A method for constructing an animal model of sarcopenia, comprising a recombinant AGR2 protein and its related gene fragments, a recombinant vector, its uses and drugs.

Abstract

This invention belongs to the field of pharmaceutical technology, specifically relating to a recombinant AGR2 protein and its related gene fragments, a recombinant vector, its uses and drugs, and a method for constructing an animal model of sarcopenia. The amino acid sequence of the recombinant AGR2 protein is shown in SEQ ID NO.2. Animal experiments have demonstrated that the recombinant AGR2 protein of this invention, without affecting body weight or food intake, can reduce fat mass, increase lean meat mass, increase gastrocnemius and / or quadriceps muscle mass, improve muscle strength, improve muscle function, and effectively improve the sarcopenia-like phenotype.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a recombinant AGR2 protein and its related gene fragments, recombinant vector, uses and drugs, and a method for constructing an animal model of sarcopenia. Background Technology

[0002] Sarcopenia is an age-related syndrome characterized by a decrease in skeletal muscle mass, strength, and / or function. It primarily affects older adults and is a significant factor contributing to mobility impairment, reduced quality of life, and increased risk of falls. The pathogenesis of sarcopenia is complex, involving multiple factors including neuroendocrine changes, inadequate nutrient intake, chronic inflammation, oxidative stress, hormonal fluctuations, and reduced physical activity. Early identification and intervention for sarcopenia are crucial for improving the quality of life and preventing related complications in older adults.

[0003] Regarding treatment strategies, there are currently no specific drugs, and intervention remains centered on exercise and nutrition. Exercise intervention is considered the most effective non-pharmacological approach, with resistance training being the most crucial, significantly improving muscle strength and cross-sectional area. Aerobic exercise can improve mitochondrial function and metabolic status, and the combination of the two often yields better results. In terms of nutritional intervention, adequate protein intake (especially high-quality protein rich in leucine) can promote muscle protein synthesis; vitamin D supplementation also plays a role in improving muscle function and reducing the risk of falls; in addition, nutritional supplements such as omega-3 fatty acids and creatine are also being studied as potential adjunctive measures. In recent years, drug development for sarcopenia has also been actively progressing. For example, anti-MSTN antibodies and muscle growth factor-related signaling modulators have shown some potential; selective androgen receptor modulators (SARMs) and GLP-1 receptor agonists are also being explored in clinical trials. However, there are currently no reports of functional proteins effectively treating sarcopenia. Summary of the Invention

[0004] To address the above problems, the present invention provides a recombinant AGR2 protein, the amino acid sequence of which is shown in SEQ ID NO.2.

[0005] The present invention also provides a gene fragment that encodes the aforementioned recombinant AGR2 protein.

[0006] Furthermore, its nucleotide sequence is shown in SEQ ID NO.1.

[0007] The present invention also provides a recombinant vector, which is a plasmid containing the aforementioned gene fragment.

[0008] The present invention also provides a recombinant bacterium, which is Escherichia coli comprising the aforementioned recombinant vector.

[0009] The present invention also provides the use of the aforementioned recombinant AGR2 protein in the preparation of a medicament for treating sarcopenia.

[0010] Furthermore, the drug is a drug that reduces fat mass and increases muscle mass.

[0011] Furthermore, the drug is a drug that increases the gastrocnemius and / or quadriceps muscles.

[0012] Furthermore, the drug is a drug that increases muscle mass, improves muscle strength, and / or enhances muscle function.

[0013] The present invention also provides a medicament for treating sarcopenia, which is a formulation prepared by using the aforementioned recombinant AGR2 protein as the active ingredient and adding pharmaceutically acceptable excipients; the formulation includes an injectable form.

[0014] Finally, this invention provides a method for constructing an animal model of sarcopenia, the method being for constructing... AGR2 Gene knockout animals; the aforementioned AGR2 The gene's accession number on NCBI is NM_011783.2; the animals mentioned include mice. AGR2 Gene knockout animals can be prepared using the following methods: 1) After female mice undergo superovulation, they are allowed to mate naturally with male mice, and fertilized eggs are then obtained from the oviduct; 2) Inject the CRISPR / Cas9 editing solution into the fertilized eggs obtained in step 1) and culture them to the 2-cell stage to obtain embryos; 3) The embryos obtained in step 2) are transferred into pseudopregnant mice for gestation; 4) DNA was extracted from the tail tissue of mice after birth for identification; The CRISPR / Cas9 editing solution is a mixture containing Cas9 mRNA and sgRNA; The nucleotide sequence of the sgRNA is shown in SEQ ID NO.3~4.

[0015] This invention first discovered a correlation between the AGR2 protein and sarcopenia, and then constructed... AGR2 Gene knockout animal models confirmed that AGR2 protein deficiency can induce sarcopenia. Based on this, Further studies using exogenous recombinant AGR2 protein in two classic animal models of sarcopenia (aged senile sarcopenia and obese sarcopenia) showed that it effectively reduced fat mass, increased lean meat mass, and significantly increased the volume of the gastrocnemius and quadriceps muscles without affecting body weight or food intake. It also improved muscle strength and function, thereby alleviating the sarcopenia-like phenotype. Given the targeted therapeutic effect of recombinant AGR2 protein on sarcopenia, its development into a novel drug for treating sarcopenia has significant clinical translational value.

[0016] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0017] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0018] Figure 1 Elisa examination was performed to check the changes of AGR2 in the serum of patients with sarcopenia; Figure 2 Muscle strength testing in AGR2 knockout mice; (A) Knockout mice and wild-type mice; (B) Grip strength testing; (C) Treadmill test to measure total distance, exhaustion time, and final exhaustion rate; Figure 3 AGR2 knockout reduces muscle mass. (A) Body composition analysis; (B) Images of the heart, kidneys, and muscles of various parts of AGR2 wild-type and knockout mice; (C) Organ-to-body weight ratio.

[0019] Figure 4 Statistics on the area of ​​quadriceps muscle fibers in AGR2 knockout mice; (A) H&E staining; (B) CSA statistics.

[0020] Figure 5 rAGR2 improves sarcopenia in aged mice; (A) Experimental flowchart; (B) Body weight; (CD) MRI analysis of body composition, lean meat mass (C) and fat mass (D); (E) Grip strength test; (F) Organ and tissue images; (G) Muscle mass; (H) Kidney and heart mass; Figure 6 Statistical analysis of quadriceps muscle fiber area in aged mice treated with rAGR2; (A) H&E staining; (B) CSA statistics.

[0021] Figure 7rAGR2 injection in HFD mice did not affect body weight, but reduced fat mass and increased lean meat mass; (A) Mouse injection procedure; (B) Body weight; (C) Food intake; (D) Body composition analysis; Figure 8 Changes in organ weight in rAGR2-injected HFD mice; (A) Representative images of organ tissues after mouse sacrifice; (B) Tissue-to-body weight ratio. Detailed Implementation

[0022] The raw materials and equipment used in the specific embodiments of the present invention are all known products, obtained by purchasing commercially available products.

[0023] Example 1: Study on the effect of AGR2 protein on sarcopenia 1. Changes in AGR2 expression in patients with sarcopenia Serum samples were collected from normal individuals and patients with sarcopenia. ELISA was used to detect changes in AGR2 protein expression in serum. The ELISA kit was purchased from Elabscience (E-EL-H0298). Results showed that AGR2 levels were decreased in patients with sarcopenia, suggesting that it may be involved in regulating the progression of sarcopenia. Figure 1 ).

[0024] 2. Effects of AGR2 knockout on muscle tissue Systemic AGR2 knockout mice were constructed using the CRISPR-Cas9 method. Firstly, based on... AGR2 Genetically designed sgRNAs were used, with primer sequences as follows: gRNA-A1 (SEQ ID NO.3): TTAGGGTAACCGTTTCCGGAAGG, gRNA-A2 (SEQ ID NO.4): AAAAGCGTGTGCGCCTTCCAGGG. After standardized procedures (i.e., female mice undergoing superovulation were naturally mated with male mice, fertilized eggs were obtained from the oviduct, microinjected into a CRISPR / Cas9 editing system, cultured to the 2-cell stage, and then transplanted into pseudopregnant recipient mice for gestation), offspring mice were obtained. DNA was extracted from the tail tissue of the offspring mice, and PCR was used to confirm successful deletion. Further hybridization of the resulting female and male heterozygous mice yielded AGR2 knockout mice, which were then propagated to obtain a sufficient number of knockout and wild-type mice. Figure 2 A).

[0025] Using AGR2 knockout mice to assess muscle function, it was found that after AGR2 knockout, the grip strength of the mice was significantly reduced when using a grip strength meter for grip strength testing. Figure 2 B); The Treadmill experiment showed that the total distance, exhaustion time, and exhaustion rate of AGR2 knockout mice were significantly lower than those of wild-type mice (B). Figure 2 C) indicates that muscle strength is significantly reduced after knocking out AGR2.

[0026] Body composition analysis of mice using nuclear magnetic resonance MRI showed that knockout mice had significantly reduced lean meat and significantly increased fat. Figure 3 A). Dissection of the mice revealed a decrease in body weight, but no significant differences were observed in organs such as the heart and kidneys. Figure 3 B), while muscle mass in various parts of the body is significantly reduced ( Figure 3 BC, Figure 4 (AB), including the quadriceps femoris, gastrocnemius and tibialis anterior muscles, suggesting that knocking out AGR2 caused a sarcopenia-like phenotype.

[0027] The above results indicate that the expression level of AGR2 protein in the body is closely related to sarcopenia. AGR2 protein deficiency leads to a significant decrease in muscle mass and marked decline in muscle function, resulting in typical clinical symptoms of sarcopenia. Based on this mechanism, further exploration of methods to treat sarcopenia using AGR2 protein is of great value.

[0028] Example 2: Application Study of Recombinant AGR2 Protein in the Treatment of Sarcopenia 1. Preparation of recombinant AGR2 protein 1.1 Materials and Reagents pET28a plasmid (commercial off-the-shelf plasmid), DH5α and BL21 (DE3) (purchased from Shanghai Sangon Biotech), high-fidelity enzyme KOD (purchased from Toyobo (Shanghai) Biotechnology Co., Ltd.), kanamycin powder (purchased from BioFroxx Biotechnology), IPTG powder (purchased from Abbkine Biotechnology Co., Ltd.), and HFD feed (purchased from Research diets).

[0029] 1.2 Reorganization AGR2 Gene design Mice were found in the NCBI database. AGR2 The coding region of gene (NM_011783.2) was optimized. AGR2 Gene recombination expression fragments constitute recombination AGR2 The gene, whose nucleotide sequence is shown in SEQ ID NO.1: aaagacaccacagtcaaatctggagccaaaaaggacccaaaggactctcggcccaaactacctcagacactctccagaggttggggcgatcagctcatctggactcagacatacga agaagctttatacagatccaagacaagcaacagacccttgatggtcattcatcacttggacgaatgcccacacagtcaagccttaaagaaagtgtttgctgaacataaagaaatcc agaaattggcagagcagtttgttctcctcaacctggtctatgaaacaactgacaagcacctttctcctgatggccagtatgtccccagaattgtgtttgtagacccatccctgacg gtgagggcagacatcactggacgatactcaaaccggctctacgcttatgaaccttctgacacagctctgttgtacgacaacatgaagaaagctctcaagctgctaaagacagaattg Reorganization AGR2 The amino acid sequence of the recombinant AGR2 protein (rAGR2) expressed by the gene is shown in SEQ ID NO.2: KDTTVKSGAKKDPKDSRPKLPQTLSRGWGDQLIWTQTYEEALYRSKTSNRPLMVIHHLDECPHSQALKKVFAEHKEIQKLAEQFVLLNLVYETTDKHLSPDGQYVPRIVFVDPSLTVRADITGRYSNRLYAYEPSDTALLYDNMKKALKLLKTEL 1.3 Construction of recombinant strains expressing rAGR2 PCR amplification was performed using mouse small intestinal tissue cDNA as a template to obtain the above recombinant DNA. AGR2 The amplified DNA sequence was ligated into the pET28a plasmid vector. The ligation product was transformed into DH5α competent E. coli cells. Single clones were picked, cultured, and identified by colony PCR. Positive clones were screened and sent to the company for sequencing verification to obtain the correctly constructed recombinant plasmid pET28a-mAGR2. The recombinant plasmid pET28a-mAGR2 was then introduced into E. coli BL21(DE3) via transformation. AGR2 The recombinant strain of the gene was named pET28a-mAGR2-BL21.

[0030] 1.4 Detection of rAGR2 expression in recombinant strain The pET28a-mAGR2-BL21 strain was inoculated into 1 L LB medium containing kanamycin and cultured until OD500. 600 IPTG was added to a final concentration of 0.5 mM at a pH of 0.6 for induction culture at 16℃ for 18 h. After induction, based on the properties of the recombinant AGR2 protein, a lysis buffer: PBS (pH=7.4) was prepared. The bacterial cells were resuspended in the above lysis buffer and homogenized using a high-pressure homogenizer. Subsequently, the rAGR2 protein was purified using a His affinity chromatography column under low temperature conditions. Finally, the protein was eluted with imidazole. SDS-PAGE gel analysis showed that the rAGR2 protein had good solubility and high purity. The purified protein underwent endotoxin removal treatment, and the endotoxin content was measured. Only after meeting the standards was it used for in vivo injection in mice.

[0031] 2. The role of rAGR2 in sarcopenia and sarcopenia-like phenotype 2.1 Effects of rAGR2 protein on senile sarcopenia 18-month-old C57BL / 6 mice were intraperitoneally injected with PBS and rAGR2, respectively, every other day, at a concentration of 1 mg / kg for 45 consecutive days. Figure 5 A). There was no significant difference in body weight between the two groups. Figure 5 B), body composition analysis using nuclear magnetic resonance MRI showed a significant increase in lean meat content in the rAGR2 treatment group (B), Figure 5 C), fat content decreased ( Figure 5 D). Grip strength was measured using a grip strength meter, and the results showed that the grip strength of mice treated with rAGR2 was significantly increased ( Figure 5 E). After sacrificing mice, it was found that the quadriceps and gastrocnemius muscles of the treated group mice were significantly enlarged (E). Figure 5 FG), while there were no significant changes in the heart and kidneys ( Figure 5 H). The quadriceps femoris muscle of mice was fixed, embedded, sectioned, and stained with H&E. Figure 6 A), the area of ​​muscle fibers was statistically analyzed, and the results showed that the number of large-area muscle fibers increased and the number of small-area muscle fibers decreased in the muscle tissue of mice treated with AGR2 recombinant protein. Figure 6 B). These results indicate that the AGR2 recombinant protein significantly improves the sarcopenic phenotype in aged mice.

[0032] 2.2 Effects of rAGR2 on obesity-related sarcopenia Eight-week-old male C57BL / 6J mice were fed with HFD for 8 weeks and then divided into two groups: an experimental group and a control group. The experimental group was intraperitoneally injected with rAGR2 at a concentration of 1 mg / kg for 12 weeks, while the control group was injected with PBS solution (…). Figure 7 During the period, the body weight of the mice was measured, and it was found that there was no significant difference in body weight and food intake between the control group and the group injected with AGR2 recombinant protein. Figure 7 BC). Body composition analysis using MRI 11 weeks later revealed a significant decrease in fat mass and a significant increase in lean meat mass in mice injected with rAGR2. Figure 7 D). Mice were sacrificed after 12 weeks, and tissues were collected and their weights quantified. Weighing of the mice and their organs revealed that although the mice's body weight remained unchanged, muscle mass significantly improved. Figure 8 A) Increased gastrocnemius and quadriceps muscles, and significantly reduced fat mass in various areas ( Figure 8 B).

[0033] The above results indicate that in the treatment of sarcopenia, supplementation with exogenous recombinant AGR2 protein can effectively alleviate muscle loss, increase muscle strength, and improve muscle function without adversely affecting body weight and food intake.

[0034] In summary, this invention first discovered a correlation between AGR2 protein and sarcopenia in clinical research, and then confirmed through animal experiments that AGR2 protein deficiency can induce sarcopenia. Based on this, supplementing with exogenous recombinant AGR2 protein can reduce fat mass, increase lean meat mass, and enlarge the gastrocnemius and quadriceps muscles without adversely affecting body weight or food intake, while simultaneously enhancing muscle strength and function, thereby effectively improving the sarcopenia-like phenotype. Therefore, developing recombinant AGR2 protein into a drug for treating sarcopenia has significant clinical value.

Claims

1. The use of recombinant AGR2 protein in the preparation of drugs for treating sarcopenia, characterized in that: The amino acid sequence of the recombinant AGR2 protein is shown in SEQ ID NO.

2.

2. The use according to claim 1, characterized in that: The drug in question is a medication that reduces body fat.

3. The use according to claim 1, characterized in that: The drug mentioned is a drug that increases lean meat content.

4. The use according to claim 1, characterized in that: The drug is a drug that increases the strength of the gastrocnemius and / or quadriceps muscles.

5. The use according to claim 1, characterized in that: The drug in question is a drug that increases muscle mass.

6. The use according to claim 1, characterized in that: The drug in question is a medication that enhances muscle strength.

7. The use according to claim 1, characterized in that: The drug is a medication that improves muscle function.

8. A drug for treating sarcopenia, characterized in that: It is a formulation prepared with recombinant AGR2 protein as the active ingredient and pharmaceutically acceptable excipients; the formulation includes injectable formulations; The amino acid sequence of the recombinant AGR2 protein is shown in SEQ ID NO.

2.

9. A method for constructing an animal model of sarcopenia, characterized in that: The method is to construct AGR2 Gene knockout animals; the aforementioned AGR2 The gene's accession number on NCBI is NM_011783.2; the animals mentioned include mice.

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