Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
  • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
  • C07K14/4702—Regulators; Modulating activity
  • C07K14/4705—Regulators; Modulating activity stimulating, promoting or activating activity
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans

Definitions

• 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.
• 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.
• 83/02123 discloses a method for determining ⁇ -1 , 3-glucan specifically by using
• composition for specific determination of ⁇ -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.
• 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)).
• 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)).
• 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
• composition comprising prophenoloxidase for detecting ⁇ -1 , 3-
• PCT/KR01/00196 can be used to diagnose a fungal infection
• the present invention relates to a novel protein, which is a component in
• composition for fungal infection diagnosis activated by ⁇ -1 , 3-glucan.
• the present invention relates to a gene coding the novel protein, which is
• 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 Ca 2+ , 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 Ca 2+ 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.
• the present invention relates to a novel protein, which is a component in
• composition for fungal infection diagnosis related to the prophenoloxidase
• amino acid sequence As used herein, the term 'amino acid sequence', 'polypeptide' or 'protein'
• polypeptide or a protein.
• base sequence mutant refers to an altered
• insects refers to an insect or insects that has a phenoloxidase system in the body, and preferably a holometabola insect.
• insects include those belonging to Crustaceans such as lobsters and shrimps and Coleoptera.
• prophenoloxidase is present and is activated to phenoloxidase by a cascade reaction, which is activated by beta-1 , 3-glucan or lipopolysaccharide.
• phenoloxidase system refers to a system that
• phenoloxidase composition refers to a composition that comprises all or some components of the phenoloxidase system and detects ⁇ -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.
• 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.
• the body fluid of an insect such as plasma or hemocyte can be used as a specimen.
• 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.
• 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
• 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.
• NBI National center for biotechnology information
• the 45 kDa protein has an unique structure wherein serine residue is substituted with glycine in the active site of the serine protease.
• 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).
• 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.
• the 45 kDa protein can be mass produced by using the recombinant microorganisms transformed with the gene coding the 45 kDa protein.
• 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
• the protein named 35 kDa protein in the present description is obtained by cutting off the N-terminal end of the 45kDa protein.
• the 35 kDa protein is obtained by cutting between Arg-99 and Glu-100 of the 45 kDa
• the 35 kDa protein has a similar function as the 45 kDa protein.
• the present invention relates to a gene coding the 45 kDa protein, its
• the gene according to the present invention is composed of a DNA
• the gene of the present invention is composed of 40 ⁇
• the present invention provides a gene coding the 45 kDa protein by
• the obtained gene has an open reading frame of 1245 bp corresponding to 415
• the present invention relates to a gene coding the 35 kDa protein, its
• the gene according to the present invention is composed of a DNA
• the gene of the present invention is composed of
• fractions can be used to produce 45 kDa protein or the 35 kDa protein, its mutant or its fractions by the recombinant microorganisms.
• 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.
• 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.
• the 45 kDa protein is directly related to the activation of the phenoloxidase system and is a component of the phenoloxidase composition.
• 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.
• Anticoagulation buffer solution pH 4.6: trisodium citrate 30 mM, citric acid 26 mM, EDTA 20 mM, sodium chloride 15mM ⁇ -1 , 3-glucan solution: a solution prepared by mixing 10 ⁇ l of the solution,
• 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
• Wash buffer solution (x 1) for antibody purification a solution prepared by adding 0.5 % of 2 mM NaN 3 in Ix wash buffer solution
• TBS 20 mM Tris, 153 mM NaCI
• TTBS 20 mM Tris, 153 mM NaCI, 0.1 %Tween 20
• 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%
• AP buffer solution 30 ml 1 M Tris-HCI (pH 9.5), 1.74 g NaCI, 1.5ml 1 M MgSO 4 quantity sufficient with DDW to 300 ml
• Color development reaction solution 100 ml AP buffer solution, 1320 ⁇ l of
• 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 MgSO 4 .7H 2 O, 5 g yeast extract, 10 g NZ amine, 15 g bactoagar, 1 L DDW
• NZY plate top agar: 5 g NaCI, 2 g MgS0 4 .7H 2 0, 5 g yeast extract, 10 g NZ amine, 7 g bactoagar, 1L DDW
• SM buffer solution 5.8 g NaCI, 2 g MgS0 4 .7H 2 O, 50 ml of 1 M Tris-HCI (pH 7.5), 5 ml of 2 % gelatin quantity sufficient with DDW to 1 L
• hemocytes were collected. The collected hemocytes were stored at -80 °C for
• hemocytes were centrifuged for 20 min at 4 °C at 22,000 x g. The supernatant
• 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
• 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 Ca 2+ 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
• 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
• Example 5 After culturing upon shaking XL-1-Blue in the mixed solution of 5 ml LB
• the solution was mixed with 3.5 ml top agar, which was preheated to 48 °C, and
• IPTG treated filter was placed on the plate and cultured for 8 more hours at 37 °C.
• the plate was stored for 20 min at 4 °C, and
• the filter was placed in 3 % gelatin solution for blocking for 30 min at 27 °C.
• the Color was developed from the prepared filter by immersing the filter in
• 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.
• the secondary positive clone was selected by following the same steps as in the selection of the primary selection.
• phage was put into a 50 ml Felcon tube and cultured upon shaking. After adding
• 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)
• column 3 1 ⁇ g 45 kDa protein
• column 4 1 ⁇ g PPAF-I
• column 5 2 ⁇ g
• lane 2 PPAF-I (2 ⁇ g)
• lane 3 45kDa protein (2 ⁇ g)
• the result shows that the 45 kDa protein is cleaved to 35 kDa in size by the activated PPAF-I in the absence of Ca 2+ .
• 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.
• 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.
• the gene according to the present invention can be used in mass-producing the protein necessary to prepare the composition for diagnosing fungal infections.

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• 2000
  • 2000-08-24 KR KR10-2000-0049207A patent/KR100456006B1/ko not_active IP Right Cessation
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  • 2001-08-24 AU AU2001282640A patent/AU2001282640A1/en not_active Abandoned
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