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Porcine parvovirus and Japanese encephalitis bivalent subunit vaccine as well as preparation method and application thereof

A parvovirus, Japanese encephalitis technology, applied in biochemical equipment and methods, vaccines, viruses, etc., can solve the problems of increasing the stimulation of pigs, increasing the number of immunizations, and the virulence of vaccines, and achieving retention of immunogenicity , the effect of improving the secretion expression level and the high expression amount

Active Publication Date: 2021-01-05
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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  • Porcine parvovirus and Japanese encephalitis bivalent subunit vaccine as well as preparation method and application thereof
  • Porcine parvovirus and Japanese encephalitis bivalent subunit vaccine as well as preparation method and application thereof
  • Porcine parvovirus and Japanese encephalitis bivalent subunit vaccine as well as preparation method and application thereof

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] ①Enz...

Embodiment 2

[0059] (1) Preparation of porcine parvovirus VP2 and Japanese encephalitis virus E protein duplex genetic engineering vaccine

[0060] In this example, the PPV VP2 and JEV E proteins purified in Example 1 were measured for concentration, filtered, and the two were mixed in equal amounts, and then emulsified with ISA 15VG adjuvant (purchased from SEPPIC) at a ratio of 4:1 to prepare a Unit double vaccine, so that the content of PPV VP2 and JEV E antigens in each milliliter of vaccine is 40ug, and stored at 4°C for later use.

[0061] (2) Safety evaluation of porcine parvovirus VP2 and Japanese encephalitis virus E protein duplex genetically engineered vaccine

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

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Abstract

The invention relates to a porcine parvovirus and Japanese encephalitis bivalent subunit vaccine as well as a preparation method and application thereof. The nucleotide sequences for encoding the porcine parvovirus VP2 protein and the Japanese encephalitis E protein are optimized and matched with the signal peptide GP64, and then an insect-baculovirus eukaryotic expression system is used for expression, so that the obtained porcine parvovirus VP2 protein and Japanese encephalitis E protein are completely consistent in structure, function and natural state, and the expression quantity is remarkably increased, and the immunogenicity is vaccine is maintained to the maximum degree; after the bivalent subunit vaccine is prepared from the bivalent subunit vaccine and an adjuvant, a relatively good immune protection effect can be achieved on the porcine parvovirus and the Japanese encephalitis at the same time, and the protection effect exceeds that of an existing porcine parvovirus inactivated vaccine and an existing Japanese encephalitis virus attenuated vaccine. Compared with a monovalent vaccine, the bivalent subunit vaccine has the advantages of simplicity, convenience, economy, practicability 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 Applications(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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