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CN-122011208-A - Self-assembled nano-particle vaccine of double antigen of orf virus, preparation and application thereof

CN122011208ACN 122011208 ACN122011208 ACN 122011208ACN-122011208-A

Abstract

The invention provides an orf virus double-antigen self-assembled nanoparticle, which is a fusion protein containing an orf virus protein and ferritin, wherein the orf virus protein comprises an orf virus protein ORFV050 and an orf virus protein ORFV 086. The ORFV050-ORFV086-Fer fusion protein prepared by the invention is self-assembled into nano particles in vitro, can induce the production of high-titer serum antibodies of organisms and cytokines such as IFN-gamma, TNF-alpha, IL-6 and the like, and can be further used for preparing vaccines for preventing and/or treating the oral ulcer. Based on ORFV050-ORFV086-Fer fusion protein, the self-assembled protein nanoparticle vaccine provided by the invention is safe and effective, can induce immunized mice to generate lasting immune protection, has good immune efficacy, and can be used for clinically preventing and/or treating the infection of the aphtha virus.

Inventors

  • ZHAO KUI
  • SUN YIRAN
  • WU YIGUANG
  • Xu Mengshi
  • LV LIJUN
  • GUAN JIYU
  • HE WENQI
  • GAO FENG

Assignees

  • 吉林大学

Dates

Publication Date
20260512
Application Date
20260126

Claims (10)

  1. 1. An orf virus double antigen self-assembled nanoparticle, the nanoparticle being a fusion protein comprising an orf virus protein and ferritin, wherein the orf virus protein comprises an orf virus protein ORFV 050 and an orf virus protein ORFV 086.
  2. 2. The method of claim 1, wherein the fusion protein comprises an ORFV 050 protein, an ORFV 086 protein and a monomeric ferritin subunit which are sequentially connected in series, and a connecting peptide is respectively arranged between the C end of the ORFV 050 protein, the N end of the ORFV 050 protein and the C end of the ORFV 086 protein, and between the N end of the ORFV 086 protein and the C end of the monomeric ferritin subunit.
  3. 3. The orf virus double antigen self-assembled nanoparticle as in claim 1 or 2, wherein the orf virus protein ORFV050 has an amino acid sequence as shown in SEQ ID No. 6.
  4. 4. The orf virus double antigen self-assembled nanoparticle as in claim 1 or 2, wherein the orf virus protein ORFV086 has an amino acid sequence as shown in SEQ ID No. 7.
  5. 5. The orf virus double antigen self-assembled nanoparticle as in claim 1 or 2, wherein the ferritin has an amino acid sequence as shown in SEQ ID No. 5.
  6. 6. The vaccine of the orf virus dual antigen self-assembled nanoparticle of any one of claims 1-5.
  7. 7. The method of any one of claims 1-5, wherein the nucleic acid encoding the fusion protein comprising the orf virus protein ORFV050 and the orf virus protein ORFV086 and the ferritin is inserted into an expression vector, and the fusion protein self-assembles to form a nanoparticle after recombinant expression, wherein the fusion protein is designated as ORFV050-ORFV086-Fer.
  8. 8. The method according to claim 7, wherein the nucleotide encoding the ORFV050-ORFV086-Fer comprises the nucleotide encoding the ORFV050 protein shown in SEQ ID NO. 2, or the nucleotide encoding the ORFV086-Fer comprises the nucleotide encoding the ORFV086 protein shown in SEQ ID NO. 3.
  9. 9. The nucleotide encoding an orf virus dual antigen self-assembled nanoparticle of any one of claims 1-5.
  10. 10. Use of the orf virus double antigen self-assembled nanoparticle according to any one of claims 1-5 in the preparation of a vaccine for treating and/or preventing orf.

Description

Self-assembled nano-particle vaccine of double antigen of orf virus, preparation and application thereof Technical Field The invention relates to the veterinary field and the medical technical field, in particular to a preparation method and application of a double-antigen self-assembled nanoparticle vaccine carrying an orf virus ORFV050 protein and an ORFV086 protein. Background The Orf (Orf) is also called as contagious impetigo of sheep or contagious dermatitis of sheep, and is an acute and high contagious infectious disease of small ruminants such as sheep and goats caused by Orf virus (ORFV) infection. ORFV is a representative member of the parapoxviridae family of poxviridae and has a high degree of epitheliotropism, and the affected animals are characterized by papules, blisters, pustules, ulcers and warty crusts formed on the skin/mucous membranes at the lips, perinases, eyelids, earroots, breasts and other sites, resulting in feeding disorders of the affected animals, causing a decline in their growth/reproductive performance and even death, severely impeding the development of the sheep industry and related industries. People can also directly or indirectly contact the bacteria to cause infection, and the susceptible groups mainly comprise herd, veterinarian, butcher, breeder, fur processor, sacrifice personnel and the like, thereby potentially threatening public health and safety. The ORFV genome is large, containing about 134 genes in total, and consists of end regions (ORFs 001-008 and ORFs 112-134) and intermediate core regions (ORFs 009-111). The viral core region genes are conserved and mainly encode factors associated with viral morphogenesis, transcription, replication, etc., while the terminal regions are mainly responsible for encoding proteins associated with viral virulence, immunomodulation and host range. The ORFV 050 gene and the ORFV 086 gene are located in the core region of the viral genome and are conserved among different strains. The virus particle core protein VP8 encoded by the ORFV 050 gene is an advanced protein necessary for virus assembly, while the protein encoded by the ORFV 086 gene and the hydrolysate are both important structural proteins for assembling mature virus particles. The results of the letter analysis for the ORFV 050 and ORFV 086 proteins show that both are capsid proteins, are proteins with high stability and hydrophilicity, and have no transmembrane domain and signal peptide. Because of the lack of specific drugs for the control of the aphtha epidemic disease, vaccination is still an important means of preventing the disease. The conventional inactivated vaccine has the defects that the exposure of the antigenic determinant is incomplete, the antigenic determinant is easy to change in the inactivation process, and the like, so that the vaccine has lower immunity protection capability, and the attenuated vaccine has potential safety hazards such as virulence return and the like. Subunit vaccine has the advantages of high safety, T, B cell immunity induction and the like because the subunit vaccine does not contain viral genome, and becomes an important direction for researching and developing novel, safe and efficient ORFV vaccine. Ferritin is an emerging carrier platform, has high stability, good biocompatibility and unique self-assembly capability, and is a flexible and reliable vaccine carrier platform. Typically, 24 subunits are arranged in octahedral symmetry around a hollow core, with 8 triple and 6 quadruple axes of symmetry on the surface. The N-terminus of each subunit is exposed at the outer surface of the globular structure and can be used to display antigen proteins. The ferritin is taken as a carrier, and antigen protein is fixed on ferritin to prepare ferritin nanoparticle vaccine, which is an important technical means for creating novel subunit vaccine. Disclosure of Invention The invention aims to overcome the defects of the prior art, based on a self-assembled helicobacter pylori ferritin (Ferritin) twenty-four polymer, the fusion antigen carrying the orf virus ORFV050 protein and the ORFV086 protein is connected in series by a connecting peptide GGGGS and fused at the N end of ferritin, self-assembled nano particles with good assembly form are prepared, and lasting immune protection is induced in mice after immunization, so that the self-assembled nano particles are hopeful to be developed into novel nano particle vaccines for the orf virus. The invention provides a double-antigen self-assembled ferritin nanoparticle vaccine carrying orf virus ORFV050 protein and ORFV086 protein, a preparation method and application thereof. The orf capsid proteins ORFV050 and ORFV086 were concatenated with the linker peptide GGGGS and fused to the ferritin N-terminus. Preferably, the fusion protein gene is inserted into an expression vector (preferably, pET-28a vector), a pET-28a-ORFV050-ORFV086-Fer protein recombinant plasmid is constructed, and the f