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

A technology of staphylococcus intestinal and immune superiority, applied in the application field of medical biotechnology, can solve the problems of long test period, incomplete antigen B cell epitopes, complicated steps, etc., and achieves no omission of epitopes, good application prospects, and methods Easy and fast effects

Inactive Publication Date: 2015-06-10
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, and the identified B cell epitopes of antigens are incomplete, and immunodominant epitopes cannot be identified

Method used

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  • B cell immunodominance epitope peptide of staphylococcus aureus enterotoxin B and preparation method and application thereof
  • B cell immunodominance epitope peptide of staphylococcus aureus enterotoxin B and preparation method and application thereof
  • B cell immunodominance 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 (number Q6GFN0) derived from Staphylococcus aureus MRSA252 was searched in the UniProt protein database, and a short peptide of 18 amino acids with overlapping steps was synthesized from the first 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 protei...

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 positions (L45R, Y89A, Y94A) in SEB were mutated, pGEX6p-2 vector was selected, and recombinant SEB protein (rSEB) was cloned and expressed. After the rSEB protein was purified by GST column (protein purity greater than 90%), It was identified by Western Blot. (Mutation site reference: 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 adjuvan...

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 immunodominance epitope peptide of staphylococcus aureus enterotoxin B and a preparation method and application thereof, and particularly provides the B cell immunodominance epitope peptide of the staphylococcus aureus enterotoxin. The amino acid sequence of the B cell immunodominance epitope peptide is shown in SEQ ID NO:42, and the amino acid sequence of core epitope corresponding to the B cell immunodominance epitope peptide is shown in SEQ ID NO:69. According to the B cell immunodominance epitope peptide of the staphylococcus aureus enterotoxin B and the preparation method and application thereof, an overlapping peptide combined titrimetric method is utilized to identify the B cell immunodominance epitope peptide of the staphylococcus aureus enterotoxin B, the screening method is simple, convenient, rapid and accurate, and no omission exists. The immunodominance epitope peptide does not contain unnecessary or even harmful parts, so that using risks of the prepared vaccine is reduced, a larger vaccine application value is achieved, and the B cell immunodominance epitope peptide of the staphylococcus aureus enterotoxin B and the preparation method and application thereof can be used for preparing the epitope vaccine or the preventive vaccine 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 cocci widely existing in nature, and it is also an important pathogenic bacteria that causes nosocomial 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 addi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/31G01N33/68A61K39/085A61P31/04
CPCC07K14/31A61K39/085G01N33/68
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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