Antigenic epitope simulative peptide of classical swine fever virus (CSFV) E2 protein, and preparation method and application thereof

A swine fever virus and peptide-mimicking technology, applied in viral peptides, biochemical equipment and methods, viruses, etc., can solve the problems of complex expression of E2 protein, and achieve process safety, short production cycle, and high sensitivity. Effect

Inactive Publication Date: 2016-12-07
WUHAN KEQIAN BIOLOGY CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the process of expressing E2 protein is still relatively complicated, and the ex...

Method used

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  • Antigenic epitope simulative peptide of classical swine fever virus (CSFV) E2 protein, and preparation method and application thereof
  • Antigenic epitope simulative peptide of classical swine fever virus (CSFV) E2 protein, and preparation method and application thereof
  • Antigenic epitope simulative peptide of classical swine fever virus (CSFV) E2 protein, and preparation method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0032] A preparation method of an antigenic epitope mimetic peptide of swine fever virus E2 protein, the steps are:

[0033] A. Purification and identification of E2 monoclonal antibody:

[0034] The 4A7 monoclonal antibody was purified by caprylic acid-ammonium sulfate method (Jin Boquan, Cellular and Molecular Immunology Technology. Xi'an: Fourth Military Medical University Press, 2002) (hybridoma 4A7 preservation number: CCTCC NO.C2014229, preserved in Wuhan in 2014 University China Type Culture Collection Center), and the purification effect was identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The specific steps were slightly changed, and the method was as follows: add For four parts of 0.06 mol / L (pH=5.0) acetic acid buffer solution, adjust the pH value to 4.5 with 0.1 mol / L hydrochloric acid. At room temperature (15° C. to 25° C., the same below), 3.3% octanoic acid by volume of the original ascitic fluid was slowly added, and stirred ...

Embodiment 2

[0048] A kind of analog peptide of classical swine fever virus E2 protein is used in the preparation treatment or prevention swine fever ELSIA antibody drug detection (kit), and its steps are:

[0049] A. Antigen-coated plate preparation in the swine fever ELISA antibody detection kit:

[0050] According to the 12-peptide sequence shown in SEQ ID NO: 1 of the present invention and conventional chemical synthesis methods in the art, a short peptide with a purity greater than 95% is prepared, and this short peptide is the detection antigen. Dissolve the antigen with carbonate buffer and dilute it to 2 μg / mL, then add 100 μL per well into a 96-well ELISA plate, and place it overnight at 4°C to allow the antigen to adsorb on the ELISA plate. The next day, the liquid in the wells was discarded, and 150 μL of phosphate buffered saline (containing 0.5% BSA) was added to each well, placed in a 37° C. incubator for 2 hours, and the liquid in the wells was discarded. Pat dry.

[0051]...

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Abstract

The invention discloses an antigenic epitope simulative peptide of classical swine fever virus (CSFV) E2 protein, and a preparation method and an application thereof. The preparation method includes the steps of: A) immunizing a BALB/C mouse with the CSFV E2 protein that is expressed by baculovirus to prepare monoclonal antibodies aiming to the E2 protein, and screening and identifying the monoclonal antibodies to obtain a monoclonal antibody 4A7 that has a blocking effect; B) purifying the monoclonal antibody 4A7 and coating an ELISA plate with the monoclonal antibody, and performing screening with a phage 12 peptide library, wherein the screening stress is increased round by round, thereby finally obtaining positive clone having high affinity; and C) performing DNA sequencing and sequence comparison analysis, which prove that the positive clone product is not completely consistent with the sequence of the original CSFV E2 protein; and D) performing chemical synthesis to the twelve amino acid sequences that are screened before according to a peptide synthetic technology. The invention also provides an application of the simulative peptide of the CSFV E2 protein in preparation of an ELISA antibody medicine detection kit for treating or preventing swine fever. The synthesized short peptide is used for detection of the swine fever antibody as a coating antigen. The simulative peptide has low cost and high purity in large-scale production.

Description

technical field [0001] The invention belongs to the field of biotechnology. More specifically, it relates to an antigenic epitope mimic peptide of the classical swine fever virus E2 protein, and also relates to a preparation method of the antigenic epitope mimic peptide of the classical swine fever virus E2 protein, and also relates to an antigenic epitope of the classical swine fever virus E2 protein Use of mimetic peptides. Background technique [0002] Classical swine fever virus (CSFV) is a member of the genus Pestivirus in the Flaviviridae family. The swine fever caused by CSFV is a severe infectious disease that is currently harmful to the pig industry in my country. Serological studies on the disease Monitoring helps to control the occurrence of swine fever. The earliest detection of swine fever was the indirect hemagglutination test, which required culturing the virus, preparing virus antigens, and sheep red blood cells for the detection, which was a complicated and ...

Claims

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Application Information

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IPC IPC(8): C07K14/18G01N33/68G01N33/569
CPCC07K14/005C12N2770/24322G01N33/56983G01N33/68G01N2333/183
Inventor 但汉并王艳伟董晓辉徐高原金梅林陈焕春
Owner WUHAN KEQIAN BIOLOGY CO LTD
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