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African swine fever virus p54 protein epitope peptide and application thereof

An African swine fever virus and antigenic epitope technology, applied in the field of genetic engineering, to avoid non-specific reactions, high antigen purity, and simplify screening steps

Inactive Publication Date: 2021-04-30
ZHENGZHOU UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are few reports on the N-terminus of p54 protein

Method used

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  • African swine fever virus p54 protein epitope peptide and application thereof
  • African swine fever virus p54 protein epitope peptide and application thereof
  • African swine fever virus p54 protein epitope peptide and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Preparation and identification of embodiment 1 anti-ASFV p54 protein monoclonal antibody

[0040] 1. Synthesis of complete antigen of N-terminal polypeptide of ASFV p54 protein

[0041] (1) Taking the p54 protein sequence of the ASFV strain SY18 in the first ASF outbreak reported in China as a reference, the N-terminal region of the p54 protein can be simulated by solid-phase synthetic peptide technology ( 1 MDSEFFQPVYPRHYGECLSPVTTPSFFST 29 ) polypeptide, named p54N 29 .

[0042] (2) Polypeptide-coupled carrier protein BSA (bovine serum albumin) or ovalbumin (OVA): the polypeptide p54N 29 Coupling with BSA and OVA respectively, and obtaining the immunogen BSA-p54N after dialysis 29 and enveloped original OVA-p54N 29 .

[0043] 2. Immunization of BALB / c mice

[0044] Immunogen BSA-p54N 29 Freund's complete adjuvant was added to it, and after emulsification, 2 female BALB / c mice aged 4 to 8 weeks were immunized by multi-point subcutaneous injection on the back, wi...

Embodiment 2

[0053] Example 2 Identification of key amino acid sequences of antigenic epitopes

[0054] 1. Design of overlapping peptides

[0055] The polypeptide scanning method was used to design and synthesize three polypeptides, covering the entire NTD, and adjacent polypeptides overlapped by 7 amino acids. The amino acid sequence of the polypeptide segment is shown in Table 1.

[0056] Table 1

[0057] name position amino acid sequence P1 1-15aa MDSEFFQPVYPRHYG P2 9-23aa VYPRHYGECLSPVTT P3 18-29aa LSPVTTPSFFST

[0058] 2. Indirect ELISA screening for short peptides that can bind to antibodies

[0059] Dilute the polypeptide with CBS buffer, coat the microtiter plate, 37°C, 2h; wash the plate 2-3 times with PBST; block with 5% skimmed milk, 37°C, 2h; use the monoclonal antibody obtained in Example 1 of the present invention as The primary antibody was used for indirect ELISA, and incubated at 37°C for 1 hour; after washing the plate, add HR...

Embodiment 3

[0068]Example 3 Application of Epitope Peptide in the Preparation of African Swine Fever Virus Specific Antibody

[0069] In the specific preparation process of the antibody, the fusion protein obtained by coupling the epitope peptide provided by the present invention with the carrier protein is used as an immunogen to immunize animals, so as to obtain the specific antibody of the African swine fever virus p54 protein. The carrier protein is BSA (bovine serum albumin) or ovalbumin (OVA).

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Abstract

The invention relates to an African swine fever virus p54 protein epitope peptide and application thereof. According to the invention, a new African swine fever virus p54 protein epitope is selected out through screening, the identification of the p54 protein N-terminal B cell epitope is completed, and the antigen epitope map of the p54 protein is improved. The selected antigen epitope (2DSEFFQPV9) is discovered for the first time, is highly conservative among various strains of African swine fever, and has important significance for serological detection of ASFV. The antigen epitope peptide and the antigen epitope peptide derived from the antigen epitope peptide and having the same function can be used for preparing a specific antibody of the African swine fever virus p54 protein, preparing a reagent for detecting or identifying the ASFVp54 protein, and laying a foundation for researching and developing vaccines for preventing and / or treating African swine fever virus infection.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to an African swine fever virus p54 protein antigenic epitope peptide and an application thereof. Background technique [0002] African swine fever virus (ASFV), a large and complex DNA virus, is the causative agent of African swine fever (ASF). ASF is a highly contagious hemorrhagic disease in domestic and wild boars that has caused huge economic losses in recent years. Clinical signs and mortality due to the different virulence of the virus vary in different hosts. In domestic and wild boars, infection by highly virulent isolates results in an acute form of ASF with morbidity and mortality of up to 100%. Viruses of moderate or low virulence produce weaker clinical signs in their subacute and chronic forms, but survive longer. African wild pigs (warthogs, bush pigs, and forest pigs) can be infected without any clinical symptoms and thus serve as reservoir...

Claims

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Application Information

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IPC IPC(8): C07K14/01C07K16/08G01N33/577G01N33/569
CPCC07K14/005C07K16/081G01N33/577G01N33/56983C12N2710/12022G01N2333/01G01N2469/20
Inventor 王爱萍张改平陈玉梅蒋敏刘燕凯周景明王彦伟冯景
Owner ZHENGZHOU UNIV
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