Application of trichinella spiralis pulp in inhibiting osteoclast differentiation and preventing and treating bone destruction

Abstract

This invention relates to the field of biomedicine, specifically to the application of the Trichinella spiralis-derived RANKL-binding protein TsPULP in inhibiting osteoclast differentiation and preventing bone destruction. A single protein (Trichinella spiralis polyubiquitin-like protein, TsPULP) was screened from the excretory and secretory antigens of Trichinella spiralis muscle larvae. TsPULP binds to RANKL (receptor activator of nuclear factor κB) ligand and competitively inhibits the binding of RANKL to its receptor, thereby inhibiting downstream signaling pathways in osteoclast differentiation, reducing osteoclast production, and alleviating osteoporosis or rheumatoid arthritis and other bone-destructive diseases. TsPULP exhibits no significant cytotoxicity or hepatotoxicity and can be directly expressed and produced in Escherichia coli. It is stable, has a high yield, and low production cost, providing a candidate molecule for the development of novel, highly effective, and low-toxicity anti-bone-destructive drugs.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to the application of Trichinella spiralis-derived RANKL-binding protein TsPULP in inhibiting osteoclast differentiation and preventing bone destruction. Background Technology

[0002] Bone destruction refers to the loss of localized bone tissue due to factors such as immune or metabolic imbalances, leading to changes in skeletal structure and functional impairment. Clinically, numerous diseases can cause bone destruction, such as osteoporosis and rheumatoid arthritis. The normal maintenance of the skeletal system relies on the dynamic balance between bone resorption by osteoclasts and bone formation by osteoblasts. Osteoclasts are the only cells in the body that perform bone resorption; they are tissue-specific multinucleated giant cells formed by the differentiation and fusion of mononuclear / macrophages located on the bone surface. Excessive activation of osteoclasts leading to hyperactive bone resorption is a significant cause of bone destruction. Targeting osteoclasts has become a major approach to preventing and treating bone destruction, such as with bisphosphonates. The RANKL / RANK / OPG axis is a major factor regulating osteoclast differentiation. Receptor activator of nuclear factor-κB (RANK) on the surface of osteoclast precursor cells binds to the RANK ligand (RANKL) produced by osteoblasts, initiating a downstream signaling cascade that promotes osteoclast maturation, degradation, and resorption of bone matrix. OPG, an osteoproteger and decoy protein for RANKL, binds to RANKL, inhibiting the binding of RANKL to RANK, thereby blocking the signaling pathway for osteoclast differentiation and reducing osteoclast production. Drugs targeting this pathway mainly include recombinant OPG protein and the RANKL monoclonal antibody Denosumab. Denosumab is currently used clinically to treat patients with osteoporosis or rheumatoid arthritis.

[0003] Although various drugs are currently available for treating bone-destructive diseases, most have significant side effects. Long-term use of the RANKL monoclonal antibody Denosumab can suppress RANKL function, leading to the accumulation of osteoclast precursor cells. Upon discontinuation of the drug, a rebound increase in osteoclast formation and activation can occur, resulting in multiple fractures. Bisphosphonates, bone resorption inhibitors, can easily cause cardiovascular disease, breast cancer, atypical femoral fractures, and osteonecrosis of the jaw. Furthermore, these drugs are usually expensive, and patients with osteoporosis or arthritis often require long-term medication, placing a significant financial burden on them. Therefore, there is still a need to develop more effective, safe, and low-cost drugs. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the present invention aims to provide the application of Trichinella spiralis-derived RANKL-targeting protein TsPULP in inhibiting osteoclast differentiation and preventing bone destruction. A single protein (Trichinella spiralis polyubiquitin-like protein, TsPULP) was screened from Trichinella spiralis excretory secretory antigens (TsMES) that can bind to nuclear factor κB receptor activator ligand (RANKL) and competitively inhibit its binding to its receptor, nuclear factor κB receptor activator (RANK), thereby inhibiting downstream signaling pathways of osteoclast differentiation, reducing osteoclast production, and alleviating osteoporosis or rheumatoid arthritis and other bone-destructive diseases. Furthermore, it exhibits no significant cytotoxicity or hepatotoxicity, providing a new treatment option for bone-destructive diseases.

[0005] To solve the above-mentioned technical problems, the technical solution provided by the present invention is as follows:

[0006] Applications of the Trichinella spiralis-derived RANKL-targeting protein TsPULP in any of the following aspects.

[0007] 1) Inhibits osteoclast differentiation;

[0008] 2) Prevention and treatment of bone destruction;

[0009] 3) Alleviate bone loss caused by osteoporosis;

[0010] 4) Relieves bone damage caused by rheumatoid arthritis and arthritis.

[0011] Furthermore, the amino acid sequence of the Trichinella spiralis-derived RANKL-targeting protein TsPULP is any one of the following:

[0012] (1) The amino acid sequence as shown in SEQ ID NO.1;

[0013] (2) Proteins with the same function but with one or more amino acid residues substituted and / or deleted and / or added as shown in SEQ ID NO.1;

[0014] (3) An amino acid sequence that can hybridize with the coding sequence of the amino acid sequence shown in SEQ ID NO.1 under moderately stringent conditions and encode a protein with the same function.

[0015] Furthermore, this is achieved by competitively binding the Trichinella spiralis-derived RANKL-targeting protein TsPULP to RANKL and inhibiting the binding of RANK to RANKL.

[0016] Furthermore, the Trichinella spiralis-derived RANKL-binding protein TsPULP inhibits bone resorption or osteoclast activity by suppressing the activation of downstream signaling pathways in osteoclasts and inhibiting the expression of osteoclast-related proteins.

[0017] Application of Trichinella spiralis-derived RANKL-binding protein TsPULP in the preparation of drugs for treating osteoclast-induced bone disease.

[0018] Furthermore, the drug includes drugs that inhibit osteoporosis caused by osteoclasts.

[0019] Furthermore, the drug that inhibits osteoclast-induced osteoporosis is a drug used to treat rheumatoid arthritis and bone-destructive diseases caused by arthritis.

[0020] Application of Trichinella spiralis-derived RANKL-binding protein TsPULP in the preparation of drugs for inhibiting osteoclast differentiation in vitro.

[0021] Furthermore, the Trichinella spiralis-derived RANKL-binding protein TsPULP is used to prepare a drug that inhibits bone loss in vivo due to excessive activation of osteoclasts.

[0022] Furthermore, the method for preparing the Trichinella spiralis-derived RANKL-targeting protein TsPULP is as follows:

[0023] 1) Preparation of Trichinella spiralis excretory and secretory antigen TsMES;

[0024] 2) Based on the number of unique peptides, the number of times unique peptides were detected, and the amino acid coverage, proteins that bind to RANKL in the excretory and secretory antigens of Trichinella spiralis were preliminarily screened, and proteins that can bind to RANKL, such as TsPULP, Uncharacterized protein, and Tubulin alpha chain, were screened.

[0025] 3) Verify the binding affinity of the target proteins TsPULP, Uncharacterized protein, and Tubulinalphachain selected in 2) to RANKL, and confirm that only TsPULP can bind directly to RANKL;

[0026] 4) Extract mRNA from Trichinella spiralis;

[0027] 5) Using the mRNA obtained in step 4) as a template, cDNA is obtained by reverse transcription;

[0028] 6) Using the reverse transcribed cDNA obtained in step 5) as a template, the target gene TsPULP as shown in SEQ ID NO.2 was amplified by PCR. The primer sequences are as follows:

[0029] Upstream primer: F-GGAATTCTTGATTTTTGCTGGAAAGCAATTAGAA (SEQ ID NO.3)

[0030] Downstream primer: R-CCAAGCTTTTACTTCGCAGAGAAATTTCCACGGT (SEQ ID NO.4)

[0031] 7) The target gene obtained in step 6) was digested with XhoⅠ and HindⅢ and then ligated into the PET-28a vector containing the His tag to construct the recombinant vector PET-28a-TsPULP;

[0032] 8) The recombinant vector PET-28a-TsPULP obtained in step 7) was transformed into DH5α competent cells. After sequencing and alignment, a positive plasmid was obtained and then transformed into DE3 competent cells. The cells were screened with 50 ng / ml kanamycin to obtain positive bacteria expressing TsPULP protein.

[0033] 9) Induce expression of the recombinant positive plasmid obtained in step 8);

[0034] 10) The recombinant Trichinella spiralis TsPULP protein expressed by induction was purified to obtain a single Trichinella spiralis-derived RANKL-binding protein TsPULP.

[0035] The beneficial effects of this invention are:

[0036] This invention verifies that TsPULP can effectively bind to human and mouse RANKL and competitively inhibit the binding of RANKL to RANK, thereby reducing osteoclast production and alleviating bone loss in animal models of bone-destructive diseases such as osteoporosis or arthritis, without significant cytotoxicity or hepatotoxicity. TsPULP can be directly expressed and produced in Escherichia coli, is stable, has a high yield, and has low production costs. Attached Figure Description

[0037] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used in conjunction with embodiments of the invention to explain the invention and do not constitute a limitation thereof. In the drawings:

[0038] Figure 1 The results of TsMES protein binding to murine RANKL (mRANKL) are shown below: a. Pulldown assay to verify proteins that can bind to mRANKL as screened by proteomic analysis; b. ELISA assay to verify that the binding of TsPULP to mRANKL is concentration-dependent; c. SPR assay to detect the affinity of TsPULP to mRANKL.

[0039] Figure 2 The results show the binding of TsPULP to human RANKL (hRANKL) in TsMES: a. Pulldown assay to verify proteins that can bind to hRANKL; b. ELISA assay to verify that the binding of TsPULP to hRANKL is concentration-dependent; c. SPR assay to detect the affinity of TsPULP to hRANKL.

[0040] Figure 3 Diagram showing TsPULP competitively inhibiting the binding of RANKL to RANK; a. Schematic diagram of the TsPULP-mRANKL binding complex predicted by AlphaFold2, where red amino acid residues indicate the interaction distance between the two proteins as analyzed by UCSF Chimera. The amino acid residues; b. The structure diagram of the mRANKL-mRANK complex, where the red amino acid residues are those analyzed by UCSF Chimera, indicating that the interaction distance between the two proteins is less than [missing information]. The diagram shows the amino acid residues of mRANKL; c. a combined diagram of mRANKL binding to TsPULP and mRANK, where the red amino acid residues are those where mRANKL binds to both TsPULP and mRANK; d. a schematic diagram of the TsPULP-hRANKL complex predicted by AlphaFold2, where the red amino acid residues are those where the interaction distance between the two proteins is less than [value missing] as analyzed by UCSF Chimera. The amino acid residues; the structural diagram of the e.hRANKL-hRANK complex, where the red amino acid residues are those analyzed by UCSF Chimera, indicating that the interaction distance between the two proteins is less than [missing information]. f. Combined diagram of hRANKL binding to TsPULP and hRANK, where the red amino acid residues are the amino acid residues that hRANKL binds to both TsPULP and hRANK simultaneously; g. Competitive SPR experiment results verifying that TsPULP inhibits the binding of mRANKL to mRANK; h. Competitive SPR experiment results verifying that TsPULP inhibits the binding of hRANKL to hRANK.

[0041] Figure 4The diagram shows the in vitro inhibition of mRANKL-induced osteoclast differentiation in mouse BMDMs by TsPULP; a. Western blot results verifying the expression levels of osteoclast differentiation-related proteins in mouse BMDMs induced by TsPULP inhibition of mRANKL; b. Western blot grayscale value statistics; c. Typical diagram verifying TsPULP inhibition of mRANKL-induced osteoclast formation in mouse BMDMs by TRAP staining; d. Statistics on the number of TRAP-positive cells.

[0042] Figure 5 The diagram shows the in vitro inhibition of hRANKL-induced osteoclast differentiation in human PBMCs by TsPULP; a. Western blot results verifying the expression levels of osteoclast differentiation-related proteins in hRANKL-induced human PBMCs by TsPULP; b. Western blot grayscale statistics; c. Typical diagram verifying the inhibition of hRANKL-induced osteoclast formation in human PBMCs by TRAP staining; d. Statistics on the number of TRAP-positive cells.

[0043] Figure 6 Figures showing the results of TsPULP alleviating bone loss in OVX mice; a. Typical micro-CT scan images of the femur in each group of mice; b. Statistical graph of femoral parameters in each group of mice; c. Typical image of TRAP staining verifying TsPULP inhibiting osteoclast formation in the femur of OVX mice; d. Statistical graph of the number of TRAP-positive cells in the femur of mice.

[0044] Figure 7 Images showing TsPULP alleviating bone erosion in CIA mice; a. Typical micro-CT scan images of small paw bones in each group; b. Statistical graph of paw bone parameters in each group of mice; c. Typical image of TRAP staining verifying TsPULP inhibiting local osteoclast formation in the paws of CIA mice; d. Statistical graph of the number of TRAP-positive cells in mouse paws.

[0045] Figure 8 Figure 1 shows the toxicity test results of TsPULP; a. Effect of CCK8 assay on the viability of mouse BMDMs and human PBMCs; c. Effect of HE staining on mouse liver tissue; d. Effect of HE staining on mouse kidney tissue. Detailed Implementation

[0046] The preferred embodiments of the present invention will be described below with reference to the accompanying drawings. It should be understood that the following embodiments are given for illustrative purposes only and are not intended to limit the scope of the present invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and essence.

[0047] Example 1. Preparation of Trichinella spiralis excretory secretion antigen TsMES

[0048] After euthanizing ICR mice used for breeding, the skin, internal organs, and adipose tissue were removed. The mice were then chopped and placed in a meat grinder with 200 ml of distilled water. The meat was ground for 2 minutes, paused for 1 minute, and repeated three times. The mixture was then poured out, and 1% pepsin and hydrochloric acid were added. After stirring well, the mixture was placed in a 37°C water bath for 4 hours for digestion. After digestion, 1.7 g of sodium chloride was added to stop the process. The mixture was then filtered through a 100-mesh sieve and allowed to stand for 40 minutes to allow the worms to settle to the bottom of the beaker. Suspended impurities were discarded. The worm sediment at the bottom was collected by centrifugation into a 15 ml tube and washed 20 times with 2% penicillin and streptomycin in a biosafety cabinet. The worms were then soaked in 1640 medium at a rate of 3000-5000 worms / ml and incubated at 37°C with 5% CO2 for 48 hours. After 48 hours, the liquid was filtered through a 200-mesh sieve and collected into a 15 ml tube. The mixture was centrifuged at 5000 rpm at 4°C for 10 minutes. The supernatant contained the excretory and secretory antigens. The supernatant was transferred to a 3KD concentration tube and concentrated by centrifugation at 4000 rpm and 4°C for 40 min. The supernatant was then replaced with phosphate-buffered saline (PBS). Concentration was stopped when 1 ml of liquid remained, thus obtaining the Trichinella spiralis excretory secretion antigen.

[0049] Example 2. Screening of mRANKL-binding proteins in Trichinella spiralis excretory and secretory antigens.

[0050] 5 μg of Trichinella spiralis excretory antigen and 5 μg of mouse RANKL were incubated at 4°C for 4-6 h. RANKL antibody was added at a 1:200 ratio, and the mixture was incubated at 4°C for another 4-6 h. Finally, Protein A / G agarose beads were added. After incubation, the mixture was centrifuged at 3000 rpm for 4 min at 4°C. The bottom agarose beads were collected and washed 3-4 times with PBS. SDS-PAGE electrophoresis was then performed, and the gel strips were analyzed for protein spectrometry to determine if any proteins could bind to mouse RANKL. Based on the number of unique peptides, the frequency of detection of unique peptides, and amino acid coverage, several proteins that might bind to mRANKL were preliminarily identified (Table 1). The top three proteins in terms of the combined number of unique peptides, the frequency of detection of unique peptides, and amino acid coverage—Polyubiquitin-like protein, uncharacterized protein, and Tubulin alpha chain—were selected for further identification to determine which single protein could bind to RANKL.

[0051] Table 1

[0052]

[0053] Example 3. Expression and purification of a single Trichinella spiralis-derived protein bound to mRANKL

[0054] First, *Trichinella spiralis* mRNA was extracted using the Trizol method, and cDNA was obtained after reverse transcription. The target genes (Polyubiquitin-like protein, uncharacterized protein, and Tubulin alpha chain) were amplified by PCR, digested with XhoI and HindIII, and ligated into the PET-28a (His-tagged) vector. The recombinant vector PET-28a-Polyubiquitin-like protein, PET-28a-Uncharacterized protein, and PET-28a-Tubulin alpha chain were transformed into DH5α competent cells. Positive plasmids were obtained after sequencing alignment and then transformed into DE3 competent cells. Selection was performed using kanamycin (50 ng / ml) to complete the construction of positive expression bacteria. The positive expression bacteria were inoculated into 1 L LB medium, and the bacterial count was increased to OD100. 600When the concentration is 0.6-0.8, add IPTG, induce at 16℃ for 12-16 h, centrifuge, collect the bacterial pellet, resuspend, sonicate, and centrifuge at high speed. Since all three proteins are inclusion bodies, discard the supernatant, dissolve the protein pellet in binding buffer containing 6M guanidine hydrochloride, sonicate at 30% intensity for 10 min, centrifuge at 12,000 rpm for 30 min, collect the supernatant, and mix with Ni 2+ After incubation in affinity chromatography medium for 2 hours, the protein was washed with 50 mM imidazole and eluted with 500 mM imidazole to obtain the target protein. The protein was then dialyzed overnight at 4°C in 20 mM Tris-HCl, centrifuged at 12,000 rpm for 15 min, and the protein precipitate was collected. The precipitate was then refolded using a protein refolding kit to obtain a soluble protein solution. The purity of the purified target protein was assessed by SDS-PAGE gel electrophoresis, and its concentration was determined by the BCA method.

[0055] Example 4. Expression and purification of mouse and human RANKL and RANK

[0056] (1) Expression and purification of mouse and human RANKL

[0057] Vectors were constructed according to method 3 above. Murine RANKL (mRANKL) and human RANKL (hRANKL) were constructed into the plasmid PEGX-6P-1 (expressing the GST tag), and positive clones were screened for protein expression induction. Expression was induced for 12-16 hours, followed by centrifugation. The bacterial pellet was collected, resuspended in binding buffer, and sonicated. After high-speed centrifugation, the supernatant was collected and incubated with GST-binding resin for 2 hours. The target protein was eluted with elution buffer containing 10 mM reduced glutathione. The protein was then replaced with GST-tagged protease digestion buffer by dialysis. Protease was added at a ratio of 2 U per 100 μg of protein, and digestion was performed at 4°C for 4-8 hours. The protein was then added to glutathione agarose resin for binding, centrifuged, and the supernatant (containing the target protein) was collected. The purity of the purified target proteins (mRANKL and hRANKL) was detected by SDS-PAGE gel electrophoresis, and their concentration was determined by the BCA method.

[0058] (2) Expression and purification of mouse and human RANK

[0059] The murine RANK (mRANK) and human RANK (hRANK) were constructed into the PET-28a vector and expressed according to method 3. Since they are also inclusion bodies, they were purified and refolded according to method 3 to obtain the target proteins (mRANK and mRANKL), and the protein concentration was determined by BCA method.

[0060] Example 5. Verification of the binding of the screened target proteins, polyyubiquitin-like protein, uncharacterized protein, and Tubulin alpha chain, to mRANKL.

[0061] (1) Pull-down experiments to verify the protein that can bind to mRANKL

[0062] The cloned and expressed 5 μg GST-mRANKL fusion protein was bound to glutathione agarose beads at 4 °C for 4–6 h. Then, 5 μg of the top three proteins (Polyubiquitin-like protein, uncharacterized protein, and Tubulin alpha chain) were added and incubated for 2 h. The precipitate was collected by centrifugation, washed three times with PBS to remove non-specifically bound proteins, and then resuspended in 50 μl PBS. After electrophoresis, Western blot was performed to detect whether these three proteins could directly bind to mRANKL. Figure 1 a indicates that only the Trichinella spiralis polyubiquitin-like protein (TsPULP) can directly bind to mRANKL.

[0063] (2) ELISA experiment to verify the binding of TsPULP and mRANKL

[0064] TsPULP protein was coated onto the bottom of ELISA plates at different doses (0, 0.25, 0.5, 1, 2, 4, 8, 16 μg / ml) overnight at 4°C. After washing three times with PBST and blocking, GST-mRANKL was added at a dose of 1 μg / well, along with an equal amount of BSA as a negative control. The plates were incubated at room temperature for 2 h, washed, and then GST antibody was added, followed by HRP-labeled goat anti-mouse secondary antibody. TMB was added for color development for 10-15 min, and stop solution was added to terminate the color development. The absorbance of each group was measured at 450 nm using a microplate reader to observe the changes in the binding ability of mRANK and mRANKL with the increase of TsPULP content. Figure 1 b shows that, compared to the negative control BSA, the absorbance value of GST-mRANKL increased with the increase of the amount of TsPULP protein coated on the bottom of the plate, indicating that the binding of TsPULP to mRANKL is concentration-dependent.

[0065] (3) Observation of the affinity between TsPULP and mRANKL using surface plasmon resonance (SPR) technique

[0066] mRANKL was immobilized on a CM5 chip. TsPULP protein solutions of different concentrations (15.625 nM, 31.25 nM, 62.5 nM, 15.625 nM, 125 nM, 250 nM, and 500 nM) were used as the mobile phase and flowed through the channel surface immobilized with RANKL. The dissociation constant (KD) value (dissociation constant Kd / binding constant Ka) was calculated based on the final change in protein surface refractive index, and the affinity of TsPULP binding was observed. Figure 1 c shows that the affinity between the two is 3.27 nM, indicating that TsPULP and mRANKL have a strong binding force.

[0067] (4) Verify the binding ability of TsPULP and hRANKL.

[0068] 1) Pull-down experiments verify the binding of TsPULP and hRANKL.

[0069] The pull-down test was performed according to the method in Example 5(1). Figure 2 The study showed that TsPULP can bind directly to hRANKL in vitro.

[0070] 2) ELISA experiment to verify the binding of TsPULP and hRANKL

[0071] ELISA detection was performed according to the method in Example 5(2). Figure 2 b shows that the binding of TsPULP to hRANKL is concentration-dependent.

[0072] 3) Surface plasmon resonance (SPR) technique was used to observe the affinity of TsPULP to hRANKL. SPR was performed according to the method in Example 5 (3) to detect the affinity of TsPULP to hRANKL. Figure 2 c shows that the affinity between the two is 62.4 nM, indicating that the binding force between TsPULP and hRANKL is relatively strong.

[0073] Example 6. TsPULP competitively inhibits the binding of human and mouse RANKL to RANK.

[0074] (1) Comparison of RANKL binding sites with TsPULP and RANK

[0075] The amino acid sequences of mRANKL and TsPULP, and hRANKL and TsPULP, respectively, were input into the molecular docking algorithm AlphaFold2 (https: / / colab.research.google.com / github / sokrypton).

[0076] In the file / ColabFold / blob / main / AlphaFold2.ipynbAlphaFold), the protein structure and interaction sites were predicted. The model with the highest score was selected and imported into the UCSF Chimera tool to analyze the interaction distance between the two proteins. The amino acids were identified to predict amino acid sites in RANKL that could interact with TsPULP, and these sites were compared with the resolved structures. Figure 3 The af data showed that both human and mouse RANKL contained sites that simultaneously bind to TsPULP and RANK, indicating that human and mouse RANK can bind to both TsPULP and RANK at the same time.

[0077] (2) Competitive SPR experiments verified the ability of TsPULP to inhibit the binding of RANKL and RANK.

[0078] mRANK or hRANK was immobilized on a CM5 chip. Different concentrations of 0, 0.5 μM, 1 μM, and 2 μM TsPULP 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 through the surface of the channel immobilized with RANK. The ability of TsPULP to competitively inhibit the binding of RANKL and RANK was observed by the change in the RU value of RANKL binding to RANK. Figure 3 The results showed that as the concentration of TsPULP increased, the RU value of human and mouse RANKL binding to RANK decreased, indicating that TsPULP can inhibit the binding of human and mouse RANKL to RANK.

[0079] Example 7. TsPULP inhibits the differentiation of mouse bone marrow macrophages into osteoclasts in vitro.

[0080] (1) Induction of mouse bone marrow macrophages (BMDMs) into osteoclasts

[0081] After euthanizing C57 mice by cervical dislocation, the femur and tibia were harvested. Bone marrow cells were washed away with PBS and placed in DMEM complete medium (containing 1% penicillin and streptomycin and 10% fetal bovine serum) and incubated overnight at 37°C in a 5% CO2 incubator. The next day, the cells were centrifuged at 500g for 5 min, and the supernatant containing adherent cells was collected and placed in DMEM complete medium containing 30 ng / ml M-CSF. The cells were then cultured in an incubator. The following day, 50 ng / ml RANKL was added for induction. The medium was changed every three days, maintaining the concentrations of M-CSF and RANKL. BMDMs were obtained after 5-7 days of induction.

[0082] (2) Western blot verification showed that TsPULP inhibited the activation of the mouse osteoclast signaling pathway.

[0083] The mouse BMDMs obtained above were seeded into 6-well plates. After cell adhesion, 50 ng / ml mRANKL and TsPULP were added. The cells were divided into the following groups: ① PBS group; ② mRANKL group; ③ mRANKL+TsPULP 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 4 ab showed that, compared with the mRANKL group, the levels of osteoclast-related molecules (p-p65, p-iκB, p-p38, p-ERK, p-JNK, NFAT) decreased after the addition of TsPULP, indicating that TsPULP can significantly inhibit the expression of mRANKL-induced osteoclast-related proteins in mouse BMDM.

[0084] (3) TRAP staining to observe the number of osteoclasts formed

[0085] Mature osteoclasts express a large amount of tartrate-resistant alkaline phosphatase (TRAP), which stains purple-red or wine-red upon TRAP staining. Therefore, TRAP staining was used to analyze the effect of TsPULP on osteoclast differentiation in vitro. The mouse bone marrow BMDMs obtained above were processed at 5 × 10⁻⁶... 4 Cells were seeded in 48-well plates and cultured in DMEM complete medium containing 30 ng / ml M-CSF and 50 ng / ml mRANKL, along with TsPULP. Cells were grouped as described above. Cell confluence was observed daily. After 5-7 days of confluence, the supernatant was discarded, and the cells were fixed in 4% paraformaldehyde at room temperature for 10 min. Osteoclasts were stained using a TRAP staining kit, rinsed with double-distilled water, and the nuclei were stained with hematoxylin. After the staining with tap water, the cells were photographed under a light microscope. Cells with three or more nuclei and cytoplasm stained wine-red or purplish-red were identified as osteoclasts. The number of osteoclasts in each well was counted to observe the effect of TsPULP on mRANKL-induced osteoclast formation in mice. Figure 4 The CD results showed that, compared with the mRANKL group, the number of TRPA-positive osteoclasts was significantly reduced after the addition of TsPULP, indicating that TsPULP can significantly inhibit the formation of mouse osteoclasts in vitro.

[0086] Example 8. TsPULP inhibits the differentiation of human peripheral blood mononuclear cells into osteoclasts in vitro.

[0087] (1) Induction of human peripheral blood mononuclear cells (PBMCs) into osteoclasts

[0088] Human peripheral blood was placed in human peripheral blood mononuclear cell separation medium and centrifuged at differential speed (3000 rpm, 30 min, acceleration / deceleration at speed 3) to extract mononuclear cells (PBMCs). CD14 was then extracted from human peripheral blood using the CD14 MicroBead Kit mononuclear cell sorting kit (Miltenyi, Germany) according to the kit instructions via immunomagnetic bead separation. + Mononuclear cells were seeded in cell culture dishes and αMEM complete medium containing 30 ng / ml M-CSF were added to induce them into macrophages.

[0089] (2) Western blot verification showed that TsPULP inhibited the activation of human osteoclast signaling pathway.

[0090] The human macrophages obtained above were seeded into 6-well plates. After cell adhesion, 50 ng / ml RANKL and TsPULP were added. The cells were divided into the following groups: ① PBS group; ② hRANKL group; ③ hRANKL+TsPULP 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 5 ab showed that, compared with the hRANKL group, the levels of osteoclast-related molecules (p-p65, p-iκB, p-p38, p-ERK, p-JNK, NFAT) decreased after the addition of TsPULP, and TsPULP could significantly inhibit the expression of osteoclast-related proteins induced by hRANKL in human PBMCs.

[0091] (3) TRAP staining to observe the number of osteoclasts formed

[0092] The human macrophages obtained above were processed at a ratio of 5 × 10⁶ 4 / wells were seeded in 48-well plates, and DMEM complete medium containing 30 ng / ml M-CSF and 50 ng / ml hRANKL was added. TsPULP was also added, and the cells were grouped as above. The cell fusion status was observed daily. After cell fusion at 10-14 days, TRAP staining was performed according to the method in Example 7(3), and the number of osteoclasts was counted to observe the effect of TsPULP on the differentiation of human peripheral blood mononuclear cells into osteoclasts induced by hRANKL in vitro. Figure 5 The CD results showed that, compared with the hRANKL group, the number of TRPA-positive osteoclasts was significantly reduced after the addition of TsPULP, indicating that TsPULP can significantly inhibit the formation of osteoclasts in human PBMCs in vitro.

[0093] Example 9. TsPULP alleviates bone loss in ovariectomized osteoporotic (OVX) mice.

[0094] Ovariectomized (OVX) osteoporosis model was established in female C57BL / 6 mice using ovariectomy. Mice were randomly divided into four groups of eight mice each: ① Sham group; ② Ovariectomy alone group (OVX group); ③ OVX + TsPULP (1 mg / kg) group; ④ OVX + TsPULP (2 mg / kg) group. Mice in each group were anesthetized with 3% sodium pentobarbital via intraperitoneal injection at a dose of 0.1 ml / 100 g. Skin preparation was performed on the lateral aspect of the back, and the area was disinfected with iodine. In the OVX group, a longitudinal incision of approximately 1 cm was made on the lateral aspect of the back, the skin and muscle were separated, the uterus was ligated at the distal end, and both ovaries were removed. In the sham group, only the adipose tissue surrounding the ovaries was removed, and the mice were sutured and fed routinely for one week. The OVX + TsPULP group received TsPULP via intraperitoneal injection one week later. Mice were sacrificed eight weeks after modeling to observe the effect of TsPULP on bone destruction in osteoporotic mice.

[0095] (1) Micro-CT and 3D modeling techniques were used to observe the degree of bone loss in osteoporotic mice.

[0096] Bilateral femurs of mice were collected and fixed in 4% paraformaldehyde. Micro-CT tomography scans of the fixed femurs were performed using a Scanco Micro-CT 50 imaging system (Scanco, Switzerland). The results were analyzed using 3D analysis software (VGStudioMAX) to measure the bone volume / tissue volume percentage (BV / TV), bone mineral density (BMD), number of trabeculae (Tb.N), and trabecular thickness (Tb.Th) of the femurs in each group of mice. The effect of TsPULP on bone loss in osteoporotic mice was observed. Figure 6 ab showed that, compared with the OVX group, intraperitoneal injection of TsPULP significantly increased BV / TV, BMD, Tb.N, Tb.Th, indicating that TsPULP can significantly alleviate bone loss in OVX mice.

[0097] (2) TRAP staining to detect osteoclast activation level in the femur of osteoporotic mice

[0098] After fixation, the femur was decalcified and prepared into paraffin sections. After dewaxing, the sections were immersed in 0.1M CaCl2-MgCl2 solution to activate tartrate-resistant acid-alkaline phosphatase (TRAP). After rinsing with PBS, the staining solution was prepared according to the TRAP staining kit instructions, and TRAP staining was performed at 37℃ for 45 min. After rinsing with double-distilled water, the nuclei were stained with hematoxylin and then blued with tap water. After drying and mounting, osteoclasts were counted under a light microscope to observe the effect of TsPULP on the number of osteoclasts in osteoporotic mice. Figure 6CD results showed that, compared with the OVX group, intraperitoneal injection of TsPULP significantly reduced the number of osteoclasts in the femur of mice, indicating that TsPULP can inhibit osteoclast formation in OVX mice.

[0099] Example 10. TsPULP alleviates bone destruction in mice with collagen-induced arthritis (CIA).

[0100] Male DBA / 1 mice were randomly divided into four groups: ① Control group; ② CIA group; ③ CIA+TsPULP group (1 mg / kg); ④ CIA+TsPULP group (2 mg / kg). Bovine type II collagen (CII) and complete Freund's adjuvant (CFA) were mixed and emulsified (100 μg CII, 100 μL / mouse), and administered intradermally 1.5 cm from the base of the tail. Day 0 was designated as the first collagen induction. On day 21, an equal volume of incomplete Freund's adjuvant was mixed with CII, and a booster immunization was performed using the same method to establish the CIA model. The CIA+TsPULP group received TsPULP via intraperitoneal injection starting from day 0. After the second booster immunization, the morbidity rate and joint scores of mice in each group were observed every other day. Mice were sacrificed on day 49 to observe the effect of TsPULP on bone erosion in CIA mice.

[0101] (1) Micro-CT and 3D modeling techniques were used to observe the degree of bone destruction in CIA mice.

[0102] Mouse paws were taken, fixed in 4% paraformaldehyde, and Micro-CT scans were performed according to the method in Example 9(1). Bone parameters such as BV / TV and Tb.Th were calculated to observe the effect of TsPULP on bone destruction in CIA mice. Figure 7 ab showed that, compared with the CIA group, intraperitoneal injection of TsPULP significantly increased indicators such as BV / TV and Tb.Th, indicating that TsPULP can significantly alleviate bone destruction in CIA mice.

[0103] (2) TRAP staining to detect the activation level of osteoclasts in the joints of CIA mice

[0104] After the fixed femur was decalcified, it was prepared into paraffin sections and stained with TRAP according to the method in Example 9(2) to observe the effect of TsPULP on the number of osteoclasts in osteoporotic mice. Figure 7 The CD results showed that, compared with the CIA group, the number of osteoclasts in the femur of mice was significantly reduced after intraperitoneal injection of TsPULP, indicating that TsPULP can inhibit the formation of osteoclasts in the joints of CIA mice.

[0105] Example 11. Toxicity detection of TsPULP

[0106] (1) CCK8 assay for TsPULP cytotoxicity

[0107] The mouse BMDM obtained in Example 7 and the human PBMCs obtained in Example 8 were respectively divided into 1×10 4 After being seeded in 96-well plates and allowed to adhere overnight, TsPULP was added at serial dilutions of 0, 1, 2, 4, 8, and 16 μg / ml. After incubation for 24 h, CCK8 reagent was added, and the absorbance was measured at 450 nm using a microplate reader after 4 h to observe the cytotoxicity of TsPULP. Figure 8 The results showed that TsPULP had no significant cytotoxicity against mouse BMDM and human PBMCs.

[0108] (2) HE staining to detect the hepatotoxicity and nephrotoxicity of TsPULP

[0109] To investigate the toxicity of TsPULP to mouse liver and kidney tissues, C57 mice were divided into three groups: PBS group, low-dose TsPULP (1 mg / kg), and high-dose TsPULP (2 mg / kg). TsPULP was injected once daily for 8 weeks. After 8 weeks, the livers and kidneys of the mice were harvested, fixed overnight in 4% paraformaldehyde, embedded, sectioned, and stained with hematoxylin and eosin (HE) to observe the effects of TsPULP on the liver and kidneys of mice. Figure 8 b shows that after intraperitoneal injection of TsPULP, there was no obvious inflammatory cell infiltration in the liver tissue, the hepatocytes were arranged regularly, and the liver lobule structure was normal. There was no significant change compared with the non-injection group. Figure 8 The results showed that after intraperitoneal injection of TsPULP, the renal tubular epithelial cells were neatly arranged, the interstitial tissue showed no edema, fibrosis, or inflammation, and the glomerular basement membrane remained intact without atrophy or sclerosis, showing no significant changes compared to the uninjected group. These results indicate that TsPULP did not exhibit significant hepatotoxicity or nephrotoxicity in vivo and had minimal side effects.

[0110] The contents not described in detail in this specification are existing technologies known to those skilled in the art.

[0111] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Trichinella spiralis-derived RANKL targeting protein Ts The application of PULP in the preparation of drugs for treating rheumatoid arthritis and osteoporosis is characterized by, The Trichinella-derived RANKL targeting protein Ts The amino acid sequence of PULP is shown in SEQ ID NO.1; The drug inhibits bone loss caused by osteoclast differentiation.

2. The application according to claim 1, characterized in that, Through the Trichinella-derived RANKL targeting protein Ts PULP competitively binds to RANKL, the ligand of nuclear factor κB receptor activator, and inhibits the binding of RANK to RANKL.

3. The application according to claim 1, characterized in that, The Trichinella-derived RANKL targeting protein Ts PULP inhibits bone resorption or osteoclast activity by suppressing the activation of downstream signaling pathways in osteoclasts and inhibiting the expression of osteoclast-related proteins.

4. The application according to claim 1, characterized in that, Preparation of Trichinella spiralis-derived RANKL targeting protein Ts The PULP method is as follows: 1) Preparation of Trichinella spiralis excretory secretion antigen TsMES; 2) Based on the number of unique peptides, the number of times unique peptides were detected, and the amino acid coverage, proteins in the excretory and secretory antigens of Trichinella spiralis that bind to RANKL were preliminarily screened, and proteins that can bind to RANKL were further screened. Ts PULP, Uncharacterized protein, Tubulin alpha chain; 3) Validate the target protein screened in 2) Ts The binding affinity of PULP, uncharacterized protein, and Tubulin alpha chain to RANKL was confirmed to be only... Ts PULP can be directly combined with RANKL; 4) Extract mRNA from Trichinella spiralis; 5) Using the mRNA obtained in step 4) as a template, cDNA is obtained through reverse transcription; 6) Using the reverse transcribed cDNA obtained in step 5) as a template, PCR amplify the target gene as shown in SEQ ID NO.

2. Ts PULP; 7) The target gene obtained in step 6) was digested with XhoI and HindIII and then ligated into the His-tagged PET-28a vector to construct the recombinant vector PET-28a- Ts PULP; 8) The recombinant vector PET-28a- obtained in step 7) Ts PULP was transformed into DH5α competent cells, and positive plasmids were obtained after sequencing alignment. These plasmids were then transformed into DE3 competent cells, and selection was performed using 50 ng / ml kanamycin to obtain plasmids expressing the desired plasmids. Ts PULP protein-positive bacteria; 9) Induce expression of the recombinant positive plasmid obtained in step 8); 10) Trichinella spiralis expressed by induction Ts The PULP recombinant protein was purified to obtain a single Trichinella spiralis-derived RANKL-binding protein. Ts PULP.

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