CN118684781A - GnRH-VLP recombinant castration vaccine and preparation method thereof - Google Patents

Abstract

The present invention relates to the field of biotechnology, and in particular to a GnRH-VLP recombinant castration vaccine and a preparation method thereof. The present invention provides a variety of GnRH-VLP recombinant proteins with high purity and good specificity. The vaccine antigens prepared using these recombinant proteins have high purity, good safety, and good castration effects, and can be used to castrate a variety of animals.

Description

GnRH-VLP recombinant castration vaccine and preparation method thereof

Technical Field

The present invention relates to the field of biotechnology, in particular to a GnRH-VLP recombinant castration vaccine and a preparation method thereof.

Background Art

Castration is the removal of the reproductive system of an animal or the loss of its sexual function by external means, including the removal of the testes from male individuals and the removal of the ovaries from females, which are collectively called gonadectomy. In addition to surgical removal of the gonads, local radiation irradiation and chemical treatment can also be used for castration. After castration, vertebrates lose the source of sex hormone secretion, and under its influence, sometimes the appendages of the genitals, secondary sexual characteristics and tertiary sexual characteristics degenerate.

Gonadotropin-releasing hormone (GnRH) is an endogenous polypeptide hormone of animals. Physiological doses of GnRH-I can increase the concentration of gonadotropins (such as a slight increase in FSH and a significant increase in LH), promote the synthesis and secretion of gonadal hormones (such as estradiol, progesterone and testosterone), promote the maturation of follicles and ovulation or testicular development and sperm maturation, and produce and maintain secondary sexual characteristics. In addition, GnRH-I can also directly affect the gonads, regulate the synthesis and secretion of gonadal steroid hormones, and promote gamete formation.

Self-antigenic proteins are often difficult to induce antibody responses against self-antigens. One way to improve the efficiency of vaccination is to increase the reproducibility of the antigens applied. Unlike isolated proteins, viruses can induce rapid and effective immune responses in the absence of any adjuvants and with and without T cell help. Compared to a few proteins, they are able to trigger much stronger immune responses than their isolated components. For B cell responses, it is well known that a key factor in viral immunogenicity is the reproducibility and order of surface epitopes. Many viruses exhibit quasi-crystalline surfaces with regularly arranged epitopes that can efficiently cross-link epitope-specific immunoglobulins on B cells. This cross-linking of immunoglobulins on the surface of B cells is a strong activation signal that directly induces cell cycle progression and the production of IgM antibodies. In addition, such triggered B cells are able to activate T helper cells, which in turn induces the conversion of IgM antibodies to IgG antibodies in B cells and the production of long-lived B cell memory targets for any vaccination. Viral structures have even been implicated in the production of anti-antibodies in autoimmune diseases and are part of the natural response to pathogens. Therefore, antigens presented by highly organized viral surfaces are able to induce strong antibody responses against antigens.

Virus-like particles are the main capsid proteins of an RNA bacteriophage, which are self-assembled from multiple monomers into a new type of highly immunogenic virus-like particles (VLPs). These VLPs do not contain the bacteriophage RNA genome and cannot replicate. However, studies have found that a variety of polypeptides can be fused to the N- or C-terminus of the virus-like particle protein, and the resulting fusion protein forms virus-like particles when expressed in a host, typically and preferably in Escherichia coli. In addition, it has been found that if the polypeptide contains at least one antigen, the antigen or at least one antigenic site of the antigen is displayed on the outer surface of the assembled VLP, and the assembled VLP can effectively improve the immunogenicity of the target antigen. In addition to the virus-like particles AP205 and Qβ that have been reported, there are also a variety of VLP platforms such as CuMV and tobacco mosaic virus VLP, all of which have the potential to be developed into castration vaccines.

Summary of the invention

In view of this, the technical problem to be solved by the present invention is to provide a GnRH-VLP recombinant castration vaccine and a preparation method thereof. The present invention provides a variety of recombinant VLP castration vaccines based on gonadotropin-releasing hormone, which can solve the problem that it is usually difficult to induce antibody responses against self-antigens.

The present invention provides a GnRH-VLP recombinant protein, which comprises GnRH and a virus-like particle protein; the virus-like particle protein comprises MS2, T4, TMV, CPMV, CMV, PapMV, FHV and/or EILV.

Wherein, the GnRH has an amino acid sequence as shown below: QHWSYGLRPG (SEQ ID NO: 1);

or a sequence in which one or more amino acids are substituted, deleted, added and/or replaced based on the amino acid sequence shown in SEQ ID NO: 1;

Or a sequence having a homology of more than 90% with the amino acid sequence shown in SEQ ID NO:1.

In the present invention, the GnRH-VLP recombinant protein is GnRH-MS2. The MS2 is derived from Escherichia coli MS2 bacteriophage. The amino acid sequence of the MS2 includes:

The coat protein has the following amino acid sequence:

MASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVTC SVRQSSAQNRKYTIKVEVPKVATQTVGGVELPVAAWRSYLNMELTIPIFATN SDCELIVKAMQGLLKDGNPIPSAIAANSGIY(SEQ ID NO: 2);

or a sequence in which one or more amino acids are substituted, deleted, added and/or replaced based on the amino acid sequence shown in SEQ ID NO: 2;

Or a sequence with more than 90% homology to the amino acid sequence shown in SEQ ID NO: 2.

The mature enzyme protein has the amino acid sequence shown below:

MRAFSTLDRENETFVPSVRVYADGETEDNSFSLKYRSNWTPGRFNSTGAKTKQWHYPSPYSRGALSVTSIDQGAYKRSGSSWGRPYEEKAGFGFSLDARSCYSLFPVSQNLTYIEVPQNVANRASTEVLQKVTQGNFNLGVALAEARSTASQLATQTIALVKAYTAARRGNWRQALRYLALNEDRKFRSKHVAGRWLELQFGWLPLMSDI QGAYEMLTKVHLQEFLPMRAVRQVGTNIKLDGRLSYPAANFQTTCNISRRIVIWFYINDARLAWLSSLGILNPLGIVWEKVPFSFVVDWLLPVGNMLEGLTAPVGCSYMSGTVTDVITGESIISVDAPYGWTVERQGTAKAQISAMHRGVQSVWPTTGAYVKSPFSMVHTLDALALIRQRLSR(SEQ ID NO: 3);

or a sequence in which one or more amino acids are substituted, deleted, added and/or replaced based on the amino acid sequence shown in SEQ ID NO: 3;

Or a sequence with more than 90% homology to the amino acid sequence shown in SEQ ID NO:3.

In the GnRH-MS2 recombinant protein, GnRH and MS2 are directly constructed into a vector and packaged into VLP. The preparation method comprises: cloning the MS2 5' non-coding sequence, mature enzyme protein, coat protein and GnRH gene sequence into an expression vector to obtain a recombinant expression vector, and then transferring the recombinant expression vector into a host and expressing it.

Correspondingly, the present invention also provides:

1), a nucleic acid encoding the GnRH-VLP recombinant protein;

II), an expression unit containing a nucleic acid encoding GnRH-VLP;

III), a recombinant vector containing a nucleic acid encoding GnRH-VLP or an expression unit as described above;

IV), host cells transformed or transfected with an expression vector of GnRH-VLP;

V) The culture product of the host cell as described above.

The present invention also provides a vaccine containing GnRH-VLP. The vaccine also includes an adjuvant. In the GnRH-VLP vaccine of the present invention, the adjuvant is not limited. In some embodiments, the GnRH-VLP vaccine is prepared using an aluminum adjuvant. The concentration of GnRH-VLP in the vaccine is 800, 400, 200, 100 μg/mL, preferably, the concentration is 400 μg/mL.

Immunizing animals with this vaccine can achieve the effect of castration. Therefore, the present invention also provides the use of the above-mentioned vaccine in animal castration. Correspondingly, the present invention also provides a method for castrating an animal, comprising administering the vaccine as described above. The dose administered is 200 μg. 10 to 100 days after immunization, the anti-GnRH recombinant protein antibody titer and testosterone level decreased. In some tests, the anti-GnRH recombinant protein antibody titer decreased by 50% to 90%, specifically 50% to 70%, 60% to 80%, 70% to 90%, more specifically 50% to 60%, 60% to 70%, 70% to 80%, 80% to 90%. In some tests, testosterone levels are reduced to 50% to 90% of the original level, specifically 50% to 70%, 60% to 80%, 70% to 90%, more specifically 50% to 60%, 60% to 70%, 70% to 80%, 80% to 90%.

In the present invention, the GnRH-VLP recombinant protein is GnRH-T4. Wherein, the T4 is from the capsid of bacteriophage T4. The amino acid sequence of the T4 includes:

Soc has the amino acid sequence shown below:

MASTRGYVNIKTFEQKLDGNKKIEGKEISVAFPLYSDVHKISGAHYQT FPSEKAAYSTVYEENQRTEWIAANEDLWKVTG(SEQ ID NO: 4);

or a sequence in which one or more amino acids are substituted, deleted, added and/or replaced based on the amino acid sequence shown in SEQ ID NO: 4;

Or a sequence having a homology of 90% or more to the amino acid sequence shown in SEQ ID NO:4.

Hoc has the following amino acid sequence:

MTFTVDITPKTPTGVIDETKQFTATPSGQTGGGTITYAWSVDNVPQDGAEATFSYVLKGPAGQKTIKVVATNTLSEGGPETAEATTTITVKNKTQTTTLAVTPASPAAGVIGTPVQFTAALASQPDGASATYQWYVDDSQVGGETNSTFSYTPTTSGVKRIKCVAQVTATDYDALSVTSNEVSLTVNKKTMNPQVTLTPPSINVQQDASAT FTANVTGAPEEAQITYSWKKDSSPVEGSTNVYTVDTSSVGSQTIEVTATVTAADYNPVTVTKTGNVTVTAKVAPEPEGELPYVHPLPHRSSAYIWCGWWVMDEIQKMTEEGKDWKTDDPDSKYYLHRYTLQKMMKDYPEVDVQESRNGYIIHKTALETGIIYTYP(SEQ ID NO: 5);

or a sequence in which one or more amino acids are substituted, deleted, added and/or replaced based on the amino acid sequence shown in SEQ ID NO: 5;

Or a sequence with more than 90% homology to the amino acid sequence shown in SEQ ID NO:5.

In the GnRH-T4 recombinant protein, GnRH and T4 are directly constructed into a vector and packaged into VLP. The preparation method includes: cloning the T4Soc or Hoc gene sequence and the GnRH gene sequence into an expression vector to obtain a recombinant expression vector, and transferring the recombinant expression vector into a host and expressing it.

Correspondingly, the present invention also provides:

1), a nucleic acid encoding the GnRH-VLP recombinant protein;

II), an expression unit containing a nucleic acid encoding GnRH-VLP;

III), a recombinant vector containing a nucleic acid encoding GnRH-VLP or an expression unit as described above;

IV), host cells transformed or transfected with an expression vector of GnRH-VLP;

V) The culture product of the host cell as described above.

The present invention also provides a vaccine containing GnRH-VLP. The vaccine also includes an adjuvant. In the GnRH-VLP vaccine of the present invention, the adjuvant is not limited. In some embodiments, the GnRH-VLP vaccine is prepared using an aluminum adjuvant. The concentration of GnRH-VLP in the vaccine is 800, 400, 200, 100 μg/mL, preferably, the concentration is 400 μg/mL.

Immunizing animals with this vaccine can achieve the effect of castration. Therefore, the present invention also provides the use of the above-mentioned vaccine in animal castration. Correspondingly, the present invention also provides a method for castrating an animal, comprising administering the vaccine as described above. The dose administered is 200 μg. 10 to 100 days after immunization, the anti-GnRH recombinant protein antibody titer and testosterone level decreased. In some tests, the anti-GnRH recombinant protein antibody titer decreased by 50% to 90%, specifically 50% to 70%, 60% to 80%, 70% to 90%, more specifically 50% to 60%, 60% to 70%, 70% to 80%, 80% to 90%. In some tests, testosterone levels are reduced to 50% to 90% of the original level, specifically 50% to 70%, 60% to 80%, 70% to 90%, more specifically 50% to 60%, 60% to 70%, 70% to 80%, 80% to 90%.

In the present invention, the GnRH-VLP recombinant protein is GnRH-TMV, and the TMV is derived from tobacco mosaic virus. The amino acid sequence of the TMV is:

MSYSITTPSQFVFLSSAWADPIELINLCTNALGNQFQTQQARTVVQRQFSEVWKPSPQVTVRFPDSDFKVYRYNAVLDPLVTALLGAFDTRNRIIEVENQANPTTAETLDATRRVDDATVAIRSAINNLIVELIRGTGSYNRSSFESSSGLVWTSGPAT(SEQ ID NO: 6);

or a sequence in which one or more amino acids are substituted, deleted, added and/or replaced based on the amino acid sequence shown in SEQ ID NO: 6;

Or a sequence having a homology of more than 90% with the amino acid sequence shown in SEQ ID NO:6.

In the GnRH-TMV recombinant protein, GnRH and TMV are connected by chemical coupling. The preparation method thereof comprises: obtaining the recombinant protein by chemical coupling of GnRH protein and TMV-NtK.

Correspondingly, the present invention also provides:

1), a nucleic acid encoding the GnRH-VLP recombinant protein;

II), an expression unit containing a nucleic acid encoding GnRH-VLP;

III), a recombinant vector containing a nucleic acid encoding GnRH-VLP or an expression unit as described above;

IV), host cells transformed or transfected with an expression vector of GnRH-VLP;

V) The culture product of the host cell as described above.

The present invention also provides a vaccine containing GnRH-VLP. The vaccine also includes an adjuvant. In the GnRH-VLP vaccine of the present invention, the adjuvant is not limited. In some embodiments, the GnRH-VLP vaccine is prepared using an aluminum adjuvant. The concentration of GnRH-VLP in the vaccine is 800, 400, 200, 100 μg/mL, preferably, the concentration is 400 μg/mL.

Immunizing animals with this vaccine can achieve the effect of castration. Therefore, the present invention also provides the use of the above-mentioned vaccine in animal castration. Correspondingly, the present invention also provides a method for castrating an animal, comprising administering the vaccine as described above. The dose administered is 200 μg. 10 to 100 days after immunization, the anti-GnRH recombinant protein antibody titer and testosterone level decreased. In some tests, the anti-GnRH recombinant protein antibody titer decreased by 50% to 90%, specifically 50% to 70%, 60% to 80%, 70% to 90%, more specifically 50% to 60%, 60% to 70%, 70% to 80%, 80% to 90%. In some tests, testosterone levels are reduced to 50% to 90% of the original level, specifically 50% to 70%, 60% to 80%, 70% to 90%, more specifically 50% to 60%, 60% to 70%, 70% to 80%, 80% to 90%.

In the present invention, the GnRH-VLP recombinant protein is GnRH-CPMV. Wherein, the CPMV is from cowpea mosaic virus. The amino acid sequence of the CPMV is:

MSGLFHHRTKPRKKRVFPMATRLTKKQLAQAIQNTLPNPPRRRRRAKRRAAQVPKPTQAGVSMAPIAQGAMVKLRPPMLRSSMDVTILSHCELSTELAVTDSIVVSSELVMPFTVGTWLRGVAQNWSKYAWVAIRYTYLPSCPTTTSGAIHMGFQYDMADTLPSVNQLSNLKGYVTGPVWEGQSGLCFVNNSKCSDTSRAITIALDTNEV SEKRYPFKTATDYTTAVGVNANIGNILVPARLVIAMEGGSSSTAVNTGRLYASYTIRLIEPIAAALNL (SEQ ID NO: 7);

or a sequence in which one or more amino acids are substituted, deleted, added and/or replaced based on the amino acid sequence shown in SEQ ID NO: 7;

Or a sequence with more than 90% homology to the amino acid sequence shown in SEQ ID NO:7.

In the GnRH-CPMV recombinant protein, GnRH and CPMV are connected by chemical coupling. The preparation method thereof includes: obtaining the protein by coupling CPMV with GnRH polypeptide.

Correspondingly, the present invention also provides:

1), a nucleic acid encoding the GnRH-VLP recombinant protein;

II), an expression unit containing a nucleic acid encoding GnRH-VLP;

III), a recombinant vector containing a nucleic acid encoding GnRH-VLP or an expression unit as described above;

IV), host cells transformed or transfected with an expression vector of GnRH-VLP;

V) The culture product of the host cell as described above.

The present invention also provides a vaccine containing GnRH-VLP. The vaccine also includes an adjuvant. In the GnRH-VLP vaccine of the present invention, the adjuvant is not limited. In some embodiments, the GnRH-VLP vaccine is prepared using an aluminum adjuvant. The concentration of GnRH-VLP in the vaccine is 800, 400, 200, 100 μg/mL, preferably, the concentration is 400 μg/mL.

Immunizing animals with this vaccine can achieve the effect of castration. Therefore, the present invention also provides the use of the above-mentioned vaccine in animal castration. Correspondingly, the present invention also provides a method for castrating an animal, comprising administering the vaccine as described above. The dose administered is 200 μg. 10 to 100 days after immunization, the anti-GnRH recombinant protein antibody titer and testosterone level decreased. In some tests, the anti-GnRH recombinant protein antibody titer decreased by 50% to 90%, specifically 50% to 70%, 60% to 80%, 70% to 90%, more specifically 50% to 60%, 60% to 70%, 70% to 80%, 80% to 90%. In some tests, testosterone levels are reduced to 50% to 90% of the original level, specifically 50% to 70%, 60% to 80%, 70% to 90%, more specifically 50% to 60%, 60% to 70%, 70% to 80%, 80% to 90%.

In the present invention, the GnRH-VLP recombinant protein is GnRH-CMV. Wherein, the CMV is from cucumber mosaic virus. The amino acid sequence of the CMV is:

MGQYIKANSKFIGITERRRRPRRGSRSAPSSADANFRVLSQQLSRLNKTLAAGRPTINHPTFVGSERCKPGYTFTSITLKPPKIDRGSYYGKRLLLPDSVTEYDKKLVSRIQIRVNPLPKFDSTVWVTVRKVPASSDLSVAAISAMFADGASPVLVYQYAASGVQANNKLLYDLSAMRADIGDMRKYAVLVYSKDDALETDELVLHVDVEHQRIPTS GVLPV (SEQ ID NO: 8);

or a sequence in which one or more amino acids are substituted, deleted, added and/or replaced based on the amino acid sequence shown in SEQ ID NO: 8;

Or a sequence having a homology of 90% or more to the amino acid sequence shown in SEQ ID NO:8.

In the GnRH-CMV recombinant protein, GnRH and CMV are connected by chemical coupling. The preparation method thereof comprises: obtaining the protein by chemical coupling of GnRH protein and CMV-VLP.

Correspondingly, the present invention also provides:

1), a nucleic acid encoding the GnRH-VLP recombinant protein;

II), an expression unit containing a nucleic acid encoding GnRH-VLP;

III), a recombinant vector containing a nucleic acid encoding GnRH-VLP or an expression unit as described above;

IV), host cells transformed or transfected with an expression vector of GnRH-VLP;

V) The culture product of the host cell as described above.

The present invention also provides a vaccine containing GnRH-VLP. The vaccine also includes an adjuvant. In the GnRH-VLP vaccine of the present invention, the adjuvant is not limited. In some embodiments, the GnRH-VLP vaccine is prepared using an aluminum adjuvant. The concentration of GnRH-VLP in the vaccine is 800, 400, 200, 100 μg/mL, preferably, the concentration is 400 μg/mL.

Immunizing animals with this vaccine can achieve the effect of castration. Therefore, the present invention also provides the use of the above-mentioned vaccine in animal castration. Correspondingly, the present invention also provides a method for castrating an animal, comprising administering the vaccine as described above. The dose administered is 200 μg. 10 to 100 days after immunization, the anti-GnRH recombinant protein antibody titer and testosterone level decreased. In some tests, the anti-GnRH recombinant protein antibody titer decreased by 50% to 90%, specifically 50% to 70%, 60% to 80%, 70% to 90%, more specifically 50% to 60%, 60% to 70%, 70% to 80%, 80% to 90%. In some tests, testosterone levels are reduced to 50% to 90% of the original level, specifically 50% to 70%, 60% to 80%, 70% to 90%, more specifically 50% to 60%, 60% to 70%, 70% to 80%, 80% to 90%.

In the present invention, the GnRH-VLP recombinant protein is GnRH-PapMV. Wherein, the PapMV is derived from papaya mosaic virus. The amino acid sequence of the PapMV is:

MSKSSMSTPNIAFPAITQEQMSSIKVDPTSNLLPSQEQLKSVSTLMVAAKVPAASVTTVALELVNFCYDNGSSAYTTVTGPSSIPEISLAQLASIVKASGTSLRKFCRYFAPIIWNLRTDKMAPANWEASGYKPSAKFAAFDFFDGVENPAAMQPPSGLIRSPTQEERIANATNKQVHLFQAAAQDNNFTSNSAFITKGQISGSTPTIQFLPPPE(S EQID NO: 9);

or a sequence in which one or more amino acids are substituted, deleted, added and/or replaced based on the amino acid sequence shown in SEQ ID NO: 9;

Or a sequence with more than 90% homology to the amino acid sequence shown in SEQ ID NO:9.

In the GnRH-PapMV recombinant protein, GnRH and PapMV are connected by chemical coupling. The preparation method thereof comprises: obtaining the protein by chemical coupling of GnRH protein and PapMV-VLP.

Correspondingly, the present invention also provides:

1), a nucleic acid encoding the GnRH-VLP recombinant protein;

II), an expression unit containing a nucleic acid encoding GnRH-VLP;

III), a recombinant vector containing a nucleic acid encoding GnRH-VLP or an expression unit as described above;

IV), host cells transformed or transfected with an expression vector of GnRH-VLP;

V) The culture product of the host cell as described above.

The present invention also provides a vaccine containing GnRH-VLP. The vaccine also includes an adjuvant. In the GnRH-VLP vaccine of the present invention, the adjuvant is not limited. In some embodiments, the GnRH-VLP vaccine is prepared using an aluminum adjuvant. The concentration of GnRH-VLP in the vaccine is 800, 400, 200, 100 μg/mL, preferably, the concentration is 400 μg/mL.

Immunizing animals with this vaccine can achieve the effect of castration. Therefore, the present invention also provides the use of the above-mentioned vaccine in animal castration. Correspondingly, the present invention also provides a method for castrating an animal, comprising administering the vaccine as described above. The dose administered is 200 μg. 10 to 100 days after immunization, the anti-GnRH recombinant protein antibody titer and testosterone level decreased. In some tests, the anti-GnRH recombinant protein antibody titer decreased by 50% to 90%, specifically 50% to 70%, 60% to 80%, 70% to 90%, more specifically 50% to 60%, 60% to 70%, 70% to 80%, 80% to 90%. In some tests, testosterone levels are reduced to 50% to 90% of the original level, specifically 50% to 70%, 60% to 80%, 70% to 90%, more specifically 50% to 60%, 60% to 70%, 70% to 80%, 80% to 90%.

In the present invention, the GnRH-VLP recombinant protein is GnRH-FHV. Wherein, the FHV is derived from a kennel virus. The amino acid sequence of the FHV is:

MVNNIKPKRQRSQRVVVTTTQTAPIPQQNVPRNGRRRRNRARRNRRRGRGMNMAALSRLSQPGLAFLKCAFAPPDFNTDPGKGIPDRFEGKVVSRKDVLNQSISFESGKDTFILIAPTPGVAYWSASVDAGSFPTSTTTTFTPTNYPGFTSMFGTTATSRSDQVSSFRYASMNVGIYPTSNLMQFAGSITVWKCPIKLTTAQFPVSTVPA TSSLVHSLVGLDGVLAVGPDNFSESFIKGVFSQSACNEPDFEFSDILEGIQILPPLNVDIGSTGQPFTLSAGNENTSGIVGWGNMDTIVIRVSAPTGAVNSAILKAWSCIEYRPNPNAMLYQFGHDSPPLDEVALQEYRTVARSLPVAVIAAQNASMWERVKAIIKSSLAAASNIPGPIGVAASGISGLSALFEGFGF (SEQ ID NO: 10);

or a sequence in which one or more amino acids are substituted, deleted, added and/or replaced based on the amino acid sequence shown in SEQ ID NO: 10;

Or a sequence having 90% or more homology to the amino acid sequence shown in SEQ ID NO: 10.

In the GnRH-FHV recombinant protein, GnRH and FHV are directly constructed into a vector and packaged into VLP. The preparation method comprises: cloning the N-terminal part of the FHV coat protein, GnRH and the N-terminal part of the GnRH coat protein gene sequence into an expression vector to obtain a recombinant expression vector, transfecting the recombinant expression vector into a host, and obtaining it through expression.

Correspondingly, the present invention also provides:

1), a nucleic acid encoding the GnRH-VLP recombinant protein;

II), an expression unit containing a nucleic acid encoding GnRH-VLP;

III), a recombinant vector containing a nucleic acid encoding GnRH-VLP or an expression unit as described above;

IV), host cells transformed or transfected with an expression vector of GnRH-VLP;

V) The culture product of the host cell as described above.

The present invention also provides a vaccine containing GnRH-VLP. The vaccine also includes an adjuvant. In the GnRH-VLP vaccine of the present invention, the adjuvant is not limited. In some embodiments, the GnRH-VLP vaccine is prepared using an aluminum adjuvant. The concentration of GnRH-VLP in the vaccine is 800, 400, 200, 100 μg/mL, preferably, the concentration is 400 μg/mL.

Immunizing animals with this vaccine can achieve the effect of castration. Therefore, the present invention also provides the use of the above-mentioned vaccine in animal castration. Correspondingly, the present invention also provides a method for castrating an animal, comprising administering the vaccine as described above. The dose administered is 200 μg. 10 to 100 days after immunization, the anti-GnRH recombinant protein antibody titer and testosterone level decreased. In some tests, the anti-GnRH recombinant protein antibody titer decreased by 50% to 90%, specifically 50% to 70%, 60% to 80%, 70% to 90%, more specifically 50% to 60%, 60% to 70%, 70% to 80%, 80% to 90%. In some tests, testosterone levels are reduced to 50% to 90% of the original level, specifically 50% to 70%, 60% to 80%, 70% to 90%, more specifically 50% to 60%, 60% to 70%, 70% to 80%, 80% to 90%.

In the present invention, the GnRH-VLP recombinant protein is GnRH-EILV. Wherein, the EILV is from Eilat virus. The amino acid sequence of the EILV is:

MEKPTVNVDVDPQSPFVLQLQKHFPQFEIVANMVTPNDHANARAFSHCASKLIEAEVPVTTPIIDIGSAPARRMYSEHRYHCVCPMKCPEDPDRLTTYANRLVENATKIANKRLDAKLQDLKQVLETPDIETDSICFHDDATCRWVAEVSVMQDVYIDAPSSIYHQALKGIRKIYWIGFDTTPFMFKALAGSYPSYNTNWADEKVL EARNIGLCSTTLSEGSTGKLSIMRKKRLLPGAQVYFSVGSTLYPENR

SNLMSWHLPSVFHLKGRNAFTCRCDTVVNCDGYVVKKITISPNLIGTPAGY

AVTNNSEGFLLCKVTDTVRGERVSFPVCMSIPATICDQMTGILATDINPEDA

QKLLVGLNQRIVVNGKTNRNVNTMQNHLLPAVAQGFSKWAKERKADGDD

EKHLGTRERSLTFGCLWAFRTKKVHSFYRPPGTQTIVKVESVFTASPLAIPIR

QTSLPISLRLKLKMAIAKKQNNPIATITQTQITNAIEFQKEATETARAVELNN

ALPPLRATEQDPPTPSVEHVVCEVEELSDDIGGALVETPRGHVRILPQPTDVK

VGNYLVISPQAVLRNDKLSRLHPLAEQIKVITHTGRKGRYEVAPYSGKMLL

PCGTSVPWPQFCALAESATLVFNEREMIDRKLAYIAQHGPALNTDEEQYKV

IKASAADSEYVFDIDRMRCVPTKEANGLVLVGELTQPPYHELAMQGLYTRP

AAPYPIETIGVIGTPGSGKSAIIKNTVTTKDLVTSGKKENCKEIETDVLRLRN

LVIKSRTVDSVLLNGCTQEVDVLHVDEAFACHAGTLLALIAIVKPRCKVVL

YGDPKQCGFFNLMQIKVHFNNPEVDVCSQLHYKYISRRCILPVTAIVSSIHY

DGKMRTTNTADQRIEIDTTGTSKPKPTDLILTCFRGWVKQLQLEYPRNEVM

TAAASQGLTRKRVYAVRYKVNENPLYAFTSEHVNVLLTRTEHTLVWKTLQG

DPWIKHLSNVPKGNFSATVDEWHAEHERIMNAIRMPTPEVNAFSCKTNVC

WAKALVPVLATAGLKLSGAQWTELFPQFERDEPHSATFALDVLCIKYFGM

DLTSGIFAKPTVPLTFHPVSRYHPQAHWDNANGEQRYGFDPDIAKALARRF

PVFSQAAKGHAISPILGTTHTLSSRDNYVPVNRIVPHTLKGEYTYVKQDSL

ASVLSAVQAFSVLVVSSEPIASATKQITWVAPLGTAGCIHTHRLPWGFPKMS

LHDAVAVNMETEYRGHHYQQCEDHVAILKTLGKSALANLRPGGTLILRTY

GYADRNSENVITALARKFARVTAVRSSNPSSNTEIYLIFRKFDNNRSRQFTLH

HLNRAISALYESPCDPDGVGAAPSYSVIRGDITATNSHAIVVPVTPERKDGV

YRACSKKWGPLPRLEWTEGATLFSPGSPATLQVCVPSLQNTDTTSTQQAYR

AIAKVVVDEQIPSLSLPVLTMKKTGTADTVSESLNHLVTALDQTDANVTIY

CLDKSRLIKIKEVIARKEAVTELIDDDLEIDEELTWVHPDSCLRNRTGFSTDK

GKLYSYLEGTKFHQMAKDFAERSLFPDEMEANEHICSLILGETIDGIRERCP

VTDNPPSSPPKTVPCLCMYAMTPERALRLKSNSVTQITVCSSFVLKKHHIK

GVQKIQCTAPMLFNPTPLTSRTVRTPPQVSARAALDLPPVAPMPSVPAPVSL

TPTRRAPPPPLTKRPVVVRPSTPPPPPPPVRQTPTPVLAPRTGSTAAPTPTPRLS

LSTDQPSVDISFGDFSPAETMSLMLSSPGSDTASITFGDFDEDEVESIVGREY

WLTGAGGYIFSSDTGSGHLQQRSVLQNRTTETIIERVTHDRIHAPQLNEARE

EVLKLKYQMYPSDANKSRYRARKVENQKAICISRLTAGSRSYSFGTTEAEC

YRETYPAVMYSSSLPSSYSAPTTAVAVCNAYLAANYPTVASYQITDEYDAYL

DMVDGTMACLDTASFNPSKLRSFPKVHKYLEPTIRSAVPSPFQNTLQNVLT

AATKRNCNVTQMRELPTLDSAAFNVECFRKYACNNDYWQEYADKPIRITT

EYVTAYVAKLKGPKAAALFSKTHDLPALGEVPMDRFVMDMKRDVKVTPG

SKHTEERPKVQVIQAAEPLATAYLCGIHRELVRRLTAALLPNIHTLFDMSAE

DFDATLAHHFKKGDPVLETDIASFDKSQDDALALTGLMILEDLGVDQPLM

DLIEAAFGDITSTHLPTGARFRFGAMMKSGMFLTLFINTVLNVVIASRVLED

KLTHSACAAFIGDDNIIHGVISDRIMADRCATWMNMEVKIIDAVMGDYPPY

FCGGFLIIDSVTNTACRVADPLKRLFKLGKPLTADDDHDDDRRRALEDETK

AWFRVGIREGITAAVSSRYEVDNILPVLLALRTFALSTRNFSALRGTLKTLYN(SEQ ID NO: 11);

or a sequence in which one or more amino acids are substituted, deleted, added and/or replaced based on the amino acid sequence shown in SEQ ID NO: 11;

Or a sequence having 90% or more homology to the amino acid sequence shown in SEQ ID NO: 11.

In the GnRH-EILV recombinant protein, GnRH and EILV are directly constructed into a vector and packaged into VLP. The preparation method includes: cloning the GnRH gene sequence into the EILV virus cDNA plasmid vector to obtain a recombinant expression vector, transfecting the recombinant expression vector into a host cell, and obtaining the protein through expression.

Correspondingly, the present invention also provides:

1), a nucleic acid encoding the GnRH-VLP recombinant protein;

II), an expression unit containing a nucleic acid encoding GnRH-VLP;

III), a recombinant vector containing a nucleic acid encoding GnRH-VLP or an expression unit as described above;

IV), host cells transformed or transfected with an expression vector of GnRH-VLP;

V) The culture product of the host cell as described above.

The present invention also provides a vaccine containing GnRH-VLP. The vaccine also includes an adjuvant. In the GnRH-VLP vaccine of the present invention, the adjuvant is not limited. In some embodiments, the GnRH-VLP vaccine is prepared using an aluminum adjuvant. The concentration of GnRH-VLP in the vaccine is 800, 400, 200, 100 μg/mL, preferably, the concentration is 400 μg/mL.

Immunizing animals with this vaccine can achieve the effect of castration. Therefore, the present invention also provides the use of the above-mentioned vaccine in animal castration. Correspondingly, the present invention also provides a method for castrating an animal, comprising administering the vaccine as described above. The dose administered is 200 μg. 10 to 100 days after immunization, the anti-GnRH recombinant protein antibody titer and testosterone level decreased. In some tests, the anti-GnRH recombinant protein antibody titer decreased by 50% to 90%, specifically 50% to 70%, 60% to 80%, 70% to 90%, more specifically 50% to 60%, 60% to 70%, 70% to 80%, 80% to 90%. In some tests, testosterone levels are reduced to 50% to 90% of the original level, specifically 50% to 70%, 60% to 80%, 70% to 90%, more specifically 50% to 60%, 60% to 70%, 70% to 80%, 80% to 90%.

In the present invention, the GnRH-VLP recombinant protein is GnRH-HBsAg. Wherein, the HBsAg is derived from hepatitis B surface antigen. The amino acid sequence of the HBsAg is: mgqnlstsnplgffpdhqld pafrantanpdwdfnpnkdtwpdankvgagafglgftpphggllgwspqaqgilqtlpanpppastnrqsgrqptplspplrnthpqamqwnsttfhqtlqdprvrglyfpaggsssgtvnpvlttasplssifsrigdpalnmenitsgflgpllvlqagfflltriltipqsldswwtslnflggt tvclgqnsqsptsnhsptscpptcpgyrwmclrrfiiflfilllclifllvlldyqgmlpvcplipgssttstgpcrtcmttaqgtsmypsccctkpsdgnctcipipsswafgkflwewasarfswlsllvpfvqwfvglsptvwlsviwmmwywgpslysilspflpllpiffclwvyi(SEQ ID NO: 12);

or a sequence in which one or more amino acids are substituted, deleted, added and/or replaced based on the amino acid sequence shown in SEQ ID NO: 12;

Or a sequence having a homology of 90% or more to the amino acid sequence shown in SEQ ID NO: 12.

In the GnRH-HBsAg recombinant protein, GnRH and HBsAg are directly constructed into a vector and packaged into VLP. The preparation method includes: cloning and inserting the gene sequences of GnRH and HBs into a plasmid vector to obtain a recombinant expression vector, transfecting the recombinant expression vector into a host cell, and obtaining the VLP through expression.

Correspondingly, the present invention also provides:

1), a nucleic acid encoding the GnRH-HBsAg recombinant protein;

II), an expression unit containing a nucleic acid encoding GnRH-HBsAg;

III), a recombinant vector containing a nucleic acid encoding GnRH-HBsAg or an expression unit as described above;

IV), host cells transformed or transfected with an expression vector of GnRH-HBsAg;

V) The culture product of the host cell as described above.

The present invention also provides a vaccine containing GnRH-VLP. The vaccine also includes an adjuvant. In the GnRH-VLP vaccine of the present invention, the adjuvant is not limited. In some embodiments, the GnRH-VLP vaccine is prepared using an aluminum adjuvant. The concentration of GnRH-VLP in the vaccine is 800, 400, 200, 100 μg/mL, preferably, the concentration is 400 μg/mL.

Immunizing animals with this vaccine can achieve the effect of castration. Therefore, the present invention also provides the use of the above-mentioned vaccine in animal castration. Correspondingly, the present invention also provides a method for castrating an animal, comprising administering the vaccine as described above. The dose administered is 200 μg. 10 to 100 days after immunization, the anti-GnRH recombinant protein antibody titer and testosterone level decreased. In some tests, the anti-GnRH recombinant protein antibody titer decreased by 50% to 90%, specifically 50% to 70%, 60% to 80%, 70% to 90%, more specifically 50% to 60%, 60% to 70%, 70% to 80%, 80% to 90%. In some tests, testosterone levels are reduced to 50% to 90% of the original level, specifically 50% to 70%, 60% to 80%, 70% to 90%, more specifically 50% to 60%, 60% to 70%, 70% to 80%, 80% to 90%.

In the present invention, the GnRH-VLP recombinant protein is GnRH-M1. Wherein, the M1 is the virus core protein influenza matrix protein. The amino acid sequence of the M1 is: mslltevetyvlsivpsgplkaeiaqrledvfagkntdlealmewlktrpilspltkgilgfvftltvpserglqrrrfvqnalngngdpnnmdravklyrklkreitfhgakeialsysagalascmgliynrmgavttesafglicatceqiadsqhkshrqmvtttnplirhenrmvlasttakameqmagsseqaaeamevasqarqmvqamraigthpssstglkndllenlqayqkrmgvqmqrfk (SEQ ID NO: 13);

or a sequence in which one or more amino acids are substituted, deleted, added and/or replaced based on the amino acid sequence shown in SEQ ID NO: 13;

Or a sequence having a homology of 90% or more to the amino acid sequence shown in SEQ ID NO: 13.

In the GnRH-M1 recombinant protein, a vector constructed by GnRH and HA is mixed with a vector constructed by M1 and packaged into VLP. The preparation method comprises: cloning and inserting the gene sequences of GnRH and HA into a plasmid vector, and cloning and inserting the gene sequence of M1 into a plasmid vector to obtain two recombinant expression vectors, and co-transfecting the two recombinant expression vectors into a host cell to obtain the VLP through expression.

Correspondingly, the present invention also provides:

1), a nucleic acid encoding the GnRH-M1 recombinant protein;

II), an expression unit containing a nucleic acid encoding GnRH-M1;

III), a recombinant vector containing a nucleic acid encoding GnRH-M1 or an expression unit as described above;

IV), host cells transformed or transfected with an expression vector of GnRH-M1;

V) The culture product of the host cell as described above.

The present invention also provides a vaccine containing GnRH-VLP. The vaccine also includes an adjuvant. In the GnRH-VLP vaccine of the present invention, the adjuvant is not limited. In some embodiments, the GnRH-VLP vaccine is prepared using an aluminum adjuvant. The concentration of GnRH-VLP in the vaccine is 800, 400, 200, 100 μg/mL, preferably, the concentration is 400 μg/mL.

Immunizing animals with this vaccine can achieve the effect of castration. Therefore, the present invention also provides the use of the above-mentioned vaccine in animal castration. Correspondingly, the present invention also provides a method for castrating an animal, comprising administering the vaccine as described above. The dose administered is 200 μg. 10 to 100 days after immunization, the anti-GnRH recombinant protein antibody titer and testosterone level decreased. In some tests, the anti-GnRH recombinant protein antibody titer decreased by 50% to 90%, specifically 50% to 70%, 60% to 80%, 70% to 90%, more specifically 50% to 60%, 60% to 70%, 70% to 80%, 80% to 90%. In some tests, testosterone levels are reduced to 50% to 90% of the original level, specifically 50% to 70%, 60% to 80%, 70% to 90%, more specifically 50% to 60%, 60% to 70%, 70% to 80%, 80% to 90%.

The present invention provides a method for preparing the recombinant protein, comprising culturing the host cell to obtain a culture product containing the recombinant protein.

The invention provides a composition comprising the recombinant protein, GnRH antigen or any antigen capable of making animals infertile.

Furthermore, the composition also includes an acceptable adjuvant.

The present invention provides application of the recombinant protein, the biological material or the composition in preparing a subunit vaccine.

The present invention provides a GnRH-VLP subunit vaccine, which comprises the recombinant protein or the composition described in the present invention.

In some embodiments of the present invention, the recombinant protein can be GnRH-MS2 recombinant protein, GnRH-T4 recombinant protein, GnRH-TMV recombinant protein or GnRH-CPMV recombinant protein, or it can be GnRH-CMV recombinant protein, GnRH-PapMV recombinant protein, GnRH-FHV recombinant protein GnRH-EILV recombinant protein, GnRH-HBsAg recombinant protein or GnRH-M1 recombinant protein, and the present invention is not limited to this.

The GnRH-VLP subunit vaccine provided by the present invention has a variety of VLP platforms, does not limit the types of VLP, and increases the types and quantities of castration vaccines.

The present invention also provides a method for preparing the subunit vaccine, comprising mixing the recombinant protein with an adjuvant; or mixing the composition with a buffer.

The present invention provides a method for castration of an animal, comprising administering the subunit vaccine of the present invention.

Animals that can be castrated include birds and mammals.

The subunit vaccine is prepared using the multiple recombinant proteins expressed by the present invention, which has high antigen purity, good safety, and is non-pathogenic to animals such as mice, and is easy to pass safety evaluation. In addition, the vaccine has an efficient antigen presentation method and can effectively induce the immune system to produce an immune protection response.

DETAILED DESCRIPTION

The present invention provides a GnRH-VLP recombinant castration vaccine and a method for preparing the same. A person skilled in the art can refer to the contents of this article and appropriately improve the process parameters to achieve the same. It should be particularly noted that all similar substitutions and modifications are obvious to a person 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 of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

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

In the present invention, the nucleic acid can be DNA, RNA, cDNA or PNA. 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, for example, a part of a vector (e.g., 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. The gene sequence can use a wild-type sequence, or it can be codon-optimized, and the present invention does not limit this.

In the present invention, the expression unit comprises an expression unit composed of a single or multiple tandem forms of the nucleic acid of the present invention, a promoter and a terminator, and the present invention is not limited to this.

In the present invention, the recombinant expression vector refers to a nucleic acid vector, which is a recombinant DNA molecule that contains a desired coding sequence and an appropriate nucleic acid sequence or element that is essential for the expression of an operably linked coding gene in a specific host organism. The nucleic acid sequence or element necessary for expression in bacteria includes a promoter, a ribosome binding site and possible other sequences. The recombinant expression vector is selected according to the host. The expression vector described in the present invention can be circular or linear, and the present invention does not limit this.

Furthermore, the host described in the present invention includes bacteria, fungi, viruses or animals. The bacteria include Gram-positive bacteria and Gram-negative bacteria; the Gram-positive bacteria include but are not limited to Escherichia coli. The fungi include molds, yeasts, and mushrooms; the yeasts include Saccharomyces cerevisiae, Saccharomyces cerevisiae, Pichia pastoris, and Candida. The viruses include but are not limited to adenoviruses, adeno-associated viruses, lentiviruses, and prions. The animals include humans, mice, rabbits, pigs, zebrafish, and the like. The expression of the nucleic acid encoding the recombinant protein in the host can be either integrated or episomal, and the present invention does not limit this.

Taking the prokaryotic host as an example, it can be Escherichia coli, Bacillus, Streptomyces or cyanobacteria, etc.; its plasmid can be pET series plasmid, pGEX series plasmid, pKBP series plasmid or pcDNA series plasmid.

The adjuvant of the present invention is an immunomodulator or an immunopotentiator, including aluminum salt adjuvants, protein adjuvants, nucleic acid adjuvants, lipid-containing adjuvants, mixed adjuvants or aggregate structure adjuvants.

In some embodiments of the present invention, the adjuvant is an aluminum hydroxide adjuvant.

The present invention will be further described below in conjunction with embodiments:

Example 1 GnRH-MS2 subunit vaccine

Escherichia coli MS2 bacteriophage is a positive polarity single-stranded RNA spherical virus with a genome length of 3659bp, encoding four protein molecules, including mature enzyme protein, coat protein, replicase protein and lytic protein. The study found that in vitro, the genes of the mature enzyme protein and coat protein of MS2 bacteriophage and the 5' non-coding sequence containing gene regulatory elements were cloned into an expression vector, and the induced expression of the protein can self-assemble into mature virus-like particles (VLPs), and inserting several genes at specific sites of its coat protein gene can express it into VLPs displaying exogenous amino acids.

1. Acquisition of GnRH-MS2 target protein

(1) Construction of recombinant plasmid: The MS2 5' non-coding sequence, mature enzyme protein, coat protein and GnRH gene sequence were directly synthesized into the pET28a plasmid vector by a gene company to obtain the recombinant plasmid GnRH-MS2-pET28a.

(2) Transfer the recombinant plasmid into the expression strain: The recombinant plasmid GnRH-MS2-pET28a was transferred into the BL21 (DE3) Escherichia coli expression strain to obtain the GnRH-MS2-pET28a-BL21 recombinant expression strain.

(3) Preparation of recombinant plasmid GnRH-MS2-pET28a: 5uL of the glycerol culture containing the plasmid was inoculated into 5mL of LB medium (containing 50ug/ml kanamycin) using a pipette, and cultured at 37℃ with shaking for 14-16 hours. After culture, 1mL of the bacterial solution was taken for sequencing. The remaining bacterial solution was extracted with a plasmid extraction kit, and the nucleic acid concentration was detected by Nanodrop2000 nucleic acid detector.

(4) Bacterial culture and GnRH-MS2 VLP expression: Transform the BL21 (DE3) expression strain, shake the bacteria and concentrate by centrifugation, spread on kanamycin-resistant plates, culture at 37°C overnight, scrape the colonies, inoculate 10 mL of LB medium containing kanamycin, and shake the bacteria at 37°C at 200 r to a bacterial liquid OD value of about 0.8. Transfer it to 500 mL of LB medium containing kanamycin, and shake the bacteria at 37°C at 180 r to a bacterial liquid OD value of about 0.8. Add IPTG inducer and express overnight at 30°C. On the third day, centrifuge at 4500 rpm for 15 min to collect the bacteria. Ultrasonicate the bacteria for 30 min. After the end of ultrasound, collect the supernatant at 9500 rpm for 20 min at 4°C.

(5) Purification of GnRH-MS2 VLPs: The supernatant was centrifuged and purified by nickel column. The nickel column was equilibrated with PBS for 5 column volumes. The sample was loaded and washed with PBS for 5 column volumes, 25 nM imidazole for 2 column volumes, and then washed with 25 mM imidazole for 5 column volumes. Then, 50, 100, 150, 200, and 250 mM imidazole were used to elute for 5 column volumes. The effluent from loading to 500 mM imidazole was collected.

Take 8 μL from each collection solution, add 2 μL 5× protein electrophoresis loading buffer, and heat at 100°C for 5 min. Perform protein electrophoresis and stain to observe the concentration of the collection solution where the target band is located. Ultrafilter the target protein using an ultrafiltration tube and replace it with PBS solvent.

2. GnRH-MS2 VLP subunit vaccine immunization method

GnRH-MS2 virus-like particles were mixed with aluminum hydroxide adjuvant for immunization. Eight-week-old male C57BL/6 mice (five mice per group) were immunized with 50 μg of GnRH-MS2 virus-like particle vaccine on day 0, day 14, and day 28. GnRH-MS2 without adjuvant group, adjuvant pbs negative group, simple MS2 experimental group, and simple GnRH experimental group were set up at the same time. Anti-GnRH recombinant protein antibody titers and testosterone levels of these mice were measured. On the 70th day after immunization, the mice were killed and the testicular weight was measured.

3. Determination of mouse anti-GnRH antibody titer

At different time points during the experiment, sera were collected from immunized mice and control mice. Anti-GnRH IgG antibody titers were determined by ELISA as follows:

96-well plates were coated with 2μg/mL GnRH-BSA overnight at 4℃, 100μL per well, washed 5 times with PBST the next day, blocked with 300μL 2% skim milk, blocked at 37℃ for 2h, then washed 5 times with 1/1000 PBST, mouse serum was diluted with 1:500 as the initial double dilution, 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, 1:128000, 1:256000, and 2% skim milk as the diluent. From high to low dilution, 100ul per well, incubated at room temperature with shaking for 45min. Washed 5 times with PBST. HRP-labeled goat anti-mouse polyclonal antibody was used as the secondary antibody, with a dilution of 1:5000, and 1% skim milk powder was used as the diluent, 100ul per well, and incubated at room temperature for 45min with shaking. TMB color development was performed for 5-10min, and the stop solution was used to stop the color development, and the wavelength was directly read at 450nm. The optical density (OD) was measured at 450nm ELISA reader (BioRad Benchmark). The maximum OD450 serum dilution was calculated using these data. The anti-GnRH IgG antibody titer was 50% to 90%, preferably 60% to 80%.

Example 2 GnRH-T4 subunit vaccine

The bacteriophage T4 capsid contains two non-essential external proteins, Soc and Hoc, which allow for high-density arrangement of antigenic epitopes in the form of peptides, domains, full-length proteins, and even multi-subunit complexes. Bacteriophage T4 VLPs are highly immunogenic, do not require adjuvants, and provide complete protection against bacterial and viral pathogens. The T4 capsid gene sequence Soc or Hoc and the GnRH gene sequence are cloned into a prokaryotic expression vector to obtain a recombinant expression vector, and the GnRH VLP subunit vaccine can be obtained by expression and purification in an E. coli expression system.

1. Acquisition of GnRH-T4 target protein

(1) Construction of recombinant plasmid: T4Soc or Hoc gene sequence and GnRH gene sequence were directly synthesized into pET28a plasmid vector by a gene company to obtain recombinant plasmid GnRH-T4-pET28a.

(2) Transform the recombinant plasmid into the expression strain: Transform the recombinant plasmid GnRH-T4-pET28a into the BL21 (DE3) Escherichia coli expression strain to obtain the GnRH-T4-pET28a-BL21 recombinant expression strain.

(3) Preparation of recombinant plasmid GnRH-T4-pET28a: 5ul of the glycerol culture containing the plasmid was inoculated into 5mL of LB medium (containing 50ug/mL kanamycin) using a pipette, and cultured at 37℃ with shaking for 14-16 hours. After culture, 1mL of the bacterial solution was taken for sequencing. The remaining bacterial solution was extracted with a plasmid extraction kit, and the nucleic acid concentration was detected with a Nanodrop2000 nucleic acid detector.

(4) Bacterial culture and GnRH-T4 VLP expression: Transform the BL21 (DE3) expression strain, shake the bacteria and concentrate by centrifugation, spread on kanamycin-resistant plates, culture at 37°C overnight, scrape the colonies, inoculate 10 mL of LB medium containing kanamycin, and shake the bacteria at 37°C 200 r to a bacterial liquid OD value of about 0.8. Transfer it to 500 mL of LB medium containing kanamycin, and shake the bacteria at 37°C 180 r to a bacterial liquid OD value of about 0.8. Add IPTG inducer and express overnight at 30°C. On the third day, centrifuge at 4500 rpm for 15 min to collect the bacteria. Ultrasonicate the bacteria for 30 min. After the end of ultrasound, centrifuge at 9500 rpm for 20 min at 4°C to collect the supernatant.

(5) Purification of GnRH-T4 VLPs: The supernatant was centrifuged and purified by nickel column. The nickel column was equilibrated with PBS for 5 column volumes. The sample was loaded and washed with PBS for 5 column volumes, 25 nM imidazole for 2 column volumes, and then washed with 25 mM imidazole for 5 column volumes. Then, 50, 100, 150, 200, and 250 mM imidazole were used to elute for 5 column volumes. The effluent from loading to 500 mM imidazole was collected.

Take 8 μL from each collection solution, add 2 μL 5× protein electrophoresis loading buffer, and heat at 100°C for 5 min. Perform protein electrophoresis and stain to observe the concentration of the collection solution where the target band is located. Ultrafilter the target protein using an ultrafiltration tube and replace it with PBS solvent.

2. GnRH-T4 VLP subunit vaccine immunization method

GnRH-T4 virus-like particles were mixed with aluminum hydroxide adjuvant for immunization. Eight-week-old male C57BL/6 mice (five mice per group) were immunized with 50 μg of GnRH-T4 virus-like particle vaccine on day 0, day 14, and day 28. GnRH-T4 without adjuvant group, adjuvant pbs negative group, simple T4 experimental group, and simple GnRH experimental group were set up at the same time. Anti-GnRH recombinant protein antibody titers and testosterone levels of these mice were measured. On the 70th day after immunization, the mice were killed and the testicular weight was measured.

3. Determination of mouse anti-GnRH antibody titer

At different time points during the experiment, sera were collected from immunized mice and control mice. Anti-GnRH IgG antibody titers were determined by ELISA as follows:

96-well plates were coated with 2μg/mL GnRH-BSA overnight at 4℃, 100μL per well, washed 5 times with PBST the next day, blocked with 300μl 2% skim milk, blocked at 37℃ for 2h, then washed 5 times with 1/1000 PBST, mouse serum was diluted with 1:500 as the initial double dilution, 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, 1:128000, 1:256000, and 2% skim milk as the dilution solution. From high to low dilution, 100ul per well, incubated at room temperature with shaking for 45min. Washed 5 times with PBST. HRP-labeled goat anti-mouse polyclonal antibody was used as the secondary antibody, with a dilution of 1:5000, and 1% skim milk powder was used as the diluent, 100uL per well, and incubated at room temperature for 45min with shaking. TMB color development was performed for 5-10min, and the stop solution was used to stop the color development, and the wavelength was directly read at 450nm. The optical density (OD) was measured at 450nm ELISA reader (BioRad Benchmark). The maximum OD450 serum dilution was calculated using these data. The anti-GnRH IgG antibody titer was 50% to 90%, preferably 60% to 80%.

Example 3 GnRH-TMV subunit vaccine

Tobacco mosaic virus (TMV) is a plant virus that cannot infect animal cells, but has been shown to interact with and stimulate mammalian dendritic cells. The GnRH gene sequence was cloned into a prokaryotic expression vector to obtain a recombinant expression vector, and the GnRH protein was expressed and purified to obtain the GnRH protein. The GnRH protein was chemically coupled to the surface of TMV to prepare a GnRH-TMV-VLP subunit vaccine.

1. Obtaining GnRH-TMV target protein

(1) Construction of recombinant plasmid: The GnRH gene sequence was directly synthesized into the pKBP121 expression vector by a gene company to obtain the recombinant plasmid GnRH-TMV-pKBP121.

(2) Transfer the recombinant plasmid into the plant expression system: The recombinant plasmid GnRH-TMV-pKBP121 was transiently transfected into tobacco (Nicotiana benthamiana, Nb).

(3) Purification of GnRH-TMV-pKBP121: The transfected plants were harvested, the soluble protein fractions were separated, and GnRH was purified by a combination of protein A affinity and anion exchange chromatography.

(4) By infecting wild-type Nb plants with TMV N-terminal lysine mutation (NtK) virus, modified TMV-NtK with N-terminal lysine mutation was generated. Infected tissues were collected, soluble proteins were isolated, and viral particles were purified by a combination of Capto-Q and Capto-Core 700 chromatography. In an ISO 5 environment, inactivation was confirmed by exposing the virus to ultraviolet light at 254 nm (UV254), 5142 J/m2.

(5) Chemical coupling of GnRH-TMV and TMV-NtK: TMV-NtK was mixed with GnRH-TMV in a 1:1 molar ratio in 100 mM 2-(N-morpholino)ethanesulfonic acid and 500 mM NaCl solution at pH 6. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) was added, followed by 5 mM N-hydroxysulfosuccinimide (NHS). The EDC concentration was optimized based on the time and concentration of EDC to produce no "free" protein antigen, which varied depending on the protein. The coupling was terminated with 1 mM methylamine, and the coupled vaccine was then dialyzed against PBS overnight (Slidalyzer, 10 kD MCO). The TMV-Ag conjugate was visualized using 8-16% SDS-PAGE gels (BioRad). The final dialyzed vaccine concentration was determined by BCA analysis (BioRad).

2. GnRH-TMV VLP subunit vaccine immunization method

GnRH-TMV virus-like particles were mixed with aluminum hydroxide adjuvant for immunization. On day 0, day 14, and day 28, eight-week-old male C57BL/6 mice (five mice in each group) were immunized with 50 μg of GnRH-TMV virus-like particle vaccine, and a GnRH-TMV without adjuvant group, an adjuvant pbs negative group, a simple TMV experimental group, and a simple GnRH experimental group were set up. The anti-GnRH recombinant protein antibody titer and testosterone levels of these mice were measured. On day 70 after immunization, the mice were killed and the testicular weight was measured.

3. Determination of mouse anti-GnRH antibody titer

At different time points during the experiment, sera were collected from immunized mice and control mice. Anti-GnRH IgG antibody titers were determined by ELISA as follows:

96-well plates were coated with 2μg/mL GnRH-BSA overnight at 4℃, 100μL per well, washed 5 times with PBST the next day, blocked with 300μL 2% skim milk, blocked at 37℃ for 2h, then washed 5 times with 1/1000 PBST, mouse serum was diluted with 1:500 as the initial double dilution, 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, 1:128000, 1:256000, and 2% skim milk as the diluent. From high to low dilution, 100ul per well, incubated at room temperature with shaking for 45min. Washed 5 times with PBST. HRP-labeled goat anti-mouse polyclonal antibody was used as the secondary antibody, with a dilution of 1:5000, and 1% skim milk powder was used as the diluent, 100uL per well, and incubated at room temperature for 45min with shaking. TMB color development was performed for 5-10min, and the stop solution was used to stop the color development, and the wavelength was directly read at 450nm. The optical density (OD) was measured at 450nm ELISA reader (BioRad Benchmark). The maximum OD450 serum dilution was calculated using these data. The anti-GnRH IgG antibody titer was 50% to 90%, preferably 60% to 80%.

Example 4 GnRH-CPMV subunit vaccine

Cowpea Mosaic Virus (CPMV) can display foreign peptides on the surface of virus particles, which is very suitable for development into subunit vaccines. CPMV RNA and CPMV-GnRH integration plasmids were constructed respectively, and GnRH-VLP was expressed and purified through a plant expression system to obtain a GnRH-CPMV-VLP subunit vaccine.

1. Acquisition of GnRH-CPMV target protein

(1) GnRH peptide synthesis: The GnRH gene sequence is directly sent to the company to synthesize GnRH peptide;

(2) GnRH polypeptide is coupled with CPMV to obtain GnRH-CPMV VLP;

(3) Purification of GnRH-CPMV VLPs: GnRH-CPMV VLPs were purified by ultracentrifugation.

2. GnRH-CPMV VLP subunit vaccine immunization method

GnRH-CPMV virus-like particles were mixed with aluminum hydroxide adjuvant for immunization. On day 0, day 14, and day 28, eight-week-old male C57BL/6 mice (five mice in each group) were immunized with 50 μg of GnRH-CPMV virus-like particle vaccine, and a GnRH-CPMV unadjuvanted group, an adjuvant pbs-negative group, a simple CPMV experimental group, and a simple GnRH experimental group were set up. The anti-GnRH recombinant protein antibody titer and testosterone levels of these mice were measured. On day 70 after immunization, the mice were killed and the testicular weight was measured.

3. Determination of mouse anti-GnRH antibody titer

At different time points during the experiment, sera were collected from immunized mice and control mice. Anti-GnRH IgG antibody titers were determined by ELISA as follows:

96-well plates were coated with 2μg/mL GnRH-BSA overnight at 4℃, 100μL per well, washed 5 times with PBST the next day, blocked with 300μL 2% skim milk, blocked at 37℃ for 2h, then washed 5 times with 1/1000 PBST, mouse serum was diluted with 1:500 as the initial double dilution, 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, 1:128000, 1:256000, and 2% skim milk as the diluent. From high to low dilution, 100ul per well, incubated at room temperature with shaking for 45min. Washed 5 times with PBST. HRP-labeled goat anti-mouse polyclonal antibody was used as the secondary antibody, with a dilution of 1:5000, and 1% skim milk powder was used as the diluent, 100uL per well, and incubated at room temperature for 45min with shaking. TMB color development was performed for 5-10min, and the stop solution was used to stop the color development, and the wavelength was directly read at 450nm. The optical density (OD) was measured at 450nm ELISA reader (BioRad Benchmark). The maximum OD450 serum dilution was calculated using these data. The anti-GnRH IgG antibody titer was 50% to 90%, preferably 60% to 80%.

Example 5 GnRH-CMV subunit vaccine

Cucumber Mosaic Virus (CMV) VLP has a CMV modified capsid protein with an inserted T cell stimulatory epitope. The target protein can be coupled to the CMV capsid protein (G4SLPET) by site-directed catalysis of the SorteaseA enzyme to produce immunogenic virus-like particles. GnRH and CMV VLP are expressed and purified in a prokaryotic expression system, and then catalyzed by the SorteaseA enzyme to prepare a GnRH-CMV-VLP subunit vaccine.

1. Acquisition of GnRH-CMV target protein

(1) Construction of recombinant plasmid: The GnRH gene sequence was directly synthesized into the pcDNA3.1 expression vector by a gene company to obtain the recombinant plasmid GnRH-pcDNA3.1.

(2) Transfer the recombinant plasmid into the mammalian expression system: Transiently transfect the recombinant plasmid GnRH-pcDNA3.1 into 293F cells.

(3) Purification of GnRH-pcDNA3.1: harvest the supernatant of transfected cells and purify GnRH using a Ni column.

(4) Purification of CMV-VLP: Escherichia coli C2566 was transformed with the plasmid pET-CMV containing the CMV coat protein (CP) gene and induced with IPTG. CMV-VLP was purified by sucrose gradient centrifugation.

(5) Chemical coupling of GnRH-pcDNA3.1 and CMV-VLP: CMV-VLP was mixed with GnRH-pcDNA3.1 at a molar ratio of 1:1 in 100 mM 2-(N-morpholino)ethanesulfonic acid and 500 mM NaCl solution at Ph = 6. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) was added, followed by 5 mM N-hydroxysulfosuccinimide (NHS). The EDC concentration was optimized based on the time and concentration of EDC to produce no "free" protein antigen, which varied depending on the protein. The coupling was terminated with 1 mM methylamine, and the coupled vaccine was then dialyzed against PBS overnight (Slidalyzer, 10 kD MCO). The conjugate was visualized using 8-16% SDS-PAGE gels (BioRad). The final dialyzed vaccine concentration was determined by BCA analysis (BioRad).

2. GnRH-CMV VLP subunit vaccine immunization method

GnRH-CMV virus-like particles were mixed with aluminum hydroxide adjuvant for immunization. On day 0, day 14, and day 28, eight-week-old male C57BL/6 mice (five mice per group) were immunized with 50 μg of GnRH-CMV virus-like particle vaccine, and a GnRH-CMV without adjuvant group, an adjuvant pbs negative group, a simple CMV experimental group, and a simple GnRH experimental group were set up. The anti-GnRH recombinant protein antibody titer and testosterone level of these mice were measured. On day 70 after immunization, the mice were killed and the testicular weight was measured.

3. Determination of mouse anti-GnRH antibody titer

At different time points during the experiment, sera were collected from immunized mice and control mice. Anti-GnRH IgG antibody titers were determined by ELISA as follows:

96-well plates were coated with 2μg/mL GnRH BSA overnight at 4℃, 100μL per well, washed 5 times with PBST the next day, blocked with 300μL 2% skim milk, blocked at 37℃ for 2h, then washed 5 times with 1/1000 PBST, mouse serum was diluted with 1:500 as the initial double dilution, 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, 1:128000, 1:256000, and the diluent was 2% skim milk. From high to low dilution, 100ul per well, incubated at room temperature with shaking for 45min. Washed 5 times with PBST. HRP-labeled goat anti-mouse polyclonal antibody was used as the secondary antibody, with a dilution of 1:5000, and 1% skim milk powder was used as the diluent, 100ul per well, and incubated at room temperature for 45min with shaking. TMB color development was performed for 5-10min, and the stop solution was used to stop the color development, and the wavelength was directly read at 450nm. The optical density (OD) was measured at 450nm by an ELISA reader (BioRad Benchmark). The maximum OD450 serum dilution was calculated using these data. The anti-GnRH IgG antibody titer was 50% to 90%, preferably 60% to 80%.

Example 6 GnRH-PapMV subunit vaccine

The GnRH gene sequence is inserted into the papaya mosaic virus (PapMV) coat protein gene sequence to form a recombinant PapMV coat protein sequence containing an antigenic peptide fusion. After expression and purification by a suitable expression vector (Escherichia coli, yeast, mammalian cells), these recombinant coat proteins can be organized into VLPs.

1. Acquisition of GnRH-PapMV target protein

(1) Construction of recombinant plasmid: The GnRH gene sequence was directly synthesized into the pcDNA3.1 expression vector by a gene company to obtain the recombinant plasmid GnRH-pcDNA3.1.

(2) Transfer of the recombinant plasmid into the mammalian expression system: The recombinant plasmid GnRH-pcDNA3.1 was transiently transfected into 293F cells.

(3) Purification of GnRH-pcDNA3.1: harvest the supernatant of transfected cells and purify GnRH using a Ni column.

(4) PapMV-VLP purification: Escherichia coli C2566 was transformed with the plasmid pET-PapMV containing the PapMV coat protein (CP) gene and induced with IPTG. PapMV-VLP was purified by sucrose gradient centrifugation.

(5) Chemical coupling of GnRH-pcDNA3.1 and PapMV-VLP: PapMV-VLP was mixed with GnRH-pcDNA3.1 at a molar ratio of 1:1 in 100 mM 2-(N-morpholino)ethanesulfonic acid and 500 mM NaCl solution at pH 6. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) was added, followed by 5 mM N-hydroxysulfosuccinimide (NHS). The EDC concentration was optimized based on the time and concentration of EDC to produce no "free" protein antigen, which varied depending on the protein. The coupling was terminated with 1 mM methylamine, and the coupled vaccine was then dialyzed against PBS overnight (Slidalyzer, 10 kD MCO). The conjugate was visualized using 8-16% SDS-PAGE gels (BioRad). The final dialyzed vaccine concentration was determined by BCA analysis (BioRad).

2. GnRH-PapMV VLP subunit vaccine immunization method

GnRH-PapMV virus-like particles were mixed with aluminum hydroxide adjuvant for immunization. On day 0, day 14, and day 28, eight-week-old male C57BL/6 mice (five mice in each group) were immunized with 50 μg of GnRH-PapMV virus-like particle vaccine, and a GnRH-PapMV without adjuvant group, an adjuvant pbs negative group, a simple PapMV experimental group, and a simple GnRH experimental group were set up. The anti-GnRH recombinant protein antibody titer and testosterone levels of these mice were measured. On day 70 after immunization, the mice were killed and the testicular weight was measured.

3. Determination of mouse anti-GnRH antibody titer

At different time points during the experiment, sera were collected from immunized mice and control mice. Anti-GnRH IgG antibody titers were determined by ELISA as follows:

96-well plates were coated with 2μg/mL GnRH BSA at 4℃ overnight, 100μL per well, washed 5 times with PBST the next day, blocked with 300μL 2% skim milk, blocked at 37℃ for 2h, then washed 5 times with 1/1000 PBST, mouse serum was diluted with 1:500 as the initial double dilution, 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, 1:128000, 1:256000, and 2% skim milk as the dilution solution. From high to low dilution, 100ul per well, incubated at room temperature with shaking for 45min. Washed 5 times with PBST. HRP-labeled goat anti-mouse polyclonal antibody was used as the secondary antibody, with a dilution of 1:5000, and 1% skim milk powder was used as the diluent, 100uL per well, and incubated at room temperature for 45min with shaking. TMB color development was performed for 5-10min, and the stop solution was used to stop the color development, and the wavelength was directly read at 450nm. The optical density (OD) was measured at 450nm by an ELISA reader (BioRad Benchmark). The maximum OD450 serum dilution was calculated using these data. The anti-GnRH IgG antibody titer was 50% to 90%, preferably 60% to 80%.

Example 7 GnRH-FHV subunit vaccine

Flock House Virus (FHV), a non-enveloped insect RNA virus, has an icosahedral capsid composed of 180 identical coat protein subunits. Based on its genetic simplicity, exceptional yield and high physicochemical stability, and because there are no known safety issues with the use of FHV in animals and humans, the virus is very suitable for various biotechnological applications. The GnRH gene sequence is cloned into a prokaryotic expression vector to obtain a recombinant expression vector, and the GnRH protein is expressed and purified to obtain the GnRH protein. FHV infects insect cells to produce and collect virus particles, and the GnRH protein is coupled to the surface of the FHV particles by non-covalent or covalent means to prepare a subunit vaccine.

1. Acquisition of GnRH-FHV target protein

(1) Construction of recombinant plasmid: The N-terminal part of FHV coat protein, GnRH and the C-terminal part of coat protein gene sequences were directly synthesized into the pBacPAK9 plasmid vector by a gene company to obtain the recombinant plasmid GnRH-FHV-pBacPAK9;

(2) Transformation of recombinant plasmid into insect cells: transiently transfect the recombinant plasmid GnRH-FHV-pBacPAK9 into insect cells, collect the supernatant, and use the supernatant to infect insect cells again;

(3) Purification of GnRH-FHV VLPs: The supernatant was collected and VLPs in the supernatant were precipitated by adding NaCl at a final concentration of 0.2 M and polyethylene glycol 8000 at a final concentration of 8% (w/v) and stirring the mixture at 4°C for 1 hour. VLPs were purified by ultracentrifugation and sucrose density gradient centrifugation.

2. GnRH-FHV VLP subunit vaccine immunization method

GnRH-FHV virus-like particles were mixed with aluminum hydroxide adjuvant for immunization. On day 0, day 14, and day 28, eight-week-old male C57BL/6 mice (five mice per group) were immunized with 50 μg of GnRH-FHV virus-like particle vaccine, and a GnRH-FHV without adjuvant group, an adjuvant pbs negative group, a simple FHV experimental group, and a simple GnRH experimental group were set up. The anti-GnRH recombinant protein antibody titer and testosterone levels of these mice were measured. On day 70 after immunization, the mice were killed and the testicular weight was measured.

3. Determination of mouse anti-GnRH antibody titer

At different time points during the experiment, sera were collected from immunized mice and control mice. Anti-GnRH IgG antibody titers were determined by ELISA as follows:

96-well plates were coated with 2μg/mL GnRH BSA at 4℃ overnight, 100μL per well, washed 5 times with PBST the next day, blocked with 300μL 2% skim milk, blocked at 37℃ for 2h, then washed 5 times with 1/1000PBST, mouse serum was diluted with 1:500 as the initial double dilution, 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, 1:128000, 1:256000, and 2% skim milk as the diluent. From high to low dilution, 100uL per well, incubated at room temperature with shaking for 45min. Wash the plate 5 times with PBST. HRP-labeled goat anti-mouse polyclonal antibody was used as the secondary antibody, with a dilution of 1:5000, and 1% skim milk powder was used as the diluent, 100ul per well, and incubated at room temperature for 45min with shaking. TMB color development was performed for 5-10min, and the stop solution was used to stop the color development, and the wavelength was directly read at 450nm. The optical density (OD) was measured at 450nm by an ELISA reader (BioRad Benchmark). The maximum OD450 serum dilution was calculated using these data. The anti-GnRH IgG antibody titer was 50% to 90%, preferably 60% to 80%.

Example 8 GnRH-EILV subunit vaccine

The EILV virus is the only described insect-specific alphavirus and is fundamentally and completely defective in infecting vertebrate cells, even after it is artificially delivered into the cytoplasm, due to its inability to enter cells and replicate its RNA genome. The GnRH gene sequence was constructed onto a plasmid with the EILV cDNA gene sequence to form an EILV/GnRH chimeric plasmid, and transfected into mosquito cells (C7/10) by a suitable method, the supernatant was collected to infect the cells again, and the virus was collected and purified to obtain the GnRH-EILV VLP subunit vaccine.

1. Obtaining the target protein of GnRH-EILV

(1) Construction of recombinant plasmid: The GnRH gene sequence was directly synthesized into the EILV virus cDNA plasmid vector by a gene company to obtain the recombinant plasmid EILV-GnRH.

(2) Transformation of recombinant plasmid into insect cells: The recombinant plasmid EILV-GnRH was transiently transfected into C7/10 cells, the supernatant was collected, and the supernatant was used to infect insect cells again.

(3) Purification of EILV-GnRH VLPs: The supernatant was collected and VLPs in the supernatant were precipitated by adding NaCl at a final concentration of 0.2 M and polyethylene glycol 8000 at a final concentration of 8% (w/v) and stirring the mixture at 4°C for 1 hour. VLPs were purified by ultracentrifugation and sucrose density gradient centrifugation.

2. GnRH-EILV VLP subunit vaccine immunization method

GnRH-EILV virus-like particles were mixed with aluminum hydroxide adjuvant for immunization. On day 0, day 14, and day 28, eight-week-old male C57BL/6 mice (five mice per group) were immunized with 50 μg of EILV-GnRH virus-like particle vaccine, and an EILV-GnRH no adjuvant group, an adjuvant pbs negative group, a pure EILV experimental group, and a pure GnRH experimental group were set up. The anti-GnRH recombinant protein antibody titer and testosterone levels of these mice were measured. On day 70 after immunization, the mice were killed and the testicular weight was measured.

3. Determination of mouse anti-GnRH antibody titer

At different time points during the experiment, sera were collected from immunized mice and control mice. Anti-GnRH IgG antibody titers were determined by ELISA as follows:

96-well plates were coated with 2μg/mL GnRH BSA at 4℃ overnight, 100μL per well, washed 5 times with PBST the next day, blocked with 300μL 2% skim milk, blocked at 37℃ for 2h, then washed 5 times with 1/1000PBST, mouse serum was diluted with 1:500 as the initial double dilution, 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, 1:128000, 1:256000, and 2% skim milk as the diluent. From high to low dilution, 100uL per well, incubated at room temperature with shaking for 45min. Wash the plate 5 times with PBST. HRP-labeled goat anti-mouse polyclonal antibody was used as the secondary antibody, with a dilution of 1:5000, and 1% skim milk powder was used as the diluent, 100ul per well, and incubated at room temperature for 45min with shaking. TMB color development was performed for 5-10min, and the stop solution was used to stop the color development, and the wavelength was directly read at 450nm. The optical density (OD) was measured at 450nm by an ELISA reader (BioRad Benchmark). The maximum OD450 serum dilution was calculated using these data. The anti-GnRH IgG antibody titer was 50% to 90%, preferably 60% to 80%.

Example 9 GnRH-HBsAg subunit vaccine

Hepatitis B vaccines are based on the major hepatitis B surface antigen (HBsAg), a protein that is able to assemble into so-called virus-like particles (VLPs).

1. Obtaining GnRH-HBsAg target protein

(1) Construction of recombinant plasmid

The HBs and GnRH gene sequences were directly synthesized into the pET28a plasmid vector by a gene company to obtain the recombinant plasmid GnRH-HBs-pET28a;

(2) Preparation of recombinant plasmid GnRH-HBs-pET28a

Use a pipette to take 5ul of the glycerol bacteria containing the plasmid and inoculate it into 5mL of LB medium (containing 50ug/ml kanamycin), shake and culture at 37℃ for 14-16 hours, and take 1mL of the bacterial solution for sequencing. Use a plasmid extraction kit to extract the plasmid from the remaining bacterial solution, and use the Nanodrop2000 nucleic acid detector to detect the nucleic acid concentration.

(3) Transfer the recombinant plasmid into the expression strain

The recombinant plasmid GnRH-HBs-pET28a was transformed into the BL21 (DE3) Escherichia coli expression strain to obtain the GnRH-HBs-pET28a-BL21 recombinant expression strain;

(4) Bacterial culture and GnRH-HBsAg VLP purification

After shaking the recombinant strain, centrifuge and concentrate, apply kanamycin resistance plates, culture overnight at 37℃, scrape the colony, inoculate 10mL LB medium containing kanamycin, and shake at 37℃ 200r until the OD value of the bacterial solution is about 0.8. Transfer it to 500mL LB medium containing kanamycin, shake at 37℃ 180r until the OD value of the bacterial solution is about 0.8. Add IPTG inducer and express overnight at 30℃. On the third day, centrifuge at 4500rpm for 15min to collect the bacteria. Ultrasonicate the bacteria for 30min. After the end of ultrasound, collect the supernatant at 9500rpm for 20min at 4℃.

The supernatant was centrifuged and purified by nickel column. The nickel column was equilibrated with PBS for 5 column volumes. Sample was loaded, washed with PBS for 5 column volumes, washed with 25nM imidazole for 2 column volumes, and then washed with 25mM imidazole for 5 column volumes. Then, eluted with 50, 100, 150, 200, and 250mM imidazole for 5 column volumes. The effluent from loading to 500mM imidazole was collected.

Take 8 μL from each collection solution, add 2 μL 5× protein electrophoresis loading buffer, and heat at 100°C for 5 min. Perform protein electrophoresis and stain to observe the concentration of the collection solution where the target band is located. Ultrafilter the target protein using an ultrafiltration tube and replace it with PBS solvent.

2. GnRH-HBsAg VLP subunit vaccine immunization method

GnRH-HBsAg VLPs were mixed with aluminum hydroxide adjuvant for immunization. Eight-week-old male C57BL/6 mice (five mice per group) were immunized with 50 μg of GnRH-HBsAg virus-like particle vaccine on day 0, day 14, and day 28. GnRH-HBsAg without adjuvant group, adjuvant pbs negative group, HBsAg alone experimental group, and GnRH alone experimental group were set up at the same time. Anti-GnRH recombinant protein antibody titers and testosterone levels of these mice were measured. On day 70 after immunization, mice were killed and testicular weights were measured.

3. Determination of mouse anti-GnRH antibody titer

At different time points during the experiment, sera were collected from immunized mice and control mice. Anti-GnRH IgG antibody titers were determined by ELISA as follows. 96-well plates were coated with 2 μg/ml GnRH-BSA overnight at 4°C, 100 μl per well, and the plates were washed 5 times with PBST the next day. 300 μl 2% skim milk was added to block the plates, and the plates were blocked at 37°C for 2 h. After that, the plates were washed 5 times with 1/1000 PBST. The mouse sera were diluted in a gradient of 1:500 as the initial double dilution, 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, 1:128000, and 1:256000, and the dilution was diluted with 2% skim milk. From high to low dilutions, 100 ul per well was incubated at room temperature with shaking for 45 min. The plates were washed 5 times with PBST. HRP-labeled goat anti-mouse polyclonal antibody was used as the secondary antibody, with a dilution of 1:5000, and 1% skim milk powder was used as the diluent, 100ul per well, and incubated at room temperature for 45min. TMB color development was performed for 5-10min, and the stop solution was used to stop the reaction, and the wavelength was directly read at 450nm. The optical density (OD) was measured by a 450nm ELISA reader (BioRad Benchmark). The maximum OD450 serum dilution was calculated using these data.

Example 10 GnRH-M1 subunit vaccine

The viral core protein influenza matrix protein (M1) can be assembled into enveloped virus-like particles (VLPs) when expressed simultaneously with antigens containing influenza hemagglutinin (HA) and neuraminidase (NA).

1. Acquisition of GnRH-CMV target protein

(1) Construction of recombinant plasmid

The GnRH gene sequence and HA gene sequence were synthesized into the pcDNA3.1 expression vector by a gene company to obtain the recombinant plasmid GnRH-HA-pcDNA3.1, and the M1 gene sequence was synthesized into the pcDNA3.1 expression vector to obtain the recombinant plasmid M1-pcDNA3.1.

(2) Transfer of recombinant plasmid into mammalian expression system

The recombinant plasmids GnRH-HA-pcDNA3.1 and M1-pcDNA3.1 were transiently transfected into 293F cells.

(3) GnRH-M1 VLP purification

After 48 h, the supernatant of transfected cells was harvested and GnRH VLPs were purified by ultrafiltration. Ultracentrifugal filter devices (Merck, Darmstadt, Germany) were used for concentration. The concentrated samples were subjected to ultracentrifugation at 135,000 × g, 4°C for 4 h through 20% glycerol buffer. The cell pellet containing GnRH-M1 VLPs was harvested and resuspended with 500 μL of phosphate-buffered saline (PBS).

2. GnRH-M1 VLP subunit vaccine immunization method

GnRH-M1 VLP virus-like particles were mixed with aluminum hydroxide adjuvant for immunization. Eight-week-old male C57BL/6 mice (five mice per group) were immunized with 50 μg of GnRH-M1 VLP vaccine on day 0, day 14, and day 28. A GnRH-M1 VLP without adjuvant group, an adjuvant pbs-negative group, a CMV-only experimental group, and a GnRH-only experimental group were also set up. The anti-GnRH recombinant protein antibody titers and testosterone levels of these mice were measured. On day 70 after immunization, the mice were killed and the testicular weights were measured.

3. Determination of mouse anti-GnRH antibody titer

At different time points during the experiment, sera were collected from immunized mice and control mice. Anti-GnRH IgG antibody titers were determined by ELISA as follows:

96-well plates were coated with 2μg/mL GnRH BSA overnight at 4℃, 100μL per well, washed 5 times with PBST the next day, blocked with 300μL 2% skim milk, blocked at 37℃ for 2h, then washed 5 times with 1/1000 PBST, mouse serum was diluted with 1:500 as the initial double dilution, 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, 1:128000, 1:256000, and 2% skim milk as the diluent. From high to low dilution, 100ul per well, incubated at room temperature with shaking for 45min. Washed 5 times with PBST. HRP-labeled goat anti-mouse polyclonal antibody was used as the secondary antibody, with a dilution of 1:5000, and 1% skim milk powder was used as the diluent, 100ul per well, and incubated at room temperature for 45min with shaking. TMB color development was performed for 5-10min, and the stop solution was used to stop the color development, and the wavelength was directly read at 450nm. The optical density (OD) was measured at 450nm by an ELISA reader (BioRad Benchmark). The maximum OD450 serum dilution was calculated using these data. The anti-GnRH IgG antibody titer was 50% to 90%, preferably 60% to 80%

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 (10)

1. GnRH-VLP recombinant protein, characterized in that it comprises GnRH and virus-like particle protein; the virus-like particle protein comprises MS2, T4, TMV, CPMV, CMV, PapMV, FHV, EILV, HBsAg and/or M1. 2. The GnRH-VLP recombinant protein according to claim 1, characterized in that the GnRH protein has the following amino acid sequence: (1) the amino acid sequence shown in SEQ ID NO: 1; or (2) A sequence in which one or more amino acids are substituted, deleted, added and/or replaced based on the amino acid sequence shown in (1); or (3) a sequence having a homology of more than 90% with the amino acid sequence shown in (1); The MS2 is derived from Escherichia coli MS2 bacteriophage; The T4 is derived from the bacteriophage T4 capsid; The TMV is derived from tobacco mosaic virus; The CPMV is derived from cowpea mosaic virus; The CMV is derived from cucumber mosaic virus; The PapMV is derived from papaya mosaic virus; The FHV is derived from a Furry Shed Virus; The EILV is derived from the Eilat virus; The HBsAg is derived from hepatitis B surface antigen; The M1 is derived from influenza matrix protein. 3. Biomaterial, characterized in that it includes any of the following: 1), a nucleic acid encoding the recombinant protein according to claim 1; II), an expression unit containing the nucleic acid described in I); III), a recombinant vector containing the nucleic acid described in I) or the expression unit described in II); IV), a host cell transformed or transfected with the expression vector described in III); V) and IV) the culture products of the host cells. 4. A method for preparing the recombinant protein according to claim 1 or 2, characterized in that it comprises culturing the host cell according to claim 3 to obtain a culture product containing the recombinant protein according to claim 1. 5. A composition, characterized in that it comprises the recombinant protein according to claim 1 or 2, GnRH antigen or any antigen that can make animals infertile. 6. The composition according to claim 5, characterized in that it also includes an acceptable adjuvant; the adjuvant includes an aluminum salt adjuvant, a protein adjuvant, a nucleic acid adjuvant, a lipid adjuvant, a mixed adjuvant or an aggregate structure adjuvant. 7. Use of the recombinant protein according to claim 1 or 2, the biological material according to claim 3, or the composition according to claim 5 or 6 in the preparation of a subunit vaccine. 8. GnRH-VLP subunit vaccine, characterized in that it comprises the recombinant protein described in claim 1 or 2 or the composition described in claim 5 or 6. 9. The method for preparing the subunit vaccine according to claim 8, characterized in that it comprises mixing the recombinant protein according to claim 1 or 2 with an adjuvant; or mixing the composition according to claim 5 or 6 with a buffer. 10. A method for castration of an animal, comprising administering the subunit vaccine according to claim 8.