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Group-I type-4 aviadenovirus DNA vaccine, and preparation method and application thereof

A DNA vaccine and avian adenovirus technology, applied in the field of DNA vaccines, can solve unseen problems and achieve the effects of low cost, convenient use and simple preparation

Active Publication Date: 2017-12-05
安徽东方帝维生物制品股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no relevant report on the penton gene DNA vaccine of group I adenovirus type 4

Method used

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  • Group-I type-4 aviadenovirus DNA vaccine, and preparation method and application thereof
  • Group-I type-4 aviadenovirus DNA vaccine, and preparation method and application thereof
  • Group-I type-4 aviadenovirus DNA vaccine, and preparation method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0033] A kind of I group 4 type avian adenovirus DNA vaccine of the present embodiment contains I group 4 type avian adenovirus penton gene, the sequence of the I group 4 type avian adenovirus penton gene is shown in SEQ ID No.1 Show.

Embodiment 2

[0035] The preparation method of a kind of I group 4 type poultry adenovirus DNA vaccine of the present embodiment comprises the following steps:

[0036] (1) Proliferation of group I adenovirus type 4

[0037] The virulent strain of avian adenovirus type 4 of Group I was isolated from diseased broilers; the virus was inoculated on LMH cells grown into a single layer, adsorbed for 1 hour at 37°C, added DMEM maintenance solution with 5% serum concentration, and continued to cultivate until 60- After 64 hours, when the cells appear round shrinkage, detachment and other lesions, collect the poison and store at -70°C for later use;

[0038] (2) Primer design and synthesis

[0039]①According to the genome sequence of group I group 4 avian adenovirus (Gene ID: 10399473), select the penton gene (penton) sequence, and use Oligo7 software to design a pair of specific primers for the penton gene:

[0040] Primer 1: 5'-CGGAATTCATGTGGGGGTTGCAGCCG-3',

[0041] Primer 2: 5'-GTCGACGTCGCGG...

Embodiment 3

[0053] The safety and immune effect detection of a kind of I group type 4 avian adenovirus DNA vaccine of the present embodiment comprises the following steps:

[0054] (1) Vaccine safety testing

[0055] Use 20 3-week-old SPF chickens, 10 chickens in the first group, inject 200 μg of the pCI-penton vaccine prepared in the above examples into the leg muscles (100 μg for each of the two legs), and 10 chickens in the second group as a control In the group, 200 μg of plasmid pCI was injected intramuscularly into the legs, and observed continuously for 2 weeks to observe whether the immunized chickens had any local and systemic adverse reactions caused by the injection of the vaccine.

[0056] The results showed that the spirit and diet of chickens in the two groups were normal after immunization, and there was no abnormal reaction such as redness and swelling at the injection site.

[0057] (2) Detection of vaccine immune effect

[0058] 20 2-week-old SPF chickens were randomly...

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Abstract

The invention discloses a group-I type-4 aviadenovirus DNA (Deoxyribonucleic Acid) vaccine. The antigen of the group-I type-4 aviadenovirus DNA vaccine contains a group-I type-4 aviadenovirus penton gene. The invention also discloses a preparation method and application of the group-I type-4 aviadenovirus DNA vaccine. The preparation method comprises the following steps of (1) designing a group-I type-4 aviadenovirus penton gene primer with EcoR I and Sal I restriction enzyme cutting sites, and amplifying the target gene fragment by using the group-I type-4 aviadenovirus DNA as a template; (2) connecting the penton gene fragment onto a pMD-18T vector; (3) connecting the penton gene to an eukaryotic expression vector pCI; (4) transfecting pCI-penton into an LMH cell; and (5) performing amplification and extracting and purifying pCI-penton. The group-I type-4 aviadenovirus DNA vaccine has the advantages that the preparation is simple; the use is convenient; the cost is low; the humoral immunity and the cellular immunity can be excited at the same time; the comprehensive protection is effectively provided for SPF chickens; and the toxicity enhancing risk does not exist.

Description

technical field [0001] The invention relates to the technical field of DNA vaccines, in particular to a group I type 4 poultry adenovirus DNA vaccine and a preparation method and application thereof. Background technique [0002] Fowl adenovirus (Fowl adenovirus, FAdV) belongs to the genus Fowl Adenoviridae and is divided into three groups according to their antigenicity: Group I, Group II and Group III. In 1987, a disease characterized by pericardial effusion broke out in broiler chicken farms in the Ankara region of Pakistan, which brought huge losses to the local chicken industry, followed by broiler flocks in Germany, India, Iran, Mexico and Japan. Diseases characterized by pericardial effusion-inclusion body hepatitis syndrome have broken out one after another, and the mortality rate can reach more than 80%. Studies have confirmed that the pericardial effusion-inclusion body hepatitis syndrome in these areas is caused by group I type 4 poultry caused by adenovirus. Gr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/235A61P1/16A61P31/14C12N15/34C12N15/79
CPCA61K39/12A61K2039/53A61K2039/552A61K2039/57A61K2039/575C07K14/005C12N15/79C12N2710/10222C12N2710/10234
Inventor 张星刘渝李成山刘涛汪鑫鑫商俊
Owner 安徽东方帝维生物制品股份有限公司
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