CN1443774A - Leptospira hemolysin and its code sequence - Google Patents

Abstract

The present invention provides a kind of new leptospiral hemolysin, polynucleotide for coding said hemolysin and method for producing said hemolysin by using recombination technique. Said invention also discloses the application of the hemolysin and its code sequence.

Description

Leptospira hemolysin and encoding sequence thereof

Technical field

The invention belongs to biotechnology and medical field, specifically, the present invention relates to the polynucleotide of new coding Leptospira hemolysin, and the polypeptide of this polynucleotide encoding.The invention still further relates to the purposes and the preparation of these polynucleotide and polypeptide.

Background technology

Leptospirosis is a kind of widely distributed Amphixenosis, mainly is distributed in developing countries and regions such as southern china and South East Asia, South Asia.In China, this disease nineteen ninety-five is listed Notifiable disease in.1955-1993, annual sickness rate are 7/100000ths, and the annual mortality ratio accounts for 1.02% of number of the infected.

China leptospirosis popular flora based on jaundice hemorrhage group and Bo Mona group.The jaundice hemorrhage group has 15 serotypes, wherein to rely type the most common.

Though it is China has obtained great success aspect the preventing and treating of this disease, still unresolved to the problems such as efficient protection of the mechanism of the pathogenic immunity of this disease, vaccine.

Leptospira (Leptospira sp.) belongs to gram-negative bacteria.Jaundice appears in Leptospira patient in varying degrees, have simultaneously or do not have hemorrhage or hepatic and renal function impaired, the degree of jaundice is not relevant with hepatic necrosis usually, it is normal that the state of an illness is recovered the back liver function.It is generally acknowledged that this and red corpuscle are impaired relevant, red corpuscle is subjected to after the effect of toxin such as hemolysin haemolysis taking place, and a large amount of bilirubin enters in the tissue, and liver metabolism and cause jaundice fully is continuous and high bilirubin symptom takes place.

Yet, so far, Leptospira hemolysin was not still disclosed.Therefore, this area presses for the new Leptospira hemolysin of exploitation, so that be that mechanism of causing a disease and the diagnosis of studying leptospirosis provides the foundation.

Summary of the invention

The purpose of this invention is to provide new Leptospira hemolysin with and fragment, analogue and derivative.

Another object of the present invention provides the polynucleotide of these polypeptide of coding.

Another object of the present invention provides the method for these polypeptide of production and the purposes of this polypeptide and encoding sequence.

In a first aspect of the present invention, novel isolated hemolysin polypeptide is provided, it comprises; Polypeptide or its conservative property variation polypeptide or its active fragments or its reactive derivative with SEQID NO:2,4,6,8,10,12,14,16 aminoacid sequences.Preferably, this polypeptide is the polypeptide with SEQ ID NO:2,4,6,8,10,12,14,16 aminoacid sequences.More preferably this polypeptide does not contain signal peptide sequence.

In a second aspect of the present invention, the polynucleotide of separated coding aforementioned polypeptides are provided, these polynucleotide comprise a nucleotide sequence, and this nucleotide sequence is shown at least 70% homogeny with a kind of nucleotides sequence that is selected from down group: (a) polynucleotide of the above-mentioned Leptospira hemolysin polypeptide of coding; (b) with polynucleotide (a) complementary polynucleotide.Preferably, this polynucleotide encoding has the polypeptide of aminoacid sequence shown in the SEQ ID NO:2,4,6,8,10,12,14,16.More preferably, the sequence of these polynucleotide is be selected from down group a kind of: (a) have SEQ ID NO:1,3,5,7,9,11,13,15.

In a third aspect of the present invention, the carrier that contains above-mentioned polynucleotide is provided, and has been transformed or host cell of transduceing or the host cell that is directly transformed or transduce by above-mentioned polynucleotide by this carrier.

In a fourth aspect of the present invention, the method for preparing the Leptospira hemolysin polypeptide is provided, this method comprises: (a) under the condition that is fit to the expression Leptospira hemolysin, cultivate the above-mentioned host cell that is transformed or transduce; (b) from culture, isolate the Leptospira hemolysin polypeptide.

In a fifth aspect of the present invention, provide and above-mentioned Leptospira hemolysin polypeptid specificity bonded antibody.The nucleic acid molecule that can be used for detecting also is provided.

In a sixth aspect of the present invention, the method that whether has hemolysin in the test sample is provided, it comprises: sample is contacted with the specific antibody of hemolysin, observe whether form antibody complex, formed antibody complex and just represented to exist in the sample hemolysin.

In a seventh aspect of the present invention, provide the purposes of polypeptide of the present invention and encoding sequence.For example the encoding sequence of Leptospira hemolysin 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.

In a eighth aspect of the present invention, provide a kind of detection leptospiral test kit, it is characterized in that, it contain the specific detection Leptospira hemolysin primer and/or with Leptospira hemolysin specificity bonded antibody.

In a ninth aspect of the present invention, a kind of vaccine composition is provided, it contains Leptospira hemolysin polypeptide of the present invention or its fragment and the pharmaceutically acceptable carrier of safe and effective amount.

Others of the present invention are because disclosing of the technology of this paper is conspicuous to those skilled in the art.

Description of drawings

Following accompanying drawing is used to illustrate specific embodiments of the present invention, and is not used in qualification by the scope of the invention that claims defined.

Fig. 1 has shown the functional domain of each hemolysin of the present invention.It is as follows respectively to number implication among the figure:

PD011673 is PRECURSOR HYDROLASE SIGNAL SPHINGOMYELINASE HEMOLYSIS SMASEPHOSPHODIESTERASE B

PD447657 is PRECURSOR HYDROLASE SIGNAL SPHINGOMYELINASE HEMOLYSIS SMASEPHOSPHODIESTERASE B

PD041204 is C SPHINGOMYELINASE PRECURSOR SMASE HEMOLYTIC SIGNAL HYDROLASEPHOSPHODIESTERASE

PD314750 be Leptospira borgpetersenii serovar hardjo type hardjobovis (TrEMBLQ9RMG4, HEM)

PD191426 is HEMOLYTIC HEMOLYSIN HLPA From Prevotella intermediaTrEMBL 068620, HEM; TrEMBL Q9ZGK8-LEPBO lipopolysaccharide O-antigen; TrEMBL Q9X5J4-MYCAV, HEM)

PD007579 is COMPLETE PROTEOME HEMOLYSIN TLYA HAEMOLYSIN HEMOLYSIN-LIKE U0247AMEMBRANE HEMOLYSIS MJ1257 From Tr051459-BORBU, HEM; TrQ9PQ54-UREPA, HEM; HLYA-TREHY, HEM

PD406054 is PROTEOME COMPLETE HEMOLYSIN HLYA from TrEMBLQ9F6Y3-CHLAU, TrEMBLP74319-SYNY3; TrEMBLQ9X1R2-THEMA

PD002457 is COMPLETE PROTEOME INTEGRAL MEMBRANE TRANSMEMBRANE CBS DOMAIN REPEATHEMOLYSIN PLASMID

PD002327 is COMPLETE PROTEOME HEMOLYSIN CBS DOMAIN MAGNESIUM COBALT REPEATTRANSPORT EFFLUX

PD005611 is PROTEOME COMPLETE HEMOLYSIN CBS DOM TrEMBL066507-AQUAE, HEM

Embodiment

The inventor is through extensive and deep research, relies type to rely the strain separation and purification and identified the new hemolysin of a class from leptospira interrogans (Leptospirainerrogans) jaundice hemorrhage group.Finished the present invention on this basis.

In the present invention, term " Leptospira hemolysin polypeptide " or " Leptospira hemolysin " are used interchangeably, all refer to have the Leptospira hemolysin aminoacid sequence albumen or the polypeptide of (SEQ ID NO:2,4,6,8,10,12,14,16).They comprise the Leptospira hemolysin that contains or do not contain initial methionine, and the hemolysin that contains or do not contain signal peptide.

As used herein, " isolating " is meant that material separates (if natural substance, primal environment promptly is a natural surroundings) from its primal environment.For example polynucleotide and the polypeptide under the native state in the active somatic cell do not have separation and purification, 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 hemolysin or polypeptide " is meant that the hemolysin 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 hemolysin of standard.

Polypeptide of the present invention can be recombinant polypeptide, natural polypeptides, synthetic polypeptide, preferred recombinant polypeptide.Polypeptide of the present invention can be the product of natural purifying, or the product of chemosynthesis, or uses recombinant technology to produce from protokaryon or eucaryon host (for example, bacterium, yeast, higher plant, insect and mammalian cell).The host used according to the recombinant production scheme, polypeptide of the present invention can be glycosylated, but preferably nonglycosylated.

The present invention also comprises fragment, derivative and the analogue of Leptospira hemolysin.As used herein, term " fragment ", " derivative " are meant with " analogue " and keep identical biological function of natural Leptospira hemolysin of the present invention or active polypeptide basically.Polypeptide fragment of the present invention, derivative or analogue can be that (i) has one or more conservative or substituted polypeptide of non-conservation amino-acid residue (preferred conservative amino acid residue), and the amino-acid residue of such replacement can be also can not encoded by genetic code, or (ii) in one or more amino-acid residues, has a polypeptide of substituted radical, or (iii) mature polypeptide and another compound (such as the compound that prolongs the polypeptide transformation period, polyoxyethylene glycol for example) merges formed polypeptide, or (iv) additional aminoacid sequence is fused to this peptide sequence and the polypeptide that forms (as leader sequence or secretion sequence or be used for the sequence or the proteinogen sequence of this polypeptide of purifying, or with the fusion rotein of the segmental formation of antigen I gG).According to the instruction of this paper, these fragments, derivative and analogue belong to the known scope of those skilled in the art.

In the present invention, term " Leptospira hemolysin polypeptide " refers to have the active or immunogenic SEQ ID NO:2 of Leptospira hemolysin, 4,6,8,10,12,14,16 polypeptide of sequence.This term also comprises having and the variant form Leptospira hemolysin identical function, above-mentioned 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 at C-terminal and/or N-terminal interpolation one or several (being generally in 10, more preferably is in 5) amino acid.This term also comprises the active fragments and the reactive derivative of Leptospira hemolysin.

The variant form of this polypeptide comprises: homologous sequence, conservative property varient, allelic variant, natural mutation, induced mutation body, under high or low tight degree condition can with the coded albumen of the DNA of Leptospira hemolysin DNA hybridization and the polypeptide or the albumen that utilize the antiserum(antisera) of anti-Leptospira hemolysin polypeptide to obtain.The present invention also provides other polypeptide, as comprises Leptospira hemolysin polypeptide or its segmental fusion rotein.Except the polypeptide of total length almost, the present invention has also comprised the soluble fragments of Leptospira hemolysin polypeptide.Usually, this fragment have the Leptospira hemolysin peptide sequence at least about 10 continuous amino acids, usually at least about 30 continuous amino acids, preferably at least about 50 continuous amino acids, more preferably at least about 80 continuous amino acids, best at least about 100 continuous amino acids.These fragments also can be used as immunogen to induce at leptospiral immune response.

Invention also provides the analogue of Leptospira hemolysin or polypeptide.The difference of these analogues and natural Leptospira hemolysin polypeptide can be the difference on the aminoacid sequence, also can be the difference that does not influence on the modified forms of sequence, perhaps haves both at the same time.These polypeptide comprise natural or the inductive genetic variant.The induce variation body can obtain by various technology, as by radiation or be exposed to mutagenic compound and produce random mutagenesis, also can pass through site-directed mutagenesis method or the biological technology of other known moleculars.Analogue also comprises having the analogue that is different from the amino acid whose residue of natural L-(as D-amino acid), and has non-natural analogue that exist or synthetic amino acid (as β, gamma-amino acid).Should be understood that polypeptide of the present invention is not limited to the above-mentioned representational polypeptide that exemplifies.

(the not changing primary structure usually) form of modification comprises: the chemically derived form such as the acetylize or carboxylated of the polypeptide that body is interior or external.Modification also comprises glycosylation, carries out glycosylation modified and polypeptide that produce in the procedure of processing as those in the synthetic and processing of polypeptide or further.Modified forms also comprises have the phosphorylated amino acid residue sequence of (as Tyrosine O-phosphate, phosphoserine, phosphothreonine).Thereby also comprise the polypeptide that has been improved its anti-proteolysis performance or optimized solubility property by modifying.

In the present invention, " Leptospira hemolysin conservative property variation polypeptide " refers to compare with SEQ ID NO:2,4,6,8,10,12,14,16 aminoacid sequence, 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.With hemolysin lep919_12 is example, and 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, be example with hemolysin lep919_12, " 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 IDNO:1.The rest may be inferred for other albumen of the present invention.Nucleotide sequence of the present invention can be according to the corresponding relation of codon, goes out according to SEQ ID NO:2,4,6,8,10,12,14,16 deduced amino acid.

The polynucleotide of encoding mature polypeptide 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.

Term " polynucleotide of coded polypeptide " can be the polynucleotide that comprise this polypeptide of encoding, and also can be the polynucleotide that also comprise additional code and/or non-coding sequence.

The invention still further relates to the varient of above-mentioned polynucleotide, its coding has the polypeptide of identical aminoacid sequence or fragment, analogue and the derivative of polypeptide with the present invention.The varient of these polynucleotide can be the allelic variant of natural generation or the varient that non-natural takes place.These nucleotide diversity bodies comprise and replace varient, deletion mutation body and insert varient.As known in the art, allelic variant is the replacement form of polynucleotide, and it may be replacement, disappearance or the insertion of one or more Nucleotide, but can be from not changing the function of its encoded polypeptides in fact.

The invention still further relates to and above-mentioned sequence hybridization and two sequences between have at least 50%, preferably at least 70%, the polynucleotide of at least 80% homogeny more preferably.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 sophisticated Leptospira hemolysin.

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 to separate the polynucleotide of coding hemolysin.

Leptospira hemolysin 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.

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 hemolysin encoding 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 hemolysin polypeptide of reorganization.In general following steps are arranged;

(1). with the polynucleotide (or varient) of coding Leptospira hemolysin 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, the Leptospira hemolysin 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.The carrier of Shi Yonging includes but not limited in the present invention: the expression vector based on T7 of expressing in bacterium; The pMSXND expression vector of in mammalian cell, expressing and at the carrier that derives from baculovirus of expressed in insect cells.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 Leptospira hemolysin 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.Expression vector also comprises ribosome bind site and the transcription terminator that translation initiation is used.

In addition, expression vector preferably comprises one or more selected markers, to be provided for selecting the phenotypic character of transformed host cells, cultivate Tetrahydrofolate dehydrogenase, neomycin resistance and the green fluorescent protein (GFP) of usefulness as eukaryotic cell, or be used for colibacillary tsiklomitsin or amicillin resistance.

Comprise the carrier of above-mentioned suitable dna sequence dna and suitable promotor or control sequence, can be used to transform appropriate host cell, so that it can marking protein.

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 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 Leptospira hemolysin or the polypeptide of reorganization are of use in many ways, comprising (but being not limited to): some fragment of hemolysin merged to constitute have immunogenicity and do not have toxic fused antigen, as immunogen.

On the other hand, the present invention also comprises Leptospira hemolysin 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 Leptospira hemolysin gene product or fragment.Preferably, refer to that those can combine with Leptospira hemolysin gene product or fragment but nonrecognition and be incorporated into the antibody of other irrelevant antigen molecule.The present invention also comprise those can with modify or without the Leptospira hemolysin gene product bonded antibody of modified forms.

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; 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 Leptospira hemolysin gene product of purifying or its have antigenic fragment, can be applied to animal to induce the generation of polyclonal antibody.Similarly, expressing Leptospira hemolysin 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.

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 people such as normal condition such as Sambrook, molecular cloning: laboratory manual (New York:ColdSpring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.

Embodiment 1

Yeast culture

1) substratum: use the EMJH liquid nutrient medium, preparation 5 * 1000 milliliters of EMJH substratum, take by weighing BSA50g.Sodium acetate, anhydrous 0.5g, Sodium.alpha.-ketopropionate 1g, ferrous sulfate 0.25g, 1.5% magnesium chloride 3.5ml, 0.4% zinc sulfate 5ml, 0.15 copper sulfate 1ml, Sodium phosphate dibasic 12.6g, potassium primary phosphate 1.5g, sodium-chlor 5g, ammonium chloride 1.25g, 0.05% VB ₁₂2ml, VB ₁0.025g accent pH is 7.2-7.6, be settled to 1000ml respectively with 0.7,0.45, the nitrocellulose filter filtration sterilization in 0.2nm aperture.During use, with its 5 times of dilutions of sterilized water.

2) culture condition: 30 ℃, 100 rev/mins of wave and culture 40 hours.

3) bacterial strain: leptospira interrogans jaundice hemorrhage group relies type to rely strain.

Embodiment 2

The nucleotide sequence of hemolysin and aminoacid sequence

With ordinary method extracting Leptospira DNA, make up the library.Measure genom sequence.According to Computer Analysis, synthetic primer is a template with extractive Leptospira DNA, carries out pcr amplification, obtains the ORF sequence of hemolysin.Sequence verification conforms to genome sequence once more.These ORF codings have the polypeptide of aminoacid sequence shown in the SEQ ID NO:2,4,6,8,10,12,14,16.The basic condition of 8 kinds of hemolysins of the present invention is as shown in the table.

The nucleotide sequence of Table A hemolysin and aminoacid sequence

	Title	ORF sequence SEQ ID NO:	Length (bp)	The SEQ ID NO of protein sequence:	Length (aa)
						Hemolysin No.1	?lep919_12	????1	??720	????2	????239

Hemolysin No.2	?lep923-82	????3	????1677	????4	????558
						Hemolysin No.3	?lep928_20	????5	????1794	????6	????597
Hemolysin No.4	?lep928_23	????7	????1872	????8	????623
						Hemolysin No.5	?lep904_14	????9	????1335	????10	????444
Hemolysin No.6	?lep925_128	????11	????831	????12	????276
						Hemolysin No.7	?lep925_54	????13	????1179	????14	????392
Hemolysin No.8	?lep924-101	????15	????942	????16	????313

The hemolysin of Leptospira hemolysin No.1-8 of the present invention and other bacteriums all has certain homology, and has hemolysin its specific structure territory (Fig. 1).

Embodiment 3

Protein expression

With conventional gene engineering method fusion expression vector pET28b (Novagene company) is gone in the Leptospira hemolysin gene clone, the recipient bacterium of amalgamation and expression is selected e. coli bl21 (DE3) for use.After the normal condition fermentation, extract the reorganization tropina, carry out the SDS-PAGE electrophoresis then, molecular weight and predictor are basic identical.The hemolysin of expression in escherichia coli (fusion rotein that has His-tag) by the Ni-NTA column separating purification, obtains pure Leptospira hemolysin (fusion rotein).

Embodiment 4

Preparation antibody

With the purifying that obtains among the embodiment 3, reorganization His-fusion rotein is used for immune animal to produce antibody, concrete grammar is as follows: with the complete Freund ' s adjuvant emulsion of fusion rotein with equivalent.With the albumen of 200 μ g emulsifications, rabbit is carried out the lymphoglandula injection.After one month,, rabbit is carried out the lymphoglandula injection with booster immunization with the dosage of 200 μ g with the same antigen of the non-complete Freund ' s adjuvant emulsion of equivalent.Carried out booster immunization one time every 14 days, carry out at least three times.The sero-fast specific reaction activity that obtains is checked antibody titers with ELISA.Found that antibody can combine with albumen of the present invention specifically.

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.

＜110〉＜120〉＜130〉021333＜160〉16＜170〉PatentIn version3.1＜210〉1＜211〉720＜212〉DNA＜213〉 ( Leptospira interrogans )＜400〉1atgaaaatga tacaaaatat tttgcgaaaa gaaaaagtta aaacatggaa tcaattgaaa 60ggctctaaat ttttcagatg ttgttgtctg tttattattt tatttttttt cgattgttta 120cctgatttac agtcttctga aaataattta tttttatctt tgctttctct tccaaaaaat 180caaggtataa tattaagaaa cgataaatct attgcaaagt ctaaaacgac acgaaaatac 240ggctgggtta cttttacatc tcaagtaacc ggaaaaaaaa tccaagcaga tcctgaacga 300cctgacggat ggttaagagt gaatgcttcg agtaacacgg attttacgac gttcacttta 360tatcaggatg gaaaagatcc agattgtatc aaaggcggcc ctatccgggt tgagcctact 420gcgtatcgaa attattattg gaattggtgg cttggaggag gtgcgggtaa ttacgcctat 480tatcctaaat ataaagatgg ttctaacaaa cttcaaattt acgttttaaa agtcagcggc 540tgtttggaat caggagatag agtcttattt tcggattatg atactataac tcaggatgat 600tattttgtca tagattggga tggaggcagc tggaatgagt atctttttct ttggtataaa 660tttcctaagg ttcagagagg atatttttat gttcaattga atgaaggccc tgaggaataa 720＜210〉2＜211〉239＜212〉PRT＜213〉 ( Leptospira interrogans )＜400〉2Met Lys Met Ile Gln Asn Ile Leu Arg Lys Glu Lys Val Lys Thr Trp1 5 10 15Asn Gln Leu Lys Gly Ser Lys Phe Phe Arg Cys Cys Cys Leu Phe Ile

20??????????????????25??????????????????30Ile?Leu?Phe?Phe?Phe?Asp?Cys?Leu?Pro?Asp?Leu?Gln?Ser?Ser?Glu?Asn

35??????????????????40??????????????????45Asn?Leu?Phe?Leu?Ser?Leu?Leu?Ser?Leu?Pro?Lys?Asn?Gln?Gly?Ile?Ile

50??????????????????55??????????????????60Leu?Arg?Asn?Asp?Lys?Ser?Ile?Ala?Lys?Ser?Lys?Thr?Thr?Arg?Lys?Tyr65??????????????????70??????????????????75??????????????????80Gly?Trp?Val?Thr?Phe?Thr?Ser?Gln?Val?Thr?Gly?Lys?Lys?Ile?Gln?Ala

85??????????????????90??????????????????95Asp?Pro?Glu?Arg?Pro?Asp?Gly?Trp?Leu?Arg?Val?Asn?Ala?Ser?Ser?Asn

100?????????????????105?????????????????110Thr?Asp?Phe?Thr?Thr?Phe?Thr?Leu?Tyr?Gln?Asp?Gly?Lys?Asp?Pro?Asp

115?????????????????120?????????????????125Cys?Ile?Lys?Gly?Gly?Pro?Ile?Arg?Val?Glu?Pro?Thr?Ala?Tyr?Arg?Asn

130?????????????????135?????????????????140Tyr?Tyr?Trp?Asn?Trp?Trp?Leu?Gly?Gly?Gly?Ala?Gly?Asn?Tyr?Ala?Tyr145?????????????????150?????????????????155?????????????????160Tyr?Pro?Lys?Tyr?Lys?Asp?Gly?Ser?Asn?Lys?Leu?Gln?Ile?Tyr?Val?Leu

165?????????????????170?????????????????175Lys?Val?Ser?Gly?Cys?Leu?Glu?Ser?Gly?Asp?Arg?Val?Leu?Phe?Ser?Asp

180?????????????????185?????????????????190Tyr?Asp?Thr?Ile?Thr?Gln?Asp?Asp?Tyr?Phe?Val?Ile?Asp?Trp?Asp?Gly

195?????????????????200?????????????????205Gly?Ser?Trp?Asn?Glu?Tyr?Leu?Phe?Leu?Trp?Tyr?Lys?Phe?Pro?Lys?Val

210 215 220Gln Arg Gly Tyr Phe Tyr Val Gln Leu Asn Glu Gly Pro Glu Glu225 230 235＜210〉3＜211〉1677＜212〉DNA＜213〉 ( Leptospira interrogans )＜400〉3atgaaaacaa aacgaaatcc ccttctaaac aaatgcaaaa aaccaaacaa atttaaatat 60agaaacctat taattgggct tttactatgt ttattccctc taaattgtac atttaaagat 120tcattagtat acaataaatt tttaatgtat atttcatttt cacataagaa tttaaatttt 180gaattagaga acatcaattc aactagaaag acccttttac caaagtcaga aatagattta 240aatattctca gttataatct ttttttatat tcaaaagaag atatatattt tggatattgg 300gaagaagaaa aaagggcgga gcttcttgga aaatcaaaat ttacaaaaaa tcaggacgtc 360attgtactca gcagagtgtt tgatacaaat gcacgaaata ctcttctaga caatcttaat 420ttagaatatc ctgaccaaac cgacgtaatc ggaaaaacta agtatggttg ggatcaaact 480ctcggagatt ttagacagca gatcaacaac ggaggagttg tcattttaag taaatggcct 540attcaagaaa agatacagta tatcttcaaa aatcatggat gtggtaatga cacattctac 600aataagggtt ttgcttacgt aaaaattaaa aaaggaagtc agatcattca tattgtagga 660accgatactc aatcggaaga ttcgacatgt tccgatttag gagtaaatgc aagaatcaat 720cagctcactg aaattaaaaa atttatcgat tcaaaacgta tatccaacaa agaaatcgta 780ttgatcgcgg gagctttgaa cgtagacaaa agtaatcaat ctgaatataa gaatatgctt 840aatatactag aagtaaatga accaaattat gcaggaattc catttacatg ggacacgaaa 900aaaaacaaga tcgctgctta taataatatc tattactcct ggaaccagac ctcaaattac 960ggggaatata tactcgtttc caaaaatcac tttcaacttc cgatttggca aaatttagcg 1020tatgatccga tttcaccgac tacgtggaaa agaaaaaatg gatatacaag ttatgaattt 1080tctgatcatt tcccaattta tggttttgtt tacgccgacc cttccactcc tacaaagtca 1140ggacacagaa gaaaatacga tcaagtatcc ttaatagcta aatataccgg aaaagcaata 1200caagtggatc ataatagacc agacggatgg ttaaaagcag atggaactgc aaaagaaaaa 1260ggaacagaat ttacaaaatt caatttactg caagaatacg accctgattc agacaccttt 1320tgtatgttag gtgggcgtgt aagaatcgaa tcttctcaat atttaaatta cttttggact 1380tggtggctta gaggaggagg aggtaactat gcctattatc ctaaatttga tgatagctcc 1440aaactactcg aaatgataat aatacgtcaa ggatgtttag aagacgaaag tttggtcgta 1500ttcaaagatt ttgatacata cgggaaatac tattattttc ttgcagtctg ggaaaatgga 1560agttggaaag attatattta tctttggtac acaaatgccc aaccaaattc gtactttatt 1620gctaaattaa atacctcacc tgaaagagat tggagcaagg atctaattta tcgttag 1677＜210〉4＜211〉558＜212〉PRT＜213〉 ( Leptospira interrogans )＜400〉4Met Lys Thr Lys Arg Asn Pro Leu Leu Asn Lys Cys Lys Lys Pro Asn1 5 10 15Lys Phe Lys Tyr Arg Asn Leu Leu Ile Gly Leu Leu Leu Cys Leu Phe

20??????????????????25??????????????????30Pro?Leu?Asn?Cys?Thr?Phe?Lys?Asp?Ser?Leu?Val?Tyr?Asn?Lys?Phe?Leu

35??????????????????40??????????????????45Met?Tyr?Ile?Ser?Phe?Ser?His?Lys?Asn?Leu?Asn?Phe?Glu?Leu?Glu?Asn

50??????????????????55??????????????????60Ile?Asn?Ser?Thr?Arg?Lys?Thr?Leu?Leu?Pro?Lys?Ser?Glu?Ile?Asp?Leu65??????????????????70??????????????????75??????????????????80Asn?Ile?Leu?Ser?Tyr?Asn?Leu?Phe?Leu?Tyr?Ser?Lys?Glu?Asp?Ile?Tyr

85??????????????????90??????????????????95Phe?Gly?Tyr?Trp?Glu?Glu?Glu?Lys?Arg?Ala?Glu?Leu?Leu?Gly?Lys?Ser

100?????????????????105?????????????????110Lys?Phe?Thr?Lys?Asn?Gln?Asp?Val?Ile?Val?Leu?Ser?Arg?Val?Phe?Asp

115?????????????????120?????????????????125Thr?Asn?Ala?Arg?Asn?Thr?Leu?Leu?Asp?Asn?Leu?Asn?Leu?Glu?Tyr?Pro

130?????????????????135?????????????????140Asp?Gln?Thr?Asp?Val?Ile?Gly?Lys?Thr?Lys?Tyr?Gly?Trp?Asp?Gln?Thr145?????????????????150?????????????????155?????????????????160Leu?Gly?Asp?Phe?Arg?Gln?Gln?Ile?Asn?Asn?Gly?Gly?Val?Val?Ile?Leu

165?????????????????170?????????????????175Ser?Lys?Trp?Pro?Ile?Gln?Glu?Lys?Ile?Gln?Tyr?Ile?Phe?Lys?Asn?His

180?????????????????185?????????????????190Gly?Cys?Gly?Asn?Asp?Thr?Phe?Tyr?Asn?Lys?Gly?Phe?Ala?Tyr?Val?Lys

195?????????????????200?????????????????205Ile?Lys?Lys?Gly?Ser?Gln?Ile?Ile?His?Ile?Val?Gly?Thr?Asp?Thr?Gln

210?????????????????215?????????????????220Ser?Glu?Asp?Ser?Thr?Cys?Ser?Asp?Leu?Gly?Val?Asn?Ala?Arg?Ile?Asn225?????????????????230?????????????????235?????????????????240Gln?Leu?Thr?Glu?Ile?Lys?Lys?Phe?Ile?Asp?Ser?Lys?Arg?Ile?Ser?Asn

245?????????????????250?????????????????255Lys?Glu?Ile?Val?Leu?Ile?Ala?Gly?Ala?Leu?Asn?Val?Asp?Lys?Ser?Asn

260?????????????????265?????????????????270Gln?Ser?Glu?Tyr?Lys?Asn?Met?Leu?Asn?Ile?Leu?Glu?Val?Asn?Glu?Pro

275?????????????????280?????????????????285Asn?Tyr?Ala?Gly?Ile?Pro?Phe?Thr?Trp?Asp?Thr?Lys?Lys?Asn?Lys?Ile

290?????????????????295?????????????????300Ala?Ala?Tyr?Asn?Asn?Ile?Tyr?Tyr?Ser?Trp?Asn?Gln?Thr?Ser?Asn?Tyr305?????????????????310?????????????????315?????????????????320Gly?Glu?Tyr?Ile?Leu?Val?Ser?Lys?Asn?His?Phe?Gln?Leu?Pro?Ile?Trp

325?????????????????330?????????????????335Gln?Asn?Leu?Ala?Tyr?Asp?Pro?Ile?Ser?Pro?Thr?Thr?Trp?Lys?Arg?Lys

340?????????????????345?????????????????350Asn?Gly?Tyr?Thr?Ser?Tyr?Glu?Phe?Ser?Asp?His?Phe?Pro?Ile?Tyr?Gly

355?????????????????360?????????????????365Phe?Val?Tyr?Ala?Asp?Pro?Ser?Thr?Pro?Thr?Lys?Ser?Gly?His?Arg?Arg

370?????????????????375?????????????????380Lys?Tyr?Asp?Gln?Val?Ser?Leu?Ile?Ala?Lys?Tyr?Thr?Gly?Lys?Ala?Ile385?????????????????390?????????????????395?????????????????400Gln?Val?Asp?His?Asn?Arg?Pro?Asp?Gly?Trp?Leu?Lys?Ala?Asp?Gly?Thr

405?????????????????410?????????????????415Ala?Lys?Glu?Lys?Gly?Thr?Glu?Phe?Thr?Lys?Phe?Asn?Leu?Leu?Gln?Glu

420?????????????????425?????????????????430Tyr?Asp?Pro?Asp?Ser?Asp?Thr?Phe?Cys?Met?Leu?Gly?Gly?Arg?Val?Arg

435?????????????????440?????????????????445Ile?Glu?Ser?Ser?Gln?Tyr?Leu?Asn?Tyr?Phe?Trp?Thr?Trp?Trp?Leu?Arg

450?????????????????455?????????????????460Gly?Gly?Gly?Gly?Asn?Tyr?Ala?Tyr?Tyr?Pro?Lys?Phe?Asp?Asp?Ser?Ser465?????????????????470?????????????????475?????????????????480Lys?Leu?Leu?Glu?Met?Ile?Ile?Ile?Arg?Gln?Gly?Cys?Leu?Glu?Asp?Glu

485?????????????????490?????????????????495Ser?Leu?Val?Val?Phe?Lys?Asp?Phe?Asp?Thr?Tyr?Gly?Lys?Tyr?Tyr?Tyr

500?????????????????505?????????????????510Phe?Leu?Ala?Val?Trp?Glu?Asn?Gly?Ser?Trp?Lys?Asp?Tyr?Ile?Tyr?Leu

515?????????????????520?????????????????525Trp?Tyr?Thr?Asn?Ala?Gln?Pro?Asn?Ser?Tyr?Phe?Ile?Ala?Lys?Leu?Asn

530 535 540Thr Ser Pro Glu Arg Asp Trp Ser Lys Asp Leu Ile Tyr Arg545 550 555＜210〉5＜211〉1794＜212〉DNA＜213〉 ( Leptospira interrogans )＜400〉5atgatcacaa agcgaaatat accaccttgt aaaaaaaact ggaaatataa gaaaaaatct 60ataagtttaa cactgattac aatttgttac atgtttcttt tccttacaag ttgtaaacct 120ggcaaaaaaa attccataaa tcttctatta ctcttactga acactttaga taacaaaaat 180gtaaatgaga aaattgaaga ttcaacaaac acagatccta gttcaaatgt aaatgaggaa 240gatgaaaatt caataaacgc aaatgccaac gacaacgccc cttcggattc cgactcttca 300aatccacgct ctccagacaa aaatcctgta aatcctactt ctccaaattc aagctctgca 360gatataggaa ttaaaatttt aagccacagc atatttatgg cacctacgaa tctttcaagt 420tggggcgatt tgggacaaga agaaagggca caaagaattg caagttcaag ttatataaaa 480aatcaagaca ttattgtgtt tgaagggtta tctcataaca acgctgagaa aattctctta 540gaaaaaattc gttccgaata cccataccaa accaacgtag taggtagaac aaaaaaaggt 600tggaatgcaa ctcttggagc ttatacaact tctccaatgg ctaacggagg agttatcatc 660gttagtaaat ggcctatcga agaaaaagtt caatatatat tcaacaattc aaattgtggt 720caagaccagt attataacaa aggatttgct tatgtgaaaa tcaacaaaga tggaaaaaaa 780tttcatgtta tcggaactca gctacaagct cgtgaacctg actgctttaa ttctggagaa 840accatacgta aacttcaact caatgatatt aaaagtttta ttgattctaa agacattcca 900aaagatgaaa ccgttttgat tacgggtgat ttaaacatta tcaaaggaag taatgaatat 960ttcgatatga tctctaaact aaatgtcaac gaacctagat atgtcggagt tccttttact 1020ttagatacaa aaacaaacgc acttgccgca tactattacg aaaaggaaaa accgatttac 1080ctggattata ttctcgtttc aaaattacac gctcaaccac cggtttggca aaatttagct 1140tacgatccaa tttcaaatac aacctggaaa agatctgatg gatatacaag ttacgaattt 1200tcagatcgtt atccggtata cggttttata tatgcggatt cttccactcc tacaaaatct 1260ggacataaaa gaaaatacga tcaggtttct tttcaatcga cctttaatcg taaattcatt 1320caagcagatc ataataaaaa agacggatgg ttaaaagcgg atacaagaat aaaaacagat 1380tttactaaat tcaatttatt gcaagagaat gtttccgaat ctaatccgtc ttgtatgaat 1440agtgggtcag tcagaattga atcttcctat tatttaaatt attattggaa ttggtttatt 1500ggcgcagcca gcggtgacta cggctattat acgaaattta ataacggctc cgatagctta 1560ggtattaaaa atttagacaa tggatgtcta aaagatggta gtagagttgc cttctatgat 1620tgggatacaa ttggtggtgg ctattattac ctaacagttt gggacaaagg aagctggaaa 1680gaacatttgt ttctttgggt acaatccttt cttagctcaa gagagatttt ttacttacat 1740ctagactcta acccacccaa agactggagt aaagatttaa tctatcatca ttag 1794＜210〉6＜211〉597＜212〉PRT＜213〉 ( Leptospira interrogans )＜400〉6Met Ile Thr Lys Arg Asn Ile Pro Pro Cys Lys Lys Asn Trp Lys Tyr1 5 10 15Lys Lys Lys Ser Ile Ser Leu Thr Leu Ile Thr Ile Cys Tyr Met Phe

20??????????????????25??????????????????30Leu?Phe?Leu?Thr?Ser?Cys?Lys?Pro?Gly?Lys?Lys?Asn?Ser?Ile?Asn?Leu

35??????????????????40??????????????????45Leu?Leu?Leu?Leu?Leu?Asn?Thr?Leu?Asp?Asn?Lys?Asn?Val?Asn?Glu?Lys

50??????????????????55??????????????????60Ile?Glu?Asp?Ser?Thr?Asn?Thr?Asp?Pro?Ser?Ser?Asn?Val?Asn?Glu?Glu65??????????????????70??????????????????75??????????????????80Asp?Glu?Asn?Ser?Ile?Asn?Ala?Asn?Ala?Asn?Asp?Asn?Ala?Pro?Ser?Asp

85??????????????????90??????????????????95Ser?Asp?Ser?Ser?Asn?Pro?Arg?Ser?Pro?Asp?Lys?Asn?Pro?Val?Asn?Pro

100?????????????????105?????????????????110Thr?Ser?Pro?Asn?Ser?Ser?Ser?Ala?Asp?Ile?Gly?Ile?Lys?Ile?Leu?Ser

115?????????????????120?????????????????125His?Ser?Ile?Phe?Met?Ala?Pro?Thr?Asn?Leu?Ser?Ser?Trp?Gly?Asp?Leu

130?????????????????135?????????????????140Gly?Gln?Glu?Glu?Arg?Ala?Gln?Arg?Ile?Ala?Ser?Ser?Ser?Tyr?Ile?Lys145?????????????????150?????????????????155?????????????????160Asn?Gln?Asp?Ile?Ile?Val?Phe?Glu?Gly?Leu?Ser?His?Asn?Asn?Ala?Glu

165?????????????????170?????????????????175Lys?Ile?Leu?Leu?Glu?Lys?Ile?Arg?Ser?Glu?Tyr?Pro?Tyr?Gln?Thr?Asn

180?????????????????185?????????????????190Val?Val?Gly?Arg?Thr?Lys?Lys?Gly?Trp?Asn?Ala?Thr?Leu?Gly?Ala?Tyr

195?????????????????200?????????????????205Thr?Thr?Ser?Pro?Met?Ala?Asn?Gly?Gly?Val?Ile?Ile?Val?Ser?Lys?Trp

210?????????????????215?????????????????220Pro?Ile?Glu?Glu?Lys?Val?Gln?Tyr?Ile?Phe?Asn?Asn?Ser?Asn?Cys?Gly225?????????????????230?????????????????235?????????????????240Gln?Asp?Gln?Tyr?Tyr?Asn?Lys?Gly?Phe?Ala?Tyr?Val?Lys?Ile?Asn?Lys

245?????????????????250?????????????????255Asp?Gly?Lys?Lys?Phe?His?Val?Ile?Gly?Thr?Gln?Leu?Gln?Ala?Arg?Glu

260?????????????????265?????????????????270Pro?Asp?Cys?Phe?Asn?Ser?Gly?Glu?Thr?Ile?Arg?Lys?Leu?Gln?Leu?Asn

275?????????????????280?????????????????285Asp?Ile?Lys?Ser?Phe?Ile?Asp?Ser?Lys?Asp?Ile?Pro?Lys?Asp?Glu?Thr

290?????????????????295?????????????????300Val?Leu?Ile?Thr?Gly?Asp?Leu?Asn?Ile?Ile?Lys?Gly?Ser?Asn?Glu?Tyr305?????????????????310?????????????????315?????????????????320Phe?Asp?Met?Ile?Ser?Lys?Leu?Asn?Val?Asn?Glu?Pro?Arg?Tyr?Val?Gly

325?????????????????330?????????????????335Val?Pro?Phe?Thr?Leu?Asp?Thr?Lys?Thr?Asn?Ala?Leu?Ala?Ala?Tyr?Tyr

340?????????????????345?????????????????350Tyr?Glu?Lys?Glu?Lys?Pro?Ile?Tyr?Leu?Asp?Tyr?Ile?Leu?Val?Ser?Lys

355?????????????????360?????????????????365Leu?His?Ala?Gln?Pro?Pro?Val?Trp?Gln?Asn?Leu?Ala?Tyr?Asp?Pro?Ile

370?????????????????375?????????????????380Ser?Asn?Thr?Thr?Trp?Lys?Arg?Ser?Asp?Gly?Tyr?Thr?Ser?Tyr?Glu?Phe385?????????????????390?????????????????395?????????????????400Ser?Asp?Arg?Tyr?Pro?Val?Tyr?Gly?Phe?Ile?Tyr?Ala?Asp?Ser?Ser?Thr

405?????????????????410?????????????????415Pro?Thr?Lys?Ser?Gly?His?Lys?Arg?Lys?Tyr?Asp?Gln?Val?Ser?Phe?Gln

420?????????????????425?????????????????430Ser?Thr?Phe?Asn?Arg?Lys?Phe?Ile?Gln?Ala?Asp?His?Asn?Lys?Lys?Asp

435?????????????????440?????????????????445Gly?Trp?Leu?Lys?Ala?Asp?Thr?Arg?Ile?Lys?Thr?Asp?Phe?Thr?Lys?Phe

450?????????????????455?????????????????460Asn?Leu?Leu?Gln?Glu?Asn?Val?Ser?Glu?Ser?Asn?Pro?Ser?Cys?Met?Asn465?????????????????470?????????????????475?????????????????480Ser?Gly?Ser?Val?Arg?Ile?Glu?Ser?Ser?Tyr?Tyr?Leu?Asn?Tyr?Tyr?Trp

485?????????????????490?????????????????495Asn?Trp?Phe?Ile?Gly?Ala?Ala?Ser?Gly?Asp?Tyr?Gly?Tyr?Tyr?Thr?Lys

500?????????????????505?????????????????510Phe?Asn?Asn?Gly?Ser?Asp?Ser?Leu?Gly?Ile?Lys?Asn?Leu?Asp?Asn?Gly

515?????????????????520?????????????????525Cys?Leu?Lys?Asp?Gly?Ser?Arg?Val?Ala?Phe?Tyr?Asp?Trp?Asp?Thr?Ile

530?????????????????535?????????????????540Gly?Gly?Gly?Tyr?Tyr?Tyr?Leu?Thr?Val?Trp?Asp?Lys?Gly?Ser?Trp?Lys545?????????????????550?????????????????555?????????????????560Glu?His?Leu?Phe?Leu?Trp?Val?Gln?Ser?Phe?Leu?Ser?Ser?Arg?Glu?Ile

565?????????????????570?????????????????575Phe?Tyr?Leu?His?Leu?Asp?Ser?Asn?Pro?Pro?Lys?Asp?Trp?Ser?Lys?Asp

580?????????????????585?????????????????590Leu?Ile?Tyr?His?His

595＜210〉7＜211〉1872＜212〉DNA＜213〉 ( Leptospira interrogans )＜400〉7atgataaaca aaataacaaa accaaaacta cttataggtt attacctatt gttattctct 60ttgatacgtt gtttacccga aaaagaatcc tcatataagg atttatttac ttcgttatta 120ttcctcccca accaaacaaa tagcaatcaa gtaaattcgg tttccataaa caacgacccc 180gcaaacccaa atccagttaa cccggcatcc gcaaataaca atcaagtaaa cgcagttcca 240gaaaatgacg atccagcaaa cctaaatcca gttaatccgg catctgcaaa tagcaatcaa 300gtaaatgcag ctccagaaaa tggcagcccc gcagatccaa atccagccaa cctagcatct 360gcaaataata atcaagtaaa tgcagttcca gctaacaatt attttacgaa agaagattcc 420tcaaataata ttcctaaaaa agtaaattct aaaaatgtag aaataaaagt actgagtcac 480aacgtattta tgttgcccac aaatcttcca cgatggggaa atttgggaca cgatgaaaga 540gcaaaacgaa tttcaaaatc agattacgta aaaaatcagg acgttatcgt gtttgaagaa 600gccttcgaca caagtgcaag aaaaattctt ttagacaatc ttcgagaaga atatccgtat 660caaacagatg tggtcggaag aacaaaaaaa aattgggacg caagcttagg caattttaga 720tcttattcgc tcgttaacgg aggagttgta atcctaagta aatggcctat cgaagaaaag 780attcaatata tctttaacga ctcaggttgt ggagcggatt ggtttgcaaa taaaggattt 840gtttatgtaa aaatcaacaa agaaggaaaa aaattccatg tcatcggaac ccacgctcag 900tctcaggatc agaactgttc aaatctagga ataccaaaca gagctaatca atttgatgac 960attagaaatt tcatatattc taaaaatatt ccaaaagatg aaaccgtttt aatcgtgggt 1020gatttaaacg ttatcaaaga aagtaacgaa tattacgata tgatttctag gttaaatgtt 1080aacgaaccta gatatgttgg agttcctttt acttgggatg caaaaacaaa cgagattgct 1140gcatactatt acgaaaacga agaaccggtt tacttagatt atattttcgt ttcaaaatca 1200cacgctcaac caccggtttg gcaaaattta gcttacgatc cggtttctaa acaaacttgg 1260accgtatctg gatatacaag cgatgaattt tcggatcact atccgatata cggttttgta 1320tacgcggatc cttctactcc tacaaagtcc ggacataaaa aaaaatacga tcaagtctct 1380tttcaatcag ctgctaatgg taaatacatt caagcagatc ctaatagaaa aaacggatgg 1440ttaaaagcgg acgctgtaat agaaacggat tttactaaat tcaacctatt gcaagaagga 1500aatttaaatc cttcctgcat aaaaaatggt ctcgttagaa ttgaatcttc ccgtttttta 1560aactactttt ggaactggtg gttaggcgga ggttccggta actacggcta ttactctaag 1620tttaatgacg cttccaatca acttgaaatt atcaatttga gtgatgaatg cctagaaaac 1680ggtagtaaaa tcgtgttcaa agattatgat acttattcta gaaaccatta ctatctcacc 1740gtttgggaca aaggaaattg gaatgaacat ctttatcttt ggaaagattc gatcagccag 1800agagaaatct tttatctcaa attaaattct actccggtaa gaaattggag tgcggatctt 1860atttatcgct aa 1872＜210〉8＜211〉623＜212〉PRT＜213〉 ( Leptospira interrogans )＜400〉8Met Ile Asn Lys Ile Thr Lys Pro Lys Leu Leu Ile Gly Tyr Tyr Leu1 5 10 15Leu Leu Phe Ser Leu Ile Arg Cys Leu Pro Glu Lys Glu Ser Ser Tyr

20??????????????????25??????????????????30Lys?Asp?Leu?Phe?Thr?Ser?Leu?Leu?Phe?Leu?Pro?Asn?Gln?Thr?Asn?Ser

35??????????????????40??????????????????45Asn?Gln?Val?Asn?Ser?Val?Ser?Ile?Asn?Asn?Asp?Pro?Ala?Asn?Pro?Asn

50??????????????????55??????????????????60Pro?Val?Asn?Pro?Ala?Ser?Ala?Asn?Asn?Asn?Gln?Val?Asn?Ala?Val?Pro65??????????????????70??????????????????75??????????????????80Glu?Asn?Asp?Asp?Pro?Ala?Asn?Leu?Asn?Pro?Val?Asn?Pro?Ala?Ser?Ala

85??????????????????90??????????????????95Asn?Ser?Asn?Gln?Val?Asn?Ala?Ala?Pro?Glu?Asn?Gly?Ser?Pro?Ala?Asp

100?????????????????105?????????????????110Pro?Asn?Pro?Ala?Asn?Leu?Ala?Ser?Ala?Asn?Asn?Asn?Gln?Val?Asn?Ala

115?????????????????120?????????????????125Val?Pro?Ala?Asn?Asn?Tyr?Phe?Thr?Lys?Glu?Asp?Ser?Ser?Asn?Asn?Ile

130?????????????????135?????????????????140Pro?Lys?Lys?Val?Asn?Ser?Lys?Asn?Val?Glu?Ile?Lys?Val?Leu?Ser?His145?????????????????150?????????????????155?????????????????160Asn?Val?Phe?Met?Leu?Pro?Thr?Asn?Leu?Pro?Arg?Trp?Gly?Asn?Leu?Gly

165?????????????????170?????????????????175His?Asp?Glu?Arg?Ala?Lys?Arg?Ile?Ser?Lys?Ser?Asp?Tyr?Val?Lys?Asn

180?????????????????185?????????????????190Gln?Asp?Val?Ile?Val?Phe?Glu?Glu?Ala?Phe?Asp?Thr?Ser?Ala?Arg?Lys

195?????????????????200?????????????????205Ile?Leu?Leu?Asp?Asn?Leu?Arg?Glu?Glu?Tyr?Pro?Tyr?Gln?Thr?Asp?Val

210?????????????????215?????????????????220Val?Gly?Arg?Thr?Lys?Lys?Asn?Trp?Asp?Ala?Ser?Leu?Gly?Asn?Phe?Arg225?????????????????230?????????????????235?????????????????240Ser?Tyr?Ser?Leu?Val?Asn?Gly?Gly?Val?Val?Ile?Leu?Ser?Lys?Trp?Pro

245?????????????????250?????????????????255Ile?Glu?Glu?Lys?Ile?Gln?Tyr?Ile?Phe?Asn?Asp?Ser?Gly?Cys?Gly?Ala

260?????????????????265?????????????????270Asp?Trp?Phe?Ala?Asn?Lys?Gly?Phe?Val?Tyr?Val?Lys?Ile?Asn?Lys?Glu

275?????????????????280?????????????????285Gly?Lys?Lys?Phe?His?Val?Ile?Gly?Thr?His?Ala?Gln?Ser?Gln?Asp?Gln

290?????????????????295?????????????????300Asn?Cys?Ser?Asn?Leu?Gly?Ile?Pro?Asn?Arg?Ala?Asn?Gln?Phe?Asp?Asp305?????????????????310?????????????????315?????????????????320Ile?Arg?Asn?Phe?Ile?Tyr?Ser?Lys?Asn?Ile?Pro?Lys?Asp?Glu?Thr?Val

325?????????????????330?????????????????335Leu?Ile?Val?Gly?Asp?Leu?Asn?Val?Ile?Lys?Glu?Ser?Asn?Glu?Tyr?Tyr

340?????????????????345?????????????????350Asp?Met?Ile?Ser?Arg?Leu?Asn?Val?Asn?Glu?Pro?Arg?Tyr?Val?Gly?Val

355?????????????????360?????????????????365Pro?Phe?Thr?Trp?Asp?Ala?Lys?Thr?Asn?Glu?Ile?Ala?Ala?Tyr?Tyr?Tyr

370?????????????????375?????????????????380Glu?Asn?Glu?Glu?Pro?Val?Tyr?Leu?Asp?Tyr?Ile?Phe?Val?Ser?Lys?Ser385?????????????????390?????????????????395?????????????????400His?Ala?Gln?Pro?Pro?Val?Trp?Gln?Asn?Leu?Ala?Tyr?Asp?Pro?Val?Ser

405?????????????????410?????????????????415Lys?Gln?Thr?Trp?Thr?Val?Ser?Gly?Tyr?Thr?Ser?Asp?Glu?Phe?Ser?Asp

420?????????????????425?????????????????430His?Tyr?Pro?Ile?Tyr?Gly?Phe?Val?Tyr?Ala?Asp?Pro?Ser?Thr?Pro?Thr

435?????????????????440?????????????????445Lys?Ser?Gly?His?Lys?Lys?Lys?Tyr?Asp?Gln?Val?Ser?Phe?Gln?Ser?Ala

450?????????????????455?????????????????460Ala?Asn?Gly?Lys?Tyr?Ile?Gln?Ala?Asp?Pro?Asn?Arg?Lys?Asn?Gly?Trp465?????????????????470?????????????????475?????????????????480Leu?Lys?Ala?Asp?Ala?Val?Ile?Glu?Thr?Asp?Phe?Thr?Lys?Phe?Asn?Leu

485?????????????????490?????????????????495Leu?Gln?Glu?Gly?Asn?Leu?Asn?Pro?Ser?Cys?Ile?Lys?Asn?Gly?Leu?Val

500?????????????????505?????????????????510Arg?Ile?Glu?Ser?Ser?Arg?Phe?Leu?Asn?Tyr?Phe?Trp?Asn?Trp?Trp?Leu

515?????????????????520?????????????????525Gly?Gly?Gly?Ser?Gly?Asn?Tyr?Gly?Tyr?Tyr?Ser?Lys?Phe?Asn?Asp?Ala

530?????????????????535?????????????????540Ser?Asn?Gln?Leu?Glu?Ile?Ile?Asn?Leu?Ser?Asp?Glu?Cys?Leu?Glu?Asn545?????????????????550?????????????????555?????????????????560Gly?Ser?Lys?Ile?Val?Phe?Lys?Asp?Tyr?Asp?Thr?Tyr?Ser?Arg?Asn?His

565?????????????????570?????????????????575Tyr?Tyr?Leu?Thr?Val?Trp?Asp?Lys?Gly?Asn?Trp?Asn?Glu?His?Leu?Tyr

580?????????????????585?????????????????590Leu?Trp?Lys?Asp?Ser?Ile?Ser?Gln?Arg?Glu?Ile?Phe?Tyr?Leu?Lys?Leu

595?????????????????600?????????????????605Asn?Ser?Thr?Pro?Val?Arg?Asn?Trp?Ser?Ala?Asp?Leu?Ile?Tyr?Arg

610 615 620＜210〉9＜211〉1335＜212〉DNA＜213〉 ( Leptospira interrogans )＜400〉9atggaattaa tcggcttttt tataatcgtt ttacttatat ttgcgaacgg atttttcgtt 60tccgcagagt ttgctttggt ttcgattcgt ccttctcgtt tagaggaatt gatcaaggaa 120aataaacctc ttgcatttat taccaaacgc gcggctcaaa aactcaatga catgttatcc 180gtatgtcagg taggaattac catcgccagt cttttgttag gttgggtggg ggaaggttac 240gtttctagat ggcttatttt tcttttggaa atgtttggct attccgcaaa cgaagcaact 300gttcacggtt tggcgattac tatttcgttt acgattatta cgtttctaca tatactttta 360ggagaacttc ttcctaagac agtagcaatt cagaacacgg aaacgattgc actttttatt 420agcattcctt tattcttctt ttattatcta ttttatccaa ttactttctt cttaaatgaa 480atgacatctt ttcttttgaa gctgattgga atcgaagcaa acaaaagcag aatgatgcat 540tctcctgaag aattgatgat catcatagaa gagcaaaaca aacaaggtaa gatcgatcag 600gaagaatttc aaatcattca gaatacgttt cagttttcag aacatcaagc aaaggatgtg 660atgactcatc gtttgagtat cattgggatt ccgcatgata cttctatgga ttctttgatt 720tctatcattg cagaacacca tttttctaga tatcctattt atgaaggcag tacagataaa 780attattggaa tcattcatgt tcagacgtat cttacttggt tgtctaattc taaaaaaggt 840agaaaagaaa aagttaccgc aattatgcaa cctccgattt ttgttcccga aggtctttcg 900atagaaaagg tgatgcaaaa actccgagaa aacaaacaac acatggcgat cgttatagat 960gagtacggtg gagttgcggg tcttcttaca ttagaggata ttatcgaaga aatttttgga 1020cagatccgag atgaaacgga cgatcatgag acggatccgt tccctactca acattcggac 1080agttttacga tcgacggcga agcggagtta gacgatctaa aagaaattct tgtaggagtc 1140caagaagaag agatcaaaga tattcgtacg attgcaggtt ttatcttagg tcgtttagaa 1200gatatgccgg aagaaggttc tacaatttct cttcaaacag gaactcttac cgttgagaaa 1260atggaaggta ataaaattct ttcggttcgt tttacaagag ttagcttaaa caatcgagct 1320cagtctaaaa aatga 1335＜210〉10＜211〉444＜212〉PRT＜213〉 ( Leptospira interrogans )＜400〉10Met Glu Leu Ile Gly Phe Phe Ile Ile Val Leu Leu Ile Phe Ala Asn1 5 10 15Gly Phe Phe Val Ser Ala Glu Phe Ala Leu Val Ser Ile Arg Pro Ser

20??????????????????25??????????????????30Arg?Leu?Glu?Glu?Leu?Ile?Lys?Glu?Asn?Lys?Pro?Leu?Ala?Phe?Ile?Thr

35??????????????????40??????????????????45Lys?Arg?Ala?Ala?Gln?Lys?Leu?Asn?Asp?Met?Leu?Ser?Val?Cys?Gln?Val

50??????????????????55??????????????????60Gly?Ile?Thr?Ile?Ala?Ser?Leu?Leu?Leu?Gly?Trp?Val?Gly?Glu?Gly?Tyr65??????????????????70??????????????????75??????????????????80Val?Ser?Arg?Trp?Leu?Ile?Phe?Leu?Leu?Glu?Met?Phe?Gly?Tyr?Ser?Ala

85??????????????????90??????????????????95Asn?Glu?Ala?Thr?Val?His?Gly?Leu?Ala?Ile?Thr?Ile?Ser?Phe?Thr?Ile

100?????????????????105?????????????????110Ile?Thr?Phe?Leu?His?Ile?Leu?Leu?Gly?Glu?Leu?Leu?Pro?Lys?Thr?Val

115????????????????120?????????????????125Ala?Ile?Gln?Asn?Thr?Glu?Thr?Ile?Ala?Leu?Phe?Ile?Ser?Ile?Pro?Leu

130?????????????????135?????????????????140Phe?Phe?Phe?Tyr?Tyr?Leu?Phe?Tyr?Pro?Ile?Thr?Phe?Phe?Leu?Asn?Glu145?????????????????150?????????????????155?????????????????160Met?Thr?Ser?Phe?Leu?Leu?Lys?Leu?Ile?Gly?Ile?Glu?Ala?Asn?Lys?Ser

165?????????????????170?????????????????175Arg?Met?Met?His?Ser?Pro?Glu?Glu?Leu?Met?Ile?Ile?Ile?Glu?Glu?Gln

180?????????????????185?????????????????190Asn?Lys?Gln?Gly?Lys?Ile?Asp?Gln?Glu?Glu?Phe?Gln?Ile?Ile?Gln?Asn

195?????????????????200?????????????????205Thr?Phe?Gln?Phe?Ser?Glu?His?Gln?Ala?Lys?Asp?Val?Met?Thr?His?Arg

210?????????????????215?????????????????220Leu?Ser?Ile?Ile?Gly?Ile?Pro?His?Asp?Thr?Ser?Met?Asp?Ser?Leu?Ile225?????????????????230?????????????????235?????????????????240Ser?Ile?Ile?Ala?Glu?His?His?Phe?Ser?Arg?Tyr?Pro?Ile?Tyr?Glu?Gly

245?????????????????250?????????????????255Ser?Thr?Asp?Lys?Ile?Ile?Gly?Ile?Ile?His?Val?Gln?Thr?Tyr?Leu?Thr

260?????????????????265?????????????????270Trp?Leu?Ser?Asn?Ser?Lys?Lys?Gly?Arg?Lys?Glu?Lys?Val?Thr?Ala?Ile

275?????????????????280?????????????????285Met?Gln?Pro?Pro?Ile?Phe?Val?Pro?Glu?Gly?Leu?Ser?Ile?Glu?Lys?Val

290?????????????????295?????????????????300Met?Gln?Lys?Leu?Arg?Glu?Asn?Lys?Gln?His?Met?Ala?Ile?Val?Ile?Asp305?????????????????310?????????????????315?????????????????320Glu?Tyr?Gly?Gly?Val?Ala?Gly?Leu?Leu?Thr?Leu?Glu?Asp?Ile?Ile?Glu

325?????????????????330?????????????????335Glu?Ile?Phe?Gly?Gln?Ile?Arg?Asp?Glu?Thr?Asp?Asp?His?Glu?Thr?Asp

340?????????????????345?????????????????350Pro?Phe?Pro?Thr?Gln?His?Ser?Asp?Ser?Phe?Thr?Ile?Asp?Gly?Glu?Ala

355?????????????????360?????????????????365Glu?Leu?Asp?Asp?Leu?Lys?Glu?Ile?Leu?Val?Gly?Val?Gln?Glu?Glu?Glu

370?????????????????375?????????????????380Ile?Lys?Asp?Ile?Arg?Thr?Ile?Ala?Gly?Phe?Ile?Leu?Gly?Arg?Leu?Glu385?????????????????390?????????????????395?????????????????400Asp?Met?Pro?Glu?Glu?Gly?Ser?Thr?Ile?Ser?Leu?Gln?Thr?Gly?Thr?Leu

405?????????????????410?????????????????415Thr?Val?Glu?Lys?Met?Glu?Gly?Asn?Lys?Ile?Leu?Ser?Val?Arg?Phe?Thr

420?????????????????425?????????????????430Arg?Val?Ser?Leu?Asn?Asn?Arg?Ala?Gln?Ser?Lys?Lys

435 440＜210〉11＜211〉831＜212〉DNA＜213〉 ( Leptospira interrogans )＜400〉11ttgtttccac agaagaagaa caaacattct ttcaggagct tccgatttac attggccaga 60gaaaaaatta ggctcgatgt tcttcttttt gaaaggggat ttgccgattc tctggaaaaa 120gcgaaaagtt taattctttc gggctctgta ttagtaaacg aacaaaaggt tactaaagta 180ggattcaaat ttccaaaaga ttctgaaatc agaattttaa acatcattcc agaatatgta 240agcagaggag tttataaact gttaaaagcc tttgaggttt ttcctttaca agttgatgga 300aaactttgta tagatttggg tgcttctacg ggaggattta cgcaagtgct tttagaaaaa 360ggggcttgga aagtttttgc ttgtgatgtg ggttatggtc agcttgcaga aaagttaaga 420aatcattctt ctgtgatcgt aaaagatcgt tttcatctga aaaatttatc tgctttagaa 480atcgactggg aaaacaatcg gtttcaaaca cctcatccgg aagcgatcgt aatcgtaatg 540gacttgagtt ttatttctct ccgatccgtt tttccagtga tccaaaaatt gagaaaagaa 600aaggacattc caaaattaga atgtgtttct ttgataaaac cacaatttga agccaatcgg 660aatgaccttg tcaaaggtat tttaaaagat tctaaaattc gatttcaaat tgtactttct 720ctttgtaggt atcttaaaaa ggaaattgga ggtttcgttt taggtttgga atggtctccg 780atagaaggta gggacggaaa taaagaaatc cttttgtttt ggaacttata g 831＜210〉12＜211〉276＜212〉PRT＜213〉 ( Leptospira interrogans )＜400〉12Leu Phe Pro Gln Lys Lys Asn Lys His Ser Phe Arg Ser Phe Arg Phe1 5 10 15Thr Leu Ala Arg Glu Lys Ile Arg Leu Asp Val Leu Leu Phe Glu Arg

20??????????????????25??????????????????30Gly?Phe?Ala?Asp?Ser?Leu?Glu?Lys?Ala?Lys?Ser?Leu?Ile?Leu?Ser?Gly

35??????????????????40??????????????????45Ser?Val?Leu?Val?Asn?Glu?Gln?Lys?Val?Thr?Lys?Val?Gly?Phe?Lys?Phe

50??????????????????55??????????????????60Pro?Lys?Asp?Ser?Glu?Ile?Arg?Ile?Leu?Asn?Ile?Ile?Pro?Glu?Tyr?Val65??????????????????70??????????????????75??????????????????80Ser?Arg?Gly?Val?Tyr?Lys?Leu?Leu?Lys?Ala?Phe?Glu?Val?Phe?Pro?Leu

85??????????????????90??????????????????95Gln?Val?Asp?Gly?Lys?Leu?Cys?Ile?Asp?Leu?Gly?Ala?Ser?Thr?Gly?Gly

100?????????????????105?????????????????110Phe?Thr?Gln?Val?Leu?Leu?Glu?Lys?Gly?Ala?Trp?Lys?Val?Phe?Ala?Cys

115?????????????????120?????????????????125Asp?Val?Gly?Tyr?Gly?Gln?Leu?Ala?Glu?Lys?Leu?Arg?Asn?His?Ser?Ser

130?????????????????135?????????????????140Val?Ile?Val?Lys?Asp?Arg?Phe?His?Leu?Lys?Asn?Leu?Ser?Ala?Leu?Glu145?????????????????150?????????????????155?????????????????160Ile?Asp?Trp?Glu?Asn?Asn?Arg?Phe?Gln?Thr?Pro?His?Pro?Glu?Ala?Ile

165?????????????????170?????????????????175Val?Ile?Val?Met?Asp?Leu?Ser?Phe?Ile?Ser?Leu?Arg?Ser?Val?Phe?Pro

180?????????????????185?????????????????190Val?Ile?Gln?Lys?Leu?Arg?Lys?Glu?Lys?Asp?Ile?Pro?Lys?Leu?Glu?Cys

195?????????????????200?????????????????205Val?Ser?Leu?Ile?Lys?Pro?Gln?Phe?Glu?Ala?Asn?Arg?Asn?Asp?Leu?Val

210?????????????????215?????????????????220Lys?Gly?Ile?Leu?Lys?Asp?Ser?Lys?Ile?Arg?Phe?Gln?Ile?Val?Leu?Ser225?????????????????230?????????????????235?????????????????240Leu?Cys?Arg?Tyr?Leu?Lys?Lys?Glu?Ile?Gly?Gly?Phe?Val?Leu?Gly?Leu

245?????????????????250?????????????????255Glu?Trp?Ser?Pro?Ile?Glu?Gly?Arg?Asp?Gly?Asn?Lys?Glu?Ile?Leu?Leu

260?????????????????265?????????????????270Phe?Trp?Asn?Leu

275＜210〉13＜211〉1179＜212〉DNA＜213〉 ( Leptospira interrogans )＜400〉13ttggtcgaag cgctgtctgt ctcggatctc cggaggaaat ccatgaaccg ttctatcata 60ttaattacag gatttttatt tatctgcgcc ggccttttaa ccgcagtgta tcaaattacg 120attcaagacg aagattccaa acgaaagaac gttctggaaa aaattaaaga aggcgaggaa 180tacttaaaac aaaccaattc aaaagctgca gaaaaggctg tggatatatt ctctgaactt 240tccgccagag agattccaga agaacattct ttccgtgtta agtacgatat gggaagagca 300cttgaaagaa accaagatag tcttttagcg ctggggattt atagagaatt aaatcaaaaa 360gaaggccttt ccagagacga acgctcaaaa gttgcgtatt ccatgggaaa tcttctttta 420caactcaatc gagacgaaga aggaaaaggt catttagaag aggttcttag aatttcagcg 480gattctaaac ttcgttcaaa cgcgttatct gcaattgctg actattatat gaaaaaggga 540aattatgatc tttctcgcaa aaattacgtc ctcgccctcc aggaagatcc tgaaaatgta 600aaagccagag ttcgttgggg aaaatcttta cgcagaatgg gtaaggattg gtccgcctac 660gatgtttatg acgattacgc tcaagctgga ttctactttg atccagaaaa agaaaaagta 720agttccgagt ttagaagtgg tatcttagag aaagcaagac aactctacgt tcgtaaacag 780tactatggcg ctatcgatac ttttaaaaaa gcccttgata tgggcgtcag ctcgaaagcg 840gaagaacaag ctttgtttta tattgcagaa agttacgaag cgattggtaa atccgattcc 900gctcttcaat acctaaatcg agttttagga aatcaagatg gctctttaga tcaaacggct 960ctttttcgca aaggtacaat ctattttaaa agtggtaaat atgaaaaggc tgcagcctta 1020tttcaagaag ccacggataa atatccggat tctcccgttg gtagaaaggc aagcgcttgg 1080aaaaaagaat ccttggatca agtggaagat aatcttcatt acaagcagga agataaagaa 1140aaaagtaaag aggatctaga aaccgaaaaa ttggattga 1179＜210〉14＜211〉392＜212〉PRT＜213〉 ( Leptospira interrogans )＜400〉14Leu Val Glu Ala Leu Ser Val Ser Asp Leu Arg Arg Lys Ser Met Asn1 5 10 15Arg Ser Ile Ile Leu Ile Thr Gly Phe Leu Phe Ile Cys Ala Gly Leu

20??????????????????25??????????????????30Leu?Thr?Ala?Val?Tyr?Gln?Ile?Thr?Ile?Gln?Asp?Glu?Asp?Ser?Lys?Arg

35??????????????????40??????????????????45Lys?Asn?Val?Leu?Glu?Lys?Ile?Lys?Glu?Gly?Glu?Glu?Tyr?Leu?Lys?Gln

50??????????????????55??????????????????60Thr?Asn?Ser?Lys?Ala?Ala?Glu?Lys?Ala?Val?Asp?Ile?Phe?Ser?Glu?Leu65??????????????????70??????????????????75??????????????????80Ser?Ala?Arg?Glu?Ile?Pro?Glu?Glu?His?Ser?Phe?Arg?Val?Lys?Tyr?Asp

85??????????????????90??????????????????95Met?Gly?Arg?Ala?Leu?Glu?Arg?Asn?Gln?Asp?Ser?Leu?Leu?Ala?Leu?Gly

100?????????????????105?????????????????110Ile?Tyr?Arg?Glu?Leu?Asn?Gln?Lys?Glu?Gly?Leu?Ser?Arg?Asp?Glu?Arg

115?????????????????120?????????????????125Ser?Lys?Val?Ala?Tyr?Ser?Met?Gly?Asn?Leu?Leu?Leu?Gln?Leu?Asn?Arg

130?????????????????135?????????????????140Asp?Glu?Glu?Gly?Lys?Gly?His?Leu?Glu?Glu?Val?Leu?Arg?Ile?Ser?Ala145?????????????????150?????????????????155?????????????????160Asp?Ser?Lys?Leu?Arg?Ser?Asn?Ala?Leu?Ser?Ala?Ile?Ala?Asp?Tyr?Tyr

165?????????????????170?????????????????175Met?Lys?Lys?Gly?Asn?Tyr?Asp?Leu?Ser?Arg?Lys?Asn?Tyr?Val?Leu?Ala

180?????????????????185?????????????????190Leu?Gln?Glu?Asp?Pro?Glu?Asn?Val?Lys?Ala?Arg?Val?Arg?Trp?Gly?Lys

195?????????????????200?????????????????205Ser?Leu?Arg?Arg?Met?Gly?Lys?Asp?Trp?Ser?Ala?Tyr?Asp?Val?Tyr?Asp

210?????????????????215?????????????????220Asp?Tyr?Ala?Gln?Ala?Gly?Phe?Tyr?Phe?Asp?Pro?Glu?Lys?Glu?Lys?Val225?????????????????230?????????????????235?????????????????240Ser?Ser?Glu?Phe?Arg?Ser?Gly?Ile?Leu?Glu?Lys?Ala?Arg?Gln?Leu?Tyr

245?????????????????250?????????????????255Val?Arg?Lys?Gln?Tyr?Tyr?Gly?Ala?Ile?Asp?Thr?Phe?Lys?Lys?Ala?Leu

260?????????????????265?????????????????270Asp?Met?Gly?Val?Ser?Ser?Lys?Ala?Glu?Glu?Gln?Ala?Leu?Phe?Tyr?Ile

275?????????????????280?????????????????285Ala?Glu?Ser?Tyr?Glu?Ala?Ile?Gly?Lys?Ser?Asp?Ser?Ala?Leu?Gln?Tyr

290?????????????????295?????????????????300Leu?Asn?Arg?Val?Leu?Gly?Asn?Gln?Asp?Gly?Ser?Leu?Asp?Gln?Thr?Ala305?????????????????310?????????????????315?????????????????320Leu?Phe?Arg?Lys?Gly?Thr?Ile?Tyr?Phe?Lys?Ser?Gly?Lys?Tyr?Glu?Lys

325?????????????????330?????????????????335Ala?Ala?Ala?Leu?Phe?Gln?Glu?Ala?Thr?Asp?Lys?Tyr?Pro?Asp?Ser?Pro

340?????????????????345?????????????????350Val?Gly?Arg?Lys?Ala?Ser?Ala?Trp?Lys?Lys?Glu?Ser?Leu?Asp?Gln?Val

355?????????????????360?????????????????365Glu?Asp?Asn?Leu?His?Tyr?Lys?Gln?Glu?Asp?Lys?Glu?Lys?Ser?Lys?Glu

370 375 380Asp Leu Glu Thr Glu Lys Leu Asp385 390＜210〉15＜211〉942＜212〉DNA＜213〉 ( Leptospira interrogans )＜400〉15atgtcattag ctccgattgc actttttgtt tataatagac cggaacatac aaaaagaacg 60atcgattctt tatcaaacaa cgattatgcg gaacagtctg aattatttgt attttgtgat 120ggacctaagc ccaatacaaa tatggataag attagagaag ttagatcgat tgtcaaacaa 180gcgactggtt ttaaaaaagt tatcgtttat gaaaaagaaa acaatgtagg tttagcaaaa 240tctattatat ctggagtgac tgaactagta acaaaacatg gaaaaattat cgttttagaa 300gacgatatga taacttcgaa atattttttg acttatttaa atcatggatt agaatattat 360gaaaatttaa atcaggtagt ttctatacac gcttatcgat tgccgtttac cgcaaaaact 420cccgagacgt attttttaag aggtgctgat tgttggggct gggcgacttg gaaaaggggc 480tgggatctat tcgaggtcga tgggcaaaaa cttttggata aattaatttc agaaaaacta 540atttatcaat ttgatatgca agggtcttat ccttataccc aaatgttaaa agatcagatt 600gcaggtaaga atgattcctg ggcgataaga tggtatgctt ccactttttt acaaaataaa 660ttaactttat atcctgcaag atctttagtg tttaatattg gattggatgc ttccggaaca 720cattgtgaag ttacaaacga atacgatgtg gatttgagtt cgattccgat tcggattgaa 780acgattcaaa ttgaagagaa cgtttacatt agaaatttat atagagatta ttttcgcaaa 840ctcagtcaat cagatttaag aactcgaatt tttggtcgat tgaaacaact tttagaacgt 900ataaaaacga gaatttggcc agacaaatct aatttgctct ag 942＜210〉16＜211〉313＜212〉PRT＜213〉 ( Leptospira interrogans )＜400〉16Met Ser Leu Ala Pro Ile Ala Leu Phe Val Tyr Asn Arg Pro Glu His1 5 10 15Thr Lys Arg Thr Ile Asp Ser Leu Ser Asn Asn Asp Tyr Ala Glu Gln

20??????????????????25??????????????????30Ser?Glu?Leu?Phe?Val?Phe?Cys?Asp?Gly?Pro?Lys?Pro?Asn?Thr?Asn?Met

35??????????????????40??????????????????45Asp?Lys?Ile?Arg?Glu?Val?Arg?Ser?Ile?Val?Lys?Gln?Ala?Thr?Gly?Phe

50??????????????55??????????????????????60Lys?Lys?Val?Ile?Val?Tyr?Glu?Lys?Glu?Asn?Asn?Val?Gly?Leu?Ala?Lys65??????????????70??????????????????????75??????????????????80Ser?Ile?Ile?Ser?Gly?Val?Thr?Glu?Leu?Val?Thr?Lys?His?Gly?Lys?Ile

85??????????????????90??????????????????95Ile?Val?Leu?Glu?Asp?Asp?Met?Ile?Thr?Ser?Lys?Tyr?Phe?Leu?Thr?Tyr

100?????????????????105?????????????????110Leu?Asn?His?Gly?Leu?Glu?Tyr?Tyr?Glu?Asn?Leu?Asn?Gln?Val?Val?Ser

115?????????????????120?????????????????125Ile?His?Ala?Tyr?Arg?Leu?Pro?Phe?Thr?Ala?Lys?Thr?Pro?Glu?Thr?Tyr

130?????????????????135?????????????????140Phe?Leu?Arg?Gly?Ala?Asp?Cys?Trp?Gly?Trp?Ala?Thr?Trp?Lys?Arg?Gly145?????????????????150?????????????????155?????????????????160Trp?Asp?Leu?Phe?Glu?Val?Asp?Gly?Gln?Lys?Leu?Leu?Asp?Lys?Leu?Ile

165?????????????????170?????????????????175Ser?Glu?Lys?Leu?Ile?Tyr?Gln?Phe?Asp?Met?Gln?Gly?Ser?Tyr?Pro?Tyr

180?????????????????185?????????????????190Thr?Gln?Met?Leu?Lys?Asp?Gln?Ile?Ala?Gly?Lys?Asn?Asp?Ser?Trp?Ala

195?????????????????200?????????????????205Ile?Arg?Trp?Tyr?Ala?Ser?Thr?Phe?Leu?Gln?Asn?Lys?Leu?Thr?Leu?Tyr

210?????????????????215?????????????????220Pro?Ala?Arg?Ser?Leu?Val?Phe?Asn?Ile?Gly?Leu?Asp?Ala?Ser?Gly?Thr225?????????????????230?????????????????235?????????????????240His?Cys?Glu?Val?Thr?Asn?Glu?Tyr?Asp?Val?Asp?Leu?Ser?Ser?Ile?Pro

245?????????????????250?????????????????255Ile?Arg?Ile?Glu?Thr?Ile?Gln?Ile?Glu?Glu?Asn?Val?Tyr?Ile?Arg?Asn

260?????????????????265?????????????????270Leu?Tyr?Arg?Asp?Tyr?Phe?Arg?Lys?Leu?Ser?Gln?Ser?Asp?Leu?Arg?Thr

275?????????????????280?????????????????285Arg?Ile?Phe?Gly?Arg?Leu?Lys?Gln?Leu?Leu?Glu?Arg?Ile?Lys?Thr?Arg

290?????????????????295?????????????????300Ile?Trp?Pro?Asp?Lys?Ser?Asn?Leu?Leu305?????????????????310

Claims (10)

1. isolating Leptospira hemolysin polypeptide, it is characterized in that it comprises: have the polypeptide or its conservative property variation polypeptide or its active fragments or its reactive derivative that are selected from SEQID NO:2,4,6,8,10,12,14,16 aminoacid sequence.

2. polypeptide as claimed in claim 1 is characterized in that, this polypeptide is the polypeptide with SEQ ID NO:2, aminoacid sequence of 4,6,8,10,12,14,16.

3. isolating polynucleotide is characterized in that, it comprises a nucleotide sequence, and this nucleotide sequence is shown at least 70% homogeny with a kind of nucleotides sequence that 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:2,4,6,8,10,12,14,16.

5. polynucleotide as claimed in claim 3 is characterized in that, the sequence of these polynucleotide is selected from down group:

(a) has SEQ ID NO:1,3,5,7,9,11,13,15.

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

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

8. the preparation method of a Leptospira hemolysin polypeptide is characterized in that, this method comprises:

(a) being fit to express under the condition of Leptospira hemolysin, cultivate the described host cell of claim 7;

(b) from culture, isolate the Leptospira hemolysin polypeptide.

9. energy and the described Leptospira hemolysin specificity of claim 1 bonded antibody.

10. one kind is detected leptospiral test kit, it is characterized in that, it contain the specific detection Leptospira hemolysin primer and/or with Leptospira hemolysin specificity bonded antibody.

Images