JPH06105686A - Porphyrin monoclonal antibody structural gene - Google Patents

Abstract

PURPOSE:To provide a new structural gene useful for the mass-production of porphyrin monoclonal antibody. CONSTITUTION:A structural gene of a monoclonal antibody capable of specifically recognizing porphyrin of formula I (R<1> to R<4> are independently H or CO2H; R<1> to R<4> are not H at the same time). The gene contains a base sequence of e.g. formula II in the VL and CL regions of L-chain. It can be prepared by producing a cell capable of producing a mouse hybridoma antibody, preparing an mRNA from various kinds of cells, synthesizing the 1st chain of cDNA, treating the H chain and L chain with PCR and restriction enzyme, cloning with an E.coli plasmid vector and determining the base sequence.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a gene for a porphyrin monoclonal antibody and a porphyrin monoclonal antibody.

[0002]

2. Description of the Related Art In recent years, much research has been conducted on molecular recognition. However, the currently synthesized host molecules recognize only very simple low molecules and ions, and therefore, larger and more complex molecules and macromolecules can be recognized. Host molecules that recognize and respond are desired. In a biological system, recognition of a substrate by an enzyme and recognition by a polymer such as an antigen-antibody reaction are carried out, and it has a very high recognition specificity. The immune system is used in many ways due to its high specificity and diversity. Recently, an antibody (Catalytic Antibody) that exhibits a catalytic action using the immune system has been produced. Some of the monoclonal antibodies against porphyrin have an absorption spectrum that shifts to a longer wavelength by binding with a metal complex of porphyrin, and also exhibits oxidase activity. Is expected to be used.

Porphyrin metal complexes are widely present in the body as constituents of blood proteins that carry oxidation such as hemoglobin and enzymes such as catalase b and P450, and play an important role in electron transport and decomposition of hydrogen peroxide. Is fulfilling. If it is possible to artificially produce substances having these functions, it is expected to be applied to various uses. On the other hand, an antibody having a catalytic activity called an antibody enzyme (Catalytic Antibody) has been reported by Lana et al. Or Schertz et al. And has been attracting attention (Science 234, 1566 (1986)). The reason for this is that the antibody enzyme is several steps superior to the ordinary protein enzyme in terms of substrate specificity, superior in stability, and the reaction or substrate catalyzed by using an appropriate hapten. The uniqueness can be freely designed. Most of these are antibodies that can be obtained by using the transition state analog of the reaction as a hapten, but a cofactor such as a coenzyme can also be obtained as a hapten. According to the idea of the latter method, if an antibody that can specifically bind to the metal complex of porphyrin is obtained, the antibody may have the above-mentioned function.

The inventors of the present invention have already used Mesotetrakis (4-
Several kinds of monoclonal antibodies against carboxyphenyl) porphyrin (abbreviated as TCPP) were prepared, and it was reported that the antibody could bind to Zn (II) complex of TCPP (Chem. Lett., 917 (1990). After that, as a result of examining its function and structure, one of those antibodies was found to bind to TCPP, causing the Soret band of TCPP to shift to a long wavelength in the absorption spectrum, and causing the induced cotton effect in the CD spectrum. We have found an antibody that expresses oxidase activity by binding to a metal complex of synthetic porphyrin (Japanese Patent Application No. 3-332513).
These antibodies are obtained by the following method. That is, TCPP which is an antigen and keyhole limpet hemocyanin which is a hapten (abbreviated as KLH) or BSA (Bo
vine Serum albuminn) is covalently bound, and this is administered to mice, and the spleen cells of the immunized mice are taken out and fused with myeloma cells to prepare fused cells. It can be produced by screening a clone producing a desired antibody that recognizes the porphyrin represented by the general formula (I) from the fused cells and culturing the clone.

The antibody-producing strain monocloned by the above-mentioned method was injected into the abdominal cavity of Balb / C mice that had been previously injected with blistane about 1 to 2 weeks before the ascites was collected 2 weeks later. Purify by salting out with ammonium sulfate and various chromatographies. As another method, there is a method in which antibody-producing cells are expanded and cultured in a serum-free medium to prepare antibodies. However, each of these preparation methods has the disadvantage that it takes time and the amount of antibody produced is limited, making it difficult to produce the antibody on a practical scale. Therefore, the present inventors have continued to study microbial production by gene manipulation technology as a method capable of producing a large amount of antibody in a short time in a large amount and having stable quality.

[0006]

SUMMARY OF THE INVENTION An object of the present invention is to develop a porphyrin monoclonal antibody which is necessary for developing a new method for producing a porphyrin monoclonal antibody economically and in large quantities using recombinant DNA technology. It is an object of the present invention to provide a structural gene of an antibody and at the same time provide a basic structural gene necessary for modifying the properties of a porphyrin monoclonal antibody by a protein engineering technique.

[0007]

The present invention has the general formula (I):

[Chemical 2](In the formula, R¹, R², R³And R^FourIs H or COOH
Is. However, R¹= R ²= R³= R^FourExcept when = H
Monochrome that specifically recognizes porphyrin
This is the structural gene for the lonal antibody.

The present invention also provides a porphyrin monoclonal antibody containing the amino acid sequence set forth in SEQ ID NO: 1 in the VL and CL regions of the L chain, or the VH and CH1 regions of the H chain and set forth in SEQ ID NO: 2. Porphyrin Monoclonal Antibody or Fab Containing Amino Acid Sequence
A porphyrin monoclonal antibody containing the amino acid sequence of SEQ ID NO: 1 and the amino acid sequence of SEQ ID NO: 2 in a fragment. The primary structure of immunoglobulin G (IgG) is shown in FIG.

The immune system is generally considered as a defense mechanism of higher animals against pathogenic bacteria, but an immunoglobulin (Ig antibody) which specifically binds a non-self substance (immunogen, antigen) when it enters the living body. Is produced. The immunoglobulins all have basically similar structures,
It is composed of a heavy chain (H chain) and two light chains (L chain) of the main chain. The binding site to the antigen for both H and L chains is from the N-terminal
The amino acid sequence from the 100th to 110th amino acids varies depending on the specificity of the antibody. Therefore, this part is called a variable region (V region: VL, VH). The amino acid sequence after the V region is constant depending on the antibody class and is called the constant region (C region: CL, CH) (see FIG. 1). The root portion of the V-shaped structure which is divided into two branches is called a hinge region and can be easily cleaved with an enzyme such as papain or trypsin. The two fragments having the antigen-binding site obtained at this time are called Fab fragments.
The part of the Ig molecule that is directly involved in antigen recognition is considered to be the variable region, and in particular, the hypervariable region within that region.

The porphyrin monoclonal antibody of the present invention is a monoclonal antibody that specifically recognizes TCCP and is produced by the following method. That is, TCPP, which is an antigen, and KLH, which is a hapten, are covalently bound, and this is administered to mice to extract spleen cells from the immunized mouse to prepare fused cells with myeloma cells. It can be produced by screening a clone producing a desired antibody that recognizes the porphyrin represented by the general formula (I) from the fused cells and culturing the clone. In this method, a single cloned antibody-producing strain was previously injected with blistane about 1-2 weeks before Balb /
It is injected into the abdominal cavity of C mouse, and after 2 weeks, ascites fluid is collected, salted out with ammonium sulfate and purified by various chromatographies. As another method, there is a method in which antibody-producing cells are expanded and cultured in a serum-free medium to prepare antibodies. This fused cell is prepared to obtain a monoclonal antibody that recognizes the porphyrin represented by the general formula (I).

[0011] These antibodies are recognized to have different properties such as dissociation constants of porphyrins and oxidase activities. In the present invention, the following steps are carried out in obtaining the structural gene and determining the nucleotide sequence of one of these porphyrin monoclonal antibodies (produced by the 13-1 strain).

(A) Acquisition of mouse hybridoma antibody-producing cells (b) Preparation of mRNA from various cells (c) Synthesis of cDNA first strand (d) PCR of H and L chains (e) Treatment with restriction enzyme (e) f) Cloning and nucleotide sequencing with E. coli plasmid vector

The above steps will be described in detail below. (a) Acquisition of mouse hybridoma antibody-producing cells Monoclonal antibody-producing cells against porphyrin are prepared, and antibody-producing cells are prepared by the following steps in order to determine the structural gene of the antibody from the cells. TCPP is used as a hapten in preparing antibody-producing cells. First, this porphyrin was bound to the side chain of KLH, which is a carrier protein, and this was used as an adjuvant (Complete Freunds Adj.
uvant) and mixed into an emulsion, and injected as an antigen into the mouse (Balb / c) intraperitoneally. Thus, the spleen cells of the immunized mouse were taken out, the myeloma cells were cell-fused, and the antibody recognizing the porphyrin used as the hapten from the fused cells was treated with TCPP as an antigen.
Found by Linked Immunosorbent Assay (ELISA). Each antibody-producing cell is grown in the abdominal cavity of a mouse, and the ascites is cryopreserved (-80 ° C). At the time of mRNA preparation, each ascites is thawed and cultured in a serum-free medium for tissue culture to collect cells.

(B) Preparation of mRNA From cultured cells, a conventional method (for example, Molecular Cloning page 191 (1
982)) and extract total RNA and prepare poly (A) + RNA. That is, culturing cells are guanidinium thiocyanate (abbreviated as GTC) in order to dissociate RNA by activating RNase of the culturing cells and denaturing the protein.
And add N-lauroyl sarcosine and homogenize. The cell lysate is centrifuged to remove proteins and
A supernatant containing NA is obtained. Next, mRNA is prepared from this. RNA is purified by specific adsorption and desorption using a column having a poly A structure existing at the 3'end of mRNA and a oligo sequence dT structure complementary to the poly A structure as a ligand.
That is, a supernatant containing crude RNA is adsorbed on an oligo (dT) column that has been activated in advance and then eluted to obtain poly (A) + RNA.

(C) Synthesis of cDNA First Strand For synthesis of cDNA first strand, oligo dT is added to the obtained poly (A) + RNA, and this is used as a primer to act a reverse transcriptase. A DNA-RNA hybrid is synthesized and heat-denatured to obtain a DNA chain.

(D) Amplification of H and L chains (PCR: Pol
ymerase Chain Reaction) The first strand of the cDNA obtained in (c) above is a gene encoding the L chain and H chain of the antibody. In recent years, antibody studies have revealed that the L chain and the H chain have regions that form a well-conserved amino acid sequence in all antibodies, regardless of the type of monoclonal antibody.
Utilizing this finding, it has become possible to select monoclonal antibodies from blood cells and tissues from the immunized spleen and lymph nodes by a gene manipulation method. That is, the following method is usually used for screening for producing monoclonal antibody-producing cells. Mouse and human lymphocytes are purified, cells are thawed, and then mRNA is isolated. Separation m
A cDNA chain is synthesized from RNA using an oligo (dT) primer. Next, an amplification primer specific to the H and L chains was used to
CR is performed to amplify the cDNA fragment. The H chain amplification product thus amplified was pooled and the H chain vector (Stratacyt
e company). Similarly, the L chain amplification product is pooled and ligated to the L chain vector. This is subjected to lambda phage packaging and plated on a petri dish to obtain a library. Fage DNA from the H chain library is cut with a restriction enzyme (EcoRI, HindIII in the case of Stratagene). Similarly, L chain library
The phage DNA from E. coli is digested with restriction enzymes (EcoRI, MluI in the case of Stratagene). After combining the H chain and L chain products to create a combination library, packaging for screening for the antigen-binding protein,
This is a method of screening by expressing through an operation of plating (ImmunoZap ™ Cloninng
and Expression System, Stratacyte). This method
Since 10 million clones are screened within one week, the number of screened clones can be significantly increased as compared with the conventional hybridoma method.

In the present invention, H chain and L chain are amplified using this method. That is, 5'for amplification of the L chain
PCR was carried out using VL (κ) (described in Table 3) as the side primer and CL (κ) (described in Table 3) as the 3 ′ side primer. For amplification of H chain, CH1 was used as a 3'-side primer.
(γ1) (described in Table 4) was used, and PCR was carried out by a usual method using four types of primers (described in Table 4) of VHa, VHb, VHc, and VHd as 5′-side primers.

(E) Restriction enzyme treatment An enzyme having restriction enzyme sites on both sides of the L chain region in the DNA chain amplified by the PCR method (for example, SacI and Xba).
After treatment with I), the cleavage reaction product is subjected to agarose electrophoresis to extract a DNA fragment of about 700 base pairs (see FIG. 2).
The extracted fragment DNA is ligated with the ImmunoZapL vector and subjected to packaging using Giga Pack Gold Kit (Stratagene). Next, Escherichia coli XL-1Blue was transduced to prepare phagemid DNA by alkali extraction,
Retreat with a restriction enzyme (eg, SacI-XbaI). The cleavage reaction product is subjected to agarose electrophoresis and the excised DNA fragment is extracted. Similarly, for the H chain, the H chain in the DNA fragment amplified by the PCR method is treated with a suitable restriction enzyme (for example, XhoI and SpeI) to cut it out (see FIG. 3). This digestion reaction product is then subjected to agarose electrophoresis to extract a DNA fragment. Furthermore, by ligating with ImmunoZap H vector, packaging, E. coli XL-Blue transduction, phagemid extraction, DNA alkaline extraction, restriction enzyme (eg XhoI and SpeI) treatment, agarose electrolysis as with L chain. Isolate the DNA fragment by electrophoresis.

(F) Escherichia coli plasmid vector cloning and nucleotide sequence determination For both the H chain and L chain clones, a conventional sequencing method is used. In this case, M13mp18 and M13mp19 are subcloned into a single chain sequence. The nucleotide sequence is determined by the Kens method.

[0020]

INDUSTRIAL APPLICABILITY The structural gene of the present invention is inserted into an expression vector to prepare a recombinant plasmid that can be expressed in a suitable host cell, and the host cell is transformed to obtain a recombinant host cell (transformant). The recombinant host cell (transformant) can be cultured under conditions capable of expressing the structural gene of the present invention to produce and recover a porphyrin monoclonal antibody. That is, by using a structural gene of the porphyrin monoclonal antibody of the present invention, a transformant obtained by transforming a host cell with a recombinant plasmid containing the gene, the porphyrin monoclonal antibody is artificially controlled under conditions. It is possible to stably produce the cells under the conditions described below, and it is possible to significantly shorten the number of culture days or significantly increase the productivity. Further, the structural gene of the present invention can be modified into an antibody having desired properties by changing the nucleotide sequence.

[0021]

EXAMPLES The present invention will be described in detail below with reference to examples. Example 1 (1) Culture of hybridoma cells (porphyrin monoclonal antibody-producing cells) Anti-porphyrin monoclonal antibody-producing cells were prepared by the method described above. That is, TCPP was used as a hapten. First, this porphyrin was attached to the side chain of the carrier protein KLH.
Covalently bonded using 1,1'-carbonyldiimidazole,
This was mixed with complete Freund's adjuvant, and the suspension was used as an antigen to intraperitoneally inject into mice (Balb / C). Thus, the splenocytes of the immunized mouse were taken out, fused with myeloma cells, and the antibody that recognizes the porphyrin used as the hapten from the fused cells was TCPP.
5 types were found by ELISA using as an antigen, and each was frozen and stored. Here, one of the anti-porphyrin antibody-producing cells, strain 13-1 (-800C, frozen storage) was cultured by the following method. That is, about 1 ml of the antibody-producing cells which had been frozen and stored in the serum tube were thawed in a 37 ° C warm bath. Next, this was transferred to a sterilized centrifuge tube, mixed with 10 ml of a cell glosser-H (tissue culture serum-free medium, manufactured by Sumitomo Pharmaceutical Co., Ltd.) solution containing 50 μg / ml of gentamicin sulfate, and then centrifuged (1000 rpm, 5 minutes), The supernatant was removed with an aspirator. After repeating this operation again, the cell glosser H
Add 30 ml of the solution, and add about 10 ml to each of 3 plates (for tissue culture, outer diameter x height = 94 x 21: Iwaki Glass 25020 DISH100
N) and incubate at 37 ° C, 5% CO ₂ for 3 days.
I got it. Further, the culture medium was subcultured to a new dish, and the culture medium of the plate in which the cells were sufficiently grown was removed by an aspirator to remove the floating dead cells. Based on these cells, about 10 ml of CellGlosser H was added to the plate.
The solution was added, and the plates were expanded to 16 plates. When all plates were fully grown, cells were harvested by centrifugation.

(2) Preparation of mRNA (1) Cell disruption and extraction of total RNA To the cells recovered by centrifugation, 1.5 ml of extraction buffer (guanidinium thiocyanate, N-lauroyl sarcosine) was added to give 26- The solution was homogenized by putting the solution in and out with a syringe equipped with a G needle. Add the elution buffer (10 mM Tris-HCl (pH 7.
4), 1 mM EDTA) 3 ml was added, briefly homogenized, transferred to a sterilized polypropylene tube, and centrifuged (1
The supernatant was collected by performing (000 rpm, 5 minutes).

(2) Purification of mRNA The total RNA extract obtained by the above method was charged on an oligo (dT) -cellulose span column (Quick PrepTM mRNA purification kit, Pharmacia), and then the column was purified. The head was covered and shaken for 10 minutes. Then centrifuge (350G, 2
Minutes) and the supernatant was removed by decantation. High salt buffer (10mM Tris-HCl (pH7.4), 1mM EDTA, 0.5MN
aCl) was injected, and the mixture was gently shaken and then centrifuged (350 G, 2 minutes), and the supernatant was removed by decantation. Then remove the lid from the column and remove the low salt buffer solution (10 mM Tris-HCl (pH7.4), 1 mM ED
Inject 3 ml of TA, O.1M NaCl), shake gently, and then centrifuge.
(350 G, 2 minutes), and the buffer solution was removed. Then sterilized 1.
A 5 ml screw top Eppendorf tube was placed in a centrifuge tube, placed so that the lower part of the column entered the opening of the Eppendorf tube, and the elution buffer (10 mM) that had been incubated at 65 ° C. Tris-HCl (pH7.4), 1 mM EDTA) 250
μl was injected from the top of the column, centrifuged (350 G, 2 minutes) and eluted. The operation was performed twice more. M recovered
RNA quantification was performed by measuring the absorbance at 260 nμ (A26
0) was measured. [RNA concentration] = (A260) × 40 μg / ml The prepared sample was a glycogen solution (10 mg / ml).
10 μl of DEPC treated water and 1/10 volume of potassium acetate solution (2.
5M potassium acetate (pH 5.0)) and 2- to 2.5-fold amount of ethanol were added, and the mixture was stored at -80 ° C.

The amount of RNA obtained by this method is as follows.

[Table 1]

(3) cDNA synthesis RNA (8.3 μg) obtained from strain 13-1 was dissolved by adding distilled water in an Eppendorf tube (completely autoclaved) to prepare oligo (dT) 18 primer. -4 μl (400 mg) was added, and 6
After heating at 5 ° C for 5 minutes, the mixture was left at room temperature for 45 minutes. Furthermore, the following reagents were further added to this mixed solution. 10 × 1 strand cDNA synthesis buffer 10 μl 40 U / μl Human placenta-derived ribonuclease inhibitor 10 μl (400 units) 10 mM dNTP 5 μl (50 nmol) Reverse transcriptase (Life Sciences AMVRTxL) 2 μl

This reaction mixture was heated at 41 ° C. for 90 minutes to carry out an enzymatic reaction. The estimation of the amount of synthesized cDNA was performed as follows. That is, 5 μl of the reaction solution was taken, and 0.5 μl of [ ³² P] dCTP was added thereto to carry out the reaction.
After the reaction, the reaction was stopped by cooling to 40 ° C. The measurement of [ ³² P] dCTP uptake for monitoring the progress of the reaction was carried out by dropping 1 μl of the reaction solution after completion of the reaction onto each of two Whatman GF / C filters, and
After air-drying the sheets, soak them in scintillation liquid and all [ ³² P] d
CTP was counted. The other one is 5% TCA / 20 mM
The filter was washed with sodium pyrophosphate, distilled water and ethanol in this order to incorporate the cDNA [ ³²
The progress of the reaction was estimated by counting P] dCTP. The amount of cDNA synthesized was 0.17 μg.

(4) Amplification by PCR Next, amplification of the first strand of the prepared cDNA by PCR was performed using an ImmunoZap ™ cloning kit (Stratatacyte). (1) Confirmation of the antibody genes first Monokurona - for Le antibody gene is confirmed that it is obtained by the above method, the primer well highly conserved CH _2, CH ₃ region in the antibody gene structure as a control experiment -CH _2, CH ₃ (primer for IgG1 -) was confirmed to be amplified using (Table 2).

The following composition was used as a reaction composition for amplification. First-strand synthesis reaction solution 5 μl 2 mM dNTPs 10 μl Primer (CH ₂ , CH ₃ ) 1.5 μl 10 x Reprisem buffer 10 μl distilled water 71.5 μl This reaction mixture was treated at 94 ° C. for 5 minutes, then at 54 ° C. 5
Incubate for minutes, then 0.5 μl of ReprisemTM (25 Units) (Ep
Add icentre's thermostable DNA polymerase, and add (72
After repeating 39 cycles of treatment at ℃ for 3 minutes, treatment at 93 ℃ for 1.5 minutes and treatment at 54 ℃ for 2.5 minutes, the mixture was left at 72 ℃ for 10 minutes and cooled to 40 ℃. As a result of amplification by this PCR, for the 13-1 strain, PCR of 595 bp CH ₂ -CH ₃ was performed.
A band considered to be a product was confirmed, and it was revealed that the antibody gene was obtained.

[0029]

[Table 2]

(2) Amplification of L chain The PCR of the L chain was carried out under the same conditions as the experiment for confirming the antibody gene. That is, PCR was performed using VL (κ) (described in Table 3) as the 5′-side primer and CL (κ) (described in Table 3) as the 3′-side primer. Since the N-terminal amino acid sequence of the antibody protein of the 13-1 strain has been determined, the 5'-primer oligomer VL (κ) · 13-1 (see Table 3) is shown below. PCR was performed using As a result, amplification of the DNA fragment in the target region was observed.

[0031]

[Table 3]

(3) Amplification of H Chain Amplification of the H chain was also carried out in the same manner as the confirmation experiment of the antibody gene.
As a result of PCR using CH1 (γ1) (described in Table 4) as the 3′-side primer and four kinds of primers VHa to VHd (described in Table 4) as the 5′-side primers, VHb was obtained.
Amplification of the antibody gene was observed when amplified with the -CH1 (γ1) primer (described in Table 4).

[0033]

[Table 4]

(5) Restriction enzyme treatment and cloning (1) Restriction enzyme treatment and cloning of L chain amplification product PCR product obtained by amplification of L chain (total amount of PCR reaction solution extracted with phenol / chloroform) , Ethanol precipitation was performed, electrophoresis was performed, and the target band was recovered with the Gene Clean II kit and dissolved in distilled water) and digested with a restriction enzyme under the following conditions. L chain PCR product 25 μl 10 × T buffer 20 μl O.1% BSA 20 μl SacI (12 u / ul) 6 μl (60 U) XbaI (8 u / ul) 7.5 μl (60 U) Distilled water 147.5 μl After a total of 200 μl of the above reaction mixture was reacted at 37 ° C. for 20 minutes, the reaction mixture was subjected to 1.5% agarose gel electrophoresis. After the electrophoresis, a DNA fragment of about 700 bp was extracted with a gel extraction kit (BIO-101, Gene CleanII kit).

Next, the prepared DNA fragment was subjected to Immu by the following procedure.
The noZAP L vector (Fig. 2) was ligated. ImmunoZAP L vector (1 μg / μl) 1 μl (1 μg) XbaI-SacI DNA fragment (0.1 μg / μl) 0.3 μl (0.03 μg) T4 DNA ligase (1 U / μl) 0.5 μl (0.5 U) 5 × ligation buffer 2 μl Distilled water 6.2 μl Total 10 μl

The reaction mixture was reacted overnight at a reaction temperature of 4 ° C. After the reaction, the cells were packaged using Lambda DNA in vitro packaging kit (Giga Pack Gold Kit, stratagene), and then infected with E. coli XL-1 Blue,
The obtained plaque was treated with SM buffer (NaCl 5.8 g / l, MgSO ₄
2.0g / l, 1M Tris-HCl (pH7.5) 50ml / l, 2% gelatin 5ml /
l): Chloroform = 25: 1 was suspended in 500 μl to obtain a stock of λ phage. 50 for E. coli XL-1 Blue
μl of the phage stock solution (1 × 10 ⁵ pfu) was double-infected with helper phage R408 (1 × 10 ³ pfu) and incubated at 37 ° C. for 3 hours. This culture solution was treated at 70 ° C. for 20 minutes and centrifuged, and a single chain of Bluescript was obtained in the supernatant. E. coli XL-Bl using this single-stranded blue script
ue was transformed to obtain cells having a double-stranded blue script. Phagemid DNA from the cells according to the alkaline method
Was extracted. The prepared DNA was reconverted into Xba I and Sac
The reaction solution treated with I was subjected to 1.5% agarose electrophoresis in the same manner as above to extract a DNA fragment of about 700 bp (see FIG. 2).

(2) Restriction enzyme treatment and cloning of H chain amplification product
The PCR product obtained by amplifying the Ning H chain was digested with a restriction enzyme under the following conditions. H chain PCR product 25 μl 10 × K buffer 20 μl XhoI (12u / ul) 5 μl (60 U) SpeI (10 u / ul) 6 μl (60 U) Distilled water 144 μl Total 200 μl Similar to the case of L chain, reaction temperature 37 ° C
And reacted for 2 hours. After the reaction, about 700b as in the case of L chain
The DNA fragment of p was extracted by gel, ligated to the Immuno ZAP H vector (Fig. 3), and then subjected to the Lambda DNA in vitro packaging kit (Giga Pack Gold Kit, strat).
agene) and then E. coli XL-
1 Blue was infected and 5 ml of the culture solution was centrifuged, and the supernatant was subjected to a phage. Thereafter, cells having a double-stranded blue script were obtained in the same manner as the restriction enzyme treatment and cloning of the L chain amplification product. According to the alkaline method, the phagemid D was obtained from the cells.
NA was extracted. This prepared DNA was again converted into XhoI and SpeI.
Using the reaction solution treated with step 1, 1.5% agarose electrophoresis was performed as described above to extract a DNA fragment of about 700 bp (see FIG. 3).

(6) Subcloning into E. coli plasmid vector and sequence (1) Subcloning into Escherichia coli plasmid vector For L chain, 5- (1) The prepared DNA fragment was M13mp18,
Subcloning was performed on the XbaI-SacI site of M13mp19 according to a conventional method. Regarding the H chain, the DNA fragment prepared in 5- (2) was also subcloned into the XhoI-SpeI sites of M13mp18 and M13mp19 in the same manner. The nucleotide sequence of the thus subcloned plasmid was determined by the single-stranded sequence method. Sequence is Sequence 2.0.-Deaza-dGTP
It was determined using a kit (USB) and a T7 sequencing kit (Pharmacia).

A base sequence was obtained by the above method, and
As shown in FIG. 3, a region well conserved in the variable region and the constant region was observed in both the L chain and H chain.

[0040]

[Sequence list]

SEQ ID NO: 1 Sequence length: 612 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA to mRNA Origin Biological name: Mouse Strain name: Porphyrin monoclonal antibody-producing cell 13-
1 strain Sequence characteristics: Characteristic symbol: domain Location: 1..291 Method of determining characteristic: E Other information: VL domain Characteristic symbol: domain Location: 316..624 Method of determining characteristic : E Other information: CL domain sequence GCT TCT TTG GCT GTG TCT CTG GGG 24 Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 1 5 10 15 CAG AGG GCC ACC ATC TCA TGC AGG GCC AGC AAA AGT GTC AGT GCA TCT 72 Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Lys Ser Val Ser Ala Ser 20 25 30 GGC TAT ATT TAT ATG CAC TGG TAC CAA CAG AAA CCA GGA CAG CCA CCC 120 Gly Tyr Ile Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 AAA CTC CTC ATC TCT CTT GCA ACC AAC CTA GAA TCT GGG GTC CCT GCC 168 Lys Leu Leu Ile Ser Leu Ala Thr Asn Leu Glu Ser Gly Val Pro Ala 50 55 60 AGG TTC AGT GGC TCT GGG TCT GGG ACA GAC TTC ACC CTC AAC ATC CAT 216 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His 65 70 75 80 CCT GTG GAG GAG GAG GAT GTT GCA ACC TAT TAC TGT CA G CAC AGT AGG 264 Pro Val Glu Glu Glu Asp Val Ala Thr Tyr Tyr Cys Gln His Ser Arg 85 90 95 GAG CTT CCG CTC ACG TTC GGT GCT GGG ACC AAG CTG GAG CTG AAA CGG 312 Glu Leu Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg 100 105 110 GCT GAT GCT GCA CCA ACT GTA TCC ATC TTC CCA CCA TCC AGT GAG CAG 360 Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln 115 120 125 TTA ACA TCT GGA GGT GCC TCA GTC GTG TGC TTC TTG AAC AAC TTC TAC 408 Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr 130 135 140 CCC AAA GAC ATC AAT GTC AAG TGG AAG ATT GAT GGC AGT GAA CGA CAA 456 Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln 145 150 155 160 AAT GGC GTC CTG AAC AGT TGG ACT GAT CAG GAC AGC AAA GAC AGC ACC 504 Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr 165 170 175 TAC AGC ATG AGC AGC ACC CTC ACG TTG ACC AAG GAC GAG TAT GAA CGA 552 Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg 180 185 190 CAT AAC AGC TAT ACC TGT GAG GCC ACT CAC A AG ACA TCA ACT TCA CCC 600 His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro 195 200 205 ATT GTC AAG AGC 612 Ile Val Lys Ser 210

SEQ ID NO: 2 Sequence length: 615 Sequence type: Nucleic acid Number of strands: Double strand Topology-: Linear Sequence type: cDNA to mRNA Origin organism name: Mouse strain name: Porphyrin monoclonal Antibody-producing cell 13-
1 strain Sequence features Characteristic symbol: domain Location: 1..315 Method of determining feature: E Other information: VH domain Characteristic symbol: Location: 316..615 Method of determining feature: E Other information: CH domain sequence GGA GGC TTG GTG AGG CCT GGA AAT 24 Gly Gly Leu Val Arg Pro Gly Asn 5 TCT CTG AAA CTC TCC TGT CTT ACC TCG GGG TTC ACT TTC AGT AAC TAC 72 Ser Leu Lys Leu Ser Cys Leu Thr Ser Gly Phe Thr Phe Ser Asn Tyr 10 15 20 CGG ATG CAC TGG CTT CGC CAG CCT CCA GGG AAG AGG CTG GAG TGG ATT 120 Arg Met His Trp Leu Arg Gln Pro Pro Gly Lys Arg Leu Glu Trp Ile 25 30 35 40 GCT GTA ATT ACA GTC AAA TCT GAT AAT TAT GGA GCA AAA TAT GCA GAG 168 Ala Val Ile Thr Val Lys Ser Asp Asn Tyr Gly Ala Lys Tyr Ala Glu 45 50 55 TCT GTG AGA GGC AGA TTC ACT ATT TCA AGA GAT GAT TCA AAA AGC AGT 216 Ser Val Arg Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser 60 65 70 GTC TAC CTG CAG ATG AAC AGA TTA AGA GAG GAA GAC ACT GCC ACT TAT 264 Val Tyr Leu Gln Met Asn Arg Leu Arg Glu Glu Asp Thr Ala Thr Tyr 75 80 85 TAC TGT TGT AGA ACC CCC TGG GTC TAT GCT ATG GAC TGC TGG GGT CAA 312 Tyr Cys Cys Arg Thr Pro Trp Val Tyr Ala Met Asp Cys Trp Gly Gln 90 95 100 GGA ACC TCA GTC ATC GTC TCC TCA GCC AAA ACG ACA CCC CCA TCT GTC 360 Gly Thr Ser Val Ile Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val 105 110 115 120 TAT CCA CTG GCC CCT GGA TCT GCT GCC CAA ACT AAC TCC ATG GTG ACC 408 Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr 125 130 135 CTG GGA TGC CTG GTC AAG GGC TAT TTC CCT GAG CCA GTG ACA GTG ACC 456 Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr 140 145 150 TGG AAC TCT GGA TCC CTG TCC AGC GGT GTG CAC ACC TTC CCA GCT GTC 504 Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val 155 160 165 CTG CAG TCT GAC CTC TAC ACT CTG AGC AGC TCA GTG ACT GTC CCC TCC 552 Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser 170 175 180 AGC ACC TGG CCC AGC GAG ACC GTC ACC TGC AAC GTT GCC CAC CCG GCC 600 Ser Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala His Pro Ala 185 190 195 200 AGC AGC ACC AAG GTG 615 Ser Ser Thr Lys Val 205

[Brief description of drawings]

FIG. 1 shows the primary structure of immunoglobulin G (IgG).

FIG. 2 shows the Immuno ZAP L vector.

FIG. 3 shows the Immuno ZAP H vector.

Continuation of front page (51) Int.Cl. ⁵ Identification number Office reference number FI Technical display location C07K 15/28 8517-4H C12N 5/20 15/06 (C12N 15/12 C12R 1:91) (C12P 21 / 08 C12R 1:91) 8931-4B C12N 15/00 C

Claims (6)

[Claims] 1. A compound represented by the general formula (I):(In the formula, R¹, R², R³And R^FourIs H or COOH
Is. However, R¹= R ²= R³= R^FourExcept when = H
Monochrome that specifically recognizes porphyrin
Structural gene of lonal antibody. 2. The monoclonal antibody structural gene according to claim 1, which contains the nucleotide sequence of SEQ ID NO: 1 in the VL and CL regions of the L chain. 3. The monoclonal antibody structural gene according to claim 1, which contains the nucleotide sequence set forth in SEQ ID NO: 2 in the VH and CH1 regions of the H chain. 4. A porphyrin monoclonal antibody containing the amino acid sequence set forth in SEQ ID NO: 1 in the VL and CL regions of the L chain. 5. A porphyrin monoclonal antibody containing the amino acid sequence set forth in SEQ ID NO: 2 in the VH and CH1 regions of the H chain. 6. A porphyrin monoclonal antibody containing the amino acid sequence set forth in SEQ ID NO: 1 and the amino acid sequence set forth in SEQ ID NO: 2 in a Fab fragment.