JP2832885B2 - Cedar pollen allergen - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a novel cedar pollen allergen, particularly, Ala-
The present invention relates to a cedar pollen allergen having the sequence of Ile-Asn-Ile-Phe-Asn-.

[0002]

2. Description of the Related Art Japanese cedar hay fever is an allergic disease caused by pollen flying at the time of flowering of Japanese cedar. recent years,
In Japan, the number of cedar pollinosis patients is gradually increasing with the increase in the area of plantation of cedar, and even though it is a seasonal occurrence, it is not negligible in public health. Conventionally, cedar hay fever has been treated with steroid hormones, sodium cromoglycate, or the like, but these are all symptomatic treatments, and only temporarily relieve the symptoms of the patient. On the other hand, attempts have been made to cure cedar pollinosis by administering cedar pollen allergen itself, which is a causative substance of cedar pollinosis, to reduce sensitization.

[0003] However, there is no known cedar pollen allergen that can be suitably used in such desensitization therapy.

[0004]

DISCLOSURE OF THE INVENTION The present inventors have focused on new cedar pollen allergens for the purpose of preventing or treating cedar pollinosis, and have conducted intensive studies with the aim of establishing a new desensitizer. In the process, Japanese Patent Application Laid-Open No. 1-156926 has already filed an application for a desensitizer in which a cedar pollen allergen and a saccharide are covalently bonded.

[0005] However, although this desensitizer has been found to relieve the symptoms of most patients, it has been found that some patients are still difficult to desensitize with this desensitizer. Therefore, further research has been continued on the allergen itself contained in cedar pollen, and search for a new allergen and intensive research have been continued for its use.

[0006]

DISCLOSURE OF THE INVENTION The present invention has been made to solve the above-mentioned problems, and is directed to a novel cedar pollen allergen, in particular, Ala-I as a partial amino acid sequence.
The pollen allergen having the sequence of le-Asn-Ile-Phe-Asn- was found to be present in a small amount in pollen, and this new pollen allergen was disclosed in JP-A-1-156.
No. 926 discloses a partial amino acid sequence of As
It has been found that the pollen allergen having the sequence of p-Asn-Pro-Ile-Asp-Ser- is present in an amount of about 1/50 to 1/5, and the use thereof has been established and the present invention has been established. completed.

The cedar pollen allergen referred to in the present invention is an allergen prepared from pollen of Japanese cedar (Omotesugi, Urasugi), and its structure is represented by Ala-Ile-Asn-Ile-Phe-Asn as a partial amino acid sequence.
And more preferably Ala-Ile-A
sn-Ile-Phe-Asn-Val-Glu-Ly
Allergens having the s-Tyr- sequence are preferred.

[0008] The saccharides referred to in the present invention include, for example, starch, amylose, dextran, polysucrose, pullulan, ercinan, curdlan, gum arabic, tragacanth gum, guar gum, xanthan gum, carrageenan,
Pectin, cellulose, glucomannan, chitosan,
Various simple or complex polysaccharides such as lipopolysaccharide, polysaccharide derivatives thereof, or partial hydrolysates of these polysaccharides can be freely used, and the average molecular weight thereof is usually 500 to 10,000,000, especially 10 , 0
Those in the range of 00 to 1,000,000 are desirable.

Above all, the use of a water-soluble neutral polysaccharide having a maltotriose residue as a repeating unit, such as pullulan, ercinan or a partial hydrolyzate thereof, can be achieved by covalently binding to a cedar pollen allergen. While preventing anaphylaxis due to allergens,
This is advantageous because a more effective cedar pollinosis reducing sensitizer can be easily produced.

The use of complex polysaccharides such as lipopolysaccharides derived from microorganisms such as Escherichia coli, Salmonella, and Serratia or partial hydrolysates thereof can be achieved by covalently binding to cedar pollen allergen to bind to mucosa and the like. Because of its excellent properties, it is convenient as a transdermal or transmucosal desensitizer.

As the covalent bonding method referred to in the present invention, various methods capable of covalently bonding the cedar pollen allergen of the present invention to carbohydrates can be employed, for example, a diazo method, a peptide method, an alkylation method, a cross-linking method, an amide bond. Method, periodate oxidation method, SS bond method and the like. Examples of the diazo method include p-aminobenzyl, p-aminobenzoyl, m-aminobenzyl, m-aminobenzoyl, m-aminoanisole, m-aminobenzyloxymethyl, and 3- (p-aminobenzyl).
Aminophenoxy) -2-hydroxypropionyl group,
An activated sugar obtained by introducing an aromatic amino group such as a 3- (p-amino-m-methylanilino) -5-chlorotriazinyl group by a known method may be reacted with cedar pollen allergen. .

In the peptide method, for example, a carbohydrate having a carboxyl group is converted into a derivative such as azide, acid chloride, carbodiimide, isocyanate or the like, and an activated carbohydrate such as a cyanogen bromide-activated carbohydrate. What is necessary is just to react with cedar pollen allergen.

As an alkylation method, for example, a saccharide halogenated alkyl derivative into which a chloroacetyl group, a bromoacetyl group, an iodoacetyl group, a triazinyl halide group or the like has been introduced may be reacted with a cedar pollen allergen.

As the crosslinking method, for example, glutaraldehyde, glyoxal, succinaldehyde, hexamethylene diisocyanate, toluene-2,4-diisocyanate, bisazobenzidine, coexistence of cedar pollen allergen and carbohydrate A crosslinking reagent such as N, N'-ethylenebismaleimide may be reacted.

As an amide bonding method, for example, a saccharide having an amino group is converted to, for example, bromoacetyl bromide,
The activated saccharide obtained by reacting with a haloacyl halide such as chlorobutyryl chloride, fluoropropionyl fluoride and iodovaleryl iodide may be reacted with cedar pollen allergen.

The weight ratio of the cedar pollen allergen to the saccharide during the reaction is usually selected from the range of 1: 0.001 to 1,000, preferably 1: 0.01 to 100. The reaction conditions are
The resulting cedar pollen allergen carbohydrate conjugates significantly reduce the ability to produce anaphylaxis and allergy-induced immunoglobulin E antibodies without substantially reducing the natural immunoglobulin G and immunoglobulin M antibody production ability of the cedar pollen allergen. Any conditions can be used as long as the temperature is about 0 to 100 ° C., the pH is about 3 to 12, and the pH is about 0.1 to 5
A range of 0 hours is chosen.

The cedar pollen allergen saccharide conjugate thus produced is usually filtered, washed, centrifuged,
After separation and purification by methods such as salting out, dialysis, adsorption / desorption, gel filtration, ion exchange chromatography, affinity chromatography, and electrophoresis, concentrate and collect in the form of a solution or syrup, or dry and powder. Collect to obtain a desensitizer for cedar pollinosis.

The desensitizing agent is used as it is or, if necessary, with the addition of stabilizers, preservatives, adjuvants, excipients and the like, and is advantageously used as a prophylactic or therapeutic agent for cedar pollinosis. it can.

The desensitizer thus produced significantly increases the production of immunoglobulin G and immunoglobulin M antibodies specific to the allergen, as compared with conventionally used cedar pollen allergens. In addition, the production of an immunoglobulin E antibody specific to the allergen causing allergy or anaphylaxis can be significantly reduced. In addition, the amount of immunoglobulin E antibody specific to the allergen can be significantly reduced by administration to cedar pollinosis patients.

When the cedar pollen allergen of the present invention is covalently bonded to a saccharide, there is no loss of adsorption to a glass container, a metal device or the like, and the stability is good. The desensitizer of the present invention in which a cedar pollen allergen and a carbohydrate are covalently bonded can be administered without fear of anaphylaxis, and the time required for desensitization is about 1 hour.
It has the advantage that it can be reduced to / 3 to 1/200. Further, such a desensitizing agent is, for example, a lyophilized injection,
Manufactured as injections such as solution injections, intradermal, subcutaneous,
About 0.01 to 10 per adult once in muscle, intraperitoneal, etc.
The purpose of desensitization can be achieved by administering an amount selected from the range of 000 ng once or twice a week for about 1 to 12 months.

Further, the above desensitizing agent is also produced as a transdermal or transdermal drug such as a troche, sublingual tablet, eye drops, intranasal spray, poultice, cream, lotion, etc. The purpose of the desensitization can be advantageously achieved by appropriately selecting the dose, the administration frequency and the like.

In the case of this topical administration method, the above-mentioned desensitizing agent can also inhibit the binding between immunoglobulin E and allergen bound to the tissue of a Japanese cedar pollinosis patient. Is immediate and topical,
Immediate relief of the patient's distress.

The desensitizer prepared using the cedar pollen allergen of the present invention is advantageously used not only as a preventive or therapeutic agent for cedar pollinosis but also as a preventive or therapeutic agent for cypress pollinosis. it can. Further, such a desensitizing agent is disclosed in
By using in combination with the desensitizing agent disclosed in JP-A-56926, it is also possible to advantageously carry out the application to a wider range of patients. Hereinafter, the present invention will be described in detail by experiments.

[0024]

[Experiment I-1] <Preparation of cedar pollen allergen> A cedar pollen collected from a cedar (Omotesugi) produced in Chiba Prefecture was added with about a 15-fold amount by weight of a 0.125 M sodium hydrogencarbonate aqueous solution (pH 8.0).
, And extracted slowly with stirring at 4 ° C. for 1 hour, followed by centrifugation to obtain a supernatant. The residue was again extracted and centrifuged. These supernatants were salted out with ammonium sulfate (80% saturation), the salted-out product was dialyzed and filtered, and the filtrate was applied to a DEAE-Sephadex column to collect a non-adsorbed fraction, which was applied to a CM-Sephadex column. , Phosphate buffer (pH 7.0)
, And then applied to a Mono S column, and separated and eluted with a sodium chloride concentration gradient in the presence of Tris-HCl buffer (pH 7.0) to strongly bind to the immunoglobulin E antibody from human hay fever patients and the anti-cedar pollen allergen mouse antibody. A purified cedar pollen allergen solution exhibiting affinity was obtained at a yield of about 0.001% per solid based on the raw material cedar pollen.

In the method for preparing an allergen of the present invention, the cedar pollen allergen disclosed in JP-A-1-156926 was eluted at a sodium chloride concentration of about 0.25 M upon separation and elution from a Mono S column. On the other hand, the cedar pollen allergen of the present invention was eluted at a sodium chloride concentration of about 0.40 M, and the yield was
It was about 1/20 of the cedar pollen allergen disclosed in Japanese Patent No. 56926.

The product was measured by SDS-PAGE and found to have a molecular weight of about 40,000 ± 5,000 and an isoelectric point of about 9.5.

Using this product, The Journal of Biological Chemistry (The Journal)
of Biological Chemistr
y), 256, 7990-7997 (1981)
According to the content described in (1), the protein was decomposed with a gas-phase protein sequencer, identified by high-performance liquid chromatography, and the partial amino acid sequence was examined. Alanine-isorosine-asparagine-isoleucine-phenylalanine-asparagine-valine- It was found to have a glutamic acid-lysine-tyrosine- sequence.
All of the amino acid residues constituting this sequence are in the L-form with optical isomerism. In this specification, Ala-Ile-Asn
-Ile-Phe-Asn-Val-Glu-Lys-
It may be indicated by Tyr- and an abbreviation symbol.

[0028]

[Experiment I-2] <Preparation of covalent bond of cedar pollen allergen and pullulan> 2 w / v of pullulan (average molecular weight 200,000)
2 ml of 1.7 w / v% cyanuric chloride solution was added to 100 ml of a 100% aqueous solution, and kept at 5 ° C. or lower in ice water while maintaining the pH at 7.0 with a 5% aqueous sodium carbonate solution.
After reacting for an hour, dialyzed overnight at 4 ° C. water while maintaining the pH to obtain an activated pullulan solution. To 30 ml of the activated pullulan solution, 40 ml of the purified cedar pollen allergen solution obtained by the method of Experiment I-1 (containing about 1 mg of allergen per ml) was added, and the mixture was maintained at pH 7.0 and 37 ° C. for 5 hours with stirring. The mixture was left at 5 ° C. overnight, 6 g of glycine was added thereto, and the mixture was kept for 10 hours while stirring. This was dialyzed against a 0.01 M acetate buffer (pH 5.0), then applied to a CM-Sephadex column, and the non-adsorbed fraction was subjected to microfiltration to obtain a covalently bound allergen pullulan. The yield was about 60% per allergen protein.

Unlike the cedar pollen allergen itself, this product has no loss of adsorption to glass containers and metal appliances, has good stability and is easy to handle.

[0030]

[Experiment I-3] <Test of administration to animals>

[Experiment I-3-1] <Prophylactic agent test> Allergen pullulan covalently conjugate obtained by the method of Experiment I-2 using mice (BALB / c, 10 to 12 weeks old, female, 6 mice per group) 1 as an allergen
0.2 ml of physiological saline containing μg was intraperitoneally administered once a week for 3 weeks, and one week later, 1 μg of cedar pollen allergen obtained by the method of Experiment I-1 and aluminum hydroxide as an adjuvant 0.2 ml of physiological saline containing 4 mg of the mixture was similarly administered.

The amounts of immunoglobulin G, immunoglobulin M antibody and immunoglobulin E antibody specific to cedar pollen allergen were measured immediately before administration of the mixture of allergen and aluminum hydroxide and one week after the end of administration. As a control, the same procedure was carried out except that a mixture of 1 μg of cedar pollen allergen prepared by the method of Experiment I-1 and 40 μg of pullulan used in Experiment I-2 was used instead of the covalent conjugate of allergen pullulan. The amounts of the immunoglobulin G and the immunoglobulin M antibody are determined by referring to The Journal of Biochemistry (The Journal).
of Biochemistry), Vol. 92, No. 14,
The amount of immunoglobulin E antibody is determined by Life Science (Life S) based on the antibody titer by enzyme immunosorbent assay (EIA) reported on pages 13 to 1424 (1982).
Science, Vol. 8, Part II, pp. 813-820 (1969). The results are shown in Table 1.

[0032]

[Table 1]

As is evident from the results in Table 1, the covalent bond of cedar pollen allergen and pullulan of the present invention, unlike a mere mixture thereof, is suitable as a desensitizer for preventing cedar pollinosis. is there.

[0034]

[Experiment I-3-2] <Therapeutic agent test> Using 1 μg of cedar pollen allergen obtained by the method of Experiment I-1, using a mouse (BALB / c, 10 to 12 weeks old, female, 6 mice per group) 0.2 ml of physiological saline containing a mixture of 4 mg of aluminum hydroxide as an adjuvant was administered intraperitoneally once a week for three weeks, and two weeks later, the allergen pullulan obtained by the method of Experiment I-2 was used. 0.2 ml of physiological saline containing 1 μg of the covalent conjugate as an allergen was similarly administered three times a week for three weeks.

Further, re-induction of immunoglobulin E antibody production was similarly performed using the allergen aluminum hydroxide mixture.

Immunoglobulin G, immunoglobulin M
The amount of the antibody and the amount of the immunoglobulin E antibody were determined immediately before the administration of the covalent conjugate of the allergen and one week after the administration.
Further, one week after re-induction of the immunoglobulin E antibody, blood was collected and measured.

In the control experiment, as in Experiment I-3-1, a mixture of cedar pollen allergen and pullulan was used instead of the covalent conjugate of allergen pullulan. The results are shown in Table 2.

[0038]

[Table 2]

As is evident from Table 2, the covalent conjugate of cedar pollen allergen and pullulan, unlike a mere mixture thereof, is suitable as a desensitizer for treating cedar pollinosis.

In addition, a guinea pig, rat or mouse, which has been sensitized in advance by administration of an allergen aluminum hydroxide mixture, and which has been confirmed to produce immunoglobulin E, is subjected to oral administration of a physiological saline solution containing the above-mentioned covalently bound allergen pullulan conjugate. Administered intranasally, intranasally, intradermally or subcutaneously,
One hour later, cedar pollen allergen was given in the oral cavity, nasal cavity,
When administered intradermally or subcutaneously, no allergic reaction that should occur was observed.

As described above, the covalently bonded cedar pollen allergen and saccharide of the present invention exerts a high desensitizing effect without fear of anaphylaxis. Here, there is utility of the cedar pollen allergen of the present invention.

[0042]

[Experiment II-1] <Preparation of covalent bond of cedar pollen allergen and lipopolysaccharide> Lipopolysaccharide 10 derived from Escherichia coli
mg to 1 ml of 10 mM calcium phosphate solution containing 1 mg
Add 60 μl of 00 mM sodium periodate and add
After reacting for 0 minutes to cleave the lipopolysaccharide sugar chain, 1M-glycine hydrochloride buffer (pH 4.4) at 4 ° C.
Dialyzed overnight to remove excess sodium periodate,
A 0.1 M sodium bicarbonate buffer solution was added thereto, and p
H was adjusted to about 9.5.

1 ml of a phosphate buffer (pH 9.5)
In addition, purified cedar pollen allergen 1 obtained by the method of Experiment I-1
0 mg was dissolved therein, and the above lipopolysaccharide solution was mixed with the solution to form a Schiff base.

Further, sodium borohydride was added to complete the complex formation reaction, and then Sephadex G-1
A fraction containing a covalent allergen lipopolysaccharide was collected and subjected to microfiltration to obtain a covalent allergen lipopolysaccharide. The yield was about 40% per allergen protein.

Unlike the cedar pollen allergen itself, the product has no loss of adsorption to a glass container or the like, has good stability, and is easy to handle.

[0046]

[Experiment II-2] <Test of administration to animals>

<Experiment II-2-1><Therapeutic agent test> Using mice (BALB / c, 10 to 12 weeks old, female, 6 mice per group), sharing allergen lipopolysaccharide obtained by the method of Experiment II-1 1 ml of physiological saline containing 10 μg of the conjugate as an allergen was orally administered to mice three times a week for three weeks, and one week after the administration, blood was collected and immunoglobulin A specific to cedar pollen allergen was added. The amounts of immunoglobulin G antibody and immunoglobulin E antibody were measured.

A control experiment was carried out in the same manner as the above experiment except that a mixture of an allergen and a lipopolysaccharide was used instead of the allergen lipopolysaccharide covalent bond. The amounts of the immunoglobulin A and the immunoglobulin G antibody can be determined by the Journal of Immunological Methods (Journal of Immunolo).
gical Methods), Volume 6, 355-36.
Based on the antibody titer by the EIA method reported on page 2 (1975), the amount of immunoglobulin E antibody was determined by the experiment I-3-1.
And the antibody titer by the PCA reaction used in the above. Result is,
It is shown in Table 3.

[0048]

[Table 3]

As is evident from the results in Table 3, the covalent lipopolysaccharide conjugate prepared using the cedar pollen allergen of the present invention, unlike the mere mixture thereof, prevents or prevents cedar pollinosis. It is suitable as a desensitizing agent to be treated.

Further, using a mouse which has been sensitized in advance by administration of an allergen aluminum hydroxide mixture and which has been confirmed to produce immunoglobulin E, a physiological saline containing the covalently bound lipopolysaccharide conjugate is intraorally and intracellularly. When the cedar pollen allergen was sprayed into the oral cavity and the nasal cavity 30 minutes after spraying into the nasal cavity, no allergic reaction which should originally occur was observed.

In general, the covalent bond between a cedar pollen allergen such as an allergen lipopolysaccharide covalent bond and a complex polysaccharide is the same as the covalent bond between a cedar pollen allergen such as an allergen pullulan covalent bond and a simple polysaccharide. In comparison, it has superior mucosal binding properties and a long time to stay locally, so it has a high absorption rate in mucous membranes and is more effective as a transdermal and transmucosal desensitizer such as oral and nasal cavities Demonstrate.

Hereinafter, examples and reference examples of the present invention will be described.

[0053]

【Example】

<Preparation of cedar pollen allergen> Purified cedar pollen allergen solution was prepared using cedar pollen collected from cedar (Urasugi) produced in Akita Prefecture in accordance with the method of Experiment I-1. In a yield of about 0.001% per solid. The product was measured by SDS-PAGE and found to have a molecular weight of about 40,000 ± 5,000 and an isoelectric point of about 9.5.

When the partial amino acid sequence of this product was examined in accordance with the method of Experiment I-1. And the result was the same as the partial amino acid sequence of the pollen allergen.

[0055]

[Reference Example 1] <Desensitizer> Pullulan (average molecular weight about 140,000)
5 g was dissolved in 400 ml of water, the pH was adjusted to 10.7 with a 1N sodium hydroxide solution, and 3 g of cyanogen bromide was gradually added while maintaining the pH, followed by a reaction for 1 hour. Then 1
After adjusting the pH to 5.0 with N-hydrochloric acid, the solution was dialyzed against cold water while maintaining this pH to prepare an activated pullulan solution.

To this activated pullulan solution, 200 ml of a pollen allergen solution derived from uragi prepared by the method of Example was added and reacted at room temperature for 24 hours. After the reaction, a three-fold amount of acetone was added to the reaction solution, and the precipitate was collected.
It is dissolved in a 01M acetate buffer (pH 5.0), centrifuged to remove insolubles, the supernatant is applied to a CM-Sephadex column, the non-adsorbed fraction is subjected to microfiltration, filled in an ampoule, and shared with allergen pullulan. A liquid desensitizer containing the conjugate was obtained. The yield was about 70% per allergen protein.

This product has no fear of anaphylaxis,
Since it exhibits a high desensitizing effect, it is suitable as a preventive or therapeutic agent for cedar pollinosis. Also, unlike the case of cedar pollen allergen, this product has no loss of adsorption to glass containers and metal appliances, has good stability and is easy to handle.

[0058]

[Reference Example 2] Pullulan partially hydrolyzed product (average molecular weight: about 1)
(000) 52 g of dimethylformamide 110 ml
Dissolve while heating to room temperature, then cool to room temperature and add
To the mixture were added 1.0 g of 4-nitrobenzoyl chloride with stirring, and the mixture was kept at room temperature for 17 hours. To this was added twice the volume of n-propyl alcohol, and the precipitate was collected and dissolved in dimethylformamide. This operation was repeated three times, and the resulting precipitate was dissolved in 100 ml of a 5% w / v sodium hydrosulfite aqueous solution.
C. for 30 minutes, decolorize with activated charcoal, and double the volume of n
-What was precipitated with propyl alcohol was dissolved in water,
Dialysis against tap water overnight. Cool this solution to below 2 ° C,
With stirring, hydrochloric acid is added to a final concentration of about 0.1 N, then sodium nitrite is added to a concentration of about 1 w / v%, and the mixture is reacted for 30 minutes. Activated pullulan partial hydrolyzate was obtained by dialysis for an hour.

To this activated pullulan partial hydrolyzate solution was added 20 ml of pollen allergen solution derived from Japanese cedar, obtained by the method of Experiment I-1.
After maintaining the mixture at 4 ° C. for 2 hours while stirring, the mixture was purified in the same manner as in Example 1 and packed in an ampoule to obtain a liquid desensitizer containing a covalently bound partial hydrolyzate of allergen pullulan. . The yield is about 60% per allergen protein
Met. This product is suitable as a prophylactic or therapeutic agent for cedar pollinosis as in the case of the examples, has no loss of adsorption to glass containers and metal appliances, has good stability, and is easy to handle.

[0060]

Reference Example 3 Elsinane (average molecular weight of about 200,00
0) Dissolve 10 g in 200 ml distilled water while heating,
Then, the mixture was cooled to room temperature, 5 g of hexamethylenediamine was added thereto, and the pH was adjusted to 1 with a 1N sodium hydroxide solution.
1.0. 5 g of cyanogen bromide was added thereto while maintaining the pH at 20 ° C. or lower in ice water and maintaining the pH, and the mixture was reacted for 30 minutes with stirring, and subsequently dialyzed with distilled water at 4 ° C. for 1 hour to obtain an activated ercinan solution. did. To this activated elsinan solution, 2 ml of a 25% w / v glutaraldehyde solution and 60 ml of a cedar pollen allergen solution derived from Japanese cedar prepared by the method of Experiment I-1 were added, and 10 ml of a 1 M acetate buffer (pH 5.0) was further added. The binding reaction was carried out with stirring at 4 ° C. for 24 hours, then glycine was added to a concentration of 1 M, kept at room temperature for 24 hours, and further centrifuged.
The supernatant was subjected to gel filtration to collect a fraction of covalently bound allergen ercinan. This is concentrated, microfiltered, bottled,
After freeze-drying and cap sealing, a solid desensitizer containing a covalently attached allergen ercinan was obtained. The yield was about 50% per allergen protein. This product is suitable as a prophylactic or therapeutic agent for cedar pollinosis as in the case of the examples, and its handling is easy.

[0061]

REFERENCE EXAMPLE 4 1-w / v% carboxymethylcellulose (average molecular weight: about 20,000) aqueous solution
After adding 2 g of ethyl-3- (3-dimethylaminopropyl) carbodiimide-methiodide, maintaining the pH at 4.0 with 1N-hydrochloric acid, and reacting at room temperature for 2 hours with stirring, the mixture was dialyzed against distilled water overnight. Then, an activated carboxymethyl cellulose solution was prepared. To this activated carboxymethylcellulose solution was added 50 ml of a ragweed-derived pollen allergen solution prepared by the method of Example, and the mixture was allowed to react at room temperature with stirring at pH 4.5 while stirring. The mixture was purified in the same manner as described above and packed in an ampoule to obtain a liquid desensitizer containing a covalently bonded allergen carboxymethylcellulose. The yield was about 30% per allergen protein. This product has immunoglobulin G specific for cedar pollen allergen and immunoglobulin M antibody production amount slightly lower than those of covalent conjugates of allergen pullulan and allergen ercinan, but immunoglobulins specific to the allergen Since it does not produce E antibody, it can be used as a prophylactic or therapeutic agent for cedar pollinosis, and its handling is easy.

[0062]

[Reference Example 5] 100 mg of lipopolysaccharide derived from Salmonella was subjected to 25 m of 50% saturated sodium acetate at about 4 ° C.
l, and the pH is adjusted using 0.5 N sodium hydroxide.
9.0, and 20 µl of bromoacetyl bromide
Mixed solution of water and 1 ml of anhydrous dioxane (pH about 8.5)
Was slowly added. Next, the pH was adjusted to about 4.5 using 6N-hydrochloric acid, and dialyzed against water at 4 ° C. for 5 days to prepare an activated lipopolysaccharide solution. To this activated lipopolysaccharide solution, 40 ml of a pollen allergen solution derived from uragi prepared by the method of Example was added, and the pH was adjusted to 4.
After reacting at 25 ° C. for 2 days while stirring while maintaining at 5, the mixture was purified in the same manner as in Reference Example 2 and filled in an ampoule to obtain a liquid desensitizer containing a covalently bonded allergen lipopolysaccharide. . The yield was about 35% per allergen protein. This product is suitable as a prophylactic or therapeutic agent for Japanese cedar pollinosis, and its handling is easy. In addition, this product, which uses lipopolysaccharide as a carbohydrate, has excellent mucosal binding properties and a long time to stay locally. It exerts a better effect as a sensitizer.

[0063]

As is evident from the above description, the novel cedar pollen allergen of the present invention, in particular, the partial amino acid sequence of Ala-Ile-Asn-Ile-Phe
A desensitizer in which a pollen allergen having an -Asn- sequence and a carbohydrate are covalently bonded to each other significantly increases the production of immunoglobulin G and immunoglobulin M antibodies specific to cedar pollen allergen, And the production of immunoglobulin E antibody specific to the allergen causing anaphylaxis can be significantly reduced, so that administration can be carried out without fear of anaphylaxis, and the period required for desensitization is about 1 hour.
/ 3 to 1/200.

The cedar pollen allergen of the present invention can be used for immunoglobulin E bound to the tissue of a cedar pollinosis patient.
It has been possible to provide a desensitizing agent that can block the antigen-antibody reaction between lipase and pollen allergens and immediately alleviate local pain in patients. Such a desensitizing agent can be advantageously used as a topical administration agent such as a transdermal or transdermal drug, and has great industrial significance.

──────────────────────────────────────────────────続 き Continued on the front page (58) Field surveyed (Int.Cl. ⁶ , DB name) A61K 39/36 BIOTECHABS (STN) CA (STN) MEDLINE (STN)

Claims (4)

(57) [Claims] 1. A partial amino acid sequence of Ala-Il
A cedar pollen allergen having the sequence of e-Asn-Ile-Phe-Asn-. 2. A partial amino acid sequence of Ala-Il
e-Asn-Ile-Phe-Asn-Val-Glu
The cedar pollen allergen according to claim 1, which has a sequence of -Lys-Tyr-. 3. The molecular weight measured by SDS-PAGE is about 40,000 ± 5,000 and the isoelectric point is about 9.9.
The cedar pollen allergen according to claim 1 or 2, which is 5. 4. The cedar pollen allergen according to claim 1, 2 or 3, wherein the cedar pollen allergen is prepared from pollen of Nippon Sugi.