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Chicken infectious rhinitis subunit vaccine and preparing method thereof

A chicken infectious rhinitis and subunit vaccine technology, applied in the field of poultry infectious disease prevention, can solve problems such as difficulty in antigenic epitopes, and achieve the effects of good immune protection activity and excellent protection effect

Active Publication Date: 2016-12-14
BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, when designing recombinant antigens for the preparation of vaccines, it is very difficult to screen the antigenic epitopes shared by the three serotypes of Apg, A, B, and C, which have neutralizing activity. , Reports on the protective antigens of three serotypes of Avibacterium paragallinarum infection

Method used

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  • Chicken infectious rhinitis subunit vaccine and preparing method thereof
  • Chicken infectious rhinitis subunit vaccine and preparing method thereof
  • Chicken infectious rhinitis subunit vaccine and preparing method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1, Prediction of Antigenic Epitopes of the Outer Membrane Protein of Aviola paragallinarum

[0054] Outer membrane protein: its amino acid sequence is shown in Seq ID No.27, which is derived from the outer membrane protein coding gene of GenBank No. KJ867498.1 Avibacterium paragallinarum 221 strain.

[0055] Epitope prediction: use the epitope prediction software geneious (Biomatters.Ltd, website http: / / www.geneious.com / ) to predict the epitope of the outer membrane protein, and analyze the secondary structure, hydrophilicity and hydrophobicity of the outer membrane protein. Antigenicity and other information ( Figure 7 ).

[0056] Sequence analysis and splicing: comprehensive software analysis, according to the antigenicity of the protein sequence, considering the difficulty of protein expression and purification, avoiding the signal peptide region, and selecting the appropriate epitope sequence or epitope concentrated region for sequence splicing; Based on ...

Embodiment 2

[0060] Embodiment 2, prokaryotic expression of the recombinant antigenic protein of Avian bacillus paragallinarum

[0061] 1. Materials

[0062] 1.1 Plasmids and strains

[0063] The pET28a plasmid (Pharmacia company product purchased from Beijing Bailingke Biotechnology Co., Ltd.) used in this example; the pET28a plasmid map is as follows figure 1 shown.

[0064] The competent Escherichia coli BL21 (DE3) was purchased from Beijing Tiangen Biotechnology Co., Ltd.

[0065] The strains of Aviobacterium paragallinum used in the present invention are Hp8, 221, 0083, BJ, 222, Modesto, and 668 strains, which are purchased from China Veterinary Drug Control Institute in Beijing, China, and belong to commercial strains.

[0066] 1.2 Antigen protein of Avibacterium paragallinarum

[0067] The five recombinant antigen proteins p1, p2, p3, p4 and p5 of Avibacterium paragallinarum obtained by prediction, analysis and splicing by bioinformatics software in Example 1.

[0068] 1.3 Mai...

Embodiment 3

[0144] Embodiment 3, characteristic analysis of recombinant antigenic protein

[0145] 1. Analyze the antigenicity of recombinant antigenic protein by Western-blot method

[0146] Carry out SDS-PAGE electrophoresis with the above-mentioned purified recombinant antigenic protein p1, p2, p3, p4, p5 according to conventional methods, the steps are:

[0147] 1) Transfer: Cut out 6 pieces of Whatman 3M filter paper and 1 piece of nitrocellulose membrane (NC membrane). The size of the filter paper and membrane should be exactly the same as the size of the gel or slightly smaller than the size of the gel. Mark the corner of the filter membrane with a pencil. Ensure the relative direction of the membrane and gel after transfer; soak the nitrocellulose membrane in purified water for 5 minutes; add a small amount of transfer buffer to another shallow tray, and soak 6 pieces of Whatman 3M filter paper in it. Then install the transfer electrophoresis tank according to the following steps...

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Abstract

The invention relates to the field of poultry infectious disease epidemic prevention, in particular to a chicken infectious rhinitis subunit vaccine and a preparing method thereof. Provided avibacterium paragallinarum immunoprotecive antigen protein is characterized in that the amino acid sequence includes sixteen antigen epitopes or an epitope field shown from Seq ID No.1-16, preferably, the amino acid sequence is selected from one of Seq ID No. 17, 18, 19, 20 and 21. The invention further provides the chicken infectious rhinitis subunit vaccine. The immunity active component comprises the avibacterium paragallinarum immunoprotecive antigen protein. The vaccine can obviously improve resistance to A,B,C serotype type avibacterium paragallinarum of chicken, the protective effect of the vaccine is better than that of a whole cell inactivated vaccine, and the vaccine can be used for preventing chicken infectious rhinitis caused by avibacterium paragallinarum of different serotype types.

Description

technical field [0001] The invention relates to the field of epidemic prevention of poultry infectious diseases, in particular to a chicken infectious rhinitis subunit vaccine and a preparation method thereof. Background technique [0002] Avian infectious coryza is one of the most important respiratory diseases caused by Avibacterium paragallinarum (Apg) infection. Chickens with avian infectious rhinitis mainly present clinical symptoms such as runny nose, swollen eyelids and watery eyes. Chicken infectious rhinitis can lead to the delay of laying eggs and the decline of egg production, thus causing a lot of economic losses. [0003] Apg is a short Gram-negative bacilli belonging to the family Pasteurellaceae. Page et al. divided Apg into three serotypes, A, B, and C. Studies have shown that Apg of the three serotypes A, B, and C have different degrees of pathogenicity, but the inactivated bacteria of the three do not exist cross-immunity. In order to prevent chicken in...

Claims

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Application Information

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IPC IPC(8): C07K14/285C12N15/31A61K39/102A61P31/04
CPCA61K39/102A61K2039/552C07K14/285
Inventor 王宏俊李淑芳陈小玲张培君龚玉梅李桂萍
Owner BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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