KR101702601B1 - Portable Diagnostic Kit for Detecting Cancer Existence Using Enzyme Composition - Google Patents

Abstract

The present invention relates to a portable cancer diagnosing kit for diagnosing presence of cancer and metabolic diseases by detecting a concentration of a cancer biomarker, tyrosine, existing in urine. The portable cancer diagnosing kit of the present invention comprises: a case (10) having a first sample chamber (11) and a second sample chamber (12) formed therein; an inspection window (15) made of a transparent material, and formed in a position corresponding to the second sample chamber on one surface of the case; a comparison window (16) made of a transparent material, and formed in a position corresponding to the first sample chamber (11) on another surface of the case; a separation member installed to be longitudinally moved by passing through the top end part of the case (10); a punching portion (50) installed to upwardly protrude on the top surface of the second sample chamber (12) of the case (10); an enzyme storing pack (40) installed at the top of the top surface of the second sample chamber (12) to be longitudinally moved, and accommodating a tyrosine-detecting enzyme composition (30) for converting L-tyrosine existing in urine into melanin; an elastic member (60) for separating the bottom end part of the enzyme storing pack (40) from the punching portion (50) by applying elastic force to the top side of the enzyme storing pack (40) with respect to the top surface of the second sample chamber (12); and a pressing portion (25) installed at the top of the separation member, and downwardly pressing the enzyme storing pack (40) as the separation member descends.

Description

[0001] The present invention relates to a portable cancer diagnostic kit using an enzyme composition,

The present invention relates to a kit for diagnosing the presence or absence of cancer and a disease, and more particularly to a kit for detecting the presence of cancer biomarker tyrosine (Tyrosinase), a dipachrome mutant enzyme The present invention relates to a portable cancer diagnostic kit for diagnosing the presence or absence of cancer and metabolic diseases through the color change of urine by mixing an enzyme composition containing Dopachrome tautomerase (DCT) and tyrosinase-related protein 1 (Tyrp1) with a predetermined amount of urine .

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, The enzyme composition for detection of cancer biomarkers that can detect the presence of cancer and metabolic diseases is used in environmentally friendly manner and the testee injects a predetermined amount of urine into the kit easily and easily The present invention provides a portable cancer diagnostic kit that can be used as a diagnostic tool.

Another object of the present invention is to provide a portable cancer diagnostic kit which can directly compare the color of the urine with the enzyme composition and the color of the urine with the enzyme composition injected, .

According to another aspect of the present invention, there is provided a portable cancer diagnostic kit including a first sample chamber having an inlet for introducing urine into one side of an upper end of the first sample chamber, a second sample chamber formed at one side of the first sample chamber, And a partition wall having a passage dividing the first sample chamber and the second sample chamber and connecting the first sample chamber and the second sample chamber to each other at a lower end thereof; A transparent inspection window formed on a surface of the case at a position corresponding to the second sample chamber; A transparent window formed on one surface of the case at a position corresponding to the first sample chamber; A separating member which is installed to be slidable up and down with respect to the partition wall through the upper end of the case to fill a predetermined amount of urine in the second sample chamber and then descend by a user's operation to close the passage of the partition wall; A perforation protruding upward from an upper surface of the second sample chamber of the case, the perforation being formed at a central portion thereof so as to penetrate the channel in a vertical direction and having a pointed perforation protrusion at an upper end thereof; An enzyme composition for detecting tyrosine, which is provided on the upper side of the upper surface of the second sample chamber so as to be movable upward and downward and converts tyrosine (L-Tyrosine) present in the urine into melanin, A tubular enzyme storage pack closed by a sealing film perforable by perforation; An elastic member for applying an elastic force upward to the enzyme storage pack on the upper surface of the second sample chamber to separate the sealing film of the enzyme storage pack from perforation; And a pressing unit installed on the separating member to press the enzyme storage pack downward as the separating member descends.

The enzyme composition of the present invention is characterized in that it forms black melanin from the tyrosine to distinguish it from the urine color of the sample.

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 the enzyme composition for detecting tyrosine in the enzyme storage pack is mixed with the urine, the enzyme composition reacts with the cancer biomarker L-tyrosine present in the urine, Since the color changes from brown to black, the occurrence of cancer and metabolic diseases can be immediately confirmed with the naked eye.

Therefore, it is possible to easily recognize the occurrence of cancer and metabolic diseases in a home, not a specialized institution such as a hospital.

Particularly, according to the present invention, when a predetermined amount of urine flows into the sample chamber of the case, the inlet is automatically closed by the plotter, so that a certain amount of urine can be injected into the case. Therefore, the amount of the enzyme composition stored in the enzyme storage pack and the amount of urine can be tested at a predetermined weight ratio, preferably 1: 1 to 3: 1.

In addition, the space in which the urine is received after the urine is injected into the case is divided into a space where the enzyme composition is injected and a space in which the enzyme composition is not injected, and a transparent window is formed in each space so that the enzyme composition is not injected into the urine And the color of the urine in the state where the enzyme composition is injected into the urine can be directly compared with each other, so that the color change of the urine can be clearly recognized.

1 is a perspective view illustrating a portable cancer diagnostic kit according to an embodiment of the present invention.
FIGS. 2 to 4 are sectional views sequentially showing operation examples of the cancer diagnostic kit of FIG. 1. FIG.
Fig. 5 is a plan view of the cancer diagnostic kit of Fig. 1;
FIG. 6 is a control color chart for measuring the color of the eumelanin produced by the embodiment of the enzyme composition for detecting tyrosine 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 for detecting tyrosine 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.

1 to 5 illustrate a cancer diagnosis kit according to an embodiment of the present invention. The cancer diagnosis kit of the present invention includes a first sample chamber 11 and a second sample chamber 12 in which a first sample chamber 11 and a second sample chamber 12 are formed (10); a transparent window (15) formed on one surface of the case (10) at a position corresponding to the second sample chamber (12) A separating member provided so as to be able to move up and down through the upper end of the case 10 and a separating member provided at a position corresponding to the second sample chamber 11 of the case 10, 12), an upper surface of the upper surface of the second sample chamber 12, which is provided so as to be movable upward and downward, and a tyrosine (L-Tyrosine ) Into melanin was housed in an enzyme storage pack (" An elastic member for upwardly urging the enzyme storage pack 40 against the upper surface of the second sample chamber 12 to separate the lower end of the enzyme storage pack 40 from the perforations 50; And a pressurizing unit 25 installed on the separating member and pressing the enzyme storage pack 40 downward as the separating member descends.

The case 10 has a cylindrical shape such as a cylinder or a rectangular parallelepiped and has a cylindrical shape such that the color of the urine inside the first sample chamber 11 and the second sample chamber 12 can be visually observed 10 is provided with a transparent window 16 and an inspection window 15 at positions corresponding to the first sample chamber 11 and the second sample chamber 12, respectively. When the urine reaches the predetermined water level of the first sample chamber 11, a phrase or pattern indicating the full water level, for example, "Full" may be displayed in the comparison window 16 so that urine is no longer injected .

A colourimetric color table 17 is additionally displayed near the examination window 15 of the case 10 so as to discriminate the presence or absence of cancer or metabolic disease through the color of the urine observed through the examination window 15 .

A partition wall 13 separating the first sample chamber 11 and the second sample chamber 12 is provided in the case 10 so as to extend in the vertical direction. A passage 14 connecting the first sample chamber 11 and the second sample chamber 12 is formed at a lower end of the partition wall 13. The passage 14 may be closed closed by a sealing pad 22 formed at the lower end of the separating member.

A guide groove 13a is formed in the partition wall 13 so that the separating member is vertically movable.

A valve plate 18 having a valve hole 18a through which a urine flowing through the inlet 11a passes is formed at a middle portion of the first sample chamber 11 of the case 10, Is installed across the top of the chamber (11). In the first sample chamber 11, a floater 70 floating by the urine flowing into the first sample chamber 11 is provided and the urine is filled up to a certain height in the first sample chamber 11 The plotter 70 closes the valve hole 18a of the valve plate 18 so that only a predetermined amount of urine can be filled in the first sample chamber 11 and the second sample chamber 12. [

The plotter 70 is made of synthetic resin such as plastic of hollow sphere and floated inside the first sample chamber 11 by urine introduced into the first sample chamber 11.

The separating member includes a slide bar 21 slidably installed inside the guide groove 13a of the partition wall 13 and provided with the pressing portion 25 at an upper portion thereof, A sealing pad 22 installed on the upper surface of the partition wall 13 to close a passage of the lower part of the partition wall 13 and an upper end portion of the slide bar 21 exposed in the outside of the upper surface of the case 10, And a pushing control protrusion 24 protruding radially outward from the outer surface of the operation cap 23. The pushing protrusion 24 protrudes radially outward. An operation groove 19 is formed at the upper end of the case 10 so as to have an inner diameter larger than the diameter of the operation cap 23 and into which the operation cap 23 can be inserted. ) Is formed in a radial direction on one side of the press-fit recess (19a).

Therefore, even if the user presses the operation cap 23 in a state where the pushing projection 24 and the press-permitting groove 19a do not coincide with each other, the pushing control projection 24 is caught by the outer upper end of the operation groove 19 The lowering of the operation cap 23 is restricted and the operation cap 23 is rotated to press the operation cap 23 in a state where the press control projection 24 and the press- And the pushing control protrusion 24 are inserted into the operating groove 19 and the press-permitting groove 19a, respectively.

The pressing operation of the operating cap 23 is controlled by the pressing control projection 24 of the operating cap 23 and the pressing allowing groove 19a of the operating groove 19 so that the case 10 is filled with urine It is possible to prevent the enzyme storage pack 40 from being punctured by the operation cap 23 being pushed due to a mistake of the user or an impact with another object.

The enzyme storage pack 40 is made of a plastic or metal tubular material having a lower surface open and accommodates the enzyme composition 30 therein. The lower surface of the enzyme storage pack 40 is closed by a sealing film 41 made of a thin aluminum foil or resin so that the enzyme composition 30 is safely stored in the enzyme storage pack 40.

The sealing film 41 is pierced by the perforation 50 to be stored in the enzyme storage pack 40 when the enzyme storage pack 40 is pressed downward by the pressing unit 25 connected to the separating member. The enzyme composition 30 in the second sample chamber 12 can be introduced into the second sample chamber 12 through the perforation 50.

The perforation 50 is formed to protrude upward at the center of the upper surface of the second sample chamber 12, the channel 52 is vertically penetrated, and a sealing film 41 is formed at the upper end. And a perforated protrusion (51) of a pointed shape is provided so as to be torn and perforated.

The elastic member 60 is disposed between the upper surface of the second sample chamber 12 and the lower surface of the enzyme storage pack 40 so that the upper surface of the second sample chamber 12 The sealing film 41 of the enzyme storage pack 40 is separated from the perforations 50 by applying an elastic force upward to the sealing film 41 of the enzyme storage pack 40 so that the user does not lower the separating member. Is not punctured by the perforation (50).

Meanwhile, the enzyme composition 30 is a liquid composition contained in the enzyme storage pack 40, and includes a tyrosinase, a Dopachrome tautomerase (DCT), and a tyrosinase-related protein 1 (Tyrp1), converts tyrosine (L-Tyrosine) present in the urine into melanin to change the color of the urine.

The amount of the enzyme composition 30 stored in the enzyme storage pack 40 and the amount of urine stored in the second sample chamber 12 are preferably in a ratio of 1: 1 to 3: 1 by weight, Do.

When the enzyme composition 30 is mixed with the urine in the second sample chamber 12, it is possible to know whether the enzyme composition 30 has a disease such as cancer through a change in urine color. As described above, A transparent inspection window 15 is provided on one side of the case 10 so that the inside of the second sample chamber 12 can be seen.

Since the urine color changes from brown to black as the tyrosine concentration in the urine increases, the color of the reference color table 17 displayed on the case 10 is also changed between brown and black The colors are displayed.

The components and the action of the enzyme composition (30) will be described in more detail below.

The portable cancer diagnostic kit of the present invention configured as above operates as follows.

First, urine of a subject is collected in a separate container (for example, a paper cup or the like), and urine is injected through an inlet 11a formed in the upper end of the case 10, And flows into the first sample chamber 11 through the valve hole 18a of the plate 18. [

The urine introduced into the first sample chamber 11 flows into the second sample chamber 12 through the passage 14 formed at the lower end of the partition wall 13 as shown in FIG. The sample chamber 11 and the second sample chamber 12 are simultaneously filled with urine. At this time, the floater 70 in the first sample chamber 11 floats up by the urine.

The urine is filled up to a predetermined level in the first sample chamber 11 and the second sample chamber 12 and the plotter 70 is inserted into the valve hole 18a of the valve plate 18 provided in the first sample chamber 11 To close the valve hole 18a, the user stops the injection of the urine through the inlet port 11a.

Then, the user rotates the operation cap 23 in the direction of the arrow in Fig. 5 so that the pushing control protrusion 24 is aligned with the pushing recess 19a of the operation groove 19, 23, the slide bar 21 descends along the guide groove 13a of the partition wall 13. As shown in Fig.

The sealing pad 22 provided at the lower end of the slide bar 21 completely closes the passage 14 of the partition wall 13 to completely separate the first sample chamber 11 and the second sample chamber 12 At the same time, the pressing portion 25 on the slide bar 21 presses the upper surface of the enzyme storage pack 40 downward.

When the pressurizing unit 25 presses the enzyme storage pack 40 downward, the elastic storage member 40 supporting the enzyme storage pack 40 is compressed and the enzyme storage pack 40 is lowered. The sealing film 41 at the lower end of the pack 40 is torn and punctured by the perforation 50. [

The enzyme composition 30 contained in the enzyme storage pack 40 is lowered along the flow path 52 of the perforation 50 and mixed with the urine in the second sample chamber 12.

As described above, when the enzyme composition 30 reacts with tyrosine in the urine and the urine color changes to black, it can be judged that cancer or disease is present. At this time, since the enzyme composition 30 is not mixed in the urine of the first sample chamber 11 and the enzyme composition 30 is mixed only in the urine of the second sample chamber 12, By comparing the color of the initial urine observed and the color of the urine observed through the examination window 15, it is possible to reliably recognize whether there is a change in color of the urine.

As described above, when the enzyme composition 30 is mixed with the urine contained in the second sample chamber 12 of the case 10, the enzyme composition 30 is mixed with the cancer biomarker tyrosine (L-Tyrosine) To melanin, so that the user can easily diagnose cancer or disease.

The enzyme composition 30 for detecting the tyrosine includes an enzyme that inhibits tyrosine kinases such as tyrosinase, Dopachrome tautomerase (DCT) and 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 melanin and measuring the concentration through the color of the melanin.

In particular, in the present invention, in the conversion of L-tyrosine into melanin using the enzyme composition (30), the melanin is characterized by being 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, when measuring the concentration of tyrosine present in the urine through the hue of the produced eumelanin, the color of the produced eumelanin is compared with that of the control color table (17) to confirm the concentration of tyrosine in the urine . This makes it possible to easily diagnose the occurrence of cancer and metabolic diseases.

Hereinafter, the tyrosine detecting enzyme composition (30) of the present invention will be described in more detail by way of 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: First sample chamber
11a: Inlet port 12: Second sample chamber
13: partition wall 13a: guide groove
14: passage 15: inspection window
16: Comparison window 17: Contrast color table
18: valve plate 18a: valve hole
19: operating groove 19a: pressing allowance groove
21: Slide bar 22: Sealing pad
23: operation cap 24: pushing control projection
25: Pressurizing part 30: Enzyme composition
40: Enzyme storage pack 41: Sealing film
50: Perforation 51: Perforation projection
52: channel 60: elastic member
70: Plotter

<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 (6)

A second sample chamber formed on one side of the first sample chamber, and a second sample chamber formed on the other side of the first sample chamber and the second sample chamber, A case having a partition wall having a passage for connecting the first sample chamber and the second sample chamber to each other;
A transparent inspection window formed on a surface of the case at a position corresponding to the second sample chamber;
A transparent window formed on one surface of the case at a position corresponding to the first sample chamber;
A separating member which is installed to be slidable up and down with respect to the partition wall through the upper end of the case to fill a predetermined amount of urine in the second sample chamber and then descend by a user's operation to close the passage of the partition wall;
A perforation protruding upward from an upper surface of the second sample chamber of the case, the perforation being formed at a central portion thereof so as to penetrate the channel in a vertical direction and having a pointed perforation protrusion at an upper end thereof;
An enzyme composition for detecting tyrosine, which is provided on the upper side of the upper surface of the second sample chamber so as to be movable upward and downward and converts tyrosine (L-Tyrosine) present in the urine into melanin, A tubular enzyme storage pack closed by a sealing film perforable by perforation;
An elastic member for applying an elastic force upward to the enzyme storage pack on the upper surface of the second sample chamber to separate the sealing film of the enzyme storage pack from perforation;
A pressing unit installed on the separating member to press the enzyme storage pack downward as the separating member descends;
Wherein the diagnostic kit comprises: The method according to claim 1, wherein the enzyme composition comprises a tyrosinase, a Dopachrome tautomerase (DCT), and a tyrosinase-related protein 1 (Tyrp1) Kits. [3] The enzyme composition according to claim 2, 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 an amount of 0.5 to 2 by weight, wherein the color of the urine is changed from brown to black as the tyrosine concentration is increased by converting L-tyrosine into Eumelanin. Cancer diagnosis kit. The enzyme composition for detecting tyrosine according to claim 2 or 3, wherein the enzyme composition for detecting tyrosine is a phosphate buffer or a Tris-HCl buffer solution having a final concentration of 50 to 100 mM, % Tween 20, and distilled water, and the liquid is made to be a liquid having a final reaction pH of 7.0 to 8.5, and is encapsulated in an enzyme storage pack. [2] The apparatus according to claim 1, wherein a valve plate, through which a valve hole through which the urine flowing through the inlet port passes, is installed across the upper part of the first sample chamber, Wherein a plotter floating by urine is provided in the sample chamber and the plotter closes the valve hole of the valve plate when the urine is filled up to a predetermined height in the first sample chamber. The slide bar according to claim 1, wherein the separating member comprises: a slide bar slidably installed inside the guide groove formed in the up-and-down direction within the partition wall and having the pressing portion provided thereon; A working cap rotatably installed at an upper end of a slide bar exposed outside the upper surface of the case in a cylindrical form with a lower surface opened; And a pushing protrusion formed to protrude outward in the direction,
Wherein an operating cavity is formed at an upper end of the case so as to have an inner diameter larger than the diameter of the operating cap and into which the operating cap can be inserted, and a press- In the state where the pressing projection and the pressing allowance groove are not aligned with each other, the pressing projection is caught by the upper end of the operating groove to restrict the lowering of the actuating cap. When the pressing projection and the press- And the control projection is lowered while being inserted into the operating groove and the pressing allowance groove, respectively.

Images