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Preparing method of porcine pseudorabies virus subunit vaccine, vaccine composition and application

A technique for porcine pseudorabies and vaccine composition, which is applied in the field of preparation method and vaccine composition prepared by the same, can solve the problems of application limitation, immunogenicity inferior to attenuated vaccine and inactivated vaccine, and high production cost

Active Publication Date: 2017-01-04
PU LIKE BIO ENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Subunit vaccines do not contain nucleic acid substances, are relatively safe, and will not produce persistent or latent infections after vaccination. The immune response produced can be distinguished from wild virus infections, which is conducive to the control and elimination of diseases. The production cost of rabies virus subunit vaccine is high, the immunogenicity is not as good as attenuated vaccine and inactivated vaccine, and the application is limited

Method used

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  • Preparing method of porcine pseudorabies virus subunit vaccine, vaccine composition and application
  • Preparing method of porcine pseudorabies virus subunit vaccine, vaccine composition and application
  • Preparing method of porcine pseudorabies virus subunit vaccine, vaccine composition and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Example 1 Tandem expression of porcine pseudorabies virus gB protein fragment and gD protein

[0060] 1. Amplification of porcine pseudorabies virus gB protein fragment

[0061]The PRV HN1201 virus was inoculated on the well-growing PK15 cells, and 200 μL of the harvested virus liquid was taken, and the PRV genomic DNA was extracted according to the instructions of the viral nucleic acid extraction kit II kit from geneaid company. Use primers gBF1 and gBR1 to expand gB gene 62-148aa, use primers gBF2 and gBR2 to expand gB gene 546-700aa, use Overlapping PCR to amplify the two together, and use the primer design to add GGSG linking amino acids between the two. See Table 1 for the primers, Table 2 for the PCR system, and Table 3 for the PCR reaction conditions.

[0062] Table 1 Primers for amplification of gB protein fragments

[0063]

[0064] Table 2 PCR system

[0065] 2×PrimeSTAR GC buffer 25 μL PRV gDNA 1μL primers (10pM) 1μL / 1μL ...

Embodiment 2

[0086] Example 2 Preparation of porcine pseudorabies virus gB protein

[0087] 1. Amplification of porcine pseudorabies virus gB gene

[0088] Using the PRV genomic DNA extracted in Example 1 as a template, primers gBF and gBR were used to amplify HNgB gene 62-752aa. See Table 6 for the primers, Table 2 for the PCR system, and Table 3 for the reaction conditions.

[0089] Table 6 gB gene amplification primers

[0090]

[0091]

[0092] 2. Construction of Donor Plasmids

[0093] Referring to the method for constructing the donor plasmid in Example 1, the PCR product amplified in step 1 was recovered to construct the donor plasmid, which was identified and named pFastBac-HNgB correctly.

[0094] 3. Construction of recombined Bacmid

[0095] Referring to the construction method of the recombinant Bacmid in Example 1, the donor plasmid obtained in step 2 was used to construct the recombinant Bacmid, and the identified correct recombinant Bacmid was named Bac-HNgB.

[00...

Embodiment 3

[0100] Example 3 Preparation of porcine pseudorabies virus gD protein

[0101] 1. Amplification of porcine pseudorabies virus gD gene

[0102] Using the PRV genomic DNA extracted in Example 1 as a template, the primers gD18F and gD353R in Table 4 in Example 1 were used to amplify the gD gene. The PCR system is based on Table 2, and the reaction conditions are based on Table 3.

[0103] 2. Construction of Donor Plasmids

[0104] Referring to the construction method of the donor plasmid in Example 1, the PCR product amplified in step 1 was recovered to construct the donor plasmid, which was identified as pFastBac-HNgD correctly.

[0105] 3. Construction of recombined Bacmid

[0106] Referring to the construction method of the recombinant Bacmid in Example 1, the donor plasmid obtained in step 2 was used to construct the recombinant Bacmid, and the identified correct recombinant Bacmid was named Bac-HNgD.

[0107] 4. Acquisition and passage of recombinant baculovirus

[0108...

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Abstract

The invention relates to a preparing method of porcine pseudorabies virus subunit vaccine composition. The preparing method includes the steps that 1, a gB protein fragment gene and a gD protein gene are cloned and amplified respectively; 2, the amplified gB protein fragment gene and the amplified gD protein gene are used for constructing plasmid of tandem expression gB protein and gD protein; 3, gB+gD recombinant protein is expressed through the obtained plasmid of tandem expression gB protein and gD protein and purified, adjuvant is added, and emulsification is carried out. The preparing method is simple, porcine pseudorabies virus gB and gD protein can be prepared in quantity, shorter time is spent, the expression amount is large, the production cost is greatly reduced, which is beneficial for large-scale production. The virus subunit vaccine containing the prepared gB and gD protein is good in immune effect and small in immune amount and capable of effectively preventing diseases related to the porcine pseudorabies virus and related infectious diseases caused by the porcine pseudorabies virus.

Description

technical field [0001] The invention belongs to the field of veterinary biological products, and in particular relates to a preparation method of a porcine pseudorabies virus subunit vaccine, a vaccine composition prepared therefrom, and an application of the vaccine composition. Background technique [0002] Pseudorabies, also known as Aujeszky's disease, is a disease caused by porcine herpesvirus type Ⅰ (Suid herpesvirus 1) pseudorabies virus (PRV) in pigs, cattle, sheep, etc An acute infectious disease of a variety of domestic animals, poultry and wild animals with fever, severe itching (except pigs) and encephalomyelitis as the main symptoms. PRV has strong pantropicity, neurotropicity, and latent infection characteristics. It can be latently infected in the peripheral nervous system for a long time. When the latent virus is activated and becomes an infectious virus, the latently infected host will develop the disease. Pseudorabies in pigs is widespread in our country a...

Claims

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Application Information

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IPC IPC(8): A61K39/245C12N15/85A61P31/22
Inventor 田克恭王同燕孙进忠张许科
Owner PU LIKE BIO ENG
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