KR20030037856A - Polyclonal antibody to ribosomal protein S3 and Diagnostic Kit of cancer using the same - Google Patents

Abstract

PURPOSE: A polyclonal antibody to ribosomal protein small subunit 3(rpS3) and a diagnostic kit of cancer using the same are provided. The polyclonal antibody can specifically recognize amino acids from 164 to 243 in the amino acid sequence of rpS3, so that it can be useful for diagnosis of leukemia or various cancers. CONSTITUTION: A polyclonal antibody specifically recognizing ribosomal protein S3 is provided, wherein the polyclonal antibody derived from an immunized rabbit is capable of specifically recognize amino acids from 164 to 243 in the amino acid sequence of rpS3 represented by SEQ ID NO: 1; and the polyclonal antibody is produced by inserting the rpS3 gene into a vector pMAL-c, transforming E. coli BL-21 with the rpS3-containing pMAL-c and expressing maltose-rpS3 from the transformed E. coli BL-21; injecting the maltose-rpS3 to a rabbit; fusing 80 C-terminal amino acids of rpS3 into a vector pGEX-5X-1; transforming E. coli BL-21 with the recombinant vector; expressing protein from the transformed E. coli BL-21; and reacting the serum of the immunized rabbit with a SDS-PAGE developed protein of E. coli BL-21.

Description

Polyclonal antibody to ribosomal protein S3 and Diagnostic Kit of cancer using the same}

The present invention relates to a polyclonal antibody against ribosomal protein S3 and a method for diagnosing cancer using the same. More specifically, polyclonal antibodies (hereinafter abbreviated as S3CA) which can be usefully used for diagnosing leukemia or various cancers by specifically recognizing sequences 164 to 243 of amino acids of the rpS3 protein, and diagnostic methods thereof It is about.

Life is constantly being damaged by exposure to chemicals and ultraviolet rays from outside that damage DNA. In addition, oxidative DNA damage caused by highly reactive oxygen compounds in vivo as a result of oxygen respiration causes a wide variety of damage to DNA. Repairing it is a DNA repair enzyme (Repair enzyme), their abnormalities will lead to degenerative diseases such as aging and cancer.

The rpS3 gene expresses UV endonuclease III, which is missing or modified in XP (xeroderma pigmentosum) group D cells, a disease susceptible to skin cancer.

UV (ultraviolet light) endonuclease refers to an excision DNA repair enzyme that cuts and repairs damaged DNA after being irradiated with ultraviolet light. These are usually bi-functional enzymes that cut damaged bases with DNA glycosylase activity, form AP (apurinic / apyrimidinic) sites, and then cut AP sites using AP endonuclease activity, or DNA produced by UV irradiation. These enzymes have excision endonuclease activity that recognizes and cleaves bulky sites of damage.

Xeroderma pigmentosum (XP) is a hereditary disease in humans that is medically sensitive to sunlight and ultraviolet rays and is autosomal recessive and causes skin cancer and malignant melanoma induced by sunlight. The frequency is increased, leading to premature age 20, in some cases the disease leads to progressive neurodegeneration (Kraemer dt al., 1984 Carcinogenesis 5, 511-514). Cultured cells from XP patients show increased susceptibility to death by UV and removal of many sites of chemically induced bulky DNA damages as well as an inefficient reduction in the level of UV-induced DNA repair synthesis. In particular, it is known that failure to remove damaged DNA such as pyrimidine dimer does not normally perform excision repair. Eight complementarity groups were defined based on the recovery of UV-induced repair synthesis by cell fusion. Seven complementarity groups (A-G) perform abnormal excision repair, and one deformed group (XP-variant) exhibits a normal level of excision repair, although the post replication repair is incorrect.

Human cells from the XP-D complementarity group were found to lack or modify human AP endonuclease I. This AP endonuclease I and UV endonuclease III were found to be the same enzyme. In addition, the mechanism of AP endonuclease of this enzyme was found to be lyase (or AP endonuclease) through β-elimination. In substrate specificity, the phosphodiester bond of the site where pyrimidine dimer was generated by UV light was detected. There was also a mechanism to break (Kim et al., 1995 J. Biol. Chem. 270, 13620-13629).

Purification and cloning of this protein revealed ribosomal protein S3, a protein located on the outer surface of the 40S subunit, a product of the rpS3 gene. Expression of cloned rpS3 gene in E. coli revealed UV endonuclease activity, revealing that the three proteins of AP endonuclease I, UV endonuclease III and ribosomal protein S3 are identical proteins Recombinant S3 was also found to have the activity of breaking the phosphodiester bond of the pyrimidine dimer (Kim et al., 1995 J. Biol. Chem. 270, 13620-13629; Kuhnlein et al. , 1978 Nucleic Acids Res. 5, 951-960; Deutsch et al., 1997 J. Biol. Chem. 272, 32857-32860).

The rpS3 gene is located on 11q13.3-q13.5 of human chromosome 11. Of particular note is that the rpS3 gene is overexpressed in patients with colorectal cancer. Therefore, the gene product of rpS3 is either missing or modified in XP-D, overexpressed in colorectal cancer, and most likely related to other cancers. In addition, studies of the 11q13.3-q13.5 region frequently result in structural abnormality, amplification of genes, multiple endocrine neoplasia type I, and breast carcinoma. And overexpression of human cancers such as B cell neoplastic tumors (Pogue-Geile et al., 1991 Mol. Cell. Biol. 11, 3842-3849).

The present inventors studied the rpS3 gene, while 1-26 amino acids at the N-terminus of the rpS3 protein exhibited apoptosis activity, and amino acid positions 164-243 were DNA repair and regulatory domains. Patent application was filed (Korean Patent Application No. 2001-0063504). Further research has revealed that amino acids 164-243 are the sites where cells interact with other proteins, and their three-dimensional structure changes depending on the state of the cell.

The inventors of the present invention focus on the above-described production of a polyclonal antibody that specifically recognizes the sequence of 164-243 amino acids of the rpS3 protein, the antibody can specifically recognize and diagnose leukemia and cancer cells The present invention was completed by revealing the following.

Accordingly, an object of the present invention is to provide a polyclonal (S3CA) antibody that specifically recognizes human ribosomal protein small subunit 3 (rpS3) and an effective method for producing the antibody.

Another object of the present invention to provide a method for diagnosing leukemia or cancer cells using the S3CA antibody.

1 is a schematic diagram of a plasmid in which the rpS3 gene (732bp) is inserted using EcoR I- Sal I in a multi cloning site (MCS) of pMAL-c for use as an immunogen. Antibodies were prepared by injecting toss-rpS3 fusion proteins into rabbits.

FIG. 2 is a schematic diagram of a recombinant vector in which 80 amino acids of C-terminal of rpS3 are fused to GST using EcoR I and Sal I of MCS in pGEX-5X-1 vector to purify an antibody.

3 is a result of Western blot using the antibody of the present invention.

Figure 4 is a comparison of the expression pattern of rpS3 in peripheral blood monocytes (PBMC) of leukemia patients using the antibody of the present invention.

(A: Western blot using rabbit anti-rpS3 polyclonal antibody, B: mouse anti-beta actin antibody to quantify protein amount Using Western blot.)

Figure 5 is a result of comparing the expression of rpS3 in cancer cell lines using the antibody of the present invention.

In the present invention, in order to prepare a polyclonal antibody of rpS3, first inserting the rpS3 gene (genbank, acession number U14992; SEQ ID NO: 1) into the vector pMAL-c (New England Bio-Lab Cat. # N8076S) and then maltose- rpS3 is expressed and used as an immunogen.

PGEX-5X-1 (Amersham Pharmacia Biotech Cat. # 27) to purify only polyclonal antibodies that specifically recognize amino acid number 164-243 sequence of rpS3 protein from rabbits immunized with maltose-rpS3. 80 C-terminal amino acids of rpS3 are used in fusion to GST.

The present invention provides a diagnostic kit for diagnosing cancer and leukemia. Specifically, the cancer and leukemia diagnostic kit of the present invention includes a polyclonal antibody that specifically recognizes the amino acid number 164-243 sequence of the rpS3 protein.

The polyclonal antibody to the ribosome protein S3 of the present invention and a diagnostic method using the same can be applied to early diagnosis and prediction of cancer and leukemia.

Hereinafter, the present invention will be described in detail through examples. However, the following examples are only for illustrating the present invention is not limited thereto.

EXAMPLES Preparation of Polyclonal Antibodies of Ribosome Protein S3 of the Invention

Step 1: Preparation of the Antigen

Human rpS3 (ribosomal protein small subunit 3 genbank, acession number U14992; SEQ ID NO: 1) gene (SEQ ID NO: 1) using EcoR I- Sal I in the multicloning site (MCS) of pMAL-c (New England Bio-Lab Cat. # N8076S) 732 bp) was inserted to construct the plasmid (FIG. 1). The plasmid was transformed into BL-21 (Amersham Pharmacia Biotech Cat. # 27-1542-01), a type of E. coli strain, incubated for 3 hours at 37 ° C in a liquid medium, and then again 0.5 mM IPTG. Was added and incubated at 30 ° C. for 3 hours. The cultured BL-21 was subjected to cell lysis using a sonicator, and then the supernatant was poured into an amylose resin column, and then maltose was added to obtain a fused protein.

Stage 2: Immunity

The maltose-rpS3 fusion protein obtained in step 1 was mixed with an adjuvant at a ratio of 3: 7, and then intravenously injected into rabbits of about 20 weeks of age to 10 mg / mL, and the second inoculation was performed in the first phase. Two weeks after the inoculation, the third dose was given three weeks after the second inoculation.

Step 3: Purification of the Antibody

In order to purify the serum isolated from the immunized rabbit (rabbit) was carried out as follows.

First, as shown in FIG. 2, 80 C-terminal amino acids of rpS3 are fused to GST using EcoR I and Sal I in MCS of pGEX-5X-1 (Amersham Pharmacia Biotech Cat. # 27-4584-01) vector The plasmid was then transformed into BL-21 (Amersham Pharmacia Biotech Cat. # 27-1542-01), a type of E. coli strain, incubated at 37 ° C. for 3 hours in a liquid medium, and then again 0.5. mM IPTG was added and incubated at 30 ° C. for 3 hours. The cultivated BL-21 was subjected to cell lysis using a sonicator, and GST beads were added to the supernatant to obtain a fused protein. The protein thus obtained was developed on 12% SDS-PAGE, transferred to a nitrocellulose membrane, and the membrane was reacted with a blocking solution for 2 hours, and then immunized rabbit serum was added at 4 ° C. React for one day. The antibody-attached nitrocellulose was washed three times with 1 × TBST, and then reacted with 0.2M Glycine-HCl (pH2.8) at room temperature for 15 minutes to drop the antibody, followed by neutralization with 2M Tris-HCl (pH9.4). .

Experimental Example 1 Implementation of Western Blot Using the Present Polyclonal Antibody

Western blotting confirmed that the polyclonal antibody purified in the above example can detect endogeneous rpS3 (about 30 kDa) isolated from mice and humans.

Each cell lysate was dissolved in Tris buffer containing a Protease Inhibitor Cocktail to obtain protein by sonication. Each protein was quantified to 40 µg and then subjected to 12% SDS-PAGE. After electrophoresis, it was transferred to nitrocellulose. Samples transferred to nitrocellulose were reacted with a blocking solution made of skim milk for 1 hour, and then the rabbit-polyclonal antibody to the purified rpS3 c-terminus was 0.25 µg / ml. After treatment at room temperature for 1 hour, HRP conjugated goat anti-rabbit IgG serum conjugated with horseradish peroxidase was treated at room temperature for 1 hour and then washed three times with 1 × PBST. After the substrate (Chemiluminescence Blotting substrate, Roche Dignostics cat. # 1 501 399) was treated to X-ray film.

As shown in FIG. 3, the lysate (lane 1) derived from the human body and the lysate (lane 2) derived from the mouse and the rabbit anti-rpS3 polyclonal antibody of the present invention (rabbit anti) It was confirmed that endogeneous rpS3 (about 30 kDa) can be detected by specifically binding -rpS3 polyclonal antibody).

Experimental Example 2: Investigation of Expression of rpS3 in PBMC of Leukemia Patients Using Antibody of the Present Invention

In order to compare the expression pattern of rpS3 in peripheral blood mononuclear cells of leukemia patients and peripheral blood mononuclear cells (PBMC) of normal humans, the expression patterns of peripheral blood monocytes of 19 leukemia patients were investigated. .

Peripheral blood monocytes obtained from each patient were dissolved in Tris buffer containing a Protease inhibitor cocktail, and then mixed with glass beads. After being allowed to stand at 4 ° C. for 10 minutes, centrifugation was performed to obtain proteins. This was quantified and electrophoresed by 30 μg on 12% SDS-PAGE and transferred to nitrocellulose membrane. The transferred nitrocellulose was Western blotted using the S3CA antibody prepared in Example and a mouse antibody against β-Actin (Santa Cruz cat. # Sc-8432).

In Fig. 4, 1 (A) and 1 (B) PCs represent the cell lysate of plasmacytoma MPC11 of mouse cancer cell line as a positive control, and NC represents negative control. ) Represents a cell lysate derived from normal PBMCs, and numbers 1 to 18 represent cell lysates of PBMCs in each leukemia patient. 1 (A) is a Western blot treated with rabbit anti-rpS3 polyclonal antibody, and 1 (B) is a mouse for quantifying the amount of protein in each lane. It was treated with a mouse anti-beta actin antibody.

As shown in FIG. 4, the expression of rpS3 was increased in PBMCs of 14 leukemia patients compared to PBMCs of normal subjects (about <74%). However, when seven normal subjects were examined, there was no increase in expression of even one.

These results indicate that although the protein is present in normally slow-growing cells, it is hardly recognized by the S3CA antibody and strongly recognized in peripheral blood monocytes of rapidly proliferating cancer cells or leukemia cells. Or early in the cell, the 80 amino acid region of the C-terminal is exposed due to the change of the tertiary structure, the recognition specificity for the S3CA antibody is thought to increase.

Experimental Example 3: Investigation of Expression of rpS3 in Cancer Cell Lines Using Antibodies of the Present Invention

Western blot was performed in the same manner as in Experiment 2 to investigate the expression pattern of rpS3 in cancer cell lines.

Lane 1 represents the degradation product of Molt-4, a human acute T lymphoblast, lane 2 represents the degradation product of MPC11, mouse plasmacytomas, and lane 3 represents the oncogene for transformation. As a cell line transfected with (oncogene), a digested product of NIH3T3, a mouse embryonic fibroblast, and lane 4 is a F-65 cell line, and a digested product of normal fibroblasts derived from skin as a sample. will be.

As a result, as shown in FIG. 5, the cell lines (lane 3) transfected with cancer cell lines (lane 1 and lane 2) and the oncogene at the position of 30 kDa were darker than the normal cell lines, indicating that the rpS3 was overexpressed. Could.

Therefore, it was found that overexpression of rpS3 gene correlated with the incidence of leukemia and cancer. In particular, the rpS3 gene was an important target gene and it was confirmed for the first time that its antibody can be used for the diagnosis and prevention of leukemia and cancer. .

As described above, the S3CA antibody of the present invention is a very useful invention in the pharmaceutical industry because it can diagnose leukemia or various cancers by specifically recognizing the sequence of 164-243 amino acids of the rpS3 protein.

Claims (3)

An antibody that specifically recognizes human ribosomal protein small subunit 3 (rpS3). The rabbit-polyclonal antibody of claim 1, which specifically recognizes amino acid number 164-243 sequence of rpS3 protein. A diagnostic kit for leukemia or cancer cells, comprising the antibody of claim 1 or 2 as an active ingredient.