CN1583788A - Tanscription activating factor genes of candida albicans and their use - Google Patents
Tanscription activating factor genes of candida albicans and their use Download PDFInfo
- Publication number
- CN1583788A CN1583788A CN 03150427 CN03150427A CN1583788A CN 1583788 A CN1583788 A CN 1583788A CN 03150427 CN03150427 CN 03150427 CN 03150427 A CN03150427 A CN 03150427A CN 1583788 A CN1583788 A CN 1583788A
- Authority
- CN
- China
- Prior art keywords
- leu
- gln
- ser
- caswi1
- asn
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Peptides Or Proteins (AREA)
Abstract
This invention provides new candida albicans activating transcription factor gene CaSWI1 and coded protain CaSwi1, which participates in candida albicans' pathopoiesis. It also provides protein thereof and its coding sequence's application. The product CaSwi1 protein of this gene coding is the important hypha growth activating factor and toxic factor and which participates in hypha growth and pathopoiesis for candida albicans.
Description
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, nitrogenous source utilization and oxygen are 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 in Candida albicans, also has retarding effect, it mainly is the signal pathway of Tup1 mediation, rely on supressor Rfg1, Nrg1 waits play a role (Kadosh D et al.Mol.Cell.Biol.21:2496-2505: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.).ScSwi1p 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, ScSwi1p 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 P
LPromotor; 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 pH8.0,1mM 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 (pH8.0) 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.
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 T4 dna ligase (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.
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 caswil 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
<130>034049
<160>4
<170>PatentIn?version?3.1
<210>1
<211>3802
<212>DNA
＜213〉Candida albicans (Candida albicans)
<220>
<221>CDS
<222>(554)..(3514)
<223>
<400>1
gatcagagtg?gtcggagatg?ctcttggata?tatggtggga?ccctgctatg?tacgaacaaa???60
tgcacatgca?atgggaacac?aaagaacagg?acgctctaga?aaccttgtat?tcaacacaag??120
cttggataag?agagagaatt?gcgtttttac?ctttacgcca?aatcaatgcg?ttcccaccag??180
gcgcttgttc?agaccaagct?gacgatccac?agtatttttt?ccaaaaccat?gactttgtag??240
tgaatatggc?agggtgtgaa?tggggtagag?attgctgggg?tgaaatggaa?cattataagg??300
cgttgtcgaa?aaagcttcat?aagagatggt?ggaagttttg?ggagtagtag?aattgtactt??360
tgtagtgtgt?ttttttttaa?tttacatagt?tactttaaca?gccgttagga?aacagcgaca??420
aagaagagaa?aaaaaaaact?tcgagttcaa?gagagagaga?aaaaaaaaat?caaaacaaca??480
aaaaactgag?gaaacggaaa?agttctattc?aaaggtcaac?cataaatcta?ggtttagcga??540
aatataataa?atc?atg?tct?gat?tgg?ttg?aat?gaa?aat?gca?ttt?gca?gat?????589
Met?Ser?Asp?Trp?Leu?Asn?Glu?Asn?Ala?Phe?Ala?Asp
1???????????????5???????????????????10
agc?aac?agc?aat?gac?gat?ttt?ttg?aat?tcc?att?ttt?gat?cag?agc?caa????637
Ser?Asn?Ser?Asn?Asp?Asp?Phe?Leu?Asn?Ser?Ile?Phe?Asp?Gln?Ser?Gln
15??????????????????20??????????????????25
gga?gag?caa?caa?gct?cca?cag?gtt?gcg?cag?gtt?tct?aca?tca?atg?tca????685
Gly?Glu?Gln?Gln?Ala?Pro?Gln?Val?Ala?Gln?Val?Ser?Thr?Ser?Met?Ser
30??????????????????35??????????????????40
aac?cca?cca?ctt?caa?tca?caa?tca?gcc?ctg?tct?act?tca?aga?atc?tca????733
Asn?Pro?Pro?Leu?Gln?Ser?Gln?Ser?Ala?Leu?Ser?Thr?Ser?Arg?Ile?Ser
45??????????????????50??????????????????55??????????????????60
caa?gca?cac?act?cca?atg?tac?caa?cag?tct?cct?gtt?act?gct?cac?act????781
Gln?Ala?His?Thr?Pro?Met?Tyr?Gln?Gln?Ser?Pro?Val?Thr?Ala?His?Thr
65??????????????????70??????????????????75
ata?ccg?caa?aac?agt?cct?caa?agt?atg?ccg?aat?caa?gta?gca?cag?cct????829
Ile?Pro?Gln?Asn?Ser?Pro?Gln?Ser?Met?Pro?Asn?Gln?Val?Ala?Gln?Pro
80??????????????????85??????????????????90
caa?caa?caa?ata?cca?cca?ccc?cct?ctg?caa?cac?tta?caa?cag?aca?acc????877
Gln?Gln?Gln?Ile?Pro?Pro?Pro?Pro?Leu?Gln?His?Leu?Gln?Gln?Thr?Thr
95??????????????????100?????????????????105
gcc?caa?atg?ctt?cct?caa?cag?caa?cag?caa?cag?caa?cag?caa?caa?cag????925
Ala?Gln?Met?Leu?Pro?Gln?Gln?Gln?Gln?Gln?Gln?Gln?Gln?Gln?Gln?Gln
110?????????????????115?????????????????120
cag?caa?caa?aaa?caa?gag?cag?cta?tac?aga?atg?aag?caa?cag?att?tac????973
Gln?Gln?Gln?Lys?Gln?Glu?Gln?Leu?Tyr?Arg?Met?Lys?Gln?Gln?Ile?Tyr
125?????????????????130?????????????????135?????????????????140
caa?cag?cag?atg?cta?aaa?aag?cag?caa?gaa?aac?atg?tct?cgt?cag?cct????1021
Gln?Gln?Gln?Met?Leu?Lys?Lys?Gln?Gln?Glu?Asn?Met?Ser?Arg?Gln?Pro
145?????????????????150?????????????????155
tct?ccc?atg?aat?tca?gct?ggg?cat?aac?aca?caa?caa?aat?acg?cca?atc????1069
Ser?Pro?Met?Asn?Ser?Ala?Gly?His?Asn?Thr?Gln?Gln?Asn?Thr?Pro?Ile
160?????????????????165?????????????????170
acc?cag?aat?gca?aaa?aca?cct?caa?aat?aat?tca?aaa?ttg?caa?tca?atg????1117
Thr?Gln?Asn?Ala?Lys?Thr?Pro?Gln?Asn?Asn?Ser?Lys?Leu?Gln?Ser?Met
175?????????????????180?????????????????185
caa?atg?gaa?ttg?ttt?ttt?tct?gtt?ttg?tat?gac?ttt?ttg?cag?cgg?agc????1165
Gln?Met?Glu?Leu?Phe?Phe?Ser?Val?Leu?Tyr?Asp?Phe?Leu?Gln?Arg?Ser
190?????????????????195?????????????????200
ggg?atc?tcc?att?ccc?caa?tca?tta?gct?atc?aat?ggt?aag?aga?gtc?aat????1213
Gly?Ile?Ser?Ile?Pro?Gln?Ser?Leu?Ala?Ile?Asn?Gly?Lys?Arg?Val?Asn
205?????????????????210?????????????????215?????????????????220
ttg?ttc?att?ttg?tat?atc?ttg?agt?caa?aga?ttg?ggt?gga?tac?agg?gtc????1261
Leu?Phe?Ile?Leu?Tyr?Ile?Leu?Ser?Gln?Arg?Leu?Gly?Gly?Tyr?Arg?Val
225?????????????????230?????????????????235
atg?aag?gct?ttt?cta?ctt?atg?tca?cca?gag?cag?cag?cgt?atg?cag?caa????1309
Met?Lys?Ala?Phe?Leu?Leu?Met?Ser?Pro?Glu?Gln?Gln?Arg?Met?Gln?Gln
240?????????????????245?????????????????250
cag?aat?cct?tgg?gca?cta?atg?gcc?gac?aaa?atg?ggg?ttt?cat?gat?aaa????1357
Gln?Asn?Pro?Trp?Ala?Leu?Met?Ala?Asp?Lys?Met?Gly?Phe?His?Asp?Lys
255?????????????????260?????????????????265
ggg?gag?gac?ccc?atg?gcc?aaa?gaa?aga?att?gct?aga?gaa?ctt?tgc?aat????1405
Gly?Glu?Asp?Pro?Met?Ala?Lys?Glu?Arg?Ile?Ala?Arg?Glu?Leu?Cys?Asn
270?????????????????275?????????????????280
tgc?tat?agt?aac?ttt?att?tta?cca?tat?gaa?gag?tat?tat?gcc?aca?cct????1453
Cys?Tyr?Ser?Asn?Phe?Ile?Leu?Pro?Tyr?Glu?Glu?Tyr?Tyr?Ala?Thr?Pro
285?????????????????290?????????????????295?????????????????300
gaa?ggg?aat?aag?gat?att?gaa?gct?agc?aaa?cat?aga?ttc?caa?cag?caa????1501
Glu?Gly?Asn?Lys?Asp?Ile?Glu?Ala?Ser?Lys?His?Arg?Phe?Gln?Gln?Gln
305?????????????????310?????????????????315
att?gtt?cag?aaa?tat?ctg?tcc?aat?gtc?ccc?aca?ccg?gct?cct?aat?caa????1549
Ile?Val?Gln?Lys?Tyr?Leu?Ser?Asn?Val?Pro?Thr?Pro?Ala?Pro?Asn?Gln
320?????????????????325?????????????????330
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
335?????????????????340?????????????????345
tta?tct?cgt?act?agc?aac?tcg?gtg?aat?aac?tca?cca?aat?gtc?agc?tct????1645
Leu?Ser?Arg?Thr?Ser?Asn?Ser?Val?Asn?Asn?Ser?Pro?Asn?Val?Ser?Ser
350?????????????????355?????????????????360
cct?ttc?cat?aac?acc?cct?cga?caa?gtt?cca?gca?cca?gca?ccg?gca?ccg????1693
Pro?Phe?His?Asn?Thr?Pro?Arg?Gln?Val?Pro?Ala?Pro?Ala?Pro?Ala?Pro
365?????????????????370?????????????????375?????????????????380
gta?caa?acg?gcc?gcg?tct?atg?cct?ccg?cca?aca?cct?gtt?aca?gcc?aaa????1741
Val?Gln?Thr?Ala?Ala?Ser?Met?Pro?Pro?Pro?Thr?Pro?Val?Thr?Ala?Lys
385?????????????????390?????????????????395
tct?caa?aca?aca?aaa?gca?gat?gcc?cat?att?ttg?aaa?aat?tat?act?cct????1789
Ser?Gln?Thr?Thr?Lys?Ala?Asp?Ala?His?Ile?Leu?Lys?Asn?Tyr?Thr?Pro
400?????????????????405?????????????????410
ttc?aaa?aag?att?gta?gaa?acg?cat?ggt?cca?ttc?aat?atc?aaa?gag?ctc????1837
Phe?Lys?Lys?Ile?Val?Glu?Thr?His?Gly?Pro?Phe?Asn?Ile?Lys?Glu?Leu
415?????????????????420?????????????????425
tcg?caa?ttg?tct?act?gaa?atc?gag?gtg?aca?aaa?cca?gtt?tat?ctt?ttt????1885
Ser?Gln?Leu?Ser?Thr?Glu?Ile?Glu?Val?Thr?Lys?Pro?Val?Tyr?Leu?Phe
430?????????????????435?????????????????440
gct?cca?gaa?tta?gga?atc?atc?aac?ctc?cag?gct?ttg?aca?atg?tca?gta????1933
Ala?Pro?Glu?Leu?Gly?Ile?Ile?Asn?Leu?Gln?Ala?Leu?Thr?Met?Ser?Val
445?????????????????450?????????????????455?????????????????460
aaa?agc?aat?agt?ggg?atc?caa?agc?tca?gaa?gtt?gtg?aac?gcc?ctt?aac????1981
Lys?Ser?Asn?Ser?Gly?Ile?Gln?Ser?Ser?Glu?Val?Val?Asn?Ala?Leu?Asn
465?????????????????470?????????????????475
aca?ttg?ttg?gtt?gct?aca?tca?gac?gtt?aat?tat?gct?ttt?caa?ata?aag????2029
Thr?Leu?Leu?Val?Ala?Thr?Ser?Asp?Val?Asn?Tyr?Ala?Phe?Gln?Ile?Lys
480?????????????????485?????????????????490
gat?atc?atg?gag?tta?tta?gac?gca?tta?tct?tct?ttg?ggg?aag?gat?gtt????2077
Asp?Ile?Met?Glu?Leu?Leu?Asp?Ala?Leu?Ser?Ser?Leu?Gly?Lys?Asp?Val
495?????????????????500?????????????????505
ttg?aac?aaa?att?gta?gga?gtc?aac?aca?gaa?gag?gat?tgc?gtg?aat?gct????2125
Leu?Asn?Lys?Ile?Val?Gly?Val?Asn?Thr?Glu?Glu?Asp?Cys?Val?Asn?Ala
510?????????????????515?????????????????520
gat?gtg?aca?aaa?ttg?tct?gcc?aat?ggt?cgg?att?gat?gat?att?ttt?aat????2173
Asp?Val?Thr?Lys?Leu?Ser?Ala?Asn?Gly?Arg?Ile?Asp?Asp?Ile?Phe?Asn
525?????????????????530?????????????????535?????????????????540
cgt?tat?gtt?aaa?gga?caa?ggc?gaa?gat?ata?ctg?tat?gtg?gtt?aat?tcg????2221
Arg?Tyr?Val?Lys?Gly?Gln?Gly?Glu?Asp?Ile?Leu?Tyr?Val?Val?Asn?Ser
545?????????????????550?????????????????555
ctt?aca?ggg?gaa?gtt?gtt?tct?gac?gat?gag?gat?atc?gag?gaa?ctt?ttt????2269
Leu?Thr?Gly?Glu?Val?Val?Ser?Asp?Asp?Glu?Asp?Ile?Glu?Glu?Leu?Phe
560?????????????????565?????????????????570
tca?gta?tgt?gat?gac?ggg?aat?ctg?gaa?tct?cct?att?gac?aag?ctg?act????2317
Ser?Val?Cys?Asp?Asp?Gly?Asn?Leu?Glu?Ser?Pro?Ile?Asp?Lys?Leu?Thr
575?????????????????580?????????????????585
gat?ctg?tta?gat?gtg?ctt?gag?ttt?cat?ttg?gat?gat?tat?ctt?aca?gca????2365
Asp?Leu?Leu?Asp?Val?Leu?Glu?Phe?His?Leu?Asp?Asp?Tyr?Leu?Thr?Ala
590?????????????????595?????????????????600
ttg?aag?aat?ttc?aaa?tct?gaa?aac?aaa?cac?cat?ttt?agc?aaa?ctt?caa????2413
Leu?Lys?Asn?Phe?Lys?Ser?Glu?Asn?Lys?His?His?Phe?Ser?Lys?Leu?Gln
605?????????????????610?????????????????615?????????????????620
aca?cga?agt?gcc?acg?gat?gat?caa?ata?ttg?tta?gtc?gat?gaa?ttg?ata????2461
Thr?Arg?Ser?Ala?Thr?Asp?Asp?Gln?Ile?Leu?Leu?Val?Asp?Glu?Leu?Ile
625?????????????????630?????????????????635
act?ata?aca?atg?atc?ttg?aga?aat?att?tct?ttt?gct?gaa?tat?aat?aaa????2509
Thr?Ile?Thr?Met?Ile?Leu?Arg?Asn?Ile?Ser?Phe?Ala?Glu?Tyr?Asn?Lys
640?????????????????645?????????????????650
gaa?cct?atg?gca?gga?aat?cgt?ctt?ttc?aag?gat?tta?ttg?ttt?tca?act????2557
Glu?Pro?Met?Ala?Gly?Asn?Arg?Leu?Phe?Lys?Asp?Leu?Leu?Phe?Ser?Thr
655?????????????????660?????????????????665
gtc?aaa?agc?gtt?gct?tta?aac?aat?gac?aaa?ttt?gtg?ttt?agc?cgc?aaa????2605
Val?Lys?Ser?Val?Ala?Leu?Asn?Asn?Asp?Lys?Phe?Val?Phe?Ser?Arg?Lys
670?????????????????675?????????????????680
cgg?tta?tgt?tta?ctc?aag?gat?tgt?ctt?tta?atg?cta?gac?aat?att?tct????2653
Arg?Leu?Cys?Leu?Leu?Lys?Asp?Cys?Leu?Leu?Met?Leu?Asp?Asn?Ile?Ser
685?????????????????690?????????????????695?????????????????700
ttg?ttt?act?cat?ttg?cat?act?tta?gag?gaa?gca?ttt?tta?tca?ttt?gtt????2701
Leu?Phe?Thr?His?Leu?His?Thr?Leu?Glu?Glu?Ala?Phe?Leu?Ser?Phe?Val
705?????????????????710?????????????????715
tta?gta?gca?tcc?ttt?gga?cct?aag?att?gaa?gat?caa?tac?aag?atc?cca????2749
Leu?Val?Ala?Ser?Phe?Gly?Pro?Lys?Ile?Glu?Asp?Gln?Tyr?Lys?Ile?Pro
720?????????????????725?????????????????730
cga?tgc?aat?att?gaa?acc?cat?tcg?tat?ttt?gcc?ttt?ggt?ctt?gat?gcc????2797
Arg?Cys?Asn?Ile?Glu?Thr?His?Ser?Tyr?Phe?Ala?Phe?Gly?Leu?Asp?Ala
735?????????????????740?????????????????745
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
atg?agt?ata?ttg?cct?ttt?gat?gcc?aac?aca?ttt?gaa?ttg?tca?aaa?ttc????3037
Met?Ser?Ile?Leu?Pro?Phe?Asp?Ala?Asn?Thr?Phe?Glu?Leu?Ser?Lys?Phe
815?????????????????820?????????????????825
atc?ttc?atg?agg?agt?cca?aca?att?tca?cag?atg?tta?ttt?ggg?gcc?aaa????3085
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?Ser?Ala?Leu?Ser?Thr?Ser?Arg?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 protein-specific bonded antibody.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB031504272A CN100341891C (en) | 2003-08-20 | 2003-08-20 | Tanscription activating factor genes of candida albicans and their use |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB031504272A CN100341891C (en) | 2003-08-20 | 2003-08-20 | Tanscription activating factor genes of candida albicans and their use |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1583788A true CN1583788A (en) | 2005-02-23 |
| CN100341891C CN100341891C (en) | 2007-10-10 |
Family
ID=34597520
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB031504272A Expired - Fee Related CN100341891C (en) | 2003-08-20 | 2003-08-20 | Tanscription activating factor genes of candida albicans and their use |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN100341891C (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1948335B (en) * | 2005-10-14 | 2010-05-12 | 中国科学院上海生命科学研究院 | Candida albicans mycellium regulating and controlling factor gene and its use |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1061092C (en) * | 1998-02-27 | 2001-01-24 | 中国科学院上海生物化学研究所 | Component of white monilia with reverse transcription transposons structure |
-
2003
- 2003-08-20 CN CNB031504272A patent/CN100341891C/en not_active Expired - Fee Related
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1948335B (en) * | 2005-10-14 | 2010-05-12 | 中国科学院上海生命科学研究院 | Candida albicans mycellium regulating and controlling factor gene and its use |
Also Published As
| Publication number | Publication date |
|---|---|
| CN100341891C (en) | 2007-10-10 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN1617927A (en) | Pyroglutamyl peptidase and its gene | |
| CN101037699A (en) | Glycerol-3-phosphate dehydrogenase gene and use thereof | |
| CN1932015A (en) | Branched-chain amino acid aminotransferase gene and use thereof | |
| CN1301307A (en) | Methods for producing polypeptides in filamentous fungal mutant cells | |
| CN1650006A (en) | Glucose dehydrogenase beta-subunit and DNA encoding the same | |
| CN1289523C (en) | Paddy rice potassium, sodium ion transport gene and its application | |
| CN1629188A (en) | Specific antigen of Japanese blood fluke and its use | |
| CN100341891C (en) | Tanscription activating factor genes of candida albicans and their use | |
| CN1517436A (en) | Oxide-reductase | |
| CN1902218A (en) | Yeast strains with improved fructose fermentation capacity | |
| CN1286973C (en) | Histone methyl transferase and its preparing method | |
| CN1170850C (en) | Human angiogenin-like protein and coding sequence and application thereof | |
| CN1948335A (en) | Candida albicans mycellium regulating and controlling factor gene and its use | |
| CN1948339A (en) | Preparation and purification of telomerase activity inhibition protein | |
| CN1303102C (en) | Method for diagnosing and curing alopecia utilizing the Rhor gene of human and rat and the encoding products | |
| CN1948334A (en) | Candida albicans cell wall adhesion factor gene and its use | |
| CN1566146A (en) | Paddy rice stalk extension gene, coded protein and application thereof | |
| CN1763086A (en) | ANK protein for controlling fungus colony growth and pathogenicity and its coding gene and utilization | |
| CN1756841A (en) | Koji mold-origin phospholipase A2 | |
| CN1170848C (en) | Novel human hepatoma associated protein and coding sequence thereof | |
| CN1590405A (en) | White candidas transcription factor gene and its use | |
| CN1769436A (en) | Nanjing bass 3-hydroxyl-3-methyl glutaryl coenzyme A reductase protein encoding sequence | |
| CN1281962C (en) | Tumor relevant secretory protein as a liver cancer marker and uses thereof | |
| CN1216989C (en) | A novel penicillin G acylase and use thereof | |
| CN1232530C (en) | Ethane cyclic amp receptor protein of wheat and its coding sequence |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C17 | Cessation of patent right | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20071010 Termination date: 20120820 |