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Avian adenovirus fiber protein subunit vaccine

A subunit vaccine and poultry adenovirus technology, applied in the direction of vaccines, viruses, and viral peptides, can solve the problems of strong virulence of attenuated bacterial strains, hidden infection, and interference with the efficacy of attenuated vaccines, and achieve convenient and accurate detection methods. High stability and strong specific effect

Inactive Publication Date: 2019-08-16
YEBIO BIOENG OF QINGDAO
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Existing poultry adenovirus vaccines use the complete pathogen as the antigen for vaccine production, but the antigenicity of the strains used to manufacture vaccines and the clinically popular strains is quite different, which affects the protective effect of the virus challenge and restricts the elimination of the virus. Use of Live Vaccines
Moreover, the effectiveness of the attenuated vaccine is easily interfered by maternal antibodies and antibiotics, and the attenuated strain has the possibility of returning to stronger virulence and inducing recessive infection

Method used

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  • Avian adenovirus fiber protein subunit vaccine
  • Avian adenovirus fiber protein subunit vaccine
  • Avian adenovirus fiber protein subunit vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] Embodiment 1: Amplification and sequence analysis of fiber gene

[0016] Suspected pericardial effusion syndrome occurred in a chicken farm in Yantai, Shandong Province in 2017. After clinical investigation and laboratory testing, it was initially diagnosed as avian adenovirus infection. After investigation, it is believed that the above-mentioned affected chickens have been injected with poultry adenovirus vaccine before. It is suspected that avian adenovirus has mutated under the selection pressure of the vaccine, and the virus was successfully isolated from the liver of the affected chicken and used as a template for amplification.

[0017] 1. Amplify the fiber gene of avian adenovirus

[0018] According to the avian adenovirus fiber gene sequence published in NCBI, primers were designed and synthesized. The sequence information of the primers is as follows:

[0019] primer1: 5′-ATGCTCCGAGCCCCTAAAAG-3′;

[0020] primer2: 5'-TTACGGGAGGGAGCCCGCTG-3'.

[0021] The i...

Embodiment 2

[0025] Embodiment 2, the construction of expression fiber gene recombinant plasmid

[0026] 2.1 Enzyme digestion reaction

[0027] 2.1.1 Mark the 1.5mL EP tube to be used, add and mix the sample in the 1.5mL EP tube according to the following table: the reaction system is 50 μL, and the sample addition is as follows:

[0028]

[0029] 2.1.2 Place the 1.5mL EP tube in step 2.2.1 in a constant temperature water bath at 37°C for 2-3 hours.

[0030] 2.1.3 Gel recovery of double enzyme digestion products

[0031] Take out the above-mentioned double digestion system and perform agarose gel electrophoresis to recover the DNA fragments therein.

[0032] (1) Mark the sample collection EP tube, adsorption column and collection tube.

[0033] (2) Weigh the marked empty EP tube and record the value.

[0034] (3) Carefully cut out a single target DNA band from the agarose gel on a gel cutter with a scalpel and put it into a clean 1.5mL centrifuge tube.

[0035] (4) Add 600 μL PC bu...

Embodiment 3

[0094] Expression, purification and identification of the protein of embodiment 3

[0095] 3.1 Expression and identification Inoculate the sequencing-positive strains on LB plates containing kanamycin sulfate and culture them overnight at 37°C. Pick colonies and culture them in LB culture medium containing kanamycin sulfate and Escherichia coli culture medium until the OD600 of the bacteria liquid is 0.6-0.8, add lactose with a final concentration of 0.02M, and conduct induction culture for 5-7 hours , and the cells were collected by centrifugation. The bacteria sludge was suspended and crushed with PBS. Add SDS-PAGE loading buffer [50mM Tris-HCl (pH6.8), 100mM Dithiothreitol (DTT), 2% SDS, 0.05% Bromophemol blue, 10% Glycerol], boil for 5min, use 12% separating gel, 5% concentration The gel was subjected to polyacrylamide gel electrophoresis, 100 volts for about 2.5 hours, Coomassie brilliant blue R250 staining, it was found that the unoptimized fiber gene was not expressed...

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Abstract

The invention provides an avian adenovirus fiber protein subunit vaccine. A new-type antigen fiber protein of an avian adenovirus is used, after a sequence is optimized, the fiber protein acquires soluble expression in escherichia coli, and an expression product is used for preparing the subunit vaccine, wherein an amino acid sequence of the avian adenovirus fiber antigen protein is SEQ ID NO: 4,and a nucleotide sequence of an encoding gene thereof is SEQ ID NO: 3. The subunit vaccine prepared by the avian adenovirus fiber protein has the characteristics of high antigen stability, high purity, strong specificity, no generation of other uncorrelated antibodies, and convenient and accurate detection method. A firm foundation is established for industrially producing the avian adenovirus subunit vaccine and diagnostic reagent.

Description

technical field [0001] The invention belongs to the technical field of veterinary biological products, in particular to an adenovirus fiber protein subunit vaccine. Background technique [0002] Fowl Adenovirus (Fowl Adenovirus, FAdV) belongs to Adenoviridae, and is divided into 5 species (A-E) and 12 serotypes; FAdV has been reported all over the world. FAdV infection generally causes subclinical conditions, while acute infection mainly causes inclusion body hepatitis, pericardial effusion, and gizzard erosion. Since 2013, cases of inclusion body hepatitis and pericardial effusion caused by FAdV in domestic chicken flocks have gradually increased. By 2015, FAdV infection broke out in chicken flocks in several provinces in China. At present, the outbreak of FAdV not only occurs in 3-4 week-old broiler chickens, but also occurs in 10-20 week-old laying hens, which has caused serious economic losses to the domestic chicken industry. [0003] The avian adenovirus is a non-en...

Claims

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Application Information

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IPC IPC(8): C07K14/075C12N15/34A61K39/235A61P31/20
CPCA61K39/12A61K2039/552A61P31/20C07K14/005C12N2710/10222C12N2710/10234
Inventor 郭伟伟向银辉窦小龙范根成杜元钊
Owner YEBIO BIOENG OF QINGDAO
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