Co-expressed molecular adjuvant enhanced divalent foot and mouth disease protein engineering vaccine

A foot-and-mouth disease, enhanced technology, applied in the field of biotechnology genetic engineering, can solve problems such as inconsistency, lack of 6th or 7th residues, etc.

Active Publication Date: 2012-04-11
QINGDAO MINGQIN BIOLOGICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, Asian type 1 VP4 proteins occasionally lack residues 6 or 7, which is inconsistent with A, O, and SAT FMD (Reddy GR, 1999; Stram Y, 1994)

Method used

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  • Co-expressed molecular adjuvant enhanced divalent foot and mouth disease protein engineering vaccine
  • Co-expressed molecular adjuvant enhanced divalent foot and mouth disease protein engineering vaccine
  • Co-expressed molecular adjuvant enhanced divalent foot and mouth disease protein engineering vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Example 1 Molecular adjuvants enhance the design idea of ​​polypeptide-encoded protein in Asian type 1 and type A foot-and-mouth disease bivalent protein engineering vaccine

[0069] Integrating the genome sequences and antigenic structures of multiple Asian type 1 and type A foot-and-mouth disease virus epidemic strains AF / 72, LC / 96, AKT / 03, Asia-1-JSL, KZ / 03, HeNzk / 06 and other strains in China and Southeast Asia , epidemiological research progress, optimized the design of recombinant foot-and-mouth disease bivalent protein engineering vaccine. The present invention uses bioinformatics software to analyze the hydrophilicity, antigenicity, plasticity, surface accessibility and Garnier-Robson secondary structure of its main outer membrane proteins VP1 and VP2, and predict possible B cell antigen epitopes And on the basis of the killer T cell epitope, according to the similarity of the epitope position and amino acid sequence, analyze the common and specific antigenic ep...

Embodiment 2

[0071] Embodiment two Escherichia coli expression vector and the construction of expression bacterial strain

[0072] The nucleotide encoding the polypeptide designed in Example 1 was sent to Shanghai Handsome Biotechnology Co., Ltd. for synthesis, and BamH I (5' end) and HindIII (3' end) restriction enzyme sites were designed at both ends of the nucleotide fragment At the same time, the two segments of multi-epitope tandem nucleotide fragments without molecular adjuvant were also designed with EcoRI (5' end) and HindIII (3' end) restriction enzyme sites, and sent to Shanghai Handsome Biotechnology Co., Ltd. synthesized as a control. After the two fragments were synthesized, 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 named pMD18T-The / A-Asia1B / Tc and pMD18T-IFNα-The / A-Asia1B / Tc, respectively. The two p...

Embodiment 3

[0076] Example 3 Fermentation, purification and emulsification of engineering bacteria

[0077] Fermentation Take the production strains, inoculate them in 2ml LB liquid medium (containing 100μg / ml ampicillin), and culture at 37°C with shaking at 180rpm 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 medium used for fermentation is a semi-synthetic medium prepared with distilled water and does not contain any antibiotics. 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 is 37.0±0.1°C, the dissolved oxygen is controlled at about 20%, and the pH is controlled at 7.0. When the OD6...

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Abstract

The invention relates to preparation and application of a molecular adjuvant enhanced Asia 1 and A foot and mouth disease divalent vaccine. The vaccine comprises a molecular adjuvant interferon-alpha (IFN-alpha) polypeptide, a two-section T cell auxiliary antigenic epitope polypeptide, a six-section antigenic epitope polypeptide related to Asia 1 and A foot and mouth disease major outer membrane protein VP1 and VP2, and a killer T cell epitope polypeptide. The invention also relates to a preparation method and a using method for the vaccine. An animal experiment proves that the virus attack protective efficacy of the molecular adjuvant enhanced divalent foot and mouth disease protein engineering vaccine is obviously higher than that of a non-molecular adjuvant enhanced vaccine. By the recommended immunizing dose of the molecular adjuvant enhanced vaccine, a tested animal can resist attack of 10,000 median infective doses (ID50) of virulent Asia 1 and A foot and mouth disease virus, and the efficacy experiment also proves that the molecular adjuvant enhanced divalent vaccine for each animal at least contains 4 median protective doses (PD50).

Description

technical field [0001] The invention belongs to the field of biotechnology genetic engineering, and mainly relates to the preparation and application of a protein engineering vaccine co-expressed with a molecular adjuvant for preventing Asian type 1 and type A foot-and-mouth disease. Specifically, using gene recombination technology, multiple B cell antigen epitopes, killer T cell antigen epitopes (CTL epitopes), multiple T cell helper epitopes, and multiple T cell epitopes of different Asian type 1 and A type FMD main outer membrane proteins VP1 and VP2 The antigenic epitope (Th epitope) is connected in series with the molecular adjuvant polypeptide (IFNα), and cloned into the vector, transformed into the host bacteria, prepared by fermentation, purification, and emulsification processes, and the co-expressed immune effect-enhanced bivalent protein engineering vaccine for foot-and-mouth disease is obtained And the application of the vaccine in preventing foot-and-mouth diseas...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/135A61K39/39C12N15/12C12N15/42C12N15/63C12N1/21C12N15/62C07K19/00A61P31/14
Inventor 李殿明蒲勤李毅齐春梅田春辉赵明顾富香任百亮张导春牛纪涛刘甜甜刘祯
Owner QINGDAO MINGQIN BIOLOGICAL TECH CO LTD
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