Castration vaccine having engineered ferritin and gnrh expressed in tandem, and use

Abstract

The present invention relates to the technical fields of molecular biology, virology, immunology and medicine, and in particular to a castration vaccine having engineered ferritin and GnRH expressed in tandem, and a use. In the present invention, the engineered ferritin and different quantities of GnRH are linked in tandem and then recombined, so as to construct a GnRH-ferritin nanoparticle castration vaccine. The vaccine is used for castration of animals. Experimental results show that the GnRH and mutant ferritin recombined vaccine has a significantly enhanced immune effect and can significantly reduce testosterone levels and testicular weight, and compared with other combination methods, the nanoparticle castration vaccine having four GnRHs and the mutant ferritin linked in tandem has higher expression, stronger immunogenicity, faster immune response, and significant advantages in effect, and can be widely applied in the technical field of castration of animals.

Description

Castration vaccine expressing modified ferritin and GnRH in tandem and its application Technical Field

[0001] The present invention relates to the fields of molecular biology, virology, immunology and medical technology, and in particular to a castration vaccine expressing modified ferritin and GnRH in series and its application. Background Art

[0002] Animal castration involves the removal of an animal's sexual organs or reproductive capacity. It's used in livestock farming to reduce animal aggression, improve meat quality, and remove mutton odor. It's also used by pets to reduce sexual behavior in dogs and cats, making them more docile. Currently, surgical castration is the only method used, often causing significant harm to the animal and increasing the risk of infection and death. Castration vaccines utilize immunological methods to induce the body to produce antibodies against sex-related hormones, thereby neutralizing these hormones and preventing the animal from engaging in sexual behavior, ultimately achieving the purpose of castration. Compared to traditional surgical castration, vaccine castration offers advantages such as less stress on animals, increased safety, and labor savings.

[0003] Gonadotropin-releasing hormone (GnRH) is a crucial reproductive hormone in animals, primarily regulating reproductive processes and physiological functions. GnRH was discovered in the hypothalamus of pigs and sheep in 1971, and its molecular structure determined. Currently, nine GnRH variants have been identified, including two in chicken, four in fish (salmon, sturgeon, mullet, and sea bream), and two in lamprey. The GnRH molecule is composed of 10 amino acids, with amino acids 1-4 (the N-terminus) and 9-10 (the C-terminus) being highly conserved. The 8th amino acid shows the greatest variation, followed by amino acids 5, 7, and 6. However, its biological effects are virtually identical across mammals. At least 24 GnRH families have been identified, and the GnRH amino acid sequence is highly conserved across all mammals. Its amino acid sequence is pyroglutamic acid-histidine-tryptophan-serine-tyrosine-glycine-leucine-arginine-proline-glycine, i.e. PGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2, and its molecular weight is 1.181KDW.

[0004] At least two types of GnRH are synthesized in the vertebrate brain. One type, called GnRH-I, is secreted from the hypothalamus and acts on the pituitary gland. At least one other type, called GnRH-II or GnRH-III, appears in brain tissues outside the hypothalamus and may not directly regulate the synthesis and secretion of gonadotropins. The GnRH commonly referred to is GnRH-I.

[0005] However, GnRH is a substance naturally present in the body, making it difficult to induce an antibody response against it. GnRH is a small, 10-peptide molecule that is not easily recognized by the body and leads to antibody production. Therefore, developing a GnRH vaccine that facilitates recognition and improves its immunogenicity has important research and application value in the field of animal castration.

[0006] Summary of the Invention

[0007] In view of this, the technical problem to be solved by the present invention is to provide a castration vaccine expressing modified ferritin and GnRH in tandem and its application.

[0008] The present invention provides a recombinant protein comprising n(GnRH) and ferritin;

[0009] Said n is any integer between 1 and 10;

[0010] The GnRH has at least one of the amino acid sequences shown in a) to c) below:

[0011] a) having the amino acid sequence shown in SEQ ID NO: 1; or

[0012] b) an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence described in (a), and having the same function as the amino acid sequence described in (a); or

[0013] c) an amino acid sequence having at least 80% sequence identity with the amino acid sequence described in (a) or (b).

[0014] The ferritin has at least one of the amino acid sequences shown in the following i) to iii):

[0015] i) having the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 3; or

[0016] ii) an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence described in (i), and having the same function as the amino acid sequence described in (i); or

[0017] iii) an amino acid sequence having at least 80% sequence identity with the amino acid sequence described in (i) or (ii).

[0018] The at least 80% may specifically include 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and non-integer percentages between 80% and 100%.

[0019] Furthermore, in the recombinant protein of the present invention,

[0020] Each GnRH is connected by a connecting peptide 1, and the connecting peptide 1 is m(G3S), wherein m is any integer between 1 and 10;

[0021] The n(GnRH) and ferritin are connected via a connecting peptide 2;

[0022] The connecting peptide 2 is z(G3S), wherein z is any integer between 1 and 10;

[0023] The amino acid sequences of the connecting peptide 1 and the connecting peptide 2 may be the same or different.

[0024] The connecting peptide of the present invention may also be other flexible polypeptides composed of GS forms, including GxSy, wherein x is any integer, y is any integer, and x and y may be the same or different.

[0025] The recombinant protein of the present invention comprises a plurality of GnRHs in series, and the plurality can be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In a specific embodiment of the present invention, the plurality is 1, 2, 4, 5, 6, 7 or 10.

[0026] Furthermore, the amino acid sequence of the recombinant protein of the present invention has at least one of the following amino acid sequences d) to f):

[0027] d) having an amino acid sequence as shown in SEQ ID NO: 4 to SEQ ID NO: 13; or

[0028] e) an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence described in (d), and having the same function as the amino acid sequence described in (d); or

[0029] f) an amino acid sequence having at least 80% sequence identity with the amino acid sequence described in (d) or (e).

[0030] The at least 80% may specifically include 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and non-integer percentages between 80% and 100%.

[0031] In some specific embodiments of the present invention, the recombinant protein includes multiple GnRHs in series and unmutated Helicobacter pylori ferritin (nFerritin), and the amino acid sequence of the unmutated Helicobacter pylori ferritin is shown in SEQ ID NO: 2; experimental results show that 1, 2, and 4 GnRHs in series are expressed in ferritin at higher soluble levels in Escherichia coli, while 5, 7, and 10 GnRHs in series are expressed in lower soluble levels in Escherichia coli; when applied to animal castration, 4 GnRHs in series on ferritin have the best immunogenicity and the highest serum titer, and can significantly reduce the testosterone content in serum and reduce the specific gravity of testicles.

[0032] In other embodiments of the present invention, the recombinant protein includes multiple GnRHs in series and a mutant Helicobacter pylori ferritin (rFerritin). The mutant Helicobacter pylori ferritin is obtained by mutating the aspartic acid D at position 5 of the amino acid sequence shown in SEQ ID NO:2 to proline P, and mutating the glutamic acid E at position 147 to proline P. The stability of the mutated amino acids is further enhanced. Specifically, the mutated amino acid sequence is shown in SEQ ID NO:3. Experimental results show that, compared with the recombinant protein recombined with unmutated ferritin and GnRH, the mutated ferritin recombinant protein has a higher serum titer, a greater reduction in serum testosterone content and testicular specific gravity, and better performance. At the same time, the recombinant protein composed of four GnRHs and mutant ferritin (4(GnRH)-rFerritin) performs better than other combinations.

[0033] In a specific embodiment of the present invention,

[0034] The amino acid sequence of one GnRH and unmutated ferritin in tandem is shown in SEQ ID NO: 4;

[0035] The amino acid sequence of the two GnRHs and the unmutated ferritin in series is shown in SEQ ID NO: 5;

[0036] The amino acid sequence of the four GnRHs and the unmutated ferritin in series is shown in SEQ ID NO: 6;

[0037] The amino acid sequence of the five GnRHs and the unmutated ferritin in series is shown in SEQ ID NO: 7;

[0038] The amino acid sequence of the 7 GnRHs concatenated with the unmutated ferritin is shown in SEQ ID NO:8;

[0039] The amino acid sequence of 10 GnRHs concatenated with unmutated ferritin is shown in SEQ ID NO: 9;

[0040] The amino acid sequence of 1 GnRH and the mutant ferritin in tandem is shown in SEQ ID NO: 10;

[0041] The amino acid sequence of the two GnRHs and the mutant ferritin in series is shown in SEQ ID NO: 11;

[0042] The amino acid sequence of the four GnRHs and the mutant ferritin in series is shown in SEQ ID NO: 12;

[0043] The amino acid sequence of the 6 GnRHs and the mutant ferritin in series is shown in SEQ ID NO:13.

[0044] The ferritin in the recombinant protein of the present invention can self-assemble into nanoparticles. In this invention, it is recombined with the GnRH antigen as an antigen carrier. The ferritin in the present invention can be derived from bacteria, plants, algae, insects, fungi, or mammals, and can be wild-type or mutant.

[0045] Furthermore, the connecting peptide of the recombinant protein of the present invention maintains appropriate steric hindrance of each element of the recombinant protein of the present invention while maintaining the maximum performance of the overall immune performance.

[0046] The present invention provides a biomaterial comprising any one of the following items A) to E):

[0047] A), a nucleic acid encoding the recombinant protein of the present invention;

[0048] B), an expression unit comprising the nucleic acid described in A);

[0049] C) a recombinant vector containing the nucleic acid described in A) or the expression unit described in B);

[0050] D) transforming or transfecting a host cell with the recombinant vector described in C);

[0051] E) and D) the culture products of the host cells.

[0052] The nucleic acid of the present invention comprises at least one of the nucleic acid molecules shown in F) to J) below:

[0053] F), having a nucleotide sequence as shown in SEQ ID NO: 14 to SEQ ID NO: 23; or

[0054] G), a nucleic acid molecule complementary to the nucleic acid molecule shown in F); or

[0055] H), a nucleic acid molecule that encodes the same protein as the nucleic acid molecule of F) or G), but is different from the nucleic acid molecule of F) or G) due to the degeneracy of the genetic code; or

[0056] I), a nucleic acid molecule obtained by substituting, deleting or adding one or more bases to the nucleic acid molecule shown in F), G) or H), and having the same or similar functions as the nucleic acid molecule shown in F), G) or H); or

[0057] J) a nucleic acid molecule having at least 80% sequence identity with the nucleotide sequence described in F), G), H) or I).

[0058] The at least 80% may specifically include 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and non-integer percentages between 80% and 100%.

[0059] In a specific embodiment of the present invention, the codon-optimized nucleotide sequence of GnRH-rFerritin is shown in SEQ ID NO: 10; the codon-optimized nucleotide sequence of 4(GnRH)-rFerritin is shown in SEQ ID NO: 12; and the codon-optimized nucleotide sequence of 4(GnRH)-nFerritin is shown in SEQ ID NO: 6.

[0060] In the present invention, the nucleic acid can be DNA, RNA, cDNA or PNA. In an embodiment of the present invention, the nucleic acid is in the form of DNA. The DNA form includes cDNA, genomic DNA or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. Nucleic acid can include nucleotide sequences with different functions, such as coding regions and non-coding regions such as regulatory sequences (e.g., promoters or transcription terminators). Nucleic acid can be linear or circular in topology. Nucleic acid can be a part of a vector (such as an expression or cloning vector), or a fragment. The nucleic acid can be obtained directly from a natural source, or can be prepared with the assistance of recombination, enzymatic methods or chemical techniques. The RNA form is mRNA obtained by gene transcription, etc.

[0061] In the present invention, the nucleic acid may be optimized or unoptimized, and the optimization includes, but is not limited to, codon usage bias, elimination of secondary structures that are detrimental to expression (e.g., hairpin structures), changes in GC content, CpG dinucleotide content, mRNA secondary structure, cryptic splice sites, early polyadenylation sites, internal ribosome entry sites and binding sites, negative CpG islands, RNA instability regions, repetitive sequences (direct repeats, inverted repeats, etc.), and restriction sites that may affect cloning. In some specific embodiments of the present invention, the nucleic acid of the present invention is optimized for codon usage bias.

[0062] The recombinant vector of the present invention comprises a vector backbone and the nucleic acid of the present invention. The vector backbone may be derived from plants, animals, bacteria, fungi, phages, or viruses, but the present invention does not limit this.

[0063] The recombinant vectors described herein refer to recombinant nucleic acid vectors, which are recombinant DNA molecules that contain the desired coding sequence and appropriate nucleic acid sequences or elements necessary for expression of the operably linked coding gene in a specific host organism. Nucleic acid sequences or elements necessary for expression in prokaryotes include promoters, ribosome binding sites, and possibly other sequences. Prokaryotes are known to utilize promoters, enhancers, and terminators. Once transformed into a suitable host, the vector can replicate and function independently of the host genome, or, in some cases, integrate into the genome itself. In this specification, the terms "plasmid" and "vector" are sometimes used interchangeably, as plasmids are the most commonly used vector form. However, the present invention is intended to encompass other forms of expression vectors that function equivalently and are known or will become known in the art, including but not limited to plasmids, phage particles, viral vectors, and / or potential genomic inserts alone. In specific embodiments, nucleic acids encoding the fusion proteins provided herein can be constructed into various prokaryotic expression vectors, for example, pET28a.

[0064] The host cell of the present invention is transfected or transformed with the recombinant vector of the present invention.

[0065] Furthermore, the transformation methods include chemical transformation and electroporation; the transfection methods include calcium phosphate coprecipitation, artificial liposome method, and viral transfection. The viral transfection methods include adenovirus transfection, adeno-associated virus transfection, lentivirus transfection, etc.

[0066] The host cells of the present invention may be derived from plants, animals, bacteria, fungi, bacteriophages, or viruses, but are not limited thereto. The present invention uses vectors constructed using recombinant DNA technology to transform or transfect host cells, so that the transformed host cells are capable of replicating protein-encoding vectors or expressing desired proteins.

[0067] In a specific embodiment of the present invention, the host cell is Escherichia coli, more specifically Escherichia coli T7 shuffle.

[0068] The present invention provides a method for preparing the recombinant protein, which comprises culturing the host cell of the present invention to obtain a culture containing the recombinant protein.

[0069] The present invention provides a composition comprising the recombinant protein of the present invention and a drug for animal castration.

[0070] The animal trend drug includes polypeptide, protein or chemical drug.

[0071] The present invention provides the use of any one of the following I) to V in the preparation of a product for reducing testosterone content, reducing testicular weight and / or castration of animals:

[0072] 1) the recombinant antigen of the present invention;

[0073] II), the biomaterial of the present invention;

[0074] III), a culture containing the recombinant protein obtained by the method for preparing the recombinant protein of the present invention;

[0075] IV), the composition of the present invention;

[0076] The present invention provides a vaccine comprising any one of the recombinant protein or the composition of the present invention and a pharmaceutically acceptable adjuvant.

[0077] Furthermore, the excipient is used to maintain the activity of the vaccine or help the vaccine to work, and the excipient includes at least one of an adjuvant, a buffer, a surfactant and / or an antioxidant.

[0078] Furthermore, the adjuvant includes an aluminum salt adjuvant, a protein adjuvant, a nucleic acid adjuvant, a lipid-containing adjuvant, a mixed adjuvant, or an aggregate structure adjuvant; the aluminum salt adjuvant includes at least one of aluminum phosphate, aluminum hydroxide, or potassium aluminum sulfate. In a specific embodiment of the present invention, the adjuvant is aluminum oxide, and the volume ratio of aluminum oxide to recombinant antigen protein is 1:1. The buffer includes PBS buffer, KCl buffer, NaCl buffer, Tris buffer, etc., and the antioxidant includes but is not limited to DTT or β-mercaptoethanol.

[0079] The present invention provides a method for preparing the vaccine, which comprises mixing an adjuvant with the recombinant protein of the present invention or the composition of the present invention.

[0080] More specifically, in some specific embodiments of the present invention, the vaccine is a n(GnRH)-rFerritin virus-like particle vaccine, and its preparation method specifically comprises the following steps:

[0081] (1) The 10-peptide gene of n GnRH was directly cloned into the pET28a prokaryotic expression vector by connecting the 10-peptide gene of n GnRH with the mutant Helicobacter pylori Ferritin protein gene through a flexible amino acid linker to obtain the positive recombinant plasmid n(GnRH)-rFerritin-pET28a;

[0082] (2) The recombinant plasmid with correct sequence determination was transformed into Escherichia coli T7shuffle to obtain the recombinant expression strain n(GnRH)-rFerritin-pET28a-T7shuffle;

[0083] (3) Fermentation culture of the recombinant expression strain n(GnRH)-rFerritin-pET28a-T7shuffle, then adding IPTG to induce expression, breaking the bacteria and taking the supernatant to obtain the n(GnRH)-rFerritin recombinant protein.

[0084] (4) The vaccine is obtained by mixing the n(GnRH)-rFerritin recombinant protein with an adjuvant.

[0085] Among them, the nanoparticle vaccine prepared when n is 4 has the best effect, namely 4(GnRH)-rFerritin nanoparticle vaccine.

[0086] The n(GnRH)-rFerritin or n(GnRH)-nFerritin nanoparticle castration vaccine provided by the present invention, wherein the n(GnRH)-rFerritin or n(GnRH)-nFerritin recombinant protein is produced by bacteria, specifically Escherichia coli, is easy to produce and has a high expression level, making it convenient for industrial production. In addition, the vaccine of the present invention has strong immunogenicity and a rapid immune response, and has greater promotion value and can be widely used in the field of animal castration.

[0087] The present invention provides a method for reducing testosterone levels, reducing testicle weight and / or castrating animals, which comprises administering the vaccine of the present invention, wherein the administration may comprise injection.

[0088] The present invention constructs a GnRH-ferritin nanoparticle castration vaccine by combining modified ferritin with different numbers of GnRH in series. The vaccine is used for animal castration. Experimental results show that the immune effect of the vaccine recombined with GnRH and mutant ferritin is significantly enhanced, and can significantly reduce testosterone levels and testicular weight. At the same time, compared with other combinations, the nanoparticle castration vaccine with four GnRHs and mutant ferritin in series has a higher expression level, stronger immunogenicity, and faster immune response, with significant advantages in effect, and can be widely used in the field of animal castration technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0089] 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. 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.

[0090] FIG1 is a schematic diagram showing a recombinant plasmid after connecting n(GnRH) and nFerritin or rFerritin genes with pET28a plasmid provided in an embodiment of the present invention;

[0091] FIG2 is a schematic diagram of SDS-PAGE of GnRH-rFerritin and GnRH-nFerritin nanoparticle proteins provided in an embodiment of the present invention;

[0092] FIG3 shows a schematic diagram of SDS-PAGE of 2(GnRH)-rFerritin, 4(GnRH)-rFerritin, and 6(GnRH)-rFerritin nanoparticle proteins. DETAILED DESCRIPTION

[0093] The present invention provides a modified castration vaccine expressing ferritin and GnRH in tandem and its applications. Those skilled in the art can refer to the contents herein and appropriately improve the process parameters to achieve the desired results. It should be noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications herein without departing from the content, spirit, and scope of the present invention to implement and apply the technology of the present invention.

[0094] Ferritin is an ancient and large protein family that is widely present in animals, plants and microorganisms. Naturally synthesized ferritin is mainly composed of a mineral core and a protein shell. The protein shell is a spherical hollow nanocage structure composed of 24 ferritin subunits, in which every 3 subunits form a trimer subunit, and 8 trimer subunits are assembled into a protein shell with an outer diameter of 12nm and an inner diameter of 8nm. Ferritin is very stable and can withstand high temperatures and various denaturants without affecting its natural protein structure. The N-terminus of ferritin extends to the outer surface and is easy to be genetically modified and fused to protein polypeptides. The present invention uses ferritin nanoparticles from Helicobacter pylori to display antigens through screening, which can significantly enhance the immunogenicity of the antigen and induce stronger humoral and cellular immune responses.

[0095] Natural ferritin easily assembles into nanoparticles. Coupling to exogenous proteins or polypeptides can affect their assembly and stability. Furthermore, more GnRH on the ferritin surface can interfere with each other, hindering recognition by the immune system. Therefore, the present invention compared the assembly rate, expression level, and immunogenicity of different numbers of GnRH coupled to ferritin. Comparative experiments showed that 1, 2, or 4 GnRHs conjugated to ferritin resulted in higher soluble expression levels in E. coli, while 5, 7, and 10 GnRHs conjugated to ferritin resulted in lower soluble expression levels in E. coli. Four GnRHs conjugated to ferritin exhibited the best immunogenicity.

[0096] At the same time, the present invention mutated ferritin, and the mutated ferritin was more stable. Experimental results showed that the nanoparticle vaccine prepared by recombining the mutated ferritin with GnRH was more effective than the nanoparticle vaccine prepared by recombining the unmutated GnRH, and the immunogenicity of the nanoparticle vaccine recombined with 4 GnRHs and the mutated ferritin was the best.

[0097] The ferritin in the present invention may be derived from bacteria, plants, algae, insects, fungi or mammals, and may be wild type or mutant, and the present invention is not limited thereto. In some specific embodiments of the present invention, the ferritin is derived from Helicobacter pylori, and its amino acid sequence is shown in SEQ ID NO: 2. The amino acid sequence of the mutated ferritin is shown in SEQ ID NO: 3.

[0098] GnRH is a small 10-peptide molecule that is difficult for the body to recognize and produce antibodies. In this experiment, GnRH was first linked in series using flexible amino acids, typically several glycine (G) and serine (S) chains. The tandem GnRH was then linked to the N-terminus of ferritin using flexible amino acids for tandem expression. After ferritin spontaneously formed nanoparticles, the tandem GnRH was displayed on the nanoparticle surface.

[0099] The GnRH tandem connection described herein refers to a tandem connection of two or more GnRHs, preferably a tandem connection of four GnRHs. In the present invention, the GnRH amino acid sequence used is PGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2, or an amino acid sequence having at least 70% sequence identity thereto.

[0100] The expression system of the present invention includes Escherichia coli expression system, baculovirus, yeast or other protein expression systems.

[0101] The n(GnRH)-nFerritin or n(GnRH)-rFerritin nanoparticle vaccine of the present invention comprises n(GnRH)-nFerritin or n(GnRH)-rFerritin recombinant protein and an adjuvant, wherein the adjuvant includes but is not limited to aluminum hydroxide adjuvant, oil adjuvant, water adjuvant, biphasic adjuvant, etc.

[0102] The amino acid sequence of GnRH is: EHWSYGLRPG (SEQ ID NO: 1);

[0103] The amino acid sequence of unmutated ferritin (nFerritin) is: MLSKDIIKLLNEQVNKEMNSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIVFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKGKDHATFNFLQWYVSEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS (SEQ ID NO: 2);

[0104] The amino acid sequence of the mutant ferritin (rFerritin) is: MLSKBIIKLLNEQVNKEMNSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIVFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKGKDHATFNFLQWYVSEQHEEEVLFKDILDKIELIGNPNHGLYLADQYVKGIAKSRKS (SEQ ID NO: 3);

[0105] The amino acid sequence of GnRH-nFerritin is: MEHWSYGLRPGGGGSLSKDIIKLLNEQVNKEMNSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIVFLNENNVP VQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKGKDHATFNFLQWYVSEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS(SEQ ID NO:4);

[0106] The amino acid sequence of 2(GnRH)-nFerritin is: MEHWSYGLRPGGGGSEHWSYGLRPGGGGSLSKDIIKLLNEQVNKEMNSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIVFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKGKDHATFNFLQWYVSEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS (SEQ ID NO:5);

[0107] The amino acid sequence of 4(GnRH)-nFerritin is: MEHWSYGLRPGGGGSEHWSYGLRPGGGGSEHWSYGLRPGGGGSEHWSYGLRPGGGGSLSKDIIKLLNEQVNKEMNSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIVFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKGKDHATFNFLQWYVSEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS (SEQ ID NO:6);

[0108] The amino acid sequence of 5(GnRH)-nFerritin is: MEHWSYGLRPGGGGSEHWSYGLRPGGGGSEHWSYGLRPGGGGSEHWSYGLRPGGGGSEHWSYGLRPGGGGSLSKDIIKLLNEQVNKEMNSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIVFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKGKDHATFNFLQWYVSEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS (SEQ ID NO:7);

[0109] The amino acid sequence of 7(GnRH)-nFerritin is: MEHWSYGLRPGGGGSEHWSYGLRPGGGGSEHWSYGLRPGGGGSEHWSYGLRPGGGGSEHWSYGLRPGGGGSEHWSYGLRPGGGGSEHWSYGLRPGGGGSEHWSYGLRPGGGGSEHWSYGLRPGGGGSEHWSYGLRPGGGGSLSKDIIKLLNEQVNKEMNSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIVFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKGKDHATFNFLQWYVSEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS (SEQ ID NO:8);

[0110] The amino acid sequence of 10(GnRH)-nFerritin is: MEHWSYGLRPGGGGSEHWSYGLRPGGGGSEHWSYGLRPGGGGSEHWSYGLRPGGGGSEHWSYGLRPGGGGSEHWSYGLRPGGGGSEHWSYGLRPGGGGSEHWSYGLRPGGGGSEHWSYGLRPGGGGSEHWSYGLRPGGGGSEHWSYGLRPGGGGSLSKDIIKLLNEQVNKEMNSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIVFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKGKDHATFNFLQWYVSEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS (SEQ ID NO:9)

[0111] The amino acid sequence of GnRH-rFerritin is: MEHWSYGLRPGGGGSLSKPIIKLLNEQVNKEMNSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIVFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKGKDHATFNFLQWYVSEQHEE EVLFKDILDKIELIGNPNHGLYLADQYVKGIAKSRKS (SEQ ID NO:10);

[0112] The amino acid sequence of 2(GnRH)-rFerritin is: MEHWSYGLRPGGGGSEHWSYGLRPGGGGSLSKPIIKLLNEQVNKEMNSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIVFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKGKDHATFNFLQWYVSEQHEEEVLFKDILDKIELIGNPNHGLYLADQYVKGIAKSRKS (SEQ ID NO:11);

[0113] The amino acid sequence of 4(GnRH)-rFerritin is: MEHWSYGLRPGGGGSEHWSYGLRPGGGGSEHWSYGLRPGGGGSEHWSYGLRPGGGGSLSKPIIKLLNEQVNKEMNSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIVFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKGKDHATFNFLQWYVSEQHEEEVLFKDILDKIELIGNPNHGLYLADQYVKGIAKSRKS (SEQ ID NO:12);

[0114] The amino acid sequence of 6(GnRH)-rFerritin is: MEHWSYGLRPGGGGSEHWSYGLRPGGGGSEHWSYGLRPGGGGSEHWSYGLRPGGGGSEHWSYGLRPGGGGSEHWSYGLRPGGGGSLSKPIIKLLNEQVNKEMNSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIVFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKGKDHATFNFLQWYVSEQHEEEVLFKDILDKIELIGNPNHGLYLADQYVKGIAKSRKS (SEQ ID NO:13);

[0115] The nucleotide sequence after codon optimization of GnRH-nFerritin is: atggaacactggtcttacggtctgcgtccgggtggtggtggttctctgtctaaagacatcatcaaactgctgaacgaacaggttaacaaagaaatgaactcttctaacctgtacatgtctatgtcttcttggtgctacacccactctctggacggtgctggtctgttcctgttcgaccacgctgctgaagaatacgaacacgctaaaaaactgatcgttttcctgaacgaaaacaacgttccggttcagctgacctctatctctgctccggaacacaaattcgaaggtctgacccagatcttccagaaagcttacgaacacgaacagcacatctctgaatctatcaacaacatcgttgaccacgctatcaaaggtaaagaccacgctaccttcaacttcctgcagtggtacgtttctgaacagcacgaagaagaagttctgttcaaagacatcctggacaaaatcgaactgatcggtaacgaaaaccacggtctgtacctggctgaccagtacgttaaaggtatcgctaaatctcgtaaatct(SEQ ID NO:14);

[0116] The nucleotide sequence after codon optimization of 2(GnRH)-nFerritin is: atggaacactggtcttacggtctgcgtccgggtggtggtggttctgaacactggtcttacggtctgcgtccgggtggtggtggttctctgtctaaagacatcatcaaactgctgaacgaacaggttaacaaagaaatgaactcttctaacctgtacatgtctatgtcttcttggtgctacacccactctctggacggtgctggtctgttcctgttcgaccacgctgctgaagaatacgaacacgctaaaaaactgatcgttttcctgaacgaaaacaacgttccggttcagctgacctctatctctgctccggaacacaaattcgaaggtctgacccagatcttccagaaagcttacgaacacgaacagcacatctctgaatctatcaacaacatcgttgaccacgctatcaaaggtaaagaccacgctaccttcaacttcctgcagtggtacgtttctgaacagcacgaagaagaagttctgttcaaagacatcctggacaaaatcgaactgatcggtaacgaaaaccacggtctgtacctggctgaccagtacgttaaaggtatcgctaaatctcgtaaatct(SEQ ID NO:15);

[0117] The nucleotide sequence after codon optimization of 4(GnRH)-nFerritin is: atggaacactggtcttacggtctgcgtccgggtggtggtggttctgaacactggtcttacggtctgcgtccgggtggtggtggttctgaacactggtcttacggtctgcgtccgggtggtggtggttctgaacactggtcttacggtctgcgtccgggtggtggtggttctctgtctaaagacatcatcaaactgctgaacgaacaggttaacaaagaaatgaactcttctaacctgtacatgtctatgtcttcttggtgctacacccactctctggacggtgctggtctgttcctgttcgaccacgctgctgaagaatacgaacacgctaaaaaactgatcgttttcctgaacgaaaacaacgttccggttcagctgacctctatctctgctccggaacacaaattcgaaggtctgacccagatcttccagaaagcttacgaacacgaacagcacatctctgaatctatcaacaacatcgttgaccacgctatcaaaggtaaagaccacgctaccttcaacttcctgcagtggtacgtttctgaacagcacgaagaagaagttctgttcaaagacatcctggacaaaatcgaactgatcggtaacgaaaaccacggtctgtacctggctgaccagtacgttaaaggtatcgctaaatctcgtaaatct(SEQ ID NO:16);

[0118] The nucleotide sequence after codon optimization of 5(GnRH)-nFerritin is: atggaacactggtcttacggtctgcgtccgggtggtggtggttctgaacactggtcttacggtctgcgtccgggtggtggtggttctgaacactggtcttacggtctgcgtccgggtggtggtggttctgaacactggtcttacggtctgcgtccgggtggtggtggttctgaacactggtcttacggtctgcgtccgggtggtggtggttctctgtctaaagacatcatcaaactgctgaacgaacaggttaacaaagaaatgaactcttctaacctgtacatgtctatgtcttcttggtgctacacccactctctggacggtgctggtctgttcctgttcgaccacgctgctgaagaatacgaacacgctaaaaaactgatcgttttcctgaacgaaaacaacgttccggttcagctgacctctatctctgctccggaacacaaattcgaaggtctgacccagatcttccagaaagcttacgaacacgaacagcacatctctgaatctatcaacaacatcgttgaccacgctatcaaaggtaaagaccacgctaccttcaacttcctgcagtggtacgtttctgaacagcacgaagaagaa gttctgttcaaagacatcctggacaaaatcgaactgatcggtaacgaaaaccacggtctgtacctggctgaccagtacgttaaaggtatcgctaaatctcgtaaatct(SEQ ID NO:17);

[0119] The nucleotide sequence after codon optimization of 7(GnRH)-nFerritin is as follows: atggaacactggtcttacggtctgcgtccgggtggtggtggttctgaacactggtcttacggtctgcgtccgggtggtggtggttctgaacactggtcttacggtctgcgtccgggtggtggtggttctgaacactggtcttacggtctgcgtccgggtggtggtggttctgaacactggtcttacggtctgcgtccgggtggtggtggttctgaacactggtcttacggtctgcgtccgggtggtggtggttctctgtctaaagacatcatcaaactgctgaacgaacaggttaacaaagaaatgaactcttctaacctgtacatgtctatgtcttcttggtgctacacccactctctggacggtgctggtctgttcctgttcgaccacgctgctgaagaatacgaacacgctaaaaaactgatcgttttcctgaacgaaaacaacgttccggttcagctgacctctatctctgctccggaacacaaattcgaaggtctgacccagatcttccagaaagcttacgaacacgaacagcacatctctgaatctatcaacaacatcgttgaccacgctatcaaaggtaaagaccacgctaccttcaacttcctgcagtggtacgtttctgaacagcacgaagaagaagttctgttcaaagacatcctggacaaaatcgaactgatcggtaacgaaaaccacggtctgtacctggctgaccagtacgttaaaggtatcgctaaatctcgtaaatct(SEQ ID NO:18);

[0120] The nucleotide sequence after codon optimization of 10(GnRH)-nFerritin is: atggaacactggtcttacggtctgcgtccgggtggtggtggttctgaacactggtcttacggtctgcgtccgggtggtggtggttctgaacactggtcttacggtctgcgtccgggtggtggtggttctgaacactggtcttacggtctgcgtccgggtggtggtggttctgaacactggtcttacggtctgcgtccgggtggtggtggttctgaacactggtcttacggtctgcgtccgggtggtggtggttctgaacactggtcttacggtctgcgtccgggtggtggtggttctgaacactggtcttacggtctgcgtccgggtggtggtggttctgaacactggtcttacggtctgcgtccgggtggtggtggttctctgtctaaagacatcatcaaactgctgaacgaacaggttaacaaagaaatgaactcttctaacctgtacatgtctatgtcttcttggtgctacacccactctctggacggtgctggtctgttcctgttcgaccacgctgctgaagaatacgaacacgctaaaaaactgatcgttttcctgaacgaaaacaacgttccggttcagctgacctctatctctgctccggaacacaaattcgaaggtctgacccagatcttccagaaagcttacgaacacgaacagcacatctctgaatctatcaacaacatcgttgaccacgctatcaaaggtaaagaccacgctaccttcaacttcctgcagtggtacgtttctgaacagcacgaagaagaagttctgttcaaagacatcctggacaaaatcgaactgatcggtaacgaaaaccacggtctgtacctggctgaccagtacgttaaaggtatcgctaaatctcgtaaatct(SEQ ID NO:19)

[0121] The nucleotide sequence after codon optimization of GnRH-rFerritin is: atggaacactggtcttacggtctgcgtccgggtggtggtggttctctgtctaaaccgatcatcaaactgctgaacgaacaggttaacaaagaaatgaactcttctaacctgtacatgtctatgtcttcttggtgctacacccactctctggacggtgctggtctgttcctgttcgaccacgctgctgaagaatacgaacacgctaaaaaactgatcgttttcctgaacgaaaacaacgttccggttcagctgacctctatctctgctccggaacacaaattcgaaggtctgacccagatcttccagaaagcttacgaacacgaacagcacatctctgaatctatcaacaacatcgttgaccacgctatcaaaggtaaagaccacgctaccttcaacttcctgcagtggtacgtttctgaacagcacgaagaagaagttctgttcaaagacatcctggacaaaatcgaactgatcggtaacccgaaccacggtctgtacctggctgaccagtacgttaaaggtatcgctaaatctcgtaaatct(SEQ ID NO:20);

[0122] The nucleotide sequence after codon optimization of 2(GnRH)-rFerritin is: atggaacactggtcttacggtctgcgtccgggtggtggtggttctgaacactggtcttacggtctgcgtccgggtggtggtggttctctgtctaaaccgatcatcaaactgctgaacgaacaggttaacaaagaaatgaactcttctaacctgtacatgtctatgtcttcttggtgctacacccactctctggacggtgctggtctgttcctgttcgaccacgctgctgaagaatacgaacacgctaaaaaactgatcgttttcctgaacgaaaacaacgttccggttcagctgacctctatctctgctccggaacacaaattcgaaggtctgacccagatcttccagaaagcttacgaacacgaacagcacatctctgaatctatcaacaacatcgttgaccacgctatcaaaggtaaagaccacgctaccttcaacttcctgcagtggtacgtttctgaacagcacgaagaagaagttctgttcaaagacatcctggacaaaatcgaactgatcggtaacccgaaccacggtctgtacctggctgaccagtacgttaaaggtatcgctaaatctcgtaaatct(SEQ ID NO:21);

[0123] The nucleotide sequence after codon optimization of 4(GnRH)-rFerritin is: atggaacactggtcttacggtctgcgtccgggtggtggtggttctgaacactggtcttacggtctgcgtccgggtggtggtggttctgaacactggtcttacggtctgcgtccgggtggtggtggttctgaacactggtcttacggtctgcgtccgggtggtggtggttctctgtctaaaccgatcatcaaactgctgaacgaacaggttaacaaagaaatgaactcttctaacctgtacatgtctatgtcttcttggtgctacacccactctctggacggtgctggtctgttcctgttcgaccacgctgctgaagaatacgaacacgctaaaaaactgatcgttttcctgaacgaaaacaacgttccggttcagctgacctctatctctgctccggaacacaaattcgaaggtctgacccagatcttccagaaagcttacgaacacgaacagcacatctctgaatctatcaacaacatcgttgaccacgctatcaaaggtaaagaccacgctaccttcaacttcctgcagtggtacgtttctgaacagcacgaagaagaagttctgttcaaagacatcctggacaaaatcgaactgatcggtaacccgaaccacggtctgtacctggctgaccagtacgttaaaggtatcgctaaatctcgtaaatct(SEQ ID NO:22);

[0124] The nucleotide sequence of 6(GnRH)-rFerritin after codon optimization is: (SEQ ID NO: 23);

[0125] The test materials used in the present invention are all common commercial products and can be purchased in the market.

[0126] The present invention will be further described below in conjunction with the embodiments:

[0127] Example 1 Preparation of GnRH-GnRH-Ferritin Nanoparticle Protein

[0128] The molecular biology experimental methods used in the examples of the present invention, such as enzyme digestion and homologous recombination ligation, can be performed with reference to the second edition of Molecular Cloning. The basic materials for preparing the GnRH-ferritin nanoparticles of the present invention include: the amino acid sequence of the Helicobacter pylori ferritin protein, the GnRH amino acid sequence, the pET28a plasmid, the T7shuffle Escherichia coli strain, a plasmid miniprep kit, LB medium, IPTG, kanamycin, a nickel column, and imidazole. The codon-optimized nucleotide sequence was sent to BGI for synthesis of the corresponding pET28a recombinant plasmid. The recombinant plasmid design is shown in Figure 1.

[0129] 1. rFerritin amino acid design

[0130] The Helicobacter pylori ferritin protein consists of 167 amino acids, including five α-helices (A, B, C, D, and E), as shown in Table 1. Each α-helix is ​​connected by a varying number of amino acids. The E-α-helix is ​​smaller and has less interaction with other α-helices, potentially oscillating. Foreign proteins are typically attached to the front end of the A-α-helix, which also tends to oscillate, hindering the stability of the recombinant protein. In this study, the amino acid at position 5 of the A-α-helix (aspartic acid D) was mutated to proline P (a rigid amino acid), and the amino acid at position 147 of the E-α-helix (glutamic acid E) was mutated to proline P (a rigid amino acid), which improved protein stability. After conjugation to GnRH, the protein's instability index (Instability Index) was reduced from 38.64 to 37.54 using the online protein property prediction website https: / / web.expasy.org / protparam / , as shown in Table 1.

[0131] Table 1. Ferritin protein α helix and position

[0132] 2. Preparation of GnRH-rFerritin Nanoparticles

[0133] (1) Target protein gene design and synthesis: GnRH and rFerritin proteins were linked by three glycines and one serine (GGGS). After codon optimization, the nucleic acid sequence was codon-cleaved with BamHI and XhoI restriction sites added at both ends. The synthesis was then commissioned to Shenzhen BGI Genomics Co., Ltd.

[0134] (2) Construction of recombinant plasmid: The plasmid containing the target gene synthesized by BGI was double-digested with BamHI and XhoI enzymes, the target gene was recovered, and ligated with the pET28a plasmid double-digested with BamHI and XhoI enzymes. The plasmid was then transfected into a DH5α competent cell for amplification, and the recombinant plasmid was extracted to obtain the GnRH-rFerritin-pET28a recombinant plasmid.

[0135] (3) Transferring the recombinant plasmid into the expression strain: The recombinant plasmid GnRH-rFerritin-pET28a was transferred into the Escherichia coli T7shuffle expression strain to obtain the GnRH-rFerritin-pET28a-T7shuffle recombinant expression strain.

[0136] (4) Cultivation of recombinant bacteria and induction of target protein expression: The GnRH-rFerritin-pET28a-T7shuffle recombinant expression strain was spread on a plate containing 100 μg / ml kanamycin resistance and cultured overnight at 37°C. A single colony was picked and inoculated into 10 mL of LB medium containing 100 μg / ml kanamycin and shaken at 37°C at 200 rpm until the OD600 of the bacterial solution was 0.8-2.0. The colony was then transferred into 500 mL of LB medium containing 100 μg / ml kanamycin and shaken at 37°C at 200 rpm until the OD600 of the bacterial solution was about 2.0-4.0. β-D-galactoside (IPTG) inducer was added at a final concentration of 0.5 mmol / L and expression was induced at 28°C overnight.

[0137] (5) Bacterial cell collection and disruption: The bacterial cell suspension was centrifuged at 8000 rpm and the cells were collected. The cells were resuspended in 0.01 mol / L PBS solution at a weight to volume ratio of 1:10. The cells were then homogenized twice at 2-8°C using a high-pressure homogenizer at 900 bar.

[0138] (7) Endotoxin Removal: After bacterial cell disruption, centrifuge at 8000 rpm to collect the supernatant. Add Triton X-114 to a final concentration of 1.5% and stir at 2-8°C for 1 hour. After treatment, return the sample temperature to 30°C and maintain for 40 minutes. Centrifuge to remove the precipitate and collect the supernatant. Repeat the above process twice.

[0139] (8) Protein purification: The protein solution after endotoxin removal was concentrated and replaced with a 100KD membrane, and then purified by chromatography using an S300 molecular sieve chromatography filler. The target protein peak was collected, which was the purified GnRH-rFerritin nanoparticle protein.

[0140] II. Preparation of GnRH-nFerritin, 2(GnRH)-nFerritin, 4(GnRH)-nFerritin, 5(GnRH)-nFerritin, 7(GnRH)-nFerritin, 10(GnRH)-nFerritin, 2(GnRH)-rFerritin, 4(GnRH)-rFerritin, and 6(GnRH)-rFerritin Nanoparticle Proteins

[0141] According to the same method for preparing the GnRH-rFerritin nanoparticle protein in step 1, expression vectors for expressing GnRH-nFerritin, 2(GnRH)-nFerritin, 4(GnRH)-nFerritin, 5(GnRH)-nFerritin, 7(GnRH)-nFerritin, 10(GnRH)-nFerritin, 2(GnRH)-rFerritin, 4(GnRH)-rFerritin, and 6(GnRH)-rFerritin nanoparticle proteins are designed, and then the protein expression and purification are performed to obtain the nanoparticle protein.

[0142] The expressed GnRH-rFerritin protein and GnRH-nFerritin protein were subjected to SDS-PAGE protein gel electrophoresis. As shown in Figure 2, the expression level of GnRH-rFerritin protein was significantly higher than that of GnRH-nFerritin protein. Protein gel electrophoresis was performed on 2(GnRH)-rFerritin, 4(GnRH)-rFerritin, 6(GnRH)-rFerritin and 2(GnRH)-nFerritin, 4(GnRH)-nFerritin, 5(GnRH)-nFerritin, 7(GnRH)-nFerritin, and 10(GnRH)-nFerritin. It can be seen that the expression levels of 2(GnRH)-rFerritin and 4(GnRH)-rFerritin proteins were higher than those of 6(GnRH)-rFerritin proteins, which may be due to the increase in the amount of GnRH. , affecting the correct folding and assembly of Ferritin protein (the results are shown in Figure 3); the trends of 2(GnRH)-nFerritin, 4(GnRH)-nFerritin, 5(GnRH)-nFerritin, 7(GnRH)-nFerritin, and 10(GnRH)-nFerritin were roughly the same, and the protein expression levels of 2(GnRH)-nFerritin and 4(GnRH)-nFerritin were higher than those of 5(GnRH)-nFerritin, 7(GnRH)-nFerritin, and 10(GnRH)-nFerritin.

[0143] Example 2 Immunogenicity Detection of Ferritin Virus-like Particle Vaccines with Different Amounts of GnRH

[0144] The vaccine was prepared by mixing GnRH-nFerritin nanoparticle protein, 2(GnRH)-nFerritin nanoparticle protein, 4(GnRH)-nFerritin nanoparticle protein, 5(GnRH)-nFerritin nanoparticle protein, 7(GnRH)-nFerritin nanoparticle protein, 10(GnRH)-nFerritin nanoparticle protein, GnRH-rFerritin nanoparticle protein, 2(GnRH)-rFerritin nanoparticle protein, 4(GnRH)-rFerritin nanoparticle protein, 6(GnRH)-rFerritin nanoparticles, and GnRH polypeptide (synthesized by Shenzhen BGI Genomics Co., Ltd.) virus-like particles with aluminum hydroxide adjuvant in a volume ratio of 1:1. Eight-week-old male C57BV / 6 mice were divided into 12 groups of 5 mice each and immunized with 0.5 ml of the above-mentioned vaccine, GnRH peptide, and saline on days 0, 14, and 28. Anti-GnRH antibody titers and testosterone levels were measured on days 0, 14, 28, 42, and 70. Mice were sacrificed on day 70, and testicular weights were measured.

[0145] Antibody testing results showed that in male mice immunized with the GnRH-rFerritin nanoparticle vaccine and the GnRH-nFerritin nanoparticle vaccine, the GnRH-rFerritin nanoparticle vaccine had slightly higher antibody levels than the GnRH-nFerritin nanoparticle vaccine. The 4(GnRH)-rFerritin nanoparticle vaccine had higher antibody levels than the GnRH-rFerritin nanoparticle vaccine, the 2(GnRH)-rFerritin nanoparticle vaccine, the 6(GnRH)-rFerritin nanoparticle vaccine, and the unmutated ferritin nanoparticle group (nFerritin). All nanoparticle vaccines had significantly higher antibody levels than the GnRH peptide vaccine group and the control group, as shown in Table 2.

[0146] Table 2. GnRH antibody titer test results

[0147] Post-immunization testosterone levels in mice receiving the GnRH-rFerritin nanoparticle vaccine were slightly lower than those in mice receiving the GnRH-nFerritin nanoparticle vaccine. Testosterone concentrations in mice receiving both the 4(GnRH)-nFerritin and 4(GnRH)-rFerritin nanoparticle vaccines were lower, but the differences were not significant (P>0.05). Testosterone concentrations in all nanoparticle vaccine groups were lower than those in the GnRH polypeptide and control groups. The results are shown in Table 3.

[0148] Table 3. Test results of testosterone levels in mouse serum

[0149] On day 70, mice were sacrificed, and testicles were removed and weighed. Testicular weights in the GnRH-rFerritin nanoparticle vaccine group were slightly lower than those in the GnRH-nFerritin nanoparticle vaccine group. Testicular weights in the 4(GnRH)-rFerritin nanoparticle vaccine group were the lowest, significantly different from those in the other groups (P < 0.05). Testicular weights in all nanoparticle vaccine groups were lower than those in the GnRH peptide and control groups. The results are shown in Table 4.

[0150] Table 4. Mouse testis weight test results

[0151] The above are only preferred embodiments of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A recombinant protein, characterized in that Including n(GnRH) and ferritin; The n is any integer between 1 and 10; The GnRH has an amino acid sequence as shown in SEQ ID NO: 1; The ferritin has an amino acid sequence as shown in SEQ ID NO: 2 or SEQ ID NO:

3.

2. The recombinant protein according to claim 1, characterized in that Each GnRH is connected by a connecting peptide 1, and the connecting peptide 1 is m(G3S), wherein m is any integer between 1 and 10; The n(GnRH) and ferritin are connected via a connecting peptide 2; The connecting peptide 2 is z(G3S), wherein z is any integer between 1 and 10.

3. The recombinant protein according to claim 1 or 2, characterized in that The recombinant protein has at least one of the amino acid sequences shown in SEQ ID NO:4 to SEQ ID NO:

13.

4. Biomaterial, characterized in that Including any one of the following A) to E): A), a nucleic acid encoding the recombinant protein according to any one of claims 1 to 3; B), an expression unit comprising the nucleic acid described in A); C), a recombinant vector containing the nucleic acid described in A) or the expression unit described in B); D), a host cell transformed or transfected with the recombinant vector described in C); E) and D) the culture products of the host cells.

5. The method for preparing the recombinant protein according to any one of claims 1 to 3, characterized in that: The method comprises culturing the host cell according to claim 4 to obtain a culture containing the recombinant protein.

6. A composition, characterized in that A drug comprising the recombinant protein according to any one of claims 1 to 3 and animal castration.

7. Use of any one of I) to V below in the preparation of a product for reducing testosterone content, reducing testicle specific gravity and / or animal castration: I), the recombinant antigen according to any one of claims 1 to 3; II), the biomaterial according to claim 4; III), a culture containing the recombinant protein obtained by the preparation method according to claim 5; IV) The composition according to claim 6.

8. A vaccine, characterized in that The invention comprises the recombinant protein according to any one of claims 1 to 3 or the composition according to claim 6 and a pharmaceutically acceptable excipient.

9. The vaccine according to claim 8, characterized in that The auxiliary material includes at least one of an adjuvant, a buffer, a surfactant and / or an antioxidant.

10. A method for reducing testosterone levels, reducing testicular specific gravity and / or castration of animals, characterized in that: Comprising administration of the vaccine of claim 8 or 9.

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