JP2001037481A - Gene conding for variable region of anti-chrysanthemic acid monoclonal antibody - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new gene which codes for the heavy-chain variable region of an anti-chrysanthemic acid monoclonal antibody having a specific amino acid sequence, and can be used for producing the antibody which allows rapid and accurate detection of chrysanthemic acid having the basic structure of pyrethoroid insecticides. SOLUTION: This is a new gene which codes for the heavy-chain variable region of an anti-chrysanthemic acid monoclonal antibody, wherein the region has an amino acid sequence selected from the group consisting of the amino acid sequence having amino acid residues from no. 1 to no. 112 shown by formula Ι, the amino acid sequence having amino acid residues from no. 1 to no. 112 shown by formula, Π, and amino acid sequences obtained by deleting, substituting, or adding gone or more amino acid residue(s) from, in, or to those amino acid sequences, allows interacting with chrysanthemic acid by forming an antibody-binding site, and can be used for producing the antibody which allows repaid and accurate detection of chrysanthemic acid having the basic structure of pyrethgroid insecticides. This gene is obtained by cloning cDNA of a chrysanthemic acid monoclonal antibody-producting hybridoma.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a gene encoding the variable region of an anti-chrysanthemic acid monoclonal antibody, that is, an antigen-binding site. The present invention further provides a vector containing the gene, a host cell containing the expression vector, and a method for producing the variable region of the anti-Chrysmic acid monoclonal antibody by culturing the host cell.

[0002]

2. Description of the Related Art Chrysanthemic acid has the following formula (1):

[0003]

Embedded image

[0004] It is a compound having a structure represented by the following formula and constituting the basic skeleton of pyrethroid insecticides such as pyrethrin and allethrin. In particular, esters of dichlorovinyl chrysanthemic acid and pyrethroid alcohol are used, for example, as household insecticides because of their high insecticidal activity and relatively low toxicity to warm-blooded animals.

[0005] In recent years, there has been great social interest in pesticide residues in the environment such as soil, water, and the atmosphere, and post-harvest pesticides in imported agricultural products, which have recently increased in particular. In order to ensure the safety of the environment and food, it is necessary to measure the amount of chrysanthemic acid contained therein quickly and accurately.

[0006] The immunological measurement method is a method for detecting an antigen based on an antigen-antibody reaction in which the antibody specifically recognizes the antigen. Due to its excellent accuracy, simplicity, rapidity, and economic efficiency, it has recently been used. It's getting attention. In immunoassays, a wide variety of labels have been applied as detection methods, for example enzymes, radiotracers, chemiluminescent or fluorescent substances, metal atoms, sols, latexes and bacteriophages.

[0007] Among immunoassays, enzyme immunoassay (EIA) using an enzyme has been widely used as a particularly excellent one. An excellent review of enzyme immunoassays can be found in Tijssen P, "Practic.
e and theoryof enzyme imm
unoassays "in Laboratory
technologies in biochemistr
y and molecular biology,
Elsevier Amsterdam New Yo
rk, Oxford ISBN 0-7204-42
00-1 (1990).

[0008] Chrysanthemic acid is described in, for example, JP-A-9-37.
No. 783, JP-A-9-37784, Pest
ic. Sci. 1998, 54, 189-194 and "Reactivity and Structure of Anti-Chrysanthemic Acid Monoclonal Antibody"
(The 2nd meeting of the Society for Immunochemical Assays (1997), the academic meeting (June 7, 1997), the abstract of the lecture, page 17)
(Both are incorporated by reference herein), an antibody is prepared against chrysanthenic acid and a pyrethroid-based compound containing chrysanthemic acid as an antigen using a polymer compound bound to the terminal carboxyl group of chrysanthemic acid. It is described that it was done. Specifically, these documents disclose KCA190 and KCA226 as monoclonal antibodies against chrysanthemic acid. The monoclonal antibody KCA226 can be obtained, for example, from a hybridoma deposited at the National Institute of Bioscience and Biotechnology at the depository number FERM P-15051.

However, before the present invention, the amino acid sequence of the antibody against chrysanthemic acid has not been elucidated, and the gene encoding the antibody has not been obtained. Therefore, a region important for the binding of the antibody to the antigen is not clear, and the antibody or a fragment thereof capable of binding to the antigen (for example, _Fab fragment or the like) cannot be produced by genetic engineering.

[0010]

SUMMARY OF THE INVENTION An object of the present invention is to provide a gene encoding a heavy chain variable region of an anti-chrysanthemic acid monoclonal antibody.

Another object of the present invention is to provide a gene encoding the variable region of the light chain of the anti-chrysanthemic acid monoclonal antibody. Another object of the present invention is to provide a vector containing a gene encoding the light chain variable region of the anti-chrysanthemic acid monoclonal antibody, and a host cell transformed with the vector.

It is still another object of the present invention to provide a method for producing the variable region of the heavy and / or light chain of the anti-chrysanthemic acid monoclonal antibody by genetic engineering by culturing the host cell. And

[0013]

Means for Solving the Problems The present inventors have made intensive studies for the purpose of solving the above problems, and as a result, isolated a gene encoding an anti-chrysanthemic acid monoclonal antibody and arrived at the present invention. The details will be described below.

In general, an immunoglobulin constituting an antibody is composed of a heavy chain having a molecular weight of 50,000 to 70,000 (also called an H chain) and a light chain having a molecular weight of 20,000 to 30,000 (also called an L chain). The peptide chain structure of the basic structure of immunoglobulin is 2
One heavy chain and two light chains are linked by disulfide and non-covalent bonds to give a molecular weight of 150,000-19
It is ten thousand. The regions located at the N-terminus of both heavy and light chains are:
Even if a sample is from the same class (subclass) of the same animal, its amino acid sequence is not constant and is called a variable region. On the other hand, the amino acid sequence at the C-terminal side is substantially constant for each class and is called a constant region (or “Fc region”).

The antigen-binding site of an antibody is composed of heavy and light chain variable regions, and the specificity of binding depends on the amino acid sequence of this site. The part of the variable region that has a remarkable difference is called a hypervariable region. The present invention focuses on the variable regions of the heavy and light chains of the anti-chrysanthemic acid monoclonal antibody, and provides genes thereof.

Specifically, the present invention provides a gene encoding the variable region of the heavy chain of an anti-chrysanthemic acid monoclonal antibody. In the present invention, the heavy chain variable region of the anti-chrysanthemic acid monoclonal antibody comprises the following i) -iii): i) an amino acid sequence having amino acid residues 1-112 of SEQ ID NO: 1; ii) an amino acid residue of SEQ ID NO: 3 An amino acid sequence selected from the group consisting of: an amino acid sequence having 1-112; and iii) an amino acid sequence in which one or more amino acid residues have been deleted, substituted or added in the amino acid sequence of i) or ii). Have
In addition, it is possible to form an antigen-binding site and interact with chrysanthemic acid.

Preferably, the heavy chain variable region of the anti-chrysanthemic acid monoclonal antibody encoded by the gene of the present invention comprises:
I) or ii) below: i) an amino acid sequence having amino acid residues 1-112 of SEQ ID NO: 1; or ii) an amino acid sequence having amino acid residues 1-112 of SEQ ID NO: 3.

More preferably, the gene of the present invention has the nucleotide sequence 1-336 of SEQ ID NO: 1 or the nucleotide sequence 1-336 of SEQ ID NO: 3. The present invention also provides a gene encoding the light chain variable region of the anti-chrysanthemic acid monoclonal antibody. In the present invention, the light chain variable region of the anti-chrysanthemic acid monoclonal antibody comprises the following i) -iii): i) an amino acid sequence having amino acid residues 1-99 of SEQ ID NO: 2; ii) an amino acid residue of SEQ ID NO: 4 An amino acid sequence having a group 1-99; and iii) an amino acid sequence in which one or more amino acid residues are deleted, substituted or added in the amino acid sequence of the above i) or ii); Has,
In addition, it is possible to form an antigen-binding site and interact with chrysanthemic acid.

Preferably, the light chain variable region of the anti-chrysanthemic acid monoclonal antibody encoded by the gene of the present invention comprises:
I) or ii) below: i) an amino acid sequence having amino acid residues 1 to 99 of SEQ ID NO: 1; or ii) an amino acid sequence having amino acid residues 1 to 99 of SEQ ID NO: 3.

More preferably, the gene of the present invention has the nucleotide sequence 1-297 of SEQ ID NO: 2 or the nucleotide sequence 1-297 of SEQ ID NO: 4. The above-described gene of the present invention can be obtained by a known method based on the disclosure of the present specification. For example, but not by way of limitation, the genes of the present invention can be obtained from hybridomas producing anti-chrysanthemic acid monoclonal antibodies. First, mRNA is prepared from these hybridomas by a known method, and based on the mRNA, a single-stranded cDNA is synthesized by a reverse transcriptase, and then the heavy chain or the light chain of the anti-chrysmic acid monoclonal antibody disclosed herein is synthesized. The gene of the present invention can be selectively obtained by using a PCR method, a hybridization method, or the like based on the amino acid sequence or base sequence of the variable region. Such methods are well known, and those skilled in the art can easily isolate the gene of the present invention based on the disclosure of the present specification.

Hybridomas producing an anti-chrysanthemic acid monoclonal antibody include, but are not limited to, for example, KC, deposited at the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology mentioned above.
A266 can be used. Alternatively, for example, JP-A-9-37784 and Pestic. Sc
i. 1998, 54, 189-194.

The gene of the present invention may be chemically synthesized using known techniques based on the description in the present specification. Examples of the sequence of the gene of the present invention are disclosed in the sequence listing described later. SEQ ID Nos. 1 and 2 in the sequence listing correspond to the KCA
1 shows the nucleotide sequence and deduced amino acid sequence of a gene encoding 190 heavy chain variable regions and light chain variable regions. SEQ ID NOs: 3 and 4 show the nucleotide sequence and deduced amino acid sequence of the gene encoding the heavy chain variable region and light chain variable region of KCA266, respectively.

Amino acid residues 27-37, 52-61 and 10 in the heavy chain of KCA190 and KCA266
1-109 corresponds to the hypervariable region and is called H1, H2 and H3, respectively. Similarly, KCA190 and KCA
Amino acid residues 18-25, 41- in the 266 light chain
46 and 80-87 correspond to the hypervariable regions,
1, referred to as L2 and L3. Generally, in an immunoglobulin, the hypervariable regions are close to each other on a three-dimensional structure and assemble antibody active groups, and this region determines the specificity for the antigen to be bound. KCA190 and KC
In the light chain of A266, the amino acid sequence differs only at amino acid residue 89: leucine for KCA190 and phenylalanine for KCA266. Other sequences were identical. KCA190 and K
The amino acid sequences of the heavy chains of CA266 differed by about 35% in total, and in particular, by 63% in H1-H3. Thus, the affinity of two antibodies to chrysanthemum for chrysanthemum is
Differences in the amino acid sequence of the variable region of the heavy chain, especially in the H3 region on the C-terminal side where the difference is significant, are considered to be significantly different.

The variable region of the heavy or light chain of the antichrysanthemic acid monoclonal antibody having the amino acid sequence encoded by the gene of the present invention is limited to those having the amino acid sequences specifically described in the sequence listing and drawings. In addition, the sequence has an amino acid sequence in which one or more amino acid residues have been deleted, substituted or added, and is capable of forming an antigen-binding site and interacting with chrysanthemum acid, Also includes those that retain the function as antibody variable regions. Such genes also include those that have a higher ability to bind chrysanthemic acid than the amino acid sequences specifically described herein. Such a gene may be derived from a hybridoma producing an antibody other than KCA190 and KCA266 as an antibody against chrysanthemic acid. Alternatively, the KCA190 and KCA2 described herein
DNA prepared by synthesis and conjugation of oligonucleotides or by site-directed mutagenesis techniques based on 66 base and / or amino acid sequences
It may be synthesized from the construct. For example, a gene of the invention can be used under stringent conditions (eg, 2 × SSC
Hybridization at 50 ° C. overnight in medium; 2 × SSC
Medium, washed at 50 ° C.) A gene encoding a peptide capable of hybridizing with DNA having any one of the nucleotide sequences of SEQ ID NOs: 1 to 4 and forming an antigen-binding site and interacting with chrysanthemic acid Including.

In general, amino acid sequence substitutions must be made conservatively, ie, the most preferred substituted amino acids form an antigen-binding site in a manner substantially equivalent to KCA190 or KCA266 to form Are amino acids that do not affect the ability of the variable region to interact with Examples of conservative substitutions include, for example, KCA190 or KCA
Amino acid substitutions that do not alter the secondary and / or tertiary structure of 266 are included. As a further example, Il
replacing one aliphatic residue with another such as replacing e, Val, Leu or Ala with each other, or Lys and Arg; Glu and As
p; or replacing one polar residue with another polar residue, such as a substitution between Gln and Asn. Other such conservative substitutions are well known, for example, replacement of an entire region with similar hydrophobic properties.

As another example, to cause a Cys residue to be deleted or replaced by another amino acid,
The sequence encoding the Cys residue can be altered to prevent the formation of inappropriate intramolecular disulfide bridges during regeneration. The amino acids to be replaced are tryptophan, serine,
Selected from the group consisting of aspartic acid and lysine,
Most preferably, the amino acid is tryptophan.

Similarly, amino acid sequences can be deleted or added by taking into account the potential effects of the deletion or insertion on biological activity. For example, the use of a yeast expression system expresses a homologous carbohydrate-reduced analog, but the N-glycosylation site can be modified to eliminate glucosylation. Glycosylation sites in eukaryotic polypeptides are amino acid triplets, As
n-XY (where X is any amino acid except Pro and Y is Ser or Thr).
Appropriate modifications of the nucleotide sequence encoding this triplet will result in substitutions, additions or deletions that prevent the attachment of carbohydrate residues on the Asn side chain. Alternatively, the sequence encoding dibasic amino acid residues can be modified to enhance expression in yeast systems where KEX2 protease activity is present. Expression of Variable Region of Anti-Chrysanthenic Acid Monoclonal Antibody By inserting the gene of the present invention into an appropriate expression vector and culturing host cells transformed with the expression vector, the variable region of anti-chrysanthemic acid monoclonal antibody is genetically engineered. It is possible to cause the expression.

Generally, the variable regions of the heavy and light chains of an antibody contain an antigen-binding site, and the DNAs of both regions are connected by a linker and expressed in a host cell, whereby a single-chain Fv with antigen-binding ability is expressed. It is known that a protein (sFv) can be obtained. Therefore, for example, by expressing genes encoding both heavy and light chain variable regions of the present invention in host cells such as Escherichia coli, it is possible to obtain a large amount of a protein capable of binding chrysanthemic acid. (FIG. 5). This is cheaper than maintaining monoclonal antibodies obtained by culturing in a medium requiring serum, and is expected to be applied to enzyme immunoassays. Alternatively, animals and plants resistant to pyrethroid insecticides can be produced by introducing a vector containing the gene of the present invention into animals and plants.

[0029] Recombinant expression vectors for expressing the variable region (hereinafter, simply referred to as "variable region" depending on the context) of the anti-chrysanthemic acid monoclonal antibody by recombinant genetic engineering techniques include mammals, microorganisms, and viruses. Or a cDNA sequence encoding a variable region, operably linked to an appropriate transcriptional or translational regulatory nucleotide sequence, such as that derived from an insect gene. Examples of regulatory sequences include sequences having a regulatory role in gene expression (eg, regulated promoters or enhancers), operator sequences to control transcription, if desired, mRNA,
Sequences encoding the ribosome binding site and appropriate sequences to control initiation and termination of transcription and translation are included.

[0030] Nucleotide sequences are operably linked when the regulatory sequence is operatively associated with the variable region DNA sequence. For example, if the promoter nucleotide sequence controls the transcription of the variable region DNA sequence, then the promoter nucleotide sequence is operable to
It is linked to the A sequence. Still further, if a ribosome binding site is located in the vector to facilitate translation, the ribosome binding site will be operably linked to the variable region polypeptide sequence. Furthermore, a sequence encoding a signal peptide can be incorporated into an expression vector. For example, the DNA sequence of a signal peptide (secretory leader) can be
It can be linked to an NA sequence.

Suitable host cells for expressing the variable region polypeptides include prokaryotes, yeast or higher eukaryotic cells. Prokaryotes include gram negative or gram positive organisms, such as E. coli or Bacilli. Prokaryotic host cells suitable for transformation include, for example, E. coli, Bacillus subtilis, Salmonella type.
himurium and various other species of the genus Pseudomonas, Streptomyces and Saccharomyces. Higher eukaryotic cells include established cell lines of mammalian origin. Cell-free translation systems can also be used to produce variable region polypeptides using RNA obtained based on the DNA constructs disclosed herein. Cloning and expression vectors suitable for use in bacterial, fungal, yeast and mammalian cell hosts are described, for example, in Pouwels et al.
loaning Vector: A Laborator
yManual, Elsevier, New York
(1985).

The expression vector carrying the recombinant variable region DNA sequence is transfected or transformed into a substantially homogeneous culture of a suitable host microorganism or mammalian cell line. A transformed host cell is a cell that has been transformed or transfected with a nucleotide sequence encoding a variable region polypeptide, and that expresses the variable region polypeptide. The expressed variable region polypeptide will be located within the host cell and / or secreted into the culture supernatant fluid, depending on the nature of the host cell and the genetic construct inserted into the host cell.

[0033] Expression vectors transfected into prokaryotic host cells generally contain one or more selectable phenotypic markers. Selectable phenotypic markers are, for example, genes encoding proteins that confer antibody resistance or supply auxotrophy, and an origin of replication recognized by the host to ensure amplification within the host. . Other useful expression vectors for prokaryotic host cells include selectable markers of bacterial origin derived from commercially available plasmids. This selectable marker can include the genetic elements of the cloning vector pBR322 (ATCC 37017). pBR322 contains genes for ampicillin and tetracycline resistance, and thus provides a simple means to identify transformed cells. pBR322 “backbone (backb
one) "portion is ligated with an appropriate promoter and variable region DNA sequence. Other commercially available vectors include, for example, pKK223-3 (Pharm
Acia Fine Chemicals, Uppsa
la, Sweden) and pGEM1 (Promega)
a Biotec, Madison, WI, USA).

[0034] Promoter sequences are commonly used in recombinant prokaryotic host cell expression vectors. Common promoter sequences include β-lactamase (penicillinase), lactose promoter system (Chang et al., Na
cure, 275: 615, 1978; and Goed.
del et al., Nature, 281: 544, 197.
9), tryptophan (trp) promoter system (Go
eddel et al., Nucl. Acids Res. , 8:
4057, 1980; and EP-A-36776),
And tac promoter (Maniatis, Mol
eco Cloning: A Laborato
ry Manual, Cold Spring Harb
or Laboratory, 412, 1982). Particularly useful prokaryotic host cell expression systems employ the phage λPL promoter and the cI857ts thermotolerant repressor sequence. Plasmid vectors incorporating derivatives of the λ PL promoter available from the American Type Culture Collection (ATCC) include plasmid pHUB2 [resident in E. coli strain JMB9 (ATCC 37092)] and p
PLc28 [resident in E. coli RR1 (ATCC53082)].

The variable region may be of the genus Saccharomyces (eg,
S. cerevisiae) can also be expressed in yeast host cells. Other yeast genera such as Pichia or Kluyveromyces can also be used.
Yeast vectors will sometimes include a replication initiation sequence, an autonomously replicating sequence (ARS), a promoter region, a polyadenylation sequence, and a transcription termination sequence from a 2μ yeast plasmid. Desirably, the yeast vector contains a replication initiation sequence and a selectable marker. Suitable yeast vector promoter sequences include metallothionein, 3-
Phosphoglycerate kinase (Hitzeman et al.,
J. Biol. Chem. , 255: 2073, 198.
0) or other glycolytic enzymes (Hess et al.,
J. Adv. Enzyme Reg. , 7: 149, 1
968; and Holland et al., Biochem. ,
17: 4900, 1978), for example, enolase, glyceraldehyde-3-phosphate dehydrogenase,
Hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triose phosphate isomerase,
Phosphoglucose isomerase, glucokinase and the like. Other suitable vectors and promoters for use in yeast expression are also available from Hitzema
n, EPA-73,657.

[0036] Mammalian or insect host cell culture systems can also be used to express recombinant variable region polypeptides. Examples of suitable mammalian host cell lines include the COS-7 line of monkey kidney cells (ATCC C
RL1651) (Gluzman et al., Cell, 23:
175, 1981), L cells, C127 cells, 3T3 cells (ATCC CCL163), Chinese hamster ovary (CHO) cells, HeLa cells and BHK (A
TCC CRL10) cell lines are included. Suitable mammalian expression vectors include non-transcribed elements, such as origins of replication, promoter sequences, enhancers, etc., linked to the structural gene; ribosome binding sites, polyadenylation sites, splice donor and acceptor sites, and transcription termination sequences, etc. 5 ′ or 3 ′ contiguous untranslated sequence;

The transcriptional and translational control sequences of the mammalian host cell expression vector can be excised from the viral genome. For example, commonly used mammalian cell promoter and enhancer sequences include polyomavirus, adenovirus 2, simian virus 40 (S
V40) and human cytomegalovirus. A DNA sequence derived from the SV40 virus genome,
For example, the SV40 origin, early and late promoters, enhancers, splices, and polyadenylation sites can be used to provide other genetic elements required for expression of structural gene sequences in mammalian host cells.

[0038] Mammalian expression vectors include, for example, Oka
yama and Berg (Mol. Cell Bio
l. , 3: 280, 1983). Cosman et al., N
Nature, 312: 768, 1984;
PMLSV N1 / N4, a useful high expression vector
Has been deposited as ATCC 39890. A further useful mammalian expression vector is EP-A-0367.
556, and US patent application Ser. No. 07 / 701,41.
5, filed May 16, 1991, incorporated herein by reference.

Although not limited, it is preferred that the heavy and light chain variable regions are expressed in tandem with a linker sequence between them. By expressing a plurality of chains including a heavy chain and a light chain in close proximity, the ability to bind to the antibody can be increased. Utilizing the gene of the present invention, it is possible to bind the heavy chain and the tandem in tandem and express them in large amounts. The linker sequence is not particularly limited, but the length is preferably about 6 to 20 bases. A commercially available linker sequence can be used, for example, Linker Primer Mix of Pharmacia Biotech can be used (FIG. 5).

The gene encoding the variable region of the present invention may be expressed by binding to the gene encoding the variable region such that the variable region is covalently bound or aggregated with other proteins or polypeptides. Such fusions include, for example, the N-terminal region or the C-terminus of the variable region polypeptide involved in transporting the complex from its synthesis site, either simultaneously or after translation, to a site inside or outside the cell membrane or cell wall. Region signal or leader polypeptide (eg, Saccharomyces alpha factor leader). Alternatively, the variable region polypeptide fusion may be a fusion with a polypeptide (eg, poly-His) added to facilitate purification and identification of the variable region.

[0041] Fusion proteins can be prepared using conventional techniques of enzymatic cleavage and ligation of fragments from the desired sequence. PCR techniques using synthetic oligonucleotides can be used to prepare and / or amplify the desired fragment. Synthetic oligonucleotides exhibiting the desired sequence can also be used to prepare DNA constructs encoding the fusion protein. Also, the fusion protein has a variable region,
As well as leader (or signal peptide) sequences, oligomerization region (eg, leucine zipper moieties or suitable zipper moieties) linker sequences, and highly immunogenic that provide a means for easily purifying or rapidly detecting the fusion protein. Including sequences that code the parts,
One or more additional sequences can be included.

The signal peptide promotes secretion of the protein from the cell. Flag® octapeptide (Hopp et al., Bio / Technology,
6: 1204, 1988) are reversible by specific monoclonal antibodies that do not alter the biological activity of the fusion protein, have high immunogenicity, and allow for rapid detection and easy purification of the expressed fusion protein. To provide an epitope that is bound to. The Flag® sequence is also activated by bovine mucosal enterokinase.
Fusion proteins cleaved specifically at the residue immediately following the Asp-Lys pair and capped with this peptide are also resistant to intracellular degradation in E. coli. Flag
A murine monoclonal antibody that binds to ® is deposited with the ATCC (accession number HB925).
9); A method for purifying a fusion protein containing a Flag® sequence using an antibody is described in US Pat.
No. 1,912, incorporated herein by reference.

The gene encoding the variable region of the present invention is
The antibody may be expressed by binding to a gene encoding a constant region (hereinafter, referred to as “Fc region”) of the antibody. The constant region may be the same as the monoclonal antibody from which the variable region is derived, or may be from a different monoclonal antibody. Suitable Fc regions can bind to protein A or protein G, or can be recognized by antibodies that can be used to purify or detect fusion proteins containing the Fc region. As the Fc region, for example, a known human IgG1 or murine IgG1 Fc region can be used. A fragment of an appropriate Fc region, for example, a human IgG, wherein the sequence of amino acids responsive to binding to protein A has been deleted, such that the fragment binds to protein G but does not bind to protein A.
One Fc region can also be used.

In addition, the immunological measurement method using an antibody may generally cause problems such as sensitivity, specificity, and resistance to organic solvents. Such a problem can be solved by expressing the gene of the present invention as a fusion protein with another protein or peptide. For example, but not limited to, for performing measurements at elevated temperatures for the purpose of increasing specificity, Tth DNA polymerase
and a fusion protein with a thermostable enzyme such as restriction enzyme Taq I. Recombinant variable region polypeptide The gene of the present invention can be used to produce the variable region of the heavy and / or light chain of the anti-Chrysanthemum monoclonal antibody. Specifically, host cells transformed with a vector containing the gene of the present invention are cultured under conditions that promote the expression of the heavy and / or light chain variable regions of the anti-chrysanthemic acid monoclonal antibody, and The heavy chain and / or light chain variable region of the anti-chrysanthemic acid monoclonal antibody is recovered from the culture.

[0045] The variable region polypeptides can be prepared by culturing the transformed host cells under the culture conditions necessary to express the variable region polypeptide. Next, the resulting expressed polypeptide can be purified from the culture solution or cell extract. If desired, the variable region polypeptide can be converted to a commercially available protein concentration filter, such as Am
icon or Millipore Pellicon
It can be concentrated using an ultrafiltration unit. After the concentration process, the concentrate can be loaded onto a purification matrix, such as a gel filtration medium. Alternatively, an anion exchange resin such as free diethylaminoethyl (D
A matrix or substrate having EAE) groups can be used. The matrix can be acrylamide, agarose, dextran, cellulose or other types commonly used for protein purification. Alternatively, a cation exchanger can be used.
Suitable cation exchangers include various insoluble matrices containing sulfopropyl or carboxymethyl groups. Sulfopropyl groups are preferred.

Finally, to purify the variable region further, one or more steps using a hydrophobic RP-HPLC medium (eg, silica gel with free methyl or other aliphatic groups). Phase high performance liquid chromatography (RP-HPLC) can be used.
Some or all of the foregoing purification means may be variously combined and used to provide a substantially homogeneous recombinant protein.

In order to affinity-purify the expressed variable region polypeptide, an affinity column to which chrysanthemic acid is bound can be used. The variable region polypeptide can be removed from the affinity column with a high salt elution buffer and then dialyzed against the lower salt buffer used.

The recombinant protein produced in bacterial culture is
Usually, first disruption of the host cells, centrifugation, extraction from the cell pellet for insoluble polypeptides or from the supernatant for soluble polypeptides, and one or more steps of concentration, salting Isolated by precipitation, ion exchange, affinity purification or ultrafiltration chromatography. Finally, RP-HPLC can be used for the final purification step. Microbial cells can be disrupted according to any conventional method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents.

The transformed yeast host cells are desirably used to express the variable region as a secreted polypeptide. This simplifies purification. The recombinant polypeptide secreted from the yeast host cell fermentation is Ur
dal et al. (J. Chromatog., 296: 17).
1, 1984). Urdal et al. Describe a two-step reverse-phase HPLC performed sequentially to purify human recombinant IL-2 on a preparative HPLC column.

Hereinafter, the present invention will be described specifically with reference to Examples, but these are not intended to limit the technical scope of the present invention. Those skilled in the art can easily modify and change the present invention based on the description in the present specification, and they are included in the technical scope of the present invention.

[0051]

EXAMPLES Example (1) Partial Purification of Anti-Chrysanthemic Acid mRNA mRNA Anti-Chrysanthemic Acid Monoclonal Antibodies KCA190 and KCA2
66 producing hybridoma cells (mouse) (KCA
266 is deposited as FERM P-15051 at the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology).
⁶ was suspended in 1 ml of an RNA preparation solution (ISOGEN solution, purchased from Wako Pure Chemical Industries) to destroy cells. After allowing the mixture to stand at room temperature for 5 minutes, 0.2 ml of chloroform was added thereto, the mixture was stirred with a vortex for 15 seconds, and then allowed to stand for 3 minutes to extract RNA from the cells. 12.0 using Hitachi Centrifuge R15
After centrifugation at 00 rpm for 3 minutes, RNA was recovered in the supernatant. The supernatant was transferred to a new tube, and 0.5 ml of isopropanol was added to precipitate RNA. 12.0 with the above centrifuge
The mixture was centrifuged at 00 rpm for 10 minutes to collect the RNA as a precipitate. After washing with a 75% ethanol solution, the precipitate was dried. The RNA precipitate was dissolved in water, and the concentration was determined from the absorbance at 260 nm.

Next, about 200 μg of the obtained RNA was added to an extraction buffer (10 mM Tris-HCl pH 7.5,
Oli equivalent to 1 mM EDTA, 0.1% SDS)
200 μl of a gotex-dT30 latex (purchased from Takara Shuzo) solution was added, and the mixture was heated at 65 ° C. for 5 minutes. Thereafter, the mixture was rapidly cooled in ice for 3 minutes, and 1/10 of a 5M sodium chloride solution was added thereto, followed by heating at 37 ° C. for 10 minutes to bind only mRNA to Oligo-dT30.
The mixture was centrifuged at 12,000 rpm for 3 minutes in the above centrifuge. After 200 μl of sterilized water was added to the precipitated Oligo-dT30, the mixture was heated at 65 ° C. for 5 minutes and then rapidly cooled in ice for 3 minutes to dissociate the mRNA from the resin. The mRNA was recovered as a supernatant by centrifugation again. (2) Synthesis of variable region cDNA for heavy and light chains of anti-chrysanthemic acid monoclonal antibody cDNA was synthesized by Mouse ScFv Modul.
e / Recombinant Page Antib
ody System (purchased from Pharmacia Biotech) was used. About 800n each for light chain and heavy chain mRNA
g, 21 μl was heated at 65 ° C. for 10 minutes and then rapidly cooled in ice to destroy the secondary structure of mRNA. Then Prime
d First Strand Mix (purchased from Pharmacia Biotech) solution 1 μl and DDT solution 1 μl
Was added and reacted at 37 ° C. for 1 hour. Lig for light chains
HT Primer Mix (purchased from Pharmacia Biotech), 2 μl, sterile water 64 μl, H for heavy chain
2 μl of Heavy Primer 1 and Heavy P
2 μl of primer 2 (purchased from Pharmacia Biotech) was added to 62 μl of sterilized water, and heated at 95 ° C. for 5 minutes. After that, AmpriTaq DNA Polymer
ase (purchased from PerkinElmer), add 1 μl, and add 9
5 ° C for 1 minute, 55 ° C for 2 minutes, 72 ° C for 2 minutes for 30 minutes
Repeated times. Thus, cDNA fragments for the variable regions of the heavy and light chains of the anti-chrysanthemic acid monoclonal antibody were obtained.
Each fragment was analyzed by GE from 1.5% agarose after electrophoresis.
Extracted using NECREAN II (purchased from BIO101). (3) Construction of Plasmid Containing Each Variable Region cDNA of Anti-Chrysanthemic Acid Monoclonal Antibody Heavy and Light Chains 50 ng of each obtained cDNA was used as Linker Pri.
mer Mix (purchased from Pharmacia Biotech) 4
μl, 10xPfu buffer (STRATAGENE
CLONING SYSTEMS) 5 μl, 2
0 mM dNTP (purchased from Pharmacia Biotech)
2.5 μl, 25 mM magnesium chloride 5 μl, P
fu DNA Polymerase (STRATA
(Purchased from GENECLINGING SYSTEMS) 5
U 1 μl, make up to 50 μl with sterile water, and
After repeating 7 times at 63 ° C. for 4 minutes for 7 minutes, Pfu D
NA Polymerase 1μl, Buffer for 10xPfu (Stratagene Cloning SY
5 μl, 20 mM dNTP (purchased from Pharmacia Biotech), 1 μl, PS Pri
mer Mix (purchased from Pharmacia Biotech) 4
μl and 39 μl of sterilized water, and then again at 95 ° C. for 1 minute.
55 ° C. for 2 minutes and 72 ° C. for 2 minutes were repeated 30 times.
The DNA fragment obtained by linking the heavy chain and light chain variable region cDNA fragments with a linker was obtained from 1.5% agarose after electrophoresis using the GENECLEAN II Kit (BIO).
(Purchased from No. 101). This fragment is Hin
pUC1 cleaved at one place so as to be blunt-ended by cII
Ligated to 9 plasmids. That is, 3 pm
ol DNA fragment, 0.003 pmol of the pUC19HincII-cleaved fragment and a 6-fold amount of a ligation kit (purchased from Takara Shuzo) A solution DNA solution and an equal amount of solution B,
The reaction was performed at 6 ° C. overnight. After ethanol precipitation of the reaction solution, 1
The cells were dissolved in 0 μl of sterile water, added to JM109 competent cells (purchased from Toyobo), and transformed. (4) Determination of base sequence of cDNA insert and amino acid sequence of each variable region of heavy chain and light chain of anti-chrysanthemic acid monoclonal antibody The whole base sequence of cDNA insert was determined by dideoxy method. As a primer, a universal primer for pUC (Forward, Reverse)
(Purchased from BRL) and a primer (S
3, S4) (purchased from Pharmacia Biotech) using the Dyterminator Cycle Sequ
ncing FS Ready Reaction K
D by it (purchased from PerkinElmer Japan)
The reaction was performed using NA thermal Cycler 480 (purchased from PerkinElmer Japan).

FIG. 5 shows the structure of the insert, and FIG. 1 to FIG. 4 show the nucleotide sequences of the variable regions of the heavy and light chains and the amino acid sequences thereof. The resulting heavy chain variable region was KCA19
Both 0 and KCA226 consist of 336 bases and 112 amino acid residues, and the light chain variable region has 297 bases and 9 amino acids.
There were 9 residues. The obtained amino acid sequence showed 70% or more homology with the variable regions of two or more mouse antibody genes registered in the protein database, and was confirmed to be an antibody gene.

[0054]

According to the present invention, a gene encoding the variable region of the heavy and light chains of the anti-chrysanthemic acid monoclonal antibody is provided, and the amino acid sequence and the base sequence of the variable region have been clarified. In the present invention, particularly by analyzing the genes of two types of anti-chrysanthemic acid monoclonal antibodies, the variable regions, in particular, the regions considered to be involved in binding to an antigen, were compared in detail.

By expressing in a host cell using the gene provided by the present invention, it became possible to obtain a large amount of a protein capable of binding to chrysanthemic acid. This is cheaper than maintaining monoclonal antibodies obtained by culturing in a medium requiring serum, and can be applied to enzyme immunoassay. In addition, by introducing the gene of the present invention into animals and plants, animals and plants resistant to pyrethroid insecticides can be produced. The gene of the present invention is further modified, and fused with an antibody or a thermostable enzyme having more excellent binding ability, so as to be a general problem in performing an enzyme immunoassay having an antibody, sensitivity, specificity, Overcoming organic solvent resistance is also possible.

[0056]

[Sequence List] SEQUENCE LISTING <110> Institute for Environmental Immunology <120> Gene encoding variable region of anti-Chrysanthemum monoclonal antibody <130> 990370 <160> 4 <170> JIS <210> 1 <211> 336 < 212> DNA <400> 1 atg gcc cag gtc aag ctg cag gag tca gga cct gag ctg gtg aag 45 Met Ala Gln Val Lys Leu Gln Glu Ser Gly Pro Glu Leu Val Lys 1 5 10 15 cct ggg gct tca gtg aag ata tcc tgc aag gct tct ggc tac tcc 90 Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser 20 25 30 ttc att gac cac tat ata aac tgg gtg aag cag aag cct gga cag 135 Phe Ile Asp His Tyr Ile Asn Trp Val Lys Gln Lys Pro Gly Gln 35 40 45 gga ctt gag tgg att gga tgc ttt ttt cct gga agc ggt aat agt 180 Gly Leu Glu Trp Ile Gly Cys Phe Phe Pro Gly Ser Gly Asn Ser 50 55 60 aag tac att gag aac ttc agg ggc aag gcc aca ttg act gta gac 225 Lys Tyr Ile Glu Asn Phe Arg Gly Lys Ala Thr Leu Thr Val Asp 65 70 75 aca tcc tcc agt aca gcc tac atg cag ctc agc agt ctg gca tct 270 Thr Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu A la Ser 80 85 90 gag gac act gct gtc tat ttc tgt gca agg gat gat tcc gac gga 315 Glu Asp Thr Ala Val Tyr Phe Cys Ala Arg Asp Asp Ser Asp Gly 95 100 105 gct atg gac tac tgg ggc caa 336 Ala Met Asp Tyr Trp Gly Gln 110 112 <210> 2 <211> 297 <212> DNA <400> 2 gca atc atg tct gca tct cca ggg gag agg gtc acc atg acc tgc 45 Ala Ile Met Ser Ala Ser Pro Gly Glu Arg Val Thr Met Thr Cys 1 5 10 15 agt gcc agc tca agt ata cgt tac ata tat tgg tac caa cag aag 90 Ser Ala Ser Ser Ser Ile Arg Tyr Ile Tyr Trp Tyr Gln Gln Lys 20 25 30 cct gga tcc tcc ccc aga ctc ctg att tat gac aca tcc aac gtg 135 Pro Gly Ser Ser Pro Arg Leu Leu Ile Tyr Asp Thr Ser Asn Val 35 40 45 gct cct gga gtc cct ttt cgc ttc agt ggc agt ggg tct ggg acc 180 Ala Pro Gly Val Pro Phe Arg Phe Ser Gly Ser Gly Ser Gly Thr 50 55 60 tct tat tct ctc aca atc aac cga atg gag gct gag gat gct gcc 225 Ser Tyr Ser Leu Thr Ile Asn Arg Met Glu Ala Glu Asp Ala Ala 65 70 75 act tat tac tgc cag gag tgg agt ggt tat ccg tac acg ctc gga 270 Thr Tyr Tyr Cys G ln Glu Trp Ser Gly Tyr Pro Tyr Thr Leu Gly 80 85 90 ggg ggg acc aag ctg gaa atc aaa cgg 297 Gly Gly Thr Lys Leu Glu Ile Lys Arg 95 99 <210> 3 <211> 336 <212> DNA <400> 3 atg gcc cag gtg aaa ctg cag gag tca gga act gag gtg gtg aag 45 Met Ala Gln Val Lys Leu Gln Glu Ser Gly Thr Glu Val Val Lys 1 5 10 15 cct ggg gct tca gtg aag ttg tcc tgc aag gct tct ggc tac atc 90 Pro Gly Ala Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Ile 20 25 30 ttc aca agt tat gat ata gac tgg gtg agg cag acg cct gaa cag 135 Phe Thr Ser Tyr Asp Ile Asp Trp Val Arg Gln Thr Pro Glu Gln 35 40 45 gga ctt gag tgg att gga tgg att ttt cct gga gag ggg agt act 180 Gly Leu Glu Trp Ile Gly Trp Ile Phe Pro Gly Glu Gly Ser Thr 50 55 60 gaa tac aat gag aag ttc aag ggc agg gcc aca ctg agt gta gac 225 Glu Tyr Asn Glu Lys Phe Lys Gly Arg Ala Thr Leu Ser Val Asp 65 70 75 aag tcc tcc agc aca gcc tat atg gag ctc act agg ctg aca tct 270 Lys Ser Ser Ser Thr Ala Tyr Met Glu Leu Thr Arg Leu Thr Ser 80 85 90 gag gac tct gct gtc tat ttc tgt g ct aga ggg gac tac tat agg 315 Glu Asp Ser Ala Val Tyr Phe Cys Ala Arg Gly Asp Tyr Tyr Arg 95 100 105 cgc tac ttt gac ttg tgg ggc 336 Arg Tyr Phe Asp Leu Trp Gly 110 112 <210> 4 <211> 336 <212> DNA <400> 4 gca atc atg tct gca tct cca ggg gag agg gtc acc atg acc tgc 45 Ala Ile Met Ser Ala Ser Pro Gly Glu Arg Val Thr Met Thr Cys 1 5 10 15 agt gcc agc tca agt ata cgt tac ata tat tgg tac caa cag aag 90 Ser Ala Ser Ser Ser Ile Arg Tyr Ile Tyr Trp Tyr Gln Gln Lys 20 25 30 cct gga tcc tcc ccc aga ctc ctg att tat gac aca tcc aac gtg 135 Pro Gly Ser Ser Pro Arg Leu Leu Ile Tyr Asp Thr Ser Asn Val 35 40 45 gct cct gga gtc cct ttt cgc ttc agt ggc agt ggg tct ggg acc 180 Ala Pro Gly Val Pro Phe Arg Phe Ser Gly Ser Gly Ser Gly Thr 50 55 60 tct tat tct ctc aca atc aac cga atg gag gct gag gat gct gcc 225 Ser Tyr Ser Leu Thr Ile Asn Arg Met Glu Ala Glu Asp Ala Ala 65 70 75 act tat tac tgc cag gag tgg agt ggt tat ccg tac acg ttc gga 270 Thr Tyr Tyr Cys Gln Glu Trp Ser Gly Tyr Pro Tyr Thr Phe Gly 80 85 90 ggg ggg acc aag ctg gaa ata aaa cgt 297 Gly Gly Thr Lys Leu Glu Ile Lys Arg 95 99

[Brief description of the drawings]

FIG. 1 shows the nucleotide sequence of the heavy chain variable region of anti-chrysanthemic acid monoclonal antibody 190 and the amino acid sequence deduced therefrom.

FIG. 2 shows the nucleotide sequence of the light chain variable region of anti-chrysanthemic acid monoclonal antibody 190 and the amino acid sequence deduced therefrom.

FIG. 3 shows the nucleotide sequence of the heavy chain variable region of anti-chrysanthemic acid monoclonal antibody 226 and the amino acid sequence deduced therefrom.

FIG. 3 shows the nucleotide sequence of the light chain variable region of the anti-chrysanthemic acid monoclonal antibody 226 and the amino acid sequence deduced therefrom.

FIG. 5 shows the configuration of pKCA109 and 226, in which black bars and shaded portions indicate the DNA regions of the heavy chain and light chain variable regions, respectively. Restriction enzyme Sfi on both ends
There are I and NotI cleavage sites, and the gene can be cut out. The thin line represents the base sequence of pUC19.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12N 5/10 C12P 21/08 C12P 21/08 G01N 33/53 D G01N 33/53 33/577 B 33 / 577 C12N 5/00 A F-term (reference) 4B024 AA11 BA41 CA04 DA02 DA03 DA05 DA11 EA04 4B064 AG27 CA02 CA05 CA10 CA11 CA19 CC24 DA13 4B065 AA01X AA57X AA88X AA90X AA91Y AB01 AC14 BA02 CA25 CA46 4H045 FA10

Claims (11)

[Claims] 1. A gene encoding a heavy chain variable region of an anti-chrysanthemic acid monoclonal antibody, wherein the heavy chain variable region of the anti-chrysanthemic acid monoclonal antibody has the following i) -iii): i) SEQ ID NO: 1. An amino acid sequence having amino acid residues 1-112; ii) an amino acid sequence having amino acid residues 1-112 of SEQ ID NO: 3; and iii) a deletion of one or more amino acid residues in the amino acid sequence of i) or ii) above. An amino acid sequence selected from the group consisting of a lost, substituted or added amino acid sequence;
And said gene capable of forming an antigen-binding site and interacting with chrysanthemic acid. 2. The heavy chain variable region of the anti-chrysanthemic acid monoclonal antibody has the following i) or ii): i) an amino acid sequence having amino acid residues 1-112 of SEQ ID NO: 1; or ii) an amino acid of SEQ ID NO: 3 The gene according to claim 1, which has an amino acid sequence of amino acid sequence having residues 1-112. 3. The gene according to claim 1, which has the nucleotide sequence 1-336 of SEQ ID NO: 1 or the nucleotide sequence 1-336 of SEQ ID NO: 3. 4. A gene encoding a light chain variable region of an anti-Chrysanthenic acid monoclonal antibody, wherein the light chain variable region of the anti-Chrysanthemic acid monoclonal antibody has the following i) -iii): i) SEQ ID NO: 2 An amino acid sequence having amino acid residues 1-99; ii) an amino acid sequence having amino acid residues 1-99 of SEQ ID NO: 4; and iii) one or more amino acid residues in the amino acid sequence of i) or ii) above are missing. An amino acid sequence selected from the group consisting of a lost, substituted or added amino acid sequence;
And said gene capable of forming an antigen-binding site and interacting with chrysanthemic acid. 5. The method according to claim 5, wherein the light chain variable region of the anti-chrysanthemic acid monoclonal antibody has the following i) or ii): i) an amino acid sequence having amino acid residues 1 to 99 of SEQ ID NO: 1; The gene according to claim 4, which has an amino acid sequence of the amino acid sequence having residues 1 to 99. 6. The gene according to claim 4, which has the nucleotide sequence 1-297 of SEQ ID NO: 2 or the nucleotide sequence 1-297 of SEQ ID NO: 4. A vector comprising the gene according to any one of claims 1 to 6. 8. The vector according to claim 7, which is a plasmid. 9. The vector according to claim 6, which is capable of self-propagation in a host cell. [10] A host cell transformed with the vector according to any one of [7] to [9]. 11. A method for producing a heavy chain and / or light chain variable region of an antichrysanthemic acid monoclonal antibody, which is a host cell transformed with the vector according to any one of claims 7 to 9. Is cultured under conditions that promote the expression of the variable region of the heavy and / or light chain of the anti-oxalate monoclonal antibody, and the variable of heavy and / or light chain A method comprising retrieving an area.