JPH06201700A - Antibody against product of tumor-represser gene mcc - Google Patents

Abstract

PURPOSE:To develop the study of cancers and tumors so as to quickly and easily diagnose them by using an antibody obtained by using the link of a antigenic peptide having a specific amino acid sequence and carrier protein as an antigen. CONSTITUTION:Antigenic peptide having a specific amino acid sequence and carrier protein are linked with each other by a well known method and an antibody is obtained by giving the link to an animal, such as mouse, etc., as an antigen with the purpose of causing immunity in the bodies of the animal. By using the obtained refined antibody and alkaline phosphastase-binding anti-rabbit IgG antibody, the product of a tumor-represser gene MCC is detected. As a result, the existence of a protein having a prescribed molecular weight is discovered from a semian fibroblast strain CV-1 expressing the tumor-represser gene MCC. Since such an antibody specifically recognizes the product of the tumor-represser gene, it can be utilized for analyzing the occurrence of cancers and tumors and diagnosing the cancers and tumors. In addition, since the antibody is manufactured by using an antigen composed only of the peptide which has a short chain and other substances prepared by using methods which are well known in the field of biological engineering, it can be manufactured easily.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to an antibody, and more particularly to an antibody against the product of the tumor suppressor gene MCC, which can be used for the diagnosis of cancer and tumor.

[0002]

2. Description of the Related Art Hybridomas obtained by fusing normal cells and cancer cells show the same shape value as normal cells and in inherited tumors such as retinoblastoma, Wilms tumor, and neuroblastoma. Since an abnormality is found at a specific site on the chromosome, it has been considered that the abnormality of the tumor suppressor gene is involved in the development of hereditary tumor. fact,
Due to advances in molecular biology, tumor suppressor genes for hereditary tumors such as retinoblastoma and Wilms tumor have been cloned one after another, their structures have been elucidated, and abnormalities in the genes have been confirmed. Has arrived. Furthermore, at present, not only the hereditary tumors mentioned above but also lung cancer, breast cancer,
It is becoming clear that abnormalities of tumor suppressor genes play an important role in the development of general tumors such as gastric cancer and colon cancer.

[0003]

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention In view of the above-mentioned conventional techniques, it is essential to study tumor suppressor genes, especially their products, in order to elucidate the development of cancer and tumors. Therefore, it is an object of the present invention to provide an antibody against the product of a tumor suppressor gene, thereby developing research on the development of various cancers and tumors, and eventually opening the way to use in the diagnosis of cancers and tumors. .

[0004]

[Means and Actions for Solving the Problems and Achieving the Objects] According to the present invention, the above-mentioned problems are solved in that the amino acid sequence is Cys-Glu-Asn-Ser-Arg-Pro-His-Thr-Asn-Glu-Thr-Ser- Le
An antibody obtained by using a conjugate of an antigenic peptide which is u and a carrier protein as an antigen, wherein the immunoglobulin class is IgG, the molecular weight is about 150,000, and the molecular extinction coefficient [E ^1% (1 cm),
(280 nm)] is 1.40, and the above object is achieved while being solved by an antibody against the product of the tumor suppressor gene MCC.

In the present invention, the reason why the peptide having the above specific amino acid sequence is adopted as the antigenic peptide is that it is part of the putative structure of the cancer suppressor gene MCC for colorectal cancer and has a high antigenicity. Because it was estimated. As the carrier protein to be bound to this antigenic peptide, various kinds can be used, and examples thereof include keyhole limpet hemocyanin (KLH) and bovine serum albumin. The binding between the antigenic peptide and the carrier protein is a method known per se, for example, a method using succinimide [T. Kitagawa et al.
iochem. ”Vol. 79, p. 233 (1976)].

The obtained conjugate is administered as an antigen to animals such as mice, rabbits, rats and sheep for the purpose of immunization by a conventional method. As a method for obtaining the antibody according to the present invention from an immunized animal, a conventional method can be adopted.
For example, it can be performed by collecting blood from the immunized animal and preparing an antiserum according to a conventional method. [In this case, the presence or absence of the antibody can be determined by cells expressing the tumor suppressor gene MCC, for example, normal monkey fibroblasts. Strain CV-1 can be solubilized and analyzed by Western blotting using the above antisera to determine whether the product (molecular weight 100000) of the tumor suppressor gene MCC (molecular weight 100000) is detected], or immunized animals Can be obtained as a monoclonal antibody by preparing a hybridoma by collecting the lymphocytes of Escherichia coli and fusing with a myeloma cell, identifying a hybridoma that specifically produces an antibody by screening, and culturing the antibody-producing hybridoma. The desired antibody can also be obtained by transplanting the above-described antibody-producing hybridoma into an animal to produce the antibody, and then collecting and isolating by collecting blood or the like.

[0007]

EXAMPLES Next, the present invention will be described in more detail and specifically with reference to antibody production examples and confirmation test examples.

Production Example (A) Preparation of Antigenic Peptide A putative structure that has already been reported for the product of the tumor suppressor gene MCC for colorectal cancer was examined, and a peptide presumed to have high antigenicity A peptide having an amino acid sequence was synthesized by a solid phase method using an automatic peptide synthesizer (Beckman 990B type). Cys-Glu-Asn-Ser-Arg-Pro-His-Thr-Asn-Glu-Thr-Ser-Le
u Try the synthetic peptide above at 0 ° C for 30 minutes.
Peel off the solid phase by treatment with 75% hydrogen fluoride / 25% anisole, equilibrate with 0.05 M ammonium acetate buffer (pH 7.0) containing 1 mM dithiothreitol, and add SP.
It was adsorbed on a Sephadex column (2.5 x 50 cm).
The adsorbate in the column was fractionally eluted with a gradient of 500 ml of the above equilibration buffer and 500 ml of 0.5 M acetic acid (pH 7.0) containing 1 mM dithiothreitol. The peptide-containing fractions were collected and concentrated by checking each fraction with Fluororescamine, and the concentrate was concentrated on a Sephadex G-10 column (1.0 x 50%) equilibrated with a 30% acetic acid solution.
cm) and the peptide-containing fractions were collected, concentrated and dried in the same manner as above to obtain the desired antigenic peptide. The peptide was immersed in a 1N hydrochloric acid solution at 120 ° C. overnight for hydrolysis, and the amino acid composition was examined by an amino acid analyzer. The results were as follows. Cys 1.0; Glu 1.9; Asn 2.0; Ser 2.0; Arg 0.9; Pro
1.1; His 1.0; Thr 1.9; Leu 1.0 (%)

(B) Preparation of antigen (binding to carrier protein) Keyhole limpet hemocyanin dissolved in 10 mM phosphate buffer at a rate of 10 mg / ml and 10 m at a rate of 15 mg / ml
63 μl of n-maleimido-n-hydroxysuccinimide ester dissolved in M phosphate buffer was mixed and allowed to react for 30 minutes at room temperature. 0.1M phosphate buffer (pH
The keyhole limpet hemocyanin was activated by chromatographing the above reaction solution at a temperature of 4 ° C. using a Sephadex G-25 column equilibrated with 6.0). 2.3 ml of this activated keyhole limpet hemocyanin and 10 mM / ml of 10 mM phosphate buffer (pH 7.3) containing 5 mM EDTA was added to the solution of the antigenic peptide obtained in (a) above. Solution 0.1 ml
And are mixed, the pH is adjusted to 6.5 and then at room temperature 4
By mixing for a time, a desired antigen (a conjugate of an antigenic peptide and a carrier protein, keyhole limpet hemocyanin) was obtained. The formation of this bound product was confirmed by SDS-gel electrophoresis analysis.

(C) Preparation of antibody 200 μg of the antigen obtained in (b) above was injected into the palm of a rabbit together with Freund's complete adjuvant. After that, immunization was performed by further injecting the antigen 200 μg four times subcutaneously at intervals of 3 weeks. From the last dose
Blood was collected on the 10th day and serum was collected. Centrifuge this serum
A saturated ammonium sulfate solution (pH 7.4) was added to the supernatant obtained by (10000 xg) to adjust the ammonium sulfate concentration to 14%. The solution was stirred under ice-cooling overnight and then centrifuged at 10000 xg for 10 minutes to collect a precipitate. The obtained precipitate was dissolved in distilled water and dialyzed against 500 times amount of 0.15M NaCl for 48 hours to obtain an antiserum solution.

DEAE-cellulose (Whatman DE32) column equilibrated with 10 mM phosphate buffer (pH 7.2)
(1.0 x 15 cm) was poured with 2 ml of the above antiserum solution to obtain an immunoglobulin IgG fraction as a flow-through fraction. This IgG
Yield is 24 mg. The collected IgG was dialyzed against 0.1 M sodium carbonate buffer (pH 8.0). On the other hand, activated CH-Sepharose 4B (Pharmacia) 1
g was suspended in 5 μl of 0.1 M sodium carbonate buffer (pH 8.0), and the antigenic peptide obtained in (a) above was added to the suspension.
Peptide-bound CH-Sepharose 4B was prepared by adding mg to the column (0.5
x 2.0 cm), and apply the dialyzed Ig above to this column.
Charge the G fraction, wash thoroughly with 0.15M NaCl / 0.002M sodium carbonate buffer (pH 8.0), and then elute the peptide antibody adsorbed on the column with 0.17M glycine-hydrochloric acid buffer (pH 2.3). Let 0.15 collected eluate
It was dialyzed against M NaCl and then ultrafiltered to obtain 0.5 ml of a solution containing the desired IgG antibody.

Confirmation Test Example (1) Determination of Immunoglobulin Class Using the anti-rabbit IgG antibody, anti-rabbit IgM antibody and anti-rabbit IgA + IgM + IgG antibody manufactured by Kappel Co. Immunoprecipitation test of the obtained antibody (purified antibody against the product of the tumor suppressor gene APC) was carried out to determine the immunoglobulin class.
That is, in the center of the hole formed in the 1% agar gel, the above 3
Put the anti-rabbit antibody of the species, and in the hole formed around it, put the purified antibody according to the production example, which was diluted from 1/20 volume to these anti-rabbit antibody by 2 times, and leave it at 0 ° C overnight. Later, when the formed sedimentation edge was observed, the purified antibody was found to be anti-rabbit IgG antibody and anti-rabbit IgA + IgM + Ig.
It was confirmed that the immunoglobulin class was IgG since it showed immunoprecipitation by the G antibody.

(2) Determination of molecular weight The molecular weight of the purified antibody obtained in Production Example was determined by Sephadex G-100.
Was determined by gel filtration method. That is, Sephadex G equilibrated with 0.02M sodium carbonate buffer.
-100 column (1.0 x 100 cm) with the purified antibody obtained in the production example.
Charge 1 mg, develop with the above buffer solution, measure the absorbance at 280 nm to detect the eluted protein, and detect the molecular weight marker [Bio-Rad Molecular Weight Measurement Kit (No. 151-1
901) and thyroglobulin (molecular weight 670000), γ-globulin (molecular weight 158000), ovalbumin (molecular weight 44
000), myoglobin (molecular weight 17,000) and vitamin B ₁₂
(The molecular weight of 1350 is preset), and the molecular weight of the purified antibody was about 150.
It turned out to be 000.

(3) Molecular extinction coefficient 1 mg of the purified antibody obtained in the Production Example was dissolved in 1 ml of sodium carbonate buffer (pH 9.0), and the absorbance at 280 nm was measured to be 1.40. Therefore the molecular extinction coefficient is E ^1% (1 cm)
= 1.40.

(4) Immunospecificity Monkey fibroblast cell line CV expressing the tumor suppressor gene MCC
-1 was solubilized with RIPA buffer (1% NP-40, 0.1% sodium deoxycholate, 0.5% NaCl and 1 mM phenylmethylsulfonyl fluoride and 50 mM Tris-HCl buffer, pH 7.4) At 10000 xg
Centrifuge for 30 minutes to collect the supernatant.
It was separated by 10% SDS-gel electrophoresis according to the method of Emmli). After Western transfer of each of the obtained isolated proteins to a nitrocellulose filter by a conventional method, detection of the tumor suppressor gene MCC product was performed using the purified antibody obtained by the production example and an alkaline phosphatase-conjugated anti-rabbit IgG antibody (manufactured by Kappel). I went. As a result, the presence of a protein with a molecular weight of 100000 was confirmed in the monkey fibroblast cell line CV-1 expressing the tumor suppressor gene MCC.
In addition, when the antigenic peptide described in the item (a) of Production Example was added to absorb the antibody and the same test as above was performed, a protein having a molecular weight of 100000 could not be detected. These facts indicate that the antibody obtained by the production example specifically recognizes the product of the tumor suppressor gene MCC.

[0016]

The antibody according to the present invention specifically recognizes the product of the tumor suppressor gene. Since it is becoming clear that the tumor suppressor gene is involved in the development of various types of general cancers and tumors as well as hereditary special cancers and tumors, the antibody according to the present invention is used in the development of cancers and tumors. It can be used for elucidation and diagnosis. Furthermore, the antibody according to the present invention has a short chain length,
Therefore, only by using a peptide that is easy to synthesize as an antigen material,
Others can be prepared by a method known per se in the field of biotechnology, and are therefore easy to manufacture.

[Sequence list]

 SEQ ID NO: 1 Sequence length: 13 Sequence type: Amino acid Topology: Linear Sequence type: Synthetic peptide Sequence Cys Glu Asn Ser Arg Pro His Thr Asn Glu Thr Ser Leu 1 5 10

Claims (1)

[Claims] 1. Amino acid sequence of Cys-Glu-Asn-Ser-Arg-Pro-His-Thr-Asn-Glu-Thr-Ser-Le
An antibody obtained by using a conjugate of an antigenic peptide which is u and a carrier protein as an antigen, wherein the immunoglobulin class is IgG, the molecular weight is about 150,000, and the molecular extinction coefficient [E ^1% (1 cm),
(280 nm)] is 1.40, which is an antibody against the product of the tumor suppressor gene MCC.