The receptor for follicle-stimulating hormone (fsh) human dna, vector, method of production, the pharmaceutical composition

 

(57) Abstract:

The invention relates to the field of molecular biology and genetic engineering and can be used in the pharmaceutical industry and in medical practice to reduce endogenous activity of follicle stimulating hormone (FSH). By screening cDNA libraries identified the complete nucleotide sequence encoding the FSH receptor of human rights. The inclusion of this sequence in the expression vector and transfection of the received vector host cells has allowed the isolation and purification of functionally active recombinant forms of the receptor. Along with full-length polypeptide FSH-receptor human recombinant DNA obtained shortened from the C-end of the fragments that retain the ability to bind FSH. The proposed farmcampsite to reduce endogenous FSH activity, comprising as active principle recombinant FSH receptor of human rights or its active fragment. 5 C. p. F.-ly, 8 ill., table 1.

The proposed application is a partial continuation application of U.S. reg. 07/670085 filed March 15, 1991, for which priority is claimed.

Prior techniques is the study of techniques of recombinant DNA.

Follicle-stimulating hormone (FSH) is a heterodimeric glycoprotein hormone produced by the pituitary gland and has a structure similar to the structure of the luteinizing hormone (LH), thyroid-stimulating hormone (TSH), both of which are secreted by the pituitary gland, and human chorionic gonadotropin (HCG), which is produced in the placenta. These hormones are relatively large (28-38 kDa) and consist of ecovalence related-subunit, which is the same for all of these hormones, and-subunits, which all hormones is different, and which are responsible for the binding specificity of the receptor.

It is known that cellular receptors for these hormones belong to the class of G protein-coupled membrane receptors, which when activated, is capable of stimulating increased activity of adenylylcyclase. This leads to increased levels of secondary mediator - adenosine 3', 5' - monophosphate (CAMR), which in turn increases the level of synthesis and secretion of steroids. Chart hygroscopic amino acid sequences of these receptors revealed three main areas: 1) hydrophilic aminobenzene region, called the amino-terminal in brannum domain; and 3) carboxy-terminal region containing potential sites of phosphorylation (serine, threonine and the tyrosine residues), and called carboxy-terminal intracellular or cytoplasmic domain. This family of receptors glycoprotein hormones differs from other G protein-coupled receptors (such as2- adrenergichesky, adoptedby receptor and the receptor substance To a) the large size of their hydrophilic amino-terminal domains, which are involved in the binding of hormones.

Receptor FC expressed in the Sertoli cells of the testis and granular cells of the ovary. Although the need for mainly pure receptor FS person quite obvious; however, treatment of natural drugs is practically disadvantageous procedure, and moreover, it is unlikely that such a treatment will be sufficient to determine the amino acid sequence. Recently, one group of researchers was cloned cDNA encoding the receptor of rat FSH; the deduced amino acid sequence of this receptor, which was expressed in mammalian cells (Sprengel, Mol. Endocrunol. 4: 525, 1990). Another group of researchers tried to clone FS is eaten. FSH (Parmentier, Science 246:1620, 1389).

Brief description of the invention

The invention relates to a mostly pure receptor of human FSH, or its fragment, or mutant, able to bind FSH, and also cDNA that encodes a specified receptor, fragment, or mutant; the expression vectors containing the indicated DNA, to cells, transfected with the indicated expression vectors; and to methods for producing a specified receptor, fragment or mutant by culturing these transfected cells. The invention also relates to pharmaceutical compositions containing the specified receptor, fragment, or mutant, and to methods of treatment using such compositions in order to reduce the bioactivity of endogenous FSH. In the proposed application discloses improved analysis of people. FSH using receptor, fragment or mutant of the invention.

Brief description of drawings

In Fig. 1 presents a map of cDNA clones for receptor FS person. The figure shows a partial restriction map, which was constructed by combining data on DNA sequences obtained from portions of each of the five clones. On the map marked uvelicheniya below the restriction map. Designations and approximate sizes of the clones (in KB) are indicated to the left and to the right of each solid lines, respectively. Dashed arrows above the restriction map shows the approximate location of the amino-terminal intracellular domain, a transmembrane domain, and a carboxy-terminal intracellular domain of the encoded protein. Position insertions (probably intron or portion of the intron) in the clone 5-10 shows frame.

In Fig. 2A and 2B shows the strategy used to construct cDNA "full" FSH-receptor person for expression of the encoded protein in cell lines mammal.

In Fig. 3 is a diagram of plasmid expression vector used to produce cell lines Cho-DICK, stably transfected people FC receptor.

In Fig.4 is a diagram of plasmid expression vector used to produce cell lines Y1, stably transfected people FC receptor.

In Fig. 5 illustrates the levels of intracellular CAMR measured in Cho-DUKX cell line GHOH EP 40-13, after treatment with different doses of either FSH (circles) or LH (triangles). Each point represents redrawn progesterone, measured in culture medium Y1 cell line Y1HFSHR4-38, after treatment with different doses FS. Vertical lines indicated by the standard deviation (cf. square Osh.) when measuring progesterone for each dose FS.

Detailed description of the invention

Considered in the description of the receptor people. FS, and his FS-binding fragments or mutants are polypeptides having the ability to recognize and selectively contact person FSH, as well as to produce slight immune response in humans. Thus, the invention relates to a receptor people. FS having the amino acid sequence depicted in SEQ ID 2 and FSH-binding fragments or mutants that retain a high degree of homology (at least 95% identity) with the specified amino acid sequence, or at least its extracellular part. Scope of the invention includes such fragments people. FC-receptor, which remain after the division of the cytoplasmic and/or transmembrane domain of the full polypeptide. Particularly preferred fragment of the invention is aminobenzene extracellular portion containing approximately amino acids, which extend over the entire width of the transmembrane regions (shown in SEQ ID 2, in the section, "distinctive features") are extracellular, and they can be custom made (by means of respective spacer elements molecules) with a fragment containing amino Kontsevoy extracellular region, to improve binding. This polypeptide can be glycosylase, as in the natural receptor, or it may be partially or fully deglycosylated.

In addition, it is assumed that the above-described amino-terminal extracellular region, only part of it can be effectively used for PS binding, since the probability that for this purpose do not require the full extracellular domain, is very high. Thus, the fragment, which is shorter than 349 amino acids of the full extracellular domain, can be easily produced and analyzed for effective binding to FSH. Because the area FS-binding extracellular domain entirely preserved, the full length of the fragment is not a critical value. For this reason, you can expect to either end of the extracellular domain, or a FSH-binding fragment can also be added amino acids, not Britanie refers to the fragment people. FC-receptor, which comprises, basically, a part of the extracellular domain, and which largely retains the same ability to FS-binding, and that the full extracellular domain.

Scope of the invention also includes a mutant form of the above receptor and its fragments. Such mutants can be obtained by using conservative substitutions in 1-10 amino acid residues; and the localization and the nature of these substitutions is chosen so that they do not adversely affect FC-binding properties of modified receptor or its fragment.

Mostly pure people FC-receptor, or a fragment, or mutant obtained by selection and klonirovania DNA encoding the indicated receptor, fragment, or mutant, from cDNA or genomic library with the following legirovaniem this DNA into a vector, transferowania host cells with this vector, culturing the transfected host cell under conditions conducive to expression of the receptor, fragment or mutant, and the selection of the receptor, fragment or mutant of culture.

DNA used to obtain expression vectors may be genome DNA or cDNA that encodes the ed is ture, enhancers, etc., This DNA can be easily modified by substitution, deletion or insertion of nucleotides (for example, by site-specific mutagenesis) so that these mutations did not adversely affect the biological activity or the ability of FSH-binding of the expressed protein. For example, conservative substitutions (mutations) that change in 1-10 amino acids, can be implemented without adverse effects on the complete structure and activity of the expressed protein (mutein). In addition, some parts of the DNA, for example parts that encode cytoplasmic and/or transmembrane domains, can be deleterows so that expressively only one protein fragment, such as soluble extracellular domain. Receptor people. FSH or FSH-binding fragment or mutant can also be expressed in the form of a hybrid protein. One such hybrid proteins can contain a polypeptide at the carboxy-end, which has properties that facilitate protein purification or immobilization of purified protein on a solid substrate for use in the analysis of FSH or cleaning procedures FSH. Another such hybrid protein mo is="ptx2">

Receptor people. FSH produced in accordance with the invention is mainly clean, which means that it mainly contains no undesirable biological inclusions, usually associated with FC-receptor extracted from natural sources such as bacteria, viruses, and other proteins. The specified receptor may be included in a pharmaceutical composition by mixing with a suitable pharmaceutical acceptable carriers well known in the art.

Generally speaking, the pharmaceutical compositions can be formulated for oral, parenteral (subcutaneous, intramuscular and intravenous), vaginal, rectal, buccal (e.g., sublingual), transdermal or intranasal administration. Compositions for parenteral administration are generally made in the form of a liquid solution, dispersion or emulsion, preferably in the form of isotonic; for vaginal or rectal administration is in the form of creams or suppositories, for oral or buccal, in the form of tablets or capsules; and for intranasal administration is in the form of a powder, drops for transmission in the nose, or aerosols. This can be used medicinal it to be introduced into the polymer matrix, liposomes and microspheres in order to regulate the delivery of drugs to specific tissues or organs.

These compositions can be entered as discrete dosage forms, which can be obtained in accordance with conventional pharmaceutical techniques. Compositions for parenteral administration may contain as excipients sterile water or saline; alkalophile, such as propylene glycol; polyalkylene glycols such as polyethylene glycol; oils of vegetable origin, hydrogenated naphthalenes, etc., Compositions for vaginal or rectal administration, such as suppositories may contain as excipients, for example, polyalkylene glycols, vaseline, cocoa butter, etc., Compositions for insertion through the nose, made in the form of powders, can as fillers contain lactose or dextran; or they can be made in the form of aqueous or oil solutions and introduced through the nose in the form of drops or spray dosing solution. For TRANS-bugelnogo the introduction of standard solvents are sugar, calcium stearate, starch, swelling in cold water, etc. To the solution or powder composition can be Gouverneto-active substances (surfactants) are such salts, which retain the ability to increased absorption of peptide and surfactant properties, and do not have undesirable effects on the composition, and do not have other undesirable properties.

The dose of the active ingredients, as well as the method and mode of administration depend on the specific data of the patient's condition and the desired therapeutic effect, and can be determined by the treating physician.

Pharmaceutical compositions containing the receptor people. FSH or FSH-binding fragments or mutants, can be administered to the patient in doses that are therapeutically effective for binding to endogenous pirkwieser in the body of the patient follicle-stimulating hormone (FSH), in order to regulate an acceptable level of bioactive FSH. For example, the pharmaceutical compositions of the invention can be effectively used to reduce endogenous FSH bioactivity. For example, women may be assigned to treatment with use of the above compositions in order to prevent the growth and maturation of follicles, and thus prevent pregnancy. Men such treatment may be prescribed to prevent spermatogenesis. Especially preferred farmacevticheskogo FSH, containing the amino-terminal extracellular domain or a substantial part, and with mostly the same capacity for FSH-binding, and that the complete receptor.

Mostly pure receptor people. FSH can also be successfully used in the standard analysis for FSH, which, for example, described Reichert in Endocrinology 94: 483, 1974. Substitution in pure receptor of the invention, or fragment, capable of FSH-binding, will significantly improve conduct these analyses. A fragment containing the amino-terminal extracellular domain or FSH-binding portion and/or the hybrid can also be affine associated with the column for the allocation of FSH from liquids, extracts, etc.

The specified receptor may be inserted into a stable cell line, preferably a line of eukaryotic cells, and most preferably a line of mammalian cells, which are capable of producing detectable biological response upon stimulation of the receptor. Measurement of cellular response in the presence of FSH in the analysis (e.g., serum, plasma, culture medium, tissue homogenates) will serve as an indication of biological activity, and thus significant value of diagnostics order to assess substances, that can interact with FSH receptor, or the tested peptides or small proteins for their ability to bind to the FSH-receptor in srinilaya system with high productivity. An example of such highly productive srinilaya system is a system in which the binding of the ligand with recombinant FSH receptor contributes to the production or block the formation of the evaluated product, such as CAMR or progesterone. In addition, this system can be enhanced by appropriate linking easily detectable marker, for example bioluminescense agent (e.g., gene luciferase), with signal-transducers site of the receptor (for example, using a item CAMR response).

Mostly pure receptor people. FSH or SDF-binding fragments or mutants can also be used in x-ray crystallographic analysis to develop molecular models. Such models can be used to determine the tertiary structure of the hormone-binding domain of people. FC-receptor. Thus obtained information could make a significant contribution to the study of the structure of important areas, providing interaction between FSH and ehooy activity.

Recombinant technique used for producing proteins and DNA of the invention includes methods of identifying relevant mutations, vectors, host cells, culturing conditions, etc. is well known in the art and are adequately described, for example, in U.S. patent 4761371 and WO 88/09819, the disclosure of which is introduced in the proposed description by reference. Experimental scheme, bacterial and bacteriophobia culture media and chemical solutions used in the examples below (if it is not specifically mentioned), described in detail in Sambrook "Molecular Cloning: A Laboratory Manual". 2nd ed., Cold Spring Harbor Laboratory Press, 1989.

Example 1. Isolation and characterization of cDNA clones receptor people. FSH

Screening of the library

cDNA clone of the rat receptor FSH, similar to that described Sprengel in Japan. Endocrinol". 4: 525, 1990, received from Dr.William Moyle University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School. This cDNA clone was insertional in late expressing vector R SVI SV40 (Pharmacia LKB, product number: 27-4509-01) and the resulting vector was designated p SVLFSUR. the 2.1 - KB fragment of DNA containing the region corresponding to the region that encodes a full-receptor of rat FSH, cut out from the plasmid using site b - a fragment of the receptor of rat FSH was purified using electroelution with gel. This purified DNA fragment was used as a probe for screening the library to identify cDNA clones receptor people. FSH.

cDNA library lambda gtII constructed from RNA extracted from the testicles of a man, was obtained from Clontech, Palo AltO, CA (cat.the number HL 101b), and to use amplified. 20 aliquot of amplified libraries, which corresponds to approximately 7,5104plaque-forming units (PFU) were adsorbed, about 0.5 ml suspension for cultivation of strain YI088 E. Li. This suspension was obtained by culturing overnight culture YI088 at 37oIn NZY or L-medium, supplemented with 0.2% maltose and 10 mm gSO4and deposition of cells with their subsequent resuspending in 10 mm MgSO40, N600=0,5. Then for each suspension of phage/cell) was added to about 6.5 ml of molten top NZY - agarose (0.7 percent) at a temperature of 48oC, and the resulting mixture was poured into one of 20 cups (150 mm) with NZY - agar, preheated to 42oC. the Total number of phage, passaged for primary screening was approximately 1,5106. After 4-hour incubation at 42oC follow atnah nitrocellulose filters (Millipore) in accordance with the procedures described by Benton and Davies (Science 196: 180, 1977). Using the procedure of priming using the oligonucleotide described by Feinberg and Vogelstein (Anal. Biochem. 137: 266, 1983), slice - matrix DNA receptor of rat FSH, described in the previous paragraph, generated 32P - labeled sample with a specific activity 1-2109them. in minutes /micrograms (mcg). Prehybridization phage layers on nitrocellulose filters was performed in a buffer containing 50% formamide, 5SS [ISSC=0.15 M sodium chloride, of 0.015 M sodium citrate], 20 mm phosphate-sodium chloride buffer (pH 7,2), 10 denhardt's reagent (beagent denhardt's = 1% Ficoll, 1% polyvinylpyrrolidon, and 1% bovine serum albumin), and 100 μg/ml tRNA at 37oWith about 6 hours. Hybridization of the filters was carried out in the same buffer, except that the sample DNA rat FSH receptor was added at a concentration of about 3106them/min per ml of buffer. After hybridization at 37oC for 16-24 hours, the excess sample was washed with filter in 2SS, 0,1% - ordinator at room temperature for 30 minutes and then 0.2 security standards 0,1% - ordinator at 37oC for 60 minutes. Then the filters were exposed to x-ray plexi (R Kodak) overnight at -70oC. 6 duplicate positives identifitsirovali removed from 150 mm - cups in those places, where there was a positive clones. The phage was suirable by immersing these layers in SM. Suspended phage then seeded onto 150 mm plates with NZY - agar, as described above for the primary screening. Cups containing about 500 PFU/ Cup, was selected for secondary screening. The procedures used to obtain filters and hybridization for the secondary screening were similar to the procedures described above for the primary screening.

After secondary screening 5 alleged people. FSH-receptor-positive clones were identified and allocated in the form of purified bacteriophage clones gtII. These clones were identified: 1-5, 5-10, 11-11, 13-9 and 15-6.

Determination of DNA sequences alleged cDNA clones receptor people. FSH

Phage DNA was obtained from each isolate cDNA gtII using the method of lysis in the cups described by Sambrook in Molecular Cloning: A Laboratory Manual, "2nd ed. , Cold Spring Harbor Laboratory Press, 1989, page 2.118. Phage DNA was digested restricteduse the endonuclease EcoRI, and then akcionirovanie by size in agarose gel for purification of insertion of the cDNA fragments. Purified cDNA insert was subcloned into the RI website U18 in order to facilitate subsequent manipulation by cloning and m method of small-scale alkaline lysis (Sambrook, ibid, see above, pp. 1,25), and then purified by passing through an Elutip-d column (Schleicher, & Schuell Keene, NH) in accordance with the instructions of the manufacturers. After this half of the plasmid DNA obtained from each drug was denaturiruet 0.2 N. NaOH, 0.2 mm EDTA) in a volume of 20 μl at room temperature for 10 minutes. Denatured plasmid DNA was neutralized and precipitated with ethanol by the addition of 7.5 μl of 7.5 M ammonium acetate and 110 μl of 100% ethanol, after which the mixture was cooled in liquid nitrogen. The DNA precipitate was besieged for 10 minutes on microcentrifuge. The DNA precipitate was washed in 70% ethanol and dried off. The sequencing reaction was performed using DNA-T7 polymerase(United States Biochemical) according to the instruction by the manufacturer. Preliminary sequencing reaction was performed using forward and reverse Sequeira primers (Pharmacia L) for polylinker region U18. The data obtained in the preliminary sequencing, was used to construct people. FSH - receptor-specific Sequeira primers. These primers or synthesized on a DNA synthesizer model 391 plied Biosystem, or ordered in National Biosciences Ihc, Hamel, MN. Some data on DNA sequences were polulitrovye using forward and reverse primers.

Preliminary sequencing data showed that none of the five cDNA isolates does not represent a region encoding a full person FC - receptor, however, the combined data sequencing in five clones can be used to output the full protein sequence. Schematic diagram of the relative locations of each of the five clones as compared to the full cDNA sequence that encodes people FSH-receptor presented (see below) in Fig.1. cDNA sequence full of people FC - receptor, obtained by combining the data sequence carried out in accordance with the computer program of the Assembly of fragments (Genetic Computer Group (GCG), depicted in SEQ ID 1, and the deduced amino acid sequence depicted in SEQ ID 2. Analysis of DNA sequences people. FC - receptor allowed us to identify long open-reading frames of 2085 nucleotides, which encodes a protein in the 695 amino acids. Receptor human FSH by 3 amino acids longer than the receptor of rat FSH. Full % similarity between the DNA of rat and human FSH and protein sequences, which were determined using the program GGG stfit, was 87% and 90%, respectively. On the estimation of the 87% similarity with the corresponding region of the rat FSH. The seven transmembrane domains of these two species, which are connected bridge connection of three extracellular and three intracellular loops, have the identity of 95%, and the carboxy-terminal intracellular region have only 81% identity. Partial amino acid sequence published Parmentier, Science 246:1620-1622, 1989, corresponds to amino acids 399-525 in SEQ ID 2.

Clone 5-10 was a mutant and had the insertion of 0.25 KB after nucleotide T at position 446 in SEQ ID 1. DNA sequence insertions has no similarities with any part of a DNA sequence Krasnogo FSH or human FSH receptor, and not similar to any known sequence in the gene Bank or databases of DNA sequences in EMBL. The corresponding region in the clone 11-11 does not contain the specified insertion. cDNA clones of the LH receptor isolated from a cDNA library thyroid gland of man, contain a similar mutation (Frazier, Mol.Endocrinol. 4:1264-1276, 1990). These mutations, obviously, occur as a result of incomplete and/or aberrant splicing RNA molecules. This explanation is confirmed by the presence of 3' - spliceimage consensus (G'G) in the 3' region of the connection insertions 5-10.

Plasmids U18 containing cDNA inserts: 10-11 (boccia type culture (ATS, Rockville, MD), 1 Marr 1991, and have the following rooms admission: ATSC, ATSC and ATSC respectively. The specified Deposit was made in accordance with all requirements of the Budapest agreement.

Example 2. Construction of vectors for expression of the full receptor people. FSH in mammalian cells

The strategy of constructing DNA encoding the receptor people. FSH, for expression in mammalian cells is shown in Fig.2A and 2B. In Fig. 2A, NSiI-AMN - a fragment of the 5'-691 base pairs (p. O.), containing the start of the ATS, was isolated from FS HR II-II and was subcloned into the U18, digested > PST and VMN. The resulting plasmid, FSHR II-IInb, linearizable with SphI. The end was a small mistake by processing the fragment maple DNA polymerase I (New England Biolobs, Beverly, MA). After ligating hol - linkers (New England Biolabs, Beverly, MA) with blunt ends of the plasmid, and the mixture was digested hoI and BamHI and 5'-fragment of the receptor people. FSH, approximately 700 p. O., was purified. A fragment from the middle region cDNA people FSH receptor was isolated from FSHR II-II by digestion with BamHI and Shl and were gel-purification of the fragment of approx. in 734 p. O. 3' - fragment people. FSH-receptor containing the stop codon TAA, was isolated from pHFSHR15-6 by first digesting the plasmid which can be the digested SphI and XhoI, and 3' - SphI-XhoI - fragment of approximately 690 p. O.

In Fig. 2B, 5'-700 p. O. - hoI - AMN - and middle 734. p. O. BamHI-SphI fragments of cDNA people FSH receptor was subcloned into pUCI8 - XhoI (manufactured by transformation Smal site polylinker pUC18 in XhI website using XhoI-linkers), digested hoI and ShI. The obtained plasmid (pHFSHR 1.4 XS) was digested hoI and SphI. Digested DNA was fractionally by size using electrophoresis in 5-6% polyacrylamide gel, and the fragment (about 1400 p. O.), containing 5' - and middle region cDNA people FC-receptor, cut out and purified by electroelution. 3-SphI-XhoI fragment containing the stop codon TAA, was subcloned into the pUC18-XhoI, SphI digested and XhoI. The obtained plasmid was digested SphI and EcoRI. Digested DNA was fractionally 5-6% polyacrylamide gel, and the piece about 700 p. O. cut out and purified by electroelution. Then carried out the Assembly region that encodes a full person FSH receptor, combining the 5'-1400 p. O. - XhoI-SphI fragment from the 3'-700 p. O.-SphI-EcoRI fragment when legirovanii with UC18-hoI, digested hoI and cRI. The correctness of the construction indicated FSHRX, was checked by digestion restricteduse endonucleases and DNA sequencing. Fully constructed the spine and 12 p. O. the 3'non-coding sequences in addition to the region that encodes a full person FSH receptor, carved from pHFSHRX by hoI-digestion. Digested DNA was fractionally by size by electrophoresis in a 0.7% agarose gel. the 2.1 KB fragment was cut out from the gel, purified by electroelution in Little lueTankTM(ISCO) using procedures recommended by the manufacturer, and inserted into XhoI sites of plasmid cloning vectors for expression in the cell of a mammal. Plasmids containing the insert in the proper orientation for transcription of mRNA people FSH-receptor, was selected by restriction analysis. Before introduction into mammalian cells plasmid DNA expression vector was purified either by using two successive treatments of high-speed centrifugation in density gradient of cesium chloride, or by using plasmid set, maxi kit (Qiagen, Chatsworth, CA) according to the manufacturer's instructions.

For the expression of people MGF - receptor may be used any suitable expressing vector mammalian cells. The plasmid vector has the following prerequisites: eukaryotic promoter such as the promoter of the mouse metallothionein I (MMT-I), a promotion is To people. FSH-receptor; a marker gene such as a gene of resistance to neomycin (neo) gene dihydrofolate (DFR), or the gene encoding multidrug resistance (DR), for selection of transfected cells, polyadenylation signal (poly A), such as an early site of the SV40 polyadenylation to the 3'-processing of the RNA transcript of the receptor; and bacterial replication scope and resistance gene to an antibiotic, such as ori pBP322 and the gene for resistance to ampicillin; the presence of these components is necessary for growth and propagation of the plasmid vector in an appropriate strain of E. coli. For some cell lines may be necessary introduction of Nitron in the region, which is transcribed into mRNA, encoding people. FSH receptor. This intron is preferably placed between the promoter and the cDNA insert people. FSH-receptor, which is consistent with its localization in the 5'-netransliruemoi region of the RNA transcript people. FSH receptor. For these purposes may be used any suitable intron. In these experiments, as the intron was used XbaI- > PST - part (2 kV) of the intron in the gene-subunit of human glycoprotein (Fiddes, J. Mol. Appl. Genet. 1: 13, 1931). In the expression vectors that part of the intron was inserted magogo splanirovano fragment, but donor splanirovano fragment was supplied synthetic oligonucleotide.

Chart expressing vector people. FSH-receptor used for transfection Cho cells, namely DHFSHRX (people FSH receptor in LH3AXSV2DHFRh IVS ), shown in Fig.3, a chart expressing vector people. FSH-receptor used for stable transfection Y1-cells, namely pNHFSHRX (people FSH receptor in CL3SV2 NEOh IVS), shown in Fig. 4. Sequence MMT-1-gene (nucleotides 4542-7514 in Fig.3, and nucleotides 5192-8164 in Fig.4), including the promoter region, similar to the sequences shown "upstream" from hoI website design CLH3X (Reddy, DNA, 6: 461, 1987) Fragment of intron And gene-subunit people glycoprotein, described in the previous paragraph, is located between nucleotides 7514 and 9514 in Fig. 3 and between nucleotides 8164-10164 in Fig. 4. "Downstream" from h01-site, MMT-1-introns and sequences, polyadenylation signal was removed and replaced early site SV40 polyadenylation (nucleotides 11614-11857 in Fig.3 and nucleotides 12264-12508 in Fig. 4) ML region (Lusky, Nature 293:79, 1981) contains bacterial site of initiation of replication and the gene for resistance to ampicillin from RVN 322 (nucleotides 1925-4542 in Fig.3, and nucleotides 2575-nego SV40 promoter, small T-intron, and sequences early region polyadenylation corresponds vuII-BamHI-fragment in SV2DHFR (Subramani, Mol Cell Biol., 1:854, 1981). PvuII site was turned into a BamHI site before the introduction of this fragment in the plasmid expression vector. Transcription neo gene (nucleotides 1-2575 in Fig.4) was synthesized by replacing the DHFR cDNA (nucleotides 342-1077 in Fig.3) HindIII-SmaI fragment (1385 p. O.), derived from pSV2-neo (Southern, J. NoI, Appl. Genet, 1:327, 1982). SmaI site was turned into AMN website before the introduction of this fragment in expressing plasmid vector.

For transfection of the most stable cell lines mammals can be used plasmid pNhFSHRX, which was deposited in ATSC 19 November 1991, the number of admission ADS. This deposition was carried out in accordance with all requirements of the Budapest Treaty. Plasmid pDhFSHRX is most suitable for transfection of cell lines, which lack activity dihydrofolate (DHFR).

The technique of cloning and constructing DNA can be used by any expert in order to modify expressing design DNA people. FSH receptor, so that was coded fragments FSH binding. These modified is used for transfection of mammalian cells in order to obtain lines, secreting soluble fragments of FSH-binding people. MGF-receptor, such as the amino-terminal extracellular cement or FSH-binding fragment.

Example 3. Obtaining cell lines mammal, which stably Express a functional including recombinant FSH receptor or FSH-binding fragment or mutant

Suitable cell lines mammal for expression of recombinant people. FSH receptor and its derivatives are the cells of the Chinese hamster ovary (Cho), mouse adenocarcinoma Y1, rat pituitary GH3, breast carcinoma human MSG, and cells primary human kidney 293. This example describes the use of cells Y1 and SNO.

Cells Y1 represent the count of clonogenic strain steroidsbritish cells derived from tumors of mouse cortical substance of adrenal glands (Yasumura, Cancer Res. 26:529-536, 1966). For this experiment, these cells were obtained from cell Bank of ATSS ADS CCL 79), and then they were cultivated in the medium HamF10, supplemented with 15% horse serum (HS), 2,5 FBS, and 1% L-glutamine (growth medium for Y1).

Cho-DUKX cells represent a count of clonogenic mutant cells Chinese hamster ovary in which OTS is supporting environment - alpha (MEM - ), supplemented with 10% FS and 1% L-glutamine (growth medium for Cho.

The cultivation of all cells was performed at 37oWith 5% CO2in wet conditions.

Transfection using calcium phosphate

Scheme transfection used in this experiment was a modification of a published method (Craham, Virology, 52:456, 1973). Approximately 24 hours before transfection cells were seeded at a Cup with a diameter of 100 mm at a density 7105cells per Cup (SNO-DU) or 1106cells per Cup (Y1). For transfection to 0.5 ml transfection buffer was added to 10 μg of DNA plasmid vector (DHFSHRX) for cells Cho-DUKX and pNHFSHR for cells Y1), Transfection buffer was prepared by mixing 4 grams (g) NCl; 0,185 g KCl; 0.05 g of Na2HPO4; 0.5 g of dextrose, and 2.5 g of HEPES in serialno distilled water and brought volume up to 500 ml, and the pH to 7.5. To the mixture transfection buffer and DNA was added 31 μl of 2 M Cal2. The resulting solution was mixed using a vibration mixer, and for 45 minutes at room temperature to precipitate the DNA. After removal of culture medium precipitated DNA was applied on top of the cells. After keeping at room temperature for 20 minutes was added is within 3.5 minutes in 3-4 ml transfection buffer, containing 15% glycerol. After that, the cells were twice washed in phosphate-buffer solution, and then added 10 ml of medium growth. Approximately 48 hours after transfection cells SNO-DU has subculturally when the index dilution 1:10, and then added to the selection medium. After transfection of Y1 cells were cultured for 72 hours, and then subculturally when the index dilution 1:5 in the selective medium. Selective medium for the cells Y1 contained growing environment for cells Y1 with 80 µg/ml S. MTX-resistant colonies of cells SNO-DU and G418-resistant colonies of cells Y1, formed after about 2-3 weeks, collected trimmed and cultivated until then, until the number of cells is not sufficient for cryopreservation and evaluation on the reactivity of the hormone.

Illustration of a bio-responsiveness people recombinant FSH receptor in stably transfected cells SNO

To determine whether cDNA - design people FC-receptor to produce biologically functional protein in stably transfected cells SNO, colony MTX - resistant cells SNO treated people recombinant FSH, and measured the levels of intracellular charm. For analysis at the level of tamr cells were subcloned into the 12-lo cultivation of each well was washed with 1.5 ml of warm growth medium. Then each washed well was added 300 μl passionately growth medium containing 0.1 mm 3-isobutyl-1-methylxanthines (Sigma). After a 15-minute cultivation to the medium in each well was added people recombinant FSH so that the final concentration was approximately 335 ng (2413 mIU) per ml After 30 minutes of cultivation analysis was completed by the fact that cells were subjected to four cycles of rapid freeze - thawing. 50 μl - aliquot of each cell lysate was injected in 1.5 ml tube for protein determination. For each remaining sample of lysate was added cold ethanol (300 μl), and then the sample was transferred into a separate 1.5 ml tubes. All samples of the lysates were centrifuged for 15 minutes at 13,000 g to remove cellular debris. Determination of the full content of soluble protein was carried out using the test kit proteins obtained from SIV-Rad (cat 500-0002) in accordance with the instructions of the manufacturers. Treated with ethanol samples of the lysates were liofilizovane in the aliquot 5-100 ál and resuspendable in 100 μl of buffer for analysis obtained in the form of a test kit CAMR Dupont/NEN (Medical Product, cat. N-033). The content of CIDA in samples resuspending lysates was determined according the samples were not diluted. In most cell lines Cho, transfected people FSH receptor, higher levels of intracellular CAMR was detected in cells after FSH stimulation than in the control cells (retransferring cells CHO-DUK and cells SNO-DUK, transfected LH3AX SV2DHFRh1VS). On average, the response in these cells is approximately 16 times greater than the response in the control cells.

For the analysis of affect dose used one nacionalnog cell line, FSHR 4Q-13. When this was repeated the procedure described in the previous paragraph, except that cells were treated either people recombinant FSH or LH in a concentration of about 0-10 000 mIU/ml Each dose were analyzed in three duplicates. The number of produced intracellular CAMR expressed in nanomolar (nm) to milligrams (mg) soluble protein, and the curve of the dependence of the response of the dose was built for the concentrations of FSH and LH. These curves of the dose-response to FSH and LH are shown in Fig.5. The results showed that the SNO cell line CHOHFSHR4Q-13 was found saturable increase in intracellular CAMR in response to FSH stimulation, but in response to LH-stimulation of this increase was not observed, 50% of the maximum FSH stimulation (ED50) biologically functional people. FSH receptor in Cho-cells, which mediate specific CAMR-response to FSH, and do not respond to LH.

The content of the FSH receptor in cell lines CHOHFSHR4Q-13 or in the same line, which produces recombinantly people FSH receptor or FSH-binding fragments or mutants may be increased by gradually increasing MTX concentration. This leads to amplification of the number of copies of the DHFR cDNA together with the attached sequences, which include cDNA people FSH receptor, which indirectly contributes to the synthesis of the encoded proteins. It can also be implemented, if necessary, to increase the sensitivity bioanalysis. In addition, mammalian cells, which Express high levels of people FSH receptor or its FC-binding fragments or mutants can also be used to produce large quantities of protein people. FSH-receptor for therapeutic purposes, or for radioimmunoassay receptors.

Line CHOHFSHR 4Q-13 or a similar line can also be used in the analysis to evaluate the in vitro bioactivity of pharmaceutical preparations containing FSH or FSH-like substance. Gene-reporter such as luciferase, can be sootvetsvenno using non-radioactive method, for example, using bioluminescence, in order to determine FSH bioactivity.

Line CHOHFSHR 4Q-13 was deposited in ATSC 19 November 1991, the number of admission ADS CRL 10921. This deposition was carried out in accordance with the requirements of the Budapest Treaty.

Illustration of bioactivity of recombinant people. FSH receptor in stably transfected cells Y1 mouse adrenal gland.

To determine what can the cDNA-design people FSH receptor to produce biologically functional protein in stably transfected cells Y1, colonies of G418-esistenti of Y1 cells were treated people recombinant FSH, and culture medium was analyzed for the content of progesterone. For the analysis of progesterone cells subculturally 6-hole plates (35 mm diameter, each hole) at a density of 4105cells per well in selective medium for Y1 cells. Ambassador 2-day cultivation selective medium was removed and replaced with 3 ml of medium mF 10, supplemented with 5% horse serum, 0.8% of amniotic bovine serum (FBS), 1% L-glutamine, 80 μg/ml G418 (environment analysis). At this time, the estimated average number of cells per well for each cell line by-eskay environment, containing 100 mIU/ml of human recombinant FSH. After incubation cups with culture was placed on ice, and the culture medium from each well was transferred into a separate laboratory glass test tubes (1275). Tubes containing culture medium, were placed for 10 minutes in a bath of boiling water, and then centrifuged at 1100 g at 4oC. Supernatant brought in a clean laboratory glass test tubes (1275) and kept overnight at -20oC. then determined the levels of progesterone using the finished product Serono Diagnostics Progesterone, MAA, 12274, supplied by Ciba Corning, Medfield, MA. The analysis was carried out in accordance with the manufacturer's instructions, except that the samples of culture medium and progesterone for the standard curve were diluted in phosphate buffer solution containing 0.1% bovine serum albumin and 0.6% of sodium azide (instead of the diluted buffer, provided in the kit). Progesterone to plot a standard curve was obtained from Sa iochem (cat. 5341) and before use, diluted to a concentration of 15 μg/ml in 100% ethanol. The mother liquor was stored at -70oC. Final concentration in the dilution of progesterone for the standard curve were the same as recommended in the Aha="ptx2">

Several FSH-treated lines Y1, stably transfected people FSH receptor, secretively elevated levels of progesterone compared with control cells (same cell line, but not processed FSH, atransferrinemia cells Y1, Y1 cells, transfected CL 3SV2NEOh IVS). One nacionalnog line, YIHFSHR4-38 was chosen to analyze the dependence of the response on the dose. In this case, repeating the procedure described in the previous paragraph, except that cells were treated with different concentrations of FSH, 0-100 mIU/ml Each dose duplicated. The amount of progesterone produced FSH-stimulated cells Y1, normalized to 1106cells, and built the curves of dependence of the response on the concentration of FSH. These curves are presented in Fig. 6. In this experiment, a 25-fold increase in progesterone activity was observed at the dose of 20 mIU/ml people. recombinant FSH. The linear range FSH ranged from 2.5 to 20 mIU/ml FSH, and ED50=6,4 mIU/ml FSH. The results showed that in Y1 cells, stably transfected with a recombinant people. FSH receptor, observed FSH dose-dependent increase in the secretion of progesterone. Therefore, when the expression in the cells Y1 mouse adrenal gland is, what do YIHFHR4-38 - lines, or from the same cell lines, can be used to develop a sensitive in vitro assays for FSH or FSH-like substances. Other in vitro tests FSH, currently used, such as bioanalysis aromatase of granular cells and the analysis of aromatase of Sertoli cells, require a primary cell cultures from rats when conducting each analysis. Then, as for bioassays using new cells Y1 and SNO are stable transformed cell lines. Thanks to new methods of implementation in vitro bioassays clearly superior to presently known methods in terms of simplicity, accuracy and reliability.

Example 4

Testing of biological activity in vitro FSH human cells, stably transfected with a receptor FSH man

Mammalian cells, stably transfected with the FSH receptor (folliculo-stimulating hormone) of a person described in example 3, were used to test the samples for FSH activity. Details of the methods using the cell line Cho and Y1, described below.

Tests on cyclic AMP

Resistant to methotrexate cell line CHO-DUKX, stabil the face-to-face tablets at a density of 25 thousand cells per 1 well and cultured for 48 hours at 37oC. Cells of each well was washed with 3 ml of warm culture medium and then preincubated for 15 minutes at 37oWith 400 μl of culture medium without serum, containing 0.1 mm 3-isobutyl-1-methylxanthines (Sigma, St.Louis, MO). Highly purified FSH or LH (luteinizing hormone) man added at final concentrations of from 0.01 to 12000 Millie./ml and then continue incubation for 30 minutes at 37oC. Test end up holding four rapid cycles "freeze-thaw". The resulting lysate divided into aliquots of 50 μl, which is centrifuged atoWith, 13000g for 15 minutes. The supernatant fraction used for protein determination using a set of reagents BioRad Protein Assay kit. The remaining lysate add 300 ál of ethanol and the resulting mixture was centrifuged at 4oWith, 13000g for 15 minutes. Aliquots (5-100 ál) obtained supernatant fraction of cell lysate test for the presence of camp (cyclic amp) using a set of reagents DuPont/NEN Medical Products kit.

Tests for progesterone

Of Y1 cells, stably transfected with a receptor FSH person (cells Y1-hFSHR) subculturally 6-hole tablets when potkulturne selective for cells Y1 medium was removed and replaced with 3 ml of medium Ham''s F10 with 5% horse serum, 0,8% fetal calf serum, 1% L-glutamine and 80 μg/ml G418. After 24 hours, culture medium was removed and cells washed twice in 2 ml of the test environment (environment Ham''s F10 with the addition of 1 mg/ml bovine serum albumin, 1% L-glutamine and 80 μg/ml G418). The average number of cells per well estimated by sampling and direct counting of cells of the three test holes. The cells are then cultured for 4 hours in 1 ml of the test medium containing from 0.01 to 2500 Millie. /ml recombinant FSH or LH man. After incubation, the culture medium is transferred into a glass tube, boiled for 10 minutes and centrifuged at 1100g at 4oC. Levels of progesterone are determined using a set of reagents Serono Diagnostics Progesterone MAIA kit (obtained from the distributor Ciba Corning, Medfield, MA). The test is conducted in accordance with the manufacturer's recommendations, except that the samples of culture medium and progesterone used to construct a calibration curve (Cal Biochem, La Jolla, CA), diluted in phosphate-buffered saline containing 0.1% bovine serum albumin and 0.6% of sodium azide instead of cultivation in the buffer, which is included in the specified set of reagents.

Data analysis dependent on the dose of FSH and LH

As shown in Fig.7 (A, B)exact camp and secreted progesterone, respectively, then, when cultured in the presence of increasing concentrations of recombinant human FSH. However, neither cell line did not respond to LH. Has also been a change in cell morphology Y1-hFSHR: their epithelia-like phenotype was changed to a rounded shape in response to the influence of FSH. Similar morphological changes have been described for cells Y1, stimulirovalis the action of ACTH (Yasumura et al., 1966, Cancer Res., 26, 529-535), and Y1 cells that were stimulated TSH and microinjection mRNA receptor TSH (Parmentier et al., 1989, Science, 246, 1620-1622). The amount of recombinant human FSH required for half of the maximum (ED50) stimulate production of camp in CHO cells-hFSHR, amounted to 140 Millie./ml, which is significantly above the value of the ED50for the characteristic of the level of secretion of progesterone (4 Millie./ml) in cell line Y1-hFSHR. These data are in good agreement with previously published data (Fritz et al. , 1978, Can. J. Biochem., 56, 875-879; Fritz et al., 1978; Natl. Cancer Inst. Monogr., 48, 381-382): this indicates that a higher concentration of FSH is necessary for conditioning to maximize production of camp in isolated cultured Sertoli cells and significantly lower concentrations needed to achieve the Maxim substrate androgen to estradiol.

Example 5

Construction of mutant FSH receptor gene of the person encoding the extracellular domain (ECD)

Using the method of polymerase chain reaction (PCR) for constructing mutant variants of the ECD of the receptor FSH

The method of forming vector expressing the extracellular domain (ECD) of the receptor gonadotropin, was proposed by Xie et al., 1990, J. Biol. Chem., 265, 21411-21414. Briefly, the method of polymerase chain reaction (PCR) with respect to overlapping "ledges" (But et al., 1989, Gene, 77, 51-59) was used for separation of the transmembrane domain of the receptor structure LH-CG with the formation of the fragment that encodes domain ECD, truncated at residue alanine-338. Specialist in the art can easily use the same approach with the aim of obtaining a fragment encoding domain ECD of the receptor FSH, which would have been shortened by residue arginine-349 in accordance with the sequence SEQ ID NO 2. The same method can be applied to generate DNA fragments that encode smaller fragments domain ECD of the receptor FSH person, which can be conducted by changing the nucleotide sequences of the seed in accordance with 5'- and 3'-end of DNA encoding amino acid posledovatelnostyakh cassettes can be used to obtain mutant variants, encoding different variants of the ECD. Examples of this approach are described below.

Construction of the mutant receptor FSH person with the purpose of expression of full-domain ECD using splitting restrictase and cassette mutagenesis

The mutant variant, with the complete corresponding decoded extracellular domain of the FSH receptor person, includes a signal segment secretion (from methionine-[17] to glycine-[1] according to SEQ ID NO 2) and 349 amino acid residues of the Mature protein receptor from cysteine-1 to arginine-349 according to the sequence SEQ ID NO 2. The total number of amino acid residues encoded by the DNA fragment used for ekspressirovali in eukaryotic cells mutant variant is, therefore, 366.

With the aim of constructing 366-amino acid mutant variant domain ECD of two cDNA fragment of the receptor FSH person - 5'end 700-BP XhoI/BamHI fragment and the median 734-BP BamHI/-SphI fragment (example 2; Fig. 2.) - was subcloned into the composition of the plasmid pUC18-XhoI, pre-split restrictase XhoI and SphI. The vector pUC18-XhoI was formed by converting a restriction site SmaI as part of polylinker plasmid pUC18 at the XhoI site with XhoI-linkers. PLA is m right vzaimoraschetyi, outlined pHFSHR1.4xs. This plasmid was digested with restrictase EcoRV and SphI and the resulting fragment length 3700 nucleotides were purified in the gel.

Two complementary synthetic oligonucleotide - muta3a and muta3b - design so that they coded amino acid from isoleucine-342 to arginine-349 in accordance with the sequence SEQ ID NO 2, and exegetically C-terminal "tail" and a stop codon. After annealing these oligonucleotides form a cartridge, which can be Legerova on 3700-BP EcoRV/SphI fragment of the composition plasmids pHFSHR1.4xs. In the resulting design is retained the restriction sites EcoRV and SphI. In the sequence of the oligonucleotides contribute AseI restriction site required for screening designs, bearing this synthetic tape. The resulting cassette denote pHFSHR1.1XA. Sequencing of the DNA sequence is used to confirm the accuracy of coding 366-amino acid domain ECD of FSH plus exiisting "tail" and enable the stop codon TAA. It is clear that exegetically "tail" is only included in order to facilitate purification of the protein, i.e., exactly the same design can be created without the inclusion of histidine codons in the domain ECD of the receptor FSH person by giving benefits" available restriction sites

Four mutant variant domain ECD of the receptor FSH person, marked "a", "b", "C" and "d" design based on the unique recognition sites restrictedly available in full-size cDNA sequence that encodes the receptor for FSH person in accordance with is shown in Fig.8A. Construction of mutant variants of the receptor FSH person is carried out by sublimirovanny cDNA fragments of their composition plasmids hFSHRX, including the corresponding full-size cDNA (Fig.2) the plasmid rcwm. Plasmid rcwm is derived from the plasmid pUC cloning vector having polylinker sequence, designed to include unique restriction sites along with the start-codon and the stop codon, in accordance with the shown in Fig.8B. Subclavian part polylinker plot plasmids rkvm provides the required stop codon and the start codon in each of the generated DNA structures.

The mutant variant "a" is obtained by ligating the purified gel 686-BP XhoI/XbaI fragment of the composition hFSHRx on gel purified plasmid rkvm treated restrictase XhoI and NheI. Similarly, the mutant variant "b" receive the Ministry of foreign Affairs rqum, treated with restriction enzyme XhoI. The mutant variant "C" is obtained by ligating the purified gel 966-BP XhoI/BbsI fragment of the composition hFSHRx. The mutant variant "d" obtained by ligating the purified gel 1074-BP XhoI/NsiI fragment of the composition hFSHRx on gel purified plasmid rkvm treated restrictase XhoI and > PST. The resulting mutant variants "a", "b", "C" and "d" begin with the start codon of the signal segment (methionine-[17]) and are truncated at amino acid residues, respectively, 206, 231, 302 and 335 according to the sequence SEQ ID NO 2. Codons that controls the stop broadcast, contained in the composition of polylinker plasmids rcwm. DNA sequencing is carried out in respect of all mutant variants to verify the expected domain structure ECD.

Example 6

The formation of cell lines that Express mutant ECD-receptor polypeptides FSH man

Construction of plasmid vectors with the aim of ekspressirovali mutant variants of the ECD of the receptor FSH man

To obtain 366-amino acid mutant variant domain ECD of the receptor FSH person SphI site of plasmid pHFSHR1.1XA can be converted either into the EcoRI-site or in the XhoI site by the enzyme maple DNA polymerase I of Escherichia coli and add in ligation reaction of suitable linkers. After ligating the mixture is treated with a suitable restriction enzyme (either EcoRI or XhoI) and 1100-nucleotide fragment of clear gel and built by XhoI or EcoRI site expressing vector so that was obtained plasmid, such as, for example, shown in Fig.3.

Similarly, mutant variants "a", "b", "C" and "d" can be cut in the form XhoI fragments, suitable for embedding in a composition suitable expressing vectors. If selected expressing the vector contains cloning sites, recognizable restrictase EcoRI or XhoI, it can be used linkers that will ensure the conversion of either of the ends of the fragment, or the cloning site in the vector in the correct site. In one embodiment, expressing the vector is suitable for expression in mammalian cells: their organization is similar to that described in example 2. In another embodiment expressing vector is a baculovirus vector in accordance with the previously described (Luckow &Summers, 1989, Virology, 170, 31-39). Can also be formed by vectors and other expression systems such as yeast and bacteria.

Expression of mutant ECD variants of the receptor FSH person in the ka, used as described in examples 2 and 3 in order to obtain the culture supernatant fractions and cells containing biologically active protein.

Expression of mutant ECD variants of the receptor FSH person in insect cells

Baculovirus vectors encoding domain ECD of the receptor FSH person, used for co-transfection with linearized DNA purified by the material of the genome of AcMNPV virus that causes multiple polyhedron (poliedro) cores butterfly Autographa californica. Routine methods of cultivation of insect cells and production of viral titer is carried out as described in summers and Smith (Summers & Smith, 1988, "A Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedures", Texas Agricult. .. Stat., Bull. N 1555, College Station, TX). The vector carrying the gene ECD, and DNA virus AcMNPV used in an amount of 10 μg and 1 mg, respectively. Cells Spodooptera frugiperda (Sf) (American type culture collection Rockville, MD) transferout using the method with calcium phosphate (example 3), and the culture supernatant fraction was collected after 4 days. Fresh monolayer cell culture Sf infect a series of ten-fold dilution of the culture supernatant and their cover of 1.5% low-melting agarose (SeaPlaq Cup cover environment of Grace, including 10% fetal calf serum, and cultured for 4 days at 27oC. After the formation of "plaques" allocate some of recombinant colonies (negative occlusion). Conduct two rounds of purification colonies. Purified recombinantly virus from these colonies amplified thus, to obtain the virions for further infections. The structure of the recombinant virus is confirmed with the use of analysis methods PCR and southern blotting. The culture supernatant fractions containing biologically active fragments domain ECD of the receptor FSH man, produced by seeding cells in Sf as monolayers in flasks for culturing tissues. The Sf cells cultivated in an environment of Grace with the addition of 10% fetal calf serum, infect recombinant virus and then cultured for 4 days at 27oC.

Example 7

Analysis of binding parameters FSH mutant variants of the ECD of the receptor FSH man

Analysis of the binding of FSH to assess the activity of the protein ECD in conditioned culture medium and cell fractions

Specific binding125I-labeled FSH to determine cell clot and fractions of the cell lysate, as well as for generouse buffer (25 M sucrose, 0.05 M HEPES, pH 7.5, 5 mm MgCl2and 5 mm CaCl2), containing 0.1% (V/o) bovine serum albumin. In the variants with cell clot and fractions of cell lysate 500 thousand cells suspended in 1 ml of homogenization buffer and then homogenized using a device dounce". The suspension is centrifuged at 16000g for 30 minutes at 4oC. One 40-μl aliquot of the supernatant fraction used to determine protein content (BioRad Protein Assay kit) and two 90-ál aliquot used for analysis of binding of FSH. Cell clot resuspending 200 μl homogenization buffer and divided into 90-ál aliquots for analysis on the binding of FSH.

In the test for linkage 90 µl of the test sample (which is diluted culture supernatant, cell lysate or solubilizing cell clot), 10 ál 125I-FSH (96000 pulse/min, 1,1 nm) and 10 ál of water or 1.2 μm recombinant FSH people unite in faleconosco polypropylene tube h mm the resulting mixture was incubated at 37oC for 45 minutes. The solution was placed on ice and add 50 µl of 0.5% (V/o) gamma-globulin buffer (50 mm Tris pH 7,35). After 5 minutes in each sample add 160 ál of 21% (V/o) solution of PEG-8000 (50 mm Tris - R is of the share of non-specific binding to less than 15% of total binding spend the secondary precipitation. Sucked off the supernatant fraction and the clot is dissolved in 1 ml of 0.1% (V/o) Triton X-100 FSB, diluted with 0.5 ml of 0.5% gamma-globulin buffer followed by the addition of 1.5 ml of a solution of PEG-8000. The mixture is left on ice for 30 minutes, then centrifuged at 3000g, and the supernatant fraction is removed by suction. Clots hitting you on the counter. Indicators labeled specific binding125I-FSH expressed in counts/min, calculate the total binding125I-FSH minus indicator of nonspecific binding.

Parameters specific binding125I-FSH defined for fractions of cells with mutant variants structures "a", "b", "C", "d" and 366-ECD amino acid receptor FSH (example 5), expressed in cells SNO shown in the table. Cells SNO expressing full-receptor FSH person and retrospectively the original cell line Cho included as controls.

The results show that in the case of mutant variants of "b", "C", "d" and 366 main part of the binding125I-FSH accounts for the fraction of homogenized cell lysate. In the case of mutant variant "and" binding125I-FSH is similar to that observed in and either normally not expressed, or has no significant activity on the binding of FSH. In the variant with full-receptor FSH main binding is localized in fractions of the cell clot: this indicates its contents, as expected, associated with membrane receptors.

The analysis method Scatchard carried out to determine the affinity of binding of FSH mutant variants of "b", "C", "d" and 366 in comparison with the full-length receptor. The results show that the Kd values for options "C", "d", and 366, respectively 1,1510-10, 0,810-10and 3,3310-10i.e. similar to the index Kd, specific for full-length receptor - 2,2710-10. The mutant variant "b" is characterized by approximately an order of magnitude higher Kd value, component 2,410-9.

The conclusion that emerges from the analysis, indicates that portions of the domain ECD of the receptor FSH person, which include amino acids 1-231 in accordance with SEQ ID NO 2, sufficient for binding of FSH with high affinity. However, the fragments, which consist of amino acids 1-302 according to SEQ ID NO 2, showing the highest affinity equivalent to those that are characteristic of intact the>/BR>Purification of mutant variants of the ECD of the receptor FSH person, including exegetically "tail" (exegetical)

For mutant variants of domain ECD of the receptor FSH man, designed in such a way that they With-the ends were exegetically "tail" was applied to affinity purification using immobilization metal-affinity column. Column includes Nickel, attached as a chelating agent to the resin, which is connected nitryltriacetic acid. Four of the six sites of chelation on this column occupied by Nickel, and the two sites are free and can gelatinous histidine. Elution is provided either by lowering the pH or by the addition of imidazole. Acidic pH values lead to the protonation of histidine, resulting in its dissociation from nitryltriacetic acid. The imidazole competes with histidine for nitryltriacetic acid, which also provides the dissociation of the protein from the resin. The efficiency of the washing and elution nitryltriacetic acid control via analysis by electrophoresis in SDS-page to visualize proteins.

Purification of mutant variants of the ECD of the receptor FSH on FSH-affinity column

To clean mutandi mutant variants, including exegetically "tail", prepare affinity resin, which immobilized FSH. FSH covalently binds to cyanopolyyne resin with primary amines of the protein. To prevent the destruction of the column due to the dissociation of the subunits of FSH in the harsh conditions of washing or elution method of forming cross-links - and-subunits of FSH before FSH immobilized on the resin. Presumably the cross-linking occurs between primary amines and carboxyl groups, which are adjacent to each other in type, as is the case in non-covalent salt "bridges", presumably involved in the formation of heterodimer. Cross-stitched material is then separated from the reactants and from unimolecular stitched FSH using the method of gel filtration, and the effectiveness of cross-stitching determined by electrophoresis in SDS-page under conditions that ensure dissociation seamless cross-way of the material. Purified cross-stitched material is then fixed on cyanopolyyne resin for use in the purification of the receptor.

Example 9

Testing of biological activity in vitro mutant variants of ECD Retz is s 3 and 4) can be used to assess biological activity in vitro mutant variants of the ECD of the receptor FSH person in the test for competitive binding with human FSH. Used essentially the same procedure that was described in example 4, in connection with the testing for progesterone. Prepare serial dilution mutant polypeptide ECD of the receptor FSH person and added to a constant concentration of FSH (for example, the concentration of which in the absence of the antagonist provides 80% of the maximum stimulation in this test). Biologically active polypeptide ECD of the receptor FSH person prevents induced FSH secretion of progesterone in effective concentrations and over her, as determined using serial dilutions.

Example 10

Testing of biological activity in vivo mutant variants of the ECD of the receptor FSH person on the model rats

Well studied in vivo model based on female and male rats Sprague-Dawley, are used to assess the biological activity of gonadotropins: accordingly, they are suitable for testing activity of mutant variants of the ECD of the receptor FSH person.

Testing in vivo biological activity of mutant variants of the ECD of the receptor FSH person using female rats

Females rats Sprague-Dawley at the age of 25 days was intraperitoneally injected with human FSH (5-10%), ECD Retz is of determined experimentally) or just filler. After 48 hours the animals were killed and they were isolated ovaries. Selected tissue samples and put them in 4% formalin for subsequent histological analysis.

For morphometric analysis, the ovaries were left fixed for 12 hours in 4% formalin, embedded in paraffin and prepared ultra-thin sections with a thickness of 3 μm. Serial sections were on on slides for every 50 μm. The intervals of this size (50 μm) were selected based on the diameter of the oocytes (80 μm): as a result, each follicle can be analyzed without redundancy. Histological preparations were stained with a mixture of hematoxylin and eosin. Normal and atrioticheskie (neprevychnye) follicles was calculated based on the following morphological criteria: the appearance of the oocyte, the presence of a layer of granular cells and the number of layers of granular cells in accordance with the criteria Woodruff et al. (Woodruff et al., 1989, In "Growth Factors and the Ovary", ed. A. N. Hirschfield, pp. 291-295, New York, Plenum Press; Osman, 1985, J. Reprod. Fertil., 73, 261-270).

To determine the proliferation index ovaries taken from animals that had been injected labeled3N-tamilin, fix as well as non-radioactive samples, and prepared ultra-thin sections with a thickness of 3 moulali and stained. Then counted the number of nuclei containing granules of silver.

The diameter of the follicles was determined using an image analyzer BioquantTM. Measured in two directions that are at right angles to each other at the level of "bridge" granular cells, leading to the Cumulus and oocyte. Repeated measurements were excluded because the image of the first slice was kept in the computer's memory and then superimposed on the image of the subsequent slice.

The size of the follicle is an indicator of fertility: the size of the follicle increases in size during puberty and becomes ready for ovulation, i.e., thus, is a morphological marker of the Mature oocyte. The results show that the ovaries treated filler, and the ovaries, which is characterized by an optimal ratio of concentrations of FSH and biologically active polypeptide ECD of the receptor FSH mainly contain oocytes trophoblastic growth (their diameter is less than 250 microns). When the effect on animals of pure FSH noted the presence of small follicles (250 µm) and large follicles (>500 μm). These data confirm that the domain ECD of the receptor FSH person also decided is by introducing into the growing follicles3H-thymidine. In prepubertal follicles after exposure to only filler revealed a low level of inclusion in the parietal granular cells, cells Teka and cells discus proligerus (i.e., Cumulus cells and the adjacent cells). This group was marked not all follicles. Impact FSH cause the stimulation of the growth of follicles with consistent reinforcement include3H-thymidine in granular cells and cells Teka (meningeal cells); however, when combined impact. FSH with ECD domain of the FSH receptor include labeled3H-thymidine in granular cells and cells Teka does not occur. In contrast to the effect of "clear" filler, in the form of impact FSH most of the follicles, with different size (growth stage) include a radioactive label. Morphologically large follicles in animals, which simultaneously affected FSH and ECD domain of the FSH receptor, are atleticheskii. These data are further evidence that the domain ECD of the receptor FSH person is biologically active and blocks the activity itself FSH model in vivo.

Testing in vivo biological activity of mutant variants of the ECD of the receptor is 50 g) were subcutaneously injected with the ECD polypeptide receptor FSH person for 2 days every 6 hours. Control animals were injected only filler. The effective doses were determined experimentally. Later, 2 days after the initial injection the animals were immobilized testes removed, weighed them and to prepare sections for histological analysis.

The data obtained showed that the introduction of rats domain ECD of the receptor FSH causes degeneration semennikova tissue and a significant slowdown in the growth of the testes. In contrast, the control animals were not observed neither the slowdown in the growth of the testes, not tissue degeneration in them. These data show that the domain ECD of the receptor FSH is able to neutralize the activity of endogenous FSH, which is necessary for normal growth and development of the testes during puberty.

Example 11

Pharmaceutical use of the ECD of the receptor FSH person to treat patients

As discussed in the section "Detailed description of the invention, a purified preparation of the FSH receptor can be used to bind to endogenous FSH and neutralizing its activity. In male patients the introduction of the polypeptide ECD of the human FSH receptor should lead to a decrease in the intensity or blocking spumigena or block the development of follicles and the development of estrogen. This will cause infertility, i.e., this effect can be used as a method of contraception. Other therapeutic applications may include application of the ECD of the receptor FSH person to reduce the production of estrogen by the ovaries. This can be valuable in the treatment of diseases such as breast cancer and endometriosis. The ECD polypeptide receptor FSH can also be used to reduce the number of developing follicles in the process of artificial insemination. This may find particular use in the treatment of patients with a diagnosis of polycystic ovaries, which are prone to the development of ovarian hyperstimulation syndrome.

Suitable purified mutant variants of the ECD of the receptor FSH person prepared in the form of pharmaceutical compositions intended for administration to patients. Examples of such pharmaceutical compositions is described in the section "Detailed description of the invention".

Dosage

Dose of a medicinal product intended for administration to a patient, determined in accordance with clinical practice and will vary depending on such factors as the patient's age, height, body weight, sex, medical history and overall SOS is RNO 10 mg/kg (in terms of body weight of the patient), daily or several times (for example, 3 times) a week, although you may be used and the higher and lower doses.

In particular, the dose is determined based on pharmacological parameters excretion entered ECD mutant variant of the FSH receptor, which uses standard pharmacological approaches known in the art. Methods of preparing such dosage forms are known and are obvious to experts: see, e.g., A. R. Gennaro (ed.), "Remington's Pharmaceutical Sciences, 18th ed., Mack, Easton, Pa, 1990.

The concentration of the molecules according to the invention in pharmaceutical compositions is not critical, but typically is in the range from about 1 μg/ml to about 20 mg/ml

Monitoring of male patients according to the available biological effects is carried out using criteria such as counting the number of sperm, the determination of motility and viability. Observation of patients-women with regard to the possible biological effects associated with the monitoring of follicular development, which uses high-resolution ultrasound sonography, as well as control over the content of estrogen in the blood. The impact of the introduction of biologically asset is in (oligozoospermia), the decrease in motility, viability and the violation of their morphology, while in women-women are the result of such exposure is associated with a decrease in serum estrogen suppression of follicular development and the absence of ovulation.

The list of sequences is given at the end of the description.

1. The polypeptide having the properties of the receptor folliculo-stimulating hormone (FSH) human produced by cells transfected with a recombinant vector containing a DNA nucleotide sequence shown in SEQ ID NO: 1, or its fragment, deleteriously with 3'-end, ensuring the expression of the polypeptide that retains the ability to bind human FSH.

2. The DNA molecule encoding the polypeptide under item 1, characterized by the nucleotide sequence shown in SEQ ID NO: 1 or the corresponding fragment.

3. The vector expression of the polypeptide under item 1 in mammalian cells, containing operatively linked to the DNA molecule, as described in paragraph 2 is functionally capable in mammalian cells the promoter and acceptable marker gene.

4. The method of producing the polypeptide under item 1, characterized by the fact that cultured cells, transposase to reduce the biological activity of endogenous FSH, containing as active ingredient an effective amount of the polypeptide having the properties of FSH receptor human, and pharmaceutically acceptable carrier, characterized in that the active ingredient is represented by the polypeptide described in paragraph 1.

 

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Peptide derivatives // 2187511
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