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Vibrio-splendidus-resistant egg yolk antibody and preparation method thereof

A technology of vibrio brilliant and egg yolk antibody, which is applied in the fields of antibodies, food preparation, antibacterial drugs, etc., can solve the problems that there is no preparation method, potency detection method and application of anti vibrio brilliant egg yolk antibody, and is suitable for large-scale The effect of large-scale production, high production cost, and easy industrialization

Active Publication Date: 2015-12-30
DALIAN UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the patents applied for publication and authorization in aquatic products include anti-prawn virus disease, anti-Cryptosporidiosis, anti-vibriosis parahaemolyticus, anti-Aeromonas hydrophila, and anti-eel vibriosis. There are no relevant research reports on anti-Vibrio splendidus yolk antibody and its preparation method, titer detection method and application

Method used

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  • Vibrio-splendidus-resistant egg yolk antibody and preparation method thereof
  • Vibrio-splendidus-resistant egg yolk antibody and preparation method thereof
  • Vibrio-splendidus-resistant egg yolk antibody and preparation method thereof

Examples

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Embodiment 1

[0027] Example 1 Preparation of anti-Vibrio brilliant yolk antibody

[0028] (1) Preparation of Vibrio brilliant antigen

[0029] Vibrio splendidus AP622 (Vibriosplendidus) was donated by Professor Li Hua of Dalian Ocean University, and it is preserved in the Korean Collection for Type Cultures. The bacterium was cultured on TSA solid agar medium at 28°C for 24 hours, then a single colony was picked and inoculated in 2216E liquid medium, and cultured on a shaker at 28°C and 140 rpm for 12 hours until the bacterial concentration was 10 8 CFU / mL, and then centrifuged to obtain the precipitate and supernatant containing Vibrio splendidus, the precipitate was mixed with sterile PBS to obtain 10 9 CFU / mL bacterial suspension of Vibrio brilliant.

[0030] (2) Inactivation of Vibrio brilliant antigens

[0031] Add formaldehyde to the bacterial suspension so that the final volume concentration of formaldehyde is 0.5%. After inactivation on a shaking table at 37°C for 24 hours, cen...

Embodiment 2

[0038] The titer detection of embodiment 2 egg yolk antibody

[0039] The titer was detected by enzyme-linked immunosorbent assay, with 10 9 CFU / mL bacteria concentration of Vibrio splendidus coated ELISA plate, 100 μL per well, coated overnight at 4 °C, shake the plate, wash 3 times with washing solution, add blocking solution containing 1% BSA and bathe in water at 37 °C for 2 h. After the water bath, shake the plate, wash 3 times, and then add 100 μL of anti-Vibrio splendidus yolk antibody (or crude egg yolk antibody extract) diluted in conjugate diluent containing 1‰BSA to each well, and set negative control and blank control at the same time, 37 ℃ water bath for 2h. After the water bath, wash 3 times, add 100 μL of horseradish peroxidase-labeled rabbit anti-chicken antibody (IgG-HRP) diluted in conjugate diluent to each well, and bathe in water at 37°C for 1 hour. Wash 5 times after the reaction, add 100 μL of TMB chromogenic solution to each well, develop color at room...

Embodiment 3

[0040] Example 3: Identification of the purity of egg yolk antibody

[0041] Get the product obtained in step (4) step (5) in the embodiment 1 and carry out yolk antibody purity identification, use non-reducing SDS-PAGE gel electrophoresis detection, the result is as follows figure 2 , M: Protein Maker; 1: crude extract of egg yolk antibody; 2: solution obtained after ammonium sulfate salting out; 3: solution obtained after sodium sulfate salting out; 4: solution obtained after ultrafiltration and concentration; 5: egg yolk antibody freeze-dried powder . Detected by reducing SDS-PAGE gel electrophoresis, the results are as follows image 3 , where M: protein maker; 1: crude extract of yolk antibody; 2: solution obtained after ammonium sulfate salting out; 3: solution obtained after sodium sulfate salting out; 4: solution obtained after ultrafiltration and concentration; 5: lyophilized egg yolk antibody pink. figure 2 The results show that the molecular weight of IgY is 18...

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Abstract

The invention discloses a vibrio-splendidus-resistant egg yolk antibody and a preparation method thereof. The vibrio-splendidus-resistant egg yolk antibody is obtained from a vibrio splendidus immunized laying hens, has features of good specificity and high titer, is used for preventing and treating diseases caused by vibrio splendidus and immunological detection, and has good effects. The vibrio-splendidus-resistant egg yolk antibody is obtained by extracting hyperimmune eggs laid by the vibrio splendidus immunized laying hens, blood sampling is not required, immunized animals are not injured, and modern animal rights protection rules are met; and each egg contains more than 100mg of the vibrio-splendidus-resistant egg yolk antibody approximately, and 3g of the vibrio-splendidus-resistant egg yolk antibody can be obtained in a month, and is 10-20 times greater than an antibody extracted from blood of an immunized rabbit. The vibrio-splendidus-resistant egg yolk antibody has the advantages of safety, environmental protection, high efficiency, low cost and the like, and is easy to industrialize.

Description

technical field [0001] The invention relates to the field of biological product technology and application, in particular to an anti-vibrio brilliant yolk antibody and its preparation method and application. Background technique [0002] Vibrio splendidus is a pathogenic halophilic bacterium widely distributed in marine and salt lake environments, belonging to the genus Vibrio in the Vibrio family. Vibrio splendidus is an important pathogenic bacterium of marine animals, which can cause various diseases of sea cucumbers, turbot, flounder, Atlantic salmon, trout, perch and other aquatic animals. Vibrio splendidus can cause "rotting skin syndrome" of sea cucumbers, and the mortality rate is as high as more than 90%, which brings huge losses to the aquaculture industry. [0003] At present, there are mainly the following methods for the prevention and treatment of diseases caused by Vibrio splendidus: antibiotics and chemical drugs, Chinese herbal medicines, inactivated vaccin...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/12C07K16/02A61K39/395A61P31/04A23L1/305A23K1/16G01N33/569
Inventor 李晓宇靖凯霖徐永平王丽丽李淑英
Owner DALIAN UNIV OF TECH
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