JP2811095B2 - Gene fragment encoding the constant region of feline immunoglobulin kappa chain and mouse x feline chimeric antibody - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a novel feline monoclonal antibody which can be expected for diagnosis, treatment and prevention of feline diseases, particularly infectious diseases. More specifically, the present invention relates to a gene fragment encoding a constant region of a feline immunoglobulin κ chain constituting a feline monoclonal antibody, and a mouse x feline chimeric antibody using the same.

Background of the Invention Cats have long been loved by humans as pets, but in recent years, cats and cats have been referred to as "companion species" in Europe and the United States. It is getting. On the other hand, medicine,
The contribution of laboratory animals from pharmacy, animal husbandry, veterinary medicine to psychology has been great, but in recent years, minimal desease cats have been used in drug effect tests and safety tests.
Under such names, the degree of contribution has further increased. In each case, of course, more and more knowledge about these cat diseases, especially epidemics, is needed.
It is required that methods for diagnosis, treatment and prevention be established.

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 feline serum materials, and this therapy has become unusable even if one wishes to use it. 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 here is not a conventional mouse monoclonal antibody because it eliminates 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 × (cat × mouse) hybridoma using a mouse heterohybridoma as a parent strain, (5) a chimeric monoclonal antibody (the variable (V) region to which the antigen binds 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 successful cases of the method have been reported.

Here, (1) that the fusion efficiency is low or that there is no suitable myeloma parent strain, (2) that there is no suitable virus corresponding to the EB virus in humans and that there is no suitable chemical agent, (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.

Object of the Invention Under these circumstances, the present inventors have conducted repeated studies to isolate the gene encoding the amino acid sequence of the constant region of feline immunoglobulin, and have succeeded in isolating this gene. did. That is, the present invention provides a gene encoding the constant region of a feline immunoglobulin κ chain, which has not been reported at all, and thereby enables the production of a feline chimeric antibody. The feline chimeric antibody produced by using the gene encoding the feline immunoglobulin κ chain of the present invention can be applied to diagnostics, therapeutics and prophylactics that have no side effects on feline diseases, especially infectious diseases. Things.

Configuration and Effect of the Invention As the immunoglobulin kappa chain, human and mouse [PAHieter et al., Cell, 22, p197 (1980); H. Sakano
Et al., Nature 280, p288-294 (1979)], and furthermore, in the kappa chains of other animal species, rabbits [L. Emorine et al.
Roc. Natl. Acad. Sci. USA, 80, p5709-5713 (1983)]. However, there has been no report that any analysis has been performed on the cat immunoglobulin κ chain gene which is the subject of the present invention.

On the other hand, it has been found that the type of feline immunoglobulin L chain is mainly a λ chain [L. Hood et al., Cold Sprin
g Harbor Symp. Quant. Biol. 32, p133-146 (1967)].
Therefore, it is thought that there are very few lymphocytes expressing the κ chain, and the messenger RNA of antibody-producing cells is converted to cDNA.
It was considered difficult to obtain the target gene by the cloning method. However, when a mouse x feline chimeric antibody is produced by genetic recombination, since a mouse κ chain variable region gene is usually used as the L chain variable region gene, the κ chain constant region of the feline immunoglobulin L chain is used. It is necessary to obtain the gene of

The present inventors have repeated studies to isolate a gene fragment encoding a cat immunoglobulin κ chain constant region of interest from various types of probes and various hybridization conditions from chromosomal DNA of cat liver cells. However,
We succeeded in obtaining a gene fragment that seems to encode the feline immunoglobulin constant region.

As a result of comparing the amino acid sequence predicted from the salt sound sequence of the gene fragment thus cloned with the sequence of the C region gene of an immunoglobulin of another animal species and performing gene analysis, the result obtained by the present invention was obtained. Gene fragment is κ
It was found to be a gene fragment encoding an immunoglobulin constant region belonging to the chain.

The gene fragment encoding the constant region of the feline immunoglobulin κ chain of the present invention is a DNA sequence encoding a peptide consisting of 109 amino acids, and the four amino acid sequences from the carboxy terminus of the constant region are -Cys-Gln -Arg-Glu is one of its features. That is, in the amino acid sequences of the human and mouse κ chain constant regions reported so far, the carboxy terminus is Cys (cysteine).
It is known that, as in the feline immunoglobulin κ chain constant region gene obtained in the present invention, the C-terminal side of Cys present on the carboxy-terminal side is followed by three more amino acids. The constant region consisting of an amino acid sequence is a rare example to be hated, and can be said to be a sequence characteristic of the cat immunoglobulin κ chain constant region of the present invention.

Further, the gene encoding the constant region of the feline immunoglobulin κ chain of the present invention has a gene fragment represented by a restriction enzyme cleavage map shown below.

A preferred example of the gene encoding the cat immunoglobulin κ chain constant region of the present invention is a gene fragment encoding the amino acid sequence described below.

There is no report on such an amino acid sequence or the acid-base sequence encoding it, and it is disclosed for the first time by the present invention.

The gene fragment encoding the feline immunoglobulin κ chain constant region of the present invention is not limited to the gene fragment encoding the above amino acid sequence, but also includes genes having different allotypes in which amino acids are partially substituted. . The presence of such genes having different allotypes has been reported in the gene analysis of immunoglobulin κ chains of humans, rabbits, etc., in which the presence of a gene encoding a peptide having one to several amino acids different from each other is reported. It is expected from this.

Examples of the specific nucleic acid base sequence of the gene encoding the C region of the feline immunoglobulin κ chain of the present invention include:
The base sequence shown in FIG. 4 is exemplified.

A mouse-cat chimeric antibody can be prepared by directly using the cat immunoglobulin kappa chain C region gene of the present invention, and using this as a lobe, an immunoglobulin kappa chain C region cDNA can be obtained from a cDNA library of cat antibody producing cells. It can also be cloned and used to produce chimeric antibodies. The method for producing the chimeric antibody is the method already shown for the mouse-human chimeric antibody [Watanabe et al., Cancer Research, 47,
p999-1005 (1987)]. That is, the chimeric antibody gene basically consists of the V region gene and the C region.
It is constructed by joining two types of gene fragments of a region gene. Furthermore, depending on the method of gene isolation, there are mainly combinations of the two bonds. That is, chromosomal DNA
And a combination of V and C region genes isolated from cDNA and V and C region genes isolated from cDNA.

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 a promoter and an enhancer necessary for expression. Preferably. However, the promoter, enhancer and the like do not need to be derived from mouse, and may be derived from cat, human or mouse. The promoter is preferably located 5 'upstream of the V region, and the enhancer is preferably located between the V region gene and the C region gene. However, 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 (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 the transformation is carried out using a plasmid having both the H chain and L chain chimeric antibody genes, only 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. If the cells transformed in this manner are cultured under the same appropriate conditions as ordinary hybridomas (for example, in RPMI 1640 medium containing 10% bovine fat serum), the cells can be converted into ordinary hybridomas. A mouse-cat chimeric antibody is secreted and produced in the same manner as the antibody produced by the hybridoma, and the chimeric antibody can be purified by a method similar to that for a normal antibody.

The gene fragment encoding the feline immunoglobulin provided by the present invention discloses a specific amino acid sequence or DNA sequence of the C region of the feline immunoglobulin κ chain. The resulting mouse-cat chimeric antibody can be a substantially effective diagnostic, prophylactic and therapeutic agent for cat diseases that has never been seen before.

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

Example (1) Conditions for cross-hybridization In order to clone a cat κ chain gene by a cross-hybridization method, conditions for cross-hybridization with a human κ chain were examined. The gene containing the human Cκ region used here was cloned from human cultured cell ARH77 strain [ATCC CRL 162],
It was distributed by Professor Takeshi Watanabe of Kyushu University, Institute for Biomedical Defense and Medicine [Kudo et al., Gene, 33, p181 (1985); Nishimura et al., Cancer Res., 47, p999 (1987)]. This human C
An EcoRI-EcoRI fragment containing the CK exon was cut out from the κ gene and used as a probe.

From cat liver cells, the method of N. Blin and DWStafford [Nu
c. Acids. Res., 3, p2303 (1976)], chromosomal DNA was isolated, and 10 μg of each chromosomal DNA was digested with restriction enzyme EcoRI (Takara Shuzo; hereinafter, unless otherwise noted, the reagents used in this example were Takara Shuzo or (Using Toyobo). The restriction enzyme-cleaved DNA was electrophoresed on a 0.7% agarose gel, transferred to a nitrocellulose membrane filter (S & S: BA85), and then subjected to Southern hybridization with a [ ³² P] -labeled DNA probe containing a human Cκ region. went. Southern hybridization conditions were 6xSSC
_{[0.09M Na 3 C 6 H 5} O 7 · 2H 2 O, 0.9M NaCl], 10mM EDTA [ Dojin Kagaku], 65 ° C. in a solution of 0.5% SDS [Bio-Rad]
Went overnight. The final washing condition of the filter is 0.1xSS
C, in a solution of 0.1% SDS at 45 ° C. for 15 minutes. The result of applying this filter to autoradiography is
A single binder of about 5.5 kb was formed as shown. The size of the molecule was calculated from the marker DNA obtained by cutting λ phage DNA with HindIII. Since this 5.5 kb DNA fragment seems to contain a cat κ chain gene, it was used as a target for cloning.

(2) Isolation of kappa chain gene After 100 µg of chromosomal DNA of cat liver was completely digested with EcoRI, the DNA fragment corresponding to 5.5 kb was subjected to sucrose density gradient centrifugation [sucrose 10 to 40% (wt / vol), 26000 rpm, 18 hours, 15
° C]. Next, this DNA fragment and the EcoRI arm of λgt11 vector DNA (Stratagene) were ligated by T4 DNA ligase, and packaging was performed in vitro using a Stratagene kit, and the κ chain gene library of cat liver cells was obtained. I got it. From this library, plaque hybridization [WDBenton, RDavis, Science, 196, p180 (197
7)], and the clone CEκ8 containing feline Cκ chain exon
a was selected. FIG. 3 shows a restriction enzyme cleavage map of this clone. The EcoRI insert of this clone was
Davis method [M. Thomas, RW Davis, J. Mol. Biol., 91, p31.
5 (1974)].
It was subcloned into the EcoRI site of the pUC18 vector.

(3) Southern and Northern Blot Analysis Using CEκ8a First, Southern blot analysis using this CEκ8a was performed. 10μg of chromosomal DNA from cat liver cells
The DNA is electrophoresed on a 0.7% agarose gel and transferred to a nylon membrane filter (Genescreen Plus, NEN Research Products).
Including feline Cκ chain region ^[32 P] standard CEκ8a probe Southern hybridization was performed. The method of Southern hybridization followed the protocol of the manual attached to GeneScreen Plus. As a result of comparing the detected band pattern with the cross-hybridization pattern using a human Cκ chain probe, a band was found at exactly the same position (about 5.5 kb). The molecular size was calculated from the marker DNA obtained by cutting λ phage DNA with HindIII. From these results, it was presumed that the feline Cκ region gene was one type of gene having no other subtype. This is the case for humans and mice [PAHieter et al., Cell, 22, p197 (198
9); EEMax et al., Proc. Natl. Acad. Sci. USA, 76, P3450 (19
79)].

Next, Northern blot analysis was performed. For the RNA used for hybridization, total RNA was extracted from cat spleen cells using the guanidium thiocyanate method [JMGhingwin et al., Biochemi
stry, 18, p5294 (1979)] and oligo d
Purified into poly A + RNA using a t-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] standard CEκ8a probe. The method of Northern hybridization followed the protocol of the manual attached to Zinscreen Plus. With this probe, a band was detected at about 1.3 kb (FIG. 2). This size is almost the same as the size of the immunoglobulin kappa chain gene known in mice and humans.

From these two results, this CEκ8a is a functional cat C
It was presumed to be an active gene that produces the κ region.

(4) Nucleotide base sequence and amino acid sequence of CEκ8a In order to examine the nucleobase sequence of the cat Cκ region, an approximately 2.0 kb DNA fragment (Xval-
AvaI fragment) was isolated and recloned into the pUC18 vector at the XvaI-AvaI site. This plasmid is prepared by a conventional method [for example, T. Maniatis “Molecular Cloning” Cold Spring Ha
rbor Lab. (1982)], and from this Xva I-Ava I fragment, Xba I-Dde I, Dde I-Hae
III, Hae III-Hinf I and Xba I-Rsa I small DNA fragments were prepared. After changing each of these small fragments to blunt ends using T4-DNA polymerase, the M13mp19 vector S
It was inserted into the ma I site using a treasure ligation kit. According to the method of Toyobo Instruction Manual, JM
101 competent cells were prepared, transformed with 1M3mp19 DNA into which the Cκ region gene was inserted, and single-stranded DNA was extracted and purified. Furthermore, the nucleobase sequencing of this single-stranded DNA
The measurement was performed using Takara M13 sequencing kit and Fuji Genser Gel System. The direction in which the nucleic acid base sequence was determined is shown in FIG. As a result of nucleobase sequencing,
A Cκ gene consisting of one exon was identified. 4th
The figure shows the results. Furthermore, when this nucleic acid base sequence was converted to amino acids, it was shown that this gene had an open reading frame and was not a pseudogene (FIG. 5).

Based on the CEκ8a nucleic acid base sequence, LASL and EM were analyzed using gene analysis software (Genetxy: software development company).
A homology search of the BL database showed high homology with human and mouse immunoglobulin kappa chains, and no homology with genes other than immunoglobulin kappa chain genes. When the homology between the Cκ region of the CEκ8a gene and the mouse and human Cκ regions is 73.3% for mouse and 73.0% for human at the acid-expanding level, 54.7% for mouse and 59.6% for human at the amino acid level there were.

From the above results, the CEκ8a gene was arguably a gene belonging to the cat κ chain, and was considered to be a gene capable of producing a mouse-cat chimeric antibody.

Incidentally, the present inventors have proposed such a cat immunoglobulin κ
Escherichia coli having a vector in which the gene encoding the constant region of the chain has been incorporated is used to prepare Escherichia coli FCK-CEK8.
It has been deposited as A, Microorganisms Kenkyu No. 10985.

(5) Isolation of mouse immunoglobulin κ chain variable (Vκ) region gene Chromosomal DNA was isolated from anti-CPV antibody-producing hybridoma JP2 (γ1, κ), and 100 μg of the chromosomal DNA was purified with Hind III.
(It is known that a monoclonal antibody that neutralizes canine parvovirus can also neutralize nuco parvovirus). Next, this DNA fragment and λL47 vector DNA
(Stratagene) was ligated with T4 DNA ligase and
A chromosomal DNA library of P2 cells was obtained. From this library, the plaque hybridization method [WDBe
A clone Jp2gL411 containing a Vκ region gene of an anti-CPV antibody was selected using a mouse Jκ probe according to nton, RW Davis, Science, 196 (see p180 (1977)), and FIG. From this gene fragment, Vκ
A BamH I-Hind III fragment containing an exon part was prepared,
The following cat-mouse chimeric antibody L chain gene material was used.

(6) Mouse-cat chimeric antibody L chain gene (pSV2-EPLC
Preparation of Cκ) The plasmid pCEκ8aXA obtained in (4) was digested with HindIII and EcoRI to prepare a 2 kb HindIII-EcoRI DNA fragment containing the feline immunoglobulin Cκ gene. on the other hand,
Rasmid pJP2gL411 containing the mouse immunoglobulin Vκ chain gene obtained in (5) was digested with BamHI and HindIII to obtain a 4.4 kb mouse immunoglobulin Vκ-Jκ region gene. These genes were digested with EcoR I and BamHI and pSV2-neo vector [PJSouthern et al., J. Mol. Appl.
Genet., 1, p327 (1982)] using the treasure ligation kit to obtain plasmid pSV2-PLCCκ. Next, a 1.0 kb EcoRI containing the enhancer region of human γ chain was used.
-EcoR I DNA fragment [THRabbits et al. Nature 306, p80
6 (1983)] using T4-DNA to change the ends to blunt ends using a polymerase, ligating a BamHI linker (manufactured by Takara) to both ends using a Takara ligation kit, and using BamHI at both ends.
A site-modified human gamma chain enhancer region gene was obtained. This gene was ligated to the plasmid pSV2-PLCCκ
The plasmid was inserted into the mHI site to prepare a plasmid pSV2-EPLCCκ. (Fig. 7)

[Brief description of the drawings]

FIG. 1 shows that the chromosomal DNA of cat liver cells was converted to the restriction enzyme EcoR I.
FIG. 3 is a schematic diagram showing the results of Southern hybridization performed with a [ ³² P] -labeled probe containing a human Cκ chain region. FIG. 2 shows poly A + RNA extracted from cat spleen cells and [
FIG. 2 is a schematic diagram of Northern hybridization with a [ ³² P] -labeled CEκ8a probe. FIG. 3 shows a DNA fragment encoding a feline immunoglobulin κ chain constant region cloned in the present invention (CEκ8
The restriction map of a) and the region (→) subjected to nucleotide sequence analysis are shown. FIG. 4 shows the DNA fragment CEκ8a cloned in the present invention.
1 shows the DNA base sequence encoding the feline immunoglobulin kappa chain constant region present in. FIG. 5 shows the DNA fragment C cloned in the present invention.
1 shows the entire amino acid sequence of the cat immunoglobulin κ chain constant region encoded in Eκ8a. FIG. 6 shows a restriction enzyme cleavage map of clone JP2gL411 containing the Vκ region gene of the anti-CPV antibody prepared in Example (5). FIG. 7 shows a construction diagram of the gene (pSV2-EPLCCκ) expressing the anti-CPV mouse × cat chimeric antibody L chain constructed in Example (6).

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ identification symbol FI (C12N 15/09 ZNA C12R 1:91) (58) Investigated field (Int.Cl. ⁶ , DB name) C12N 15/00- 15/90 BIOSIS (DIALOG) WPI (DIALOG) EPAT (QUESTEL)

Claims (5)

(57) [Claims] 1. A gene fragment containing a DNA sequence encoding the following polypeptide (a) or (b). (A) A feline immunoglobulin κ chain constant region polypeptide having the following amino acid sequence: (B) a polypeptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence (a), and having a function of a cat immunoglobulin κ chain constant region. 2. 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 a function of a cat immunoglobulin κ chain constant region. 3. The following (a) or (b) is located on the 3 'side of the gene fragment encoding the variable region of the mouse immunoglobulin L chain.
A recombinant DNA molecule encoding a mouse x feline chimeric antibody L chain, wherein a gene fragment encoding the constant region of the feline immunoglobulin κ chain is connected. (A) A feline immunoglobulin κ chain constant region polypeptide having the following amino acid sequence: (B) a polypeptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence (a), and having a function of a cat immunoglobulin κ chain constant region. 4. A gene fragment encoding a variable region of a mouse immunoglobulin L chain, and a gene fragment of the following (a) or (b) encoding a constant region of a feline immunoglobulin κ chain is connected to the 3 'side of the gene fragment. A recombinant DNA molecule encoding a mouse x feline chimeric antibody L chain. (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 a function of a cat immunoglobulin κ chain constant region. 5. The following (a) or (b) is located on the 3 'side of the gene fragment encoding the variable region of the mouse immunoglobulin L chain.
A cell transformed with a cell expression vector incorporating a recombinant DNA molecule encoding a mouse x feline chimeric antibody L chain, wherein a gene fragment encoding the constant region of a feline immunoglobulin κ chain is connected. The expressed mouse x feline chimeric antibody L chain peptide. (A) A feline immunoglobulin κ chain constant region polypeptide having the following amino acid sequence: (B) a polypeptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence (a), and having a function of a cat immunoglobulin κ chain constant region.