Cloning and sequencing of allergens of dermatophagoides (house dust mite)

a technology of dermatophagoides and allergens, which is applied in the field of cloning and sequencing of allergens of dermatophagoides (house dust mites), can solve the problems of considerable effort and achieve the effect of desensitizing the individual and reducing sensitivity or desensitizing

Inactive Publication Date: 2006-01-12
MERCK PATENT GMBH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0006]Dermatophagoides DNA, proteins and peptides of the present invention are useful for diagnostic and therapeutic purposes. For example, isolated D. farinae protein or peptide can be used to detect sensitivity in an individual to house dust mites and can be used to treat sensitivity (reduce sensitivity or desensitize) in an individual, to whom therapeutically effective quantities of the D. farinae protein or peptide is administered. For example, isolated D. farinae protein allergen, such as Der f I or Der f II can be administered periodically, using standard techniques, to an individual in order to desensitize the individual. Alternatively, a peptide w

Problems solved by technology

Two species, D. pteronyssinus and D. farinae, predominate and, as a result, considerable e

Method used

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  • Cloning and sequencing of allergens of dermatophagoides (house dust mite)
  • Cloning and sequencing of allergens of dermatophagoides (house dust mite)
  • Cloning and sequencing of allergens of dermatophagoides (house dust mite)

Examples

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example 1

Isolation and Characterization of cDNA Coding for Der f I

Materials and Methods

Dermatophagoides Farinae Culture

[0046] Mites were purchased from Commonwealth Serum Laboratories, Parkville, Australia.

Construction of the D. farinae cDNA λgt11 Library

[0047] Polyadenylated mRNA was isolated from live D. farinae mites and cDNA was synthesized by the RNase H method (Gubler, V. and B. J. Hoffman, Gene, 25:263-269 (1983)) using a kit (Amersham International, Bucks.). After the addition of EcoRI linkers (New England Biolabs, Beverly, Mass.) the cDNA was ligated to alkaline phosphatase treated λgt11 arms (Promega, Madison, Wis.). The ligated DNA was packaged and plated in E. coli Y1090 (r-) to produce a library of 2×104 recombinants.

Isolation of Der f I cDNA Clones from the D. farinae cDNA λgt11 Library

[0048] Screening of the library was performed by hybridization with two probes comprising the two Der p I cDNA BamHI fragments 1-348 and 349-857 generated by BamHI digestion of a deri...

example 2

Isolation and Characterization of cDNA Coding for Der f II

Materials and Methods

Amino Acid Sequence Analysis

Preparation of λgt11 D. farinae cDNA Ligations

[0059] D. farinae was purchased from Commonwealth Serum Laboratories, Parkville, Australia, and used to prepare mRNA (polyadenylated RNA) as described (Stewart, G. A. and W. R. Thomas, Int. Arch. Allergy Appl Immunol., 83:384-389 (1987)). The mRNA was suspended at approximately 0.5 μg / μl and 5 μg used to prepare cDNA by the RNase H method. (Gubler, U. and Hoffman, B. J., Gene. 25:263-269 (1983)) using a kit (Amersham International, Bucks). EcoRI linkers (Amersham, GGAATTCC) were attached according to the method described by Huynh et al., Constructing and screening cDNA libraries in gt10 and gt11, In: Glover, DNA Cloning vol. A practical approach pp. 47-78 IRL Press, Oxford (1985)). The DNA was then digested with EcoRI and recovered from an agarose gel purification by electrophoresis into a DEAE membrane (Schleicher and Schue...

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Abstract

Isolated DNA encoding allergens of Dermatophagoides (house dust mites) particularly of the species Dermatophagoides farinae and Dermatophagolides pteronyssinus, which are protein allergens or peptides which include at least one epitope of the protein allergen. In particular, DNA encoding two major D. farinae allergens, Der f I and Der f II and DNA encoding a D. pteronyssinus allergen, Der p I. In addition, the proteins or peptides encoded by the isolated DNA, their use as diagnostic and therapeutic reagents and methods of diagnosing and treating sensitivity to house dust mite allergens.

Description

RELATED APPLICATION [0001] This application is a Continuation-In-Part of U.S. Ser. No. 458,642 entitled “Cloning of Mite Allergens”, by Wayne Robert Thomas, Geoffrey Alexander, Stewart Keven James Turner and Richard John Simpson (filed Feb. 13, 1990). The teachings of application U.S. Ser. No. 45.8,642 are incorporated herein by reference.FUNDING [0002] Work described herein was funded by grants from the Princess Margaret Children's Medical Research Foundation, the Australian Health and Medical Research Council and the Asthma Foundation of Australia. BACKGROUND [0003] Recent reports have documented the importance of responses to the Group I and Group II allergens in house dust mite allergy. For example, it has been documented that over 60% of patients have at least 50% of their anti-mite antibodies directed towards these proteins (Lind, P. et al., Allergy, 39:259-274 (1984); van der Zee, J. S. et al., J. Allergy Clin. Immunol., 81:884-896 (1988)). It is possible that children show a...

Claims

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Application Information

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IPC IPC(8): C12P21/00A61K39/35A61K38/00A61K39/36A61K49/00A61P37/08C07H21/04C07K14/00C07K14/435C07K14/54C12N15/09C12N15/12C12P21/02G01N33/53G01N33/569
CPCA61K38/00C07K14/5403C07K14/43531A61P37/08C12N15/11
Inventor THOMAS, WAYNECHUA, KAW-YAN
Owner MERCK PATENT GMBH
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