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Polypeptide vaccine for resisting multiple hypotype avian influenza viruses and preparation method thereof

An avian influenza virus and polypeptide vaccine technology, which is applied in the directions of antiviral agents, chemical instruments and methods, and medical preparations containing active ingredients, etc., to achieve the effects of long validity period, convenient transportation and storage, and simple production process

Inactive Publication Date: 2010-12-08
XIAMEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Previously, several research groups have successively studied the fusion gene expression and immunogenicity of M2e and HBcAg, and achieved good immune protection after immunizing animals (Marina De Filette, WillyMin Jou, Ashley Birkett, Katie Lyons, Brian Schultz, Anne Tonkyro, Stephanie Resch, Walter Fiers. Universal influenza A vaccine: Optimization of M2-based constructs [J]. Virology, 2005, 337: 149-161), the M2e gene fragment of avian influenza virus was simultaneously fused into the HBcAg gene There is no literature report on the study of the 5′ end and MIR region of

Method used

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  • Polypeptide vaccine for resisting multiple hypotype avian influenza viruses and preparation method thereof
  • Polypeptide vaccine for resisting multiple hypotype avian influenza viruses and preparation method thereof
  • Polypeptide vaccine for resisting multiple hypotype avian influenza viruses and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1: Obtaining of M2e Fragment and HBcAg Fusion Gene

[0033] The OE-PCR (overlap extension PCR) method was used to construct the fusion gene, and the following primers were synthesized:

[0034] M2eF: 5′-GGCGTCGACATGAGTCTTTCTAACCGAGG-3′

[0035] M2eR: 5′-GTCAATGTCAGGATCACTTGAATCGCTG-3′

[0036] M2eHF: 5′-AGTGATCCTGACATTGACCCGTATAAAG-3′

[0037] M2eH 1R: 5′-CCAACCGTTACGGGTTGGGGGTTTCCACTTCGGTCAGCAGGCTTGCTGGGTCTTCCAAATTAC-3′

[0038] M2eH2F: 5′-ACCCGTAACGGTTGGGAATGCCGTTGCAGCGATAGCAGCGATTCCAGGGAATTAGTAGTCAG-3′

[0039] M2eHR: 5′-GGCAAGCTTCTAACATTGAGATTCCCGAG-3′

[0040]In the primers, 18 bases are complementary between M2eR and M2eHF, and 15 bases are complementary between M2eH1R and M2eH2F. Primer M2eF has a restriction site Sal I, and M2eHR is designed with a restriction site HindIII, so that the restriction fragment can be ligated into the prokaryotic expression vector pMALc2x.

[0041] The fusion gene construction of the M2e fragment connected to the 5' end...

Embodiment 2

[0043] Embodiment 2: the prokaryotic expression vector construction of M2eHBc+ fusion gene

[0044] After the M2eHBc+ fusion gene fragment was digested by Sal I and HindIII, it was ligated with the prokaryotic expression vector pMALc2x purified by the same digestion, and the ligation product was transformed into DH5α. The white clone picked from the resistance plate was digested and identified by sequencing. The correct clones were named pMALc2x-M2eHBc+, respectively.

Embodiment 3

[0045] Example 3: Expression, identification and purification of M2eHBc+ fusion protein

[0046] Transform the expression strain BL21(DE3) with the correctly sequenced prokaryotic expression vector pMALc2x-M2eHBc+, and inoculate the clones identified as positive in the pMAL special medium added with ampicillin and resistance, and culture with shaking until OD 600 = about 0.6, add IPTG to a final concentration of 0.8mmol / L to induce fusion gene expression. The induction conditions are: temperature 37°C, rotation speed 160r / min, induction 4h. After the induction, the bacterial cells were collected by centrifugation, and the protein expression was detected by SDS-PAGE electrophoresis (for the protein expression results, see figure 2 ). SDS-PAGE electrophoresis was performed on the bacterial protein induced by IPTG and the uninduced bacterial control. After the electrophoresis, the protein was transferred to a nylon membrane, and the rabbit anti-H5N1M2 protein antibody was used...

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Abstract

The invention discloses to an anti-multi-subtype avian influenza virus peptide vaccine and a preparation method thereof, which relates to the peptide vaccine and provides the anti-multi-subtype avian influenza virus peptide vaccine that is safe, reliable and can simultaneously effectively prevent a plurality of subtype avian influenza viruses and the preparation method. The peptide vaccine existsin a form of virus-like particles, and avian influenza virus M2e epitope is effectively presented on the surfaces of the virus-like particles, thus constituting the amino acid sequence of the subunitof the virus-like particles and coding the DNA sequence of the amino acid sequence. A corresponding primer is designed to obtain the DNA sequence; the primer is further inserted in the multi-cloning region of a vector pMAL-c2x to form an expression vector plasmid pMALc2x-M2eHBc plus of the peptide vaccine, recombinant plasmid is transformed into escherichia coli, thus obtaining recombinant genetic engineering bacteria; inoculation and culture are carried out, IPTG is added, the induction of the target gene expression is carried out, cells are centrifugally collected, freeze-thaw is carried out, PBS buffer liquid is added for resuspending bacteria, supernatant liquid is centrifugally taken, the filtration and the purification are further carried out.

Description

technical field [0001] The present invention relates to a polypeptide vaccine, in particular to the preparation of an in vitro expressed virus-like granule anti-multi-subtype avian influenza virus polypeptide vaccine, the protein molecule amino acid sequence and the corresponding gene sequence constituting the virus-like granule , and the preparation method and application of the virus-like particle. Background technique [0002] Avian Influenza Virus (AIV) belongs to the family Orthomyxoviridae, the genus Influenzavirus, and is a type A influenza virus. Avian Influenza (Avian Influenza, AI) is a poultry infection and disease syndrome caused by the virus (Calker BW. Avian Disease [M]. Beijing: Beijing Agricultural University Press, 1991). Both the Animal Epidemiology Organization (IOE) of the International Veterinary Bureau and my country's "Livestock and Poultry Epidemic Prevention Regulations" have listed the disease as a Class A severe infectious disease. At present, th...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K39/145C12N15/62C12N15/63C07K19/00A61P31/16
Inventor 陈亮张国广张辉煌李东霄曾雅明张红心郭小玲
Owner XIAMEN UNIV
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