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Porcine pseudorabies virus strain and application thereof

A technology for porcine pseudorabies and virus strains, which can be applied in the directions of antiviral agents, viruses/phages, and medical preparations containing active ingredients, etc.

Inactive Publication Date: 2015-05-20
INST OF ANIMAL SCI & VETERINARY HUBEI ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The immune porcine pseudorabies vaccines prepared at home and abroad are mainly gene-deleted attenuated live vaccines such as pseudorabies virus strains Bartha-K61 and BUK, but in actual production, the disease has not been effectively controlled, and large-scale outbreaks have occurred in individual areas It still exists, whether the epidemic strains have mutated, it must be further isolated and studied

Method used

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  • Porcine pseudorabies virus strain and application thereof
  • Porcine pseudorabies virus strain and application thereof
  • Porcine pseudorabies virus strain and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0013] Embodiment 1: the big-eared white rabbit enhanced isolation method of porcine pseudorabies virus

[0014] A case of suspected porcine pseudorabies symptoms occurred in a pig farm in Hubei Province, and the pathogenic test was positive for porcine pseudorabies virus. Therefore, sick pigs with typical symptoms were selected for virus isolation. The specific steps are as follows:

[0015] 1. Aseptically collect the brain and spleen of diseased pigs, weigh them and add sterilized saline at a weight ratio of 1:5, homogenate, and then undergo ultrasonic crushing, freeze and thaw repeatedly 3 times, and then centrifuge at 3000r / min for 15min. Take the supernatant and add penicillin (strep)mycin double antibody to 2000IU / ml, put it in refrigerated overnight at 4°C, after passing the sterility test, subcutaneously inoculate five 1.5kg big-eared white rabbits, 5ml each. The big-eared white rabbits that died within 24 hours were discarded, and observed every 12 hours thereafter fo...

Embodiment 2

[0018] Embodiment 2: Identification of pseudorabies virus HBWH / 2014 virus strain

[0019] 1. Electron microscope negative stain observation

[0020] After culturing for 5-7 days, the supernatant of the diseased cells was ultracentrifuged on a sucrose gradient, and the virus particles were separated for negative staining observation by electron microscope.

[0021] The results showed: irregular round virus particles with capsids and envelopes, with a diameter of about 110-180nm, see figure 2 (A: magnified 150,000 times; B: magnified 100,000 times).

[0022] 2. The tissue culture median lethal dose (TCID) of the virus 50 ) determination

[0023] Take the 5th-generation cell culture of the isolate that caused CPE and freeze-thaw it repeatedly 3 times, and use the maintenance solution to make the virus 10 times respectively. -1 ~10 -9 Serial dilutions were inoculated in 96-well cell culture plates full of BHK-21 monolayer cells, each dilution was inoculated into 6 wells, 0.1...

Embodiment 3

[0035] Embodiment 3: porcine pseudorabies virus gE gene sequencing detection

[0036] 1. Design and synthesis of primers

[0037] Design with reference to the gE gene sequence of porcine pseudorabies virus reported in GenBank,

[0038] Upstream primer P1: 5'GCGGCCCTTTTCTGCTGCG3';

[0039] Downstream primer P2: 5'AGCGGGGCAGGACATCAA3'.

[0040] Primers were synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.

[0041] 2. PCR amplification

[0042] The volume of the PCR reaction system is 50 μL, including 1 μL of the upper and lower primers at a concentration of 10 μmol / L, 5 μL of the DNA template, 25 μL of 2×EasyTaq PCRMix, and the remaining ddH 2 0 to 50 μL. Reaction program: 97°C for 10 min, 72°C for 4 min; 95°C for 1 min, 65°C for 2 min, 72°C for 2.5 min, 34 cycles; 72°C for 10 min.

[0043] 3. PCR product cloning and sequencing

[0044] Cloning and Identification of PCR Products The ligation reaction system was 10 μL, and 5 μL of PCR products, 1 μL of pMD18-T vec...

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Abstract

The invention provides a novel porcine pseudorabies virus strain separated from the attacked swinery. The code of the novel porcine pseudorabies virus strain is PRV-HBWH / 2014; the virus strain is biologically collected in China Center for Type Culture Collection in June of 2014, with the microorganism collection number CCTCC NO: V201439. According to the molecular biological technical analysis, partial nucleotidesequences of the gE gene of the virus strain are as shown in SEQ ID NO: 1 in the sequence table. The separated virus strain is the major epidemicstrain of Chinese PRV and can be used as a virus seed for preparing a porcine pseudorabies vaccine.

Description

technical field [0001] The invention belongs to the technical field of animal virus isolation, and in particular relates to a porcine pseudorabies virus HBWH / 2014 and the application of the virus strain in preparing vaccines. Background technique [0002] Pseudorabies (PR) is an acute infectious disease caused by pseudorabies virus (PRV) that can infect a variety of domestic and wild animals, mainly including fever, abortion, stillbirth, itching (except pigs) and encephalomyelitis Symptoms, of which pigs are the most harmful, and pigs are the only natural host. Once the pigs have survived the infection, they will be recessively infected and carry and detoxify for a long time, becoming the main source of infection in the pig farm. At present, pseudorabies is considered to be one of the main diseases that threaten pig production and cause death. China has listed it as a second-class infectious disease, and OIE has listed it as a B-class polyzoan zoonosis of legally reported a...

Claims

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Application Information

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IPC IPC(8): C12N7/00A61K39/245A61P31/22C12R1/93
Inventor 郭锐田永祥周丹娜杨克礼段正赢刘泽文袁芳艳刘威
Owner INST OF ANIMAL SCI & VETERINARY HUBEI ACADEMY OF AGRI SCI
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