Pig pseudorabies virus subunit vaccine and preparation method and application thereof

A technology of porcine pseudorabies virus and subunit vaccines, which is applied in the fields of botany equipment and methods, biochemical equipment and methods, applications, etc., can solve the problem of immunogenicity inferior to attenuated vaccines and inactivated vaccines, lack of safe and effective vaccines, Problems such as high production cost, to achieve good immune effect, conducive to large-scale production, and low cost

Inactive Publication Date: 2017-12-19
扬州优邦生物药品有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

5% carbolic acid was inactivated by 2min, but 0.5% carbolic acid was still infectious after 32 days
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

Method used

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  • Pig pseudorabies virus subunit vaccine and preparation method and application thereof
  • Pig pseudorabies virus subunit vaccine and preparation method and application thereof
  • Pig pseudorabies virus subunit vaccine and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Expression of embodiment 1 fusion protein gB-STLT-gC and gB-gC

[0040] 1. Amplification of porcine pseudorabies virus gB protein fragment and gB-STLT amplification

[0041] Take 200 μL of virus liquid (stored by Yangzhou Youbang Biopharmaceutical Co., Ltd.), and extract the total PRV DNA according to the instructions of DNAisoReagent reagent from TaKaRa Company. Use primers gBF and gBR to amplify the 529-744 amino acid fragment of gB gene, use the STLT gene synthesized by Nanjing GenScript, and use overlap PCR to amplify the two together (primers are gBF and STLTR). The primers are shown in Table 1, the PCR reaction system is shown in Table 2, and the PCR reaction conditions are shown in Table 3.

[0042]The primers gBF and gBR' were used to amplify the fragment encoding 529-744 amino acids of the gB' gene. The primers are shown in Table 1, the PCR reaction system is shown in Table 2, and the PCR reaction conditions are shown in Table 3.

[0043] Table 1 PCR primer l...

Embodiment 2

[0067] The preparation of embodiment 2 porcine pseudorabies virus subunit vaccine

[0068] Take the purified gB-STLT-gC and gB-gC recombinant proteins obtained in Example 1, add an adjuvant to emulsify, mix well, and store at 4°C. See Tables 5 and 6 for specific vaccine ratios.

[0069] Table 5 Composition ratio of porcine pseudorabies virus virus subunit vaccine gB-STLT-gC

[0070]

[0071] Table 6 The composition ratio of gB-gC of porcine pseudorabies virus virus subunit vaccine

[0072]

Embodiment 3

[0073] Example 3 Porcine pseudorabies virus subunit vaccine immunogenicity test

[0074] Forty 28-day-old weaned piglets with negative gE antibody and positive gB antibody were selected, randomly divided into 8 groups, 5 pigs in each group, and kept in isolation. Groups 1 to 6 were respectively immunized with vaccine 1, vaccine 2, vaccine 3, vaccine 4, vaccine 5 and vaccine 6 prepared in Example 2, and groups 7 and 8 were injected with the same dose of sterile PBS. Twenty-eight days after immunization, the pig pseudorabies mutant strain (isolated by Yangzhou Youbang Biopharmaceutical Co., Ltd. in 2015 and sent to a pig farm in Jiangxi for inspection, named JX-15) was used to challenge the virus in groups 1 to 7 with nasal drops. The attack dose is 1×10 8.0 TICD 50 / mL. Piglets were observed clinically every day after challenge, and rectal temperature was measured and recorded.

[0075] Results Under this challenge dose, the 5 pigs in the first group developed fever and mil...

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Abstract

The invention discloses a pig pseudorabies virus subunit vaccine and a preparation method and an application thereof, which belong to the field of a biological product used for animal. The method comprises the following steps: 1) respectively cloning and amplifying a gB protein fragment gene and a gC protein fragment gene; 2) performing artificial synthesis of an escherichia coli enterotoxin gene STLT; 3) constructing plasmid for expressing fusion protein gB-STLT-gC; and 4) expressing to obtain a gB-STLT-gC recombinant protein, purifying the recombinant protein, adding an adjuvant, and performing emulsification to obtain the pig pseudorabies virus subunit vaccine. The preparation method is simple, the antigen protein of pig pseudorabies virus is massively prepared, the method has the advantages of short time consuming, high expression level, and greatly reduced production cost, and is in favor of large scale production, the obtained subunit vaccine has the advantages of good immunization effect and small immunization dosage, and can effectively prevent the pig pseudorabies virus-related disease and disease related to infection due to pig pseudorabies virus.

Description

technical field [0001] The invention relates to a porcine pseudorabies virus subunit vaccine and a preparation method and application thereof, belonging to the field of veterinary biological products. Background technique [0002] Pseudorabies virus belongs to the family Herpesviridae and the genus Porcine Herpesvirus. Virus particles are round, with a diameter of 150-180 nm and a nucleocapsid diameter of 105-110 nm. The outermost layer of the virion is the viral envelope, which is a lipid bilayer structure derived from the host cell. On the surface of the capsule, there are fibrous processes about 8-10 nm in radial arrangement. [0003] Pseudorabies virus is one of the more resistant herpesviridae. The half-life at 37°C is 7 hours, it can survive for 46 days at 8°C, and it can survive for 10-30 days on dry grass, branches, and food at 25°C. However, when storing the virus for a short period of time, freezing at 4°C is better than freezing at -15°C and -20°C Save better. ...

Claims

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Application Information

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IPC IPC(8): A61K39/245A61K39/39A61P31/22C12N15/62C12N15/866C12N5/10C07K19/00
CPCA61K39/12A61K39/39A61K2039/552C07K14/005C07K2319/55C12N15/86C12N2710/14043C12N2710/16722C12N2710/16734
Inventor 丁国伟宋庆庆李玉安叶正琴许兆君杨豫蒙
Owner 扬州优邦生物药品有限公司
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