WO2004087914A1 - Method of constructing antibody - Google Patents

Abstract

It is intended to provide a method of constructing an antibody by which a monoclonal antibody can be prepared within a short time at a high efficiency and which is commonly usable. Namely, a method of constructing an antibody comprising the following steps (a) to (d): (a) the step of bringing a labeled antigen prepared by labeling an antigen recognized by a target antibody into contact with cells containing a target cell producing the above antibody to thereby bonding the labeled antigen to the target cell; (b) the step of separating the labeled target cell obtained in the step (a); (c) the step of using the labeled target cell thus separated to prepare an antibody gene carried by it; and (d) the step of expressing the antibody gene thus prepared with the use of an expression vector.

Description

 Specification

Antibody production method

Technical field

 The present invention relates to a method for producing an antibody. Specifically, the present invention relates to a method for producing an antibody using a genetic engineering technique, and an application thereof.

Background art

The antibody-related industry has traditionally developed based on the technology for producing monoclonal antibodies by cell fusion. Many antibodies have been developed and sold as research reagents, diagnostic reagents, and reagents for monitoring various substances, and the development and production of therapeutic antibodies are underway. In recent years, an antibody library production technology using a phage display system has been developed, and has exerted great power in the isolation of human antibodies. The present inventors and collaborators have used the phage display system as a basic technology to isolate human antibodies against various infectious diseases that are clinically useful. As a result, we succeeded in producing several human antibodies each showing very strong neutralizing activity against influenza virus and varicella-zoster virus (presented at the 24th Annual Meeting of the Molecular Biology Society of Japan). “Isolation of human anti-influenza virus neutralizing antibody from antibody library”, presented at the 50th Virological Society of Japan “Isolation of varicella-zoster virus neutralizing antibody from human antibody library”). In addition, as a reagent for protein function analysis in the post-genome era, we have been conducting systematic isolation and preparation of antibodies (presented at the 25th Annual Meeting of the Molecular Biology Society of Japan using antibodies against proteins derived from C. elegans. Expression pattern analysis ”,“ Isolation of monoclonal antibodies against rc. Elegans-derived proteins ”). Through this series of studies, we successfully cloned the gene encoding the antibody present in the animal body, including humans. It was shown that the antibody produced in the E. coli production system and the antibody produced in the animal were completely identical in terms of antigen binding (at the 25th Annual Meeting of the Molecular Biology Society of Japan). Presentations: “Expression pattern analysis using antibodies against proteins derived from C. elegans”, Isolation of monoclonal antibodies against proteins derived from rc. While phage display technology has various advantages, its low efficiency has been a problem. In vivo, a heavy chain (H chain) and a light chain (L chain) exist as a set. To reproduce this in a phage display system, first, each of the H chain and L chain must be grouped. After library consolidation, each library must be combined to produce a large antibody library. Therefore, for example, in order to cover “10,000 kinds of antibodies”, it is necessary to construct a large antibody library of 10,000 × 100,000 = 100 million. Although the library thus prepared contains the target antibody, most of the antibody consists of a combination of the H chain and the L chain that does not exist originally. And considerable technical proficiency is required. In particular, there is no guarantee that the resulting clone will be the same combination (H and L) as the antibody the animal originally made. On the other hand, the classical method for producing a monoclonal antibody using cell fusion involves a problem that, first of all, a great deal of labor and cost is required until isolation of a monoclonal antibody-producing cell. In addition, the probability that an antibody-producing cell can be fused with a myeloma cell and the obtained hybridoma can be cloned is low, and the types of hybridoma finally obtained are extremely small. Therefore, it is extremely difficult to obtain the desired antibody-producing cells. Disclosure of the invention The present invention has been made under the above background, and it is an object of the present invention to provide a highly versatile antibody production method capable of preparing a monoclonal antibody in a short period of time and efficiently. Main purpose. Means for Solving the Problems The present inventors have conducted intensive studies in order to achieve the above object, and have reached the following configurations.

 [1] An antibody production method comprising the following steps (a) to (d):

 (a) contacting a labeled antigen obtained by labeling an antigen recognized by a target antibody with a cell population containing a target cell producing the antibody, and binding the labeled antigen to the target cell;

 (b) separating the labeled target cells obtained in step (a);

(c) using the separated labeled target cells to prepare an antibody gene carried by the cells; and

 (A step of expressing the prepared antibody gene using an expression vector.

[2] The method for producing an antibody according to [1], wherein one labeled target cell is separated in the step (b).

[3] The method for producing an antibody according to [1] or [2], wherein the step (b) is performed by manipulation, [5] extra dilution method, or flow cytometry.

[4] The antibody production according to any one of [1] to [3], wherein the step (c) is carried out by a nucleic acid amplification reaction using the genomic DNA of the labeled evening get cell as a type III. Method. [5] The method for producing an antibody according to any one of [1] to [4], wherein the step (c) is performed directly using the labeled target cells separated in the step (b).

[6] The method for producing an antibody according to any one of [1] to [5], wherein the step (c) includes the following steps:

 (C-1) A set of primers capable of amplifying H chain genes, a set of primers capable of amplifying L chain genes, and a reagent kit containing the separated labeled target cells. Performing a nucleic acid amplification reaction after adding the nucleotide triphosphate;

 (c-2) a step of removing a part of the reaction solution after step (c-1), adding a primer set capable of amplifying the H chain gene thereto, and then performing a nucleic acid amplification reaction; and

 (c-3) A step of removing a part of the reaction solution after step (c-1), adding a primer set capable of amplifying an L chain gene thereto, and then performing a nucleic acid amplification reaction.

[7] The method for producing an antibody according to [6], wherein the nucleic acid amplification reaction in the step (c-1) is a PCR of 5 cycles or more. [8] The antibody production method according to [6] or [7], wherein the nucleic acid amplification reaction in the steps (c-2) and (c-3) is PCR of 20 cycles or more.

[9] The antibody production method according to any one of [1] to [8], wherein in the step (c), an H chain variable region gene and an L chain variable region gene are prepared. [10] The expression vector has a linker sequence,

 The method for producing an antibody according to [9], wherein in the step (d), the H chain variable region gene and the L chain variable region gene are inserted into the expression vector so as to be linked via the linker sequence.

[11] In any one of [1] to [8], in the step (c), an H chain variable region gene and an L chain gene encoding an L chain variable region and an L chain constant region are prepared. The method for producing an antibody according to any of the above items.

[12] In the step (c), an H chain variable region gene and an L chain variable region gene are prepared,

 The expression vector has a first sequence encoding an H chain constant region, and a second sequence encoding an L chain constant region,

 In the step (d), the H chain variable region gene is inserted into the expression vector so as to be linked to the first sequence, and the L chain variable region gene is so linked as to be linked to the second sequence. The method for producing an antibody according to any one of [1] to [8], which is inserted into an expression vector.

[13] In the step (c), an H chain variable region gene and an L chain gene encoding an L chain variable region and an L chain constant region are prepared,

 The expression vector has a sequence encoding a heavy chain constant region,

In the step (d), the H chain variable region gene is inserted into the expression vector so as to be linked to the sequence, and the L chain gene is inserted into the vector. The method for producing an antibody according to any one of the above. [14] In the step (c), an H chain variable region gene and an L chain variable region chain gene are prepared,

 The method for producing an antibody according to any one of [1] to [8], wherein the step (d) includes the following steps:

 Inserting the H chain variable region gene into a first expression vector having an H chain constant region gene, and expressing an H chain in which the H chain variable region and the H chain constant region are connected;

 Inserting the L chain variable region gene into a second expression vector having an L chain constant region gene to express an L chain in which the L chain variable region and the L chain constant region are connected.

[15] In the step (c), an H chain variable region gene and an L chain gene encoding an L chain variable region and an L chain constant region are prepared,

 The method for producing an antibody according to any one of [1] to [8], wherein the step (d) includes the following steps:

 Inserting the H chain variable region gene into a first expression vector having an H chain constant region gene to express an H chain in which the H chain variable region and the H chain constant region are connected; and

 Inserting the L chain gene into a second expression vector to express an L chain in which the L chain variable region and the L chain constant region are connected.

[16] In the step (c), an H chain variable region gene is prepared.

 [5] The method for producing an antibody according to any of [5].

[17] In the step (c), an L chain variable region gene is prepared.

The method for producing an antibody according to any of [5]. [18] The antibody production method according to any one of [12] to [15], wherein an IgG class antibody is constructed in the step (d). [19] The method for producing an antibody according to any one of [1] to [18], wherein the evening get cells are human cells.

[20] The method for producing an antibody according to any one of [1] to [19], wherein the labeled antigen is an antigen labeled with a fluorescent substance, bitin, or magnetic beads.

[21] An antibody obtained by the antibody production method according to any one of [1] to [20].

[22] A method for preparing an antibody gene, comprising the following steps (a) to (c):

 (a) contacting a labeled antigen obtained by labeling an antigen recognized by an antibody of interest with a cell population containing an overnight getter cell producing the antibody, and binding the labeled antigen to the evening getter cell; Step of causing;

 (b) separating the labeled target cells obtained in step (a);

 (c) A step of using the separated labeled target cells to prepare an antibody gene carried by the labeled target cells.

[23] Gene preparation method comprising the following steps (i) to (iii):

 (i) labeling cells having a particular property in the cell population;

 (ii) separating the labeled cells obtained by step (i); and

(iii) a step of preparing a target gene using the separated labeled cells. [24] The gene preparation method according to [23], wherein one labeled cell is separated in the step (ii). BRIEF DESCRIPTION OF THE FIGURES

 FIG. 1 is a diagram showing the results of fluorescent staining of spleen cells of mice immunized with lysozyme. The left panel is a stained image with Alexa488-labeled lysozyme, and the right panel is a stained image with Alexa546-labeled anti-mouse IgG antibody.

 FIG. 2 is a diagram showing the results of fluorescent staining of spleen cells of mice immunized with GFP. The left column is a stained image with GFP, and the right column is a stained image with Alexa546-labeled anti-mouse IgG antibody.

 Figure 3 shows the expected fluorescent staining image when the fusion protein is used as an antigen. Cells expressing antibodies to the target protein (VHH) are stained with Alexa488 (green fluorescence) (Fig. 3, lower left), and cells expressing antibodies to the tag protein (MBP) are Alexa488 (green fluorescence) and It is considered to be stained with Alexa546 (red fluorescence) (Fig. 3, lower right).

 FIG. 4 shows the results of actual fluorescent staining of spleen cells of mice immunized with the fusion protein (MBP-VHH). The left column is a stained image with AI exa488-labeled MBP-VHH, and the right column is a stained image with Alexa546-labeled MBP.

 FIG. 5 is a diagram showing the results of agarose gel electrophoresis of the PCR product in Example 4. The samples in each lane are as follows.

 λΗ; DNA size marker

 Α50 cycles of cells (40 cycles after 10 cycles)

 After 10 cycles of A cells, 48 cycles of〗 ii i with the reaction solution for H chain amplification

After 10 cycles of A cells, 48 cycles of 1 / I with the reaction solution for light chain amplification 50 cycles of B cells (40 cycles after 10 cycles)

 After 10 cycles of B cells, 48 cycles of I with the reaction solution for H chain amplification.After 10 cycles of B cells, 48 cycles of 1 μ | with the reaction solution of L chain amplification.

 After 10 cycles of C cells, 48 cycles of l I I in the reaction solution for H chain amplification After 10 cycles of C cells, 48 cycles of 1 I in the reaction solution for L chain amplification

10; 50 cycles with the reaction solution for D cell H chain amplification (40 cycles after 10 cycles)

 11; After 10 cycles with the D cell H chain amplification reaction solution, 48 cycles of 1 I with the H chain amplification reaction solution

 FIG. 6 is a flowchart showing the procedure for constructing the vector PSCCA4-E8d.

 FIG. 7 is a diagram showing the configuration (partial sequence) of the vector scNcopFCAH9-E8VHdVLd. FIG. 8 is a diagram showing the configuration of the vector sclkopFCAH9-E8VHdVLd (continuation of the sequence shown in FIG. 7).

 FIG. 9 is a diagram showing the configuration (partial sequence) of the vector PSCCA4-E8d.

 FIG. 10 is a diagram showing the configuration of the vector PSCCA4-E8d (continuation of the sequence shown in FIG. 9).

 FIG. 11 is a graph showing the results of a color development test in Example 8. The absorbance (wavelength 492) for each sample is shown. PBS is a negative control. FIG. 12 is a diagram showing the results of fluorescent staining of human antibody-producing cells. The left column is a stained image with the AI exa488-labeled influenza vaccine, and the right column is a stained image with the PE-labeled anti-human IgG antibody.

 FIG. 13 is a flowchart showing the procedure for constructing the expression vector pSMAP.

FIG. 14 is a diagram showing the configuration (partial sequence) of vector pSMAP expressing human Fab-PP type antibody. FIG. 15 is a diagram showing the structure of the vector pSMAP expressing the human Fab-PP type antibody (continuation of the sequence shown in FIG. 14).

 FIG. 16 is a graph showing the results of a color development test (ELISA) in Example 15. The absorbance (wavelength 492) of each sample is shown.

 FIG. 17 is a graph showing the results of ELISA (Example 16) using the purified antibody. The absorbance (wavelength 492) of each sample is shown. BEST MODE FOR CARRYING OUT THE INVENTION

 (Labeling step)

 In the antibody production method of the present invention, as a first step, a labeled antigen obtained by labeling an antigen recognized by an objective antibody is brought into contact with a cell population including an evening-producing cell producing the antibody, A step (Step (a)) of binding the labeled antigen to the target cell is performed. By this step, cells (also referred to herein as “target cells”) that produce the target antibody (also referred to as “evening antibody” in the present specification) will be labeled.

 The origin of the target antibody (evening antibody) in the present invention is not particularly limited. For example, antibodies derived from various species, such as mice, rats, rabbits, egrets, guinea pigs, rabbits, bush, higgins, giants, sal, chimpanzees, and humans.

The “antigen recognized by the target antibody” refers to a molecule specifically recognized by the overnight antibody. In the following description, unless otherwise specified, “antigen” means an antigen recognized by a target antibody. In the present invention, various kinds of antigens can be adopted. The antigen is typically a peptide or protein, but may be variously modified as long as it has a function as an antigen. For example, it has another peptide or protein part in addition to the functional part as an antigen, or Any fusion protein can be used as an antigen in the present invention. In addition, those to which a sugar chain, a fatty chain or the like is added may be used as the antigen.

The “cell population containing target cells” is prepared from animal tissues, organs, and the like that are expected to contain target cells in the evening. For example, such a cell population can be prepared from a tissue or organ in which antibody-producing cells are present in an animal immunized with an antigen (an antigen recognized by a target antibody). Specific examples of the tissue in which the antibody-producing cells are present include spleen, bone marrow, peripheral blood, cord blood, and tonsils. Examples of animals serving as a source of the cell population include mice, rats, rabbits, guinea pigs, guinea pigs, pigs, pigs, sheep, goats, monkeys, chimpanzees, and humans, but are not limited thereto. An appropriate one is selected according to the antibody. In addition, immunization is not essential. That is, when an animal that produces or possesses a target cell is available, it is possible to prepare a target cell population from the animal without immunization. For example, when an antibody associated with a disease is used as a target antibody, it is expected that an animal suffering from the disease will produce the target antibody (ie, the presence of target cells). In an animal, a cell population containing target cells can be prepared from tissues or organs in which antibody-producing cells are likely to be present. Labeling substances used for labeling antigens include, for example, fluorescein isothiocyanate (FITC), tetramethyl rhodamine isothiocyanate (TRITC) europium, Alexa488 (trade name), Alexa546 Fluorescent substances such as (trade name), chemiluminescent substances such as luminol, isorluminol, and acridinium derivatives, coenzymes such as NAD, bistin, and magnet beads can be used. Labeling with a labeling substance can be performed by a conventional method, for example, Molecular Cloning, Third Editi. on, Cold Spring Laboratory Press, New York. The contact between the labeled antigen and the cell population can be performed in a solution appropriate for the binding between the labeled antigen and the target cells. The contact between the labeled antigen and the cell population is preferably performed under conditions that cause less damage to target cells in the cell population. In particular, it is preferable to carry out the treatment under such conditions that the cell membrane-bound antibody of the target cell is not removed or its original function is not lost. By reacting under such conditions, the target cells and the labeled antigen are well bound, so that the evening cells can be efficiently recovered, and the evening cells having a good survival state can be obtained. Can be obtained. Examples of conditions that cause less damage to the target cells include conditions in which the reaction is performed in a low-temperature environment (for example, on ice). Prior to the labeling step described above, the cell population is contacted with an unlabeled antigen, and the cells that have bound to the antigen are recovered, thereby reducing the proportion of target cells in the cell population that is subjected to the labeling step. It is preferable to keep it high. By performing such a pretreatment, labeling in the labeling step becomes more efficient. The pre-processing is performed, for example, in the following procedure. First, an unlabeled antigen is fixed to a support such as a plate. Next, a solution in which the cell population is suspended is brought into contact with the antigen immobilized on the support. Then, wash and remove non-specifically adsorbed components (eg, cells). Finally, cells bound to the antigen are recovered by pipetting or the like.

(Cell separation step)

In this step, the labeled target cells obtained by the labeling step are separated (step (b)). For separation of labeled target cells, Appropriate methods are used depending on the type of substance. For example, when a fluorescent substance is used as the labeling substance, the labeled target cells can be observed with a fluorescence microscope. That is, visualization by fluorescence is possible, and cell separation can be performed using this. On the other hand, the flow cytometry (cell sorter) also enables cell separation using fluorescence as an index. According to flow cytometry, efficient and highly accurate cell separation is possible. Further, even when biotin is used as the labeling substance, good separation can be achieved by utilizing the binding reaction with avidin. Similarly, when magnet beads are used, good separation using magnets is possible. In addition, it is also possible to perform this step by using the manipulation ultra dilution method.

The above-described cell separation operation is performed using an apparatus (cell saw, etc.) and equipment appropriate for each method. However, since such an apparatus is commercially available, it can be easily used as desired. It is possible to carry out a separation operation. In addition, for the specific operation method, it is possible to refer to the instruction manual (for example, the instruction manual of Beckman Coulter Flow Cytometry EP IC ALTRATy pel V) attached to the equipment to be used, etc. . When an antigen having an additional portion such as a fusion protein is used, some of the cells separated and recovered as being bound to the labeled antigen express an antibody against the additional portion in addition to the target cell of interest. Cells (unintended cells) may be mixed. In such a case, it is necessary to separate target cells from unintended cells. The following methods (first method and second method) can be used to separate target cells from unintended cells, for example. The first method uses two types of labeling substances in the labeling step. In this method, first, an antigen (including an additional portion) labeled with one labeling substance (labeled antigen) and an antigen (only the additional portion labeled with a different labeling substance) (labeled additional portion) are prepared. Then, in the labeling step, the labeled antigen and the labeled additional portion are simultaneously brought into contact with the cell population. As a result of this treatment, both the labeled antigen and the labeled additional portion bind to cells expressing an antibody capable of binding to the additional portion, and two types of labeling signals are observed. . On the other hand, only the labeled antigen binds to the target cell, and only one labeling signal is observed. Therefore, sorting using the label signal as an index (that is, collection of target cells) becomes possible. The second method is to use the labeled additional portion alone. In this method, first, only the additional portion is prepared and labeled. The binding between the label-added portion thus obtained and the cells recovered by the labeling step is examined. Then, since the cells showing the binding are cells expressing an antibody recognizing the added portion, the target cells are selected by removing them. In the cell separation step, it is preferable to separate one labeled target cell. By performing such a separation operation, it becomes possible to obtain the H chain gene and the L chain gene derived from one cell in the antibody gene preparation step J described later, and the H chain of the antibody finally obtained. And the L chain combination originally existed, that is, the combination of the H chain and the L chain can produce a natural antibody. The information on the combination of is very useful when analyzing the obtained antibody or when modifying it by genetic engineering, etc. In addition, methods for separating one labeled target cell include: Manipulation, ultra-dilution, flow cytometry, etc. can be used.

(Antibody gene preparation step)

In this step, using the separated labeled target cells, the antibody gene carried by the cells is prepared (step (c)). Specifically, for labeled target cells The target antibody gene existing therein is prepared (isolated) using the nucleic acid. It is also possible to synthesize cDNA using the mRNA in the labeled target cell as type III and to prepare an antibody gene.However, since the RNA molecule is unstable, care must be taken when handling it. It is difficult to use the cells unless they are living cells or when the living cells are rapidly frozen. Therefore, in the present invention, it is preferable to prepare a target antibody gene using genomic DNA as a template. For example, such a preparation can be performed using a nucleic acid amplification reaction such as PCR.

 The use of genomic DNA has the advantage that various problems that occur when using RNA are solved, and that a desired antibody gene can be prepared from tissue sections and the like. The reason that the desired antibody gene can be prepared from the genomic DNA is that the antibody gene expressed in the cell undergoes DNA rearrangement.

It is preferable to directly subject the separated labeled target cells to a nucleic acid amplification reaction to prepare an antibody gene. If the nucleic acid amplification reaction is directly performed, the antibody gene can be prepared by simple operations. Also, the required time can be reduced. Here, “directly” means that the step of preparing a nucleic acid, such as RMA or cDNA, that functions as type I when amplifying is not performed. Therefore, for the purpose of enabling the nucleic acid amplification reaction, a crushing treatment or the like (for example, a heat treatment) of the labeled target cell may be performed prior to the nucleic acid amplification reaction. In addition, as shown in Examples described later, when the labeled target cells were directly subjected to PCR, favorable amplification of the antibody gene was confirmed. In this step, an H chain gene and / or an L chain gene are prepared. The term “chain gene” as used herein refers to a gene (H chain variable region gene) encoding only the H chain variable region (V _H DJ _H ), and a constant region (partly (A gene encoding an H-chain constant region is referred to as an H-chain constant region gene.). Similarly, the term “light chain gene” refers to the light chain variable region (V _L J _L ) is used as an expression that includes a gene that encodes only (VL chain variable region gene) and a gene that encodes a constant region (even if it is a part) in addition to the L chain variable region.

In one particular embodiment of this step, H chain variable region (V _H DJ _H) gene and L chain variable region gene (V _L J _L) is prepared. According to this embodiment, in the gene expression step J described later, an Fv antibody molecule, scFv antibody, Fab antibody, F (ab ') ₂ antibody having an ability to bind to an antigen, or a separately prepared H chain antibody Antibodies such as IgG class, IgA class, IgD class and IgE class can be obtained by using the normal region gene and the L chain constant region gene together.

In another embodiment, a gene encoding an H chain variable region gene as the H chain gene and a gene encoding a constant region in addition to the L chain variable region as the L chain gene is prepared. According to this embodiment, an antibody having an L-chain constant region is prepared without separately preparing an L-chain constant region in the “Step of expressing an antibody” described below. Here, considering that there are two types of L chain called κ chain and λ chain, if the L chain is reconstructed by combining the separately prepared L chain constant regions, In some cases, the types of the variable region and the constant region do not match, and the obtained antibody may not have the expected activity. By preparing an L chain gene that also covers the constant region as described above and using it to express the L chain, there is no fear of this type of mismatch. When the Η-chain gene and the L-chain gene are prepared in this step, both are prepared simultaneously or separately. A specific example of the latter case is shown below. First, a primer set for amplifying a short-chain gene, a primer set for amplifying a short-chain gene, and genomic xynucleotide triphosphate are added to a solution containing the separated labeled overnight getter cells, and then added. Cycles to several tens of cycles (specifically, for example, 5, 6, 7, 8, 9, 10, 15, 20, 30, (0 cycle) PCR (1st stage PCR). Subsequently, the reaction solution is separated for H chain gene and L chain gene. Then, after adding the primer set for amplifying the H chain gene to the reaction solution for the H chain gene, perform PCR until sufficient amplification is obtained (2nd stage PCR). For example, several cycles to several tens of cycles, preferably 20 cycles or more (eg, 20 cycles, 25 cycles), more preferably 30 cycles or more (eg, 30 cycles, 35 cycles), and still more preferably 40 cycles or more (eg, 40 cycles) (45 cycles, 50 cycles, 60 cycles) (2nd stage PCR). On the other hand, a primer set for amplifying the L chain gene is added to the reaction solution for the L chain gene, and PCR is performed in the same manner. The H-chain gene amplification primers and L-chain amplification primers added during the second step PCR are partially or entirely different even if they are the same as those used during the first step PCR. You may.

Primer sets for H chain gene amplification and L chain gene amplification are known (for example, Japanese Patent Application Laid-Open Nos. Hei 3 — 52081 and No. Hei 4 — 506,071). , W001 / 62907, Campbell, MJ et a, Mo I. Immuno I., 29, 193-203 (1992), Marks, JD et al., J. Mol. Biol. 222, 581-597 (1991). ), Phage Display AI aborato ry manua I, Carlos F. Barbas III, Dennis R. Burton, Jamie K. Scott, Gregg J. Silverman, Cold Spring Harbor Laboratory Press, or MRC homepage "V-basej: http: // http: // www. mrc-cpe. cam. ac. uk / PRIMERS. php? menu = [Primers disclosed or disclosed in Ci3) or modified versions of these may be used. The prepared antibody gene may be subcloned and amplified, and in this case, typically, the prepared antibody gene is integrated into an appropriate cloning vector (gene expression step). In this step, the prepared antibody gene is expressed using an expression vector (step (d)). First, the antibody gene obtained in the “antibody gene preparation step” is inserted (incorporated) into the expression vector. The configuration of the expression vector is not particularly limited as long as it is suitable for expression of the antibody gene. For example, SV40 vs. based vector, EB vs. based vector, BPV (Papi's mouth virus) ba.sed vector, PCMV vector, and those obtained by modifying these vectors can be used. The term "modification" used herein includes, for example, changing or adding a promoter sequence, an enhancer sequence, etc., in order to enhance expression efficiency, and adding a selectable marker gene. Insertion of an antibody gene into an expression vector can be performed by a well-known method using restriction enzymes and DNA ligase (for example, see Molecular Cloning, Third Edition, 1.84, Cold Spring Harbor Laboratory Press, New York). This can be done by: '

When expressing the H chain and the L chain simultaneously, the H chain gene and the L chain gene are inserted into the same expression vector or separate expression vectors. In the latter case, an expression vector for expressing the H chain gene and an expression vector for expressing the L chain gene are used in combination, and the H chain gene and the L chain gene prepared in the above “antibody gene preparation step” are used. Each is inserted into the corresponding vector. Thereafter, the H chain and the L chain are expressed using these two types of recombinant vectors. Preferably, the host is co-transformed with these two recombinant vectors, and the H chain and L chain are expressed in the same cell. When an H-chain variable region gene and an L-chain variable region gene are used as the antibody genes to be inserted, those having a linker sequence can be used as an expression vector. This linker sequence contains the inserted heavy chain in the expression vector. It is located at the position that bridges the variable region gene and the light chain variable region gene. In other words, the expression vector is designed to have an H chain variable region gene insertion site and an L chain variable region gene insertion site so as to sandwich the linker sequence. By performing expression manipulation using such an expression vector, a scFv antibody in which the H chain variable region and the L chain variable region are linked by a linker can be obtained. By inserting another sequence (a sequence encoding a part of the H chain or a sequence encoding a part of the L chain) in the expression vector in advance, the region encoded by the other sequence is inserted into the expression vector. An antibody (including only H chain and L chain) in which the region encoding the antibody gene obtained in the above antibody gene preparation step J is linked. Using an expression vector having a constant region gene and an insertion site adjacent to the gene, insert the H chain variable region gene prepared in the above “antibody gene preparation step”, and then express in an appropriate host Then, an H chain having a variable region and a constant region is obtained. Note that an intervening sequence may be present between the H chain constant region and the insertion site in the expression vector, as long as an expression product that appropriately functions as an H chain is obtained.

 In addition, using an expression vector having an H chain constant region gene, an insertion site for the H chain variable region gene adjacent to the gene, and an insertion site for the L chain gene, the above “antibody gene preparation step” was used. After inserting the prepared H chain variable region gene and L chain gene, and expressing them in an appropriate host, H chain and L chain having a variable region and a constant region can be obtained.

When inserting the antibody gene prepared in the “antibody gene preparation step” into the expression vector instead of using an expression vector into which other sequences have been inserted in advance, the other sequence is simultaneously expressed in the expression vector. By inserting the DNA into the above, an antibody to which the region encoded by the other sequence is linked can be expressed. It should be noted that “simultaneously J” here does not mean that strict temporal synchronization is required. Insertion of another sequence may be performed later, or vice versa. As the other sequences described above, those suitable for the purpose are selected and used. For example, when the human H chain variable region gene and the human L chain gene covering the variable region and the constant region are prepared in the “antibody gene preparation step”, the constant region gene of human lgG1 or the human The constant region gene of human lgG4 or the constant region gene of human lgA1 can be used as the “other sequence”. Of course, not only the antibody gene derived from the same species as the antibody gene prepared in the “antibody gene preparation step” but also an antibody gene derived from a different species can be used as another sequence. For example, a chimeric antibody can be obtained by preparing a mouse variable region gene in the “antibody gene preparation step” and combining a human constant region gene as another sequence. Various types of antibodies can be expressed depending on the type of antibody gene to be inserted and the configuration of the expression vector. Examples of antibodies that can be expressed include Fv antibody, scFv antibody, dsFv antibody, Fab antibody, F (ab ') ₂ antibody, IgG class, IgA class, and lgD class. It is also possible to obtain an antibody that does not have a complete constant region (ie, has only a part of the constant region).

Here, the r _SC Fv antibody ", an Fv consisting of H chain variable region and L-chain Allowed ^ region, by coupling the C-terminal and the other of the N-terminus of one strand with a suitable peptide linker This is a single-chain antibody fragment. As the peptide linker, for example, (GGGGS) ₃ having high flexibility can be used. On the other hand, the `` dsFv antibody '' is an H chain variable region and a Cys residue introduced at an appropriate position in the chain variable region, and the H chain variable region and the L chain variable region are stabilized by disulfide bonds. Fv fragment. The position of introduction of Cys residue in each chain can be determined based on the conformation predicted by molecular modeling. it can. The vector (recombinant vector) into which the antibody gene has been inserted is used for transformation of an appropriate host. The host is not particularly limited as long as it is capable of retaining the introduced antibody gene in an expressible state by being transformed with the recombinant vector. For example, CH0 cells (Chinese hamster ovary) (A. Wright & SLMorrison, J. Immunol. 160, 3393-3402 (1998)), SP2 / 0 cells (mouse myeoma) (K. Motmans et a, Eur. J. Cancer Prev. 5, 512-519 (1996), RP Junghans et a, Cancer Re s. 50, 1495-1502 (1990)) and other animal cells, eukaryotic cells such as yeast, and Escherichia coli. Prokaryotic cells can be used as hosts.

 Transformation of a host with a recombinant vector can be performed, for example, by the lipofectin method (RW Mai one et al., Proc. Nat I. Acad. Sc. USA 86, 6077 (1989), PL Feigner et al., Pro. c. Natl. Acad. Sci. USA 84,7413 (1987), Elect-portion method, calcium phosphate method (FL Graham & AJvan der Eb, Virology 52, 456-467 (1973)), DEAE-Dext The ran method is used, etc. By culturing the transformant obtained as described above under conditions in which the antibody gene carried by the transformant is expressed, the transformant can be used in the cells of the transformant or in the culture medium. Antibodies can be collected by methods such as centrifugation, ammonium sulfate fractionation, salting out, ultrafiltration, affinity chromatography, ion exchange chromatography, and gel filtration chromatography. It can be performed in an appropriate combination.

For expression, a fusion protein expression system can be used. That is, an expression system in which a target antibody is prepared as a fusion protein with GST (glutathion S-transferase), MBP (maltose binding protein) or the like may be used. On the other hand, a modified antibody can be obtained by binding a low-molecular compound, a labeling substance, etc. Wear. The labeling substance, a radioactive substance such as ^{U 5} I, pel old Kishida Ichize, beta - D - moth Lac Toshida Ichize, micro pel old Kishida Ichize, horseradish peroxide old Kishida - peptidase (HR P :), full Resin isothiocyanate (FITC), rhodamine isothiocyanate (RITC), alkaline phosphatase, and biotin can be used. The antibody production method of the present invention can be used for production of various antibodies. For example, it can be used to produce test (diagnostic) antibodies and therapeutic antibodies. An antibody prepared in connection with a specific disease can be used as a diagnostic agent as it is, or it can be used as a therapeutic agent if it shows a neutralizing activity. In addition, by cloning antibodies produced in the patient's body, diagnostic and therapeutic antibodies can be produced.Furthermore, by cloning a large number of antibodies, the types of antibodies produced in the patient's body can be determined. Information about the whole picture is obtained.

There are various foreign substances that affect the human body, such as toxins secreted by bacteria, snake venom, toxins contained in foods, viruses, etc., but patients affected by these produce specific antibodies during recovery. are doing. The method of the present invention is applicable to all these examples. For example, it can be used for monocloning antibodies exhibiting specific properties, such as hepatitis C virus neutralizing antibodies, from human blood. In the antibody production method of the present invention described above, an antibody gene is obtained in the process. That is, an antibody gene is obtained as a result of a series of operations including a “labeling step”, a “cell separation step”, and an “antibody gene preparation step”. The present inventors have further studied with attention to this point. As a result, they came to the knowledge that these steps can be applied not only to the preparation of antibody genes but also to the preparation of various genes by making some modifications without changing the nature of the steps. Based on such knowledge, the second aspect of the present invention provides the following configuration. [23] A gene preparation method including the following steps (i) to (H i): ()) a step of labeling cells having specific properties in a cell population; (Η) a step obtained by the step (i). Separating the labeled cells obtained; (Hi) preparing a target gene using the separated labeled cells.

 [24] The method of preparing a gene according to [23], wherein one labeled cell is separated in the step (ii). The second aspect of the present invention includes, for example, the preparation of virus genes (isolation), the preparation of autoantibody genes (isolation), the preparation of cancer or tumor suppressor genes (isolation), and the attack of tumor cells. It can be used for gene preparation (isolation). With regard to Τcell receptors, it has been reported that MHC + -old lygopeptides can be tetramerized to recognize them. し た Expression of cell receptors ΤIt is reported that cells can be labeled (identified). According to the method disclosed in the aspect, the ot, / 3 gene can be cloned by converting it into one cell. In this case, if the cell receptor (α, j8) gene, which recognizes a specific MHC + oligopeptide (cancer-specific), is cloned, it can be used to construct a composition for cancer treatment, or It becomes possible to construct a method for treating cancer. In addition, terms and matters that are not particularly described below are the same as those in the above-mentioned “antibody production method”.

(Step (ί))

In step (i), cells having a particular property in the cell population are labeled. This step (ί) corresponds to the “labeling step” described above. Sources of cell populations include specific tissues and organs of animals (eg, liver, guinea pig, monkey, monkey, chimpanzee, human, etc.). Organ, spleen, skin, muscle, fat, bone marrow, peripheral blood, etc.). “Specific properties” refers to properties related to the gene of interest. Typically, it refers to a property resulting from possession of a gene other than its own genome, or a property resulting from mutation of a target gene from a normal state. Specific examples of “cells having specific properties” include cells infected with a virus, autoantibody-producing cells, cancerous cells, and T cells.

(Step (i ί))

 In step (ii), the labeled cells obtained in step (i) are separated. This step (ii) corresponds to the above-mentioned “cell separation step” and is carried out in a similar procedure.

(Step (ίίί))

 In step (ii), the target gene is prepared using the separated labeled cells. This step ({i}) corresponds to the “antibody gene preparation step” described above, and is performed in the same procedure. The genes prepared in the above series of steps are analyzed by themselves, detected and diagnosed using them, detected and diagnosed using their expression products, and used as diagnostics and prophylaxis using the information. It can be used for pitfalls of drugs or therapeutic drugs. Thus, the second aspect of the present invention includes the following methods (25 to 27).

 [25] A method for analyzing a virus mutation, comprising the following steps:

 (1) labeling virus-infected cells in the cell population;

 (2) separating the labeled virus-infected cells;

(3) preparing a viral gene from the separated labeled virus-infected cells; and (4) a step of analyzing the obtained virus gene to detect a virus mutation (variation in antigenicity); Here, "Virus J is a virus having the ability to mutate and is of value in analyzing the mutation. For example, influenza virus and HIV are analyzed.

 For the labeling in step (1), for example, a labeled nucleic acid having a specific binding ability to a viral nucleic acid (DNA, RNA) is used. Preferably, one labeled virus-infected cell is separated in step (2).

 By applying the above “method for analyzing mutations in viruses”, for example, it is possible to design a universally effective vaccine by completely tracing the changes in antigenicity that occur in the body of a person infected with HIV. In addition, isolation of neutralizing antibodies induced in response to the antigenic fluctuations (antigen- drt) caused by influenza every year, isolation of escape mutants using them, and production of next-generation vaccines It can also be applied to other applications.

[26] A method for preparing an autoantibody gene, comprising the following steps:

 (1) labeling autoantibody-producing cells in the cell population;

 (2) separating labeled autoantibody-producing cells;

 (3) Step of preparing an autoantibody gene from the separated autoantibody-producing cells.

The autoantibody gene obtained by the above method can be used for suppressing the production of autoantibodies. Specifically, for example, the obtained sequence information of the autoantibody gene is analyzed, and based on the obtained information, a method of suppressing the expression of the gene (or the activity of the antibody encoded by the gene) is constructed, Alternatively, a method for detecting an autoantibody can be constructed. In other words, it is possible to develop treatments and diagnostics for various autoimmune diseases It becomes.

 Preferably, one labeled autoantibody-producing cell is separated in step (2).

[27] A method for preparing a causative gene for cancer, comprising the following steps:

 (1) preparing a cell population containing cancerous lymphoid leukocytes;

 (2) separating cancerous lymphoid leukocytes from the cell population;

 (3) A step of preparing a gene associated with canceration from the isolated cancerous lymphoid leukocytes.

 By comparing the gene obtained by the above method with the corresponding normal gene (identity detection), the cancerous state of lymphoid leukocytes can be detected and analyzed. That is, it can be applied to the development of a very early diagnosis method for lymphoid leukocytes (using the clonal indicated by tumor cells). Preferably, one cancerous lymphoid leukocyte is separated in step (2).

<Example 1> Labeling test using lysozyme as antigen

Spleen purified antigen (lysozyme) from immunized mice was taken out, after disjoint cells through a mesh crush, 1% BSA, washed with PBS containing 0.1% MAN _3. This was added to a lysozyme sensitization plate (a solution prepared by dissolving lysozyme in PBS to 25 tg / ml) in a microcup (Nunc-1 mtnunomodu Ie F8 maxi sorp loose (manufactured by Nunc)). after standing overnight was added in the solution discarded, 5% BSA, was added to those flops locking) placed further overnight with PBS containing 0.1% NaN _3, slowly Ri carefully 1% BSA, 0.1 % NaN ₃ were washed twice with PBS containing, 1% BSA, the cells were harvested bound by PBS Depipette queuing containing 0.1% NaN _3. Among them, 2.6 x 10 ⁵ cells were labeled with an AI exa488 label using the Alexa488 protein labeling kit (MoIecu Iar probes). Reaction of Zodium with Alexa546-labeled anti-mouse IgG antibody (Molecular probes) at a final concentration of 500-fold resulted in 99% of cells stained with AI exa546-labeled anti-mouse IgG antibody (red fluorescence). Was also stained with Alexa488-labeled lysozyme (green fluorescence) (Figure 1). Example 2> Labeling test using GFP as antigen

The spleen was removed from the mouse immunized with GFP, crushed, passed through a mesh to dissociate the cells, and then washed with PBS containing 1% BSA and 0.1% NaN ₃ . Apply this solution to a GFP-sensitized plate (solution prepared by dissolving GFP in PBS to 25 g / ml to a micro force (Nunc-1 mmunomodu IeF8 maxisorp loose (Nunc)) in 100 μi increments! ], Place it in “ ¹ ”, discard the solution, add overnight to PBS containing 5% BSA and 0.1% NaN ₃ and block it), and slowly and carefully add 1% BSA and 0.1% After washing twice with PBS containing NaN ₃ , the bound cells were collected by pipetting with PBS containing 1% BSA and 0.1% MaN ₃ . When 8.4X10 ⁴ cells were reacted with GFP 3.7 S and Alexa546-labeled anti-mouse IgG antibody (Molecular probes) at a final concentration of 500-fold, A Iexa546-labeled anti-mouse IgG antibody (red) 92.6% of the cells stained with (fluorescence) were also stained with GFP (green fluorescence) (Fig. 2).

<Example 3> Labeling test using fusion protein as antigen

When producing an antigen, the expression level is often low or insolubilized unless a fusion protein is used. In the case of using an antigen that can be obtained only in large amounts as such a fusion protein, in order to mark the target antibody-producing cells, only the evening protein portion (MBP) is purified and Alexa546 protein labeling kit ( The fusion protein (MBP-VHH) was labeled green using an Alexa488 protein labeling kit (MoIecu Iar probes) using red (Molecular probes). From purified antigen (MBP-VHH) were immunized mice, spleens were removed, after disjoint cells passed through a mesh crush, 1% BSA, washed with PBS containing 0.1% NaN _3. This was added to a MBP-VHH sensitization plate (solution prepared by dissolving MBP-VHH in PBS to 20 ig / ml) in a Microcup (Nunc-lmmunomodu IeF8maxisorp loose (Nunc)). After adding At I and leaving overnight, discard the solution, add it to PBS containing 5% BSA and 0.1% MaN ₃ and then add it overnight. % N aN ₃ washed twice with PBS containing, 1% BSA, the cells were harvested bound by pipetting with PBS containing 0.1% NaN _3. Among them, with respect to 7.0X10 ⁴ cells, Alexa488-labeled MBP-VHH 1.2; and ag, was reacted with Alexa546-labeled MBP 1.2 ^ g. As expected, as shown in Figure 3, cells expressing antibodies to the protein of interest (VHH) were stained with Ale xa488 (green fluorescence) (lower left in Figure 3) and expressed antibodies to the tag protein (MBP). The cells that are present will be stained with Alexa488 (green fluorescence) and Alexa546 (red fluorescence) (Fig. 3, lower right). Figure 4 shows the actual results. The lower cells, which are thought to express the antibody to the tag protein BP, stain both green and red, and the upper cells, which are thought to express the antibody to the desired VHH, only green Stained. The weak green fluorescence in cells that are thought to be expressing antibodies to MBP may be due to competition with the red labeled MBP. The above results indicate that cells expressing antibodies to the target protein can be distinguished from antigens that can be obtained in large amounts only with the fusion protein.

<Example 4> Separation of labeled cells

 The cells (anti-lysozyme antibody-producing cells) marked with the labeled antigen (anti-lysozyme antibody-producing cells) were converted into one cell by manipulation. The cells were suspended in a 20 I PCR buffer, and a reaction solution having the following composition was added.

10X PCR buffer (supplied with HotStarTaq DMA polymarase kit): 3 μI 10m dNTP: 1 μI

 Primer _mVH mix (200pmol / t I): Ο. Βμ I

 Primer m river mix (200praol // i I): 0.5 I

 Primer mVK mix (200pmol / t I): 0.5μ I

 Primer mJ K mix (200pmol / I): 0.5 I

 Q-solut ion (supplied with HotStarTaq DNA polymarase kit): 10μI

 Distilled water: 13μΙ

 HotStarTaq DNA polymarase: 1 μ. I

 30 μI total

(Primer IIIVH πι: An equal mixture of the following 17 types. Note that (a / g) and (g / t) represent a two-base mix.)

 Name: → 3 '

 mVH1 A: act tac tcg egg ccc age egg cca tgg ccg a (g / t) g tgc age ttc agg agt cag g (52mer) (SEQ ID NO: 9, SEQ ID NO: 10)

 mVHIBI: act tac tcg egg ccc age egg cca tgg ccc agg tgc age tga agg ag t cag g (52mer) (SEQ ID NO: 11)

 mVH1B2: act tac tcg egg ccc age egg cca tgg ccc agg tgc age tga age ag t cag g (52mer) (SEQ ID NO: 12)

 mVH2A1: act tac tcg egg ccc age egg cca tgg ccg agg tec age tgc a (a / g) ca (a / g) t ctg g (52mer) (SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, (SEQ ID NO: 16)

 mVH2A2: act tac tcg egg ccc age egg cca tgg ccg agg ttc age tgc age ag t ctg g (52mer) (SEQ ID NO: 17)

mVH2Bl: act tac tcg egg ccc age egg cca tgg ccc agg tec aac tgc age ag c ctg g (52mer) (SEQ ID NO: 18)

 mVH2B2: act tac tcg egg ccc age egg cca tgg ccc agg tec acc tgc age ag t ctg g (52mer) (SEQ ID NO: 19)

 mVH3A: act tac tcg egg ccc age egg cca tgg ccg agg tga age tgg tgg a (a / g) t ctg g (52mer) (SEQ ID NO: 20; SEQ ID NO: 21)

 mVH3B: ac t tac tcg egg ccc age egg cca tgg ccg agg tga age ttc t g agt ctg g (52mer) (SEQ ID NO: 22)

 mVH3C1: act tac tcg egg ccc age egg cca tgg ccg aag tga age t tg agg ag t ctg g (52raer) (SEQ ID NO: 23)

 mVH3C2: act tac tcg egg ccc age egg cca tgg ccg agg tga age tgg atg ag a ctg g (52raer) (SEQ ID NO: 24)

 mVH3C3: act tac tcg egg ccc age egg cca tgg ccg aag tga age tgg tgg ag t ctg a (52raer) (SEQ ID NO: 25)

 mVH3D1: act tac tcg egg ccc age egg cca tgg ccg aag tgc age tgg tgg ag t ctg g (52mer) (SEQ ID NO: 26)

 mVH3D2: act tac tcg egg ccc age egg cca tgg ccg aag tga tgc tgg tgg ag t ctg g (52mer) (SEQ ID NO: 27)

 mVH3D3: act tac tcg egg ccc age egg cca tgg ccg aag tga age tgg tgg ag t ctg g (52mer) (SEQ ID NO: 28)

 mVH5A1: act tac tcg egg ccc age egg cca tgg ccg agg ttc age ttc age ag t ctg g (52mer) (SEQ ID NO: 29)

mVH5A2: act tac tcg egg ccc age egg cca tgg ccc agg tec age tgc age ag t ctg g (52mer) (SEQ ID NO: 30) (primer mJH mix: A mixture of the following four equivalents) Name: 5 '→ 3'

 mJHIXho: cgt 111 ggc get cga gac ggt gac cgt ggt ccc tgc g (37mer) (SEQ ID NO: 31)

 mJH2Xho: cgt ttt ggc get cga gac tgt gag agt ggt gcc t tg (37mer) (SEQ ID NO: 32)

 mJH3Xho: cgt ttt ggc get cga gac agt gac cag agt ccc t tgg (37mer) (SEQ ID NO: 33)

 mJH4Xho: cgt ttt ggc get cga gac ggt gac tga ggt tec ttga (37mer) (SEQ ID NO: 34)

(Primer mV / em: An equal mixture of the following 21 types. In addition, (g / t), (c / g), and (a / c) represent a two-base mix.)

 Name: 5 '→ 3'

 mVKIA: gcc cag cca gcc atg gcc gac att gtg atg aca cag tct cc (41mer) (SEQ ID NO: 35)

 raVKIB: gcc cag cca gcc atg gcc gac att gtg atg tea cag tct cc (41mer) (SEQ ID NO: 36)

 raVK2A: gcc cag cca gcc atg gcc gat gtt gtg atg acc caa act cc (41mer) (SEQ ID NO: 37)

 mVK2B: gcc cag cca gcc atg gcc gat gtt t tg atg acc caa act cc (41mer) (SEQ ID NO: 38)

 mVKZC: gcc cag cca gcc atg gcc gat att gtg atg ac (g / t) cag get gc (41mer) (SEQ ID NO: 39, SEQ ID NO: 40)

mVK2D: gcc cag cca gcc atg gcc gat att gtg ata acc cag gat ga (.41mer) (SEQ ID NO: 41) mVK3A: gcc cag cca gcc a tg gcc gac a 11 gtg c tg acc caa tct cc (41mer) (SEQ ID NO: 42)

 mVK3B: gcc cag cca gcc a tg gcc gac a 11 gtg ctg aca cag tct cc (41mer) (SEQ ID NO: 43)

 mVK3C: gcc cag cca gcc atg gcc aaa at t gtg ctg acc caa tct cc (41mer) (SEQ ID NO: 44).

 mVK4A: gcc cag cca gcc atg gcc gaa aat gtg ct (c / g) acc cag tct cc (41m er) (SEQ ID NO: 45, SEQ ID NO: 46)

 mVK4B: gcc cag cca gcc atg gcc gaa att gtg etc acc cag tct cc (41mer) (SEQ ID NO: 47)

 mVK4C: gcc cag cca gcc atg gcc caa att gt t etc acc cag tct cc (41mer) (SEQ ID NO: 48)

 mVK5A: gcc cag cca gcc atg gcc gat ate cag atg aca cag act ac (41mer) (SEQ ID NO: 49)

 mVK5B: gcc cag cca gcc atg gcc gac ate cag atg ac (a / c) cag tct cc (41mer) (SEQ ID NO: 50, SEQ ID NO: 51)

 mVK5C: gcc cag cca gcc atg gcc gacate aag atg acc cag tct cc (.41mer (SEQ ID NO: 52)

 mVK5D: gcc cag cca gcc atg gcc gac att cag atg acc cag tct cc (41mer) (SEQ ID NO: 53)

 mVK5E: gcc cag cca gcc atg gcc gac att gtg atg acc cag tct ca (41mer) (SEQ ID NO: 54)

 mVK5F: gcc cag cca gcc atg gcc agt att gtg atg acc cag act cc (41mer) (SEQ ID NO: 55)

mVK5G: gcc cag cca gcc atg gcc gac ate t tg ctg act cag tct cc (41raer) (SEQ ID NO: 56)

 mVK5H: gcc cag cca gcc atg gcc aac att gta atg acc caa tct cc (41mer) (SEQ ID NO: 57)

 mVK6A: gcc cag cca gcc atg gcc caa att gt t etc tec cag tct cc (41mer). (SEQ ID NO: 58)

(Primer 1 mJ / cm: Equivalent mixture of the following 4 types)

 Name: 5 '→ 3'

 mJKlAsc: GGA GTC GAC TGG CGC GCC GAA CGT TTG ATT TCC AGC TTG GT (41mer) (SEQ ID NO: 59)

 mJK2Asc: GGA GTC GAC TGG CGC GCC GAA CGT TTT ATT TCC AGC TTG GT (41raer) (SEQ ID NO: 60)

 mJK4Asc: GGA GTC GAC TGG CGC GCC GAA CGT TTT ATT TCC AAC TTT GT (41mer) (SEQ ID NO: 61)

 mJK5Asc: GGA GTC GAC TGG CGC GCC GAA CGT TTC AGC TCC AGC TTG GT (41mer) (SEQ ID NO: 62) Next, add a drop of mineral oil, heat at 95 ° C for 10 minutes, 94 ° C for 30 seconds, 60 ° 50 cycles of PCR were performed for 30 seconds at C and 1 minute at 72 ° C. However, the gene of interest was not amplified. Therefore, after performing the above-mentioned reaction for 10 cycles, fractionate 1 μ | at a time, add Η chain amplification reaction solution I to one side, and / c chain amplification reaction solution 49 μ I to the other side. Was subjected to a further 48 cycles of the PCR reaction.

 (Reaction solution for chain amplification)

10X PCR buffer (included with HotStarTaq DNA ρο marase kit): 5Ai I lOmM dNTP: 1 u I Primer iiiVH mix (200pmol / D: 0.5 ^ 1 '

 Primer mJH mix (200pmol / M I): 0.5 / x I

 Q-solution (included with HotStarTaq DMA po I ymarase kit): 10 I

 Distilled water: 32μΙ

 HotStarTaq DNA po I ymarase: 1 μ. I

 Total 50 1

(Reaction solution for K chain amplification)

 10X PCR buffer (supplied with HotStarTaq DNA polymarase kit): 5i

 10 mM dNTP: 1 μI

 Primer mV / mix (200pmol / I): 0.5 I

 Primer mJ c mix (200pmoΙ / I): 0.5 μI

 Q-solution (supplied with HotStarTaq DNA po I ymarase k): 10 z I

 Distilled water: 32 μI

 HotStarTaq DNA po I ymarase: 1 μ. I

 Total 50 ^ I

As a result of the PCR reaction, gene amplification was observed for both the H and κ chains (FIG. 5).

<Example 5> Construction of expression vector pSCCA4-E8d

An expression vector was constructed according to the following procedure (see Fig. 6). scNcopFCAH9-E8VHdVLd Vector 1 (See W001 / 96401. The nucleotide sequence of the vector (SEQ ID NO: 7) is shown in Figure 7 and Figure 8) (1 μΙ), Rev Primer (100 pmol / ml) 1 μΙ, SfiR primer-(100 pmol / ml) 1 μI, 10XLA PCR buffer 10 I, 25 mM MgCI ₂ 10 μΙ, 2.5 mM d NTP mix 16 At U sterile distilled water 60 II, TaKaRa LA Taq Polymerase (5U / I Takara Shuzo) The mixture was mixed, two drops of mineral oil were added, the mixture was kept at 95 ° C for 2 minutes, and the reaction was repeated at 94 ° C for 1 minute, 58 ° C for 2 minutes, and 72 ° C for 1 minute for 16 cycles. After confirming the obtained PCR product by agarose gel electrophoresis, it was cut out, treated with phenol, precipitated with EtOH and concentrated to 101 (fragment A). Similarly, scNcopFCAH9-E8VHdVLd 1 / i (1 ^ 1) .SfiF primer (100 praol / ml) 1 I, BstR primer (100 pmol / ml) 1 μI 10XLA PCR buffer 10 / i I, 25 mM MgCI ₂ 10 iU 2.5mM dNTP mix 16 / zl, sterile distilled water 60μΙ, Ta KaRa LA Taq Po I ymerase (5U / ^ I Takara Shuzo) 1 I, mix, add 2 drops of mineral oil, 95 ° After 2 minutes of incubation at C, the reaction was repeated at 94 ° C for 1 minute, 58 ° C for 2 minutes, and 72 ° C for 1 minute for 16 cycles. After confirming the obtained PCR product by agarose gel electrophoresis, it was cut out, treated with phenol, precipitated with EtOH and concentrated to 10 I (fragment B). Furthermore, scNcopFCA H9-E8VHdVLd lg (1 xl), BstF primer one (100pmol / ml) 1 / ^ 1, cp3R primer - (100pmol / ml) 1 z and _{10XLA PCR buffer 10μ U 25mM HgCI 2} 10μ and 2.5 mM d NTP mix 16μ I, sterile distilled water 60μ Ι, TaKaRa LA Taq Po I ymerase (5U / i I Takara Shuzo) 1 il, add 2 drops of mineral oil, incubate at 95 ° C for 2 minutes, 94 ° The reaction was repeated for 16 minutes at C〗, 58 ° C for 2 minutes, and 72 ° C for 1 minute. After confirming the obtained PCR product by agarose gel electrophoresis, it was cut out, treated with phenol, precipitated with EtOH, and concentrated to 10 µM (C fragment). Α Fragment 5 / il, B fragment 5 ^ 1, Rev primer (10Opmo I / m I) 1β and BstR primer (100pmol / ml) 1 ^ 1, 10XLA PCR buffer 10 I, 25mM MgCl ₂ 10μΙ, 2.5 mM dNTP mix 16 / ti K Sterile distilled water 51μ, mix with TaKaRa LA Taq Polymerase e (5U / I Takara Shuzo) 1 il, add 2 drops of mineral oil, incubate at 95 ° C for 1 minute, 94 ° C1 The reaction was repeated for 17 minutes at 58 ° C for 4 minutes and at 72 ° C for 2 minutes. After confirming the obtained PCR product by agarose gel electrophoresis, it was cut out, treated with phenol, precipitated with EtOH, and concentrated to I (D fragment). C fragment 5 I, D fragment 5 l, Rev primer (100 pmol / ml) 1 μΙ, cp3R primer (100 pmol / ml) 1 I, 10 XLA PCR buffer ίθμ L 25 m MgCI ₂ 10 M K 2.5 mM dNTP mix 16 μ Sterile distilled water 51μΙ, Ta KaRa LA Taq Po I yraerase (5υ / I Takara Shuzo) Mix 1 t I, add 2 drops of mineral oil, incubate at 95 ° C for 1 minute, 94 ° C for 1 minute, 58 ° C for 4 minutes, 72 ° C The 2-minute reaction was repeated for 17 cycles. After confirming the obtained PCR product by agarose gel electrophoresis, it was cut out, treated with phenol, precipitated with EtOH, and concentrated to 20 µI. Of these, 7 μΙ, 10ΧΜ buffer 10 I, sterile distilled water 78, and Hindi II (10 U / I Takara Shuzo) 5 I were mixed and reacted at 37 ° C. for 2 hours. To this was added 1 μL of 5 M NaCI and 5 μI of BstPI I (Takara Shuzo), and the mixture was further reacted at 37 ° C for 2 hours. After confirming this by agarose gel electrophoresis, it was cut out, treated with phenol, precipitated with EtOH, and concentrated to 15 I (insert fragment). Similarly, mix scNcopFCAH9-E8VHdVLdlg (1iil), 10μM buffer, 10μ, sterile distilled water 86, and Hindi II (10U / I Takara Shuzo) 3μI, and react at 37 ° C for 2 hours. Was. To this, 5 M NaCI 1 iI and BstPI (5 U / I Takara Shuzo) 3 μI were added, and the mixture was further reacted at 37 ° C. for 2 hours. After confirming this by agarose gel electrophoresis, it was excised, treated with phenol, precipitated with EtOH and concentrated to 15 I (vector fragment). Insert fragment 2 μ.I, vector fragment 1 I, 10 μl ligation buffer 1.5 U 10 mM ATP 1.5 μI, D 8 μL T4 DNA I igase (350 units / μ.I Takara Shuzo) 1 μI The mixture was mixed and incubated at 16 ° C for 3 hours. After ethanol precipitation, it was dissolved in TE diluted 10-fold with sterile distilled water, and half of the solution was used to transform DH12S. Plasmid was extracted from each of the resulting 24 transformants and subjected to agarose gel electrophoresis to confirm the nucleotide sequence of the clone containing the insert. A clone containing the expected substitution was selected and designated as PSCCA4-E8d (the nucleotide sequence (SEQ ID NO: 8) is shown in FIGS. 9 and 10). The primers used in the above are as follows.

Rev "primer: 5'-AGGAAACAGCTATGACCATG-3 '(20mer) (SEQ ID NO: 1)

SfiR primer: 5'-CGGCTGGGCCGCGAGTAA-3 '(18mer) (SEQ ID NO: 2) Sf iF primer: 5'-TTACTCGCGGCCCAGCCGGCCCCTGACATCTGAGGACACT-3 '(40mer) (SEQ ID NO: 3)

 BstF primer: 5'-GGTCACCGTCTCGAGCGGCGGTGG-3 '(24mer) (SEQ ID NO: 4) BstR primer: 5'-CCACCGCCGCTCGAGACGGTGACC-3' (24mer) (SEQ ID NO: 5) cp3R primer: 5'-GCCAGCATTGACAGGAGGTTG-3 '(21mer) ) (SEQ ID NO: 6)

<Example 6> Preparation of L chain gene

Among the PCR products obtained in Example 4, the L chain was first treated with phenol-cloth form, precipitated with ethanol, and dried. Then, distilled water was added to 84 μl, and 10ΧΜΕΒ4 buffer (supplied with Ascl manufactured by 製 Co.) 10 μl Then, 3 l of restriction enzyme Ncol (10 units / iK, manufactured by Takara Shuzo) and 3 μm of restriction enzyme Ascl (IOunits / μ, manufactured by NEB) were added and reacted at 37 ° C. for 2 hours. After the reaction, fractionation was performed by agarose gel electrophoresis, and the L chain gene was excised from the gel and purified. For purification after the excision, QIAquick Gel Extraction kit manufactured by Qiagen was used. The excised L chain gene NcoI Ascl fragment was ethanol precipitated, dried, and suspended in TE buffer {μ} diluted 10-fold with sterile distilled water. Similarly, vector-DNA pSCCA4-E8d (see FIGS. 6, 9 and 10) 2 xg (2μ, 1), distilled water 8 μμ. I, 10XNEB4 buffer (supplied with Asc I manufactured by NEB) 10μ Ι, 3 μΙ of restriction enzyme Ncol (10 units / MK, manufactured by Takara Shuzo) and 3I of restriction enzyme Ascl (10 units / ^ L, manufactured by NEB) were added and reacted at 37 ° C. for 2 hours. After the reaction, fractionation was performed by agarose gel electrophoresis, and the vector DNA pSCCA4-E8d Ncotyl Asc I fragment was cut out from the gel and purified. The QIAquick Gel Extraction kit manufactured by Qiagen was used for purification after the excision. The excised vector DNA pSCCA4-E8d Ncol-Ascl fragment was precipitated with ethanol, dried, and suspended in TE buffer 10 iI diluted 10-fold with sterile distilled water. Dissolve the N-chain Ascl fragment of the L chain gene and μI in 10.5 tI of distilled water, and add the vector DNA pSCCA4-E8d Ncol-Ascl fragment 1 μU 10ΧΤ4 DNA Iigase buffer 1.5 x I and T4 DNA Iase 1 I. Addition I The igase reaction was performed at 16 ° C for 2 hours. This was precipitated with ethanol, dried, and suspended in 13 μl of TE buffer diluted 10-fold with sterile distilled water. 1.5% of this was added to Elect Max DH12S (Escherichia coli) 20I, and elect opening was performed. The Escherichia coli after the electoral poration was placed in 1 ml of 2XTY medium and cultured at 37 ° C. for an hour. Of this, 100 l was spread on an LBGA plate and cultured at 30 ° C for 1 晚. Plasmids were prepared from the thus obtained knees using a plasmid extractor P50 manufactured by KURAB0, and the base sequence of the L chain gene was analyzed using a DNA sequencer CEQ2000XL manufactured by Beckman Coulter. A clone having the correct gene without any deletion was selected. Example 7> Preparation of H chain gene

Next, the excised H chain gene was incorporated into the clone selected in Example 6. The H-chain PCR product was treated with phenol-cloth form, precipitated with ethanol, and dried, and then distilled water ff.5 (i \, 10XNEB2 buffer (supplied with NEB SfiI) 10 / iI, 10XBSA (N 10 I, restriction enzyme Sf M (8 units / ^ I, manufactured by NEB) 2.5 / ζ 付 属 were added, and reacted at 50 ° C for 2 hours. The restriction enzyme Xhol (8 units / ^ U manufactured by Takara Shuzo) was added and reacted for 2 hours at 37 ° C. After the reaction, fractionation was performed by agarose gel electrophoresis, and the H chain gene was cut out from the gel. A QIAquick Gel Extraction kit manufactured by Qiagen was used for the excision and purification The excised H chain gene Sf protein Xhol fragment was precipitated with ethanol, dried, and diluted 10-fold with sterile distilled water. In the same manner, 2 ig (5 / x I) of the clone selected in Example 6 was treated with phenol-cloth form, and suspended in ethanol. After precipitation and drying, 77.5 μl of distilled water was added and 10ΧΝΕΒ2 buffer (supplied with Sf iI manufactured by ΝΕΒ) 10μI, 10XBSA (Νsupplied with SfίI manufactured by EB) 10 し2.5 uI (8 units / MI, NEB) was added and reacted for 2 hours at 50 ° C. Then, 3 μΙ of Xhol (8 units / I, Takara Shuzo) was added. The reaction was carried out at 37 ° C for 2 hours. After electrophoresis, fractionation was performed, and the Sfil Xhol fragment of the clone selected in Example 6 was cut out from the gel and purified. For excision and purification, QIAquick Gel Extraction kit manufactured by Qiagen was used. The excised SfM-Xhol fragment of the clone selected in Example 6 was precipitated with ethanol, dried, and suspended in 10 I of TE buffer diluted 10-fold with sterile distilled water. Dissolve 1 tl of Η chain gene Sfil Xhol fragment in 10.5 / il of distilled water and add SfM-Xhol fragment I, 10XT4 DNA I igase buffer 1.5 fi I and T4 DNA I igase 1 n I of the clone selected in Example 6. The Iigase reaction was performed at 16 ° C for 2 hours. This was precipitated with ethanol, dried, and suspended in TE buffer 3I diluted 10-fold. Of these, 1.5 ^ 1 was added to Electromax DH12S (Escherichia coli) 20 i I and electroporation was performed. E. coli after electroporation was placed in 1 ml of 2XTY medium and cultured at 37 ° C for an hour. Of these, 100 I was spread on an LBGA plate and cultured at 30 ° C for 1 晚. Plasmids were prepared from the obtained colonies using Plasmid Extractor P50 manufactured by KURAB0, and the nucleotide sequence of the H chain gene was analyzed using a DNA sequencer CEQ2000XL manufactured by Beckman Coal Yuichi. A clone with the correct gene without any deletion was selected.

<Example 8> Expression of antibody

 The clone selected in Example 7 was first cultured in 3 ml of TYGA medium— 晚. This culture solution 30 ^ I was cultured in 2XTY medium containing 0.1% glucose and lOO g / ml ampicillin for 2 hours and 30 minutes, and 0.1M IPTG (manufactured by Wako Pure Chemical Industries for Biochemistry) was added to express the antibody. Was led. 100 l of culture supernatant was sensitized to lysozyme sensitization plate (25 g / ml solution of lysozyme dissolved in PBS

After adding 100 μl to a Roccup (Nunc-lmmunomodule F8 maxi sorp loose (manufactured by Nunc)), letting it stand overnight, discard the solution, and further add PBS with 5% BSA and 0.1% NaN3 to PBS. And blocked at 37 ° C for 1 hour, then 0.05% Washed four times with Tween20-PBS. The washed plate was added with ΙΟΟμ I of anti-cp3 antibody (manufactured by MBL) 2, / ml, reacted at 37 ° C for 1 hour, and washed four times with 0.05% Tween20-PBS. To the washed plate, add 100 I of a persian oxidase-labeled anti-Peacock IgG (H + L) antibody (diluted 5,000-fold, manufactured by MBL), and react at 37 ° C for 1 hour. Washed 4 times with% Tween20-PBS. To the plate after washing, 100 I of a solution of orthodiene diamine and hydrogen peroxide was added to cause a temporary reaction, and then 2N sulfuric acid 100 AtI was added to stop the reaction, and the absorbance at a wavelength of 492 nm was measured. As a result, as shown in FIG. 11, the reactivity with the antigen was confirmed (Lyso2-UHL-13x10, Lyso2-13HL-13x1 negative control). From the above results, it was shown that an antibody reacting with an antigen can be produced using an antibody gene obtained from one cell.

<Example 9> Labeling of human antibody-producing cells

Similarly, it was examined whether human antibody-producing cells could be marked with a labeled antigen or the like. First, the influenza vaccine (2000) was labeled with Alexa 488 labeling kit (Molecular probe). This labeled influenza vaccine and PE-labeled anti-human IgG antibody were reacted with human peripheral blood (5 mM EDTA). No cells were found to be stained in peripheral blood obtained from healthy individuals, but in peripheral blood obtained from a subject who had contracted influenza one month ago, staining was observed as shown in Figure 12. From these results, it was confirmed that the human antibody-producing cells can be marked with the labeled antigen in the same manner as in the mouse. If one cell with this mark is separated and the antibody gene is prepared and expressed in the same manner as in the above example, an antibody that binds to influenza virus can be obtained. It is thought that antibodies that neutralize the virus can be obtained. <Example 10> Labeling test using botulinum toxoid type A as an antigen Lymphocytes were collected from a blood sample of a human to which the purified antigen (botulinum toxoid A) was administered. 3 g of Alexa488-labeled botulinum toxoid labeled with Alexa488 protein labeling kit (Molecular probes) on 1.3 × 10 ⁷ cells, and Alexa546-labeled anti-human IgG antibody (Mo After reaction with Iecu Iar probes (99%), 99% of the cells stained with Alexa546-labeled anti-human IgG antibody (red fluorescence) were also stained with Alexa488-labeled botulinum toxoid (green fluorescence).

<Example 11> Amplification of antibody gene from isolated cells

 Cells marked with a labeled antigen (anti-botulinum toxoid type A antibody-producing cells) were transformed into one cell with a cell saw. The cells were suspended in a 20 I PCR buffer, and 30 I of a reaction solution having the following composition was added.

 10X PCR buffer (supplied with HotStarTaq DNA polymarase kit): 3μ Ι 10mM dNTP: 1μ. I

 Primer hVH ηΠχ (100ριτ) θΙ / μ I): 0.5μ I

 Primer h river mixOOOpmol / i I): 0, I

 Primer hVK m (100 pmol / I): 1 I

 Primer hVA ηιίχ (100ριιιοΙ / μ I): 1 μ. I

 Primer hCK Asc (100pmol / l): Ι Ι

 Primer hCA Asc (100pmol / j J): Ι μ Ι

 Distilled water: 20μ I

 HotStarTaq DNA polymarase: 1 ιχ I

 30 μI total

Human VH primer

hVH1a: GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCC CAGGTGCAGCTGGTGCAGTCTGG (sequence number No. 6 3)

hVH2a-2: GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCC CAGRTCACCTTGAAGGAGTCTGGTCC (Torimi column number 6 4)

hVH3a: GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCC GAGGTGCAGCTGGTGGAGTGTGG (SEQ ID NO: 65)

hVH4a: GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCC CAGGTGCAGCTGCAGGAGTCGGG (SEQ ID NO: 66)

hVH4a-2: GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCC CAGGTGCAGCTACAGCAGTGGGG (SEQ ID NO: 67)

hVH6a: GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCC CAGGTACAGCTGCAGCAGTCAGG (SEQ ID NO: 68)

Country 7: GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCC CAGGTGCAGCTGGTGCAATCTGGGTCTGAGT (SEQ ID NO: 69)

 A mixture of the above primers as follows was used as a primer hVH mix. hVH1a: hVH2a-2: hVH3a: hVH4a: hVH4a-2: hVH6a: hVH7 = 2: 2: 4: 1: 1: 1: 1

Human JH primer

hJH1-2: GGTGGAGGCACTCGAGACGGTGACCAGGGTGC (Sequence No. 70)

hJH3: GGTGGAGGCACTCGAGACGGTGACCATTGTCC (SEQ ID NO: 71)

hJH4-5: GGTGGAGGCACTCGAGACGGTGACCAGGGTTC (SEQ ID NO: 72)

hJH6: GGTGGAGGCACTCGAGACGGTGACCGTGGTCC (SEQ ID NO: 73)

A mixture of equal amounts of the above primers was designated as primer hJH m ₍ Human Vκ pnmei huVKIa: GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCC GACATCCAGATGACCCAGTCTCC (SEQ ID NO: 74)

huVK2a: GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCC GATGTTGTGATGACTCAGTCTCC (SEQ ID NO: 75)

huVK3a: GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCC GAAATTGTGTTGACGCAGTCTCC (SEQ ID NO: 76)

huVK4a: GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCC GACATCGTGATGACCCAGTCTCC (SEQ ID NO: 77)

hVK5-2: GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCC GAAACGACACTCACGCAGTCTCCAGCATT (SEQ ID NO: 78)

hVK6-2: GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCC GAAATTGTGCTGACTCAGTCTCCAGACTT (SEQ ID NO: 79)

hVK6-3: GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCC GATGTTGTGATGACACAGTCTCCAGCTTT (SEQ ID NO: 80)

 A mixture of the above primers as follows was defined as a primer-hV / mix. huVKIa: huVK2a: huVK3a: huVK4a: hVK5-2: hVK6-2: hVK6-3 = 6: 3: 6: 3: 1: 1: 1 hC c Asc: TCGACTGGCGCGCCGAACACTCTCCCCTGTTGAAGCTCTTTGTG (SEQ ID NO: 8 1)

Human V λ rimer

 V then 1: GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCC CAGTCTGTGTTGACGCAGCCGCC (SEQ ID NO: 82)

V then 2: GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCC CAGTCTGCCCTGACTCAGCCTGC (SEQ ID NO: 83) VL2-2: GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCC CAGTCTGCCCTGACTCAGCCTC (SEQ ID NO: 84)

 VL3a-2: GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCC TCCTATGAGCTGACTCAGCCAC (SEQ ID NO: 85)

VL3b: GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCC TCTTCTGAGCTGACTCAGGACCC (SEQ ID NO:

8 6)

 VL4-a: GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCC CAGCCTGTGCTGACTCAATCATC (sequence number 87)

 VL4-b: GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCC CAGCTTGTGCTGACTCAATCGCC (SEQ ID NO: 88)

 VL4-C: GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCC CTGCCTGTGCTGACTCAGCCCCC (sequence number 89)

 VL5: GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCC CAGGCTGTGCTCACTCAGCCGTC (SEQ ID NO:

9 0)

VL5-abd e: GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCC CAGCCTGTGCTGACTCAGCCAHCTTCC (SEQ ID NO: 91)

 VL5-C: GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCC CAGGCTGTGCTGACTCAGCCGGCTTCC (SEQ ID NO: 92)

 VL6: GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCC AATTTTAtGCTGACTCAGCCCCA (SEQ ID NO: 93)

 VL7a b: GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCC CAGRCTGTGGTGACTCAGGAGCCCTCACTG (SEQ ID NO: 94)

 VL8a: GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCC CAGACTGTGGTGACCCAGGAGCCATCGTTC (SEQ ID NO: 95)

VL9a: GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCC CAGCCTGTGCTGACTCAGCCACCTTCTGCA (Distribution (Column number 9 6)

 VL10a: GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCC CAGGCAGGGCTGACTCAGCCACCCTCGGTG (SEQ ID NO: 97) A mixture of the above primers as follows was used as a primer hVA mix.

VL1: VL2: VL2-2: VL3a-2: VL3b: VL4-a: VL4-b: VL4-C: VL5: VL5-abde: VL5-C: VL7ab: VL6: VL8a: VL9a: VL10a = 12: 3 : 3: 3: 3: 1: 1: 1: 1: 1: 1: 6: 1.5: 1.5: 1.5: 1.5 hCAAsc: TCGACTGGCGCGCCGAACATTCTGTAGGGGCCACTGTCTTCTC (SEQ ID NO: 98) Next, add a drop of mineral oil and place at 95 ° C. After heating for 10 minutes, 13 cycles of PCR were performed at 94 ° C for 30 seconds, 63 ° C for 30 seconds, and 72 ° C for 1 minute. Then, 1 l each of the above PCR product was taken, and the reaction mixture for H chain amplification, the reaction mixture for K chain amplification 49 I and the reaction mixture for λ chain amplification 49 I were added. Was served. (Reaction solution for H chain amplification)

 10X PCR primer (supplied with HotStarTaq DNA polymerase kit): 5 i I 10 mM dNTP: 1 I

 Primer hVH nUx (100pmol / i I): ΰ. Βμ. I

 Primer hJH m (100pmol / M i): 0.5μ \

 Distilled water: 41 I

 HotStarTaq DMA polymerase: 1 μ. I

Total 50 μI (reaction solution for c-chain amplification) 10XPCR / offer (included with HotStarTaq DMA polymerase kit): 5 ^

 10mM dNTP: 1 β I

 Primer hVK nUx (IOOpmol / μ I): 1 β I

 Primer hCKAsc (100 pmol / ju. I): 1 μ. I

 Distilled water: 40μΙ

 HotStarTaq DNA polymerase: 1 μ. I

 50 l total

(Reaction solution for λ chain amplification)

 10XPCR buffer (supplied with HotStarTaq DNA ρο merase kit): 5μ '

 10 mM dNTP: 1 μ. I

 Primer hVAmix (lOOpmo I / I): 1 I

 Primer hCAAsc (100 pmol / Ai I): 1 μ. I

 Distilled water: 40jti l

 HotStarTaq DNA polymerase: 1 I

 Total 50μI

As a result of the PCR reaction, amplification of H chain, κ chain and λ chain genes was observed.

<Example 1 2> Construction of expression vector pSMAP

 An expression vector pSMAP capable of expressing a Fab-PP antibody was prepared as follows. At that time, the SfiI cleavage site was removed in the PCR reaction. Figure 13 shows the outline of the construction.

PFCAH9-E8d vector 1 (see 01/62907) 1με (1μΙ), hCHIBX primer (100pmol / ml) 1μ and PelBNco primer (10Opmo l / m I) \ μ. \, 10XLA PCR buff 10 ml 25niM MgCI ₂ 10L 2.5raM dNTP mix 16μ, mix 60 I of sterile distilled water and TaKaRa LA Taq Polymerase (5U / I Takara Shuzo) 1 1 and add 2 drops of mineral oil, 95 ° C After 2 minutes of incubation at 94 ° C, the reaction was repeated at 94 ° C for 1 minute, 58 ° C for 2 minutes, and 72 ° C for 1 minute for 16 cycles.

 hCHIBX: 5'-CACCACGGTCACCGTCTCGAGCGCCTC-3 '(27mer) (SEQ ID NO: 99)

PeI BNco: 5'-TAGTGGCCATGGCTGGCTGGGCAGCGAGT-3 '(29mer) (SEQ ID NO: 100) After confirming the obtained PCR product by agarose gel electrophoresis, it was cut out, treated with phenol, precipitated with EtOH and concentrated to 10 I. To 5 µl of the mixture, 10 µl of buffer (10 µl), 5 il of col (10 U / ZI Takara Shuzo) and 5 xl of BstPI (5 U / I Takara Shuzo) were added, and reacted at 37 ° C for 2 hours. After confirming this by agarose gel electrophoresis, it was cut out, treated with phenol, precipitated with EtOH, and concentrated to 15 µI (insert fragment). Similarly, pSCCA4-E8d (see Example 5) 1 fi g or 1), 10 l of 10XH buffer, 10 l of sterile distilled water Ncol (10 U / I Takara Shuzo) 5 il, BstPI (5 U / MI Takara Shuzo) Was added and reacted at 37 ° C. for 2 hours. After confirming this by agarose gel electrophoresis, it was cut out, treated with phenol, precipitated with EtOH and concentrated to 15 I (vector fragment). Insert fragment 2I, vector fragment 1 / i and 10X Iigation buffer (supplied with T4 DNA Iigase) 1.5μΙ, DW 9.5μμ, Τ4 DNA Iigase (350 units / μ, I Takara Shuzo) 1 ^ I In addition, they were mixed and incubated at 16 ° C for 3 hours. After ethanol precipitation, the precipitate was dissolved in 3 µl of TE diluted 10-fold with sterile distilled water, and half of the solution was used to transform DH12S. Plasmids were extracted from each of the 24 transformants obtained and subjected to agarose gel electrophoresis to confirm the nucleotide sequence of the clone containing the insert. Clones containing the expected substitutions were selected. This was digested with Sal I, and the cp3 region was omitted. Since the cp3 region was sandwiched between Sal I sites, it was cut with Sal I and self-fried. Mix PFCAH9-E8d vector with 15 g (15μ 1), 10xH Buffer (supplied with Sal I) 2 I, Sal I (1511 / Shiba Takara Shuzo) 2 The reaction was performed at 37 degrees for 1 hour. The reaction solution was electrophoresed on a agarose gel, the target band was cut out, treated with phenol, precipitated with EtOH, and dissolved in sterile distilled water (12.5MI). This was mixed with 1 μl of T4 Ligase (350 U / u.I, manufactured by Takara Shuzo) and 1.5 MI of 10 × T4 Ligase Buffer (supplied with T4 DNAIigase) and reacted at 16 ° C. for 3 hours. After ethanol precipitation, it was dissolved in TE3I diluted 10-fold with sterile distilled water, and half of the solution was used to transform DH12S. The cells were spread on an LBGA plate and cultured at 30 ° C overnight. Plasmid was extracted from 12 of the resulting transformants and subjected to agarose gel electrophoresis to confirm the nucleotide sequence of the clone containing the insert. As expected, clones without the cp3 region were selected. This was used as a vector pSMAP for expressing a human Fab-PP type antibody. The nucleotide sequence (SEQ ID NO: 101) and amino acid sequence (SEQ ID NO: 102) of pSMAP are shown in FIGS. 14 and 15.

<Example 13> Preparation of L chain gene

Of the PCR products obtained in Example 11, the L chain was first treated with phenol-cloth-form, ethanol-precipitated, and dried.Then, distilled water was added to lOXNEBuffer 4 (supplied with NEB Ascl). 10) Restriction enzyme Ncol (10 units / U, manufactured by Takara Shuzo) 3I and restriction enzyme Ascl dOunits / U NEB (3 μM) were added, and the mixture was reacted at 37 ° C. for 2 hours. After the reaction, fractionation was performed by agarose gel electrophoresis, and the L chain gene was excised from the gel and purified. For purification after the excision, QIAquick Gel Extraction kit manufactured by Qiagen was used. The cut out L chain gene NcoI Ascl fragment was ethanol precipitated, dried, and suspended in 10 I TE buffer diluted 10-fold with sterile distilled water. Similarly, the vector pSMAP 2 g (2 μl, distilled water 82 μl and lOXNEBuffer 4 (supplied with NEB Ascl) 10 μ U restriction enzyme Ncol (10 units // z L Takara Shuzo) 3 I 3 μ As of the enzyme Ascl (10 units / l, manufactured by NEB) was added and reacted for 2 hours at 37 ° C. After the reaction, fractionation was performed by agarose gel electrophoresis, and the vector PSMAP Ncot Ascl Cut fragments from gel And purified. For purification after the excision, QIAquick Gel Extraction kit manufactured by Qiagen was used. The excised vector pSMAP Ncol-Ascl fragment was precipitated with ethanol, dried, and suspended in 10 tI of TE buffer diluted 10-fold with sterile distilled water. L chain gene N coat Ascl fragment 1 vector pSMAP Ncol-Ascl fragment 1 i Distilled water 10.5 tI, 10 XT4 DNA I igase buffer 1.5 μI, Τ4 DNA I igase 1 μ, I The reaction was performed at 16 ° C for 2 hours. This was precipitated with ethanol, dried, and suspended in TE buffer 3I diluted 10-fold with sterile distilled water. Of these, 1 ^ 1 was added to Elect Max Max DH12S (Escherichia coli) 20ii I, electroporation was carried out, spread on LBGA plates, and cultured at 30 ° C for 1 min. Plasmids were extracted from 24 of the obtained transformants and subjected to agarose gel electrophoresis, and the nucleotide sequence of the clone containing the insert was confirmed using a DNA sequencer CEQ2000XL manufactured by Beckman Coulter, Inc. A clone having the correct gene without any deletion was selected.

<Example 14> Preparation of H chain gene

Next, the cut H chain gene was incorporated into the clone selected in Example 13. The H chain PCR product is treated with phenol-chloroform, precipitated with ethanol, and dried. Distilled water is used. 10XNEBuffer 2 (supplied with NEB SfII) 10μI, 10XBSA (supplied with NEB Sfil) 1 nl Then, 3 μl of restriction enzyme Sfil (8 units / μL manufactured by NEB) was added, and the mixture was reacted at 50 ° C. for 2 hours. Subsequently, 3 MI restriction enzyme Xhol (8 units / l, manufactured by Takara Shuzo) was added, and reacted at 37 ° C for 2 hours. After the reaction, fractionation was performed by agarose gel electrophoresis, and the H chain gene was excised from the gel and purified. For excision and purification, QIAquick Gel Extraction kit manufactured by Qiagen was used. The excised H chain gene SfU-Xhoi fragment was precipitated with ethanol, dried, and suspended in TE buffer 10 I diluted 10-fold with sterile distilled water. Similarly, 2 g (5 / I) of the clone selected in Example 13 was treated with phenol-cloth form, precipitated with ethanol, and dried. K 10XNEBuf fer 2 (supplied with NEB Sfil) 10μΙ, 10XBSA (supplied with NEB SfM) 1 / il, 3 l of restriction enzyme Sfil (8 units / K NEB), The reaction was performed at 50 ° C for 2 hours. Subsequently, a restriction enzyme Xhol (8 units / k, manufactured by Takara Shuzo) 3 I was added thereto, and the mixture was reacted at 37 ° C for 2 hours. After the reaction, fractionation was performed by agarose gel electrophoresis, and the SfiI-XhoI fragment of the clone selected in Example 13 was cut out from the gel and purified. For cutting and purification, QIAquick Gel Extract® on kit manufactured by Qiagen was used. The cut-out Sfitro Xhol fragment of the clone selected in Example 13 was precipitated with ethanol, dried, and suspended in 10 M TE buffer diluted 10-fold with sterile distilled water. H chain gene Sf protein Xho I fragment 1 At and Sf M-Xhol fragment of the clone selected in Example 13 1 nK 10 4 DNA I igase buffer 1.5 I, T4 DNA I igase 1 and sterile distilled water 1 The mixture was mixed with 0.5MI, and the Iigase reaction was performed at 16 ° C for 2 hours. This was precipitated with ethanol, dried, and suspended in 10-fold diluted TE buffer I. Of these, 1 I was added to 20 μI of ElectMouth Max DH12S (E. coli), and electroporation was performed. E. coli after electoral poration was spread on LBGA plates, and cultured at 30 ° C for 1 晚. Plasmid was extracted from 24 of the obtained transformants and subjected to agarose gel electrophoresis, and the nucleotide sequence of the clone containing the insert was confirmed using DNA sequencer CEQ2000XL manufactured by Beckman Coal Yuichi. A clone having the correct gene without any deletion was selected.

<Example 15> Expression of antibody

The clones selected in Example 14 were first inoculated into a 96-well plate containing 200 μl of TYGA medium and cultured at 30 ° C. overnight. This culture solution 2I was cultured in 2XTY medium 100I containing 0.1% glucose and 100 g / ml ampicillin at 37 ° C for 2 hours, and 0.1M IPTG (manufactured by Wako Pure Chemical Industries for Biochemistry) was added. To induce antibody expression. The culture supernatant (μμ) cultured at 30 ° C was botulinum toxin type 感 sensitized plate (Bollinu) A solution prepared by dissolving Stoxoid type A in PBS to 4 Aig / ml is added to a microcup (Nunc-1 Oki unomodule F8 maxisorp Iose (manufactured by Nunc)) at 100 I each, and the solution is left for a while. Discarded, and blocked four times with PBS containing 5% BSA and 0.1% NaN3 overnight and blocked), allowed to react at room temperature for 1.5 hours, and washed four times with PBS. To the washed plate, 100 μl of a 1000-fold diluted human Fc region (manufactured by MBL) labeled with peroxidase was added, reacted at room temperature for 1 hour, and washed 4 times with PBS. After adding a solution of orthophenylenediamine and hydrogen peroxide (100 I) and reacting for a while, the sulfuric acid was stopped by adding 2-sulfuric acid (00 I), and the absorbance at a wavelength of 492 nm was measured. As a result, as shown in FIG. 16, the reactivity with the antigen was confirmed.

<Example 16> Purification of antibody

 The antibody Fab-PP reacting with the antigen in Example 15 was expressed in large amounts in E. coli and purified using an IgG Sepharose column (manufactured by Amersham Pharmacia) and a Ni-MTA Agarose column (manufactured by Qiagen). Incubate the clone expressing the antibody DNA that reacts with the antigen at 30 ° C in 2xYT medium at 50 Oml.If E. coli increases to 0D600 = 0.5, add IPTG to the final concentration ImM to induce antibody expression, and culture at 30 ° C for 1 晚. did. The culture was centrifuged, 175 g of ammonium sulfate was added to the supernatant, and the mixture was centrifuged again. The pellet was dissolved in 30 ml of PBS containing Protease Inhibitor Complete (Roche), placed in a dialysis tube, and pulverized with 5 L of PBS.

The IgG Sepharoselml was packed in the column, washed with 10 ml of PBS, and the sample supernatant concentrated with ammonium sulfate was applied to the IgG Sepharose column. After washing with 20 ml of PBS, the column was eluted with 0.2 M Glycine-HCI pH 2.7 and immediately neutralized with 1 M Tris-HCI pH 8.9. 1 ml of Ni-NTA Agarose was packed in the column, washed with PBSIOml, and all the samples eluted from the IgG Sepharose column were applied to the Ni-NTA Agarose column. After washing with 20 ml of PBS, elution was performed with 0.1 M Imidazol-HCI PH7.5. Replace the eluted sample solution with PBS and use Araicon Ultra It was concentrated at 10,000 (Millipore). Botulinum toxoid type A sensitizing plate (A solution of botulinum toxoid type A dissolved in PBS to 10 Atg / ml) was placed in a microcup (Nunc-lnimunoniodul e F8 max i sorp Ioose (Nunc)). After 100 l of soy sauce and put on a plate, discard the solution, place it in PBS containing 5% BSA and 0.1% NaN3, place it at a further 4 ° C, and block it. A microcup containing 4 g / ml of PBS dissolved in PBS was also prepared as a negative control.) The purified antibody and H / IIOOl were added (PBS negative control) and incubated at room temperature. After reacting for 1.5 hours, the plate was washed four times with PBS. After washing the plate, a peroxidase-labeled human Fc region (manufactured by MBL) diluted 1000-fold was added to 100 / xI, reacted at room temperature for 1 hour, and washed 4 times with PBS. After adding 100 μl of a solution of orthophenylenediamine and hydrogen peroxide and reacting at room temperature for 3 minutes, the reaction was stopped by adding 100 μl of 2-sulfuric acid, and the absorbance at a wavelength of 492 nra was measured. As a result, as shown in FIG. 17, the reactivity with the antigen was confirmed. The above results indicate that an antibody that reacts with an antigen can be produced using an antibody gene obtained from one human antibody-producing cell. The present invention is not limited to the description of the embodiment and the example of the above invention. Various modifications are included in the present invention without departing from the scope of the claims and within the scope of those skilled in the art. The contents of articles, published patent gazettes, and patent gazettes specified in this specification are all incorporated by reference. Industrial potential

Since the antibody production method of the present invention does not involve cell fusion or screening from the antibody library, the desired antibody can be produced efficiently. In addition, each The time required for steps is short, and the desired antibody can be obtained in a short period of time. Furthermore, antibodies derived from various species (for example, mouse, rat, magpie, human, etc.) can be produced, and their versatility is high. In particular, its utility is extremely high in that it is possible to produce an antibody derived from an animal, which was difficult to produce using a conventional method.

In conventional monoclonal isolation techniques, animals are immunized with an antigen, the spleen is removed, and B lymphocytes are treated as a population and immortalized by fusion with cancer cells. When an antibody library is prepared using a phage display system, the H chain and L chain are prepared as a library as a whole, and then combined to produce a large antibody library. In both methods, a clone producing the desired antibody is selected from the population. In the antibody production method provided by the present invention, cells that produce an antibody that binds to an antigen of interest are separated using an antigen-antibody reaction, and a target antibody gene is obtained from the cells, and expressed. By doing so, the desired antibody is prepared. The method of the present invention including such a series of steps is superior to the conventional method in the following various points. First, the method of the present invention has the advantage that antibodies of various animals can be targeted. The conventional method involving cell fusion (cell fusion method) mainly uses a mouse rat, and involves many difficulties when the method is applied to other animals including humans. On the other hand, in the point that antibodies present in blood of various animal species can be monoclonally cloned, the method of the present invention is equivalent to the production of an antibody library using the phage display system (library one method), but the efficiency is high. In the aspect of the present invention, the present invention is far superior. In the case of the phage display system, if a monoclonal antibody of interest is to be surely cloned, a large library is inevitably produced, and the implementation scale must be large. On the other hand, in the method of the present invention, since only the target antibody-producing cells need to be targeted, the scale may be small. This also has the effect of shortening the time required for antibody preparation. On the other hand, antibodies with excellent performance are produced in vivo by maturation of the antibodies, but the present invention has the same superiority as the cell fusion method in cloning the antibodies themselves. In this respect, the phage display method is inefficient. In addition, the method of the present invention is superior to hitherto known methods in that the type of antibody obtained can sufficiently control the type of antibody present in the living body.

Claims

The scope of the claims 1. An antibody production method comprising the following steps (a) to (d):  (a) contacting a labeled antigen obtained by labeling an antigen recognized by an objective antibody with a cell population containing a target cell that produces the antibody, and binding the labeled antigen to the target cell;  (b) separating the labeled target cells obtained in step (a); (c) using the separated labeled target cells to prepare an antibody gene carried by the cells; and  and (d) expressing the prepared antibody gene using an expression vector. 2. The method for producing an antibody according to claim 1, wherein one labeled target cell is separated in the step (b). 3. The method for producing an antibody according to claim 1, wherein the step (b) is performed by manipulation, ultradilution, or flow cytometry. 4. The antibody preparation according to any one of claims 1 to 3, wherein the step (c) is performed by a nucleic acid amplification reaction using genomic DNA of the labeled target cell as a type III. Method. 5. The method for producing an antibody according to any one of claims 1 to 4, wherein the step (c) is performed directly using the labeled target cells separated in the step (b). Law. 6. The method for producing an antibody according to any one of claims 1 to 5, wherein the step (c) includes the following steps:  (c-1) A primer set capable of amplifying the H chain gene, a primer set capable of amplifying the L chain gene, and a deoxynucleotide in a solution containing the separated labeled target cells. Performing a nucleic acid amplification reaction after adding triphosphate;  (C-2) Step of performing a nucleic acid amplification reaction after removing a part of the reaction solution after the step (c-1), adding a set of primers capable of amplifying the H chain gene thereto, as well as  (C-3) Take a part of the reaction solution after step (c-1), add a primer set capable of amplifying L chain gene, and then perform nucleic acid amplification reaction . 7. The antibody production method according to claim 6, wherein the nucleic acid amplification reaction in the step (c-1) is a PCR of 5 cycles or more. 8. The antibody production method according to claim 6, wherein the nucleic acid amplification reaction in the steps (c-2) and (c-3) is PCR of 20 cycles or more. 9. The antibody production method according to any one of claims 1 to 8, wherein in the step (c), an H chain variable region gene and an L chain variable region gene are prepared. 10. The expression vector has a linker sequence, In the step (), the H chain variable region gene and the L chain variable region gene are inserted into the expression vector so as to be linked via the linker sequence. 10. The method for producing an antibody according to claim 9. 11. In step (c), an H chain variable region gene and an L chain gene encoding an L chain variable region and an L chain constant region are prepared. 9. The method for producing an antibody according to any one of clauses 8. 12. In the step (c), the H chain variable region gene and the L chain variable region gene are prepared,  The expression vector has a first sequence encoding an H chain constant region, and a second sequence encoding an L chain constant region,  In the step (d), the H chain variable region gene is inserted into the expression vector so as to be linked to the first sequence, and the L chain variable region gene is linked to the second sequence. The method for producing an antibody according to any one of claims 1 to 8, wherein the antibody is inserted into the expression vector. 13. In the step (c), an H chain variable region gene and an L chain gene encoding an L chain variable region and an L chain constant region are prepared,  The expression vector has a sequence encoding an H chain constant region,  2. The method according to claim 1, wherein in the step (d), the H chain variable region gene is inserted into the expression vector so as to be linked to the sequence, and the L chain gene is inserted into the vector. 9. The method for producing an antibody according to any one of items 8. 14. In the step (c), an H chain variable region gene and an L chain variable region chain gene are prepared, 2. The method according to claim 1, wherein said step (d) includes the following steps. 9. The method for producing an antibody according to any of items 8 to 8:  Inserting the H chain variable region gene into a first expression vector having an H chain constant region gene, and expressing an H chain in which the H chain variable region and the H chain constant region are connected;  Inserting the L chain variable region gene into a second expression vector having an L chain constant region gene, and expressing an L chain in which the L chain variable region and the L chain constant region are connected. 15. In the step (c), an H chain variable region gene and an L chain gene encoding an L chain variable region and an L chain constant region are prepared,  The method for producing an antibody according to any one of claims 1 to 8, wherein the step (d) includes the following steps:  Inserting the H chain variable region gene into a first expression vector having an H chain constant region gene, and expressing an H chain in which the H chain variable region and the H chain constant region are connected;  Inserting the L chain gene into a second expression vector to express an L chain in which the L chain variable region and the L chain constant region are connected. 1, 6. The antibody production method according to any one of claims 1 to 5, wherein in the step (c), an H chain variable region gene is prepared. 17. The antibody production method according to any one of claims 1 to 5, wherein in the step (c), an L chain variable region gene is prepared. 18. The method for producing an antibody according to any one of claims 12 to 15, wherein an IgG class antibody is constructed in the step (d). 19. The method for producing an antibody according to any one of claims 1 to 18, wherein the target cells are human cells. 20. The method for producing an antibody according to any one of claims 1 to 19, wherein the labeled antigen is an antigen labeled with a fluorescent substance, bitin, or magnetic beads. 21. An antibody obtained by the antibody production method according to any one of claims 1 to 20. 2 2. A method for preparing an antibody gene, comprising the following steps (a) to (c):  (a) A labeled antigen obtained by labeling an antigen recognized by an objective antibody is brought into contact with a cell population containing the target cells producing the antibody, and the labeled antigen is bound to the target cells. Steps;  (b) separating the labeled target cells obtained in step (a); and  (c) using the separated labeled target cells to prepare an antibody gene carried by the labeled target cells. 2 3. Gene preparation method including the following steps (i) to (i ~ ί):  (Labeling cells having a particular property in a cell population;  (Ii) separating the labeled cells obtained in step (i); and (Η ί) a step of preparing a target gene using the separated labeled cells. 2 4. In the step (i ί), one labeled cell is separated. Gene preparation method _c described in section