JPH07188289A - Polypeptide, its production and use - Google Patents

Abstract

PURPOSE:To obtain a new polypeptide, having specific physical properties, capable of causing pollinosis and useful as a desensitizer, etc. CONSTITUTION:This polypeptide has the following physico-chemical properties: molecular weight: 55000+ or -5000 daltons (measured by the SDS-polyacrylamide gel electrophoresis); isoelectric point: 9.0+ or -0.5 (measured by the isoelectric focusing); amino acid sequence at the N-terminal: having an amino acid sequence expressed by the formula at the N-terminal; ultraviolet absorption spectrum: the absorption maximum at about 280nm; solubility in solvents: soluble in water, a physiological saline solution and a phosphoric acid buffer solution; biological action: capable of binding to an immunoglobulin E antibody collected from blood of a patient suffering from pollinosis and causing the pollinosis and stability: inactivated by heating at 100 deg.C for 1 min in an aqueous solution (pH7.2) without being substantially inactivated even by allowing the polypeptide to stand at 4 deg.C in an aqueous solution (pH7.2) for 1 month. Furthermore. the polypeptide is obtained by culturing a transformant containing a DNA capable of coding the polypeptide in a culture medium for culturing and collecting the produced polypeptide from the resultant culture.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel polypeptide causing pollinosis, a method for producing the same, and use as a desensitizing agent.

[0002]

2. Description of the Related Art In recent decades, the number of people complaining of rhinitis and conjunctivitis due to pollinosis has been increasing in Japan in early spring. Due to the large number of patients and the onset of various events in the early spring, it has been frequently taken up in the media and is now one of the issues that cannot be ignored in public health.

It is said that hay fever is a kind of allergic disease, and its main cause is an antigenic substance in cedar pollen, that is, cedar pollen allergen. When cedar pollen scattered in the air enters the human body, an immunoglobulin E antibody against the cedar pollen allergen is produced. When cedar pollen invades next in this state, the allergen in the pollen immunoreacts with the immunoglobulin E antibody, and allergic symptoms are exhibited.

At present, it is known that cedar pollen has at least two kinds of allergens having different antigenicities. One of them is Yasueda et al.
Allergic and Clinical Immunology ", Vol. 71, No. 1, pp. 77-86 (1983), and today, this is" Cryj.
It is called "I". The other is Tanyei et al., "F.B.S.Letters", Volume 239, Volume 2.
No. 329-332 (1988) and Sakaguchi et al., "Allergy", No. 45, 309-312 (19).
90) and is today referred to as "Cry j II". Cry j I and Cry j II are usually present in cedar pollen at a ratio of about 50: 1 to 5: 1, and most of the sera collected from pollinosis patients have Cry j I and Cry j I.
It is said to react to I as well. Sawaya et al., "Allergy", Vol. 42, No. 6, 738-747 (199).
3 years), Cry jII was used for intradermal tests and RAS
It is reported that the T-test exhibits the same antigenicity as Cry j I.

As described above, several cedar pollen allergens have already been isolated and their properties and properties have been elucidated to some extent. Therefore, administration of purified cedar pollen allergen to humans to desensitize them causes pollinosis. There is a prospect of treatment and prevention. Recently, some desensitizing agents have been devised for that purpose. For example, in JP-A-1-156926 and JP-A-3-93730, Asp-A is added to the N-terminal.
sn-Pro-Ile-Asp-Ser- or Ala-
A proposal has been made to covalently bind pullulan, which is a type of polysaccharide, to an allergen having an amino acid sequence represented by Ile-Asn-Ile-Phe-Asn-, and administer the resulting complex to humans as a desensitizing agent. There is. However, a large amount of high-purity allergen is usually required for the diagnosis of allergic disease and desensitization therapy, and the allergen in cedar pollen is scarce and its stability is low, and it is a diagnostic agent for pollinosis. It is expected that there will be a great deal of difficulty in covering the desensitizing agent with cedar pollen alone.

On the other hand, recent progress in recombinant DNA technology is remarkable. Nowadays, even for a polypeptide whose entire amino acid sequence has not been elucidated, a gene encoding the same can be isolated, and if its nucleotide sequence can be elucidated, a recombinant DNA containing the DNA encoding the polypeptide can be prepared, By culturing the transformant obtained by introducing this into cells of microorganisms and animals and plants, it has become possible to obtain a desired amount of polypeptide relatively easily.

From the above circumstances, it has been awaited that all the genes encoding allergens in Japanese cedar pollen are isolated and their nucleotide sequences are elucidated as soon as possible. Recently published International Patent Application Publication No. 93/01213 discloses a nucleotide sequence encoding Cry j I, which is one of such allergens. As described above, the allergen causing pollinosis is Cry. not only j I,
For accurate diagnosis and effective hyposensitization therapy, it is an urgent need in the art to isolate the genes encoding the other allergens and to elucidate their nucleotide sequences.

[0008]

In view of such a situation, an object of the present invention is to provide a novel polypeptide which causes pollinosis.

Another object of the present invention is to provide a method for producing the polypeptide by applying recombinant DNA technology.

Still another object of the present invention is to provide the use of the polypeptide as a desensitizing agent.

[0011]

The present invention solves the first problem by a polypeptide having the following physicochemical properties. (1) Molecular weight 55,000 ± 5,000 daltons (SDS-polyacrylamide gel electrophoresis) (2) Isoelectric point 9.0 ± 0.5 (isoelectric focusing) (3) N-terminal amino acid sequence N-terminal Ala-Met-Lys-Phe-Ile-A
la-Pro-Met-Ala-Phe-Val-Al
a-Met-Gln-Leu-Ile-Ile-Met
It has an amino acid sequence represented by -Ala-. (4) Ultraviolet absorption spectrum It has an absorption maximum near 280 nm. (5) Solubility in solvent Soluble in water, physiological saline and phosphate buffer. (6) Biological action It binds to immunoglobulin E antibody collected from the blood of patients with hay fever. Causes hay fever. (7) Stability Deactivates when heated at 100 ° C. for 10 minutes in an aqueous solution (pH 7.2). Even if left in an aqueous solution (pH 7.2) at 4 ° C. for 1 month, it is not substantially deactivated.

The present invention solves the second problem by a method comprising culturing a transformant containing a DNA encoding the polypeptide in a culture medium and collecting the polypeptide produced from the culture. Is.

The present invention solves the third problem by a desensitizing agent containing the polypeptide as an active ingredient.

[0014]

The polypeptide of the present invention is a novel substance having unique physicochemical properties, which is not found in conventionally known cedar pollen allergens.

The production method of the present invention makes it possible to produce a desired amount of such a polypeptide by recombinant DNA technology.

The desensitizing agent of the present invention exerts a desensitizing effect when administered to mammals including humans.

The present invention will be described below based on experimental examples, examples, etc. The present invention relates to a novel polypeptide causing pollinosis. The present inventor has been studying allergens in cedar pollen, and has found that the novel allergens that have been unknown so far are included. When this allergen was isolated by combining various purification methods centered on column chromatography and its properties and properties were investigated, it was found that its essence is a polypeptide and has the following physicochemical properties. . (1) Molecular weight 40,000 ± 5,000 daltons (SDS-polyacrylamide gel electrophoresis) (2) Isoelectric point 9.0 to 10.0 (isoelectric focusing) (3) Partial amino acid sequence His-Asp -Ala-Ile-Asn-Ile-P
he-Asn-Val-Glu-Lys, His-AS
p-Cys-Thr-Glu-Ala-Phe-Ser
-Thr-Ala-Trp-Gln-Ala-Ala-
Cys-Lys, Asn-Thr-Ile-Gly-T
hr-Gly-Asp-Asp-Cys-Val-Al
a-Ile-Gly-Thr-Gly-Ser-Ser
-Asn-Ile-Val-Ile-Glu-ASp-
Leu-Ile-Cys-Gly-Pro-Gly, or Ile-Ala-Ser-Cys-Leu-Asn-.
Asp-Asn-Ala-Asn-Gly-Tyr-P
he-Ser-Gly-His-Val-Ile-Pr
It has a partial amino acid sequence represented by o-Ala. (4) Ultraviolet absorption spectrum It has an absorption maximum near 280 nm. (5) Solubility in solvent Soluble in water, physiological saline and phosphate buffer. (6) Biological action Causes hay fever. It binds to immunoglobulin E antibody collected from the blood of patients with hay fever. (7) Stability Deactivates when heated at 100 ° C. for 10 minutes in an aqueous solution (pH 7.2). Even if left in an aqueous solution (pH 7.2) at 4 ° C. for 1 month, it is not substantially deactivated.

The polypeptide having such physicochemical properties has not been known yet, and it is judged to be a novel substance.

Then, the present inventor succeeded in isolating mRNA from cedar pollen as a result of intensive screening of the gene encoding this polypeptide. This mR
D obtained by reverse transcriptase reaction with NA as a template to prepare cDNA, and gene amplification of the cDNA by PCR method
When the NA fragment was analyzed, the target gene was deciphered from the 5 sequence shown in Chemical formula 3 below, and it was found that the polypeptide was composed of a maximum of 514 amino acids as shown in Chemical formula 4. . Next, a series of experiments leading to elucidation of the nucleotide sequences or amino acid sequences shown in Chemical formulas 3 and 4 will be described.

[0020]

[Chemical 3]

[0021]

[Chemical 4]

[0022]

[Experimental Example 1 Purification of Polypeptide] About 16 parts by weight of 0.1 parts by weight per 1 part by weight of pollen collected from male flowers of Urasugi from Akita
A 25 M sodium hydrogencarbonate aqueous solution (pH 8.2) was added, and the mixture was extracted with stirring at 4 ° C. for 1 hour and then centrifuged to collect the supernatant. The residue was reprocessed in the same manner, and the obtained supernatant and the supernatant obtained in the first extraction were pooled, and ammonium sulfate was added to this to 80% saturation for salting out. A 50 mM Tris-hydrochloric acid buffer solution (pH 7.
It was dialyzed against 8) for 10 hours, filtered, and then subjected to DEAE-Sephadex column chromatography preliminarily equilibrated with 50 mM Tris-hydrochloric acid buffer (pH 7.8) to collect a non-adsorbed fraction.

Thereafter, acetic acid was added to the non-adsorbed fraction to pH.
After adjusting to 5.0, 10 mM acetate buffer (pH
5.0) and equilibrated with CM-Sephadex, 10 mM acetate buffer (pH 5.0) was passed through to wash the column, and then 0.1 M phosphate buffer (pH 7.
0) and 0.3 M sodium chloride were passed through the column to elute the peptide component. A fraction containing the polypeptide is collected and preliminarily collected in 10 mM phosphate buffer (pH
The column was loaded onto a Mono-S column that had been equilibrated with 5.0), and the column was subjected to 10 mM Tris-HCl buffer (pH) under a concentration gradient of sodium chloride increasing from 0M to 0.5M.
7.0), the sodium chloride concentration was 0.
The polypeptide was eluted at 4M.

The purified polypeptide obtained in this manner remarkably reacted with immunoglobulin E antibody collected from patients with pollinosis and anti-cedar pollen allergen derived from mouse.
The yield is about 0.006 in terms of the solid content of cedar pollen as a raw material.
%Met.

[0025]

Experimental Example 2 Physicochemical Properties of Polypeptide In this experimental example, the physicochemical properties of the purified polypeptide obtained in Experimental Example 1 were examined.

[0026]

[Experimental Example 2-1 Molecular Weight] U.K. Lamuri, "Nature", Vol. 227, pp. 680-685 (1970)
S.) purified polypeptide according to the method reported in
When subjected to DS-polyacrylamide gel electrophoresis, a band was observed at a position corresponding to a molecular weight of 40,000 ± 5,000 daltons. The molecular weight markers used at this time were bovine serum albumin (67,000 daltons), ovalbumin (45,000 daltons), carbonic anhydrolase (30,000 daltons), chymotrypsinogen A (25,000 daltons) and cytochrome C. (12,000 Dalton).

A component having a molecular weight of 40,000 ± 5,000 daltons was transferred from the gel onto a nitrocellulose membrane, and an anti-cedar pollen allergen antibody derived from mouse and an anti-mouse immunoglobulin antibody derived from goat labeled with horseradish peroxidase were obtained. Upon actuation, significant immunostaining was observed. This suggests that this component is a kind of cedar pollen allergen.

[0028]

[Experimental Example 2-2 Isoelectric Point] The isoelectric point of the purified polypeptide was 9.0 to 1 as measured by the isoelectric focusing method.
It was 0.0.

[0029]

[Experimental example 2-3 Partial amino acid sequence] The purified polypeptide was digested with trypsin, and the digested product was subjected to chromatography column "Hi-Pore PR-318" manufactured by Bio-Rad.
Separation by high performance liquid chromatography using "type" gave peptide fragments of four kinds of names. When analyzed using an amino acid sequencer, these peptide fragments were (a) His-Asp-Ala-Ile-Asn.
-Ile-Phe-Asn-Val-Glu-Lys,
(B) His-Asp-Cys-Thr-Glu-A
la-Phe-Ser-Thr-Ala-Trp-Gl
n-Ala-Ala-Cys-Lys, (c) Asn
-Thr-Ile-Gly-Thr-Gly-Asp-
Asp-Cys-Val-Ala-Ile-Gly-T
hr-Gly-Ser-Ser-Asn-Ile-Va
l-Ile-Glu-Asp-Leu-Ile-Cys
-Gly-Pro-Gly, or (d) Ile-Al
a-Ser-Cys-Leu-Asn-Asp-Asn
-Ala-Asn-Gly-Tyr-Phe-Ser-
It was found to have an amino acid sequence represented by Gly-His-Val-Ile-Pro-Ala.

[0030]

[Experimental Example 2-4 Ultraviolet absorption spectrum] When the ultraviolet absorption spectrum in an aqueous solution was measured using a spectrophotometer, the purified polypeptide showed an absorption maximum near a wavelength of 280 nm.

[0031]

Experimental Example 2-5 Solubility in Solvent When tested by a conventional method, the purified polypeptide was soluble in water, physiological saline and phosphate buffer.

[0032]

[Experimental Example 2-6 Biological Action] The purified polypeptide, when tested by the method described below, has the property of binding to an immunoglobulin E antibody collected from the blood of a patient with hay fever and T that specifically reacts with the polypeptide. It shows the property of inducing cell proliferation.

[0033]

[Experimental Example 2-6 (a) Immunoglobulin E antibody binding test] 1 μl / well of purified polypeptide was adsorbed on a 96-well microplate, and 1 μl / well of serum collected from a pollinator patient or a healthy person was adsorbed. After adding each, the mixture was incubated at 37 ° C. for 2 hours. next,
After washing the microplate with distilled water to remove unbound serum, 1 μl of the anti-human immunoglobulin E antibody derived from horse mackerel-derived peroxidase derived from Oki was used.
Each well was added and incubated at 37 ° C. for another 2 hours. The microplate was washed with distilled water to remove unbound antibody, a small amount of hydrogen peroxide solution containing orthophenylenediamine was added to each well, and after color development, the absorbance at 492 nm was measured.

As a result, while the absorbance of the system using the serum of a healthy person was about 0.1, the absorbance of the system using the serum of the hay fever patient reached about 2.0. This indicates that the purified polypeptide significantly binds to the immunoglobulin E antibody contained in the blood of pollinosis patients. This supports the fact that the purified polypeptide is one of the causative agents of hay fever, that is, has the property of inducing hay fever.

[0035]

[Experimental Example 2-6 (b) T cell proliferation induction test] Mononuclear cells were separated from heparinized peripheral blood of pollinosis patients by Ficoll-Hypaque gravity centrifugation. 10% mononuclear cells
(V / v) Suspended in RPMI1640 medium (pH 7.0) supplemented with AB serum at a concentration of 1 × 10 ⁶ cells / ml, and purified polypeptide obtained in Experimental Example 1 was added at 20 μg / ml.
After adding ml, the cells were cultured at 37 ° C for 7 days in a 5% CO ₂ incubator. The cultured mononuclear cells were washed with fresh RPMI1640 medium (pH 7.0) and then resuspended in the same medium at 1 × 10 ⁶ cells / ml. Recombinant human interleukin-2 was added to the culture medium at 5 units / ml, and the mixture was further cultured for 3 days in the same manner as above. The mononuclear cells thus pretreated were subjected to the T cell proliferation test described below.

10% in 96 well microplate
(V / v) 2 × 10 ⁴ cultured mononuclear cells suspended in RPMI1640 medium (pH 7.0) supplemented with AB serum
Cells / well, 2 μCi of ³ H-thymidine, and 100 μg / ml of the purified polypeptide, and added with saline to 200
μl / well. The cells in the wells were cultured in a 10% CO ₂ incubator at 37 ° C. for 3 days, and the amount of ³ H-thymidine incorporated into the cells was measured by a scintillation counter. At the same time, a system using physiological saline containing no polypeptide was provided, and this was treated in the same manner as above to serve as a control.

As a result, about 300 cpm in the control system
Was observed, the system in which the purified polypeptide was added showed a remarkable uptake of about 6,500 cpm per 100 μg / ml of the polypeptide. This indicates that the purified polypeptide has antigenicity.

[0038]

Experimental Example 2-7 Stability When the purified polypeptide was incubated in an aqueous solution (pH 7.2) at 100 ° C. for 10 minutes, no residual activity was observed. On the other hand, when the purified polypeptide was stored in an aqueous solution (pH 7.2) at 4 ° C for 1 month, no substantial decrease in activity was observed.

The polypeptide having the above physicochemical properties has not been known yet, and it is judged to be a novel substance.

Next, the experiments carried out to elucidate the nucleotide sequence encoding this polypeptide and the entire amino acid sequence will be described. In summary, the amino acid sequence of the polypeptide according to the present invention is subjected to the following series of operations. It has been clarified through. (1) A pollen extract collected from a Japanese cedar male flower was purified by a purification method centered on chromatography to isolate a highly pure polypeptide. (2) This polypeptide was digested with trypsin, and four kinds of peptide fragments were isolated from the digested product and the amino acid sequence thereof was determined. (3) An oligonucleotide presumed to encode these partial amino acid sequences was chemically synthesized, and RT-P
Separately extracted from Japanese cedar pollen using CR primer
Gene amplification was performed using RNA as a template. (4) A plurality of DNA fragments encoding the polypeptide, which are chemically synthesized based on the partial amino acid sequence in the same manner and hybridized with an isotope-labeled oligonucleotide as a probe and the DNA fragment obtained by gene amplification. Was selected. (5) The base sequence of the selected DNA fragment is determined, and the amino acid sequence decoded from the base sequence is compared with the partial amino acid sequence to confirm that the estimated base sequence encodes the polypeptide. confirmed.

[0041]

[Experimental Example 3 Determination of the entire amino acid sequence of a polypeptide and the nucleotide sequence encoding the same] In this Experimental Example, the nucleotide sequence encoding the polypeptide and the entire amino acid sequence will be clarified. The method itself used in this experimental example is known in the art, and for example, T. Manatis, et al., "Molecular Cloning a Laboratory Manual," 1
Published by Cold Spring Harbor in 989,
Masami Muramatsu "Lab Manual Genetic Engineering", 1988,
It is also detailed in the publication of Maruzen Publishing Co., Ltd.

[0042]

[Experimental Example 3-1 Preparation of total RNA] 10 g of cedar pollen pre-freeze-treated with liquid nitrogen was added to 10 mM tris-hydrochloric acid buffer (pH 7.4), 200 mM sodium chloride, 6 mM magnesium chloride and 1% (w / v) sarcosyl. And 40 ml of a phenol / chloroform / isoamyl alcohol solution were added, and the mixture was treated with Polytron to crush pollen. Treated product 10,000 × g, 4 ℃
Centrifuge for 10 minutes, collect the separated aqueous layer, add an equal volume of fresh phenol / chloroform / isoaminoalcohol solution, extract in the same manner as above, and centrifuge again to collect the aqueous layer. After adding 2.5 volumes of ethanol to this, it was left at −20 ° C. for 2 hours to precipitate total RNA.

Total RNA was washed with 75% (v / v) ethanol, dried and dissolved in 29 ml of sterile distilled water to give 300
After adding mM phosphate buffer (pH 6.8),
It was loaded onto a hydroxyapatite column that had been equilibrated with M phosphate buffer (pH 6.8). 10 mM column
After washing with phosphate buffer (pH 6.8), 300 mM phosphate buffer (pH 6.8) was passed to elute total RNA, and 2.5 times volume of 75% (v / v) ethanol was added to the eluate. In addition, total RNA was precipitated by standing at -20 ° C for 2 hours.
The precipitated total RNA was washed with 75% (v / v) ethanol, dried, dissolved in 0.5 ml of sterilized distilled water, and used for selection of clones SC09, SC31, SC216, SC50 and SC362 described below. The total RNA recovered was about 500 μg.

[0044]

[Experimental Example 3-2 Preparation of First-Stranded cDNA] 4 μl of 25 mM magnesium chloride was added to 1 μg of total RNA obtained in Experimental Example 3-1, and 10 × PCR buffer (10 mM
Tris-HCl buffer (pH 8.3), 500 mM potassium chloride) 2 μl, 1 mM dNTP mix 8 μm
l, 1 unit / μl RNase inhibitor 1 μl, 2.5
Unit / µl Reverse transcriptase (1 µl), 2.5 µM random hexamer (1 µl) were added, and the volume was adjusted to 20 µl with sterile distilled water.
By a conventional method, the mixture was incubated in a 0.5 ml reaction tube at 25 ° C. for 10 minutes, 42 ° C. for 30 minutes, 99 ° C. for 5 minutes, and 5 ° C. for 5 minutes to carry out reverse transcriptase reaction to give first strand cDNA. A solution containing was obtained.

[0045]

[Experimental Example 3-3 Selection of SC09 clone and determination of partial base sequence] First strand c obtained in Experimental Example 3-2
4 μm of 25 mM magnesium chloride to 20 μl of DNA solution
l, 10x PCR buffer (100 mM Tris-HCl buffer (pH 8.3), 500 mM potassium chloride) 8μ
1, 2.5 units / μl 0.5 μl of amplitac DNA polymerase, 1 pmol of the following primer 1 as a sense primer and 1 pmol of the following primer 2 as an antisense primer were added, respectively, to make 100 μl with sterile distilled water. Then, by a conventional method, the mixture was repeatedly reacted 40 times in a cycle of 94 ° C. for 1 minute, 45 ° C. for 2 minutes, and 72 ° C. for 3 minutes to amplify a DNA fragment partially encoding the polypeptide using cDNA as a template. . In addition, as for the primer 1 and the primer 2, Experimental example 2-
An oligonucleotide chemically synthesized based on the amino acid sequence represented by His-Asp-Ala-Ile-Asn-Ile or Ala-Trp-Gln-Ala-Ala in the partial amino acid sequences (a) and (b) shown in 3 Yes, it had a base sequence shown in Table 1 or Table 2 below. As the PCR reagent, "GeneAmp RNA PCR Kit" manufactured by Takara Shuzo was mainly used.

[0046]

[Table 1]

[0047]

[Table 2]

A portion of the obtained PCR product was taken, fractionated by electrophoresis on a 2% agarose gel by a conventional method, pressure blotted on a nylon membrane, and then fixed with 0.4N sodium hydroxide. . The nylon membrane was washed with 2 × SSC, air dried, 5 × SSPE, 5 × Denhardt's solution, 0.5% (w
/ V) SDS and 100 μg / ml denatured salmon sperm DNA
Dip into a pre-hybridization mixture containing
Incubated at ℃ for 3 hours. Separately, as the probe 1, Phe-Asn-Val-Glu-L in the partial amino acid sequence (a) shown in Experimental Example 2-3 was prepared by a conventional method.
Oligonucleotides having the nucleotide sequences shown in Table 3 below, which are presumed to encode ys, were chemically synthesized and labeled with [γ- ³² P] ATP by T4 nucleotide kinase. 1 pmol of this probe 1 was taken, and 5x SSPE and 5x
The nylon membrane was immersed in a mixed solution containing Denhardt's solution and 0.5% (w / v) SDS, incubated at 45 ° C. for 24 hours for hybridization, and then the nylon membrane was 6 × SS.
After washing with C and autoradiography, it was confirmed that the PCR product contained the target DNA fragment.

[0049]

[Table 3]

Next, 50 ng of the plasmid vector "pT7 Blue T" manufactured by Takara Shuzo Co., Ltd., an appropriate amount of T4 DNA ligase and 100 mM ATP were added to the remaining PCR products to a final concentration of 1 mM, and the mixture was incubated at 16 ° C. for 24 hours to prepare the vector. Recombinant D obtained by inserting DNA fragment
NA was introduced into E. coli NoVa Blue strain obtained from Pharmacia by the competent cell method. The transformant was bactotryptone 10 g / l, sodium chloride 1
1 g / l, Bacto agar 15 g / l, ampicillin 10
0 mg / l, X-Gal 40 mg / l and isopropyl-β-D-thiogalactopyranoside (hereinafter referred to as “IPT
Abbreviated as "G". ) A plate medium containing 23.8 mg / l was inoculated and incubated at 37 ° C. for 24 hours to form colonies. The nylon membrane was placed on the culture medium, allowed to stand for 30 seconds, then the nylon membrane was peeled off, and immersed in a mixed solution containing 0.5 M sodium hydroxide and 1.5 M sodium chloride for 7 minutes. The nylon membrane was taken out and 0.5 M Tris-hydrochloric acid buffer solution (pH 7.
2) for 3 minutes, washed with 2 × SSC, washed with 0.4M sodium hydroxide for 20 minutes, washed with 5 × SSC, air-dried, 5 × SSPE, 5 × Denhardt's solution, 0.5
% (W / v) SDS, 100 μg / ml denatured salmon sperm D
It was immersed in a prehybridization mixture containing NA and incubated at 65 ° C. for 3 hours. After that, the nylon membrane was hybridized with the probe 1 in the same manner as above, and autoradiography was performed. Based on the results,
Clone SC09 containing the original DNA fragment was selected from the plate medium.

The amphibilin 10
L-broth medium (pH 7.2) containing 0 μg / ml was inoculated and cultured at 37 ° C. for 18 hours, the bacterial cells were collected from the culture, crushed, and then extracted by a usual alkaline method to give recombinant D
I got NA. When the DNA fragment in this recombinant DNA was decoded by the dideoki method using the DNA sequencing kit "Sequenase version 2.0" manufactured by Toyobo Co., Ltd., it had the nucleotide sequence from the 173rd position to the 240th position in the chemical formula 3. It turned out.

[0052]

[Experimental Example 3-4 Selection of Clone SC31 and Determination of Partial Nucleotide Sequence] In this experimental example, D different from clone SC09 was used.
A clone SC31 containing the NA fragment is selected and its partial base sequence is determined. Unless otherwise specified, each step in this experimental example is exactly the same as in Experimental example 3-3.

By the conventional method, primer 3 having a nucleotide sequence shown in Table 4 below arbitrarily selected from the partial nucleotide sequences of clone SC09 selected in Experimental Example 3-3 and the partial amino acid sequence shown in Experimental Example 2-3 ( Asn-Ile- in c)
A primer 4 having the nucleotide sequence shown in Table 5 below, which is presumed to encode Val-Ile-Glu, was chemically synthesized.
A portion of the first-strand cDNA solution obtained in Experimental Example 3-2 was taken, and this was subjected to gene amplification in the presence of Primer 3 and Primer 4 in the same manner as in Experimental Example 3-3, and clone SC09 was obtained from the obtained DNA fragment. Probe 2 having the nucleotide sequence shown in Table 6 below, which is arbitrarily selected from the partial nucleotide sequence of
Was used to collect a further DNA fragment partially encoding the polypeptide. Then, in the same manner as in Experimental Example 3-3, the pT7 blue T vector into which this DNA fragment was inserted was introduced into Escherichia coli NoVa Blue strain, and probe 2 was used from a colony obtained by culturing this E. coli strain in a plate medium. Clone SC31 was selected. This clone SC31 is the 210th nucleotide in the nucleotide sequence of Chemical formula 3.
It had a nucleotide sequence corresponding to the 795th position to the 795th position.

[0054]

[Table 4]

[0055]

[Table 5]

[0056]

[Table 6]

[0057]

[Experimental Example 3-5 Selection of Clone SC216 and Determination of Partial Base Sequence] A primer having the base sequence shown in Table 7 below arbitrarily selected from the partial base sequence of the clone SC31 selected in Experimental Example 3-4 by a conventional method. 5 and Asn-Asp in the partial amino acid sequence (d) shown in Experimental Example 2-3.
-Asn-Ala-Asn-Gly was presumed to code and primer 6 of the base sequence shown in the following Table 8 was chemically synthesized. First strand c obtained in Experimental Example 3-2
A portion of the DNA solution was taken, and this was subjected to gene amplification in the presence of the primer 5 and the primer 6 in the same manner as in Experimental Example 3-3, and the DNA fragment thus obtained was arbitrarily selected from the partial base sequence of the clone SC31. Using the probe 3 having the nucleotide sequence shown in Table 9 above, another DNA fragment partially encoding the polypeptide was collected. After that, Experimental Example 3
In the same manner as in -3, the pT7 blue T vector containing this DNA fragment was introduced into E. coli NoVaBlue strain,
Clone SC216 was selected from the colonies obtained by culturing this E. coli strain in a plate medium using probe 3. This clone SC216 had a nucleotide sequence corresponding to the 763rd position to the 1st and 233nd positions in the nucleotide sequence of Chemical formula 3.

[0058]

[Table 7]

[0059]

[Table 8]

[0060]

[Table 9]

[0061]

[Experimental example 3-6 Selection of clone SC50 and determination of partial base sequence] A part of the total RNA obtained in Experimental example 3-1 was taken,
This was subjected to a reverse transcriptase reaction in the presence of primer 7 having a base sequence shown in Table 10 below, which was arbitrarily selected from the partial base sequence of clone SC09 according to the method of Experimental Example 3-2. To the solution containing the obtained first strand cDNA, 4 μl of 5 × Tailing buffer, 4 μl of dATP,
After adding 10 units of deoxyribonucleotide terminal transferase and making it 20 μl with sterile distilled water, 37 ° C., 70 ° C.,
Incubated at 4 ° C. for 5 minutes each. Remove excess primer from the reaction with a spin filter,
After concentration, purified polydA-added first strand c
DNA Flowman et al., "Proceedings of National Academy of Sciences USA", Vol. 85, No. 8,998.
-10, 9002 (1988) and 10 pmol of primer 8 having the nucleotide sequence shown in Table 11 below.
0μ PCR buffer 10μl, dNTP mix 1μ
1, 2.5 units / μl 0.5 μl of Amplitac DNA Polymerase was added to 100 μl of sterile distilled water, and then incubated at 72 ° C. for 30 minutes, 99 ° C. for 5 minutes, and 4 ° C. for 5 minutes in this order. Second strand cdN
A was synthesized. After removing excess primer from the reaction product with a spin filter and concentrating, it was arbitrarily selected from Primer 9 having the base sequence shown in Table 12 below reported by Flowman et al. And a partial base sequence of SC09. 10 pmol of the primer 10 having the nucleotide sequence shown in Table 13 below, 10 μl of 10 × PCR buffer, 1 mM, respectively
After adding 1 µl of dNTP mix and 0.5 µl of 2.5 units / µl amplitac DNA polymerase to 100 µl with sterilized distilled water, gene amplification was carried out in the same manner as in Example 3-3, and the obtained DNA fragment was used to clone clone SC09. Using the probe 4 having the nucleotide sequence shown in Table 14 below, which was arbitrarily selected from the partial nucleotide sequence of, the additional DNA fragment partially encoding the polypeptide was collected. afterwards,
In the same manner as in Experimental Example 3-3, the pT7 blue T vector having this DNA fragment inserted was introduced into Escherichia coli NoVa Blue strain, and cloned using a probe 4 from a colony obtained by culturing the E. coli strain in a plate medium. SC5
0 was selected. This clone SC50 had a base sequence corresponding to the 1st to 198th bases in the base sequence of Chemical formula 3.

[0062]

[Table 10]

[0063]

[Table 11]

[0064]

[Table 12]

[0065]

[Table 13]

[0066]

[Table 14]

[0067]

[Experimental Example 3-7 Selection of Clone SC362 and Determination of Partial Base Sequence] 20 μl of the total RNA obtained in Experimental Example 3-1 was taken, and primer 8 was replaced by 0.5 with a random hexamer.
A reverse transcriptase reaction was carried out in the same manner as in Experimental Example 3-2 except that μg was used to obtain a solution containing first-strand cDNA. This solution was prepared by using primer 9 and clone S shown in Table 12.
Table 1 below arbitrarily selected from the partial base sequence of C216
Experimental Example 3 in the presence of the primer 11 having the nucleotide sequence shown in 5
Gene amplification by the method of No. -3, and using the probe 5 having the nucleotide sequence shown in Table 16 below, which is arbitrarily selected from the partial nucleotide sequence of the clone SC216 from the obtained DNA fragment, is used to partially encode the polypeptide. The DNA fragment of Then, in the same manner as in Experimental Example 3-3, the pT7 blue T vector into which this DNA fragment was inserted was introduced into Escherichia coli NoVaBlue strain, and this Escherichia coli strain was cultured in a plate medium. Clone SC362 was selected. This clone S
C362 had a base sequence corresponding to the 1,216th to the 1,548th bases in the base sequence of Chemical formula 3.

[0068]

[Table 15]

[0069]

[Table 16]

[0070]

[Experimental example 3-8 All nucleotide sequences encoding polypeptide and all amino acid sequences of polypeptide] Experimental examples 3-3 to 3
As a result of comprehensive analysis of the results of -7, it was revealed that the gene encoding the polypeptide has the base sequence from the 5'end shown in Chemical formula 3. As a result of decoding this base sequence, the polypeptide is N
It has an amino acid sequence from the end, is composed of a maximum of 514 amino acid residues, and naturally contains all four types of partial amino acid sequences shown in Experimental Example 2-3.

Thus, the polypeptide of this invention comprises:
The present inventor has discovered the first result in the world as a result of many years of research, and has unique physicochemical properties and amino acid sequences that are not found in conventionally known cedar pollen allergens. However, on the other hand, recombinant DNA
Advances in technology have allowed the art to substitute one or more of its constituent amino acids with other amino acids without substantially altering the biological activity of the polypeptide. In view of such state of the art, the polypeptide referred to in the present invention is not limited to the one having the amino acid sequence shown in Chemical formula 4 as it is, and the polypeptide in the amino acid sequence shown in Chemical formula 4 while retaining a specific N-terminal amino acid sequence. It is intended to include all homologous variants in which one or more amino acids have been replaced by other amino acids.

Next, the method for producing the polypeptide according to the present invention will be specifically described with reference to Examples and the like.

The polypeptide of the present invention is of natural origin or artificially, so long as it has the amino acid sequence shown in Chemical formula 4 or a sequence homologous to the amino acid sequence. It does not matter whether they are synthesized. Examples of the natural source include cedar trees belonging to the genus Cedar (Cryptomeria japonica). From the pollen and male flowers, as described above, the polypeptide of the present invention and the DNA encoding the same should be collected. Is possible. To artificially synthesize the polypeptide of the present invention, for example, it is chemically synthesized based on the amino acid sequence shown in Chemical formula 4, or DNA having the nucleotide sequence of Chemical formula 3 is appropriately inserted into an expression vector and recombined. A transformant obtained by using DNA as a host and appropriately introducing it into a host may be cultured.

As mentioned above, the present invention provides recombinant DNA.
It also provides a method for producing the polypeptide by applying the technique. The method according to the present invention comprises culturing a transformant containing a DNA encoding the polypeptide in a culture medium and collecting the polypeptide produced from the culture. The transformant used in the present invention is usually
It includes a replicable recombinant DNA having a DNA encoding the polypeptide inserted therein. Such recombinant DNA usually comprises a cDNA encoding the polypeptide and a replicable expression vector, and can be prepared relatively easily by ordinary recombinant DNA technology if the cDNA is available. Examples of such expression vectors include pKK223-3, pRL-λ, pBTrp2D.
NA, pUB110, YEp13, Ti plasmid, R
i plasmid, pBI121 and the like. Among them, pKK223 can be used to express the DNA encoding the polypeptide in microorganisms such as Escherichia coli, Bacillus subtilis and yeast.
-3, pRL-λ, pBTrp2DNA, pUB110
And YEp13 are preferable, and Ti plasmid and Ri plasmid are preferable for expressing in plant cells.

To insert the DNA into the expression vector, a method generally used in the art can be adopted. For example, the cDNA encoding the polypeptide is appropriately cleaved with a restriction enzyme, while the replicable appropriate vector is cleaved with the same enzyme. Then, by ligating the obtained DNA fragment and vector fragment with DNA ligase, recombinant D capable of replication can be obtained.
NA is obtained. By introducing such recombinant DNA into microorganisms such as Escherichia coli, Bacillus subtilis, actinomycetes, yeast, and cells of higher animals and plants by the competent cell method, protoblast method, electroporation method, etc. When the obtained transformant is cultured in a culture medium, the polypeptide is produced inside or outside the cells.

The produced polypeptide can be purified by a method generally used in the art. For example, a transformant culture and / or cells or cells are collected, and if necessary, the cells or cells are disrupted, and the resulting crude polypeptide is salted out, dialyzed, filtered, concentrated, and centrifuged. It may be purified by separation, gel filtration chromatography, ion exchange chromatography, affinity chromatography, electrophoresis, isoelectric focusing and the like. In particular, the monoclonal antibody specific to Cry j II reported in T. Kawashima et al., "International Archives of Allergy and Immunology", Vol. 98, pp. 110-117 (1992), is the It is extremely useful for purifying peptides, and a high-purity polypeptide can be obtained with a minimum of labor and time by affinity chromatography using this monoclonal antibody bound to a water-insoluble carrier. The partially purified or purified polypeptide thus obtained is a desensitizing agent for diagnosing or treating / preventing hay fever, and an immunological assay such as enzyme immunoassay or radioimmunoassay for diagnosing hay fever. It has a wide range of uses in methods, and further in academic research for elucidating the pathogenic mechanism of allergic diseases in general including hay fever.

Next, the method for producing the polypeptide according to the present invention will be specifically described based on Examples.

[0078]

Example A-1 Production of Polypeptide In this example, a plasmid vector was used to introduce DNA encoding the polypeptide into Escherichia coli to form a transformant, and the transformant was cultured. To produce the polypeptide. It is needless to say that the method can be modified in various ways in the art, and this example is only one example, and the manufacturing method of the present invention is not limited to this example.

[0079]

[Example A-1 (a) Preparation of transformant] Experimental example 3-
1 μg of the total RNA obtained in 1 above was used as a sense primer or an antisense primer and arbitrarily selected from the partial base sequence of clone SC50. The primer 12 having the base sequence shown in Table 17 below and the partial base sequence of clone SC216 were arbitrarily selected. Experimental Examples 3-2 and 3-except that the selected primer 13 having the nucleotide sequence shown in Table 18 below was used
Gene amplification was carried out in the same manner as 3. After that, according to the usual method,
The PCR product containing the DNA fragment was digested with restriction enzymes EcoRI and N
cleaved with coI, containing the start codon ATG, EcoRI
A DNA fragment having a cleavage site and an NcoI cleavage site added was obtained.

[0080]

[Table 17]

[0081]

[Table 18]

Separately, 1 μl of total RNA obtained in Experimental Example 3-1 was used.
g, Experimental Example 3 except that Primer 14 and Primer 15 having the base sequences shown in Table 19 or Table 20 below arbitrarily selected from the partial base sequences of clone SC216 and clone SC362, respectively, were used as sense primers or antisense primers. Gene amplification was performed in the same manner as in -2 and 3-3. Then, the PCR product containing the DNA fragment is cleaved with the restriction enzymes PstI and NcoI by a conventional method to contain the termination codon TGA and the PstI cleavage site and NcoI.
A DNA fragment having a coI cleavage site added was obtained.

[0083]

[Table 19]

[0084]

[Table 20]

10 μg of each of the two kinds of DNA fragments thus obtained was added to a suitable amount of sterile distilled water in advance with the restriction enzyme Eco.
10 ng of the plasmid vector pKK223-3 manufactured by Pharmacia, which had been treated with RI and PstI, and an appropriate amount of T.
4 DNA ligase and 100 mM ATP were added to a final concentration of 1 mM, and the mixture was incubated at 16 ° C. for 18 hours. The obtained recombinant DNA was transformed into Escherichia coli JM109 strain (ATCC 5332 by the competent cell method).
Introduced into 3) and transformed. This transformant was inoculated into an L-broth medium (pH 7.2) containing 100 μg / ml of ampicillin by a conventional method, cultured at 37 ° C. for 18 hours, and then a recombinant DNA was extracted by a usual alkaline method.
This recombinant DNA was named "pKCJ2-5", and its structure was analyzed by the dideoxy method. As shown in FIG. 1, in this pKCJ2-5, the cDNA region KCJ cDNA having the nucleotide sequence of Chemical formula 3 was obtained. Is Tac
It was linked downstream of the promoter PTac.

[0086]

Example A-1 (b) Production of Polypeptide Recombinant D
Example A-1 (a) in which NA pKCJ2-5 was introduced.
Of the transformant containing 100 μg / ml of ampicillin
After inoculation into broth medium (pH 7.2) and culturing at 37 ° C. for 18 hours, 0.1 ml of the culture was taken, transferred to the same fresh culture medium, and further cultured at 37 ° C. Then, the absorbance of the culture medium at a wavelength of 600 nm is 0.3.
IPTG was added to a final concentration of 1 mM when the temperature reached, and the cells were further cultured for 4 hours. Then, the bacterial cells collected from the culture by centrifugation were suspended in cold distilled water, and the bacterial cells were ultrasonically disrupted and then centrifuged to collect the supernatant.

After the supernatant was concentrated, it was loaded on a mAb N26 sepharose column binding a monoclonal anti-Cry j II antibody which had been equilibrated with 10 mM phosphate buffer (pH 7.0) containing 0.14 M sodium chloride in advance, Then, 0.1 M glycine-hydrochloric acid buffer solution (pH 2.
3) The solution was passed through to elute the peptide component. A fraction containing the polypeptide was collected from the eluted fraction, neutralized by adding 1 M phosphate buffer (pH 7.0), and dialyzed against 10 mM phosphate buffer (pH 7.0) for 10 hours. Then, the dialyzed solution was collected, concentrated and lyophilized by a conventional method to obtain a purified polypeptide solid product. The yield was about 3 mg per liter of culture medium.

[0088]

Example A-1 (d) Physicochemical Properties of Polypeptide
When the purified polypeptide obtained by the method of Example A-1 (c) was tested by the method of Experimental Example 2, it had the following physicochemical properties. (1) Molecular weight When tested by SDS-polyacrylamide gel electrophoresis in the same manner as in Experimental Example 2-1, the purified polypeptide showed a major band at a position corresponding to a molecular weight of 55,000 ± 5,000 daltons. . (2) Isoelectric point When tested by an isoelectric focusing method in the same manner as in Experimental Example 2-2, the purified polypeptide had an isoelectric point of 9.0 ± 0.5. (3) N-terminal amino acid sequence When analyzed by an ordinary method using an amino acid sequencer "470A type" manufactured by Applied Biosystems, the purified polypeptide was found to have Ala-M at the N-terminal.
et-Lys-Phe-Ile-Ala-Pro-Me
t-Ala-Phe-Val-Ala-Met-Gln
It had an amino acid sequence represented by -Leu-Ile-Ile-Met-Ala-. (4) Ultraviolet absorption spectrum When an ultraviolet absorption spectrum in an aqueous solution was measured using a spectrophotometer, the purified polypeptide showed a wavelength of 280.
The absorption maximum was shown in the vicinity of nm. (5) Solubility in solvent When tested by a conventional method, purified polypeptide was found to be water,
It was soluble in saline and phosphate buffer. (6) Biological action When tested by the method of Experimental Example 2-6 (a), the absorbance in the system using the serum of a healthy person was about 0.1, while the serum of a pollinator patient was used. In this system, the absorbance reached about 2.0, indicating that the polypeptide was significantly bound to the immunoglobulin E antibody contained in the blood of patients with pollinosis. And this is
This proves that the polypeptide is one of the causative agents of hay fever, that is, has the property of inducing hay fever. On the other hand, when tested by the method of Experimental Example 2-7 (b), uptake of about 200 cpm of ³ H-thymidine was observed in the control system to which physiological saline containing no polypeptide was administered. In the system to which the polypeptide was added, a remarkable uptake of about 8,500 cpm was observed per 50 μg / mg of the polypeptide.
This indicates that the polypeptide has antigenicity.

Next, the use of the polypeptide according to the present invention will be specifically described with reference to Examples and Experimental Examples.

Since the polypeptide of the present invention is one of the causative agents of pollinosis, it has a wide range of uses as a desensitizing agent for treating, preventing and diagnosing pollinosis. The desensitizing agent of the present invention contains, as an active ingredient, the polypeptide or a complex of the polypeptide and a specific sugar described below. A desensitizing agent for the purpose of diagnosing hay fever usually contains a partially purified or purified polypeptide obtained by the method as described above as it is, while it is used for treatment / prevention of hay fever. Is
Prior to formulation, a specific sugar is covalently bound to the polypeptide to form a complex.

The saccharide as referred to in the present invention is one that can be covalently bound to the polypeptide and enhances or reduces the desensitizing effect and / or side effect of the polypeptide by forming a complex. Say. Examples of such sugars include simple or complex starch, amylose, dextran, polysucrose, pullulan, elucinan, curdlan, gum arabic, gum tragacanth, guar gum, xanthan gum, carrageenan, cellulose, glucomannan, chitosan, lipopolysaccharide and the like. Polysaccharides and derivatives and partial hydrolysates of these sugars are included, and the average molecular weight thereof is usually about 500 to 10,000,000.
Daltons, preferably about 10,000 to 100
It is in the range of 10,000 Daltons. Among the above-mentioned saccharides, a complex with pullulan, erucinan, or a partial hydrolyzate thereof, which essentially has maltotriose as a repeating unit, is effective for desensitization when administered to mammals including humans. While the immunoglobulin G antibody and the immunoglobulin M antibody are produced in a considerable amount, the immunoglobulin E antibody, which is the main cause of unwanted side effects including anaphylactic shock, is difficult to produce. This is extremely convenient for safely and effectively carrying out the desensitization therapy which requires repeated administration.

Further, for example, lipopolysaccharides derived from microorganisms such as Escherichia coli, Salmonella, Serratia, etc. and partial hydrolysates thereof can be made into a complex with the polypeptide to give mucosa of the mammal. It has the property of increasing the affinity for and significantly improving the intake efficiency. From this, the complex with these sugars is particularly useful in a desensitizing agent which is premised on transdermal or transmucous administration.

Such a complex is usually obtained by reacting the polypeptide with an activated sugar, or by reacting the polypeptide with 2 in one molecule.
The above-mentioned reagent having an active functional group may be used to crosslink the polypeptide and the carbohydrate. Examples of the individual reaction methods include a diazo method, a peptide method, an alkylation method, a crosslinking method, an amide bond method, a periodate oxidation method, a disulfide bond method, and the like, but these reaction methods themselves are known in the art. For example, Japanese Patent Application Laid-Open No. 3-93730 discloses a typical method in detail. The weight ratio of the polypeptide to the carbohydrate at the start of the reaction is usually about 1: 0.001 to 1: 1,000, preferably about 1: 0.01 to 1: 100. Be done. Depending on the reaction method, if it is below this range, the binding of the peptides will be remarkable, and if it is above this range, the binding of the carbohydrates will be remarkable. In any case, it brings about a decrease in the efficiency of the reaction and the purification after the reaction, and the above range was made the best. The temperature, pH and reaction time during the reaction should be set so that the polypeptide is not easily deactivated or decomposed, and undesired side reactions are minimized. Usually, the temperature is about 0 to 100. It is preferable to complete the reaction in about 0.1 to 50 hours at a temperature of about 0.1 to 12 at pH.

The complex produced by the reaction can be used in, for example, dialysis, salting out, filtration, concentration, centrifugation, gel filtration chromatography, ion exchange chromatography, affinity chromatography, electrophoresis, isoelectric focusing, and the like. Can be purified by a general method generally used in, and if necessary, these methods may be appropriately combined. And
Depending on the final use form, the purified complex may be concentrated or lyophilized to form a liquid or a solid.

The desensitizing agent of the present invention is not only in the form of the polypeptide and / or complex obtained as described above, but also in other physiologically acceptable forms such as carriers and excipients. , Diluents, immunostimulants, stabilizers, and even
If necessary, the form as a composition with one or more other drugs containing an anti-inflammatory agent such as a steroid hormone or sodium glymoglycate or an antihistamine is included. Further, the desensitizing agent of the present invention also includes a drug in a dosage unit form, and the drug in a dosage unit form means the polypeptide and / or the complex of the present invention, for example, a daily dose or an integer thereof. Mean drug in physically separate, unitary dosage forms suitable for administration, containing an amount equivalent to double (up to 4 times) or submultiples thereof (up to 1/40).
Drugs in such dosage forms include powders, fine granules, granules, pills, tablets, capsules, troches, syrups, emulsions, ointments, plasters, poultices, suppositories, eye drops,
Examples include nasal drops, sprays and injections.

The method of using the desensitizing agent according to the present invention will be described. The desensitizing agent of the present invention can be used in the same manner as a general desensitizing agent containing a cedar pollen allergen. That is, in order to diagnose hay fever with the desensitizing agent of the present invention, a usual test method known as a scratch test or an intradermal test is adopted, and first, a scratch on the skin surface of the subject is attached to a degree that does not bleed. An appropriate amount of the diagnostic desensitizing agent according to the present invention is dropped, or an appropriate amount of the diagnostic desensitizing agent according to the present invention is intradermally administered. After 15 to 30 minutes have passed, the presence and size of wheal is examined, and when the size of wheal is a certain value or more, it is determined to be positive.

In the treatment with the desensitizing agent of the present invention, the appropriate dose and dosage are usually determined based on the above-mentioned diagnostic results. A subject with a positive diagnostic result is orally or parenterally administered with the desensitizing agent of the present invention containing a complex of the polypeptide and a specific carbohydrate. Symptoms,
Although it varies depending on the usage and the like, specifically, it is usually about 0.0001 to 100,000 ng per adult, preferably about 0.001 to 10 while observing the patient's symptoms and the course after administration. As a guideline, 10,000 ng, once a week to once a month, for about a month to a year, usually
Repeated doses may be given intradermally, subcutaneously, intramuscularly, intraperitoneally or transmucousally while increasing the dose. For the prevention of hay fever, the same dose and usage may be used, and usually about 0.0001 to 100,000 ng per adult, preferably about 0.001 while observing the health condition of the subject and the course after administration. As a guideline, once a week or once a month for about 1 to 6 months, the dose is usually increased and administered repeatedly intradermally, subcutaneously, intramuscularly or transmucousally. When the desensitizing agent according to the present invention is regularly administered repeatedly from autumn to early spring of the following year, allergic symptoms in the onset season of the following year can be minimized or eliminated.

Next, regarding the desensitizing agent according to the present invention, 2
Specific examples will be described with reference to Examples 3 to 3.

[0099]

[Example B-1 Dry injection] Average molecular weight of about 200,0
2 g of 00 dalton purified pullulan was dissolved in 100 ml of distilled water, and 2 ml of a 1.7% (w / v) acetone solution of cyanuric chloride was added. Then, while maintaining the pH of the solution at around 7 with a 5% (w / v) sodium carbonate aqueous solution, the mixture was allowed to stand for 2 hours at 4 ° C. or lower in an ice bath for reaction. Example A-1 was added to the solution containing the activated pullulan thus obtained.
200 mg of purified polypeptide obtained by the method of (b)
In addition, the mixture was reacted with stirring at pH 7.0 and 37 ° C. for 5 hours. Then, 1% (w / v) of glycine was added to the reaction product,
After stirring at 37 ° C for 1 hour with stirring to block unreacted active groups, 0.01M acetate buffer (pH
5.0) for 5 hours and then 0.01
CM equilibrated with M acetate buffer (pH 5.0)
-By Sephadex column chromatography, a complex of the polypeptide and pullulan was collected from the non-adsorbed portion. Then, the complex was dissolved in a physiological saline containing 1% (w / v) human serum albumin as a stabilizer to a final polypeptide concentration of about 100 ng / ml by a conventional method, sterilized by filtration, and then sterilized. 2 ml was dispensed into vials, freeze-dried and sealed.

Prior to administration, this product is used by first adding 1 ml of distilled water for injection or the like into a vial and then uniformly dissolving the contents. The product, which has excellent stability and contains a complex of the polypeptide and pullulan as an active ingredient, is useful as a dry injection for treating / preventing hay fever.

[0101]

Example B-2 Injection: 1 g of CM-cellulose having an average molecular weight of about 20,000 daltons was dissolved in 200 ml of distilled water, and 2 g of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide-methiozide was added to the solution. After
While maintaining the pH of the solution near 4 with 1N hydrochloric acid, the mixture was reacted at room temperature for 2 hours with stirring. The reaction product was dialyzed against distilled water for 24 hours, and the dialyzed solution was collected to give Example A-1.
30 mg of the purified polypeptide obtained by the method of (b) was added, and the mixture was allowed to react at room temperature for 15 hours at pH 4.5 for 15 hours. Thereafter, the complex in the reaction product was purified in the same manner as in Example B-1, concentrated, dissolved in 50% (v / v) glycerin aqueous solution, sterile filtered, and dispensed in 2 ml aliquots into a sterile vial. , Sealed.

Prior to administration, this product was prepared by adding 100 to 100,000-fold volume of 50% (v / v) glycerin aqueous solution to a vial, referring to the diagnostic results of a scratch test or an intradermal test. Then, the contents are diluted evenly before use. The polypeptide and CM as active ingredients
-The product containing a complex with cellulose is useful as a hyposensitizing injection for treating / preventing hay fever.

[0103]

Example B-3 Liquid Solution 100 mg of purified lipopolysaccharide derived from Salmonella was dissolved in 25 ml of 50% saturated aqueous sodium acetate solution at about 4 ° C., and the pH of the solution was adjusted to 9.0 with 0.5N sodium hydroxide. While maintaining the pH at about 8.5, 1 ml of anhydrous dioxane containing 20 μl of bromoacetyl bromide was added dropwise. Then, the pH of the reaction product was adjusted to about 4.5 with 6N hydrochloric acid, and dialyzed against distilled water at 4 ° C. for 48 hours to obtain an aqueous solution containing activated lipopolysaccharide. After adding 40 mg of the purified polypeptide obtained by the method of Example A-1 (b) to this aqueous solution, the pH of the solution
Was maintained at room temperature for about 4.5 hours and allowed to react at room temperature for 48 hours. Then, the reaction product was purified in the same manner as in Example B-1, concentrated, and lyophilized to obtain a solid complex of the polypeptide and lipopolysaccharide. Then, this solid was added as a stabilizer at 1% so that the final concentration was 100 ng / ml.
(W / v) Dissolved in distilled water containing purified gelatin, and sterilized by a conventional method to obtain a liquid preparation.

The product containing a complex of the polypeptide and lipopolysaccharide as an active ingredient is useful as an eye drop, a nasal drop and a liquid for oral spray for treating / preventing hay fever.

[0105]

Example B-4 Sublingual agent Average molecular weight about 200,000
Dalton's purified erucinan was uniformly dissolved in 400 ml of distilled water, and the pH of the solution was adjusted with 1N sodium hydroxide aqueous solution.
Was adjusted to 10.7, 3 g of cyanogen bromide was gradually added while the pH of the aqueous solution was maintained at about 10.0, and the reaction was carried out for 1 hour. After adjusting the pH of the reaction product to 5.0 with 1N hydrochloric acid,
While maintaining this pH, dialysis was performed against cold water for 10 hours to obtain an aqueous solution containing activated erucinan. Next, 20 mg of the purified polypeptide obtained by the method of Example A-1 (b) was added to this aqueous solution, and the mixture was allowed to stand at room temperature for 24 hours for reaction. After completion of the reaction, 3 volumes of acetone was added to the reaction product, the formed precipitate was collected, dissolved in 0.01M acetate buffer (pH 5.0), and the insoluble material was removed by centrifugation, and then 0 0.01 M acetate buffer (pH 5.
The fraction containing the complex of the polypeptide and erucinan was collected from the non-adsorbed fraction by subjecting it to CM-Sephadex column chromatography that had been equilibrated in 0).
Then, by a conventional method, this fraction is sterilized by filtration, concentrated,
After freeze-drying and pulverization, anhydrous crystalline α-maltose powder "Finetose" manufactured by Hayashibara was uniformly mixed, and the mixture was tabletted by a conventional method to obtain a tablet containing 100 ng of polypeptide per tablet (200 mg) of the product. It was

The product which is excellent in ingestibility and stability is useful as a sublingual agent for treating / preventing hay fever.

[0107]

Example B-5 Diagnostic Agent 100 mg of the purified polypeptide obtained by the method of Example A-1 (b) was added to physiological saline 20.
After dissolving in ml and sterilizing filtration by a conventional method, 1 ml each was dispensed into a sterilized vial bottle, lyophilized, and sealed to obtain a diagnostic agent.

This product is dissolved in 1 ml of distilled water for injection and then further diluted 10-fold with the same distilled water to be used for diagnosis of pollinosis by a scratch test or an intradermal test.

[0109]

Example B-6 Diagnostic Agent 1 mg of the purified polypeptide obtained by the method of Example A-1 (b) was added to 50% (v / v) glycerin aqueous solution containing 5% (w / v) sodium chloride 2
After dissolving in 0 ml and performing sterile filtration by a conventional method, 1 ml was dispensed into a sterilized vial bottle.

This product is diluted 20 times with a 50% (v / v) glycerin aqueous solution and used for diagnosis of hay fever by a scratch test or an intradermal test.

Next, the effects and the like of the desensitizing agent according to the present invention will be described based on a few experimental examples.

[0112]

Experimental Example 3 Animal Experiment In this experimental example, the complex of the polypeptide of the present invention and a specific carbohydrate is treated / prevented for pollinosis by actually administering the desensitizing agent of the present invention to an experimental animal. It is to prove that it is effective.

[0113]

[Experimental Example 3-1 Preventive effect] Female BA aged 10 to 12 weeks
In one group consisting of 6 LB / c mice, the desensitizing agent obtained by the method of Example B-1 was converted into the amount of polypeptide to be 1
It was intraperitoneally injected once a week at a rate of μg / animal for 3 weeks. One week after the final administration of the desensitizing agent, 1 μg of the purified polypeptide obtained by the method of Example A-1 (b) as an antigen and 4 mg of aluminum hydroxide as an immunostimulant were included in order to induce pollinosis. Physiological saline 0.2m
1 was injected as above. Then, immediately before the antigen administration and one week after the antigen administration, the blood of the mouse was collected, and the amount of immunoglobulin E antibody, immunoglobulin G antibody and immunoglobulin M antibody specific to the polypeptide contained therein was examined. .

Separately, in place of the desensitizing agent, Example A-1
A system for administering a mixture containing the purified polypeptide obtained by the method of (b) and the purified pullulan having an average molecular weight of about 200,000 daltons in a weight ratio of 1:15 was prepared, and treated in the same manner as above. Was used as a control. The amount of the immunoglobulin E antibody specific to the polypeptide is determined by "Life Sciences", I Motor et al., Vol.
No. 6, Part II, pp. 813-820 (1969)
The amount of immunoglobulin G antibody and immunoglobulin M antibody specific to the polypeptide by the PCA reaction reported in S. Yoshitake et al., "The Journal of Biochemistry", Vol. 92, No. 5,
The measurement was performed by the enzyme immunosorbent assay (EIA) reported on pages 1,413 to 1,424 (1982), and each was represented by the average antibody titer of 6 mice. The results are shown in Table 21.
Shown in.

[0115]

[Table 21]

As is clear from the results shown in Table 21, in comparison with the control, the system in which the desensitizing agent of the present invention containing the complex of the polypeptide and pullulan of the present invention was administered in advance, was desensitized. While the immunoglobulin G antibody and the immunoglobulin M antibody which are effective for E. coli were produced in a considerable amount, the production of the immunoglobulin E antibody was suppressed to a substantially nonexistent level. Immunoglobulin E antibody is said to be the main cause of unwanted side effects such as anaphylactic shock, and it is a complex fact that administration of the desensitizing agent of the present invention to mice significantly suppressed its production. It means that the desensitizing agent of the present invention containing the body can be used safely and effectively for preventing pollinosis in mammals including humans.

[0117]

[Experimental example 3-2 Therapeutic effect of parenteral administration] A group of 6 female BALB / c mice aged 10 to 12 weeks was prepared.
As an antigen, 1 μg of the purified polypeptide obtained by the method of Example A-1 (b) was added once a week to 0.2 ml of physiological saline containing 4 mg of aluminum hydroxide as an immunostimulant.
It was injected intraperitoneally for 3 weeks to induce hay fever. One week after the final administration of the antigen, the desensitizing agent obtained by the method of Example B-1 was converted into a polypeptide amount of 10
Injection was performed once a week at a dose of 0 ng / animal for 3 weeks in the same manner as above. One week after the final administration of the desensitizing agent, the antigen alone was again administered in the same manner as above to perform the treatment for inducing the production of immunoglobulin E antibody. Immediately before the administration of the desensitizing agent, one week after the final administration of the desensitizing agent, and one week after the re-induction of immunoglobulin E antibody, blood was collected from the mouse and specific for the polypeptide contained in the sonico. The amounts of immunoglobulin E antibody, immunoglobulin G antibody and immunoglobulin M antibody were examined by the same method as in Example 3-1.

Separately, instead of the desensitizing agent, Example A-1
A system for administering a mixture containing the purified polypeptide obtained by the method of (b) and the purified pullulan having an average molecular weight of about 200,000 daltons in a weight ratio of 1:15 was prepared, and treated in the same manner as above. Was used as a control. The results are shown in Table 22.

[0119]

[Table 22]

As is clear from the results shown in Table 22, as compared with the control, in the system to which the desensitizing agent of the present invention containing the complex of the polypeptide and pullulan was administered in advance, the desensitizing agent was Even after administration of
Significant amount of immunoglobulin G even after antibody re-induction
An antibody and an immunoglobulin M antibody were recognized. As for the immunoglobulin E antibody, production was suppressed to virtually no level after administration of the desensitizing agent and even after reinduction of the immunoglobulin E antibody. These results mean that parenteral administration of the desensitizing agent of the present invention containing the complex can safely and effectively treat hay fever in mammals including humans.

[0121]

[Experimental example 3-3 Therapeutic effect by oral administration] 10 to 1
A physiological saline containing 1 μg of the purified polypeptide obtained by the method of Example A-1 (b) as an antigen and 4 mg of aluminum hydroxide as an immunostimulant in a group of 6 2-week-old female BALB / c mice. 0.2 ml of water once a week for 3
It was injected intraperitoneally for a week to induce hay fever.
One week after the final administration of the antigen, the sublingual preparation obtained by the method of Example B-4 was pulverized and orally administered at a dose of 10 μg / animal once a week for 3 weeks in terms of the amount of polypeptide. Was administered. One week after the final administration of the sublingual agent, blood was collected from the mice, and the amounts of immunoglobulin A antibody, immunoglobulin G antibody and immunoglobulin E antibody specific to the polypeptide contained therein were examined.

Separately, instead of the sublingual agent, Example A-1
A system was provided for oral administration of a solid mixture containing the purified polypeptide obtained by the method of (b) and the purified lipopolysaccharide derived from Salmonella in a weight ratio of 1:15, and the system was treated in the same manner as above. Was used as a control. The amount of immunoglobulin A antibody and immunoglobulin G antibody specific to the polypeptide can be determined by the method described by Earl Meolini et al.
Immunological Methods, Volume 6, 355-36
According to the EIA method reported on page 2 (1975),
In addition, immunoglobulin E specific for the polypeptide
The amount of antibody was measured by the same method as in Experimental Example 3-1,
Each is represented by the average antibody titer of 6 mice. The results are shown in Table 23.

[0123]

[Table 23]

As is clear from the results shown in Table 23, when compared with the control, in the system to which the desensitizing agent of the present invention containing the complex of the polypeptide and lipopolysaccharide was previously administered, the immunoglobulin was While the A antibody and the immunoglobunoline M antibody were produced in a considerable amount, the production of the immunoglobulin E antibody was suppressed to a substantially zero level. This indicates that the desensitizing agent of the present invention containing the complex can safely and effectively treat pollinosis in mammals including human even by oral administration.

Although data are not shown, the methods of Examples B-1 to B-
4. The desensitizing agent obtained by the method of 4 is administered by intradermal, subcutaneous, intramuscular or intraperitoneal injection in mice, rats and guinea pigs, or in the form of eye drops, nasal drops or oral sprays. Upon transmucosal administration, all of the above desensitizing agents exhibited significant effects in the treatment and prevention of hay fever without causing serious side effects. This becomes particularly remarkable in a complex of the polypeptide with pullulan, erucinan, etc., and a carbohydrate essentially having maltotriose as a repeating unit, and in this case, as compared with other complexes, It was found that the dose and administration period required to achieve the desired desensitization can be further reduced or shortened.

[0126]

[Experimental example 3-4 Acute toxicity test] 20
The therapeutic / preventive desensitizing agents obtained by the methods of Examples B-1 to B-4 were orally or intraperitoneally administered to the mice on the day. As a result, it was found that these desensitizing agents have LD50 of 1,000,000 ng or more by any administration route. This indicates that the desensitizing agent of the present invention containing a complex of the polypeptide and a specific sugar can be safely compounded and used in a drug to be administered to mammals including humans.

[0127]

As described above, the polypeptide according to the present invention is a novel causative agent of pollinosis. Since the polypeptide of the present invention has the property of inducing pollinosis in mammals including humans, it can be used not only for desensitizing agents for diagnosing or treating / preventing pollinosis but also for enzyme immunoassay and radioimmunoassay. It has a wide range of uses in the diagnosis of hay fever and also in academic research to elucidate the pathogenic mechanism of allergic diseases in general. In particular, the complex of the polypeptide of the present invention and a specific carbohydrate produces a significant amount of immunoglobulin antibody effective for desensitization when administered to mammals including human, while causing unwanted side effects including anaphylactic shock. It has a property that it does not substantially produce immunoglobulin E antibody, which is the main cause. Due to this property, when treating or preventing pollinosis using the desensitizing agent according to the present invention,
The dose and administration period of the antigen required for treatment and prevention can be significantly reduced or shortened. The polypeptide, which is also useful as described above, can be produced in a desired amount relatively easily by the method of the present invention applying recombinant DNA technology.

The present invention exerts such remarkable actions and effects, and it can be said that the invention has a great significance to contribute to the field.

[Brief description of drawings]

FIG. 1 Recombinant DNA pKCJ2 used in the present invention
It is a figure which shows the structure of -5.

[Explanation of symbols]

KCJ cDNA DNA encoding the polypeptide of this invention PTac Tac promoter rrnBT1T2 Transcription termination region of liposome RNA operon AmpR Ampicillin resistance gene ori Origin of replication in E. coli

─────────────────────────────────────────────────── ───

[Procedure amendment]

[Submission date] April 12, 1994

[Procedure Amendment 1]

[Document name to be amended] Statement

[Correction target item name] Full text

[Correction method] Change

[Correction content]

[Document name] Statement

Title: Polypeptide, production method and use thereof

[Claims]

[Chemical 1]

[Chemical 2]

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel polypeptide causing pollinosis, a method for producing the same, and use as a desensitizing agent.

[0002]

2. Description of the Related Art In recent decades, the number of people complaining of rhinitis and conjunctivitis due to pollinosis has been increasing in Japan in early spring. Due to the large number of patients and the onset of various events in the early spring, it has been frequently taken up in the media and is now one of the issues that cannot be ignored in public health.

It is said that hay fever is a kind of allergic disease, and its main cause is an antigenic substance in cedar pollen, that is, cedar pollen allergen. When cedar pollen scattered in the air enters the human body, an immunoglobulin E antibody against the cedar pollen allergen is produced. When cedar pollen invades next in this state, the allergen in the pollen immunoreacts with the immunoglobulin E antibody, and allergic symptoms are exhibited.

At present, it is known that cedar pollen has at least two kinds of allergens having different antigenicities. One of them is Yasueda et al.
Allergic and Clinical Immunology ", Vol. 71, No. 1, pp. 77-86 (1983), and today, this is" Cryj.
It is called "I". The other is Tanyei et al., "F.B.S.Letters", Volume 239, Volume 2.
No. 329-332 (1988) and Sakaguchi et al., "Allergy", No. 45, 309-312 (19).
90) and is today referred to as "Cry j II". Cry j I and Cry j II are usually present in cedar pollen at a ratio of about 50: 1 to 5: 1, and most of the sera collected from pollinosis patients have Cry j I and Cry j I.
It is said to react to I as well. Sawaya et al., "Allergy", Vol. 42, No. 6, 738-747 (199).
3 years), Cry jII was used for intradermal tests and RAS
It is reported that the T-test exhibits the same antigenicity as Cry j I.

As described above, several cedar pollen allergens have already been isolated and their properties and properties have been elucidated to some extent. Therefore, administration of purified cedar pollen allergen to humans to desensitize them causes pollinosis. There is a prospect of treatment and prevention. Recently, some desensitizing agents have been devised for that purpose. For example, in JP-A-1-156926 and JP-A-3-93730, Asp-A is added to the N-terminal.
sn-Pro-Ile-Asp-Ser- or Ala-
It has been proposed to covalently bind pullulan, which is one of the polysaccharides, to an allergen having an amino acid sequence represented by Ile-Asn-Ile-Phe-Asn-, and administer the resulting complex to humans as a desensitizing agent. ing. However, a large amount of high-purity allergen is usually required for the diagnosis of allergic disease and desensitization therapy, and the allergen in cedar pollen is scarce and its stability is low, and it is a diagnostic agent for pollinosis. It is expected that there will be a great deal of difficulty in covering the desensitizing agent with cedar pollen alone.

On the other hand, recent progress in recombinant DNA technology is remarkable. Nowadays, even for a polypeptide whose entire amino acid sequence has not been elucidated, a gene encoding the same can be isolated, and if its nucleotide sequence can be elucidated, a recombinant DNA containing the DNA encoding the polypeptide can be prepared, By culturing the transformant obtained by introducing this into cells of microorganisms and animals and plants, it has become possible to obtain a desired amount of polypeptide relatively easily.

From the above circumstances, it has been awaited that all the genes encoding allergens in Japanese cedar pollen are isolated and their nucleotide sequences are elucidated as soon as possible. Recently published International Patent Application Publication No. 93/01213 discloses a nucleotide sequence encoding Cry j I, which is one of such allergens. As described above, the allergen causing pollinosis is Cry. not only j I,
For accurate diagnosis and effective hyposensitization therapy, it is an urgent need in the art to isolate the genes encoding the other allergens and to elucidate their nucleotide sequences.

[0008]

In view of such a situation, an object of the present invention is to provide a novel polypeptide which causes pollinosis.

Another object of the present invention is to provide a method for producing the polypeptide by applying recombinant DNA technology.

Still another object of the present invention is to provide the use of the polypeptide as a desensitizing agent.

[0011]

The present invention solves the first problem by a polypeptide having the following physicochemical properties. (1) Molecular weight 55,000 ± 5,000 daltons (SDS-polyacrylamide gel electrophoresis) (2) Isoelectric point 9.0 ± 0.5 (isoelectric focusing) (3) N-terminal amino acid sequence N-terminal Ala-Met-Lys-Phe-Ile-A
la-Pro-Met-Ala-Phe-Val-Al
a-Met-Gln-Leu-Ile-Ile-Met
It has an amino acid sequence represented by -Ala-. (4) Ultraviolet absorption spectrum It has an absorption maximum near 280 nm. (5) Solubility in solvent Soluble in water, physiological saline and phosphate buffer. (6) Biological action It binds to immunoglobulin E antibody collected from the blood of patients with hay fever. Causes hay fever. (7) Stability Deactivates when heated at 100 ° C. for 10 minutes in an aqueous solution (pH 7.2). Even if left in an aqueous solution (pH 7.2) at 4 ° C. for 1 month, it is not substantially deactivated.

The present invention solves the second problem by a method comprising culturing a transformant containing a DNA encoding the polypeptide in a culture medium and collecting the polypeptide produced from the culture. Is.

The present invention solves the third problem by a desensitizing agent containing the polypeptide as an active ingredient.

[0014]

The polypeptide of the present invention is a novel substance having unique physicochemical properties, which is not found in conventionally known cedar pollen allergens.

The production method of the present invention makes it possible to produce a desired amount of such a polypeptide by recombinant DNA technology.

The desensitizing agent of the present invention exerts a desensitizing effect when administered to mammals including humans.

The present invention will be described below based on experimental examples, examples, etc. The present invention relates to a novel polypeptide causing pollinosis. The present inventor has been studying allergens in cedar pollen, and has found that the novel allergens that have been unknown so far are included. When this allergen was isolated by combining various purification methods centered on column chromatography and its properties and properties were investigated, it was found that its essence is a polypeptide and has the following physicochemical properties. . (1) Molecular weight 40,000 ± 5,000 daltons (SDS-polyacrylamide gel electrophoresis) (2) Isoelectric point 9.5 ± 0.5 (isoelectric focusing) (3) Partial amino acid sequence His-Asp -Ala-Ile-Asn-Ile-P
he-Asn-Val-Glu-Lys, His-AS
p-Cys-Thr-Glu-Ala-Phe-Ser
-Thr-Ala-Trp-Gln-Ala-Ala-
Cys-Lys, Asn-Thr-Ile-Gly-T
hr-Gly-Asp-Asp-Cys-Val-Al
a-Ile-Gly-Thr-Gly-Ser-Ser
-Asn-Ile-Val-Ile-Glu-ASp-
Leu-Ile-Cys-Gly-Pro-Gly, or Ile-Ala-Ser-Cys-Leu-Asn-.
Asp-Asn-Ala-Asn-Gly-Tyr-P
he-Ser-Gly-His-Val-Ile-Pr
It has a partial amino acid sequence represented by o-Ala. (4) Ultraviolet absorption spectrum It has an absorption maximum near 280 nm. (5) Solubility in solvent Soluble in water, physiological saline and phosphate buffer. (6) Biological action Causes hay fever. It binds to immunoglobulin E antibody collected from the blood of patients with hay fever. (7) Stability Deactivates when heated at 100 ° C. for 10 minutes in an aqueous solution (pH 7.2). Even if left in an aqueous solution (pH 7.2) at 4 ° C. for 1 month, it is not substantially deactivated.

The polypeptide having such physicochemical properties has not been known yet, and it is judged to be a novel substance.

Then, the present inventor succeeded in isolating mRNA from cedar pollen as a result of intensive screening of the gene encoding this polypeptide. This mR
D obtained by reverse transcriptase reaction with NA as a template to prepare cDNA, and gene amplification of the cDNA by PCR method
Analysis of the NA fragment revealed that the gene of interest contained the base sequence from the 5'end shown in Chemical formula 3 below. Then, when the base sequence was decoded, it was found that the polypeptide was composed of a maximum of 514 amino acids as shown in Chemical formula 4. Next, a series of experiments leading to elucidation of physicochemical properties of polypeptides will be described.

[0020]

[Chemical 3]

[0021]

[Chemical 4]

[0022]

[Experimental Example 1 Purification of Polypeptide] About 16 parts by weight of 0.1 parts by weight per 1 part by weight of pollen collected from male flowers of Urasugi from Akita
A 25 M sodium hydrogencarbonate aqueous solution (pH 8.2) was added, and the mixture was extracted with stirring at 4 ° C. for 1 hour and then centrifuged to collect the supernatant. The residue was reprocessed in the same manner, and the obtained supernatant and the supernatant obtained in the first extraction were pooled, and ammonium sulfate was added to this to 80% saturation for salting out. A 50 mM Tris-hydrochloric acid buffer solution (pH 7.
It was dialyzed against 8) for 10 hours, filtered, and then subjected to DEAE-Sephadex column chromatography preliminarily equilibrated with 50 mM Tris-hydrochloric acid buffer (pH 7.8) to collect a non-adsorbed fraction.

Thereafter, acetic acid was added to the non-adsorbed fraction to pH.
After adjusting to 5.0, 10 mM acetate buffer (pH
5.0) was loaded onto CM-Sephadex, which was equilibrated with 10 mM acetate buffer (pH 5.0) to wash the column, and then 0.1 M phosphate buffer (pH 7.
0) and 0.3 M sodium chloride were passed through the column to elute the peptide component. A fraction containing the polypeptide is collected and preliminarily collected in 10 mM phosphate buffer (pH
The column was loaded onto a Mono-S column that had been equilibrated with 5.0), and the column was subjected to 10 mM Tris-HCl buffer (pH) under a concentration gradient of sodium chloride increasing from 0M to 0.5M.
7.0), the sodium chloride concentration was 0.
The polypeptide was eluted at 4M.

The purified polypeptide obtained in this manner remarkably reacted with immunoglobulin E antibody collected from patients with pollinosis and anti-cedar pollen allergen derived from mouse.
The yield is about 0.006 in terms of the solid content of cedar pollen as a raw material.
%Met.

[0025]

Experimental Example 2 Physicochemical Properties of Polypeptide In this experimental example, the physicochemical properties of the purified polypeptide obtained in Experimental Example 1 were examined.

[0026]

[Experimental Example 2-1 Molecular Weight] U.K. Lamuri, "Nature", Vol. 227, pp. 680-685 (1970)
S.) purified polypeptide according to the method reported in
When subjected to DS-polyacrylamide gel electrophoresis, a band was observed at a position corresponding to a molecular weight of 40,000 ± 5,000 daltons. The molecular weight markers used at this time were bovine serum albumin (67,000 daltons), ovalbumin (45,000 daltons), carbonic anhydrolase (30,000 daltons), chymotrypsinogen A (25,000 daltons) and cytochrome C. (12,000 Dalton).

A component having a molecular weight of 40,000 ± 5,000 daltons was transferred from a gel to a nitrocellulose membrane, and a mouse-derived anti-cedar pollen allergen antibody and horseradish-derived peroxidase-labeled goat-derived anti-mouse immunoglobulin antibody were labeled. When it was allowed to act, a remarkable immunostaining was observed. This suggests that this component is a kind of cedar pollen allergen.

[0028]

[Experimental example 2-2 Isoelectric point] The isoelectric point of the purified polypeptide was 9.5 ± 0.
It was 5.

[0029]

[Experimental example 2-3 Partial amino acid sequence] The purified polypeptide was digested with trypsin, and the digested product was subjected to chromatography column "Hi-Pore PR-318" manufactured by Bio-Rad.
Separation by high performance liquid chromatography using "type" gave peptide fragments of four kinds of names. When analyzed using an amino acid sequencer, these peptide fragments were (a) His-Asp-Ala-Ile-Asn.
-Ile-Phe-Asn-Val-Glu-Lys,
(B) His-Asp-Cys-Thr-Glu-A
la-Phe-Ser-Thr-Ala-Trp-Gl
n-Ala-Ala-Cys-Lys, (c) Asn
-Thr-Ile-Gly-Thr-Gly-Asp-
Asp-Cys-Val-Ala-Ile-Gly-T
hr-Gly-Ser-Ser-Asn-Ile-Va
l-Ile-Glu-Asp-Leu-Ile-Cys
-Gly-Pro-Gly, or (d) Ile-Al
a-Ser-Cys-Leu-Asn-Asp-Asn
-Ala-Asn-Gly-Tyr-Phe-Ser-
It was found to have an amino acid sequence represented by Gly-His-Val-Ile-Pro-Ala.

[0030]

[Experimental Example 2-4 Ultraviolet absorption spectrum] When the ultraviolet absorption spectrum in an aqueous solution was measured using a spectrophotometer, the purified polypeptide showed an absorption maximum near a wavelength of 280 nm.

[0031]

Experimental Example 2-5 Solubility in Solvent When tested by a conventional method, the purified polypeptide was soluble in water, physiological saline and phosphate buffer.

[0032]

[Experimental Example 2-6 Biological Action] The purified polypeptide, when tested by the method described below, has the property of binding to an immunoglobulin E antibody collected from the blood of a patient with hay fever and T that specifically reacts with the polypeptide. It shows the property of inducing cell proliferation.

[0033]

[Experimental Example 2-6 (a) Immunoglobulin E antibody binding test] 1 μl / well of purified polypeptide was adsorbed on a 96-well microplate, and 1 μl / well of serum collected from a pollinator patient or a healthy person was adsorbed. After adding each, the mixture was incubated at 37 ° C. for 2 hours. next,
After washing the microplate with distilled water to remove unbound serum, 1 μl of goat-derived anti-human immunoglobulin E antibody labeled with horseradish-derived peroxidase was used.
Each well was added and incubated at 37 ° C. for another 2 hours. The microplate was washed with distilled water to remove unbound antibody, a small amount of hydrogen peroxide solution containing orthophenylenediamine was added to each well, and after color development, the absorbance at 492 nm was measured.

As a result, while the absorbance of the system using the serum of a healthy person was about 0.1, the absorbance of the system using the serum of the hay fever patient reached about 2.0. This indicates that the purified polypeptide significantly binds to the immunoglobulin E antibody contained in the blood of pollinosis patients. This supports the fact that the purified polypeptide is one of the causative agents of hay fever, that is, has the property of inducing hay fever.

[0035]

[Experimental Example 2-6 (b) T cell proliferation induction test] Mononuclear cells were separated from heparinized peripheral blood of pollinosis patients by Ficoll-Hypaque gravity centrifugation. 10% mononuclear cells
(V / v) Suspended in RPMI1640 medium (pH 7.0) supplemented with AB serum at a concentration of 1 × 10 ⁶ cells / ml, and purified polypeptide obtained in Experimental Example 1 was added at 20 μg / ml.
After adding ml, the cells were cultured at 37 ° C for 7 days in a 5% CO ₂ incubator. The cultured mononuclear cells were washed with fresh RPMI1640 medium (pH 7.0) and then resuspended in the same medium at 1 × 10 ⁶ cells / ml. Recombinant human interleukin-2 was added to the culture medium at 5 units / ml, and the mixture was further cultured for 3 days in the same manner as above. The mononuclear cells thus pretreated were subjected to the T cell proliferation test described below.

10% in 96 well microplate
(V / v) 2 × 10 ⁴ cultured mononuclear cells suspended in RPMI1640 medium (pH 7.0) supplemented with AB serum
Cells / well, 2 μCi of ³ H-thymidine, and 100 μg / ml of the purified polypeptide, and added with saline to 200
μl / well. The cells in the wells were cultured in a 10% CO ₂ incubator at 37 ° C. for 3 days, and the amount of ³ H-thymidine incorporated into the cells was measured by a scintillation counter. At the same time, a system using physiological saline containing no polypeptide was provided, and this was treated in the same manner as above to serve as a control.

As a result, about 300 cpm in the control system
Was observed, the system in which the purified polypeptide was added showed a remarkable uptake of about 6,500 cpm per 100 μg / ml of the polypeptide. This indicates that the purified polypeptide has antigenicity.

[0038]

Experimental Example 2-7 Stability When the purified polypeptide was incubated in an aqueous solution (pH 7.2) at 100 ° C. for 10 minutes, no residual activity was observed. On the other hand, when the purified polypeptide was stored in an aqueous solution (pH 7.2) at 4 ° C for 1 month, no substantial decrease in activity was observed.

The polypeptide having the above physicochemical properties has not been known yet, and it is judged to be a novel substance.

Next, the experiments carried out to elucidate the nucleotide sequence encoding this polypeptide and the entire amino acid sequence will be described. In summary, the amino acid sequence of the polypeptide according to the present invention is subjected to the following series of operations. It has been clarified through. (1) A pollen extract collected from a Japanese cedar male flower was purified by a purification method centered on chromatography to isolate a highly pure polypeptide. (2) This polypeptide was digested with trypsin, and four kinds of peptide fragments were isolated from the digested product and the amino acid sequence thereof was determined. (3) An oligonucleotide presumed to encode these partial amino acid sequences was chemically synthesized, and RT-P
Separately extracted from Japanese cedar pollen using CR primer
Gene amplification was performed using RNA as a template. (4) A plurality of DNA fragments encoding the polypeptide, which are chemically synthesized based on the partial amino acid sequence in the same manner and hybridized with an isotope-labeled oligonucleotide as a probe and the DNA fragment obtained by gene amplification. Was selected. (5) The base sequence of the selected DNA fragment is determined, and the amino acid sequence decoded from the base sequence is compared with the partial amino acid sequence to confirm that the estimated base sequence encodes the polypeptide. confirmed.

[0041]

[Experimental Example 3 Determination of the entire amino acid sequence of a polypeptide and the nucleotide sequence encoding the same] In this Experimental Example, the nucleotide sequence encoding the polypeptide and the entire amino acid sequence will be clarified. The method itself used in this experimental example is known in the art, and for example, T. Manatis, et al., "Molecular Cloning a Laboratory Manual," 1
Published by Cold Spring Harbor in 989,
Masami Muramatsu "Lab Manual Genetic Engineering", 1988,
It is also detailed in the publication of Maruzen Publishing Co., Ltd.

[0042]

[Experimental Example 3-1 Preparation of total RNA] 10 g of cedar pollen pre-freeze-treated with liquid nitrogen was added to 10 mM tris-hydrochloric acid buffer (pH 7.4), 200 mM sodium chloride, 6 mM magnesium chloride and 1% (w / v) sarcosyl. And 40 ml of a phenol / chloroform / isoamyl alcohol solution were added, and the mixture was treated with Polytron to crush pollen. Treated product 10,000 × g, 4 ℃
Centrifuge for 10 minutes, collect the separated aqueous layer, add an equal volume of fresh phenol / chloroform / isoaminoalcohol solution, extract in the same manner as above, and centrifuge again to collect the aqueous layer. After adding 2.5 volumes of ethanol to this, it was left at −20 ° C. for 2 hours to precipitate total RNA.

Total RNA was washed with 75% (v / v) ethanol, dried and dissolved in 29 ml of sterile distilled water to give 300
After adding mM phosphate buffer (pH 6.8),
It was loaded onto a hydroxyapatite column that had been equilibrated with M phosphate buffer (pH 6.8). 10 mM column
After washing with phosphate buffer (pH 6.8), 300 mM phosphate buffer (pH 6.8) was passed to elute total RNA, and 2.5 times volume of 75% (v / v) ethanol was added to the eluate. In addition, total RNA was precipitated by standing at -20 ° C for 2 hours.
The precipitated total RNA was washed with 75% (v / v) ethanol, dried, dissolved in 0.5 ml of sterilized distilled water, and used for selection of clones SC09, SC31, SC216, SC50 and SC362 described below. The total RNA recovered was about 500 μg.

[0044]

[Experimental Example 3-2 Preparation of First-Stranded cDNA] 4 μl of 25 mM magnesium chloride was added to 1 μg of total RNA obtained in Experimental Example 3-1, and 10 × PCR buffer (10 mM
Tris-HCl buffer (pH 8.3), 500 mM potassium chloride) 2 μl, 1 mM dNTP mix 8 μm
l, 1 unit / μl RNase inhibitor 1 μl, 2.5
Unit / µl Reverse transcriptase (1 µl), 2.5 µM random hexamer (1 µl) were added, and the volume was adjusted to 20 µl with sterile distilled water.
By a conventional method, the mixture was incubated in a 0.5 ml reaction tube at 25 ° C. for 10 minutes, 42 ° C. for 30 minutes, 99 ° C. for 5 minutes, and 5 ° C. for 5 minutes to carry out reverse transcriptase reaction to give first strand cDNA. A solution containing was obtained.

[0045]

[Experimental Example 3-3 Selection of SC09 clone and determination of partial base sequence] First strand c obtained in Experimental Example 3-2
4 μm of 25 mM magnesium chloride to 20 μl of DNA solution
l, 10x PCR buffer (100 mM Tris-HCl buffer (pH 8.3), 500 mM potassium chloride) 8μ
1, 2.5 units / μl 0.5 μl of amplitac DNA polymerase, 1 pmol of the following primer 1 as a sense primer and 1 pmol of the following primer 2 as an antisense primer were added, respectively, to make 100 μl with sterile distilled water. Then, by a conventional method, the mixture was repeatedly reacted 40 times in a cycle of 94 ° C. for 1 minute, 45 ° C. for 2 minutes, and 72 ° C. for 3 minutes to amplify a DNA fragment partially encoding the polypeptide using cDNA as a template. . In addition, as for the primer 1 and the primer 2, Experimental example 2-
Oligonucleotide chemically synthesized based on the amino acid sequence represented by His-Asp-Ala-Ile-Asn-Ile or Ala-Trp-Gln-Ala-Ala in the partial amino acid sequences (a) and (b) shown in 3 And had the base sequences shown in Table 1 or Table 2 below. As the PCR reagent, "GeneAmp RNA PCR Kit" manufactured by Takara Shuzo Co., Ltd. was mainly used.

[0046]

[Table 1]

[0047]

[Table 2]

A portion of the obtained PCR product was taken, fractionated by electrophoresis on a 2% agarose gel by a conventional method, pressure blotted on a nylon membrane, and then fixed with 0.4N sodium hydroxide. . The nylon membrane was washed with 2 × SSC, air dried, 5 × SSPE, 5 × Denhardt's solution, 0.5% (w
/ V) SDS and 100 μg / ml denatured salmon sperm DNA
Dip into a pre-hybridization mixture containing
Incubated at ℃ for 3 hours. Separately, as the probe 1, Phe-Asn-Val-Glu-L in the partial amino acid sequence (a) shown in Experimental Example 2-3 was prepared by a conventional method.
Oligonucleotides having the nucleotide sequences shown in Table 3 below, which are presumed to encode ys, were chemically synthesized and labeled with [γ- ³² P] ATP by T4 nucleotide kinase. 1 pmol of this probe 1 was taken, and 5x SSPE and 5x
The nylon membrane was immersed in a mixed solution containing Denhardt's solution and 0.5% (w / v) SDS, incubated at 45 ° C. for 24 hours for hybridization, and then the nylon membrane was 6 × SS.
After washing with C and autoradiography, it was confirmed that the PCR product contained the target DNA fragment.

[0049]

[Table 3]

Next, the remaining PCR product was added with 50 ng of a plasmid vector "pT7 Blue T" manufactured by Takara Shuzo Co., Ltd., an appropriate amount of T4 DNA ligase and 100 mM ATP to a final concentration of 1 mM, and then incubated at 16 ° C. for 24 hours. The DNA fragment was inserted into Escherichia coli NoVa Blue strain obtained from Takara Shuzo Co., Ltd. by the competent cell method.
The transformants were bactotryptone 10 g / l, sodium chloride 11 g / l, bacto agar 15 g / l, ampicillin 100 mg / l, X-Gal 40 mg / l and isopropyl-β-D-thiogalactopyranoside (hereinafter,
Abbreviated as "IPTG". ) A plate medium containing 23.8 mg / l was inoculated and incubated at 37 ° C. for 24 hours to form colonies. Place the nylon membrane on the culture medium, leave it for 30 seconds, then peel off the nylon membrane,
It was immersed for 7 minutes in a mixed solution containing 0.5 M sodium hydroxide and 1.5 M sodium chloride. Take out the nylon membrane, 1.
After being immersed in 0.5 M Tris-hydrochloric acid buffer solution (pH 7.2) containing 5 M sodium chloride for 3 minutes and washed with 2 × SSC,
Immerse in 0.4M sodium hydroxide for 20 minutes, 5 × SS
After washing with C and air-drying, it was immersed in a pre-hybridization mixture containing 5 × SSPE, 5 × Denhardt's solution, 0.5% (w / v) SDS, 100 μg / ml denatured salmon sperm DNA, and incubated at 65 ° C. for 3 days. Incubated for hours. Then, probe 1 was hybridized to the nylon membrane and autoradiographed in the same manner as above, and based on the result, clone SC09 containing the initial DNA fragment was selected from the plate medium.

This clone SC09 was treated with ampicillin 10
L-broth medium (pH 7.2) containing 0 μg / ml was inoculated and cultured at 37 ° C. for 18 hours, the bacterial cells were collected from the culture, crushed, and then extracted by a usual alkaline method to give recombinant D
I got NA. When the DNA fragment in this recombinant DNA was decoded by the dideoki method using the DNA sequencing kit "Sequenase version 2.0" manufactured by Toyobo Co., Ltd., it had the nucleotide sequence from the 173rd position to the 240th position in the chemical formula 3. It turned out.

[0052]

[Experimental Example 3-4 Selection of Clone SC31 and Determination of Partial Nucleotide Sequence] In this experimental example, D different from clone SC09 was used.
A clone SC31 containing the NA fragment is selected and its partial base sequence is determined. Unless otherwise specified, each step in this experimental example is exactly the same as in Experimental example 3-3.

By the conventional method, primer 3 having a nucleotide sequence shown in Table 4 below arbitrarily selected from the partial nucleotide sequences of clone SC09 selected in Experimental Example 3-3 and the partial amino acid sequence shown in Experimental Example 2-3 ( Asn-Ile- in c)
A primer 4 having the nucleotide sequence shown in Table 5 below, which is presumed to encode Val-Ile-Glu, was chemically synthesized.
A portion of the first-strand cDNA solution obtained in Experimental Example 3-2 was taken, and this was subjected to gene amplification in the presence of Primer 3 and Primer 4 in the same manner as in Experimental Example 3-3, and clone SC09 was obtained from the obtained DNA fragment. Probe 2 having the nucleotide sequence shown in Table 6 below, which is arbitrarily selected from the partial nucleotide sequence of
Was used to collect a further DNA fragment partially encoding the polypeptide. Then, in the same manner as in Experimental Example 3-3, the pT7 blue T vector into which this DNA fragment was inserted was introduced into Escherichia coli NoVa Blue strain, and probe 2 was used from a colony obtained by culturing this E. coli strain in a plate medium. Clone SC31 was selected. This clone SC31 is the 210th nucleotide in the nucleotide sequence of Chemical formula 3.
It had a nucleotide sequence corresponding to the 795th position to the 795th position.

[0054]

[Table 4]

[0055]

[Table 5]

[0056]

[Table 6]

[0057]

[Experimental Example 3-5 Selection of Clone SC216 and Determination of Partial Base Sequence] A primer having the base sequence shown in Table 7 below arbitrarily selected from the partial base sequence of the clone SC31 selected in Experimental Example 3-4 by a conventional method. 5 and Asn-Asp in the partial amino acid sequence (d) shown in Experimental Example 2-3.
-Asn-Ala-Asn-Gly was presumed to code and primer 6 of the base sequence shown in the following Table 8 was chemically synthesized. First strand c obtained in Experimental Example 3-2
A portion of the DNA solution was taken, and this was subjected to gene amplification in the presence of the primer 5 and the primer 6 in the same manner as in Experimental Example 3-3, and the DNA fragment thus obtained was arbitrarily selected from the partial base sequence of the clone SC31. Using the probe 3 having the nucleotide sequence shown in Table 9 above, another DNA fragment partially encoding the polypeptide was collected. After that, Experimental Example 3
In the same manner as in -3, the pT7 blue T vector containing this DNA fragment was introduced into E. coli NoVaBlue strain,
Clone SC216 was selected from the colonies obtained by culturing this E. coli strain in a plate medium using probe 3. This clone SC216 had a nucleotide sequence corresponding to the 763rd position to the 1st and 233nd positions in the nucleotide sequence of Chemical formula 3.

[0058]

[Table 7]

[0059]

[Table 8]

[0060]

[Table 9]

[0061]

[Experimental example 3-6 Selection of clone SC50 and determination of partial base sequence] A part of the total RNA obtained in Experimental example 3-1 was taken,
This was subjected to a reverse transcriptase reaction in the presence of primer 7 having a base sequence shown in Table 10 below, which was arbitrarily selected from the partial base sequence of clone SC09 according to the method of Experimental Example 3-2. To the solution containing the obtained first strand cDNA, 4 μl of 5 × Tailing buffer, 4 μl of dATP,
After adding 10 units of deoxyribonucleotide terminal transferase and making it 20 μl with sterile distilled water, 37 ° C., 70 ° C.,
Incubated at 4 ° C. for 5 minutes each. Remove excess primer from the reaction with a spin filter,
After concentration, purified polydA-added first strand c
DNA Flowman et al., "Proceedings of National Academy of Sciences USA", Vol. 85, No. 8,998.
-10, 9002 (1988) and 10 pmol of primer 8 having the nucleotide sequence shown in Table 11 below.
0μ PCR buffer 10μl, dNTP mix 1μ
1, 2.5 units / μl 0.5 μl of Amplitac DNA Polymerase was added to 100 μl of sterile distilled water, and then incubated at 72 ° C. for 30 minutes, 99 ° C. for 5 minutes, and 4 ° C. for 5 minutes in this order. Second strand cdN
A was synthesized. After removing excess primer from the reaction product with a spin filter and concentrating, it was arbitrarily selected from Primer 9 having the base sequence shown in Table 12 below reported by Flowman et al. And a partial base sequence of SC09. 10 pmol of the primer 10 having the nucleotide sequence shown in Table 13 below, 10 μl of 10 × PCR buffer, 1 mM, respectively
After adding 1 µl of dNTP mix and 0.5 µl of 2.5 units / µl amplitac DNA polymerase to 100 µl with sterilized distilled water, gene amplification was carried out in the same manner as in Example 3-3, and the obtained DNA fragment was used to clone clone SC09. Using the probe 4 having the nucleotide sequence shown in Table 14 below, which was arbitrarily selected from the partial nucleotide sequence of, the additional DNA fragment partially encoding the polypeptide was collected. afterwards,
In the same manner as in Experimental Example 3-3, the pT7 blue T vector having this DNA fragment inserted was introduced into Escherichia coli NoVa Blue strain, and cloned using a probe 4 from a colony obtained by culturing the E. coli strain in a plate medium. SC5
0 was selected. This clone SC50 had a base sequence corresponding to the 1st to 198th bases in the base sequence of Chemical formula 3.

[0062]

[Table 10]

[0063]

[Table 11]

[0064]

[Table 12]

[0065]

[Table 13]

[0066]

[Table 14]

[0067]

[Experimental Example 3-7 Selection of Clone SC362 and Determination of Partial Base Sequence] 20 μl of the total RNA obtained in Experimental Example 3-1 was taken, and primer 8 was replaced by 0.5 with a random hexamer.
A reverse transcriptase reaction was carried out in the same manner as in Experimental Example 3-2 except that μg was used to obtain a solution containing first-strand cDNA. This solution was prepared by using primer 9 and clone S shown in Table 12.
Table 1 below arbitrarily selected from the partial base sequence of C216
Experimental Example 3 in the presence of the primer 11 having the nucleotide sequence shown in 5
Gene amplification by the method of No. -3, and using the probe 5 having the nucleotide sequence shown in Table 16 below, which is arbitrarily selected from the partial nucleotide sequence of the clone SC216 from the obtained DNA fragment, is used to partially encode the polypeptide. The DNA fragment of Then, in the same manner as in Experimental Example 3-3, the pT7 blue T vector into which this DNA fragment was inserted was introduced into Escherichia coli NoVaBlue strain, and this Escherichia coli strain was cultured in a plate medium. Clone SC362 was selected. This clone S
C362 had a base sequence corresponding to the 1,216th to the 1,545th bases in the base sequence of Chemical formula 3.

[0068]

[Table 15]

[0069]

[Table 16]

[0070]

[Experimental example 3-8 All nucleotide sequences encoding polypeptide and all amino acid sequences of polypeptide] Experimental examples 3-3 to 3
As a result of comprehensive analysis of the results of -7, it was revealed that the gene encoding the polypeptide has a base sequence from the 5'end shown in Chemical formula 3. As a result of decoding this base sequence, the polypeptide is N
It has an amino acid sequence from the end, is composed of a maximum of 514 amino acid residues, and naturally contains all four types of partial amino acid sequences shown in Experimental Example 2-3.

Thus, the polypeptide of this invention comprises:
The present inventor has discovered the first result in the world as a result of many years of research, and has unique physicochemical properties and amino acid sequences that are not found in conventionally known cedar pollen allergens. However, on the other hand, recombinant DNA
Advances in technology have allowed the art to substitute one or more of its constituent amino acids with other amino acids without substantially altering the biological activity of the polypeptide. In view of such state of the art, the polypeptide referred to in the present invention is not limited to the one having the amino acid sequence shown in Chemical formula 4 as it is, and the polypeptide in the amino acid sequence shown in Chemical formula 4 while retaining a specific N-terminal amino acid sequence. It is intended to include all homologous variants in which one or more amino acids have been replaced by other amino acids.

Next, the method for producing the polypeptide according to the present invention will be specifically described with reference to Examples and the like.

The polypeptide of the present invention is of natural origin or artificially, so long as it has the amino acid sequence shown in Chemical formula 4 or a sequence homologous to the amino acid sequence. It does not matter whether they are synthesized. Examples of the natural source include cedar trees belonging to the genus Cedar (Cryptomeria japonica). From the pollen and male flowers, as described above, the polypeptide of the present invention and the DNA encoding the same should be collected. Is possible. To artificially synthesize the polypeptide of the present invention, for example, it is chemically synthesized based on the amino acid sequence shown in Chemical formula 4, or DNA having the nucleotide sequence of Chemical formula 3 is appropriately inserted into an expression vector and recombined. A transformant obtained by using DNA as a host and appropriately introducing it into a host may be cultured.

As mentioned above, the present invention provides recombinant DNA.
It also provides a method for producing the polypeptide by applying the technique. The method according to the present invention comprises culturing a transformant containing a DNA encoding the polypeptide in a culture medium and collecting the polypeptide produced from the culture. The transformant used in the present invention is usually
It includes a replicable recombinant DNA having a DNA encoding the polypeptide inserted therein. Such recombinant DNA usually comprises a cDNA encoding the polypeptide and a replicable expression vector, and can be prepared relatively easily by ordinary recombinant DNA technology if the cDNA is available. Examples of such expression vectors include pKK223-3, pRL-λ, pBTrp2D.
NA, pUB110, YEp13, Ti plasmid, R
i plasmid, pBI121 and the like. Among them, pKK223 can be used to express the DNA encoding the polypeptide in microorganisms such as Escherichia coli, Bacillus subtilis and yeast.
-3, pRL-λ, pBTrp2DNA, pUB110
And YEp13 are preferable, and Ti plasmid and Ri plasmid are preferable for expressing in plant cells.

To insert the DNA into the expression vector, a method generally used in the art can be adopted. For example, the cDNA encoding the polypeptide is appropriately cleaved with a restriction enzyme, while the replicable appropriate vector is cleaved with the same enzyme. Then, by ligating the obtained DNA fragment and vector fragment with DNA ligase, recombinant D capable of replication can be obtained.
NA is obtained. By introducing such recombinant DNA into microorganisms such as Escherichia coli, Bacillus subtilis, actinomycetes, yeast, and cells of higher animals and plants by the competent cell method, protoblast method, electroporation method, etc. When the obtained transformant is cultured in a culture medium, the polypeptide is produced inside or outside the cells.

The produced polypeptide can be purified by a method generally used in the art. For example, a transformant culture and / or cells or cells are collected, and if necessary, the cells or cells are disrupted, and the resulting crude polypeptide is salted out, dialyzed, filtered, concentrated, and centrifuged. It may be purified by separation, gel filtration chromatography, ion exchange chromatography, affinity chromatography, gel electrophoresis, isoelectric focusing and the like.
Above all, Tea International
Archives of Allergy and Immunology ”, Vol. 98, pp. 110-117 (1992), the monoclonal antibody specific to Cry j II is extremely useful for purification of the polypeptide, By affinity chromatography using this monoclonal antibody bound to a water-insoluble carrier, a highly pure polypeptide can be obtained with a minimum of labor and time. The partially purified or purified polypeptide thus obtained is a desensitizing agent for diagnosing or treating / preventing hay fever, and an immunological assay such as enzyme immunoassay or radioimmunoassay for diagnosing hay fever. It has a wide range of uses in methods, and further in academic research for elucidating the pathogenic mechanism of allergic diseases in general including hay fever.

Next, the method for producing the polypeptide according to the present invention will be specifically described based on Examples.

[0078]

Example A-1 Production of Polypeptide In this example, a plasmid vector was used to introduce DNA encoding the polypeptide into Escherichia coli to form a transformant, and the transformant was cultured. To produce the polypeptide. It is needless to say that the method can be modified in various ways in the art, and this example is only one example, and the manufacturing method of the present invention is not limited to this example.

[0079]

[Example A-1 (a) Preparation of transformant] Experimental example 3-
1 μg of the total RNA obtained in 1 above was used as a sense primer or an antisense primer and arbitrarily selected from the partial base sequence of clone SC50. The primer 12 having the base sequence shown in Table 17 below and the partial base sequence of clone SC216 were arbitrarily selected. Experimental Examples 3-2 and 3-except that the selected primer 13 having the nucleotide sequence shown in Table 18 below was used
Gene amplification was carried out in the same manner as 3. After that, according to the usual method,
The PCR product containing the DNA fragment was digested with restriction enzymes EcoRI and N
cleaved with coI, containing the start codon ATG, EcoRI
A DNA fragment having a cleavage site and an NcoI cleavage site added was obtained.

[0080]

[Table 17]

[0081]

[Table 18]

Separately, 1 μl of total RNA obtained in Experimental Example 3-1 was used.
g, Experimental Example 3 except that Primer 14 and Primer 15 having the base sequences shown in Table 19 or Table 20 below arbitrarily selected from the partial base sequences of clone SC216 and clone SC362, respectively, were used as sense primers or antisense primers. Gene amplification was performed in the same manner as in -2 and 3-3. Then, the PCR product containing the DNA fragment is cleaved with the restriction enzymes PstI and NcoI by a conventional method to contain the termination codon TGA and the PstI cleavage site and NcoI.
A DNA fragment having a coI cleavage site added was obtained.

[0083]

[Table 19]

[0084]

[Table 20]

10 μg of each of the two kinds of DNA fragments thus obtained was added to a suitable amount of sterile distilled water in advance with the restriction enzyme Eco.
10 ng of the plasmid vector pKK223-3 manufactured by Pharmacia, which had been treated with RI and PstI, and an appropriate amount of T.
4 DNA ligase and 100 mM ATP were added to a final concentration of 1 mM, and the mixture was incubated at 16 ° C. for 18 hours. The obtained recombinant DNA was transformed into Escherichia coli JM109 strain (ATCC 5332 by the competent cell method).
Introduced into 3) and transformed. This transformant was inoculated into an L-broth medium (pH 7.2) containing 100 μg / ml of ampicillin by a conventional method, cultured at 37 ° C. for 18 hours, and then a recombinant DNA was extracted by a usual alkaline method.
This recombinant DNA was named "pKCJ2-5", and its structure was analyzed by the dideoxy method. As shown in FIG. 1, in this pKCJ2-5, the cDNA region KCJ cDNA having the nucleotide sequence of Chemical formula 3 was obtained. Is Tac
It was linked downstream of the promoter Ptac.

[0086]

Example A-1 (b) Production of Polypeptide Recombinant D
Example A-1 (a) in which NA pKCJ2-5 was introduced.
Of the transformant containing 100 μg / ml of ampicillin
After inoculation into broth medium (pH 7.2) and culturing at 37 ° C. for 18 hours, 0.1 ml of the culture was taken, transferred to the same fresh culture medium, and further cultured at 37 ° C. Then, the absorbance of the culture medium at a wavelength of 600 nm is 0.3.
IPTG was added to a final concentration of 1 mM when the temperature reached, and the cells were further cultured for 4 hours. Then, the bacterial cells collected from the culture by centrifugation were suspended in cold distilled water, and the bacterial cells were ultrasonically disrupted and then centrifuged to collect the supernatant.

After the supernatant was concentrated, it was loaded on a mAb N26 sepharose column binding a monoclonal anti-Cry j II antibody which had been equilibrated with 10 mM phosphate buffer (pH 7.0) containing 0.14 M sodium chloride in advance, Then, 0.1 M glycine-hydrochloric acid buffer solution (pH 2.
3) The solution was passed through to elute the peptide component. A fraction containing the polypeptide was collected from the eluted fraction, neutralized by adding 1 M phosphate buffer (pH 7.0), and dialyzed against 10 mM phosphate buffer (pH 7.0) for 10 hours. Then, the dialyzed solution was collected, concentrated and lyophilized by a conventional method to obtain a purified polypeptide solid product. The yield was about 3 mg per liter of culture medium.

[0088]

Example A-1 (d) Physicochemical Properties of Polypeptide
When the purified polypeptide obtained by the method of Example A-1 (c) was tested by the method of Experimental Example 2, it had the following physicochemical properties. (1) Molecular weight When tested by SDS-polyacrylamide gel electrophoresis in the same manner as in Experimental Example 2-1, the purified polypeptide showed a major band at a position corresponding to a molecular weight of 55,000 ± 5,000 daltons. . (2) Isoelectric point When tested by an isoelectric focusing method in the same manner as in Experimental Example 2-2, the purified polypeptide had an isoelectric point of 9.0 ± 0.5. (3) N-terminal amino acid sequence When analyzed by an ordinary method using an amino acid sequencer "470A type" manufactured by Applied Biosystems, the purified polypeptide was found to have Ala-M at the N-terminal.
et-Lys-Phe-Ile-Ala-Pro-Me
t-Ala-Phe-Val-Ala-Met-Gln
It had an amino acid sequence represented by -Leu-Ile-Ile-Met-Ala-. (4) Ultraviolet absorption spectrum When an ultraviolet absorption spectrum in an aqueous solution was measured using a spectrophotometer, the purified polypeptide showed a wavelength of 280.
The absorption maximum was shown in the vicinity of nm. (5) Solubility in solvent When tested by a conventional method, purified polypeptide was found to be water,
It was soluble in saline and phosphate buffer. (6) Biological action When tested by the method of Experimental Example 2-6 (a), the absorbance in the system using the serum of a healthy person was about 0.1, while the serum of a pollinator patient was used. In this system, the absorbance reached about 2.0, indicating that the polypeptide was significantly bound to the immunoglobulin E antibody contained in the blood of patients with pollinosis. And this is
This proves that the polypeptide is one of the causative agents of hay fever, that is, has the property of inducing hay fever. On the other hand, when tested by the method of Experimental Example 2-7 (b), uptake of about 200 cpm of ³ H-thymidine was observed in the control system to which physiological saline containing no polypeptide was administered. In the system to which the polypeptide was added, a remarkable uptake of about 8,500 cpm was observed per 50 μg / mg of the polypeptide.
This indicates that the polypeptide has antigenicity.

Next, the use of the polypeptide according to the present invention will be specifically described with reference to Examples and Experimental Examples.

Since the polypeptide of the present invention is one of the causative agents of pollinosis, it has a wide range of uses as a desensitizing agent for treating, preventing and diagnosing pollinosis. The desensitizing agent of the present invention contains, as an active ingredient, the polypeptide or a complex of the polypeptide and a specific sugar described below. A desensitizing agent for the purpose of diagnosing hay fever usually contains a partially purified or purified polypeptide obtained by the method as described above as it is, while it is used for treatment / prevention of hay fever. Is
Prior to formulation, a specific sugar is covalently bound to the polypeptide to form a complex.

The saccharide as referred to in the present invention is one that can be covalently bound to the polypeptide and enhances or reduces the desensitizing effect and / or side effect of the polypeptide by forming a complex. Say. Examples of such sugars include simple or complex starch, amylose, dextran, polysucrose, pullulan, elucinan, curdlan, gum arabic, gum tragacanth, guar gum, xanthan gum, carrageenan, cellulose, glucomannan, chitosan, lipopolysaccharide and the like. Polysaccharides and derivatives and partial hydrolysates of these sugars are included, and the average molecular weight thereof is usually about 500 to 10,000,000.
Daltons, preferably about 10,000 to 100
It is in the range of 10,000 Daltons. Among the above-mentioned saccharides, a complex with pullulan, erucinan, or a partial hydrolyzate thereof, which essentially has maltotriose as a repeating unit, is effective for desensitization when administered to mammals including humans. While the immunoglobulin G antibody and the immunoglobulin M antibody are produced in a considerable amount, the immunoglobulin E antibody, which is the main cause of unwanted side effects including anaphylactic shock, is difficult to produce. This is extremely convenient for safely and effectively carrying out the desensitization therapy which requires repeated administration.

Further, for example, lipopolysaccharides derived from microorganisms such as Escherichia coli, Salmonella, Serratia, etc. and partial hydrolysates thereof can be made into a complex with the polypeptide to give mucosa of the mammal. It has the property of increasing the affinity for and significantly improving the intake efficiency. From this, the complex with these sugars is particularly useful in a desensitizing agent which is premised on transdermal or transmucous administration.

Such a complex is usually obtained by reacting the polypeptide with an activated sugar, or by reacting the polypeptide with 2 in one molecule.
The above-mentioned reagent having an active functional group may be used to crosslink the polypeptide and the carbohydrate. Examples of the individual reaction methods include a diazo method, a peptide method, an alkylation method, a crosslinking method, an amide bond method, a periodate oxidation method, a disulfide bond method, and the like, but these reaction methods themselves are known in the art. For example, Japanese Patent Application Laid-Open No. 3-93730 discloses a typical method in detail. The weight ratio of the polypeptide to the carbohydrate at the start of the reaction is usually about 1: 0.001 to 1: 1,000, preferably about 1: 0.01 to 1: 100. Be done. Depending on the reaction method, if it is below this range, the binding of the peptides will be remarkable, and if it is above this range, the binding of the carbohydrates will be remarkable. In any case, it brings about a decrease in the efficiency of the reaction and the purification after the reaction, and the above range was made the best. The temperature, pH and reaction time during the reaction should be set so that the polypeptide is not easily deactivated or decomposed, and undesired side reactions are minimized. Usually, the temperature is about 0 to 100. It is preferable to complete the reaction in about 0.1 to 50 hours at a temperature of about 0.1 to 12 at pH.

The complex formed by the reaction is subjected to, for example, dialysis, salting out, filtration, concentration, centrifugation, gel filtration chromatography, ion exchange chromatography, affinity chromatography, gel electrophoresis, isoelectric focusing and the like. Can be purified by methods commonly used in the art,
If necessary, these methods may be combined appropriately. Then, depending on the final use form, the purified complex may be concentrated or lyophilized to be in a liquid or solid state.

The desensitizing agent of the present invention is not only in the form of the polypeptide and / or complex obtained as described above, but also in other physiologically acceptable forms such as carriers and excipients. , Diluents, immunostimulants, stabilizers, and even
If necessary, the form as a composition with one or more other drugs containing an anti-inflammatory agent such as a steroid hormone or sodium glymoglycate or an antihistamine is included. Further, the desensitizing agent of the present invention also includes a drug in a dosage unit form, and the drug in a dosage unit form means the polypeptide and / or the complex of the present invention, for example, a daily dose or an integer thereof. Mean drug in physically separate, unitary dosage forms suitable for administration, containing an amount equivalent to double (up to 4 times) or submultiples thereof (up to 1/40).
Drugs in such dosage forms include powders, fine granules, granules, pills, tablets, capsules, troches, syrups, emulsions, ointments, plasters, poultices, suppositories, eye drops,
Examples include nasal drops, sprays and injections.

The method of using the desensitizing agent according to the present invention will be described. The desensitizing agent of the present invention can be used in the same manner as a general desensitizing agent containing a cedar pollen allergen. That is, in order to diagnose hay fever with the desensitizing agent of the present invention, a usual test method known as a scratch test or an intradermal test is adopted, and first, a scratch on the skin surface of the subject is attached to a degree that does not bleed. An appropriate amount of the diagnostic desensitizing agent according to the present invention is dropped, or an appropriate amount of the diagnostic desensitizing agent according to the present invention is intradermally administered. After 15 to 30 minutes have passed, the presence and size of wheal is examined, and when the size of wheal is a certain value or more, it is determined to be positive.

In the treatment with the desensitizing agent of the present invention, the appropriate dose and dosage are usually determined based on the above-mentioned diagnostic results. A subject with a positive diagnostic result is orally or parenterally administered with the desensitizing agent of the present invention containing a complex of the polypeptide and a specific carbohydrate. Symptoms,
Although it varies depending on the usage and the like, specifically, it is usually about 0.0001 to 100,000 ng per adult, preferably about 0.001 to 10 while observing the patient's symptoms and the course after administration. As a guideline, 10,000 ng, once a week to once a month, for about a month to a year, usually
Repeated doses may be given intradermally, subcutaneously, intramuscularly, intraperitoneally or transmucousally while increasing the dose. For the prevention of hay fever, the same dose and usage may be used, and usually about 0.0001 to 100,000 ng per adult, preferably about 0.001 while observing the health condition of the subject and the course after administration. As a guideline, once a week or once a month for about 1 to 6 months, the dose is usually increased and administered repeatedly intradermally, subcutaneously, intramuscularly or transmucousally. When the desensitizing agent according to the present invention is regularly administered repeatedly from autumn to early spring of the following year, allergic symptoms in the onset season of the following year can be minimized or eliminated.

Next, regarding the desensitizing agent according to the present invention, 2
Specific examples will be described with reference to Examples 3 to 3.

[0099]

[Example B-1 Dry injection] Average molecular weight of about 200,0
2 g of 00 dalton purified pullulan was dissolved in 100 ml of distilled water, and 2 ml of a 1.7% (w / v) acetone solution of cyanuric chloride was added. Then, while maintaining the pH of the solution at around 7 with a 5% (w / v) sodium carbonate aqueous solution, the mixture was allowed to stand for 2 hours at 4 ° C. or lower in an ice bath for reaction. Example A-1 was added to the solution containing the activated pullulan thus obtained.
200 mg of purified polypeptide obtained by the method of (b)
In addition, the mixture was reacted with stirring at pH 7.0 and 37 ° C. for 5 hours. Then, 1% (w / v) of glycine was added to the reaction product,
After stirring at 37 ° C for 1 hour with stirring to block unreacted active groups, 0.01M acetate buffer (pH
5.0) for 5 hours and then 0.01
CM equilibrated with M acetate buffer (pH 5.0)
-By Sephadex column chromatography, a complex of the polypeptide and pullulan was collected from the non-adsorbed portion. Then, the complex was dissolved in a physiological saline containing 1% (w / v) human serum albumin as a stabilizer to a final polypeptide concentration of about 100 ng / ml by a conventional method, sterilized by filtration, and then sterilized. 2 ml was dispensed into vials, freeze-dried and sealed.

Prior to administration, this product is used by first adding 1 ml of distilled water for injection or the like into a vial and then uniformly dissolving the contents. The product, which has excellent stability and contains a complex of the polypeptide and pullulan as an active ingredient, is useful as a dry injection for treating / preventing hay fever.

[0101]

Example B-2 Injection: 1 g of CM-cellulose having an average molecular weight of about 20,000 daltons was dissolved in 200 ml of distilled water, and 2 g of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide-methiozide was added to the solution. After
While maintaining the pH of the solution near 4 with 1N hydrochloric acid, the mixture was reacted at room temperature for 2 hours with stirring. The reaction product was dialyzed against distilled water for 24 hours, and the dialyzed solution was collected to give Example A-1.
30 mg of the purified polypeptide obtained by the method of (b) was added, and the mixture was allowed to react at room temperature for 15 hours at pH 4.5 for 15 hours. Thereafter, the complex in the reaction product was purified in the same manner as in Example B-1, concentrated, dissolved in 50% (v / v) glycerin aqueous solution, sterile filtered, and dispensed in 2 ml aliquots into a sterile vial. , Sealed.

Prior to administration, this product was prepared by adding 100 to 100,000-fold volume of 50% (v / v) glycerin aqueous solution to a vial, referring to the diagnostic results of a scratch test or an intradermal test. Then, the contents are diluted evenly before use. The polypeptide and CM as active ingredients
-The product containing a complex with cellulose is useful as a hyposensitizing injection for treating / preventing hay fever.

[0103]

Example B-3 Liquid Solution 100 mg of purified lipopolysaccharide derived from Salmonella was dissolved in 25 ml of 50% saturated aqueous sodium acetate solution at about 4 ° C., and the pH of the solution was adjusted to 9.0 with 0.5N sodium hydroxide. While maintaining the pH at about 8.5, 1 ml of anhydrous dioxane containing 20 μl of bromoacetyl bromide was added dropwise. Then, the pH of the reaction product was adjusted to about 4.5 with 6N hydrochloric acid, and dialyzed against distilled water at 4 ° C. for 48 hours to obtain an aqueous solution containing activated lipopolysaccharide. After adding 40 mg of the purified polypeptide obtained by the method of Example A-1 (b) to this aqueous solution, the pH of the solution
Was maintained at room temperature for about 4.5 hours and allowed to react at room temperature for 48 hours. Then, the reaction product was purified in the same manner as in Example B-1, concentrated, and lyophilized to obtain a solid complex of the polypeptide and lipopolysaccharide. Then, this solid was added as a stabilizer at 1% so that the final concentration was 100 ng / ml.
(W / v) Dissolved in distilled water containing purified gelatin, and sterilized by a conventional method to obtain a liquid preparation.

The product containing a complex of the polypeptide and lipopolysaccharide as an active ingredient is useful as an eye drop, a nasal drop and a liquid for oral spray for treating / preventing hay fever.

[0105]

Example B-4 Sublingual agent Average molecular weight about 200,000
Dalton's purified erucinan was uniformly dissolved in 400 ml of distilled water, and the pH of the solution was adjusted with 1N sodium hydroxide aqueous solution.
Was adjusted to 10.7, 3 g of cyanogen bromide was gradually added while the pH of the aqueous solution was maintained at about 10.0, and the reaction was carried out for 1 hour. After adjusting the pH of the reaction product to 5.0 with 1N hydrochloric acid,
While maintaining this pH, dialysis was performed against cold water for 10 hours to obtain an aqueous solution containing activated erucinan. Next, 20 mg of the purified polypeptide obtained by the method of Example A-1 (b) was added to this aqueous solution, and the mixture was allowed to stand at room temperature for 24 hours for reaction. After completion of the reaction, 3 volumes of acetone was added to the reaction product, the formed precipitate was collected, dissolved in 0.01M acetate buffer (pH 5.0), and the insoluble material was removed by centrifugation, and then 0 0.01 M acetate buffer (pH 5.
The fraction containing the complex of the polypeptide and erucinan was collected from the non-adsorbed fraction by subjecting it to CM-Sephadex column chromatography that had been equilibrated in 0).
Then, by a conventional method, this fraction is sterilized by filtration, concentrated,
After freeze-drying and crushing, anhydrous crystalline α-maltose powder “Finetose” manufactured by Hayashibara Co., Ltd. was uniformly mixed, and the mixture was tabletted by a conventional method to give a tablet containing 100 ng of polypeptide per 1 tablet (200 mg) of the product. Got

The product which is excellent in ingestibility and stability is useful as a sublingual agent for treating / preventing hay fever.

[0107]

Example B-5 Diagnostic Agent 100 mg of the purified polypeptide obtained by the method of Example A-1 (b) was added to physiological saline 20.
After dissolving in ml and sterilizing filtration by a conventional method, 1 ml each was dispensed into a sterilized vial bottle, lyophilized, and sealed to obtain a diagnostic agent.

This product is dissolved in 1 ml of distilled water for injection and then further diluted 10-fold with the same distilled water to be used for diagnosis of pollinosis by a scratch test or an intradermal test.

[0109]

Example B-6 Diagnostic Agent 1 mg of the purified polypeptide obtained by the method of Example A-1 (b) was added to 50% (v / v) glycerin aqueous solution containing 5% (w / v) sodium chloride 2
After dissolving in 0 ml and performing sterile filtration by a conventional method, 1 ml was dispensed into a sterilized vial bottle.

This product is diluted 20 times with a 50% (v / v) glycerin aqueous solution and used for diagnosis of hay fever by a scratch test or an intradermal test.

Next, the effects and the like of the desensitizing agent according to the present invention will be described based on a few experimental examples.

[0112]

Experimental Example 3 Animal Experiment In this experimental example, the complex of the polypeptide of the present invention and a specific carbohydrate is treated / prevented for pollinosis by actually administering the desensitizing agent of the present invention to an experimental animal. It is to prove that it is effective.

[0113]

[Experimental Example 3-1 Preventive effect] Female BA aged 10 to 12 weeks
In one group consisting of 6 LB / c mice, the desensitizing agent obtained by the method of Example B-1 was converted into the amount of polypeptide to be 1
It was intraperitoneally injected once a week at a rate of μg / animal for 3 weeks. One week after the final administration of the desensitizing agent, 1 μg of the purified polypeptide obtained by the method of Example A-1 (b) as an antigen and 4 mg of aluminum hydroxide as an immunostimulant were included in order to induce pollinosis. Physiological saline 0.2m
1 was injected as above. Then, immediately before the antigen administration and one week after the antigen administration, the blood of the mouse was collected, and the amount of immunoglobulin E antibody, immunoglobulin G antibody and immunoglobulin M antibody specific to the polypeptide contained therein was examined. .

Separately, in place of the desensitizing agent, Example A-1
A system for administering a mixture containing the purified polypeptide obtained by the method of (b) and the purified pullulan having an average molecular weight of about 200,000 daltons in a weight ratio of 1:15 was prepared, and treated in the same manner as above. Was used as a control. The amount of the immunoglobulin E antibody specific to the polypeptide is determined by "Life Sciences", I Motor et al., Vol.
No. 6, Part II, pp. 813-820 (1969)
The amount of immunoglobulin G antibody and immunoglobulin M antibody specific to the polypeptide by the PCA reaction reported in S. Yoshitake et al., "The Journal of Biochemistry", Vol. 92, No. 5,
The measurement was performed by the enzyme immunosorbent assay (EIA) reported on pages 1,413 to 1,424 (1982), and each was represented by the average antibody titer of 6 mice. The results are shown in Table 21.
Shown in.

[0115]

[Table 21]

As is clear from the results shown in Table 21, in comparison with the control, the system in which the desensitizing agent of the present invention containing the complex of the polypeptide and pullulan of the present invention was administered in advance, was desensitized. While the immunoglobulin G antibody and the immunoglobulin M antibody which are effective for E. coli were produced in a considerable amount, the production of the immunoglobulin E antibody was suppressed to a substantially nonexistent level. Immunoglobulin E antibody is said to be the main cause of unwanted side effects such as anaphylactic shock, and it is a complex fact that administration of the desensitizing agent of the present invention to mice significantly suppressed its production. It means that the desensitizing agent of the present invention containing the body can be used safely and effectively for preventing pollinosis in mammals including humans.

[0117]

[Experimental example 3-2 Therapeutic effect of parenteral administration] A group of 6 female BALB / c mice aged 10 to 12 weeks was prepared.
As an antigen, 1 μg of the purified polypeptide obtained by the method of Example A-1 (b) was added once a week to 0.2 ml of physiological saline containing 4 mg of aluminum hydroxide as an immunostimulant.
It was injected intraperitoneally for 3 weeks to induce hay fever. One week after the final administration of the antigen, the desensitizing agent obtained by the method of Example B-1 was converted into a polypeptide amount of 10
Injection was performed once a week at a dose of 0 ng / animal for 3 weeks in the same manner as above. One week after the final administration of the desensitizing agent, the antigen alone was again administered in the same manner as above to perform the treatment for inducing the production of immunoglobulin E antibody. Immediately before the administration of the desensitizing agent, one week after the final administration of the desensitizing agent, and one week after the re-induction of immunoglobulin E antibody, blood was collected from the mouse and specific for the polypeptide contained in the sonico. The amounts of the immunoglobulin E antibody, immunoglobulin G antibody and immunoglobulin M antibody were examined by the same method as in Experimental Example 3-1.

Separately, instead of the desensitizing agent, Example A-1
A system for administering a mixture containing the purified polypeptide obtained by the method of (b) and the purified pullulan having an average molecular weight of about 200,000 daltons in a weight ratio of 1:15 was prepared, and treated in the same manner as above. Was used as a control. The results are shown in Table 22.

[0119]

[Table 22]

As is clear from the results shown in Table 22, as compared with the control, in the system to which the desensitizing agent of the present invention containing the complex of the polypeptide and pullulan was administered in advance, the desensitizing agent was Even after administration of
Significant amount of immunoglobulin G even after antibody re-induction
An antibody and an immunoglobulin M antibody were recognized. As for the immunoglobulin E antibody, production was suppressed to virtually no level after administration of the desensitizing agent and even after reinduction of the immunoglobulin E antibody. These results mean that parenteral administration of the desensitizing agent of the present invention containing the complex can safely and effectively treat hay fever in mammals including humans.

[0121]

[Experimental example 3-3 Therapeutic effect by oral administration] 10 to 1
A physiological saline containing 1 μg of the purified polypeptide obtained by the method of Example A-1 (b) as an antigen and 4 mg of aluminum hydroxide as an immunostimulant in a group of 6 2-week-old female BALB / c mice. 0.2 ml of water once a week for 3
It was injected intraperitoneally for a week to induce hay fever.
One week after the final administration of the antigen, the sublingual preparation obtained by the method of Example B-4 was pulverized and orally administered at a dose of 10 μg / animal once a week for 3 weeks in terms of the amount of polypeptide. Was administered. One week after the final administration of the sublingual agent, blood was collected from the mice, and the amounts of immunoglobulin A antibody, immunoglobulin G antibody and immunoglobulin E antibody specific to the polypeptide contained therein were examined.

Separately, instead of the sublingual agent, Example A-1
A system was provided for oral administration of a solid mixture containing the purified polypeptide obtained by the method of (b) and the purified lipopolysaccharide derived from Salmonella in a weight ratio of 1:15, and the system was treated in the same manner as above. Was used as a control. The amount of immunoglobulin A antibody and immunoglobulin G antibody specific to the polypeptide can be determined by the method described by Earl Meolini et al.
Immunological Methods, Volume 6, 355-36
According to the EIA method reported on page 2 (1975),
In addition, immunoglobulin E specific for the polypeptide
The amount of antibody was measured by the same method as in Experimental Example 3-1,
Each is represented by the average antibody titer of 6 mice. The results are shown in Table 23.

[0123]

[Table 23]

As is clear from the results shown in Table 23, when compared with the control, in the system to which the desensitizing agent of the present invention containing the complex of the polypeptide and lipopolysaccharide was previously administered, the immunoglobulin was While the A antibody and the immunoglobunoline M antibody were produced in a considerable amount, the production of the immunoglobulin E antibody was suppressed to a substantially zero level. This indicates that the desensitizing agent of the present invention containing the complex can safely and effectively treat pollinosis in mammals including human even by oral administration.

Although data are not shown, the methods of Examples B-1 to B-
4. The desensitizing agent obtained by the method of 4 is administered by intradermal, subcutaneous, intramuscular or intraperitoneal injection in mice, rats and guinea pigs, or in the form of eye drops, nasal drops or oral sprays. Upon transmucosal administration, all of the above desensitizing agents exhibited significant effects in the treatment and prevention of hay fever without causing serious side effects. This becomes particularly remarkable in a complex of the polypeptide with pullulan, erucinan, etc., and a carbohydrate essentially having maltotriose as a repeating unit, and in this case, as compared with other complexes, It was found that the dose and administration period required to achieve the desired desensitization can be further reduced or shortened.

[0126]

[Experimental example 3-4 Acute toxicity test] 20
The therapeutic / preventive desensitizing agents obtained by the methods of Examples B-1 to B-4 were orally or intraperitoneally administered to the mice on the day. As a result, it was found that these desensitizing agents have LD50 of 1,000,000 ng or more by any administration route. This indicates that the desensitizing agent of the present invention containing a complex of the polypeptide and a specific sugar can be safely compounded and used in a drug to be administered to mammals including humans.

[0127]

As described above, the polypeptide according to the present invention is a novel causative agent of pollinosis. Since the polypeptide of the present invention has the property of inducing pollinosis in mammals including humans, it can be used not only for desensitizing agents for diagnosing or treating / preventing pollinosis but also for enzyme immunoassay and radioimmunoassay. It has a wide range of uses in the diagnosis of hay fever and also in academic research to elucidate the pathogenic mechanism of allergic diseases in general. In particular, the complex of the polypeptide of the present invention and a specific carbohydrate produces a significant amount of immunoglobulin antibody effective for desensitization when administered to mammals including human, while causing unwanted side effects including anaphylactic shock. It has a property that it does not substantially produce immunoglobulin E antibody, which is the main cause. Due to this property, when treating or preventing pollinosis using the desensitizing agent according to the present invention,
The dose and period of administration of the antigen required for treatment / prevention can be significantly reduced or shortened. The polypeptide, which is also useful as described above, can be produced in a desired amount relatively easily by the method of the present invention applying recombinant DNA technology.

The present invention exerts such remarkable actions and effects, and it can be said that the invention has a great significance to contribute to the field.

[Brief description of drawings]

FIG. 1 Recombinant DNA pKCJ2 used in the present invention
It is a figure which shows the structure of -5.

[Description of symbols] KCJ cDNA DNA encoding the polypeptide of the present invention Ptac Tac promoter rrnBT1T2 Liposomal RNA operon transcription termination region AmpR Ampicillin resistance gene ori Origin of replication in E. coli

[Procedure amendment]

[Submission date] August 4, 1994

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be corrected] Claim 17

[Correction method] Change

[Correction content]

[Procedure Amendment 2]

[Document name to be amended] Statement

[Name of item to be corrected] 0005

[Correction method] Change

[Correction content]

As described above, several cedar pollen allergens have already been isolated and their properties and properties have been elucidated to some extent. Therefore, administration of purified cedar pollen allergen to humans to desensitize them causes pollinosis. There is a prospect of treatment and prevention. Recently, some desensitizing agents have been devised for that purpose. For example, in JP-A-1-156926 and JP-A-3-93730, Asp-A is added to the N-terminal.
sn-Pro-Ile-Asp-Ser- or Ala-
It has been proposed to covalently bind pullulan, which is one of the polysaccharides, to an allergen having an amino acid sequence represented by Ile-Asn-Ile-Phe-Asn-, and administer the resulting complex to humans as a desensitizing agent. ing. However, a large amount of high-purity allergen is usually required for the diagnosis of allergic disease and desensitization therapy, and the allergen in cedar pollen is scarce and its stability is low, and it is a diagnostic agent for pollinosis. It is expected that there will be a great deal of difficulty if the cedar pollen is used as the only desensitizing agent and the desensitizing agent.

[Procedure 3]

[Document name to be amended] Statement

[Name of item to be corrected] 0023

[Correction method] Change

[Correction content]

Thereafter, acetic acid was added to the non-adsorbed fraction to pH.
After adjusting to 5.0, 10 mM acetate buffer (pH
The sample was loaded on a CM-Sephadex column that had been equilibrated with 5.0), 10 mM acetate buffer (pH 5.0) was passed through to wash the column, and then 0.1 M phosphate buffer (pH) was added.
The peptide component was eluted by passing an eluent consisting of 7.0) and 0.3 M sodium chloride through the column. Fractions containing the polypeptide were collected, loaded onto a Mono-S column that had been equilibrated with 10 mM phosphate buffer (pH 5.0) in advance, and subjected to a concentration gradient of sodium chloride increasing from 0 M to 0.5 M. When a 10 mM Tris-hydrochloric acid buffer solution (pH 7.0) was passed through the column, the polypeptide was eluted when the sodium chloride concentration was 0.4M.

[Procedure amendment 4]

[Document name to be amended] Statement

[Name of item to be corrected] 0029

[Correction method] Change

[Correction content]

[0029]

[Experimental example 2-3 Partial amino acid sequence] The purified polypeptide was digested with trypsin, and the digested product was subjected to chromatography column "Hi-Pore PR-318" manufactured by Bio-Rad.
Separation by high performance liquid chromatography using "type" gave four types of peptide fragments. When analyzed using an amino acid sequencer, these peptide fragments were (a) His-Asp-Ala-Ile-As.
n-Ile-Phe-Asn-Val-Glu-Ly
s, (b) His-Asp-Cys-Thr-Glu
-Ala-Phe-Ser-Thr-Ala-Trp-
Gln-Ala-Ala-Cys-Lys, (c) A
sn-Thr-Ile-Gly-Thr-Gly-As
p-Asp-Cys-Val-Ala-Ile-Gly
-Thr-Gly-Ser-Ser-Asn-Ile-
Val-Ile-Glu-Asp-Leu-Ile-C
ys-Gly-Pro-Gly, or (d) Ile-
Ala-Ser-Cys-Leu-Asn-Asp-A
sn-Ala-Asn-Gly-Tyr-Phe-Se
r-Gly-His-Val-Ile-Pro-Ala
It was found to have an amino acid sequence represented by

[Procedure Amendment 5]

[Document name to be amended] Statement

[Correction target item name] 0061

[Correction method] Change

[Correction content]

[0061]

[Experimental example 3-6 Selection of clone SC50 and determination of partial base sequence] A part of the total RNA obtained in Experimental example 3-1 was taken,
This was subjected to a reverse transcriptase reaction in the presence of primer 7 having a base sequence shown in Table 10 below, which was arbitrarily selected from the partial base sequence of clone SC09 according to the method of Experimental Example 3-2. To the solution containing the obtained first strand cDNA, 4 μl of 5 × Tailing buffer, 4 μl of dATP,
After adding 10 units of deoxyribonucleotide terminal transferase and making it 20 μl with sterile distilled water, 37 ° C., 70 ° C.,
Incubated at 4 ° C. for 5 minutes each. Remove excess primer from the reaction with a spin filter,
After concentration, poly dA-added first strand c produced
DNA Flowman et al., "Proceedings of National Academy of Sciences USA", Vol. 85, No. 8,998.
-10, 9002 (1988) and 10 pmol of primer 8 having the nucleotide sequence shown in Table 11 below.
0μ PCR buffer 10μl, dNTP mix 1μ
1, 2.5 units / μl 0.5 μl of Amplitac DNA Polymerase was added to 100 μl of sterile distilled water, and then incubated at 72 ° C. for 30 minutes, 99 ° C. for 5 minutes, and 4 ° C. for 5 minutes in this order. Second strand cdN
A was synthesized. After removing excess primer from the reaction product with a spin filter and concentrating, it was arbitrarily selected from Primer 9 having the base sequence shown in Table 12 below reported by Flowman et al. And a partial base sequence of SC09. 10 pmol of primer 10 having the nucleotide sequence shown in Table 13 below, 10 μl of 10 × PCR buffer, 1 m
1 μl of MdNTP mix and 0.5 μl of 2.5 units / μl amplitac DNA polymerase were added to make 100 μl with sterile distilled water, and then gene amplification was carried out in the same manner as in Experimental Example 3-3, and a clone was obtained from the obtained DNA fragment. SC09
Using the probe 4 having the nucleotide sequence shown in Table 14 below, which was arbitrarily selected from the partial nucleotide sequence of, the additional DNA fragment partially encoding the polypeptide was collected. Then, in the same manner as in Experimental Example 3-3, the pT7 blue T vector into which this DNA fragment was inserted was transformed into Escherichia coli NoVa Blu.
clone S using the probe 4 from the colony obtained by introducing this E. coli strain into a plate medium and culturing this E. coli strain in a plate medium.
C50 was selected. This clone SC50 had a base sequence corresponding to the 1st to 198th bases in the base sequence of Chemical formula 3.

[Procedure correction 6]

[Document name to be amended] Statement

[Correction target item name] 0075

[Correction method] Change

[Correction content]

To insert the DNA into the expression vector, a method generally used in the art can be adopted. For example, the DNA encoding the polypeptide is appropriately cleaved with a restriction enzyme, while the vector capable of autonomous replication is cleaved with the same enzyme. Then, the obtained DNA fragment and the vector fragment are ligated with a DNA ligase to obtain a replicable recombinant DNA. Obtained by introducing such recombinant DNA into microorganisms such as Escherichia coli, Bacillus subtilis, actinomycetes, yeast, and cells of higher animals and plants by the competent cell method, protoplast method, electroporation method, etc. When the transformant obtained is cultured in a culture medium, it produces the polypeptide inside or outside the cells.

[Procedure Amendment 7]

[Document name to be amended] Statement

[Correction target item name] 0088

[Correction method] Change

[Correction content]

[0088]

Example A-1 (c) Physicochemical properties of polypeptide
When the purified polypeptide obtained by the method of Example A-1 (b) was tested by the method of Experimental Example 2, it had the following physicochemical properties. (1) Molecular weight When tested by SDS-polyacrylamide gel electrophoresis in the same manner as in Experimental Example 2-1, the purified polypeptide showed a major band at a position corresponding to a molecular weight of 55,000 ± 5,000 daltons. . (2) Isoelectric point When tested by an isoelectric focusing method in the same manner as in Experimental Example 2-2, the purified polypeptide had an isoelectric point of 9.0 ± 0.5. (3) N-terminal amino acid sequence When analyzed using an amino acid sequencer "470A type" manufactured by Applied Biosystems by a general method, the purified polypeptide was found to have Ala-Me at the N-terminal.
t-Lys-Phe-Ile-Ala-Pro-Met
-Ala-Phe-Val-Ala-Met-Gln-
It had an amino acid sequence represented by Leu-Ile-Ile-Met-Ala-. (4) Ultraviolet absorption spectrum When an ultraviolet absorption spectrum in an aqueous solution was measured using a spectrophotometer, the purified polypeptide showed a wavelength of 280.
The absorption maximum was shown in the vicinity of nm. (5) Solubility in solvent When tested by a conventional method, purified polypeptide was found to be water,
It was soluble in saline and phosphate buffer. (6) Biological action When tested by the method of Experimental Example 2-6 (a), the absorbance in the system using the serum of a healthy person was about 0.1, while the serum of a pollinator patient was used. In this system, the absorbance reached about 2.0, indicating that the polypeptide was significantly bound to the immunoglobulin E antibody contained in the blood of patients with pollinosis. And this is
This proves that the polypeptide is one of the causative agents of hay fever, that is, has the property of inducing hay fever. On the other hand, when tested by the method of Experimental Example 2-6 (b), uptake of about 200 cpm of ³ H-thymidine was observed in the control system to which physiological saline containing no polypeptide was administered. In the system to which the polypeptide was added, a remarkable uptake of about 8,500 cpm was observed per 50 μg / ml of the polypeptide.
This indicates that the polypeptide has antigenicity.

[Procedure Amendment 8]

[Document name to be amended] Statement

[Correction target item name] 0095

[Correction method] Change

[Correction content]

The desensitizing agent of the present invention is not only in the form of the polypeptide and / or complex obtained as described above, but also in other physiologically acceptable forms such as carriers and excipients. , Diluents, immunostimulants, stabilizers, and even
As needed, the form as a composition with one or more other drugs containing an anti-inflammatory agent such as a steroid hormone or sodium cromoglycate or an antihistamine is included. Further, the desensitizing agent of the present invention also includes a drug in a dosage unit form, which means the polypeptide and / or complex of the present invention, for example, a daily dose or a dose thereof. It means a drug, which is contained in an amount that is an integral multiple (up to 4 times) or a multiple thereof (up to 1/40) and is in a physically separated and unitary dosage form suitable for administration. Drugs in such dosage forms include powders, fine granules, granules, pills, tablets, capsules, troches, syrups, emulsions, ointments, plasters, poultices, suppositories, eye drops, Examples include nasal drops, sprays and injections.

[Procedure Amendment 9]

[Document name to be amended] Statement

[Correction target item name] 0103

[Correction method] Change

[Correction content]

[0103]

Example B-3 Liquid Solution 100 mg of purified lipopolysaccharide derived from Salmonella was dissolved in 25 ml of 50% saturated sodium acetate aqueous solution at about 4 ° C., and the pH of the solution was adjusted to 9.0 with 0.5N sodium hydroxide. While maintaining the pH at about 8.5, 1 ml of anhydrous dioxane containing 20 μl of bromoacetyl bromide was added dropwise. Then, the pH of the reaction product was adjusted to about 4.5 with 6N hydrochloric acid, and dialyzed against distilled water at 4 ° C. for 48 hours to obtain an aqueous solution containing activated lipopolysaccharide. After adding 40 mg of the purified polypeptide obtained by the method of Example A-1 (b) to this aqueous solution, the pH of the solution
Was maintained at room temperature for about 4.5 hours and allowed to react at room temperature for 48 hours. Then, the reaction product was purified in the same manner as in Example B-1, concentrated, and lyophilized to obtain a solid complex of the polypeptide and lipopolysaccharide. Then, this solid was added as a stabilizer at 1% so that the final concentration was 100 ng / ml.
(W / v) Dissolved in distilled water containing purified gelatin, and sterilized by a conventional method to obtain a liquid preparation.

[Procedure Amendment 10]

[Document name to be amended] Statement

[Correction target item name] 0105

[Correction method] Change

[Correction content]

[0105]

Example B-4 Sublingual agent Average molecular weight about 200,000
2 g of Dalton's purified erucinan was uniformly dissolved in 400 ml of distilled water, the pH of the solution was adjusted to 10.7 with a 1N sodium hydroxide aqueous solution, and 3 g of cyanogen bromide was gradually added while keeping the pH of the aqueous solution near 10.0. In addition, the reaction was carried out for 1 hour. The pH of the reaction product was adjusted to 5.0 with 1N hydrochloric acid, and then dialyzed against cold water for 10 hours while maintaining this pH to obtain an aqueous solution containing activated erucinan.
Next, 20 mg of the purified polypeptide obtained by the method of Example A-1 (b) was added to this aqueous solution, and the mixture was allowed to stand at room temperature for 24 hours for reaction. After the reaction was completed, 3 volumes of acetone was added to the reaction product, and the formed precipitate was collected and was added to 0.01M.
It is dissolved in an acetate buffer (pH 5.0), insoluble matter is removed by centrifugation, and then 0.01 M acetate buffer (pH
It was subjected to CM-Sephadex column chromatography which had been equilibrated with 5.0), and a fraction containing a complex of the polypeptide and erucinan was collected from the non-adsorbed fraction. Then, by a conventional method, this fraction was sterilized by filtration, concentrated, freeze-dried, pulverized, and then anhydrous crystals α- manufactured by Hayashibara Co., Ltd.
Maltose powder "Fine Tose" was uniformly mixed, and the mixture was tabletted by a conventional method to give a tablet containing 100 ng of the polypeptide per product (200 mg).

[Procedure Amendment 11]

[Document name to be amended] Statement

[Name of item to be corrected] 0112

[Correction method] Change

[Correction content]

[0112]

[Experimental Example 4 Animal Experiment] In this experimental example, the complex of the polypeptide of the present invention and a specific carbohydrate is treated or prevented for pollinosis by actually administering the desensitizing agent of the present invention to an experimental animal. It is to prove that it is effective.

[Procedure Amendment 12]

[Document name to be amended] Statement

[Name of item to be corrected] 0113

[Correction method] Change

[Correction content]

[0113]

[Experimental Example 4-1 Preventive effect] Female BA aged 10 to 12 weeks
In one group consisting of 6 LB / c mice, the desensitizing agent obtained by the method of Example B-1 was converted into the amount of polypeptide to be 1
It was intraperitoneally injected once a week at a rate of μg / animal for 3 weeks. One week after the final administration of the desensitizing agent, 1 μg of the purified polypeptide obtained by the method of Example A-1 (b) as an antigen and 4 mg of aluminum hydroxide as an immunostimulant were included in order to induce pollinosis. Physiological saline 0.2m
1 was injected as above. Then, immediately before the antigen administration and one week after the antigen administration, the blood of the mouse was collected, and the amount of immunoglobulin E antibody, immunoglobulin G antibody and immunoglobulin M antibody specific to the polypeptide contained therein was examined. .

[Procedure Amendment 13]

[Document name to be amended] Statement

[Correction target item name] 0117

[Correction method] Change

[Correction content]

[0117]

[Experimental example 4-2 Therapeutic effect of parenteral administration] A group of 6 female BALB / c mice aged 10 to 12 weeks was prepared.
As an antigen, 0.2 ml of physiological saline containing 1 μg of the purified polypeptide obtained by the method of Example A-1 (b) and 4 mg of aluminum hydroxide as an immunostimulant, once a week,
It was injected intraperitoneally for 3 weeks to induce hay fever. One week after the final administration of the antigen, the desensitizing agent obtained by the method of Example B-1 was converted into a polypeptide amount of 10
Injection was performed once a week at a dose of 0 ng / animal for 3 weeks in the same manner as above. One week after the final administration of the desensitizing agent, the antigen alone was again administered in the same manner as above to perform the treatment for inducing the production of immunoglobulin E antibody. Immediately before the administration of the desensitizing agent, one week after the final administration of the desensitizing agent, and one week after the re-induction of immunoglobulin E antibody, blood was collected from the mouse, and an immunogen specific to the polypeptide contained therein was collected. The amounts of the globulin E antibody, the immunoglobulin G antibody and the immunoglobulin M antibody were examined by the same method as in Experimental Example 4-1.

[Procedure Amendment 14]

[Document name to be amended] Statement

[Correction target item name] 0121

[Correction method] Change

[Correction content]

[0121]

[Experimental example 4-3 Therapeutic effect by oral administration] 10 to 1
A physiological saline containing 1 μg of the purified polypeptide obtained by the method of Example A-1 (b) as an antigen and 4 mg of aluminum hydroxide as an immunostimulant in a group of 6 2-week-old female BALB / c mice. 0.2 ml of water once a week for 3
It was injected intraperitoneally for a week to induce hay fever.
One week after the final administration of the antigen, the sublingual preparation obtained by the method of Example B-4 was pulverized and orally administered at a dose of 10 μg / animal once a week for 3 weeks in terms of the amount of polypeptide. Was administered. One week after the final administration of the sublingual agent, blood was collected from the mice, and the amounts of immunoglobulin A antibody, immunoglobulin G antibody and immunoglobulin E antibody specific to the polypeptide contained therein were examined.

[Procedure Amendment 15]

[Document name to be amended] Statement

[Name of item to be corrected] 0122

[Correction method] Change

[Correction content]

Separately, instead of the sublingual agent, Example A-1
A system was provided for oral administration of a solid mixture containing the purified polypeptide obtained by the method of (b) and the purified lipopolysaccharide derived from Salmonella in a ratio of 1:15 by weight, and treated in the same manner as above. Served as a control. The amount of immunoglobulin A antibody and immunoglobulin G antibody specific to the polypeptide can be determined by the method described by Earl Meolini et al.
Immunological Methods, Volume 6, 355-36
According to the EIA method reported on page 2 (1975),
In addition, immunoglobulin E specific for the polypeptide
The amount of antibody was measured by the same method as in Experimental Example 4-1,
Each is represented by the average antibody titer of 6 mice. The results are shown in Table 23.

[Procedure Amendment 16]

[Document name to be amended] Statement

[Name of item to be corrected] 0126

[Correction method] Change

[Correction content]

[0126]

[Experimental example 4-4 Acute toxicity test] 20
The therapeutic / preventive desensitizing agents obtained by the methods of Examples B-1 to B-4 were orally or intraperitoneally administered to the mice on the day. As a result, these desensitizing agents showed LD5 of 1,000,000 ng / kg or more by any route of administration.
It turned out to be 0. This indicates that the desensitizing agent of the present invention containing a complex of the polypeptide and a specific sugar can be safely compounded and used in a drug to be administered to mammals including humans.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Office reference number FI technical display location // (C12P 21/02 C12R 1:19)

Claims (17)

[Claims] 1. A polypeptide having the following physicochemical properties. (1) Molecular weight 55,000 ± 5,000 daltons (SDS-polyacrylamide gel electrophoresis) (2) Isoelectric point 9.0 ± 0.5 (isoelectric focusing) (3) N-terminal amino acid sequence N-terminal Ala-Met-Lys-Phe-Ile-A
la-Pro-Met-Ala-Phe-Val-Al
a-Met-Gln-Leu-Ile-Ile-Met
It has an amino acid sequence represented by -Ala-. (4) Ultraviolet absorption spectrum It has an absorption maximum near 280 nm. (5) Solubility in solvent Soluble in water, physiological saline and phosphate buffer. (6) Biological action It binds to immunoglobulin E antibody collected from the blood of patients with hay fever. Causes hay fever. (7) Stability Deactivates when heated at 100 ° C. for 10 minutes in an aqueous solution (pH 7.2). Even if left in an aqueous solution (pH 7.2) at 4 ° C. for 1 month, it is not substantially deactivated. 2. The polypeptide according to claim 1, which has the amino acid sequence from the N-terminus shown in the following chemical formula 1 or an amino acid sequence homologous thereto. [Chemical 1] 3. The polypeptide according to claim 1, which is derived from cedar pollen. 4. A D encoding the polypeptide of claim 1.
A method for producing a polypeptide, which comprises culturing a transformant containing NA in a culture medium and collecting a polypeptide produced from the culture. 5. The method for producing the polypeptide according to claim 4, wherein the polypeptide has the amino acid sequence of Chemical formula 1 or an amino acid sequence homologous thereto. 6. The method for producing the polypeptide according to claim 4 or 5, wherein the DNA has a base sequence from the 5'end shown in Chemical formula 2 below. [Chemical 2] 7. The DNA has a base sequence in which one or more of the bases in Chemical formula 2 are replaced with other bases without changing the amino acid sequence shown in Chemical formula 1, based on the degeneracy of the genetic code. Item 7. A method for producing the polypeptide according to Item 4, 5 or 6. 8. The method for producing the polypeptide according to claim 4, 5, 6 or 7, wherein the DNA is derived from cedar pollen or male cedar flowers. 9. The method for producing the polypeptide according to claim 4, 5, 6, 7 or 8, wherein the host in the transformant is a microorganism. 10. The vector in the transformant is pKK.
223-3, The manufacturing method of the polypeptide of Claim 4, 5, 6, 7, 8 or 9. 11. A polypeptide produced from a culture is selected from salting out, dialysis, filtration, concentration, centrifugation, gel filtration chromatography, ion exchange chromatography, affinity chromatography, electrophoresis and isoelectric focusing. Collecting by one or more methods,
A method for producing the polypeptide according to 5, 6, 7, 8, 9 or 10. 12. A desensitizing agent containing the polypeptide according to claim 1 as an active ingredient. 13. The desensitizing agent according to claim 12, wherein the polypeptide has the amino acid sequence shown in Chemical formula 1 or an amino acid sequence homologous thereto. 14. The polypeptide is obtained by culturing a transformant containing the DNA encoding the polypeptide of claim 1 in a culture medium and collecting the culture.
The desensitizing agent according to 2 or 13. 15. The desensitizing agent according to claim 12, 13 or 14, wherein a carbohydrate is covalently bound to the polypeptide. 16. The desensitizing agent according to claim 15, wherein the sugar essentially has maltotriose as a repeating unit. 17. The method according to claim 13, 14, 15 or 16 containing serum albumin and / or gelatin as a stabilizer.
The desensitizing agent described in.