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Anti-reovirus oral recombinant spore vaccine for grass carp and preparation method of anti-reovirus oral recombinant spore vaccine

A technology of reovirus and Bacillus subtilis, applied in antiviral agents, viral antigen components, recombinant DNA technology, etc., can solve the problems of loss of immune induction and safety risks

Inactive Publication Date: 2017-09-01
INST OF AQUATIC LIFE ACAD SINICA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The main reason is that the recombinant spores prepared by the traditional spore surface display technology can germinate and form vegetative cells in the intestinal tract of animals, resulting in the loss of immune induction[11,12]
In addition, recombinant spores contain antibiotic resistance gene markers for screening recombinant strains, and there are certain safety risks

Method used

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  • Anti-reovirus oral recombinant spore vaccine for grass carp and preparation method of anti-reovirus oral recombinant spore vaccine
  • Anti-reovirus oral recombinant spore vaccine for grass carp and preparation method of anti-reovirus oral recombinant spore vaccine
  • Anti-reovirus oral recombinant spore vaccine for grass carp and preparation method of anti-reovirus oral recombinant spore vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Preparation of Oral Recombinant Spore Vaccine Displaying GCRV Antigen Vp7 Using gerKB as Integration Site

[0055] 1. Molecular Biology Operations

[0056] 1.1 Extraction of Bacillus subtilis chromosome

[0057] Centrifuge to collect 10mL Bacillus subtilis 168 (trp - ) culture, add 0.5mL TE to suspend the pellet. Add 30 μL lysozyme (100 mg / mL) to each microcentrifuge tube, and react at 37°C for 1 h; add 50 μL 10% sodium dodecylsulfonate (SDS) and 20 μl 20 mg / mL proteinase K, shake evenly, and React at ℃ for 2 hours, add an equal volume of phenol and chloroform to extract, take the supernatant, add 2 times the volume of ethanol, and after 2 hours at room temperature, centrifuge at 12,000g for 10 minutes, discard the supernatant, wash the DNA precipitate with 500 μL of 75% ethanol, and Remove inorganic salt ions. After the DNA precipitate is dried, add 30-50 μL TE or ddH 2 O dissolved the DNA and stored it at -20°C for later use.

[0058] 1.2 Molecular biology techn...

Embodiment 2

[0094] Preparation of Oral Recombinant Spore Vaccine Displaying GCRV Antigen Vp7 Using gerAA as Integration Site

[0095] 1. Molecular biology manipulation and bacterial transformation

[0096] Same as the method described in 1. and 2. in Example 1.

[0097] 2. Construction of an integrative vector with gerAA as the integration site

[0098] 2.1 Construction of an integrative platform vector with gerAA as the integration site

[0099] Using gerAA-1 and gerAA-2 as primers, B. subtilis 168 chromosome as a template, PCR amplified gerAA gene fragment, T4 DNA polymerase filled in and cloned into pUC18 plasmid pJS313 (NdeI restriction site was destroyed, Molecular Cloning : Experiment Manual "Second Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989) at the PvuII site, the resulting recombinant plasmid is pJS1482. PstI and SacI restriction sites exist in the gerAA fragment in the pJS1482 plasmid. Km-nif1 and Km-nif2 primers containing nif sequence, pUB110...

Embodiment 3

[0111] Recombinant Bacillus subtilis spores displaying Vp7 on the surface with gerBB as integration site without antibiotic resistance gene marker

[0112] 1. Molecular biology manipulation and bacterial transformation

[0113] Same as the method described in 1. and 2. in Example 1.

[0114] 2. Construction of an integrative vector with gerBB as the integration site

[0115] 2.1 Construction of an integrative platform vector with gerBB as the integration site

[0116] Using gerBB-1 and gerBB-2 as primers, B. subtilis 168 chromosome as a template, PCR amplified gerBB gene fragment, T4 DNA polymerase filled in and cloned into pUC18 plasmid pJS313 (NdeI restriction site was destroyed, Molecular Cloning : Experiment Manual "Second Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989) at the PvuII site, the resulting recombinant plasmid is pJS1881g. Using gerBB-P and gerBB-S as primers and pJS1881g as a template, the inverse PCR amplification product was di...

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Abstract

The invention relates to a preparation method of an anti-reovirus oral recombinant bacillus subtilis spore vaccine for a grass carp. The anti-reovirus oral recombinant bacillus subtilis spore vaccine is prepared in the way that recombinant bacillus subtilis spore on the surface of which a grass carp reovirus immunoprotecive antigen is displayed is coated with a grass carp granulated feed, and the spore has a germination defect and no antibiotic resistance genetic markers. The preparation method comprises the steps that the recombinant bacillus subtilis spore having the germination defect and no antibiotic resistance genetic markers and displaying the grass carp reovirus immunoprotecive antigen on the surface are built, the anti-reovirus oral recombinant spore vaccine in which the grass carp granulated feed coats the recombinant spore is prepared, and the anti-reovirus oral recombinant bacillus subtilis spore vaccine for the grass carp is used for preventing and treating grass carp hemorragic disease aroused by reovirus.

Description

technical field [0001] The invention belongs to the field of oral vaccines for aquatic animals. The invention relates to an oral recombinant spore vaccine with germination defect and no antibiotic resistance gene marker which displays the protective antigen of grass carp reovirus on the surface and its preparation method. Background technique [0002] Grass carp reovirus (GCRV) is one of the most virulent fish viruses discovered so far. It can not only infect grass carp and cause severe hemorrhagic disease, but also infect herring, wheat ear carp, and rare goby carp. , causing significant economic losses to aquaculture. GCRV is also one of the best-studied fish viruses so far. In GCRV virus particles, virus surface antigens such as Vp5 and Vp7 can induce grass carp to produce specific protective antibodies, which are important antigens for the development of GCRV vaccines [1]. At present, inactivated cell vaccines and live attenuated vaccines for grass carp hemorrhagic di...

Claims

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Application Information

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IPC IPC(8): A61K39/15A61P31/14C12N15/75C12N15/62
Inventor 宁德刚张磊费倩韦瑶
Owner INST OF AQUATIC LIFE ACAD SINICA