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Recombinant H9N2 subtype avian influenza enhanced multi-epitope vaccine

An avian influenza and multi-epitope technology, applied in the field of biotechnology genetic engineering, can solve the problems of high cost of chicken embryo multiplication, fuzzy species boundaries, interference with epidemiological monitoring, etc.

Active Publication Date: 2015-07-29
GUANGZHOU PUTAI BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the vaccines used to prevent and control avian influenza are mainly inactivated vaccines produced by the proliferation of chicken embryos. Although the traditional inactivated vaccines have many advantages, they still have many shortcomings, such as interfering with epidemiological monitoring and relying on chicken embryos. The cost of reproduction is high, it is easy to mutate in chicken embryos, and the burning of chicken embryo residues pollutes the environment, etc., and inactivated vaccines and live virus vaccines cannot effectively cope with the rapid mutation of influenza viruses, the coexistence of multiple subtypes of influenza, and the boundaries of species increasingly ambiguous trend

Method used

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  • Recombinant H9N2 subtype avian influenza enhanced multi-epitope vaccine
  • Recombinant H9N2 subtype avian influenza enhanced multi-epitope vaccine
  • Recombinant H9N2 subtype avian influenza enhanced multi-epitope vaccine

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Embodiment 1

[0026] Example 1 Design Idea of ​​H9N2 Subtype Avian Influenza Multi-epitope Vaccine Protein

[0027] According to the amino acid sequences of structural proteins hemagglutinin (HA), neuraminidase (NA), nucleocapsid protein (NP) and matrix protein 2 (M2) of the main epidemic strains of domestic H9N2 subtype avian influenza, the present invention utilizes related biological Informatics software DNASTAR, BIMAS and SYFPEITHI analyzed the neutralizing epitope, Th epitope, CTL epitope and B cell epitope of the epidemic strains, and introduced avian cytokine interleukin 18 (chIL-18) as an adjuvant molecule. The designed epitope and interleukin-18 molecule polypeptide were co-expressed in Escherichia coli in series, and after fermentation, purification, emulsification and other processes, the H9N2 subtype avian influenza enhanced multi-epitope vaccine with ideal immunogenicity was obtained. The vaccine prepared by the invention can effectively prevent H9N2 subtype bird flu.

[0028]...

Embodiment 2

[0030] Embodiment two Escherichia coli expression vector and the construction of expression bacterial strain

[0031] The designed polypeptide-encoding nucleotides were sent to Shanghai Handsome Biotechnology Co., Ltd. for synthesis. BamH I (5' end) and HindIII (3' end) restriction enzyme sites were designed at both ends of the nucleotide fragment. After synthesis, they were respectively cloned into the pMD18T vector, and sequence determination confirmed that the inserted gene fragment was consistent with the designed sequence (see the sequence list). The recombinant plasmids were respectively named pMD18T-chIL18-AIV(H9N2). Digest the plasmid with the corresponding restriction endonuclease. The Escherichia coli expression vector is the pRSETB plasmid from Invitrogen Company, which is also treated with the same restriction endonuclease. Digestion conditions: 10 μl reaction system, add 2 μl plasmid into the system , 5 activity units of restriction endonuclease (New England Biol...

Embodiment 3

[0035] Fermentation, purification and emulsification of embodiment three engineering bacteria

[0036] The production strains were taken for fermentation, inoculated in 2 mL of LB liquid medium (containing 100 μg / mL ampicillin), and cultured at 37° C. with shaking at 200 rpm for 12 hours to activate the strains. Then inoculate the shake flask with an inoculation amount of 1:100, shake and culture at 37°C until OD600=3, and then inoculate it into a fermenter at a ratio of 10%. The fermentation medium is a semi-synthetic medium prepared with distilled water. Calibrate the dissolved oxygen and pH electrodes, start the tank to stir, the rotation speed is 300rpm, and sterilize the tank on-line. When the temperature of the culture solution in the tank drops to 37.0°C, calibrate the pH and dissolved oxygen (OD) zero point. The fermentation temperature was 37.0±0.1°C, the dissolved oxygen was controlled at about 40%, and the pH was controlled at 7.0. After inoculation, when the OD600...

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Abstract

The invention relates to preparation and application of a recombinant H9N2 subtype avian influenza (Avian influenza H9N2) enhanced multi-epitope vaccine. The vaccine takes neutralizing epitope, Th epitope, CTL epitope and B cell epitope of the major structural protein hemagglutinin (HA), neuraminidase (NA), nucleocapsid protein (NP) and matrix protein 2 (M2) of H9N2 subtype avian influenza virus as the frame structure, after being in flexible linker connection and series connection with cell factor interleukin 18 (chIL-18), the frame structure is then cloned into a pRSETB carrier to be transformed into escherichia coli, and then processes like fermentation, purification and emulsification are carried out, so that an avian influenza enhanced multi-epitope vaccine with ideal immunogenicity is achieved. Animal experiments indicate that the recombinant H9N2 subtype avian influenza enhanced multi-epitope vaccine is not only good in safety, but also can activate effective humoral immune and cellular immune responses.

Description

technical field [0001] The invention belongs to the field of biotechnology genetic engineering, and mainly relates to the preparation and application of a recombinant H9N2 subtype avian influenza enhanced multi-epitope vaccine. Specifically, using genetic recombination technology, the main structural proteins: hemagglutinin (HA), neuraminidase (NA), nucleocapsid protein (NP) and matrix protein 2 (M2) neutralizing epitope, Th epitope The recombinant H9N2 subtype avian influenza multi-epitope vaccine and the recombinant H9N2 subtype avian influenza multi-epitope vaccine and the The application of vaccine in the prevention of avian infectious disease H9N2 subtype avian influenza. Background technique [0002] Influenza virus can be divided into A, B and C three types. Among them, the distribution range of type B and type C influenza virus is small, and the harm is relatively light, while the harm of type A influenza virus is the most serious, in addition to infecting humans, ...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62A61K39/145A61P31/16
Inventor 李殿明蒲勤张毓金齐春梅田春辉刘甜甜任百亮张导春党将将吴启凡冯鑫
Owner GUANGZHOU PUTAI BIOTECH
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