CN103059119A - Dust mite allergen Derf28 and genes and application thereof - Google Patents

Abstract

The invention relates to dust mite allergen Derf28 and genes and an application thereof, and belongs to the field of biomedicine. A new natural allergen Derf28 in a manner of separating and purifying from the dust mites is obtained, and encoding genes of the allergen are cloned. The dust mite allergen Derf28 disclosed by the invention comprises 659 amino acids. The first-stage structure of the amino acid sequence of the allergen is shown in SEQ ID No.1. The sequences of the encoding genes of the allergens comprise 1980 nucleotides, wherein the serial number of the gene nucleotide sequence in GenBank is KC305502. The dust mite allergen has the following beneficial effects that the dust mite allergen Derf28 and the genes thereof are provided, and the dust mite allergen Derf28 can be applied to preparation of drugs for treating dust mite anaphylactic diseases.

Description

Dust mite allergen Der f 28 and gene and application

Technical field:

The present invention relates to a kind of dust mite allergen Der f 28 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 invention is to a kind of dust mite allergen Der f 28 and gene and application.

Technical scheme of the present invention comprises preparation and the determined amino acid sequence of dust mite allergen Der f 28, the clone of dust mite allergen Der f 28 gene orders, evaluation three partial contents of Der f 28 allergenicities.

Der f 28 is our separation and purification obtains from the homogenate of dirt mite first a kind of new allergen proteins, and it belongs to heat-shocked-70 protein family (Heat shock protein 70), and molecular weight is 70 kDa approximately.Der f 28 is comprised of 659 amino acid, its aminoacid sequence following (SEQ ID NO:1):

MSKTPAIGIDLGTTYSCVGVFQHGKVEIIANDQGNRTTPSYVGFTDTERLIGDAAKNQVA 60

MNPSNTVFDAKRLIGRKFDETTVQADMKHWPFKVIEKGNKPAIEVEFKGETKQFIPEEIS 120

SMVLVKMRETAEAYLGGTVNNAVITVPAYFNDSQRQATKDSGLIAGLNVLRIINEPTAAA 180

IAYGLDKKGGAGERNVLIFDLGGGTFDVSLLTIEEGIFEVKSTAGDTHLGGEDFDNRLVN 240

HFVKEFKRKHKKDLTTNARALRRLRTSCERAKRTLSSAAQTSIEIDSLFEGIDFYTSITR 300

ARFEELCADLFRSTMEPVERVLRDAKTDKSSVNEIVLVGGSTRIPKIQRLVADFFNKDPN 360

KSINPDEAIAYGAAVQASILSGDTSSKSTNEILLLDVAPLSLGIETAGGVMTALIKRDTT 420

IPTRSPRLSLPTPTTSLVSRFRSTRVSVLAPRTTTSLVSSSSPVSPCSSWCSSDRGHFDV 480

DANGIMNVGAVEKGTGKTNKITITNDKGRLSKEEIERMLAEAEKYKAEDEAEAARIHAKN 540

GPESYAYSLRNTVNEGKLSISDSDKEKLRARLTRLSTGSTTTRPPARRSTTLNRRSSRVL 600

PTPSFWLPTVVLAVVVPPLVVTPVLVALVVLTRLRSALRSLTKKVFNGVFLFPVLFDVS 659

Dust mite allergen Der f 28 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 KC305502) of dust mite allergen Der f 28 is comprised of 1980 Nucleotide, and 5 ' end to 3 ' terminal sequence of this gene is (nucleotides sequence is classified SEQ ID NO:2 as):

atgagtaaaa ctcctgctat cggtattgat ctgggcacaa cttactcctg tgttggtgtg 60

ttccagcacg gaaaagtcga aatcatcgcc aacgaccagg gaaaccgaac cacgcctagc 120

tatgtgggat tcacggacac cgaaaggctt attggtgatg ccgctaaaaa tcaggtagcg 180

atgaatccgt ctaacaccgt cttcgacgcc aagcgtctga tcggtcgtaa gtttgatgag 240

actacggtcc aggctgacat gaagcactgg cccttcaagg tcatcgagaa gggcaacaag 300

cccgctatcg aggttgagtt caagggtgag accaagcagt tcattcccga ggagatctct 360

tccatggtcc tggtcaagat gcgtgagacc gctgaggctt acctcggtgg caccgtcaac 420

aacgccgtca tcactgtccc cgcctacttc aacgactctc agcgccaggc caccaaggac 480

tctggtctca tcgccggtct taacgtcctc cgtatcatca acgagcccac cgctgccgcc 540

attgcctacg gtctcgacaa gaagggcggc gctggcgagc gcaacgtcct gatcttcgac 600

ttgggtggtg gtactttcga tgtttctctc ttgaccattg aggagggtat cttcgaggtc 660

aagtctaccg ctggtgacac tcacttgggt ggtgaggact tcgacaaccg tctcgtcaac 720

cacttcgtca aggagttcaa gcgtaagcac aagaaggacc tgaccaccaa cgctcgtgct 780

ctccgtcgct tgcgtacctc ttgcgagcgt gcgaagcgta ccctctcttc cgccgcccag 840

acctccattg agatcgactc tctcttcgag ggtatcgact tctacacctc catcacccgt 900

gcccgtttcg aggaactctg cgcagacctc ttccgctcca ccatggagcc cgtcgagcgt 960

gtcctccgtg acgctaagac cgacaagtct tccgtcaacg agattgtcct cgtcggtggt 1020

tctactcgta ttcccaagat ccagcgtctc gtcgccgact tcttcaacaa ggaccccaac 1080

aagtctatca accctgacga ggctattgcc tacggtgctg ccgtccaggc ctccatcctg 1140

tccggtgaca cttcctccaa gtcgaccaac gagattctcc tgctcgatgt tgccccgctt 1200

tctctcggta tcgagaccgc tggtggtgtc atgaccgccc tcatcaagcg tgacaccact 1260

atccctacca gaagtccgag actttctcta cctactccga caaccagcct ggtgtctcga 1320

ttcaggtcta cgagggtgag cgtgctcgca ccaaggacaa caacctcctt ggtaagttcg 1380

agctctccgg tatccccctg ctcctcgtgg tgttcctcag atcgaggtca cttcgatgtc 1440

gacgccaacg gtatcatgaa cgtcggcgct gttgagaagg gcactggcaa gaccaacaag 1500

atcaccatca ccaacgacaa gggccgtctt tcgaaggagg agattgagcg catgcttgct 1560

gaggccgaga agtacaaggc tgaagatgag gctgaggctg ctcgtatcca cgccaagaac 1620

ggtcccgagt cctacgctta ctccctgaga aacaccgtca acgagggcaa gctcagcatc 1680

agcgactccg acaaggaaaa gctcagggca agattgacga gactgtcaac tggctcgaca 1740

acaaccagac cgccagcaag gaggagtacg actctcaaca gaaggagctc gagggtattg 1800

ccaaccccat cattttggct gcctacggtg gtgctggcgg tggtggtccc gcccctggtg 1860

gtgacgccgg tgctggtggc actcgtggtg ctgacgaggt tgaggagcgc cctgaggagc 1920

ttgactaaaa aggtttttaa tggtgttttt ctttttcctg ttctctttga cgtttcttga 1980

Der f 28 Western blot by being purified to detect and find, from 41 parts of dust mite allergy patients serums, Der f 28 is positive with wherein 28 parts, and the positive reaction rate is 68.3%.Elisa detects discovery, and positive group of absorption value at the 450nm place is approximately 3.7 times of negative control group.Suppress experiment by Elisa and find, Der f 28 dependence modes in gradient suppress dust mite allergy patients serum IgE is combined with coated dirt mite homogenate.Clinical skin acupuncture experiment shows, for 10 dust mite allergy patients, Der f 28 is positive with the skin acupuncture experiment of 7 wherein, and the positive reaction rate is 70%.In the basophilic cell activation experiment, the percentage that Der f 28 acts on CD63 that dust mite allergy patient peripheral blood basophilic cell induces and the two positive cells of CCR3 is approximately 4.8 times of negative control group.

Above-mentioned anaphylactogen detects to be found, Der f 28 has stronger allergenicity, is the main allergen 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 allergen Der f 28 and gene thereof are provided, and dust mite allergen Der f 28 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 after the peak of anion-exchange column Resource Q type figure from dirt mite homogenate molecular sieve Sephadex-G75 II peak.

Fig. 1-C be II peak in Fig. 1-B after the peak of Resource Q type figure, what insert is Der f 28 electrophorograms.

Fig. 2-A is Der f 28 and wherein 9 dust mite allergy patients serums and 1 negative control Western blot result.

The Elisa result that Fig. 2-B is Der f 28 and dust mite allergy patients serum IgE effect, wherein Group1 serum used is from Healthy People, and as negative control, Group2 serum used is from the dust mite allergy patient.

The basophilic cell activation results that Fig. 3-A to Fig. 3-B is Der f 28, wherein:

Fig. 3-A basophilic cell used is from Der f 28 irritated patients' peripheral blood, Group1 is not used as negative control with Der f 28 activation, Fig. 3-B basophilic cell used is from the peripheral blood of Healthy People, and Group1 is not used as negative control with Der f 28 activation.

Its mid point 9 of Western blot that Fig. 4 is dirt mite homogenate Sephadex-G75 II peak and 10 is Der f 28.

Embodiment:

Embodiment mono-: evaluation and the determined amino acid sequence thereof of dust mite allergen Der f 28

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 Der f 28

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 II peak, as shown in Figure 1B, the II peak in Figure 1B continued Resource Q to peak type figure, and as shown in Figure 1 C, what in Fig. 1 C, insert is Der f 28 electrophorograms to peak type figure.

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 clone of dust mite allergen Der f 28 genes

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, dust mite allergen Der f 28 gene order amplification and mensuration:

Aminoacid sequence according to separation and purification gained dust mite allergen Der f 28 in embodiment mono-, we have designed a degenerated primer Der f 28-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 28-F: 5’-AA(A/G)TT(T/C)GA(T/C)GA(A/G)AC(A/T/C/G)AC(A/T/C/G)GT

(A/T/C/G)CA(A/G)-3’。

Wherein, the base in bracket means degenerated primer.

Der f 28-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 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 28 is comprised of 1980 Nucleotide, and 5 ' end to 3 ' terminal sequence is:

1 ATGAGTAAAACTCCTGCTATCGGTATTGATCTGGGCACAACTTACTCCTGTGTTGGTGTG

1 M S K T P A I G I D L G T T Y S C V G V

61 TTCCAGCACGGAAAAGTCGAAATCATCGCCAACGACCAGGGAAACCGAACCACGCCTAGC

21 F Q H G K V E I I A N D Q G N R T T P S

121 TATGTGGGATTCACGGACACCGAAAGGCTTATTGGTGATGCCGCTAAAAATCAGGTAGCG

41 Y V G F T D T E R L I G D A A K N Q V A

181 ATGAATCCGTCTAACACCGTCTTCGACGCCAAGCGTCTGATCGGTCGTAAGTTTGATGAG

61 M N P S N T V F D A K R L I G R K F D E

241 ACTACGGTCCAGGCTGACATGAAGCACTGGCCCTTCAAGGTCATCGAGAAGGGCAACAAG

81 T T V Q A D M K H W P F K V I E K G N K

301 CCCGCTATCGAGGTTGAGTTCAAGGGTGAGACCAAGCAGTTCATTCCCGAGGAGATCTCT

101 P A I E V E F K G E T K Q F I P E E I S

361 TCCATGGTCCTGGTCAAGATGCGTGAGACCGCTGAGGCTTACCTCGGTGGCACCGTCAAC

121 S M V L V K M R E T A E A Y L G G T V N

421 AACGCCGTCATCACTGTCCCCGCCTACTTCAACGACTCTCAGCGCCAGGCCACCAAGGAC

141 N A V I T V P A Y F N D S Q R Q A T K D

481 TCTGGTCTCATCGCCGGTCTTAACGTCCTCCGTATCATCAACGAGCCCACCGCTGCCGCC

161 S G L I A G L N V L R I I N E P T A A A

541 ATTGCCTACGGTCTCGACAAGAAGGGCGGCGCTGGCGAGCGCAACGTCCTGATCTTCGAC

181 I A Y G L D K K G G A G E R N V L I F D

601 TTGGGTGGTGGTACTTTCGATGTTTCTCTCTTGACCATTGAGGAGGGTATCTTCGAGGTC

201 L G G G T F D V S L L T I E E G I F E V

661 AAGTCTACCGCTGGTGACACTCACTTGGGTGGTGAGGACTTCGACAACCGTCTCGTCAAC

221 K S T A G D T H L G G E D F D N R L V N

721 CACTTCGTCAAGGAGTTCAAGCGTAAGCACAAGAAGGACCTGACCACCAACGCTCGTGCT

241 H F V K E F K R K H K K D L T T N A R A

781 CTCCGTCGCTTGCGTACCTCTTGCGAGCGTGCGAAGCGTACCCTCTCTTCCGCCGCCCAG

261 L R R L R T S C E R A K R T L S S A A Q

841 ACCTCCATTGAGATCGACTCTCTCTTCGAGGGTATCGACTTCTACACCTCCATCACCCGT

281 T S I E I D S L F E G I D F Y T S I T R

901 GCCCGTTTCGAGGAACTCTGCGCAGACCTCTTCCGCTCCACCATGGAGCCCGTCGAGCGT

301 A R F E E L C A D L F R S T M E P V E R

961 GTCCTCCGTGACGCTAAGACCGACAAGTCTTCCGTCAACGAGATTGTCCTCGTCGGTGGT

321 V L R D A K T D K S S V N E I V L V G G

1021 TCTACTCGTATTCCCAAGATCCAGCGTCTCGTCGCCGACTTCTTCAACAAGGACCCCAAC

341 S T R I P K I Q R L V A D F F N K D P N

1081 AAGTCTATCAACCCTGACGAGGCTATTGCCTACGGTGCTGCCGTCCAGGCCTCCATCCTG

361 K S I N P D E A I A Y G A A V Q A S I L

1141 TCCGGTGACACTTCCTCCAAGTCGACCAACGAGATTCTCCTGCTCGATGTTGCCCCGCTT

381 S G D T S S K S T N E I L L L D V A P L

1201 TCTCTCGGTATCGAGACCGCTGGTGGTGTCATGACCGCCCTCATCAAGCGTGACACCACT

401 S L G I E T A G G V M T A L I K R D T T

1261 ATCCCTACCAGAAGTCCGAGACTTTCTCTACCTACTCCGACAACCAGCCTGGTGTCTCGA

421 I P T R S P R L S L P T P T T S L V S R

1321 TTCAGGTCTACGAGGGTGAGCGTGCTCGCACCAAGGACAACAACCTCCTTGGTAAGTTCG

441 F R S T R V S V L A P R T T T S L V S S

1381 AGCTCTCCGGTATCCCCCTGCTCCTCGTGGTGTTCCTCAGATCGAGGTCACTTCGATGTC

461 S S P V S P C S S W C S S D R G H F D V

1441 GACGCCAACGGTATCATGAACGTCGGCGCTGTTGAGAAGGGCACTGGCAAGACCAACAAG

481 D A N G I M N V G A V E K G T G K T N K

1501 ATCACCATCACCAACGACAAGGGCCGTCTTTCGAAGGAGGAGATTGAGCGCATGCTTGCT

501 I T I T N D K G R L S K E E I E R M L A

1561 GAGGCCGAGAAGTACAAGGCTGAAGATGAGGCTGAGGCTGCTCGTATCCACGCCAAGAAC

521 E A E K Y K A E D E A E A A R I H A K N

1621 GGTCCCGAGTCCTACGCTTACTCCCTGAGAAACACCGTCAACGAGGGCAAGCTCAGCATC

541 G P E S Y A Y S L R N T V N E G K L S I

1681 AGCGACTCCGACAAGGAAAAGCTCAGGGCAAGATTGACGAGACTGTCAACTGGCTCGACA

561 S D S D K E K L R A R L T R L S T G S T

1741 ACAACCAGACCGCCAGCAAGGAGGAGTACGACTCTCAACAGAAGGAGCTCGAGGGTATTG

581 T T R P P A R R S T T L N R R S S R V L

1801 CCAACCCCATCATTTTGGCTGCCTACGGTGGTGCTGGCGGTGGTGGTCCCGCCCCTGGTG

601 P T P S F W L P T V V L A V V V P P L V

1861 GTGACGCCGGTGCTGGTGGCACTCGTGGTGCTGACGAGGTTGAGGAGCGCCCTGAGGAGC

621 V T P V L V A L V V L T R L R S A L R S

1921 TTGACTAAAAAGGTTTTTAATGGTGTTTTTCTTTTTCCTGTTCTCTTTGACGTTTCTTGA

641 L T K K V F N G V F L F P V L F D V S *

Wherein, the part of underscore and the Der f 28 be purified to carry out the sequence that amino acid sequencing obtains and are complementary.* be terminator codon.

Embodiment tri-: the allergenicity of dust mite allergen Der f 28 detects

The dust mite allergen Der f 28 that separation and purification is obtained carries out following allergenicity detection.

One, the Western blot of Der f 28 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 28 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 the dust mite allergy patients 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, the Elisa of Der f 28 experiment

With coating buffer, Der f 28 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 0D450 nm.

Three, the competitive Elisa of Der f 28 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 Der f 28 room temperature effect 1 hour, two anti-1:2000 dilution, the absorption values of detection OD450 of pressing.

Four, Der f 28 induces 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 28, 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 be used the common size statement of CD63 and the two positive basophilic cells of CCR3.

Five, the skin acupuncture of Der f 28 experiment

Drop in patient's forearm palmar skin with the Der f 28 of physiological saline solution, pass Der f 28 drop prick skin with special pricking needles, a small amount of Der f 28 is entered in skin, dry the Der f 28 left over, read result after 15 min, make feminine gender and positive control with physiological saline and histamine respectively.Result is as table 1.

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

SEQUENCE LISTING

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

<120 > dust mite allergen Der f 28 and gene thereof and application

<130> 1

<160> 2

<170> PatentIn version 3.3

<210> 1

<211> 659

<212> PRT

<213> Dermatophagoides farinae

<400> 1

Met Ser Lys Thr Pro Ala Ile Gly Ile Asp Leu Gly Thr Thr Tyr Ser

1 5 10 15

Cys Val Gly Val Phe Gln His Gly Lys Val Glu Ile Ile Ala Asn Asp

20 25 30

Gln Gly Asn Arg Thr Thr Pro Ser Tyr Val Gly Phe Thr Asp Thr Glu

35 40 45

Arg Leu Ile Gly Asp Ala Ala Lys Asn Gln Val Ala Met Asn Pro Ser

50 55 60

Asn Thr Val Phe Asp Ala Lys Arg Leu Ile Gly Arg Lys Phe Asp Glu

65 70 75 80

Thr Thr Val Gln Ala Asp Met Lys His Trp Pro Phe Lys Val Ile Glu

85 90 95

Lys Gly Asn Lys Pro Ala Ile Glu Val Glu Phe Lys Gly Glu Thr Lys

100 105 110

Gln Phe Ile Pro Glu Glu Ile Ser Ser Met Val Leu Val Lys Met Arg

115 120 125

Glu Thr Ala Glu Ala Tyr Leu Gly Gly Thr Val Asn Asn Ala Val Ile

130 135 140

Thr Val Pro Ala Tyr Phe Asn Asp Ser Gln Arg Gln Ala Thr Lys Asp

145 150 155 160

Ser Gly Leu Ile Ala Gly Leu Asn Val Leu Arg Ile Ile Asn Glu Pro

165 170 175

Thr Ala Ala Ala Ile Ala Tyr Gly Leu Asp Lys Lys Gly Gly Ala Gly

180 185 190

Glu Arg Asn Val Leu Ile Phe Asp Leu Gly Gly Gly Thr Phe Asp Val

195 200 205

Ser Leu Leu Thr Ile Glu Glu Gly Ile Phe Glu Val Lys Ser Thr Ala

210 215 220

Gly Asp Thr His Leu Gly Gly Glu Asp Phe Asp Asn Arg Leu Val Asn

225 230 235 240

His Phe Val Lys Glu Phe Lys Arg Lys His Lys Lys Asp Leu Thr Thr

245 250 255

Asn Ala Arg Ala Leu Arg Arg Leu Arg Thr Ser Cys Glu Arg Ala Lys

260 265 270

Arg Thr Leu Ser Ser Ala Ala Gln Thr Ser Ile Glu Ile Asp Ser Leu

275 280 285

Phe Glu Gly Ile Asp Phe Tyr Thr Ser Ile Thr Arg Ala Arg Phe Glu

290 295 300

Glu Leu Cys Ala Asp Leu Phe Arg Ser Thr Met Glu Pro Val Glu Arg

305 310 315 320

Val Leu Arg Asp Ala Lys Thr Asp Lys Ser Ser Val Asn Glu Ile Val

325 330 335

Leu Val Gly Gly Ser Thr Arg Ile Pro Lys Ile Gln Arg Leu Val Ala

340 345 350

Asp Phe Phe Asn Lys Asp Pro Asn Lys Ser Ile Asn Pro Asp Glu Ala

355 360 365

Ile Ala Tyr Gly Ala Ala Val Gln Ala Ser Ile Leu Ser Gly Asp Thr

370 375 380

Ser Ser Lys Ser Thr Asn Glu Ile Leu Leu Leu Asp Val Ala Pro Leu

385 390 395 400

Ser Leu Gly Ile Glu Thr Ala Gly Gly Val Met Thr Ala Leu Ile Lys

405 410 415

Arg Asp Thr Thr Ile Pro Thr Arg Ser Pro Arg Leu Ser Leu Pro Thr

420 425 430

Pro Thr Thr Ser Leu Val Ser Arg Phe Arg Ser Thr Arg Val Ser Val

435 440 445

Leu Ala Pro Arg Thr Thr Thr Ser Leu Val Ser Ser Ser Ser Pro Val

450 455 460

Ser Pro Cys Ser Ser Trp Cys Ser Ser Asp Arg Gly His Phe Asp Val

465 470 475 480

Asp Ala Asn Gly Ile Met Asn Val Gly Ala Val Glu Lys Gly Thr Gly

485 490 495

Lys Thr Asn Lys Ile Thr Ile Thr Asn Asp Lys Gly Arg Leu Ser Lys

500 505 510

Glu Glu Ile Glu Arg Met Leu Ala Glu Ala Glu Lys Tyr Lys Ala Glu

515 520 525

Asp Glu Ala Glu Ala Ala Arg Ile His Ala Lys Asn Gly Pro Glu Ser

530 535 540

Tyr Ala Tyr Ser Leu Arg Asn Thr Val Asn Glu Gly Lys Leu Ser Ile

545 550 555 560

Ser Asp Ser Asp Lys Glu Lys Leu Arg Ala Arg Leu Thr Arg Leu Ser

565 570 575

Thr Gly Ser Thr Thr Thr Arg Pro Pro Ala Arg Arg Ser Thr Thr Leu

580 585 590

Asn Arg Arg Ser Ser Arg Val Leu Pro Thr Pro Ser Phe Trp Leu Pro

595 600 605

Thr Val Val Leu Ala Val Val Val Pro Pro Leu Val Val Thr Pro Val

610 615 620

Leu Val Ala Leu Val Val Leu Thr Arg Leu Arg Ser Ala Leu Arg Ser

625 630 635 640

Leu Thr Lys Lys Val Phe Asn Gly Val Phe Leu Phe Pro Val Leu Phe

645 650 655

Asp Val Ser

<210> 2

<211> 1980

<212> DNA

<213> Dermatophagoides farinae

<400> 2

atgagtaaaa ctcctgctat cggtattgat ctgggcacaa cttactcctg tgttggtgtg 60

ttccagcacg gaaaagtcga aatcatcgcc aacgaccagg gaaaccgaac cacgcctagc 120

tatgtgggat tcacggacac cgaaaggctt attggtgatg ccgctaaaaa tcaggtagcg 180

atgaatccgt ctaacaccgt cttcgacgcc aagcgtctga tcggtcgtaa gtttgatgag 240

actacggtcc aggctgacat gaagcactgg cccttcaagg tcatcgagaa gggcaacaag 300

cccgctatcg aggttgagtt caagggtgag accaagcagt tcattcccga ggagatctct 360

tccatggtcc tggtcaagat gcgtgagacc gctgaggctt acctcggtgg caccgtcaac 420

aacgccgtca tcactgtccc cgcctacttc aacgactctc agcgccaggc caccaaggac 480

tctggtctca tcgccggtct taacgtcctc cgtatcatca acgagcccac cgctgccgcc 540

attgcctacg gtctcgacaa gaagggcggc gctggcgagc gcaacgtcct gatcttcgac 600

ttgggtggtg gtactttcga tgtttctctc ttgaccattg aggagggtat cttcgaggtc 660

aagtctaccg ctggtgacac tcacttgggt ggtgaggact tcgacaaccg tctcgtcaac 720

cacttcgtca aggagttcaa gcgtaagcac aagaaggacc tgaccaccaa cgctcgtgct 780

ctccgtcgct tgcgtacctc ttgcgagcgt gcgaagcgta ccctctcttc cgccgcccag 840

acctccattg agatcgactc tctcttcgag ggtatcgact tctacacctc catcacccgt 900

gcccgtttcg aggaactctg cgcagacctc ttccgctcca ccatggagcc cgtcgagcgt 960

gtcctccgtg acgctaagac cgacaagtct tccgtcaacg agattgtcct cgtcggtggt 1020

tctactcgta ttcccaagat ccagcgtctc gtcgccgact tcttcaacaa ggaccccaac 1080

aagtctatca accctgacga ggctattgcc tacggtgctg ccgtccaggc ctccatcctg 1140

tccggtgaca cttcctccaa gtcgaccaac gagattctcc tgctcgatgt tgccccgctt 1200

tctctcggta tcgagaccgc tggtggtgtc atgaccgccc tcatcaagcg tgacaccact 1260

atccctacca gaagtccgag actttctcta cctactccga caaccagcct ggtgtctcga 1320

ttcaggtcta cgagggtgag cgtgctcgca ccaaggacaa caacctcctt ggtaagttcg 1380

agctctccgg tatccccctg ctcctcgtgg tgttcctcag atcgaggtca cttcgatgtc 1440

gacgccaacg gtatcatgaa cgtcggcgct gttgagaagg gcactggcaa gaccaacaag 1500

atcaccatca ccaacgacaa gggccgtctt tcgaaggagg agattgagcg catgcttgct 1560

gaggccgaga agtacaaggc tgaagatgag gctgaggctg ctcgtatcca cgccaagaac 1620

ggtcccgagt cctacgctta ctccctgaga aacaccgtca acgagggcaa gctcagcatc 1680

agcgactccg acaaggaaaa gctcagggca agattgacga gactgtcaac tggctcgaca 1740

acaaccagac cgccagcaag gaggagtacg actctcaaca gaaggagctc gagggtattg 1800

ccaaccccat cattttggct gcctacggtg gtgctggcgg tggtggtccc gcccctggtg 1860

gtgacgccgg tgctggtggc actcgtggtg ctgacgaggt tgaggagcgc cctgaggagc 1920

ttgactaaaa aggtttttaa tggtgttttt ctttttcctg ttctctttga cgtttcttga 1980

Claims (3)

1. dust mite allergen Der f 28, is characterized in that this albumen is to separate a kind of new allergen albumen obtained from the homogenate of dirt mite, by 659 amino acid, formed, and molecular weight 70kDa, its aminoacid sequence is shown in SEQ ID NO:1.

2. the gene of coding dust mite allergen Der f 28, is characterized in that GenBank accession KC305502 is comprised of 1980 nucleotide sequences, and its nucleotides sequence is classified as shown in SEQ ID NO:2.

3. the application of the dust mite allergen Der f 28 of claim 1 in preparation treatment dust mite allergy disease medicament.

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