WO2000034472A1 - Proteine intervenant dans la spermatogenese - Google Patents

Proteine intervenant dans la spermatogenese Download PDF

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Publication number
WO2000034472A1
WO2000034472A1 PCT/DE1999/003972 DE9903972W WO0034472A1 WO 2000034472 A1 WO2000034472 A1 WO 2000034472A1 DE 9903972 W DE9903972 W DE 9903972W WO 0034472 A1 WO0034472 A1 WO 0034472A1
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WO
WIPO (PCT)
Prior art keywords
dna
protein
spermatogenesis
amino acid
acid sequence
Prior art date
Application number
PCT/DE1999/003972
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German (de)
English (en)
Inventor
Zdenek Sedlacek
Annemarie Poustka
Original Assignee
Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts filed Critical Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts
Priority to EP99965394A priority Critical patent/EP1137772A1/fr
Priority to AU20918/00A priority patent/AU2091800A/en
Publication of WO2000034472A1 publication Critical patent/WO2000034472A1/fr

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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/08—Drugs for genital or sexual disorders; Contraceptives for gonadal disorders or for enhancing fertility, e.g. inducers of ovulation or of spermatogenesis
    • 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 spermatogenesis protein, a DNA coding for such a protein and a method for producing such a protein.
  • the invention further relates to antibodies directed against the protein and to the use of the DNA and the protein for examining or influencing spermatogenesis.
  • Spermatogenesis is the formation of sperm in mammals or humans. This formation takes place in the testicles. During spermatogenesis, especially the pachytene stage of meiosis, the X and Y chromosomes attach to each other and form a so-called sex body. In this the X and Y chromosomes are inactive, i.e. they are not transcribed.
  • the present invention is therefore based on the object of providing a means by which spermatogenesis can be examined and, if necessary, ways can be shown by means of which it is possible to intervene in spermatogenesis.
  • the present invention is based on the applicant's knowledge that the X-chromosomally localized gene XAP-5 has a partner gene which is autosomally localized and is expressed in many tissues. For example, the expression can be found in the testes, where it is particularly strong in spermatogenesis, in particular in the stages of the primary and secondary spermatocytes and the round spermatids.
  • the partner gene is designated X5L and is located in the human genome on chromosome 6 in the 6pter region. The applicant has isolated and characterized X5L on the PAC clone LLNLp 704K12294Q13.
  • the DNA comprises a coding sequence and an intron and leads to an approximately 1.6 kb cDNA.
  • the applicant has also recognized that mutations in the X5L protein can impair spermatogenesis.
  • a spermatogenesis protein comprising the amino acid sequence of FIG. 1 or an amino acid sequence different therefrom by one or more amino acids, with a homology between the latter amino acid sequence and the amino acid sequence of FIG. 1 of at least 80%.
  • an amino acid sequence different from one or more amino acids encompasses any amino acid sequence coding for an X5 protein which has a homology of at least 80% to that of Fig. 1.
  • the amino acid sequence can differ from that of Fig. 1 by additions, deletions , Substitutions and / or inversions of individual amino acids, in particular the amino acid sequence can be that of FIG.
  • nucleic acid which codes for an X5L product.
  • the nucleic acid can be an RNA or a DNA, e.g. B. a cDNA, his.
  • a DNA is preferred which comprises the following:
  • a DNA different by one or more base pairs encompasses any DNA sequence coding for an X5L protein that hybridizes with the DNA of FIG. 1 and codes for an X5L protein whose amino acid sequence has a homology of at least 80% that of Fig. 1.
  • the DNA sequence can differ from the DNA of FIG. 1 by additions, deletions, substitutions and / or inversions of individual base pairs. In particular, the DNA sequence can be that of FIGS. 2-4.
  • hybridization indicates hybridization under normal conditions, in particular at 20 ° C. below the melting point of the DNA sequence.
  • the DNAs of FIGS. 1-4 were h-X5L-c, h-X5L-g, m-X5L-c and m-X5L-g at DSMZ (German Collection of Microorganisms and Cell Cultures) under DSM 12550, DSM 12553 , DSM 12552 and DSM 12551 on November 26, 1998.
  • DSMZ German Collection of Microorganisms and Cell Cultures
  • a DNA according to the invention can be present as such or in combination with any other DNA.
  • a DNA according to the invention coding for an X5L protein can be present in an expression vector.
  • examples of such are known to the person skilled in the art.
  • an expression vector for E. coli these are, for example, pGEMEX, pUC derivatives, pGEX-2T, pET3b and pQE-8.
  • yeast for example, pY100 and Ycpadl are to be mentioned
  • animal cells for example, p CR, pEFBOS, cDM8 and pCEV4 are to be mentioned.
  • the bacculovirus expression vector pAcSGHisNT-A is particularly suitable.
  • suitable cells in order to express the DNA according to the invention which is present in an expression vector.
  • suitable cells include the E. coli strains HB101, DH1, xl776, JM101, JM 109, BL21 and SG 13009, the yeast strain Saccharomyces cerevisiae and the animal cells L, NIH 3T3, FM3A, CHO, COS, Vero and HeLa and the insect cells sf9.
  • 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.
  • Another object of the present invention is an antibody directed against an above protein or fusion protein.
  • Such an antibody can be produced by conventional methods. It can be polyclonal or monoclonal. For its production, it is favorable to immunize animals, in particular rabbits or chickens for a polyclonal and mice for a monoclonal antibody, with an above (fusion) rotein or fragments thereof. Further "boosters" of the animals can be carried out with the same (fusion) protein or fragments thereof. The polyclonal antibody can then be obtained from the serum or egg yolk of the animals. For the monoclonal antibody, animal spleen cells are fused with myeloma cells.
  • Kit Such comprises one or more of the following components:
  • auxiliaries such as carriers, buffers, solvents, controls, etc.
  • the present invention makes it possible to study spermatogenesis.
  • An X5L protein can be detected with an antibody according to the invention. A relationship between an X5L protein and processes in spermatogenesis can be established. An X5L protein can also be used to detect an autoantibody directed against this protein. Both detections can be carried out by conventional methods, in particular a Western blot, an ELISA, immunoprecipitation or by immunofluorescence. Furthermore, the organization and expression of the gene coding for an X5L protein can be detected with a nucleic acid according to the invention, in particular a DNA and primers derived therefrom. This detection can be carried out in the usual way, in particular in a Southern blot, via in situ hybridization or by PCR.
  • the present invention is suitable for taking measures to inhibit or increase the activity of an X5L protein in people.
  • An X5L protein can be inhibited with an antibody according to the invention.
  • an X5L protein especially after coupling to a protein that is not considered foreign by the body, for example transferrin or BSA, the amount of an X5L protein can be increased in people become.
  • a nucleic acid according to the invention in particular a DNA, which is placed under the control of a constitutive or inducible promoter and after its expression to provide an X5L protein in the person or in certain tissues, eg testicles, this leads.
  • the present invention thus represents means for investigating spermatogenesis and intervening in a regulating manner.
  • the latter includes both their activation and their inhibition.
  • the present invention provides means for diagnosing and treating disorders of spermatogenesis.
  • FIG. 1 shows the DNA and amino acid sequences of a spermatogenesis protein (X5L protein) comprising 325 amino acids according to the invention.
  • Sequence is a human cDNA.
  • the DNA comes from the human genome.
  • the cDNA of Fig. 1 begins on
  • Base pair 739 There is an intron between base pairs 828 and 1129.
  • a polyadenylation site is located at base pair 2658.
  • 3 shows the DNA and amino acid sequences of a 334
  • X5L protein comprising amino acids.
  • the DNA sequence is a mouse cDNA.
  • the cDNA of Figure 3 begins at base pair 445 of Figure 4 (A). There is an intron between base pairs 492-1232 of Fig. 4 (A). Between Base pairs 1-1136 of FIG. 4 (B has an intron. A polyadenylation sequence is located on base pair 2306 of FIG. 4 (B).
  • FIG. 6 shows the detection of mRNA of an X5L protein in testes.
  • the presence of such mRNA is limited to tubules (Fig. 6 (A)).
  • mRNA of an X5L protein is present in the stages of the primary and secondary spermatocytes (stars) and round spermatids (RS). Mature sperm are marked with (MS) and sperm togoni with (arrowheads). Sertoli cells (arrows) and Leydig-
  • Cells (L) have no mRNA of an X5L protein.
  • RNA blots from human tissues such as pancreas, adrenal medulla, thyroid, adrenal cortex, testicles, thymus, small intestine, stomach, fetal brain, fetal lungs, fetal liver and fetal kidney obtained from Clontech become one
  • Hybridized A [ ⁇ 32 P] dCTP-labeled X5L protein-specific DNA which lies between base pairs 1073-1409 of the DNA of FIG. 1 is used as the hybridization sample. The hybridization takes place overnight with subsequent washing steps under normal conditions. As a control, the blots are also hybridized with a radioactive ⁇ -actin sample (cf. FIG. 5).
  • mRNA in an X5 L protein is expressed in a wide variety of tissues.
  • the size of the expanded mRNA is 1, 7 and. 4, 3 kb, was on different polyadenylation signals of the DNA of Fig. 1 is due.
  • the expression of mRNA of an X5L protein is shown to be strongest in testes.
  • RNA in situ hybridization to mouse testis tissue is carried out. For this, the method of Wilkinson, D.G. (1992), Oxford University Press, New York. "Antisense” or “sense” RNA samples are used which correspond to base pairs 5-1169 of the DNA from FIG. 1.
  • mRNA of an X5L protein in testicular tissue. It also shows that expression is limited to tubules. At the cellular level, expression of mRNA of an X5L protein is shown in the stages of the primary and secondary spermatocytes as well as the round spermatids. Spermatogonia, mature Sertoli cells and Leyding cells, on the other hand, show no expression of mRNA of an X5L protein.
  • Example 2 Production and purification of a spermatogenesis protein (X5L protein) according to the invention
  • the DNA of FIG. 1 is provided with BAMHI linkers, cleaved with BamHI and inserted into the expression vector pQE-8 (Qiagen) cleaved with BamHI.
  • the expression plasmid pQE-8 / X5L is obtained.
  • pQE-8 / X5L is used to transform E.coli SG 13009 (see Gottesman, S. et al., J. Bacteriol. 148, (1981), 265-273).
  • the bacteria are cultivated in an LB medium with 100 ⁇ g / ml ampicillin and 25 / g / ml kanamycin and induced for 4 h with 60 ⁇ M isopropyl- ⁇ -D-thiogalactopyranoside (IPTG).
  • Lysis of the bacteria is achieved by adding 6 M guanidine hydrochloride, followed by chromatography (Ni-NTA resin) in the presence of 8 M with the lysate Urea according to the manufacturer's (Qiagen) specifications of the chromatography material.
  • the bound fusion protein is eluted in a pH 3.5 buffer. After neutralization, the fusion protein is subjected to 18% SDS-polyacrylamide gel electrophoresis and stained with Coomassie blue (cf. Thomas, JO and Kornberg, RD, J. Mol. Biol. 149 (1975), 709-733).
  • Example 3 Production and detection of an antibody according to the invention
  • a fusion protein according to the invention from Example 2 is subjected to an 18% SDS-polyacrylamide gel electrophoresis. After staining the gel with 4 M sodium acetate, an approximately 37 kD band is cut out of the gel and incubated in phosphate-buffered saline. Gel pieces are sedimented before the protein concentration of the supernatant is determined by SDS-polyacrylamide gel electrophoresis, which is followed by a Coomassie blue staining. Animals are immunized with the gel-purified fusion protein as follows:
  • 35 ⁇ g of gel-purified fusion protein in 0.7 ml of PBS and 0.7 ml of complete or incomplete Freund's adjuvant are used per immunization.
  • a fusion protein according to the invention from Example 1 is subjected to SDS polyacrylate gel electrophoresis and transferred to a nitrocellulose filter (cf. Khyse-Andersen, J., J. Biochem. Biophys. Meth. 10, (1984), 203-209). Western blot analysis was performed as in Bock, C.-T. et al.
  • the nitrocellulose filter is incubated for one hour at 37 ° C. with a first antibody.
  • This antibody is rabbit serum (1: 10000 in PBS).
  • the nitrocellulose filter is incubated with a second antibody.
  • This antibody is an alkaline phosphatase-linked monoclonal goat anti-rabbit IgG antibody (Dianova) (1: 5000) in PBS.
  • Antibodies are extracted from egg yolk and tested in a Western blot. There are polyclonal antibodies according to the invention followed up.

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Reproductive Health (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Veterinary Medicine (AREA)
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  • Pregnancy & Childbirth (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
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Abstract

L'invention concerne une protéine intervenant dans la spermatogenèse, un ADN codant une telle protéine et un procédé de production d'une telle protéine. L'invention concerne en outre des anticorps dirigés contre cette protéine ainsi que l'utilisation de cet ADN et de cette protéine pour étudier la spermatogenèse ou influer sur cette dernière.
PCT/DE1999/003972 1998-12-10 1999-12-08 Proteine intervenant dans la spermatogenese WO2000034472A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
EP99965394A EP1137772A1 (fr) 1998-12-10 1999-12-08 Proteine intervenant dans la spermatogenese
AU20918/00A AU2091800A (en) 1998-12-10 1999-12-08 Spermatogenesis protein

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE19856882.7 1998-12-10
DE19856882A DE19856882C1 (de) 1998-12-10 1998-12-10 Spermatogenese-Protein

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WO2000034472A1 true WO2000034472A1 (fr) 2000-06-15

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EP (1) EP1137772A1 (fr)
AU (1) AU2091800A (fr)
DE (1) DE19856882C1 (fr)
WO (1) WO2000034472A1 (fr)

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CN108424909B (zh) * 2018-03-14 2021-11-30 江汉大学 一种纤毛组装调控基因及其编码蛋白、获取方法和应用

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997022625A1 (fr) * 1995-12-20 1997-06-26 Indiana University Foundation Compositions de ghrh-rp et procedes

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997022625A1 (fr) * 1995-12-20 1997-06-26 Indiana University Foundation Compositions de ghrh-rp et procedes

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
SEDLACK Z. ET AL.: "X5L gene; XAP-5-like protein.", EMBL DATABASE, ACCESSION NUMBER Y18503, 30 June 1999 (1999-06-30), XP002135681 *
TOYODA A ET AL.: "Human HXC-26 gene", EMBL DATABASE ; ACCESSION NUMBER D83389, 19 February 1997 (1997-02-19), XP002135680 *

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EP1137772A1 (fr) 2001-10-04
DE19856882C1 (de) 2000-05-04
AU2091800A (en) 2000-06-26

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