CN1252269C - Nucleotide peculiar to 0-antigen of 058 type bacillus coli and 5 type Sh. dysenterae - Google Patents

Abstract

The present invention provides a specific nucleotide to the O-antigens of Escherichia coli O58 and Shigella dysenteriae 5. The specific nucleotide is the complete nucleotide sequence of gene clusters controlling the synthesis of O-antigens in the Escherichia coli O58 and the Shigella dysenteriae 5, such as a separated nucleotide with the total length of 17769 basic groups shown as SEQ ID NO: 1 or the nucleotide of SEQ ID NO: 1 which has one or a plurality of inserted, deleted or substituted basic groups and can simultaneously keep the functions of the separated nucleotide. The specific nucleotide also comprises the oligonucleotide of oligosaccharide unit processing genes in O-antigen gene clusters stemmed from the escherichia coli O58 and the Shigella dysenteriae 5. The present invention verifies that the oligonucleotide has high specificity to all of the O-antigens of the Escherichia coli O58 and the Shigella dysenteriae 5 through PCR. The present invention also discloses a method for using the oligonucleotide to detect and identify the Escherichia coli O58 and the Shigella dysenteriae 5 in the human body and the environment.

Description

Nucleotide to the O-antigen-specific of intestinal bacteria O58 and shigella dysenteriae 5 types

Technical field

The present invention relates to the complete nucleotide sequence of control O-antigen synthetic gene cluster in intestinal bacteria O58 (Escherichia coli O58) and shigella dysenteriae 5 types (Shigella bodyii 5), particularly relate in intestinal bacteria O58 and shigella dysenteriae 5 types oligonucleotide in the control O-antigen synthetic gene cluster, can utilize these oligonucleotide of O-antigen-specific intestinal bacteria O58 and shigella dysenteriae 5 types in human body and the environment and identify O-antigen in these pathogenic bacterium quickly and accurately.

Background technology

Shigellae is the pathogenic bacterium that grow up along with the human evolution, can attack colon film epithelial cell, causes self limiting pyogenic infection focus, causes human bacillary dysentery.Human have higher susceptibility to Shigellae, only need be less than the infection that ten bacterium just can cause the people, children and adult easy infection, particularly children, easily cause acute poisoning dysentery, and the O-antigen of Shigellae is the one of the main reasons that Shigellae causes disease.

Intestinal bacteria O58 also is pathogenic bacterium, it belongs to STEC (Shiga toxin-producingEscherichia coli), promptly produce the intestinal bacteria of Shiga toxin, it is important pathogenic bacteria [Steven P et al (2001) " Virulence Properties and Serotypesof Shiga Toxin-Producing Escherichia coli from Healthy AustralianSlaughter-Age Sheep " the .Journal of Clinical Microbiology that causes livestock disease, Vol.39, No.5,2017-2021]; This external Middle East and North Africa finds that also intestinal bacteria O58 can cause the diarrhoea [Leonard F.Perusk I et al (1999) " Phenotypic Diversity ofEnterotoxigenic Escherichia coli Strains from a Community-Based Studyof Pediatric Diarrhea in Periurban Egypt " JOURNAL OF CLINICALMICROBIOLOGY.2974 2978 Vol.37, No.9] of children below three years old.Therefore the detection to intestinal bacteria O58 and shigella dysenteriae 5 types is important, needs the method that can detect intestinal bacteria O58 and shigella dysenteriae 5 types quickly and accurately.

The lipopolysaccharides that is positioned at the intestinal bacteria surface is the morbific inducements of intestinal bacteria, and O-antigen is lipopolysaccharides outermost layer structure, is the target of immune system recognition and the site of phage absorption.The antigenic disappearance of O-can cause the serum sensitivity of many pathogenic agent, perhaps seriously undermines the virulence [Frank etal (1987) " The function of antibody and complement in the lysis ofbacteria " .Rev Infect Dis 177:1750-1753.Pluschke G et al " Role ofthe capsule and the O-antigen in resistance of O18:Kl Escherichia colito complement-mediated king " .J Bacteriol 42:907-913] of pathogenic agent.Intestinal bacteria are kinds, and the bacterial strain in planting is generally identified by O-antigen and H-antigen (sometimes by K-antigen).Wherein O-antigen has the height diversity, intestinal bacteria have 166 kinds of different O-antigens, the antigenic variation of O-may be colibacillary origin and keep its multifarious major cause [Reeves, P.R (1992) " Variation in antigens; niche specific selection and bacteria1populations " .FEMS Microbiol.Lett, 100:509-516].

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 o-antigen transhipment enzyme again, then aggregate into polysaccharide by polysaccharase, be connected to again and form lipopolysaccharide molecule [Whitfield, C. (1995) " Biosynthesis of lipopolysaccharide Oantigens " .Trends in Microbiology.3:178-185 on the glycolipid molecule; Schnaitman, C.A.andJ.D.Klena. (1993) " Genetics of lipopolysaccharide biosynthesis inentericbacteria " .Microbiological Reviews, 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. (1996) " Bacterial polysaccharide synthesis and genenomenclature " Trends in Microbiology, 4:495-503].In intestinal bacteria, Shigellae and Salmonellas, O-antigen gene [Lei Wang.et al (2001) " Sequence analysis of four Shigella boydii O-antigen loci:implicationfor Escherichia coli and Shigella relationships " .Infection andImmunity, 11:6923-6930 bunch between galF and gnd gene; Lei Wang and Peter Reeves (2000) " The Escherichiacoli O111 and Salimonella enterica O35 gene clusters:gene clusters encodingthe same colitose-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 o-antigen 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 ofabequose and paratose synthase genes (rfb) by polymerase chain reactionfor identification of S.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.1996, Paton, the A.W et.al serotype [" Molecularmicrobiological investigation of an outbreak of Hemolytic-UremicSyndrome 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 of Escherichia 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.

The antigenic structure of O-of intestinal bacteria O58 and shigella dysenteriae 5 types is known.Their structure is duplicate, [B.A.Dmitriev.V.L et al (1977) " Somatic antigens of Shigella.The strucuture of the specificpolysaccharide chain of Shigella dysenteriae type 5lipopolysaccharide " the .European Journal of Biochemistry of repeating unit that it is made up of 4 sugar, Vol 78,381-387] .[B.A.Dmitriev.V.L et al (1977), European Journal ofBiochemistry, Vol 79,111-115].

Summary of the invention

The Nucleotide that the purpose of this invention is to provide a kind of O-antigen-specific to intestinal bacteria O58 and shigella dysenteriae 5 types.It is the Nucleotide in the O-antigen gene bunch of intestinal bacteria O58 and shigella dysenteriae 5 types, is the special Nucleotide that comes from o-antigen transhipment enzyme gene, pol gene and glycosyltransferase gene.

An object of the present invention is to provide the full length nucleotide sequence of the O-antigen gene bunch of intestinal bacteria O58 and shigella dysenteriae 5 types.

A time purpose of the present invention has provided the gene of the O-antigen gene bunch that constitutes intestinal bacteria O58 and shigella dysenteriae 5 types: transhipment enzyme gene 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; Glycosyltransferase gene comprises orf6, orf9, orf11, orf12 gene; Sugar synthesis path gene comprises rmlB, rmlD, rmlA, rmlC, manB, manC, orf7, orf8 gene.Their zero positions in O-antigen gene bunch and final position and nucleotide sequence all are listed in the table 4.

Another purpose of the present invention has provided oligonucleotide, and they come from the gene of coding transhipment enzyme in the O-antigen gene bunch of intestinal bacteria O58 and shigella dysenteriae 5 types respectively, comprises the wzx gene or with wzx the gene of identity function is arranged; Come from the gene of coding polysaccharase, comprise the wzy gene or the gene of identity function is arranged with wzy; Come from the gene of encoding glycosyl transferring enzyme, comprise orf6, orf9, orf11, orf12 gene.They are the oligonucleotide in the said gene, and length is at 10-20nt; They are special to the O-antigen of intestinal bacteria O58 and shigella dysenteriae 5 types; The oligonucleotide of the gene of especially listing in the table 1 that comes from coding transhipment enzyme and the gene of polysaccharase, they are high specials to the O-antigen of intestinal bacteria O58 and shigella dysenteriae 5 types, and these oligonucleotide are also reconfigurable, and the oligonucleotide after the combination also is a high special to the O-antigen of intestinal bacteria O58 and shigella dysenteriae 5 types.

The above-mentioned oligonucleotide that another 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 detect O-antigen and detection and identification of escherichia coli O58 and shigella dysenteriae 5 types with identification of escherichia coli O58 and shigella dysenteriae 5 types by these methods.

A further object of the present invention has provided the method for complete sequence of the O-antigen gene bunch of separating Escherichia coli O58 and shigella dysenteriae 5 types.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 characterized in that the Nucleotide of the O-antigen-specific of intestinal bacteria O58 and shigella dysenteriae 5 types, and it is the isolating intestinal bacteria O58 shown in SEQ ID NO:1 and the Nucleotide of shigella dysenteriae 5 types, 17769 bases of total length; The base that perhaps has one or more insertions, disappearance or replacement keeps the intestinal bacteria O58 of SEQ ID NO:1 of described isolating functional nucleotide and the Nucleotide of shigella dysenteriae 5 types simultaneously.

The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O58 and shigella dysenteriae 5 types is characterized in that, comprising called after rmlB, rmlD, rmlA, rmlC, wzx, orf6, orf7, orf8, orf9, wzy, orf11, orfl2, manC, 14 genomic constitutions of manB are all between galF gene and gnd gene.

The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O58 and shigella dysenteriae 5 types is characterized in that described gene comprises: transhipment enzyme gene comprises the wzx gene or with wzx the gene of identity function is arranged; Pol gene comprises the wzy gene or with wzy the gene of identity function is arranged; Glycosyltransferase gene comprises orf6, orf9, orf11, orf12 gene.Wherein said transhipment enzyme gene is the Nucleotide of 4728 to 5915 bases among the SEQ ID NO:1; Described pol gene is the Nucleotide of 9803 to 11134 bases among the SEQ IDNO:1; Described orf6 gene is the Nucleotide of 5905 to 6513 bases among the SEQ ID NO:1; The orf9 gene is the Nucleotide of 8977 to 9798 bases among the SEQ ID NO:1; The orf11 gene is the Nucleotide of 11127 to 12251 bases among the SEQ ID NO:1; The orf12 gene is the Nucleotide of 12333 to 13442 bases among the SEQ ID NO:1.

The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O58 and shigella dysenteriae 5 types is characterized in that it comes from the oligonucleotide of described wzx gene or wzy gene or glycosyltransferase gene; And their mixing or their reorganization.

The Nucleotide of aforesaid O-antigen high special to intestinal bacteria O58 and shigella dysenteriae 5 types, it is characterized in that, the oligonucleotide of the described wzx of coming from gene is to being: the Nucleotide of 4967 to 4984 bases among the SEQ ID NO:1 and the Nucleotide of 5336 to 5353 bases, the Nucleotide of 5341 to 5358 bases among the SEQ ID NO:1 and the Nucleotide of 5839 to 5856 bases; The oligonucleotide of the described wzy of coming from gene is to being: the Nucleotide of 10475 to 10492 bases among the SEQ ID NO:1 and the Nucleotide of 11855 to 10872 bases, the Nucleotide of 10110 to 10127 bases among the SEQ ID NO:1 and the Nucleotide of 10932 to 10949 bases.

The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O58 and shigella dysenteriae 5 types is in the application that detects other polysaccharide antigen of expressing the antigenic bacterium of O-, the O-antigen of identifying bacterium and bacterium.

The recombinant molecule of the Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O58 and shigella dysenteriae 5 types is providing the O-antigen of expressing intestinal bacteria O58 and shigella dysenteriae 5 types by inserting to express, and the application in the preparation bacterial vaccine.

The application of the Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O58 and shigella dysenteriae 5 types, it is characterized in that, it is used for PCR, is used for hybridization and fluoroscopic examination or is used to make gene chip or microarray as probe as primer, for bacterial detection.

The separation method of the Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O58 and shigella dysenteriae 5 is characterized in that, comprises the steps:

(1) genomic extraction: in substratum, cultivate intestinal bacteria O58 and shigella dysenteriae 5 types, centrifugal collecting cell; The genomic dna that obtains detects by agarose gel electrophoresis;

(2) by the O-antigen gene in pcr amplification intestinal bacteria O58 and shigella dysenteriae 5 types bunch: with the genome of intestinal bacteria O58 and shigella dysenteriae 5 types is that template is passed through its O-antigen gene of Long pcr amplification bunch, with the PCR product that obtains, detect the size and the specificity thereof of PCR product with agarose gel electrophoresis, merge this long PCR product, and with DNA purification kit purified pcr product;

(3) make up O-antigen gene bunch library: Long PCR purified product is used shotgun make up O-antigen gene bunch library;

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

(5) splicing of nucleotide sequence and analysis: applying biological information science software splicing and edit all sequences, thus obtain the Nucleotide full length sequence of the O-antigen gene bunch of intestinal bacteria O58 and shigella dysenteriae 5 types;

(6) screening of specific gene: at wzx, the wzy gene design primer in the O-antigen gene of intestinal bacteria O58 and shigella dysenteriae 5 types 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, determines the antigenic high degree of specificity of O-of wzx, wzy gene pairs intestinal bacteria O58 and shigella dysenteriae 5 types;

(7) detection of primer sensitivity: cultivate intestinal bacteria O58 and shigella dysenteriae 5, after the bacterial count respectively with 5 * 10 ³, 5 * 10 ², 5 * 10 ¹5 and 0 viable bacteria join in a certain amount of certain thing to be detected, sneak into the thing to be detected of bacterium and use sample as detecting, sample is added the LB substratum, getting the LB substratum that some and sample mix cross filters, filtered liquid is cultivated, carried out the PCR reaction as pcr template with oligonucleotide after the peek milliliter is handled from cultured bacterium liquid, detect its sensitivity intestinal bacteria O58 and shigella dysenteriae 5.

The separation method of the Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O58 and shigella dysenteriae 5 types is characterized in that, comprises the steps:

(1) genomic extraction: 37 ℃ of incubated overnight intestinal bacteria O58 and shigella dysenteriae 5 types in the LB of 5mL substratum, centrifugal collecting cell.With 500ul 50mM Tris-HCl (pH8.0) and 10ul0.4M 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 liquor and use isopyknic phenol again: chloroform: twice of primary isoamyl alcohol (25: 24: 1) mixing solutions extracting, get supernatant liquor, again with isopyknic ether extracting to remove remaining phenol, supernatant liquor is with 2 times of volume ethanol deposit D NA, roll out DNA and wash DNA with 70% ethanol with glass yarn, at last DNA is resuspended among the 30ul TE, genomic dna detects by 0.4% agarose gel electrophoresis;

(2) by the O-antigen gene in pcr amplification intestinal bacteria O58 and shigella dysenteriae 5 types bunch: with the genome of intestinal bacteria O58 and shigella dysenteriae 5 types is that template is passed through its O-antigen gene of Long pcr amplification bunch; At first according to the galF gene design upstream primer (5 ' ATT GTG GCT GCA GGG ATC AAA GAA ATC-3 ') that often is found in O-antigen gene bunch upstream, again according to the gnd gene design downstream primer (5 '-TAG TCG CGG TGN GCC TGG ATT AAGTTC GC-3 ') 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 is as follows: 94 ℃ of pre-sex change 2 minutes, 94 ℃ of sex change were 10 seconds then, 60 ℃ of annealing 30 seconds, 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 6 pipe long PCR 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; Enzyme is cut the dna fragmentation size is concentrated between the 1kb-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), 1.25ul the T4DNA polysaccharase of 100mM DTT and 5 units, 11 ℃ were reacted 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 ℃ of reactions 20 minutes make 3 of DNA ' end add the dA tail, and 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 24 hours in 16 ℃, cumulative volume is 90ul, and the 10 * 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 Bio-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 Bio-Rad company and shock by electricity, voltage is 2.5 kilovolts, 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, then bacterium is coated in and contains penbritin, 37 ℃ of incubated overnight on the LB solid medium of X-Gal and IPTG, 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, extract plasmid and cut the segmental size of evaluation insertion wherein with the EcoRI enzyme from each clone simultaneously, the white that obtains clone group has constituted the O-antigen gene bunch library of intestinal bacteria O58 and shigella dysenteriae 5 types;

(4) to the cloning and sequencing in the library: from the library, select insert 100 clones of fragment more than 1000bp by Shanghai biotechnology company limited with ABI377 type automatic dna sequencer to unidirectional order-checking of insertion fragment in cloning, make sequence reach 80% fraction of coverage, carry out backward sequencing by the sequence that will interrelate again and survey logically obtaining remaining 20% sequence, thereby obtain 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 ResearchCouncil) Molecular Biology Lab and edit all sequences, thereby obtain the Nucleotide full length sequence of the O-antigen gene bunch of intestinal bacteria O58 and shigella dysenteriae 5 types, the quality of sequence is mainly guaranteed by two aspects: 1) genome of intestinal bacteria O58 and shigella dysenteriae 5 types is done 6 Long PCR reactions, mix these products then to produce the library.2), guarantee high-quality fraction of coverage more than 3 to each base; Behind the nucleotide sequence of the O-antigen gene that obtains intestinal bacteria O58 and shigella dysenteriae 5 types bunch, with American National biotechnology information science center (The National Center for BiotechnologyInformation, NCBI) orffinder finds gene, find the reading frame of 14 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 O58 and shigella dysenteriae 5 types at last;

(6) screening of specific gene: at wzx, the wzy gene design primer in the O-antigen gene of intestinal bacteria O58 and shigella dysenteriae 5 types bunch; Respectively designed two pairs of primers in each gene, the difference that every pair of primer is distributed in the corresponding gene is local to guarantee its specificity; Is that template is carried out PCR with these primers with the intestinal bacteria of 166 kinds of serotypes and the genome of 43 strain Shigellaes, all primers all obtain positive findings in intestinal bacteria O58 and shigella dysenteriae 5 types, the correct band of any size does not all increase in other groups, that is to say, do not obtaining any PCR product band in the array mostly, though in the minority group, obtain PCR product band, but its size does not meet the expection size, so wzx, wzy gene pairs intestinal bacteria O58 and shigella dysenteriae 5 types and O-antigen thereof all are high specials.

(7) detection of primer sensitivity: buy the live pig meat stuffing on the market, stir, be divided into the 20g portion, exist in-40 ℃ of refrigerators standby.The frozen bacterium liquid of 10 μ l intestinal bacteria O58 and shigella dysenteriae 5 types is inoculated in the triangular flask of 20ml LB substratum, in 37 ℃, 200 rev/mins, cultivate 12 hours to saturated, the cultured bacterium liquid that takes a morsel is done 106 and 107 times dilution, remaining bacterium liquid is put in 4 ℃ the refrigerator standby, get 50 μ, 1 dilution bacterium liquid coating LB agar plate, 37 degree are cultivated 12h, to being coated with plate count, calculate viable bacteria concentration in the stoste.In 5 portions of live pig meat stuffings, mix 5 * 10 respectively ³, 5 * 10 ², 5 * 10 ¹, 5 and 0 viable bacteria stir, and add 200ml LB substratum, and through 6 layers of filtered through gauze, filtered liquid 200 rev/mins, is cultivated 12h in 37 ℃.Get 3ml bacterium liquid in 6 from cultured bacterium liquid, centrifugal 5 minutes of 000g removes supernatant, adds 100 μ l MQ ultrapure waters and blows precipitation and mixing open, puts into 100 degree boiling water and boils 15 minutes, and lysate is in 12, and centrifugal 8 minutes of 000g gets 1 μ supernatant as pcr template.Right with 4 pairs of oligonucleotide, the Nucleotide of 4967 to 4984 bases among the SEQ ID NO:1 and the Nucleotide of 5336 to 5353 bases, the Nucleotide of 5341 to 5358 bases among the SEQ ID NO:1 and the Nucleotide of 5839 to 5856 bases, the Nucleotide of 10475 to 10492 bases among the SEQ ID NO:1 and the Nucleotide of 11855 to 10872 bases, the Nucleotide of 10110 to 10127 bases among the SEQ ID NO:1 and the Nucleotide of 10932 to 10949 bases, carry out the PCR reaction, the PCR reaction system is as follows: MQ:15.7 μ l, Mg ²⁺: 2.5 μ l, Buffer:2.5 μ l, dNTP:1 μ l, Taq enzyme: 0.3 μ l, P1:1 μ l, P2:1 μ l, template DNA: 1 μ l.The PCR reaction conditions is: 95 ℃: 5 ', 95 ℃: 30 ", 56 ℃: 45 ", 72 ℃: 1 ', 72 ℃: 5 ', totally 30 circulations.Reaction is got 10 μ l reaction product electrophoresis after finishing, if the amplified band that conforms to the expection size is arranged, then the result is positive, if do not have, then the result is negative.Participated in 5 * 10 ³, 5 * 10 ², 5 * 10 ¹And every part of pork filling of 5 viable bacterias all obtains positive findings in the PCR of 4 pairs of primers reaction.The pork filling that participates in 0 viable bacteria obtains negative findings in the PCR of 4 pairs of primers reaction.Illustrate that these 4 pairs of primers are 0.25 bacterium/g to the detection sensitivity of intestinal bacteria O58 in the pork filling and shigella dysenteriae 5 types when using aforesaid method.

Just, first aspect of the present invention provides the full length nucleotide sequence of the O-antigen gene bunch of intestinal bacteria O58 and shigella dysenteriae 5 types, its complete sequence shown in SEQ ID NO:1,17769 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 O58 and shigella dysenteriae 5 types by method of the present invention, as described in Table 3, it comprises called after rmlB, rmlD, rmlA, rmlC, wzx, orf6, orf7, orf8, orf9, wzy, orf11, orf12, manC, 14 genomic constitutions of manB are 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 O58 and shigella dysenteriae 5 types, 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); Glycosyltransferase gene comprises orf6, orf9, orf11, orf12 gene; Special sugared synthesis path gene in the bacterial polysaccharides antigen comprises rmlB, rmlD, rmlA, rmlC, manB, manC, orf7, orf8 gene.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 o-antigen 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 special, and the o-antigen that the present invention relates to transhipment enzyme gene, pol gene and glycosyltransferase gene are special to the O-antigen of intestinal bacteria O58 and shigella dysenteriae 5 types.

The 3rd aspect of the present invention, wzy gene in the O-antigen gene bunch that comes from intestinal bacteria O58 and shigella dysenteriae 5 types is provided or the gene of identity function and wzx gene is arranged or with wzx the oligonucleotide of gene of identity function and the oligonucleotide that glycosyltransferase gene comprises orf6, orf9, orf11, orf12 gene are arranged with wzy, they are any one section oligonucleotide in these genes.But, be to list in the wzy gene in the O-antigen gene bunch that comes from intestinal bacteria O58 and shigella dysenteriae 5 types in the table 1 or the gene, wzx gene of identity function arranged or have the oligonucleotide of gene of identity function right preferentially with wzx with wzy 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 being to obtain expecting the product of size in the pcr amplification that carries out of template with intestinal bacteria O58 and shigella dysenteriae 5 types only, and are all not obtain expecting the product of size in the pcr amplification that carries out of template other bacterium listed with table 2.In more detail, with these oligonucleotide to being that PCR that primer is done is reflected at and does not all obtain spawn in most of bacteriums, so, can determine these primers promptly the listed oligonucleotide of table 1 be high special to intestinal bacteria O58 and shigella dysenteriae 5 types and their O-antigen.

Described Nucleotide to the intestinal bacteria O58 and the O-antigen-specific of shigella dysenteriae 5 types separate and authentication method comprises the steps: 1) genomic extraction; 2) the O-antigen gene in pcr amplification intestinal bacteria O58 and shigella dysenteriae 5 types bunch; 3) structure in O-antigen gene bunch library; 4) to the cloning and sequencing in the library; 5) splicing of nucleotide sequence and analysis finally obtain the structure of O-antigen gene bunch; 6) screening of specific gene; 7) detection of primer sensitivity.

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

As described herein, " oligonucleotide " mainly is meant gene, the gene of coding polysaccharase and one section nucleic acid molecule in the encoding glycosyl transferase gene of the coding transhipment enzyme that derives from the O-antigen gene bunch, they can change on length, generally change in 10 to 20 Nucleotide scopes.Especially the oligonucleotide that comes from wzx gene (nucleotide position is the Nucleotide of 4728 to 5915 bases from SEQ ID NO:1) and the wzy gene (nucleotide position is 9803 to 11134 bases from SEQ ID NO:1) all is a high special to intestinal bacteria O58 and shigella dysenteriae 5 types.

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 transhipment enzyme gene, comprise the wzx gene or with wzx the gene of identity function are arranged; Come from pol gene, comprise the wzy gene or the gene of identity function is arranged with wzy; Come from glycosyltransferase gene, comprise orf6, orf9, orf11, orf12 gene.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 transhipment enzyme gene, come from pol gene and come from the combination of the oligonucleotide in the glycosyltransferase gene.

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) coding transhipment enzyme gene, comprise the wzx gene or (ii) the encode gene of polysaccharase of the gene of identity function is arranged, comprise the wzy gene or the gene of identity function is arranged with wzy with wzx.(iii) the encoding glycosyl transferase gene comprises orf6, orf9, orf11, orf12 gene.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 O58 and shigella dysenteriae 5 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.

The present inventor 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 and comes from that coding transhipment enzyme gene comprises the wzx gene or have the gene of gene, the coding polysaccharase of identity function to comprise the wzy gene with wzx or with wzy the oligonucleotide of gene of identity function and the oligonucleotide that the encoding glycosyl transferase gene comprises orf6, orf9, orf11, orf12 gene are arranged.This cover oligonucleotide is special to the O-antigen of a special bacterium, and this special bacterium O-antigen is expressed by intestinal bacteria O58 and shigella dysenteriae 5 types.

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 coding transhipment enzyme, comprise the wzx gene or (ii) the encode gene of polysaccharase of the gene of identity function is arranged with wzx, the gene that comprises the wzy gene or identity function arranged with wzy is the encoding glycosyl transferase gene (iii), comprises orf6, orf9, orf11, orf12 gene.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 O58 and shigella dysenteriae 5 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 and be used to detect the many to oligonucleotide of the inventive method, many here is that the gene that comes from coding transhipment enzyme comprises the wzx gene or with wzx the gene of identity function arranged to oligonucleotide; The gene that comes from the coding polysaccharase comprises the wzy gene or with wzy the gene of identity function is arranged; The gene that comes from the encoding glycosyl transferring enzyme comprises orf6, orf9, orf11, orf12 gene.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 coding transhipment enzyme, comprise the wzx gene or (ii) the encode gene of polysaccharase of the gene of identity function is arranged with wzx, the gene that comprises the wzy gene or identity function arranged with wzy is the encoding glycosyl transferase gene (iii), comprises orf6, orf9, orf11, orf12 gene.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 O58 and shigella dysenteriae 5 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 the wzx gene or the gene of identity function and wzy gene are arranged or have the gene of identity function and glycosyltransferase gene to comprise on the sequence of orf6, orf9, orf11, orf12 gene with wzy with wzx 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-.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 O58 and shigella dysenteriae 5 types 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 O58 and shigella dysenteriae 5 types 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 O58 and shigella dysenteriae 5 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 liquor, use isopyknic phenol again: chloroform: primary isoamyl alcohol (25: 24: 1) solution extracting twice, get supernatant liquor, again with isopyknic ether extracting to remove remaining phenol.Supernatant liquor rolls out DNA and washes DNA with 70% ethanol with glass yarn with 2 times of volume ethanol deposit D NA, at last DNA is resuspended among the 30ul TE.Genomic dna detects by 0.4% agarose gel electrophoresis.

Embodiment 2: by O-antigen gene in pcr amplification intestinal bacteria O58 and shigella dysenteriae 5 types bunch.

With the genome of intestinal bacteria O58 and shigella dysenteriae 5 types is that template is passed through its O-antigen gene of Long pcr amplification bunch.At first according to the galF gene design upstream primer (5 ' ATT GTG GCT GCA GGG ATC AAA GAA ATC-3 ') that often is found in O-antigen gene bunch upstream, again according to the gnd gene design downstream primer (5 '-TAG TCG CGC TGN GCC TGG ATTAAG TTC GC-3 ') 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, 61 ℃ of annealing 30 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 6 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, the DNaseI of the 1mg/ml of 1ul dilution in 1: 2000, reaction is carried out at room temperature.Enzyme is cut the dna fragmentation size is concentrated between the 1kb-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 ℃ were reacted 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 24 hours in 16 ℃, cumulative volume is 90ul.10 * the bufffer 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 the competent cell bacillus coli DH 5 alpha.Get a ring bacillus coli DH 5 alpha list bacterium colony 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 liquor, 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 liquor, 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 alpha 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 O58 and shigella dysenteriae 5 types with the EcoRI enzyme.

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

From the library, select insert 100 clones of fragment more than 1000bp by Shanghai biotechnology company limited with ABI377 type automatic dna sequencer to unidirectional order-checking of insertion fragment in cloning, make sequence reach 80% fraction of coverage.Residue 20% sequence is surveyed logical obtaining by backward sequencing and with some sequence again, obtains all sequences of O-antigen gene bunch at last.

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 O58 and shigella dysenteriae 5 types obtained.The quality of sequence is mainly guaranteed by two aspects: 1) genome of intestinal bacteria O58 and shigella dysenteriae 5 types is done 6 Long PCR reactions, mix these products then to produce the library.2), guarantee high-quality fraction of coverage more than 3 to each base.Behind the nucleotide sequence of the O-antigen gene that obtains intestinal bacteria O58 and shigella dysenteriae 5 types bunch, with American National biotechnology information science center (The National Center for Biotechnology Information, NCBI) orffinder finds gene, find the reading frame of 14 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 O58 and shigella dysenteriae 5 types at last, as shown in table 3.

By retrieving and relatively, finding the dTDP-D-glucose-4 of orf1 encoded protein and Shigella boyii, the 6-desaturase has 98% homogeny in 361 amino acid, 98% similarity.DTDP-D-glucose-4,6-desaturase are by the rmlB genes encoding, and the homogeny of height shows that orf1 also is the rmlB gene, called after rmlB.The dTDP-D-glucose of Orf2 encoded protein and Escherjchiacoli-4-rhamnosyl reductase enzyme has 98% homogeny, 99% similarity in 299 amino acid.DTDP-D-glucose-4-rhamnosyl reductase enzyme is by the rmlD genes encoding, and the homogeny of height shows that orf2 also is the rmlD gene, called after rmlD.The Cori ester thymidine transferring enzyme of Orf3 encoded protein and Shigella boydii has 97% homogeny, 98% similarity in 291 amino acid.Cori ester thymidine transferring enzyme is by the rmlA genes encoding, and the homogeny of height shows that orf3 also is the rmlA gene, called after rmlA.The dTDP-6-DDG 3 of Orf4 encoded protein and Escherichia coli, the 5-mutase has 88% homogeny in 173 amino acid, 89% similarity.DTDP-D-glucose 4,6-desaturase are by the rmlC genes encoding, and higher homogeny shows that orf4 also is the rmlC gene, called after rmlC.The synthetic jointly rhamnosyl of these four genes.Blast comparison shows that the O-antigen transhipment enzyme Wzx of orf5 encoded protein and Plesiomona shigelloides has 23% homogeny, 44% similarity in 393 amino acid whose sequences.And algorithm [Eisenberg by people such as Eisenberg, D, Schwarz, E.etal (1984) .Analysis of membrane andsurface protein sequences with the hydrophobic momentplot.J.Mol.Biol.179:125-142] find that orf5 has 10 potential transmembrane domains, this is the proteic characteristic feature of Wzx, the proteic aminoterminal of Wzx has about 40 amino acid whose conservative motifs, so can determine orf5 is the wzx gene, called after wzx.The acetyltransferase of the exocellular polysaccharide of Orf6 encoded protein and Chromobacterium violaceum has 44% homogeny in 152 amino acid; 57% similarity; therefore infer that orf6 also is an acetyltransferase gene, with the temporary called after orf6 of orf6.The pyruvyl-transferase of Orf7 encoded protein and Bacillusanthracis has 24% homogeny in 77 amino acid, 49% similarity infers that orf7 also is a gene that produces pyruvyl-transferase, called after orf7 temporarily.The coenzyme F 420-reduction desaturase of Orf8 encoded protein and Methanothermobacterthermautotrophicus has 29% homogeny, 48% similarity in 399 amino acid.By search, find that orf8 encoded protein and the E value of the motif PF00535 that guards are 3.4 * e to Pfam protein-based order sequenced data storehouse ^-35, infer that therefore orf8 also is the gene of a coding reduction desaturase, with the temporary called after orf8 of orf8.The glycosyltransferase of Orf9 encoded protein and Ureaplasma urealyticum has 28% homogeny in 100 amino acid, 31% similarity is inferred the orf9 glycosyltransferase of also encoding, with the temporary called after orf9 of orf9.The O-antigen polysaccharase of Orf10 encoded protein and Vibrio cholerae has 30% homogeny, 43% similarity in 198 amino acid whose sequences.And algorithm [Eisenberg by people such as Eisenberg, D, Schwarz, E.etal (1984) .Analysis of membrane andsurface protein sequences with the hydrophobic momentplot.J.Mol.Biol.179:125-142] find that the orf10 encoded protein has 11 potential transmembrane domains, it has similar secondary structure to many Wzy albumen, and hydrophilic loop (loop) in the big kytoplasm is arranged, feature with typical O-antigen polysaccharase, so determine that orf10 is the wzy gene, called after wzy.The glycosyltransferase of Orf11 encoded protein and Escherichia coli has 27% homogeny in 335 amino acid, 48% similarity is inferred the Orf11 glycosyltransferase of also encoding, with the temporary called after Orf11 of orf11.The glycosyltransferase of Orf12 encoded protein and Bacteroides thetaiotaomicron has 46% homogeny in 369 amino acid, 65% similarity, by search, find that the E value of the conservative motif PF00535 of orf12 encoded protein and glycosyltransferase family is 1.5 * e to Pfam protein-based order sequenced data storehouse ^-35, therefore infer that orf12 also is a glycosyltransferase gene, temporarily called after orf12.The mannose-1-phosphate guanyltransferase of the manC genes encoding of rf13 encoded protein and Escherichia coli O157:H7 has 70% homogeny in 472 amino acid, 86% similarity, the homogeny of height shows that orf13 also is a manC gene, therefore with orf13 called after manC.The phosphomannomutase of the manB genes encoding of Orf14 encoded protein and Escherichia coli has 99% homogeny in 456 amino acid, 99% similarity, the homogeny of height shows that orf14 also is a manB gene, therefore with orf14 called after manB.

Embodiment 6: the screening of specific gene

At wzx, wzy gene design primer in the O-antigen gene of intestinal bacteria O58 and shigella dysenteriae 5 types bunch, the position of these genes in nucleotide sequence sees Table 1.

Transhipment enzyme gene, pol gene and their function corresponding and the size of the O antigen gene bunch of intestinal bacteria O58 and shigella dysenteriae 5 types in table 1, 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 ID NO: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.

Mdh (malate dehydrogenase) gene is to be present in all colibacillary genomes and a gene of high conservative, so we according to the mdh gene design primer (5 '-TTC ATC CTA AACTCC TTA TT-3 ') and (5 '-TAA TCG CAG GGG AAA GCA GG-3 '), extract genome then from the intestinal bacteria of 166 kinds of serotypes, method as previously mentioned.With this to primer from the colibacillary genome of 166 kinds of serotypes PCR with identification of escherichia coli and detect its genomic quality.

Table 2 is intestinal bacteria and 43 strain Shigellaes and their sources that are used to screen 166 kinds of serotypes of specific gene, and for the convenience that detects, we are divided into one group with their every 12-19 bacterium.All list in the table in their source.

Contain intestinal bacteria O58 in the 3rd group, the genomic dna that contains shigella dysenteriae 5 types in the 9th group is as positive control.Being the genomic dna that does not contain intestinal bacteria O58 in the 13rd group, is the genomic dna that does not contain shigella dysenteriae 5 types in the 14th group, as negative 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 2 minutes, 95 ℃ of sex change 15 seconds, annealing temperature is because of the difference different (with reference to table 1) of primer, annealing time is 50 seconds, and 72 ℃ were extended 2 minutes, and carried out 30 circulations like this.Continue to extend 10 minutes at 72 ℃ at last, reaction system is 25ul.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, and every pair of primer has obtained except be PCR in the 3rd group after the correct band of expection size, and the correct band of any size does not all increase in other groups.So wzx, wzy gene pairs intestinal bacteria O58 and shigella dysenteriae 5 types and O-antigen thereof all are high specials.

At last, from intestinal bacteria O58 and shigella dysenteriae 5 types, screen gene by PCR: wzx, wzy gene to the O-antigen high special of intestinal bacteria O58 and shigella dysenteriae 5 types.And the oligonucleotide of these intragenic any one section 10-20nt is special to the O-antigen of intestinal bacteria O58 and shigella dysenteriae 5 types, and the primer in especially above-mentioned each gene is that oligonucleotide is high specials to detecting the back confirmation through PCR to intestinal bacteria O58 and shigella dysenteriae 5 types.These all oligonucleotide all can be used for intestinal bacteria O58 and shigella dysenteriae 5 types in the human body and environment rapidly and accurately, and can identify their O-antigen.

Embodiment 7: the detection of primer sensitivity.

Buy the live pig meat stuffing on the market, stir, be divided into the 20g portion, exist in-40 ℃ of refrigerators standby.The frozen bacterium liquid of 10 μ l intestinal bacteria O58 and shigella dysenteriae 5 types is inoculated in the triangular flask of 20ml LB substratum, in 37 ℃, 200 rev/mins, cultivate 12 hours to saturated, the cultured bacterium liquid that takes a morsel does 10 ⁶With 10 ⁷Dilution doubly, remaining bacterium liquid are put in 4 ℃ the refrigerator standby, get 50 μ l dilution bacterium liquid coating LB agar plate, and 37 degree are cultivated 12h, to being coated with plate count, calculate viable bacteria concentration in the stoste.In 5 portions of live pig meat stuffings, mix 5 * 10 respectively ³, 5 * 10 ², 5 * 10 ¹, 5 and 0 viable bacteria stir, and add 200ml LB substratum, and through 6 layers of filtered through gauze, filtered liquid 200 rev/mins, is cultivated 12h in 37 ℃.Get 3ml bacterium liquid in 6 from cultured bacterium liquid, centrifugal 5 minutes of 000g removes supernatant, adds 100 μ l MQ ultrapure waters and blows precipitation and mixing open, puts into 100 degree boiling water and boils 15 minutes, and lysate is in 12, and centrifugal 8 minutes of 000g gets 1 μ supernatant as pcr template.Right with 4 pairs of oligonucleotide, the Nucleotide of 4967 to 4984 bases among the SEQ ID NO:1 and the Nucleotide of 5336 to 5353 bases, the Nucleotide of 5341 to 5358 bases among the SEQ ID NO:1 and the Nucleotide of 5839 to 5856 bases, the Nucleotide of 10475 to 10492 bases among the SEQ ID NO:1 and the Nucleotide of 11855 to 10872 bases, the Nucleotide of 10110 to 10127 bases among the SEQ ID NO:1 and the Nucleotide of 10932 to 10949 bases, carry out the PCR reaction, the PCR reaction system is as follows: MQ:15.7 μ l, Mg ²⁺: 2.5 μ l, Buffer:2.5 μ l, dNTP:1 μ l, Taq enzyme: 0.3 μ l, P1:1 μ l, P2:1 μ l, template DNA: 1 μ l.The PCR reaction conditions is: 95 ℃: 5 ', 95 ℃: 30 ", 56 ℃: 45 ", 72 ℃: 1 ', 72 ℃: 5 ', totally 30 circulations.Reaction is got 10 μ l reaction product electrophoresis after finishing, if the amplified band that conforms to the expection size is arranged, then the result is positive, if do not have, then the result is negative.Participated in 5 * 10 ³, 5 * 10 ², 5 * 10 ¹And every part of pork filling of 5 viable bacterias all obtains positive findings in the PCR of 4 pairs of primers reaction.The pork filling that participates in 0 viable bacteria obtains negative findings in the PCR of 4 pairs of primers reaction.Illustrate that these 4 pairs of primers are 0.25 bacterium/g to the detection sensitivity of intestinal bacteria O58 in the pork filling and shigella dysenteriae 5 types when using aforesaid method.

By clone and the expression in the vaccine strains of attenuation, can set up recombiant vaccine to O antigen gene bunch.O antigen is the surface antigen of topmost Gram-negative bacteria, can cause the intensive immune response, is one of best target molecule of making recombiant vaccine.Viret laboratory success in 1993 the O antigen gene of Shigellae Sonnei bunch is expressed in a strain Salmonellas Tyziai vaccine bacterium, experimentation on animals proof can cause rabbit immune response (Molecular Microbiology1993,7:239-252).The group of China Military Medical Science Institute also similarly works being engaged in the Viret laboratory.Bunch express the O antigen gene of intestinal bacteria O111 success in 1999 in the Wang Lei laboratory in salmonella vaccine STM-1, and the bacterial strain set up of proof can cause the blood of mouse and humoral response (Microbial Pathogenesis 1999,27:55-59).So the O antigen-specific gene order of intestinal bacteria O58 of the present invention and shigella dysenteriae 5 types can be applied to set up recombiant vaccine.

Nucleotide sequence (shown in the SEQ ID NO:1) according to the O-antigen-specific to intestinal bacteria O58 and shigella dysenteriae 5 types of the present invention, structure specific nucleic acid probe, be fixed on the carrier of chip and make biochip, after the sample that will detect is suitably handled, carry out hybridization with biochip, utilize the biochip signal analysis equipment just can obtain corresponding bacteria situation in the sample then.The DNA chip that this intestinal bacteria O antigen is identified can be directly used in clinical and other check place (as food-processing and production industry, the Micro biological Tests of animal and veterinary industry customs quarantine control etc.).This chip only need enlarge output, just can industrialization under identical condition.

Table 3 is structural tables of the O-antigen gene bunch of intestinal bacteria O58 and shigella dysenteriae 5 types.Listed the structure of the O-antigen gene bunch of intestinal bacteria O58 and shigella dysenteriae 5 types in table, their structure is duplicate, altogether by 14 genomic constitutions, and each gene box indicating, and in square frame, write the title of gene.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 location tables of the gene in the O-antigen gene bunch of intestinal bacteria O58 and shigella dysenteriae 5 types, in table, listed the accurate position of all open reading frame in complete sequence in the O-antigen gene bunch of intestinal bacteria O58 and shigella dysenteriae 5 types, 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 bacterium: ATG and GTG.

Sequence list

SEQUENCE LISTING

＜110〉Tianjin Biochip Technology Co., Ltd

＜120〉to the Nucleotide of the O-antigen-specific of intestinal bacteria O58 and shigella dysenteriae 5 types

<160>1

<170>PatentIn version 3.1

<210>1

<211>17769

<212>DNA

<213>Escherichia coli and shigella

<400>1

attgtggctg cagggatcaa agaaatcctc ctggtaactc acgcgtccaa gaacgcggtc 60

gaaaaccact tcgacacctc ttatgaatta gaatctctcc ttgagcagcg cgtgaagcgt 120

caactgcttg cggaagtgca gtccatctgt ccaccgggcg tgaccattat gaacgtgcgt 180

cagggcgaac ctttaggttt gggccgctcc attttatgtg cacgacctgc cattggtgac 240

aacccatttg tcgtggtgct gccagacgtt gtgatcgatg acgccagcgc cgacccgctg 300

cgctacaacc ttgctgccat gattgcgcgc ttcaacgaaa cgggccgcag ccaggtgctg 360

gcaaaacgta tgccgggtga cctctctgaa tactctgtca tccagaccaa agagccgctg 420

gatcgtgagg gtaaagtcag ccgcattgtt gaatttattg aaaaaccaga tcagccgcag 480

acgctggact cagacatcat ggccgttggt cgctatgtgc tttctgcaga tatttggccg 540

gaacttgaac gcactcagcc tggtgcatgg gggcgtattc agctgactga tgctattgcc 600

gaactggcta aaaaacagtc tgttgatgca atgctgatga caggcgacag ctacgactgc 660

ggtaaaaaaa tgggctatat acaggcgttt gtgaagtatg gactgcgtaa cctaaaagaa 720

ggggcgaagt tccggaaagg gattgagaag ctgttaagcg aataatgaaa atctgaccgg 780

atgtaacggt tgataagaaa attataacgg cagtgaagat tcgtggcgaa agtaatttgt 840

tgcgaatctt cctgccgttg ttttatataa acaatcagaa taacaatgag ttagcaatag 900

gattttagtc aaagttttcc aggattttcc ttgtttccag agtggattgg taagacaatt 960

agcgtttgaa tttttcgggt ttagcgcgag taggtaatgc tcgtcacatc gtcggcatgc 1020

atgcagtgct ctggtagctg taaagcca

g ggcggtagcg tgcattaata cctctattaa 1080

tcaaactgag agccgcttat ttcacagcat gctctgaagt aatatggaat aataaagtga 1140

a

atacttgt tactggtggc gcaggattta ttggttct c tgtagttcgt cacattataa 1200

ataatacgca

gatagtgtt gttaatgtcg ataaattaac gtacgccgga a ctggaat 1260

c ttgctga tgtttc

at tctgaacgct atgtttttga acatgcggat

tttgcgatg 1320

cagctg

aat ggcacggatt tttgctcagc atcagccgga tgcagtgatg cacctggctg 1380

ctgaaagcca tgt

gatcgt tcaattacag g ctgcggc atttattgaa accaatattg 1440

ttggtac ta tgtccttttg gaagc

ctc gcaattactg gtctgctctt gat

cgaca 1500

agaaaaatag cttccgtttt catcatattt ctactgacga agtctatggt gatttgcctc 1560

atcctgacga agtaaataat aaaga

caat tacccctctt tactgagacg acagcttacg 1620

cgccaag

ag

cc

tattc gc

tc

aag catcaagcga tcatttagtc cg

cttgga 1680

aacgtaccta tggtttaccg accattgtga cta ttgttc gaataactac ggtccttatc 1740

actttccgga aaaattgatt ccactagtaa ttcttaatgc tctggaaggt aaggcattac 1800

ctatttatgg caaaggggat caaattcgtg actggctgta tgttgaagat catgcgcgtg 1860

cgttatatac cgtcgtaacc gaaggtaaag cgggtgaaac ttataacatt ggtggacaca 1920

acgaaaagaa aaacatcgat gtagtgctca ctatttgtga tttgttggat gagattgtac 1980

cgaaagagaa atcttaccgc gagcaaatta cttatgttgc cgatcgcccg ggacacgatc 2040

gccgttatgc gattgatgca gagaagatta gccgcgaatt gggctggaaa ccgcaggaaa 2100

cgtttgagag cgggattcgt aaaacggtgg aatggtacct gtccaataca aaatgggttg 2160

ataatgtgaa aagtggtgac tatcaatcgt ggattgaaca gaactatgag ggccgccagt 2220

aatgaatatc ctcctttttg gcaaaacagg gcaggtaggt tgggaactac agcgtgctct 2280

ggcacctctg ggtaatttga ttgctcttga tgttcactcc actgattact gtggtgattt 2340

tagtaatcct gaaggtgtag ctgaaaccgt aagaagcatt cggcctgata ttattgtcaa 2400

cgcagccgct cacaccgcag tagacaaagc agaatcagaa ccggagtttg cacaattact 2460

taacgcaaca agtgtcgaag cgattgcgaa agcagcaaat gaagttggag cttgggttat 2520

ccattactcg actgattacg tcttccctgg aaatggcgac atgccatggc tggagacgga 2580

tgcaaccgca ccgc

aaatg tttacggtga aaccaagtta gctggagaaa aagcattaca 2640

agagcattgt gcgaagcacc taattttccg tacaagctgg gtctatgcag gtaaaggaaa 2700

taacttcgcc aaaacgatgt tgcgtctggc aaaagagcgt gaagaattag ccgttattaa 2760

tgatcagttt ggtgcgccaa caggtgctga actgctggct gattgtacgg cacatgcaat 2820

tcgtgtggca ctgaataaac cagaagtcgc aggcttgtat catctggtag ccagtggtac 2880

cacaacctgg cacgattatg ctgcgctggt ttttgaagag gcgcgcaaag caggcattcc 2940

ccttgcactc aacaagctca atgcagtacc aacaacagcc tatcctacac cagcccgtcg 3000

cccccataac tctcgcctta atacagaaaa atttcagcag aactttgcgc ttgtcttgcc 3060

tgactggcag gttggtgtga aacgaatgct caacgaatta tttacgacta cagcaattta 3120

atagtttttg catcttgttc gtgatggtgg agcaagatga atcaaaagga at atgaaat 3180

gaaaacgcgt aaaggtacta ttttagcggg tggttctggt actcgtcttt atcctgtgac 3240

tatggctgtc agtaaacagc tattacctat ttatgataag ccgatgatct attacccgct 3300

ttctacactg atgttagcgg gtattcgcga tattctgatt atcagtacgc cgcaggatac 3360

tcctcgtttt caacaactgc tgggtgacgg gagccagtgg gggctaaatc ttcagtataa 3420

agtgcaaccg agtccagatg gtcttgcgca ggcatttatc atcggtgaag agtttatcgg 3480

tggtgatgat cgtgctttgg ttcttggtga taatatcttc tacggtcatg atctgccgaa 3540

gttaatggat gtcgctgtta acaaagaaag tggtgcaacg gtatttgcct atcacgttaa 3600

tgatcctgaa cgctacggtg tcgttgagtt tgataaaaat ggtacggcga tcagcctgga 3660

agaaaaaccg ctacaaccaa aaagtaatta tgcggtaacc gggctttatt tctatgataa 3720

cgacgttg g gaaatggcga aaaaccttaa gccttctgcc cgcggtgaac tggaaattac 3780

cgatattaac cgtatttata tggaacaggg gcgtttatct gttgccatga tgggccgtgg 3840

ttatgcatgg ctggacacgg ggacacatca aagcctgatt gaggcaagca acttcattgc 3900

aacaattgaa gaacgtcagg ggctgaaagt ttcctgcccg gaagaaattg cttaccgtaa 3960

agggtttatt gatgctgaga aggtgaaagt attagctgaa ccgctgaaaa aaaatgctta 4020

tggtcagtat ctgctgaaaa tgattaaagg ttagtaataa aatgaacgtt attaaaacag 4080

aaattccaga cgtattaatt ttcgaaccga aaatttttgg tgacgagcgc ggtttctttt 4140

tcgagagctt taaccagaag gtttttgagg aagctgtagg ccgcaaagtt gaatttgttc 4200

aggataatca ttcgaagtct tgcaaaggtg ttttacgcgg actgcattat cagttagaac 4260

cttatgctca aggaaaactg gtacgctgtg ttgttggtga agtttttgat gttgcggttg 4320

atattcgtaa atcgtcacct acctttggca aatgggtagg ggtgaattta tctgctgaga 4380

ataagcgcca gttatggatc ccggaggggt ttgcgcatgg gttcataacg ttgtcaaata 4440

tagccgaatt tctttataaa acaacagatt tttattctcc aaaacatgag gtttgcattc 4500

ggtatgatga tgagagcatt ggtatacaat ggcctaaagc aactgacaag atgttgatat 4560

cttcaaaaga taaaaaaggt ttggatttta cttctttaat gtgagttatt atatgctaat 4620

tttatatcta ttaatttctt cgcatgcatc tgaaaattta ttatagctgg ctcaatggac 4680

attacattga tttctattaa tggatggttt tactcaaagt aaatataatg aaaataaatg 4740

cttgtttgtc atttgg

aca acattatgta agatgatcac tggtcttctc tttttggcaa 4800

tcttatcgca ttcaataaat aatgctgaat atagcattat tgtaacatcg attttttttg 4860

cccagttact ctccgttttt gttgatggtg ggatcaataa tgaagttctc tcactaacta 4920

ataagtcatt agataacgat attgatggat atcagaaact atcaatgaat ggagcagtta 4980

ggctagttgt tcatttgttt ttttcttttg tgttatcaac ttattttatg tttgctgatg 5040

gatggagtgc tgggggggtg tttttttt g gatacatttc tggggtacta acctcaatat 5100

tagaaacata ctctataaaa ttaaaaacag aatataaata tattgaggat tttattttaa 5160

atattttttg cagtctgttt gttattttat catctttttt tatttacata tttcctcaat 5220

gtcttatgtt atatcttgtt ttttttagat taatcccttt ggctatatat agaaaaccat 5280

tgcaagtatt taaaatgata agagaaatag attataaatt aataagggat aattatatcg 5340

cacggaaaca ttac

ctatc gattcgattg cgacaaacat taatttacaa ttagactcgt 5400

tttttctatt gttattattc ggtagagata tgtatgcata ttatcaacca ttgaatcggt 5460

tatataattc atgtataggg ttgtcggcag cagttgtatc atttgctatt ccatatgcac 5520

atttactgac aagtagaata aagaagatat actttttagt tttcttgttt tcatcatcag 5580

caattataat ttcattatca tattattttt tttctagaga tgtcgtgatg gtattctttg 5640

gtgaaaaatt ttctatggaa agacaatata tatttctatt tagtttattg atttttatgc 5700

gatatttatg cgcctcattt ggttcatatt taatga

aaa tggagagcaa aaaaaaagag 5760

cactcataaa tcttactatt actattttat gcattccatt tatattaatt tctgaaacgt 5820

attctggaat attg

tggtg ataattggag ggcaaatatt aatatcatac ttttacgtac 5880

taatgatcaa aagaggagtt ggtcatgata aataaaaccc atatcatttt tctgaagttt 5940

atatgtcgtg ttctcattaa aaaaaatcaa tttgaaagtt gtatattgag acgattatat 6000

ataaaactat atgatattga tattggtatg tatagctatg gttgttttga ccctaaaaga 6060

gtgccaccta aaacaaaaat aggaagatat tgttcgtttg caccaacagc atatatattt 6120

ggacgaaatc atggggttga atttgtctcg ttgcatccat atttatataa ttcagagttg 6180

ggtttagtaa aacaagacac catagcaata acttcacgag tgatagaaga cgacgtttgg 6240

tttgggcata atagtgtaat ttcacctgcg gtaaaattta tagggagagg ggct ttatc 6300

gctactggag cagttgtcac tagcgacgtt cctagatatg cgattgttgc aggagttccg 6360

gcaaagatca tcaaatatag atttgaaccg agagtgataa atattattga aaagagtaaa 6420

tggtggttaa gaacgaaatc tgaattacaa aaaatgatta aattagatag tgggtttata 6480

tttacaccgg ggtattttga tgaaaccaat taaaatttgc ttagcatggc acaatattaa 6540

ttcgactaac tatggcgtta gtgctcttgc agttgcacaa gttgctcttt tagttgagtc 6600

tgcgagtcgt tcaaaagttg ctattgaatt ggagactttt ggtacaccgt ttgttaatga 6660

gttatcaatt agaaaggaag tagagcaaag atttgcagtt aaattaacac atagagattt 6720

ctcattaaaa aagtttattg ttgattttgc aaaacttgat ttttctggac ttagattatt 6780

tgacaaatac gatattgtaa tggacattgg tgagggtgac agttttacgg atatatatgg 6840

aattaagaga tttattacat tctcaattac aaagtattta gcgttaagag ttaaaaagaa 6900

actcattctt tcacctcaaa caatcggacc ttttaatagc agaatatcat atataatagc 6960

gcgttatttg atttcgaaat gtgattcagt tttttctcgt gactataaat ctacgaaatt 7020

catacatgac atgggattga attgtgaaga agtatcagat gtagcattta ctctaccata 7080

tgatactttg cccaagatag ataacagtat tggtatcaac atttcaggtt tgttatggaa 7140

tggtggttac acaggcgata atcagtttgg attgacagtt gattataagt tattcgtcaa 7200

agagattatt gaattattta gagttagagg gaaggaagtt catttgattg gacatgtcat 7260

tgcagaccat ctacctgtgg aagatgacta tcgtgtttgc aatcaaatta aagaaatgtt 7320

ttcattagat aatggagtaa tagtagcacc taaatttaca tcacctatcc aagctaaaac 7380

ttatatgagt cagttaacat tttttactgg atcacgtatg catgcaacca ttggggcact 7440

atctgcagga gttgtaacag tacctatagc gtatagtcga aaattctctg gagtatttgg 7500

tagtttggat tatcgtttta ctttggatgc ttatacatta gataccaaaa gtttagtgac 7560

aaaattattt gagtattatg ataataattt tgaagaaatg caatctgcaa tgattaatgc 7620

aagaattaaa gcaaatcaaa gaaacgaaaa atatgttggt tatcttcagg aattattaag 7680

caatgaaaaa aatagcacaa gtcattgaat ctggaatgtg tgtgggttgt ggattttgca 7740

ctgaaaaccc caatggtatg gatattaata aagaaggata ttatagacct atttctttca 7800

ttgatgattc attatcacag ctagtatgcc ctggtaaatc tatttcccat aataacagta 7860

tggctcctta taacctttta tgggggccgg tagtgagttg tgagagtgga aatgcagttg 7920

acccagatat acgtcataag gggtcttctg gtggggtttt aaccgccatt gcagtttatc 7980

ttgttgattc tggattggtt gatgcaataa tacaggttgg tgtttcagtt gataatccaa 8040

tacgcaatgt aacatatata atgaagagtc aggaagatat attaaaatgt gctggctctc 8100

gctattcacc ttcatcacca cttagtgtca ttcgttcatt attaggtaat ggtacacggt 8160

acgctgttat tggtaaaccc tgtgatattg cggctatgcg cacattggtt aatagtcggc 8220

aagaatttca agagcagttt ccatacctac tttcatttat gtgtgctgga gtgccaagtg 8280

aagaaggtac aagaaatatt cttgagcgat ggcacataaa acatgaacat ttaatctcgt 8340

ttcgttatcg aggagacgga tggccaggat tgacaaaagc tattactgat gatggtgaag 8400

agtttactat gacttataat gaatcatggg gcggtgtact taatcgctac ttacagcctc 8460

gttgcaaact atgtgctgac ggtattggtg aagctgctga tattgtatgt gcagacgctt 8520

ggtattctac tactaacggc tatcctagtt ttatagagaa ggagggacgt agtttaacaa 8580

ttgctcgtac acttaaaggc cgtcaattgt tggatcttgc tttaaataaa aatgtaatat 8640

cacttacacc ttttaatatt tctgatcttg agaaaattca accataccaa gctaatcgta 8700

aacagacggc aaatgttcgt cgctgggctg ttatgcttct ggggggaagt gtgccgaatt 8760

ttaaaggtta tagtcttaat aaattaatgt ttagagcccc aataaaaatt actttaaagg 8820

cattttttgg tacattgata agaaaaatga agggacgtat ttgaaaacta attatatatt 8880

cttcggtttt tttgtgagca ttgtacaatc atcactga t ataaataacc gcaataggaa 8940

ttattaaacg tgttgaataa caatatggta ttttaaatga ttaaagtaaa tgttattgtt 9000

gcactttatt atccaaatta ctatcagagg gttagggaag agattatttc ttttctgcag 9060

ggatttgatt attttatagt gtttgtggac aataatagaa cggtcaaacc aaatatagag 9120

aataataaaa atgtaacgtg gataataggg agtaatattg gtggtgaatt ttctgcttgg 9180

gatgaaggtt atgacctcct ggttaatatg aaaaatccgt cagatgatga aattatagtt 9240

tttattaatg atacattttg tcatcaccat ttttttacat gtttcgatag gtatttatat 9300

aggaaagcaa tatctacatg tgaagataat aacatttatg gtgatgttaa cagtataggc 9360

gaatttttta gtgtttatga tcggcatttt tcatcttggg tatcttctta tttttttcta 9420

ggtagaaaaa aaaacattga taaactttta cctcttaata aacaaagtgg tatggacgca 9480

gaatatatga gatatcttag taatgcattg gtatctaagc gcgttgatgt tcctatcttt 9540

tcggataaat tgaatcagca tctcacgaat tggttatttc cagttaatag caagggatgg 9600

tatggcgcaa gaaacgtatc acagcaactt attctgttta aactgaatgc aataattaat 9660

gaaaaattgc tcacttataa tataattgaa catgatcttt tattaacaaa tatttatcat 9720

ggaaaaataa gaacgcttta caattctatg cgtaacaagc tatatttttt ttgcaaaaat 9780

aataagttga tagggtgata taatgtatgc aaataaagca atgcccaaaa gacatacgag 9840

agaaagagtg tttttaccac tattactttc gatgattttc atttgcatga taataaatga 9900

tttgccacta caacaatatc ttggaacttt aggtgcaagt cctatgtggg ggggggcatt 9960

atttattttt atgttgattg ttattcgtag ccgctttgtt ttaaatcttg attctaccag 10020

taaatatttt ctctattttt acctattaac atttactata tcgcttttac aatgtttcta 10080

ttatgccatc tctagaggtt ctgtagaaaa cgagtatggt ggattgattt ttggtaagct 10140

tgtatttgct tcgacgtatt atatcgttta tttttttact atatacactg ccattttttt 10200

ggctagaaga ataacatcct ctgctcttaa attttgtatc ataaatgctt ctgttttgtt 10260

gcttcttctt cttattttgg agtttttttc gccaaactca ttgagtttat ttcataaaag 10320

tatggatggc tatggctttg gatttagaca aaggttattg tcccctgaac catcaatggc 10380

tgcgtttact ttaaatattt tcttgttgat aagcatagta ctggttaact caaagatcgc 10440

taaactattt atgggaggtg cgcttgtagt aggcaaccta tttattggct ctaaagcttc 10500

gctcatatta attttgatga gtggtgttat tgtgttttat cttaatatga acttaaccca 10560

aaaaataaaa tcgttactta tattaattcc tgtatcaatt gcagtttttt atgtgttttt 10620

aaatacgatt cttccagcac tgattgtaga tgtagataaa ttcacaagtg ttagcactcg 10680

tttaataaca gcccttgcgg ctctgctgag tttaatatac tatccattgg gtgagggcta 10740

tggtacctat agttcttatc ttttgggacc tcttgattta gctgttcaat tagcagaaga 10800

actattgcct tttacattaa atgtaagcga ggtcaataaa atgctaatga ctggtgagtc 10860

gttagcagca aagtcaggta ttcttttttc tgttatccag agtggagtcg tttccctcat 10920

atttttttac gtgatttatc gaaatgcgtt tagaaaactc tgtgctgcca ggctatctaa 10980

ttataacaag ataatgatgc ggttggttct atggtattca ttgttatcaa tcatgtttgc 11040

tgttaatatt gaagttgtct atgctttttt attgccgttt attataattg atcattatgc 11100

tataaattat atgcgatctt atgattatga ataaaatttt atatatctcg ctctctcttg 11160

atgagaaaaa ctatggtggc agcatcgtgt ctagaaataa tttaaaagca ttgagggcac 11220

ttgaagatag tgaggtgaag gaagttgcaa tagttaagaa attaaaaggg atatatgaat 11280

atgaactaca aactaatgtg tcaaaagtaa aaatagccat agataacctg aagggatatg 11340

ctggacggtt aaatcgcgat tgtttattga aaattaaaaa tattataaaa gtctttgagc 11400

ccaaattcgt ttatcttgac tccagtttgc ttgggggaat agctgaa

ta tctaaagaga 11460

tatctacaga cataaaagtt attacatttt ttcataatgt cgagattgat tttgaattag 11520

aaagattgaa gtcaggggga atattgtact tgccttctct tttgccatct tatttagcgg 11580

aaaaaaaagc tataaaatat agtgatataa taatttcact tcataaaaat gatagtgcta 11640

ggcttaagtg catatatgga cgttcgtctg atttttgtgt cccagtttgt atagaagatg 11700

acttgagtaa cgaggtaata ttacataaaa ataatagtac agatagtata tttagagttg 11760

gttttattgg aactgcattt ttcgcgaata ttaaagctgc agagttcatt tcgaaaaaat 11820

tatcacctag gtttttgaat aataaacaaa tagaatttat tattgcgggt aatggtttcg 11880

ataagtactc aaaattactt aatcgtaaaa atgtaaaa

c tctgggctat attaattcaa11940

ttgaagactt ttataatgaa gttgatgtta taatatctcc cgtcctgacg ggagcaggca 12000

tgaaagtgaa aatagcggaa gcgattaaat ataataagaa ggtaattgcc tcgtcatttt 12060

cattaattgg atatgagact atgttaaata gtcaaaatat tttttcttgc attactctag 12120

atgattatgc gatagcaatt aaaaagttga gcacacatcc tatgactcac tctgatactc 12180

gtgaatatta tttgaaattt ttctcaagtc aagcatgtat taactatttt aaatatattt 12240

tgaactccta atagttaaaa tgctacttct atgtgtgttt attgtgttga gttttttgtt 12300

ttactgcttt tatgtagtag aggatgatgc tgatgaaggt attgcaagtt agtaagtttt 12360

acccacctgt acatggagga attgagcaag tcgcgtttga tatcagtgaa ggcatgtttg 12420

aagaacatat acaggctgtt gatattttat gtgtagatcc attgggacct cgtattgatg 12480

ataggaatta taaatatcga gtttttcgac aaaagacttt tattgttcta ttttccactc 12540

cgatttcaat atcatttata aataaatggc gaaaaataaa agataattat gatattatcc 12600

acgtacattt acctaaccct ttagctgttc tcgctatata tctttttcct ccaaaaggta 12660

aaattgtatt acattggcat agtgatattg ttaagcaaaa aaaattattc agattatttt 12720

tacctttaca aagatggatt ttggataggt gcacttatat aattgtgact agtccagtgt 12780

atggtcaatc atctccatcg ctgcaacaat atcaaaataa attaatttgt attcctatag 12840

gtgtagatac cagtgttatg cctgttgacc tacaagaaga aaaattaatt aaaagtaagt 12900

ataaagataa aaaaataata ttttcattag ggcgattggt ttattataaa ggtatggata 12960

ttttaattga ttcagcgaaa tatttacctg atgattacat catattaatt gggggggggg 13020

gcgcattatt aaaaaaatta aggaggcaga tttatgataa taacttatca cacaaggttg 13080

ttgttttggg aagtatagat tataaatgct tagcttcata ttataaatgc tgtgatgtat 13140

tttgtctccc atctatacat gagtctgaag cgtttggtgt ggtacaatta gaagcaatga 13200

gtttttcaaa gccattagtt tctacgaata tcccccgaag tggtgttcca tgggttaacg 13260

aaaatggaac atcaggaatt gttgttgagc caaggagtgc tatagctctt gctgaaggga 13320

ttgttaaaac tattgctaat agtgaaaaat tcagtaaagg agcaaaagat agatttgata 13380

tgctctttac taagagatta atggttaata atgtatacaa attatattta agtataaagt 13440

gagtcaaaat atgtctttac ttcctgttat cattgctggt ggtaccggta gtcgtttgtg 13500

gcctttatca cgagttaagc atccaaagca atttctccat ttaggggatg atggaacaat 13560

gttacagacg acattaaatc gtcttcaagg gttaaagtgt gataacccaa ttgtaatttg 13620

caatgagcag catagattta tagttgctga gcaacttcga caattaaata aactcactca 13680

gaatattatt cttgagcccg tcggccggaa tactgcacca gctgtaactc tcgctgcact 13740

gaatgcaata cgtaataagt caaaacaatc aaaattaatt ttggttcttg cagctgatca 13800

tattataaaa gacgaagatg cattttgtag aagtgtgctg agtgctattc catatgcaaa 13860

caaaggaaaa ttaatcacat ttggtatagt gcctaatagt cccgaaacag gctatggata 13920

tataaaaaga ggtcatttat gtagtggcaa taatgctaat ttagcttttg aagtggctga 13980

gttcgttgaa aaacctaaca tagatacagc tcaagagttt ctttcatctg gtaactatta 14040

ttggaatagt ggtatgtttt tatttcgagc tgataggtat ttagatgaat taaaaaaata 14100

tagaccagac atacttgagg cctgtaaaaa atcaatgatt gaacttaatg gagatcttga 14160

ttttattcgt atcaataaag atgctttttg cgcttgccca gatgagtcaa ttgattatgc 14220

cgttatggag aaaacgaatg atgcggttgt tatcccaatg gatgctggct ggagtgatgt 14280

ggggtcatgg tcatctttat gggaaatgag taataaaacc attgaagaaa atgtaatagt 14340

tggtgatgta taacttatg attctgacag cagctacctc cgttcag

tt cagggctgct 14400

tgctacagtt ggggtaaaag atcttgttgt tgttcaaaca aaagatgctg tgttagtagc 14460

taataggaat tcagttcaga atgtaaaaaa aattgtcgaa aggcttaaat cagaaaatcg 14520

tagtgaagtt tttacgcatc ttgaagttta tcgtccttgg ggtaaatatg agtctatcga 14580

taatggtgaa cgctatgaag ttaaacgaat ttccgtaaaa cctggagagg ggatttcatt 14640

gcaaatgcat caccatcgtt ctgaacattg gataatagtt tcgggtactg caaaagtaac 14700

aatttgtgat gaaacaagaa ttctcagtga aaatgaatct atttatatac ctgtcggggc 14760

gaaacattgt ttagagaatc cgggaaaaat tatgttggag cttatagaag ttcgctccgg 14820

ctcctatcta ggagaagatg atgtcatccg ttttgccgac agatatggaa gaacataaat 14880

gcacaataag atcatcctag ataaattaac ttgctttaaa gcctacgata ttcgcggaaa 14940

actaggcgag gaactgaatg aagatatcgc

tggcgcatt ggtcgcgcct atggcgaatt 15000

tctcaaaccg aaaaccattg tgttaggcgg cgatgtccgc ctcaccagcg aaaccttaaa 15060

actggcgctg gcgaaaggtt tacaggatgc gggcgtcgat gtgctggata tcggcatgtc 15120

cggcaccgaa gagatttatt tcgccacgtt ccatctcggc gtggatggcg gcattgaagt 15180

taccgccagc cataatccga tggattataa cggcatgaag ctggtgcgcg aaggggctcg 15240

cccgatcagc ggcgataccg gactgcgcga cgtccagcgt ctggcagaag ccaacgactt 15300

tcctcccgtc gatgaaacca aacgcggtcg ctatcagcaa atcaatctgc gtgacgctta 15360

cgttgatcac ctgttcggtt atatcaacgt caaaaacctc acgccgctca agctggtgat 15420

taactccggg aacggcgcgg cgggtccggt ggtggacgcc attgaagccc gctttaaagc 15480

cctcggcgca cctgtggaat taatcaaagt acacaacacg ccggacggca atttccccaa 15540

cggtattcct aacccgctgc tgccggaatg ccgcgacgac acccgcaatg cagtcatcaa 15600

acacggcgcg gatatgggca ttgcctttga tggcgatttt gaccgctgtt tcctgtttga 15660

tgaaaaaggg cagtttatcg agggctacta cattgtcggt cttctggcag aagcgttcct 15720

cgaaaaaaat cccggcgcga agatcatcca cgatccacgt ctctcctgga acaccgttga 15780

tgtggtgacc gccgcgggcg gcactccggt gatgtcgaaa accggacacg cctttattaa 15840

agaacgtatg cgcaaggaag acgccatcta cggtggcgaa atgagcgccc accactattt 15900

ccgtgatttc gcttactgcg acagcggcat gatcccgtgg ctgctggtcg ccgaactggt 15960

gtgtctgaaa ggaaaaacgc tgggcgaact ggtgcgcgac cggatggcgg cgtttccggc 16020

aagcggtgag atcaacagca aactggcgca ccccgttgag gcgattaatc gcgtcgaaca 16080

gcattttagc cgtgaggcgc tggcggtgga tcgcaccgat ggcatcagca tgacctttgc 16140

cgactggcgc tttaacctgc gctcctccaa caccgaaccg gtggtgcggt tgaatgtgga 16200

atcgcgcggt gatgtaccgc tgatggaaga aaagacaaaa cttatccttg agttactgaa 16260

caagtaattc agtaatttca tataaatggg ttttaaaaaa cggaaaagat gagatatccg 16320

gtgtggtata gccaaggtaa tgctattcag tatctctatg agtgagttaa catctatacc 16380

acatttaagc cgcacacttg gcggtaacca cccctgacag gagtaaacaa tgtcaaagca 16440

acagatcggc gtcgtcggta tggcagtgat gggacgcaac ctcgcgctca acatcgaaag 16500

ccgtggttat accgtctcta ttttcaaccg ttcccgtgat aagacggaag aaattattgc 16560

cgaaaatcca ggcaagaaac tggttcctta ctatacggtg aaagaattcg ttgaatctct 16620

tgaaacgcct cgtcgcatcc tgttaatggt gaaagcaggt gcaggcacgg atgctgctat 16680

tgattccctt aagccatacc tcgataaagg tgacatcatc attgatggtg gtaatacctt 16740

cttccaggac accattcgtc gtaaccgtga gctttctgcc gaaggtttta acttcatcgg 16800

taccggtgtt tccggcggtg aagagggggc gctgaaaggg ccttccatca tgcctggtgg 16860

ccagaaagaa gcctatgaac tggttgcgcc gatcctgacc aaaatcgccg ccgttgctga 16920

agatggcgaa ccgtgcgtta cctatatcgg tgccgatggc gcgggtcact atgtgaagat 16980

ggttcacaac ggtattgaat acggtgatat gcaactgatt gctgaagcct attctctcct 17040

gaaaggcggc ctgaatctct ctaacgaaga actggcacag acctttaccg agtggaataa 17100

cggtgaactg agcagctacc tgatcgacat caccaaagat atcttcacca aaaaagatga 17160

agacggtaac tatctggttg atgtgatcct ggatgaagcg gctaacaaag gtaccggtaa 17220

atggaccagc cagagcgcgc tggatctcgg cgaaccgctg tcgttgatta ctgagtcagt 17280

gtttgcacgt tatatctcgt ctctgaaaga tcagcgcgtg gccgcgtcta aagttctctc 17340

tggtccgcaa gcacagccag ctggcgataa agctgagttc atcgagaaag ttcgtcgtgc 17400

gctgtatctg ggcaaaatcg tttcttacgc tcaggg ttc tctcaactgc gtgcggcgtc 17460

tgaagagtac aactgggatc tgaactacgg cgaaatcgcg aagattttcc gtgctggctg 17520

cat atccgt gcgcagttcc tgcagaaaat caccgatgc

tatgccgaaa atccgcagat 17580

cgctaacctg ctgctggctc cgtacttcaa gcaaattgcc gatgactacc agcaggcgct 17640

gcgtgatgtc gttgcttatg cagtacagaa cggtatcccg gttccgacct tcgctgctgc 17700

ggttgcctat tatgacagct accgtgccgc tgttctgcct gcgaacctga ttcaggccca 17760

gcgtgacta 17769

It in the sequence list nucleotide sequence of the O antigen gene bunch of intestinal bacteria O58, it is the same that the nucleotides sequence of the nucleotide sequence of the O antigen gene of shigella dysenteriae 5 types bunch and the O antigen gene of intestinal bacteria O58 bunch shows 99.76%, different base box indicating is the Nucleotide of shigella dysenteriae 5 types above this base.

Wzx gene, wzy gene and wherein primer and PCR data in the O antigen gene of table 1 intestinal bacteria O58 and shigella dysenteriae 5 types bunch

Gene	Function	The base position of gene	The forward primer position	The reverse primer position	PCR product length	Produce the group number of correct big or small electrophoresis band	The annealing temperature of PCR (℃)
								wzx wzy	O-antigen transhipment enzyme O-antigen polysaccharase	4728-5915 9803-11134	4967-4984 5341-5358 10475-10492 10110-10127	5336-5353 5839-5856 11855-10872 10932-10949	387bp 516bp 398bp 840bp	1* 1* 1** 1*	58 58 58 58

1* also produces the electrophoresis band of correct size except producing the electrophoresis band of correct size in the 9th group in the 3rd group.These two groups all is positive controls

1** also produces the electrophoresis band of correct size except producing the electrophoresis band of correct size in the 9th group in the 3rd group.An incorrect electrophoresis band of size is arranged in the 6th group

The intestinal bacteria of 166 kinds of serotypes of table 2 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 shigella dysenteriae 10, Shigella bogdii 11, shigella flexneri 12, wild-type e. coli 13, wild-type e. coli 14, wild-type e. coli	O1, O2, O5, O7, O8, O9, O12, O13, O14, O15, O16, O17, O18, O19ab, O20, O21, O22, O23, O24 O4, O10, O25, O26, O27, O28, O29, O30, O32, O33, O34, O35, O36, O37, O38, O40, O41, O42, O43 O6, O44, O45, O46, O48, O49, O50, O51, O52, O54, O55, O56, O57, O58, O60, O61, O62, O53 O63, O65, O66, O69, O70, O71, O74, O75, O76, O77, O78, O79, O80, O81, O82, O83, O68 O84, O85, O86, O87, O88, O89, O90, O91, O92, O98, O99, O101, O102, O103, O104, O105, O106, O97, O107, O108, O109, O110, O111, O112ab, O112ac, O113, O115, O116, O118, O120, O123, O125, O126, O128, O117 O129, O130, O131, O132, O133, O134, O135, O58, O137, O138, O139, O141, O142, O143, O144, O145, O140 O146, O147, O148, O150, O152, O154, O156, O157, O158, O159, O160, O161, O163, O164, O165, O166, O153 O168, O169, O170, O171, O172, O173, D1, D2, D3, D4, D5, D6, D7, D8, D9, D10, D11, D12, D13 B1, B2, B3, B4, B6, B7, B8, B9, B10, B11, B12, B13, B14, B15, B16, B17, B18 F1a, F1b, F2a, F2b, F3, F4a, F4b, F5 (v:4), F5 (v:7), F6, DS, DR O3, O11, O39, O59, O64, O73, O96, O95, O100, O114, O151, O155, O124, O167, O162, O121, O127, O149, O119 remove the 9th group of bacterium of the 3rd group of bacterium removal shigella dysenteriae 5 types of intestinal bacteria O58	IMVS a IMVS a IMVS a IMVS a IMVS a IMVS a IMVS a IMVS a b c d d d IMVS a

For the convenience that detects, every 12-19 bacterium is divided into one group, and 12 groups altogether, the 13rd group and the 14th group are as negative control

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. China Preventive Medicial Science Institute's epidemiological study institute

Table 3 is structural tables of the O-antigen gene bunch of intestinal bacteria O58 and shigella dysenteriae 5 types

galF FmlB rmlDrmlArmlC wzxorf7 orf8 orf9 orf10 wzy orf12 orf13 manC manB gnd

1kb

Table 4 is location tables of the gene in the O-antigen gene bunch of intestinal bacteria O58 and shigella dysenteriae 5 types

ATTGTGGCTG CAGGGATCAA AGAAATCCTC CTGGTAACTC ACGCGTCCAA GAACGCGGTC 60

GAAAACCACT TCGACACCTC TTATGAATTA GAATCTCTCC TTGAGCAGCG CGTGAAGCGT 120

CAACTGCTTG CGGAAGTGCA GTCCATCTGT CCACCGGGCG TGACCATTAT GAACGTGCGT 180

CAGGGCGAAC CTTTAGGTTT GGGCCGCTCC ATTTTATGTG CACGACCTGC CATTGGTGAC 240

AACCCATTTG TCGTGGTGCT GCCAGACGTT GTGATCGATG ACGCCAGCGC CGACCCGCTG 300

CGCTACAACC TTGCTGCCAT GATTGCGCGC TTCAACGAAA CGGGCCGCAG CCAGGTGCTG 360

GCAAAACGTA TGCCGGGTGA CCTCTCTGAA TACTCTGTCA TCCAGACCAA AGAGCCGCTG 420

GATCGTGAGG GTAAAGTCAG CCGCATTGTT GAATTTATTG AAAAACCAGA TCAGCCGCAG 480

ACGCTGGACT CAGACATCAT GGCCGTTGGT CGCTATGTGC TTTCTGCAGA TATTTGGCCG 540

GAACTTGAAC GCACTCAGCC TGGTGCATGG GGGCGTATTC AGCTGACTGA TGCTATTGCC 600

GAACTGGCTA AAAAACAGTC TGTTGATGCA ATGCTGATGA CAGGCGACAG CTACGACTGC 660

GGTAAAAAAA TGGGCTATAT ACAGGCGTTT GTGAAGTATG GACTGCGTAA CCTAAAAGAA 720

GGGGCGAAGT TCCGGAAAGG GATTGAGAAG CTGTTAAGCG AATAATGAAA ATCTGACCGG 780

ATGTAACGGT TGATAAGAAA ATTATAACGG CAGTGAAGAT TCGTGGCGAA AGTAATTTGT 840

TGCGAATCTT CCTGCCGTTG TTTTATATAA ACAATCAGAA TAACAATGAG TTAGCAATAG 900

GATTTTAGTC AAAGTTTTCC AGGATTTTCC TTGTTTCCAG AGTGGATTGG TAAGACAATT 960

AGCGTTTGAA TTTTTCGGGT TTAGCGCGAG TAGGTAATGC TCGTCACATC GTCGGCATGC 1020

ATGCAGTGCT CTGGTAGCTG TAAAGCCA

G GGCGGTAGCG TGCATTAATA CCTCTATTAA 1080

Orf1's is initial

TCAAACTGAG AGCCGCTTAT TTCACAGCAT GCTCTGAAGT AATATGGAAT AATAAA GTGA 1140

A ATACTTGT TACTGGTGGC GCAGGATTTA TTGGTTCT

C TGTAGTTCGT CACATTATAA 1200

ATAATACGCA GATAGTGTT GTTAATGTCG ATAAATTAAC GTACGCCGGA

A CTGGAAT 1260

C

CTTGCTGA TGTTTC

GAT TCTGAACGCT ATGTTTTTGA ACATGCGGAT

TTTGCGATG 1320

CAGCTG

AAT GGCACGGATT TTTGCTCAGC ATCAGCCGGA TGCAGTGATG CACCTGGCTG 1380

CTGAAAGCCA TGT

GATCGT TCAATTACAG G

CTGCGGC ATTTATTGAA ACCAATATTG 1440

TTGGTAC TA TGTCCTTTTG GAAGC

GCTC GCAATTACTG GTCTGCTCTT GAT

GCGACA 1500

AGAAAAATAG CTTCCGTTTT CATCATATTT CTACTGACGA AGTCTATGGT GATTTGCCTC 1560

ATCCTGACGA AGTAAATAAT AAAGA

CAAT TACCCCTCTT TACTGAGACG ACAGCTTACG 1620

CGCCAAG

AG

CC

TATTC

GC

TC

AAG CATCAAGCGA TCATTTAGTC CG

GCTTGGA 1680

AACGTACCTA TGGTTTACCG ACCATTGTGA CTA

TTGTTC GAATAACTAC GGTCCTTATC 1740

ACTTTCCGGA AAAATTGATT CCACTAGTAA TTCTTAATGC TCTGGAAGGT AAGGCATTAC 1800

CTATTTATGG CAAAGGGGAT CAAATTCGTG ACTGGCTGTA TGTTGAAGAT CATGCGCGTG 1860

CGTTATATAC CGTCGTAACC GAAGGTAAAG CGGGTGAAAC TTATAACATT GGTGGACACA 1920

ACGAAAAGAA AAACATCGAT GTAGTGCTCA CTATTTGTGA TTTGTTGGAT GAGATTGTAC 1980

CGAAAGAGAA ATCTTACCGC GAGCAAATTA CTTATGTTGC CGATCGCCCG GGACACGATC 2040

GCCGTTATGC GATTGATGCA GAGAAGATTA GCCGCGAATT GGGCTGGAAA CCGCAGGAAA 2100

CGTTTGAGAG CGGGATTCGT AAAACGGTGG AATGGTACCT GTCCAATACA AAATGGGTTG 2160

ATAATGTGAA AAGTGGTGAC TATCAATCGT GGATTGAACA GAACTATGAG GGCCGCCAGT 2220

The termination of the initial Orf1 of Orf2

A ATGAATATC CTCCTTTTTG GCAAAACAGG GCAGGTAGGT TGGGAACTAC AGCGTGCTCT 2280

GGCACCTCTG GGTAATTTGA TTGCTCTTGA TGTTCACTCC ACTGATTACT GTGGTGATTT 2340

TAGTAATCCT GAAGGTGTAG CTGAAACCGT AAGAAGCATT CGGCCTGATA TTATTGTCAA 2400

CGCAGCCGCT CACACCGCAG TAGACAAAGC AGAATCAGAA CCGGAGTTTG CACAATTACT 2460

TAACGCAACA AGTGTCGAAG CGATTGCGAA AGCAGCAAAT GAAGTTGGAG CTTGGGTTAT 2520

CCATTACTCG ACTGATTACG TCTTCCCTGG AAATGGCGAC ATGCCATGGC TGGAGACGGA 2580

TGCAACCGCA CCGC AAATG TTTACGGTGA AACCAAGTTA GCTGGAGAAA AAGCATTACA 2640

AGAGCATTGT GCGAAGCACC TAATTTTCCG TACAAGCTGG GTCTATGCAG GTAAAGGAAA 2700

TAACTTCGCC AAAACGATGT TGCGTCTGGC AAAAGAGCGT GAAGAATTAG CCGTTATTAA 2760

TGATCAGTTT GGTGCGCCAA CAGGTGCTGA ACTGCTGGCT GATTGTACGG CACATGCAAT 2820

TCGTGTGGCA CTGAATAAAC CAGAAGTCGC AGGCTTGTAT CATCTGGTAG CCAGTGGTAC 2880

CACAACCTGG CACGATTATG CTGCGCTGGT TTTTGAAGAG GCGCGCAAAG CAGGCATTCC 2940

CCTTGCACTC AACAAGCTCA ATGCAGTACC AACAACAGCC TATCCTACAC CAGCCCGTCG 3000

CCCCCATAAC TCTCGCCTTA ATACAGAAAA ATTTCAGCAG AACTTTGCGC TTGTCTTGCC 3060

The termination of Orf2

TGACTGGCAG GTTGGTGTGA AACGAATGCT CAACGAATTA TTTACGACTA CAGCAATT TA 3120

Orf3's is initial

ATAGTTTTTG CATCTTGTTC GTGATGGTGG AGCAAGATGA AT

AAAAGGA ATGATGAA AT 3180

GAAAACGCGT AAAGGTACTA TTTTAGCGGG TGGTTCTGGT ACTCGTCTTT ATCCTGTGAC 3240

TATGGCTGTC AGTAAACAGC TATTACCTAT TTATGATAAG CCGATGATCT ATTACCCGCT 3300

TTCTACACTG ATGTTAGCGG GTATTCGCGA TATTCTGATT ATCAGTACGC CGCAGGATAC 3360

TCCTCGTTTT CAACAACTGC TGGGTGACGG GAGCCAGTGG GGGCTAAATC TTCAGTATAA 3420

AGTGCAACCG AGTCCAGATG GTCTTGCGCA GGCATTTATC ATCGGTGAAG AGTTTATCGG 3480

TGGTGATGAT CGTGCTTTGG TTCTTGGTGA TAATATCTTC TACGGTCATG ATCTGCCGAA 3540

GTTAATGGAT GTCGCTGTTA ACAAAGAAAG TGGTGCAACG GTATTTGCCT ATCACGTTAA 3600

TGATCCTGAA CGCTACGGTG TCGTTGAGTT TGATAAAAAT GGTACGGCGA TCAGCCTGGA 3660

AGAAAAACCG CTACAACCAA AAAGTAATTA TGCGGTAACC GGGCTTTATT TCTATGATAA 3720

CGACGTTG

G GAAATGGCGA AAAACCTTAA GCCTTCTGCC CGCGGTGAAC TGGAAATTAC 3780

CGATATTAAC CGTATTTATA TGGAACAGGG GCGTTTATCT GTTGCCATGA TGGGCCGTGG 3840

TTATGCATGG CTGGACACGG GGACACATCA AAGCCTGATT GAGGCAAGCA ACTTCATTGC 3900

AACAATTGAA GAACGTCAGG GGCTGAAAGT TTCCTGCCCG GAAGAAATTG CTTACCGTAA 3960

AGGGTTTATT GATGCTGAGA AGGTGAAAGT ATTAGCTGAA CCGCTG AAA AAAATGCTTA 4020

The termination Orf4's of Orf3 is initial

TGGTCAGTAT CTGCTGAAAA TGATTAAAGG T TAGTAATAA A ATGAACGTT ATTAAAACAG4080

AAATTCCAGA CGTATTAATT TTCGAACCGA AAATTTTTGG TGACGAGCGC GGTTTCTTTT 4140

TCGAGAGCTT TAACCAGAAG GTTTTTGAGG AAGCTGTAGG CCGCAAAGTT GAATTTGTTC 4200

AGGATAATCA TTCGAAGTCT TGCAAAGGTG TTTTACGCGG ACTGCATTAT CAGTTAGAAC 4260

CTTATGCTCA AGGAAAACTG GTACGCTGTG TTGTTGGTGA AGTTTTTGAT GTTGCGGTTG 4320

ATATTCGTAA ATCGTCACCT ACCTTTGGCA AATGGGTAGG GGTGAATTTA TCTGCTGAGA 4380

ATAAGCGCCA GTTATGGATC CCGGAGGGGT TTGCGCATGG GTTCATAACG TTGTCAAATA 4440

TAGCCGAATT TCTTTATAAA ACAACAGATT TTTATTCTCC AAAACATGAG GTTTGCATTC 4500

GGTATGATGA TGAGAGCATT GGTATACAAT GGCCTAAAGC AACTGACAAG ATGTTGATAT 4560

The termination of Orf4

CTTCAAAAGA TAAAAAAGGT TTGGATTTTA CTTCTTTAAT G TGAGTTATT ATATGCTAAT 4620

TTTATATCTA TTAATTTCTT CGCATGCATC TGAAAATTTA TTATAGCTGG CTCAATGGAC 4680

Orf5's is initial

ATTACATTGA TTTCTATTAA TGGATGGTTT TACTCAAAGT AAATATA ATG AAAATAAATG 4740

CTTGTTTGTC ATTTGC ACA ACATTATGTA AGATGATCAC TGGTCTTCTC TTTTTGGCAA 4800

TCTTATCGCA TTCAATAAAT AATGCTGAAT ATAGCATTAT TGTAACATCG ATTTTTTTTG 4860

CCCAGTTACT CTCCGTTTTT GTTGATGGTG GGATCAATAA TGAAGTTCTC TCACTAACTA 4920

ATAAGTCATT AGATAACGAT ATTGATGGAT ATCAGAAACT ATCAATGAAT GGAGCAGTTA 4980

GGCTAGTTGT TCATTTGTTT TTTTCTTTTG TGTTATCAAC TTATTTTATG TTTGCTGATG 5040

GATGGAGTGC TGGGGGGGTG TTTTTTTT

G GATACATTTC TGGGGTACTA ACCTCAATAT 5100

TAGAAACATA CTCTATAAAA TTAAAAACAG AATATAAATA TATTGAGGAT TTTATTTTAA 5160

ATATTTTTTG CAGTCTGTTT GTTATTTTAT CATCTTTTTT TATTTACATA TTTCCTCAAT 5220

GTCTTATGTT ATATCTTGTT TTTTTTAGAT TAATCCCTTT GGCTATATAT AGAAAACCAT 5280

TGCAAGTATT TAAAATGATA AGAGAAATAG ATTATAAATT AATAAGGGAT AATTATATCG 5340

CACGGAAACA TTAC

CTATC GATTCGATTG CGACAAACAT TAATTTACAA TTAGACTCGT 5400

TTTTTCTATT GTTATTATTC GGTAGAGATA TGTATGCATA TTATCAACCA TTGAATCGGT 5460

TATATAATTC ATGTATAGGG TTGTCGGCAG CAGTTGTATC ATTTGCTATT CCATATGCAC 5520

ATTTACTGAC AAGTAGAATA AAGAAGATAT ACTTTTTAGT TTTCTTGTTT TCATCATCAG 5580

CAATTATAAT TTCATTATCA TATTATTTTT TTTCTAGAGA TGTCGTGATG GTATTCTTTG 5640

GTGAAAAATT TTCTATGGAA AGACAATATA TATTTCTATT TAGTTTATTG ATTTTTATGC 5700

GATATTTATG CGCCTCATTT GGTTCATATT TAATGA

AAA TGGAGAGCAA AAAAAAAGAG 5760

CACTCATAAA TCTTACTATT ACTATTTTAT GCATTCCATT TATATTAATT TCTGAAACGT 5820

ATTCTGGAAT ATTG

TGGTG ATAATTGGAG GGCAAATATT AATATCATAC TTTTACGTAC 5880

The termination of the initial Orf5 of Orf6

TAATGATCAA AAGAGGAGTT GGTC ATGATA AA TAAAACCC ATATCATTTT TCTGAAGTTT5940

ATATGTCGTG TTCTCATTAA AAAAAATCAA TTTGAAAGTT GTATATTGAG ACGATTATAT 6000

ATAAAACTAT ATGATATTGA TATTGGTATG TATAGCTATG GTTGTTTTGA CCCTAAAAGA 6060

GTGCCACCTA AAACAAAAAT AGGAAGATAT TGTTCGTTTG CACCAACAGC ATATATATTT 6120

GGACGAAATC ATGGGGTTGA ATTTGTCTCG TTGCATCCAT ATTTATATAA TTCAGAGTTG 6180

GGTTTAGTAA AACAAGACAC CATAGCAATA ACTTCACGAG TGATAGAAGA CGACGTTTGG 6240

TTTGGGCATA ATAGTGTAAT TTCACCTGCG GTAAAATTTA TAGGGAGAGG GGCT

TTATC 6300

GCTACTGGAG CAGTTGTCAC TAGCGACGTT CCTAGATATG CGATTGTTGC AGGAGTTCCG 6360

GCAAAGATCA TCAAATATAG ATTTGAACCG AGAGTGATAA ATATTATTGA AAAGAGTAAA 6420

TGGTGGTTAA GAACGAAATC TGAATTACAA AAAATGATTA AATTAGATAG TGGGTTTATA 6480

The termination of the initial Orf6 of Orf7

TTTACACCGG GGTATTTTG A TGAAACCAAT TAAAATTTGC TTAGCATGGC ACAATATTAA 6540

TTCGACTAAC TATGGCGTTA GTGCTCTTGC AGTTGCACAA GTTGCTCTTT TAGTTGAGTC 6600

TGCGAGTCGT TCAAAAGTTG CTATTGAATT GGAGACTTTT GGTACACCGT TTGTTAATGA 6660

GTTATCAATT AGAAAGGAAG TAGAGCAAAG ATTTGCAGTT AAATTAACAC ATAGAGATTT 6720

CTCATTAAAA AAGTTTATTG TTGATTTTGC AAAACTTGAT TTTTCTGGAC TTAGATTATT 6780

TGACAAATAC GATATTGTAA TGGACATTGG TGAGGGTGAC AGTTTTACGG ATATATATGG 6840

AATTAAGAGA TTTATTACAT TCTCAATTAC AAAGTATTTA GCGTTAAGAG TTAAAAAGAA 6900

ACTCATTCTT TCACCTCAAA CAATCGGACC TTTTAATAGC AGAATATCAT ATATAATAGC 6960

GCGTTATTTG ATTTCGAAAT GTGATTCAGT TTTTTCTCGT GACTATAAAT CTACGAAATT 7020

CATACATGAC ATGGGATTGA ATTGTGAAGA AGTATCAGAT GTAGCATTTA CTCTACCATA 7080

TGATACTTTG CCCAAGATAG ATAACAGTAT TGGTATCAAC ATTTCAGGTT TGTTATGGAA 7140

TGGTGGTTAC ACAGGCGATA ATCAGTTTGG ATTGACAGTT GATTATAAGT TATTCGTCAA 7200

AGAGATTATT GAATTATTTA GAGTTAGAGG GAAGGAAGTT CATTTGATTG GACATGTCAT 7260

TGCAGACCAT CTACCTGTGG AAGATGACTA TCGTGTTTGC AATCAAAATT AAGAAATGTT 7320

TTCATTAGAT AATGGAGTAA TAGTAGCACC TAAATTTACA TCACCTATCC AAGCTAAAAC 7380

TTATATGAGT CAGTTAACAT TTTTTACTGG ATCACGTATG CATGCAACCA TTGGGGCACT 7440

ATCTGCAGGA GTTGTAACAG TACCTATAGC GTATAGTCGA AAATTCTCTG GAGTATTTGG 7500

TAGTTTGGAT TATCGTTTTA CTTTGGATGC TTATACATTA GATACCAAAA GTTTAGTGAC 7560

AAAATTATTT GAGTATTATG ATAATAATTT TGAAGAAATG CAATCTGCAA TGATTAATGC 7620

AAGAATTAAA GCAAATCAAA GAAACGAAAA ATATGTTGGT TATCTTCAGG AATTATTAAG 7680

The termination of the initial Orf7 of Orf8

CA ATGAAAAA AATAGCACAA GTCAT TGAAT CTGGAATGTG TGTGGGTTGT GGATTTTGCA7740

CTGAAAACCC CAATGGTATG GATATTAATA AAGAAGGATA TTATAGACCT ATTTCTTTCA 7800

TTGATGATTC ATTATCACAG CTAGTATGCC CTGGTAAATC TATTTCCCAT AATAACAGTA 7860

TGGCTCCTTA TAACCTTTTA TGGGGGCCGG TAGTGAGTTG TGAGAGTGGA AATGCAGTTG 7920

ACCCAGATAT ACGTCATAAG GGGTCTTCTG GTGGGGTTTT AACCGCCATT GCAGTTTATC 7980

TTGTTGATTC TGGATTGGTT GATGCAATAA TACAGGTTGG TGTTTCAGTT GATAATCCAA 8040

TACGCAATGT AACATATATA ATGAAGAGTC AGGAAGATAT ATTAAAATGT GCTGGCTCTC 8100

GCTATTCACC TTCATCACCA CTTAGTGTCA TTCGTTCATT ATTAGGTAAT GGTACACGGT 8160

ACGCTGTTAT TGGTAAACCC TGTGATATTG CGGCTATGCG CACATTGGTT AATAGTCGGC 8220

AAGAATTTCA AGAGCAGTTT CCATACCTAC TTTCATTTAT GTGTGCTGGA GTGCCAAGTG 8280

AAGAAGGTAC AAGAAATATT CTTGAGCGAT GGCACATAAA ACATGAACAT TTAATCTCGT 8340

TTCGTTATCG AGGAGACGGA TGGCCAGGAT TGACAAAAGC TATTACTGAT GATGGTGAAG 8400

AGTTTACTAT GACTTATAAT GAATCATGGG GCGGTGTACT TAATCGCTAC TTACAGCCTC 8460

GTTGCAAACT ATGTGCTGAC GGTATTGGTG AAGCTGCTGA TATTGTATGT GCAGACGCTT 8520

GGTATTCTAC TACTAACGGC TATCCTAGTT TTATAGAGAA GGAGGGACGT AGTTTAACAA 8580

TTGCTCGTAC ACTTAAAGGC CGTCAATTGT TGGATCTTGC TTTAAATAAA AATGTAATAT 8640

CACTTACACC TTTTAATATT TCTGATCTTG AGAAAATTCA ACCATACCAA GCTAATCGTA 8700

AACAGACGGC AAATGTTCGT CGCTGGGCTG TTATGCTTCT GGGGGGAAGT GTGCCGAATT 8760

TTAAAGGTTA TAGTCTTAAT AAATTAATGT TTAGAGCCCC AATAAAAATT ACTTTAAAGG 8820

The termination of Orf8

CATTTTTTGG TACATTGATA AGAAAAATGA AGGGACGTAT T TGAAAACTA ATTATATATT 8880

CTTCGGTTTT TTTGTGAGCA TTGTACAATC ATCACTGA

T ATAAATAACC GCAATAGGAA 8940

Orf9's is initial

TTATTAAACG TGTTGAATAA CAATATGGTA TTTTAA ATGA TTAAAGTAAA TGTTATTGTT 9000

GCACTTTATT ATCCAAATTA CTATCAGAGG GTTAGGGAAG AGATTATTTC TTTTCTGCAG 9060

GGATTTGATT ATTTTATAGT GTTTGTGGAC AATAATAGAA CGGTCAAACC AAATATAGAG 9120

AATAATAAAA ATGTAACGTG GATAATAGGG AGTAATATTG GTGGTGAATT TTCTGCTTGG 9180

GATGAAGGTT ATGACCTCCT GGTTAATATG AAAAATCCGT CAGATGATGA AATTATAGTT 9240

TTTATTAATG ATACATTTTG TCATCACCAT TTTTTTACAT GTTTCGATAG GTATTTATAT 9300

AGGAAAGCAA TATCTACATG TGAAGATAAT AACATTTATG GTGATGTTAA CAGTATAGGC 9360

GAATTTTTTA GTGTTTATGA TCGGCATTTT TCATCTTGGG TATCTTCTTA TTTTTTTCTA 9420

GGTAGAAAAA AAAACATTGA TAAACTTTTA CCTCTTAATA AACAAAGTGG TATGGACGCA 9480

GAATATATGA GATATCTTAG TAATGCATTG GTATCTAAGC GCGTTGATGT TCCTATCTTT 9540

TCGGATAAAT TGAATCAGCA TCTCACGAAT TGGTTATTTC CAGTTAATAG CAAGGGATGG 9600

TATGGCGCAA GAAACGTATC ACAGCAACTT ATTCTGTTTA AACTGAATGC AATAATTAAT 9660

GAAAAATTGC TCACTTATAA TATAATTGAA CATGATCTTT TATTAACAAA TATTTATCAT 9720

GGAAAAATAA GAACGCTTTA CAATTCTATG CGTAACAAGC TATATTTTTT TTGCAAAAAT 9780

The termination Orf10's of Orf9 is initial

AATAAGTTGA TAGGG TGATA TA ATGTATGC AAATAAAGCA ATGCCCAAAA GACATACGAG9840

AGAAAGAGTG TTTTTACCAC TATTACTTTC GATGATTTTC ATTTGCATGA TAATAAATGA 9900

TTTGCCACTA CAACAATATC TTGGAACTTT AGGTGCAAGT CCTATGTGGG GGGGGGCATT 9960

ATTTATTTTT ATGTTGATTG TTATTCGTAG CCGCTTTGTT TTAAATCTTG ATTCTACCAG 10020

TAAATATTTT CTCTATTTTT ACCTATTAAC ATTTACTATA TCGCTTTTAC AATGTTTCTA 10080

TTATGCCATC TCTAGAGGTT CTGTAGAAAA CGAGTATGGT GGATTGATTT TTGGTAAGCT 10140

TGTATTTGCT TCGACGTATT ATATCGTTTA TTTTTTTACT ATATACACTG CCATTTTTTT 10200

GGCTAGAAGA ATAACATCCT CTGCTCTTAA ATTTTGTATC ATAAATGCTT CTGTTTTGTT 10260

GCTTCTTCTT CTTATTTTGG AGTTTTTTTC GCCAAACTCA TTGAGTTTAT TTCATAAAAG 10320

TATGGATGGC TATGGCTTTG GATTTAGACA AAGGTTATTG TCCCCTGAAC CATCAATGGC 10380

TGCGTTTACT TTAAATATTT TCTTGTTGAT AAGCATAGTA CTGGTTAACT CAAAGATCGC 10440

TAAACTATTT ATGGGAGGTG CGCTTGTAGT AGGCAACCTA TTTATTGGCT CTAAAGCTTC 10500

GCTCATATTA ATTTTGATGA GTGGTGTTAT TGTGTTTTAT CTTAATATGA ACTTAACCCA 10560

AAAAATAAAA TCGTTACTTA TATTAATTCC TGTATCAATT GCAGTTTTTT ATGTGTTTTT 10620

AAATACGATT CTTCCAGCAC TGATTGTAGA TGTAGATAAA TTCACAAGTG TTAGCACTCG 10680

TTTAATAACA GCCCTTGCGG CTCTGCTGAG TTTAATATAC TATCCATTGG GTGAGGGCTA 10740

TGGTACCTAT AGTTCTTATC TTTTGGGACC TCTTGATTTA GCTGTTCAAT TAGCAGAAGA 10800

ACTATTGCCT TTTACATTAA ATGTAAGCGA GGTCAATAAA ATGCTAATGA CTGGTGAGTC 10860

GTTAGCAGCA AAGTCAGGTA TTCTTTTTTC TGTTATCCAG AGTGGAGTCG TTTCCCTCAT 10920

ATTTTTTTAC GTGATTTATC GAAATGCGTT TAGAAAACTC TGTGCTGCCA GGCTATCTAA 10980

TTATAACAAG ATAATGATGC GGTTGGTTCT ATGGTATTCA TTGTTATCAA TCATGTTTGC 11040

TGTTAATATT GAAGTTGTCT ATGCTTTTTT ATTGCCGTTT ATTATAATTG ATCATTATGC 11100

The termination of the initial Orf10 of Orf11

TATAAATTAT ATGCGATCTT ATGATT ATGA A TAAAATTTT ATATATCTCG CTCTCTCTTG11160

ATGAGAAAAA CTATGGTGGC AGCATCGTGT CTAGAAATAA TTTAAAAGCA TTGAGGGCAC 11220

TTGAAGATAG TGAGGTGAAG GAAGTTGCAA TAGTTAAGAA ATTAAAAGGG ATATATGAAT 11280

ATGAACTACA AACTAATGTG TCAAAAGTAA AAATAGCCAT AGATAACCTG AAGGGATATG 11340

CTGGACGGTT AAATCGCGAT TGTTTATTGA AAATTAAAAA TATTATAAAA GTCTTTGAGC 11400

CCAAATTCGT TTATCTTGAC TCCAGTTTGC TTGGGGGAAT AGCTGAA

TA TCTAAAGAGA11460

TATCTACAGA CATAAAAGTT ATTACATTTT TTCATAATGT CGAGATTGAT TTTGAATTAG 11520

AAAGATTGAA GTCAGGGGGA ATATTGTACT TGCCTTCTCT TTTGCCATCT TATTTAGCGG 11580

AAAAAAAAGC TATAAAATAT AGTGATATAA TAATTTCACT TCATAAAAAT GATAGTGCTA 11640

GGCTTAAGTG CATATATGGA CGTTCGTCTG ATTTTTGTGT CCCAGTTTGT ATAGAAGATG 11700

ACTTGAGTAA CGAGGTAATA TTACATAAAA ATAATAGTAC AGATAGTATA TTTAGAGTTG 11760

GTTTTATTGG AACTGCATTT TTCGCGAATA TTAAAGCTGC AGAGTTCATT TCGAAAAAAT 11820

TATCACCTAG GTTTTTGAAT AATAAACAAA TAGAATTTAT TATTGCGGGT AATGGTTTCG 11880

ATAAGTACTC AAAATTACTT AATCGTAAAA ATGTAAAA

CTCTGGGCTAT ATTAATTCAA 11940

TTGAAGACTT TTATAATGAA GTTGATGTTA TAATATCTCC CGTCCTGACG GGAGCAGGCA 12000

TGAAAGTGAA AATAGCGGAA GCGATTAAAT ATAATAAGAA GGTAATTGCC TCGTCATTTT 12060

CATTAATTGG ATATGAGACT ATGTTAAATA GTCAAAATAT TTTTTCTTGC ATTACTCTAG 12120

ATGATTATGC GATAGCAATT AAAAAGTTGA GCACACATCC TATGACTCAC TCTGATACTC 12180

GTGAATATTA TTTGAAATTT TTCTCAAGTC AAGCATGTAT TAACTATTTT AAATATATTT 12240

The termination of Orf11

TGAACTCC TA ATAGTTAAAA TGCTACTTCT ATGTGTGTTT ATTGTGTTGA GTTTTTTGTT 12300

Orf12's is initial

TTACTGCTTT TATGTAGTAG AGGATGATGC TG ATGAAGGT ATTGCAAGTT AGTAAGTTTT 12360

ACCCACCTGT ACATGGAGGA ATTGAGCAAG TCGCGTTTGA TATCAGTGAA GGCATGTTTG 12420

AAGAACATAT ACAGGCTGTT GATATTTTAT GTGTAGATCC ATTGGGACCT CGTATTGATG 12480

ATAGGAATTA TAAATATCGA GTTTTTCGAC AAAAGACTTT TATTGTTCTA TTTTCCACTC 12540

CGATTTCAAT ATCATTTATA AATAAATGGC GAAAAATAAA AGATAATTAT GATATTATCC 12600

ACGTACATTT ACCTAACCCT TTAGCTGTTC TCGCTATATA TCTTTTTCCT CCAAAAGGTA 12660

AAATTGTATT ACATTGGCAT AGTGATATTG TTAAGCAAAA AAAATTATTC AGATTATTTT 12720

TACCTTTACA AAGATGGATT TTGGATAGGT GCACTTATAT AATTGTGACT AGTCCAGTGT 12780

ATGGTCAATC ATCTCCATCG CTGCAACAAT ATCAAAATAA ATTAATTTGT ATTCCTATAG 12840

GTGTAGATAC CAGTGTTATG CCTGTTGACC TACAAGAAGA AAAATTAATT AAAAGTAAGT 12900

ATAAAGATAA AAAAATAATA TTTTCATTAG GGCGATTGGT TTATTATAAA GGTATGGATA 12960

TTTTAATTGA TTCAGCGAAA TATTTACCTG ATGATTACAT CATATTAATT GGGGGGGGGG 13020

GCGCATTATT AAAAAAATTA AGGAGGCAGA TTTATGATAA TAACTTATCA CACAAGGTTG 13080

TTGTTTTGGG AAGTATAGAT TATAAATGCT TAGCTTCATA TTATAAATGC TGTGATGTAT 13140

TTTGTCTCCC ATCTATACAT GAGTCTGAAG CGTTTGGTGT GGTACAATTA GAAGCAATGA 13200

GTTTTTCAAA GCCATTAGTT TCTACGAATA TCCCCCGAAG TGGTGTTCCA TGGGTTAACG 13260

AAAATGGAAC ATCAGGAATT GTTGTTGAGC CAAGGAGTGC TATAGCTCTT GCTGAAGGGA 13320

TTGTTAAAAC TATTGCTAAT AGTGAAAAAT TCAGTAAAGG AGCAAAAGAT AGATTTGATA 13380

TGCTCTTTAC TAAGAGATTA ATGGTTAATA ATGTATACAA ATTATATTTA AGTATAAAG T 13440

The termination Orf13's of Orf12 is initial

GAGTCAAAAT ATGTCTTTAC TTCCTGTTAT CATTGCTGGT GGTACCGGTA GTCGTTTGTG13500

GCCTTTATCA CGAGTTAAGC ATCCAAAGCA ATTTCTCCAT TTAGGGGATG ATGGAACAAT 13560

GTTACAGACG ACATTAAATC GTCTTCAAGG GTTAAAGTGT GATAACCCAA TTGTAATTTG 13620

CAATGAGCAG CATAGATTTA TAGTTGCTGA GCAACTTCGA CAATTAAATA AACTCACTCA 13680

GAATATTATT CTTGAGCCCG TCGGCCGGAA TACTGCACCA GCTGTAACTC TCGCTGCACT 13740

GAATGCAATA CGTAATAAGT CAAAACAATC AAAATTAATT TTGGTTCTTG CAGCTGATCA 13800

TATTATAAAA GACGAAGATG CATTTTGTAG AAGTGTGCTG AGTGCTATTC CATATGCAAA 13860

CAAAGGAAAA TTAATCACAT TTGGTATAGT GCCTAATAGT CCCGAAACAG GCTATGGATA 13920

TATAAAAAGA GGTCATTTAT GTAGTGGCAA TAATGCTAAT TTAGCTTTTG AAGTGGCTGA 13980

GTTCGTTGAA AAACCTAACA TAGATACAGC TCAAGAGTTT CTTTCATCTG GTAACTATTA 14040

TTGGAATAGT GGTATGTTTT TATTTCGAGC TGATAGGTAT TTAGATGAAT TAAAAAAATA 14100

TAGACCAGAC ATACTTGAGG CCTGTAAAAA ATCAATGATT GAACTTAATG GAGATCTTGA 14160

TTTTATTCGT ATCAATAAAG ATGCTTTTTG CGCTTGCCCA GATGAGTCAA TTGATTATGC 14220

CGTTATGGAG AAAACGAATG ATGCGGTTGT TATCCCAATG GATGCTGGCT GGAGTGATGT 14280

GGGGTCATGG TCATCTTTAT GGGAAATGAG TAATAAAACC ATTGAAGAAA ATGTAATAGT 14340

TGGTGATGTA TAACTTATG ATTCTGACAG CAGCTACCTC CGTTCAG

TT CAGGGCTGCT 14400

TGCTACAGTT GGGGTAAAAG ATCTTGTTGT TGTTCAAACA AAAGATGCTG TGTTAGTAGC 14460

TAATAGGAAT TCAGTTCAGA ATGTAAAAAA AATTGTCGAA AGGCTTAAAT CAGAAAATCG 14520

TAGTGAAGTT TTTACGCATC TTGAAGTTTA TCGTCCTTGG GGTAAATATG AGTCTATCGA 14580

TAATGGTGAA CGCTATGAAG TTAAACGAAT TTCCGTAAAA CCTGGAGAGG GGATTTCATT 14640

GCAAATGCAT CACCATCGTT CTGAACATTG GATAATAGTT TCGGGTACTG CAAAAGTAAC 14700

AATTTGTGAT GAAACAAGAA TTCTCAGTGA AAATGAATCT ATTTATATAC CTGTCGGGGC 14760

GAAACATTGT TTAGAGAATC CGGGAAAAAT TATGTTGGAG CTTATAGAAG TTCGCTCCGG 14820

The termination Orf14's of Orf13 is initial

CTCCTATCTA GGAGAAGATG ATGTCATCCG TTTTGCCGAC AGATATGGAA GAACA TAAAT 14880

GCACAATAAG ATCATCCTAG ATAAATTAAC TTGCTTTAAA GCCTACGATA TTCGCGGAAA 14940

ACTAGGCGAG GAACTGAATG AAGATATCGC

TGGCGCATT GGTCGCGCCT ATGGCGAATT 15000

TCTCAAACCG AAAACCATTG TGTTAGGCGG CGATGTCCGC CTCACCAGCG AAACCTTAAA 15060

ACTGGCGCTG GCGAAAGGTT TACAGGATGC GGGCGTCGAT GTGCTGGATA TCGGCATGTC 15120

CGGCACCGAA GAGATTTATT TCGCCACGTT CCATCTCGGC GTGGATGGCG GCATTGAAGT 15180

TACCGCCAGC CATAATCCGA TGGATTATAA CGGCATGAAG CTGGTGCGCG AAGGGGCTCG 15240

CCCGATCAGC GGCGATACCG GACTGCGCGA CGTCCAGCGT CTGGCAGAAG CCAACGACTT 15300

TCCTCCCGTC GATGAAACCA AACGCGGTCG CTATCAGCAA ATCAATCTGC GTGACGCTTA 15360

CGTTGATCAC CTGTTCGGTT ATATCAACGT CAAAAACCTC ACGCCGCTCA AGCTGGTGAT 15420

TAACTCCGGG AACGGCGCGG CGGGTCCGGT GGTGGACGCC ATTGAAGCCC GCTTTAAAGC 15480

CCTCGGCGCA CCTGTGGAAT TAATCAAAGT ACACAACACG CCGGACGGCA ATTTCCCCAA 15540

CGGTATTCCT AACCCGCTGC TGCCGGAATG CCGCGACGAC ACCCGCAATG CAGTCATCAA 15600

ACACGGCGCG GATATGGGCA TTGCCTTTGA TGGCGATTTT GACCGCTGTT TCCTGTTTGA 15660

TGAAAAAGGG CAGTTTATCG AGGGCTACTA CATTGTCGGT CTTCTGGCAG AAGCGTTCCT 15720

CGAAAAAAAT CCCGGCGCGA AGATCATCCA CGATCCACGT CTCTCCTGGA ACACCGTTGA 15780

TGTGGTGACC GCCGCGGGCG GCACTCCGGT GATGTCGAAA ACCGGACACG CCTTTATTAA 15840

AGAACGTATG CGCAAGGAAG ACGCCATCTA CGGTGGCGAA ATGAGCGCCC ACCACTATTT 15900

CCGTGATTTC GCTTACTGCG ACAGCGGCAT GATCCCGTGG CTGCTGGTCG CCGAACTGGT 15960

GTGTCTGAAA GGAAAAACGC TGGGCGAACT GGTGCGCGAC CGGATGGCGG CGTTTCCGGC 16020

AAGCGGTGAG ATCAACAGCA AACTGGCGCA CCCCGTTGAG GCGATTAATC GCGTCGAACA 16080

GCATTTTAGC CGTGAGGCGC TGGCGGTGGA TCGCACCGAT GGCATCAGCA TGACCTTTGC 16140

CGACTGGCGC TTTAACCTGC GCTCCTCCAA CACCGAACCG GTGGTGCGGT TGAATGTGGA 16200

ATCGCGCGGT GATGTACCGC TGATGGAAGA AAAGACAAAA CTTATCCTTG AGTTACTGAA 16260

The termination of Orf144

CAAG TAATTC AGTAATTTCA TATAAATGGG TTTTAAAAAA CGGAAAAGAT GAGATATCCG 16320

GTGTGGTATA GCCAAGGTAA TGCTATTCAG TATCTCTATG AGTGAGTTAA CATCTATACC 16380

ACATTTAAGC CGCACACTTG GCGGTAACCA CCCCTGACAG GAGTAAACAA TGTCAAAGCA 16440

ACAGATCGGC GTCGTCGGTA TGGCAGTGAT GGGACGCAAC CTCGCGCTCA ACATCGAAAG 16500

CCGTGGTTAT ACCGTCTCTA TTTTCAACCG TTCCCGTGAT AAGACGGAAG AAATTATTGC 16560

CGAAAATCCA GGCAAGAAAC TGGTTCCTTA CTATACGGTG AAAGAATTCG TTGAATCTCT 16620

TGAAACGCCT CGTCGCATCC TGTTAATGGT GAAAGCAGGT GCAGGCACGG ATGCTGCTAT 16680

TGATTCCCTT AAGCCATACC TCGATAAAGG TGACATCATC ATTGATGGTG GTAATACCTT 16740

CTTCCAGGAC ACCATTCGTC GTAACCGTGA GCTTTCTGCC GAAGGTTTTA ACTTCATCGG 16800

TACCGGTGTT TCCGGCGGTG AAGAGGGGGC GCTGAAAGGG CCTTCCATCA TGCCTGGTGG 16860

CCAGAAAGAA GCCTATGAAC TGGTTGCGCC GATCCTGACC AAAATCGCCG CCGTTGCTGA 16920

AGATGGCGAA CCGTGCGTTA CCTATATCGG TGCCGATGGC GCGGGTCACT ATGTGAAGAT 16980

GGTTCACAAC GGTATTGAAT ACGGTGATAT GCAACTGATT GCTGAAGCCT ATTCTCTCCT 17040

GAAAGGCGGC CTGAATCTCT CTAACGAAGA ACTGGCACAG ACCTTTACCG AGTGGAATAA 17100

CGGTGAACTG AGCAGCTACC TGATCGACAT CACCAAAGAT ATCTTCACCA AAAAAGATGA 17160

AGACGGTAAC TATCTGGTTG ATGTGATCCT GGATGAAGCG GCTAACAAAG GTACCGGTAA 17220

ATGGACCAGC CAGAGCGCGC TGGATCTCGG CGAACCGCTG TCGTTGATTA CTGAGTCAGT 17280

GTTTGCACGT TATATCTCGT CTCTGAAAGA TCAGCGCGTG GCCGCGTCTA AAGTTCTCTC 17340

TGGTCCGCAA GCACAGCCAG CTGGCGATAA AGCTGAGTTC ATCGAGAAAG TTCGTCGTGC 17400

GCTGTATCTG GGCAAAATCG TTTCTTACGC TCAGGG TTC TCTCAACTGC GTGCGGCGTC 17460

TGAAGAGTAC AACTGGGATC TGAACTACGG CGAAATCGCG AAGATTTTCC GTGCTGGCTG 17520

CAT TCCGT GCGCAGTTCC TGCAGAAAAT CACCGATGC

TATGCCGAAA ATCCGCAGAT17580

CGCTAACCTG CTGCTGGCTC CGTACTTCAA GCAAATTGCC GATGACTACC AGCAGGCGCT 17640

GCGTGATGTC GTTGCTTATG CAGTACAGAA CGGTATCCCG GTTCCGACCT TCGCTGCTGC 17700

GGTTGCCTAT TATGACAGCT ACCGTGCCGC TGTTCTGCCT GCGAACCTGA TTCAGGCCCA 17760

GCGTGACTA 17769

Sequence in the table is the nucleotide sequence of the O antigen gene bunch of intestinal bacteria O58, it is the same that the nucleotides sequence of the nucleotide sequence of the O antigen gene of shigella dysenteriae 5 types bunch and the O antigen gene of intestinal bacteria O58 bunch shows 99.76%, different base box indicating is the Nucleotide of shigella dysenteriae 5 types above this base.The initial sum terminated position of each gene of the O antigen gene of intestinal bacteria O58 and shigella dysenteriae 5 types bunch all is the same as can be known from Table 4.

Only being preferred embodiment of the present invention below, is not that the present invention is imposed any restrictions, all according to the technology of the present invention essence to above embodiment make an amendment, equivalent variations and modification, all belong in the scope of technical solution of the present invention.

Claims (3)

1, a kind of oligonucleotide is characterized in that, is selected from the Nucleotide of 4967 to 4984 bases among the SEQ ID NO:1; The Nucleotide of 5336 to 5353 bases among the SEQ ID NO:1; The Nucleotide of 5341 to 5358 bases among the SEQ ID NO:1; The Nucleotide of 5839 to 5856 bases among the SEQ ID NO:1; The Nucleotide of 10110 to 10127 bases among the SEQ ID NO:1; The perhaps Nucleotide of 10932 to 10949 bases among the SEQ ID NO:1.

2, a kind of oligonucleotide is right, it is characterized in that, is selected from the Nucleotide of 4967 to 4984 bases among the SEQ ID NO:1 and the Nucleotide of 5336 to 5353 bases among the SEQ ID NO:1; The Nucleotide of 5839 to 5856 bases among the Nucleotide of 5341 to 5358 bases among the SEQ ID NO:1 and the SEQ ID NO:1; The perhaps Nucleotide of 10932 to 10949 bases among the Nucleotide of 10110 to 10127 bases among the SEQ ID NO:1 and the SEQ ID NO:1.

3, the application of the nucleotide pair of the Nucleotide of claim 1 or claim 2, it is characterized in that, it is used for PCR as primer, perhaps is used for hybridization and fluoroscopic examination, makes gene chip or microarray as probe, for detecting intestinal bacteria O58 and shigella dysenteriae 5 types.