WO2002016425A1

WO2002016425A1 - Protein of phenoloxidase system and gene coding the same

Info

Publication number: WO2002016425A1
Application number: PCT/KR2001/001435
Authority: WO
Inventors: Bok Luel Lee; Chong Jin Park; Seung-Suh Hong; Hyun-Soo Lee
Original assignee: Samyang Genex Corporation
Priority date: 2000-08-24
Filing date: 2001-08-24
Publication date: 2002-02-28
Also published as: AU2001282640A1; KR20020016079A; KR100456006B1

Abstract

The present invention relates to a protein of the phenoloxidase activation system by beta-1,3-glucan, a gene coding the same and a method for diagnosing fungus by using the same. The protein of the present invention is a component of a composition for detecting beta-1,3-glucan derived from insects and can be used to reconstitute the same composition.

Description

PROTEIN OF PHENOLOXIDASE SYSTEM AND GENE CODING THE SAME

BACKGROUND ART OF THE INVENTION

Invertebrates including insects with open blood-vascular system have a fast and efficient defense system to prevent the loss of body fluid and to remove the invading foreign molecules after injury. Prophenoloxidase system, a melanin production system by foreign bodies, is well known as a defense system. (Ashida, M., & Brey, P. (1998), Molecular Mechanisms of Immune Responses in Insects pp.135-172 Chapman & Hall, London). It has been reported that prophenoloxidase activation by the activation system of prophenoloxidase is carried out by a series of cascade reactions initiated by the fungal or bacterial cell wall components such as beta-1 , 3-glucan, lipopolysaccharide and peptidoglycan.

Among immuno-compromised cancer patients, organ transplantation patients and AIDS patients, increase of systemic fungal infection has become a serious medical problem. The death rate due to such an infection is increasing.

Major symptoms of the systemic fungal infections include candidiasis, aspergillosis and cryptococcal meningitis. These fungal infections can be controlled and treated by the human immune system in a healthy person. There is no ability, however, to fight against the infections for the immuno-compromised patients such as cancer patents, bone marrow transplantation patient, organic transplantation patient, burn victims or AIDS patients. It is important, therefore, to diagnose and to treat the fungal infection at an early stage of the infection by administering anti-fungal drugs. Currently, however, early diagnosis for fungal infection is yet to be achieved.

To diagnose a fungal infection in patients, the standard method in mycrobiology, i.e., a method wherein blood is drawn from a patient and cultivated to diagnose the fungal infection has been used. The method has a shortcoming in that the treatment may not be done on time since the result of the diagnosis can be obtained only after cultivation period of 2-5 days. To improve these shortcomings, research has been widely performed to find a system to recognize

accurately the infinitesimal amount of one of the fungal cell wall components, β-

1 , 3-glucan existing in the patient's blood. US patent 5,266,461 discloses a

reagent for determining β-1 , 3-glucan by using limulus hemocyte lysate without

the interference of endotoxins (Clinical Pathology Vol. 33, 639-644 (1985)). There is a shortcoming, however, that the stability of the reagent is low. WO

83/02123 discloses a method for determining β-1 , 3-glucan specifically by using

the hemocyte lysate of crayfish. It is essential, however, to add a purified anti-

endotoxin from the hemocyte lysate to determine β-1 , 3-glucan by this method.

The composition for specific determination of β-1 , 3-glucan, by isolating

the hemolymph of silkworm, coleoptesra, Holotrichia diomphalia, is well known. The involved factors and reaction mechanism are yet to be determined. Recently, the present inventors have determined the structure of a protein related to the phenoloxidase cascade reaction in Holotrichia diomphalia. This protein has a serine protease activity and is the activated prophenoloxidase activation factor-l (activated PPAF-I)) (European Journal of Biotechnology vol. 257, 615-619 (1998)). Also the present inventors have determined the structures of prophenoloxidase and phenoloxidase that are related to phenoloxidase cascade reaction (Mol. Cells. Vol. 7, No. 1 90-97 (1997)). Also the present inventors

have introduced a composition comprising prophenoloxidase for detecting β-1 , 3-

glucan in insect plasma and a preparation method thereof and shown that they

can be used to diagnose a fungal infection (PCT/KR01/00196).

The reaction system composed of a series of cascade reaction steps,

however, is activated easily by foreign pathogens or other molecules from outer

system, or inherent factors induced by the degranulation reaction of the insect's

own hemocytes. As a result, prophenoloxidase is converted to phenoloxidase to

produce melanin by using catecholamines. Therefore, it has been difficult to carry

out this reaction system under in vitro conditions. Therefore, it is necessary to

elucidate the factors in the system and the correlation between these factors to

understand the prophenoloxidase activation system in a molecular level.

SUMMARY OF THE INVENTION

The present invention relates to a novel protein, which is a component in

the composition for fungal infection diagnosis activated by β-1 , 3-glucan.

The present invention relates to a gene coding the novel protein, which is

a component in the composition for fungal infection diagnosis activated by β-1 , 3-

glucan.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a graph showing the phenoloxidase activity by beta-1 , 3-glucan in G solution and the hemocyte lysate;

Figure 2 is a result of the Western blot analysis exhibiting the presence of 45 kDa protein according to the present invention;

Figure 3 is a graph showing the phenoloxidase activity according to the presence of Ca²⁺, 45 kDa protein, prophenoloxidase and active PPAF-I;

Figure 4 is an electrophoresis photograph showing the changes in the 45 kDa protein by 45 kDa protein, prophenoloxidase and active PPAF-I according to the presence of Ca²⁺ ion;

Figure 5 is an electrophoresis photograph showing the changes in the 45 kDa protein by active PPAF-I;

Figure 6 is graph showing the phenoloxidase activity by beta-1 , 3-glucan in G solution supplemented with 45 kDa protein.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention relates to a novel protein, which is a component in

the composition for fungal infection diagnosis related to the prophenoloxidase

activation and activated by β-1 , 3-glucan, a gene coding the same and the method

for diagnosing fungal infection using the same.

The terminologies and techniques mentioned herein have the generally

accepted meanings in the field that the present invention belongs to. Also the

literatures mentioned in the description of the present invention are included in the present description to illustrate the present invention.

As used herein, the term 'amino acid sequence', 'polypeptide' or 'protein'

is not limited to a perfectly natural amino acid sequence of a peptide, a

polypeptide or a protein.

As used herein, the term 'base sequence mutant' refers to an altered

base sequence due to substitution, deletion or addition of one or more bases in

the gene of the present invention while maintaining biological or immunological

activity.

As used herein, the term 'amino acid sequence mutant' refers to an

altered amino acid sequence due to substitution, deletion or addition of one or

more amino acids of the protein of the present invention while maintaining

biological or immunological activity.

As used herein, the term "an insect" or "insects" refers to an insect or insects that has a phenoloxidase system in the body, and preferably a holometabola insect. The examples of insects include those belonging to Crustaceans such as lobsters and shrimps and Coleoptera. In the body of these insects, prophenoloxidase is present and is activated to phenoloxidase by a cascade reaction, which is activated by beta-1 , 3-glucan or lipopolysaccharide.

As used herein, the term "phenoloxidase system" refers to a system that

activates a prophenoloxidase to a phenoloxidase by a series of reactions by β-

1 , 3-glucan in insects.

As used herein, the term "phenoloxidase composition" refers to a composition that comprises all or some components of the phenoloxidase system and detects β-1 , 3-glucan in the presence of calcium ions.

The present invention relates to a protein composed of 1 ~ 415 amino acid residues of the SEQ. ID. No. 2, its mutant and the fragment of the protein or the mutant. In the present invention, the present inventors have isolated and solved the structure of a novel protein related to the phenoloxidase activation. The novel 45 kDa protein is an essential component in the phenoloxidase system and a newly identified prophenoloxidase activation factor. The 45 kDa protein has a molecular weight of approximately 45 ~ 50 kDa by SDS-PAGE. To isolate the 45 kDa protein, the body fluid of an insect such as plasma or hemocyte can be used as a specimen. In the process of isolating the body fluid of an insect, a buffer solution containing a chelating agent can prevent coagulation and adsorption of the blood cells instead of adding serine protease inhibitor separately. The 45 kDa protein according to the present invention can be used to obtain the antibody against the 45 kDa protein. Also the antibody can be used to screen 45 kDa protein like in the body fluid of other insects.

The 45 kDa protein of the present invention can be used to prepare the substrate of the phenoloxidase system or the composition to detect beta-1 , 3- glucan based on the phenoloxidase system. In other words, the 45 kDa protein is a factor constituting the phenoloxidase system of the cascade reactions and is cleaved by an activated PPAF-I protein, which is an activation factor in the phenoloxidase system. Therefore, it is possible to design a substrate containing portions of the 45 kDa protein sequence such as a peptide that is recognized and

cleaved by the composition for detecting beta-1 , 3-glucan. The homology of the 45 kDa protein of the present invention has been investigated by using the protein sequence database of National center for biotechnology information (NCBI). The result shows that the 45 kDa protein is a serine protease homologue with an N-terminal domain and an active domain. In the 45 kDa protein, histidine and asparaginic acid residues exist among the three active residues in ordinary serine protease. The 45 kDa protein has an unique structure wherein serine residue is substituted with glycine in the active site of the serine protease. Also six cysteins, which make three disulfide bonds, are preserved in the 45 kDa protein. And there are three potential N-glycosylation sites (Asn-Xaa-Ser/Thr).

It is expected that proteins functioning similar to the 45 kDa protein exist in the body fluid of the insects with the phenoloxidase system activated by beta- 1 , 3-glucan besides the body fluid df the larvae of Holotrichia diomphalia used according to the present invention. It is also expected that the antibodies of 45 kDa protein according to the present invention can be used to detect and to isolate the 45 kDa protein like proteins existing in the body fluid of other insects.

The 45 kDa protein can be mass produced by using the expression system with an aid of the molecular biological technique. For instance, the 45 kDa protein can be mass produced by using the recombinant microorganisms transformed with the gene coding the 45 kDa protein.

Also the present invention relates to a protein composed of 100 ~ 415 amino acid residues of the SEQ. ID. No. 2, its mutant and the fragment of the

protein or the mutant.

The protein named 35 kDa protein in the present description is obtained by cutting off the N-terminal end of the 45kDa protein. In other words, the 35 kDa protein is obtained by cutting between Arg-99 and Glu-100 of the 45 kDa

protein. The 35 kDa protein has a similar function as the 45 kDa protein.

In vitro experiment shows that the 45 kDa protein can not be cleaved by

activated PPAF-I in the presence of Ca²⁺ ion, but is cleaved to 35 kDa by

activated PPAF-l in the absence of Ca²⁺ ion (Figure 5).

Also the present invention relates to a gene coding the 45 kDa protein, its

mutant or its fraction.

The gene according to the present invention is composed of a DNA

sequence coding amino acid residues of the SEQ. ID. No. 2, its mutant or its

fraction. As an example the gene of the present invention is composed of 40 ~

1284 bp of the SEQ. ID. No. 1.

The present invention provides a gene coding the 45 kDa protein by

immunoscreening with the antibody against the 45 kDa protein prepared by the

purified 45 kDa protein after constructing the cDNA library of the insect, the DNA

sequence of the gene and the amino acid sequence coded by the same gene.

The obtained gene has an open reading frame of 1245 bp corresponding to 415

amino acids.

Also the present invention relates to a gene coding the 35 kDa protein, its

mutant and the fragment of the protein or the mutant.

The gene according to the present invention is composed of a DNA

sequence coding amino acid residues 100 ~ 415 of the SEQ. ID. No. 2, its mutant

or its fraction. As an example the gene of the present invention is composed of

DNA sequence of 337 ~ 1284 bp of the SEQ. ID. No. 1.

The gene coding the 45 kDa protein or the 35 kDa protein, its mutant or its

fractions can be used to produce 45 kDa protein or the 35 kDa protein, its mutant or its fractions by the recombinant microorganisms. In the above process, the base sequence can be changed appropriately considering codon usage of the host microorganisms.

The present inventors have identified the fact that the 45 kDa protein has been purified by SDS-PAGE under reducing conditions. The present inventors have also confirmed that the 45 kDa protein is a component of the phenoloxidase composition by determining the phenoloxidase activity with a purified prophenoloxidase, purified active PPAF-I and the 45 kDa protein. Also the in vitro reconstitution experiment shows that the purified prophenoloxidase and activated PPAF-I does not show phenoloxidase activity in the presence of beta- 1 , 3-glucan. Only when the 45 kDa protein is added along with the purified prophenoloxidase and activated PPAF-I, the phenoloxidase activity is exhibited in the presence of beta-1 , 3-glucan. From these results, it is confirmed that the 45 kDa protein is directly related to the activation of the phenoloxidase system and is a component of the phenoloxidase composition.

If the purified 45 kDa protein is added to the phenoloxidase composition, the sensitivity of the phenoloxidase activation reaction by beta-1 , 3-glucan increases more. The 45 kDa protein for the above activation reaction can be selected from the complete 45 kDa protein or its fraction, can be mass produced by using the recombinant microorganism system, and can be partially or completely purified. The 35 kDa protein of the present invention is expected to have same effects that the 45 kDa protein has.

The buffer solutions and the method to determine the phenoloxidase

activity used in the present invention are as follows. Anticoagulation buffer solution (pH 4.6): trisodium citrate 30 mM, citric acid 26 mM, EDTA 20 mM, sodium chloride 15mM β-1 , 3-glucan solution: a solution prepared by mixing 10 μl of the solution,

made by mixing 1 mg of β-1, 3-glucan (curdlan, Wako Pure Chemical Industries,

Ltd., Japan) in 1 ml of 0.1 N NaOH, and 990 μl of 20 mM Tris buffer solution (pH

8.0)

Skim milk solution for antibody purification (pH 7.9): a solution prepared by dissolving 2.5 gram of skim milk in 50 ml of 20 mM Tris/HCI buffer solution (pH 7.9) Wash buffer solution (x 10) for antibody purification: 100 mM Tris, 10 mM

EDTA, 1 % Triton X-100, 1.5 M NaCI

Wash buffer solution (x 1) for antibody purification: a solution prepared by adding 0.5 % of 2 mM NaN₃ in Ix wash buffer solution

TBS: 20 mM Tris, 153 mM NaCI TTBS: 20 mM Tris, 153 mM NaCI, 0.1 %Tween 20

Filter for IPTG treatment: 190.6 mg IPTG, 40ml of distilled and deionized (DDW) water high-TBST solution: 30 ml 1M Tris-HCI (pH 7.9), 5.25 g NaCI, 15 ml 20%

Tween 20 quantity sufficient with DDW to 600 ml low-TBST solution: 20 ml 1 M Tris-HCI (pH 7.9), 17.5 g NaCI, 5 ml 20%

Tween 20 quantity sufficient with DDW to 2 L

3 % Gelatin solution: 12 g gelatin, 800 μl 10% NaN₃ quantity sufficient with

low-TBST to 400 ml

Primary antiserum solution: 2 ml purified antibody, 2 ml 3 % gelatin

solution, 60 μl of E. coli lysate (10 mg/ml), 60 μl 10% NaN₃, 1.88 ml DDW Secondary antiserum solution; 1.7 ml 3 % gelatin solution in 5 ml of goat

anti-rabbit IgG (H+L)-AP conjugate (Bio-Rad Company) (5 μl)

AP buffer solution: 30 ml 1 M Tris-HCI (pH 9.5), 1.74 g NaCI, 1.5ml 1 M MgSO₄ quantity sufficient with DDW to 300 ml

Color development reaction solution: 100 ml AP buffer solution, 1320 μl of

50 mg/ml NBT in 70 % DMF, 660 μl of 50 mg/ml BCIP in DMF

LB liquid medium: 10 g NaCI, 10 g Trypton, 5 g yeast extract quantity sufficient with DDW to 1 L

NZY plate: 5 g NaCI, 2 g MgSO₄.7H₂O, 5 g yeast extract, 10 g NZ amine, 15 g bactoagar, 1 L DDW

NZY plate (top agar): 5 g NaCI, 2 g MgS0₄.7H₂0, 5 g yeast extract, 10 g NZ amine, 7 g bactoagar, 1L DDW

SM buffer solution: 5.8 g NaCI, 2 g MgS0₄.7H₂O, 50 ml of 1 M Tris-HCI (pH 7.5), 5 ml of 2 % gelatin quantity sufficient with DDW to 1 L

Determination of phenoloxidase activity

After reacting 85 μl of 20 mM Tris buffer solution (pH 8.0) containing 1 μg

of beta-1 , 3-glucan and 5 μl of G solution (corresponding to 50 μg pf protein) with

10 μl of hemocyte lysate (corresponding to 200 μg of protein) or 10 μl of purified

45 kDa protein (1 μg) at 30 °C for 10 min as a pretreatment, 400 μl of the

substrate solution ( 20 mM Tris buffer solution (pH 8.0) supplemented with 5 mM CaCI₂, 1 mM 4-methylcatechol(MC), 2 mM 4-hydroxyproline ethylester(HP)) was

added to react at 30 °C for 10 min. To this reaction mixture, 500 μl of 20 % acetic acid was added to terminate the reaction. And the absorbance was measured at 520 nm. One unit of the phenoloxidase activity means 0,1 increase of absorbance at 520 nm.

The present invention is further illustrated by way of the following

Examples, but by no means limited thereto.

Example 1.

Approximately 400 larvae of Holotrichia diomphalia were collected and anesthetized on ice. Hemolymph was collected in a test tube on ice from each of larvae by inserting 1 ml of the anticoagulation buffer solution through a 25 G needle connected to a 5 ml sterile syringe and by dissecting the abdomen of the

larvae. After centrifuging the collected hemolymph for 10 min at 4 °C at 420 x g

and washing it subsequently with the anticoagulation buffer solution, the

hemocytes were collected. The collected hemocytes were stored at -80 °C for

further experiments. Approximately 0.5 g of the hemocytes was suspended into 5ml of the buffer solution A (Tris buffer solution (pH6.5, 50mM + 1mM EDTA)) and homogenized by sonicating for 5 seconds five times. The sonicated

hemocytes were centrifuged for 20 min at 4 °C at 22,000 x g. The supernatant

was used as the hemocyte lysate.

The plasma was collected from the supernatant after centrifuging the hemolymph and used for further experiments by adjusting pH to pH 4.6 by adding

1 M citric acid and by storing at -80 °C. 40 ml of the supernatant, obtained by

centrifuging 45 ml of the plasma for 4 h at 4 °C at 203,006 x g, was concentrated to 3 ml by ultrafiltration (cut off: 10,000). After packing Toyopearl HW-55S resin into a 1.4 x 50 cm column, the column was equilibrated with a sufficient amount of 50 mM Tris-HCI/20mM EDTA buffer solution (pH6.5). The concentrated sample was loaded into the equilibrated column. The solution was eluted at 0.15 ml/min flow rate with 50 mM Tris~HCI/20mM EDTA buffer solution (pH6.5). The concentration of the protein was determined by collecting 3.5 ml fractions and by measuring the absorbance at 280 nm. The phenoloxidase composition was obtained by collecting the fractions exhibiting the phenoloxidase activity by adding calcium ion and beta-1 , 3-glucan. This phenoloxidase composition was named G solution.

Example 2.

To examine the effect of the hemocyte lysate on the phenoloxidase activation, the phenoloxidase activity of the G solution and hemocyte lysate by beta-1 , 3-glucan was determined. The result is shown in Figure 1 (columnl : substrate solution (5 mM CaCI₂, 50 mM Tris buffer solution, 1 mM 4-MC, 2 mM

4-HP), column 2: 5 μl G solution (corresponding to 50 μg protein) + 1 μg beta-

1 , 3-glucan + substrate solution complemented with CaCI₂, column 3: 10 μl

hemocyte lysate 10ul (200 μg protein) + 1 μg beta-1 , 3-glucan + substrate solution

complemented with CaCI₂, column 4: 5 μl G solution I (50 μg protein) + hemocyte

lysate 10ul (200 μg protein) + 1 μg beta-1 , 3-glucan + substrate solution without

CaCI₂, column 5: 5 μl G solution (50 μg protein) + hemocyte lysate 10ul (200 μg

protein) + 1 μg beta-1 , 3-glucan + substrate solution complemented with CaCI₂). Example 3.

The 45 kDa protein was isolated from the hemocyte lysate by the following procedure. Approximately 3 ml of the hemocyte lysate obtained from Example 1 was loaded into the Blue-Sepharose column (1.0X5.2 cm) equilibrated with buffer solution A. The column was washed at a flow rate of 0.2 ml/min. The eluant was collected and concentrated by ultrafiltration (cut off: 10,000). The concentrated sample (2 ml, corresponding to 50 mg protein) was loaded into the Sephacryl S-200 (1.5X120cm) column equilibrated with buffer solution B (50 mM Tris buffer solution (pH 6.5), 1 mM EDTA, 0.1 M NaCI). The fractions were obtained by eluting the column with the same buffer solution. Each fraction was reacted with the substrate containing purified prophenoloxidase, activated PPAF- I and Ca²⁺to collect the fractions with the phenoloxidase activity. The obtained fractions were loaded into Phenyl Sepharose column (0.5X7cm) equilibrated with buffer solution C (50 mM phosphate buffer solution (pH7.0), 1.7 M ammonium sulfate) and eluted with a linear gradient of 1.7 ~ 0 M ammonium sulfate gradient at a flow rate of 0.3 ml/min. The fractions containing the 45 kDa protein were identified by SDS-PAGE under reducing conditions and concentrated by ultrafiltration (cut off: 10,000). The concentrated sample was loaded into Superdex-200 FPLC column equilibrated with buffer solution B and eluted by using the same buffer solution. The fractions exhibiting the phenoloxidase activity was collected and loaded into mono-Q column equilibrated with buffer solution D (20 mM Tris buffer solution (pH 7.4)) and eluted by applying linear gradient of 0 ~ 1 M Sodium Chloride at a flow rate of 0.4ml/min. A single protein band at ca. 45 kDa was obtained by performing 10 % SDS-PAGE under reducing and non-reducing conditions from the fractions with phenoloxidase activity. The purified 45 kDa protein from Example 4 was used to prepare polyclonal antibody from rabbits. It was confirmed that the purified 45 kDa protein from the hemocyte lysate is a component of the G solution by performing Western blotting experiment using the obtained antibody. The result is shown in Figure 2 (lane 1: size marker, lane 2: hemocyte lysate, lane 3: G solution, lane 4: isolated purified 45 kDa protein).

The purified 45 kDa protein was digested by trypsin to determine partial

amino acid sequences. In the solution containing 30 μg of the sample, equal

volume of 0.4 M NH₄HCO₃ buffer solution containing 8 M urea was added to dissolve the sample. 45 mM DTT solution corresponding to 10 % of the total

volume was added and reacted for 15 min at 50 °C to reduce the protein. 100

mM iodoacetamide solution corresponding to 10 % of the total volume was added to the reduced solution and reacted for 15 min at room temperature to alkylate the reduced cysteine residues. The product was precipitated by adding 10 % TCA, and the obtained protein residues were dissolved with 0.4 M NH₄HCO₃ buffer

solution containing 8 M urea. The proteins were fragmented by adding 2 μg

trypsin and reacted for 13 hours at 37 °C. The fragmented proteins were

separated with a C18 reverse phase HPLC column (chemcosorb 5-ODS-H

2.1x 150/W) under linear concentration gradient system for 90 min until the

concentration of solvent B reached up to 90 % at a flow rate of 0.2 ml/min. The partial amino acid sequence was determined by Edman degradation method. Partial amino acid sequence AGEWDTLTEKERLPY PLVQADNI FVLDQTFVXAGGE

Example 4.

The antibody of the 45 kDa protein was obtained by injecting the purified 45 kDa protein obtained in Example 3 into a rabbit (white, male, 2.7 kg). To confirm the antibody formation, the 45 kDa protein was separated by

10 % SDS-PAGE under reducing conditions and transferred to a PVDF

membrane in the transfer buffer solution at 280 mM at 4 °C for 4 hours. The

membrane was washed with TBS, and nonspecific reaction was inhibited by incubating upon shaking the membrane in TBS supplemented with 5 % skim milk

and 1 % horse serum at 4 °C overnight. The antiserum separated from the rabbit

was incubated upon shaking with 100 times diluted TTBS containing 2.5 % skim milk at room temperature for 2 hours. After washing five times with TTBS, the secondary antibody (anti-rabbit Ig. horseradish peroxidase-conjugated donkey antibody) was incubated with 100 times diluted TTBS at room temperature for 30 min. After washing five times with TTBS, ECL blot was performed for 1 min under semi-dark conditions to photosensitize the developed fluorescence. It was confirmed that this antibody specifically recognizes the 45 kDa protein.

Example 5. After culturing upon shaking XL-1-Blue in the mixed solution of 5 ml LB

medium, 50 μl of 20 % maltose, and 50 μl of 1 M MgSO₄, 10 mM MgS0₄ was

added into the precipitated cells to adjust OD₆₀₀ to 0.5. According to the method

described by Moon et al. (J. Biochem. (1994) (Tokyo)1 16, 53-58), cDNA library of Holotrichia diomphalia larva was prepared. Into the cDNA library solution,

200 μl of XL-1-blue was added and cultured at 37 °C for 15 min upon shaking.

The solution was mixed with 3.5 ml top agar, which was preheated to 48 °C, and

the mixture was spread onto NZY plate. After cultivating for 4 hours at 42 °C,

IPTG treated filter was placed on the plate and cultured for 8 more hours at 37 °C.

After the termination of the reaction, the plate was stored for 20 min at 4 °C, and

the filter was dried completely in air. The dried filter was washed with high-TBST

solution at room temperature for 10 min and with low-TBST solution twice at room

temperature for 10 min. Water was removed completely from the washed filter.

The filter was placed in 3 % gelatin solution for blocking for 30 min at 27 °C.

Subsequently, the filter was reacted with the antibody solution against the 45 kDa

protein for 2.5 hours at 27 °C. After the termination of the reaction, the filter was

washed three times with low-TBST solution for 30 min at 27 °C. This filter was

washed three times with low-TBST solution to carry out the color development

reaction as follows.

The Color was developed from the prepared filter by immersing the filter in

the color development reaction solution, or 100ml of AP buffer containing 1320 μl

of 50mg/ml nitro blue tetrazolium chloride (NBT, Bio-Rad 170-6532) and 660 μl of

50 mg/ml 5-bromo-4-chloro-3-indoylphosphate-p-toluidine salt (BCIP, Bio-Rad

170-6539). The filter was dried completely in air. The clone exhibiting remarkably purple color was considered to be primary positive clone. The primary positive clone was diluted appropriately with LB liquid medium. In the

quantity assumed to produce 100 ~ 200 plaques, 200 μl XL-1-Blue (OD₆₀₀=0.5)

was added and cultured for 15 min at 37 °C upon shaking. In this solution, 3 ml

of the preheated top agar was added and spread onto the NZY plate. The secondary positive clone was selected by following the same steps as in the selection of the primary selection.

Two hundred microliters XL-1-Blue (OD₆₀₀=1.0), 200 μl positive plaque

solution obtained from the secondary selection process and 1 μl ExAssist helper

phage was put into a 50 ml Felcon tube and cultured upon shaking. After adding

3 ml LB liquid medium was added and culturing for 3 hours at 37 °C upon shaking,

the solution was thermally treated for 20 min at 70 °C. The thermally treated

solution was centrifuged for 15 min at 1 ,000^χ g, and 15 ml of the supernatant was

drawn and stored at 4 °C.

In 200 μl SOLR cells (OD₆₀₀=1.0), 2 μl or 10 μl each of phagemid diluted

10 times with LB liquid medium was added and cultured for 15 min at 37 °C upon

shaking. Fifty microliters of the solution was taken and spread onto the

LB/ampicillin plate. After culturing for 12 hours at 37 °C, single colony was

isolated and cultured by adding 5 ml LB liquid medium and 10 μl ampicillin (50

mg/ml) for 12 hours at 37 °C. The plasmid with the inserted gene for 45 kDa

protein was isolated with a DNA purification kit (Promega, wizard plus sv

minipreps).

The gene sequence of the 45kDa protein was doubly determined from the 5' and 3' ends (SEQ. ID. No. 1). From the base sequence, the amino acid sequence was deduced (SEQ. ID. No. 2)

Example 6.

In the reconstitution experiment with the purified 45 kDa protein, prophenoloxidase and the activated PPAF-I, the phenoloxidase activity was determined. Prophenoloxidase and activated PPAF-I were purified as described earlier (Lee et al. (1998) Eur. J. Biochem. 254, 50-57, Kwon et al. (1997) Mol. Cells. 7(1). 90-97). The result of the phenoloxidase activation experiment is shown in Figure 3 with or without Ca ⁺, 45 kDa protein, prophenoloxidase,

activated PPAF-I (column 1 : substrate solution, column 2: 2 μg prophenoloxidase,

column 3: 1 μg 45 kDa protein, column 4: 1 μg PPAF-I, column 5: 2 μg

prophenoloxidase + 1 μg 45 kDa protein, column 6: 2 μg prophenoloxidase + 1 μg

PPAF-I, column 7: 1 μg 45 kDa protein + 1 μg PPAF-I, column 8: 2 μg

prophenoloxidase + 1 μg 45k Da protein + 1 μg PPAF-I (without CaCI₂), column 9:

2 μg prophenoloxidase + 1 μg 45 kDa protein + 1 μg PPAF-I). When the 45 kDa

protein was reacted with prophenoloxidase and the activated PPAF-I, the phenoloxidase activity was observed regardless of Ca²⁺ concentration (column 8 and 9). The phenoloxidase activity was not observed, however, when prophenoloxidase was reacted with only one of the 45 kDa protein or the activated PPAF-I (column 6 and 7).

The result of the electrophoresis experiment of the above mixture exhibiting the phenoloxidase activity under reducing conditions is shown in Figure

4 ((A), lane 1 : prophenoloxidase(2 μg), lane 2: 45kDa protein(2 μg), lane 3: PPAF-I (2 μg), lane 4: prophenoloxidase(2 μg), 45kDa protein (2 μg) and PPAF-

1(2 μg) without adding Ca²⁺, for 60 min at 30 °C, (B) reacted under the same condition with Ca²⁺). The result shows that the 45 kDa protein is cleaved to a 35 kDa in size when the reaction is carried out without Ca ⁺.

Example 7.

To confirm the production of the 35 kDa protein, the activated PPAF-I and the 45 kDa protein was reacted with or without Ca²⁺. The result of the

electrophoresis experiment is shown in Figure 5 ((A), lane 1 : 45kDa protein (2 μg),

lane 2: PPAF-I (2 μg), lane 3: 45kDa protein (2 μg) and PPAF-I (2 μg) without

adding Ca²⁺, for 60 min at 30 °C, (B) reacted under the same condition with Ca ⁺).

The result shows that the 45 kDa protein is cleaved to 35 kDa in size by the activated PPAF-I in the absence of Ca²⁺. By transferring the 35 kDa protein onto a PVDF membrane after electrophoresis and determining the amino acid sequence, it was confirmed that the 45 kDa protein was cleaved between Arg-99 and Glu-100.

Example 8

The phenoloxidase activity more increased by adding G solution and the purified 45 kDa protein in the beta-1 , 3-glucan solution. The result is shown in Figure 6. EFFECT OF THE INVENTION

The protein according to the present invention is one of the phenoloxidase activation factors and is a part of the effective composition for diagnosing fungal infections. The protein of the present invention can be used to prepare the composition for diagnosing fungal infections. Also the gene according to the present invention can be used in mass-producing the protein necessary to prepare the composition for diagnosing fungal infections.

Claims

1. A protein comprising amino acid residues 1 - 415 of the SEQ. ID. No. 2, its mutant or its fraction. 2. A protein comprising amino acid residues 100 - 415 of the SEQ. ID. No.

2, its mutant or its fraction.

3. A DNA sequence coding the protein comprising amino acid residues 1 - 415 of the SEQ. ID. No. 2, its mutant or its fraction.

4. A DNA sequence coding the protein comprising amino acid residues 100 - 415 of the SEQ. ID. No. 2, its mutant or its fraction.