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CN-122011212-A - Nanoparticle antigen based on ferritin carrier, recombinant expression carrier, recombinant lentiviral carrier, and preparation method and application thereof

CN122011212ACN 122011212 ACN122011212 ACN 122011212ACN-122011212-A

Abstract

The invention discloses a nanoparticle antigen based on ferritin carrier, a recombinant expression carrier, a recombinant lentiviral carrier, a preparation method and application thereof, wherein the nanoparticle antigen comprises a recombinant protein formed by fusion of a transmissible gastroenteritis virus spike protein receptor binding domain and ferritin subunit through a flexible connecting peptide, and the recombinant protein is self-assembled to form the spherical nanoparticle antigen. The invention successfully constructs eukaryotic expression plasmid and lentiviral packaging plasmid, and realizes high-efficiency expression and purification of fusion protein in HEK293T cells and ExpiCHO cells. The purified recombinant protein can be spontaneously assembled in vitro to form spherical nano particles with uniform structure and regular morphology. Transmission electron microscopy confirmed good structural integrity and high density multivalent antigen display capability. Has the advantages of high safety, strong immunogenicity, easy industrialization and the like, and provides a new technical path for the development of TGEV and related coronavirus vaccines.

Inventors

  • Cao Shinuo
  • ZHOU MO
  • ZHANG LI
  • Nie Nannan
  • CHEN CHANGCHUN
  • ZHU SHANYUAN

Assignees

  • 江苏农牧科技职业学院

Dates

Publication Date
20260512
Application Date
20260226

Claims (10)

  1. 1. A nanoparticle antigen based on ferritin carrier, wherein the nanoparticle antigen comprises a recombinant protein formed by fusion of a transmissible gastroenteritis virus spike protein receptor binding domain and ferritin subunit through a flexible connecting peptide, and the recombinant protein self-assembles to form the spherical nanoparticle antigen.
  2. 2. The nanoparticle antigen of claim 1, wherein the amino acid sequence of the transmissible gastroenteritis virus spike protein receptor binding domain is set forth in SEQ ID No. 2; And/or the amino acid sequence of the ferritin is shown as SEQ ID No. 6.
  3. 3. The nanoparticle antigen of claim 1, wherein the flexible linker peptide is an SGG flexible linker peptide; Preferably, the amino acid sequence of the SGG flexible connecting peptide is shown as SEQ ID No.3 or SEQ ID No. 5; More preferably, the N-terminal of the receptor binding domain of the spike protein of the transmissible gastroenteritis virus is sequentially connected with a Kozak sequence shown as SEQ ID No. 7, a signal peptide shown as SEQ ID No. 1 and a His tag shown as SEQ ID No. 8.
  4. 4. A nanoparticle antigen according to any one of claims 1 to 3, wherein the ferritin self-assembles from 24 identical subunits to form a highly symmetrical octahedral cage structure; Preferably, the nanoparticle antigen has an average diameter of 10-30nm.
  5. 5. A recombinant expression vector encoding the nanoparticle antigen of any one of claims 1-4, wherein the recombinant expression vector comprises a eukaryotic expression vector and a gene encoding the nanoparticle antigen of any one of claims 1-4; preferably, the eukaryotic expression vector is selected from the group consisting of pCAGGS vectors.
  6. 6. A recombinant lentiviral vector encoding the nanoparticle antigen of any one of claims 1-4, wherein the recombinant lentiviral vector comprises a lentiviral vector and a gene encoding the nanoparticle antigen of any one of claims 1-4; preferably, the lentiviral vector is selected from pLVX vectors.
  7. 7. A recombinant cell line expressing the nanoparticle antigen of any one of claims 1-4, wherein the recombinant cell line is a HEK293T cell or ExpiCHO cell transduced with the recombinant expression vector of claim 5 or the recombinant lentiviral vector of claim 6.
  8. 8. A method for preparing a nanoparticle antigen according to any one of claims 1 to 4, comprising culturing eukaryotic cells transfected or transduced with the recombinant expression vector according to claim 5 or the recombinant lentiviral vector according to claim 6, collecting the cell culture, purifying, and self-assembling the purified protein in a renaturation buffer to obtain the nanoparticle antigen; Preferably, the purification process is His tag affinity purification using HisSep Ni-NTA MagBeads magnetic bead purification system.
  9. 9. Use of a nanoparticle antigen according to any one of claims 1-4 for the preparation of a swine transmissible gastroenteritis virus vaccine.
  10. 10. Use of the nanoparticle antigen of any one of claims 1-4 for the preparation of a serodiagnostic reagent for transmissible gastroenteritis virus.

Description

Nanoparticle antigen based on ferritin carrier, recombinant expression carrier, recombinant lentiviral carrier, and preparation method and application thereof Technical Field The invention relates to the technical field of biological products and nanometer vaccines for livestock, in particular to nanoparticle antigens based on ferritin vectors, recombinant expression vectors, recombinant lentiviral vectors, and a preparation method and application thereof. Background The transmissible gastroenteritis (Transmissible gastroenteritis, TGE) is a highly contagious, acute intestinal infectious disease caused by transmissible gastroenteritis virus (Transmissible gastroenteritis virus, TGEV), mainly infects newborn and weaned piglets, clinically presents as severe watery diarrhea, vomiting, dehydration and emaciation, has a piglet mortality rate of up to 80-100%, and causes continuous and huge economic loss to the pig industry. With the popularization of intensive breeding, TGEV infection is widely spread worldwide, and often mixed with other enterocoronaviruses such as Porcine Epidemic Diarrhea Virus (PEDV) and porcine delta coronavirus (PDCoV) to infect, clinical symptoms are highly similar, and diagnosis and prevention and control difficulties are increased. Thus, the development of safe, efficient and easily industrializable TGEV vaccines has become a critical issue in the current veterinary field that needs to be addressed. TGEV is an enveloped single-stranded positive-stranded RNA virus, with a genome length of about 28.5 kb, encoding a variety of structural proteins, of which Spike protein (S protein) plays a central role in viral infection and immune response. The S protein is divided into S1 and S2 subunits, and the Receptor-binding domain (RBD) in the S1 subunit is responsible for specifically recognizing host cell receptors such as porcine aminopeptidase N (pacn), thereby mediating viral entry into cells. Thus, the RBD region is a core target for TGEV vaccine and diagnostic reagent development. The existing TGEV vaccine mainly comprises attenuated live vaccine and inactivated vaccine. Attenuated live vaccine can induce stronger immune protection, but has the risk of virulence reversion and biological safety, and inactivated vaccine has higher safety, but has weaker immunogenicity, usually needs adjuvant assistance, and is difficult to induce effective mucosal immunity. When the traditional vaccines face virus variation and mixed infection, the protection efficiency is limited, and the requirements of the modern pig industry on efficient and accurate prevention and control cannot be met. In recent years, subunit vaccines based on RBD have been attracting attention due to advantages such as clear composition, high safety, and easiness in quality control. However, RBD-only proteins are less immunogenic and are difficult to activate B cells efficiently and induce high titers of neutralizing antibodies. To enhance its efficacy, nanoparticle carrier technology was introduced into the antigen display field, where ferritin nanoparticles became an ideal antigen presentation platform due to their self-assembly into 24-mer cage structures, excellent stability and biocompatibility. The platform can realize multivalent display of antigen through gene fusion, simulate the natural conformation of virus, and thus enhance immune response. However, in the prior art, the integration of TGEV RBD into ferritin platform still has the problems of low expression efficiency, non-uniform self-assembly, poor structural stability, insufficient immunological evaluation and the like, and the industrialization application of the TGEV RBD is limited. Among the numerous protein nanoplatforms, ferritin (Ferritin) is widely used for multivalent display of viral antigens due to its ability to self-assemble naturally into 24-mer cage structures, good thermal/pH stability, mature expression purification processes, and compatibility in a variety of expression systems. The target antigen is connected with the ferritin subunit in a gene fusion mode, so that high-density and uniform display of the antigen can be realized in a self-assembly process, and compared with a chemical coupling method, the method has the advantages of better conformation retention, controllable loading proportion, high batch-to-batch consistency and the like. However, successful integration of the RBD region of TGEV S protein into ferritin nanoplatforms still faces a number of key technical challenges including the impact of fusion site and linker peptide design on correct folding of RBD and exposure of key epitopes, selection of expression system for regulation of potential glycosylation modification, soluble expression of fusion protein, self-assembly efficiency and particle uniformity control, physicochemical stability of nanoparticles during storage and use. Thus, there is a need for a novel ferritin-based TGEV RBD nanoparticle antigen construction strategy to overco