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Bigeminal multi-epitope vaccine for chicken infectious laryngotracheitis and egg drop syndrome

A technology of sex and fusion protein, which is applied in the field of biotechnology and genetic engineering, can solve problems that have not yet been achieved, and achieve the effects of infection prevention, simple and easy-to-operate immunization methods, and high immunogenicity

Active Publication Date: 2019-08-06
QINGDAO MINGQIN BIOLOGICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] At present, there is no domestic or international registered ILT and EDS dual genetic engineering vaccine

Method used

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  • Bigeminal multi-epitope vaccine for chicken infectious laryngotracheitis and egg drop syndrome
  • Bigeminal multi-epitope vaccine for chicken infectious laryngotracheitis and egg drop syndrome
  • Bigeminal multi-epitope vaccine for chicken infectious laryngotracheitis and egg drop syndrome

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 The source of the fusion protein gene

[0039] The present invention comprehensively analyzes the gene sequence, antigen structure and epidemiological research progress of main epidemic strains of chicken infectious laryngotracheitis and egg drop syndrome at home and abroad, according to ILTV main structural proteins gB, gD, gE and EDSV Penton, Fiber proteins Amino acid sequence, using bioinformatics software to analyze its hydrophilicity, antigenicity, plasticity, surface accessibility and secondary structure, predict possible B-cell antigenic epitopes and T-cell antigenic epitopes, and then determine 8 segments of ILTV Antigen epitope polypeptide and 6-segment EDSV antigen epitope polypeptide. The vaccine skeleton structure is formed through flexible Linker connection and then connected in series with IL-8. The overall structure of the vaccine is:

[0040] ILTV-1 - ILTV-2 - ILTV-3 - ILTV-4 - ILTV-5 - ILTV-6 - ILTV-7 - ILTV-8 -EDSV-1 - EDSV-2 - EDSV-3 - EDSV...

Embodiment 2

[0041] Example 2 Construction of Escherichia coli expression vector and expression strain

[0042] The polypeptide coding nucleotides designed in Example 1 were sent to Shanghai Handsome Biotechnology Co., Ltd. for synthesis. BamHI (5' end) and EcoRI (3' end) restriction enzyme sites were designed at both ends of the nucleotide fragment respectively. The synthesized fragment was cloned into the pMD18T vector, and sequence determination confirmed that the inserted gene fragment was consistent with the involved sequence (see the sequence list). The recombinant plasmid was named pMD18T-ILTV / EDSV-IL-8, and the plasmid was digested with the corresponding restriction endonuclease. The Escherichia coli expression vector was pRSETB plasmid from Invitrogen Company, and the same restriction endonuclease was also used Treatment, digestion conditions: 10 μl reaction system, 2 μl plasmid, 5 activity units of restriction endonuclease (New England biolabs), 1 μl 10× buffer, supplemented with...

Embodiment 3

[0046] Example 3 Fermentation, purification and emulsification of engineering bacteria

[0047] Fermentation Inoculate the production strains into 2ml of LB liquid medium containing 100μl / ml ampicillin, and culture at 37°C with shaking at 180rpm 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 the 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 OD...

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Abstract

The invention relates to a bigeminal multi-epitope vaccine for chicken infectious laryngotracheitis and egg drop syndrome. Specifically, a gene recombination technology is used, 8 antigen epitope polypeptides related to chicken infectious laryngotracheitis virus gB, gD, and gE proteins as well as 6 antigen epitope polypeptides related to egg drop syndrome virus Penton and Fiber protein are connected with a molecular adjuvant IL-8, then are connected to a vector, transformed into host bacteria, and prepared by fermentation, purification and emulsification processes to obtain the bigeminal multi-epitope vaccine. The animal experiments show that the vaccine is safe and easy to use, and can induce significant humoral immunity and cellular immune responses in the body, the persistent period ofan antibody is long, and the infection of the infectious laryngotracheitis virus and egg drop syndrome virus can be effectively prevented.

Description

technical field [0001] The invention belongs to the field of biotechnology genetic engineering and relates to a fusion protein used for preventing chicken infectious laryngotracheitis and egg drop syndrome. Specifically, using gene recombination technology, 8 segments of antigenic epitope polypeptides related to chicken infectious laryngotracheitis virus gB, gD, gE proteins and 6 segments of antigenic epitope polypeptides related to egg drop syndrome virus Penton and Fiber proteins , connected in series with molecular adjuvant IL-8, connected to a carrier, transformed into host bacteria, prepared through fermentation, purification and emulsification processes, to obtain a chicken infectious laryngotracheitis and egg drop syndrome dual multi-epitope vaccine. Animal experiments show that the vaccine has high safety, is easy to use, can induce significant humoral immunity and cellular immune response, and has a long duration of antibody, which can effectively prevent the infectio...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62A61K39/235A61K39/255A61K39/295A61P31/20A61P31/22
CPCC07K14/005A61K39/12A61P31/20A61P31/22C12N2710/16022C12N2710/16034C12N2710/10222C12N2710/10234C07K2319/40A61K2039/552A61K2039/70A61K2039/572A61K2039/575
Inventor 李殿明蒲勤张晓丹田春辉齐春梅刘甜甜任百亮张导春党将将
Owner QINGDAO MINGQIN BIOLOGICAL TECH CO LTD
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