CN116058335A

CN116058335A - Construction method and application of spontaneous ankylosing spondylitis model

Info

Publication number: CN116058335A
Application number: CN202211496951.3A
Authority: CN
Inventors: 王晓建; 马铭婕; 杨伟宪; 李天骐; 纪爽; 俞莉萍
Original assignee: Fuwai Hospital of CAMS and PUMC
Current assignee: Fuwai Hospital of CAMS and PUMC
Priority date: 2022-11-25
Filing date: 2022-11-25
Publication date: 2023-05-05

Abstract

The invention discloses a construction method and application of a spontaneous ankylosing spondylitis model, wherein the ankylosing spondylitis model is a non-human animal model, and the spontaneous ankylosing spondylitis non-human animal model is constructed by expressing mutant BMP9 protein. The invention also provides methods for identifying a therapeutic agent for treating ankylosing spondylitis in a non-human animal model of spontaneous ankylosing spondylitis. The invention also provides the use of a non-human animal model of spontaneous ankylosing spondylitis in assessing the effect of a therapeutic agent in the treatment or prevention of ankylosing spondylitis.

Description

Construction method and application of spontaneous ankylosing spondylitis model

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to a construction method and application of a spontaneous ankylosing spondylitis model.

Background

Ankylosing spondylitis (ankylosing spondylitis, AS) is a systemic disease that is dominated by chronic inflammation of the medial axis joint, mainly involving the sacroiliac joint and the spinal joint, and advanced stages can lead to joint deformity and "bamboo-like" changes in the spine, causing serious physical deformity and disability. The prevalence of AS in China is about 0.3%, and the number of patients is estimated to be more than 400 ten thousand. AS mostly developed in young and young (20-40 years), the average age of onset of chinese patients is 29.2 years, with men being higher than women (men: women=2.8:1). AS is a chronic progressive disease, and existing drugs cannot block the disease process. Once a patient develops a disease, the patient often loses work capacity gradually over a period of ten years. Early onset, high onset and high disability of AS bring great pain to patients and families, and become a heavy health burden of the society of China.

AS mainly involves the axises and joints, and human tissues are difficult to obtain. Therefore, AS experimental animal models are extremely important for our deep understanding of pathogenesis and development of new therapeutic strategies. In the past two decades, researchers have constructed a variety of AS-related animal models, including HLA-B27 transgenic rat/mouse models, inflammation-related models, ankylosing spondylitis models, and the like. These experimental animal models make an important contribution to our understanding of the pathological mechanisms of AS complex diseases, but there are still drawbacks and limitations. Taking the most widely used AS animal model HLA-B27/h beta 2m double transgenic rat AS an example, the model can generate the similar spondylitis and hindpaw arthritis AS human AS, but has the problems of long modeling period (the rat needs to be fed until 7-9 months of age), low modeling rate (the rat generates the spondylitis only by 30-50% at 9 months of age), light symptoms, clinical ankylosing spondylitis and the like. To date, no animal model has been able to fully mimic the clinical manifestations and pathological progression of human AS. Therefore, it is important to accelerate the construction of experimental animal models which spontaneously produce AS and are more pathologically close to clinical patients.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a method for constructing an animal model of spontaneous ankylosing spondylitis and application thereof.

In order to achieve the above purpose, the present invention adopts the following technical scheme:

in a first aspect, the invention provides a method of constructing a non-human animal model of spontaneous ankylosing spondylitis, which expresses a mutated BMP9 protein.

Further, the non-human animal model refers to a non-human animal that has or exhibits characteristics of a disease or condition.

Further, the amino acid sequence of the mutant BMP9 protein is shown as SEQ ID NO. 1.

Further, the method for expressing mutated BMP9 protein in ankylosing spondylitis non-human animal model comprises the following steps: introducing a nucleic acid molecule encoding said mutated BMP9 protein into a single cell embryo or embryonic stem cell of a non-human animal.

Further, the method for expressing mutated BMP9 protein in ankylosing spondylitis non-human animal model comprises the following steps: introducing an expression vector comprising a nucleic acid molecule of said mutated BMP9 protein into a single cell embryo or embryonic stem cell of a non-human animal.

Further, the nucleic acid molecule comprises the following sequence: a nucleotide sequence formed by mutating 948 th nucleotide from G to T on a protein Coding region (CDS) of a wild type BMP9 gene (gene ID: 2658).

Further, the expression vectors include, but are not limited to, linear polynucleotides, plasmids, and viral vectors.

Further, the viral vectors include, but are not limited to, lentiviral vectors, retroviral vectors, adenoviral vectors, adeno-associated viral vectors.

Further, the expression vector introduction method includes electroporation, calcium phosphate method, liposome method, DEAE dextran method, microinjection, viral infection or liposome transfection.

Further, the non-human animal is a rodent.

Further, the rodent includes the following options:

1) The hamster family such as hamster, barter, gu Shili hamster, cricetin, hao Shili hamster, barin, chu Shili hamster, glatiramer hamster;

2) Hamster family such as black line hamster a field mouse and a crown mouse;

3) Murine species such as black, brown, sandy, new world, old world, norway species, borilysia, tree, cotton, wood, stick, rice, kangaroo, and climbing;

4) The equine island murine species such as longtail giant mice, south african cyst mice, african giant mice, ma Daobai tail mice;

5) The spiny mouse is selected from spiny mouse and pig tail mouse;

6) Mole murine such as mole, bamboo rat, zokor;

7) Acanthaceae such as acanthus;

8) The family of the species Amanidae, such as African rats.

Further, the ankylosing spondylitis non-human animal model exhibits one or more symptoms of scoliosis, sternal herniation, joint stiffness, tail stiffness, anterior limb deformity, posterior limb sacroiliac joint deformity, spinal hyperosteogeny, spinal fusion, significant reduction in bone density, and significant reduction in bone trabecula.

In a second aspect, the invention provides a cell line derived from a non-human animal model expressing mutant BMP9 protein ankylosing spondylitis prepared by the construction method described in the first aspect of the invention.

In a third aspect, the invention provides an embryonic stem cell derived from a ankylosing spondylitis non-human animal model prepared by the construction method of the first aspect of the invention.

In a fourth aspect, the invention provides a method of identifying a therapeutic agent for the treatment of ankylosing spondylitis, the method comprising the steps of:

1) The medicament is administered to the ankylosing spondylitis non-human animal prepared by the construction method described above.

2) One or more assays are performed to determine whether the agent has a therapeutic effect on one or more abnormal symptoms associated with ankylosing spondylitis.

3) The agent is identified as a therapeutic agent when the agent has a therapeutic effect on one or more abnormal symptoms associated with ankylosing spondylitis.

Further, the therapeutic agents include non-steroidal anti-inflammatory agents, hormonal agents, targeted small molecule agents, proteasome inhibitors, immunosuppressants, tumor necrosis inhibitors, cytokines, activators of co-stimulatory molecules, inhibitors of inhibitory molecules.

In a fifth aspect, the present invention provides the use of a ankylosing spondylitis non-human animal model, which is a model prepared using the construction method described above, in the screening of a medicament for the treatment or prevention of ankylosing spondylitis.

Further, the medicament includes one or more pharmaceutically acceptable excipients.

Further, the excipient includes binders, fillers, disintegrants, lubricants, ointments, preservatives, antioxidants, flavoring agents, fragrances, co-solvents, emulsifiers, solubilizers, osmotic pressure regulators, colorants.

In a sixth aspect, the present invention provides the use of a ankylosing spondylitis non-human animal model, which is a model prepared using the construction method described above, for assessing the therapeutic effect of a product for the treatment of ankylosing spondylitis.

The seventh aspect of the invention provides application of a mutated BMP9 protein or a nucleotide sequence for synthesizing the mutated BMP9 protein in constructing a non-human animal model of spontaneous ankylosing spondylitis, wherein the amino acid sequence of the mutated BMP9 protein is shown as SEQ ID NO. 1.

Further, the nucleotide sequence of the synthetic mutant BMP9 protein comprises the sequence: the 948 th nucleotide of CDS sequence of wild BMP9 gene is changed from G to T.

The invention has the beneficial effects that:

the invention provides a ankylosing spondylitis non-human animal model, which has clear genetic background and is closer to the real symptoms of diseases, solves the problem of larger difference between the traditional animal model and clinical diseases, and has important significance for clinical research of ankylosing spondylitis.

Drawings

FIG. 1 is a schematic representation of the translation, cleavage, maturation process and mutation positions of BMP9 protein; wherein A: BMP9 protein translation, cleavage, and maturation; b: different mutated positions of BMP 9;

FIG. 2 is a diagram showing Western blot detection of the expression of BMP9 protein by 6 mutant plasmids; wherein A: the condition that the BMP9 protein is expressed by 6 mutant plasmids in the cell lysate; b: the condition that the BMP9 protein is expressed by 6 mutant plasmids in cell supernatant;

FIG. 3 is a diagram of PCR identification of Tg-BMP9-MUT rat genotypes; wherein, NC: a negative control; PC: a positive control; m: a nucleic acid marker; BLK: blank control; tg-MUT: tg-BMP9-MUT mutant transgenic positive rats;

FIG. 4 is a graph of the results of a gene sequencing of the Tg-BMP9-MUT rat, wherein the red arrow points to the mutation site BMP9 c.948G > T, p.Arg316Ser;

FIG. 5 is a graph showing Western Blot results of BMP9 expression in lung and liver tissues of WT rats and Tg-BMP9-MUT rats, wherein A: two rat lung tissue Western Blot results, B: western Blot results of two rat liver tissues;

FIG. 6 is a dorsal view of WT rats, BMP9 knockout rats (BMP 9-KO), wild type BMP9 overexpressing rats (Tg-BMP 9), tg-BMP9-MUT overexpressing male rats, wherein A: WT rat, B: BMP9 knockout rat (BMP 9-KO), C: wild-type BMP9 overexpressing rats (Tg-BMP 9), D: tg-BMP9-MUT overexpressing male rats;

FIG. 7 is a chest diagram of WT rats, tg-BMP9-MUT overexpressing male rats, wherein A: WT rat, B: tg-BMP9-MUT overexpressing male rats;

FIG. 8 is a graph of WT rats, tg-BMP9-MUT overexpressing male rat tail, wherein A: WT rat, B: tg-BMP9-MUT overexpressing male rats;

FIG. 9 is a foot map of WT rats, tg-BMP9-MUT overexpressing male rats, wherein A: WT rat, B: tg-BMP9-MUT overexpressing male rats;

FIG. 10 is a side view of a CT 3D modeling of the whole body bone of a WT rat, a male rat overexpressing Tg-BMP9-MUT, wherein A: WT rat, B: tg-BMP9-MUT overexpressing male rats;

FIG. 11 is a top view of a CT 3D model of the whole body bone of a WT rat, a Tg-BMP9-MUT overexpressing male rat, wherein A: WT rat, B: tg-BMP9-MUT overexpressing male rats;

FIG. 12 is a CT 3D modeling of the spinal column of WT rats, tg-BMP9-MUT overexpressing male rats, wherein A: WT rat, B: tg-BMP9-MUT overexpressing male rats;

FIG. 13 is a 3D image of tibial trabecular bone of a WT rat, tg-BMP9-MUT overexpressing male rat, wherein A: WT rat, B: tg-BMP9-MUT over-expressed male rats.

Detailed Description

The invention will be described in further detail below with the understanding that the terminology is intended to be in the nature of words of description rather than of limitation.

The terms "nucleic acid sequence" or "polynucleotide" are used interchangeably herein and refer to a nucleic acid molecule, DNA or RNA containing deoxyribonucleotides or ribonucleotides, respectively. The nucleic acid may be double-stranded, single-stranded, or comprise portions of double-stranded or single-stranded sequences.

The term "expression" refers to the process of transcription of a polynucleic acid into mRNA and translation into a peptide, polypeptide or protein. If the polynucleic acid is derived from genomic DNA and a suitable eukaryotic host cell or organism is selected, expression may involve splicing of mRNA.

The term "nucleic acid" includes ribonucleic acid (RNA) or deoxyribonucleic acid (DNA), which may be complementary DNA (cDNA) or genomic DNA.

The term "treatment" refers to administration of a compound or composition to control the progression of a disease. Control of disease progression is understood to achieve beneficial or desired clinical results including, but not limited to, alleviation of symptoms, diminishment of disease duration, stabilized pathological states (particularly avoiding additional exacerbations), delay of disease progression, amelioration and remission (both partial and total) of the pathological state. Control of disease progression also involves an increase in survival compared to the expected survival without treatment.

The present invention provides novel non-human animal models of ankylosing spondylitis. In fact, the inventors have discovered a new animal model of ankylosing spondylitis that reproduces the central features of human diseases.

The term "non-human animal" includes non-human vertebrates, more preferably mammals, such as domestic livestock (e.g., cattle, horses, pigs), pets (e.g., dogs, cats), or rodents. The term "rodent" refers to any and all members of a phylogenetic rodent (e.g., mice, rats, squirrels, beasts, woodchuck, ground rats, field rats, woodchuck, hamsters, guinea pigs, and thorn guinea pigs), including any offspring of all offspring derived therefrom.

The term "non-human animal model" refers to a non-human animal that has or exhibits characteristics of a disease or condition. By animal model is meant any use of the animal for studying a disease or condition, for example for studying progression or development or response to a new therapy or an existing therapy.

The gene of bone morphogenetic protein 9 (BMP 9, also known as growth differentiation factor 2, gdf 2) is located on the 10q11.22 chromosome and has a gene ID of 2658, including genes and encoded proteins and homologs thereof, mutations, and the like. The term "BMP9" encompasses full length, unprocessed genes or proteins, as well as any form of genes or proteins derived from processing in a cell. The term encompasses naturally occurring variants of the biomarker. The gene ID is available at https:// www.ncbi.nlm.nih.gov/gene.

The mutant BMP9 protein is protein expressing the amino acid sequence shown in SEQ ID No. 2, the nucleic acid molecule for synthesizing the mutant BMP9 protein is formed by changing the 948 th base of the CDS sequence of the wild BMP9 from G to T, the mutant BMP9 is a human mutant, and the mutant BMP9 is synthesized artificially.

The term "expression vector" as used herein refers to expression vectors that may contain regulatory sequences and coding sequences derived from different sources, or regulatory sequences and coding sequences derived from the same source but arranged in a manner different from that found in nature, either alone or in combination. The genetic elements of host cells that must be present on a vector to successfully transform, select and propagate a nucleic acid fragment comprising a mutant gene according to the invention are well known to those skilled in the art. The expression vector may comprise any combination of deoxyribonucleotides, ribonucleotides, or modified nucleotides, the expression vector may be transcribed to form RNA, wherein the RNA may be capable of forming double-stranded RNA and/or hairpin structures, the expression vector may be expressed in a cell, or isolated, or synthetically produced, the expression vector may further comprise a promoter or other sequence that aids in manipulation or expression of the construct.

In some embodiments, the nucleic acid sequence is operably linked to an expression vector. The expression vectors are already available commercially, such as some viral vectors, plasmids, phages.

In a preferred embodiment, the expression vector is a plasmid selected from plasmids conventionally used in the art for constructing transgenic constructs, typically having a "spacer" and multiple cloning sites or alternative sequences located on either side of the "spacer" such that one can insert the corresponding DNA sequence of the gene into the multiple cloning site or alternative sequences on it in a forward and reverse manner. The expression vector typically also contains a promoter, origin of replication, and/or marker gene, etc.

In a specific embodiment of the invention, the plasmid is pcDNA3.1-ALB.

Alternatively, the expression vector may be introduced into the cell by known methods such as electroporation, calcium phosphate, liposome, DEAE dextran, microinjection, viral infection, liposome transfection, and binding to a cell membrane permeable peptide.

In a specific embodiment of the invention, the introduction of the expression vector into the cell is by microinjection.

Alternatively, the non-human animal is a rodent.

In some embodiments, rodents of the present disclosure include, as non-limiting examples, mice, rats, and hamsters. In some embodiments, rodents of the present disclosure include, as non-limiting examples, mice and rats. In some embodiments, the rodent is selected from the general family of rats (muroide). In some embodiments, the rodents of the present disclosure are from a family selected from the group consisting of: the species of hamster (Calomyscidae) (e.g., hamster, baryotus hamster, gu Shili hamster, cricetrimus, hao Shili hamster, hamster bearded, chu Shili hamster, glatiramer hamster), the species of hamster (Cricetidae) (e.g., black line hamster, field mice, crown mice), the species of murine (Muridae) (black mice, brown mice, gerbil, new world rats, old world rats, norway species of rats, borilysia rats, tree rats, cotton rats, wood rats, stick rats, rice rats, kangarter rats, chinaroot), the species of masomidae (Nesomyidae) (long tail rats, south africa rats, african rats, ma Daobai tail rats), the species of acanthaceae (plaatacantomyidae) (e.g., acanthus, pigtail rats), and the species of spaalactaridae (e.g., mole, bamboo rats and zokorea). In some embodiments, the rodents of the present disclosure are selected from the group consisting of mice or rats (murine), gerbils, spines, and coronaries. In some embodiments, the rats of the present disclosure are from members of the murine family (Muridae).

In a specific embodiment of the invention, the rodent is a rat.

In some embodiments, the ankylosing spondylitis non-human animal model exhibits one or more symptoms of scoliosis, sternal herniation, joint stiffness, tail rigidity, anterior limb deformity, posterior limb sacroiliac joint deformity, spinal hyperosteogeny, spinal fusion, significant reduction in bone density, significant reduction in bone trabeculae.

In a specific embodiment of the invention, the ankylosing spondylitis non-human animal spontaneously develops ankylosing spondylitis after a period of growth.

The non-human animals of the invention are useful for in vivo assays. In addition, the non-human animals of the invention may be used as a source of somatic, fetal or embryonic cells, once isolated and cultured, for in vitro testing. In addition, immortalized cell lines may be prepared from the cells, if desired, using conventional techniques. Thus, in another aspect, the invention provides an isolated cell line derived from a non-human animal of the invention.

In some embodiments, the invention provides embryonic stem cells derived from the ankylosing spondylitis non-human animals described previously.

In some embodiments, the invention provides offspring of non-human animals. Offspring of the non-human animals of the invention may be obtained by conventional methods, for example, by conventional methods such as by classical hybridization techniques between the non-human animals of the invention, or by in vitro fertilization of eggs and/or sperm of the non-human animals of the invention. As used herein, the term "offspring" refers to each offspring of each generation after the original transformed non-human animal.

In some embodiments, a non-human animal modified with a mutated BMP9 protein is bred with a wild-type animal to obtain mutant BMP9 positive animal offspring.

In some embodiments, the invention provides a method of identifying a therapeutic agent for treating ankylosing spondylitis, the method comprising:

administering an agent to the ankylosing spondylitis non-human animal described previously;

performing one or more assays to determine whether the agent has an effect on one or more abnormalities associated with ankylosing spondylitis;

the agent is identified as a therapeutic agent when the agent has a therapeutic effect on one or more abnormalities associated with ankylosing spondylitis.

The agents of the invention are preferably administered in a pharmaceutically acceptable vehicle. Suitable pharmaceutical carriers are known to those skilled in the art. For parenteral administration, the compounds are typically dissolved or suspended in sterile water or saline. For enteral administration, the compounds are incorporated into inert carriers in the form of tablets, liquids or capsules. Suitable carriers may be starches or sugars and include lubricants, flavoring agents, binders and other materials of the same nature. The compounds may also be topically applied by topical application of solutions, creams, gels or polymeric materials (e.g., pluronicTM, BASF).

Alternatively, the compound may be administered in liposomes or microspheres (or microparticles). Methods of preparing liposomes and microspheres for administration to a patient are known to those skilled in the art. Essentially, the material is dissolved in an aqueous solution, if necessary, the appropriate phospholipids and lipids are added together with the surfactant, and the material is dialyzed or sonicated as necessary. Microspheres formed from polymers or proteins are well known to those skilled in the art and can be tailored to pass through the gastrointestinal tract directly into the blood stream. Alternatively, the compound may be incorporated and the microsphere or complex of microspheres implanted for slow release over a period of days to months.

The methods of the invention are preferably used to identify agents that alleviate such symptoms or signs.

In another embodiment, the invention relates to a method of assessing the effectiveness of a treatment for ankylosing spondylitis, the method comprising the steps of:

1) Providing a pharmaceutical composition or compound to be tested to a non-human animal model according to the invention;

2) The effect observed on the model of treatment with the pharmaceutical composition or compound was evaluated.

According to a preferred embodiment of the invention, said effect to be observed is referred to as a physiological pathological change. The physiological and pathological changes to be detected in the animal model of the invention refer to any improvement of the physiological and pathological changes present in the animal model as previously described.

In another embodiment, the invention provides the use of an animal model according to the invention or a cell line according to the invention or an embryonic stem cell according to the invention for the screening of a medicament for the treatment or prevention of ankylosing spondylitis.

Candidate compounds or drugs for use in the methods of the invention may include all different types of organic or inorganic molecules, including peptides, oligosaccharides or polysaccharides, fatty acids, steroids, and the like. Moreover, possible compounds to be screened include, for example, hematopoietic stem cells, enzymes, and gene therapy products, e.g., recombinant vectors, and the like. These compounds may be administered alone or in combination with each other.

Screening using animal models, candidate compounds can be administered before, during, or after the appearance of a particular disease phenotype. Diagnostic tests known to those skilled in the art can be used to monitor symptoms of disease progression or regression. Methods of monitoring symptoms of disease progression or regression are well known to those skilled in the art. Such as doppler ultrasound, pathology detection, etc.

The invention is further illustrated below in connection with specific embodiments. It should be understood that the particular embodiments described herein are presented by way of example and not limitation. The principal features of the invention may be used in various embodiments without departing from the scope of the invention.

EXAMPLE 1 Carrier transfection and detection of muteins

1. Vector construction

7 BMP9 eukaryotic expression vectors, namely a BMP9 wild type plasmid, six mutant plasmids (p.R316S, S320C, V109L, S282 frame shift mutation, A353T and V423M) were constructed, transfected into eukaryotic cells HEK 293EBNA, the cells themselves and cell supernatants were harvested, and the expression of the mutant proteins was detected using BMP9 antibodies.

2. Results

The BMP9 protein is 429 amino acids in total, comprising 3 domains: n-terminal Signal peptide domain (amino acids 1-22), intermediate precursor domain (Pro-domain, amino acids 23-319) and C-terminal Mature BMP9 (amino acids 320-429). After the full-length protein Pre-Pro-BMP9 is translated in vivo, the protein is secreted out of the cell under guidance of a signal peptide. The originally linked Pro-domain and Mature-BMP9 are cleaved at the 316-319 amino acid (RXR) position by Fusion enzyme, then covalently bound to form dimers, and transported throughout the blood circulation as shown in FIG. 1A.

BMP9 protein 316-319 amino acids (RXXR) are highly conserved across all BMP family proteins, and are important for proper protein shear maturation and function. The BMP9 mutation p.r316s of interest in this patent is located at position 1 of the "RXXR" domain, converting "R (arginine)" to "S (serine)" as shown in fig. 1B.

To clarify the effect of BMP9 mutation p.r316s on protein, we constructed eukaryotic expression vectors with R316S and 5 additional different BMP9 mutations (S320C, V109L, S282 frame shift mutations, a353T and V423M) and transferred into 293 cells. Western blot detects the BMP9 protein expression in cell supernatants, and none of the 6 mutant plasmid supernatants detected the substance-BMP 9 protein as shown in the blue frame line of FIG. 2A, but R316S expressed a large amount of uncleaved mutant protein as shown in the red frame line of FIG. 2B, suggesting that the R316S mutation not only reduced substance-BMP 9, but also produced a large amount of exogenously uncleaved mutant protein.

Example 2 construction and detection of spontaneous ankylosing spondylitis model

1. Construction of vectors

1. Artificially synthesizing human mutant BMP9 full-length CDS; this mutant differs from wild-type BMP9 (GeneID: 2658) only by one nucleotide in the CDS sequence c.G948T.

2. Construction of a transgenic plasmid containing the full-length CDS of the humanized mutant BMP was designated pcDNA3.1-ALB-BMP9-MUT. The plasmid is constructed autonomously in a laboratory, and the ALB gene promoter sequence drives the humanized mutant BMP9 gene to be highly expressed in the liver.

3. The full length of the pcDNA3.1-ALB-BMP9-MUT plasmid was 8574bp, and the nucleic acid was confirmed to be correct by Sanger sequencing.

4. The amino acid sequence expressed by the humanized mutant BMP9 is shown as SEQ ID NO. 1, and the specific mutation is that the 316 th amino acid is changed from arginine to serine.

2. Microinjection

1. Ligating male mice: vasally ligated SD male mice.

2. Superovulation: 10 SD mice of 3-4 weeks of age were injected with hormone for supervoling.

3. Fertilized egg injection: about 100 fertilized eggs were taken for injection.

4. Preparation of recipient mice: female rats with a age of 8 weeks were mated with male ligatured rats, and male rats with a thrombus were selected.

5. Embryo transfer: the fertilized eggs after injection are transplanted to the ampulla of the oviduct of the recipient mouse.

3. Genotyping

1. Tail number cutting: rats born for 7-10 days were numbered by cutting off the toes and tail tips.

2. Genomic DNA extraction: rat genomic DNA was extracted using the genome DNA extraction kit (EE 101-12) from the whole gold (Transgen) company.

3. And (3) PCR detection: synthetic PCR genotyping primers (Table 1) the rats were genotyped using the TaKaRa RR042A kit according to the reagent ratios shown in Table 2 and the reaction conditions shown in Table 3.

TABLE 1 identification of Tg-BMP9-MUT genotype primer sequences

R-BMP9-Mut-F	5’-TCCAGATGGCAAACATACGC-3’	SEQ ID NO:2
			R-BMP9-Mut-R	5’-GCTCCACCCTTGTCTTATCCTG-3’	SEQ ID NO:3
Target fragment	554bp

TABLE 2 identification of Tg-BMP9-MUT genotype PCR reaction System

10×LA PCR BufferⅡ(Mg ²⁺ Plus)	2.0μl
		dNTP Mixture(2.5μM)	1.6μl
primer-S (50. Mu.M)	0.2μl
		primer-A (50. Mu.M)	0.2μl
Template DNA	1.0μl
		LA Taq	0.2μl
Add ddH ₂ O to total volume	20.0μl

TABLE 3 identification of Tg-BMP9-MUT genotype PCR amplification procedure

4. Analysis of results:

after completion of PCR, the PCR reaction was stopped with a 6×loading buffer, and electrophoresis was performed on a 1% agarose gel, and only rats carrying the BMP9 transgenic fragment could amplify 554bp positive band, while the same-litter negative rats had no band (FIG. 3). For the amplified PCR product, the presence of the mutation site, BMP9 c.948G > T, p.arg316Ser, was verified by Sanger sequencing (FIG. 4). Western Blot examined the expression of BMP9 in lung and liver tissues of WT and Tg-BMP9-MUT rats (FIG. 5), endogenous BMP9 protein was detected in both lung and liver tissues of both rats (blue arrow), but both lung and liver tissues of Tg-BMP9-MUT rats expressed a mutant BMP9 protein (red arrow), which was present only in mutant rats and had a molecular weight of 60KD, which was significantly greater than endogenous BMP9 protein (55 KD).

4. Breeding offspring:

1. and mating the F0 generation rat with the wild SD rat to obtain the F1 generation rat, and identifying the F1 generation Tg-BMP9-MUT rat by PCR.

2. Male mice heterozygous for each generation were male-female 1:2 proportion and wild female mice are matched in a cage to obtain the next generation of rats.

3. By 2022, 6 months, rats had been passaged to F5 generation, were genotype stable, had a proportion of 23-83% positive rats per generation and an average positive rate of 50%, conforming to mendelian's law of genetics (table 4).

TABLE 4 statistical results of breeding of Tg-BMP9-MUT rats

5. Spontaneous ankylosing spondylitis onset phenotyping of Tg-BMP9-MUT rats

1. Wild type rats (WT), BMP9 knockout rats (BMP 9-KO), wild type BMP9 transgenic rats (Tg-BMP 9), and Tg-BMP9-MUT male rats, which were not modified by any gene, had a substantially normal phenotype before 1 month of age, with no obvious abnormalities in spinal column, bone, etc.

2. Tg-BMP9-MUT male rats develop spontaneous skeletal abnormalities from 1.5 months of age. 100% of male Tg-BMP9-MUT rats developed skeletal abnormalities by 3 months of age (FIG. 6D), WT wild-type rats without any genetic modification (FIG. 6A), BMP9 knockout rats BMP9-KO (FIG. 6B), and wild BMP9 transgenic rats Tg-BMP9 (FIG. 6C) did not develop skeletal abnormalities. Therefore, WT wild-type rats without any genetic modification were used as control rats in subsequent experiments.

The following phenotypes were specifically observed in 3 month old WT wild-type rats without any genetic modification as a control group and 3 month old male Tg-BMP9-MUT male rats as an experimental group:

1) The control group rats had no abnormalities in the spine and hind legs (fig. 6A); the rats in the experimental group were scoliotic and the right hind paw everted (fig. 6D).

2) No sternal abnormality in rats in the control group (fig. 7A); the sternum of the rats in the experimental group were protruded, and the left front leg and the right rear leg were not bendable (fig. 7B).

3) Rat tail of control group was not abnormal (fig. 8A); the experimental group rats had strong tails and were unable to droop naturally (fig. 8B).

4) The hind legs of the rats in the control group are free of abnormalities and flexible in joints (fig. 9A); the hind legs of the rats in the experimental group were swollen, joint stiffness (fig. 9B).

3. The 3 month old WT wild type rats without any genetic modification are used as a control group, the 3 month old male Tg-BMP9-MUT male rats are used as an experimental group, and CT detection results are consistent with the abnormal phenotype observed in appearance.

The whole body bone CT 3D modeling shows that the bones of rats in the control group are normal, and the limbs are normal to bend after anesthesia (figure 10A); the rats in the experimental group had a spinal column bulge, a forelimb deformity, a posterior sacroiliac joint deformity, and neither the right forelimb nor the hindlimb were able to bend normally after anesthesia (fig. 10B).

CT 3D modeling at the top view shows that the spinal column of the rats in the control group is normal (FIG. 11A); the spinal column of the rats of the experimental group was obviously laterally bent (fig. 11B).

Local fine CT detection of the spinal column shows that the spinal column of the rat in the control group is normal (figure 12A); the experimental group rats had hyperosteogeny, bone fusion occurred, and typical "bamboo-like" lesions occurred (fig. 12B).

4. 3D imaging analysis was performed on the tibial trabeculae of the rats using 3 month old WT wild-type rats without any genetic modification as a control group and 3 month old male Tg-BMP9-MUT male rats as an experimental group, and the analysis results showed that the bone density of the experimental group Tg-BMP9-MUT rats was significantly reduced relative to the control group rats (FIG. 13).

The quantitative analysis results of bone density are shown in Table 5, the tibial bone density of the experimental group Tg-BMP9-MUT rat is only 52 percent (158+/-40 vs 304+/-8, P < 0.001) of the tibial bone density of the control group WT rat, the femoral bone density of the experimental group Tg-BMP9-MUT rat is only 28 percent (63+/-25 vs 229+/-12, P < 0.001) of the femoral bone density of the control group WT rat, and the number of the bone trabeculae of the experimental group Tg-BMP9-MUT rat is only 69 percent (P < 0.05) of the bone trabeculae of the control group WT rat, which indicates that the bone density and the bone trabeculae of the experimental group Tg-BMP9-MUT rat are obviously reduced.

TABLE 5 detection results of tibial bone Density, femoral bone Density, trabecular bone count

Note that: sh: tibia; th: femur; sp: a spine; BMD: bone mineral density; BVF: bone volume fraction; BS/BV: bone surface area to bone volume ratio; tb.Th: bone trabecular thickness; nub: bone small Liang Shu; tb.sp: bone trabecular septum; tb. Pf: bone trabecular model factor

The above description of the embodiments is only for the understanding of the method of the present invention and its core ideas. It should be noted that it will be apparent to those skilled in the art that several improvements and modifications can be made to the present invention without departing from the principle of the invention, and these improvements and modifications will fall within the scope of the claims of the invention.

Claims

1. A method for constructing a spontaneous ankylosing spondylitis non-human animal model, which is characterized in that the ankylosing spondylitis non-human animal model expresses mutant BMP9 protein;

preferably, the non-human animal model refers to a non-human animal having or exhibiting characteristics of a disease or condition;

preferably, the amino acid sequence of the mutant BMP9 protein is shown in SEQ ID NO. 1.

2. The method of claim 1, wherein the method for expressing mutated BMP9 protein in the ankylosing spondylitis non-human animal model comprises the steps of: introducing a nucleic acid molecule encoding said mutant BMP9 protein into a single cell embryo or embryonic stem cell of a non-human animal;

preferably, the method for expressing mutated BMP9 protein in the ankylosing spondylitis non-human animal model comprises the following steps: introducing an expression vector comprising a nucleic acid molecule of said mutated BMP9 protein into a single cell embryo or embryonic stem cell of a non-human animal;

preferably, the nucleic acid molecule comprises the following sequence: a nucleotide sequence formed by mutating 948 th nucleotide from G to T on CDS sequence of wild BMP9 gene;

preferably, the expression vectors include, but are not limited to, linear polynucleotides, plasmids, and viral vectors;

preferably, the viral vectors include, but are not limited to, lentiviral vectors, retroviral vectors, adenoviral vectors, adeno-associated viral vectors.

3. The construction method according to claim 2, wherein the expression vector introduction method comprises electroporation, calcium phosphate method, liposome method, DEAE dextran method, microinjection, viral infection or liposome transfection.

4. A method of construction according to any one of claims 1 to 3, wherein the non-human animal is a rodent.

5. The method of construction of claim 4, wherein the rodent comprises the following options:

2) Hamster family such as black line hamster, voles and coronaries;

5) The spiny mouse is selected from spiny mouse and pig tail mouse;

6) Mole murine such as mole, bamboo rat, zokor;

7) Acanthaceae such as acanthus;

8) The family of the species Amanidae, such as African rats.

6. The method of claim 1, wherein the ankylosing spondylitis non-human animal model exhibits one or more symptoms of scoliosis, sternal herniation, joint stiffness, tail stiffness, anterior limb deformity, posterior sacroiliac joint deformity, spinal hyperosteogeny, spinal fusion, significant reduction in bone density, significant reduction in bone trabeculae.

7. A method of identifying a therapeutic agent for treating ankylosing spondylitis, said method comprising the steps of:

1) Administering an agent to a ankylosing spondylitis non-human animal prepared by the construction method of claim 1;

2) Performing one or more assays to determine whether the agent has a therapeutic effect on one or more abnormal symptoms associated with ankylosing spondylitis;

3) Identifying the agent as a therapeutic agent when the agent has a therapeutic effect on one or more abnormal symptoms associated with ankylosing spondylitis;

preferably, the therapeutic agent comprises a non-steroidal anti-inflammatory agent, a hormonal preparation, a targeted small molecule preparation, a proteasome inhibitor, an immunosuppressant, a tumor necrosis inhibitor, a cytokine, an activator of co-stimulatory molecules, an inhibitor of inhibitory molecules.

8. Use of a ankylosing spondylitis non-human animal model for screening a medicament for treating or preventing ankylosing spondylitis, characterized in that the ankylosing spondylitis non-human animal model is a model prepared using the construction method of claim 1;

preferably, the medicament comprises one or more pharmaceutically acceptable excipients;

preferably, the excipients include binders, fillers, disintegrants, lubricants, ointments, preservatives, antioxidants, flavoring agents, fragrances, co-solvents, emulsifiers, solubilizers, osmotic pressure regulators, colorants.

9. Use of a ankylosing spondylitis non-human animal model for evaluating the therapeutic effect of a product for treating ankylosing spondylitis, characterized in that the ankylosing spondylitis non-human animal model is a model prepared using the construction method of claim 1.

10. The application of mutant BMP9 protein or the nucleotide sequence for synthesizing the same in constructing a non-human animal model of spontaneous ankylosing spondylitis is characterized in that the amino acid sequence of the mutant BMP9 protein is shown as SEQ ID NO. 1;

preferably, the nucleotide sequence of the synthetic mutated BMP9 protein comprises the sequence: the 948 th nucleotide of CDS sequence of wild BMP9 gene is changed from G to T.