JP2012090571A - Method for immunizing animal, composition for immunization, method for producing antibody, and method for producing hybridoma - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for allowing induction of a response of humoral immunity with higher efficiency in a shorter period of time when an antibody against an antigenic protein is prepared according to gene immunization.SOLUTION: There is provided the method for immunizing an animal including immunizing a non-human animal with an antigen; and thereby inducing the response of the humoral immunity to a desired membrane protein in the non-human animal. The method for immunizing the animal includes administering a fusion gene obtained by linking a gene encoding the full length or a part of flagellin to a gene encoding the full length or a part of the membrane protein to the non-human animal; thereby expressing the fusion gene in the non-human animal; and inducing the humoral immunity to the membrane protein. In addition, there are provided a composition for immunization used in the method, a method for producing an antibody utilizing the method, and a method for producing a hybridoma.

Description

  The present invention relates to an animal immunization method, an immunization composition, an antibody production method, and a hybridoma production method. More specifically, the present invention relates to a fusion gene between a gene encoding a membrane protein and a gene encoding flagellin. Immunization method for animals that induces humoral immunity response by administration to human animals, immunization composition for use in the immunization method, antibody production method using the immunization method, immunization by the immunization method The present invention relates to a method for producing a hybridoma in which B lymphocytes and myeloma of a non-human animal are fused, and an antibody production method using the hybridoma.

  Antibodies play a major role in humoral immunity and play an important role in biological defense mechanisms together with sensitized lymphocytes. On the other hand, antibodies are frequently used in various techniques utilizing specific affinity with the antigen, such as affinity chromatography and immunoassay. That is, since an antibody specifically and strongly binds to a specific antigen, the antibody is useful as an analysis tool for identifying and quantifying a specific antigen or for a protein whose function is unknown. For these purposes, antibodies are widely used in the fields of clinical examination and biotechnology.

  A typical method for producing an antibody against a specific antigen is to induce a humoral immunity by administering a desired protein serving as an antigen to the animal, and collecting antiserum from the bodily fluid of the animal. That is, the antigen protein itself is used as an immunogen to be administered (immunized) to animals. In this case, as an antigen protein used as an immunogen, for example, a protein isolated and purified from a biological sample or the like is used. In addition, a recombinant antigen protein obtained by using recombinant DNA technology or a peptide corresponding to a part of the antigen protein is chemically synthesized and administered to an animal as an immunogen. .

  On the other hand, there is a technique called gene immunization that uses not the antigen protein itself but a gene encoding the antigen protein as an immunogen. When performing gene immunization, for example, a gene encoding an antigen protein is incorporated into an appropriate expression vector, and the animal is inoculated with the expression vector. Then, the gene incorporated in the expression vector is expressed in the animal body, and the antigen protein is synthesized. As a result, an immune response is induced by the antigen protein synthesized in the animal body.

As described above, there are various techniques for producing an antibody against a specific antigen, which can be selected according to the purpose. For example, gene immunization is appropriate in the following cases.
First, according to gene immunization, immunization can be performed as long as a gene encoding an antigen protein is isolated, and it is not necessary to isolate and purify the antigen protein. Therefore, for gene immunization, if you want to simplify the process by omitting the preparation of antigen protein, antigen protein for which purification method has not been established, antigen protein that is difficult to purify, unknown antigen protein for which only gene is known, etc. This is suitable for the production of antibodies against.
In addition, in order for a protein to exhibit its function in vivo, it is necessary to form a correct three-dimensional structure, but gene immunization is suitable for obtaining an antibody that recognizes the correct three-dimensional structure in a protein. That is, in gene immunization, genes administered to animals are transcribed and translated in vivo, which is useful for obtaining antibodies against antigenic proteins having higher order structures.
Furthermore, in the case of gene immunization, an immune response can be induced even if the amount of antigen protein expressed and synthesized in the animal body is much smaller than the amount required for direct administration of the antigen protein. There is also an advantage. Furthermore, even when it is difficult to prepare an antigen protein by recombinant DNA technology, it is not necessary to synthesize a peptide antigen separately as in the conventional method, and only the full length of the gene is introduced into the animal. There is also an advantage that it is good.

As described above, genetic immunization has advantages not found in conventional methods, but depending on the type of antigen protein, a humoral immunity response may not be induced and antibodies may not be produced.
For example, if the antigen protein is extremely homologous to the protein inherently possessed by the immunized animal, the antigen protein is not recognized as a foreign substance even if it is synthesized in the animal body. May not be induced.
In addition, when the antigen protein is unstable in the animal body, the amount of the antigen protein in the animal body decreases, and a humoral immune response may not be induced. Further, when the antigen protein mainly induces cellular immunity, humoral immunity is hardly induced.
In addition, as a problem specific to gene immunity, antigen protein is poor even when the antigen protein gene is poorly introduced into the animal or when the antigen protein gene has a low expression level in the animal body. May be reduced and a humoral immune response may not be induced.

  In order to solve the above-described problems of gene immunity, various devices have been proposed. For example, when inducing an immune response against urokinase, the urokinase gene is not administered alone, but in the form of a fusion gene with a transmembrane domain gene, thereby inducing a high immune response against urokinase. There is an example of obtaining an antibody against (Patent Document 1). It is considered that the high immune response obtained here occurred because the urokinase moiety was forced to be placed on the cell surface in the fusion protein, which is the expression product of the fusion gene.

In the field of vaccines, there is an example in which a fusion gene (chimeric nucleic acid) of a gene encoding HSP70, which is a heat shock protein, and a gene encoding an antigen protein is used as a DNA vaccine (Patent Document 2). In this example, a gene encoding HSP70 derived from Mycobacterium tuberculosis is used. However, this technique was able to induce antigen-specific cellular immunity (killer T cells), but not humoral immunity, ie antibody production. On the other hand, when a fusion protein of Mycobacterium tuberculosis-derived HSP70 and an antigen protein is used as an immunogen, there is a report that HSP70 functions as a suitable adjuvant and antibody production against the antigen protein is induced (Patent Document 3).
These means that the immune response mechanism in the antigen-presenting cell differs between the immune response by the fusion protein of HSP70 and the antigen protein and the immune response by the fusion gene of the HSP70 gene and the antigen protein gene. . Thus, when it is desired to induce a humoral immune response to a certain antigen protein, immunization using the antigen protein gene does not always induce a desired immune response. Rather, it is more likely that the desired immune response against the antigen protein cannot be induced.

International Publication No. 02/08416 International Publication No.01 / 29233 International Publication No. 94/29459

  As described above, there are many unknown parts of gene immunity, and there is still no area for trial and error. Therefore, in order to produce an antibody against a desired antigen protein by gene immunization, a technique capable of inducing a humoral immunity response with high reproducibility regardless of the type of antigen protein is required. In view of the above-mentioned present situation, the present invention aims to provide various techniques capable of inducing a humoral immune response in a shorter period of time and with higher efficiency when producing an antibody against an antigen protein by gene immunization. And

  The present inventors have studied various measures for solving the above problems. As a result, by using a gene encoding flagellin, which is a protein that constitutes bacterial flagella, and is also a type of Toll-like receptors (hereinafter abbreviated as “TLRs”), It has been found that a humoral immune response to a desired antigen protein, particularly a membrane protein, can be induced in a shorter period of time and with higher efficiency. Based on this new knowledge, a series of techniques such as a novel animal immunization method and antibody production method were established, and the present invention was completed. That is, the present invention provided to solve the above problems is as follows.

  The invention according to claim 1 is an animal immunization method for inducing a humoral immunity response to a desired membrane protein in the non-human animal by immunizing the non-human animal with an antigen. By administering a fusion gene obtained by linking a gene encoding the full length or a part of flagellin to a gene encoding the full length or a part thereof to the non-human animal, the fusion gene is expressed in the non-human animal, An animal immunization method characterized by inducing humoral immunity against a membrane protein.

  The immunization method of the present invention belongs to gene immunity, and administers to a non-human animal a fusion gene obtained by linking a gene encoding the full length or part of a membrane protein to a gene encoding the full length or part of flagellin. To do. Then, the fusion gene is expressed in the non-human animal body, and a fusion protein obtained by linking the full length or part of flagellin to the full length or part of the membrane protein is synthesized. As a result, humoral immunity against the membrane protein is induced. According to the animal immunization method of the present invention, the activity of antigen-presenting cells via TLR of flagellin, even if it is a membrane protein that cannot induce a humoral immune response even if a gene encoding the membrane protein alone is administered. The humoral immunity response can be induced by the conversion, and an antibody against the membrane protein can be produced.

  The flagellins may include Salmonella, Escherichia, Bordetella, Shigella, Legionella, Burkholderia, Pseudomonas, Helicobacter (Pseudomonas) It is derived from microorganisms belonging to the genus Helicobacter, Vibrio, Serratia, Calobacter, Listeria, Clostridium, or Borrelia (Claim 2).

  The membrane protein is preferably a G protein-coupled receptor, an ion channel receptor, a tyrosine kinase receptor, a CD antigen, a cell adhesion molecule, or a cancer antigen.

  The non-human animal is preferably a mouse, rat, rabbit, cow, horse, dog, cat, goat, sheep, pig, chicken, duck, or turkey (Claim 4).

  The invention according to claim 5 is an immunizing composition for use in the animal immunization method according to any one of claims 1 to 4 and administered to a non-human animal, wherein It is a composition for immunization characterized by containing a fusion gene obtained by linking a gene encoding the full length or a part of flagellin to a gene encoding a region.

  When carrying out the above-described animal immunization method of the present invention, a composition in which the fusion gene is dissolved in an appropriate solvent can be prepared, and the composition can be administered to the animal. The immunizing composition of the present invention is an immunizing composition for use in the animal immunization method of the present invention, and encodes the full length or part of flagellin in the gene encoding the full length or part of the membrane protein. It contains a fusion gene formed by linking genes. According to the immunizing composition of the present invention, the fusion gene can be administered to animals by a method such as injection. Moreover, the dosage of a fusion gene can be accurately adjusted by adjusting the concentration of the fusion gene in the composition.

  Invention of Claim 6 acquires the antibody with respect to the said membrane protein from the non-human animal immunized by the animal immunization method in any one of Claims 1-4, The manufacturing method of the antibody characterized by the above-mentioned It is.

  The antibody production method of the present invention is a method of collecting an antibody against a membrane protein from a non-human animal immunized by the animal immunization method of the present invention. According to the method for producing an antibody of the present invention, a membrane protein for which no purification method has been established, a membrane protein that is difficult to purify, a membrane protein that is difficult to reconstitute into a lipid membrane, an unknown membrane that is known only for genes Proteins and recombinant DNA techniques can be used to obtain antibodies against membrane proteins that have a low gene expression level and that do not produce antibodies when the gene is administered alone. In addition, when acquiring an antibody from the bodily fluid of an animal, the said antibody becomes a polyclonal thing.

  The invention according to claim 7 is a membrane protein wherein B lymphocytes collected from a non-human animal immunized by the animal immunization method according to any one of claims 1 to 4 and myeloma are cell-fused. A method for producing a hybridoma comprising obtaining a hybridoma that produces an antibody against.

  In the method for producing a hybridoma of the present invention, B lymphocytes collected from a non-human animal whose humoral immunity response has been induced by the above-described animal immunization method of the present invention, and myeloma are cell-fused. A hybridoma producing an antibody against a membrane protein is prepared. With this configuration, a hybridoma that produces a monoclonal antibody against a membrane protein that does not produce an antibody even when the membrane protein itself or its gene is administered alone can be obtained.

  The invention according to claim 8 is a method for producing an antibody, wherein an antibody against the membrane protein is obtained from the hybridoma produced by the method for producing a hybridoma according to claim 7.

  In the method for producing an antibody of the present invention, an antibody against the membrane protein is obtained from the hybridoma produced by the above-described method for producing a hybridoma of the present invention. With such a configuration, it is possible to obtain an antibody (monoclonal antibody) against a membrane protein that does not produce an antibody even when the membrane protein itself or its gene is administered alone.

  According to the animal immunization method and immunization composition of the present invention, when genetic immunization is performed, a membrane protein that cannot induce a humoral immune response even if the gene is inoculated alone into the animal, Can also induce a humoral immune response.

  According to the method for producing an antibody of the present invention, even when a gene is immunized, even if it is a membrane protein that cannot induce a humoral immunity response even if the gene is inoculated alone, Can be produced.

  According to the method for producing a hybridoma of the present invention, even when the gene is immunized, even if it is a membrane protein that cannot induce a humoral immune response even if the gene is inoculated alone into an animal, the membrane protein Hybridomas producing monoclonal antibodies against can be produced.

It is the figure which analyzed the expression of flagellin fusion mETAR in the cell surface with the flow cytometer, (a) shows the case of 293T cell which introduce | transduced flagellin fusion mETAR gene, (b) shows the case of 293T cell. It is a graph which shows the amount of interleukin 4 (IL-4) secretion from the T cell extract | collected from the mouse | mouth of each gene immunity group. It is a graph which shows the amount of interferon gamma (IFN-gamma) secretion from the T cell extract | collected from the mouse | mouth of each gene immunity group. It is a graph which shows the result of having analyzed the titer of the serum extract | collected from the mouse | mouth of each gene immunity group with the flow cytometer.

  Hereinafter, embodiments of the present invention will be specifically described. Prior to the description of the embodiments, a general description of flagellin and Toll-like receptors (TLRs) will be given.

  Toll-like receptors (TLRs) play an important role in early innate immunity by detecting microorganisms. TLRs are expressed as membrane proteins on the cell surface and in endosomes, and TLRs 1 to 9 have been identified so far. These receptors recognize structural motifs that are highly conserved only in pathogenic microorganisms called pathogen-associated molecular patterns (PAMPs). PAMPs include lipopolysaccharide (LPS), peptidoglycan, lipoprotein, glycoprotein, virus-derived double-stranded RNA, unmethylated CpG DNA, and the like. Stimulation of TLRs by PAMPs initiates signaling cascades such as MyD88 and TRIF (Medzhitov R et al., Nature, 1997, 388, 6640, p. 394-397). . These signaling cascades are said to induce activation of transcription factors such as AP-1, NF-κB and IRF, thereby inducing the production of inflammatory cytokines and effector cytokines, leading to acquired immunity. It has also become clear that the innate immune response via these receptors is important for the induction of acquired immune responses centered on T cell responses (Ishii KJ et al., Journal of・ Clinical Immunology (Journal of Clinical Immunology), 2007, Vol. 27, p.363-371).

  Flagellin is a globular protein that forms the helical fiber of bacterial flagella. The molecular weight of flagellin varies considerably (30,000 to 70,000) depending on its origin (bacterial species). Flagellin induces TLR5-dependent inflammation in both animals and plants. Flagellin plays an important role in the pathogenicity of bacteria itself by promoting migration and adhesion to infected hosts, and is also known to be involved in immune responses to antigen-specific T cells. .

  In the field of vaccines, attempts have been made to use flagellin as an adjuvant that enhances antigenicity (Japanese Patent Publication No. 2007-535924, International Publication No. 2005/042564). Flagellin is a ligand for TLR5 and is known to bind to and activate TLR5 on antigen-presenting cells such as macrophages and dendritic cells. Flagellin has also been reported to be an effective adjuvant that allows T cells to enhance immune responses in vivo (McSorley SJ et al., Journal of Immunology). (Journal of Immunology), 2002, 169, p. 3914-3919). However, the effectiveness of these methods is limited only to soluble antigens. This is the case where humoral immunity is induced using the action of flagellin for membrane proteins including G protein-coupled receptors. It has not been reported until.

  Subsequently, an embodiment of the present invention will be described. In the following description, unless otherwise specified, “membrane protein gene” refers to “a gene encoding the full length or part of the membrane protein”. Similarly, “flagellin gene” refers to “a gene encoding the full length or part of flagellin”.

  The animal immunization method of the present invention induces a humoral immune response to a desired membrane protein in a non-human animal by immunizing the non-human animal with an antigen. In the animal immunization method of the present invention, “a fusion gene obtained by linking a gene encoding the full length or part of flagellin to a gene encoding the full length or part of the membrane protein” is administered to a non-human animal. To express the fusion gene in the non-human animal and induce humoral immunity against the membrane protein.

  In the animal immunization method of the present invention, both the gene encoding the “full length” of the membrane protein and the gene encoding the “part” of the membrane protein can be used, and can be used properly according to the purpose. When a gene encoding the full length of the membrane protein is used, for example, it is suitable for producing an antibody against each of a plurality of epitopes possessed by the membrane protein or an antibody that recognizes the three-dimensional structure of the membrane protein. On the other hand, when a gene encoding a part of a membrane protein is used, for example, it is suitable for producing an antibody against a specific epitope.

  As a mode of linking the “gene encoding the full length or part of the membrane protein” (membrane protein gene) and the “gene encoding the full length or part of flagellin” (flagellin gene), the 3 ′ end of the membrane protein gene And the 5 ′ end of the flagellin gene and the 5 ′ end of the membrane protein gene and the 3 ′ end of the flagellin gene are both included. Moreover, the membrane protein gene and the flagellin gene may be indirectly linked via a sequence such as a spacer.

  Furthermore, an embodiment in which the flagellin gene is “inserted” at a place other than the end of the membrane protein gene (that is, inside) is also included in the present invention. This embodiment can also be interpreted as the end of the flagellin gene linked to the end of the gene encoding a part of the membrane protein. Similarly, an embodiment in which the membrane protein gene is “inserted” at a place other than the end of the flagellin gene (ie, inside) is also included in the present invention. This embodiment can also be interpreted as the end of the membrane protein gene linked to the end of the gene encoding a part of the flagellin gene.

  “A fusion gene obtained by linking a gene encoding the full length or part of flagellin to a gene encoding the full length or part of the membrane protein” may include genes other than the membrane protein gene and the flagellin gene. . For example, in addition to the intervening sequence such as the spacer described above, a sequence having a specific function or the like can be added to the 3 ′ end or 5 ′ end of the fusion gene, or can be inserted inside.

  The type (origin) of flagellin employed in the present invention is not particularly limited, and any flagellin can be used. Examples of the origin of flagellin include the genus Salmonella such as Salmonella typhimurium; the genus Escherichia such as Escherichia coli; the genus Bordetella such as Bordetella pertussis; Shigella genus such as Shigella; Legionella genus; Burkholderia genus; Pseudomonas genus; Helicobacter genus; Vibrio genus; Serratia genus; (Caulobacter) genus; Listeria genus; Clostridium genus; Borrelia genus, etc.

  As an example of flagellin, Salmonella typhilium-derived flagellin will be described. Salmonella typhilium-derived flagellin is a protein having a mass of about 52 kDa. The flagellar skeleton of Salmonella typhilium is formed by polymerizing the flagellins. The gene for Salmonella typhilium-derived flagellin is known, and its nucleotide sequence is also known (for example, GeneBank # D13689; SEQ ID NO: 5). For example, a primer set is designed based on the information of the base sequence shown in SEQ ID NO: 5, and PCR is performed using a nucleic acid (genomic DNA, vector, plasmid, etc.) containing a Salmonella typhilium-derived flagellin gene as a template to obtain a desired The flagellin gene can be obtained. Moreover, the gene of Salmonella typhilium-derived flagellin can also be obtained by chemical synthesis.

  The “gene encoding the full length or part of flagellin” (flagellin gene) in the animal immunization method of the present invention includes the same gene derived from natural flagellin as well as the gene encoding the full length of natural flagellin. A gene encoding a protein having activity is included. Examples include a gene encoding a mutant flagellin obtained by modifying natural flagellin by amino acid substitution / insertion or the like. In addition, there are a gene encoding a protein consisting of a domain of a natural type or a part of the mutant flagellin, and a gene encoding a protein lacking a part of the domain of the natural type or the mutant flagellin. Can be mentioned.

  The membrane protein employed in the animal immunization method of the present invention is not particularly limited. Examples include G protein-coupled receptor (GPCR), ion channel receptor, tyrosine kinase receptor, CD antigen, cell adhesion molecule, cancer antigen and the like. In particular, GPCR has high species homology between humans and non-human animals, and is a useful protein for drug development. Therefore, obtaining an antibody against GPCR using the animal immunization method of the present invention is useful for construction of an assay system, functional analysis, and the like.

  Examples of GPCRs include endothelin receptor, rhodopsin, muscarinic acetylcholine receptor, adrenergic receptor, adenosine receptor, angiotensin receptor, dopamine receptor, glucagon receptor, histamine receptor, opioid receptor, GABA receptor, Gastrin receptor, serotonin receptor, P2Y receptor, cannabinoid receptor, cholecystokinin receptor, olfactory receptor, secretin receptor, somatostatin receptor, bradykinin receptor, C-C chemokine receptor, CXC chemokine receptor, Examples include galanin receptor, leukotriene receptor, melanin-concentrating hormone receptor, prostanoid receptor, orexin receptor.

  As a preferred embodiment, a fusion gene in which a flagellin gene is inserted between a DNA sequence encoding a signal sequence of a membrane protein and a gene encoding an extracellular domain from the 5 ′ end (coding a signal sequence of a 5′-membrane protein) DNA sequence encoding -flagellin gene-gene encoding extracellular domain-3 '), or DNA sequence encoding foreign signal sequence such as Igκ leader sequence from the 5' end and flagellin gene and membrane protein gene or extracellular membrane protein Examples include the use of a fusion gene of a gene encoding a domain (5'-DNA sequence encoding a foreign signal sequence-flagellin gene-membrane protein gene or gene-3 'encoding an extracellular domain of a membrane protein). With this configuration, flagellin is presented on the cell membrane when the fusion gene is expressed, and macrophages and dendritic cells expressing TLR5 can be activated.

  The non-human animal used in the animal immunization method of the present invention is not particularly limited, but mammals and birds are preferably used from the viewpoint of ease of handling. Examples of mammals include mice, rats, rabbits, cows, horses, dogs, cats, goats, sheep and pigs. Examples of birds include chickens, ducks or turkeys.

In the animal immunization method of the present invention, an embodiment in which a fusion gene is incorporated into an expression vector and its expression is regulated by a promoter is preferred. With this configuration, the fusion gene is reliably expressed in the animal body, and humoral immunity is more reliably induced.
The expression vector may be any expression vector that can be replicated in animal cells. Expression vectors such as pCI, pSI, pAdVantage, pTriEX, pKA1, pCDM8, pSVK3, pMSG, pSVL, pBK-CMV, pBK-RSV, and EBV Can be mentioned.
In addition, the promoter on the expression vector may be any one that functions in animal cells. For example, the CMV promoter of Cytomegarovirus (CMV); the AML promoter of Adenovirus Major Late (AML); the SV40 promoter of Simian Virus 40 (SV40); the fusion of SV40 and HTLV-1 LTR SRα promoter which is a promoter; EF-1α promoter of elongation factor (EF), and the like.

  Furthermore, the expression vector may contain an enhancer that enhances promoter activity. Further, the expression vector may contain a CpG motif. The CpG motif may be in any position on the expression vector, and may be only one place or a plurality of places.

  The method for administering the fusion gene in the animal immunization method of the present invention is not particularly limited, and can be performed by a known technique such as subcutaneous injection, intramuscular injection, intravenous injection or the like. Administration by particle gun is also applicable. The dose of the fusion gene may be appropriately determined according to the expression vector used, the type of promoter, etc., but as a guideline, it is generally 1 to 3 mg / kg body weight per time, which is 25 to 25 for mice. 100 μg / time. Further, the administration may be performed once, but a higher humoral immunity response can be induced by performing multiple administrations at regular intervals.

Next, the immunizing composition of the present invention will be described. The immunizing composition of the present invention is used in the animal immunization method of the present invention and is administered to a non-human animal. The gene encoding the full length or part of the membrane protein contains the full length or part of flagellin. It contains a fusion gene formed by linking genes to be encoded. As a typical shape of the immunizing composition of the present invention, the fusion gene is dissolved in an isotonic solution. Examples of isotonic solutions include saline and phosphate buffered saline (PBS). In addition, various buffers can be used as the solvent. It is also effective to add a metal ion such as Mg ²⁺ to the isotonic solution in order to increase the antibody production ability. Furthermore, GM-CSF, TNFα, and IL-4, which are cytokines that induce differentiation of T _H 2 helper T cells that induce humoral immunity, are added to isotonic solutions, and genes encoding these cytokines It is also effective to increase the antibody production ability. Furthermore, IL-1, IL-2, IL-4, IL-5, IL-6, IL, which are cytokines that induce activation, division, and differentiation into antibody-producing cells, which are the main players in antibody production It is also possible to increase antibody production by adding -10 or genes encoding them to isotonic solutions. The concentration of the fusion gene in the immunizing composition of the present invention is, for example, about 10 to 500 μg / mL.

  Furthermore, the immunizing composition of the present invention may contain an oligonucleotide composed of a CpG motif. In this case, the oligonucleotide functions as an adjuvant and can also be used as a vaccine for inducing a higher immune response.

Next, a method for producing the antibody of the present invention will be described. The method for producing an antibody of the present invention includes two aspects. One aspect is to obtain antibodies against membrane proteins from non-human animals immunized by the animal immunization method of the present invention. As a specific example, partial blood collection is periodically performed from an animal after immunization to measure the antibody titer and monitor the production state of the antibody. Then, when the antibody titer reaches the maximum, whole blood is collected to prepare serum. Then, an antibody is obtained from the obtained serum. In this case, the obtained antibody is a polyclonal antibody. In addition, as a method for isolating and purifying an antibody from serum, a method generally used for antibody purification can be used. For example, affinity chromatography using protein A can be used.
Other aspects of the antibody production method of the present invention will be described later.

  Next, the manufacturing method of the hybridoma of this invention is demonstrated. The method for producing a hybridoma of the present invention obtains a hybridoma that produces an antibody against a membrane protein by fusing B lymphocytes collected from a non-human animal immunized by the animal immunization method of the present invention with myeloma. Is. Known methods can be used as they are for cell fusion, hybridoma selection and cloning. For example, cell fusion can be performed by the method of Kohler and Milstein. The selection of hybridomas can be performed by culturing using a HAT selection medium. Furthermore, the hybridoma can be cloned by a limiting dilution method.

  By culturing the hybridoma thus cloned, an antibody against a membrane protein (monoclonal antibody) can be produced. That is, another aspect of the antibody production method of the present invention is to obtain an antibody against a membrane protein from the hybridoma produced by the hybridoma production method of the present invention. For example, the hybridoma can be cultured, and a desired antibody can be obtained from the culture. Hybridoma culture may be carried out in the peritoneal cavity of an animal such as a mouse or in vitro using a dish or the like. When hybridomas are cultured in the abdominal cavity of animals such as mice, ascites can be collected, and antibodies can be isolated and purified from the ascites. When cultured in vitro, the antibody can be isolated and purified from the culture solution. As a method for purifying an antibody, in the case of an antibody whose subclass is IgG, for example, it can be performed by affinity chromatography using the above-mentioned protein A.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to the examples.

(1) Isolation and preparation of mouse-derived endothelin A receptor (mETAR) gene Mouse-derived endothelin A receptor (mETAR) (NM_010332.2) gene as a model antigen protein, mouse lung cDNA library (Takara Bio Inc.) PCR was performed using the oligonucleotide having the nucleotide sequence shown in SEQ ID NOs: 1 and 2 as a primer pair, and a DNA fragment (DNA fragment A) containing the mETAR gene having the nucleotide sequence shown in SEQ ID NO: 3 was obtained. In DNA fragment A, a sequence encoding a NheI site at the 5 ′ end and 2 stop codons (TAATAG) at the 5 ′ end and a SalI site were introduced into the DNA fragment A. In addition, a DNA fragment containing the mETAR gene from which the signal sequence (ss) was removed by PCR using the mouse lung cDNA library as a template and the oligonucleotides having the nucleotide sequences shown in SEQ ID NOs: 2 and 4 as primer pairs (DNA fragment B, mETAR (Δs.s.)) was obtained. The DNA fragment B (mETAR (Δs.s.)) has a sequence derived from a primer, a HindIII-NheI site at the 5 ′ end, a sequence encoding two stop codons (TAATAG) at the 3 ′ end, and a SalI site. Was introduced.

(2) Preparation of flagellin gene From Salmonella typhilium-derived flagellin gene (FliC) having the base sequence shown in SEQ ID NO: 5 (obtained from the information of GeneBank # D13689), HindIII at the 5 ′ end and mETAR derived from mETAR shown in SEQ ID NO: 3 A gene having a signal sequence (ss) and a flag tag sequence shown in SEQ ID NO: 6 and a SalI site introduced at the 3 ′ end (hereinafter “ss-flag-FliC”) Was synthesized). s. s. -Flag-FliC was integrated into the subcloning vector pUC57, and the plasmid pUC57-s. s. -Flag-FliC was obtained.

(3) Construction of a vector for gene immunization that expresses a fusion protein in which flagellin is linked to a mouse-derived endothelin A receptor Mammalian expression vector pCI Mammalian Expression Vector (Promega) is digested with restriction enzymes NheI and SalI, and derived from Baclia After dephosphorylating the end with alkaline phosphatase (BAP), the DNA fragment A prepared in (1) above was inserted to obtain vector pCI-mETAR. Further, the vector pCI-mETAR was digested with NotI and SalI, the end was dephosphorylated with BAP, and the DNA fragment B prepared in (1) above was inserted, and the vector pCI-mETAR (Δs.s.) was inserted. Built. Further, pCI-mETAR (Δs.s.) and pUC57-s. s. -Flag-FliC was digested with NotI and NheI, and the end was dephosphorylated with BAP, followed by s. s. -Flag-FliC is inserted and pCI-s. s. -Flag-FliC-mETAR (Δs.s.) was constructed. That is, the vector pCI-s. s. -Flag-FliC-mETAR (Δs.s.) has a fusion gene in which a flagellin gene is linked to the 5 ′ end of the mETAR gene. On the other hand, the vector pCI-mETAR has only the mETAR gene.

(4) Preparation of mouse-derived endothelin A receptor stably expressing cells After digesting the vector pCI-mETAR constructed in (3) with NheI and SalI and dephosphorylating the ends with BAP, pCIneo (Promega) The vector pCIneo-mETAR was constructed at the NheI-XhoI site.

37.5 μL of Lipofectamine 2000 (Invitrogen) solution, 625 μL of OPTI-MEM (Gibco) medium, and 625 μL of OPTI-MEMI medium containing 20 μg of pCIneo-mETAR were mixed. Using this mixed solution, pCIneo-mETAR was introduced into 2 × 10 ⁵ CHO-K1 cells (DS Pharma). The CHO-K1 cell into which the gene was introduced was cultured in Ham'sF12K (Wako Pure Chemical Industries) + 10% FBS medium (ICN) for 30 hours. Further, each cell was detached, suspended, seeded with 5 × 10 ⁵ cells in a 100 mm dish, and treated with Ham's F12K + 10% FBS medium containing antibiotic G418 (Promega) at a concentration of 0.8 mg / mL for 2 weeks. Was done. After drug treatment, antibiotic resistant cells were cloned by limiting dilution.

(5) Cell surface expression of flag-Flic-mETAR Vector pCI-s. s. -Flag-FliC-mETAR (Δs.s.) was introduced into 293T cells (DS Pharma) using Lipofectamine 2000 (hereinafter referred to as “gene-transferred 293T cells”). The gene-transferred 293T cells were cultured in Mem-α (Wako Pure Chemical Industries, Ltd.) + 10% FBS medium for 48 hours. The cultured cells were peeled and collected, washed with PBS, Anti-FLAG M2 antibody (Sigma) was added as a primary antibody, and reacted at room temperature. Further, the cells are washed with PBS, and a phycoerythrin-labeled anti-mouse IgG antibody (Beckman Coulter, Inc.) is used as a secondary antibody on the cell membrane of the gene-transferred 293T cell, “a fusion gene in which the flagellin gene is linked to the mETAR gene. ”(Flagellin-fused mETAR) was analyzed. As a control, the measurement was performed under the same conditions on 293T cells of only the host into which the gene was not introduced. The results are shown in FIG.

  As shown in FIG. 1 (a), fluorescence derived from phycoerythrin was detected in the transgenic 293T cells. On the other hand, as shown in FIG. 1B, almost no phycoerythrin-derived fluorescence was detected in the control 293T cells. From this, it was shown that the flagellin-fused mETAR is expressed on the cell membrane in the gene-transferred 293T cells.

(6) Gene immunization The vector pCI-s. s. -Flag-FliC-mETAR (Δs.s.) was dissolved to a concentration of 500 μg / mL to prepare a composition for immunization. This composition for immunization was immunized by injecting 0.10 mL each into both thigh muscles of 8-week-old mouse BALB / c (female) (day 0). As a result, pCI-s. s. -Flag-FliC-mETAR (Δs.s.) was administered to both legs at 50 μg each, that is, 100 μg per animal. Thereafter, immunization was repeated in the same manner on the 7th, 21st and 28th days. Blood was collected on days 0, 21, and 28 to prepare serum. As a control, evaluation was performed using a mouse (control A) administered with only PBS containing no vector and a mouse (control B) administered with the vector pCI-mETAR expressing mETAR alone.

(7) Cytokine ELISA measurement In order to examine whether the lymphocytes of the mice immunized in (6) induce humoral immunity, T cells were isolated and cytokine IL-4 that induces humoral immunity was measured. . Specifically, CD4 ⁺ T cells were sorted from the spleen and lymph nodes of mice immunized in (6) using MACS CD4 beads (Milteny Biotech). The isolated CD4 ⁺ T cells are suspended in RPMI-1640 (Sigma) + 10% FBS so as to be 2 × 10 ⁶ cells / mL, and previously coated with an anti-αβTCR antibody (Becton Dickinson) at 30 μg / mL. Added to the prepared plate to stimulate the cells. Culturing was performed at 37 ° C. for 24 hours in a CO ₂ incubator, and the culture supernatant was collected.

  Anti-IL-4 antibody diluted to 2 μg / mL using a Cytokine ELISA Kit (Becton Dickinson) was plated on an ELISA plate and immobilized at 4 ° C. After washing with a washing solution (PBST) in which PBS and Tween 20 were mixed, a blocking solution (Nacalai Tesque) was added to perform blocking at room temperature. After washing with PBST, the culture supernatant collected in (7) was added and allowed to react at room temperature. Further, it was washed with PBST, and a biotin-labeled anti-IL-4 antibody diluted to 1 μg / mL was added. After reaction at room temperature, the plate was washed with PBST, and further reacted by adding horseradish peroxidase-labeled streptavidin. Finally, after washing with PBST, TMB (Betil) was added to perform a colorimetric reaction. Thereafter, the reaction was stopped with 1N sulfuric acid (Wako Pure Chemical Industries, Ltd.). Thereafter, IL-4 was measured with a microplate reader (Bio-Rad) at a wavelength of 450 nm (FIG. 2).

  As shown in FIG. 2, IL-4 was hardly secreted in the T cells of mice (control A) administered with PBS alone. Also, a very small amount of IL-4 was secreted from the T cells of mice (control B) administered with only the mETAR gene. In contrast, IL-4 was secreted from T cells of mice administered with the flagellin-fused mETAR gene. This indicated that flagellin induced humoral immunity.

  IFN-γ was measured in place of IL-4 by the above operation (FIG. 3). As shown in FIG. 3, IFN-γ was hardly secreted by T cells of mice (control A) administered with PBS alone. Further, only a very small amount of IFN-γ was secreted from the T cells of mice administered with only the mETAR gene (control B). In contrast, IFN-γ was secreted from T cells of mice administered with the flagellin-fused mETAR gene. This indicated that flagellin induced IFN-γ secretion from T cells.

(9) Evaluation of antibody binding in serum to mETAR by flow cytometry CHO-K1 cells (hereinafter referred to as “mETAR gene-introduced CHO-K1 cells”) into which pCIneo-mETAR has been introduced and stable expression has been confirmed, and pCIneo The introduced CHO-K1 cells (hereinafter referred to as “control CHO-K1 cells”) were washed with PBS. Serum on day 42 after immunization was diluted 100-fold and incubated with each cell. Further, each cell was washed with PBS, phycoerythrin-labeled anti-mouse IgG antibody was added as a secondary antibody, and then the interaction between each cell and anti-mETAR antibody in serum was analyzed with a flow cytometer FACScalibur. The results are shown in FIG.

  As shown in FIG. 4, when mETAR gene-introduced CHO-K1 cells were used, serum from mice before gene immunization, serum from PBS-administered mice (control A), and mETAR gene-administered mice (control B) No phycoerythrin-derived fluorescence was detected from any of the sera. This indicated that humoral immunity against mETAR was not induced in Control A and Control B mice. On the other hand, fluorescence derived from phycoerythrin was detected from the serum of mice administered with the flagellin-fused mETAR gene. This indicated that mice administered with the flagellin-fused mETAR gene produced an anti-mETAR antibody, and the anti-mETAR antibody bound to mETAR gene-introduced CHO-K1 cells.

When control CHO-K1 cells were used, no phycoerythrin-derived fluorescence was detected in any mouse serum. This indicated that the anti-mETAR antibody did not bind to control CHO-K1 cells.
From the above, the vector pCI-s. s. -The production of antibodies specifically recognizing the extracellular domain of mETAR in mouse serum could be induced by genetic immunization with flag-FliC-mETAR (Δs.s.).

Claims (8)

An animal immunization method for inducing a humoral immunity response to a desired membrane protein in the non-human animal by immunizing the non-human animal with an antigen,
By administering to a non-human animal a fusion gene obtained by linking a gene encoding the full-length or part of flagellin to a gene encoding the full-length or part of the membrane protein, the fusion gene is transferred within the non-human animal. An animal immunization method characterized by causing expression and inducing humoral immunity against the membrane protein.   The flagellins may include Salmonella, Escherichia, Bordetella, Shigella, Legionella, Burkholderia, Pseudomonas, Helicobacter (Pseudomonas) It is derived from microorganisms belonging to the genus Helicobacter, Vibrio, Serratia, Calobacter, Listeria, Clostridium, or Borrelia The animal immunization method according to claim 1.   3. The membrane protein according to claim 1, wherein the membrane protein is a G protein-coupled receptor, an ion channel receptor, a tyrosine kinase receptor, a CD antigen, a cell adhesion molecule, or a cancer antigen. Animal immunization method.   The said non-human animal is a mouse, a rat, a rabbit, a cow, a horse, a dog, a cat, a goat, a sheep, a pig, a chicken, a duck, or a turkey. Animal immunization method. A composition for immunization used in the animal immunization method according to any one of claims 1 to 4 and administered to a non-human animal,
A composition for immunization comprising a fusion gene obtained by linking a gene encoding the full length or part of flagellin to a gene encoding the full length or part of the membrane protein.   An antibody production method, comprising obtaining an antibody against the membrane protein from a non-human animal immunized by the animal immunization method according to claim 1.   A hybridoma producing an antibody against the membrane protein is obtained by fusing B lymphocytes collected from a non-human animal immunized by the animal immunization method according to any one of claims 1 to 4 and myeloma. A method for producing a hybridoma, comprising:   A method for producing an antibody, comprising obtaining an antibody against the membrane protein from a hybridoma produced by the method for producing a hybridoma according to claim 7.

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