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CN-120647731-B - Recombinant 2.1d subtype swine fever E2 protein, subunit vaccine and application thereof

CN120647731BCN 120647731 BCN120647731 BCN 120647731BCN-120647731-B

Abstract

The invention discloses a recombinant 2.1d subtype swine fever E2 protein, subunit vaccine and application thereof, and belongs to the technical field of genetic engineering. The invention removes 344-375 amino acids of 2.1d subtype swine fever E2 protein, retains 1-343 amino acids, can retain the most important epitope with protective effect of 2.1d subtype swine fever E2 protein, basically does not influence the space structure of recombinant 2.1d subtype swine fever E2 protein, can retain the immunogenicity to the greatest extent, can efficiently express the recombinant 2.1d subtype swine fever E2 protein in a prokaryotic expression system, and further can remarkably improve the soluble expression level of the recombinant 2.1d subtype swine fever E2 protein through coexpression molecular chaperones, and has better protective effect on infection of 2.1d subtype swine fever virus after the recombinant 2.1d subtype swine fever E2 protein is prepared into subunit vaccine.

Inventors

  • LI GUOPAN
  • RONG JUN
  • SUN HAOYU
  • RONG XUENING
  • KUANG HONGYAN

Assignees

  • 长江大学
  • 荆州市长新生物技术有限公司

Dates

Publication Date
20260508
Application Date
20250611

Claims (6)

  1. 1. A method for preparing recombinant 2.1d subtype swine fever E2 protein, which is characterized by comprising the following steps: Culturing the recombinant cells, and obtaining a culture through induced expression; isolating recombinant subtype 2.1d classical swine fever E2 protein from said culture; The recombinant cell comprises a nucleic acid molecule encoding the recombinant 2.1d subtype swine fever E2 protein or a recombinant vector comprising a nucleic acid molecule encoding the recombinant 2.1d subtype swine fever E2 protein and at least one of pTf, pG-KJE vectors; the recombinant 2.1d subtype swine fever E2 protein is selected from any one of the following: a1 The amino acid sequence is shown as SEQ ID NO. 3; A4 Amino acid sequences obtained by ligating a tag or a signal peptide to the N-terminal and/or C-terminal of the amino acid sequence defined in A1).
  2. 2. The method of claim 1, wherein the nucleic acid molecule is selected from any one of the following: B1 A nucleic acid molecule with a nucleotide sequence shown as SEQ ID NO. 4.
  3. 3. The recombinant 2.1d subtype swine fever E2 protein subunit vaccine is characterized by comprising the recombinant 2.1d subtype swine fever E2 protein prepared by the preparation method of claim 1 and an adjuvant.
  4. 4. A method of preparing a recombinant subtype 2.1d swine fever E2 protein subunit vaccine of claim 3, comprising the steps of: Adding an inactivating agent into the recombinant 2.1d subtype swine fever E2 protein for inactivation treatment to obtain an inactivated recombinant 2.1d subtype swine fever E2 protein; Mixing the inactivated recombinant 2.1d subtype swine fever E2 protein with an adjuvant, and emulsifying to obtain the recombinant 2.1d subtype swine fever E2 protein subunit vaccine.
  5. 5. The method for preparing the recombinant 2.1d subtype swine fever E2 protein subunit vaccine of claim 4, wherein the inactivating agent comprises formaldehyde, the final concentration of the added inactivating agent is 0.05-0.15%, and the inactivating treatment comprises inactivating for 60-80 hours at a temperature of 0-4 ℃; the content of the recombinant 2.1d subtype swine fever E2 protein in the recombinant 2.1d subtype swine fever E2 protein subunit vaccine is 200-400 mug/mL.
  6. 6. Use of a recombinant 2.1d subtype swine fever E2 protein prepared by a preparation method according to any one of claims 1-2, a recombinant 2.1d subtype swine fever E2 protein subunit vaccine according to claim 3, a recombinant 2.1d subtype swine fever E2 protein subunit vaccine prepared by a preparation method according to any one of claims 4-5 for the preparation of a medicament for the prevention and/or treatment of infection by a 2.1d subtype swine fever virus.

Description

Recombinant 2.1d subtype swine fever E2 protein, subunit vaccine and application thereof Technical Field The invention belongs to the technical field of genetic engineering, and particularly relates to a recombinant 2.1d subtype swine fever E2 protein, subunit vaccine and application thereof. Background Swine Fever (CSF) is a contagious disease characterized by immune suppression, high fever, multiple organ hemorrhage, and high morbidity and mortality due to the classical swine fever virus (CSFV, CLASSICAL SWINE FEVER virus), which is a group of animal epidemic diseases in our country. Although large-scale swine fever epidemic situation does not occur in China in recent years, the problems of chronic infection, subclinical infection and the like are continuously increased due to the fact that the conditions of clinical symptoms, infection, epidemic forms and the like of swine fever viruses are more complicated, and the difficulties of diagnosis, prevention and control of swine fever are greatly increased due to the fact that the conditions of mixed infection of various diseases and the like are continuously increased. The E2 protein is envelope glycoprotein with the size of about 55kDa, is the most important protective antigen protein of CSFV and is also the main antigen for inducing the body to generate neutralizing antibodies. The E2 protein has low conservation and large variation coefficient, and has 4 different antigen regions (A, B, C, D), wherein the A region is divided into A1, A2 and A3 sub-regions, the A1 and A2 are relatively conserved and difficult to vary, the A3 and B, C, D are relatively easy to vary, and the antigen epitopes of the A1 and B, C regions are important factors for generating protective immune response of organisms. Based on the E2 gene sequence CSFV is divided into 3 genotypes (1, 2, 3) and 11 genotypes (1.1, 1.2, 1.3, 1.4, 2.1, 2.2, 2.3, 3.1, 3.2, 3.3, 3.4), wherein the 2.1 subtype can be further divided into 2.1a, 2.1b, 2.1c, 2.1d. China reported CSFV of subtype 2.1d for the first time in 2015 and became one of the main epidemic strains in China. The subtype 2.1d E2 protein has common molecular characteristics on amino acids at five positions R 32、S35、W183、K207、K306, and has low homology with the standard virulent strain Shimen strain and attenuated vaccine strain C strain of China. This difference in antigenicity allows CSFV of subtype 2.1d to escape the immunoprotection of the C-strain vaccine, which is an important cause of frequent outbreaks of swine fever of subtype 2.1d in recent years. Vaccines are an important means of control of CSFV. The HCLV (swine fever lapinized virus vaccine) developed in China is one of the most widely applied vaccine strains at present, but the vaccine cannot well distinguish wild virus infection after immunization, and the problem of insufficient cross protection of 2.1d subtype CSFV exists. The recombinant E2 protein expressed by utilizing the genetic engineering has the advantages of good antigenicity, high expression quantity, easy purification, large-scale production and capability of carrying out serotype marking, and the most commonly used insect cell-baculovirus expression system has the defects of high production cost, strict production technical requirements and long production period. The E2 antigen is produced by using an escherichia coli expression system, and is particularly suitable for commercial production and utilization aiming at the 2.1d subtype CSFV E2 protein which is mainly popular at present, but the efficient and soluble expression of the 2.1d subtype CSFV E2 by escherichia coli and the correct conformation and immunogenicity are one of the difficulties in the whole industry at present. Disclosure of Invention The invention aims to provide a recombinant 2.1d subtype swine fever E2 protein, subunit vaccine and application thereof. The method is used for solving the problems of poor solubility, low expression quantity and poor immunogenicity of the E2 antigen protein expressed in an escherichia coli expression system in the preparation process of the existing E2 protein genetic engineering subunit vaccine. In a first aspect, the invention provides a recombinant 2.1d subtype swine fever E2 protein, wherein the recombinant 2.1d subtype swine fever E2 protein is selected from any one of A1) an amino acid sequence shown as SEQ ID NO. 3, A2) an amino acid sequence with substitution, deletion or addition of one or more amino acids compared with the amino acid sequence defined by A1), A3) an amino acid sequence with more than 80% sequence identity compared with the amino acid sequence defined by A1) or A2), and A4) an amino acid sequence obtained by connecting a tag or a signal peptide to the N-terminal and/or the C-terminal of the amino acid sequence defined by A1) or A2) or A3). The recombinant 2.1d subtype E2 protein provided by the invention can be a natural, recombinant or synthetic active polypeptide, and the active polype