CN103073636A - Dermtophagoides farinae allergens Der f 29 and Der f 30, and genes and applications thereof - Google Patents

Abstract

The invention relates to dermtophagoides farinae allergens Der f 29 and Der f 30, and genes and applications of the Der f 29 and the Der f 30, and belongs to the technical field of biomedicine. The Der f 29 and the Der f 30 are formed by amino acid sequences as shown in SEQ ID NO:1 (sequence identification number:1) and SEQ ID NO:3 respectively. The genes coding the Der f 29 and the Der f 30 are formed by nucleotide sequences as shown in SEQ ID NO:2 and SEQ ID NO:4 respectively. The Der f 29 and the Der f 30 are applied in preparing a medicine for treating a dermtophagoides farinae anaphylactic disease. The Der f 29 and the Der f 30 and the genes of the Der f 29 and the Der f 30 are provided, so that the Der f 29 and the Der f 30 can be applied in preparing the medicine for treating the dermtophagoides farinae anaphylactic disease.

Description

Dust mite allergen Derf29 and Derf30 and gene thereof and application

Technical field:

The present invention relates to a kind of dust mite allergen Der f 29 and Der f 30 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 that the dirt mite contains nearly more than 30 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 29 and Der f 30 and gene and application.

Technical scheme of the present invention is as follows:

Der f 29 is our separation and purification obtains from the homogenate of dirt mite first a kind of new allergen proteins, and it belongs to a class Peptidyl-prolyl cis-trans isomerase protein family, the about 15kDa of molecular weight.Der f 29 is comprised of 164 amino acid, its aminoacid sequence following (SEQ ID NO:1):

MALPRVFFDIAADNQPLGRIVIELRSDVVPKTAENFRALCTGEKGFGFKSSSFHRIIPNF 60

MIQGGDFTNHNGTGGKSIYGNKFADENFTLQHTGPGIMSMANAGPNTNGSQFFITTVKTT 120

WLDGKHVVFGSVVEGMDIVKKVESYGSQSGKPSKKVTIANCGQL 164

Dust mite allergen Der f 29 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 AY283280.1) of dust mite allergen Der f 29 is comprised of 495 Nucleotide, and 5 ' end to 3 ' terminal sequence of this gene is (nucleotides sequence is classified SEQ ID NO:2 as):

atggcacttc ctcgtgtttt tttcgatatt gctgccgata atcaaccatt gggtcgtatt 60

gtcattgagc tccgtagtga tgttgtgccc aaaacagcgg aaaatttccg tgcactttgc 120

actggtgaaa aaggatttgg ttttaaatca tcctcatttc atcgtatcat acccaatttt 180

atgatccaag gcggtgattt cactaaccat aatggtactg gtggtaaatc catctatggt 240

aacaaatttg ccgatgaaaa tttcacactt caacacaccg gacctggtat catgtccatg 300

gcaaatgccg gtccaaacac caatggttca cagtttttca ttacaaccgt aaagactacc 360

tggttggatg gcaaacacgt tgtttttggt tcggttgtcg aaggaatgga cattgtaaaa 420

aaggtggaaa gctatggctc acaatcgggt aaaccatcca agaaagtgac cattgctaat 480

tgtggtcagc tttaa 495

Der f 30 belongs to iron-binding protein family (Ferritin), and molecular weight is 15 kDa approximately.Der f 30 is comprised of 171 amino acid, its aminoacid sequence following (SEQ ID NO:3):

MAANPESTTKTSRVRMNIQINLEFYASYVYQQMAYHFNRDDVALPGFEKFFDVSSKEERE 60

HAERFMKLQNQRGGRIVLDDIHKPQQQDWSSGLEAMRAALELEKTVNQALLDLHAVATKH 120

NDAQFADFIETHYLTEQVEAIKKLADYITNLERCGPGLGEYLFDRHTLHSS 171

Dust mite allergen Der f 30 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 KC305503) of dust mite allergen Der f 30 is comprised of 516 Nucleotide, and 5 ' end to 3 ' terminal sequence of this gene is (nucleotides sequence is classified SEQ ID NO:4 as):

atggctgcta atcctgaatc aacaaccaaa acttcacgtg tacgaatgaa tattcaaatt 60

aatttggagt tctatgcatc ctatgtatat caacagatgg cctatcattt taatcgtgat 120

gatgttgcat tgcctggttt tgaaaaattt ttcgatgtat catccaaaga agaacgtgaa 180

cacgctgaac gttttatgaa attacagaat caacgtggtg gacgtattgt attggatgat 240

attcataaac cgcaacaaca agattggtca tcaggattgg aagcaatgcg tgctgcattg 300

gaattggaaa aaacagtcaa tcaggcattg ttggatttgc atgccgttgc caccaaacac 360

aatgatgcac aatttgctga ttttattgaa acacattatc taactgaaca agtggaagcc 420

atcaagaaat tggctgatta tattaccaat ttggaacgtt gtggccccgg acttggtgaa 480

tatctttttg atcgtcatac attgcattca tcgtaa 516

Western blot detects discovery, and from 41 parts of dust mite allergy patients serums, Der f 29 is positive with wherein 35 parts, and the positive reaction rate is 85.6%; Der f 30 is positive with wherein 26 parts, and the positive reaction rate is 63.4%.Elisa detects discovery, and positive group of absorption value at the 450nm place of Der f 29 and Der f 30 is respectively 4.6 and 5.1 times of negative control group.Clinical skin acupuncture experiment shows, for 10 dust mite allergy patients, Der f 29 is positive with the skin acupuncture experiment of 7 wherein, and the positive reaction rate is 70%; Der f 30 is positive with the skin acupuncture experiment of 6 wherein, and the positive reaction rate is 60%.In the basophilic cell activation experiment, the percentage that Der f 29 acts on CD63 that dust mite allergy patient peripheral blood basophilic cell induces and the two positive cells of CCR3 is approximately 7.1 times of negative control group; The percentage that Der f 30 acts on CD63 that dust mite allergy patient peripheral blood basophilic cell induces and the two positive cells of CCR3 is approximately 5.9 times of negative control group.

Above-mentioned anaphylactogen detects to be found, Der f 29 and Der f 30 have stronger allergenicity, are the main allergens from the dirt mite, can be as the application of preparation treatment dust mite allergy disease medicament.

Beneficial effect of the present invention is: dust mite allergy Der f 29 and Der f 30 and gene thereof are provided, and dust mite allergen Der f 29 and Der f 30 can be as the application of preparation treatment dust mite allergy disease medicament.

The accompanying drawing explanation:

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

Fig. 1-B is that dirt mite homogenate molecular sieve Sephadex-G75 III peak is after the peak of anion-exchange column Resource Q type figure.

Fig. 1-C is peak type figure and the electrophorogram that Der f 29 crosses Resource Q.

Fig. 1-D is peak type figure and the electrophorogram that Der f 30 crosses Resource Q.

Fig. 2-A is Der f 29 and wherein 9 dust mite allergy patients serums and 1 negative control Western blot result.

Fig. 2-B is Der f 30 and wherein 9 dust mite allergy patients serums and 1 negative control Western blot result.

Fig. 2-C is the Elisa result of Der f 29 and dust mite allergy patients serum IgE effect, wherein organizes 1 use serum from Healthy People, as negative control, organizes 2 serum used from the dust mite allergy patient.

Fig. 2-D is the Elisa result of Der f 29 and dust mite allergy patients serum IgE effect, wherein organizes 1 serum used from Healthy People, as negative control, organizes 2 serum used from the dust mite allergy patient.

Fig. 3-A to Fig. 3-D is the basophilic cell activation results of Der f 29.Wherein:

Fig. 3-A basophilic cell used, from Der f 29 irritated patients' peripheral blood, is organized 1 and with Der f 29 activation, is not used as negative control; Fig. 3-B basophilic cell used is from the peripheral blood of Healthy People, and organizing 1 is also with Der f 29 activation, not to be used as negative control; Fig. 3-C basophilic cell used is from Der f 30 irritated patients' peripheral blood, group 1 is not used as negative control with Der f 30 activation, Fig. 3-D basophilic cell used is from the peripheral blood of Healthy People, and organizing 1 is also with Der f 30 activation, not to be used as negative control.

The Western blot figure that Fig. 4-A is dirt mite homogenate Sephadex-G75 III peak, its mid point 1 is Der f 29.

The Western blot figure that Fig. 4-B is dirt mite homogenate Sephadex-G75 III peak, its mid point 1 is Der f 30.

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 dust mite allergen

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 1A.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.

To continue anion-exchange column Resource Q from molecular sieve gel chromatography Sephadex-G75 III peak, peak type figure is as shown in Fig. 1-B, 2 peaks in Figure 1B continued the peak type figure of Resource Q again as shown in Fig. 1-C and Fig. 1-D, and what insert is Der f 29 and Der f 30 electrophorograms.

Three, two-dimensional electrophoresis and Western blot thereof detect

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.

D. the Western blot of two-dimensional electrophoresis: need to run two two-dimensional electrophoresis for same sample, wherein one for substantive dyeing, one is then used in Western blot, 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.

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 29 in embodiment mono-, we have designed a degenerated primer Der f 29-F and Der f 30-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’-ATTCTAGAGGCCGAGGCGGCCATG-3’。

Der f 29-F: 5’-ATGGC(A/T/C/G)CT(A/T/C/G)CC(A/T/C/G)AG(A/G)GT(A/T/C/G)

TT(T/C)TT(T/C) -3’。

Der f 30-F: 5’-ATGGC(A/T/C/G)GC(A/T/C/G)AA(T/C)CC(A/T/C/G)GA(A/G)

AG(T/C)AC(A/T/C/G) -3’。

Wherein, the base in bracket means degenerated primer.

Degenerated primer 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 30 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 29 is comprised of 495 Nucleotide, and the cDNA sequence of allergen Der f 30 is comprised of 516 Nucleotide.

Embodiment tri-: the allergenicity of dust mite allergen Der f 29 detects

The dust mite allergen Der f 29 that separation and purification is obtained carries out following allergenicity detection.

One, Western blot experiment

The SDS-PAGE glue that the preparation gum concentration is 12%, at the reductive condition electrophoresis.After electrophoresis, utilize the transferring film groove that the Der f 29 on PAGE glue is gone to PVDF upper, the skim-milk with 5% seals 2 hr, and washings adds 4 ℃ of overnight incubation of primary antibodie (primary antibodie is Yao horsefly allergic disease human serum, presses the 1:80 dilution) after washing 3 times.Hatch 1 hr(bis-and resist for the sheep anti human IgE with adding two anti-normal temperature after washings washing 3 times), after again washing 3 times, available two anti-connected horseradish peroxidase substrates develop.

Two, Elisa experiment

With coating buffer, anaphylactogen is diluted to 10 μ g/ml, spends the night with 4 ℃ of 50 μ l are coated, the patients serum press the 1:50 dilution, two anti-be to dilute by 1:2000, finally survey the absorbance value of OD450 nm.

Three, competitive Elisa suppresses experiment

Detailed process: the dirt mite crude extract that is 50 μ g/ml by 50 μ l concentration is coated with 96 orifice plates, and primary antibodie is pressed the 1:30 dilution with 3% BSA, with final concentration, is then 30 to 3 * 10 respectively ^-4the crude extract of μ g/ml and the effect of anaphylactogen room temperature 1 hour, two anti-1:2000 dilution, the absorption values of detection OD450 of pressing.

Four, induce the basophilic cell activation experiment

Utilize the ficoll-hypaque method to separate the peripheral blood lymphocytes (PBMC) in the irritated patients blood of Der f 29, PBMC contains lymphocyte, monocyte and basophilic cell.Anaphylactogen and PBMC that the final concentration of take is 1 μ g/ml are hatched 30 min at 37 ℃, with 37 ℃ of antibody FITC-anti-CD63 and PE-anti-CD193 (CCR3), hatch 20 minutes after washing 3 times again, with flow cytometer, detect after washing 3 times.That wherein positive control is used is anti-IgE, and that negative control is used is wash buffer (cell PBS).The anti-CD193 of PE mark (CCR3) stably express on the basophilic cell film, it can be used as the marker of basophilic cell.On the streaming figure of PBMC, basophilic cell is positioned at lymphocyte and monocytic intersection, utilizes negative control and PE-anti-CD193 (CCR3) that this part cell is irised out (gating).The basophilic cell activation results can mean with the percentage of CD63 and the two positive basophilic cells of CCR3.

Five, skin acupuncture experiment

Drop in patient's forearm palmar skin with the anaphylactogen of physiological saline solution, by special pricking needles prick skin, a small amount of anaphylactogen is entered in skin, dry the anaphylactogen of leaving over, read result after 15 min, make feminine gender and positive control with physiological saline and histamine respectively.Der f 29 results are as table 1, and Der f 29 is as table 2.

29 couples of 10 dust mite allergy patients' of table 1:Der f skin acupuncture experimental result

30 couples of 10 dust mite allergy patients' of table 2:Der f skin acupuncture experimental result

SEQUENCE LISTING

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

<120 > dust mite allergen Der f 29 and Der f 30 and gene and application

<130> 1

<160> 4

<170> PatentIn version 3.3

<210> 1

<211> 164

<212> PRT

<213> Dermatophagoides farinae

<400> 1

Met Ala Leu Pro Arg Val Phe Phe Asp Ile Ala Ala Asp Asn Gln Pro

1 5 10 15

Leu Gly Arg Ile Val Ile Glu Leu Arg Ser Asp Val Val Pro Lys Thr

20 25 30

Ala Glu Asn Phe Arg Ala Leu Cys Thr Gly Glu Lys Gly Phe Gly Phe

35 40 45

Lys Ser Ser Ser Phe His Arg Ile Ile Pro Asn Phe Met Ile Gln Gly

50 55 60

Gly Asp Phe Thr Asn His Asn Gly Thr Gly Gly Lys Ser Ile Tyr Gly

65 70 75 80

Asn Lys Phe Ala Asp Glu Asn Phe Thr Leu Gln His Thr Gly Pro Gly

85 90 95

Ile Met Ser Met Ala Asn Ala Gly Pro Asn Thr Asn Gly Ser Gln Phe

100 105 110

Phe Ile Thr Thr Val Lys Thr Thr Trp Leu Asp Gly Lys His Val Val

115 120 125

Phe Gly Ser Val Val Glu Gly Met Asp Ile Val Lys Lys Val Glu Ser

130 135 140

Tyr Gly Ser Gln Ser Gly Lys Pro Ser Lys Lys Val Thr Ile Ala Asn

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Cys Gly Gln Leu

<210> 2

<211> 495

<212> DNA

<213> Dermatophagoides farinae

<400> 2

atggcacttc ctcgtgtttt tttcgatatt gctgccgata atcaaccatt gggtcgtatt 60

gtcattgagc tccgtagtga tgttgtgccc aaaacagcgg aaaatttccg tgcactttgc 120

actggtgaaa aaggatttgg ttttaaatca tcctcatttc atcgtatcat acccaatttt 180

atgatccaag gcggtgattt cactaaccat aatggtactg gtggtaaatc catctatggt 240

aacaaatttg ccgatgaaaa tttcacactt caacacaccg gacctggtat catgtccatg 300

gcaaatgccg gtccaaacac caatggttca cagtttttca ttacaaccgt aaagactacc 360

tggttggatg gcaaacacgt tgtttttggt tcggttgtcg aaggaatgga cattgtaaaa 420

aaggtggaaa gctatggctc acaatcgggt aaaccatcca agaaagtgac cattgctaat 480

tgtggtcagc tttaa 495

<210> 3

<211> 171

<212> PRT

<213> Dermatophagoides farinae

<400> 3

Met Ala Ala Asn Pro Glu Ser Thr Thr Lys Thr Ser Arg Val Arg Met

1 5 10 15

Asn Ile Gln Ile Asn Leu Glu Phe Tyr Ala Ser Tyr Val Tyr Gln Gln

20 25 30

Met Ala Tyr His Phe Asn Arg Asp Asp Val Ala Leu Pro Gly Phe Glu

35 40 45

Lys Phe Phe Asp Val Ser Ser Lys Glu Glu Arg Glu His Ala Glu Arg

50 55 60

Phe Met Lys Leu Gln Asn Gln Arg Gly Gly Arg Ile Val Leu Asp Asp

65 70 75 80

Ile His Lys Pro Gln Gln Gln Asp Trp Ser Ser Gly Leu Glu Ala Met

85 90 95

Arg Ala Ala Leu Glu Leu Glu Lys Thr Val Asn Gln Ala Leu Leu Asp

100 105 110

Leu His Ala Val Ala Thr Lys His Asn Asp Ala Gln Phe Ala Asp Phe

115 120 125

Ile Glu Thr His Tyr Leu Thr Glu Gln Val Glu Ala Ile Lys Lys Leu

130 135 140

Ala Asp Tyr Ile Thr Asn Leu Glu Arg Cys Gly Pro Gly Leu Gly Glu

145 150 155 160

Tyr Leu Phe Asp Arg His Thr Leu His Ser Ser

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<210> 4

<211> 516

<212> DNA

<213> Dermatophagoides farinae

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atggctgcta atcctgaatc aacaaccaaa acttcacgtg tacgaatgaa tattcaaatt 60

aatttggagt tctatgcatc ctatgtatat caacagatgg cctatcattt taatcgtgat 120

gatgttgcat tgcctggttt tgaaaaattt ttcgatgtat catccaaaga agaacgtgaa 180

cacgctgaac gttttatgaa attacagaat caacgtggtg gacgtattgt attggatgat 240

attcataaac cgcaacaaca agattggtca tcaggattgg aagcaatgcg tgctgcattg 300

gaattggaaa aaacagtcaa tcaggcattg ttggatttgc atgccgttgc caccaaacac 360

aatgatgcac aatttgctga ttttattgaa acacattatc taactgaaca agtggaagcc 420

atcaagaaat tggctgatta tattaccaat ttggaacgtt gtggccccgg acttggtgaa 480

tatctttttg atcgtcatac attgcattca tcgtaa 516

Claims (3)

1. Dust mite allergen Der f 29 and Der f 30, 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 29 and Der f 30, 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 29 claimed in claim 1 and Der f 30 application in preparation treatment dust mite allergy disease medicament.

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