MRNA (messenger ribonucleic acid) medicine capable of expressing horse follicle-stimulating hormone as well as preparation method and application of mRNA medicine

Abstract

The invention discloses an mRNA drug capable of expressing horse follicle-stimulating hormone, a preparation method and application thereof, and belongs to the technical field of therapeutic drugs, the mRNA drug comprises an mRNA molecule and a drug carrier, the mRNA molecule comprises a 5'terminal untranslated region, a coding RNA gene of horse follicle-stimulating hormone protein, a 3 'terminal untranslated region and polyadenylic acid, and the drug carrier comprises lipid nanoparticles; compared with the prior art, the invention develops a novel follicle-stimulating hormone supplementing method by taking mRNA as a way, and has the characteristics of high flexibility, safety and efficiency.

Description

Technical Field

[0001] The invention belongs to the technical field of therapeutic drugs, and specifically relates to an mRNA drug capable of expressing equine follicle-stimulating hormone, a preparation method and application thereof. Background Art

[0002] Follicle-stimulating hormone is a glycoprotein hormone secreted by the anterior pituitary gland. Its main function is to promote the growth and development of follicles in the ovaries and the secretion of estrogen.

[0003] Follicle-stimulating hormone is often used in assisted reproductive technology to promote the simultaneous maturation of multiple follicles to improve the pregnancy rate. It is also often used to treat ovarian dysfunction, such as ovarian quiescence, persistent corpus luteum and follicle development arrest. With the development of assisted reproductive technology and the increasing demand for improving animal reproductive efficiency, the production of follicle-stimulating hormone has become particularly important.

[0004] At present, the production method of follicle-stimulating hormone is generally to extract and purify the blood collected from pregnant mares. This method has a single production technology and unstable safety.

[0005] In view of this, an mRNA drug capable of expressing equine follicle-stimulating hormone, a preparation method and an application thereof are designed to solve the above problems. Summary of the invention

[0006] In order to solve the problems raised in the above background technology, the present invention provides an mRNA drug capable of expressing equine follicle-stimulating hormone, a preparation method and application thereof, which has the characteristics of high flexibility, safety and efficiency.

[0007] Another object of the present invention is to provide a method for preparing an mRNA drug capable of expressing equine follicle-stimulating hormone and its application.

[0008] To achieve the above-mentioned purpose, the present invention provides the following technical scheme: an mRNA drug capable of expressing equine follicle-stimulating hormone, comprising: an mRNA molecule and a drug carrier, wherein the mRNA molecule comprises a 5' untranslated region, an RNA gene encoding equine follicle-stimulating hormone protein, a 3' untranslated region and polyadenylic acid, and the drug carrier comprises lipid nanoparticles.

[0009] Furthermore, the RNA gene encoding the horse follicle-stimulating hormone protein includes an RNA gene encoding the α subunit of the horse follicle-stimulating hormone protein and an RNA gene encoding the β subunit of the horse follicle-stimulating hormone protein.

[0010] Furthermore, the sequence of the 5' non-translated region is shown as SEQ ID NO.1, the sequence of the RNA gene encoding the α subunit of horse follicle-stimulating hormone protein is shown as SEQ ID NO.7, the sequence of the RNA gene encoding the β subunit of horse follicle-stimulating hormone protein is shown as SEQ ID NO.10, the sequence of the 3' non-translated region is shown as SEQ ID NO.2, and the sequence of polyadenylation is shown as SEQ ID NO.3.

[0011] The method for preparing the mRNA drug capable of expressing equine follicle-stimulating hormone comprises the following steps:

[0012] S1: Construction of the mRNA drug expression vector plasmid of equine follicle-stimulating hormone;

[0013] S2: The plasmid is digested with Bsa I restriction endonuclease to linearize the circular DNA, and then recovered using a DNA fragment recovery kit;

[0014] S3: The linearized DNA molecules were transcribed in vitro using T7 High Yield RNA Transcription Kit to obtain mRNA with N1-Me-Pseudo UTP modification;

[0015] S4: The mRNA with N1-Me-Pseudo UTP modification is capped by Cap101;

[0016] S5: Extraction of capped mRNA molecules by lithium chloride precipitation;

[0017] S6: The cap-containing mRNA molecules are packaged into lipid nanoparticles to prepare LNP-mRNA drugs, which are mRNA drugs that can express equine follicle-stimulating hormone.

[0018] The invention discloses an application of the mRNA drug capable of expressing horse follicle-stimulating hormone in drugs for increasing the content of follicle-stimulating hormone.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The present invention develops a novel follicle-stimulating hormone supplementation method using mRNA as a pathway, which is highly flexible, safe, and efficient compared to the prior art. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a graph showing the results of agarose gel electrophoresis of in vitro transcribed mRNA of the present invention;

[0022] Figure 2This is a diagram of the Western Blot detection result in the in vitro transcribed mRNA protein expression verification experiment of the present invention;

[0023] Figure 3 This is a graph showing the particle size analysis results after LNP-mRNA packaging of the present invention;

[0024] Figure 4 This is a graph showing the results of testing the serum estradiol content in mice immunized with the FSH mRNA drug of the present invention;

[0025] Figure 5 This is a diagram showing the size of the uterus of mice immunized with the FSH mRNA drug of the present invention;

[0026] Figure 6 These are the pathological sections of different tissues after mice were immunized with the FSH mRNA drug of the present invention. DETAILED DESCRIPTION

[0027] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0028] An mRNA drug capable of expressing follicle-stimulating hormone (FSH) comprises an mRNA molecule and a drug carrier, wherein the mRNA molecule comprises a 5' non-translated region, an RNA gene encoding follicle-stimulating hormone (FSH) protein, a 3' non-translated region and polyadenylic acid, and the drug carrier comprises lipid nanoparticles.

[0029] Specifically, the RNA gene encoding the equine follitropin protein includes the RNA gene encoding the α subunit of the equine follitropin protein and the RNA gene encoding the β subunit of the equine follitropin protein.

[0030] Specifically, the sequence of the 5' non-translated region is shown as SEQ ID NO.1, the sequence of the RNA gene encoding the α subunit of horse follicle-stimulating hormone protein is shown as SEQ ID NO.7, the sequence of the RNA gene encoding the β subunit of horse follicle-stimulating hormone protein is shown as SEQ ID NO.10, the sequence of the 3' non-translated region is shown as SEQ ID NO.2, and the sequence of polyadenylation is shown as SEQ ID NO.3.

[0031] A method for preparing an mRNA drug capable of expressing equine follicle-stimulating hormone comprises the following steps:

[0032] S1: Construction of the mRNA drug expression vector plasmid of equine follicle-stimulating hormone;

[0033] S2: The plasmid is digested with Bsa I restriction endonuclease to linearize the circular DNA, and then recovered using a DNA fragment recovery kit;

[0034] S3: The linearized DNA molecules were transcribed in vitro using T7 High Yield RNA Transcription Kit to obtain mRNA with N1-Me-Pseudo UTP modification;

[0035] S4: The mRNA with N1-Me-Pseudo UTP modification is capped by Cap101;

[0036] S5: Extraction of capped mRNA molecules by lithium chloride precipitation;

[0037] S6: The cap-containing mRNA molecules are packaged into lipid nanoparticles to prepare LNP-mRNA drugs, which are mRNA drugs that can express equine follicle-stimulating hormone.

[0038] The invention discloses an mRNA drug capable of expressing horse follicle-stimulating hormone for use in a drug for increasing follicle-stimulating hormone content. Specific embodiments

[0040] Construction of mRNA drug expression vector of horse follicle-stimulating hormone:

[0041] Construction of antigen expression vector plasmid:

[0042] 1.1. Entrust Shanghai Sangon Biotechnology Co., Ltd. to synthesize the following gene sequences:

[0043] From 5' to 3' end, they are T7 promoter, 5' untranslated region, FSH-α, 3' untranslated region and polyadenylic acid, wherein the sequences of T7 promoter, 5' untranslated region, FSH-α, 3' untranslated region and polyadenylic acid are shown in SEQ ID NO.4, SEQ ID NO.1, SEQ ID NO.5, SEQ ID NO.2 and SEQ ID NO.3 respectively;

[0044] From 5' to 3' end, they are T7 promoter, 5' untranslated region, FSH-β, 3' untranslated region and polyadenylic acid, wherein the sequences of T7 promoter, 5' untranslated region, FSH-β, 3' untranslated region and polyadenylic acid are shown in SEQ ID NO.4, SEQ ID NO.1, SEQ ID NO.8, SEQ ID NO.2 and SEQ ID NO.3 respectively;

[0045] 1.2. Beijing Qingke Biotechnology Co., Ltd. was commissioned to synthesize the following primers, see Table 1:

[0046] Table 1: Primer sequence list

[0047]

[0048] The above plasmid was used as a template for PCR amplification reaction to obtain the gene sequence of horse follicle stimulating hormone, which is FSH. Hereinafter, FSH is used to represent horse follicle stimulating hormone. The gene sequence of horse follicle stimulating hormone is the FSH gene sequence. The enzyme of the reaction system is PrimeSTAR Max DNA Polymerase from TAKARA. The reaction system is shown in Table 2:

[0049] Table 2: PCR reaction system table

[0050]

[0051]

[0052] 1.3. Double digestion: XhoⅠ and KpnⅠ restriction endonucleases from TransGen were used to recover the DNA fragment containing the FSH gene and the pUC57 vector fragment by agarose gel electrophoresis;

[0053] 1.4. DNA was recovered from PCR products using 1% agarose gel electrophoresis, where the gel recovery kit was selected from TransGen;

[0054] 1.5. Homologous recombination: Takara's T4 DNA ligase was used to carry out ligation reaction using the pUC57 vector fragment recovered in 1.3 and the FSH fragment recovered in 1.4. The conditions were set at 25°C for 15 min. After the reaction, it was added to DH5α competent cells. The competent cells were selected from Beijing Qingke Biotechnology Co., Ltd. After ice bathing for 30 min, it was immediately heat-shocked at 42°C for 90 s, and then immediately ice bathed for 2 min. After the reaction, 1 mL of LB culture solution was added, and the cells were shaken at 37°C and 200 rpm for 30 min in a shaker, followed by centrifugation at 2000 rpm for 10 min. Finally, the cells were resuspended in 100 μL LB and spread on solid agar culture plates. The culture plates were selected from kanamycin resistance (50 μg / mL). They were incubated at 37°C until the next day, and colonies were picked.

[0055] 1.6. Perform plasmid mini-extraction, extract the plasmid from 2 mL of bacterial solution, use the DNA mini-extraction kit from TransGen, use XhoⅠ and KpnⅠ restriction endonucleases for double enzyme digestion identification, and sequence the recombinant plasmid using Beijing Qingke Company to verify that the sequence is correct;

[0056] Synthesize horse follicle-stimulating hormone mRNA:

[0057] The plasmid obtained above was digested with Bsa I restriction endonuclease from Yisheng Biotechnology Co., Ltd. This process will lead to linearization of the plasmid. The reaction system is shown in Table 3. DNA was recovered after reacting at 37.0°C for 1 hour. The kit was selected from TransGen Co., Ltd.

[0058] Table 3: Plasmid linearization reaction system

[0059]

[0060] The in vitro transcription was performed using the T7 Transcription Kit product of Yisheng Biotechnology Co., Ltd. The reaction system is shown in Table 4. Cap analogs were added to the synthesized mRNA, Cap101 was added to the reaction system, and the synthesis conditions were set at 37°C for 2h. After the reaction, 7.5M lithium chloride with a volume of 1.5 times the reaction system was added to the synthesized product to precipitate the mRNA. The mRNA was resuspended in DEPC water, and the RNA concentration was measured. The final mRNA product was stored at -80°C;

[0061] Table 4: In vitro transcription reaction system

[0062]

[0063]

[0064] Verify the quality of in vitro transcribed mRNA by 1% agarose gel electrophoresis. Figure 1 As shown, Marker represents RNA marker 6000, 1 represents FSH-β mRNA, and 2 represents FSH-α mRNA. The results show that the in vitro transcribed mRNA band size is correct and clear and single, proving that the synthesized mRNA has good quality. The mRNA sequences are shown in SEQ ID NO.7 and SEQ ID NO.10;

[0065] The DNA sequence of the 5' untranslated region is shown in SEQ ID NO.1:

[0066] GAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCGCTAGCCTCGAG

[0067] The DNA sequence of the 3' untranslated region is shown in SEQ ID NO.2:

[0068] GATATCTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGC CCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTG

[0069] The sequence of polyadenylation is shown in SEQ ID NO.3, which contains 104 bases A:

[0070] AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[0071] The DNA sequence of the T7 promoter is shown in SEQ ID NO.4:

[0072] TAATACGACTCACTATAGG

[0073] The DNA sequence of FSH-α is shown in SEQ ID NO.5:

[0074] ATGGACTACTATAGAAACACGCCGCCGTCATCCTTGCTACACTTAGTGTGTTCCTCCACATACTTCATTCCTTCCCTGACGGAGAATTTACTACACAGGATTGTCCTGAGTGTAAGTTGCGCGAGAACAAGTACTTTTTTAAGCTTGGCGTACCCATCTATCAGTGTAAGGGCTGCTGCTTTTCAAGGGCCT ATCCAACACCAGCTAGATCAAGAAAGACTATGCTTGTACCGAAGAATATCACCTCTGAAAGCACTTGCTGCGTCGCGAAAGCCTTTATTAGGGTTACCGTGATGGGAAACATTAAGCTCGAAAATCACACCCAGTGCTACTGTAGTACCTGCTATCACCACAAGATAGATTACAAAGATGATGACGACAAGTAA

[0075] The amino acid sequence of FSH-α is shown in SEQ ID NO.6:

[0076] MDYYRKHAAVILATLSVFLHILHSFPDGEFTTQDCPECKLRENKYFFKLGVPIYQCKGCCFSRA YPTPARSRKTMLVPKNITSESTCCVAKAFIRVTVMGNIKLENHTQCYCSTCYHHKIDYKDDDDK

[0077] The mRNA sequence of FSH-α is shown in SEQ ID NO.7:

[0078] GAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCGCUAGCCUCGAGGCCACCAUGGACUACUAUAGAAAACACGCCGCCGUCAUCCUUGCUACACUUAGUGUGUUCCUCCACAUACUUCAUUCCUUCCCUGACGGAGAAUUUACUACACAGGAUUGUCCUGAGUGUAAGUUGCGCGAGAACAAGUACUUUUUUAAGCUUGGCGUACCCAUCUAUCAGUGUAAGGGCUGCUGCUUUUCAAGGGCCUAUCCAACACCAGCUAGAUCAAGAAAGACUAUGCUUGUACCGAAGAAUAUCACCUCUGAAAGCACUUGCUGCGUCGCGAAAGCCUUUAUUAGGGUUACCGUGAUGGGAAACAUUAAGCUCGAAAAUCACACCCAGUGCUACUGUAGUACCUGCUAUCACCACAAGAUAGAUUACAAAGAUGAUGACGACAAGUAAGGUACCGAUAUCUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAUCUAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[0079] The DNA sequence of FSH-β is shown in SEQ ID NO.8:

[0080] ATGAAAAGTGTTCAGTTTTGCTTCCTTTTTTGCTGCTGGAAAGCCGTGTGCTGTAACAGCTGCGAGCTGACAAATATAACCATCGCAGTAGAGAAGGAAGAGTGCGGCTTCTGCATCAGTATCAACACTACATGGTGCGCCGGCTACTGTTATACCAGGGATCTGGTGTATAAAGATCCAGCCCGGCCAAATATCCAGAAAACTTGCACCTTCAAAGAGCTCGTATATGAGACAGTGAAGGTCCCTGGTTGCGCACATCATGCCGACTCCCTCTACACATACCCTGTGGCAACAGCATGTCATTGCGGAAAGTGTAATTCCGACTCCACTGACTGCACAGTGCGAGGTCTGGGACCTAGTTACTGCTCTTTTGGCGACATGAAGGAAGATTATAAGGACGACGATGACAAATAA

[0081] The amino acid sequence of FSH-β is shown in SEQ ID NO.9:

[0082] MKSVQFCFLFCCWKAVCCNSCELTNITIAVEKEECGFCISINTTWCAGYCYTRDLVYKDPARPNIQKTCTFKELVYETVKVPGCAHHADSLYTYPVATACHCGKCNSDSTDCTVRGLGPSYCSFGDMKEDYKDDDDK

[0083] The mRNA sequence of FSH-β is shown in SEQ ID NO.10:

[0084] ;

[0085] Preparation of mRNA drugs capable of expressing equine follicle-stimulating hormone:

[0086] The plasmid obtained during the construction of the expression vector was digested with Bsa I restriction endonuclease to linearize the circular DNA, which was then recovered using a DNA fragment recovery kit;

[0087] The linearized DNA molecules were transcribed in vitro, and the T7 High Yield RNA Transcription Kit (N1-Me-Pseudo UTP) product was selected from Yisheng Company, and the obtained mRNA was modified with N1-Me-Pseudo UTP;

[0088] At the same time, Cap101 was added to cap the in vitro transcribed mRNA. Cap101 was selected from Cangzhou Weixin Biological Company.

[0089] Cap-containing mRNA molecules were extracted by lithium chloride precipitation;

[0090] The obtained mRNA molecules are packaged into lipid nanoparticles to prepare LNP-mRNA drugs;

[0091] The experimental operation is as follows:

[0092] The alcohol phase was prepared by dissolving the lipids in anhydrous ethanol in a ratio of cationic lipid (SM102): distearoylphosphatidylcholine (DSPC): cholesterol: DMG-2000 = 50:10:38.5:1.5 (mass ratio);

[0093] For the aqueous phase preparation, citric acid buffer (50 mM) with pH = 4.0 was selected as the solute for dissolving mRNA;

[0094] The mRNA was packaged using a microfluidic device with an alcohol phase:water phase (volume ratio of 1:3);

[0095] Use RNase-free PBS buffer for dilution and 30kDa ultrafiltration tube for concentration.

[0096] On this basis, an equal volume of PBS solution with a sucrose concentration of 20% was added to adjust the mRNA concentration to 60 μg / ml and the sucrose concentration to 10%, and the mixture was filtered using a 0.22 μm filter membrane to finally obtain the FSH LNP-mRNA drug, which was then packaged and stored at -20°C.

[0097] The above liposome nanoparticles contain at least one of a cationic lipid, distearoylphosphatidylcholine, cholesterol and DMG-2000;

[0098] The dynamic light detection particle size distribution test results are shown in the attached Figure 2 As shown, Marker represents protein marker, 1 represents blank control, 2 represents FSH-α mRNA, and 3 represents FSH-β mRNA. The results show that the prepared FSH LNP-mRNA drug particles are uniform, the particle size of FSH-α LNP-mRNA is about 120nm, and the particle size of FSH-β LNP-mRNA is about 100nm.

[0099] Expression verification of horse follicle-stimulating hormone protein:

[0100] The in vitro transcribed mRNA was transfected into HEK-293T cells for expression verification, and the transfection reagent was selected from TransGen Company;

[0101] 293T cells were evenly plated in a 24-well plate, and RNA was transfected using PEI. 0.8 μg mRNA was transfected into each well. After 48 hours, transfected cells were collected, and loading buffer was added to treat the samples. SDS-PAGE electrophoresis was performed. The antibodies used were mouse anti-FLAG tag antibody from Jinbiology and HRP-labeled goat anti-mouse secondary antibody. After incubation, color development was performed, and the Western Blot results were obtained as shown in the attached figure. Figure 3 As shown, A represents the particle size result of FSH-αmRNA-LNP, and B represents the particle size result of FSH-βmRNA-LNP. The results show that the size of the protein band after transfection of FSH mRNA is consistent with the expectation, indicating that FSH mRNA can be expressed in cells.

[0102] Mouse immune evaluation experiment:

[0103] The FSH mRNA prepared in the process of FSH protein expression verification was used to immunize BALB / c mice, and the immune effect was observed and evaluated;

[0104] Fifteen 4-week-old SPF Kunming mice were purchased from Liaoning Changsheng Biotechnology Co., Ltd. and randomly divided into 3 groups, 5 mice / group. 20 μg mRNA drugs or 100 μL PBS were injected into the leg muscles for immunization, and 20 U horse chorionic gonadotropin (PMSG) was injected into the positive control group intraperitoneally. The increase of PMSG and FSH levels will significantly promote the content of sex hormones in the body.

[0105] On the first day and 7 days after immunization, the blood of mice was collected and the serum was separated. The estradiol content in the serum was detected by ELISA. The estradiol ELISA kit was selected from Shanghai ELISA Biological. The results are shown in the attached figure. Figure 4 As shown, A represents the serum estradiol content one day after immunization, B represents the serum estradiol content seven days after immunization, * represents P<0.05, ** represents P<0.01. The results show that the estradiol content in the serum of mice increased significantly on the first day after mRNA and PMSG immunization of mice. The serum estradiol content of mice in the normal group was about 25pmol / L, and that of the positive control group was about 34pmol / L, which was higher than that of the blank control group (P<0.01), while that of the FSH immunization group could reach about 37pmol / L, which was significantly higher than that of the positive control group (P<0.05), indicating that the mRNA effect was significant.

[0106] On the 7th day, the estradiol levels in the blank control group and the positive control group were both about 25pmol / L, while that in the FSH mRNA group was up to 31pmol / L, which was significantly higher than that in the two groups (P<0.05), indicating that the duration of the effect of FSH mRNA was longer than that of PMSG.

[0107] The above results indicate that FSH mRNA can express FSH in mice and can maintain it for a long time;

[0108] Fourteen days after the injection, the mice were killed and their ovarian tissues were isolated and photographed. The results are shown in the attached figure. Figure 5 As shown, compared with the blank control, the uterus size of mice increased significantly after injection of mRNA-LNP, indicating that FSH can significantly promote the sexual maturity of mice;

[0109] Further observations on the safety of mRNA were conducted by observing pathological sections of different tissues. Figure 6 As shown, after mice were immunized with FSH mRNA, no obvious damage was observed to the liver, spleen, kidney and other organs of the mice, indicating that the safety of FSH mRNA drugs is relatively high.

[0110] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. An mRNA drug capable of expressing equine follicle-stimulating hormone, characterized in that: include: mRNA molecules and drug carriers, wherein the mRNA molecules include a 5' untranslated region, an RNA gene encoding follicle-stimulating hormone protein, a 3' untranslated region and polyadenylic acid, and the drug carrier includes lipid nanoparticles.

2. The mRNA drug capable of expressing equine follicle-stimulating hormone according to claim 1, characterized in that: The RNA gene encoding the horse follicle stimulating hormone protein includes an RNA gene encoding the α subunit of the horse follicle stimulating hormone protein and an RNA gene encoding the β subunit of the horse follicle stimulating hormone protein.

3. The mRNA drug capable of expressing equine follicle-stimulating hormone according to claim 2, characterized in that: The sequence of the 5' non-translated region is shown as SEQ ID NO.1, the sequence of the RNA gene encoding the α subunit of horse follicle-stimulating hormone protein is shown as SEQ ID NO.7, the sequence of the RNA gene encoding the β subunit of horse follicle-stimulating hormone protein is shown as SEQ ID NO.10, the sequence of the 3' non-translated region is shown as SEQ ID NO.2, and the sequence of polyadenylation is shown as SEQ ID NO.

3.

4. A method for preparing an mRNA drug capable of expressing equine follicle-stimulating hormone according to any one of claims 1 to 3, characterized in that: The following steps are involved: S1: Construction of the mRNA drug expression vector plasmid of equine follicle-stimulating hormone; S2: The plasmid is digested with Bsa I restriction endonuclease to linearize the circular DNA, and then recovered using a DNA fragment recovery kit; S3: The linearized DNA molecules were transcribed in vitro using T7 High Yield RNA Transcription Kit to obtain mRNA with N1-Me-Pseudo UTP modification; S4: The mRNA with N1-Me-Pseudo UTP modification is capped by Cap101; S5: Extraction of capped mRNA molecules by lithium chloride precipitation; S6: The cap-containing mRNA molecules are packaged into lipid nanoparticles to prepare LNP-mRNA drugs, which are mRNA drugs that can express equine follicle-stimulating hormone.

5. Use of an mRNA drug capable of expressing equine follicle-stimulating hormone according to any one of claims 1 to 3 in a drug for increasing the content of follicle-stimulating hormone.

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