WO1996037607A2 - Acide nucleique pourvu du domaine polymorphe d'un gene de recepteur de bradykinine b¿2? - Google Patents

Acide nucleique pourvu du domaine polymorphe d'un gene de recepteur de bradykinine b¿2? Download PDF

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Publication number
WO1996037607A2
WO1996037607A2 PCT/DE1996/000891 DE9600891W WO9637607A2 WO 1996037607 A2 WO1996037607 A2 WO 1996037607A2 DE 9600891 W DE9600891 W DE 9600891W WO 9637607 A2 WO9637607 A2 WO 9637607A2
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WO
WIPO (PCT)
Prior art keywords
dna
bradykinin receptor
polymorphic
receptor gene
polymorphic region
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Application number
PCT/DE1996/000891
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German (de)
English (en)
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WO1996037607A3 (fr
Inventor
Andreas Braun
Stefan Kammerer
Original Assignee
Roscher, Adelbert
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Application filed by Roscher, Adelbert filed Critical Roscher, Adelbert
Publication of WO1996037607A2 publication Critical patent/WO1996037607A2/fr
Publication of WO1996037607A3 publication Critical patent/WO1996037607A3/fr

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    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to a nucleic acid comprising a polymorphic region of a B 2 bradykinin receptor gene, a method for producing such a nucleic acid and the use thereof.
  • Bradykinin is a vasoactive nonapeptide of the kinin family, which is released from kininogens by proteolytic activity of kallikrein. Bradykinin is involved in various physiological processes such as cardiovascular hemostasis, neurotransmission, cell proliferation or bronchopulmonary contraction.
  • bradykinin works via a cell surface receptor, which is called the B 2 bradykinin receptor. This receptor binds to G proteins and triggers, among other things, the activation of phospholipase C and / or phospholipase A.
  • Studies of the genomic structure of the human B 2 bradykinin receptor gene show that it comprises 3 exons.
  • bradykinin plays a role in various diseases and is present in different amounts. For example, the amount of bradykinin increases in heart disease and asthma and decreases in allergies.
  • the present invention is therefore based on the object of providing a means by which it can be determined whether the B 2 bradykinin receptor is present in different forms in the above diseases. According to the invention, this is achieved by the subject matter in the claims.
  • the present invention thus relates to a nucleic acid comprising a polymorphic region of a B 2 bradykinin receptor gene.
  • polymorphic region of a B 2 bradykinin receptor gene encompasses any region of a B 2 bradykinin receptor gene which can have a sequence polymorphism in individuals of a population, for example humans.
  • nucleic acid encompasses any type of nucleic acid, e.g. RNA and DNA, the latter being preferred.
  • RNA and DNA the latter being preferred.
  • the present invention will now be described by way of example for DNA.
  • the present invention is based on the applicant's knowledge that the B 2 - bradykinin receptor gene has polymorphic regions. This is evident from the applicant's recent work in which DNA from blood donors was examined for the B 2 bradykinin receptor gene. Frequently found polymorphic areas are shown in FIGS. 1-4.
  • Figure 1 describes a DNA comprising a polymorphic region of exon 1 of a B 2 bradykinin receptor gene.
  • the polymorphic region is located at nucleotide position 12 downstream of the transcription start site. It represents a tandem repeat polymorphism and is expressed, for example, in the alleles BE1-2G, BE1 -3G and BE1 -3T.
  • BE1 -2G there are two units of the repeat GGTGGGGAC, while BE1-3G has three of these units.
  • BE1-3T has three repeat units, two of which correspond to the above repeat and one has the sequence GGTGGTGAC.
  • Figure 2 (A) describes a DNA comprising a polymorphic region of exon 2 of a B 2 bradykinin receptor gene.
  • the polymorphic region is located at nucleotide position 181 of the cDNA. It represents a base polymorphism and is expressed, for example, in the alleles BE2-C and BE2-T.
  • BE2-C there is a C, which leads to the codon CGT (Arg), while BE2-T has a T, which leads to the codon TGT (Cys).
  • Figure 2 (B) shows the open reading frame (ORF) of the alleles BE2-C and BE2-T.
  • Figure 3 describes a DNA comprising a polymorphic region of exon 3 of a B 2 bradykinin receptor gene.
  • the polymorphic region is located in the 3'-untranslated region of exon 3. It represents a polymorphism of repeats with a consensus sequence, TGGA (A) GGGCTAGAACC, and is expressed, for example, in the alleles BE3-R48 and BE3-R35 off. There are 48 repeats in BE3-R48, while BE3-R35 has 35 repeats.
  • Figure 4 describes a DNA comprising a polymorphic region of the exon 1 promoter region.
  • the polymorphic region is located at nucleotide positions 675 and 1 1 53. It is a base polymorphism and is expressed e.g. in the BP-TC and BP-CT alleles.
  • BP-TC has a T (625) and a Cd 153
  • BP-CT has a C (675) and a T (1 1 53).
  • the DNAs of Figures 1-4 are preferred embodiments of the present invention.
  • the DNA of Fig. 3 was BE3-R35 / 1 or BE3-R48 / 1 and the DNA of Fig. 1 as BE1 -3T (Xba 7.0 / 2) at the DSM (German Collection of Microorganisms and Cell Cultures) filed under DSM 9927, DSM 9928 and DSM 9929 on April 20, 1995.
  • DSM German Collection of Microorganisms and Cell Cultures
  • a DNA according to the invention can be present in a vector or expression vector.
  • examples of such are known to the person skilled in the art.
  • these are e.g. pGEMEX, pUC derivatives, pGEX-2T and pET3b.
  • yeast e.g. to call pY100 and Ycpadl
  • animal cells e.g. pKCR, pEFBOS, cDM8 and pCEV4 must be specified.
  • suitable cells around one, in an expression vector to express the present DNA according to the invention.
  • suitable cells include the E. coli strains HB101, DH1, x1776, JM101, JM109 and BL21, the latter being preferred, the yeast strain Saccharomyces cerevisiae and the animal cells L, 3T3, FM3A, CHO, COS, Vero and HeLa .
  • DNA according to the invention has to be inserted into an expression vector. He is also aware that this DNA can be inserted in conjunction with a DNA coding for another protein or peptide, so that the DNA according to the invention can be expressed in the form of a fusion protein.
  • polymorphic regions can be detected in a B 2 bradykinin receptor gene. It is thus possible, for example, to determine whether the B 2 bradykinin receptor is present in different forms in various diseases, such as cardiac diseases, asthma and allergies.
  • the above areas can be demonstrated by conventional methods. Reference is made to the example below.
  • the polymorphic areas of Figures 1-3 are detected in B 2 -Bradykinin receptor genes from blood donors. A customary PCR method is carried out for this. The amplified DNA fragments are then compared with one another in the customary manner, for example by means of gel electrophoresis. The above polymorphic areas are detected at different frequencies.
  • B 2 bradykinin receptors can be found which have changed in their gene or protein structure. This can be done via a DNA according to the invention or a polypeptide according to the invention.
  • the receptors can then be tested for their activity in conventional methods. Such methods can also include the use of drugs such as angiotensin converting enzyme inhibitors, kinin receptor agonists or antagonists and bradykinin agonists or antagonists, whereby their effect on altered B 2 bradykinin receptors can be examined.
  • drugs such as angiotensin converting enzyme inhibitors, kinin receptor agonists or antagonists and bradykinin agonists or antagonists, whereby their effect on altered B 2 bradykinin receptors can be examined.
  • Antibodies to modified B 2 bradykinin receptors can also be produced. These antibodies are suitable for both diagnostic and therapeutic purposes. Polypeptides according to the invention can be used in a conventional manner for their production.
  • the invention is illustrated by the following example.
  • Genomic DNA was isolated from 179 randomly selected blood donors. This genomic DNA was used to detect polymorphic areas in the respective B 2 bradykinin receptor genes. For this purpose, a PCR method was carried out, the primer pairs chosen being those which enable the polymorphic regions indicated in FIGS. 1-3 to be amplified. The following primer pairs were used: for exon 1: BE 1 F (5 '-G CCCTTCAAAGATGAG CTG-3') and BE 1 R (5 '-
  • BE2F (5'-CCATTTCTCCTCCCTGCTCGAG-3') and BE2R (5'-
  • the PCR reaction had a total volume of 50 ⁇ ⁇ and contained 1 ⁇ g of genomic DNA, 50 ng of forward (F) and reverse (R) primer, 1, 25 U Taq polymerase, 200 ⁇ mol of each of the dNTPs and 1.5 mM MgCl 2 .
  • the cycling conditions were initially 5 minutes at 94 ° C, followed by 1 minute at 94 ° C, 1 minute at 53 ° C and 1 minute at 72 ° C for 40 cycles and an end extension time of 5 minutes at 72 ° C.
  • PCR products obtained were subjected to single-strand conformation polymorphism electrophoresis (SSCP electrophoresis).
  • 5 ⁇ ⁇ of the PCR mixture were diluted with 15 ⁇ ⁇ formamide and denatured at 95 ° C. for 5 minutes.
  • the SSCP gel was a 12% polyacrylamide gel buffered with 1 x TBE (85 mM Tris, 90 mM boric acid, 2 mM EDTA) and the electrophoresis was carried out in 1x TBE, at room temperature, with constant 120 volts for 24 hours.
  • the polymorphic region of FIG. 1 is detected in the form of the alleles 2G, 3G and 3T.
  • the PCR products (10 l) obtained were digested with 4 U Taql at 65 ° C. for 1 hour. This was followed by electrophoretic separation on a 2.5% MetaPhor TM agarose gel with 1 ⁇ TBE buffer containing 35 g / ml ethidium bromide at 100 V for 2 hours. The separation was made visible under UV light.
  • the polymorphic region of FIG. 2 is detected in the form of two alleles.
  • the one allele (C at nucleotide position 181) was cleaved twice by Taql, while the other allele (T at nucleotide position 181) is cleaved only once (cf. FIG. 6).
  • the PCR products obtained (10 ⁇ ⁇ ) were applied directly to a 1% agarose gel and electrophoretically with 1 ⁇ TBE buffer containing 35 ⁇ g / ml ethidium bromide at 100 V 2 Hours separated. The separation was made visible under UV light. 7 shows the separation.
  • the polymorphic region of FIG. 3 is detected in the form of the alleles R48, R35, RV1 and RV2.
  • the latter alleles have an intermediate number of repeats and are underrepresented.

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Biophysics (AREA)
  • Medicinal Chemistry (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Immunology (AREA)
  • Biochemistry (AREA)
  • Cell Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Toxicology (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Endocrinology (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne un acide nucléique pourvu d'un domaine polymorphe d'un gène de récepteur de bradykinine B2, le procédé de préparation d'un tel acide nucléique et son utilisation.
PCT/DE1996/000891 1995-05-23 1996-05-22 Acide nucleique pourvu du domaine polymorphe d'un gene de recepteur de bradykinine b¿2? WO1996037607A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE19518931.0 1995-05-23
DE1995118931 DE19518931C1 (de) 1995-05-23 1995-05-23 Nukleinsäure mit einem polymorphen Bereich eines B¶2¶-Bradykinin-Rezeptor-Gens

Publications (2)

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WO1996037607A2 true WO1996037607A2 (fr) 1996-11-28
WO1996037607A3 WO1996037607A3 (fr) 1997-01-16

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DE (1) DE19518931C1 (fr)
WO (1) WO1996037607A2 (fr)

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996003495A1 (fr) * 1994-07-27 1996-02-08 Merck & Co., Inc. Animaux transgeniques modifies par le recepteur b2 de la bradykinine

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996003495A1 (fr) * 1994-07-27 1996-02-08 Merck & Co., Inc. Animaux transgeniques modifies par le recepteur b2 de la bradykinine

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, Bd. 184, Nr. 1, 1992, ORLANDO, FL US, Seiten 260-268, XP002019028 HESS J.F., BORKOWSKI J.A., YOUNG G.S., STRADER C.D., RANSOM R.W.: "Cloning and pharmacological characterization of a human bradykinin (BK-2) receptor" *
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, Bd. 211, Nr. 1, 6.Juni 1995, ORLANDO, FL US, Seiten 226-233, XP002019026 KAMMERER S., BRAUN A., ARNOLD N., ROSCHER A.A.: "The human bradykinin B2 receptor gene: full length cDNA, genomic organization and identification of the regulatory region" *
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, Bd. 211, Nr. 1, 6.Juni 1995, ORLANDO, FL US, Seiten 234-240, XP002019027 BRAUN A., KAMMERER S., BOHME E., MULLER B., ROSCHER A.A.: "Identification of polymorphic sites of the human bradykinin B2 receptor gene" *
GENOMICS, Bd. 15, 1993, Seiten 435-438, XP000610048 POWELL S.J., SLYNN G., THOMAS C., HOPKINS B., BRIGGS I., GRAHAM A: "Human bradykinin B2 receptor: nucleotide sequence analysis and assignment to chromosome 14." *

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DE19518931C1 (de) 1996-09-19
WO1996037607A3 (fr) 1997-01-16

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