JP2008048601A - Production methods and systems for products useful for treatment - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a technique suitable for the production of a therapeutically useful gene product (for example, Hsp72, HLA, etc.). <P>SOLUTION: The method of producing a therapeutically useful gene product comprises: (A) the step of providing a material capable of producing the gene product; (B) the step of subjecting the material to a treatment temperature at which a donor of the material can fully exert the effect of a thermal therapy; and (C) the step of harvesting the gene product from the material. The system relating to the method is also provided. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a technique for producing a product useful for treatment.

  Conventionally, surgical treatment, radiation therapy, chemotherapy, and the like have been used to treat cancer, but these are invasive treatment methods that damage normal tissues of the human body. Damage is done. Moreover, the present condition is that there is no appropriate treatment method for intractable infectious diseases such as HIV. On the other hand, in recent years, using the difference in temperature sensitivity between normal cells and cancer cells and viruses, the affected area is heated to 41 ° C. to 43 ° C. to protect cancer cells and viruses while killing cancer cells and viruses. (Hyperthermia) has attracted attention as a non-invasive treatment method. Moreover, it has been confirmed that the therapeutic effect is enhanced by using hyperthermia in combination with radiation therapy or chemotherapy. And it is predicted that various gene products are involved in the therapeutic effect.

  The present invention is disclosed in JP 2003-126135 A (Patent Document 1), JP 2003-126138 A (Patent Document 2), and JP 2001-299798 A (Patent Document 3). It can be applied to intractable infectious diseases such as HIV, and deep body temperature is raised to the treatment temperature in a short time without exposing the donor to an unnecessarily high temperature. A thermotherapy device capable of treatment was provided.

  The temperature used for these thermotherapy was predicted to be useful for the production of various gene products involved in thermotherapy.

  However, the optimum temperature of the donor for maximizing the donor's thermotherapy effect varies depending on the individual donor, and even in the same donor, it may vary from day to day. The treatment temperature was set at a preferred temperature range.

  For this reason, there is a disadvantage that the heat treatment effect is not improved for the donor and the physical strength of the donor is consumed. For example, if the donor's treatment temperature is lower than the optimum temperature, the effect is not improved, and the burden time on the donor becomes longer. If the donor's treatment temperature is higher than the optimum temperature, the number of granulocytes is increased. There were drawbacks. It has also been reported that in some cases even harmful effects such as degeneration, necrosis, or disintegration of granulocytes are observed even when treated in a generally desirable temperature range.

There are not a few examples in which high-efficiency production is not always possible even when a temperature generally used for hyperthermia treatment is used to produce a gene product preferable for treatment. Accordingly, it has been awaited to provide a technique suitable for producing a gene product preferable for treatment.
JP 2003-126135 A JP 2003-126138 A JP 2001-299798 A

  The present invention has been made to eliminate the above-mentioned drawbacks, and the object of the present invention is to provide gene products useful for therapy (for example, Hsp72, HLA, Hsp10, Hsp27, Hsp47, Hsp40, Hsp60, Hsp90, Hsp73). , Hsp110, etc.).

  The present inventor has solved the above problems by finding that a gene product useful for treatment can be produced by using an optimum temperature for maximizing the effect of thermotherapy.

  Accordingly, the present invention provides the following.

In one aspect, the present invention is a method of producing a gene product useful for therapy comprising:
A) providing a producer having the ability to produce the gene product;
B) subjecting the producer to an optimal temperature for maximizing the thermotherapy effect of the producer's donor; and C) obtaining the gene product from the producer;
A method comprising:

  In one embodiment, the gene product is selected from the group consisting of Hsp72, HLA, Hsp10, Hsp27, Hsp47, Hsp40, Hsp60, Hsp90, Hsp73 and Hsp110.

  In one embodiment, the gene product is Hsp72.

  In one embodiment, the producer is a cell, tissue, organ or organism.

  In one embodiment, the producer comprises blood or lymph.

  In one embodiment, the producer is an artificial producer.

  In one embodiment, the producer comprises a nucleic acid sequence encoding the gene product.

  In one embodiment, the producer comprises cells transformed with a nucleic acid sequence encoding the gene product.

  In one embodiment, the gene product comprises SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19 , Encoded by the sequence set forth in SEQ ID NO: 21 or SEQ ID NO: 23.

In one embodiment, the optimum temperature is the following of the donor:
a) Expression level and function of Hsp72 in donor blood cells,
b) changes in donor blood pressure,
c) Donor heart rate,
d) donor blood flow,
e) pH of donor blood,
f) the amount of HLA expressed in the donor's blood cells, and g) the number of lymphocytes in the donor's blood,
It is determined based on a biological change selected from the group consisting of:

  In one embodiment, obtaining the gene product is accomplished by using affinity purification.

  In one embodiment, the affinity purification uses an antibody.

  In one embodiment, the method further includes the step of specifying the optimum temperature.

In one embodiment, at the specific time, the following:
a) Expression level and function of Hsp72 in donor blood cells,
b) changes in donor blood pressure,
c) Donor heart rate,
d) donor blood flow,
e) pH of donor blood,
f) the amount of HLA expressed in the donor's blood cells, and g) the number of lymphocytes in the donor's blood,
A step of observing a biological change selected from the group consisting of:

  In another aspect, the present invention relates to a gene product produced by the method of the present invention.

In another aspect, the invention provides a system for producing a gene product useful for therapy comprising:
A) a producer having the ability to produce the gene product;
B) means for subjecting the producer to an optimal temperature for maximizing the thermotherapy effect of the producer's donor; and C) means for obtaining the gene product from the producer;
A method comprising:

  In one embodiment, the system of the present invention further comprises D) means for identifying the optimum temperature.

  In one embodiment of the system of the invention, the gene product is selected from the group consisting of Hsp72, HLA, Hsp10, Hsp27, Hsp47, Hsp40, Hsp60, Hsp90, Hsp73 and Hsp110.

  In one embodiment of the system of the present invention, the gene product is Hsp72.

  In one embodiment of the system of the present invention, the producer is a cell, tissue, organ or organism.

  In one embodiment of the system of the invention, the producer comprises blood or lymph.

  In one embodiment of the system of the present invention, the producer is an artificial producer.

  In one embodiment of the system of the present invention, the producer comprises a nucleic acid sequence encoding the gene product.

  In one embodiment of the system of the present invention, the producer comprises a cell transformed with a nucleic acid sequence encoding the gene product.

  In one embodiment of the system of the present invention, the gene product comprises SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, encoded by the sequence set forth in SEQ ID NO: 19, SEQ ID NO: 21 or SEQ ID NO: 23.

In one embodiment of the system of the present invention, the optimum temperature is the following of the donor:
a) Expression level and function of Hsp72 in donor blood cells,
b) changes in donor blood pressure,
c) Donor heart rate,
d) donor blood flow,
e) pH of donor blood,
f) the amount of HLA expressed in the donor's blood cells, and g) the number of lymphocytes in the donor's blood,
The system of claim 16, wherein the system is determined based on a biotransformation selected from the group consisting of:

  In one embodiment of the system of the present invention, the means for obtaining the gene product includes an affinity purification means.

  In one embodiment of the system of the present invention, the affinity purification means comprises an antibody specific for the gene product.

In one embodiment of the system of the present invention, the means for identifying is:
a) Means for measuring the expression level and function of Hsp72 in donor blood cells,
b) means for measuring changes in the blood pressure of the donor,
c) means for measuring the heart rate of the donor,
d) means for measuring the blood flow of the donor,
e) means for measuring the pH of the donor's blood;
f) means for measuring the expression level of HLA in the donor's blood cells; and g) means for measuring the number of lymphocytes in the donor's blood.
Means for observing a biological change selected from the group consisting of:

  The above object, other objects, features and advantages of the present invention will become more apparent from the following detailed description of embodiments of the present invention with reference to the drawings.

  The present invention provides a technique for producing gene products useful for treatment in a natural form, in a normal form, and with an unprecedented high efficiency. Such techniques are useful in producing the treatment itself or a medicament for use in the treatment. Therefore, unlike in the case of expression in cells not derived from a donor such as E. coli, it has a high probability of being effective and has high safety because it does not contain extra peptides.

  Preferred embodiments of the present invention will be shown below, but those skilled in the art can appropriately implement the embodiments and the like from the description of the present invention and well-known and common techniques in the field, and the effects and effects of the present invention can be easily achieved. It should be recognized to understand.

  The invention will now be described by way of example, with reference to the accompanying drawings, where appropriate. Throughout this specification, it should be understood that the singular forms also include the plural concept unless specifically stated otherwise. Therefore, it should be understood that the expression of the singular includes the concept of the plural unless specifically stated otherwise. In addition, it is to be understood that the terms used in the present specification are used in the meaning normally used in the art unless otherwise specified. Thus, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control.

  The embodiments provided below are provided for a better understanding of the present invention, and it is understood that the scope of the present invention should not be limited to the following description. Therefore, it is obvious that those skilled in the art can make appropriate modifications within the scope of the present invention with reference to the description in the present specification.

(Definition)
(Biochemistry)
As used herein, the terms “protein”, “polypeptide”, “oligopeptide” and “peptide” are used interchangeably herein to refer to polymers of amino acids of any length and variants thereof. Say. This polymer may be linear, branched, or cyclic. The amino acid may be natural or non-natural and may be a modified amino acid. The term also includes what can be assembled into a complex of multiple polypeptide chains. The term also encompasses natural or artificially modified amino acid polymers. Such modifications include, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation or any other manipulation or modification (eg, conjugation with a labeling component). This definition also includes, for example, polypeptides containing one or more analogs of amino acids (eg, including unnatural amino acids, etc.), peptide-like compounds (eg, peptoids) and other modifications known in the art. Is included.

  In this specification, the comparison of similarity, identity and homology between amino acid sequences and base sequences is calculated using default parameters using BLAST, which is a sequence analysis tool.

  In the present specification, the “amino acid” may be natural or non-natural. “Derivative amino acid” or “amino acid analog” refers to an amino acid that is different from a naturally occurring amino acid but has the same function as the original amino acid. Such derivative amino acids and amino acid analogs are well known in the art.

  The term “natural amino acid” refers to the L-isomer of a natural amino acid. Natural amino acids are glycine, alanine, valine, leucine, isoleucine, serine, methionine, threonine, phenylalanine, tyrosine, tryptophan, cysteine, proline, histidine, aspartic acid, asparagine, glutamic acid, glutamine, γ-carboxyglutamic acid, arginine, ornithine , And lysine. Unless otherwise indicated, all amino acids referred to herein are L-forms, but forms using D-form amino acids are also within the scope of the present invention.

  As used herein, “amino acid variant” refers to a molecule that is not a natural amino acid but is similar to the physical properties and / or functions of a natural amino acid. Examples of amino acid modifications include those in which an alkyl group, a halo group, a nitro group, or the like is bonded to phenylalanine benzyl side chain (para-position, meta-position, ortho-position, etc.), ethionine, canavanine, 2-methylglutamine and the like. It is done. In the present invention, it is understood that amino acid variants may include unnatural amino acids and amino acid mimetics.

  As used herein, “unnatural amino acid” means an amino acid that is not normally found naturally in proteins. Examples of non-natural amino acids include norleucine, para-nitrophenylalanine, homophenylalanine, para-fluorophenylalanine, 3-amino-2-benzylpropionic acid, homo-arginine D-form or L-form and D-phenylalanine.

  Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides may also be referred to by a generally recognized one letter code.

The character codes are as follows.
3 letter code of amino acid 1 letter code Meaning Ala A alanine Cys C cysteine Asp D aspartic acid Glu E glutamic acid Phe F phenylalanine Gly G glycine His H histidine Ile I isoleucine Lys K lysine Leu L leucine Met M methionine Asl G Glutamine Arg R Arginine Ser S Serine Thr T Threonine Val V Valine Trp W Tryptophan Tyr Y Tyrosine Asx Asparagine or Aspartate Glx Glutamine or Glutamate Xaa Unknown or other amino acids.

Base symbol Meaning
a adenine g guanine c cytosine t thymine u uracil r guanine or adenine purine y thymine / uracil or cytosine pyrimidine m adenine or cytosine amino group k guanine or thymine / uracil keto group s guanine or cytosine w adenine or thymine / uracil b guanine or cytosine Thymine / uracil d adenine or guanine or thymine / uracil h adenine or cytosine or thymine / uracil v adenine or guanine or cytosine n adenine or guanine or cytosine or thymine / uracil, unknown or other base.

  In the present specification, the “corresponding” amino acid or nucleic acid has or has the same action as a predetermined amino acid in a reference polypeptide or polynucleotide in a polypeptide molecule or polynucleotide molecule, respectively. In particular, in the case of an enzyme molecule, it means an amino acid that is present at the same position in the active site and contributes to the catalytic activity in the same way. For example, an antisense molecule of a polynucleotide can be a similar part in an ortholog corresponding to a particular part of the antisense molecule. In the case of the peptide of the present invention, a specific sequence in the human cell growth factor is used. In the specific sequence in the cell growth factor of other species of animals, the portion corresponding to the peptide of the present invention corresponds to the “corresponding amino acid”. Is understood.

  In the present specification, the “corresponding” gene refers to a gene having, or expected to have, the same action as that of a predetermined gene in a species as a reference for comparison in a certain species. When there are a plurality of genes having the same, those having the same origin evolutionarily. Thus, the corresponding gene of a gene can be an ortholog of that gene. For example, the gene corresponding to mouse cell growth factor is human cell growth factor.

  In the present specification, the “fragment” refers to a polypeptide or polynucleotide having a sequence length of 1 to n−1 with respect to a full-length polypeptide or polynucleotide (length is n). The length of the fragment can be appropriately changed according to the purpose. For example, the lower limit of the length is 3, 4, 5, 6, 7, 8, 9, 10, Examples include 15, 20, 25, 30, 40, 50 and more amino acids, and lengths expressed in integers not specifically listed here (eg, 11 etc.) are also suitable as lower limits. obtain. In the case of polynucleotides, examples include 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100 and more nucleotides. Non-integer lengths (eg, 11 etc.) may also be appropriate as a lower limit. In the present invention, when a polypeptide or a polynucleotide is used as a biomolecule, such a fragment is also used in the same manner as the full-length one as long as a desired purpose (for example, a cell attracting effect) is achieved. It is understood that it can be done.

  In the present specification, the lengths of polypeptides and polynucleotides can be represented by the number of amino acids or nucleic acids, respectively, as described above, but the above numbers are not absolute, and the upper limit is not limited as long as they have the same function. Alternatively, the above-mentioned number as an adjustment is intended to include a number above and below the number (or, for example, 10% above and below). In order to express such intention, in this specification, “about” may be added before the number. However, it should be understood herein that the presence or absence of “about” does not affect the interpretation of the value.

  As used herein, “biological activity” refers to an activity that a certain factor (eg, polypeptide or protein) may have in vivo, and includes an activity that exhibits various functions. For example, when a certain factor is an antisense molecule, its biological activity includes binding to a nucleic acid molecule of interest, thereby suppressing expression. For example, when a factor is an enzyme, the biological activity includes the enzyme activity. In another example, when an agent is a ligand, the ligand includes binding to the corresponding receptor. Such biological activity can be measured by techniques well known in the art.

  As used herein, the terms “polynucleotide”, “oligonucleotide”, and “nucleic acid” are used interchangeably herein and refer to a polymer of nucleotides of any length. The term also includes “derivative oligonucleotide” or “derivative polynucleotide”. A “derivative oligonucleotide” or “derivative polynucleotide” refers to an oligonucleotide or polynucleotide that includes a derivative of a nucleotide or that has an unusual linkage between nucleotides and is used interchangeably. Specific examples of such an oligonucleotide include, for example, 2′-O-methyl-ribonucleotide, a derivative oligonucleotide in which a phosphodiester bond in an oligonucleotide is converted to a phosphorothioate bond, and a phosphodiester bond in an oligonucleotide. Is an N3′-P5 ′ phosphoramidate bond derivative oligonucleotide, a ribose and phosphodiester bond in the oligonucleotide converted to a peptide nucleic acid bond, uracil in the oligonucleotide is C− A derivative oligonucleotide substituted with 5-propynyluracil, a derivative oligonucleotide where uracil in the oligonucleotide is substituted with C-5 thiazole uracil, and cytosine in the oligonucleotide is C-5 propynylcytosine Substituted derivative oligonucleotides, derivative oligonucleotides in which the cytosine in the oligonucleotide is replaced with phenoxazine-modified cytosine, derivative oligonucleotides in which the ribose in the DNA is replaced with 2'-O-propylribose Examples thereof include derivative oligonucleotides in which ribose in the oligonucleotide is substituted with 2′-methoxyethoxyribose. Unless otherwise indicated, a particular nucleic acid sequence may also be conservatively modified (eg, degenerate codon substitutes) and complementary sequences, as well as those explicitly indicated. Is contemplated. Specifically, a degenerate codon substitute creates a sequence in which the third position of one or more selected (or all) codons is replaced with a mixed base and / or deoxyinosine residue. (Batzer et al., Nucleic Acid Res. 19: 5081 (1991); Ohtsuka et al., J. Biol. Chem. 260: 2605-2608 (1985); Rossolini et al., Mol. Cell. Probes 8: 91-). 98 (1994)).

  As used herein, “nucleic acid molecule” is used interchangeably with nucleic acid, oligonucleotide, and polynucleotide, and includes cDNA, mRNA, genomic DNA, and the like. Nucleic acid molecules can be provided in circular (eg, circular vectors, plasmids, etc.) or linear (eg, PCR fragments). As used herein, nucleic acids and nucleic acid molecules can be included within the concept of the term “gene”. Nucleic acid molecules encoding certain gene sequences also include “splice variants”. Similarly, a particular protein encoded by a nucleic acid encompasses any protein encoded by a splice variant of that nucleic acid. As the name suggests, a “splice variant” is the product of alternative splicing of a gene. After transcription, the initial nucleic acid transcript can be spliced such that different (another) nucleic acid splice products encode different polypeptides. The production mechanism of splice variants varies, but includes exon alternative splicing. Other polypeptides derived from the same nucleic acid by read-through transcription are also encompassed by this definition. Any product of a splicing reaction (including recombinant forms of splice products) is included in this definition.

  As used herein, “gene” refers to a factor that defines a genetic trait. Usually arranged in a certain order on the chromosome. Those that define the primary structure of a protein are called structural genes, and those that influence the expression are called regulatory genes (for example, promoters). In the present specification, genes include structural genes and regulatory genes unless otherwise specified. As used herein, “gene” may refer to “polynucleotide”, “oligonucleotide” and “nucleic acid” and / or “protein”, “polypeptide”, “oligopeptide” and “peptide”. As used herein, “gene product” also refers to “polynucleotide”, “oligonucleotide” and “nucleic acid” and / or “protein” “polypeptide”, “oligopeptide” and “peptide” expressed by a gene. Is included. A person skilled in the art can understand what a gene product is, depending on the situation. It is understood that the gene product of the present invention refers to all such gene products.

  As used herein, “homology” of genes (eg, nucleic acid sequences, amino acid sequences, etc.) refers to the degree of identity of two or more gene sequences with each other. Therefore, the higher the homology between two genes, the higher the sequence identity or similarity. Whether two genes have homology can be examined by direct sequence comparison or, in the case of nucleic acids, hybridization methods under stringent conditions. When directly comparing two gene sequences, the DNA sequence between the gene sequences is typically at least 50% identical, preferably at least 70% identical, more preferably at least 80%, 90% , 95%, 96%, 97%, 98% or 99% are identical, the genes are homologous. In the present specification, “similarity” of genes (for example, nucleic acid sequences, amino acid sequences, etc.) refers to two or more gene sequences when the conservative substitution is regarded as positive (identical) in the above homology. The degree of identity with each other. Thus, if there is a conservative substitution, identity and similarity differ depending on the presence of the conservative substitution. When there is no conservative substitution, identity and similarity indicate the same numerical value.

  In this specification, the comparison of similarity, identity and homology between amino acid sequences and base sequences is calculated using default parameters using BLAST, which is a sequence analysis tool.

  As used herein, “terminator” is a sequence that is located downstream of a region encoding a protein of a gene and is involved in termination of transcription when DNA is transcribed into mRNA, and addition of a poly A sequence. It is known that the terminator is involved in the stability of mRNA and affects the expression level of the gene.

  In this specification, “promoter” refers to a region on DNA that determines the transcription start site of a gene and directly regulates its frequency, and is usually a base sequence that initiates transcription upon binding of RNA polymerase. . Therefore, in this specification, a part having a promoter function of a gene is referred to as a “promoter part”. Since the promoter region is usually a region within about 2 kbp upstream of the first exon of the putative protein coding region, if the protein coding region in the genomic base sequence is predicted using DNA analysis software, The promoter region can be estimated. The putative promoter region varies for each structural gene, but is usually upstream of the structural gene, but is not limited thereto, and may be downstream of the structural gene. Preferably, the putative promoter region is present within about 2 kbp upstream from the first exon translation start point. In the present specification, for example, a T7 promoter or the like can be used.

  As used herein, “enhancer” refers to a sequence used to increase the expression efficiency of a target gene. Such enhancers are well known in the art. A plurality of enhancers may be used, but one may be used or may not be used.

  As used herein, “operably linked” refers to a transcriptional translational regulatory sequence (eg, promoter, enhancer, etc.) or a translational regulatory sequence that has the expression (operation) of a desired sequence. That means. In order for a promoter to be operably linked to a gene, the promoter is usually placed immediately upstream of the gene, but need not necessarily be adjacent.

  In this specification, any technique may be used for introducing a nucleic acid molecule into a cell, and examples thereof include transformation, transduction, and transfection. Such a technique for introducing a nucleic acid molecule is well known in the art and commonly used. For example, Ausubel F. et al. A. (1988), Current Protocols in Molecular Biology, Wiley, New York, NY; Sambrook J et al. (1987) Molecular Cloning: A Laboratory Manual, 2nd Ed. And its third edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, a separate volume of experimental medicine “Gene Transfer & Expression Analysis Experimental Method” Yodosha, 1997, and the like. Gene transfer can be confirmed using the methods described herein, such as Northern blot, Western blot analysis, or other well-known conventional techniques.

  Moreover, as a method for introducing a vector, any of the above-described methods for introducing DNA into cells can be used. For example, transfection, transduction, transformation, etc. (for example, calcium phosphate method, liposome method, DEAE dextran method, electroporation method, method using particle gun (gene gun), etc.).

  In the present specification, when referring to a gene, “vector” or “recombinant vector” refers to a vector capable of transferring a target polynucleotide sequence to a target cell. Such vectors can be autonomously replicated in donor cells such as prokaryotic cells, yeast, animal cells, plant cells, insect cells, individual animals, and individual plants, or can be integrated into chromosomes. Examples include those containing a promoter at a position suitable for nucleotide transcription. Among vectors, a vector suitable for cloning is called “cloning vector”. Such cloning vectors usually contain multiple cloning sites that contain multiple restriction enzyme sites. Such restriction enzyme sites and multiple cloning sites are well known in the art, and those skilled in the art can appropriately select and use them according to the purpose. Such techniques are described in the literature described herein (eg, Sambrook et al., Supra).

  As used herein, the term “expression vector” refers to a nucleic acid sequence in which various regulatory elements are operably linked in a donor cell in addition to a structural gene and a promoter that regulates its expression. Regulatory elements can preferably include terminators, selectable markers such as drug resistance genes, and enhancers. It is well known to those skilled in the art that the type of organism (eg, animal) expression vector and the type of regulatory element used can vary depending on the donor cell or protein expression system.

  As used herein, “transformant” refers to all or part of a living organism such as a cell produced by transformation. Examples of the transformant include prokaryotic cells, yeast, animal cells, plant cells, insect cells and the like. A transformant is also referred to as a transformed cell, transformed tissue, transformed donor, or the like depending on the subject. The cell used in the present invention may be a transformant.

  When prokaryotic cells are used in genetic manipulation or the like in the present invention, prokaryotic cells include, for example, prokaryotic cells belonging to the genus Escherichia, Serratia, Bacillus, Brevibacterium, Corynebacterium, Microbacterium, Pseudomonas, etc. Escherichia coli XL1-Blue, Escherichia coli XL2-Blue, Escherichia coli DH1 are exemplified.

  As used herein, “detection” or “quantification” of gene expression (eg, mRNA expression, protein expression) can be accomplished using suitable methods including, for example, mRNA measurement and immunoassay methods. Examples of molecular biological measurement methods include Northern blotting, dot blotting, and PCR. Examples of the immunological measurement method include an ELISA method using a microtiter plate, an RIA method, a fluorescent antibody method, a Western blot method, and an immunohistochemical staining method. Examples of the quantitative method include an ELISA method and an RIA method. It can also be performed by a gene analysis method using an array (eg, DNA array, protein array). The DNA array is widely outlined in (edited by Shujunsha, separate volume of cell engineering "DNA microarray and latest PCR method"). For protein arrays, see Nat Genet. 2002 Dec; 32 Suppl: 526-32. Examples of gene expression analysis methods include, but are not limited to, RT-PCR, RACE method, SSCP method, immunoprecipitation method, two-hybrid system, in vitro translation and the like. Such further analysis methods are described in, for example, Genome Analysis Experimental Method / Yusuke Nakamura Lab Manual, Editing / Yusuke Nakamura Yodosha (2002), etc., all of which are incorporated herein by reference. Is done.

  In the present specification, the “variant” refers to a substance in which a part of the original substance such as a polypeptide or polynucleotide is changed. Such variants include substitutional variants, addition variants, deletion variants, truncated variants, allelic variants, and the like. Such a variant can also be used as the cell growth factor of the present invention as long as the desired purpose can be achieved. An allele refers to genetic variants that belong to the same locus and are distinguished from each other. Therefore, an “allelic variant” refers to a variant having an allelic relationship with a certain gene. Such allelic variants usually have the same or very similar sequence as their corresponding alleles, usually have nearly the same biological activity, but rarely have different biological activities. May have. “Species homologue or homolog” means homology (preferably at least 60% homology, more preferably at least 80%, at a certain amino acid level or nucleotide level within a certain species. 85% or higher, 90% or higher, 95% or higher homology). The method for obtaining such species homologues will be apparent from the description herein. “Ortholog” is also called an orthologous gene, which is a gene derived from speciation from a common ancestor with two genes. For example, taking the hemoglobin gene family with multiple gene structures as an example, the human and mouse alpha hemoglobin genes are orthologs, but the human alpha and beta hemoglobin genes are paralogs (genes generated by gene duplication). . Orthologs are useful for estimating molecular phylogenetic trees. Orthologs of genes useful for the treatment of the present invention can also be useful in the present invention, since orthologs can usually perform the same function as the original species in another species.

  As used herein, “substitution, addition or deletion” of a polypeptide or polynucleotide is a substitution of an amino acid or a substitute thereof, or a nucleotide or a substitute thereof, respectively, with respect to the original polypeptide or polynucleotide. , Adding or removing. Such substitution, addition, or deletion techniques are well known in the art, and examples of such techniques include site-directed mutagenesis techniques. There may be any number of substitutions, additions or deletions, as long as it is one or more, and such numbers may be used for the desired function (eg, angiogenesis, cell regeneration) in a variant having that substitution, addition or deletion. Etc.) as long as it is retained. For example, such a number can be one or several, and preferably can be within 20%, within 10%, or less than 100, less than 50, less than 25, etc. of the total length.

(Gene products useful for treatment)
As used herein, a “gene product useful for treatment” refers to any gene product known to play a positive role in the healing of the body. Examples of such gene products include Hsp (for example, Hsp72, Hsp10, Hsp27, Hsp47, Hsp40, Hsp60, Hsp90 (for example, Hsp90α or Hsp90β), Hsp73, Hsp110, etc.), HLA, etc. It is not limited to.

  As used herein, “Hsp” refers to a heat shock protein. Hsp is a general term for a group of proteins whose synthesis is induced when a cell or an individual undergoes a temperature change higher than the normal temperature. Representative Hsps include, in addition to Hsp72, for example, Hsp70, Hsp10, Hsp27, Hsp47, Hsp40, Hsp60, Hsp90 (eg, Hsp90α or Hsp90β), Hsp73 and Hsp110, ubiquitin and Hsp26, etc. Not. “Heat shock protein” or “Hsp” refers to a group of proteins whose synthesis is induced when cells / tissues or individuals are exposed to a temperature (heat shock) of 5 to 10 ° C. higher than a physiological temperature. It is a typical example of stress protein and is said to be common to all living organisms from E. coli to plants and higher vertebrates. Hsp is expressed with a number of 1000 units of its molecular weight. For example, a typical heat shock protein having a molecular weight of 70,000 is referred to as Hsp70. Strictly speaking, this nomenclature may mark up to one place. The synthesis of heat shock proteins is induced not only by high temperatures but also by stress on various cells such as heavy metals, ethanol, and oxygen deficiency. Stress, including high temperature, denatures cellular proteins and forms insoluble precipitates that can cause fatal damage to cells. Heat shock proteins have a function to eliminate this harm. It is divided into the following two types according to the mechanism of action. (1) A protein that binds to a protein having an abnormal structure, prevents precipitation, and at the same time assists its regeneration. Hsp70 (DnaK in E. coli) and Hsp60 (GroEL) correspond to this. (2) An enzyme-based protein that selectively degrades denatured protein. Ubiquitin, which is commonly found in eukaryotes and binds to denatured proteins and serves as a label for degradation. Expression of a gene encoding a heat shock protein is caused by the function of a heat shock activated transcription factor (HSF). This induction is mainly due to the regulation of the transcription level, but it also involves the mechanism of the translation level, and may be synthesized in large quantities and accumulated in cells in a short time. In addition, the function of assisting regeneration of the denatured protein possessed by the heat shock protein is essential for normal cells, and therefore it is said that there are many genes that encode this gene that need to be expressed without stress. Many genes of heat shock proteins overlap during the evolution process and show isotypes. Even if they encode proteins with the same function, expression is induced only by heat shock, and other genes are constitutively expressed. ing.

  In the present specification, the heat shock protein is also referred to as a molecular chaperone. Proteins are linear polymers of amino acids but are folded into a three-dimensional structure unique to individual proteins. A protein that catalytically assists this folding is called a chaperone. If a protein shows such an action, its property is called a chaperone action. The chaperone does not interact with and is not a component of the folded protein. The chaperone binds to the denatured protein and prevents the denatured proteins from aggregating and irreversibly detaching from the original folding pathway, thus increasing the correct folding yield. It has been. Examples of chaperones include Hsp100 (ClpA, B), Hsp90 (HtpG), Hsp70 (DnaK, Bip), Hsp60 (GroEL, chaperonin), Hsp40 (DnaJ), Hsp10 (GroES, cpn10), etc. Groups, but not limited to. Many are heat shock proteins and can be used redundantly herein unless otherwise noted. When the cells are exposed to high temperatures and the protein is about to denature, the increased production chaperone binds to it and prevents aggregation. In addition, when the protein is transported and passes through the membrane, it is transported in an elongated string shape, so that the chaperone is bound and the protein is drawn beforehand. In this case, it is not a folding promotion but a pause, but this can also be included in the chaperone action. ATP is required for actions such as Hsp60 and Hsp70. Hsp60 can assist in the folding of a very wide variety of proteins, while there are chaperones that only work on collagen, such as Hsp47. Among the chaperones, Hsp60 is particularly important and is present in cells of all living organisms, and this deletion mutation is lethal.

  As used herein, “Hsp72” refers to a heat shock protein typically represented by SEQ ID NO: 1 (nucleic acid) and SEQ ID NO: 2 (amino acid). As used herein, it is understood that variants of the above sequences can also be used as long as they have the action of Hsp72 (eg, molecular chaperones, etc.).

  As used herein, “HLA” refers to human leukocyte antigen. HLA antigens are roughly divided into two groups, class I antigens and class II antigens. Examples of class I antigens include HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G. Class I antigens are expressed on almost all nucleated cells. Class I antigens form complexes with intracellularly produced peptides and present the complexes to the antigen-specific T cell receptor of CD8 positive cytotoxic T cells. Examples of class II antigens include HLA-DR, HLA-DQ, HLA-DP, and the like. Class II antigens are expressed only on cells such as macrophages, B cells, activated T cells, dendritic cells, and thymic epithelial cells, and present foreign antigen-derived peptides to CD4-positive helper T cells. Class II antigens are known to be involved in positive and negative selection of T cells in the thymus as well as antigen presentation. “HLA” is a human major histocompatibility antigen gene complex, which is located in a gene region existing on the short arm of chromosome 6 over a region of 1.8 cM in length. To date, at least six loci or regions of HLA-A, B, C, DR, DP, and DQ have been identified. The HLA-A, B, and C genes encode HLA-A, B, and C antigens, respectively. Each gene exhibits a very high degree of polymorphism and there are dozens of alleles. In the HLA-DR, DP, and DQ regions, there are structural loci of the α chain and β chain of the HLA-D antigen (= Ia antigen), and usually the α chain and β chain gene products in the same region bind to each other. To make D antigen (DR, DP, DQ antigen). There are one α chain and β chain functional genes in the DP and DQ regions, respectively, but one α chain gene and at least three β chain genes have been identified in the DR region. There are also many alleles at each of these loci. One set of specific allele combinations of the HLA-A, B, C, DR, DP, and DQ loci is referred to as an HLA haplotype. Thus, a single human will have two HLA haplotypes derived from the father and mother. There is a strong linkage disequilibrium between specific alleles at each HLA locus, and certain HLA haplotypes are frequently found in certain populations. In particular, it has been found that there are significant racial differences in linkage disequilibrium between HLA loci and the frequency of alleles at each locus. In addition, a statistically significant correlation was found between a specific allele of the HLA gene and several intractable refractory diseases (eg, HLA-B27 and ankylosing myelitis). The relationship can be used to diagnose a disease. Exemplary gene sequences can include, but are not limited to, SEQ ID NOs: 3 and 4 (nucleic acid and amino acid sequences, respectively).

  As used herein, “Hsp10” refers to a heat shock protein typically represented by SEQ ID NO: 5 (nucleic acid) and SEQ ID NO: 6 (amino acid). In the present specification, it is understood that variants of the above sequences can also be used as long as they have the action of Hsp10 (eg, molecular chaperones).

  As used herein, “Hsp27” refers to a heat shock protein typically represented by SEQ ID NO: 7 (nucleic acid) and SEQ ID NO: 8 (amino acid). In the present specification, it is understood that variants of the above sequences can also be used as long as they have the action of Hsp27 (eg, molecular chaperones).

  As used herein, “Hsp40” refers to a heat shock protein typically represented by SEQ ID NO: 9 (nucleic acid) and SEQ ID NO: 10 (amino acid). As used herein, it is understood that variants of the above sequences can also be used as long as they have the action of Hsp40 (eg, molecular chaperones, etc.).

  As used herein, “Hsp47” (also referred to as CBP2) refers to a heat shock protein typically represented by SEQ ID NO: 11 (nucleic acid) and SEQ ID NO: 12 (amino acid). It is understood herein that variants of the above sequences can also be used so long as they have Hsp47 activity (eg, molecular chaperones, etc.).

  As used herein, “Hsp60” refers to a heat shock protein typically represented by SEQ ID NO: 13 (nucleic acid) and SEQ ID NO: 14 (amino acid). As used herein, it is understood that variants of the above sequences can also be used as long as they have the action of Hsp60 (eg, molecular chaperones, etc.).

  As used herein, “Hsp70” refers to a heat shock protein typically represented by SEQ ID NO: 15 (nucleic acid) and SEQ ID NO: 16 (amino acid). As used herein, it is understood that variants of the above sequences can also be used so long as they have Hsp70 activity (eg, molecular chaperones).

  As used herein, “Hsp73” refers to a heat shock protein typically represented by SEQ ID NO: 17 (nucleic acid) and SEQ ID NO: 18 (amino acid). In the present specification, it is understood that variants of the above sequences can also be used as long as they have the function of Hsp73 (for example, molecular chaperones).

  As used herein, “Hsp90” is representative of SEQ ID NO: 19 (nucleic acid = Hsp90α) and SEQ ID NO: 20 (amino acid = Hsp90α), and SEQ ID NO: 21 (nucleic acid = Hsp90β) and SEQ ID NO: 22 (amino acid = Hsp90β). It refers to the heat shock protein shown in As used herein, it is understood that variants of the above sequences can also be used so long as they have the action of Hsp90 (eg, molecular chaperones).

  As used herein, “Hsp110” refers to a heat shock protein typically represented by SEQ ID NO: 23 (nucleic acid) and SEQ ID NO: 24 (amino acid). As used herein, it is understood that variants of the above sequences can also be used so long as they have the action of Hsp110 (eg, molecular chaperones).

  As used herein, “producer” refers to all or part of an organism that can produce a gene product, or an artificial product thereof. Examples of such include, but are not limited to, cells, tissues, organs or organisms. Alternatively, examples of artificial products include, but are not limited to, cell-free systems, transformed cells, tissues, organs, or organisms. Preferably, such producers can be provided in a naturally occurring state. Thus, for example, it is understood that body fluids, blood, lymph, etc. can also be used as producers of the present invention.

  As used herein, “donor” refers to a subject from which the producer of the present invention is derived. As the donor, any organism having a blood system can be used. Preferably, vertebrates are used, and more preferably mammals (eg, primates, rodents, etc.) are used. More preferably, primates are used. Most preferably, a human is used.

  As used herein, “optimum temperature (also referred to as set point)” refers to a) Hsp72, HLA, Hsp10, Hsp27, Hsp47, Hsp40, Hsp60, Hsp90, Hsp73 in donor blood cells and Expression level and function of proteins such as Hsp110, b) changes in donor blood pressure, c) donor heart rate, d) donor blood flow, e) donor blood pH, f) HLA levels in donor blood cells Expression level, g) Refers to the donor's body temperature at which changes such as the number of lymphocytes in the donor's blood occur in the donor's blood. The “optimum temperature” is generally in the range of 39 ° C. ± 1.0 ° C. and varies depending on the individual and the health condition of the individual. The donor's body temperature can be measured, for example, in the armpit, sublingual, rectum, deep subcutaneous, intraarterial, tympanic membrane, and esophagus. Donor body temperature can preferably be measured in the sublingual and rectal, and most preferably in the rectum.

  When the donor is gradually heated, the following changes occur around the optimum temperature.

The expression level of Hsp72 gradually increases until the body temperature reaches the optimum temperature, and decreases when the body temperature exceeds the optimum temperature. The blood pressure gradually increases until the body temperature reaches the optimum temperature, and decreases when the body temperature exceeds the optimum temperature. The heart rate gradually increases until the body temperature reaches the optimum temperature, and decreases when the body temperature exceeds the optimum temperature.
The blood flow gradually increases until the body temperature reaches the optimum temperature, and decreases when the body temperature exceeds the optimum temperature.

  When the body temperature decreases until the body temperature reaches the optimum temperature, the blood pH increases when the body temperature exceeds the optimum temperature, and when the body temperature increases until the body temperature reaches the optimum temperature, the body temperature exceeds the optimum temperature. And decrease.

  The expression level of HLA gradually increases until the body temperature reaches the optimum temperature, and then reaches a plateau.

  It was unexpectedly found that the temperature at which the parameter changes in vitro and the temperature at which the parameter changes in vivo are almost the same. Therefore, it is not necessary to observe the change of the parameter in vivo. The optimum temperature of the donor is determined by using Hsp72 or HLA, cell size, and cell density produced by varying the temperature of peripheral blood leukocytes collected from the donor in vitro.

(Assay for detecting and quantifying the expression of Hsp72 and HLA in peripheral blood leukocytes)
For expression of Hsp72, HLA, Hsp10, Hsp27, Hsp47, Hsp40, Hsp60, Hsp90, Hsp73, Hsp110, etc. in peripheral blood leukocytes, use conventional Western blotting, ELISA, real-time RT-PCR, Northern blotting, etc. Can be detected and quantified. The number of peripheral blood cells required for this is easily determined by those skilled in the art, but is preferably 8 × 10 ⁵ to 10 × 10 ⁵ cells. HLA expression on peripheral blood leukocyte cell membranes can be detected and quantified by using conventional FACS analysis, fluorescence microscopy, and the like. The number of peripheral blood cells required for this is easily determined by those skilled in the art, but is preferably 5 × 10 ⁵ to 10 × 10 ⁵ cells.

  The means (A) for specifying the optimum temperature of the donor may be directly connected to the control device (D) so as to input a signal of the optimum temperature of the donor to the control device in real time, or the control device. Or a signal of the optimum temperature may be input to the control device indirectly.

  The temperature raising program for specifying the optimum temperature of the donor is created, for example, with the following points in mind.

  When the difference between the chamber (hot water temperature) and the body temperature is large, the rate of temperature increase is fast, and when the difference between the chamber (hot water temperature) and the body temperature is small, the temperature increase rate is set to be slow.

  From the point where the rectal temperature rises rapidly (the point where the infiltration temperature from the body surface coincides with the temperature rise due to blood circulation), the temperature control is switched to one with a small specific heat.

  A temperature slightly lower than the target temperature (for example, when the target rectal temperature is 42.0 ° C., the hot water temperature is 41.8 ° C.) is maintained. The reason for this is the use of the heat pool (preheating) effect. The hot water temperature is 41.8 ° C, but the rectal temperature is 42.0 ° C.

  The holding time takes into account the balance between the heat release rate (1) due to the temperature difference from the body surface temperature to the hot water temperature and the acceleration of rectal temperature rise (2). The case where the hot water temperature is set low is when the speed (2) is higher than the speed (1). The balance of speed (1) and speed (2) will reverse from equilibrium over time. In this case, the hot water temperature may be heated to suppress the speed (1), or the speed (2) may be increased in advance so as not to reverse.

  Thus, based on the difference between the chamber (hot water temperature) and body temperature, the temperature is adjusted by adjusting the balance between the speed (1) and the speed (2) by calculation.

  A series of operations from the start of treatment of the main body mechanism to the end of treatment will be described, for example, as follows.

  First, the optimal temperature of the donor for maximizing the donor's thermotherapy effect is specified. It is preferable that the means (A) for specifying the optimum temperature of the donor is performed using Hsp72 or hHLA, whose production amount varies by changing the temperature of peripheral blood leukocytes collected from the donor in vitro, as an index. In this case, specifically, the donor's blood (2-3 ml) is collected and, as shown in FIG. It is also called a set point). In this way, the optimum heating condition for each donor is determined (that is, an in vivo treatment program can be created), and the control device is controlled based on the simulation of the heating effect.

  As used herein, a protein producer includes a translation enzyme (typically ribosomal RNA = rRNA or a ribosome containing the same) and other regulatory elements for translating a set of tRNA and mRNA necessary for translation.

  As used herein, “transcription” is used in the same meaning as commonly used in the art, and refers to a reaction in which the nucleotide sequence of a gene DNA is copied as complementary RNA. This reaction is usually performed in vivo (intracellular), but may be performed in a cell-free system as long as necessary materials are present. Such cell-free systems can include DNA-dependent RNA polymerases and their cofactors and / or regulators known in the art, as well as the necessary nucleotides and the like. The reverse transcription reaction (DNA synthesis reaction using RNA as a template) is referred to as “reverse transcription” in the present specification.

  In the present specification, “translation” refers to a process of reading a base sequence on mRNA and selecting an amino acid corresponding to the information to form a peptide chain during protein biosynthesis or artificial synthesis. Translation is also usually performed in vivo (intracellular), but can also be performed in a cell-free system as long as the necessary materials are present. Such a cell-free system is referred to herein as a “protein synthesis system”. When performed in vivo, mRNA binds to ribosomes, and amino acids are sequentially linked by aminoacyl-tRNAs each having a structure corresponding to a codon (triple base) on the mRNA. Cell-free systems include ribosome systems removed from cells and other necessary amino acids, and these systems can be performed in vitro.

  As used herein, “isolation” of a substance (eg, a biological factor such as a cell, nucleic acid or protein) refers to other substance (eg, a biological factor in the cell in which the biological factor naturally occurs) In the case of a sugar chain or sugar chain-containing substance, a factor other than the sugar chain or sugar chain-containing substance, or a sugar chain or sugar chain-containing substance other than the target sugar chain or sugar chain-containing substance; Nucleic acid containing nucleic acid sequence other than nucleic acid and non-target nucleic acid; in the case of protein, substantially separated or purified from non-protein factor and protein containing amino acid sequence other than target protein) Say. The “isolated” sugar chain or sugar chain-containing substance includes a sugar chain or sugar chain-containing substance purified by the purification method of the present invention. Therefore, an isolated sugar chain or sugar chain-containing substance includes a chemically synthesized sugar chain or sugar chain-containing substance.

  In the present specification, “purification” of a substance (for example, a biological factor such as a sugar chain, a nucleic acid, or a protein) refers to removing at least a part of the factor that naturally accompanies the substance. Therefore, purification and separation partially overlap in form. Therefore, the purity of the substance in a purified substance (eg, a biological agent such as a sugar chain or a sugar chain-containing substance) is usually higher (ie, concentrated) than the state in which the substance is normally present. However, as long as naturally associated factors are reduced, unconcentrated states are also included in the concept of “purification”.

  In this specification, “concentration” of a substance (for example, a biological agent such as a sugar chain or a sugar chain-containing substance) means that the substance is raised to a concentration higher than the content contained in the sample before concentration. The act of letting go. Concentration therefore also overlaps in part with the concept of purification and separation. Therefore, a concentrated substance (eg, a biological agent such as a sugar chain or a sugar chain-containing substance) is usually intended to have a reduced content of impurities in the state in which the substance is normally present. As long as the content of the substance is increased, certain impurities may be increased, and the state of not being “purified” is also included in the concept of “concentration”.

(Producing method of protein when using cell system)
Derived from a producer having the ability to produce a gene product useful for the treatment of the present invention, or an animal cell having a recombinant vector incorporating a DNA encoding the gene product useful for the treatment, if necessary. The transformant is cultured according to a normal culture method, the protein of the present invention is produced and accumulated, and the protein of the present invention is collected from the culture of the present invention to produce the protein or glycoprotein of the present invention. it can.

  The method for culturing the product of the present invention in a medium can be carried out according to a usual method used for culturing donor-derived cells. As a medium for culturing cells, any medium can be used as long as it contains a carbon source, a nitrogen source, inorganic salts, etc. that can be assimilated by the organism of the present invention, and can efficiently culture transformants. May be used.

  Any carbon source may be used as long as it can be assimilated by each microorganism. Glucose, fructose, sucrose, molasses containing these, carbohydrates such as starch or starch hydrolysate, organic acids such as acetic acid and propionic acid, ethanol Alcohols such as propanol can be used.

  Examples of nitrogen sources include ammonium salts of various inorganic or organic acids such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, and ammonium phosphate, other nitrogen-containing substances, and peptone, meat extract, yeast extract, corn steep liquor, and casein. A hydrolyzate, soybean meal, soybean meal hydrolyzate, various fermented cells, digested products thereof, and the like can be used.

  As the inorganic salt, monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate and the like can be used. The culture is performed under aerobic conditions such as shaking culture or deep aeration stirring culture.

  The culture temperature is usually 15 to 40 ° C., but in the present invention, it is set to the optimum temperature (set point) for maximizing the thermotherapy effect of the donor of the producer. It is important to. By maintaining the producer of the present invention at this optimum temperature, it was unexpectedly shown that the present invention improves gene product production capacity. For example, the level of improvement is 2 to 10 times at 37 ° C., about 100 times, or depending on the gene product, 0 may be 1 (that is, an unexpressed product will be expressed). . The culture time is usually 5 hours to 7 days. During the culture, the pH is maintained at 3.0 to 9.0. The pH is adjusted using an inorganic or organic acid, an alkaline solution, urea, calcium carbonate, ammonia or the like. Moreover, you may add antibiotics, such as an ampicillin or tetracycline, to a culture medium as needed during culture | cultivation.

  In a specific embodiment, when a promoter is used, an inducer may be added to the medium as necessary when culturing a microorganism transformed with an expression vector using an inducible promoter as the promoter. For example, when cultivating a microorganism transformed with an expression vector using the lac promoter, isopropyl-β-D-thiogalactopyranoside or the like is used for culturing a microorganism transformed with an expression vector using the trp promoter. An acid or the like may be added to the medium.

  For example, when animal cells are used, the culture medium for culturing the cells of the present invention may be RPMI1640 medium [The Journal of the American Medical Association, 199, 519 (1967)], Eagle's MEM medium [Science, 122 , 501 (1952)], DMEM medium [Virology, 8, 396 (1959)], 199 medium [Proceedings of the Society for the Biological Medicine, 73, 1 (1950)], or fetal bovine serum or the like was added to these media A medium or the like is used.

  The culture is usually performed for 1 to 7 days under conditions such as pH 6 to 8, 25 to 40 ° C., and 5% CO 2. Moreover, you may add antibiotics, such as kanamycin, penicillin, streptomycin, to a culture medium as needed during culture | cultivation.

  Even when a tissue, organ, or organism is used, the optimum temperature for maximizing the therapeutic effect of the donor of the producer using the conditions under which such tissue, organ, or organism normally grows, except for the temperature. Production can be achieved by setting (set point). By maintaining the producer of the present invention at this optimum temperature, it was unexpectedly shown that the present invention improves gene product production capacity. For example, the level of improvement is 2 to 10 times at 37 ° C., about 100 times, or depending on the gene product, 0 may be 1 (that is, an unexpressed product will be expressed). .

(Purification and isolation of protein)
A protein of the present invention from a culture of a producer having the ability to produce a gene product useful for the treatment of the present invention or a transformant transformed with a nucleic acid sequence encoding a gene product useful for the treatment, if necessary. In order to isolate or purify, conventional enzyme isolation or purification methods commonly used in the art can be used. For example, when the gene product useful for the treatment of the present invention is secreted outside the cells of the producer, the culture is treated by a technique such as centrifugation to obtain a soluble fraction. From the soluble fraction, anion exchange chromatography using a resin such as a solvent extraction method, a salting out method using ammonium sulfate, a precipitation method using an organic solvent, diethylaminoethyl (DEAE) -Sepharose, DIAION HPA-75 (Mitsubishi Chemical), etc. Chromatography method, cation exchange chromatography method using resin such as S-Sepharose FF (Pharmacia), hydrophobic chromatography method using resin such as butyl sepharose, phenyl sepharose, gel filtration method using molecular sieve, affinity chromatography A purified sample can be obtained by using a technique such as a chromatography method, a chromatofocusing method, an electrophoresis method such as isoelectric focusing.

  When the gene product useful for the treatment of the present invention accumulates in the production body in a dissolved state, the culture is centrifuged to collect cells in the culture, and after washing the cells, an ultrasonic crusher Then, the cells are disrupted with a French press, a Manton Gaurin homogenizer, a dynomill or the like to obtain a cell-free extract. From the supernatant obtained by centrifuging the cell-free extract, a solvent extraction method, a salting-out method using ammonium sulfate, a desalting method, a precipitation method using an organic solvent, diethylaminoethyl (DEAE) -Sepharose, DIAION HPA-75 (Mitsubishi Chemical) anion exchange chromatography using a resin, cation exchange chromatography using a resin such as S-Sepharose FF (Pharmacia), hydrophobic chromatography using a resin such as butyl sepharose and phenyl sepharose When the insoluble matter is formed in the production cell using a method such as a chromatography method, gel filtration using molecular sieve, affinity chromatography method, chromatofocusing method, electrophoresis method such as isoelectric focusing etc. Similarly, collect cells and crush them, then centrifuge. From the obtained precipitate fraction, the protein of the present invention is recovered by an ordinary method, and the insoluble matter of the protein is solubilized with a protein denaturant. After diluting or solubilizing this solubilized solution into a solution that does not contain a protein denaturant or the concentration of the protein denaturant does not denature the protein, the protein of the present invention is constituted into a normal three-dimensional structure, A purified sample can be obtained by the same isolation and purification method as described above.

  Further, a conventional protein purification method [J. Evan. Sadler et al .: Methods in Enzymology, 83, 458].

  Furthermore, it can also be purified by affinity chromatography using an antibody against the protein of the present invention itself. The protein of the present invention can be obtained by known methods [J. Biomolecular NMR, 6,129-134, Science, 242, 1162-1164, J. Biol. Biochem. , 110, 166-168 (1991)], can be produced using an in vitro transcription / translation system.

  The structural analysis of the purified protein of the present invention can be carried out by a method usually used in protein chemistry, for example, a method described in Protein Structural Analysis for Gene Cloning (Hirano Hisashi, Tokyo Kagaku Dojin, 1993). . The physiological activity of the protein of the present invention can be measured according to a known measurement method depending on the protein.

(General techniques used in this specification)
The techniques used herein are within the technical scope of the art, unless specifically indicated otherwise, microfluidics, microfabrication, organic chemistry, biochemistry, genetic engineering, molecular biology, Use well-known and conventional techniques in microbiology, genetics and related fields. Such techniques are well described, for example, in the documents listed below and in references cited elsewhere herein.

  For microfabrication, see, for example, Campbell, S .; A. (1996). The Science and Engineering of Microelectronic Fabrication, Oxford University Press; V. (1996). Micromicroarray Fabrication: a Practical Guide to Semiconductor Processing, Semiconductor Services; Madou, M .; J. et al. (1997). Fundamentals of Microfabrication, CRC1 5 Press; Rai-Chudhury, P .; (1997). Handbook of Microlithography, Micromachining, & Microfabrication: Microlithography, etc., which are incorporated by reference in the relevant portions herein.

Molecular biological techniques, biochemical techniques, and microbiological techniques used herein are well known and commonly used in the art, and are described in, for example, Maniatis, T. et al. et al. (1989). Molecular Cloning: A Laboratory Manual, Cold Spring Harbor and its 3rd Ed. (2001); Ausubel, F .; M.M. , Et al. eds, Current Protocols in Molecular Biology, John Wiley & Sons Inc. NY, 10158 (2000); Innis, M .; A. (1990). PCR Protocols: A Guide to Methods
and Applications, Academic Press; Innis, M .; A. et al. (1995). PCR Strategies, Academic Press; Sinsky, J. et al. J. et al. et al. (1999). PCR Applications: Protocols for Functional Genomics, Academic Press; Gait, M .; J. et al. (1985). Oligonucleotide Synthesis: A Practical Approach, IRL Press; Gait, M .; J. et al. (1990). Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein, F .; (1991). Oligonucleotides and Analogues: A Practical Approc, IRL Press; Adams, R .; L. et al. (1992). The Biochemistry of the Nucleic Acids, Chapman &Hall; Shabarova, Z .; et al. (1994). Advanced Organic Chemistry of Nucleic Acids, Weinheim; Blackburn, G .; M.M. et al. (1996). Nucleic Acids in Chemistry and Biology, Oxford University Press; Hermanson, G .; T.A. (1996). Bioconjugate Techniques, Academic Press; Method in Enzymology 230, 242, 247, Academic Press, 1994; Related parts (which may be all) are incorporated by reference.

(Medicine, cosmetics, etc., and treatment and prevention using the same)
In another aspect, the present invention relates to pharmaceuticals and cosmetics. The medicament and cosmetic may further comprise a pharmaceutically acceptable carrier and the like. Examples of the pharmaceutically acceptable carrier contained in the medicament of the present invention include any substance known in the art.

  Such suitable formulation materials or pharmaceutically acceptable carriers include antioxidants, preservatives, colorants, flavors, and diluents, emulsifiers, suspending agents, solvents, fillers, bulking agents, buffers. Include, but are not limited to, agents, delivery vehicles, diluents, excipients and / or pharmaceutical adjuvants. Typically, the medicament of the present invention is a composition comprising isolated pluripotent stem cells, or a variant or derivative thereof, together with one or more physiologically acceptable carriers, excipients or diluents. It is administered in the form of For example, a suitable vehicle can be water for injection, physiological solution, or artificial cerebrospinal fluid, which can be supplemented with other materials common to compositions for parenteral delivery. .

  Acceptable carriers, excipients or stabilizers used herein are non-toxic to the recipient and are preferably inert at the dosages and concentrations used and Phosphate, citrate, or other organic acids; ascorbic acid, α-tocopherol; low molecular weight polypeptides; proteins (eg, serum albumin, gelatin or immunoglobulin); hydrophilic polymers (eg, polyvinyl Amino acids (eg, glycine, glutamine, asparagine, arginine or lysine); monosaccharides, disaccharides and other carbohydrates (including glucose, mannose, or dextrin); chelating agents (eg, EDTA); sugar alcohols (eg, EDTA); Mannitol or sorbitol ; Salt-forming counterions (such as sodium); and / or non-ionic surface-active agents (e.g., Tween, Pluronic (pluronic) or polyethylene glycol (PEG)).

  Exemplary suitable carriers include neutral buffered saline or saline mixed with serum albumin. Preferably, the product is formulated as a lyophilizer using a suitable excipient (eg, sucrose). Other standard carriers, diluents and excipients may be included as desired. Other exemplary compositions include pH 7.0-8.5 Tris buffer or pH 4.0-5.5 acetate buffer, which may further include sorbitol or a suitable substitute thereof. .

  The medicament of the present invention can be administered orally or parenterally. Alternatively, the medicament of the present invention can be administered intravenously or subcutaneously. When administered systemically, the medicament used in the present invention may be in the form of a pharmaceutically acceptable aqueous solution free of pyrogens. Such a pharmaceutically acceptable composition can be easily prepared by those skilled in the art by considering pH, isotonicity, stability and the like. In this specification, the administration method includes oral administration, parenteral administration (for example, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, mucosal administration, intrarectal administration, intravaginal administration, local administration to the affected area, Skin administration, etc.). Formulations for such administration can be provided in any dosage form. Examples of such a pharmaceutical form include a liquid, an injection, and a sustained release agent.

  The medicament of the present invention may be a physiologically acceptable carrier, excipient or stabilizer (Japanese Pharmacopoeia 14th edition or its latest edition, Remington's Pharmaceutical Sciences, 18th Edition, AR, as required). Gennaro, ed., Mack Publishing Company, 1990, etc.) can be prepared and stored in the form of a lyophilized cake or aqueous solution by mixing a sugar chain composition having a desired degree of purity.

  The amount of the composition used in the treatment method of the present invention takes into consideration the purpose of use, target disease (type, severity, etc.), donor age, weight, sex, past history, cell morphology or type, and the like. Can be easily determined by those skilled in the art. The frequency with which the treatment method of the present invention is applied to a subject (or donor) also depends on the purpose of use, target disease (type, severity, etc.), donor age, weight, gender, medical history, treatment course, etc. In view of this, it can be easily determined by those skilled in the art. Examples of the frequency include administration every day to once every several months (for example, once a week to once a month). It is preferable to administer once a week to once a month while observing the course.

  When the present invention is used as a cosmetic product, the cosmetic product can also be prepared while complying with the regulations stipulated by the authorities.

(Pesticide)
The composition of the present invention can also be used as an agrochemical component. When formulated as an agrochemical composition, it may contain agriculturally acceptable carriers, excipients, stabilizers and the like, if desired.

  When the composition of the present invention is used as an agrochemical, herbicide (such as pyrazolate), insecticide / acaricide (such as diazinon), fungicide (such as probenazole), plant growth regulator (eg, paclobutrazole) ), Nematicides (eg, benomyl), synergists (eg, piperonyl butoxide), attractants (eg, eugenol), repellents (eg, creosote), pigments (eg, , Food Blue No. 1, etc.), fertilizers (eg, urea, etc.), etc. can also be mixed as required.

(Health and food)
The present invention can also be used in the health / food field. In such a case, the points to be noted when used as an oral medicine as described above should be considered as necessary. In particular, when used as a functional food / health food such as a specific health food, it is preferable to treat it according to a medicine.

  Hereinafter, preferred embodiments of the present invention will be described as examples. The embodiments provided below are provided for a better understanding of the present invention, and it is understood that the scope of the present invention should not be limited to the following description. Therefore, it is obvious that those skilled in the art can make appropriate modifications within the scope of the present invention with reference to the description in the present specification.

  References such as scientific literature, patents and patent applications cited herein are hereby incorporated by reference in their entirety to the same extent as if each was specifically described.

  As mentioned above, although this invention has been illustrated using preferable embodiment of this invention, this invention should not be limited and limited to this embodiment. It is understood that the scope of the present invention should be construed only by the claims. It is understood that those skilled in the art can implement an equivalent range based on the description of the present invention and the common general technical knowledge from the description of specific preferred embodiments of the present invention. Patents, patent applications, and documents cited herein should be incorporated by reference in their entirety, as if the contents themselves were specifically described herein. Understood.

  Hereinafter, although an example explains the composition of the present invention in detail, the present invention is not limited to this. The reagents used in the following were commercially available from Wako Pure Chemicals and Promega unless otherwise specified.

(Example 1: Hsp (heat shock protein) production method by heating to extraction / purification method of produced Hsp)
(I. Production of monoclonal antibody against Hsp70)
A GST fusion protein (GST-Hsp72) is prepared using GST Gene Fusion System Kit (manufactured by Amersham Bioscience). Hsp72 cDNA is ligated with the pGEX vector, and E. coli BL21 is transformed with the prepared plasmid. Thereafter, a clone having the target plasmid is selected. The selected E. coli is cultured and expression is confirmed. For confirmation, any known method such as Northern blot analysis is used. The target GST protein is solubilized (in accordance with the following donor cell extraction procedure) and purified with an anti-GST antibody-binding column. The purified GST protein is digested with the above Factor Xa of Kit and applied again to the anti-GST antibody column to obtain a flow-through fraction (Hsp72). . The thus prepared recombinant Hsp (rHsp) (including SEQ ID NO: 2) is administered and immunized (3 to 4 times) into the skin of a mouse (Balb / c mouse female, CLEA Japan) together with an adjuvant. The antibody titer is evaluated by ELISA using rHsp70 and Western blotting. ELISA and Western blotting can be performed using a kit commercially available from Promega.

  Again, rHsp is used to fuse the spleen extracted from boosted mice with myeloma cells (available from the Human Science Research Resource Bank). The fused cells are selected by limiting dilution.

  Anti-Hsp antibody producing hybridomas are screened using rHsp coated 96 well microtiter plates. The selected hybridoma is administered intraperitoneally to the mouse and proliferated to obtain a large amount of ascites. An equal amount of saturated ammonium sulfate is added to the antibody-containing ascites to precipitate the antibody fraction (partially purified antibody).

  The partially purified antibody is purified using a protein A or protamine column. The purified antibody is reacted with CNBr activated Sepharose beads to bind the antibody. This is used as an anti-Hsp antibody binding affinity column.

(II. Hsp extraction / purification)
(Determination of the optimum temperature using the expression level of Hsp72 as an index)
4 ml of peripheral blood was collected from the subject. Peripheral blood mononuclear cells were separated from peripheral blood by Lymphoprep (Nycommed Pharma AS, Oslo, Norway). About 1 × 10 ⁶ peripheral blood mononuclear cells were lysed in a lysis buffer (Funakoshi Co., Ltd., included in Hsp70 ELISA kit). The solution was slowly warmed starting at 37 ° C. Aliquots were removed from this solution every 0.5 ° C. Proteins were extracted from the extracted solution aliquots by Funakoshi Co., Ltd., Hsp70 ELISA kit, and the expression level of Hsp72 was quantified by ELISA using Hsp72 ELISA Kit (Stressgen Biotechnologies, Sydney, Canada). The temperature at which the expression level of Hsp72 turned from an increase to a decrease was determined as the optimum temperature for thermotreating this subject. That is, the expression level of Hsp72 was the maximum at this optimum temperature.

(Determination of optimum temperature using HLA-DR expression level as an index)
4 ml of peripheral blood was collected from the subject. Peripheral blood mononuclear cells were separated from peripheral blood by Lymphoprep (Nycommed Pharma As, Oslo, Norway). About 1 × 10 ⁶ peripheral blood mononuclear cells were double-stained with an antibody against CD4, CD8, CD16, CD56 or CD57 and an anti-HLA antibody, and expression of HLA-DR for each cell population by FACS analysis The amount was quantified. The temperature at which the expression level of HLA-DR when the peripheral blood of this subject was warmed in vitro turned from an increase to a decrease was determined as the optimum temperature for thermotherapy of this subject. That is, the expression level of HLA-DR was the maximum at this optimum temperature.

(Determination of optimum temperature using donor index)
Information about each of the following parameters was obtained from the subject (human):
a) Expression level and function of Hsp72 in donor blood cells,
b) changes in donor blood pressure,
c) Donor heart rate,
d) donor blood flow,
e) pH of donor blood,
f) Expression level of HLA in donor blood cells, and g) Number of lymphocytes in donor blood.

  Of these, the optimum temperature was determined under the following conditions.

a) Expression level and function of Hsp72 in donor blood cells: Temperature that induces maximum expression level b) Change in donor blood pressure: Temperature at which dilation pressure begins to rise c) Donor heart rate: Increased heart rate D) Donor blood flow: temperature at which increased blood flow begins to decrease e) Donor blood pH: temperature at which the change in the peripheral blood pH of the donor (rise or fall) reaches a plateau f) Donor Expression level of HLA in blood cells: temperature showing maximum expression level on cell membrane surface g) Number of lymphocytes in donor's blood: temperature showing increase in maximum lymphatic series (Processing of producer at optimal temperature) )
The optimum temperatures obtained as described above were 39 ° C. for the subject 1, 38.5 ° C. for the subject 2, and 39.5 ° C. for the subject 3. Lymph fluid and blood removed from each subject were processed using a system that strictly maintains these temperatures.

  As a control experiment, in addition to those conducted uniformly at 37 ° C. and 40 ° C., those having an optimum temperature of 38.5 ° C. were conducted at 39 ° C. or vice versa.

(Purification)
Lymph fluid and blood are each subjected to the following treatment in an amount of 500 mL. It will be appreciated that this amount can be varied as appropriate and that the following processes can also be scaled up and down.

1 mM chaps-containing 10 mM Tris-HCl buffer pH 7.4 [150 mM NaCl, 10 μg / ml leupeptin, 1 μg / ml pepstatin, 20 μg / ml soybean inhibitor, 10 μM p-aminophenyl-phenylmethylsulfonylfluoride (PMSF)] is added (at a rate of 10 ⁸ cells / ml). The resulting mixture is sonicated (in ice for 5 minutes) to disrupt the cells uniformly.

  The resulting mixture is ultracentrifuged (100,000 × g, 1 hour).

  Further, the supernatant is filtered (0.45 μm pore size filter).

  The filtrate is applied to the affinity column (anti-Hsp antibody binding affinity column) prepared as described above.

The column is washed with 10 mM Tris-HCl buffer pH 7.4 (containing 150 mM NaCl, 10 mM PMSF) containing 1% Chaps. Washing is monitored by absorbance at 280 nm (OD ₂₈₀ ).

  Desorb Hsp in 0.2 M acetic acid solution pH 2.5.

  The adsorbed fraction is neutralized with 1M Tris-HCl pH 8.0.

  The neutralized fraction is dialyzed thoroughly against phosphate buffer (at 4 ° C.). Repeat the dialysate exchange. Thereby, purified Hsp is obtained.

  When the purified amount was measured, Hsp72 increased by 10 times or more at 37 ° C and 40 ° C at each optimum temperature. In addition, when the temperature is 38.5 ° C., when the culture is performed at 39 ° C., the production amount is halved. When the optimal temperature is 39.0 ° C. and the culture is performed at 38.5 ° C., the production amount is reduced to 30%. Thus, it is understood that maintaining the producer at the optimum temperature is important for the production of the gene product.

(Example 2: Examples in tissues, organs and organisms)
Mononuclear cells from peripheral blood are isolated and sorted using lymphoprep (# 11145545, Axis-Shield POC. As, Oslo, Norway), or liver tissue is subjected to collagenase treatment to obtain hepatocytes. . The cells are heated at the optimum temperature determined in Example 1 to induce an increase in the expression level of Hsp72. As a control experiment, an experiment at another temperature described in Example 1 is also performed.

  After the induction of the protein, separation and purification are performed and quantified by an affinity column to which a monoclonal antibody is bound.

  When purification is performed in the same manner as described in Example 1 and the amount purified is measured, it can be seen that the expression level of Hsp72 increases at each optimum temperature compared to 37 ° C and 40 ° C.

(Example 3: Recombination experiment)
HeLa cells or CHO cells were transformed with a vector (pGEX) containing the nucleotide described in SEQ ID NO: 1. This transformant is cultured at the optimum temperature in the same manner as in Example 1.

  As mentioned above, although this invention has been illustrated using preferable embodiment of this invention, this invention should not be limited and limited to this embodiment. It is understood that the scope of the present invention should be construed only by the claims. It is understood that those skilled in the art can implement an equivalent range based on the description of the present invention and the common general technical knowledge from the description of specific preferred embodiments of the present invention. Patents, patent applications, and documents cited herein should be incorporated by reference in their entirety, as if the contents themselves were specifically described herein. Understood.

  According to the present invention, a protein useful for treatment (for example, Hsp72) that can be used in medicines, agricultural chemicals, foods, and the like can be easily produced with high efficiency. Accordingly, it is understood that the application covers various fields.

(Description of array)
SEQ ID NO: 1 shows the nucleic acid sequence of Hsp72 used in the present invention.
SEQ ID NO: 2 This shows the amino acid sequence of Hsp72 used in the present invention.
SEQ ID NO: 3 shows the nucleic acid sequence of HLA-B used in the present invention.
SEQ ID NO: 4 shows the amino acid sequence of HLA-B used in the present invention.
SEQ ID NO: 5: This shows the nucleic acid sequence of Hsp10 used in the present invention.
SEQ ID NO: 6: This shows the amino acid sequence of Hsp10 used in the present invention.
SEQ ID NO: 7: This shows the nucleic acid sequence of Hsp27 used in the present invention.
SEQ ID NO: 8: This shows the amino acid sequence of Hsp27 used in the present invention.
SEQ ID NO: 9: This shows the nucleic acid sequence of Hsp40 used in the present invention.
SEQ ID NO: 10: This shows the amino acid sequence of Hsp40 used in the present invention.
SEQ ID NO: 11: This shows the nucleic acid sequence of Hsp47 used in the present invention.
SEQ ID NO: 12 This shows the amino acid sequence of Hsp47 used in the present invention.
SEQ ID NO: 13: This shows the nucleic acid sequence of Hsp60 used in the present invention.
SEQ ID NO: 14: This shows the amino acid sequence of Hsp60 used in the present invention.
SEQ ID NO: 15: This shows the nucleic acid sequence of Hsp70 used in the present invention.
SEQ ID NO: 16 This shows the amino acid sequence of Hsp70 used in the present invention.
SEQ ID NO: 17: This shows the nucleic acid sequence of Hsp73 used in the present invention.
SEQ ID NO: 18: This shows the amino acid sequence of Hsp73 used in the present invention.
SEQ ID NO: 19: This shows the nucleic acid sequence of Hsp90α used in the present invention.
SEQ ID NO: 20: This shows the amino acid sequence of Hsp90α used in the present invention.
SEQ ID NO: 21: This shows the nucleic acid sequence of Hsp90β used in the present invention.
SEQ ID NO: 22: This shows the amino acid sequence of Hsp90β used in the present invention.
SEQ ID NO: 23: This shows the nucleic acid sequence of Hsp110 used in the present invention.
SEQ ID NO: 24: This shows the amino acid sequence of Hsp110 used in the present invention.

Claims (29)

A method of producing a gene product useful for therapy comprising:
A) providing a producer having the ability to produce the gene product;
B) subjecting the producer to an optimal temperature for maximizing the thermotherapy effect of the producer's donor; and C) obtaining the gene product from the producer;
Including the method. 2. The method of claim 1, wherein the gene product is selected from the group consisting of Hsp72, HLA, Hsp10, Hsp27, Hsp47, Hsp40, Hsp60, Hsp90, Hsp73 and Hsp110. The method of claim 1, wherein the gene product is Hsp72. The method according to claim 1, wherein the producer is a cell, tissue, organ or organism. The method of claim 1, wherein the producer comprises blood or lymph. The method according to claim 1, wherein the producer is an artificial producer. The method of claim 1, wherein the producer comprises a nucleic acid sequence encoding the gene product. The method of claim 1, wherein the producer comprises a cell transformed with a nucleic acid sequence encoding the gene product. The gene product is SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21 or SEQ ID NO: The method of claim 1, encoded by the sequence of number 23. The optimum temperature is the following for the donor:
a) Expression level and function of Hsp72 in donor blood cells,
b) changes in donor blood pressure,
c) Donor heart rate,
d) donor blood flow,
e) pH of donor blood,
f) the amount of HLA expressed in the donor's blood cells, and g) the number of lymphocytes in the donor's blood,
The method of claim 1, wherein the method is determined based on a biotransformation selected from the group consisting of: The method of claim 1, wherein the step of obtaining a gene product is accomplished by using affinity purification. The method according to claim 11, wherein the affinity purification uses an antibody. The method of claim 1, further comprising identifying the optimum temperature. In the specific case, the following:
a) Expression level and function of Hsp72 in donor blood cells,
b) changes in donor blood pressure,
c) Donor heart rate,
d) donor blood flow,
e) pH of donor blood,
f) the amount of HLA expressed in the donor's blood cells, and g) the number of lymphocytes in the donor's blood,
The method according to claim 13, further comprising observing a biological change selected from the group consisting of: A gene product produced by the method of claim 1. A system for producing gene products useful for therapy,
A) a producer having the ability to produce the gene product;
B) means for subjecting the producer to an optimal temperature for maximizing the thermotherapy effect of the producer's donor; and C) means for obtaining the gene product from the producer;
Including the method. The system of claim 16, further comprising: D) means for identifying the optimum temperature. 17. The system of claim 16, wherein the gene product is selected from the group consisting of Hsp72, HLA, Hsp10, Hsp27, Hsp47, Hsp40, Hsp60, Hsp90, Hsp73 and Hsp110. The system of claim 16, wherein the gene product is Hsp72. The system according to claim 16, wherein the producer is a cell, tissue, organ or organism. The system of claim 16, wherein the producer comprises blood or lymph. The system of claim 16, wherein the producer is an artificial producer. The system of claim 16, wherein the producer comprises a nucleic acid sequence encoding the gene product. 17. The system of claim 16, wherein the producer comprises a cell transformed with a nucleic acid sequence encoding the gene product. The gene product is SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21 or SEQ ID NO: The system of claim 16, encoded by the sequence of number 23. The optimum temperature is the following for the donor:
a) Expression level and function of Hsp72 in donor blood cells,
b) changes in donor blood pressure,
c) Donor heart rate,
d) donor blood flow,
e) pH of donor blood,
f) the amount of HLA expressed in the donor's blood cells, and g) the number of lymphocytes in the donor's blood,
The system of claim 16, wherein the system is determined based on a biotransformation selected from the group consisting of: The system according to claim 16, wherein the means for obtaining the gene product comprises an affinity purification means. 28. The system of claim 27, wherein the affinity purification means comprises an antibody specific for the gene product. The means for specifying is as follows:
a) Means for measuring the expression level and function of Hsp72 in donor blood cells,
b) means for measuring changes in the blood pressure of the donor,
c) means for measuring the heart rate of the donor,
d) means for measuring the blood flow of the donor,
e) means for measuring the pH of the donor's blood;
f) means for measuring the expression level of HLA in the donor's blood cells; and g) means for measuring the number of lymphocytes in the donor's blood.
The system according to claim 17, further comprising means for observing a biological change selected from the group consisting of: