CN1261445C - Nucleotide specific for escherichia coli 0156 O-antigen - Google Patents

Abstract

The present invention provides a nucleotide specific to an O-antigen of Escherichia coli O156, which is a complete nucleotide sequence of a gene cluster for controlling the synthesis of the O-antigen in Escherichia coli 0140, such as the separated nucleotide disclosed in SEQ ID NO: 1 with the full length of 15390 basic groups, or the nucleotide of SEQ ID NO: 1 comprising one or a plurality of inserted, deleted or substituted basic groups and maintaining the functions of the separated nucleotide simultaneously. The present invention also comprises an oligonucleotide of a glycosyltransferase gene and an oligosaccharide unit processing gene derived from an O-antigen gene cluster of Escherichia coli O156. In the present invention, PCR indicates that the oligonucleotide has high specificity on the O-antigen of Escherichia coli O156. The present invention also discloses a method for detecting and identifying Escherichia coli O156 in human bodies and in environments by the oligonucleotide of the present invention.

Description

Nucleotide to the O-antigen-specific of intestinal bacteria O156 type

Technical field

The present invention relates to the complete nucleotide sequence of control O-antigen synthetic gene cluster in the intestinal bacteria O156 type (Escherichia coli O156), particularly relate in the intestinal bacteria O156 type oligonucleotide in the control O-antigen synthetic gene cluster, can utilize these the oligonucleotide of the O-antigen-specific intestinal bacteria O156 type in human body and the environment and identify O-antigen in these pathogenic bacterium quickly and accurately.

Background technology

O-antigen is the O specific polysaccharide composition in the gram negative bacterium lipopolysaccharides, and it is made up of many multiple oligosaccharide unit.The antigenic building-up process of O-is studied clearlyer: by glycosyltransferase nucleoside diphosphate monose is transferred on the fat molecule that is fixed on the cell inner membrance earlier, then in the inboard synthesis of oligose unit of inner membrance, the antigenic oligosaccharide unit of O-is transferred to the inner membrance outside by the transhipment enzyme again, then aggregate into polysaccharide by polysaccharase, be connected to again and form lipopolysaccharide molecule [Whitfield, C. (1995) " Biosynthesis of lipopolysaccharide O antigens " .Trends inMicrobiology.3:178-185 on the glycolipid molecule; Schnaitman, C.A.and J.D.Klena. (1993) " Genetics oflipopolysaccharide biosynthesis in entericbacteria " .MicrobiologicalReviews, 57 (3): 655-682].Coding is responsible for the generally adjacent arrangement on karyomit(e) of gene of all enzyme molecules of O-antigen synthetic, form a gene cluster [Reeves, P.R., et al. (1935) " Bacterialpolysaccharide synthesis and gene nomenclature " Trends in Microbiology, 4:495-503].In Shigellae, intestinal bacteria and Salmonellas, O-antigen gene [Lei Wang.et al (2001) " Sequence analysis of four Shigella boydii O-antigenloci:implication for Escherichia coli and Shigella relationships " .Infection andImmunity, 11:6923-6930 bunch between galF and gnd gene; Lei Wang and Peter Reeves (2000) " The Escherichia coliO111 and Salmonella enterica O156 gene clusters:gene clusters encoding the samecolitose-containing O antigen are highly conserved " .Journal ofBacteriology.182:5256-5261].The O-antigen gene bunch contains three genoids: sugared synthesis path gene, glycosyltransferase gene, oligosaccharide unit treatment gene, the required nucleoside diphosphate monose of enzymic synthesis O-antigen of wherein sugared synthesis path genes encoding; Thereby the enzyme of glycosyltransferase gene coding forwards nucleoside diphosphate monose and other molecule to and makes monose aggregate into oligosaccharide unit on the monose; The oligosaccharide unit treatment gene comprises transhipment enzyme gene and pol gene, and they transfer to the bacterium inner membrance outside with oligosaccharide unit, and repolymerization becomes polysaccharide.Glycosyltransferase gene and oligosaccharide unit treatment gene only are present in the gene cluster of carrying these genes.The difference of monose in the O-antigen, between monose between the difference of link button and the oligosaccharide unit difference of link button constituted the antigenic diversity of O-, and the composition of monose, the link button between monose and the link button between the oligosaccharide unit are by the Gene Handling in the O-antigen gene bunch, so the O-antigen gene bunch has determined O-antigenic synthetic, has also determined the antigenic diversity of O-.

Because O-antigen is extremely strong antigen, be one of important paathogenic factor of intestinal bacteria, it has extremely strong diversity again simultaneously, and this enlightens us can study a kind of intestinal bacteria and good, highly sensitive method of the antigenic specificity of O-thereof of detecting quickly and accurately.With surperficial polysaccharide is that the serology immune response of target has been used to somatotype and the evaluation to bacterium always since the thirties in last century, is unique means of identifying pathogenic bacterium.This diagnostic method needs a large amount of antiserum(antisera)s, and the antiserum(antisera) general classes is incomplete, quantity not sufficient, and also there are some difficulties in a large amount of antiserum(antisera)s in preparation with in storing.On the other hand this method length consuming time, sensitivity is low, loss is high, poor accuracy, so, generally believe that now this traditional serology detection method will be that the modern molecular biology method replaces.1993, Luk, J.M.C et.al has identified the O-antigen [Luk of Salmonellas with the specific nucleotide sequence of Salmonellas (S.enterica) O-antigen gene bunch by PCR method, J.M.C.et.al. (1993) " Selective amplification of abequose andparatose synthase genes (rfb) by polymerase chain reaction for identification ofS.enterica major serogroups (A; B; C2; andD) ", J.Clin.Microbiol.31:2118-2123].Luk, the method for et.al is with corresponding to Salmonellas serotype E 1, D1 obtains the oligonucleotide special to the Salmonellas of different serotypes after the nucleotide sequence of the CDP-abequose in the A, the O-antigen of B and C2 and the synthetic gene of CDP-tyvelose is arranged.Nineteen thirty-five, Paton, the A.W et.al serotype [" Molecular microbiological investigation of an outbreak of Hemolytic-Uremic Syndrome caused by dry fermented sausage contaminated with Shiga-liketoxin producing Escherichia coli " .J.Clin.Microbiol.34:1622-1627] of the oligonucleotide that comes from the wbdI gene of the O-antigen-specific of E.coli O111 having been identified the toxogenic E.coli O111 of a strain, but afterwards studies show that Paton, the usefulness of A.W et.al comes from the oligonucleotide of wbdI gene and identifies that the method for the serotype of E.coli O111 has false positive results to occur.Bastin D.A.and Reeves, P.R. think, this is because the wbdI gene is sugared synthesis path gene [the Bastin D.A.andReeves of a supposition, P.R. (1995) Sequence and analysis of the O antigen gene (rfb) cluster ofEscherichia coli O111.Gene 164:17-23], and have this sugar in the antigenic structure of the O-of other bacterium, so sugared synthesis path gene is not a high special for O-antigen yet

Shigellae has 46 kinds of serotypes, but have only 33 kinds of different O-antigens, intestinal bacteria have 166 kinds of different O-antigen [Reeves, P.R (1992) " Variation in O antigens; niche specificselection and bacterial populations " .FEMS Microbiol.Lett, 100:509-516], the two sibship is very near, and there are 12 kinds to be intestinal bacteria and the total [Ewing of Shigellae, W.H. (1986) " Edwards and Ewing ' s identification ofthe Enterobacteriaceae " .Elsevier SciencePublishers, Amsterdam, The Netherlands; T.cheasty, et al. (1983) " Antigenicrelationships between the enteroinvasive Escherichia coli antigensO28ac; O122ac; O124; O136, O143, O144; O152 and and Shigella O antigens " J.clinMicrobiol, 17 (4): 681-684]

Summary of the invention

The Nucleotide that the purpose of this invention is to provide a kind of O-antigen-specific to intestinal bacteria O156 type.It is the Nucleotide in the O-antigen gene bunch of intestinal bacteria O156 type, is the special Nucleotide that comes from glycosyltransferase gene and transhipment enzyme gene and pol gene.

A time purpose of the present invention has provided the full length nucleotide sequence of the O-antigen gene bunch of intestinal bacteria O156 type.

Another object of the present invention has provided the gene of the O-antigen gene bunch that constitutes intestinal bacteria O156 type: the gene of transhipment enzyme is the wzx gene or with wzx the gene of identity function is arranged; Pol gene is the wzy gene or with wzy the gene of identity function is arranged; Fucose synthase gene manBC, gmd.fcl or and manBC, gmd.fcl has the gene of identity function; Seminose hydrolase gene gmm or the gene of identity function is arranged with gmm; Pyruvy1 transferase gene orf2 or the gene of identity function is arranged with orf2; Glycosyltransferase gene comprises orf3, orf5, orf6, the orf11 gene.

Another purpose of the present invention has provided oligonucleotide, and the gene that they come from encoding glycosyl transferring enzyme in the O-antigen gene bunch of intestinal bacteria O156 type respectively comprises off3, orf5, orf6, the orf11 gene; The gene that coming from coding transhipment enzyme is the wzx gene or with wzx the gene of identity function is arranged; The gene that comes from the coding polysaccharase is the wzy gene or with wzy the gene of identity function is arranged; Come from Fucose synthase gene manBC, gmd.fcl or and manBC, gmd.fcl has the gene of identity function; They are the oligonucleotide in the said gene, and length is at 10-20nt; They are special to the O-antigen of intestinal bacteria O156 type; Especially the oligonucleotide of listing in the table 1, they are high specials to the O-antigen of intestinal bacteria O156 type, and these oligonucleotide are also reconfigurable, the oligonucleotide after the combination also is a high special to the O-antigen of intestinal bacteria O156 type.

The above-mentioned oligonucleotide that a further object of the present invention provides can be used as primer and is used for nucleic acid amplification reaction, perhaps be used for hybridization as probe, perhaps be used to make gene chip or microarray, thereby pass through O-antigen and the detection and the identification of escherichia coli O156 type of these methods detections and identification of escherichia coli O156 type.

An also purpose of the present invention has provided the method for complete sequence of the O-antigen gene bunch of separating Escherichia coli O156 type.Can obtain the complete sequence of the O-antigen gene bunch of other bacteriums according to present method operation, the complete sequence of the gene cluster of the bacterium of other polysaccharide antigens that also can obtain to encode.

The objective of the invention is to realize by following technical scheme.

The present invention is to the Nucleotide of the O-antigen-specific of intestinal bacteria O156 type, and it is the isolating Nucleotide shown in SEQ ID NO:1,15390 bases of total length; The base that perhaps has one or more insertions, disappearance or replacement keeps the Nucleotide of the SEQ ID NO:1 of described isolating functional nucleotide simultaneously.

The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O156 type, it is by 12 genomic constitutions, all between galF gene and gnd gene.

The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O156 type, wherein said gene is: the gene of transhipment enzyme comprises the wzx gene or with wzx the gene of identity function is arranged; Pol gene wzy gene or the gene of identity function is arranged with wzy; Fucose synthase gene manBC, gmd.fcl or and manBC, gmd.fcl has the gene of identity function; Seminose hydrolase gene gmm or the gene of identity function is arranged with gmm; Pyruvy1 transferase gene orf2 or the gene of identity function is arranged with orf2; Glycosyltransferase gene comprises orf3, orf5, orf6, the orf11 gene; Wherein said gene: wzx is the Nucleotide of 1366 to 2505 bases among the SEQID NO:1; Wzy is the Nucleotide of 4783 to 5988 bases among the SEQ ID NO:1; ManBC, gmd.fcl are respectively 12459 to 13883,10301 to 11707,7744 to 8862,8866 to 9831 among the SEQ ID NO:1; Orf2 is the Nucleotide of 8866 to 9831 bases among the SEQ ID NO:1; Gmm is the Nucleotide of 9834 to 10295 bases among the SEQ ID NO:1; Glycosyltransferase gene comprises orf3, orf5, orf6, and the orf11 gene is respectively the Nucleotide of 3532 to 4773,5960 to 6667,6664 to 7743,11707 to 12453 bases among the SEQ ID NO:1.

The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O156 type, wherein it is to come from described wzx gene, wzy gene, Fucose synthase gene manBC, gmd.fcl or and manBC, gmd.fcl has the gene of identity function; Seminose hydrolase gene gmm or the gene of identity function is arranged with gmm; Pyruvyl transferase gene orf2 or the gene of identity function is arranged with orf2; Glycosyltransferase gene comprises orf3, orf5, orf6, the orf11 gene; And their mixing or their reorganization.

The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O156 type, the oligonucleotide of the described wzx of coming from gene is to being: the Nucleotide of 1512 to 1532 bases among the SEQ ID NO:1 and the Nucleotide of 1846 to 1864 bases; The Nucleotide of 1851 to 1869 bases among the SEQ ID NO:1 and the Nucleotide of 2229 to 2247 bases.The oligonucleotide that comes from the wzy gene is to being: the Nucleotide of 5209 to 5227 bases among the SEQ ID NO:1 and the Nucleotide of 5840 to 5858 bases; The Nucleotide of 5594 to 5614 bases among the SEQ ID NO:1 and the Nucleotide of 5865 to 5885 bases.

The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O156 type is detecting the application of expressing the antigenic bacterium of O-, identify other polysaccharide antigen of the O-antigen of bacterium and bacterium in diagnosis.

The recombinant molecule of the Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O156 type, and can provide the O-antigen of expressing intestinal bacteria O156 type by inserting to express, and become bacterial vaccine.

The application of the Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O156 type, wherein it is used for PCR, is used for hybridization and fluoroscopic examination or is used to make gene chip or microarray, the bacterium in human body and the environment as probe as primer.

The separation method of the Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O156 type is characterized in that it comprises the steps:

(1) genomic extraction: 37 ℃ of incubated overnight intestinal bacteria O156 types in the LB of 5mL substratum, centrifugal collecting cell.With 500ul 50mM Tris-HCl (pH8.0) and 10ul 0.4M EDTA re-suspended cell, 37 ℃ of incubations 20 minutes, the N,O-Diacetylmuramidase that adds 10ul 10mg/ml then continues insulation 20 minutes.The Proteinase K, the 15ul 10%SDS that add 3ul 20mg/ml afterwards, 50 ℃ of incubations 2 hours, the RNase that adds 3ul 10mg/ml again, 65 ℃ of incubations 30 minutes, add equal-volume phenol extracting mixture, get supernatant and use isopyknic phenol again: chloroform: primary isoamyl alcohol (25: 24: 1) mixing solutions extracting twice, get supernatant again with isopyknic ether extracting to remove remaining phenol.Supernatant rolls out DNA and washes DNA with 70% ethanol with glass yarn with 2 times of volume ethanol deposit D NA, and DNA is resuspended among the 30ul TE; Genomic dna detects by 0.4% agarose gel electrophoresis;

(2) by the O-antigen gene in the pcr amplification intestinal bacteria O156 type bunch: with the genome of intestinal bacteria O156 type is that template is passed through its O-antigen gene of Long pcr amplification bunch, at first according to the JumpStart sequences Design upstream primer (#1523-ATT GTGGCT GCA GGG ATC AAA GAA AT) that often is found in O-antigen gene bunch promoter region, again according to the gnd gene design downstream primer (#1524-TAG TCG CGT GNG CCT GGA TTA AGT TCG C) in O-antigen gene bunch downstream; With the Expand Long Template PCR method of Boehringer Mannheim company amplification O-antigen gene bunch, the PCR response procedures was as follows: 94 ℃ of pre-sex change 2 minutes; 94 ℃ of sex change are 10 seconds then, annealed 15 seconds for 60 ℃, 68 ℃ were extended 15 minutes, carry out 30 circulations like this, last, continue to extend 7 minutes at 68 ℃, obtain the PCR product, detect the size and the specificity thereof of PCR product with 0.8% agarose gel electrophoresis, merge 5 pipe longPCR products, and with the Wizard PCR Preps purification kit purified pcr product of Promega company;

(3) make up O-antigen gene bunch library: make up O-antigen gene bunch library with the Novagen DNaseI shot gun method that is modified, reaction system is a 300ng PCR purified product, 0.9ul 0.1M MnCl ₂, the DNaseI of the 1mg/ml of 1ul dilution in 1: 2000, reaction is carried out at room temperature, and enzyme is cut the dna fragmentation size is concentrated between the 1.5kb-3kb, then adds 2ul 0.1M EDTA termination reaction.Merge the same reaction system of 4 pipes, with isopyknic phenol extracting once, use isopyknic phenol: chloroform: primary isoamyl alcohol (25: 24: 1) mixing solutions extracting once, after using isopyknic ether extracting once again, dehydrated alcohol deposit D NA with 2.5 times of volumes, and wash precipitation with 70% ethanol, be resuspended at last in the 18ul water, in this mixture, add 2.5ul dNTP (1mMdCTP, 1mMdGTP, 1mMdTTP subsequently, 10mMdATP), 1.25ul the T4DNA polysaccharase of 100mM DTT and 5 units, 11 ℃ 30 minutes, enzyme is cut product mends into flush end, after 75 ℃ of termination reactions, add the Tth archaeal dna polymerase of 5 units and corresponding damping fluid thereof and system is expanded as 80ul, 70 ℃ of reactions 20 minutes make 3 of DNA ' end add the dA tail.This mixture is through the equal-volume chloroform: after primary isoamyl alcohol (24: 1) mixing solutions extracting and the extracting of equal-volume ether with 3 * 10 of Promega company ^-3The pGEM-T-Easy carrier connect 10 hours in 16 ℃, cumulative volume is 90ul.10 * the buffer of 9ul and the T4DNA ligase enzyme of 25 units are wherein arranged, use the dehydrated alcohol precipitation of the 3M NaAc (pH5.2) of 1/10 volume and 2 times of volumes to be connected mixture at last, wash precipitation with 70% ethanol again, be dissolved in after the drying and obtain connecting product in the 30ul water; Preparation method with the electric transformed competence colibacillus cell of Bi0-Rad company prepares the competence e.colidh5, get after 2-3ul connects product and 50ul competence bacillus coli DH 5 alpha mixes, forward in the electric shock cup of 0.2cm of Bi0-Rad company and shock by electricity, voltage is 2.5 kilovolts, time is 5.0 milliseconds to 6.0 milliseconds, the SOC substratum that adds 1ml after the electric shock immediately in cup makes the bacterium recovery, then bacterium is coated in and contains penbritin, on the LB solid medium of X-Gal and IPTG, 37 ℃ of incubated overnight, obtain blue white bacterium colony next day, with the white colony that obtains promptly the white clone forward on the LB solid medium that contains penbritin and cultivate, from each clone, extract plasmid simultaneously, and cutting the segmental size of evaluation insertion wherein with the EcoRI enzyme, the white that obtains clone group has constituted the O-antigen gene bunch library of intestinal bacteria O156 type;

(4) to the cloning and sequencing in the library: from the library, select 96 clones of insertion fragment more than 1kb and the insertion fragment among the clone is checked order with this lab A BI3730 type automatic dna sequencer, sequence reaches 100% fraction of coverage, thereby obtains all sequences of O-antigen gene bunch;

(5) splicing of nucleotide sequence and analysis: the Pregap4 and the splicing of Gap4 software of the Staden package software package of publishing with Britain Camb MRC (Medical Research Council) Molecular Biology Lab and edit all sequences, thus obtain the Nucleotide full length sequence of the O-antigen gene bunch of intestinal bacteria O156 type; The quality of sequence is mainly guaranteed by two aspects: 1) genome of intestinal bacteria O156 type is done 5 Long PCR reactions, mix these products then to produce the library, 2) to each base, guarantee high-quality fraction of coverage more than 3, after obtaining the nucleotide sequence of intestinal bacteria O156 type O-antigen gene bunch, with American National biotechnology information science center (The National Center forBiotechnology Information, NCBI) orffinder finds gene, find the reading frame of 12 openings, determine also that with the function of finding the reading frame that these are open what gene they are with the genetic comparison among the blast groupware and the GenBank, finish gene annotation with the Artemis software at Britain sanger center again, do accurate comparison between DNA and protein sequence with Clustral W software, obtain the structure of the O-antigen gene bunch of intestinal bacteria O156 type at last;

(6) specific gene screening: at wzx, the wzy gene design primer in the O-antigen gene of dysentery intestinal bacteria O156 type bunch; Respectively designed two pairs of primers in each gene, every pair of primer is distributed in the different places in the corresponding gene, to guarantee its specificity; Is that template is carried out PCR with these primers with the genomes of 166 strain intestinal bacteria and 43 strain Shigellaes, except that a band that in the 13rd group, has obtained the expection size, any product that all do not increase in other groups is so the O-antigen of wzx, wzy gene pairs intestinal bacteria O156 type all is high special.

Just, first aspect of the present invention provides the full length nucleotide sequence of the O-antigen gene bunch of intestinal bacteria O156 type, its complete sequence shown in SEQ ID NO:1,15390 bases of total length; The base that perhaps has one or more insertions, disappearance or replacement keeps the Nucleotide of the SEQ ID NO:1 of described isolating functional nucleotide simultaneously.Obtained the structure of the O-antigen gene bunch of intestinal bacteria O156 type by method of the present invention, as shown in table 3, it is altogether by 12 genomic constitutions, all between galF gene and gnd gene.

Second aspect of the present invention provides the gene in the O-antigen gene bunch of intestinal bacteria O156 type, promptly transports enzyme gene (wzx gene or the gene of identity function arranged with wzx); Pol gene (wzy gene or the gene of identity function arranged with wzy); The Fucose synthase gene (manBC, gmd.fcl or and manBC, gmd.fcl has the gene of identity function); Seminose hydrolase gene (gmm or the gene of identity function arranged with gmm); Pyruvyl transferase gene (orf2 or the gene of identity function arranged with orf2); Glycosyltransferase gene, (comprise orf3, orf5, orf6, the orf11 gene or with orf3, orf5, orf6, orf11 has the gene of identity function).Their zero positions in O-antigen gene bunch and final position and nucleotide sequence all are listed in the table 4; The invention particularly relates to glycosyltransferase gene, transhipment enzyme gene and pol gene, because sugared synthesis path gene is that the gene of synthetic nucleosides bisphosphate monose is common, common by indication to more exocellular polysaccharide now, O-antigen to bacterium is not very special, and the glycosyltransferase gene that the present invention relates to, transhipment enzyme gene and pol gene are high specials to the O-antigen of intestinal bacteria O156 type.

The 3rd aspect of the present invention, wzx gene in the O-antigen gene bunch that comes from intestinal bacteria O156 type is provided or the gene, wzy gene of identity function is arranged or gene, the Fucose synthase gene manBC of identity function are arranged with wzy with wzx, gmd.fcl or and manBC, gmd.fcl has the gene of identity function; Seminose hydrolase gene gmm or the gene of identity function is arranged with gmm; Pyruvyl transferase gene orf2 or the gene of identity function is arranged with orf2; Glycosyltransferase gene comprises orf3, orf3, orf6, the orf11 gene; They are any one section oligonucleotide in these genes.But, be that the oligonucleotide of listing in the table 1 is right preferentially by usefulness, in table 1, also listed these oligonucleotide to the position in O-antigen gene bunch and with these oligonucleotide to being the size of the product of the PCR reaction done of primer, the annealing temperature in these PCR reaction free lists is carried out.These primers are except that a band that has obtained the expection size in the 13rd group, and any product that all do not increase in other groups is so the O-antigen of wzx, wzy gene pairs intestinal bacteria O156 type all is high special.

The separation method of the Nucleotide of described O-antigen-specific to intestinal bacteria O156 type comprises the steps: 1) genomic extraction; 2) the O-antigen gene in the pcr amplification intestinal bacteria O156 type bunch; 3) make up O-antigen gene bunch library; 4) to the cloning and sequencing in the library; 5) splicing of nucleotide sequence and analysis; 6) screening of specific gene.

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

As used herein, " oligonucleotide " mainly refers to derive from gene, the gene of coding transhipment enzyme and intragenic one section nucleic acid molecule of coding polysaccharase of the encoding glycosyl transferring enzyme in the O-antigen gene bunch, they can change on length, generally change in 10 to 20 Nucleotide scopes; More definite these oligonucleotide of saying are to come from wzx gene (nucleotide position is the Nucleotide of 1366 to 2505 bases from SEQ ID NO:1); Wzy gene (nucleotide position is the Nucleotide of 4783 to 5988 bases from SEQ ID NO:1); Fucose synthase gene manBC, gmd.fcl (being respectively the Nucleotide of 12459 to 13883,10301 to 11707,7744 to 8862,8866 to 9831 bases among the SEQ ID NO:1); Seminose hydrolase gene gmm (being the Nucleotide of 9834 to 10295 bases among the SEQ ID NO:1); Orf2 (being the Nucleotide of 8866 to 9831 bases among the SEQ ID NO:1); Glycosyltransferase gene comprises orf3, orf5, orf6, orf11 gene (being respectively the Nucleotide of 3532 to 4773,5960 to 6667,6664 to 7743,11707 to 12453 bases among the SEQ ID NO:1); Coming from above intragenic oligonucleotide is high special to intestinal bacteria O156 type.

In addition, the antigenic gene cluster of the different O-of the coding of two genetic resemblances produces new O-antigen by gene recombination or sudden change sometimes, thereby produces new bacteria types, new mutant strain.In this environment, need filter out many specificitys that oligonucleotide is detected with raising with recombination hybridization.Therefore, the invention provides a whole set of many mixtures to oligonucleotide, they come from glycosyltransferase gene; Come from transhipment enzyme and pol gene, comprise the wzx gene or the gene, wzy gene of identity function arranged or the gene of identity function is arranged with wzy with wzx.The mixture of these genes is special to a special bacterial polysaccharides antigen, is special thereby make this cover oligonucleotide to the polysaccharide antigen of this bacterium.More particularly, the mixture of these oligonucleotide is to come from glycosyltransferase gene, wzx gene or the gene, wzy gene of identity function arranged or with wzy the combination of the oligonucleotide in the gene of identity function is arranged with wzx.

On the other hand, the present invention relates to the evaluation of oligonucleotide, they can be used for detecting the O-antigen of expressing the antigenic bacterium of O-and identifying bacterium in diagnosis.

The present invention relates to a kind of antigenic method of one or more bacterial polysaccharideses that detects in the food, these antigens can make sample can with the oligonucleotide specific hybrid of following at least one gene, these genes are: (i) gene of the encoding glycosyl transferring enzyme gene of transhipment enzyme and polysaccharase of (ii) encoding comprises the wzx gene or the gene, wzy gene of identity function is arranged or with wzy the gene of identity function is arranged with wzx.At least one oligonucleotide can be hybridized with at least one more than one such gene specific of expressing the special antigenic bacterium of O-under the situation of condition permission, and these bacteriums are intestinal bacteria O156 types.Available PCR method detects, more can with behind the Nucleotide mark in the inventive method as probe by hybridization such as southern-blot or fluoroscopic examination, perhaps by antigen and bacterium in gene chip or the microarray assay sample.

Planner of the present invention considers following situation: when one special oligonucleotide detects when invalid, the mixture of oligonucleotide can with the target region specific hybrid with test sample.Therefore the invention provides a cover oligonucleotide and be used for detection method of the present invention.Here said oligonucleotide is meant the gene that comes from the encoding glycosyl transferring enzyme, the gene of coding transhipment enzyme and the gene of polysaccharase, comprises the wzx gene or the gene, wzy gene of identity function is arranged or with wzy the oligonucleotide of the gene of identity function is arranged with wzx.This cover oligonucleotide is special to the O-antigen of a special bacterium, and this special bacterium O-antigen is expressed by intestinal bacteria O156 type.

On the other hand, the present invention relates to a kind of antigenic method of one or more bacterial polysaccharideses that detects in the movement, these antigens can make sample can with the oligonucleotide specific hybrid of following at least one gene, these genes are: (i) gene of the encoding glycosyl transferring enzyme gene of transhipment enzyme and polysaccharase of (ii) encoding comprises the wzx gene or the gene, wzy gene of identity function is arranged or with wzy the gene of identity function is arranged with wzx.At least one oligonucleotide can be expressed more than one such gene specific hybridization of the special antigenic bacterium of O-with at least one under the situation of condition permission.These bacteriums are intestinal bacteria O156 types.Oligonucleotide among available the present invention is made the method test sample of primer by PCR, also can with behind the oligonucleotide molecules mark among the present invention as probe by hybridization such as southern-blot or fluoroscopic examination, perhaps by antigen and bacterium in gene chip or the microarray assay sample.

General a pair of oligonucleotide may with same gene recombination also can with different gene recombinations, but must have in them an oligonucleotide can specific hybrid to the distinguished sequence of special antigenic type, another oligonucleotide can be hybridized in non-specific zone.Therefore, when the oligonucleotide in the special polysaccharide antigen gene cluster is reconfigured, can select specific gene mixture hybridization in a pair of oligonucleotide and the polysaccharide antigen gene cluster at least, perhaps select many mixture hybridization oligonucleotide and specific gene.Even even when all genes were all unique in the specific genes bunch, this method also can be applied to discern the nucleic acid molecule of the gene mixture in this gene cluster.Therefore the invention provides a whole set of is used to detect the many to oligonucleotide of the inventive method, many here is that the gene of the gene that comes from the encoding glycosyl transferring enzyme, coding transhipment enzyme and polysaccharase comprises the wzx gene or the gene, wzy gene of identity function arranged or with wzy the gene of identity function is arranged with wzx to oligonucleotide, this cover oligonucleotide is special to a special bacterial polysaccharides, and this cover oligonucleotide may be the Nucleotide of necessary gene during sugar synthesizes.

On the other hand, the present invention also relates to the antigenic method of one or more bacterial polysaccharideses in the sample that a kind of detection comes from patient.One or more bacterial polysaccharides antigens in the sample can make sample can with a specific hybrid in a pair of oligonucleotide in following at least one gene, these genes are: (i) gene of the encoding glycosyl transferring enzyme gene of transhipment enzyme and polysaccharase of (ii) encoding comprises the wzx gene or the gene, wzy gene of identity function is arranged or with wzy the gene of identity function is arranged with wzx.Under the situation of condition permission at least one oligonucleotide can with sample at least one express more than one such gene specific hybridization of the special antigenic bacterium of O-, these bacteriums are intestinal bacteria O156 types.Oligonucleotide among available the present invention is made the method test sample of primer by PCR, also can will pass through hybridization as probe behind the oligonucleotide mark among the present invention, perhaps by antigen and bacterium in gene chip or the microarray assay sample.

In more detail, method described above can be understood as when oligonucleotide when being used, it is not to derive from glycosyltransferase gene, wzx gene or with wzx the gene, wzy gene of identity function arranged or have on the sequence of gene of identity function with wzy that one of them oligonucleotide molecules can hybridize to one.In addition, when two oligonucleotide can both be hybridized, they may be hybridized in same gene and also may hybridize on the different genes.Also promptly, when cross reaction goes wrong, can select the mixture of oligonucleotide to detect the blended gene so that the specificity of detection to be provided.

The present inventor believes that the present invention is not necessarily limited to the above nucleotide sequence coded specific O-antigen of carrying, and is widely used in detecting all expression O-antigens and identifies the antigenic bacterium of O-.And because O-antigen is synthetic and the similarity of other polysaccharide antigens (as bacterium born of the same parents exoantigen) between synthesizing, method of the present invention and molecule also are applied to these other polysaccharide antigen.

The present invention discloses the full length sequence of the O-antigen gene bunch of intestinal bacteria O156 type first, and can from the sequence of this total length gene cluster of not cloned, produce recombinant molecule, can produce the O-antigen of expressing intestinal bacteria O156 type by inserting to express, and become useful vaccine.

Embodiment

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

Embodiment 1: genomic extraction:

37 ℃ of incubated overnight intestinal bacteria O156 types in the LB of 5mL substratum, centrifugal collecting cell.With 500ul 50mM Tris-HCl (pH8.0) and 10ul 0.4M EDTA re-suspended cell, 37 ℃ of incubations 20 minutes, the N,O-Diacetylmuramidase that adds 10ul 10mg/ml then continues insulation 20 minutes.The Proteinase K, the 15ul 10%SDS that add 3ul 20mg/ml afterwards, 50 ℃ of incubations 2 hours add the RNase of 3ul 10mg/ml again, 65 ℃ of incubations 30 minutes.Add equal-volume phenol extracting mixture, get supernatant and use isopyknic phenol again: chloroform: primary isoamyl alcohol is taken out (25: 24: 1) mixing solutions and is carried twice, get supernatant again with isopyknic ether extracting to remove remaining phenol, supernatant is with 2 times of volume ethanol deposit D NA, roll out DNA and wash DNA with glass yarn, at last DNA is resuspended among the 30ul TE with 70% ethanol.Genomic dna detects by 0.4% agarose gel electrophoresis.

Embodiment 2: by the O-antigen gene in the pcr amplification intestinal bacteria O156 type bunch:

With the genome of intestinal bacteria O156 type is that template is passed through its O-antigen gene of Long pcr amplification bunch.At first according to the JumpStart sequences Design upstream primer (#1523-ATT GTG GCT GCA GGG ATC AAA GAA AT) that often is found in O-antigen gene bunch promoter region, again according to the gnd gene design downstream primer (#1524-TAG TCG CGT GNG CCTGGA TTA AGT TCG C) in O-antigen gene bunch downstream; With the Expand Long TemplatePCR method of Boehringer Mannheim company amplification O-antigen gene bunch, the PCR response procedures was as follows: 94 ℃ of pre-sex change 2 minutes; 94 ℃ of sex change are 10 seconds then, 60 ℃ of annealing 15 seconds, and 68 ℃ were extended 15 minutes, and carried out 30 circulations like this.At last, continue to extend 7 minutes at 68 ℃, obtain the PCR product, the agarose gel electrophoresis with 0.8% detects the size and the specificity thereof of PCR product.Merge 5 pipe long PCR products, and with the Wizard PCR Preps purification kit purified pcr product of Promega company.

Embodiment 3: make up O-antigen gene bunch library:

At first be the acquisition that connects product: make up O-antigen gene bunch library with the Novagen DNaseI shot gun method that is modified.Reaction system is a 300ng PCR purified product, 0.9ul 0.1M MnCl ₂, 1ul1: the DNaseI of the 1mg/ml of 2000 dilutions, reaction is carried out at room temperature.Enzyme is cut the dna fragmentation size is concentrated between the 1.5kb-3kb, then adds 2ul 0.1M EDTA termination reaction.Merge the same reaction system of 4 pipes, with isopyknic phenol extracting once, use isopyknic phenol: chloroform: primary isoamyl alcohol (25: 24: 1) mixing solutions extracting once, after using isopyknic ether extracting once again, dehydrated alcohol deposit D NA with 2.5 times of volumes, and wash precipitation with 70% ethanol, be resuspended at last in the 18ul water.In this mixture, add 2.5ul dNTP (1mMdCTP subsequently, 1mMdGTP, 1mMdTTP, 10mMdATP), the T4DNA polysaccharase of 1.25ul 100mM DTT and 5 units, 11 ℃ 30 minutes, enzyme is cut product mend into flush end, after 75 ℃ of termination reactions, add the Tth archaeal dna polymerase of 5 units and corresponding damping fluid thereof and system is expanded as 80ul, 70 ℃ were reacted 20 minutes, made 3 of DNA ' end add the dA tail.This mixture is through the equal-volume chloroform: after primary isoamyl alcohol (24: 1) mixing solutions extracting and the extracting of equal-volume ether with 3 * 10 of Promega company ^-3The pGEM-T-Easy carrier connect 10 hours in 16 ℃, cumulative volume is 90ul.10 * the buffer of 9ul and the T4DNA ligase enzyme of 25 units are wherein arranged.Use the dehydrated alcohol precipitation of the 3M NaAc (pH5.2) of 1/10 volume and 2 times of volumes to be connected mixture at last, wash precipitation with 70% ethanol again, be dissolved in after the drying and obtain connecting product in the 30ul water.

Next is the preparation of competent cell: the method that provides with reference to Bio-Rad company prepares competent cell bacillus coli DH 5.Get the single bacterium colony of a ring bacillus coli DH 5 in the LB of 5ml substratum, 180rpm cultivated after 10 hours, got in the LB substratum that the 2ml culture is transferred to 200ml, and 37 ℃ of 250rpm thermal agitations are cultivated OD600 about 0.5, ice bath cooling was 20 minutes then, in centrifugal 15 minutes of 4 ℃ of 4000rpm.Confide all supernatant, dispel thalline, in centrifugal 15 minutes of 4 ℃ of 4000rpm with the deionization aqua sterilisa 200ml of cold ice precooling.Deionization aqua sterilisa 100ml with cold ice precooling dispelled thalline again, in centrifugal 15 minutes of 4 ℃ of 4000rpm.With 10% glycerine suspension cell of cold ice precooling, centrifugal 10 minutes of 4 ℃ of 6000rpm abandon supernatant, precipitate 10% glycerine suspension cell with the precooling of 1ml ice at last, are competent cell.The competent cell that makes is packed as 50ul one pipe ,-70 ℃ of preservations.

Be electric transformed competence colibacillus cell at last: get after 2-3ul connects product and 50ul competence bacillus coli DH 5 mixes, forward in the electric shock cup of 0.2cm of Bio-Rad company and shock by electricity, voltage is 2.5 kilovolts, and the time is 5.0 milliseconds-6.0 milliseconds.The SOC substratum that adds 1ml after the electric shock immediately in cup makes the bacterium recovery.Immediately bacterium is coated in 37 ℃ of inversion incubated overnight on the LB solid medium that contains penbritin, X-Gal and IPTG then, obtains blue white bacterium colony next day.With the white colony that obtains promptly the white clone forward on the LB solid medium that contains penbritin and cultivate, from each clone, extract plasmid and cut the segmental size of evaluation insertion wherein simultaneously, obtain the O-antigen gene bunch library that white clone group has constituted intestinal bacteria O156 type with the EcoRI enzyme.

Embodiment 4: to the cloning and sequencing in the library:

From the library, select insert 96 clones of fragment more than 1kb with this lab A BI3730 type automatic dna sequencer to unidirectional order-checking of insertion fragment among the clone, make sequence reach 100% fraction of coverage, thus all sequences of acquisition O-antigen gene bunch.

Embodiment 5: the splicing of nucleotide sequence and analysis:

The Pregap4 and the splicing of Gap4 software of the Staden package software package of publishing with Britain Camb MRC (Medical Research Council) Molecular Biology Lab and edit all sequences, thus the Nucleotide full length sequence (seeing sequence list) of the O-antigen gene bunch of intestinal bacteria O156 type obtained.The quality of sequence is mainly guaranteed by two aspects: 1) genome of intestinal bacteria O156 type is done 5 Long PCR reactions, mix these products then to produce the library.2), guarantee high-quality fraction of coverage more than 3 to each base.After obtaining the nucleotide sequence of intestinal bacteria O156 type O-antigen gene bunch, with American National biotechnology information science center (The National Center for BiotechnologyInformation, NCBI) orffinder finds gene, find the reading frame of 12 openings, determine also that with the function of finding the reading frame that these are open what gene they are with the genetic comparison among the blast groupware and the GenBank, finish gene annotation with the Artemis software at Britain sanger center again, do accurate comparison between DNA and protein sequence with ClustralW software, obtain the structure of the O-antigen gene bunch of intestinal bacteria O156 type at last, as shown in table 3.

By retrieving and comparing, find orf7, Fucose synthase gene manBC in 8,10,12 encoded protein and the intestinal bacteria-antigen gene bunch, the albumen of gmd.fcl genes encoding has very high consensus amino acid sequence, manBC, gmd.fcl gene are responsible for the synthetic of Fucose, and Fucose is a kind of rare monose in the O-antigen, therefore other monose is responsible for the genes encoding of its synthetic enzyme by other positions of genome, and these genes do not repeat in O-antigen gene bunch.We are with orf7, and 8,9,12 distinguish called after gmd.fcl and manBC, and the existence of Fucose is arranged in the O-antigen of supposition intestinal bacteria O156, and the existence of Fucose is arranged in its O-antigen, and the analytical results of gene cluster can be corresponding with its structure.

Orfl and orf4 are the proteic genes that there is transmembrane segment in only two codings of intestinal bacteria O156 kind.The O-antigen transferring enzyme of Orf1 encoded protein and Yersinia pestis bacterium has 47% sequence identity, and it contains 12 uniform transmembrane segments by the proteic topology discovery of HMMTOP2.0 programanalysis, and this is the proteic characteristic feature of Wzx.So name orf1 is wzx.Orf4 encoded protein and intestinal bacteria O-antigen polysaccharase have 28% consistence, 45% similarity, it contains 9 transmembrane segments by the proteic topology discovery of HMMTOP2.0 programanalysis, and hydrophilic loop (loop) in big (61 an amino acid) kytoplasm is arranged, and this is the proteic characteristic feature of Wzy.So name orf4 is wzy.

Orf3, the albumen of 5,6,11 4 genes encodings and other known glycosyltransferases have the sequence identity of 24-36% and the sequence similarity of 44-53%.By the search to glycosyltransferase motif database among the Pfam, the homology desired value of the consensus sequence of the albumen of these four genes encodings and known glycosyltransferase family 1 and 2 is 1 * e ^-12To 5 * e ^-17, so we infer this four genes encoding glycosyltransferases, and because each glycosyltransferase specificity catalysis forms a kind of disaccharide bond, so we infer that the antigenic oligosaccharide unit of O-of intestinal bacteria O156 may be made up of four monose.Because the definite function of these four genes can't be determined, so we are with the temporary called after orf3 of these four genes, orf5, orf6, orf11.

Embodiment 6: the screening of specific gene:

At wzx, wzy gene design primer in the O-antigen gene of intestinal bacteria O156 type bunch, the position of these genes in nucleotide sequence sees Table 1.

Table 1 has been listed glycosyltransferase gene and oligosaccharide unit treatment gene and intragenic primer and PCR data in the O antigen gene bunch of intestinal bacteria O156 type.Glycosyltransferase gene, transhipment enzyme gene and pol gene and their function corresponding and the size of the O antigen gene bunch of intestinal bacteria O156 type in table, have been listed.In each gene, we have respectively designed two pairs of primers, and the difference that every pair of primer is distributed in the corresponding gene is local to guarantee its specificity.In table, also listed position and the size of each primer in SEQ IDNO:1.Is that template carry out PCR with listed corresponding annealing temperature in the table with the genomes of all bacterium in the table 2 with every pair of primer, has obtained corresponding PCR product, and its size is also listed in the table.

Table 2 is 166 strain intestinal bacteria and 43 strain Shigellaes and their sources that are used to screen specific gene, and for the convenience that detects, we are divided into one group with their every 8-10 bacterium, and 13 groups altogether, all list in the table in their source.

The genomic dna that contains intestinal bacteria O156 type in the 13rd group is as positive control.Do template with every group of bacterium, be PCR by following condition with every pair in the table 1 primer: 95 ℃ of pre-sex change after 5 minutes, 95 ℃ of sex change 30 seconds, annealing time is 30 seconds, temperature sees Table 1,72 ℃ extended 2 minutes, carried out 25 circulations like this.Continue to extend 5 minutes at 72 ℃ at last, reaction system is 25ul.Template is dilution in 1: 20, gets 1 μ l.After reaction finishes, get the 10ulPCR product and detect the fragment that amplifies by 0.8% agarose gel electrophoresis.

For wzx, wzy gene, each gene all has two pairs of primers detected, every pair of primer has obtained except be PCR in the 13rd group after the correct band of expection size, the correct band of any size does not all increase in other groups, that is to say, not obtaining any PCR product band in the array mostly, so wzx, wzy gene pairs intestinal bacteria O156 type and O-antigen thereof are high specials.

At last, from intestinal bacteria O156 type, screen gene by PCR: wzx, wzy and three glycosyltransferase genes to the O-antigen high special of intestinal bacteria O156 type.And the oligonucleotide of these intragenic any one section 10-20nt is special to the O-antigen of intestinal bacteria O156 type, and the primer in especially above-mentioned each gene is that oligonucleotide is high special to detecting the back confirmation through PCR to intestinal bacteria O156 type.These all oligonucleotide all can be used for the intestinal bacteria O156 type in the human body and environment rapidly and accurately, and can identify their O-antigen.

Table 3 is structural tables of the O-antigen gene bunch of intestinal bacteria O156 type, in table, listed the structure of the O-antigen gene bunch of intestinal bacteria O156 type, altogether by 12 genomic constitutions, each gene box indicating, and in square frame, write the title of gene, numeral be the order of the open reading frame (orf) in the O-antigen gene bunch.Two ends at O-antigen gene bunch are galF gene and gnd gene, and they do not belong to O-antigen gene bunch, and we are just with the increase full length sequence of O-antigen gene bunch of their one section sequences Design primer.

Table 4 is site plan of the gene in the O-antigen gene bunch of intestinal bacteria O156 type, listed the accurate position of all open reading frame in complete sequence in the O-antigen gene bunch of intestinal bacteria O156 type in the drawings, at the underscoring of the initiator codon and the terminator codon of each open reading frame.The initiator codon of open reading frame has two in intestinal bacteria: ATG and GTG.

SEQUENCE LISTING

＜110〉Nankai University

＜120〉to the Nucleotide of the O antigen-specific of intestinal bacteria O156 type

＜130〉to the Nucleotide of the O antigen-specific of intestinal bacteria O156 type

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<170>PatentIn version 3.2

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<211>15390

<212>DNA

<213>Escherichia coli

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attgtggctg cagggatcaa agaaatcatt gtggctgcag ggattaaaga aatcattgtg 60

gctgcaggga tcaaagaaat cctcctggta actcacgcgt ccaagaacgc ggtcgaaaac 120

cacttcgaca cctcttatga attagaatct ctccttgagc agcgcgcaaa gcgtcaacta 180

ctggcggaag tacagtccat ttgcccgccg ggcgtgacca ttatgaacgt gcgtcagggc 240

gaacccttag gtttaggcca ctccattttg tgtgcacgac ccgccattgg tgacaaccca 300

tttgtcgtgg tattgccaga cgttgtgatc gacgacgcca gcgccgaccc gctgcgctac 360

aaccttgctg ccatgattgc gcgtttcaat gaaacgggcc gtagccaggt gctggcaaaa 420

cgtatgccgg gtgacctctc tgaatactcc gttattcaga ccaaagaacc actggatcgt 480

gaaggtaaag tcagccgcat tgttgaattt atcgaaaaac cggatcagcc gcagacgctg 540

gactcagaca tcatggccgt tggtcgctat gtgctttctg cagatatttg gccggaacta 600

gaacgcactc agcctggtgc atgggggcgt attcagctga cagatgccat cgctgaactg 660

gcgaaaaaac agtccgttga tgccatgctg atgacaggtg acagctacga ctgcggtaaa 720

aaaatgggtt atatgcaggc atttgtgaag tatggactac gcaacctgaa agaaggggcg 780

aagttccgta aagggattga gaatctgtta agggattaat gaaaatctga ccggatgtaa 840

cggttgataa gaaaattata acggcagtga agattcgtgg cgaaagtaat ttgttgcgaa 900

tattcctgcc gttgttttat ataaacaatc agaataacaa cgagttagca ataggatttt 960

agtcaaagtt ttccaggatt ttccttgttt ccagagcgga ttggtaagac aattagcgtt 1020

tgaatttttc gggtttagcg cgagtgggta acgctcgtca catcgtaggc atgcatgcag 1080

tgctctggta gctgtaaagc caggtgcggt agcgtgggta atgtttacta cctatatagt 1140

tacgtgcaat gaaaaataag aataagtgct gaatgatttt gattatgaat attttagatt 1200

ttttataagg aggatattat aaatatagga actaaaatct ctatttatgc ggcattatct 1260

agaggtctta aaatattaag tggacccata ataattcttg ttgtttcaaa gacattaagt 1320

tcagaggaaa ttggatttta ttatactttt ttcagcttag taactatgca gcagttagct 1380

gaattaggtt tagggcaaac attaagattg catattgctc atgcgtttaa aatggacgag 1440

tcgggatggt gttttaactc aaaaaaagaa attaagcaat actattctct aagtgtaaaa 1500

tggttcttgg tcgttgcgtt atgtactatc attattattg gtcctgcggg tatttattat 1560

tatcattctt accaagggga tattatttgg gaaggcccat gggtatcatt agtattggtt 1620

acagcactaa caattataat aacgcctata caaatcacaa ttgaatctct acaacatcaa 1680

atttcagtat ttaaagcaca gacatatgca tcaattatca atagtatatc attatgtctg 1740

ttattgtttt ctggatttgg tctatatgca attgctttaa gcctatttat cagtaactta 1800

acagtcttaa tatttttatg ggggaaaata aaatggctga gaaataacct gatgcctgtt 1860

cttgttgata caaatttttt tgttgttttt aaagagatct ggccgctgtt ttctcgtgta 1920

gctgttgtat ctgtatttgg tttccttttt tggaatggat ttaatctaat tgcatttaaa 1980

gtatatgagc cctcatatgc aggacaagta attttatcca taaccttagc gagagctggc 2040

tatggatttt ctgagtcaat tttatatggg caaataacat acatatctaa tttgatttcg 2100

aacaaaaaat ataatgaggc tatgatagtt tataaaaaaa atacatatat agcactaacg 2160

attttgttaa taggatatgt gtttttttta cttcttatgt atcttttccc agaattctac 2220

ttatttaaaa aaatagtagg ctcaagctat ttaattagta tatttgtatt ttttataata 2280

gttctttttt taacctcagg aaataactat gtcagatgct ttaaaacaga accatttgta 2340

aaagtgtcca tctttcacgg ggtcatggta ccaacattat tttatttatc atcagttttt 2400

tatgagggtt attatttata tccatgttca ttggtgttga ttgtttctct aattttatca 2460

aaaagaatat caagcaagta ttttagaatg tccggcatta attaataata ggtgaaaagt 2520

gaataagaaa gataactttg tgaatatttt tgacacaagt attgcatcat tgaatgttgg 2580

tgattacatt attatggatg ctgtgaccaa acagttatct acaataagta atttaccgca 2640

gactgttaca ttaccaacgc atgaccattt tggtagagaa gggcgacgtt tacttagctt 2700

ggctaaatat tctattgttg gcggcactaa tttattatca tcgcatgtta cacaatatcg 2760

ccaatggcgg tttcgtatga cagatctttt atttttaaaa aaatgcatat taatgggagt 2820

tggttggtgg caatatcaag ataaaccaga cagagttaca aaatttattt tgaagcatat 2880

attacacaat gaaatgttac actcagttcg tgattcattt acattgaaac aacttcattc 2940

cattggcatt actaatgtca taaataccgg ttgtccgaca atgtgggatt tgacaccaga 3000

gcactgttgc aatattccaa gagaaaaggg aagacgtgta ttatttacat taacagatta 3060

taatcaagac ttatcagcgg atgcattatt aatcaatact cttaaaagac attatgatga 3120

ggtattgttt tggccacagg ggagtgaaga tattcactat atgaatacat tcagtacaga 3180

tattagaaat gggcttaaaa ttttacgacc atcattatct gaacttaatg caaatttaat 3240

gcttaaggaa actgattatg ttgggacacg attacatgcg gggattcgtg cattgcaact 3300

tcgcgcaaga actataatta taggtgttga caatagagca atagagaaag cgaaagactt 3360

taatcttcct gtattaaaac gagatgctat tttggagctg gaaaatatga tagttgaacc 3420

acttattact caaattaata tgccgtgggg aaatattgaa aagtggaaag atcaatttta 3480

atattattct tttttgttca gtagagtttt ttaagataat ttagggaccg gatgaaaata 3540

ttaatattga atcattcaga caatgttggc ggggctgcta gggcagccta ccgattgcat 3600

atgctattta agcaaaaatt tgctaatagt gagatgtttg ttagagtaaa aaaaagtgat 3660

agctcaagtg tatttacccc aaagggcatt tattcttttt tatcaaagat acgataccca 3720

cttagcaagt ttattttgaa aaaattatta catatctgga agcaggattt aattagttta 3780

aatatgatcc catcttcttt gtcaagaata attaatgaat ccgatgcgga tattgtaaat 3840

attcattgga ttgctggaga aacaatatca tttaaagacc tatcacgaat aaaaaaacct 3900

ttagtctgga ccatgcatga tatgtggcca ttcattggcg ctgaacatta catagacgag 3960

tcaagaactg attggttgtg tgggtataaa aacctattcg gaaagtttgg tataataaat 4020

aggtacttgt ggagaaaaaa actaaaaact tacagtcgac taaatatatg tttcgtatct 4080

ccaagcaaat ggcttgcaga aaaagctaag ctgagcggtc tattacaagg tatgcgaatt 4140

gaagtaattc ctaatcctat aaatttcgac atatttaagc ctctagataa agagttttcg 4200

agaaaagcat taaatctatc tctatcatca aagataattt tatttggtgc tgtaggaggt 4260

gttaatgatc agcgtaaagg ttacactcta ctatatgaat ccttgatgca cctttcggat 4320

aaaataagta ataaagaaaa tattgagtgt gttttttttg gacagagtga acagccaaaa 4380

gagtttgctc cacttccatt taaatgtacc tggttcggtg tggtcaacga tgatgtttca 4440

ttatcgttgt tatacaacgc tgctgatatt actgttgtgc catcacgaat ggagaacttg 4500

ccacagatat gtacagaagc acaatcgtgt ggatgccctg tcgttgcatt caacactact 4560

ggaataagtg aaacaattga acacaataaa acaggttatt tggcaaatgc atttgataca 4620

ttagacttat ctaaagcaat gtatacactc ctttttaata atgatttgaa caaaatgttt 4680

tctgcaaatg caagcgaaag agcgcgtttt ctttggaatg gtgatagtat tgccaaaaaa 4740

tatgaatcca ttttctcaag tttaaaggag tgatctgagt aagtggaaaa tcaacacgtt 4800

tatttttata agaaatcaaa taaagcaaat gtttcaattg ttgccttgat ttttacatat 4860

atctcaatta ttggttggag cccttttttt agtggatatt tagctgtacc attatttcta 4920

acatatcata tacccttttt attattgctt gcttacttta tgttaacaca aaaaataagt 4980

agtgctttaa tttcattatc gataataatg attatactta tttctgcact aacattgtta 5040

acagaatcat taattaattt taatcgttat atttttgaac cgctgacctt tttgctttgc 5100

tattattgta cagtaagtag aaaacttgcc gaaaaaatat gcacatatgc atcatgtgtt 5160

gtaatcatag gcattgtgct atcagtaatt tctttttgtt atgcgtatgc aggaggtaac 5220

gaaatattaa cttttcataa tgtcgatgga cgattgaata gtttatatct aacttcattt 5280

agtaatttta aaataggaaa tattatccga ccatcattta tatatgatga acccggggca 5340

ttcagttttg tcatatgttt tgtcgttatt ttaagggagg ttttgggaag aaaatataat 5400

tgggtttacc ttcttggagg catgattaca ttaagtttaa ctcaatttat catattatca 5460

ctatatatta tagttaaaaa ttggaaaaat attaaacaaa tccttgtgtt gtgttttgca 5520

ttattcgggt tgataatgtt tacatataat aataatgaat tcgatttttt ctataataga 5580

ttcaatacgg aaaatggaac atttagcggt gataatagaa gcatgcagat ctcaaattat 5640

ttaaatgttg tcaatgagaa tatgtttctt ctgggggact ataagtgtca tgataacata 5700

aatcagagat gtgaaagtca tggtgatatt tcatcaagta tagtaactcc aatatacaaa 5760

ggcggtattc tacaactatt actacaaatt attactcatg caataatatt ttggttggca 5820

ctccgagaaa gaaaactcat attcccagcc ttagtattaa caatactact gcttcaaaga 5880

cccttttttg atgtagtggg atattcagtt atgatctatt attctatttt tttaatgttt 5940

aattttaaag tgtttaaaga tgattattat cacagtcgta aaaaatgatc cagatggact 6000

tttaaagacc tataattctg taatatctga aattgggagt ggattaaagt gcagtcggtg 6060

gattattaaa ctaagcagcg atgcagatgt ctctttaaaa aagattatca atctgcctga 6120

ttattgccat gtatatactg attatgataa aggtatttac tcagctttta accaagcaat 6180

ttcaaaaatt ccatcatcgc atgaaaatga atatgtaatg tttttaaatg ctggtgatga 6240

aattataaat ggacaagaga taaataagct tcttgatctg caaagagata acatcgatat 6300

ttttattgct tgtgctaaaa taccaggtaa agaaaaaata atatatccaa tctcatcttt 6360

aatcggcgtt ggaggattaa atttttcatt ccagtctatt ttataccgag taaaaatatt 6420

gaaagagatt ccttttccag aaaaatataa aatagcagga gattatgcgc attatttaaa 6480

aatacaagac cgagataaga ttatatgtac tttgattatt tctacttttt ctctagatgg 6540

tttatcaggg cgtagtgttt ataaaactcg gctagaaaat ttcaaagctg gtttcgagca 6600

taatggtata agagaaatat ttaattttgt caaaatgatt attataagag tattggataa 6660

aatatgattg ctttttttat tggaccatca actggaaaag ttacaggtca atcaatagcc 6720

ttcaatataa tatatgataa ctattcaggt aaaaaatatc ttataaatta tccagatcca 6780

gatgaagggt ttttatcttt atttaaagga tatattcaat cgttttttgt tttcattttc 6840

ataaatttgt tatttttttt taaaaaaaag actgtttata taacaagcag tcgaactctg 6900

attggttttt ttcgtgatgc tatttatatt atagtcggga agttattttt tgcaaaagtt 6960

gttaatcatt tgcatggtgc tgattttaaa tattttagaa atgagcaatg ttatataatt 7020

aaaaaaataa ttgattatgt ttataataag atagatgttt caattgttct cactgcaagg 7080

atgaaggaac aatataacat atatcggaag atgaaaatca aagtaatagg taactgttat 7140

tttggtctta gttacaggca agagaagaaa atttttaata atgaaacgct aaaactagtt 7200

tttctttcta atttattata ctccaaaggg atcgtacatc taataaatgt ggtaaaaaga 7260

ataaataatg aaggtataaa ggtaagtctc gaaattgcgg gaaaaatatt gagtgatgaa 7320

tatgttgatt cgtgtaaatt gaaagaaata ttagaggcta aaattgaaaa ttgtccatat 7380

attaaatatc atggacccct gaattctgaa aagaaaagtg ctttactaaa cagatctgat 7440

gtgtttgtgt tgccaacatt ctatcgctct gaagctcagc ctatttctgt tcttgaagca 7500

atgtattttg gttgtgtgat tgtaacgacg gctcataatt atatgccgga ttttgttacg 7560

gcgaagaatg gtatcattat taatcctgat gatgaggacg atttatacga agctttatta 7620

aaaatcaata atatgaagag tatattttct gatgttgcag catataatgc ctgctatgtt 7680

aaaaaaaatt tttctccaga aaattatata aacaaaatta cttcagtaat aatagaggat 7740

taaatgaaaa aagcatttat tacaggcatc accgggcaag atggctctta tttggctgaa 7800

tttcttcttg acaaaggcta tgaagtgcat ggcattaagc gtcgagcatc ttcgtttaac 7860

acagaacgtg ttgatcatat ttatcaagat cgccacaacc aaaatccaaa tttctttctt 7920

cattatggtg acttaacaga tacatcaaac ctgatacgtc taatcaaaga aatccaacca 7980

gacgaagttt ataaccttgg cgctcaatct catgtagcgg tttcattcga atcacctgaa 8040

tatactgctg acgtagatgc gatggggact ttacgtctgc tggaagcgat tcgtatttgt 8100

gggctagaga aaaaaacacg tttttatcag gcatcaactt ctgaactctt cggcttagtt 8160

caggaaatcc cgcagcgaga aacaacacca ttctaccctc gttcgcctta tgctgttgcg 8220

aagatgtatg catactggat tactgtaaac tatcgtgaat cctatggaat gtatgcctgt 8280

aacggtattc tcttcaacca tgaatccccg cgtcgtggtg aaacattcgt aacacgtaaa 8340

atcactcgtg ctattgcaaa tatttcgcag ggaatcgaaa aatgtctcta tcttggcaat 8400

atggattcac tgcgcgactg ggggacatgc gaaagattac gtgcgtatgc agtgatgatg 8460

ttgcagcaag atcacccaga ggattttgtt attgcaacag gcaaacaaat ttccgtacgt 8520

gaatttgttc gtatgtcagc taaagaagtc ggtctggagt tagaattctc cggacaaggt 8580

gttgatgaaa tagcaactgt tgtgaataaa acatctgact gtgctattgg tgttagtatt 8640

ggggatgtga tcgttcgtgt tgatcctcgt tatttccgtc ctgcagaggt tgaaactctt 8700

cttggtgatc cagcaaaagc gaaaagagta ttgggttggg aaccagagat tacagttgaa 8760

gagatgtgtg cagaaatggt tgccagtgat ctggcgaaag caaaacagca tgcactactg 8820

aaaagccatg gttacgatgt tgcagtttct ctggagcggt aaggtatgaa aaaaaaacgt 8880

atctacgttg ctggtcaccg aggcatggtt ggctctgcta tttgccgtca attatcactg 8940

cgtgatgata tcgaattagt ggtcaaaaca cacaaagaac tcgatctaac cgtacagaag 9000

gatgttgatg cattttttgc gcaagagaaa attgatcagg tttatcttgc tgcggctaaa 9060

gttggtggca tttatgccaa caatacattt ccggctgaat tcatctatca gaatctcatg 9120

attgagagta atattattca ttcagctcac aaggccggaa ttcaaaaatt acttttttta 9180

ggctcaagct gtatttatcc taaattcgca gagcagccga tgaacgagtc tgcgctttta 9240

acgggcatac ttgagccaac taatgagcca tacgcaattg ctaaaattgc gggcataaaa 9300

ttatgtgaat cttataaccg tcaatatggc cgtgattatc gcagtgtaat gcctactaat 9360

ctttatggca taaatgataa tttccacccc gaaaactctc atgttattcc ggcactcatg 9420

cgtagattcc atgaggcaaa agagagtggt gcaccagaag ttattgtctg gggaacagga 9480

acaccgatgc gtgagttttt atatgttgat gatatggctg ccgcatctgt tcatgtaatg 9540

gaacttgacg aagcaattta tcaacaaaat tcacagccta tgttatctca tattaatgtt 9600

ggtacgggtg tagattgttc catacgtgaa atggctgaaa caatggcctc tgtggtgggt 9660

tatcaaggta aaattgtttt tgatgttact aaacctgatg gcactccgcg taaacttatg 9720

gacgttaccc ggctcaaaaa cttgggctgg caatatcaat ataatttaca tgaaggctta 9780

tcattaacat ataaatggtt tattgagaat attaattctt ttcggggata gttatgaata 9840

agagattgga acgtgagcta tttaaaacaa tagttgagca tactcctcta atctcgattg 9900

atctcataat tagaaacgat aaaggagagg cgctgctggg gcagcgcctg aatcgcccag 9960

cacaaaatta ttggtttgtg ccaggagggc gaatttataa ggatgagtca ttcgagattg 10020

catttaagcg gataacattt gaagaggtgg gcgttcaaat tagtcttgat gatgccttat 10080

ttctaggggt gtatgaacat ttttacaatg ataatttttc tgaagcagaa ttttctacac 10140

actatgtagt gcatggatat gaaatccaac ttaaacctca gcaacttcac ctaccaacgg 10200

tccagcataa ttcctacaag tggtttgatg tagtaacgtt gcttaatagc actacagttc 10260

atcaatatac caaaaattat tttataccaa ggtaatagat atgctacttc ccgttgtcat 10320

ggccggtggt tctggtacca gattatggcc tctttcacgt acactttatc cgaaacaatt 10380

tctatcttta aatagtcgtt taaccatgtt gcaagagaca ttgcggcggc ttgacaaggt 10440

agaacataaa cccgctttgg tcatttgtaa cgaatcacat cgctttatcg ttgctgaaca 10500

attgcgtaaa gagggtttaa agcatagcgg tattttgctt gagcctgttg gccgtaatac 10560

tgcgcctgct gtagcactcg cagcacttca ggctatggta actggagatg atcctattct 10620

gttggttctt gctgctgatc atgaaattca gaatgaggat aatttcattg atgcagttct 10680

tgcagccaag aattttgcag aacagggtaa gcttgttacg tttggtattg ttccaacatc 10740

cccagagact ggctatggtt acattaagtc aggtgagtat ctggatggaa aaggttacaa 10800

agttgcggct tttgttgaaa aaccagagct tcacgtagct cagcagtata tatcagatgg 10860

cggttatctt tggaatagtg ggatgtttat gttcagagca tctgtattta tcaatgaact 10920

gaaaaaattc cgaccagata ttttagccag ttgccaacgc tccttgtcct cttcgataca 10980

agatttagat tttatccgtc tggataatgc ttcattttct tgctgtcctg aagagtctat 11040

tgactatgca gttatggaaa aaacagccga agctgtcgtc gttcccttaa atgcacaatg 11100

gagtgatgtt gggtcatggt ctgcattgtg ggaaataagt tcaaaagatc aaagcggtaa 11160

tgccattcgt ggtgatgtat tggttgaaga tgctacagat agttatcttt attcgcagca 11220

tagacttatt ggtgctgtgg gcataaagga tttggttgtt gttgaaacga aagatgccgt 11280

attagttgct cataaagata aagttcagca agttaaaaac atcgtcgctc aacttaaaaa 11340

gaataatcgc acagaatatt tacagcatcg ggaaatattt cggccttggg gcagtcatga 11400

tactatagct gaagggccgc gctttcaagt gaaacatgtg attgtattac ctggccacat 11460

taccgctaag cagattcatt accatcgtac tgagcattgg attgttgtct cggggacagc 11520

taaagtccat cttgaagata agacttacct tgtttctgaa aatgaatcaa catatatacc 11580

tgttggtgtt ccacatgcca ttgaaaatcc tggcaagatc ccgcttgaaa taatcgaggt 11640

tagatcagga gtctacctgg aagaggatga tgttataaga gtttcttctt ctggagtagg 11700

atactaatgc gaatttctat tatcacagct acttataata gtgaaaaaac tctccttgat 11760

acattacttt ctctagaaaa acaaacgtat ccagatatcg aaatatagt tgtagatgga 11820

gcatcgaacg ataatacaat taagctgatc aagggtaatt gtacaaaagt ttcaaaaatc 11880

atttgcgaac ccgataaggg catttatgat gctctgaata aaggtattca agcagcttcg 11940

ggtgatgtta ttggtttttt acattctgat gatttattac cctatgatga tgctattgca 12000

gatatagcaa aaacatttga aattacaggg tgtgatgcta tttatggcga tttggagtat 12060

gttgcccaaa atgatacgac taaacgtatt agattatgga aaagtggttc atttagtcgt 12120

ttcaagatga aagtgggttg gatgccgcca cacccatcat tttatatgaa acgcaaatgt 12180

tataggcagt ttggttgttt ttctttagat tatcgaatat ctgctgatta tgactcatta 12240

ttacgatata ttttaaaaca acgtatttca acagaatatt taccaaaagt cttagtgaag 12300

atgcgtgttg gtggaattag caatcgttca gtatcttcaa tggtaaagaa gtcaatggaa 12360

gacattcgtg ttatgaacca gaatggtatt atctggccaa tagctttagt gtataaaaat 12420

atatccaaac ttcctcaatt tattaaaaag taatcatcat gttaaatgtt aaaaaaatca 12480

ttaacgatag taatattgcg ttcggtacta gtggtgctcg cggtttagtg atcgattttt 12540

cccatgatgt ttgtgctgcg ttcactcatg cgtttctttc tgttattgat gacaaataca 12600

attttaataa agttgcctta gcaattgata accgcccaag cagttacgaa atcgctcagg 12660

catgcgcctt cgctatcaaa caacatgggt tcactgtcga atatcatggt gtaattccga 12720

ctcccgcatt agctcattat tcgatgcaga aaaacattcc ctgcataatg gtcacaggga 12780

gtcatatccc ttttgatcgt aatggcttaa aattctacag acctgatggt gaaatcacta 12840

aagaggatga gctcgcaatt ataaatagtg agtatacatt ttcgcctgta ggtgtattac 12900

ctcatcttga aacaagcact gaaggtgcag actactacct tgagcgttat gtttctcttt 12960

ttaatcctga tattttaaag gggaaaagaa tagggctata tgaacattct agtgcaggac 13020

gcgatttata tgctcctgtt tttaatcaat tgggtgcaga ggtcatttcc ttaggtagaa 13080

gtgatgagtt cgttcctatt gatacggaag cagtaagcga tgaagatcgc atacttgcaa 13140

gagagtggtc taaaaaatat aatcttgatg ctattttctc aacagatggc gatggtgatc 13200

gtcctttagt tgccgatgaa aatggtgaat ggctaagagg cgatattctg ggattactta 13260

ctgctattga acttaatatc aaggcgttgg ctattccagt gagttgtaat acagcaattg 13320

aacagtctaa taaatttgca agtatacaac gaacgaaaat aggttctcct tatgtaattg 13380

cagcgtttac agatcttgct aagcaattta attcagtcgc tggttttgaa gctaatggtg 13440

gctttctcct tgcctccgat ttacaaatta atggcaagga attaaaatcg ttacctacac 13500

gagatgctgt gttaccagca ttaatgctct taatagcttc tcgcaatagt actatttctc 13560

aactgattaa taatcttcct cagcgattca cttggtcaga tagagttaaa aacttccctt 13620

cagattcaag tcaacaaatt ataaaaaatg ccatatcgtc acccaataat ttctttaata 13680

gtttaggtta tgaatcatta tcctgttccg ctattgatga aacggatggt gcaagattta 13740

ctttaaataa tggtgatatt atacatctcc gtccttccgg taatgctcca gaactccgtt 13800

gttatgctga ggccagtgat gaaaatcagg ctaagcaata tgttacgaat gtgctgggaa 13860

atattacctc tttgatttct tgatgttata ggtttatcta cgcttatacg tgtgcgtagg 13920

tttgattaca cgtagatgcc ggtatacaga attgaagaac ggtatttgtt gcattaatga 13980

aattcagcac tacacacatt cgtgcaactt gagataacat ctcaatcata tccaagtcgc 14040

gcatacatcg cggtgaacac cccctgacag gagtaaacaa tgtcaaagca acagattggt 14100

gtcgtcggta tggcagtgat ggggcgcaac cttgcgctca acatcgaaag ccgtggttat 14160

accgtctcta ttttcaaccg ttcccgtgag aagacggaag aagtgattgc cgaaaatcca 14220

ggcaaaaaat tggttcctta ctttacggtg aaagggtttg ttgaatctct ggaaacgcct 14280

cgtcgcatcc tgttaatggt gaaagcaggt gcaggcacgg atgctgcaat tgattccctc 14340

aaaccatatc tcgataaagg tgacatcatc attgatggtg gtaatacctt cttccaggac 14400

accattcgtc gtaaccgtga gctttctgct gaaggcttta acttcattgg taccggtgtt 14460

tccggtggtg aagagggcgc gctgaaaggg ccttccatca tgcctggtgg ccagaaagaa 14520

gcctatgaac tggttgcgcc gatcctgacc aaaatcgccg ccgttgctga agatggcgaa 14580

ccgtgcgtta catatattgg tgccgatggt gcgggtcact atgtgaaaat ggttcacaac 14640

ggtattgaat acggtgatat gcaactgatt gctgaagcct attctctgct taaaggcggc 14700

ctgaatctct ctaacgaaga actggcacag acctttaccg agtggaataa cggtgaactg 14760

agcagctacc tgatcgacat caccaaagat atcttcacca aaaaagatga agacggtaac 14820

tatctggttg atgtgatcct ggatgaagcg gctaacaaag gtaccggtaa atggaccagc 14880

cagagcgcgc tggatctcgg cgaaccgctg tcgttgatta ctgagtctgt gtttgcacgt 14940

tatatctcgt ctctgaaaga tcagcgcgtg gccgcatcta aagttctctc tggcccgcaa 15000

gcacagccag caggcgacaa ggctgagttc atcgaaaaag ttcgccgtgc gctgtatctg 15060

ggcaaaatcg tttcttacgc tcagggcttt tctcagctgc gtgctgcgtc tgaagagtac 15120

aactgggatc tgaactacgg cgaaatcgca aagattttcc gtgctggctg catcatccgt 15180

gcgcagttcc tgcagaaaat caccgatgca tatgccgaaa atccgcagat cgctaacctg 15240

ctgctggctc cgtacttcaa gcaaattgcc gatgactacc agcaggcgct gcgcgatgtc 15300

gtcgctcatg cagtacagaa cggtatcccg gttccgacct tcgccgctgc ggttgcctat 15360

tacgatagct accgtgccgc tgttctgcct 15390

Glycosyltransferase gene in the O antigen gene of table 1 intestinal bacteria O156 type bunch and oligosaccharide unit treatment gene and wherein Primer and PCR data

Gene	Function	The base position of gene	Forward primer	Reverse primer	The length of PCR product	Produce the group number of correct big or small electrophoresis band	The annealing temperature of PCR (℃)
								Wzx	O-antigen transhipment enzyme	1366-2505	1512-1532	1846-1864	353bp	0 *	56
			1851-1869	2229-2247	397bp	0 *	55
								Wzy	O-antigen polysaccharase	4783-5988	5209-5227	5840-5858	650bp	0 *	60
			5594-5614	5865-5885	292bp	0 *	56

^*Only in intestinal bacteria O156 type, obtain a correct band

Table 2 166 strain intestinal bacteria and 43 strain Shigellaes and their source

Group number	The bacterial strain that contains in this group	The source
			1, wild-type e. coli 2, wild-type e. coli 3, wild-type e. coli 4, wild-type e. coli 5, wild-type e. coli 6, wild-type e. coli 7, wild-type e. coli 8, wild-type e. coli 9, wild-type e. coli 10, wild-type e. coli 11, wild-type e. coli 12, wild-type e. coli 13, the 8th group of bacterial strain adds the escherichia coli reference culture	O1, O2, O5, O7, O12, O13, O14, O15, O16, O17, O19ab, O20, O21, O22, O23, O24, O59 O25, O26, O27, O28, O29, O30, O32, O33, O35, O36, O37, O38, O40, O41, O42, O43 O44, O45, O46, O48, O49, O50, O51, O52, O54, O55, O56, O57, O58, O60, O61, O62 O63, O65, O66, O69, O70, O71, O74, O75, O76, O77, O78, O79, O80, O81, O82, O83 O84, O85, O86, O87, O88, O89, O91, O92, O98, O99, O101, O102, O103, O104, O106 O107, O108, O109, O110, O111, O112ab, O112ac, O113, O115, O116, O118, O120, O123, O125, O126, O128 O129, O130, O131, O132, O133, O134, O135, O136, O137, O138, O139, O141, O142, O143, O144, O145 O146, O147, O148, O150, O152, O154, O155, O157, O158, O159, O160, O161, O163, O164, O165, O166 O168, O169, O170, O171, O172, O173, D1, D2, D3, D4, D5, D6, D7, D8, D9, D10, D11, D12 B1, B2, B3, B4, B6, B7, B8, B9, B10, B11, B12, B13, B14, B15, B16, B17, B18 F1a, F1b, F2a, F2b, F3, F4b, F5 (v:4), F5 (v:7), F6, FX becomes, and FY becomes, DS, DR, O3, O11, O39, O59, O64, O73, O96, O95, O100, O114, O151, O155, O124 0156	IMVS a IMVS a IMVS a IMVS a IMVS a IMVS a IMVS a IMVS a b c d d d IMVS a IMVS

^*For the convenience that detects, we are divided into one group with every 13-17 bacterium, 13 groups altogether

a.Institude of Medical and Veterinary Science，Anelaide，Australia

b.Statens Serum Institut，Copenhagen，Denmark

C.O172 and O173 come from Statens Serum Institut, Copenhagen, and Denmark, all the other come from IMVS

D.d. China Preventive Medicial Science Institute's epidemiological study institute

Table 3 colibacillus O156 type O antigen gene structure iron

E.coli O156 O-antigen gene cluster

#orf galF wzx orf2 orf3 wzy orf5 orf6 gmd fcl gmm manC orf11 manB gnd

％G+C 30 34 31 28 29 28 43 39 38 41 33 38

Table 4 intestinal bacteria O156 type O antigen gene cluster gene position

ATTGTGGCTG CAGGGATCAA AGAAATCATT GTGGCTGCAG GGATTAAAGA AATCATTGTG 60

GCTGCAGGGA TCAAAGAAAT CCTCCTGGTA ACTCACGCGT CCAAGAACGC GGTCGAAAAC 120

CACTTCGACA CCTCTTATGA ATTAGAATCT CTCCTTGAGC AGCGCGCAAA GCGTCAACTA 180

CTGGCGGAAG TACAGTCCAT TTGCCCGCCG GGCGTGACCA TTATGAACGT GCGTCAGGGC 240

GAACCCTTAG GTTTAGGCCA CTCCATTTTG TGTGCACGAC CCGCCATTGG TGACAACCCA 300

TTTGTCGTGG TATTGCCAGA CGTTGTGATC GACGACGCCA GCGCCGACCC GCTGCGCTAC 360

AACCTTGCTG CCATGATTGC GCGTTTCAAT GAAACGGGCC GTAGCCAGGT GCTGGCAAAA 420

CGTATGCCGG GTGACCTCTC TGAATACTCC GTTATTCAGA CCAAAGAACC ACTGGATCGT 480

GAAGGTAAAG TCAGCCGCAT TGTTGAATTT ATCGAAAAAC CGGATCAGCC GCAGACGCTG 540

GACTCAGACA TCATGGCCGT TGGTCGCTAT GTGCTTTCTG CAGATATTTG GCCGGAACTA 600

GAACGCACTC AGCCTGGTGC ATGGGGGCGT ATTCAGCTGA CAGATGCCAT CGCTGAACTG 660

GCGAAAAAAC AGTCCGTTGA TGCCATGCTG ATGACAGGTG ACAGCTACGA CTGCGGTAAA 720

AAAATGGGTT ATATGCAGGC ATTTGTGAAG TATGGACTAC GCAACCTGAA AGAAGGGGCG 780

AAGTTCCGTA AAGGGATTGA GAATCTGTTA AGGGATTAAT GAAAATCTGA CCGGATGTAA 840

CGGTTGATAA GAAAATTATA ACGGCAGTGA AGATTCGTGG CGAAAGTAAT TTGTTGCGAA 900

TATTCCTGCC GTTGTTTTAT ATAAACAATC AGAATAACAA CGAGTTAGCA ATAGGATTTT 960

AGTCAAAGTT TTCCAGGATT TTCCTTGTTT CCAGAGCGGA TTGGTAAGAC AATTAGCGTT 1020

TGAATTTTTC GGGTTTAGCG CGAGTGGGTA ACGCTCGTCA CATCGTAGGC ATGCATGCAG 1080

TGCTCTGGTA GCTGTAAAGC CAGGTGCGGT AGCGTGGGTA ATGTTTACTA CCTATATAGT 1140

TACGTGCAAT GAAAAATAAG AATAAGTGCT GAATGATTTT GATTATGAAT ATTTTAGATT 1200

TTTTATAAGG AGGATATTAT AAATATAGGA ACTAAAATCT CTATTTATGC GGCATTATCT 1260

AGAGGTCTTA AAATATTAAG TGGACCCATA ATAATTCTTG TTGTTTCAAA GACATTAAGT 1320

Orf1 is initial

TCAGAGGAAA TTGGATTTTA TTATACTTTT TTCAGCTTAG TAACT ATGCA GCAGTTAGCT 1380

GAATTAGGTT TAGGGCAAAC ATTAAGATTG CATATTGCTC ATGCGTTTAA AATGGACGAG 1440

TCGGGATGGT GTTTTAACTC AAAAAAAGAA ATTAAGCAAT ACTATTCTCT AAGTGTAAAA 1500

TGGTTCTTGG TCGTTGCGTT ATGTACTATC ATTATTATTG GTCCTGCGGG TATTTATTAT 1560

TATCATTCTT ACCAAGGGGA TATTATTTGG GAAGGCCCAT GGGTATCATT AGTATTGGTT 1620

ACAGCACTAA CAATTATAAT AACGCCTATA CAAATCACAA TTGAATCTCT ACAACATCAA 1680

ATTTCAGTAT TTAAAGCACA GACATATGCA TCAATTATCA ATAGTATATC ATTATGTCTG 1740

TTATTGTTTT CTGGATTTGG TCTATATGCA ATTGCTTTAA GCCTATTTAT CAGTAACTTA 1800

ACAGTCTTAA TATTTTTATG GGGGAAAATA AAATGGCTGA GAAATAACCT GATGCCTGTT 1860

CTTGTTGATA CAAATTTTTT TGTTGTTTTT AAAGAGATCT GGCCGCTGTT TTCTCGTGTA 1920

GCTGTTGTAT CTGTATTTGG TTTCCTTTTT TGGAATGGAT TTAATCTAAT TGCATTTAAA 1980

GTATATGAGC CCTCATATGC AGGACAAGTA ATTTTATCCA TAACCTTAGC GAGAGCTGGC 2040

TATGGATTTT CTGAGTCAAT TTTATATGGG CAAATAACAT ACATATCTAA TTTGATTTCG 2100

AACAAAAAAT ATAATGAGGC TATGATAGTT TATAAAAAAA ATACATATAT AGCACTAACG 2160

ATTTTGTTAA TAGGATATGT GTTTTTTTTA CTTCTTATGT ATCTTTTCCC AGAATTCTAC 2220

TTATTTAAAA AAATAGTAGG CTCAAGCTAT TTAATTAGTA TATTTGTATT TTTTATAATA 2280

GTTCTTTTTT TAACCTCAGG AAATAACTAT GTCAGATGCT TTAAAACAGA ACCATTTGTA 2340

AAAGTGTCCA TCTTTCACGG GGTCATGGTA CCAACATTAT TTTATTTATC ATCAGTTTTT 2400

TATGAGGGTT ATTATTTATA TCCATGTTCA TTGGTGTTGA TTGTTTCTCT AATTTTATCA 2460

It is initial that orf1 stops orf2

AAAAGAATAT CAAGCAAGTA TTTTAGAATG TCCGGCATTA AT TAATAATA GGTGAAAA GT 2520

GAATAAGAAA GATAACTTTG TGAATATTTT TGACACAAGT ATTGCATCAT TGAATGTTGG 2580

TGATTACATT ATTATGGATG CTGTGACCAA ACAGTTATCT ACAATAAGTA ATTTACCGCA 2640

GACTGTTACA TTACCAACGC ATGACCATTT TGGTAGAGAA GGGCGACGTT TACTTAGCTT 2700

GGCTAAATAT TCTATTGTTG GCGGCACTAA TTTATTATCA TCGCATGTTA CACAATATCG 2760

CCAATGGCGG TTTCGTATGA CAGATCTTTT ATTTTTAAAA AAATGCATAT TAATGGGAGT 2820

TGGTTGGTGG CAATATCAAG ATAAACCAGA CAGAGTTACA AAATTTATTT TGAAGCATAT 2880

ATTACACAAT GAAATGTTAC ACTCAGTTCG TGATTCATTT ACATTGAAAC AACTTCATTC 2940

CATTGGCATT ACTAATGTCA TAAATACCGG TTGTCCGACA ATGTGGGATT TGACACCAGA 3000

GCACTGTTGC AATATTCCAA GAGAAAAGGG AAGACGTGTA TTATTTACAT TAACAGATTA 3060

TAATCAAGAC TTATCAGCGG ATGCATTATT AATCAATACT CTTAAAAGAC ATTATGATGA 3120

GGTATTGTTT TGGCCACAGG GGAGTGAAGA TATTCACTAT ATGAATACAT TCAGTACAGA 3180

TATTAGAAAT GGGCTTAAAA TTTTACGACC ATCATTATCT GAACTTAATG CAAATTTAAT 3240

GCTTAAGGAA ACTGATTATG TTGGGACACG ATTACATGCG GGGATTCGTG CATTGCAACT 3300

TCGCGCAAGA ACTATAATTA TAGGTGTTGA CAATAGAGCA ATAGAGAAAG CGAAAGACTT 3360

TAATCTTCCT GTATTAAAAC GAGATGCTAT TTTGGAGCTG GAAAATATGA TAGTTGAACC 3420

Orf2 stops

ACTTATTACT CAAATTAATA TGCCGTGGGG AAATATTGAA AAGTGGAAAG ATCAATTT TA 3480

Orf3 is initial

ATATTATTCT TTTTTGTTCA GTAGAGTTTT TTAAGATAAT TTAGGGACCG G ATGAAAATA 3540

TTAATATTGA ATCATTCAGA CAATGTTGGC GGGGCTGCTA GGGCAGCCTA CCGATTGCAT 3600

ATGCTATTTA AGCAAAAATT TGCTAATAGT GAGATGTTTG TTAGAGTAAA AAAAAGTGAT 3660

AGCTCAAGTG TATTTACCCC AAAGGGCATT TATTCTTTTT TATCAAAGAT ACGATACCCA 3720

CTTAGCAAGT TTATTTTGAA AAAATTATTA CATATCTGGA AGCAGGATTT AATTAGTTTA 3780

AATATGATCC CATCTTCTTT GTCAAGAATA ATTAATGAAT CCGATGCGGA TATTGTAAAT 3840

ATTCATTGGA TTGCTGGAGA AACAATATCA TTTAAAGACC TATCACGAAT AAAAAAACCT 3900

TTAGTCTGGA CCATGCATGA TATGTGGCCA TTCATTGGCG CTGAACATTA CATAGACGAG 3960

TCAAGAACTG ATTGGTTGTG TGGGTATAAA AACCTATTCG GAAAGTTTGG TATAATAAAT 4020

AGGTACTTGT GGAGAAAAAA ACTAAAAACT TACAGTCGAC TAAATATATG TTTCGTATCT 4080

CCAAGCAAAT GGCTTGCAGA AAAAGCTAAG CTGAGCGGTC TATTACAAGG TATGCGAATT 4140

GAAGTAATTC CTAATCCTAT AAATTTCGAC ATATTTAAGC CTCTAGATAA AGAGTTTTCG 4200

AGAAAAGCAT TAAATCTATC TCTATCATCA AAGATAATTT TATTTGGTGC TGTAGGAGGT 4260

GTTAATGATC AGCGTAAAGG TTACACTCTA CTATATGAAT CCTTGATGCA CCTTTCGGAT 4320

AAAATAAGTA ATAAAGAAAA TATTGAGTGT GTTTTTTTTG GACAGAGTGA ACAGCCAAAA 4380

GAGTTTGCTC CACTTCCATT TAAATGTACC TGGTTCGGTG TGGTCAACGA TGATGTTTCA 4440

TTATCGTTGT TATACAACGC TGCTGATATT ACTGTTGTGC CATCACGAAT GGAGAACTTG 4500

CCACAGATAT GTACAGAAGC ACAATCGTGT GGATGCCCTG TCGTTGCATT CAACACTACT 4560

GGAATAAGTG AAACAATTGA ACACAATAAA ACAGGTTATT TGGCAAATGC ATTTGATACA 4620

TTAGACTTAT CTAAAGCAAT GTATACACTC CTTTTTAATA ATGATTTGAA CAAAATGTTT 4680

TCTGCAAATG CAAGCGAAAG AGCGCGTTTT CTTTGGAATG GTGATAGTAT TGCCAAAAAA 4740

It is initial that orf3 stops orf4

TATGAATCCA TTTTCTCAAG TTTAAAGGAG TGATCTGAGT AA GTGGAAAA TCAACACGTT 4800

TATTTTTATA AGAAATCAAA TAAAGCAAAT GTTTCAATTG TTGCCTTGAT TTTTACATAT 4860

ATCTCAATTA TTGGTTGGAG CCCTTTTTTT AGTGGATATT TAGCTGTACC ATTATTTCTA 4920

ACATATCATA TACCCTTTTT ATTATTGCTT GCTTACTTTA TGTTAACACA AAAAATAAGT 4980

AGTGCTTTAA TTTCATTATC GATAATAATG ATTATACTTA TTTCTGCACT AACATTGTTA 5040

ACAGAATCAT TAATTAATTT TAATCGTTAT ATTTTTGAAC CGCTGACCTT TTTGCTTTGC 5100

TATTATTGTA CAGTAAGTAG AAAACTTGCC GAAAAAATAT GCACATATGC ATCATGTGTT 5160

GTAATCATAG GCATTGTGCT ATCAGTAATT TCTTTTTGTT ATGCGTATGC AGGAGGTAAC 5220

GAAATATTAA CTTTTCATAA TGTCGATGGA CGATTGAATA GTTTATATCT AACTTCATTT 5280

AGTAATTTTA AAATAGGAAA TATTATCCGA CCATCATTTA TATATGATGA ACCCGGGGCA 5340

TTCAGTTTTG TCATATGTTT TGTCGTTATT TTAAGGGAGG TTTTGGGAAG AAAATATAAT 5400

TGGGTTTACC TTCTTGGAGG CATGATTACA TTAAGTTTAA CTCAATTTAT CATATTATCA 5460

CTATATATTA TAGTTAAAAA TTGGAAAAAT ATTAAACAAA TCCTTGTGTT GTGTTTTGCA 5520

TTATTCGGGT TGATAATGTT TACATATAAT AATAATGAAT TCGATTTTTT CTATAATAGA 5580

TTCAATACGG AAAATGGAAC ATTTAGCGGT GATAATAGAA GCATGCAGAT CTCAAATTAT 5640

TTAAATGTTG TCAATGAGAA TATGTTTCTT CTGGGGGACT ATAAGTGTCA TGATAACATA 5700

AATCAGAGAT GTGAAAGTCA TGGTGATATT TCATCAAGTA TAGTAACTCC AATATACAAA 5760

GGCGGTATTC TACAACTATT ACTACAAATT ATTACTCATG CAATAATATT TTGGTTGGCA 5820

CTCCGAGAAA GAAAACTCAT ATTCCCAGCC TTAGTATTAA CAATACTACT GCTTCAAAGA 5880

CCCTTTTTTG ATGTAGTGGG ATATTCAGTT ATGATCTATT ATTCTATTTT TTTAATGTTT 5940

The initial orf4 of orf5 stops

AATTTTAAAG TGTTTAAAG A TGATTATTAT CACAGTCGTA AAAAA TGATC CAGATGGACT 6000

TTTAAAGACC TATAATTCTG TAATATCTGA AATTGGGAGT GGATTAAAGT GCAGTCGGTG 6060

GATTATTAAA CTAAGCAGCG ATGCAGATGT CTCTTTAAAA AAGATTATCA ATCTGCCTGA 6120

TTATTGCCAT GTATATACTG ATTATGATAA AGGTATTTAC TCAGCTTTTA ACCAAGCAAT 6180

TTCAAAAATT CCATCATCGC ATGAAAATGA ATATGTAATG TTTTTAAATG CTGGTGATGA 6240

AATTATAAAT GGACAAGAGA TAAATAAGCT TCTTGATCTG CAAAGAGATA ACATCGATAT 6300

TTTTATTGCT TGTGCTAAAA TACCAGGTAA AGAAAAAATA ATATATCCAA TCTCATCTTT 6360

AATCGGCGTT GGAGGATTAA ATTTTTGTCA CCAGTCTATT TTATACCGAG TAAAAATATT 6420

GAAAGAGATT CCTTTTCCAG AAAAATATAA AATAGCAGGA GATTATGCGC ATTATTTAAA 6480

AATACAAGAC CGAGATAAGA TTATATGTAC TTTGATTATT TCTACTTTTT CTCTAGATGG 6540

TTTATCAGGG CGTAGTGTTT ATAAAACTCG GCTAGAAAAT TTCAAAGCTG GTTTCGAGCA 6600

TAATGGTATA AGAGAAATAT TTAATTTTGT CAAAATGATT ATTATAAGAG TATTGGATAA 6660

The initial orf5 of orf6 stops

AAT ATGATTG CTTTTTTTAT TGGACCATCA ACTGGAAAAG TTACAGGTCA ATCAATAGCC 6720

TTCAATATAA TATATGATAA CTATTCAGGT AAAAAATATC TTATAAATTA TCCAGATCCA 6780

GATGAAGGGT TTTTATCTTT ATTTAAAGGA TATATTCAAT CGTTTTTTGT TTTCATTTTC 6840

ATAAATTTGT TATTTTTTTT TAAAAAAAAG ACTGTTTATA TAACAAGCAG TCGAACTCTG 6900

ATTGGTTTTT TTCGTGATGC TATTTATATT ATAGTCGGGA AGTTATTTTT TGCAAAAGTT 6960

GTTAATCATT TGCATGGTGC TGATTTTAAA TATTTTAGAA ATGAGCAATG TTATATAATT 7020

AAAAAAATAA TTGATTATGT TTATAATAAG ATAGATGTTT CAATTGTTCT CACTGCAAGG 7080

ATGAAGGAAC AATATAACAT ATATCGGAAG ATGAAAATCA AAGTAATAGG TAACTGTTAT 7140

TTTGGTCTTA GTTACAGGCA AGAGAAGAAA ATTTTTAATA ATGAAACGCT AAAACTAGTT 7200

TTTCTTTCTA ATTTATTATA CTCCAAAGGG ATCGTACATC TAATAAATGT GGTAAAAAGA 7260

ATAAATAATG AAGGTATAAA GGTAAGTCTC GAAATTGCGG GAAAAATATT GAGTGATGAA 7320

TATGTTGATT CGTGTAAATT GAAAGAAATA TTAGAGGCTA AAATTGAAAA TTGTCCATAT 7380

ATTAAATATC ATGGACCCCT GAATTCTGAA AAGAAAAGTG CTTTACTAAA CAGATCTGAT 7440

GTGTTTGTGT TGCCAACATT CTATCGCTCT GAAGCTCAGC CTATTTCTGT TCTTGAAGCA 7500

ATGTATTTTG GTTGTGTGAT TGTAACGACG GCTCATAATT ATATGCCGGA TTTTGTTACG 7560

GCGAAGAATG GTATCATTAT TAATCCTGAT GATGAGGACG ATTTATACGA AGCTTTATTA 7620

AAAATCAATA ATATGAAGAG TATATTTTCT GATGTTGCAG CATATAATGC CTGCTATGTT 7680

AAAAAAAATT TTTCTCCAGA AAATTATATA AACAAAATTA CTTCAGTAAT AATAGAGGAT 7740

It is initial that orf6 stops orf7

TAAATGAAAA AAGCATTTAT TACAGGCATC ACCGGGCAAG ATGGCTCTTA TTTGGCTGAA 7800

TTTCTTCTTG ACAAAGGCTA TGAAGTGCAT GGCATTAAGC GTCGAGCATC TTCGTTTAAC 7860

ACAGAACGTG TTGATCATAT TTATCAAGAT CGCCACAACC AAAATCCAAA TTTCTTTCTT 7920

CATTATGGTG ACTTAACAGA TACATCAAAC CTGATACGTC TAATCAAAGA AATCCAACCA 7980

GACGAAGTTT ATAACCTTGG CGCTCAATCT CATGTAGCGG TTTCATTCGA ATCACCTGAA 8040

TATACTGCTG ACGTAGATGC GATGGGGACT TTACGTCTGC TGGAAGCGAT TCGTATTTGT 8100

GGGCTAGAGA AAAAAACACG TTTTTATCAG GCATCAACTT CTGAACTCTT CGGCTTAGTT 8160

CAGGAAATCC CGCAGCGAGA AACAACACCA TTCTACCCTC GTTCGCCTTA TGCTGTTGCG 8220

AAGATGTATG CATACTGGAT TACTGTAAAC TATCGTGAAT CCTATGGAAT GTATGCCTGT 8280

AACGGTATTC TCTTCAACCA TGAATCCCCG CGTCGTGGTG AAACATTCGT AACACGTAAA 8340

ATCACTCGTG CTATTGCAAA TATTTCGCAG GGAATCGAAA AATGTCTCTA TCTTGGCAAT 8400

ATGGATTCAC TGCGCGACTG GGGGACATGC GAAAGATTAC GTGCGTATGC AGTGATGATG 8460

TTGCAGCAAG ATCACCCAGA GGATTTTGTT ATTGCAACAG GCAAACAAAT TTCCGTACGT 8520

GAATTTGTTC GTATGTCAGC TAAAGAAGTC GGTCTGGAGT TAGAATTCTC CGGACAAGGT 8580

GTTGATGAAA TAGCAACTGT TGTGAATAAA ACATCTGACT GTGCTATTGG TGTTAGTATT 8640

GGGGATGTGA TCGTTCGTGT TGATCCTCGT TATTTCCGTC CTGCAGAGGT TGAAACTCTT 8700

CTTGGTGATC CAGCAAAAGC GAAAAGAGTA TTGGGTTGGG AACCAGAGAT TACAGTTGAA 8760

GAGATGTGTG CAGAAATGGT TGCCAGTGAT CTGGCGAAAG CAAAACAGCA TGCACTACTG 8820

It is initial that orf7 stops orf8

AAAAGCCATG GTTACGATGT TGCAGTTTCT CTGGAGCGG T AAGGT ATGAA AAAAAAACGT 8880

ATCTACGTTG CTGGTCACCG AGGCATGGTT GGCTCTGCTA TTTGCCGTCA ATTATCACTG 8940

CGTGATGATA TCGAATTAGT GGTCAAAACA CACAAAGAAC TCGATCTAAC CGTACAGAAG 9000

GATGTTGATG CATTTTTTGC GCAAGAGAAA ATTGATCAGG TTTATCTTGC TGCGGCTAAA 9060

GTTGGTGGCA TTTATGCCAA CAATACATTT CCGGCTGAAT TCATCTATCA GAATCTCATG 9120

ATTGAGAGTA ATATTATTCA TTCAGCTCAC AAGGCCGGAA TTCAAAAATT ACTTTTTTTA 9180

GGCTCAAGCT GTATTTATCC TAAATTCGCA GAGCAGCCGA TGAACGAGTC TGCGCTTTTA 9240

ACGGGCATAC TTGAGCCAAC TAATGAGCCA TACGCAATTG CTAAAATTGC GGGCATAAAA 9300

ITATGTGAAT CTTATAACCG TCAATATGGC CGTGATTATC GCAGTGTAAT GCCTACTAAT 9360

CTTTATGGCA TAAATGATAA TTTCCACCCC GAAAACTCTC ATGTTATTCC GGCACTCATG 9420

CGTAGATTCC ATGAGGCAAA AGAGAGTGGT GCACCAGAAG TTATTGTCTG GGGAACAGGA 9480

ACACCGATGC GTGAGTTTTT ATATGTTGAT GATATGGCTG CCGCATCTGT TCATGTAATG 9540

GAACTTGACG AAGCAATTTA TCAACAAAAT TCACAGCCTA TGTTATCTCA TATTAATGTT 9600

GGTACGGGTG TAGATTGTTC CATACGTGAA ATGGCTGAAA CAATGGCCTC TGTGGTGGGT 9660

TATCAAGGTA AAATTGTTTT TGATGTTACT AAACCTGATG GCACTCCGCG TAAACTTATG 9720

GACGTTACCC GGCTCAAAAA CTTGGGCTGG CAATATCAAT ATAATTTACA TGAAGGCTTA 9780

It is initial that orf8 stops orf9

TCATTAACAT ATAAATGGTT TATTGAGAAT ATTAATTCTT TTCGGGGA TA GTT ATGAATA 9840

AGAGATTGGA ACGTGAGCTA TTTAAAACAA TAGTTGAGCA TACTCCTCTA ATCTCGATTG 9900

ATCTCATAAT TAGAAACGAT AAAGGAGAGG CGCTGCTGGG GCAGCGCCTG AATCGCCCAG 9960

CACAAAATTA TTGGTTTGTG CCAGGAGGGC GAATTTATAA GGATGAGTCA TTCGAGATTG 10020

CATTTAAGCG GATAACATTT GAAGAGGTGG GCGTTCAAAT TAGTCTTGAT GATGCCTTAT 10080

TTCTAGGGGT GTATGAACAT TTTTACAATG ATAATTTTTC TGAAGCAGAA TTTTCTACAC 10140

ACTATGTAGT GCATGGATAT GAAATCCAAC TTAAACCTCA GCAACTTCAC CTACCAACGG 10200

TCCAGCATAA TTCCTACAAG TGGTTTGATG TAGTAACGTT GCTTAATAGC ACTACAGTTC 10260

It is initial that orf9 stops orf10

ATCAATATAC CAAAAATTAT TTTATACCAA GG TAATAGAT ATGCTACTTC CCGTTGTCAT 10320

GGCCGGTGGT TCTGGTACCA GATTATGGCC TCTTTCACGT ACACTTTATC CGAAACAATT 10380

TCTATCTTTA AATAGTCGTT TAACCATGTT GCAAGAGACA TTGCGGCGGC TTGACAAGGT 10440

AGAACATAAA CCCGCTTTGG TCATTTGTAA CGAATCACAT CGCTTTATCG TTGCTGAACA 10500

ATTGCGTAAA GAGGGTTTAA AGCATAGCGG TATTTTGCTT GAGCCTGTTG GCCGTAATAC 10560

TGCGCCTGCT GTAGCACTCG CAGCACTTCA GGCTATGGTA ACTGGAGATG ATCCTATTCT 10620

GTTGGTTCTT GCTGCTGATC ATGAAATTCA GAATGAGGAT AATTTCATTG ATGCAGTTCT 10680

TGCAGCCAAG AATTTTGCAG AACAGGGTAA GCTTGTTACG TTTGGTATTG TTCCAACATC 10740

CCCAGAGACT GGCTATGGTT ACATTAAGTC AGGTGAGTAT CTGGATGGAA AAGGTTACAA 10800

AGTTGCGGCT TTTGTTGAAA AACCAGAGCT TCACGTAGCT CAGCAGTATA TATCAGATGG 10860

CGGTTATCTT TGGAATAGTG GGATGTTTAT GTTCAGAGCA TCTGTATTTA TCAATGAACT 10920

GAAAAAATTC CGACCAGATA TTTTAGCCAG TTGCCAACGC TCCTTGTCCT CTTCGATACA 10980

AGATTTAGAT TTTATCCGTC TGGATAATGC TTCATTTTCT TGCTGTCCTG AAGAGTCTAT 11040

TGACTATGCA GTTATGGAAA AAACAGCCGA AGCTGTCGTC GTTCCCTTAA ATGCACAATG 11100

GAGTGATGTT GGGTCATGGT CTGCATTGTG GGAAATAAGT TCAAAAGATC AAAGCGGTAA 11160

TGCCATTCGT GGTGATGTAT TGGTTGAAGA TGCTACAGAT AGTTATCTTT ATTCGCAGCA 11220

TAGACTTATT GGTGCTGTGG GCATAAAGGA TTTGGTTGTT GTTGAAACGA AAGATGCCGT 11280

ATTAGTTGCT CATAAAGATA AAGTTCAGCA AGTTAAAAAC ATCGTCGCTC AACTTAAAAA 11340

GAATAATCGC ACAGAATATT TACAGCATCG GGAAATATTT CGGCCTTGGG GCAGTCATGA 11400

TACTATAGCT GAAGGGCCGC GCTTTCAAGT GAAACATGTG ATTGTATTAC CTGGCCACAT 11460

TACCGCTAAG CAGATTCATT ACCATCGTAC TGAGCATTGG ATTGTTGTCT CGGGGACAGC 11520

TAAAGTCCAT CTTGAAGATA AGACTTACCT TGTTTCTGAA AATGAATCAA CATATATACC 11580

TGTTGGTGTT CCACATGCCA TTGAAAATCC TGGCAAGATC CCGCTTGAAA TAATCGAGGT 11640

TAGATCAGGA GTCTACCTGG AAGAGGATGA TGTTATAAGA GTTTCTTCTT CTGGAGTAGG 11700

It is initial that orf10 stops orf11

ATAC TAATGC GAATTTCTAT TATCACAGCT ACTTATAATA GTGAAAAAAC TCTCCTTGAT 11760

ACATTACTTT CTCTAGAAAA ACAAACGTAT CCAGATATCG AATATATAGT TGTAGATGGA 11820

GCATCGAACG ATAATACAAT TAAGCTGATC AAGGGTAATT GTACAAAAGT TTCAAAAATC 11880

ATTTGCGAAC CCGATAAGGG CATTTATGAT GCTCTGAATA AAGGTATTCA AGCAGCTTCG 11940

GGTGATGTTA TTGGTTTTTT ACATTCTGAT GATTTATTAC CCTATGATGA TGCTATTGCA 12000

GATATAGCAA AAACATTTGA AATTACAGGG TGTGATGCTA TTTATGGCGA TTTGGAGTAT 12060

GTTGCCCAAA ATGATACGAC TAAACGTATT AGATTATGGA AAAGTGGTTC ATTTAGTCGT 12120

TTCAAGATGA AAGTGGGTTG GATGCCGCCA CACCCATCAT TTTATATGAA ACGCAAATGT 12180

TATAGGCAGT TTGGTTGTTT TTCTTTAGAT TATCGAATAT CTGCTGATTA TGACTCATTA 12240

TTACGATATA TTTTAAAACA ACGTATTTCA ACAGAATATT TACCAAAAGT CTTAGTGAAG 12300

ATGCGTGTTG GTGGAATTAG CAATCGTTCA GTATCTTCAA TGGTAAAGAA GTCAATGGAA 12360

GACATTCGTG TTATGAACCA GAATGGTATT ATCTGGCCAA TAGCTTTAGT GTATAAAAAT 12420

It is initial that orf11 stops orf12

ATATCCAAAC TTCCTCAATT TATTAAAAAG TAATCATCAT GTTAA ATGTT AAAAAAATCA 12480

TTAACGATAG TAATATTGCG TTCGGTACTA GTGGTGCTCG CGGTTTAGTG ATCGATTTTT 12540

CCCATGATGT TTGTGCTGCG TTCACTCATG CGTTTCTTTC TGTTATTGAT GACAAATACA 12600

ATTTTAATAA AGTTGCCTTA GCAATTGATA ACCGCCCAAG CAGTTACGAA ATCGCTCAGG 12660

CATGCGCCTT CGCTATCAAA CAACATGGGT TCACTGTCGA ATATCATGGT GTAATTCCGA 12720

CTCCCGCATT AGCTCATTAT TCGATGCAGA AAAACATTCC CTGCATAATG GTCACAGGGA 12780

GTCATATCCC TTTTGATCGT AATGGCTTAA AATTCTACAG ACCTGATGGT GAAATCACTA 12840

AAGAGGATGA GCTCGCAATT ATAAATAGTG AGTATACATT TTCGCCTGTA GGTGTATTAC 12900

CTCATCTTGA AACAAGCACT GAAGGTGCAG ACTACTACCT TGAGCGTTAT GTTTCTCTTT 12960

TTAATCCTGA TATTTTAAAG GGGAAAAGAA TAGGGCTATA TGAACATTCT AGTGCAGGAC 13020

GCGATTTATA TGCTCCTGTT TTTAATCAAT TGGGTGCAGA GGTCATTTCC TTAGGTAGAA 13080

GTGATGAGTT CGTTCCTATT GATACGGAAG CAGTAAGCGA TGAAGATCGC ATACTTGCAA 13140

GAGAGTGGTC TAAAAAATAT AATCTTGATG CTATTTTCTC AACAGATGGC GATGGTGATC 13200

GTCCTTTAGT TGCCGATGAA AATGGTGAAT GGCTAAGAGG CGATATTCTG GGATTACTTA 13260

CTGCTATTGA ACTTAATATC AAGGCGTTGG CTATTCCAGT GAGTTGTAAT ACAGCAATTG 13320

AACAGTCTAA TAAATTTGCA AGTATACAAC GAACGAAAAT AGGTTCTCCT TATGTAATTG 13380

CAGCGTTTAC AGATCTTGCT AAGCAATTTA ATTCAGTCGC TGGTTTTGAA GCTAATGGTG 13440

GCTTTCTCCT TGCCTCCGAT TTACAAATTA ATGGCAAGGA ATTAAAATCG TTACCTACAC 13500

GAGATGCTGT GTTACCAGCA TTAATGCTCT TAATAGCTTC TCGCAATAGT ACTATTTCTC 13560

AACTGATTAA TAATCTTCCT CAGCGATTCA CTTGGTCAGA TAGAGTTAAA AACTTCCCTT 13620

CAGATTCAAG TCAACAAATT ATAAAAAATG CCATATCGTC ACCCAATAAT TTCTTTAATA 13680

GTTTAGGTTA TGAATCATTA TCCTGTTCCG CTATTGATGA AACGGATGGT GCAAGATTTA 13740

CTTTAAATAA TGGTGATATT ATACATCTCC GTCCTTCCGG TAATGCTCCA GAACTCCGTT 13800

GTTATGCTGA GGCCAGTGAT GAAAATCAGG CTAAGCAATA TGTTACGAAT GTGCTGGGAA 13860

Orf12 stops

ATATTACCTC TTTGATTTCT TGATGTTATA GGTTTATCTA CGCTTATACG TGTGCGTAGG 13920

TTTGATTACA CGTAGATGCC GGTATACAGA ATTGAAGAAC GGTATTTGTT GCATTAATGA 13980

AATTCAGCAC TACACACATT CGTGCAACTT GAGATAACAT CTCAATCATA TCCAAGTCGC 14040

GCATACATCG CGGTGAACAC CCCCTGACAG GAGTAAACAA TGTCAAAGCA ACAGATTGGT 14100

GTCGTCGGTA TGGCAGTGAT GGGGCGCAAC CTTGCGCTCA ACATCGAAAG CCGTGGTTAT 14160

ACCGTCTCTA TTTTCAACCG TTCCCGTGAG AAGACGGAAG AAGTGATTGC CGAAAATCCA 14220

GGCAAAAAAT TGGTTCCTTA CTTTACGGTG AAAGGGTTTG TTGAATCTCT GGAAACGCCT 14280

CGTCGCATCC TGTTAATGGT GAAAGCAGGT GCAGGCACGG ATGCTGCAAT TGATTCCCTC 14340

AAACCATATC TCGATAAAGG TGACATCATC ATTGATGGTG GTAATACCTT CTTCCAGGAC 14400

ACCATTCGTC GTAACCGTGA GCTTTCTGCT GAAGGCTTTA ACTTCATTGG TACCGGTGTT 14460

TCCGGTGGTG AAGAGGGCGC GCTGAAAGGG CCTTCCATCA TGCCTGGTGG CCAGAAAGAA 14520

GCCTATGAAC TGGTTGCGCC GATCCTGACC AAAATCGCCG CCGTTGCTGA AGATGGCGAA 14580

CCGTGCGTTA CATATATTGG TGCCGATGGT GCGGGTCACT ATGTGAAAAT GGTTCACAAC 14640

GGTATTGAAT ACGGTGATAT GCAACTGATT GCTGAAGCCT ATTCTCTGCT TAAAGGCGGC 14700

CTGAATCTCT CTAACGAAGA ACTGGCACAG ACCTTTACCG AGTGGAATAA CGGTGAACTG 14760

AGCAGCTACC TGATCGACAT CACCAAAGAT ATCTTCACCA AAAAAGATGA AGACGGTAAC 14820

TATCTGGTTG ATGTGATCCT GGATGAAGCG GCTAACAAAG GTACCGGTAA ATGGACCAGC 14880

CAGAGCGCGC TGGATCTCGG CGAACCGCTG TCGTTGATTA CTGAGTCTGT GTTTGCACGT 14940

TATATCTCGT CTCTGAAAGA TCAGCGCGTG GCCGCATCTA AAGTTCTCTC TGGCCCGCAA 15000

GCACAGCCAG CAGGCGACAA GGCTGAGTTC ATCGAAAAAG TTCGCCGTGC GCTGTATCTG 15060

GGCAAAATCG TTTCTTACGC TCAGGGCTTT TCTCAGCTGC GTGCTGCGTC TGAAGAGTAC 15120

AACTGGGATC TGAACTACGG CGAAATCGCA AAGATTTTCC GTGCTGGCTG CATCATCCGT 15180

GCGCAGTTCC TGCAGAAAAT CACCGATGCA TATGCCGAAA ATCCGCAGAT CGCTAACCTG 15240

CTGCTGGCTC CGTACTTCAA GCAAATTGCC GATGACTACC AGCAGGCGCT GCGCGATGTC 15300

GTCGCTCATG CAGTACAGAA CGGTATCCCG GTTCCGACCT TCGCCGCTGC GGTTGCCTAT 15360

TACGATAGCT ACCGTGCCGC TGTTCTGCCT 15390

The above, it only is preferred embodiment of the present invention, be not that the present invention is done any pro forma restriction, every foundation technical spirit of the present invention all still belongs in the scope of technical solution of the present invention any simple modification, equivalent variations and modification that above embodiment did.

Claims (2)

1, a kind of oligonucleotide of the O-antigen-specific to intestinal bacteria O156 type is right, and it is characterized in that: described oligonucleotide is to being: the Nucleotide of 1512 to 1532 bases among the SEQ ID NO:1 and the Nucleotide of 1846 to 1864 bases; The Nucleotide of 1851 to 1869 bases among the SEQ ID NO:1 and the Nucleotide of 2229 to 2247 bases; The Nucleotide of 5209 to 5227 bases among the SEQ ID NO:1 and the Nucleotide of 5840 to 5858 bases; Or the Nucleotide of the Nucleotide of 5594 to 5614 bases among the SEQ ID NO:1 and 5865 to 5885 bases.

2, the right application of the described oligonucleotide of claim 1 is characterized in that it is used for PCR as primer, perhaps is used for hybridization and fluoroscopic examination, makes gene chip or microarray confession detection intestinal bacteria O156 type as probe.