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Recombinant serum type-4 fowl adenovirus for expressing serum type-8 fowl adenovirus spike protein and a preparation method thereof

A kind of avian adenovirus, fiber spike protein technology, applied in the field of genetic engineering

Active Publication Date: 2021-07-02
YANGZHOU UNIV
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

However, there are currently no commercial vaccines for FAdV-4 and FAdV-8

Method used

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  • Recombinant serum type-4 fowl adenovirus for expressing serum type-8 fowl adenovirus spike protein and a preparation method thereof
  • Recombinant serum type-4 fowl adenovirus for expressing serum type-8 fowl adenovirus spike protein and a preparation method thereof
  • Recombinant serum type-4 fowl adenovirus for expressing serum type-8 fowl adenovirus spike protein and a preparation method thereof

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Experimental program
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Embodiment

[0040] 1. Isolation of serum type 8 avian adenovirus: get the liver of the sick dead chicken suspected of avian adenovirus infection, grind it and add PBS in a ratio of 1:5 to make a suspension; 5000r / min centrifugal 15min, get the supernatant; Add 1000 IU / ml of penicillin and streptomycin, react at 37°C for 30 minutes; filter through a 0.45um microporous filter, inoculate 8-day-old SPF chicken embryos through the allantoic cavity at a dose of 0.2ml / embryo, and inoculate 96-120 The allantoic fluid was collected after 1 hour; the allantoic fluid was identified as avian adenovirus and stored at -20°C.

[0041] 2. Preparation of serum type 8 avian adenovirus genome: Take 400uL of the virus supernatant of the isolated strain of avian adenovirus in a 1.5mL finger tube, add 400uL of cell lysate (50mmol / L Tris-HCl pH 8.0, 20mmol / L EDTA pH 8.0, 2% SDS and proteinase K), mix well and place in a 56°C water bath for 3 hours; add 400uL Tris to balance phenol, mix well and centrifuge at 10...

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Abstract

The invention relates to a recombinant serum type-4 fowl adenovirus for expressing serum type-8 fowl adenovirus spike protein and a preparation method thereof, and the principle and the core key technology of the invention are that sgRNA is used for replacing EGFP and then virus purification is carried out to obtain the recombinant fowl adenovirus capable of stably amplifying the fowl adenovirus type 8 spike protein. The recombinant FAdV-4 for expressing the fowl adenovirus type-8 spike protein is successfully obtained by inserting the fowl adenovirus type-8 spike protein gene F which is popular at present as a carrier, and an in-vitro experiment result shows that the replication capacity of the FAdV4-F1-F-F2 recombinant virus is almost the same as that of a FAdV-4 wild virus, and the recombinant virus can be used as an inactivated recombinant multivalent vaccine for preventing and controlling FAdV-4 and FAdV-8.

Description

technical field [0001] The invention relates to a recombinant serotype 4 avian adenovirus expressing the spike protein of serotype 8 avian adenovirus and a preparation method thereof, belonging to the technical field of genetic engineering. Background technique [0002] Fowl Adenovirus (Fowl Adenovirus, FAdV) belongs to Adenoviridae, and is divided into 5 species (A-E) and 12 serotypes (FAdV-1~8a, FAdV-8b~11). Although it 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, the cases of inclusion body hepatitis and pericardial effusion syndrome caused by FAdVs in domestic chickens have gradually increased. By 2015, FAdV infection broke out in chicken flocks in several provinces in China. At present, FAdV outbreaks not only occur in 3-4 week-old broiler chickens, but also occur in 10-20 week-old laying hens, causing se...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01C12N15/861C12N15/113C12N15/34C12R1/93
CPCC12N7/00C12N15/86C12N15/113C07K14/005C12N2710/10222C12N2710/10243Y02A50/30
Inventor 叶建强路浩谢泉连明君王伟康李拓凡秦爱建邵红霞
Owner YANGZHOU UNIV
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