JP2003026700A - Antibody recognizing serine-threonine protein phosphorylation enzyme - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for detecting a genome having the function of a serine-threonine protein phosphorylation enzyme playing an important action of an information-transmitting system. SOLUTION: A synthetic peptide based on the amino acid sequence of a sub-domain VIB portion having the highest similarity between the serine- threonine protein phosphorylation enzymes, among 11 sub-domains having commonality between the enzymes is used as an antigen to prepare the antibody which recognizes many serine-threonine protein phosphorylation enzymes.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an antibody reactive with serine-threonine protein kinase and a method for detecting or measuring serine-threonine protein kinase using the antibody.

[0002]

2. Description of the Related Art A cell signal transduction system begins when a stimulus transmitted mainly to the cell membrane activates a receptor or a channel protein on the cell membrane. Then, the stimuli are sequentially transmitted into the cells by increasing / decreasing second messengers such as calcium ions and cAMP, and activating and deactivating various protein kinases (Cano, E., and Mahadevan, L . (1995) Parallel sig
nal processing among mammalian MAPKs. Trends Bioch
em. Sci. 20, 117-122).

It is said that more than 1,000 kinds of protein phosphorylases, which play an important role in the series of signal transduction systems, exist in animal and plant cells. The primary structure has been determined and reported. These protein kinases are roughly divided into two. One is a protein that phosphorylates the Tyr residue of the target protein, which is called Tyr protein kinase. This phosphatase is often the growth factor receptor itself. The other is serine, which phosphorylates the Ser or Thr residue of the target protein.
It is a threonine protein kinase. Typical examples of serine-threonine protein kinases include protein kinase A, protein kinase C, and multifunctional Ca ²⁺ / calmodulin-dependent protein kinase.
(CaMKII, CaMKIV), MAP kinase and the like.

By comparing the primary structures of serine-threonine protein kinases reported so far, it is known that there are eleven subdomain structures conserved among protein kinases (Hanks). , S.
K., and Hunter, T. (1995) The eukaryotic protein ki
nase superfamily: kinase (catalytic) domain struct
ure and classification. FASEB J. 9, 576-596). In conventional studies, it has been actively conducted to prepare an antibody having high specificity for a specific enzyme, and investigate the intracellular trend of the target enzyme or the distribution thereof. However, recently, in the post-genome era, it is required to discover various functions of the genome. In such a situation, it is very useful to detect a genome having the function of serine-threonine protein kinase, which plays an important role in the signal transduction system as described above, from a large number of genomes. . However, a detection method that can be used practically has not been supplied yet.

[0005]

DISCLOSURE OF THE INVENTION An object of the present invention is to provide an antibody reactive with serine-threonine protein phosphorylating enzyme and a method for detecting or measuring serine-threonine protein phosphorylating enzyme using the antibody. .

[0006]

Means for Solving the Problems As a result of intensive studies made by the present inventors in view of the above-mentioned problems of the prior art, there is a commonality between serine-threonine protein kinases.
Of the subdomains of several sites, an antibody is prepared using a synthetic peptide based on the amino acid sequence of the VIB part of the subdomain with the highest similarity between enzymes as an antigen, and the antibody recognizes various serine-threonine protein kinases This has led to the completion of the present invention.

That is, the present invention provides the following antibody, detection or measurement method. Item 1. An antibody against a peptide having the amino acid sequence represented by SEQ ID NO: 1. Item 2. Furthermore, the antibody according to item 1, which is reactive with at least one protein selected from CaMKII, CaMKIV, protein kinase A, protein kinase C, MAP kinase, and MAP kinase kinase. Item 3. Item 2. The antibody according to Item 1, which is obtained by immunizing warm-blooded animals other than humans with the peptide represented by SEQ ID NO: 1 or a carrier protein conjugate thereof as an immunogen. Item 4. Item 4. The antibody according to Item 3, which is a rabbit antiserum. Item 5. Item 4. The antibody according to Item 3, which is a polyclonal antibody. Item 6. A method for detecting or measuring serine-threonine protein kinase, which comprises performing an immunological assay using the antibody according to Item 1.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION In the present specification, abbreviations for amino acids, peptides, base sequences, nucleic acids, etc. indicate IUPA.
The provisions of C and IUB, “Guidelines for preparing specifications including base sequences or amino acid sequences” (Edited by the Patent Office), and symbols conventionally used in the field shall be followed.

The antibody according to the present invention can be produced in accordance with a general antibody production method using a peptide having the amino acid sequence represented by SEQ ID NO: 1 or an immunizing antigen containing the peptide. The antibodies include polyclonal antibodies and monoclonal antibodies such as antisera of warm-blooded animals and chicken egg antibodies. Further, the antibody of the present invention has reactivity with the peptide having the amino acid sequence represented by SEQ ID NO: 1, and further has reactivity with serine-threonine protein kinase.

The antibody of the present invention having such characteristics can be obtained by immunizing a warm-blooded animal (excluding human) with a specific immunizing antigen, and the method or method can be according to a conventional method. For example, by immunizing a warm-blooded animal with the peptide represented by SEQ ID NO: 1 serving as an immunizing antigen,
The antibody can be produced in a body fluid such as blood of the warm-blooded animal or in an egg laid by the warm-blooded animal and obtained from them.

As the immunizing antigen, a peptide or protein having the amino acid sequence represented by SEQ ID NO: 1 is used. The peptide can be produced by peptide synthesis according to a conventional method, and the protein is available as serine-threonine protein kinase. In addition, the carrier-protein conjugate of the peptide represented by SEQ ID NO: 1 is prepared by adding cysteine to the peptide according to a conventional method, for example, by adding cysteine to the N-terminus of the peptide, and then adding to this a keyhole limpet hemocyanin (keyhole limpet hemocyanin) ( KLH) and is conjugated with KLH). As the carrier protein, any of various carrier proteins commonly used in the art for enhancing the immunogenicity of the antigen or hapten can be used. Examples thereof include various animal proteins such as albumin, globulin, hemocyanin, and artificial polypeptides such as polylysine. These carrier proteins can be made into a conjugate with the peptide represented by SEQ ID NO: 1 by a condensation reaction or the like using a commonly used reagent. Examples of the reagent include 1-ethyl-3- (3-
Dimethylaminopropyl) carbodiimide hydrochloride (EC
Water-soluble carbodiimides such as DI) and condensing agents such as dimethylsperimidate (DMS) are used. The carrier protein conjugate obtained as described above has a desired immunogenicity and can be used as an immunogen for producing the antibody of the present invention.

[0012] The immunization using the above-mentioned immunizing antigen and the operation for obtaining the desired antibody can also be carried out according to a conventional method. For example, in the production of polyclonal antibodies, warm-blooded animals such as rabbits, sheep, guinea pigs, and chickens are immunized one or more times with the above-mentioned immunizing antigen, usually with Freund's complete adjuvant, to produce antiserum. It can be carried out by collecting and collecting antiserum according to a conventional method. When a chicken is used as a warm-blooded animal, the immunizing antigen is immunized one or more times to cause the chicken egg laying eggs to produce immunoglobulin Y (IgY), and the egg yolk of the chicken egg A polyclonal antibody can also be obtained by obtaining IgY according to the method.

The antibody of the present invention can be obtained as a monoclonal antibody according to a conventional method. The monoclonal antibody is produced by, for example, immunizing a mouse by immunizing the above immunizing antigen with Freund's complete adjuvant one or more times to immunize the mouse, and producing antibody by the conventional method such as cell fusion method and bone marrow. It can be obtained by fusing with spleen cells and cloning, isolating a monoclonal cell producing the desired antibody, and culturing the cell.

The antibody of the present invention thus obtained is
If desired, it can be further purified by a conventional method such as salting out with ammonium sulfate, ion exchange chromatography, gel chromatography, affinity chromatography and the like.

When obtaining the antibody of the present invention,
Immunization was performed, for example, by testing the reactivity of the obtained antibody with the peptide represented by SEQ ID NO: 1 and further by testing the reactivity with serine-threonine protein kinase. It can be carried out appropriately between individual warm-blooded animals or between cloned single clones. The reactivity test is not particularly limited as long as the immunoreactivity between the antibody and the test substance can be confirmed, and can be favorably carried out, for example, according to the assay method described in Examples below.

A general immunological assay using the thus-obtained antibody of the present invention enables detection or measurement of serine-threonine protein phosphatase. Examples of the immunological assay include enzyme immunoassay (EIA) and enzyme immunometric assay (ELI).
SA), fluorescent immunoassay, luminescent immunoassay and the like can be used without limitation. These assays are
It can be carried out according to a general competitive method or sandwich method. Preferably EIA is used.

In the EIA method, the antibody of the present invention is used as a solid-phased antibody, and the carrier to be solid-phased is not particularly limited, and any one commonly used in the EIA method can be used. For example, a 96-well microtiter plate made of polystyrene can be mentioned. To immobilize the antibody, for example, a buffer solution containing the antibody may be placed on a carrier and allowed to stand at 4 ° C. overnight. The composition and concentration of the buffer solution may be according to a conventional method. The antigen of the antibody of the present invention is bound with an enzyme to form an enzyme conjugate. The method for binding is not particularly limited as long as it is a method for binding the enzyme and the antigen, and a conventional method may be used. The EIA method using the immobilized antibody thus obtained and the enzyme-labeled antigen can detect and measure serine-threonine protein phosphatase.

In measuring and detecting the serine-threonine protein phosphatase, it is convenient to use a measurement kit containing the antibody of the present invention as an active ingredient. Such a kit contains, in addition to the antibody of the present invention,
Any other reagent necessary for carrying out the measurement / detection can be further included. Examples thereof include the above-mentioned labeled antigen and / or standard antigen solution, and assay buffer solution.

[0019]

The present invention will be described in more detail with reference to the following examples, which are not intended to limit the technical scope of the present invention. Those skilled in the art can easily make modifications and changes to the present invention based on the description of the present specification, and those are included in the technical scope of the present invention.

(I) The peptide (CVVHRDLKPENLLLAS) in which cysteine is added to the N-terminal of the amino acid sequence at positions 132 to 146 of Ca ²⁺ / calmodulin-dependent protein kinase II is Shimadzu's solid-phase peptide synthesizer PSSM8. It was used and synthesized by the fluorenylmethoxycarbonyl peptide (fmoc) synthesis method. The peptide was purified by high performance liquid chromatography using reverse phase column chromatography (ODS-80Tm, manufactured by Tosoh Corporation). A FAB mass spectrometer was used to determine the molecular weight of the peptide.

(Ii) CaMKII was purified from rat cerebral cortex. CaMKIV and protein kinase C were purified from rat brain. Protein kinase A (catalytic subunit) was purified from bovine heart. MAP kinase and MEK (MAP kinase kinase) were purchased from Upstate Biotechnology.

(Iii) Western blotting was performed by an existing method (Harlow, E., and Lane, D. (1988) Anti.
bodies: a laboratory manual. Cold Spring Harbor La
boratory Press).

Example 1 The cysteine at the N-terminus of the synthetic peptide synthesized in (i) above was replaced with the carrier protein keyhole limpet hemocyanin (KL).
In order to bind to H)) and increase the antigenicity, the following operations were performed. KLH was dissolved in 10 mM sodium phosphate buffer (pH 7.2) to prepare 546 μl of a 14.5 (mg / ml) solution. N- (6-maleimidocaproyloxy) succinimide (N- (6-maleimid
56 μl of a solution (24.5 mg / ml) of ocaproyloxy) succinimide) (manufactured by Dojindo Kagaku) in dimethylformamide was added, and the mixture was stirred at room temperature for 30 minutes. After centrifugation at 10,000 rpm for 5 minutes, the supernatant was subjected to gel filtration chromatography Sephadex G50 (Amersham
Pharmacia Biotech) and loaded with 50 mM sodium phosphate buffer (pH 6.0) to elute, and a fraction having a peak at 280 nm was collected. 1 mM sodium acetate buffer (pH 4.0)
Add a solution of 5 mg of synthetic peptide dissolved in 1 ml,
The mixture was stirred at room temperature for 3 hours to prepare an antigen solution. Freund's complete adju
vant) (FCA) and the antigen solution were stirred to prepare an emulsion, and a rabbit (Japanese white) was immunized. After that, a total of four immunizations were performed every two weeks. However, from the second immunization, instead of FCA, incomplete Freund's adjuvant (Freund's inc
omplete adjuvant) was used. A small amount of blood was collected from the immunized rabbit, and CaMKII purified from rat cerebral cortex was used to perform dot blot analysis to confirm that the antibody titer was increased. After that, a large amount of blood was collected from the rabbit.
The blood was left at room temperature for 3.5 hours and then left in a low temperature room for 48 hours to form a blood clot, and then centrifuged at 3000 g to obtain serum. This serum was mixed with 50% saturated ammonium sulfate and stirred for 4.5 hours, and then the precipitate was collected and stored at 4 ° C. and used as an antibody (hereinafter referred to as KI98 antibody).

Example 2 In order to examine the reaction specificity of the antibody KI98 obtained in Example 1, purified serine-threonine protein phosphatase was subjected to SDS electrophoresis, followed by protein staining and Western blotting. (Fig. 1). KI98 is the CaMKI I checked
I, CaMKIV, protein kinase A, protein kinase C, and MAP kinase could all be detected. Although not shown, it was confirmed that MEK (MAP kinase kinase) can be detected.

Next, in order to investigate the limit of the amount of antigen that can be detected by this antibody, Dot immunobinding assay (Hawkes, R., Niday,
E., and Gordon, J. (1982) A dot-immunobinding assa
y for monoclonal and other antibodies. Anal. Bioch
em. 119, 142-147). In this method, various amounts of antigen proteins (CaMKIV, protein kinase A, MAP kinase) on nitrocellulose filter were changed.
Was spotted, and detection was attempted with the KI98 antibody. As shown in FIG. 2, CaMKIV, protein kinase A, and MA investigated
Minimal amount of spotted antigen (0.2 ng) with P kinase
It turned out that it was detectable up to.

Example 3 Next, various serine-threonine protein phosphatases contained in the cell extract were detected by Western blotting using the KI98 antibody. Rat cerebrum, cerebellum, spleen, liver, kidney, lung, heart soluble fraction (cytoplasmic protein) and granular fraction (membrane protein, nuclear protein, etc.)
After separation by SDS electrophoresis, analysis by Western blotting revealed that different stained bands were detected depending on the organ as shown in FIG. CaMKII (Knighton, DR, known to be abundant in rat brain)
Zheng, J., Eyck, LFT, Xuong, NH., Taylor,
SS, and Sowadski, JM (1991) Structure of ap
eptide inhibitor bound to the catalytic subunit of
cyclic adenosine monophosphate-dependent proteink
inase. Science 253, 414-420), the α subunit and β subunit were found at the predicted molecular weight positions of 50 kDa and 60 kDa in the cerebral extract, and the β subunit of CaMKII was found in the cerebellar extract. A band that seems to be a unit was clearly observed. In addition, since various different bands are observed in other organs, it is confirmed that various serine-threonine protein kinases are detected by this antibody.

[0027]

INDUSTRIAL APPLICABILITY A subdomain in which primary structure is retained among various serine-threonine protein kinases
Even in VIB, the amino acid sequences differ slightly depending on the enzyme. However, the KI98 antibody obtained this time can recognize various serine-threonine protein kinases. Therefore, in expression cloning of many genomes, unknown serine-threonine protein phosphatase can be used as an important detection tool for catching up the analysis. As described above, the KI98 antibody is considered to be a very useful tool for widely detecting serine-threonine protein kinase.

[0028]

[Sequence list]                                SEQUENCE LISTING <110> National Institute of Advanced Industrial Science <120> Antibody recognizing serine-threonine kinase <130> 113MS0288 <140> <141> <160> 6 <170> PatentIn Ver. 2.1 <210> 1 <211> 15 <212> PRT <213> Human <400> 1 Val Val His Arg Asp Leu Lys Pro Glu Asn Leu Leu Leu Ala Ser   1 5 10 15 <210> 2 <211> 15 <212> PRT <213> Human <400> 2 Ile Val His Arg Asp Leu Lys Pro Glu Asn Leu Leu Tyr Ala Thr   1 5 10 15 <210> 3 <211> 15 <212> PRT <213> Human <400> 3 Leu Ile Tyr Arg Asp Leu Lys Pro Glu Asn Leu Leu Ile Asp Gln   1 5 10 15 <210> 4 <211> 15 <212> PRT <213> Human <400> 4 Ile Ile Tyr Arg Asp Leu Lys Leu Asp Asn Val Met Leu Asp Ser   1 5 10 15 <210> 5 <211> 15 <212> PRT <213> Human <400> 5 Val Leu His Arg Asp Leu Lys Pro Ser Asn Ser Ser Leu Asn Thr   1 5 10 15 <210> 6 <211> 15 <212> PRT <213> Human <400> 6 Ile Met His Arg Asp Val Lys Pro Ser Asn Ile Leu Val Asn Ser   1 5 10 15

[Brief description of drawings]

FIG. 1 shows protein staining (A) and Western blotting (B) performed in Example 2. A
In, M1 represents a high molecular weight marker protein,
M2 indicates a low molecular weight marker protein, lane 1 indicates
Lane 2 shows CaM kinase IV (50 ng), lane 3 shows protein kinase A (100 ng).
(50 ng), lane 4 shows protein kinase C (10
0 ng) and lane 5 shows GST-MAP kinase (20 ng). In B, KI98 antiserum (1/200 dilution) was used as the primary antibody, peroxidase-labeled anti-rabbit antibody (1/5000 dilution) was used as the secondary antibody, and SuperSigna was used for detection.
1 (PEIRCE) was used, Lane 1 was CaM kinase II (40n
g), lane 2 shows CaM kinase IV (20 ng),
Lane 3 shows protein kinase A (10 ng), lane 4 shows protein kinase C (100 ng), and lane 5 shows GST-MAP kinase (20 ng).

FIG. 2 shows the results of detection sensitivity of serine-threonine protein kinase for Example 2. CaM kinase IV (P
KIV), protein kinase A (PKA), MAP kinase (M
0.5 μl of each apk) was spotted on a nitrocellulose membrane, Dot immunobinding assay was performed, KI98 antiserum (1/200 dilution) was used as the primary antibody, and peroxidase-labeled anti-rabbit antibody (1 (/ 5000 dilution) was used, and SuperSignal (PIERCE) was used for detection.

FIG. 3 shows protein staining (A: Coomassie blue staining) and Western blotting (B) performed in Example 3. Lanes 1 to 7 show the soluble fractions (15 μg) of rat cerebrum, cerebellum, spleen, liver, kidney, lung and heart, and lanes 8 to 14 show rat cerebrum, cerebellum, spleen, liver and kidney, respectively. , Pulmonary and heart granular fractions (15 μg), lane 15 shows prestained markers, KI98 antiserum (1/200 dilution) was used as the primary antibody, and peroxidase-labeled anti-rabbit antibody as the secondary antibody. (1/5000 dilution) is used, and SuperSigna is used for detection.
l (PIERCE) was used.

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Atsuhiko Ishida             2-1, 1-1, Midorigaoka Higashi, Asahikawa, Hokkaido               Asahikawa Medical University (72) Inventor Shun Kinoshita             2393 Iketo, Miki-cho, Kida-gun, Kagawa Prefecture Kagawa Univ.             Faculty of Agriculture (72) Inventor Isamu Kameshita             2393 Iketo, Miki-cho, Kida-gun, Kagawa Prefecture Kagawa Univ.             Faculty of Agriculture F-term (reference) 4H045 AA11 CA42 DA75 EA50 FA74

Claims (6)

[Claims] 1. An antibody against a peptide having the amino acid sequence represented by SEQ ID NO: 1. 2. CaMKII, CaMKIV, protein kinase A, protein kinase C, MAP kinase and MAP
The antibody according to claim 1, which is reactive with at least one protein selected from kinases. 3. The antibody according to claim 1, which is obtained by immunizing warm-blooded animals other than humans with the peptide represented by SEQ ID NO: 1 or a carrier protein conjugate thereof as an immunizing antigen. 4. The antibody according to claim 3, which is a rabbit antiserum. 5. The antibody according to claim 3, which is a polyclonal antibody. 6. A method for detecting or measuring serine-threonine protein phosphatase, which comprises performing an immunological assay using the antibody according to claim 1.