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Aviadenovirus Hexon and chicken infectious bursal disease virus VP2 fusion antigen and subunit vaccine, and preparation method of aviadenovirus Hexon and chicken infectious bursal disease virus VP2 fusion antigen and subunit vaccine

A subunit vaccine, chicken infectious technology, applied in the biological field, can solve the problem of lack of immune protection of Hexon and chicken VP2 fusion protein and vaccine research, Hexon genetic engineering vaccine is in the basic research stage, differences in antigenicity and hydrophilicity, etc. problem, to achieve the effect of protecting infection, reducing repetitive work, and good immunogenicity

Active Publication Date: 2017-12-19
RINGPU (BAODING) BIOLOGICAL PHARMACEUTICAL CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The current genetic engineering vaccines for the two proteins have the following disadvantages: (1) Only the VP2 genetic engineering vaccine is commercially produced, while the Hexon genetic engineering vaccine is still in the basic research stage
(2) Two proteins are expressed separately and mixed to prepare a double vaccine, which leads to low production efficiency and duplication of labor
(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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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 amplified the gene fragment of the fusion protein of avian adenovirus Hexon and chicken infectious bursal disease virus antigen VP2; using SOC-PCR method, the specificity of chicken infectious bursal disease virus VP2 and avian adenovirus Hexon was obtained 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] Primer pair 1 (F1, R1) was designed according to the conserved base region P1 and P2 sequences of avian adenovirus Hexon protein, and primer pair 2 (F2, R2) was designed according to the hypervariable region sequence of chicken infectious b...

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 SOC-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 avian 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 aden...

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 an aviadenovirus Hexon and infectious bursal disease virus VP2 fusion antigen and subunit vaccine, and a preparation method and application of the aviadenovirus Hexon and chicken infectious bursal disease virus VP2 fusion antigen and subunit vaccine. The aviadenovirus Hexon and chicken infectious bursal disease virus VP2 fusion antigen comprises an aviadenovirus Hexon protein and an chicken infectious bursal disease virus VP2 protein. The fusion antigen has the following amino acid sequence: (1) a protein formed by an amino acid sequence as shown in SEQ ID No.1; or (2) an amino acid sequence of a functional protein with the same code and homology being 95 percent to 100 percent as the protein amino acid sequence limited by the sequence SEQ ID No.1. A protective antigen of the subunit vaccine prepared through the invention is the fusion antigen, and the fusion antigen is prepared by adopting a gene engineering fermentation method, 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. Broiler hydropericardium syndrome caused by this 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) Protei...

Claims

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Application Information

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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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