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Porcine pseudorabies virus gene-deleted strain, vaccine composition and their preparation method and application

A technology for porcine pseudorabies and vaccine composition, which is applied in the field of animal virology and can solve the problems of inability to identify wild virus strains and vaccine strain infections and the like

Inactive Publication Date: 2016-03-02
PULIKE BIOLOGICAL ENG INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The variant strain inactivated vaccine of porcine pseudorabies provided by the patent application CN103305474A has considerable limitations in production practice because it cannot identify wild strains and vaccine strain infections

Method used

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  • Porcine pseudorabies virus gene-deleted strain, vaccine composition and their preparation method and application
  • Porcine pseudorabies virus gene-deleted strain, vaccine composition and their preparation method and application
  • Porcine pseudorabies virus gene-deleted strain, vaccine composition and their preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Embodiment 1, collection and isolation of virus

[0058] Isolate the sample from the suspected porcine pseudorabies infection sample from Henan, collect pig brain tissue aseptically, add MEM culture medium at 1:10 (volume ratio), grind, prepare tissue suspension, after repeated freezing and thawing 3 times, 2000r Centrifuge at 1 / min for 15 min, collect the supernatant, and then filter through a 0.2 μm membrane filter, subculture on PK-15 cells at 37°C for 1 hour, replace with MEM medium containing 2% calf serum, and culture at 37°C for 5 days. Harvest the poisonous culture medium, freeze and thaw twice, collect the poison, and add MEM culture medium containing 2% calf serum. Porcine pseudorabies virus PCR detection kit (Beijing Century Yuanheng Animal Epidemic Prevention Technology Co., Ltd.) was used to detect porcine pseudorabies virus, and the result was positive; the isolated virus was detected by using PCR kit to detect exogenous virus (porcine blue ear disease Vi...

Embodiment 2

[0060] Embodiment 2, the genetic characteristic of isolated virus

[0061] The genetic characteristics of the virus isolated in Example 1 were determined by genetic analysis. The genomic DNA of porcine pseudorabies virus isolated on PK15 cells was used as a template, and the primers shown in Table 1 were used for PCR. Primer Premier 5.0 was used to design primer sequences for amplifying gB, gC, and gD genes, respectively.

[0062] Using the extracted genomic DNA as a template, prepare the PCR amplification system as follows: template DNA 100 μg, PrimerSTARHSDNAPolymerase (2.5U / μl) 0.5μl, 2×PrimerSTARGCBuffer 25μl, upstream and downstream primers 1μl (10pmol / μl), dNTPMix (2.5mMeach) 4μl, And the volume was made up to 50 μl with distilled water. A two-step PCR reaction was carried out: denaturation at 98°C for 10 sec, and then annealing and extension at 68°C (the required time was calculated according to 1 kb / min), a total of 30 cycles. The PCR reaction was terminated at 4°C....

Embodiment 3

[0065] Embodiment 3, the pathogenicity test of virus

[0066] 3.1 Pathogenicity of piglets of different ages

[0067] Six 34- to 35-day-old swine pseudorabies antibody-negative piglets were randomly divided into 2 groups, 4 pigs / group (test group) and 2 pigs / group (control group), and inoculated with porcine pseudorabies virus HN1201 strain (challenge dose 2×10 8.0 TCID 50 / head), the control group was inoculated with DMEM medium; at the same time, four 49-day-old piglets were inoculated with the virulent HN1201 strain (the challenge dose was 2×10 8.0 TCID 50 per head), and 35-day-old piglets were still used as the control. After the virus inoculation, the body temperature of the piglets was measured every day, and the clinical symptoms and death were observed. The specific results are shown in Table 2.

[0068] Table 2 Pathogenicity of porcine pseudorabies virulent HN1201 strain to piglets of different ages

[0069]

[0070]

[0071] The results showed that inoculat...

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Abstract

The invention provides a vaccine composition, comprising porcine pseudorabies virus gE gene-deleted strain of immunizing dose, or an attenuated live totivirus antigen or inactivated totivirus antigen of the culture thereof. The vaccine composition is effective in inducing generation of antibodies to provide effective protection for swine and can be used as a marker vaccine to effectively distinguish wild strains and vaccine strains.

Description

[0001] This application is a divisional application of patent application 201410059931.9 technical field [0002] The invention relates to a porcine pseudorabies virus gene deletion strain, a prepared vaccine composition thereof, a preparation method and application thereof, and belongs to the field of animal virology. Background technique [0003] Pseudorabies, also known as Aujeszky's disease, is a kind of pig, cattle, sheep and other domestic animals, poultry and wild animals caused by porcine herpesvirus type Ⅰ (Suidherpesvirus 1 strain) in the α subfamily of Herpesviridae (Herpesviridae) An acute infectious disease characterized by fever, severe itching (except for pigs) and encephalomyelitis. Pseudorabies in pigs is widespread in our country and is seriously harmful. It is one of the main diseases restricting the production of large-scale pig farms. It can cause abortion in pregnant sows, stillbirth or mummified fetuses and piglets with neurological symptoms, paralysi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00A61K39/245A61P31/22C12R1/93
Inventor 张许科孙进忠田克恭谭菲菲
Owner PULIKE BIOLOGICAL ENG INC
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