WO2002072628A1

WO2002072628A1 - Physiologically active polypeptide and polynucleotide encoding the same

Info

Publication number: WO2002072628A1
Application number: PCT/JP2002/002184
Authority: WO
Inventors: Naohisa Ishikawa; Hidetsugu Murakami
Original assignee: Japan Science And Technology Corporation
Priority date: 2001-03-08
Filing date: 2002-03-08
Publication date: 2002-09-19
Also published as: JPWO2002072628A1

Abstract

A rat-origin polypeptide having the amino acid sequence of SEQ ID NO:2 and having a physiological activity similar to the verotoxin of enteropathogenic Escherichia coli; and a polynucleotide encoding this polypeptide and having the base sequence of SEQ ID NO:1. This polypeptide is useful in clarifying the cytotoxicity of the verotoxin and developing therapeutic methods for infection with enteropathogenic E. coli. Moreover, the physiological activity of this polypeptide closely relates to neurodegeneration as observed in motor depression and apoptosis. Thus, this polypeptide largely contributes to the clarification of the onset mechanism of neurodegenerative diseases and development of remedies therefor. Furthermore, this polypeptide occurs in vascular endothelial cells and has an effect of elevating blood pressure. Thus, it is also usable in clarifying the activity mechanism of essential hypertension and developing novel hypotensive drugs.

Description

Description: A polypeptide having a physiological activity and

A polynucleotide encoding the polypeptide of

TECHNICAL FIELD The invention of this application relates to a polypeptide having a physiological activity and a polynucleotide encoding the polypeptide. More specifically, the invention of this application relates to a novel bioactive polypeptide having a physiological activity similar to the pathogenic bacterium toxin and useful for elucidation of cytotoxicity and development of therapeutic drugs, and a large amount of this polypeptide. It relates to a polynucleotide useful for production and the like.

BACKGROUND ART Intestinal hemorrhagic Escherichia coli 0157, a verotoxin (VT) produced by this bacterium causes hemolytic uremic syndrome, and has recently become a major social problem.

The inventors of the present application have developed a method for isolating and purifying the toxin (VT1, VT2) of Escherichia coli 0157 and testing for its infection (j. Mass Spectr. 32: 1140-). 1 142, 1997; Aichi Medical Journal No. 1483: 3-5, 1996; Aichi Prefectural Institute of Public Health annual report dai25: 36, 1997). Using these toxins, we have also succeeded in producing anti-VT1 and anti-VT2 antibodies from rabbits (The Japanese Biochemical Society, Biochemistry, Vol. 72, No. 8, 2000). Infection of pathogenic Escherichia coli can cause dramatic changes in infected individuals even if the amount of venom toxin produced is extremely small. The existence of specific receptors for this substance has been postulated. If the functions of these physiological substances and their receptors are elucidated, the cytotoxicity of verotoxin It is expected that it will be possible to understand the effects and develop a fundamental treatment for the pathogenic Escherichia coli infection. However, at present, there is no known bioactive substance or receptor. The invention of this application has been made in view of the circumstances described above, and comprises a novel in vivo physiologically active substance having a physiological activity similar to that of verotoxin such as pathogenic Escherichia coli 0157, and encoding this substance. It is an object to provide a polynucleotide.

DISCLOSURE OF THE INVENTION This application provides the following inventions (1) to (7) as inventions for solving the above-mentioned inventions.

(1) A purified rat derived from a rat having the amino acid sequence of SEQ ID NO: 2 and having biological activity.

(2) A purified polynucleotide encoding the polypeptide of the invention (1).

(3) The polynucleotide according to the invention (2), having the nucleotide sequence constituting the protein translation region of SEQ ID NO: 1.

(4) A polynucleotide derived from a non-rat animal in which a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1 or a partial sequence thereof hybridizes under stringent conditions. (5) A polypeptide expressed from the polynucleotide of the invention (4) and having 50% or more homology with the polypeptide of the invention (1).

(6) A synthetic oligonucleotide having the amino acid sequence of SEQ ID NO: 3, 4, or 5, and having biological activity. (7) An antibody against the polypeptide of the invention (1).

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a graph showing the amount of mRNA transcribed from the polynucleotide of the present invention for each organ of a rat. FIG. 2 shows the results of in situ hybridization in which the amount of mRNA of the polynucleotide of the present invention in rat liver was measured. FIG. 3 shows the state of a rat to which the synthetic oligopeptide of the present invention was administered intraventricularly. FIG. 4 shows the change in blood pressure of a rat injected intravenously with the synthetic rigopeptide of the present invention. FIG. 5 shows the passage change of cell morphology cultured in the presence of the oligopeptide of the present invention.

ί Figure 6 shows an electrophoretic image of DNA extracted from the cultured cells shown in Figure 5.

BEST MODE FOR CARRYING OUT THE INVENTION The polypeptide of the invention (1) is an isolated and purified rat protein having an amino acid sequence of SEQ ID NO: 2, which is similar to or similar to a toxin produced by pathogenic Escherichia coli. It has the same physiological activity (cytotoxicity). The polypeptide of the invention (1) may be isolated from a rat tissue, prepared by a peptide by chemical synthesis based on the amino acid sequence of SEQ ID NO: 2, or obtained by encoding a polynucleotide encoding the amino acid sequence of SEQ ID NO: 2. Produced by using recombinant DNA technology Although it can be obtained by a method or the like, a method of obtaining by recombinant DNA technology is preferably used. For example, RNA is prepared by in vitro transcription from a vector having the polynucleotide (the protein translation region of cDNA: ORF) of the invention (3), and the resulting RNA is subjected to invitro translation by using this as a cyclized form. The protein can be expressed in the mouth. When the polynucleotide is recombined into an appropriate expression vector by a known method, the polynucleotide encodes in prokaryotic cells such as Escherichia coli and Bacillus subtilis, and eukaryotic cells such as yeast, insect cells, and mammalian cells. Protein can be expressed in large amounts. When the polypeptide of the invention (1) is produced by expressing DNA by in vitro translation, the polynucleotide of the invention (3) is inserted into a vector having an RNA polymerase promoter to prepare a recombinant vector, When this vector is added to an in vitro translation system such as a heron reticulocyte lysate or a wheat germ extract containing an RNA polymerase corresponding to the promoter, the polypeptide of the invention (1) is produced at the in vitro mouth. be able to.

Examples of the RNA polymerase promoter include T7, T3, SP6 and the like. Examples of vectors containing these RNA polymerase promoters include pCMV-SPORT, pKA1, pCDM8, pT3 / T718, ρΤ7 / 319, pBluescript II and the like. When the polypeptide of the invention (1) is produced by expressing DNA in a microorganism such as Escherichia coli, expression having an origin, a promoter, a ribosome binding site, a DNA cloning site, a terminator and the like which can be replicated in the microorganism is used. An expression vector in which the polynucleotide of the invention (3) is recombined is prepared in a vector, the host cell is transformed with the expression vector, and the resulting transformant is cultured. Polypeptides encoding nucleotides can be mass-produced in microorganisms. At this time, a protein fragment containing an arbitrary region can be obtained by adding a start codon and a stop codon before and after the arbitrary translation region for expression. Alternatively, it can be expressed as a fusion protein with another protein. By cleaving the fusion protein with an appropriate protease, only the protein portion encoded by the polynucleotide can be obtained. Examples of expression vectors for Escherichia coli include a pUC system, a pBluescript I pET expression system, a pGEX expression system, and a pQE expression system. When the polypeptide of the invention (1) is produced by expressing DNA in a eukaryotic cell, the polynucleotide of the invention (3) has a promoter, a splicing region, a poly (A) addition site, and the like. If the recombinant vector is prepared by inserting it into an eukaryotic cell expression vector and introduced into eukaryotic cells, the polypeptide of the invention (1) can be produced in eukaryotic cells. Examples of expression vectors include pKA1, pCDM8, pSVK3, pMSG, pSV and pBK-CMV, pBK-RSV, EBV vector, pRS, pYES2, and the like. If plND / V5-His, pFLAG-CMV-2, pEGFP-N1, pEGFP-C1, etc. are used as expression vectors, they can be expressed as fusion proteins with various tags such as His tag, FLAG tag, and GFP. Can also. As eukaryotic cells, monkey kidney cells COS7, cultured mammalian cells such as Chinese hamster ovary cells CHO, budding yeast, fission yeast, silkworm cells, African toad frog egg cells and the like are generally used, and the polypeptide of the invention (1) is used. Any eukaryotic cell can be used as long as it can express E. coli. In order to introduce the expression vector into eukaryotic cells, known methods such as an electroporation method, a calcium phosphate method, a liposome method, and a DEAE dextran method can be used. After expressing the polypeptide of the invention (1) in prokaryotic cells or eukaryotic cells, the target polypeptide can be isolated and purified from the culture by a combination of known separation procedures. For example, treatment with a denaturant such as urea or a surfactant, ultrasonic treatment, enzyme elimination, salting-out / solvent precipitation, dialysis, centrifugation, ultrafiltration, gel filtration, SDS-PAGE, isoelectric focusing Examples include electrophoresis, ion exchange chromatography, hydrophobic chromatography, affinity chromatography, and reversed-phase chromatography. The polypeptide of the invention (1) includes a peptide fragment (5 or more amino acid residues) containing any partial amino acid sequence of the amino acid sequence represented by SEQ ID NO: 2. These peptide fragments can be used as antigens for producing antibodies. The polypeptide of the invention (1) also includes a fusion protein with any other protein. For example, a protein fusion with Dalphin Chin-S-transferase (GST) and green fluorescent protein (GFP) can be exemplified. Further, the polypeptide of the invention (1) may undergo various modifications in the cell after being translated. Therefore, modified proteins are also included in the scope of the protein of the invention (1). Such post-translational modifications include removal of the N-terminal methine ninine, Examples include N-terminal acetylation, sugar chain addition, limited degradation by intracellular protease, myristylation, isoprenylation, phosphorylation and the like. Invention (2) is a purified polynucleotide encoding the polypeptide of the invention (1), and includes a rat genomic DNA, its mRNA and cDNA (specifically, a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1), Their complementary strands are included. The polynucleotide of the invention (3) is a polynucleotide (cDNA) containing a base sequence constituting the translation region (ORF) of SEQ ID NO: 1. Since the polypeptide of the invention (.1) is expressed in any cell, a rat cDNA library prepared from rat cells is screened using an oligonucleotide probe synthesized based on the nucleotide sequence of SEQ ID NO: 1. By doing so, the same clone as the polynucleotide of the invention (3) can be easily obtained. Alternatively, the target cDNA can also be synthesized by RT-PCR using these oligonucleotides as primers and using mRNA isolated from rat cells as type III. In general, polymorphism due to individual differences is frequently observed in mammalian genes. Therefore, in the nucleotide sequence of SEQ ID NO: 1, a polynucleotide in which one or more nucleotides have been added, deleted, and substituted with Z or another nucleotide is also included in the scope of the polynucleotide of the invention (3). Similarly, polypeptides in which one or more amino acids have been added, deleted and / or substituted by other amino acids resulting from these changes also have the same physiological activity as Vero toxin. It is included in the range of the polypeptide of (1). Invention (4) is a polynucleotide in which the polynucleotide consisting of SEQ ID NO: 1 or a partially continuous sequence thereof is hybridized under stringent conditions, and comprises all mammals, including humans, other than rats. Some regions of genomic DNA (genomic DNA fragments), including their mRNA and cDNA. Here, the stringent condition includes the nucleotide sequence of SEQ ID NO: 1 or a partially continuous sequence thereof (for example, 10 bp or more). Conditions that allow for selective and detectable specific binding of the polynucleotide to genomic DNA. Stringent conditions are defined by salt concentration, organic solvent (eg, formamide), temperature, and other known conditions. That is, the stringency is increased by decreasing the salt concentration, increasing the concentration of the organic solvent, or increasing the hybridization temperature. For example, a stringent salt concentration is usually about 750 mM or less NaCI and about 75 mM or less trisodium citrate, more preferably about 500 mM or less NaCI and about 50 mM or less trisodium citrate, Most preferably, it is about 250 mM or less for NaCI and about 25 mM or less for trisodium citrate. A stringent organic solvent concentration is at least about 35% formamide, most preferably at least about 50%. Stringent temperature conditions are about 30 ° C. or higher, more preferably about 37 ° C. or higher, and most preferably about 42 ° C. or higher. Other conditions include hybridization time, detergent (eg, SDS) concentration, and the presence or absence of carrier DNA, and various stringencies can be set by combining these conditions. Can be. Also, the washing conditions after the pre-digestion affect the stringency. The washing conditions are also defined by the salt concentration and the temperature, and the stringency of washing increases with decreasing salt concentration and increasing temperature. For example, stringent salt conditions for washing are preferably about 30 mM NaCI or less and about 3 mM trisodium citrate, most preferably about 15 mM NaCI or less and about 1.5 mM trisodium citrate. It is. Stringent temperature conditions for washing are about 25 ° C. or higher, more preferably about 42 ° C. or higher, and most preferably about 68 ° C. or higher. The polynucleotide of the invention (4) may be, for example, a genome prepared from genomic DNA other than rat by the above-described stringing method using the above-mentioned polynucleotide as a probe, It can be isolated by screening libraries and cDNA libraries. The polypeptide of the invention (5) is a non-rat animal-derived polypeptide produced from the polynucleotide of the invention (4), and is 50% or more of the rat-derived 'polypeptide of the invention (1), Preferably, the polypeptide has an amino acid homology of 75% or more, most preferably 90% or more, and has the same physiological activity as the polypeptide of the invention (1). The polypeptide of the invention (5) can be obtained by the same known recombinant DNA technique as that of the polypeptide of the invention (1). Can be obtained. The oligopeptide of the invention (6) is a part of the polypeptide of the invention (1) and is a peptide consisting of 8 amino acid residues of SEQ ID NOS: 3, 4 and 5, respectively. These oligonucleotides have the same physiological activity as the polypeptide of the invention (1), as shown in Examples described later, and can be used for elucidation of cytotoxicity and development of drugs and the like. These oligopeptides can be prepared by a known peptide synthesis method. The antibody of the invention (7) can be obtained from serum after immunizing an animal using the polypeptide of the invention (1) as an antigen. As the antigen, a peptide chemically synthesized based on the amino acid sequence of SEQ ID NO: 2 or a polypeptide protein expressed in eukaryotic cells or prokaryotic cells can be used. Alternatively, it can be prepared by introducing the above expression vector for eukaryotic cells into an animal muscle or skin by injection or gene gun, and then collecting serum (for example, see Japanese Patent Application Laid-Open No. 7-313187). No. invention). As the animal, a mouse, a rat, a heron, a goat, a chicken, and the like are used. By fusing B cells collected from the spleen of the immunized animal with myeloma to produce a hybridoma, a monoclonal antibody against the polypeptide can be produced. Hereinafter, the invention of this application will be described in more detail and specifically with reference to examples, but the invention of this application is not limited to the following examples.

Example

Example 1

Cloning of cDNA Purified cDNA of interest was prepared according to the following procedure.

CDNA was prepared by converting mRNA isolated from rat brain into type II, and this was converted to double strand Then, it was integrated into an Agt11 vector to create a rat brain cDNA library. Next, after packaging this cDNA library, the cells are infected into host Escherichia coli and cultured on an agar medium to form plaques, and the proteins contained in the plaques are transferred to a double-cell membrane to create replicas. The replica was reacted with an anti-VT2 rabbit antibody, and the procedure of detecting an anti-VT2 antibody-positive plaque using a peroxidase-labeled anti-rabbit IgG goat antibody was repeated at least three times to purify the cDNA. The purified cDNA was amplified and its nucleotide sequence was determined.

As a result, it was confirmed that the obtained cDNA clone had the nucleotide sequence of SEQ ID NO: 1.

Example 2

Production of polypeptide by in vitro translation mRNA was prepared from the cDNA cloned in Example 1.ゥ Using a lysate of heron reticulocytes, a protein translated from the above mRNA was obtained in a mammalian cell-free protein synthesis system. After SDS-electrophoresis and Western plot of the translated protein, a peptide having a molecular weight of 5 kDa ′, which reacts with the anti-VT2 antibody, was confirmed.

Furthermore, the sequence of 6 amino acid residues from the N-terminus of the obtained 5 kDa peptide was determined, and the ORF region of the cloned cDNA was determined.

As a result, the theoretical molecular weight of the polypeptide consisting of 43 amino acid residues encoded in the ORF region of the cDNA was consistent with the molecular weight obtained by SDS-electrophoresis.

Example 3

Preparation of Antibody to Polypeptide The amino acid sequence of the polypeptide encoded by the cDNA clone obtained in Example 1 (based on SEQ ID NO: 2, a Cys residue was added to the C-terminal of the amino acid sequences of SEQ ID NOs: 3, 4, and 5). The added oligopeptide was chemically synthesized. Next, using maleimide, each of the synthetic oligopeptides was covalently bound to octabutene (acetylated pepsin serum albumin) to prepare three types of antigen molecules. These antigen molecules were mixed with Freund's complete adjuvant, and immunized by inoculating <ゥ egrets. The immunization schedule consisted of three inoculations of three antigens every four weeks. After confirming the production of Egret antibody by the ELISA method, finally, three kinds of antigens were inoculated into each Egret, boosted, and antiserum was collected. From this antiserum, the IgG fraction was purified by affinity chromatography using protein G-Sepharose to obtain a rabbit antibody to each of the rival peptides of SEQ ID NOS: 3, 4, and 5.

Each of the obtained antibodies recognizes the N-terminal side, the central part, and the C-terminal side of the polypeptide having the amino acid sequence of SEQ ID NO: 2. It is possible to confirm more accurately.

Example 4

Polypeptide expression distribution A PCR primer was synthesized based on the nucleotide sequence (sequence number 1) of the polynucleotide obtained in Example 1, and the mRNA expression level in various rat organs was determined by the well-known RT-PCT method. did. Figure 1 shows the data obtained by quantifying one actin as a control and standardizing the value to 1.0.

As is evident from the results in Fig. 1, this polypeptide is expressed systemically in rats, but is highly expressed in the central nervous system (cerebrum, hypothalamus, cerebellum, and medulla oblongata), especially in the spinal cord. Was confirmed. Following the liver and kidney, the least expressed site was the skeletal muscle.

In addition, the expression level increased with aging. Sprague-Dawley rats were bred for a long period of time, and liver specimens of rats 2-4 months, 6 months, 12 months, 18 months, 24 months and 30 months old were prepared. In order to compare the presence or absence of mRNA expression in these pathological specimens ('In situ hybridization was performed on each specimen.

The results are shown in Fig. 2, and the expression level increased with aging. 24 months old Was significantly expressed in hepatic vascular endothelial cells

Example 5

Synthesis old Rigopepuchido of in vivo bioactivity SEQ ID NO: 3, 4 and 5 oligo peptide was synthesized in, respectively, 10- ^S M a Sairi Gopepuchido solution 0.075ml of concentration, pen Bok Barupitaru anesthetized rats Bokudai 4 Brain Room.

Figure 3 shows the rat after waking from anesthesia. The rat was unable to move around, remained immobile for the next two weeks, and lost weight due to inability to eat and drink. However, in the third week, motor function recovered.

Furthermore, when the three synthetic peptides were injected intravenously into rats, a dose-dependent increase in blood pressure was observed as shown in FIG. These elevated blood pressures were abolished by L-NAME (NO synthase inhibitor) pretreatment.

Example 6

In vitro bioactivity of synthetic human lipopeptideSynthetic human rigopeptides of SEQ ID NOs: 3, 4 and 5 were added to the culture medium of rat pheochromocytoma cell line PC12 and human cervical cancer cell line HeLa cells. .

The results are shown in Fig. 5. In PC12 cells in the presence of nerve cell growth factor (Dif-PC12), dendrites disappeared according to passage (p-4; p-19; p-36). The number also decreased in the 19s (p-19). The number of PC12 cells in the absence of nerve cell growth factor also decreased in the 19th generation. On the other hand, in human-derived HeLa cells, the number of cells decreased at the fourth and 19th generations. When DNA was extracted from the subcultured cells and subjected to electrophoresis, a "ladder-like" band was observed as shown in Fig. 6, confirming that DNA fragmentation due to apoptosis occurred. . INDUSTRIAL APPLICABILITY The invention of this application provides a novel polypeptide having a bioactivity equivalent or similar to that of verotoxin of pathogenic Escherichia coli, and a polynucleotide encoding this polypeptide. This polypeptide is useful for elucidating the cytotoxicity of Vero toxin and developing therapeutics against pathogenic Escherichia coli infection. Furthermore, the physiological activity of this polypeptide is deeply involved in neurodegeneration, as shown by the inhibition of motor function and neuronal apoptosis. For this reason, this polypeptide greatly contributes to elucidation of the pathogenesis of neurodegenerative diseases and development of therapeutic drugs. In addition, since this polypeptide is present in vascular endothelial cells and has an effect of increasing blood pressure, it can be used for elucidation of the active mechanism of essential hypertension and for development of new antihypertensive agents.

Claims

The scope of the claims

1. A purified rat derived from a rat having the amino acid sequence of SEQ ID NO: 2 and having biological activity.

2. A purified polynucleotide encoding the polypeptide of claim 1.

3. The polynucleotide according to claim 2, which has a nucleotide sequence constituting a protein translation region of SEQ ID NO: 1.

4. A polynucleotide derived from a non-rat animal, wherein the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1 or a partial sequence thereof is hybridized under stringent conditions.

5. A polypeptide expressed from the polynucleotide of claim 4 and having 50% or more homology with the polypeptide of claim 1.

6. A synthetic oligonucleotide having the amino acid sequence of SEQ ID NO: 3, 4, or 5, and having biological activity. '7. An antibody against the polypeptide of claim 1.