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A kind of heterogeneous gibel crucian carp anti-CYHV-2 oral recombinant spore vaccine and its preparation method

A heterogeneous gibel crucian carp, cyhv-2 technology, applied in recombinant DNA technology, antiviral agents, pharmaceutical formulations, etc., can solve problems such as safety risks and loss of immune induction

Inactive Publication Date: 2019-10-11
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 recombinant spores prepared by traditional spore surface display technology can germinate and form vegetative cells in the intestinal tract of animals, resulting in the loss of immune induction[9]
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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  • A kind of heterogeneous gibel crucian carp anti-CYHV-2 oral recombinant spore vaccine and its preparation method
  • A kind of heterogeneous gibel crucian carp anti-CYHV-2 oral recombinant spore vaccine and its preparation method
  • A kind of heterogeneous gibel crucian carp anti-CYHV-2 oral recombinant spore vaccine and its preparation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Preparation of Oral Recombinant Spore Vaccine Displaying CyHV-2 Antigen ORF25 Using gerKB as Integration Site

[0055] 1. Molecular Biology Operations

[0056] 1.1 Extraction of Bacillus subtilis chromosome

[0057] Collect 10mL B.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 technical operation

...

Embodiment 2

[0096] Preparation of Oral Recombinant Spore Vaccine Displaying CyHV-2 Antigen ORF25 Using gerAA as Integration Site

[0097] 1. Molecular biology manipulation and bacterial transformation

[0098] The operation method described in 1. and 2. in Example 1 is the same.

[0099] 2. Construction of an integrative vector with gerAA as the integration site

[0100] 2.1 Construction of an integrative platform vector with gerAA as the integration site

[0101] Using gerA-1 and gerA-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 gerA fragment of pJS1482 plasmid. Km-nif1 and Km-nif2 primers containing nif seque...

Embodiment 3

[0113] Recombinant Bacillus subtilis spores displaying ORF25 with gerBB as integration site without antibiotic resistance gene marker

[0114] 1. Molecular biology manipulation and bacterial transformation

[0115] Same as the method described in 1. and 2. in Example 1.

[0116] 2. Construction of an integrative vector with gerBB as the integration site

[0117] 2.1 Construction of an integrative platform vector with gerBB as the integration site

[0118] 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 digested with P...

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Abstract

The invention relates to a Carassius auratus gibelio anti-Cyprinid herpesvirus 2 (CyHV-2) oral recombinant Bacillus subtilis spore vaccine and a preparation method thereof. The vaccine is prepared by coating recombinant Bacillus subtilis spore CyHV-2 having a surface showing a CyHV-2 immune protective antigen with a Carassius auratus gibelio granular feed; and the above spore is a spore with germination defects and no antibiotic-resistant gene markers. The preparation method comprises the following steps: constructing the recombinant Bacillus subtilis spore CyHV-2 having germination defects, having no antibiotic resistance gene markers and having a surface showing the CyHV-2 immune protective antigen, and preparing the Carassius auratus gibelio granular feed coated CyHV-2 oral recombinant Bacillus subtilis spore vaccine used for preventing and treating CyHV-2 induced Carassius auratus gibelio hemorrhage. The oral recombinant Bacillus subtilis spore vaccine having germination defects, having no antibiotic resistance gene markers and having a surface showing the CyHV-2 immune protective antigen, prepared in the invention, has the characteristics of safety and high efficiency.

Description

technical field [0001] The invention belongs to the field of oral vaccines for aquatic animals. The oral recombinant spore vaccine related to germination defect, without antibiotic resistance gene marker and displaying CyHV-2 protective antigen on the surface and its preparation method. Background technique [0002] Herpesvirus type II (CyHV-2) is the pathogen of heterogeneous gibel carp hematopoietic organ necrosis, with strong infectivity, high lethality (over 90%), and wide prevalence [1]. CyHV-2 is a double-stranded DNA virus with a genome size of 290,304bp, encoding 150 viral proteins [2]. Among them, the genes orf25, orf25B and orf146 encode viral envelope proteins, which, like other viral envelope proteins, can induce specific immune protection in host animals [2]. CyHV-2-induced hematopoietic organ necrosis of heterogeneous gibel crucian carp has no effective drugs yet. Vaccination is the most effective measure to induce acquired immunity and prevent and control v...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K39/245C12N15/75A61P31/22
Inventor 宁德刚韦瑶张磊费倩
Owner INST OF AQUATIC LIFE ACAD SINICA
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