KR890007749A - Antimicrobial protein composition containing the same and its use - Google Patents

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Antimicrobial protein composition containing the same and its use

Since this is an open matter, no full text was included.

FIG. 1: Subcellular distribution of the bactericidal activity in human neutrophils, (A) is a marker for azolofil granules (spinal cord peroxidase), specific granules (vitamin B12 binding protein) and plasma membrane (alkaline phosphate) And (B) is the profile of the bactericidal activity, each fraction extracted with 0.05 M glycine-Hcl pH 2.0 and centrifuged at 10,000 g for 20 minutes and the supernatant at a protein concentration of 5 micrograms / ml. . Incubated with E. coli K12. Bacterial lethality after incubation at 37 ° C. for 30 minutes was indicated, with the degree of insertion (C) being the purified adjuvants required to yield a 50% reduction in bacterial colony-forming units (LD ₅₀ ). ●) and tablets The amount of protein from specific granules (Δ) is shown.

FIG. 2: HL60 Postnucleate Supernatant Percoll Gradients of bacterium activity and bactericidal activity, dotted line (…) indicates turbidity (OP ₄₅₀ ), and thick bar lines indicate bactericidal activity The hatched bar line indicates the bactericidal activity.

FIG. 3: Graph of membrane association of azurophil-induced bactericidal factor (ADBF). Azulope granules separated in buffer (pH7.3) were ground and centrifuged by freeze-thaw / sonic. Supernatant (■), pellet material (●) and overall granules (Δ) were extracted at pH 2.0 and tested for bactericidal activity.

Claims (100)

Consists of at least two polypeptides selected from the group consisting of human polymorphonuclear leukocyte polypeptides having an apparent molecular weight of 3,500 Daltons, 13,000 Daltons, 18,000 Daltons, 29,000 Daltons, and 54,000 Daltons, under conditions of pH 5.0-8.0, 10 mM Respiratory rupture-independent bactericidal activity against bacteria and fungi at below calcium ion concentration, bactericidal activity against bacteria at sodium chloride concentration below 0.3M, bactericidal activity against fungi at sodium chloride concentration below 0.15M A composition useful as a fungicide with a. The composition of claim 1, wherein the polypeptides of human polymorphonuclear leukocytes are derived from HL-60 cells. The human polymorphonuclear leukocyte polypeptides in the composition of claim 1 derived from KG-1 cells. The composition of claim 1, wherein the human polymorphonuclear leukocyte polypeptides are derived from azurophil granules. 2. The composition of claim 1, wherein the human polymorphonuclear leukocyte polypeptides having an apparent molecular weight of about 3500 Daltons are defensin. The composition according to claim 1, wherein the human polymorphonuclear leukocyte polypeptides having an apparent molecular weight of 13000 Daltons contain a polypeptide of 12867 Daltons from amino acid 105 to amino acid 220 shown in FIG. The composition of claim 1 comprising a human polymorphic leukocyte polypeptide having an apparent molecular weight of about 18000 Daltons present in the azurophil granules extract. The composition of claim 1 comprising a human polymorphonuclear leukocyte polypeptide having a molecular weight of about 29000 daltons present in the azure cell granule extract. 9. The composition of claim 8, wherein the human polymorphonuclear leukocyte polypeptide having an apparent molecular weight of about 29000 Daltons is elastase. The composition of claim 8, wherein the human polymorphonuclear leukocyte polypeptide having an apparent molecular weight of about 29000 Daltons is Catepsin G. The composition of claim 1 comprising a human polymorphonuclear leukocyte polypeptide having an apparent molecular weight of about 54000 daltons present in the azure cell granule extract. The human polymorphonuclear leukocyte polypeptide of claim 11 having an apparent molecular weight of about 54000 Daltons has an N-terminal amino acid sequence Val-Asn-Pro-Gly-Val-Val-Arg-Ile-Ser-Gln-Lys-Gly- Leu-Asp-Tyr-Ala-Ser-Gln-Gln-Gly-Thr-Ala-Ala-Leu-Gln-XX-Leu-Lys-His-Ile-Lys-Ile-Pro-Asp-Tyr-Leu Composition. The composition according to claim 1, wherein the bacteria are Gram-positive bacteria or Gram-negative bacteria. The composition according to claim 3, wherein the Gram-positive bacteria is Staphylococcus aureus, β-hemolytic streptococci, or Listeria, and the Gram-negative bacteria are Pseudomonas aeryuzinosa, Escherichia coli, or Salmonella ripimurium. The composition of claim 1 wherein the fungus is yeast. The composition of claim 15 wherein the yeast is Candida albicans. A method for sterilizing bacteria or fungi comprising contacting bacteria or fungi with a sterilizing effective amount of the composition of claim 1. A pharmaceutical composition effective for treating a bacterial or fungal infection containing the composition of claim 1 and a pharmaceutically applicable carrier. A method of treating a patient with bacterial or fungal sensitization comprising administering to a patient an amount of the pharmaceutical composition of claim 18 effective to sterilize the bacterium or fungus. Among the methods for preparing a composition effective as a bactericide: (a) sufficient isolation of polymorphonuclear leukocytes from blood to obtain a sample rich in polymorphonuclear leukocytes: (b) suspending the sample rich in polymorphonuclear leukocytes in a suitable buffer: (c) Treating this polymorphonuclear leukocyte suspension with an appropriate protease inhibitor: (d) treating the polymorphonuclear leukocyte suspension to dissolve suspended leukocytes: (e) treating the dissolved leukocyte suspension above with an appropriate chelating agent: (f) The treated lysed leukocyte suspension was centrifuged to obtain nuclear / non-destructive cell phase and postnuclear supernatant: (g) recovery of postnuclear supernatant: (h) postnuclear according to density gradient. The supernatant is fractionated: (i) a fraction of the density gradient comprising the azure cell granules is collected: (j) the collected azure cell granules have a pH of about 7 Suspension in the sap solution: (k) The suspension of the azure cell granules obtained as a result of the above was treated with an extraction solvent having a pH of 8.0 or less and capable of dissolving the azure cell membrane protein to obtain an extraction solvent / azure cell membrane suspension: (l) separating the extractant / azur apoptotic cell suspension to form a solid and a supernatant: (m) a method comprising recovering the supernatant. The method of claim 20, wherein the protease inhibitor is diisopropylfluorophosphate. The method of claim 20, wherein the blood sample is obtained from a human. The method of claim 20, wherein the sample rich in polymorphonuclear leukocytes contains at least about 95% polymorphonuclear leukocytes and at most about 3% eosinophils. The method of claim 20, wherein the polymorphonuclear leukocyte suspension is treated with nitrogen vacuum to obtain a dissolved leukocyte suspension. The method of claim 20, wherein the density gradient comprises a discrete Percoll density gradient. 21. The method of claim 20, wherein the fraction of density gradient is separated from the azure cell granules prior to performing the process of (j). 21. The method of claim 20, wherein the azure cell granule suspension is treated to dissolve the azure apoptotic cell granules prior to the process of (k), the cell membrane fragments are recovered, and treated with an extractant to extract / Azur apoptotic membrane suspension. How to get it. The method of claim 20, wherein the pH of the extractant is 4.0 or less. The method of claim 28, wherein the pH of the extractant is about 2.0. 21. The method of claim 20, wherein the extractant has a pH of about 70. and is a nonionic detergent. A purified polypeptide effective as a fungicide comprising a polymorphonuclear leukocyte polypeptide of human having an inferred molecular weight of 24823 Daltons and having a fragment derived therefrom or including a fragment derived therefrom. A DNA molecule encoding a polypeptide of claim 31 comprising at least a portion of the nucleic acid sequence shown in FIG. 20. 32. The purified polypeptide of claim 31, having an apparent molecular weight of about 13000 Daltons. The purified polypeptide of claim 33, wherein the inferred molecular weight is 12867 daltons. 35. The purified polypeptide of claim 34 comprising an amino acid sequence from amino acid 105 to amino acid 220 shown in FIG. A DNA molecule encoding a polypeptide of claim 35 comprising a DNA sequence from nucleic acid salt 369 to nucleic acid salt 716 shown in FIG. 32. The purified polypeptide of claim 31 having an apparent molecular weight of about 18000 Daltons. A DNA molecule encoding the polypeptide of claim 37. A method of sterilizing the polypeptide of claim 31 by contacting it with a bactericidal effective amount. A vector comprising the DNA molecule of claim 38. 41. The vector of claim 40 comprising a plasmid. A host vector system comprising the plasmid of claim 41 in a suitable host and producing a human polymorphonuclear leukocyte polypeptide or fragment thereof having an inferred molecular weight of 24823 daltons. Production of human polymorphonuclear leukocyte polypeptides or fragments thereof having a molecular weight of 24823 Daltons including the method of growing the host vector system of claim 42 under suitable conditions capable of producing human polymorphonuclear leukocyte polypeptides and resulting human polymorphisms A method of recovering a nuclear leukocyte polypeptide. 32. A method for producing a purified polypeptide of claim 31: (a) culturing neutrophil progenitor cells: (b) harvesting cultured neutrophil progenitor cells: (c) culturing the neutrophil progenitor cells obtained as (b). Suspended in a suitable buffer: (d) the resulting neutrophil progenitor suspension was treated to obtain a lysed neutrophil progenitor suspension: (e) dissolved to obtain nuclear and non-destructive cell phases and postnuclear supernatants. Isolate neutrophil progenitor suspensions: (f) Recover postnucleate supernatant: (g) Extract postnucleate supernatant with an extraction solvent that has a pH of about 8.0 or less and can dissolve cell membrane proteins. Obtain insoluble cell membrane phase: (h) separate extractant phase from insoluble cell membrane phase to obtain soluble protein phase and insoluble cell membrane phase: (i) recover soluble protein phase: (j ) Purifying the recovered soluble protein phase to obtain purified 24823 Daltons polypeptide or fragment thereof. The method of claim 44, wherein the neutrophil progenitor cells are HL-60 cells. 45. The method of claim 44, wherein prior to performing step (d), the neutrophil progenitor suspension is treated with a suitable protease inhibitor. 45. The method of claim 44, wherein the suspension of dissolved neutrophils is treated with a suitable chelating agent before performing step (e). 45. The method of claim 44, wherein the soluble protein phase is purified by ion exchange chromatography. 45. The method of claim 44, wherein the soluble protein phase is purified by size exclusion chromatography. 45. The method of claim 44, wherein the soluble protein phase is purified by affinity chromatography. 45. The method of claim 44, wherein the soluble protein phase is purified by immuno-affinity chromatography. A pharmaceutical composition useful for the treatment of a bacterial infection containing a sterile effective amount of the purified polypeptide of claim 21 and a pharmaceutically applicable carrier. A method of treating a bacterial infection patient comprising administering to the patient a sterile effective amount of the pharmaceutical composition of claim 52. A purified polypeptide useful as a fungicide comprising a human polymorphonuclear leukocyte polypeptide having an apparent molecular weight of about 18000 Daltons. 55. The method of claim 54, wherein there is no respiratory rupture-dependency, bactericidal activity against bacteria and fungi at a calcium ion concentration of at least 10 mM and a pH of 5.0 to 8.0, and bactercidal activity at a sodium chloride concentration of at least about 0.3 M. , A purified polypeptide having fungicidal activity at a sodium chloride concentration of at least about 0.15 M. A DNA molecule encoding the polypeptide of claim 54. A method of disinfecting a microorganism by contacting the microorganism with a bactericidal effective amount of the polypeptide of claim 54. A vector comprising the DNA molecule of claim 56. 59. The vector of claim 58 comprising a plasmid. A host vector system comprising the plasmid of claim 59 in a suitable host for producing a human polymorphonuclear leukocyte polypeptide having an apparent molecular weight of about 18000 Daltons. A method of producing a human polymorphonuclear leukocyte polypeptide having an apparent molecular weight of about 18000 Daltons, including growth of the host vector system of claim 60, under conditions suitable for producing human polymorphonuclear leukocyte polypeptides and the resulting human polymorphonuclear leukocytes A method of recovering a polypeptide. 55. A method for preparing a purified polypeptide of claim 54: (a) culturing neutrophil progenitor cells: (b) harvesting the cultured neutrophil progenitor cells: (c) suspending the cultured neutrophil progenitor cells of (b) in a suitable buffer. And (d) treating the suspension of neutrophil progenitors in (c) to obtain a suspension of lysed neutrophil progenitors, and (e) separating the suspension of treated lysed neutrophil progenitors into the phase and post of the nuclear and non-destructive cells. Obtain a postnuclear supernatant: (f) Recover the postnuclear supernatant: (g) Treat the post nucleus supernatant with an extraction solvent that has a pH of 8.0 or less and can dissolve cell membrane proteins. And (h) separating the extractant phase from the insoluble cell membrane phase to obtain a soluble protein phase and an insoluble cell membrane phase: (i) recovering the soluble protein phase and then (j) recovering Sea component manufacturing method comprising a process for producing a polypeptide of the 18,000 dalton protein purification Purification of the phase. The method of claim 62, wherein the neutrophil progenitor cells are HL-60 cells. 63. The method of claim 62, wherein prior to performing step (d), the neutrophil progenitor suspension is treated with a suitable protease yesterday. 63. The method of claim 62, wherein the suspension of dissolved neutrophils is treated with a suitable chelating agent prior to performing step (e). 63. The method of claim 62, wherein the soluble protein phase is purified by ion exchange chromatography. 63. The method of claim 62, wherein the soluble protein phase is purified by size exclusion chromatography. 63. The method of claim 62, wherein the soluble protein phase is purified by affinity chromatography. 63. The method of claim 62, wherein the soluble protein is purified by immuno-affinity chromatography. A pharmaceutical composition effective for treating an infection of a bacterium or fungus comprising a sterile effective amount of the bacterium or fungus in an amount of the purified polypeptide of claim 54 and a pharmaceutically applicable carrier. A method of treating a patient infected with a bacterium or fungus comprising administering the pharmaceutical composition of claim 70 to a patient in an amount effective to sterilize the bacterium or fungus. Polymorphonuclear humans with no respiratory burst-independent, bactericidal activity at calcium ion concentrations of at least about 10 mM and sodium chloride concentrations of at least 0.3M, pHs of about 5.0 to 8.0, and apparent molecular weights of about 54000 daltons Purified polypeptides useful as fungicides comprising leukocyte polypeptides. 73. The purified polypeptide of claim 72 having bactericidal activity against fungi at a pH of about 5.0 to 8.0, a calcium ion concentration of at least about 10 mM and a sodium chloride concentration of about 0.15 M. A DNA molecule encoding the polypeptide of claim 72. 73. The N-terminal amino acid sequence Val-Asn-Pro-Gly-Val-Val-Val-Arg-Ile-Ser-Gln-Lys-Gly-Leu-Asp-Tyr-Ala-Ser-Gln-Gln Purified polypeptide comprising Gly-Thr-Ala-Ala-Leu-Gln-XX-Leu-Lys-His-Ile-Lys-Ile-Pro-Asp-Tyr-Leu. 75. A method of sterilizing bacteria or fungi comprising contacting the polypeptide of claim 73 with a bacterium or fungus in an amount effective to sterilize the bacterium or fungus. A vector comprising the DNA molecule of claim 74. 78. The vector of claim 77 comprising a plasmid. Suitable host for the production of polymorphonuclear leukocyte polypeptides in humans having an apparent molecular weight of about 54000 Daltons, which are not respirable rupture-dependent and have bactericidal activity at a pH of about 5.0 to 8.0, at least about 10 mM calcium ion and at least about 0.3 M sodium chloride. A host vector system comprising the plasmid of claim 78 therein. In the manufacturing method for the production of polymorphonuclear leukocyte polypeptide of human with no respirable rupture-dependency, bactericidal activity at pH about 5.0 to 8.0, calcium ion concentration over 10mM and sodium chloride concentration over 0.3M and apparent molecular weight 54000 Daltons 79. A method comprising growing the host vector system of claim 79 and recovering the human polymorphonuclear leukocyte polypeptide obtained therefrom under conditions suitable for producing human polymorphonuclear leukocyte polypeptide. 73. A method of preparing a purified polypeptide of claim 72, comprising: (a) culturing neutrophil progenitor cells: (b) harvesting cultivated posterior neutrophil progenitor cells; and (c) neutrophil progenitor cells harvested in (b). Suspended in: (d) treated with neutrophil progenitors in (c) to obtain a suspension of lysed neutrophil progenitors; and (e) treated with lysed neutrophil progenitor suspensions. Obtain a postnuclear supernatant: (f) Recover the postnuclear supernatant: (g) Treat the postnuclear supernatant with an extraction solvent that has a pH of about 8.0 or less and can dissolve cell membrane proteins. Obtaining the cell membrane phase: (h) Isolating the extractant phase from the insoluble cell membrane phase to obtain the soluble protein phase and the insoluble cell membrane phase: (i) Recovering the soluble protein phase: (j) 54000 Daltons Production process which comprises the purification of the recovered soluble protein to obtain the polypeptide. 82. The method of claim 81, wherein the neutrophil precursor cells are HL-60 cells. 82. The method of claim 81, wherein prior to performing step (d), the neutrophil progenitor suspension is treated with a suitable protease inhibitor. 82. The process of claim 81, wherein prior to performing step (e), the dissolved neutrophil suspension is treated with a suitable chelating agent. 82. The method of claim 81, wherein the soluble protein phase is purified by ion exchange chromatography. 84. The method of claim 81, wherein the dissolved protein phase is purified by size exclusion chromatography. 82. The method of claim 81, wherein the soluble protein phase is purified by affinity chromatography. 82. The method of claim 81, wherein the soluble protein phase is purified by immuno-affinity chromatography. A pharmaceutical composition useful for treating a bacterial infection comprising a sterile effective amount of the purified polypeptide of claim 72 and a pharmaceutically applicable carrier. 90. A method of treating a bacterially infected patient comprising administering to the patient a sterile effective amount of the pharmaceutical composition of claim 89. A pharmaceutical composition useful for the treatment of a bacterial or fungal infection comprising a sterile effective amount of the bacterium or fungal micropeptide of claim 73 and a pharmaceutically applicable carrier. 92. A method of treating a patient with a bacterial or fungal infection comprising administering to the patient a sterile effective amount of the pharmaceutical composition of claim 91. A method of sterilizing fungi comprising contacting a fungalidal fungus with a polymorphonuclear leukocyte polypeptide of human having an apparent molecular weight of about 29000 Daltons. 94. The method of claim 93, wherein the human polymorphonuclear leukocyte polypeptide having an apparent molecular weight of about 29000 daltons is present in the azure cell granule extract. 95. The method of claim 93, wherein the human polymorphonuclear leukocyte polypeptide having an apparent molecular weight of about 29000 Daltons is elastase. 94. The method of claim 93, wherein the human polymorphonuclear leukocyte polypeptide having an apparent molecular weight of about 29000 Daltons is cathepsin. A pharmaceutical composition comprising a human polymorphonuclear leukocyte polypeptide having an apparent molecular weight of about 29000 Daltons and a pharmaceutically applicable carrier. 98. A method of treating a patient with a bacterial infection comprising administering to the patient a sterile effective amount of the pharmaceutical composition of claim 97. The method of treating a patient with a fungal infection comprising administering the pharmaceutical composition of claim 97 to a patient in a sterile effective amount of the fungus. No respiratory rupture-dependent, bactericidal effect against bacteria and fungi at pH of about 5.0 to 8.0, calcium ion concentration of about 10 mM or more, bactericidal activity against bacteria at sodium chloride concentration of about 0.3M or more, about 0.15M A purified polypeptide effective as a fungicide comprising a human polymorphonuclear leukocyte polypeptide having an apparent molecular weight of about 25000 Daltons having fungicidal activity against fungi at a sodium chloride concentration above. ※ Note: The disclosure is based on the initial application.