MRNA vaccine for regulating and controlling reproductive capacity of animals and application of mRNA vaccine

Abstract

The invention discloses an mRNA vaccine for regulating and controlling the reproductive capacity of animals and application of the mRNA vaccine, the antigen of the mRNA vaccine is a GnRH fusion protein antigen formed by coupling GnRH and Fc protein or Flden protein, the immunogenicity and stability of GnRH are improved, and experiments prove that the prepared mRNA vaccine can well control the level of sex hormones so as to regulate and control the reproductive capacity of the animals, so that the animal reproductive capacity can be regulated and controlled. The safety is good; experiments further show that the level of antibodies induced by the GnRH-Fc mRNA vaccine is higher than that of a GnRH-Folden mRNA vaccine group, and the GnRH-Fc mRNA vaccine can reduce the hormone level more effectively compared with the GnRH-Folden mRNA vaccine, so that the reproductive capacity of animals can be better controlled.

Description

Technical Field

[0001] This invention belongs to the field of viral vaccine technology, and relates to an mRNA vaccine and its application, specifically an mRNA vaccine for regulating animal reproductive capacity and its application. Background Technology

[0002] Veterinary castration is a veterinary surgical procedure that eliminates the sexual desire and reproductive capacity of livestock through surgery or medication. Its main purposes include making livestock more docile and hardworking, easier to manage; improving meat quality and increasing yield; controlling mating behavior within the herd, which is beneficial for the breeding and selection of superior breeds. Surgical castration is a traditional method that involves surgically removing the testes of male animals to rapidly lower serum testosterone levels. However, it is irreversible. Furthermore, traditional surgical castration methods may cause pain and stress to animals, as well as a long recovery period and the risk of postoperative infection. Medical castration, on the other hand, achieves its therapeutic purpose by inhibiting the production of androgens from the male testes and adrenal glands, or by suppressing the secretion of estrogen from the uterus in female animals. Medical castration is reversible and allows for better control of treatment side effects.

[0003] Veterinary castration injections are a common method of medical castration. They are administered directly to the testicular parenchyma or ovary, causing necrosis of the spermatocytes and spermatids or ceasing estrogen secretion from the ovary, thus achieving castration. This includes the use of gonadotropin-releasing hormone (GnRH) agonists or antagonists. Compared to traditional surgical castration, GnRH castration has several advantages, particularly in terms of animal welfare. Surgical castration can cause pain, infection risks, and stress in animals, while GnRH castration avoids these problems. GnRH injection castration is a method of controlling animal reproductive function through injection. In summary, GnRH injection castration, as an emerging castration method, is not only effective but also more in line with animal welfare requirements, and shows promise as an alternative to traditional surgical castration.

[0004] GnRH castration involves actively injecting the animal to induce the production of anti-GnRH antibodies. These antibodies neutralize GnRH in the body, thereby inhibiting or terminating the function of the pituitary-gonadal axis. This leads to gonadal atrophy, terminating gameteogenesis and achieving castration. Studies have shown that GnRH castration can significantly reduce the levels of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) in animals, causing gonadal degeneration and atrophy, affecting reproductive function, and thus achieving castration. The mechanism of GnRH castration involves the KiSS-1 / GPR54 system, an important regulatory system for reproductive system development. GnRH neurons are directly regulated by kisspeptins, which bind to their receptor GPR54, activating GnRH neurons, stimulating the release of GnRH, and subsequently regulating the release of LH and FSH. Active injection can reduce the levels of these hormones, thereby achieving castration. Furthermore, the reversibility of GnRH castration is also an important aspect of research. Studies have shown that after animals are actively injected with GnRH, the anti-GnRH antibodies produced begin to decline after a certain period of time, and after a certain point in time, almost no antibodies remain. This suggests that the effect of GnRH castration may gradually weaken over time.

[0005] mRNA vaccines possess immunogenicity, potentially reducing reliance on traditional adjuvants and thus mitigating the side effects of immune responses. Furthermore, since GnRH-based castration is somewhat reversible, precise regulation of reproductive function can be achieved by adjusting the dosage and frequency of mRNA vaccine immunizations. Therefore, developing an mRNA vaccine for GnRH-based castration to reduce the safety risks of current surgical methods and to effectively control reproductive function through dosage and frequency of immunizations has become a current research trend. Summary of the Invention

[0006] The purpose of this invention is to provide an mRNA vaccine for regulating animal reproductive capacity and its application, which can increase the immunogenicity and stability of GnRH, thereby effectively controlling the level of sex hormones and thus regulating reproductive capacity, and has good safety.

[0007] To achieve the above objectives, a specific embodiment of the present invention provides the following technical solution:

[0008] On one hand, a polynucleotide includes a first nucleotide encoding a GnRH fusion protein, said GnRH fusion protein including a GnRH protein or an immunogenic fragment thereof, and further including a tag protein or an immunogenic fragment thereof coupled to said GnRH protein or immunogenic fragment thereof, said GnRH protein or immunogenic fragment thereof having an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology with SEQ ID NO:1.

[0009] In one or more embodiments of the present invention, the tag protein is an Fc protein or a Folden protein, the amino acid sequence of the Fc protein is shown in SEQ ID NO:2, and the amino acid sequence of the Folden protein is shown in SEQ ID NO:3.

[0010] In one or more embodiments of the present invention, the GnRH protein or its immunogenic fragment is coupled to the tag protein or its immunogenic fragment via a linker, wherein the linker has an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology with SEQ ID NO:15.

[0011] In one or more embodiments of the present invention, when the tag protein is an Fc protein, the amino acid sequence of the GnRH fusion protein has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology with SEQ ID NO:4, or

[0012] When the tag protein is a Folden protein, the amino acid sequence of the GnRH fusion protein has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology with SEQ ID NO:5.

[0013] In one or more embodiments of the present invention, when the tag protein is an Fc protein, the sequence of the first nucleotide has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology with SEQ ID NO: 6 or 7; or

[0014] When the tagged protein is a Folden protein, the sequence of the first nucleotide has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology with SEQ ID NO:8 or 9.

[0015] In one or more embodiments of the present invention, the 5' end of the first nucleotide is further connected to a second nucleotide sequence encoding a signal peptide;

[0016] Preferably, the signal peptide is tPA, IL-2, IL-6 or a neuropeptide, and the neuropeptide is preferably VIP, CGRP or NPY;

[0017] More preferably, the 5' end of the second nucleotide sequence is connected to a 5' UTR sequence, the nucleotide sequence of which is preferably as shown in SEQ ID NO:10, or

[0018] The first nucleotide is further connected to a 3' UTR sequence at its 3' end, and the end of the 3' UTR sequence is further connected to a PolyA sequence. The nucleotide sequence of the 3' UTR sequence is preferably as shown in SEQ ID NO:11.

[0019] Preferably, the 5' end of the 5' UTR sequence is provided with a T7 promoter, the nucleotide sequence of which is shown in SEQ ID NO:12, and the polynucleotide is preferably provided with a FLAG tag.

[0020] In one or more embodiments of the present invention, the polynucleotide is DNA or RNA;

[0021] Preferably, when the polynucleotide is RNA, the 5' end of the 5' UTR sequence of the RNA is further provided with a 5' capping structure, which is preferably Cap1 (methylation of the ribose of the neighboring nucleotide of m7GpppN), Cap2 (methylation of the ribose of the second nucleotide downstream of m7GpppN), Cap3 (methylation of the ribose of the third nucleotide downstream of m7GpppN), Cap4 (methylation of the ribose of the fourth nucleotide downstream of m7GpppN) or Cap 101.

[0022] On the other hand, the present invention provides a carrier constructed from the above-mentioned polynucleotides.

[0023] In another aspect, the present invention provides a cell derived from the aforementioned carrier.

[0024] In another aspect, the present invention provides a composition for regulating reproductive capacity, comprising the above-described polynucleotide or mRNA transcribed from the above-described vector.

[0025] In one or more embodiments of the present invention, the composition is an mRNA vaccine comprising mRNA expressing a GnRH fusion protein.

[0026] In one or more embodiments of the present invention, the mRNA sequence expressing the GnRH fusion protein has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology with SEQ ID NO:7 or SEQ ID NO:9.

[0027] In one or more embodiments of the present invention, the composition further includes a pharmaceutically acceptable carrier, preferably a liposome, wherein the mRNA in the mRNA vaccine is encapsulated in liposome nanoparticles of the liposome, and the particle size of the liposome nanoparticles is preferably 50-200 nm.

[0028] Preferably, the liposome nanoparticles are selected from at least one of cationic lipids, distearate phosphatidylcholine (DSPC), cholesterol, and DMG-2000;

[0029] More preferably, the liposome nanoparticles are composed of the following components in parts by weight: 30-80 parts cationic lipids, 5-15 parts distearate phosphatidylcholine (DSPC), 24-52 parts cholesterol, and 1-2 parts DMG-2000.

[0030] More preferably, the liposome nanoparticles are composed of the following components in parts by weight: 50 parts cationic lipids, 10 parts distearate phosphatidylcholine (DSPC), 38.5 parts cholesterol, and 1.5 parts DMG-2000.

[0031] In another aspect, the present invention also provides the use of the above-mentioned polynucleotide, the carrier of claim 7, or the composition in the preparation of a drug for regulating animal sex hormone levels.

[0032] In another aspect, the present invention also provides a method for using the above-mentioned polynucleotide, vector, composition or mRNA vaccine to regulate reproductive capacity, comprising administering an appropriate amount of polynucleotide, vector or composition to a desired host, said host may be human or animals such as mice, rats, cats, dogs, cattle, sheep, goats, pigs, rabbits, horses, chickens, ducks etc.

[0033] Specifically, it can be administered orally, parenterally, via inhalation spray, topically, rectally, nasally, orally, via vaginally, or through an implanted reservoir.

[0034] Compared with existing technologies, the mRNA vaccine for regulating animal reproductive capacity of the present invention and its application, by coupling a series of tag proteins with GnRH to form GnRH fusion proteins, unexpectedly discovered that GnRH fusion proteins formed by coupling Fc protein or Folden protein with GnRH can increase the immunogenicity and stability of GnRH; and the mRNA vaccine prepared therefrom has been experimentally demonstrated to effectively control the level of sex hormones, thereby regulating reproductive capacity, and has good safety; further experiments show that the antibody level induced by the GnRH-Fc mRNA vaccine is higher than that of the GnRH-Folden mRNA vaccine group, and the GnRH-Fc mRNA vaccine can more effectively reduce hormone levels than the GnRH-Folden mRNA vaccine, thereby better controlling the reproductive capacity of animals;

[0035] This mRNA vaccine effectively controls reproductive function by adjusting the dosage and number of immunizations. It overcomes the problem in existing technologies where the effect of GnRH castration may gradually weaken over time after injecting GnRH antibodies. Furthermore, it is safe to use and is unlikely to cause side effects. Attached Figure Description

[0036] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0037] Figure 1 This is a gel electrophoresis image of the in vitro transcribed mRNA in Example 2 of the present invention, wherein the marker is RNAmarker6000, 1 is GnRH-Folden mRNA, and 2 is GnRH-Fc mRNA;

[0038] Figure 2 This is the result of the verification of protein expression in cells of mRNA in Example 3 of the present invention, wherein Marker is a protein marker, 1 is the denatured GnRH-Folden protein, 2 is the non-denatured GnRH-Folden protein, 3 is the denatured GnRH-Fc protein, and 4 is the non-denatured GnRH-Fc protein.

[0039] Figure 3A This is a graph showing the particle size distribution of GnRH-Fc mRNA-LNP after mRNA encapsulation in Example 4 of this invention.

[0040] Figure 3BThis is a graph showing the particle size distribution of GnRH-Folden mRNA-LNP after mRNA encapsulation in Example 4 of this invention.

[0041] Figure 4 This is a titer diagram of the specific IgG antibody induced in mice after injection of mRNA drug in Example 5 of the present invention;

[0042] Figure 5 This is a graph showing the changes in serum estrogen levels in mice after injection of mRNA drug in Example 5 of the present invention;

[0043] Figure 6 This is an in situ photograph of the uterus of mice after mRNA drug injection in Example 5 of the present invention;

[0044] Figure 7 This is a photograph of the uterus of a mouse after injection of mRNA drug in Example 5 of the present invention;

[0045] Figure 8 This is a statistical chart of uterine weight in mice after mRNA drug injection in Example 5 of the present invention;

[0046] Figure 9 These are photographs of mouse uterine pathological sections from Example 5 of the present invention;

[0047] Figure 10 These are photographs of mouse ovarian pathological sections from Example 5 of the present invention;

[0048] Figure 11 This is a titer diagram of the specific IgG antibody induced in cats after injection of mRNA drug in Example 6 of the present invention;

[0049] Figure 12 This is a graph showing the change in serum estrogen levels in cats after injection of mRNA drug in Example 6 of the present invention. Detailed Implementation

[0050] To enable those skilled in the art to better understand the technical solutions of this invention, the technical solutions of the embodiments of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of this invention.

[0051] The term "mRNA" refers to messenger RNA, which is a type of single-stranded ribonucleic acid transcribed from one strand of DNA as a template. It carries genetic information and can guide protein synthesis.

[0052] The term "3'-UTR" refers to the "3'-untranslated region" or "3'UTR," which refers to the region located at the 3' end of a gene, downstream of the stop codon in a protein-coding region, and which is transcribed but not translated into an amino acid sequence, or the corresponding region in an RNA molecule. The 3'-UTR typically extends from the stop codon of the translation product to a poly(A) sequence that usually attaches after transcription. The 3'-UTR of mammalian mRNA typically has a homologous region known as the AAUAAA hexanucleotide sequence. This sequence may be a poly(A) attachment signal and is often located 10 to 30 bases upstream of the poly(A) attachment site. The 3'-UTR may contain one or more inverted repeats that can fold to create stem-loop structures that act as barriers to ribonucleases or interact with proteins known to enhance RNA stability, such as RNA-binding proteins.

[0053] The term "polyA" refers to a "polyadenylated nucleotide sequence," "poly(A) sequence," or "poly(A) tail," which is a sequence of adenosine residues typically located at the 3' end of an RNA molecule. This invention allows such sequences to attach during RNA transcription via a DNA template based on repeating thymidine residues in a strand complementary to the coding strand; however, these sequences are not normally encoded in DNA but are attached to the free 3' end of RNA post-transcriptional in the cell nucleus by template-independent RNA polymerase.

[0054] The terms “transformation,” “transfection,” and “transduction” have the meanings generally understood by those skilled in the art: the process of introducing exogenous DNA or RNA into a host.

[0055] Unless otherwise specified, the "reagents" or "materials" mentioned in this article are all available from regular commercial channels.

[0056] Example 1. Obtaining the protein expression vector for mRNA drugs

[0057] To improve the translation efficiency of the target mRNA vaccine, we attempted to further optimize the gene sequence of the GnRH protein antigen (amino acid sequence SEQ ID NO: 1) through codon optimization, resulting in an optimized gene sequence of the GnRH protein antigen.

[0058] The designed sequence was then synthesized, ligated to the DNA sequence of the tag protein, and used to construct the antigen expression vector for the mRNA vaccine. Specifically, the following gene sequences were synthesized by Shanghai Sangon Biotech Co., Ltd.: from 5' to 3' ends, they were: T7 promoter (SEQ ID NO:12), 5'UTR (SEQ ID NO:10), kozak sequence (GCCACC), tPA signal peptide sequence (SEQ ID NO:13), GnRH-Fc nucleotide sequence (SEQ ID NO:6) or GnRH-Folden nucleotide sequence (SEQ ID NO:8) or GnRH-GCN4 nucleotide sequence (SEQ ID NO:18) or GnRH-Ferrtin nucleotide sequence (SEQ ID NO:19), 3'UTR (SEQ ID NO:11), and polyA (SEQ ID NO:14). A Bsa I restriction site was introduced after the polyA sequence to facilitate plasmid linearization. Using genetic engineering, the above target gene sequences were constructed into the cloning vector pUC57, and plasmids pUC57-GnRH-Fc, pUC57-GnRH-Folden, pUC57-GnRH-GCN4, and pUC57-GnRH-Ferrtin were constructed. These plasmids were then transformed into competent E. coli cells, and large-scale culture of the two bacteria and large-scale extraction of the two plasmids were performed.

[0059] The second nucleotide sequence has a 5' UTR sequence attached to its 5' end, and the nucleotide sequence of the 5' UTR sequence is preferably as shown in SEQ ID NO:10, or

[0060] The first nucleotide is further connected to a 3' UTR sequence at its 3' end, and the end of the 3' UTR sequence is further connected to a PolyA sequence. The nucleotide sequence of the 3' UTR sequence is preferably as shown in SEQ ID NO:11.

[0061] Preferably, the 5' end of the 5' UTR sequence is provided with a T7 promoter, the nucleotide sequence of which is shown in SEQ ID NO:12, and the polynucleotide is preferably provided with a FLAG tag.

[0062] The DNA sequence of the 5' untranslated region (5'UTR) is shown in SEQ ID NO:10:

[0063] GAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCGCTAGCCTCGAG

[0064] The DNA sequence of the 3' untranslated region (3'UTR) is shown in SEQ ID NO:11:

[0065] GGTACCGATATCTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCC CAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTG

[0066] Polyadenylate (polyA) has the sequence shown in SEQ ID NO:14 and contains 104 bases A:

[0067] AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[0068] The DNA sequence of the T7 promoter is shown in SEQ ID NO:12:

[0069] TAATACGACTCACTATAGG

[0070] The DNA sequence of the signal peptide tPA is shown in SEQ ID NO:13:

[0071] ATGGACGCCATGAAGAGAGGCCTGTGCTGTGTGTTGCTGCTGTGCGGCGCAGTGTTCGTGTCCGCCTCC

[0072] The DNA sequence of the GnRH-Fc protein is shown in SEQ ID NO:6:

[0073] GAGCACTGGAGCTACGGCTTGAGGCCTGGAGGCGGAGGTGGAAGTGGAGGCGGTGGATCTGGAGGTGGCGGAAGCGACAAGACACACACCTGTCCACCTTGTCCAGCACCTGAGCTGTTGGGAGGTCCATCCGTGTTTCTGTTCCCTCCAAAGCCGAAAGACACACTGATGATCTCTCGGACACCTGAAGTGACATGCGTGGTGGTGGACGTGTCTCACGAAGATCCAGAGGTGAAGTTCAACTGGTACGTGGACGGTGTGGAAGTGCACAACGCCAAGACCAAGCCAAGGGAAGAACAGTACAACAGCACCTACAGGGTGGTGAGCGTGCTGACAGTGCTGCACCAAGACTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGAGCAACAAGGCTCTGCCTGCACCTATCGAGAAGACCATCTCCAAGGCCAAAGGCCAGCCTAGAGAGCCTCAGGTGTACACACTGCCACCTTCTCGCGAAGAGATGACCAAGAACCAGGTGAGCCTGACATGCCTGGTGAAGGGCTTCTACCCTAGCGACATTGCCGTGGAATGGGAGTCCAATGGCCAGCCTGAGAACAACTACAAGACCACACCTCCAGTTCTGGACAGCGATGGCTCTTTCTTTCTGTACAGCAAGCTGACCGTGGACAAGTCTCGGTGGCAGCAGGGAAACGTGTTCAGCTGCTCTGTGATGCACGAGGCTTTGCACAACCACTACACACAGAAGTCTCTCTCTCTGTCTCCAGGCAAAGACTACAAAGACGACGACGACAAGTAA

[0074] The amino acid sequence of the GnRH-Fc protein is shown in SEQ ID NO:4:

[0075]

[0076] The italicized and underlined portion is the amino acid sequence of the GnRH protein (SEQ ID NO:1), the regular and bold portion is the amino acid sequence of the Folden protein (SEQ ID NO:2), and the middle portion is the linker (SEQ ID NO:15).

[0077] The mRNA sequence of GnRH-Fc protein is shown in SEQ ID NO:7:

[0078]

[0079] The protein DNA sequence of GnRH-Folden is shown in SEQ ID NO:8:

[0080] GAGCACTGGAGCTACGGCTTGAGGCCTGGAGGTGGCGGTGGATCAGGTGGTGGAGGTTCCGGTGGTGGTGGTTCAGGTGGAGGTGGATCCGGCTACATACCAGAGGCACCTAGAGATGGCCAAGCTTACGTGAGAAAGGACGGCGAATGGGTGCTGCTGTCTACCTTTCTCGACTACAAAGACGACGATGATAAGTAA

[0081] The amino acid sequence of the GnRH-Folden protein is shown in SEQ ID NO:5:

[0082] EHWSYGLRPG GGGGSGGGGSGGGGS GGGGSGYIPEAPRDGQAYVRKDGEWVLLSTFLDYKDDDDK The italicized and underlined portion is the amino acid sequence of the GnRH protein (SEQ ID NO:1), the regular and bold portion is the amino acid sequence of the Folden protein (SEQ ID NO:3), and the middle portion is the linker (SEQ ID NO:15).

[0083] The mRNA sequence of GnRH-Folden protein is shown in SEQ ID NO:9:

[0084] GAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCGCUAGCCUCGAGGCCACCAUGGACGCCAUGAAGAGGGGGCUGUGCUGCGUGCUGCUGCUGUGCGGAGCCGUGUUCGUGAGCGCCUCCAUGGACGCCAUGAAGAGAGGCCUGUGCUGUGUGUUGCUGCUGUGCGGCGCAGUGUUCGUGUCCGCCUCCGAGCACUGGAGCUACGGCUUGAGGCCUGGAGGUGGCGGUGGAUCAGGUGGUGGAGGUUCCGGUGGUGGUGGUUCAGGUGGAGGUGGAUCCGGCUACAUACCAGAGGCACCUAGAGAUGGCCAAGCUUACGUGAGAAAGGACGGCGAAUGGGUGCUGCUGUCUACCUUUCUCGACUACAAAGACGACGAUGAUAAGUAAGGUACCGAUAUCUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[0085] The amino acid sequence of the GnRH-GCN4 protein is shown in SEQ ID NO:16:

[0086] EHWSYGLRPGGGGGSGGGGSGGGGSRMKQIEDKIEEILSKQYHIENEIARIKKLIGDYKDDDDK

[0087] The DNA sequence of the GnRH-GCN4 protein is shown in SEQ ID NO:18:

[0088] GAGCACTGGAGCTACGGCTTGAGGCCTGGAGGTGGTGGAGGTTCCGGTGGTGGTGGTTCAGGTGGAGGTGGATCCCGCATGAAGCAGATCGAGGACAAGATCGAAGAGATTCTGAGCAAGCAGTACCACATCGAGAACGAGATCGCCAGAATCAAGAAACTGATTGGTGACTATAAAGACGATGATGACAAG

[0089] The amino acid sequence of GnRH-Ferrtin protein is shown in SEQ ID NO:17:

[0090] EHWSYGLRPGGGGGSGGGGSGGGGSDIIKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFNDILYKIELIGNENHGLYLADQYVKGIAKSRKSDYKDDDDK

[0091] The DNA sequence of GnRH-Ferrtin protein is shown in SEQ ID NO:19:

[0092] .

[0093] Example 2. mRNA transcription verification experiment

[0094] The plasmids pUC57-GnRH-Fc and pUC57-GnRH-Folden obtained in Example 1 were digested with Bsa I restriction endonuclease from Novizan Biosciences and linearized by reacting at 37°C for 1 hour. The DNA template was recovered using a DNA recovery kit from TransGen. The linearization system is shown in Table 1.

[0095] Table 1. Plasmid linearization reaction system

[0096]

[0097] mRNA was synthesized in vitro using the T7 transcription kit from Yisheng Biotechnology, and a capping structure was added to the mRNA using Cap101 from Cangzhou Weikexin Biotechnology. The reaction was carried out at 37℃ for 3 hours, with specific reaction conditions as shown in Table 2. After the reaction, 1.5 volumes of lithium chloride solution were added to the system, and the mixture was incubated at -20℃ for 1 hour to precipitate the mRNA. Subsequently, the mixture was centrifuged at 10000 rpm for 10 minutes, the supernatant was removed, and the mRNA was redissolved in DEPC-treated water. The RNA concentration was determined using a Nanodrop device and stored at -80℃. A 1% agarose gel was prepared and electrophoresis was performed to evaluate the quality of the synthesized RNA. The results are shown below. Figure 1 As shown. From Figure 1 It can be seen that all five in vitro synthesized mRNA bands were clear, single, and consistent with the expected size.

[0098] Table 2 In vitro transcription reaction system

[0099]

[0100] Example 3. Protein Expression Validation Assay

[0101] The purified in vitro transcribed mRNA was transfected into HEK-293T cells using TransGen's PEI reagent for expression verification. 293T cells were evenly seeded in six-well plates, and 0.8 μg of mRNA was transfected into each well using PEI. After 48 h of transfection, cells were collected. Denaturing gels were loaded with denaturing loading buffer and boiled for 5 minutes before loading. Non-denaturing gels were loaded directly with non-denaturing loading buffer without boiling. After SDS-PAGE electrophoresis, the cells were incubated sequentially with TransGen's anti-FLAG protein monoclonal antibody and TransGen's HRP-labeled goat anti-rabbit IgG antibody, followed by ECL staining to obtain Western blotting results. The results are shown below. Figure 2 As shown.

[0102] from Figure 2 As can be seen, compared with the control group, in the experimental groups transfected with two mRNAs, denaturing gels showed protein bands of the expected size, indicating that the mRNA could be expressed in cells. The Fc tag promoted the formation of protein dimers, and the Folden tag promoted the formation of protein trimers. Non-denaturing gels showed bands of the expected size, indicating that the expressed protein could maintain its expected structure.

[0103] Example 4. Preparation of mRNA vaccine

[0104] The mRNA package obtained in Example 2 was prepared as an LNP-mRNA vaccine.

[0105] The specific experimental steps are as follows: First, an alcohol phase was prepared by dissolving lipids in anhydrous ethanol at a mass ratio of cationic lipid (SM102):distearate phosphatidylcholine (DSPC):cholesterol:DMG-2000 = 50:10:38.5:1.5. Next, an aqueous phase was prepared using 50mM citrate buffer (pH 4.0) as the medium for dissolving mRNA. Then, mRNA was packaged using a microfluidic device with an alcohol phase:aqueous phase (volume ratio 1:3). Next, the mRNA was diluted with RNase-free PBS buffer and concentrated using a 30kDa ultrafiltration tube. An equal volume of 20% sucrose PBS solution was added to adjust the mRNA concentration to 60 μg / ml, and the sucrose concentration to 10%. Finally, after filtration through a 0.22μm filter membrane, the prepared LNP-mRNA vaccines, including GnRH-Fc mRNA vaccine (GnRH-Fc mRNA-LNP) and GnRH-Folden mRNA vaccine (GnRH-Folden mRNA-LNP), are obtained and stored at -20℃ after aliquoting.

[0106] The liposome nanoparticles contain at least one of cationic lipids, distearate phosphatidylcholine (DSPC), cholesterol, and DMG-2000.

[0107] The particle size distribution was detected by dynamic light, and the results are shown in Figure 3. As can be seen from Figure 3, the prepared LNP-mRNA vaccine particles are uniform, with a particle size of approximately 140 nm.

[0108] The GnRH-GCN4 mRNA vaccine and the GnRH-Ferrtin mRNA vaccine were obtained using the same methods as in Examples 2-4.

[0109] Example 5. Mouse Injection Evaluation Experiment

[0110] The efficacy of the mRNA vaccine prepared in Example 4 was evaluated in mice. Fifteen 8-week-old SPF female Kunming mice were randomly divided into three groups of five each. Each group was injected with 10 μg of GnRH-Fc mRNA vaccine, 10 μg of GnRH-Folden mRNA vaccine, and 100 μL of PBS, respectively. A booster injection was given two weeks after the initial injection. Two weeks after the booster injection, the mice were sacrificed, their organs were photographed, the size of the uterus was weighed, and blood was collected and serum was separated for analysis. The changes in antibody and estradiol levels in mouse serum were detected using an ELISA kit from Shanghai Enzyme-Linked Biotechnology Co., Ltd. The GnRH-GCN4 mRNA vaccine and the GnRH-Ferrtin mRNA vaccine did not affect the changes in antibody and estradiol levels in mouse serum. The experimental results for the GnRH-Fc mRNA vaccine and the GnRH-Folden mRNA vaccine are as follows: Figure 4 As shown.

[0111] from Figure 4 It can be seen that after injection, the serum titers of specific anti-GnRH antibodies in mice from both mRNA vaccine groups were significantly higher than those in the blank control group. This indicates that the mRNA drug can effectively induce high levels of specific anti-GnRH IgG antibodies and effectively stimulate the body's humoral injection response. Furthermore, the antibody levels induced in the GnRH-Fc mRNA vaccine group were higher than those in the GnRH-Folden mRNA vaccine group.

[0112] Subsequently, the changes in estradiol (E2) levels in mouse serum were further examined, and the results were as follows: Figure 5 As shown, consistent with expectations, estradiol levels decreased in both groups of mice after drug injection, with the GnRH-Fc mRNA vaccine group showing a greater reduction than the GnRH-Folden mRNA vaccine group.

[0113] The size of the mouse's uterus was further observed, and the results were as follows: Figure 6-7 As shown. From Figure 6-7 It can be seen that the uterine size of mice in both drug groups was significantly reduced, indicating that the drugs can inhibit uterine development in mice. The uterine weight was recorded, and the results are as follows: Figure 8 As shown, the uterine weight statistics are consistent with the image results.

[0114] Finally, HE staining was performed on the mouse uterus and ovaries, and the results are as follows: Figure 9-10 As shown. From Figure 9 It can be seen that, compared with the normal control group, the endometrial thickness of mice in both drug groups was significantly reduced, indicating that the endometrium underwent atrophic changes. From Figure 10As can be seen from the pathological sections of the ovaries, the content of corpus luteum in the ovaries of mice in both drug groups was significantly increased compared with that in the control group. This could lead to a decline in ovarian function, resulting in a decrease in the levels of estrogen and progesterone secreted by the ovaries, thus causing a decline in ovarian function. In addition, the number of primary and secondary oocytes in the ovaries of mice in the drug group was significantly reduced.

[0115] The above results indicate that the two mRNA vaccine drugs disrupted the development of the reproductive organs in female mice after injection, thereby reducing their reproductive capacity. Furthermore, the GnRH-Fc mRNA vaccine reduced reproductive capacity to a greater extent than the GnRH-Folden mRNA vaccine group.

[0116] Example 6. Evaluation Experiment of Cat Injection

[0117] The efficacy of the mRNA drug prepared in Example 4 was evaluated in cats. Nine 5-month-old female cats were randomly divided into three groups of three each. Each group received either 100 μg of GnRH-Fc mRNA drug, 100 μg of GnRH-Folden mRNA drug, or 1 mL of PBS as a blank control. A booster injection was given two weeks after the initial injection. Blood samples were collected two weeks after the booster injection, and serum was separated for analysis. The changes in serum antibody and estradiol levels were detected using an ELISA kit from Shanghai Enzyme-Linked Biotechnology Co., Ltd. The results are as follows: Figure 11-12 As shown.

[0118] from Figure 11 It can be seen that after injection, the specific anti-GnRH antibody titers in the cat serum of the two mRNA vaccine groups were significantly higher than those in the blank control group. This indicates that the mRNA drug can effectively induce high levels of specific anti-GnRH IgG antibodies and effectively stimulate the body's humoral injection response. Figure 11 Furthermore, the antibody levels induced by the GnRH-Fc mRNA vaccine group were higher than those induced by the GnRH-Folden mRNA vaccine group. Subsequent analysis of changes in estradiol levels in feline serum revealed... Figure 12 As shown in the figure. Consistent with expectations, after drug injection, the levels of estradiol (E2) in both groups of mice decreased, and the decrease was greater in the GnRH-FcmRNA vaccine group than in the GnRH-FoldenmRNA vaccine group.

[0119] The above results indicate that the two groups of mRNA vaccine drugs reduced hormone levels in cats, thereby regulating their reproductive capacity, and the reduction was greater in the GnRH-Fc mRNA vaccine group than in the GnRH-Folden mRNA vaccine group.

[0120] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, all variations falling within the meaning and scope of equivalents of the claims are intended to be included within the present invention. No reference numerals in the claims should be construed as limiting the scope of the claims.

[0121] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A polynucleotide comprising a first nucleotide encoding a GnRH fusion protein, said GnRH fusion protein comprising a GnRH protein or an immunogenic fragment thereof, further comprising a tag protein or an immunogenic fragment thereof coupled to said GnRH protein or immunogenic fragment thereof, said GnRH protein or immunogenic fragment thereof having an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology to SEQ ID NO:

1.

2. The polynucleotide according to claim 1, characterized in that, The tag protein is an Fc protein or a Folden protein, the amino acid sequence of which is shown in SEQ ID NO:2 and the amino acid sequence of which is shown in SEQ ID NO:

3.

3. The polynucleotide according to claim 2, characterized in that, The GnRH protein or its immunogenic fragment is coupled to the tag protein or its immunogenic fragment via a linker, wherein the amino acid sequence of the linker has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology with SEQ ID NO:

15. Preferably, when the tag protein is an Fc protein, the amino acid sequence of the GnRH fusion protein has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology with SEQ ID NO:4, or When the tag protein is a Folden protein, the amino acid sequence of the GnRH fusion protein has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology with SEQ ID NO:

5.

4. The polynucleotide according to claim 3, characterized in that, When the tag protein is an Fc protein, the sequence of the first nucleotide has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology with SEQ ID NO: 6 or 7. or When the tagged protein is a Folden protein, the sequence of the first nucleotide has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology with SEQ ID NO:8 or 9.

5. The polynucleotide according to claim 4, characterized in that, The 5' end of the first nucleotide is also attached to a second nucleotide sequence encoding a signal peptide; Preferably, the signal peptide is tPA, IL-2, IL-6 or a neuropeptide, and the neuropeptide is preferably VIP, CGRP or NPY; More preferably, the 5' end of the second nucleotide sequence is connected to a 5' UTR sequence, the nucleotide sequence of which is preferably as shown in SEQ ID NO:10, or The first nucleotide is further connected to a 3' UTR sequence at its 3' end, and the end of the 3' UTR sequence is further connected to a PolyA sequence. The nucleotide sequence of the 3' UTR sequence is preferably as shown in SEQ ID NO:

11. Preferably, the 5' end of the 5' UTR sequence is provided with a T7 promoter, the nucleotide sequence of which is shown in SEQ ID NO:12, and the polynucleotide is preferably provided with a FLAG tag.

6. The polynucleotide according to any one of claims 1 to 5, characterized in that, The polynucleotide is DNA or RNA; Preferably, when the polynucleotide is RNA, the 5' end of the 5' UTR sequence of the RNA is further provided with a 5' capping structure, and the 5' capping structure is preferably Cap1, Cap2, Cap3, Cap4 or Cap101.

7. A vector constructed from any one of the polynucleotides of claims 1 to 6.

8. A cell derived from the vector of claim 7.

9. A composition for regulating animal reproductive capacity, comprising the polynucleotide of any one of claims 1 to 6 or mRNA transcribed from the vector of claim 7.

10. The composition according to claim 9, characterized in that, The composition is an mRNA vaccine, which comprises mRNA expressing a GnRH fusion protein.

11. The composition according to claim 10, characterized in that, The mRNA sequence expressing the GnRH fusion protein has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology with SEQ ID NO:7 or SEQ ID NO:

9.

12. The composition according to claim 11, characterized in that, The composition further includes a pharmaceutically acceptable carrier, preferably a liposome, wherein the mRNA in the mRNA vaccine is encapsulated in liposome nanoparticles, and the particle size of the liposome nanoparticles is preferably 50-200 nm. Preferably, the liposome nanoparticles are selected from at least one of cationic lipids, distearate phosphatidylcholine (DSPC), cholesterol, and DMG-2000; More preferably, the liposome nanoparticles are composed of the following components in parts by weight: 30-80 parts cationic lipids, 5-15 parts distearate phosphatidylcholine (DSPC), 24-52 parts cholesterol, and 1-2 parts DMG-2000. More preferably, the liposome nanoparticles are composed of the following components in parts by weight: 50 parts cationic lipids, 10 parts distearate phosphatidylcholine (DSPC), 38.5 parts cholesterol, and 1.5 parts DMG-2000.

13. The use of the polynucleotide according to any one of claims 1 to 6, the carrier according to claim 7, or the composition according to any one of claims 9 to 12 in the preparation of a medicament for regulating animal reproductive capacity.

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