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Avian adenovirus hexon and chicken infectious bursal disease virus vp2 fusion antigen, subunit vaccine and preparation method thereof

A subunit vaccine, chicken infectivity technology, applied in the biological field, can solve the problems of Hexon genetic engineering vaccine in the basic research stage, low production efficiency, difference in antigenicity and hydrophilicity, etc.

Active Publication Date: 2021-04-30
RINGPU (BAODING) BIOLOGICAL PHARMACEUTICAL CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the genetic engineering vaccines of the two proteins have the following disadvantages: (1) Only VP2 genetic engineering vaccines are commercially produced, while Hexon genetic engineering vaccines are still in the basic research stage
(2) Two kinds of proteins are expressed separately, and the double vaccine is prepared after mixing. The production efficiency is low and the work is repeated.
(3) During the production of viral subunit vaccines, the two proteins are directly fused and expressed in tandem, which destroys the original tertiary conformation of the protein, resulting in differences in antigenicity and hydrophilicity
(4) At present, there is no antigenic research on the fusion protein of avian adenovirus Hexon and chicken infectious bursal disease virus VP2, and there is no immune protection and vaccine research on the fusion protein of Hexon and chicken VP2

Method used

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  • Avian adenovirus hexon and chicken infectious bursal disease virus vp2 fusion antigen, subunit vaccine and preparation method thereof
  • Avian adenovirus hexon and chicken infectious bursal disease virus vp2 fusion antigen, subunit vaccine and preparation method thereof
  • Avian adenovirus hexon and chicken infectious bursal disease virus vp2 fusion antigen, subunit vaccine and preparation method thereof

Examples

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preparation example Construction

[0045] The preparation method of the fusion antigen of avian adenovirus Hexon and chicken infectious bursal disease virus VP2 of the present invention comprises the following steps:

[0046] (1) PCR amplifies the gene fragment of the fusion protein of avian adenovirus Hexon and chicken infectious bursal disease virus antigen VP2; adopts SOE-PCR method to obtain the specificity of chicken infectious bursal disease virus VP2 and avian adenovirus Hexon A nucleotide fragment, which inserts a specific connecting peptide fragment between two genes, and the amino acid sequence of the specific connecting peptide fragment is SEQ ID NO.2;

[0047] Cloning of avian adenovirus Hexon gene and chicken infectious bursal disease virus VP2 gene:

[0048] Design primer pair 1 (F1, R1) according to the conserved base region P1, P2 sequence of avian adenovirus Hexon protein, and design primer pair 2 (F2, R2) according to the hypervariable region sequence of chicken infectious bursal disease virus...

Embodiment 1

[0059] This example describes the preparation method of the fusion gene nucleotide fragment of avian adenovirus Hexon and chicken infectious bursal disease virus VP2 provided by the present invention. The SOE-PCR method is used for gene amplification, and the connecting peptide is added between the avian adenovirus Hexon protein and the chicken infectious bursal disease virus VP2 protein by adding a connecting peptide sequence to the primer sequence. Include the following steps:

[0060] (1) Extraction of Chicken Infectious Bursal Disease Virus and Poultry Adenovirus Genomes

[0061] Take chicken infectious bursal disease virus and avian adenovirus liquid 200ul respectively, and extract chicken infectious bursal disease virus and avian adenovirus genomic RNA / DNA according to the instructions of TaKaRa's virus DNA / RNA extraction kit.

[0062] (2) Cloning of fusion gene of avian adenovirus Hexon and chicken infectious bursal disease virus VP2

[0063] According to the avian ad...

Embodiment 2

[0079] This example describes the construction of the recombinant plasmid pET-28a-Hexon-VP2 and the recombinant strain.

[0080] The fusion gene fragment of Hexon and VP2 described in Example 1 and the expression vector pET-28a were double-digested with restriction endonucleases EcoRI and XhoI, the digested products were recovered and purified, and the fusion gene fragment was ligated into the pET-28a vector. The recombinant plasmid pET-28a-Hexon-VP2 was obtained.

[0081] Transform the recombinant vector into DH5α competent cells, pick a single colony for culture, extract the recombinant plasmid pET-28a-Hexon-VP2 for sequencing analysis, and transform the correctly identified recombinant plasmid pET-28a-Hexon-VP2 into Escherichia coli Rosseta competent cells, pick a single colony for culture, extract the recombinant plasmid and carry out EcoRI and XhoI double enzyme digestion identification analysis to obtain the recombinant strain.

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Abstract

The invention discloses a fusion antigen of avian adenovirus Hexon and chicken infectious bursal disease virus VP2, a subunit vaccine, a preparation method and application thereof. The invention relates to a fusion antigen of avian adenovirus Hexon and chicken infectious bursal disease virus VP2, wherein the fusion antigen includes avian adenovirus Hexon protein and chicken infectious bursal disease virus VP2 protein. The fusion antigen has the following amino acid sequence: (1) a protein consisting of the amino acid sequence shown in SEQ ID No.1; or (2) a homology of 95% to 100 to the amino acid sequence defined by SEQ ID No.1 % amino acid sequence encoding the same functional protein. The protective antigen of the subunit vaccine prepared by the invention is a fusion antigen, which is produced by genetic engineering fermentation, and has the advantages of low cost, high antigen purity, good immunogenicity and high safety.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a fusion antigen of avian adenovirus Hexon and chicken infectious bursal disease virus VP2, a subunit vaccine and a preparation method thereof. Background technique [0002] Avian adenovirus (FAV) is distributed worldwide and has many serotypes. The broiler hydropericardium syndrome caused by the virus is caused by adenovirus-C serotype 4 infection. It has been reported all over the country, and It presents a rising trend of morbidity and seriously endangers the healthy development of my country's poultry industry. Hexon protein is the main structural protein of avian adenovirus, which has immunogenicity. [0003] Infectious bursal disease of chickens is an acute contagious disease of young chickens. It is often prevalent in local areas and brings great economic losses to the poultry industry. VP2 protein is the main structure of infectious bursal disease virus (IBDV) Pro...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/70A61K39/295A61K39/235A61K39/12A61P31/20A61P31/14
CPCA61K39/12A61K2039/5256A61K2039/552A61K2039/70C07K14/005C07K2319/00C12N15/70C12N2710/10222C12N2710/10234C12N2720/00022C12N2720/10034
Inventor 刘云涛郑朝朝何平有孙颖吴亚婷郁宏伟鲍恩东朱秀同杨保收
Owner RINGPU (BAODING) BIOLOGICAL PHARMACEUTICAL CO LTD
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