Af-abc gene of aspergillus fumigatus abc transporter and application thereof

Abstract

The application adopts a gene knockout method of homologous recombination to knockout a gene sequence of an ABC transport protein coding gene Af-ABC in Aspergillus fumigatus, and has very important scientific significance and application value for studying growth, virulence and sensitivity to voriconazole of Aspergillus fumigatus, and researching new targeted drugs for the gene and the coded protein, and screening aspergillosis treatment drugs.

Description

Technical Field

[0001] This invention belongs to the technical field of ABC transporter proteins, specifically relating to the Af-ABC gene of Aspergillus fumigatus ABC transporter protein and its application. Background Technology

[0002] In recent years, with the increase in hematological diseases, malignant tumors, HIV / AIDS, and autoimmune diseases, the overuse of broad-spectrum antibiotics, and the widespread use of glucocorticoids and immunosuppressants, the incidence of invasive aspergillosis has increased significantly. Even with today's rapid advancements in modern medicine, invasive aspergillosis remains a major cause of death among these patients, with a mortality rate as high as 60% to 100%.

[0003] Currently, the number and variety of antifungal drugs are very limited, resulting in far from ideal efficacy against invasive aspergillosis. The efficacy rate of voriconazole, a first-line treatment, is only 52.8%, while that of the classic antifungal drug amphotericin B is only 31.6%. Furthermore, the resistance of the main pathogen, *Aspergillus fumigatus*, to existing antifungal drugs further restricts the treatment of invasive aspergillosis. In European countries, approximately 7% of clinically isolated *Aspergillus fumigatus* strains are triazole-resistant, and this proportion is on the rise. More importantly, among these resistant strains, over 80% exhibit cross-resistance to itraconazole, voriconazole, and posaconazole. Therefore, developing novel antifungal drug targets and identifying ways to enhance the fungicidal activity of existing antifungal drugs are crucial for improving clinical outcomes.

[0004] ABC transporters are ATP-transporting enzymes found on cell membranes in all organisms, from microorganisms to humans. ABC transporters facilitate the transport of various molecules across the cell membrane by binding to and hydrolyzing ATP.

[0005] In fungal cells, ABC transporters are key proteins for the entry and exit of molecules and compounds. To study the function of ABC transporters and their impact on drug sensitivity and related phenotypes in Aspergillus fumigatus, it is necessary to identify the genes that influence drug sensitivity of Aspergillus fumigatus ABC transporters. This information will be used to study novel antifungal drug targets, explore ways to enhance the fungicidal activity of existing antifungal drugs, and develop novel targeted drugs against this gene and its encoded protein. Summary of the Invention

[0006] The purpose of this invention is to provide an Af-ABC gene of Aspergillus fumigatus ABC transporter and its application.

[0007] The technical solution of this invention is:

[0008] An Af-ABC gene of an ABC transporter from Aspergillus fumigatus, characterized in that: the nucleotide sequence of the Aspergillus fumigatus Af-ABC gene is shown in Sequence 1; and the protein it encodes consists of 1604 amino acid residues as shown in Sequence 2.

[0009] The Af-ABC gene of the Aspergillus fumigatus ABC transporter can be used as a target in the design of targeted drugs.

[0010] The Af-ABC gene of the Aspergillus fumigatus ABC transporter can be used in screening drugs for the treatment of aspergillosis.

[0011] The advantages of this invention are:

[0012] This invention employs a homologous recombination gene knockout method to obtain a gene knockout strain in Aspergillus fumigatus encoding an ABC transporter protein with the sequence Af-ABC. This is of great scientific significance and application value for studying the growth, virulence, and sensitivity to voriconazole of Aspergillus fumigatus, as well as for developing novel targeted drugs against this gene and its encoded protein and screening drugs for the treatment of aspergillosis. Attached Figure Description

[0013] Figure 1 The PCR gel run diagrams of the upstream and downstream products show that the gene bands are approximately 1.2 kb in length.

[0014] Figure 2 The PCR gel image of the product pyrG, which is about 1.5kb in length, shows the knockout of the desired intermediate segment.

[0015] Figure 3 The image shows the PCR gel run of the fusion of the upper, middle and lower product segments, with a length of approximately 4.0 kb.

[0016] Figure 4 The positive transformant △AfABC obtained after knockout was verified by PCR gel electrophoresis, with a length of approximately 1.5kb.

[0017] Figure 5 Growth curves of positive transformant △AfABC and wild-type strain AF293.

[0018] Figure 6 Colony diameters of positive transformant △AfABC and wild-type strain AF293.

[0019] Figure 7 Results of Etest drug susceptibility testing between positive transformant △AfABC and wild-type strain AF293. Specific implementation methods

[0020] The following description, with reference to the accompanying drawings and examples, illustrates the specific implementation methods of the present invention.

[0021] The applicant obtained the Af-ABC gene of the Aspergillus fumigatus ABC transporter through the following method:

[0022] The inventors used high-throughput gene knockout from an efflux pump gene library to screen for a predicted gene in Aspergillus fumigatus that affects drug sensitivity, namely the Af-ABC gene. The full-length gene fragment was identified as 4923 bp in the Aspergillus fumigatus genome (https: / / www.ncbi.nlm.nih.gov / assembly / GCF_000002655.1), with a coding region of 4812 bp. The gene is identified as gene number 351122 in the gene library.

[0023] To verify that knocking out the Af-ABC gene increases the sensitivity of Aspergillus fumigatus to voriconazole and regulates the growth rate of Aspergillus fumigatus, the applicant conducted the following experiments:

[0024] Experiment 1: Constructing the knockout box, the method is as follows:

[0025] The experimental strain used was Aspergillus fumigatus strain A1160 (ΔKU80,pyrG-), purchased from (http: / / www.fgsc.net / asperg.html).

[0026] The Af-ABC gene was constructed using a highly efficient homologous recombination method. The knockout cassette for this gene was constructed as follows:

[0027] 1) First, genomic DNA of Aspergillus fumigatus AF293 was extracted (OMEGA® Fungal DNA Kit). Then, using 5'-gaccCGGACCTTGTATTAGCCCTAT-3' as the upstream primer and 5'-tagtTCTGTTACCGAGCCGGGCCATCTTTGTGCTTCGCTGTCTTC-3' as the downstream primer, the flanking sequence upstream of the Af-ABC gene coding region was amplified, approximately 1.2 kb. The gene sequence is shown in Sequence 3 of the sequence listing. After verification by 1% agarose gel electrophoresis, the product was purified (MolPure® PCR Purification Kit) and stored at -20℃. The amplification conditions were: 94℃, 4.5 min; (94℃, 30 s; 54℃, 30 s; 72℃, 1 min) 30 cycles; 72℃, 10 min. The PCR system consisted of: 2 μl upstream primer, 2 μl downstream primer, 6 μl ddH2O, 15 μl DNA, and 25 μl 2×Hieff® PCR Master Mix.

[0028] 2) Using 5'-tggtCGCCTGTAGTTCGATAAGTCT-3' as the upstream primer and 5'-gctcTGAACGATATGCTCCAACCTTTCCTTCTCCGAAATGAGCTCAC-3' as the downstream primer, the flanking sequence downstream of the Af-ABC gene coding region was amplified, approximately 1.2 kb. The gene sequence is shown in Sequence 4 of the sequence listing. After gel electrophoresis verification, the product was purified (as above) and stored at -20℃. The amplification conditions were: 94℃, 4.5 min; (94℃, 30 s; 54℃, 30 s; 72℃, 1 min) 30 cycles; 72℃, 10 min. The PCR system consisted of: 2 μl upstream primer, 2 μl downstream primer, 6 μl ddH2O, 15 μl DNA, and 25 μl 2×Hieff® PCR MasterMix.

[0029] 3) Using 5'-ccggCTCGGTAACAGAACTACCGCAGACAATGCTCTCTATC-3' as the upstream primer and 5'-gttgGAGCATATCGTTCAGAGCAATACCGTTACACATTTCCA-3' as the downstream primer, and plasmid pLAX223 as the DNA template (MolPure® Plasmid Mini Kit), the pyrG encoding gene fragment, approximately 1.3kb, was amplified. The gene sequence is shown in Sequence Listing 5. After gel electrophoresis verification and purification (as above), it was stored at -20℃. The amplification conditions were: 95℃, 2 min; (95℃, 30 s; 55℃, 30 s; 68℃, 1.5 min) 35 cycles; 68℃, 5 min. The PCR system consisted of: 2 μl upstream primer, 2 μl downstream primer, 6 μl ddH2O, 15 μl DNA, and 25 μl 2×HieffPCR® Master Mix.

[0030] 4) Perform fusion PCR on the three purified products mentioned above. Using 5'-taccAACCCTGGACGTCTACTCTCA-3' as the upstream primer and 5'-atgaGCCCCGAAGTCATTGCGATTA-3' as the downstream primer, the three gene fragments were fused by PCR to obtain the knockout cassette. The gene sequence is shown in Sequence 6 of the sequence listing for the fusion product. The fusion product was verified by gel electrophoresis and was approximately 4.0 kb in length, which is the knockout cassette of the Af-ABC gene. After gel extraction and purification (using the MolPure® Gel Extraction Kit), it was stored at -20°C. The amplification conditions were: 95°C, 2 min; (95°C, 30 s; 70°C, 1 s; 55°C, 30 s; 72°C, 3 min 50 s) 35 cycles; 72°C, 5 min. The PCR system consisted of: 5 μl of upstream flanking gene fragment, 5 μl of downstream flanking gene fragment, 5 μl of pyrG fragment, 2 μl of upstream primer, 2 μl of downstream primer, 6 μl of ddH2O, and 25 μl of 2×Hieff Canace® Gold PCR Master Mix.

[0031] 5) The lengths of the upstream and downstream flanking gene fragments, the pyrG gene fragment, and the three fusion product fragments were verified by 1% agarose gel electrophoresis, as shown in the attached figure. Figure 1 To be continued Figure 3 As shown in Figure 1, lane 1 is the GoldBand 5000 DNA Marker, and lanes 2 and 3 are the upstream and downstream flanking gene fragments, respectively, each approximately 1.2 kb in length. Figure 2 In the image, lane 1 contains the GoldBand 5000 DNA Marker, and lane 2 contains the pyrG gene fragment, approximately 2.2 kb in length. (See attached image.) Figure 3 In the diagram, lane 1 contains the GoldBand 5000 DNA Marker, and lane 2 contains the PCR product of three gene fragment fusions, with each fragment approximately 5.0 kb in length. This confirms the successful acquisition of the knockout cassette.

[0032] Example 2: Construction of protoplasts

[0033] 1) In this invention, the intermediate host strain A1160 (ΔKU80,pyrG-) specifically used for transformation was purchased from (http: / / www.fgsc.net / asperg.html). This strain is a uracil auxotrophic strain. Due to the lack of the gene fragment pyrG, this strain cannot grow on Czapek's solid medium (CZA Qingdao Haibo Biotechnology No. HB0231-1) without uracil. Therefore, the inventors used CZA without uracil for screening, and the positive transformant ΔAfABC with the knockout cassette successfully introduced can grow in this medium.

[0034] 2) The solutions required for the experiment are briefly described below:

[0035] KCl / C6H8O7·H2O solution: Accurately weigh 16.4g KCl and 4.2g C6H8O7·H2O into a beaker, add 1700ml deionized distilled water, adjust the pH of the mixture to 5.8, then add deionized distilled water to bring the volume to 2L, autoclave, and store at 4℃.

[0036] Protoplast solution: Take 20 ml of KCl / C6H8O7·H2O solution in a beaker, add 0.5 g of compound enzyme preparation LYSISELEVAGE (SAS SOFRALAB OENOFRANCE, batch number 83377), 5 ml of liquid pectinase (Xiasheng Beijing Biotechnology Development Co., Ltd., batch number 12112105), and 1 g of cellulase (Xiasheng Beijing Biotechnology Development Co., Ltd., batch number 2200837). Mix thoroughly and filter through a disposable sterilized syringe-driven 0.22-μm PVDF filter into a 250 ml autoclaved flask. Within 30 minutes before use, mix thoroughly with an equal volume of Sabouraud broth (SAB, Beijing Solarbio Science & Technology Co., Ltd., product number L8300).

[0037] KCl / CaCl2 solution: Weigh 4.47g KCl and 0.74g CaCl2·2H2O into a beaker, add 100ml deionized distilled water and mix well, autoclave and store at room temperature.

[0038] PEG solution: Weigh 400g of PEG3350 (Biosharp Biosharp catalog number BS203) solid powder into a beaker, add 800ml of KCl / CaCl2 solution and mix until completely dissolved. Make up the volume to 1L with KCl / CaCl2 solution. Autoclave and store at room temperature. (Before use, filter the solution through a 0.22-μm PVDF filter using a disposable sterile syringe into the autoclaved flask.)

[0039] 3) The protoplast construction method is briefly described as follows: Fresh Aspergillus fumigatus A1160 (grown in CZA containing uracil) was taken from a 37℃ incubator. Spores were collected using sterile saline containing 0.03% Tween 80, and the concentration of the bacterial suspension was adjusted to 1×10⁹ spores / ml. 150µl of the above bacterial suspension was added to 50 ml of Sabouraud dextrose medium and cultured for 16 hours. The hyphae were collected by filtration using sterile cloth (purchased from Sigma, catalog number: 308623), then treated with protoplast solution and cultured at 30℃ for 4 hours; finally, protoplasts were obtained by stepwise washing and centrifugation with KCl / CaCl₂ solution.

[0040] Experiment 3: Obtaining positive transformants △AfABC

[0041] 1) Take 10µl and 20µl of the knockout cassette product obtained after fusion into 1.5ml sterile EP tubes, and add 20µl of filtered PEG solution and 50µl of Aspergillus fumigatus strain A1160 to each tube respectively. Mix the mixture by pipetting with a sterile pipette tip, and incubate on ice for 30min. Finally, add 100µl of filtered PEG solution to each tube, mix again by pipetting with a sterile pipette tip, and incubate on ice for 5min. Spread the mixture after the second incubation evenly on CZA solid medium without uracil using sterile agar beads (Sangon Biotech Shanghai Co., Ltd., model B529319-0025), ensuring that the medium has sufficient oxygen. Incubate at room temperature for 24h, and then transfer to a 37℃ incubator for 3-5 days to allow the strain to grow sufficiently. A saline group without the knockout cassette was set up as a control group.

[0042] 2) After the incubation time is reached, the culture medium in the 37℃ incubator is taken out for observation. If the control group culture medium has bacterial growth, it means that the culture medium is contaminated by other bacterial strains, and the results of this experiment are not accurate. If no bacterial strain grows in the control group culture medium, but bacterial strains grow in the 10µl knockout vector product group and the 20µl knockout vector product group, then positive transformants are obtained.

[0043] 3) Verification of positive transformants △AfABC: The obtained positive transformants were transferred to CZA for secondary culture and DNA extraction. Verification PCR was performed using 5'-AAGACATGATGGCGGTTCTCCA-3' and 5'-GACCCGGACCTTGTATTAGCCCTAT-3' as upstream primers, and 5'-TCCTGCCGGGCGAAGGGATCG-3' and 5'-TGGTCGCCTGTAGTTCGATAAGTCT-3' as downstream primers. The gene sequence was obtained by 1% agarose gel electrophoresis, yielding a length of approximately 2 kb. Results are attached. Figure 4As shown, lane 1 is the GoldBand 5000 DNA Marker, and lanes 2 and 3 are validation bands. The obtained products were purified and stored at -20°C, and then sent to a sequencing company (Shanghai Sangon Biotech) for sequencing and alignment. Amplification conditions were: 94°C, 4.5 min; (94°C, 30 s; 54°C, 30 s; 72°C, 2.5 min) 30 cycles; 72°C, 10 min. The PCR system consisted of: 2 μl upstream primer, 2 μl downstream primer, 6 μl ddH2O, 15 μl DNA, and 25 μl 2×Hieff® PCR Master Mix.

[0044] 3) After PCR and sequencing verification, a PCR product containing the upstream flanking sequence of the coding region (which is not included in the three-segment fusion knockout box) and the pyrG fragment was obtained, thus confirming the successful acquisition of the positive transformant △AfABC.

[0045] Experiment 5: Related Phenotypic Studies

[0046] 1) Determination of growth curves: Transformant △AfABC (at least 2×10⁴ CFU / ml) and wild-type strain AF293 bacterial suspensions were taken. Fourteen 1.5ml EP tubes were taken from each tube, and equal volumes of bacterial suspension and SAB liquid medium (200 μl each) were added to each tube. The EP tubes were placed in a shaking incubator at 37℃ and 200 rpm for 48 h. Then, 100 μl of bacterial suspensions from each of the shaking times (0, 2, 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, and 48 h) were spread onto SDA solid medium and incubated for 48 h. Spores were counted and analyzed. Compared with wild-type Aspergillus fumigatus strain AF293, the knockout strain △AfABC showed significantly lighter colony color, significantly slower spore growth, and reduced sporulation (see attached image). Figure 5 .

[0047] 2) Measurement of colony diameter: 5 μl of a bacterial suspension with a concentration of 1×10⁶ CFU / mL was dropped into the middle of CZA solid medium and incubated at 37℃ for 48 h. The colony diameter was measured and recorded at 24 h and 48 h of incubation. Compared with the wild-type Aspergillus fumigatus strain AF293, the knockout strain △AfABC showed a significantly smaller colony diameter (see attached figure). Figure 6 .

[0048] 3) E-test susceptibility test: A bacterial suspension with a concentration of 2×10⁶ CFU / ml was spread onto 1640 solid medium. A VOR E-test strip (Guangzhou Yiman Biotechnology Co., Ltd.) was applied to the center of the medium using a sterile cotton swab. The medium was then incubated at 37℃ for 48 hours before the results were read. Compared with the wild-type Aspergillus fumigatus strain AF293, the knockout strain △AfABC showed significantly increased susceptibility to voriconazole, with the minimum inhibitory concentration of voriconazole decreasing by 4 times (see Appendix). Figure 7 .

[0049] Thus, the above experiments clearly show that knocking out the Af-ABC gene increases the sensitivity of Aspergillus fumigatus to voriconazole, slows its growth, and reduces sporulation, which can be used in the research of developing targeted drugs and screening new antifungal drugs.

[0050] The application of the Af-ABC gene of the Aspergillus fumigatus ABC transporter is as follows:

[0051] 1. Design of targeted drugs targeting gene Af-ABC. This invention aims to achieve targeted therapy for aspergillosis patients by designing targeted drugs. Targeted drugs are medications or formulations that target specific pathogens, releasing effective therapeutic components at the target site. In this invention, gene Af-ABC serves as an effective therapeutic target. Drugs are designed to inhibit the expression of gene Af-ABC or antagonize the expression protein of gene Af-ABC. Such drugs targeting gene Af-ABC will significantly increase the sensitivity of the pathogen in aspergillosis patients to voriconazole, thereby improving the efficiency of clinical treatment.

[0052] 2. Screening for novel antifungal drugs. To screen for drugs that can effectively treat Aspergillus fumigatus infection, the gene Af-ABC was used as a screening marker. Novel drugs were selected to act on Aspergillus fumigatus alone or in combination with voriconazole. By monitoring the expression level of the Af-ABC gene, drugs that reduce or eliminate the expression level of the Af-ABC gene were screened, which are drugs that are sensitive to Aspergillus fumigatus or can improve the sensitivity of Aspergillus fumigatus to voriconazole. sequence list <110> Jingzhou Central Hospital (Jingzhou Hospital Affiliated to Yangtze University) <120> A gene for the Af-ABC transporter of Aspergillus fumigatus and its application <141> 2022-06-08 <160> 6 <170> SIPOSequenceListing 1.0 <210> 1 <211> 4815 <212> DNA <213> Aspergillus fumigatus <400> 1 atgttgaatc tcttccttag gcagatttgg acgctgacag tcaagaatct gctcctcgtc 60 tttgtgcgcc cagcagttac aacaaccctc agggccctgg tattgccagt gattttcgtt 120 gctttcatat cctacgcaaa gaaccttttc atccctccgt cagagtatgg tattggcagt 180 cccactcccc tcagttcgct aggagctgcg ctcggtgccg tctcagggaa tagagacaag 240 ttggtattcg tccacaatgg cctgactggc ggcgatgttg agcaagtcat ccaccgagtt 300 gcggatcctg caaaggccag cgcaaaacaa gtacatattc tttcgtcgga ggatgagctg 360 cgagagttgt gtcgaacatc gcttcgtgga gtgtcatcat gcatagctgc agctatcttt 420 tactcttctc ccacggaagg gccaaacgaa tactggaatt actcaattcg cacggatggt 480 tcgctaggtg ttgggatcaa agtcaaccga aatgacaatg atcaggagat ctacctcctt 540 ccattccagc attcgattga ctgggcgata gcgcaggtca actcaaccaa caacggtaac 600 gcgctaccga gtcacgttga ggaatacccg ttcacgtccc tttcccaaga ggaacgaaag 660 gaacagatcc gaacccgcta catgggcgcc atcattgaca tcattgctgt ggctatcttc 720 attgaaattg tgggtgtcac gtaccagctt actgggctta tagcgatgga aagagagctc 780 ggaatgagcc agttgattga ttgcatgttg cccaacacct ctcactggca atcacaagct 840 gctcggttca ttgcagcaca cctggctttg gatatcgtgt attgcccagc atggattatc 900 tccggtgcaa tcctgaaatt tggggtgtac ggcaagacct cggctggaat agtcataatc 960 tacaatatcc tggccggtct cgcgttgtcc tccttttcca tattcggcgc ctcatttttc 1020 aggaaggctc agctcagcgg tatcactgtc gttatccttt gtttgatttt gggagtaatc 1080 gcgcaactgg cacctgcttc gacaaacggt gctgttgtca ttctcagtct cctatttcca 1140 cccatgaact ttgtttattt ctttgttctc atggctcgat gggagagaca gggtctcgct 1200 acggaccttg tgcgcgcagc tccggagaat ccgtggacag ttcctggtat agccctctgg 1260 attttactga ttgttcaaat catcgtctat cctctactgg ccgccgtagt cgagcgagat 1320 ctttatggca ctaagtccaa gagtcgaagg acagtgacct ctggcggctc gatcgctgtg 1380 cgtttgaatg aatttaccaa agaatatcag ccgagctggt tctaccgaaa ggtagctcca 1440 tggtttggaa gcagccgaca gagtgtacgg gccgttaacg acctctccat ggacgtcaga 1500 aaaggtgaaa ttgtggtcct cctcggtgca aacgggtcgg gcaaatctac cacactggat 1560 gccatctccg gtctaacgac aatcacctcc gggagtatcg acgtagacta cggcgactcc 1620 ggcggacggt ttggtctctg cccacagaag aacgttctct gggataagct tacagtgaaa 1680 gagcatataa agatattcaa caggctgaag agtaccggag aagtggacag tattgagcaa 1740 cttctcaaat tgctaaacga ctgtgactta gctcagaaag tgagctcgct cacgaagacg 1800 ctctcagggg gccagaaacg aaaagttcaa ttggccatga tgcttaccgg cggctcatcg 1860 atttgcgctg tcgatgaagt ctcatctgga attgatccaa ttgcacgcgc caaaatatgg 1920 gacatccttc ttgcagagag aggatcacgg acaattcttt tgactactca ttttctagat 1980 gaagcagacc ttctcgccga tcacatcaca atcctatcca agggttcgct caaagcacag 2040 ggctcgagcg ttgagctaaa ggatagattg ggttcgggtt accgaattca tgtgctcaac 2100 gttcctggct cagagaaggt agccggtctg cagttcgaca gtattgaaaa agaggtccat 2160 tttgatgata ccgtctactc cgcgaaagat tctacggaag cttctcgtct gatgtcagtg 2220 ttggagaaa agggtgtcac agaatatcgt gtcagcgggc cgacgatcga agatgttttt 2280 ctctcagttg cagaagagtt agactcggac agactacgag aagagcctaa catcgcactg 2340 aaggggacgg attacgctga aaagaatcct ggaagcagtg acagcgaggg gcttcggcta 2400 ttgactggaa aacgcattag catgttcctt cagtcgtggt atttgtttcg caagagggtc 2460 acaatattgc gccgcaaccc tatcccatac ttggccgctt tgctcatccc agtcgttgcg 2520 gccggacttg ttactctttt cctgaagggt gctacgaagg caggttgttc cggtgatagc 2580 gcgtatcgtg catctggctt tgagtcactg gcgtctctgg aaaacttga atttgtggtt 2640 ggtcctcgag acaggctgtc tatctccgct cttgagagct tcattggctc cctatctggt 2700 ttgatacggt cgactcggaa tacgtctttg aatctcgact ctcgatttca cctagtaaac 2760 agtctcacgg agtttgaaga ttatatatct caacatata ccaacataac ccctggcggt 2820 ttctacctgg gagacgcaaa ttctgcgcct acattcgcgt ggaagggaga caacagtcac 2880 ttcccgttat ccgctatcac tcagaatgcc ctggataact ttctgaccaa cgtgcccatc 2940 tctttccact tccagttttt cgacattcca tggcagtcag attctgggaa ggcactccag 3000 ctgattgtct attttggact tgctatgtcg gtataccctg ctttgtttgc tctctatccc 3060 accgttgagc gcttgaagaa tgtccgagcg ctgcatttca gtaacggggt gagaggcgcc 3120 tcgctctggc tagcatacct gacgttcgac ttctgtatcg tcgtcgcatc tagcgtcctt 3180 gcaatagtca tattcagggc cgcgtcggat gtatggtacc atgccgaata tctcttcgtc 3240 gttttcttcc tctacgggtt gtgcacaacc atactatcat atgtggtttc cctcgtctcg 3300 aagtctcagc ttgcagcttt cgcaattgca gctggcggac agtgcgtctt gttcttgatt 3360 tacttcatcg cctacatgtc ggtattgaca tacgcaccta cccagaaagt ggatgagtac 3420 cttgagatta cgcactttac catcggactc attgcgccat ccggaaatct tcttcgagca 3480 atgttcactt ccctgaatac gttctccatc ctatgtcgag gacgagagat cgcctcatac 3540 ccgggtgaga tcactctcta cggaggccca atcccttatc tgatctgtca atcctttttc 3600 ctgtttggat tgctgctttg gattgatgga gggcctgtgc tttccatgat gaggaaa 3660 gtcaagcagg accacgtgga agaagaac actgtcgatg gagatgttgc cgctgaatta 3720 gcacgagtgg aagggtcaac cgacggacta cgggttctgc acctgacgaa gaattcaag 3780 aagtctgtcg cggttgatga cgtgacattc ggggtaccga aatctcaagt ttttgctctc 3840 ttagggccaa atggagcagg gaaaccacc acaatctctc tcatccgcgg tgatatggta 3900 ccttccaaca atgggaaa tattttcgtg aacaatattt cggttctgag gaaccgtgca 3960 gcagcacgct cccaccttgg ggtctgtcca cagtttgatg caatggatca aatgaccgtc 4020 atcgagcatc tcgagttcta tgcccgcatc cgtggtgtga ccgatgtgaa gcacaatgtc 4080 aggagtaa tgcgtgctgt gggccttctt cccttccagc accgcatggc aacaaaactc 4140 tcaggaggta ataaacgcaa gctttctctc ggtattgctc taatgggcaa ccctctcgtc 4200 ttgctgctgg atgaacctag ctccggcatg gacgcagcct cgaagcgtgt gatgtggaag 4260 acgttggctt ctgtagtccc tggacgctcc attgtgctga cgacccactc catggaggag 4320 gcagatgcac tggcactcg ggcaggaatc atggccaagc gtatgctggc gctggggaca 4380 actgatgacc tgcggaagg atatgtaac aaatatcatg tccatctagt ccattcacag 4440 gcaccgcaca ctacggacga aactatggaa cgcatccggg attgggtgca aagaacttt 4500 cctggtgccg tgatagagca gaagacctat catgggcaga taaggttcag tattcctgca 4560 acagcggcat cttcatctcc aaaggcggaa atcactgaat atgctgcggg tcctattgcc 4620 gaggagaag agctcggacc cggacccaga tatcaatccg tcacagag catcgtcagc 4680 tccatattct cgaaattgga gcagagcaag actgatctag gcgtccagta ttactctgtc 4740 agccaaacta cattggacca agtctccctc gccattgttg gatacgggag 4800 gagaatgctg gagag 4815 <210> 7 <211> 1596 <212> PRT <213> Aspergillus fumigatus <400> 7 With Leu Asn Leu Phe Leu Arg Gln Ile Trp Thr Leu Thr Val Lys Asn 1 5 10 15 Leu Leu Leu Val Phe Val Arg Pro Ala Val Thr Thr Thr Leu Arg Ala 20 25 30 Leu Val Leu Pro Val Ile Phe Val Ala Phe Ile Ser Tyr Ala Lys Asn 35 40 45 Leu Phe Ile Pro Pro Ser Glu Tyr Gly Ile Gly Ser Pro Thr Pro Leu 50 55 60 Ser Ser Leu Gly Ala Ala Leu Gly Ala Val Ser Gly Asn Arg Asp Lys 65 70 75 80 Leu Val Phe His Asn Gly Leu Thr Gly Gly Asp Val Glu Gln Val Ile 85 90 95 His Arg Val Ala Asp Pro Ala Lys Ala Ser Ala Lys Gln Val His Ile 100 105 110 Leu Ser Ser Glu Asp Glu Leu Arg Glu Leu Cys Arg Thr Ser Leu Arg 115 120 125 Gly Val Ser Ser Cys Ile Ala Ala Ala Ile Phe Tyr Ser Ser Pro Thr 130 135 140 Glu Gly Pro Asn Glu Tyr Trp Asn Tyr Ser Ile Arg Thr Asp Gly Ser 145 150 155 160 Leu Gly Val Gly Ile Lys Val Asn Arg Asn Asp Asn Asp Gln Glu Ile 165 170 175 Tyr Leu Leu Pro Phe Gln His Ser Ile Asp Trp Ala Ile Ala Gln Val 180 185 190 Asn Ser Thr Asn Asn Gly Asn Ala Leu Pro Ser His Val Glu Glu Tyr 195 200 205 Pro Phe Thr Ser Leu Ser Gln Glu Glu Arg Lys Glu Gln Ile Arg Thr 210 215 220 Arg Tyr Met Gly Ala Ile Ile Asp Ile Ile Ala Val Ala Ile Phe Ile 225 230 235 240 Glu Ile Val Gly Val Thr Tyr Gln Leu Thr Gly Leu Ile Ala Met Glu 245 250 255 Arg Glu Leu Gly Met Ser Gln Leu Ile Asp Cys Met Leu Pro Asn Thr 260 265 270 Ser His Trp Gln Ser Gln Ala Ala Arg Phe Ile Ala Ala His Leu Ala 275 280 285 Leu Asp Ile Val Tyr Cys Pro Ala Trp Ile Ile Ser Gly Ala Ile Leu 290 295 300 Lys Phe Gly Val Tyr Gly Lys Thr Ser Ala Gly Ile Val Ile Ile Tyr 305 310 315 320 Asn Ile Leu Ala Gly Leu Ala Leu Ser Ser Phe Ser Ile Phe Gly Ala 325 330 335 Ser Phe Phe Arg Lys Ala Gln Leu Ser Gly Ile Thr Val Val Ile Leu 340 345 350 Cys Leu Ile Leu Gly Val Ile Ala Gln Leu Ala Pro Ala Ser Thr Asn 355 360 365 Gly Ala Val Val Ile Leu Ser Leu Leu Phe Pro Pro Met Asn Phe Val 370 375 380 Tyr Phe Phe Val Leu Met Ala Arg Trp Glu Arg Gln Gly Leu Ala Thr 385 390 395 400 Asp Leu Val Arg Ala Ala Pro Glu Asn Pro Trp Thr Val Pro Gly Ile 405 410 415 Ala Leu Trp Ile Leu Leu Ile Val Gln Ile Ile Val Tyr Pro Leu Leu 420 425 430 Ala Ala Val Val Glu Arg Asp Leu Tyr Gly Thr Lys Ser Lys Ser Arg 435 440 445 Arg Thr Val Thr Ser Gly Gly Ser Ile Ala Val Arg Leu Asn Glu Phe 450 455 460 Thr Lys Glu Tyr Gln Pro Ser Trp Phe Tyr Arg Lys Val Ala Pro Trp 465 470 475 480 Phe Gly Ser Ser Arg Gln Ser Val Arg Ala Val Asn Asp Leu Ser Met 485 490 495 Asp Val Arg Lys Gly Glu Ile Val Val Leu Leu Gly Ala Asn Gly Ser 500 505 510 Gly Lys Ser Thr Thr Leu Asp Ala Ile Ser Gly Leu Thr Thr Ile Thr 515 520 525 Ser Gly Ser Ile Asp Val Asp Tyr Gly Asp Ser Gly Gly Arg Phe Gly 530 535 540 Leu Cys Pro Gln Lys Asn Val Leu Trp Asp Lys Leu Thr Val Lys Glu 545 550 555 560 His Ile Lys Ile Phe Asn Arg Leu Lys Ser Thr Gly Glu Val Asp Ser 565 570 575 Ile Glu Gln Leu Leu Lys Leu Leu Asn Asp Cys Asp Leu Ala Gln Lys 580 585 590 Val Ser Ser Leu Thr Lys Thr Leu Ser Gly Gly Gln Lys Arg Lys Val 595 600 605 Gln Leu Ala Met Met Leu Thr Gly Gly Ser Ser Ile Cys Ala Val Asp 610 615 620 Glu Val Ser Ser Gly Ile Asp Pro Ile Ala Arg Ala Lys Ile Trp Asp 625 630 635 640 Ile Leu Leu Ala Glu Arg Gly Ser Arg Thr Ile Leu Leu Thr Thr His 645 650 655 Phe Leu Asp Glu Ala Asp Leu Leu Ala Asp His Ile Thr Ile Leu Ser 660 665 670 Lys Gly Ser Leu Lys Ala Gln Gly Ser Ser Val Glu Leu Lys Asp Arg 675 680 685 Leu Gly Ser Gly Tyr Arg Ile His Val Leu Asn Val Pro Gly Ser Glu 690 695 700 Lys Val Ala Gly Leu Gln Phe Asp Ser Ile Glu Lys Glu Val His Phe 705 710 715 720 Asp Asp Thr Val Tyr Ser Ala Lys Asp Ser Thr Glu Ala Ser Arg Leu 725 730 735 Met Ser Val Leu Glu Glu Lys Gly Val Thr Glu Tyr Arg Val Ser Gly 740 745 750 Pro Thr Ile Glu Asp Val Phe Leu Ser Val Ala Glu Glu Leu Asp Ser 755 760 765 Asp Arg Leu Arg Glu Glu Pro Asn Ile Ala Leu Lys Gly Thr Asp Tyr 770 775 780 Ala Glu Lys Asn Pro Gly Ser Ser Asp Ser Glu Gly Leu Arg Leu Leu 785 790 795 800 Thr Gly Lys Arg Ile Ser Met Phe Leu Gln Ser Trp Tyr Leu Phe Arg 805 810 815 Lys Arg Val Thr Ile Leu Arg Arg Asn Pro Ile Pro Tyr Leu Ala Ala 820 825 830 Leu Leu Ile Pro Val Val Ala Ala Gly Thr Leu Phe Leu Lys Gly Ala 835 840 845 Thr Lys Ala Gly Cys Ser Gly Asp Ser Ala Tyr Arg Ala Ser Gly Phe 850 855 860 Glu Ser Leu Ala Ser Leu Glu Asn Phe Glu Phe Val Val Gly Pro Arg 865 870 875 880 Asp Arg Leu Ser Ile Ser Ala Leu Glu Ser Phe Ile Gly Ser Leu Ser 885 890 895 Gly Leu Ile Arg Ser Thr Arg Asn Thr Ser Leu Asn Leu Asp Ser Arg 900 905 910 Phe His Leu Val Asn Ser Leu Thr Glu Phe Glu Asp Tyr Ile Ser His 915 920 925 Asn Tyr Thr Asn Ile Thr Pro Gly Gly Phe Tyr Leu Gly Asp Ala Asn 930 935 940 Ser Ala Pro Thr Phe Ala Trp Lys Gly Asp Asn Ser His Phe Pro Leu 945 950 955 960 Ser Ala Ile Thr Gln Asn Ala Leu Asp Asn Phe Leu Thr Asn Val Pro 965 970 975 Ile Ser Phe His Phe Gln Phe Phe Asp Ile Pro Trp Gln Ser Asp Ser 980 985 990 Gly Lys Ala Leu Gln Leu Ile Val Tyr Phe Gly Leu Ala Met Ser Val 995 1000 1005 Tyr Pro Ala Leu Phe Ala Leu Tyr Pro Thr Val Glu Arg Leu Lys Asn 1010 1015 1020 Val Arg Ala Leu His Phe Ser Asn Gly Val Arg Gly Ala Ser Leu Trp 1025 1030 1035 1040 Leu Ala Tyr Leu Thr Phe Asp Phe Cys Ile Val Val Ala Ser Ser Val 1045 1050 1055 Val Phe Arg Ala Ala Ser Asp Val Trp Tyr His Ala Glu Tyr Leu Phe 1060 1065 1070 Val Val Phe Phe Leu Tyr Gly Leu Cys Thr Thr Ile Leu Ser Tyr Val 1075 1080 1085 Val Ser Leu Val Ser Lys Ser Gln Leu Ala Ala Phe Ala Ile Ala Ala 1090 1095 1100 Gly Gly Gln Cys Val Leu Phe Leu Ile Tyr Phe Ile Ala Tyr Met Ser 1105 1110 1115 1120 Val Leu Thr Tyr Ala Pro Thr Gln Lys Val Asp Glu Tyr Leu Glu Ile 1125 1130 1135 Thr His Phe Thr Ile Gly Leu Ile Ala Pro Ser Gly Asn Leu Leu Arg 1140 1145 1150 Ala Met Phe Thr Ser Leu Asn Thr Phe Ser Ile Leu Cys Arg Gly Arg 1155 1160 1165 Glu Ile Ala Ser Tyr Pro Gly Glu Ile Thr Leu Tyr Gly Gly Pro Ile 1170 1175 1180 Leu Tyr Leu Ile Cys Gln Ser Phe Phe Leu Phe Gly Leu Leu Leu Trp 1185 1190 1195 1200 Ile Asp Gly Gly Pro Val Leu Ser Met Met Arg Arg Lys Val Lys Gln 1205 1210 1215 Asp His Val Glu Glu Lys Asn Thr Val Asp Gly Asp Val Ala Ala Glu 1220 1225 1230 Leu Ala Arg Val Glu Gly Ser Thr Asp Gly Leu Arg Val Leu His Leu 1235 1240 1245 Thr Lys Lys Phe Lys Lys Ser Val Ala Val Asp Asp Val Thr Phe Gly 1250 1255 1260 Val Pro Lys Ser Gln Val Phe Ala Leu Leu Gly Pro Asn Gly Ala Gly 1265 1270 1275 1280 Lys Thr Thr Thr Ile Ser Leu Ile Arg Gly Asp Met Val Pro Ser Asn 1285 1290 1295 Asn Gly Gly Asn Ile Phe Val Asn Asn Ile Ser Val Leu Arg Asn Arg 1300 1305 1310 Ala Ala Ala Arg Ser His Leu Gly Val Cys Pro Gln Phe Asp Ala Met 1315 1320 1325 Asp Gln Met Thr Val Ile Glu His Leu Glu Phe Tyr Ala Arg Ile Arg 1330 1335 1340 Gly Val Thr Asp Val Lys His Asn Val Thr Glu Val Met Arg Ala Val 1345 1350 1355 1360 Gly Leu Leu Pro Phe Gln His Arg Met Ala Thr Lys Leu Ser Gly Gly 1365 1370 1375 Asn Lys Arg Lys Leu Ser Leu Gly Ile Ala Leu Met Gly Asn Pro Leu 1380 1385 1390 Val Leu Leu Leu Asp Glu Pro Ser Ser Gly Met Asp Ala Ala Ser Lys 1395 1400 1405 Arg Val Met Trp Lys Thr Leu Ala Ser Val Val Pro Gly Arg Ser Ile 1410 1415 1420 Val Leu Thr Thr His Ser Met Glu Glu Ala Asp Ala Leu Ala Thr Arg 1425 1430 1435 1440 Ala Gly Ile Met Ala Lys Arg Met Leu Ala Leu Gly Thr Thr Asp Asp 1445 1450 1455 Leu Arg Lys Arg Tyr Gly Asn Lys Tyr His Val His Leu Val His Ser 1460 1465 1470 Gln Ala Pro His Thr Thr Asp Glu Thr Met Glu Arg Ile Arg Asp Trp 1475 1480 1485 Val Gln Lys Asn Phe Pro Gly Ala Val Ile Glu Gln Lys Thr Tyr His 1490 1495 1500 Gly Gln Ile Arg Phe Ser Ile Pro Ala Thr Ala Ala Ser Ser Ser Pro 1505 1510 1515 1520 Lys Ala Glu Ile Thr Glu Tyr Ala Ala Gly Pro Ile Ala Glu Glu Glu 1525 1530 1535 Glu Leu Gly Pro Gly Pro Arg Tyr Gln Ser Val Asn Lys Ser Ile Val 1540 1545 1550 Ser Ser Ile Phe Ser Lys Leu Glu Gln Ser Lys Thr Asp Leu Gly Val 1555 1560 1565 Gln Tyr Tyr Ser Val Ser Gln Thr Thr Leu Asp Gln Val Phe Leu Ala 1570 1575 1580 Ile Val Gly His Asn Ile Arg Glu Glu Asn Ala Gly 1585 1590 1595 <210> 3 <211> 1201 <212> DNA <213> Aspergillus fumigatus <400> 3 gacccggacc ttgtattagc cctatcggtt gattatttca ttgcttgtct ttcatttcat 60 ctgtgtttga taccaaccct ggacgtctac tctcaatccc tcctgtgatc atttaattct 120 cctacaatat atttctgtat atatgaacct gaactactgg gacatagaag catttggcca 180 gtgtgcatga agcacagggg cacatctttt ccttcttctg ctgtctatga ctagaccaat 240 atcacaatct cccaccgaag accccccacg ccggtagtac cctgacccca ggccatcccc 300 cacctccgct gctgatcgtt tttttctcca cgtctccata aacttcatcc cttcatcaac 360 tcggcagttc taaaaccatc gacctcatgt tattaacgca cgtatcaaga ccaactgaac 420 ctgaatgaaa gagtcataac tggcgcatcg gccacggaat ctcgacgatt tccacctatc 480 atctgccaga cgaggatgcc acgccttgtt ctcggtctta ccccgactgc tcgactactc 540 cgcaccggga atgtcaccct gcccatcacc gcatatctca gatcttgacc aatgatccag 660. cgaaccaacg aattctttgc gctggtgctg gcattctccc cgaaggcccc tgaatcgctc aatggtcatc cacaccgcta tatgacgatg ttgaccccca cgccgctacc aagtactcac 720. tggtttgtag ctttcaaccc cgcaaaatat gccgggagca aggccgaggc tgtgagctcc 780 aacctggggt agggtagaat gtttgtctcg cataacgtgg ggtggatatg ctttagcgtt 840 gatacttcct gatgtggaag ctacacttgg gacgattatc caacgcctcc tgtggccatg atatgagcat ctgggcaatg aggtctccgc tttggctttg gggtccatcc aaactatgcc attcaatac caactggatt cagcttctcc agacataggt tctgagacgt ctatattttt agctgttcat cggtaccatc gctgacttgc aagcaagaaa caacgaaag gcaatatggg 1080 tgatagaact accgagccac agcatggctc caaaatgcag tctgcagaga cccctaccat catacacagc gaaaatactc agctcgaacc aggatggaag acagcgaagc acaaagatgg c 1201 <210> 4 <211> 1201 <212> DNA <213> Aspergillus fumigatus <400> 4 ctttccttct ccgaaatgag ctcacgaggg ttggtgtgga gatggccgtg aaggtcttca 60 gtccaaatgt cgagagtgtg gaaattgtac agaggacgga gaagaggaag agaagagcaa 120 gactgtacta catgaggtat gtactagcat ctggtgaccg gcaccaccag gaagaatgtt 180 cctcctgtgc agtgaagcta aaattttgct ctctcgaatt cacaggaagc ccaagcacga 240 tatgggaagc gtcgagaaca ttgtgtcgaa ctacctgcgg cagaagtcgg tccttactgg 300 tcagcgttca gccggtgcac ggagacagaa acggtgaatt ggagcaattc ctattctgtc 360 tgtattatga ttttggcgaa atgtactata ctactatact tgcacatgga tattcgggaa 420 tcgacagttt ttggcgcaca cattgagtcc ggaacacagg ccatttcaca tcagcatcta 480 ctgcttggtg atggaaacta ttgtttgggg agtacatcga acttcagata atcccaaata 540 gtcctacaaa aacgagagat catgatcggt gatcggtact ctattctccc gttcccgcga 600 gcttgtccct cagcaatgtc tctcggaaag gagtcgccaa tcctaccgtc caacctgatt 660 tatgaaacac tatgctaccc ggataagtgg acagacggaa caatcttgca attgtagcaa 720 tgtagacaaa agatctatag tgactagtgc tatctcgtat gacgacacca tatggtagct 780 agaagtgcgt cctgtgaaga atctttgaac tcgtggcttc gcttataagg tggctgaatg 840 ttgtattggg gaatttctcc gacggcttct gggcgccaac ccaatctgtc ttgattggcg 900 cgatctgtta tggtacattg cccacccagt ccaatgacag ccgttgaatg aatcgatcgc 960 aggggcagtg ccgggcacat agattgacgt gccaggaaat ccaagaaaca ccagtctccg 1020 ccgtcttgcg ccgaggtcgc ttggacatca tcagtctggt tgccgtcttg cgctaatcgc 1080 aagatgtttc tataataatc gcaatgactt cggggctcat caatttcgat cttaaagctt 1140 actcttactt ctcctgggat cgaggatgag ctccgaagac ttatcgaact acaggcgacc 1200 a 1201 <210> 5 <211> 1370 <212> DNA <213> Aspergillus fumigatus <400> 5 ccgcagacaa tgctctctat cctggtggca ggcgtcaagt acccagaggc agcagcgggc 60 ttaggagcgg cctgggttgt tctccgcacc ctctacatgc tgggctatat ttatagcgac 120 aagccgaacg gcaccggcag gtacaatggt tcgctgtact tgcttgcgca agcgggtctt 180 tggggattga gcgcatttgg tgttgcaaag gatttgatgt aaatgtagtc gacatcttag 240 cacagagggg agagttgata aaatgtggtc tgtttgaatg atagtcgggt tcgtgaccta 300 tattcgtgat agtggagata ggtctgcgcc tatcttatcg ggccggagca aaaattccac 360 cgcagcgggg tgagttttcg ttatacagcc atcccacttc cagcttcaaa ttgtcagttt 420 aatccagccc aattcaatca ttggagaacc gccatcatgt cttcgaagtc ccacctcccc 480 tacgcaattc gcgcaaccaa ccatcccaac cctttaacat ctaaactctt ctccatcgcc 540 gaggagaaga aaaccaacgt caccgtctcc gcagacgtta ctacttccgc cgagctcctc 600 gatcttgctg accgtacatc ctgcaccaat gcccctccag gataacaaat agctgatgcg 660 tagtgagtac aggcctaggc ccctatatcg cagttctgaa aacccacatc gacatcctca 720 ccgatctcac cccgtcgacc ctttcctcgc tccaatccct cgcgacaaag cacaacttcc 780 tcatctttga ggaccgcaag ttcatcgaca tcggcaacac cgtgcaaaag cagtaccacg 840 gtggcgctct ccgcatctcc gaatgggcac acatcatcaa ctgcgccatc ctgccgggcg 900 aagggatcgt cgaggccctc gcacagacaa ccaagtctcc tgactttaaa gacgcgaatc 960 aacgaggtct cctgattctt gccgagatga cgagtaaggg atctcttgcg acaggggagt 1020 cacaggcacg ctcggttgag tacgcgcgga agtataaggg gtttgtgatg ggattcgtga 1080 gtacaagggc gttgagtgag gtgctgcccg aacagaaaga ggagagcgag gattttgtcg 1140 tctttacgac tggggtgaat ctgtcggata agggggataa gctggggcag cagtatcaga 1200 cacctgggtc ggcggttggg cgaggtgcgg actttatcat tgcgggtagg ggcatctata 1260 aggcggacga tccagtcgag gcggttcaga ggtaccggga ggaaggctgg aaagcttacg 1320 agaaaagagt tggactttga gtgtgagtgg aaatgtgtaa cggtattgac 1370 <210> 6 <211> 3772 <212> DNA <213> Aspergillus fumigatus <400> 6 gacccggacc ttgtattagc cctatcggtt gattatttca ttgcttgtct ttcatttcat 60 ctgtgtttga taccaaccct ggacgtctac tctcaatccc tcctgtgatc atttaattct 120 cctacaatat atttctgtat atatgaacct gaactactgg gacatagaag catttggcca 180 gtgtgcatga agcacagggg cacatctttt ccttcttctg ctgtctatga ctagaccaat 240 atcacaatct cccaccgaag accccccacg ccggtagtac cctgacccca ggccatcccc 300 cacctccgct gctgatcgtt tttttctcca cgtctccata aacttcatcc cttcatcaac 360 tcggcagttc taaaaccatc gacctcatgt tattaacgca cgtatcaaga ccaactgaac 420 ctgaatgaaa gagtcataac tggcgcatcg gccacggaat ctcgacgatt tccacctatc 480 atctgccaga cgaggatgcc acgccttgtt ctcggtctta ccccgactgc tcgactactc 540 cgcaccggga atgtcaccct gcccatcacc gcatatctca gatcttgacc aatgatccag 600 ccaaccaacg aattctttgc gctggtgctg gcattctccc cgaaggcccc tgaatcgctc 660 aatggtcatc cacaccgcta tatgacgatg ttgaccccca cgccgctacc aagtactcac 720 tggtttgtag ctttcaaccc cgcaaaatat gccgggagca aggccgaggc tgtgagctcc 780 aacctggggt agggtagaat gtttgtctcg cataacgtgg ggtggatatg ctttagcgtt 840 gatacttcct gatgtggaag ctacacttgg gacgattatc caacgcctcc tgtggccatg atatgagcat ctgggcaatg aggtctccgc tttggctttg gggtccatcc aaactatgcc attcaatac caactggatt cagcttctcc agacataggt tctgagacgt ctatattttt agctgttcat cggtaccatc gctgacttgc aagcaagaaa caacgaaag gcaatatggg 1080 tgatagaact accgagccac agcatggctc caaaatgcag tctgcagaga cccctaccat catacacagc gaaaatactc agctcgaacc aggatggaag acagcgaagc acaaagatgg cccgcagaca atgctctcta tcctggtggc aggcgtcaag tacccagagg cagcagcggg 1260 cttaggagcg gcctgggttg ttctccgcac cctctacatg ctgggctata tttatagcga 1320. caagccgaac ggcaccggca ggtacaatgg ttcgctgtac ttgcttgcgc aagcgggtct ttggggattg agcgcatttg gtgttgcaaa ggatttgatg taaatgtagt cgacatctta gcacagaggg gagagttgat aaaatgtggt ctgtttgaat gatagtcggg ttcgtgacct 1500 atattcgtga tagtggagat aggtctgcgc ctatcttatc gggccggagc aaaaattcca 1560 ccgcagcggg gtgagttttc gttatacagc catcccactt ccagcttcaa attgtcagtt 1620 taatccagcc caattcaatc attggagaac cgccatcatg tcttcgaagt cccacctccc 1680 ctacgcaatt cgcgcaacca accatcccaa ccctttaaca tctaaactct tctccatcgc 1740 cgaggagaag aaaaccaacg tcaccgtctc cgcagacgtt actacttccg ccgagctcct 1800 cgatcttgct gaccgtacat cctgcaccaa tgcccctcca ggataacaaa tagctgatgc 1860 gtagtgagta caggcctagg cccctatatc gcagttctga aaacccacat cgacatcctc 1920 accgatctca ccccgtcgac cctttcctcg ctccaatccc tcgcgacaaa gcacaacttc 1980 ctcatctttg aggaccgcaa gttcatcgac atcggcaaca ccgtgcaaaa gcagtaccac 2040 ggtggcgctc tccgcatctc cgaatgggca cacatcatca actgcgccat cctgccgggc 2100 gaagggatcg tcgaggccct cgcacagaca accaagtctc ctgactttaa agacgcgaat 2160 caacgaggtc tcctgattct tgccgagatg acgagtaagg gatctcttgc gacaggggag 2220 tcacaggcac gctcggttga gtacgcgcgg aagtataagg ggtttgtgat gggattcgtg 2280 agtacaaggg cgttgagtga ggtgctgccc gaacagaaag aggagagcga ggattttgtc 2340 gtctttacga ctggggtgaa tctgtcggat aagggggata agctggggca gcagtatcag 2400 acacctgggt cggcggttgg gcgaggtgcg gactttatca ttgcgggtag gggcatctat 2460 aaggcggacg atccagtcga ggcggttcag aggtaccggg aggaaggctg gaaagcttac 2520 gagaaaagag ttggactttg agtgtgagtg gaaatgtgta acggtattga cctttccttc 2580 tccgaaatga gctcacgagg gttggtgtgg agatggccgt gaaggtcttc agtccaaatg 2640 tcgagagtgt ggaaattgta cagaggacgg agaagaggaa gagaagagca agactgtact 2700 acatgaggta tgtactagca tctggtgacc ggcaccacca ggaagaatgt tcctcctgtg 2760 cagtgaagct aaaattttgc tctctcgaat tcacaggaag cccaagcacg atatgggaag 2820 cgtcgagaac attgtgtcga actacctgcg gcagaagtcg gtccttactg gtcagcgttc 2880 agccggtgca cggagacaga aacggtgaat tggagcaatt cctattctgt ctgtattatg 2940 attttggcga aatgtactat actactatac ttgcacatgg atattcggga atcgacagtt 3000 tttggcgcac acattgagtc cggaacacag gccatttcac atcagcatct actgcttggt 3060 gatggaaact attgtttggg gagtacatcg aacttcagat aatcccaaat agtcctacaa 3120 aaacgagaga tcatgatcgg tgatcggtac tctattctcc cgttcccgcg agcttgtccc 3180 tcagcaatgt ctctcggaaa ggagtcgcca atcctaccgt ccaacctgat ttatgaaaca 3240 ctatgctacc cggataagtg gacagacgga acaatcttgc aattgtagca atgtagacaa 3300 aagatctata gtgactagtg ctatctcgta tgacgacacc atatggtagc tagaagtgcg 3360 tcctgtgaag aatctttgaa ctcgtggctt cgcttataag gtggctgaat gttgtattgg 3420 ggaatttctc cgacggcttc tgggcgccaa cccaatctgt cttgattggc gcgatctgtt 3480 atggtacatt gcccacccag tccaatgaca gccgttgaat gaatcgatcg caggggcagt 3540 gccgggcaca tagattgacg tgccaggaaa tccaagaaac accagtctcc gccgtcttgc 3600 gccgaggtcg cttggacatc atcagtctgg ttgccgtctt gcgctaatcg caagatgttt 3660 ctataataat cgcaatgact tcggggctca tcaatttcga tcttaaagct tactcttact 3720 tctcctggga tcgaggatga gctccgaaga cttatcgaac tacaggcgac ca 3772

Claims

1. The application of the Aspergillus fumigatus Af-ABC gene in the preparation of targeted drugs that enhance the sensitivity of Aspergillus fumigatus to voriconazole, or in the preparation of targeted drugs that enhance the efficacy of voriconazole against Aspergillus fumigatus; characterized in that, The nucleotide sequence of the Aspergillus fumigatus Af-ABC gene is shown in Sequence 1; the targeted drug is designed with the Aspergillus fumigatus Af-ABC gene as the target; the application is to increase the sensitivity of Aspergillus fumigatus to voriconazole by inhibiting the expression of the Af-ABC gene, thereby reducing the minimum inhibitory concentration of voriconazole by 4 times.

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