuvant (CFA) were mixed and emulsified (100 μg CII, 100 μL / mouse), and intradermally immunized at 1.5 cm from the base of the mouse tail. The first collagen induction was set as day 0, and on day 21, it was changed to an equal volume mixture of incomplete Freund's adjuvant and CII, and boosted immunization was performed in the same method to prepare the CIA model. The CIA + TsPRTP21 group was treated with TsPRTP21 by intraperitoneal injection starting from day 0. After the second booster immunization, the incidence and joint scores of mice in each group were observed every other day. The mice were sacrificed on day 49 to observe the effect of TsPRTP21 on bone erosion in CIA mice.

[0092] (1) Observation of bone destruction degree in CIA mice by Micro-CT and 3D modeling technology

[0093] The mouse paw was taken and fixed in 4% paraformaldehyde, and Micro-CT scanning was performed according to the method in 5(1), and bone parameter indexes such as BV / TV and Tb.Th were calculated to observe the effect of TsPRTP21 on bone destruction in CIA mice. Figure 16 As shown in a and b, compared with the CIA group, after intraperitoneal injection of TsPRTP21, BV / TV and Tb.Th were significantly increased, indicating that TsPRTP21 can significantly relieve bone destruction in CIA mice.

[0094] 2) Detection of the activation level of local osteoclasts in the joints of CIA mice by TRAP staining

[0095] After decalcifying the fixed femur, paraffin sections were made, and TRAP staining was performed according to the method in 9(2) to observe the effect of TsPRTP21 on the number of osteoclasts in osteoporotic mice. Figure 17 As shown in a and b, compared with the CIA group, after intraperitoneal injection of TsPRTP21, the number of osteoclasts in the mouse femur was significantly reduced, indicating that TsPRTP21 can inhibit the formation of local osteoclasts in the joints of CIA mice.

[0096] 7. Toxicity detection of TsPRTP21

[0097] (1) Detection of the cytotoxicity of TsPRTP21 by CCK8

[0098] The mouse BMDMs and human PBMCs obtained in 1(6)① and 1(7)① were respectively at 1×10 4Inoculate into a 96-well plate. After overnight adherence, add TsPRTP21 with different concentration gradients by serial dilution. After incubation for 24 h, add CCK8 reagent. After 4 h, measure the absorbance at 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader to observe the cytotoxicity of TsPRTP21. Figure 18 As shown in a, TsPRTP21 has no obvious cytotoxicity to mouse BMDMs and human PBMCs.

[0099] (2) Detect the hepatotoxicity and nephrotoxicity of TsPULP by HE staining

[0100] To detect the toxicity of TsPRTP21 to mouse liver and kidney tissues, divide C57 mice into three groups: PBS group, low-dose TsPRTP21 (2 mg / kg), and high-dose TsPRTP21 (4 mg / kg). Inject TsPRTP21 once a day for 8 consecutive weeks. After 8 weeks, take the livers and kidneys of the mice, fix them in 4% paraformaldehyde overnight, embed them, section them, and then perform HE staining to observe the effects of TsPRTP21 on mouse livers and kidneys. Figure 18 As shown in b, after intraperitoneal injection of TsPRTP21, there is no obvious infiltration of inflammatory cells in the liver tissue, the hepatocytes are arranged regularly, and the hepatic lobule structure is normal. There is no obvious change compared with the non-injected group. Figure 18 As shown in c, after intraperitoneal injection of TsPRTP21, the renal tubular epithelial cells in the kidney tissue are arranged neatly, the interstitial tissue has no edema, fibrosis or inflammation, the glomerular basement membrane is intact, and there is no atrophy or sclerosis. There is also no obvious change compared with the non-injected group. The above results indicate that TsPRTP21 does not show obvious hepatotoxicity and nephrotoxicity in vivo and has fewer side effects.

[0101] The content not described in detail in this specification belongs to the prior art well-known to those of ordinary skill in the art.

Claims

1. A Trichinella spiralis-derived RANKL targeting polypeptide TsPRTP21, characterized in that: The amino acid sequence of the Trichinella spiralis-derived RANKL targeting polypeptide TsPRTP21 is shown in SEQ ID NO.

1.

2. The use of a Trichinella spiralis-derived RANKL targeting polypeptide TsPRTP21 as claimed in claim 1, characterized in that: The polypeptide TsPRTP21 can inhibit the binding of RANKL to its receptor RANK by competitively binding to RANKL, thereby inhibiting the downstream pathway of osteoclast differentiation and reducing the formation and activation of osteoclasts.

3. The use according to claim 2, characterized in that: The polypeptide TsPRTP21 can inhibit the differentiation of mouse bone marrow macrophages into osteoclasts induced by mouse-derived RANKL.

4. The use according to claim 2, characterized in that: The polypeptide TsPRTP21 can inhibit the differentiation of human peripheral blood monocytes into osteoclasts induced by human RANKL.

5. Application of a Trichinella spiralis-derived RANKL targeting peptide TsPRTP21 in the development of drugs for the prevention and treatment of bone destructive diseases.

6. An application of a Trichinella spiralis polyubiquitin-like protein TsPULP to bind to specific sites of human and mouse RANKL, characterized in that: The specific site can be used to bind to both mouse-derived and human-derived RANKL, thereby inhibiting the downstream pathway of osteoclast differentiation, reducing the generation of osteoclasts, and thereby designing the RANKL-targeting peptide TsPRTP21; The specific sites are located at amino acids 36, 38, 40 and 46 of the sequence shown in SEQ ID NO.

4.

7. An application of a Trichinella spiralis polyubiquitin-like protein TsPULP to bind to a specific site of mouse-derived RANKL, characterized in that: The specific amino acid sites in TsPULP can bind to mouse-derived RANKL, thereby inhibiting the activation of osteoclast signaling pathways and reducing the differentiation of mouse bone marrow macrophages into osteoclasts induced by mouse-derived RANKL. The specific sites are located at amino acids 2, 5, 6, 7, 36, 38, 40, 44 and 46 of the sequence shown in SEQ ID NO.

4.

8. An application of a Trichinella spiralis polyubiquitin-like protein TsPULP and a human RANKL binding specific site, characterized in that: The specific amino acid sites in TsPULP can bind to human RANKL, thereby inhibiting the activation of osteoclast signaling pathways and reducing the differentiation of human peripheral blood monocytes into osteoclasts induced by human RANKL. The specific sites are located at amino acids 36, 38, 40, 46, 98 and 100 of the sequence shown in SEQ ID NO.4.

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