Bovine viral diarrhea virus (BVDV) E2 protein antigen multi-epitope fusion peptide as well as preparation and application thereof

An antigenic epitope and multi-epitope technology, applied in the direction of viral peptides, fusion polypeptides, viruses, etc., to achieve good specificity, increase immune protective antigenic epitopes, and small coefficient of variation

Active Publication Date: 2018-10-02
SHANDONG NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no research on the vaccine of BVDV E2 protein antigen multi-epitope (E2 protein epitope of BVDV-1 and BVDV-2) fusion peptide and the diagnostic kit for detecting BVDV E2 protein antibody at home and abroad

Method used

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  • Bovine viral diarrhea virus (BVDV) E2 protein antigen multi-epitope fusion peptide as well as preparation and application thereof
  • Bovine viral diarrhea virus (BVDV) E2 protein antigen multi-epitope fusion peptide as well as preparation and application thereof
  • Bovine viral diarrhea virus (BVDV) E2 protein antigen multi-epitope fusion peptide as well as preparation and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Example 1 Screening of BVDV E2 antigenic epitope peptides

[0058] 1. Prediction of BVDV E2 epitope and preparation of epitope peptide

[0059] The gene sequences of BVDV-1 and BVDV-2E2 in GenBank were analyzed by online software for antigen epitopes, and a total of 35 B cell antigen sites were obtained, of which the B cell antigen epitopes of BVDV-1E2 and BVDV-2E2 were respectively 16 According to the sequence position and sequence similarity, 14 linear antigenic epitopes (Table 1) were selected for chemical synthesis of the polypeptides. In order to increase the immunogenicity of the polypeptides, the polypeptides were coupled with KLH respectively.

[0060] Table 1 Synthesized BVDV epitope peptides

[0061]

[0062] 2. Immunization of Antigenic Epitope Peptides

[0063] The above-mentioned KLH-coupled epitope peptides were immunized with 6-8 week-old Balb / c mice respectively. For the first immunization, KLH-antigen epitope peptides prepared with Freund's complet...

Embodiment 2

[0069] Example 2 Preparation of BVDV E2 antigen multi-epitope fusion peptide

[0070] 1. Construction of pET28a(+)-E2 recombinant vector

[0071] The above four antigenic epitope peptides with good immunogenicity screened are connected by flexible chains to make them function completely. The nucleotide sequence is: Capital letters are protective bases, underlines are restriction endonucleases BamH I and EcoR I restriction sites respectively, bold sequences are flexible chain connection sequences, and other sequences are antigenic epitope sequences. The synthetic E2 antigen epitope sequence was ligated by enzyme digestion, transformed into Escherichia coli DH5α, screened with blue and white spots, picked white colonies, extracted plasmid DNA, and carried out enzyme digestion verification. Cultivate the positive recombinant bacteria overnight in LB, extract the plasmid DNA with the kit, sequence the plasmid, culture the recombinant bacteria with correct sequencing, extract ...

Embodiment 3

[0078] Example 3 Application of E2 antigen multi-epitope fusion peptide in BVDV epitope vaccine

[0079] 1. Selection of immunized animals and preparation of antiserum: select 6 female New Zealand rabbits weighing 1.5kg, including 3 rabbits in the immunization group and 3 rabbits in the control group, inject subcutaneously at multiple points on the back, and the immune dose of E2 multi-epitope fusion peptide is 200ug / rat , the control group used PBS instead of multi-epitope fusion peptide; the first immunization was emulsified with Freund's complete adjuvant, the second immunization and the third immunization were emulsified with Freund's incomplete adjuvant; immunization was performed every 14 days, on the day of the first immunization and after immunization Blood is collected from the ear vein every 7 days, and 14 days after the third immunization, the antibody in the serum is tested. The fourth immunization can only be performed when the antibody titer is >1:10000. After the...

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Abstract

The invention discloses a bovine viral diarrhea virus (BVDV) E2 multi-epitope fusion peptide and a preparation method thereof. The preparation method comprises the following steps: performing gene synthesis on two respective epitope tandem sequences of optimized E2 proteins such as BVDV-1 and BVDV-2, cloning the sequences to a pET-28a (+) vector, transforming Escherichia coli DH-5alpha, transforming Escherichia coli BL21 from an accurate recombinant vector, performing IPTG inducible expression, and performing Ni-NTA purification, so as to obtain the BVDV E2 multi-epitope fusion peptide. Afterbeing subjected to animal immunization, the E2 multi-epitope fusion peptide is capable of producing a neutralizing antibody of the BVDV, and can be used for research and development of BVDV epitope vaccines; an ELISA (Enzyme-linked Immuno Sorbent Assay) detection kit prepared by utilizing the E2 multi-epitope fusion peptide can be used for BVDV antibody detection of cattle, and has the advantagesof being high in specificity, high in sensitivity, excellent in repeatability and easy to operate.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to BVDV E2 protein antigen multi-epitope fusion peptide and its preparation and application, in particular to BVDV-1 type and BVDV-2 type E2 protein antigen multi-epitope fusion peptide and its preparation method, and the Application of antigenic multi-epitope fusion peptide of BVDV-1 type and BVDV-2 type E2 protein in BVDV epitope vaccine and diagnostic reagent. Background technique [0002] Bovine viral diarrhea-mucosal disease is an important infectious disease caused by bovine viral diarrhea virus (BVDV), clinically characterized by fever, diarrhea, mucosal erosion, ulcer, leukopenia, persistent infection, immunosuppression , Abortion of pregnant cows, stillbirth and deformed fetus or fatal mucosal disease are the main features. The disease is distributed worldwide and has caused huge economic losses to the development of the global cattle industry. At present, more than 20 province...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/569G01N33/535G01N33/543C07K19/00C12N15/62
CPCC07K14/005C07K2319/00C12N2770/24322G01N33/535G01N33/543G01N33/56983G01N33/6854G01N2333/14
Inventor 何洪彬王洪梅何成强李书霞侯佩莉赵贵民
Owner SHANDONG NORMAL UNIV
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