JP2003252899A - Epitope having immunologic reactivity for farm animal serum albumin - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an epitope (peptide) of bovine serum albumin or sheep serum albumin, having immunologic reactivity for the human or a pet animal, and to provide a method for determining an epitope having immunologic reactivity of farm animal serum albumin. <P>SOLUTION: The method for determining an epitope having immunologic reactivity comprises comparing the amino acid sequence of human serum albumin with a farm animal serum albumin, preparing the peptide corresponding to a different part thereof, referring the obtained to an immunological test and finding out a peptide having immunologic reactivity. And an epitope having immunologic reactivity, of bovine serum albumin or sheep serum albumin, and determined by the method is provided. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD [0001] The present invention relates to an epitope (peptide) of bovine serum albumin (hereinafter referred to as BSA) or sheep serum albumin (hereinafter referred to as SSA) having immunological reactivity to humans or pet animals, and livestock serum albumin. And a method for determining an epitope having immunological reactivity.

[0002]

2. Description of the Related Art In recent years, the number of patients with food allergy has been increasing, and so-called five major allergens (milk, eggs, wheat, buckwheat, peanuts) are known. Although not as major as the five major allergens, meat may also cause allergies. For example, beef is allergenic. And it is known from the study of beef allergy that BSA is the main allergen of beef. Since BSA is also contained in milk, beef allergic patients also develop allergies when ingesting milk. Similarly, milk allergic patients also develop allergies when ingesting beef. It is known that pet animals such as dogs and cats are allergic to beef. As one of the methods for testing allergens, there is an immunological test method using IgE antibody in patient serum. However, it is difficult to obtain a large amount of patient serum, and thus there are limits to the implementation of this method. Therefore, there is a need for an immunological test method that can be performed universally. In addition, an antibody that specifically recognizes an allergen is desirable as an antibody to be subjected to an immunological test method, and a substance that causes an allergy, its constituent components (often,
A protein) or its amino acid sequence (epitope having immunological reactivity) is specified, and an antibody produced using it as an antigen is desirable. As a method for identifying an immunologically reactive epitope, 1) a method for investigating the reactivity of a limited degradation product prepared by enzymatic or chemical treatment of an allergen with a patient IgE antibody, 2) synthesis with reference to an amino acid sequence database Although a method for examining the reactivity of the overlapping peptide with the IgE antibody of the patient is known, it is a very laborious and inefficient method. On the other hand, as described above, the major allergen of beef was known to be BSA, but the immunologically reactive epitope in BSA was not known. In addition, it is said that there is an epitope in the amino acid sequence of the protein exhibiting allergenicity, which binds to the receptor of T cell and gives immunological stimulation to T cell, but this BSA has not examined it. It was

[0003]

SUMMARY OF THE INVENTION As described above, the conventional epitope determination method requires a considerable amount of time. Based on such a problem, the inventors have investigated a novel method for determining the epitope structure of livestock serum albumin having immunological reactivity, and as a result, compared the amino acid sequences of human serum albumin and livestock serum albumin, and found that the amino acid sequences differed. It was found that the epitope having immunological reactivity can be easily determined by preparing peptides at a certain position and subjecting them to an immunological test to find peptides having immunological reactivity. The present invention provides an immunologically reactive epitope of BSA and SSA determined by such a method, and a method of determining an immunologically reactive epitope. The immunological reactivity here means IgE antibody (in other words, B cell).
And / or T cell reactivity with the receptor.

[0004]

Means for Solving the Problems The present invention, which has been made to solve the above problems, provides (1) the following amino acid sequence or an amino acid sequence in which 1 to 5 amino acids are added to its N-terminal and / or C-terminal Having an immunological reactivity with BSA having: Ser-Pro-Asp-Leu-Pro-Lys-Leu-Lys-Pro-Asp-Pro-Asn-Th
r-Leu- Tyr-Ala-Asn-Lys-Tyr-Asn-Gly-Val-Phe-Gln- Glu-Tyr-Ala-Val-Ser-Val- Ala-Cys-Tyr-Thr-Ser-Val-Phe- Asp-Lys-Leu-Lys-His-Le
u-Val-Asp- Leu-Ile-Leu-Asn-Arg- Glu-Ser-Lys- Ala-Phe-Asp-Glu-Lys-Leu- Pro-Asp-Thr-Glu-Lys- Glu-Asn-Phe- Val- (2) An immunologically reactive epitope of SSA having the following amino acid sequence or an amino acid sequence in which 1 to 5 amino acids are added to its N-terminal and / or C-terminal; Tyr-Ala-Asn-Lys -Tyr-Asn-Gly-Val-Phe-Gln- Glu-Tyr-Ala-Val-Ser-Val- Leu-Ile-Leu-Asn-Arg- Pro-Asp-Thr-Glu-Lys- Glu-Asn-Phe -Val- (3) Compare the amino acid sequence of livestock serum albumin with the amino acid sequence of human or pet animal serum albumin,
Animal sera in which peptides having amino acid sequences having different amino acid sequences are subjected to immunological tests to test immunological reactivity and peptides having immunological reactivity are determined as epitopes having immunological reactivity And a method for determining an immunologically reactive epitope of albumin.

[0005]

BEST MODE FOR CARRYING OUT THE INVENTION As described above, it is known that the major allergen of beef is BSA. In a skin prick test, Fiocchi et al. Report that all children with beef allergy respond to BSA. However, to date there has been little practical information about the immunologically reactive epitopes. Serum albumin is one of the most widely studied and utilized proteins biochemically. It accounts for about 60% of serum proteins. The homology between BSA and human serum albumin (hereinafter referred to as HSA) is about 76%, and the repeating pattern of disulfide bonds is strictly preserved. It is surprising that the major constituents of serum act as allergens.

Therefore, the immunological reactivity of beef allergy patients to BSA was examined. Preliminary experiments have shown that IgE antibodies of patients with beef allergens sensitive to BSA do not bind HSA. Therefore, when the amino acid sequences of BSA and HSA were compared in detail, 15 epitopes having very different amino acid sequences were found. Therefore, these peptides were synthesized to produce IgE in patients with meat allergy.
The reactivity with the antibody (in other words, B cell) and the reactivity with the peripheral blood mononuclear cell fraction (in other words, T cell) were examined.

As a result, the following amino acid sequence or its N
A peptide having an amino acid sequence in which 1 to 5 amino acids, preferably homologous amino acids, were added to the terminal and / or C-terminal was identified as an immunologically reactive epitope in the BSA molecule. The above-mentioned homologous amino acid means, among the amino acid sequence numbers of BSA, 1) an amino acid having the sequence number located at the N-terminal and / or C-terminal of the following amino acid sequence, and 2) between BSA and HSA It refers to the amino acids that are common to the amino acid sequences (same for SSA). Here, the 1 to 5 amino acids added to both ends have a role of a spacer that connects to the plate, and as long as it has immunological reactivity, changing this portion did not affect the binding property. . Ser-Pro-Asp-Leu-Pro-Lys-Leu-Lys-Pro-Asp-Pro-Asn-Th
r-Leu- Tyr-Ala-Asn-Lys-Tyr-Asn-Gly-Val-Phe-Gln- Glu-Tyr-Ala-Val-Ser-Val- Ala-Cys-Tyr-Thr-Ser-Val-Phe- Asp-Lys-Leu-Lys-His-Le
u-Val-Asp- Leu-Ile-Leu-Asn-Arg- Glu-Ser-Lys- Ala-Phe-Asp-Glu-Lys-Leu- Pro-Asp-Thr-Glu-Lys- Glu-Asn-Phe- Val-Antibodies prepared using these peptides as antigens are useful for assaying BSA in foods eaten by beef allergy patients, and are useful for preventing food allergy in beef allergy patients.

When SSA was examined in the same manner, the following amino acid sequence or its N-terminal and / or
Alternatively, a peptide having an amino acid sequence in which 1 to 5 amino acids, preferably homologous amino acids, are added to the C-terminal is
It was identified as an immunologically reactive epitope in the A molecule. Tyr-Ala-Asn-Lys-Tyr-Asn-Gly-Val-Phe-Gln- Glu-Tyr-Ala-Val-Ser-Val- Leu-Ile-Leu-Asn-Arg- Pro-Asp-Thr-Glu- Lys- Glu-Asn-Phe-Val-

[0009]

The present invention will be described in more detail based on the following examples, but the invention is not intended to be limited to these examples. The test methods and materials described below are summarized below.

(1) Twelve beef allergy patients obtained by obtaining test serum informed consent (CAP-RAST score: meat 4 to 6; milk 3 to
6) and one healthy adult were tested. BSA (Sigma
Manufactured by SDS-PAGE in the usual way and transferred to PVDF membrane
When the membrane was reacted with the test patient serum and Western blotting was carried out using an anti-human IgE antibody as a secondary antibody, the test patient serum reacted with BSA. On the other hand, the test healthy adult serum treated in the same manner did not react with BSA.

(2) Synthesis of peptide NCBI (National Center for Biotechnology Informatio
The amino acid sequences of BSA and HSA were compared with reference to the protein data bank of n). The sequence of BSA is NC
BI accession number 229552 and accession number P02769 (due to incomplete individual data), and HSA sequence based on NCBI accession number 5821846. As shown in Table 1, a peptide corresponding to the amino acid sequence of BSA was synthesized with a peptide synthesizer (PSSM-8), the synthesized peptide was subjected to reverse phase HPLC, the main peaks were collected, freeze-dried, and DMSO (dimethyl sulfoxide). It was dissolved at a concentration of 5 mM in and stored at -80 ° C. In Table 1, for the sake of convenience, amino acids are represented by 1 according to the conventional method.
It is shown in letter notation (same for Table 2).

[0012]

[Table 1]

The amino acid sequences of BSA and SSA were compared with reference to the NCBI protein data bank. The data of BSA are two sequences (registration number 229552 and P02769), SSA
Is based on registration number 113582. As you can see here, BS
A and SSA had completely the same epitope except peptide No. 4 (see Table 2). Also in peptide No. 4, Tyr-Al which is an immunologically reactive epitope
a-Asn-Lys-Tyr-Asn-Gly-Val-Phe-Gln- were in agreement.
Furthermore, the epitopes found here can be used as immunologically reactive epitopes of ruminant goats as well as cattle and sheep.

[0014]

[Table 2]

Similarly, by referring to the protein data bank of NCBI and comparing the above-mentioned BSA data with amino acid sequence data of serum albumin of pet dogs and cats, the same as in the case of BSA and HSA, B for cats and cats
Epitopes with immunological reactivity of SA can be found.

(3) Reactive peptide solution of IgE antibody against synthetic peptide 4 times volume of 0.1M carbonate buffer (pH 9.6)
100 μl was fixed to the wells of the plate by the method of Gregorius et al., Blocked with 200 μl of 1% HSA, and then treated with patient serum. The peptide-bound IgE antibody is a biotinylated anti-human IgE antibody (Kirkegaard & P
erry Lab. Inc., USA) and streptavidin-peroxidase complex (Boehringer Mannheim, Germany)
It was detected with 0-phenylenediamine (Wako Pharmaceutical Co., Ltd.). The result is shown in FIG. In conclusion, the peptide was found to have immunological reactivity with the IgE antibody of beef allergy patients (in other words, the B cells of the patient).

(4) Reactivity of T cells to synthetic peptide Production of allergen-specific IgE antibody by B cells is controlled by allergen-specific T cells. Therefore, the reactivity of the synthetic peptide No. 11 in Table 1 with T cells of beef allergy patients was examined. Peripheral mononuclear cell fractions were prepared from blood collected from 4 beef allergy patients according to a conventional method and seeded on a culture plate (Nunc) at 1 × 10 ⁵ cells / well, in the presence of synthetic peptide (50 mM) and The cells were cultured in the absence, and the uptake of 5-bromo-2'-deoxyuridine was measured to examine the proliferation of T cells, that is, the reactivity with T cells. As a result, it was confirmed that the peptide reacts with T cells of beef allergy patients.

[0018]

INDUSTRIAL APPLICABILITY As described above, since the immunologically reactive epitope of the present invention specifically reacts with the IgE antibody (that is, B cell) of a beef allergy patient, an antibody for detecting an allergen of BSA is prepared. Can be used as an antigen for. In addition, using the immunologically reactive epitope of the present invention as an index, it is possible to reduce the allergens of beef and milk and processed foods containing them. Furthermore,
Among the immunologically reactive epitopes of the present invention, there are epitopes that specifically react with T cells. By using this epitope as an index, it is possible to control the reactivity of beef and milk and processed foods containing them to T cells. Further, according to the method for determining an immunologically reactive epitope of the present invention, an immunologically reactive epitope in livestock serum albumin can be simply and quickly determined.

[0019]

[Sequence list] <110> Nippon Meat Packers, Inc. <120> Immuno-reactive epitope of livestock serum albumin <160> 9 <210> 1 <211> 14 <212> PRT <213> Artificial Sequence <400> 1 Ser-Pro-Asp-Leu-Pro-Lys-Leu-Lys-Pro-Asp-Pro-Asn-Thr-Leu-   1 5 10 <210> 2 <211> 10 <212> PRT <213> Artificial Sequence <400> 2 Tyr-Ala-Asn-Lys-Tyr-Asn-Gly-Val-Phe-Gln-   1 5 10 <210> 3 <211> 6 <212> PRT <213> Artificial Sequence <400> 3 Glu-Tyr-Ala-Val-Ser-Val-   1 5 <210> 4 <211> 15 <212> PRT <213> Artificial Sequence <400> 4 Ala-Cys-Tyr-Thr-Ser-Val-Phe-Asp-Lys-Leu-Lys-His-Leu-Val-Asp-   1 5 10 15 <210> 5 <211> 5 <212> PRT <213> Artificial Sequence <400> 5 Leu-Ile-Leu-Asn-Arg-   1 5 <210> 6 <211> 3 <212> PRT <213> Artificial Sequence <400> 6 Glu-Ser-Lys-   1 <210> 7 <211> 6 <212> PRT <213> Artificial Sequence <400> 7 Ala-Phe-Asp-Glu-Lys-Leu-   1 5 <210> 8 <211> 5 <212> PRT <213> Artificial Sequence <400> 8 Pro-Asp-Thr-Glu-Lys-   1 5 <210> 9 <211> 4 <212> PRT <213> Artificial Sequence <400> 9 Glu-Asn-Phe-Val-   1

[Brief description of drawings]

FIG. 1 shows the binding properties of IgE to synthetic peptides. The synthetic peptides (No. 1 to 16) were fixed on an immunoplate and subjected to ELISA using diluted pooled serum. Note that nd means non-detection.

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Norihisa Takahata             3-3 Midorigahara, Tsukuba, Ibaraki Japan             Central Research Laboratory, Ham Co., Ltd. (72) Inventor Fumitake Morimatsu             3-3 Midorigahara, Tsukuba, Ibaraki Japan             Central Research Laboratory, Ham Co., Ltd. F-term (reference) 4H045 AA11 BA13 BA14 BA15 BA16                       BA17 CA42 DA70 DA86 EA01                       EA50 FA10

Claims (3)

[Claims] 1. An immunologically-reactive epitope of bovine serum albumin having the following amino acid sequence or an amino acid sequence in which 1 to 5 amino acids are added to its N-terminal and / or C-terminal. Ser-Pro-Asp-Leu-Pro-Lys-Leu-Lys-Pro-Asp-Pro-Asn-Th
r-Leu- Tyr-Ala-Asn-Lys-Tyr-Asn-Gly-Val-Phe-Gln- Glu-Tyr-Ala-Val-Ser-Val- Ala-Cys-Tyr-Thr-Ser-Val-Phe- Asp-Lys-Leu-Lys-His-Le
u-Val-Asp- Leu-Ile-Leu-Asn-Arg- Glu-Ser-Lys- Ala-Phe-Asp-Glu-Lys-Leu- Pro-Asp-Thr-Glu-Lys- Glu-Asn-Phe- Val- 2. An immunologically-reactive epitope of sheep serum albumin having the following amino acid sequence or an amino acid sequence in which 1 to 5 amino acids are added to its N-terminal and / or C-terminal. Tyr-Ala-Asn-Lys-Tyr-Asn-Gly-Val-Phe-Gln- Glu-Tyr-Ala-Val-Ser-Val- Leu-Ile-Leu-Asn-Arg- Pro-Asp-Thr-Glu- Lys- Glu-Asn-Phe-Val- 3. A method for comparing the amino acid sequence of domestic animal serum albumin with the amino acid sequence of human or pet animal serum albumin, and subjecting the peptides of the amino acid sequences having different amino acid sequences to immunological tests. A method for determining an immunologically-reactive epitope of livestock serum albumin, which tests reactivity and determines an immunologically-reactive peptide as an immunologically-reactive epitope.