H7N9 subtype avian influenza virus-like particle vaccine preparation as well as preparation and application thereof

A bird flu virus and granule technology, which is applied in the direction of antiviral agents, double-stranded DNA viruses, antisense single-stranded RNA viruses, etc., to achieve the effect of facilitating large-scale production, increasing the cost of use, and inhibiting the detoxification of chickens

Active Publication Date: 2021-12-24
SOUTH CHINA AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In view of the problems faced in the development of avian influenza virus-like particle vaccines, it is urgent to develop an auxiliary preparation that can improve the cross-protection ability of the vaccine, so as to make up for the insufficient cross-protection efficacy of avian influenza virus-like particle vaccines against heterologous strains or mutant strains. Prevention and Control of H7N9 Avian Influenza Virus

Method used

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  • H7N9 subtype avian influenza virus-like particle vaccine preparation as well as preparation and application thereof
  • H7N9 subtype avian influenza virus-like particle vaccine preparation as well as preparation and application thereof
  • H7N9 subtype avian influenza virus-like particle vaccine preparation as well as preparation and application thereof

Examples

Experimental program
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Embodiment 1

[0045] The preparation of embodiment 1 avian influenza virus-like particles (H7N9-VLP)

[0046] (1) Construction of HA, NA, and M1 gene recombination transfer plasmids

[0047] (1) In this embodiment, the nucleotide sequences of the HA, NA, and M1 genes of the avian influenza virus have been codon-optimized, biased towards insect cell expression, and 6x his tags are added to the C-terminals of the HA, NA, and M1 genes; Artificially synthesized to obtain the nucleotide sequences of the codon-optimized HA, NA, and M1 genes, and respectively link them into the PUC57 vector to obtain the corresponding recombinant plasmids (Beijing Liuhe Huada Gene Technology Co., Ltd.); wherein, the codon-optimized The nucleotide and amino acid sequences of the HA gene, NA gene, and M1 gene are shown below.

[0048] Nucleotide sequence of HA gene after codon optimization:

[0049] ATGAACACTCAGATCCTGGTCTTCGCTCTGATCGCTATCATCCCCACTAACGCCGACAAGATCTGCCTGGGTCACCACGCTGTGAGCAACGGCACTAAGGTCAACACTCTGACTGA...

Embodiment 2

[0078] Example 2 Construction of Recombinant Baculovirus Plasmid Bacmid-HMN

[0079] (1) The present embodiment designs a recombinant chimeric protein HMN (amino acid sequence such as SEQ ID NO: 9) based on the conservative epitope of the avian influenza virus. The recombinant chimeric protein HMN is expressed by the conservative antigenic epitope of the avian influenza virus The conserved antigenic epitope of avian influenza virus includes amino acid residues 76-130 of hemagglutinin protein HA2 (HA2 76-130), matrix protein M2 Amino acid residues 2-24 of the extracellular domain (M2e 2-24) and amino acid residues 55-69 of the nucleoprotein NP (NP 55-69), the amino acid sequences of which are shown in SEQ ID NO: 1-3 , the amino acid sequence of bee venom signal peptide and 6x-His tag protein is shown in SEQ ID NO: 4-5; wherein, M2e 2-24 has four copies of tandem repeats, and each copy is connected by a flexible linker linker1 ; NP 55-69 has eight copies of tandem repeats, each...

Embodiment 3

[0088] The acquisition of embodiment 3 recombinant baculovirus BV-HMN

[0089] (1) Using the conventional liposome-mediated transfection method, the recombinant bacmid-HMN prepared in Example 2 was transfected into sf9 insect cells (Invitrogen). The specific steps of transfection are:

[0090] ①Confirm that the Sf9 cells in the six-well plate to be treated are in the logarithmic phase (1.0~2.5×10 6 cell / mL), and the cell viability is higher than 95%;

[0091] ② Take 1 μL of recombinant bacmid Bacmid-HMN (concentration above 1000ng / μL) and dilute it in 100 μL of Grace’s solution (Thermo Fisher Scientific), mix gently; mix before use II liposome, draw 6-8 μL into 100 μL Grace’s solution, briefly vortex and mix well; mix the diluted recombinant baculovirus plasmid Bacmid-HMN with the diluted II mix (total volume about 210μL), mix gently and incubate at room temperature for 15-30min to obtain DNA-liposome mixture;

[0092] ③ Pipette 800 μL of Grace's solution to the DNA-lipo...

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Abstract

The invention belongs to the technical field of biology and particularly relates to an H7N9 subtype avian influenza virus-like particle vaccine preparation as well as preparation and application thereof. The H7N9 subtype avian influenza virus-like particle vaccine preparation provided by the invention comprises a recombinant chimeric protein HMN and H7N9 subtype avian influenza virus-like particles, wherein the recombinant chimeric protein HMN is formed by chimeric combination of avian influenza virus conservative epitopes, bee venom signal peptides and 6x-His tag proteins. The recombinant chimeric protein HMN is combined with an H7N9 subtype avian influenza virus-like particle antigen, so that the defect that an avian influenza virus-like particle vaccine is insufficient in cross protection effect on variant strains is overcome, complete protection is provided for attack of the H7N9 subtype variant strains, and thus, the avian influenza epidemic situation is more effectively prevented and controlled.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a H7N9 subtype avian influenza virus-like particle vaccine preparation and its preparation and application. Background technique [0002] Avian Influenza virus (AIV) is an enveloped, segmented, negative-strand RNA virus belonging to the family Orthomyxoviridae and the genus Influenzavirus. Highly pathogenic avian influenza (HPAI) caused by the H7N9 subtype avian influenza virus has caused great harm to the poultry farming industry. What's more serious is that the H7N9 subtype avian influenza virus has broken through the interspecies barrier between birds and mammals and can cause human infection. Because the H7N9 subtype avian influenza virus poses a double threat to poultry farming and human health, the prevention and control of H7N9 subtype avian influenza can not only restore huge economic losses, but also has important public health significance. The current prevent...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/145A61K39/39A61K39/385A61P31/16C07K19/00C07K14/11C12N15/62C12N15/866C12N7/01
CPCA61K39/12A61K39/39A61K39/385A61P31/16C07K14/005C12N15/86C12N7/00C07K2319/02C07K2319/21C12N2760/16122C12N2760/16123C12N2760/16134C12N2710/14021C12N2710/14043C12N2800/105A61K2039/552A61K2039/6031Y02A50/30
Inventor 樊惠英孔德鑫廖明陈陶然胡小龙
Owner SOUTH CHINA AGRI UNIV
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