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A kind of B cell epitope of type 1 duck hepatitis A virus vp3 protein and its identification method and application

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

Active Publication Date: 2019-03-08
SICHUAN AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, the detection and prevention methods for duck hepatitis A virus type 1 still need to be improved and perfected. The current detection methods are mainly traditional serological detection based on whole virus or molecular biological detection based on viral genome sequence. Therefore, it is urgent to develop new and effective methods. Diagnostic reagents

Method used

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  • A kind of B cell epitope of type 1 duck hepatitis A virus vp3 protein and its identification method and application
  • A kind of B cell epitope of type 1 duck hepatitis A virus vp3 protein and its identification method and application
  • A kind of B cell epitope of type 1 duck hepatitis A virus vp3 protein and its identification method and application

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Experimental program
Comparison scheme
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Embodiment 1

[0056] 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.

[0057] 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.

[0058] 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.

[0059] 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.

[0060] A kind of identification method of type 1 duck hepatitis A virus VP3 protein B cell epitope, comprises the following steps:

[...

Embodiment 2

[0107] The expression analysis of embodiment 2 recombinant protein and the optimization of expression condition

[0108] experiment method:

[0109] 1. Expression analysis of recombinant protein

[0110] 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 / L IPTG, induce expression at 37°C for 4h, after the end, centrifuge the bacterial solution at 8000r / min for 5min to collect the bacterial pellet, and add 20mmol / L Tris at a volume ratio of 1:10 (Tris-Cl solution:bacterial solution) -Cl (pH8.0) solution was suspended, and the bacterial cells were ultrasonically broken in an ice-water bath for 60 s with an interval of 60 s, repeated until ...

Embodiment 3

[0121] Embodiment 3 A kind of identification experiment result of type 1 duck hepatitis A virus VP3 protein B cell epitope

[0122] 1. Preparation and potency determination of VP3 recombinant protein polyclonal antibody

[0123] 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

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a B cell epitope of type 1 duck hepatitis A virus VP3 protein, an identification method and application thereof. Background technique [0002] Duck hepatitis, also known as duck viral hepatitis (Duck viral hepatitis, DVH), is a highly contagious and fatal duck viral infectious disease caused by duck hepatitis virus (Duckhepatitis virus, DHV), which is characterized by acute onset, The spread is fast, the course of disease is short, and the case fatality rate is high. The necropsy of sick ducks shows hepatomegaly, dendritic congestion or hemorrhagic spots on the surface. The disease was first discovered in the United States in 1945. DHV was initially divided into three serotypes (DHV-1, DHV-2 and DHV-3), and later DHV-2 and DHV-3 were classified into the Astroviridae family, and there was no antigenic correlation among the three serotypes. Now DHV-1 has been na...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/10G01N33/68A61K39/12A61P31/14
CPCA61K39/12C07K14/005C12N2770/32422G01N33/5768G01N2333/10
Inventor 程安春汪铭书沈友林杨乔朱德康陈孝跃
Owner SICHUAN AGRI UNIV
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