JP2811102B2 - Gene fragment encoding the constant region of feline immunoglobulin gamma 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 γ chain constituting a feline monoclonal antibody, and a mouse x feline chimeric antibody using the same.

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, medicine,
The contribution of laboratory animals ranging from pharmacy, animal husbandry, veterinary medicine to psychology has been great, but in recent years, minimal desease cats have been used for drug effect detection and safety testing.
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 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 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 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 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 a human monoclonal antibody production example (for example, safety 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 the antibody molecule capable of binding to the antibody 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 a constant region of a feline immunoglobulin γ chain, which has not been reported at all, and thereby enables the production of a feline chimeric antibody. The feline chimeric antibody produced using the gene encoding the feline immunoglobulin γ 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.

Structure and Effect of the Invention As the immunoglobulin gamma chain, human and mouse [for example, A. Shimizu et al., Cell, 29, p121 (1982); N. Taka
hashi et al., Cell, 29, p671 (1982)], and furthermore, in the γ-chain of other animal species, a rabbit [CLMartens
USA, 79, p6018 (1982)], cattle [KLKnight et al., J. Immunol, 140, p3654 (1988)], etc., which are related to feline immunoglobulin γ chains. There are no reports yet.

The present inventors have succeeded in obtaining, from chromosomal DNA of cat liver cells, a gene fragment which seems to encode a cat immunoglobulin constant region using a human immunoglobulin gene as a probe. Further, using this as a probe, immunoglobulin γ
The chain C region cDNA was cloned. An amino acid sequence predicted from the nucleotide sequence of the cloned gene fragment,
The gene fragment obtained by the present invention was compared with the sequence of the immunoglobulin C region gene of another animal species, and the gene fragment obtained by the present invention was a gene fragment encoding an immunoglobulin constant region belonging to the γ chain. There was found.

The obtained feline immunoglobulin gamma chain was analyzed for the nucleotide sequence of the DNA fragment encoding the constant region, and the amino acid sequence of the constant region was found. This was used to report the immunoglobulin gamma of human, mouse, rabbit, etc. When compared with the amino acid sequence of the chain constant region, as an amino acid sequence specific to the feline immunoglobulin γ chain constant region, the amino acid sequence near the first cysteine from the N-terminal side of the CH1 domain of the γ chain constant region is as follows: The amino acid sequence of (A) was found.

The present inventors have compared the amino acid sequence of the feline immunoglobulin gamma-chain constant region analyzed according to the present invention and the amino acid sequence of the immunoglobulin gamma-chain constant region of various animals that have been analyzed so far. It has been found that the -Ser-Cys-Gly-Thr- region existing near the cysteine has a different amino acid sequence depending on the species of cat, mouse, human and the like. At the same time, this region was also found to be extremely well conserved between subclasses, for example, as the amino acid sequence of the human γ chain constant region, and the above-described sequence revealed by the present invention is a cat immunoglobulin γ The sequence was assumed to be unique to the chain constant region.

In addition, the vicinity of cysteine on the C-terminal side of the CH1 domain (the following amino acid sequence (B)), the N-terminal side of the sugar chain binding site (Asn) of the CH2 domain (the following amino acid sequence (C)),
In the vicinity of cysteine on the N-terminal side of the H3 domain (the following amino acid sequence (D)), a similar sequence unique to the feline immunoglobulin γ chain constant region was also found.

The amino acid sequence of this region cloned in the present invention is as follows, and this amino acid sequence is the same as that of the unique amino acid sequences (B), (C) and (D) present in the feline immunoglobulin γ chain constant region. A preferred example is given.

In the gene fragment encoding the feline immunoglobulin γ chain constant region of the present invention, the above (A), (B), (C)
Alternatively, a DNA sequence encoding the amino acid sequence of (D) is included in a part thereof. Such γ above
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 γ chain, and was first revealed by the present invention.

Feline immunoglobulin γ containing these amino acid sequences
One preferred example of chain constant is the amino acid sequence shown below.

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

Examples of the specific nucleic acid base sequence of the gene encoding the C region of the feline immunoglobulin γ chain of the present invention include:
The base sequence shown in FIG. 6 is exemplified.

In addition, as a result of Southern hybridization with the feline chromosomal DNA using the gamma chain gene of the present invention, in addition to the gamma chain of the present invention, there are several C region genes of other subclasses belonging to the same feline gamma chain. It was shown that. Examples of human and mouse [for example, A. shimizu et al., Cell, 2
9, p121 (1982); N. Takahashi et al., Cell, 29, p671 (198
2)], it seems that there are several subclasses in the feline gamma chain. It is also known that cat gamma chain has at least two subclasses serologically [J.
Michael Kehoe et al., J. Immunology Vol. 109, No. 3, p511-5.
16 (1972)], the genes of the present invention appear to encode either of these two subclasses. Therefore, it is possible to clone the remaining subclass C region genes using the gene of the present invention.

It should be noted that, even within the same subclass, the presence of genes having different allotypes in which one to several amino acids have been substituted exists in human and rabbit immunoglobulin γ.
It is also expected from the results of gene analysis of the chains. The gene fragment encoding the feline immunoglobulin γ chain constant region of the present invention is not limited to the gene fragment encoding the above-described amino acid sequence, and also includes such genes having different allotypes in which amino acids are partially substituted. I do.

The method for producing the chimeric antibody was the method already described for the mouse-human chimeric antibody [Watanabe et al., Cancer Research, 47, p999.
-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. A 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. The method for preparing and expressing the chimeric antibody L chain gene has been previously filed by the present applicant (Japanese Patent Application No. 1-255424) and is described in detail therein. To obtain a mouse-cat chimeric antibody,
It is necessary to transform host animal cells with the thus prepared plasmid containing the chimeric antibody gene. Examples of the host animal cells include immortalized mouse and other animal cells, preferably a B lymphocyte cell line [eg, P3X63Ag8 · 653 (ATCC CRL 1580), P3X62Ag8U · 1 (A
TCC CRL 1597), P3 / NS1 / 1 Ag4-1 (ATCC CRL18), Sp
2 / 0-Ag / 12 (ATCC CRL 1581) and other plasmacytomas, hybridomas]. Methods for transforming cells with DNA include methods such as DEAE-dextran method, calcium phosphate coprecipitation method, protoprost fusion method, and electroporation method [for example, see BD Hames et al.
nd Translation "IRL Press (1984)], and any method may be used. When 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 light and light chains, two types of markers are required, in which case a double transformation method is used in which transformation is performed with one plasmid and then with another plasmid. If the cells thus transformed are cultured under the same suitable conditions as a normal hybridoma (for example, in a PRMI1640 medium containing 10% fetal bovine serum), the normal hybridoma production from the cells is obtained. Mice as well as antibodies
A feline chimeric antibody is secreted and produced. This chimeric antibody can be purified by the same method as for a normal antibody.

The feline immunoglobulin-encoding gene fragment provided by the present invention starts a specific amino acid sequence or DNA sequence of the C region of the feline immunoglobulin γ chain, and the gene is used as described above. 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 γ-chain gene by a cross-hybridization method, conditions for cross-hybridization with a human γ-chain were examined. The gene containing the human Cγ region used here was cloned from a human cultured cell strain ARH77 [ATCC CRL 1621] and distributed by Professor Takeshi Watanabe of Kyushu University, Institute of Bioregulation Medicine. [Kudo et al., Gene, 33, p181 (1985);
Nishimura et al., Cancer Res., 47, p999 (1987)]. As a result of homology analysis between a mouse, a human, and a rabbit γ chain using a computer, it was shown that the region containing the CH2-CH3 exon has a very high homology, particularly. Therefore, from this human Cγ gene, BamHI containing CH2-CH3 exon
The -HpaI fragment was excised and used as a probe.

From cat liver cells, the method of N. Blin and DWStafford [Nu
c. Acids. Res., 3, p2303 (1976)], and chromosomal DNA was isolated by 10 μg of polysomal DNA using the restriction enzyme BamHI (manufactured by Takara Shuzo; hereinafter, unless otherwise specified, the reagents used in this example were (Using Toyobo). The restriction enzyme-digested DNA is electrophoresed on a 0.7% agarose gel, and the nitrocellulose membrane filter (S & S: BA
85) After transcription, [ ³² P] -labeled DNA containing human Cγ region
Southern hybridization was performed with the probe. Southern hybridization conditions were 6xSSC [0.09MN
_{a 3 C 6 H 5 O 7} · 2H 2 O, 0.9M NaCl], 10mM EDTA [ Dojin Kagaku], it was carried out 65 ° C. overnight in a solution of 0.5% SDS [Bio-Rad. The final washing condition of the filter is 0.1xSSC,
Performed at 45 ° C. for 15 minutes in a solution of 0.1% SDS. As a result of subjecting this filter to autoradiography, two bands of about 5.8 kb and 5.5 kb were formed as shown in FIG.
The size of the molecule was calculated from the marker DNA obtained by cutting λ phage DNA with HindIII. These two bands were used as targets for cloning.

(2) Isolation of γ chain gene After 100 µg of chromosomal DNA of cat liver was completely digested with BamHI, the DNA fragments corresponding to 5.8 and 5.5 kb were subjected to sucrose density gradient centrifugation [sucrose 10 to 40% (wt / vol), 26000 rpm, 18 hours, 15 ° C.]. Next, this DNA fragment and Charomi
d 9-42 vector DNA (Nippon Gene) was ligated with BamHI digested DNA using T4 DNA ligase, and in vitro packaging was carried out using a Straterdin kit, to infect LE392 E. coli (Stratadin). Thus, a gamma chain gene library of cat liver cells was obtained. From this library, colony hybridization was performed using the human Cγ probe under the same conditions as the above-mentioned cross-hybridization (latest: Handbook for Genetic Engineering Experiments, p426).
25γ7c was selected. The size of this clone is about 5.8kb
And one of the two bands previously shown in Southern blot analysis. FIG. 2 shows a map of the restriction enzyme cleavage points. This plasmid clone is prepared by a conventional method [for example, T. Maniat
is “Molecular Cloning” Cold Spring Harbor Lab. (198
2)] and the BamHI insert was isolated. After cutting with Sac I for easier use,
A DNA fragment (SacI fragment) of about 1.3 kb which seems to contain the Cγ exon was isolated and recloned into the SacI site of the pUC18 vector.

(3) Southern and Northern Blot Analysis Using CB25γ7c First, Southern blot analysis using this CB25γ7c was performed. 10 μg of chromosomal DNA from cat liver cells
After digestion with I, this DNA was electrophoresed on a 0.7% agarose gel, transferred to a nylon membrane filter (GeneScreen Plus, NEN Research Products), and [ ³² P] -labeled CB25γ7c containing a feline Cγ chain region. Southern hybridization was performed with the probe. The method of Southern hybridization followed the protocol of the manual attached to GeneScreen Plus.
As a result of comparing the pattern of the detected band with the pattern of the cross-hybridization using the human Cγ chain probe, the band was found at exactly the same position (about 5.8 and 5.5 kb) (same as FIG. 1). result). The molecular size was calculated from the marker DNA obtained by cutting λ phage DNA with HindIII. This result indicated that there were several Cγ region genes of other subclasses belonging to the same cat γ chain other than CB25γ7c. As described above, it has been reported that the cat γ chain also has two subclasses, and this CB25γ7c gene seemed to encode any of these two subclasses.

Next, Northern blot analysis was performed. The RNA used for hybridization was cat spleen cells and cat x mouse heterohybridoma FM-T1 cells established by the present inventors (Microtechnical Laboratories No. 2947 (Original Deposits: Microtechnical Laboratories No. 10077)
No.) and deposited by the guanidium thiocyanate method [JMGhingwin et al., Bioc.
hemistry, 18, p5294 (1979)], and further purified to poly A + RNA using an oligo dT column (Pharmacia). 2 μg of this RNA was electrophoresed for 3 μg.
After spreading on a 0.75% agarose gel containing% formaldehyde and transferring to a nylon membrane filter (Genescreen Plus), Northern hybridization was performed with a [ ³² P] -labeled CB25γ7c probe. The method of Northern hybridization followed the protocol of the manual attached to GeneScreen Plus. With this probe, a band was detected at about 1.8 kb in the RNAs of both cells (FIG. 3). This size is almost the same as the size of the immunoglobulin gamma chain gene known in mice and humans.

From these two results, it was determined that CB25γ7c was an active gene containing a functional cat Cγ region. still,
Escherichia coli having this CB25γ7c-integrated plasmid was used as Escherichia coli CCG-CB25G [Microtechnical Lab.
No.] has been deposited by the applicant.

(4) Isolation of cDNA clones The cat Cγ region was prepared from exon /
It is expected that it has an intron structure. Cat C
To know the nucleobase sequence of the exon part of the γ region,
Feline immunoglobulin gamma-chain mRNA after splicing
It is necessary to clone the cDNA made from. As described above, cat x mouse heterohybridoma
FM-T1 cells hybridize with CB25γ7c
It synthesizes mRNA. Therefore, a cDNA library was constructed from FM-T1 cells, and the cat γ chain cDNA was probed using CB25γ7c as a probe.
Was attempted.

Using a cDNA synthesis system plus (Amersham) from poly A + RNA extracted and purified from FM-T1 cells,
cDNA was synthesized. EcoRI site of the synthesized cDNA
After methylation using methylase, an EcoRI linker was added using T4 DNA ligase. Further, this cDNA was completely digested with a restriction enzyme EcoR I, and an EcoR I linker was added using a biogel A-50 m column (Bio-Rad).
The cDNA was purified. Next, this cDNA and λgt11 vector DNA
(Stratagene) EcoRI arm was ligated with T4 DNA ligase, in vitro packaging was carried out using a Stratagene kit, and the cDNA of FM-T1 cells was
I got the library.

From this library, the plaque hybridization method of Benton and Davis [WDBenton, RWDavis, Scie
nce, 196, p180 (1977)], a clone T1CB containing a feline immunoglobulin Cγ chain was selected using a CB25γ7c probe. The size of this clone is 1.3 kb and its restriction enzyme cleavage map is shown in FIG. The EcoRI insert of this clone was cloned by the method of Thomas and Davis [M. Thomas, RDavis, J. et al.
Mol. Biol., 91, p315 (1974)], and recloned into the EcoRI site of pUC18.

(5) Nucleotide base sequence and amino acid sequence of T1CB In order to examine the nucleobase sequence of cat Cγ region, Pst I-Pst I, Pst I-Rsa I, Pst I-Hind
III, SacI-SmaI, EcoRI-SacI and HindIII-EcoRI were prepared. After changing each of these small fragments to blunt ends using T4-DNA polymerase, M13m
It was inserted into the Sma I site of the p19 vector using a treasure ligation kit. According to the method of the Toyobo Instruction Manual, JM101 competent cells were prepared, transformed with M13mp19 DNA into which the Cγ region 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 the Takara M13 sequencing kit and Fuji
Performed using the Genser 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 γ gene consisting of VDJC was confirmed. FIG. 5 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 (see FIG. 6).

Based on this T1CB nucleic acid base sequence, gene analysis software (Ge
Using netyx (manufactured by Software Development Co., Ltd.), the homology search of the LASL and EMBL databases showed high homology with human and mouse immunoglobulin γ chains and no homology with genes other than immunoglobulin γ chain genes. Did not. When the homology between the Cγ region of the T1CB gene and the mouse and human Cγ regions is 70.2% for mouse γ1 and 76.8% for human G1 at the nucleic acid level, 61.0% for mouse γ1 and 61.0% for human G1 at the amino acid level 69.7%.

From the above results, the CB25γ7c and T1CB genes were arguably genes belonging to the feline γ chain, and seemed to be genes that would enable the production of a mouse-feline chimeric antibody.

(5) Isolation of mouse immunoglobulin γ-chain variable (VH) region gene Chromosomal DNA was isolated from anti-CPV antibody-producing hybridoma JP2 (γ1, κ), and 100 μg of chromosomal DNA was subjected to restriction enzyme Hind III.
(It is known that a monoclonal antibody that neutralizes canine parvovirus can also neutralize feline 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
nton, RWDavis, Science, 196, p180 (1977)], and using a mouse JH probe, a clone JP2gH211 containing the VH region gene of the anti-CPV antibody was selected. FIG. 7 is a map of the restriction enzyme cleavage points. An EcoR I-Sac I fragment containing the VH exon portion was prepared from this gene fragment, and used as a material for the following mouse mouse chimeric antibody H chain gene. In addition, Escherichia coli having a plasmid in which the V region gene of the mouse immunoglobulin H chain having anti-canine parvovirus activity was incorporated was used as Escheichia coli MVH-JP2 [Microtechnical Lab.
No. 11167] has been deposited by the applicant. In addition, anti-canine parvovirus mouse immunoglobulin L cloned from the same JP2 cells using a mouse Jκ probe.
The V region gene of the chain has been deposited by the Applicant as Escherichia Coli MVK-JP2 [No.

(6) Mouse-cat chimeric antibody H chain gene (pSV2-PHCC
Preparation of γ) The plasmid pCB25γ7c obtained in (4) was digested with BamHI to obtain a 5.8 kb B fragment containing the feline immunoglobulin Cγ chain gene.
The amHI fragment was prepared. This gene was cut with BamHI
The SV2-gpt vector was ligated together using a treasure ligation kit to obtain a plasmid pSV2-CCγ. Next, (5)
Both ends of the EcoR I-Sac I fragment of pJP2gH211 obtained in T4-
Change to blunt ends using DNA polymerase, and use the treasure ligation kit to obtain Hp of plasmid pSV2-CCγ as described above.
The plasmid was inserted into the aI site to prepare a plasmid pSV2-PHCCγ. (See Fig. 8)

[Brief description of the drawings]

FIG. 1 shows that the chromosomal DNA of cat liver cells was converted to restriction enzyme BamHI
FIG. 3 is a schematic diagram showing the results of Southern hybridization performed with a [ ³² P] -labeled probe containing a human Cγ chain region. FIG. 2 shows a chromosomal DNA fragment encoding a feline immunoglobulin γ chain constant region (C
1 shows a restriction map of B25γ7c). FIG. 3 is a schematic diagram of Northern hybridization between poly A + RNA extracted from cat spleen cells (lane 1) and a cat antibody-producing heterohybridoma FM-T1 (lane 2) and a [ ³² P] -labeled CB25γ7c probe. FIG. 4 shows the cDNA fragment T1CB1a cloned in the present invention.
1 shows a restriction enzyme cleavage map and a region (→) subjected to nucleotide sequence analysis. FIG. 5 shows the cDNA fragment T1CB1a cloned in the present invention.
2 shows the DNA base sequence encoding the feline immunoglobulin γ chain constant region present in. FIG. 6 shows the cDNA fragment T1CB1a cloned in the present invention.
1 shows the entire amino acid sequence of the feline immunoglobulin γ chain constant region encoded therein. The underlined amino acid sequences indicate the regions of the amino acid sequences (A) to (D) unique to the feline immunoglobulin gamma chain constant region described in the detailed description of the invention. FIG. 7 shows a restriction enzyme cleavage map of clone JP2gH211 containing the VH region gene of the anti-CPV antibody prepared in Example (5). FIG. 8 shows a construction diagram of the gene (pSV2-PHCCγ) expressing the anti-CPV mouse × cat chimeric antibody H 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 (4)

(57) [Claims] 1. A gene fragment containing a DNA sequence encoding the following polypeptide (a) or (b). (A) Feline immunoglobulin γ-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 which has a function of a cat immunoglobulin γ chain constant region. 2. The feline immunoglobulin γ according to the above (1).
Mouse x feline chimeric antibody H, wherein a gene fragment encoding the constant region of the chain is connected to the 3 'side of the gene fragment encoding the variable region of the mouse immunoglobulin H chain.
A recombinant DNA molecule that encodes a strand. 3. Incorporating the recombinant DNA molecule according to the above (2) into an expression vector, culturing cells transformed with the recombinant vector, and recovering the expressed mouse x feline chimeric antibody H chain. A method for producing a mouse x feline chimeric antibody H chain, characterized by the following: 4. A mouse x feline chimeric antibody heavy chain peptide obtained by the method for producing a mouse x feline chimeric antibody heavy chain according to item (3).