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Vaccine utilizing vibrio anguillarum flagellin and application thereof

A technology of flagellin and Vibrio eel, applied in the field of vaccinology, can solve the problems of reducing bacterial infectivity, the possibility of flagellin research has not been carried out, and achieve the effect of high-efficiency immune protection effect.

Inactive Publication Date: 2013-03-27
INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Studies have shown that mutations of FlaA, FlaD and FlaE genes can reduce bacterial infection ability, while mutations of FlaB and FlaC have no significant effect on bacterial infection, so it is speculated that FlaA, FlaD and FlaE may be involved in bacterial virulence
At present, the research on Vibrio anguillarum flagella is mainly focused on pathogenicity, and the possibility of flagellin as a vaccine has not yet been carried out.

Method used

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  • Vaccine utilizing vibrio anguillarum flagellin and application thereof
  • Vaccine utilizing vibrio anguillarum flagellin and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Construction method of Vibrio anguillarum flagellin expression vector:

[0018] Construction of plasmid pEFB1: PCR amplification of Vibrio anguillarum flagellin FlaB gene (the gene sequence has been reported. See Naka H, ​​Dias GM, Thompson CC, Dubay C , Thompson FL, Crosa JH. Complete genome sequence of the marine fish pathogen Vibrio anguillarum harboring the pJM1 virulence plasmamid and genomic comparison with other virulent strains of V. anguillarum and V. ordalii. Infect Immun 2011;79:2889–900). The PCR conditions are: 94°C60s pre-denatured template DNA, then 94°C40s, 50°C60s, 72°C60s, 5 cycles; then 94°C40s, 62°C60s, 72°C60s, 25 cycles and then 72°C C extension reaction 10min. The PCR product was purified with Tiangen DNA Product Purification Kit.

[0019] The vector pET259 (see Zheng, W.J., Hu, Y.H., Sun, L., 2010. Cloning and analysis of a ferritin subunit from turbot (Scophthalmus maximus). Fish. Shellfish. Immunol. 28, 829–836 for the construction process of...

Embodiment 2

[0023] Induced expression and purification of flagellin: The above-mentioned plasmid pEFB1 was transformed into Escherichia coli BL21(DE3) (purchased from "Tiangen Biochemical Technology Co., Ltd.", Beijing) by conventional methods, and contained kanamycin (30ug / ml) Cultured on LB solid medium for 18-24 hours, picked a transformant and named it BL21 / pEFB1. Cultivate BL21 / pEFB1 overnight in LB liquid medium containing kanamycin (30ug / ml); take 1ml of overnight culture solution, add 100ml fresh LB liquid medium containing kanamycin (30ug / ml) culture medium at 37°C with shaking at 200rpm until OD 600 If the concentration is 0.6, add IPTG with a final concentration of 0.3mM, continue shaking culture at 160rpm at 30°C for 5-6h, then centrifuge at 5000g, 4°C for 10min, collect the bacterial liquid, add 5ml of lysate, and shake slowly on a shaker at room temperature 1-2 hours until the bacterial suspension becomes clear. The bacterial solution was centrifuged at 10000g at 4°C for 3...

Embodiment 3

[0025] Application of flagellin as a vaccine

[0026] Step 1) Preparation of adjuvant and vaccine mixture.

[0027] Adjuvant preparation: 5% (mass ratio) NaOH and 5% (mass ratio) Al 2 (SO 4 ) 3 Mix in a 2:5 volume ratio and centrifuge the mixture at 10,000g for 5 minutes. The pellet was suspended in PBS to 0.2 mg / ml.

[0028] Vaccine mixture preparation: Dilute the vaccine protein purified in the above example to 300ug / ml in PBS; mix the diluted vaccine protein with an adjuvant in equal volume. Preparation of adjuvant control solution: mix equal volumes of PBS and adjuvant. The composition of the PBS is by weight percentage: 0.8% NaCl, 0.02% KCl, 0.358% Na 2 HPO 4 .12H 2 O, 0.024% NaH 2 PO 4 , and the balance is water.

[0029] Step 2) Immunization application of the vaccine. 60 flounder (each weighing about 12.7g) were randomly divided into 2 groups, 30 in each group. These 2 groups are named as Group A and Group B, respectively. Each fish in group A was injecte...

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Abstract

The invention relates to the field of vaccinology, and particularly relates to a vaccine utilizing vibrio anguillarum flagellin and an application thereof. According to the invention, vibrio anguillarum is taken as a template and F1 / R1 is taken as a primer to obtain a PCR (polymerase chain reaction) amplification product which is connected with pET259, and after connection transformation, plasmid is split to be mixed with an adjuvant, so as to obtain the vaccine utilizing vibrio anguillarum flagellin, wherein the primer is F1: 5'-CCCGGGATGCCTAAGGAGATCAATATG-3' and R1: 5'-CCCGGGACCCAATAGACTAAGAGCT-3'. The vaccine has the immune protection efficiency reaching 78% on vibrio anguillarum.

Description

technical field [0001] The invention relates to the field of vaccinology, in particular to a vaccine utilizing Vibrio anguillarum flagellin and its application. Background technique [0002] Vibrio anguillarum is a Gram-negative bacterium that can infect a variety of aquatic animals, including fish, shrimp, and shellfish. Vibrio eel infection can lead to vibriosis, which is an epidemic disease in the world and can occur in any season. It has caused serious economic losses to the world's industrial breeding for many years. Genome sequence analysis showed that the Vibrio anguillarum genome contains multiple flagellin genes, encoding flagellin FlaA, FlaB, FlaC, FlaD, and FlaE, respectively. Studies have shown that mutations of FlaA, FlaD and FlaE genes can reduce bacterial infection ability, while mutations of FlaB and FlaC have no significant effect on bacterial infection, so it is speculated that FlaA, FlaD and FlaE may be involved in bacterial virulence. At present, the re...

Claims

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Application Information

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IPC IPC(8): A61K39/106A61P31/04C12N15/70C12R1/63
Inventor 孙黎贾盼盼
Owner INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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