Recombinant protein factor inhibiting lipogenesis, dna, its encoding, the expression vector, a recombinant strain of mammalian cells cos 1-producing factor inhibiting lipogenesis, its production method and a composition having activity of inhibiting lipogenesis

 

(57) Abstract:

The invention relates to biotechnology. The obtained recombinant protein factor inhibiting lipogenesis with a molecular weight of 23 kDa, installed the nucleotide sequence of the DNA fragment encoding a protein obtained. Protein obtained by the cultivation of mammalian cells, COS 1, transformed with the expression vector pSR-20-2, set containing the DNA fragment. Recombinant protein is used in a composition having activity of inhibiting lipogenesis. The invention is able to provide therapy and diagnosis of various cytopenias. 6 C. and 4 h.p. f-crystals, 3 ill., 9 table.

The technical field to which the invention relates

The invention concerns a previously unknown individual protein, which is able to suppress cell differentiation to adipocytes, are able to suppress lipoproteina the lipase and which (simultaneously or separately) is able to induce colony-stimulating factor. The present invention also concerns DNA, which encodes the mentioned protein, expression vector recombinant DNA comprising the DNA of the host, which is transformed according to the above vector expre the LASS="ptx2">

Prior art

It is well known that the existence of stromal bone marrow cells, which support the flow of homopause, in addition to hematopoietic receiver cells and hematopoietic factors, or, in other words, the hematopoietic microenvironment is necessary for the occurrence of disorders in a living organism. People in the moment of their birth the active haematopoiesis occurs in the bone marrow throughout the body. However, when hoopoes no longer be necessary with the growth of his or her bone marrow is gradually occupied by adipocytes, extending from the edges of the limbs as a result of proliferation of brain space. The bone marrow in which hoopoes, called red bone marrow and bone marrow, where he was replaced by adipocytes, is called the yellow bone marrow. When you accelerate the flow of homopause due to bleeding or hemolysis haematopoiesis resumes in yellow bone marrow (Kashiwamura, M. (1988), "IGAKUNO AYOMI (History of Medicine)", 146, 264-268).

Cells that have hematopoietic function in the hematopoietic microenvironment are largely preadipocyte. It is known that the differentiation of these cells into adipocytes, they lose Sanogo bone marrow is able to support the proliferation of hematopoietic receiver cells. However, the differentiation of these cells into adipocytes, this activity decreases (Kodama, H. et al. (1984), J. Cell. Physiol. 118, 233-240). It is known that blast cell megakaryocyte and mikrofalowe colonies can induction by co-cultivation of cells PA6 cells and murine bone marrow (Kodama, H. (1987), "YIKKEN IGAKU" (Experimental medicine)", 5, 826-830). Thus, a protein capable of inhibiting lipogenesis, affects preadipocytes and adipocytes, bone marrow, promotora their hematopoietic capacity, or, in other words, he promotiom their ability to induce leukocytes, macrophages and platelets (resulting in megakaryocytes).

In addition, reported that the amount of colony stimulating factor produced in preadipocyte cell line H-I/A, originating from murine bone marrow, decreased during the differentiation of these cells into adipocytes (Nakamura M. et al. (1985), Proc. Soc. Exp. Biol. Med., 179, 283-287).

As will be shown below, inhibiting lipogenesis factor that meets the present invention, the effect on the cell line H-I/A, accelerating the production of makrofagov colony-stimulating factor. In addition to stimulating effects in respect of monocytes and macrophages macrofamily colony-stimulating factor about Cloning, 8, 245-252). It is also known that macrofamily colony-stimulating factor shows the ability to act on monocytes, accelerating the production of their megakaryocyte potentiating factor. Moreover, from the data obtained in clinical therapy, it follows that macrofamily colony-stimulating factor promotes recovery from neutropenia and thrombocytopenia associated with conducting carcinomas chemotherapy (Motoyoshi, K. (1986), " GAN TO KAGAKURYOHO (Cancer and chemotherapy)", 16, 3531-3536).

Thus, inhibiting lipogenesis factor that meets the present invention, the effect on the bone marrow or peripheral preadipocytes and adipocytes, increasing their ability to maintain the flow of hemopoiesis.

Examples of proteins that have been known until the present invention suppresses adipocyte differentiation and lipoproteina the lipase in adipocytes, is a tumor necrosis factor (Price, S. R. et al. (1986), Arch. Biochem. Biophys., 251, 738-746 and Kawakami, M. et al. (1987), J. Biochem., 101, 331-338), interleukin-I (Beutler, A. B. and Cerami, A. (1985), J. Immunol., 135, 3969-3971), interferons (Keay, S. and Grossberg, S. E. (1980), Proc. Natl. Acad. Sci. USA, 77, 4099-4103, and Patton, J. S. et al., (1986), Proc. Natl. Acad. Sci. USA, 83, 8313-8317) and leukemia inhibitory factor (Mori, M. et al. (1989), Biochem. Biophys.SS="ptx2">

In addition, although before the priority date of the present invention and a message describing the isolation and expression of cDNA predecessor monkey interleukin-II, along with disclosure of the nature of the activity in relation to incentive plasmacytoma growth and activity in relation to education megakaryocyte colonies in relation to the supernatant culture fluids (Proc. Natl. Acad. Sci. USA. 87, 7512-7516 (1990)), there is no direct correlation with the present invention, because the message is not clearly made in the identification monkey interleukin-II, and vaguely confirmed the expression and activity of human interleukin-II.

Disclosure of the substance of the invention

The present inventors have uncovered a previously unknown individual protein, mainly with activity against suppression of cell differentiation to adipocytes, in relation to suppress leakage lipoproteidna lipase in adipocytes, but also in relation to the induction of colony-stimulating factor (factor inducing colony-stimulating factor), and thus were able to get large quantities mentioned protein using the method of genetic engineering, resulting in aura, inhibiting lipogenesis. As such, the above mentioned factor can be effectively used in the treatment and diagnosis of various types of anemia and other blood diseases. For example, the factor can be used in case of aplastic anemia, leukopenia caused by a toxin or radiation, infection caused by viruses, bacteria and parasites, cytopenia, occurring after bone marrow transplantation, and cytopenia caused by chemotherapy cancerstricken tools, etc. in Addition, the above mentioned factor can also be applied to the conservation of components transfused blood that now finds wide use. Moreover, in addition to these immunologic-enhancing effects mentioned factor can also be used to prevent obesity, preventing the normal functioning of the body, or as a therapeutic drug in the treatment of disease.

The term "cytopenia" used in the present invention, applies to all these diseases, and various diseases of the immune system, sometimes caused by these diseases. The term "cytopenias ameliorant (the breed)" is the generic name of these CLASS="ptx2">

In addition, at least one of the types of activity, including inhibition of morphological differentiation preadipocyte cells to adipocytes, suppression lipoproteidna lipase in adipocytes and the maintenance and promotion of the ability of preadipocytes to produce colony-stimulating factor can be named as "inhibiting lipogenesis activity and the protein having such activity, can be named as "inhibiting lipogenesis factor".

The present invention relates to the following aspects.

1. The invention relates to a protein obtained by the method of genetic engineering, which substantially does not contain other proteins of human origin, has activity against inhibition of lipogenesis and has the properties listed below:

1) has an apparent molecular weight of about 23000 established by electrophoresis in polyacrylamide gel with sodium dodecyl sulfate under reducing conditions using a 12.5% gel;

2) it has N-terminal amino acid sequence in the form of a number of amino acids 1 to 10, indicated in sequence No. 2 of the sequence listing, and

3) it undergoes Noah chromatography under the following conditions:

(a) in column used substance CM-toyopearl 650M (2,2 20 cm, firm "Tosoh Corporation"),

b) for the preparation of an eluting buffer solution using solution a (solution of boric acid with a concentration of 10 mm sodium hydroxide (pH 9,0) content 13 mm potassium chloride) and solution B (solution A containing 300 mm sodium chloride),

C) the flow rate is 3 ml/min,

g) the concentration gradient in the conduct of said chromatography is set linearly changing with the transition for 50 min from A solution with a concentration of 100% to solution B with a concentration of 100%.

2. The invention relates to a protein obtained by the method of genetic engineering, which substantially does not contain other proteins of human origin, has activity against inhibition of lipogenesis and consists of an amino acid sequence composed of amino acid numbers 1 - 178 with the formation of the sequence shown in the amino acid sequence 2 of the sequence listing, or is a substance with the same properties as mentioned protein in which one or more amino acid residues deleted, inserted or substituted in one or more sory substantially does not contain other proteins of human origin consists of an amino acid sequence composed of amino acid numbers 1 - 178 with the formation of the sequence shown in the amino acid sequence 2 of the sequence listing, and has activity against inhibition of lipogenesis.

4. The invention relates to a protein on the PP. 1, 2 or 3, containing the hydrogen atom or the group Met (methyl group) in the N-terminal position.

5. The invention relates to DNA coding for a protein on PP. 1, 2, 3, or 4.

6. The invention relates to DNA coding for the protein under item 1 or p. 2 consisting of the nucleotide sequence with nucleotides from 81 to 614 with the formation of the sequence shown in the nucleotide sequence of the number 1 of the sequence listing, or a sequence having equivalent properties.

7. The invention relates to DNA coding for the protein under item 1 or p. 3 consisting of the nucleotide sequence with nucleotides from 81 to 614 with the formation of the sequence shown in the nucleotide sequence of the number 1 of the sequence listing.

8. The invention relates to DNA with a group of ATG in the 5'-terminal position of the DNA by the PP. 5, 6 or 7.

10. The invention relates to a host transformed by a recombinant expression vector DNA under item 9.

11. The invention relates to a method of producing a protein, which comprises culturing a host transformed under the influence of the expression vector recombinant DNA comprising DNA coding for a protein on PP. 1, 2, 3 or 4, in which case mentioned DNA can be expressed and replication, and the allocation of the mentioned protein from the cell extract or solution culture.

12. The invention relates to a pharmaceutical composition containing a therapeutically effective amount of a protein based on PP. 1, 2, 3 and (simultaneously or separately) 4 together with a pharmaceutically acceptable carrier or excipient.

13. The invention relates to cytopenias ameliorant containing a therapeutically effective amount of a protein based on PP. 1, 2, 3 and (simultaneously or separately) 4 together with a pharmaceutically acceptable carrier or excipient.

14. The invention concerns a drug against obesity, containing a therapeutically effective amount of a protein based on PP. 1, 2, 3 and (simultaneously or separately) 4 together with a pharmaceutically acceptable suppression of differentiation preadipocytes on adipocytes by administration of a therapeutically effective amount of a protein based on PP. 1, 2, 3 and (simultaneously or separately) 4 patients with cytopenia.

16. The invention relates to a therapeutic or preventive method of suppressing the differentiation of preadipocytes on adipocytes by administration of a therapeutically effective amount of a protein based on PP. 1, 2, 3 and (simultaneously or separately) 4 to patients suffering from obesity.

Preferably, the protein responsible present invention, represented the protein under item 2 or p. 3, and better under item 3.

While it is desirable that DNA was responsible DNA coding for the protein under item 2 or p. 3, preferably a DNA coding for the protein under item 3.

Determination of protein by p. 1 may be different.

17. The invention relates to a protein obtained by the method of genetic engineering, which substantially does not contain other proteins of human origin, has activity against inhibition of lipogenesis and has the properties listed below:

1) it detects the apparent molecular weight of about 23000 established by electrophoresis in polyacrylamide gel with sodium dodecyl sulfate under reducing conditions using a 12.5% gel;

2) it has N-terminal amino acid posledovati in the region from 220 to 290 mm in the case of weak cation exchange chromatography in the following conditions:

(a) in column used substance CM-toyopearl 650M (2,2 20 cm, firm "Tosoh Corporation"),

b) for the preparation of an eluting buffer solution using solution a (solution of boric acid with a concentration of 10 mm sodium hydroxide (pH 9,0) content 13 mm potassium chloride) and solution B (solution A containing 300 mm sodium chloride),

C) the flow rate is 3 ml/min,

g) the concentration gradient in the conduct of said chromatography is set linearly changing with the transition for 50 min from A solution with a concentration of 100% to solution B with a concentration of 100%.

Determination of protein in p. 2 may be different.

18. The invention relates to a protein obtained by the method of genetic engineering, which substantially does not contain other proteins of human origin, has activity against inhibition of lipogenesis and consists of amino acid sequence with the following General formula (I), provided at the end of the description, or applies to substances with the same properties as mentioned protein, in which one or more amino acid residues deleted, inserted or substituted in one or more sites mentioned protein.

Description Bel is which substantially does not contain other proteins of human origin has activity against inhibition of lipogenesis and consists of amino acid sequence with the following General formula (I), provided at the end of the description.

Description DNA on p. 6 may be different.

20. The invention relates to DNA coding for the protein under item 17 or p. 18 consisting of a nucleotide sequence according to the following General formula (II), provided at the end of the description, or the nucleotide sequence with equivalent properties.

Description DNA under item 7 may be different.

21. The invention relates to DNA coding for the protein under item 17 or p. 19, leading to the formation of the nucleotide sequence with the following General formula (II), provided at the end of the description.

Acid DNA that meets the present invention, receive, for example, by preparing mRNA encoding a protein having activity against inhibition of lipogenesis, making it from mammalian cells, which have the ability to produce the aforementioned protein, with subsequent conversion of mRNA into double-stranded cDNA using the known method. Although animal cells, which serve as the source of the above-mentioned mRNA that meets the present invention, and before the nd Handa, H. (1985), Proc. Natl. Acad. Sci. USA, 82, 3477-3480), can be used not only tumor cell strains, but can also be used in cells and tissue that can be isolated from mammals, or other established cell lines.

Although when carrying out the extraction of mRNA and can be used guanidine thiocyanate hot phenol method, or guanidine thiocyanates-guanidine hydrochloric acid method, preference should be given guanidine thiocyanato chloritization method.

Since it is known that a large part of the mRNA present in the cytoplasm of eukaryotic cells, has a poly-A sequence at the 3'end region, mRNA adsorb in oligo (dT)-cellulose column, with the advantage of using this feature, and then purified by elution. This mRNA may be further subjected to fractionation by centrifugation in a density gradient of sucrose, etc.

To be sure that the thus obtained mRNA encodes a protein having activity against inhibition of lipogenesis, mRNA can be translated into protein by checking its physiological activity or protein can be a way to identify the t to be carried out by injection of mRNA in oocytes of the African tree frog (Xenopus Iaevis), that translates mRNA into protein (Gurdon, J. B. et al., (1972), Nature, 233, 177-182), or by using the translational reaction, in which case use reticulocyte listnow system or a system with an extract from wheat seeds (Schleif, R. F. and Wensink, P. C. (1981), "Practical Method in Molecular Biology", Springer-Verlag, NY).

Test for activity against inhibition of lipogenesis can be carried out by measuring the activity in relation to morphological changes-reduction transition preadipocytes in adipocytes, which is achieved by using, for example, a mouse cell line 3T3-LI and so on, or by measuring lipoproteidna lipase activity (Beutler, B. A. et al. (1985), J. Immunol., 135, 3972-2977).

After synthesis of single-stranded cDNA from the thus obtained mRNA by reverse transcriptase, where mRNA plays the role of a matrix of single-stranded cDNA synthesized double-stranded cDNA. Although the methods that can be used for this synthesis, and include SI-nukleazy way (Efstratiadis, A. et al. (1976), Cell, 7, 279-288), the method Landa (Land, H. et al. (1981), Nucleic Acids. Res., 9, 2251-2266) and the way About. Jun SW (Yoo, O, J. et al. (1983), Proc. Natl. Acad. Sci. USA, 79, 10449-1053), preference should be given to the method of Okayama-Berg (Okayama, H. and Berg, P. (1982), Mol. Cell. Biol., 2, 161-170), if govori plasmids injected into the culture of Escherichia coli, for example, in strain DH5 that is necessary for transformation mentioned owner. The desired recombinant can be selectively obtained with the use of tetracycline resistance or ampicillinbuy immunity as an index. For example, when the host cell is a culture of Escherichia coli, transformation of host cells can be produced using the method of Hanahan (Hanahan, D. (1983), J. Mol. Biol., 166, 557-580). More precisely, the transformation can be carried out by adding the aforementioned recombinant DNA to the competent cells prepared in the presence of CaCl2, MgCl2or RbCl. Incidentally, the phage vector lambda strain, etc. can be also used as a vector instead of plasmids.

For breeding strain with the ability of DNA-coding in relation to the desired factor, inhibiting lipogenesis, from a transformed host, obtained as indicated above, can be used in different ways, listed below.

1. The selection method using a synthetic oligonucleotide samples

In the case when the amino acid sequence of the desired protein is fully or partially known (some area of rebuildability), synthesize an oligonucleotide probe that corresponds to the above amino acid (nucleotide sequence may be constructed on the frequency of codon usage, or it may consist of a large number of nucleotide sequences, where the nucleotide sequence can be mixed; in the latter case, the number of the mentioned sequences can be reduced by incorporating inosine). This oligonucleotide is used as a sample (labeled with phosphorus-32 and sulfur-35) by hybridization using denatured DNA of the transformed strain, which was subjected to immobilization on nitrocellulose filter, and then conduct the search and selection of the resulting positive strain.

2. Selection using samples prepared by conducting polymerase chain reaction

In the case when the amino acid sequence of the target protein is fully or partially known, synthesize oligonucleotides helices one and opposite directions, corresponding to a protein of the above-mentioned amino acids. Polymerase chain reaction (Saiki, R. K. et al., (1988), Science, 239, 487-491) is conducted using these oligonucleotides for amplification of the DNA fragment encoding the requirement of the e produce a factor inhibiting lipogenesis, or genomic DNA can be used for DNA templates used in this way. After the introduction of labels in the form of phosphorus-32 and sulfur-35 prepared in this way DNA fragment of the desired clone is subjected to selection, conducting coloniaivu hybridization, or blaskova hybridization using a DNA fragment labeled as samples.

3. Selection with production factor, inhibiting lipogenesis, when using other animal cells

The strain of acid cDNA that encodes a desired factor, inhibiting lipogenesis, choose from the original transformed cell strains, or cultivating the transformed strain, amplificare gene transfective gene in animal cells (in this case, utilize semireflective the plasmids containing transcriptional promoter region, or a plasmid, which can be introduced into an animal cell chromosome), thus producing a protein encoded gene, and measuring the inhibitory lipogenesis activity in the supernatant liquid culture or in a cell extract, or, as an option, detecting inhibiting lipogenesis factor, using the antibody suitable for this factor.

4. Selecci is proactively introducing the cDNA into the expression vector, induce protein within transformants and detecting transformant producing factor, inhibiting lipogenesis, using antibody to factor that inhibits lipogenesis, and the secondary antibody to the above-mentioned antibodies.

5. The method using selective hybridization PA system

In this method, after applying spots of cDNA obtained from transformants on nitrocellulose filter or something similar, which is necessary for the hybridization of mRNA or of cells producing factor, inhibiting lipogenesis, mRNA associated with the cDNA is subjected to dissociation and extraction. The extracted mRNA is then either Inuktitut in protein translation system such as the oocytes of the African tree frog, or broadcast in protein using the free cell system such as krolikovs reticulocyte lysate or wheat grain extract, what are you doing to test the activity of this protein in relation to inhibition of lipogenesis, or, as an option, the factor inhibiting lipogenesis, can be detected by using antibodies to the above-mentioned factor, which ensures the selection of the desired strain.

As will be described in Neu samples designed to reflect the existence of AUUUA-motif (Shaw, G. and Kamen, R. (1986) Cell, 46, 659-667) present along with acids mRNA of the cytokines that is done before applying the above methods required of transformant can also be selected by using the above method 3 in relation to the selected positive transformant, or rather, the production of inhibiting lipogenesis factor in other animal cells, followed by selection.

A method of obtaining a DNA that encodes a factor inhibiting lipogenesis, from the thus obtained transformants of interest may be in accord with the known method (Maniatis, T. et al., (1982), "Molecular Cloning - A Laboratory Manual", Cold Spring Harbor Laboratory, NY). For example, this can be achieved by selection of plasmid DNA from cells and cDNA selection-region of the above-mentioned plasmid DNA.

The sequence present in the DNA obtained in this way can be determined, for example, the use of chemical modified method of Maxima-Gilbert (Maxam, A. M. and Gilbert, W. (1980), "Methods in Enzymology", 65, 499-559) or using the method of completing dideoxynucleotide chain through the application of phage M13 (Messing, J. and Vieira, J. (1982), Gene, 19, 269-276).

And decibe into an appropriate vector DNA, you can transform the host cells in other prokaryotes and eukaryotes. Moreover, through the introduction of an appropriate promoter sequence associated with the expression of a gene in these vectors, the gene may be expressed in appropriate host cells.

Examples of prokaryotic cell owners are, for example, culture of Escherichia coli and Bacillus subtilis. In order to effect expression of a gene of interest in these host cells, the host cells can be transformed with the participation of replicon selected from particles that is compatible with the owner, or, in other words with a plasmid vector containing a replication origin and a regulatory sequence. In addition, it is desirable that the vector had the sequence, is able to give the transformed cells with a selective expression character (phenotype).

For example, strain K12 culture of E. coli is often used to obtain the host, whereas the plasmid pBR322 and pUC typically used for vector. However, the present invention is not limited to the use of these formations, and can be used any of various known types of bacterial strains and vectors. Examples of promoters are tryptophan is ramotar bacteriophagous origin and promoter in the form factor TU with straightened polypeptide chain, in the culture of Eschirichia coli. Any of these promoters can be used in the production of inhibiting lipogenesis factor that meets the present invention.

For example, the strain 207-25 it is desirable to use for the culture of Bacillus subtilis and pTUB228 (Ohmura, K. et al. (1984), J. Biochem., 95, 87-93), etc. to use for the vector. However, the present invention is not limited to these entities. Often for promoter use regulatory sequence of a-amylase gene of a culture of Bacillus subtilis. Secretion outside of the bacteria may be further carried out by using a DNA sequence that encodes a signal peptide sequence at-amylase, as necessary.

For eukaryotic host cells are cells of vertebrates, insects, yeast, etc. Though COS-cells, which are cells of the monkey (Gluzman, Y. (1981), Cell, 23, 175-182), and the strain with insufficient content reductase Polevoy acid from ovarian cells of the Chinese hamster (Urlaub, G. and Chasin, L. A. (1980), Proc. Natl. Acad. Sci. USA, 77, 4216-4220), etc. are often used as vertebrate cells, the present invention is not limited to these cells.

Vectors with the promoter, located above the gene must be expressed, RNA with alsomany for vector expression in vertebrate cells. This vector can also be replicating the beginning, as necessary. The expression vector can be illustrated by the formation of pSV2dhfr with early SV40 promoter (Subramani, S. et al. (1981). Mol. Cell. Biol., 1, 854-864). However, the present invention is not limited to the aforementioned expression vector.

Meanwhile, yeast culture in a typical use case for eukaryotic host microorganism. In particular, it is desirable to use yeast culture type Sheremetiev, such as Saccharomyces cerevisiae. As expression vectors for eukaryotic microorganisms such as yeast, can be, for example, used alcoholic dehydrogenase gene promoter (Bennetzen, J. L. and Hall, B. D. (1982), J. Biol. Chem., 257, 3018-3025) or kislotnyi fosfatazy gene promoter (Miyanohara, A. et al. (1983), Proc. Natl. Acad. Sci. USA., 80, 1-5).

As host cells for the expression vector, if, for example, to talk about COS-cells, can be used vector capable of Autonomous undergo replication in COS-cells with SV40-start replication, transcription and RNA-binding site. The above-mentioned expression vector may be introduced into COS cells using DEAE-dextranomer method (Luthman, H. and Magnusson, G. (1983), Nucleic Acids Res., 11, 1295-1308), calc is ω (Neumann, E. et al. (1982), EMBO J., 1, 841-845), thus obtaining the desired transformed cells. Meanwhile, in the case of ovarian cells of the Chinese hamster cell host transformed cells that stably produce the factor, inhibiting lipogenesis, can be obtained by cotransfection vector that can Express Neogene functioning, as in the case of drug-resistant marker G418: such as pRSVneo (Sambrook, J. et al. (1989), "Molecular Cloning-A Laboratory Manual", Cold Spring Harbor Laboratory, NY) or pSV2-neo (Southern, P. J. and Berg, P. (1982), J. Mol. Appl. Cenet. 1, 327-341), along with the expression vector, followed by selection for G418-resistant colonies.

Required of transformant obtained in this way can be cultivated in the usual way, and thus the factor inhibiting lipogenesis, can be produced either inside or outside the cells. The environment used to carry out the above-mentioned cultivation, can be successfully selected from those different types, which are generally taken into account the nature of the host cell. Examples of environments that can be used when the above-mentioned COS-cells are used as host cells are medium RPMI-1640 and minimal nutrient medium Needle (Eagle), modifica cow fetus.

Thus, the factor inhibiting lipogenesis, which is produced either inside or outside the cells of the transformants can be isolated and purified in a variety of known methods of allocation used with regard to physical properties, chemical properties, etc. factor, inhibiting lipogenesis. Practical implementations of the above methods include the processing of standard protein precipitants, ultrafiltration, various types of liquid chromatography, such as chromatography on a molecular sieve (gel filtration), adsorption chromatography, ion exchange chromatography, chromatography on affinity and high-performance liquid chromatography, dialysis, and the use of these methods in combination.

According to the above method, the desired factor, inhibiting lipogenesis, can be easily produced on an industrial scale, with high yield and with high purity. Various physical properties of the recombinant enzyme inhibition of lipogenesis factor that meets the present invention, which is obtained in this way are described in the following examples. This factor is useful in various fields, mentioned earlier, which follows from its biological activity.

Examples of such a protein that exhibits activity against inhibition of lipogenesis, are proteins consisting of 178 amino acids with amino acid Pro (Proline), which represents the amino acid number 1 in the amino acid sequence shown in sequence No. 2 of the sequence listing, and is N-terminal, or, in other words, this includes those proteins that are thought to represent factors clearly show activity against inhibition of lipogenesis. Incidentally, this protein is oblist sodium under reducing conditions.

In addition to the above, DNA encoding the aforementioned developed factor with activity against inhibition of lipogenesis, is also an example of DNA that meets the present invention. More preferably, the aforementioned DNA represented DNA with numbers from 81 to 614 from the nucleotide sequence indicated by the sequence number 1 of the sequence listing.

In the General case, consider that genes in eukaryotes exhibit polymorphism, of the type known in the case of gene interferon, and so on (see, e.g., Nishi, T. et al. (1985), J. Biochem., 97, 153 to 159). When the manifestation of this polymorphism in the form of a substitution of one or several amino acids appear when the different amino acids remain the same, despite the change in nucleotide sequence.

Even those proteins that have one or more amino acid residues deleted, entered or substituted at least at one site amino acid sequence of the precursor of factor inhibitory activity against lipogenesis, consisting of amino acid numbers from -21 to +178 the amino acid sequence specified by the sequence number 2 in the sequence listing, and so is the Windows from +1 to +178 the amino acid sequence, the specified sequence number 2, may also have activity against inhibition of lipogenesis. These proteins are referred to here as the equivalent factor with inhibitory activity against lipogenesis.

For example, it is known that the protein obtained by converting the nucleotide sequence corresponding to cysteine gene interleukin-2, the nucleotide sequence responsible Serino support their IL-2 activity (Wang, A et al., (1984), Science, 224, 1431-1433). For this reason, while such proteins or natural or synthetic origin have activity against inhibition of lipogenesis, they all fall under the present invention.

In addition to the above, DNA encoding these proteins consisting of the nucleotide sequences with equivalent properties fall under the present invention.

Different types of DNA that meets the present invention can be manufactured nukleinovokisly by chemical synthesis using conventional methods, such as postit-Threepenny method (Hunkapiller, M. et al., (1984),

Nature, 310, 105-111), based on data about the above-mentioned factor, inhibiting lipogenesis.

Incidentally, for the, and it can be determined according to conventional methods (Grantham, R. et al., (1981), Nucleic Acids Res. , 9, 43-74) by considering, for example, the frequency of codon usage have taken the owner. Partial change codons in these nucleotide sequences may be produced in the usual way, such as specific site mutagenesis (Mark, D. F. et al. (1984), Proc. Natl. Acad. Sci. USA, 81, 5662-5666), in which case use the sample consists of the synthesized oligonucleotides that encode the desired change.

Brief description of drawings

In Fig. 1 shows the map of enzyme restrictions from cDNA that encodes a factor inhibiting lipogenesis, which designation ORF characterizes the region, allowing the reading.

In Fig. 2 schematically shows the structure of the plasmid, which expresses the factor inhibiting lipogenesis, in accordance with example 6 of the present description.

In Fig. 3 shows the image obtained in the case of the analysis by carrying out electrophoresis in polyacrylamide gel with sodium dodecyl sulfate, and the picture obtained by carrying out spot analysis Western (Western) using used for preparing antipeptide antibodies under reducing conditions with respect to the total proteins.

The best option of carrying out the invention

The present invention will be described more specific by looking at examples, not limiting the scope of the invention, and the preparative examples. First of all it should be noted that the method applied to measure activity against inhibition of lipogenesis, will be explained in the comparative examples.

Comparative example

The method used in the present invention for measuring the activity of factor inhibiting lipogenesis, is the following. Cells used for measurement of, is an embryonic fibroblastoma cells mouse line ZTZ-LI (Green, H. and Kehinde, O. (1974), Cell, 1, 113-116), which were purchased from the American type culture collection of microorganisms. Culture all cells were subjected to temperatures in the 37oC when placed in a humidified gas mixture containing 10% carbon dioxide and 90% air. Cell culture were processed using medium A (DMEM (minimum nutrient medium Needle, modified Dulbecco) with glucose 4.5 g/l, which produces a firm "Gibco"), containing 10% immobilizovannoi bovine serum p is opacity induced according to the method, described by Rubin and others (Rubin, C. et al. (1978), J. Biol. Chem., 253, 7570-7578).

The dimension associated with the inhibiting effect of morphological changes of the cell line ZTZ-on L1 adipocytes

The cultivation of the cell line ZTZ-L1 conducted by suspendirovanie mentioned cells in medium A at density 1,0104cells in 1 ml, and then the pipette was collected 0.5 ml cell suspension and this amount was placed in a recess in the plate, which was recesses 48 (production firm "Costar"). Cells became suitable for draining after three days of cultivation. The medium was then replaced by fresh medium A, and after culturing for two days, the medium was replaced with medium B, inducing lipogen differentiation (DMEM (minimum nutrient medium Needle, modified Dulbecco, glucose 4.5 g/l) containing 10 mm HEPES (pH of 7.2), 3% serum fetal cow, 5 μg/ml bovine insulin (manufactured by Sigma), 8 µg/ml Biotin (manufactured by Sigma), 4 mcg/ml Pantothenic acid (manufactured by Sigma), and 1.0 μm dexamethasone (manufactured by Sigma) and 0.5 mm isobutylmethylxanthine (the production company "Aldrich")). At this time, were simultaneously added to the sample. Later Wednesday, the replacement is of ASCA, made for the first time, the medium was replaced with medium C, necessary to confirm the existence of adipocytes (DMEM (minimum nutrient medium Needle, modified Dulbecco, glucose 4.5 g/l) containing 5% immobilizovannoi serum fetal cows, 10 mm HEPES (pH of 7.2) and 100 ng/ml bovine insulin).

After culturing in medium C for two days the cells were fixed with 5% formaldehyde solution. Droplets of fat that have accumulated in the cells and cell nuclei, respectively, were stained with oil red O dye and hematoxylin. After the invasion of macrophages counted the number of cells in which there was red staining droplets of fat that was seen on the photos that did, along with counting the number okleinowany cells, stained with hematoxyline. Speed lipogenic differentiation was then calculated by the formula below:

< / BR>
Incidentally, the fixation of the cells, staining with oil red O dye and hematoxylineosin staining was performed in accordance with the requirements of experimental management (Mitomo, and Y. Takayama, S. "RINSHOKENSAKOZA (clinical Testing Seminar)", Vol. 12, "Pathology" (1982), Ishiyaku Publishing).

Measurement of activity in respect of odavlennuliine and other (Beutler B. A. et al., (1985), J. Immunol., 135, 3972-3977). Fat cell adipocytes transformed cell line ZTZ-LI was prepared by the method described in the section titled "Measurement associated with the inhibiting effect of morphological changes of the cell line ZTZ-on L1 adipocytes". However, it was not added to the sample during the induction of differentiation into adipocytes. The medium was then replaced with fresh medium C, and there was added to the sample. After culturing for 18 h, the medium was removed and cells washed twice PBS(-) (phosphate-saline buffer solution; production company "Nissui Saaki"). Then in each recess was added 300 μl of medium D (DMEM (minimum nutrient medium Needle, modified Dulbecco, glucose 4.5 g/l) containing 5% immobilizovannoi serum fetal cows, 10 mm HEPES (pH of 7.2), 100 ng/ml bovine insulin and 10% on 1 Il sodium heparin (manufactured by "Novo industries")). After culturing for one hour to collect 100 µl of the supernatant from the culture, and the liquid used for measurement lipoproteidna lipase activity. Measurement of the activity in relation to suppress lipoproteidna lipase was carried out on three samples of each sample, after which three maintenance by way of Nelson-Ile and SOCA (Nilson-Ehle, P. and Schotz, M. C. (1976), J. Lipid Res., 17, 536-541). For this purpose, 100 l of the supernatant liquid culture, prepared by the method described above, was mixed with an equal volume of substrate solution (13 mm glycerate[9,10(n)-3H]oleic acid (51.8% of cbec/mmol, manufactured by "Amersham"), 1.3 mg/ml L-phosphatidylglycerol (manufactured by Sigma), 20 mg/ml bovine serum albumin (manufactured by Sigma), 135 mm Tris-hydrochloride (Tris-HCl, pH 8,1, manufactured by Sigma), 16,5% (volume by volume) of glycerol and 16.5% (volume by volume) immobilizovannoi serum fetal cows) and left to react at 37oC for 120 minutes the Reaction was stopped by the addition of 1.05 ml of 0.1 M carbonatecoreg solution of boric acid (buffer solution with pH of 10.5) and 3.25 ml of a mixture of methanol, chloroform and heptane, taken by volume in the ratio 141:125:100. After vigorous stirring, the reaction mixture was centrifuged at 3000 G for 15 minutes by tritium in water-methanol layer was performed using a liquid scintillation counter. Lipoproteine lipase activity in 1 unit was defined as the amount of activity produced 1 μmol of fatty acid per 1 min. by the way, 13 μm glycerate[9,1 UP>3H]oleic acid (manufactured by "Amersham", 370 GBC/mol) triolein (manufactured by Sigma), and then carried out the purification by chromatography on a column of silica gel.

Example 1

The selection of poly(A)-+RNA from KM-102 cells

Cell line KM-102 were cultured for 36 plastic cups for cultivation (15 cm diameter) using modified minimum nutrient medium Iscove (Iscove) (production firm "Boeringer-Mannheim) containing 10% serum fetal cows. After growing the cells to confluence were added formalparameterlist and calcium ionophor A23187, bringing their concentrations up to 10 ng/ml and 0.2 μm. Cultivation was then continued, and during this period the cells 12 cups at certain points in time literally guanidiniocarbonyl solution (4 M guanidinoacetate, 1% sarkosyl, 20 mm ethylenediaminetetraacetic acid, 25 mm sodium citrate (pH 7.0), 100 mm 2-mercaptoethanol and 0.1% antifoam A) that did respectively after 3, 6 and 14 hours; then the solution was removed.

The selection of poly(A)+-RNA is mainly carried out as described in the manuals (Maniatis, T. et al. (1982) "Molecular Cloning-A Laboratory Manual", pp. 196-198). More precisely, the selection is conducted is poured, that was done several times using a syringe of 10 ml, respectively provided with needles gauge 21G. The solution containing 5,7 M chloride CESI and 0.1 M ethylenediaminetetraacetic acid (pH 7.5) was added in an amount of 3 ml each pollonaruwa centrifuge tube, compatible with rotary basket centrifuge type RPS-40T, manufactured by "Hitachi Koki", then the above lysed cell solution was added, pouring from the top to the previously added to the solution, which made up until the tubes are not filled. After centrifugation of these solutions for 18 h at 20oC at a speed of 30,000 rpm, the resulting precipitation was dissolved in 400 μl of distilled water, and then spent the precipitation with ethanol; and there was added an equal volume mixture of chloroform with 1-butanol (taken in the ratio 4:1), after which it was carried out by stirring. The aqueous layer was then extracted, making separation by centrifugation. After deposition from aqueous ethanol layer deposited sediments were dissolved in 600 μl of distilled water than was provided for obtaining total RNA. From each sample subjected to PMAA23187 for 3, 6 and 14 h were obtained in each case approximately 4.5 mg of total RNA.

This amount is Gali chromatography on oligo(dT)cellulose column, what was achieved the purification of poly(A)+-RNA. In other words, after dilution total RNA in the adsorption buffer solution (0.5 M sodium chloride, 20 mm Tris-HCl (pH 7.5), 1 mm ethylenediaminetetraacetic acid, and 0.1% sodium dodecyl sulfate) and heating at 65oC for 5 min, the resulting solution was applied to an oligo(dT)cellulose column (manufactured by Pharmacia, type 7) filled with the same solution. Acid poly(A)+The RNA was recovered by elution with eluent (10 mm Tris-HCl (pH 7.5), 1 mm ethylenediaminetetraacetic acid and 0.05% sodium dodecyl sulfate), which received 100 µg of poly(A)+-RNA.

Example 2

Getting library data for cDNA

Library data for cDNA was obtained according to the method of Okayama-Berg. Specifically, 5 μg of poly(A)+-RNA and 24 units of reverse transcriptase were brought into interaction in the reaction mixture (50 mm Tris-HCl (pH 8,3), 8 mm magnesium chloride, 30 mm KCl, 0.3 mm of dithiothreitol, 2 mm dATP, 2 mm DSTF, 2 mm dCTP, 2 mm dTTP, 10 µci [-32P] dCTP and 1.4 μg of the vector sample DNA (3'-oligo(dT)-terminal pcDV-1; manufactured by Pharmacia) when 42oC for 60 minutes

After the interruption of the interaction by adding 2 μl of 0.25 M ethylenediaminetetraacetic acid, and 1 ál 1 is forma. To the aqueous layer obtained by centrifugation, was added 20 μl of 4 M ammonium acetate and 80 μl of ethanol, after which the solution was cooled at -70oC within 15 minutes Later, the precipitate was collected by conducting separation by centrifugation, and the precipitate was washed with 75% ethanol, and then dried under reduced pressure.

Then the dried residue was dissolved in 15 µl of the end transferase reaction mixture (140 mm cacodylate potassium, 30 mm Tris-HCl (pH 6,8), 1 mm CoCl2, 0.5 mm dithiothreitol, and 0.2 μg of poly-A (polyadenylic acid) and 100 mm dCTP). After keeping the reaction mixture at 37oC for 3 min there was added 18% deoxynucleotidyl transferase and the mixture was allowed to react for 5 minutes Then after the interruption of the interaction by adding 1 ál of 0.25 M ethylenediaminetetraacetic acid, and 0.5 ál of 10% sodium dodecyl sulfate protein was extracted with a mixture of phenol and chloroform. After centrifugation of the reaction mixture, the aqueous layer was separated and there was added 15 μl of 4 M ammonium acetate and 60 μl of ethanol, and then carried out a thorough mixing. After cooling the mixture at -70oC for 15 min thus obtained precipitate was collected by centrifugation.

The residue is then dissolved in 10 the deposits and 1 mm dithiothreitol), and there was added 2.5 units restrictive enzyme HindIII, so that the splitting of the sediment, which was carried out at 37oC for approximately 1 h after removal of proteins by treatment with a mixture of phenol and chloroform were precipitation with ethanol. After cooling the reaction mixture at -70oC for 15 min the precipitate was collected by centrifugation and dissolved in 10 μl TE buffer solution (10 mm Tris-HCl (pH 7.5) and 1 mm ethylenediaminetetraacetic acid). To 9 ál of reaction mixture (10 mm Tris-HCl (pH 7.5), 1 mm ethylenediaminetetraacetic acid, and 100 mm sodium chloride) was added 1 µl of this solution, which was added 10 ng of oligo(dG)-terminal binding DNA (3'-oligo(dG)-leaf binder of the type pL-1 Hind 3; manufactured by Pharmacia), and then was heated at 65oC for 5 minutes the Reaction mixture was then left to stand at 42oC for 30 minutes After cooling the reaction mixture in water with ice, there was added 10 μl of 10x-ligase buffer solution (10 mm ATP 660 mm Tris-HCl (pH 7.5), 66 mm magnesium chloride and 100 mm dithiothreitol), 78 μl of distilled water and 8 units of T4-DNA-ligase, after it was left to stand for 12oC at night.

Then add 10 ál of 1 M KCl, 1 unit of ribonuclease H, 33 units of DNA polymerase 1, 4 units of DNA ligase is mine with a concentration of 50 µg/µl, and the mixture was stirred for 12oC for 1 h and then at 25oC for 1 h After a five-fold dilution of the reaction mixture with distilled water strain DH5 culture of Escherichia coli immediately transformed, making it by way Hanahan (Hanahan, D. (1983), J. Mol. Biol. 166, 557-580), resulting in the received library data for cDNA cell line KM-102.

Example 3

Preparation of oligonucleotide samples

The oligonucleotide of 15 bases of the 5'-TAAATAAATAAATAA-3', designated as ATT-3, chemically synthesized based on the sequence AUUUA stored in the untranslated 3'region of the mRNA of cytokines. The synthesis was performed using an automatic DNA synthesizer type 380B produced by the firm "Aplaud of Biosystems" that was done according to the procedures given in the manual. This method is based on the principle described by Carterton and others (Metteucci, M. D. and Caruthers, M. H. (1981), J. Am. Chem. Soc. 103, 3185-3191), and he called fostamatinib way. After synthesis 15 nucleotides they were released by separation from the base resin, and the resultant mixture was subjected to drying by freezing, the result that was obtained by oligonucleotide powder. The powder was dissolved in distilled water and until ispolzovat

On the nitrocellulose filter were fixed 6500 recombinant strains acids DNA taken from the above-mentioned cDNA libraries for cell line KM-102, which was done according to the method described by Grunstein and Hogness (Grunstein, M. and Hogness, D. S. (1975), Proc. Natl. Acad. Sci. USA, 72, 3961-3965). Next, coloniaivu hybridization was performed after injection of the label at the 5'-end of the test (ATT-3) using phosphorus-32, what did the usual procedure (see manual "Molecular Cloning-A Laboratory Manual"). Prehybridization carried out in a solution containing the composition of 6 X SSC (1 X SSC is composed of 150 mm sodium chloride and 15 mm traintravel citrate), 1-X is the solution of Denhardt (Denhardt), 0.25% sodium dodecyl sulfate, 0.05% sodium pyrophosphate and 100 μg/ml denatured DNA salmon sperm that was done at 37oC for 3 h, after which hybridization was performed in a reaction mixture consisting of a composition of 6 X SSC containing a sample of a radioactive label in the form of phosphorus-32 (ATT-3), 1-X is the solution of Denhardt, 17 μg/ml yeast tRNA and 0.05% sodium pyrophosphate, what he was doing at the 31oC during the night. After completion of the reaction nitrocellulose filter was washed with a solution of 6 X SSC containing 0.05% sodium pyrophosphate, which was done at room temperature for two hours, then spent the autoradiograph. In R. what Itanium conventional methods, randomly chose several clones, and then spent the composition of the private nucleotide sequence of the cDNA, which was used by dideoxy method. Using your personal computer, then conducted a search for homologues of the nucleotide sequences whose nucleotide sequence registered in GenBank data Bank or laboratory EMBL. It was thereby determined that the area subjected to hybridization under the influence of ATT-3-sample, is homologous to some members of the AIu-repeated sequences (Schmid, C. W. and Jelinek, W. R. (1982), Science 216, 1065-1070).

Therefore, typing the label in the form of phosphorus-32 in the DNA fragment containing AIu-repeated sequence obtained from human genomic DNA, when conducting colonial hybridization mentioned above 33 clones using this snippet for education sample, found that 12 clones have AIu-repeated sequences. When examining the length of the cDNA insert the remaining 21 clones found that the inserts contain from 50 to 3600 grounds. After mapping the restrictive enzymes acid cDNA 21 clones were partially determined nucleotide sequence of the mentioned is she in the above-mentioned data Bank for reason, and were selected clones with new sequences that are not registered in the specified Bank data bases.

Since these plasmids contain early SV40-promoter and replication start, they are acceptable for ekspressirovali cDNA in COS-1 cells. For this reason, the plasmid DNA was introduced into cells COS-1, making it for clones selected as talked about this above. Transfection of cells COS-1 was performed by electroporation using the implant device type GTE-1 firm "Shimadzu". In other words, cells COS-1 were grown in a flask to poluchivshego state, and the cells were collected by processing them tribenuronmethyl acid and twice washed with phosphate-saline buffer (-) solution (manufactured by "Nissui Seiyaku"). Further, these cells suspended in phosphate-saline buffer (-) solution at a concentration of 6107cells in 1 ml on the other hand, each plasmid DNA, prepared by the cesium chloride method, brought to a concentration of 200 μg/ml by adding phosphate-saline buffer (-) solution. Together mixed in 20 μl each of the above cell suspension and DNA solution and then placed in the camera in which the distance between the electrodes sostavleniya camera at 4oC for about 5 min the mixture of cells and DNA in the camera, was added to 10 ml of DMEM (minimum nutrient medium Needle, modified Dulbecco) containing 10% serum fetal cows. The resulting mixture was then transferred to a Cup and cultured in the presence of 5% CO2at 37oC during the night. Then the supernatant from the culture was removed, cells were washed free of serum-DMEM (minimum nutrient medium Needle, modified Dulbecco) and added 10 ml of DMEM, and then were cultured for three days. After removing the supernatant fluid from a culture of a cell culture obtained by this method, the supernatant was determined by the previously mentioned activity in inhibition of morphological changes in adipocytes.

In the above adiposity inhibiting activity was determined in the supernatant culture in the case of cells, COS-1, transfection the plasmid (pcD-20-2) clone number 20-2. Some activity against inhibition lipogenesis transformation are given in table. 1.

Education pUC-CAT is a plasmid obtained by the introduction of chlorophenylacetylene is it the promoter, which can operate in mammalian cells. The transfection was performed in the same way as in the case of plasmid pcD-20-2. As can be seen from the table. 1, the activity, which inhibits the morphological change of the cell line ZTZ-LI on adipocytes, is detected in the supernatant from the culture of COS-1 cells, transfection the plasmid pcD-20-2. In addition, regarding the cell line ZTZ-LI, forced to differentiation to adipocytes (see tab. 2) there is ignominiously lipoproteine lipase activity. The values presented in table 2 are relative (expressed in percent), and they are derived from considerations that lipoproteine lipase activity 100% (blank experiment) occurs only when using DMEM without serum.

In accordance with the foregoing, was built restrictive enzyme map for cDNA inserted into plasmid pcD-20-2 (see Fig. 1). Next, dimethoxymethane was determined the entire nucleotide sequence of the cDNA was obtained nucleotide sequence with a sequence number 1 in the sequence listing. It was found that the cDNA is introduced into a plasmid pcD-20-2, consists of 1065 grounds and tail poly(A)-h is of 199 amino acids and beginning with methionine. When comparing the resulting nucleotide sequence with sequences available in the data Bank on the grounds of the laboratory EMBL and those in GenBank, did not have homology with any known sequences. In addition, when comparing the amino acid sequence coded for the interval of the open reading with sequences contained in the NBPF and SWISS Bank data on proteins, it was also noted the lack of homology to known sequences. In view of the above, it was confirmed that the interval of the open reading available cDNA inserted into plasmid pcD-20-2, encodes a previously unknown polypeptide. Amino acid sequence encoded by this open interval reading, specified consistently with the number 2 in the sequence listing.

The area containing highly hydrophobic amino acids (signal peptide), observed in various secretory proteins, is located in the N-terminal region, which begins with a methionine. It follows that this protein is a secretory protein.

Example 5

The synthesis of the peptide and the preparation used for preparing antipeptide antisera

A peptide corresponding AB>2-Gly-Pro-Pro-Arg-Val-Ser-Pro-Asp-Pro-Arg-Ala-Glu-Leu-Asp-Ser-COOH) was synthesized to confirm the considerations that previously unknown peptide encoded by the open interval reading the cDNA insert of the plasmid pcD-20-2, is a secretory protein, and detecting the factor that inhibits lipogenesis, during the cleaning of such a factor, described below. Such synthesized peptide used as antigen for immunization of a rabbit and the preparation of antisera. The peptide synthesis was carried out according tBOC-amino acid solid-phase method (Merrifield, R. B. (1963), J. Am. Chem. Soc. , 85, 2149-2154), which was accompanied by the use of standard automatic program synthesis using a peptide synthesizer type 430A, manufactured by the firm "Apld of Biosystems". After blocking and removing the resin peptide was purified on anion-exchange column. Confirmation of the nature of the spectrum of the main peak was obtained by carrying out high-performance liquid chromatography using a column with reversed phase. Further, by conducting the subsequent concentration of the peptide by means of freeze-drying, the peaks were separated and the substance were collected, making it way high-performance liquid chromatography using what if analysis method of high performance liquid chromatography and the determination of amino acid composition. Amino acid composition was determined using the Pico-Tag system, representing an automated amino acid analyzer manufactured by "Waters".

Zamochenny the limpet hemocyanin (pigment mollusk saucer-shaped cap from the keyhole) that serves as a protein carrier, combined with the resulting peptide through glutaraldehyde method (Konopka, J. B. et al. (1984), J. Virol., 51, 223-232). This formation was used as antigen in the case krolikovs immobilization. The homogeneous antigen suspended in full stimulator Freud and rabbits were immunized by injection of the suspension under the skin of the back. Whole blood was collected after the introduction of the suspension three times at intervals of about two weeks. Measurement of the antibody titer in the serum of the rabbit was performed using the ELISA test. Immunoglobulin IgG fraction was prepared from the resulting antisera, using protein A-separato 6MB-column manufactured by Pharmacia. This formation is then used as the primary antibodies.

Example 6

Production and purification inhibiting lipogenesis factor in COS-1 cells

1. Design vector high exprest consisting of 1240 base pairs and containing a cDNA insert, and subjected to cleaning. Meanwhile, spending splitting vector high expression pcDL-SR 296 (Takebe, J. et al. (1988), Mol. Cell. Biol., 8, 466-472) via BamHI, received a fragment of 3400 base pairs, containing the promoter SR , and BamHI-fragment containing the cDNA, was annexed by conducting the reaction using T4 DNA ligase. This DNA is then used for transformation of strain DH5 culture of Escherichia coli, and conducted analysis of the plasmids from resulting transformants. Chose a strain in which the direction of cDNA transcription was the same as the direction of the promoter SR , and this plasmid was called pSR-20-2 (see Fig. 2).

Incidentally, the promoter SR includes early SV40 promoter and a sequence of R-U5 long terminal duplications education HTLV-1 and detects the promotor activity, 10-100 times higher than that in the early promoter of SV40.

Next, the cells COS-1 transformed resulting plasmid pSR-20-2, using the antibody obtained in example 5, to ensure that the factor inhibiting lipogenesis, is secreted in the supernatant culture. Transfection of cells COS-1 held either electroporation way, or DEAE-dextranomer method (Luthman, H. and Magnusson, G. (1983), Nucleic Acids Res., 11, 1295-1308). Each noderole the plasmid pcDL-SR-296, not containing cDNA, and plasmid pSR-20-2 expressing factor, inhibiting lipogenesis, respectively, were processed by taking the liquid 160 µl trichloroacetic acid to protein deposition, and then, separated by centrifugation,

received sludge. After washing the precipitate with acetone, cooled with ice, and air drying the precipitate was dissolved in a test buffer solution containing 2-mercaptoethanol that was necessary for electrophoresis in polyacrylamide gel with sodium dodecyl sulfate, after which the analysis of this method was performed in reducing conditions using a 12.5% gel. Detection of protein bands associated with electrophoresis, was performed by silver staining using sivest-Stein produced by the firm "Nacalai Task" (see the plate on the left side of Fig. 3). The only band with a specific pattern has been observed at the place indicated by the arrow (molecular weight approximately 23000) using the supernatant from the cell culture COS-1, transfection the plasmid pSR-20-2.

In addition, after the point of application of protein bands, similarly arise when conducting electrophoresis in polyacrylamide gel, nitrocap KS-8441), that was done by the method Tobin and others (Towbin, H. et al. (1979), Proc. Natl. Acad. Sci. USA 76, 4350-4354), conducted a case analysis on Western (Western) using primary antibodies prepared in example 5. The operation point of application Westernu was performed using Immun-Blot-test kit (GAR-HRP), manufactured by Bio-Rad, acting according to the manufacturer's instructions (see the plate on the right side of Fig. 3). Only a specific band detected by silver staining (molecular weight about 23000), was characterized by interaction with antipeptide antibody, thus indicating that the protein encoded by the open interval read from cDNA inserted into plasmid pcD-20-2, is a secretory protein.

Next, we conducted the survey behavioural activity against inhibition lipogenesis differentiation using the supernatant of the cell culture COS-1 transformed with plasmid pSR-20-2. Some activity is shown in table. 3.

Although the activity against inhibition lipogenesis differentiation was not observed in the supernatant from the cell culture COS-1 transformed with plasmid pSDL-SR296, activity was detected in the case of plasmid pSR-20-2. In addition, lipoproteine lipase activity was also strongly suppressed in the case of cell line ZTZ-L1, which were induced to undergo differentiation into adipocytes (see tab. 4). Inhibiting activity, specified in tab. 3 and table. 4, was significantly stronger than in the case of plasmid pcD-20-2 (PL. 1 and table. 2).

II. Purification and analysis of N-terminal amino acid sequence,

After dialysis for 7 l free from serum supernatant from the culture obtained by transfection of cells COS-1 plasmid pSR-20-2 by the method described above, at 20 times the volume of dialysis buffer (10 mm boric acid, sodium hydroxide (pH 9,0) and 13 mm potassium chloride), which was done at 4oC for 15 h, were weak cation-exchange chromatography using a high performance liquid chromatograph manufactured by the company "pharmacy"; it was done under the following conditions:

column - substance CM-toyopearl 650M (2,2 20 cm, manufactured by "Task");

eluting buffer solution -

solution A: a solution of boric acid with a concentration of 10 mm sodium hydroxide (pH 9,0) content 13 mm potassium chloride,

Solution B: solution A containing 300 mm chloride is many concentration gradient with the transition for 50 min from solution A (100%) solution B (100%).

Bitmap drawing Westernu carried out for each of the fractions, which were obtained that were made used for preparing antipeptide antibodies prepared in example 5, identifies the fractions containing factor inhibiting lipogenesis (numbers 35 - 45). All peak fraction (number 41), containing the largest number of factor that inhibits lipogenesis, which was eleirovania using 260 mm sodium chloride, concentrated, putting a precipitation treatment trichloroacetic acid, and analyzed by carrying out electrophoresis in polyacrylamide gel with sodium dodecyl sulfate when using 12.5% gel in reducing conditions. After electrophoresis, protein bands from polyacrylamide gel point was applied to a PVDF membrane (manufactured by "Millipore", material immobilon), making it at the current density of 0.8 mA/cm2using helium membrane device for the application of points (firm "Marisol", type KS-8441) in transfer buffer solution (0.02% sodium dodecyl sulfate, 20% methanol and 25 mm triborate (pH 9,5)), which was carried out at 4oC for 15 h After washing the membrane with points within 5 min 10 mm retrievability BA for 5 min, the membrane was air-dried. Further, the part that was transferred to the factor, inhibiting lipogenesis, was isolated from the membrane, and the sequence number up to ten amino acid residues counting from the N-Terminus was determined using gas-phase protein sequencer (manufactured by "Aplaud of Biosystems", model 475A).

Phenylthiohydantoin-amino acids obtained in the respective reaction cycles, were separated and identified by high pressure liquid chromatography on a column of reversed phase chromatograph manufactured by "Aplaud of Biosystems", model 120A). Amino acid sequence set described below:

Pro-Gly-Pro-Pro-Pro-Gly-Pro-Pro-Arg-Val-.

It follows that the human factor, inhibiting lipogenesis, is secreted outside the cells in the Mature form, starting with Pro-balance, what happens after biosynthesis of the precursor, which consists of 199 amino acids, and losing signal peptide consisting of 21 amino acids located at the N-end, due to the stiffness.

Example 7

Biological activity of purified factor that inhibits lipogenesis

Factor inhibiting lipogenesis, was purified from free from serum supernatant is combined, such as cation exchange chromatography, hydrophobic chromatography and gel filtration column chromatography and so on, and the above-mentioned factor was detected as a single band that did the analysis by polyacrylamide gel electrophoresis with sodium dodecyl sulfate. Was discovered amazing activity that is installed in the study of activity against inhibition lipogenesis differentiation and activity in relation to suppress lipoproteidna lipase that was carried out with the use of this purified factor that inhibits lipogenesis.

Thus, it was concluded that the factor inhibiting lipogenesis, which meets the present invention, is a consequence of the presence of certain protein that has activity against inhibition of lipogenesis.

Example 8

Secretory production of a factor that inhibits lipogenesis transformation in ovarian cells of Chinese hamster

Ovarian cells of the Chinese hamster in the logarithmic growth phase, were cotransfection, spending callaborate DNA coprecipitation using pSR-20-2 and pSRneo when DNA ratio of 5:1. Selection of transformed stammo is g/ml of the substance G418 (firm "Gibco Oriental") and 10% serum fetal cow, what happened in the last 9 days. After selection of the 15 strains resulting from resistant colonies colonies were transferred to a plate with 24 holes that were made using cloning rings, and the cultivation was continued. Then those cells that have reached the merged state, moved in square bottles and subcultural; 15 strains producing factor, inhibiting lipogenesis, was studied using the above-mentioned method of the point of application Westernu.

The result said was the choice of one clone showing the highest production factor, inhibiting lipogenesis. This clone was then cultured by the method of limiting dilution using a plate 96 of the recess, and 4 were selected clone produced from this single cell. After transfer of these four strains of cells in the Cup for cultivation and cultivation to the state of merging a number of factors, inhibiting lipogenesis, was subjected to the study using the point method Western that was done for each of the supernatant liquid culture. Secretory produced number of factors, inhibiting lipogenesis, were stable and there was no dosadasnjih liquids with culture, previously obtained when lipoproteidna lipase activity of the cell line ZTZ-L1, for all four clones from the supernatant of the liquid culture was determined activity against suppression lipoproteidna lipase. Next, one of the four strains were cultured in free serum medium (complete nutritional system CHO-1, manufactured by the firm "Ventrex"). After culturing the strain in free serum medium for two days subcultivation conducted by processing the trypsin-ethylenediaminetetraacetic acid, and then the cultivation was continued for another three days. When removing the supernatant from the culture and the study of it by using the point method Western was established, the factor inhibiting lipogenesis, stably produced.

Example 9

Inhibitory effect on lipogen differentiation of murine bone marrow; dedicated preadipocyte cell line H-I/A

Preadipocyte cell line H-I/A, isolated from murine bone marrow (Nakamura, M. et al. (1985), Soc. Exp. Biol. Med., 179, 283-287), suspended at a density 2,0104cells in 1 ml medium E (Wednesday Fischer (Fischer) (production firm "Gibco"), containing 10% of immob the pipette on the plate Celtic C-1 plate 48F (manufactured by Sumitomo bakelite") in the amount of 0.5 ml to deepen and cultured at 33oC in humidified gaseous environment containing 5% carbon dioxide and 95% air. After three days the medium was replaced with medium F (medium E containing 1 μm hydrocortisone (manufactured by Sigma)), what was done with the intention of inducing lipogenesis differentiation. Then the supernatant from the cell culture COS-1 transformed with plasmid pSR-20-2, supernatant from cell culture COS-1, transfection the plasmid pSDL-SR296, or free from the serum DMEM (minimum nutrient medium Needle, modified Dulbecco) was added in Wednesday. The medium was replaced with fresh medium F, and the supernatant from the cell culture COS-1 and DMEM was added again, which made every four days. After fixation of cells in 5% formaldehyde, did on the 26th day after adding the environment F, fat droplets, which were accumulated in the cells, and cell nuclei were stained respectively with dye oil red O and hematoxylin. Speed lipogenic differentiation was then calculated using the same method as used for the evaluation of inhibitory effects on lipogen differentiation of the cell line ZTZ-LI. As shown in the table. 5, the supernatant from the cell culture COS-1, transfectional fact, the purified factor inhibiting lipogenesis, which was purified from the supernatant from the cell culture COS-1, transfection the plasmid pSR-20-2 significantly inhibits lipogen differentiation of the cell line H-I/A.

Example 10

Impact-induced colony-stimulating factor on bone marrow; dedicated preadipocyte cell line H-I/A

Cell line H-I/A is suspended at a density 1,0104cells per 1 ml in the environment E and the pipette was transferred into a Cup for cultivation on 6 holes (the production company "Kowsar") in an amount of 3 ml per deepening. Cells were cultured at 33oC in humidified gas mixture containing 5% carbon dioxide and 95% air. After culturing cells for six days the medium was replaced with fresh medium E. was added to the supernatant from the cell culture COS-1, transfection the plasmid pSR-20-2, supernatant from cell culture COS-1, transfection the plasmid pcDL-SR 296, or free from the serum DMEM (minimum nutrient medium Needle, modified Dulbecco), and then were grown for 5 h

Then, after three times washing the cells free from sivaram within 14 days; and the supernatant from the culture was collected from each recess. Thus obtained supernatant from the culture was filtered using milekovic GV-filter with a pore size of 0.22 μm (manufactured by Nippon Millipore industries"), and the filtrate is then used to test the colony-stimulating activity. The test colony-stimulating activity was carried out according to the method proposed by Kubota and others (Kubota, K. et al., (1981), Cancer Res., 41, 3052-3057). Cells femoral bone marrow taken from the female mouse line AWN He/N (purchased from the firm "Japan Charles river), suspended at a density 1,0105cells in 1 ml RPMI medium 1640 (manufactured by "Gibco"), to which was added 0.3% baktagir (the production company "Difco"), 20% serum fetal cows and 10% supernatant from the cell culture H-I/A. Each of these suspensions were collected in the amount of 1 ml and placed in a plastic Cup: 35 mm 10 mm (manufactured by "lax"). After culturing the cells at 37oC in humidified gas mixture containing 5% carbon dioxide and 95% air for seven days was measured respectively the number of colonies formed in the cups.

The supernatant from the cell culture is echinopanax the plasmid pSR-20-2, found a significantly higher colony-stimulating activity (p<0.01) in comparison with the supernatant fluid from a culture of cells H-I/A, processed by the environment E, to which was added the supernatant from the cell culture COS-1, transfection the plasmid pcDL-SR 296, or free from serum DMEM (minimum nutrient medium Needle, modified Dulbecco). The results obtained are summarized in table. 6.

Cell line H-I/A, taken in an amount 5,0106, suspended in 50 ml of medium E and cultured in the flask Axel (AcceI) (production firm "Costar") within five days. Next, the medium was replaced with fresh medium E, and simultaneously added to the supernatant from the cell culture COS-1, transfection the plasmid pSR-20-2, supernatant from cell culture COS-1, transfection the plasmid pcDL-SR296, or free from the serum DMEM (minimum nutrient medium Needle, modified Dulbecco). After culturing the mixture for 18 h, total RNA was isolated from cells, in doing so, as it was done in example 1. Some amount of mRNA makrofagov colony-stimulating factor analyzed Norderney (Northern hybridization method (Thomas, P. (1980). Proc. Natl. Acad. Sci. USA. , 77, 5 the irradiation factor obtained by conducting polymerase chain reaction (Ladner, M. B. et al., (1988), 85, 6706-6710), was labelled with phosphorus-32, for which we used the system Multiprime DNA labelling systems (production company "Amersham"), and this formation was used as a sample. The number of hybridized samples labeled as phosphorus-32 was installed using the image analyzer (manufactured by the Fuji photo film). The sample labeled as phosphorus-32 was hybridisable with RNA, the amount of which was approximately 2500 bases and approximately 4,500 grounds. When you compare the total amounts of samples hybridized with RNA from these dimensions, it was found that the amount of mRNA makrofagov colony-stimulating factor per unit of total RNA is increased in the case of cell line H-I/A, processed by the environment E, to which was added 5% supernatant from the cell culture COS-1, transfection the plasmid pSR-20-2 when compared with the case processing environment E, to which was added 5% supernatant from the cell culture COS-1, transfection the plasmid pcDL-SR296 (see tab. 7).

Example 11

Impact-induced colony-stimulating factor on cell line H-I/A, differenziali at the 33oC for 12 days in the flask Axel (AcceI). During this time of cultivation, the medium was replaced with fresh medium F once in every four days. Next, the medium was replaced with medium E, and the cultivation was continued for another three days. The result was noticed that oily droplets accumulate in the 40% tax or more cells, indicating the course of differentiation into adipocytes. The medium was then replaced with environment E, to which was added 5% supernatant from the cell culture COS-1, transfection the plasmid pSR-20-2, supernatant fluid from a culture of cells, COS-1, transfection the plasmid pcDL-SR296, or free from serum-DMEM (minimum nutrient medium Needle, modified Dulbecco), after which the cultivation was carried out for 18 hours After cultivation, the medium was removed and cells were washed twice in phosphate-saline buffer (-), after which was added 25 ml of medium E, to which was added sodium heparin, brought to a final concentration of 10 units per 1 ml of the Mixture was cultured at 33oC for 15 minutes In the measurement lipoproteidna lipase activity in the supernatant culture significant lipoproteine lipase inhibition was observed only in (C).

Next, in the same way as in example 1, was isolated total RNA from cells prepared as described previously. The amount of mRNA in makrofagov colony-stimulating factor was then determined in the same manner as in example 10, except that every time used 10 µg total RNA. Cell line H-I/A-treated medium E containing 5% supernatant from the cell culture COS-1, transfection the plasmid pSR-20-2, was found a significant increase in the number of mRNA makrofagov colony-stimulating factor per unit of total RNA, if to compare with the case of processing medium E containing 5% supernatant from the cell culture COS-1, transfection the plasmid pcDL-SR296 (see tab. 9).

Preparative example 1

Preparation of granules with intersolubility shell containing 50% factor, inhibiting lipogenesis

Weighed 70 parts (by weight; the same is true hereinafter) of purified factor that inhibits lipogenesis, which was obtained in example 8 10 parts of lactose and 20 parts of microcrystalline cellulose and thoroughly mixed in a mortar, then mixed with sufficient amount of water. The pounded mixture then was subjected to what was necessary for the formation of granules. The granules were passed through maruerite (MARUMERIZER) than was provided for obtaining granules. After drying, these granules at 40oC for two hours in a drying Cabinet with air circulation resulting dry granules are classified by passing through sieves caliber 35 mesh (0.5 mm in particle size scale of the American society for testing and materials). The granules, the size of which exceeded 35 mesh (0.5 mm) was used for the manufacture of granules with intersolubility shell. First of all, the usual procedure was to prepare a solution of 5% hydroxypropylmethylcellulose equal amounts of methylene chloride and ethanol, and 100 parts of the above-mentioned dry granules were coated by spraying 100 parts of this solution, what was I doing in device for covering. Further, processed in such a way that the granules were coated by spraying, making it as before, 300 parts of a solution containing 10% hydroxypropylmethylcellulose phthalate (manufactured by "Shin-Etsu chemical", mark HP-55), 1.5% of stearic acid, 50% acetone and 38.5% ethanol, which was prepared in the usual manner. Coated granules obtained in this way was dried at 40oC for one hour in a drying Cabinet with air circulation, resulting in received EN paginas.

Preparative example 2

Capsules

The above-mentioned granules with intersolubility coating containing 50% factor, inhibiting lipogenesis, took 200 mg and placed in capsules No. 3, resulting in the received hard capsules containing 100 mg of factor inhibiting lipogenesis, per ampoule.

Preparative example 3

Injection

Factor inhibiting lipogenesis, can be used in the form of ampoules, sterile solution containing factor, dissolved or suspended in water or other pharmaceutically acceptable liquids. Or ampoule containing a sterile powdered drug (preference should be given to the factor, the inhibitory lipogenesis, which is subjected to drying by freezing), may be when using full pharmaceutically acceptable liquid.

Industrial application

Factor inhibiting lipogenesis, which meets the present invention is administered either as such or together with other therapeutic drugs in the treatment of aplastic anemia, leukopenia caused by toxins or radiation, infections caused by viruses, bacteria and parasites, cytopenia that occur after t is logical disorders. Factor inhibiting lipogenesis, used either as such or in combination with other therapeutic drugs for the prevention and treatment of morbid obesity.

The composition cytopenias ameliorant or composition of the drug against obesity, which meets the present invention and used in the treatment of the above conditions include a mixture of acceptable medically carrier and a therapeutically effective amount of a factor that inhibits lipogenesis. The above composition can be introduced in various forms, such as tablets, capsules, granules, powders or syrups intended for oral administration and injection, intravenous drip, or in the form of a suppository intended for parenteral administration.

In the case of the introduction by injection or by intravenous drip infusion of a therapeutic composition that meets the present invention is in the form of pyrogen free and no parenterally acceptable aqueous solution. The question of preparing parenterally acceptable protein solution in regard to pH, isotonicity and stability is within the normal capabilities of the special is use States can be determined by the doctor by taking into account those factors, which affect the action of the above-mentioned drug, such as the patient's condition, body weight, sex, age, diet, the influence of other infections, the time of administration, and other factors that are clinically important. Usually in oral introduction day in an adult patient may be introduced from about 0.01 to 1000 mg, which can be done either at once or in several parts. Meanwhile, in the case of not oral introduction at a time can be injection from about 0.01 to 100 mg, which can be done either subcutaneously or intramuscularly, or intravenously.

The composition cytopenias ameliorant that meets the present invention may be used in combination with other hematopoietic factors, such as interleukins from IL-I to IL-10, factor, leukemia inhibitory, the receiver cell factor, colony-stimulating factors GM-CSF, G-CSF, M-CSF, Meg-CSF, tumor necrosis factor, interferon or erythropoetin. In this case, although other gemopoeticescoe factors and can act as one of the mixed components cytopenias ameliorating composition conforming to the present invention, they can be introduced back into the patient in the form of a separate composition. Additional IP is my using only one other gomeopaticheskih factors.

Meanwhile, the composition used against obesity, which meets the present invention may be used in combination with other acceptable drugs used against obesity, such as anorectic, inhibitors of intestinal absorption, digestive enzyme inhibitors, metabolic accelerator hormone drugs, inhibitors of fat synthesis or inhibitors of insulin secretion. In addition, it can also be used in combination with diet therapy and physical therapy. In this case, the composition used against obesity may promote the activity or to increase the effectiveness of therapy, achieved with the use of only one other protivovospolitelnyh drugs, and to increase the effectiveness of therapy reached in the event of only one other protivovospolitelnyh therapies.

Because cytopenias ameliorant or protivovospalitelnye drugs used in the present invention are of biological origin, or genetic recombinants, they are characterized by low levels of toxicity. According to the results of observations carried out within five days after oral administration of adult male ossea the invention, does not detect toxicity at levels of 500 mg/kg or less.

1. Recombinant protein factor inhibiting lipogenesis, with activity consisting in the suppression of morphological differentiation preadipocytes in adipocytes, in the suppression of adipocyte lipoprotein lipase having the amino acid sequence set on the basis of the coding DNA sequence of the formula given in the graphics part,

and a molecular weight of about 23,000 set by electrophoresis in polyacrylamide gel with sodium dodecyl sulfate reducing conditions using a 12.5% gel.

2. Recombinant protein under item 1 with the following properties: eluted at NaCl concentration in the range of 220-290 mm in the case of chromatography on a weak cation-exchanger under the following conditions: (a) in column used CM Toyopearl Pack 650 M (2.2 x 20 cm, firm Tosoh Corporation); C) as an eluting buffer solutions using solution A (10 mm boric acid - NaOH, pH 9,0, 13mm - Cl) and solution b (solution a containing 300 mm NaCl); (c) the flow rate is 3 ml/min; d) the concentration gradient in the conduct of said chromatography is adapted for linear gradient from 100% solution a to 100% solution In lnasty his predecessor SEQ ID N2, listed in the description.

4. Protein under item 1, characterized in that it has an amino acid sequence that corresponds presented in the sequence N 2 list of sequences with non-amino acid residues from -1 to +178, or a protein that has not all the specified sequence, but having activity against inhibition of lipogenesis.

5. DNA encoding a recombinant protein in PP.1 to 4 and having the nucleotide sequence shown in the graphics part.

6. DNA under item 5, having a nucleotide sequence from 81 to 614 in the nucleotide sequence of SEQ ID No. 1, is given in the description.

7. Recombinant vector pSR-20-2 for expression of factor inhibiting lipogenesis, containing Bam HI fragment of 3.4 kb vector pSDL-SR 296 and Bam - fragment of 1.2 kb, which includes the nucleotide sequence corresponding to SEQ ID No. 1.

8. Recombinant strain of mammalian cells, COS-1, producing a factor of inhibiting lipogenesis, characterized by the fact that cells COS-1 transformed with plasmid pSR-20-2.

9. A method of obtaining a recombinant protein in PP.1 to 4, providing for the transformation of mammalian cells recombinant overovanie transformed cells, characterized in that the expression vector contains a DNA fragment on PP.5 and 6, the block clean.

10. The composition having activity of inhibiting lipogenesis, containing a therapeutically effective amount of protein and a pharmaceutically acceptable excipient or carrier, wherein the protein having above mentioned activity, is a protein according to any one of paragraphs.1 to 4.

 

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