KR101702596B1 - Self-detectable Diagnostic Kit for Detecting Cancer Existence Using Enzyme Composition - Google Patents

Abstract

The present invention relates to an autonomous color detecting cancer diagnosing kit for diagnosing the presence of cancer through a change in a color of urine by mixing the urine with an enzyme composition for detecting a concentration of a cancer biomarker, tyrosine, existing in urine, wherein the change in a color of urine can be automatically detected by using a color detecting sensor. The autonomous color detecting cancer diagnosing kit according to the present invention comprises: a case having a feeding hole for feeding urine; an enzyme accommodating unit formed in the case; an enzyme composition accommodated in the enzyme accommodating unit, and converting L-tyrosine existing in urine into melanin; a color detecting sensor installed in the enzyme accommodating unit, and detecting a color of urine in the enzyme accommodating unit after urine is fed; a light source for emitting light to the enzyme accommodating unit; and an information processing unit for determining the presence of cancer by analyzing color information detected by the color detecting sensor, and outputting a determined result to the outside.

Description

[0001] The present invention relates to a self-detectable diagnostic kit for detecting cancer,

The present invention relates to a kit for diagnosing the presence or absence of cancer and a disease, and more particularly, to an enzyme composition for detecting the concentration of cancer biomarker tyrosine present in a specimen, The present invention relates to a self-color detecting type cancer diagnostic kit which can detect the presence of cancer and metabolic diseases through a change and automatically detect a change in color of a sample using a color detecting sensor.

Cancer markers are substances that occur in the malignant tumor cells themselves or are caused by reactions of normal tissues against cancer and exhibit abnormally high concentrations in the blood, urine or tissues, and the cancer marker concentration in the blood, urine or tissue You can diagnose, screen, and track progress and degeneration.

In general, the measurement or detection of the concentration of the cancer marker is carried out through the blood, but the blood has a problem that it is difficult for the general person to collect it.

There has been developed a method for diagnosing cancer by detecting the concentration of cancer biomarker present in the urine by using urine which is not easy to obtain and which is not a biomaterial such as blood or tissue.

On the other hand, it is known that the tyrosine can be used as the cancer biomarker because the concentration of tyrosine present in the urine is high in cancer patients as compared with the general public.

Korean Patent No. 10-0033547 relates to a mixed reagent for diagnosing cancer by detecting aromatic amine (Tyrosine) commonly present in urine of a cancer patient, wherein the mixed reagent is composed of mercury, nickel, nitric acid and distilled water .

However, the above-mentioned mixed reagent contains mercury and is currently prohibited because it has a great influence on the environment and human body.

Therefore, there is an urgent need to develop a composition for diagnosing cancer by eco-friendly detection of the concentration of cancer biomarker tyrosine (Tyrosine) present in the urine by using urine which is not easy to obtain but is not easy to obtain.

Korean Patent No. 10-1323371 discloses a prostate cancer diagnostic kit capable of semi-quantitatively measuring the amount of whole prostate-specific antigen in the urine using an immunochromatography method. This patented prostate cancer diagnostic kit is designed to diagnose prostate cancer visually through the color of antigens and antibody responses of prostate specific antigen (PSA), a serine protease that is expressed at high levels in the prostate epidermis .

However, the prostate cancer diagnosis kit of the above-mentioned patent can diagnose prostate cancer only to a limited extent, and it is not possible to diagnose whether cancer other than prostate cancer is present.

In addition, a cancer diagnostic kit in which cancer is diagnosed by collecting the existing urine may not be accurately inspected if the amount of urine injected into the kit exceeds or falls below a predetermined amount. In the case where a user (examinee) It is very difficult to take the urine and inject the exact amount required for the test into the kit.

Korean Patent No. 10-0033547 Korean Patent No. 10-1323371

DISCLOSURE OF THE INVENTION The present invention has been made to solve the above-mentioned problems, and it is an object of the present invention to provide a method for producing L-tyrosine, which comprises the enzymes which are characterized in converting L- tyrosine into Eumelanin, By using an enzyme composition for detection of cancer biomarkers that can detect the presence of cancer in an environmentally friendly manner and automatically detecting the color change of urine, it is possible to diagnose the presence of cancer, And can easily perform an inspection.

According to another aspect of the present invention, there is provided a self-color detection type cancer diagnostic kit including: a case having a charging port into which urine is injected; An enzyme accommodating portion formed inside the case; An enzyme composition which is contained in the enzyme-accommodating portion and converts tyrosine (L-Tyrosine) present in the urine into melanin; A color detecting sensor installed in the enzyme receiving part and detecting the color of urine in the enzyme receiving part after the urine is inputted; A light source that emits light to the enzyme containing portion; And an information processor for analyzing the color information detected by the color detection sensor to determine the presence of the cancer and outputting the determination result to the outside.

According to the present invention, the enzyme composition for detecting tyrosine contains enzymes that are characterized in converting L-tyrosine into Eumelanin. Therefore, when a certain amount of urine is injected through the inlet formed in the case and mixed with the enzyme composition in the enzyme storage portion, the enzyme composition reacts with the cancer biomarker L-tyrosine present in the urine and the urine color changes from brown to black depending on the concentration of tyrosine Since the color is discolored, the color can be detected and the occurrence of cancer can be confirmed immediately.

In particular, the color detection type cancer diagnostic kit of the present invention detects the color of urine by detecting the color of the urine when the user inputs urine into the enzyme receiving portion in the case, It is possible to diagnose the presence of cancer more accurately and conveniently than when detecting a color change.

Therefore, the user can easily confirm the occurrence of cancer even in a home, not a professional institution such as a hospital.

1 is a perspective view illustrating a cancer diagnostic kit according to an embodiment of the present invention.
2 and 3 are sectional views of the cancer diagnosis kit of FIG. 1, FIG. 2 shows a state before urine is injected, and FIG. 3 shows a state where urine is inspected after urine is injected.
4 is an enlarged view of a portion A in Fig.
5 is a block diagram schematically showing the configuration of an information processing unit constituting the cancer diagnostic kit of the present invention.
FIG. 6 is a control color chart for measuring the color of the eumelanin produced by the embodiment of the enzyme composition in the cancer diagnostic kit of the present invention. FIG.
FIG. 7 is a photograph comparing the color of melanin produced by the conventional enzyme composition with the embodiment of the enzyme composition formed in the cancer diagnostic kit of the present invention.

Hereinafter, preferred embodiments of the cancer diagnostic kit according to the present invention will be described in detail with reference to the accompanying drawings.

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1 to 5 illustrate a cancer diagnostic kit according to an embodiment of the present invention. The cancer diagnostic kit of the present invention includes a case 10 having a charging port 11 into which urine is injected, And an enzyme composition 30 for converting tyrosine (L-Tyrosine) contained in the urine into melanin by being accommodated in the enzyme accommodating portion 20, A color detection sensor 40 installed at an upper portion of the enzyme accommodating portion 20 to detect the color of urine in the enzyme accommodating portion 20 after the urine is injected, And an information processing unit 60 for analyzing color information detected by the color detection sensor 40 to determine the presence of the cancer and outputting the determination result to the outside.

In the case 10, a charging port 11 for urine is formed to penetrate in the up and down direction. The upper end of the charging port 11 is preferably formed so that the outer periphery thereof is inclined downward so that the urine can be smoothly guided to the inside of the charging port 11 and prevented from flowing out. In order to prevent the enzyme composition 30 from flowing out to the outside, a cap 15 for opening and closing the charging port 11 may be provided at the upper end of the charging port 11. [ The stopper (15) is inserted into the inlet (11) so as to open and close the inlet (11). It is of course possible to open and close the charging port 11 by using a tape or a known sealing mechanism without using the cap 15 differently from this embodiment.

An operation button 65 for allowing the user to operate the color detection sensor 40 and a result of the diagnosis by the color detection sensor 40 and the information processing unit 60 are displayed on the upper portion or the side portion of the case 10 And a result notification unit 63 for outputting the result to the outside.

A transparent window 13 may be provided on one side of the case 10 so that the user can visually confirm the amount of urine introduced into the enzyme accommodating portion 20 through the inlet 11. Therefore, the user can input urine by a predetermined amount while viewing the transparent window 13.

The result notification unit 63 may include a plurality of LEDs for indicating the diagnosis result as lights of different colors. For example, a red LED 63a that emits red light when it is determined that there is cancer, a green LED 63b that emits green light when it is determined that there is no cancer, And a yellow LED 63c that emits yellow light when it is determined that the LEDs 63a, 63b, and 63c are arranged in a line.

In addition, an LCD display may be applied as the result notification unit 63, or a speaker capable of outputting the result as a voice or sound may be applied.

Although not shown, a power supply unit including a battery for supplying power to operate the color detecting sensor 40, the light source 50, and the information processing unit 60 is installed in the case 10.

The enzyme storage part 20 has a tubular shape in which an upper portion of the inlet 22 communicates with a lower end of the inlet 11 and functions as a container for containing the enzyme composition 30 in the liquid. The enzyme accommodating portion 20 is integrally formed inside the case 10.

It is preferable that the amount of the enzyme composition 30 in the enzyme accommodating portion 20 and the amount of urine injected into the enzyme accommodating portion 20 are in the ratio of 1: 1 to 3: 1 by weight in view of the accuracy of the test. Therefore, in order to prevent the urine from being injected when a certain amount of urine is injected into the enzyme accommodating portion 20, the inner portion of the inlet 22 through which the lower end of the inlet 11 communicates with the enzyme accommodating portion 20 Shaped valve body 71 is rotatably provided around the hinge axis 72 so that when the urine reaches a certain level of the enzyme accommodating portion 20, the valve body 71 rotates to rotate the inlet 22 Closing. A floater 73 floating by urine is provided at an end of the valve body 71 so that the valve body 71 rotates as the floater 73 floats by the urine. A coupling piece 74 made of a metal or a magnet is mounted on the upper surface of the floater 73 and the coupling piece 74 is fixed around the entrance 22 of the enzyme accommodating portion 20 by a magnetic force A magnet 75 is provided. When the valve body 71 closes the inlet 22 of the enzyme accommodating portion 20, the engaging piece 74 of the plotter 73 is firmly engaged with the magnet 75, It is possible to stably maintain the state in which the upper inlet 22 of the enzyme accommodating portion 20 is closed.

The enzyme composition (30) converts the cancer biomarker tyrosine (L-Tyrosine) present in the urine into eumelanin. As the tyrosine concentration in the urine increases, the amount of the eumelanin increases, To black. Therefore, the color detection sensor 40 can detect the color of the urine reacted with the enzyme composition 30, thereby measuring the tyrosine concentration, thereby detecting the presence or absence of cancer.

The color detection sensor 40 may be a known color sensor, an image sensor, or the like, and may detect color by capturing the colors of urine in the enzyme accommodating portion 20. In order to allow the color detection sensor 40 to detect color of urine with high accuracy, the light source 50 includes a white light source for emitting white light and an ultraviolet light source for emitting ultraviolet light in a wavelength band of 300 to 380 nm A polarizing filter 41 and an ultraviolet cutoff filter 42 for blocking ultraviolet rays are installed in the lower part of the color detecting sensor 40 to remove irregular reflection due to light reflected from the urine.

When ultraviolet light is irradiated to the urine and the color detection sensor 40 photographs only the visible light using the ultraviolet cut filter 42 at the lower part of the color detection sensor 40, the ultraviolet light is excited by ultraviolet rays, Which is known to be the most common. The above-mentioned eumelanin absorbs ultraviolet light and has a characteristic of darkening color. Therefore, when ultraviolet rays are used together as a light source and the ultraviolet ray blocking filter 42 is disposed directly below the color detecting sensor 40, the color of the euromelanine in the urine can be detected more clearly.

The information processing unit 60 includes a color detecting unit 61 for detecting a color by receiving a signal from the color detecting sensor 40 and a color detecting unit 61 for detecting a color detected by the color detecting unit 61, And a result notification unit 63 for outputting the result determined by the determination unit 62 to the outside.

The color detection unit 61 and / or the determination unit 62 may be configured in a microprocessor.

The result notification unit 63 may include a plurality of LEDs 63a, 63b, and 63c that emit light of different colors for each determination result as described above.

The thus configured cancer diagnostic kit operates as follows.

The user opens the cap 15 and inputs the collected urine through the inlet 11 using a syringe type injector or other known injection device. The urine introduced through the inlet 11 is injected into the enzyme accommodating portion 20 through the inlet 22 formed at the upper end of the enzyme accommodating portion 20.

At this time, as the urine is supplied into the enzyme accommodating portion 20 and the water level is increased, the floater 73 is gradually floated by the urine so that the valve body 71 rotates, and the enzyme accommodating portion 20 When the urine is filled up to the vicinity of the upper inlet 22, the valve body 71 closes the inlet 22 of the enzyme accommodating portion 20. The engaging piece 74 attached to the floater 73 is attached to the magnet 75 so that the valve body 71 maintains the state in which the inlet 22 of the enzyme accommodating portion 20 is closed.

When the amount of urine is filled in the enzyme accommodating portion 20 by a predetermined amount, the user stops the injection of urine through the inlet 11 and closes the inlet 11 with the stopper 15.

The urine supplied into the enzyme accommodating portion 20 reacts with the enzyme composition 30 in the enzyme accommodating portion 20 of the tyrosine contained therein to produce eumelanin, and the color changes.

When the user presses the operation button 65 provided at the upper end of the case 10, the light source 50 emits white light and ultraviolet light while power is supplied to the light source 50 and the color detection sensor 40, The sensor 40 captures urine to detect the color.

The color detecting unit 61 of the information processing unit 60 receives the signal of the color of the urine obtained by the color detecting sensor 40 and determines the color of the urine. The judging unit 62 compares the color of the urine detected by the color detecting unit 61 with the stored color-cancer information data to determine whether or not the cancer is cancerous.

The diagnosis result determined by the determination unit 62 is visually and / or audibly transmitted to the user through the result notification unit 63. [ For example, when the determination unit 62 determines that there is cancer, the red LED 63a is operated to emit red light. When it is determined that there is no cancer, the green LED 63b is operated. If it is not clear, but the cancer is likely to be present, turn on the amber LED (63c).

As described above, when the urine is injected into the enzyme receiving portion 20 in the case 10, the color detecting sensor 40 detects the color of the urine and automatically detects the presence or absence of the cancer It is possible to diagnose the presence of cancer more accurately and conveniently than when the user perceives the color change of the urine with the naked eye.

As described above, the self-color detecting type cancer diagnostic kit of the present invention can be applied to the case where urine is mixed into the enzyme composition 30 in the enzyme-accommodating portion 20, and the enzyme composition 30 is mixed with the cancer biomarker tyrosine (L-Tyrosine) into melanin, thereby allowing the user to easily diagnose cancer.

The enzyme composition 30 for the detection of tyrosine includes a tyrosinase, a Dopachrome tautomerase (DCT) and a tyrosinase-related protein 1 (Tyrp1) It acts to detect the concentration of marker tyrosine (L-Tyrosine).

More specifically, the enzyme composition (30) comprises an enzyme composition in which tyrosinase is supplemented with a Dopachrome tautomerase (DCT) and tyrosinase-related protein 1 (Tyrp1) Cancer can be diagnosed by converting the cancer biomarker tyrosine (L-Tyrosine) present in the urine into eumelanin phosphorus and measuring the concentration through the color of the eumelanin.

Since the enzyme composition (30) according to the present invention is intended to diagnose cancer using urine, the concentration of the enzyme composition (30) can be determined through the use of Pheomelanin which is yellowish brown or mixed melanin which is brown When measuring, it may be difficult to measure the concentration because it is similar to the color of the urine. Therefore, it is preferable that the enzyme composition (30) according to the present invention measures the concentration through black colored eumelanin.

By adding DCT and Tyrp1 enzyme to tyrosinase, the enzyme composition (30) not only accelerates the pathway of eumelanin biosynthesis, but also induces the color of the resulting eumelanin to a more blackish path.

Since the concentration of tyrosine present in the urine can be measured through the color of the eumelanin produced by the reaction with the enzyme composition (30), when the color of the urine caused by the produced eumelanin is close to black, Is high.

The detection of tyrosine by the enzyme composition 30 can be carried out using a phosphate buffer or a Tris-HCl buffer solution having a final concentration of 50 to 100 mM and a Tween 20 having a final concentration of 0.1 to 0.5% It is preferred that the final reaction is measured under the condition of pH 7.0 to 8.5.

For example, if the phosphate buffer solution contained in the enzyme composition 30 is 200 mM, and the total volume of 500 μl of the enzyme composition and 500 μl of the urine sample is 1000 μl, the tyrosine detection reaction can be performed at a final concentration of 50 mM .

In one embodiment of the present invention, the enzyme composition (30) comprises tyrosinase, DCT, and Tyrp1, and is supplemented with phosphate buffer or Tris-HCl buffer solution, Tween20 and distilled water .

Preferably, the enzyme composition (30) comprises 40 parts by weight of tyrosinase, 1 part by weight of tyrosinase, 0.5 to 2 parts by weight of DCT and 0.5 to 2 parts by weight of Tyrp1.

If the above content is not satisfied, it may be difficult to measure the concentration of tyrosine because the color of the produced melanin may appear to be a yellowish brown color and be mixed with the urine color.

As described above, the enzyme composition (30) accelerates the pathway of eumelanin biosynthesis by tyrosinase to DCT and Tyrp1 enzyme, and induces the color of the resulting eumelanin to a more black-colored pathway.

Referring to Reaction Scheme 1 below, L-tyrosine is oxidized by tyrosinase to form 3,4-dihydroxyphenylalanine (DOPA) and then oxidized by tyrosinase to form DOPA quinone , And Leucodopachrome to form Dopachrome.

The dopachrome is a melanin precursor and is tautomerized by DCT to form 5,6-dihydroxyindole-2-carboxylic acid (DHICA).

The 5,6-dihydroxyindole-2-carboxylic acid is oxidized by Tyrp 1 to form indole-5,6-quinonecarboxylic acid and the indole-5,6-quinonecarboxylic acid is polymerized to form uamelanin .

[Reaction Scheme 1]

The tyrosinase is a tyrosinase hydroxylase and a copper-containing enzyme having catalytic activity of dopa oxidase, which can be found in microorganisms and plant and organism tissues. In particular, the tyrosinase promotes the production of melanin and other pigments by oxidation of phenols such as tyrosine.

The Dopachrome tautomerase (DCT), also known as tyrosinase-related protein 2 (Tyrp 2), is a protein that is characterized by the binding of tyrosinase and Tyrp1 to convert L-tyrosine to melanin in melanocytes It is a melanin cell specific enzyme.

The tyrosinase-related protein 1 (Tyrp1) plays a role in stabilizing tyrosinase.

The enzyme composition (30) according to the present invention is characterized in that the dopa chromium is converted to indole-5,6-dihydroxyindole via 5,6-dihydroxyindole by tyrosinase, quinone) to form melanin near the brown color (see the following reaction formula 2), and is tautomerized by DCT and oxidized by Tyrp 1 to form melanin close to black to distinguish it from urine color of the sample So that the concentration measurement can be effectively performed.

[Reaction Scheme 2]

In one embodiment of the present invention, the tyrosine concentration in the urine can be confirmed from the color information of the eumelanin detected by the color detection sensor 40. This makes it easy to diagnose the occurrence of cancer.

Hereinafter, the enzyme composition (30) of the present invention will be described in more detail by examples. It is to be understood by those skilled in the art that these embodiments are for illustrative purpose only and that the scope of the present invention is not limited to these embodiments.

Production Example 1: Preparation of each component of the enzyme composition

The tyrosinase enzyme was purchased from Sigma.

DCT and Tyrp1 were obtained from mouse blood by reference to the following steps and purified after protein expression in E. coli.

(i) RNA extraction from mouse blood

RiboEx RNA extraction solution kit (Jinol Biotechnology, Korea) was used in this embodiment. The process is as follows.

a. 300 μl of mouse blood and 600 μl of RiboEx RNA extraction solution (Jeolol Biotechnology, Korea) were mixed from the mouse blood, mixed strongly and left at room temperature for 5 ~ 10 minutes to dissolve the cell membrane.

b. 200 μl of chloroform was added and mixed vigorously. The mixture was centrifuged at 13,000 rpm at 4 ° C for 10 minutes using a centrifuge to separate the upper and lower layers.

c. After collecting only the upper layer, the same amount of the column-binding buffer was added and mixed well. Then, the solution was added to the column provided in the kit and spin-down to bind only RNA to the column.

d. The wash buffer provided in the kit was added to the column, and then the material other than RNA was removed by spin-down. The remaining wash buffer was removed by centrifugation for an additional 1 minute.

e. The elution buffer was added to recover the RNA bound to the column, and the RNA was recovered by spinning 5 minutes later.

(ii) preparing cDNA from the obtained RNA

 1 μg RNA, 5 units of reverse transcriptase (reverse transcriptase), oligo dT-50 pmol, 0.1 M DTT- 2 μl, 10x reaction buffer - 2 μl, water - about 20 μl; The mixture was prepared in the above composition and reacted at 45 ° C for 10 minutes and at 38 ° C for 3 hours or longer in a PCR instrument.

(iii) securing the gene (DCT, Tyrp1) from cDNA

a. In order to amplify the gene from the cDNA generated in the above step, the mixture was prepared with the following composition, and the gene amplification reaction (PCR) was performed under the following conditions.

Mixture composition: 2 μl of cDNA solution, 5 units of pfu DNA amplification enzyme, 2 μl of 10 mM dNTP, 4 μl of 10 × reaction buffer, 2 pmol of each primer pair, and water - about 40 μl.

Amplification reaction conditions: 95 ° C 4 min - 1 time; 95 ° C for 20 seconds, 60 ° C for 20 seconds, 72 ° C for 1 minute and 50 seconds - 35 times; 72 占 폚 10 min - 1 time; Continue at 4 ℃.

The information of the primer pair used in the above reaction is shown in Table 1 below.

division Base sequence Primer for DCT amplification Forward primer Atgggccttgtgggatggg (SEQ ID NO: 1) Reverse primer Ctaggcttcctccgtgtatctcttgc (SEQ ID NO: 2) Tyrp1 primer for amplification Forward primer Atgaaatcttacaacgtcctccccct (SEQ ID NO: 3) Reverse primer Tcagaccatggagtggttaggattcg (SEQ ID NO: 4)

The DCT and Tyrp1 enzymes were prepared by inserting the genes obtained by the above method into the pET28a vector and constructing a fusion protein in which 6x His was attached to the N-terminus and transformed into BL21 (DE3) Escherichia coli. The process is as follows.

(i) the step of culturing the transformed E. coli

a. NdeI of the pET28a vector, and DCT or Tyrp1 gene into the XhoI restriction enzyme site, thereby preparing a recombinant DNA which produces 6x His ~ protein.

b. E. coli transformed with BL21 (DE3) was prepared by heat shock transformation.

c. The E. coli strain was cultured in 3 L of Luria broth at 37 DEG C and 200 rpm at OD600 ~ 0.7, followed by treatment with 0.1M IPTG to induce protein production.

d. After further culturing at 30 DEG C for 4 hours, E. coli was precipitated by centrifugation.

   (ii) Escherichia coli disruption step

a. The precipitated E. coli was resuspended in 200 ml of 1x PBS and washed by centrifugation.

b. The precipitated E. coli was suspended in 40 ml of 0.5 M NaCl, 5 mM imidazole, 20 mM Tris-HCl, pH 7.9; Lt; / RTI >

c. Escherichia coli was disrupted in an ultrasonic cell disruptor under the condition of Energy 38% max, total cell breaking time 4 min (2 second sonic treatment / 4 sec pause). The bottle containing the E. coli sample remained ice-filled during the cell disruption process.

d. The shredded E. coli was centrifuged at 13,000 rpm, 30 minutes, and 4 ° C.

e. Only the supernatant was collected in a clean conical tube.

   (iii) Purification of his tag-fusion protein using Ni-NTA agarose resin

a. The water-soluble protein solution obtained in the above step was mixed with Ni-NTA agarose resin and shaken for 30 minutes at 4 ° C to induce binding of the his tag-fusion protein to the Ni-NTA agarose resin.

b. The protein solution and the resin mixture were put into the column, and the column cock was opened to extract the liquid.

c. 400 ml of washing buffer (0.5 M NaCl, 60 mM imidazole, 20 mM Tris-HCl, pH 7.9) was passed through to remove proteins other than impurities and fusion proteins.

d. 3 ml of elution buffer (0.25 M NaCl, 500 mM imidazole, 20 mM Tris-HCl, pH 7.9) was added and allowed to stand for 10 minutes.

e. d process was repeated twice.

(iii) Protein purification using FPLC-size exclusion method

a. The recovered proteins were loaded onto FPLC equipped with a superdex S200 (GE healthcare) column and the proteins were separated by size.

b. Among the obtained fraction samples, fractions corresponding to his tag fusion proteins were identified by polyacrylamide gel electrophoresis and finally the desired his tag fusion protein was obtained.

c. Abs 280 values were measured, and protein concentrations were converted from the obtained values, and they are shown in Table 2 below.

division Protein concentration (OD ₂₈₀ nm) 1 mg DCT / ml 1.65 1 mg Tyrp1 / ml 1.42

Example 1

40 [micro] l of DCT having an activity of 1000 units / ml prepared in Preparation Example 1 and 40 [micro] l of Tyrp1 having an activity of 1000 units / ml were added to 40 [micro] l of tyrosinase enzyme having activity of 1000 unit / ml, 250 μl of 200 mM Tris buffer pH 8, 25 μl of 10% Tween 20, and 105 μl of distilled water were added to prepare an enzyme composition (stock solution).

Stock solution Volumn (500 μl) 200 mM Tris buffer pH8 250 μl 10% Tween 20 25 μl Tyrosinase (> = 1000 unit / ml) 40 μl DCT (> = 1000 unit / ml) 40 μl TYRP1 (> = 1000 unit / ml) 40 μl D.W 105 μl

Next, urine samples were collected from normal and cancer patients. The method of collecting the urine samples was done by taking 30 ml of sample from the urine without taking the first urine from the urethra in the first urine collection after the morning wake.

In order to measure the concentration of tyrosine present in a certain amount, the same amount of 500 μl of each of the urine collected from the normal person and the cancer patient was reacted with the enzyme composition of Example 1 (total 1000 μl each).

Then, the color of the urine produced by each of the produced eumelanins is compared with the control color table of FIG. 6, and is shown in Table 4 below.

Color of urine by eumelanin Urine of a normal person Yellow to tan Urine of cancer patient black

As shown in Table 4, the urine color produced by the reaction of the normal human urine with the enzyme composition of the present invention was yellow to yellowish brown, while the urine of the cancer patient was reacted with the enzyme composition, The color of the urine was confirmed to be black.

This shows that tyrosine is present in large amounts in urine of cancer patients.

Example 2

When the enzyme composition (30) of the present invention, which additionally contains DCT and Tyrp1, is used as a tyrosinase compared to the conventional enzyme composition containing only tyrosinase, the color of the urine produced by the produced uromelain appears more black The following experiment was carried out.

A mixture (A) in which 500 μl of normal human urine collected in the same manner as in Example 1 was mixed with 40 parts of tyrosinase, 20 units of DCT and 20 units of Tyrp1, A mixture (B) in which 500 μl of the composition was mixed with an existing enzyme composition containing only 40 units of tyrosinase, and 500 μl of urine of a cancer patient were mixed with 40 units of tyrosinase, 20 units of DCT and 20 units of Tyrp1 The mixture (C) mixed with the enzyme composition was reacted at 37 DEG C for 30 minutes, respectively.

7, the mixture (A) in which the enzyme composition according to the present invention and the urine of the normal person are reacted shows a yellow color, whereas the mixture (B) and (C) in which the urine of the cancer patient is reacted shows a darker tan or black Respectively.

Especially, as a result of urine detection of a cancer patient, it was confirmed that the color of the mixture (C) containing DCT and Tyrp 1 is more black than the mixture (B) containing only tyrosinase.

Therefore, by adding the DCT and Tyrp1 enzyme to the tyrosinase, the enzyme composition according to the present invention not only accelerates the pathway of eumelanin biosynthesis, but also can induce the color of the resulting eumelanin to a more black-colored pathway Respectively.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the present invention as defined by the appended claims. And it is to be understood that such modified embodiments belong to the scope of protection of the present invention defined by the appended claims.

10: Case 11:
15: Stopper 20: Enzyme receptacle
22: inlet 30: enzyme composition
40: Color detection sensor 41: Polarization filter
42: ultraviolet cut filter 50: light source
60: information processor 61: color detector
62: Judgment section 63: Result notification section
71: valve body 72: hinge shaft
73: Plotter 74: Coupling piece
75: Magnet

<110> CubeBio <120> Enzyme Compositions for Detecting Cancer Biomarker Tyrosine <130> DP18030 <160> 7 <170> KoPatentin 3.0 <210> 1 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> primer <400> 1 atgggccttg tgggatggg 19 <210> 2 <211> 26 <212> RNA <213> Artificial Sequence <220> <223> primer <400> 2 ctaggcttcc tccgtgtatc tcttgc 26 <210> 3 <211> 26 <212> RNA <213> Artificial Sequence <220> <223> primer <400> 3 atgaaatctt acaacgtcct ccccct 26 <210> 4 <211> 26 <212> RNA <213> Artificial Sequence <220> <223> primer <400> 4 tcagaccatg gagtggttag gattcg 26 <210> 5 <211> 556 <212> PRT <213> Agaricus bisporus <400> 5 Met Ser Leu Ile Ala Thr Val Gly Pro Thr Gly Gly Val Lys Asn Arg   1 5 10 15 Leu Asn Ile Val Asp Phe Val Lys Asn Glu Lys Phe Phe Thr Leu Tyr              20 25 30 Val Arg Ser Leu Glu Leu Leu Gln Ala Lys Glu Gln His Asp Tyr Ser          35 40 45 Ser Phe Phe Gln Leu Ala Gly Ile His Gly Leu Pro Phe Thr Glu Trp      50 55 60 Ala Lys Glu Arg Pro Ser Met Asn Leu Tyr Lys Ala Gly Tyr Cys Thr  65 70 75 80 His Gly Gln Val Leu Phe Pro Thr Trp His Arg Thr Tyr Leu Ser Val                  85 90 95 Phe Glu Gln Ile Leu Gln Gly Ala Ala Ile Glu Val Ala Asn Lys Phe             100 105 110 Thr Ser Asn Gln Thr Asp Trp Ile Gln Ala Ala Gln Asp Leu Arg Gln         115 120 125 Pro Tyr Trp Asp Trp Gly Phe Glu Leu Met Pro Pro Asp Glu Val Ile     130 135 140 Lys Asn Glu Glu Val Asn Ile Thr Asn Tyr Asp Gly Lys Lys Ile Ser 145 150 155 160 Val Lys Asn Pro Ile Leu Arg Tyr His Phe His Pro Ile Asp Pro Ser                 165 170 175 Phe Lys Pro Tyr Gly Asp Phe Ala Thr Trp Arg Thr Thr Val Arg Asn             180 185 190 Pro Asp Arg Asn Arg Arg Glu Asp Ile Pro Gly Leu Ile Lys Lys Met         195 200 205 Arg Leu Glu Glu Gly Gln Ile Arg Glu Lys Thr Tyr Asn Met Leu Lys     210 215 220 Phe Asn Asp Ala Trp Glu Arg Phe Ser Asn His Gly Ile Ser Asp Asp 225 230 235 240 Gln His Ala Asn Ser Leu Glu Ser Val His Asp Asp Ile His Val Met                 245 250 255 Val Gly Tyr Gly Lys Ile Glu Gly His Met Asp His Pro Phe Phe Ala             260 265 270 Ala Phe Asp Pro Ile Phe Trp Leu His His Thr Asn Val Asp Arg Leu         275 280 285 Leu Ser Leu Trp Lys Ala Ile Asn Pro Asp Val Trp Val Thr Ser Gly     290 295 300 Arg Asn Arg Asp Gly Thr Met Gly Ile Ala Pro Asn Ala Gln Ile Asn 305 310 315 320 Asp Glu Thr Pro Leu Glu Pro Phe Tyr Gln Ser Glu Asp Lys Val Trp                 325 330 335 Thr Ser Ala Ser Leu Ala Asp Thr Ala Arg Leu Gly Tyr Ser Tyr Pro             340 345 350 Asp Phe Asp Lys Leu Val Gly Gly Thr Lys Glu Leu Ile Arg Asp Ala         355 360 365 Ile Asp Asp Leu Ile Asp Glu Arg Tyr Gly Ser Lys Pro Ser Ser Gly     370 375 380 Ala Arg Asn Thr Ala Phe Asp Leu Leu Ala Asp Phe Lys Gly Ile Thr 385 390 395 400 Lys Glu His Lys Glu Asp Leu Lys Met Tyr Asp Trp Thr Ile His Val                 405 410 415 Ala Phe Lys Lys Phe Glu Leu Lys Glu Ser Phe Ser Leu Leu Phe Tyr             420 425 430 Phe Ala Ser Asp Gly Gly Asp Tyr Asp Gln Glu Asn Cys Phe Val Gly         435 440 445 Ser Ile Asn Ala Phe Arg Gly Thr Thr Pro Glu Thr Cys Ala Asn Cys     450 455 460 Gln Asp Asn Glu Asn Leu Ile Gln Glu Gly Phe Ile His Leu Asn His 465 470 475 480 Tyr Leu Ala Arg Asp Leu Glu Ser Phe Glu Pro Gln Asp Val His Lys                 485 490 495 Phe Leu Lys Glu Lys Gly Leu Ser Tyr Lys Leu Tyr Ser Arg Glu Asp             500 505 510 Lys Ser Leu Thr Ser Leu Ser Val Lys Ile Glu Gly Arg Pro Leu His         515 520 525 Leu Pro Pro Gly Glu His Arg Pro Lys Tyr Asp His Thr Gln Asp Arg     530 535 540 Val Val Phe Asp Asp Val Ala Val His Val Ile Asn 545 550 555 <210> 6 <211> 517 <212> PRT <213> Mus musculus <400> 6 Met Gly Leu Val Gly Trp Gly Leu Leu Leu Gly Cys Leu Gly Cys Gly   1 5 10 15 Ile Leu Leu Arg Ala Arg Ala Gln Phe Pro Arg Val Cys Met Thr Leu              20 25 30 Asp Gly Val Leu Asn Lys Glu Cys Cys Pro Pro Leu Gly Pro Glu Ala          35 40 45 Thr Asn Ile Cys Gly Phe Leu Glu Gly Arg Gly Gln Cys Ala Glu Val      50 55 60 Gln Thr Asp Thr Arg Pro Trp Ser Gly Pro Tyr Ile Leu Arg Asn Gln  65 70 75 80 Asp Asp Arg Glu Gln Trp Pro Arg Lys Phe Phe Asn Arg Thr Cys Lys                  85 90 95 Cys Thr Gly Asn Phe Ala Gly Tyr Asn Cys Gly Gly Cys Lys Phe Gly             100 105 110 Trp Thr Gly Pro Asp Cys Asn Arg Lys Lys Pro Ala Ile Leu Arg Arg         115 120 125 Asn Ile His Ser Leu Thr Ala Gln Glu Arg Glu Gln Phe Leu Gly Ala     130 135 140 Leu Asp Leu Ala Lys Lys Ser Ile His Pro Asp Tyr Val Ile Thr Thr 145 150 155 160 Gln His Trp Leu Gly Leu Leu Gly Pro Asn Gly Thr Gln Pro Gln Ile                 165 170 175 Ala Asn Cys Ser Val Tyr Asp Phe Phe Val Trp Leu His Tyr Tyr Ser             180 185 190 Val Arg Asp Thr Leu Leu Gly Pro Gly Arg Pro Tyr Lys Ala Ile Asp         195 200 205 Phe Ser His Gln Gly Pro Ala Phe Val Thr Trp His Arg Tyr His Leu     210 215 220 Leu Trp Leu Glu Arg Glu Leu Gln Arg Leu Thr Gly Asn Glu Ser Phe 225 230 235 240 Ala Leu Pro Tyr Trp Asn Phe Ala Thr Gly Lys Asn Glu Cys Asp Val                 245 250 255 Cys Thr Asp Glu Leu Leu Gly Ala Ala Arg Gln Asp Asp Pro Thr Leu             260 265 270 Ile Ser Arg Asn Ser Arg Phe Ser Thr Trp Glu Ile Val Cys Asp Ser         275 280 285 Leu Asp Asp Tyr Asn Arg Arg Val Thr Leu Cys Asn Gly Thr Tyr Glu     290 295 300 Gly Leu Leu Arg Arg Asn Lys Val Gly Arg Asn Asn Glu Lys Leu Pro 305 310 315 320 Thr Leu Lys Asn Val Gln Asp Cys Leu Ser Leu Gln Lys Phe Asp Ser                 325 330 335 Pro Pro Phe Phe Gln Asn Ser Thr Phe Ser Phe Arg Asn Ala Leu Glu             340 345 350 Gly Phe Asp Lys Ala Asp Gly Thr Leu Asp Ser Gln Val Met Asn Leu         355 360 365 His Asn Leu Ala His Ser Phe Leu Asn Gly Thr Asn Ala Leu Pro His     370 375 380 Ser Ala Asn Asp Pro Val Phe Val Val Leu His Ser Phe Thr Asp 385 390 395 400 Ala Ile Phe Asp Glu Trp Leu Lys Arg Asn Asn Pro Ser Thr Asp Ala                 405 410 415 Trp Pro Gln Glu Leu Ala Pro Ile Gly His Asn Arg Met Tyr Asn Met             420 425 430 Val Pro Phe Pro Pro Val Thr Asn Glu Glu Leu Phe Leu Thr Ala         435 440 445 Glu Gln Leu Gly Tyr Asn Tyr Ala Val Asp Leu Ser Glu Glu Glu Ala     450 455 460 Pro Val Trp Ser Thr Thr Leu Ser Val Val Ile Gly Ile Leu Gly Ala 465 470 475 480 Phe Val Leu Leu Leu Gly Leu Leu Ala Phe Leu Gln Tyr Arg Arg Leu                 485 490 495 Arg Lys Gly Tyr Ala Pro Leu Met Glu Thr Gly Leu Ser Ser Lys Arg             500 505 510 Tyr Thr Glu Glu Ala         515 <210> 7 <211> 537 <212> PRT <213> Mus musculus <400> 7 Met Lys Ser Tyr Asn Val Leu Pro Leu Ala Tyr Ile Ser Leu Phe Leu   1 5 10 15 Met Leu Phe Tyr Gln Val Trp Ala Gln Phe Pro Arg Glu Cys Ala Asn              20 25 30 Ile Glu Ala Leu Arg Arg Gly Val Cys Cys Pro Asp Leu Leu Pro Ser          35 40 45 Ser Gly Pro Gly Thr Asp Pro Cys Gly Ser Ser Ser Gly Arg Gly Arg      50 55 60 Cys Val Ala Val Ile Ala Asp Ser Arg Pro His Ser Arg His Tyr Pro  65 70 75 80 His Asp Gly Lys Asp Asp Arg Glu Ala Trp Pro Leu Arg Phe Phe Asn                  85 90 95 Arg Thr Cys Gln Cys Asn Asp Asn Phe Ser Gly His Asn Cys Gly Thr             100 105 110 Cys Arg Pro Gly Trp Arg Gly Ala Ala Cys Asn Gln Lys Ile Leu Thr         115 120 125 Val Arg Arg Asn Leu Leu Asp Leu Ser Pro Glu Glu Lys Ser His Phe     130 135 140 Val Arg Ala Leu Asp Met Ala Lys Arg Thr Thr His Pro Gln Phe Val 145 150 155 160 Ile Ala Thr Arg Arg Leu Glu Asp Ile Leu Gly Pro Asp Gly Asn Thr                 165 170 175 Pro Gln Phe Glu Asn Ile Ser Val Tyr Asn Tyr Phe Val Trp Thr His             180 185 190 Tyr Tyr Ser Val Lys Lys Thr Phe Leu Gly Thr Gly Gln Glu Ser Phe         195 200 205 Gly Asp Val Asp Phe Ser His Glu Gly Pro Ala Phe Leu Thr Trp His     210 215 220 Arg Tyr His Leu Leu Gln Leu Glu Arg Asp Met Gln Glu Met Leu Gln 225 230 235 240 Glu Pro Ser Phe Ser Leu Pro Tyr Trp Asn Phe Ala Thr Gly Lys Asn                 245 250 255 Val Cys Asp Val Cys Thr Asp Asp Leu Met Gly Ser Arg Ser Asn Phe             260 265 270 Asp Ser Thr Leu Ile Ser Pro Asn Ser Val Phe Ser Gln Trp Arg Val         275 280 285 Val Cys Glu Ser Leu Glu Glu Tyr Asp Thr Leu Gly Thr Leu Cys Asn     290 295 300 Ser Thr Glu Gly Gly Pro Ile Arg Arg Asn Pro Ala Gly Asn Val Gly 305 310 315 320 Arg Pro Ala Val Gln Arg Leu Pro Glu Pro Gln Asp Val Thr Gln Cys                 325 330 335 Leu Glu Val Arg Phe Asp Thr Pro Pro Phe Tyr Ser Asn Ser Thr             340 345 350 Asp Ser Phe Arg Asn Thr Val Glu Gly Tyr Ser Ala Pro Thr Gly Lys         355 360 365 Tyr Asp Pro Ala Val Arg Ser Leu His Asn Leu Ala His Leu Phe Leu     370 375 380 Asn Gly Thr Gly Gly Gln Thr His Leu Ser Pro Asn Asp Pro Ile Phe 385 390 395 400 Val Leu Leu His Thr Phe Thr Asp Ala Val Phe Asp Glu Trp Leu Arg                 405 410 415 Arg Tyr Asn Ala Asp Ile Ser Thr Phe Pro Leu Glu Asn Ala Pro Ile             420 425 430 Gly His Asn Arg Gln Tyr Asn Met Val Pro Phe Trp Pro Pro Val Thr         435 440 445 Asn Thr Glu Met Phe Val Thr Ala Pro Asp Asn Leu Gly Tyr Ala Tyr     450 455 460 Glu Val Gln Trp Pro Gly Gln Glu Phe Thr Val Ser Glu Ile Ile Thr 465 470 475 480 Ile Ala Val Ala Ala Leu Leu Ala Val Ala Ile Phe Gly Val                 485 490 495 Ala Ser Cys Leu Ile Arg Ser Ser Ser Thr Lys Asn Glu Ala Asn Gln             500 505 510 Pro Leu Leu Thr Asp His Tyr Gln Arg Tyr Ala Glu Asp Tyr Glu Glu         515 520 525 Leu Pro Asn Pro Asn His Ser Met Val     530 535

Claims (8)

A case 10 having an inlet 11 into which urine is injected;
An enzyme accommodating portion 20 formed inside the case 10;
An enzyme composition (30) accommodated in the enzyme accommodating portion (20) and converting tyrosine (L-Tyrosine) present in the urine into melanin;
A color detecting sensor 40 for detecting the color of urine in the enzyme accommodating unit 20 after the urine is input into the enzyme accommodating unit 20;
A light source (50) for emitting light to the enzyme storage part (20);
An information processing unit (60) for analyzing color information detected by the color detection sensor (40) to determine the presence of cancer and outputting the determination result to the outside;
/ RTI &gt;
Wherein said enzyme composition comprises tyrosinase, Dopachrome tautomerase (DCT), and tyrosinase-related protein 1 (Tyrp1). delete The image processing apparatus according to claim 1, wherein the information processing unit (60) comprises: a color detecting unit (61) for receiving a color signal from the color detecting sensor (40) And a result notification unit 63 for outputting the result determined by the determination unit 62 to the outside. Self color detection type cancer diagnosis kit. 4. The apparatus of claim 3, wherein the light source (50) is a white light source for emitting white light and an ultraviolet light source for emitting ultraviolet light,
And a polarizing filter (41) and an ultraviolet cut filter (42) are installed under the color detecting sensor (40). The apparatus according to claim 1, wherein the urine is provided at an inlet (22) communicating with the inlet (11) and the enzyme accommodating portion (20) so as to be rotatable about a hinge axis (72) A floater 73 installed on the valve body 71 and floated by urine and a metal or magnet mounted on the floater 73. [ , And a magnet (75) for fixing the coupling piece (74) by a magnetic force. delete [Claim 2] The method according to claim 1, wherein the enzymatic composition is prepared by mixing 40 units of tyrosinase in a weight ratio of 1: 0.5 to 2: 1 by weight of the tyrosinase-conjugated enzyme (DCT) and tyrosinase-related protein 1 (Tyrp1) in a ratio of 0.5 to 2 by weight, wherein the color of urine is changed from brown to black as the concentration of tyrosine is increased by converting L-Tyrosine into Eumelanin Color detection type cancer diagnosis kit. The enzyme composition according to claim 1 or 7, wherein the enzyme composition is a phosphate buffer or a Tris-HCl buffer solution having a final concentration of 50 to 100 mM, a Tween 20 , And distilled water, and is made into a liquid having a final reaction pH of 7.0 to 8.5.

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