Edwardsiella tarda subunit oral microencapsule vaccine for aquatic product

A subunit vaccine and microcapsule technology, which is applied in the direction of microcapsules, vaccines, and capsule delivery, can solve the problems of physiological damage to fish bodies and difficulty in implementing small-scale fish bodies, and achieve good immune effects, good immune enhancement effects, and convenient delivery. The effect of large-scale promotion and application

Inactive Publication Date: 2016-09-07
MARINE BIOLOGY INST OF SHANDONG PROVINCE
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

It takes a lot of manpower and time to vaccinate by injection in a large-scale intensive farming model. At the same time, injec...

Method used

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  • Edwardsiella tarda subunit oral microencapsule vaccine for aquatic product
  • Edwardsiella tarda subunit oral microencapsule vaccine for aquatic product
  • Edwardsiella tarda subunit oral microencapsule vaccine for aquatic product

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Example 1: Isolation of Edwardsiella tarda strains and analysis of their virulence characteristics

[0021] (1) The diseased flounder was taken from Shandong Huangdao Fish Farm. The average weight of the diseased fish was (200±5) g, and the average body length was (20±5) cm. The main symptoms were ascites, protruding anus, liver and kidney swelling.

[0022] (2) Take dying diseased fish with typical ascites symptoms, pick some tissues from liver, kidney, ascites and other lesions under aseptic conditions, streak them on a common nutrient agar plate, culture them at 28°C for 24 hours, and pick out those with the same shape. The dominant colonies were purified and cultured until pure cultures were obtained. The formula of ordinary nutrient agar medium is: yeast extract 3g, peptone 10g, sodium chloride 15g, distilled water 1L, pH 7.6-7.8, agar 20g.

[0023] (3) The isolated pure cultures of each bacterial strain were expanded on ordinary nutrient agar plates. After cultu...

Embodiment 2

[0028] Embodiment 2: the prokaryotic recombinant expression of GAPDH, outer membrane protein ompW and flagellin fliC of Edwardsiella tarda bacterial strain ET-1008

[0029] (1) Sequence amplification of the target expressed protein: according to the gene sequences of Genbank blunt Edwardian GAPDH, ompW and fliC, respectively design their specific primers (see Table 1). Using the bacterial genome extraction kit, the extracted bacterial genomic DNA was used as a template, and the forward and reverse primers of each gene were used for PCR amplification. The PCR reaction system and reaction conditions were: 50 μl reaction system, containing 37 μl H 2 O, 5 μl 10×Taq buffer, 4 μl dNTP (2.5 mM), 1 μl each of forward and reverse primers, 1 μl TaqDNA Polymerase and 1 μl DNA template; Extend for 1 min at °C for 30 cycles, and finally extend for 8 min at 72 °C. The PCR product was detected by 1% agarose gel electrophoresis, and the gel imaging system was photographed and observed. The r...

Embodiment 3

[0036] Embodiment 3: the extraction of Edwardsiella tarda lipopolysaccharide (LPS)

[0037] (1) Bacterial culture: use a sterile inoculation loop to pick a small amount of preserved ET-1008 colonies and streak them on a common nutrient agar plate, wash the well-grown bacterial lawn with a liquid medium, and transfer it to a medium containing 40ml In a 100ml Erlenmeyer flask, culture overnight at 28°C with shaking (160rpm). Add 7ml of bacterial solution into a 10ml centrifuge tube, centrifuge at 5000r / min for 10min at 4°C to collect the bacteria, wash with normal saline three times, wash with 7ml of 50mmol / L phosphate buffer (pH 7.0, containing 5mmol / L EDTA, 20mmol / L MgCl 2 ) to suspend bacteria at a concentration of 1×10 8 CFU / ml.

[0038] (2) Preparation of LPS by hot phenol water method: Ultrasonic crushing was carried out under ice bath conditions, and the set time was 5 minutes, of which 4s were on and 4s were off; the bacterial liquid was transferred to a 250ml beaker,...

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Abstract

The invention relates to an edwardsiella tarda subunit oral microencapsule vaccine for an aquatic product. The edwardsiella tarda subunit oral microencapsule vaccine comprises a calcium alginate-chitosan release-controlled microcapsule and three recombinant protein-lipopolysaccharide conjugates which are encapsulated in the microcapsule, wherein the three recombinant protein-lipopolysaccharide conjugates include edwardsiella tarda glyceraldehyde-3-phosphate dehydrogenases, outer membrane protein and flagellin which are mixed with astragalus polysaccharide. The oral microencapsule is characterized in that the encapsulation efficiency is 78%, the protein load is 102mg/g, and the grain size is 150+/-10 microns. The prepared oral microencapsule vaccine is convenient to use, proper in grain size, high in slow release performance and high in antigen protection performance, and can effectively irritate a fish intestinal immune system to perform immune response so as to enable a fish to gain the specific immunity protection capacity for edwardsiella tarda; and as a result, the edwardsiella tarda subunit oral microencapsule vaccine is applicable to prevention and treatment of fish edwardsiella tarda disease.

Description

technical field [0001] The invention belongs to the technical field of aquaculture vaccines, and in particular relates to an oral subunit microcapsule vaccine of Edwardsiella tarda in aquatic products. Background technique [0002] With the rapid development of aquaculture in recent years, the problem of aquaculture animal diseases has become increasingly prominent, among which bacterial diseases have caused huge losses to the fishery economy worldwide. Edwardsiella tarda is an important pathogenic bacterium of aquatic animals, which can infect a variety of fish including freshwater and seawater, and can cause a large number of deaths in a short period of time, which has brought huge damage to my country's aquaculture industry. Economic losses. The use of vaccines can improve the specific immunity level of farmed fish, so that fish can develop immunity against infection by specific pathogenic microorganisms, without polluting the environment, and without drug residues. It is...

Claims

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Application Information

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IPC IPC(8): A61K39/02A61K9/50A61K47/36A61P31/04A23K20/00C07K14/195C12N1/20C12R1/01
CPCA61K9/5036A61K39/025A61K2039/542A61K2039/552C07K14/195C12N1/205C12R2001/01
Inventor 刁菁叶海斌王晓璐于晓清李乐
Owner MARINE BIOLOGY INST OF SHANDONG PROVINCE
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