KR100883530B1 - Protein marker adenosylhomocysteinase for colon cancer diagnosis and diagnosis kit for colon cancer using antibodies against the same - Google Patents

Abstract

The present invention relates to a diagnostic kit for colorectal cancer comprising a protein marker Adenosylhomocysteinase and an antibody thereof, capable of diagnosing colorectal cancer. Adenosyl homocysteinase selected as a protein marker to diagnose colorectal cancer due to its specific increase, and a colon cancer diagnostic kit comprising an antibody that selectively recognizes the same, and an antigen-antibody combining reaction The present invention relates to a method for detecting adenosyl homocysteine in plasma. The colorectal cancer diagnostic kit comprising an antibody against adenosyl homocysteinase selected as a marker for colorectal cancer diagnosis according to the present invention not only has high sensitivity and accuracy of diagnosis, but also very easily diagnoses colorectal cancer using the patient's plasma. It can be useful for the early diagnosis of colorectal cancer.

Colorectal cancer, Diagnostic marker, Adenosyl homocysteine

Description

PROTIN MARKER ADENOSYLHOMOCYSTEINASE FOR COLON CANCER DIAGNOSIS AND DIAGNOSIS KIT FOR COLON CANCER USING ANTIBODIES AGAINST THE SAME}

The present invention relates to a colorectal cancer diagnostic kit comprising a protein marker adenosyl homocysteinase and an antibody that specifically recognizes it, which can selectively diagnose the presence or absence of colorectal cancer in the blood.

Colon cancer is mostly adenocarcinoma (adenocarcinoma) in pathology, and largely divided into colon cancer and rectal cancer. The incidence of site-specific incidence occurs most often in the lower colon, or rectum, at about 50%. Recent studies have shown that changes in dietary habits have significantly increased the incidence and mortality of colorectal cancer in Korea, and the incidence of colorectal cancer increased 420% from 1995 to 2002, making it the number one in cancer. (The 2003 Health Insurance Statistics Yearbook, published by the National Health Insurance Service).

The cause of colorectal cancer is still unknown, but genetic factors, dietary habits associated with eating high-fat and low-fiber foods, and inflammatory bowel disease are being considered. Colorectal cancer can occur in all age groups, but as the age increases, the incidence increases and occurs frequently in the 50s and 60s. The incidence of men and women is higher in colon cancer in women and rectal cancer in men.

Treatment of colorectal cancer is performed by chemotherapy and radiotherapy based on surgical resection. Despite advances in surgical therapies, chemotherapy and radiotherapy, mortality rates are very high when people miss the point of surgery because they are metastasized to other organs due to the characteristics of colorectal cancer that progress without specific symptoms. The 5-year average survival rate is at least 90% in stage 1, at least 70% in stage 2, at least 50% in stage 3 and below 5% in stage 4.However, the earlier the early detection and treatment of colorectal cancer, Significantly increased (2004 Cancer Information, published by the National Cancer Center). Therefore, the development of a method for effectively and early diagnosis of colorectal cancer is urgently required.

The diagnosis of colorectal cancer is performed mainly by rectal digital examination, fecal occult blood test and colonography in patients with colon-related symptoms. The inspection is done. Although fecal blood tests or endoscopic examinations are currently used to diagnose colon cancer at the earliest stages, detection of fecal blood typically refers to the development of a tumor of considerable size and the occult blood standard (guaiac). The sensitivity of the fecal test to 26% is so low that 74% of patients with malignant lesions may not be detected (Ahlquist et al . , Gastroenterol . Clin . North) . Am . 26: 41-55, 1997; Korean Patent Publication No. 2006-0027893; Korean Patent Publication No. 2005-0114099). In addition, visualization of precancerous and cancerous lesions is the best approach for early detection, but colonoscopy can be painful and risky to patients as well as significant cost (Silvis et al., JAMA 235: 928-930, 1976; Geenen et al. , Am. J. Dig . Dis . 20: 231-235, 1975; Anderson et al . , J. Natl . Cancer Institute 94: 1126-1133, 2002).

Conventional methods as described above are not only accurate in diagnosis, but also early diagnosis of patients before colorectal cancer is not possible, which causes inconveniences to the subjects. In order to make up for the shortcomings of these conventional diagnostic methods, attempts have been made to diagnose colorectal cancer by measuring the concentration of tumor markers in the patient's plasma (Clinton et al . , Biomed . Sci . Instrum . 39: 408-414, 2003; Rui et al., Proteomics 3: 433-439, 2003; Soletormos et al., Dan . Med . Bull . 48: 229-255, 2001).

Currently, only diagnostic plasma tests based on carcinoembryonic antigen (CEA), tumor-associated glycoproteins are available for diagnostic assistance in the field of colorectal cancer. CEA is increased in 95% of tissues obtained from patients with colorectal cancer, gastric cancer, pancreatic cancer and the majority of breast carcinoma, lung carcinoma and head and neck carcinoma (Goldenberg et al . , J. Natl . Cancer Inst . (Bethesda) 57: 11-22, 1976). However, elevated CEA levels have also been reported in patients with non-malignant disease, showing normal CEA levels in the serum of most patients with colorectal cancer, especially during the early stages of the disease (Carriquiry et al . , Dis . Colon Rectum 42: 921-929, 1999; Herrera, etc., Ann Surg 183: 5-9, 1976 ; Wanebo etc., N. Engl J. Med 299:. ... 48-451, 1978). The use of CEA measured from serum or plasma in detection recurrence is thus controversial and not yet widely applied (Martell et al . , Int . J. Biol . Markers 13: 145-149, 1998; Moertel et al., JAMA 270: 943-947, 1993; International Patent Publication No. WO 2004/057335)

Meanwhile, adenosylhomocysteinase is a catalytic enzyme that reversibly hydrolyzes adenosylhomocysteine to adenosine and homocysteine (Turner et al., Cell. Biochem . Biophys . 33: 101-125, 2000). The adenosyl homocysteine is formed by transferring a methyl group to adenosylhomomethionine. Accumulation of adenosyl homocysteine is essential for methylation by inhibiting the function of methyltransferase. Live deletions lacking the adenosyl homocysteinase gene showed a high mortality rate in early embryos (Miller et al., EMBO J. 13: 1806-1816, 1994), and deficiency of adenosyl homocysteinase in humans was severe. Restriction and malformation were induced (Baric et al . , Proc . Natl . Acad . Sci . USA 101: 4234-4239, 2004). Adenosyl homocysteinase was known to be present in the cytoplasm, but recent transfer of adenosyl homocysteinase from the cytoplasm to the nucleus has been reported during cell culture and during early embryonic development of the nail frog ( X. laevis ). Radomski et al . , Mol . Biol . Cell 10: 4283-4298, 1999). In addition, the enzyme has been reported to form complexes with mRNA methyltransferase and RNA polymerase II (Radomski et al . , Biochim . Biophys . Acta . 1590: 93-102, 2002 ). This indicates that adenosyl homocysteinase performs important functions not only as a biological enzyme but also in embryonic development (Cooper et al ., J. Biol . Chem . 281: 22471-22484, 2006). In addition, adenosine produced from adenosyl homocysteine by reversible hydrolysis of adenosyl homocysteine regulates cell differentiation (Barry et al., Cancer Res . 60: 1887-1894, 2000; Ethier et al . , Am . J. Physiol . 265: H131-H138, 1993), it has been reported to induce apoptosis (Dawicki et al., Am . J. Physiol . 273: L485-L494, 1997; Harrington et al . , Am . J. Physiol . 279: L733- L742, 2000; Rounds et al . , Am. J. Physiol . 275: L379-L388, 1998; Saitoh et al . , Biochem . Pharmacol . 67: 2005-2011, 2004; Wen et al . , Br . J. Pharmacol . 140: 1009-1018 , 2003). In addition, it has been reported that adenosyl homocysteine modulates adenosine-induced apoptosis as described above (Hermes et al . , Exp . Cell) . Res . 313: 264-283, 2007).

Studies of adenosyl homocysteinase associated with cancer have reported increased expression of this gene in ovarian cancer (Peters et al., Cancer Eidermiol . Biomarker . 14: 1717-1723, 2005), after treatment with the anticancer drug 5-Fluoruracil in colorectal cancer cell lines, this gene was reported to be included in the altered gene list (Paula et al . , Mol . Cancer 5: 20, 2006). However, only studies on the amount of gene expression of adenosyl homocysteinase have been reported. To date, no changes in protein expression and protein markers of adenosyl homocysteinase have been performed in relation to disease, and there have been no reports of changes in blood, particularly in relation to cancer.

Therefore, the inventors of the present invention, while continuing to research to solve the problems of the prior art related to the early diagnosis of colorectal cancer, to find a protein that specifically changes in a biological sample obtained from colorectal cancer patients and early diagnosis of colorectal cancer As a result of intensive research efforts to develop a method for the use, the adenosyl homocysteinase protein was found to be present in an increased amount in the plasma of colorectal cancer patients in comparison with the non-patient control group. The present invention was completed by confirming that colon cancer can be easily diagnosed using the immunochemical method.

Accordingly, it is an object of the present invention to provide a method for easily diagnosing colon cancer using adenosyl homocysteine present in plasma.

In order to achieve the above object, the present invention provides adenosyl homocysteine protein marker for colon cancer diagnosis having an amino acid sequence of SEQ ID NO: 6 .

The present invention also provides a diagnostic kit for colon cancer comprising an antibody that selectively recognizes the adenosyl homocysteinase protein marker.

In addition, the present invention provides a method for detecting an adenosyl homocysteine protein marker through an antigen-antibody binding reaction in a sample.

Colorectal cancer diagnostic kit using an antibody that specifically recognizes adenosyl homocysteinase selected as a marker protein for colorectal cancer diagnosis in the present invention uses a plasma that is relatively easy to collect as a sample for biopsy. Unlike the method for diagnosing colorectal cancer, colon cancer can be diagnosed very easily without burdening the patient, and the diagnosis can be useful for early diagnosis of colorectal cancer.

According to one aspect of the invention, the present invention provides an adenosyl homocysteine protein marker having an amino acid sequence of SEQ ID NO: 6 as a protein marker for colon cancer diagnosis.

In order to select a marker protein exhibiting a specific change in colorectal cancer, the present invention is directed to DIGE (Difference In Gel Electrophoresis) two-dimensional protein body of normal tissue without cancer cells at the same surgical site as the extracted cancer tissue after surgery. Comparison and analysis were performed using two-dimensional electrophoresis (Alfonso et al., Proteomics 5: 2602-2611, 2005; Friedman et al., Proteomics 4 : 793-811, 2004; Katayama et al . , Surg . Today 36: 1085-1093, 2006; Tan et al., J. Proteome Res . 5: 1098-1106, 2006). DIGE is a new proteomics technique that compensates for the shortcomings of the existing two-dimensional electrophoresis. The same fluorescence color label is labeled on each sample and the labeled samples are mixed with each other to achieve the same IEF (isoelectricfocusing) and SDS-polyacrylamide electrophoresis. Separate according to isoelectric point and mass. This enables the simultaneous analysis of two samples in one gel, which compensates for the differences between the gels and the difficulty of statistical processing, which has been a disadvantage of the conventional two-dimensional electrophoresis. The separated proteins are compared and analyzed by computer program, and the protein spots with specific changes are compared with normal tissues. To identify the selected proteins, they were trypsinized and separated into a number of peptide fragments. The masses of each fragment and the daughter ions derived from the fragments were then measured by tandem mass spectrometry and a computer analysis program was used. Search the database. As a result, the tryptic peptides having an amino acid sequence of SEQ ID NO: 2 to 21 detected with an increase in expression in colorectal cancer tissues specifically have adenosine having an amino acid sequence of SEQ ID NO: 1 with an isoelectric point of 5.74 and a molecular weight of 48 kDa. It is identified as being a real homostinase (see FIGS . 1-2 ).

MRNA and protein expression levels of adenosyl homocysteinase, which have been shown to specifically increase in colorectal cancer tissue, are examined in tissues by reverse transcriptase-polymerase chain reaction and immunoblotting. As a result, mRNA expression of adenosyl homocysteinase was significantly increased in colorectal cancer tissues, and protein concentrations measured using antibodies that selectively recognized them also specifically increased in colorectal cancer tissues. Check (see FIGS . 3 and 4 ).

In addition, cancer tissues extracted from the colon cancer patient group and normal tissues surrounding them were subjected to immunohistochemical staining to confirm that adenosyl homocysteinase is increased specifically in cancer tissues (see FIG. 5 ).

As described above, in order to confirm whether adenosyl homocysteinase, which specifically increases in colorectal cancer tissues, can be usefully used for analysis of blood, the presence or absence of expression in plasma is confirmed by a tandem mass spectrometer. Blotting is used to detect the concentration of adenosyl homocysteine in plasma.

First, plasma and non-patient plasma of colon cancer patients are collected separately to prepare a sample of colorectal cancer patients and non-patient control plasma. Since few proteins make up the majority of plasma protein concentrations in human plasma, relatively large amounts of proteins must first be removed to identify small amounts of proteins (Anderson NL. Et al., Mol . Cell . Proteomics , 1:..... 845-867 , 2002; Somiari RI , etc., J. Chromatogr B Analyt Technol Biomed Life Sci . , 815: 215-225, 2005). Relatively high amounts of proteins, such as albumin, immunoglobulin A, immunoglobulin G, heptoglobin, transferrin, trypsin inhibitors, and the like, from plasma samples of prepared colon cancer patients and non-patient controls. Liquid chromatography is performed using multiple affinity columns to remove. Protein compartments not bound to the column are tracked with ultraviolet absorbance at 280 nm to collect and remove proteins bound to the column. Thus, the relatively large amounts of the six proteins are mostly concentrated plasma protein compartments are removed, and then subjected to electrophoresis using SDS polyacrylamide gel with colon cancer tissue. A 48 kDa sized spot corresponding to the molecular weight of adenosyl homocysteine was selected, treated with trypsin, and analyzed by a tampon mass spectrometer to identify adenosyl homocysteine having an amino acid sequence of SEQ ID NO: 1 . (See FIG. 6 ). After confirming the presence of adenosyl homocysteinase in the plasma, the plasma protein was electrophoresed using SDS polyacrylamide gel, the separated plasma protein was transferred to PVDF membrane, and the anti-adenosyl homocysteinase antibody And then mouse IgG-HRP, which binds to immunoglobulin G of the mouse. Thereafter, the PVDF membrane is reacted with a chromophoric substrate and then exposed to an X-ray film to determine the concentration of adenosyl homocysteine antigen.

As a result, the adenosyl homocysteinase protein exhibits the same expression pattern as that in colorectal cancer tissues, and the expression of adenosyl homocysteinase is increased by 1.88 times in the plasma of colorectal cancer patients compared to the plasma of the non-patient control group. (See FIGS. 7 and 8 ). This result is also confirmed in the ROC graph with sensitivity and accuracy (see FIG . 9 ). From the above results, the concentration of each antigen was measured using an antibody that selectively recognizes adenosyl homocysteinase in plasma, and a test subject diagnosed as colorectal cancer showed a relatively increased value compared to the non-patient control group. Make sure you can.

The present invention is the first to examine the change in the expression level in the blood as well as tissues in relation to the progression of adenosyl homocysteinase and intracellular cancer, and to use this as a diagnostic marker for colorectal cancer. In addition, since the protein can be detected in plasma, unlike the conventional protein, which can be diagnosed only through biopsy, the protein can be used to diagnose colorectal cancer in a simple manner without causing inconvenience to the patient.

According to one embodiment of the present invention, colon cancer is collected by detecting a plasma sample of a subject and detecting the adenosyl homocysteinase, which is the colon cancer diagnostic marker contained in the plasma sample of the subject, using an antibody that selectively recognizes It is possible to check the onset of the disease.

Therefore, the method of confirming the onset of colorectal cancer using an antibody that selectively recognizes the atenosyl homocysteinase of the present invention is a novel immunological diagnostic tool using the patient's plasma, and has excellent sensitivity and plasma without biopsy. Can be easily analyzed in the target, it can be useful for early diagnosis of colorectal cancer.

Accordingly, according to another aspect of the present invention, the present invention provides a colorectal cancer diagnostic kit comprising an antibody that selectively binds to adenosyl homocysteine, a marker protein for colorectal cancer diagnosis.

In order to prepare antibodies that selectively bind to adenosyl homocysteine marker proteins, the acquisition of adenosyl homocysteinase must be preceded by the amino acids of the adenosyl homocysteinase protein of SEQ ID NO : 1 . It can be synthesized using sequences, produced in microorganisms using genetic recombination, or prepared separately from plasma.

For the purposes of the present invention, the antibody may include both polyclonal and monoclonal antibodies of adenosyl homocysteine, but monoclonal antibodies capable of more specifically binding to antigens are preferred.

Polyclonal antibodies can be prepared by injecting an adenosyl homocysteinase marker protein or fragment thereof into an external host with an immunogen according to conventional methods known to those skilled in the art. Examples of such external hosts include mammals such as mice, rats, sheep and rabbits, and immunogens are generally administered by intramuscular, intraperitoneal or subcutaneous injection, together with an adjuvant to increase antigenicity. Is administered to immunize an external host. Serum from the immunized external host can be routinely collected to collect serum showing enhanced titers and specificity for antigens or to isolate and purify antibodies therefrom to produce polyclonal antibodies specific for adenosyl homocysteinase. .

Monoclonal antibodies can be prepared using methods of generating immortalized cell lines by fusion known to those skilled in the art (Koeher and Milstein, Nature 256: 495, 1975). Briefly, the method is first immunized with a pure adenosyl homocysteinase marker protein or fragment thereof, or a peptide thereof is synthesized and combined with bovine serum albumin to immunize the mouse. Antigen-producing B lymphocytes isolated from immunized mice are fused with myeloma cells of human or mouse to produce immortalized hybridoma cells. Subsequently, by examining the production of monoclonal antibodies in hybridoma cells by enzyme-linked immunosorbent assay (ELISA), positive clones were selected and cultured, and the antibodies were isolated, purified or injected into the abdominal cavity of mice. Ascites may be harvested to prepare monoclonal antibodies specific for adenosyl homocysteine.

Antibodies used in the detection of the adenosyl homocysteinase proteins of the invention include functional fragments of antibody molecules as well as complete forms having two full length light chains and two full length heavy chains. A functional fragment of an antibody molecule means a fragment having at least antigen binding ability and includes Fab, F (ab '), F (ab') ₂ , Fv, and the like.

The colorectal cancer diagnostic kit of the present invention includes not only antibodies that selectively recognize adenosyl homocysteinase, but also tools, reagents, and the like, which are generally used for immunological analysis in the art.

According to one embodiment of the invention, the colorectal cancer diagnostic kit is an antibody specific for adenosyl homocysteinase protein; Secondary antibody conjugates to which a label that is developed by reaction with a substrate is conjugated; A color substrate solution to be colored and reacted with the label; Washing liquid; And it may include a solution for stopping the enzyme reaction.

In addition, the colorectal cancer diagnostic kit of the present invention may further include a positive control comprising an adenosyl homocysteinase standard antigen and a negative control comprising an antiserum of an animal not injected with the antigen.

The colorectal cancer diagnostic kit of the present invention can diagnose colorectal cancer by quantitatively or qualitatively analyzing the antigen to the antibody protein through an antigen-antibody binding reaction, and the antigen-antibody binding reaction is a conventional enzyme immunoassay (ELISA). ), Radioimmunoassay (RIA), sandwich assay, western blotting, immunoprecipitation, immunohistochemical staining, fluorescence immunoassay, enzyme substrate coloring, antigen-antibody aggregation, etc. It can be measured using. For example, the diagnostic kit may be provided to perform an enzyme immunoassay that reacts with a recombinant monoclonal antibody protein using a 96-well microtiter plate coated with a sample and a control surface.

As a fixture for antigen-antibody coupling reaction, a nitrocellulose membrane, a PVDF membrane, a well plate synthesized with a polyvinyl resin or a polystyrene resin, a slide glass made of glass, or the like may be used. .

As for the label of the secondary antibody, a conventional color developing agent having a color reaction is preferable, and horseradish peroxidase (HRP), basic alkaline phosphatase, colloid gold, poly L-lysine-fluorescein isothiocyanate (FITC), Labels such as fluorescents such as rhodamine-B-isothiocyanate (RITC) and dyes may be used.

Color substrate to induce color development is preferably used according to the labeling reaction, TMB (3,3 ', 5,5'-tetramethyl benzidine), ABTS [2,2'-azino-bis (3) -ethylbenzothiazoline-6-sulfonic acid), and OPD (o-phenylenediamine) can be used. At this time, the color substrate is more preferably provided in a dissolved state in a buffer solution (0.1 M NaAc, pH 5.5). A chromogenic substrate such as TMB is decomposed by HRP used as a marker of the secondary antibody conjugate to generate a chromosome deposit, and the presence or absence of adenosyl homocysteinase protein antigen is visually confirmed by the deposition degree of the chromosome deposit. Detect.

The wash preferably comprises phosphate buffer, NaCl and Tween 20, more preferably a buffer consisting of 0.02 M phosphate buffer, 0.13 M NaCl, and 0.05% Tween 20 (PBST). The wash solution is washed 3 to 6 times by reacting the secondary antibody to the antigen-antibody conjugate after the antigen-antibody binding reaction, and then adding an appropriate amount to the fixture. As the reaction terminating solution, sulfuric acid solution (H ₂ SO ₄ ) may be preferably used.

In addition, another aspect of the present invention provides a method for detecting adenosyl homocysteinase, which is a marker protein for colorectal cancer diagnosis, in a sample using an antigen-antibody binding reaction.

The detection method is a protein-immobilized or separated by electrophoresis (SDS-PAGE) and then transferred to the PVDF membrane and contacted with an antibody that selectively recognizes adenosyl homocysteine through an antigen-antibody binding reaction. Indirectly identifying the presence of adenosyl homocysteinase protein. The antigen-antibody binding reaction includes enzyme immunoassay (ELISA), radioimmunoassay (RIA), sandwich measurement, western blotting, immunoprecipitation, immunohistochemical staining, fluorescence immunoassay, enzyme substrate coloration, and antigen-antibody aggregation. Etc. can be mentioned. Serum or plasma may be used as a sample, and plasma is more preferable.

According to a preferred embodiment of the present invention, the detection method,

1) coating the sample and the protein of the control to the fixture;

2) performing an antigen-antibody binding reaction by adding an antibody specific for adenosyl homocysteinase to the fixture;

3) detecting the antigen-antibody binding reactant produced through the antigen-antibody binding reaction using a secondary antibody conjugate and a color substrate solution; And

4) comparing the detection results for the sample and the control.

Specifically, the plasma protein is separated from the sample according to the molecular weight. Since adenosyl homocysteinase has a molecular weight of about 48 kDa, electrophoresis is performed using a 10% polyacrylamide gel, and the proteins separated therefrom are fixed by transferring to a fixture such as a PVDF membrane. Subsequently, an antigen-antibody binding reaction is performed by adding an antibody specific for adenosyl homocysteinase to the immobilized protein antigen. If the adenosyl homocysteinase protein is present in the sample, an antigen-antibody binding reaction occurs when an antibody is added to the membrane to which the protein is immobilized. In order to measure the degree of binding of adenosyl homocysteinase and antibodies to the antibody, a step of binding to a secondary antibody having an affinity for each antibody is performed, wherein the horseradish peroxidase (HRP) conjugated to the secondary antibody is an ECL substrate. The presence of adenosyl homocysteine protein, a diagnostic marker for colorectal cancer, in the sample sample is detected by comparing the extent of the color development reaction with enhanced chemiluminescence and the control group.

Meanwhile, a biological microchip capable of detecting an antigen for the antibody protein by immobilizing an antibody specific for adenosyl homocysteinase on a biological microchip and then reacting with a sample sample isolated from the subject. Using an automated microarray system has the advantage of analyzing a large number of samples in one analysis.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention more specifically, but the scope of the present invention is not limited by these examples.

Example  1: colorectal cancer specific diagnosis As a marker Of adenosyl homo moss stenaze  Sympathy

In order to select marker proteins showing specific changes in colorectal cancer, the present inventors performed DIOL two-dimensional electrophoresis on protein bodies of normal tissues without cancer cells at the same surgical site as the extracted cancer tissues after surgery for six colon cancer patients. Separated. The separated proteins were compared and analyzed by a decyder computer program, and protein spots with specific changes in cancer tissues were selected as compared to normal tissues ( FIG. 1 ). To identify the selected protein, it was treated with trypsin (10 ng / μl) for 12 hours at 37 ° C, separated into a number of peptide fragments, and the masses of each fragment and the daughter ions derived from the fragments were examined using a tandem mass spectrometer. Measured and analyzed using a computer analysis program. As a result, 20 tryptic peptides having the amino acid sequence set forth in SEQ ID NO: 2 to 21 have an amino acid sequence of SEQ ID NO: 1 and have an isoelectric point of 5.74 and a molecular weight of 48 kDa adenosyl homocyste. It was identified as a naase protein.

2 Tandem mass spectrometry results of the peptide of SEQ ID NO: 6, which is one of the peptides obtained by trypsin cleavage in the above process, show that the protein corresponding to the spot identified by the mass spectrometer is adenosyl homocyste. It can be confirmed that it is Inazase.

Example  2: Reverse transcription -Polymerase chain reaction ( RT - PCR )and Immunoblotting  Within the organization Of adenosyl homo moss stenaze  detection

MRNA and protein expression levels of adenosyl homocysteinase, a specific quantitative change in colorectal cancer, were examined in tissues by reverse transcriptase-polymerase chain reaction and immunoblotting. Reverse transcriptase-polymerase chain reaction was performed after extraction of mRNA from cancer tissues and normal tissues from 11 colon cancer patients.

Briefly, first, 1 μg of total RNA was synthesized using random hexamers (Random hexamer, Invitrogen) and M-MLV reverse transcriptase (Invitrogen) according to the manufacturer's instructions, followed by final synthesis. The volume of cDNA solution was adjusted to 50 μl. Forward and reverse primers ( SEQ ID NOs: 22 and 23 ), 10 mM dATP, dGTP for 1.5 mM MgCl ₂ , 20 pM of adenosyl homocysteinase, respectively, to a total of 20 μl with 2 μl of cDNA solution as a template , PCR was performed by adding dTTP and dCTP, 1 × PCR buffer, and 1 U polymerase (AmpliTaq Gold with GeneAmp polymerase, Applied Biosystems). At this time, PCR was performed by adding forward and reverse primers ( SEQ ID NOs: 24 and 25 ) to 2 to 10 pM of β-actin as an internal control for RNA quality. The PCR reaction was premodified at 95 ° C. for 10 minutes, followed by repeating the reaction for 25 seconds at 30 ° C. and 30 seconds at 55 ° C. for 25 seconds and finally amplifying at 72 ° C. for 30 seconds. After the reaction was completed, the PCR product was quantified by electrophoresis on 1.5 to 2% agarose gel to which ethidium bromide was added, and the gene was compared with the amount of β-actin PCR product. The expression level of was evaluated.

Immunoblotting for adenosyl homocysteinase was performed on six samples used in the DIGE secondary electrophoresis method of Example 1. Electrophoresis was performed using a 10% SDS polyacrylamide gel to separate proteins in the sample according to molecular weight. The isolated protein was transferred to a PVDF membrane in a solution containing 50 mM Tris-HCl, 130 mM glycine, 0.05% SDS and 12.5% methanol and the transferred membrane was 5% skim milk. The reaction was carried out for 1 hour in a TBST containing Tri-buffered saline Tween 20. After preparing the PVDF membrane in the same manner as above, the antibody of adenosyl homocysteinase (1: 500, Abnova) was reacted with PVDF membrane at 4 ° C. for 12 hours, washed three times with TBST, and then treated with mouse immunoglobulin. It was reacted with binding mouse IgG-HRP (1: 4000, GE Healthcare) for 1 hour at 25 ℃. Thereafter, the PVDF membrane was reacted with ECL (enhanced chemiluminescence, Amersham Bioscience, Inc.), and then subjected to X-ray film to measure the respective protein concentrations.

Figure 3 mRNA expression amount of adenosyl homocysteine RT-PCR results show a 2.5-fold increase in colorectal cancer tissue compared to normal tissue of colon cancer patients, FIG. In immunoblotting analysis using an antibody that selectively recognizes adenosyl homocysteine, the concentration of protein is increased three times in colorectal cancer tissues compared to normal tissues of colorectal cancer patients.

Example  3: in tissue using immunohistochemical staining Of adenosyl homo moss stenaze  detection

Immunohistochemical staining was performed to determine the expression level of adenosyl homocysteinase in cancer tissues and control tissues of colon cancer patients.

First, the collected tissues were placed in 4% paraformaldehyde (PFA) fixative and fixed at 4 ° C. for 24 hours, and then the fixed tissues were made into paraffin blocks using a microtome (Rotary microtome, LEICA, Germany) to 4 μm. Thick tissue sections were prepared. After the tissue sections slide prepared by attaching the tissue sections to the slide coated with triethoxy silane (Coated) for 1 hour at 60 ℃ pre-deparaffin to remove a portion of the paraffin (pre-deparaffin), Treatment with xylene and alcohol (Alcohol) completely removed paraffin. The tissue section slides were washed twice with 3 distilled water and allowed to stand for 10 minutes in phosphate buffered saline (PBS) and then diluted to adenosyl homocysteine at a ratio of 1:50 as the primary antibody. Antibody (Abnova) was added to the slide and reacted for 30 minutes at room temperature. ChemMate Envision Kit (DAKO) was used to inhibit the activity of peroxidase in tissues to prevent binding of nonspecific proteins. The tissue section slides were rinsed with S3006 washing solution in the kit, washed for 10 minutes with PBS solution, and then subjected to immunohistochemical staining at room temperature for 30 minutes by dropping the Envision / HRP solution. After the reaction was completed, the tissue section slide was rinsed again with S3006 washing solution and washed with PBS solution for 10 minutes, and then reacted with substrate-chromogen solution at room temperature for 20 minutes. The tissue sections slides of which the reaction was terminated were contrast-stained with hematoxylin (Hematoxylin, S3309, DACO), and then treated with alcohol, acetone, and xylene for 10 minutes, respectively, and observed with an optical microscope.

5 is As a result of immunohistochemical staining of cancer tissues and their surrounding normal tissues from colon cancer patients, adenosyl homocysteinase was stained strongly (dark brown) in cancer tissues of colon cancer patients. On the other hand, normal tissue cells around the cancer cells can be seen not to be stained by the anti-adenosyl homocysteinase antibody is blue.

Example  4: Immunoblotting  In plasma used Of adenosyl homo moss stenaze  detection

Take 30 μl of plasma from each of 10 colon cancer patients and 10 non-patient controls, and remove the six proteins, albumin, immunoglobulin A, immunoglobulin G, transferrin, trypsin inhibitor, and heptoglibin, which make up most of the plasma proteins. In order to perform the chromatography equipped with a MARS (multiple affinity removal system, Agilent) column. Protein compartments not bound to the column were tracked with ultraviolet absorbance at 280 nm and collected to remove proteins bound to the column. Thus, the plasma protein samples of the colorectal cancer patient group and the non-patient control group were prepared by concentrating only the proteins having a size of 3 kDa or more by collecting the partitions from which most of the six proteins were removed.

The plasma protein of the colon cancer patient group prepared above was subjected to electrophoresis using SDS polyacrylamide gel along with colorectal cancer tissue. 48 kDa-sized spots corresponding to the molecular weight of adenosyl homocysteinase were screened and treated with trypsin (10 ng / μl), and peptides obtained therefrom were analyzed by a tamdom mass spectrometer and they were amino acid sequence of SEQ ID NO: 1 . It was confirmed that the adenosyl homocysteine protein having ( Fig. 6 ). 10% SDS polyacrylamide gel after mixing 5 ㎍ of each of the plasma protein sample of the colon cancer patient group confirmed the presence of adenosyl homocysteinase and the buffer protein sample of the non-patient control group with 1% SDS The electrophoresis was performed using. The separated plasma proteins were transferred to PVDF membrane in a solution containing 50 mM Tris-HCl, 130 mM glycine, 0.05% SDS and 12.5% methanol, and 5% degreasing of the transferred membrane. The reaction was carried out in TBST (Tris-buffered saline Tween 20) containing milk powder for 1 hour.

After preparing two PVDF membranes, antibodies to adenosyl homocysteinase (1: 500, Abnova) were separately reacted with each PVDF membrane at 4 ° C. for 12 hours, washed three times with TBST, and then mice. Mouse IgG-HRP (1: 4000, GE Healthcare, Inc.) that binds to immunoglobulins was reacted at 25 ° C. for 1 hour. After the reaction was terminated, the PVDF membrane was reacted with ECL (enhanced chemiluminescence, Amersham Bioscience) and photosensitive on an X-ray film to determine the respective protein concentration.

FIG. 6 shows that the tissues and plasma of colorectal cancer patients were analyzed by SDS-polyacrylamide electrophoresis, respectively, followed by trypsin treatment with adenosyl homocysteinase spots, and peptides of SEQ ID NO: 6 from the peptides obtained therefrom. Shows the spectrum obtained by analyzing the tandem mass spectrometry (tandem mass spectrometry). The table at the bottom of the spectrum shows the mass values of the daughter ions that can be theoretically derived from the peptides of SEQ ID NO: 6.If the mass values of daughter ions from tandem mass spectrometry match the theoretical values, It is shown in bold font. While the spectrum is very clear in tissues, many daughter ions agree with the theory, while in plasma images of the spectrum are not clear, as a result of quantitative differences in adenosyl homocysteine, resulting in accurate peptide fragments and The mass of daughter ions derived from the fragments was identical in both colorectal cancer tissue and plasma, and was identified as adenosyl homocysteinase of SEQ ID NO: 1 .

FIG. 7 is a graph showing the results of immunoblotting with anti-adenosyl homocysteinase antibody and the antigen concentrations of the non-patient plasma and the colorectal cancer patient groups after electrophoresis analysis. 8 is a result showing the statistical processing of the antigen by the mean concentration measured by immune blotting of the FIG. In FIG. 7 , to reduce the deviation between the two gels subjected to immunoblotting, a control mixture of 10 non-patient controls with the same concentration and a colorectal cancer mixture of 10 patients with the same concentration of plasma cancer were added. The reliability of the immunoblotting results was increased. As shown in Figures 7 and 8 , adenosyl homocysteinase protein in the plasma of the colorectal cancer patient group shows the same expression pattern as in the colorectal cancer tissue, adenosine in the plasma of colorectal cancer patients compared to the plasma of the non-patient control group The expression of silmocysteine increased 1.88 fold.

FIG. 9 shows the concentration of the antigen measured in FIG. 7 in a receiver operating characteristic (ROC) graph. The ROC graph is a graph of all susceptibility / specificity pairs obtained by continuously changing judgment thresholds over the full range of observed data, primarily indicating the accuracy of the test (Zweig et al . , Clin . Chem . 39: 561-577, 1993). . AUC (area under the curve) of adenosyl homocysteine in the ROC graph is 0.859, indicating that the assay is both sensitive and accurate.

From the above results, the concentration of adenosyl homocysteinase protein was increased by measuring the concentration of each antigen using an antibody that selectively recognizes adenosyl homocysteinase in plasma and comparing it with the non-patient control group. The test subjects were confirmed to be diagnosed with colorectal cancer.

FIG. 1 shows the results of a single spot with increased expression in colorectal cancer tissues by labeling cancer tissues extracted from a group of colorectal cancer patients and normal tissues surrounding them with fluorescence, and comparing and analyzing them by two-dimensional electrophoresis. ,

2 is different Tryptic peptides from among SEQ ID NO: obtained by processing a single spot is colon cancer tissue-specific expression of an increase in the Fig. 1 with trypsin: obtained by analyzing a peptide in 6 to tandem mass spectrometry (tandem mass spectrometry) Spectral results,

3 is a mRNA of adenosyl homocysteinase using quantitative reverse transcription polymerase chain reaction (RT-PCR) targeting cancer tissues extracted from a group of colorectal cancer patients and normal tissues around them. Results of comparing and analyzing expression levels,

Figure 4 shows the immunoblot with anti-adenosyl homocysteine antibody after analyzing the cancer tissue and the surrounding normal tissue extracted from the colon cancer patient group by SDS-polyacrylamide gel electrophoresis Is the result of

FIG. 5 is a result of comparing and analyzing the expression levels of adenosyl homocysteinase using immunohistochemical staining in cancer tissues extracted from a group of colorectal cancer patients and surrounding normal tissues,

Figure 6 shows the tandem of the peptide of SEQ ID NO: 6 obtained by analyzing the cancer tissue and plasma of colorectal cancer patients respectively by SDS-polyacrylamide electrophoresis and then trypsinized each site corresponding to the size of adenosyl homocysteinase Is the result of mass spectrometry analysis,

FIG. 7 shows the results of immunoblotting with anti-adenosyl homocysteinase antibody after analysis of plasma of the non-patient control group and the colorectal cancer patient group by SDS-polyacrylamide electrophoresis, respectively. Is a graph of

8 will shown by statistical processing with the average of the adenosyl Homo upon antigen concentration in the stacking or the plasma in the plasma of colorectal cancer patients in a non-patient control group as determined by immune blot of Figure 7,

FIG. 9 shows the antigen concentration of the adenosyl homocysteinase obtained in FIG. 7 in a receiver operating characteristic (ROC) graph.

<110> Korea Institute of Science and Technology <120> PROTEIN MARKER ADENOSYLHOMOCYSTEINASE FOR COLON CANCER DIAGNOSIS          AND DIAGNOSIS KIT FOR COLON CANCER USING ANTIBODIES AGAINST THE          SAME <130> KC071182 <160> 25 <170> KopatentIn 1.71 <210> 1 <211> 432 <212> PRT <213> human adenosylhomocysteinase <400> 1 Met Ser Asp Lys Leu Pro Tyr Lys Val Ala Asp Ile Gly Leu Ala Ala   1 5 10 15 Trp Gly Arg Lys Ala Leu Asp Ile Ala Glu Asn Glu Met Pro Gly Leu              20 25 30 Met Arg Met Arg Glu Arg Tyr Ser Ala Ser Lys Pro Leu Lys Gly Ala          35 40 45 Arg Ile Ala Gly Cys Leu His Met Thr Val Glu Thr Ala Val Leu Ile      50 55 60 Glu Thr Leu Val Thr Leu Gly Ala Glu Val Gln Trp Ser Ser Cys Asn  65 70 75 80 Ile Phe Ser Thr Gln Asp His Ala Ala Ala Ala Ile Ala Lys Ala Gly                  85 90 95 Ile Pro Val Tyr Ala Trp Lys Gly Glu Thr Asp Glu Glu Tyr Leu Trp             100 105 110 Cys Ile Glu Gln Thr Leu Tyr Phe Lys Asp Gly Pro Leu Asn Met Ile         115 120 125 Leu Asp Asp Gly Gly Asp Leu Thr Asn Leu Ile His Thr Lys Tyr Pro     130 135 140 Gln Leu Leu Pro Gly Ile Arg Gly Ile Ser Glu Glu Thr Thr Thr Gly 145 150 155 160 Val His Asn Leu Tyr Lys Met Met Ala Asn Gly Ile Leu Lys Val Pro                 165 170 175 Ala Ile Asn Val Asn Asp Ser Val Thr Lys Ser Lys Phe Asp Asn Leu             180 185 190 Tyr Gly Cys Arg Glu Ser Leu Ile Asp Gly Ile Lys Arg Ala Thr Asp         195 200 205 Val Met Ile Ala Gly Lys Val Ala Val Val Ala Gly Tyr Gly Asp Val     210 215 220 Gly Lys Gly Cys Ala Gln Ala Leu Arg Gly Phe Gly Ala Arg Val Ile 225 230 235 240 Ile Thr Glu Ile Asp Pro Ile Asn Ala Leu Gln Ala Ala Met Glu Gly                 245 250 255 Tyr Glu Val Thr Thr Met Asp Glu Ala Cys Gln Glu Gly Asn Ile Phe             260 265 270 Val Thr Thr Thr Gly Cys Ile Asp Ile Ile Leu Gly Arg His Phe Glu         275 280 285 Gln Met Lys Asp Asp Ala Ile Val Cys Asn Ile Gly His Phe Asp Val     290 295 300 Glu Ile Asp Val Lys Trp Leu Asn Glu Asn Ala Val Glu Lys Val Asn 305 310 315 320 Ile Lys Pro Gln Val Asp Arg Tyr Arg Leu Lys Asn Gly Arg Arg Ile                 325 330 335 Ile Leu Leu Ala Glu Gly Arg Leu Val Asn Leu Gly Cys Ala Met Gly             340 345 350 His Pro Ser Phe Val Met Ser Asn Ser Phe Thr Asn Gln Val Met Ala         355 360 365 Gln Ile Glu Leu Trp Thr His Pro Asp Lys Tyr Pro Val Gly Val His     370 375 380 Phe Leu Pro Lys Lys Leu Asp Glu Ala Val Ala Glu Ala His Leu Gly 385 390 395 400 Lys Leu Asn Val Lys Leu Thr Lys Leu Thr Glu Lys Gln Ala Gln Tyr                 405 410 415 Leu Gly Met Ser Cys Asp Gly Pro Phe Lys Pro Asp His Tyr Arg Tyr             420 425 430 <210> 2 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> tryptic peptide <400> 2 Gly Ile Ser Glu Glu Thr Thr Thr Gly Val His Asn Leu Tyr Lys   1 5 10 15 <210> 3 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> tryptic peptide <400> 3 Met Met Ala Asn Gly Ile Leu Lys Val Pro Ala Ile Asn Val Asn Asp   1 5 10 15 Ser Val Thr Lys              20 <210> 4 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> tryptic peptide <400> 4 Val Ala Asp Ile Gly Leu Ala Ala Trp Gly Arg   1 5 10 <210> 5 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> tryptic peptide <400> 5 Ala Leu Asp Ile Ala Glu Asn Glu Met Pro Gly Leu Met Arg   1 5 10 <210> 6 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> tryptic peptide <400> 6 Val Ala Val Val Ala Gly Tyr Gly Asp Val Gly Lys   1 5 10 <210> 7 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> tryptic peptide <400> 7 Lys Leu Asp Glu Ala Val Ala Glu Ala His Leu Gly Lys   1 5 10 <210> 8 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> tryptic peptide <400> 8 Ser Lys Phe Asp Asn Leu Tyr Gly Cys Arg   1 5 10 <210> 9 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> tryptic peptide <400> 9 Leu Asp Glu Ala Val Ala Glu Ala His Leu Gly Lys   1 5 10 <210> 10 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> tryptic peptide <400> 10 Ala Thr Asp Val Met Ile Ala Gly Lys   1 5 <210> 11 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> tryptic peptide <400> 11 Tyr Pro Gln Leu Leu Pro Gly Ile Arg   1 5 <210> 12 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> tryptic peptide <400> 12 Lys Ala Leu Asp Ile Ala Glu Asn Glu Met Pro Gly Leu Met Arg   1 5 10 15 <210> 13 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> tryptic peptide <400> 13 Phe Asp Asn Leu Tyr Gly Cys Arg   1 5 <210> 14 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> tryptic peptide <400> 14 Arg Ile Ile Leu Leu Ala Glu Gly Arg   1 5 <210> 15 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> tryptic peptide <400> 15 Trp Leu Asn Glu Asn Ala Val Glu Lys   1 5 <210> 16 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> tryptic peptide <400> 16 Arg Ala Thr Asp Val Met Ile Ala Gly Lys   1 5 10 <210> 17 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> tryptic peptide <400> 17 Ala Gly Ile Pro Val Tyr Ala Trp Lys   1 5 <210> 18 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> tryptic peptide <400> 18 Gln Ala Gln Tyr Leu Gly Met Ser Cys Asp Gly Pro Phe Lys Pro Asp   1 5 10 15 His Tyr Arg Tyr              20 <210> 19 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> tryptic peptide <400> 19 Val Pro Ala Ile Asn Val Asn Asp Ser Val Thr Lys   1 5 10 <210> 20 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> tryptic peptide <400> 20 Val Asn Ile Lys Pro Gln Val Asp Arg   1 5 <210> 21 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> tryptic peptide <400> 21 Asp Gly Pro Leu Asn Met Ile Leu Asp Asp Gly Gly Asp Leu Thr Asn   1 5 10 15 Leu Ile His Thr Lys              20 <210> 22 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> forward primer specific for adenosylhomocysteinase <400> 22 accctgggtg ctgaggtg 18 <210> 23 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer specific for adenosylhomocysteinase <400> 23 gtggatgagg ttggtgaggt 20 <210> 24 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> forward primer specific for beta-actin <400> 24 acagagtact tgcgctcagg ag 22 <210> 25 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> reverse primer specific for beta-actin <400> 25 tgtatgcctc tggtcgtacc ac 22  

Claims (12)

A colorectal cancer diagnostic kit comprising an antibody specific for Adenosylhomocysteinase having an amino acid sequence of SEQ ID NO: 1 , which is a colorectal cancer diagnostic protein marker. The method of claim 1, Colorectal cancer diagnostic kit, characterized in that the antibody is a polyclonal antibody or a monoclonal antibody. The method of claim 2, Secondary antibody conjugates to which a label that is developed by reaction with a substrate is conjugated; A color substrate solution to be colored and reacted with the label; Washing liquid; And a colorectal cancer diagnostic kit further comprising an enzyme reaction stopper. The method of claim 3, Diagnostic kit characterized in that the label of the secondary antibody conjugate is selected from the group consisting of horseradish peroxidase (HRP), basic alkaline phosphatase, colloid gold, fluorescent (fluorescein) and dye (dye) . The method of claim 3, Group consisting of TMB (3,3 ', 5,5'-tetramethyl benzidine), ABTS [2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)] and OPD (o-phenylenediamine) The diagnostic kit, characterized in that selected from. A method for detecting adenosyl homocysteinase, a protein marker for colorectal cancer diagnosis, using an antigen-antibody binding reaction from a sample. The method of claim 6, 1) coating the sample and the protein of the control to the fixture; 2) performing an antigen-antibody binding reaction by adding an antibody specific for adenosyl homocysteinase to the fixture; 3) detecting the antigen-antibody binding reactant produced through the antigen-antibody binding reaction using a secondary antibody conjugate and a color substrate solution; And 4) comparing the detection results for the sample and the control. The method of claim 7, wherein The sample of step 1) is characterized in that the serum or plasma. The method of claim 7, wherein The fixture of step 1) is selected from the group consisting of nitrocellulose membranes, PVDF membranes, well plates synthesized from polyvinyl or polystyrene resins and slide glass of glass. The method of claim 9, The antigen-antibody binding reaction of step 2) is performed by enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), sandwich assay, western blotting, immunoprecipitation method, immune tissue Method selected from the group consisting of chemical staining (immunohistochemical staining), fluorescence immunoassay, enzyme substrate coloration, antigen-antibody aggregation method. The method of claim 7, wherein The label of the secondary antibody conjugate of step 3) is selected from the group consisting of HRP, basic dephosphatase, colloidal gold, fluorescent substance and pigment. The method of claim 7, wherein The chromogenic substrate of step 3) is selected from the group consisting of TMB, ABTS and OPD.

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