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Preparation of infectious bursal disease (IBD) protein engineering vaccine

A capsule protein and vaccine technology, applied in the field of biotechnology genetic engineering, can solve the problems of subclinical secondary infection, mutant strains, high virulence, etc.

Active Publication Date: 2015-05-20
QINGDAO MINGQIN BIOLOGICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because attenuated strain vaccines are often not fully attenuated, they may cause certain pathological damage to the lymphoid follicles of the Bursa of Fabricius after immunization, and some may even cause immunosuppression (Rautenschlein et al., 2005), which can also lead to hypervirulence. Emergence of strains or mutants (Snyder, 1990)
Furthermore, live vaccines against some IBDV variants lead to immunosuppression and subclinical secondary infection (Fussell, 1998; Donnelly et al., 2000)

Method used

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  • Preparation of infectious bursal disease (IBD) protein engineering vaccine
  • Preparation of infectious bursal disease (IBD) protein engineering vaccine
  • Preparation of infectious bursal disease (IBD) protein engineering vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1 Design Idea of ​​Bursal Protein Engineering Vaccine Protein

[0023] According to the amino acid sequences of the structural proteins VP2 and VP3 of the main prevailing strains of Bursa Fabricius at present, the present invention uses relevant bioinformatics software DNASTAR, BIMAS and SYFPEITHI to analyze the B cell neutralizing epitopes and T cell epitopes of the prevailing strains, and introduces poultry The cytokine interleukin 15 (IL-15) acts as an adjuvant molecule. The designed bursal virus B cell and T cell epitopes and interleukin 15 molecular polypeptide were co-expressed in Escherichia coli in series, and after fermentation, purification, emulsification and other processes, the bursal protein engineering vaccine with ideal immunogenicity was obtained. The vaccine prepared by the invention can effectively prevent pseudobursal disease.

[0024] Comprehensive analysis of domestic bursal virus epidemic strain genome sequence, antigen structure, epidemi...

Embodiment 2

[0026] Example 2 Construction of Escherichia coli expression vector and expression strain

[0027] 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 named pMD18T-IBDV(VP2 / 3)-chIL15, respectively. The two plasmids were digested with the corresponding restriction enzymes. The E. coli expression vector was the pRSETB plasmid from Invitrogen Company, and the same restriction enzymes were also used for treatment. Digestion conditions: 10 μl reaction system, adding 2 μl of plasmid, 5 activity units of restriction endonuclease (New England Biolabs), 1 μl of 10× buffer wa...

Embodiment 3

[0031] Example 3 Fermentation, purification and emulsification of engineering bacteria

[0032] Fermentation Take the production strains, inoculate them in 2ml LB liquid medium (containing 100μg / ml ampicillin), and culture them at 37°C with shaking at 200rpm 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 40%, and the pH is controlled at 7.0. When th...

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Abstract

The invention relates to preparation and application of an infectious bursal disease (IBD) protein engineering vaccine. The preparation method comprises the following steps: connecting B cell neutralizing epitope and T cell immunizing epitope of IBDV (infectious bursal disease virus) primary structure proteins VP2 and VP3 as a vaccine frame structure through a flexible linker, connecting with cell factor poultry interleukin 15(chIL-15) in series, cloning into a pRSETB vector, transforming Escherichia coli, fermenting, purifying, emulsifying and the like to obtain the IBD protein engineering vaccine with ideal immunogenicity. The invention also relates to an application method of the vaccine. The animal experiment indicates that the IBD protein engineering vaccine has equivalent effects to the live virus vaccine on the humoral immunity and cellular immunity level, and can stimulate the generation of T lymphopoiesis immunoreaction on the cellular level and the generation of antibody immunoreaction with virus neutralizing activity on the humoral level. The attacking protection test indicates that the IBD protein engineering vaccine group has obviously better effects than the live vaccine group.

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 bursal disease protein engineering vaccine. Specifically, using genetic recombination technology, the epitopes of the main structural proteins VP2 and VP3 are connected in series with the cytokine avian IL-15, cloned into the vector, transformed into the host bacteria, and prepared by fermentation, purification and emulsification to obtain the recombinant bursa A protein engineering vaccine and the application of the vaccine in the prevention of poultry infectious disease Bursal virus disease. Background technique [0002] Chicken infectious bursal disease (Infeetious Bursal Disease, IBD) is an acute, highly contagious infectious disease caused by infectious bursal virus IBDV, and is one of the most serious infectious diseases that endanger the chicken industry ( Ikuta et al., 2001; van den Berg, 2000). The v...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/70C12N1/21A61K39/12A61K38/20A61P31/14
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
Owner QINGDAO MINGQIN BIOLOGICAL TECH CO LTD
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