CN100341891C - Tanscription activating factor genes of candida albicans and their use - Google Patents

Abstract

The present invention provides novel candida albicans transcription activating factor gene CaSWI1 participating in the pathogenic action of candida albicans and encoding protein CaSwi1 thereof. The present invention also provides the purpose of the protein and an encoding sequence thereof. The product of protein CaSwi1 encoded by the gene is an important mycelial growth activating factor and a toxic factor and participates in the mycelial growth and the pathogenic procedure of the candida albicans.

Description

Candida albicans activating transcription factor gene and uses thereof

Technical field

The present invention relates to Candida albicans mycelial growth genes involved and Disease-causing gene.Particularly, relate to and from the genomic library of Candida albicans, be cloned into Candida albicans activating transcription factor gene C aSWI1 and uses thereof.The product C aSwi1 albumen of this genes encoding is important hypha growth incitant and virulence factor, participates in the mycelial growth and the pathogenic course of Candida albicans.

Background technology

Candida albicans (Candida albicans) is a kind of a kind of opportunistic human body cause illness fungi that is separated to clinically, special in the patient of immune down regulation, as the organ transplantation patient, HIV patient etc., can cause superficial part and the infection of deep system widely, infection site comprises the oral cavity, and vagina etc. cause white mouth, vaginitis etc., also can invade epidermis and endotheliocyte and enter blood and arrive internal organs, as kidney, brain etc., cause septicemia, seriously can cause death (Odds, F.C.1994.J.Am.Acad, Dermatol.31:S2-S5.).

Candida albicans has different growthhabits under different growth conditionss, comprise thalline (yeastform), pseudohypha (pseudohyphae) and mycelia (hyphae).This form transformation ability directly influences pathogenecity (Odds, the F.C.1985.Crit Rev Microbiol.12:45-93 of Candida albicans; Brown, A.J.P.et al.1999.Trends Microbiol.7:334-338.), its system's infection ability of the bacterial strain of mycelial growth defective descends greatly or disappears (Lo, H.J.et al, Cell 90:939-949.).Many culture condition can cause the modality of Candida albicans, comprise serum, temperature, and the pH value, the ammonia source utilizes and oxygen is pressed or the like.

The interior molecular mechanism of cell of regulating the modality of Candida albicans mainly contains MAPK approach (mitoge-actlvaied protein kinase pathway) and cAMP/PKA approach (cAMP-dependentprotein kinase A pathway).Also find what Cph2 mediated simultaneously, relevant (Stoldt, V.R., et al.EMBOJ.16:1982-1991 also all take place with the form of Candida albicans in Efg1 signal pathway mediation and that pH replys; El Barkani A et al.Mol.Cell.Biol.20:4635-4647.).The signal pathway that also has retarding effect in Candida albicans mainly is the signal pathway of Tup1 mediation, relies on supressor Rfg1, and Nrg1 waits (the Kadosh D et al.Mol.Cell.Biol.21:2496-2505 that plays a role; Braun BR et al EMBO J.20:4753-4761.).

Yeast saccharomyces cerevisiae (Sacchromyces cerevisiae) SWI1 base prisoner (ScSWI1) is at first according to relevant (Michael Stern et al.J.Mol.Biol.1984, the 178:853-868 that obtains that clone of its correct expression with haploid cell HO gene and alcohol dehydrogenase 2 gene (ADHII); Aileen K.et al.Genet.1987,116:523-530.).ScSwilp has BRIGHT/ARID (AT-richinteractive domain) structural domain, be a nuclear locating sequence (Aileen K.et al.Genet.1987,116:523-530.).Knocking out of ScSWI1 gene, conversion in haploid cell between a type capable of blocking and the α type gonotype, between double somatocyte, cause the reduction division defective, cause growth defect, to non-fermenting carbohydrate (as semi-lactosi, sucrose, cotton in sugar, glycerine etc.) defective and a that utilizes, α type cell-specific gene is (as STE6, MCM1, MAT α 1 etc.) and other and metabolism related gene (as SUC2, INO1, ADHI, PHO85 etc.) downward modulation of Biao Daing (Aileen K.et al.Genet.1987,116:523-530; Craig L.Peterson et al.Cell.1992,68:573-583; Priya Sudarsanam etal.PANS.2000,97 (7): 3364-3369.).Hereditary and biochemical evidence shows in yeast cell, ScSwilp is a component of the SWI/SNF mixture that is grouped into by 11 one-tenth, under the condition that ATP exists, this mixture can with rna plymerase ii holoenzyme (RNA polymerase holoenzyme), activating transcription factor (transcriptional activator), histone on the chromatin, nonhistones composition and other supressor interact, change STRUCTURE OF CHROMATIN, remove the inhibition factor, the rna plymerase ii holoenzyme is raised on the promotor of institute's regulatory gene, thereby activated transcription (Loree GriffinBurns et al.Mol.Cell.Biol.1997,17 (8): 4811-4819; Kruger et al.Genes.Dev.1995,9:2770-2779; Igor M et al.EMBO.J.1997,16 (20): 6263-6271; Christopher J et al.Cell.1996,84:235-244; Kristen E.Neely et al.Mol.Cell.Biol.2002,22 (6): 1615-1625; Natalya Yudkovsky et al.GenesDev.1999,13:2369-2374.), also might make chromatin Structure more closely suppress to transcribe (Joseph A et al.Genes Dev, 2002,16:2231-2236.).

In sum, the disease that causes in view of Candida albicans gets more and more, so this area presses for exploitation and pathogenic relevant gene of Candida albicans and albumen.

Summary of the invention

The purpose of this invention is to provide a kind of new Candida albicans activating transcription factor gene and proteins encoded thereof that participates in the Candida albicans pathogenic effects.

Another object of the present invention provides the purposes of this polypeptide and encoding sequence thereof.

In a first aspect of the present invention, a kind of isolating Candida albicans CaSWI1 polypeptide is provided, this polypeptide is selected from down group:

(a) has the polypeptide of SEQ ID NO:2 aminoacid sequence;

(b) replacement, disappearance or the interpolation of SEQ ID NO:2 aminoacid sequence through one or more (1-15, preferably 1-10) amino-acid residue formed, and have transcriptional activation function by (a) polypeptides derived.

In another preference, described polypeptide has the aminoacid sequence shown in the SEQ ID NO:2.

In a second aspect of the present invention, a kind of isolating polynucleotide are provided, it comprises a nucleotide sequence, and this nucleotide sequence is selected from down group:

(a) coding is as the polynucleotide of polypeptide as described in claim 1 and 2;

(b) with polynucleotide (a) complementary polynucleotide.

In another preference, described polynucleotide encoding has the polypeptide of aminoacid sequence shown in the SEQ ID NO:2.

In another preference, the sequence of described polynucleotide has 554-3514 position among the SEQ ID NO:1.

In a third aspect of the present invention, a kind of carrier is provided, it contains the polynucleotide of the above-mentioned polynucleotide of the present invention, and a kind of host cell, and it contains above-mentioned carrier.

In fourth aspect present invention, provide a kind of can with Candida albicans CaSWI1 protein-specific bonded antibody of the present invention.

In a third aspect of the present invention, provide the carrier that contains polynucleotide of the present invention.

In a fourth aspect of the present invention, provide a kind of can with Candida albicans CaSwi1 protein-specific bonded antibody of the present invention.

In a fifth aspect of the present invention, preparation CaSwi1 is provided proteic method, this method comprises: (a) under expression condition, cultivate the above-mentioned host cell that is transformed or transduce; (b) from culture, isolate CaSwi1 albumen.

In a sixth aspect of the present invention, the nucleic acid molecule that can be used for detecting is provided, it contains a successive 15-3802 Nucleotide in the above-mentioned polynucleotide.

In a seventh aspect of the present invention, provide and whether had the proteic method of CaSwi1 in the test sample, it comprises: sample is contacted with the proteic specific antibody of CaSwi1, observe whether form antibody complex, formed antibody complex and just represented to exist in the sample CaSwi1 albumen.

In a eighth aspect of the present invention, provide the purposes of polypeptide of the present invention and encoding sequence.Polypeptide for example of the present invention can be used to screen the agonist that promotes the CaSwi1 polypeptide active, and perhaps screening suppresses the antagonist of CaSwi1 polypeptide active.The proteic encoding sequence of CaSwi1 of the present invention or its fragment can be used as primer and be used for pcr amplification reaction, perhaps are used for hybridization as probe, perhaps are used to make gene chip or microarray.

Description of drawings

Fig. 1 has shown Candida albicans CaSWI1 full length gene nucleotide sequence.

Fig. 2 has shown the nucleotide sequence and the deduced amino acid of Candida albicans CaSWI1 gene open reading frame.

Fig. 3 A has shown Candida albicans CaSwi1 albumen and the proteic aminoacid sequence of Saccharomyces Cerevisiae in S cSwi1 relatively.Black part is divided the expression same amino acid.

Fig. 3 B has shown that CaSWI1 can the growth defect of the complementary scswi1 mutant strain of part on glycerin medium.The left side is the YPD substratum, the complementary phenomenon of no obvious growth defective, and the right is to be the SD substratum of sole carbon source with glycerine, CaSWI1 can the complementary scswi1 growth defect of part phenomenon.

Fig. 4 A has shown the colonial morphology of wild bacterium of Candida albicans and CaSWI1 high expression level bacterial strain.Culture condition is the SD substratum, 37 ℃, and 7 days.CaSWI1 high expression level bacterial strain forms the bacterium colony of surface ruffle.

Fig. 4 B has shown the cellular form of scraping wild bacterium of the Candida albicans of getting and CaSWI1 high expression level bacterial strain from Fig. 4 A, and bacterial strain has the cell and the pseudohypha of obvious elongation.

Fig. 4 C has shown the Northern hybridization analysis, detects CaSWI1 expression of gene situation in wild bacterium of Candida albicans and the CaSWI1 high expression level bacterial strain.

Fig. 5 has shown that knocking out of CaSWI1 gene causes albicans growth slow.Culture condition is the YPD solid medium, 37 ℃, and 3 days.

Fig. 6 has shown that knocking out of Candida albicans CaSWI1 gene causes Candida albicans mycelial growth defective.A. solid blood serum medium and Lee ' s substratum, 37 ℃ of cultivations.B. liquid YPD adds 10% serum and Lee ' s substratum, 37 ℃ of cultivations.

Fig. 7 has shown the Northern hybridization analysis, detects the regulation and control of CaSWI1 gene pairs mycelial growth genetic transcription.The expression of caswi1 disappearance strain blocking-up mycelia specific gene HWP1 and ECE1.

Fig. 8 has shown the infection experiment of mouse system, and knocking out of CaSWI1 gene causes Candida albicans toxicity to descend.High is 1 * 10 ⁷/ ml high-concentration bacterial liquid; Low is 1 * 10 ⁶/ ml lower concentration bacterium liquid, every mouse tail vein injection 100 μ l bacterium liquid.

Embodiment

The inventor is through extensive and deep research, screening obtains the new activating transcription factor gene C aSWI1 (SEQ ID NO:1) of a 3.8kb from Candida albicans (Candida albicans) genomic library, its open reading frame contains 2964 Nucleotide, 987 the amino acid whose protein (SEQ ID NO:2) of encoding.

Test shows that (1) CaSwi1 can the growth defect phenotype of the complementary yeast saccharomyces cerevisiae scswi1 of part mutant strain on glycerin medium.In Candida albicans, CaSwi1 is constitutive expression, and knocking out of CaSWI1 gene causes the albicans growth form to change, and comprises the mycelial growth defective, and the speed of growth descends.(2) the CaSWI1 gene can be regulated and control the expression of some mycelia specific genes such as HWP1, ECE1 on molecular level, and high expression level CaSWI1 gene can activate mycelial growth to a certain extent in Candida albicans.(3) disappearance of CaSWI1 gene has been blocked the formation of Candida albicans mycelia really.The disappearance of CaSWI1 gene has influenced the mycelial growth Expression of Related Genes really.(4) knocking out of CaSWI1 gene causes Candida albicans mycelial growth defective, even make Candida albicans also can't form mycelia under intensive mycelia inductive condition.(5) find that by the infection experiment of mouse system the strain of CaSWI1 gene knockout does not have toxicity.Therefore, activating transcription factor CaSwi1 albumen is important hypha growth incitant and virulence factor in the Candida albicans, participates in the pathogenic course of Candida albicans.Finished the present invention on this basis.

In the present invention, term " CaSwi1 albumen ", " CaSwi1 polypeptide " or " Candida albicans CaSwi1 polypeptide " are used interchangeably, all refer to the to have Candida albicans CaSwi1 aminoacid sequence albumen or the polypeptide of (SEQ ID NO:2), and SEQ ID NO:2 aminoacid sequence formed through replacement, disappearance or the interpolation of one or more amino-acid residues, and have transcriptional activation function by (a) polypeptides derived.

As used herein, " isolating " is meant that material separates (if natural substance, primal environment promptly is a natural surroundings) from its primal environment.Do not have separation and purification as polynucleotide under the native state in the active somatic cell and polypeptide, but same polynucleotide or polypeptide as from native state with in other materials that exist separately, then for separation and purification.

As used herein, " isolating Candida albicans CaSwi1 albumen or polypeptide " is meant that Candida albicans CaSwi1 polypeptide is substantially free of natural relative other albumen, lipid, carbohydrate or other material.Those skilled in the art can use the purified technology of protein purifying Candida albicans CaSwi1 albumen of standard.Basically pure polypeptide can produce single master tape on non-reduced polyacrylamide gel.

In the present invention, term " Candida albicans CaSwi1 polypeptide " refers to have the SEQ ID NO.2 polypeptide of sequence of Candida albicans CaSwi1 protein-active.This term also comprises having and variant form Candida albicans CaSwi1 albumen identical function, SEQ ID NO.2 sequence.These variant forms comprise (but being not limited to): several (are generally 1-50, preferably 1-30, more preferably 1-20,1-10 best) amino acid whose disappearance, insertion and/or replacement, and add one or several at C-terminal and/or N-terminal and (be generally in 20, preferably being in 10, more preferably is in 5) amino acid.For example, in the art, when replacing, can not change proteinic function usually with the close or similar amino acid of performance.Again such as, add one or several amino acid at C-terminal and/or N-terminal and also can not change proteinic function usually.This term also comprises proteic active fragments of Candida albicans CaSwi1 and reactive derivative.

In the present invention, " Candida albicans CaSwi1 albumen conservative property variation polypeptide " refers to compare with the aminoacid sequence of SEQ ID NO:2, there are 10 at the most, preferably at the most 8, more preferably at the most 5,3 amino acid is replaced by similar performance or close amino acid and is formed polypeptide at the most best.These conservative property variation polypeptide preferably carry out the amino acid replacement according to table 1 and produce.

Table 1

Initial residue	Representational replacement	The preferred replacement
			Ala(A)	Val；Leu；Ile	Val
Arg(R)	Lys；Gln；Asn	Lys
			Asn(N)	Gln；His；Lys；Arg	Gln
Asp(D)	Glu	Glu
			Cys(C)	Ser	Ser
Gln(Q)	Asn	Asn
			Glu(E)	Asp	Asp
Gly(G)	Pro；Ala	Ala
			His(H)	Asn；Gln；Lys；Arg	Arg
Ile(I)	Leu；Val；Met；Ala；Phe	Leu
			Leu(L)	Ile；Val；Met；Ala；Phe	Ile
Lys(K)	Arg；Gln；Asn	Arg
			Met(M)	Leu；Phe；Ile	Leu
Phe(F)	Leu；Val；Ile；Ala；Tyr	Leu
			Pro(P)	Ala	Ala
Ser(S)	Thr	Thr
			Thr(T)	Ser	Ser
Trp(W)	Tyr；Phe	Tyr
			Tyr(Y)	Trp；Phe；Thr；Ser	Phe
Val(V)	Ile；Leu；Met；Phe：Ala	Leu

Polynucleotide of the present invention can be dna form or rna form.Dna form comprises the DNA of cDNA, genomic dna or synthetic.DNA can be strand or double-stranded.DNA can be coding strand or noncoding strand.The coding region sequence of encoding mature polypeptide can be identical with the coding region sequence shown in the SEQ ID NO:1 or the varient of degeneracy.As used herein, " varient of degeneracy " is meant that in the present invention coding has the protein of SEQ ID NO:2, but with the differentiated nucleotide sequence of coding region sequence shown in the SEQ ID NO:1.

The polynucleotide of the mature polypeptide of coding SEQ ID NO:2 comprise: the encoding sequence of an encoding mature polypeptide; The encoding sequence of mature polypeptide and various additional code sequence; Encoding sequence of mature polypeptide (with optional additional code sequence) and non-coding sequence.

The invention still further relates to and above-mentioned sequence hybridization and two sequences between have at least 50%, preferably at least 70%, more preferably at least 80%, the polynucleotide of at least 90% homogeny best.The present invention be more particularly directed under stringent condition and the interfertile polynucleotide of polynucleotide of the present invention.In the present invention, " stringent condition " is meant: (1) than hybridization under low ionic strength and the comparatively high temps and wash-out, as 0.2 * SSC, and 0.1%SDS, 60 ℃; Or (2) hybridization the time is added with denaturing agent, as 50% (v/v) methane amide, 0.1% calf serum/0.1%Ficoll, 42 ℃ etc.; Or (3) only at the homogeny between the two sequences at least more than 90%, be more preferably 95% and just hybridize when above.And the polypeptide of interfertile polynucleotide encoding has identical biological function and activity with the mature polypeptide shown in the SEQ ID NO:2.

The invention still further relates to nucleic acid fragment with above-mentioned sequence hybridization.As used herein, the length of " nucleic acid fragment " contains 15 Nucleotide at least, better is at least 30 Nucleotide, is more preferably at least 50 Nucleotide, preferably more than at least 100 Nucleotide.Nucleic acid fragment can be used for the amplification technique (as PCR) of nucleic acid to determine and/or the proteic polynucleotide of separation coding Candida albicans CaSwi1.

Candida albicans CaSwi1 Nucleotide full length sequence of the present invention or its fragment can obtain with the method for pcr amplification method, recombination method or synthetic usually.For the pcr amplification method, can be disclosed according to the present invention about nucleotide sequence, especially open reading frame sequence designs primer, and with commercially available dna library or by the prepared dna library of ordinary method well known by persons skilled in the art as template, amplification and must relevant sequence.When sequence is longer, usually needs to carry out twice or pcr amplification repeatedly, and then the fragment that each time amplifies is stitched together by proper order.

In case obtained relevant sequence, just can obtain relevant sequence in large quantity with recombination method.This normally is cloned into carrier with it, changes cell again over to, separates obtaining relevant sequence then from the host cell after the propagation by ordinary method.

In addition, also the method for available synthetic is synthesized relevant sequence, especially fragment length more in short-term.Usually, by first synthetic a plurality of small segments, and then connect and to obtain the very long fragment of sequence.

At present, can be fully obtain the dna sequence dna of code book invention albumen (or its fragment, or derivatives thereof) by chemosynthesis.This dna sequence dna can be introduced in various existing dna moleculars as known in the art (or as carrier) and the cell then.In addition, also can will suddenly change and introduce in the protein sequence of the present invention by chemosynthesis.

Use method (Saiki, the et al.Science1985 of round pcr DNA amplification/RNA; 230:1350-1354) be optimized for acquisition gene of the present invention.The primer that is used for PCR can suitably be selected according to sequence information of the present invention disclosed herein, and available ordinary method is synthetic.Available ordinary method is as the DNA/RNA fragment by gel electrophoresis separation and purifying amplification.

The present invention also relates to comprise the carrier of polynucleotide of the present invention, and the host cell that produces through genetically engineered with carrier of the present invention or Candida albicans CaSwi1 albumen coded sequence, and the method that produces polypeptide of the present invention through recombinant technology.

Recombinant DNA technology (Science, 1984 by routine; 224:1431), can utilize polymerized nucleoside acid sequence of the present invention to can be used to express or produce the CaSwi1 polypeptide of reorganization.In general following steps are arranged:

(1). with the polynucleotide (or varient) of coding Candida albicans CaSwi1 polypeptide of the present invention, or transform or the transduction proper host cell with the recombinant expression vector that contains these polynucleotide;

(2). the host cell of in suitable medium, cultivating;

(3). separation, protein purification from substratum or cell.

Among the present invention, Candida albicans CaSwi1 polynucleotide sequence can be inserted in the recombinant expression vector.Term " recombinant expression vector " refers to that bacterial plasmid well known in the art, phage, yeast plasmid, vegetable cell virus, mammalian cell virus are as adenovirus, retrovirus or other carriers.In a word, as long as can duplicate in host and stablize, any plasmid and carrier can be used.A key character of expression vector is to contain replication orgin, promotor, marker gene and translation controlling elements usually.

Method well-known to those having ordinary skill in the art can be used to make up and contains Candida albicans CaSwi1 DNA sequences encoding and suitable transcribing/the translate expression vector of control signal.These methods comprise extracorporeal recombinant DNA technology, DNA synthetic technology, the interior recombinant technology of body etc.Described dna sequence dna can effectively be connected on the suitable promotor in the expression vector, and is synthetic to instruct mRNA.The representative example of these promotors has: colibacillary lac or trp promotor; Lambda particles phage PL promotor; Eukaryotic promoter comprises LTRs and some other known may command gene expression promoter in protokaryon or eukaryotic cell or its virus of CMV immediate early promoter, HSV thymidine kinase promoter, early stage and late period SV40 promotor, retrovirus.Expression vector also comprises ribosome bind site and the transcription terminator that translation initiation is used.

Host cell can be a prokaryotic cell prokaryocyte, as bacterial cell; Or eukaryotic cell such as low, as yeast cell; Or higher eucaryotic cells, as mammalian cell.Representative example has: intestinal bacteria, streptomyces; The bacterial cell of Salmonella typhimurium; Fungal cell such as yeast; Vegetable cell; The insect cell of fruit bat S2 or Sf9; The zooblast of CHO, COS, 293 cells or Bowes melanoma cells etc.

Can carry out with routine techniques well known to those skilled in the art with the recombinant DNA transformed host cell.When the host was prokaryotic organism such as intestinal bacteria, the competent cell that can absorb DNA can be used CaCl in exponential growth after date results ₂Method is handled, and used step is well-known in this area.Another kind method is to use MgCl ₂If desired, transforming also the method for available electroporation carries out.When the host is an eukaryote, can select following DNA transfection method for use: coprecipitation of calcium phosphate method, conventional mechanical method such as microinjection, electroporation, liposome packing etc.

The transformant that obtains can be cultivated with ordinary method, expresses the polypeptide of coded by said gene of the present invention.According to used host cell, used substratum can be selected from various conventional substratum in the cultivation.Under the condition that is suitable for the host cell growth, cultivate.After host cell grows into suitable cell density, induce the promotor of selection with suitable method (as temperature transition or chemical induction), cell is cultivated for some time again.

The extracellular can be expressed or be secreted into to recombinant polypeptide in the above methods in cell or on cytolemma.If desired, can utilize its physics, the separating by various separation methods with other characteristic and the albumen of purification of Recombinant of chemistry.These methods are well-known to those skilled in the art.The example of these methods includes, but are not limited to: conventional renaturation handles, with protein precipitant handle (salt analysis method), centrifugal, the broken bacterium of infiltration, superly handle, the combination of super centrifugal, sieve chromatography (gel-filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography (LC) technology and these methods.

The Candida albicans CaSwi1 albumen or the polypeptide of reorganization are of use in many ways.These purposes include, but is not limited to: be used to screen and promote or to antibody, polypeptide or other part of anti-candida albicans CaSwi1 protein function.The peptide molecule that can suppress or stimulate Candida albicans CaSwi1 protein function that can be used for seeking therapeutic value with the reorganization Candida albicans CaSwi1 protein screening peptide library of expressing.

On the other hand, the present invention also comprises Candida albicans CaSwi1 DNA or the polypeptide of its fragment coding has specific polyclonal antibody and monoclonal antibody, especially monoclonal antibody.Here, " specificity " is meant that antibody capable is incorporated into Candida albicans CaSwi1 gene product or fragment.Preferably, refer to that those can combine with Candida albicans CaSwi1 gene product or fragment but nonrecognition and be incorporated into the antibody of other irrelevant antigen molecule.

The present invention not only comprises complete mono-clonal or polyclonal antibody, but also comprises having immunocompetent antibody fragment, as Fab ' or (Fab) ₂Fragment; Heavy chain of antibody; Light chain of antibody; Genetically engineered strand Fv molecule; Or chimeric antibody.Antibody of the present invention can be prepared by the known various technology of those skilled in that art.For example, the Candida albicans CaSwi1 gene product of purifying or its have antigenic fragment, can be applied to animal to induce the generation of polyclonal antibody.Similarly, expressing Candida albicans CaSwi1 albumen or its has antigenic segmental cell and can be used to immune animal and produce antibody.Antibody of the present invention also can be monoclonal antibody.This type of monoclonal antibody can utilize hybridoma technology to prepare.

The proteic antibody of anti-candida albicans CaSwi1 can be used in the immunohistochemistry technology, detects the Candida albicans CaSwi1 albumen in the biopsy specimen.

Utilize albumen of the present invention,, can filter out with Candida albicans CaSwi1 albumen interactional material takes place, as acceptor, inhibitor, agonist or antagonist etc. by various conventional screening methods.

The invention still further relates to the diagnostic testing process of quantitative and detection and localization Candida albicans CaSwi1 protein level.These tests are known in the art.

Whether having the proteic method of Candida albicans CaSwi1 in a kind of detection test sample is to utilize the proteic specific antibody of Candida albicans CaSwi1 to detect, and it comprises: sample is contacted with Candida albicans CaSwi1 protein specific antibody; Observe whether form antibody complex, formed antibody complex and just represented to exist in the sample Candida albicans CaSwi1 albumen.

Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, people such as Sambrook for example, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.

Universal method:

Southern analyzes

Candida albicans gene group DNA method for extracting and Southern hybridization analysis method are according to document operation (Chen et al.J.Bacteriol.174:5624-56312.).Genomic dna PvuII (available from GIBCO company) complete degestion, agarose gel after electrophoresis is finished soaks 45min with sex change liquid and neutralizer respectively, and nylon membrane soaks into distilled water or 10xSSC, puts up and changes the film platform, glue and intermembranous with the Parafilm sealing adds a 500g counterweight., be to change the transfer of film liquid to spend the night with 10xSSC, rinsing in second day is dried crosslinked.Film after the crosslinked hybrid pipe of packing into adds 10ml prehybridization solution (6xSSC, 5xdenhardt ' s Reagent, 0.5%SDS, 100 μ g/ml milt DNA are in 42 ℃ of prehybridization 12h, and probe adds 42 ℃ of hybridization of 150 μ l (probe of about 1/3 mark) 10-16h at 100 ℃ of sex change 5min.0.1xSSC 0.1%SDS washes two to three times, each 40min takes out film, dries room temperature or-70 ℃ of compressing tablets.Probe mark is operated with the random primer labelling test kit of invitrogene and according to its explanation.The solution that will contain 25ng DNA is in 100 ℃ of heat denatured 5-10min, place ice bath, add Random Primers Buffer Mixture more successively, 2 μ l dCTP, 2 μ l dGTP, 2 μ l dTTP, 3 μ l[α-32P]-dATP (10 μ Ci/ μ l), add 1 μ l Klenow enzyme behind the mixing, in 25 ℃ of insulation 1h, with the centrifugal 4min of 3000rpm, collect centrifugate with Sephadex G-25 post, be the probe solution behind the purifying.Probe adds in the hybridization system after 100 ℃ of sex change 5min cooled on ice.

Northern analyzes

With the total RNA of hot phenol method extracting Candida albicans, and Northern hybridization analysis method reference literature (Greenberg, M.E.1987, Current protocols in molecular biology, vol.1.Wiley Interscience, New York, N.Y.).Particularly method is as follows: each bacterial strain is at YPD, is cultured to the logarithmic phase later stage in 30 ℃, is forwarded to YPD+10% serum, perhaps Lee ' s substratum is 1, initial OD=0.05, and under relevant temperature, cultivate the fixed time, with the total RNA of hot phenol method extracting Candida albicans.20 μ l applied sample amounts comprise RNA sample 12 μ l (25 μ g), 4 μ l formaldehyde, 2 μ l 10xMOPS, 2 μ l sample-loading buffers, 65 ℃ of heating 10min, 0 ℃ of cooling 2min, centrifugal 5s, last sample.Electrophoresis carries out (87ml DEPC treating water, the cold slightly back of heating and melting adds 2.7ml 37% formaldehyde for 1g agarose, 10ml 10xMOPS) containing on the sex change glue of formaldehyde.Deposition condition is 100mA, and 50-70V is same with capillary method commentaries on classics film after the 5-7h electrophoresis is finished, and changes film liquid 5xSSC.After the commentaries on classics film spends the night, take out film, film is parched, UV-crosslinked with Bio-Ra GS Gene Linker C3protocol, add the 10ml prehybridization solution, 65 ℃, be replaced by the 10ml hybridization solution behind the 2h, add 65 ℃ of hybridization of probe and spend the night, use 2xSSC, 0.1%SDS washes film twice for 65 ℃, each 30min.The film that hybridization is good does not dry, and wraps-70 ℃ of compressing tablets in back with preservative film.Heavily film 0.5%SDS is washed in hybridization, and 90-100 ℃ of heating 5-10min, or recommend use 0.5%SDS according to Clontech takes out standbyly behind 90-100 ℃ of its naturally cooling of heating 10min relief 10min, as if a usefulness immediately not, wrap with preservative film and to be placed on-20 ℃ of preservations.

The conversion of Candida albicans and yeast saccharomyces cerevisiae

Prepare PEG/LiAc solution (pH 7.5) 10ml; In 1.5mI Eppendof pipe, add plasmid (if change yeast saccharomyces cerevisiae, plasmid 0. μ g is if change Candida albicans, the about 5 μ g of linearization plasmid) successively, 10 μ l10mg/ml milt DNA, mixing; Add 0.1ml competent cell and 0.6ml PEG/LiAc, the votex mixing; 30 ℃ of 200rpm cultivate 30min; Add 70 μ l DMSO, mixings gently; 42 ℃ of thermal shocking 15min, during shake up gently frequently; Ice bath～2min: high speed centrifugation 15s sops up supernatant, with 0.2ml TE re-suspended cell; Coat on the suitable nutrition screening SD flat board.

The infection experiment of mouse system

Is experimental subjects with body weight at 16-18g ICR male mice, by tail vein injection 100 each bacterial strain of μ l Candida albicans, and cultivates observation 25 days.Two concentration of each injection height of each bacterial strain, high-concentration bacterial liquid is 5 * 10 ⁷Cell/ml, lower concentration bacterium liquid is 5 * 10 ⁶Cell/ml.

Embodiment 1:

The clone of CaSWI1 gene

The chromogene storehouse of the Candida albicans that will make up with ordinary method changes yeast saccharomyces cerevisiae monoploid flo8 disappearance strain (can available from Yeast Genetics Stock Culture Center of the American Type CultureCollection) over to, on the SD-ura flat board, grow, obtain 30,000 bacterium colonies, after the water flushing, remaining altogether 40 bacterium colonies on the flat board.These bacterium colony complementations flo8 lack caused intussusception defective, can carry out intussusception.

Extracting yeast plasmid by the following method: inoculate single bacterium colony in 5ml SD ura-liquid nutrient medium, 30 ℃ of shaking culture 20h, the centrifugal 3min of room temperature 5000r/min, abandon supernatant, add 200 μ l yeast lysate (2%Triton X-100,1%SDS, 100mmol/L NaCl, 10mmol/L Tris-HCl pH 8.0,1mM EDTA), resuspended yeast vibrates, add 0.3mg pickling glass pearl (425～600 μ m), 200 μ l phenol: chloroform: primary isoamyl alcohol (25: 24: 1), the 5min that fully vibrates, liquid nitrogen freezes 10min, room temperature is melted, the 5min that fully vibrates again, the centrifugal 10min of room temperature 12000r/min is transferred to new 1.5ml centrifuge tube with supernatant liquor, add 20 μ l 3mol/L NaAc, 500 μ l dehydrated alcohols ,-70 ℃ of frozen 1h, 4 ℃ of centrifugal 10min of 12500r/min, abandon supernatant, 70% washing with alcohol precipitation is drained, and adds 20 μ l TE (pH 8.0) dissolving.

Plasmid that will extracting goes out from these bacterium colonies, change intestinal bacteria over to, extractive plasmid imports monoploid and the strain of amphiploid flo8 disappearance respectively from intestinal bacteria, observe the intussusception and the filamentous growth phenotype of bacterium colony, finds No. 37 plasmids to the strain of yeast saccharomyces cerevisiae flo8 disappearance pseudohypha grow and intussusception has very strong promoter action.With No. 37 plasmid order-checkings, and compare analysis by NCBI, obtain the CaSWI1 gene, 5 ' and 3 ' the end non-coding region that comprises open reading frame, total length has 3802 Nucleotide (Fig. 1 and SEQ ID NO:1), its ORF is positioned at the 554-3314 position, the albumen (Fig. 2 and SEQ ID NO:2) of coding total length 987aa.

Utilize the search of BLAST homology, find that the coded product of this gene and the SWI1 gene encoding production of yeast saccharomyces cerevisiae (Sacchromycescerevisiae) have the highest homology (23%identity), therefore be this unnamed gene CaSWI1, the albumen of CaSWI1 genes encoding is called CaSwi1.

Embodiment 2

Candida albicans CaSWI1 gene can the complementary ScSWI1 functional defect of part

The CaSWI1 gene can be proofreaied and correct ste7/ste7 in Expression in Saccharomyces Cerevisiae, ste12/ste12, and tec1/tec1, the defective of flo8/flo8 and phd1/ste12 disappearance strain mycelial growth illustrates that CaSWI1 has the ability that mycelia forms that activates in yeast saccharomyces cerevisiae.

Pass through domain analyses, find that CaSwi1 and ScSwi1 have a tangible BRIGHT/ARID structural domain, this structural domain has nonspecific DNA in conjunction with character, protein with this structural domain can be incorporated into and contain the more DNA zone of AT (Deborah Wilsker et al.Cell.Growth.Differentiation.2002,13:95-106.), this structural domain has diversity on 26S Proteasome Structure and Function.At both BRIGHT/ARID structural domain N ends the zone of being rich in glutamine is arranged all, this regional supposition can combine with some transcription factors.Also have a zinc fingers at the C of ScSwi1 end, in CaSwi1, do not have (Fig. 3 A).According to this zinc fingers proof ScSwi1 be a nuclear locating sequence (Aileen K.et al.Genet.1987,116:523-530.).

The disappearance strain scswi1 of ScSWI1 gene shows defective (Aileen K.et al.Genet.1987, the 116:523-530 that non-sugar fermentation is utilized; Craig L.Peterson et al.Cell.1992 68:573-583.), can suppress this defective and import ScSWI1 again.Candida albicans CaSwi1 and Saccharomyces Cerevisiae in S cSwi1 have similarity on sequence and structural domain, but in the utilization of non-sugar fermentation, only the growth defect that CaSWI1 can the strain of the complementary scswi1 disappearance of part in glycerin medium.Be on the substratum of carbon source with fermentable sugars glucose, scswi1 disappearance strain and wild strain speed of growth difference are little, but with semi-lactosi, sucrose, non-sugar fermentation such as raffinose is on the substratum of carbon source, the scswi1 disappearance strain speed of growth obviously descends, and imports and cross to express the speed of growth that CaSWI1 can not improve the disappearance strain, and its speed of growth is similar with the strain of scswi1 disappearance basically.And on the SD substratum that with glycerine is sole carbon source, the strain of scswi1 disappearance imports and can improve its speed of growth behind the CaSWI1 and grow in glycerin medium, explanation is being in the utilization of carbon source with glycerine, the defective (Fig. 3 B) of the complementary ScSWI1 function of CaSWI1 energy part, and with other non-sugar fermentation be on the substratum of carbon source CaSWI1 can not be complementary the defective of scswi1 disappearance strain growth, therefore CaSwi1 and ScSwi1 have certain similarity on sequence, and the complementarity of part is also arranged on function.

Embodiment 3

The high expression level of CaSWI1 promotes the Candida albicans mycelial growth

1.CaSWI1 the structure of high-expression plasmid

The CaSWI1 encoder block obtains from the wild type strain pcr amplification with pfu archaeal dna polymerase (available from ancient cooking vessel state company), cuts 1 hour with 37 ℃ of enzymes of KpnI (available from GIBCO company).Candida albicans high-expression vector BA1 (Feng, Q.et al.J.Bacteriol.181:6339-6346) cuts with the KpnI enzyme and with alkaline phosphatase (CIP, available from BioLab company) 37 ℃ handled back 1 hour, be connected 1 hour with the section of PCR enzyme is disconnected for 16 ℃ with T4DNA ligase enzyme (available from GIBCO company), transformed into escherichia coli, the picking mono-clonal, the extraction plasmid is cut evaluation with KpnI and EcoRV (all available from GIBCO company) enzyme and is obtained the CaSWI1 high-expression plasmid.The used primer of amplification CaSWI1 encoder block is primer 1:GCT GGT ACC ATG TCT GAT TGG TTG AAT GAA AAT GC (SEQID NO:3) and primer 2: GTC GGT ACC CTA GAT CCC CTC ACA AGC CTT TAA (SEQ ID NO:4).

The proteic molecular weight of CaSwi1 of expressing is about 110KD, conforms to predictor.

2.CaSWI1 high expression level promote the Candida albicans mycelial growth

The mycelia that the high expression level of CaSWI1 in the strain of yeast saccharomyces cerevisiae disappearance can activate the disappearance strain forms, can activate mycelial growth in order to study the high expression level of CaSWI1 in Candida albicans, the ADE2 site integrating vector of utilization under the ADHI promotor is handled imports CaSWI1 in the Candida albicans, find the high expression level of CaSWI1, can promote the formation of Candida albicans mycelia to a certain extent.Wild type strain is at the SD substratum, cultivate after 7 days for 37 ℃, form smooth surface, the bacterium colony of regular edges, and the high expression level bacterial strain surface of CaSWI1 begins to form gauffer, and the edge is irregular, and forms in a spot of mycelia intrusion substratum (Fig. 4 A), scrape the bacterium that takes a morsel and examine under a microscope, find that the high expression level bacterial strain of CaSWI1 forms more pseudohypha (Fig. 4 B) than wild type strain.Northern experiment confirm CaSWI1 in this bacterial strain really high expression level (Fig. 4 C).

These results show that CaSWI1 has certain mycelia activation at the intravital high expression level of Candida albicans.

Embodiment 4

Knocking out of Candida albicans CaSWI1 gene

In order to study the function of Candida albicans CaSWI1 gene, in the present embodiment, in Candida albicans, knocked out this gene.External structure CaSWI1 gene knockout plasmid, CaSWI1 open reading frame (openreading frame, ORF) 1.8kb BclI segment substitutes with HisG-URA3-HisG in, method by homologous recombination, can replace BclI fragment in the CaSWI1 gene on the karyomit(e) with HisG-URA3-HisG, thus the CaSWII gene on the destruction karyomit(e).Screening sign URA3 and a HisG sequence can contain 5-fluororotic acid (5-fluoro-orotic acid, 5-FOA) dull and stereotyped go up by two in the same way the reorganization of HisG homologous sequence on same karyomit(e) encircle out, the recon of having lost URA3 screening sign can obtain (Boeke et al.Mol.Gen.Genet.197:345-346) by the forward screening on the 5-FOA flat board, thereby can carry out the conversion of next round and then destroy CaSWI1 gene on another karyomit(e), the recon genotype detects with Southern hybridization and identifies.

The result:

1, the phenotype of Candida albicans CaSWI1 disappearance strain

Knocking out of CaSWI1 gene caused the variation of a series of cellular fories of Candida albicans and colonial morphology.Knocking out of CaSWI1 gene causes the albicans growth defective, and the importing again of CaSWI1 then can be replied the speed of growth (Fig. 5).With sucrose, the non-sugar fermentation of semi-lactosi and raffinose is that the substratum of carbon source and the speed of growth on the substratum that is carbon source do not have significant difference with glucose, but be on the substratum of carbon source with glycerine, the disappearance strain speed of growth is slightly decline on the substratum of carbon source than with glucose, illustrates that CaSWI1 has effect utilizing on the pathways metabolism of glycerine really.The CaSWI1 gene knock out the variation that causes the albicans cell form.The strain of caswi1 disappearance is in YPD or SD substratum, under 30 ℃ of culture condition, several to dozens of cells can be formed with branched streptocyte, in these cell masses, often middle initial parent cell can form bigger spherule cell, and chain intermediary daughter cell tends to elongation, some daughter cell can grow several daughter cells at different sites, terminal daughter cell major part all is a spheric, and a part also has slight elongation, and this cellular form is very similar with the pseudohypha in the Candida albicans.And wild type strain presents typical dispersive spherule cell growth under same culture condition.The cellular form of this chain-like growth all has discovery (Mark D.McNemaret al.J.Bacteriology.Apr.2002, p:2058-206 in the disappearance strain in the disappearance strain of other Candida albicans and yeast saccharomyces cerevisiae; Eric S.Bensen et al.Eukaryotic Cell, Oct.2002, p:787-798.), the division of the normal formation of septin ring or septum separates the generation problem after the conversion of prompting possibility G2/M phase or the mitotic division, and hint CaSwi1 may have effect in these processes.On YPD or SD substratum, the colonial morphology of caswi1 disappearance strain also changes.By the original slick surface that has, the rule edge transition be the surface and the irregular edge of gauffer, and between bacterium colony and substratum and the adhesion between bacterium and the bacterium obviously descend.And do not cause in the disappearance of Saccharomyces Cerevisiae in S cSWI1 and the variation of cell and colonial morphology to have illustrated that CaSWI1 may bring into play different effects in vivo with ScSWI1 yet.At Candida albicans CBK1, FKH2 disappearance strain and yeast saccharomyces cerevisiae FKH1 also can form similar cell and colonial morphology (Mark D.McNemar etal.J.Bacteriology.Apr.2002, p:2058-206 in the strain of the two disappearance of FKH2; Peter C.Hollenhorst etal.Genetics.2000.154:1533-1548; Eric S.Bensen et al.EukaryoticCell, Oct.2002, p:787-798.).

2, the CaSWI1 gene knocks out the influence that the Candida albicans mycelia is formed

It is essential to its intrusion and infection that the Candida albicans mycelia forms ability.The knocking out of CaSWI1 gene greatly reduces the Candida albicans mycelia and forms ability.Containing on the solid Agar plate of serum, through 37 ℃ of cultivations in 5 days, wild type strain and answer bacterial strain all form mycelia consumingly, and the disappearance strain forms less circular bacterium colony, and mycelia forms seriously be obstructed (Fig. 6 A).This mycelial growth blocking-up phenomenon is more obvious on solid Lee ' s flat board.Through 37 ℃ of cultivations in 7 days, wild type strain and answer bacterial strain all form mycelia consumingly, and caswi1 disappearance strain mycelia formation ability is blocked fully, though the disappearance strain grows up to the irregular bacterium colony in edge on Lee ' s solid board, but the edge but is slick, does not see the formation (Fig. 6 A) of mycelia.In the liquid medium within (Fig. 6 B), YPD adds 10% calf serum, through 37 ℃, after cultivating in 3 hours, can see wild type strain and reply bacterial strain all forming typical mycelia (hyphae), the tubulose cell of prolongation and the cell walls of parallel growth are not significantly hung between the cell and are contracted, the strain of caswi1 disappearance then can not form this typical mycelia, remains chain between the cell and links to each other.In Lee ' s liquid nutrient medium, cellular form and YPD add 10% serum cultivate in basically identical, illustrate that the disappearance of CaSWI1 gene blocked the formation of Candida albicans mycelia really.

3, the disappearance of CaSWI1 gene causes Candida albicans mycelia specific gene change of Expression

The disappearance of Candida albicans CaSWI1 gene caused the variation of a series of cells and colonial morphology, detected phenotype that CaSWI1 genetically deficient causes and change that the change that is by which genetic expression realizes on gene level.HWP1, ECE1 (Staab et al.J.Biol.Chem.271:6298-6305; Birse et al.Infect.Immun.61:3648-3655.) be Candida albicans mycelial growth specific gene, under the mycelial growth condition, these genes all present high expression level, and under the yeast growth condition, these genes are not expressed.Can see by the Northern hybridization analysis, under the mycelia inductive condition, the disappearance of CaSWI1 gene is not induced the expression of HWP1 and ECE1, and other bacterial strain that can be induced into mycelial growth all has the high expression level (Fig. 7) of HWP1 and ECE1, illustrates that the disappearance of CaSWI1 gene has influenced the mycelial growth Expression of Related Genes really.

4, the disappearance of CaSWI1 gene is to the toxic influence of Candida albicans

Knocking out of CaSWI1 gene causes Candida albicans mycelial growth defective, even make Candida albicans under intensive mycelia inductive condition, also can't form mycelia, carry out system's infection experiment by the mouse tail vein injection albicans strain and detect knocking out of CaSWI1 gene and whether caused bacterial strain toxicity to descend.The result shows that wild type strain is toxic, and no matter caswi1 disappearance strain is in high density (5 * 10 ⁷Cell/ml) still at lower concentration (5 * 10 ⁶All do not have toxicity (Fig. 8) under the cell/ml), illustrate that activating transcription factor CaSwi1 is a virulence factor important in the Candida albicans, participate in the pathogenic course of Candida albicans.

All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.

Sequence table

＜110〉Shanghai Inst. of Life Science, CAS

＜120〉Candida albicans activating transcription factor gene and uses thereof

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Met Ser Asp Trp Leu Asn Glu Asn Ala Phe Ala Asp

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Ser Asn Ser Asn Asp Asp Phe Leu Asn Ser Ile Phe Asp Gln Ser Gln

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Gly Glu Gln Gln Ala Pro Gln Val Ala Gln Val Ser Thr Ser Met Ser

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Asn Pro Pro Leu Gln Ser Gln Ser Ala Leu Ser Thr Ser Arg Ile Ser

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Gln Ala His Thr Pro Met Tyr Gln Gln Ser Pro Val Thr Ala His Thr

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Ile Pro Gln Asn Ser Pro Gln Ser Met Pro Asn Gln Val Ala Gln Pro

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Gln Gln Gln Ile Pro Pro Pro Pro Leu Gln His Leu Gln Gln Thr Thr

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Ala Gln Met Leu Pro Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln

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Gln Gln Gln Lys Gln Glu Gln Leu Tyr Arg Met Lys Gln Gln Ile Tyr

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Gln Gln Gln Met Leu Lys Lys Gln Gln Glu Asn Met Ser Arg Gln Pro

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Ser Pro Met Asn Ser Ala Gly His Asn Thr Gln Gln Asn Thr Pro Ile

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Thr Gln Asn Ala Lys Thr Pro Gln Asn Asn Ser Lys Leu Gln Ser Met

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Gln Met Glu Leu Phe Phe Ser Val Leu Tyr Asp Phe Leu Gln Arg Ser

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

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Leu Phe Ile Leu Tyr Ile Leu Ser Gln Arg Leu Gly Gly Tyr Arg Val

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Met Lys Ala Phe Leu Leu Met Ser Pro Glu Gln Gln Arg Met Gln Gln

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Gly Glu Asp Pro Met Ala Lys Glu Arg Ile Ala Arg Glu Leu Cys Asn

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Cys Tyr Ser Asn Phe Ile Leu Pro Tyr Glu Glu Tyr Tyr Ala Thr Pro

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Glu Gly Asn Lys Asp Ile Glu Ala Ser Lys His Arg Phe Gln Gln Gln

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Ile Val Gln Lys Tyr Leu Ser Asn Val Pro Thr Pro Ala Pro Asn Gln

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caa agt cca atg ata agt agc gct cca aca cca cac caa caa agg aag 1597

Gln Ser Pro Met Ile Ser Ser Ala Pro Thr Pro His Gln Gln Arg Lys

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Leu Ser Arg Thr Ser Asn Ser Val Asn Asn Ser Pro Asn Val Ser Ser

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Pro Phe His Asn Thr Pro Arg Gln Val Pro Ala Pro Ala Pro Ala Pro

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Phe Lys Lys Ile Val Glu Thr His Gly Pro Phe Asn Ile Lys Glu Leu

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Ser Gln Leu Ser Thr Glu Ile Glu Val Thr Lys Pro Val Tyr Leu Phe

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Thr Leu Leu Val Ala Thr Ser Asp Val Asn Tyr Ala Phe Gln Ile Lys

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

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Leu Asn Lys Ile Val Gly Val Asn Thr Glu Glu Asp Cys Val Asn Ala

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

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Leu Thr Gly Glu Val Val Ser Asp Asp Glu Asp Ile Glu Glu Leu Phe

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

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Asp Leu Leu Asp Val Leu Glu Phe His Leu Asp Asp Tyr Leu Thr Ala

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Leu Lys Asn Phe Lys Ser Glu Asn Lys His His Phe Ser Lys Leu Gln

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Glu Pro Met Ala Gly Asn Arg Leu Phe Lys Asp Leu Leu Phe Ser Thr

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Arg Leu Cys Leu Leu Lys Asp Cys Leu Leu Met Leu Asp Asn Ile Ser

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Leu Phe Thr His Leu His Thr Leu Glu Glu Ala Phe Leu Ser Phe Val

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Leu Val Ala Ser Phe Gly Pro Lys Ile Glu Asp Gln Tyr Lys Ile Pro

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Arg Cys Asn Ile Glu Thr His Ser Tyr Phe Ala Phe Gly Leu Asp Ala

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ttc acc aag ttg atg gtc aga gag cca tac aat aga tca tta att cag 2845

Phe Thr Lys Leu Met Val Arg Glu Pro Tyr Asn Arg Ser Leu Ile Gln

750 755 760

gca gtt ttg aat gga act ttg aat tcc agt atg aca gga tat tct gtc 2893

Ala Val Leu Asn Gly Thr Leu Asn Ser Ser Met Thr Gly Tyr Ser Val

765 770 775 780

agt tta cag gat caa gag tac aca aga aag tta atc aag gct tac aac 2941

Ser Leu Gln Asp Gln Glu Tyr Thr Arg Lys Leu Ile Lys Ala Tyr Asn

785 790 795

aaa gat tat aag tca gca tca ctt ttg agt caa gct ttc cag atg tat 2989

Lys Asp Tyr Lys Ser Ala Ser Leu Leu Ser Gln Ala Phe Gln Met Tyr

800 805 810

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Met Ser Ile Leu Pro Phe Asp Ala Asn Thr Phe Glu Leu Ser Lys Phe

815 820 825

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Ile Phe Met Arg Ser Pro Thr Ile Ser Gln Met Leu Phe Gly Ala Lys

830 835 840

ttg tta ata gac atg gtt cca gtt gat gat ttg aac aca cat cat aac 3133

Leu Leu Ile Asp Met Val Pro Val Asp Asp Leu Asn Thr His His Asn

845 850 855 860

aag ttg tct ctt tat tgg cta ttg gaa aac cgg gag tta tta ttg ggt 3181

Lys Leu Ser Leu Tyr Trp Leu Leu Glu Asn Arg Glu Leu Leu Leu Gly

865 870 875

aat ttt gcc agg ata gtc gtt gct tta tct aca gaa act gga aaa ttc 3229

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

880 885 890

cca cgc gaa tcg cct gag cac aag gtt ttg tcg ctg gta tta cgc aag 3277

Pro Arg Glu Ser Pro Glu His Lys Val Leu Ser Leu Val Leu Arg Lys

895 900 905

gcc ctc gtt gtt atc aat tca ttg gtc gac aat gca gtt ttg gct aag 3325

Ala Leu Val Val Ile Asn Ser Leu Val Asp Asn Ala Val Leu Ala Lys

910 915 920

gaa ata gga gac ctg cga cac acc gac ttg atg gag agt ttg aca gat 3373

Glu Ile Gly Asp Leu Arg His Thr Asp Leu Met Glu Ser Leu Thr Asp

925 930 935 940

tgt ttg gct ttc cca aga ata ata ccc gat gcc att ttg aca ttg gac 3421

Cys Leu Ala Phe Pro Arg Ile Ile Pro Asp Ala Ile Leu Thr Leu Asp

945 950 955

acg ttt ttg gca cct acc att gat acc aat tta ggt aaa gaa gtt gtc 3469

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

960 965 970

aga tta ctt agg tat ttg aag gat tta aag gct tgt gag ggg atc 3514

Arg Leu Leu Arg Tyr Leu Lys Asp Leu Lys Ala Cys Glu Gly Ile

975 980 985

tagatgtatc ttcggtttgg tttttttttt tgtgatttgt tcttatctat aattgttgtt 3574

gtgtattgtt tttattttgt aaattttaat gtataattaa tcaaagcgca agtgcgcaag 3634

aatataattt tgttgcaatc atgtaagcac ctttatctgt agattgcaaa tattacactt 3694

tcctaattat taatgccctg atgtgtattc gttatctttt tgcttgattg cttgtgcttg 3754

tattccctcc ccctctttat catgtttacc aatctgggta taacgatc 3802

<210>2

<211>987

<212>PRT

＜213〉Candida albicans (Candida albicans)

<400>2

Met Ser Asp Trp Leu Asn Glu Asn Ala Phe Ala Asp Ser Asn Ser Asn

1 5 10 15

Asp Asp Phe Leu Asn Ser Ile Phe Asp Gln Ser Gln Gly Glu Gln Gln

20 25 30

Ala Pro Gln Val Ala Gln Val Ser Thr Ser Met Ser Asn Pro Pro Leu

35 40 45

Gln Ser Gln S6r Ala Leu Ser Thr SerArg Ile Ser Gln Ala His Thr

50 55 60

Pro Met Tyr Gln Gln Ser Pro Val Thr Ala His Thr Ile Pro Gln Asn

65 70 75 80

Ser Pro Gln Ser Met Pro Asn Gln Val Ala Gln Pro Gln Gln Gln Ile

85 90 95

Pro Pro Pro Pro Leu Gln His Leu Gln Gln Thr Thr Ala Gln Met Leu

100 105 110

Pro Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Lys

115 120 125

Gln Glu Gln Leu Tyr Arg Met Lys Gln Gln Ile Tyr Gln Gln Gln Met

130 135 140

Leu Lys Lys Gln Gln Glu Asn Met Ser Arg Gln Pro Ser Pro Met Asn

145 150 155 160

Ser Ala Gly His Asn Thr Gln Gln Asn Thr Pro Ile Thr Gln Asn Ala

165 170 175

Lys Thr Pro Gln Asn Asn Ser Lys Leu Gln Ser Met Gln Met Glu Leu

180 185 190

Phe Phe Ser Val Leu Tyr Asp Phe Leu Gln Arg Ser Gly Ile Ser Ile

195 200 205

Pro Gln Ser Leu Ala Ile Asn Gly Lys Arg Val Asn Leu Phe Ile Leu

210 215 220

Tyr Ile Leu Ser Gln Arg Leu Gly Gly Tyr Arg Val Met Lys Ala Phe

225 230 235 240

Leu Leu Met Ser Pro Glu Gln Gln Arg Met Gln Gln Gln Asn Pro Trp

245 250 255

Ala Leu Met Ala Asp Lys Met Gly Phe His Asp Lys Gly Glu Asp Pro

260 265 270

Met Ala Lys Glu Arg Ile Ala Arg Glu Leu Cys Asn Cys Tyr Ser Asn

275 280 285

Phe Ile Leu Pro Tyr Glu Glu Tyr Tyr Ala Thr Pro Glu Gly Asn Lys

290 295 300

Asp Ile Glu Ala Ser Lys His Arg Phe Gln Gln Gln Ile Val Gln Lys

305 310 315 320

Tyr Leu Ser Asn Val Pro Thr Pro Ala Pro Asn Gln Gln Ser Pro Met

325 330 335

Ile Ser Ser Ala Pro Thr Pro His Gln Gln Arg Lys Leu Ser Arg Thr

340 345 350

Ser Asn Ser Val Asn Asn Ser Pro Asn Val Ser Ser Pro Phe His Asn

355 360 365

Thr Pro Arg Gln Val Pro Ala Pro Ala Pro Ala Pro Val Gln Thr Ala

370 375 380

Ala Ser Met Pro Pro Pro Thr Pro Val Thr Ala Lys Ser Gln Thr Thr

385 390 395 400

Lys Ala Asp Ala His Ile Leu Lys Asn Tyr Thr Pro Phe Lys Lys Ile

405 410 415

Val Glu Thr His Gly Pro Phe Asn Ile Lys Glu Leu Ser Gln Leu Ser

420 425 430

Thr Glu Ile Glu Val Thr Lys Pro Val Tyr Leu Phe Ala Pro Glu Leu

435 440 445

Gly Ile Ile Asn Leu Gln Ala Leu Thr Met Ser Val Lys Ser Asn Ser

450 455 460

Gly Ile Gln Ser Ser Glu Val Val Asn Ala Leu Asn Thr Leu Leu Val

465 470 475 480

Ala Thr Ser Asp Val Asn Tyr Ala Phe Gln Ile Lys Asp Ile Met Glu

485 490 495

Leu Leu Asp Ala Leu Ser Ser Leu Gly Lys Asp Val Leu Asn Lys Ile

500 505 510

Val Gly Val Asn Thr Glu Glu Asp Cys Val Asn Ala Asp Val Thr Lys

515 520 525

Leu Ser Ala Asn Gly Arg Ile Asp Asp Ile Phe Asn Arg Tyr Val Lys

530 535 540

Gly Gln Gly Glu Asp Ile Leu Tyr Val Val Asn Ser Leu Thr Gly Glu

545 550 555 560

Val Val Ser Asp Asp Glu Asp Ile Glu Glu Leu Phe Ser Val Cys Asp

565 570 575

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

580 585 590

Val Leu Glu Phe His Leu Asp Asp Tyr Leu Thr Ala Leu Lys Asn Phe

595 600 605

Lys Ser Glu Asn Lys His His Phe Ser Lys Leu Gln Thr Arg Ser Ala

610 615 620

Thr Asp Asp Gln Ile Leu Leu Val Asp Glu Leu Ile Thr Ile Thr Met

625 630 635 640

Ile Leu Arg Asn Ile Ser Phe Ala Glu Tyr Asn Lys Glu Pro Met Ala

645 650 655

Gly Asn Arg Leu Phe Lys Asp Leu Leu Phe Ser Thr Val Lys Ser Val

660 665 670

Ala Leu Asn Asn Asp Lys Phe Val Phe Ser Arg Lys Arg Leu Cys Leu

675 680 685

Leu Lys Asp Cys Leu Leu Met Leu Asp Asn Ile Ser Leu Phe Thr His

690 695 700

Leu His Thr Leu Glu Glu Ala Phe Leu Ser Phe Val Leu Val Ala Ser

705 710 715 720

Phe Gly Pro Lys Ile Glu Asp Gln Tyr Lys Ile Pro Arg Cys Asn Ile

725 730 735

Glu Thr His Ser Tyr Phe Ala Phe Gly Leu Asp Ala Phe Thr Lys Leu

740 745 750

Met Val Arg Glu Pro Tyr Asn Arg Ser Leu Ile Gln Ala Val Leu Asn

755 760 765

Gly Thr Leu Asn Ser Ser Met Thr Gly Tyr Ser Val Ser Leu Gln Asp

770 775 780

Gln Glu Tyr Thr Arg Lys Leu Ile Lys Ala Tyr Asn Lys Asp Tyr Lys

785 790 795 800

Ser Ala Ser Leu Leu Ser Gln Ala Phe Gln Met Tyr Met Ser Ile Leu

805 810 815

Pro Phe Asp Ala Asn Thr Phe Glu Leu Ser Lys Phe Ile Phe Met Arg

820 825 830

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

835 840 845

Met Val Pro Val Asp Asp Leu Asn Thr His His Asn Lys Leu Ser Leu

850 855 860

Tyr Trp Leu Leu Glu Asn Arg Glu Leu Leu Leu Gly Asn Phe Ala Arg

865 870 875 880

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

885 890 895

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

900 905 910

Ile Asn Ser Leu Val Asp Asn Ala Val Leu Ala Lys Glu Ile Gly Asp

915 920 925

Leu Arg His Thr Asp Leu Met Glu Ser Leu Thr Asp Cys Leu Ala Phe

930 935 940

Pro Arg Ile Ile Pro Asp Ala Ile Leu Thr Leu Asp Thr Phe Leu Ala

945 950 955 960

Pro Thr Ile Asp Thr Asn Leu Gly Lys Glu Val Val Arg Leu Leu Arg

965 970 975

Tyr Leu Lys Asp Leu Lys Ala Cys Glu Gly Ile

980 985

<210>3

<211>35

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>(1)..(35)

＜223〉primer

<400>3

gctggtacca tgtctgattg gttgaatgaa aatgc 35

<210>4

<211>33

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>(1)..(33)

＜223〉primer

<400>4

gtcggtaccc tagatcccct cacaagcctt taa 33

Claims (8)

1. isolating Candida albicans CaSWI1 polypeptide is characterized in that this polypeptide is selected from down group:

(a) has the polypeptide of SEQ ID NO:2 aminoacid sequence;

(b) SEQ ID NO:2 aminoacid sequence is formed through replacement, disappearance or the interpolation of one or more amino-acid residues, and have transcriptional activation function by (a) polypeptides derived.

2. polypeptide as claimed in claim 1 is characterized in that, it has the aminoacid sequence shown in the SEQ ID NO:2.

3. isolating polynucleotide is characterized in that, it comprises a nucleotide sequence, and this nucleotide sequence is selected from down group:

(a) coding is as the polynucleotide of polypeptide as described in claim 1 and 2;

(b) with polynucleotide (a) complementary polynucleotide.

4. polynucleotide as claimed in claim 3 is characterized in that this polynucleotide encoding has the polypeptide of aminoacid sequence shown in the SEQ IDNO:2.

5. polynucleotide as claimed in claim 4 is characterized in that, the sequence of these polynucleotide has 554-3514 position among the SEQID NO:1.

6. a carrier is characterized in that, it contains the described polynucleotide of claim 3.

7. a host cell is characterized in that, it contains the described carrier of claim 6.

8. energy and the described Candida albicans CaSWI1 of claim 1 polypeptid specificity bonded antibody.

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