B-cell antigenic multi-epitope peptide linked in tandem in OmpU of vibrio mimicus, making method and application thereof

Abstract

The present invention relates to an antigenic B-cell multi-epitope peptide linked in tandem in the outer membrane protein(Omp) U gene of vibrio mimicus, a making method and an application thereof, which belong to the field of molecular immunology. The B-cell multi-epitope peptide linked in tandem can induce fish to make protective immunity response to vibrio mimicus infection. In the present invention, the amino acid sequence of the antigenic B-cell multi-epitope peptide linked in tandem in the OmpU is shown in SEQIDNO:10. The B-cell multi-epitope peptide linked in tandem in the OmpU of vibrio mimicus is made by a genetic engineering technique. The verification of immune blotting analysis, specific antibody detection and immune animal protective experiments shows that the peptide can elicit efficient and specific protective humoral immunity response to vibrio mimicus infection. The B-cell multi-epitope peptide linked in tandem can be used for the immune diagnosis and the immune prevention and treatment of aquatic animal ascitic diseases. The present invention has high social benefit and economic benefit.

Description

technical field

[0001] The invention belongs to the field of molecular immunology, and specifically relates to an OmpU antigen B cell multi-epitope tandem peptide capable of inducing a fish body to produce a protective immune response against Vibrio mimicus infection, a construction method and application thereof. Background technique

[0002] Vibrio mimicus (Vibrio mimicus) is a pathogenic bacteria commensal to humans and aquatic animals. It can not only cause severe ascites in aquatic animals, but also infect humans through water bodies and aquatic products, causing diarrhea and food poisoning in humans. According to incomplete statistics, in recent years, the average case fatality rate of fish ascites disease has reached 30%-40%, and there is an upward trend, which has become a bottleneck for the sustainable development of aquaculture industry. Therefore, it is very necessary to establish a rapid diagnosis method for the disease and to develop a new and efficient vaccine ...

Examples

Embodiment 1

[0025] Example 1 Screening of B cell antigen epitopes of Vibrio mimicus OmpU protein and design of multi-epitope tandem peptides

[0026] 1. Materials

[0027] Amino acid sequence of OmpU gene of Vibrio mimicus Anhui isolate; epitope analysis software (DNAStar software).

[0028] 2. Methods and results

[0029] 1. Screening of B-cell epitopes of Vibrio mimicus OmpU protein

[0030] DNAStar software was used to comprehensively analyze the secondary structure, flexible region, hydrophilicity, surface possibility and antigenic index of the outer membrane protein OmpU of Vibrio mimeticus, and to screen its B cell epitopes. Select regions with hydrophilic index ≥ 0, surface possibility index ≥ 1, and antigenic index ≥ 1 in the OmpU protein structure, and further exclude the segments where the secondary structure is located in the α-helix and β-sheet regions that are not easy to form epitopes, and the remaining Segments identified as OmpU protein B-cell epitopes.

[0031] Test r...

Embodiment 2

[0037] Example 2 Construction method of Vibrio mimicus OmpU protein B cell multi-epitope tandem peptide

[0038] 1. Materials

[0039] 1. Plasmids, engineering bacteria and reagents,

[0040] Expression plasmid pAML-c4x, Escherichia coli E.coli TB1 strain, Amylose chromatography column and rabbit anti-MBP serum were purchased from NEB Biological Co., Ltd.

[0041] Nucleic acid restriction enzymes (PstI, EcoRI), Taq DNA polymerase and T 4 DNA ligase was purchased from Dalian TaKaRa Biotechnology Engineering Co., Ltd.

[0042] HRP-labeled goat anti-rabbit IgG (HRP-IgG), polyvinylidene fluoride membrane (PVDF), low molecular weight standard protein, Marker DL2000 and Marker DL15000 are products of MBI Company in the United States

[0043] DNA gel recovery kit and plasmid extraction kit were purchased from Hangzhou Weitejie Biotechnology Co., Ltd.

[0044] Agarose, ethidium bromide, ampicillin, Coomassie brilliant blue R 250 and isopropylthio-β-D-galactoside (IPTG) were purch...

Embodiment 3

[0129] Example 3 Preparation of multi-epitope tandem peptide vaccine and its immune protection effect

[0130] 1. Materials

[0131] The multi-epitope tandem peptide was prepared according to the above examples; healthy grass carp (about 100 g / tail) was purchased from Bingzi Fishing Farm, Feixi County, Anhui Province; Freund's adjuvant was a product of MBI Company in the United States.

[0132] 2. Methods and results

[0133] 1. Preparation of multi-epitope tandem peptide vaccine: the purified recombinant multi-epitope tandem peptide is mixed with Freund's adjuvant complete adjuvant or incomplete adjuvant in equal volume, emulsified into immunogen (vaccine)

[0134] 2. Immunization and specific antibody detection of multi-epitope tandem peptide vaccine: 30 healthy grass carp were randomly divided into three groups, 10 in each group. The fins of the first group were intramuscularly injected with the multi-epitope tandem peptide vaccine, the fins of the second group were injec...

technical field

[0001] The invention belongs to the field of molecular immunology, and specifically relates to an OmpU antigen B cell multi-epitope tandem peptide capable of inducing a fish body to produce a protective immune response against Vibrio mimicus infection, a construction method and application thereof. Background technique

[0002] Vibrio mimicus (Vibrio mimicus) is a pathogenic bacteria commensal to humans and aquatic animals. It can not only cause severe ascites in aquatic animals, but also infect humans through water bodies and aquatic products, causing diarrhea and food poisoning in humans. According to incomplete statistics, in recent years, the average case fatality rate of fish ascites disease has reached 30%-40%, and there is an upward trend, which has become a bottleneck for the sustainable development of aquaculture industry. Therefore, it is very necessary to establish a rapid diagnosis method for the disease and to develop a new and efficient vaccine ...

Examples

Embodiment 1

[0025] Example 1 Screening of B cell antigen epitopes of Vibrio mimicus OmpU protein and design of multi-epitope tandem peptides

[0026] 1. Materials

[0027] Amino acid sequence of OmpU gene of Vibrio mimicus Anhui isolate; epitope analysis software (DNAStar software).

[0028] 2. Methods and results

[0029] 1. Screening of B-cell epitopes of Vibrio mimicus OmpU protein

[0030] DNAStar software was used to comprehensively analyze the secondary structure, flexible region, hydrophilicity, surface possibility and antigenic index of the outer membrane protein OmpU of Vibrio mimeticus, and to screen its B cell epitopes. Select regions with hydrophilic index ≥ 0, surface possibility index ≥ 1, and antigenic index ≥ 1 in the OmpU protein structure, and further exclude the segments where the secondary structure is located in the α-helix and β-sheet regions that are not easy to form epitopes, and the remaining Segments identified as OmpU protein B-cell epitopes.

[0031] Test r...

Embodiment 2

[0037] Example 2 Construction method of Vibrio mimicus OmpU protein B cell multi-epitope tandem peptide

[0038] 1. Materials

[0039] 1. Plasmids, engineering bacteria and reagents,

[0040] Expression plasmid pAML-c4x, Escherichia coli E.coli TB1 strain, Amylose chromatography column and rabbit anti-MBP serum were purchased from NEB Biological Co., Ltd.

[0041] Nucleic acid restriction enzymes (PstI, EcoRI), Taq DNA polymerase and T 4 DNA ligase was purchased from Dalian TaKaRa Biotechnology Engineering Co., Ltd.

[0042] HRP-labeled goat anti-rabbit IgG (HRP-IgG), polyvinylidene fluoride membrane (PVDF), low molecular weight standard protein, Marker DL2000 and Marker DL15000 are products of MBI Company in the United States

[0043] DNA gel recovery kit and plasmid extraction kit were purchased from Hangzhou Weitejie Biotechnology Co., Ltd.

[0044] Agarose, ethidium bromide, ampicillin, Coomassie brilliant blue R 250 and isopropylthio-β-D-galactoside (IPTG) were purch...

Embodiment 3

[0129] Example 3 Preparation of multi-epitope tandem peptide vaccine and its immune protection effect

[0130] 1. Materials

[0131] The multi-epitope tandem peptide was prepared according to the above examples; healthy grass carp (about 100 g / tail) was purchased from Bingzi Fishing Farm, Feixi County, Anhui Province; Freund's adjuvant was a product of MBI Company in the United States.

[0132] 2. Methods and results

[0133] 1. Preparation of multi-epitope tandem peptide vaccine: the purified recombinant multi-epitope tandem peptide is mixed with Freund's adjuvant complete adjuvant or incomplete adjuvant in equal volume, emulsified into immunogen (vaccine)

[0134] 2. Immunization and specific antibody detection of multi-epitope tandem peptide vaccine: 30 healthy grass carp were randomly divided into three groups, 10 in each group. The fins of the first group were intramuscularly injected with the multi-epitope tandem peptide vaccine, the fins of the second group were injec...

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