Casein allergen Tyr p12 coding gene, recombinant protein and application thereof
1. A tyrophagus putrescentiae allergen Tyrp12 coding gene is characterized in that the nucleotide sequence of the coding gene is shown in SEQ ID No: 1 is shown.
2. An artificial gene encoding a recombinant protein of tyrophagus putrescentiae allergen Tyrp12, comprising the gene encoding tyrophagus putrescentiae allergen Tyrp12 of claim 1.
3. The artificially synthesized gene according to claim 2, which has a nucleotide sequence shown in SEQ ID No: 2, respectively.
4. Use of the artificially synthesized gene of claim 2 or 3 in the preparation of a tyrophagus putrescentiae allergen Tyr p12 recombinant protein.
5. The use of claim 4, wherein the nucleotide sequence of the synthetic gene is cloned into a prokaryotic expression vector.
6. A Tyrp12 recombinant protein of Tyrophagus putrescentiae allergen has an amino acid sequence shown in SEQ ID No: 3, respectively.
7. Use of the recombinant protein according to claim 6 for the preparation of a reagent for the accurate medical examination of patients with allergic diseases.
Background
Dust mites are one of the most important allergen organisms causing allergic diseases, are widely distributed in indoor mattresses, sofas, pillows, carpets, summer sleeping mats and the like, and are common in the types of dust mites, house dust mites, tropical acarus, Tyrophagus putrescentiae and the like. As an organism, the dust mite extract contains multiple allergen components, which are named as component 1 and component 2 … … in the order of discovery, and nearly 40 components have been discovered. According to the international allergen nomenclature rules, the name of an allergen is the first three letters of its source, generic name (e.g. Dermatophagoides, the first three letters being Der), blank space, the first letter of the species name (e.g. the first letter of pteronyssinus), blank space, and finally the order in which the allergen is found or its clinical meaning is indicated by Arabic numerals, e.g. Derp 1 is named for Derma 1 component of Dermatophagoides allergen 1.
Extracts of individual dust mites are a mixture of allergen components, but the patient's etiology may be related to only one or some of the components. Furthermore, the dust mite extract is of poor quality, such as insufficient content of major components, unstable types and content of contained components, containing non-allergenic substances, and low immunogenicity of one or some of the components. Thus, the use of dust mite extracts in clinical practice causes a number of serious and even life-threatening side effects, so that it is necessary to carry out cumbersome subsequent treatments for their side effects. Therefore, there is a need to test single component allergens whose reagents definitely cause allergic diseases.
According to the WHO/IUIS allergen nomenclature Commission (http:// www.allergen.org /), the reported dust mite allergen components comprise Der f 1-8, Der f 10-11, Der f 13-18 and Der f20-39, the house dust mite allergen components comprise Der p 1-11, Der 13-15, Der 18, Der 20-21, Der 23-26, Der 28-33, Der 36 and Der 37-38, the putrescent butterfish allergen components comprise Tyrp 1-3, 7-8, 10, 13, 20, 28 and 34-36, and the tropical claw-free mite allergen components comprise Blot 1-8, Blot 10-13, Blot 19 and Blot 21. With the rapid development of high-throughput sequencing technology, the sequencing of mitochondrial nuclear genomes and whole genomes of a plurality of mite species is completed, and a means is provided for discovering new allergen components. Although the complete genetic data for dust mites and house dust mites have been published, the 12 th component-encoding gene was not found. At present, only the 12 th component encoding gene of the tropicalis jacobia allergen is published in the official website (www.allergen.org) of the international allergen nomenclature committee.
Disclosure of Invention
The invention aims at providing a tyrophagus putrescentiae allergen Tyrp12 coding gene.
Still another object of the present invention is to provide an artificially synthesized gene encoding tyrophagus putrescentiae allergen Tyrp12 recombinant protein.
The invention further aims to provide application of the artificial synthetic gene in preparation of the tyrophagus putrescentiae allergen Tyr p12 recombinant protein.
Another object of the present invention is to provide a Tyrp12 recombinant protein, a Tyrp allergen, of Tyrophagus putrescentiae.
The invention is realized in such a way that a tyrophagus putrescentiae allergen Tyrp12 coding gene has a nucleotide sequence shown as SEQ ID No: 1 is shown.
The invention further discloses an artificial synthetic gene for coding a tyrophagus putrescentiae allergen Tyrp12 recombinant protein, which comprises the coding gene of the tyrophagus putrescentiae allergen Tyrp12 in claim 1.
Preferably, the nucleotide sequence of the artificially synthesized gene is as shown in SEQ ID No: 2, respectively.
The invention further discloses an application of the artificially synthesized gene in preparation of tyrophagus putrescentiae allergen Tyrp12 recombinant protein.
Preferably, the nucleotide sequence of the artificially synthesized gene is cloned into a prokaryotic expression vector.
The invention further discloses a tyrophagus putrescentiae allergen Tyrp12 recombinant protein, the amino acid sequence of which is shown as SEQ ID No: 3, respectively.
The invention further discloses application of the recombinant protein in preparation of a test reagent for testing patients with allergic diseases in precise medical tests.
The invention overcomes the defects of the prior art and provides a Tyrp12 encoding gene of a Tyrophagus putrescentiae allergen, a recombinant protein and application thereof. Tyrophagus putrescentiae (Tyrophagus putrescentiae) is a storage mite, is also commonly found in indoor dust, is an important inhalant allergen, and causes allergic asthma and/or rhinitis. The National Center for Biotechnology Information, NCBI has published the Whole Genome sequencing of Tyrophagus putrescentiae (BioProjects: PRJNA598686, white Genome Shotgun: INSDC: JAAALH000000000.1), the invention discovers that a section of gene has homology with the 12 th component Blot t 12 of the Tropidotium tropicalis when the Genome is subjected to functional gene annotation, according to the sequence, the invention amplifies the section of code from Tyrophagus putrescentiae Total RNA by using RT-PCR, the sequencing verification confirms the coding gene of Tyr p12, the gene is subjected to codon optimization, artificial synthesis of gene fragment, cloning into pET28a (+) vector, transformation of Escherichia coli, IPTG induced expression and nickel affinity purification in sequence, finally obtains recombinant protein, and the recombinant protein is applied to clinical detection of the positive serum of the Tyrophagus putrescentiae and has the positive rate of 27.27% (3/11).
Compared with the defects and shortcomings of the prior art, the invention has the following beneficial effects: the invention firstly obtains the coding gene of the Tyrp12, and the recombinant protein prepared by the coding gene is used for preparing a test reagent for testing patients with allergic diseases in precise medical science.
Drawings
FIG. 1 is a electrophoresis picture of Tyrophagus putrescentiae Total RNA; wherein, Lane M is DL 2,000DNA Marker, Lane 1 is Tyrophagus putrescentiae Total RNA;
FIG. 2 is a Tyr p12 PCR product agarose gel; wherein lane M is DL 2,000DNA Marker, lane 1 is Tyrp12 gene PCR product (+), and lane 2 is Tyrp12 gene PCR product (-);
FIG. 3 shows the result of agarose gel electrophoresis of the cleavage product of pET28a plasmid, wherein lane M is lambda-Hind III digest, lane 1 is pET28a plasmid, and lane 2 is pET28a-Nde I/EcoR I;
FIG. 4 shows the results of SDS-PAGE identification of the expression of the full-length pET-28a (+) -Tyrp 12 gene; wherein, lane M is Protein MW marker (Broad, lane 1 is pET28a carrier holoprotein, lane 2 is pET28a carrier supernatant, lane 3 is pET28a carrier precipitate, lane 4 is Tyrp12 holoprotein, lane 5 is Tyrp12 supernatant, lane 6 is Tyrp12 precipitate;
FIG. 5 is a TyrP12 SDS-PAGE result; wherein, lane M is Protein MW marker (Broad), lane 1 is pET28a carrier holoprotein, lane 2 is pET28a carrier supernatant, lane 3 is pET28a carrier precipitate, lane 4 is Tyrp12 holoprotein, lane 5 is Tyrp12 supernatant, lane 6 is Tyrp12 precipitate;
FIG. 6 is a Tyrp12 Westernblotting graph; wherein, lane M1 is Precision Plus Protein Standards, lane 1 is pET28a carrier whole Protein, lane 2 is pET28a carrier supernatant, lane 3 is pET28a carrier precipitate, lane 4 is Tyrp12 whole Protein, lane 5 is Tyrp12 supernatant, lane 6 is Tyrp12 precipitate, lane M2 is perfect Protein marker;
FIG. 7 is an electrophoretogram of Tyr P12 supernatant on a Ni column; wherein M is from small to large: 10. 15, 20, 35, 45, 60, 75, 100, 140, 180 KDa;
FIG. 8 is a SDS-PAGE of Tyrp12 protein purification; wherein, the purity of electrophoresis software analysis (gray level analysis) is more than 90 percent;
FIG. 9 shows the positive rate of the recombinant allergen rTyrp 12 detected by IgE-Western Blotting; wherein, Lane M is a prestained protein molecular weight marker, Lanes 1-11 are sera of patients allergic to crude extract of Tyrp, Lane 1-3 are sera of patients detecting rTyrp 12 positive by IgE-Western Blotting, and Lane 12-15 are healthy controls.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Firstly, gene cloning, expression, plasmid construction, sequencing verification
1. Total RNA extraction
Total RNA extraction was performed on Tyrophagus putrescentiae material using RNAioso Plus (Code No.9108), and 1. mu.l of the Total RNA was subjected to 1% agarose gel electrophoresis, as shown in FIG. 1, indicating that the Total RNA extracted can satisfy the requirements of the subsequent experiments.
2. Acquisition of target Gene
2.1 primer design and Synthesis
And designing a primer according to the obtained Tyr p12 fragment sequence.
The upstream primer F1: 5'-CGCGCGGCAGCCATATGAATCTACCAGTTTTTCTCTTGTCCGTC-3' the flow of the air in the air conditioner,
the downstream primer R1: 5'-GACGGAGCTCGAATTCTTATTCGGTAATCTTGGCATTAAATTTGAG-3' are provided.
2.2 reverse transcription
Using TaKaRa PrimeScriptTMRT-PCR Kit (Code No. RR014A) synthesized cDNA, and a negative control was set up. Reaction Total RNA1(μ g, Random 6mers (20 μ M)1 μ L, Oligo dT Primer (2.5 μ M)1 μ L, dNTP mix (10mM each)1 μ L, RNase Free dH2O up to 10. mu.L, reaction condition 65 ℃ for 5min, and standing on ice for 2 min. Then, 4. mu.L of 5 XPrimeScript RT Buffer, 0.5. mu.L of RNase Inhibitor (40U/. mu.L), 0.5. mu.L of PrimeScript RTase (for 2Step), RNase Free dH were added2O5 μ L, placing in a PCR instrument, and carrying out reaction conditions: 30 ℃ for 10min, 45 ℃ for 30min and 70 ℃ for 15 min.
2.3 PCR amplification
2.3.1, PCR amplification using TaKaRa Tks Gflex DNApolymerase (Code No. R060A). PCR reaction system, 1. mu.L of the reverse transcription reaction solution and 2 XGflex PCR Buffer (Mg)2+，dNTPplus)25μL、Tks Gflex DNAPolymerase(1.25units/μL)1μL、INF Primer(20μM)1μL、INR Primer(20μM)1μL、dH2O21. mu.L, total 50. mu.L, reaction conditions: 94 ℃ for 1min1 cycles, then 30 cycles at 98 ℃ for 10sec, 60 ℃ for 15sec, and 68 ℃ for 30 sec.
2.3.2, 5 μ L of each PCR product is subjected to 1% agarose gel electrophoresis, and the electrophoresis detection result shows that: the fragments of interest were amplified to a size consistent with the expected length and the results are shown in FIG. 2.
2.4 PCR product purification
The band of the desired amplification product of about 0.5kbp in length in the above 1 st lane was recovered and purified by cutting the Gel using TaKaRa MiniBEST Agarose Gel DNA Extraction Kit Ver.4.0(Code No. 9762).
2.5 sequencing analysis
The PCR purified product was sequenced using primer F, R:
the upstream primer F2: 5'-ATGAATCTACCAGTTTTTCTCTTGTCCGTC-3' the flow of the air in the air conditioner,
the downstream primer R2: 5'-TTATTCGGTAATCTTGGCATTAAATTTGAG-3', respectively;
the sequencing result is shown in SEQ ID No: 1, the sequencing result is consistent with the sequence of the CDS region overlapping region of the Tyrp12 gene, and subsequent cloning experiments are continued.
3. Vector DNA preparation
3.1 vector restriction
The plasmid pET28a was digested simultaneously with Nde I/EcoR I, and the reaction system pET28a plasmid 1. mu.g, 10 XQuickCut Buffer 5. mu.L, Nde I (10U/. mu.L) 1. mu. L, EcoRI (10U/. mu.L) 1. mu.L, at 37 ℃ for 2 hours. 10. mu.L of the digested product was subjected to 1% agarose gel electrophoresis, and the results are shown in FIG. 3.
3.2 purification of the vector
The digested product of about 5.3kbp in length in lane 2 was recovered and purified by cutting with TaKaRa MiniBESTAgarose Gel DNAextraction KitVer.4.0(Code No.9762), and named Vector DNA.
4. In-Fusion, transformation, positive clone screening and plasmid sequencing
4.1, useHD Cloning Kit (Clontech Code No.639650) In-Fusion reaction of the PCR product with Vector DNA (ca. 50 ng/. mu.L), 2. mu. L, PCR product (ca. 50 ng/. mu.L), 1. mu.L, 5 Xin-Fusion HD Enzyme Premix 2. mu. L, dH2O Up to 10. mu.L, reaction conditions: 15min at 50 ℃.
4.2, 2. mu.L of the In-Fusion reaction solution was thermally transferred to E.coli component Cells JM109(Code No.9052), plated, and cultured overnight at 37 ℃. Selecting positive bacterial colony bacteria, extracting plasmids, sequencing the plasmids by using a primer X '5'-GCTAGT TAT TGC TCA GCG G-3'', and comparing the sequencing result with the sequencing result of a PCR product to be consistent.
5. Tyrp12 protein expression
5.1 seed culture
20 μ L of Tyrp12 glycerol was added to liquid medium of 2mLLB/Kan (50 ng/. mu.L) + Cm (34 ng/. mu.L), and cultured at 37 ℃ under O/N.
5.2 Main culture and Induction
5mL of LB/Kan (50 ng/. mu.L) medium was added to the Glass tube, and 100. mu.L of the seed culture broth was added thereto. Cultured at 37 ℃ until OD600nm became about 0.6, induced by addition of 150mM IPTG 33. mu.L (final 1mM IPTG), and cultured at 37 ℃ for 4 hours. The absorbance before induction was 0.60, and the absorbance before collection was 0.2.
5.3 protein extraction
After the collection, the cells corresponding to 2.0OD were suspended in 320. mu.L PBS and then disrupted by ultrasonication, and the disrupted cell solution was centrifuged (12,000 Xrpm, 5 min).
5.4 electrophoresis of the extracts
Each extract (total protein, supernatant, precipitate) was taken in an amount of 8. mu.L (equivalent to 0.05 OD), and subjected to SDS-PAGE by adding 2. mu.L of 5 XSDS Loading Buffer and heating at 99 ℃ for 10 minutes. Electrophoresis conditions; (c.c.)25 mA/piece, about 70 minutes, using gel; after electrophoresis of 10%, 12.5% and 15% polyacrylamide gels, CBB-R250 was stained, and the stained solution was used for destaining, and the results of SDS-PAGE/CBB staining are shown in FIG. 4.
5.5, Experimental results
According to the result of SDS-PAGE, no expression is obtained, and then codon optimization and artificial synthesis are carried out to express the gene again.
Second, artificially synthesized Gene expression
1. Preparation of DNA fragment
Synthesizing single-chain small-fragment DNA according to the DNA sequence, and splicing the single-chain small-fragment DNA into a complete double-chain DNA fragment by a PCR method. After signal peptide of a Tyrp12 full-length gene is removed and codon optimization is carried out, the gene, 408bp, with the sequence shown as SEQ ID No: 2, the protein sequence coded by the gene is shown as SEQ ID No: 3, respectively.
2. Ligation reaction
2.1 preparing a reaction solution in a Microtube
Insert DNA 1. mu.L (about 80ng), Vector DNA (pET28a (+) -Nde I/EcoR I) 1. mu.L (about 50ng), dH2O6μL、5×In-Fusion HD Enzyme Premix*2μL。
Note:component of HD Cloning Kit (Clontech Code No. 639650).
2.2, 50 ℃ for 15 minutes
2.5. mu.L of the In-Fusion product was transformed into E.coli component Cell JM109(TaKaRa Code No. 9052).
3. Positive clone detection and plasmid DNA extraction
The colonies on the plate were subjected to PCR using the primer "5'-GCT AGT TAT TGC TCA GCG G-3'" to detect the length of the insert in the contained plasmid, and the colonies were subjected to plasmid DNA extraction.
4. DNA sequencing
5. The experimental results are as follows: the insert in the plasmid was completely correct.
6、WesternBlotting
6.1 transformation
mu.L of the plasmid was transferred to a complent cell BL21(DE3), plated with Kan/antibiotic Kan (50 ng/. mu.L) plates, plated with 50. mu.L of the transformation solution, and cultured at 37 ℃ under O/N. The same procedure was performed for the empty vector pET28a +.
6.2 cultivation and Induction
Single colonies containing active fractions of pET-28a (+) -Tyrp 12 were picked up in 2mL LB/Kan (50 ng/. mu.L) medium and cultured O/N at 37 ℃. The same operation was performed with the empty vector. 5mL of LB/Kan (50 ng/. mu.L) medium was added to the Glass tube, and 100. mu.L of the seed culture broth was added thereto. The OD600nm value was about 0.6 in the 37 ℃ culture, and the induction was carried out by adding 150mM IPTG 33. mu.L (final 1mM IPTG), and the culture was carried out at 37 ℃ for 4 hours.
6.3 protein extraction
After the collection, the cells corresponding to 2.0OD were suspended in 320. mu.L PBS and then disrupted by ultrasonication, and the disrupted cell solution was centrifuged (12,000 Xrpm, 5 min).
6.4 electrophoresis of the extracts
Each extract (total protein, supernatant, precipitate) was taken in an amount of 8. mu.L (equivalent to 0.05 OD), and subjected to SDS-PAGE by adding 2. mu.L of 5 XSDS Loading Buffer and heating at 99 ℃ for 10 minutes. Electrophoresis conditions; (c.c.) gel was used at 25 mA/piece, about 70 points; after the 15% polyacrylamide gel electrophoresis is finished, CBB-R250 is dyed, and is decolored by using a decoloration solution. The results of SDS-PAGE/CBB staining are shown in FIG. 5.
6.5、Western Blotting
Respectively shearing a PVDF membrane and filter paper into the same size as the gel, sequentially placing the PVDF membrane, the gel and the filter paper between electrode plates of a membrane rotating instrument after the PVDF membrane and the filter paper are treated by using a membrane rotating buffer solution, starting to rotate the membrane, and setting parameters of the membrane rotating instrument: the current 50mA transfers for 80 min. PVDF membrane was placed in 10mL Blocking buffer containing 1.5% BSA and blocked by placing it flat overnight at 4 ℃. An Antibody reaction was carried out for 1 hour using 9mL of the diluted Penta-His Antibody solution. TBST buffer (20mL) 2 washes; wash TBS buffer washes 3 times. A secondary antibody reaction was performed for 1 hour using 9mL of a diluted HRP-Rabbit Anti-Mouse IgG antibody solution. TBST buffer (20mL) 2 washes; wash TBS buffer washes 3 times. 2mL of TrueBlue Peroxidase Substrate was developed for 1 min. The PVDF membrane after color development is shown in FIG. 6.
6.6 Experimental results
The target protein is expressed but is basically insoluble as judged by WesternBlotting.
Purification of Tyrp12 recombinant protein
1. Buffer solution preparation
And (3) an equilibrium buffer: 20mM Tris, 300mM NaCl, pH8.0, prewash buffer: 20mM Tris, 300mM NaCl, pH8.0, elution buffer 1: 20mM Tris, 300mM NaCl, 20mM Imi, pH8.0, elution buffer 2: 20mM Tris, 300mM NaCl, 80mM Imi, pH8.0 elution buffer 3: 20mM Tris, 300mM NaCl, 500mM Imi, pH8.0, dialysis buffer: 20mM Tris, pH8.0.
2. Crushing of thallus
And (3) breaking the bacteria buffer solution: 20mM Tris, 300mM NaCl, pH 8.0. And (3) bacteria breaking conditions: 400w, stop for 6s after exceeding 4s, and break the bacteria for 10min in an ice bath at 4 ℃. After crushing, centrifuging the crushed bacteria liquid, and carrying out conditions as follows: 12000rpm, 4 ℃, 15min, supernatant to be purified.
3. Purifying by column chromatography
A GE Ni Sepharose 6Fast Flow (Code No.10257810) chromatography pad was used in a volume of 3.0 mL. The packing was equilibrated with 10 column volumes of 30mL of deionized water and 10 column volumes of 30m of equilibration buffer at a flow rate of 0.5 mL/min. After equilibration, the sample was loaded at a flow rate of 0.5 mL/min. After protein adsorption, the packing was washed with 30mL of 10 column volumes of prewash buffer at a flow rate of 0.5 mL/min. And sequentially eluting the Ni filler step by step according to the sequence of the elution buffer solutions 1, 2 and 3, collecting eluent of each part, tracking the protein elution condition by using a Coomassie brilliant blue method in the elution process, and stopping collecting when the color is changed to negative control.
4. SDS-PAGE electrophoresis
mu.L of the sample was taken, 5. mu.L of 5 XSDS Loading Buffer was added thereto, and the mixture was heated at 100 ℃ for 10min to carry out SDS-PAGE.
Electrophoresis conditions: voltage 120V, time 80 min. A 15% separation gel was used. After electrophoresis, the R250 is dyed by dye solution, and then is decolored by decoloration solution, and an electrophoretogram is shown in figure 7.
5. Dialysis
Buffer dialysis was performed 3 times using 20mM Tris, pH 8.0. Dialysis buffer r 20mM Tris, pH8.0, substitution rate: 1:1000, dialysis membrane: membra-cel md443.5 × 500clr μ lotno.300811349), electrophoresis gel: the results of 15% SDS PAGE/Coomassie brilliant blue staining, SDS-PAGE of dialyzed samples, are shown in FIG. 8.
6. Experimental results quantitative analysis was performed using Nanodrop-1000: the protein concentration of 3.0mL of the volume is 0.88mg/mL, the total amount of the protein is 2.64mg, and the protein purity is more than 90 percent.
Fourth, IgE-Western Blotting detection rTyr p12 positive rate
1. Serum
Ig-western blotting serum was derived from the previous epidemiological survey (Unpublished) of the present invention, and has 11 positive and 4 negative serums, and the basic data are shown in Table 1.
TABLE 1 serum epidemiology questionnaire
In table 1, the serial numbers 1-11 are positive serum of crude extract of Tyrophagus putrescentiae, and the serial numbers 12-15 are negative serum of crude extract of Tyrophagus putrescentiae.
2. IgE-WB detection recombinant protein rTyrp 12 positive rate
mu.L of the recombinant protein (equivalent to 0.05 OD) was taken, 2. mu.L of 5 XSDS Loading Buffer was added, and the mixture was heated at 95 ℃ for 10min to carry out SDS-PAGE. Electrophoresis conditions: 25 mA/piece, 70 min. After electrophoresis with 12.5% polyacrylamide gel, CBB-250 was stained and destained with destaining solution. Respectively shearing a PVDF membrane and filter paper into a size the same as that of the gel, sequentially placing the PVDF membrane, the gel and the filter paper between electrode plates of a membrane rotating instrument after the PVDF membrane and the filter paper are treated by using a membrane rotating buffer solution, simultaneously starting to rotate the membranes by 2 membranes, and setting the parameters of the membrane rotating instrument as follows: current 54mA, transfer time 80 min. PVDF membrane was placed in 12mL Blocking buffer containing 1.5% BSA and blocked by keeping it flat at 37 ℃ for 1 h. The following compositions were used: 10mL of 10-diluted serum solution was subjected to a primary antibody reaction for 1 hour. TBST buffer (25mL) 1 wash; wash TBS buffer 2 times. A secondary antibody reaction was performed for 1 hour using 9mL of a 1:2500 diluted Goatanti-human IgE (HRP) antibody solution. TBST buffer (25mL) 1 wash; wash TBS buffer 2 times. 1mL of TrueBlue Peroxidase substrate was developed for 1 min. According to the IgE-WB results, the positive rate of the Tyrp12 was 27.27%, which is shown in FIG. 9.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Sequence listing
<110> tin-free city children hospital
<120> casomophytes pythium allergen Tyr p12 coding gene, recombinant protein and application thereof
<141> 2021-05-08
<160> 8
<170> SIPOSequenceListing 1.0
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atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60
atgaatctac cagtttttct cttgtccgtc tttttgctgg tccttctgac tggcccaact 120
gtgaatggta aactggtcac tctttgtgat tctccacatg ggacctttca gtcagttagc 180
atcttaaact gcaaaaacac ggatcgattt tgcgtgttta agaagaacac caatgtttcc 240
attgaagtaa actttgtgcc caattacgca gcaacgtctg tccaaacgaa aatcattggc 300
gatgtagccg gcgtgccgat tccctttccc gtcaacccaa aggaggcatg cggcaactac 360
ggtctcaact gtccactgac caccggtgac aagacccaat ttaaaatgga acttcccatc 420
aaggctgcct atcctgccat tgccgttggc gtgactatca aacttgtcga tgagagtagt 480
gctaacctcg tctgtctcaa atttaatgcc aagattaccg aataagaatt cgagctccgt 540
cgacaagctt gcggccgcac tcgagcacca ccaccaccac cactgagatc cggctgctaa 600
caaagcccga aaggaagctg agttggctgc tgccaccgct ga 642
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<212> DNA
<213> Tyrophagus putrescentiae
<400> 2
catatgaaac tggttaccct gtgcgactct ccgcacggta ccttccagtc tgtttctatc 60
ctgaactgca aaaacaccga ccgtttctgc gttttcaaaa aaaacaccaa cgtttctatc 120
gaagttaact tcgttccgaa ctacgctgct acctctgttc agaccaaaat catcggtgac 180
gttgctggtg ttccgatccc gttcccggtt aacccgaaag aagcttgcgg taactacggt 240
ctgaactgcc cgctgaccac cggtgacaaa acccagttca aaatggaact gccgatcaaa 300
gctgcttacc cggctatcgc tgttggtgtt accatcaaac tggttgacga atcttctgct 360
aacctggttt gcctgaaatt caacgctaaa atcaccgaat aagaattc 408
<210> 3
<211> 152
<212> PRT
<213> Tyrophagus putrescentiae
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Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro
1 5 10 15
Arg Gly Ser His Met Lys Leu Val Thr Leu Cys Asp Ser Pro His Gly
20 25 30
Thr Phe Gln Ser Val Ser Ile Leu Asn Cys Lys Asn Thr Asp Arg Phe
35 40 45
Cys Val Phe Lys Lys Asn Thr Asn Val Ser Ile Glu Val Asn Phe Val
50 55 60
Pro Asn Tyr Ala Ala Thr Ser Val Gln Thr Lys Ile Ile Gly Asp Val
65 70 75 80
Ala Gly Val Pro Ile Pro Phe Pro Val Asn Pro Lys Glu Ala Cys Gly
85 90 95
Asn Tyr Gly Leu Asn Cys Pro Leu Thr Thr Gly Asp Lys Thr Gln Phe
100 105 110
Lys Met Glu Leu Pro Ile Lys Ala Ala Tyr Pro Ala Ile Ala Val Gly
115 120 125
Val Thr Ile Lys Leu Val Asp Glu Ser Ser Ala Asn Leu Val Cys Leu
130 135 140
Lys Phe Asn Ala Lys Ile Thr Glu
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<213> Artificial Sequence
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cgcgcggcag ccatatgaat ctaccagttt ttctcttgtc cgtc 44
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atgaatctac cagtttttct cttgtccgtc 30
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ttattcggta atcttggcat taaatttgag 30
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<213> Artificial Sequence
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gctagttatt gctcagcgg 19
