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Staphylococcus aureus FnBPA-A protein mimic epitope peptides having immunizing protection, mimic epitope peptide composition, and applications of mimic epitope peptides and mimic epitope peptide composition

A Staphylococcus, protein simulation technology, applied in the direction of antibody mimics/scaffolds, medical preparations containing active ingredients, combined chemistry, etc., can solve the difficulty of mastitis in dairy cows, the hazards of the dairy industry, and affect the quality and safety of milk and dairy products human health issues

Active Publication Date: 2017-03-08
ANHUI AGRICULTURAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

For a long time, due to the irrational use of antibiotics in veterinary clinics, the emergence of animal-derived methicillin-resistant Staphylococcus aureus strains has been induced, which not only makes it very difficult to prevent and treat mastitis in dairy cows, but also brings serious harm to the dairy industry. Quality and Safety of Dairy Products and Human Health

Method used

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  • Staphylococcus aureus FnBPA-A protein mimic epitope peptides having immunizing protection, mimic epitope peptide composition, and applications of mimic epitope peptides and mimic epitope peptide composition
  • Staphylococcus aureus FnBPA-A protein mimic epitope peptides having immunizing protection, mimic epitope peptide composition, and applications of mimic epitope peptides and mimic epitope peptide composition
  • Staphylococcus aureus FnBPA-A protein mimic epitope peptides having immunizing protection, mimic epitope peptide composition, and applications of mimic epitope peptides and mimic epitope peptide composition

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1: Preparation and purification of rabbit anti-FnBPA-A protein antibody

[0043] 1.1 Antibody Preparation

[0044] Mix 1 mL of rFnBPA-A purified protein (prepared in our laboratory) with a concentration of 1 mg / mL and 42 μL of Tween-80 as the water phase, and then add 3126 μL of white oil and shake it repeatedly on a vortex shaker to emulsify. The emulsified immunogen was subcutaneously injected into the neck and back of experimental rabbits at multiple points, and the immunization dose was 1 mg / rabbit. The second immunization was performed on the 14th day after the first immunization, and the immunization dose and immunization route were the same. Three immunizations were performed on the 10th day after the second immunization, and no immune adjuvant was added during the third immunization. The immunization dose and immunization route were the same as those of the first immunization. Rabbit blood was collected before immunization and on the 32nd day after imm...

example 2

[0048] Example 2: Epitope screening and identification of FnBPA-A protein

[0049] 2.1 Panning of phage random dodecapeptide library with rabbit anti-FnBPA-A purified antibody

[0050] Phage random dodecapeptide library kit was purchased from NEB Company, and the library capacity was 2.7×10 9 transformants with a titer of 1.5 × 10 13 pfu / μL, Escherichia coli ER2537 is the host strain of the peptide library. The panning was carried out according to the kit instructions, and the brief process was as follows: use 0.1mol / L pH8.6 NaHCO 3 Solution The purified rabbit anti-FnBPA-A antibody was diluted to 100 μg / mL, coated on a 96-well plate, and kept at 4°C overnight. The next day, washed 6 times with TBST solution (TBS+0.5% Tween-20), added 5% BSA, and blocked for 1 h at 37°C. Discard the blocking solution and add 10 μL of the original peptide library (1.0×10 11 PFU), 195 μL of TBST and 195 μL of purified negative serum were mixed and added to enzyme-labeled wells, 100 μL / well,...

example 3

[0065] Example 3: Detection of immune activity of FnBPA-A protein mimic epitope

[0066] 3.1 Immunization of experimental mice and detection of immunogenicity of mimetope peptides

[0067] Kunming mice weighing 18-20 g were randomly divided into 12 groups, 20 in each group. The first group is the rFnBPA-A protein immunization group; the second group is the phage M13 immunization group; the 3-8 groups are the C2, C8, C10, C15, C19 and C23 positive phage clone immunization groups respectively; the 9-11 groups are the mock table Different ratios of peptides P1 and P2 were mixed group; the 12th group was the normal saline control group. The mice in the rFnBPA-A protein immunization group were injected with rFnBPA-A protein emulsified in Freund's adjuvant, 30 μg / mouse; the mice in the positive phage clone immunization group were injected with phage clone, 1×10 12 pfu / mouse; the mice in the mimotope peptide mixed group were injected with the mixed peptide emulsified in Freund's ad...

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Abstract

The invention relates to staphylococcus aureus FnBPA-A protein mimic epitope peptides having immunizing protection, a mimic epitope peptide composition, and applications of the mimic epitope peptides and the mimic epitope peptide composition. Two immunoprotective mimic epitope peptides provided by the invention have the amino acid sequences respectively shown in SEQ ID NO:1 and SEQ ID NO:2, the mimic epitope peptide composition consists of two polypeptides shown in SEQ ID NO:1 and SEQ ID NO:2, and the mass ratio of the polypeptide shown in SEQ ID NO:1 to the polypeptide shown in SEQ ID NO:2 is 2 to 1. Experiment animal immunoprotective tests show that the two mimic epitope peptides can stimulate a body to produce high-level specific antibodies and have a certain degree of immunizing protection; moreover, the mimic epitope peptide composition has the immunoprotective effect on staphylococcus aureus infection better than that of an FnBPA-A holoprotein. Therefore, the mimic epitope peptides and the composition thereof can be used as effective components for development of multi-epitope vaccines of staphylococcus aureus and prevention of cow mastitis caused by staphylococcus aureus.

Description

technical field [0001] The present invention relates to antigenic epitope peptides, in particular to mimetic epitope peptides of Staphylococcus aureus FnBPA-A protein with immune protection, mimetic epitope peptide compositions and their application in the preparation of Staphylococcus aureus multi-epitope vaccines . Background technique [0002] Staphylococcus aureus (S.aureus) is an important zoonotic pathogen, which can not only cause various human diseases, but also often cause clinical and recessive mastitis in cows. For a long time, due to the irrational use of antibiotics in veterinary clinics, the emergence of animal-derived methicillin-resistant Staphylococcus aureus strains has been induced, which not only makes it very difficult to prevent and treat mastitis in dairy cows, but also brings serious harm to the dairy industry. Quality and safety of dairy products and human health. Therefore, selecting the appropriate target antigen to develop a new type of Staphylo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/08C12N15/11C07K19/00A61K39/085A61P31/04C40B30/04
CPCA61K39/085A61K2039/55566C07K7/08C07K2319/00C40B30/04
Inventor 李槿年周欢欢赵雨婷刘雪兰夏生林
Owner ANHUI AGRICULTURAL UNIVERSITY
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