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Fish broad-spectrum vibrio subunit vaccine and preparation method

A broad-spectrum vibrio sub-vaccine technology, applied in the biological field, to avoid the effect of degradation

Inactive Publication Date: 2012-06-27
FUZHOU UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Currently, there is no vaccine that is effective in preventing and controlling fish infections caused by various pathogenic Vibrio species

Method used

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  • Fish broad-spectrum vibrio subunit vaccine and preparation method
  • Fish broad-spectrum vibrio subunit vaccine and preparation method
  • Fish broad-spectrum vibrio subunit vaccine and preparation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1 Preparation of FlaA-OmpK subunit vaccine

[0030] (1) Vibrio parahaemolyticus fla A and ompK Cloning of target gene

[0031] design clone fla A Gene primers:

[0032] Upstream: 5-GGATCCATGGCGATTAACGTT-3 (BamH I)

[0033] Downstream: 5-CTCGAGGCCCAACAAGCTTAg-3 (Xho I)

[0034] design clone ompK Gene primers:

[0035] Upstream: 5-CGGGATCCGCAGATTACTCTGACGGCGATAT-3 (BanH I)

[0036] Downstream: 5-CCCAAGCTTTTAGAACTTGTAAGTTACTGCGA-3 (Hind III)

[0037] Using the total DNA of Vibrio parahaemolyticus (standard strain ATCC17802) as a template, PCR amplification of flagellin A gene fragment and outer membrane protein K gene fragment was carried out with designed primers respectively. PCR products were analyzed by electrophoresis, and obvious specific bands could be observed. use fla A The PCR product of the primers is about 1150bp, using ompK The PCR product of the primer is about 730bp, and the electrophoresis result is as follows figure 1 shown.

...

Embodiment 2

[0061] Example 2 Preparation of FlaA-OmpK oral enteric-coated microsphere vaccine

[0062] Weigh 100mg of FlaA-OmpK protein and dissolve it in 20mL TE-buffer, and dissolve 15g of acrylic resin No. Ⅱ in 250mL of ethanol. The two solutions are magnetically stirred and mixed evenly to serve as the internal oil phase.

[0063] Slowly add the inner oil phase to 1000mL liquid paraffin containing 0.5mL lecithin and 0.35% Span80 under high-speed stirring (≥10000r / min), emulsify for 30min, then change to magnetic stirring (600r / min), and keep stirring The ethanol is completely volatilized, and the microspheres are solidified. The precipitate was collected by centrifugation, washed several times with petroleum ether, and dried under reduced pressure for 12 hours to obtain the final product.

[0064] The prepared enteric-coated microspheres had a drug loading of 1%, and an encapsulation efficiency of 77.5%±3.7%. Electron microscope observation shows that the particles are nearly sphe...

Embodiment 3

[0066] Example 3 Study on Immunogenicity of FlaA-OmpK Subunit Vaccine to Mariculture Black Grouper

[0067] 3.1 Study on the immunogenicity of FlaA-OmpK subunit vaccine to marine cultured black grouper

[0068] Healthy American black grouper (100±10g / tail) was randomly selected and grouped, and after fully adapting to the environment of the breeding pond, the immune test was carried out.

[0069] Injection immunization method, divided into 4 immunization groups (OmpK group, fusion protein FlaA-OmpK high-dose group, fusion protein FlaA-OmpK low-dose group and mixed protein group (OmpK +FlaA), 25 tails in each group. The above experimental groups The dosage (0.2ml) per fish was OmpK (100μg), FlaA-OmpK (230μg), FlaA-OmpK (100μg), OmpK+FlaA equimolar mixture (100μg). Freud's complete adjuvant (first immunization) / Freund's incomplete adjuvant (second immunization) were mixed at a ratio of 1:1, and after complete emulsification, intraperitoneal injection. In the immunization grou...

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Abstract

The invention discloses a broad-spectrum vibrio subunit vaccine for preventing fish pathogenic vibrio infection and a preparation method, belonging to the technical field of biology. The fusion protein OmpK-FlaA of an outer membrane protein OmpK with a conserved structure and a flagellin protein FlaA on the vibrio parahaemolyticus surface serves as an antigen component of the vaccine. The method is characterized by comprising the following steps of: superposing and extending Ppolymerase chain reaction (PCR) to the ompK and flaA genes of the vibrio parahaemolyticus to obtain a fusion protein gene flaA-ompK; constructing flaA-ompK-pET-28a recombinant plasmids; and obtaining the fusion FlaA-OmpK with high purity through exogenous induction expression and purification. The vaccine prepared bythe invention is safe and nontoxic and has no side effects; injection immune can be adopted; and the immune can also be realized by taking enteric microsphere vaccine orally; cross immunity protection can be acted to fish pathogenic vibrio (vibrio parahaemolyticus, vibrio alginolyticus, vibro harveyi, vibrio anguillarum and vibrio vulnificus).

Description

technical field [0001] The invention relates to a fish broad-spectrum Vibrio subunit vaccine and a preparation method thereof, belonging to the field of biotechnology. Background technique [0002] In recent years, the scale of artificial fish farming has expanded rapidly, and the degree of production intensification has continued to increase. Diseases have become a bottleneck restricting the healthy development of aquaculture. According to statistics, the national aquaculture disease incidence rate reaches more than 50%, the loss rate is about 30%, and the annual direct loss is as high as tens of billions of dollars. Among numerous bacterial diseases, vibriosis is recognized as one of the most serious diseases in fish farming. The main pathogenic Vibrio include: Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio harveii, Vibrio anguillarum and Vibrio vulnificus. [0003] With the abuse of microbial diseases in aquaculture, the abuse of antibiotics has become more and ...

Claims

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Application Information

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IPC IPC(8): A61K39/39A61K39/106A61K9/16A61K9/08A61K47/32A61P31/04C12N15/70C07K19/00C07K1/22C12R1/19
Inventor 林海英郑磊郭养浩唐凤翔石贤爱郑允权
Owner FUZHOU UNIVERSITY
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