CN105859851A

CN105859851A - Aspergillus fumigatus Asp f 4 linear antigenic epitope minimum base sequence peptide Asp f 4104-109 and extended oligopeptides thereof

Info

Publication number: CN105859851A
Application number: CN201610366595.1A
Authority: CN
Inventors: 顾少华; 霍克克; 徐万祥; 石晶; 高岩; 季朝能; 谢毅
Original assignee: Fudan University
Current assignee: Fudan University
Priority date: 2016-05-30
Filing date: 2016-05-30
Publication date: 2016-08-17

Abstract

The invention belongs to the technical field of biological detection, and particularly relates to an Aspergillus fumigatus Asp f 4 linear antigenic epitope minimum base sequence peptide and extended oligopeptides thereof. The Aspergillus fumigatus Asp f 4 linear antigenic epitope minimum base sequence peptide is Asp f 4104-109, and the extended oligopeptides of the minimum base sequence peptide are Asp f 4101-109, Asp f4102-110, Asp f 4103-111 and Asp f 4104-112 of which the amino acid sequences are disclosed as SEQ ID NO.1-SEQ ID NO.5. The Aspergillus fumigatus Asp f 4 linear antigenic epitope minimum base sequence peptide and extended oligopeptides thereof can be used as an antigen independently or combinedly for specifically detecting the serum of the Aspergillus-fumigatus-infected patient.

Description

Linear epitope minimum motif peptide Asp f4 of Aspergillus fumigatus Asp f4104-109And extended short peptides thereof

Technical Field

The invention belongs to the technical field of biological detection, and particularly relates to a linear B Cell Epitope (BCE) of main antigen protein Asp f4 of aspergillus fumigatus causing invasive aspergillosis, allergic bronchitis and aspergillus swelling, a minimum motif peptide thereof, and application of the epitope motif peptide.

Background

Aspergillus fumigatus (Aspergillus fumigatus) is a pathogenic bacterium widely existing in nature, and susceptible people inhale spores thereof and colonize the lung, thereby producing three aspergillosis: aspergillus swelling, which leads to hemoptysis, allergic bronchitis, which leads to pulmonary fibrosis, and invasive aspergillosis with a high mortality rate (Warris A, et al, The biology of pulmony aspergillus infestations. J Impect, 69 Suppl 1: S36-41,2014).

Serum immunological markers are important for the diagnosis of aspergillosis (Chabi ML, et al, Pulmonyaspastrillosis. Diagn Interv Imaging, 96(5):435-42, 2015). GM (Galactomannan ) and BG ((1, 3) -beta-D-glucans, (1, 3) -beta-D-glucans) are polysaccharide components of the cell wall of Aspergillus fumigatus, enter the blood circulation of a host along with the germination of spores and the growth of mycelia, so that GM and BG monoclonal antibodies can be used for detecting corresponding circulating antigens in serum respectively, and are the current common methods for clinically diagnosing Aspergillus fumigatus infection, but have certain limitations in specificity and sensitivity (Liao Wan Qing. laboratory diagnosis of invasive fungal infection. test medicine, 25 (7): 503-. Previous studies have shown that the use of Recombinant proteins in vitro, which are highly immunogenic in Aspergillus fumigatus, allows the detection of circulating antibodies in the serum of infected individuals, but that the individual Recombinant proteins also have certain limitations in specificity and sensitivity (Sarfatij, et al. Recombinant antibodies as diagnostic markers for agriculture. diagnostic infection Dis. 55(4):279-91, 2006). The long epitope peptide of one protein antigen may cross-react with antibodies of other antigen proteins, so that identification of the minimal motif of the protein epitope may improve the specificity of detection; the detection sensitivity can be improved by combining the minimum motifs of a plurality of protein epitopes into a chimeric peptide.

Asp f4 as one of the allergens is a functionally unknown protein, the name of which is given by the World Health Organization and the International society for immunization, the allergen nomenclature division Commission (World Health Organization)and International Union of Immunological Societies (WHO/IUIS) Allergen Nomeculation Sub-committee) approved consent (http:// www.allergen.org/search. php. After infection of humans with Aspergillus fumigatus, the immune system produces corresponding IgG, IgE and IgA antibodies against Asp f4 (Kurup VP, et al. specific antibodies to recombinant antibodies of Aspergillus fumigatus tissues with ABPA. clin Mol Allergy,4:11,2006). The minimal epitope motif of the Asp f4 is identified, and the minimal epitope motif of the Asp f4 and the minimal epitope motif of other Aspergillus fumigatus antigen epitope peptides are used for constructing and detecting serum chimeric peptides, which is favorable for improving the specificity and sensitivity of diagnosing Aspergillus fumigatus infection. Thus, we used E.coli to express Asp f4^15-322And purifying by a nickel column, and immunizing a New Zealand white rabbit to obtain Asp f4 antiserum. Expression of overlapping 9 amino acid residues, covering Asp f4, with improved biosynthetic peptide strategy^15-322Full-length 18 peptide fusion protein, followed by immunoblotting with Asp f4 antiserum to identify positive 18 peptide; a9-peptide fusion protein of 8 amino acid residues overlapping each other was further constructed for the positive 18 peptides, and the minimum epitope motif was identified (Xu, et al. minor motif mapping of a knock down epitope on manazona pellucida protein-4 using peptide biology strategy, J reprodImmunel, 81: 9-16, 2009).

Disclosure of Invention

The invention aims to provide an epitope minimum motif peptide of an Aspergillus fumigatus Asp f4 protein, an extended short peptide containing the minimum motif peptide and application of the short peptide containing the motif peptide.

The amino acid sequence of the linear epitope minimum motif peptide of the Aspergillus fumigatus Asp f4 protein provided by the invention is shown in SEQ ID No.1 and is marked as Asp f4^104-109The amino acid sequence of the first letter is: sadwtn.

The invention also provides a linear epitope minimum motif peptide Asp f4 of the Aspergillus fumigatus Asp f4 protein^104-109The extended short peptide (9 peptide) of (1), the sequence thereof is as followsSEQ ID No.2- -SEQ ID No.5, wherein:

SEQ. ID No.2：X₁X₂X₃sadwtn, denoted Asp f4^101-109。

Wherein X₁Is S, X₂Is G, X₃Is V, is the minimal motif Asp f4^104-109The extended residues, where the 9-peptide fusion protein is chemically synthesized or fusion expressed, may be added or deleted or replaced with other residues.

SEQ. ID No.3：X₁X₂sadwtnX₃Is denoted as Asp f4^102-110. Wherein X₁Is G, X₂Is V, X₃Is T, is the minimal motif Asp f4^104-109The extended residues, where the 9-peptide fusion protein is chemically synthesized or fusion expressed, may be added or deleted or replaced with other residues.

SEQ. ID No.4：X₁sadwtnX₂X₃Is denoted as Asp f4^103-111. Wherein X₁Is V, X₂Is T, X₃Is P, is the smallest motif Asp f4^104-109The extended residues, where the 9-peptide fusion protein is chemically synthesized or fusion expressed, may be added or deleted or replaced with other residues.

SEQ. ID No.5：sadwtn X₁X₂X₃Is denoted as Asp f4^104-112. Wherein X₁Is T, X₂Is P, X₃Is A, minimal motif Asp f4^104-109The extended residues, where the 9-peptide fusion protein is chemically synthesized or fusion expressed, may be added or deleted or replaced with other residues.

The content of the invention is further described in detail as follows:

1. according to the Asp f4 (NCBI accession No: XP-749515) full-length 322aa protein sequence of the Aspergillus fumigatus Af293 strain, the Signal peptide is 1-14aa predicted by Signal P software. Removal of the Asp f4 signal peptide 1-14aa coding sequence, according to E.coli codon biasGood, Total Synthesis of Asp f4^15-322The coding gene was cloned from EcoR I and Sal I cleavage sites in pET-22(b +) plasmid to construct pET-22(b) -Asp f4^15-322Recombinant plasmid pET-22(b) -Asp f4 with 6 × His label at C end^15-322Transferred into escherichia coli BL21(DE3), and induced expression is carried out to obtain recombinant protein Asp f4¹⁵ ^-322(ii) a Obtaining purified Asp f4 after nickel column affinity chromatography^15-322(ii) a Purification of Asp f4^15-322Immunizing New Zealand white rabbits to obtain high specificity rabbit anti-Asp f4 antiserum (figure 1);

2. construction of a plasmid for fusion expression of pXXGST-1, overlapping 9 amino acid residues with each other, covering Asp f4^15-322Full-length GST-18 peptide fusion proteins (Xu, et al, minimum kinetic mapping of a knock down epitope on human phage protein-4 using peptide biosynthesis strategy. J reprodImmunol, 81: 9-16, 2009). Immunoblotting was then performed with rabbit Asp f4 antiserum and one of the positive 18 peptides was identified: asp f4^96-113dssss sgvsadwtnte (FIG. 2). Construction of a covering Asp f4 overlapping each other by 8 amino acid residues^96-113Full length and Asp f4^104-112Adjacent Asp f4^105-113GST-9 peptide fusion protein, and 4 positive 9 peptides are obtained by immunoblot identification: asp f4^101-109sgvsadwtn、Asp f 4^102-110gvsadwtnT、Asp f 4^103- ¹¹¹vsadwtnTP and Asp f4^104-112sadwtnptpa, consensus Asp f4 thereof^104-109sadwtn is the Asp f4 antigen minimal epitope motif peptide (FIG. 3).

The epitope minimum motif peptide Asp f4 of the invention^104-109Can be used for preparing serum recognition epitope markers for clinically diagnosing invasive aspergillosis, allergic bronchitis and aspergillus swelling caused by aspergillus fumigatus infection.

The extended short peptide Asp f4 of the epitope minimum motif peptide^101-109、Asp f 4^102-110、Asp f4^103-111And Asp f4^104-112Used alone or in combination for the preparation of a medicament for clinical diagnosis of invasive aspergillosis caused by Aspergillus fumigatus infection,Allergic bronchitis and aspergillus serum recognize epitope markers.

Drawings

FIG. 1 results of rabbit anti-Asp f4 antiserum ELISA. Asp f4 immune rabbit sera No.1, No.2, No.3 and No.4, pre-immune rabbit sera as controls.

FIG. 2 Asp f 418 peptide fusion protein electrophoresis and immunoblotting image. The upper blue strip is an electrophoresis pattern, and the lower black strip is an immunoblotting pattern. Lane 0 is GST188 expressed in pXXGST-2, and lanes 1 to 34 are transdomain Asp f4^15-32218 peptide overlapping 9 amino acid residues with GST188 fusion protein due to Asp f4^15-322Full length 308aa, Asp f4 in lane 34^312-322. (ii) a Wherein the positive immunoblot band in lane 10 is Asp f4^96-113。

FIG. 3 Asp f4^104-109Minimal epitope peptide motif immunoblot. 10-5 denotes Asp f4^100-10810-6 denotes Asp f4^101-10910-7 denotes Asp f4^102-11010-8 denotes Asp f4^103-11110-9 denotes Asp f4^104-11211-1 represents Asp f4^105-113And the arrow indicates a positive band of the immunoblot reaction. The minimal epitope peptide motif is Asp f4^104- ¹⁰⁹sadwtn。

Detailed Description

The invention can be further described by the following examples. These examples are not meant to limit the scope of the invention, which has been fully set forth in the foregoing description. The following examples are experimental methods without specific conditions being noted, all according to conventional conditions and the third edition "molecular cloning" of the Huangpetang et al translation, edited by [ Mei ] J. sambrook and D.W. Lassel: the procedures described in the laboratory manuals "(scientific Press, 2002) and the" antibody technology laboratory Manual "(scientific Press, 2002) by American E. Harlo and D. ryan, eds Shenyi et al translation, or according to the conditions suggested by the manufacturer.

Materials and methods:

1. pET-22(b +) plasmid and E.coli BL21(DE3) strain were purchased from Novagen, Germany, and the heat-inducible expression plasmid pXXGST-1 and the control plasmid pXXGST-2 were constructed by the present inventors (patent No. 200710173305.2);

2. restriction enzymes EcoR I, BamH I, Sal I were purchased from New England Biolabs, Inc., USA, DL2000DNA molecular weight standards were purchased from Beijing kang, century Biotechnology Inc., T4 DNA ligase and prestained protein molecular weight standards were purchased from Thermo-Fermentas, Inc., USA, mouse 6 XHis monoclonal antibody and Freund's complete adjuvant and Freund's incomplete adjuvant were purchased from Sigma, horseradish peroxidase (HRP) -labeled goat anti-mouse IgG and horseradish peroxidase (HRP) -labeled goat anti-rabbit IgG were purchased from Santa Cruz Biotechnology, USA;

3. the Gel Extraction Gel recovery kit and the Plasmid Mini Plasmid small-scale Extraction kit are purchased from Omega company in the United states;

4. new Zealand white rabbits were purchased from Shanghai Sphall-BiKai laboratory animals Co., Ltd;

5. Asp f 4^15-322the coding gene was synthesized by Shanghai Asahi-crown Biotech development Co., Ltd. Two ends are respectively a BamH I cohesive end and a Sal I cohesive end, and the middle is covered by Asp f4^15-322A positive-strand DNA fragment of the 18-peptide-encoding DNA sequence plus TAA stop codon, which is full-length and overlaps with each other by 9 amino acid residues, and a positive-strand DNA fragment of the 9-peptide-encoding DNA sequence plus TAA stop codon, which covers 18 peptides of the immunoblotting reaction and overlaps with each other by 8 amino acid residues, were synthesized by Shanghai-Li Biotechnology Ltd. DNA recombinants sequencing by Shanghai Ruidi Biotech limited was accomplished.

Asp f 4^104-109The specific steps for identification of the minimal epitope peptide motif are as follows:

1. tobacco kojiMould Asp f4^15-322Expression and purification of

1.1 expression vector pET-22(b) -Asp f4^15-322Construction of

The Signal peptide was 1-14aa predicted by Signal P software based on the protein sequence of Asp f4 (NCBI accession No: XP-749515) of A.fumigatus Af293 strain. Removing the 1-14aa coding sequence of Asp f4 signal peptide, and fully synthesizing Asp f4 according to the codon preference of Escherichia coli^15-322The coding gene was cloned from EcoR I and Sal I cleavage sites in pET-22(b +) plasmid to construct pET-22(b) -Asp f4^15-322Recombinant plasmid, Asp f4^15-322The total length is 308 amino acids.

1.2 recombinant Asp f4^15-322Expression of

The recombinant plasmid pET-22(b) -Asp f4 with 6 × His label at the C end^15-322Taking 1 mu L (about 100 ng/mu L) and transforming the 1 mu L into 10 mu L escherichia coli BL21(DE3) competent cells, carrying out heat shock at 42 ℃ for 90s after ice bath for 30min, cooling the cells on ice for 5min, adding 900 mu L LB liquid culture medium for resuscitation at 37 ℃ for 40 min-60 min, finally centrifuging the cells at 10000r/min for 1min, discarding about 800 mu L culture medium, and coating ampicillin-resistant LB flat plates after the rest are uniformly mixed. And after the bacterial liquid is absorbed by the culture medium, performing inverted culture at 37 ℃ for 16-24 h.

Single colonies were picked and cultured overnight at 37 ℃ in 3mL LB liquid medium containing 50. mu.g/mL ampicillin. The next day, the overnight bacteria are transferred to a new 3mL LB liquid culture medium according to the proportion of 1:50, the working concentration of ampicillin is unchanged, after the overnight bacteria are cultured at 37 ℃ for about 2.5-3 h (OD 600 is 0.6-0.8), 1mL bacteria liquid sample is taken as a control before induction, isopropyl thiogalactoside (IPTG) with the final concentration of 1mM is added into the rest bacteria liquid to induce the expression of the recombinant protein, and the expression condition is cultured for 4h at 37 ℃. And (3) taking a 500 mu L induced bacterial liquid sample, centrifuging for 5min at 10000r/min, collecting thalli, dissolving the thalli by using 150 mu L1 XPBS Buffer solution, adding 50 mu L4 XPSDS-PAGE Loading Buffer, mixing uniformly, and boiling the sample in water for 7-10 min for storage for later use.

2 identical 12% SDS-PAGE gels were prepared in the same order (egg)White molecular weight marker → whole bacterial protein before induction → whole bacterial protein after induction) and the same loading amount (5 mu L) are subjected to SDS-PAGE gel electrophoresis in the same electrophoresis system, wherein one piece of gel is used for staining and analyzing an induction expression strip by Coomassie brilliant blue R250 after electrophoresis is finished, the other piece of gel is used for carrying out Western-blot verification by using a mouse 6 × His monoclonal antibody as a primary antibody and using a goat anti-mouse IgG marked by HRP as a secondary antibody, and the experimental result shows that the recombinant protein Asp f4 is successfully expressed^15-322。

1.3 recombinant protein Asp f4^15-322Purification of (2)

Selecting a monoclonal strain with high expression quantity as an inoculum, inoculating 10 mu L of the inoculum into 50mL of LB liquid culture medium containing 50ug/mL of ampicillin at 37 ℃ for overnight culture, transferring the bacterial liquid into 500mL of LB liquid culture medium containing 50 mu g/mL of ampicillin according to the proportion of 1:50 the next day, inducing for 4h by using 1mM IPTG, centrifuging at 4 ℃ of 5000r/min for 5min, discarding the culture medium, collecting thalli, weighing the thalli (weighing the plastic bottle with 500mL in advance), adding 1 × startbuffer with the thalli weight of 10 cosmetology (adding 10mL 1 × startbuffer to 1g of thalli, and adding 50mM Na buffer to the startbuffer₂HPO₄/NaH₂PO₄pH8.0, 300mM NaCl, 10mM imidazole, 1mM β -mercaptoethanol), resuspending the cells, disrupting the cells under appropriate pressure using a high-pressure cell disrupter, repeating the disruption 3 times, centrifuging at 15000rpm for 30 minutes at 4 ℃ to collect the supernatant, washing the 5mL His Trap HP prepacked column with 5 column volumes of purified water and balancing with 5 column volumes of Start Buffer, washing the column with StartBuffer after loading, removing unadsorbed proteins, washing the column with 40 mM imidazole concentration Start Buffer, removing as much contaminating proteins as possible, and then setting a length of 15mL, 12% -100% precipitation Buffer (50 mM Na₂HPO₄/NaH₂PO₄300mM NaCl, 250mM imidazole, pH 8.0) and collecting the target protein. Meanwhile, pET-22(b +) empty plasmid is used as a negative control and experimental recombinant plasmid pET-22(b) -Asp f4^15-322Parallel experiments are carried out, and two thalli before induction of empty plasmids and thalli after induction of IPTG for 4 hours are collected and used for SDS-PAGE electrophoresis.

Respectively collecting 20uL of the whole bacterial liquid, the supernatant and the purified product, adding SDS Loading buffer Loading with the working concentration of 1 xSDS, uniformly mixing, boiling for 5min, cooling, taking 10 mu L of sample for detecting the denatured SDS-PAGE electrophoresis. Protein bands were matched to expected molecular weight, samples with purity above 80% were pooled, ultrafiltered, concentrated, and the purified protein concentration was estimated by Bradford method. The purity of the detailed purified protein was estimated using pixel calculation software.

2. Preparation of Rabbit anti-recombinant protein Asp f4 antiserum

1mg of Asp f4 is taken^15-322Emulsifying the purified protein with an equivalent volume of Freund complete adjuvant, and injecting a New Zealand white rabbit subcutaneously for primary immunization; after 3 weeks 0.5mg Asp f4 was taken^15-322Emulsifying the purified protein with an equal volume of Freund incomplete adjuvant, and injecting a New Zealand white rabbit subcutaneously for first boosting immunization; after 2 weeks 0.5mg Asp f4 was taken^15-322Emulsifying the purified protein with an equal volume of Freund incomplete adjuvant, and injecting a New Zealand white rabbit subcutaneously for second boosting immunization; and after one week of second boosting immunization, taking about 3-5 mL of blood from the ear marginal vein of the experimental rabbit, centrifuging to obtain antiserum, and detecting the antibody titer by an ELISA method for later use.

3. Asp f 4^104-109Identification of minimal epitope peptide motifs

3.1 Asp f 4^96-113Positive epitope peptide identification

Designing a spanning Asp f4 based on published information of the Asp f4 gene sequence^15-322The 18 peptides of 9 amino acid residues overlapped with each other in the whole sequence encode DNA plus and minus chain segments (plus 5 '-gatcc at the 5' -end of the plus chain, plus tag-3 'at the 3' -end, plus 5 '-tcgactta at the 5' -end of the minus chain, plus g-3 'at the 3' -end), and carry out DNA synthesis. The complementary plus and minus strand fragments of 1 or 2 ODs were treated with ddH₂Dissolving O into 20 mu mol/mu L storage liquid (according to single data of a DNA synthesis report), and respectively taking 10 mu L storage liquid and 20 ddH₂O is heated in a water bath at 94 ℃ for 5min in a 1.5 mLEppendorf tube, 2 muL of annealing fragments and about 200 ng/muL of 1 muL of annealing fragments are sucked into a 15 muL reaction volume and subjected to BamH I and SThe al I double-enzyme digested pXXGST-1 plasmid, 1 mu L T4 DNA ligase and 1.5 mu L buffer solution thereof are connected overnight; the ligation liquid is transformed into a competent BL21(DE3) host bacterium, spread on an LB plate containing Amp, and cultured overnight at 37 ℃; on the next day, single clones growing on ampicillin LB plates were picked and transferred to 3mL LB culture medium for induction expression, GST188 protein expressed by pXXGST-2 was used as a control, each expressed GST188-18 peptide fusion protein was confirmed by 15% SDS-PAGE analysis (the difference between the electrophoretic mobility of the control protein and that of the control protein was about 2 kDa), and each recombinant clone was picked and sent out for DNA sequencing. Inoculating the clone with correct sequencing result of the insert into 3mL LB culture solution of Amp, shaking and culturing at 30 ℃ overnight, transferring to fresh LB culture solution containing Amp according to 1/50 in the next morning, shaking and culturing at 30 ℃ for 2-3 h until the thallus concentration reaches 0.6-0.8 OD, then raising the temperature to 42 ℃ for thermal induction for 4h, centrifugally collecting the thallus, adding a sample lysate, boiling for 5min, and performing 15% SDS-PAGE. Immunoblotting was performed using rabbit anti-Asp f4 antiserum as the primary antibody and goat anti-rabbit IgG/HRP as the secondary antibody. Asp f4^96-113Is one of the positive epitope peptides.

3.2 Asp f 4^104-109Identification of minimum epitope motif peptides

Construction of expression spanning Asp f4 by using pXXGST-1 as expression vector^96-113Mutually overlapping by 8 amino acid residues and with Asp f4^104-112Adjacent Asp f4^105-113A series of 9 peptide GST188 fusion proteins are prepared by taking rabbit anti-Asp f4 antiserum as a primary antibody and goat anti-rabbit IgG/HRP as a secondary antibody, performing immunoblotting, and performing Asp f4^101-109sgvsadwtn、Asp f 4^102-110gvsadwtnT、Asp f 4^103-111vsadwtnTP and Asp f4^104-112sadwtnTPA is positive epitope peptide, and the consensus sequence thereof is Asp f4¹⁰⁴ ^-109sadwtn is the Asp f4 antigen minimal epitope motif peptide.

Application example

Using Asp f4^101-109、Asp f 4^102-110、Asp f 4^103-111And Asp f4^104-112Detection of Asp f4 antibodies in serum

1. With Asp f4^15-322Serum is taken after the protein immunization of New Zealand white rabbits, the serum before the immunization is taken as a control, and the Asp f4 antibody titer of 4 experimental rabbits reaches 1.28 × 10⁵Above (fig. 1), for subsequent immunoblotting reactions;

2. expression of Asp f4 using pXXGST-1 as vector^100-108、Asp f 4^101-109、Asp f 4^102-110、Asp f 4¹⁰³ ^-111、Asp f 4^104-112And Asp f4^105-113A fusion protein;

3. expressed Asp f4^100-108、Asp f 4^101-109、Asp f 4^102-110、Asp f 4^103-111、Asp f 4^104-112And Asp f4^105-113Performing 15% SDS-PAGE electrophoresis on the fusion protein;

4. transferring the electrophoresis gel protein strip to a PVDF membrane in an electrified way, sealing a blotting membrane by using a sealing solution (TBSTM), washing the membrane by using a sealing buffer solution (TBST), then taking experimental rabbit serum diluted in a certain proportion as a primary antibody, and carrying out oscillation incubation at 4 ℃ for overnight;

5. the next day, after washing the membrane with TBST, the blotting membrane was placed in TBSTM, and a certain proportion of diluted HRP-labeled goat anti-rabbit IgG was added as a secondary antibody, and incubated with shaking at room temperature for 1.5 h. Washing the membrane with TBST for 3 times, placing the membrane in TBS buffer solution, adding ECL chemiluminescence agent, exposing after about 1min, and determining the detection result. Contains the minimal epitope motif peptide Asp f4^104-109Asp f4 of sadwtn^101-109、Asp f 4^102-110、Asp f 4^103-111And Asp f4^104-112The blotting reaction is positive; and Asp f4^100- ¹⁰⁸sSgvsadwt and Asp f4^105-113adwtnTPAE is due to the lack of the peptide Asp f4 contained in the minimal epitope motif, respectively^104-109Residue 109 and residue 104, residue S in (1), the blotting reaction was negative (fig. 3).

Although the present invention describes the epitope minimummotif Asp f4 of the Asp f4 protein^104-109sadwtn, and antigenic peptides developed with 9 peptides or 9 peptide fusion proteins containing minimal motifs alone and/or in combination for use in detecting human A.fumigatus infection ((ii))Or chemically synthesized or fusion expressed), it will be apparent to those skilled in the relevant art that various modifications can be made to the epitope peptide motif and the extended minimal motif-containing 9 peptide fusion protein of the present invention without departing from the spirit and scope of the invention. It is, therefore, intended that the appended claims cover all such modifications that are within the scope of this present invention.

SEQUENCE LISTING

<110> university of Compound Dan

<120>Linear epitope minimum motif peptide Asp f4 of Aspergillus fumigatus Asp f4^104-109And extended short peptides thereof

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<170>PatentIn version 3.3

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Claims

1. A minimal epitope motif peptide of Aspergillus fumigatus Asp f4 protein is characterized by having an amino acid sequence shown in SEQ.ID No. l and marked as Asp f4^104-109The abbreviated amino acid sequence is sadwtn.

2. An extended short peptide comprising the minimal epitope motif peptide of the Asp f4 protein of aspergillus fumigatus of claim 1, wherein:

has an amino acid sequence shown as SEQ ID No.2 and is marked as Asp f4^101-109Extended 9 peptide-abbreviated amino acidsThe sequence is sgvsadwtn; or,

has an amino acid sequence shown as SEQ ID No.3 and is marked as Asp f4^102-110The short-term amino acid sequence of the extension 9 peptide is gvsadwtnT; or

Has an amino acid sequence shown as SEQ ID No.4 and is marked as Asp f4^103-111The short-word amino acid sequence of the extension 9 peptide is vsadwtntP; or,

has an amino acid sequence shown as SEQ ID No.5 and is marked as Asp f4^104-112The short-term amino acid sequence of the extension 9 peptide is sadwtnTPA;

where the 9-peptide fusion protein is chemically synthesized or fusion expressed, the extended residue portion may be deleted or replaced with another residue.

3. Use of the epitope minimum motif peptide according to claim 1 for the preparation of serum recognition epitope markers for the clinical diagnosis of invasive aspergillosis, allergic bronchitis and aspergillosis caused by aspergillus fumigatus infection.

4. Use of the extended short peptides of the epitope minimum motif peptide according to claim 2, alone or in combination, for the preparation of epitope markers for serum recognition of invasive aspergillosis, allergic bronchitis and aspergillosis caused by aspergillus fumigatus infection as clinical diagnosis.