A kind of subunit vaccine for preventing type 4 avian adenovirus and its preparation method and application

A subunit vaccine and poultry adenovirus technology, applied in antiviral immunoglobulin, botanical equipment and methods, biochemical equipment and methods, etc., can solve the problems of neutralizing immune deficiency, etc. degree, to ensure the effect of immune effect

Active Publication Date: 2019-07-02
扬州优邦生物药品有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the function of the main structural protein Hexon protein (Hexon) of avian adenovirus has been studied a lot. This protein has major genus and subgenus-specific epitopes and minor species-specific epitopes. Can cause a strong neutralizing response, when it is replaced or mutated it will lead to the loss of neutralizing immunity

Method used

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  • A kind of subunit vaccine for preventing type 4 avian adenovirus and its preparation method and application
  • A kind of subunit vaccine for preventing type 4 avian adenovirus and its preparation method and application
  • A kind of subunit vaccine for preventing type 4 avian adenovirus and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Embodiment 1: Construction of recombinant baculovirus

[0031] 1. Extraction of avian adenovirus genome: extract the total DNA of SD strain of avian adenovirus type 4 by traditional method;

[0032] 2. Design and synthesis of primers: Using the self-isolated genome of avian adenovirus type 4 SD strain of group I as a template to analyze the antigenicity, hydrophilicity and surface display probability of the protein encoded by the FAVⅠhexon gene, select 494 amino acids at the N-terminus and 239 at the C-terminus One amino acid and two structural domains for baculovirus eukaryotic expression. A pair of specific primers were designed respectively according to the C-terminal and N-terminal domain gene sequences of the avian adenovirus Hexon gene published in GenBank, and Xho I and Sph I restriction sites were respectively added at both ends of the C-terminal primers, And the two ends of the N-terminal were added with BamH I and HindⅢ restriction sites respectively, and the...

Embodiment 2

[0049] Example 2: Preparation of C-terminal and N-terminal domain proteins of recombinant Hexon protein

[0050] 1. Recombinant baculovirus amplification: Inoculate the recombinant baculovirus rBacHexon-P10-C-PH-N into insect cells sf9, culture at 27°C for 4 days, collect the culture, centrifuge and take the supernatant to obtain the F2 generation recombinant rod virus;

[0051] 2. Identification of expressed protein:

[0052] (1) Insert the above-mentioned F2 generation recombinant baculovirus into insect cell sf9 with an inoculum amount of MOI=5-10, culture at 27°C and 110 rpm for 4 days, collect the culture, centrifuge and take the supernatant to obtain recombinant Hexon protein C -terminal and N-terminal domain proteins;

[0053] (2) SDS-PAGE identification: The above supernatant was subjected to SDS-PAGE electrophoresis; after electrophoresis, after staining and decolorization, it was found that the C-terminus was at about 27kDa and the N-terminus was at about 55kDa, an...

Embodiment 3

[0058] Embodiment 3: vaccine preparation

[0059] 1. Inactivation: Add the C-terminal and / or N-terminal domain protein of the recombinant Hexon protein prepared in large quantities in Example 2 into the inactivation tank, and add the final concentration of 0.2% to 0.5% inactivator BEI, Inactivate at 37°C for 24h.

[0060] 2. Inspection of semi-finished products

[0061] (1) Sterility test: carry out sterility test according to the appendix of the current "Chinese Veterinary Pharmacopoeia".

[0062] (2) Determination of protein content: the protein content was detected by Bradford method.

[0063] (3) Inactivation test: Insect cell sf9 was taken from the inactivated protein liquid, and cultured at 27° C. for 72 hours. No lesions were observed, and the inactivation test was judged to be qualified.

[0064] 3. Preparation of recombinant subunit vaccine:

[0065] The semi-finished protein antigen after passing the inspection is used for vaccine preparation (the liquid compone...

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Abstract

The invention discloses a subunit vaccine for preventing type-IV aviadenovirus as well as a preparation method and application thereof, which belongs to the field of veterinary biological products. The preparation method comprises the following steps: cloning and amplifying a C-end and an N-end structural domain genes of FadV 4C Hexon genes; establishing a recombinant baculovirus rBac-HEXON-P10-C-PH-N capable of co-expressing two antigen proteins by using an insect cell-baculovirus bidirectional expression system, wherein the recombinant virus efficiently expresses the FAdV 4C Hexon-C and Hexon-N antigen proteins in the insect cell HF; and extracting, purifying, BEI inactivation, and emulsifying by adding adjuvant, thus obtaining the vaccine. The preparation method is simple, a great amount of FAdV 4C Hexon-C and Hexon-N proteins can be simultaneously prepared, the consumed time is short, the expression amount is high, the production cost is greatly reduced, and the mass production canbe realized. The obtained recombinant subunit vaccine is good in immunity effect, small in immunity dose and capable of preventing the infection of the aviadenovirus.

Description

technical field [0001] The invention relates to a subunit vaccine for preventing type 4 avian adenovirus and its preparation method and application, belonging to the field of veterinary biological products. Background technique [0002] Fowl adenovirus (Fowl adenovirus, FAdV) can be divided into 3 groups, the well-known virus strains of group I include chicken embryo lethal orphan virus, quail bronchitis virus and chicken inclusion body hepatitis virus, group II fowl adenovirus includes turkey hemorrhage Enteritis virus, marble spleen virus and chicken spleen virus, group III adenovirus only includes egg drop syndrome virus. Group I adenoviruses can be divided into 12 serotypes. According to serum analysis in high-incidence areas, they are mainly C (serum type 4) and E (serum type 8). Ankara disease (hydropericardium-hepatitis syndrome) is caused by serum Caused by type 4, the disease is more common in broiler chickens. Recently, it has also begun to infect chickens of Ma c...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/075C07K16/08C12N15/34C12N15/866A61K39/235A61P31/20G01N33/569
CPCA61K39/12A61K2039/552A61P31/20C07K14/005C07K16/081C12N15/86C12N2710/10222C12N2710/10234C12N2710/14043G01N33/56983
Inventor 叶正琴范娟钱钟丁国伟潘杰李玉和宋庆庆杨振董昌海李玉安
Owner 扬州优邦生物药品有限公司
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