CN101062942A - Aspergillus fumigatus original active oxygen lethality related protein and its coding gene - Google Patents

Abstract

The invention discloses a protein related to active oxygen kill and came from aspergillus fumigatus and coding gene, which is characterized by the following: comprising (a) and (b) protein; choosing amino acid residue sequence from sequences 2 in sequence table as the protein (a); replacing or deleting or adding amino acid residue sequence from sequences 2 trough one or several amino acid residue sequence; relating to active oxygen kill; deriving from the protein (a) as the protein (b). This protein is a new type antifungal working target point, which lay the foundation for development of antifungal.

Description

Derive from Aspergillus fumigatus with active oxygen lethality related protein and encoding gene thereof

Technical field

The present invention relates to a kind of that derive from Aspergillus fumigatus and active oxygen lethality related protein and encoding gene thereof.

Background technology

Along with the antibiotic widespread use of high-efficiency broad spectrum, carrying out in a deep going way of antineoplaston, the continuous enforcement of organ transplantation and other interventional therapy of surgery, the widespread use of corticosteroid hormone, and being on the increase of AIDS patient, aggressive aspergillosis (Invasive aspergillosis, IA) present ascendant trend, and the state of an illness is very serious, mortality ratio in severe immunocompromised host (immunocompromised) bodies such as leukemia and bone marrow transplantation receptor often surpasses 90%, has become this group patient main causes of death take place.The appearance of this situation, pathogenesis main and IA is not fully aware of, and the diagnostic means of IA is very inaccurate reliable, most importantly not good enough with the IA medication effect, and confidential relation is arranged.

IA is caused by infection by Aspergillus fumigatus that mainly Aspergillus fumigatus is a kind of haploid filamentous fungus; Because Aspergillus fumigatus is to the great effect of human health, the U.S. and Britain scholar unite and have launched its genomic mensuration work, and finish the end of the year 2005; The result shows that Aspergillus fumigatus has 8 karyomit(e)s; Total size is 29.2Mb; Through comparing with other species gene group sequence, find possible albumen coded sequence 9922CDS, wherein make an appointment with half to belong to known protein family (www.sanger.ac.uk), half then belongs to new gene in addition.Although can obtain the data of Aspergillus fumigatus genome aspect by retrieval www.tigr.org, but the Aspergillus fumigatus specific gene corresponding to concrete which part of these gene orders, but remain numerous scientific workers and make great efforts the important content understanding and decode, and illustrate of the enforcement of the function of these gene orders for pathogenesis, diagnostic means and the treatment measure of IA, have great importance.

The medicine that is approved for treatment aggressive aspergillus infection at present has amphotericin B, itraconazole, voriconazole (Voriconazole), Caspofungin and Mi Kafen clean.

(reactive oxygen species ROS) is meant chemically reactive some special oxidation state or some oxygenatedchemicals far above ground state oxygen to reactivity keto, not only comprises oxygen center free radical such as O ₂-, and OH, and comprise the non-free radical derivative of some oxygen, as H ₂O ₂, singlet oxygen and hypochlorous acid, even also comprise the epoxy metabolite of superoxide, hydroperoxide and endogenous lipid and foreign compound, because they all contain the active oxygen functional group that contains of chemical property.Vitamin k4 (also claiming vitamin K3) is by producing super-oxide indirect oxidation glutathione active oxygen.

Summary of the invention

The purpose of this invention is to provide a kind of that derive from Aspergillus fumigatus and active oxygen lethality related protein and encoding gene thereof.

Derive from Aspergillus fumigatus and active oxygen lethality related protein provided by the present invention, name is called FAP1, derives from Aspergillus fumigatus (Aspergillus fumigatus), is following (a) or protein (b):

(a) protein of forming by the amino acid residue sequence of sequence in the sequence table 2;

(b) with the amino acid residue sequence of sequence in the sequence table 2 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and relevant with active oxygen lethality by (a) deutero-protein.

Wherein, the sequence in the sequence table 2 is made up of 615 amino-acid residues.

Experiment showed, the polypeptide of forming from N-terminal the 1st to 316 amino acids residue sequence (name is called FAP1R) by sequence in the sequence table 2, also relevant with active oxygen lethality, be the active fragments of FAP1.

The replacement of described one or several amino-acid residue and/or disappearance and/or interpolation are meant replacing and/or lack and/or add in sequence 2 outside the 1st to 316 of N-terminal.

In order to make the FAP1 in (a) be convenient to purifying, proteinic N end or C end that can the amino acid residue sequence of sequence 2 is formed in by sequence table connect label as shown in table 1.

The sequence of table 1. label

Label	Residue	Sequence
			Poly-Arg	5-6 (being generally 5)	RRRRR
Poly-His	2-10 (being generally 6)	HHHHHH
			FLAG	8	DYKDDDDK
Strep-tag II	8	WSHPQFEK
			c-myc	11	EQKLISEEDL

Above-mentioned (b) but in the FAP1 synthetic, also can synthesize its encoding gene earlier, carry out biology again and express and to obtain.The encoding gene of FAP1 in above-mentioned (b) can pass through SEQ ID № in the sequence table: the codon of one or several amino-acid residue of disappearance in 1 the dna sequence dna, and/or carry out the missense mutation of one or several base pair, and/or obtain at the encoding sequence that its 5 ' end and/or 3 ' end connects the label shown in the table 1.

Above-mentioned deriving from Aspergillus fumigatus also belongs to protection scope of the present invention with encoding gene active oxygen lethality related protein (FAP1).

Describedly derive from Aspergillus fumigatus and encoding gene active oxygen lethality related protein, its nucleotide sequence is the proteinic polynucleotide of sequence 2 in the code sequence tabulation.

Described derive from the encoding sequence with encoding gene active oxygen lethality related protein Aspergillus fumigatus can be sequence 1 in the sequence table from 5 ' nucleotide sequence that terminal the 1st to 1848 deoxyribonucleotides are formed.

Describedly derive from Aspergillus fumigatus and encoding gene active oxygen lethality related protein, specifically can be following 1) or 2) gene:

1) its nucleotide sequence be sequence 1 in the sequence table or the sequence 3 in the sequence table from the 1476th to 3464 deoxyribonucleotide of 5 ' end; 2) under stringent condition with 1) the dna sequence dna hybridization that limits and encode described derive from Aspergillus fumigatus and dna molecular active oxygen lethality related protein.

Described stringent condition can be 0.1 * SSPE (or 0.1 * SSC), in the solution of 0.1%SDS,, and wash film with this solution 65 ℃ of hybridization down.

Sequence 1 in the sequence table is the cDNA sequence of FAP1.Sequence 3 in the sequence table is genomic dna sequence and flanking sequences thereof of FAP1.

Contain and above-mentionedly derive from recombinant expression vector, transgenic cell line and the transformed host bacterium with encoding gene active oxygen lethality related protein Aspergillus fumigatus and also belong to protection scope of the present invention.

After aspergillus fumigatus spores sucks the body alveolar, when interacting with immune defense system, producing opposing and further escape the body neutrophil leucocyte and the process of killing and wounding of the ROS that pulmonary alveolar macrophage discharges, is the crucial link that causes that finally aggressive infects.The present invention confirms, the destruction of Aspergillus fumigatus FAP1 gene causes Aspergillus fumigatus to ROS tetchiness in the environment, it is closely related to illustrate that ROS is decomposed in the opposing of FAP1 and encoding gene thereof and Aspergillus fumigatus, prompting FAP1 and encoding gene thereof have participated in that pulmonary alveolar macrophage and neutrophil leucocyte discharge the process that ROS kills and wounds in the Aspergillus fumigatus opposing host natural defending system, are important novel virulence factors.Experimental results show that the destruction of FAP1 gene, make Aspergillus fumigatus to the ROS tetchiness, but do not influence the susceptibility of Aspergillus fumigatus to antifungal drug commonly used, illustrate by destroying the FAP1 gene, the inhibition materials such as ROS that FAP1 caused suppress the process of increased activity to Aspergillus fumigatus, different with the mechanism of tradition antifungal drug commonly used, so Aspergillus fumigatus FAP1 and active fragments thereof, and/or their encoding gene is a novel antifungal drug action target spot, for finding novel research of antifungal targets, the antifungal drug (particularly preventing and/or treating the medicine of aggressive aspergillosis) of development high-efficiency low-toxicity is laid a good foundation.

Description of drawings

Figure 1A is that primer p1 and p2PCR identify the positive transformant that the FAP1 gene is knocked out

Figure 1B is that primer p3 and p4PCR identify the positive transformant that the FAP1 gene is knocked out

Fig. 2 A is that spotting method is measured FAP1 gene disruption Aspergillus fumigatus (Δ fap1), FAP1 gene complementation strain (MC), FAP1 active fragments strain (TR) to H ₂O ₂Susceptibility

Fig. 2 B is that spotting method is measured Δ fap1, MC, the TR susceptibility to Menadione

Embodiment

Experimental technique among the following embodiment if no special instructions, is ordinary method.

The albumen relevant of embodiment 1, Aspergillus fumigatus and the acquisition of encoding gene thereof with active oxygen lethality

The contriver utilizes bioinformatics method to retrieve the possible AP-1 gene of Aspergillus fumigatus with the AP-1 dna homolog of Candida albicans, yeast saccharomyces cerevisiae, fission yeast from Aspergillus fumigatus genome (www.tigr.org/tdb/e2k1/afu1/), find that the possible AP-1 gene (being the FAP1 gene) of Aspergillus fumigatus is positioned on No. 5 karyomit(e), its nucleotide sequence is the sequence 1 in the sequence table, and amino acid sequence coded is the sequence 2 in the sequence table.

On this basis, the contriver from the encoding sequence upstream of the possible AP-1 gene of Aspergillus fumigatus promptly 5 ' hold about 1.0kb and its downstream are 3 ' hold about 1.0kb place to design primer: FAP1u-F:

5 '-gtca GGGCCCGAGGACGTGGTGGTTTCAGT-3 ' and FAP1u-R:

5 '-tgag GGGCCCGCTGCTGAAGCTATCCCAAC-3 ' (lowercase is a restriction enzyme site protection sequence); Genomic dna with Aspergillus fumigatus (Aspergillus fumigatus) AF293 (#1100 is available from U.S. Fungal Genetics StockCenter) is a template, carries out polymerase chain reaction (PCR), and the size of the amplified production that obtains is about 4.8kb.Then, with this amplified production strategy through the DNA reorganization, recombinate with plasmid pBluescript SK+ (available from Stratagene), sequencing, sequencing result shows that the nucleotide sequence of this pcr amplification product is the sequence 3 in the sequence table.From 5 of sequence 3 ' the 1st to 1475 deoxyribonucleotide of end is FAP1 gene 5 ' flanking sequence, from 5 of sequence 3 ' the 1476th to 3464 deoxyribonucleotide of end is FAP1 gene ORF sequence, from 5 of sequence 3 ' the 3465th to 4812 deoxyribonucleotide of end is FAP1 gene flanking sequence, from 5 of sequence 3 ' the 1st to 20 deoxyribonucleotide of end is primer sequence, is primer sequence from 5 of sequence 3 ' the 4893rd to 4812 deoxyribonucleotide of end.

Confirm the AP-1 gene height homology of FAP1 gene and Candida albicans, yeast saccharomyces cerevisiae, fission yeast.

The cDNA that obtains with the total RNA reverse transcription by Aspergillus fumigatus (Aspergillus fumigatus) AF293 (#1100 is available from U.S. FungalGenetics Stock Center) is a template, utilizes primers F AP1-cDNA-F:

5’-TCAGGTACCATGGCGGACTACAATACTCT-3’，FAP1-cDNA-R：

5 '-ATTTGACGCGACCCATGA-3 ' carries out polymerase chain reaction (PCR), amplified production carries out agarose electrophoresis to be separated, reclaim and be cloned on the pMD 18-T Vector carrier, carry out sequencing analysis, sequencing result shows, this pcr amplification product has the nucleotide sequence of sequence 1 in the sequence table, is the cDNA sequence of FAP1.The cDNA sequence length of FAP1 is 1848bp.The encoding sequence of FAP1 is the 1st to the 1845th deoxynucleotide (1846-1848 is a terminator codon) of 5 of sequence 1 in sequence table ' end, 615 amino acid (sequence 2 in the sequence table) of encoding.

The checking of embodiment 2, the protein function relevant with active oxygen lethality

One, Aspergillus fumigatus FAP1 gene is destroyed, concrete grammar is as follows:

1, the structure of FAP1 gene disruption carrier

(1) contains the structure of the recombinant plasmid pfap1 of FAP1 gene

Genomic dna with Aspergillus fumigatus (Aspergillus fumigatus) AF293 (#1100 is available from U.S. Fungal GeneticsStock Center) is a template, pcr amplification FAP1 gene fragment.This FAP1 gene fragment is made up of for (1348bp) 3 ' flanking sequence (1475bp), FAP1 ORF (1989bp) and 5 ' flanking sequence length.The upstream and downstream primer is all introduced the ApaI restriction enzyme site.Primer sequence is as follows:

FAP1u-F：5’-GTCA GGGCCCGAGGACGTGGTGGTTTCAGT-3’，

FAP1u-R：5’-TGAG GGGCCCGCTGCTGAAGCTATCCCAAC-3’。

PCR product size is about 4.8kb.Plasmid pBluescript SK+ (Stratgene) and PCR product carry out enzyme with ApaI respectively to be cut, and then enzyme is cut product T ₄Dna ligase connects, and transformed competence colibacillus intestinal bacteria DH10B.Select positive transformant, with the ApaI enzyme cut, pcr amplification and order-checking identify to confirm the PCR product cloning to the ApaI site of pBluescript SK+ (Stratgene), the sequence of PCR product is shown in sequence in the sequence table 3; The plasmid called after pFAP1 of reorganization.

(2) amplification of pyrG gene and with the reorganization of pFAP1 plasmid: the genomic dna with Aspergillus nidulans A90 (U.S. FungalGenetics Stock Center) is a template, its pyrG gene of pcr amplification, bilateral primer are all introduced NcoI restriction enzyme site (the black underscore shows):

pyrG u-F：5’-ACT CCATGGGTCAATACCGTTACACATTTCCA-3’

pyrG u-R：5’-TCA CCATGGCCGCAGACAATGCTCTCTATC-3’。Then with NcoI respectively enzyme cut this PCR product and plasmid pFAP1, enzyme is cut product T ₄Dna ligase connects, and transformed competence colibacillus intestinal bacteria DH10B.Select positive transformant, with enzyme cut, pcr amplification and order-checking identify to confirm that the PCR product recombinated on the pFAP1 plasmid called after pfap1:pyrG of reorganization.

2, the destruction of Aspergillus fumigatus FAP1 gene:

Aspergillus fumigatus AF293.1 (#1137, available from U.S. Fungal Genetics Stock Center) for deriving from the Aspergillus fumigatus AF293 (#1100 that carries out gene sequencing, available from U.S. Fungal Genetics Stock Center) the strain of uridylic auxotrophy, this bacterial strain must contain on the substratum of uridylic, or when having replenished the pyrG gene, could grow, can on the basic medium that does not contain uridylic, screen positive transformant when therefore, the plasmid pfap1:pyrG that contains the pyrG gene transforms Aspergillus fumigatus AF293.1.

(20g/L Fructus Hordei Germinatus soaks juice to the rich medium MAG liquid that contains uridylic and uridine with Aspergillus fumigatus uridylic auxotrophy strain AF293.1 spore inoculating, the 2g/L peptone, 1g/L glucose, the 1mg/L pyroxamine, 8.8mg/L riboflavin), hatch the budding period (Germination phase), have the enzyme of digestion to handle with pair cell walls such as Glucanex then and prepare protoplastis; With recombinant plasmid pfap1:pyrG, be transformed in the Aspergillus fumigatus uridylic auxotrophy strain AF293.1 protoplastis with polyoxyethylene glycol-glycerine method after, do not containing basic medium MM (glucose 10g/L, the NaNO of uridylic ₃300g/L, KCl 26g/L, MgSO ₄: 7H ₂O24.65g/L, KH ₂PO ₄68g/L, K ₂HPO ₄87.1g/L, agar 20g/L) go up and screen the Aspergillus fumigatus AF293.1 (can in MM, grow) that obtains positive transformant-conversion pfap1:pyrG.

3, the checking of positive transformant----PCR method

Extract the genomic dna of Aspergillus fumigatus positive transformant, identify with the PCR method whether this positive transformant has obtained the FAP1 gene disruption.The transformant of homologous recombination takes place in the screening of pcr amplification method.1. design a pair of primer (p1, p2), the dna fragmentation of the one section 548bp (from 5 of sequence 3 ' the 2156th to 2703 deoxyribonucleotides of end) on the amplification FAP1 gene, if the FAP1 gene is knocked out, this dna fragmentation then can not be amplified out (Figure 1A).The PCR primer is: p1:5 '-GGACGATGGAGAGTCTGAGC-3 ' and P2:

5’-CCAAGAGAACGGACTTCTGC-3’。2. design 1 pair of primer (p3, p4), this primer centering has a primer to anneal on the chromosome sequence beyond the FAP1 gene knockout box, and another primer is annealed on selection markers pyrG.Remove as the FAP1 clpp gene, then this should be able to amplify product (Figure 1B) to primer.Checking FAP1 knocks out and uses primer: p3:5 '-TCAACTCTCCCCTCTGTGCT-3 ', and p4:

5 '-CAGCTTCTCCAGCCACTACC-3 ', the product size is 2407bp.

The result shows certain Aspergillus fumigatus strain (fap1 represents with Δ) that has obtained the FAP1 gene disruption shown in Figure 1A and 1B.Among Figure 1A, 1.Marker (100ladder DNA marker, the rich biomedical technology company limited that steps) 2. is wild strain Aspergillus fumigatus AF293 contrast, and 3,4 for not knocking out the transformant of FAP1, and 5 for knocking out the transformant of FAP1.Among Figure 1B, 1 is the strain of FAP1 gene disruption, and 2,3 is the not destroyed transformant of FAP1, and 4 is (100ladder DNA marker, the rich biomedical technology company limited that steps).

Two, the checking of the protein function that the FAP1 gene is relevant with active oxygen lethality

1, positive transformant is to the mensuration of ROS susceptibility

Measure the Aspergillus fumigatus positive transformant of FAP1 gene disruption and Aspergillus fumigatus AF293 (#1100 is available from U.S. Fungal Genetics Stock Center) respectively to vitamin k4 (menadione) and H with spot laboratory method (spot assay) ₂O ₂The susceptibility of these two kinds of ROS.Concrete experimental technique is as follows: at the H that contains just like different concns shown in Fig. 2 A ₂O ₂Or shown in Fig. 2 B MM substratum (glucose 10g/L, the NaNO of the Menadione of different concns ₃300g/L, KCl 26g/L, MgSO ₄: 7H ₂O 24.65g/L, KH ₂PO ₄68g/L, K ₂HPO ₄87.1g/L, agar 20g/L) and central inoculating spores 5 μ l (1 * 10 ⁵Individual/ml), culture dish is hatched 72h at 37 ℃.The result is shown in Fig. 2 A and 2B, and wild strain (Aspergillus fumigatus AF293 represents with WT in Fig. 2 A and 2B) is at the H of 2.0mM ₂O ₂Or the Menadione of 15 μ M can be survived when existing; And the Aspergillus fumigatus positive transformant of FAP1 gene disruption (in Fig. 2 A and 2B, representing with Δ fap1), at the H of 2.0mM ₂O ₂Or the Menadione of 10 μ M all is killed when existing and can not survives.

The Aspergillus fumigatus positive transformant of measuring the FAP1 gene disruption with micro-liquid base dilution method is to antifungal drug amphotericin B (Amphotericin B commonly used, AMB), itraconazole (Itraconazole, ITC), voriconazole (Voriconazole, VOR), Caspofungin (Caspofungin, CAS) and common ROS vitamin k4 (Menadione), H ₂O ₂Susceptibility.Concrete experimental technique is as follows: and the M38-A scheme laboratory standard council (CLSI) clinical with reference to the U.S. carried out.Substratum is RPMI 1640 liquid nutrient mediums, and the MOPS that with the final concentration is 0.165mol/l is a damping fluid, and adjusting substratum pH value in the time of 25 ℃ is 7.0 ± 0.1.The working concentration of various antifungal drugs and ROS is as follows, AMB, ITC, VOR and CAS:16-0.03 μ g/ml, H ₂O ₂: 16-0.03mM, Menadione:16-0.03 μ M.The aspergillus spore bacteria suspension is adjusted concentration to 2 * 10 ⁶Individual spore/ml is again with 50 times of RPMI1640 dilutions.Get 100 μ l bacterium liquid and be inoculated into 96 orifice plates, the spore amount of every hole inoculation is 2 * 10 on the final 96 hole drug sensitive plates ⁴Individual spore/ml.Drug sensitive plate is hatched 48h and is read the result under 35 ℃; MIC is defined as the lowest concentration of drug of the no fungal growth that naked eyes observe directly.The result is as shown in table 1, shows that the FAP1 gene disruption does not influence the susceptibility of Aspergillus fumigatus to antifungal drug commonly used, has only improved the susceptibility of Aspergillus fumigatus to ROS.In the table 1, the concentration unit of antifungal drug amphotericin B (AMB), itraconazole (ITC), voriconazole (VOR) and Caspofungin (CAS) is μ g/ml, H ₂O ₂Concentration unit is mmol/L, and the Menadione concentration unit is μ mol/L.In the table 1, wild strain (Aspergillus fumigatus AF293) represents that with WT the Aspergillus fumigatus positive transformant of FAP1 gene disruption is represented with Δ fap1.

Various antifungal drugs of table 1. and oxygenant are to the minimal inhibitory concentration of Aspergillus fumigatus Δ fap1 strain

	AMB	ITC	VOR	CAS(MIC/MEC)	H 2O 2	Menadione
							WT Δfap1	0.5 0.5	0.25 0.25	1 1	＞16/≤0.12 ＞16/≤0.12	4 0.25	1.25 0.6

2, the complementation of the FAP1 gene of FAP1 gene disruption Aspergillus fumigatus

Genomic dna with Aspergillus fumigatus AF293 (#1100 is available from U.S. Fungal Genetics Stock Center) is a template, utilizes FAP1u-F and FAP1u-R pcr amplification FAP1 gene fragment.This FAP1 gene fragment is made up of for (1475bp) 3 ' flanking sequence (1348bp), FAP1 ORF (1989bp) and 5 ' flanking sequence length.The upstream and downstream primer is introduced NotI and SmalI restriction enzyme site respectively.Primer sequence is as follows:

FAP1u-F：5’-GTCA GGGCCCGAGGACGTGGTGGTTTCAGT-3’，

FAP1u-R：5’-TGAG GGGCCCGCTGCTGAAGCTATCCCAAC-3’。

After the PCR product that obtains and pRG3-AMA1-NotI (available from U.S. Fungal Genetics StockCenter) carrier cut with NotI and SmalI enzyme respectively, use T ₄Dna ligase connects, and transformed competence colibacillus intestinal bacteria DH10B (available from Stratgene).Select positive transformant, with enzyme cut, pcr amplification and order-checking identify to confirm that this PCR product recombinated on the pRG3-AMA1-NotI carrier (AMA1 guarantee this carrier self-replicating but unconformability to karyomit(e)) the plasmid called after pRG3-AMA1-FAP1 of reorganization.

Because Δ fap1 can not tolerate 1mM H ₂O ₂, wild strain Aspergillus fumigatus AF293 then can, so select to contain 1mMH ₂O ₂Substratum as screening culture medium, pRG3-AMA1-FAP1 is transformed into Δ fap1.Preparation Aspergillus fumigatus Δ fap1 strain protoplastis method, removing substratum is that other prepares with Aspergillus fumigatus AF293.1 protoplastis outside the MAG (20g/L Fructus Hordei Germinatus soaks juice, 2g/L peptone, 1g/L glucose, 1mg/L pyroxamine, 8.8mg/L riboflavin).Method is as follows: get 50 μ l Δ fap1 protoplastiss and 5 μ g pRG3-AMA1-FAP1, join 50 μ l conversion fluids, place 20min on ice; Add 500 μ l conversion fluids again, room temperature is placed 30min; Above bacterium liquid is joined 3ml contain 1mM H ₂O ₂And the basic nutrient agar of 1mol/l sucrose (minimal medium, MM) in (for preventing to solidify, substratum keeps 47 ℃ in water-bath), be poured in the MM culture dish that contains 0.2mol/l sucrose 37 ℃ of cultivation 48h behind the mixing again.Compare with the Δ fap1 that transforms pRG3-AMA1-NotI.

The transformant that obtains (called after MC, E respectively) that pRG3-AMA1-FAP1 and pRG3-AMA1-NotI transform Δ fap1 goes down to posterity to and contains the H of 2.0mM ₂O ₂On the basic medium of the Menadione of 10 μ M, the result shows the Aspergillus fumigatus Δ fap1 well-grown that has transformed pRG3-AMA1-FAP1, and the Aspergillus fumigatus that has transformed pRG3-AMA1-NotI can not grow.

Aspergillus fumigatus gene transformation system is research Aspergillus fumigatus mechanism of causing a disease and the research of development novel anti fungi-medicine very useful molecular genetics means and the most active field; This technology comprises a carrier that has selection marker, and the Transformation Program that this carrier is imported the host bacterium.The applied selection marker of Aspergillus fumigatus (Aspergillus fumigatus) gene transformation system has the dominance selection marker at present, as Totomycin (hygromycin) and phleomycin, and auxotrophy sign such as pyrG gene and niaD gene etc.The contriver finds, is recipient bacterium with the Aspergillus fumigatus of destroying the FAP1 gene, with the positive selection marker of Aspergillus fumigatus FAP1 gene, with H ₂O ₂Perhaps Menadione (vitamin K3) is a screening reagent, can develop a kind of novel Aspergillus fumigatus gene transformation system, for new approach has been opened up in the molecular genetics operation of Aspergillus fumigatus.

The clone and the functional verification in embodiment 2, FAP1 protein-active zone

With Aspergillus fumigatus (Aspergillus fumigatus) AF293 (#1100, available from U.S. Fungal GeneticsStock Center) genomic dna be template, the active region FAP1-R that utilizes primers F ap1-TR-F and Fap1-TR-R pcr amplification FAP1 gene is (from 5 of sequence 3 ' the 1476th to 2805 deoxyribonucleotides of end, the 1-316 amino acid of coding FAP1) and upstream flanking sequence (from 5 of sequence 3 ' the 423rd to 2805 deoxyribonucleotides of end) increase, be cloned between the BamHI and SphI restriction enzyme site of shuttle plasmid pRG3-AMA1-NotI (available from U.S. Fungal Genetics Stock Center) that can self-replicating the plasmid called after pRG3-AMAI-NotI-TR of successful connection again.Wherein, Fap1-TR-F:

5’-ATTA GGATCCGATTTCTAAGCACCGGGTCA-3’，Fap 1-TR-R：

5 '-GCTATAGGT GCATGCTTAGGGAGTGCTACTCT-3 ' (upstream and downstream is introduced BamHI, SphI restriction enzyme site respectively).Wherein downstream primer is introduced terminator codon TAA.This plasmid is transformed into Aspergillus fumigatus Δ fap1, the transformant called after TR that obtains.

Measure following bacterial strain respectively to vitamin k4 (menadione) and H with spot laboratory method (spot assay) ₂O ₂The susceptibility of these two kinds of ROS: Aspergillus fumigatus AF293 (representing with WT), changes the Δ fap1 transformant MC of pRG3-AMA1-FAP1 over to and changes Δ fap1 transformant E and the transformant TR of pRG3-AMA1-NotI over to the Aspergillus fumigatus positive transformant (fap1 represents with Δ) of the FAP1 gene disruption among the embodiment 1.Concrete experimental technique is as follows: at the H that contains just like different concns shown in Fig. 2 A ₂O ₂Or shown in Fig. 2 B MM substratum (glucose 10g/L, the NaNO of the Menadione of different concns ₃300g/L, KCl 26g/L, MgSO ₄: 7H ₂O 24.65g/L, KH ₂PO ₄68g/L, K ₂HPO ₄87.1g/L, agar 20g/L) and central inoculating spores 5 μ l (1 * 10 ⁵Individual/ml), culture dish is hatched 72h at 37 ℃.The result shows the H of wild strain (Aspergillus fumigatus AF293 represents with WT) at 2.0mM shown in Fig. 2 A and 2B ₂O ₂Or the Menadione of 15 μ M can be survived when existing; And the Aspergillus fumigatus (representing with E) of the Aspergillus fumigatus positive transformant Δ fap1 of FAP1 gene disruption and the FAP1 gene disruption that changes blank carrier pRG3-AMA1-NotI over to is at the H of 2.0mM ₂O ₂Or the Menadione of 10 μ M all is killed when existing and can not survives; Change the positive transformant TR of pRG3-AMAI-NotI-TR over to and change the H of the Δ fap1 transformant MC of pRG3-AMA1-FAP1 at 2.5mM ₂O ₂Or the Menadione of 30 μ M can survive when existing (Fig. 2 A and 2B).Illustrate that the Aspergillus fumigatus that contains a plurality of copy FAP1 genes, FAP1 gene activity zone strengthens above-mentioned oxygenant tolerance.

Sequence table

<160>3

<210>1

<211>1848

<212>DNA

＜213〉Aspergillus fumigatus (Aspergillus fumigatus)

<400>1

atggcggact acaatactct ttatcaacaa ggtctttatc tctcccccga tcaacaggac 60

ctcctcctcg cagctctttc atcgaacaac ccgactcaga aacaacaaac agtgacgcac 120

aactccgaag ccaaccagaa cctcaatcac acacccggcc atgcttcttc cggtagcttc 180

agtgtttctc cccccagtgg tttggacggc tcggtgaatc agtcaactac tttcggctac 240

gaagatagtc cttacctgga tctgaatccc gacttcgacc ttgattttct gggcaacgag 300

agcttgattg gtgatctgcc cccgagcttg ccttcgactg aagactatga gcctggtgat 360

aagcgaaagg atatcgatgg acaagtaaat gacaaagagg attcgggcaa gaagcgacgg 420

gagagcgatg agaaagcagc caagaagcct ggtagaaagc cactgacctc ggaacctact 480

tcgaagcgca aggctcagaa tcgcgcagct cagcgagcct tccgggagcg gaaagaaaag 540

cacctgaagg atctggaggc caaggtggag gagctacaga aagcttctga taatgcaaac 600

caagagaacg gacttctgcg cgctcaggtg gagcgtttac aactagagct caaggagtat 660

cgcaagcgtc tctcctgggt aacgagcacc agcggcctct ctcctgttaa tgctatccca 720

ggtgcatact ccaaaggcat gtatggtctg aacaataatg agttcatgtt cgacttcccc 780

aagttcgggg atctgcccgg ttcacacttg ttcaccaata cgcaaacaag caagtcgaat 840

cagaacaaag cgaaggacaa cccgacagca actccacgta gcgaagctca ggtccccggt 900

gtcctcaacc gcaacgatct gaaaatctcg agccccaacg gcctttccaa cggaccatca 960

cccgccaagt ccacaccaag cggccagaca ccgaattcgc aaacatctac acgacctggc 1020

tctggtacat tgaacggagc cgttgataac aatggagctg ccaggggcta ccaggtcaat 1080

tcgtcgtaca gcgcgagcac aaagcaggca acccacgata cccctagctc agactctcca 1140

tcgtcctctt cagattcgca ccagagccag ctgctctctt ccaatgggac ctcacctgag 1200

ccatctttgc actcgcctgc cgtaaaggcg actgagagta gcactcccca tgcatgcacc 1260

tataccacca tcaacggtga ggaatccttc tgcgctcagc ttagtatggc atgtggcaac 1320

atcaataatc ccattccagc tgtcagacaa aatagcgaaa gcgcgtcaaa cacccctagc 1380

catgccaata gttccgacaa agctcttggt ctggattttt tcgcccaaca gaatggaggt 1440

cagttcgacc ctgtcctgtt cggtgattgg cgtgagcctc aagatgctat tttgtcacaa 1500

gactttggta catttttcga tgatgcgttc cctctccccg acttgggaag cccatcccac 1560

aattttagcg aggcgaccaa gcaaccagca gcgccaaaga aggatcttat cgctgaaatt 1620

gacagtaaac tggatgaaga tgaggaggtt gtgcctggcg aggacaagtc gcaaatgctc 1680

acctgcaata agatatggga tcgtctgcaa tccatggaga agttccgtaa tggcgagatc 1740

gacgtggaca atctctgttc agaattgcgc accaaagcca gatgctctga aggcggtgta 1800

gtcgtcaatc agaaagatgt cgaggacatc atgggtcgcg tcaaatag 1848

<210>2

<211>615

<212>PRT

＜213〉Aspergillus fumigatus (Aspergillus fumigatus)

<400>2

Met Ala Asp Tyr Asn Thr Leu Tyr Gln Gln Gly Leu Tyr Leu Ser Pro

1 5 10 15

Asp Gln Gln Asp Leu Leu Leu Ala Ala Leu Ser Ser Asn Asn Pro Thr

20 25 30

Gln Lys Gln Gln Thr Val Thr His Asn Ser Glu Ala Asn Gln Asn Leu

35 40 45

Asn His Thr Pro Gly His Ala Ser Ser Gly Ser Phe Ser Val Ser Pro

50 55 60

Pro Ser Gly Leu Asp Gly Ser Val Asn Gln Ser Thr Thr Phe Gly Tyr

65 70 75 80

Glu Asp Ser Pro Tyr Leu Asp Leu Asn Pro Asp Phe Asp Leu Asp Phe

85 90 95

Leu Gly Asn Glu Ser Leu Ile Gly Asp Leu Pro Pro Ser Leu Pro Ser

100 105 110

Thr Glu Asp Tyr Glu Pro Gly Asp Lys Arg Lys Asp Ile Asp Gly Gln

115 120 125

Val Asn Asp Lys Glu Asp Ser Gly Lys Lys Arg Arg Glu Ser Asp Glu

130 135 140

Lys Ala Ala Lys Lys Pro Gly Arg Lys Pro Leu Thr Ser Glu Pro Thr

145 150 155 160

Ser Lys Arg Lys Ala Gln Asn Arg Ala Ala Gln Arg Ala Phe Arg Glu

165 170 175

Arg Lys Glu Lys His Leu Lys Asp Leu Glu Ala Lys Val Glu Glu Leu

180 185 190

Gln Lys Ala Ser Asp Asn Ala Asn Gln Glu Asn Gly Leu Leu Arg Ala

195 200 205

Gln Val Glu Arg Leu Gln Leu Glu Leu Lys Glu Tyr Arg Lys Arg Leu

210 215 220

Ser Trp Val Thr Ser Thr Ser Gly Leu Ser Pro Val Asn Ala Ile Pro

225 230 235 240

Gly Ala Tyr Ser Lys Gly Met Tyr Gly Leu Asn Asn Asn Glu Phe Met

245 250 255

Phe Asp Phe Pro Lys Phe Gly Asp Leu Pro Gly Ser His Leu Phe Thr

260 265 270

Asn Thr Gln Thr Ser Lys Ser Asn Gln Asn Lys Ala Lys Asp Asn Pro

275 280 285

Thr Ala Thr Pro Arg Ser Glu Ala Gln Val Pro Gly Val Leu Asn Arg

290 295 300

Asn Asp Leu Lys Ile Ser Ser Pro Asn Gly Leu Ser Asn Gly Pro Ser

305 310 315 320

Pro Ala Lys Ser Thr Pro Ser Gly Gln Thr Pro Asn Ser Gln Thr Ser

325 330 335

Thr Arg Pro Gly Ser Gly Thr Leu Asn Gly Ala Val Asp Asn Asn Gly

340 345 350

Ala Ala Arg Gly Tyr Gln Val Asn Ser Ser Tyr Ser Ala Ser Thr Lys

355 360 365

Gln Ala Thr His Asp Thr Pro Ser Ser Asp Ser Pro Ser Ser Ser Ser

370 375 380

Asp Ser His Gln Ser Gln Leu Leu Ser Ser Asn Gly Thr Ser Pro Glu

385 390 395 400

Pro Ser Leu His Ser Pro Ala Val Lys Ala Thr Glu Ser Ser Thr Pro

405 410 415

His Ala Cys Thr Tyr Thr Thr Ile Asn Gly Glu Glu Ser Phe Cys Ala

420 425 430

Gln Leu Ser Met Ala Cys Gly Asn Ile Asn Asn Pro Ile Pro Ala Val

435 440 445

Arg Gln Asn Ser Glu Ser Ala Ser Asn Thr Pro Ser His Ala Asn Ser

450 455 460

Ser Asp Lys Ala Leu Gly Leu Asp Phe Phe Ala Gln Gln Asn Gly Gly

465 470 475 480

Gln Phe Asp Pro Val Leu Phe Gly Asp Trp Arg Glu Pro Gln Asp Ala

485 490 495

Ile Leu Ser Gln Asp Phe Gly Thr Phe Phe Asp Asp Ala Phe Pro Leu

500 505 510

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

515 520 525

Pro Ala Ala Pro Lys Lys Asp LeuIle Ala Glu Ile Asp Ser Lys Leu

530 535 540

Asp Glu Asp Glu Glu Val Val Pro Gly Glu Asp Lys Ser Gln Met Leu

545 550 555 560

Thr Cys Asn Lys Ile Trp Asp Arg Leu Gln Ser Met Glu Lys Phe Arg

565 570 575

Asn Gly Glu Ile Asp Val Asp Asn Leu Cys Ser Glu Leu Arg Thr Lys

580 585 590

Ala Arg Cys Ser Glu Gly Gly Val Val Val Asn Gln Lys Asp Val Glu

595 600 605

Asp Ile Met Gly Arg Val Lys

610 615

<210>3

<211>4812

<212>DNA

＜213〉Aspergillus fumigatus (Aspergillus fumigatus)

<400>3

gctgctgaag ctatcccaac tatccctcca gagcaggtag tagatacaga acaacgccct 60

caacgggcac ccccacgctg cagtgactgc catattctag gccataggcg attgcaatgt 120

ccgcagcgca agaataacta gatttagtaa taaaatcatg ttttaggggt tcaaaatagc 180

ctccaatttc ggccgcggcc aaattctatg gtatggtgat ccgctcggtt gcgtgatccg 240

ctcgcttacc gattacgtta attagtaact aagcaagcaa gtttaatttc ccccggttga 300

aagagggacc attgcacgga agtagcaagt cagcttctga ctgagcattt ctatggctgg 360

aactagataa tatggcgcgt ggaacttcgt cggcacggag agaaccaata tccggccaaa 420

tgtgatttct aagcaccggg tcactcaaaa tttccgctct aaagcagcgg gagccatcag 480

atacaaccat tggcttccat ctatctactg gcccttgttc aaccaataaa agctatcgac 540

aattgacgct gcctttctgc acccaatttg ccccccatga tcctatggaa acacccatcc 600

taagactggc ctaaggcgtt gattgattgc cttgtgaacc gctccatcgc cgcccccgtc 660

ttcttggaaa agtggaaaat gatggccttc ggtttcatcc ttgtaccacc tcccctctca 720

gccacgtctc tctcacttca cctattctca tcagtagaat cccctgtctc aattcccact 780

tcgaatctct tcatctccgc aggcttcttc ctggcctgaa aatcaatcac tcatatccaa 840

cgacctattc tcaatcggac cctgatttgc cgctttcggc agcccctcat cgccagctat 900

caagcggtac cgctgcgcca ttccactcta tacgacatct ttccatctga cactaggcca 960

ctaggccaca ctctggcatc agctcttggc cgctgcaacc ttttatctta atctcaccac 1020

cttttttttg accctttcgc tgccactgta cttctcatcg actctcctcc gtcatcaatt 1080

ctttcgctca tattgtgata tctcattcga ctcgcttgga gagcctggaa tccgcttgac 1140

tgtatctttt ctgattgtcc cacattccct tttgtcttca catcaacggg attgatgcct 1200

tgttgaagcg gggtggaatc ataagagctt tagtggcctt tccgtcttca tcagcattta 1260

caaactcagc actttgacac atttcttggg ctctccatcg aaaagataaa cgggctcaaa 1320

gcccggcctt ttccggttga ggacaccgat aagccgacaa cctaaacatt gcattgtatt 1380

cgaacaagcc gacagcgggt taaacaatca ctctcagttg acccgaatca tcctactgtt 1440

attcacagcg ataccccgct tcgttttcag ttgccatggc ggactacaat actctttatc 1500

aacaaggtct ttatctctcc cccgatcaac aggacctcct cctcgcagct ctttcatcga 1560

acaacccgac tcagaaacaa caaacagtga cgcacaactc cgaagccaac cagaacctca 1620

atcacacacc cggccatgct tcttccggta gcttcagtgt ttctcccccc agtggtttgg 1680

acggctcggt gaatcagtca actactttcg gctacgaaga tagtccttac ctggatctga 1740

atcccgactt cgaccttgat tttctgggca acgagagctt gattggtgat ctgcccccga 1800

gcttgccttc gactgaagac tatgagcctg gtgataagcg aaaggatatc gatggacaag 1860

taaatgacaa agaggattcg ggcaagaagc gacgggagag cgatgagaaa gcagccaaga 1920

agcctggtag aaagccactg acctcggaac ctacttcggt atgttttctg tgaccctttt 1980

cctacgtgca ggtcaatgtg taggtttttt gactctcggg ggcgctaacc tgttggacag 2040

aagcgcaagg ctcagaatcg cgcagctcag cgagccttcc gggagcggaa agaaaagcac 2100

ctgaaggatc tggaggccaa ggtggaggag ctacagaaag cttctgataa tgcaaaccaa 2160

gagaacggac ttctgcgcgc tcaggtggag cgtttacaac tagagctcaa ggagtatcgc 2220

aagcgtctct cctgggtaac gagcaccagc ggcctctctc ctgttaatgc tatcccaggt 2280

gcatactcca aaggcatgta tggtctgaac aataatgagt tcatgttcga cttccccaag 2340

ttcggggatc tgcccggttc acacttgttc accaatacgc aaacaagcaa gtcgaatcag 2400

aacaaagcga aggacaaccc gacagcaact ccacgtagcg aagctcaggt ccccggtgtc 2460

ctcaaccgca acgatctgaa aatctcgagc cccaacggcc tttccaacgg accatcaccc 2520

gccaagtcca caccaagcgg ccagacaccg aattcgcaaa catctacacg acctggctct 2580

ggtacattga acggagccgt tgataacaat ggagctgcca ggggctacca ggtcaattcg 2640

tcgtacagcg cgagcacaaa gcaggcaacc cacgataccc ctagctcaga ctctccatcg 2700

tcctcttcag attcgcacca gagccagctg ctctcttcca atgggacctc acctgagcca 2760

tctttgcact cgcctgccgt aaaggcgact gagagtagca ctccccatgc atgcacctat 2820

accaccatca acggtgagga atccttctgc gctcagctta gtatggcatg tggcaacatc 2880

aataatccca ttccagctgt cagacaaaat agcgaaagcg cgtcaaacac ccctagccat 2940

gccaatagtt ccgacaaagc tcttggtctg gattttttcg cccaacagaa tggaggtcag 3000

ttcgaccctg tcctgttcgg tgattggcgt gagcctcaag atgctatttt gtcacaagac 3060

tttggtacat ttttcgatga tgcgttccct ctccccgact tgggaagccc atcccacaat 3120

tttagcgagg cgaccaagca accagcagcg ccaaagaagg atcttatcgc tgaaattgac 3180

agtaaactgg atgaagatga ggaggttgtg cctggcgagg acaagtcgca aatgctcacc 3240

tgcaataaga tatggtatgc ttaatctcat cccttaccgc tctccactct cttgctaact 3300

acttgtgtcg tagggatcgt ctgcaatcca tggagaagtt ccgtaatggc gagatcgacg 3360

tggacaatct ctgttcagaa ttgcgcacca aagccagatg ctctgaaggc ggtgtagtcg 3420

tcaatcagaa agatgtcgag gacatcatgg gtcgcgtcaa atagacggcc tttgagtgaa 3480

gtcgatacga cggtaaagac agtcgacatc ttgtacaatt tccggccaat tttatacaca 3540

cccttcggcg aattcgaaga cagataccca tgttaaacga cgctgcaatt gatgttacca 3600

cgaacaattt ttaggtcttt aaaaggaagt ttgggtggat ttcatgggtc gttcattctg 3660

attactttcc caatcttgag taatccttgt tgctttcgaa tcttcgcttc aaggcagcca 3720

ttctcgttca ggatttgtct cgctttaccc actttctttc attgtgaatc tttcctatcc 3780

ccgcttaatg gcgcacttga ggcacggagt cgggaatcgc tactggggct ttggtttgga 3840

aagactgtac atcagcaaca gccctcactt cagtcggcca ttcagccaca tccggatctg 3900

gagtatttgt gttcttcttg ttttcggact ggagcttcgg attcttgctt ctcaaagaac 3960

gggaccgtag tcattgcaat tgcttgttgc tcagctctaa ctttgttcaa agcagtagta 4020

cacgagaaat gtatattgag tacaattgtt gaatcctcgc atacaagcca ttgttctacc 4080

tctaacctgt gttaccgtac ccagacaaac tcatgtgctg cacaattgca tctatagaag 4140

aaaagcgaag ctgaagctac aagggtatcg atattggtgc gtgccaggaa agacttagaa 4200

tacacaaaag gtcgctgttg gtgccctttc ctgtctgctg ttcttgttct tgttaatggt 4260

ttcgtttttt tttttctttt aattgcttcc ttcgtctctt ttttttccaa ttgtcatccg 4320

caccaaagtt ggcagcttcc tctctcttgc ggaggggcac cttctgctcc aacaacttcc 4380

atactcgcca gtcgaagggg accgaatgat aaggtcttct actagagtgt aagaaatgtc 4440

catgtgcatc tcgctcagaa acgaagaaaa taagtgaaga aaagaagctg aaaggaggag 4500

aaagtaaagg ctttgcaaat atcacaagag aagaaattaa caaccatgag agactaattg 4560

aacacagcgt gccggggaaa ttggaaggtg gcaaggacat aacacagaag aacaggcaaa 4620

tgaggaaatg atatcacgct caggactctc caccagaagg ggtgggcttg tcaccggagg 4680

gtgtcgatgt cgatgctgca ggagagtccg aggtgcctcc cttgccgaag ccaccacggc 4740

cacctcttcc accacgtcca ccgcggtcgg taccacggcc gcgtccgcgt ccactgaaac 4800

caccacgtcc tc。4812

Claims (10)

1, that derive from Aspergillus fumigatus and active oxygen lethality related protein is following (a) or protein (b):

(a) protein of forming by the amino acid residue sequence of sequence in the sequence table 2;

(b) with the amino acid residue sequence of sequence in the sequence table 2 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and relevant with active oxygen lethality by (a) deutero-protein.

2, albumen according to claim 1 is characterized in that: the described polypeptide of forming from N-terminal the 1st to 316 amino acids residue sequence that derives from Aspergillus fumigatus and active fragments active oxygen lethality related protein by sequence in the sequence table 2.

3, claim 1 is described derives from Aspergillus fumigatus and encoding gene active oxygen lethality related protein.

4, gene according to claim 3 is characterized in that: describedly derive from Aspergillus fumigatus and encoding gene active oxygen lethality related protein, be following 1) or 2) gene:

1) its nucleotide sequence be sequence 1 in the sequence table or the sequence 3 in the sequence table from 1476 to 3464 deoxyribonucleotides of 5 ' end;

2) under stringent condition with 1) the dna sequence dna hybridization that limits and encode described derive from Aspergillus fumigatus and dna molecular active oxygen lethality related protein.

5, contain claim 3 or 4 and describedly derive from recombinant expression vector, transgenic cell line or transformed host bacterium Aspergillus fumigatus and encoding gene active oxygen lethality related protein.

6, described derive from Aspergillus fumigatus and the active oxygen lethality related protein of claim 1, and/or claim 1 is described derive from Aspergillus fumigatus with the application of active oxygen lethality related protein encoding gene in the screening antifungal drug.

7, application according to claim 6 is characterized in that: described antifungal drug is the medicine that prevents and/or treats the aggressive aspergillosis.

8, claim 1 is described derives from Aspergillus fumigatus and active fragments active oxygen lethality related protein, by the polypeptide of forming from N-terminal the 1st to 316 amino acids residue sequence of sequence in the sequence table 2.

9, the encoding gene of the described active fragments of claim 8.

10, the encoding gene of described active fragments of claim 8 and/or the described active fragments of claim 8, the application in the screening antifungal drug; Described antifungal drug is the medicine that prevents and/or treats the aggressive aspergillosis.

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