KR101413689B1 - Monoclonal Antibody Specifically Binding to Human Tescalcin Protein and Its Use - Google Patents

Abstract

The present invention provides a hybridoma cell having a deposit number of KCTC 11790BP, which is characterized by producing a monoclonal antibody to a human TESC protein, a monoclonal antibody against a human TESC protein produced by the hybridoma cell, A composition for diagnosing cancer comprising an antibody as an active ingredient, a cancer diagnostic kit, and a protein chip for cancer diagnosis. Since the monoclonal antibody to the human TESC of the present invention has a specific binding ability of high affinity to human TESC protein, it can be useful for the diagnosis of cancer and is also very useful for the production of a cancer diagnostic kit or a protein chip.

Description

Monoclonal Antibodies Specifically Binding to Human TESC Proteins and Their Use [

The present invention relates to the diagnostic use of a monoclonal antibody that specifically binds to a human TESC (Tescalcin) protein. More particularly, the present invention relates to a monoclonal antibody against human TESC, a hybridoma cell line that produces the antibody, a cancer diagnostic composition comprising the monoclonal antibody as an effective ingredient, a cancer diagnostic kit, and a cancer diagnostic diagnostic protein chip.

Cancer is a disease caused by abnormal cell division and abnormal division and cell division, resulting in about 6 million patients worldwide and about 1.4 million patients in the US every year. Approximately 600,000 patients die and are the second leading cause of death following heart disease. Current research interests of academia, bio-industry, and governmental organizations are currently being studied, and more rapid and effective development of anti-cancer treatment methods is being actively pursued through studies on the identification of carcinogenic factors and mechanisms of cancer progression.

The types of cancer mainly include carcinomas (85% of total cancer) arising from the epithelium of the tissue and sarcomas (6% of total cancer) produced by the connective tissues such as muscles, bones, Lymphoma (5% of all cancers) and brain tumors are found in the tissues where leukocytes are formed, such as bone marrow, and the like. .

According to statistics of the Central Cancer Registry in Korea, the average number of cancer cases in 2003-2005 was 132,941, 72,952 in males and 59,989 in females. The average annual incidence was 274.1 cases per 100,000 population, with 300.0 cases for men and 248.2 cases for women. In 2003-2005, the most common cancer was stomach cancer, followed by lung cancer, colorectal cancer, liver cancer, thyroid cancer, breast cancer, and cervical cancer. In the case of men, gastric cancer occurred in the order of lung cancer, liver cancer, colon cancer, and prostate cancer in the order of breast cancer, thyroid cancer, stomach cancer, colon cancer and lung cancer. A total of 65,909 people died of cancer in 2006, of which 27.0% died from cancer. The most common cancer deaths in 2006 were lung cancer (14.4%), followed by liver cancer (16.6%), stomach cancer (16.4%), colorectal cancer (9.5%) and pancreatic cancer (Statistical Yearbook of Cause of Death in 2006, National Statistical Office). Cancer with the highest mortality rate compared to 1996 is lung cancer, followed by colon cancer, prostate cancer, and pancreatic cancer. On the other hand, the cancer with the highest mortality rate was stomach cancer, followed by uterine cancer. As a result, the number of cancer deaths and the number of deaths are increasing year by year, and most cancer is not good and many people die from cancer.

Methods of diagnosing cancer in modern medicine are variously performed such as radiography, histopathology, ultrasonography, and blood test. The most easy to collect and manage, and easy to test, is the screening test for cancer using blood. This is to prevent or detect cancer early, and if it is already discovered and treated, It is used for the purpose. Many types of cancer markers are now known, but many types of cancer markers may be detected or increased without cancer, which is not sufficient for cancer diagnosis. In addition, the types of cancer markers vary greatly depending on the type of cancer, and there is no cancer marker that can detect all cancers that occur in humans with one test, and there are many cancers that do not have cancer markers. For this reason, research is needed to find more specific and sensitive cancer markers for cancer. Several types of cancer marker candidate proteins and genes have been reported by research on genomics and proteomics.

The inventors of the present invention have applied for a patent by finding a series of genes that are specifically overexpressed in cancer and suggesting that these genes can be utilized as diagnostic markers of cancer in the previous research (Patent Application No. 10-2008- 0120096). As a result of the above-described studies by the present inventors, TESC has been disclosed as a gene that can be utilized as a marker of cancer.

The TESC gene (NM_017899) is located at chromosome 12q24.22. The transcriptionally expressed protein (214 amino acids, 25 Kda) interacts with the sodium / hydrogen exchanger (NHE1) Encodes EF-hand calcium binding protein. TESC is known to be expressed only in testes that are not ovaries in the critical period when testes and ovaries are determined. Currently, TESC has not been reported with diseases involving cancer, nor is there a significant lack of research on molecular biology.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The inventors of the present invention confirmed that TESC (Tescalcin) gene is overexpressed in cancer cells more specifically than normal cells and confirmed its usefulness as a cancer marker. In a biological sample, a monoclonal antibody capable of specifically detecting TESC protein We have tried diligently to develop antibodies. As a result, the present inventors completed the present invention by successfully producing a monoclonal antibody-producing hybridoma cell line that specifically binds to TESC protein through the cell fusion method using the over-expressing recombinant TESC protein and isolating and purifying it .

Accordingly, it is an object of the present invention to provide a hybridoma cell line having the accession number KCTC 11790BP for producing a monoclonal antibody against a human TESC protein.

Another object of the present invention is to provide monoclonal antibodies against human TESC proteins produced by hybridoma cells with accession number KCTC 11790BP.

It is still another object of the present invention to provide a cancer-detecting composition comprising the monoclonal antibody as an active ingredient.

It is still another object of the present invention to provide a cancer diagnostic kit comprising the monoclonal antibody as an active ingredient.

It is still another object of the present invention to provide a cancer chip for cancer diagnosis comprising the monoclonal antibody as an active ingredient.

The objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, there is provided a hybridoma cell line, wherein the accession number is KCTC 11790BP, which is characterized by producing a monoclonal antibody against a human TESC protein.

According to another aspect of the present invention, the present invention provides monoclonal antibodies against human TESC proteins produced by hybridoma cells with accession number KCTC 11790BP.

The monoclonal antibody of the present invention specifically binds to human TESC (Tescalcin) protein with high affinity.

According to a preferred embodiment of the present invention, the monoclonal antibody of the present invention specifically binds to the 51-100th amino acid sequence region of the TESC protein of SEQ ID NO: 13.

The monoclonal antibody of the present invention is an antibody produced by a hybridoma cell line deposited with KCTC 11790BP on Oct. 20, 2010 in KCTC (Korean Collection for Type Cultures).

As used herein, the term " antibody "refers to a specific antibody to a human TESC (Tescalcin) protein, including the complete antibody form as well as the antigen binding fragment of the antibody molecule.

A complete antibody is a structure having two full-length light chains and two full-length heavy chains, each light chain linked by a disulfide bond with a heavy chain. The heavy chain constant region has gamma (gamma), mu (mu), alpha (alpha), delta (delta) and epsilon (epsilon) types and subclasses gamma 1 (gamma 1), gamma 2 ), Gamma 4 (gamma 4), alpha 1 (alpha 1) and alpha 2 (alpha 2). The constant region of the light chain has the kappa and lambda types (Cellular and Molecular Immunology, Wonsiewicz, MJ, Ed., Chapter 45, pp. 41-50, WB Saunders Co. Philadelphia, PA (1991); Nisonoff, A., Introduction to Molecular Immunology, 2nd Ed., Chapter 4, pp. 45-65, Sinauer Associates, Inc., Sunderland, MA (1984)).

An antigen binding fragment of an antibody molecule means a fragment having an antigen binding function and includes Fab, F (ab ') 2, F (ab') 2, Fv and the like. Fabs in the antibody fragment have one antigen-binding site in a structure having a variable region of a light chain and a heavy chain, a constant region of a light chain, and a first constant region (CH1) of a heavy chain. Fab 'differs from Fab in that it has a hinge region that contains at least one cysteine residue at the C-terminus of the heavy chain CH1 domain. The F (ab ') 2 antibody is produced when the cysteine residue of the hinge region of the Fab' forms a disulfide bond. Recombinant techniques for generating Fv fragments with minimal antibody fragments having only heavy chain variable regions and light chain variable regions are described in PCT International Publication Nos. WO88 / 10649, WO 88/106630, WO 88/07085, WO 88/07086 and WO 88/09344. The double-chain Fv is a non-covalent bond, and the variable region of the heavy chain and the light chain variable region are connected to each other. The single-chain Fv generally shares the variable region of the heavy chain and the variable region of the short chain through the peptide linker Or directly connected at the C-terminus to form a dimer-like structure like the double-stranded Fv. Such an antibody fragment can be obtained using a protein hydrolyzing enzyme (for example, a Fab can be obtained by restriction of the whole antibody to papain, and F (ab ') 2 fragment can be obtained by cleavage with pepsin) Can be produced through recombinant DNA technology.

In the present invention, the antibody is preferably in the form of a Fab or a complete antibody form. In addition, the heavy chain constant region may be selected from any one of gamma (gamma), mu (mu), alpha (alpha), delta (delta) or epsilon (epsilon). Preferably, the constant region is gamma 1 (IgGl), gamma 3 (IgG3) and gamma 4 (IgG4), and most preferably is gamma 1 (IgGl) isotype. The light chain constant region may be a kappa or lambda form, preferably a kappa form. Thus, it is understood that the preferred antibody of the present invention is an Fab or IgG1 form having a kappa light chain and a gamma 1 (gamma 1) heavy chain.

As used herein, the term " heavy chain " refers to a variable region domain VH comprising an amino acid sequence having a sufficient variable region sequence to confer specificity to an antigen and a full length heavy chain comprising three constant region domains CH1, CH2 and CH3, It means all fragments.

As used herein, the term " light chain " refers to both the full length light chain and its fragments, including the variable region domain VL and the constant region domain CL, including the amino acid sequence with sufficient variable region sequence to confer specificity to the antigen.

As used herein, the term " monoclonal antibody " refers to a single molecule composition of an antibody molecule obtained in a substantially identical population of antibodies, wherein the monoclonal antibody exhibits a single binding specificity and affinity for a particular epitope.

Monoclonal antibodies of the invention can be obtained from hybridoma cells produced by cell fusion methods known in the art. Generally, hybridoma cells that secrete monoclonal antibodies are made by fusing immune cells and cancer cell lines from immunologically appropriate host animals, such as mice injected with antigen proteins. The fusion of these two cells is accomplished by fusion using a method known in the art using polyethyleneglycol, and the antibody producing cells are proliferated by a standard culture method. After subcloning by limited dilution to obtain a uniform cell population, hybridoma cells capable of producing an antigen-specific antibody are cultured in vitro or in vivo.

Examples of myeloma cells used for cell fusion include mouse-derived p3 / x63-Ag8, p3-U1, NS-1, MPC-11, SP-2/0, F0, P3x63 Ag8, V653, S194, A variety of cell lines can be used. The cell line used in the specific embodiment of the present invention is myeloma cell NS-1.

The monoclonal antibody produced by the above hybridoma cells can be used in an unpurified state and can be further purified by various conventional methods such as dialysis, salt precipitation, ion exchange chromatography, size exclusion chromatography, affinity chromatography And the like.

(RIA), enzyme immunoassay (ELISA), immunofluorescence, Western blotting, and the like, in order to select a single clone that selectively recognizes TESC protein. And flow cytometry can be used, but the present invention is not limited thereto. According to one specific embodiment of the present invention, a single clone is selected by enzyme immunoassay (ELISA).

According to another aspect of the present invention, there is provided a cancer diagnostic composition comprising the monoclonal antibody against the human TESC (Tescalcin) protein as an active ingredient.

Monoclonal antibodies that specifically bind to human TESC proteins of the invention can be used to diagnose cancer by applying to biological samples.

"Cancer" to be diagnosed in the present invention includes, for example, lung cancer, non-small cell lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, skin or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, gastric cancer, The present invention relates to a method of treating cancer in a patient suffering from cancer, colon cancer, colorectal cancer, breast cancer, fallopian tube carcinoma, endometrial carcinoma, cervical cancer, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, Cancer of the central nervous system (CNS), primary central nervous system (CNS) neoplasia, renal cell carcinoma, renal pelvic carcinoma, central nervous system (CNS) tumor, prostate cancer, prostate cancer, soft tissue sarcoma, Lymphoma, spinal cord tumor, brainstem glioma, pituitary adenoma, and the like.

The term " biological sample " as used herein refers to tissues, cells, whole blood, serum, plasma, tissue autopsy samples (brain, skin, lymph nodes, spinal cord etc.), cell culture supernatant, ruptured eukaryotic cells, But is not limited thereto. Information on the occurrence of cancer can be obtained by detecting the formation of an antigen-antibody complex by reacting these biological samples with the antibody of the present invention in a manipulated or untreated state.

The formation of the above-mentioned antigen-antibody complex can be performed by a colorimetric method, an electrochemical method, a fluorimetric method, a luminometry method, a particle counting method, a visual assessment method, Can be detected by the scintillation counting method.

As used herein, " detection " is intended to detect an antigen-antibody complex and may be carried out using various markers. Specific examples of the label include an enzyme, a minerals, a ligand, a luminescent material, a microparticle or a radioactive isotope. Examples of the enzyme used as the detection label include acetylcholinesterase, alkaline phosphatase,? -D-galactosidase, horseradish peroxidase,? -Lactamase, and the like, Eu ^{3 +} , Eu ^{3 +} chelate, or cryptate. Ligands include biotin derivatives and the like. Emitters include acridinium esters and isoluminol derivatives. Fine particles include colloid Gold and colored latex, and the radioactive isotopes include ⁵⁷ Co, ³ H, ¹²⁵ I and ¹²⁵ I-Bonton Hunter reagents and the like.

Preferably, the antigen-antibody complexes can be detected using enzyme immunoassay (ELISA). Enzyme immunoassay (ELISA) includes direct ELISA using a labeled antibody that recognizes an antigen attached to a solid support, indirect ELISA using a labeled secondary antibody that recognizes the capture antibody in a complex of an antibody recognizing the antigen attached to a solid support , A direct sandwich ELISA using another labeled antibody that recognizes the antigen in the complex of the antibody and the antigen attached to the solid support, and the other antibody recognizing the antigen in the complex of the antibody and the antigen attached to the solid support. And indirect sandwich ELISA using labeled secondary antibodies recognizing the antibody. The antibody of the present invention can have a detection label, and when it does not have a detection label, it can be identified by treating another antibody capable of capturing the antibody of the present invention and having a detection label.

According to another aspect of the present invention, the present invention provides a cancer diagnostic kit comprising the monoclonal antibody against the human TESC as an active ingredient.

The cancer diagnostic kit of the present invention may contain a substrate, a suitable buffer solution, a secondary antibody labeled with a chromogenic enzyme or a fluorescent substance, a chromogenic substrate and the like for immunological detection of the antibody. The substrate may be a nitrocellulose membrane, a 96-well plate synthesized with a polyvinyl resin, a 96-well plate synthesized with a polystyrene resin, a glass slide glass or the like, and the coloring enzyme may be a peroxidase, Alkaline phosphatase may be used, and fluorescent materials such as FITC and RITC may be used. The coloring substrate solution may be ABTS (2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid ) Or OPD (o-phenylenediamine), TMB (tetramethylbenzidine) can be used.

According to still another aspect of the present invention, there is provided a cancer chip for diagnostic cancer comprising a monoclonal antibody against human TESC as an active ingredient.

The monoclonal antibody is arranged at a predetermined position on the substrate and is immobilized at a high density. A method of analyzing a sample using a protein chip is a method of separating a protein from a sample, hybridizing the separated protein with a protein chip to form an antigen-antibody complex, reading the protein, It is possible to confirm whether the cancer has developed or not.

The advantages of the present invention are summarized as follows.

(I) a monoclonal antibody against a TESC (Tescalcin) protein that is overexpressed in human cancer, and a hybridoma cell line that produces this antibody.

(Ii) The monoclonal antibody against human TESC of the present invention has a specific binding ability of high affinity to human TESC protein, and thus can be useful for diagnosis of cancer.

(Iii) Monoclonal antibodies against human TESC of the present invention are very useful for the production of cancer diagnostic kits or protein chips.

As described in detail above, the present invention provides a hybridoma cell having a deposit number of KCTC 11790BP, which is characterized by producing a monoclonal antibody to a human TESC protein, a human TESC protein produced by the hybridoma cell A monoclonal antibody for cancer, a cancer diagnostic composition comprising the monoclonal antibody as an active ingredient, a cancer diagnostic kit, and a protein chip for cancer diagnosis. Since the monoclonal antibody to the human TESC of the present invention has a specific binding ability of high affinity to human TESC protein, it can be useful for the diagnosis of cancer and is also very useful for the production of a cancer diagnostic kit or a protein chip.

FIG. 1 shows the result of electrophoresis after confirming the expression of recombinant proteins of His-TESC after transforming E. coli with pET22b-TESC and purifying the expressed protein using an affinity column (Ni-NTA column).
FIG. 2 shows the results of enzyme immunoassay (ELISA) in which the antibody produced in the fusion cell line obtained after the injection of the His-TESC recombinant protein as an antigen was tested for the reactivity with the recombinant antigen expressed from pET22b-TESC.
FIG. 3 shows the results of electrophoresis of the expressed TESC protein and Western blotting using the culture supernatant of a TESC 69-18-11 clone.
FIG. 4 shows the result of determining the isotype by selecting a clone of TESC 69-18-11 which was confirmed to secrete a TESC-specific monoclonal antibody. TESC 69-18-11 clones secrete IgG1 isotype-type antibodies.
FIG. 5 is a graph showing the results of immunohistochemical analysis of TESC-expressing cell line lysates by electrophoresis and separating protein vials. Using purified antibodies (monoclonal antibodies) of TESC 69-18-11 clones and rabbits (polyclonal antibodies) The results were compared by Western blotting.
FIG. 6 shows Western blotting of tissue samples of colon cancer cell line and colon cancer patients using the monoclonal antibody against human TESC of the present invention.
FIG. 7A is a schematic view showing a region where TESC protein is divided into four parts in order to epitope map the TESC protein region bound by TESC 69-18-11 clone monoclonal antibody. FIG.
FIG. 7B is a diagram showing the construction of a pET22b expression vector in which four portions of TESC protein are cloned. FIG.
Figure 7c shows that the TESC 69-18-11 clone monoclonal antibody does not bind to 5.5kDa (1-150bp, 1-50aa). 11.1 kDa (1-300 bp, 1-100 aa).

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Example  One: TESC of cDNA  Expression vector production

ATGGGCGCTGCCCACTCC-3 '(SEQ ID No. 1) and 5'-GTGGCAGAGGGCCATGGTT-3' (SEQ ID NO: 2) as a template and a cDNA library of cells expressing the TESC gene (GenBank Accession Number: NM_017899) ) Was performed for 35 cycles (1 cycle: 94 ° C. for 40 seconds, 57 ° C. for 40 seconds, and 72 ° C. for 1 minute) for TESC by polymerase chain reaction (RT-PCR). Then, the TESC gene of the obtained PCR product of 645 bp was recovered, cloned into a pTOPO vector, and the base sequence was confirmed to confirm that it was a TESC gene. The TESC gene cloned into the pTOPO vector was amplified using the primer pair of 5'-CGGGATCCGATGGGCGCTGCCCACTCC-3 '(SEQ ID No. 3) and 5'-CGAAGCTTGTGGCAGAGGGCCATGGTT-3' (SEQ ID No. 4) To 214th amino acid was amplified by PCR and then cut with Bam HI and Hind III and ligated to the E. coli expression vector pET22b vector.

Example 2 Expression Analysis of Recombinant TESC in E. coli Cells

The pET22b-TESC plasmid containing the TESC gene containing the 1-214 amino acid sequence of the TESC protein was transformed into BL21 DE3 Escherichia coli to confirm that the TESC protein was normally expressed. The transformant was cultured in a 500 mL Erlenmeyer flask with 1 mM IPTG induction for 4 hours to induce TESC protein expression. Escherichia coli was harvested by centrifugation, lysis buffer was added, cells were disrupted with a sonicator, and a supernatant was obtained by centrifugation. The supernatant was treated with Ni-NTA column to separate and purify the His-TESC protein, and the expression and purification of His-TESC protein was analyzed by SDS-PAGE.

Example  3: Antigen immunization of mice

In order to obtain immunized mice necessary for the development of hybridoma cell lines, 50 μg of recombinant His-TESC fusion protein and the same amount of MPS (MPL-TDM Adjuvant) were mixed and incubated at 40-45 ° C. for 30 minutes And injected intraperitoneally into Balb / c mice 4-6 weeks old. After 2 weeks, 25 μg of the recombinant His-TESC fusion protein and the same amount of the immunoadjuvant antigen (MPL + TDM Adjuvant) were mixed well and injected into the abdominal cavity of the mice. After 4 - 5 days, a small amount of blood was drawn from the mouse eye vein to confirm titer, and TESC protein was injected into the abdominal cavity 3 days before the cell fusion experiment.

Example 4: Preparation of hybridoma cells by cell fusion

In order to carry out the cell fusion required for the preparation of hybridoma cells, 10 ⁷ splenocytes and 10 ⁶ myeloma cells of mice obtained by injecting the prepared immune-mediated His-TESC fusion protein were inoculated into a 50 ml test tube Gathered. P3 / NS / 1-Ag4-1 (NS-1) was used as the parent cell of cell fusion. The cells were maintained at a maximum density of 5 × 10 ⁵ / ㎖ in DMEM medium containing 10% FBS.

The mice immunized in Example 3 were removed from the cervical vertebra, and the spleen located on the left side of the torso was taken out and finely grinded into a mesh to prepare a suspension. The obtained splenocytes were centrifuged in a 50 ml centrifuge tube, Was repeated twice to sufficiently wash the spleen cells. After resuspending splenocytes and NS-1 cells in 10 ml each, count the number of cells and mix them in a 50 ml centrifuge tube so that the ratio of splenocytes and NS-1 cells is 10: 1. And precipitated by centrifugation. The centrifuged precipitate was dispersed by tapping with a finger, maintained at 37 ° C for 1 minute, and then 1 ml of 45% polyethylene glycol (PEG) fusogen in Hans's buffer (HBSS) And then slightly shaken for another minute. Then, 5 ml of culture medium (DMEM) was added over 1 minute, and DMEM was gradually added while shaking until 30 ml. After the suspension was centrifuged again, the cell precipitate was resuspended in a separation medium (HAT medium) at a rate of 1 - 2 × 10 ⁵ / ml, 0.2 ml was added to each well of a 96-well microtiter plate, and the cells were incubated at 37 ° C. in a humidified carbon dioxide incubator Lt; / RTI >

Example  5: Producing Monoclonal Antibodies Hybridoma  Cell sorting

Among the hybridoma cell groups prepared in Example 4, screening was carried out by ELISA using a TESC antigen in order to select hybridoma cells that specifically react only with TESC antigen. Each 100 μl (1 μg / ml) of TESC antigen was added to the microtiter plate and attached to the plate surface. Unreacted antigen was washed with phosphate buffer solution-Tween 20 (PBST) (Bovine Serum Albumin). After incubation at room temperature for one hour, the cells were washed with PBST solution. 100 μl of the culture solution of hybridoma cells was added to each well, reacted at room temperature for 2 hours, and then washed with PBST solution to remove unreacted culture. Mouse IgG-horseradish peroxidase (goat anti-mouse IgG-HRP) was added thereto, followed by reaction at room temperature for 1 hour, followed by washing with PBST solution. Subsequently, a substrate solution (OPD) of peroxidase was added and reacted, and the degree of the reaction was measured by absorbance at 490 nm. As a result, hybridoma cell lines secreting an antibody having a specific binding specificity to TESC antigen were selected, and a hybridoma cell line that specifically reacted with TESC antigen was selected through several repeated experiments, Lt; / RTI > to yield a monoclonal antibody. At this time, one clone, TESC 69-18-11, was finally obtained. The hybridoma cell line TESC 69-18-11 was deposited with KCTC 11790BP on October 20, 2010 in KCTC (Korean Collection for Type Cultures, KCTC), 52, Eun-dong, Yuseong-gu, Daejeon,

These clones were cloned and frozen, and the supernatant was assayed for enzyme activity by enzyme immunoassay, and the subclass type was confirmed. The monoclonal TESC 69-18-11 was found to be IgG1 and showed a high specific binding to TESC. TESC 69-18-11 clones were injected into the mice to prepare multiple solutions.

Example  6: Mass production of monoclonal antibodies

In order to mass-produce the monoclonal antibody from the continuously hybridizing hybridoma cells obtained in Example 5, 0.5 ml of Incomplete Freund Adjuvant was intraperitoneally injected into a mouse (Balb / c) . One week later, each hybridoma was injected into the abdominal cavity of the experimental rats at 2 × 10 ⁶ per mouse, and multiple liquids were collected from the experimental rats whose abdominal cavity was swollen after 7 to 10 days. To obtain high concentration hybridoma cells contained in this multiple liquid, the plural liquids are centrifuged at 10,000 rpm to take the supernatant, and some of them are stored at -70 ° C. until they are purified, and some of them are subjected to affinity column G agarose column).

Example  7: Specificity test of monoclonal antibody

The specificity of the monoclonal antibody obtained in Example 6 was compared with that of the polyclonal antibody (FIG. 5). First, 30 μg of the cell lysate was subjected to SDS-PAGE electrophoresis to separate the proteins. After transferring the proteins to the PVDF membrane, the monoclonal antibody and the polyclonal antibody against TESC were added to 0.1 μg / ml and allowed to react at room temperature for 2 hours. HRP-immobilized mouse secondary antibody was subjected to secondary reaction, and sensitized to X-ray film to compare the expression of TESC protein. As can be seen from the results of FIG. 5, the monoclonal antibody reacted at the site of the original TESC protein (25 kD), but in the case of the polyclonal antibody, the monoclonal antibody also reacted with the protein at other sites than the TESC protein site.

Example  8: Use of Monoclonal Antibodies in Colorectal Cancer Cell Lines and Colon Cancer Tissue TESC  Protein expression study

The expression of TESC protein was analyzed in colon cancer cell line and colon cancer tissue using monoclonal antibody TESC 69-18-11. First, 30 μg of the cell line and tissue extract were subjected to SDS-PAGE electrophoresis to separate proteins, and the proteins were transferred to a PVDF membrane. Monoclonal antibody against TESC was added to the PVDF membrane at a concentration of 0.1 μg / ml And allowed to react at room temperature for 2 hours. HRP-immobilized mouse secondary antibody was subjected to secondary reaction, and sensitized to X-ray film to compare the expression of TESC protein. TESC protein was expressed in the colon cancer cell line, and was found to be overexpressed in cancerous tissues of colon cancer patients (FIG. 6).

Example  9: of monoclonal antibody Epitope Mapping  ( Epitope mapping )

Epitope mapping was performed to determine which part of the TESC protein the monoclonal antibody reacted with. The TESC protein was digested in four fractions, 5.5 kDa (1-150 bp, 1-50 aa), 11.1 kDa (1-300 bp, 1-100 aa), 16.5 kDa (1-450 bp, 1-150 aa), 25 kDa 214aa). The recombinant proteins were prepared as follows, and the binding sites of the antibodies were identified. Expression of the TESC partial recombinant protein was performed by the following method. First, 645 bp of the TESC coding sequence was divided into 4 equal parts and cloned into pET22b expression vector with a length of 1-150 bp, 1-300 bp, 1-450 bp, 1-645 bp (see FIGS. 7A and 7B).

Bam HI and Hind III were used as restriction enzymes, and the following primer pairs were prepared and polymerase chain reaction (RT-PCR) was carried out. The obtained PCR products were recovered and cloned:

1-150 bp: primer pair 5'-CGGGATCCGATGGGCGCTGCCCACTCC-3 '(SEQ ID No. 5), 5'-CGAAGCTTGACATTGTTCAAGTTCTCCT-3' (SEQ ID No. 6)

1-300 bp: primer pair 5'-CGGGATCCGATGGGCGCTGCCCACTCC-3 '(SEQ ID No. 7), 5'-CGAAGCTTGGTGTCGATGGGCCGGAAGT-3' (SEQ ID No. 8)

1-450 bp: primer pair 5'-CGGGATCCGATGGGCGCTGCCCACTCC-3 '(SEQ ID No. 9), 5'-CGAAGCTTCTCGATGTGAGGGTTTCCCG-3' (SEQ ID No. 10)

1-645bp: primer pair 5'-CGGGATCCGATGGGCGCTGCCCACTCC-3 '(SEQ ID No. 11), 5'-CGAAGCTTGTGGCAGAGGGCCATGGTT-3' (SEQ ID No. 12)

Using the above primer pair, DNA encoding a protein coding for each site was amplified by 35 cycles of PCR (1 cycle: 94 ° C for 40 seconds, 57 ° C for 40 seconds, 72 ° C for 1 minute) And cloned into the pET22b expression vector using the enzyme (see Fig. 7B). The cloned pET22b-TESC vector was transformed into BL21 competent cells. The grown colonies were cultured in LB culture medium and treated with 1 mM IPTG at 37 DEG C, followed by incubation for 4 hours to express the protein. For each TESC partial protein expressed, Western blotting was performed using the monoclonal antibody of the present invention. As a result, as shown in FIG. 7C, the monoclonal antibody TESC 69-18-11 of the present invention was found to bind at 151 to 300 bp (51-100 aa) of the TESC protein.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Korea Biotechnology Research Institute KCTC11790 20101020

<110> Korea Research Institute of Bioscience and Biotechnology <120> Monoclonal Antibody Specific Binding to Human Tescalin          Protein and Its Use <160> 13 <170> Kopatentin 1.71 <210> 1 <211> 18 <212> DNA <213> Homo sapiens <400> 1 atgggcgctg cccactcc 18 <210> 2 <211> 19 <212> DNA <213> Homo sapiens <400> 2 gtggcagagg gccatggtt 19 <210> 3 <211> 27 <212> DNA <213> Homo sapiens <400> 3 cgggatccga tgggcgctgc ccactcc 27 <210> 4 <211> 27 <212> DNA <213> Homo sapiens <400> 4 cgaagcttgt ggcagagggc catggtt 27 <210> 5 <211> 27 <212> DNA <213> Homo sapiens <400> 5 cgggatccga tgggcgctgc ccactcc 27 <210> 6 <211> 28 <212> DNA <213> Homo sapiens <400> 6 cgaagcttga cattgttcaa gttctcct 28 <210> 7 <211> 27 <212> DNA <213> Homo sapiens <400> 7 cgggatccga tgggcgctgc ccactcc 27 <210> 8 <211> 28 <212> DNA <213> Homo sapiens <400> 8 cgaagcttgg tgtcgatggg ccggaagt 28 <210> 9 <211> 27 <212> DNA <213> Homo sapiens <400> 9 cgggatccga tgggcgctgc ccactcc 27 <210> 10 <211> 28 <212> DNA <213> Homo sapiens <400> 10 cgaagcttct cgatgtgagg gtttcccg 28 <210> 11 <211> 27 <212> DNA <213> Homo sapiens <400> 11 cgggatccga tgggcgctgc ccactcc 27 <210> 12 <211> 27 <212> DNA <213> Homo sapiens <400> 12 cgaagcttgt ggcagagggc catggtt 27 <210> 13 <211> 214 <212> PRT <213> Homo sapiens <400> 13 Met Gly Ala Ala His Ser Ala Ser Glu Glu Val Arg Glu Leu Glu Gly   1 5 10 15 Lys Thr Gly Phe Ser Ser Asp Gln Ile Glu Gln Leu His Arg Arg Phe              20 25 30 Lys Gln Leu Ser Gly Asp Gln Pro Thr Ile Arg Lys Glu Asn Phe Asn          35 40 45 Asn Val Pro Asp Leu Glu Leu Asn Pro Ile Arg Ser Ser Lys Ile Val Arg      50 55 60 Ala Phe Phe Asp Asn Arg Asn Leu Arg Lys Gly Pro Ser Gly Leu Ala  65 70 75 80 Asp Glu Ile Asn Phe Glu Asp Phe Leu Thr Ile Met Ser Tyr Phe Arg                  85 90 95 Pro Ile Asp Thr Thr Met Asp Glu Glu Gln Val Glu Leu Ser Arg Lys             100 105 110 Glu Lys Leu Arg Phe Leu Phe His Met Tyr Asp Ser Asp Ser Asp Gly         115 120 125 Arg Ile Thr Leu Glu Glu Tyr Arg Asn Val Val Glu Glu Leu Leu Ser     130 135 140 Gly Asn Pro His Ile Glu Lys Glu Ser Ala Arg Ser Ile Ala Asp Gly 145 150 155 160 Ala Met Met Glu Ala Ala Ser Val Cys Met Gly Gln Met Glu Pro Asp                 165 170 175 Gln Val Tyr Glu Gly Ile Thr Phe Glu Asp Phe Leu Lys Ile Trp Gln             180 185 190 Gly Ile Asp Ile Glu Thr Lys Met His Val Arg Phe Leu Asn Met Glu         195 200 205 Thr Met Ala Leu Cys His     210

Claims (11)

A hybridoma cell having a deposit number of KCTC 11790BP, characterized by producing a monoclonal antibody against a human TESC (Tescalcin) protein.
Monoclonal antibody to human TESC (Tescalcin) protein produced by hybridoma cells with accession number KCTC 11790BP.
3. The antibody of claim 2, wherein said monoclonal antibody specifically binds to the 51-100th amino acid sequence of the TESC (Tescalcin) protein of Sequence Listing 13.
A composition for diagnosing colorectal cancer comprising the monoclonal antibody of claim 2 or 3 as an active ingredient.
delete A diagnostic kit for colon cancer comprising the monoclonal antibody of claim 2 or 3 as an active ingredient.
delete A protein chip for diagnosing colorectal cancer comprising the monoclonal antibody of claim 2 or 3.
delete Human TESC (Tescalcin) protein comprising the steps of contacting the monoclonal antibody of claim 2 or 3 with a biological sample isolated from human body and then measuring the antigen-antibody complex to provide information on diagnosis of colon cancer / RTI &gt;
delete

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