JPH10295384A - Purified immunoglobulin comprising monoclonal antibody against p86 subunit of human dna helicase 2 - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new purified immunoglobulin capable of being specifically reacted with a subunit antigen having a specific molecular weight of human DNA helicase 2, comprising a monoclonal antibody capable of recognizing the subunit, capable of analyzing intracellular localization of helicase in a cell cycle. SOLUTION: This new purified immunoglobulin comprises a monoclonal antibody which is capable of being specifically reacted with a subunit antigen having 86 kDa molecular weight of human DNA helicase 2 and of recognizing the subunit. The immunoglobulin is capable of readily visualizing and analyzing intracellular localization of a DNA helicase, which is a DNA dependent adenosine triphosphatase having activity of rewinding double strand of DNA and is indispensable in correction and restoration of DNA, in each period of cell cycle by fluorescent antibody method, etc. The immunoglobulin is obtained by immunizing a mouse against a HeLa cell in a mitotic phase, fusing a spleen cell obtained from the mouse with a myeloma cell, screening the formed hybridoma cell, selecting a cell to be strongly reacted with a cell in a mitotic phase and culture in an abdominal cavity of mouse after the cloning.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a novel monoclonal antibody against human DNA helicase 2, especially human D
The present invention relates to a monoclonal antibody against the p86 subunit of NA helicase 2.

[0002]

2. Description of the Related Art DNA helicase is a DNA-dependent adenosine triphosphatase (DNA dependent ATPase) having an activity of unwinding a DNA double strand, and is involved in processes of DNA replication, transcription, recombination and repair. (K. Guider et al., "Annual Review of Biochemistry," Vol. 50: 233-260.
1981). Numerous DNA helicases have been isolated from various organisms, and the properties of several DNA helicases obtained from viruses, prokaryotes or yeast have been investigated in detail (SW Mattson et al. "Annual Review of Biochemistry, 59: 289-329, 1990). However, the functions of DNA helicases obtained from mammals, particularly humans, in vivo have hardly been clarified.

[0003] Human DNA helicase 2 (hereinafter "HDH")
2 ") is a helicase identified from extracts of HeLa cells. This helicase may be a DNA fragment at the end of a DNA fragment or a single-stranded nick or gapped cyclic DN.
A. (T. Mimori et al., “The Journal.
Of Biological Chemistry "Volume 261, 22
No .: 10375-10379, 1986; R.
Brier et al., Ibid., Volume 268, Issue 10: 7594-
7601, 1993; Falzon et al.
268, 4: 10546-10552, 1993
Year). It has the activity of unwinding the partially unwound DNA duplex in the direction from the 3 'to the 5' end. HDH2 also binds to a 350 kDa peptide to form a DNA-dependent serine / threonine protein kinase (TM Gottlieb et al., Cell 72).
Volume: 131-142, 1993). HDH2 is a previously reported autoimmune antigen, Ku autoanti.
gen. (N. Chuteya et al., The Embo Journal, Vol. 13, No. 20, 4991-50.)
01, 1994). Ku has been reported in patients with scleroderma / polymyositis multiple syndrome (T. Mimori et al., "The Journal of Clinical Investigation", Vol. 68: 6).
11-620, 1981), and later reported from patients with other rheumatic diseases (T. Mimori et al.,
"The Journal of Biological Chemistry," Vol. 261, No. 5, pp. 2274-2278, 1986
Year).

[0004] HDH2 (Ku) has a molecular weight of 70 kDa.
And 86 kDa subunits (hereinafter p70 and p
86, abbreviated as p7
0 and p86 are non-covalently bonded. Both subunits have a leucine zipper in the amino acid sequence,
This is thought to bind to DNA, and p70
It has been shown that it can bind non-specifically to the end of double-stranded DNA (T. Mimori et al., "The Journal of Biological Chemistry", vol. 261: 22).
10375-10379, 1986). Further p7
0 has been shown to bind to the nuclear matrix within the nucleus (JK Vishwanasa et al., "Molecular and Cellular Biochemistry" 146).
Volume: 121-126, 1995). On the other hand, although p86 is mainly localized in the nucleus, it has been reported that p86 is also present on the cell surface in human T cells (RG Dargir et al., "Autoimmunity", Vol. 13, No. 13: 265-
267, 1992).

[0005] It has also been found that the receptor for somatostatin-14 obtained from human HGT-1 cells is p86 (ML Romancer et al., "The Journal.
Of Biological Chemistry "Volume 269, 26
No .: 17664-17468, 1994). Both subunits have been shown by immunoblotting to be present in small amounts in the cytoplasm of HeLa cells in logarithmic growth phase (JK Vishwanasa et al., Supra).
However, the detailed functions of both p70 and p86 have not been clarified.

[0006]

In such studies, monoclonal antibodies and polyclonal antibodies against HDH2 have conventionally existed, but they can be obtained using a fusion protein obtained by incorporating a helicase gene into Escherichia coli and expressing it as an immunogen. Thus, production of monoclonal antibodies produced using all proteins constituting cells as an immunogen has not yet been realized.

[0007] HDH2 is considered to be a regulatory factor that plays an important role in performing tasks such as DNA replication and repair. Antibodies against HDH2 are used at each stage of the helicase cell cycle, particularly in the collapse and reconstitution of the nuclear envelope. It is indispensable to easily visualize and analyze the intracellular localization at the mitotic stage in which HDH2 occurs, in order to elucidate the function of HDH2 in vivo. Nevertheless, antibodies against HDH2 have not been commercially available, and researchers in this area obtain antibodies by exchanging personal ties between researchers or exchanging letters. Is the current situation. However, these antibodies were different, and individual researchers used these different antibodies to publish their own research results, resulting in qualitative confusion in the results. Therefore, if an anti-helicase monoclonal antibody prepared using all proteins constituting a cell as an immunogen is provided, it can be expected that epoch-making results will be achieved in DNA replication and the like.

[0008]

SUMMARY OF THE INVENTION The present inventor has proposed that mitotic H
The mice were immunized with eLa cells to generate antibodies that were particularly reactive with cells in the mitotic phase. Among the antibodies prepared so far, it was revealed that an antibody (referred to as HmH2) that specifically reacts with a component having a molecular weight of 86 kDa recognized p86 of HDH2. Therefore, the present inventor has proposed this antibody H
Using mH2, the subcellular localization of p86 in the cell cycle of HmH2 was analyzed in detail, and the present invention was achieved.

That is, the present invention provides a human DNA helicase 2
Is a purified immunoglobulin consisting of a monoclonal antibody that specifically reacts with and recognizes a subunit having a molecular weight of 86 kDa.

[0010]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The progress of achievement of the present invention will be described with reference to the following examples.

[0011]

【Example】

[Materials and Methods] [Materials] Nocodazole and pristane, phenylenediamine for Sigma, Eagle's minimum essential medium (Eagle's MEM) for Nissui Pharmaceutical, PVDF membrane Immobilon-P and Immobilon -P ^sq (Immobilon-P ^sq ) is Millipore (M
(ILLIPORE) was used. Rhodamine-labeled anti-mouse IgG, biotin-labeled anti-mouse IgG, and peroxidase-labeled anti-mouse IgG are available from Capel (CAPPE).
L), Vectorstain ABC Kit (Ve)
ctstein ABC kit) and Vector Shield (VECTASHIELD) are available from Vector Laboratories,
Ampure PA Kit (Ampure ^™ PA ki)
t) was from Amersham.

[Preparation of mitotic cells] HeLa cells (RIB0007, RIKEN, derived from human cervical cancer tissue)
Was cultured on a culture dish (245 × 245 cm: Sumitomo Bakelite) using Eagle's minimum essential medium containing 10% FCS (fetal calf serum). Mitotic cells can be obtained by the method of Yamashiro et al. (S. Yamashiro et al., Nature, 344: 6).
75-678, 1990).
After treatment with / ml nocodazole for 16 hours, the medium was agitated with a 10 ml pipette to separate mitotic cells. The mitotic cells were collected by low-speed centrifugation and washed with PBS.

[Antigen and Immunity] The mitotic cells are 0.9% Na
It was suspended in an aqueous solution of Cl to a concentration of 5 × 10 ⁷ cells / ml. An equal volume of complete Freund's adjuvant was added to the suspension and Branso Ultrasonics (Bran
son Ultrasonics) Sonifier ^R
Model 450 (Sonifier ^R Model 45)
0) for 20 seconds and sonication with output2. B
ALB / c mice (female, 8 weeks old)
1 was injected intraperitoneally. After 15, 31, and 34 days, the emulsion prepared using incomplete Freund's adjuvant instead of complete Freund's adjuvant was injected intraperitoneally.

[Preparation of Monoclonal Antibody HmH2] Monoclonal antibodies were prepared by the method of Gulfure et al. (G. Gulfre et al., "Nature", Vol. 550-552, 1977)
Was performed according to 1.7 × 10 ⁸ cels obtained from mice
Is spleen cells were fused with 4.0 x 10 ⁷ cells SP2 / 0-Ag14 myeloma cells using 50% (w / v) polyethylene glycol 4000. Hybridoma cells are cultured in the ELISA supernatant described below.
Using A method, indirect immunofluorescent antibody method, and immunoblot method, those that strongly reacted with dividing cells were screened.

[0015] BALB / c mice (female, 7 weeks old) were intraperitoneally injected with 0.5 ml of pristane.
mH2 producing hybridoma cells were cultured in 4 × 10 ⁶ cells
It was injected intraperitoneally. Three weeks later, the abdomen of the mouse was enlarged, and a syringe needle (NEOLUS18Gx11 / 2, manufactured by Terumo Corporation) was stabbed into the abdominal cavity to collect ascites. Collected ascites centrifugation (9000xg, 4 ℃, 30 minutes), Ann Pure ^TM PA kit from the supernatant (Ampure ^TM PA k
It) was used to purify the IgG fraction. Purified IgG
Was confirmed to be an HmH2 antibody by an immunoblotting method and an indirect immunofluorescence antibody method. The isotype of this monoclonal antibody is Amersham mouse monoclonal antibody isotyping
Kit (Amersham mouse monocl)
onal antibodyisotyping ki
Determined using t).

[Solid Phase ELISA Method] Solid-pha
se enzyme-linked immunoso
rvent assay (ELISA) is described in the method of Kotani et al. (M. Kotani et al., Journal of Immunological Methods, Vol. 157, pp. 241-252, 199).
3 years). Poly L lysine (1 mg / ml)
Was added to a 96-well microplate (MS8296F: Sumitomo Bakelite) pre-incubated for 1 hour at room temperature, and 2 × 10 ⁴ cells / dividing HeLa cells were added.
The well was added and attached to the plate. Cells were fixed with 3.5% formaldehyde in PBS for 10 minutes. PBS
After washing three times with, 100 μl of the hybridoma culture supernatant was added to each well and allowed to stand for 60 minutes. Thereafter, the wells were washed, and 50 μl of peroxidase-labeled anti-mouse IgG was added and left for 60 minutes. After washing again, 100 μl of phenylenediamine (1 μg
/ Ml) was added. Fifteen minutes after the addition of the chromogenic substrate, the absorbance of each well was measured using a microplate reader (MPR-A4i, Tosoh Corporation).

[Electrophoresis and immunoblot] SDS-P
AGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) is a buffer-only method (U.
K. Remley, "Nature" Vol. 227, 680-68
5 (1970), and otherwise, the method of Bratler (DP Bratler et al., Journal of Chromatography, Vol. 64, pp. 147-155, 19).
72 years), 7.5% and 12% polyacrylamide gels were used.

The sample contains a 1/4 volume (v / v) of a 5-fold concentrated SDS sample buffer [final concentration 1% SDS,
40 mM Tris-HCl (pH 6.8), 4.5%
Glycerol, 50 mM DDT, O.D. 003% BP
B]. Bio-Rad (B
io-Rad), SDS-PAGE molecular
Weight Standers, Broad Range (SDS-
PAGE Molecular Weight Sta
ndards, Broad Range).
The gel was stained with a CBB staining solution (0.15% CBB R-
250 (w / v), 50% methanol, 10% acetic acid), and destained with an aqueous solution of 7.5% methanol and 7.5% acetic acid.

The transfer of the protein in the gel to the PVDF membrane was performed by the method of Tobin et al. (H. Tobin et al., "Proceedings of the National Academy of Sciences of the United States of the United States." U.S.A., Vol. 76, No. 9, pages 4350-4354,
1979). The gel after SDS-PAGE was applied to a PVDF membrane [Immobilon-P (Immobilo).
nP)] Semi-dry transfer device KS
-8460, [manufactured by Marysol] (100 mA constant flow, 2 hours). 3% PVDF membrane
After blocking with BSA-containing PBS (room temperature, 1 hour), it was reacted with the HmH2 antibody. After the reaction, 0.05% T
The plate was washed three times with PBS containing ween 20 for 10 minutes. Next, biotin-labeled anti-mouse IgG (diluted 200-fold with PBS)
And washed as above. Next, the PVDF membrane was transferred to an ABC reaction solution (Vectastain ABC kit),
After reacting for 30 minutes, it was washed in the same manner as above. Color development 0.5 mg / ml of 3,3'-diaminobenzidine tetrahydrochloride (DAB), were performed in 0.07% H ₂ O ₂ solution.

[Indirect immunofluorescence antibody method] HeLa cells were cultured on a cover glass. Put the cover glass in PBS
And then fixed with methanol (−20 ° C., 2 minutes). After washing 4 times with PBS for 5 minutes, 3% BSA
Blocking (room temperature, 1 hour) was carried out with PBS containing PBS.
After reacting with the HmH2 culture supernatant in a wet box (room temperature, 1 hour), it was washed with PBS (5 times, 3 times). It was allowed to react with rhodamine-labeled mouse IgG (diluted 200-fold with PBS) in a light-shielded wet box (room temperature, 30 minutes). Then, the plate was washed with PBS (3 times for 5 minutes), and about 20 μl of vector shield and 5 μl of 4,6-
Diamino-2-phenylindole (DAPI) was loaded. A cover glass was placed on top of it, and the area was closed with nail polish. Specimens were prepared using a fluorescence microscope (OPTIPHO).
T, Nikon ^™ ) and photographed (FX-35
A, Nikon ^™ ). Shooting with a confocal microscope
Performed on a Zeiss ^™ .

[Identification of HmH2 antigen] 5 ml of F buffer [20 mM Tris-HCl] was added to about 5 ml (about 5 × 10 ^7-8 cells) of HeLa cells collected with PBS.
(PH 7.5), 2 mM MgCl ₂ , 1 mM EGT
A, 200 mM NaCl, 0.1 mM DTT, 1 m
M PMSF, 10 μg / ml leupeptin]
Homogenized in ice. Ultracentrifuge (5000)
0xg, 2 ° C, 30 minutes), and the supernatant was filtered (EB-
DISK25, 0.45 μm). Sephacryl S-300 (Sephyl) equilibrated with F buffer
acryl S-300) [Pharmacia (Pharm
aci)], and the mixture was eluted with an F buffer and fractionated into 100 tubes of 3 ml each. The column chromatography was performed at 4 ° C.

HmH2 antibody obtained from mouse ascites was converted to a binding buffer (AmpurePA kit).
Was coupled to a protein A column (same kit) equilibrated in step 1. After coupling, the column was washed twice with 5 ml of binding buffer (same kit). The fraction containing the antigen obtained by the above gel filtration column was added to the protein A column to which the HmH2 antibody was bound. The column was washed twice and eluted with 3 ml of an elution buffer (same kit). Since the eluate has a low pH, 1 ml of 1 M Tris-HCl (pH 9.
0) was added to bring the pH to approximately neutral.

Protein fragmentation by endoproteinase Glu-C in SDS-PAGE was performed according to the method of Cleveland et al. (DW Cleveland et al., "The Journal of Biological Chemistry" 252).
Vol. 3, No. 1102-1106, 1977). A band of about 85 kDa, which is an HmH2 antigen, was cut out from the gel, and immersed in SDS sample buffer for 30 minutes. Thereafter, SDS-PAGE was performed on a 12% polyacrylamide gel together with endoproteinase Glu-C.
did. When the electrophoresis tip reached the running gel, electrophoresis was stopped to perform an enzymatic reaction with endoproteinase Glu-C, and electrophoresis was restarted 30 minutes later.

HmH2 antigen endoproteinase Gl
The uC digested fragment V was obtained from PVDF membrane Immobilon-P ^SQ (I
mmobilon-P ^SQ ) and stained with CBB staining solution. After destaining with 90% methanol and 7% acetic acid aqueous solution, a major band was cut out, and a protein automatic sequencer (Model 473A, Perkin-El) was used.
The amino acid sequence was determined using mer Applied Biosystems. The revealed sequence was subjected to homology search with a known protein at the Japan DNA Data Bank (DDBJ).

[Concentration of protein] Deoxycholic acid sodium salt was added to the sample [final concentration: 0.017%]
(W / v)], followed by vigorous stirring and then left at room temperature for 15 minutes. Add trichloroacetic acid to the sample [final concentration: 6
% (V / v)] at room temperature for 15 minutes, followed by centrifugation (9%).
000 xg, room temperature, 30 minutes). The obtained precipitate was dissolved in an SDS sample buffer, and an appropriate amount of 2.5 M Tris-HCl (pH: 8.8) was added for pH adjustment.

[Results] [Intracellular distribution of HmH2 antigen] The HmH2 antibody has been screened as an antibody that reacts particularly strongly with mitotic cells. Thus, using HemH2 antibody, HeLa
The cells were subjected to a fluorescent antibody method to observe the distribution of the HmH2 antigen in the cells during the interphase and the division. The photographs are shown in FIG. In FIG. 1, a to c are photographs showing indirect immunofluorescent antibody images of HeLa cells by the HmH2 antibody, and each photograph is an image in the same visual field. a is a fluorescent image by the HmH2 antibody, b is a DNA stained image by DAPI, c
Is a Nomarski differential interference image. Cells in the mitotic phase are visible near the center and lower right of each photograph, indicating that the HmH2 antigen is distributed throughout the cells.

In interphase cells, the HmH2 antigen is distributed in the nucleus (a and b). In contrast, in cells in the mitotic phase, the HmH2 antigen is distributed throughout the cells (a and c). In the cell at the center of the photograph, nuclear reconstitution seems to have begun, but the HmH2 antibody strongly reacts near the nucleus, suggesting that the HmH2 antigen converges on the nucleus at this time. As can be seen from these, it was revealed that HmH2 antigen distributed in the nucleus during interphase is distributed throughout the cell during mitosis.

[Immunoblot for crude cell extract]
Since the distribution of the HmH2 antigen greatly changes between interphase and mitosis, the present inventor considered that there is some change in the antigen itself. Therefore, the crude extracts of the cells in the interphase and the mitotic phase in which the total protein amount was uniform were immunoblotted with the HmH2 antibody to examine the quantitative and qualitative changes of the HmH2 antigen.

FIG. 2 is an immunoblot of the HmH2 antibody at interphase and mitosis. Crude extracts of interphase and mitotic phase HeLa cells with the same total protein amount were subjected to SDS-P
AGEs and immunoblotting. The leftmost lane is a molecular weight marker, and the number on the left indicates kDa.
Lane 1 is a crude extract of interphase cells, and lane 2 is a crude extract of mitotic cells. As a result, the HmH2 antigen
It was found that almost the same amount was present regardless of the interphase (lane 1) and the mitotic phase (lane 2). From this, Hm
It has been ruled out that the H2 antigen is a substance that is slightly present in the nucleus during interphase and is abundant during mitosis.
In addition, no change was observed in the molecular weight of the HmH2 antigen in each of the interphase and the mitosis.

[Distribution of HmH2 antigen in each phase of mitosis] The HmH2 antigen is distributed in the nucleus during interphase and throughout the cell during mitosis (Fig. 1a to 1c). Therefore, the localization of the HmH2 antigen in each phase of the mitosis was examined in more detail.
The photograph in FIG. 3 shows the Hm in each phase of HeLa cell division.
The distribution of H2 antigen is shown, ac is interphase, df is early period,
g to i are metaphase cells, j to l are late cells, m to r are terminal cells, (a, d, g, j, m, p) are Nomarski differential interference images, and (b, e, h, k, n, q) is DAPI by DAPI
The NA-stained image, (c, f, i, l, o, r) is a fluorescent image with the HmH2 antibody. Arrowheads (ac) indicate nuclei.

As shown in FIG. 3, antigens (ac) localized in the nucleus in the interphase spread throughout the cell (df) in the mitotic phase. This suggests that during the interphase, the antigen distributed in the nucleus or on the surface of the nucleus spreads throughout the cell when the nuclear membrane enters and enters the mitosis. From g to r, it was found that the cells continued to be distributed throughout the cells throughout the mitosis.

[Intracellular Distribution of HmH2 Antigen Observed with Confocal Microscope] In order to examine the localization of HmH2 antigen inside the cell in more detail, observation was performed using a confocal laser scanning microscope. The observed distribution of HmH2 antigen is shown in FIG. In the figure, a to c are interphase cells, and the part of the nucleus shown in b that is not recognized by the HmH2 antibody is considered to be a nucleolus. d to f show the terminal cells, and e shows that the HmH2 antigen is not distributed on the chromosome (see f), the spindle and the contractile ring.
gi are cells in the latter half of cytokinesis. (A, d, g)
Is the Nomarski differential interference image, (b, e, h) is HmH2
The distribution of the antigen, (c, f, i), is a DNA stained image with DAPI.

As described above, in the interphase, the HmH2 antigen exists in the nucleus and is widely distributed in nucleoplasm other than the nucleolus (FIG. 4, b). In the mitotic, H _m H2 antigen, chromosome, not distributed in spindle and contractile ring, widely distributed in the cytoplasm until immediately before the end mitotic (Figure 4, h) revealed.

[Identification of HmH2 antigen] First, the crude extract of HeLa cells was fractionated using a gel filtration column, and the fraction in which the HmH2 antigen was present was confirmed by immunoblotting. The HmH2 antigen in this fraction was converted to a protein A column (Ampure P
A kit). The fraction thus obtained was electrophoresed, and it was confirmed by immunoblotting that HmH2 antigen was obtained (FIG. 5).

FIG. 5 shows that the fractions obtained from the protein A column were subjected to SDS-PA on a 7.5% polyacrylamide gel.
GE was transferred to a PVDF membrane and subjected to immunoblotting. Lane 1 is CBB-stained, lane 2 is a primary antibody using HmH2, lane 3 is a primary antibody reaction was not performed, and the secondary antibody was reacted. The leftmost lane is a molecular weight marker, Indicates kDa. Arrows indicate HmH2 antigen.

The band indicated by the arrow in lane 2 in FIG. 5 indicates that the HmH2 antibody has specifically reacted.
In lane 3, it can be seen that no reaction occurs unless the HmH2 antibody is used. From this, the band indicated by the arrow is HmH
Two antigens were determined.

The HmH2 antigen thus obtained was excised from the gel and fragmented with endoproteinase Glu-C. FIG. 6 shows the result of electrophoresis of the HmH2 antigen fragmented by the endoproteinase Glu-C.
That is, the HmH2 antigen obtained from the eluate of the protein A column was partially digested with endoproteinase Glu-C, and subjected to SDS-PAG on a 12% polyacrylamide gel.
E, Transfer to PVDF membrane (Immobilon-P ^sq ), CBB
It is stained. a band is the original H not digested by endoproteinase Glu-C.
1 shows the mH2 antigen. Major bands of b to e were obtained as digested fragments. The leftmost lane is a molecular weight marker, and the number to the left indicates kDa.

Thus, the amino acid sequence of each N-terminal was determined, and homology search was performed with a known protein. That is, the N-terminal amino acid sequence of the digested fragment of FIG.
Human DNA determined to have high homology by BJ
The amino acid sequence of the 86 kDa subunit of helicase (p86) is shown in the following sequence listing.

[0039]

[Sequence list]

In the above arrangement, the upper row b,
N-terminal amino acid sequence of the digested fragment from which c, d, and e were obtained;
P8 in which the lower p86 was determined to have high homology
6 is part of the amino acid sequence. The numbers below indicate the number of residues from the N-terminus. It shows that the sequence enclosed by a frame is a common sequence. From the above amino acid sequence, the HmH2 antigen was identified as p86.

[0041]

The monoclonal antibody produced by the present invention, ie, mouse immunoglobulin G1, specifically binds to and recognizes the p86 subunit of human DNA helicase 2 in cells or cell extracts. .
Therefore, the intracellular localization of helicase essential for DNA replication and repair at each stage of the cell cycle can be easily visualized and analyzed by a fluorescent antibody method or the like.

Furthermore, despite the fact that they are indispensable regulatory factors for DNA replication and the like as described above, hitherto, researchers in this area have not obtained sufficient antibodies against human DNA helicase 2 and Confusion arises in that different researchers use different antibodies to present their own research results, but the commercialization and widespread use of the monoclonal antibodies of the present invention will lead to future research results. A qualitative improvement over time is expected. Further, since the antibody is a "monoclonal antibody" instead of a "polyclonal antibody", there is an advantage that antibodies having the same properties can be easily produced in large quantities.

[Brief description of the drawings]

FIG. 1 is a photograph showing an indirect immunofluorescent antibody image of HeLa cells by an HmH2 antibody.

FIG. 2 is an immunoblot of HmH2 antibody in interphase and mitosis.

FIG. 3 is a photograph showing the distribution of HmH2 antigen at each stage of the mitotic phase of HeLa cells.

FIG. 4 is a photograph obtained by observing the distribution of HmH2 antigen using a confocal laser scanning microscope.

FIG. 5 is an immunoblot showing the presence of HmH2 antigen in a crude extract of HeLa cells.

FIG. 6 shows a band obtained by subjecting HmH2 antigen fragmented by endoproteinase Glu-C to SDS-PAGE on a 12% polyacrylamide gel, transferring the PVD membrane, and performing CBB staining.

[Explanation of symbols]

In FIG. 1, a is a fluorescent image by the HmH2 antibody, and b is D
DNA staining image by API, c is Nomarski differential interference image. Lane M at the left end of FIG. 2 is a molecular weight marker,
The number to the left indicates kDa. Lane 1 is a crude extract of interphase cells, and lane 2 is a crude extract of mitotic cells. In FIG. 3, a to c indicate interphase, d to f indicate early stage, g to i indicate middle stage, j to l indicate late stage, and m to r indicate end stage cells. Also,
a, d, g, j, m, and p are Nomarski differential interference images,
b, e, h, k, n, and q are DNA staining images by DAPI, and c, f, i, l, o, and r are fluorescence images by the HmH2 antibody. Arrowheads a to c indicate nuclei. 4a to 4c show interphase cells, d to f show end-phase cells, and g to c.
i is a cell in the latter half of cytokinesis. a, d, g are Nomarski differential interference images, b, e, h are distributions of HmH2 antigen,
c, f, and i are DNA staining images by DAPI. FIG.
Lane 1 was obtained by CBB staining, lane 2 was obtained by using HmH2 as a primary antibody, lane 3 was reacted by a secondary antibody without performing a primary antibody reaction, and lane M on the left end was a molecular weight marker. The numbers on the left indicate kDa.
Arrows indicate HmH2 antigen. FIG. 6 a shows the band of the original HmH2 antigen not digested by endoproteinase Glu-C, be to major bands as digested fragments, lane M at the left end is a molecular weight marker, and the number on the left is kDa. Show.

──────────────────────────────────────────────────の Continued on front page (51) Int.Cl. ⁶ Identification code FI (C12P 21/08 C12R 1:91)

Claims (1)

[Claims] 1. The molecular weight of human DNA helicase 2 is 86 k
A purified immunoglobulin comprising a monoclonal antibody that specifically reacts with and recognizes the Da subunit antigen.