B cell immunodominant epitope peptide of staphylococcus aureus enterotoxin B and preparation method and application thereof

A staphylococcal enteric and immunodominant technology, applied in the field of medical biotechnology, can solve the problems of incomplete antigen B cell epitopes, cumbersome steps, long test cycle, etc., achieve good application prospects, simple and fast method, epitope no-miss effect

Inactive Publication Date: 2014-05-07
ARMY MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Screening B cell epitopes of antigens with monoclonal antibodies is cumbersome, the test cycle is long, ...

Method used

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  • B cell immunodominant epitope peptide of staphylococcus aureus enterotoxin B and preparation method and application thereof
  • B cell immunodominant epitope peptide of staphylococcus aureus enterotoxin B and preparation method and application thereof
  • B cell immunodominant epitope peptide of staphylococcus aureus enterotoxin B and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Example 1 Synthesis of Short Peptides Containing 18 Amino Acids with Walking Overlap

[0065] SEB is Staphylococcus aureus enterotoxin B, and its sequence is conserved among various strains, with a total length of 261 amino acids, of which 1-27 amino acids are signal peptides. The overlapping peptides constructed by recombination are 18 peptides between 1-261, which are derived from the MRSA252 international standard strain. The SEB protein sequence (No. P57839) derived from Staphylococcus aureus MRSA252 was searched in the UniProt protein database, and a short peptide with 18 amino acid steps was synthesized from the 1st amino acid (synthesized with the assistance of Shanghai Qiangyao Company), a total of 42 , with a purity greater than 90%. The synthetic peptide information is shown in Table 1. The synthesized peptides were dissolved in DMSO to a storage concentration of 0.5 mg / mL, and stored at -70°C after aliquoting.

[0066] UniProt protein database search URL...

Embodiment 2

[0072] Example 2 Screening and Identification of Antigen Dominant Epitope Peptides

[0073] 1. With the assistance of aluminum salt adjuvant, the protective effect analysis of antigen immunization and the collection of antiserum

[0074] (1) Preparation and identification of recombinant mutant SEB:

[0075] According to literature reports, three amino acid sites (L45R, Y89A, Y94A) in SEB were mutated, pGEX6p-2 vector was selected, recombinant SEB protein (rSEB) was cloned and expressed, and rSEB protein was purified by GST column (protein purity greater than 90%), It was identified by Western Blot. (Reference for mutation site: Oral Vaccine Formulations Stimulate Mucosal and Systemic Antibody Responses against Staphylococcal Enterotoxin B in a Piglet Model. Clinical and Vaccine Immunology, 2010 (17) 8: 1163-1169)

[0076] (2) Animal immunity:

[0077] Animals in aluminum salt adjuvant group were immunized by intramuscular injection, and animals in Freund's adjuvant group we...

Embodiment 3

[0091] Example 3 Synthesis of truncated peptides corresponding to step-overlapping immunodominant peptides

[0092] For the immunodominant epitope peptide sequence screened in Example 2, 2 amino acids were truncated sequentially from the N-terminal and C-terminal respectively, and the corresponding truncated peptides covering the full length of each immunodominant epitope peptide were synthesized; the recombinantly constructed overlapping peptides There are three groups of truncated peptides, each group corresponds to an immunodominant epitope peptide, and the difference between adjacent truncated peptides in each group is 2 amino acids, and the synthetic purity is greater than 90% (synthesized with the assistance of Shanghai Qiangyao Company). The synthetic truncated peptide information is shown in Table 3. The synthesized peptides were dissolved in DMSO to a storage concentration of 0.5 mg / mL, and stored at -70°C after aliquoting.

[0093] The amino acid sequence informat...

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Abstract

The invention relates to a B cell immunodominant epitope peptide of staphylococcus aureus enterotoxin B and a preparation method and application thereof, and concretely provides a B cell immunodominant epitope peptide of staphylococcus aureus enterotoxin B. Amino acid sequences of the B cell immunodominant epitope peptide are shown in SEQ ID NO: 17, 35 and 42. Corresponding amino acid sequences of core epitopes of the B cell immunodominant epitope peptide are respectively shown in SEQ ID NO: 48, 54 and 69. According to the preparation method, the B cell immunodominant epitope peptide of the staphylococcus aureus enterotoxin B is assayed by an overlapping peptide binding titration method, so that the screening method is simple, convenient, quick and accurate and is free from omission. The immunodominant epitope peptide does not contain unnecessary or even harmful parts, so that the risk of vaccines prepared from the immunodominant epitope peptide during use is lowered; the immunodominant epitope peptide has a relatively high vaccine application value and can be applied to the preparation of epitope vaccines and/or prevention vaccines of the staphylococcus aureus enterotoxin B.

Description

technical field [0001] The invention relates to the field of biopharmaceuticals, in particular to three B cell immunodominant epitopes from Staphylococcus aureus enterotoxin B, a method for identifying the B cell immunodominant epitopes and its application in the field of medical biotechnology. Background technique [0002] Staphylococcus aureus (S.aureus), as a representative of Gram-positive bacteria, is a kind of pathogenic coccus that widely exists in nature, and it is also an important pathogen that causes hospital infection and community infection. Survey studies have shown that in the United States, S.aureus is the most common pathogen of community skin and soft tissue infections (about 75%); in Japan, bullous impetigo caused by S.aureus accounts for 92% of impetigo; In Africa, S. aureus accounts for 55% to 72% of the pathogens of tropical pyomyositis; in China, S. aureus is the number one pathogen of infective endocarditis (accounting for 31% to 34%). In addition, S...

Claims

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Application Information

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IPC IPC(8): C07K14/31G01N33/68A61K39/085A61P31/04
CPCA61K39/00C07K14/31G01N33/6821G01N33/6824G01N2333/31
Inventor 吴超赵卓邹全明李滨孙合强陈立章金勇魏姗姗胡健曾浩王逸麟
Owner ARMY MEDICAL UNIV
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