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CN-121554612-B - Getavirus genetic engineering subunit vaccine and preparation method and application thereof

CN121554612BCN 121554612 BCN121554612 BCN 121554612BCN-121554612-B

Abstract

The invention discloses a genetic engineering subunit vaccine of a cover tower virus, and a preparation method and application thereof, and belongs to the technical field of vaccine preparation. The invention adopts a eukaryotic expression system to obtain the recombinant protein of the Katta virus P6E, and further loads the recombinant protein onto the surface of a nanoparticle skeleton to form recombinant nanoparticles. The recombinant protein and the recombinant nano-particles are respectively compounded with an adjuvant to prepare the genetic engineering subunit vaccine. After two immunizations, the vaccines with two components can obviously induce the host to generate strong humoral immunity and cellular immunity reaction, thereby providing effective protection for GETV infection. The technical scheme of the invention provides important technical basis and application reference for research and development and optimization of the Gatta virus vaccine.

Inventors

  • SU SHUO
  • JIANG ZHIWEN
  • QIN YING
  • XING GANG
  • WANG NINGNING
  • LI DONGYAN

Assignees

  • 南京农业大学三亚研究院
  • 因卓生物技术(上海)有限公司

Dates

Publication Date
20260508
Application Date
20260126

Claims (13)

  1. 1. A recombinant protein of a cover-tower virus P6E, wherein the recombinant protein of the cover-tower virus P6E is at least one of the following (a 1) - (a 2): (a1) A protein with an amino acid sequence shown as SEQ ID NO. 2; (a2) And (b) ligating protein tags at the N-terminal and/or C-terminal of the protein of (a 1) to obtain a fusion protein with the same function.
  2. 2. A recombinant nanoparticle loaded with the recombinant P6E protein of the cover virus of claim 1, wherein the recombinant nanoparticle is obtained by displaying the recombinant P6E protein on the surface of the nanoparticle through a SpyTag003/SpyCatcher003 covalent linkage system.
  3. 3. The recombinant nanoparticle according to claim 2, wherein the nanoparticle is a SpyCatcher003-mi3 nanoparticle or a SpyCatcher 003-ferritin nanoparticle, and the amino acid sequence of the SpyCatcher003-mi3 nanoparticle is shown as SEQ ID NO. 4.
  4. 4. A genetic engineering vaccine of a kappaphycus virus, comprising a pharmaceutically acceptable adjuvant and an antigenic component selected from the group consisting of (b 1), (b 2) and (b 3): (b1) The cover virus P6E recombinant protein of claim 1; (b2) Recombinant nanoparticle loaded with a recombinant protein of the gecko virus P6E according to claim 2; (b3) A composition comprising (b 1) the recombinant protein of the gecko virus P6E and (b 2) the recombinant nanoparticle loaded with the recombinant protein of the gecko virus P6E.
  5. 5. The genetically engineered vaccine of claim 4, wherein: when the antigen component is the combination (b 1), the content of the recombinant protein of the P6E of the cover tower virus is 50-200 mug/mL; when the antigen component is the combination (b 2), the content of the recombinant nanoparticle loaded with the recombinant protein of the virus P6E of the gecko is 100-200 mug/mL; when the antigen component is the combination (b 3), the content of the recombinant protein of the virus P6E of the virus of the gecko is 50-200 mug/mL, and the content of the recombinant nanoparticle loaded with the recombinant protein of the virus P6E of the gecko is 100-200 mug/mL.
  6. 6. The genetically engineered vaccine of claim 4, wherein the adjuvant is an oil-in-water adjuvant and the volume ratio of the adjuvant to the antigen component is 1:4.
  7. 7. A nucleic acid molecule encoding the recombinant protein of the cover tower virus P6E of claim 1, wherein the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID No. 1.
  8. 8. An expression cassette, recombinant vector or host cell comprising the nucleic acid molecule of claim 7.
  9. 9. A method for preparing the recombinant nanoparticle according to claim 2, which is characterized by comprising the step of covalently coupling the purified recombinant protein of the virus P6E of the Kappaphycus alvarezier with the purified nanoparticle under in vitro conditions by a SpyTag003/SpyCatche 003 system to obtain the recombinant nanoparticle carrying the recombinant protein of the virus P6E of the Kappaphycus alvarezier.
  10. 10. Use of the recombinant protein of the kappaphycus alvarezii P6E of claim 1 or the recombinant nanoparticle of claim 2 for the preparation of a medicament for the prevention of kappaphycus alvarezii infection.
  11. 11. Use of the genetic engineering vaccine of katavirus of claim 4 in the preparation of a medicament for preventing katavirus infection.
  12. 12. Use of the nucleic acid molecule of claim 7 for the preparation of a genetic engineering vaccine against a galta virus.
  13. 13. Use of the expression cassette, recombinant vector or host cell of claim 8 in the preparation of a genetic engineering vaccine against a katavirus.

Description

Getavirus genetic engineering subunit vaccine and preparation method and application thereof Technical Field The invention belongs to the technical field of medicines, and particularly relates to a genetic engineering subunit vaccine of a cover tower virus, and a preparation method and application thereof. Background The Gatta virus (Getah virus, GETV) belongs to the genus A and is an RNA virus mainly transmitted by mosquito vectors, and has a rising trend in recent years in pig farms in China, and can cause symptoms such as fever, diarrhea of piglets, abortion of sows and the like, and cause significant losses to health of swineries and the economy of breeding industries. As GETV has a continuously expanding host range, prevention and control of the virus epidemic has become an important requirement for the aquaculture industry in China. However, the current prevention and control measures against the katavirus still mainly depend on biosafety management, and no commercial katavirus vaccine is put on the market, which severely limits the active immune protection capability of the pig herd. In recent years, genetic engineering subunit vaccines have been attracting attention because of their clear composition, high safety, virtually no host hybrid proteins, avoidance of potential biosafety risks associated with inactivation or attenuation processes, and ability to accurately present protective antigens through molecular design. However, traditional subunit vaccines are often poorly immunogenic and there is a need to enhance their immune effects by structural optimization or the use of novel delivery vehicles. The subunit vaccine based on the protein nano particles has the outstanding advantages that the protein nano particles have a highly regular and stable three-dimensional structure, can self-assemble to form uniform particles, and present multi-copy antigen epitopes on the surfaces of the protein nano particles in a highly repeated and high-density mode, so that the crosslinking effect of B cell receptors can be remarkably enhanced, the powerful humoral immune response can be promoted, dendritic cells can be effectively activated, the cellular immune response can be enhanced, and the immunogenicity of the subunit vaccine can be comprehensively improved. Thus, the design of nanoparticle presentation strategies based on optimized subunit antigens and binding proteins has become an important trend in current vaccine development. However, few studies have been reported to date to apply nanoparticle technology to the use of the katavirus subunit vaccine, and in particular, recombinant nanoparticle vaccines based on accurate antigen design and controllable assembly strategies are lacking, and only in grant No. CN117964722B, there is a report to fusion express GETV E protein and ferritin in a baculovirus system to form self-assembled nanoparticles. Therefore, on the basis of the advantages of the subunit vaccine and the protein nanoparticle carrier, a GETV gene engineering subunit platform based on structural optimization is constructed, and the characteristics of the subunit vaccine and the protein nanoparticle carrier are cooperatively integrated, so that the immunogenicity and the protection effect of GETV antigen can be further enhanced, a safer and more efficient novel vaccine technical route can be provided for the prevention and control of porcine circus-laba virus, and the novel vaccine has important scientific significance and application value. Disclosure of Invention In order to overcome the defects of the prior art in the aspect of control of the Gatta virus, the invention aims to provide a Gatta virus genetic engineering subunit vaccine, and a preparation method and application thereof. The vaccine adopts the recombinant protein of the Gattavirus P6E prepared by a mammalian cell expression system as an antigen, and the antigen is fixed on the surface of mi3 nano-particles in a covalent coupling mode, so that the recombinant nano-particles capable of displaying multiple copies of the P6E antigen are formed. And (3) respectively compounding the obtained recombinant protein of the P6E of the Gaota virus and the P6E nano particles with an adjuvant to prepare the Gaota virus subunit vaccine and the Gaota virus nano particle vaccine. Both vaccines can effectively induce the organism to generate humoral immunity and cellular immunity response aiming at GETV, thereby realizing effective prevention of the infection of the Gatta virus. The above objects of the present invention are achieved by the following technical solutions: In a first aspect, the invention provides a recombinant protein of a cover tower virus P6E, wherein the recombinant protein of the cover tower virus P6E is formed by connecting a cover tower virus P62 protein and an E1 protein after removing a transmembrane domain through a Linker sequence, and the recombinant protein of the P6E is at least one of the following proteins (a 1) to (a 2)