H5 subtype avian influenza vaccine strain capable of distinguishing immunized and infected animals, and preparation method and application thereof

A technology for avian influenza and vaccine strains, which is applied in the field of preparing vaccine strains for distinguishing immunity and infection of H5 subtype avian influenza, can solve the problems of inability to completely eliminate highly pathogenic avian influenza, antigenic drift, and antigenicity differences of vaccine strains, and achieve Highlight the significance of public health safety, avoid antigenic variation, good replication and growth effects

Active Publication Date: 2018-06-01
ZHEJIAN DIFFERENCE BIOLOGICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, the rescued A / B type NA chimeric virus may have defects in growth performance and needs to be continuously passaged in vitro to adapt
Continuous passage will introduce adaptive mutations, causing the risk of antigenic drift, resulting in a large difference in the antigenicity of the prepared vaccine strains from the original wild epidemic strains
[0004] Although the existing H5 subtype whole virus inactivated vaccine has the advantages of certain immune effect and low price, it cannot serologically distinguish immune animals from infected animals (DIVA), which seriously affects the monitoring of virus epidemics and hinders the detection of Thorough purification of H5 subtype avian influenza in farms
Since the existence of highly pathogenic avian influenza in farms cannot be completely eliminated, the highly pathogenic H5 virus has the risk of continuously threatening public health security

Method used

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  • H5 subtype avian influenza vaccine strain capable of distinguishing immunized and infected animals, and preparation method and application thereof
  • H5 subtype avian influenza vaccine strain capable of distinguishing immunized and infected animals, and preparation method and application thereof
  • H5 subtype avian influenza vaccine strain capable of distinguishing immunized and infected animals, and preparation method and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] The preparation method of embodiment 1 avian influenza vaccine strain Re-MuH5-DIVA-ΔNS virus

[0044] (1) Construction of low pathogenicity HA mutant gene

[0045] The pFlu vector is a bidirectional transcription vector, which can not only transcribe the RNA of the complete virus at the human polI promoter, but also transcribe the viral mRNA under the CMV promoter, thereby synthesizing the viral protein (Hoffmann et al., PNAS, USA 97,6108- 6113, 2000).

[0046] Artificially synthesized wild-type H5 gene (A / Duck / Hubei / 49 / 2005), the highly conserved sequence (RERRRKRGLF) in the highly pathogenic wild-type HA amino acid sequence was mutated into a low-pathogenic amino acid sequence by site-directed mutagenesis (RETRGLF) to obtain the corresponding low pathogenicity MuH5HA gene sequence. Cloning the modified MuH5HA gene into the pFlu vector through the BsmBI site to obtain the recombinant plasmid pFlu-MuH5HA, the construction principle is as follows figure 2 shown.

[...

Embodiment 2

[0054] The preparation method of embodiment 2 avian influenza vaccine strain Re-MuH5-DIVA-ΔNS virus

[0055] The preparation method in this embodiment 2 is the same as that of embodiment 1, except that figure 1When the A / B chimeric NA gene is artificially synthesized, the DNA sequence encoding the amino acid sequence of the extracellular domain protein in the influenza B virus NA is different from that in Example 1, and the others are the same as in Example 1.

[0056] In this embodiment, the DNA sequence encoding the amino acid sequence of the extracellular domain protein (SEQ ID NO: 6) in influenza B virus NA is shown in SEQ ID NO: 7, as the tag gene sequence, shown in SEQ ID NO: 7 The sequence of B is from B / Brisbane / 60 / 2008 in the Victoria group of influenza virus B (Ping J et al, PNAS, 2016, 113(51):E8296-E8305).

[0057] Next, the Re-MuH5-DIVA-ΔNS vaccine strain prepared by the present invention is further tested for its effect.

[0058] Method: the Re-MuH5-DIVA-ΔNS va...

Embodiment 3

[0064] Embodiment 3 Preparation of Re-MuH5-DIVA-ΔNS inactivated vaccine

[0065] Collect 50 ml of the allantoic fluid of Re-MuH5-DIVA-ΔNS vaccine strain F0, F1, F2 or F3 prepared in the above example, and inactivate it with formalin solution with a final concentration of 0.25% at 37°C for 24 hours. Add 2% Tween-80 to the inactivated allantoic fluid, emulsify with white oil containing 3% Span 80 after fully dissolving, the emulsification ratio is 1:3, the shear emulsification speed is 12000rpm, 3min. Through dosage form inspection, particle size inspection, viscosity inspection, and stability inspection, it is determined that the inactivated oil seedlings are milky white water-in-oil emulsion with low viscosity, uniform particle size, good stability, and suitable for injection.

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Abstract

The invention discloses an H5 subtype avian influenza vaccine strain capable of distinguishing immunized and infected animals, and a preparation method and application thereof. Highly-pathogenic H5 subtype avian influenza brings in great economic loss to the animal husbandry and poses a severe threat to public health. Although conventional H5 subtype avian influenza totivirus inactivated vaccineshave the advantages of definite effect, low cost, a wide application scope and the like, but cannot serologically distinguish immunized and infected animals, which brings in great obstacles for monitoring and purifying of avian influenza. According to the invention, the NA of B type influenza is used as a label so as to successfully establish the method for constructing the H5 subtype avian influenza vaccine strain capable of distinguishing immunized and infected animals. The H5 subtype avian influenza vaccine strain has critical application value and public health significance to prevention,control and purification of H5 subtype avian influenza.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering vaccines, and relates to a method and application for preparing vaccine strains for distinguishing immunity and infection of H5 subtype avian influenza. Background technique [0002] Avian influenza viruses belong to the family Orthomyxoviridae and the genus Influenzavirus. Influenza viruses can be divided into A, B, and C types according to their antigenicity. Type A influenza has a wide range of hosts (including birds, humans, pigs, etc.) and is more pathogenic and harmful. Influenza B only infects humans and seals, and its pathogenicity is relatively low. Type C influenza virus, found only in humans and pigs. The genomes of influenza A and B can be divided into eight gene segments: PB2, PB1, PA, NP, HA, NA, M, and NS. After the host is infected, a large number of antibodies can be produced against HA, NA, M1 and NP. Among them, HA can directly induce the main neutralizing antibo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/62C07K19/00C12N7/01C12N15/87A61K39/145A61P31/16
CPCA61K39/12A61K2039/5252A61K2039/552C07K14/005C07K2319/02C07K2319/03C12N7/00C12N15/87C12N2760/16221C12N2760/16222C12N2760/16234A61K39/145A61K2039/5254C12N2760/16121C12N2760/16122C12N2760/16134A61K2039/53C12N15/113
Inventor 宋家升
Owner ZHEJIAN DIFFERENCE BIOLOGICAL TECH CO LTD
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