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

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Problems solved by technology

Currently, there is no vaccine that is effective in preventing and co

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

Example Embodiment

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

[0030] (1) Vibrio parahaemolyticus flaA with ompK Cloning of the target gene

[0031] Design clone flaA 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, the designed primers were used to carry out PCR amplification of the flagellin A gene fragment and the outer membrane protein K gene fragment. The PCR products are analyzed by electrophoresis, and obvious specific bands can be observed. use flaA The PCR product of the primer is about 1150bp, using ompK The PCR product of the primer is about 730bp, and the electrophoresis result is as follows figure 1 Shown.

[0038] Respectively omp...

Example Embodiment

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

[0062] Weigh 100 mg of FlaA-OmpK protein and dissolve it in 20 mL of TE-buffer, and dissolve 15 g of acrylic resin No. II in 250 mL of ethanol. The two solutions are mixed evenly by magnetic stirring and used as the inner oil phase.

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

[0064] The prepared enteric-coated microspheres have a drug load of 1% and an encapsulation rate of 77.5%±3.7%. Observed by electron microscope, the particles are nearly spherical, with a...

Example Embodiment

[0066] Example 3 Study on the immunogenicity of FlaA-OmpK subunit vaccine to mariculture black grouper

[0067] 3.1 The immunogenicity of FlaA-OmpK subunit vaccine to black grouper

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

[0069] The injection immunization method is divided into 4 immunization groups (OmpK group, high-dose fusion protein FlaA-OmpK group, low-dose fusion protein FlaA-OmpK group and mixed protein group (OmpK + FlaA), each with 25 tails. The above experimental group The dosage (0.2ml) of each fish was OmpK (100μg), FlaA-OmpK (230μg), FlaA-OmpK (100μg), OmpK+FlaA equimolar mixture (100μg). When the OmpK group was immunized, the drug components were compared with Falcon The complete adjuvant of Freund's (first immunization) / Incomplete Freund's adjuvant (second immunization) was mixed at a ratio of 1:1, and a...

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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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