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A-type avibacterium paragallinarum immunizing protective antigen and application thereof

A protective antigen and poultry technology, applied in the field of poultry infectious disease vaccine preparation, can solve the problems of toxic and side effects affecting chicken egg production and growth, immune failure, etc.

Active Publication Date: 2016-09-21
BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

On the other hand, because Aviobacterium paragallinarum contains toxins such as LPS, the toxic and side effects caused by large-scale vaccination will also affect the egg production and growth of chickens, thus resulting in frequent immune failures in production.

Method used

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  • A-type avibacterium paragallinarum immunizing protective antigen and application thereof
  • A-type avibacterium paragallinarum immunizing protective antigen and application thereof
  • A-type avibacterium paragallinarum immunizing protective antigen and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Embodiment 1, the prediction of Avian bacillus paragallinarum epitope

[0059] Using online forecasting software ( http: / / www.cbs.dtu.dk / services / BepiPred / ) Predict the epitope of the outer membrane protein coding gene (GenBank No.KJ867495.1) of Aviobacterium paragallinarum 221 strain type A, optimize multiple times according to the prediction results, and screen the immunogen from hundreds of epitopes obtained Some of the epitopes are intercepted based on experience, so that the final spliced ​​antigenic protein is easy to express and has good immune activity.

[0060] The optimized antigen epitope sequences were spliced ​​to obtain the recombinant antigenic protein P41 of Avibacterium paragallinarum, the amino acid sequence of which is shown in SEQ ID NO:1.

Embodiment 2

[0061] Embodiment 2, the prokaryotic expression of Avian bacillus paragallinarum recombinant antigenic protein P41

[0062] 1. Synthesis of the target gene and its primers

[0063] The nucleotide sequence shown in SEQ ID NO: 2 was sent to Huada Technology (Beijing) Co., Ltd. for full sequence synthesis, and the target gene of the recombinant antigen protein P41 of Aviparagallinarum with a length of 1085bp was obtained.

[0064] Design upstream and downstream primers according to the sequence of the target gene, and respectively introduce NcoI and XhoI restriction sites into the upstream and downstream primers to obtain primers shown in SEQ ID NO: 3 and SEQ ID NO: 4, provided by Huada Technology ( Beijing) Co., Ltd. synthesis.

[0065] 2. Construction of recombinant expression plasmids

[0066] 2.1 PCR amplification of target protein coding gene

[0067] Using the P41 target gene artificially synthesized in step 1 as a template, perform PCR amplification according to the fol...

Embodiment 3

[0164] Embodiment 3, characteristic analysis of recombinant antigenic protein

[0165] 1. Analyze the antigenicity of recombinant antigenic protein by Western-blot method

[0166] The recombinant antigen protein P41 of above-mentioned purification is carried out SDS-PAGE electrophoresis according to conventional method, and its steps are:

[0167] 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: lay t...

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Abstract

The invention relates to an A-type avibacterium paragallinarum immunizing protective antigen and application thereof. Artificial A-type avibacterium paragallinarum immunizing protective antigen protein provided by the invention has the amino acid sequence shown as SEQ ID NO: 1, and is named as P41. The protein has very strong immunogenicity, so that a subunit vaccine prepared from the antigen protein can significantly enhance the resistance of a chicken to A-type avibacterium paragallinarum, has a protective effect superior to that of the whole-cell inactivated vaccine, and can effectively prevent the avibacterium paragallinarum from infection.

Description

technical field [0001] The invention belongs to the technical field of preparation of poultry infectious disease vaccines, and in particular relates to an immunoprotective antigen of Avian bacilli paragallily and its application. Background technique [0002] Avibacterium paragallinarum (Apg) is a short Gram-negative bacillus belonging to Pasteurellaceae. In 1932 De Blieck first reported the first isolation of a Gram-negative bacillus from chickens with acute catarrhal inflammation of the nasal passages and sinus mucosa, facial edema, and conjunctivitis. The initial research reports pointed out that the growth conditions of this pathogen require both X (heme crystal) factor and V (nicotinamide adenine dinucleotide, NAD) factor, and early researchers named it Haemophilus gallinaceus (Haemophilus gallinarum). In 1962, Page et al found that only factor V was required for growth of isolates from all IC cases. Therefore, some scholars proposed to name Haemophilus gallinarum, w...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/285C12N15/31C12N15/70C12N1/21A61K39/102A61P31/04C12R1/19
CPCA61K39/102C07K14/285
Inventor 王宏俊李淑芳龚玉梅张培君李桂萍
Owner BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES