JP2009118759A - New protein having human cancer cell-selective cytotoxic ability - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a protein giving specific damages to specific cells such as human cancer cells in active forms, and to provide a DNA encoding the protein, and the like. <P>SOLUTION: Provided is a protein comprising an amino acid sequence having a specific sequence, or a protein which comprises an amino acid sequence modified by one or more of the deletion, replacement, insertion and addition of one or more amino acids in a range having ≥70% of homology to either of the amino acid sequences, and has cytotoxic activity in an active form. Also provided is a DNA comprising a nucleotide sequence having a specific sequence, or a DNA which hybridizes with a DNA comprising a nucleotide sequence complementary to the DNA in a stringent condition, and encodes a protein having a cytotoxic activity in an active form. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

The present invention relates to a useful protein derived from a microorganism, and more specifically, cell recognition and cell destruction ability derived from Bacillus thuringiensis (hereinafter sometimes abbreviated as “BT”) M019 strain and the like. The present invention relates to an amino acid sequence of a novel protein having a nucleotide sequence, a gene encoding the same, and uses thereof.

  Bacillus thuringiensis was discovered in 1901 by a Japanese researcher Shigego Ishiwatari as a pathogenic bacterium of the moth. Since then, the toxin protein produced by this bacterium has been widely used as a microbial insecticide for the control of agricultural and sanitary pests.

  It has been known by studies so far that a crystalline protein of BT having insecticidal activity is activated by protease treatment as needed after solubilization with alkali and exhibits toxicity to insects. Crystalline proteins having insecticidal activity include (1) a Cry protein (usually having a molecular weight of 60 to 75 kDa in the activated form) that exhibits selective destruction to insect cells in the state activated by protease, and (2) insects It has been clarified that it is classified into a Cyt protein having a non-selective destructive activity against the cells and a haemolytic activity of erythrocytes (usually having a molecular weight of 22-30 kDa in the activated form). Crystalline proteins having such insecticidal activity are also used as insecticides targeting insect cells.

  The crystalline protein of BT as a cell recognition protein has high selectivity typified by Cry protein, richness of diversity based on tens of genotypes, and genetic modification because it is a prokaryotic protein It has an excellent feature that is not found in other cell recognition proteins such as antibodies.

On the other hand, only 30 to 40% of BT strains express a crystalline protein having insecticidal activity. The nature of crystalline proteins that have no insecticidal activity produced by BT strains has been unknown for a long time, but in recent years, some of such proteins have been activated by proteases after solubilization with alkali. In addition, a substance that produces a protein having cell recognition activity or cytotoxic activity has been found (Patent Documents 1 to 3). For example, Patent Document 3 discloses a protein derived from the BT1470 strain (before activation: 34 kDa, after activation: 26 kDa) that has cytotoxic activity against cancer cells and the like by treatment with proteinase K. Yes.
JP-A-11-222498 JP 2003-284568 A JP 2005-333865 A

  An object of the present invention is to provide proteins that specifically damage specific cells such as human cancer cells, DNAs encoding such proteins, and uses of these proteins and DNAs.

The present inventor has obtained a Bacillus thuringiensis strain which is isolated from soil, arable soil, forest soil, and stored dust, and does not show pathogenicity against three species of Phosporidae and two species of dipods. Used to selectively damage vertebrate cells (including cancer cells) when treated with protease (trypsin) after solubilization with alkali among toxin proteins of unknown activity derived from these strains It became clear that there was a novel protein that became like.

  Furthermore, the present inventor clarifies the amino acid sequence and the base sequence of the gene encoding the protein produced by the Bacillus thuringiensis M019 strain among such novel proteins, and completes the present invention. It came. In particular, the protein consisting of the amino acid represented by SEQ ID NO: 6 described below is a completely new protein that is dissimilar to a known crystalline protein that does not have insecticidal activity produced by the BT strain.

  That is, the present invention has a cytotoxic activity of 53 kDa to 57 kDa obtained by treating a protein consisting of the amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6, and these proteins with trypsin. Provided with a protein. Further, the present invention provides any one of amino acid deletion, substitution, insertion or addition within a range having 70% or more homology with any of the amino acid sequences represented by SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6. Proteins comprising an amino acid sequence modified by one or more types and having cytotoxic activity in an active form, and proteins having cytotoxic activity obtained by treating these proteins with trypsin are also provided.

  The present invention also relates to a protein comprising an amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6, such as a DNA comprising a nucleotide sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5. And an active form that hybridizes under stringent conditions with a DNA comprising a nucleotide sequence complementary to a DNA comprising the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5 DNA encoding a protein having cytotoxic activity is provided.

In one aspect of the present invention, the cytotoxic activity is specific to at least one cell selected from the group consisting of leukemia cells, uterine cancer cells and liver cancer cells. The protein and DNA of the present invention can be obtained as derived from the genus Bacillus, in particular, the Bacillus thuringiensis M019 strain (Accession No. FERM P-21398).

  Furthermore, the present invention relates to the invention relating to the use of the protein and DNA as described above, that is, a pharmaceutical composition containing the protein of the present invention, particularly an anticancer agent, and a vector containing the DNA of the present invention and a trait containing the vector. Provide a converter. In addition, the present invention provides a method for producing the protein of the present invention, comprising (a) culturing cells expressing the protein of the present invention; and (b) recovering the protein of the present invention. To do.

  The present invention enables research and development at a gene level and a molecular level for a protein having a cell type selective disorder activity. The present invention can also establish techniques for mass production of these proteins. Accordingly, the present invention provides DDS technology development in the pharmaceutical field, development of diagnostic agents, development of specific cells in the field of agriculture, forestry and fisheries, breeding of animals and plants by selective destruction of tissues, and novel pest control agents in the chemical industry field. This is considered to contribute greatly to a wide range of technologies such as development. The proteins of the present invention are also highly selective and are useful for specifically recognizing and / or destroying specific cells. Moreover, since the protein of the present invention has high specificity for specific cancer cells, it is useful as a pharmaceutical composition (specifically, an anticancer agent and a cell labeling agent).

  The protein consisting of the amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6 is a “protein having cytotoxic activity in an active form” obtained from Bacillus thuringiensis M019 strain.

  Further, an amino acid sequence having high homology with any of the amino acid sequences represented by SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6, usually 70% or more, preferably 85% or more, more preferably 90% or more, and still more preferably A protein comprising an amino acid sequence modified by any one or more of amino acid deletion, substitution, insertion or addition within a range having a homology of 95% or more, most preferably 99% or more is also represented by SEQ ID NO: 2, Similar to the protein consisting of the amino acid sequence shown in No. 4 or SEQ ID No. 6, it can be a protein having cytotoxic activity in the active form.

  As will be described later, a protein having the amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6, or a protein having an amino acid sequence highly homologous thereto is trypsin (on the C-terminal side of arginine or lysine). It has a function of specifically cleaving peptide bonds.) And can be activated by treatment. Therefore, the homology of the amino acid sequence as described above is within the range where the site cleaved by trypsin treatment is not affected, for example, the amino acid sequence near the 262nd from the N-terminal side of SEQ ID NOs: 2 and 4. It is desirable that the homology is particularly high.

  Here, “cytotoxic activity” refers to the ability to specifically destroy specific cells. For example, when measured by a method such as an MTT assay, if the cytotoxic activity against a specific cell is significantly higher than the cytotoxic activity against a negative control, it is considered to have cytotoxic activity against that cell. . That is, the cell in which the protein of the present invention exhibits cytotoxic activity includes: (a) a step of incubating the protein with any cell in a culture medium; and (b) the protein has cytotoxic activity against the cell. Determining whether or not.

  Examples of the negative control include any cell in which the protein of the present invention does not exhibit cytotoxic activity. For example, HC cells and NIH3T3 cells can be used. On the other hand, examples of the positive control include any cells in which the protein of the present invention exhibits cytotoxic activity. Preferably, cells arbitrarily selected from HepG2 cells and HeLa cells are used. Cells that are destroyed to the same extent or better than the positive control are more preferred as cells in which the protein of the present invention exhibits cytotoxic activity.

  Examples of cells that are specifically damaged by the protein of the present invention include human-derived uterine cancer cells, liver cancer cells, and leukemia T cells (see Examples), but the protein of the present invention is a cell other than these. It may have a hindrance activity against. In addition, the protein of the present invention may have a single disorder activity against a single cell or a combination of a plurality of cells.

  The “protein having cytotoxic activity in the active form” means, for example, treatment with an alkaline buffer for solubilizing a crystalline protein, or treatment with a protease for cleaving the protein at a predetermined site. , Refers to a protein in an inactive form (precursor) that is converted to a form having cytotoxic activity (active form) when treated under specific conditions, and the above activity even if not treated under specific conditions (Ie, proteins that are present in an active form from the beginning).

The protein consisting of the amino acid sequence of SEQ ID NO: 2 is usually present in an inactive form unless it is subjected to a specific treatment (78 kDa, “CP78A” in the examples), but under alkaline conditions or under alkaline conditions and protease (trypsin) ) Is converted to the active form of the protein (53 kDa, “T53A / B” in the examples) and has cytotoxic activity.

The protein consisting of the amino acid sequence of SEQ ID NO: 4 is usually present in an inactive form unless it is subjected to a specific treatment (78 kDa, “CP78B” in the examples), but under alkaline conditions or under alkaline conditions and protease (trypsin) ) Is converted to the active form of the protein (57 kDa, “T57B” in the examples) and is assumed to have cytotoxic activity.

  The protein consisting of the amino acid sequence of SEQ ID NO: 6 is usually present in an inactive form unless it is subjected to a specific treatment (84 kDa, “CP84” in the examples), but under alkaline conditions or under alkaline conditions and protease (trypsin) ) Is converted to the active form of the protein (57 kDa, “T57A” in the examples) and has cytotoxic activity.

  Note that the 53 kDa to 57 kDa protein having cytotoxic activity is not a single molecule of such size, but a small fragment of the peptide generated by the activation treatment is not a molecule close to such size. It may be a covalently bound form of the protein. In any case, the active form of the protein having the predetermined size according to the present invention has a size of 53 kDa to 57 kDa, for example, by SDS-PAGE and DAEA cellulose chromatography as shown in Examples described later. Anything can be confirmed.

The conditions and procedures for alkaline treatment and protease treatment for protein activation in the present invention are those that have a predetermined size (kDa) equivalent to that of the active form of the protein and that have a cytotoxic activity. If it is. In the present invention, it is particularly desirable to use trypsin as the protease. An example of a specific aspect of such treatment is as shown in the Examples, and Nagamatsu, Y., Itai, Y. Hatanaka, C., Funatsu,
References such as G., Hayashi, K. (1984) Agric. Biol. Chem. 48, 611-619 can also be referred to.

  The protein of the present invention is obtained from a cell that expresses the protein of the present invention. Examples of cells that express the protein of the present invention include, for example, cells that naturally express the protein of the present invention (preferably, Bacillus thuringiensis A1462 strain), and expression vectors encoding the DNA that expresses the protein of the present invention. The transformant containing is mentioned.

  The protein of the present invention can be produced by culturing such cells in a medium and recovering those contained in the resulting culture. At this time, since the protein of the present invention tends to be obtained as a crystalline protein inclusion body, it is solubilized by alkali treatment according to a known method, and further subjected to protease treatment and activated as necessary. It is preferable to use it.

  The medium to be used preferably contains a carbon source, an inorganic nitrogen source or an organic nitrogen source necessary for the growth of the host cell (transformant). Examples of the carbon source include glucose, dextran, soluble starch, and sucrose. Examples of the inorganic or organic nitrogen source include ammonium salts, nitrates, amino acids, corn steep liquor, peptone, casein, meat extract, large Examples include soybean cake and potato extract. If desired, it may contain other nutrients [eg, inorganic salts (eg, calcium chloride, sodium dihydrogen phosphate, magnesium chloride), vitamins, antibiotics (eg, tetracycline, neomycin, ampicillin, kanamycin, etc.]). .

  Culturing is performed by methods known in the art. Specific culture media and culture conditions used according to the host cells are exemplified below, but the culture conditions in the present invention are not limited to these.

When the host is a bacterium, actinomycetes, yeast, filamentous fungus or the like, for example, a liquid medium containing the above nutrient source is suitable. Preferably, the medium has a pH of 5-8. When the host is Escherichia coli, preferred media include LB medium, M9 medium (Miller. J., Exp. Mol. Genet, p. 431, Cold Spring Harbor Laboratory, New York (1972)) and the like. The culture can be performed usually at 14 to 43 ° C. for about 3 to 24 hours with aeration and stirring as necessary. When the host is Bacillus subtilis, it can be carried out usually at 30 to 40 ° C. for about 16 to 96 hours with aeration and stirring as necessary. When the host is Bacillus thuringiensis or a mutant thereof (preferably Bacillus thuringiensis BFR1), a preferable medium is CYS medium (Yamamoto, T., ACS Symp. Ser. 432, 46-60 (1990)). (In this case, the culture is performed, for example, at 20 ° C. for 96 hours on an agar plate). When the host is yeast, examples of the medium include Burkholder's minimal medium (Bostian. KL et al, Proc. Natl. Acad. Sci. USA, 77, 4505 (1980)), and the pH is 5-8. desirable. Culture is usually about 1 at about 20-35 ° C.
It is carried out for 4 to 144 hours, and if necessary, aeration and stirring can be performed.

When the host is an animal cell, the medium is, for example, a minimal essential medium (MEM) containing about 5 to 20% fetal calf serum (Science, 122, 501 (1952)), Dulbecco's modified minimal essential medium (DMEM) (Virology, 8, 396 (1959)), RPMI 1640 medium (J. Am. Med. Assoc., 199,
519 (1967)), 199 medium (proc. Soc. Exp. Biol. Med., 73, 1 (1950)) and the like can be used. The pH of the medium is preferably about 6 to 8, and the culture is usually carried out at about 30 to 40 ° C. for about 15 to 72 hours, and if necessary, aeration and stirring can be performed.

  The protein can be purified by appropriately combining various commonly used separation techniques. For example, salting out, solvent precipitation, dialysis, ultrafiltration, gel filtration, non-denaturing PAGE, SDS-PAGE, ion exchange chromatography, hydroxylapatite chromatography, affinity chromatography, reverse phase high performance liquid chromatography, isoelectric focusing The protein of the present invention can be obtained with high purity by appropriately selecting a known separation method such as electrophoresis.

The present invention also provides an antibody having specific affinity for the protein of the present invention. This antibody may be any fragment as long as it has not only a complete antibody molecule but also an antigen binding site (CDR) for the protein of the present invention. For example, Fab, F (ab ′) ₂ , ScFv, minibody, etc. Can be mentioned. The antibody of the present invention is labeled with, for example, horseradish peroxidase or the like, and used to detect whether or not the protein of the present invention is bound to a specific cell.

  For example, a polyclonal antibody is obtained by using the protein of the present invention or a fragment thereof as an antigen, together with a commercially available adjuvant (for example, complete or incomplete Freund's adjuvant), and subcutaneously or intraperitoneally about 2 to 4 times every 2 to 3 weeks. Administer (measure the antibody titer of the partially collected serum by a known antigen-antibody reaction and confirm its rise), collect whole blood approximately 3-10 days after the final immunization, and purify the antiserum Can be obtained. Examples of animals to which the antigen is administered include mammals such as rats, mice, rabbits, goats, guinea pigs, and hamsters.

The present invention also provides a method for identifying a substance having an affinity for the protein of the present invention. This method includes the steps of (a) contacting the protein with a test substance, and (b) determining whether the test substance has affinity for the protein. Examples of test substances that are contacted with the protein of the present invention include, but are not limited to, sugars, lipids, proteins, nucleic acids, synthetic compounds, and the like. Preferred affinity is less than 10 ⁻⁶ M, less than 10 ⁻⁷ M, less than 10 ⁻⁸ M, less than 10 ⁻⁹ M, less than 10 ⁻¹⁰ M, less than 10 ⁻¹¹ M, or less than 10 ⁻¹² M. An affinity having a dissociation constant (K _d ) is mentioned. Such affinity is measured, for example, by labeling the protein of the present invention with a radioisotope and then subjecting it to a binding assay with a test substance. A substance having an affinity for the protein of the present invention is used, for example, when the protein of the present invention is purified.

  The present invention comprises a DNA encoding a protein comprising the amino acid represented by SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6, preferably a nucleotide sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5. DNA is provided.

  A DNA that hybridizes under stringent conditions with a DNA consisting of the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5 is also a DNA that can encode the precursor protein of the present invention. Here, the “stringent condition” is a condition in which a so-called specific hybrid is formed with a DNA consisting of the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5, and a non-specific one is not formed. In the present invention, the sodium concentration is 150 to 900 mM, preferably 600 to 900 mM, and the temperature is 60 to 68 ° C, preferably 65 ° C. The stringency can be adjusted by appropriately changing the salt concentration, temperature, etc. during the hybridization reaction or washing. DNA having high homology with DNA comprising the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5, preferably 70% or more, more preferably 80% or more, still more preferably 95% or more, and most preferably 99 When DNA having a homology of at least% is targeted, for example, the sodium concentration may be set low and the temperature set within the above range.

  Furthermore, among the DNAs of the present invention, an oligonucleotide comprising at least about 20, preferably about 18, more preferably at least about 16, and most preferably at least about 15 consecutive nucleotides, or an antisense oligonucleotide thereof Is also provided by the present invention. These oligonucleotides can be used as a primer pair for amplifying the DNA of the present invention or as a DNA probe. By using this DNA probe, it is possible to screen a cDNA library or a genomic library of Bacillus thuringiensis or other organisms to obtain a homologue of the DNA of the present invention.

  The present invention also provides a recombinant vector and an expression vector containing DNA encoding the protein of the present invention. The recombinant vector of the present invention is not particularly limited as long as it can be replicated and maintained or autonomously propagated in various prokaryotic and / or eukaryotic host cells, and includes plasmid vectors and viral vectors. The recombinant vector can be conveniently obtained by adding a DNA encoding the protein of the present invention to an appropriate restriction enzyme and ligase, or, if necessary, a linker or adapter. It can be prepared by ligation using DNA. Examples of such vectors include plasmids derived from E. coli such as pBR322, pBR325, pUC18 and pUC19, yeast derived plasmids such as pSH19 and pSH15, and Bacillus subtilis derived plasmids such as pUB110, pTP5 and pC194. . Examples of the plasmid derived from Bacillus thuringiensis include pHT3101 and pHT315. Moreover, bacteriophage such as λ phage, papovirus such as SV40, bovine papilloma virus (BPV), retrovirus such as Moloney murine leukemia virus (MoMuLV), adenovirus (AdV), adeno-associated virus (AAV), Animal and insect viruses such as vaccinia virus and baculovirus are exemplified.

  In particular, the present invention provides an expression vector in which a DNA encoding the protein of the present invention is placed under the control of a promoter functional in the target host cell. As a vector to be used, a promoter region that functions in various prokaryotic and / or eukaryotic host cells and can control transcription of a gene located downstream thereof (for example, trp promoter when the host is E. coli, When the host is Bacillus subtilis, such as lac promoter, lectA promoter, etc. When the host is a yeast, SPO1 promoter, SPO2 promoter, penP promoter, etc. When the host is yeast, PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, etc. SV40-derived early promoter, MoMuLV-derived long terminal repeat, adenovirus-derived early promoter, and the like, and a transcription termination signal of the gene, that is, a terminator region, The terminator region is not particularly limited as long as it is linked via a sequence containing at least one restriction enzyme recognition site, preferably a unique restriction site that cleaves the vector only at that site. It further contains a selection marker gene for selection of transformants (a gene that confers resistance to drugs such as tetracycline, ampicillin, kanamycin, hygromycin, phosphinothricin, a gene that complements auxotrophic mutations, etc.) It is preferable. Further, when the DNA encoding the protein of the present invention does not contain an initiation codon and a termination codon, the initiation codon (ATG or GTG) and the termination codon (TAG, TGA, TAA) are respectively located downstream of the promoter region and the terminator region. A vector contained upstream of is preferably used.

  When a bacterium is used as a host cell, the expression vector generally needs to contain a replicable unit capable of autonomous replication in the host cell in addition to the promoter region and terminator region described above. The promoter region includes an operator and a Shine-Dalgarno (SD) sequence in the vicinity of the promoter.

  In the case where the protein of the present invention is secreted into the medium of the transformant and the DNA encoding the protein of the present invention does not contain the signal peptide coding sequence, an appropriate signal is used as a vector following the start codon. A secretory expression vector further comprising a codon is preferably used.

  When the DNA encoding the protein of the present invention is isolated from genomic DNA and obtained in a form containing the original promoter and terminator region, the expression vector of the present invention can be replicated or autonomously propagated in the target host cell. It can be prepared by inserting the DNA into an appropriate site of a known cloning vector.

  The present invention also provides a transformant obtained by transforming a host cell with the above expression vector. The host cell used in the present invention is not particularly limited as long as it is compatible with the above-described expression vector and can be transformed, and is a natural cell or artificially established usually used in the technical field of the present invention. Various cells such as mutant cells or recombinant cells (eg, bacteria (E. coli, Bacillus subtilis, lactic acid bacteria, Bacillus thuringiensis or mutants thereof (preferably a Bacillus thuringiensis strain that does not produce toxin protein)) And yeast (such as Saccharomyces, Pichia, and Kriveromyces), animal cells, insect cells, and the like), but prokaryotic cells are more preferable.

  Moreover, those skilled in the art can create a Bacillus thuringiensis mutant suitable for expressing the protein of the present invention. For example, by performing curing from a Bacillus thuringiensis strain that produces toxin protein and removing plasmid DNA, a Bacillus thuringiensis strain that does not produce toxin protein can be obtained. One skilled in the art can use the above technique to obtain any Bacillus thuringiensis strain that produces the protein of the present invention without producing a toxin protein. For details, see Gonzalez, J. M. et al. PLASMID 5, 351-365 (1981).

The expression vector can be introduced into a host cell by a conventionally known method. For example, in the case of bacteria, the method of Cohen et al. (Proc. Natl. Acad. Sci. USA., 69,2110 (1972)),
By the protoplast method, competent method, etc., in the case of yeast, the method of Hinnnen et al. (Proc. Natl. Acad. Sci. USA., 75, 1927 (1978)), by the lithium method, etc., in the case of animal cells, Graham (Virology, 52, 456 (1973)), etc., and in the case of insect cells, use the method of Summers et al. (Mol. Cell. Biol. 3,2156-2165 (1983)), etc. Can do.

  The present invention relates to a pharmaceutical composition comprising the protein of the present invention, an expression vector or a transformant as an active ingredient, that is, a disease associated with cells in which the protein of the present invention exhibits cytotoxic activity, determined by the identification method described above. A pharmaceutical composition that can be applied to, for example, an anticancer agent is provided. More specifically, the protein of the present invention can be used as an active ingredient of a therapeutic agent for leukemia, preferably a therapeutic agent for uterine cancer, and more preferably a therapeutic agent for liver cancer.

When the protein of the present invention is used as an anticancer agent, the protein of the present invention is effective by itself, but in order to enhance the effect, it is a complex with another anticancer agent (covalent or noncovalent). Or you may use as a concomitant agent. For example, although a complex of an immunoglobulin and an anticancer agent has been reported, the protein of the present invention can also be used as a complex with another anticancer agent. The protein of the invention can also be labeled with a radioisotope (eg, ¹³¹ I). For example, by treatment with ¹³¹ I-labeled anti-CD20 monoclonal antibody, B
Very good results have been reported in patients with cellular lymphoma. Similarly, when the protein of the present invention is labeled with ¹³¹ I, its destructive effect on cancer cells can be enhanced. The protein of the present invention can also be used as a complex with a toxin (eg, ricin). For example, there is a report that a remarkable effect was confirmed in a patient with T cell lymphoma by a complex of an anti-CD5 antibody and a ricin A chain. The protein of the present invention can also be used as a complex with ricin A chain.

  The term “composition” or “agent” as used herein refers to a substance containing a specific substance as an active ingredient. For example, the pharmaceutical composition of the present invention contains the protein of the present invention as an active ingredient and exhibits a therapeutic effect for a specific disease. Such a pharmaceutical composition or agent is a solid, semi-solid or liquid form containing the protein of the present invention as an active ingredient in a mixture with an organic or inorganic carrier or excipient suitable for oral or parenteral application. Can be used in the form of pharmaceutical preparations. The active ingredients are, for example, powders, tablets, pellets, capsules, suppositories, liquids, emulsions, suspensions, aerosols, sprays and other forms suitable for use, normal, non-toxic And can be combined with a pharmaceutically acceptable carrier. Further, auxiliary agents, stabilizers, thickeners and the like may be used if necessary. These carriers and excipients may be sterilized as necessary, or may be sterilized after preparation. The therapeutically effective dose of the protein of the invention, which is the active ingredient, will also vary depending on its purity, activity and mode of administration, and the age, health, weight, sex and type of disease of the individual patient to be treated. Those skilled in the art can appropriately determine the dose in consideration of these factors.

  The present invention also includes a method of processing on a computer using the DNA sequence information and amino acid sequence information of the present invention. For example, using the BLAST and FASTA programs of the National Center for Biotechnology Information (NCBI), it becomes possible to search for DNA sequences and amino acid sequences having a predetermined homology with the DNA sequence information and amino acid sequence information of the present invention. . Further, in molecular modeling, gene shuffling, and the like, it is possible to obtain more optimal sequence information by using the DNA sequence information and amino acid sequence information of the present invention.

The present invention will be described more specifically with reference to the following examples. However, these are merely examples and do not limit the scope of the present invention.
[material]
The cells used for the experiment were purchased from the following. HeLa cells (human cervical cancer cells), HepG2 cells (liver cancer cells), MOLT-4 cells (human leukemia T cells), Jurkat cells (human leukemia T cells), A549 cells (human lung cancer cells), CACO-2 Cells (human colon cancer cells), COS-7 cells (African green monkey kidney-derived cells), Vero cells (African green monkey kidney-derived cells) and NIH3T3 cells (mouse embryo-derived fibroblasts) were purchased from RIKEN. UtSMC cells (human normal uterine smooth muscle cells) were purchased from Takara Shuzo. HC cells (human normal hepatocytes) were purchased from Cell Systems. Sheep blood was purchased from Japan Biotest Laboratories.

[Example 1: Observation of strain morphology]
Bacillus thuringiensis M019 strain (Accession No. FERM P-21398) was placed on a 1.7% agar plate containing BT medium 1 (1% polypeptone + 1% meat extract + 0.4% salt + 4.5 μM manganese sulfate, pH 8). After culturing at 96 ° C. for 96 hours to form spores, dehydration, resin embedding and sectioning were performed according to a conventional method, followed by observation with an electron microscope. As shown in FIG. The formation of one amorphous parasporal inclusion consisting of the M019 strain crystal protein was observed in the sporangia, parallel to the spores of about 2.7 microns length.

[Example 2: Composition, structure and activation method of crystal protein]
The culture of Bacillus thuringiensis M019 strain (Accession No. FERM P-21398) was collected, and the sporangia were destroyed by ultrasonic waves to release parasporal inclusion bodies, and then washed thoroughly and lyophilized. Crystalline protein is alkaline buffer (50 mM NaHCO ₃ -Na ₂ CO ₃ + 1
It was selectively solubilized in mM EDTA + 10 mM dithiothreitol, pH 10.2) at 0 ° C. for 20 minutes. SDS-PAGE electrophoresis revealed the presence of at least two crystalline proteins with an apparent molecular weight of 84 kDa (CP84, about 30%) and 78 kDa (CP78, about 70%). In DEAE cellulose chromatography, CP84 was eluted with about 290 mM Tris (pH 8.3), and CP78 was eluted with about 370 mM Tris (pH 8.3). Both fractions were dialyzed against 200 mM Tris (pH 8.3), 2% by weight bovine pancreatic trypsin was added, and limited digestion was performed at 25 ° C. for 20 hours. From the CP84 fraction, an apparent molecular weight of 57 kDa (T57A) and from the CP78 fraction, 57 kDa (T57B, about 10%) and 53 kDa (T53, about 90%) active fragments appeared. This originally suggested the presence of three types of crystal proteins (about 30% CP84, about 63% CP78A and about 7% CP78B).

  Alternatively, trypsin activation of the total crystal protein of the M019 strain was immobilized on the DEAE cellulose column (in order to minimize the action of the M019 strain endogenous protease. Trypsin binds to DEAE cellulose. do not do). Once the active fragments that had been eluted were separated again by DEAE cellulose chromatography, T57A was about 80 mM Tris (pH 8.3), T57B was about 90 mM Tris (pH 8.3), and T57B was about 100 mM and about 120 mM Tris (pH 8.3). ) Eluted T53A and T53B, respectively. The reproducibility of the appearance amount of T53B was low.

  The results of active protein separation by SDS-PAGE analysis and chromatography are shown in FIG. SDS-PAGE electrophoresis of each fraction was transferred to a PVDF membrane (Immobilin-P, Millipore), and the N-terminal amino acid sequence was analyzed using protein sequencer Model G1000A (Hewlett-Packard). Although unidentified, the sequence of IAEPPSTGVITQFRILNDN (included in T57B N-terminal, SEQ ID NO: 4; see FIG. 8) from T57B is the same MAEPPSTGVITQFRILNDN (T53A / B N-terminal, SEQ ID NO: 2 from T53A and T53B). (See Fig. 8). From this, it was clarified that CP78 has two types of A and B having high homology with each other and gives T53 and T57B, respectively, upon activation with trypsin. The difference in structure between T53A and T53B is unknown. Re-chromatography gave a P1 fraction mainly composed of T57A component, a P2 fraction mainly composed of T53A, and a P3 fraction mainly composed of T53B, but the T57B component was not isolated.

In addition, the internal amino acid sequences of these proteins were determined. Proteins in the above P1, P2 or P3 fractions are denatured in 10% trichloroacetic acid, the precipitate is dissolved with 1 N NaOH, adjusted to pH 9 and digested with lysyl endopeptidase (EC 3.4.21.50). The resulting internal peptide was separated by C18 reverse phase HPLC. The amino acid sequence of the almost purified internal peptide was determined by the above method. From the P1 fraction, TVIPGGVIFSVTGNK (P1 # 65), IEDFNSQLNFALNSK (P1 # 61), LMNFGYSK (P1 # 44B), LDPTQINGDWK (P1 # 44A), DANAMDSFDTHPIISQK (P1 # 47A), FAIPDHK
(57k-A) and DIPNYEQVNNK (57k-B) sequences (both included in SEQ ID NO: 6) are P2
From fractions VRATYVNDYIGK (53k # 2), ALGAAGYAPNVVGVRYS (53k # 3) and YPGYK (53k # 1)
(Both included in common with SEQ ID NOs: 2 and 4. See FIG. 8) From the P3 fraction, the sequence of LHSVSAYGLSK (P3 # 32A) is included in common with SEQ ID NOs: 2 and 4. Reference) was obtained. No difference in separation pattern of internal peptides from P2 and P3 fractions by HPLC was found.

[Example 3: Cytotoxic effect of the protein of the present invention on various cell lines]
Various cell lines were cultured under the conditions shown in Table 1, and then dispensed into a 96-well multiwell plate at a concentration of 2.2 × 10 ⁵ cells / ml. The plate was further incubated for 24 hours and then used for testing. For each of these cultured cells, T57A (P1 fraction), T53A (P2 fraction), or T53B (P3 fraction) protein activated by the trypsin treatment of the present invention and all activated M019 strain proteins The cytotoxic activity of the mixture (All: containing T57A, T57B, T53A and T53B in the original crystal protein composition) was measured by observing cell shape change and cell viability by MTT method. That is, 10 μl of protein solutions of various concentrations were added to 90 μl of cultured cells in each well, and cell viability after 20 hours was measured using a commercially available CellTiter96 ^™ reagent (Promega Corp., WI, USA). . The strength of cytotoxic activity was calculated by the final protein concentration (EC ₅₀ ) required for 50% of the cells to be damaged compared to the control.

  The results are shown in Table 2. From the results shown in Table 2, the protein of the present invention is specific to specific cells such as HeLa cells (cervical cancer cells), MOLT-4 cells (leukemia T cells), HepG2 cells (liver hepatoma cells). It became clear that it showed cytotoxicity. In addition, the M019 strain crystal protein not treated with trypsin did not show cytotoxicity even at the maximum experimental concentration. The results of microscopic observation of HepG2 cells (liver cancer cells) damaged by T53A (P2 fraction) are shown in FIG.

[Example 4: Cloning of gene]
The total plasmid DNA of Bacillus thuringiensis M019 strain (Accession No. FERM P-21398) is extracted (at least 4 species with different lengths are observed) and then extracted with restriction enzymes Xba I or Sal I. digested, each of the resulting fragments were ligated to Bam HI- Xba I adapter DNA or Bam HI- Sal I adapter DNA, further creating a library and inserted into Bam HI cloning site of λEMBL3 phage DNA (Stratagene, USA) did. The probe DNA for cloning the target gene (CP84 gene, CP78A gene and CP78B gene) is digoxigenin-labeled DNA amplified by PCR combined with the mixed DNA primer created by reverse translating the amino acid sequence of the above internal peptide Used. By plaque hybridization with the above library, the XbλN53 # 13 (7.3 kb) fragment containing the upstream portion of the CP78B gene, the Xbλ # 9 (7.5 kb) fragment containing the downstream portion of the CP78B gene and the upstream portion of the CP78A gene, and the CP78A gene The Xbλ # 7 (17 kb) fragment containing the downstream part was cloned, while the XbλG3 # 7 (9.4 kb) fragment and the SalλG3 # 1 (9.3 kb) fragment containing the CP84 gene were cloned.

  FIG. 4 shows the restriction enzyme map of these M019 strain plasmid DNA fragments and the position of each gene. FIG. 5 shows the nucleotide sequence (SEQ ID NO: 1) of the ORF of the CP78A gene and its deduced amino acid sequence (SEQ ID NO: 2). The nucleotide sequence of the ORF of the CP78B gene (SEQ ID NO: 3) and its deduced amino acid sequence (SEQ ID NO: 4) are shown in FIG. FIG. 7 shows the nucleotide sequence of the ORF of the CP84 gene (SEQ ID NO: 5) and the deduced amino acid sequence (SEQ ID NO: 6). FIG. 8 shows that there is 83.5% homology between CP78A protein (SEQ ID NO: 2) and CP78B protein (SEQ ID NO: 4). The CP84 protein (SEQ ID NO: 6) is a protein of a type different from CP78A and CP78B as a whole, although only the first 50 amino acids are homologous with the CP78A and CP78B proteins.

[Example 5: Single expression of CP78A protein]
Plasmid DNA inserted between the Hin dIII and Sal I sites of pHY300PLK (Takara Shuzo), a shuttle vector of Escherichia coli and Bacillus subtilis Bacillus subtilis , containing an ORF (2,265 nucleotides) for CP78A protein (see Fig. 4). Created. With this DNA, a Bacillus thuringiensis mutant BFR1 strain that formed spores but did not produce crystal protein was transformed using electroporation. When this transformant was cultured on a BHI brain heart infusion medium (DIFCO) agar plate containing tetracycline, a cuboid crystal protein inclusion body was formed. When analyzed according to Example 2, a 78 kDa protein was produced. This protein produced a 53 kDa fragment upon activation with trypsin, and the EC50 value of cytotoxic activity against HeLa cells was 10 mg / ml. It showed the properties of CP78A protein both in structure and activity. In addition to the ORF, the 3,416 nucleotide region was found to contain a promoter, a ribosome binding region, and a transcription termination region.

It is a figure of the electron micrograph of M019 stock | strain which has formed the spore and parasporal crystal protein inclusion body. It is a figure which shows SDS-PAGE of the crystal protein of M019 strain | stump | stock, and its trypsin digest, the separation of a trypsin digest by DEAE cellulose chromatography, and the N terminal amino acid sequence of the main protein fragment in a trypsin digest. It is a figure which shows the HepG2 liver cancer cell damaged by the activation product T53A (P2 fraction) by trypsin of crystal protein CP78A of M019 strain. It is a figure which shows arrangement | positioning of the restriction enzyme map of the plasmid DNA fragment of M019 stock | strain, and a crystal protein gene. It is a figure which shows the gene nucleotide sequence and amino acid sequence of CP78A protein of M019 strain. It is a figure which shows the gene nucleotide sequence and amino acid sequence of CP78B protein of M019 strain. It is a figure which shows the gene nucleotide sequence and amino acid sequence of CP84 protein of M019 strain. It is a figure which shows that CP78A and CP78B protein of a M019 strain | stump | stock are mutually homologous proteins.

Claims (20)

  DNA consisting of the nucleotide sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5.   A DNA encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6.   A protein that hybridizes under stringent conditions with a DNA comprising a nucleotide sequence complementary to the DNA comprising the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5 and has cytotoxic activity in an active form DNA encoding   The DNA according to claim 3, wherein the cytotoxic activity is specific to at least one cell selected from the group consisting of leukemia cells, uterine cancer cells and liver cancer cells.   The DNA according to claim 3 or 4, wherein the DNA is derived from the genus Bacillus. DNA is Bacillus thuringiensis M019 strain (Accession number FERM P-21398)
The DNA according to claim 5, which is derived from   A vector containing the DNA according to any one of claims 1 to 6.   A transformant containing the vector according to claim 7.   A protein comprising the amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6.   A protein having cytotoxic activity of 53 kDa, obtained by treating a protein consisting of the amino acid sequence represented by SEQ ID NO: 2 with trypsin.   A protein having a cytotoxic activity of 57 kDa obtained by treating a protein consisting of the amino acid sequence represented by SEQ ID NO: 4 with trypsin.   A protein having a cytotoxic activity of 57 kDa, obtained by treating a protein consisting of the amino acid sequence represented by SEQ ID NO: 6 with trypsin. Modified by any one or more of amino acid deletion, substitution, insertion or addition within a range having 70% or more homology with any of the amino acid sequences shown in SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6 A protein having an amino acid sequence and having cytotoxic activity in an active form.   A protein having cytotoxic activity obtained by treating the protein of claim 13 with trypsin.   The protein according to claim 13 or 14, wherein the cytotoxic activity is specific to at least one cell selected from the group consisting of leukemia cells, uterine cancer cells and liver cancer cells.   The protein according to any one of claims 9 to 15, wherein the protein is derived from the genus Bacillus. Protein is Bacillus thuringiensis M019 strain (Accession number FERM P-213)
98) The protein according to claim 16, which is derived from 98).   The pharmaceutical composition containing the protein of any one of Claims 9-17.   The anticancer agent containing the protein of any one of Claims 9-17.   A method for producing the protein according to any one of claims 9 to 17, comprising the following steps: (a) culturing cells expressing the protein; and (b) recovering the protein; Including the method.