Antigen protein of avibacterium paragallinarum and application of antigen protein

An antigenic protein, chicken and poultry technology, applied in the field of antigenic protein of Avian bacillus paragallinarum, can solve the problems of inability to detect antibodies, time-consuming and laborious, and the difference between chicken erythrocyte batches affecting the test results, so as to reduce the detection cost and avoid false negatives. , the effect of improving sensitivity

Active Publication Date: 2018-03-13
BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method has the deficiencies in the following aspects: 1. Fresh chicken red blood cells must be used when evaluating the vaccine effect of Apg A type bacteria, so it is necessary to raise test chickens all year round, and to ensure the health of chickens for blood supply; blood collection and blood processing, etc. The process is time-consuming and labor-intensive; 2. Differences between batches of chicken red blood cells affect the test results; 3. This method ultimat...

Method used

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  • Antigen protein of avibacterium paragallinarum and application of antigen protein
  • Antigen protein of avibacterium paragallinarum and application of antigen protein
  • Antigen protein of avibacterium paragallinarum and application of antigen protein

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Experimental program
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Embodiment 1

[0053] The outer membrane protein antigen selection of embodiment 1 Avian bacilli paragallinarum

[0054] Screen the antigen-encoding genes of Aviola paragallinarum in GenBank, use the epitope prediction software geneious to predict the epitopes of the outer membrane protein, and analyze the secondary structure, hydrophilicity, hydrophobicity, antigenicity and other information of the outer membrane protein for antigen expression. bit forecast. Finally, the outer membrane protein of GenBank No. KJ867497 Avian Bacillus paragallinarum was selected as the target antigen, and comprehensive software analysis was performed. According to the antigenicity of the protein sequence, the difficulty of protein expression and purification was considered, and the signal peptide region was avoided to select the appropriate antigen. Epitope sequence or epitope concentration area; according to the prediction results and accumulated experimental experience, the selected epitope sequence is optim...

Embodiment 2

[0055] Prokaryotic expression of embodiment 2 Avian bacillus paragallinarum recombinant antigenic protein P9

[0056] 1. Sequence Synthesis

[0057] The gene coding sequence of the antigenic protein P9 is as described in Example 1, and the specific sequence is shown in SEQ ID NO.2, which can be synthesized by Huada Technology (Beijing) Co., Ltd.

[0058] Design upstream and downstream primers according to their respective base sequences, and introduce EcoRI and XhoI restriction sites into the upstream and downstream primers respectively. Huada Technology (Beijing) Co., Ltd. can be entrusted to carry out primer synthesis. The primer sequence is as follows: Seq ID No. .3 ~ 4 shown.

[0059] 2. Construction of recombinant expression plasmids

[0060] 2.1 PCR amplification of the gene encoding the antigenic protein P9

[0061] Using the synthetic product obtained in step 1 as a template, the synthetic primers were used to carry out PCR,

[0062] PCR reaction system:

[0063] ...

Embodiment 3

[0109] The preparation of embodiment 3 Avian bacillus paragallinarum vaccine preparation

[0110] 1. Preparation of vaccine and preparation of recombinant protein antiserum

[0111] The purified recombinant antigen protein P9 and MONTANIDE in Example 2 TM ISA 71VG adjuvant (product of SEPPIC company) is mixed and emulsified in a weight ratio of 3:7 (30% antigen by weight, 70% adjuvant by weight) (for the operation method, see the introduction of the product use method of SEPPIC company), so that the final concentration of antigenic protein in the vaccine 20 μg / ml. The prepared vaccine was named vP9.

[0112] Immunization method: Take 42-day-old SPF chickens and divide them into 5 groups, 10 birds / group. Among them, 3 groups are vaccine immunization groups, and 0.5mL / mouse, 0.2mL / mouse, and 0.1mL / mouse of the vP9 vaccine are injected into the chest respectively; one group is a non-antigen adjuvant control; the other group is a non-immune control group. Four weeks after the ...

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Abstract

The invention discloses an antigen protein of avibacterium paragallinarum and application of the antigen protein. Amino acid sequences of the antigen protein are shown as SEQ ID NO.1. The invention further provides a reagent kit for detecting antibodies of the avibacterium paragallinarum and a vaccine preparation for the avibacterium paragallinarum. The reagent kit can be used for detecting the antibodies induced by outer membrane proteins of A-type thalli, B-type thalli and C-type thalli of the avibacterium paragallinarum or assisting in detecting the antibodies induced by the outer membraneproteins of the A-type thalli, the B-type thalli and the C-type thalli of the avibacterium paragallinarum. The antigen protein, the application, the reagent kit and the vaccine preparation have the advantages that immune states of chicken can be judged by the aid of detection results, so that corresponding measures can be adopted by farmers in the poultry industry; the reagent kit contains negative control serum and positive control serum, and accordingly the accuracy and the reliability of the detection results can be guaranteed; the vaccine preparation can be used for being vaccinated in thechicken, and accordingly the chicken can be effectively protected against infection of the A-type, B-type and C-type avibacterium paragallinarum.

Description

technical field [0001] The invention relates to the technical field of biological detection, in particular to an antigenic protein of Avian bacillus paragallinarum and its application. Background technique [0002] Avibacterium paragallinarum (Apg) is a small Gram-negative bacterium with a length of about 1-3 μm and a width of 0.4-0.8 μm. It has no flagella and does not form spores. Virulent strains often have capsules. The culture conditions of Aviola paragallinarum are relatively harsh, and NAD needs to be added to the medium, but NAD-independent isolates were found in South Africa, indicating that NAD is not an essential nutrient. The bacteria are facultatively anaerobic and require an anaerobic environment or 3%-10% CO when cultured on a solid medium 2 . [0003] Avibacterium paragallinarum is the pathogenic bacterium of infectious coryza (IC) in chickens, which causes a respiratory infectious disease in chickens. It causes chickens to sneeze and discharge thin watery...

Claims

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Application Information

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IPC IPC(8): C07K14/195C12N15/31G01N33/68A61K39/02A61P31/04
CPCA61K39/102A61K2039/552C07K14/195G01N33/6854
Inventor 王宏俊梅晨黄明明张培君龚玉梅李淑芳李桂萍
Owner BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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