A GnRH nucleic acid vaccine

Abstract

The present invention discloses a GnRH nucleic acid vaccine; in the present invention, GnRH is linked through a specific linker to form a multimer, or GnRH or the multimer is linked to a carrier protein to form a fusion antigen, and the multimer or the fusion antigen is expressed by mRNA. Compared with conventional linkers, 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 of animals or pets, effectively controlling the fertility of animals or pets.

Description

Technical Field

[0001] The present invention belongs to the technical field of nucleic acid vaccines, and particularly relates to a GnRH nucleic acid vaccine. Background Art

[0002] Sterilization and castration of pets such as dogs and cats can avoid unwanted reproduction, control the number of offspring, make the pets more docile and easier to raise, and avoid being abandoned due to the inability to give away the offspring after breeding or the pet owner's inability or lack of time to take care of the offspring. In addition, sterilization of pet dogs can effectively avoid a series of related diseases, including reducing the risk of breast cancer in female dogs, eliminating the risk of pyometra, and avoiding diseases such as ovarian tumors, ovarian cysts, false pregnancy, and dystocia in dogs; and can reduce the probability of prostate hyperplasia and infection in male dogs, reduce the incidence of perineal hernia, prevent the occurrence of perianal adenoma, and reduce urine marking in male dogs. Sterilization surgery is the most commonly used method at present, but it can cause high stress in animals, and the wound is prone to infection. In addition, the surgery cost is relatively expensive, and postoperative care is required, increasing the burden on pet owners. Therefore, there is a high demand for sterilization methods that can replace surgery.

[0003] Gonadotropin releasing hormone (GnRH) is one of the important hormones that regulate reproductive activities in mammals. It is synthesized and secreted by the hypothalamus, stimulates the anterior pituitary gland of the body to secrete gonadotropins, and then stimulates the production and release of sex hormones. It can also directly act on the gonads and regulate animal reproduction by regulating the function of the gonads. Physiological doses of GnRH can promote the release of luteinizing hormone and follicle-stimulating hormone by the pituitary gland, act on the gonads through the blood circulation, promote the development of testes and sperm formation in male animals, promote the development of ovaries and follicle maturation in female animals, promote the synthesis and secretion of sex hormones, further promote gamete production, and maintain secondary sexual characteristics. Research shows that by actively immunizing to induce anti-GnRH antibodies and reducing GnRH levels, the synthesis of luteinizing hormone and follicle-stimulating hormone is reduced, accompanied by reduced estrus, and the disruption of testicular development and sperm production. It is expected to play a castrating role in both male and female animals.

[0004] GnRH is a small peptide containing ten amino acids and is a small molecule hapten with weak immunogenicity. It is difficult to cause an immune response by using it alone as the immunogen of a vaccine, which is a difficult point in the industry. Therefore, it is necessary to modify GnRH and then conjugate it with a macromolecular carrier protein to improve immunogenicity (see Chinese patents (application numbers 201711339036.2, 202310434223.8, 202211461291.5, 202211052856.4)). Although many studies have been carried out on the modification level of GnRH antigens in the prior art, there are still technical problems such as unremarkable immunogenicity and large side effects in both natural GnRH antigens and modified analogues.

[0005] mRNA vaccines introduce mRNA sequences encoding viral antigens into the body through a delivery system, express antigens in the body, and then stimulate an immune response in the body. They are considered the third-generation vaccine technology. mRNA vaccines break through the immune activation mode of traditional vaccines, innovatively use the body's own cells to produce antigens, activate dual-specific immunity, form immune memory, provide more persistent specific immunity, and have stronger immune effects than traditional inactivated vaccines and subunit protein vaccines. In addition, the mRNA introduced into the body by mRNA vaccines only exists in the cytoplasm and will not integrate into the host genome and can be naturally degraded in the body. mRNA vaccines do not need to be used in combination with other vaccine adjuvants, which can reduce adverse reactions caused by other substances. Therefore, they have higher safety than DNA vaccines and traditional adjuvant vaccines.

[0006] Utilizing the high immunogenicity and high safety of mRNA vaccines can well make up for the defects of existing GnRH vaccines, such as low immunogenicity and large immunization doses. Since GnRH is a short peptide composed of only 10 amino acids, when using mRNA to express single-molecule GnRH in the body, there will be low expression levels and easy degradation, resulting in low immunogenicity. Therefore, it is necessary to use a linker to link and polymerize GnRH molecules and link them to carrier proteins. Using traditional linkers, such as GGGGS, SGS, EAAAK, GPGPG, KK, AAY, etc., will also affect the mRNA expression level and antigen epitope display to varying degrees. Summary of the Invention

[0007] In view of the above-mentioned prior art, during the process of using mRNA to optimize the expression of GnRH in the present invention, a GnRH-specific linker (SEQ ID NO.1) was accidentally obtained; and the mRNA vaccine constructed with GnRH and carrier protein was studied using this specific linker, providing a GnRH multimer antigen, a GnRH carrier protein antigen, and an mRNA vaccine encoding the GnRH multimer antigen or GnRH carrier protein antigen and its application. The present invention adopts the following technical solutions:

[0008] <First aspect>

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

[0010] As an embodiment, the multimer is a dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer, nonamer, or decamer.

[0011] <Second aspect>

[0012] The present invention also relates to a recombinant engineering bacterium, which is obtained by connecting the gene containing the GnRH multimer antigen described above to an expression vector and then transforming Escherichia coli.

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

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

[0015] <The third aspect>

[0016] The present invention also relates to an Escherichia coli PS038, and its preservation number is CCTCC NO: M20242333.

[0017] <The fourth aspect>

[0018] The present invention relates to a GnRH carrier protein antigen, and the amino acid sequence of the GnRH carrier protein antigen is directly connected by the above-mentioned GnRH multimer antigen amino acid sequence and the carrier protein amino acid sequence.

[0019] As an embodiment, the carrier protein is 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 phage, the AP205 protein of phage, the VP1 protein of norovirus, the β-defensin HDP protein or the artificially designed i301 protein.

[0020] As an embodiment, the direct connection means that the GnRH multimer antigen amino acid sequence is at the amino terminus of the carrier protein amino acid, or the GnRH multimer antigen amino acid sequence is at the carboxyl terminus of the carrier protein amino acid, or the GnRH multimer antigen amino acid sequence is inserted into the carrier protein amino acid sequence.

[0021] <The fifth aspect>

[0022] The present invention relates to a ribonucleic acid (RNA) sequence, and the ribonucleic acid (RNA) sequence comprises a coding signal peptide sequence, a coding sequence of the above-mentioned GnRH multimer antigen or a coding sequence of the above-mentioned GnRH carrier protein antigen, and a termination codon sequence.

[0023] As an embodiment, the signal peptide sequence is derived from canine GnRH signal peptide sequence, feline GnRH signal peptide sequence, porcine GnRH signal peptide sequence, murine GnRH signal peptide sequence, human GnRH signal peptide sequence, human TPA signal peptide sequence, human ALB signal peptide sequence, murine kappa light chain signal peptide sequence, murine heavy chain signal peptide sequence or artificially designed signal peptide sequence.

[0024] As an embodiment, the signal peptide sequence is preferably canine GnRH signal peptide sequence.

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

[0026] As an embodiment, the termination codon sequence is preferably TAGTGATGA.

[0027] <Sixth aspect>

[0028] The present invention relates to a GnRH mRNA vaccine, which comprises the above ribonucleic acid (RNA) and a composition composed of a liposome delivery system.

[0029] <Seventh aspect>

[0030] The present invention relates to the use of the aforementioned GnRH multimer antigen, the aforementioned GnRH carrier protein antigen, the aforementioned ribonucleic acid sequence, or the aforementioned GnRH mRNA vaccine.

[0031] The use is for the preparation of drugs for regulating the reproductive ability of animals or pets. It includes castration of animals or pets; such as castration of cats and dogs.

[0032] Escherichia coli PS038 of the present invention was deposited with the China Center for Type Culture Collection on October 25, 2024. The deposit address is Wuhan University, China, and the deposit number is CCTCC NO: M20242333. It is used for the preparation of GnRH hexamer antigen plasmid and further for the preparation of nucleic acid vaccines.

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

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

[0035] Figure 1 Electrophoresis diagram of the recombinant proteins and recombinant fusion proteins expressed in Example 4 of the present invention;

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

[0037] Figure 3 In (A) and (B), the mRNA cell expression of 32 different constructs was detected using the Western Blot method;

[0038] Figure 4 The mRNA cell expression of 17 GnRH vector protein constructs was detected using a GnRH ELISA kit;

[0039] Figure 5 The mRNA cell expression of 17 GnRH vector protein constructs was detected using the Western Blot method;

[0040] Figure 6 Serum testosterone content after immunizing male C57 mice with the vaccine used in Example 7;

[0041] Figure 7 Anti-GnRH antibody titer in the serum after immunizing male C57 mice with the vaccine used in Example 7;

[0042] Figure 8 Serum testosterone content at different times after immunizing male dogs with the vaccine in Example 8;

[0043] Figure 9 Serum estradiol content at different times after immunizing female dogs with the vaccine in Example 8;

[0044] Figure 10 Anti-GnRH antibody titer in the serum at different times after immunizing dogs with the vaccine in Example 8;

[0045] Figure 11 Anti-GnRH antibody titer in the serum at different times after immunizing domestic cats with the vaccine in Example 9. Detailed implementation manners

[0046] The present invention will be described in detail below in conjunction with embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but do not limit the present invention in any form. It should be noted that for those of ordinary skill in the art, several adjustments and improvements can be made without departing from the concept of the present invention. These all belong to the protection scope of the present invention.

[0047] Example 1, mRNA construction and preparation

[0048] Unless otherwise specified, the mRNA construction and preparation involved in the present invention are circular mRNA. The circular precursor RNA of the present invention is derived from the clean PIE system, and the plasmid construction and circular mRNA preparation methods refer to the contents shown in patents CN202210200112.6 and CN202210200186.X.

[0049] 1.1 Plasmid construction

[0050] Construct a plasmid containing the GnRH multimer antigen or GnRH carrier protein antigen coding sequence. This step is entrusted to GenScript Biotech Corporation in Suzhou for gene synthesis and cloning. The DNA vector for generating mRNA is obtained by double digestion with EcoRI and BsaI of a gene fragment formed by sequentially connecting the restriction enzyme sites EcoRI, T7 promoter, intron fragment II, truncated translation initiation element fragment II, GnRH multimer antigen or GnRH carrier protein antigen coding region sequence, polyAC, truncated translation initiation element fragment I, intron fragment I, and restriction enzyme site BsaI and inserting it into the pUC57 vector. The sequences used for constructing the DNA vector are shown in Table 1.

[0051] Table 1 Plasmid construction sequences

[0052]

[0053]

[0054] 1.2 Preparation of linear plasmid template

[0055] The plasmid synthesized in the previous step is digested with BsaI alone to convert the plasmid DNA into linear cDNA. The enzyme digestion reaction system is as follows:

[0056]

[0057]

[0058] Digest overnight at 37°C. Use a universal DNA recovery kit (Tiangen Biochemical Technology Co., Ltd.) to recover the enzyme digestion product.

[0059] 1.3 In vitro transcription

[0060] Perform an in vitro transcription reaction on the recovered linear cDNA template. The in vitro transcription reaction system is as follows:

[0061]

[0062] Incubate with shaking at 37 °C and 220 rpm for 3 - 5 hours, then add 1 KU DNaseI and react at 37 °C for 15 - 30 min. The crude transcription product obtained is purified using Dynabeads from ThermoFisher TM Oligo(dT) 25 to purify the RNA. The purified RNA is analyzed by agarose gel electrophoresis to confirm that no degradation has occurred.

[0063] 1.4 Cyclization reaction

[0064] Perform a cyclization reaction on the purified linear RNA in the following system:

[0065]

[0066] Incubate at 55 °C for 15 - 30 min, then add 5 ml of 7.5 M 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 the precipitate twice with ice-cold absolute ethanol, and dissolve the precipitate in 1 ml of enzyme-free water. Quantitatively determine the mRNA using Nanodrop, and analyze the integrity and purity of the mRNA by capillary electrophoresis.

[0067] Example 2, GnRH multimer antigen coding region construct

[0068] To study the effect of the linker on the expression of GnRH multimers by mRNA, the present invention respectively uses different linkers to link GnRH monomers (SEQ ID NO.2), different signal peptides and stop codons to construct the coding region sequences of GnRH dimers, tetramers, hexamers, octamers, and decamers, forming GnRH multimer antigen coding region constructs, and prepares the corresponding mRNA for in vitro cell transfection screening or animal immunization screening through Example 1. The linkers used for screening are shown in Table 2, the signal peptides used for screening are shown in Table 3, and the GnRH multimer antigen coding region constructs generated are shown in Table 4.

[0069] Table 2 Linker sequences

[0070]

[0071]

[0072] Table 3 Signal peptide sequences

[0073]

[0074]

[0075] Table 4 GnRH multimer antigen coding region constructs

[0076]

[0077]

[0078]

[0079]

[0080] Example 3. GnRH carrier protein antigen coding region constructs

[0081] In addition to forming multimers using linkers to enhance the immune effect, GnRH vaccines are usually linked to carrier proteins to increase the solubility of the antigen and the antigen size to improve immunogenicity. In the present invention, Linker 1 or Linker 11 in Example 2 is selected to be linked to different carrier proteins to form different GnRH carrier protein antigen coding region constructs, and the corresponding mRNAs are prepared for in vitro cell transfection screening or animal immunization screening. The GnRH carrier protein antigen coding region constructs generated are shown in Table 5.

[0082] Table 5 GnRH carrier protein coding region constructs

[0083]

[0084]

[0085]

[0086] Example 4. Expression of GnRH hexamer recombinant protein and recombinant fusion protein

[0087] To better screen the linker of GnRH mRNA vaccine, this example expressed GnRH hexamer recombinant protein and recombinant fusion protein for use as a control for GnRH mRNA expression verification or an immune control for the mRNA vaccine. The GnRH hexamer recombinant protein used the conventional (GGGGS)3 linker and a 6×His tag was added to the C-terminus for purification. The amino acid sequence of the coding region of the GnRH hexamer recombinant protein was as shown in SEQ ID NO.97, and the nucleotide sequence was as shown in SEQ ID NO.98. The GnRH recombinant fusion protein was expressed by fusing GnRH hexamer with the carrier protein defensin HDP, and a 6×His tag was added to the C-terminus for purification. The amino acid sequence of the coding region of the GnRH recombinant fusion protein was as shown in SEQ ID NO.99, and the nucleotide sequence was as shown in SEQ ID NO.100. The specific operations are as follows:

[0088] (1) Gene synthesis and vector construction: The nucleotide sequence (SEQ ID NO.98) gene fragment of the coding region of the GnRH hexamer recombinant protein and the nucleotide sequence (SEQ ID NO.100) gene fragment of the coding region of the GnRH recombinant fusion protein were artificially synthesized, an NcoI restriction site was added to the 5’ end, and an EcoRI restriction site was added to the 3’. The synthesized gene fragments were inserted into the pET28a plasmid using double digestion with NcoI and EcoRI to obtain the pET28a-GnRH-six and pET28a-GnRH-HDP plasmids. The obtained plasmids were transformed into Escherichia coli DH5α, and screening was performed on a 1.5% agar solid medium. Recombinant plasmids with correct sequencing were selected, and the recombinant plasmids were transformed into Escherichia coli BL21, and a highly expressing strain was selected and named BL21 / pET28a-GnRH-six and BL21 / pET28a-GnRH-HDP, respectively. Gene artificial synthesis, PCR, restriction enzyme digestion and insertion, and strain screening and sequencing were completed by Nanjing Genscript Biotechnology Co., Ltd.

[0089] (2) Induced expression of recombinant protein: 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 °C for resuscitation. It was subcultured at an inoculation ratio of 1:100. When the culture reached an OD600 of 0.6, IPTG with a final concentration of 0.4 mM was added for induced expression. After 4 h of induced culture, the cells were collected by centrifugation at 3500 rpm for 10 min.

[0090] (3) Recombinant protein purification: After washing the collected bacterial cells twice with PBS, resuspend them in 100 ml of lysis buffer (20 mM sodium phosphate, 0.5 M NaCl, 10 mM imidazole, 1 mg / ml lysozyme, 20 μg / mg DNaseI, pH 7.4). Under ice bath conditions, use an ultrasonic disruptor to perform ultrasonic disruption for 15 min. Centrifuge at 10,000 rpm for 10 min and collect the precipitate. Add 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) to the precipitate. Under ice bath conditions, use an ultrasonic disruptor to perform ultrasonic treatment for 15 min. Centrifuge at 10,000 rpm for 10 min and collect the supernatant. The supernatant is purified by nickel column to obtain GnRH hexamer recombinant protein or GnRH recombinant fusion protein.

[0091] (4) Identification of recombinant protein: The GnRH hexamer recombinant protein or GnRH recombinant fusion protein obtained after purification is detected by SDS-PAGE electrophoresis. First, perform electrophoresis at 80 V for 20 - 30 min, and then at 120 V for 50 - 60 min. After staining and decolorization, take a photo. The results are as Figure 1 shown, indicating that the sizes of GnRH hexamer recombinant protein and GnRH recombinant fusion protein are consistent with the theory.

[0092] Example 5. In vitro expression verification of GnRH multimer antigen and carrier protein antigen mRNA

[0093] To study the expression effects of GnRH multimer antigens and carrier protein antigens formed by different linker arms and signal peptides in the form of mRNA, the coding region constructs in Example 2 and Example 3 are transfected into 293T cells at 100 ng / well through the -mRNA Transfection Kit. The transfection conditions are as follows:

[0094]

[0095] Incubate in a 96-well plate at 37 °C with 5% carbon dioxide for 48 hours, and collect the cell supernatant. Use a GnRH ELISA kit (Sangon Biotech, Shanghai) and detect according to the operating steps of the kit. At the same time, perform parallel detection on the collected cell supernatant by Western Blot to eliminate the influence of different GnRH antibodies on the detection results. For Western Blot detection, use SurePAGE TM protein precast gel with a concentration of 4 - 20% (GenScript Biotech Corporation). Use Anti-GnRH rabbit polyclonal antibody (Abcam) as the primary antibody and HRP-labeled goat anti-rabbit antibody (Thermo) as the secondary antibody.

[0096] We first compared the cellular expression of hexamers with 15 different linkers (Table 4 constructs 1 - 15) using the same signal peptide sequence. Figure 2 (A) and Figure 3 (A) shows that the expression level of the GnRH hexamer construct based on linker 11 is superior to that of constructs with other conventional linkers. The Escherichia coli strain used for plasmid preparation of the GnRH hexamer construct GL11 - S1 with linker 11 has been deposited at the China Center for Type Culture Collection. The deposit address is Wuhan University, China, and the deposit number is CCTCC NO: M20242333. On this basis, we optimized different signal peptides and stop codons (Table 4 constructs 16 - 32) to improve the impact on the expression level. Figure 2 (B) and Figure 3 (B) shows that optimizing the signal peptide and stop codon can effectively increase the expression level. We also verified the constructs formed by connecting GnRH with different carrier proteins through the linker of the present invention and expressed them via mRNA to explore the influence of the linker on the expression level and antigenic epitopes. The carrier protein constructs used in the present invention are shown in Table 5. Figure 4 and Figure 5 respectively show the expression levels of the cell supernatants of the constructs detected using a GnRH ELISA kit and by Western Blot. The results show that all constructs are expressed.

[0097] Example 6. Preparation of GnRH mRNA Vaccine

[0098] The mRNA vaccine prepared in the present invention adopts a general preparation method of cationic lipid encapsulation, referring to the content shown in Patent CN202110662426.3. The specific operation steps are as follows:

[0099] (1) Preparation of lipid solution: Dissolve cationic lipid CMAX4, cholesterol (5 - cholesten - 3β - ol), neutral lipid DSPC (distearoylphosphatidylcholine), and PEG - modified lipid PEG - DMG (polyethylene glycol - dimyristoyl glycerol) in anhydrous ethanol at a lipid molar ratio of 50:38.5:10:1.5 to prepare a 5 mg / ml CMAX4 lipid solution.

[0100] Among them, the cationic lipid CMAX4 (butyl 2-octanoate 4-[(3-{[3-({3-[bis(3-{4-[(2-butyloctanoyl)oxy]butoxy}-3-oxopropyl)amino]propyl}(methyl)amino)propyl](3-{4-[(2-butyloctanoyl)oxy]butoxy}-3-oxopropyl)amino}propoxy)oxy]butyl) was purchased from Suzhou Keruimed Biopharmaceutical Technology Co., Ltd., and cholesterol (5-cholesten-3β-ol), neutral lipid DSPC (distearoylphosphatidylcholine), and PEG-modified lipid PEG-DMG (polyethylene glycol-dimyristoyl glycerol) were purchased from AVT (Shanghai) Pharmaceutical Technology Co., Ltd.

[0101] (2) Preparation of mRNA solution: Use the construct with high cell expression verified in Example 5 to prepare the mRNA solution. Preferably, use constructs G-L1, G-L11-S1, GL11-Fer, GL11-E2, G-Qβ-L11, GL11-AP205, GL1-AP205, GL11-HDP, GL1-HDP, GL11-i301, GL1-i301. Dissolve the mRNA of the preferred construct in a 10 mM citric acid buffer saline solution with a pH of 4.0 and dilute to a final concentration of 200 μg / mL to obtain the mRNA solution.

[0102] (3) Use microfluidic technology to quickly mix the lipid solution and the mRNA solution at a volume ratio of 1:3 and use ultrafiltration technology to replace the buffer environment with PBS at pH 7.0 to remove ethanol, and prepare LNP-mRNA.

[0103] (4) Add a sucrose solution to the LNP-mRNA solution until the final sucrose concentration is 8% (w / v). Use Quant-iT TM RiboGreen TM RNA Assay Kit (Invitrogen TM R11490) kit to measure the mRNA concentration in the prepared LNP-mRNA, and dilute the LNP-mRNA solution to 50 μg / ml, aliquot and store frozen after sterile filtration to obtain the mRNA vaccine.

[0104] (5) Measure the particle size, polydispersity index PDI, and surface potential of the mRNA vaccine by dynamic light scattering method on a Zeta potential - laser particle size analyzer Malvern Zetasizer Nano-ZEN 3600 (Malvern); determine that it is within the specified range.

[0105] Example 7. Immunization of Mice with GnRH mRNA Vaccine and Antibody Detection

[0106] To study the differences in immunogenicity among GnRH mRNA vaccines with different constructs, in this example, the preferred construct mRNA vaccine prepared in Example 6 and the recombinant protein vaccine expressed in Example 4 were used to immunize male C57 mice aged 6 - 8 weeks. Five mice were immunized with each GnRH mRNA vaccine or recombinant protein vaccine construct, at a dose of 5 μg / mouse / time, with a volume of 100 μl. Immunization was performed via the hind leg muscle. At the same time, mice injected with normal saline were used as negative controls. Immunization was carried out at 0 week and 3 weeks respectively. Blood was collected 14 days after the second immunization to separate serum, and the GnRH antibody and testosterone content in the serum were detected respectively. The serum testosterone content was detected using a competitive method Testosterone ELISA kit (Sangon Biotech) according to the operating procedures in the instruction manual. The GnRH antibody in the serum was detected using an indirect ELISA method to detect the antibody titer. The main steps of the GnRH antibody indirect ELISA detection method are as follows:

[0107] (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 ELISA plate (Costar) and incubate overnight at 4°C; after washing 3 times, add 5% BSA and block for 2 hours at 37°C; pat dry the moisture, dry at 37°C, and store at 4°C for later use.

[0108] (2) Gradiently dilute the collected mouse serum 2-fold with PBS, add samples of each dilution to the 96-well plate coated in the previous step, and react at 37°C for 1 hour; after washing 3 times, add 100 μl / well of HRP-labeled rabbit anti-mouse IgG (Sigma) and react at 37°C for 1 hour; after washing 5 times, add TMB substrate (Beyotime) to develop color for 10 min, and measure the OD value at 450 nm.

[0109] (3) Antibody titer calculation: The antibody titer is the highest serum dilution multiple at which the OD value is greater than 2 times the OD value of the PBS control well.

[0110] Figure 6The serum testosterone levels of mice in 11 different GnRH mRNA vaccines, 2 protein vaccines, and the control group were shown. Compared with the control group, the testosterone levels after the second immunization on the 14th day were decreased in the GnRH mRNA vaccine group. Compared with the recombinant protein vaccine group, the testosterone levels in all GnRH mRNA vaccine groups were lower than those in the recombinant protein vaccine group. Among the GnRH mRNA vaccine groups, the testosterone levels in the mRNA vaccine groups constructed with a specific linker (linker 11) (G-L11-S1, GL11-Fer, GL11-E2, G-Qβ-L11, GL11-AP205, GL11-HDP, GL11-i301) were significantly lower than those in the mRNA vaccine groups constructed with a conventional linker (linker 1) (G-L1, GL1-AP205, GL1-HDP, GL1-i301). Figure 7 The GnRH antibody titers in the sera of each immunization group were shown. The antibody titers indicated that immune stimulation was generated in each vaccine group, and GnRH antibodies were produced. Compared with the recombinant protein vaccine group, the epitope display was more exposed and the antibody titers were higher in the GnRH mRNA vaccine group, and the stimulation of the body was more effective. The antibody titers in the mRNA vaccine groups using a specific linker (linker 11) were significantly higher than those in the mRNA vaccine groups using a conventional linker (linker 1). The immune effect of the mice showed that the GnRH mRNA vaccine constructed with a specific linker had more advantages in immune effect and was more significant in reducing the serum testosterone level.

[0111] Example 8. Evaluation of the castration effect of GnRH mRNA vaccine in dogs

[0112] Thirty healthy dogs aged 5 - 7 months (15 male dogs and 15 female dogs) were selected. Fifteen male dogs and fifteen female dogs were randomly divided into five groups respectively. In each group, 3 male dogs and 3 female dogs were co - housed in a dog pen. The first group was immunized with GL11 - Fer mRNA vaccine, the second group was immunized with GL11 - AP205 mRNA vaccine, the third group was immunized with GL11 - i301 mRNA vaccine, and the fourth group was immunized with GnRH - six recombinant protein vaccine. Each dog was immunized with 100 μg of mRNA vaccine or 100 μg of recombinant protein, and boosted once after one month. The fifth group was the control group. According to the immunization time and frequency of the immunized groups, each dog was injected with 1 ml of normal saline each time. Blood samples were collected before the first immunization, and then every two months. The 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 measurement methods of testosterone content and GnRH antibody titer were the same as those in Example 7. The estradiol content was measured using an estradiol ELISA kit (Huamei Bio) according to the instructions. During the experiment, the estrus frequency of male dogs and the litter size of female dogs in each group were observed for one year continuously.

[0113] Table 6 shows the experimental observation results after one - year immunization. The three mRNA vaccine - immunized dog groups had 100% infertility, which was significantly higher than that of the recombinant control group and the normal saline control group. Figure 8 、 Figure 9 Respectively show the changes in the content of testosterone hormone in the serum of male dogs and estradiol hormone in the serum of female dogs during the one - year period after immunization. The results show that the three mRNA vaccine - immunized groups had lower and longer - lasting control of hormone levels. Figure 10 Shows the dilution changes of GnRH antibodies in the blood of all experimental dogs during the one - year period after immunization. The results show that the three mRNA vaccine - immunized groups had higher antibody dilutions and longer - lasting antibodies.

[0114] Table 6 Evaluation of the castration effect of GnRH mRNA vaccine - immunized dogs

[0115]

[0116]

[0117] Example 9: Evaluation of the castration effect of GnRH mRNA vaccine - immunized cats

[0118] Thirty healthy domestic cats aged 4 - 6 months (15 males and 15 females) were selected. The male and female domestic cats were randomly paired, one male and one female as a pair, and each pair was kept in a separate cage. Three pairs of cats were grouped for immunization. The first group was immunized with the GL11 - FermRNA vaccine, the second group with the GL11 - AP205 mRNA vaccine, the third group with the GL11 - i301mRNA vaccine, and the fourth group with the GnRH - six recombinant protein vaccine. Each cat was immunized with 50 μg of mRNA vaccine or 50 μg of recombinant protein, and boosted once after one month. The fifth group was the control group. According to the immunization time and frequency of the immunized groups, each cat was injected with 0.5 ml of normal saline each time. Blood samples were collected before the first immunization and then once a month. The serum was separated and stored at - 80 °C. The estrus days of male cats, the parturition time and litter size of female cats in each pair were observed. Observation ended after each pair of cats gave birth. In each pair of cats, blood collection ended when the female cat was observed to be pregnant. If not pregnant, observation continued for one year. After the last blood collection, the GnRH antibody titers in the sera of all cats were measured using the same method as in Example 7.

[0119] Table 7 shows the immunocastration effects of each pair of cats within one year. The three mRNA vaccine - immunized groups significantly inhibited male cat estrus and female cats were 100% infertile, which was higher than that of the recombinant control group and the normal saline control group, and the castration effect was significant. Figure 11 It shows that the GnRH antibodies in the cats in the three mRNA vaccine groups can last for one year, and the sterilization effect can be achieved within one year after vaccination.

[0120] Table 7 Evaluation of the Immunocastration Effect of GnRH mRNA Vaccines in Cats

[0121]

[0122]

[0123] In summary, in the present invention, GnRH (SEQ ID NO.2) is linked to form a multimer through a specific linker arm (SEQ ID NO.1), or the GnRH multimer is linked to a carrier protein to form a fusion antigen, and an mRNA - expressed multimer or fusion antigen is prepared. Compared with the conventional linker arm (Table 2), the expression level is higher, and the GnRH epitope is better presented. The mRNA vaccine formed by encapsulating mRNA with liposomes can be used for animal or pet castration to effectively control the fertility of animals or pets.

[0124] The specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the above - mentioned specific embodiments, and those skilled in the art can make various modifications or alterations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A GnRH multimer antigen, wherein the amino acid sequence of the GnRH multimer antigen is the sequence shown at positions 23-196 in SEQ ID NO.25, or the sequence shown at positions 23-254 in SEQ ID NO.37, or the sequence shown at positions 23-312 in SEQ ID NO.

39.

2. A recombinant engineering bacterium, characterized in that, The recombinant engineering bacterium is obtained by connecting the gene encoding the GnRH multimer antigen as claimed in claim 1 to an expression vector and then transforming Escherichia coli.

3. An Escherichia coli ( Escherichia coli ), designated as PS038, which has been deposited under the accession number CCTCC NO: M20242333.

4. A GnRH carrier protein antigen, characterized in that, The amino acid sequence of the GnRH carrier protein antigen is directly connected by the amino acid sequence of the GnRH multimer antigen as claimed in claim 1 and the amino acid sequence of the carrier protein; The direct connection means that the amino acid sequence of the GnRH multimer antigen is at the amino terminus of the amino acid sequence of the carrier protein, or the amino acid sequence of the GnRH multimer antigen is at the carboxyl terminus of the amino acid sequence of the carrier protein; The carrier protein is ferritin from Helicobacter pylori, E2 protein from thermophilic bacteria, Qβ protein from bacteriophage, AP205 protein from bacteriophage, β-defensin HDP protein or artificially designed i301 protein; the sequence of ferritin from Helicobacter pylori is the sequence shown at positions 198-363 in SEQ ID NO.67; the sequence of E2 protein from thermophilic bacteria is the sequence shown at positions 198-439 in SEQ ID NO.69; the sequence of Qβ protein from bacteriophage is the sequence shown at positions 24-155 in SEQ ID NO.73; the sequence of AP205 protein from bacteriophage is the sequence shown at positions 198-329 in SEQ ID NO.77; the sequence of β-defensin HDP protein is the sequence shown at positions 198-274 in SEQ ID NO.83; the sequence of artificially designed i301 protein is the sequence shown at positions 198-401 in SEQ ID NO.

87.

5. A ribonucleic acid sequence, which contains a coding signal peptide sequence, a coding GnRH multimer antigen sequence as claimed in claim 1, and a termination codon sequence; or contains a coding signal peptide sequence, a coding GnRH carrier protein antigen sequence as claimed in claim 4, and a termination codon sequence; The coding signal peptide sequence is the canine GnRH signal peptide shown in SEQ ID NO.131, the feline GnRH signal peptide shown in SEQ ID NO.133, the porcine GnRH signal peptide shown in SEQ ID NO.135, the murine GnRH signal peptide shown in SEQ ID NO.137, the human GnRH signal peptide shown in SEQ ID NO.139, the human TPA signal peptide shown in SEQ ID NO.141, the murine heavy chain signal peptide shown in SEQ ID NO.147 or the artificially designed signal peptide shown in SEQ ID NO.149; The termination codon sequence is TGA, TGATGATGA, TAGTAGTAG, TAGTGATGA or TAGTGATAA.

6. A GnRH mRNA vaccine, the GnRH mRNA vaccine comprising a composition consisting of the ribonucleic acid sequence as claimed in claim 5 and a liposome delivery system.

7. Use of the GnRH multimer antigen as claimed in claim 1 in the preparation of a medicament for regulating the reproductive ability of animals or pets; the animals or pets being dogs, cats or mice.

8. Use of the GnRH carrier protein antigen as claimed in claim 4 in the preparation of a medicament for regulating the reproductive ability of animals or pets; the animals or pets being dogs, cats or mice.

9. Use of the ribonucleic acid sequence as claimed in claim 5 in the preparation of a medicament for regulating the reproductive ability of animals or pets; the animals or pets being dogs, cats or mice.

10. Use of the GnRH mRNA vaccine as claimed in claim 6 in the preparation of a medicament for regulating the reproductive ability of animals or pets; the animals or pets being dogs, cats or mice.

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