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Porcine pseudorabies virus gene deletion attenuated vaccine strain for passage via low temperature of cell and attenuation via drug screening and application thereof

A porcine pseudorabies and attenuated vaccine technology, which is applied in the application field of porcine pseudorabies virus gene deletion attenuated vaccine strains, prevention and treatment of porcine pseudorabies, can solve the problems that sheep cannot completely resist the attack of PRVHeN1 strain and cannot effectively neutralize the virus , to achieve the effects of differential diagnosis, high protection efficiency and good safety

Active Publication Date: 2017-01-04
HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

We isolated the virus from the field and named it PRV HeN1 strain. Through serum neutralization test and sheep immunization test, it was confirmed that the neutralizing antibody produced by immunized pigs with PRV vaccine strain Bartha K61 strain widely used in China could not effectively neutralize the new strain. The isolated virus PRV HeN1 strain, and the sheep immunized with Bartha K61 could not completely resist the challenge of PRV HeN1 strain

Method used

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  • Porcine pseudorabies virus gene deletion attenuated vaccine strain for passage via low temperature of cell and attenuation via drug screening and application thereof
  • Porcine pseudorabies virus gene deletion attenuated vaccine strain for passage via low temperature of cell and attenuation via drug screening and application thereof
  • Porcine pseudorabies virus gene deletion attenuated vaccine strain for passage via low temperature of cell and attenuation via drug screening and application thereof

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Experimental program
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Effect test

Embodiment 1

[0028] Embodiment 1, the acquisition and molecular characteristic identification of porcine pseudorabies virus TP strain

[0029] Porcine pseudorabies virus HeN1 strain (this virus strain has been recorded in the patent application with publication number CN102994458, and the invention name is "Porcine pseudorabies virus virulent strain, its gene deletion vaccine strain and its application" has been deposited in China Microorganism General Microbiology Center of Species Preservation Management Committee, Microbial Preservation Number is CGMCC NO.6656.) The F10 generation of Vero cells that grow into a good monolayer is inoculated, the temperature of the cell incubator is adjusted to 35 ° C, and the virus is harvested after 2-3 days of cultivation. Freeze and thaw once to continue passage. When the virus adapts to this temperature and can cause more than 80% of the cells to become diseased in about 48 hours, lower the temperature of the incubator by 1-2°C, and so on, and gradua...

Embodiment 2

[0032] Example 2, Safety Evaluation of Porcine Pseudorabies Virus TP Strain (PRV TP, CGMCC No.12300) to Susceptible Animals

[0033] 1) Pathogenicity test of porcine pseudorabies virus TP strain to Balb / c mice

[0034] 55 Balb / c mice aged 7-8 weeks, 5 in each cage. The PRV TP strain and rPRV-gE - -EGFP + Virus strain (microorganism preservation number is CGMCC NO.6657) was diluted to 10 2.0 -10 6.0 TCID 50 / 0.2mL, 5 mice (1 cage) were inoculated with each dilution of the virus, each with 0.2mL. The other 5 mice served as negative controls. Observe and record the death situation of mice every day after inoculation, the results are shown in Table 1, calculate the half lethal dose LD of two strains of viruses to mice 50 . The results showed that the median lethal dose (LD) of PRV TP strains to mice 50 ) is 10 5.17 TCID 50 , while rPRV-gE - -EGFP + LD in mice 50 for 10 3.32 TCID 50 .

[0035] Table 1 Comparison of pathogenicity of different strains to Balb / c mice ...

Embodiment 3

[0042] Embodiment 3, the immune protection efficacy test of porcine pseudorabies virus TP strain to piglets

[0043] Fifteen 21-day-old piglets that were negative for PRV by antibody detection were randomly divided into 3 groups with 5 piglets in each group. Group A each intramuscularly injects 1 head portion (every head portion virus content is 10 5.0 TCID 50 ) PRV Bartha K61 vaccine, each head of group B was intramuscularly injected with 1 head (every head virus content was 10 5.0 TCID 50 ) PRV TP strain, group C was used as the challenge control. 14 days after immunization, each pig was inoculated with 2 mL containing 10 6.0 TCID 50 F5 generation of PRV HeN1 strain. Blood was collected weekly after immunization to separate serum, and anti-PRV gB and gE antibodies were detected using IDEXX kits. Body temperature was measured daily for 14 days after challenge, and clinical responses were observed. The results are shown in Table 3. The results showed that most of the pi...

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Abstract

The invention discloses a porcine pseudorabies virus gene deletion attenuated vaccine strain for passage via low temperature of a cell and attenuation via drug screening and an application thereof. The attenuated vaccine strain is prepared through the following steps: based on a porcine pseudorabies virus variant strain (named as strain HeN1, of which the microbial preservation serial No. is CGMCC No. 6656), firstly, carrying out low-temperature passage and screening on a Vero cell to obtain large fragments of deleted viruses including gI, gE, Us9, Us2 and part of inverted repeated sequence which exist in zone US through, and then making the TK gene thereof partially deleted through drug screening. The gene-deleted attenuated vaccine strain is named as strain PRV TP, of which the microbial preservation serial No. is CGMCC No. 12300. A live vaccine or an inactivated vaccine (a single vaccine or combined vaccine) can be prepared from the attenuated vaccine strain disclosed by the invention, and can prevent porcine pseudorabies effectively, and a reagent for diagnosing or treating porcine pseudorabies can be prepared from the attenuated vaccine strain too. According to the porcine pseudorabies virus gene deletion attenuated vaccine strain for passage via low temperature of a cell and attenuation via drug screening and the application thereof, the porcine pseudorabies attenuated vaccine strain PRV TP has the advantages of good safety, efficient protection, convenient differential diagnosis and the like.

Description

technical field [0001] The invention relates to a gene-deleted attenuated vaccine strain of porcine pseudorabies virus, and also relates to the application of the vaccine strain in preventing and treating porcine pseudorabies. The invention belongs to the technical field of medicine. Background technique [0002] Porcine Pseudorabies (PR) is an acute infectious disease caused by Porcine Pseudorabies Virus (PRV). All suckling piglets infected with pseudorabies virus have fever and neurological symptoms, and the mortality rate is almost as high as 100%. Under normal circumstances, there will be no obvious symptoms of infection in adult pigs, only mild symptoms such as sneezing, coughing, and elevated body temperature; infection of pregnant sows with pseudorabies virus can lead to abortion, stillbirth, weak offspring and mummified fetuses; boar infection Can cause orchiditis. The disease is highly contagious and can easily spread through the air and through the respiratory tr...

Claims

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Application Information

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IPC IPC(8): C12N7/00A61K39/245A61P31/22C12R1/93
CPCA61K39/12A61K2039/5254A61K2039/552C12N7/00C12N2710/16721C12N2710/16734
Inventor 田志军彭金美
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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