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A dual subunit vaccine of porcine parvovirus and Japanese encephalitis and its preparation method and application

A technology of parvovirus and Japanese encephalitis, applied in biochemical equipment and methods, viruses, vaccines, etc., can solve the problems of strong vaccine virulence, increase the number of immunizations, increase pig body stimulation, etc., and achieve good immunogenicity and safety, increase the level of secreted expression, and retain the effect of immunogenicity

Active Publication Date: 2022-05-03
WUHAN KEQIAN BIOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] These two viruses are important infectious diseases that cause reproductive disorders in sows. They are prevalent in pig herds and mainly cause stillbirths, deformed fetuses and mummified fetuses in sows, causing great managerial losses to pig farms and seriously restricting pig farming. develop
At present, the prevention and control of such infectious diseases in the existing technology mainly relies on vaccination, but in recent years, the phenomenon of mixed infection of pigs with various pathogens has become more and more common, making the disease more and more complicated, which not only brings great challenges to clinical diagnosis and prevention and control. Great difficulties, but also brought huge economic losses to the pig farm
At present, the prevention and treatment of these two diseases is mainly to immunize PPV inactivated vaccine and JEV attenuated single vaccine respectively. In large-scale pig farms, the number of immunizations is increased, the stimulation to the pig body is increased, and the pig body produces a stress response; The Japanese encephalitis virus vaccine is a live vaccine, frequent and repeated use increases the possibility of the vaccine's virulence returning to strength

Method used

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  • A dual subunit vaccine of porcine parvovirus and Japanese encephalitis and its preparation method and application
  • A dual subunit vaccine of porcine parvovirus and Japanese encephalitis and its preparation method and application
  • A dual subunit vaccine of porcine parvovirus and Japanese encephalitis and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] (1) Construction of recombinant transfer vectors pFastBac-PPV VP2 and pFastBac-JEV E

[0034] 1. Obtain the target gene PPV-VP2, JEV-E

[0035] According to insect cell High Five TM artificially synthesized 1836bp PPV-VP2 and 1467bp JEV-E, and introduced the AcMNPV GP64 signal peptide sequence at the amino terminus, and introduced the 8*His tag peptide sequence at the carboxy terminus, laying the foundation for the affinity purification of the target protein . The synthetic gene sequences are shown in SEQ ID NO:1 and SEQ ID NO:2. The synthetic target genes PPV-VP2 and JEV-E were placed on the plasmid vector pUC-18 (Invitrogen Company), that is, obtained in the form of pUC-PPV VP2 and pUC-JEV E.

[0036] 2. Construction of recombinant transfer vectors pFastBac-PPV VP2 and pFastBac-JEV E

[0037] The recombinant transfer vectors pFastBac-PPV VP2 and pFastBac-JEV E were constructed using conventional recombinant plasmid construction procedures and methods:

[0038] ① En...

Embodiment 2

[0059] (1), preparation of porcine parvovirus VP2 and Japanese encephalitis virus E protein dual genetic engineering vaccine

[0060] In this example, the PPV VP2 and JEV E proteins purified in Example 1 were filtered after the concentration determination, and the two were mixed in equal amounts, and then emulsified with ISA 15VG adjuvant (purchased from SEPPIC Company) at a ratio of 4:1 to prepare sub- The unit double vaccine, so that the content of PPV VP2 and JEV E antigen in each ml of vaccine is 40ug, and stored at 4°C until use.

[0061] (2) Safety evaluation of porcine parvovirus VP2 and Japanese encephalitis virus E protein dual genetic engineering vaccine

[0062] Purchase 10 female Balb / C mice weighing 16-18g, and divide them into two groups, A and B, with 5 mice in each group. Each mouse in group A was subcutaneously injected with 0.3ml of the VP2 and E protein double vaccine prepared in step (1); each mouse in group B was injected with 0.3ml of dialysis buffer (20...

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Abstract

The invention relates to a dual subunit vaccine of porcine parvovirus and Japanese encephalitis and its preparation method and application. The present invention optimizes the nucleotide sequences encoding porcine parvovirus VP2 protein and Japanese encephalitis E protein, and expresses them using the insect-baculovirus eukaryotic expression system after adding signal peptide GP64 to obtain porcine parvovirus VP2 protein and Japanese encephalitis E protein. The structure and function of type encephalitis E protein are completely consistent with the natural state, and the expression level is significantly increased, and its immunogenicity is retained to the greatest extent. After being prepared with an adjuvant to prepare a double subunit vaccine, it can simultaneously treat porcine parvovirus and porcine parvovirus. The Japanese encephalitis plays a better immune protection effect, and the protective effect is better than the existing porcine parvovirus inactivated vaccine and Japanese encephalitis virus attenuated vaccine. Compared with monovalent vaccines, it has the advantages of simplicity, economy, practicality and stress reduction.

Description

technical field [0001] The invention relates to the technical field of veterinary biological products, in particular to a dual subunit vaccine of porcine parvovirus and Japanese encephalitis and its preparation method and application. Background technique [0002] Porcine parvovirus (PPV) is a member of the Parvovirus genus in the Parvoviridae family. It is a small non-enveloped virus with a single-stranded DNA genome structure. The diameter of the virus particle is about 20nm and the genome is about 5.2Kb. PPV has two open reading frames (ORFs), including nonstructural protein (NS1) and major structural protein (VP2). The VP2 protein is the main target for inducing neutralizing antibodies. The virus is the main disease causing reproductive disorders in pigs. The disease mainly manifests as infection and death of embryos and fetuses, especially stillbirths, deformed fetuses and mummified fetuses in primiparous sows, but the sows themselves have no obvious symptoms. The viru...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K39/295A61K39/23A61K39/12A61P31/14A61P31/20
CPCA61K39/12A61P31/20A61P31/14C12N2750/14334C12N2770/24134A61K2039/552A61K2039/70Y02A50/30
Inventor 周明光张华伟陈映瑾徐高原曾小燕朱娴静金建云陈章表
Owner WUHAN KEQIAN BIOLOGY CO LTD
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