Recombinant avian influenza subunit vaccine and preparation method thereof

A technology of subunits and recombinant antigens, applied in the fields of botanical equipment and methods, biochemical equipment and methods, recombinant DNA technology, etc., can solve the problems of long production cycle and high production cost

Active Publication Date: 2021-07-23
SHANXI ACAD OF ADVANCED RES & INNOVATION
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Its production cost is higher and the production cycle is longer

Method used

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  • Recombinant avian influenza subunit vaccine and preparation method thereof
  • Recombinant avian influenza subunit vaccine and preparation method thereof
  • Recombinant avian influenza subunit vaccine and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0084] 1. Plasmid construction of H5 subtype HA gene and NA gene:

[0085] We double digested the DNA fragment encoding HA or NA (EcoRI and XhoI restriction sites) into the PCAGGS vector (map as figure 1 ), and adding a signal peptide sequence before the coding fragment of the protein is helpful for protein secretion and expression, inserting the coding sequence of 6 histidine tags (hexa-His-tag) and the translation stop codon after the coding fragment of the protein, In this way, a recombinant protein with a histidine tag at the C-terminal will be obtained, which is convenient for subsequent protein purification. The recombinant plasmids were confirmed by PCR, enzyme digestion and sequencing to confirm that the inserted foreign fragments were completely correct. When HA is expressed in mammalian cells, sialic acid will be attached to it, resulting in no ability to bind to receptors. Therefore, NA is also expressed, and the gene of 09NA is selected, and FLAG tag is added for ...

Embodiment 2

[0098] Embodiment 2 H5 / H1-LSQ vaccine preparation

[0099] The protein concentration of H5 / H1-LSQ is 10 mg / ml, dilute the protein with PBS, mix the protein with an equal volume of Freund's complete adjuvant or Freund's incomplete adjuvant, and emulsify completely until the bath water does not dissolve for animal inoculation.

Embodiment 3

[0103] 1. Experimental animals: BALB / c female mice aged 4-6 weeks, 6 in each group, and the way of immunization is intramuscular injection.

[0104] 2. Group settings: ① Negative control (adjuvant MF59); ② Positive control (Influenza virus split vaccine TIV); ③ Experimental group H5 / H1-LSQ (vaccine prepared in Example 2); ④ Experimental group H5 / H1 (the vaccine prepared in Comparative Example 1).

[0105] 3. Experimental steps:

[0106] 3.1 Animal immunity:

[0107] First immunization: the immunization dose of the negative group and the experimental group is 100 μL for each mouse; the immunization dose of the positive group is 100 μL for each mouse.

[0108] The immunization dose of each animal in the experimental group is 20μg / 100μL / animal, dilute the protein with PBS, 6 animals in each group, mix different experimental group proteins with an equal volume of MF59 adjuvant, shake until completely emulsified until the bath water does not melt before use immune to animals.

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Abstract

The invention relates to the technical field of vaccine preparation, and especially relates to a recombinant avian influenza subunit vaccine and a preparation method thereof. A head structure in a main protective antigen HA skeleton of H5 subtype avian influenza is reserved, and a neck area of a vaccine H5 is replaced with an influenza HA neck area having broad-spectrum immune protection after structural biology design and transformation, so that the neck area can be proved to protect mutant strains of different H5 and provide broad-spectrum protection on H7 in a mouse experiment.

Description

technical field [0001] The invention relates to the technical field of vaccine preparation, in particular to a recombinant avian influenza subunit vaccine and a preparation method thereof. Background technique [0002] Influenza virus belongs to the Orthomyxoviridae family and is a segmented RNA virus with a single negative strand. According to the different antigenic determinants on nucleoprotein (NP) and matrix protein (M1), influenza viruses can be divided into four types: A, B, C, and D, among which A and B can pose a threat to human health, especially It is type A, which causes about 300,000 to 500,000 deaths each year due to global seasonal influenza and sporadic influenza, with a mortality rate as high as 60%. According to the influenza virus infection objects, viruses can be divided into groups such as human influenza virus, swine influenza virus, equine influenza virus and avian influenza virus. Avian influenza is a type of zoonotic infectious disease caused by av...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/11C07K19/00C12N15/44C12N15/62A61K39/145A61P31/16
CPCC07K14/005A61K39/12A61P31/16C07K2319/02C07K2319/21C07K2319/43C12N2760/16121C12N2760/16134
Inventor 仝舟杨佳成志敏邢潇王洋高山高福
Owner SHANXI ACAD OF ADVANCED RES & INNOVATION
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