Gnrh nucleic acid vaccine

Abstract

Provided is a GnRH nucleic acid vaccine. GnRH is linked by means of a specific linker arm to form a multimer, or the GnRH or multimer is linked to a carrier protein to form a fusion antigen. Compared with a conventional linker arm, the expression of the multimer or fusion antigen via mRNA achieves higher expression levels and provides better presentation of GnRH epitopes; and an mRNA vaccine developed by encapsulating mRNA in liposomes can be used for castrating animals or pets, thereby effectively controlling the fertility of animals or pets.

Description

A GnRH nucleic acid vaccine Technical Field

[0001] This invention belongs to the field of nucleic acid vaccine technology, specifically relating to a GnRH nucleic acid vaccine. Background Technology

[0002] Spaying / neutering pets such as dogs and cats can prevent unwanted breeding, control the number of offspring, make pets more docile and easier to care for, and prevent abandonment due to unsuitability for puppies or lack of care from owners. Furthermore, spaying / neutering dogs can effectively prevent a range of related diseases, including reducing the risk of mammary cancer in female dogs, eliminating the risk of pyometra, and preventing ovarian tumors, ovarian cysts, pseudopregnancy, and dystocia. It can also reduce the likelihood of prostatic hyperplasia and infection in male dogs, decrease the incidence of perineal hernias, prevent perianal adenomas, and reduce urine markings in male dogs. While spaying / neutering surgery is currently the most common method, it causes significant stress for animals, is prone to wound infection, and is expensive, requiring post-operative care and increasing the burden on pet owners. Therefore, alternative spaying / neutering methods are in high demand.

[0003] Gonadotropin-releasing hormone (GnRH) is one of the important hormones regulating reproductive activity in mammals. Synthesized and secreted by the hypothalamus, it stimulates the anterior pituitary gland to secrete gonadotropins, which in turn stimulate the production and release of sex hormones. It can also directly act on the gonads, regulating animal reproduction by modulating gonadal function. Physiological doses of GnRH can promote the release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) from the pituitary gland, which then act on the gonads through blood circulation. This promotes testicular development and spermatogenesis in males, and ovarian development and follicle maturation in females, thereby promoting the synthesis and secretion of sex hormones, further promoting gamete production, and maintaining secondary sexual characteristics. Studies have shown that inducing anti-GnRH antibodies through active immunization to reduce GnRH levels leads to decreased synthesis of LH and FSH, accompanied by reduced estrus, and disruption of testicular development and spermatogenesis, potentially having a castration effect in both male and female animals.

[0004] GnRH is a small peptide containing ten amino acids, a small molecule hapten with weak immunogenicity. Using it alone as an immunogen for vaccines is difficult to elicit an immune response, which is a challenge in the industry. Therefore, it is necessary to modify GnRH and then link it to a large molecular carrier protein to improve immunogenicity (see Chinese patents (application numbers 201711339036.2, 202310434223.8, 202211461291.5, 202211052856.4)). Although existing technologies have carried out a lot of research on the modification of GnRH antigens, both natural GnRH antigens and modified analogs still have technical problems such as poor immunogenicity and large side effects.

[0005] mRNA vaccines deliver mRNA sequences encoding viral antigens into the body via a delivery system, where they are expressed and produce antigens, stimulating an immune response. This is considered a third-generation vaccine technology. mRNA vaccines break through the traditional immune activation model by innovatively utilizing the body's own cells to produce antigens, thereby activating bispecific immunity, forming immune memory, and providing more durable specific immunity. They exhibit stronger immune efficacy than traditional inactivated vaccines and subunit protein vaccines. Furthermore, the mRNA delivered to the body exists only in the cytoplasm and does not integrate into the host genome, and it can be naturally degraded in the body. mRNA vaccines do not require combination with other vaccine adjuvants, reducing adverse reactions caused by other substances, thus offering a higher safety profile compared to DNA vaccines and traditional adjuvanted vaccines.

[0006] The high immunogenicity and safety of mRNA vaccines can effectively compensate for the shortcomings of existing GnRH vaccines, such as low immunogenicity and high immunization doses. Since GnRH is a short peptide composed of only 10 amino acids, expressing a single GnRH molecule in vivo using mRNA results in low expression levels and easy degradation, leading to low immunogenicity. Therefore, linker arms are needed to connect and polymerize the GnRH molecule, as well as to link it to a carrier protein. However, using traditional linker arms, such as GGGGS, SGS, EAAAK, GGPPG, KK, and AAY, can also affect mRNA expression levels and antigenic epitope display to varying degrees. Summary of the Invention

[0007] In view of the above-mentioned prior art, this invention, during the process of optimizing GnRH expression using mRNA, accidentally obtained a GnRH-specific linker arm (SEQ ID NO. 1); and used this specific linker arm to study mRNA vaccines constructed from GnRH and carrier proteins, providing a GnRH multimeric antigen, a GnRH carrier protein antigen, and an mRNA vaccine encoding either the GnRH multimeric antigen or the GnRH carrier protein antigen, as well as their applications. This invention adopts the following technical solution:

[0008] <First Aspect>

[0009] This invention relates to a GnRH multimeric antigen, the amino acid sequence of which comprises a GnRH polypeptide sequence (SEQ ID NO.2) and a specific linker polypeptide (SEQ ID NO.1).

[0010] As one implementation, the polymer is a dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer, nonamer, or decamer.

[0011] <Second aspect>

[0012] The present invention also relates to a recombinant engineered bacterium, which is obtained by ligating a gene containing the encoding the aforementioned GnRH multimer antigen into an expression vector and then transforming it into Escherichia coli.

[0013] As one embodiment of the present invention, the GnRH multimeric antigen amino acid sequence comprises a canine GnRH signal peptide as shown in SEQ ID NO.130, a GnRH polypeptide sequence as shown in SEQ ID NO.2, and a specific linker polypeptide as shown in SEQ ID NO.1.

[0014] As one embodiment of the present invention, the amino acid sequence of the GnRH multimer antigen is shown in SEQ ID NO.41.

[0015] <Third aspect>

[0016] This invention also relates to a strain of Escherichia coli, PS038, with accession number CCTCCNO:M20242333.

[0017] <Fourth Aspect>

[0018] This invention relates to a GnRH carrier protein antigen, wherein the amino acid sequence of the GnRH carrier protein antigen is directly linked from the above-mentioned GnRH multimer antigen amino acid sequence and the carrier protein amino acid sequence.

[0019] As one implementation, the carrier protein is derived from the surface protein HBsAg of hepatitis B virus, the core protein HBcAg of hepatitis B virus, the ferritin Fer of Helicobacter pylori, the E2 protein of thermophilic bacteria, the L1 protein of human papillomavirus, the Qβ protein of bacteriophage, the AP205 protein of bacteriophage, the VP1 protein of norovirus, the β-defensin HDP protein, or the artificially designed i301 protein.

[0020] As one implementation, the direct connection is either the GnRH multimeric antigen amino acid sequence at the amino terminus of the carrier protein amino acid, or the GnRH multimeric antigen amino acid sequence at the carboxyl terminus of the carrier protein amino acid, or the GnRH multimeric antigen amino acid sequence inserted into the carrier protein amino acid sequence.

[0021] <Fifth Aspect>

[0022] This invention relates to a ribonucleic acid (RNA) sequence comprising a sequence encoding a signal peptide, a sequence encoding the aforementioned GnRH multimer antigen or the aforementioned GnRH carrier protein antigen, and a stop codon sequence.

[0023] As one implementation, the signal peptide sequence is derived from canine GnRH signal peptide sequences, feline GnRH signal peptide sequences, porcine GnRH signal peptide sequences, mouse GnRH signal peptide sequences, human GnRH signal peptide sequences, human TPA signal peptide sequences, human ALB signal peptide sequences, mouse K light chain signal peptide sequences, mouse heavy chain signal peptide sequences, or artificially designed signal peptide sequences.

[0024] As one implementation, the signal peptide sequence is preferably a canine GnRH signal peptide sequence.

[0025] As one implementation, the termination codon sequence is (TAA)x(TAG)y(TGA)z, where x+y+z≥1, and x, y, and z are each independent integers greater than or equal to 0;

[0026] As one implementation, the stop codon sequence is preferably TAGTGATGA.

[0027] <Sixth Aspect>

[0028] This invention relates to a GnRH mRNA vaccine comprising a composition consisting of the aforementioned ribonucleic acid (RNA) and a liposome delivery system.

[0029] <Seventh Aspect>

[0030] This invention relates to the application of the aforementioned GnRH multimeric antigen, the aforementioned GnRH carrier protein antigen, the aforementioned ribonucleic acid sequence, or the aforementioned GnRH mRNA vaccine.

[0031] The application is in the preparation of drugs for regulating the reproductive capacity of animals or pets. This includes castration of animals or pets, such as cats and dogs.

[0032] The *Escherichia coli* PS038 strain of this invention was deposited with the China Center for Type Culture Collection (CCTCC) at Wuhan University, China on October 25, 2024, with accession number CCTCCNO:M20242333. It is used to prepare GnRH hexamer antigen plasmids, which are then used in the preparation of nucleic acid vaccines.

[0033] Compared with the prior art, the present invention constructs GnRH multimeric antigen and GnRH carrier protein antigen through a GnRH-specific linker arm (SEQ ID NO.1), which can improve the expression level of GnRH multimer and GnRH carrier protein encoded by mRNA and the display of GnRH antigen epitopes. The mRNA vaccine prepared from this mRNA has better immunogenicity, higher safety and longer antibody duration. Attached Figure Description

[0034] Other features, objects, and advantages of the present invention will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings:

[0035] Figure 1 is an electrophoresis diagram of the recombinant protein and recombinant fusion protein expressed in Example 4 of the present invention;

[0036] In Figure 2, (A) and (B) show the mRNA expression in cells of 32 different constructs detected using the GnRH ELISA kit;

[0037] In Figure 3, (A) and (B) show the mRNA expression in cells of 32 different constructs detected by Western Blot.

[0038] Figure 4 shows the mRNA expression of 17 GnRH vector protein constructs in cells, detected using a GnRH ELISA kit.

[0039] Figure 5 shows the mRNA expression in cells of 17 GnRH vector protein constructs detected by Western Blot.

[0040] Figure 6 shows the serum testosterone levels in male C57 mice immunized with the vaccine used in Example 7;

[0041] Figure 7 shows the anti-GnRH antibody titer in the serum of male C57 mice immunized with the vaccine used in Example 7;

[0042] Figure 8 shows the serum testosterone levels at different times after immunizing male dogs with the vaccine in Example 8;

[0043] Figure 9 shows the serum estradiol levels at different times after the female dogs were immunized with the vaccine in Example 8;

[0044] Figure 10 shows the anti-GnRH antibody titers in the serum of dogs immunized with the vaccine at different times in Example 8;

[0045] Figure 11 shows the anti-GnRH antibody titers in the serum of domestic cats at different time points after immunization with the vaccine in Example 9. Detailed Implementation

[0046] The present invention will be described in detail below with reference to embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make several adjustments and improvements without departing from the concept of the present invention. These all fall within the protection scope of the present invention.

[0047] Example 1: mRNA Construction and Preparation

[0048] Unless otherwise specified, all mRNA construction and preparation involved in this invention are circular mRNAs. The circularized precursor RNA of this invention is derived from the clean PIE system, and its plasmid construction and circular mRNA preparation methods are as shown in patents CN202210200112.6 and CN202210200186.X.

[0049] 1.1 Plasmid Construction

[0050] Plasmids encoding GnRH multimeric antigens or GnRH vector protein antigens were constructed. This step was outsourced to Suzhou GenScript Biotech Co., Ltd. for gene synthesis and cloning. DNA vectors producing mRNA were constructed by sequentially ligating the following restriction enzymes: EcoRI, T7 promoter, intron fragment II, translation initiation element truncation fragment II, the coding region sequence of the GnRH multimeric antigen or GnRH vector protein antigen, polyAC, translation initiation element truncation fragment I, intron fragment I, and BsaI restriction enzyme. The resulting gene fragment was then double-digested with EcoRI and BsaI and inserted into the pUC57 vector. The sequences used to construct the DNA vector are shown in Table 1.

[0051] Table 1 Plasmid construction sequences

[0052] 1.2 Preparation of linear plasmid templates

[0053] The plasmid synthesized in the previous step was digested with BsaI to convert the plasmid DNA into linear cDNA. The enzyme digestion reaction system is as follows:

[0054] The enzyme was incubated overnight at 37°C. The digested products were recovered using a universal DNA recovery kit (Tiangen Biotech Co., Ltd.).

[0055] 1.3 In vitro transcription

[0056] The recovered linear cDNA template was subjected to in vitro transcription. The in vitro transcription reaction system is as follows:

[0057] Incubate at 37°C and 220 rpm with shaking for 3-5 hours, then add 1 KU DNase I and react at 37°C for 15-30 min. Obtain the crude transcript using ThermoFisher's Dynabeads. TM Oligo(dT) 25 The RNA was then purified. The purified RNA was analyzed by agarose gel electrophoresis to confirm that no degradation had occurred.

[0058] 1.4 Cycling reaction

[0059] The purified linear RNA was cyclized in the following system:

[0060] Incubate at 55°C for 15-30 min, then add 5 ml of 7.5M lithium chloride solution and incubate overnight at -20°C. Centrifuge at 5000 rpm for 20 min at 4°C using a refrigerated centrifuge, collect the precipitate, wash twice with ice-cold anhydrous ethanol, and dissolve the precipitate in 1 ml of enzyme-free water. Quantitatively determine the mRNA using Nanodrop, and analyze by capillary electrophoresis to confirm the integrity and purity of the mRNA.

[0061] Example 2: Construct of GnRH multimer antigen coding region

[0062] To investigate the effect of linker arms on the expression of GnRH multimers in mRNA, this invention used different linker arms to connect GnRH monomers (SEQ ID NO. 2), along with different signal peptides and stop codons, to construct GnRH dimer, tetramer, hexamer, octamer, and decamer antigen coding region sequences, forming GnRH multimer antigen coding region constructs. The corresponding mRNAs were prepared according to Example 1 for in vitro cell transfection screening or animal immunization screening. The linker arms used for screening are shown in Table 2, the signal peptides used are shown in Table 3, and the constructed GnRH multimer antigen coding region constructs are shown in Table 4.

[0063] Table 2 Connecting arm sequence

[0064] Table 3 Signal peptide sequences

[0065] Table 4. Constructs of GnRH multimer antigen coding regions

[0066] Example 3: Construction of GnRH carrier protein antigen coding region

[0067] In addition to using linker arms to form polymers and enhance the immunization effect, GnRH vaccine immunization typically involves linking the antigen to a carrier protein to increase antigen solubility and size, thereby improving immunogenicity. This invention uses linker arm 1 or linker arm 11 from Example 2 to link with different carrier proteins to form different GnRH carrier protein antigen coding region constructs, and prepares corresponding mRNAs for in vitro cell transfection screening or animal immunization screening. The constructed GnRH carrier protein antigen coding region constructs are shown in Table 5.

[0068] Table 5. GnRH vector protein coding region constructs

[0069] Example 4: Expression of GnRH hexamer recombinant protein and recombinant fusion protein

[0070] To better screen the linker arms for GnRH mRNA vaccines, this embodiment expresses GnRH hexamer recombinant protein and recombinant fusion protein, serving as controls for GnRH mRNA expression validation or as immunization controls for mRNA vaccines. The GnRH hexamer recombinant protein uses a conventional (GGGGS)3 linker arm, with a 6×His tag added to the C-terminus for purification. The amino acid sequence of the coding region of the GnRH hexamer recombinant protein is shown in SEQ ID NO. 97, and the nucleotide sequence is shown in SEQ ID NO. 98. The GnRH recombinant fusion protein is expressed by fusing GnRH hexamer with the carrier protein defensin HDP, with a 6×His tag added to the C-terminus for purification. The amino acid sequence of the coding region of the GnRH recombinant fusion protein is shown in SEQ ID NO. 99, and the nucleotide sequence is shown in SEQ ID NO. 100. Specific procedures are as follows:

[0071] (1) Gene Synthesis and Vector Construction: The nucleotide sequence of the coding region of the GnRH hexamer recombinant protein (SEQ ID NO. 98) and the nucleotide sequence of the coding region of the GnRH recombinant fusion protein (SEQ ID NO. 100) were artificially synthesized. An NcoI restriction site was added at the 5' end, and an EcoRI restriction site was added at the 3' end. The synthesized gene fragments were inserted into the pET28a plasmid using double digestion with NcoI and EcoRI, resulting in pET28a-GnRH-six and pET28a-GnRH-HDP plasmids. The obtained plasmids were transformed into *E. coli* DH5α and screened on 1.5% agar solid medium. Recombinant plasmids with correct sequencing were selected and transformed into *E. coli* BL21. A high-expression strain was selected and named BL21 / pET28a-GnRH-six and BL21 / pET28a-GnRH-HDP, respectively. Gene synthesis, PCR, restriction enzyme insertion, and strain screening and sequencing were performed by Suzhou GenScript Biotech Co., Ltd.

[0072] (2) Recombinant protein induction expression: The selected BL21 / pET28a-GnRH-six or BL21 / pET28a-GnRH-HDP was inoculated into LB medium containing 30 μg / ml kanamycin and cultured overnight at 37℃ for recovery. The culture was expanded at an inoculation ratio of 1:100. When the OD600 reached 0.6, IPTG with a final concentration of 0.4 mM was added to induce expression. After 4 h of induction culture, the cells were collected by centrifugation at 3500 rpm for 10 min.

[0073] (3) Recombinant protein purification: The collected bacterial cells were washed twice with PBS and resuspended in 100 ml of lysis buffer (20 mM sodium phosphate, 0.5 M NaCl, 10 mM imidazole, 1 mg / ml lysozyme, 20 μg / mg DNase I, pH 7.4). The mixture was sonicated for 15 min on ice. The precipitate was collected by centrifugation at 10,000 rpm for 10 min. 50 ml of denaturing lysis buffer (0.1 M Tris, 0.5 M NaCl, 10 mM DTT, 10 mM imidazole, 8 M urea, pH 7.5) was added to the precipitate, and the mixture was sonicated for 15 min on ice. The supernatant was collected by centrifugation at 10,000 rpm for 10 min. The supernatant was purified by nickel column chromatography to obtain the GnRH hexamer recombinant protein or the GnRH recombinant fusion protein.

[0074] (4) Identification of recombinant proteins: The purified GnRH hexamer recombinant protein or GnRH recombinant fusion protein was detected by SDS-PAGE electrophoresis. First, electrophoresis was performed at 80V for 20-30 min, and then at 120V for 50-60 min. After staining and destaining, the results were photographed. The results are shown in Figure 1, which shows that the size of GnRH hexamer recombinant protein and GnRH recombinant fusion protein is consistent with the theory.

[0075] Example 5: In vitro expression verification of GnRH multimer antigen and carrier protein antigen mRNA

[0076] To investigate the expression effects of GnRH multimer antigens and carrier protein antigens formed by different linker arms and signal peptides via mRNA, the coding region constructs from Examples 2 and 3 were used to prepare mRNAs in Example 1. The Transfection Kit was used to transfect 293T cells at 100 ng / well. The transfection conditions were as follows:

[0077] Cells were incubated in 96-well plates at 37°C with 5% CO2 for 48 hours, and the cell supernatant was collected. The GnRH ELISA kit (Shanghai Sangon Biotech) was used for detection according to the kit's operating procedures. Simultaneously, the collected cell supernatant was analyzed in parallel using Western blotting to eliminate the influence of different GnRH antibodies on the detection results. Western blotting was performed using SurePAGE. TM Protein pregels were prepared at a concentration of 4-20% (GenScript Biotechnology Co., Ltd.). The primary antibody used was Anti-GnRH rabbit polyclonal antibody (Abcam), and the secondary antibody used was HRP-labeled goat anti-rabbit antibody (Thermo).

[0078] We first compared the cellular expression of hexamers with 15 different linkers (Constructions 4.1-15) using the same signal peptide sequence. Figures 2(A) and 3(A) show that the GnRH hexamer construct based on linker arm 11 showed superior expression compared to constructs with other conventional linkers. The *E. coli* strain used to prepare the plasmid for the GnRH hexamer construct GL11-S1 (linker arm 11) has been deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, China, accession number CCTCC NO: M20242333. Based on this, we optimized different signal peptides and stop codons (Constructions 4.16-32) to improve their impact on expression levels. Figures 2(B) and 3(B) show that optimizing the signal peptide and stop codon effectively improved expression levels. We also verified the expression of GnRH by mRNA in constructs formed by linking GnRH with different carrier proteins using the linker arms of this invention, and explored the effect of the linker arms on expression levels and antigenic epitopes. The carrier protein constructs used in this invention are shown in Table 5. Figures 4 and 5 show the expression levels in cell supernatants of the constructs detected by the GnRH ELISA kit and by Western Blost, respectively. The results show that all constructs expressed the antigen.

[0079] Example 6: Preparation of GnRH mRNA vaccine

[0080] The mRNA vaccine prepared in this invention uses a common cationic lipid encapsulation method, as described in patent CN202110662426.3. The specific steps are as follows:

[0081] (1) Preparation of lipid solution: The cationic lipid CMAX4, cholesterol (5-cholestene-3β-ol), neutral lipid DSPC (distearate phosphatidylcholine), and PEG-modified lipid PEG-DMG (polyethylene glycol-dimyristic acid glyceride) were dissolved in anhydrous ethanol at a lipid molar ratio of 50:38.5:10:1.5 to prepare a 5 mg / ml CMAX4 lipid solution.

[0082] The cationic lipid CMAX4(4-[(3-{[3-({3-[bis(3-{4-[(2-butyloctanoyl)oxy]butoxy}-3-oxypropyl)amino]propyl}(methyl)amino)propyl](3-{4-[(2-butyloctanoyl)oxy]butoxy}-3-oxypropyl)amino}propoxy)oxy]butyl-2-octanoate butyl ester) was purchased from Suzhou Kerui Med Biomedical Technology Co., Ltd., while cholesterol (5-cholestyrene-3β-ol), neutral lipid DSPC (distearate phosphatidylcholine), and PEG-modified lipid PEG-DMG (polyethylene glycol-dimyristic acid glyceride) were purchased from Aivit (Shanghai) Pharmaceutical Technology Co., Ltd.

[0083] (2) mRNA solution preparation: mRNA solution was prepared using the constructs with high cell expression levels verified in Example 5. Constructs G-L1, G-L11-S1, GL11-Fer, GL11-E2, G-Qβ-L11, GL11-AP205, GL1-AP205, GL11-HDP, GL1-HDP, GL11-i301, and GL1-i301 were preferred. The mRNA of the preferred constructs was dissolved in a 10mM citrate buffer solution at pH 4.0 and diluted to a final concentration of 200 μg / mL to obtain the mRNA solution.

[0084] (3) Using microfluidic technology, the lipid solution and mRNA solution were rapidly mixed at a volume ratio of 1:3, and the buffer environment was replaced with PBS at pH 7.0 by ultrafiltration to remove ethanol, thus preparing LNP-mRNA.

[0085] (4) Add sucrose solution to the LNP-mRNA solution until the final sucrose concentration is 8% (w / v), and use Quant-iT TM RiboGreen TM RNA Assay Kit (Invitrogen) TM The concentration of mRNA in the prepared LNP-mRNA was determined using the R11490 kit, and the LNP-mRNA solution was diluted to 50 μg / ml, sterile filtered, aliquoted and frozen to obtain the mRNA vaccine.

[0086] (5) The particle size, polydispersity index (PDI), and surface potential of the mRNA vaccine were measured using dynamic light scattering on a Malvern Zetasizer Nano-ZEN 3600 (Malvern) potential-laser particle size analyzer to ensure they were within the specified range.

[0087] Example 7: Immunization and Antibody Detection of GnRH mRNA Vaccine in Mice

[0088] To investigate the differences in immunogenicity of different GnRH mRNA vaccines, this embodiment immunized 6-8 week old male C57 mice with the preferred construct mRNA vaccine prepared in Example 6 and the recombinant protein vaccine expressed in Example 4, respectively. Five mice were immunized with each GnRH mRNA or recombinant protein vaccine, 5 μg / mouse / dose, 100 μl, via intramuscular immunization in the hind leg. Mice injected with physiological saline served as negative controls. Immunization was performed at weeks 0 and 3, respectively. Blood was collected on day 14 after the second immunization to separate serum, and the levels of GnRH antibodies and testosterone in the serum were measured. Serum testosterone levels were measured using a competitive testosterone ELISA kit (Sangon Biotech) according to the manufacturer's instructions. Serum GnRH antibody titers were detected using an indirect ELISA method. The main steps of the indirect ELISA method for GnRH antibody detection are as follows:

[0089] (1) Dilute the recombinant GnRH protein (Abcam) to 1 μg / ml with 0.1M bicarbonate buffer (pH 9.6), add 100 μl / well to a 96-well microplate (Costar) and incubate overnight at 4°C; wash 3 times, add 5% BSA and block at 37°C for 2 hours; pat dry, dry at 37°C, and store at 4°C for later use.

[0090] (2) The collected mouse serum was serially diluted with PBS at a 2-fold ratio. Each dilution of the sample was added to the 96-well plate coated in the previous step and reacted at 37°C for 1 hour. After washing 3 times, 100 μl of HRP-labeled rabbit anti-mouse IgG (Sigma) was added to each well and reacted at 37°C for 1 hour. After washing 5 times, TMB substrate (Beyotime) was added and the color was developed for 10 min. The OD value at 450 nm was measured.

[0091] (3) Antibody titer calculation: The antibody titer is the highest serum dilution factor whose OD value is greater than twice the OD value of the PBS control well.

[0092] Figure 6 shows the serum testosterone levels in mice with 11 different GnRH mRNA vaccine constructs, 2 protein vaccines, and a control group. Compared with the control group, the testosterone levels of the GnRH mRNA vaccine were lower on day 14 after two immunizations. Compared with the recombinant protein vaccine, the testosterone levels of all GnRH mRNA vaccine groups were lower than those of the recombinant protein vaccine group. Among the GnRH mRNA vaccine groups, the testosterone levels of the mRNA vaccine groups constructed using a specific linker arm (linker arm 11) (G-L11-S1, GL11-Fer, GL11-E2, G-Qβ-L11, GL11-AP205, GL11-HDP, GL11-i301) were significantly lower than those of the mRNA vaccine groups constructed using a conventional linker arm (linker arm 1) (G-L1, GL1-AP205, GL1-HDP, GL1-i301). Figure 7 shows the GnRH antibody titers in the serum of each immunization group. The antibody titers indicate that all vaccine groups induced immune stimulation and produced GnRH antibodies. Compared with the recombinant protein vaccine group, the GnRH mRNA vaccine group showed more exposed epitopes, higher antibody titers, and more effective stimulation. The antibody titers of the mRNA vaccine group using the specific linker arm (linker arm 11) were significantly higher than those of the mRNA vaccine group using the conventional linker arm (linker arm 1). Mouse immunization results show that the GnRH mRNA vaccine constructed using the specific linker arm has a superior immunization effect and a more significant effect on reducing serum testosterone levels.

[0093] Example 8: Evaluation of the effect of GnRH mRNA vaccine on castration in dogs

[0094] Thirty healthy puppies aged 5-7 months (15 males and 15 females) were selected. The 15 males and 15 females were randomly divided into five groups, with 3 males and 3 females in each group kept together in a pen. The first group was immunized with GL11-Fer mRNA vaccine, the second group with GL11-AP205 mRNA vaccine, the third group with GL11-i301 mRNA vaccine, and the fourth group with GnRH-six recombinant protein vaccine. Each dog was immunized with 100μg of mRNA vaccine or 100μg of recombinant protein, and a booster immunization was given one month later. The fifth group served as the control group. According to the immunization time and number of immunizations in the immunization groups, each dog was injected with 1ml of physiological saline each time. Blood samples were collected before the first immunization, and then every two months thereafter. Serum was separated and stored at -80°C. After the last blood collection, the testosterone content in the serum of all male dogs, the estradiol content in the serum of all female dogs, and the GnRH antibody titer in the serum of all dogs were measured. The testosterone content and GnRH antibody titer were measured using the same methods as in Example 7. The estradiol content was measured using an estradiol ELISA kit (Huamei Biotechnology) according to the instructions. During the experiment, the number of estrus cycles in male dogs and the number of litters in female dogs were observed, and the observation continued for one year.

[0095] Table 6 shows the experimental observation results one year after immunization. 100% of the dogs immunized with the three mRNA vaccine groups were infertile, significantly higher than the recombinant control group and the saline control group. Figures 8 and 9 show the changes in serum testosterone levels in male dogs and serum estradiol levels in female dogs one year after immunization, respectively. The results show that the three mRNA vaccine groups had lower hormone control levels and a longer duration of effect. Figure 10 shows the changes in the dilution of GnRH antibodies in the blood of all experimental dogs one year after immunization. The results show that the three mRNA vaccine groups had higher antibody dilution and a longer duration of antibody effect.

[0096] Table 6 Evaluation of the castration effect of GnRH mRNA vaccine in dogs

[0097] Example 9: Evaluation of the efficacy of GnRH mRNA vaccine in immunizing castrated cats

[0098] Thirty healthy domestic cats aged 4-6 months (15 males and 15 females) were selected. Male and female cats were randomly paired into one-to-one pairs and housed in individual cages. Three pairs of cats were grouped together for immunization. The first group was immunized with GL11-Fer mRNA vaccine, the second with GL11-AP205 mRNA vaccine, the third with GL11-i301 mRNA vaccine, and the fourth with GnRH-six recombinant protein vaccine. Each cat received either 50 μg of mRNA vaccine or 50 μg of recombinant protein, with a booster immunization one month later. The fifth group served as the control group, and each cat received 0.5 ml of saline solution per immunization, based on the immunization schedule and frequency. Blood samples were collected before the first immunization and monthly thereafter. Serum was separated and stored at -80℃. The number of days of estrus in male cats and the birthing time and number of kittens in female cats were observed in each pair of cats. The observation ended after each pair of cats gave birth. Blood collection was stopped when the female cat in each pair was observed to be pregnant. If she was not pregnant, the observation continued for one year. After the last blood collection, the GnRH antibody titer in the serum of all cats was measured using the same method as in Example 7.

[0099] Table 7 shows the immunization-induced castration effect for each pair of cats over one year. The three mRNA vaccine immunization groups significantly suppressed estrus in male cats and achieved 100% infertility in female cats, which was higher than that of the recombinant control group and the saline control group, indicating a significant castration effect. Figure 11 shows that GnRH antibodies in the three mRNA vaccine groups could persist in the cats for one year, and the vaccines were effective in sterilization within one year of injection.

[0100] Table 7 Evaluation of the castration effect of GnRH mRNA vaccine in cats

[0101] In summary, this invention connects GnRH (SEQ ID NO. 2) to form a multimer via a specific linker arm (SEQ ID NO. 1), or connects the GnRH multimer to a carrier protein to form a fusion antigen, preparing mRNA expression multimers or fusion antigens. Compared to conventional linker arms (Table 2), the expression level is higher, and the GnRH epitope is better displayed. The mRNA vaccine formed by encapsulating mRNA with liposomes can be used for castration in animals or pets, effectively controlling the fertility of animals or pets.

[0102] The specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art can make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A GnRH multimeric antigen, wherein the amino acid sequence of the GnRH multimeric antigen comprises a GnRH polypeptide sequence as shown in SEQ ID NO.2 and a specific linker polypeptide as shown in SEQ ID NO.

1.

2. The GnRH multimeric antigen of claim 1, wherein, The polymer is a dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer, nonamer, or decamer.

3. A recombinant engineered bacterium, characterized in that, The recombinant engineered bacteria are obtained by ligating a gene containing the GnRH multimer antigen as described in claim 1 into an expression vector and then transforming it into Escherichia coli.

4. An Escherichia coli PS038, with accession number CCTCC NO:M20242333.

5. A GnRH carrier protein antigen, characterized in that, The amino acid sequence of the GnRH carrier protein antigen is directly linked from the amino acid sequence of the GnRH multimer antigen as described in claim 1 and the amino acid sequence of the carrier protein.

6. The GnRH carrier protein antigen according to claim 5, characterized in that, It also includes at least one of the following technical features: A. The carrier protein is derived from the surface protein of hepatitis B virus, the core protein of hepatitis B virus, ferritin of Helicobacter pylori, E2 protein of thermophilic bacteria, L1 protein of human papillomavirus, Qβ protein of bacteriophage, AP205 protein of bacteriophage, VP1 protein of norovirus, β-defensin HDP protein, or artificially designed i301 protein. B. The direct connection is either the GnRH multimeric antigen amino acid sequence at the amino terminus of the carrier protein amino acid, or the GnRH multimeric antigen amino acid sequence at the carboxyl terminus of the carrier protein amino acid, or the GnRH multimeric antigen amino acid sequence inserted into the carrier protein amino acid sequence.

7. A ribonucleic acid sequence comprising a sequence encoding a signal peptide, a sequence encoding a GnRH multimer antigen as claimed in claim 1 or a GnRH carrier protein antigen as claimed in claim 5, and a stop codon sequence.

8. The ribonucleic acid sequence of claim 7, wherein, It also includes at least one of the following technical features: C. The signal peptide sequence is derived from canine GnRH signal peptide sequence, feline GnRH signal peptide sequence, porcine GnRH signal peptide sequence, mouse GnRH signal peptide sequence, human GnRH signal peptide sequence, human TPA signal peptide sequence, human ALB signal peptide sequence, mouse K light chain signal peptide sequence, mouse heavy chain signal peptide sequence, or artificially designed signal peptide sequence. D. The termination codon sequence is (TAA)x(TAG)y(TGA)z, where x+y+z≥1, and x, y, and z are independent integers greater than or equal to 0.

9. A GnRH mRNA vaccine comprising a composition consisting of the ribonucleic acid sequence as described in claim 7 and a liposome delivery system.

10. The use of a GnRH multimeric antigen as described in claim 1, or a GnRH carrier protein antigen as described in claim 5, or a ribonucleic acid sequence as described in claim 7, or a GnRH mRNA vaccine as described in claim 9 in the preparation of a drug for regulating reproductive capacity in animals or pets.

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