Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/575—Immunoassay; Biospecific binding assay; Materials therefor for cancer
• • 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
  • C07K14/475—Growth factors; Growth regulators
  • C07K14/515—Angiogenesic factors; Angiogenin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
  • G01N2333/475—Assays involving growth factors
  • G01N2333/515—Angiogenesic factors; Angiogenin
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S436/00—Chemistry: analytical and immunological testing
  • Y10S436/815—Test for named compound or class of compounds
  • Y10S436/817—Steroids or hormones
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
  • Y10S530/806—Antigenic peptides or proteins
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
  • Y10S530/827—Proteins from mammals or birds
  • Y10S530/832—Milk; colostrum

Definitions

• the present invention relates to a new protein of about 17 KD, with angiogenic action, to its method of isolation from milk of mammals, to therapeutic compositions containing it, to a method of detection and / or of assay, as well as immunological reagents for detecting and / or assaying angiogenins in mammals, their homologs and their fragments.
• TAF Tumor angiogenesis factor
• Angiogenin isolated from human tumor cells comprises a single protein chain comprising 123 amino acids and the sequence of which is as follows:
• the C-terminal amino acid is proline; three disulfide bridges link cysteines 26-81, 39-92 and 57-107.
• human angiogenin is 35% homologous to that of human pancreatic ribonuclease, in particularly with regard to the amino acids essential for ribonucleolytic activity (cf. BIOCHEMISTRY, (1985), 24, 5494-5499. KURACHI et al.).
• the activity of human angiogenin is important, since 50 ng, or 3.5 picomoles, are capable of causing vascularization of the rabbit cornea and 35 fentomoles are capable of inducing vascularization of the chicken embryo.
• the present invention has therefore set itself the aim of providing a new protein having properties similar to those of human angiogenin, obtained by inexpensive means which are easy to implement, allowing high quantitative yields. It is also an object of the invention to provide a new process for obtaining said protein, which does not have the drawbacks of the processes of the prior art; in fact, this new process makes it possible to obtain large quantities of the protein in question at a very low cost, which, in the context of the industrial manufacture of pharmaceutical compositions containing this protein, has significant advantages. It is another object of the invention to provide pharmaceutical compositions containing said protein.
• the present invention relates to a protein, characterized in that it has a sequence which comprises 125 amino acids, and corresponds to formula I below: (I) Ala 1 -Gln-Asp-Asp-Tyr 5 -Arg -Tyr-Ile-His-Phe 10- Leu-Thr-Gln-His-Tyr 15 -Asp-Ala-Lys-Pro-Lys 20 - Gly-Arg-Asn-Asp-Glu 25 -Tyr-Cys-Phe-Asn -Met 30 - Met-Lys-Asn-Arg-Arg 35 -Leu-Thr-Arg-Pro-Cys 40
• the molecular mass was evaluated by comparing the speed of electrophoretic migration of said bovine protein with that of the following controls: myoglobin (MW: 17,200), myoglobin 1 + 2 (MW: 14,600), myoglobin A (MW: 8,240), myoglobin 2 (MW: 6,380), myoglobin 3 (MW: 2,560) (Pharmacia).
• said protein 17 KD, determined after total acid hydrolysis, the following amino acids are present in the following proportions: Phe: 6, Leu: 4, Ile: 9, Met: 2, Val: 4, Pro : 7, Ser s 6, Thr: 6, Ala: 4, Tyr: 6, His: 6, Glu (Gin): 10, Asp (Asn): 16, Lys: 9, Arg: 15, Gly: 9, Cys : 6.
• said protein is obtained by extraction of milk from mammals, in particular from cows or by cloning or by synthesis.
• the present invention also relates to peptides which constitute fragments of the 17KD protein according to the invention, it covers in particular:
• a peptide which has the following amino acid sequence: Phe-Asp-Glu-Ser-Phe 1 20 -Ile-Thr-Pro-Arg-His 1 25 and which corresponds to the C terminal fragment of the 17 KD protein
• Glu-Asn 110 -Gly-Leu-Pro-Val-His 115 -Phe which aligns with the sequence 108-115 of human angiogenin, in which sequence 7 of 8 residues are identical;
• peptide having the following amino acid sequence: Arg-Tyr-Ile-His-Phe 10 -Leu-Thr-Gln-His-Tyr 15 -Asp-Ala-Lys of which 11 of 13 residues align with the sequence 5-17 human angiogenin;
• peptide which has the following amino acid sequence: Leu 70 -Arg-Ile-Ser-Lys-Ser 75 -Glu-Phe-Gln of which 8 out of 10 residues align with the sequence 69-77 of angiogenin human.
• a peptide which has the following amino acid sequence: Arg 67 -Gly-Asp, said peptide being recognized by a receptor for endothelial cells.
• the present invention also relates to a process for obtaining said protein according to the invention, characterized in that said protein is extracted from mammalian milk, in particular from cow's milk.
• the extraction of said protein is carried out by cation exchange chromatography, followed by elution with an appropriate eluent.
• the eluent is an alkaline salt of a weak organic acid, in particular sodium acetate.
• the eluted fraction is subjected to a second cation exchange chromatography.
• the protein thus isolated is purified by chromatography on a gel-filtration column.
• the protein thus purified is obtained with a yield of the order of 0.5 mg / liter of milk.
• the milk of bovine origin is subjected to delipidation.
• defatting is carried out by centrifugation.
• the centrifugation is carried out at 4000 g for 30 min and at a temperature of 4oC.
• the present invention also relates to a therapeutic composition which comprises, as active compound, the 17 KD protein and / or fragments or homologues thereof, in particular for the treatment of disorders requiring inhibition or increased growth blood vessels.
• the therapeutic compositions in accordance with the invention can be used in all pathologies in which there is a problem of vascularization, and in particular wounds, bedsores, ulcers, grafts, circulatory insufficiencies. They can also be used in cosmetology (skin, scalp). They can also be used in the veterinary field, in particular in the diagnosis of mastitis and the selection of lactating cows.
• the present invention also relates to an immunological reagent for detecting or assaying mammalian angiogenins, characterized in that it is chosen from the group which comprises anti-protein 17 KD antibodies, anti-peptide antibodies, in particular antibodies against one of the peptides defined above, which antibodies being used alone or as a mixture.
• the present invention also relates to a method for detecting and assaying mammalian angiogenins in biological fluids, characterized in that an anti-angiogenin antibody, in particular an anti-protein antibody, is reacted under appropriate conditions 17 KD or an anti-peptide antibody, in accordance with the invention with a biological fluid supposed to contain said angiogenin, the reading of the reaction being carried out by an appropriate means, such as in particular RIA, ELISA, Immunofluorescence.
• the present invention further relates to a kit for detecting and / or assaying, in biological fluids, angiogenin in mammals and in particular human angiogenin, characterized in that it comprises:
• anti-angiogenin antibody in particular of anti-protein 17 KD antibody or of anti-peptide antibody, in particular of antibodies against one of the peptides defined above ;
• buffers optionally an appropriate quantity of buffers, diluents, reagents, necessary for the implementation of said detection and / or assay.
• the invention also comprises other provisions, which will emerge from the description which follows, which refers to an example of preparation of the protein according to the invention, as well as to a report of '' experiments carried out:
• the attached Figure 1 represents the elution diagram of the 17 KD protein from the Phenyl Superose column, with the time in minutes on the abscissa, and the optical densities on the ordinate. Peak 2 contains the pure 17 KD protein.
• the yield is 25 mg of protein, ie 0.5 mg / l of defatted milk.
• the protein 17 KD of bovine origin has an additional alanine in the N-terminal position.
• bovine RNAse 17 KD of bovine origin shares a similar homology with bovine RNAse (39% of the remains being identical), to that of human angiogenin for human RNAse (34% of the remains being identical).
• pancreatic ribonuclease A the three-dimensional structure of angiogenin human has been evaluated when the sites, on which the mutations have focused, are reported on this structure; in this case, it can be observed that, with the exception of the Arg / Ile mutation at position 43, all the substitutions which occur in the spatial structure of the protein, involved in its ribonucleolytic activity, are conservative substitutions.
• the fertilized eggs are placed in an incubator at 38oC in an atmosphere at 70% humidity.
• a window is opened in the shell and is closed by a gas permeable membrane.
• the protein 17 KD of bovine origin in accordance with the invention can be used in a pharmaceutically acceptable form for the treatment of disorders requiring inhibition or increased growth of blood vessels in humans.

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• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Engineering & Computer Science (AREA)
• Immunology (AREA)
• Molecular Biology (AREA)
• Organic Chemistry (AREA)
• Urology & Nephrology (AREA)
• Biochemistry (AREA)
• General Health & Medical Sciences (AREA)
• Hematology (AREA)
• Medicinal Chemistry (AREA)
• Biomedical Technology (AREA)
• Microbiology (AREA)
• Biophysics (AREA)
• Toxicology (AREA)
• Vascular Medicine (AREA)
• Gastroenterology & Hepatology (AREA)
• Biotechnology (AREA)
• Cell Biology (AREA)
• Genetics & Genomics (AREA)
• Zoology (AREA)
• Food Science & Technology (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Physics & Mathematics (AREA)
• Analytical Chemistry (AREA)
• General Physics & Mathematics (AREA)
• Pathology (AREA)
• Peptides Or Proteins (AREA)
• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

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• 1987
  • 1987-11-19 FR FR8715984A patent/FR2623509B1/fr not_active Expired - Lifetime
• 1988
  • 1988-11-18 EP EP88910059A patent/EP0342219A1/de not_active Withdrawn
  • 1988-11-18 US US07/392,977 patent/US5171845A/en not_active Expired - Fee Related
  • 1988-11-18 WO PCT/FR1988/000566 patent/WO1989004837A1/fr not_active Ceased
• 1989
  • 1989-04-26 AU AU33701/89A patent/AU619830B2/en not_active Ceased

Non-Patent Citations (1)

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