CN103215236A - Dust mite allergens Derf8 and Derf20, and genes and applications thereof - Google Patents

Abstract

The invention relates to dust mite allergens Derf8 and Derf20, and genes and applications thereof, and belongs to the technical field of biomedicine. The dust mite allergens Derf8 and Derf20 are respectively composed of amino acid sequences represented by SEQ ID NO: 1 and SEQ ID NO: 3. The genes coding the dust mite allergens Derf8 and Derf20 are respectively composed of nucleotide sequences represented by SEQ ID NO: 2 and SEQ ID NO: 4. The invention also relates to applications of the dust mite allergens Derf8 and Derf20 in preparing medicines used for treating dust mite allergic diseases. The invention has the advantages that: the dust mite allergens Derf8 and Derf20 and the genes thereof are provided; and the applications of the dust mite allergens Derf8 and Derf20 in preparing medicines used for treating dust mite allergic diseases are provided.

Description

Dust mite allergen Derf8 and Derf20 and gene thereof and application

Technical field:

The present invention relates to a kind of dust mite allergen Der f 8 and Der f 20 and gene and application, belong to field of biomedicine technology.

Background technology:

Anaphylactic disease (as asthma, allergic rhinitis etc.) is common disease, frequently-occurring disease clinically, the total incidence of countries in the world anaphylactic disease is up to 10-30%, it is the great hygienic problems of our times, China has asthmatic patient more than 2,000 ten thousand now at present, Allergic Rhinitis more than 5,000 ten thousand, and its M & M is still in rising trend, recent two decades comes sickness rate almost to turn over one times.In causing numerous inhalant allergens of anaphylactic disease, the dirt mite is the most important factor that causes the allergic airway disease disease.Since Voorhorst and Spieksma(1964) confirm that first dirt mite and meta-bolites are the main allergen in room dirt since, countries in the world (American-European countries, Japan, China etc.) allergology worker also unanimously confirms that by a large amount of investigation the dirt mite is global main allergen.The dirt mite is the about 70-80% of positive rate in the immunodiagnosis of anaphylactic disease patient-specific.Since Noon and Freeman(1911) since first Application thimothy grass Pollen allergenic extract (allergen extract) is used for the treatment of pollinosis, desensitization treatment is the history of existing more than 90 year so far.Along with the further understanding to the pathogenetic further investigation of allergic disease and immunotherapy mechanism, WHO(1997) suggestion renames as allergen vaccine (allergen vaccine) by allergenic extract, and points out in " policy paper of the relevant immunotherapy of WHO " (1998): (1) encourages the standardized allergen vaccine of application and development; (2) high-quality allergen vaccine is depended in successful immunotherapy.Therefore, develop the efficient allergen vaccine of a new generation and be still this area research focus both at home and abroad.

Because the dirt mite is the medical science arthropods, structure and complicated, although at present people in the hundreds of albumen of dirt mite preliminary evaluation go out 16 kinds of anaphylactogen compositions, studies show that the anaphylactogen much more than 30 that the dirt mite contains plants.With regard to some dust mite allergy patients, he may only produce anaphylaxis to the class in the dirt mite or number class allergen protein.And for dust mite allergy patient diagnosis and desensitization treatment, be at present still the thick immersion liquid of dirt mite clinically, wherein contain in a large number the non-specific anaphylactogen of patient and other impurity, this has seriously hindered the standardized formulation of dust mite allergy original reagent and use clinically thereof.So further verify the definite component of dust mite allergen, will bring for the dust mite allergy patient desensitization treatment of individuation.

Summary of the invention:

The object of the present invention is to provide a kind of dust mite allergen Der f 8 and Der f 20 and gene and application.

Technical scheme of the present invention is as follows:

Der f 8 is that we identify a kind of new allergen protein obtained first from the homogenate of dirt mite, and it belongs to glutathione transferring enzyme family (Glutathione S-transferase), and molecular weight is 32 kDa approximately.Der f 8 is comprised of 197 amino acid, its aminoacid sequence following (SEQ ID NO:1):

MLAYAGVDYVDKRYNIGPNFDRSEWLNEKFNLGLDFPNLPYYMDGDVKITQSMAILRYLA 60

RKYNMDGTNEQERLRISMAEQQTHDMFMAMVRICYDPNMENLRVDYLKTLPDSLKLMEKF 120

LANHDYIAGSKISYADFYLYEYLCRMKVMVPEVYGQFENLKKFVERIESLPRIAEYIKKQ 180

KPTTFNASLAKWNGSYA 197

Dust mite allergen Der f 8 gene clone steps comprise: the total RNA of dirt mite extracts, the mRNA purifying, and mRNA reverse transcription and cDNA library build, and the design primer, utilize PCR method amplification coding gene.The gene sequencing result shows that the encoding gene (GenBank accession KC305499) of dust mite allergen Der f 8 is comprised of 595 Nucleotide, and 5 ' end to 3 ' terminal sequence of this gene is (nucleotides sequence is classified SEQ ID NO:3 as):

atgttggcat atgccggtgt tgattatgtt gataaacgtt ataatattgg tccgaatttt 1

gatcgttcag aatggttgaa tgaaaaattc aatttgggtt tagatttccc aaatttgcca 61

tattacatgg atggtgatgt taaaattacc caatcgatgg ccattcttcg ttatttggcc 121

cgtaaatata acatggatgg tactaatgaa caggaacgtt tacgaatttc aatggctgaa 181

caacaaacac atgatatgtt tatggccatg gtccgtattt gttatgatcc aaatatggaa 241

aacttacgag ttgattattt gaaaacattg cccgattcat tgaaattgat ggaaaaattt 301

ttagctaatc atgattatat tgccggttca aaaatttctt atgcagattt ttatttgtat 361

gaatatttgt gccgtatgaa agtcatggta ccagaagtat atggacaatt tgaaaatttg 421

aaaaaatttg ttgaacgcat tgaatcattg ccacgtattg ccgaatacat taagaaacaa 481

aaaccaacaa cattcaatgc atcattggct aaatggaatg gttcttatgc ctaa 595

Der f 20 belongs to a class arginine kinase (Arginine kinase), and molecular weight is 40 kDa approximately.Der f 20 is comprised of 356 amino acid, its aminoacid sequence following (SEQ ID NO:2):

MVDQATLSKLEAGFQKLQNAQDCHSLLKKYLTRDVLDQLKTKKTDMGATLLDVIQSGVEN 60

LDSGVGIYAPDAQSYKTFAALFDPIIDDYHKGFKPTDKHPQTDFGNIEHFVNVDPKNEYV 120

ISTRVRCGRSLKGYPFNPMLTEAQYKEMETKVKGQLATFEGELKGTYYPLLGMDKATQQK 180

LIDDHFLFKEGDRFLQAANACRYWPVGRGIFHNDKKTFLMWVNEEDHLRIISMQKGGDLK 240

EVFGRLVKAVKHIEQKIPFSRDDRLGYLTFCPTNLGTTIRASVHIKLPKLAADRKKLEEV 300

AARYNLQVRGTAGEHTESVGGIYDISNKRRMGLTEYQAVKEMQDGIIELIKMEKSL 356

Dust mite allergen Der f 20 gene clone steps comprise: the total RNA of dirt mite extracts, the mRNA purifying, and mRNA reverse transcription and cDNA library build, and the design primer, utilize PCR method amplification coding gene.The gene sequencing result shows that the encoding gene (GenBank accession EU106619.1) of dust mite allergen Der f 20 is comprised of 1071 Nucleotide, and 5 ' end to 3 ' terminal sequence of this gene is (nucleotides sequence is classified SEQ ID NO:4 as):

atggtcgatc aagctaccct gagtaaattg gaagccggtt tccaaaaatt acagaatgct 60

caagattgtc attcattgtt gaaaaagtat ttgactcgcg atgtgttaga tcaactcaag 120

acgaaaaaga ccgacatggg cgcaacatta ttggatgtta tccaatctgg cgtggaaaac 180

ctggacagtg gtgttggtat ctatgctcct gatgctcaat catacaaaac atttgctgca 240

ttgttcgatc caatcattga tgattaccat aaaggcttca aaccgaccga taaacatccg 300

caaactgatt tcggcaatat cgaacacttt gtcaatgttg atcctaaaaa cgaatacgtc 360

atttctactc gtgttcgatg tggccgttcg ttgaaaggct atccattcaa ccctatgttg 420

acagaggctc aatacaaaga aatggaaacc aaagtgaaag gacaattggc cacattcgaa 480

ggcgaattga aaggcaccta ttacccattg ttgggaatgg ataaagctac tcaacagaaa 540

ttgatcgacg atcatttctt gttcaaggaa ggtgatcgat tcttgcaagc tgccaatgca 600

tgtcgttact ggcctgttgg tcgtggtatc ttccacaatg acaaaaaaac attcttgatg 660

tgggtaaacg aagaagatca tttgcgtatc atttccatgc aaaaaggtgg cgatctaaaa 720

gaggtctttg gacgtttggt caaggctgtc aagcacattg aacaaaagat tccattctct 780

cgtgatgacc gtctcggtta tttgacattc tgtccaacca atcttggcac aaccatccgt 840

gcttcggttc atatcaaact tccgaaattg gccgctgacc gtaagaagtt ggaagaagtg 900

gctgctcgtt acaatcttca agtgcgtggc actgcaggtg aacataccga aagtgtgggt 960

ggtatctatg atattagtaa caaacgacga atgggtctca ccgaatacca agctgttaa1020

gaaatgcaag atggcatcat tgaattgatt aaaatggaaa aatcattgta a 1071

Detect by conventional two-dimensional electrophoresis and two-way Western blot method, find that Der f 8 and Der f 20 have stronger allergenicity, the application in preparation treatment dust mite allergy disease medicament.

Beneficial effect of the present invention is: dust mite allergen Der f 8 and Der f 20 and gene thereof are provided, and dust mite allergen Der f 8 and Der f 20 can be as the application of preparation treatment dust mite allergy disease medicament.

The accompanying drawing explanation:

Fig. 1. be the peak type figure that molecular sieve Sephadex-G75 is crossed in the homogenate of dirt mite.

The Westernblot that Fig. 2 is dirt mite homogenate Sephadex-G75 II peak, wherein: put 1 Der f 20, put 2 and 3 for Der f 8.

Fig. 3-A to Fig. 3-E is the ESI-Q-TOF mass spectrum.Wherein: the mass spectrum that Fig. 3-A to Fig. 3-B is Der f 8; The mass spectrum that Fig. 3-C to Fig. 3-E is Der f 20

Embodiment:

Embodiment mono-: the evaluation of dust mite allergen and determined amino acid sequence thereof

One, the preparation of the raising of dirt mite and collection and the homogenate of dirt mite

The dust mite of pure strain is carried out to batch as for the mouse grain that adopts the grinds powder under the condition of 25 ℃ of relative humidity 75% raises.Because the dirt mite has the life characteristic of lucifuge, the available incandescent lamp luminescence method is separated the dirt mite from feed.For the dirt mite separated, can add appropriate 20mM pH 7.8 Tris-HCL fully to grind, then at centrifugal 30 min of rotating speed 12000 * g, obtain supernatant.

Two, the separation and purification of dirt mite homogenate and two-dimensional electrophoresis and Western blot thereof detect

The supernatant liquor of above-mentioned collection of take is raw material, is splined on and uses in advance the good molecular sieve gel chromatography Sephadex-G75 of 20mM pH7.8 Tris-HCL damping fluid balance, with identical damping fluid, carries out wash-out.Flow velocity is 0.3ml/min, with the every 10min of automatic collector, collects a pipe, detects the absorption value of every pipe at 280nm and 215nm place, makes the peak type figure of absorption value variation as shown in Figure 1.Then every peak is collected to freeze-drying and concentrated separation and purification or two-dimensional electrophoresis and the Western blot detection thereof for next step.Two-dimensional electrophoresis and Western blot concrete steps thereof are as follows:

A. sample preparation: adopt the 2D clean-up of GE company the sample from each peak of molecular sieve to be carried out to the processing such as desalination and concentration, main process is as follows:

1: in will the micro centrifugal pipe of sample as for 1.5 ml containing the 60 μ g 100 μ l that have an appointment, add 300 μ l precipitation agents vibrations to stir evenly, ice bath 15 min.

2: with centrifugal 5 min of maximum speed of revolution (12000 * g), get clean supernatant as far as possible.Add 40 μ l coprecipitators, ice bath 5 min.Centrifugal 5 min of same rotational speed, remove supernatant with liquid-transfering gun again, adds 25 μ l to add 1 ml lavation buffer solution (-20 ℃ of at least precoolings 1 hour) and 5 μ l detergent additives, vibration until precipitation scatter fully.Pipe is hatched under-20 ℃ at least 30 min, every 10 min vibration 20 to 30 s.

3: with centrifugal 5 min of rotating speed 12000 * g.

4: carefully supernatant is removed, now visible white precipitate, will precipitate simply air-dry.

5: add 150 μ l hydrating fluids dissolution precipitation again, use to isoelectric focusing electrophoresis (IEF) in order to first.

B. isoelectrofocusing (IEF): will be splined on the non-linear adhesive tape of the long Immobiline DryStrip of 7 cm containing the 60 μ g sample 100 μ l hydrating fluids of having an appointment, on Ettan IPGphor III isoelectrofocusing system, focus on, the isoelectrofocusing condition is 20 ℃, the electric current of every glue is 50 μ A, and gross focusing volt hour is about 6 kVh.The adhesive tape that isoelectrofocusing is good is respectively washed 15 min with the balance liquid that contains dithiothreitol (DTT) (DTT) and iodo-acid amide respectively, on the SDS-PAGE glue face that the concentration that good adhesive tape is disposed across to prepare by balance is 12%, and seals with agarose, prepares second to electrophoresis.

C. second to electrophoresis: for the glue width, only have the blob of viscose of 7 cm to be run glue with SE260.In the condition of 20 mA/ glue, get final product next half an hour.

Embodiment bis-: the gene clone of dust mite allergen

One, the total RNA of dirt mite extracts

A. take the approximately dirt mite alive of 80 mg, add 1 mL Trizol extracting solution (American I nvitrogen company) of precooling, and add the abundant homogenate of liquid nitrogen.

B. the chloroform that adds Trizol 1/5 volume, acutely mix approximately 15 seconds, and room temperature is placed 5 minutes, and 4 ℃, centrifugal 10 minutes of 12000 rpm, get supernatant.

C. supernatant adds isopyknic Virahol, and room temperature is placed 10 minutes, and 4 ℃, centrifugal 10 minutes of 12000 rpm, precipitation is washed once with 75% ethanol, dries, and pipe end throw out is the total RNA of intestines in the Eupolyphaga Seu Steleophaga digestive tube.

Two, the purifying of dirt mite mRNA

Dirt mite mRNA separation and purification adopts the PolyATtract of U.S. PROMEGA company mRNA Isolation Systems test kit.

A. get the total RNA 500 μ g of dirt mite and be dissolved in 500 μ L DEPC water, 65 ℃ of water-bath 10 min, add Oligo (dT) probe and the 13 μ L 20 * SSC solution of people 3 μ L, mixes, and places room temperature cooling, is called A liquid.

B. the washing of magnetic bead (SA-PMP): magnetic bead is flicked and mixes, to magnetic frame absorption 30 seconds, abandon supernatant, add 0. 5 * SSC, 300 μ L, to magnetic frame absorption 30 seconds, finally add 100 μ L 0. 5 * SSC and suspend, be called B liquid.

C. A liquid is added in B liquid, room temperature was placed 10 min, to magnetic frame absorption 30 seconds, abandon supernatant, with 0. 1 * SSC washing 4 times, finally abandon supernatant, add 100 μ L DEPC aqueous suspensions, adsorb 30 s to magnetic frame, supernatant is moved to new test tube, then add 150 μ L DEPC water resuspended, adsorb 30 s to magnetic frame, move supernatant to above-mentioned test tube, be intestines mRNA in the Eupolyphaga Seu Steleophaga digestive tube of purifying.

D. the sodium acetate solution that adds pH5. 2,3 M of 1/10 volume, the equal-volume Virahol, in-70 ℃ of placements 30 minutes, 4 ℃, centrifugal 10 min of 12000 rpm, abandoned supernatant, is precipitated and dissolved in 10 μ L DEPC water.

Three, dirt mite cDNA library builds

Adopt the Creator of CLONTECH company ^tMsMART ^tMcDNA Library Construction Kit builds dirt mite cDNA library.

A. cDNA the first chain is synthetic

Add 2 μ l dirt mite mRNA, 1 μ l SMART IV primer, 1 μ l CDS III/3 ' PCR primer, 1 μ l RNA-free water in aseptic PCR pipe, make cumulative volume reach 5 μ l, mix also of short duration centrifugal.

1.72 ℃ insulation 2 min, hatch 2 min on ice.

2. add 2 μ l 5 * the first chain buffer, 1 μ l 20 mmol/L DTT, 1 μ l 10 mmol/L dNTP mixtures, 1 μ l PowerScript reversed transcriptive enzyme in above-mentioned PCR pipe, mix also of short duration centrifugal.

3., after being placed in 42 ℃ of insulation 1 hr of PCR instrument, ice bath stops the synthetic of the first chain.

B. adopt long end polymerase chain reaction (LD-PCR) method amplification cDNA the second chain

1. 1 μ l cDNA the first chain, 40 μ l deionized waters, 5 μ l 10 * Advantage 2 PCR damping fluids, 1 μ l 50 * dNTP mixture, 1 μ l 5 ' PCR primer, 1 μ l CDS III/3 ' PCR primer and 1 μ l polysaccharase are mixed in the PCR pipe.

2. in the PCR instrument, by following program, increase: 95 ℃, 20 sec; 22 circulations: 95 ℃, 5 sec; 68 ℃, 6 min.

3., after amplification finishes, synthetic double-stranded cDNA is placed in to-70 ℃ of preservations.

C. the conversion that enzyme is cut, connected and connects product:

1. add 1 μ L pMD19-T carrier (Japanese Takara company), the double-stranded solution of 4 μ L dirt mite cDNA in Eppendorf tube, full dose is 5 μ L.

2. add 5 μ L(equivalent) the ligase enzyme buffer mixture.

3. 16 ℃ are reacted 2 hours.

4. full dose (10 μ L) is added in 100 μ L DH5 α competent cells (sky, Beijing root biochemical technology company limited), ice bath 30 minutes.

5. after 42 ℃ of 90 seconds of heating, then in ice, place 1 minute.

6. add 37 ℃ of LB substratum 900 μ L of bathing of temperature, 37 ℃ of slow shaking culture 60 minutes.

7. get 200 μ L and coat on the LB substratum that contains X-Gal, IPTG, Amp 37 ℃ and cultivate 16 hours, form single bacterium colony.

8. 5 mL LB liquid nutrient medium washing bacterium colonies for each LB plate, add 30% glycerine frozen.The cDNA built is approximately containing 2 * 10 ⁶individual independent clone.

Four, the amplification of dust mite allergen gene order and mensuration:

Aminoacid sequence according to separation and purification gained dust mite allergen Der f 8 in embodiment mono-, we have designed a degenerated primer Der f 8-F and Der f 20-F, joint primer CDS III/3 ' the PCR pairing of using when building intestines cDNA library in the Eupolyphaga Seu Steleophaga digestive tube, forward and reverse primer sequence is:

CDS III/3’ PCR：5’-ATT CTA GAG GCC GAG GCG GCC ATG-3’

Der f 8-F: 5’-ATGCT(A/T/C/G)GC(A/T/C/G)TA(T/C)GC(A/T/C/G)GG(A/T/C/G)

GT(A/T/C/G)GA(T/C)-3’

Der f 20-F: ’-GG(A/T/C/G)GG(A/T/C/G)GA(T/C)CT(A/T/C/G)AA(A/G)GA(A/G)

GT(A/T/C/G)TT(T/C)GG(A/T/C/G)-3’

Wherein, the base in bracket means degenerated primer.

Degenerated primer Der f 8-F and joint primer CDS III/3 ' PCR pairing are used, and the dirt mite cDNA of take is template, carries out PCR.Its reaction conditions is: 95 ℃ of denaturation 4 min, then carry out 35 in following condition and take turns circulation, 94 ℃ of 30 sec, 55 ℃ of 30 sec, 72 ℃ of 40 sec, then 72 ℃ of 10 min.Then the PCR product is connected on the T carrier, imports bacillus coli DH 5 alpha, the screening mono-clonal is checked order.Result show the to encode cDNA sequence of allergen Der f 8 is comprised of 595 Nucleotide, and the cDNA sequence of coding allergen Der f 20 is comprised of 891 Nucleotide.

Embodiment tri-: the allergenicity of dust mite allergen Der f 8 and Der f 20 detects

Need to run two two-dimensional electrophoresis for the same sample in example one, wherein one for substantive dyeing, and one is then used in Western blot and detects its sensitization, and detailed process is as follows:

1: transferring film: the albumen on 2-D-SDS-PAGE glue is gone on pvdf membrane under the condition of 200 mA 2 h with the transferring film groove.

2: sealing: will turn the pvdf membrane that albumen is arranged and seal 2h with 5% skim-milk at normal temperature.

3: primary antibodie is hatched: the pvdf membrane sealed is washed with PBS, primary antibodie (dust mite allergy patients serum) is pressed to the 1:20 dilution with 5% skim-milk, 4 ℃ of overnight incubation.

4: two anti-hatching: the pvdf membrane that primary antibodie is hatched with the PBS washing, is pressed the 1:2000 dilution to two anti-(sheep anti human IgEs) with 5% skim-milk again, at normal temperature, hatches 1 hour.

5: develop: by two PBS washings 3 times for anti-pvdf membrane of hatching, each 5 min, add two appropriate anti-special luminous substrate on the film surface, with film, developed.

E. will mate 2-D-PAGE protein site preferably with development point on film and carry out the order-checking of ESI-Q-TOF mass spectrum.

Result is as shown in Fig. 2-B, and its mid point 1 Der f 20, put 2 and 3 for Der f 8.

SEQUENCE LISTING

<110 > Kunming Institute of Zoology, Chinese Academy of Sciences

<120 > dust mite allergen Der f 8 and Der f 20 and gene and application

<130> 1

<160> 4

<170> PatentIn version 3.3

<210> 1

<211> 197

<212> PRT

<213> Dermatophagoides farinae

<400> 1

Met Leu Ala Tyr Ala Gly Val Asp Tyr Val Asp Lys Arg Tyr Asn Ile

1 5 10 15

Gly Pro Asn Phe Asp Arg Ser Glu Trp Leu Asn Glu Lys Phe Asn Leu

20 25 30

Gly Leu Asp Phe Pro Asn Leu Pro Tyr Tyr Met Asp Gly Asp Val Lys

35 40 45

Ile Thr Gln Ser Met Ala Ile Leu Arg Tyr Leu Ala Arg Lys Tyr Asn

50 55 60

Met Asp Gly Thr Asn Glu Gln Glu Arg Leu Arg Ile Ser Met Ala Glu

65 70 75 80

Gln Gln Thr His Asp Met Phe Met Ala Met Val Arg Ile Cys Tyr Asp

85 90 95

Pro Asn Met Glu Asn Leu Arg Val Asp Tyr Leu Lys Thr Leu Pro Asp

100 105 110

Ser Leu Lys Leu Met Glu Lys Phe Leu Ala Asn His Asp Tyr Ile Ala

115 120 125

Gly Ser Lys Ile Ser Tyr Ala Asp Phe Tyr Leu Tyr Glu Tyr Leu Cys

130 135 140

Arg Met Lys Val Met Val Pro Glu Val Tyr Gly Gln Phe Glu Asn Leu

145 150 155 160

Lys Lys Phe Val Glu Arg Ile Glu Ser Leu Pro Arg Ile Ala Glu Tyr

165 170 175

Ile Lys Lys Gln Lys Pro Thr Thr Phe Asn Ala Ser Leu Ala Lys Trp

180 185 190

Asn Gly Ser Tyr Ala

195

<210> 2

<211> 594

<212> DNA

<213> Dermatophagoides farinae

<400> 2

atgttggcat atgccggtgt tgattatgtt gataaacgtt ataatattgg tccgaatttt 60

gatcgttcag aatggttgaa tgaaaaattc aatttgggtt tagatttccc aaatttgcca 120

tattacatgg atggtgatgt taaaattacc caatcgatgg ccattcttcg ttatttggcc 180

cgtaaatata acatggatgg tactaatgaa caggaacgtt tacgaatttc aatggctgaa 240

caacaaacac atgatatgtt tatggccatg gtccgtattt gttatgatcc aaatatggaa 300

aacttacgag ttgattattt gaaaacattg cccgattcat tgaaattgat ggaaaaattt 360

ttagctaatc atgattatat tgccggttca aaaatttctt atgcagattt ttatttgtat 420

gaatatttgt gccgtatgaa agtcatggta ccagaagtat atggacaatt tgaaaatttg 480

aaaaaatttg ttgaacgcat tgaatcattg ccacgtattg ccgaatacat taagaaacaa 540

aaaccaacaa cattcaatgc atcattggct aaatggaatg gttcttatgc ctaa 594

<210> 3

<211> 356

<212> PRT

<213> Dermatophagoides farinae

<400> 3

Met Val Asp Gln Ala Thr Leu Ser Lys Leu Glu Ala Gly Phe Gln Lys

1 5 10 15

Leu Gln Asn Ala Gln Asp Cys His Ser Leu Leu Lys Lys Tyr Leu Thr

20 25 30

Arg Asp Val Leu Asp Gln Leu Lys Thr Lys Lys Thr Asp Met Gly Ala

35 40 45

Thr Leu Leu Asp Val Ile Gln Ser Gly Val Glu Asn Leu Asp Ser Gly

50 55 60

Val Gly Ile Tyr Ala Pro Asp Ala Gln Ser Tyr Lys Thr Phe Ala Ala

65 70 75 80

Leu Phe Asp Pro Ile Ile Asp Asp Tyr His Lys Gly Phe Lys Pro Thr

85 90 95

Asp Lys His Pro Gln Thr Asp Phe Gly Asn Ile Glu His Phe Val Asn

100 105 110

Val Asp Pro Lys Asn Glu Tyr Val Ile Ser Thr Arg Val Arg Cys Gly

115 120 125

Arg Ser Leu Lys Gly Tyr Pro Phe Asn Pro Met Leu Thr Glu Ala Gln

130 135 140

Tyr Lys Glu Met Glu Thr Lys Val Lys Gly Gln Leu Ala Thr Phe Glu

145 150 155 160

Gly Glu Leu Lys Gly Thr Tyr Tyr Pro Leu Leu Gly Met Asp Lys Ala

165 170 175

Thr Gln Gln Lys Leu Ile Asp Asp His Phe Leu Phe Lys Glu Gly Asp

180 185 190

Arg Phe Leu Gln Ala Ala Asn Ala Cys Arg Tyr Trp Pro Val Gly Arg

195 200 205

Gly Ile Phe His Asn Asp Lys Lys Thr Phe Leu Met Trp Val Asn Glu

210 215 220

Glu Asp His Leu Arg Ile Ile Ser Met Gln Lys Gly Gly Asp Leu Lys

225 230 235 240

Glu Val Phe Gly Arg Leu Val Lys Ala Val Lys His Ile Glu Gln Lys

245 250 255

Ile Pro Phe Ser Arg Asp Asp Arg Leu Gly Tyr Leu Thr Phe Cys Pro

260 265 270

Thr Asn Leu Gly Thr Thr Ile Arg Ala Ser Val His Ile Lys Leu Pro

275 280 285

Lys Leu Ala Ala Asp Arg Lys Lys Leu Glu Glu Val Ala Ala Arg Tyr

290 295 300

Asn Leu Gln Val Arg Gly Thr Ala Gly Glu His Thr Glu Ser Val Gly

305 310 315 320

Gly Ile Tyr Asp Ile Ser Asn Lys Arg Arg Met Gly Leu Thr Glu Tyr

325 330 335

Gln Ala Val Lys Glu Met Gln Asp Gly Ile Ile Glu Leu Ile Lys Met

340 345 350

Glu Lys Ser Leu

355

<210> 4

<211> 1071

<212> DNA

<213> Dermatophagoides farinae

<400> 4

atggtcgatc aagctaccct gagtaaattg gaagccggtt tccaaaaatt acagaatgct 60

caagattgtc attcattgtt gaaaaagtat ttgactcgcg atgtgttaga tcaactcaag 120

acgaaaaaga ccgacatggg cgcaacatta ttggatgtta tccaatctgg cgtggaaaac 180

ctggacagtg gtgttggtat ctatgctcct gatgctcaat catacaaaac atttgctgca 240

ttgttcgatc caatcattga tgattaccat aaaggcttca aaccgaccga taaacatccg 300

caaactgatt tcggcaatat cgaacacttt gtcaatgttg atcctaaaaa cgaatacgtc 360

atttctactc gtgttcgatg tggccgttcg ttgaaaggct atccattcaa ccctatgttg 420

acagaggctc aatacaaaga aatggaaacc aaagtgaaag gacaattggc cacattcgaa 480

ggcgaattga aaggcaccta ttacccattg ttgggaatgg ataaagctac tcaacagaaa 540

ttgatcgacg atcatttctt gttcaaggaa ggtgatcgat tcttgcaagc tgccaatgca 600

tgtcgttact ggcctgttgg tcgtggtatc ttccacaatg acaaaaaaac attcttgatg 660

tgggtaaacg aagaagatca tttgcgtatc atttccatgc aaaaaggtgg cgatctaaaa 720

gaggtctttg gacgtttggt caaggctgtc aagcacattg aacaaaagat tccattctct 780

cgtgatgacc gtctcggtta tttgacattc tgtccaacca atcttggcac aaccatccgt 840

gcttcggttc atatcaaact tccgaaattg gccgctgacc gtaagaagtt ggaagaagtg 900

gctgctcgtt acaatcttca agtgcgtggc actgcaggtg aacataccga aagtgtgggt 960

ggtatctatg atattagtaa caaacgacga atgggtctca ccgaatacca agctgttaag 1020

gaaatgcaag atggcatcat tgaattgatt aaaatggaaa aatcattgta a 1071

Claims (3)

1. Dust mite allergen Derf 8 and Der f 20, it is characterized in that by the aminoacid sequence shown in SEQ ID NO:1 and SEQ ID NO:3, being formed respectively.
2. 2. the gene of coding dust mite allergen Der f 8 and Der f 20, is characterized in that the nucleotide sequence shown in SEQ ID NO:2 and SEQ ID NO:4, consisting of respectively.
3. 3. dust mite allergen Der f 8 claimed in claim 1 and Der f 20 application in preparation treatment dust mite allergy disease medicament.

Images