Gonadotropin releasing hormone antigen peptide, vaccine and application

Abstract

The invention relates to the technical field of vaccine preparation, and particularly discloses a gonadotropin releasing hormone antigen peptide, a vaccine and application, and the gonadotropin releasing hormone antigen peptide is obtained by forward or reverse series connection of GnRH monomers shown in SEQ ID NO.2 through a flexible Linker molecule composed of 1-3 Gly. The gonadotropin releasing hormone antigen peptide provided by the invention is used for inhibiting development or function exertion of an animal reproductive system, and the purpose of castration without a castration operation is achieved.

Description

Technical Field

[0001] This invention relates to the field of vaccine preparation technology, specifically to a gonadotropin-releasing hormone antigen peptide, a vaccine, and its applications. Background Technology

[0002] Surgical castration, also known as neutering, is a common method for animal production management and companion animal sterilization. However, surgical castration has many drawbacks, including cruelty to animals, especially since it is mostly performed without anesthesia, easily causing high stress, increased risk of surgical wound infection, increased morbidity and even death, and reduced animal productivity. In the pig farming industry alone, the economic losses caused directly or indirectly by surgical castration amount to billions of yuan annually nationwide. With increasing emphasis on animal welfare, traditional surgical castration has gradually been phased out. Finding new castration technologies to replace traditional surgical castration has become a key challenge that urgently needs to be overcome in the livestock farming industry and the field of companion animal sterilization. Gonadotropin-releasing hormone (GnRH) active immunization, as a new animal-friendly castration technology, is the most promising new method to replace traditional surgical castration. However, this technology currently faces several technical bottlenecks, including large doses of vaccine antigen (hundreds of micrograms or milligrams per injection), high costs, and the risk of immune "escape" after immunization, which severely restrict its widespread application in actual production. Therefore, it is essential to explore new GnRH antigen peptides. Summary of the Invention

[0003] To develop a novel gonadotropin-releasing hormone (GnRH) antigen peptide, this invention provides a GnRH antigen peptide, a vaccine, and its applications. The GnRH antigen peptide provided by this invention is used to inhibit the development of the animal reproductive system, achieving castration without surgical intervention.

[0004] This invention provides a gonadotropin-releasing hormone antigen peptide, which is obtained by connecting two GnRH monomers shown in SEQ ID NO.2 in forward or reverse tandem via a flexible linker molecule composed of 1 to 3 glycine residues.

[0005] The gonadotropin-releasing hormone antigen peptide provided by this invention is used to inhibit the development of the animal reproductive system, thereby achieving castration without castration surgery.

[0006] Furthermore, the gonadotropin-releasing hormone antigen peptide sequence is selected from any one of SEQ ID NO.1 and SEQ ID NO.3 to SEQ ID NO.5.

[0007] Furthermore, the gonadotropin-releasing hormone antigen peptide is also coupled to a carrier protein, with the coupling site being an additional cysteine ​​residue added to the C-terminus or N-terminus of the gonadotropin-releasing hormone antigen peptide.

[0008] Furthermore, the carrier protein is chicken ovalbumin, keyhole hemocyanin, tetanus toxoid, bovine serum albumin, or human serum albumin.

[0009] Furthermore, the gonadotropin-releasing hormone antigen peptide is synthesized using CTC Resin resin as a raw material and employing Fmoc solid-phase polypeptide synthesis.

[0010] The present invention also provides a GnRH vaccine containing the gonadotropin-releasing hormone antigen peptide described above.

[0011] Furthermore, the GnRH vaccine is prepared by emulsifying gonadotropin-releasing hormone antigen peptide with an adjuvant.

[0012] Furthermore, the adjuvant is Spector adjuvant.

[0013] This invention also provides the application of gonadotropin-releasing hormone antigen peptide or GnRH vaccine in the preparation of animal castration products.

[0014] Furthermore, the castration product is a GnRH vaccine.

[0015] Compared with the prior art, the present invention has the following beneficial effects: This invention connects GnRH monomers to a flexible linker molecule composed of 1 to 3 glycine residues. The tandem connection of the GnRH monomers does not affect their natural β-II turn conformation. The prepared GnRH antigen peptide and vaccine are used to inhibit the development of the animal reproductive system, facilitating castration without surgical intervention. This effectively solves the three major technical bottlenecks hindering the practical application and promotion of GnRH immunization and castration vaccines: large antigen dosage, high cost, and the risk of immune "escape" after immunization. Attached Figure Description

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0017] Figure 1 The figure shows the sequence information of the synthesized peptide in Example 1 of the present invention; in the figure, ① to ① represent the four cases of synthesized peptide.

[0018] Figure 2 To study the influence of the tandem arrangement of gonadotropin-releasing hormone molecules on their structure; In the diagram, A represents the native conformation of the gonadotropin-releasing hormone decapeptide molecule: B is the prediction of the conformation of gonadotropin-releasing hormone monomers using Alphfold 2; C represents the conformational image of a direct tandem conjugate of gonadotropin monomers: D to F are synergistic compounds of gonadotropin monomers linked together by a flexible linker molecule.

[0019] Figure 3 Evaluation of the immunization and castration effect of the GnRH vaccine prepared in Example 1 of this invention on male SD mice; In the diagram, A represents a schematic diagram of the immunization program for each group; B represents the effect of different immunization groups on testicular volume in male SD rats. C represents the effect of different immunization groups on the appearance of testes in male SD rats.

[0020] Figure 4 To evaluate the castration effect of GnRH vaccine containing 2.5 μg GLG antigen in immunizing male SD mice; In the figure, A is a schematic diagram of the immunization program for male SD mice immunized with GnRH vaccine containing 2.5 μg of GLG antigen; B shows the effect of immunization on the testes of male SD rats before and after immunization. From left to right, the photos are of the testes of male rats in the control group and male rats in the immunized group. Effects of GnRH vaccine (C = 2.5 μg GLG antigen) on the testes of male SD mice immunized with the testis.

[0021] Figure 5 This is an evaluation of the effect of the GnRH vaccine prepared in Example 1 of the present invention on castration in breeding boars; In the diagram, A is a schematic diagram of the immunization procedure; B represents the effect of GnRH vaccine immunization prepared in Example 1 on the size of the testes in boars. C represents the effect of GnRH vaccine immunization prepared in Example 1 on testicular volume in boars; D represents the effect of GnRH vaccine immunization prepared in Example 1 on spermatogenesis in boar testes.

[0022] Figure 6 This is an evaluation of the effect of the GnRH vaccine prepared in Example 3 of the present invention on immunization and castration in male SD rats; In the diagram, A represents a schematic diagram of the immunization program for each group; B represents the effect of GnRH vaccine immunization prepared in Example 3 on testicular development in male SD rats. C is an image showing the analysis of testicular tissue sections from male SD rats immunized with the GnRH vaccine prepared in Example 3. Detailed Implementation

[0023] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0024] GnRH is a short peptide composed of 10 amino acid residues, with the sequence shown in SEQ ID NO.2. It has a low molecular weight and no antigenic immunogenicity.

[0025] Figure 2 As shown in A, the natural conformation of GnRH monomers exhibits a unique β-II turn secondary conformation, forming a horseshoe turn, as described in the literature with PMID number 15082521. Figure 2 As shown in Figure B, the conformation of gonadotropin-releasing hormone monomers was predicted using Alphfold 2, and the prediction results were in high agreement with their native conformation. When GnRH monomers were directly tandemly synthesized chemically, the native conformation of the GnRH molecule was greatly disrupted; its specific β-II turn secondary conformation almost disappeared, as shown in Figure B. Figure 2 C.

[0026] This invention utilizes flexible linker molecules composed of 1 to 3 Gly molecules to tandemly connect GnRH monomers in either a forward or reverse manner. This significantly protects the specific conformation of the tandem GnRH monomers. The linker tandem arrangement perfectly maintains the native conformation of the gonadotropin-releasing hormone monomers within the conjugate, such as... Figure 2 D~ Figure 2 E. For example Figure 2 As shown in F, Figure 2 The sequence shown is coupled to a carrier protein via an additional amino acid at the C-terminus or N-terminus to prepare a vaccine as an antigen. Immunizing animals with this vaccine can significantly stimulate their immune system to produce anti-GnRH antibodies. These antibodies specifically bind to endogenous GnRH in the animal's body, blocking the binding of endogenous GnRH to its receptor GnRHR, thereby inhibiting gonadal development, sex hormone synthesis, and gamete production, thus achieving the purpose of immunization and castration.

[0027] The GnRH antigen peptide sequence obtained by tandem GnRH monomers in this invention is as follows: Figure 1 As shown, there are four cases, as illustrated in SEQ ID NO.1 and SEQ ID NO.3 to SEQ ID NO.5 respectively.

[0028] SEQ ID NO.1: XHWSYGLRPGGGQHWSYGLRPGC; where X represents pyroglutamic acid.

[0029] The three-letter abbreviation sequence of SEQ ID NO.1 is: pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-Gly-Gly-Gln-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-Cys.

[0030] SEQ ID NO.3: CQHWSYGLRPGGGQHWSYGLRPGX; X is an amino group; The three-letter abbreviation sequence of SEQ ID NO.3 is: Cys-Gln-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-Gly-Gly-Gln-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2.

[0031] SEQ ID NO.4: XHWSYGLRPGGGPRLGYSWHQC; where X represents pyroglutamic acid.

[0032] The three-letter abbreviation sequence of SEQ ID NO.4 is: pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-Gly-Gly-Pro-Arg-Leu-Gly-Tyr-Ser-Trp-His-Gln-Cys.

[0033] SEQ ID NO.5: XHWSYGLRPGGGGPRLGYSWHQC; where X represents pyroglutamic acid.

[0034] The three-letter abbreviation sequence of SEQ ID NO.5 is: pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-Gly-Gly-Gly-Pro-Arg-Leu-Gly-Tyr-Ser-Trp-His-Gln-Cys.

[0035] Example 1: A gonadotropin-releasing hormone antigen peptide, a vaccine, and its preparation.

[0036] I. Construction of Gonadotropin-Releasing Hormone Antigen Peptide 1. GnRH antigen peptide synthesis (1) Using CTC Resin resin as raw material, amino acid sequences such as those obtained by Fmoc solid-phase peptide synthesis were synthesized. Figure 1 The GnRH-linker-GnRH-C oligopeptide shown in ① is referred to as the synthetic peptide, and its sequence is shown in SEQ ID NO.1. The synthetic peptide was purified using reversed-phase high-performance liquid chromatography.

[0037] SEQ ID NO.1: pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-Gly-Gly-Gln-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-Cys. The underlined Gly-Gly indicates the linker, and the underlined Cys indicates the coupling site between the synthetic peptide and the carrier protein.

[0038] SEQ ID NO.2: XHWSYGLRPG, where X represents pyroglutamic acid.

[0039] The three-letter abbreviation sequence of SEQ ID NO.2 is: pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly.

[0040] (2) The synthetic peptide was coupled to the carrier protein using 3-maleimide benzoate succinimide to obtain the GnRH antigen peptide, denoted as GLG antigen. The -SH position of the cysteine ​​residue at position 23 of the synthetic peptide is the coupling site between the synthetic peptide and the carrier protein.

[0041] In this invention, gonadotropin-releasing hormone is abbreviated as GnRH; 3-maleimide benzoate succinimide ester is abbreviated as MBS; and the carrier protein is chicken egg albumin provided by Coolaber Company, abbreviated as OVA, CAS number 9006-59-1.

[0042] II. GnRH Vaccine Preparation GnRH antigen, prepared using Spector adjuvant, was emulsified to obtain a GnRH vaccine. The emulsification steps are as follows:

[0043] (1) GnRH antigen is dissolved in 0.85% NaCl to obtain an aqueous solution of antigen.

[0044] (2) Preparation of Spector emulsifier: Mix Span85 and Tween85 thoroughly at a volume ratio of 54:46 and set aside. Measure 9 times the volume of mineral oil Marcol 52 and 1 times the volume of Spector emulsifier into a 300mL glass graduated cylinder. Use a Turaxx50 homogenizer to stir at 8000rpm. Keep the homogenizer at 8000rpm and slowly add 8 times the volume of antigen aqueous solution to the emulsification cylinder with a dropper. Move the emulsification cylinder up and down during the addition process to fully emulsify the antigen. After all the antigen aqueous solution has been added to the emulsification cylinder, adjust the speed of the Turaxx50 homogenizer to 12000rpm and continue stirring for 1min. Keep the emulsification cylinder in water throughout the emulsification process to prevent the high temperature generated by stirring from damaging the antigen activity. After emulsification, quickly cool the vaccine temperature to room temperature with tap water and store it at 4℃ for use immediately.

[0045] Example 2: Evaluation of the effects of gonadotropin-releasing hormone antigen peptide and vaccine on immunization and castration.

[0046] I. Evaluation of the effect of immunization and castration on male SD rats 1. Initial immunization Forty male SD mice were randomly divided into five groups of eight each: Control, G6KT-10, G6KT-50, GLG-10, and GLG-50. The immunization status of each group is as follows: Figure 3 Group A. At 6 weeks of age, each group received their initial immunization, with 1 mL of vaccine injected intramuscularly into the leg. See below:

[0047] Control: The control group was injected with an emulsifier that did not contain GnRH antigen, known as Placeboemulsion.

[0048] G6KT-10: An existing GnRH tandem antigen vaccine, named G6K-GnRH-tandem-OVA; PMID: 9682361, with an immunization dose of 10 μg, is used as a control.

[0049] G6KT-50: An existing GnRH tandem antigen vaccine, named G6K-GnRH-tandem-OVA; PMID: 9682361, with an immunization dose of 50 μg, is used as a control.

[0050] GLG-10: Immunization with the GnRH vaccine prepared in Example 1 of this invention, with an immunization dose of 10 μg.

[0051] GLG-50: Immunization with the GnRH vaccine prepared in Example 1 of this invention, with an immunization dose of 50 μg.

[0052] 2. Strengthen immunity Male SD rats received a booster immunization four weeks after the initial injection, using the same method and dosage. Six weeks after the booster, all rats were euthanized by cervical dislocation under anesthesia. Testes were separated, weighed, and histologically analyzed. The results are shown below. Figure 3 B~ Figure 3 C.

[0053] like Figure 3 As shown, compared with the control group of male rats, immunization with vaccines containing 10 μg and 50 μg of GnRH tandem antigen in the G6KT-10 and G6KT-50 experimental groups only caused testicular atrophy in 37.5% of male rats. Figure 3 B. In the GLG-10 and GLG-50 experimental groups, immunization with the vaccine prepared in Example 1 of this invention at a dose of 10 μg caused significant testicular atrophy in 87.5% of male mice. When the immunization dose was increased to 50 μg, significant testicular atrophy was caused in all male mice. Figure 3 B.

[0054] like Figure 3 As shown in Figure C, it is visually apparent that the vaccine prepared in Example 1 of this invention significantly inhibits testicular development in male mice. Using the same immunization procedure, the dosage per male mouse was further reduced to a GnRH vaccine containing 2.5 μg of GLG antigen, and the results are as follows... Figure 4 As shown, injection of a GnRH vaccine containing only 2.5 μg of GLG antigen significantly inhibited testicular development in male SD rats.

[0055] The above results indicate that the GnRH vaccine prepared in this invention can effectively solve the three major technical bottlenecks hindering the practical promotion of GnRH immunization castration vaccines: large antigen dosage, high cost, and easy immune "escape" after immunization.

[0056] II. Evaluation of the effectiveness of castration immunization in breeding boars Ten culled Duroc boars aged 23 weeks were selected and divided into a Control group and a GLG-100 experimental group, with five culled Duroc boars in each group. See the immunization diagram below. Figure 5 As shown in A. Active immunization with GnRH vaccine in the GLG-100 experimental group: 2 mL of GnRH vaccine containing 100 μg GLG antigen was injected intramuscularly into the neck. A booster immunization was administered two weeks after the initial injection, using the same method and dosage as the initial immunization. The Control group received an emulsion without GnRH antigen as a control, immunized in the same manner as the GLG-100 experimental group. Two weeks after the booster immunizations in the Control and GLG-100 experimental groups, all experimental boars were euthanized, and the test results are shown in [Figure A]. Figure 5 .

[0057] like Figure 5As shown, compared with the control group boars, injection of 100 μg GLG antigen significantly induced testicular atrophy in breeding boars just 2 weeks after immunization. Figure 5 B and Figure 5 As shown in C. Tissue sections showed that immunization for only 2 weeks completely suppressed spermatogenesis in boar testes, such as... Figure 5 As shown in D.

[0058] The above results further demonstrate that the GnRH vaccine prepared in Example 1 of this invention can be used as a highly effective GnRH castration vaccine.

[0059] Example 3: A gonadotropin-releasing hormone antigen peptide, a vaccine, and its preparation.

[0060] I. Construction of Gonadotropin-Releasing Hormone Antigen Peptide 1. GnRH antigen peptide synthesis (1) Since the C-terminus of the GnRH monomer is a Gly residue, it can itself act as a linker molecule. Therefore, if the second GnRH monomer is reverse-tandemly linked, the amount of Gly in the linker molecule can be reduced. Based on this, the present invention designs a second GnRH monomer reverse-tandem antigen. Using CTC Resin resin as raw material, the amino acid sequence is synthesized using the Fmoc solid-phase peptide synthesis method as shown in the figure. Figure 1 The GnRH-linker-inverted GnRH-C oligopeptide shown in ③ is also known as the GLiG-C oligopeptide, or simply the synthetic peptide. The sequence of the synthetic peptide is shown in SEQ ID NO.4. The synthetic peptide was purified using reversed-phase high-performance liquid chromatography.

[0061] SEQ ID NO.4: pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-Gly-Gly-Pro-Arg-Leu-Gly-Tyr-Ser-Trp-His-Gln-Cys. The underlined Gly indicates the linker, and the underlined Cys is the site where the synthetic peptide couples with the carrier protein.

[0062] (2) The synthetic peptide was coupled to the carrier protein using 3-maleimide benzoate succinimide to obtain the GnRH antigen peptide, denoted as GLiG antigen. The -SH position of the cysteine ​​residue at position 22 of the synthetic peptide is the coupling site between the synthetic peptide and the carrier protein.

[0063] In this invention, gonadotropin-releasing hormone is abbreviated as GnRH; 3-maleimide benzoate succinimide ester is abbreviated as MBS; and the carrier protein is chicken egg albumin, abbreviated as OVA.

[0064] II. GnRH Vaccine Preparation GnRH antigen prepared by emulsification with Spector adjuvant was used to prepare GnRH vaccine.

[0065] Example 4: Evaluation of the effects of gonadotropin-releasing hormone antigen peptide and vaccine on immunization and castration.

[0066] I. Evaluation of the effect of immunization and castration on male SD rats 1. Initial immunization Sixteen male SD mice were randomly divided into two groups of eight each: Control and GLiG. The immunization status of each group is as follows: Figure 6 Group A. At 6 weeks of age, each group received their initial immunization, with 1 mL of vaccine injected intramuscularly into the leg. See below:

[0067] Control: The control group was injected with an emulsifier that did not contain GnRH antigen, known as Placeboemulsion.

[0068] GLiG-2.5: Immunization with the GnRH vaccine prepared in Example 3 of this invention, the immunization dose is GnRH antigen containing 2.5 μg GLiG.

[0069] 2. Strengthen immunity Male SD rats received a booster immunization four weeks after the initial injection, using the same method and dosage. Six weeks after the booster, all rats were euthanized by cervical dislocation under anesthesia. Testes were separated, weighed, and histologically analyzed. The results are shown below. Figure 6 B~ Figure 6 C.

[0070] like Figure 6 As shown in Figure B, compared with the control group of male rats, the GLiG experimental group showed that testicular atrophy in 87.5% of male rats immunized with a vaccine containing 2.5 μg of the second GnRH monomer reverse tandem antigen alone. Figure 6 B. Histological analysis showed that immunization with a vaccine containing GLiG antigen completely inhibited spermatogenesis in the testes of male SD rats, such as Figure 6 As shown in C.

[0071] This result confirms that by using a strategy of reverse tandem cascading of a Gly flexible linker with a second GnRH monomer to prepare GnRH antigens, the number of Gly molecules in the linker molecule can be reduced to only one.

[0072] It should also be noted that the gonadotropin-releasing hormone antigen peptide can also be obtained by connecting two or more GnRH monomers shown in SEQ ID NO.2 in forward or reverse tandem via a flexible linker molecule composed of 1 to 3 glycine residues. This is not limited to the two examples in the embodiments of this invention.

[0073] In summary, the gonadotropin-releasing hormone antigen peptide prepared by this invention significantly inhibits the development of the animal reproductive system, achieving castration without the need for castration surgery. This has significant implications for animal husbandry.

[0074] Although preferred embodiments of the invention have been described, those skilled in the art, once they have learned the basic inventive concept, can make other changes and modifications to these embodiments.

[0075] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A gonadotropin-releasing hormone antigen peptide, characterized in that, The flexible linker molecule, composed of 1 to 3 glycines, is obtained by connecting two GnRH monomers shown in SEQ ID NO.2 in forward or reverse tandem.

2. The gonadotropin-releasing hormone antigen peptide according to claim 1, characterized in that, The gonadotropin-releasing hormone antigen peptide sequence is selected from any one of SEQ ID NO.1 and SEQ ID NO.3 to SEQ ID NO.

5.

3. The gonadotropin-releasing hormone antigen peptide according to claim 1, characterized in that, The gonadotropin-releasing hormone antigen peptide is also coupled to a carrier protein, with the coupling site being an additional cysteine ​​residue added to the C-terminus or N-terminus of the gonadotropin-releasing hormone antigen peptide.

4. The gonadotropin-releasing hormone antigen peptide according to claim 3, characterized in that, The carrier protein is chicken ovalbumin, keyhole cyanin, tetanus toxoid, bovine serum albumin, or human serum albumin.

5. A GnRH vaccine, characterized in that, The GnRH vaccine contains the gonadotropin-releasing hormone antigen peptide as described in any one of claims 1 to 4.

6. The GnRH vaccine according to claim 5, characterized in that, The GnRH vaccine was prepared by emulsifying gonadotropin-releasing hormone antigen peptide with an adjuvant.

7. The GnRH vaccine according to claim 6, characterized in that, The adjuvant is Specol adjuvant.

8. The use of the gonadotropin-releasing hormone antigen peptide according to any one of claims 1 to 4 or the GnRH vaccine according to any one of claims 6 to 7 in the preparation of animal castration products.

9. The application according to claim 8, characterized in that, The castration product is a GnRH vaccine.

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