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EP-4737572-A1 - PREPARATION METHOD FOR CASTRATING AP205 VIRUS-LIKE PARTICLE SUBUNIT VACCINE

EP4737572A1EP 4737572 A1EP4737572 A1EP 4737572A1EP-4737572-A1

Abstract

The present invention relates to the fields of molecular biology, virology, immunology and medicine, and in particular to a preparation method for a castrating AP205 virus-like particle subunit vaccine.

Inventors

  • ZHA, Lisha
  • ZHOU, Yuhang
  • HUANG, QIAN
  • WU, Maobai
  • ZHENG, Qi

Assignees

  • Shenzhen Herz Life Science Technology Co., Ltd

Dates

Publication Date
20260506
Application Date
20230629

Claims (11)

  1. A nucleic acid encoding a GnRH-I-AP205 fusion protein, comprising: a nucleotide sequence of SEQ ID NO: 1; or a sequence obtained by substitution, deletion, insertion, and/or replacement of one or more nucleotides of SEQ ID NO: 1; or a sequence having more than 90% amino acid sequence identity to SEQ ID NO: 1.
  2. The nucleic acid according to claim 1, wherein the nucleotide sequence of the nucleic acid is SEQ ID NO: 1.
  3. An expression unit comprising the nucleic acid according to claim 3.
  4. A recombinant vector comprising the nucleic acid according to claim 1 or 2, or the expression unit according to claim 3.
  5. A host cell, wherein the host cell is transformed or transfected with the recombinant vector according to claim 4, or the nucleic acid according to claim 1 or 2 is integrated into the genome of the host cell.
  6. The host cell according to claim 5, wherein the host cell is Escherichia coli SHuffle ™ T7 strain.
  7. A method for preparing a GnRH-I-AP205 fusion protein, comprising culturing the host cell according to claim 5 or 6 to obtain a culture product containing the GnRH-I-AP205 fusion protein.
  8. A culture product obtained by the method according to claim 7.
  9. A vaccine comprising the culture product according to claim 8 and an adjuvant.
  10. A method for preparing the vaccine according to claim 9, comprising purifying the culture product according to claim 8 and mixing the culture product with an adjuvant.
  11. A method for animal castration, comprising administering the vaccine according to claim 10 to the animal.

Description

FIELD The present disclosure relates to the field of biology, virology, immunology and medicine, and particularly relates to a method for preparing an AP205 virus-like particle subunit vaccine for castration. BACKGROUND Castration refers to the removal of the reproductive system of an animal or the loss of sexual function by external means, including the removal of the testes from male individuals and the removal of the ovaries from female individuals and the two together are referred to as gonadectomy. In addition to surgically removing the gonads, castration can also be achieved by local radiation exposure or chemical treatment. After castration is done in a vertebrate, the source of sex hormone secretion is lost, and as a result, the accessory organs of the genital organs, as well as the secondary and tertiary sexual characteristics, may degenerate. Gonadotropin-releasing hormone (GnRH) is an endogenous polypeptide hormone of animals. A physiological dose of GnRH-I can cause an increase in the concentration of gonadotropins (such as a mild increase in FSH and a significant increase in LH), thereby promoting the synthesis and secretion of sex hormones (such as estradiol, progesterone, and testosterone), facilitating follicular development and maturation leading to ovulation, or promoting testicular development and sperm maturation, and inducing and maintaining secondary sexual characteristics. In addition, GnRH-I can also directly affect the gonads, regulating the synthesis and secretion of gonadal steroid hormones and promoting gametogenesis. Autologous antigenic proteins are generally difficult to induce antibody responses against self-antigens. A method to improve the vaccination efficiency is to increase the repetitiveness of the applied antigen. Unlike isolated proteins, viruses can induce a rapid and effective immune response both with and without the help of T cells, even in the absence of any adjuvant. Compared with a few isolated proteins, viruses can trigger a much stronger immune response than their separated components. For B-cell responses, it is well known that a key factor in viral immunogenicity is the repetitiveness and order of surface epitopes. Many viruses exhibit quasicrystal surfaces with regularly arranged epitopes that can effectively cross-link epitope-specific immunoglobulin on B cells. Such cross-linking of surface immunoglobulin on B cells serves as a strong activation signal, directly inducing cell cycle progression and the production of IgM antibodies. Moreover, these activated B cells can stimulate T helper cells, which then induce the class switching of IgM antibodies to IgG antibodies in B cells, as well as the generation of long-term B-cell memory, which is the purpose of any vaccination. The structure of viruses is even related to the production of anti-antibodies in autoimmune diseases and constitutes part of the natural response to pathogens. Therefore, antigens presented on the highly organized viral surface are capable of inducing strong antibody responses against the antigen. Virus-like particles are the major capsid proteins of RNA bacteriophages, which self-assemble from multiple monomers into a novel class of virus-like particles (VLPs) with high immunogenicity. These VLPs do not contain the RNA genome of the bacteriophage and are therefore incapable of replication. However, studies have shown that various polypeptides can be fused to the N-terminus or C-terminus of the virus-like particle protein, and the resulting fusion proteins can form 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 and the antigen or at least one antigenic epitope thereof is displayed on the outer surface of the assembled VLPs, the formation of VLPs can effectively enhance the immunogenicity of the target antigen. In addition to the reported virus-like particles AP205 and Qβ, there are also various other VLP platforms such as CuMV and tobacco mosaic virus VLPs, all of which have potential for development into castration vaccines. The Escherichia coli expression system is favorable for the efficient expression of virus-like particles. However, to ensure correct protein folding and achieve faster and more efficient expression, the codon sequence of the exogenous nucleic acid needs to be adaptively modified. SUMMARY In view of the problems of the prior art, the present disclosure provides a method for preparing an AP205OPT virus-like particle subunit vaccine for castration. The vaccine can be easily obtained through bacterial culture with high expression yield for convenient industrial production, and the vaccine exhibits strong immunogenicity and induces rapid immune response. The present disclosure provides a nucleic acid encoding a GnRH-I-AP205 fusion protein, comprising: a nucleotide sequence of SEQ ID NO: 1; ora sequence obtained by substitution, deletion, inse