CN1222937A - Pathogenic escherichia coli associated protein - Google Patents

Abstract

The present invention provides the EspA polypeptide, which is secreted by pathogenic E. coli, such as the enteropathogenic (EPEC) and enterohemorrhagic (EHEC) E. coli. Diagnosis of disease caused by such pathogenic E. coli can be performed by standard techniques, such as those based upon the use of antibodies which bind to EspA to detect the protein, as well as those based on the use of nucleic acid probes for detection of nucleic acids encoding EspA protein. The invention also provides isolated nucleic acid sequences encoding EspA, EspA polypeptide, EspA peptides, a method for producing recombinant EspA, antibodies which bind to EspA, and a kit for the detection of EspA-producing E. coli. The invention also provides a method of immunizing a host with EspA to induce a protective immune response to EspA.

Description

Pathogenic escherichia coli associated protein

The relevant application of cross-reference

The application requires the right of priority of the U.S. Provisional Patent Application 60/015,999 of submission on April 23rd, 1996.

Subsidize the statement of research about federal government

The present invention carries out under the support of Public Health Service (public health portion) appropriation AI 32074, is to obtain this appropriation from National Institutes of Health (NIH).United States Government has some right to the present invention.

Invention field

Put it briefly, the invention relates to the virulence of invasive organism, more particularly, be about with cause the relevant virulence factor of the sick bacterium of enteron aisle.

Background of invention

Microbiotic has used for many years, has successfully treated the various bacteria infection.But, the resistance of bacterial antibiotic, in the past several years in become serious day by day problem.Many pathogenic bacterias have resistance to several antibiosis now, and in some cases, the disease that they cause no longer can be used conventional antibiotic therapy.Although antibiosis have in the past successful history,, in the past two during the decade, even have, also only developed a few new microbiotic.Also existing medicine is introduced new change, still, usually at short notice, these compounds have been produced resistance again.

A lot of researchs show that if the gene of coding virulence factor is undergone mutation, the organism that contains this gene then no longer includes pathogenic.In addition, if the host is prevented the inoculation of virulence factor, then usually can stop disease to take place.But, not there are some researches show as yet, the specificity of virulence factor is suppressed to reduce the generation of disease.

Having produced the mechanism of action that infects in contratoxin, adhesion, intrusion and the cell studies.But every kind of virulence factor adopts different mechanism, and it is impossible that this just makes development wide spectrum inhibitor become.Each is considered to may be the conservative factor of treatment target, all is a two regulation system of forming.But this system is not specific to virulence factor, and is used in several bacterium house keeper system.These systems in eukaryotic cell system, have been found, if use inhibitor, with the danger that increases host toxicity in addition.In order to develop the ideal anti-infection agent, the bacterium mechanism of causing a disease of microbiotic influence should be general to many pathogenic agent, is specific to mechanism of causing a disease, and does not exist in host cell.Such system of recent findings is a bacterium III type excretory system.

The special mechanism of Gram-negative bacteria utilization discharges molecule and crosses over their duplicature and outer protoplasma, and this is one virulence factor transferred to the key process of bacterium surface, bacterium surface they could with host's interaction between component.The Gram-negative bacteria secretion has been divided into four kinds of main approach.The firstth, I type secretion is the secretion that small molecules toxin family and intestinal bacteria prototype hemolysin adopt.The secondth, II type excretory system is most of Gram-negative bacterias comprise the multiple molecule of some virulence factor in order to discharge a main output pathway; It has identical mechanism with mammiferous drug resistance.The 3rd is IV type excretory system, and it is coded in the secretory product, as the part cutting self of mechanism of secretion; Neisseria (Neisseria) IgA proteolytic enzyme is the prototype of this system.The 4th is III type approach, is the Secretory Pathway of recent findings.

It is the excretory system of secreting virulence protein Y OPs as Yersinia (Yersinia) that III type excretory system is described at first, and it is critical to the virulence of Yersinia.Thereafter in the several plant pathogenic agent, find the homology excretory system, comprised pseudomonas syringae, Pseudomonas solanacearum and xanthomonas campestris.These phytopathogens utilize this secretion path secretion to causing the needed virulence factor of plant disease (harpins and other).Though excretory system is similar, harpins and YOPs (being secreted virulence factor) are not homeopeptides.More nearest in some other pathogenic bacteria, found the necessary other several III type excretory systems of virulence.These systems comprise the system that infects that salmonella (Salmonalla) and Shigella (Shigella) are used to enter cell and cause disease.In salmonella, also found another III type excretory system that disease is played a decisive role, though the secretory product and the virulence mechanism in this path are not determined as yet.Pseudomonas aeruginosa (Pseudomonasaeruginosa) has secretion extracellular enzyme S, a kind of protein virulence factor, necessary III type excretory system.

Causing enteropathy intestinal bacteria (EPEC) is the major cause of infantile diarrhea, is first intestinal bacteria (intestinal bacteria) that discovery causes gastro-enteritis.EnteropathogenicEscherichia coli activates the epithelial signal transduction path of host, and causes the cytoskeleton rearrangement, forms end seat shape simultaneously and adheres to/surface damage venereal disease kitchen range.

Available Three-stage Model is described the pathogenesis of enteropathogenicEscherichia coli.Beginning is that the location adheres to epithelial cell, by the mediation of TV type bacterium cilium, is to activate host's epithelial cell signal transduction path subsequently, and closely attached on the host cell.The two last steps are collectively referred to as adheres to and surface damage.Signal conduction in host's epithelial cell comprises, activates the activity of host cell Tyrosylprotein kinase, causes the tyrosine phosphorylation of host's membranin Hp90 of 90 kilodaltons, and flows out intracellular phosphoinositide (IP3) and calcium.After the sort signal conduction, bacterium closely adheres to epithelial surface, has damaged the epithelial microvillus of host simultaneously, and has piled up cytoskeletal protein below bacterium.

Summary of the invention

The present invention is based in the pathogenic bacterium a kind of and virulence proteins associated matter of finding in the enteropathogenicEscherichia coli for example.

To the dna sequence analysis of intestinal cell surface damage locus between eaeA and the espB (Locus of EnterocyteEffacemant), found a gene (espA) with 25 kilodalton enteropathogenicEscherichia coli secretory protein N-terminal sequences match.The mutant that has insertion sequence in espA is not secreted this protein, can not activate epithelial cell signal conduction or cause the cytoskeleton rearrangement.But these functions can remedy by a cloning wild-type espA gene.

To two enteropathogenicEscherichia coli gene espA and the espB of coding secretion property virulence factor EspA and EspB respectively, clone and check order.Shown that these protein are relevant with the startup of host's epithelial cell signal conducting path and infection processs.Because EspA is a kind of secreted protein,, it is used for the fusion rotein that is connected with required polypeptide so being ideally suited.

III type secretion path is a potential inhibitor ideal target, because it is the specific conservative property of a virulence factor path of finding in bacterium.The invention provides a kind of method, be used to identify the inhibitor of III type excretory system.Target of the present invention is to point to the new Ceftriaxone therapy of development.Be different from other microbiotic, by the not kill pathogenic bacteria or the growth of inhibition pathogenic bacteria of compound of method discriminating of the present invention; But this compound will stop the virulence factor of secretion to causing that disease plays a decisive role.Because III type excretory system is to show the primary virulence mechanism of conservative property largely in multiple pathogenic bacteria, so some compound of differentiating by means of the inventive method will be the broad spectrum therapeutical agent.Its advantage is possible find a kind of some people that can be used for treating, the novel treatment of animal and plant disease.

Brief Description Of Drawings

Fig. 1 shows nucleotide sequence and the deduced amino acid (being called as SEQID NO:1 and SEQ ID NO:2 respectively at this) of espA.Ledger line below the possible ribosome binding site is indicated.The Nucleotide that is included in the disappearance flank that being located among primer Donne-99 and the Donne-100 make up among the pLCL121 adds shade and indicates.

Fig. 2 shows the structure of nonpolar sudden change among the espA.Primer Donne-90 and its anti-primer are used to increase and contain the fragment of gene 5 ' part, are entered pCRscript by the clone then, constitute pLCL119.Primer Donne-100 and this universal primer are used to increase and contain the fragment of 3 ' part, and this fragment is entered pCR script by the clone, constitute pLCL120.New Nru I site is mixed among Donne-99 and the Donne-100,, constitute pLCL121, the disappearance of a 150bp is arranged in its espA gene so that the Nrn of pLCL120 I-Sal I fragment can be entered pLCL119 by the clone.To enter the Nru I site of pLCL 121 from the Sma I fragment cloning of pUC 18K, constitute pLCL 122, contain the aphA-3 kalamycin resistance gene among this pUC 18K.This insertion causes the aphA-3 gene transcription to merge and the translation of espA gene 3 ' end is merged, and preserves the espA frame simultaneously.Indicated by ledger line below the ribosome bind site.

Fig. 3 represents the gene mapping of plasmid, contains RDEC-1 (A) and enteropathogenicEscherichia coli (EPEC) espA (B), espD and espB gene in the plasmid.The position (A) that terminator codon is inserted is done in the arrow indication in espA and espB, and is fabricated the phase shift mutation (B) that enters Bgl II site among the espD.Among the espB disappearance of 250 base pairs by with // mark.Solid thick line and hollow thick line are represented the open reading frame of open reading frame and prediction.Restriction enzyme is indicated with following: Bam, BamH I; Ec, the EcoR I; Bg, the Bgl II; Xb, the Xba I; Sa, the Sal I.

Fig. 4 shows the nucleotide sequence (A) (being called SEQ IDNO:3 and SEQ ID NO:4 at this) of RDEC-1 espA, and the nucleotide sequence of espB (B) (being called SEQ IDNO:5 and SEQ ID NO:6 at this).Asterisk indication terminator codon.Indicated by ledger line below the possible ribosome bind site.The EspA of prediction and EspB aminoacid sequence are at C) in be arranged in a straight line (SEQ IDNO:7-10).The shadow region is represented identical.Deposited in the Nucleotide and the aminoacid sequence of EMBL GenBank (European Molecular Bioglogy Laboratory's nucleotide sequence), and their retrieval number is as follows: RDEC-1 espA (U80908), RDEC-1 espB (U80796), enteropathogenicEscherichia coli: the espA of E2348/69 strain (Z54352), enteropathogenicEscherichia coli: the espB of E2348/69 strain (Z21555), enteropathogenicEscherichia coli: the espD of E2348/69 strain (Y09228), cause the intestinal bleeding intestinal bacteria: the espB (X96953) of EDL933 serotype 0157 strain causes the intestinal bleeding intestinal bacteria: the espB (X99670) of 413/89-1 serotype 026 strain.

Describe in detail

The invention provides the polypeptide of a kind of EspA of being called as, it is by enteropathogenic E．Coli, as causes Sick Escherichia coli (EPEC) of enteron aisle and cause that intestinal bleeding Escherichia coli (EHEC) secrete. To by But this enteropathogenic E．Coli causes the diagnosis of disease and can be undertaken by the technology of standard, for example those Based on the technology of using antibody, this antibody capable is combined with EspA and this protein is detected, and Those are based on the technology of using nucleic acid probe, for detection of the nucleic acid of coding EspA polypeptide. The present invention also The nucleotide sequence of the separation of coding EspA polypeptide is provided, and the EspA peptide is for generation of restructuring EspA's The recombination to construct method, the antibody that can be combined with EspA, and for detection of the large intestine bar that produces EspA The kit of bacterium. It is a kind of with EspA immunity host, in order to bring out EspA pre-that the present invention also provides The anti-immunoreactive method of property.

The preferred embodiments of the invention are in the following drawings and concrete narration is described in detail in detail. In case ... details.

Title " EspA " (EPEC Secreted (or signaling) Protein A, EPEC as used herein Secretion (signal) albumin A) refer to a peptide species, it is big from causing the enteron aisle disease or causing enterorrhagia The secreted protein of enterobacteria is measured the molecule with about 25 kilodaltons by SDS-PAGE Amount. EspA is the protein of enteropathogenicEscherichia coli secretion, and it conducts activating the epithelial cell signal, Close contact, and form and to adhere to and the surface damage focus, the process that activates with disease association is essential. Epithelial cell is exemplary cells.

Noun " polypeptide " as used herein, comprise its any naturally occurring allelic variation body and Artificial recombination bodily form formula. The EspA polypeptide had both comprised naturally occurring form as used herein, also comprised Recombinant form, i.e. the non-natural existence form of certain protein and peptide, it and naturally occurring EspA Peptide is fully similar, makes it to have to cause pathogenic similar functions. The example of this peptide species comprises coming From enteropathogenicEscherichia coli with cause the colibacillary EspA polypeptide of intestinal bleeding, but be not limited to this A bit. Proteins and peptides also comprises its derivative, homologue and mimicry peptide. On the other hand, EspA peptide Can carry out chemical synthesis with synthetic technology well known to those skilled in the art, preferably use automatically Peptide synthesizer engages on the resin at polyethylene glycol-styrene (PEGPS) with NaFmoc amino acid Synthesize. For example can utilize suitable joint such as peptide amide joint (PAL), form the carbon oxanamide (Carboxamide) end group.

Noun " pure basically " is used herein to describes a kind of molecule such as polypeptide (for example EspA polypeptide, perhaps its segment), wherein is substantially devoid of other protein, lipid, carbohydrate, nucleic acid and natural and other biological substance of its bonded.For example, a kind of pure basically molecule such as polypeptide can be that at least 60% dry weight is a desired molecule.The protein purification method purifying EspA polypeptide of those skilled in the art's available standards, measure the purity of this polypeptide with the method for standard, these methods comprise for example polyacrylamide gel electrophoresis (as SDS-PAGE), column chromatography (as high performance liquid chromatography (HPLC) (HPLC)), and amino terminal amino acid sequential analysis.

EspA polypeptide of the present invention can have an EspA aminoacid sequence from people or rabbit enteropathogenicEscherichia coli, for example aminoacid sequence of Fig. 1 or Fig. 4.The EspA polypeptide as the polypeptide that shows in Fig. 1 and 4, can have about 25kD molecular weight it is identified when measure with SDS-PAGE.

Have and the EspA peptide sequence EspA polypeptide among Fig. 1 and 4 for example, substantially the same polypeptide of sequence is also included among the present invention." being identical basically " aminoacid sequence, it is the sequence only be different from standard sequence by substituting of conserved amino acid, for example replace another same amino acid (for example with a hydrophobic amino acid such as Isoleucine with an amino acid, Xie Ansuan, leucine or methionine(Met), replace another hydrophobic amino acid, perhaps replace another polare Aminosaeren with a polare Aminosaeren, for example replace Methionin with arginine, replace aspartic acid with L-glutamic acid, or with glutamine replacement l-asparagine), or by one or several non-conservative substituting, disappearance, or insert and be different from the sequence of standard sequence, as long as this polypeptide also keeps at least a EspA activity specific or an EspA specificity epitope.For example, can lack one or several amino acid in the EspA polypeptide, cause polypeptide structure to change, and change its biological activity not obviously.For example the amino acid to optional N-terminal of EspA biological activity or C-terminal can be removed.This modification can cause producing less active EspA polypeptide.

Comprise that other EspA polypeptide in the present invention are, have amino acid sequence of polypeptide,, the polypeptide of at least 50% same acid sequence is arranged as the aminoacid sequence of EspA in Fig. 1 and 4 with EspA.In the mensuration of amino acid sequence homology, length relatively can be for example at least 15 amino acid, perhaps at least 20,25 or 35 amino acid.Can be with the sequence analysis software of standard (GeneticComputer Group for example, University of Wisconsin Biotechnology Center, 1710University Avenue, Madison, the sequence analysis software bag of WI 53705; Also can be referring to Ausubel etc., the same).

The present invention also comprises the EspA polypeptide fragment that keeps at least a EspA activity specific or epi-position.For example, contain 8-10 amino acid whose EspA polypeptide fragment at least, can in producing the EspA specific antibody, be used as immunogen.This fragment can contain, for example, and the aminoacid sequence of guarding among the EspA.Except being used as peptide based immunogens, above-mentioned EspA fragment can also be used for immunoassay, as ELISA, and the existence of EspA specific antibody in the test sample.

Method with any standard can obtain EspA polypeptide of the present invention.For example, the EspA polypeptide can produce (face as follows) in the recombinant expression system of standard, by chemosynthesis (this method may only be confined to the little peptide fragment of EspA), perhaps obtained (referring to for example Ausubel by purifying the tissue of natural expression therein from them, et al, the same).

The isolated nucleic acid molecule that the present invention also provides the above-mentioned EspA polypeptide of encoding with and fragment.For example, coding is included among the present invention as the nucleic acid of EspAs in Fig. 1 and 4.These nucleic acid can contain naturally occurring nucleotide sequence (seeing Fig. 1 and 4), perhaps can be the degeneracys owing to genetic code, and are different from the natural acid of the EspAs that encodes but the nucleotide sequence of the same amino acid of encoding.Nucleic acid of the present invention can contain DNA or RNA Nucleotide, the combination of the two or its modified forms.

Mean not with 5 with " isolating nucleic acid " ' and nucleic acid such as the DNA or the RNA molecule of the direct adjacency of 3 ' flanking sequence, when be present in its source organism naturally occurring genome in the time, under the normal circumstances be and the direct adjacency of this flanking sequence, so this noun is to describe the nucleic acid that for example is integrated in carrier such as plasmid or the virus vector; Be integrated into the nucleic acid of (or in the homocellular genome, still being different from its site that exists naturally) in the heterogenous cell genome; And the nucleic acid that exists as independent molecule, as the dna fragmentation that produces by pcr amplification or digestion with restriction enzyme, or the RNA molecule that produces by in-vitro transcription.This noun also is used to describe the recombinant nucleic acid of a heterozygous genes part that constitutes the auxiliary peptide sequence of coding, and this auxiliary peptide sequence can be used for for example producing fusion rotein.

Nucleic acid molecule of the present invention can be used as template in the standard method that produces EspA gene product (as EspA RNA and EspA polypeptide, seeing below).In addition, the nucleic acid molecule and relevant nucleic acid of coding EspA polypeptide (and fragment), can in method, use based on its hybridization characteristic, should for example comprise that (1) this nucleic acid contained and the nucleic acid complementary of coding EspA polypeptide by relevant nucleic acid, or the sequence that can hybridize with it, or the fragment of this nucleic acid (for example contains at least 12,15,20, or the fragment of 25 Nucleotide); And (2) this nucleic acid contain can with the sequence of following sequence hybridization, this sequence is a complementary with the nucleic acid of coding EspA polypeptide, or the fragment of this nucleic acid (for example contain at least 12,15,20, or the fragment of 25 Nucleotide).For example will be described in further detail below, some nucleic acid molecule can be used to following method like this: the PCR method that is used for synthetic EspA nucleic acid, be used for the method that there is EspA nucleic acid in test sample, be used for the examination method and the methods of treatment of the new EspA family member of identifier number nucleic acid.

The present invention also comprises the method that is used to differentiate certain nucleic acid molecule, and this nucleic acid molecule is except the EspA shown in code pattern 1 and 4, and other EspA peptide family member also encodes.In these methods, be with EspA-specific probe such as EspA-specific dna probe, to containing the sample of coding EspA polypeptide-nucleic acid, for example the cDNA nucleic acid library is carried out examination.The EspA-specific dna probe be specifically with the nucleic acid of coding EspA polypeptide, perhaps with the nucleic acid molecule of its complementary sequence hybridization (for example contain DNA or RNA nucleic acid molecule, or its combination or modified forms).Noun " EspA-specific probe ", according to method of the present invention refer to can with the nucleic acid of coding EspA polypeptide, perhaps its complementary sequence bonded probe, the nucleic acid of this binding ratio and other polypeptide of coding, but perhaps its complementary sequence in conjunction with having bigger detection level.Therefore noun " EspA specific probe " comprises nucleic acid (or its complementary sequence) the bonded probe of coding EspA polypeptide together.

The present invention helps to produce easily the EspA-specific dna probe.Can design the method that obtains this probe in proper order according to the aminoacid sequence shown in Fig. 1-3.Can use the method (referring to for example Ausubel, et al, the same) of any standard to produce this probe, it can contain at least 12, perhaps at least 15,25,35,50,100, or 150 Nucleotide.For example, preferably this probe can produce with the pcr amplification method.In this method, need design and the corresponding primer of EspA-conserved sequence, this conserved sequence can include the EspA-specific amino acid, and the PCR product of generation can be used as the probe in examination nucleic acid library such as cDNA library.Usually after this process,, can identify the nucleotide sequence of coding EspA according to analysis to EspA sequence in Fig. 1 and 4.

Known to the professional of this area, the PCR primer typically should be designed to contain at least 15 Nucleotide, for example 15-30 Nucleotide.To set forth the design to the EspA-Auele Specific Primer below, this primer contains 21 Nucleotide of 7 the amino acid EspA peptides of encoding.Preferred situation is, the EspA-conservative amino acid of all encoding of the most or whole Nucleotide in this probe comprises the EspA-specific amino acid.For example, the peptide of spendable its contained sequence encoding of primer should contain the 40%EspA-conservative amino acid at least.A kind of like this primer that contains 21 Nucleotide can comprise the sequence of 3 the EspA-conservative amino acid of encoding at least.Like this, this primer can contain at least 1 of coding, for example the sequence of 7 EspA specific amino acid.In case the EspA-specific amino acid, as chosen at the template of its design primer sequence, just can application examples such as the chemical process of standard synthesize this primer.As mentioned above, because the degeneracy of genetic code, this primer should be designed to comprise the sequence of suitable degeneracy, and those skilled in the art can easily measure like this.

The polynucleotide of coding EspA polypeptide represented in noun " espA " as used herein.These polynucleotide comprise DNA, cDNA and the RNA sequence of the EspA that encodes.Also comprise all polynucleotide of all or part of EspA that encodes at this.This polynucleotide comprise naturally occurring, synthetic and by the polynucleotide of particular procedure.For example, can make the espA polynucleotide suffer site-directed mutagenesis.This epsA polynucleotide sequence also comprises its antisense sequences.The nucleotide sequence of all degeneracys all comprises in the present invention, as long as changing function does not take place the coded EspA peptide ammino acid sequence of nucleotide sequence thus.

The present invention also comprise can with the nucleic acid molecule of multi-nucleotide hybrid of the present invention.Noun " nucleic acid " comprises RNA and strand and double-stranded DNA and cDNA as used herein.The polynucleotide of coding EspA comprise nucleotide sequence in Fig. 1 and 4, and sequence complementary nucleotide sequence therewith.Complementary sequence may comprise antisense nucleotide.When this sequence is RNA, the deoxynucleotide among Fig. 1 and 4, A, G, C and T will be respectively by Nucleotide A, G, C and U replace.The fragment of above-mentioned nucleotide sequence is also included among the present invention, and its length is at least 15 bases, this be enough to make this fragment can be optionally under physiological condition with code pattern 1 or 4 in protein DNA hybridization.

In nucleic acid hybridization reaction, the condition that is used to reach specific stringency level will have nothing in common with each other, and this depends on the character for the treatment of hybrid nucleic acid.For example, when selecting hybridization conditions, the length in nucleic acid hybridization district, complementary degree, the formation of nucleotide sequence (for example GC is to the content of AT), and the type of nucleic acid (for example RNA or DNA) all may be taken into account.Further consider be one of them nucleic acid whether by immobilization on filter membrane for example.

Be the example that improves the stringency condition gradually below: 2 * SSC/0.1%SDS at room temperature (hybridization conditions) roughly, roughly 0.2 * SSC/0.1%SDS (low stringency condition) at room temperature; 0.2 * SSC/0.1%SDS under about 42 ℃ (moderate stringency condition); In 0.1 about 68 ℃ * SSC (height stringency condition).Rinsing can be only carried out with a kind of condition wherein, for example uses height stringency condition, perhaps available wherein every kind of condition, for example by above-named order, every kind condition 10-15 minute, repeat cited any one and go on foot or all steps.But as described above, optimal conditions will have nothing in common with each other, and this depends on the specific cross reaction that is comprised, and can determine by rule of thumb.

Can obtain dna sequence dna of the present invention by several method.For example, this DNA can isolate with hybridization technique well known in the art.These technology comprise, but be not limited to (1) and make library and probe hybridization, so that detection homologous nucleotide sequence, (2) use can with required dna sequence dna annealed primer, DNA is carried out polymerase chain reaction (PCR), and (3) carry out antibody screening to expression library, so that detect the cloning dna fragmentation with same constitutional features.

The screening procedure that depends on nucleic acid hybridization makes it and might isolate any gene order from any organism, as long as there is suitable probe to utilize.The corresponding oligonucleotide probe of partial sequence with the coding desired protein can perhaps be produced by the cracking native sequences by chemosynthesis.Chemosynthesis need be known the segmental aminoacid sequence of short oligopeptides.This protein DNA sequence of encoding can be derived according to genetic code, but must consider the degeneracy of password.When this sequence is a degeneracy, just might mix addition reaction.This comprises the heterogeneity mixture of sex change distrand DNA.For this screening, preferably the distrand DNA of single-stranded dna or sex change is hybridized.When combining application, even rare expression product also can be cloned with polymerase chain reaction technique.

The invention provides the nucleotide sequence of coding EspA polypeptide, contain their carrier and host cell, and expression method.Isolate after the EspA peptide, just can isolate the nucleic acid of this peptide of coding by means of method well known in the art.These isolating nucleic acid connections can be entered carrier, and the host cell that importing is fit to is expressed.The connection of nucleic acid and be well known in the art (referring to Sambrook etc. in the method for cell inner expression, Molecular Cloning:A Laboratory Manual (molecular cloning: laboratory manual), Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989, be hereby incorporated by).

Noun " espB " and " eaeA " as used herein refer to the gene that is different from espA of coding enteropathogenicEscherichia coli secretory protein.Noun " EspB " and " EaeA " as used herein refer to respectively the protein by espB and eaeA genes encoding.

The invention provides the carrier of the polynucleotide that include coding EspA polypeptide.For example, the plasmid (pMSD2) that has a complete espA can make it recover secretion EspA protein in espA defective bacterial strain." carrier " comprises plasmid well known to those skilled in the art as used herein, DNA and rna virus vector, and baculovirus vector is used for zymic carrier and other carrier.The carrier of several types can be buied, and can be used for realizing the present invention.Be used to realize that the example of carrier of the present invention comprises that those extensively change the carrier as low copy carrier pMW118, just selecting suicide vector pCVD442, and the pBluescript II SK (+) that can buy (Stragene, La Jolle, CA).

When this carrier was plasmid, as is known to the person skilled in the art, it included multiple composition usually, comprises promotor, signal sequence, Phenotypic Selection gene, replication orgin, and other necessary composition.The promotor that is used for the prokaryotic organism carrier the most at large comprises the LacZ promoter systems, alkaline phosphatase phoA promotor, phage PL promotor (a kind of temperature sensitive promotor), tac promotor (a heterozygosis trp-lac promotor of regulating) by the lac repressor, trp promoter and phage t7 promotor.For example, the low copy carrier pMW118 under the control of LacZ promotor.

Signal sequence generally is found with 5 ' end and directly is connected with the nucleic acid of this peptide of coding, therefore will be transcribed the aminoterminal at fusion rotein.

Typical Phenotypic Selection gene is a kind of so proteinic genes of those codings, and this albumen mass-energy makes host cell have antibiotics resistance.For example, ampicillin resistance gene (amp) and tetracycline resistance gene (tet) are easy to be used for this purpose.Another example is, in order to select carrier on the kantlex flat board, the aphA-3 box clone of coding kalamycin resistance gene (kan) can be entered a carrier zone, and the polynucleotide of coding EspA polypeptide are contained in this zone.

With standardization recombinant DNA program well-known to those having ordinary skill in the art, make up the carrier that is fit to, make it to contain the polynucleotide of coding EspA polypeptide.Will be combined to form carrier, the separation polynucleotide of coding EspA polypeptide disconnect, and link together by specific order and orientation and to produce required carrier.

The present invention also provides a kind of host cell, and it contains the carrier of the polynucleotide that carry coding EspA polypeptide.In order to improve the output of expressing EspA, polynucleotide of the present invention can be used to produce conversion or cells transfected.By means of standard method well known to those skilled in the art, can from cell transformed, isolate EspA.For example can use the immunoaffinity purification effect and separate this protein.

Enter suitable host cell by DNA is shifted, the dna sequence dna of coding EspA can be expressed external." host cell " is that carrier can be bred therein, the effable cell of the DNA of carrier.This noun also comprises any filial generation of target host cell.Should be realized that all filial generations all may be inequality with parental cell, because may undergo mutation in reproduction process.But, when using noun " host cell ", also comprise this filial generation.Stable transfer means that external DNA is continued to be retained in the host cell, and the method for this stable transfer has been set up in this area.

According to the present invention, the EspA polynucleotide sequence can be inserted into in the recombinant expression vector.Noun " recombinant expression vector " refers to by inserting or mix the plasmid well known in the art that the EspA gene order is handled, virus or other vehicle.The promoter sequence that this expression vector contains can promote host cell to transcribe the gene order of insertion effectively.This expression vector contains replication orgin usually, promotor, and the specific gene that makes it to carry out cell transformed Phenotypic Selection.

The polynucleotide sequence of coding EspA can be expressed in prokaryotic organism or eukaryote.The host can be microorganism, yeast, insect and Mammals.Expressing the method for the dna sequence dna that has eukaryote sequence or viral row preface in prokaryotic organism, is well known in the art.Biological functionality virus and the plasmid DNA carrier that can express in the host and duplicate also be well known in the art.These carriers are used to mix dna sequence dna of the present invention.

Can transform host cell with recombinant DNA by means of routine techniques well known to those skilled in the art.When the host is prokaryotic organism, as intestinal bacteria, the competent cell that can absorb DNA can prepare from the cell of results exponential phase of growth, and available subsequently program well known in the art is passed through CaCl ₂Method is handled.Perhaps, also can use MgCl ₂Or RbCl.If desired, transform also and can after forming the host cell protoplastis, carry out.Another example is to use three close keying actions, and carrier heredity is imported intestinal bacteria, and particularly enteropathogenicEscherichia coli or rabbit cause the enteropathy intestinal bacteria.By being grown on the microbiotic cell transformed is selected, microbiotic is tsiklomitsin (tet) or penbritin (amp) normally, owing to have tet or amp resistant gene on the carrier, transformant have resistance to antibiosis.In specific embodiment, be to select according to the resistance of kantlex and sucrose being carried out cell.

When the host is eukaryote, can use as coprecipitation of calcium phosphate, conventional mechanical treatment technology such as microinjection, electroporation inserts and is packaged in the intravital plasmid of lipid, perhaps inserts the DNA infection protocol of virus vector etc.Also can invent the dna sequence dna of EspA with code book, and coding can be selected second kind of external dna molecular such as simple born of the same parents' rash thymidine kinase gene of phenotype, transformed eukaryotic karyocyte.Another kind method is to use Eukaryotic virus vector, as simian virus 40 (SV40) or bovine papilloma virus, instantaneous infection or transformed eukaryotic karyocyte, and express this protein (referring to for example, Eucaryotic ViralVectors (Eukaryotic virus vector), Cold Spring Harbor Laboratory, Gluzman ed, 1982).

Can comprise that preparative chromatography separates with the immunity of using monoclonal antibody or polyclonal antibody by means of the method for routine to the polypeptide of the present invention of microbial expression or its fragment is separated and purifying.

May intestinal bacteria be arranged as the prokaryotic organism of host cell; The JM101 strain, e. coli k12 294 strains (ATCC number 31,446), intestinal bacteria W3110 strain (ATCC number 27,325), intestinal bacteria X1776 (ATCC number 31,537), intestinal bacteria XL-1 Blue (Stratagene) and intestinal bacteria B; But, can also use much other coli strains, as HB101, NM522, NM538, NM539 and procaryotic other many kinds and genus.Except above-named coli strain, bacillus such as Bacillus subtillis (Bacillus subtillis), other enterobacteriaceae lactobacteriaceae such as Salmonella typhimurium (Salmonella typhimunium) or serratia marcescens (Serratiamarcesans), and various pseudomonas all can be used as host cell.In a specific embodiment, prokaryotic host cell is an enteropathogenicEscherichia coli.In another specific embodiment, prokaryotic host cell is the rabbit enteropathogenicEscherichia coli.

The eukaryote that can be used as host cell has yeast strain such as PS23-6A, W301-18A, LL20, D234-3, INVSC1, INVSC2, YJJ337.Promoter sequence and enhancer sequence also are useful as ga11 and pEFT-1.Vra-4 also can provide suitable enhancer sequence.Sequence as functional replication orgin comprises ars1 and 2 μ circular plasmids.

Gram-negative bacteria is one group of multifarious microorganism, comprise Spirochaetes (Spirochaetes) as treponema and Borrelia, gram negative bacillus such as false pseudomonas bacillus section, legion Cordycepps, enterobacteriaceae, vibrionaceae, Pasteurellaceae, Gram-negative coccus such as eisseriaceae, the anaerobism Bacteroides, and other Gram-negative bacteria such as Rickettsiae, chlamydozoan, mycoplasma.

Gram negative bacillus is very important to clinical medicine.They comprise (1) enterobacteriaceae, this is a Cordycepps of being made up of many main cause of disease Pseudomonas, (2) vibrios, Campylobacter and Helicobacterium, (3) opportunistic diseases microorganism (pseudomonas for example, Flavobacterium and some other bacterium) and (4) influenzae and Bordetella.Gram negative bacillus is at abdominal viscera, in peritonaeum and the urinary tract infection, and at respiratory tract, burn or the skin of wound and Secondary cases invasion bacterium that host resistance weakens the position in the major microorganisms found.At present, they are common causes of life-threatening septicemia.The example of pathogenic gram negative bacillus has intestinal bacteria (diarrhoea, urinary tract infection, neonatal meningitis), Shigella bacterium (dysentery), salmonella typhi (typhoid), Salmonella typhimurium (gastro-enteritis), colitis Yersinia (colitis), Yersinia pestis (black plague), vibrio cholerae (cholera), the crooked bacterium (colitis) of jejunitis, jejunitis Helicobacter pylori (gastritis, stomach ulcer), (opportunistic infection comprises burn to the false careless born of the same parents bacterium of green pus, urethra, respiratory tract, wound infection, and primary cutaneous, eye, otic infections), hemophilus influenzae (children's meningitis, epiglottitis, otitis media, sinusitis and bronchitis), and Bordetella pertussis (Whooping cough).Vibrios is active Gram-negative rod Pseudomonas (vibrionaceae).Vibrio cholerae causes people's cholera, and other kind vibrios causes Animal diseases.Escherichia coli host is in the intestines of people and warm-blooded animal, and they are parts of intestines endocommensal flora, but has the escherichia species that causes the humans and animals intestinal tract disease.They comprise in the intestines assembles intestinal bacteria (enteroaggregative intestinal bacteria) (EaggEC), enterorrhagia intestinal bacteria (EHEC), intestines are invaded intestinal bacteria (enteroinvasive intestinal bacteria) (EIEC), enteropathogenicEscherichia coli (EPEC) and enterotoxigenic escherichia coli (ETEC).Cause urinary tract infection intestinal bacteria (Uropathogenic intestinal bacteria) and (UPEC) cause urinary tract infection.Also have the scorching intestinal bacteria (NMEC) of newborn infant's after birth.Except animal being caused some infects the similarly infection with the mankind, also there are some special Animal diseases, comprising: calf septicemia, bovine mastitis, bowel oedema disease, and poultry alveolar disease.

The kind of Neisseria (Neisseria) comprises the grey matter Neisseria gonorrhoeae, Diplococcus gonorrhoeae, Diplococcus gonorrhoeae Kochii subspecies, the lactose Neisseria gonorrhoeae, Neisseria meningitidis, the polysaccharide Neisseria gonorrhoeae, the mucous membrane Neisseria gonorrhoeae, dry Neisseria gonorrhoeae, faint yellow Neisseria gonorrhoeae, the non-sugar of golden yellow Neisseria gonorrhoeae is separated kind, the cavy Neisseria gonorrhoeae, rabbit Neisseria gonorrhoeae and sheep Neisseria gonorrhoeae, some taxonomist thinks that mucositis mora Salmonella (branhamella) also belongs to Neisseria.Other relevant bacterial classification comprises Kingella, Aitken Salmonella, Simonsiella, chainlet bacterium, CDCEF-4 group and CDC M-5 group.Wei Rong Shi coccus is a Gram-negative coccus, belongs to the similar bacterium of anaerobism of Neisseria.The part that these non-active diplococcuses are oral cavity normal microfloras.

Pathogenic bacterium among the aerobic coccus group of Gram-negative comprise Neisseria gonorrhoeae, mora Salmonella (branhamella) and acinetobacter calcoaceticus.Neisseria comprises two important human pathogenic bacterias, Diplococcus gonorrhoeae (urethritis, sub-Uterine Cervix inflammation, salpingitis, rectitis, pharyngitis, conjunctivitis, pelvis inflammation venereal disease, sacroiliitis, transmissible disease), and Neisseria meningitidis (meningitis, septicemia, pneumonia, sacroiliitis, urethritis).Think some other harmless Gram-negative coccus in the past, found it is human infection's common cause recently again, comprised mucositis mora Salmonella (branhamella) (chronic lung disease patient's bronchitis and bronchopneumonia, sinusitis, otitis media).

EspA polypeptide of the present invention can also be used for producing the EspA polypeptide epitope is had immunocompetence or bonded antibody with it.Provide basically by having the antibody that the specific mixing monoclonal antibody of different epi-positions is formed, and different monoclonal antibody formulations.Monoclonal antibody can prepare (Kohler, et a1, Nature, 256:495,1975 by containing antigenic protein fragments by means of method well known in the art; Current Protocols in Molecular Biology, Auswbel, et al.ed, 1989).

In the present invention the noun of Shi Yonging " antibody " comprise can in conjunction with the complete molecule of epi-position with and fragment, as Fab, Fab ', F (ab ') ₂, and Fv.These antibody fragments kept some optionally with the ability of its antigen or receptors bind, be defined as follows: (1) Fab, the fragment that includes antibody molecule monovalent antigen bound fraction can prepare by a part that produces a complete light chain and a heavy chain with the complete antibody of papain digestion; (2) Fab ', this fragment of antibody molecule can be reduced then by with the complete antibody of pepsin, produces complete light chain and part heavy chain and obtains, and each antibody molecule can obtain two Fab ' fragments; (3) (Fab ') ₂, by with the complete antibody of pepsin, do not carry out this fragment that subsequently reductive action can obtain antibody, F (ab ') ₂Be two segmental dimers of Fab ' by two disulfide bonds; (4) Fv is defined as the genetic engineering of being expressed as two strands that contains variable region of light chain and variable region of heavy chain and makes up fragment; (5) single-chain antibody is defined as and contains variable region of light chain, and the genetic engineering of variable region of heavy chain makes up molecule, and these two variable regions are connected by suitable peptide linker, is the single chain molecule of heredity fusion.

Prepare these segmental methods and be known in the art (referring to for example, Harlow and Lane, Antibodies:A Laboratory Manual, (antibody: laboratory manual), Cold Spring HarborLaboratory, New York (latest edition) is hereby incorporated by).

Epi-position is the other position of the antigen of antibody on the antigen any antigenic determinant of bonded with it.Epi-position is made up of the chemically reactive surface group of for example amino acid or sugared side chain molecule usually, and has specific Three Dimensions Structure usually, and specific charge characteristic.

If needed, polyclonal antibody or monoclonal antibody can be further purified, and for example by being incorporated into a matrix, and go out from wash-out wherein, combine a kind of peptide on this matrix, the peptide that perhaps a kind of this antibody produces at its.Those skilled in the art is familiar with being used for the immunological technique of purifying and/or concentrated polyclonal antibody and monoclonal antibody various technology commonly used (referring to for example Coligan etc., Unit9, Current Protocols in Immunology, Wiley Interscience, latest edition is hereby incorporated by).

The present invention also provides the peptide epitopes that is used to design espA specificity nucleotide probe or anti--EspA antibody.This probe or antibody can be used for identifying may with other pathogenic agent virulence proteins associated matter or gene, including, but not limited to polypeptide or polynucleotide from Gram-negative bacteria.

Antibody of the present invention comprises polyclone and monoclonal antibody, chimeric antibody, and single-chain antibodies etc. have with the hyperimmunization specificity and are incorporated into EspA protein of the present invention, peptide or nucleotide sequence or its segmental ability.These antibody can not be labeled, perhaps by mark suitably.Antibody of the present invention can be used to for example EspA is carried out the affinity purifying.Antibody of the present invention can be used for qualitative or the detection by quantitative cell by known immunological method, group group sample, these protein or peptide in sample formulation or the liquid.Antibody of the present invention can also be used for qualitative or detection by quantitative nucleotide sequence or its protein by known immunological method.

The invention provides a kind of method that is used for test sample EspA polypeptide, comprise making contacting with antibody at the EspA polypeptide from the sample of object to be checked; And detection antibody is to the combination of EspA polypeptide.In conjunction with being the indication that has the EspA polypeptide in the sample.Noun " sample " comprises from Mammals or people's object, the perhaps material of other animal as used herein.This sample is including, but not limited to hair, skin samples, tissue sample, culturing cell, the substratum of culturing cell, and biological liquid.For example, can in HeLa cell (for example people) culture, detect the EspA polypeptide.

Refer to cell lump from the connection of people or other animal (for example CNS tissue, nervous tissue or ocular tissue) at this used noun " tissue ", comprise connection material and the fluent material relevant with this cell.For example, the rabbit enteropathogenicEscherichia coli can find in the region between the heart and the diaphragm intestines and the colon at the rabbit stomach.Noun " biological liquid " refers to the fluent material from people or other animal as used herein.These biological liquids are including, but not limited to blood, blood plasma, and serum, the serum derivative, bile, mucus, saliva, sweat, amniotic fluid, and cerebrospinal fluid (CSF) are as waist or ventricles of the brain CSF.

Noun " sample " also comprises the solution that contains this isolated polypeptide as used herein, and this polypeptide is secreted to substratum wherein, and the substratum that contains the host cell that produces this EspA polypeptide.For example, sample may be to be differentiated by SDS-PAGE, and is transferred to the protein example that nitrocellulose filter carries out the Western immunoblotting assay.Those skilled in the art can determine to obtain the needed sample size of a certain reaction by means of the experimental technique of standard.The best sample amount can be determined by serial dilution.

In one embodiment, the indication that exists the EspA polypeptide to be infected in the sample by enteropathogenicEscherichia coli.In another embodiment, exist the EspA polypeptide to be caused the indication of enterorrhagia coli-infection in the sample.

Estimate protein of the present invention, protein fragments and synthetic peptide serve many purposes, and comprise prediction, treatment, application such as diagnosis or medicinal design.Protein of the present invention, protein fragments and synthetic peptide will have the mono-clonal of specific immune response and polyclonal antibody provides the basis for preparation and protein of the present invention.In one embodiment, the invention provides a kind of method, be used for host, carry out immunity by the polypeptide that the host is given claim 1 to the disease sensitivity that causes by the intestinal bacteria that produce EspA; And induce the host that the EspA polypeptide is produced protective immune response.Therefore can prevent to produce of the infection of the microorganism of EspA to the host.In more specific embodiment, the microorganism that produces EspA is a coli strain.In more specific embodiment, this coli strain is enteropathogenicEscherichia coli or causes the enterorrhagia intestinal bacteria.

In another embodiment, the invention provides a kind of method, be used to alleviate the disease that is caused by the microorganism that produces EspA, is by with EspA polypeptide immune host, induces the immunne response of host to the EspA polypeptide.In more specific embodiment, the microorganism that produces EspA is a coli strain.In more specific embodiment, this coli strain is enteropathogenicEscherichia coli or causes the enterorrhagia intestinal bacteria.The invention provides the method for espA polynucleotide in a kind of test sample, is to suspect the sample contain the espA polynucleotide and can contact with the nuclear probe of espA multi-nucleotide hybrid by making; And detect the hybridization of this probe and espA polynucleotide.Find that hybridization is the indication that has the espA polynucleotide in the sample.

In another embodiment, the invention provides a kind of microorganism of the espA of having mutator gene.The preferred microorganism that espA gene wherein can be suddenlyd change is including, but not limited to bacterium.The bacterium that espA gene wherein can be suddenlyd change is intestinal bacteria.The intestinal bacteria that espA gene wherein can be suddenlyd change are enteropathogenicEscherichia colis and cause the enterorrhagia intestinal bacteria.

The invention provides the method that is used to produce reorganization espA polynucleotide, comprise that the nucleic acid with the coding selected marker inserts in the polynucleotide of coding EspA polypeptide.The polynucleotide encoding that produces contains the reorganization EspA polypeptide of selected marker.For example, selected marker can be simple sore exanthema virus (HSV) sign, can buy the antibody at this sign on the market.

The invention provides the method that is used to produce reorganization EspA polypeptide, is by making the host cell of the polynucleotide that contain coding EspA polypeptide, growing under the condition that allows its expression and secretion EspA polypeptide: isolate this polypeptide again.The method that produces polypeptide and peptide with recombinant technology within the scope of the present invention." allow its express and excretory condition " refers to suitable like this condition as used herein, so that nucleic acid may transcribe and translate, and the separable polypeptide that goes out such generation.The polypeptide that is produced may be secreted the protein in the substratum into.Substratum comprises liquid, material or the organism that microorganism can grow therein or microorganism can survive therein.Such environment for example can be, animal tissues or body fluid, water and other liquid, food, foodstuff products or food extracting solution, and some inanimate objects.For example, microorganism can be grown in Luria-Bertani (LB) substratum.Unnecessary this environment can promote microorganism growth, as long as it allows its existence.

The invention provides a kind of method of identifying the compound that suppresses bacterium III type excretory system, is the polynucleotide by the selected marker of will encoding, and introduces the bacterium with III type excretory system; Make bacterium allow the bacterial growth justacrine to grow under the condition of the polypeptide of polynucleotide encoding thus; Make again and infer the compound bacterium contact therewith that suppresses bacterium III type excretory system; Induce this polypeptide expression; Detect the secretion of this polypeptide then.In implementing present method, the acrinia indication is to the restraining effect of bacterium III type excretory system.The noun that uses in invention " secretion of III type " and " III type Secretory Pathway " refer to discharges the special entity that molecule is crossed over cytolemma.Discharging molecule and cross over cytolemma, can be the process of closing chain with the surface of host's interaction between component to virulence factor is transferred to.III type secretion path uses Triphosaden (ATP) to make the energy.III type secretion path is different from other secretion path of finding in the Gram-negative bacteria, though it have with flagellum and thread phagocytosis assembled base because of the homologous gene.It is different from any Mammals path.It is often relevant with the generation of disease.By III type secretion path excretory virulence factor, between various pathogenic bacterias, have nothing in common with each other, though the parts of III type secretion mechanism, at least to Salmonellas, Shigellae and Yersinia can exchange.

And polypeptide of the present invention or nucleotide sequence can be used for identifying with their interactions (for example combining) and influence their bioactive compound or compositions.This effect comprises and suppressing or the active or secretion of activation EspA.

The invention provides a kind of method, by mutagenesis in the Nucleotide of coding EspA polypeptide, and produce the non-virulent microorganism, the nucleotide sequence insertion mutational site of coding selected marker thing; The espA polynucleotide of sudden change are imported the karyomit(e) espA gene of a kind of microorganism, and guiding is undergone mutation in karyomit(e) espA gene; Select to have the microorganism of sudden change then.Noun " sudden change " refers at gene nucleotide series as used herein, particularly the change in the polynucleotide of coding EspA polypeptide.Sudden change comprises that generation has the sudden change of different aminoacids sequence EspA polypeptide, missense mutation (comprising phase shift mutation), nonsense mutation (comprise and knock out sudden change), and the gene recombination technology that produces the fusion rotein that contains part EspA polypeptide.In one embodiment, the nucleotide sequence of coding selected marker can be encoded to the resistance of kantlex.For example, the aphA-3 box clone of coding kalamycin resistance gene (Kan) can be entered the polynucleotide of coding EspA polypeptide, be used on generation knocks out the kantlex culture plate of sudden change, selecting the espA polynucleotide of sudden change.

Implement preferred microorganism of the present invention therein including, but not limited to bacterium.In another embodiment, being used for the microorganism that polynucleotide at coding EspA polypeptide produce sudden change is intestinal bacteria.What can be transformed in intestinal bacteria is enteropathogenicEscherichia coli and causes the enterorrhagia intestinal bacteria.

The invention provides a kind of method that activates tyrosine kinase activity in the host cell, be all to join in the host cell by the espA defective type mutant bacterial that will express the Eae polypeptide and the eaeA-defective type mutant bacterial of expressing the EspA polypeptide, bacterium is combined with host cell, thereby activate the activity of Tyrosylprotein kinase in the host cell.In one embodiment, the activation of tyrosine kinase activity in the host cell has caused the tyrosine phosphorylation of host's membranin Hp90 of 90 kilodaltons, and causes endocellular phosphorus acid inositol (IP3) and calcium current to go out.For example, when these two mutant strains were used to coinfection HeLa cell, the eaeA mutant can be used for replenishing the dissemination of espA mutant.Therefore, the invention provides a kind of useful scientific approach, by means of the RESEARCH ON CELL-BIOLOGY pathogenesis.

The present invention includes a kind of test kit that includes one or more antibody of the present invention, and a kind of test kit based on Nucleotide.In one embodiment, this test kit can be used for detecting the EspA polypeptide, and it is a kind of loading appliance that is separated, and has held the container of compact package, wherein be equipped with can with EspA polypeptide bonded antibody." wrapping tool " comprises phial as used herein, and test tube etc. are equipped with a kind of isolating composition that is used for present method in each wrapping tool.In one embodiment, this can be detectable label with EspA polypeptide bonded antibody.In more specific embodiment, this marker is selected from radio isotope, bioluminescent compound, chemiluminescence compound, fluorescent chemicals, metal chelator and enzyme.

In another embodiment, this test kit can be used for detecting the EspA polynucleotide, and it is a kind of loading appliance that is separated, and has held the container of compact package, wherein be equipped with can with the nucleic acid probe of EspA multi-nucleotide hybrid.In one embodiment, this can be detectable label with the nucleic acid probe of EspA multi-nucleotide hybrid.In more specific embodiment, this marker is selected from radio isotope, bioluminescent compound, chemiluminescence compound, fluorescent chemicals, metal chelator and enzyme.

Because EspA is a kind of secretory protein, so can be used as fusion partner, it is used to clone and express other peptide and protein, for example, be blended in the proteinic EspA that studies, can produce in host cell such as intestinal bacteria by recombinant technology, this fusion rotein is secreted in the substratum that this transformed host cells grows therein.This fusion rotein can be separated by means of anti--EspA antibody, and EspA and peptide of being studied or protein are disconnected.Can identify this EspA fusion rotein with ELISA or other immunoaffinity method.The invention provides a kind of method of the EspA of generation fusion rotein, comprise host cell is grown under the condition that allows its expression and secretion fusion polypeptide, isolate fusion polypeptide then, this host cell contains the polynucleotide of the EspA that encodes, these polynucleotide be operably connected to coding the polynucleotide of the polypeptide of studying or peptide.Noun " be operably connected or in conjunction with " refers between promoter sequence and the structure gene, perhaps under the situation of fusion rotein is being the functional connection that is activated between the gene that the daughter nucleus acid sequence regulates.The promotor control that is operably connected is by the expression of building stone encoded polypeptides (as fusion rotein).

The present invention will be described to plan to use the following examples, rather than limit it.Although applied those implementation methods all are typical, can also selectively use other program well known to those skilled in the art.

Embodiment 1

The dna sequence analysis of enteropathogenicEscherichia coli espA gene

The purpose of this embodiment is in order to describe the feature of espA, and this is one and causes enteropathogenicEscherichia coli to have to adhere to and the active gene of surface damage.The secretory protein of espA genes encoding 25 kilodaltons, being positioned the enteropathogenicEscherichia coli genome, is in the intestinal cells surface damage locus between needed locus eaeA of tight adhesion and the espB (Locus of EnterocyteEffacement) at two.

Dna sequence dna to the intestinal cells surface damage locus between enteropathogenicEscherichia coli eaeA and the espB is measured.Dna sequencing is performed as follows: will extend to the Sal I-Bgl II dna fragmentation of the intestinal cells surface damage locus of espB (5 ') upstream from eaeA, the clone enters among the plasmid pBluescript that can buy, and forms plasmid pLCL109.Terminal from the closed son (closer) of plasmid pLCL109 to the eaeB gene, formed a series of nested type DNA disappearances.Use synthetic Oligonucleolide primers on demand, [a-35S] dATP, and Sequenase can be used as template with these DNA disappearances of plasmid pLCL109, measure the nucleotide sequence of dna double thigh.By means of dna sequence data being analyzed by the software package of the GeneticComputer Group of Wisconsin university exploitation.

Analysis to this dna sequence dna demonstrates three open reading frame.By the aminoacid sequence of the dna sequence dna prediction that is positioned at second open reading frame (espA) 5 ' end, identical with a kind of proteinic N-terminal sequence, this protein has the MR by about 25 kilodaltons of enteropathogenicEscherichia coli excretory.During with TEASTA retrieval Genbank database, do not detect protein with similar sequences.Therefore, this espA genes encoding enteropathogenicEscherichia coli secretory protein.

Predicted molecular weight by the intact proteins of espA coding is 20468 dalton (Fig. 1).Before the N-terminal of the secretory product of being reported, there is not leader sequence.Strong total ribosome bind site terminates in initiator codon 7 Nucleotide before.In aminoterminal 19 residues 11 are predicted to be Serine or Threonine.

Embodiment 2

Make up sudden change in the espA gene on plasmid and karyomit(e)

The purpose of present embodiment is to make up sudden change in the espA gene on the microbial staining body.The purpose of present embodiment is to make up sudden change in the espA gene on plasmid.This plasmid can be used for then producing sudden change at the espA of other microorganism gene.Made up the plasmid that in the espA gene, has nonpolar sudden change.And produced the mutant strain that in its karyomit(e) espA gene, has nonpolar sudden change.

The espA gene that has nonpolar sudden change has been described in Fig. 2.(PCR) formed the disappearance that has restriction enzyme site with polymerase chain reaction, can make the aphA-3 box of that mycin resistance of coding card be entered this disappearance district by the clone like this.In three all frames, the front of this aphA-3 box (5 ') are translation stop codon, and what be right after this box back (3 ') is total ribosome bind site and initiator codon.Design has kept the frame of espA 3 ' end when the espA gene inserts, therefore can not influence downstream (3 ') gene transcription and translation.Dna sequencing has confirmed the frame of this sudden change.

By following by means of polymerase chain reaction, make up nonpolar sudden change in plasmid espA gene: pcr template is plasmid pLCL114, and it contains the pLCL119 Cla I-Bgl II fragment that is entered pBluescrit by the clone.Use two pairs of primers, had the universal primer and the reverse primer that has Donne-100 of Donne～99.Oligonucleotide Donne-99 and Donne-100 are respectively the segmental Nucleotide 4157 to 4140 of Sal I-Bgl II and 4297 to 4324.For the purpose of cloning, all designed NruL restriction enzyme digestion position at 5 of Donne-99 and Donne-100 ' end.Oligonucleotide is to make up in the Maryland of Baltimore university biological polymer laboratory, in the partial circulating device 50 μ l samples has been carried out PCR.Carried out 30 PCR reaction cycle, 94 ℃ of DNA sex change 1 minute, 55 ℃ to primer annealing 2 minutes, extended 3 minutes at 72 ℃ of polynucleotide.The plasmid pCRscript that 2 amplified fragments that form are advanced can buy by the clone separately constitutes pLCL119 and pLCL120 respectively.With Sal I and Nru I the insertion fragment cloning of pLCL120 is entered pLCL119 then, constitute the pLCL121 that contains required disappearance.To insert the into Nru I site of pLCL121 in the aphA-3 kanamycin resistance cassette of lateral 850 base pairs in Sma I site then.

The espA allelotrope of sudden change is cloned and into just selected suicide vector PCVD442, and be imported into wild-type enteropathogenicEscherichia coli E2348/69 strain by the allelotrope exchange.Made up the espA mutant strain by following: will contain interruption the espA gene from the Sal I with aphA-3 kalamycin resistance plasmid-Sac I fragment, the clone enters the just selection suicide vector pCVD442 among the DH5 apir, be used for by three close keying actions, perhaps, in the cuvette of 0.1cm, carry out electroporation and handle and importing E2348/69 bacterial strain by with producer in the intestinal bacteria arteries and veins that is set in 1.8kV.

On the LB kantlex culture plate of revising, the espA mutant is selected.The enteropathogenicEscherichia coli mutant strain UMD872 that forms has resistance to sucrose and kantlex, to the penbritin sensitivity.With the primer Donne-52 of two sudden change flanks and the pcr amplification that Donne-73 carries out, confirmed the structure of espA sudden change.This bacterium is in 50%LB liquid nutrient medium/50% glycerine (volume/volume),-70 ℃ of storages, incubation growth on the LB agar plate or in the LB liquid nutrient medium, add paraxin (20 μ g/ml) when needing, penbritin (200 μ g/ml), nalidixic acid (50 μ g/ml), or kantlex (50 μ g/ml).

Embodiment 3

The destruction of enteropathogenicEscherichia coli espA gene has been eliminated the proteic secretion of EspA

The purpose of present embodiment is the protein of differentiating by the espA genes encoding.The proteinic N-terminal sequence data of EspA compared show that it is consistent with the espA gene order that is translated.In order to confirm this result, make espA deletion mutant UMD872 incubation growth in radiolabeled tissue culture medium (TCM).The disappearance of espA gene has caused losing the radio-labeling secretory protein of 25 kilodaltons.By using the anti--EPEC serum that reacts with the EspA secretory protein to carry out immunoprecipitation, confirmed this result.There is not the secretory protein of 25 kilodaltons with anti--Western analysis demonstration that EPEC serum carries out.Lack the limit bacterial strain when transforming espA with the plasmid (pMSD2) that has complete espA, the proteic secretion of EspA is restored.This bacterium increases generation by plasmid-encoded EspA albumen, and makes III type secretion road reduce other proteinic secretion.

Embodiment 4

The epithelial cell that enteropathogenicEscherichia coli infects cultivation needs EspA

The purpose of present embodiment be measure enteropathogenicEscherichia coli EspA albumen whether with to epithelial infect relevant.Excite host's signal transduction pathway and infect host cell and all need EspA albumen.

With parent's wild-type or espA mutant enteropathogenicEscherichia coli strain infection simple epithelium cancer cells (Hela) 3 hours.Measure the number of adherent and intracellular (promptly invading) enteropathogenicEscherichia coli bacterium.Corresponding with the speeds of growth different between mutant and the parental type enteropathogenicEscherichia coli bacterial strain, two strain bacterium are inequality to the absolute number of Hela cell adhesion, although espA mutant strain UMD872 has the adhesion of sufficient amount to epithelial cell, lack the quantity of invading.But as the plasmid pMSD2 by the complete espA gene of having encoded, behind the additional espA gene of genetic technique, the quantity of the UMD872 of intrusion individual layer HeLa cell is near the level of wild-type.

The HeLa monolayer cell has been carried out the coinfection test, be used to be determined at espA, whether the intrusion behavior defective of espB and eaeA aspect mutant strain can pass through the genetic technique trans-complementation, mediation invasion effect subsequently.But the conduction of eaeA mutant strain activation signal adheres to but lack closely.Signal conduction by the mediation of eaeA mutant strain can make the bacterial strain of espB defective enter epithelial cell, otherwise but then can not.When with eaeA and espA mutant strain coinfection HeLa cell, the eaeA mutant strain has remedied the intrusion of espA mutant strain, but the intrusion of eaeA mutant strain does not increase.Really, the signal conduction by stronger adherent eaeA mutant strain brings out causes having increased the absorption to the espA mutant strain.Do not make wherein any strain strengthen the invasion effect with espA and espB disappearance strain coinfection, show that espA and espB can not complement each other.

Coinfection experiment showed, that as espB the intrusion behavior of espA mutant strain has been remedied by eaeA, otherwise but then can not.Really, the signal conduction by stronger adherent eaeA mutant strain causes has caused increasing the absorption of expressing the espA mutant strain of intimin.On the contrary, espA or espB mutant strain all can not remedy mutually, mean that these two kinds of protein may work to the same steps as of bringing out the conduction of epithelial cell signal jointly.

Embodiment 5

Signal conduction incident is essential to EspA in the epithelial cell to bringing out

Whether the purpose of present embodiment measures EspA, and signal conduction incident is essential in the epithelial cell to bringing out.EnteropathogenicEscherichia coli brings out the tyrosine phosphorylation effect of host cell 90 kilodalton membranins, and the protein of phosphorylation takes place subsequently, and Actin muscle and other cytoskeleton composition are built up below adherent bacterium.The espA mutant strain that lacks dissemination being brought out the ability of these two kinds of signal conduction incidents of mammalian cell measures.Different with the wild-type enteropathogenicEscherichia coli, espA mutant strain UMD872 can not bring out the phosphorylation of host Hp90.By the coding proteic plasmid of EspA (pMSD2) ability of bringing out this phosphorylation event is recovered.

Adhere to invading to measure and be performed as follows: (m.o.i.1: 100) the 105 HeLa cells of cultivation in DMEM are 3 hours with different enteropathogenicEscherichia coli strain infections.In the Dulbecco ' s Minimal Eagles substratum (DMEM) that has replenished 10% (volume/volume) foetal calf serum, with 5%CO ₂Cultivate the HeLa cell for 37 ℃.It is inferior earlier monolayer cell to be given a baby a bath on the third day after its birth in phosphate-buffered saline, does cytolysis then in the 1%Triton (volume/volume) of phosphate-buffered saline preparation, and does dull and stereotyped the cultivation with serial dilutions on the LB agar plate.In order to measure intrusion, in the pair cell rinsing, dissolved cell and doing before dull and stereotyped the cultivation is with the common incubation of monolayer cell and gentamicin (100 μ g/ml) washed 1 hour, so that kill the bacterium of outside.

With wild-type or mutant enteropathogenicEscherichia coli strain infection individual layer HeLa cell 3 hours.Isolate the Triton X-100 solubility and the insoluble hide collagen of going up of HeLa cell.Protein example separates by SDS-PAGE, and is transferred on the nitrocellulose filter, surveys with anti--Tyrosine O-phosphate specific antibody then.

By following enteropathogenicEscherichia coli secretory protein and the HeLa cell protein isolated: with 106 HeLa cell inoculation tissue culturing plate overnight incubation.Before infecting, there is not the DMEM of methionine(Met)/halfcystine to replace this substratum with containing cycloheximide (100 μ g/ml).Make the HeLa cell in the DMEM that has replenished 10% (vol/vol) foetal calf serum, with 5%CO ₂37 ℃ of following incubation growth.The adding enteropathogenicEscherichia coli (m.o.i.100: 1), at 5%CO ₂Cultivated 2.5 hours for 37 ℃ in the incubator, add 200 μ g/ml's then ³⁵S halfcystine/methionine(Met) was cultivated 30 minutes.Isolate culture supernatant, and by centrifugal (18000 * g, 10 minutes) precipitation bacterium wherein.By adding ice-cold Tricholroacetic Acid (10%vol/vol) secretory protein in the supernatant liquor is precipitated, and hatched 60 minutes on ice.The same by the centrifugal protein precipitation that makes, and then be suspended in the Laemelli sample buffer.Make sample separation by means of 12%SDS-PAGE, measure the protein distribution plan, perhaps be transferred to nitrocellulose filter, survey with anti--EPEC antibody then by autoradiographic technique.

All enteropathogenicEscherichia coli bacterial strains have all demonstrated the tyrosine phosphorylated proteins Ep85 of 85 kilodaltons in insoluble component, prove to exist on monolayer cell to cause the sick E.coli bacterial strain of enteron aisle.

By following HeLa cell Tyrosine O-phosphate albumen is analyzed: earlier, in containing the 1%TritonX-100 of proteinase inhibitor, make cytolysis then with the individual layer HeLa cell of ice-cold phosphate-buffered salt washing infection 3 times.Isolate the insoluble and soluble component of Triton, be suspended in again in the Leamelli sample buffer,,, analyze Tyrosine O-phosphate albumen by means of Western immunoblotting assay method with anti--phosphotyrosine antibody.

Resist-phosphotyrosine antibody or poisonous fungus cyclic peptide of rhodamine with fluorescently-labeled, detecting the HeLa cell that infects by immunofluorescence microscopy shows, be different from the wild-type parent enteropathogenicEscherichia coli, the espA mutant strain does not make tyrosine phosphorylated proteins or actin cytoskeleton build up under the adhesion microcolony.But,, Tyrosine O-phosphate and Actin muscle accumulation effect are recovered by using the bacterial strain that on plasmid pMSD2, carries the espA gene.

Being performed as follows immunofluorescence microscopy detects: to the HaLa cell of inoculation culture on the circular lid slide, infected 3 hours with enteropathogenicEscherichia coli or mutant strain.This monolayer cell of rinsing, and fixing in 2.5% Paraformaldehyde 96, then to actin filament dyeing (with phalloidin-rhodamine), perhaps to have the anti--phosphotyrosine antibody dyeing of suitable fluorescent mark second antibody.

Embodiment 6

To rabbit enteropathogenicEscherichia coli (RDEC-1)

The characterized of secretion property virulence albumen EspA and EspB

The purpose of present embodiment is the structure of middle EspA of research rabbit enteropathogenicEscherichia coli (RDEC-1) and EspB.EspA and espB gene are cloned, and their sequence and the gene order of enteropathogenicEscherichia coli (EPEC) are made comparisons.EspA albumen shows some similarities (88.5% is identical).The EspB active site of protein is allogenic (69.8% is identical), but identical with a bacterial strain that causes intestinal bleeding intestinal bacteria (EHEC).

By following espA and espB gene are cloned and sequential analysis: the dna fragmentation of coding RDEC-1 espA and espB can obtain from the RDEC-1 chromosomal DNA by PCR, the primer of the enteropathogenicEscherichia coli gene order that using controls oneself announces, use the Vent archaeal dna polymerase to carry out PCR, from RDEC-1 and enteropathogenicEscherichia coli bacterial strain amplification chromosomal DNA.Carry out the PCR reaction cycle 30 times, 94 ℃ of sex change 1 minute, 55 ℃ of annealing 1 minute, and 72 ℃ extended 2 minutes.4.3 kilobase that form are connected product enter among the plasmid pBluescript that buys, and two strands is checked order.Be performed as follows dna sequencing: by the right dna fragmentation of this 4.3 kilobase of pcr amplification coding espA and espB gene, use primer AA01 (+) and MSll (-), with the RDEC-l chromosomal DNA as dna profiling.The blunt ends fragment that forms digests with the Sal I, and the clone enters Sal I-Sma I site of the plasmid pBluescript-IISK (+) that buys.The TaqDyeDeoxy test kit that application is buied is to dna sequence dna and the two strands of RDEC-1 espA check order (Fig. 3).

Found two open reading frame in this cloning zone, these dna sequence dnas are similar to the espA and the espB of enteropathogenicEscherichia coli.The molecular weight (192 amino acid) of the RDEC-1 EspA of prediction is 23533 dalton, and the molecular weight of RDEC-1 EspB (314 amino acid) is 33219 dalton.RDEC-1 EspA is similar a bit to the EspA of enteropathogenicEscherichia coli, has 88.5% homogeny (Fig. 4 A and 4B).

A result who does not expect is, RDEC-1 EspB albumen and nearest report, from causing enterorrhagia intestinal bacteria 413/89-1 strain, the EspB of 026 serotype is identical, but (T becomes C to have two nucleotide differences at 12 base pairs and 729 base pair positions, become T with G), this bacterial strain is to isolate from the tire ox at first, also isolates from the patient who suffers from hemolytic uremic syndrome later on.And, RDEC-1 EspB with cause comparing of enterorrhagia colon bacillus 0157 serotype and enteropathogenicEscherichia coli bacterial strain, show 70.3% and 69.8% homogeny respectively.When with the sequence of enteropathogenicEscherichia coli relatively the time, at RDEC-1 with cause among the EspB of enterorrhagia intestinal bacteria (026 and 0157 serotype), found little deletion sequence (4A-C) in same position.

These results show RDEC-1 codified espA and espB gene, and show that the EspA polypeptide of prediction is a high conservative in RDEC-1 and enteropathogenicEscherichia coli.But, this EspB with cause the enterorrhagia intestinal bacteria, rather than the EspB of enteropathogenicEscherichia coli is more similar.EspA downstream open reading frame (3 ') shows and the EspD of enteropathogenicEscherichia coli has similarity, EspD is a kind of adjusting EspB excretory secretory protein, be to excite host cell signal conducting path needed (EMBL GenBank data, query ID Y09228).These results show, espD in RDEC-1 also between espA and espB.

Embodiment 7

Characterized to RDEC-1 EspA and EspB

The purpose of present embodiment is the function of middle EspA of research rabbit enteropathogenicEscherichia coli (RDEC-1) and EspB.The sudden change of espA and espB discloses among the RDEC-1, and the gene product of RDEC-1 is essential to exciting the host signal conducting path and invading the HeLa cell.The plasmid that contains enteropathogenicEscherichia coli espA and espB remedies the effect proof to the RDEC-1 mutant strain, and they can exchange on function, though the protein mediated higher levels of intrusion of enteropathogenicEscherichia coli.And the maximum of RDEC-1 and enteropathogenicEscherichia coli secretory protein is expressed, and is to occur in them separately under host's the body temperature, and body temperature may be that enteropathogenicEscherichia coli lacks one of infective reason to rabbit.

In order to confirm RDEC-1 espA and the espB effect in host's epithelial cell signal conducting path, in espA and espB design construction nonpolar terminator codon sudden change.Made up two suicide carriers, and by replying in conjunction with being imported into the RDEC-1 wild type strain.The mutant strain AAF001 Δ A (EspA that forms ^-), AAF001 Δ B (EspB ^-) and double-mutant strain AAF001 Δ AB (EspA ^-/ EspB ^-), be proved by the digestion of Bg II.

In RDEC-1 espA and espB gene, made up the sudden change of nonpolar terminator codon by following: by pcr amplification the right intestinal cells surface damage locus dna fragmentation of 2.7 kilobase of coding espA and espB gene, use primer BK25 (+) and MSll (-), make dna profiling with pORF123B.With the blunt-ended fragment of EcoR I digestion formation, and, obtain pORF23B by EcoR I-Sma I site that the clone enters pBluescriptll SK (+) carrier.EcoR I-Bgl II dna fragmentation that 1.1 kilobase are right, it is from the pORF23B that contains espA, the clone enters EcoR I-BamH I site of pBluescript II SK (+), obtains pORF23.

In order in espA, to make up nonpolar sudden change, carried out inverse PCR, use contains the Δ espA (+) and Δ espA (-) primer of Bgl II restriction enzyme site and terminator codon, uses ring-type pORF23 to make dna profiling, PCR product flush end is connected obtain the pORF23 Δ.The plasmid that forms contains the Bagl II site at a terminator codon and 235 base pair places, an espA initiator codon downstream (3 '), has confirmed this point by dna sequencing.To contain from 1.1 kilobase of the sudden change espA of pORF23A Sal I-Sac I dna fragmentation is inserted the into same loci of suicide vector pCVD442, and obtain the PAA23 Δ, this carrier pCVD442 contains and just selects SacB gene and ampicillin resistance gene.The plasmid that forms is imported intestinal bacteria SM10 (pir, and oppositely in conjunction with entering the RDEC-1 that carries pACYC184.

For the nonpolar sudden change of structure in espB, use Δ espB (+) and Δ espB (-) primer, carried out inverse PCR with pBxb as dna profiling.PBxb contains 1.4 kilobase from pORF23B to Xba I fragment, and this pORF238 coding is entered the espB of pBluescript carrier by the clone.The PCR product self that forms connects and obtains the pBxb Δ, and it contains a terminator codon and a Bgl II site that is imported by Δ espB (-) and Δ espB (+) primer.In the pBxb Δ, the esp gene that forms is deleted 250 base pairs, Sal I-Sac I dna fragmentation that 1.1 kilobase are right inserts the into same loci of pCVD442 to start from espB initiator codon downstream 154 base pairs (3 '), obtain the pAABxb Δ, dna fragmentation wherein contains the sudden change espB from pBXbA.This plasmid transduction that forms is entered intestinal bacteria SM10 λ pir, and oppositely in conjunction with entering the RDEC-1 that carries pACYC184.In order in espA and espB, to make up two sudden changes, the pAABxb Δ can be imported AAF001 Δ A (EspA-) bacterial strain.Maintain resistance by their phenotype, and, prove three nonpolar mutant strains of RDEC-1 the penbritin sensitivity to sucrose and paraxin.In order to confirm in espA and espB, to have inserted terminator codon, can prepare chromosomal DNA from each mutant strain, carry out PCR with the primer that contains the esp gene.The PCR product that forms carries out enzyme with the Bgl II and cuts digestion, so that the restriction enzyme site of this structure of alleged occurrence.EspA is named as AAF001 Δ A (EspA respectively or/and contain the mutant strain of terminator codon among the espB ^-), AAF001 Δ B (EspB ^-) and AAF001 Δ AB (EspA ^-/ EspB ^-).

In order to confirm that suicide vector does not influence flanking region or other locus separately, can implement sudden change and turn back to wild-type (" reverse mutation ").By suicide vector pAA23 and pAABxb is reverse in conjunction with entering AAF001 Δ A (EspA ^-) and AAF001 Δ B (EspB ^-) bacterial strain, obtained two reverse mutation strains.Cut digestion by PCR and Bgl II enzyme, having confirmed formed is the reverse mutation strain, AAF003 and AAF004.Construct reverse mutation by following in EspA and EspB strain: the Sal I that 1.1 kilobase are right-Sac I dna fragmentation inserts Sal I-Sac I site of pCVD442, obtains pAA23, and dna fragmentation wherein is from the pORF23 that contains espA.The Sal I that 1.4 kilobase of pBxb are right-Sac I fragment is inserted Sal I-Sac I site of pCDD422, obtains pAAFBxb.The pAABxb of pAA23 is imported SM λ pir, and oppositely in conjunction with entering AAF001AA and AAF001AB.Having confirmed formed as mentioned above is the reverse mutation strain, is named as AAF002 (Esp ⁺) and AAF003 (EspB ⁺).

Clone causing sick E.coli espA of enteron aisle and espB by following: 2.8 kilobase by pcr amplification coding espA and espB are to dna fragmentation, using primer EespA (+) and EespB (-), is template with the chromosomal DNA of enteropathogenicEscherichia coli 2348/69.Cut this fragment of digestion with BamH I and Sal I enzyme, and under the control of lacZ promotor, be imported into the Bam[HI-of low copy carrier pMW118) Sal I site, obtain pMWespAB.Cut digestion has restriction enzyme site in the espD open reading frame pMWespAB with Bgl II enzyme, be connected with Klenow fragment flush end, self has been connected to pMW6espD then, and also available Bgl II and BamH I enzyme are cut digestion pMWespAB, self connects then to have obtained pMWespB.Cut digestion pMWespAB with Bgl II-Sal II enzyme, and fill up, self connect then and obtained pMWespA with the Klenow fragment.

RDEC-1 and its secretory component of mutant strain in tissue culture medium (TCM) are analyzed.EnteropathogenicEscherichia coli is secreted 5 kinds of protein in tissue culture medium (TCM), (EspC) of 110 kilodaltons, 40 kilodaltons, 39,000 Dao Er's, (EspB) of 37 kilodaltons and (EspA) of 25 kilodaltons.RDEC-1 shows similar secretory component, and different is that it does not secrete the protein that is equal to EspC.EnteropathogenicEscherichia coli brings out the host signal conducting path does not need EspC.Though have two secretory proteins (40 and 39 kilodaltons) to be difficult to differentiate, use distinguishable these protein of SDS-PAGE of different condition.RDEC-1 secretes two protein that have similar mobility to enteropathogenicEscherichia coli EspA and EspB.

Can be by the following RDEC-1 of producing secretory protein: the bacterial cultures of overnight incubation is done dilution in 1: 100 in DMEM, the optical density(OD) (OD600) that is cultured to then at 600nm be 1.0.Transcribe for the RDEC-1 mutant strain that contains enteropathogenicEscherichia coli espA and espB recombinant plasmid is brought out, in DMEM, added isopropylthiogalactoside (IPTG).Remove by centrifugal (18000xg, 10 minutes) and to degerm, add 10% ice-cold Tricholroacetic Acid and make the supernatant liquor precipitation, and at high-volume 1 hour on ice.After centrifugal, will precipitate and be suspended in again in the Lavemmli sample buffer, and analyze by 12%SDS-PAGE.

In the Western immunoblotting detected, EspA and EspB albumen resisted-cause the sick E.coliEspA of enteron aisle and anti--enteropathogenicEscherichia coli EspB antiserum(antisera) all has cross reaction, shows that RDEC-1 also secretes EspA and EspB albumen.In the Western trace detects, used the rabbit polyclonal antibody of anti-enteropathogenicEscherichia coli EspA and EspB.

Mutant strain AAF001 Δ A, AAF001 Δ B and AAF001 Δ AB lack secretion EspA respectively, EspB and EspA/EspB, this is according to secretory component and the judgement done of Western engram analysis to them.For mutant strain AAF00 Δ A (EspA ^-) still can secrete EspB, its assignment of genes gene mapping shows that in espA downstream (3 ') terminator codon insertion sudden change does not influence downstream gene expression.These results also prove, RDEC-1 EspA and EspB albumen are the sequence encodings that is called espA and espB by us.And, two reverse mutation strain AAF002 and AAF003 that obtain from AAF001 Δ A (EspA-) and AAF001 Δ B (EspB-) at first, can express parent's secretory protein now, show among AAF001 Δ A and the AAF001 Δ B every kind of nonpolar sudden change as predict, and do not influence the locus of downstream gene and other.Although in SDS-PAGE the mobility ratio enteropathogenicEscherichia coli of RDEC-1 EspA and EspB slightly hurry up, RDEC-1 EspA that calculates and the molecular weight ratio enteropathogenicEscherichia coli of EspB bigger.

Compare EspA with wild-type RDEC-1 strain ^-, EspB-and EspA ^-/ EspB ^-The amount of other secretory protein has reduced in the bacterial strain.And, at EspA ^-/ EspB ^-40 kilodaltons and 39 kilodalton protein can detect secretory minimizing in the bacterial strain, than at EspA ^-And EspB ^-That finds in the bacterial strain is bigger.The enteropathogenicEscherichia coli albumen of secretion except that EspC is by the mediation of the III type excretory system of Sep bunch of coding.May disturb this secretion path to the brachymemma meeting of EspA or EspB by inserting terminator codon, perhaps disturb the feedback regulation of this system, thereby influence the secretion of other III type dependency secretory protein.

Esp albumen is for exciting the host signal conducting path required.EnteropathogenicEscherichia coli EspA and EspB albumen bring out the host signal conducting path, cause tyrosine phosphorylated proteins, and actin cytoskeleton and other composition are built up below adherent bacterium.Whether in the HeLa cell, trigger these incidents, available rhodamine-phalloidin and fluorescently-labeled resisting-phosphotyrosine antibody pair cell skeletal muscle filamentous actin and tyrosine-phosphorylated protein dyeing in order to measure RDEC-1 EspA and EspB.Though RDEC-1 actin cytoskeleton that is adhering to and tyrosine phosphorylated proteins accumulation level are lower than the enteropathogenicEscherichia coli, these behaviors are difficult for distinguishing for enteropathogenicEscherichia coli.On the contrary, RDEC-1 EspA ^-, EspB ^-And EspA ^-/ EspB ^-Bacterial strain is not built up actin cytoskeleton or tyrosine-phosphorylated protein below adherent bacterium.But reverse mutation strain AAF003 is similar to its parent strain with AAF004, has built up these protein.

When the plasmid that will contain enteropathogenicEscherichia coli espA or espB, the plasmid that perhaps contains these two genes imports RDEC-1 EspA ^-, EspB ^-, and EspA ^-/ EspB ^-Bacterial strain, the accumulation of actin cytoskeleton and tyrosine phosphorylated proteins also can recover.But, when during with RDEC-1 EspB coinfection, then not recovering to bring out host signal conduction incident with the enteropathogenicEscherichia coli EspA-strain of still secreting EspB.EnteropathogenicEscherichia coli EspB can not remedy RDEC-1 EspA in coinfection.Therefore, for activating the host signal conducting path, the function of EspA and EspB is similar in RDEC-1 and enteropathogenicEscherichia coli, but need secrete this two kinds of albumen by same bacterial strain.

When infecting the HeLa cell, can detect tyrosine-phosphorylation Hp90 by means of immunoblotting with enteropathogenicEscherichia coli.The cell that infects with RDEC-1 can not detect tyrosine-phosphorylation Hp90, although can be observed tyrosine phosphorylated proteins by means of immunofluorescence technique below adhering to the RDEC-1 bacterial cell.

Judge as observation, HEp-2 and T84 cell enteropathogenicEscherichia coli are not brought out tyrosine phosphorylation according to immunofluorescence microscopy.Sequencing result shows, compares with enteropathogenicEscherichia coli, and RDEC-1 EspB more is similar to and causes the colibacillary EspB of enterorrhagia.These results in the present embodiment show that in the RDEC-1 course of infection, it is owing to the lower adhesive efficiency of RDEC-1 that less tyrosine-phosphorylated protein is built up, because the proteic heterogeneity of adhesion level difference or Esp, or the two haves both at the same time.

Adhere to and infiltration capability.In in vitro tests, the EspA of enteropathogenicEscherichia coli and EspB not only with excite the host signal conducting path relevant, and be invade necessary.EspA and the effect of EspB in adhering to and invading in order to study RDEC-1 compare RDEC-1esp mutant strain and RDEC-1 strain.EspA ^-, EspB ^-And EspA ^-/ EspB ^-The adhesive capacity of bacterial strain is similar to the adhesive capacity of wild-type RDEC-1 bacterial strain, and it is irrelevant to show that adhesion and EspA and EspB express.Though the infiltration capability of wild-type RDEC-1 hangs down about 90 times than the infiltration capability of enteropathogenicEscherichia coli, at mutant strain EspA ^-, EspB ^-And EspA ^-/ EspB ^-In, this ability will further reduce.Yet the infiltration capability of reverse mutation strain AAF002 and AAF003 has recovered, and these results show that the infiltration capability of RDEC-1 depends on EspA and EspB.

In order to measure enteropathogenicEscherichia coli EspA and EspB complementary ability to the RDEC-1 mutant strain, the various plasmids that will contain enteropathogenicEscherichia coli espA and espB gene have imported the RDEC-1 mutant strain, have carried out the comparison of invasion efficiency with wild-type RDEC-1 bacterial strain.What is interesting is, carry AAF001 Δ AB (the RDEC-1 EspA of pMWespAB ^-/ EspB ^-) (EPECEspA ⁺/ EspB ⁺) its intrusion level is higher 4 times than wild-type RDEC-1, although the amount of AAF001 Δ AB strain secretion enteropathogenicEscherichia coli EspA and EspB is lower than what normally see at RDEC-1.Therefore, the difference intrusion level of seeing between RDEC-1 and enteropathogenicEscherichia coli strain to the HeLa cell is attributable to Esp albumen, and in this tissue culture model, the EspA of enteropathogenicEscherichia coli and EspB mediation intrusion are more effective.Homology comparison shows that EspA is a high conservative in RDEC-1 and enteropathogenicEscherichia coli, but EspB than heterogeneous, the difference that shows infiltration capability between RDEC-1 and the enteropathogenicEscherichia coli may be because EspB albumen.What is interesting is, cause the enterorrhagia colon bacillus 0157 and adhere to, do not enter people's ileocecum (HCT-8) epithelial cell but do not invade.The EspB of RDEC-1 with cause the colibacillary EspB of enterorrhagia, rather than enteropathogenicEscherichia coli is more similar, this may more give prominence to the effect of EspB in intrusion.These discoveries convincingly demonstrate, and the heterogeneity of Esp influences enteropathogenicEscherichia coli, RDEC-1 and cause the colibacillary infiltration capability of enterorrhagia.

The EspD sudden change also influences EspA and EspB secretion, and enteropathogenicEscherichia coli contains an open reading frame, and espD is between espA and espB.In order to confirm the effect of espD product in secretion, made up coding enteropathogenicEscherichia coli espA, the plasmid pMWespD of Δ espD (phase shift mutation) and espD in Bgl II site, and be imported into into RDEC-1 double-mutant strain AAF001AAB.The amount of enteropathogenicEscherichia coli EspA and EspB secretory protein, than low in AAF001AAB (pMWespAB), the latter is contained the complete enteropathogenicEscherichia coli espA of coding, the fragment of espD and espB gene in AAF001AAB (pMWespD).And infiltration capability has also reduced.These results show that destroying espD can influence enteropathogenicEscherichia coli EspA and the proteic secretion of EspB.In this embodiment, we find, the amount of espA and/or other secretory protein of espB sudden change also the minimizing, and this may be because due to their the brachymemma product.Compare with espA or espB mutant strain, the secretion level of espA and espB double-mutant strain has reduced more.Therefore, owing to disturb III type excretory system, may be influenced the secretion of middle EspA of AAF001AAB (pMW6espD) and EspB by the enteropathogenicEscherichia coli EspD of brachymemma.Directly excretory system is relevant therewith it be unclear that for EspD.

EnteropathogenicEscherichia coli and RDEC-1 secretory protein are subjected to the close control of temperature, and host's body temperature that this temperature is relevant with them is corresponding.Temperature can be regulated enteropathogenicEscherichia coli and be caused the expression of enterorrhagia intestinal bacteria 413/89-1 secretory protein.And temperature is increased to 37 ℃ from 20 ℃, and the expression of EspB is increased greatly.Because EspA and EspB albumen are subjected to suitable host's thermoregulation, wild-type enteropathogenicEscherichia coli and RDEC-1 strain can be seeded among the DMEM, produce secretory protein after under different temperature, cultivating, analyze by SDS-PAEG then.The enteropathogenicEscherichia coli secretory protein be expressed in 33 ℃ just obviously as seen, 36 ℃ reach maximum secretion level.39 ℃ of expression have reduced, and not seeing for 42 ℃ has secretory protein.Different therewith, the maximum of RDEC-1 secretory protein is expressed in 39 ℃, is still expressed at 42 ℃ of these albumen.These results show, in enteropathogenicEscherichia coli and RDEC-1, proteic maximum expression of Esp is that body temperature people (37 ℃) and rabbit (39 ℃) by their relevant hosts excites.

Conclusion is to excite host signal conducting path and intrusion to need this two kinds of albumen.The complementation test of being done with enteropathogenicEscherichia coli esp gene discloses, and by the host signal conduction incident that RDEC-1 and enteropathogenicEscherichia coli excite, it seems it is to be mediated by identical secretory protein.At last, the optimum expression of RDEC-1 and enteropathogenicEscherichia coli secretory protein is associated with the body temperature of their natural hosts.This can explain the host specificity of their strictnesses, and rabbit and other animal reason of not infecting enteropathogenicEscherichia coli.Zoogenetic infection research with RDEC-1 espA and espB strain are carried out will provide the information of relevant these secretory proteins to toxicity action, and may be useful to vaccine research.

Embodiment 8

Two kinds of rabbit enteropathogenicEscherichia colis (RDEC-1)

Secretory protein EspA and EspB are virulence factors

The purpose of present embodiment is proof EspA and the effect of EspB albumen in pathogenesis.In order to study the effect of these albumen in virulence, in the espA of rabbit enteropathogenicEscherichia coli bacterial strain RDEC-1 and espB, made up sudden change.

By oral cavity stomach approach to rabbit inoculation RDEC-1 and its espA and espB mutant strain childhood.Infect the back discovery of 1 week, most RDEC-1 are in caecum and colon.But the quantity of two mutant strains is less than its parent strain greatly in these tissues.In caecum, also observe RDEC-1 and adhere to sacculus rotundus (vesica be correlated with epithelium) specifically, also visible bacterial colonisation in the caecum, the sacculus rotundus that shows caecum is the important position of settling down of this pathogenic bacteria.EspA ^-And EspB ^-Strain is littler 70 and 8000 times than parent plant to the adhesion level of sacculus rotundus.These results show, the adhesive capacity of RDEC-1 and organize taxis to depend on this two kinds of Esp secretory proteins.And as if to effect of settling down and the pathogenesis of bacterium, EspB has more important role than EspA.This proves that first enteropathogenicEscherichia coli secretory protein EspA and the EspB relevant with exciting host cell signal conducting path also are that it settles down with virulence needed.

By following animal is infected: the bacterium of centrifugal collection overnight incubation is suspended in the 1ml phosphate-buffered saline again.Make New Zealanl white rabbit (body weight 1.0-1.6kg) overnight fasting, in stomach, inoculate 2.5% aseptic sodium bicarbonate of 5ml and 1ml RDEC-1 or espA or espB bacterial strain (2.5 * 10 with the oral cavity stomach tube then ¹⁰).Every rabbit was inoculated in second day the bacterium of same dosage.

Be performed as follows clinical evaluation: every rabbit is weighed every day,, and from the excrement ball, collect the bacterium that ight soil is discharged by the rectum cotton swab.The rectum cotton swab is being contained its half surface of rolling on the Mac Conkey culture plate of nalidixic acid.From the liquid manure of every 5 excrement balls of rabbit collection or same amount, be suspended in the slow middle salt solution of 3ml phosphoric acid, each ight soil suspension is got 0.1ml and is being contained the dull and stereotyped cultivation of do on the MacConkey plate of nalidixic acid.Mark by following growing state to nalidixic acid resistance bacterium colony: 0, there is not growth; 1, sparse bacterium colony is arranged; 2, fine and close bacterium colony is arranged; 3, the bacterium colony of confluent growth is arranged.

Be performed as follows sample of tissue and preparation: after by intravenous injection chlore-ammonia ketone and overdose vetanarcol administration kill animals, cut tissue immediately.

Quantity by bacterial colonization in the following mensuration intestinal tissue: get the intestinal segment (10cm) of removing caecum, make the binode bundle, between the part that binode is pricked, cut intestinal segment, then with the ice-cold phosphate-buffered saline flushing of 10ml at its near-end and far-end.Get 1g heavy-gravity caecum inclusion, be added in the 9ml phosphate-buffered saline.With the phosphate-buffered salt aqueous suspensions dilution that forms, and containing the dull and stereotyped cultivation of do on the MacConkeg plate of nalidixic acid.

Adhere to the bacterial number of intestinal tissue by following mensuration: the cork stopper punch tool with the 9mm diameter cuts tissue sample, with phosphate-buffered salt washing 3 times, put into the ice-cold phosphate-buffered saline of 2ml, and do homogenate with homogenizer, then the sample of serial dilution is done dull and stereotyped the cultivation on the MacConkey plate.Stick to every square centimeter of structural bacterial number by following calculating:

CFU/cm ²Number of bacteria * extension rate * 2ml/～0.452 on=every block of plate.

Embodiment 9

Set up the assay method of screening bacterium III type secretion inhibitor

The purpose of present embodiment provides a kind of measuring method that is used to screen bacterium III type secretion inhibitor.

With polynucleotide and several molecule of knowing of coding EspA polypeptide, comprise that the HSV sign merges.But from enteropathogenicEscherichia coli still secretory gene fusions.A kind of plasmid contains the espA gene regions of coding EspA (mediation III type secretion is needed) N-terminal part, and it has been blended in hsv (HSV) sequence of coding maker sequence, can buy at the antibody of flag sequence.This plasmid conversion is entered a bacterial strain, and this bacterial strain contains the espA sudden change, but still other enteropathogenicEscherichia coli excretory protein of III type excretory system is used in secretion.Collection contains the supernatant liquor of these fusion roteins, and to ELISA check-out console application of sample, the ELISA that carries out standard measures.Fusion rotein has been secreted in the indication of colorimeter reading.

This plasmid is also entered by conversion and lacks III type excretory bacterial strain (being negative control).When this fusion rotein was expressed in this strain bacterium, this fusion rotein was not secreted by expression.The ELISA measurement result of doing with this mutant strain confirms that it is not secreted.

Therefore, provide easily a kind of, be used to check excretory ELLSA assay method.This detection method is easy, does not need special technique.This method also is economical, because do not need expensive reagent.This method is automatization, can be used for screening the reagent of differentiating bacterium III type secretion inhibitor.

In order to measure secretion, make bacterium static overnight incubation in having the tissue culture medium of testing compound.These conditions can cause the secretion of enteropathogenicEscherichia coli mediation.Degermed by centrifugal remove in second day, this supernatant liquor is placed 96 hole microtiter plates.The ELLSA that this supernatant liquor is carried out standard measures.If this testing compound is germ-resistant, this bacterium can not grow overnight.

Polynucleotide and several molecule of knowing of another polypeptide of coding enteropathogenicEscherichia coli excretory are merged.The polynucleotide of coding EspB polypeptide are blended in the HSV sign, can buy the antibody of this tag sequence.But enteropathogenicEscherichia coli is the secretory gene fusion product still.This plasmid conversion is entered a bacterial strain, and this bacterial strain contains the espA sudden change, and still secretes other enteropathogenicEscherichia coli secretory protein that uses III type excretory system.Collection contains the bacterium supernatant liquor of these fusions, and to ELISA check-out console application of sample, the ELISA that carries out standard with for example anti--HSV antibody measures.This sieve method can be used for measuring to have by oneself the extract of medical functions plant.The extent of dilution of 1/200-1/1000 (about 250 μ g/ml) is suitable.Can repeat screening by this ELISA secretion assay method to compound likely, so that check its reproducibility.

For referencial use invention has been described though used existing preferred embodiment, should be realized that various modification will could realize according to marrow of the present invention.Therefore, the present invention is only limited by following claim.

Claims (44)

1. isolating EspA polypeptide is characterized in that:

A) be a kind of from enteropathogenicEscherichia coli or cause the colibacillary secreted protein of enterorrhagia; And

B) comprise the aminoacid sequence of listing as in SEQ ID NO:2 or SEQ ID NO:4.

2. the isolating polynucleotide of claim 1 polypeptide of encoding.

3. one kind is selected from following isolating polynucleotide:

A) nucleotide sequence of listing among the SEQ ID NO:1;

B) nucleotide sequence of listing among the SEQ ID NO:1, T wherein becomes U;

C) and a) complementary nucleotide sequence, and

D) a), b) or c) fragment, its length is at least 15 nucleotide bases, and can be under stringent condition with coding SEQ ID NO:2 in the DNA hybridization of the polypeptide listed.

4. one kind is selected from following isolating polynucleotide:

A) nucleotide sequence of listing among the SEQ ID NO:3;

B) nucleotide sequence of listing among the SEQ ID NO:3, T wherein becomes U;

C) and a) complementary nucleotide sequence, and

D) a), b) or c) fragment, its length is at least 15 nucleotide bases, and can be under stringent condition with coding SEQ ID NO:4 in the DNA hybridization of the polypeptide listed.

5. carrier that contains claim 2 polynucleotide.

6. host cell that contains claim 5 carrier.

One kind with claim 1 polypeptide bonded anti--EspA antibody.

8. the antibody of claim 7, wherein this antibody is monoclonal antibody.

9. the antibody of claim 7, wherein this antibody is polyclonal antibody.

10. the method for EspA polypeptide in the test sample comprises:

A) sample is contacted with the antibody of claim 7; With

B) test right requires combining of 7 antibody and EspA polypeptide, and wherein this combination is the indication that has the EspA polypeptide in the sample.

11. the method for claim 10, sample wherein is a tissue.

12. the method for claim 10, sample wherein is a biological liquid.

13. the method for claim 10, the wherein indication that exists the EspA polypeptide to be infected in the sample by enteropathogenicEscherichia coli.

14. the method for claim 10 wherein exists the EspA polypeptide to be caused the indication of enterorrhagia coli-infection in the sample.

15. an immune host method, the disease sensitivity of this host to causing by the intestinal bacteria that produce EspA, this method comprises:

A) this host is given the EspA polypeptide of claim 1; With

B) bring out the protective immunological reaction of host to EspA.

16. the method for claim 15, the microorganism that wherein produces EspA is intestinal bacteria.

17. the method for claim 16, the intestinal bacteria that wherein produce EspA are enteropathogenicEscherichia colis.

18. the method for claim 16, the intestinal bacteria that wherein produce EspA are to cause the enterorrhagia intestinal bacteria.

19. the method for the disease that an alleviation is caused by the microorganism that produces EspA comprises:

A) with the polypeptide immune host of claim 1; With

B) bring out the immunne response of host, thereby alleviate the disease that causes by the infected by microbes host who produces EspA the EspA polypeptide.

20. the method for claim 19, the microorganism that wherein produces EspA is intestinal bacteria.

21. the method for claim 19, the intestinal bacteria that wherein produce EspA are enteropathogenicEscherichia colis.

22. the method for claim 19, the intestinal bacteria that wherein produce EspA are to cause the enterorrhagia intestinal bacteria.

23. the method for espA polynucleotide in the test sample comprises:

A) make and suspect the sample contain the espA polynucleotide and can contact with the nucleic acid probe of the espA multi-nucleotide hybrid of claim 2; With

B) detect the hybridization of this probe and espA polynucleotide, wherein detecting of this hybridization is the indication that has the espA polynucleotide in the sample.

24. a recombination method that is used to produce the espA polynucleotide comprises: the nucleic acid of the selected marker of will encoding inserts the polynucleotide of claim 2, makes the polynucleotide encoding of formation contain the reorganization EspA polypeptide of selected marker.

25. polynucleotide that produce by the method for claim 24.

26. host cell that contains the polynucleotide of claim 25.

27. a recombination method that is used to produce the EspA polypeptide comprises:

A) make the host cell of the polynucleotide that contain coding claim 1EspA polypeptide, incubation growth under the condition that allows its expression and secretion EspA polypeptide; With

B) separate this polypeptide.

28. a method of differentiating the compound that suppresses bacterium III type excretory system comprises:

A) polynucleotide with claim 25 import the bacterium with bacterium III type excretory system;

B) make this bacterium allow bacterial growth justacrine incubation growth under the condition of the polypeptide of polynucleotide encoding thus;

C) compound that makes suspection can suppress bacterium III type excretory system contacts with this bacterium;

D) bring out this polypeptide expression; With

E) detect the secretion of this polypeptide, wherein lacking secretion is that bacterium III type excretory system is had inhibiting indication.

29. one kind is used to produce the non-virulent method of microorganism, comprises:

A) in the polynucleotide of EspA polypeptide of coding claim 1, produce sudden change;

B) will the encode nucleotide sequence of selected marker inserts this mutational site;

C) the espA polynucleotide that suddenly change in the step b) are imported the karyomit(e) espA gene of microorganism, so that in karyomit(e) espA gene, produce sudden change; And

D) select microorganism with this sudden change.

30. the method for claim 29, the nucleic acid sequence encoding of the selected marker of wherein encoding is to the resistance of kantlex.

31. the method for claim 29, microorganism wherein is intestinal bacteria.

32. one kind produces by claim 29 method, has the microorganism of sudden change espA gene.

33. one kind is used for the test kit that test right requires 1 EspA polypeptide, comprise being separated the loading appliance that can hold one or more compact package containers, described container comprise one be equipped with can with the container of EspA polypeptide bonded antibody.

34. the test kit of claim 33, antibody wherein is detectable label.

35. the test kit of claim 34, mark wherein are to be selected from radio isotope, bioluminescent compound, chemiluminescence compound, fluorescent chemicals, metal chelator, and enzyme.

36. one kind is used for the test kit that test right requires 2 espA polynucleotide, comprise being separated the loading appliance that can hold one or more compact package containers, described container comprise one be equipped with can with the container of the nucleic acid probe of espA multi-nucleotide hybrid.

37. one kind produces by claim 34 method, has the microorganism of sudden change espA gene.

38. a test kit that is used to detect the EspA polypeptide comprises being separated the loading appliance that can hold one or more compact package containers, described container comprise one be equipped with can with the container of EspA polypeptide bonded antibody.

39. the test kit of claim 38, antibody wherein is detectable label.

40. the test kit of claim 39, mark wherein are to be selected from radio isotope, bioluminescent compound, chemiluminescence compound, fluorescent chemicals, metal chelator, and enzyme.

41. a test kit that is used to detect the espA polynucleotide comprises being separated the loading appliance that can hold one or more compact package containers, described container comprise one be equipped with can with the container of the nucleic acid probe of espA multi-nucleotide hybrid.

42. the test kit of claim 41, probe wherein is a detectable label.

43. the test kit of claim 42, mark wherein are to be selected from radio isotope, bioluminescent compound, chemiluminescence compound, fluorescent chemicals, metal chelator, and enzyme.

44. a method that produces the EspA fusion rotein comprises:

A) host cell is grown under the condition that allows its expression and secretion fusion rotein, this host cell contains the polynucleotide of the EspA that encode, these polynucleotide be operably connected to encode the polynucleotide of the polypeptide of studying or peptide; With

B) isolate this fusion rotein.

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