TWI573803B - Tumor specific antigen, recombinant protein, antibody, and primer threrof, and the method for identifying mammalian tumors - Google Patents

Description

Tumor-specific antigen, recombinant protein, antibody and Pair pair and method for detecting mammalian tumors

The present invention relates to a tumor-specific antigen, a recombinant protein thereof, an antibody and a primer pair, and a method for detecting a mammalian tumor. In particular, it relates to a melanoma tumor antigen, a recombinant protein thereof, an antibody and a primer pair, and a method for detecting a mammalian tumor using a melanoma cell tumor antigen.

Tumors are medically referred to as abnormal lesions of cells. Under the action of various carcinogenic factors, cells of local tissues lose their normal regulation of growth at the genetic level. This kind of disease causes new parts of the body to exhibit uncontrolled hyperplasia, resulting in abnormal proliferation, and many of them will accumulate into tumors. It is generally believed that all tumor cells in a tumor are descendants of a mutant cell.

Tumors are an important cause of death in civil society. Tumor diagnosis procedures and methods are thoroughly detailed, such as laboratory testing, endoscopy or imaging experience, through comprehensive, systematic medical history enquiries, followed by comprehensive analysis. However, some tumors have metastasized at a stage that has not been detected by precision instruments, often resulting in failure of subsequent treatment. Therefore, it is necessary to find abnormal lesions earlier to diagnose and treat tumors at an early stage.

At present, a variety of tumor markers have been found in medical research to detect the presence of tumors at an early stage. A tumor marker is an abnormal phenomenon in a patient's tumor, including a substance produced or secreted by the tumor cell itself, or a reaction substance or a metabolite produced by the normal cell to the tumor. Tumor markers can pass through body fluids such as blood. Ascites, urine, tissue sections and other methods were tested. Therefore, the tumor marker may be a protein, a hormone, a tumor antigen or an enzyme present in a normal cell. For example, CA125 is recognized by the US Food and Drug Administration (FDA) as a tumor marker for ovarian cancer. However, the tumor markers currently found can only be used as an indicator of one or several tumors. It is necessary to simultaneously detect multiple tumor markers in order to fully confirm whether a patient has a tumor.

Accordingly, the present invention provides a melanoma tumor antigen, a recombinant protein thereof, an antibody and a primer pair, and a method of detecting a mammalian tumor. It can quickly and comprehensively detect mammalian tumors.

A first aspect of the present invention provides an isolated melanoblastoma antigen protein having an amino acid sequence as shown in SEQ ID NO: 2.

A second aspect of the present invention is an isolated nucleic acid having a sequence as shown in SEQ ID NO: 1, which encodes a melanoblastoma antigen protein of the present invention.

A third aspect of the invention is to provide an expression vector comprising the isolated nucleic acid of the invention.

A fourth aspect of the invention provides a host cell which is transformed into an expression vector of the invention.

According to the aforementioned host cell, wherein the host cell is Escherichia coli.

A fifth aspect of the present invention provides a method for producing a recombinant melanoma tumor antigen protein comprising culturing a host cell of the present invention to obtain a recombinant melanoma tumor antigen protein.

A sixth aspect of the present invention provides a recombinant melanoma tumor antigen protein having an amino acid sequence as shown in SEQ ID NO: 9, and which is produced by the production method of the present invention.

Accordingly, the present invention provides a melanoma tumor antigen and a recombinant protein thereof which are a tumor-specific antigen. The melanoma antigen is expressed in the normal tissue only in the testis and placenta, but the expression of melanoma antigen can be found in many different tumors, which is an ideal tumor marker.

A seventh aspect of the present invention is to provide an isolated antibody having binding affinity to the recombinant melanoma tumor antigen protein of the present invention.

An eighth aspect of the present invention provides a primer pair for detecting a tumor of a mammal, which is composed of a forward primer and a reverse primer according to the nucleic acid shown in Sequence Identification No. 1, wherein The sequence of the primer is selected from the group consisting of sequence identification number 3 and sequence identification number 5, and the sequence of the reverse primer is selected from the sequence indicated by sequence identification number 4 and sequence identification number 6. Form a group of people.

A ninth aspect of the present invention provides a method for detecting a tumor of a mammal in vitro, comprising: providing a biological sample, and performing a detection method for detecting whether the biological sample contains a melanoma antigen nucleic acid Or a melanoma antigen protein.

According to the aforementioned method for in vitro detection of a tumor of a mammal, wherein the mammal is a canine ( Canis lupus ).

According to the aforementioned method for in vitro detection of a tumor of a mammal, wherein the biological sample is selected from the group consisting of tumor tissue, tumor surrounding tissue, body fluid, and exudate.

According to the aforementioned method for in vitro detection of a tumor of a mammal, wherein the melanoma antigen nucleic acid has a sequence as shown in SEQ ID NO: 1, or a sequence complementary thereto, and the melanoma antigen protein has a sequence as shown in SEQ ID NO: 2.

According to the foregoing method for detecting a tumor of a mammal in vitro, wherein the detection method may be a reverse transcription polymerase chain reaction (RT-PCR), nested polymerase chain reaction (nested PCR), immunohistochemical staining, enzyme-linked immunosorbent assay (ELISA) or Western blotting.

According to the aforementioned method for detecting a tumor of a mammal in vitro, wherein the nested PCR further comprises the steps of: extracting total RNA from the biological sample; performing RT-PCR with the total RNA using the first primer pair to obtain an RT-PCR product Wherein the first primer pair consists of a primer having the sequence shown in SEQ ID NO: 3 and a primer having the sequence shown in SEQ ID NO: 4; using a second primer pair to perform nested PCR with the RT-PCR product to obtain nested PCR a product, wherein the second primer pair consists of a primer having the sequence of SEQ ID NO: 5 and a primer having the sequence of SEQ ID NO: 6; and detecting whether the nested PCR product has the same or complementary by agar gel electrophoresis A partial nucleotide sequence of a nucleic acid as shown in Sequence Identification No. 1.

Accordingly, the present invention provides a method for detecting a tumor of a mammal, which can be accurately, rapidly and comprehensively detected by using a reverse transcription polymerase chain reaction, a nested polymerase chain reaction, an immunohistochemical staining method or a Western blot method. Mammalian tumors are used to solve the problems of traditional detection of mammalian tumors that cannot be detected early in pathological morphology, and to solve the problem of time-consuming and time-consuming for comprehensive examination of multiple tumor markers.

The Summary of the Invention is intended to provide a simplified summary of the present disclosure in order to provide a basic understanding of the disclosure. This Summary is not an extensive overview of the disclosure, and is not intended to be an

The above and other objects, features, advantages and embodiments of the present invention will become more <RTIgt; <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; The protein expression analysis chart of different expression conditions of the expression vector pET-44a/MAGE-A2 of the present invention induced by IPTG; Fig. 3 is a diagram showing the protein expression analysis of each purification step of the recombinant melanoma tumor antigen protein of the present invention; The western blot method of the antibody of the present invention is shown in Fig. 5; and Fig. 5 is an analysis diagram of the enzyme-linked immunosorbent assay of the antibody of the present invention.

The disclosure of the present specification provides a novel tumor-specific antigen, a recombinant protein designed according to the tumor-specific antigen, an antibody, a primer pair for detecting a mammalian tumor, and a method for detecting a tumor of a mammal. It is a novel tumor-specific antigen found in mammalian tumor tissues, and the expression vector containing the tumor-specific antigen is constructed by genetic engineering technology, and can be expressed in a host cell in a high yield to obtain a recombinant tumor-specific antigen protein, and The recombinant tumor-specific antigen protein is used as an antigen to prepare an antibody having affinity with the tumor-specific antigen. This tumor-specific antigen is only specifically expressed in the testis and placenta in normal tissues of mammals, but is widely expressed in various tumor tissues. Therefore, the tumor-specific antigen can be used in a mammalian tumor detection test, for example, using a tumor-specific antigen set. The detection of the nucleic acid or protein of the tumor-specific antigen is sufficient to sensitively detect a low degree of tumor and exclude most non-cancer or clinically non-meaning patients.

The term "antigen" as used herein refers to a substance that contains one or more immune systems that stimulate the host to produce bodily fluids, and/or cellular antigen-specific responses. As a result of contact with appropriate cells, the antigen induces a state of sensitivity or immune response and reacts in an arguable manner with antibodies or immune cells of the sensitized individual, either in vivo or ex vivo. Antibodies can be specifically identified and bound by antibodies within a biological individual. Antigens associated with major histocompatibility complex (MHC) can also be recognized and bound by receptors located on the surface of T lymphocytes, resulting in T-lymphocyte activation.

The term "antibody" as used herein refers to an immunoglobulin molecule having a specific amino acid sequence and which is only associated with one or a group of closely related antigens, or at least one immunologically active portion of an immunoglobulin molecule. . It is secreted by B-lymphocytes and used by the immune system to identify and neutralize foreign antigens. Examples of antibodies include IgG, IgM, IgA, IgD, and IgE. Examples of immunologically active portions of immunoglobulin molecules include Fab and F(ab)' ₂ . The antibody may be a single antibody or a plurality of antibodies.

The "tumor-specific antigen" as used in the present invention refers to one of tumor antigens. Tumor antigens generally refer to antigenic substances that are newly or overexpressed during tumorigenesis and development, and can be classified into tumor-specific antigens and tumor-associated antigens according to the specificity of the antigen. Tumor-specific antigen means that the tumor antigen is expressed only in tumor tissue and not in normal tissues. Tumor-associated antigen means that the tumor antigen is present in the tumor tissue or cells, and can also be positive. Often in tissues or cells, but in tumor tissues or cells, the amount of expression far exceeds that of normal tissues or cells.

The aforementioned "melanoma antigen" of the present invention refers to Melanoma antigen-A (MAGE-A), which is an antigen of cutaneous melanoma, which is composed of 80 amino acids and has a size of about 18 kDa. The melanoma antigen was first discovered in 1991 and contained 12 genes encoding MAGE-A: MAGE-A1 to MAGE-A12. Melanoma tumor antigen is a cancer-testis antigens (CT antigens), which is an antigen during fetal development. It is expressed only in testicular and placenta in normal tissues, but melanoma antigens can be found in many different tumors. The performance is a tumor-specific antigen and is therefore an ideal tumor marker.

The present invention will be further exemplified in the following specific examples to facilitate the general knowledge of the art to which the present invention pertains, and the present invention may be fully utilized and practiced without undue interpretation. The scope of the invention is limited, but is intended to illustrate how to practice the materials and methods of the invention.

I. Preparation of recombinant melanoma tumor antigen protein Test Example 1-1: Construction of recombinant melanoblastoma antigen protein expression vector

In this test, the first primer pair was first designed according to the reserved region of the MAGE-A related gene sequence of the dog in GenBank. The first primer pair was introduced by the sequence of sequence identification number 3 and the sequence shown by sequence identification number 4. The composition of the primer. Using the various tumor tissues of canine ( Canis lupus ) as a template, the first polymerase chain reaction was performed using the first primer pair. The polymerase chain reaction reagents and reagents were carried out in the following ratios: 2.5 μl Run-Fast Taq Master kit (Protech, Taipei, Taiwan), 5 μM primers, 1 μl and 2 μl of cDNA, and finally sterilized secondary deionized water. The total volume was adjusted to 25 μl, and the solution was mixed uniformly to carry out the first polymerase chain reaction. The reaction conditions were denaturation at 94 ° C for 5 minutes, followed by 35 cycles of thermal cycling, including denaturation at 94 ° C for 1 minute, adhesion at 57 ° C for 1 minute, extension at 72 ° C for 1.5 minutes, followed by a final extension of 72 ° C for 7 minutes. The termination temperature was 4 °C. The amplified DNA fragment was separated by electrophoresis on agar extract and the amplified DNA was purified by a recovery reagent. The amplified DNA is sequenced via an automated nucleic acid sequencer.

Please refer to FIG. 1 , which is a gel electrophoresis diagram of the melanoma antigen of the present invention, wherein “M” is a molecular weight marker of DNA. (A) is a DNA fragment amplified by the first polymerase chain reaction, lanes 1 to 3 are infectious mosaic tumor samples, lanes 4 to 5 are fibrous tissue sarcoma samples, and lane 6 is a liposarcoma sample. The results showed that the first primer pair could increase the DNA fragment of about 400 bp in size. However, when the DNA sequence of the product amplified by the first polymerase chain reaction was analyzed by the nucleic acid automatic sequencer, the products of different tumor tissue sources were found to have the variability of the DNA sequence.

Therefore, in this test example, the DNA sequence amplified by the first polymerase chain reaction is further put together to analyze the reserved region to design a second primer pair, and the second primer pair is sequenced as shown in sequence identification number 5. The primer and the primer of the sequence shown in the sequence identification number 6 are constructed. The second polymerase chain reaction was performed using the second primer pair using the DNA amplified by the first polymerase chain reaction as a template. The polymerase chain reaction reagents and reagents were carried out in the following ratios: 5 μl Run-Fast Taq Master kit, 5 μM primers, 1 μl and 2 μl of cDNA, and finally sterilized secondary deionized water, adjusted to a total volume of 25 μl. After the solution is mixed uniformly, a second polymerase chain reaction is carried out. The reaction conditions were denaturation at 94 ° C for 5 minutes, followed by 35 cycles of thermal cycling, including denaturation at 94 ° C for 1 minute, adhesion at 60 ° C for 1 minute, extension at 72 ° C for 1 minute, followed by a final extension of 72 ° C for 7 minutes. The termination temperature was 4 °C. The amplified DNA fragment sequence is shown in SEQ ID NO: 1, which is the melanocytoma antigen of the present invention, which has a restriction enzyme Nde I cleavage at the 5th end and a restriction enzyme Xho I cleavage at the 3 end, which is encoded by The sequence of the melanoma antigen protein is shown in Sequence Identification Code 2.

The third polymerase chain reaction was carried out by using the DNA amplified by the first polymerase chain reaction as a template, using a primer such as the sequence of the sequence identification number 7 and a primer of the sequence of the sequence identification number 6. The polymerase chain reaction reagents and reagents were carried out in the following ratios: 5 μl Run-Fast Taq Master kit, 5 μM primers, 1 μl and 2 μl of cDNA, and finally sterilized secondary deionized water, adjusted to a total volume of 25 μl. After the solution was mixed well, a third polymerase chain reaction was carried out. The reaction conditions were denaturation at 94 ° C for 5 minutes, followed by 35 cycles of thermal cycling, including denaturation at 94 ° C for 1 minute, adhesion at 63 ° C for 1 minute, extension at 72 ° C for 1 minute, followed by a final extension of 72 ° C for 7 minutes. The termination temperature was 4 °C. The amplified fragment has a restriction enzyme Sal I cleavage at the 5 terminus and a restriction enzyme Xho I cleavage at the 3 terminus.

Please refer to FIG. 1 again for the agarose electrophoresis pattern of the melanoma tumor antigen of the present invention, wherein "M" is a molecular weight marker of DNA. Part (B) is a DNA fragment amplified by a second polymerase chain reaction reaction, which is about 280 bp in size. Part (C) is a DNA fragment amplified by the third chain reaction, which is about 280 bp in size. And analyzing the DNA sequence of the amplified product of the second polymerase chain reaction by the nucleic acid automatic sequencer (as shown in the sequence identification number 1), and determining that the isolated nucleic acid of the present invention is different according to the sequence identification number 1. The region with the least variability of tumor tissue-derived melanoma tumor antigen, which is the region with the least variability between the 12 MAGE-A (MAGE-A1 to MAGE-A12), shows that it has multiple tumors simultaneously. Confirm the patient's potential for tumors.

In order to construct a expression vector comprising a recombinant melanoma antigen, the DNA fragment amplified by the second polymerase chain reaction is first cut with restriction enzyme Nde I/ Xho I, and then cET-44a is cleaved with the same restriction enzyme. (Novagen), ligated with T4 ligase at a suitable molar ratio to obtain a expression vector pET-44a/MAGE-A containing a recombinant melanoblastoma antigen. The DNA fragment amplified by the third polymerase chain reaction was then cleaved with the restriction enzyme Sal I/ Xho I, and the expression vector pET-44a/MAGE-A, which was cleaved by the same restriction enzyme, was used to utilize T4 ligase (T4). The DNA ligase was ligated at an appropriate molar ratio to obtain a expression vector pET-44a/MAGE-A2 which was constructed to contain a double-recombinant melanoblastoma antigen, and the sequence correctness was confirmed by a nucleic acid automatic sequencer. Wherein, after two ligation, the sequence of the insert DNA fragment of the expression vector pET-44a/MAGE-A2 is as shown in SEQ ID NO: 8, which is the recombinant melanoma antigen of the present invention, which is encoded. The sequence of the recombinant melanoma antigenic protein is shown in Sequence Identification No. 9.

Test Example 1-2: Expression of recombinant melanoma antigenic protein

The expression vector pET-44a/MAGE-A2 was transformed into E. coli strain BL21, and the obtained E. coli transformed strain was cultured in LB (Luria-Bertani) culture medium supplemented with ampicillin at 37 ° C, and the absorbance of OD ₆₀₀ was reached. Adding isopropyl-β-D-thiogalactoside (IPTG) at a final concentration of 0.8 mM from 0.6 to 1.0, IPTG can induce expression vector pET-44a/MAGE-A2 with T7 promoter to express recombinant melanoma The antigenic protein is in the cytoplasm. After induction by IPTG, the bacterial solution was cultured in an incubator at 28 ° C or 37 ° C for 5 to 16 hours.

The protein performance of the expression vector pET-44a/MAGE-A2 induced by IPTG was analyzed by electrophoresis and Western blotting. The primary antibody used in the Western blotting method is a His-tag antibody. Please refer to Figure 2, part (A) is the protein electrophoresis map, where "M" is the egg The molecular weight of the white matter is marked, and the part (B) is the western blotting map. Lane 1 is a negative control group not induced by IPTG, Lane 2 is a group cultured at 28 ° C for 5 hours after induction, Lane 3 is a group cultured at 28 ° C for 10 hours after induction, and Lane 4 is at 28 ° C after induction. In the group cultured overnight, lane 5 was a group cultured at 37 ° C for 5 hours after induction, lane 6 was a group cultured at 37 ° C for 10 hours after induction, and lane 7 was a group cultured overnight at 37 ° C after induction. The results showed that the negative control group induced by IPTG did not have the expression of recombinant melanoma tumor antigen protein, while the group induced by IPTG could express recombinant melanoma tumor antigen protein under 6 different growth conditions.

Test Example 1-3: Purification of recombinant melanoma tumor antigen protein

The culture solution was centrifuged and the supernatant was discarded to collect the bacterial mass, and the bacterial mass was dissolved into the suspended cells by 10 ml of a binding buffer solution (50 mM Tris, 10 mM imidazole), and then the cells were lysed by a freeze-thaw method. The cell wall and 200 μl of lysozyme at a concentration of 20 mg/ml were added to help the cell wall of the cell. After the bacteria solution was placed at 37 ° C for 20 minutes, the chromosomal DNA was interrupted by ultrasonic vibration. Add 1ml of 5M NaCl to the bacterial solution, centrifuge at 10865×g for 10 minutes at 4°C, and then retain the supernatant containing the recombinant melanoblastoma antigen protein in the original state, and the precipitate contains denatured. The melanoblastoma antigen protein was recombined, and the precipitate was reconstituted with 10 ml of a lysis buffer solution (50 mM Tris-HCl, pH 7.4, 0.5 M NaCl, 10 mM imidazole, 6 M urea), which was shocked by ultrasonic waves and then centrifuged at 10,865 × g. Retention of recombinant melanin containing denaturation after centrifugation at 4 ° C for 20 minutes The supernatant of the cell tumor antigen protein. The supernatant of the recombinant melanoma antigenic protein containing the original state and the denaturing was purified by affinity column chromatography. Since the C-terminus of the recombinant melanoma antigen protein has 6 histidines, the purification method is to add an appropriate amount of the supernatant to a nickel-chelating nitrilotriacetic acid (Ni-NTA) containing 10 mM imidazole. Affinity column chromatography was carried out, and the reaction was uniformly mixed at 4 ° C for 2 hours, and then washed with a washing buffer (50 mM Tris-HCl, pH 7.4, 0.5 M NaCl, 20 mM imidazole), unless specifically bound. Finally, it was washed twice with a washing buffer containing 100 mM imidazole, and then twice with a washing buffer containing 400 mM imidazole, and the recombinant melanocytoma antigen protein bound to the resin was washed out. Purified recombinant melanoma antigenic protein was collected and dialyzed against dialysis buffer [PBS containing 4 M urea and 0.1 mM benzylsulfonated fluorochemical (PMSF)] to remove excess salts. The purified recombinant melanoma tumor antigen protein was confirmed by protein electrophoresis analysis.

Please refer to Fig. 3, which is a protein expression analysis diagram of each purification step of the recombinant melanoma tumor antigen protein, wherein "M" is a molecular weight marker of the protein. (A) is a partially denatured recombinant melanoma antigenic protein sample, and (B) is a native recombinant black tumor cell antigen protein sample. Lane 1 is the crude bacterial suspension induced by IPTG, lane 2 is the crude bacterial suspension induced by IPTG, lane 3 is the bacterial suspension after ultrasonic shock, and lane 4 is the bacterial suspension and affinity column. After the combined influent, lane 5 is the effluent for the first wash, and lane 6 is the wash buffer containing 100 mM imidazole. The rinsed effluent, lanes 7 to 8 are effluent flushed with wash buffer containing 400 mM imidazole. The results showed that the recombinant melanoma tumor antigen protein was only expressed in a denatured form, whereas the original recombinant melanoma tumor antigen showed less performance, but was still obtained after elution through a washing buffer containing 400 mM imidazole.

Second, the preparation of antibodies Test Example 2-1: Immunization procedure

The mice were immunized with emulsion-type antigen intramuscular injection. The test animals were three female BALA/c mice. The purified dialysis 25 μg of recombinant melanoma tumor antigen protein was added to Freund's Adjuvant (Complete) to prepare an emulsion, which was intramuscularly injected every 2 weeks, and mouse serum was collected on days 11, 25 and 39. Serum was stored at -20 °C.

Test Example 2-2: Analysis of antibodies

To test whether the prepared antibody has the ability to recognize recombinant melanoblastoma antigenic proteins, the collected serum was confirmed as a primary antibody by Western blot analysis. The serum dilutions of the mice collected on the 11th and 25th days were 1:20000, and the serum dilutions of the mice collected on the 39th day were 1:500000.

Please refer to Figure 4 for the western blot of antibodies. Sections (A) to (C) are for the first-antibody group using the mouse serum collected on the 11th day, (D) to (F) The serum of the mice collected on the 25th day is the first grade. In the group of antibodies, the parts (G) to (I) are the group in which the serum of the mouse collected on the 39th day is the primary antibody. Lane 1 is a crude bacterial suspension that has not been induced by IPTG, and Lane 2 is a crude bacterial suspension induced by IPTG. The results showed that the mouse sera collected on the 11th, 25th and 39th days had the ability to recognize the recombinant melanocytoma antigen protein, and the antibody potency was very good. The serum of the mice collected on the 11th and 25th days only needed 1 Recombinant melanoma antigen protein can be identified by /20000, and the serum antibody collected on day 39 is more potent, and only 1/500,000 can identify the recombinant melanoma antigen protein.

The antibody titer of the mouse serum collected on the 25th day was analyzed by enzyme-linked immunosorbent assay (ELISA). Different concentrations of recombinant melanocytoma antigen protein were coated on the surface of the test disc as antigen, and the antigen was diluted by 2 times, and the concentrations were 0.97, 1.95, 3.91, 7.81, 15.63, 31.25, 62.5, 125, 250, 500 and 1000ng/well. The mouse serum collected on the 25th day was used as a primary antibody and diluted in a 2-fold sequence at dilution ratios of 1:500, 1:1000, 1:2000, 1:4000, 1:8000, and 1:16000, at room temperature. After the reaction for 1 hour, it was washed 3 times with a washing buffer solution (PBS containing 0.05% Tween 20), and then an antibody (HRP-conjugated antibody) containing a binding enzyme was added at room temperature for 1 hour, and then washed with a washing buffer solution. Three times, 10 to 15 minutes after the addition of TMB coloring agent (Clinical Science Laboratory, Inc), a stop solution (2N H ₂ SO ₄ ) was added to determine the absorbance of OD ₄₅₀ by an enzyme immunoassay tester. Please refer to Fig. 5 for the analysis of the enzyme-free liquid-adsorption adsorption method of the antibody of the present invention. The results show that the maximum dilution: lower (116,000) still have the ability to 1ng recombinant protein in the cell melanoma antigen (OD ₄₅₀ of 0.48) detect, once again proved that antibodies of the invention having a high antibody titer.

Third, the detection method

In the present invention, a method for detecting a tumor of a mammal in vitro is carried out by using the first primer pair and/or the second primer pair described above, and detecting whether the sample contains melanoma antigen nucleic acid by RT-PCR or nested PCR. Or using the above antibody, the immunohistochemical staining method, the enzyme-linked liquid-free adsorption method or the Western blot method to detect whether the sample contains melanoma antigen protein. When a melanoma antigenic nucleic acid or protein is detected, it is characterized by the presence of a tumor.

Test Example 3-1: Polymerase chain reaction

This test example includes the use of RT-PCR or nested PCR to detect whether a sample of a mammal has a nucleic acid having a melanoma antigen as a basis for having a mammalian tumor. The samples tested included 10 normal testiculars of dogs, 4 fibrous tissue sarcomas, 5 liposarcoma, 3 malignant peripheral schwannomas, 3 infectious sacral tumors, 2 angiosarcomas, 5 malignant melanomas, 4 Lymphoma, 1 histiocytic sarcoma, 5 mast cell tumors, 2 mucinous sarcomas, 4 osteosarcoma, 3 plasmacytomas, and 3 malignant squamous cell tumors. The total RNA of the above tissues is extracted by a commercial kit or a conventional extraction method, and the first primer pair or the second primer pair is subjected to a reverse transcription polymerase chain reaction, and the reactants and reagents of the reverse transcription reaction are as follows. Proportion: 4 μg RNA, 2 μl of any primer with a concentration of 5 μM, 4 μl of a dNTP mixture with a concentration of 2.5 mM, 4 μl of 5X first standard buffer, 1 μl of DTT at a concentration of 0.1 M, 1 μl of RNase inhibitor and 1 μl of inverse The transcriptase was finally added to sterile deionized water and the final volume was adjusted to 15 μl. The solution was uniformly mixed, reacted at 50 ° C for 60 minutes, and then deactivated at 70 ° C for 15 minutes. The cDNA obtained by the reaction was stored in a refrigerator at -20 ° C for use in the subsequent polymerase chain reaction. The polymerase chain reaction is a primer with a first primer pair or a second primer pair, and the steps are the same as those shown in Test Example 1-1, and will not be described herein. The agarose gel electrophoresis is used to detect whether the RT-PCR product has a partial nucleotide sequence having the same or complementary nucleic acid as shown in SEQ ID NO: 1.

The reaction procedure of detecting the nucleic acid of melanoma antigen by using nested PCR is as follows: using the above-mentioned total RNA extracted from each tissue as a template, RT-PCR is performed on the total RNA with the first primer pair to obtain an RT-PCR product. . The second primer pair is used for nested PCR with the RT-PCR product to obtain the nested PCR product. Finally, the agarose gel electrophoresis is used to detect whether the nested PCR product has a partial or complementary nucleic acid having the same or complementary nucleic acid sequence number 1. Glycosidic acid sequence.

Please refer to Table 1 for the detection of the expression rate of melanoma antigen nucleic acid by polymerase chain reaction. The results showed that the detection method of the present invention was positive in the test results of the testicles and 13 different types of tumors, among which the sputum pills, malignant melanoma, angiosarcoma, malignant peripheral schwannomas, infection The detection rate of squamous cell tumor, fibrosarcoma sarcoma, histiocytic sarcoma, mucinous sarcoma and osteosarcoma is 100%, the rate of detection of liposarcoma and mast cell tumor is 80%, and that of lymphoma can reach 75%. Tumor and malignant squamous cell tumors can reach 66.66%.

Test Example 3-2: Immunohistochemical staining

Immunohistochemical staining method is to embed different normal tissues and different types of tumor tissues in paraffin, and cut the slicer into 5mm thickness slides, then remove the wax, return water, renaturation, etc. The antibody of the present invention was inserted as a primary antibody and reacted at room temperature for 10 to 60 minutes, and the dilution ratio of the primary antibody was 1:2000. Rinse three times with PBS. Secondary resistance The cells were reacted at room temperature for 20 minutes, washed three times with PBS, and stained with 3', 3'-diaminobenzendine (DAB) at a ratio of 1:100 for 10 minutes, and then stained with hematoxylin for 1 minute and then mounted.

Please refer to Table 2 for the detection of protein expression of melanocytoma antigen in normal tissues by immunohistochemical staining. The results show that in normal tissues, only the testis can detect the protein expression of melanoma antigen, while in other normal tissues. No protein expression of melanoma antigens was detected in the medium.

Please refer to Table 3 for the detection of protein expression of melanoma antigens in tumor tissues by immunohistochemical staining. The results showed that the detection method of the present invention was positive in 13 different types of tumor detection, and the detection rate of angiosarcoma, malignant peripheral schwannomas, infectious sacral tumor and osteosarcoma was over 80%. The detection rate of malignant melanoma, malignant squamous cell, fibroblastic sarcoma, plasmacytoma, mast cell tumor, lymphoma, histiocytic sarcoma and mucinous sarcoma can reach more than 60%, and the detection rate of liposarcoma can reach 40%. .

According to the above, the melanoma tumor antigen of the present invention is expressed only in the testicular and placenta in normal tissues, but is expressed in a variety of different tumors, and is an ideal tumor marker, and the melanoblastoma antigen of the present invention is 12 kinds of MAGE-A. (MAGE-A1 to MAGE-A12) The region with the least variability between each other, and further proved by the above test examples, by detecting the presence or absence of the melanoblastoma antigen nucleic acid or protein of the present invention, simultaneous detection A variety of tumors have the potential to be applied in medicine for accurate, rapid and comprehensive detection of the presence or absence of tumors in mammalian individuals. The recombinant melanoma tumor antigen protein of the present invention is the fusion protein of the melanocytoma antigen of the present invention, and the recombinant melanoma tumor antigen protein is used as an antigen for producing the antibody, thereby increasing the recognition degree of the antigen, so that the protein can be produced. An antibody with high antibody valence and high specificity.

Due to the nature of the polymerase chain reaction itself, even if there is variability in the sequence between the primer and the template to be amplified, the specific nucleic acid fragment can be synthesized by adjusting the reaction temperature of the bonding step in the polymerase chain reaction, so the technique of the present invention According to the disclosure of the present invention, it is possible to design different primers according to the nucleic acid fragment to be amplified. Therefore, any primers formed by base substitution, addition or reduction for the individual primers described in the embodiments of the present invention may still be associated with the primers described in the embodiments of the present invention. The fragment shown in the sequence identification number 1 is added to the primer pair without departing from the scope of the present invention.

In addition to the above, those having ordinary knowledge in the art to which the present invention pertains, according to the disclosure of the present invention, use any form to evaluate the analysis of the nucleic acid or protein of the melanoma antigen of the present invention without departing from the scope of the present invention. . For example, nucleic acids or proteins can be tested from a biological sample such as fresh or frozen, tissue fixed by formalin, and body fluids of blood, plasma, serum, urine or saliva using techniques known in the art to which the present invention pertains. Is separated and analyzed. Methods for analyzing specific nucleic acids, in addition to RT-PCR and nested PCR as described in the examples of the present invention, including PCR, in situ hybridization, northern blotting, high density performance arrays, microarrays, etc. are also contemplated techniques . In the method of analyzing a specific protein, in addition to the Western blotting method and the immunohistochemical staining method described in the examples of the present invention, the enzyme-containing fluid-free adsorption method and the enzyme assay are also expected techniques.

The present invention has been disclosed in the above embodiments, but it is not intended to limit the invention, and the scope of the present invention can be varied and modified without departing from the spirit and scope of the invention. It is subject to the definition of the scope of the patent application attached.

<110> National Chung Hsing University

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<400> 8

<210> 9

<211> 161

<212> PRT

<213> Canis lupus

<223> Amino acid sequence of recombinant melanoma antigen

<400> 9

Claims (17)

An isolated melanoma antigen protein having a sequence as shown in SEQ ID NO: 2. An isolated nucleic acid having the sequence shown in SEQ ID NO: 1, encoding the melanoma antigen protein of claim 1. A performance vector comprising the nucleic acid of claim 2. A host cell transformed into the expression vector of claim 3. The host cell of claim 4, which is Escherichia coli. A method for producing a recombinant melanoma tumor antigen protein comprising culturing a host cell as claimed in claim 4 or 5 to obtain a recombinant melanoma tumor antigen protein. A recombinant melanoma antigenic protein having an amino acid sequence as shown in SEQ ID NO: 9, prepared by the method of claim 6. An isolated antibody that is as heavy as described in claim 7 The group of melanoma antigenic proteins has binding affinity. A primer pair for detecting a tumor of a mammal is composed of a forward primer and a reverse primer designed according to the nucleic acid shown in Sequence Identification No. 1, wherein the sequence of the forward primer is, for example, sequence identification number 5 As shown, the sequence of the reverse primer is shown as sequence identification number 6. A method for detecting a tumor of a canine ( Canis lupus ) in vitro comprises: providing a biological sample; and performing a detection method to detect whether the biological sample contains a melanoma antigen nucleic acid or a melanoma antigen A protein, wherein the melanoblastoma antigen nucleic acid has a sequence as shown in SEQ ID NO: 1, the melanoblastoma antigen protein having a sequence as shown in SEQ ID NO: 2. The method for detecting a canine tumor in vitro as described in claim 10, wherein the biological sample is selected from the group consisting of tumor tissue, tumor surrounding tissue, body fluid, and exudate. A method for detecting a canine tumor in vitro as described in claim 10, wherein the detection method is a reverse transcription polymerase chain reaction (RT-PCR). A method for detecting a canine tumor in vitro as described in claim 10, wherein the detection method is nested polymerase chain reaction (nested PCR). The method for detecting a canine tumor in vitro according to claim 13, further comprising the steps of: extracting a total RNA from the biological sample; performing RT-PCR on the total RNA using a first primer pair, Obtaining an RT-PCR product, wherein the first primer pair consists of a primer having the sequence shown in SEQ ID NO: 3 and a primer having the sequence shown in SEQ ID NO: 4; using a second primer pair and the RT-PCR The product is subjected to nested PCR to obtain a nested PCR product, wherein the second primer pair consists of a primer having the sequence shown in SEQ ID NO: 5 and a primer having the sequence indicated by SEQ ID NO: 6; and using agar gel electrophoresis The nested PCR product is tested for a partial nucleotide sequence having the same or complementary DNA as shown in SEQ ID NO: 1. A method for detecting a canine tumor in vitro as described in claim 10, wherein the detection method is immunohistochemical staining. A method for detecting a canine tumor in vitro as described in claim 10, wherein the detection method is a Western blot method. The method for detecting a canine tumor in vitro as described in claim 10, wherein the detection method is an enzyme-linked immunosorbent assay (ELISA).