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Preparation method and application of Vibrio parahaemolyticus toxoid vaccine

A technology for preparing hemolytic Vibrio and vaccines, which is applied in the field of preparation of Vibrio parahaemolyticus toxoid vaccine and Vibrio parahaemolyticus toxoid vaccine, and can solve the problem that there are few studies on the immune effect of aquatic economic animals.

Inactive Publication Date: 2011-12-14
HUAIHAI INST OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Because Vibrio parahaemolyticus can cause food poisoning in humans, the research on Vibrio parahaemolyticus at home and abroad is more concentrated on the medical field, and there is very little research on the immune effect of aquatic economic animals.

Method used

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  • Preparation method and application of Vibrio parahaemolyticus toxoid vaccine
  • Preparation method and application of Vibrio parahaemolyticus toxoid vaccine
  • Preparation method and application of Vibrio parahaemolyticus toxoid vaccine

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Embodiment 1

[0033] Embodiment 1, a kind of preparation method of Vibrio parahaemolyticus toxoid vaccine, its steps are as follows:

[0034] (1) Amplify the target gene TT :

[0035] Primer 1: 5'-GCGAATTC ATGAAGTACCGATATTTTGCA-3',

[0036] Primer 2: 5'-GCAAGCTTTTATTGTTGATGTTTACATTC-3',

[0037] Introduce EcoRI and HindIII enzyme cutting sites at the 5' ends of primers 1 and 2 respectively; use Vibrio parahaemolyticus genomic DNA as a template to amplify the hemolytic toxin gene of Vibrio parahaemolyticus TT ; The reaction system is 50 μl, each primer is 200 nmol / L, each dNTP is 50 μmol / L, template is 50 ng, pre-denatured at 95°C for 5 minutes, and then cycled for 35 seconds at 94°C for 30 seconds, 53°C for 40 seconds, and 72°C for 50 seconds The second time, the extension was terminated at 72°C for 10 minutes, and the product was recovered by tapping rubber to obtain the target gene fragment TT ;

[0038] (2) Clone expression: the target gene fragment TT Cloned into the vecto...

Embodiment 2

[0042] Example 2, experiment on the preparation method of Vibrio parahaemolyticus toxoid vaccine.

[0043] 1 Materials and methods

[0044] 1.1 Materials

[0045] 1.1.1 Experimental materials

[0046] Vibrio parahaemolyticus ( Vibrio parahaemotytifus , VP) strain (200027) was purchased from Jiangsu Provincial Center for Disease Control;

[0047] The pET-28 plasmid and Escherichia coli BL21 were kept in this laboratory; healthy red sea bream (average 250 g) was purchased from Lianyungang Ganyu Dongfang Aquaculture Co., Ltd., feeding conditions: water temperature: 14-16 ℃; light: indoor low light; Salinity: 25 / 1000; recirculating aquaculture, artificial soft pellet feed (purchased from Shandong Hualong Biotechnology Co., Ltd.) was fed once a day in the evening, and the feeding rate was 3% (relative to body weight).

[0048] 1.1.2 Reagents

[0049] pMD18-T-Vector, DNAMarker, ProteinMarker, restriction endonuclease, DNA ligase, purchased from Dalian Baobio Co., Ltd. ...

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Abstract

The present invention is a preparation method of Vibrio parahaemolyticus toxoid vaccine, which is characterized in that: first amplifying the target gene tdh; then cloning the target gene fragment tdh into the vector pET-28 to construct the expression vector pET-28-TDH, The pET-28-TDH plasmid was transferred into the expression strain BL21; after culturing and induced expression, the cloned expression product was obtained; the cloned expression product was detected by dot blot reaction, and the Vibrio parahaemolyticus toxoid was obtained; the Vibrio parahaemolyticus toxoid was mixed with An equal volume of complete Freund's adjuvant was mixed evenly to obtain the Vibrio parahaemolyticus toxoid vaccine. The vibrio parahaemolyticus toxoid vaccine prepared by the method of the invention can be used as an immune drug for marine fishes attacked by the vibrio parahaemolyticus, and the immune protection rate can reach about 50%.

Description

technical field [0001] The present invention relates to an interdisciplinary field of marine microorganisms and mariculture, in particular to a method for preparing a Vibrio parahaemolyticus toxoid vaccine; the present invention also relates to the application of the Vibrio parahaemolyticus toxoid vaccine prepared by the aforementioned method. Background technique [0002] Vibrio parahaemolyticus ( Vibrio parahaemotytifus, Vp) is a gram-negative bacterium, belonging to Vibrio family Vibrio genus, the shape is club-shaped, rod-shaped, arc-shaped, filamentous and other pleomorphic, scattered and arranged, often densely stained at both ends, without spores, bacteria There is a single flagella at one end of the body, and the movement is lively. It is a halophilic bacterium and a zoonotic bacterium. It mainly exists in marine products such as seawater near the coast, seabed sediments, fish and shellfish, etc., and can cause food poisoning and diarrhea. Aquatic animals attacked...

Claims

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Application Information

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IPC IPC(8): A61K39/106C12N15/31C12N15/70C07K14/28A61P31/04
Inventor 王洪斌成明李信书李士虎
Owner HUAIHAI INST OF TECH
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