KR100448488B1 - A Diagnostic System Employing Apolipoprotein A-1 as a Marker of Behcet's Disease - Google Patents

Abstract

The present invention provides a system for diagnosing Behcet's disease and hydrolysis of apolipoprotein A-1 comprising electrophoresis of a protein obtained from plasma or serum, and then confirming the presence of hydrolyzed apolipoprotein A-1. The present invention relates to a method of screening a drug for inhibiting Behcet's disease by selecting an inhibitor. According to the present invention, a protein including all or a portion of apolipoprotein A-1 is used as a disease marker, and the difference between the plasma protein in patients with Behcet's disease and those in non-Behcet's disease is used to diagnose and treat Behcet's disease. We will be able to observe the prognosis, develop a cure for Behcet's disease.

Description

A diagnostic system using apolipoprotein A-1 as a marker of Behcet's disease

The present invention relates to a diagnostic system using apolipoprotein A-1 (Apolipoprotein A-1) as a disease marker of Behcet's disease. More specifically, the present invention provides a system for diagnosing Behcet's disease and apolipoprotein A-1 comprising electrophoresis of a protein obtained from plasma, and then confirming the presence of hydrolyzed apolipoprotein A-1. The present invention relates to a method for searching for a therapeutic agent for Behcet's disease, comprising selecting a substance that inhibits hydrolysis.

In the 5th century BC, Hippocrates reported skin diseases that result from frequent mouth breaks, genital areas, and repeated inflammation in the eyes. Later, in 1937, Turkey's dermatologist, Hulusi Behcet (1889-1948) defined the above symptoms as a unique syndrome and suggested the cause of the virus. The symptom is known as clinical syndrome with three main symptoms of recurrent oral and genital ulcers and ocular lesions.

Currently, Behcet's disease is recognized as a disease that occurs in various organs, and the main symptoms are oral cavity, genital ulcer, eye lesions and skin lesions, mainly cardiovascular system, respiratory system, digestive system, central nervous system, urinary system, etc. It is showing symptoms. Behcet's disease is reported worldwide, but relatively rarely in Western Europe and the United States. It is said to have a connection with Mediterranean road countries such as Turkey, Greece, Italy, Lebanon, and Arabia, and Korea, Japan, and China.

Looking at the gender distribution, the male-to-female ratio is Israel (3.8: 1) in the Middle East, Turkey (3.3: 1), Iraq (3.8: 1) and Greece (4.3: 1) and Italy (4.8: 1) along the Mediterranean coast. ), The incidence of males was high, while female incidence was high in Japan (0.95: 1) and China (0.75: 1). However, the prevalence of HLA-B5 in this disease is common among racially different countries such as Japan, the United Kingdom, Turkey, France, Israel, and Korea, and it is believed that environmental and cultural similarities are also involved.

According to domestic reports, the age distribution of patients was most common in their twenties, males were office workers, females were housewives, and family history was about 15% of patients. The causes of the disease are viral, allergic to streptococcal antigens, heavy metal poisoning, genetically related, and immune mechanisms, but there is no definite diagnosis. Doing.

The present inventors have compared a protein including all or a portion of apolipoprotein A-1 as a disease marker to compare protein differences in plasma of patients with Behcet's disease and non-Behcet's disease to diagnose, treat, and observe the disease. It was confirmed that the present invention can be completed.

After all, the main object of the present invention is to provide a diagnostic system using hydrolyzed apolipoprotein A-1 as a disease marker of Behcet's disease.

Another object of the present invention relates to a method for screening a drug for inhibiting Behcet's disease by selecting a substance that inhibits the hydrolysis of apolipoprotein A-1.

1 is a gel photograph showing the results of two-dimensional gel electrophoresis of plasma proteins isolated from patients with Behcet's disease.

Figure 2 is a gel photograph showing the results of two-dimensional gel electrophoresis of isolated plasma proteins of non-Behcet's disease.

Figure 3 is a photograph showing the results of SDS-PAGE that can be seen the plasma protein distribution of non-Behcet's disease between pI 4-5, molecular weight 10-20kDa and patients with Behcet's disease.

Figure 4 is a photograph showing the results of immunoblotting after SDS-PAGE of plasma proteins with an anti-apolipoprotein A-1 antibody.

Hereinafter, a diagnosis system using apolipoprotein A-1 (apolipoprotein A-1) as a disease marker of Behcet's disease of the present invention will be described in detail.

Blood was collected from patients with Behcet's disease and non-Behcet's disease and centrifuged to obtain plasma only. The proteins were separated by isoelectric point and mass by IEF (isoelectric focusing) analysis and SDS-PAGE electrophoresis. This was again stained with silver nitrate and the isolated protein was analyzed by Melanie 3 program. As a result, it was found at pI 4.6 and MW 14kDa, which did not appear at all in non-Behcet's disease and at all in Behcet's disease patients (hereinafter referred to as 'B-1'). For more detailed analysis, two-dimensional gel electrophoresis was repeated and stained with Coomassie Brilliant Blue G250 to cut out the spots and cut them with tyrosine, followed by MALDI-TOF (Matrix- Analysis by assisted laser desorption ionization time-of-flight mass spectrometry (Micromass, UK) revealed the amino acid sequence of apolipoprotein A-1. In order to confirm this again, the difference was transferred to the PVDF membrane (Polyvinylidene Fluoride Transfer), and after N-terminal amino acid sequencing, DETTQSPWD was found to be consistent with the N-terminus of apolipoprotein A-1. .

Thus, when the plasma of patients with Behcet's disease was immunoblotted with anti-apolipoprotein A-1 antibody by SDS-PAGE, it was found that proteins were stained by anti-apolipoprotein antibodies at molecular weights of 6,000 to 30,000. The B-1 protein position, ie 14kDa position, seen in Youngdong was also stained, but in non-Behcet's disease, it was not stained at all at a molecular weight of less than 30,000. Therefore, in the case of Behcet's disease, apolipoprotein A-1 was specifically hydrolyzed, and A-1 was found to be a fragment including an N-terminal site.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited to these examples.

Example 1 Diagnosis of Patients with Behcet's Disease by Two-Dimensional Gel Electrophoresis

Two-dimensional gel electrophoresis was performed to determine the differences in plasma proteins in patients with Behcet's disease and non-Behcet's disease. Found.

Example 1-1 Two-Dimensional Gel Electrophoresis of Plasma Proteins in Patients with Behcet's Disease and Non-Behcet's Disease

Two-dimensional gel electrophoresis was performed to determine the difference in protein between patients with and without Behcet's disease:

Protein concentrations of each plasma were determined by protein quantification of plasma from 20 patients with Behcet's disease, 200 normal subjects, and 10 non-Behcet's skin diseases. The total protein concentration of each plasma sample was 150 μg, mixed with 50 μl of SDS / DTT lysis solution (0.3% SDS, 3% dithiothreitol, 50 mM Tris-HCl, pH 8.0) for 5 minutes at 95 ° C. Incubated. Enhanced solublising solution (7M urea, 2M thiourea, 4% CHAPS (3-[(3-cholamidopropyl) dimethylammonio] -1-propan-sulfonate), 40mM Tris-HCl, 0.002% bromophenol blue , 2 mM tributylphosphine) was added thereto, followed by centrifugation at 17,000 rpm and 4 ° C. for 15 minutes. Only 500 μl of supernatant was taken and placed in an IPG dry strip re-expansion tray, and a 18 cm pH 3 to 10 linear IPG dry strip was covered over the solution and rehydrated for 16 hours.

The rehydrated strips were subjected to isoelectric focusing (IEF) analysis by applying 100,000 V · hr of electricity in a multiphor (Amersham-Pharmacia Biotech, Sweden) to move proteins in the plasma to the pH portion corresponding to the pI value. To make it possible. At the end of the IEF, the strips were placed in a lidded glass test tube and stored in a -80 ° C. cryogenic freezer until a gel for two-dimensional gel electrophoresis was made.

5 X Tris-HCl (1.875M, pH 8.8) 90 ml, 40% acrylamide solution 90 ml, 10% SDS 4.5 ml, 100 mM sodium thiosulfate 90 µl, 10% ammonium persulfate 740 µl, TEMED 74 µl, 270 ml of distilled water were mixed to prepare an 8% acrylamide gel solution, 90 ml of 5 X Tris-HCl (pH 8.8), 40 ml of acrylamide solution 180 ml, 50% glycerol 90 ml, 10% SDS 4.5 ml, 100 mM sodium thiosulfate 90 16 μl, 10% ammonium persulfate 740 μl, TEMED 74 μl, and 90 ml of distilled water were mixed to prepare a 16% acrylamide gel solution. The gel solution prepared as described above was made of SDS-PAGE gel with 8% to 16% concentration gradient of 1.5 mm in thickness and 18 cm in width. Strips stored in -80 ° C. cryogenic freezer after IEF were placed into TBP equilibration buffer (0.2 mM tributyl phosphine, 6M urea, 2% SDS, 375 mM Tris buffer (pH 8.8), 20% glycerol and 2.5% Acrylamide) 10ml was added and equilibrated with gentle stirring for 15 minutes. The strip was equilibrated onto the resulting gel and the agarose embedding solution (agarose, 1% bromophenol blue, 1 × SDS development buffer) was poured to the top of the glass plate so that the strip was fixed to the gel. An 8% to 16% gradient gradient gel with a fixed strip was placed in a pail containing SDS-PAGE development buffer made of a mixture of glycine, SDS and Trisbase, followed by electrophoresis at 200 mA for 12 hours.

Example 1-2 Staining Silver Nitrate

To confirm the SDS-PAGE results, silver nitrate was stained:

After the SDS-PAGE finished gel was removed from the glass plate, soaked in a plastic box with tertiary distilled water to be dyed and washed with gentle stirring for 1 minute. After removing the tertiary distilled water, a solution containing 25 mL of acetic acid, 100 mL of methyl alcohol and 125 mL of tertiary distilled water was added thereto, and the mixture was fixed twice with 15 minutes with gentle stirring. Then, the solution was carefully removed, a solution containing 75 mL of methyl alcohol, 10 mL of 5% sodium thiosulfate, 17 g of sodium acetate, and 165 mL of distilled water was added thereto, followed by sensitization with gentle shaking for 30 minutes. The solution was removed and washed three times with gentle stirring for 5 minutes with distilled water. 2.5% silver nitrate solution was added 25ml and tertiary distilled water 225mL mixed solution, gently stirred for 20 minutes, the solution was removed and washed twice with 1 minute gently stirred with 250mL of distilled water. 6.25 g of sodium carbonate, 100 μl of formaldehyde, and 250 mL of tertiary distilled water were added thereto, followed by stirring until the protein site appeared well. When the brown protein site appeared completely on the gel, the solution was immediately removed. The solution was mixed with EDTA 3.65 g in tertiary distilled water, and the reaction was stopped while stirring gently for 10 minutes. After 10 minutes, the solution was removed, and distilled water was added thereto, washed three times with gentle stirring for 5 minutes to complete dyeing. At this time, the mixed solution required for each step and the third distilled water at the time of washing was used 250ml per gel. Protein patterns of non-Bhette's disease patients and non-Bhette's disease patients who stained were compared and analyzed (see FIGS. 1, 2, and 3).

1 is a photograph showing the results of two-dimensional gel electrophoresis gel isolated from plasma proteins of patients with Behcet's disease, Figure 2 is a photograph showing the results of two-dimensional gel electrophoresis gel from plasma proteins of non-Behcet's disease patients, Figure 3 is a photograph showing the results of SDS-PAGE that can be seen the plasma protein distribution of non-Behcet's disease between pI 4-5, molecular weight 10-20kDa and patients with Behcet's disease. Compared with the two-dimensional gel electrophoresis pattern of plasma of Behcet's disease and the general protein pattern of two-dimensional gel electrophoresis of non-Behcet's disease, Behcet's disease was found in the plasma of Behcet's disease patients at about pI 4.6 and MW 13-14kDa. A new protein spot was found that did not appear in nonhumans.

As shown in Figs. 1 to 3, the protein point B-1 could not be found in the plasma of non-Behcet's disease, but it was seen in the plasma of Behcet's disease patients. It can be seen that.

Example 2 Identification of B-1 Proteins

Plasma of patients with Behcet's disease was subjected to two-dimensional gel electrophoresis to isolate and analyze the B-1 protein, thereby confirming that the B-1 protein is a hydrolyzed form of apolipoprotein A-1.

Example 2-1 : Identification of B-1 Protein by MALDI-TOF Analysis

MALDI-TOF analysis of the B-1 protein identified the B-1 protein, confirming that the B-1 protein is a hydrolyzed form of apolipoprotein A-1:

Behcet's disease patient specific protein point B-1 shown in Example 1 was analyzed by MALDI-TOF. Secondary electrophoresis gel of plasma of patients with Behcet's disease was stained with Brilliant Blue G250, and the protein point B-1 found in this experiment was cut (1 mm x 1 mm) at about pH 4.6, MW 13-14 kDa on the gel. ), Into a microtube. 200 µl of a desalting solution (Tris-HCl pH 8.5, acetonitrile and tertiary distilled water) was added thereto, and the mixture was gently stirred in a 37 ° C. incubator for 30 minutes. After 15 minutes centrifugation, all supernatant was removed and the gel was completely dried. 8 μl of digestion solution (tris-HCl pH 8.5, trypsin) was added and rehydrated by shaking gently at 37 ° C. for 16 hours. 8 µl of the extraction solution (acetonitrile, trifluoroacetic acid and tertiary distilled water) was added thereto, followed by ultrasonic grinding for 10 minutes. Take 1 μl of sample and place in MALDI plate (Micromass, UK) and add 10 mg / ml matrix solution (α-cyano-4-hydroxycinnamic acid) with 0.1% TFA and 50% v / v aceto. 1 μl of a solution dissolved in nitrile was added and mixed, followed by drying for 30 minutes to prepare a sample. Plates containing these peptides were analyzed by MALDI-TOF to determine the molecular weight of the peptides digested with trypsin, and related proteins were found in databases such as NCBI and SwissProt. At this time, the programs used for analysis are Msfit and Profound. After hydrolysis of B-1 protein with trypsin and MALDI analysis, the protein had an apolipoprotein A-1 site.

Example 2-2 Identification of B-1 Proteins by N-Terminal Sequencing

In order to determine the protein (B-1) found through two-dimensional gel electrophoresis, N-terminal sequencing of the protein point using Edman degradation revealed that B-1 protein was apolipoprotein A. Confirmed to be the hydrolyzed form of -1:

After two-dimensional gel electrophoresis, the gel was immersed in tertiary distilled water for 1 minute, and the N-terminal sequencing portion of the gel was cut out, which was then gelled in 10 mM CAPS (cyclohexylaminopropane sulfonic acid, pH 11) for 30 minutes. After equilibrating, the PVDF membrane was also wetted with methyl alcohol for 1 minute, and equilibrated with 10 mM CAPS (pH 11) and methyl alcohol (10% v / v) for 30 minutes. Using a Xcell-II electroblotting kit (Novex, USA), the absorbent paper, gel, PVDF membrane, and wettable paper were stacked between sponges in the order of transition buffer (10 mM CAPS (pH 11) / methyl alcohol (10%). v / v)) was blotted at 15 ℃ 90V for 3 hours. After electroblotting, the PVDF membrane was dyed for 15 minutes in a solution containing Coomassie Brilliant Blue R250 (0.1% w / v) and methyl alcohol (50% v / v), methyl alcohol (40% v / v), After desalting with a mixture of acetic acid (10% v / v), the sequence of the N-terminal amino acid was determined using a protein analyzer (Protein Sequencer, Applied Biosystems Inc., USA). As a result of analyzing the N-terminal amino acid sequence of the protein, the sequence of D E T T Q S P W D appeared to confirm that it was apolipoprotein A-1.

Example 3 Western Blotting of Hydrolyzed Apolipoprotein A-1

Using the apolipoprotein A-1 antibody (Rockland, U.S.A.), electrophoresis of 14 kDa apolipoprotein A-1 in plasma of patients with Behcet's disease was confirmed by Western blot method:

10-20% Tris-tricin gel (Novex, U.S.A.) was electrophoresed with plasma of 20 μg of protein concentration in patients with Behcet's disease. Using an Xcell-II Electroblotting Kit (Novex, USA), the absorbent paper, the gel, the nitrocellulose membrane, and the absorbent paper were stacked between sponges in the order of transfer buffer (39 mM glycine, 48 mM Tris-base, 0.03% SDS, 20). % Methyl alcohol) was added, and the protein was transferred to the nitrocellulose membrane by applying 200 mA of electricity for 30 minutes. Whether all the proteins were moved can be confirmed by walking the prestained SDS-PAGE standard markers (broad range, Bio-Rad, U.S.A.) upon electrophoresis. Protein-transferred membranes were washed with TBST buffer (10 mM Tris-HCl (pH 8.0), 150 mM NaCl 0.05% Triton X-100) and blocked for at least 1 hour with a solution of BSA (5%, bovine serum albumin) dissolved in TBST. (blocking) Wash again with TBST buffer, add a solution mixed with TBST buffer and affinity purified anti-apolipoprotein A-1 (Affinity purified anti-apolipoprotein, Goat) at a ratio of 2,000: 1, and stir the antibody for 1 hour with gentle stirring. Reacts only to specific proteins. After washing three times for 10 minutes with light stirring with TBST buffer, the solution was mixed with TBST buffer and anti-goat Ig G conjugated horse radish peroxidase at a ratio of 1,000: 1, and gently shaken for 30 minutes. After attaching the secondary antibody, when a coloring reagent was added, only a specific protein was reacted to develop on the film. The solution was washed three times for 10 minutes with gentle stirring with TBST buffer, and A and B of the ECL solution were sprayed onto the membrane with the same volume so as to cover the entire membrane. The film was covered with a wrap, and the film was placed thereon for 1 minute 30 seconds and then developed. As a result, non-Behcet's disease was not stained at the molecular weight of less than 30,000, but in the plasma of patients with Behcet's disease, several polypeptides less than 30,000 were blotted with anti-apolipoprotein A-1 antibody. : Figure 4). This indicates that apolipoprotein A-1 is hydrolyzed in patients with Behcet's disease, and Western blot of plasma of patients with Behcet's disease can be used as a standard for diagnosing Behcet's disease. Could know.

As described and demonstrated in detail above, the present invention provides a diagnostic system using apolipoprotein A-1 as a disease marker of Behcet's disease. According to the present invention, a protein including all or a portion of apolipoprotein A-1 is used as a disease marker, and the difference between the plasma protein in patients with Behcet's disease and those in non-Behcet's disease is used to diagnose and treat Behcet's disease. We will be able to observe the prognosis, develop a cure for Behcet's disease.

Claims (4)

A system for diagnosing Behcet's disease, comprising the step of confirming the presence of hydrolyzed apolipoprotein A-1 in a protein obtained from plasma or serum. The method of claim 1, After electrophoresis of the protein obtained from plasma or serum, it was confirmed that the anti-apolipoprotein A-1 antibody binds to the protein having a molecular weight of 6,000 to 30,000, and to determine the presence of the hydrolyzed apolipoprotein A-1. Characterized in that Behcet's disease diagnostic system. The method of claim 1, Wherein the hydrolyzed apolipoprotein A-1 comprises protein point B-1 Behcet's disease diagnostic system. delete