KR100456006B1 - Protein of phenoloxidase system and gene coding the same - Google Patents

Abstract

The present invention relates to a protein involved in the activation system of phenol oxidase by β-1,3-glucan, a gene thereof, and a fungal diagnosis method using the protein.

The protein of the present invention can be used to reconstitute the composition as one component of a composition capable of detecting β-1,3-glucan derived from insects.

Description

Protein of phenoloxidase system and gene coding the same

Invertebrates, including insects, which are open vascular systems, have a fast and effective defense mechanism to prevent fluid leakage and invasive foreign bodies after wounding, and among them, a prophenol oxidase activation system, which is a melanogenesis reaction by foreign foreign substances, is known. (Ashida, M., & Brey, P. (1998), Molecular Mechanisms of Immune Responses in Insects pp. 135-172 Chapman & Hall, London). Activation of prophenoloxides by the prophenoloxidase activation system proceeds by a series of chain reactions initiated by the fungal or bacterial cell wall components β-1,3-glucan, lipopolysaccharide, peptidoglycan and the like. Has been reported.

Increasing systemic fungal infections in patients with compromised immune function, such as cancer patients, organ transplant patients, and AIDS infections, are becoming a serious problem in the medical community, resulting in increasing mortality. The main forms of systemic fungal infections are candidiasis, aspergillosis and cryptococcal meningitis. These organisms are controlled by the body's immune system and healed completely in healthy people, but fight against these infections when the immune system is suppressed, such as in patients with cancer treatment or bone marrow transplants, organ transplants, burn patients or AIDS patients. Loss of ability It is important for these patients to determine early fungal infections and administer appropriate antifungal agents, but there are currently difficulties in determining early fungal infections.

As a fungal infection diagnosis method, until now, the blood of the patient is collected and the microbiological identification method is used to determine whether the infection is necessary. To improve this, research has been conducted to find a system that recognizes fungal infections by measuring β-1,3-glucan, a fungal cell wall component present in the blood of fungal infection patients. There is a method of specifically detecting β-1,3-glucan without disrupting endotoxin using limulus hemolysate, but it has the disadvantage of low stability of reagents (Clinical Pathology Vol. 33, 639-644 ( 1985)). WO 83/02123 describes a method for specifically measuring β-1,3-glucan using hemolysates of crayfish. The process of purifying and adding endotoxin factor is essential.

There are known compositions that specifically detect β-1,3-glucan by separating body fluids, such as silkworms, beetles, and Korean beetle beetles, but the mechanisms involved and reaction mechanisms are not well known. The inventors of the present invention recently made a true black beetleHolotrichia diomphalia) Revealed the structure of a protein having serine protease activity, ie, activated prophenoloxidase activating factor-I (activated PPAF-I), among proteins involved in the phenoloxidase cascade reaction (European Journel of Biotechnology vol. 257, 615-619 (1998). In addition, the structures of prophenoloxidase and phenoloxidase, which are other proteins involved in the phenoloxidase cascade reaction, were revealed (Mol. Cells. Vol. 7, No. 1 90-97 (1997)). The present inventors also found that a composition capable of specifically detecting β-1,3-glucan, including prophenoloxides in an insect plasma, a method for preparing the same, and the composition can be used for diagnosis of fungal infections. (Korean Patent Application No. 2000-2547).

However, this reaction system, which consists of a series of cascade steps, is easily activated by internal factors induced by externally invading pathogens, foreign substances, or degranulation reactions of their blood cells, and is converted into phenol oxidases to use catecholamines. Since melanin is formed, there are process difficulties in separating the reaction system in vitro. Therefore, in order to understand the prophenoloxide activity system at the molecular level, it is necessary to identify each of the factors constituting the system and the relationship between them.

The present invention relates to a novel protein that is a component of a fungal infection diagnostic composition activated by β-1,3-glucan.

The present invention relates to a gene encoding a novel protein which is a component of a fungal infection diagnostic composition activated by β-1,3-glucan.

1 shows phenoloxidase activity by β-1,3-glucan in G solution and hemolysate.

Figure 2 is a Western blotting experiment showing the presence of 45kDa protein according to the present invention.

Figure 3 shows the results of phenol oxidase activation depending on the presence of Ca ²⁺ , 45kDa protein, prophenol oxidase and active PPAF-I.

Figure 4 is an electrophoresis picture showing the results of the change in the 45 kDa protein, the propphenol oxidase and active PPAF-I according to the presence of Ca ²⁺ 45 kDa protein.

Figure 5 is an electrophoresis picture showing the results of 45 kDa protein change by the active PPAF-I.

Fig. 6 shows phenol oxides activity by β-1,3-glucan in G solution to which 45 kDa protein was added.

The present invention relates to a novel protein, a gene thereof, and a method for diagnosing a fungal infection using the same, which is a component of a fungal infection diagnostic composition activated by β-1,3-glucan, which is involved in the activation of prophenoloxides. will be.

The terms, techniques, and the like described in this specification are used in the meanings generally used in the technical field to which the present invention belongs unless there is a specific limitation. In addition, all the documents mentioned in this specification are included in this specification as the documents for demonstrating this invention.

An "amino acid sequence", "polypeptide" or "protein" is not limited to fully native amino acid sequences.

"Variants of the base sequence" means a base sequence in which one or more bases of the gene sequences of the present invention are replaced, deleted or added while maintaining the biological or immunological activity of the protein of the present invention.

A “variant of amino acid sequence” refers to an amino acid sequence that has been altered by deletion, deletion or addition of one or more amino acids of the amino acid sequences of the proteins of the invention while maintaining biological or immunological activity.

Insects in the present invention means insects that have a phenol oxidase system in the body, the morphological insects are preferred. Examples include crustaceans such as lobsters and shrimps, coleoptera, etc., and prophenol oxidases exist in their living bodies, and phenoloxy is produced by a cascade reaction activated by β-1,3-glucan or lipopolysaccharide. Activated by Days.

In the present invention, the phenol oxidase system means a system in which prophenol oxidase is activated to phenol oxidase through a series of reactions by the presence of β-1,3-glucan present in an insect.

The phenol oxidase composition in the present invention means a composition containing all or part of the phenol oxidase system components, and capable of detecting β-1,3-glucan in the presence of calcium ions.

The present invention relates to a protein consisting of amino acid residues 1 to 415 of SEQ ID NO: 3, or variants thereof, or a portion thereof.

The present invention isolates and reveals the structure of new proteins involved in the activation of phenoloxidase, revealing that the 45 kDa protein is an essential component of the phenoloxidase system and a novel prophenoloxidase activator. The 45 kDa protein has a molecular weight of about 45-50 kDa molecular weight by SDS-PAGE.

In order to isolate the 45kDa protein, the body fluid of an insect, such as plasma or blood cells, may be used as a sample. Instead of adding a separate serineprotease inhibitor in insect body fluid separation process, a buffer containing chelating agent can be used to suppress blood cell aggregation and adsorption.

The 45kDa protein according to the present invention can be used to obtain antibodies against 45kDa protein and to use it to screen 45kDa protein-like proteins present in the body fluids of other insects.

The 45 kDa protein of the invention can be used to prepare a phenol oxidase system or a substrate of a composition capable of detecting β-1,3-glucan based on this system. That is, the 45 kDa protein is a factor constituting the phenol oxidase system which proceeds through a cascade reaction, and is cleaved by activated PPAF-I protein which is an activating factor in the phenol oxidase system. Thus, one can design a substrate, such as a peptide, of a composition capable of detecting β-1,3-glucan comprising a 45 kDa protein sequence portion thus recognized and cleaved.

The protein sequence database of the National Center for Biotechnology Information (NCBI) examined the homology of the 45kDa protein to known proteins, indicating that the 45kDa protein is a serine protease analog with two domains, the N-terminal domain and the active domain. (homologue). In the 45 kDa protein, histidine and aspartic acid are still present among the three activating residues present in the normal serine protease, but the serine residues involved in the activating site of the serine protease are replaced with glycine. The 45 kDa protein also has six cysteines that make up three disulfide bonds and three potential N-glycosylation sites (Asn-Xaa-Ser / Thr).

In addition to the body fluids of Korean black beetle larvae used in the present invention, it is assumed that there is a similar protein that acts as a 45 kDa protein in the body fluids of insects having a phenol oxidase system activated by β-1,3-glucan. The antibody to 45 kDa protein according to the invention can be used to detect and isolate 45 kDa protein-like proteins present in the body fluids of other insects.

The 45 kDa protein can be produced in large quantities from recombinant microorganisms introduced with the 45 kDa protein gene, for example, using an expression system using molecular biological techniques.

The present invention also relates to a protein consisting of amino acid residues 100-415 of SEQ ID NO: 3, or a variant thereof, or part thereof.

This protein, designated herein as 35 kDa protein, is obtained by cleaving a portion of the N-terminal amino acid sequence of the 45 kDa protein, ie, between Arg-99 and Glu-100 of the 45 kDa protein, and has a similar use as the 45 kDa protein.

In vitro experiment, 45kDa protein but not cut by activated PPAF-1 in the state where the addition of Ca ^2+, is cut into 35kDa by activated PPAF-I in the state not in the addition of Ca ²⁺ (Fig. 5 ).

The invention also relates to a gene encoding a 45kDa protein, variant thereof or part thereof.

The gene according to the present invention consists of a DNA sequence encoding the amino acid sequence of SEQ ID NO: 3 or a variant thereof, or part thereof, and, for example, the DNA sequence of bases 40 to 1284 of SEQ ID NO: 1.

In the present invention, a cDNA library of an insect is prepared, the gene of the 45kDa protein is obtained through immunos screening using an antibody of the 45kDa protein prepared using the purified 45kDa protein, and the sequence thereof and the sequence of the amino acid encoded by the same are presented. . The resulting gene has an open reading frame of 1245 bases corresponding to 415 amino acids.

The invention also relates to a gene encoding a 35kDa protein, variant thereof or part thereof.

The gene according to the present invention consists of a DNA sequence encoding amino acid sequence 100-415 of SEQ ID NO: 3 or a variant thereof, or a part thereof, and, for example, consists of a DNA sequence of bases 337-1284 of SEQ ID NO: 1.

45kDa protein or 35kDa protein, variants thereof, or a portion of a gene thereof according to the present invention may be used to produce 45kDa protein or 35kDa protein, variants thereof, or part thereof using recombinant microorganisms, and may be used in codons of host microorganisms. The base can be appropriately modified in consideration of the tendency of use.

The present inventors were able to confirm that the 45 kDa protein was purified purely by SDS-PAGE under reduced conditions, and the activity of phenol oxides was determined using purified pro-PO, purified PPAF-I, and 45 kDa proteins. Investigations confirmed that the 45 kDa protein was a component of the phenol oxidase composition. In addition, in vitro reconstitution experiments, purified prophenoloxidase and activated PPAF-I alone did not show the activity of phenoloxidase in the presence of β-1,3-glucan, and only 45 kDa protein should be added together with the above two proteins. Confirming that the activity of phenoloxidase is shown, it was confirmed that the 45kDa protein is a component directly involved in phenoloxidase activation and also constitute the phenoloxidase composition.

The addition of purified 45 kDa protein to the phenol oxidase composition increases the sensitivity of the activation reaction of phenol oxidase by β-1,3-glucan. The 45 kDa protein for this may be a complete 45 kDa protein or part thereof, can be mass produced using a recombinant microbial system, and the 45 kDa protein can be used partially or fully purified. 35kDa protein of the present invention will also be able to exert the same effect of 45kDa protein.

The buffer and phenol oxidase activity measuring method used in the present invention are as follows.

Anticoagulant buffer (pH 4.6): 30 mM trisodium citrate, 26 mM citric acid, 20 mM EDTA, 15 mM sodium chloride)

β-1,3-glucan solution: made by mixing 10 μl of 1 mg of β-1,3-glucan (Curdlan, Wako Pure Chemical Industries, LTd.) in 1 ml of 0.1 N NaOH and 990ul of 20 mM Tris buffer (pH 8.0) solution

Scheme milk solution (pH 7.9) used for antibody purification: 2.5 g of scheme milk was dissolved in 50 ml of 20 mM Tris / HCl (pH 7.9).

10 × wash buffer used for antibody purification: Tris 100 mM, EDTA 10 mM, Triton X-100 1%, NaCl 1.5M

Wash buffer used for antibody purification: 0.5% skim milk in 1 × wash buffer, NaN ₃ 2 mM

TBS: Tris 20 mM, NaCl 153 mM

TTBS: Tris 20mM, NaCl 153mM, Tween 20 0.1%

IPTG Filter: IPTG 190.6mg, Tertiary Distilled Water 40ml

high-TBST solution: in 600 ml of solution, 30 ml of 1M Tris-HCl (pH7.9), 5.25 g NaCl, 15 ml of 20% Tween 20, residual amount of tertiary distilled water

low-TBST solution: In 2 L of solution, 20 ml of 1M Tris-HCl (pH 7.9), 17.5 g NaCl, 5 ml of 20% Tween 20, residual distilled water in tertiary

3% gelatin solution: in 400 ml of solution, 12 g of gelatin, 800 μl of 10% NaN ₃ , low-TBST residual

Primary antiserum solution: 2 ml of purified antibody, 2 ml of 3% gelatin solution, 60 μl E. coli hemolyte (10 mg / ml), 60 μl 10% NaN3 1.88 ml of tertiary distilled water

Secondary antisera solution: 5 ml of goat anti-rabbit IgG (H = L) -AP conjugated (Bio-Rad) solution 5 ml, 1.7 ml of 3% gelatin solution

AP buffer: In 300 ml of solution, 30 ml of 1 M Tris-HCl (pH 9.5), 1.74 g NaCl, 1.5 ml of 1 M MgSO ₄ , 3 mL residual distilled water

Color reaction: 100 ml of AP buffer, 1320 μl of 50 mg / ml NBT in 70% DMF, 660 μl of 50 mg / ml BCIP in DMF

LB liquid medium: In 1 L of medium, 10 g of NaCl, 10 g of tryptone, 5 g of yeast extract, residual amount of tertiary distilled water

NZY _{Plate: NaCl 5g, MgSO 4 .7H 2} O 2g, 5g yeast extract, 10g NZ amine, Bacto agar 15 g, distilled water 1L 3

NZY plate (top agar): 5g NaCl, 2g MgSO ₄ .7H ₂ O, 5g yeast extract, 10g NZ amine, Bacto agar 7g, 3 deionized water 1L

SM buffer solution: in the solution _{1L, NaCl 5.8g, MgSO 4 .7H} 2 O 2g, 1M Tris-HCl (pH 7.5) 50ml, 2% gelatin 5ml, 3 distilled water remaining amount

Determination of Phenolic Oxidase Activity

85 μL of 20 mM Tris buffer (pH 8.0) containing 5 μg of β-1,3-glucan, 5 μL of G solution (50 μg protein) and 10 μl of hemolysate (200 μg protein) or 10 μl of purified 45 kDa protein (1 μg) at 30 ° C. Pre-react for 10 minutes and add 400ul of substrate solution (20mM Tris buffer (pH8.0) with 5mM CaCl ₂ , 1mM of 4-methylcatechol (MC), 2mM of 4-hydroxyproline ethyl ester (HP)) After reacting for 10 minutes at 30 ℃ and put 500ul of 20% acetic acid to stop the reaction and measure the absorbance at 520nm. One unit of phenol oxides is defined as the amount of increase in absorbance of 0.1 at 520 nm.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention more specifically, and the scope of the present invention is not limited to these examples.

Example 1.

About 400 Korean locust beetles ( Holotrichia diomphlia ) larvae were anesthetized on ice, and a 5 ml sterile syringe with a 25G needle was filled with anticoagulant buffer and 1 ml was injected, and the larvae were cut to collect fluids in a test tube on ice. The collected body fluid was centrifuged at 420xg for 10 minutes at 4 ° C, washed with anticoagulant buffer to obtain blood cells, and stored at -80 ° C. Approximately 0.5 g of blood cells are suspended in 5 mL of buffer A (Tris buffer (pH6.5 50 mM + 1 mM EDTA)), homogenized by crushing the blood cells five times for 5 seconds with an ultrasonic grinder and centrifuged at 4 ° C. and 22,000 × g for 20 minutes. The supernatant was used as hemolysate.

The supernatant after centrifugation of the body fluid was obtained by plasma, adjusted to pH4.6 with 1M citric acid solution, and stored at -70 ° C and used for the experiment. 40 ml of the supernatant obtained by centrifuging 45 ml of plasma at 4 ° C. for 4 hours at 203,006 g was concentrated to 3 ml by ultrafiltration (10,000 off). It was loaded on a Toyopearl HW-55S column (1.4 × 50 cm) equilibrated with 50 mM Tris-HCl / 20 mM EDTA buffer (pH 6.5). Protein concentration was examined by measuring the absorbance at 280 nm in 3.5 ml fractions while eluting with 50 mM Tris-HCl / 20 mM EDTA buffer (pH6.5) at a rate of 1.5 mL / hour. When calcium and β-1,3-glucan solution were added, only the fractions showing phenol oxidase activity were collected to obtain a phenol oxidase composition, which was named as G solution.

Example 2.

In order to investigate the effect of hemolysate on phenol oxidase activation, phenol oxidase activity by β-1,3-glucan was measured on G solution and hemolysate and the result is shown in FIG. : Substrate solution (CaCl ₂ 5mM, Tris buffer 50mM, 4-MC 1mM, 4-HP 2mM), column 2: G solution 5ul (50ug protein) + β-1,3-glucan 1ug + CaCl ₂ containing substrate solution, column 3: hemolysate 10ul (200ug protein) + β-1,3-glucan 1ug + CaCl ₂ -containing substrate solution, column 4: G solution 5ul (50ug protein) + hemolysate 10ul (200ug protein) + β-1, 3 glucan 1 ug + substrate solution without CaCl ₂ , column 5: G solution 5 ul (50 ug protein) + hemolysate 10 ul ( ₂₀₀ ug protein) + β-1,3-glucan 1 ug + CaCl ₂ -containing substrate solution)

Example 3.

45kDa protein was isolated from hemolysates in the following manner.

About 3 ml of the hemolysate obtained in Example 1 was loaded on a Blue-Sepharose column (1.0 × 5.2 cm) equilibrated with Buffer A, washed at a rate of 0.2 ml / min, and the washing solution was collected to collect an ultrafiltration device (cut off 10,000). Concentrated). The concentrated sample (2 ml, 50 mg protein) was loaded into Sephacryl S-200 (1.5 × 120 cm) equilibrated with buffer B (50 mM tris buffer (pH6.5), 1 mM EDTA, 0.1 NaCl) and flowed into the same buffer to obtain a fraction. . Each fraction was reacted with a substrate containing purified prophenoloxides, activated PPAF-I and Ca ²⁺ , and the fractions showing phenoloxidase activity were collected and buffer C (phosphate buffer (pH7.0) 50 mM, 1.7 M ammonia). The phenyl sepharose column (0.5 × 7 cm) equilibrated with Um Sulfate) was loaded, and the ammonium sulfate solution was eluted with a linear gradient of 1.7-0 M at a rate of 0.3 ml / min. Confirmed by reduced SDS-PAGE fractions containing about 45kDa protein were collected and concentrated by ultrafiltration. The concentrated sample was loaded onto a Superdex-200 FPLC column equilibrated with Buffer B and eluted with the same buffer. The phenol oxidase activity was measured and the fractions showing the activity were collected and loaded into a mono-Q column equilibrated with buffer D (tris buffer (pH7.4) 20 mM), and the sodium chloride solution was added at 0 to 1 M at a rate of 0.4 ml / min. The linear gradient of was eluted. Fractions with phenoloxidase activity were collected and subjected to 10% SDS-PAGE under reducing and non-reducing conditions to obtain a protein having a single band at about 45 kDa. Polyclonal antibodies were prepared from rabbits using 45 kDa protein purified purely in Example 4. Western blotting experiments using the obtained antibody confirmed that the 45 kDa protein purified from the lysate was one component of the G solution. The results are shown in FIG. (Lane 1: size marker, lane 2: hemolysate, lane 3: G solution, lane 4: isolated 45 kDa protein).

The purified 45kDa protein was cut with trypsin to determine partial amino acid sequence. A solution containing 30 µg of sample was dissolved by adding 0.4 M NH ₄ HCO ₃ buffer containing the same amount of 8 M urea, and then reacted at 50 ° C. for 15 minutes by adding a 45 mM DTT solution corresponding to 10% of the total volume. After the reduction, the 100 mM iodoacetamide solution in an amount corresponding to 10% of the total volume was added and reacted for 15 minutes at room temperature to alkylate the reduced cysteine residue. This was precipitated by 10% TCA precipitation method, and the obtained protein residue was dissolved in 0.4 M NH ₄ HCO ₃ buffer containing 8 M urea, and then 2 μg of trypsin was added and reacted at 37 ° C. for 13 hours to fragment. The fragmented proteins were subjected to a linear concentration gradient system [A; using a C18 reversed phase HPLC column (chemcosorb 5-ODS-H 2.1 × 150 / W). 0% CH ₃ CN in 0.06% TFA, B; 80% CH ₃ CN in 0.052% TFA], while fragmenting the protein at a flow rate of 0.2 ml per minute while increasing the solvent B to 90% for 90 minutes, the partial amino acid sequence was determined as follows by Edman digestion. It was.

Partial amino acid sequence

AGEWDTLTEKERLPY

PLVQADNI

FVLDQTFVXAGGE

Example 4.

The purified 45 kDa protein obtained in Example 3 was injected into rabbit (white, male, 2.7 kg) to obtain an antibody of 45 kDa protein.

In order to determine whether the antibody was produced, 45 kDa protein was separated on 10% SDS-PAGE under reducing conditions, and transferred to PVDF membrane by reacting at 280 mA for 4 hours at 280 mA in transition buffer. The membrane was washed with TBS, blocked with nonspecific reaction by shaking culture at 4 ° C. with TBS containing 5% skim milk and 1% horse serum overnight, and then the antisera isolated from rabbit 2.5% skim milk. Shake incubation for 2 hours at room temperature with a solution diluted 100-fold with TTBS containing. After washing 5 times with TTBS, the secondary antibody (anti-rabbit Ig. Horseradish peroxidase-linked donkey total antibody) was reacted with a solution diluted 100-fold with TTBS for 30 minutes at room temperature, and then again washed 5 times with TTBS. Washed. The fluorescence generated by performing ECL blot for 1 minute under semi-dark conditions was exposed to the film. This antibody was found to selectively recognize 45 kDa protein.

Example 5.

After shaking the culture of XL-1-Blue in a mixture of 5 ml of LB medium, 50 μl of 20% maltose and 50 μl of 1 M-MgSO ₄ at 37 ° C., 10 mM-MgSO ₄ was added to the recovered bacterial precipitate and OD ₆₀₀ was 0.5. Was adjusted to Moon et al. (1994) J. Biochem. The cDNA library of Korean black beetle larvae was prepared according to the method described in (Tokyo) 116, 53-58. 200 μl of XL-1-Blue was added to the cDNA library solution, followed by shaking culture for 15 minutes at 37 ° C., followed by mixing with 3.5 ml of top agar preheated to 48 ° C. to apply the solution to the NZY plate. After 4 hours of incubation at 42 ° C., the filter treated with IPTG was placed on a plate and further incubated at 37 ° C. for 8 hours. After the reaction was completed, the mixture was left at 4 ° C. for 20 minutes, and the filter was completely dried in air. The dried filter was washed for 10 minutes at room temperature with a high-TBST solution, followed by two more 10-minute washes at room temperature with a low-TBST solution. The washed filter was completely drained and then placed in 3% gelatin solution and blocked at 27 ° C. for 30 minutes, and then reacted with an antibody solution of 45 kDa protein at 27 ° C. for 2.5 hours. After the reaction, the filter was washed three times with a low-TBST solution, and then reacted in a secondary antibody solution at 27 ° C. for 30 minutes. The filter was washed three times with a low-TBST solution and then a color reaction was carried out as follows.

The filter prepared above was prepared in 100 ml of AP buffer with 1320 μl of 50 mg / ml NBT (nitroblue tetrazolium chloride, Bio-rad 170-6532) and 50 mg / ml BCIP (5-bromo-4-chloro-3-indoyl phosphate). 660 μl of -p-toluidine salt Bio-rad 170-6539) was added to the color reaction solution, followed by color development and drying in air. Purple clones that were distinguished from the surroundings were determined as primary positive clones. Properly dilute the primary positive clone with LB liquid medium, add 200 μl of XL-1-Blue (OD ₆₀₀ = 0.5) to the amount expected to produce 100 to 200 plaques and shake at 37 ° C. for 15 minutes. Incubated. The preheated 3 ml top agar was added to the NZY plate, and the experiments were then conducted in the same manner as the primary screening, and the positive clones were screened secondarily.

200 μl of XL-1-Blue (OD ₆₀₀ = 1.0) and 200 μl of positive plaque solution (2 × 10 ⁵ phage) identified as a result of secondary screening, 1 μl of ExAssist helper phage were placed in a 50 ml Falcon tube for 15 minutes at 37 ° C. After incubation, 3 ml of LB liquid medium was added thereto, followed by shaking culture for 3 hours at 37 ° C., followed by further heat treatment at 70 ° C. for 20 minutes. The heat-treated solution was centrifuged at 1,000 x g for 15 minutes and the supernatant was taken in a 15 ml Falcon tube and stored at 4 ° C.

10 μl of phagemid diluted 10-fold with LB liquid medium was added to 200 μl of SOLR cells (OD ₆₀₀ = 1.0), followed by shaking culture for 15 minutes at 37 ° C., followed by 50 μl of LB / Ampicillin plate. Smeared. After incubating at 37 ° C. for 12 hours, single colonies were taken, and 5 ml of LB liquid medium and 10 μl of ampicillin (50 mg / ml) were added to a 50 ml Falcon tube, followed by incubation at 37 ° C. for 12 hours. Plasmid into which the gene of 45kDa protein was inserted was isolated using a DNA purification kit (Promega, wizard plus sv minipreps).

The obtained plasmid DNA nucleotide sequence was determined in double in both directions from the 5 'direction and the 3' direction using an automatic DNA sequencing device (SEQ ID NO: 1). The protein sequence was inferred from the nucleotide sequence (SEQ ID NO: 3).

Example 6.

Phenoloxidase activity was measured in reconstitution experiments using purified 45 kDa protein, propphenoloxides, and activated PPAF-I. Prophenoloxides and activated PPAF-I were purified according to conventional methods (Lee et al. (1998) Eur. J. Biochem. 254, 50-57, Kwon et al. (1997) Mol. Cells. 7 ( 1) .90-97). The result of phenol oxidase activation according to the presence of Ca ²⁺ , 45 kDa protein, prophenol oxidase, and active PPAF-I is shown in FIG. 3 (column 1: substrate solution, column 2: 2 ug of propenol oxidase, column 3: 1 ug of 45 kDa protein, column 4: 1 ug of PPAF-I, column 5: 2 ug of prophenoloxidase + 1 ug of 45 kDa protein, column 6: 2 ug of prophenoloxidase + 1 ug of PPAF-I, column 7: 1 ug of 45 kDa protein + PPAF- Iug, column 8: ₂ ug of prophenoloxidase + 1 ug of 45 kDa protein + 1 ug of PPAF-I (without addition of CaCl ₂ ), column 9: ₂ ug of prophenoloxidase + 1 ug + 1 ug of PPAF-I). When 45kDa protein was reacted with profeoloxidase and activated PPAF-I, it showed phenoloxidase activity regardless of Ca ²⁺ concentration (columns 8 and 9). When reacted with only one of PPAF-I, phenol oxidase activity was not observed (columns 6 and 7).

The result of electrophoresis of the above reaction mixture showing phenol oxidase activity under reducing conditions is shown in FIG. 4 ((A), lane 1: propenol oxidase (2 ug), lane 2: 45 kDa protein (2 ug), lane 3: PPAF-I (2ug), Lane 4: Prophenoloxides (2ug), 45kDa protein (2ug) and PPAF-I (2ug) were reacted at 30 ° C. for 60 minutes without adding Ca ²⁺ , (B) Reacts under the same conditions with Ca ²⁺ added). 45kDa protein can be seen cut to the size of 35kDa when reacted in the absence of Ca ²⁺ .

Example 7.

In order to confirm the production of 35kDa protein, the activated PPAF-I and 45kDa protein were reacted with or without Ca ²⁺ , followed by electrophoresis and the results are shown in FIG. 5 ((A), lanes). 1: 45 kDa protein (2 ug), lane 2: PPAF-I (2 ug), lane 3: 45 kDa protein (2 ug) and PPAF-I (2 ug) were reacted at 30 ° C. for 60 minutes without addition of Ca ²⁺ , ( B) under the same conditions with Ca ²⁺ added). The 45 kDa protein is cut to 35 kDa by activated PPAF-I in the absence of Ca ²⁺ . As a result of determining the amino acid sequence after electrophoresis of the cleaved 35kDa protein to the PVDF membrane, it was confirmed that the 45kDa protein was cleaved between Arg-99 and Glu-100.

Example 8.

Increased phenol oxides activity was obtained by adding G solution and purified 45 kDa protein to β-1,3-glucan solution. The result is shown in FIG.

The protein according to the invention is one of the phenoloxidase activating factors in insects and part of a composition effective for the diagnosis of fungal infections. The protein of the present invention can be used for the preparation of a fungal infection diagnostic composition, and the gene according to the present invention can be used to produce a large amount of proteins required for the production of a fungal infection diagnostic composition.

<110> Samyang Genex Co. Ltd. <120> Component of phenoloxidase system and gene coding the same <130> I-117 <160> 3 <170> KOPATIN 1.5 <210> 1 <211> 1437 <212> DNA <213> Holotrichia diomphalia <220> <221> CDS <222> (40) .. (1284) <400> 1 gggctgcagg aatcggcacc agcgaaacct cctttagca atg aaa cgc ttg ttc 54 Met Lys Arg Leu Phe 1 5 gtt ata act gcc ttt ttt cta ttc ggc gct gaa gcc caa aat agc gtt 102 Val Ile Thr Ala Phe Phe Leu Phe Gly Ala Glu Ala Gln Asn Ser Val 10 15 20 att gat gcg gca gtc gtg aat ata ttt gga aac gct tcc gag tac ata 150 Ile Asp Ala Ala Val Val Asn Ile Phe Gly Asn Ala Ser Glu Tyr Ile 25 30 35 cca cct ggc tac gaa ata gtc acc aag gct cct tta gga gcc cta aca 198 Pro Pro Gly Tyr Glu Ile Val Thr Lys Ala Pro Leu Gly Ala Leu T hr 40 45 50 gcc tta ccc aga tgt gga acg ggt gcc gat caa ggc aaa aaa gtg tgt 246 Ala Leu Pro Arg Cys Gly Thr Gly Ala Asp Gln Gly Lys Lys Val Cys 55 60 65 att gtt tat cat aga tgc gat gga gtc acg aat aca gta aca cca gaa 294 Ile Val Tyr His Arg Cys Asp Gly Val Thr Asn Thr Val Thr Pro Glu 70 75 80 85 gaa gtt ata aac acc aca gga gaa ggc att ttc gat atc agg gaa aac 342 Glu Val Ile Asn Thr Thr Gly Glu Gly Ile Phe Asp Ile Arg Glu Asn 90 95 100 gcc aat gaa tgc gaa agc tat ttg gac gtg tgt tgt ggc ctc cca gaa 390 Ala Asn Glu Cys Glu Ser Tyr Leu Asp Val Cys Cys Gly Leu Pro Glu 105 110 115 ggc ggt gta ttg cca act cct tcg ccc act cct cca gta gta cca gtt 438 Gly Gly Val Leu Pro Thr Pro Ser Pro Thr Pro Pro Val Val Pro Val 120 125 130 ctg aaa ccg agc ttc tgc ggc atc agg aac gaa cgt ggt ttg gac ttt 486 Leu Lys Pro Ser Phe Cys Gly Ile Arg Asn Glu Arg Gly Leu Asp Phe 135 140 145 aaa ata aca ggt cag acc aat gag gcc gaa tat ggg gaa ttt ccg tgg 534 Lys Ile Thr Gly Gln Thr Asn Glu Ala Glu Tyr Gly Glu Phe Pro Trp 150 155 160 165 atg gtg gca gta ttg aaa gcc aac gtt att ccc ggt tct ggg gaa gag 582 Met Val Ala Val Leu Lys Ala Asn Val Ile Pro Gly Ser Gly Glu Glu 170 175 180 caa ttg gta tgc ggg gga tcc ttg atc gct cca agc gtt gta tta act 630 Gln Leu Val Cys Gly Gly Ser Leu Ile Ala Pro Ser Val Val Leu Thr 185 190 195 gga gct cat tgc gtc aat agt tac caa agc aac tt a gac gcc atc aaa 678 Gly Ala His Cys Val Asn Ser Tyr Gln Ser Asn Leu Asp Ala Ile Lys 200 205 210 att cgt gca ggc gaa tgg gac acc cta acc gaa aag gaa cga ctg cca 726 Ile Arg Ala Gly Glu Trp Asp Thr Leu Thr Glu Lys Glu Arg Leu Pro 215 220 225 tac caa gaa agg aaa atc aga caa gtg atc atc cac agt aac ttc aac 774 Tyr Gln Glu Arg Lys Ile Arg Gln Val Ile Ile His Ser Asn Phe Asn 230 235 240 245 cct aag acc gtc gta aac gat gtc gcc tta tta ctg ctg gat agg cca 822 Pro Lys Thr Val Val Asn Asp Val Ala Leu Leu Leu Leu Asp Arg Pro 250 255 260 ctc gtt caa gcc gat aac atc gga acg att tgc tta cct cag caa agc 870 Leu Val Gln Ala Asp Asn Ile Gly Thr Ile Cys Leu Pro Gln Gln Ser 265 270 275 c ag ata ttc gat tcc aca gaa tgt ttc gcc agt gga tgg gga aag aag 918 Gln Ile Phe Asp Ser Thr Glu Cys Phe Ala Ser Gly Trp Gly Lys Lys 280 285 290 gaa ttc gga agc aga cat aga tat tca aat atc att ataa atc caa 966 Glu Phe Gly Ser Arg His Arg Tyr Ser Asn Ile Leu Lys Lys Ile Gln 295 300 305 tta ccg aca gtg gat cgt gat aag tgt cag gca gat ttg agg aat acc 1014 Leu Pro Thr Val Asp Arg Asp Lys Cys Gln Ala Asp Leu Arg Asn Thr 310 315 320 325 aga tta gga ctc aaa ttc gtc ctt gac caa acg ttt gtc tgt gct gga 1062 Arg Leu Gly Leu Lys Phe Val Leu Asp Gln Thr Phe Val Cys Ala Gly 330 335 340 ggt gaa caa ggc aaa gac acc tgc acc ggt gac gga gga agt cca ttg 1110 Gly Glu Gln Gly Lys Asp Thr Cys Thr Gly Asp Gly Gly Ser Pro Leu 345 350 355 ttc tgt cca gat cct cga aac cct tcg agg tat atg caa atg ggt ata 1158 Phe Cys Pro Asp Pro Arg Asn Pro Ser Arg Tyr Met Gln Met Gly Ile 360 365 370 gtg gct tgg ggc atc gga tgt ggt gat gag aat gtt ccg gga gtc tac 1206 Val Ala Trp Gly Ile Gly Cys Gly Asp Glu Asn Val Pro Gly Val Tyr 375 380 385 gca aat gtt gca cat ttc cgc aac tgg ata gac cag gag atg cag gca 1254 Ala Asn Val Ala His Phe Arg Asn Trp Ile Asp Gln Glu Met Gln Ala 390 395 400 405 aaa ggt tta agt acg aca ccc tac gtc gaa tagggc agacaaagaa 1300 Lys Gly Leu Ser Thr Thr Pro Tyr Val Glu 410 415 tgtattatcg aatcgtagag acattttgac ttaatgagtg ttattattacacat tatta aagaacgat ttgagatgga 1420 aaaaaaaaaa aaaaaaa 1437 <210> 2 <211> 415 <212> PRT <213> Holotrichia diomphalia <400> 2 Met Lys Arg Leu Phe Val Ile Thr Ala Phe Phe Leu Phe Gly Ala Glu 1 5 10 15 Ala Gln Asn Ser Val Ile Asp Ala Ala Val Val Asn Ile Phe Gly Asn 20 25 30 Ala Ser Glu Tyr Ile Pro Pro Gly Tyr Glu Ile Val Thr Lys Ala Pro 35 40 45 Leu Gly Ala Leu Thr Ala Leu Pro Arg Cys Gly Thr Gly Ala Asp Gln 50 55 60 Gly Lys Lys Val Cys Ile Val Tyr His Arg Cys Asp Gly Val Thr Asn 65 70 75 80 Thr Val Thr Pro Glu Glu Val Ile Asn Thr Thr Gly Glu Gly Ile Phe 85 90 95 Asp Ile Arg Glu Asn Ala Asn Glu Cys Glu Ser Tyr Leu Asp V al Cys 100 105 110 Cys Gly Leu Pro Glu Gly Gly Val Leu Pro Thr Pro Ser Pro Thr Pro 115 120 125 Pro Val Val Pro Val Leu Lys Pro Ser Phe Cys Gly Ile Arg Asn Glu 130 135 140 Arg Gly Leu Asp Phe Lys Ile Thr Gly Gln Thr Asn Glu Ala Glu Tyr 145 150 155 160 Gly Glu Phe Pro Trp Met Val Ala Val Leu Lys Ala Asn Val Ile Pro 165 170 175 Gly Ser Gly Glu Glu Gln Leu Val Cys Gly Gly Ser Leu Ile Ala Pro 180 185 190 Ser Val Val Leu Thr Gly Ala His Cys Val Asn Ser Tyr Gln Ser Asn 195 200 205 Leu Asp Ala Ile Lys Ile Arg Ala Gly Glu Trp Asp Thr Leu Thr Glu 210 215 220 Lys Glu Arg Leu Pro Tyr Gln Glu A rg Lys Ile Arg Gln Val Ile Ile 225 230 235 240 His Ser Asn Phe Asn Pro Lys Thr Val Val Asn Asp Val Ala Leu Leu 245 250 255 Leu Leu Asp Arg Pro Leu Val Gln Ala Asp Asn Ile Gly Thr Ile Cys 260 265 270 Leu Pro Gln Gln Ser Gln Ile Phe Asp Ser Thr Glu Cys Phe Ala Ser 275 280 285 Gly Trp Gly Lys Lys Glu Phe Gly Ser Arg His Arg Tyr Ser Asn Ile 290 295 300 Leu Lys Lys Ile Gln Leu Pro Thr Val Asp Arg Asp Lys Cys Gln Ala 305 310 315 320 Asp Leu Arg Asn Thr Arg Leu Gly Leu Lys Phe Val Leu Asp Gln Thr 325 330 335 Phe Val Cys Ala Gly Gly Glu Gln Gly Lys Asp Thr Cys Thr Gly Asp 340 345 350 Gly Gly S er Pro Leu Phe Cys Pro Asp Pro Arg Asn Pro Ser Arg Tyr 355 360 365 Met Gln Met Gly Ile Val Ala Trp Gly Ile Gly Cys Gly Asp Glu Asn 370 375 380 Val Pro Gly Val Tyr Ala Asn Val Ala His Phe Arg Asn Trp Ile Asp 385 390 395 400 Gln Glu Met Gln Ala Lys Gly Leu Ser Thr Thr Pro Tyr Val Glu 405 410 415 <210> 3 <211> 415 <212> PRT <213> Holotrichia diomphalia <400> 3 Met Lys Arg Leu Phe Val Ile Thr Ala Phe Phe Leu Phe Gly Ala Glu 1 5 10 15 Ala Gln Asn Ser Val Ile Asp Ala Ala Val Val Asn Ile Phe Gly Asn 20 25 30 Ala Ser Glu Tyr Ile Pro Pro Gly Tyr Glu Ile Val Thr Lys Ala Pro 35 40 45 Leu Gly Ala Leu Thr Ala Leu Pro Arg Cys G ly Thr Gly Ala Asp Gln 50 55 60 Gly Lys Lys Val Cys Ile Val Tyr His Arg Cys Asp Gly Val Thr Asn 65 70 75 80 Thr Val Thr Pro Glu Glu Val Ile Asn Thr Thr Gly Glu Gly Ile Phe 85 90 95 Asp Ile Arg Glu Asn Ala Asn Glu Cys Glu Ser Tyr Leu Asp Val Cys 100 105 110 Cys Gly Leu Pro Glu Gly Gly Val Leu Pro Thr Pro Ser Pro Thr Pro 115 120 125 Pro Val Val Pro Val Leu Lys Pro Ser Phe Cys Gly Ile Arg Asn Glu 130 135 140 Arg Gly Leu Asp Phe Lys Ile Thr Gly Gln Thr Asn Glu Ala Glu Tyr 145 150 155 160 Gly Glu Phe Pro Trp Met Val Ala Val Leu Lys Ala Asn Val Ile Pro 165 170 175 Gly Ser Gly Glu G lu Gln Leu Val Cys Gly Gly Ser Leu Ile Ala Pro 180 185 190 Ser Val Val Leu Thr Gly Ala His Cys Val Asn Ser Tyr Gln Ser Asn 195 200 205 Leu Asp Ala Ile Lys Ile Arg Ala Gly Glu Trp Asp Thr Leu Thr Glu 210 215 220 Lys Glu Arg Leu Pro Tyr Gln Glu Arg Lys Ile Arg Gln Val Ile Ile 225 230 235 240 His Ser Asn Phe Asn Pro Lys Thr Val Val Asn Asp Val Ala Leu Leu 245 250 255 Leu Leu Asp Arg Pro Leu Val Gln Ala Asp Asn Ile Gly Thr Ile Cys 260 265 270 Leu Pro Gln Gln Ser Gln Ile Phe Asp Ser Thr Glu Cys Phe Ala Ser 275 280 285 Gly Trp Gly Lys Lys Glu Phe Gly Ser Arg His Arg Tyr Ser Asn Ile 290 295 300 Leu Lys Lys Ile Gln Leu Pro Thr Val Asp Arg Asp Lys Cys Gln Ala 305 310 315 320 Asp Leu Arg Asn Thr Arg Leu Gly Leu Lys Phe Val Leu Asp Gln Thr 325 330 335 Phe Val Cys Ala Gly Gly Glu Gln Gly Lys Asp Thr Cys Thr Gly Asp 340 345 350 Gly Gly Ser Pro Leu Phe Cys Pro Asp Pro Arg Asn Pro Ser Arg Tyr 355 360 365 Met Gln Met Gly Ile Val Ala Trp Gly Ile Gly Cys Gly Asp Glu Asn 370 375 380 Val Pro Gly Val Tyr Ala Asn Val Ala His Phe Arg Asn Trp Ile Asp 385 390 395 400 Gln Glu Met Gln Ala Lys Gly Leu Ser Thr Thr Pro Tyr Val Glu 405 410 415

Claims (4)

A protein consisting of amino acid residues 1 to 415 of SEQ ID NO. A protein consisting of amino acid residues 100-415 of SEQ ID NO: 3. DNA sequence encoding a protein consisting of amino acid residues 1 to 415 of SEQ ID NO: 3. DNA sequence encoding a protein consisting of amino acid residues 100-415 of SEQ ID NO: 3.