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

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 greater degree of reactivity (Thompson, P. J. et al., Immunology, 64:311-314 (1988)). Allergy to mites of the genus Dermatophagoides (D.) is associated with conditions such as asthma, rhinitis and ectopic dermatitis. Two species, D. pteronyssinus and D. farinae, predominate and, as a result, considerable effort has been expended in trying to identify the allergens produced by these two species. D. pteronyssinus mites are the most common Dermatophagoides species in house dust in Western Europe and Australia. The species D. farinae predominates in other countries, such as, North America and Japan (Wharton, G. W., J. Medical Entom, 12:577-621 (1976)). It has long been recognized that allergy to mites of this genus is associated with diseases such as asthma, rhinitis and atopic dermatitis. It is still not clear what allergens produced by these mites are responsible for the allergic response and associated conditions.

SUMMARY OF THE INVENTION

[0004] The present invention relates to isolated DNA which encodes a protein allergen of Dermatophagoides (D.) house dust mite) or a peptide which includes at least one epitope of a protein allergen of a house dust mite of the genus Dermatophagoides. It particularly relates to DNA encoding major allergens of the species D. farinae, designated Der f I and Der f II, or portions of these major allergens (i.e., peptides which include at least one epitope of Der f I or of Der f II). It also particularly relates to DNA encoding major allergens of D. pteronyssinus, designated Der p I and Der p II, or portions of these major allergens (i.e., peptides which include at least one epitope of Der p I or of Der p II.

[0005] The present invention further relates to proteins and peptides encoded by the isolated Dermatophagoides (e.g., D. farinae, D. pteronyssinus) DNA. Peptides of the present invention include at least one epitope of a D. farinae allergen (e.g., at least one epitope of Der f I or of Der f II) or at least one epitope of a D. pteronyssinus allergen (e.g., at least one epitope of Der p I or of Der p II). It also relates to antibodies specific for D. farinae proteins or peptides and to antibodies specific for D. pteronyssinus proteins or peptides.

[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 which includes at least one epitope of Der f I or of Der f II can be administered for this purpose. Isolated D. pteronyssinus protein allergen, such as Der p I or Der p II, can be administered as described for Der f I or Der f II. Similarly a peptide which includes at least one Der p I epitope or at least one Der p II epitope can be administered for this purpose. A combination of these proteins or peptides (e.g., Der f I and Der f II; Der p I and Der p II; or a mixture of both Der f and Der p proteins) can also be administered. The use of such isolated proteins or peptides provides a means of desensitizing individuals to important house dust mite allergens.

BRIEF DESCRIPTION OF THE DRAWINGS

[0007] FIG. 1 is a restriction map of the cDNA insert of clone .lamda.gt11 f 1, including a schematic representation of the strategy of DNA sequencing. Arrows indicate directions in which sequences were read.

[0008] FIG. 2 is the nucleotide sequence and the predicted amino acid sequence of cDNA .lamda.gt11 f 1. Numbers above are nucleotide positions; numbers to the left are amino acid positions. Positive amino acid residue numbers correspond to the sequence of the mature excreted Der f I beginning with threonine. Negative sequence numbers refer to the signal peptide and the proenzyme regions of Der f I. The arrows indicate the beginning of the proenzyme sequence and the mature Der f I, respectively. The underlined residues -81 to -78 make up the proposed cleavage site for the proenzyme formation, while the underlined residues 53-55 represent a potential N-glycosylation site. The termination TGA codon and the adjacent polyadenylation signal are also underlined. Amino acid residues 1-28 correspond to a known tryptic peptide sequence determined by conventional amino acid sequencing analysis.

[0009] FIG. 3 is a composite alignment of the amino acid sequences of the mature Der p I and Der f I proteins. The numbering above the sequence refers to Der p I. The asterisk denotes the gap that was introduced for maximal alignment. The symbol (.) is used to indicate that the amino acid residue of Der f I at that position is identical to the corresponding amino acid residue of Der p I. The arrows indicate those residues making up the active site of Der p I and Der f I.

[0010] FIG. 4 is a comparison of the amino acid sequence in the pre- and pro-peptide regions of Der f I with those of rat cathepsin H. rat cathepsin L, papain, aleurain, CP1, CP2, rat cathepsin B, CTLA-2, MCP, Der p I and actinidin. Gaps, denoted by dashes, were added for maximal alignment. Double asterisks denote conserved amino acid residues which are shared by greater than 80% of the proenzymes; single asterisks show residues which are conserved in greater than 55% of the sequences. The symbol (.) is used to denote semiconserved equivalent amino acids which are shared by greater than 90% of the proenzyme regions.

[0011] FIG. 5 is a hydrophilicity plot of the Der p I mature protein and a hydrophilicity plot of the Der f I mature protein produced using the Hopp-Woods algorithm computed with the Mac Vector Sequence Analysis Software (IBI, New Haven) using a 6 residue window. Positive values indicate relative hydrophilicity and negative values indicating relative hydrophobicity.

[0012] FIG. 6 is the nucleotide sequence and the predicted amino acid sequence of Der f II cDNA. Numbers to the right are nucleotide positions and numbers above are amino acid residues. The stop (TAA) signal is underlined. The first 8 nucleotides are from the oligonucleotide primer used to generate the cDNA, based on the Der p II sequence.

[0013] FIG. 7 is the nucleotide sequence and predicted amino acid sequence of cDNA .lamda.gt11 p1(13T). Numbers to the right are nucleotides and numbers above are amino acid positions. Positive amino acids correspond to the sequence of mature excreted Der p I beginning with threonine.

[0014] FIG. 8 is a restriction map of Der f II cDNA, which was generated by computer from the sequence data. A map of Der p II similarly generated is shown for comparison. There are few common restriction enzyme sites conserved. Sites marked with an asterisk were introduced by cloning procedures.

[0015] FIG. 9 shows the alignment of Der f II and Der p II cDNA sequences. Numbers to the right are nucleotide position and numbers above and amino acid residues. The top line gives the Der p II nucleotide sequence and the second the Der p II amino acid residue's. The next two lines show differences of Der f II to these sequences.

[0016] FIG. 10 is a hydrophilicity plot of Der f II and Der p II using the Hopp-Woods algorithm computed with the Mac Vector Sequence Analysis Software (IBI, New Haven) using a 6-residue window.

DETAILED DESCRIPTION OF THE INVENTION

[0017] The present invention relates to a nucleotide sequence coding for an allergen from the house dust mite Dermatophagoides and to the encoded Dermatophagoides protein or peptide which includes at least one epitope of the Dermatophagoides allergen. It particularly relates to a nucleotide sequence capable of expression in an appropriate host of a major allergen of D. farinae, such as Der f I or Der f II, or of a peptide which includes at least one epitope of Der f I or of Der f II. It also particularly relates to a nucleotide sequence capable of expression in an appropriate host of a major allergen of D. pteronyssinus, such as Der p I or Der p II, or of a peptide which includes at least one epitope of Der p I or of Der p II. The Dermatophagoides nucleotide sequence is useful as a probe for identifying additional nucleotide sequences which hybridize to it and encode other mite allergens, particularly D. farinae or D. pteronyssinus allergens. Further, the present invention relates to nucleotide sequences which hybridize to a D. farinae protein-encoding nucleotide sequence or a D. pteronyssinus protein-encoding nucleotide sequence but which encode a protein from another species or type of house dust mite, such as D. microceras (e.g., Der m I and Der m II).

[0018] The encoded Dermatophagoides mite allergen or peptide which includes at least one Dermatophagoides (Der f I or Der f II; Der p I or Der p II) epitope can be used for diagnostic purposes (e.g., as an antigen) and for therapeutic purposes (e.g., to desensitize an individual). Alternatively, the encoded house dust mite allergen can be a protein or peptide, such as a D. microceras protein or peptide, which displays the antigenicity of or is cross-reactive with a Der f or a Der p allergen; generally, these have a high degree of amino acid homology.

[0019] Accordingly, the present invention also relates to compositions which include a Dermatophagoides allergen (e.g., Der f I or allergen, Der f II allergen; Der p I or Der p II allergen or other D. allergen cross-reactive therewith) or a peptide which includes at least one epitope of a Dermatophagoides allergen (Der f I, Der f II, Der p I, Der p II or other D. allergen cross-reactive therewith) individually or in combination, and which can be used for therapeutic applications (e.g., desensitization). As is described below, DNA coding for major allergens from house dust mites have been isolated and sequenced. In particular, and as is described in greater detail in the Examples, two cDNA clones, coding, respectively, for Der f I allergen and Der f II allergen, have been isolated and sequenced. The nucleotide sequences of Der p I and Der p II have also been determined. The nucleotide sequence of each of these clones has been compared with that of the homologous allergen from the related mite D. pteronyssinus (Der p I and Der p II, respectively), as has the predicted amino acid sequence of each.

[0020] The following is a description of isolation and sequencing of the two cDNA clones coding for Der f allergens and their comparison with the corresponding D. pteronyssinus allergen and a description of use of the nucleotide sequences and encoded products in a diagnostic or a therapeutic context.

Isolation and Sequence Analysis of Der f I

[0021] A cDNA clone coding for Der f I, a major allergen from the house dust mite D. farinae, has been isolated and sequenced. A restriction map of the cDNA insert of the clone is represented in FIG. 1, as is the strategy of DNA sequencing. This Der f I cDNA clone contains a 1.1-kb cDNA insert encoding a typical signal peptide, a proenzyme region and the mature Der f I protein. The product is 321 amino acid residues: a putative 18 residue signal peptide, an 80 residue proenzyme (pro-peptide) region, and a 223 residue mature enzyme region. The derived molecular weight is 25,191. The nucleotide sequence and the predicted amino acid sequence of the Der f I cDNA are represented in FIG. 2. The deduced amino acid sequence shows significant homology to other cysteine proteases in the pro-region, as well as in the mature protein. Sequence alignment of the mature Der f I protein with the homologous allergen Der p I from the related mite D. pteronyssinus (FIG. 3) revealed a high degree of homology (81%) between the two proteins, as predicted by previous sequencing at the protein level. In particular, the residues comprising the active site of these enzymes were conserved and a potential N-glycosylation site was present at equivalent positions in both mite allergens.

[0022] Conserved cysteine residue pairs (31, 71) and (65, 103), where the numbering refers to Der p I, are apparently involved in disulphide bond formation on the basis of the assumed similarity of the three dimensional structure of Der p I and Der f I to that of papain and actinidin, which also have an additional disulphide bridge. The fifth and final cysteine residue for which there is a homologous cysteine residue in papain and actinidin is the active site cysteine (residue 35 in Der f I). It is not unlikely that the two extra cysteine residues present in Der p I and Der f I may be involved in forming a third disulphide bridge.

[0023] The potential N-glycosylation site in Der p I is also present at the equivalent position in Der f I, with conservation of the crucial first and last residues of the tripeptide site. The degree of glycosylation of Der f I and Der p I has yet to be determined. Carbohydrates, including mannose, galactose, N-acetylglucosamine and N-acetylgalactosamine, have been reported in purified preparations of these mite allergens (Chapman, M. D., J. Immunol., 125:587-592 (1980); Wolden,; S. et al., Int. Arch. Allergy Appl. Immunol., 68:144-151 (1982)).

[0024] Given the degree of homology over the first thirty N-terminal amino acid residues between mature Der p I and Der m I (70%) and mature Der f I and Der m I (97%) with the Der m I residues determined by conventional amino acid sequencing (Platts-Mills TAE et al., In: Mite Allergy, a World-Wide Problem, 27-29 (1988); Lind, P. and N. Horn, In: Mite Allergy, a World-Wide Problem, 30-34 (1988)), it is probable that the full mature Der m I sequence will confirm an overall 70-80% homology between the Group I mite allergens. Der m I is an allergen from D. microceras. High homology between the proenzyme moieties of Der p I and Der f I (91%) over the residues -23 to -1 and the structural analysis of Der f I suggests that the Group I allergens are likely to have N-terminal extension peptides of the mature protein of homologous structure and, at least for the pro-peptide, composition.

[0025] Studies on the fine structure of the design of signal sequences have identified three structurally dissimilar regions so far: a positively charged N-terminal (n) region, a central hydrophobic (h) region and a more polar C-terminal (c) region that seems to define the cleavage site (Von Heijne, G., EMBO J., 3:2315-2323 (1984); Eur. J. Biochem., 133:17-21 (1983); J. Mol. Biol., 184:99-105 (1985)). Analysis of the signal peptide of Der f I revealed that it, too, contained these regions (FIG. 4). The n-region is extremely variable in length and composition, but its net charge does not vary appreciably with the overall length, and has a mean value of about +1.7. The n-region of the Der f I signal peptide, with a length of two residues, has a net charge of +2 contributed by the initiator methionine (which is unformylated and hence positively charged in eukaryotes) and the adjacent lysine (Lys) residue. The h-region of Der f I is enriched with hydrophobic residues, the characteristic feature of this region, with only one hydrophilic residue serine (Ser) present which can be tolerated. The overall amino acid composition of the Der f I c-region is more polar than that of the h-region as is found in signal sequences with the h/c boundary located between residues -6 and -5, which is its mean position in eukaryotes. Thus, the Der f I pre-peptide sequence appears to fulfill the requirements to which a functional signal sequence must conform.

[0026] While the signal sequence of Der f I and other cysteine proteases share structural homology, all being composed of the n,h and c-regions, they are highly variable with respect to overall length and amino acid sequence, as is clear in FIG. 4. However, significant sequence homology has been shown between the pro-regions of cysteine protease precursors (Ishidoh, K. et al., FEBS Letters, 226:33-37 (1987)). Alignment of the proenzyme regions of Der f I and a number of other cysteine proteases (FIG. 4) indicated that these proregions share a number of very conserved residues as well as semi-conserved residues which were present in over half of the sequences. This homology was increased if conservative amino acids such as valine (Val), isoleucine (Ile) and leucine (Leu) (small hydrophobic residues) or arginine (Arg) and Lys (positively charged residues) were regarded as identical. The Der f I proregion possessed six out of the seven highly conserved amino acids and all the residues at sites of conservative changes. The homology at less conserved sites was lower. Homology in the pro-peptide, in particular the highly conserved residues, may be important when considering the function of the pro-peptide in the processing of these enzymes, since it indicates that these sequences probably have structural and functional similarities.

[0027] Highly cross-reactive B cell epitopes on Der f I and Der p I have been demonstrated using anti-bodies present in mouse, rabbit and human sera (Heymann, P. W. et al., J. Immunol. 137:2841-2847 (1986); Platts-Mills, TAE et al., J. Allergy Clin. Immunol., 78:398-407 (1986)). However, species-specific epitopes have also been defined in these systems. Murine monoclonal antibodies bound pre-dominantly to species-specific determinants (Platts-Mills TAE et al., J. Immunol., 139:1479-1484 (1987). Some 40% of rabbit anti-Der p I reactivity was accounted for by epitopes unique to Der p I (Platts-Mills, TAE et al., J. Allergy Clin. Immunol., 78:398-407 (1986)), and some species-specific binding of antibodies from allergic humans was observed, although the majority bind to cross-reactive epitopes (Platts-Mills TAE et al., J. Immunol., 139:1479-1484 (1987).

[0028] The recombinant DNA strategy of gene fragmentation aid expression was used (Greene, W. K. et al., Immunol. (1990)) to define five antigenic regions of recombinant Der p I which contained B cell epitopes recognized by a rabbit anti-Der p I antiserum. Using the technique of immunoabsorption, three of these putative epitopes were shown to be shared with Der f I (located on regions containing amino acid residues 34-47, 60-72 and 166-194) while two appeared to be specific for Der p I (regions 82-99 and 112-140). Differences in the reactivity of these peptides to rabbit anti-D. farinae supported the above division into cross-reactive and species-specific epitopes. The sequence differences shown between the Der p I and the Der f I proteins are primarily located in the N and C terminal regions, as well as in an extended surface loop (residues 85-136) linking the two domains of the enzyme that includes helix D (residues 127-136), as predicted from the secondary and tertiary structures of papain and actinidin (Baker,. E. N. and J. Drenth, In: Biological Macromolecules and Assemblies, Vol. 3, pp. 314-368, John Wiley and Sons, NY. (1987)). The surface location of these residues is supported by the hydrophilicity plots of Der p I and Der f I in FIG. 5, which illustrate the predominantly hydrophilic nature of this region that predicts surface exposure. This region also contains the two species-specific B cell epitopes recognized by the rabbit anti-Der p I serum (see above). Analysis of the sequences in the regions containing the cross-reactive epitopes showed that two of the cross-reactive epitopes (located in regions 34-47 and 60-72) are completely conserved between Der p I and Der f I, while the majority of residues in a third cross-reactive epitope-containing region (residues region 166-194) were conserved.

[0029] Expression of results in production of pre-pro-Der f I protein in E. coli a recombinant protein of greater solubility, stability and antigenicity than that of recombinant Der p I. Protein encoded by Der f I cDNA has been expressed using a pGEX vector and has been shown by radioimmune assay to react with rabbit anti-D. farinae antibodies. The availability of high yields of soluble Der f I allergen and antigenic derivatives will facilitate the development of diagnostic and therapeutic agents and the mapping of B and T cell antigenic determinants.

[0030] With the availability of the complete amino acid sequence of recombinant Der f I, mapping of the epitopes recognized by both the B and T cell compartments of the immune system can be carried out. The use of techniques such as the screening of overlapping synthetic peptides, the use of mono-clonal antibodies and gene fragmentation and expression should enable the identification of both the continuous and topographical epitopes of Der f I. It will be particularly useful to determine whether allergenic (IgE-binding) determinants have common features and are intrinsically different from antigenic (IgG-binding) determinants and whether T cells recognize unique epitopes different from those recognized by B cells. Studies to identify the Der f I epitopes reactive with mite allergic human IgE antibodies and the division of these into determinants cross-reactive with Der p I and determinants unique to Der f I can also be carried out. B cell (and T cell) epitopes'specific for either species can be used to provide useful diagnostic reagents for determining reactivity to the different mite species, while cross-reacting epitopes are candidates for a common immuno-therapeutic agent.

[0031] As described in co-pending application U.S. Ser. No. 458,643, incorporated herein by reference, the molecular cloning of mite allergens resulted in the isolation of a cDNA clone coding for Der p I which contained a 0.8-kb cDNA insert. Sequence analysis revealed that the 222 amino acid residue mature recombinant Der p I protein showed significant homology with a group of cysteine proteases, including actinidin, papain, cathepsin H and cathepsin B.

Isolation and Sequence Analysis of Der f II

[0032] A cDNA clone coding for Der f II, a major allergen from the house dust mite D. farinae, has been isolated and sequenced, as described in Example 2. The nucleotide sequence and the predicted amino acid sequence of the Der f II cDNA are represented in FIG. 6. A restriction map of the cDNA insert of a clone coding for Der f II is represented in FIG. 8.

[0033] FIG. 9 shows the alignment of Der f II and Der p II cDNA sequences.

[0034] The homology of the sequence of Der f II with Der p II (88%) is higher than the 81% homology found with Der p I and Der f I, which is significantly different (p<0.05) using the chi.sup.2 distribution. The reason for this may simply be that the Group I allergens are larger and each residue may be less critical for the structure and function of the molecule. It is known; for example, that assuming they adopt a similar conformation to other cysteine proteases, many of the amino acid differences in Der p I and Der f I lie in residues linking the two domain structures of the molecules. The 6 cysteine molecules are conserved between the group II allergens, suggesting a similar disulphide bonding, although this may be expected, given the high overall homology. Another indication of the conservation of these proteins is that 34/55 of the nucleotide changes of the coding sequence are in the third base of a codon, which usually does not change the amino acid. Residues that may be of importance in the function of the molecule are Ser 57 where all three bases are changed but the amino acid is conserved. A similar phenomenon exists at residue 88, where a complete codon change has conserved a small aliphatic residue. Again, like Der p II, the Der f II cDNA clone does not have a poly A tail, although the 3' non-coding region is rich in adenosine and has two possible polyadenylation signals ATAA. The nucleotides encoding the first four residues are from the PCR primer which was designed from the known homology of Der p II and Der f II from N-terminal amino acid sequencing. A primer based on the C-terminal sequence can now be used to determine these bases, as well as the signal sequence.

Uses of the Subject Allergenic Proteins/Peptides and DNA Encoding Same

[0035] The materials resulting from the work described herein, as well as compositions containing these materials, can be used in methods of diagnosing, treating and preventing allergic responses to mite allergens, particularly to mites of the genus Dermatophagoides, such as D. farinae and other species (e.g., D. pteronyssinus and D. microceras). In addition, the cDNA (or the mRNA from which it was transcribed) can be used to identify other similar sequences. This can be carried out, for example, under conditions of low stringency and those sequences having sufficient homology (generally greater than 40%) can be selected for further assessment using the method described herein. Alternatively, high stringency conditions can be used. In this manner, DNA of the present invention can be used to identify sequences coding for mite allergens having amino acid sequences similar to that of Der f I, Der f II, Der p I or Der p II. Thus, the present invention includes not only Der f and Der p II, but other mite allergens as well (e.g., other mite allergens encoded by DNA which hybridizes to DNA of the present invention).

[0036] Proteins or peptides encoded by the cDNA of the present invention can be used, for example, as "purified" allergens. Such purified allergens are useful in the standardization of allergen extracts or preparations which can be used as reagents for the diagnosis and treatment of allergy to h use dust mites. Through use of the peptides of the present invention, allergen preparations of consistent, well-defined composition and biological activity can be made and administered for therapeutic purposes (e.g., to modify the allergic response of a house dust mite-sensitive individual). Der f I or Der f II peptides or proteins (or modified versions thereof, such as are described below) may, for example, modify B-cell response to Der f I or Der f II, T-cell response to Der f I and Der f II, or both responses. Similarly, Der p I or Der p II proteins or peptides may be used to modify B-cell and/or T-cell response to. Der p I or Der p II. Purified allergens can also be used to study the mechanism of immunotherapy of allergy to house dust mites, particularly to Der f I, Der f II, Der p I and Der p II, and to design modified derivatives or analogues which are more useful in immunotherapy than are the unmodified ("naturally-occurring") peptides.

[0037] In those instances in which there are epitopes which are cross-reactive, such as the three epitopes described herein which are shared by Der f I and Der p I, the area(s) of the molecule which contain the cross-reactive epitopes can be used as common immunotherapeutic peptides to be administered in treating allergy to the two (or more) mite species which share the epitope. For example, the cross-reactive epitopes could be used to induce IgG blocking antibody against both allergens (e.g., Der f I and Der p I allergen). A peptide containing a univalent antibody epitope can be used, rather than the entire molecule, and may prove advantagious because the univalent antibody epitope cannot crosslink mast cells and cause adverse reactions during desensitizing treatments. It is also possible to attach a B cell epitope to a carrier molecule to direct T cell control of allergic responses.

[0038] Alternatively, it may be desirable or necessary to have peptides which are specific to a selected Dermatophagoides allergen. As described herein, two epitopes which are apparently Der p I-specific have been identified. A similar approach can be used to identify other species-specific epitopes (e.g., Der p I or II, Der f I or II. The presence in an individual of antibodies to the species-specific epitopes can be used as a quick serological test to determine which mite species is causing the allergic response. This would make it possible to specifically target therapy provided to an individual to the causative species and, thus, enhance the therapeutic effect.

[0039] Work by others has shown that high doses of allergens generally produce the best results (i.e., best symptom relief). However, many people are unable to tolerate large doses of allergens because of allergic reactions to the allergens. Modification of naturally-occurring allergens can be designed in such a manner that modified peptides or modified, allergens which have the same or enhanced therapeutic properties as the corresponding naturally-occurring allergen but have reduced side effects (especially, anaphylactic reactions) can be produced. These can be, for example, a peptide of the present invention (e.g., one having all or a portion of the amino acid sequence of Der f I or Der f II, Der p I or Der p II). Alternatively, a combination of peptides can be administered. A modified peptide or peptide analogue (e.g., a peptide in which the amino acid sequence has been altered to modify immunogenicity and/or reduce allergenicity or to which a component has been added for the same purpose) can be used for desensitization therapy.

[0040] Administration of the peptides of the present invention to an individual to be desensitized can be carried out using known techniques. A peptide or combination of different peptides can be administered to an individual in a composition which includes, for example, an appropriate buffer, a carrier and/or an adjuvant. Such compositions will generally be administered by injection, inhalation, transdermal application or rectal administration. Using the information now available, it is possible to design a Der f I or Der f II peptide which, when administered to a sensitive individual in sufficient quantities, will modify the individual's allergic response to a Der f I and/or Der f II. This can be done, for example, by examining the structures of Der f I or Der f II, producing peptides to be examined for their ability to influence B-cell and/or T-cell responses in house dust mite-sensitive individuals and selecting appropriate epitopes recognized by the cells. Synthetic amino acid sequences which mimic those of the epitopes and which are capable of down regulating allergic response to Der f I or Der f II allergen can be made. Proteins, peptides or antibodies of the present invention can also be used, in known methods, for detecting and diagnosing allergic response to Der f I or Der f II. For example, this can be done by combining blood obtained from an individual to be assessed for sensitivity to one of these allergens with an isolated allergenic peptide of house dust mite, under conditions appropriate for binding of or stimulating components (e.g., antibodies, T cells, B cells) in the blood with the peptide and determining the extent to which such binding occurs. The Der p I and Der p II proteins or peptides can be used in a similar manner for desensitization and diagnosis of sensitivity. Der f and Der p proteins or peptides can be administered together to treat an individual sensitive to both allergen types.

[0041] It is now also possible to design an agent or a drug capable of blocking or inhibiting the ability of Der f I or Der f II to induce an allergic reaction in house dust mite-sensitive individuals. Such agents could be designed, for example, in such a manner that they would bind to relevant anti-Der f I or anti-Der f II IgEs, thus preventing IgE-allergen binding and subsequent mast cell degranulation. Alternatively, such agents could bind to cellular components of the immune system, resulting in suppression or desensitization of the allergic response to these allergens. A non-restrictive example of this is the use of appropriate B- and T-cell epitope peptide's, or modifications thereof, based on the cDNA/protein structures of the present invention to suppress the allergic response to Der f I or Der f II allergens. This can be carried out by defining the structures of B- and T-cell epitope peptides which affect B- and T-cell function in in vitro studies with blood cells from Der f I or Der f II-sensitive individuals. This can also be applied to Der p I or Der p II, in order to block allergic response to these allergens.

[0042] The cDNA encoding Der f I or Der f II or peptide including at least one epitope can be used to produce additional peptides, using known techniques such as gene cloning. A method of producing a protein or a peptide of the present invention can include, for example, culturing a host cell containing an expression vector which, in turn, contains DNA encoding all or a portion of a selected allergenic protein or peptide (e.g., Der f I, Der f II or a peptide including at least one epitope). Cells are cultured under conditions appropriate for expression of the DNA insert (production of the encoded protein or peptide). The expressed product is then recovered, using known techniques. Alternatively, the allergen or portion thereof can be synthesized using known mechanical or chemical techniques. As used herein, the term protein or peptide refers to proteins or peptides made by any of these techniques. The resulting peptide can, in turn, bee used as described previously.

[0043] DNA to be used in any embodiment of this invention can be cDNA obtained as described herein or, alternatively, can be any oligodeoxynucleotide sequence having all or a portion of the sequence represented in FIGS. 2 and 6, or their functional equivalent. Such oligodeoxynucleotide sequences can be produced chemically or mechanically, using known techniques. A functional equivalent of an oligonucleotide sequence is one which is capable of hybridizing to a complementary oligonucleotide sequence to which the sequence (or corresponding sequence portions) of FIGS. 2 and 6 hybridizes and/or which encodes a product (e.g., a polypeptide or peptide) having the same functional characteristics of the product encoded by the sequence (or corresponding sequence portion) represented in these figures. Whether a functional equivalent must meet one or both criteria will depend on its use (e.g., if it is to be used only as an oligoprobe, it need meet only the first criterion and if it is to be used to produce house dust mite allergen, it need only meet the second criterion).

[0044] The structural information now available (e.g., DNA, protein/peptide sequences) can also be used to identify or define T cell epitope peptides and/or B cell epitope peptides which are of importance in allergic reactions to D. farinae allergens and to elucidate the mediators or mechanisms (e.g., interleukin-2, interleukin-4, gamma interferon) by which these reactions occur. This knowledge should make it-possible to design peptide-based house dust mite therapeutic agents or drugs which can be used to modulate these responses.

[0045] The present invention will now be further illustrated by the following Examples, which are not intended to be limiting in any way.

EXAMPLES

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 .lamda.gt11 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 .lamda.gt11 arms (Promega, Madison, Wis.). The ligated DNA was packaged and plated in E. coli Y1090 (r-) to produce a library of 2.times.10.sup.4 recombinants.

Isolation of Der f I cDNA Clones from the D. farinae cDNA .lamda.gt11 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 derivative of the Der p I cDNA which has had two BamHI restriction sites inserted between amino acid residues -1 and 1 and between residues 116 and 117 by site-directed mutagenesis (Chua, K. Y. et al., J. Exp. Med. 167:175-182 (1988)). The probes were radiolabelled with .sup.32P by nick translation. Phage were plated at 20,000 pfu per 150 mm petri dish and plaques were lifted onto nitrocellulose (Schleicher and Schull, Dassel, FRG), denatured and baked (Maniatis, T. et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1982)). Prehybridizations were performed for 2 hours at 42.degree. C. in 50% formamide/5.times.SSCE/1x Denhardt's/poly C (0.1 mg/ml)/poly U(0.1 mg/ml) with hybridization overnight at 42.degree. C. at 10 cpm/ml. Post hybridization washes consisted of 15 min washes at room temperature with 2.times. sodium chloride citrate (SSC)/0.1% sodium dodecylsulphate (SDS), 0.5.times.SSC/0.1% SDS, 0.1.times.SSC/0.1% SDS successively and a final wash at 50.degree. C. for 30 min in 0.1.times.SSC/1% SDS.

[0049] sIsolation of DNA from .lamda.gt11 f1 cDNA Clones

[0050] Phage DNA from .lamda.gt11 f 1 clones was prepared by a rapid isolation procedure. Clarified phage plate lysate (1 ml) was mixed with 270 .mu.L of 25% wt/vol polyethylene glycol (PEG 6000) in 2.5M NaCl and incubated at room temperature for 15 min. The mixture was then spun for 5 min in a microfuge (Eppendorf, FRG), and the supernatant was removed. The pellet was dissolved in 100 .mu.L of 10 mM Tris/HCl pH8.0 containing 1 mM EDTA and 100 mM NaCl (TE). This DNA preparation was extracted with phenol/TE, the phenol phase was washed with 100 .mu.l TE, the pooled aqueous phases were then extracted another 2 times with phenol/TE, 2 times with Leder phenol (phenol/chloroform/isoamylalcohol; 25:24:1), once with chloroform and the DNA was precipitated by ethanol.

DNA Sequencing

[0051] To obtain clones for DNA sequence analysis, the .lamda.gt11 f1 phage DNA was digested with EcoRI restriction enzyme (Pharmacia, Uppsala, Sweden) and the DNA insert was ligated to EcoRI-digested M13-derived sequencing vectors mp18 and mp19 (Maniatis, T. et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1982)). Transformation was carried out using E. coli TG-1 and sequencing was performed by the dideoxynucleotide chain termination method (Sanger, F. et al., Proc. Natl. Acad. Sci. USA, 74:5463-5467 (1977)) using the Sequenase version 2.0 DNA sequencing kit (U.S.B., Cleveland, Ohio).

Polymerase Chain Reaction (PCR)

[0052] PCR was performed by the Taq DNA polymerase method (Saiki, R. K. et al., Science. 239:487-491 (1988)) using the TaqPaq kit (Biotech International, Bentley, Wash.) and the conditions recommended by the supplier with long of target DNA and 10 pmol of .lamda.gt11 primers (New England BioLabs, Beverly, Mass.).

Results

Isolation of Der f I cDNA Clones

[0053] Two clones expressing the major mite allergen Der f I were isolated from the D. farinae cDNA .lamda.gt11 library by their ability to hybridize with both of the Der p I cDNA probes (nucleotides 1-348 and 349-857). This approach was adopted because amino acid sequencing had shown high homology (80%) between these two allergens (Thomas, W. R. et al., Advances in the Biosciences, 14:139-147 (1989)). Digestion of the .lamda.gt11 f1 clone DNA with EcoRI restriction enzyme to release the cDNA insert produced three Der f I cDNA EcoRI fragments: one approximately 800 bases long and a doublet approximately 150 bases long. The Der f I cDNA insert was also amplified from the phage DNA by the polymerase chain reaction (PCR) resulting in a PCR product of approximately 1.1-kb. Each Der f I cDNA fragment was cloned separately into the M13-derived sequencing vectors mp18 and mp19 and sequenced.

DNA Sequence Analysis

[0054] The nucleotide sequence of the Der f I cDNA was determined using the sequencing strategy shown in FIG. 1. The complete sequence was shown to be 1084 bases long and included a 335-base long 5' proximal end sequence, a coding region for the entire native Der f I protein of 223 amino acids with a derived molecular weight of 25,191 and an 80-base long 3' noncoding region (FIG. 2). The assignment of the threonine residue at position 1 as the NH.sub.2-terminal amino acid of Der f I was based on data obtained by NH.sub.2-terminal amino acid sequencing of the native protein and the predicted amino acid sequence of recombinant Der p I (Chua, K. Y. et al. J. Exit. Med., 167:175-182 (1988)). The predicted amino acid sequence of the Der f I cDNA in the NH.sub.2-terminal region matched completely with that determined at the protein level (FIG. 2).

[0055] The complete mature protein is coded by a single open reading frame terminating at the TGA stop codon at nucleotide position 1007-1009. The first ATG codon at nucleotide position 42-44 is presumed to be the translation initiation site since the subsequent sequence codes for a typical signal peptide sequence.

Amino Acid Sequence Analysis

[0056] The amino acid sequence predicted by nucleotide analysis is shown in FIG. 2. As shown in the composite alignment of the amino acid sequence of mature Der p I and Der f I (FIG. 3), high homology was observed between the two proteins. Sequence homology analysis revealed that the Der f I protein showed 81% homology with the Der p I protein as predicted by previous conventional amino acid sequencing. In particular, the residues making up the active site of Der p I, based on those determined for papain, actinidin, cathepsin H, and cathepsin B, are also conserved in the Der f I protein. The residues are glutamine (residue 29), glycine, serine and cysteine (residues 33-35), histidine (residue 171) and asparagine, serine and tryptophan (residues 191-193) where the numbering refers to Der f I. The predicted mature Der f I amino acid sequence contains a potential N-glycosylation site (Asn-Thr-Ser) at position 53-55 which is also present as Asn-Gln-Ser at the equivalent position in Der p I.

[0057] Analysis of the predicted amino acid sequence of the entire Der f I cDNA insert has shown that, as for other cysteine proteases (FIG. 4), the Der f I protein has pre- and proform intermediates. As previously mentioned, the methionine residue at position -98 is presumed to be the initiation methionine. This assumption is based on the fact that firstly, the 5' proximal end sequence from residues -98 to -81 is composed predominantly of hydrophobic amino acid residues (72%), which is the characteristic feature of signal peptides (Von Heijne, G., EMBO J., 3:2315-2323 (1984)). Secondly, the lengths of the presumptive pre-(18 amino acid residues) and pro-peptides (80 residues) are similar to those for other cysteine proteases (FIG. 4). Most cysteine proteases examined have about 120 preproenzyme residues (of which an average of 19 residues form the signal peptide) with cathepsin B the smallest with 80 (Ishidoh, K. et al., FEBS Letters, 226:32-37 (1987)). Der f I falls within this range with a total of 98 preproenzyme residues.

[0058] By following the method for predicting signal-sequence cleavage sites outlined in Von Heijne, it is proposed that cleavage from the pre-Der f I sequence for proenzyme formation occurs at the signal peptidase cleavage site lying between Ala (-81) and Arg (-80) (Von Heijne, G., Eur. J. Biochem., 133:17-21 (1988) and J. Mol. Biol., 184:99-105 (1985)). Thus, the sequence from residues -98 to -81 codes for the leader peptide while the proenzyme moiety of Der f I begins at residue Arg (-80) and ends at residue Glu (-1).

Example 2

Isolation and Characterization of cDNA Coding for Der f II

Materials and Methods

Amino Acid Sequence Analysis

Preparation of .lamda.gt11 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 .mu.g/.mu.l and 5 .mu.g 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 Schuell, Dass 1, FRG, NA-45) according to protocol 6.24 of Sambrook et al., (Sambrook et al., Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory Press (1989)), except 0.5M arginine base was used for elution. The cDNA was then ligated in .lamda.gt10 and .lamda.gt11 at an arms to insert ratio of 2:1. Some was packaged for plaque libraries and an aliquot retained for isolating sequences by polymerase chain reaction as described below.

Isolation of Der f II cDNA by Polymerase Chain Reaction

[0060] To isolate Der f II cDNA, an oligonucleotide primer based on the N-terminal sequence of Der p II was made because their amino acid residues are identical in these regions (Heymann, P. W. et al., J. Allergy Clin. Immunol., 83:1055-1087 (1989)). The primer GGATCCGATCAACTCGATGC-3' was used. The first GGATCC encodes a BamH1 site and the following sequence GAT . . . encodes the first 4 residues of Der p II. For the other primer the .lamda.gt11 TTGACACCAGACCAACTGGTAATG-3' reverse primer flanking the EcoRI cloning site was used (New England Biolabs, Beverly, Mass.). The Der p II primer was designed to have approximately 50-60% G-C and to end on the first or second, rather than the third, base of a codon (Gould, S. J. et al., Proc. Natl. Acad. Sci., 86:1934-1938 (1989); Summer, R. and D. Tautz, Nucleic Acid Res., 17:6749 (1989)).

[0061] The PCR reactions were carried out in a final reaction volume of 25 .mu.l containing 67mM Tris-HCL (pH8.8 at 25.degree. C.), 16.6 mM (NH.sub.4).sub.2SO.sub.4, 40 .mu.M dNTPs, 5 mM 2-mercaptoethanol, 6 .mu.M EDTA, 0.2 mg/ml gelatin, 2 mM MgCl.sub.2, 10 pmoles of each primer and 2 units of Taq polymerase. Approximately 0.001 .mu.g of target DNA was added and the contents of the tube were mixed and overlayed with paraffin oil. The tubes were initially denatured at 95.degree. C. for 6 minutes, then annealed at 55.degree. C. for 1 minute and extended at 72.degree. C. for 2 minutes. Thereafter for 38 cycles, denaturing was carried out for 30 seconds and annealing and extension as before. In the final (40th) cycle, the extension reaction was increased to 10 minutes to ensure that all amplified products were full length. The annealing temperature was deliberately set slightly lower than the Tm of the oligonucleotide primers (determined by the formula Tm-69.3+0.41(G+C%)-650/oligo length) to allow for mismatches in the N-terminal primer.

[0062] 5 .mu.l of the reaction was then checked for amplified bands on a 1% agarose gel. The remainder of the reaction mixture was extracted with chloroform to remove all of the paraffin oil and ethanol precipitated prior to purification of the amplified product on a low melting point agarose gel (Bio-Rad, Richmond, Calif.).

Subcloning of PCR Product

[0063] The ends of the purified PCR product were filled in a reaction containing 10 mM Tris HCl, 10 mM MgCl.sub.2, 50 mM NaCl, 0.025 mM dNTP and 1 .mu.l of Klenow enzyme in a final volume of 100 .mu.l. The reaction was carried out at 37.degree. C. for 15 minutes and heat inactivated at 70.degree. C. for 10 minutes. The mixture was Leder phenol extracted before ethanol precipitation. The resulting blunt ended DNA was ligated into M13mp18 digested with Sma I in a reaction containing 0.5M ATP, 1.times. ligase buffer and 1 unit of T.sub.4 ligase at 15.degree. C. for 24 hrs and transformed into E. coli TG1 made competent by the CaCl.sub.2 method. The transformed cells were plated out as a lawn on L+G plates and grown overnight at 37.degree. C.

Preparation of Single-Stranded DNA Template for Sequencing

[0064] Isolated white plaques were picked using an orange stick into 2.5 ml of an overnight culture of TG1 cells diluted 1 in 100 in 2.times. TY broth, and grown at 37.degree. C. for 6 hours. The cultures were pelleted and the supernatant removed to a fresh tube. To a 1 ml aliquot of this supernatant 270 .mu.l of 20% polyethylene glycol, 2.5M NaCl was added and the tube was vortexed before allowing it to stand at RT for 15 minutes. This was then spun down again and all traces of the supernatant were removed from the tube. The pellet was then resuspended in 100 .mu.l of 1.times. TE buffer. At least 2 phenol:TE extractions were done, followed by 1 Leder phenol extraction and a CHCL.sub.3 extraction. The DNA was precipitated in ethanol and resuspended in a final volume of 20 .mu.l of TE buffer.

DNA Analysis

[0065] DNA sequencing was performed with the dideoxynucleotide chain termination (Sanger, F. et al., Proc. Nat. Acad. Sci., 74:5463-5467 (1977)) using DNA produced from M13 derived vectors mp18 and mp19 in E. coli TG1 and T4 DNA polymerase (Sequenase version 2.0, USB Corp., Cleveland, Ohio; Restriction endonucleases were from Toyobo, (Osaka, Japan). All general procedures were by standard techniques (Sambrook, J. et al., A Laboratory Manual, 2d Ed. Cold Spring Harbor Laboratory Press (1989)). The sequence analysis was performed using the Mac Vector Software (IBI, New Haven, Conn.).

Results

[0066] D. farinae cDNA ligated in .lamda.gt11 was used to amplify a sequence using an oligonucleotide primer with homology to nucleotides coding for the 4 N terminal residues of Der p II and a reverse primer for the .lamda.gt11 sequence flanking the coding site. Two major bands of about 500 bp and 300 bp were obtained when the product was gel electrophoresed. These were ligated into M13 mp18 and a number of clones containing the 500 bp fragment were analyzed by DNA sequencing. Three clones produced sequence data from the N-terminal primer end and one from the other orientation. Where the sequence data from the two directions overlapped, a complete match was found. One of the clones read from the N-terminal primer, contained a one-base deletion which shifted the reading frome. It was deduced to be a copying error, as the translated sequence from the other two clones matched the protein sequence for the first 20 amino acid residues of the allergen.

[0067] The sequence of the clones showing consensus and producing a correct reading frame is shown in FIG. 6, along with the inferred amino acid sequence. It coded for a 129 residue protein with no N-glycosylation site and a calculated molecular weight of 14,021 kD. No homology was found when compared to other proteins on the GenBank data base (61.0 release). It did, however, show 88% amino acid residue homology with Der p II shown in the alignment in FIG. 9. Seven out of the 16 changes were conservative. The conserved residues also include all the cysteines present at positions 8, 21, 27, 73, 78 and119. There was also considerable nucleotide homology, although the restriction enzymes map generated from the sequence data for commonly used enzymes is different from Der p II (FIG. 8). The hydrophobicity plots of the translated sequence of Der f II and Der p II shown in FIG. 10 are almost identical.

Equivalents

[0068] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Sequence CWU 1

1

22 1 1084 DNA Dermatophagoides sp. CDS (42)..(1004) mat_peptide (282) 1 gaattccgtt ttcttccatc aaaattaaaa attcatcaaa a atg aaa ttc gtt ttg 56 Met Lys Phe Val Leu -80 gcc att gcc tct ttg ttg gta ttg agc act gtt tat gct cgt cca gct 104 Ala Ile Ala Ser Leu Leu Val Leu Ser Thr Val Tyr Ala Arg Pro Ala -75 -70 -65 -60 tca atc aaa act ttt gaa gaa ttc aaa aaa gcc ttc aac aaa aac tat 152 Ser Ile Lys Thr Phe Glu Glu Phe Lys Lys Ala Phe Asn Lys Asn Tyr -55 -50 -45 gcc acc gtt gaa gag gaa gaa gtt gcc cgt aaa aac ttt ttg gaa tca 200 Ala Thr Val Glu Glu Glu Glu Val Ala Arg Lys Asn Phe Leu Glu Ser -40 -35 -30 ttg aaa tat gtt gaa gct aac aaa ggt gcc atc aac cat ttg tcc gat 248 Leu Lys Tyr Val Glu Ala Asn Lys Gly Ala Ile Asn His Leu Ser Asp -25 -20 -15 ttg tca ttg gat gaa ttc aaa aac cgt tat ttg atg agt gct gaa gct 296 Leu Ser Leu Asp Glu Phe Lys Asn Arg Tyr Leu Met Ser Ala Glu Ala -10 -5 -1 1 5 ttt gaa caa ctc aaa act caa ttc gat ttg aat gcc gaa aca agc gct 344 Phe Glu Gln Leu Lys Thr Gln Phe Asp Leu Asn Ala Glu Thr Ser Ala 10 15 20 tgc cgt atc aat tcg gtt aac gtt cca tcg gaa ttg gat tta cga tca 392 Cys Arg Ile Asn Ser Val Asn Val Pro Ser Glu Leu Asp Leu Arg Ser 25 30 35 ctg cga act gtc act cca atc cgt atg caa gga ggc tgt ggt tca tgt 440 Leu Arg Thr Val Thr Pro Ile Arg Met Gln Gly Gly Cys Gly Ser Cys 40 45 50 tgg gct ttc tct ggt gtt gcc gca act gaa tca gct tat ttg gcc tac 488 Trp Ala Phe Ser Gly Val Ala Ala Thr Glu Ser Ala Tyr Leu Ala Tyr 55 60 65 cgt aac acg tct ttg gat ctt tct gaa cag gaa ctc gtc gat tgc gca 536 Arg Asn Thr Ser Leu Asp Leu Ser Glu Gln Glu Leu Val Asp Cys Ala 70 75 80 85 tct caa cac gga tgt cac ggc gat aca ata cca aga ggc atc gaa tac 584 Ser Gln His Gly Cys His Gly Asp Thr Ile Pro Arg Gly Ile Glu Tyr 90 95 100 atc caa caa aat ggt gtc gtt gaa gaa aga agc tat cca tac gtt gca 632 Ile Gln Gln Asn Gly Val Val Glu Glu Arg Ser Tyr Pro Tyr Val Ala 105 110 115 cga gaa caa cga tgc cga cga cca aat tcg caa cat tac ggt atc tca 680 Arg Glu Gln Arg Cys Arg Arg Pro Asn Ser Gln His Tyr Gly Ile Ser 120 125 130 aac tac tgc caa att tat cca cca gat gtg aaa caa atc cgt gaa gct 728 Asn Tyr Cys Gln Ile Tyr Pro Pro Asp Val Lys Gln Ile Arg Glu Ala 135 140 145 ttg act caa aca cac aca gct att gcc gtc att att ggc atc aaa gat 776 Leu Thr Gln Thr His Thr Ala Ile Ala Val Ile Ile Gly Ile Lys Asp 150 155 160 165 ttg aga gct ttc caa cat tat gat gga cga aca atc att caa cat gac 824 Leu Arg Ala Phe Gln His Tyr Asp Gly Arg Thr Ile Ile Gln His Asp 170 175 180 aat ggt tat caa cca aac tat cat gcc gtc aac att gtc ggt tac gga 872 Asn Gly Tyr Gln Pro Asn Tyr His Ala Val Asn Ile Val Gly Tyr Gly 185 190 195 agt aca caa ggc gac gat tat tgg atc gta cga aac agt tgg gat act 920 Ser Thr Gln Gly Asp Asp Tyr Trp Ile Val Arg Asn Ser Trp Asp Thr 200 205 210 acc tgg gga gat agc gga tac gga tat ttc caa gcc gga aac aac ctc 968 Thr Trp Gly Asp Ser Gly Tyr Gly Tyr Phe Gln Ala Gly Asn Asn Leu 215 220 225 atg atg atc gaa caa tat cca tat gtt gta atc atg tgaacatttg 1014 Met Met Ile Glu Gln Tyr Pro Tyr Val Val Ile Met 230 235 240 aaattgaata tatttatttg ttttcaaaat aaaaacaact actcttgcga gtatttttta 1074 ctcggaattc 1084 2 222 PRT Dermatophagoides sp. 2 Thr Asn Ala Cys Ser Ile Asn Gly Asn Ala Pro Ala Glu Ile Asp Leu 1 5 10 15 Arg Gln Met Arg Thr Val Thr Pro Ile Arg Met Gln Gly Gly Cys Gly 20 25 30 Ser Cys Trp Ala Phe Ser Gly Val Ala Ala Thr Glu Ser Ala Tyr Leu 35 40 45 Ala His Arg Asn Gln Ser Leu Asp Leu Ala Glu Gln Glu Leu Val Asp 50 55 60 Cys Ala Ser Gln His Gly Cys His Gly Asp Thr Ile Pro Arg Gly Ile 65 70 75 80 Glu Tyr Ile Gln His Asn Gly Val Val Gln Glu Ser Tyr Tyr Arg Tyr 85 90 95 Val Ala Arg Glu Gln Ser Cys Arg Arg Pro Asn Ala Gln Arg Phe Gly 100 105 110 Ile Ser Asn Tyr Cys Gln Ile Tyr Pro Pro Asn Ala Asn Lys Ile Arg 115 120 125 Glu Ala Leu Ala Gln Thr His Ser Ala Ile Ala Val Ile Ile Gly Ile 130 135 140 Lys Asp Leu Asp Ala Phe Arg His Tyr Asp Gly Arg Thr Ile Ile Gln 145 150 155 160 Arg Asp Asn Gly Tyr Gln Pro Asn Tyr His Ala Val Asn Ile Val Gly 165 170 175 Tyr Ser Asn Ala Gln Gly Val Asp Tyr Trp Ile Val Arg Asn Ser Trp 180 185 190 Asp Thr Asn Trp Gly Asp Asn Gly Tyr Gly Tyr Phe Ala Ala Asn Ile 195 200 205 Asp Leu Met Met Ile Glu Glu Tyr Pro Tyr Val Val Ile Leu 210 215 220 3 223 PRT Dermatophagoides sp. 3 Thr Ser Ala Cys Arg Ile Asn Ser Val Asn Val Pro Ser Glu Leu Asp 1 5 10 15 Leu Arg Ser Leu Arg Thr Val Thr Pro Ile Arg Met Gln Gly Gly Cys 20 25 30 Gly Ser Cys Trp Ala Phe Ser Gly Val Ala Ala Thr Glu Ser Ala Tyr 35 40 45 Leu Ala Tyr Arg Asn Thr Ser Leu Asp Leu Ser Glu Gln Glu Leu Val 50 55 60 Asp Cys Ala Ser Gln His Gly Cys His Gly Asp Thr Ile Pro Arg Gly 65 70 75 80 Ile Glu Tyr Ile Gln Gln Asn Gly Val Val Glu Glu Arg Ser Tyr Pro 85 90 95 Tyr Val Ala Arg Glu Gln Arg Cys Arg Arg Pro Asn Ser Gln His Tyr 100 105 110 Gly Ile Ser Asn Tyr Cys Gln Ile Tyr Pro Pro Asp Val Lys Gln Ile 115 120 125 Arg Glu Ala Leu Thr Gln Thr His Thr Ala Ile Ala Val Ile Ile Gly 130 135 140 Ile Lys Asp Leu Arg Ala Phe Gln His Tyr Asp Gly Arg Thr Ile Ile 145 150 155 160 Gln His Asp Asn Gly Tyr Gln Pro Asn Tyr His Ala Val Asn Ile Val 165 170 175 Gly Tyr Gly Ser Thr Gln Gly Asp Asp Tyr Trp Ile Val Arg Asn Ser 180 185 190 Trp Asp Thr Thr Trp Gly Asp Ser Gly Tyr Gly Tyr Phe Gln Ala Gly 195 200 205 Asn Asn Leu Met Met Ile Glu Gln Tyr Pro Tyr Val Val Ile Met 210 215 220 4 112 PRT Dermatophagoides sp. 4 Met Trp Thr Ala Leu Pro Leu Leu Cys Ala Gly Ala Trp Leu Leu Ser 1 5 10 15 Ala Gly Ala Thr Ala Glu Leu Thr Tyr Asn Ala Ile Glu Lys Phe His 20 25 30 Phe Thr Ser Trp Met Lys Gln His Lys Thr Tyr Ser Ser Arg Glu Tyr 35 40 45 Ser His Arg Leu Gln Val Phe Ala Asn Asn Trp Arg Lys Ile Gln Ala 50 55 60 His Asn Gln Arg Asn His Thr Phe Lys Met Gly Leu Asn Gln Phe Ser 65 70 75 80 Asp Met Ser Phe Ala Glu Ile Lys Ile Lys Tyr Leu Trp Ser Glu Pro 85 90 95 Gln Asn Cys Ser Ala Thr Lys Ser Asn Tyr Leu Arg Gly Thr Gly Pro 100 105 110 5 113 PRT Dermatophagoides sp. 5 Met Thr Pro Leu Leu Leu Leu Ala Val Leu Cys Leu Gly Thr Ala Leu 1 5 10 15 Ala Thr Pro Lys Phe Asp Gln Thr Phe Asn Ala Gln Trp His Gln Trp 20 25 30 Lys Ser Thr His Arg Arg Leu Tyr Gly Thr Asn Glu Glu Glu Trp Arg 35 40 45 Arg Ala Val Trp Glu Lys Asn Met Arg Met Ile Gln Ile His Asn Gly 50 55 60 Glu Tyr Ser Asn Gly Lys His Gly Phe Ile His Glu Met Asn Ala Phe 65 70 75 80 Gly Asp Met Thr Asn Glu Glu Phe Arg Gln Ile Val Asn Gly Tyr Arg 85 90 95 His Gln Lys His Lys Lys Gly Arg Leu Phe Gln Glu Pro Leu Met Leu 100 105 110 Gln 6 133 PRT Dermatophagoides sp. 6 Met Ala Met Ile Pro Ser Ile Ser Lys Leu Leu Phe Val Ala Ile Cys 1 5 10 15 Leu Phe Val Tyr Met Gly Leu Ser Phe Gly Asp Phe Ser Ile Val Gly 20 25 30 Tyr Ser Gln Asn Asp Leu Thr Ser Thr Glu Arg Leu Ile Gln Leu Phe 35 40 45 Glu Ser Trp Met Leu Lys His Asn Lys Ile Tyr Lys Asn Ile Asp Glu 50 55 60 Lys Ile Tyr Arg Phe Glu Ile Phe Lys Asp Asn Leu Lys Tyr Ile Asp 65 70 75 80 Glu Thr Asn Lys Lys Asn Asn Ser Tyr Trp Leu Gly Leu Asn Val Phe 85 90 95 Ala Asp Met Ser Asn Asp Glu Phe Lys Glu Lys Tyr Thr Gly Ser Ile 100 105 110 Ala Gly Asn Tyr Thr Thr Thr Glu Leu Ser Tyr Glu Glu Val Leu Asn 115 120 125 Asp Gly Asp Val Asn 130 7 143 PRT Dermatophagoides sp. 7 Met Ala His Ala Arg Val Leu Leu Leu Ala Leu Ala Val Leu Ala Thr 1 5 10 15 Ala Ala Val Ala Tyr Ala Ser Ser Ser Ser Phe Ala Asp Ser Asn Pro 20 25 30 Ile Arg Pro Val Thr Asp Arg Ala Ala Ser Thr Leu Glu Ser Ala Val 35 40 45 Leu Gly Ala Leu Gly Arg Thr Arg His Ala Leu Arg Phe Ala Arg Phe 50 55 60 Ala Val Arg Tyr Gly Lys Ser Tyr Glu Ser Ala Ala Glu Val Arg Arg 65 70 75 80 Arg Phe Arg Ile Phe Ser Glu Ser Leu Glu Glu Val Arg Ser Thr Asn 85 90 95 Arg Lys Gly Leu Pro Tyr Arg Leu Gly Ile Asn Arg Phe Ser Asp Met 100 105 110 Ser Trp Glu Glu Phe Gln Ala Thr Arg Leu Gly Ala Ala Gln Thr Cys 115 120 125 Ser Ala Thr Leu Ala Gly Asn His Leu Met Arg Asp Ala Ala Ala 130 135 140 8 117 PRT Dermatophagoides sp. 8 Met Lys Val Ile Leu Leu Phe Val Leu Ala Val Phe Thr Val Phe Val 1 5 10 15 Ser Ser Arg Gly Ile Pro Pro Glu Glu Gln Ser Gln Phe Leu Glu Phe 20 25 30 Gln Asp Lys Phe Asn Lys Lys Tyr Ser His Glu Glu Tyr Leu Glu Arg 35 40 45 Phe Glu Ile Phe Lys Ser Asn Leu Gly Lys Ile Glu Glu Leu Asn Leu 50 55 60 Ile Ala Ile Asn His Lys Ala Asp Thr Lys Phe Gly Val Asn Lys Phe 65 70 75 80 Ala Asp Leu Ser Ser Asp Glu Phe Lys Asn Tyr Tyr Leu Asn Asn Lys 85 90 95 Glu Ala Ile Phe Thr Asp Asp Leu Pro Val Ala Asp Tyr Leu Asp Asp 100 105 110 Glu Phe Ile Asn Ser 115 9 122 PRT Dermatophagoides sp. 9 Met Arg Leu Leu Val Phe Leu Ile Leu Leu Ile Phe Val Asn Phe Ser 1 5 10 15 Phe Ala Asn Val Arg Pro Asn Gly Arg Arg Phe Ser Glu Ser Gln Tyr 20 25 30 Arg Thr Ala Phe Thr Glu Trp Thr Leu Lys Phe Asn Arg Gln Tyr Ser 35 40 45 Ser Ser Glu Phe Ser Asn Arg Tyr Ser Ile Phe Lys Ser Asn Met Asp 50 55 60 Tyr Val Asp Asn Trp Asn Ser Lys Gly Asp Ser Gln Thr Val Leu Gly 65 70 75 80 Leu Asn Asn Phe Ala Asp Ile Thr Asn Glu Glu Tyr Arg Lys Thr Tyr 85 90 95 Leu Gly Thr Arg Val Asn Ala His Ser Tyr Asn Gly Tyr Asp Gly Arg 100 105 110 Glu Val Leu Asn Val Glu Asp Leu Gln Thr 115 120 10 79 PRT Dermatophagoides sp. 10 Met Trp Trp Ser Leu Ile Pro Leu Ser Cys Leu Leu Ala Leu Thr Ser 1 5 10 15 Ala His Asp Lys Pro Ser Phe His Pro Leu Ser Asp Asp Met Ile Asn 20 25 30 Tyr Ile Asn Lys Gln Asn Thr Thr Trp Gln Ala Gly Arg Asn Glu Tyr 35 40 45 Asn Val Asp Ile Ser Tyr Leu Lys Lys Pro Cys Gly Thr Val Leu Gly 50 55 60 Gly Pro Lys Leu Pro Glu Arg Val Gly Phe Ser Glu Asp Ile Asn 65 70 75 11 113 PRT Dermatophagoides sp. 11 Met Val Ser Ile Cys Glu Gln Lys Leu Gln His Phe Ser Ala Val Phe 1 5 10 15 Leu Leu Ile Leu Cys Leu Gly Met Met Ser Ala Ala Pro Pro Pro Asp 20 25 30 Pro Ser Leu Asp Asn Glu Trp Lys Glu Trp Lys Thr Lys Phe Ala Lys 35 40 45 Ala Tyr Asn Leu Asn Asn Glu Glu Arg His Arg Arg Leu Val Trp Glu 50 55 60 Glu Asn Lys Lys Lys Ile Glu Ala His Asn Ala Asp Tyr Glu Gln Gly 65 70 75 80 Lys Thr Ser Phe Tyr Met Gly Leu Asn Gln Phe Ser Asp Leu Thr Pro 85 90 95 Glu Glu Phe Lys Thr Asn Cys Tyr Gly Asn Ser Leu Asn Arg Gly Glu 100 105 110 Met 12 113 PRT Dermatophagoides sp. 12 Met Val Ser Ile Cys Glu Gln Lys Leu Gln His Phe Ser Ala Val Phe 1 5 10 15 Leu Leu Ile Leu Cys Leu Gly Met Met Ser Ala Ala Pro Ser Pro Asp 20 25 30 Pro Ser Leu Asp Asn Glu Trp Lys Glu Trp Lys Thr Thr Phe Ala Lys 35 40 45 Ala Tyr Ser Leu Asp Asp Glu Glu Arg His Arg Arg Leu Met Trp Glu 50 55 60 Glu Asn Lys Lys Lys Ile Glu Ala His Asn Ala Asp Tyr Glu Arg Gly 65 70 75 80 Lys Thr Ser Phe Tyr Met Gly Leu Asn Gln Phe Ser Asp Leu Thr Pro 85 90 95 Glu Glu Phe Arg Thr Asn Cys Cys Gly Ser Ser Met Cys Arg Gly Glu 100 105 110 Met 13 100 PRT Dermatophagoides sp. 13 Asn Leu Leu Leu Leu Ala Val Leu Cys Leu Gly Thr Ala Leu Ala Thr 1 5 10 15 Pro Lys Phe Asp Gln Thr Phe Ser Ala Glu Trp His Gln Trp Lys Ser 20 25 30 Thr His Arg Arg Leu Tyr Gly Thr Asn Glu Glu Glu Trp Arg Arg Ala 35 40 45 Ile Trp Glu Lys Asn Met Arg Met Ile Gln Leu His Asn Gly Glu Tyr 50 55 60 Ser Asn Gly Gln His Gly Phe Ser Met Glu Met Asn Ala Phe Gly Asp 65 70 75 80 Met Thr Asn Glu Glu Phe Arg Gln Val Val Asn Gly Tyr Arg His Gln 85 90 95 Lys His Lys Lys 100 14 98 PRT Dermatophagoides sp. 14 Met Lys Phe Val Leu Ala Ile Ala Ser Leu Leu Val Leu Ser Thr Val 1 5 10 15 Tyr Ala Arg Pro Ala Ser Ile Lys Thr Phe Glu Glu Phe Lys Lys Ala 20 25 30 Phe Asn Lys Asn Tyr Ala Thr Val Glu Glu Glu Glu Val Ala Arg Lys 35 40 45 Asn Phe Leu Glu Ser Leu Lys Tyr Val Glu Ala Asn Lys Gly Ala Ile 50 55 60 Asn His Leu Ser Asp Leu Ser Leu Asp Glu Phe Lys Asn Arg Tyr Leu 65 70 75 80 Met Ser Ala Glu Ala Phe Glu Gln Leu Lys Thr Gln Phe Asp Leu Asn 85 90 95 Ala Glu 15 23 PRT Dermatophagoides sp. 15 Lys Asn Arg Phe Leu Met Ser Ala Glu Ala Phe Glu His Leu Lys Thr 1 5 10 15 Gln Phe Arg Leu Asn Ala Glu 20 16 36 PRT Dermatophagoides sp. 16 Leu Arg Phe Ile Asp Glu His Asn Ala Asp Thr Asn Arg Ser Tyr Lys 1 5 10 15 Val Gly Leu Asn Gln Phe Ala Asp Leu Thr Gly Glu Glu Phe Arg Ser 20 25 30 Thr Tyr Leu Gly 35 17 491 DNA Dermatophagoides sp. CDS (1)..(387) 17 gat caa gtc gat gtt aaa gat tgt gcc aac aat gaa atc aaa aaa gta 48 Asp Gln Val Asp Val Lys Asp Cys Ala Asn Asn Glu Ile Lys Lys Val 1 5 10 15 atg gtc gat ggt tgc cat ggt tct gat cca tgc atc atc cat cgt ggt 96 Met Val Asp Gly Cys His Gly Ser Asp Pro Cys Ile Ile His Arg Gly 20 25 30 aaa cca ttc act ttg gaa gcc tta ttc gat gcc aac caa aac act aaa 144 Lys Pro Phe Thr Leu Glu Ala

Leu Phe Asp Ala Asn Gln Asn Thr Lys 35 40 45 acc gct aaa act gaa atc aaa gcc agc ctc gat ggt ctt gaa att gat 192 Thr Ala Lys Thr Glu Ile Lys Ala Ser Leu Asp Gly Leu Glu Ile Asp 50 55 60 gtt ccc ggt att gat acc aat gct tgc cat ttt atg aaa tgt cca ttg 240 Val Pro Gly Ile Asp Thr Asn Ala Cys His Phe Met Lys Cys Pro Leu 65 70 75 80 gtt aaa ggt caa caa tat gat gcc aaa tat aca tgg aat gtg ccg aaa 288 Val Lys Gly Gln Gln Tyr Asp Ala Lys Tyr Thr Trp Asn Val Pro Lys 85 90 95 att gca cca aaa tct gaa aac gtt gtc gtt aca gtc aaa ctt gtt ggt 336 Ile Ala Pro Lys Ser Glu Asn Val Val Val Thr Val Lys Leu Val Gly 100 105 110 gat aat ggt gtt ttg gct tgc gct att gct acc cac gct aaa atc cgt 384 Asp Asn Gly Val Leu Ala Cys Ala Ile Ala Thr His Ala Lys Ile Arg 115 120 125 gat taaaaaaaaa aaataaatat gaaaattttc accaacatcg aacaaaattc 437 Asp aataaccaaa atttgaatca aaaacggaat tccaagctga gcgccggtcg ctac 491 18 857 DNA Dermatophagoides sp. CDS (1)..(735) mat_peptide (22) 18 aaa aac cga ttt ttg atg agt gca gaa gct ttt gaa cac ctc aaa act 48 Lys Asn Arg Phe Leu Met Ser Ala Glu Ala Phe Glu His Leu Lys Thr -5 -1 1 5 caa ttc gat ttg aat gct gaa act aac gcc tgc agt atc aat gga aat 96 Gln Phe Asp Leu Asn Ala Glu Thr Asn Ala Cys Ser Ile Asn Gly Asn 10 15 20 25 gct cca gct gaa atc gat ttg cga caa atg cga act gtc act ccc att 144 Ala Pro Ala Glu Ile Asp Leu Arg Gln Met Arg Thr Val Thr Pro Ile 30 35 40 cgt atg caa gga ggc tgt ggt tca tgt tgg gct ttc tct ggt gtt gcc 192 Arg Met Gln Gly Gly Cys Gly Ser Cys Trp Ala Phe Ser Gly Val Ala 45 50 55 gca act gaa tca gct tat ttg gct cac cgt aat caa tca ttg gat ctt 240 Ala Thr Glu Ser Ala Tyr Leu Ala His Arg Asn Gln Ser Leu Asp Leu 60 65 70 gct gaa caa gaa tta gtc gat tgt gct tcc caa cac ggt tgt cat ggt 288 Ala Glu Gln Glu Leu Val Asp Cys Ala Ser Gln His Gly Cys His Gly 75 80 85 gat acc att cca cgt ggt att gaa tac atc caa cat aat ggt gtc gtc 336 Asp Thr Ile Pro Arg Gly Ile Glu Tyr Ile Gln His Asn Gly Val Val 90 95 100 105 caa gaa agc tac tat cga tac gtt gca cga gaa caa tca tgc cga cga 384 Gln Glu Ser Tyr Tyr Arg Tyr Val Ala Arg Glu Gln Ser Cys Arg Arg 110 115 120 cca aat gca caa cgt ttc ggt atc tca aac tat tgc caa att tac cca 432 Pro Asn Ala Gln Arg Phe Gly Ile Ser Asn Tyr Cys Gln Ile Tyr Pro 125 130 135 cca aat gca aac aaa att cgt gaa gct ttg gct caa acc cac agc gct 480 Pro Asn Ala Asn Lys Ile Arg Glu Ala Leu Ala Gln Thr His Ser Ala 140 145 150 att gcc gtc att att ggc atc aaa gat tta gac gca ttc cgt cat tat 528 Ile Ala Val Ile Ile Gly Ile Lys Asp Leu Asp Ala Phe Arg His Tyr 155 160 165 gat ggc cga aca atc att caa cgc gat aat ggt tac caa cca aac tat 576 Asp Gly Arg Thr Ile Ile Gln Arg Asp Asn Gly Tyr Gln Pro Asn Tyr 170 175 180 185 cac gct gtc aac att gtt ggt tac agt aac gca caa ggt gtc gat tat 624 His Ala Val Asn Ile Val Gly Tyr Ser Asn Ala Gln Gly Val Asp Tyr 190 195 200 tgg atc gta cga aac agt tgg gat acc aat tgg ggt gat aat ggt tac 672 Trp Ile Val Arg Asn Ser Trp Asp Thr Asn Trp Gly Asp Asn Gly Tyr 205 210 215 ggt tat ttt gct gcc aac atc gat ttg atg atg att gaa gaa tat cca 720 Gly Tyr Phe Ala Ala Asn Ile Asp Leu Met Met Ile Glu Glu Tyr Pro 220 225 230 tat gtt gtc att ctc taaacaaaaa gacaatttct tatatgattg tcactaattt 775 Tyr Val Val Ile Leu 235 atttaaaatc aaaattttta gaaaatgaat aaattcattc acaaaaatta aaaaaaaaaa 835 aaaaaaaaaa aaaaaaaaaa aa 857 19 591 DNA Dermatophagoides sp. CDS (72)..(506) mat_peptide (120) 19 cacaaattct tctttcttcc ttactactga tcattaatct gaaaacaaaa ccaaacaaac 60 cattcaaaat g atg tac aaa att ttg tgt ctt tca ttg ttg gtc gca gcc 110 Met Tyr Lys Ile Leu Cys Leu Ser Leu Leu Val Ala Ala -15 -10 -5 gtt gct cgt gat caa gtc gat gtc aaa gat tgt gcc aat cat gaa atc 158 Val Ala Arg Asp Gln Val Asp Val Lys Asp Cys Ala Asn His Glu Ile -1 1 5 10 aaa aaa gtt ttg gta cca gga tgc cat ggt tca gaa cca tgt atc att 206 Lys Lys Val Leu Val Pro Gly Cys His Gly Ser Glu Pro Cys Ile Ile 15 20 25 cat cgt ggt aaa cca ttc caa ttg gaa gcc gtt ttc gaa gcc aac caa 254 His Arg Gly Lys Pro Phe Gln Leu Glu Ala Val Phe Glu Ala Asn Gln 30 35 40 45 aac aca aaa acg gct aaa att gaa atc aaa gcc tca atc gat ggt tta 302 Asn Thr Lys Thr Ala Lys Ile Glu Ile Lys Ala Ser Ile Asp Gly Leu 50 55 60 gaa gtt gat gtt ccc ggt atc gat cca aat gca tgc cat tac atg aaa 350 Glu Val Asp Val Pro Gly Ile Asp Pro Asn Ala Cys His Tyr Met Lys 65 70 75 tgc cca ttg gtt aaa gga caa caa tat gat att aaa tat aca tgg aat 398 Cys Pro Leu Val Lys Gly Gln Gln Tyr Asp Ile Lys Tyr Thr Trp Asn 80 85 90 gtt ccg aaa att gca cca aaa tct gaa aat gtt gtc gtc act gtt aaa 446 Val Pro Lys Ile Ala Pro Lys Ser Glu Asn Val Val Val Thr Val Lys 95 100 105 gtt atg ggt gat gat ggt gtt ttg gcc tgt gct att gct act cat gct 494 Val Met Gly Asp Asp Gly Val Leu Ala Cys Ala Ile Ala Thr His Ala 110 115 120 125 aaa atc cgc gat taaatcaaac aaaatttatt gattttgtaa tcacaaatga 546 Lys Ile Arg Asp ttgattttct ttccaaaaaa aaaataaata aaattttggg aattc 591 20 591 DNA Dermatophagoides sp. CDS (72)..(506) 20 cacaaattct tctttcttcc ttactactga tcattaatct gaaaacaaaa ccaaacaaac 60 cattcaaaat g atg tac aaa att ttg tgt ctt tca ttg ttg gtc gca gcc 110 Met Tyr Lys Ile Leu Cys Leu Ser Leu Leu Val Ala Ala 1 5 10 gtt gct cgt gat caa gtc gat gtt aaa gat tgt gcc aac aat gaa atc 158 Val Ala Arg Asp Gln Val Asp Val Lys Asp Cys Ala Asn Asn Glu Ile 15 20 25 aaa aaa gta atg gtc gat ggt tgc cat ggt tct gat cca tgc atc atc 206 Lys Lys Val Met Val Asp Gly Cys His Gly Ser Asp Pro Cys Ile Ile 30 35 40 45 cat cgt ggt aaa cca ttc act ttg gaa gcc tta ttc gat gcc aac caa 254 His Arg Gly Lys Pro Phe Thr Leu Glu Ala Leu Phe Asp Ala Asn Gln 50 55 60 aac act aaa acc gct aaa act gaa atc aaa gcc agc ctc gat ggt ctt 302 Asn Thr Lys Thr Ala Lys Thr Glu Ile Lys Ala Ser Leu Asp Gly Leu 65 70 75 gaa att gat gtt ccc ggt att gat acc aat gct tgc cat ttt atg aaa 350 Glu Ile Asp Val Pro Gly Ile Asp Thr Asn Ala Cys His Phe Met Lys 80 85 90 tgt cca ttg gtt aaa ggt caa caa tat gat gcc aaa tat aca tgg aat 398 Cys Pro Leu Val Lys Gly Gln Gln Tyr Asp Ala Lys Tyr Thr Trp Asn 95 100 105 gtt ccg aaa att gca cca aaa tct gaa aac gtt gtc gtt aca gtc aaa 446 Val Pro Lys Ile Ala Pro Lys Ser Glu Asn Val Val Val Thr Val Lys 110 115 120 125 ctt gtt ggt gat aat ggt gtt ttg gct tgc gct att gct acc cac gct 494 Leu Val Gly Asp Asn Gly Val Leu Ala Cys Ala Ile Ala Thr His Ala 130 135 140 aaa atc cgt gat taaaaaaaaa aaataaatat gaaaattttc accaacatcg 546 Lys Ile Arg Asp 145 aacaaaattc aataaccaaa atttgaatca aaaacttggg aattc 591 21 20 DNA Dermatophagoides sp. 21 ggatccgatc aactcgatgc 20 22 24 DNA Dermatophagoides sp. 22 ttgacaccag accaactggt aatg 24

Claims

1. (canceled)

2. A method of producing an isolated protein of Dermatophagoides pteronyssinus comprising the steps of: a) culturing a host cell transformed with a nucleic acid encoding a protein allergen of Dermatophagoides pteronyssinus, Der p I, comprising the amino acid sequence shown in FIG. 7, in an appropriate medium to produce a mixture of cells and medium containing said protein allergen; and b) purifying said mixture to produce isolated Der p I protein allergen.

3. The method of claim 2 wherein the nucleic acid comprises a nucleotide sequence selected from the group consisting of the nucleotide sequence shown in FIG. 7, the coding region of the nucleotide sequence shown in FIG. 7, and a nucleotide sequence which hybridizes under high or low stringency conditions to a complementary strand of the nucleotide sequence shown in FIG. 7.

4. The method of claim 3, wherein the high stringency conditions comprise hybridization at Tm-20 followed by at least one post-hybridization wash at Tm-12.

5. The method of claim 2, wherein the nucleic acid comprises the cDNA insert of phage clone .lamda.gt11 p1(13T), ATCC Deposit No. 69338.

6. A method of producing an isolated protein of Dermatophagoides pteronyssinus comprising the steps of: a) culturing a host cell transformed with a nucleic acid encoding a protein allergen of Dermatophagoides pteronyssinus, Der p I, and selected from the group consisting of the nucleotide sequence shown in FIG. 7, the coding region of the nucleotide sequence shown in FIG. 7, and a nucleotide sequence which hybridizes under high or low stringency conditions to a complementary strand of the nucleotide sequence shown in FIG. 7, in an appropriate medium to produce a mixture of cells and medium containing said protein allergen; and b) purifying said mixture to produce isolated Der p I protein allergen.

7. A method of producing an isolated antigenic peptide of Dermatophagoides pteronyssinus comprising the steps of: a) culturing a host cell transformed with a nucleic acid encoding an antigenic peptide of Dermatophagoides pteronyssinus, Der p I, comprising a portion of the amino acid sequence shown in FIG. 7, in an appropriate medium to produce a mixture of cells and medium containing said peptide; and b) purifying said mixture to produce isolated Der p I peptide.

8. The method of claim 7 wherein the nucleic acid comprises a portion of a nucleotide sequence selected from the group consisting of the nucleotide sequence shown in FIG. 7, the coding region of the nucleotide sequence shown in FIG. 7, and a nucleotide sequence which hybridizes under high or low stringency conditions to a complementary strand of the nucleotide sequence shown in FIG. 7.

9. The method of claim 7, wherein the high stringency conditions comprise hybridization at Tm-20 followed by at least one post-hybridization wash at Tm-12.

10. The method of claim 7, wherein the nucleic acid comprises a portion of the cDNA insert of phage clone .lamda.gt11 p1(13T), ATCC Deposit No. 69338.

11. A method of producing an isolated protein of Dermatophagoides pteronyssinus comprising the steps of: a) culturing a host cell transformed with a nucleic acid encoding an antigenic peptide of Dermatophagoides pteronyssinus, Der p I, and selected from the group consisting of a portion of the nucleotide sequence shown in FIG. 7, a portion of the coding region of the nucleotide sequence shown in FIG. 7, and a portion of the a nucleotide sequence which hybridizes under high or low stringency conditions to a complementary strand of the nucleotide sequence shown in FIG. 7, in an appropriate medium to produce a mixture of cells and medium containing said protein allergen; and b) purifying said mixture to produce isolated Der p I protein allergen.