B-cell epitope of VP(viral protein)3 of DHAV (duck hepatitis A virus)-1 as well as identification method and application of B-cell epitope

A technology of duck hepatitis A virus and its identification method, applied in the field of B cell epitope of VP3 protein of type 1 duck hepatitis A virus and its identification

Active Publication Date: 2015-12-30
SICHUAN AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This patented inventor describes an improved way that allows researchers to test if there are specific types of viruses called Hepatoid Nijhoff disease (also known as HN) caused by bacteria like Yellow Fever with their own unique DNA structure. These tests help identify those who have been infected but cannot yet develop symptoms such as chickenpox fever without causing any harm from other diseases associated with this condition.

Problems solved by technology

This patented describes various techniques used during studying the role played by different types of Hepatoid Virus Type C particles called HCV. These tests involve testing specific parts of these particles with antigonucletoxoids attached onto them. By measuring changes in this part, we aim to identify important epimicrobium components associated with each particle involved in the process.

Method used

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  • B-cell epitope of VP(viral protein)3 of DHAV (duck hepatitis A virus)-1 as well as identification method and application of B-cell epitope
  • B-cell epitope of VP(viral protein)3 of DHAV (duck hepatitis A virus)-1 as well as identification method and application of B-cell epitope
  • B-cell epitope of VP(viral protein)3 of DHAV (duck hepatitis A virus)-1 as well as identification method and application of B-cell epitope

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Embodiment 1

[0054] A B cell epitope of type 1 duck hepatitis A virus VP3 protein, the amino acid sequence of which is shown in SEQ ID NO:4 or SEQ ID NO:5.

[0055] The use of the B cell epitope of the VP3 protein of type 1 duck hepatitis A virus can be applied to a kit for detecting the infection of type 1 duck hepatitis A virus or the level of vaccine antibodies.

[0056] Further, the B cell epitope of the VP3 protein of duck hepatitis A virus type 1 is used as an antigen in the kit components for detecting the infection of duck hepatitis A virus type 1 or the antibody level of the vaccine.

[0057] A use of the B cell epitope of the VP3 protein of the type 1 duck hepatitis A virus, wherein the B cell epitope of the VP3 protein of the type 1 duck hepatitis A virus is used in the preparation of a vaccine for preventing the type 1 duck hepatitis A virus.

[0058] A kind of identification method of type 1 duck hepatitis A virus VP3 protein B cell epitope, comprises the following steps:

[...

Embodiment 2

[0105] The expression analysis of embodiment 2 recombinant protein and the optimization of expression condition

[0106] experiment method:

[0107] 1. Expression analysis of recombinant protein

[0108] Escherichia coli BL21(DE3) positive bacteria transformed with the recombinant expression plasmid pGEX-VP3 were inoculated into LB(Amp+) liquid medium, and activated overnight at 37°C. Take the activated bacterial solution and inoculate LB (Amp+) liquid medium at a volume ratio of 1:100, and expand the culture at 37°C for 3 to 4 hours until the A600 is about 0.6. Add a final volume of 0.4mmol / LIPTG, and induce expression at 37°C for 4 hours. After the end, the bacterial solution was centrifuged at 8000r / min for 5min to collect the bacterial precipitate, and 20mmol / LTris- Suspend in Cl (pH 8.0) solution, break up the bacterial cells in an ice-water bath with ultrasonic waves for 60 s, with an interval of 60 s, and repeat until the bacterial liquid is clear and transparent. Th...

Embodiment 3

[0119] Embodiment 3 A kind of identification experiment result of type 1 duck hepatitis A virus VP3 protein B cell epitope

[0120] 1. Preparation and potency determination of VP3 recombinant protein polyclonal antibody

[0121] Use the purified VP3 recombinant protein to immunize male rabbits to prepare antiserum. The specific steps are: firstly, use 0.5 mg of VP3 recombinant protein solution and an equal volume of Freund's complete adjuvant to emulsify into water-in-oil state, and then apply multiple points intradermally and subcutaneously on the back of the neck. Inject rabbits; 11 days after the first immunization, use 1 mg of VP3 recombinant protein solution and an equal volume of Freund's incomplete adjuvant to emulsify into a water-in-oil state, and inject rabbits subcutaneously at multiple points on the back of the neck for the second immunization; 22 days after the first immunization, use 1 mg After the VP3 recombinant protein was emulsified into a water-in-oil state ...

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Abstract

The invention belongs to the technical field of bioengineering and particularly relates to a B-cell epitope of VP(viral protein)3 of DHAV (duck hepatitis A virus)-1 as well as an identification method and an application of the B-cell epitope. The identification method of the B-cell epitope comprises the following steps: obtaining a target fragment of the VP3; constructing recombinant expression plasmid pG-EX-VP3, preparing VP3 recombinant protein, preparing a polyclonal antibody of the VP3 recombinant protein, determining the median lethal dose ELD50 of the DHAV-1 on chicken embryos or duck embryos, determining the neutralizing titer of the polyclonal antibody of the VP3 recombinant protein and identifying the B-cell epitope on the VP3. The linear B-cell epitope of the VP3 is predicted with comprehensive application of bioinformatics software, epitope peptide is artificially synthesized, identification is performed on the synthetic epitope peptide in combination with an indirect ELISA (enzyme linked immunosorbent assay) method, and reference can be provided for deep development of follow-up DHAV-1 immunological research, construction of novel diagnostic preparations and development of polypeptide vaccines.

Description

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Claims

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Application Information

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Owner SICHUAN AGRI UNIV
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