JP4243387B2 - Mast cell surface antigen DNA - Google Patents

Description

【図面の簡単な説明】
【図１】 共生培養前後の肥満細胞についてキマーゼ陽性の肥満細胞の割合とトリプターゼ濃度を測定した結果を表すグラフである。
【図２】 Ｂｃｓｃ−１とＭＡＳＡ−１との塩基配列を比較した説明図である。
【図３】 ｃＤＮＡ発現ベクターＢＣＭＧＳＮｅｏの構造説明図である。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a DNA of mast cell surface antigen.
[0002]
[Prior art]
In allergic diseases such as bronchial asthma and cedar pollinosis, first the induction of IgE production specific to the antigen occurs, and the histamine / eosinophil migration factor from mast cells and basophils activated by this induced IgE. Allergic inflammation is induced by production and release of various chemical mediators such as (ECF-A), leukotriene, platelet activating factor (PAF), and thromboxane. Particularly in tissues, mast cells release these chemical mediators, thereby playing an important role in the formation of allergic lesions.
[0003]
Human mast cells are classified into tryptase positive cells (hereinafter referred to as MC-T) and tryptase and chymase positive cells (hereinafter referred to as MC-TC) according to the composition of proteolytic enzymes contained in the granule in the mast cells. . MC-T is mainly distributed in the lung and gastrointestinal mucosal tissue, whereas MC-TC is mainly distributed in the skin tissue. Unlike other blood cells, these mast cells leave the bone marrow as pluripotent stem cells, reach the peripheral environment, and become MC-T or MC-TC after adherence to lung or skin fibroblasts . Since such mast cells are thought to play a major role in the pathogenesis of allergic diseases, it is necessary to specifically detect and isolate mast cells in elucidating the physiological role of mast cells. .
[0004]
[Problems to be solved by the invention]
However, conventionally, cell surface antigens specific to mast cells have not been known. In addition, if antibodies against cell surface antigens specific to mast cells can be used, mast cells can be specifically removed or eliminated, and cell surface antigens specific to these mast cells should be clarified. Not only elucidates the pathology of allergic diseases, but also has important implications in treatment.
[0005]
This invention is made | formed in view of the said subject, and it aims at providing DNA of a mast cell surface antigen.
[0006]
[Means for Solving the Problems and Effects of the Invention]
Mast cell surface antigens are those comprising the amino acid sequence of SEQ ID NO: 1. This amino acid sequence is a translation of the coding region of the mast cell surface antigen DNA. The base sequence of mast cell surface antigen DNA was determined by the following procedure. That is, as described in detail in the Examples section, after obtaining mast cells from cord blood mononuclear cells, mRNA is extracted from the cell extract of the mast cells, and a cDNA library is prepared from the mRNA. The resulting positive clone was confirmed for its base sequence using a DNA sequencer. Of the base sequence of SEQ ID NO: 2, the sequence of base numbers 36 to 38, that is, ATG, is the start codon, and the sequence of base numbers 2394 to 2396, that is, TGA, is the stop codon. That is, base numbers 36 to 2396 are coding regions, and the base sequence in this section encodes the amino acid sequence of SEQ ID NO: 1. The elucidation of the amino acid sequence of this mast cell antigen makes it possible to elucidate the role of mast cells in the pathogenesis of allergic diseases, and to easily obtain antibodies that specifically react with mast cells. became.
[0007]
An antibody against a mast cell surface antigen is, for example, immunized with a mammal (except human) using a mast cell surface antigen containing the amino acid sequence of SEQ ID NO: 1, and antibody-producing cells obtained from the immunized mammal Is fused with myeloma cells to produce fused cells, and from these fused cells, a clone producing an antibody that specifically binds to a mast cell surface antigen is selected and cultured, and the selected clone is cultured. It can be obtained by purifying the supernatant. By using this antibody, mast cells can be specifically removed or eliminated, so that allergic diseases can be treated. That is, this antibody is expected as a therapeutic agent for allergic diseases.
[0008]
The antibody-producing cell is prepared by, for example, immunizing a mammal (except a human) with a mast cell surface antigen containing the amino acid sequence of SEQ ID NO: 1, and producing the antibody obtained from the immunized mammal. It can be obtained by fusing cells with myeloma cells to produce fused cells, and selecting and culturing clones producing antibodies that specifically bind to mast cell surface antigens from the fused cells. .
[0009]
【Example】
[1] MC-TC cultured heparin-treated umbilical cord blood is layered on Ficoll Hipack solution (specific gravity 1.077, Sigma) and centrifuged at 300 × g for 30 minutes at room temperature to separate mononuclear cells. It was suspended in RPMI 1640 medium (Nissui Pharmaceutical) supplemented with 50% FBS (Gibco), 50 μM 2-mercaptoethanol, 4 mM L-glutamine, 100 U / ml penicillin and 50 μg / ml streptomycin. The mononuclear cell number concentration of the suspension was adjusted to 5 × 10 ⁶ / ml, seeded on a collagen-coated 10 cm culture dish (Iwaki glass), SCF (100 ng / ml, Peprotech) and IL-6 (50 ng / ml). ml, Peprotech Inc.) and cultured for 2 weeks to obtain 2-week cultured cells containing neutrophil cells, lymphocytes, macrophages, basophils and mast cell progenitor cells. SCF is a factor involved in the differentiation and proliferation of mast cells expressed on fibroblasts, and is an abbreviation for stem cell factor.
[0010]
Umbilical cord blood mononuclear cells obtained above were cultured for 6 weeks in the presence of SCF 100 ng / ml and IL-6 50 ng / ml, and when human mast cells accounted for the majority, that is, on the order of 10 ⁶ human mast cells. Thus, co-cultivation of human fibroblasts with primary cultured cells was performed. That is, the human mast cells were transferred to a monolayer culture of human skin-derived or lung-derived fibroblasts and cultured in the presence of 50 ng / ml SCF for 2 months.
[0011]
For the mast cells before and after co-cultivation, the ratio of chymase-positive mast cells and the tryptase concentration were measured. The result is shown in FIG. In FIG. 1, in the mast cells before symbiotic culture, that is, human mast cells cultured for 10 to 16 weeks in the presence of SCF and IL-6 (the left end of the graph in FIG. 1), both the ratio of chymase-positive mast cells and the tryptase concentration. Only a few were recognized. In contrast, human mast cells after symbiotic culture with dermal fibroblasts for 6-8 weeks (middle of the graph in FIG. 1) and after co-cultivation with pulmonary fibroblasts (right end of the graph in FIG. 1) are both apparent. There was a significant increase, particularly in the case of human mast cells co-cultured with dermal fibroblasts.
[0012]
Mast cells cultured for 15 weeks in the presence of SCF and IL-6, and mast cells cultured for 2 months with dermal fibroblasts after 6 weeks of culture, stained with antibodies to tryptase and antibodies to chymase As a result, staining for tryptase showed staining of both cells as a whole, whereas staining for chymase showed staining of most cells in cells co-cultured with fibroblasts, while symbiosis. Only a portion of the cells that were not cultured were stained. As a result, it was found that MC-T was differentiated into MC-TC by symbiotic culture.
[0013]
As described above, MC-TC, which is a connective tissue type human mast cell, cultures umbilical cord blood mononuclear cells in the presence of SCF and IL-6, and further performs symbiotic culture with primary cells of human dermal fibroblasts. Was obtained.
[2] Preparation of mRNA According to the instruction manual of “Quick-Prep Micro mRNA Purification Kit” of Pharmacia, poly A is obtained from the above cell extract of MC-TC. The mRNA marked with was extracted by the following procedure.
1) After centrifuging 2 × 10 ⁶ MC-TCs and washing twice with PBS, adding 400 μL of the elution buffer attached to the above kit and stirring well with a vortex mixer (rotary stirrer), 800 μL of elution buffer was added, and the mixture was stirred with a vortex mixer, transferred to an assist tube (Assist), which is a polypropylene tube, and centrifuged at 14,000 rpm for 5 minutes to remove the precipitate.
2) Meanwhile, 1 mL of oligo dT cellulose was centrifuged at 14,000 rpm, and the supernatant was removed.
3) Mix the cell extract from 1) and oligo dT cellulose from 2), mix well by inverting the assist tube for 3 minutes, and then centrifuge at 14,000 rpm for 5 seconds to remove the supernatant and 1 mL of high salt concentration After washing 5 times with 1 mL of buffer, it was washed twice with low salt concentration buffer.
4) The precipitate was suspended in 300 μL of a low salt buffer, placed in the microspin column attached to the above kit, centrifuged for 5 seconds, and washed 3 times with 500 μL of low salt buffer.
5) Add 200 μL of elution buffer warmed to 65 ° C, add 10 μL glycogen solution and 400 μL potassium acetate solution, add 1 mL of 95% ethanol, centrifuge at 14,000 rpm for 1 hour at 4 ° C, and dry under reduced pressure did.
[0014]
[3] Preparation of cDNA library cDNA library from mRNA obtained in [2] above using “Zap-cDNA synthesis kit” of Stratagene and according to the instruction manual of this kit Was made. Size fractionation was performed using the CL-2B gel attached to this kit.
[0015]
[4] Immunoscreening The phage solution of the cDNA library prepared in the above [3] was added to SM buffer (50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 10 mM MgSO ₄ .H ₂ O,. (Contained with 01% gelatin) and adjusted to 2.5-4 × 10 ⁵ pfu phage / mL. Dispense 100 μL of this solution into 10 centrifuge tubes, and culture overnight in E. coli (XL-1 blue) (cultured overnight in 5 mL of NZY medium and adjusted to OD ₆₀₀ = 1.5). 500 μL was added, stirred with a vortex mixer, and incubated at 37 ° C. for 15 minutes. To this, 7 mL of soft agarose (electrophoretic agarose dissolved in a medium to make 0.6%) was added, stirred with a vortex mixer, and spread on a square petri dish of 14 × 10 cm. After the agarose had solidified, it was immersed in 20 mM IPTG and dried on a nitrocellulose filter, and incubated at 37 ° C. for 4 hours. The NZY medium contains 5 g NaCl, 2 g MgSO ₄ .7H ₂ 0, 5 g yeast extract (yeast extract), and 5 g NZamine (casein hydrolyzate) in 1 L, and added to NaOH. After adjusting to pH 7.5, 15 g of agar was added and sterilized and melted in an autoclave.
[0016]
After incubation, the filter is removed and the plate is washed by shaking for 15 minutes in 300 mL of PBS. After repeating this twice, the PBS is discarded, and 300 mL of PBS (PBS-SM) supplemented with 0.5% skim milk is added at room temperature. Shake for 1 hour to block. Rat antiserum diluted 400-fold with PBS-SM (antiserum from the rat that provided spleen cells for the preparation of fusion cells ahMC5C12 (accession number FERM BP-6070) described later) was added, and the mixture was shaken at room temperature for 1 hour, The solution was discarded and washed with 500 mL of PBS (PBS-T) containing 0.05% Tween 20 at room temperature for 5 minutes, and this was repeated 4 times. Next, 200 mL of peroxidase-labeled anti-rat IgG (Medical and Biological Laboratories) diluted 1000-fold with PBS-SM was added, shaken at room temperature for 1 hour, then washed with 4 shakes of 500 mL of PBS-T, and further with 100 mL of PBS. Washed twice with shaking.
[0017]
Drain the filter and immerse it in a chromogenic substrate solution (diaminobenzidine 12 mg / PBS 25 mL with 2.5% cobalt chloride and 2% nickel sulfate 50 μL each), and then add 80 μL 30% hydrogen peroxide solution. In addition, color was developed, and the corresponding clone was selected from the position of color development.
[0018]
[5] Confirmation of nucleotide sequence According to the instruction manual of the above-mentioned “Zap-cDNA synthesis kit” (Stratagene), two positive clones obtained by immunoscreening were obtained from the Uni-ZAP XR vector in vivo. And then subcloned into pBluescript phagemid and propagated in E. coli, and then each clone was analyzed by PerkinElmer ABI PRISM377.
The nucleotide sequence was confirmed using a DNA sequencer.
[0019]
A homology search of the nucleotide sequence obtained here by BLAST from the database revealed that one clone was almost identical to tryptase III and the other one clone was breast cancer suppressor candidate-1 (Breast cancer suppressor candidate-1). -1 and hereinafter referred to as Bcsc-1) (FIG. 2). In FIG. 2, the sequence from 1 ′ to 2552 ′ represents Bcsc-1, the sequence from 1 ″ to 2571 ″ represents the clone obtained this time, and “*” represents the coincidence point. BLAST is an abbreviation for basic local alignment search tool.
[0020]
For clones almost identical to tryptase III, tryptase III was a well-known enzyme present in mast cell granules, so it was judged to be unrelated to the target cell surface antigen. Therefore, it was examined whether or not the clone almost identical to Bcsc-1 was the target cell surface protein.
[0021]
[6] Transfection into BHK cells Among the cDNAs obtained above, the expression vector BCMGSNeo prepared according to the method of Hatakeyama et al. Was designated and designated as the region encoding the Bcsc-1 protein registered in the database. 3, Karasuyama, H. & Melchers, F .: Eur. J. Immunol., 18, 97-104, 1988, Karasuyama, H., Tohyama, N. & Tada, T .: J. Exp. 169, 13-35, 1989, Yagita, H., Nakamura, T., Karasuyama, H. & Okumura, K.,: Proc. Natl. Acad. Sci. USA, 86, 645-649, 1989, Karasuyama, H. et al. Kudo, A .; & Melchers, F .: J. Exp. Med., 172, 969-972, 1990). Specifically, the introduction of restriction sites for XhoI and NotI into Bcsc-1 was carried out using cagCTCGGAtgggagggctctgggg (SEQ ID NO: 3) as a 5′XhoI primer, tctggatGCGGCCGCtcaagagcag (sequence number 4) using 3 ′ NotI primer. As PCR conditions, after heating at 94 ° C. for 4 minutes, react at 95 ° C. for 1 minute 15 seconds, 60 ° C. for 1 minute 10 seconds, 72 ° C. for 3 minutes, repeat this 39 times, then react at 72 ° C. for 5 minutes It was done by letting. Then, Bcsc-1 introduced with XhoI and NotI sites was treated with XhoI and NotI, and then incorporated into BCMGSNeo treated with XhoI and NotI.
[0022]
BHK cells were introduced into Bcsc-1 by the lipofection method and transformed. That is, BHK cells were seeded in a DMEM medium (Nissui Pharmaceutical) with 10% FCS to a density of 50% to 70%, cultured overnight, then replaced with a fresh medium and maintained at 37 ° C. for 2 hours. Cultured. After washing 3 times with serum-free DMEM, 800 μL of serum-free DMEM is mixed with 100 μL of serum-free DMEM containing BCMGSNeo into which 2.5 μL (2.5 μg) of Bcsc-1 has been introduced and 100 μL of serum-free DMEM containing 100 μL of lipofectamine. 200 μL was added and cultured at 37 ° C. for 3 hours.
[0023]
[7] Preparation of anti-Bcsc-1 antibody The pET-28a vector introduced with Bcsc-1 was expressed in Escherichia coli as a fusion protein with His-tag, and the fusion protein was purified using a nickel chelate column. The rabbit was immunized according to a conventional method to obtain antiserum, ie, anti-Bcsc-1 polyclonal antibody. Introduction of Bcsc-1 into the pET-28a vector was performed as follows. Specifically, EcoRI and XhoI restriction enzyme cleavage sites were introduced into Bcsc-1, and then this Bcsc-1 was treated with XhoI and EcoRI, and then incorporated into a pET-28a vector similarly treated with XhoI and EcoRI. In addition, the restriction enzyme cleavage site of EcoRI and XhoI was introduced into Bcsc-1 by using tcagGAATTCatgaggagctctt (SEQ ID NO: 5) as a 5 ′ EcoRI primer and using gggTACTCGAGaagaggaagatagc (SEQ ID NO: 6) as a 3 ′ XhoI primer. By amplification.
[0024]
[8] Cell staining and Western blotting BHK cells obtained by the above [6] (expressed by incorporating the Bcsc-1 gene) in a culture solution in which 500 μg / mL G418 was added to 10% FCS DMEM. Cultivate overnight, remove the culture medium, place the anti-Bcsc-1 polyclonal antibody (10 ³ fold dilution) obtained in [7] above, react at 37 ° C. for 1 hour, rinse with PBS, and FITC-labeled anti-rabbit IgG (H + L) (Medical and Biological Laboratories) (100-fold dilution) was reacted at 37 ° C. for 1 hour, and intracellular localization was confirmed with a fluorescence microscope.
[0025]
Similarly, as an antigen, an extract of mast cells, an extract of BHK cells introduced with Bcsc-1, an extract of eosinophils induced to differentiate using IL-5, an extract of peripheral blood cells, an extract of peripheral blood The protein recognized by this anti-Bcsc-1 polyclonal antibody was searched by Western blotting using an extract of cell line HL-60 and an extract of eosinophils differentiated from HL-60.
[0026]
As a result, when the mast cell extract was stained, diffuse fluorescence was observed throughout the cytoplasm. From this, it was considered that the Bcsc-1 gene is not directed against the mast cell surface antigen. In Western blotting, when BHK cells into which Bcsc-1 was introduced were used as antigens, a band was detected at about 45 kD, which almost coincided with the molecular weight expected from the inserted Bcsc-1. When mast cells were used as the antigen, a band was observed at a position of about 90 kD, and the results differed between the two cell extracts. This supports that the Bcsc-1 gene is not directed against a mast cell surface antigen.
[0027]
[9] Confirmation of nucleotide sequence The gene sequence found by the present inventors was compared with the gene sequence of Bcsc-1 registered on the database (see FIG. 2). As a result, in the base sequence obtained by the present inventors, base A (base number 943 '' in FIG. 2) or 140 bp upstream of the start codon of Bcsc-1 (ATG of base numbers 1055 ′ to 1057 ′ in FIG. 2) There was one extra base number 943 of SEQ ID NO: 2. Thereby, the start codon of the base sequence obtained by the present inventors (base numbers 36 ″ to 38 ″ in FIG. 2 or base number 36 of SEQ ID NO: 2). -38) was found to go back approximately 1000 bp upstream of the Bcsc-1 start codon, and the region containing the new start codon found here is a typical transcription start sequence (ACCATGG). The protein encoded by the base sequence obtained by the present inventors was named MASA-1.
[0028]
[10] Introduction of MASA-1 into BHK cells Transfection of BHK cells with MASA-1 was carried out in the same manner as in [6] above. However, tcttgcCTCGAGatgggtgacttctgtgg (SEQ ID NO: 7) was used as a 5′XhoI primer.
[0029]
[11] Confirmation of characteristics of MASA-1 Cell staining and Western blotting were carried out in substantially the same manner as in [8] above, using BHK cells introduced with MASA-1 instead of BHK cells introduced with Bcsc-1. Went. However, the cell staining was performed using the monoclonal antibody 5C12 produced by the fused cell ahMC5C12 described below as the primary antibody. As a result, when Western blotting was performed using BHK cells into which MASA-1 had been introduced as an antigen, a band was observed at a position of about 90 kD, as in the case of using mast cells. Was recognized. Expression was also observed in rat mast cell RBL-2H3, but expression was observed in umbilical cord blood, eosinophils, peripheral blood lymphocytes (PBL), and peripheral blood cell line HL-60. I couldn't. From these results, it was confirmed that the protein encoded by MASA-1 was a specific protein on the surface of mast cells.
[0030]
[12] Fusion cell ahMC5C12 (Accession number FERM BP-6070)
Here, an example of a method for producing the fused cell ahMC5C12 will be described. First, a 4 week-old rat infant was injected intraperitoneally with the cord blood obtained in Example 1 above for 2 weeks (10 ⁶ cells / 0.1 ml). The ability to induce antibody production against all antigens was lost. After 1.5 months, MC-TC (10 ⁶ cell / 0.10 ml) obtained in [1] above was intraperitoneally injected with a complete adjuvant for sensitization. Furthermore, the cells were injected intraperitoneally alone twice every two weeks thereafter. On the 4th day after the final immunization, the spleen was taken out and cell fusion was performed as shown below.
[0031]
The excised rat spleen cells and mouse myeloma cells were mixed at a ratio of 10: 1, and cell fusion was performed using 50% polyethylene glycol 1500 as a fusion accelerator. The fused cells are suspended in a HAT medium containing 10% bovine serum so that the cell concentration is 5 × 10 ⁵ cells / ml per spleen cell, and 200 μl per well is added to a 96-well microtiter plate (Nunk). Dispensed. The fused cells were cultured in a CO ₂ incubator (5% CO ₂ , 37 ° C.), and the cells were expanded by changing the medium in a HAT medium to screen for fused cells composed of spleen cells and myeloma cells. Then, it was acclimatized in HAT medium and further acclimatized with IMDM medium of 10% FCS (fetal calf serum) Iscove. As an antibody in the supernatant of the fused cell culture, MC-TC isolated from mast cytoma skin lesion was used as an antigen, and a clone producing an antibody that reacts with the antigen was selected by the fluorescent antibody method and named ahMC5C12. The obtained clone cells were suspended in 90% bovine serum each containing 10% DMSO and stored in liquid nitrogen. The anti-mast cell surface antigen monoclonal antibody produced by the clone was obtained by growing ahMC5C12 in the abdominal cavity of nude mice and purifying it from the ascites.
[0032]
[Sequence Listing]

[Brief description of the drawings]
FIG. 1 is a graph showing the results of measuring the ratio of chymase-positive mast cells and tryptase concentration in mast cells before and after co-cultivation.
FIG. 2 is an explanatory diagram comparing the base sequences of Bcsc-1 and MASA-1.
FIG. 3 is an explanatory diagram of the structure of a cDNA expression vector BCMGSNeo.

Claims (2)

A DNA having a base sequence encoding a mast cell surface antigen comprising the amino acid sequence of SEQ ID NO: 1. The DNA according to claim 1, which has the base sequence of SEQ ID NO: 2 or the base sequence represented by base numbers 36 to 2396 of the base sequence of SEQ ID NO: 2.

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