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B cell antigen epitope polypeptide of encephalitis B virus E protein and uses thereof

A technology of Japanese encephalitis virus and antigenic epitope, applied in the field of molecular immunology, can solve problems such as weak heat resistance

Inactive Publication Date: 2008-12-31
HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The virus has weak resistance to heat and can be inactivated at 56°C for 30 minutes or 100°C for 2 minutes, but it is highly resistant to low temperature and drying

Method used

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  • B cell antigen epitope polypeptide of encephalitis B virus E protein and uses thereof
  • B cell antigen epitope polypeptide of encephalitis B virus E protein and uses thereof
  • B cell antigen epitope polypeptide of encephalitis B virus E protein and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1 Overlapping (over lapping) polypeptide fusion expression series of JEV E protein antigen polypeptide fragments and indirect ELISA screening of JEVE protein B cell antigen epitopes

[0023] According to the amino acid sequence of JEV E protein, design a series of 36 polypeptides in total, these polypeptides overlap each other, and cover the 1-296 amino acid residue region of JEV E protein, which contains E protein domain I (Domain I) and domain II (Domain II). According to the amino acid sequence of each polypeptide, a pair of DNA strands are designed and synthesized, and the DNA strands are inserted into the expression vector pGEX-6p-1 and GST for fusion expression. Indirect ELISA analysis was performed on the fusion expressed series of recombinant proteins and anti-JEV serum to analyze the epitope of JEV E protein. The specific process is as follows:

[0024] Design of polypeptide sequence→synthesis of DNA chain encoding polypeptide sequence→construction of...

Embodiment 2

[0028] Example 2 Synthesis of Japanese encephalitis virus JEV E protein B cell antigen epitope

[0029] According to the amino acid sequence of the B cell epitope of JEV E protein, the solid-phase peptide synthesis technology is applied, and the automatic peptide synthesizer or artificial peptide synthesis method is adopted. The solid-phase resin adopts Fmoc-protected amino acid Wang resin or other resins. After the resin is swollen with DMF and piperidine glue Fmoc-protected, according to the sequence of amino acids, Fmoc-protected amino acids are added, and the acylation reaction is carried out in the presence of HBTU. After the acylation reaction is completed, after washing, the second Fmoc-amino acid is added for acylation reaction and washed. In such a cycle, a complete polypeptide chain is synthesized sequentially from the C-terminus of the polypeptide sequence to the N-terminus. After the synthesis is completed, select an appropriate reagent according to the amino acid...

Embodiment 3

[0031] Example 3 Chemical Modification of Japanese Encephalitis Virus JEV E Protein B Cell Antigen Epitope

[0032] The polypeptide fragment synthesized according to the method of Example 3 has a natural amino modification at its N-terminus and a natural carboxyl modification at its C-terminus.

[0033] N-terminal acetylation modification method: Synthesize the polypeptide according to the above introduction until the coupling of the last Fmoc protected amino acid is completed, and remove the N-terminal Fmoc protecting group, and then perform the following operations. Dissolve 150 μl acetic anhydride and 20 μL EIPEA in 4.8 ml DMF, mix well and cool in an ice bath. Then add the above mixed solution into the peptide synthesis reaction tube and react for 5 minutes. Then wash with 5ml DMF and dichloromethane (DCM) twice, each time for 5min. After drying with nitrogen gas, the removal, cleavage, purification and identification of side chain protecting groups were carried out acco...

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Abstract

The invention discloses a polypeptides epitope in Japanese encephalitis virus E protein neutralizing B cell, also discloses the application of the polypeptides epitope in the prevention, treatment and diagnosis of Japanese encephalitis, belonging to the field of molecular immunology. The amino acid sequence of the polypeptides epitope disclosed in the invention is selected from any of the amino acid shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 5. After being used as immunogen or vaccine immune animal organisms, the JEV E protein neutralizing B cell polypeptides epitope of the invention can produce neutralizing antibody against Japanese encephalitis virus, and can neutralize the Japanese encephalitis virus in vivo or in vitro so as to prevent the virus infecting animal organisms. The polypeptides epitope of the invention or the conjugate of the polypeptides epitope can be used as the reagents for detecting the anti-Japanese encephalitis virus antibodies or anti-Japanese encephalitis virus polypeptide antibodies.

Description

technical field [0001] The present invention relates to antigenic epitope polypeptides, especially to Japanese encephalitis virus E protein neutralizing B cell antigenic epitope polypeptides, and the invention also relates to the application of the antigenic epitope polypeptides in preventing and diagnosing Japanese encephalitis virus, belonging to field of molecular immunology. Background technique [0002] Japanese encephalitis Japanese encephalitis is an important mosquito-borne zoonotic disease caused by Japanese encephalitis virus (JEV). The virus was first isolated from the brain tissue of patients in Japan in 1953, so it is called Japanese encephalitis virus, and the disease caused by it is called Japanese encephalitis B in Japan. In my country, the virus was first isolated in 1940. In order to distinguish it from Japanese encephalitis, it was named Japanese encephalitis, or Japanese encephalitis for short. Worldwide, there are 30,000-50,000 JE cases every year, abo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/08C12N15/40A61K39/12A61P31/14G01N33/569G01N33/53
CPCY02A50/30
Inventor 华荣虹童光志步志高
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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