JP2701005B2 - Desensitizer - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a desensitizer containing a new Japanese cedar pollen allergen derived from Nippon Sugi as an active ingredient.

[0002]

2. Description of the Related Art Japanese cedar pollinosis is an allergic disease caused by pollen scattered in the air at the time of flowering of Japanese cedar. In Japan, the number of cedar pollinosis patients has recently increased as the area of planted cedar has increased. Despite the seasonal onset,
Now, it is one of the problems that cannot be ignored in public health.

[0003] Conventionally, symptomatic treatment is the main treatment for Japanese cedar pollinosis, and anti-inflammatory and anti-allergic agents such as steroid hormones and sodium cromoglycate have been frequently used. These anti-inflammatory and anti-allergic agents only temporarily alleviate the allergic symptoms caused by cedar pollinosis and do not cure the cedar pollinosis itself.In addition, steroid hormones may be severe in some patients. May cause adverse side effects.

On the other hand, as one of the attempts to diagnose cedar pollinosis and to cure cedar pollinosis, diagnosis and treatment of hyposensitization by administering cedar pollen allergen itself to a cedar pollinosis patient have been receiving attention. I have. However, in the current desensitization therapy or the like, an extremely crude cedar pollen allergen obtained by extracting Japanese cedar pollen in an aqueous medium and appropriately diluting the same is still administered to a patient. Such an extract has the disadvantage that the composition and the medicinal effect are liable to fluctuate greatly depending on the pollen of the raw material, and a certain desensitizing effect cannot be obtained even with the same extraction method.

[0005] Therefore, in order to accurately diagnose cedar pollinosis and to treat cedar pollinosis safely and effectively, all cedar pollen allergens are isolated and their properties and properties are clarified. It is an urgent need in the world to do so.

[0006]

DISCLOSURE OF THE INVENTION In view of such circumstances, an object of the present invention is to provide a desensitizing agent containing a cedar pollen allergen as an active ingredient, which has been partially elucidated in the amino acid sequence.

[0007]

Means for Solving the Problems The present invention solves the above-mentioned problems by using a desensitizer containing as an active ingredient a Japanese cedar-derived cedar pollen allergen having an amino acid sequence represented by SEQ ID NO: 1 in the sequence listing at the N-terminus. Is what you do.

[0008]

The cedar pollen allergen of the present invention elucidated to a part of the amino acid sequence exerts a stable desensitizing effect on cedar pollen allergen when administered to mammals including humans.

Such a cedar pollen allergen can be relatively easily produced in a desired amount by using pollen of Nippon Sugi as a raw material.

Hereinafter, the present invention will be described with reference to Examples and the like. The cedar pollen allergen of the present invention has an N-terminal amino acid sequence represented by SEQ ID NO: 1 in the sequence listing, usually an amino acid sequence represented by SEQ ID NO: 2 in the sequence listing. have.
The cedar pollen allergen of the present invention is a Japanese cedar (scientific name: "Cryptomeria japonica (Cry)" referred to by the common name of "Omotesugi" or "Urasugi" depending on the place of production.
ptomeria japonica))). That is, the operation of suspending the pollen of Nippon Sugi or a male flower containing the pollen in an aqueous medium and extracting it for about one hour while maintaining the temperature at about 4 ° C. is repeated once or twice or more. The extract is then subjected to salting-out, dialysis, filtration, concentration, gel filtration chromatography, ion exchange chromatography, affinity chromatography, high performance liquid chromatography, gel electrophoresis, isoelectric focusing, etc. to purify biologically active substances. Appropriate combinations of conventional methods in the art for isolation are applied, and a fraction having a molecular weight of about 50,000 daltons is collected by SDS-polyacrylamide gel electrophoresis. Specifically, for example, the extract obtained as above is salted out with ammonium sulfate, and column chromatography using DEAE-Sephadex and CM-Sephadex is applied to the precipitate containing the protein component. After purification, it is further fractionated by Mono S column chromatography and subjected to SDS-polyacrylamide gel electrophoresis in the absence of a reducing agent to a molecular weight of about 50,000 daltons, usually from 49,000 to 53,000.
The fraction showing 000 daltons is collected. When high performance liquid chromatography is combined with Mono S column chromatography, the desired fraction can be obtained more easily.

The cedar pollen allergen of the present invention thus obtained has a property of causing cedar pollinosis in mammals including humans. Due to this property, the cedar pollen allergen of the present invention exerts a remarkable desensitizing effect on cedar pollen allergen when administered to mammals similarly to a conventionally known cedar pollen extract, and diagnoses and treats cedar pollinosis Has widespread use as a desensitizing agent for

When used in combination with a desensitizer for treating cedar pollinosis, the cedar pollen allergen of the present invention is desirably formed into a complex with a saccharide.
The cedar pollen allergen of the present invention has a molecular weight of about 500 to 1
0,000,000 daltons, preferably about 10,000
00 to 1,000,000 dalton starch, amylose, dextran, polysucrose, pullulan, ercinan, curdlan, gum arabic, tragacanth gum,
For simple carbohydrates, complex carbohydrates or derivatives or partial hydrolysates thereof including guar gum, xanthan gum, carrageenan, pectin, cellulose, glucomannan, chitosan and lipopolysaccharide, for example, diazo method, peptide method, alkylation method, cross-linking It can be obtained by a covalent bond by a method, a periodate oxidation method, a disulfide bond method, or the like. The reaction method for covalently binding a saccharide to an antigenic protein is described in detail in Japanese Patent Publication No. 57-8090.

When the cedar pollen allergen of the present invention is used in the form of such a complex, not only can it be possible to substantially eliminate undesired side effects such as anaphylactic shock that may accompany the administration of the allergen, but also to use a glass container,
Since it does not easily adhere to a metal container or the like, handling is further facilitated. These effects are even more remarkable in a complex with a water-soluble neutral polysaccharide having maltotriose as a repeating unit, such as pullulan and ercinan. Lipopolysaccharide and its partial hydrolyzate derived from microorganisms such as Escherichia coli, Salmonella, and Serratia have a property of enhancing the binding to the mucosa in mammals when covalently bonded to the cedar pollen allergen of the present invention. Therefore, such a complex with a carbohydrate is extremely useful in a desensitizer administered transdermally or transmucosally.

Next, production and physicochemical properties of the cedar pollen allergen of the present invention will be described based on examples.

[0015]

[Example 1 Production of cedar pollen allergen and physicochemical properties]

[0016]

Example 1-1 Production of Japanese cedar pollen allergen Japanese cedar pollen collected from a Japanese cedar, Chiba Prefecture, was about 15 times the weight of a 0.125 M aqueous sodium hydrogen carbonate solution (pH 8.0).
0) and extracted at 4 ° C for 1 hour with gentle stirring. The above extraction operation is applied again to the residue obtained by centrifuging the extract, the obtained supernatant and the first supernatant are pooled, ammonium sulfate is added to 80% saturation, and the mixture is added at 4 ° C. overnight. After standing, the protein components were salted out.

The precipitate in the salted out product was collected,
M Tris-HCl buffer (pH 7.8), dialyzed against fresh buffer, filtered, and DE previously equilibrated with 0.05 M Tris-HCl buffer (pH 7.8)
The solution was passed through an AE-Sephadex column, and a non-adsorbed fraction was collected. This fraction was previously purified with a 0.01 M acetate buffer (pH
After loading the column on the CM-Sephadex equilibrated in 5.0), the column was washed by passing 0.01 M acetate buffer (pH 5.0), and then the column was washed with 0.1 M containing 0.3 M sodium chloride. After passing through a 1 M phosphate buffer (pH 7.0), a fraction containing a protein component was collected. Then, this fraction is
Load the Mono S column equilibrated with 0 mM Tris-HCl buffer (pH 7.8)
When a tris-hydrochloric acid buffer (pH 7.0) having a concentration gradient rising from M to 100 mM was passed, the solution containing the cedar pollen allergen of the present invention showed a yield of about 0.02% per solid cedar pollen solid content. Was obtained.

[0018]

Example 1-2 <Physicochemical properties of cedar pollen allergen>

[0019]

Example 1-2 (a) <Biological action> A part of the cedar pollen allergen obtained in Example 1-1 was taken, and an immunoglobulin E antibody collected from the blood of a hay fever patient and a mouse-derived substance were taken according to a conventional method. When the affinity for the monoclonal anti-cedar pollen allergen antibody was examined, it showed strong affinity for all. This suggests that the cedar pollen allergen of the present invention is one of the causative substances of cedar pollinosis, that is, induces cedar pollinosis in mammals including humans.

[0020]

Example 1-2 (b) <Molecular weight> A part of the cedar pollen allergen obtained in Example 1-1 was taken,
In the absence of a reducing agent, U.K.Laemli, Nature, Vol. 227, pp. 680-685 (1970)
And SDS-polyacrylamide gel electrophoresis according to the method reported in
A major band was observed around 00 Dalton.

[0021]

Example 1-2 (c) <Isoelectric point> A part of the cedar pollen allergen obtained in Example 1-1 was subjected to an isoelectric focusing gel "Ampholine Pug" manufactured by Pharmacia according to a conventional method. The plate was subjected to isoelectric focusing, showing an isoelectric point near 8.8.

[0022]

Example 1-2 (d) <N-terminal amino acid sequence> A part of the cedar pollen allergen obtained in Example 1-1 was taken,
"Journal of Biological Chemistry", 256, 7,990-7,997 (1
981) according to the method described in
When the amino acids generated by decomposition with a sequencer were identified by high performance liquid chromatography, the cedar pollen allergen of the present invention was found to have an amino acid sequence represented by SEQ ID NO: 1 at the N-terminal, specifically SEQ ID NO: 2 in the sequence table. Has the amino acid sequence shown in FIG. However, in SEQ ID NO: 2, “Xaa” represents serine, cysteine, threonine or histidine.

The cedar pollen allergen derived from Nippon Sugi having the above-mentioned physicochemical properties has not yet been known and is considered to be a novel substance.

[0024]

Example 2 <Production of cedar pollen allergen> In the same manner as in Example 1, cedar pollen collected from uragi from Akita Prefecture was extracted, and cedar pollen allergen contained in the extract was purified and fractionated. Cedar pollen allergen showing strong affinity for immunoglobulin E antibody collected from hay fever patients and monoclonal anti-cedar pollen allergen antibody derived from mouse, is approximately 0.1% per raw cedar pollen solids.
Obtained in a yield of 015%.

A part of the cedar pollen allergen was taken and tested in the same manner as in Example 1-2. The molecular weight was determined to be about 50,000 daltons by SDS-polyacrylamide gel electrophoresis, and the isoelectric focusing electrophoresis was performed. Showed an isoelectric point near 8.8. Further, in the same manner as in Example 1-2 (d),
When the amino acid sequence from the N-terminal to the 20th amino acid was examined, it was consistent with the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing.

[0026]

As described above, the present invention is based on the discovery of a new cedar pollen allergen derived from Nippon Sugi having a specific amino acid sequence at the N-terminus.
The present invention provides a cedar pollen allergen in which the amino acid sequence has been partially elucidated, and such cedar pollen allergen is administered to mammals including humans.
It exerts a stable desensitizing effect on cedar pollen allergens. Therefore, the cedar pollen allergen of the present invention
It can be advantageously used as a desensitizing agent for diagnosing and treating cedar pollinosis, and is also useful as a standard substance of a desensitizing agent for diagnosing and treating cedar pollinosis.

[0027]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 6 Sequence type: amino acid Topology: Linear Sequence type: Protein Fragment type: N-terminal fragment Sequence Asp Asn Pro Ile Asp Ser 15

SEQ ID NO: 2 Sequence length: 20 Sequence type: amino acid Topology: linear Sequence type: protein Fragment type: N-terminal fragment Sequence Asp Asn Pro Ile Asp Ser Xaa Trp Arg Gly Asp Ser Asn Trp Ala Gln Asn 15 10 15 Arg Met Lys 20

Claims (5)

(57) [Claims] 1. A desensitizing agent comprising as an active ingredient a Japanese cedar-derived cedar pollen allergen having an amino acid sequence represented by SEQ ID NO: 1 in the sequence listing at the N-terminus. 2. The cedar pollen allergen has an amino acid sequence represented by SEQ ID NO: 2 (provided that “X
“aa” shall represent serine, cysteine, threonine or histidine. 2. The desensitizing agent according to claim 1, wherein 3. The cedar pollen allergen has a molecular weight of about 50,000 daltons when measured by SDS-polyacrylamide gel electrophoresis and an isoelectric point near 8.8 when measured by isoelectric focusing. The desensitizing agent according to claim 1 or 2, characterized in that: 4. The desensitizing agent according to claim 1, wherein the cedar pollen allergen is obtained by extracting pollen of Nippon Sugi in an aqueous medium and collecting from the extract. 5. The desensitizing agent according to claim 4, wherein the cedar pollen allergen is obtained from the extract by a step including a fractionation step by Mono S column chromatography.