JPH09131191A - Equine interleukin 1 peptide, dna coding for the same, recombined vector containing the same, transformant containing the recombined vector and production of equine interleukin 1-resistant antibody - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new equine interleukin 1 peptide containing a specific amino acid sequence, capable of being produced by a genetic engineering method, and useful for the cytokine therapy of the cytokine, arthritis, laminitis, etc., of horse and as an antigen for preparing an antibody. SOLUTION: This new equine interleukin 1 peptide contains a sequence comprising at least five continuous amino acids in a peptide having an amino acid sequence represented by formula I or II, and is useful for the cytokine therapy of inflammatory diseases such as the cytokine, arthritis, laminitis, etc., of horse and useful as an antigen for preparing an antibody. The peptide is obtained by stratifying the peripheral blood of a healthy thoroughbred horse on HICOLL- HYPAQUE, centrifuging the blood, isolating mRNA from the obtained peripheral blood mononuclear fraction by a conventional method, synthesizing cDNA with the mRNA, cloning the cDNA in the presence of a primer comprising a part of human interleukin 1 gene by a PCR method, and subsequently expressing the obtained equine interleukin 1 gene in a host cell.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to equine interleukin 1 peptide, DNA encoding the same, and DNA thereof.
And a transformant containing the recombinant vector and a method for producing an anti-horse interleukin 1 antibody.

[0002]

2. Description of the Related Art In horses, inflammatory diseases such as pneumonia, arthritis and laminitis have become clinically important problems, and measurement of cytokines and basic research on cytokine therapy have become important problems. On the other hand, inflammatory cytokines are known as endogenous pyrogenic factors and chondrocyte destruction factors.
Interleukin 1 is this inflammatory cytokine,
Involved in cell activation. Two types of interleukin 1 are known, α type and β type.

To date, the structure of interleukin 1 has been determined in humans, mice, etc. ("P. T. Lomedico et al., Nature, 312, 458, pp. 19").
84 (PT Lomedico et al., Nature, Vol.312, p.458 (1
984)) '' and `` CJ March et al., Nature, 315, 641, 1985 (CJ March et al., Nature,
Vol.315, p.641 (1985)) "). However, the structure for equine interleukin 1 has not been determined. This is an obstacle in studying the relationship between equine interleukin 1 and the above-mentioned inflammatory diseases, and further in developing a diagnostic agent for inflammatory diseases in view of the relationship with equine interleukin 1. It is a road.

[0004]

The invention according to claim 1 provides an equine interleukin-1 peptide which is important for studying the relationship between equine inflammatory disease and equine interleukin-1. The invention according to claim 2 is
In addition to the effect of the invention of claim 1, an α-type equine interleukin 1 peptide important for studying the relationship between equine inflammatory disease and equine interleukin 1 subtype (α-type) is provided. is there. In addition to the effect of the invention described in claim 1, the invention described in claim 3 is a β-type horseinter which is important for studying the relationship between inflammatory diseases of horses and equine interleukin 1 subtype (β type). It provides a leukin 1 peptide.

The invention according to claim 4 provides a DNA encoding a horse interleukin-1 peptide or a DNA complementary thereto, which is important for studying the relationship between equine inflammatory disease and equine interleukin-1. Is. In addition to the effect of the invention described in claim 4, the invention described in claim 5 has an equine inflammatory disease and equine interleukin-1.
The present invention provides a DNA encoding an α-type equine interleukin 1 peptide, which is important for studying the relationship between the subtypes of α-type. In addition to the effect of the invention of claim 4, the invention of claim 6 is a β-type horse, which is important for studying the relationship between inflammatory diseases of horses and equine interleukin 1 subtype (β type). It provides a DNA encoding the interleukin 1 peptide.

The invention according to claim 7 is a recombinant containing a DNA encoding a horse interleukin 1 peptide or a DNA complementary thereto, which is important for studying the relationship between equine inflammatory disease and equine interleukin 1. It provides a vector. In addition to the effect of the invention of claim 7, the invention of claim 8 is an α-type horse, which is important for studying the relationship between equine inflammatory disease and equine interleukin-1 subtype (α type). The present invention provides a recombinant vector containing a DNA encoding an interleukin 1 peptide. The invention according to claim 9 is the invention according to claim 7.
In addition to the effects of the described invention, a group containing a DNA encoding a β-type horse interleukin 1 peptide, which is important for studying the relationship between equine inflammatory disease and equine interleukin 1 subtype (β type) It provides a replacement vector.

The invention according to claim 10 is a transformation containing a DNA encoding a horse interleukin-1 peptide or a DNA complementary thereto, which is important for studying the relationship between equine inflammatory disease and equine interleukin-1. It provides the body. In addition to the effect of the invention of claim 10, the invention of claim 11 is an α-type horse, which is important for studying the relationship between inflammatory diseases of horses and equine interleukin-1 subtype (α type). The present invention provides a transformant containing a DNA encoding an interleukin 1 peptide. The invention according to claim 12 is the invention according to claim 1.
In addition to the effect of the invention described in 0, it contains a DNA encoding a β-type horse interleukin 1 peptide, which is important for studying the relationship between equine inflammatory disease and equine interleukin 1 subtype (β type) The present invention provides a transformant. The invention according to claim 13 provides a method for producing an anti-equine interleukin 1 antibody, which is important for studying the relationship between equine inflammatory disease and equine interleukin 1.

[0008]

Means for Solving the Problems The present invention includes the following (1) to
It relates to (13). (1) A horse interleukin 1 peptide containing a sequence consisting of at least 5 consecutive amino acids in the peptide represented by SEQ ID NO: 1 or 2. (2) The peptide according to (1) above, which is the peptide represented by SEQ ID NO: 1. (3) The peptide according to (1) above, which is the peptide represented by SEQ ID NO: 2. (4) A DNA encoding the peptide according to any one of (1) to (3) or a DNA complementary thereto. (5) The DNA according to (4) above, which has the base sequence represented by SEQ ID NO: 3. (6) The DNA according to (4) above, which has the nucleotide sequence represented by SEQ ID NO: 4.

(7) A recombinant vector containing the DNA according to any one of (4) to (6) above. (8) The recombinant vector according to (7) above, which is the plasmid pCEα. (9) The recombinant vector according to (8) above, which is the plasmid pEβ5. (10) A transformant containing the recombinant vector according to any one of (7) to (9). (11) The transformant according to the above (10), which has been deposited as FERM P-15142. (12) The transformant according to (10) above, which has been deposited as FERM P-15143. (13) A method for producing an anti-horse interleukin 1 antibody, which comprises using the peptide according to any one of (1) to (3) above as an antigen.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail. Horse interleukin 1 Interleukin 1 is an inflammatory cytokine as described above, and is involved in cell activation.
There are types. The α-type equine interleukin 1 is the peptide shown by SEQ ID NO: 1, and the β-type equine interleukin 1 is the peptide shown by SEQ ID NO: 2. In the sequence listing, the amino acid sequence has the amino acid at the terminal amino group as number 1 (the same applies hereinafter).

Horse interleukin 1 peptide The horse interleukin 1 peptide in the present invention is a generic term for the horse interleukin 1 or a part thereof, or a peptide containing horse interleukin 1 or a part thereof. The equine interleukin 1 peptide is required to possess the antigenicity as equine interleukin 1.

From the viewpoint of the minimum size of equine interleukin 1 having equine interleukin 1, the equine interleukin 1 peptide has a sequence consisting of at least 5 consecutive amino acids in the peptide represented by SEQ ID NO: 1 or 2. It is preferably a peptide containing peptide (hereinafter referred to as peptide A). Examples of peptide A include the peptide represented by SEQ ID NO: 1, which is the entire amino acid sequence of α-type equine interleukin 1. The peptide A also includes the peptide represented by SEQ ID NO: 2, which is the entire amino acid sequence of β-type equine interleukin 1. These peptides possess the antigenicity as equine interleukin 1.

A longer equine interleukin 1-derived amino acid sequence contained in the peptide chain of equine interleukin 1 peptide can be expected to give a highly sensitive antigen-antibody reaction, and a high biological activity (destruction of chondrocytes). Therefore, as peptide A, two consecutive peptides in the peptide shown in SEQ ID NO: 1 can be expected.
A peptide containing a sequence consisting of 0 or more amino acids is preferable, and a peptide containing a sequence consisting of 200 or more amino acids is more preferable. Examples of such a peptide include the peptide represented by SEQ ID NO: 1 and the peptide represented by SEQ ID NO: 2 described above.

From the viewpoint of enhancing the antigenicity, the peptide A is preferably a peptide having a sequence in which a sequence consisting of at least 5 consecutive amino acids in the peptide represented by SEQ ID NO: 1 is repeated. . The sequence serving as a repeating unit is, for example, SEQ ID NO: 1 above.
And the amino acid sequence of the peptide shown in SEQ ID NO: 2. The number of repetitions is not particularly limited, but is usually 1 to 10 times. The repeating units are bound to each other directly or via an intervening substance. Examples of the intervening substance include an amino acid sequence. The number of amino acids contained in the amino acid sequence is not particularly limited, but is usually 1 to 50, and specific examples of the amino acid sequence include an amino acid sequence consisting of alanine-alanine-alanine.

As long as it retains the antigenicity as equine interleukin 1, peptide A is SEQ ID NO: 1 or 2.
From the peptide represented by
Individual) may be missing. If the number of missing amino acids is too large, the antigenicity of peptide A as equine interleukin 1 tends to be impaired. When the number of missing amino acids is large (for example, 5 or more), the antigenicity of equine interleukin 1 is likely to decrease. Therefore, in order to minimize this decrease, the peptide shown in SEQ ID NO: 1 or 2 should be omitted. It is preferable that the amino acids are continuous.

Further, as long as the antigenicity of equine interleukin 1 is retained, as peptide A, a peptide containing an amino acid sequence other than equine interleukin 1-derived amino acid sequence can be used. , This peptide is required to have no or low antigenicity as a compound other than equine interleukin 1 against the antigenicity as equine interleukin 1. Examples of such a peptide include a peptide in which the amino acid in the peptide represented by SEQ ID NO: 1 or 2 is substituted with another amino acid, or an amino acid inserted in the peptide represented by SEQ ID NO: 1 or 2. Peptide, a peptide in which an amino acid or another peptide is bound to the sequence consisting of at least 5 consecutive amino acids in the peptide represented by SEQ ID NO: 1 or 2 directly or via an intervening substance.

When the number of amino acids to be substituted or inserted is large (for example, 5 or more), and when the number of amino acids contained in other peptides to be bound is too large (for example, 1,
000 or more), the antigenicity of equine interleukin 1 is likely to decrease. Therefore, in order to minimize this decrease, the amino acids to be substituted or inserted in the peptide represented by SEQ ID NO: 1 or 2 are consecutive. It is preferable that the other peptide to be bound is a sequence consisting of less than 1,000 amino acids.

The amino acid to be replaced preferably has similar properties, and examples thereof include substitution of glycine with alanine. Examples of the amino acid or other peptide to be bound include alanine, alanine-alanine-alanine, β-galactosidase and the like. The inclusions include those mentioned above. The intervening substance, amino acid or other peptide is bound to either the amino group terminal side or the carboxyl group terminal side of the sequence consisting of at least 5 consecutive amino acids in the peptide shown in SEQ ID NO: 1 or 2. Good.

Method for Producing Horse Interleukin 1 Peptide Examples of the method for producing the horse interleukin 1 peptide in the present invention include chemical synthesis method and gene recombination method. As a chemical synthesis method, for example, there is a map (Multiple Antigen Peptide, MAP) method.
It is suitable for synthesizing peptides consisting of not more than individual amino acid sequences and can be synthesized using a commercially available peptide synthesizer. Peptides can be repeatedly synthesized by the map method. Examples of the gene recombination method include D encoding the equine interleukin 1 peptide of the present invention.
There is a method in which NA is inserted into a vector to construct a recombinant vector, which is inserted into a host to prepare a transformant, and the desired peptide is purified from the transformant.

The DNA encoding the equine interleukin 1 peptide of the present invention will be described later. Vectors include, for example, plasmids and phages. Examples of the host include Escherichia coli, Bacillus subtilis, yeast and the like. Hereinafter, a method for producing a transformant and a method for purifying a target peptide using the transformant will be described in detail.

The recombinant vector containing the DNA of the present invention is
DNA encoding the horse interleukin 1 peptide
It can be prepared by inserting (described below) into an existing plasmid vector, phage vector or the like by a conventional method. that time,
Use linkers if needed. When the inserted DNA is expressed, the DNA insertion site must be downstream of the promoter region. Examples of existing plasmid vectors include pBR322, pUC18,
pUC19, pCR2 (Invitrogen)
(US) trade name, Bluescript SK (-) (Stratagene (USA) trade name) and the like. Examples of phage vectors include λgt10 and λgt11.
Etc. All of these vectors are commercially available, and a recombinant vector corresponding to the parent vector used can be obtained. The recombinant vector containing the DNA of the present invention includes:
As will be described later, pCEα, pEβ5 and the like can be mentioned.

The resulting recombinant vector is put into a host to prepare a transformant. When a plasmid derived from Escherichia coli or λ phage is used, Escherichia coli can be used as a host, and for example, Escherichia coli HB101 strain can be used. The host is usually treated and used so as to be a competent cell. Competent cells obtained by treating Escherichia coli HB101 are sold by Takara Shuzo Co., Ltd. and others. As a method for producing a transformant using E. coli, for example, E. coli in the first half of the logarithmic growth phase is
A method of washing with 0 mM calcium chloride solution, suspending Escherichia coli in the calcium chloride solution, adding the recombinant vector thereto, and incubating at 42 ° C for 1 to 2 minutes can be adopted.

The recombinant vector and the transformant can also be prepared by using a commercially available kit. As such a kit, TA-Cloning Kit manufactured by Invitrogen Co., USA can be used. And Qiagen's Quregen Plasmid Kit (Qiag)
en Plasmid Kit) etc. A general method for producing transformants using recombinant vectors is "Edited by Sam Block et al., Molecular Cloning 2nd Edition (Cold Spring Harbor Laboratory) (1989)".
(J. Samblook et al., Molecular Cloning 2nded., Col
d Spring Harbor Laboratory Press (1989), below,
This document is referred to as the document "Molecular Cloning")
It is described in.

As a method of culturing the transformant, for example, a method of shaking the incubator at an appropriate temperature until the equine interleukin 1 peptide is sufficiently accumulated in the transformant in a medium in which the transformant can grow. Can be adopted. Examples of the medium include LB medium, TB medium, 2 ×
TY medium or the like can be used. The culture temperature may be, for example, 35 to 42 ° C. when Escherichia coli is used as the host. If the recombinant vector is capable of expressing an antibiotic resistance gene, it is preferable to add the antibiotic to the medium from the viewpoint of suppressing the growth of transformants that do not carry the recombinant vector. Examples of such antibiotics include ampicillin. The culturing time varies depending on the type of the host, the culturing device, the culturing temperature, the concentration of the transformant in the culturing solution at the start of culturing, etc., but when culturing with shaking at 37 ° C. in LB medium, it is usually overnight Is. A general method for preparing and culturing LB medium, TB medium, 2 × TY medium and the like is described in the document “Molecular Cloning”.

As a method for disrupting the cultured transformants, for example, the transformants are collected by centrifugation, suspended in a buffer solution to prepare a suspension, and the suspension is subjected to physical shock. The method of giving can be adopted. As the buffer solution, for example, a TE buffer solution can be used. As a method of giving a physical impact to the suspension, for example,
A method of irradiating the suspension with ultrasonic waves can be used. When the transformant is Escherichia coli, instead of giving a physical shock to the suspension, for example, lysozyme is added to the suspension and a TE buffer containing sodium dodecyl sulfate (SDS) is added and stirred. Therefore, a method of dissolving the transformant can also be adopted. After crushing or lysing the transformant, centrifugation is performed to remove cell debris,
Obtain the supernatant.

When the target peptide is a fusion protein having a signal peptide bound to its amino terminal side (that is, the target peptide can be secreted extracellularly), the culture solution is A method of obtaining a supernatant by centrifugation can be used.

As a method for purifying the peptide, for example, each step of removing a nucleic acid by adding streptomycin sulfate and obtaining a protein by adding ammonium sulfate can be used. As the step of removing nucleic acid by adding streptomycin sulfate, for example, streptomycin sulfate is added to any of the above supernatants, stirred for a while, and centrifuged to remove the nucleic acid as a precipitate, The procedure of obtaining the supernatant can be used.
As the step of adding ammonium sulfate to obtain a protein, for example, an operation of adding ammonium sulfate to the supernatant after removing the nucleic acid as a precipitate, stirring and centrifuging can be used. Usually, a precipitate is obtained, but since the target peptide may be contained in the supernatant, it is preferable to sample and confirm the presence or absence of the target peptide.

Subsequently, a step of obtaining a fraction containing equine interleukin 1 peptide is carried out. As this process,
For example, a method in which the precipitate is dissolved in a small amount of buffer or the supernatant is fractionated by liquid chromatography to identify the fraction containing equine interleukin 1 peptide can be used. In order to identify the fraction containing the equine interleukin 1 peptide, it is possible to use, for example, electrophoresis using the molecular weight as an index or bioassay using the bioactivity such as the chondrocyte destructive ability as an index. it can. The molecular weight of equine interleukin 1 peptide can be determined from its amino acid sequence. Specific methods for removing residues such as cell membranes, removing streptomycin sulfate-added nucleic acids and ammonium sulfate-added proteins are described in the literature “Molecular.
Cloning ".

When the vector contained in the transformant can produce the peptide encoded by the inserted DNA as a fusion protein with another peptide, this "other peptide" The horse interleukin 1 peptide can be purified by utilizing the property.

DNA Encoding Horse Interleukin 1 Peptide In the present invention, DNA encoding horse interleukin 1 peptide is DNA encoding peptide A. This DNA has the amino acid sequence of peptide A as the triplet code table. Is a DNA selected from the group of DNAs when amino acids have been replaced with nucleotide sequences according to the above. In the present invention, the DNA encoding the peptide represented by SEQ ID NO: 1 or 2 means the peptide represented by SEQ ID NO: 1 or 2 by the triplet code table (each amino acid is assigned 1 to 6 nucleotide sequences). A DNA group in which an amino acid is read as a nucleotide sequence according to the above (including DNAs having the nucleotide sequences shown in SEQ ID NO: 3 or 4, respectively).

Examples of the peptide A include those described in the above-mentioned equine interleukin 1 peptide section, and examples of the DNA encoding the peptide A include those having a nucleotide sequence corresponding to the amino acid sequences of these peptides. In the sequence listing, the base sequence has the base at the 5'end as number 1 (the same applies hereinafter).

The DNA encoding the peptide A can be prepared by a chemical synthesis method or a gene recombination method. The chemical synthesis method includes, for example, the phosphoamidide method, which is suitable for synthesizing a DNA having a base sequence having a total length of 100 bases or less, and can be chemically synthesized by a commercially available DNA synthesizer. In order to prepare DNA having a total length of more than 100 bases, a gene recombination method described later can be used, but it can also be prepared as follows. That is,
Divide the base sequence into less than 100 bases, chemically synthesize a DNA fragment consisting of each base sequence as described above,
These DNA fragments are mixed and ligated with T4-DNA ligase. At that time, a DNA fragment of a complementary strand is also synthesized, and when the DNA fragments are paired with each other so that a protruding end is generated, the protruding end serves as a sticky end, so that a target DNA is easily obtained.

As the gene recombination method, for example, as described below, a complementary DNA (cDNA) is prepared using equine end blood monocyte mononuclear cells (PBMC), and the gene of interleukin 1 of other species is prepared. And a method of amplifying a cDNA encoding a horse interleukin 1 peptide using a primer prepared based on the nucleotide sequence conserved in 1. The gene recombination method can also produce a long DNA of 100 bases or more. The base sequence of DNA can be determined by a conventional method. The nucleotide sequence can be determined by, for example, the dideoxy termination method, and a commercially available kit can be used. Such a kit is sold by Pharmacia of Sweden.

Next, the method for obtaining the cDNA encoding the equine interleukin 1 peptide from equine PBMC will be described in detail. First, blood (peripheral blood or the like) is obtained from a horse (Thoroughbred or the like), a PBMC fraction is obtained by centrifugation or the like, and this fraction is cultured. As a culture method, for example, in the presence of carbon dioxide (for example, 5% (v
/ V)), fetal bovine serum, gentamicin and lipopolysaccharide in a medium for animal cell culture containing a PBMC fraction at a suitable temperature (for example, 37 ° C) for 24 hours. As a medium for animal cell culture,
For example, RPMI1640 medium (trade name of Nissui Pharmaceutical Co., Ltd.)
Can be used.

Subsequently, PBMC cells are collected by centrifugation or the like, and the PBMC cells are lysed to obtain a cell extract. As a method for lysing cells to obtain a cell extract, for example, a method in which cells are frozen in liquid nitrogen, suspended in a lysis buffer, and kept warm can be used. Examples of the lysis buffer include Tris-hydrochloric acid (10 mM, pH 7.4) containing proteinase K (20 μg / ml), ethylenediaminetetraacetic acid (EDTA, 1 mM) and sodium dodecyl sulfate (SDS, 0.5%). Can be mentioned.

Polyadenylic acid-bound RNA (hereinafter referred to as P
oly (A) + RNA) is obtained. From cell to P
A general method for obtaining oly (A) + RNA is described in the literature "
Although it is described in "Molecular cloning", a kit sold by Invitrogen, etc. can be used. Poly (A) obtained according to a conventional method
CDNA is prepared from + RNA. A general method for preparing cDNA is described in the document "Molecular Cloning", but a kit sold by Pharmacia can also be used.

Next, a method of amplifying a cDNA encoding a horse interleukin 1 peptide using a primer prepared based on a nucleotide sequence conserved in the gene of interleukin 1 of another organism and obtaining it Will be explained. Examples of the method for amplifying the cDNA include a method in which a primer, a polymerase (tack polymerase, etc.) and deoxyribonucleotides are added to the obtained cDNA, and heating, cooling, and heat retaining steps are repeated. As a primer, a nucleotide sequence that is well conserved between human and mouse interleukin 1 genes ("P.T. Romedico et al., Nature, 312
Volume, p. 458, 1984 (PT Lomedico et al., Nature, Vo
L.312, p.458 (1984)) and CJ March et al., Nature, 315, 641, 1985 (CJ March et al.
al., Nature, Vol.315, p.641 (1985)) ”or a DNA fragment having a base sequence complementary thereto can be used. Examples of such DNA include SEQ ID NOs: 5 to 5.
A DNA fragment having the base sequence of 8 is mentioned.

The nucleotide sequence of SEQ ID NO: 5 is the nucleotide sequence located upstream of the coding region of the peptide among the nucleotide sequences of human and mouse interleukin 1α gene,
The base sequence of SEQ ID NO: 6 is a base sequence complementary to the base sequence located on the downstream side of this coding region. Therefore, the amplified cDNA is expected to contain the entire coding region of the horse interleukin 1α peptide. On the other hand, the base sequences of SEQ ID NOs: 7 and 8 are the base sequences well conserved in human and mouse interleukin 1β gene and the base sequences complementary thereto, respectively. However, judging from the positional relationship of these base sequences, it is predicted that the amplified cDNA does not contain the entire coding region of equine interleukin 1β peptide, but is a part thereof. The general method of this DNA amplification method is Polymerase Chain Reaction.
in reaction (PCR) method, the details of which are described in the document "Rule cloning".

When the obtained cDNA does not contain the entire coding region of equine interleukin 1 peptide, genome walking or the like can be further performed to obtain the DNA containing the entire coding region. A commercially available kit can also be used for genome walking.

Once the DNA encoding the equine interleukin 1 peptide is obtained, the DNA can be replicated by utilizing the gene recombination method or the PCR method described above. The operation of re-acquiring the DNA encoding the leukin 1 peptide is unnecessary.

The method using the gene recombination method is, for example,
It can be done as follows. First, a recombinant vector is prepared by inserting the obtained DNA encoding the equine interleukin 1 peptide into an existing plasmid vector, phage vector or the like as described above, and inserting the recombinant vector into a host to obtain a transformant. And to transform the transformant. Since the recombinant vector is amplified in the transformant by this culture, the amplified recombinant vector is obtained from the transformant, and the DNA encoding the horse interleukin 1 peptide is cut out using a restriction enzyme. The operation of obtaining the amplified recombinant vector from the transformant can be performed, for example, as follows. When the recombinant vector is a plasmid, the transformant is crushed and subjected to phenol / chloroform treatment and ethanol precipitation treatment to obtain DNA. The obtained DNA is subjected to ultracentrifugation using cesium chloride containing ethidium bromide to obtain the recombinant vector as a plasmid DNA. On the other hand, when the recombinant vector is a phage, the transformant is disrupted to obtain phage particles, the phage particles are lysed, and the recombinant vector is obtained as phage DNA.

As a method utilizing the PCR method, for example, a primer DNA is prepared by a chemical synthesis method based on the nucleotide sequences at both ends of the DNA to be amplified, and DNA encoding the horse interleukin 1 peptide is prepared. A method of performing the PCR method can be used as the template DNA. A general method for replicating DNA using the gene recombination method or the PCR method is described in the document "Molecular Cloning".

Method for Producing Anti-Equine Interleukin 1 Antibody The anti-equine interleukin 1 antibody of the present invention can be obtained, for example, by using the equine interleukin 1 peptide of the present invention as an antigen. It can be obtained as a leukin 1 antibody. As a method of producing antiserum, for example, a method of immunizing animals such as rabbits and mice with the equine interleukin 1 peptide of the present invention as an antigen and obtaining the serum can be used. As a method for isolating and producing an anti-horse interleukin 1 antibody, for example, a rabbit or a mouse is immunized with the horse interleukin 1 peptide of the present invention as an antigen, and the spleen cells thereof are fused with myeloma cells to form a hybridoma. A method in which a hybridoma that recognizes the horse interleukin 1 peptide described above is prepared, selected, cultured, and the culture supernatant is obtained can be used. In order to enhance the antigenicity, an equine interleukin 1 peptide of the present invention may be bound with a suitable carrier protein as an immunogen, if necessary.

Various adjuvants can be used for immunization, but Freund's complete adjuvant (F
CA) and incomplete Freund's adjuvant (FIA) are preferred. As myeloma cells, for example, P3X63A
g8.653 (ATCC (American Type Culture Coll
section) CRL-1580) or P3 / NSI / 1-A
g4-1 (ATCC TIB-18) can be used. As an animal to be immunized with the antigen, rabbit, mouse, rat, cow, sheep, goat, chicken and the like can be used, but not limited to the animal species exemplified here. The anti-horse interleukin 1 antibody can be produced according to a known general method of immunizing an animal to obtain an antibody, except that the equine interleukin 1 peptide of the present invention is used as an antigen. Antibodies include polyclonal and monoclonal antibodies.

The antiserum and the isolated and produced anti-horse interleukin 1 antibody may be modified with various enzymes, colloids and the like. The obtained antiserum and the isolated and produced anti-horse interleukin 1 antibody can be used to measure the concentration of equine interleukin 1 in blood, body fluid, urine, etc. Not only can it be used as a research reagent for studying the relationship of leukin 1, but it can also be expected to find application in the diagnosis of equine health and inflammatory diseases.

[0046]

The present invention will be described in detail below with reference to examples. Example 1 Preparation of Horse Peripheral Blood Mononuclear Cell (PBMC) cDNA Healthy Thoroughbred peripheral blood was treated with Ficoll-Hypaque (Lymphoprep).
(Norway) (trade name) and subjected to centrifugation to obtain a peripheral blood mononuclear cell fraction. Peripheral blood mononuclear cell fraction was converted to 10% fetal bovine serum, gentamicin (50 μg / m
l), RPM containing lipopolysaccharide (5 μg / ml)
I1640 medium (trade name of Nissui Pharmaceutical Co., Ltd.) was suspended at a concentration of 1 × 10 ⁶ cells / ml, and this suspension was kept at 37 ° C. in the presence of 5% (v / v) carbon dioxide gas. . 24 hours later,
Cells were harvested by centrifugation and frozen in liquid nitrogen. P from cells frozen in liquid nitrogen using Fast Track mRNA Isolation Kit (trade name of Invitrogen)
oly (A) + RNA was extracted. Using a cDNA synthesis kit (Pharmacia (Sweden)), Pol
cDNA was synthesized from y (A) + RNA.

Example 2 Acquisition of cDNA of equine interleukin-1α In order to obtain the entire coding region of equine interleukin-1α using the PCR method, the nucleotide sequences of the primers used in the PCR method are human and mouse. Among the nucleotide sequences that are well conserved in the interleukin 1α gene, the nucleotide sequence located upstream of the peptide coding region (the nucleotide sequence represented by SEQ ID NO: 5) and the nucleotide sequence located downstream of this coding region A complementary base sequence (base sequence represented by SEQ ID NO: 6) was used.

D of the nucleotide sequences shown in SEQ ID NOs: 5 and 6
NA was chemically synthesized with a DNA synthesizer and used as primers. 0.4 mM of each of these primers, 1.5 units of tack polymerase and deoxyribonucleotides were added to the cDNA prepared in Example 1 to make 100 μl, and amplification by the PCR method was performed. As the amplification operation,
Denaturation by heating (94 ° C, 1 minute), cooling (37 ° C, 1 minute)
30 minutes, and annealing (72 ° C, 1 minute) for polymerization, 30 cycles of each step.
Repeated times. After the amplification operation, the reaction solution was subjected to ethidium bromide-containing 2% agarose gel electrophoresis to obtain DN in the reaction solution.
A was separated by the difference in molecular weight (the smaller the molecular weight, the longer the migration distance), and amplified cDNA was obtained. The obtained cDNA was ligated with a linker of restriction enzyme EcoRI,
CDNA using a A-cloning kit (TA-Cloning Kit) (trade name of Invitrogen), to which a linker is bound
Was ligated with the plasmid pCR2 attached to this kit to obtain a ligation mixture. Using Escherichia coli (DH5α (trade name of BRL (USA)) as a host, the suspension of E. coli and the above ligation mixture were mixed. This mixed solution contains ampicillin (50 mg / ml), 5-bromo-4-chloro-3-indolyl-β-D-galactoside (36 mg / ml) and isopropyl-β-D-thiogalactoside (40 mg / ml). It was seeded on a TY agar plate and cultured. Furthermore, using a CUREGEN plasmid kit (trade name of CUREGEN), E. coli containing a recombinant vector having a cDNA insertion is selected as a white colony,
A colony was obtained, and a recombinant plasmid vector having the equine interleukin-1α cDNA inserted therein was obtained from Escherichia coli in the colony. The obtained recombinant plasmid vector was named pCEα.

Example 3 Nucleotide Sequence Analysis of Equine Interleukin-1α cDNA Kilo Sequence Deletion Kit (Takara Shuzo
A deletion mutant was prepared from the plasmid obtained in Example 2 using Exonuclease III and Mung Bean Nuclease (trade name). Auto
Using the Lead Sequencing Kit (Pharmacia's trade name) and following the dideoxy termination method,
The nucleotide sequence of the equine interleukin 1α cDNA inserted into the plasmid was determined. As a result, the obtained equine interleukin 1α cDNA had the nucleotide sequence of SEQ ID NO: 3. As a result of analyzing this base sequence, it was found that the base sequence of SEQ ID NO: 3 encodes the amino acid sequence of SEQ ID NO: 1.

Example 4 Acquisition of cDNA of equine interleukin-1β In order to obtain the coding region of equine interleukin-1β using the PCR method, the nucleotide sequences of the primers used in the PCR method are human and mouse interleukin-1β. Among the well-conserved base sequences of the leukin 1β gene, a base sequence located relatively upstream (base sequence represented by SEQ ID NO: 7) and a base sequence complementary to a base sequence located relatively downstream (SEQ ID NO: 7) The base sequence shown in 8) was used. DNAs having the nucleotide sequences shown in SEQ ID NOS: 7 and 8 were chemically synthesized by a DNA synthesizer and used as primers.

CDNA was amplified according to the procedure described in Example 2 except that the DNA having the nucleotide sequence of SEQ ID NO: 7 and the DNA having the nucleotide sequence of SEQ ID NO: 8 were used as primers. However, the amplified cDNA was judged to be equine interleukin-based on the nucleotide sequence of the primer used.
Since it seems that it does not contain the entire 1β coding region, the following operation was performed to acquire this entire coding region. That is, the restriction enzyme Eco was added to the cDNA obtained in Example 1.
RI / NotI adapter was ligated and ligated with the phage vector LambdaZap-II that had been digested with EcoRI in advance, and it was added to the phage shell using Gigapack Plus (trade name of Stratagene (USA)). Enclosed. This was seeded on an NZY plate and cultured. The obtained plaque was transferred to a nylon membrane filter "Hybond-N" (trade name of Amersham International (UK)).

On the other hand, the above-mentioned amplified cDNA was converted into α- ³²
It was labeled with P and used as a probe. 50% formamide, 1
% Sodium dodecyl sulfate (SDS), 5% Irish Cream, 4 × SSPE (where
1 × SSPE is 0.18 M NaCl, 0.01 M sodium phosphate and 1 mM ethylenediaminetetraacetic acid (E
An aqueous solution containing DTA))) and 100 mg / ml
Hybridization was carried out by adding the probe to a solution containing denatured salmon sperm DNA, immersing the filter in the solution, and incubating at 37 ° C. for 18 hours. The filter is 4 × SSC (where 1 × SSC is 0.1
It was washed with an aqueous solution containing 5M NaCl and 0.015M citric acid) and an aqueous solution containing 0.1% SDS at 37 ° C. for 3 hours, and autoradiography was performed at −70 ° C. By selecting a phage that gives a positive signal and adding R408 helper phage (trade name of Stratagene (USA)) to this, the insertion portion of the phage giving a positive signal is a plasmid vector "Pe bluescript" (pBluescript).
A recombinant plasmid vector inserted into E. coli, that is, a recombinant plasmid vector containing the equine interleukin-1β cDNA was obtained. The resulting recombinant plasmid vector was named pEβ5.

Example 5 Nucleotide Sequence Analysis of Horse Interleukin-1β cDNA The nucleotide sequence of the horse interleukin-1β cDNA inserted into the plasmid was determined in the same manner as in Example 3.
As a result, the equine interleukin 1β cDNA had the nucleotide sequence of SEQ ID NO: 4. As a result of analysis of this base sequence, it was found that the base sequence of SEQ ID NO: 4 encodes the amino acid sequence of SEQ ID NO: 2.

Example 6 Preparation of Transformant with Different Hosts The plasmid pCEα obtained in Example 2 and the plasmid pEβ5 obtained in Example 4 were respectively inserted into Escherichia coli JM109 strain to transform the host. Was produced. The obtained transformants have been deposited at the Institute of Biotechnology, Institute of Industrial Science, under the accession numbers FERM P-15142 and FERM P-15143, respectively.

[0055]

The peptide according to claim 1 is important for studying the relationship between inflammatory diseases of horses and interleukin 1 of horses, and is useful as a major component of reagents for this study. The peptide according to claim 2 exerts the effect of the peptide according to claim 1, and is important in studying the relationship between inflammatory diseases of horses and equine interleukin 1 subtype (α type). It is useful as a major component of research reagents. The peptide according to claim 3 exerts the effect of the peptide according to claim 1, and is important for studying the relationship between inflammatory diseases of horses and equine interleukin 1 subtype (β type). It is useful as a major component of research reagents.

The DNA of claim 4 is important for studying the relationship between equine inflammatory disease and equine interleukin 1, and is useful as a main component of a reagent for this study. The DNA according to claim 5 is the DNA according to claim 4.
It has the effect of A and is important for studying the relationship between equine inflammatory disease and equine interleukin 1 subtype (alpha type), and is useful as a main component of a reagent for this study. The DNA according to claim 6 exerts the effect of the DNA according to claim 4, and is important for studying the relationship between inflammatory diseases of horses and equine interleukin 1 subtype (β type). It is useful as a major component of research reagents.

The recombinant vector according to claim 7 is important for studying the relationship between equine inflammatory disease and equine interleukin 1, and can be used for production of equine interleukin 1 peptide. The recombinant vector according to claim 8 exhibits the effects of the recombinant vector according to claim 7, and is important for studying the relationship between inflammatory diseases of horses and equine interleukin 1 subtype (α type). And can be used for the production of equine interleukin 1α peptide. The recombinant vector according to claim 9 exhibits the effects of the recombinant vector according to claim 7, and is important for studying the relationship between equine inflammatory disease and equine interleukin-1 subtype (β type). And can be used for the production of equine interleukin 1β peptide.

The transformant according to claim 10 is important for studying the relationship between equine inflammatory disease and equine interleukin 1, and can be used for production of equine interleukin 1 peptide. The transformant according to claim 11 exhibits the effect of the transformant according to claim 10,
Furthermore, it is important for studying the relationship between inflammatory diseases of horses and equine interleukin 1 subtype (α type), and can be used for production of equine interleukin 1α peptide. The transformant according to claim 12 exerts the effect of the transformant according to claim 10, and is important for studying the relationship between inflammatory diseases of horses and equine interleukin 1 subtype (β type). And can be used for the production of equine interleukin 1β peptide. The method for producing an antibody according to claim 13 is important for studying the relationship between equine inflammatory disease and equine interleukin-1, and the anti-equine interleukin-1 antibody useful as a main component of a reagent for this study is obtained. Suitable for manufacturing.

[0059]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 270 Sequence type: Amino acid Sequence type: Peptide sequence Met Ala Lys Val Pro Asp Leu Phe Glu Asp Leu Lys Asn Cys Tyr Ser 1 5 10 15 Glu Asn Glu Asp Tyr Ser Ser Glu Ile Asp His Leu Ser Leu Thr Gln 20 25 30 Lys Ser Phe Tyr Asp Ala Ser Tyr Asp Pro Leu Pro Glu Asp Cys Met 35 40 45 Asp Thr Phe Met Ser Leu Ser Thr Ser Glu Thr Ser Lys Thr Ser Lys 50 55 60 Leu Asn Phe Lys Glu Ser Val Val Leu Val Ala Ala Asn Gly Lys Thr 65 70 75 80 Leu Lys Lys Arg Arg Leu Ser Leu Asn Gln Phe Ile Thr Asn Asp Asp 85 90 95 Leu Glu Ala Ile Ala Asn Asp Pro Glu Glu Gly Ile Ile Arg Pro Arg 100 105 110 Ser Val His Tyr Asn Phe Gln Ser Asn Thr Lys Tyr Asn Phe Met Arg 115 120 125 Ile Val Asn His Gln Cys Thr Leu Asn Asp Ala Leu Asn Gln Ser Val 130 135 140 Ile Arg Asp Thr Ser Gly Gln Tyr Leu Ala Thr Ala Ala Leu Asn Asn 145 150 155 160 Leu Asp Asp Ala Val Lys Phe Asp Met Gly Ala Tyr Thr Ser Glu Glu 165 170 175 Asp Ser Gln Leu Pro Val Thr Leu Arg Ile Ser Lys Thr Arg Leu Phe 180 1 85 190 Val Ser Ala Gln Asn Glu Asp Glu Pro Val Leu Leu Lys Glu Met Pro 195 200 205 Asp Thr Pro Lys Thr Ile Lys Asp Glu Thr Asn Leu Leu Phe Phe Trp 210 215 220 Glu Arg His Gly Ser Lys Asn Tyr Phe Lys Ser Val Ala His Pro Lys 225 230 235 240 Leu Phe Ile Ala Thr Lys Gln Gly Lys Leu Val His Met Ala Arg Gly 245 250 255 Gln Pro Ser Ile Thr Asp Phe Gln Ile Leu Asp Asn Gln Phe 260 265 270

SEQ ID NO: 2 Sequence length: 268 Sequence type: Amino acid Sequence type: Peptide sequence Met Ala Ala Val Pro Asp Thr Ser Asp Met Met Thr Tyr Cys Ser Gly 1 5 10 15 Asn Glu Asn Asp Leu Phe Phe Glu Glu Asp Gly Pro Lys Gln Met Lys 20 25 30 Gly Ser Phe Gln Asp Leu Asp Leu Ser Ser Met Gly Asp Gly Gly Ile 35 40 45 Gln Leu Gln Phe Ser His His Leu Tyr Asn Lys Thr Phe Lys His Ala 50 55 60 Met Ser Ile Ile Val Ala Val Glu Lys Leu Lys Lys Ile Pro Val Pro 65 70 75 80 Cys Ser Gln Ala Phe Gln Asp Asp Asp Leu Arg Ser Leu Phe Ser Val 85 90 95 Ile Phe Glu Glu Glu Pro Ile Ile Cys Asp Asn Trp Asp Glu Gly Tyr 100 105 110 Val Cys Asp Ala Ala Met His Ser Val Asn Cys Arg Leu Arg Asp Ile 115 120 125 Tyr His Lys Ser Leu Val Leu Ser Gly Ala Cys Glu Leu Gln Ala Val 130 135 140 His Leu Asn Gly Glu Asn Thr Asn Gln Gln Val Val Phe Cys Met Ser 145 150 155 160 Phe Val Gln Gly Glu Glu Glu Thr Asp Lys Ile Pro Val Ala Leu Gly 165 170 175 Leu Lys Glu Lys Asn Leu Tyr Leu Ser Cys Gly Met Lys Asp Gly Lys 180 185 190 Pro Thr Leu Gln Leu Glu Thr Val Asp Pro Asn Thr Tyr Pro Lys Arg 195 200 205 Lys Met Glu Lys Arg Phe Val Phe Asn Lys Met Glu Ile Lys Gly Asn 210 215 220 Val Glu Phe Glu Ser Ala Met Tyr Pro Asn Trp Tyr Ile Ser Thr Ser 225 230 235 240 Gln Ala Glu Lys Ser Pro Val Phe Leu Gly Asn Thr Arg Gly Gly Arg 245 250 255 Asp Ile Thr Asp Phe Ile Met Glu Ile Thr Ser Ala 260 265 268

SEQ ID NO: 3 Sequence length: 810 Sequence type: Nucleic acid Number of strands: Double-stranded Sequence type: cDNA to mRNA sequence ATG GCG AAA GTC CCT GAC CTC TTT GAA GAC CTG AAG AAC TGT TAC AGT 48 Met Ala Lys Val Pro Asp Leu Phe Glu Asp Leu Lys Asn Cys Tyr Ser 1 5 10 15 GAA AAT GAA GAC TAC AGT TCT GAA ATT GAC CAT CTC TCT CTG ACT CAG 96 Glu Asn Glu Asp Tyr Ser Ser Glu Ile Asp His Leu Ser Leu Thr Gln 20 25 30 AAA TCC TTC TAT GAT GCA AGC TAT GAC CCA CTT CCT GAG GAC TGC ATG 144 Lys Ser Phe Tyr Asp Ala Ser Tyr Asp Pro Leu Pro Glu Asp Cys Met 35 40 45 GAT ACA TTT ATG TCT CTG AGC ACC TCT GAA ACC TCT AAG ACA TCC AAG 192 Asp Thr Phe Met Ser Leu Ser Thr Ser Glu Thr Ser Lys Thr Ser Lys 50 55 60 CTG AAC TTC AAG GAG AGC GTG GTG CTG GTG GCA GCC AAC GGG AAG ACT 240 Leu Asn Phe Lys Glu Ser Val Val Leu Val Ala Ala Asn Gly Lys Thr 65 70 75 80 CTG AAG AAG AGA CGG TTG AGT TTA AAT CAG TTC ATC ACC AAT GAT GAC 288 Leu Lys Lys Arg Arg Leu Ser Leu Asn Gln Phe Ile Thr Asn Asp Asp 85 90 95 CTG GAA GCC A TT GCC AAT GAT CCA GAA GAA GGA ATC ATC AGG CCC CGA 336 Leu Glu Ala Ile Ala Asn Asp Pro Glu Glu Gly Ile Ile Arg Pro Arg 100 105 110 TCA GTA CAT TAC AAC TTC CAG AGC AAT ACA AAA TAC AAC TTT ATG AGG 384 Ser Val His Tyr Asn Phe Gln Ser Asn Thr Lys Tyr Asn Phe Met Arg 115 120 125 ATC GTC AAC CAC CAG TGT ACT CTG AAT GAT GCC CTC AAT CAA AGT GTA 432 Ile Val Asn His Gln Cys Thr Leu Asn Asp Ala Leu Asn Gln Ser Val 130 135 140 ATT CGA GAC ACA TCA GGT CAA TAT CTT GCG ACT GCT GCA TTA AAT AAT 480 Ile Arg Asp Thr Ser Gly Gln Tyr Leu Ala Thr Ala Ala Leu Asn Asn 145 150 155 160 CTG GAC GAC GCA GTG AAA TTT GAC ATG GGT GCT TAT ACA TCA GAA GAG 528 Leu Asp Asp Ala Val Lys Phe Asp Met Gly Ala Tyr Thr Ser Glu Glu 165 170 175 GAT TCT CAA CTT CCT GTG ACT CTA AGA ATC TCA AAA ACT CGA CTG TTT 576 Asp Ser Gln Leu Pro Val Thr Leu Arg Ile Ser Lys Thr Arg Leu Phe 180 185 190 GTG AGT GCC CAA AAT GAA GAT GAA CCC GTA CTG CTA AAG GAG ATG CCT 624 Val Ser Ala Gln Asn Glu Asp Glu Pro Val Leu Leu Lys Glu Met Pro 195 200 205 G AC ACA CCC AAA ACT ATC AAA GAT GAG ACC AAC CTC CTC TTC TTC TGG 672 Asp Thr Pro Lys Thr Ile Lys Asp Glu Thr Asn Leu Leu Phe Phe Trp 210 215 220 GAA CGT CAC GGC TCT AAG AAC TAC TTC AAA TCG GTT GCC CAT CCA AAG 720 Glu Arg His Gly Ser Lys Asn Tyr Phe Lys Ser Val Ala His Pro Lys 225 230 235 240 TTG TTT ATT GCC ACA AAG CAG GGA AAA CTG GTG CAC ATG GCA AGG GGG 768 Leu Phe Ile Ala Thr Lys Gln Gly Lys Leu Val His Met Ala Arg Gly 245 250 255 CAA CCC TCT ATC ACT GAC TTT CAG ATA TTG GAC AAC CAG TTT 810 Gln Pro Ser Ile Thr Asp Phe Gln Ile Leu Asp Asn Gln Phe 260 265 270

SEQ ID NO: 4 Sequence length: 804 Sequence type: Nucleic acid Number of strands: Double stranded Sequence type: cDNA to mRNA sequence ATG GCA GCA GTA CCC GAC ACC AGT GAC ATG ATG ACT TAC TGC AGC GGC 48 Met Ala Ala Val Pro Asp Thr Ser Asp Met Met Thr Tyr Cys Ser Gly 1 5 10 15 AAT GAG AAT GAC CTG TTC TTT GAG GAG GAT GGC CCA AAA CAG ATG AAG 96 Asn Glu Asn Asp Leu Phe Phe Glu Glu Asp Gly Pro Lys Gln Met Lys 20 25 30 GGC AGC TTC CAA GAC CTG GAC CTC AGC TCC ATG GGC GAT GGG GGC ATC 144 Gly Ser Phe Gln Asp Leu Asp Leu Ser Ser Met Gly Asp Gly Gly Ile 35 40 45 CAG CTT CAA TTC TCC CAC CAC CTC TAC AAC AAG ACT TTC AAA CAT GCC 192 Gln Leu Gln Phe Ser His His Leu Tyr Asn Lys Thr Phe Lys His Ala 50 55 60 ATG TCA ATC ATT GTG GCT GTG GAG AAG CTG AAG AAG ATA CCC GTT CCC 240 Met Ser Ile Ile Val Ala Val Glu Lys Leu Lys Lys Ile Pro Val Pro 65 70 75 80 TGC TCA CAG GCC TTC CAG GAT GAT GAC TTG AGG AGC CTC TTT TCT GTC 288 Cys Ser Gln Ala Phe Gln Asp Asp Asp Leu Arg Ser Leu Phe Ser Val 85 90 95 ATC TTT GAA G AA GAA CCC ATC ATC TGT GAC AAC TGG GAT GAA GGT TAT 336 Ile Phe Glu Glu Glu Pro Ile Ile Cys Asp Asn Trp Asp Glu Gly Tyr 100 105 110 GTA TGT GAT GCA GCC ATG CAT TCA GTG AAC TGC AGA CTC CGG GAC ATA 384 Val Cys Asp Ala Ala Met His Ser Val Asn Cys Arg Leu Arg Asp Ile 115 120 125 TAC CAT AAA TCC CTG GTG CTG TCC GGT GCA TGT GAG CTG CAG GCT GTC 432 Tyr His Lys Ser Leu Val Leu Ser Gly Ala Cys Glu Leu Gln Ala Val 130 135 140 CAC CTC AAT GGA GAG AAT ACA AAC CAA CAA GTG GTG TTC TGC ATG AGC 480 His Leu Asn Gly Glu Asn Thr Asn Gln Gln Val Val Phe Cys Met Ser 145 150 155 160 TTT GTG CAA GGA GAA GAA GAG ACT GAC AAG ATA CCT GTG GCC TTG GGC 528 Phe Val Gln Gly Glu Glu Glu Thr Asp Lys Ile Pro Val Ala Leu Gly 165 170 175 CTC AAG GAA AAG AAC CTG TAC CTG TCT TGT GGG ATG AAA GAT GGG AAG 576 Leu Lys Glu Lys Asn Leu Tyr Leu Ser Cys Gly Met Lys Asp Gly Lys 180 185 190 CCC ACC CTA CAG CTG GAG ACA GTA GAC CCC AAT ACT TAC CCA AAG AGG 624 Pro Thr Leu Gln Leu Glu Thr Val Asp Pro Asn Thr Tyr Pro Lys Arg 195 200 205 A AA ATG GAA AAG CGA TTT GTC TTC AAC AAG ATG GAA ATC AAG GGC AAC 672 Lys Met Glu Lys Arg Phe Val Phe Asn Lys Met Glu Ile Lys Gly Asn 210 215 220 GTG GAA TTT GAG TCT GCA ATG TAC CCC AAC TGG TAC ATC AGC ACC TCT 720 Val Glu Phe Glu Ser Ala Met Tyr Pro Asn Trp Tyr Ile Ser Thr Ser 225 230 235 240 CAA GCA GAA AAA AGC CCT GTC TTC CTA GGA AAT ACC AGA GGC GGC CGG 768 Gln Ala Glu Lys Ser Pro Val Phe Leu Gly Asn Thr Arg Gly Gly Arg 245 250 255 GAC ATA ACT GAC TTC ATC ATG GAA ATC ACC TCT GCC 804 Asp Ile Thr Asp Phe Ile Met Glu Ile Thr Ser Ala 260 265 268

SEQ ID NO: 5 Sequence length: 21 Sequence type: Nucleic acid Number of strands: Single strand Sequence type: Other nucleic acid Synthetic DNA Sequence TCATTGGCGT TTGAGTCAGC A 21

SEQ ID NO: 6 Sequence length: 22 Sequence type: Nucleic acid Number of strands: Single stranded Sequence type: Other nucleic acid Synthetic DNA Sequence GTAAACATTC ATTTAGAATT AC 22

SEQ ID NO: 7 Sequence length: 24 Sequence type: Nucleic acid Number of strands: Single strand Sequence type: Other nucleic acid Synthetic DNA Sequence ATGAGGATGA CTTGTTCTTT GAAG
24

SEQ ID NO: 8 Sequence length: 22 Sequence type: Nucleic acid Number of strands: Single strand Sequence type: Other nucleic acid Synthetic DNA Sequence GAGGTGCTGA TGTACCAGTT GG
22

Continuation of the front page (51) Int.Cl. ⁶ Identification code Reference number in the agency FI Technical display location // A61K 38/00 ABB A61K 37/02 ABB (C12N 15/09 ZNA C12R 1:91) (C12N 1/21 (C12R 1:19) (C12P 21/02 C12R 1:19) (72) Inventor Shigumi Takagi 5092-3 Naruse, Machida City, Tokyo Este Squeer Naruse Ichibankan 601 (72) Inventor Toshihiro Watanabe Hadano 2-2-2-304 Minamigaoka (72) Inventor Moto Tsujimoto 5-4-24 Narita Higashi, Suginami-ku, Tokyo (72) Inventor Atsuhiko Hasegawa 4-7-16 Kitacho, Kichijoji, Musashino-shi, Tokyo

Claims (13)

[Claims] 1. A horse interleukin 1 peptide comprising a sequence consisting of at least 5 consecutive amino acids in the peptide represented by SEQ ID NO: 1 or 2. 2. A peptide represented by SEQ ID NO: 1,
The peptide according to claim 1. 3. A peptide represented by SEQ ID NO: 2,
The peptide according to claim 1. 4. A DNA encoding the peptide according to any one of claims 1 to 3 or a DNA complementary thereto. 5. The DNA according to claim 4, which has the nucleotide sequence represented by SEQ ID NO: 3. 6. The DNA according to claim 4, which has the nucleotide sequence represented by SEQ ID NO: 4. 7. The DNA according to any one of claims 4 to 6.
A recombinant vector containing. 8. The recombinant vector according to claim 7, which is the plasmid pCEα. 9. The recombinant vector according to claim 8, which is the plasmid pEβ5. 10. A transformant containing the recombinant vector according to claim 7. 11. The transformant according to claim 10, which has been deposited as FERM P-15142. 12. The transformant according to claim 10, which has been deposited as FERM P-15143. 13. A method for producing an anti-horse interleukin 1 antibody, which comprises using the peptide according to any one of claims 1 to 3 as an antigen.