Recombinant baculovirus expressing senecavirus VP2 gene and preparation method and application thereof

A technology for recombining baculovirus and virus, applied in the biological field, can solve the problems of poor safety, strong return, insufficient inactivation, etc., and achieve the effects of high safety, low cost, and simple and efficient preparation method.

Active Publication Date: 2019-10-22
WUHAN KEQIAN BIOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The Chinese patent with application number 201810003888.2 discloses a method for preparing an inactivated vaccine using whole Seneca virus. However, the safety of the inactivated vaccine is poor in the production and application process, and the inactivation may be insufficient and may return to the animal body. strong potential danger

Method used

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  • Recombinant baculovirus expressing senecavirus VP2 gene and preparation method and application thereof
  • Recombinant baculovirus expressing senecavirus VP2 gene and preparation method and application thereof
  • Recombinant baculovirus expressing senecavirus VP2 gene and preparation method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0049] Example 1 Construction of recombinant baculovirus carrying porcine seneca virus VP2 protein coding gene

[0050] (1) Construction of recombinant transfer vector pFastdual-VP2

[0051] 1. Codon optimization of VP2 protein coding gene

[0052] The amino acid sequence (as shown in SEQ ID NO.1) and the nucleotide sequence of the coding gene (as shown in SEQ ID NO.2) of the porcine Seneca virus VP2 protein are obtained from the database. In order to better realize the high-level and high-purity expression of the VP2 protein, the present invention first optimizes the codons of the porcine Seneca virus VP2 protein according to the codon preference of the baculovirus. During the codon optimization process, the present invention found that the codon optimization of VP2 by using conventional codon optimization software or simply following the codon preference of the insect-baculovirus expression system, the expression level and purity of the coding gene sequence obtained It can...

Embodiment 2

[0071] Expression and purification of embodiment 2 target protein VP2

[0072] 1. Expression of the target protein VP2:

[0073] The recombinant baculoviruses Ac-VP2-1, Ac-VP2-2, and Ac-VP2-3 harvested in Example 1 were inoculated into suspension cultured insect cells High FiveTM (purchased from Invitrogen), and the inoculation dose was 0.01 MOI, The cell density is 0.8*10 6 / ml, the cell volume is 400ml. After 72-96h, the cell culture supernatant was harvested and tested by SDS-PAGE. The results were as follows: figure 2 As shown, the results showed that the VP2 expression level of Ac-VP2-3 was significantly higher than that of Ac-VP2-1 and Ac-VP2-2. Further combined with Western blotting to verify that the target protein is indeed VP2 protein. The above results show that the sequence shown in SEQ ID NO.8 obtained by specific artificial codon optimization and adding signal peptide and tag peptide sequences in the present invention can significantly increase the expressio...

Embodiment 3

[0076] Preparation of Example 3 Porcine Seneca Virus VP2 Subunit Vaccine

[0077]The VP2 protein purified in Example 2 was filtered after its concentration was measured, and emulsified with sterile MONTANIDETM ISA201VG adjuvant (purchased from SEPPIC Company) according to a mass ratio of 3:1 to prepare a porcine Seneca virus subunit vaccine, so that in each milliliter of vaccine The content of the VP2 antigen was 40 μg and stored at 4°C until use.

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Abstract

The invention relates to the field of biotechnology, in particular to recombinant baculovirus expressing senecavirus VP2 gene and a preparation method and application thereof. The recombinant baculovirus comprises one or more copies of senecavirus VP2 protein encoding gene. The invention further provides a senecavirus subunit vaccine which comprises the recombinant baculovirus expressed senecavirus VP2 protein. Accordingly, by means of artificial codon optimization, high-level and high-purity expression of senecavirus VP2 protein is achieved, and the immunogenicity of the VP2 protein is retained to the maximum extend. The senecavirus subunit vaccine has the excellent immunogenicity and high safety, and an ideal immune protection effect is achieved for infection of senecavirus.

Description

technical field [0001] The present invention relates to the field of biotechnology, in particular to a recombinant baculovirus expressing the VP2 gene of porcine Seneca virus type A, a preparation method and application thereof, a subunit vaccine containing the VP2 protein of porcine Seneca virus type A and its Preparation. Background technique [0002] Type A porcine Seneca virus (Senecavirus A, SVA) belongs to Picornaviridae (Picornaviridae) Senecavirus virus genus. SVA virus particles are composed of four structural proteins, namely VP1, VP2, VP3 and VP4, in which VP1, VP2 and VP3 proteins are exposed on the surface of the virus particle, and VP4 protein is embedded inside the virus particle. SVA can cause blisters and festering wounds at the nose and hoof crowns of pigs, resulting in lameness and even death. The mortality rate of infected piglets (within 4 days old) is as high as 30% to 70%. The infection and spread of the virus have a very negative impact on the pig b...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01C12N15/866C12N5/10A61K39/125A61P31/14
CPCA61K39/12A61K2039/552A61P31/14C07K14/005C12N15/86C12N2710/14043C12N2770/32022C12N2770/32034
Inventor 徐高原张华伟孙芳周明光郝根喜曾小燕朱娴静
Owner WUHAN KEQIAN BIOLOGY CO LTD
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