Gonadotrophin derived from invertebrates and its synthesis

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology, in particular to production of hormones and can be used for culturing invertebrates. Gonadotrophin, selected from the invertebrate Asterina pectinifera is a peptide with molecular weight 4500-4900, it has two subunits, the protein structure of which is combined with SS-bridges, formed between residues of SH cysteine contained in the subunits.

EFFECT: interfusion and oxidation of these two subunits after synthesis allow producing gonadotrophin having gonadal promoting activity.

4 cl, 8 dwg, 1 tbl, 2 ex

 

The technical field to which the invention relates

The invention relates to gonadotropic hormone derived from invertebrates, such as Asterina pectinifera and the like, and its synthesis.

The level of technology

There are two types of gonadotropic hormones of vertebrates that control the development of sexual organs and the development, maturation and release of eggs and sperm. Made significant progress in the study of gonadotropic hormones of vertebrates, for example, fish, etc. the Structural similarity of these hormones is well preserved from fish to humans, and these hormones are protein hormones with heterodimeric structure, including α-subunit and β-subunit.

It was confirmed that the gonadotropic hormones of vertebrates have interspecific effects (non-patent reference 1, patent reference 1 and 2) in vertebrates, but not in invertebrates.

Gonadotropic hormones invertebrates have not been studied so as gonadotropic hormones of vertebrates. Gonadotropin-releasing hormone (GSS) was extracted from the radial nerve Asterina pectinifera, i.e. bespozvonochnykh, and, as was confirmed that caused the multiplication of the ovarian component (non-patent references 2 and 3). It is reported that gonadotropic hormone stimulates ovarian follicular cells surrounding the oocyte in which isnike, the synthesis and secretion of the hormone, inducing maturation of the oocyte (1-methyladenine)that directly affect egg (non-patent reference 4).

Patent reference 1: Japan patent No. 2967945.

Patent reference 2: Japan patent Application Public Disclosure No. H06-107689.

Non-patent reference 1: Ed., The Japanese society for comparative endocrinology, "Biological Science of Hormone 5, Hormone and Reproduction (II)", Japan Scientific Societies Press, pp. 41-47, 1979.

Non-patent reference 2: Ed., The Japanese society for zoology, "Problems in Modern Zoology 4, Oocytes and Sperms", Tokyo University Press, pp. 21-37, 1975.

Non-patent reference 3: Shirai H., Gonad-Stimulating and Maturation-Inducing Substance, "Method in Cell Biology" Academic Press, vol. 27, pp. 73-88, 1986.

Non-patent reference 4: Mita M. & Nagahama Y., Involvement of G-proteins and adenylate cyclase in the action of gonad-stimulating substance on starfish ovarian follicle cells, Developmental Biology, 1991, 144, 262-8.

The problems solved by the present invention

As gonadotropic hormones have a stimulating effect on maturation, it is important to apply for cultivation mother of a living organism. The present invention is based on innovative disclosure patterns gonadotropic hormone bespozvonochnykh and may find potential application in the cultivation of invertebrates, such as shrimps, crabs, sea shells, etc.

Ways of solving problems

The inventors have successfully analyzed the structure of the gonad-stimulating hormone secreted by nerve cells Asterina pectinifera. Inventors forward in the new revealed what gonadotropic hormone is a peptide with a molecular mass of 4500-4900 consisting of subunits with a molecular mass of 2000-2400 and 2400-2600, and that its structure is formed SS-bridges formed between SH-remnants cysteines contained in these two subunits, and confirmed that the peptide obtained by mixing and oxidation of two synthesized subunits had a gonad-stimulating activity. The result analyzed by the authors of the present invention, can open new opportunities for a wide range of application areas, in order to establish the mass production of the hormone and other

Namely, the present invention is a gonadotropic hormone bespozvonochnykh, including the following two peptide in which SS-bridges formed between 6 cysteine these two peptides:

(a) a peptide having the amino acid sequence of SEQ ID NO: 1, or amino acid sequence comprising a deletion, substitution or addition of one or several amino acids (i.e. 2-3) in the specified amino acid sequence in addition to amino acids 4 and 16 (Cys) of the specified amino acid sequence, and having the gonad-stimulating activity;

(b) a peptide having the amino acid sequence of SEQ ID NO: 2, or amino acid sequence comprising a deletion, substitution or addition of one or n is how many (i.e. 2-3) amino acids in the specified amino acid sequence in addition to 10 amino acids, 11, 15 and 24 (Cys) of the specified amino acid sequence, and having the gonad-stimulating activity.

Also the present invention is a gonadotropic hormone bespozvonochnykh obtained by mixing and oxidation of two of the following peptides:

(a) a peptide having the amino acid sequence of SEQ ID NO: 1, or amino acid sequence comprising a deletion, substitution or addition of one or more (i.e. 2-3) amino acids in the specified amino acid sequence (preferably amino acids except Cys); and with the gonad-stimulating activity;

(b) a peptide having the amino acid sequence of SEQ ID NO: 2, or amino acid sequence comprising a deletion, substitution or addition of one or more (i.e. 2-3) amino acids in the specified amino acid sequence (preferably amino acids except Cys), and with the gonad-stimulating activity.

These two peptide can be obtained by treating (a) the nerve of the body, isolated from bespozvonochnykh, or (b) a specific authority, which reportedly has the gonad-stimulating activity, based on gonad-stimulating activity bespozvonochnykh as an indicator.

In addition, the present invention is a method of producing peptides of gonad-stimulating the ith activity, including mixing and oxidation of these two peptides. These two peptide can be obtained by any method, that is, they can be chemically synthesized or can be obtained using genetic engineering.

The present invention is a DNA encoding any of these two peptides ((c) and (d)). Peptides isolated from the organisms, the host or environment of cultivation, while these organisms are the owners transform the vector containing the two aforementioned DNA, cultivated and grown in culture medium. The peptide with the gonad-stimulating activity can be obtained by mixing and oxidation of peptides.

In addition, the present invention is a DNA which contains a DNA encoding these two peptide ((c) and (d)), with more than 70% homology with the nucleotide sequence SEQ ID NO: 3. Peptides c of gonad-stimulating activity can be obtained by culturing and growing the host body, transformed by a vector containing DNA. In addition, peptides can be isolated from organisms of the owners or their environment cultivation, the peptides can be mixed and oxidized.

Brief description of drawings

Figure 1 - photograph showing the criteria of gonad-stimulating activity. The right side shows that the egg was born and gonad-stimulating the ith activity is present.

Figure 2 shows the profile of purification of gonadotropin-releasing hormone high-performance liquid chromatography.

Figure 3 shows the profile of purification of gonadotropin-releasing hormone high-performance liquid chromatography.

Figure 4 shows the profile of purification of gonadotropin-releasing hormone microgateway high-performance liquid chromatography.

Figure 5 shows the profile of purification of gonadotropin-releasing hormone microgateway high-performance liquid chromatography.

Fig.6 shows the result of analysis of the purified gonadotropin-releasing hormone (heterodimer) on the mass spectrometer.

Fig.7 shows the result of analysis of the subunits of gonadotropic hormone in the mass spectrometer.

Fig shows the profile of the purification of the synthesized gonadotropic hormone of high-performance liquid chromatography.

Detailed description of the invention

The DNA sequence of the gene gonadotropic hormone bespozvonochnykh according to the present invention includes the nucleotide sequence (351) SEQ ID NO:3, DNA encoding these two peptide ((c) and (d)), or of a nucleotide sequence that is homologous to more than 70% nucleotide sequence of SEQ ID NO:3.

The report on the draft analysis of the human genome consortium human genome (Science 2001, 291 (5507), 1304-1351) proposed a criterion automatic is Natali gene in genome sequences using computer programs (Otto system), with this criterion, based on homology to the polynucleotide sequences with known genetic information, should have more than 92%identity in order to accurately determine the region of the gene in the genome. In the same report suggested that the criterion of homology of polynucleotide 70%, for finding genomes, increased gene duplication (later evolution could enter the gene and could contribute to partial changes in nucleotide sequence). Therefore, the nucleotide sequence with a homology of more than 70%,and preferably more than 92%to SEQ ID NO:3 in the present invention can function as a gene gonadotropic hormone bespozvonochnykh, which is applied to a closely related but different species of animals.

Amino acid sequence of gonadotropin-releasing hormone bespozvonochnykh according to the present invention includes a 116 amino acids shown in SEQ ID NO: 4, or amino acid sequence in which one or more (e.g., 2-3) of amino acids (preferably amino acids except Cys) deleterow, replaced or added. In the amino acid sequence (SEQ ID NO: 4) sequence from amino acid 1, methionine (M), to amino acid 29, glycine (G), is the signal sequence, the sequence starting from and what inoculate 30, glutamine (E), to amino acid 48, serine (S), is a subunit And (GSS-A), the sequence starting from the amino acids 49, lysine (K), to amino acid 92, arginine (R), is a subunit C (GSS-C), and the sequence starting from the amino acid 93, serine (S), to amino acids 116, cysteine (C), is a subunit B (GSS-B).

Gonadotropic hormone bespozvonochnykh according to the present invention contains a peptide formed by linking GSS-A and GSS-B 1 to 1, and the peptide contains SS-bridges, oxidative formed between cysteine (amino acids 4 and 16 of SEQ ID NO: 1 and amino acids 10, 11, 15 and 24 of SEQ ID NO: 2)contained in each peptide. The maximum formation of SS-bridges between GSS-A and GSS-B is 12.

Heterodimeric structure with a bridging structure between peptides GSS-A and GSS-B can be collected in the form of gonadotropin-releasing hormone after removal of the signal sequence of the chain of a peptide synthesized on the basis of the gene, with subsequent final destruction of the GSS-C side from the bridge structure. However, GSS-C part still may also have physiological activity according to a recent scientific publication.

Since all of gonadotropic hormones invertebrates according to the present invention are secreted from the nerve tissue, the source of gonadotropic hormones invertebrates from other species, except Asterina pectinifera was restricted to nervous tissue source species. If other species of animals, as reported, have the gonads-stimulating activity in other tissues and organs, these tissues and organs can be used as sources for extraction.

Invertebrates include many useful marine species, such as coelenterates, such as corals, echinoderms, such as sea urchins and sea slugs, clams, such as octopus and squid, and crustaceans, such as shrimps and crabs.

The peptides can be purified from these sources by extraction with the use of gonad-stimulating activity as a marker.

Cleaning can be carried out by any means, can be used as liquid chromatography and two-phase water method of separation. However, for high purification is preferably used high-performance liquid chromatography. Gel-filtration, ion-exchange chromatography or reverse-phase chromatography can be used as columns. Thus, the fractions separated by the method of purification, subjected to selection using as a marker of gonad-stimulating activity.

The gonad-stimulating activity can be measured by the following methods:

Those individuals, oocytes are very sensitive to the hormone that stimulates the maturation of the oocyte, are selected from Mature females invertebrate and separated as control animals.

The control specimens extracted ovaries in sea water and cut into small pieces. Analytical tablet for a large number of samples tested is filled with 200 µl of sea water. Two µl of the samples investigated in the gonad-stimulating activity, diluted 200 times, mixing with 398 ml sea water. Half of the sample (200 ál) was diluted twice by mixing with 200 ál of sea water on analytical tablet. Half of the sample (200 µl) was then diluted twice by mixing with 200 ál of sea water on analytical tablet. Repetition of the procedure results in a series of twofold dilutions of 200-fold dilution of up to 102,400-fold dilution. A small portion of the ovary is added to each diluted sample and left at a temperature of 25°C. Gonad-stimulating activity judged by the formation of Mature oocytes from cut off a small part of the ovary after 1 hour. Relative gonad-stimulating activity is estimated by the degree of dilution.

Gonadotropic hormone has many functions, namely long and short periods (long-term: development of gonads, short term: induction of release of eggs). Since the analysis depends on short-term options, control specimens must bear Mature oocytes with the ability to produce eggs. It is preferable to select the control individuals in their early spawning period in order to support them in a laboratory aquarium. Not always all collected specimens are so Mature to produce eggs. Also, if ever a species are capable of producing eggs, the degree of their maturity slightly different, and usually sensitivity to the hormone varies depending on individuals. Therefore, for estimation of gonad-stimulating activity, you must select Mature individuals (those who bear the oocytes with the ability to produce eggs) and, in addition, prepare a few individuals with the same sensitivity to the hormone.

Gonadotropic hormone stimulates ovarian follicle cells, and the cells of the follicle secrete another hormone - the hormone that stimulates the maturation of the oocyte, which stimulates oocyte to the production of caviar. Because the hormone that stimulates the maturation of the oocyte, directly affects the oocyte, one of the short-term function and gonadotropin-releasing hormone is the induction of the formation of hormone maturation of the oocyte. To assess the sensitivity can be used commercially available hormone that stimulates the maturation of the oocyte.

The above treatment can be repeated several times. As a result can be obtained unique peptide with a molecular mass of 4500-4900.

As shown in the following Examples, the peptide consists of two subunits (GSS-A and GSS-B), where the molecular mass of the subunits - 2000-2400 and 2400-2600. These subjeci the Itza can be obtained by recovery of the above mentioned peptide with high molecular weight. Various reductants are used for reduction reactions, while preferably used such moderate reducing agents, as dithiothreitol, 2-mercaptoethanol, thioglycolate acid, thiophenol, paratheatrical and others.

On the contrary, mixing and oxidation of these two peptides lead to the formation of the peptide (i.e. gonadotropic hormone of the present invention) with a higher molecular weight than the above-described peptides. Because each of these two subunits contains cysteine, oxidation gives SS-bridges between SH-groups cysteines contained in the subunits to associate these two subunits.

The above-mentioned oxidation can be performed in the following oxidants in addition to the method shown in the Examples:

- o-Iodobenzoic acid, iodine

- Manganese dioxide, potassium permanganate

- Peroxide

- Free oxygen (this reagent is used in the following Examples, the oxidation reaction of free oxygen can be accelerated in the presence of trace quantities of ion and copper ion).

In addition, it is possible to increase the yield for the formation of the correct SS-bridges between the peptides in the presence of the following reagents, which are used for refolding SS-bridges of the protein during the oxidation of synthetic peptides:

- thioredoxin (protein used for oxidation is vosstanovitelnogo interaction in a living system),

protein the disulfide isomerase (enzyme in the metabolism of protein disulfides in living organisms),

- BMC, (±)-TRANS-1,2-bis(2-mercaptoacetate)cyclohexane,

- 4-mercaptophenylacetic.

The peptide according to the present invention can also be obtained by genetic recombination. For example, a vector containing the DNA (i.e. the base 88-144 and 277-348 of SEQ ID NO: 3)encoding the amino acid sequence of SEQ ID NOs: 1 and 2 or the amino acid sequence in which one or more (i.e. 2-3) amino acids except Cys deleterows, replaced or added, or a vector containing a DNA in which the nucleotide sequence of more than 70% homologous to the nucleotide sequence of SEQ ID NO: 3, create, introducing organisms are the owners of with the purpose of transformation. Then the transformed organisms owners cultivate and grow. Target peptides isolated from organisms-owners or their culture media. Sometimes the resulting peptides are peptides with communication between the two subunits (GSS-A and GSS-B). However, oxidation of the resulting peptides (two subunits) in the presence of these oxidants leads to peptides of gonad-stimulating activity.

Ways to induce acceleration of maturation and ovulation with the help of these peptides with the gonad-stimulating activity include the introduction of these peptides directly in the WTO the ranks cavity or ovary invertebrates by adding sea water in the aquarium or mix with food.

The following Examples are provided to illustrate the present invention but are not intended to limit its scope.

In the following Examples of gonad-stimulating activity was investigated in the following way:

Two or three individuals carrying the oocyte with high sensitivity to gonadotropin-releasing hormone (1-methyladenine), otbiraut of several dozen Mature females Asterina pectinifera (or Asterias amurensis) and separated as control animals.

The selection was made as follows:

The hormone that stimulates the maturation of the oocytes was dissolved in sea water in 6 different concentrations, i.e. the 10-6M, 3×10-7M, 10-7M, 3×10-8M, 10-8M and 3×10-9M, with a small piece of ovary was added to various concentrations of the calling maturation hormone and sustained 60 minutes at room temperature. Maturation of the oocytes were examined under a microscope after 60 minutes, to see whether the structure of nuclei in the oocyte is degenerated to prepare for the next insemination. Was tested concentration of the hormone that stimulates the maturation of the oocyte during maturation, and those individuals who Mature at less than 10-7M concentration was used as control animals.

Ovaries (with pochobradsky morphology) were extracted from control animals in sea water and divided into fragments RA is Merom about 5 mm. Analytical tablet for a large number of samples tested was filled with 200 μl of sea water. Two µl of the samples investigated in the gonad-stimulating activity was diluted 200 times, mixing with 398 ml sea water. Half of the sample (200 ál) was diluted twice by mixing with 200 ál of sea water on analytical tablet. Half of the sample (200 ál) was then diluted twice by mixing with 200 ál of sea water on analytical tablet. Repetition of the procedure yields a series of twofold dilutions from 200-fold to 102,400-fold dilution. A small portion of the ovary is added to each diluted sample and left at a temperature of 25°C. Gonad-stimulating activity judged by the formation of Mature oocytes from cut off a small part of the ovary after 1 hour. Relative gonad-stimulating activity is assessed according to the degree of dilution.

Example 1

The fabric of the radial nerve has been extracted from Asterina pectinifera tongs, frozen on dry ice and stored. Nervous tissue wet weight 126,3 g were selected at 5550 starfish. Liquid nitrogen filled gun and tissue radial nerve stored in a frozen state, were crushed into powder. Powdered nervous tissue were added to 600 ml of 10 mm aqueous solution of ammonium acetate (containing inhibitors of proteases, i.e. the 1 μM past is Tina, 0.5 mg/l leupeptin, 0.2 mm 4-(2-amino-ethyl) phenolsulfonephthalein) in three steps and were further homogenized to fine powder electrogalvanization (Physcotron). Extracts from homogenized tissues were tsentrifugirovanie at 22,500xg at 4°C for 30 min, followed by separation of the supernatant. The sediment was homogenized with 200 ml of 10 mm solution of ammonium acetate and centrifuged at 22,500xg at 4°C for 30 minutes the Supernatant was separated and mixed with the previous supernatant. Obtained at 22,500xg supernatant was ultracentrifuged at 100,000xg at 4°C for 1 hour, and the separated supernatant. The supernatant had a gonad-stimulating activity.

After the obtained supernatant was frozen and dried, it was dissolved in 100 ml of 0.15 M ammonium carbonate. After undissolved residues were removed by centrifugation at 27,500xg at 4°C for 30 minutes, the solution was applied to a desalting column PD-10 (Amersham Biotech Co.), balanced solution of ammonium carbonate, and allocated a fraction of the eluate with high molecular weight (fraction PD-10), with the gonad-stimulating activity.

After the fraction of the eluate with high molecular weight (fraction PD-10) was frozen and dried, it was dissolved in 150 ml of 10 mm sodium phosphate (pH 7.0). The solution was put in the three receiving portions 50 ml column of Sephadex G-50 (500 cm3), ur is Novichenko phosphate sodium, and allocated fraction (fraction without the proteins of high molecular mass) of gonad-stimulating activity (fraction G-50, the output amounted to approximately 600 ml).

Approximately 415 ml fractions G-50 were treated with high-performance liquid chromatography (Shimazu Corporation, Type LC-6AD) in 26 receptions. Then the active fractions were suirvey linear concentration gradient from 10 mm sodium phosphate (pH 7.0) and 30% acetonitrile/10 mm phosphate with column Develosil RP-Aqueous AR5 (CH mm, Nomura Science). Fraction of gonad-stimulating activity were suirvey within acetonitrile 18-19% (1st fraction HPLC, figure 2).

1st HPLC fraction was concentrated to 45 ml under reduced pressure, and the concentrate was treated with high-performance liquid chromatography (Shimazu Corporation, Type LC-6AD) in 9 receptions. Then, the active fractions were suirvey in a linear concentration gradient from 20% acetonitrile/10 mm trimethylacetate (pH 4.0) is up to 25% acetonitrile/10 mm trimethylacetate with column Develosil RP-Aqueous AR5 (10×250 mm, Nomura Science). Fraction of gonad-stimulating activity were suirvey within acetonitrile 21-22% (2nd fraction HPLC, figure 3).

2nd HPLC fraction was concentrated under reduced pressure. Active fractions were suirvey in a linear concentration gradient of 16.5% acetonitrile/10 mm sodium phosphate (pH 6,0) to 17.5% acetonic the sludge/10 mm sodium phosphate (pH 6,0) with column Develosil RP-Aqueous AR5 (10× 250 mm, Nomura Science). Fraction of gonad-stimulating activity were suirvey within acetonitrile 17% (3rd SMART fraction, figure 4).

Active fractions were suirvey in a linear concentration gradient of 15% acetonitrile/10 mm sodium phosphate (pH 6,0) up to 30% acetonitrile/10 mm sodium phosphate (pH 6,0) with column Develosil RP-Aqueous AR5 (10×250 mm, Nomura Science). Fraction of gonad-stimulating activity were suirvey within acetonitrile 18% (4th SMART fraction, figure 5).

The last part of the purified fraction (4th SMART fraction) was abessolo using Ziptip (Milipore Co.) and analyzed on a mass spectrometer type MALDI-TOF (Bruker Daltonics Co., ReflexIII type). The result showed the presence of a component with a molecular mass 4737. In addition, the last fraction was recovered 50 mm dithiothreitol at room temperature for 1 hour and was again analyzed by a mass spectrometer. The result showed the presence of two signals with masses 2236 and 2507 instead 4737. Moreover, the last fraction is treated with reducing agent and an alkylating agent such as 0.2 M of iodoacetamide at room temperature for 24 hours. As molecular weight peaks 2236 and 2507 were increased by 57 (in accordance with 1 remainder SH) and 114 mass units (in accordance with 2 residues SH), respectively, the remains of SH could be specific alkylated.

Analysis 4th SMART fraction of seque what ATOR protein (ABI, Procise Type 494HT) gave two signals that indicate a mixture of two peptides. As the above results show that the component of the gonad-stimulating activity contained in the latter fraction was a polypeptide with a molecular mass 4737, and the components were divided into two component recovery, the active component has a heterodimeric structure, comprising two peptide subunits with a molecular mass 2236 and 2507, where the dimeric structure is sensitive to the reducing agent and, as shown, is SS-bridge between cysteine residues.

Amino acid analysis of a mixture of peptides of molecular weight 2236 and 2507 obtained by reduction of the polypeptide with a molecular mass 4737, was performed using mass spectrometry mass spectrometer of the type Q-TOF (Micromass Co.) (Fig.6 and Fig.7).

Amino acid sequences of these two peptides were:

GSS-A: EKYCDDDFHMAVFRTCAVS (SEQ ID NO: 1) (19 amino acid residues, molecular weight 2236);

GSS-B: SEYSGIASYCCLHGCTPSELSVVC (SEQ ID NO: 2) (24 amino acid residue molecular weight 2507).

From the results of mass spectrometer analysis and protein sequencing machine, it was found that these peptides are released from any chemical modification (intracellular post-translational modification of protein and peptide) in the side chains and terminal amino acids after biosynthesis in the nervous cells starfish the drive.

Gene GSS was cloned on the basis of amino acid sequence GSS-A and GSS-b Were synthesized based on the amino acid sequences GSS-A and GSS-B: 5'-primer DF1 (SEQ ID NO: 5) and 5'-primer DF2 (SEQ ID NO: 6), respectively; 3'-primer DR1 (SEQ ID NO: 7) and 3'-primer DR2 (SEQ ID NO: 8), respectively.

Genomic DNA was isolated and purified from testis Asterina pectinifera using QIAGEN(R) Genomic-tip.

Part of the gene sequence GSS was amplified using nested PCR using a sample of the genome as the template and synthesized degenerate primers and the nucleotide sequence was decoded DNA-sequencing machine. Based on the newly obtained sequences were synthesized 5'-primer GR (SEQ ID NO: 9) and 3'-primer GF (SEQ ID NO: 10). 5'straight and 3'-reverse DNA sequences were transcribed using primers, genomic DNA and sets CLONTECH Genome Walker (TM). Based on the obtained sequences were synthesized 5'-primer MF1 (SEQ ID NO: 11) and 5'-primer MF2 (SEQ ID NO: 12); 3'-primer MR1 (SEQ ID NO: 13) and 3'-primer MR2 (SEQ ID NO: 14).

Total RNA was isolated and purified from nerves, ambulacral legs, hepatopancreas, testes and ovaries Asterina pectinifera using NIPPON GENE ISOGEN or QUIAGEN (R) QIAzol (TM). cDNA was synthesized from purified total RNA using QIAGEN (R) Omniscript (TM) RT.

The cDNA sequence GSS was amplified using cDNA as matrix and synthesized primers (5'-primers MF1, 5'-primer MF2, 3'-primer MR1, 3'-primer MR2) using nested-PCR and cDNA sequence GSS was deciphered DNA-sequencing machine.

Thus was obtained GSS gene shown in SEQ ID NO: 3. The nucleotide sequence was translated into amino acid sequence of SEQ ID NO: 4. Sequence GSS-A and GSS-B, analyzed mass spectrometry and protein sequencing machine, are located in areas amino acids 30-48 (GSS-A) and 93-116 (GSS-B) amino acid sequence of SEQ ID NO: 4.

Amino acids 1-29 of the amino acid sequence of SEQ ID NO: 4 are characteristic signal sequence of secretory proteins, and amino acids 49-92 of the amino acid sequences are composed of 116 amino acid sequence (designated as GSS-C), cut after biosynthesis of the amino acid sequence. The sequence KR, which specifically and animations deleted after biosynthesis, is located on both ends of the sequence GSS-C.

Example 2

Based on the result of Example 1 were synthesized two peptide chains (GSS-A and GSS-B) (purity > 99.5%pure).

Two of the synthesized peptide, dissolved in 20 mm Tris-buffer at concentrations of 0.4 mm or 1 mm (each in an equimolar amount), reacted at room temperature for 3 or 20 days in the presence of oxidants with the constant p is remesiana. 99,999%gaseous oxygen or 0.1 M oxidized form of glutathione was used as oxidants. After the reaction, the reactants were separated microgateway high-performance liquid chromatography, it was estimated gonad-stimulating activity of each peak fraction.

The gonad-stimulating activity was detected in a relatively small peak fractions from the peak area of 4.2%. In addition, molecular weight 4737 (GSS-A/B), which indicates a complex structure and is the same as the molecular mass of the natural hormone, was discovered only in the peak fraction of gonad-stimulating activity according to the analysis of each peak by mass spectrometry. Other peaks without gonad-stimulating activity showed the molecular weight 2236 or 2507 (Fig).

Was estimated gonad-stimulating activity of these peptides. The results are shown in the table.

PeptideEC50(nM)
Asterina pectiniferaAsterias amurensis
GSS-ANo activityNo activity
GSS-BNo activityNo activity
GSS-A/B0,6-3,62-10
naturalof 0.7 to 4.0-

Synthesized peptide (GSS-A or GSS-B) separately showed no gonad-stimulating activity, and only complex (GSS-A/B) with a molecular mass 4737 synthesized by the oxidation reaction, showed activity. Synthesized in the reaction of oxidation hormone (GSS-A/B) showed hormonal function not only in Asterina pectinifera, but also in such related forms as Asterias amurensis.

The present invention allows mass production of gonad-stimulating hormone bespozvonochnykh and to increase the production of aquatic invertebrates type of crabs, shrimps, sea urchins, sea cucumbers and clams, as well as to develop new valuable species.

In addition, the artificial control of the number of starfish in the sea the field of water management through supply of fertilized eggs Asterina pectinifera and Asterias amurensis or fast artificial breeding can improve water quality by accelerating the biological degradability of forage accumulated on the seabed. Since these two species of sea stars - dominant species in the marine area of water resources, and their habitat covers the entire area from Hokkaido to Kyushu, can be expected extensive use.

1. Gonadotropic hormone bespozvonochnykh, including the following two peptide in which SS-bridges formed between 6 cysteine these two PE is the Chida:

(a) a peptide having the amino acid sequence of SEQ ID NO:1,

(b) a peptide having the amino acid sequence of SEQ ID NO:2.

2. Gonadotropic hormone bespozvonochnykh, obtained by mixing and oxidation of the following two peptides:

(a) a peptide having the amino acid sequence of SEQ ID NO:1,

(b) a peptide having the amino acid sequence of SEQ ID NO:2.

3. Gonadotropic hormone bespozvonochnykh according to claim 1 or 2, where the two peptide obtained by the treatment (a) nervous body, isolated from bespozvonochnykh or (b) a specific authority, about which it is known that he has the gonads-stimulating activity, based on gonad-stimulating activity bespozvonochnykh as the indicator.

4. A method of obtaining a gonadotropic hormone, comprising (1) culturing and growing the host body, transformed by a vector containing two DNA, each of which encodes the following peptides:

(a) a peptide having the amino acid sequence of SEQ ID NO:1,

(b) a peptide having the amino acid sequence of SEQ ID NO:2,

(2) mixing and oxidation of peptides derived from the host body or from the culture medium of the culture of the host body, and

(3) isolation of the product.



 

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1 tbl, 4 dwg, 6 ex

The invention relates to medicine and relates to humanized antibodies that recognize the verotoxin II, and producing their cell line

The invention relates to a method for cyclosporine And high purity by purification of the crude product containing cyclosporiasis complex by multi-step chromatography on silica gel at high load columns from 10 to 52%, using as eluent a mixture of toluene with acetone in an amount of from 10 to 30 vol.% or toluene with ethyl acetate in an amount of from 10 to 35 vol.%, cyclosporine And high purity with content cyclosporine L, U and D less than 0.05% and the content of cyclosporine and < 0,02% vol., industrial method of purification of cyclosporin a from a crude product containing complex cyclosporiasis

Cyclosporine // 2085589

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to biotechnology, in particular to hepatic cells production, and may be used in medical science. From the whole liver or resected part thereof, a cell population enriched with living cells of human liver, including hepatic stem cells/precursor cells, is obtained. Cell population contains functional hepatocytes and biliary cells expressing cytokeratin 19 (CK19), but not expressing albumin, as well as hepatic stem cells/precursor cells 9 to 13 mcm in diameter and expressing EP-CAM, CD 133 markers. Resulting cell population is used for hepatotherapy.

EFFECT: production of living population of hepatic cells sufficiently efficient for regeneration.

60 cl, 16 dwg

FIELD: medicine; biotechnologies.

SUBSTANCE: adenoviral vector carrying in the genome structure a human lactoferrin gene, administer into an allantois of the 9-10 day chicken embryoses. The subsequent planting is performed by egg incubation at temperature of 37°C within 70-75 hours. Then allocate the recombinant protein from the allantoic liquid of a chicken embryos.

EFFECT: depression of expenses and obtaining simplification of the recombinant human lactoferrin.

FIELD: medicine, biotechnologies.

SUBSTANCE: invention can be used for obtaining of the factor VII of blood coagulation. Derivatives of a polypeptide of the factor VII with amino-acid replacements Q250C, R396C and P406C are obtained or with Cysteinum attached to the S-end of native sequence of the factor VII. Obtain derivatives with use of transgene technologies in eucariotic cells-owners of mammals.

EFFECT: invention allows obtaining derivatives of the factor VII with the kept activity of the coagulative factor VII and with increased ability conjugate with PEG, in comparison with the natural form of a polypeptide.

20 cl, 2 dwg, 8 ex

FIELD: chemistry, biotechnology.

SUBSTANCE: invention relates to biotechnology. Method includes addition to fermentation broth or homogenate from E. coli of efficient quantity of ethacridinlactate solution for sedimentation of contamination from host-cells in conditions, when greater part of polypeptide remains dissolved, and isolation of heterological polypeptide from broth or homogenate.

EFFECT: simplification of target polypeptide purification and obtaining it with high degree purity.

23 cl, 15 dwg, 3 tbl

FIELD: chemistry, biotechnology.

SUBSTANCE: invention relates to field of biotechnology and preparation chemistry and can be used in biopharmacology and medicine. Cells of yeast P.pastoris are successively transformed by two different genetic structures, containing gene of human serum albumin (HAS) precursor. Obtained strain-producent is cultivated in nutrient medium. Recombinant HAS is isolated from cultural medium by clarification of said medium, as well as carrying out stages of successive centrifuging at 2000 and 10000 g, ultrafiltration, dialysis and cation-exchanging chromatography on column Source S. Target product represents eluate, including recombinant human serum albumin, 50 mM phosphate buffer, containing 400 mM of sodium chloride, with pH 9. Application of said iclaimed invention allows to extend arsenal of means, directed at production of recombinant HAS, and to obtain recombinant HAS in form of product, which in addition to recombinant HAS contains 50 mM phosphate buffer, containing 400 mM of sodium chloride and has pH 9.

EFFECT: extension of arsenal of means directed at obtaining recombinant HAS.

2 cl, 7 dwg

FIELD: chemistry.

SUBSTANCE: invention concerns biotechnology, specifically production of new polypeptides regulating carbohydrate metabolism, and can be used in medicine. New polypeptides reacting as both GLP-1 receptor agonists and glucagon receptor antagonists. Polypeptides and coding nucleic acids are used as components of pharmaceutical compositions for treatment of diabetes type 2 and metabolism disorders.

EFFECT: production of compounds providing effective glucose homeostasis for patients, suffering from carbohydrate metabolism disorders.

18 cl, 1 dwg, 4 tbl, 20 ex

FIELD: chemistry.

SUBSTANCE: invention relates to genetic engineering, namely, to obtaining inhibitors of TGF-β1 and can be used in medicine. Obtained peptides are capable of binding with transforming growth factor TGF-β1 and are potential inhibitors of biological activity of TGF-β1, binding with this cytokine directly. Peptides are obtained by recombinant method using transformed host-cell, by cultivating host-cell in conditions which ensure production of the said peptide, and its separation. The invention allows for efficiently treatment of diseases or pathological disorders connected with hyperexpression or disregulated expression of TGF-β1.

EFFECT: possibility to efficiently treat diseases or pathological disorders connected with hyperexpression or disregulated expression of TGF-β1.

12 cl, 6 dwg, 4 tbl, 4 ex

FIELD: technological processes; pharmacology.

SUBSTANCE: antagonist of human interleukine-1 receptor is prepared with the help of recombinant strain E. coli that contains plasmid, which provides production of this protein. For this purpose the strain is cultivated. Then from bacterial cells the target product is separated with application of three-stage chromatography and concentration on hydrophobic sorbent. At that the first and the third stage of mentioned chromatographic purification is performed on cation-exchanging resin, and in the second stage of chromatography the anion-exchanging resin is used.

EFFECT: application of invention allows to prepare antagonist of human receptor of high purity.

7 cl, 1 tbl, 4 ex

FIELD: technological processes; medicine.

SUBSTANCE: invention is related to preparation of recombinant analogues of human gamma-interferon and may be used in medicine for prophylactics and treatment of oncological diseases, neoplasms and inflammatory processes of humans. Highly pure genetically engineered analogue of human gamma-interferon - deltaferon with molecular mass of 16.2 kilo Daltons is produced by microbiological synthesis with further chromatographic purification on "KM"-sepharose - cationic-exchange sorbent with high linear speed of flow and suitable for preparative loads, at different pH values. On the basis of deltaferon, which contains by data of polyacrylamide gel-sodium dodecyl sulfate electrophoresis at least 98% of main substance in the form of monomer that possesses antiproliferative and anti-inflammatory activities inherent in gamma-interferon, medical product is prepared, which also includes low molecular polymer filler-stabiliser (reopolyglukine or polyvinylpyrrolidone) and salt buffer system with pH 7.0-7.1.

EFFECT: highly active and stable preparation of deltaferon is produced.

2 cl, 6 dwg, 1 tbl, 3 ex

FIELD: technological processes.

SUBSTANCE: method suggests protein of adipocyte plasma membrane, method of its preparation and complex based on this protein. Protein has molecular mass of 115 kilodaltons and has the ability to start-up tyr-phosphorylation of insulin-receptor proteins substrate in adipocyte. Method of protein preparation provides for adipocytes preparation out of rat, mouse or human tissues and plasma membranes extraction out of them. Then plenty of domains are isolated with high content of cholesterol hcDIG, which are treated with solution trypsin/NaCl. Centrifugation is done and protein fraction SDS-polyacrylamide gel is segregated with electrophoresis. Prepared protein fraction in amount of 115 kilodaltons is eluated from this gel. Complex constitutes activated protein and is formed during its combination with one of compounds from group: YCN-PIG, YMN-PIG, YCN or lcGcel.

EFFECT: protein in its activated form allows regulating glucose utilization bypassing insulin signal chain.

7 cl, 20 dwg, 1 tbl

FIELD: biotechnology, gene engineering, medicine, pharmaceutical industry.

SUBSTANCE: plasmide pBSH2EGF DNA providing expression of human epidermal growth factor (hEGF) in E.coli strains, containing polymerase T7 gene, under controlling of t7 promoter, associated with seeding site of lac-repressor, is synthesized. Also disclosed is method for production of human epidermal growth factor including inducible expression thereof in cells of Escherichia coli strain BL21(DE3) transformed with plasmide pBSH2EGF DNA followed by isolation of target product from inclusion bodies.

EFFECT: effective agent for gene engineering, medicine, pharmaceutical industry, etc.

2 cl, 3 ex, 4 dwg

FIELD: medicine, endocrinology, biochemistry, peptides.

SUBSTANCE: invention represents new peptides that act in vivo as stimulators of insulin secretion by pancreas beta-cells in glucose-dependent regimen. Such peptides as enhancers of insulin secretion stimulate insulin secretion by insula cells in rats in vitro and in vivo. Proposed peptides represent a new way for treatment of patients with reduced secretion of endogenous insulin, in particular, for treatment of diabetes mellitus type 2. In particular, invention represents polypeptide taken among the specific group VIP/PACAP-related polypeptides or their functional equivalents. Also, invention claims method for preparing both recombinant and synthetic peptides. The advantage of invention involves new peptides that can be used as stimulators of insulin secretion.

EFFECT: improved and valuable medicinal properties of peptides.

47 cl, 4 tbl, 10 dwg, 18 ex

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