Application of DAAM2 gene in preparing medicine for treating or preventing osteoporosis

Abstract

The present invention belongs to the field of biomedicine, and in particular, relates to an application of a DAAM2 gene in the preparation of a drug for treating or preventing osteoporosis, wherein the nucleotide sequence of the DAAM2 gene is shown in SEQ ID NO: 1, and the amino acid sequence of the protein encoded by the DAAM2 gene is shown in SEQ ID NO: 2. Overexpression of the DAAM2 gene can treat osteoporosis, and experimental verification shows that it exhibits a good bone protection effect in an Ovx mouse model (ovarian osteoporosis mouse model), indicating that it has the effect of treating osteoporosis.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular relates to an application of a DAAM2 gene in preparing a drug for treating or preventing osteoporosis. Background Art

[0002] Osteoporosis is a systemic bone disease caused by a variety of reasons, which leads to decreased bone density and quality, destruction of bone microstructure, increased bone brittleness, and prone to fractures.

[0003] At present, drugs used to treat osteoporosis can be mainly divided into estrogen preparations, bisphosphonate preparations and parathyroid hormone preparations. However, long-term administration of estrogen preparations may increase the incidence of breast cancer, endometrial cancer and cardiovascular diseases; bisphosphonate preparations are prone to gastrointestinal irritation. About 60% of bisphosphonate drugs entering the blood are excreted from the kidneys in their original form, so they should be used with caution in patients with abnormal renal function; and parathyroid hormone preparations generally refer to teriparatide. Although the treatment effect is very good, patients can only receive a 24-month treatment once in their lifetime. This is because teriparatide will play a PTH-like role in the body. When the amount of PTH in the body is too large, it will over-activate osteoclast activity, leading to severe bone loss, thereby aggravating the symptoms of osteoporosis.

[0004] The classical Wnt / β-catenin signaling pathway mainly activates the activity of β-catenin in cells and transfers it to the nucleus, thereby exerting its biological function. The activation of β-catenin in osteoblasts is essential for bone formation. The molecular function of the DAAM2 gene is considered to be a key regulator of the Wnt signaling pathway. It is necessary for various processes during development and plays an important role in the nervous system, such as participating in myelination, acting downstream of Wnt ligands and upstream of β-catenin (CTNNB1). At present, the research on the functional mechanism of DAAM2 is mostly focused on development and the nervous system, and the specific mechanism of DAAM2, a key susceptibility gene, in the process of osteoporosis has not been reported. Therefore, the development of new osteoporosis treatment strategies targeting DAAM2 has great innovation and clinical application prospects.

[0005] Gene therapy refers to a biological treatment method that introduces exogenous normal genes into target cells through gene transfer technology to correct or compensate for diseases caused by gene defects and abnormalities, and ultimately achieve the purpose of treatment. Since gene therapy requires the delivery of corresponding gene fragments to the human body or cells, gene therapy is inseparable from gene delivery technology, that is, vector system. At present, the vector systems used in gene therapy are mainly divided into two categories: viral vectors and non-viral vectors. The choice of viral or non-viral vectors depends largely on the target cells that need gene delivery. Traditional non-viral transfection methods include electroporation, direct injection of plasmids into cells, or the use of transfection reagents, but these strategies are usually limited to in vitro applications and can only transfect a few types of cells to meet smaller experimental scales. For differentiated cells, if cell physiology changes, such as changes in the cytoskeleton, decreased proliferation, and increased nucleases, it will make the cells difficult to be transfected. Today's more cutting-edge transfection method is to use lipid nanoparticles (LNP) to encapsulate plasmids or mRNA for gene delivery. However, this method also has limitations such as difficulty in targeting specific tissues, unstable properties, and short half-life of the delivered genetic material. For difficult-to-transfect cell types, especially in vivo systems, or experiments that require long-term expression of transgenes, viral vectors are a better choice than non-viral vectors.

[0006] Adeno-associated virus (AAV) belongs to the family of Parvoviridae and is a non-enveloped single-stranded linear DNA virus. Only with the assistance of helper viruses such as adenovirus or herpes virus can the host produce infectious AAV, so AAV is called adeno-associated virus. The AAV virus system replaces Rep and Cap with the target gene, and then co-transfects with the plasmid expressing the Rep and Cap genes to produce AAV containing the target gene. Currently, AAV has 12 wild-type serotypes and more than 100 variants. Different serotypes have different affinities for tissues or organs. Due to its high safety, low immunogenicity, and long expression cycle, AAV is considered to be the most suitable tool for studying gene function in vivo. Therefore, it is of great significance to construct a recombinant adeno-associated virus that can overexpress DAAM2 as a drug for the treatment of osteoporosis. Summary of the invention

[0007] In view of the deficiencies in the prior art, the present invention provides an application of the DAAM2 gene in the preparation of a drug for treating or preventing osteoporosis. Overexpression of the DAAM2 gene can treat osteoporosis, and the DAAM2 gene is carried by a recombinant adeno-associated virus that has been modified for bone targeting and targeted to bone cells, so that DAAM2 is overexpressed in bone cells. Experimental verification shows that it exhibits a good bone protection effect in the Ovx mouse model (ovarian osteoporosis mouse model), indicating that it has the effect of treating osteoporosis, providing strong evidence for its application in the preparation of drugs for treating or preventing osteoporosis.

[0008] The technical solution provided by the present invention is as follows:

[0009] The present invention provides an application of a DAAM2 gene in preparing a drug for treating or preventing osteoporosis, wherein the nucleotide sequence of the DAAM2 gene is shown in SEQ ID NO:1, and the amino acid sequence of the protein encoded by the DAAM2 gene is shown in SEQ ID NO:2.

[0010] Furthermore, the DAAM2 gene is carried using a bone-targeted modified adeno-associated virus as a vector.

[0011] Furthermore, the application is to insert the DAAM2 gene into a backbone vector to construct an overexpression vector for overexpressing the DAAM2 gene, and to package the overexpression vector for overexpressing the DAAM2 gene through a bone-targeted modified adeno-associated virus to obtain an adeno-associated virus for overexpressing the gene.

[0012] Furthermore, the backbone vector is a CAG-MCS-SV40 PolyA plasmid vector.

[0013] Furthermore, the method for constructing the overexpression vector for overexpressing the DAAM2 gene comprises the following steps:

[0014] Design primers to obtain the full-length sequence of the DAAM2 gene;

[0015] The DAAM2 gene was inserted between the restriction sites AgeI and SalI of the CAG-MCS-SV40 PolyA plasmid to obtain an overexpression vector for overexpressing the DAAM2 gene.

[0016] Furthermore, the sequence of the primer is:

[0017] Upstream primer: 5'-TTGGATCCCCGGGTACCGGTCGCCACCATGGCCCTCCGCAAGAGGAGTCCTCATG-3',

[0018] Downstream primer: 5′-AAGCGGCCGCCCGGGTCGACTCATTTGTCGTCATCATCCTTATAG-3′.

[0019] Furthermore, the adeno-associated virus is an AAV2 / 9 serotype AAV vector.

[0020] Furthermore, the drug also includes a pharmaceutically acceptable carrier.

[0021] Furthermore, the drug-acceptable carrier includes a diluent.

[0022] Furthermore, the drug is administered by intramuscular injection or intravenous infusion. Beneficial Effects

[0023] The present invention provides an application of a DAAM2 gene in the preparation of a drug for treating or preventing osteoporosis, which can treat osteoporosis. The DAAM2 gene is carried by a recombinant adeno-associated virus modified by bone targeting and targeted to bone cells, so that DAAM2 is overexpressed in bone cells. It has been experimentally verified that it shows a good bone protection effect in the Ovx mouse model (ovarian osteoporosis mouse model), indicating that it has the effect of treating osteoporosis. Compared with the common anti-osteoporosis drugs currently on the market, AAV-DAAM2 does not require frequent administration, greatly improves patient compliance, and reduces adverse reactions caused by multiple administrations, reduces treatment costs, and reduces the economic burden on patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Schematic diagram of the recombinant expression plasmid of the embodiment of the present invention.

[0025] Figure 2 Schematic diagram of in vitro fluorescence imaging according to an embodiment of the present invention.

[0026] Figure 3 Schematic diagram of immunofluorescence staining of frozen sections of mouse bone tissue according to an embodiment of the present invention.

[0027] Figure 4 Schematic diagram of HE staining of mouse bone tissue sections according to an embodiment of the present invention.

[0028] Figure 5 Schematic diagram of Micro-CT of mouse femur according to an embodiment of the present invention.

[0029] Figure 6 This is a Micro-CT analysis diagram of a mouse femur according to an embodiment of the present invention. DETAILED DESCRIPTION

[0030] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of protection defined by the claims of the present invention.

[0031] An embodiment of the present invention provides an application of a DAAM2 gene in the preparation of a drug for treating or preventing osteoporosis, wherein the nucleotide sequence of the DAAM2 gene is shown in SEQ ID NO:1, and the amino acid sequence of the protein encoded by the DAAM2 gene is shown in SEQ ID NO:2.

[0032] In this embodiment, the DAAM2 gene is carried by using a bone-targeted adeno-associated virus as a vector.

[0033] In this embodiment, the application is to insert the DAAM2 gene into the backbone vector to construct an overexpression vector that overexpresses the DAAM2 gene, and to package the overexpression vector that overexpresses the DAAM2 gene through a bone-targeted modified adeno-associated virus to obtain an adeno-associated virus that overexpresses the gene.

[0034] In this embodiment, the backbone vector is a CAG-MCS-SV40 PolyA plasmid vector.

[0035] In this embodiment, the method for constructing the overexpression vector for overexpressing the DAAM2 gene comprises the following steps:

[0036] Design primers to obtain the full-length sequence of the DAAM2 gene;

[0037] The DAAM2 gene was inserted between the restriction sites AgeI and SalI of the CAG-MCS-SV40 PolyA plasmid to obtain an overexpression vector for overexpressing the DAAM2 gene.

[0038] In this embodiment, the sequence of the primer is:

[0039] Upstream primer: 5'-TTGGATCCCCGGGTACCGGTCGCCACCATGGCCCTCCGCAAGAGGAGTCCTCATG-3' (SEQ ID NO. 3),

[0040] Downstream primer: 5′-AAGCGGCCGCCCGGGTCGACTCATTTGTCGTCATCATCCTTATAG-3′ (SEQ ID NO. 4).

[0041] In this embodiment, the adeno-associated virus is an AAV2 / 9 serotype AAV vector.

[0042] In this embodiment, the drug further includes a pharmaceutically acceptable carrier.

[0043] In this embodiment, the carrier accepted by the drug includes a diluent.

[0044] In this embodiment, the drug is administered by intramuscular injection or intravenous infusion. Example 1

[0045] 1. Preparation of recombinant expression plasmid

[0046] 1. Vector digestion

[0047] The vector used in this experiment was CAG-MCS-SV40 PolyA (GV526) ( Figure 1), prepare 50 µL of restriction enzyme system according to Table 1. Add various reagents in the order listed, mix them by gently blowing with a pipette, centrifuge briefly, and react at 37 ℃ for 3h or overnight. Perform agarose gel electrophoresis on the vector digestion products and recover the target band. Note: The optimal reaction temperature of most restriction endonucleases is 37 ℃, but some are not 37 ℃, such as Apa I at 25 ℃ and Bsl I at 75 ℃. Determine the corresponding reaction temperature according to the required restriction endonuclease. The restriction endonuclease should have a reaction activity of not less than 50% in the selected buffer. The specific activity of various restriction endonucleases in different buffers should refer to their instructions. The GV526 vector used in this experiment was digested by AgeI / SalI.

[0048]

[0049] 2. Acquisition of target gene fragments

[0050] 2.1 Primers

[0051]

[0052] 2.2. PCR amplification of target gene fragments

[0053] Prepare the reaction system as shown in Table 2, mix gently by pipetting, centrifuge briefly, and place in a PCR instrument for reaction.

[0054]

[0055] The reaction conditions are as follows:

[0056]

[0057] Note: The annealing temperature depends on the GC content of the primer or gene. The annealing temperature is generally set 5°C lower than the Tm of the primer. The extension time depends on the length of the PCR product. The extension time of PrimeStar HS DNA polymerase is calculated based on 1Kb / min.

[0058] 2.3 Purification of target fragment

[0059] The amplified products were analyzed by 1% agarose gel electrophoresis to check the reaction products and length, and then the amplified products were recovered and purified.

[0060] 2.4 Connection of target fragments

[0061] Prepare the reaction system shown in Table 3 in an ice-water bath. Mix by gently pipetting and centrifuge briefly to avoid bubbles. React at 37 °C for 30 min, then cool in an ice-water bath for 5 min and transform immediately.

[0062]

[0063] Note: a) The optimal molar ratio of the added linearized vector DNA and the purified PCR product is 1:2; b) The purified PCR product added to the positive control is the GAPDH gene (with the same exchange arm).

[0064] 2.5 Conversion

[0065] Add 10 µL of the exchange reaction product to 100 µL of competent cells, flick the tube several times to mix, and place on ice for 30 min. Heat shock at 42 °C for 90 s, and incubate in an ice-water bath for 2 min. Add 500 µL of LB medium and place on a 37 °C shaker for 1 h. Take an appropriate amount of bacterial solution and evenly spread it on a plate containing the corresponding antibiotics, and invert and culture it in a constant temperature incubator for 12-16 h.

[0066] 2.6 PCR identification

[0067] Prepare the reaction system as shown in Table 4, shake and mix, and centrifuge briefly. In the clean bench, use a sterile pipette tip to pick up a single colony and put it into 20 µL identification system, pipette and mix, and place it in a PCR instrument for reaction.

[0068]

[0069] PCR reaction conditions are as follows:

[0070]

[0071] Note: 1. Annealing temperature is determined by the GC content of the primer or gene. Annealing temperature is generally set 5 ℃ lower than the Tm of the primer. 2. Extension time is determined by the length of the identified PCR product. The extension time of Taq Plus DNA polymerase is calculated at 1Kb / min. Take 5µL of the amplified product and perform electrophoresis analysis on 1% agarose gel to check the reaction product and length.

[0072] 2.7 Sequencing

[0073] The identified positive clone transformants were inoculated into an appropriate amount of LB liquid culture medium containing the corresponding antibiotics, cultured at 37°C for 12-16 hours, and an appropriate amount of bacterial liquid was taken for sequencing. The sequencing results were compared with the target gene sequence. The comparison result shows that the sequencing results are completely consistent with the target sequence.

[0074] 2. Preparation of pAAV-RC Virus

[0075] Adeno-associated virus type 2 / 9 (rAAV2 / 9) was modified to obtain bone-targeted virus. As briefly described, the DNA sequence encoding the basic sequence of the bone-targeted peptide DSS (AspSerSer) 6 was codon-optimized and then inserted into the codon of the AAV9 Cap gene in the pAAV2 / 9 plasmid to obtain the Q588 capsid (DSS-588). The DSS-Nter capsid was then constructed. First, the start codon of VP2 in pAAV2 / 9 was mutated (ACG→ACC) to express only VP1 and VP3 (pAAV2 / 9.novp2). In another plasmid, the DSS sequence was fused to the N-terminus of the AAV9-VP2 ORF, and the Kozak sequence and ATG start codon were placed directly upstream of the DSS sequence, allowing the CMV promoter to drive expression (pcDNA.DSS-VP2 (AAV9)). The constructed pAAV2 / 9.novp2 and pcDNA.DSS-VP2 (AAV9) were used for the production of rAAV.

[0076] 3. Bone-targeted adeno-associated virus packaging and overexpression of DAAM2 gene

[0077] Step 1: Co-transfect the recombinant expression plasmid with pAAV-RC (carrying AAV replication and capsid genes, here the modified rAAV2 / 9-DSS-Nter) and pHelper (carrying genes derived from adeno-associated virus) into AAV-293 cells. Recombinant AAV is assembled in the packaging cells 2 to 3 days after transfection.

[0078] The specific operations are as follows:

[0079] Transfection of AAV-293 cells

[0080] In this experiment, when AAV-293 cells were transfected by calcium phosphate transfection, a product titer of >10 7 viral particles / mL was stably obtained. Note: After the transfection reagents prepared in this step are mixed, they should be added to the cell culture plate immediately. Leaving them at room temperature for too long will form large particles that are not conducive to cell phagocytosis.

[0081] 1.1. Check the host cells passaged two days ago before transfection. They should reach 70-80% confluence.

[0082] 1.2. Take out the plasmid to be co-transfected from the -20 ℃ refrigerator and adjust the concentration of the plasmid to 1 mg / mL with pH 7.5 TE buffer;

[0083] 1.3 Calculate the required transfection system and plasmid dosage according to the number of packaging plates. If packaging one plate, pipette 10 µL of each of the three plasmids into a 1.5 mL EP centrifuge tube, and then add 1 mL of 0.3M CaCl2. Mix gently;

[0084] 1.4. Pipette 1 mL of 2X HBS solution into another 15 mL conical tube. Add 1.03 mL of DNA / CaCl2 mixture (from the previous step) to it. Mix gently by turning over or repeatedly pipetting;

[0085] 1.5. Immediately add the mixed DNA / CaCl2 / HBS solution to the cell culture plate. Gently shake the cell culture plate while adding it to make the solution as evenly distributed as possible in the culture medium;

[0086] 1.6. Return the cell culture dish to the 37°C incubator for 6 hours;

[0087] Note: A negative control for viral packaging is usually set up here, which can be achieved by replacing one of the three plasmids used in transfection with TE buffer.

[0088] 1.7. After transfection, replace the culture medium in the dish with 10 mL of fresh culture medium;

[0089] 1.8. Return the culture plate to the incubator and incubate for another 66-72 hours.

[0090] Step 2: AAV viral particles are collected from infected AAV-293 cells, concentrated and purified, and desalted by ultrafiltration.

[0091] The specific operations are as follows:

[0092] 1. Harvest the virus

[0093] The extent of AAV particle packaging can be determined by observing the morphological changes of AAV-293 cells. As the virus production progresses, some cells will round up and fall off the plate, and they can be seen floating in the culture medium. Generally speaking, three days after transfection is the best time to harvest the virus.

[0094] 1.1 Prepare a dry ice ethanol bath (pour ethanol into a foam box filled with dry ice, or use liquid nitrogen instead of a dry ice ethanol bath) and a 37°C water bath;

[0095] 1.2 Collect the toxin-producing cells and the culture medium into a 15 mL centrifuge tube. When collecting the cells, tilt the culture plate at a certain angle to scrape the cells into the culture medium;

[0096] 1.3 Centrifuge at 200 × g for 3 minutes to separate cells and supernatant. Store the supernatant separately and resuspend the cells in 1 mL PBS.

[0097] 1.4 Transfer the cell suspension repeatedly between a dry ice ethanol bath and a 37°C water bath, and freeze and thaw four times. Shake slightly after each thawing. Note: Each solidification and thawing takes about ten minutes.

[0098] 1.5 Centrifuge at 10,000 × g to remove cell debris and transfer the supernatant to a new centrifuge tube.

[0099] 2. Concentration of AAV virus

[0100] 2.1 Add 40% PEG8000 to the supernatant until the final concentration is 8%. After placing on ice for 2 hours (mixing every 15 minutes), centrifuge at 2,500×g for 30 minutes. Remove the supernatant, resuspend the precipitate with PBS and combine it with the cell lysis supernatant;

[0101] 2.2 Centrifuge at 3,000 × g for 30 minutes and transfer the supernatant to another clean tube. There should be no visible cell debris in the supernatant. If there is still some debris, centrifuge again;

[0102] 2.3 Add Benzonase nuclease to digest and remove residual plasmid DNA (final concentration is 50 U / mL). Close the tube cap and invert several times to mix thoroughly. Incubate at 37°C for 30 minutes;

[0103] 2.4 Filter using a 0.45 μm filter and collect the filtrate.

[0104] 3. Purification of AAV

[0105] 3.1 Add solid CsCl to the virus concentrate until the density is 1.41 g / mL (refractive index is 1.372), which is about 6.5 g CsCl added to 10 mL of virus solution. Shake it to dissolve. CsCl will absorb heat and generate cold when it dissolves.

[0106] 3.2 Add the sample to the ultracentrifuge tube and fill the remaining space of the tube with the pre-prepared 1.41 g / mL CsCl solution;

[0107] 3.3 Centrifuge at 175,000 × g for 24 hours to form a density gradient. Collect samples of different densities step by step in sequence and take samples for titer determination. Collect the fractions enriched with AAV particles;

[0108] 3.4 Repeat the above process once.

[0109] 4. Ultrafiltration desalination

[0110] 4.1 Elution: Add 4 mL of deionized water to the Amicon-15 ultrafiltration device;

[0111] 4.2 Add the virus solution obtained by density gradient centrifugation to the ultrafiltration device. Add PBS to a total volume of 4 mL and cover the lid;

[0112] 4.3 Centrifuge at 1,500 × g for approximately 5 to 10 minutes, checking the residual volume every 5 minutes until the final volume is 200-250 µL;

[0113] 4.4 Collect the filtrate together for disinfection and put the filter membrane back into the device;.

[0114] 4.5 Add 1× PBS to dilute the concentrated virus to a volume of 4 mL;

[0115] 4.6 Repeat the above process 3 times;

[0116] 4.7 Centrifuge the ultrafiltration tube to a final volume of approximately 0.5 mL;

[0117] 4.8 Add glycerol to the virus concentrate to make the final concentration 5%. Store at -80℃ after aliquoting.

[0118] Step 3: Determine the titer of the obtained virus using quantitative PCR

[0119] The specific operations are as follows:

[0120] The viral titer was determined by quantitative PCR to detect the genome copy number of the AAV vector to determine the number of AAV viral particles.

[0121] 1.1 Prepare samples and standards, and dilute the standard plasmid and the sample to be tested to 10% of the original concentration. -5 , 10 -6 , 10 -7 , 10 -8Second-rate;

[0122] 1.2 Calculate the total volume of the reaction tube according to the number of reactions (X) (make two replicate wells for each gradient, and prepare one more well for every 10 reactions), and configure the reaction system as shown in Table 5:

[0123]

[0124] 1.3 Add 15 µL of reaction solution to each reaction well, and then add 5 µL of template;

[0125] 1.4 On the machine, set the annealing temperature to 60°C, obtain the Ct value and calculate the copy number in the AAV sample according to standard procedures.

[0126] Example 2 Bone targeting verification

[0127] In order to verify the effectiveness of bone-targeted virus in infecting bone cells, bone-targeted EGFP-encapsulated virus (AAV9-DSS-Nter-EGFP) was constructed according to the above experimental method. It was injected into mice of the same age and treated in the same way in the previous period through the tail vein at a concentration of 4×10 11 . Two weeks later, the mice were subjected to in vivo fluorescence detection and GFP immunofluorescence staining of frozen sections of bone tissue to determine whether the bone-targeted virus can reach bone cells and its efficiency.

[0128] In vivo fluorescence detection revealed that, except for liver tissue, which was highly enriched due to its own metabolic characteristics, the fluorescence intensity in bone tissue was significantly higher than that in the PBS injection group (Figure 2). Immunofluorescence detection of frozen sections of bone tissue revealed that after the bone-targeted virus was injected, the level of EGFP expression in bone cells was significantly higher than that in the PBS injection group (Figure 3). This indicates that the constructed bone-targeted virus can significantly act on bone cells and has the function of targeting bone cells.

[0129] Example 3 Anti-osteoporosis effectiveness verification

[0130] In order to determine whether bone-targeted DAAM2 has anti-osteoporosis effects, Ovx osteoporosis model mice were selected for tail vein injection at a concentration of 4×10 11 After 2 weeks, HE staining of bone tissue sections and Micro-CT histological evaluation of bone tissue were performed to determine whether bone-targeted encapsulation of DAAM2 has the effect of treating osteoporosis during aging.

[0131] The constructed bone-targeted encapsulated DAAM2 was injected into Ovx mice through the tail vein. Compared with the control group, the bone tissue sections of the AAV-DAAM2-injected mice showed thicker trabeculae and fewer fat droplets after 2 months (Figure 4). MicroCT analysis found that AAV-DAAM2 injection significantly increased the bone density of mice (Figure 5), and at the same time had a significant mitigating effect on bone microstructure. The mouse bone volume fraction (BV / TV), trabecular number (Tb.N), and trabecular thickness (Tb.Th) increased, and the trabecular spacing (Tb.Sp) decreased (Figure 6). Therefore, the present invention can have the effect of targeted overexpression of the DAAM2 gene in osteocytes, thereby promoting bone formation.

[0132] In summary, the present invention has the effect of targeting bone cells by modifying AAV9, and after encapsulating the DAAM2 plasmid, it has the effect of significantly increasing the expression level of the DAAM2 gene in bone cells and promoting bone formation, thereby having an anti-osteoporosis effect.

[0133] The above experimental examples are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention may be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be included in the scope of the claims of the present invention.

[0134] The nucleotide sequence of DAAM2 is shown as follows:

[0135]

[0136] The amino acid sequence of DAAM2 is shown below in SEQ ID NO. 2:

[0137]

[0138] The primer sequences are as follows:

[0139] Upstream primer:

[0140] Daam2(90525-1)-p1:5'-TTGGATCCCCGGGTACCGGTCGCCACCATGGCCCTCCGCAAGAGGAGTCCTCATG-3' (SEQ ID NO.3);

[0141] Downstream primer:

[0142] Daam2(90525-1)-p2:5'-AAGCGGCCGCCCGGGTCGACTCATTTGTCGTCA TCATCCTTATAG-3' (SEQ ID NO 4).

Claims

1. A use of DAAM2 gene in preparing a drug for treating or preventing osteoporosis, characterized in that: The nucleotide sequence of the DAAM2 gene is shown in SEQ ID NO:1, and the amino acid sequence of the protein encoded by the DAAM2 gene is shown in SEQ ID NO:2; the DAAM2 gene is carried by using a bone-targeted adeno-associated virus as a vector; the application is to insert the DAAM2 gene into a backbone vector to construct an overexpression vector for overexpressing the DAAM2 gene, and to package the overexpression vector for overexpressing the DAAM2 gene by the bone-targeted adeno-associated virus to obtain the adeno-associated virus for overexpressing the gene; the backbone vector is a CAG-MCS-SV40 PolyA GV526 plasmid vector; the method for constructing the overexpression vector for overexpressing the DAAM2 gene comprises the following steps: Design primers to obtain the full-length sequence of the DAAM2 gene; The DAAM2 gene was inserted between the restriction sites AgeI and SalI of the CAG-MCS-SV40 PolyA GV526 plasmid to obtain an overexpression vector for overexpressing the DAAM2 gene; The sequences of the primers are: Upstream primer: 5'-TTGGATCCCCGGGTACCGGTCGCCACCATGGCCCTCCGCAAGAGGAGTCCTCATG-3', Downstream primer: 5'-AAGCGGCCGCCCGGGTCGACTCATTTGTCGTCATCATCCTTATAG-3'; the adeno-associated virus is an AAV2 / 9 serotype AAV vector.

2. The use according to claim 1, characterized in that: The drug further includes a pharmaceutically acceptable carrier.

3. The use according to claim 2, characterized in that: The pharmaceutically acceptable carrier includes a diluent.

4. The use according to claim 3, characterized in that: The drug is administered by intramuscular injection or intravenous infusion.

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