CN116058335A - Construction method and application of spontaneous ankylosing spondylitis model - Google Patents

Abstract

The invention discloses a construction method and application of a spontaneous ankylosing spondylitis model. 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 present invention also provides a method for identifying a therapeutic agent for treating ankylosing spondylitis in a non-human animal model of spontaneous ankylosing spondylitis. The present invention also provides the application of the spontaneous ankylosing spondylitis non-human animal model in evaluating the effect of the therapeutic agent on treating or preventing ankylosing spondylitis.

Description

Construction method and application of a spontaneous ankylosing spondylitis model

technical field

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

Background technique

Ankylosing spondylitis (AS) is a systemic disease mainly characterized by chronic inflammation of the axial joints, mainly involving the sacroiliac joints and spinal joints. Severe physical deformities and disabilities. The prevalence of AS in my country is about 0.3%, and the estimated number of patients exceeds 4 million. AS mostly occurs in young adults (20-40 years old). The average age of onset in Chinese patients is 29.2 years old, and males are higher than females (male:female=2.8:1). AS is a chronic progressive disease, and existing drugs cannot block the disease process. Once the patient becomes ill, he often loses the ability to work gradually within ten years. The early onset, high incidence, and high disability of AS have brought great pain to patients and families, and have become a heavy health burden on our society.

The main organs involved in AS are axial bones and joints, and human tissues are difficult to obtain. Therefore, experimental animal models of AS are extremely important for our in-depth understanding of the pathogenesis and the 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, and ankylosing enthesitis models. These experimental animal models have made important contributions to our understanding of AS complex disease pathology, but there are still shortcomings and limitations. Take HLA-B27/hβ2m double transgenic rats, the most widely used animal model of AS at present, as an example. This model can cause spondylitis and hind paw arthritis similar to human AS, but it has a long period of modeling (it needs to be kept until adulthood). 7-9 months old rats), low modeling rate (spondylitis occurs in only 30-50% rats at the age of 9 months), mild symptoms, and differences from clinical ankylosing spondylitis. So far, no animal model can fully simulate the clinical manifestations and pathological progression of human AS. Therefore, it is very important to speed up the construction of experimental animal models that spontaneously develop AS and are pathologically closer to the clinical manifestations of patients.

Contents of the invention

The purpose of the present invention is to overcome the deficiencies of the prior art, and provide a method for constructing a spontaneous ankylosing spondylitis animal model and its application.

In order to achieve the above object, the present invention adopts the following technical solutions:

The first aspect of the present invention provides a method for constructing a non-human animal model of spontaneous ankylosing spondylitis, and the non-human animal model of ankylosing spondylitis expresses a mutant BMP9 protein.

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

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

Further, the method for expressing the mutant BMP9 protein in the non-human animal model of ankylosing spondylitis includes the following steps: introducing the nucleic acid molecule encoding the mutant BMP9 protein into single-cell embryos or embryonic stem cells of the non-human animal.

Further, the method for expressing the mutant BMP9 protein in the non-human animal model of ankylosing spondylitis includes the following steps: introducing the expression vector containing the nucleic acid molecule of the mutant BMP9 protein into single-cell embryos or embryonic stem cells of the non-human animal.

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

Further, the expression vectors include but 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, and adeno-associated viral vectors.

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

Further, the non-human animal is a rodent.

Further, the rodent includes the following options:

1) Lily hamster family, such as Lili hamster, Bashili hamster, Gushili hamster, Jinli hamster, Haoshili hamster, bearded Lili hamster, Chushili hamster, Uradley hamster;

2) Hamster family such as black-lined hamster, vole, crested mouse;

3) Muridae such as black rats, brown rats, gerbils, New World rats, Old World rats, Norwegian rats, Polynesian rats, tree rats, cotton rats, wood rats, stick rats rats, rice rats, kangaroo rats, climbing rats;

4) Falklandae rats, such as the long-tailed giant rat, South African pouch rat, African giant rat, and Falkland white-tailed rat;

5) Agoriidae, such as agouti and pigtail;

6) Molesidae, such as moles, bamboo rats, and zokors;

7) Acanthidae such as acanthus;

8) Rock rats such as African rock rats.

Further, the non-human animal model of ankylosing spondylitis exhibits scoliosis, sternum protrusion, joint stiffness, stiff tail, forelimb deformity, hindlimb sacroiliac joint deformity, spinal bone hyperplasia, spinal bone fusion, and significantly decreased bone density 1. One or more symptoms in the marked reduction of trabecular bone.

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

The third aspect of the present invention provides an embryonic stem cell derived from the non-human animal model of ankylosing spondylitis prepared by the construction method described in the first aspect of the present invention.

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

1) Administering the agent to the ankylosing spondylitis non-human animal prepared by the construction method described above.

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

Further, the therapeutic agents include non-steroidal anti-inflammatory agents, hormone preparations, targeted small molecule preparations, proteasome inhibitors, immunosuppressants, tumor necrosis inhibitors, cytokines, costimulatory molecule activators, inhibitory molecules inhibitors.

The fifth aspect of the present invention provides the application of non-human animal model of ankylosing spondylitis in the screening of drugs for the treatment or prevention of ankylosing spondylitis, said non-human animal model of ankylosing spondylitis is constructed using the aforementioned construction method Prepared model.

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

Further, the excipients include binders, fillers, disintegrants, lubricants, ointments, preservatives, antioxidants, flavoring agents, fragrances, cosolvents, emulsifiers, solubilizers, osmotic pressure regulators ,Colorant.

The sixth aspect of the present invention provides the application of the non-human animal model of ankylosing spondylitis in evaluating the therapeutic effect of products for the treatment of ankylosing spondylitis. The non-human animal model of ankylosing spondylitis is prepared using the construction method described above model.

The seventh aspect of the present invention provides mutant BMP9 protein or its synthetic nucleotide sequence in the construction of spontaneous ankylosing spondylitis non-human animal model, the amino acid sequence of the mutant BMP9 protein is shown in SEQ ID NO: 1 Show.

Further, the nucleotide sequence of the synthetic mutant BMP9 protein includes the following sequence: a nucleotide sequence formed after the 948th nucleotide on the CDS sequence of the wild-type BMP9 gene is mutated from G to T.

Beneficial effects of the present invention:

The present invention provides a non-human animal model of ankylosing spondylitis. The genetic background of the model is clear and closer to the real symptoms of the disease, which solves the problem that the existing animal models are quite different from clinical diseases. Research is of great significance.

Description of drawings

Figure 1 is a schematic diagram of BMP9 protein translation, cleavage, maturation process and mutation position; wherein, A: BMP9 protein translation, cleavage, maturation process; B: different mutation positions of BMP9;

Figure 2 is a diagram of the expression of BMP9 protein detected by six mutant plasmids by Western blot; among them, A: the expression of BMP9 protein by the six mutant plasmids in the cell lysate; B: the expression of BMP9 protein by the six mutant plasmids in the cell supernatant ;

Figure 3 is the genotype diagram of Tg-BMP9-MUT rats identified by PCR; among them, NC: negative control; PC: positive control; M: nucleic acid marker; BLK: blank control; Tg-MUT: Tg-BMP9-MUT mutation transgene positive Rat;

Figure 4 is the first-generation sequencing results of Tg-BMP9-MUT rat gene, in which the red arrow points to the mutation site BMP9c.948G>T, p.Arg316Ser;

Figure 5 is the Western Blot results of BMP9 expression in lung tissue and liver tissue of WT rats and Tg-BMP9-MUT rats, in which A: Western Blot results of two kinds of rat lung tissues, B: Western Blot results of two kinds of rat liver tissues Blot results;

Figure 6 is the back view of WT rats, BMP9 knockout rats (BMP9-KO), wild-type BMP9 overexpression rats (Tg-BMP9), and Tg-BMP9-MUT overexpression male rats, wherein A: WT rats , B: BMP9 knockout rats (BMP9-KO), C: wild-type BMP9 overexpression rats (Tg-BMP9), D: Tg-BMP9-MUT overexpression male rats;

Figure 7 is a chest map of WT rats and Tg-BMP9-MUT overexpressed male rats, wherein A: WT rats, B: Tg-BMP9-MUT overexpressed male rats;

Figure 8 is a tail view of WT rats and Tg-BMP9-MUT overexpressed male rats, wherein A: WT rats, B: Tg-BMP9-MUT overexpressed male rats;

Figure 9 is a foot map of WT rats and Tg-BMP9-MUT overexpressed male rats, wherein A: WT rats, B: Tg-BMP9-MUT overexpressed male rats;

Figure 10 is a side view of WT rats and Tg-BMP9-MUT overexpression male rats with CT 3D modeling of whole body bones, wherein A: WT rats, B: Tg-BMP9-MUT overexpression male rats;

Figure 11 is a top view of WT rats and Tg-BMP9-MUT overexpressed male rats with CT 3D modeling of whole body bones, wherein A: WT rats, B: Tg-BMP9-MUT overexpressed male rats;

Figure 12 is the CT 3D modeling of the spine of WT rats and Tg-BMP9-MUT overexpressed male rats, where A: WT rats, B: Tg-BMP9-MUT overexpressed male rats;

Figure 13 is the 3D imaging of tibial trabecular bone of WT rats and Tg-BMP9-MUT overexpressed male rats, wherein A: WT rats, B: Tg-BMP9-MUT overexpressed male rats.

Detailed ways

The present invention will be further described in detail below, and it should be understood that the terms are intended to describe rather than limit the present invention.

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. A nucleic acid can be double-stranded, single-stranded, or comprise a portion of a double-stranded or single-stranded sequence.

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 an appropriate eukaryotic host cell or organism is chosen, expression may include splicing of the 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 the administration of a compound or composition to control the progression of a disease. Control of disease progression should be understood as achieving beneficial or desired clinical outcomes, including but not limited to alleviation of symptoms, reduction of disease duration, stabilization of pathological state (especially avoiding additional exacerbations), delay of disease progression, improvement and remission of pathological state ( some and all). Control of disease progression also involves prolonging survival compared to expected survival without treatment.

The present invention provides a new non-human animal model of ankylosing spondylitis. Indeed, the inventors have discovered a new animal model of ankylosing spondylitis that recapitulates core features of the human disease.

The term "non-human animal" includes non-human vertebrates, more preferably mammals, such as domesticated livestock (eg, cows, horses, pigs), pets (eg, dogs, cats), or rodents. The term "rodent" means any and all members of the phylogenetic class Rodent (eg, mouse, rat, squirrel, beaver, woodchuck, gopher, voles, woodchuck, hamster, guinea pig, and agouti) , including any descendants of all descendants derived from it.

The term "non-human animal model" refers to a non-human animal that has or exhibits characteristics of a disease or condition. Use as an animal model refers to any use of an animal for the study of a disease or condition, eg, to study progress or development or response to new or existing therapies.

The gene of bone morphogenic protein 9 (BMP9, also called growth differentiation factor 2, GDF2) is located on chromosome 10q11.22, and the gene ID is 2658, including the gene, its encoded protein, its homologue, mutation, etc. The term "BMP9" encompasses the full-length, unprocessed gene or protein, as well as any form of the gene or protein derived from processing in the cell. The term encompasses naturally occurring variants of a biomarker. Gene IDs are available at https://www.ncbi.nlm.nih.gov/gene/.

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

The term "expression vector" as used herein refers to an expression vector that may contain regulatory and coding sequences derived from different sources, or derived from the same source but arranged in a manner different from that found in nature, such an expression vector can be independently use or in combination. Those skilled in the art are well aware of the genetic elements that must be present on the vectors to successfully transform, select and propagate host cells containing the nucleic acid fragments of the mutated genes of the present invention. The expression vector can comprise any combination of deoxyribonucleotides, ribonucleotides or modified nucleotides, the expression vector can be transcribed to form RNA, wherein the RNA can be capable of forming double-stranded RNA and/or hairpin structures, the expression vector can For expression in cells, either isolated or produced synthetically, the expression vector may also contain a promoter or other sequences to facilitate manipulation or expression of the construct.

In some embodiments, the nucleic acid sequence is operably linked into an expression vector. The expression vectors mentioned above are completely commercially available at present, such as some viral vectors, plasmids, and phages.

In a preferred embodiment, the expression vector is a plasmid, which is selected from conventional plasmids used in the art for constructing transgenic structures. Usually, there are "spacer sequences" and multiple cloning sites on both sides of the "spacer sequences" or The sequence for replacement, so that people can insert the corresponding DNA sequence of the gene into the multiple cloning site or replace the sequence for replacement on it in a forward and reverse manner. The expression vector usually also contains a promoter, an origin of replication and/or a marker gene and the like.

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

As an alternative embodiment, the expression vector can be introduced into cells by electroporation, calcium phosphate method, liposome method, DEAE dextran method, microinjection, virus infection, lipofection, and cell membrane A well-known method such as conjugation of a permeable peptide.

In a specific embodiment of the present invention, the introduction of the expression vector into the cells uses the method of microinjection.

As an alternative embodiment, the non-human animal is a rodent.

In some embodiments, rodents of the present disclosure include, by way of non-limiting examples, mice, rats, and hamsters. In some embodiments, rodents of the present disclosure include, by way of non-limiting examples, mice and rats. In some embodiments, the rodent is selected from the superfamily Muroidea. In some embodiments, the rodent of the present disclosure is from a family selected from the group consisting of: Calomyscidae (e.g., Calomyscidae, Pasteurian hamsters, Oldham hamsters, Calomyscidae hamsters, Calomyscidae hamsters, bearded hamsters Lili hamster, Chushili hamster, Uradley hamster), Cricetidae (such as black-lined hamster, vole, crested mouse), Muridae (black rat, brown rat, gerbil, New World hamster) Rat, Old World Rat, Norwegian Rat, Polynesian Rat, Tree Rat, Cotton Rat, Wood Rat, Stick Rat, Rice Rat, Kangaroo Rat, Climbing Rat), Falkland Rat Nesomyidae (giant-tailed rats, South African gopher rats, African giant rats, Falkland white-tailed rats), Platacanthomyidae (e.g., gopher rats, pig-tailed rats) and Spalacidae ( For example, moles, bamboo rats, and zokors). In some embodiments, the rodent of the present disclosure is selected from the group consisting of mice or rats (Muridae), gerbils, acanthus, and crested rats. In some embodiments, the rat of the present disclosure is from a member of the family Muridae.

In a particular embodiment of the invention, said rodent is a rat.

In some embodiments, the non-human animal model of ankylosing spondylitis exhibits scoliosis, sternum protrusion, joint stiffness, tail stiffness, forelimb deformity, hindlimb sacroiliac joint deformity, spinal hyperostosis, spinal bone fusion, One or more of the symptoms of significantly reduced bone density and significantly reduced bone trabeculae.

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

The non-human animals of the invention can be used in in vivo experiments. In addition, the non-human animals of the invention can be used as a source of somatic, fetal or embryonic cells which, once isolated and cultured, can be used for in vitro testing. Additionally, immortalized cell lines can 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 present invention provides embryonic stem cells derived from the aforementioned ankylosing spondylitis non-human animal.

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

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

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

administering the agent to the aforementioned ankylosing spondylitis non-human animal;

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

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

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 compound is usually dissolved or suspended in sterile water or saline. For enteral administration, the compound is incorporated into an inert carrier in tablet, liquid or capsule form. Suitable carriers may be starch or sugar, and include lubricants, flavourings, binders and other materials of the same nature. The compounds may also be administered topically by topical application of solutions, creams, gels or polymeric materials (eg, PluronicTM, BASF).

Alternatively, the compounds can be administered in liposomes or microspheres (or microparticles). Methods of preparing liposomes and microspheres for administration to patients are known to those skilled in the art. Essentially, the material is dissolved in an aqueous solution, the appropriate phospholipids and lipids are added together with surfactants if necessary, 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 bloodstream. Alternatively, the compound can be incorporated and implanted in microspheres or complexes of microspheres 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 evaluating the effect of ankylosing spondylitis treatment, said method comprising the steps of:

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

2) To evaluate the effect observed on said model treated with the pharmaceutical composition or compound.

According to a preferred embodiment of the present invention, said effects to be observed refer to physiopathological changes. Said physiological and pathological changes to be detected in the animal model of the present invention refer to any improvement of the previously described physiological and pathological changes present in the animal model.

In another embodiment, the present invention provides the use of the animal model according to the present invention or the cell line according to the present invention or the embryonic stem cells according to the present invention in screening drugs for treating or preventing 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, oligo- or polysaccharides, fatty acids, steroids, and the like. Furthermore, possible compounds to be screened include, for example, hematopoietic stem cells, enzymes and gene therapy products, eg, recombinant vectors, and the like. These compounds can be administered alone or in combination with one another.

Using animal models for screening, candidate compounds can be administered before, during, or after the emergence of a particular disease phenotype. Symptoms of disease progression or regression can be monitored using diagnostic tests known to those skilled in the art. Methods of monitoring symptoms of disease progression or regression are well known to those skilled in the art. For example, Doppler ultrasound, pathology detection, etc.

Below in conjunction with specific embodiment further elaborates this invention. It should be understood that the specific embodiments described herein are presented by way of example and not as limitations of the invention. The principal features of this invention can be employed in various embodiments without departing from the scope of the invention.

Vector transfection and detection of embodiment 1 mutein

1. Carrier construction

Constructed seven BMP9 eukaryotic expression vectors, one BMP9 wild-type plasmid and six mutant plasmids (p.R316S, S320C, V109L, S282framshift (frameshift mutation), A353T and V423M), and transfected into eukaryotic cells For HEK293EBNA, the cells themselves and the cell supernatant were harvested, and the expression of the mutant protein was detected with BMP9 antibody.

2. Results

The BMP9 protein has a full length of 429 amino acids and contains three structural domains: the N-terminal signal peptide domain (Signalpeptide, 1-22 amino acids), the middle precursor domain (Pro-domain, 23-319 amino acids) and the C-terminal Mature BMP9 (Mature-BMP9, amino acids 320-429). After the full-length Pre-Pro-BMP9 protein is translated in vivo, it is secreted out of cells under the guidance of a signal peptide. The originally connected Pro-domain and Mature-BMP9 are cut by Fusion enzyme at the 316-319 amino acid (RXXR) position, and then combined by covalent bonds to form a dimer, which is transported to the whole body through the blood circulation as shown in Figure 1A.

The 316-319 amino acids (RXXR) of BMP9 protein are highly conserved in all BMP family proteins, and are very important for protein shearing and maturation and normal function. The BMP9 mutation p.R316S concerned in this patent is located at position 1 of the "RXXR" domain, which changes "R (arginine)" to "S (serine)", as shown in Figure 1B.

In order to clarify the effect of the BMP9 mutation p.R316S on the protein, we constructed eukaryotic cell expression vectors with R316S and five other BMP9 mutations (S320C, V109L, S282framshift (frameshift mutation), A353T and V423M), and transformed them into 293 cells . Western blot was used to detect the expression of BMP9 protein in the supernatant of the cells. Mature-BMP9 protein was not detected in the supernatant of the six mutant plasmids, as shown in the blue frame in Figure 2A, but R316S expressed a large number of uncut mutations The red framed part of Figure 2B indicates that the R316S mutation not only reduces Mature-BMP9, but also produces a large amount of exocrine, unspliced mutant protein.

Example 2 Construction and detection of spontaneous ankylosing spondylitis model

1. Build the carrier

1. Artificially synthesized full-length CDS of human mutant BMP9; this mutant has only one nucleotide difference c.G948T in CDS sequence with wild-type BMP9 (GeneID: 2658).

2. Construction of a transgenic plasmid containing the full-length CDS of human mutant BMP named pcDNA3.1-ALB-BMP9-MUT. The plasmid is independently constructed by the laboratory, and the human mutant BMP9 gene is highly expressed in the liver driven by the promoter sequence of the ALB gene.

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

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

2. Microinjection

1. Ligated male mice: SD male mice with vasectomy.

2. Superovulation: 10 3-4 week old SD mice were injected with hormones for superovulation.

3. Injection of fertilized eggs: Take about 100 fertilized eggs for injection.

4. Preparation of recipient mice: 8-week-old SD female mice were mated with ligated male mice and selected female mice with thrombus.

5. Embryo transfer: transfer the injected fertilized eggs to the ampulla of oviduct of recipient mice.

3. Genotyping

1. Tail-cut numbering: for rats born 7-10 days old, cut off the toes and the tip of the tail for numbering.

2. Genomic DNA extraction: Rat genomic DNA was extracted using a Genomic DNA Extraction Kit (EE101-12) from Transgen.

3. PCR detection: PCR genotyping primers (Table 1) were synthesized, and the rats were genotyped using the RR042A kit from TaKaRa Company according to the reagent ratio shown in Table 2 and the reaction conditions shown in Table 3.

Table 1 identifies the Tg-BMP9-MUT genotype primer sequence

R-BMP9-Mut-F 5'-TCCAGATGGCAAACATACGC-3' SEQ ID NO:2 R-BMP9-Mut-R 5'-GCTCCACCCTTGTCTTATTCCTG-3' SEQ ID NO:3 target segment 554bp the

Table 2 Identification of Tg-BMP9-MUT genotype PCR reaction system

<![CDATA[10×LA PCR BufferⅡ(Mg²⁺Plus)]]> 2.0μl dNTP Mixture (2.5μM) 1.6μl Primer-S (50μM) 0.2μl Primer-A (50 μM) 0.2μl template DNA 1.0μl LA Taq 0.2μl <![CDATA[Add ddH₂O to total volume]]> 20.0μl

Table 3 Identification of Tg-BMP9-MUT genotype PCR amplification program

4. Result analysis:

After the PCR was completed, the PCR reaction was terminated with 6×loading buffer, and electrophoresis was performed on a 1% agarose gel. Only the rats carrying the BMP9 transgene fragment could amplify a 554bp positive band, and the negative rats in the same littermate had no band ( image 3). For the amplified PCR product, Sanger sequencing was used to verify the real existence of the mutation site ( FIG. 4 ), and the mutation site was BMP9c.948G>T, p.Arg316Ser. Western Blot was used to detect the expression of BMP9 in the lung and liver tissues of WT and Tg-BMP9-MUT rats (Fig. ), but both lung and liver tissues of Tg-BMP9-MUT rats express a mutant BMP9 protein (shown by the red arrow), which only exists in mutant rats, with a molecular weight of 60KD, which is significantly larger than Endogenous BMP9 protein (55KD).

4. Breeding offspring:

1. The obtained Founder rats were F0 generation rats mated with wild-type SD rats to obtain F1 generation, and F1 generation Tg-BMP9-MUT rats were obtained by PCR identification.

2. Each generation of heterozygous positive male mice was mated with wild-type female mice in a ratio of 1:2 to obtain the next generation of rats.

3. As of June 2022, the rats have been passed down to the F5 generation, and the genotype is stable. The proportion of positive rats in each generation is 23-83%, and the average positive rate is 50%, which conforms to the Mendelian law of inheritance (Table 4).

Table 4 Tg-BMP9-MUT rat breeding statistical results

5. Phenotype analysis of spontaneous ankylosing spondylitis in Tg-BMP9-MUT rats

1. Wild-type rats (WT), BMP9 knockout rats (BMP9-KO), wild-type BMP9 transgenic rats (Tg-BMP9), Tg-BMP9-MUT male rats without any genetic modification The pre-age phenotype was basically normal, and there were no obvious abnormalities in the spine and bones.

2. Tg-BMP9-MUT male rats gradually developed spontaneous bone abnormalities from 1.5 months old. At the age of 3 months, 100% of male Tg-BMP9-MUT male rats had skeletal abnormalities (Fig. 6D), WT wild-type rats without any genetic modification (Fig. 6B) and wild-type BMP9 transgenic rats Tg-BMP9 (Fig. 6C) had no bone abnormalities. Therefore, WT wild-type rats without any genetic modification were used as control mice in subsequent experiments.

Taking 3-month-old WT wild-type rats without any genetic modification as the control group, and 3-month-old male Tg-BMP9-MUT male rats as the experimental group, the following phenotypes were specifically observed:

1) The rats in the control group had no abnormalities in the spine and hind feet (Fig. 6A); the rats in the experimental group had scoliosis and valgus right hind feet (Fig. 6D).

2) There was no abnormality in the sternum of the rats in the control group (Fig. 7A); the sternum of the rats in the experimental group protruded, and the left foreleg and right hind leg could not be bent (Fig. 7B).

3) The tails of the rats in the control group had no abnormalities (FIG. 8A); the tails of the rats in the experimental group were stiff and unable to droop naturally (FIG. 8B).

4) The hind legs of the rats in the control group were normal and the joints were flexible (FIG. 9A); the hind legs of the rats in the experimental group were swollen and the joints were stiff (FIG. 9B).

3. The 3-month-old WT wild-type rats without any genetic modification were used as the control group, and the 3-month-old male Tg-BMP9-MUT male rats were used as the experimental group. The CT detection results were consistent with the abnormal phenotypes observed on the appearance. unanimous.

The CT 3D modeling of the whole body showed that the rats in the control group had normal bones and normal bending of the limbs after anesthesia (Fig. 10A); the rats in the experimental group had raised spines, deformed forelimbs, and deformed sacroiliac joints of the hind limbs, and the right forelimb and hindlimb were both deformed after anesthesia. Unable to bend normally (Fig. 10B).

The CT 3D modeling of the overlooking angle showed that the spines of the rats in the control group were normal (Fig. 11A); the rats in the experimental group had obvious scoliosis (Fig. 11B).

Local fine CT examination of the spine showed that the spines of the rats in the control group were normal (Fig. 12A); the rats in the experimental group had hyperosteogeny, bone fusion, and typical "bamboo-like" lesions (Fig. 12B).

4. Using 3-month-old WT wild-type rats without any genetic modification as the control group, and 3-month-old male Tg-BMP9-MUT male rats as the experimental group, 3D imaging analysis was performed on the tibial trabecular bone of the rats, and the analysis The results showed that, compared with the rats in the control group, the bone mineral density of the Tg-BMP9-MUT rats in the experimental group was significantly reduced ( FIG. 13 ).

The results of quantitative analysis of bone density are shown in Table 5. The tibia bone density of Tg-BMP9-MUT rats in the experimental group was only 52% of that of WT rats in the control group (158±40vs 304±8, p<0.001). The femur BMD of Tg-BMP9-MUT rats in the experimental group was only 28% of that of the WT rats in the control group (63±25vs 229±12, p<0.001), and the number of trabecular bone in the experimental group Tg-BMP9-MUT rats It was only 69% of the number of trabeculae in WT rats in the control group (P<0.05), indicating that the bone density and trabeculae in Tg-BMP9-MUT rats in the experimental group were significantly reduced.

Table 5 The detection results of tibial bone mineral density, femoral bone mineral density, and bone trabecular number

Note: sh: tibia; th: femur; sp: spine; BMD: bone mineral density; BVF: bone volume fraction; BS/BV: bone surface area to bone volume ratio; Tb.Th: bone trabecular thickness; Tb.Nub: bone Trabecular number; Tb.Sp: Trabecular spacing; Tb.PF: Trabecular pattern factor

The description of the above embodiments is only for understanding the method and core idea of the present invention. It should be pointed out that for those skilled in the art, without departing from the principles of the present invention, some improvements and modifications can be made to the present invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims (10)

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:

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, voles and coronaries;

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.

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.

Images