JPH06128300A - Antibody to product of cancer suppressing gene prohibitin - Google Patents

Abstract

PURPOSE:To obtain an antibody to a product of a cancer suppressing gene prohibitin utilizable for diagnosing cancer or tumor. CONSTITUTION:The antibody to a product of a cancer suppressing gene prohibitin is an amino acid sequence of a part presumed to have high antigenicity in an estimated structure related to a cancer suppressing gene prohibitin of mammary cancer. This is an antibody) obtained by using a combination of a peptide capable of coding Cys-Thr-Tyr-Leu-Pro-Ala-Gly-Gln-Ser-Val- Leu-Leu-Gln-Leu-Pro-Gln with a carrier protein as an antigen. The immunoglobulin class is IgG and its molecular weight is about 150000. The molecular extinction coefficient [E<1>% (1cm), (280nm)] is 1.40. The chain length of the peptide used as the antigen is short. Thereby, its synthesis is readily carried out. A desired antibody can be prepared by a well-known technique itself including the synthesis of this antigenic peptide.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. 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 prohibitin, 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-Thr-Tyr-Leu-Pro-Ala-Gly-Gln-Ser-Val-Leu-Leu- Gl
n-Leu-Pro-Gln is an antibody that can be obtained by using an antigenic peptide-carrier protein conjugate as an antigen. The immunoglobulin class is IgG, the molecular weight is about 150000, 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 prohibitin.

In the present invention, the reason why the peptide having the above-mentioned specific amino acid sequence is adopted as the antigenic peptide is that it is part of the putative structure of the tumor suppressor gene prohibitin for Wilms tumor and has a high antigenicity. Because it was estimated. Various types of carrier proteins can be used as the carrier protein to be bound to the antigenic peptide, for example, keyhole limpet hemocyanin (KL
H), bovine serum albumin and the like can be exemplified. The binding between the antigenic peptide and the carrier protein is a method known per se, for example, a method using succinimide [T. Kitag
Awa et al., "J. Biochem." Vol. 79, p. 233 (1976)]
Can be done at.

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 an 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 using cells expressing the tumor suppressor gene prohibitin, for example, human cervical cancer-derived cells. HeLa cells can be solubilized and analyzed by Western blotting using the above antisera to detect whether prohibitin with a molecular weight of 32000 is detected.] Alternatively, lymphocytes of immunized animals can be collected and myeloma can be collected. It can be obtained as a monoclonal antibody by preparing a hybridoma by fusing with cells, 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 The putative structure reported for the product of the tumor suppressor gene protectinin breast cancer was examined and the following amino acid A peptide having a sequence was synthesized by a solid phase method using an automatic peptide synthesizer (Model B 990B manufactured by Beckman). Cys-Thr-Tyr-Leu-Pro-Ala-Gly-Gln-Ser-Val-Leu-Leu-Gl
n-Leu-Pro-Gln The above synthetic peptide was incubated 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 0.9; Thr 0.9; Tyr 1.1; Leu 4.0; Pro 1.9; Ala
1.0; Gly 1.0; Gln 2.9; Ser 0.9, Val 1.1 (%).

(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, the antigen was further injected subcutaneously into the back four times at 200 μg every 3 weeks. After that, immunization was performed by injecting the antigen subcutaneously to the back of 200 μg four times at three-week intervals. Blood was collected 10 days after the final administration and serum was collected. The serum was centrifuged (10000 xg), and a saturated ammonium sulfate solution (pH 7.4) was added to the obtained supernatant to adjust the ammonium sulfate concentration to 14%. After stirring the solution under ice cooling overnight, 10000 x
The precipitate was collected by centrifugation at g for 10 minutes. The obtained precipitate was dissolved in distilled water and dialyzed against 500 times 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)
2 ml of the above antiserum solution was poured onto (1.5 x 15 cm) 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 the column was dialyzed as above.
Charge the gG fraction, wash thoroughly with 0.15M NaCl / 0.002M sodium carbonate buffer (pH 8.0), then elute the peptide antibody adsorbed on the column with 0.17M glycine-hydrochloric acid buffer (pH 2.3). Let Elute the collected eluate to 0.
It was dialyzed against 15M 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 anti-rabbit IgG antibody, anti-rabbit IgM antibody and anti-rabbit IgA + IgM + IgG antibody manufactured by Kappel Co., obtained by the above-mentioned production example by the Ouchterlony method. Immunoprecipitation test of the obtained antibody (purified antibody against the product of the tumor suppressor gene Prohibitin) was carried out to determine its immunoglobulin class. That is, the above 3 kinds of anti-rabbit antibodies were put in the center of the hole formed in the 1% agar gel, and the holes formed in the periphery were 1/20 to 2 times the amount of these anti-rabbit antibodies. Add the purified antibody from the above production example diluted at
After standing overnight at room temperature, the purified antibody was observed to be the anti-rabbit IgG antibody and anti-rabbit IgG antibody.
Since the immunoprecipitation was shown only by the IgA + IgM + IgG antibody, it was confirmed that the immunoglobulin class was IgG.

(2) Determination of molecular weight The molecular weight of the purified antibody (anti-prohibitin antibody) obtained in Production Example was determined by gel filtration using Sephadex G-100. That is, a Sephadex G-100 column (1.0 x 100 cm) equilibrated with 0.02 M sodium carbonate buffer was charged with 1 mg of the purified antibody obtained in the production example, developed with the above buffer, and developed at 280 nm. Absorbance is measured to detect the eluted protein, and a molecular weight marker [Bio-Rad's molecular weight measurement kit (No. 151-1901) is used for thyroglobulin (molecular weight 670000), γ-globulin (molecular weight 15800).
0), ovalbumin (molecular weight 44000), myoglobin (molecular weight 17000) and vitamin B ₁₂ (molecular weight 1350) are preset], the purified antibody has a molecular weight of about 150000. found.

(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 HeLa cells expressing the tumor suppressor gene Prohibitin
After solubilization with RIPA buffer (1% NP-40, 0.1% sodium deoxycholate, 0.5% NaCl and 1 mM phenylmethylsulfonylfluoride and 50 mM Tris-HCl buffer, pH 7.4), at 10000 xg
Centrifuge for 30 minutes and collect the supernatant.
mli) and separated by 10% SDS-gel electrophoresis. After Western transfer of each obtained separated protein to a nitrocellulose filter by a conventional method, detection of prohibitin was carried out using the purified antibody obtained in Production Example and an alkaline phosphatase-conjugated anti-rabbit IgG antibody (manufactured by Kappel). As a result, the presence of a protein having a molecular weight of 32000 was confirmed in the purified Prohibitin antibody in HeLa cells expressing Prohibitin. After the antibody was absorbed by adding the antigenic peptide described in the item (a) of Production Example, the same test as above was performed, but a protein having a molecular weight of 32000 could not be detected. These facts indicate that the antibody obtained in the Production Example specifically recognizes the product of the tumor suppressor gene prohibitin.

[0016]

INDUSTRIAL APPLICABILITY The antibody according to the present invention specifically recognizes the product of the tumor suppressor gene protectin. 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 uses only a peptide having a short chain length and therefore easy to synthesize as an antigen material, and can be prepared by other methods known per se in the field of biotechnology, and thus is easy to produce. is there.

[Sequence list]

 SEQ ID NO: 1 Sequence length: 16 Sequence type: Amino acid Topology: Linear Sequence type: Synthetic peptide Sequence Cys-Thr-Tyr-Leu-Pro-Ala-Gly-Gln-Ser-Val-Leu-Leu-Leu- Gln-Leu-Pro-Gln 1 5 10 15

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification number Office reference number FI technical display location // A61B 10/00 TC07K7 / 08 ZNA8318-4HC07K 99:00

Claims (1)

[Claims] 1. An amino acid sequence of Cys-Thr-Tyr-Leu-Pro-Ala-Gly-Gln-Ser-Val-Leu-Leu-Gl
n-Leu-Pro-Gln is an antibody that can be obtained by using an antigenic peptide-carrier protein conjugate as an antigen. The immunoglobulin class is IgG, the molecular weight is about 150000, and the molecular extinction coefficient [E ^1% (1 cm) ,
(280 nm)] is 1.40, an antibody against the product of the tumor suppressor gene prohibitin.