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Pig O/Mya98 and O/PanAsia type foot-and-mouth disease gene engineering inactivated vaccine

A technology of genetic engineering and inactivated vaccines, which is applied in the field of fusion protein and the preparation of the fusion protein, can solve the problems of incomplete inactivation in mass production, loose virus, and limited promotion

Active Publication Date: 2018-03-23
QINGDAO MINGQIN BIOLOGICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although traditional inactivated vaccines have good immunogenicity, there are risks such as incomplete inactivation in mass production and loose poison, and there are biological safety hazards in production, transportation and use, and the protection period is short
Chemically synthesized peptide vaccines are safe and effective, but expensive production costs limit their promotion

Method used

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  • Pig O/Mya98 and O/PanAsia type foot-and-mouth disease gene engineering inactivated vaccine
  • Pig O/Mya98 and O/PanAsia type foot-and-mouth disease gene engineering inactivated vaccine
  • Pig O/Mya98 and O/PanAsia type foot-and-mouth disease gene engineering inactivated vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1 The source of the fusion protein gene

[0022] The present invention comprehensively analyzes the gene sequence, antigen structure and epidemiological research progress of Burmese 98 and pan-sub-lineage O-type foot-and-mouth disease virus, according to the amino acid sequence of its main structural proteins VP1, VP2, VP3, and uses relevant bioinformatics software to analyze its relative Analysis of water content, antigenicity, plasticity, surface accessibility and secondary structure to predict possible B cell epitopes and T cell epitopes, and comprehensively related reports to determine the 4 segments of VP1 epitopes of different strains , 2 segments of VP2 antigenic epitope, 1 segment of VP3 antigenic epitope, and at the same time introduce the dominant antigenic epitope of T cell helper epitope and the dominant antigenic epitope of killer T cell. All epitopes are connected with a flexible Linker and then connected in series with the molecular adjuvant IL-2....

Embodiment 2

[0024] Example 2 Construction of Escherichia coli expression vector and expression strain

[0025] The polypeptide coding nucleotides designed in Example 1 were sent to Shanghai Handsome Biotechnology Co., Ltd. for synthesis, and EcoR I (5' end) and HindIII (3' end) restriction enzyme sites were designed at both ends of the nucleotide fragment , The synthesized fragment was cloned into the pMD18T vector, and sequence determination confirmed that the inserted gene fragment was consistent with the related sequence (see the sequence list). The recombinant plasmid was named pMD18T-FMDV-IL-2, and the corresponding restriction endonuclease was used to digest the plasmid. The Escherichia coli expression vector was pRSETA plasmid from Invitrogen Company, and the same restriction endonuclease was also used. Digestion conditions: 10 μL reaction system, 2 μL plasmid, 5 activity units of restriction endonuclease (New England biolabs), 1 μL 10× buffer, supplemented with deionized water, di...

Embodiment 3

[0029] Example 3 Fermentation, purification and emulsification of engineering bacteria

[0030] Fermentation Inoculate the production strains into 2 mL of LB liquid medium containing 100 μL / mL ampicillin, and culture at 37°C with shaking at 180 rpm for 12 hours to activate the strains. Put the activated strains into shake flasks at an inoculum size of 1:100, shake and culture at 37°C until OD600=3, and then inoculate them into fermenters at a ratio of 10%. The medium used for fermentation is a semi-synthetic medium prepared with distilled water and does not contain any antibiotics. Calibrate dissolved oxygen and pH electrodes, turn on the tank to stir at 300 rpm, and sterilize the tank on-line. When the temperature of the culture solution in the tank drops to 37°C, calibrate the pH and dissolved oxygen (OD) zero point. The fermentation temperature is 37.0°C±0.1°C, the dissolved oxygen is controlled at about 20%, and the pH is controlled at 7.0. After inoculation, when the OD6...

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Abstract

The invention relates to a fusion protein for preventing O-type foot-and-mouth disease virus, and a preparation method and application of the fusion protein. In particular, the present invention relates to a fusion protein, and the fusion protein comprises a molecular adjuvant IL-2 polypeptide, a T cell helper epitope polypeptide, a polypeptide with seven segments of epitopes related to O / Mya98 and O / PanAsia type foot-and-mouth disease virus strain main structural protein VP1, VP2 and VP3, and a killer T cell epitope polypeptide. The invention also relates to the preparation method and application of the fusion protein. The vaccine production preparation process is stable and is suitable for large-scale production. Tests show that the vaccine is safe to use and can effectively prevent infection of different O-type foot-and-mouth disease epidemic strains.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a fusion protein used for preventing O-type foot-and-mouth disease virus, a preparation method and application of the fusion protein. Specifically, using gene recombination technology, T cell helper epitope polypeptides, 7 segments of antigenic epitope polypeptides related to the main structural proteins VP1, VP2 and VP3 of O / Mya98 and O / PanAsia type foot-and-mouth disease strains, and killer T cells The epitope polypeptide is connected in series with the molecular adjuvant IL-2 polypeptide, connected to the carrier, transformed into the host bacteria, and prepared through fermentation, purification, and emulsification processes to obtain genetically engineered inactivated vaccines for porcine O / Mya98 and O / PanAsia types of foot-and-mouth disease and the vaccine in Application in prevention of O-type foot-and-mouth disease virus infection. Background technique [0002] Foot and mouth ...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62A61K39/135A61K39/39A61P31/14
CPCA61K39/12A61K39/39A61K2039/552A61K2039/55533C07K14/005C07K2319/00C12N2770/32122C12N2770/32134
Inventor 张晓丹李殿明蒲勤田春辉齐春梅刘甜甜任百亮张导春吴启凡党将将
Owner QINGDAO MINGQIN BIOLOGICAL TECH CO LTD
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