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New cedar pollen protein

A fir tree and pollen technology, applied in the field of new fir tree pollen protein, can solve the problem of ineffective allergen immunotherapy and achieve the effect of effective desensitization treatment

Active Publication Date: 2019-02-05
TAIHO PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are patient populations for whom allergen immunotherapy does not respond

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0100] The rough refinement of embodiment 1 novel fir tree pollen protein

[0101]

[0102] 50 mL of an extraction buffer (20 mM Tris-HCl, pH 9.0) was added to 2 g of cedar tree pollen, and ultrasonication was performed for 10 minutes. Next, after mixing at room temperature for 10 minutes, it was centrifuged (10,000 g, 10 minutes) to obtain a supernatant. This supernatant was again subjected to ultracentrifugation (50,000 g, 60 minutes), and the supernatant was recovered. To 17 mL of this supernatant, an equal amount of Q sepharose high performance resin (GE Healthscare) suspension equilibrated with an extraction buffer (20 mM Tris-HCl, pH 9.0) was added, followed by shaking at room temperature for 30 minutes. After the supernatant was removed by centrifugation (1,000 g, 10 minutes), the volume was adjusted to 50 mL with extraction buffer (20 mM Tris-HCl, pH 9.0), mixed by inversion, and the resin was washed by centrifugation again. Next, 10 mL of an extraction buffer (20 ...

Embodiment 2

[0105] Embodiment 2 Preparation of refined recombinant protein

[0106]

[0107] 2 g of cedar tree male flowers (anthers) frozen and pulverized in liquid nitrogen were added to a 50 mL tube containing 25 mL of Plant RNA Isolation Reagent (Invitrogen), mixed, and left to stand at room temperature for 5 minutes.

[0108] After centrifugation at 4° C. (2600×g, 5 minutes), the upper layer was filtered through a mesh with a mesh size of 100 μm by decantation. Next, 1 / 5 times the amount of 5M NaCl and 3 / 5 times the amount of chloroform were added to the recovered filtrate. After centrifugation at 4° C. (2600×g, 30 minutes), 20 mL of the upper layer was recovered, and 9 / 10 times the amount of isopropanol was added and stirred. After standing still at room temperature for 10 minutes, the RNA was precipitated by centrifugation at 4°C (2600 x g, 30 minutes). After removing the supernatant, 10 mL of 75% ethanol was added to the RNA pellet for washing. Then, after centrifugation at 4...

Embodiment 3

[0116] Example 3 Allergen Activity Confirmation of Refined Recombinant Protein

[0117] (i) Lymphocyte proliferation response in patients with cedar tree hay fever

[0118] Peripheral blood mononuclear cells were isolated from 30 mL of peripheral blood obtained from cedar hay fever patients (ImmunoCAP score ≥ 2) to become 1.11×10 6 cells / mL were suspended in RPMI-1640 medium containing 10% non-activated autologous plasma. Inoculate 180 μL of the prepared cell suspension and 20 μL of the recombinant protein solution (concentration 100 μg / mL) of the present invention on a 96-well plate (2×10 5 cells / well). At 37°C, 5% CO 2 After culturing for 3 days under the condition, add 3 For H-thymidine, the uptake amount after 16 hours was measured as radioactivity, thereby evaluating cell proliferation. Calculate the time when adding purified recombinant protein 3 The value obtained by dividing the intake of H-thymidine by the intake when it was not added was used as a stimulation i...

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Abstract

Provided are a new cedar pollen protein and a drug using this cedar pollen protein, the drug being for diagnosis, prevention, and treatment of cedar-pollen-triggered allergy diseases. The cedar pollenprotein is selected from (a) to (c), where: (a) is a protein which comprises an amino-acid sequence depicted in SEQ ID NO: 2; (b) is a protein which comprises the amino-acid sequence depicted in SEQID NO: 2, in which one or several amino acid residues are replaced, deleted, or added, and which has a cedar pollen allergenicity; and (c) is a protein which comprises amino-acid sequence having 90% or greater identity with the amino-acid sequence depicted in SEQ ID NO: 2, and which has a cedar pollen allergenicity.

Description

technical field [0001] The present invention relates to a novel protein from fir tree pollen and the application of the protein. Background technique [0002] Hay fever is caused by inhalation of pollen scattered in the air, and is an allergic disease that manifests symptoms such as allergic conjunctivitis, rhinitis, skin inflammation, and asthma such as itchy and painful eyes. Many plants are known as the cause of hay fever, but cedar tree (sugi, cedar) hay fever (an allergic disease caused by cedar tree pollen) is the most frequent hay fever in Japan. In the report of 2003, it is estimated that the incidence rate of cedar hay fever was 19.4% in 2001 (non-patent document 1), and it is estimated that at least 20 million people are afflicted by cedar hay fever, and it is not reasonable to call it a national disease. Pass. [0003] One of the more serious problems related to cedar tree hay fever can be cited the year-by-year increase in the amount of cedar tree pollen scatte...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09A61K38/02A61P37/08C07K14/415C07K16/16C12P21/08
CPCA61K38/02C07K16/16C07K14/415C12N15/09A61P37/08G01N33/6854G01N2800/24A61K38/00
Inventor 长田年弘田中悠喜
Owner TAIHO PHARMA CO LTD
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