JP2811086B2 - Cat x mouse heterohybridoma and gene fragment encoding the constant region of feline immunoglobulin lambda chain - Google Patents

Description

The present invention relates to a novel feline x mouse heterohybridoma that produces feline immunoglobulin, a method for preparing a feline immunoglobulin gene using the hybridoma, and a method for preparing a feline immunoglobulin. It relates to a gene fragment encoding a region.

Background of the Invention Cats have long been a favorite animal for humans as pets. In recent years, cats have been called "animals as companions, companions, and companions" (Companion species), and have become a member of human society. It is getting. On the other hand, the contribution as an experimental animal ranging from medicine, pharmacy, animal husbandry, veterinary medicine to psychology has been great, but in recent years, the name of a minimal disease cat has been used in drug effect tests and safety tests. The contribution is increasing further. In each case, of course, more and more knowledge about these cat diseases, especially infectious diseases, is required, and methods for their diagnosis, treatment and prevention are required. .

There are many feline viral diseases, among which diseases such as feline rhinotracheitis virus, feline parvovirus, and feline infectious peritonitis virus are acute and have a high mortality rate. Although preventive vaccines have been developed, there are still problems with current treatment methods, such as the only available treatment for cats that have become infected or affected, such as the prevention of secondary bacterial infections such as antibiotics and sulfa drugs. ing. Conventionally, hyperimmune sera and serum-derived immunoglobulins have been used as therapeutic methods and have been used effectively. However, at present, with the rise of animal welfare philosophy, it has become difficult to obtain cat serum raw materials, and it has become impossible to use this therapy even if it is desired. Therefore, if a monoclonal antibody capable of neutralizing an infectious virus can be produced instead of the conventional hyperimmune serum, it can greatly contribute to the treatment of these viral diseases.

2. Prior Art As an alternative to the above-mentioned highly immune serum, use of a monoclonal antibody having a virus neutralizing activity can be considered. The basic technology for monoclonal antibody production is
So far, it has been established mainly in mouse monoclonal antibodies. Monoclonal antibodies produced by cells such as hybridomas can be obtained in large quantities and semipermanently, which can solve the problem of lack of raw materials. However, the monoclonal antibody used here is not a conventional mouse monoclonal antibody because it has no side effects (when a mouse monoclonal antibody is used in cats, it is likely to cause side effects such as anaphylactic shock and serum sickness as a heterologous protein). Must be a feline monoclonal antibody.

The following can be considered as a method for preparing a feline monoclonal antibody as a therapeutic agent for these feline viral diseases. (1) a method using a cat x cat hybridoma,
(2) a method using cat lymphocytes transformed with a certain virus or chemical agent, (3) a method using a cat × mouse heterohybridoma, (4) a cat ×
A method using a cat x (cat x mouse) hybridoma using a mouse heterohybridoma as a parent strain, (5) a chimeric monoclonal antibody (a variable (V) region that binds to an antigen is derived from a mouse monoclonal antibody having virus neutralizing activity, The constant (C) region involved in immunogenicity and physiological activity is composed of a feline monoclonal antibody, and a mouse (V) -feline (C) chimeric monoclonal antibody) is produced by genetic recombination. No success has been reported for either method.

Here, regarding (1), the fusion efficiency is low and there is no suitable myeloma parent strain, and (2) there is no suitable virus or suitable chemical corresponding to the EB virus in humans. (3) In the method of (4), many difficulties are expected to obtain a target cat-type monoclonal antibody with high efficiency in view of the preparation example of a human-type monoclonal antibody (for example, stability problems). Therefore,
It is considered that the chimeric monoclonal antibody method (5) is a more feasible method.

This chimeric monoclonal antibody comprises a V gene cloned from a mouse × mouse hybridoma producing a mouse monoclonal antibody serving as a material for the variable (V) region, and a cat antibody producing a cat monoclonal antibody serving as a material for the constant (C) region. A plasmid vector containing a mouse (V) -cat (C) chimeric antibody gene linked to a C gene cloned from a producer cell is expressed in an animal cell (eg, mouse myeloma) host, and It is obtained. Some reports on chimeric antibodies have already been found in humans (JP-A-60-155132, JP-A-61-47500).

As described above, to prepare a feline chimeric antibody, a gene encoding the amino acid sequence of the variable (V) region of an antibody molecule capable of binding to an antigen of interest and the constant (C)
A gene encoding the amino acid sequence of the region is required.
The variable (V) region gene of the chimeric antibody is obtained from cells producing a mouse monoclonal antibody having a neutralizing activity against the various feline viruses described above, and the cells are obtained by a conventional mouse x mouse hybridoma method. It can be manufactured relatively easily. However, at present, the structure of the feline immunoglobulin C region gene serving as the constant region gene of the chimeric antibody is not known at all, and no gene has been cloned. Therefore, in order to prepare a feline chimeric antibody, finding a gene encoding the amino acid sequence of the constant (C) region of feline immunoglobulin is a very important factor.

The methods (1) to (4) have many problems in producing a monoclonal antibody having the desired specificity. However, the method (5) which is an effective material for producing a chimeric antibody ( Cell lines). That is,
This is because any cell producing a feline immunoglobulin can be a preferable material for providing the C region of the feline immunoglobulin gene for producing a chimeric antibody, regardless of its specificity.

Object of the Invention Under such circumstances, the present inventors have succeeded in producing a feline × mouse heterohybridoma producing feline immunoglobulin. Furthermore, the gene encoding the amino acid sequence of the constant region of feline immunoglobulin was successfully isolated from these cells. Further, by analyzing the nucleotide sequence of the gene fragment encoding the constant region of the feline immunoglobulin, an amino acid sequence unique to the feline immunoglobulin λ chain constant region was found, thereby completing the present invention.

That is, an object of the present invention is to provide a feline immunoglobulin-producing cat × mouse heterohybridoma which has not been reported at all. Still another object of the present invention is to provide a DNA fragment encoding a constant region of a feline immunoglobulin gene λ chain, which has not been subjected to any gene analysis. Further, the present invention provides a DNA fragment encoding the constant region of a feline immunoglobulin λ chain constituting a feline monoclonal antibody, and a feline chimeric antibody produced using this DNA fragment can be used for feline diseases, particularly infectious diseases. It enables application to diagnostics, therapeutics and prophylactics that have no side effects on the disease.

Structure and Effect of the Invention There are mainly two methods for isolating a novel feline immunoglobulin C region gene. One is to use standard methods from chromosomal DNA of cat cells [for example, T. Maniati
s “Molecular Cloning” Cold Spring Harbor Lab. (198
2)] and constructing a library and cloning the C region gene. The other method uses cat cell messenger RNA (mRNA) as a material and uses a conventional method [for example, "DNA cloning Vol. I" IRL edited by DMGlover. press (1
985)] to construct a library by synthesizing cDNA
This is a method for cloning a region gene.

In any case, it is desirable from the viewpoint of cloning efficiency to clone using cells producing a cat-type antibody protein as a material, and particularly in the latter method of synthesizing cDNA from messenger RNA, an essential condition. Becomes Several methods for establishing such an antibody-producing cell can be considered as follows. A method for producing a cat x cat hybridoma, a method for transforming a cat lymphocyte with a certain virus and a chemical agent, a method for producing a cat x mouse heterohybridoma,
A method using a cat x (cat x mouse) hybridoma using a cat x mouse heterohybridoma as a parent strain, and the like. However, as shown in the description of the prior art, the method is very difficult in practice, and the method requires a cat x mouse heterohybridoma, and consequently obtains a cat x mouse heterohybridoma. Is an important key.

Hereinafter, a method for preparing a cat x mouse heterohybridoma will be described in detail. First, a cat is immunized to activate lymphocytes for the production of cat antibodies. The immunization method may be nonspecific immunization only with adjuvant immunization, or specificity such as feline parvovirus particles (or purified antigen), feline rhinotracheitis virus particles (or purified antigen), and feline infectious peritonitis virus particles (or purified antigen). Any specific immunization using an antigen (adjuvant mixture) is possible. A spleen or lymph node is obtained from a cat immunized with nonspecific or specific immunity. Each lymphocyte obtained from the spleen or lymph node is isolated and used as a culture suspension, and activated by adding a lymphocyte activating substance such as pork weed mitogen (PWM). At this time, the specific immunity can be enhanced by adding the virus antigen together with the PWM. The cat B lymphocytes thus obtained were collected, and the lymphocytes and parental mouse myeloma cells (X63-Ag8-6.5.3, P3-X63-Ag8-U1, derived from mouse BALB / c, SP2 / 0Ag
12 mag myeloma cells) and fused with a fusion agent.
Mouse hybridomas are formed. The fusion method may be any known method, and examples of the fusion agent include polyethylene glycol and the like. Selection of the fused hybridoma is achieved in a HAT selection medium such as RPMI1640 with glutamine + 10% fetal calf serum + HAT (hypoxanthine + aminopterin + thymidine) at 37 ° C. in the presence of 5% CO ₂ . The hybridoma producing a cat IgG antibody can be confirmed by, for example, a sandwich enzyme immunoassay (EIA) method or a radioimmunoassay (RIA) method using a plate coated with an anti-cat IgG antibody. Hybridomas producing cat IgG antibodies having specificity for the specific antigen as described above (specific cat IgG antibodies) were also measured by EIA or RIA using a plate coated with the specific antigen. You can check. Thus, the selected cat IgG antibody or specific cat IgG antibody-producing cat × mouse hybridoma is monocloned by limiting dilution and established as a single clone producing a single feline IgG monoclonal antibody. In order to establish a more stable antibody-producing clone, it is necessary to repeat this cloning operation several times at an early stage. By such a method, the present inventors succeeded in establishing a cat x mouse heterohybridoma FM-T1, T2 and T3 producing a cat monoclonal antibody. As a preferred example of such a cat x mouse heterohybridoma, the applicant
T1 cells (Deposit No. 10077) have been deposited with Microdepartment. This cell is mentioned as the most desirable cell line used for the following gene preparation of the present invention.

In the case of mice and humans, the immunoglobulin gene is composed of a variable region (V region) gene, which is a binding site with an antigen, and a constant region (C region) gene having physiological activities involved in the interaction with complement and specific cells. It is well known that they are formed. Further, the V region gene is selected from one of a number of V gene fragment groups, D gene fragment groups (not yet found in the L chain) and J gene fragment groups, and linked in this order. It is formed. Further, the gene is linked to the C region gene of each class by a class switch, and becomes an immunoglobulin gene of an expression system. [Susumu Tonegawa, Nature, 307, p575 (1983); Yu Honjo, Annual Rev. Im
munol. 1, p499 (1983)]. That is, if the gene is an active C-region gene that can be expressed (transcribed into mRNA and further translated into protein), the rearrangement of the gene, which is a characteristic of immunoglobulin genes, should have been completed. Therefore, using the chromosomal DNA of the antibody-producing cell and its parent strain, a conventional method [for example, T. Maniatis “Molecular Cloning” Cold Spr.
ing Harbor Lab. (1982)] to identify an immunoglobulin gene specific to an antibody-producing cell, whereby the immunoglobulin gene including the C region gene can be determined. By using the feline antibody-producing cells in this way, the target antibody gene can be identified more quickly. Chromosome DN to target gene
When prepared from A, the gene contains an intervening sequence called an intron.

In order to isolate an antibody gene, there are mainly three screening methods available in the two cloning methods described above. Purifying an antibody protein produced by a feline antibody-producing cell, analyzing the amino acid sequence of the protein using the protein as a material, synthesizing a nucleic acid base sequence corresponding to the amino acid sequence, and using it as a probe for screening (hybridization). Method; gene fragments of mouse and human immunoglobulin genes already reported, or nucleic acid base sequences thereof [for example, Sakano et al.
Nature, 286, p676, (1980); EEMax et al., J. Biol. Chem., 2
56, p5116, (1981); JWEllison et al., Nuc. Acids. Res., 1
0, p4071, (1982); PAHeiter et al., Cell, 22, p197 (198
0)], a method of screening by cross-hybridization using a DNA or the like synthesized as a probe; expressing a feline antibody gene incorporated in an expression vector such as λgt11 in Escherichia coli or animal cells,
In this method, an expression product is screened using an antiserum (or a monoclonal antibody) raised against a feline antibody protein. Any of the methods of the cDNA cloning method can be used, and the method of the chromosomal DNA cloning method can be used.

As a result of comparing the sequence of the gene fragment encoding the C region of the feline immunoglobulin cloned in this manner with the sequence of the C region gene of the immunoglobulin of another animal species, the feline immunoglobulin C The gene fragment of the present invention encoding the region was found to be a gene fragment belonging to the λ chain.

The human [PAHiet
er et al., Nature, 294, p536 (1981); GFHollis et al., Natur
e, 296, p321 (1982)] and mice [B. Blomberg et al., Nat.
l. Acad. Sci. USA, 79, p530 (1982); J. Miller et al., Nature,
295, p428 (1982)], and in the λ chain of other animal species, rabbits [Duvoisin, MR. M. et al., J. Immunol., 13
6, p4297-4302 (1986)], but there is no report on the cat λ chain, the amino acid sequence encoding it, and the nucleic acid base sequence described in the present invention. Is disclosed for the first time.

Thus obtained by the present invention, the base sequence of the DNA fragment encoding the feline immunoglobulin λ chain constant region was analyzed, and the amino acid sequence of the constant region was found. Comparison with the amino acid sequence of the immunoglobulin λ chain constant region of mice, rabbits and the like revealed that the amino acid sequence specific to the feline immunoglobulin λ chain constant region was the first cysteine from the N-terminal side of the λ chain constant region polypeptide. Was found to be the amino acid sequence of the following (A).

The present inventors have analyzed the amino acid sequence of the cat immunoglobulin λ chain constant region analyzed by the present invention, the amino acid sequence of the canine immunoglobulin λ chain constant region separately analyzed by the present inventors, and By comparing the amino acid sequences of the immunoglobulin λ chain constant regions of various animals, the -Se existing at the N-terminal side of the cysteine described above was determined.
The r-Ala- region was found to be a region having a different amino acid sequence depending on species such as cats, mice, and humans. At the same time, it has also been found that this region is very well conserved between subtypes, for example, as the amino acid sequence of the human λ chain constant region. It was assumed that the sequence was unique to the λ chain constant region. The amino acid sequence of this region cloned in the present invention is as follows, and this amino acid sequence (B) is a preferred example of a unique amino acid sequence present in the feline immunoglobulin λ chain constant region.

The cat x mouse heterohybridoma of the present invention is characterized by producing a feline immunoglobulin containing a λ chain C region peptide having the amino acid sequence of the above (A) or (B). Furthermore, in the gene fragment encoding the feline immunoglobulin λ chain constant region of the present invention, the above (A)
Alternatively, a DNA sequence encoding the amino acid sequence of (B) is included in a part thereof. Λ above such
The amino acid sequence contained in the chain is considered to be an important amino acid sequence that determines the C region of feline immunoglobulin λ chain, and was first revealed by the present invention. Also, in the amino acid sequence of the eleventh to ninth amino acids on the N-terminal side of the second cysteine from the C-terminal of the cat λ chain constant region polypeptide,
Similarly to the above, the amino acid sequence is considered to be a region where the amino acid sequence changes due to the difference in species of cats, dogs, etc., and the feline immunoglobulin λ chain constant region of the present invention has -Pro-Asn-Glu- as its unique sequence. That was found. A preferable example of such a gene encoding the C region of the feline immunoglobulin λ chain is a gene fragment encoding the amino acid sequence of FIG. An example of a specific nucleic acid base sequence of such a gene is the base sequence shown in FIG. Such gene fragments of the present invention are not limited to those derived from feline antibody-producing cells, but also include those prepared from cells such as cat liver. However, if the aforementioned antibody-producing cells (cat x mouse heterohybridoma) are used, the antibody gene can be identified more quickly, and the C region gene can be cloned more easily. In addition, as a result of performing Southern hybridization with feline chromosomal DNA using the λ chain gene of the present invention, in addition to the λ chain of the present invention, several C region genes of other subtypes belonging to the same feline λ chain were found. It was shown to be present. Human and mouse examples [PAHiet
er et al., Nature, 294, p536 (1981); GFHollis et al., Natur
e, 296, p321 (1982); B. Blomberg et al., Proc. Natl. Acad. Sc.
i.USA, 79, p530 (1982); J. Miller et al., Nature, 295, p428.
(1982)], it seems that there are several subtypes in the cat λ chain. Feline immunoglobulin λ of the present invention
The gene fragment encoding the constant region of the chain also includes a gene fragment encoding such an amino acid sequence having a different subtype. The cloning of C genes having different subtypes can also be performed using a part of the nucleotide sequence disclosed in the present invention as a probe.

A mouse-cat chimeric antibody can be prepared directly using the feline immunoglobulin λ chain C region gene of the present invention, and further, a chromosomal DNA library can be prepared by using a part of the nucleotide sequence disclosed in the present invention as a probe. Alternatively, a genomic immunoglobulin λ chain C region gene can be cloned from the protein and a chimeric antibody can be prepared using the cloned gene. The method for producing the chimeric antibody is the method already described for the mouse-human chimeric antibody [Watanabe et al., Cancer Research, 47, p99
9-1005, (1987)]. That is, a chimeric antibody gene is basically constructed by combining two types of gene fragments, a V region gene and a C region gene. Furthermore, depending on the method of gene isolation, there are mainly combinations of the two bonds. That is, V and C region genes isolated from chromosomal DNA and V and C genes isolated from cDNA
It is a combination of region genes.

For example, when a V region gene isolated from mouse chromosomal DNA is combined with a C region gene isolated from cat chromosome DNA, the mouse V region gene contains expression control regions such as promoters and enhancers necessary for expression. Preferably. However, the promoter, enhancer and the like need not be derived from mouse, and may be derived from cat, human or virus. The promoter is located 5 'upstream of the V region, and the enhancer is
It is preferably located between the region gene and the C region gene, but the enhancer is not necessarily limited to this position. On the other hand, when a V region gene isolated from a mouse cDNA is combined with a C region gene isolated from a cat cDNA, the binding portion may have an appropriate restriction enzyme site,
If necessary, use an appropriate synthetic linker so that the amino acid sequence encoded by the V region gene and the amino acid sequence encoded by the C region gene do not shift, and the amino acid sequence of the V region and the amino acid sequence of the C region are changed. Must not be combined. Furthermore, it is necessary to add an expression control region such as an appropriate promoter or enhancer for enabling expression in animal cells to the 5 'upstream region of the gene. The chimeric antibody gene prepared in this manner is, for example, pSV2-gpt [RCMulligan et al., Proc. Natl. Acad. Sc.
i.USA, 78, p2027 (1981)], pSV2-neo [PJSouthern
, J. Mol. Appl. Genet., 1, p327 (1982)] or an appropriate vector plasmid with a selectable marker, or
It is possible to construct a chimeric antibody gene plasmid by separately or simultaneously incorporating the H chain gene and the L chain gene into a vector plasmid having a portion of a viral gene (such as papilloma virus) that can be propagated as a plasmid in a host cell. desirable. In order to obtain a mouse-cat chimeric antibody, it is necessary to transform host animal cells with the plasmid containing the chimeric antibody gene thus prepared. As host animal cells, immortalized mouse and other animal cells, preferably B lymphoid cell lines [eg, P3X63Ag8 · 653 (ATCC CRL 1580), P3X63A
g8U · 1 (ATCC CRL 1597), P3 / NS1 / 1 Ag4-1 (ATCC CRL
RL18), Sp2 / 0-Ag12 (ATCC CRL 1581), etc., hybridoma]. Methods for transforming cells with DNA include methods such as the DEAE-dextran method, the calcium phosphate coprecipitation method, the protoprost fusion method, and the electroporation method [for example, BD Trans.
iption and Translation "IRL Press (1984)], and any method may be used. When transforming with a plasmid having both the H-chain and L-chain chimeric antibody genes at the same time, one type of selection marker may be used. In the case of separate H and L chains, two types of markers are required, and in this case, a double transformation method in which transformation is performed with one plasmid and then with the other plasmid is used. The cells transformed in this manner are preferably used under the same suitable conditions as a normal hybridoma (for example, 10%
When cultured in RPMI1640 medium containing fetal calf serum), chimeric antibodies are secreted and produced from these cells in the same manner as antibodies produced by normal hybridomas. This chimeric antibody can be purified by the same method as for a normal antibody.

The mouse-cat chimeric antibody obtained as described above using the gene encoding the feline immunoglobulin λ chain C region of the present invention can provide a substantially effective diagnosis of cat diseases that has never been seen before. , Prophylactic and therapeutic agents. Further, the gene fragment encoding the feline immunoglobulin provided by the present invention may be a specific amino acid sequence or DNA of the C region of feline immunoglobulin λ chain.
Disclosed is a sequence, which will allow the isolation of C region genes of other subtypes belonging to the same λ chain in the future.

Next, examples will be described, but the present invention is not limited to these examples.

Example (1) Preparation of Feline Monoclonal Antibody-Producing Cell Immunization and Preparation of Feline Lymphocytes First, 5 ml of complete front adjuvant (CFA: manufactured by Difco) was injected subcutaneously and intraperitoneally into cats to perform nonspecific immunization. Further, the same operation was repeated several times at intervals of 2 to 3 weeks to enhance immunity. From the cat immunized in this way, spleen and lymph nodes were obtained 2-3 weeks after the final immunization. These spleens and lymph nodes were treated with 100 IU of penicillin (manufactured by Banyu Pharmaceutical Co.)-Streptomycin (manufactured by Meiji Seika).
Was thoroughly washed in an RPMI1640 medium (manufactured by Nissui Pharmaceutical Co., Ltd.) supplemented with, and then crushed into small pieces in RPMI-1640, further crushed with forceps, and made into a cell suspension by pipetting.
Next, red blood cells were removed with a red blood cell removing solution, and after several centrifugation treatments, feline lymphocytes were obtained. One spleen from one cat
Lymphocytes of 5 × 10 ⁹ cells and 1-5 × 10 ⁸ cells were obtained from lymph nodes. Further, these lymphocytes were added to L-glutamine (manufactured by Flow Laboratories) RPMI1640 + 10
5-10% in fetal bovine serum (Hyclone) in complete medium
Suspend the cells at × 10 ⁵ cells / ml, add 2.5 μg / ml of pork weed mitogen (PWM: manufactured by Gibco) at 37 ° C., 5%
The cells were stimulated and activated for 2 to 5 days in the presence of CO ₂ to activate them.

Preparation of mouse myeloma cells The myeloma cells used in the present invention have already been described by Koehler et al. In Nature, 256, p495 (1975) and Euv. J. Immunol, 6, p29.
2 (1976), in particular, the myeloma cell lines derived from mouse BALB / c, especially the sublines X63-Ag8-6.5.3 and P3-X63-Ag
8-U1, SP2 / 0Ag12. These are glutamine-added RPMI1
The cells were grown and cultured in a complete medium of 640 + 10 fetal bovine serum, collected immediately before fusion, washed twice with RPMI1640 medium, turbidized again in the same medium, and used for fusion.

Cell fusion of feline lymphocytes and mouse myeloma cells The feline lymphocyte suspension and the mouse myeloma cell suspension were mixed (cat lymphocyte: myeloma cells = 10: 1 or 5: 1).
Percentage, feline lymphocytes = 1 × 10 ⁸ or 2 × 10 ⁸ cells),
RPMI1640 was added to the cell pellet centrifuged at 1500 rpm for 5 minutes.
45% polyethylene glycol solution (manufactured by Sigma)
pH7.6 molecular weight 3650 or Selva pH7.6 molecular weight 4000) 1m
l was added at room temperature for 1 minute. After standing at 37 ° C. for 5 to 10 minutes, 40 ml of RPMI1640 was added over 6 minutes, the cells were gently turbid, and the fusion was stopped. The cells are then
After centrifugation at 10 m for 10 minutes and removing the supernatant by suction, RPMI1640 with glutamine + 10% fetal bovine serum + HAT (H: hypoxanthine 13.0 μg / ml, A: aminopterin 0.18 μg / ml, T: thymidine 3.87 μg / ml (All manufactured by Sigma) as a lymphocyte concentration of 2 to 10 × 10 ⁵ cells / ml. This is 96
The cells were sorted at 200 μ / well in a well microtiter plate and cultured at 37 ° C. in the presence of 5% CO ₂ . After 5 to 7 days, 50% medium exchange was performed with the same medium, and 10 to 10 days after fusion.
After 28 days, the medium exchange was repeated 5 to 6 times. Thus, the culture was continued until only the hybridoma grew and the screening assay was completed.

Hybridoma Screening Assay and Cloning A screening assay was performed to confirm that the growth of the hybridoma had progressed sufficiently and to detect a clone producing a cat IgG antibody. The screening assay was performed by enzyme immunoassay (EIA). That is, a 96-well plate coated with a goat anti-cat IgG antibody (manufactured by Kappel) and non-specific adsorption was blocked with bovine albumin (manufactured by Sigma) was prepared, and 50 μl of the culture supernatant from each well of the hybridoma culture plate was prepared. In addition, 37 ℃,
After incubating for 1-2 hours, PBS-T [0.01% Tween
en (manufactured by Katayama Chemical), 0.01M Phosphate, pH7.2, 0.15M
NaCl] four times, and a peroxidase-labeled goat anti-cat IgG antibody (10,000-fold dilution, manufactured by Kappel) was added in an amount of 50 µl, followed by incubation at 37 ° C for 1 hour. Then PBS-T
5 times with TMBZ substrate solution (TMBZ: Dojin Chemical 0.4
mg / ml, hydrogen peroxide solution: 0.006% by Mitsubishi Gas Chemical Company)
The color was developed by adding μ. After 10 to 15 minutes, the reaction was stopped by adding 50 µm of 0.3NH ₂ SO ₄ (Katayama Chemical Co., Ltd.), and the degree of color development was measured with a spectrophotometer (wavelength: 450). The hybridomas in the feline IgG production wells selected in this manner were subjected to single cloning (cloning) by the limiting dilution method. After the clones had grown in each well of the 96-well plate, the same enzyme immunoassay (EIA) as described above was performed to detect feline IgG-producing clones. This cloning operation was repeated at least three times to obtain a cat × mouse hybridoma clone stably producing cat IgG. Further, this hybridoma clone was sequentially expanded and cultured, and glutamine-added RPMI-1640 + HAT + 10
The cells were cryopreserved in liquid nitrogen in a cell freezing medium of% DMSO (Wako Pure Chemical).

Properties of the established hybridoma clone and the cat IgG monoclonal antibody produced by the cat.The established cat x mouse hybridoma showed a cat IgG monoclonal antibody of 1.0 to 3.0 μg over a long period of one year or more as a result of measuring the amount of the cat antibody produced by the EIA method. It is produced stably at the amount of / ml, and it has been confirmed that no abnormality is observed in the antibody production ecology.

The feline monoclonal antibody produced by the established feline × mouse hybridoma was found to be feline IgG by immunoprecipitation [see Immune Experiment Procedures, edited by the Japanese Society for Immunology]. This antibody did not form any precipitate with goat anti-mouse IgG antiserum and goat anti-human IgG antiserum,
Since the monoclonal antibody forms a precipitate only with the goat anti-cat IgG antiserum, the monoclonal antibody is determined to be a cat IgG antibody. Further, in order to prove that the monoclonal antibody was a complete cat-type monoclonal antibody, it was examined using an EIA method which is more sensitive than the immunoprecipitation method. As a result, all of the monoclonal antibodies specifically reacted only with the anti-cat IgG antibody, and the anti-human IgG antibody and the anti-mouse I
By not reacting with the gG antibody, it was proved to be a cat IgG antibody (FIG. 1). Furthermore, the monoclonal antibody was analyzed by Western blot assay [see Immune Experiment Procedures, edited by the Japanese Society of Immunology] for the antibody heavy chain (H
Chain and light chain (L chain) fragments specifically react only with the anti-cat IgG antibody, and with the anti-human IgG antibody and anti-mouse IgG antibody, neither the H chain nor the L chain fragment reacts at all. H chain, L of monoclonal antibody
Since the chain fragment is identical in molecular weight to the H chain and L chain fragments of a standard cat IgG antibody,
Complete cat I with H and L chain fragments of IgG antibody
gG monoclonal antibody (Second
Figure). In addition, the intracellular fluorescent antibody staining assay of the hybridoma clone [Immunological experiment procedure]
As a result of examining the synthesis of feline IgG antibody in the cytoplasm, all the clones were specifically stained in the cytoplasm only with the anti-feline IgG antibody, and the other anti-human IgG antibody and anti-mouse IgG were cloned.
The lack of staining with the antibody proved that the hybridoma clone synthesized a complete feline IgG monoclonal antibody in its cytoplasm. A cat x mouse heterohybridoma FM-T1 cell producing such a cat monoclonal antibody has been deposited by the present applicant as No. 2947 (Original deposit number: No. 10077, No. 10077). I have. The cells were used in the experiments described below.

(2) Construction of cDNA library Total RNA was obtained from heterohybridoma FM-T1 cells by the guanidium thiothianate method [JMGhingwin et al., Biochemist
ry, 18, p5294 (1979)].
It was purified to poly A + RNA using a column (Pharmacia). From the purified poly A + RNA, cDNA of FM-T1 cells was synthesized using cDNA synthesis system plus (Amersham).
The EcoRI site of the synthesized cDNA was methylated using EcoRI I methylase (Takara Shuzo: the reagents used in this example were manufactured by Takara Shuzo or Toyobo unless otherwise specified), and then T4 DNA ligase was used. EcoRI linker was added. The cDNA was further completely digested with the restriction enzyme EcoR I, and EcoR I was digested using a Biogel A50m column (Bio-Rad).
The cDNA with the linker added was purified. Next, this cDNA and λ
gt11 vector DNA (Stratagene) was ligated to the EcoRI arm by T4 DNA ligase, and in vitro packaging was performed using a Stratagene kit, followed by FM
-A cDNA library of T1 cells was obtained.

(3) Screening of feline immunoglobulin gene using anti-cat IgG antibody From the cDNA library of FM-T1 cells constructed as described above, one LB plate [1.5% Bacto-agar (manufactured by Gifco), 1% Bacto -Tryptone (Gifco), 0.5% B
acto-yeast extract (manufactured by Gifco), 0.25% NaCl (Wako Pure Chemical Industries, Ltd.), Kaku No. 2 Petri dish containing pH 7.5 (manufactured by Eiken Kikai Co., Ltd.)], and E. coli y1090 so that 50,000 λgt11 plaques can be formed. And inoculated with phage, and cultured at 42 ° C. for 3 hours. Then, a nitrocellulose filter (NC filter: S) impregnated with 10 mM IPTG (Wako Pure Chemical Industries, Ltd.)
& Co., Code BA85) and continue culturing at 37 ° C for 4 hours. Thereafter, the NC filter was removed from the plate, and W buffer [WB: 7 mM Tris pH 7.2, 150 mM NaCl, 0.0
05% Tween20], BLOTTO [5% skim milk, 10
μ / 100 ml Antifo amA] at 4 ° C overnight. Then 10μg
/ ml anti-cat IgG antibody (Kappel) [treated with 1% E. coli lysate (Bio-Rad) at 4 ° C overnight]
Replace with BLOTTO and react at room temperature for 2 hours. After washing 5 times with WB, peroxidase-labeled goat Ig diluted 5,000-fold
Incubation buffer [PBS pH 7.2, 0.005% Tween 20, 1% BS] containing G antibody (manufactured by Kappel) [treated with 1% E. coli lysate (manufactured by Bio-Rad) at 4 ° C overnight]
A] and react for 2 hours at room temperature. After washing five times with WB, the plate was immersed in a color developing solution containing 5% HRP color development reagent (manufactured by Bio-Rad) and 0.5% H ₂ O ₂ (Wako Pure Chemical Industries) to develop color. Phages corresponding to plaques developed on the NC filter were selected, and cloning was repeated.
In this way, a clone reacting with the anti-cat IgG antibody was selected, and finally T1-62 was obtained. This clone is about 0.7kb
From the results of the nucleic acid base sequence analysis described below, it was considered that the gene belongs to the immunoglobulin λ chain. FIG. 3 shows a map of the restriction enzyme cleavage points. Of this clone
The EcoRI insert was cloned according to the method of Thomas and Davis [M.
W. Davis, J. Mol. Biol., 91, p315 (1974)], and subcloned into the EcoRI site of the pUC18 vector.

(4) Southern Blot and Northern Blot Analysis Using T1-62 First, Southern blot analysis using this T1-62 was performed. 10 μg of chromosomal DNA from cat liver cells was digested with the restriction enzyme EcoR I
The DNA is electrophoresed on a 0.7% agarose gel and transferred to a nylon membrane filter (Genescreen Plus, NEN Research Products).
Southern hybridization was performed with a [ ³² P] -labeled T1-62 probe containing a cat Cλ chain region. The method of Southern hybridization followed the protocol of the manual attached to GeneScreen Plus. Molecular size is marker DNA obtained by cutting lambda phage DNA with Hind III
Was calculated. As a result, as shown in FIG. 4, bands were detected over various sizes up to about 2 to 20 kb. This suggests that the cat Cλ region gene is not only one subtype. Also, the fact that the Cλ region is not one type is the case of humans and mice [PAHieter et al.
ture, 294, p536 (1981); GFHollis et al., Nature, 296, p32.
1 (1982); B. Blomberg et al., Proc. Natl. Acad. Sci. USA, 79,
p53 (1982); J. Miller et al., Nature, 295, p428 (1982)]
Can also be estimated from Furthermore, it is known that the Cλ region gene is amplified in wild mice [CLScott
Nature, 300, p757 (1982)] This cat Cλ region gene may be similarly amplified.

Next, Northern blot analysis was performed. For the RNA used for hybridization, total RNA was extracted from cat spleen cells and FM-T1 cells by the guanidium thiocyanate method [JMGhing
Win et al., Biochemistry, 18, p5294 (1979)], and further purified to poly A + RNA using an oligo dT column (Pharmacia). 2 μg of this RNA was electrophoresed on a 0.75% agarose gel containing 3% formaldehyde, transferred to a nylon membrane filter (Genescreen Plus), and then subjected to Northern hybridization with a [ ³² P] -labeled T1-62 probe. The method of Northern hybridization followed the protocol of the manual attached to GeneScreen Plus.
As a result, both cells were approximately 1.3 kb with this probe.
(FIG. 5). This size is almost the same as the size of the immunoglobulin lambda chain gene known in mice and humans.

From these two results, it was estimated that this T1-62 gene was an active gene containing a functional cat Cλ region.

(5) Nucleotide base sequence and amino acid sequence of T1-62 In order to examine the nucleobase sequence of T1-62, from T1-62,
EcoR I-Sac I, Sac I-Acc II, Acc II-EcoR I, EcoR
Each small DNA fragment of I-HhaI was prepared (FIG. 3). After changing the cut surface of each of these small fragments to blunt ends using T4-DNA polymerase, the fragment was inserted into the Sma I site of the M13mp19 vector using a treasure ligation kit. According to the method of Toyobo Instruction Manual, JM101 competent cells were prepared, transformed with M13mp19 DNA into which Cλ gene was inserted, and single-stranded DNA was extracted and purified. Furthermore, the nucleic acid base sequence of this single-stranded DNA was determined using Takara M13 sequence kit and Fuji Gencer Gel System. The direction in which the nucleobase sequence was performed is shown in FIG. As a result of nucleic acid base sequence determination, a cat λ chain gene consisting of each region of VJC was confirmed. FIG. 6 shows the results. further,
Conversion of the nucleic acid base sequence into amino acids showed that the gene had an open reading frame and was not a pseudogene (FIG. 7). In addition, the applicant has deposited Escherichia coli FCL-T162 (Microcosms No. 10942) deposited with a vector incorporating the DNA fragment.

Using gene analysis software (Genetyx Ver. 6; manufactured by Software Development Company) based on the nucleic acid salt sequence of T1-62, LA
When a homology search was performed between the SL and EMBL databases,
Shows the highest homology with human immunoglobulin λ chain,
No homology was shown with genes other than the immunoglobulin λ chain gene. Comparing the Cλ region of the T1-62 gene and the Cλ region of mouse and human, the nucleic acid level is 75.8% for mouse and 81.3% for human, the amino acid level is 69.5% for mouse and 77.1% for human. Was.

From the above results, the T1-62 gene is undoubtedly a gene belonging to the cat λ chain, and is a gene that enables the production of a mouse-cat chimeric antibody.

[Brief description of the drawings]

FIG. 1 shows the results of EIA using an anti-feline antibody confirmed that the IgG produced by the feline × mouse hybridoma prepared in the present invention is a feline-type monoclonal antibody. FIG. 2 shows the results of Western blotting using an anti-cat antibody confirmed that the IgG produced by the cat × mouse hybridoma prepared in the present invention is a cat-type monoclonal antibody. FIG. 3 shows the DNA fragment encoding the feline immunoglobulin λ chain constant region (T1-6
The restriction map of 2) and the region (→) subjected to nucleotide sequence analysis are shown. FIG. 4 shows a DNA fragment encoding the feline immunoglobulin λ chain constant region cloned in the present invention (T1-6
FIG. 2 is a schematic diagram of Southern hybridization between 2) and cat liver cell chromosomal DNA (EcoRI digestion). FIG. 5 shows a DNA fragment encoding the feline immunoglobulin λ chain constant region (T1-6) cloned in the present invention.
FIG. 2 is a schematic diagram of Northern hybridization between 2) and poly-A + RNA (lane 1) or feline spleen poly-A + RNA (lane 2) of FM-T1 cells. FIG. 6 shows the DNA base sequence encoding the feline immunoglobulin λ chain constant region present in the DNA fragment (T1-62) cloned in the present invention. FIG. 7 shows the entire amino acid sequence of the feline immunoglobulin λ chain constant region encoded in the DNA fragment (T1-62) cloned in the present invention.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI C12P 21/08 C12N 15/00 C (C12N 15/09 ZNA C12R 1:91) (58) Fields surveyed (Int.Cl. ⁶ , DB name) C12N 15/00-15/90 BIOSIS (DIALOG) WPI (DIALOG) EPAT (QUESTEL)

Claims (6)

(57) [Claims] 1. A feline x mouse heterohybridoma that produces feline immunoglobulin. 2. The feline immunoglobulin L chain class is λ.
The hybridoma according to the above (1), wherein 3. The hybridoma according to (2), wherein the λ chain constant region polypeptide is a polypeptide having the following amino acid sequence (a) or (b). (A) a polypeptide having the following amino acid sequence: (B) a polypeptide comprising an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence (a), and having a function of a feline immunoglobulin λ chain constant region. 4. Synthesis of cDNA from feline immunoglobulin-producing feline × mouse heterohybridoma poly A + RNA, integration into an expression vector, expression in host cells, and screening of transformants using an anti-feline immunoglobulin antibody A method for preparing a feline immunoglobulin gene, comprising: 5. A gene fragment containing a DNA sequence encoding the following polypeptide (a) or (b). (A) A constant region polypeptide of a feline immunoglobulin λ chain having the following amino acid sequence: (B) a polypeptide comprising an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence (a), and having a function of a feline immunoglobulin λ chain constant region. 6. A gene fragment comprising the following DNA sequence (a) or (b): (A) DNA having the following nucleotide sequence. (B) D complementary to the DNA consisting of the nucleotide sequence of (a)
A DNA that hybridizes with NA under stringent conditions and encodes a polypeptide having the function of a feline immunoglobulin λ chain constant region.