JPH10259199A - Monoclonal antibody recognizing ferredoxin and hybridoma producing the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject new antibody that comprises a monoclonal antibody specifically recognizing ferredoxin and can be widely utilized in purifications including the thermal modification of the fused antigen linking to ferredoxin, a protein inducing heatresistant properties. SOLUTION: This novel monoclonal antibody specifically recognizes ferredoxin and the fused protein bearing heat-resistant ferredoxin as an expression-inducing protein also maintains resistance to heat, thus they can be widely utilized for purification and identification including the thermal modification of the expressed fused protein. This monoclonal antibody is obtained by immunizing an animal as a mouse with ferredoxin originating from Thermophilus, Theremotoga or Thermococcus, together with an adjuvant, after the antibody value increases, collecting its spleen cells, fusing them with myeloma cells to form a hybridoma, selecting the strain producing the anti- ferredoxin antibody through the enzyme immunoassay, cloning the hybridoma by the limiting dilution technique and culturing the cloned hybridoma.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a monoclonal antibody that specifically recognizes ferredoxin and a hybridoma that produces the monoclonal antibody.

[0002]

2. Description of the Related Art Advances in genetic engineering have made it possible to analyze proteins that have been purified from natural products at the gene level and artificially amplify the target protein (Itakura).
et.al, Science, 198 1056 (1977)). Thioredoxin invented thereafter (hereinafter referred to as TRX in the present specification) (Table 5) and glutathione-S
-Transferase (hereinafter referred to as GST in the present specification) (Japanese Patent Application Laid-Open No. 1-503441);
By applying the NA sequence, it is possible to express a protein that is originally difficult to express, and a technique for expressing a fusion protein has been widely used.

[0003] However, TRX and GST may have problems with the solubility of the expressed protein and nonspecific reactions derived from TRX or GST.
It has been found that ferredoxin (hereinafter referred to as FDX in the present specification) (Japanese Patent Application No. 8-356739) is useful as an expression-inducing protein replacing TRX and GST.
According to the invention, when FDX is used as an expression-inducing protein, the fusion protein to be expressed also maintains heat resistance, so that a heat denaturing means can be conveniently used in the purification process of the expressed substance. However, when purifying a large amount of protein that is easy to use industrially, in addition to the purification method using thermal denaturation, unification for applying affinity chromatography and confirming the expressed FDX fusion protein is also required. Therefore, an antibody that specifically recognizes FDX was required.

[0004]

SUMMARY OF THE INVENTION Accordingly, an object of the present invention is to provide a monoclonal antibody that specifically recognizes FDX.

[0005]

Means for Solving the Problems The present inventors have made intensive studies on antibodies specifically recognizing FDX, and
The present invention was completed by establishing a monoclonal antibody that specifically recognizes X and a hybridoma producing the monoclonal antibody.

That is, the present invention relates to a monoclonal antibody that specifically recognizes FDX and a hybridoma that produces the monoclonal antibody.

[0007] By using the monoclonal antibody of the present invention and the hybridoma producing the monoclonal antibody, F
DX and FDX fusion proteins can be directly detected,
It can be widely applied to purification of FDX and FDX fusion protein, immunoassay and the like.

Hereinafter, the present invention will be described in detail.

Since the antibody of the present invention is a monoclonal antibody that specifically recognizes FDX, it is preferable to use whole FDX or a part of FDX as an immunogen of the antibody. Not something. Preferably, FDX derived from a thermophilic bacterium, and more preferably FDX derived from Pyrococcus fuliosis,
The monoclonal antibody is not limited to these as long as it is a monoclonal antibody that can recognize FDX in a fusion protein obtained by fusing DX with a target substance.

[0010] To prepare the monoclonal antibody of the present invention, the whole or a part of FDX peptide is purified or synthesized and used as an immunogen for producing the antibody. FDX and F
Since the specificity to the DX fusion protein can be selected at the time of hybridoma screening, it is preferable to use the whole FDX as the immunogen in view of the simplicity of the technique.

[0011] The antigen thus prepared is immunized to an immunized animal such as a rabbit, a guinea pig, or a mouse. Immunization can be performed according to a conventional method, and the antigen is administered to an immunized animal alone or together with an adjuvant. After immunization, serum is collected after confirming an increase in antibody titer, but booster immunization may be performed if necessary. After confirming the increase in antibody titer, antibody-producing cells such as spleen cells and tumor cells such as myeloma cells are fused with a fusion agent such as polyethylene glycol to produce a hybridoma. The hybridomas are then selected using a selective medium such as HAT medium,
Monoclonal cells are cultured by an appropriate method such as a limiting dilution method and cultured. The culture supernatant is analyzed by a suitable immunoassay such as an enzyme immunoassay, and a clone producing the desired anti-FDX antibody is selected. Techniques for producing these monoclonal antibodies are known methods, for example, Koehler and Milstein (Nature 256 495 1975), Scherer (Natu
re 285 446 1980).
In addition, the monoclonal antibody produced by the above method
Ascites obtained by administering the hybridoma producing the monoclonal antibody to the pristane-treated mouse, or a culture supernatant of the hybridoma producing the monoclonal antibody, salt-folding, ion-exchange chromatography, gel filtration chromatography, etc. It can be recovered by means of analysis and purification.

Immunoassay for FDX and FDX fusion protein,
For purification by affinity chromatography, etc.
The present invention is particularly useful.

[0013]

EXAMPLES The present invention will be described in more detail with reference to Examples and Examples.

Reference Example 1 Preparation of FDX Examples 1 and 2 described in Japanese Patent Application No. 8-356739.
In the same manner as in the above, a recombinant FDX was prepared. Specifically, eight 53-mer primers prepared using a DNA synthesizer (Model 392, manufactured by Perkin Elmer) based on the known DNA sequence of FDX derived from P. phyllococcus friosis were used to assemble the phyllococcus phyllosis. The gene of FDX derived from Coccus furiosis was synthesized. Taq polymerase (manufactured by Toyobo Co., Ltd.) was used for the assemble PCR method.
C.-1 minute, 30 cycles, total number of bases 248 bp
Was amplified. A restriction enzyme NdeI site was added to the 5 'end, a restriction enzyme EcoRI site was added to the 3' end, and a thrombin cleavage site was added to the C end. This fragment was obtained from a 4.6 Kb pW6A vector NdeI, Ec prepared from pGEMEX-1 (Promega) and pGEX-2T (Pharmacia).
pWF6A was constructed as a vector that was integrated into the oRI site and expressed FDX.

PWF6A contains a fusion protein gene consisting of 96 amino acids including 67 amino acids derived from FDX, 10 amino acids derived from thrombin cleavage site and 19 amino acids derived from C-terminal pWF6A at NdeI and EcoRI sites. The nucleotide sequence of the inserted fragment was confirmed using a DNA sequence kit (Sequenase Kit Ver. 2.0, manufactured by Amersham United States Biochemical).

After introducing pWF6A into host E. coli, 1% bactotryptone, 0.5% yeast extract, 1%
% Sodium chloride, 50 μg / ml ampicillin, pH
After culturing in 7.5 medium (hereinafter referred to as LB medium in this specification) for 2 hours, adding 1 mM isopropylthiogalactopyranoside (hereinafter referred to as IPTG in this specification) and culturing for 2 hours To induce expression. 10 mM Tris-hydrochloric acid pH 7.5, 1 mM ethylenediaminetetraacetic acid (hereinafter, referred to as EDTA in the present specification) was added to the E. coli precipitate, and the mixture was sonicated.
% Sodium dodecyl sulfate polyacrylamide gel electrophoresis (hereinafter referred to as SDS-PAGE in the present specification). Approximately 22K by Coomassie brilliant blue staining (hereinafter referred to as CBB staining)
A band was confirmed near da, and it was recognized as FDX of Phycoccus furiosis which formed a dimer.

After introducing the produced pWF6A into host E. coli, the cells were cultured in LB medium at 37 ° C. The concentration of Escherichia coli in the culture solution was adjusted to about 1.0 at a wavelength of 600 nm in the preliminary culture.
After the turbidity of 1 mM, 1 mM IPTG was added to induce expression. After culturing for 3 hours, centrifugation was performed to collect Escherichia coli.
A 50 mM Tris-HCl buffer pH 8.
0 (hereinafter, referred to as Tris buffer in the present specification) was added, and sonication was performed under ice cooling. The expressed fusion protein was recovered in the supernatant as a soluble component after centrifugation. This supernatant was subjected to a heat treatment at 85 ° C. for 15 minutes, and 90% or more of FDX was recovered in the centrifuged supernatant after the heat treatment.

The centrifuged supernatant was subjected to ion exchange purification using a QFF anion exchange column (Pharmacia) equilibrated with Tris buffer. When eluted with a column equilibration buffer containing sodium chloride, FDX was recovered in a fraction eluted at a concentration of about 0.3 M sodium chloride. This FDX fraction was then re-equipped with Resource R equilibrated with 20 mM sodium hydroxide.
Purification was performed using a PC reversed-phase column (Pharmacia). After elution with acetonitrile, purified FDX was recovered in a fraction eluted with about 10% acetonitrile.

Reference Example 2 Preparation and Purification of TRX pWT8A prepared as a TRX-expressing vector in the same manner as pWF6A prepared in Reference Example 1 was introduced into host Escherichia coli, and then cultured in LB medium at 37 ° C. After the same expression induction as in Reference Example 1, E. coli was recovered by centrifugation. Osmotech shock was applied to the recovered E. coli, and TRX in the periplasmic fraction was extracted. Extraction TRX is 20m
The first purification was performed on a resource RPC reverse phase column (manufactured by Pharmacia) equilibrated with M sodium hydroxide. After elution with acetonitrile, TRX was recovered in the fraction eluted with about 10% to 20% acetonitrile. TR collected
X was dialyzed against 4M guanidine hydrochloride, and then subjected to a second purification using a reversed-phase column under the same conditions. About 10% ~
The purified TRX was recovered in a fraction eluted with 20% acetonitrile.

Example 1 Preparation of Monoclonal Antibody Recombinant FDX purified in Reference Example 1 was immunized by the following method. That is, using Freund's complete adjuvant (manufactured by Difco) as an adjuvant, BALB / c mice were inoculated three times by intraperitoneal injection with 100 μg per mouse. Blood was collected from the tail vein of the mouse, and an increase in serum antibody titer was confirmed by ELISA using a 96-well assay plate on which an immunogen was immobilized. The final immunization was performed by intraperitoneal administration, and three days later, the spleen was excised, and the cells were fused according to the Koehler-Milstein standard method (Nature 256, 495, 1975). The mouse myeloma cell line P3-X63-Ag8-U1 (P3U1) was used as a parent cell.
Polyethylene glycol was used as a fusing agent. The fused cells were cultured in HAT medium, and about 14 days later, the culture supernatant was subjected to primary screening by ELISA. That is, each of the recombinant FDX and the recombinant TRX prepared in Reference Example 1 and Reference Example 2 had a concentration of 2 μg.
/ Ml was immobilized on a 96-well plate, and then the culture supernatant was reacted, and its reactivity was compared with anti-mouse Ig-POD (manufactured by Dako). Wells that specifically reacted only to FDX were selected, and the cells were cloned by the limiting dilution method to establish hybridoma Fdx1. still,
This hybridoma has been deposited with the Institute of Biotechnology and Industrial Technology, and its accession number is FERM P-16046.

The thus-established hybridomas were inoculated into the abdominal cavity of mice to which 0.5 ml of pristane had been previously administered intraperitoneally at 1 × 10 ⁷ mice.
After 1 week to 10 days, the collected ascites fluid was collected, and the monoclonal antibody was purified using a Protein A column (manufactured by Bio-Rad) -MAPS-II kit buffer. A monoclonal antibody produced by hybridoma Fdx1,
It was named Fdx1 antibody.

The monoclonal antibody was isotyped by Western blotting using a kit manufactured by Amersham and found to be IgG1 · κ.

Example 3 Reactivity Test of Monoclonal Antibody Using FDX and TRX prepared in Example 1 and Reference Example 1, the specificity of the monoclonal antibody was confirmed by ELISA and Western blot.

That is, in the ELISA, the above FDX and TRX were transferred to a 96-well microplate (manufactured by Falcon).
Was sensitized by being left at 4 ° C. overnight. The sensitized plate was placed in a phosphate physiological buffer (hereinafter referred to as PB in this specification).
S) was washed with a solution containing 0.05% Tween 20 (a nonionic surfactant, manufactured by Sigma) (hereinafter, referred to as a washing solution), and 2% bovine serum albumin (hereinafter referred to as washing solution). Hereinafter, referred to as BSA in the present specification), and 150 μl of PBS containing the mixture is left at 37 ° C. for 2 hours.
The plate surface was coated with BSA.

Thereafter, the sensitized plate was washed, and 50 μl of the culture supernatant of the hybridoma Fdx1 was added.
The reaction was carried out with purified FDX and TRX for 2 hours. Thereafter, the sensitized plate was washed, and 1 part of anti-mouse Ig-POD (manufactured by Dako) diluted with a 2000-fold washing solution was added.
After adding 00 μl, the reaction was carried out at 37 ° C. for 2 hours.
After sufficiently washing with a washing solution, 0.1% 2,2'-azobis (3-ethylbenzothiazoline-6-sulfonic acid (AB
200 μl of 0.005% hydrogen peroxide solution containing TS)
After the addition and reaction at room temperature for 20 minutes, the color development at 405 nm was measured. The Fdx1 antibody specifically reacted only with FDX.

On the other hand, Western blot was performed using the FDX
And TRX were subjected to SDS-PAGE on a 12.5% polyacrylamide gel by the Lemley method, the gel subjected to electrophoresis was transferred to a nitrocellulose membrane, and PBS containing 2% BSA was added thereto. The nitrocellulose membrane was coated with BSA. After that, wash the transfer film,
The cells were cut and reacted with the supernatant of the hybridoma Fdx1 culture solution for 2 hours at room temperature. Then, the shredded transfer membrane was washed, and anti-mouse Ig-POD (manufactured by Dako) diluted 2000 times with the washing solution was used for 2 hours at room temperature. Reacted. After sufficient washing with the washing solution
Color was developed with 4-chloronaphthol containing a 0.005% hydrogen peroxide solution at room temperature. The results are shown in FIG. Fdx1
The antibody specifically reacted only with FDX.

[0027]

According to the present invention, a monoclonal antibody against FDX and a hybridoma producing the monoclonal antibody can be provided.

[Brief description of the drawings]

FIG. 1 is a diagram of a Western blot of Fdx1, FDX and TRX.

──────────────────────────────────────────────────の Continuation of the front page (51) Int.Cl. ⁶ Identification code FI C12R 1:91) (C12P 21/08 C12R 1:91) (72) Inventor Takeya Hori 2-7 Nishishinjuku, Shinjuku-ku, Tokyo No. 1 Inside Fuji Rebio Inc. (72) Inventor Satoshi Ito Inside Fuji Rebio Inc. 2-7-1 Nishi Shinjuku, Shinjuku-ku, Tokyo

Claims (8)

[Claims] 1. A monoclonal antibody that specifically recognizes ferredoxin. 2. The monoclonal antibody according to claim 1, wherein the ferredoxin is a heat-resistant protein. 3. The ferredoxin is selected from the group consisting of thermophilus, sulforobos, phyllococcus, thermotoga, thermobacillus, phyllodictium, thermococcus, thermodiscus, methanothermas, and methanococcus. The monoclonal antibody according to claim 1, which is a ferredoxin derived from a bacterium. 4. The monoclonal antibody according to claim 1, wherein the ferredoxin is a ferredoxin derived from Pyrococcus fuliosis. 5. The monoclonal antibody according to claim 1, wherein the monoclonal antibody is Fdx1. 6. A hybridoma producing the monoclonal antibody according to any one of claims 1 to 5. 7. The hybridoma according to claim 6, wherein the hybridoma is Fdx1. A method for purifying a ferredoxin fusion protein using the monoclonal antibody according to any one of claims 1 to 5.