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Vibrio mimicus oral target epitope gene vaccine and preparation method and application thereof

A technology of Vibrio mimicus and genetic vaccines, applied in vaccines, veterinary vaccines, chemical instruments and methods, etc., can solve the problems of decreased immunogenicity, achieve the effects of increasing expression, improving immune effect, and preventing nuclease degradation

Active Publication Date: 2018-07-13
ANHUI AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the "naked" epitope gene vaccine is vulnerable to nuclease degradation in the digestive tract environment after it is orally administered into the fish body. At the same time, the negative charge on the surface of the gene vaccine also limits its contact with the local antigen-presenting cell membrane of the intestinal mucosa. Therefore, "naked" epitope gene vaccines cannot be directly used for oral immunization

Method used

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  • Vibrio mimicus oral target epitope gene vaccine and preparation method and application thereof
  • Vibrio mimicus oral target epitope gene vaccine and preparation method and application thereof
  • Vibrio mimicus oral target epitope gene vaccine and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1 Design of Vibrio mimetic epitope gene vaccine target OVepis gene

[0054] Use the nucleotide sequence SEQ ID NO:8 with the linker peptide "alanine-alanine-tyrosine", the nucleotide sequence SEQ ID NO:8 with the linker peptide "glycine-glycine-glycine-glycine-serine-serine" ID NO:9 and the nucleotide sequence SEQ ID NO:9 with the linker peptide "glycine-proline-glycine", the nucleotide sequence SEQ ID NO:9 of the two B cell linear epitopes of the Vibrio mimicus adhesin protein OmpU ID NO:1 and SEQ ID NO:2, the nucleotide sequence of three B cell linear epitopes of mimic vibrio hemolysin protein VMH SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and mimic The nucleotide sequences of the two mimotopes of the vibrio hemolysin protein VMH are SEQ ID NO:6, SEQ ID NO:7 according to SEQ ID NO:1-SEQ ID NO:8-SEQ ID NO:2-SEQ ID NO :8-SEQ ID NO:3-SEQ ID NO:8-SEQ ID NO:4-SEQ ID NO:8-SEQ ID NO:5-SEQ ID NO:9-SEQ ID NO:6-SEQ ID NO:10- The sequences of SEQ ID NO:7 are concatenated to...

Embodiment 2

[0059] Example 2 Construction of eukaryotic expression recombinant plasmid carrying chimeric gene of grass carp CD74 and Vibrio mimicus epitope gene vaccine target OVepis gene

[0060] 1. Construction of eukaryotic expression recombinant plasmid pcDNA3.1(+)-CD74

[0061] 1.1 PCR amplification of CD74 protein coding gene of grass carp

[0062] The total RNA of grass carp head and kidney tissue was extracted with Trizol reagent, and cDNA was synthesized using FastQuant cDNA first-strand synthesis kit. Design a pair of specific primers according to the mRNA sequence of grass carp CD74 included in GenBank (accession number: KM369885.1), and the upstream primer is 5'-CTA GCTACG ATGGACGAGCATC-3'(SEQ ID NO:21), the underlined part is the Nhe I restriction site; the downstream primer is 5'-CCG CTCGAG TTACTCTGAGCCACACTGGG-3' (SEQ ID NO: 22), the underlined part is the Xho I restriction site. Using grass carp head kidney cDNA as a template, the CD74 protein coding gene with a size of ...

Embodiment 3

[0074] Example 3 Preparation and Safety Testing of Vibrio Mimetic Targeting Epitope Gene Vaccine

[0075] 1. Preparation of Escherichia coli DH5α slough freeze-dried powder

[0076] 1.1 Preparation of fresh slough of Escherichia coli DH5α

[0077] It will be self-constructed (the construction method belongs to the routine operation in this field, and can refer to Cai Kun, Bao Shizhong, Shi Jing, etc., preparation and preliminary research of Escherichia coli bacterial slough, Journal of the Academy of Military Medical Sciences, 2008, 32 (5): 436-438) recombinant Escherichia coli DH5α containing the temperature-controlled cleavage plasmid pBV220-LysisE was inoculated into 3 mL of LB culture medium containing ampicillin with a final concentration of 100 μg / mL, and cultured with shaking at 28°C and 180 rpm for 12 hours. Add 1 mL of the cultured bacteria solution above to 100 mL of LB culture solution containing ampicillin with a final concentration of 100 μg / mL according to the i...

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Abstract

The invention belongs to the technical field of gene vaccines and particularly relates to a vibrio mimicus oral target epitope gene vaccine. The vibrio mimicus oral target epitope gene vaccine comprises the following steps: S1, designing a vaccine target gene OVepis; S2, inserting the vaccine target gene OVepis designed in the S1 into 261-280bp of a related peptide fragment area of a II-type molecule in a protein coding gene CD74 of an immune carrier grass carp, wherein the direction is from 5'end to 3'end of a sequence; forming a CD74(1-261bp)-Ovepis-CD74(280-711bp) mosaic gene of 1020 bp, loading the mosaic gene by using a freeze-drying bacterial ghost of escherichia coli DH5a of a targeted delivery vector, and obtaining the target epitope gene vaccine. The nucleotide sequence of the mosaic gene is shown in SEQ ID NO:20. The vibrio mimicus oral target epitope gene vaccine has the beneficial effects that: the oral target epitope gene vaccine not only prevents the degradation of nuclease in vivo and increases the expression of a DNA encoding gene of the vaccine in a local antigen presenting cell of a mucous membrane, but also efficiently transfers an expressed and processed antigenpeptide to cells T and B, so that the immune effect of the vaccine is greatly improved.

Description

technical field [0001] The invention belongs to the technical field of gene vaccines, in particular to a Vibrio mimetic oral targeted epitope gene vaccine and its preparation method and application. Background technique [0002] Vibrio mimicus (Vibrio mimicus) is an intestinal pathogen that seriously endangers the healthy development of aquaculture. The bacterium mainly infects fish through the mucous membrane of the digestive tract, causing ascites disease in farmed fish. Oral vaccine-induced intestinal mucosal immune response plays a crucial role in the prevention of fish ascites disease caused by Vibrio mimicus and is the most practical vaccine formulation in aquaculture production. The epitope gene vaccine is a eukaryotic expression recombinant plasmid formed by cloning the protective antigen epitope gene to the downstream of the eukaryotic expression element. After the vaccine enters the body, it continuously expresses the antigenic protein through the transcription a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/106A61P31/04C12N15/70C12N15/62
CPCA61K39/107A61K2039/523A61K2039/542A61K2039/552C07K14/28C07K14/70596C07K2319/00C12N15/70
Inventor 李槿年曹际刘心媛刘雪兰李琳刘艳
Owner ANHUI AGRICULTURAL UNIVERSITY
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