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HVT (herpesvirus of turkey) live vector vaccine for expressing FAdV (fowl adenovirus)-4 penton protein as well as preparation and application of HVT live vector vaccine

A technology of turkey herpes virus and live vector vaccine, which is applied in the directions of virus/phage, application, vaccine, etc., can solve the problem of no prevention and control of commercial vaccines, etc.

Inactive Publication Date: 2018-07-27
HUAZHONG AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Currently, there is no commercial vaccine against the disease

Method used

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  • HVT (herpesvirus of turkey) live vector vaccine for expressing FAdV (fowl adenovirus)-4 penton protein as well as preparation and application of HVT live vector vaccine
  • HVT (herpesvirus of turkey) live vector vaccine for expressing FAdV (fowl adenovirus)-4 penton protein as well as preparation and application of HVT live vector vaccine
  • HVT (herpesvirus of turkey) live vector vaccine for expressing FAdV (fowl adenovirus)-4 penton protein as well as preparation and application of HVT live vector vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0082] Example 1: Screening and Purification of Recombinant Viruses Containing a BAC Backbone

[0083] 1. Construction of BAC backbone containing homology arms

[0084] Take the pHVTDS-pHA1 plasmid (gifted by Professor Venugopal K. Nair, UK, see the plasmid map figure 2 ) is the template amplification (L, R) of the gene fragments containing the upper and lower homology arms of the US2 region of turkey herpesvirus. Pac I restriction sites were introduced at both ends of the amplified gene fragment,

[0085] The amplification upstream primer is: ttaattaaGATGAGCTGACGTGTGGAAT;

[0086] The downstream primer is: ttaattaaACTAATATGGGCACACCCAC (the part in bold is the PacI restriction site).

[0087] PCR amplification system: Primer star 0.5 μL; 2×Primer star buffer 25 μL, dNTP 4 μL, template 1 μL, upstream and downstream primers 1 μL each, ddH 2 Make up 50 μL of O, and react according to the following procedures after mixing: pre-denaturation at 98°C for 5 min; denaturation at 9...

Embodiment 2

[0092] Example 2: Electric transfer of recombinant virus genome to Escherichia coli GS1783

[0093] 1. Preparation of GS1783 Competent Cells

[0094] Streak the Escherichia coli GS1783 stored at -80°C on an LB plate, pick a single colony and inoculate it in 5 mL of SOB medium, and cultivate overnight at 32°C with shaking at 170r / min. The GS1783 cultured overnight was inoculated into 100 mL of SOB medium at a ratio of 1:100, and cultured with shaking at 170 r / min at 32°C. After 2 hours, measure the OD600 of the bacterial liquid. When OD600 = 0.5-0.6, place it in the ice-water mixture, shake it to cool the bacterial liquid evenly, and then shake it every 5 minutes for a total of 20 minutes in ice bath. Transfer the bacterial solution to a 50mL centrifuge tube pre-cooled in an ice bath, centrifuge at 4000r / min at 4°C for 10min, discard the supernatant, and collect the bacterial cells. The bacterial cells were washed with sterilized 10% glycerol prepared with deionized water, an...

Embodiment 3

[0099] Example 3: Rescue of BAC plasmids containing the full genome of HVT

[0100] 1. Extraction of the plasmid (pHVT-BAC) containing the HVT virus genome

[0101] With BAC molecular cloning virus clone extraction plasmid, amplified egfp gene, common gel electrophoresis method preliminary identification as positive single bacterial colony, with Qiagen plasmid Midi kit kit (QIAGEN mid-quantity extraction kit, purchased from Qiagen company) a large amount Extract the plasmid. The specific steps are:

[0102] Streak the positive clones of the initially identified BAC molecular cloned virus on the resistant LB medium plate containing chloramphenicol (Cm 34 μg / mL), culture at 32°C for 24 hours, pick the positive clones, shake the vial for 16 hours and then Shake 200mL of the bacteria in a medium bottle for 12h-14h, extract the plasmid of the BAC molecular cloned virus according to the kit instructions and the improved method, use a 500mL sterilized centrifuge bottle, centrifuge ...

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Abstract

The invention discloses an HVT (herpesvirus of turkey) live vector vaccine for expressing FAdV (fowl adenovirus)-4 penton protein as well as preparation and application of the HVT live vector vaccine.The HVT live vector vaccine rHVT-Penton is obtained by inserting Penton gene into an interval between gene UL45 and gene UL46 of a genome of a HVT live vector, is used for preventing and treating Ankara disease and can realize the effect of preventing Marek's disease and HHS (hepatitis-hydropericardium syndrome) simultaneously. By parity of reasoning, two or even more diseases can be prevented and treated by an exogenous gene recombined by an HVT BAC (bacterial artificial chromosome).

Description

technical field [0001] The invention relates to the field of animal genetic engineering vaccines, in particular to a turkey herpes virus live vector vaccine expressing poultry adenovirus serum type 4 penton protein and its preparation and application. Background technique [0002] Hepatitis-hydropericardium syndrome (HHS), also known as Ankara disease, is caused by avian adenovirus serotype 4 (FAdV-4) and is characterized by pericardial effusion and inclusion body hepatitis Acute infectious diseases of poultry. In recent years, the disease has broken out in many countries and regions in the world, bringing huge threats and economic losses to the poultry industry. The virus can spread horizontally as well as vertically. The virus mainly infects 3-6 week-old broiler chickens, with a mortality rate as high as 80%; it can also infect 10-20 week-old laying hens and breeding hens; other poultry such as quail, pigeons and other infection cases have also been reported. FAdV-4 is ...

Claims

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Application Information

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IPC IPC(8): A61K39/235A61P31/20C12N15/869C12N15/34C12N15/66C12N15/65
CPCA61K39/12A61K2039/5256A61K2039/53C12N15/65C12N15/66C12N15/86C12N2710/10022C12N2710/10034C12N2710/16043
Inventor 金梅林张俊勤黄坤邹忠康超
Owner HUAZHONG AGRICULTURAL UNIVERSITY
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