GnRH nucleic acid vaccine

Abstract

The invention discloses a GnRH nucleic acid vaccine. GnRH is connected through a specific connecting arm to form a polymer, or GnRH or the polymer is connected with a carrier protein to form a fusion antigen, the polymer or the fusion antigen is expressed through mRNA, compared with a conventional connecting arm, the expression quantity is higher, GnRH epitopes are better displayed, and the mRNA vaccine formed by entrapping mRNA through lipidosome can be used for castration of animals or pets; the fertility of animals or pets is effectively controlled.

Description

Technical Field

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

[0002] Sterilization and castration of pets such as dogs and cats can avoid unnecessary reproduction, control the number of offspring, make pets more docile and easier to raise, and avoid abandonment of puppies after breeding or because the pet owners are unable or have no time to take care of the puppies. In addition, sterilization of pet dogs can effectively avoid the occurrence of a series of related diseases, including reducing the risk of breast cancer in female dogs, eliminating the risk of uterine pyometra, and avoiding ovarian tumors, ovarian cysts, false pregnancies and dystocia in dogs; it can also reduce the probability of prostate hyperplasia and infection in male dogs, reduce the incidence of perineal hernia, prevent the occurrence of perianal adenomas and reduce urine marking in male dogs. Sterilization surgery is currently the most commonly used method, but it can cause high stress in animals and the wound is prone to infection. In addition, the cost of surgery is relatively expensive and postoperative care is required, which increases the burden on pet owners. Therefore, there is a high demand for sterilization methods that replace surgery.

[0003] Gonadotropin releasing hormone (GnRH) is one of the important hormones regulating reproductive activity in mammals. It is synthesized and secreted by the hypothalamus, stimulating the anterior pituitary gland to secrete gonadotropins, which in turn stimulates the production and release of sex hormones. It can also directly act on the gonads, regulating the function of the gonads to regulate animal reproduction. Physiological doses of GnRH can promote the pituitary gland to release luteinizing hormone and follicle-stimulating hormone, which act on the gonads through the blood circulation, promote testicular development and sperm formation in male animals, promote ovarian development and follicle maturation in female animals, promote the synthesis and secretion of sex hormones, further promote gamete production, and maintain secondary sexual characteristics. Studies have shown that by inducing anti-GnRH antibodies through active immunization, reducing GnRH levels, leading to reduced synthesis of luteinizing hormone and follicle-stimulating hormone, accompanied by reduced estrus, testicular development and sperm production, it is expected to play a castration role in both male and female animals.

[0004] GnRH is a small peptide containing ten amino acids. It is a small molecule hapten with weak immunogenicity. It is difficult to induce an immune response when used alone as an immunogen for a vaccine, which is a difficulty in the industry. Therefore, it is necessary to modify GnRH and then connect it to a large molecule carrier protein to improve its immunogenicity (see Chinese patents (application numbers 201711339036.2, 202310434223.8, 202211461291.5, 202211052856.4)). Although the prior art has conducted a lot of research on the modification of GnRH antigens, both the natural GnRH antigen and the modified analogs still have technical problems such as low immunogenicity and large side effects.

[0005] mRNA vaccines are considered the third generation of vaccine technology because they introduce mRNA sequences encoding viral antigens into the body through a delivery system and express them in the body to produce antigens, which in turn stimulates an immune response in the body. mRNA vaccines break through the immune activation model of traditional vaccines and innovatively use the body's own cells to produce antigens, thereby activating dual-specific immunity, forming immune memory, and providing more lasting specific immunity. They 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 be integrated 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 are safer than DNA vaccines and traditional adjuvant vaccines.

[0006] The high immunogenicity and high safety of mRNA vaccines can well make up for the defects of low immunogenicity and large immune dose of existing GnRH vaccines. Since GnRH is a short peptide composed of only 10 amino acids, using mRNA to express a single molecule of GnRH in vivo will result in low expression and easy degradation, resulting in low immunogenicity. Therefore, it is necessary to use a linker to connect and polymerize the GnRH molecules, as well as to connect them to the carrier protein. The use of traditional linkers, such as GGGGS, SGS, EAAAK, GPGPG, KK, AAY, etc., will also affect the expression level of mRNA and the display of antigenic epitopes to varying degrees. Summary of the invention

[0007] In view of the above-mentioned prior art, the present invention accidentally obtains a GnRH-specific linker (SEQ ID NO.1) in the process of using mRNA to optimize the expression of GnRH; and uses the specific linker to study the mRNA vaccine constructed by GnRH and carrier protein, and provides a GnRH polymer antigen, a GnRH carrier protein antigen, and an mRNA vaccine encoding a GnRH polymer antigen or a 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, wherein the amino acid sequence of the GnRH multimer antigen comprises a GnRH polypeptide sequence (SEQ ID NO.2) and a specific connecting arm 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 engineered bacterium, which is obtained by connecting a gene encoding the aforementioned GnRH polymer antigen to an expression vector and then transforming Escherichia coli.

[0013] As one embodiment of the present invention, the GnRH multimer 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 arm 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] The invention also relates to an Escherichia coli PS038, whose deposit number is CCTCCNO: M20242333.

[0017] <Fourth Aspect>

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

[0019] As an embodiment, 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 an embodiment, the direct connection is 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] <Fifth Aspect>

[0022] The present invention relates to a ribonucleic acid (RNA) sequence, which comprises a coding signal peptide sequence, a coding sequence for the GnRH polymer antigen or a coding sequence for the GnRH carrier protein antigen, and a termination codon sequence.

[0023] As one embodiment, the signal peptide sequence is derived from canine GnRH signal peptide sequence, cat 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 an artificially designed signal peptide sequence.

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

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

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

[0027] <Sixth Aspect>

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

[0029] <Seventh Aspect>

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

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

[0032] The Escherichia coli PS038 of the present invention has been submitted to the China Center for Type Culture Collection for preservation on October 25, 2024, with the preservation address being Wuhan University, China, and the preservation number being CCTCCNO: M20242333. It is used to prepare a GnRH hexamer antigen plasmid, and then to prepare a nucleic acid vaccine.

[0033] Compared with the prior art, the present invention constructs GnRH polymer antigen and GnRH carrier protein antigen through a GnRH-specific linker arm (SEQ ID NO.1), which can increase the expression level of GnRH polymer 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 persistence. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

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

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

[0038] Figure 4 To detect the mRNA cellular expression of 17 GnRH carrier protein constructs using the GnRH ELISA kit;

[0039] Figure 5 To detect the mRNA cellular expression of 17 GnRH carrier protein constructs using Western Blot method;

[0040] Figure 6 is the serum testosterone content of male C57 mice immunized with the vaccine used in Example 7;

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

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

[0043] Fig. 9 The serum estradiol levels of female dogs at different times after immunization with the vaccine in Example 8;

[0044] Fig.10 is the anti-GnRH antibody titer in the serum of dogs at different times after immunization with the vaccine in Example 8;

[0045] Fig.11 The anti-GnRH antibody titers in the serum of domestic cats at different times after immunization with the vaccine in Example 9. DETAILED DESCRIPTION

[0046] The present invention is 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 are not intended to limit the present invention in any form. It should be noted that, for those of ordinary skill in the art, some adjustments and improvements can also 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 all circular mRNA. The circularized precursor RNA of the present invention is derived from the clean PIE system, and its 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] Plasmids encoding GnRH multimeric antigen or GnRH carrier protein antigen were constructed. This step was entrusted to Suzhou GenScript Biotechnology Co., Ltd. for gene synthesis and cloning. DNA vectors for producing mRNA were constructed. Gene fragments formed by sequentially connecting EcoRI restriction site, T7 promoter, intron fragment II, translation initiation element truncation fragment II, GnRH multimeric antigen or GnRH carrier protein antigen coding region sequence, ployAC, translation initiation element truncation fragment I, intron fragment I and restriction site BsaI were inserted into pUC57 vector using EcoRI and BsaI double restriction digestion. The sequences used to construct DNA vectors are shown in Table 1.

[0051] Table 1 Plasmid construction sequence

[0052]

[0053]

[0054] 1.2 Preparation of linear plasmid template

[0055] 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:

[0056]

[0057]

[0058] The enzyme digestion was carried out overnight at 37°C, and the digestion products were recovered using a universal DNA recovery kit (Tiangen Biochemical Technology Co., Ltd.).

[0059] 1.3 In vitro transcription

[0060] The recovered linear cDNA template was subjected to in vitro transcription reaction. The in vitro transcription reaction system was as follows:

[0061]

[0062] Incubate at 37°C, 220 rpm for 3-5 hours, then add 1KU DNaseI and react at 37°C for 15-30 minutes. TM Oligo(dT) 25 The purified RNA was analyzed by agarose gel electrophoresis to confirm that it was not degraded.

[0063] 1.4 Cyclization reaction

[0064] The purified linear RNA was subjected to circularization reaction 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 store at -20°C overnight. Centrifuge at 5000 rpm for 20 min at 4°C using a refrigerated centrifuge, collect the precipitate, wash the precipitate twice with ice-cold anhydrous ethanol, and dissolve the precipitate in 1 ml of enzyme-free water. Quantify mRNA using Nanodrop, and analyze by capillary electrophoresis to confirm the integrity and purity of mRNA.

[0067] Example 2: GnRH multimeric antigen coding region construct

[0068] In order to study the effect of the connecting arm on the mRNA expression of GnRH multimers, the present invention selects different connecting arms to connect GnRH monomers (SEQ ID NO.2), different signal peptides and stop codons, constructs the GnRH dimer, tetramer, hexamer, octamer, decamer antigen coding region sequences, forms GnRH multimer antigen coding region constructs, and prepares the corresponding mRNA for in vitro cell transfection screening or animal immune screening through Example 1. The connecting arms used for screening are shown in Table 2, the signal peptides used for screening are shown in Table 3, and the constructed GnRH multimer antigen coding region constructs are shown in Table 4.

[0069] Table 2 Linker sequences

[0070]

[0071]

[0072] Table 3 Signal peptide sequence

[0073]

[0074]

[0075] Table 4 GnRH multimeric antigen coding region construct

[0076]

[0077]

[0078]

[0079]

[0080] Example 3: GnRH carrier protein antigen coding region construct

[0081] In addition to using a linker to form a polymer to enhance the immune effect, GnRH vaccine immunization is usually connected to a carrier protein to increase the solubility of the antigen and the size of the antigen to improve the immunogenicity. The present invention selects the linker 1 or linker 11 in Example 2 to connect with different carrier proteins to form different GnRH carrier protein antigen coding region constructs, and prepares the corresponding mRNA for in vitro cell transfection screening or animal immune screening. The constructed GnRH carrier protein antigen coding region construct is shown in Table 5.

[0082] Table 5 GnRH carrier protein coding region construct

[0083]

[0084]

[0085]

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

[0087] In order to better screen the linker of the GnRH mRNA vaccine, this example expresses the GnRH hexamer recombinant protein and the recombinant fusion protein, which are used as controls for GnRH mRNA expression verification or immune controls for mRNA vaccines. The GnRH hexamer recombinant protein uses a conventional (GGGGS)3 linker, and adds a 6×His tag at the C-terminus for purification. The amino acid sequence of the coding region of the GnRH hexamer recombinant protein is such as SEQ ID NO.97, and the nucleotide sequence is such as SEQ ID NO.98. The GnRH recombinant fusion protein is fused and expressed by GnRH hexamer and carrier protein defensin HDP, and a 6×His tag is added to the C-terminus for purification. The amino acid sequence of the coding region of the GnRH recombinant fusion protein is such as SEQ ID NO.99, and the nucleotide sequence is such as SEQ ID NO.100. The specific operations are as follows:

[0088] (1) Gene synthesis and vector construction: Artificially synthesize the gene fragment of the nucleotide sequence (SEQ ID NO.98) of the coding region of the GnRH hexamer recombinant protein and the gene fragment of the nucleotide sequence (SEQ ID NO.100) of the coding region of the GnRH recombinant fusion protein, add the NcoI restriction site at the 5' end, add the EcoRI restriction site at the 3' end, and insert the synthesized gene fragment into the pET28a plasmid using NcoI and EcoRI double digestion to obtain pET28a-GnRH-six and pET28a-GnRH-HDP plasmids. The obtained plasmids were transformed into Escherichia coli DH5α, and screened on 1.5% agar solid medium, and the recombinant plasmids with correct sequencing were selected. The recombinant plasmids were transformed into Escherichia coli BL21, and a high-expression strain was selected, which were named BL21 / pET28a-GnRH-six and BL21 / pET28a-GnRH-HDP, respectively. Gene artificial synthesis, PCR, restriction insertion, and strain screening and sequencing were completed by Suzhou 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 at 37°C overnight for recovery. The culture was expanded at an inoculation ratio of 1:100. When the OD600 was 0.6, IPTG with a final concentration of 0.4 mM was added to induce expression. After induced culture for 4 h, the bacteria were collected by centrifugation at 3500 rpm for 10 min.

[0090] (3) Recombinant protein purification: The collected bacteria were washed twice with PBS, resuspended in 100 ml lysis buffer (20 mM sodium phosphate, 0.5 M NaCl, 10 mM imidazole, 1 mg / ml lysozyme, 20 μg / mg DNaseI pH 7.4), and ultrasonically disrupted for 15 min in an ice bath using an ultrasonic disruptor. 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, and ultrasonically disrupted for 15 min in an ice bath using an ultrasonic disruptor. Centrifuge at 10,000 rpm for 10 min and collect the supernatant. The supernatant was purified on a nickel column to obtain the GnRH hexameric recombinant protein or GnRH recombinant fusion protein.

[0091] (4) Identification of recombinant protein: The purified GnRH hexameric recombinant protein or GnRH recombinant fusion protein was detected by SDS-PAGE electrophoresis, first at 80 V for 20-30 min, then at 120 V for 50-60 min, and then stained and destained and photographed. The results are as follows: Figure 1 As shown, the sizes of GnRH hexameric recombinant protein and GnRH recombinant fusion protein are consistent with the theory.

[0092] Example 5: Verification of in vitro expression of GnRH polymer antigen and carrier protein antigen mRNA

[0093] In order to study the expression effect of GnRH multimer antigens and carrier protein antigens formed by different connecting arms and signal peptides in the form of mRNA, the mRNA prepared by Example 1 for each coding region construct in Example 2 and Example 3 was expressed by -mRNA Transfection Kit was transfected into 293T cells at 100 ng / well. The transfection conditions were as follows:

[0094]

[0095] Incubate in 96-well plates at 37°C and 5% carbon dioxide for 48 hours, and collect cell supernatants. Use a GnRH ELISA kit (Shanghai Biotech) to perform the test according to the kit's operating procedures. At the same time, Western Blot was used to perform parallel tests on the collected cell supernatants to eliminate the effects of different GnRH antibodies on the test results. Western Blot detection was performed using SurePAGE TM The protein precast gel was 4-20% (GenScript Biotech 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).

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

[0097] Example 6. Preparation of GnRH mRNA vaccine

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

[0099] (1) Preparation of lipid solution: Cationic lipid CMAX4, cholesterol (5-cholesten-3β-ol), neutral lipid DSPC (distearoylphosphatidylcholine), and PEG-modified lipid PEG-DMG (polyethylene glycol-dimyristyl 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.

[0100] Among them, 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) was purchased from Suzhou Coremade Biopharmaceutical Technology Co., Ltd., and cholesterol (5-cholesten-3β-ol), neutral lipid DSPC (distearoylphosphatidylcholine), and PEG-modified lipid PEG-DMG (polyethylene glycol-dimyristylglycerol) were purchased from Avituo (Shanghai) Pharmaceutical Technology Co., Ltd.

[0101] (2) Preparation of mRNA solution: Use the construct with high cell expression level verified in Example 5 to prepare the mRNA solution, preferably use the 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, and dissolve the mRNA of the preferred construct in 10 mM citrate buffered saline solution at pH 4.0 and dilute to a final concentration of 200 μg / mL to obtain an mRNA solution.

[0102] (3) The lipid solution and the mRNA solution were rapidly mixed at a volume ratio of 1:3 using microfluidics technology, and the buffer environment was replaced with PBS at pH 7.0 using ultrafiltration technology to remove ethanol, thereby preparing LNP-mRNA.

[0103] (4) Add sucrose solution to the LNP-mRNA solution to a final sucrose concentration of 8% (w / v) and use Quant-iT TM RiboGreen TM RNA Assay Kit (Invitrogen TM R11490) kit was used to determine the mRNA concentration in the prepared LNP-mRNA, and the LNP-mRNA solution was diluted to 50 μg / ml. After sterile filtration, the solution was divided into portions and frozen to obtain the mRNA vaccine.

[0104] (5) The particle size, polydispersity index (PDI), and surface potential of the mRNA vaccine were measured using the dynamic light scattering method on a Malvern Zetasizer Nano-ZEN 3600 (Malvern) Zeta potential laser particle size analyzer to determine if they were within the specified range.

[0105] Example 7: GnRH mRNA vaccine mouse immunization and antibody detection

[0106] In order to study the differences in immunogenicity of GnRH mRNA vaccines of different constructions, 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 6-8 week old male C57 mice, 5 mice were immunized with each constructed GnRH mRNA vaccine or recombinant protein vaccine, 5 μg / mouse / time, the volume was 100 μl, and the hind leg muscle was used for immunization. At the same time, mice injected with normal saline were used as negative controls. Immunization was performed at 0 weeks and 3 weeks, respectively. Blood was collected on the 14th day 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 testosterone (Testosterone) ELISA kit (Sangon Biotech) according to the operating procedures in the manual. The serum GnRH antibody titer was detected by indirect ELISA method. The main steps of the GnRH antibody indirect ELISA detection method are as follows:

[0107] (1) Recombinant GnRH protein (Abcam) was diluted to 1 μg / ml with 0.1 M bicarbonate buffer (pH 9.6), and 100 μl / well was added to a 96-well ELISA plate (Costar) and incubated at 4°C overnight; after washing three times, 5% BSA was added and blocked at 37°C for 2 hours; the plate was patted dry, dried at 37°C, and stored at 4°C for later use.

[0108] (2) The collected mouse serum was diluted in a 2-fold gradient using PBS, and each dilution 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 / well of HRP-labeled rabbit anti-mouse IgG (Sigma) was added, and reacted at 37°C for 1 hour; after washing 5 times, TMB substrate (Biyuntian) was added for color development for 10 minutes, and the OD value at 450 nm was measured.

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

[0110] Figure 6The testosterone levels of mice serum in 11 different constructs of GnRH mRNA vaccines and 2 protein vaccines and the control group are shown. Compared with the control group, the testosterone levels of GnRH mRNA vaccines were reduced on the 14th day 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 (G-L11-S1, GL11-Fer, GL11-E2, G-Qβ-L11, GL11-AP205, GL11-HDP, GL11-i301,) constructed using a specific linker (linker 1) were significantly lower than those of the mRNA vaccine groups (G-L1, GL1-AP205, GL1-HDP, GL1-i301) constructed using a conventional linker (linker 1). Figure 7 The antibody titers of GnRH in the serum of each immune group are shown. The antibody titers indicate that each vaccine group has produced immune stimulation to the body and produced GnRH antibodies. Compared with the recombinant protein vaccine group, the epitope display of the GnRH mRNA vaccine group is more exposed, the antibody titer is higher, and the stimulation to the body is more effective. The antibody titer of the mRNA vaccine group using a specific linker (linker 11) is significantly higher than that of the mRNA vaccine group using a conventional linker (linker 1). The immune effect of mice shows that the GnRH mRNA vaccine constructed using a specific linker has a more advantageous immune effect and is more effective in reducing serum testosterone levels.

[0111] Example 8: Evaluation of castration effects in dogs immunized with GnRH mRNA vaccine

[0112] Thirty healthy dogs aged 5-7 months were selected (15 male dogs and 15 female dogs), and 15 male dogs and 15 female dogs were randomly divided into five groups, with 3 male dogs and 3 female dogs in each group mixed 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 mRNA vaccine or 100 μg recombinant protein, and booster immunization was performed once one month later; the fifth group was the control group, and each dog was injected with 1 ml of normal saline each time according to the immunization time and number of immunization groups. Blood samples were collected before the first immunization, and blood samples were collected every two months thereafter. 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 determined; the testosterone content and the GnRH antibody titer were determined in the same manner as in Example 7, and the estradiol content was determined using an estradiol ELISA kit (Huamei Bio) according to the instructions. During the experiment, the number of estrus of male dogs in each group and the number of litters of female dogs were observed, and the observation was continued for one year.

[0113] Table 6 shows the experimental observation results after one year of immunization. The three mRNA vaccine groups immunized dogs were 100% infertile, which was significantly higher than the recombinant control group and the saline control group. Figure 8 , Fig. 9 The changes in serum testosterone levels in male dogs and serum estradiol levels in female dogs one year after immunization are shown respectively. The results show that the three mRNA vaccine groups controlled hormone levels lower and for longer periods of time. Fig.10 The results showed the changes in the dilution of GnRH antibodies in the blood of all experimental dogs one year after immunization. The results showed that the antibody dilutions of the three mRNA vaccine groups were higher and the antibody duration was longer.

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

[0115]

[0116]

[0117] Example 9: Evaluation of castration effects in cats immunized with GnRH mRNA vaccine

[0118] Thirty healthy domestic cats aged 4-6 months (15 males and 15 females) were selected, and male and female domestic cats were randomly paired into a pair of one male and one female, and the pairs were raised in one cage. Three pairs of domestic cats were immunized as a group. The first group was immunized with GL11-FermRNA vaccine, the second group was immunized with GL11-AP205 mRNA vaccine, the third group was immunized with GL11-i301mRNA vaccine, and the fourth group was immunized with GnRH-six recombinant protein vaccine. Each cat was immunized with 50μg mRNA vaccine or 50μg recombinant protein, and boosted once a month; the fifth group was the control group. According to the immunization time and number of immunization groups, each cat was injected with 0.5ml normal saline each time. Blood samples were collected before the first immunization, and blood samples were collected once a month thereafter. The separated serum was stored at -80℃. The number of days of estrus of male domestic cats, the time of littering and the number of litters of female domestic cats in each pair of cats were observed. The observation was terminated after each pair of cats gave birth. Blood collection was terminated when the female cat was observed to be pregnant in each pair of cats. If there was no pregnancy, the observation was continued for one year. After the last blood collection, the titer of GnRH antibody in the serum of all cats was determined, and the determination method was the same as the determination method in Example 7.

[0119] Table 7 shows the immune castration effect of each pair of cats within one year. The three mRNA vaccine immunization groups significantly suppressed the estrus of male cats and 100% infertility of female cats, which was higher than the recombinant control group and the saline control group, and the castration effect was significant. Fig.11 It shows that the GnRH antibodies in cats of the three mRNA vaccine groups can last for one year, and the vaccination can have a sterilization effect within one year.

[0120] Table 7 Evaluation of castration effect of GnRH mRNA vaccine immunized cats

[0121]

[0122]

[0123] In summary, the present invention connects GnRH (SEQ ID NO.2) through a specific connecting arm (SEQ ID NO.1) to form a polymer, or connects the GnRH polymer with a carrier protein to form a fusion antigen, and prepares an mRNA expression polymer or fusion antigen. Compared with the conventional connecting arm (Table 2), the expression amount 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, and effectively controls the fertility of animals or pets.

[0124] The above describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art may 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 arm polypeptide as shown in SEQ ID NO.

1.

2. The GnRH multimeric antigen according to claim 1, characterized in that The multimer is a dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer, nonamer or decamer.

3. A recombinant engineered bacterium, characterized in that: The gene encoding the GnRH multimeric antigen as claimed in claim 1 is connected to an expression vector and then transformed into Escherichia coli to obtain the recombinant engineered bacteria.

4. An Escherichia coli PS038, whose deposit number is CCTCCNO: M20242333.

5. A GnRH carrier protein antigen, characterized in that: The GnRH carrier protein antigen amino acid sequence is directly connected by the amino acid sequence of the GnRH multimer antigen as claimed in claim 1 and the carrier protein amino acid sequence.

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, the ferritin 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; B. The direct connection is that the GnRH multimer antigen amino acid sequence is at the amino end of the carrier protein amino acid, or the GnRH multimer antigen amino acid sequence is at the carboxyl end of the carrier protein amino acid, or the GnRH multimer antigen amino acid sequence is 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 sequence as claimed in claim 1 or a sequence encoding a GnRH carrier protein antigen sequence as claimed in claim 5, and a stop codon sequence.

8. The ribonucleic acid sequence according to claim 7, characterized in that It also includes at least one of the following technical features: C. The signal peptide sequence is derived from a canine GnRH signal peptide sequence, a cat GnRH signal peptide sequence, a porcine GnRH signal peptide sequence, a mouse GnRH signal peptide sequence, a human GnRH signal peptide sequence, a human TPA signal peptide sequence, a human ALB signal peptide sequence, a mouse K light chain signal peptide sequence, a mouse heavy chain signal peptide sequence or an artificially designed signal peptide sequence; D. The stop codon sequence is (TAA)x(TAG)y(TGA)z, wherein x+y+z≥1, and x, y, and z are each independently an integer greater than or equal to 0.

9. A GnRH mRNA vaccine, comprising the ribonucleic acid sequence according to claim 7 and a composition consisting of a liposome delivery system.

10. Use of the GnRH polymer antigen as claimed in claim 1, or the GnRH carrier protein antigen as claimed in claim 5, or the ribonucleic acid sequence as claimed in claim 7, or the GnRH mRNA vaccine as claimed in claim 9 in the preparation of a drug for regulating the reproductive ability of animals or pets.

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