CN1285606C - Nucleotide specific for shigella bodyii 2 O-antigen - Google Patents

Abstract

The present invention provides a specific nucleotide for the O-antigens of Shigella bodyii 2. The specific nucleotide is the nucleotide complete sequence of gene clusters in Shigella bodyii 2 for controlling the synthesis of O-antigens, such as a separated nucleotide with the total length of 13234 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 present invention also comprises the oligonucleotide of wzx genes or genes with the similar functions of wzx and wzy genes or genes with the similar functions of wzy stemmed from the O-antigen gene clusters of Shigella bodyii 2. The present invention verifies that the oligonucleotide has high specificity to all of the O-antigen genes of shigella bodyii 2 through PCR. The present invention also discloses a method for using the oligonucleotide of the present invention to detect and identify Shigella bodyii 2 in human bodies and environment.

Description

Nucleotide to the O-antigen-specific of Shigella bogdii 2 types

Technical field

The present invention relates to the complete nucleotide sequence of control O-antigen synthetic gene cluster in Shigella bogdii 2 types (Shigigella bodyii 2), particularly relate in Shigella bogdii 2 types oligonucleotide in the control O-antigen synthetic gene cluster, can utilize these oligonucleotide of O-antigen-specific Shigella bogdii 2 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.

The lipopolysaccharides that is positioned at intestinal bacteria (comprising Shigellae) surface is its morbific inducement, 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 of thecapsule and the O-antigen in resistance of O18:Kl Escherichia coli tocomplement-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 bacterialpopulations " .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 Salmonella 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.

Summary of the invention

The Nucleotide that the purpose of this invention is to provide a kind of O-antigen-specific to Shigella bogdii 2 types.It is the Nucleotide in the O-antigen gene bunch of Shigella bogdii 2 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 Shigella bogdii 2 types.

A time purpose of the present invention has provided the gene of the O-antigen gene bunch that constitutes Shigella bogdii 2 types: 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 orf5, orf8, orf9, orf10 gene; Sugar synthesis path gene comprises rmlB, rmlD, rmlA, rmlC, glf; With the synthetic relevant gene of phosphoserine, comprise orf7.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 the gene that they come from coding transhipment enzyme in the O-antigen gene bunch of Shigella bogdii 2 types respectively comprises the wzx gene or with wzx the gene of identity function arranged; The gene that comes from the coding polysaccharase comprises the wzy gene or with wzy the gene of identity function is arranged; Come from glycosyltransferase gene, comprise orf5, orf8, orf9, orf10 gene.They are the oligonucleotide in the said gene, and length is at 10-20nt; They are special to the O-antigen of Shigella bogdii 2 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 Shigella bogdii 2 types, and these oligonucleotide are also reconfigurable, and the oligonucleotide after the combination also is a high special to the O-antigen of Shigella bogdii 2 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 and identify O-antigen and the detection and evaluation Shigella bogdii 2 types of Shigella bogdii 2 types by these methods.

A further object of the present invention has provided the method for the complete sequence of the O-antigen gene bunch that separates Shigella bogdii 2 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 to the Nucleotide of the O-antigen-specific of Shigella bogdii 2 types it is the isolating Nucleotide shown in SEQ ID NO:1,13234 bases of total length; The base that perhaps has one or more insertions, disappearance or replacement keeps the Nucleotide of the SEQ ID NO:1 of described isolating functional nucleotide simultaneously.

The Nucleotide of aforesaid O-antigen-specific to Shigella bogdii 2 types is characterized in that it is by 12 genomic constitutions, all between galF gene and gnd gene.

The Nucleotide of aforesaid O-antigen-specific to Shigella bogdii 2 types is characterized in that described gene comprises: 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; Come from glycosyltransferase gene, comprise orf5, orf8, orf9, orf10 gene.Wherein said transhipment enzyme gene is the Nucleotide of 4088 to 5353 bases among the SEQ ID NO:1; Pol gene is the Nucleotide of 6127 to 7311 bases among the SEQ ID NO:1.Orf5 is the Nucleotide of 5366 to 6130 bases among the SEQ ID NO:1; Orf6 is the Nucleotide of 8092 to 8751 bases among the SEQID NO:1; Orf9 is the Nucleotide of 8748 to 9620 bases among the SEQ ID NO:1; Orf10 is the Nucleotide of 9583 to 10752 bases among the SEQ ID NO:1.

The Nucleotide of aforesaid O-antigen-specific to Shigella bogdii 2 types is characterized in that it also comprises the oligonucleotide that comes from described wzx gene or wzy gene or glycosyltransferase gene; And their mixing or their reorganization.

The Nucleotide of aforesaid O-antigen high special to Shigella bogdii 2 types, the oligonucleotide that it is characterized in that the described wzx of coming from gene is to being: the Nucleotide of 4273 to 4290 bases among the SEQ ID NO:1 and the Nucleotide of 5050 to 5072 bases; The Nucleotide of 4802 to 4819 bases among the SEQ ID NO:1 and the Nucleotide of 5286 to 5303 bases; The oligonucleotide that comes from the wzy gene is to being: the Nucleotide of 6253 to 6270 bases among the SEQID NO:1 and the Nucleotide of 6705 to 6722 bases; The Nucleotide of 6155 to 6172 bases among the SEQ IDNO:1 and the Nucleotide of 6627 to 6644 bases.

The Nucleotide of aforesaid O-antigen-specific to Shigella bogdii 2 types is detecting the application of expressing the antigenic bacterium of O-, identify other polysaccharide antigen of the O-antigen of bacterium and bacterium in diagnosis.

The recombinant molecule of the Nucleotide of aforesaid O-antigen-specific to Shigella bogdii 2 types, and can provide the O-antigen of expressing Shigella bogdii 2 types by inserting to express, and become bacterial vaccine.

The application of the Nucleotide of aforesaid O-antigen-specific to Shigella bogdii 2 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, the bacterium in available these method human body and the environment.

The separation method of the Nucleotide of aforesaid O-antigen-specific to Shigella bogdii 2 types is characterized in that, comprises the steps:

(1) genomic extraction: 37 ℃ of incubated overnight Shigella bogdii 2 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 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 Shigella bogdii 2 types bunch: with the genome of Shigella bogdii 2 types is that template is passed through its O-antigen gene of Long pcr amplification bunch; At first according to the JumpStart sequences Design upstream primer (5 '-ATT GTGGCT GCA GGG ATC AAA GAA ATC-3 ') that often is found in O-antigen gene bunch promoter region, again according to the gnd gene design downstream primer (5 '-TAG TCG CGC TGN GCC TGG ATT AAG TTC GC-3 ') in O-antigen gene bunch downstream.With the Expand Long Template PCR method of BoehringerMannheim 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, 61 ℃ 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 competence bacillus coli DH 5 cell, get after 2-3ul connects product and 50ul competence bacillus coli DH 5 mixes, forward in the electric shock cup of 0.2cm of Bio-Rad company and shock by electricity, voltage is 2.5 kilovolts, 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 Shigella bogdii 2 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 Shigella bogdii 2 types, the quality of sequence is mainly guaranteed by two aspects: 1) genome of Shigella bogdii 2 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 Shigella bogdii 2 types bunch, with American National biotechnology information science center (The NationalCenter for Biotechnology Information, NCBI) orffinder finds gene, find the reading frame of 11 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 Shigella bogdii 2 types at last;

(6) screening of specific gene: at wzx, the wzy gene design primer in the O-antigen gene of Shigella bogdii 2 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 genomes of 166 strain intestinal bacteria and 43 strain Shigellaes, all primers all obtain positive findings in Shigella bogdii 2 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 obtain PCR product band in the minority group, its size does not meet the expection size, so wzx, wzy gene pairs Shigella bogdii 2 types and O-antigen thereof all are high specials.

Just, first aspect of the present invention provides the full length nucleotide sequence of the O-antigen gene bunch of Shigella bogdii 2 types, its complete sequence shown in SEQ ID NO:1,13234 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 Shigella bogdii 2 types by method of the present invention, as described in Table 3, its 12 genes are all between galF gene and gnd gene.

Second aspect of the present invention provides the gene in the O-antigen gene bunch of Shigella bogdii 2 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 orf5, orf8, orf9, orf10; Special sugared synthesis path gene in the bacterial polysaccharides antigen comprises rmlB, rmlD, rmlA, rmlC, glf gene; Orf7 is the phosphoserine phosphatase gene, participates in the synthetic of phosphoserine.Their zero positions in O-antigen gene bunch and final position and nucleotide sequence all are listed in Fig. 3.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 very 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 Shigella bogdii 2 types.

The 3rd aspect of the present invention, wzy gene in the O-antigen gene bunch that comes from Shigella bogdii 2 types is provided or the gene of identity function and wzx gene is arranged or with wzx the gene of identity function and the oligonucleotide that glycosyltransferase gene comprises orf5, orf8, orf9, orf10 are arranged with wzy, they are any one section oligonucleotide in these genes.But, preferential being the wzy gene in the O-antigen gene that comes from Shigella bogdii 2 types bunch of listing in the table 1 or the gene of identity function and wzx gene being arranged or have the oligonucleotide of gene of identity function right by usefulness with wzx with wzy.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 Shigella bogdii 2 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, though in some bacterium, obtained PCR product band, but its size does not meet the expection size, this is that this problem can be avoided by being PCR with intragenic other primer because primer is attached to genomic other position and causes.So, can determine these primers promptly the listed oligonucleotide of table 1 be high special to Shigella bogdii 2 types and their O-antigen.

The separation method of the Nucleotide of described O-antigen-specific to Shigella bogdii 2 types comprises the steps: 1) genomic extraction; 2) the O-antigen gene in pcr amplification Shigella bogdii 2 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.

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 one section nucleic acid molecule in gene, coding polysaccharase and 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 come from wzx gene (nucleotide position is 4088 to 5353 bases from SEQ ID NO:1), the oligonucleotide of wzy gene (nucleotide position is 6127 to 7311 bases from SEQ ID NO:1) all is a high special to Shigella bogdii 2 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 and comprise orf5, orf8, orf9, orf10.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 and the combination that comes from the oligonucleotide in the pol 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 wzx because of or with wzx (ii) the encode gene of polysaccharase of the gene of identity function is arranged, the gene that comprises the wzy gene or identity function arranged with wzy is the encoding glycosyl transferase gene (iii), comprises orf5, orf8, orf9, orf10 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 Shigella bogdii 2 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 that coming from coding transhipment enzyme gene comprises the wzx gene or have the gene of identity function and the gene of coding polysaccharase to comprise the wzy gene or with wzy the gene of identity function and the oligonucleotide that the encoding glycosyl transferase gene comprises orf5, orf8, orf9, orf10 gene are arranged with wzx.This cover oligonucleotide is special to the O-antigen of a special bacterium, and this special bacterium O-antigen is expressed by Shigella bogdii 2 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 orf5, orf8, orf9, orf10 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 Shigella bogdii 2 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; Come from the encoding glycosyl transferase gene and comprise orf5, orf8, orf9, orf10 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 orf5, orf8, orf9, orf10 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 Shigella bogdii 2 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 orf5, orf8, orf9, orf10 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 Shigella bogdii 2 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 Shigella bogdii 2 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 Shigella bogdii 2 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 3ul20mg/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 the O-antigen gene in pcr amplification Shigella bogdii 2 types bunch

With the genome of Shigella bogdii 2 types is that template is passed through its O-antigen gene of Long pcr amplification bunch.At first according to the JumpStart sequences Design upstream primer (5 '-ATT GTG GCT GCA GGG ATC AAA GAA ATC-3 ') that often is found in O-antigen gene bunch promoter region, again according to the gnd gene design downstream primer (5 '-TAG TCG CGC TGN GCC TGG ATT AAG TTCGC-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 WizardPCR 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 buffer of 9ul and the T4DNA ligase enzyme of 25 units are wherein arranged.Use the dehydrated alcohol precipitation of the 3M NaAc (pH5.2) of 1/10 volume and 2 times of volumes to be connected mixture at last, wash precipitation with 70% ethanol again, be dissolved in after the drying and obtain connecting product in the 30ul water.

Next is the preparation of competent cell:

The method that provides with reference to Bio-Rad company prepares competent cell bacillus coli DH 5.Get the single bacterium colony of a ring bacillus coli DH 5 in the LB of 5ml substratum, 180rpm cultivated after 10 hours, get in the LB substratum that the 2ml culture is transferred to 200ml, 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 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 Shigella bogdii 2 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 Shigella bogdii 2 types obtained.The quality of sequence is mainly guaranteed by two aspects: 1) genome of Shigella bogdii 2 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 Shigella bogdii 2 types bunch, with American National biotechnology information science center (The National Center forBiotechnology Information, NCBI) orffinder finds gene, find the reading frame of 11 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 Shigella bogdii 2 types at last, as shown in table 3.

By retrieving and comparing, find orf1, orf2, orf3 and orf11 have very high homogeny (identity) with four synthase genes of the dTDP-rhamnosyl of the synthetic rhamnosyl precursors of many bacterium, wherein: the RmlB of orf1 and Shigella bogdii has 98% homogeny in 361 amino acid, 99% similarity; The RmlD of Orf2 and Shigella bogdii has 97% homogeny in 299 amino acid, 98% similarity; The RmlA of orf3 and Shigella bogdii has 97% homogeny in 289 amino acid, 98% similarity, the RmlC of Orf11 and Shigella bogdii has 67% homogeny in 176 amino acid, 82% similarity, illustrate that they all have high homology, can determine that orf1, orf2, orf3, Orf11 are respectively the rmlB gene of dTDP-rhamnosyl, rmlD gene, rmlA gene, rmlC gene, therefore difference called after rmlB, rmlD, rmlA, rmlC.In the O-antigenic structure of Shigella bogdii 2 types, contain rhamnosyl [V.L.L ' vov, E.l.Ramos, etal (1983) Carbohydr.Res.124,141-149], hence one can see that, and orf1, orf2, orf3, the Orf11 gene in its O-antigen gene bunch is synthetic rhamnosyl.The albumen that is similar to O-antigen transhipment enzyme of orf2 and Escherichia coli K-12 and other bacterium has 49% homogeny, 70% similarity in 406 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 orf4 has 12 potential transmembrane domains, it and many wzx protein similars, and about 50 amino acid whose conservative motifs are arranged at the proteic aminoterminal of wzx, so can determine orf4 is the wzx gene, called after wzx.The EpsF of orf5 and Streptococcusthermophilus has 41% homogeny in 226 amino acid, 61% similarity.EpsF is a glycosyltransferase, so determine that orf5 is a glycosyltransferase, called after orf5.The O-antigen polysaccharase (Wzy) of Orf6 and Shigella dysenteriae has 22% homogeny respectively, 43% similarity in 413 amino acid; In 406 amino acid, 21% homogeny is arranged respectively, 38% similarity with the O-antigen polysaccharase of Escherichia coli strain K-12; Learn that by the Eisenberg algorithm orf6 has 11 potential transmembrane domains in addition, similar secondary structure is arranged, so determine that orf6 is the wzy gene, called after wzy to other O-antigen polysaccharase.Relatively find by blast, the phosphoserine phosphatase of orf7 and Clostridium tetani E88 has 22% homogeny respectively in 204 amino acid, therefore 46% similarity infers that orf7 also is the phosphoserine phosphatase gene, temporarily called after orf7.The long agnoprotein of 276aa of the albumen of Orf8 genes encoding and Pyrococcus horikoshii has certain kinship, and both have 24% homogeny and 46% similarity in 269 amino acid.So the function of Orf8 gene is also unknown, temporarily called after orf8.Orf9 is similar to PF00535 glycosyltransferase family, and the E value is 3.3 * e ^-5Blast finds that relatively the rhamnosyltransferase I of orf9 encoded protein and Leptospira interrogans has 32% homogeny, 51% similarity in 300 amino acid; In 295 amino acid, 31% homogeny is arranged with the RfbF albumen of Actinobacillus pleuropneumoniae, 46% similarity, and RfbF albumen is the glycosyltransferase of dTDP-rhamnosyl, so infer that orf9 also is a glycosyltransferase gene, also be to shift the dTDP-rhamnosyl, temporary called after orf9.CpsF albumen among orf10 encoded protein and the Proteus mirabilis has 42% homogeny in 370 amino acid, 60% similarity, cpsF albumen is a glycosyltransferase, so orf10 also is a glycosyltransferase gene, and temporary called after orf10.The albumen of the glf genes encoding among orf12 and the Escherichia coli K-12 has 68% homogeny in 364 amino acid, 82% similarity, and they have very high homology, so orf12 also is the glf gene, called after glf.

Embodiment 6: the screening of specific gene

At wzx, wzy gene design primer in the O-antigen gene of Shigella bogdii 2 types bunch, the position of these genes in nucleotide sequence sees Table 1.

Transhipment enzyme gene and pol gene and their function corresponding and the size of the O antigen gene bunch of Shigella bogdii 2 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 166 strain intestinal bacteria, method as previously mentioned.With this to primer from the colibacillary genome of 166 strains PCR with identification of escherichia coli and detect its genomic quality.

Table 2 is 166 strain intestinal bacteria and 43 strain Shigellaes and their sources that are used to screen specific gene, and for the convenience that detects, we are divided into one group with their every 8-10 bacterium, and 27 groups altogether, all list in the table in their source.

The genomic dna that contains Shigella bogdii 2 types in the 22nd group is as positive control.Do template with every group of bacterium, be PCR by following condition with every pair in the table 1 primer: 94 ℃ of pre-sex change after 2 minutes, 94 ℃ 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 10ul PCR 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 22nd 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 Shigella bogdii 2 types and O-antigen thereof all are high specials.

At last, from Shigella bogdii 2 types, screen gene by PCR: wzx, wzy gene to the O-antigen high special of Shigella bogdii 2 types.And the oligonucleotide of these intragenic any one section 10-20nt is special to the O-antigen of Shigella bogdii 2 types, and the primer in especially above-mentioned each gene is that oligonucleotide is high specials to detecting the back confirmation through PCR to Shigella bogdii 2 types.These all oligonucleotide all can be used for Shigella bogdii 2 types in the human body and environment rapidly and accurately, and can identify their O-antigen.

Table 3 is structural tables of the O-antigen gene bunch of Shigella bogdii 2 types, has listed the structure of the O-antigen gene bunch of Shigella bogdii 2 types in table, altogether by 12 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 Shigella bogdii 2 types, in table, listed the accurate position of all open reading frame in complete sequence in the O-antigen gene bunch of Shigella bogdii 2 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〉Nankai University

＜120〉to the Nucleotide of the O-antigen-specific of Shigella bogdii 2 types

<160>1

<170>PatentIn version 3.1

<210>1

<211>13234

<212>DNA

<213>Shigella boydii

<400>1

attgtggctg cagggatcaa agaaatcctc ctggtaactc acgcgtccaa gaacgcggtc 60

gaaaaccact tcgacacctc ttatgaatta gaatctctcc ttgaacagcg cgtaaagcgt 120

caactgcttg cggaagtgca gtccatctgt ccgccgggcg tgaccattat gaacgtgcgt 180

cagggcgaac ctttaggttt gggccactcc attttatgtg cacgacctgc cattggtgac 240

aatccatttg tcgtggtgct gccagacgtt gtgatcgacg acgccagcgc cgacccgctg 300

cgttacaacc ttgctgccat gattgcacgt ttcaatgaaa cgggccgcag tcaggtgctg 360

gcaaaacgta tgccgggtga cctctctgaa tactcggtca tccagaccaa agaaccactg 420

gatcgcgaag gtaaagtcag ccgcatcgtc gaatttatcg aaaaaccgga tcagccgcag 480

acgccggact cagacatcat ggccgttggt cgctatgtgc tttctgccga tatttggccg 540

gaactggaac gtactcagcc tggtgcatgg ggacgtattc agctgactga tgccattgct 600

gaactggcga aaaaacagtc cgttgatgcc atgctgatga caggtgacag ctacgactgc 660

ggtaaaaaaa tgggctatat gcaagcgttc gtgaagtatg gactacgcaa cctgaaagaa 720

ggggcgaagt tccgtaaagg gattgagaag ttgttaagcg aataatgaaa atctgaccgg 780

atgtaacggt tgataagaaa attataacgg cagtgaagat tcgtggcgaa agtaatttgt 840

tgcgaatatt cctgccgttg ttttatataa acaatcagga taacaacgag ttagcaatag 900

gattttagtc aaagttttcc aggattttcc ttgtttccag agcggattgg taagacaatt 960

agcgtttgaa tttttcgggt ttagcgcgag tgggtaacgc tcgtcacatc gtagacatgc 1020

atgcagtgct ctggtagctg taaagccagg ggcggtagcg tgcattaata cctctattaa 1080

tcaaactgag agccgcttat ttcacagcat gctctgaagt aatatggaat aataaagtga 1140

agatacttgt tactggtggc gcaggattta ttggttctgc tgtagttcgt cacattataa 1200

ataatacgca ggatagtgtt gttaatgtcg ataaattaac gtacgccgga aacctggaat 1260

cacttgctga tgtttctgac tctgaacgct atgtttttga acatgctgat atttgcgatg 1320

ctgctgcaat ggcgcggatt tttgctcagc atcagccgga tgcagtgatg cacctggctg 1380

ctgaaagcca tgtggatcgt tcaattacag gccctgcggc atttattgaa accaatattg 1440

ttggtactta tgtccttttg gaagcggctc gcaattactg gtctgctctt gatggcgaca 1500

agaaaaatag cttccgtttt catcatattt ctactgacga agtctatggt gatttgcctc 1560

atcctgacga agtaaataat aaagaacaat tacccctctt tactgagacg acagcttacg 1620

cgcctagtag tccttattcc gcatcaaaag catccagcga tcatttagtc cgtgcgtgga 1680

aacgtaccta tggtttaccg accattgtga ctaactgttc gaataactac ggtccttatc 1740

actttccgga aaaattgatt ccactagtaa ttcttaatgc tctggaaggt aaggcattac 1800

ctatttatgg caaaggggat caaattcgtg actggctgta tgttgaagat catgcgcgtg 1860

cgttatatat cgtcgtaacc gaaggtaaag cgggtgaaac ttataacatt ggtggacaca 1920

acgaaaagaa aaacatcgat gtagtgctca ctatttgtga tttgttggat gagattgtac 1980

cgaaagagaa atcttaccgt gagcaaatta cttatgttgc cgatcgcccg ggacacgatc 2040

gccgttatgc gattgatgca gagaagatta gccgcgaatt gggatggaaa ccacaggaaa 2100

cgtttgagag cgggattcgg aagacagtgg aatggtacct gtccaataca aaatgggttg 2160

ataatgtgaa aagtggtgcc tatcaatcgt ggattgaaga gaactatgag ggccgccagt 2220

aatgaatatc ctcctttttg gcaaaacagg gcaggtaggc tgggaactac agcgtgctct 2280

cgcacctttg ggtaatttga ttgctcttga tgttcactcc actgattatt gtggtgattt 2340

tagtaatcct gaaggtgtgg ctgaaaccgt caaaaaaatt cgccctgatg ttattgttaa 2400

tgctgcggct cataccgcag tagataaggc tgagtcagaa cccgaatttg cacaattact 2460

caatgcgacc agcgttgaat caattgccaa agcggctaat gaagttgggg cctgggtaat 2520

tcattactca actgactacg tatttccggg aaccggtgaa ataccatggc tggagacgga 2580

tgcaaccgca ccgctaaatg tttacggtga aaccaagtta gccggagaaa aagcgttaca 2640

ggaacattgc gcgaagcatc ttattttccg taccagctgg gtatacgcag gtaaaggaaa 2700

taacttcgcc aaaacgatgt tgcgtctggc aaaagagcgc gaagaactgg ctgtgattaa 2760

tgatcaattt ggtgcgccaa caggtgctga gctgctggca gattgtacgg cacatgctat 2820

tcgtgtggca ctgaataaac cagaagtcgc aggcatgtac catctggtag ccagtggtac 2880

cacaacctgg cacgattatg ctgcgctggt ttttgaagag gcgcgcaaag caggtattcc 2940

ccttgcactc aacaagctca acgcagtacc agcaacagcc tatcctacac cagctcgtcg 3000

tccacataac tctcgcctta atacagaaaa atttcagcag aactttgcgc ttgttttgcc 3060

tgactggcag gttggcgtga aacgaatgct caacgaatta tttacgacta cagcaattta 3120

atagtttttg catcttgttc gtgatgatgg agcaagatga attaaaagga atgatgaaat 3180

gaaaacgcgt aaaggtatta ttttagcggg tggttctggt acacgtcttt atcctgtgac 3240

tatggctgtc agtaaacagc tattacctat ttatgataag ccgatgatct attacccgct 3300

ctctacactg atgttggcgg gtattcgcga tattctgatt attagtacgc cacaggatac 3360

tcctcgtttt caacaactgc tgggtgacgg tagccagtgg gggctaaatc ttcagtacaa 3420

agtgcaaccg actccagatg ggcttgcgca ggcgtttatt atcggtgaag agtttatcgg 3480

tggtgatgat tgtgctttgg ttcttggtga taatatcttc tacggccacg atctgccgaa 3540

gttaatggat accgctgttg acaaagaaag tggtgcaacg gtatttgcct atcacgttaa 3600

tgatcctgaa cgctacggtg tcgttgagtt tgataaaaac ggtacggcaa taagcctgga 3660

agaaaaaccg ctacaaccaa aaagtaatta tgcggtaacc gggctttatt tctatgataa 3720

cgacgttgtc gaaatggcga aaaaccttaa gccttctgcc cgtggtgaac tggaaattac 3780

cgatattaac cgtatttata tggaacaggg gcgtttatcc gttgccatga tggggcgtgg 3840

ttatgcgtgg ctggacacgg ggacacatca aagcctgatt gaggcaagca acttcattgc 3900

tacaattgaa gaacgtcagg ggttgaaagt ttcctgcccg gaagaaattg cttaccgtaa 3960

agggtttatt gattctgagc aggtgaaaat attagctgaa ccactgaaaa aaaatgctta 4020

tggtcagtat ctgctaaata tcataaataa taaaatatca taatattaac tagttgcagc 4080

tatataaata ggctcggaga ttgcattgaa gcttgttacc tctcttaata aaaatatctt 4140

ctatttagct atattgcaag gaagttattt tatactgcca ttgttaacat ttccttatct 4200

tgttagagta ttaggaccat actcttttgg tctattaggg ttttgtcaag caacaatgca 4260

atatcttgtg atggtgactg attatgggtt taattggtca gcaacacaag atgtttcgaa 4320

aagtaagaat gacaaggaaa aattaacaac aatattttgg aatgtcttct ttagtaagat 4380

tatacttgca ctttcggcaa ttgttatatt tttcggaata acatttttta ttaaggagct 4440

atattatgct caagtggtgt taatatcatt tattccaatg gtgttaggta atatattata 4500

ccctgtttgg tttttccagg gattagaaaa aatgaaatgg attacattat gtagtttaag 4560

tgcaagatta ttattaatac cattaacttt tttgttagta aaaaatccac aagatatctg 4620

gttagccgct ttgattcaag gcggcgttaa ttttatagca ggtttaattg gatttgctat 4680

tattataaat aataactgga ttgggaaatt ggtatttgat tatgatggta taatttattc 4740

attaaaaagc ggatggcata ttttcatttc aacagctgct attagcttat atacaacaac 4800

aactgttgtt gttcttgggt ttatatgtgg acctgtctct gttggctact ttaacgctgc 4860

taatacaatt agaaatgcag cccagagtct aatgaatcct atttatcaag ctgtttatcc 4920

aagaattaat tcattgtttg aagaaaatta tgcaaaaagt atgaatcttc tgaaaaaatc 4980

atttaagggc gtattattca tttcgttttc aggttcatta ctcctgtgca tactttctcc 5040

aataataata agatatggag tcggagataa atatactgag tcaattagta ttttaagaat 5100

attagccttt ttgccattta ttatttctct tagtaatttt tttggcattc aaaccatgct 5160

gacacatggt tataaaagac aatttagtaa aatactgcta atttgtggtg ttatgcatgt 5220

gataatcatt gctcctttga tcttttttgc tggggcgtat ggggctgcga tgacggtttt 5280

aattacagaa accgtagttg catcgttgat gtatcttttc ttaaaaagaa agaaaataaa 5340

tttgtttaaa taaagaggaa tattaatggc tttattcatt gcttgccata aacaaactga 5400

attacctaat catgagtgct atatccctat tcatgcgggt aagaaaatat cagggaaaat 5460

gcttgatcaa gtgttgggag atgatacagg gaataatata tcatgtaaaa ataaaaatta 5520

ttgtgagctg accgttatat attggctttg gaaaaataaa ctttttaatg atgattatat 5580

tggcttggtt cattatagga ggtattttgg aaatgtttta tcgaatttta aatttaaaga 5640

ggtgaggata tatgacagag ctaatatttc taataaaatg aaacaatatg atataattat 5700

tccggtgaaa gagagtttga agctatctgt tgttgaacac tacaggaaat tccataatgt 5760

cgaagatcta atgttggcaa aaaaaatagt tgataaatta tatcctgatt atagtgttgc 5820

attcagagaa cttttagaac aaaaaaaaat atcgctttat aatatgttta taatgaaggc 5880

tgggattttt gattgttatt gtgattggtt gttttccata ttagatgagc tggagaaaga 5940

aataaattta cgcagatacg atgaatatca atcacgtgtg ttcggcttta ttgctgagcg 6000

acttctgaat gtatggatta ataaaaataa agaagttttt aaagtcggtg aggaatttgt 6060

aattaacact gatgaatgca gtggaagagg agctcaatta atttctcaac ttagagctaa 6120

gcttaaatga acatgaccat ttttatagct ataataatgc ttatgtattc aggattatta 6180

ctgagtatta ataaagatat taaatcgccg tcggcgattc tattctttat ctgggggggg 6240

ctgttatttt tgtcgggagt aaatggagac ataactaact atacattatg tataatttta 6300

ttttcatgtt tatctttttc ggtaggggct ctactggcaa aaccttacgc tcctatattt 6360

gaacatgtat tttcctataa tataaacaat acaaaagcgt tagtattaat tacaaatatt 6420

atctcgtttg ttattttaat ctatacttta atagtttttt tatattatta taaggggggc 6480

ttcactgaat cctatataaa ttcacgtaca gaaattaatt acggagataa atcgggatta 6540

ataaaaatat atgggtatat atactatctt ttatacccat tggtatatgt atggagcttt 6600

ttatatttta aaaataaata taaaaagatt gaagaaggag agcgggcccg gttaccatat 6660

aataatcgaa cattttattt gtttttttta cttcactttt tttatgcgtt attatcaaca 6720

gctaaattaa gttttattac taattatacc aatagtgttt ttgcgattat tttttcaaaa 6780

atatctttaa aatatttact tgtcacagtg tctggtatta tatttttttt ctatttatct 6840

atgttgtttt taaataaaat tgacgactct tcaggagcat tcgaagcgtt aaaatatgga 6900

ttgatgaact atagcttggc aaacattttt gcattagatt cggttttgca aggtaaagca 6960

gtagttatag attgtaatgg agatgctaca tgtggattag ctaattttat atcatataaa 7020

gagtataaga caaatgtttt tacaatattt tattcgctaa caaaatattc tgattttttg 7080

tactcattaa ttttcttttt tgtcataggt ttctttcatt catctttgta caatgtagca 7140

aaaaaaaata agaaaacgat atcagttgtc atttgctcta ttttgtactt ccccttgtta 7200

tttcaatttt ttgaccaatt atatatgttg atgttctata tttatgcaat tgcaatgctt 7260

tatgtgatat gtttttgctc aagattaact ctaactttga agggagttta acatgctcga 7320

acatattgtt gtatttgatg tatgtggcac tctttatttg tcgaatacaa cttttgatta 7380

tgttgtattt gtacatagga aacaaaaaaa atataccaag ttaattaaat gcttattatt 7440

acgctcctta tttggaaagt tgctacataa attgcgtata tgttccttac gaaaacattt 7500

tttgaagaca ttagaaggat atgaaaagtc tactttggaa gagttggcga atttgtttta 7560

tgatgaattt ctcattttta aagaaaagaa tgaagttatg gcacttttag aacgatataa 7620

acatgagaag atcattttaa tgtctgcttc cgttgatcct gtcataaatg taattgctaa 7680

aaatttgaat gttgaggcag tatcatctaa attagaattt atcgacaata aatcaacagg 7740

gttattagaa tataatctta ctggtataaa gcatttacaa gttaataacc ctgtaagttt 7800

gattgtcact gataatttca gtgatattaa tcttattaaa cttgcagata cagcagtgat 7860

tatttcgaaa gccaaagata aaaataaatg gagagtacta ttaaatagat ttggtgtctc 7920

caaagataaa gtgaggttta tgtgatattg tatttcccat tatcatattt attcatttca 7980

agattgaaaa catggcatga aaaaattagc tggtttatta tatatccagg gtttctattg 8040

ctgtgttgtt ttttgtataa tggtagtttt gtatctttgc ttctgtcatt tatgatgacc 8100

atgtccattt atgaaatagg ctacttagac aatgactata gaccaataaa tacagaaaaa 8160

gcaccaacaa taagaggtgg agagctgcgt gatacaatta aaagtaaatt atcaagtata 8220

attttcatta gagttgttta tttttttgta tcattcgcgt tattaattta tcaaagtgat 828

atcattgctc atcactatac tgttatctat tttgtattat gtatagggtt tttaattatc 834

tccttttatt tgcataatac aatccgggat aagagaaata taatcactta tttttctttg 8400

gtgtcatgtc gttacattat tcctttgata cccactgtca catgcagtat attttttgtt 8460

tttgtcatat tattattatt ccctttaatc agaacattag aacacgcctg taaggagaaa 8520

tatggtatta aaaggttaaa ggattttatt atttaccctg atttatttag ggttaaatat 8580

tactttttct tttttgatgc tgctattgtg tatgcatata tatatcagga ctggaagatt 8640

gtattattgt cgggatattt tttgttttat cgatacataa cctttaaagt atccgataaa 8700

aattttgtaa aaaggactca tcatggatct tataagtgga ataaaaaatg aatagagtag 8760

cggtgataat tgtactatat tatcctgatg ttagtaagat caatgaaatg attgactcac 8820

tgggaaaaaa acagcgcgaa atattacttg ttgacaatac acctgagctc cacttagaaa 8880

gtgatgtttt gtgtaataac aacattcatt actatcattt aggagataac aaaggcattg 8940

cgtttgcgca gaactatggt ttcattaaag ctatcggatt aggaaagccc ttattcttta 9000

catttgacca agattcatgt atatcaaaag attatatatc atcaatgtta aaagagtatg 9060

agtacgcttg ctctctccgg agtaatattg ctgcattagg tccaacaata ataaatgaac 9120

gtaatgggaa aaagtatgat cgtgaaataa agctgggctc ccagataagt gaaacgatac 9180

atgatgttga aagtataata tcttctggtg cactcatacc cctagaggca attatcaata 9240

ttggattaaa taaagtatct tggtttatcg atttaattga catagaatgg agtttcagag 9300

ctaggaaaga aggctggaat atcctagtga cagataaagt ccttatttct cacaatatag 9360

gatctaaaga tataaatatt atggggatta aaacatttac agtttgcagt ccatttcgtt 9420

tgtattatgt ttatagaaat tggttattag ctctgagaga gcctgtattt cctttaagat 9480

ataagttaaa aaaattgata acaatgcctg tccgaatcat tatttacgct ttttgtgaac 9540

gtggaggaca acgattaaat tatattttta aggggataaa agatggactg cttggacgaa 9600

aaggatcatt taagggataa tcattcctta aaaatagttt taactgctaa tagtagttgg 9660

tatatatata attttagacg aagcacaatt aaggcactta ttaatatagg gtttgatgta 9720

ttagtggttg taccggatta tgaatataag gattctattt taaaattcgg cgctaaattt 9780

gagaaggttg atcttaaacc aaaatctatt aatattttta ctgaatgtac atctctaata 9840

tcatattata aaattataaa aaaattcaat ccacaggtta tattaacgtt tacacccaaa 9900

gctaatatat attgtggttt gcttgcttat aaatttgacg cgaaagtgat acctaatatt 9960

tctggccttg gaagtgcatt tgttaatagt aatacgatat taagtcaaat tgttaagcta 10020

ttatataaga tagcattaaa gaaagcagca ttcgtttttt ttcaaaacga ggatgatcgt 1008

cagttattga ttaattctgg ctgcgtcaat tatgagaaaa catgccgaat atttgggtct 10140

ggtgttgatt tatcaaaatt tcttccctca aataagaatt taaagcaaag atcatgtgtg 10200

aaattcatac ttgttgctag acttctatat accaaaggca tacttcacta tcttaaggcc 10260

gcagaaataa taaaatccaa atatcctaat tgttcatttt cactgttagg tgcatttgaa 10320

aaaaatgaaa agataatcac gaaggaattg attgaaggtt tttgtacgag gggtatagtt 10380

aattactatg ggacttccga tgacgttgct agtataatga agcattatga tgcaatagta 10440

ttaccatcat tttatagaga aggtgtacct aaagcgttga ttgaaggtgc ttctagtggg 10500

ttagctattt tgacaacaga taatgttggc tgtagggata tggtgcaaaa tgaagttaat 10560

ggttttttgt gtcaacctaa tgatcttgat agcttagttg acattattga aaagtatatt 10620

ctactgacag acgaaatgaa aaacacaatg tctattaagt cgagagaatt tgctgaatca 10680

cattgtgatg aaaataagat catagagaag tatactaata caattaatga cgttttaaat 10740

agaaatcact aaaggttata ataaatatga aagttcaaga cactgttctg gatggagtta 10800

aaattattga accggtcgta tatggtgaca acaggggttt ttttttagaa gtatatcaaa 10860

aacaacgtta tcaaaattta ttaaatatag aatttgattt cgtacaagat aactattcac 10920

gatcaagcaa aaatgtttta agaggattac atttccaaaa aacgaagcca caaggtaaac 10980

ttgtaagagt tgtgcgcggc gaagtttttg atgttgccgt cgatattaga aaagaatcac 11040

ctaattatgg ccaatggatt ggtgtgttac tttctgaaat gaataaaaaa caattttggg 11100

taccaccagg tttcgcacat ggttttgttg tattatctga aatggccgat tttgaatata 11160

aatgcacaga atattatgat cctgaggatg agtgttgttt attatggaat gatccagaac 11220

tcaatattca atggccttta agtaatccta tattatcaga taaagatatg aaaggttgct 11280

tacttaaaga actttaattt taactaagtt agatgacata ggaaccgcaa tgcaatatga 11340

tttcataatt ataggttcag gtttatttgg tgcggtttgc gcttatgaat taaataaaag 11400

aaataaacgt gtgttggtca ttgagaaaag ggatcatatt ggagggaata tctatactga 11460

aaaagttgac gatattcaca tacataagta tggtgctcat atatttcata caaacgatca 11520

atatatctgg aattatataa ataaattcgc ttcttttaat agattcacta attctccatt 11580

agcatatcat aaaggtaaat tatataactt acctttcaat atgaatacct tttatcagtt 11640

atgggggaca aaaactcctt ctgaagctca agataaaata gatttacaaa gaagtaaata 11700

ttctaacata actcccacaa atttagaaga acaagctctt tccctggtag gggaagatgt 11760

ttatgaaaaa ctaattaagg ggtatacgga aaaacaatgg ggacgttcat gttctgaatt 11820

accgcctttt attataaaaa gaatcccagt tcgattcact ttcgataata attatttcac 11880

cgatagattt cagggggttc ctatcggagg atacacacaa ataattgata agatgcttaa 11940

tggtgttgat cttctgctta atgttgacta tttatcagaa aaggataagt ataattctat 12000

ggcggaacgg attatatata ctgggcctat agatgcttat tttgataaaa agttgggaac 12060

tttagaatat agatccttaa agtttataac aaaaaaaatt gaggaggcta attatcaagg 12120

caatgccgta attaattata ctgaaaaaga tattcctttt actaggataa ttgagcataa 12180

gcattttgat gactcttatg aaactgaata tacctttata actgaagagt acccaataga 12240

gtggagtgag ggtcaagagc cttattatcc cattaatgat aaaaaaaata tggatatata 12300

tcataaatat agagagcttt cgcaaatgga gagtaaagta attttcggag gacgccttgc 12360

tgaatataag tattatgata tgcaccaagt tatccgttca gcactgaatt gtgtaaataa 12420

aatattacat gattaagttg ttttggcgtt attaatatta ttaattaaat atttaatgta 12480

gtaaccccct aacaggagta aacaatgtca aagcaacaga tcggcgtcgt cggtatggca 12540

gtgatggggc gcaaccttgc gctcaacatc gaaagccgtg gttataccgt ctctattttc 12600

aaccgttccc gtgagaagac ggaagaagtg attgccgaga atccaggcaa gaaactggtt 12660

ccttactata cggtgaaaga gtttgttgaa tctctggaaa cgcctcgtcg catcctgtta 12720

atggtgaaag caggtgcagg cacggatgct gctattgatt ccctcaaacc atatctcgat 12780

aaaggcgaca tcatcattga tggtggtaac accttcttcc aggacaccat tcgtcgtaat 12840

cgtgagcttt ctgccgaagg ctttaacttc attggtaccg gtgtttccgg tggtgaagaa 12900

ggtgcgctga aaggtccttc cattatgcct ggtgggcaga aagaagccta tgaactggtt 12960

gcaccgatcc tcaccaaaat cgccgcagtg gctgaagacg gtgagccatg cgttacctat 13020

attggtgccg atggcgcggg tcactatgta aaaatggttc acaacggtat tgaatacggt 13080

gatatgcagc tgattgctga agcctactct ttgcttaaag gtggcttgaa cctttccaac 13140

gaagaactgg cgcagacctt taccgagtgg aataacggtg aactgagcag ctacctgatc 13200

gacatca ccaaagatat cttcaccaaa aaagatg 13234

Wzx gene and wzy gene and wherein primer and PCR data in the O antigen gene of table 1 Shigella bogdii 2 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	O-antigen transhipment enzyme	4088-5353	4273-4290 4802-4819	5050-5072 5286-5303	800bp 502bp	0 0	60 60
wzy	O-antigen polysaccharase	6127-7311	6253-6270 6155-6172	6705-6722 6627-6644	470bp 490bp	0 0	62 60

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

Group number	The bacterial strain that contains in this group	The source
			1 2 3 4 5 6 7 8 9 10	Wild-type e. coli 01,02,03,04,010,016,018,039 wild-type e. coli 040,041,048,049,071,073,088,0100 wild-type e. coli 0102,0109,0119,0120,0121,0125,0126,0137 wild-type e. coli 0138,0139,0149,07,05,06,011,012 wild-type e. coli 013,014,015,017,019ab, 020,021,022 wild-type e. coli 023,024,025,026,027,028,029,030 wild-type e. coli 032,033,034,035,036,037,038,042 wild-type e. coli 043,044,045,046,050,051,052,053 wild-type Shigella bogdii, 2 types, 055,056,057,058,054,060,061 wild-type e. coli 062,063,064,065,066,068,069,070	IMVSa IMVS IMVS IMVS IMVS IMVS IMVS IMVS IMVS IMVS

11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27

Wild-type e. coli 074,075,076,077,078,079,080,081 wild-type e. coli 082,083,084,085,086,087,089,090 wild-type e. coli 091,092,095,096,097,098,099,0101 wild-type e. coli 0112,0162,0113,0114,0115,0116,0117,0118 wild-type e. coli 0123,0165,0166,0167,0168,0169,0170,0171 wild-type e. coli 0172,0173,0127,0128,0129,0130,0131,0132, wild-type e. coli 0133,0134,0135,0136,0140,0141,0142,0143 wild-type e. coli 0144,0145,0146,0147,0148,0150,0151,0152 wild-type e. coli 0153,0154,0155,0156,0157,0158,0159,0164 wild-type e. coli 0160,0161,0163,08,09,0124,0111 wild-type e. coli 0103,0104,0105,0106,0107,0108,0110 Shigella bogdii serotypes B 2, B4, B5, B6, B8, B9, B11, B12, B14 Shigella bogdii serotypes B 1, B3, B7, B8, B10, B13, B15, B16, B17, B18 shigella dysenteriae serotype D1, D2, D3, D4, D5, D6, D7, D8 shigella dysenteriae serum D9, D10, D11, D12, D13 shigella flexneri F6a, F1a, F1b, F2a, F2b, F3, F4a, F4b, F5 (v:7) F5 (v:4) bacillus ceylonensis A D5, DR

IMVS IMVS IMVS IMVS See b See c IMVS IMVS IMVS IMVS IMVS See d See d See d See d See d See d

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

b.0123 from IMVS；the rest from Statens Serum Institut，Copenhagen，Denmark

c.172 and 173 from Statens Serum Institut，Copenhagen，Denmark，the rest from IMVS

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

Table 3 is structural tables of the O-antigen gene bunch of Shigella bogdii 2 types

Table 4 is location tables of the gene in the O-antigen gene bunch of Shigella bogdii 2 types

ATTGTGGCTG CAGGGATCAA AGAAATCCTC CTGGTAACTC ACGCGTCCAA GAACGCGGTC 60

GAAAACCACT TCGACACCTC TTATGAATTA GAATCTCTCC TTGAACAGCG CGTAAAGCGT 120

CAACTGCTTG CGGAAGTGCA GTCCATCTGT CCGCCGGGCG TGACCATTAT GAACGTGCGT 180

CAGGGCGAAC CTTTAGGTTT GGGCCACTCC ATTTTATGTG CACGACCTGC CATTGGTGAC 240

AATCCATTTG TCGTGGTGCT GCCAGACGTT GTGATCGACG ACGCCAGCGC CGACCCGCTG 300

CGTTACAACC TTGCTGCCAT GATTGCACGT TTCAATGAAA CGGGCCGCAG TCAGGTGCTG 360

GCAAAACGTA TGCCGGGTGA CCTCTCTGAA TACTCGGTCA TCCAGACCAA AGAACCACTG 420

GATCGCGAAG GTAAAGTCAG CCGCATCGTC GAATTTATCG AAAAACCGGA TCAGCCGCAG 480

ACGCCGGACT CAGACATCAT GGCCGTTGGT CGCTATGTGC TTTCTGCCGA TATTTGGCCG 540

GAACTGGAAC GTACTCAGCC TGGTGCATGG GGACGTATTC AGCTGACTGA TGCCATTGCT 600

GAACTGGCGA AAAAACAGTC CGTTGATGCC ATGCTGATGA CAGGTGACAG CTACGACTGC 660

GGTAAAAAAA TGGGCTATAT GCAAGCGTTC GTGAAGTATG GACTACGCAA CCTGAAAGAA 720

GGGGCGAAGT TCCGTAAAGG GATTGAGAAG TTGTTAAGCG AATAATGAAA ATCTGACCGG 780

ATGTAACGGT TGATAAGAAA ATTATAACGG CAGTGAAGAT TCGTGGCGAA AGTAATTTGT 840

TGCGAATATT CCTGCCGTTG TTTTATATAA ACAATCAGGA TAACAACGAG TTAGCAATAG 900

GATTTTAGTC AAAGTTTTCC AGGATTTTCC TTGTTTCCAG AGCGGATTGG TAAGACAATT 960

AGCGTTTGAA TTTTTCGGGT TTAGCGCGAG TGGGTAACGC TCGTCACATC GTAGACATGC 1020

ATGCAGTGCT CTGGTAGCTG TAAAGCCAGG GGCGGTAGCG TGCATTAATA CCTCTATTAA 1080

TCAAACTGAG AGCCGCTTAT TTCACAGCAT GCTCTGAAGT AATATGGAAT AATAAAGTGA 1140

AGATACTTGT TACTGGTGGC GCAGGATTTA TTGGTTCTGC TGTAGTTCGT CACATTATAA 1200

ATAATACGCA GGATAGTGTT GTTAATGTCG ATAAATTAAC GTACGCCGGA AACCTGGAAT 1260

CACTTGCTGA TGTTTCTGAC TCTGAACGCT ATGTTTTTGA ACATGCTGAT ATTTGCGATG 1320

Orf1's is initial

CTGCTGCA AT GGCGCGGATT TTTGCTCAGC ATCAGCCGGA TGCAGTGATG CACCTGGCTG 1380

CTGAAAGCCA TGTGGATCGT TCAATTACAG GCCCTGCGGC ATTTATTGAA ACCAATATTG 1440

TTGGTACTTA TGTCCTTTTG GAAGCGGCTC GCAATTACTG GTCTGCTCTT GATGGCGACA 1500

AGAAAAATAG CTTCCGTTTT CATCATATTT CTACTGACGA AGTCTATGGT GATTTGCCTC 1560

ATCCTGACGA AGTAAATAAT AAAGAACAAT TACCCCTCTT TACTGAGACG ACAGCTTACG 1620

CGCCTAGTAG TCCTTATTCC GCATCAAAAG CATCCAGCGA TCATTTAGTC CGTGCGTGGA 1680

AACGTACCTA TGGTTTACCG ACCATTGTGA CTAACTGTTC GAATAACTAC GGTCCTTATC 1740

ACTTTCCGGA AAAATTGATT CCACTAGTAA TTCTTAATGC TCTGGAAGGT AAGGCATTAC 1800

CTATTTATGG CAAAGGGGAT CAAATTCGTG ACTGGCTGTA TGTTGAAGAT CATGCGCGTG 1860

CGTTATATAT CGTCGTAACC GAAGGTAAAG CGGGTGAAAC TTATAACATT GGTGGACACA 1920

ACGAAAAGAA AAACATCGAT GTAGTGCTCA CTATTTGTGA TTTGTTGGAT GAGATTGTAC 1980

CGAAAGAGAA ATCTTACCGT GAGCAAATTA CTTATGTTGC CGATCGCCCG GGACACGATC 2040

GCCGTTATGC GATTGATGCA GAGAAGATTA GCCGCGAATT GGGATGGAAA CCACAGGAAA 2100

CGTTTGAGAG CGGGATTCGG AAGACAGTGG AATGGTACCT GTCCAATACA AAATGGGTTG 2160

The termination of orf1

ATAATGTGAA AAGTGGTGCC TATCAATCGT GGATTGAAGA GAACTATGAG GGCCGCCAGT 2220

Orf2's is initial

AATGAATATC CTCCTTTTTG GCAAAACAGG GCAGGTAGGC TGGGAACTAC AGCGTGCTCT 2280

CGCACCTTTG GGTAATTTGA TTGCTCTTGA TGTTCACTCC ACTGATTATT GTGGTGATTT 2340

TAGTAATCCT GAAGGTGTGG CTGAAACCGT CAAAAAAATT CGCCCTGATG TTATTGTTAA 2400

TGCTGCGGCT CATACCGCAG TAGATAAGGC TGAGTCAGAA CCCGAATTTG CACAATTACT 2460

CAATGCGACC AGCGTTGAAT CAATTGCCAA AGCGGCTAAT GAAGTTGGGG CCTGGGTAAT 2520

TCATTACTCA ACTGACTACG TATTTCCGGG AACCGGTGAA ATACCATGGC TGGAGACGGA 2580

TGCAACCGCA CCGCTAAATG TTTACGGTGA AACCAAGTTA GCCGGAGAAA AAGCGTTACA 2640

GGAACATTGC GCGAAGCATC TTATTTTCCG TACCAGCTGG GTATACGCAG GTAAAGGAAA 2700

TAACTTCGCC AAAACGATGT TGCGTCTGGC AAAAGAGCGC GAAGAACTGG CTGTGATTAA 2760

TGATCAATTT GGTGCGCCAA CAGGTGCTGA GCTGCTGGCA GATTGTACGG CACATGCTAT 2820

TCGTGTGGCA CTGAATAAAC CAGAAGTCGC AGGCATGTAC CATCTGGTAG CCAGTGGTAC 2880

CACAACCTGG CACGATTATG CTGCGCTGGT TTTTGAAGAG GCGCGCAAAG CAGGTATTCC 2940

CCTTGCACTC AACAAGCTCA ACGCAGTACC AGCAACAGCC TATCCTACAC CAGCTCGTCG 3000

TCCACATAAC TCTCGCCTTA ATACAGAAAA ATTTCAGCAG AACTTTGCGC TTGTTTTGCC 3060

The termination of Orf2

TGACTGGCAG GTTGGCGTGA AACGAATGCT CAACGAATTA TTTACGACTA CAGCAATT TA 3120

Orf3's is initial

ATAGTTTTTG CATCTTGTTC GTGATGATGG AGCAAGATGA ATTAAAAGGA ATGATGAA AT 3180

GAAAACGCGT AAAGGTATTA TTTTAGCGGG TGGTTCTGGT ACACGTCTTT ATCCTGTGAC 3240

TATGGCTGTC AGTAAACAGC TATTACCTAT TTATGATAAG CCGATGATCT ATTACCCGCT 3300

CTCTACACTG ATGTTGGCGG GTATTCGCGA TATTCTGATT ATTAGTACGC CACAGGATAC 3360

TCCTCGTTTT CAACAACTGC TGGGTGACGG TAGCCAGTGG GGGCTAAATC TTCAGTACAA 3420

AGTGCAACCG ACTCCAGATG GGCTTGCGCA GGCGTTTATT ATCGGTGAAG AGTTTATCGG 3480

TGGTGATGAT TGTGCTTTGG TTCTTGGTGA TAATATCTTC TACGGCCACG ATCTGCCGAA 3540

GTTAATGGAT ACCGCTGTTG ACAAAGAAAG TGGTGCAACG GTATTTGCCT ATCACGTTAA 3600

TGATCCTGAA CGCTACGGTG TCGTTGAGTT TGATAAAAAC GGTACGGCAA TAAGCCTGGA 3660

AGAAAAACCG CTACAACCAA AAAGTAATTA TGCGGTAACC GGGCTTTATT TCTATGATAA 3720

CGACGTTGTC GAAATGGCGA AAAACCTTAA GCCTTCTGCC CGTGGTGAAC TGGAAATTAC 3780

CGATATTAAC CGTATTTATA TGGAACAGGG GCGTTTATCC GTTGCCATGA TGGGGCGTGG 3840

TTATGCGTGG CTGGACACGG GGACACATCA AAGCCTGATT GAGGCAAGCA ACTTCATTGC 3900

TACAATTGAA GAACGTCAGG GGTTGAAAGT TTCCTGCCCG GAAGAAATTG CTTACCGTAA 3960

AGGGTTTATT GATTCTGAGC AGGTGAAAAT ATTAGCTGAA CCACTGAAAA AAAATGCTTA 4020

The termination of Orf3

TGGTCAGTAT CTGCTAAATA TCATAAATAA TAAAATATCA TAATATTAAC TAGTTGCAGC 4080

TATATAAATA GGCTCGGAGA TTGCATTGAA GCTTGTTACC TCTCTTAATA AAAATATCTT 4140

CTATTTAGCT ATATTGCAAG GAAGTTATTT TATACTGCCA TTGTTAACAT TTCCTTATCT 4200

Orf4's is initial

TGTTAGAGTA TTAGGACCAT ACTCTTTTGG TCTATTAGGG TTTTGTCAAG CAACA ATGCA 4260

ATATCTTGTG ATGGTGACTG ATTATGGGTT TAATTGGTCA GCAACACAAG ATGTTTCGAA 4320

AAGTAAGAAT GACAAGGAAA AATTAACAAC AATATTTTGG AATGTCTTCT TTAGTAAGAT 4380

TATACTTGCA CTTTCGGCAA TTGTTATATT TTTCGGAATA ACATTTTTTA TTAAGGAGCT 4440

ATATTATGCT CAAGTGGTGT TAATATCATT TATTCCAATG GTGTTAGGTA ATATATTATA 4500

CCCTGTTTGG TTTTTCCAGG GATTAGAAAA AATGAAATGG ATTACATTAT GTAGTTTAAG 4560

TGCAAGATTA TTATTAATAC CATTAACTTT TTTGTTAGTA AAAAATCCAC AAGATATCTG 4620

GTTAGCCGCT TTGATTCAAG GCGGCGTTAA TTTTATAGCA GGTTTAATTG GATTTGCTAT 4680

TATTATAAAT AATAACTGGA TTGGGAAATT GGTATTTGAT TATGATGGTA TAATTTATTC 4740

ATTAAAAAGC GGATGGCATA TTTTCATTTC AACAGCTGCT ATTAGCTTAT ATACAACAAC 4800

AACTGTTGTT GTTCTTGGGT TTATATGTGG ACCTGTCTCT GTTGGCTACT TTAACGCTGC 4860

TAATACAATT AGAAATGCAG CCCAGAGTCT AATGAATCCT ATTTATCAAG CTGTTTATCC 4920

AAGAATTAAT TCATTGTTTG AAGAAAATTA TGCAAAAAGT ATGAATCTTC TGAAAAAATC 4980

ATTTAAGGGC GTATTATTCA TTTCGTTTTC AGGTTCATTA CTCCTGTGCA TACTTTCTCC 5040

AATAATAATA AGATATGGAG TCGGAGATAA ATATACTGAG TCAATTAGTA TTTTAAGAAT 5100

ATTAGCCTTT TTGCCATTTA TTATTTCTCT TAGTAATTTT TTTGGCATTC AAACCATGCT 5160

GACACATGGT TATAAAAGAC AATTTAGTAA AATACTGCTA ATTTGTGGTG TTATGCATGT 5220

GATAATCATT GCTCCTTTGA TCTTTTTTGC TGGGGCGTAT GGGGCTGCGA TGACGGTTTT 5280

AATTACAGAA ACCGTAGTTG CATCGTTGAT GTATCTTTTC TTAAAAAGAA AGAAAATAAA 5340

The termination Orf5's of Orf4 is initial

TTTGTTTAAA TAAAGAGGAA TATTA ATGGC TTTATTCATT GCTTGCCATA AACAAACTGA 5400

ATTACCTAAT CATGAGTGCT ATATCCCTAT TCATGCGGGT AAGAAAATAT CAGGGAAAAT 5460

GCTTGATCAA GTGTTGGGAG ATGATACAGG GAATAATATA TCATGTAAAA ATAAAAATTA 5520

TTGTGAGCTG ACCGTTATAT ATTGGCTTTG GAAAAATAAA CTTTTTAATG ATGATTATAT 5580

TGGCTTGGTT CATTATAGGA GGTATTTTGG AAATGTTTTA TCGAATTTTA AATTTAAAGA 5640

GGTGAGGATA TATGACAGAG CTAATATTTC TAATAAAATG AAACAATATG ATATAATTAT 5700

TCCGGTGAAA GAGAGTTTGA AGCTATCTGT TGTTGAACAC TACAGGAAAT TCCATAATGT 5760

CGAAGATCTA ATGTTGGCAA AAAAAATAGT TGATAAATTA TATCCTGATT ATAGTGTTGC 5820

ATTCAGAGAA CTTTTAGAAC AAAAAAAAAT ATCGCTTTAT AATATGTTTA TAATGAAGGC 5880

TGGGATTTTT GATTGTTATT GTGATTGGTT GTTTTCCATA TTAGATGAGC TGGAGAAAGA 5940

AATAAATTTA CGCAGATACG ATGAATATCA ATCACGTGTG TTCGGCTTTA TTGCTGAGCG 6000

ACTTCTGAAT GTATGGATTA ATAAAAATAA AGAAGTTTTT AAAGTCGGTG AGGAATTTGT 6060

AATTAACACT GATGAATGCA GTGGAAGAGG AGCTCAATTA ATTTCTCAAC TTAGAGCTAA 6120

The termination of the initial Orf5 of Orf6

GCTTAA ATGA ACATGACCAT TTTTATAGCT ATAATAATGC TTATGTATTC AGGATTATTA 6180

CTGAGTATTA ATAAAGATAT TAAATCGCCG TCGGCGATTC TATTCTTTAT CTGGGGGGGG 6240

CTGTTATTTT TGTCGGGAGT AAATGGAGAC ATAACTAACT ATACATTATG TATAATTTTA 6300

TTTTCATGTT TATCTTTTTC GGTAGGGGCT CTACTGGCAA AACCTTACGC TCCTATATTT 6360

GAACATGTAT TTTCCTATAA TATAAACAAT ACAAAAGCGT TAGTATTAAT TACAAATATT 6420

ATCTCGTTTG TTATTTTAAT CTATACTTTA ATAGTTTTTT TATATTATTA TAAGGGGGGC 6480

TTCACTGAAT CCTATATAAA TTCACGTACA GAAATTAATT ACGGAGATAA ATCGGGATTA 6540

ATAAAAATAT ATGGGTATAT ATACTATCTT TTATACCCAT TGGTATATGT ATGGAGCTTT 6600

TTATATTTTA AAAATAAATA TAAAAAGATT GAAGAAGGAG AGCGGGCCCG GTTACCATAT 6660

AATAATCGAA CATTTTATTT GTTTTTTTTA CTTCACTTTT TTTATGCGTT ATTATCAACA 6720

GCTAAATTAA GTTTTATTAC TAATTATACC AATAGTGTTT TTGCGATTAT TTTTTCAAAA 6780

ATATCTTTAA AATATTTACT TGTCACAGTG TCTGGTATTA TATTTTTTTT CTATTTATCT 6840

ATGTTGTTTT TAAATAAAAT TGACGACTCT TCAGGAGCAT TCGAAGCGTT AAAATATGGA 6900

TTGATGAACT ATAGCTTGGC AAACATTTTT GCATTAGATT CGGTTTTGCA AGGTAAAGCA 6960

GTAGTTATAG ATTGTAATGG AGATGCTACA TGTGGATTAG CTAATTTTAT ATCATATAAA 7020

GAGTATAAGA CAAATGTTTT TACAATATTT TATTCGCTAA CAAAATATTC TGATTTTTTG 7080

TACTCATTAA TTTTCTTTTT TGTCATAGGT TTCTTTCATT CATCTTTGTA CAATGTAGCA 7140

AAAAAAAATA AGAAAACGAT ATCAGTTGTC ATTTGCTCTA TTTTGTACTT CCCCTTGTTA 7200

TTTCAATTTT TTGACCAATT ATATATGTTG ATGTTCTATA TTTATGCAAT TGCAATGCTT 7260

The termination of the initial Orf6 of Orf7

TATGTGATAT GTTTTTGCTC AAGATTAACT CTAACTTTGA AGGG AGTT TA ACATGCTCGA 7320

ACATATTGTT GTATTTGATG TATGTGGCAC TCTTTATTTG TCGAATACAA CTTTTGATTA 7380

TGTTGTATTT GTACATAGGA AACAAAAAAA ATATACCAAG TTAATTAAAT GCTTATTATT 7440

ACGCTCCTTA TTTGGAAAGT TGCTACATAA ATTGCGTATA TGTTCCTTAC GAAAACATTT 7500

TTTGAAGACA TTAGAAGGAT ATGAAAAGTC TACTTTGGAA GAGTTGGCGA ATTTGTTTTA 7560

TGATGAATTT CTCATTTTTA AAGAAAAGAA TGAAGTTATG GCACTTTTAG AACGATATAA 7620

ACATGAGAAG ATCATTTTAA TGTCTGCTTC CGTTGATCCT GTCATAAATG TAATTGCTAA 7680

AAATTTGAAT GTTGAGGCAG TATCATCTAA ATTAGAATTT ATCGACAATA AATCAACAGG 7740

GTTATTAGAA TATAATCTTA CTGGTATAAA GCATTTACAA GTTAATAACC CTGTAAGTTT 7800

GATTGTCACT GATAATTTCA GTGATATTAA TCTTATTAAA CTTGCAGATA CAGCAGTGAT 7860

TATTTCGAAA GCCAAAGATA AAAATAAATG GAGAGTACTA TTAAATAGAT TTGGTGTCTC 7920

The termination of Orf7

CAAAGATAAA GTGAGGTTTA TG TGATATTG TATTTCCCAT TATCATATTT ATTCATTTCA 7980

AGATTGAAAA CATGGCATGA AAAAATTAGC TGGTTTATTA TATATCCAGG GTTTCTATTG 8040

Orf8's is initial

CTGTGTTGTT TTTTGTATAA TGGTAGTTTT GTATCTTTGC TTCTGTCAT T TATGATGACC 8100

ATGTCCATTT ATGAAATAGG CTACTTAGAC AATGACTATA GACCAATAAA TACAGAAAAA 8160

GCACCAACAA TAAGAGGTGG AGAGCTGCGT GATACAATTA AAAGTAAATT ATCAAGTATA 8220

ATTTTCATTA GAGTTGTTTA TTTTTTTGTA TCATTCGCGT TATTAATTTA TCAAAGTGAT 8280

ATCATTGCTC ATCACTATAC TGTTATCTAT TTTGTATTAT GTATAGGGTT TTTAATTATC 8340

TCCTTTTATT TGCATAATAC AATCCGGGAT AAGAGAAATA TAATCACTTA TTTTTCTTTG 8400

GTGTCATGTC GTTACATTAT TCCTTTGATA CCCACTGTCA CATGCAGTAT ATTTTTTGTT 8460

TTTGTCATAT TATTATTATT CCCTTTAATC AGAACATTAG AACACGCCTG TAAGGAGAAA 8520

TATGGTATTA AAAGGTTAAA GGATTTTATT ATTTACCCTG ATTTATTTAG GGTTAAATAT 8580

TACTTTTTCT TTTTTGATGC TGCTATTGTG TATGCATATA TATATCAGGA CTGGAAGATT 8640

GTATTATTGT CGGGATATTT TTTGTTTTAT CGATACATAA CCTTTAAAGT ATCCGATAAA 8700

The termination of the initial Orf8 of Orf9

AATTTTGTAA AAAGGACTCA TCATGGATCT TATAAGTGGA ATAAAAA ATG AATAGAGTAG 8760

CGGTGATAAT TGTACTATAT TATCCTGATG TTAGTAAGAT CAATGAAATG ATTGACTCAC 8820

TGGGAAAAAA ACAGCGCGAA ATATTACTTG TTGACAATAC ACCTGAGCTC CACTTAGAAA 8880

GTGATGTTTT GTGTAATAAC AACATTCATT ACTATCATTT AGGAGATAAC AAAGGCATTG 8940

CGTTTGCGCA GAACTATGGT TTCATTAAAG CTATCGGATT AGGAAAGCCC TTATTCTTTA 9000

CATTTGACCA AGATTCATGT ATATCAAAAG ATTATATATC ATCAATGTTA AAAGAGTATG 9060

AGTACGCTTG CTCTCTCCGG AGTAATATTG CTGCATTAGG TCCAACAATA ATAAATGAAC 9120

GTAATGGGAA AAAGTATGAT CGTGAAATAA AGCTGGGCTC CCAGATAAGT GAAACGATAC 9180

ATGATGTTGA AAGTATAATA TCTTCTGGTG CACTCATACC CCTAGAGGCA ATTATCAATA 9240

TTGGATTAAA TAAAGTATCT TGGTTTATCG ATTTAATTGA CATAGAATGG AGTTTCAGAG 9300

CTAGGAAAGA AGGCTGGAAT ATCCTAGTGA CAGATAAAGT CCTTATTTCT CACAATATAG 9360

GATCTAAAGA TATAAATATT ATGGGGATTA AAACATTTAC AGTTTGCAGT CCATTTCGTT 9420

TGTATTATGT TTATAGAAAT TGGTTATTAG CTCTGAGAGA GCCTGTATTT CCTTTAAGAT 9480

ATAAGTTAAA AAAATTGATA ACAATGCCTG TCCGAATCAT TATTTACGCT TTTTGTGAAC 9540

Orf10's is initial

GTGGAGGACA ACGATTAAAT TATATTTTTA AGGGGATAAA AG ATGGACTG CTTGGACGAA 9600

The termination of Orf9

AAGGATCATT TAAGGGA TAA TCATTCCTTA AAAATAGTTT TAACTGCTAA TAGTAGTTGG 9660

TATATATATA ATTTTAGACG AAGCACAATT AAGGCACTTA TTAATATAGG GTTTGATGTA 9720

TTAGTGGTTG TACCGGATTA TGAATATAAG GATTCTATTT TAAAATTCGG CGCTAAATTT 9780

GAGAAGGTTG ATCTTAAACC AAAATCTATT AATATTTTTA CTGAATGTAC ATCTCTAATA 9840

TCATATTATA AAATTATAAA AAAATTCAAT CCACAGGTTA TATTAACGTT TACACCCAAA 9900

GCTAATATAT ATTGTGGTTT GCTTGCTTAT AAATTTGACG CGAAAGTGAT ACCTAATATT 9960

TCTGGCCTTG GAAGTGCATT TGTTAATAGT AATACGATAT TAAGTCAAAT TGTTAAGCTA 10020

TTATATAAGA TAGCATTAAA GAAAGCAGCA TTCGTTTTTT TTCAAAACGA GGATGATCGT 10080

CAGTTATTGA TTAATTCTGG CTGCGTCAAT TATGAGAAAA CATGCCGAAT ATTTGGGTCT 10140

GGTGTTGATT TATCAAAATT TCTTCCCTCA AATAAGAATT TAAAGCAAAG ATCATGTGTG 10200

AAATTCATAC TTGTTGCTAG ACTTCTATAT ACCAAAGGCA TACTTCACTA TCTTAAGGCC 10260

GCAGAAATAA TAAAATCCAA ATATCCTAAT TGTTCATTTT CACTGTTAGG TGCATTTGAA 10320

AAAAATGAAA AGATAATCAC GAAGGAATTG ATTGAAGGTT TTTGTACGAG GGGTATAGTT 10380

AATTACTATG GGACTTCCGA TGACGTTGCT AGTATAATGA AGCATTATGA TGCAATAGTA 10440

TTACCATCAT TTTATAGAGA AGGTGTACCT AAAGCGTTGA TTGAAGGTGC TTCTAGTGGG 10500

TTAGCTATTT TGACAACAGA TAATGTTGGC TGTAGGGATA TGGTGCAAAA TGAAGTTAAT 10560

GGTTTTTTGT GTCAACCTAA TGATCTTGAT AGCTTAGTTG ACATTATTGA AAAGTATATT 10620

CTACTGACAG ACGAAATGAA AAACACAATG TCTATTAAGT CGAGAGAATT TGCTGAATCA 10680

CATTGTGATG AAAATAAGAT CATAGAGAAG TATACTAATA CAATTAATGA CGTTTTAAAT 10740

The termination Orf11's of Orf10 is initial

AGAAATCAC T AAAGGTTATA ATAAAT ATGA AAGTTCAAGA CACTGTTCTG GATGGAGTTA 10800

AAATTATTGA ACCGGTCGTA TATGGTGACA ACAGGGGTTT TTTTTTAGAA GTATATCAAA 10860

AACAACGTTA TCAAAATTTA TTAAATATAG AATTTGATTT CGTACAAGAT AACTATTCAC 10920

GATCAAGCAA AAATGTTTTA AGAGGATTAC ATTTCCAAAA AACGAAGCCA CAAGGTAAAC 10980

TTGTAAGAGT TGTGCGCGGC GAAGTTTTTG ATGTTGCCGT CGATATTAGA AAAGAATCAC 11040

CTAATTATGG CCAATGGATT GGTGTGTTAC TTTCTGAAAT GAATAAAAAA CAATTTTGGG 11100

TACCACCAGG TTTCGCACAT GGTTTTGTTG TATTATCTGA AATGGCCGAT TTTGAATATA 11160

AATGCACAGA ATATTATGAT CCTGAGGATG AGTGTTGTTT ATTATGGAAT GATCCAGAAC 11220

TCAATATTCA ATGGCCTTTA AGTAATCCTA TATTATCAGA TAAAGATATG AAAGGTTGCT 11280

The termination Orf12's of Orf11 is initial

TACTTAAAGA ACTT TAATTT TAACTAAGTT AGATGACATA GGAACCGCA A TGCAATATGA 11340

TTTCATAATT ATAGGTTCAG GTTTATTTGG TGCGGTTTGC GCTTATGAAT TAAATAAAAG 11400

AAATAAACGT GTGTTGGTCA TTGAGAAAAG GGATCATATT GGAGGGAATA TCTATACTGA 11460

AAAAGTTGAC GATATTCACA TACATAAGTA TGGTGCTCAT ATATTTCATA CAAACGATCA 11520

ATATATCTGG AATTATATAA ATAAATTCGC TTCTTTTAAT AGATTCACTA ATTCTCCATT 11580

AGCATATCAT AAAGGTAAAT TATATAACTT ACCTTTCAAT ATGAATACCT TTTATCAGTT 11640

ATGGGGGACA AAAACTCCTT CTGAAGCTCA AGATAAAATA GATTTACAAA GAAGTAAATA 11700

TTCTAACATA ACTCCCACAA ATTTAGAAGA ACAAGCTCTT TCCCTGGTAG GGGAAGATGT 11760

TTATGAAAAA CTAATTAAGG GGTATACGGA AAAACAATGG GGACGTTCAT GTTCTGAATT 11820

ACCGCCTTTT ATTATAAAAA GAATCCCAGT TCGATTCACT TTCGATAATA ATTATTTCAC 11880

CGATAGATTT CAGGGGGTTC CTATCGGAGG ATACACACAA ATAATTGATA AGATGCTTAA 11940

TGGTGTTGAT CTTCTGCTTA ATGTTGACTA TTTATCAGAA AAGGATAAGT ATAATTCTAT 12000

GGCGGAACGG ATTATATATA CTGGGCCTAT AGATGCTTAT TTTGATAAAA AGTTGGGAAC 12060

TTTAGAATAT AGATCCTTAA AGTTTATAAC AAAAAAAATT GAGGAGGCTA ATTATCAAGG 12120

CAATGCCGTA ATTAATTATA CTGAAAAAGA TATTCCTTTT ACTAGGATAA TTGAGCATAA 12180

GCATTTTGAT GACTCTTATG AAACTGAATA TACCTTTATA ACTGAAGAGT ACCCAATAGA 12240

GTGGAGTGAG GGTCAAGAGC CTTATTATCC CATTAATGAT AAAAAAAATA TGGATATATA 12300

TCATAAATAT AGAGAGCTTT CGCAAATGGA GAGTAAAGTA ATTTTCGGAG GACGCCTTGC 12360

TGAATATAAG TATTATGATA TGCACCAAGT TATCCGTTCA GCACTGAATT GTGTAAATAA 12420

The termination of Orf12

AATATTACAT GAT TAAGTTG TTTTGGCGTT ATTAATATTA TTAATTAAAT ATTTAATGTA 12480

GTAACCCCCT AACAGGAGTA AACAATGTCA AAGCAACAGA TCGGCGTCGT CGGTATGGCA 12540

GTGATGGGGC GCAACCTTGC GCTCAACATC GAAAGCCGTG GTTATACCGT CTCTATTTTC 12600

AACCGTTCCC GTGAGAAGAC GGAAGAAGTG ATTGCCGAGA ATCCAGGCAA GAAACTGGTT 12660

CCTTACTATA CGGTGAAAGA GTTTGTTGAA TCTCTGGAAA CGCCTCGTCG CATCCTGTTA 12720

ATGGTGAAAG CAGGTGCAGG CACGGATGCT GCTATTGATT CCCTCAAACC ATATCTCGAT 12780

AAAGGCGACA TCATCATTGA TGGTGGTAAC ACCTTCTTCC AGGACACCAT TCGTCGTAAT 12840

CGTGAGCTTT CTGCCGAAGG CTTTAACTTC ATTGGTACCG GTGTTTCCGG TGGTGAAGAA 12900

GGTGCGCTGA AAGGTCCTTC CATTATGCCT GGTGGGCAGA AAGAAGCCTA TGAACTGGTT 12960

GCACCGATCC TCACCAAAAT CGCCGCAGTG GCTGAAGACG GTGAGCCATG CGTTACCTAT 13020

ATTGGTGCCG ATGGCGCGGG TCACTATGTA AAAATGGTTC ACAACGGTAT TGAATACGGT 13080

GATATGCAGC TGATTGCTGA AGCCTACTCT TTGCTTAAAG GTGGCTTGAA CCTTTCCAAC 13140

GAAGAACTGG CGCAGACCTT TACCGAGTGG AATAACGGTG AACTGAGCAG CTACCTGATC 13200

GACATCACCA AAGATATCTT CACCAAAAAA GATG 13234

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 (2)

1, a kind of oligonucleotide of the O-antigen-specific to Shigella bogdii 2 types is right, it is characterized in that described oligonucleotide is to being: the Nucleotide of 4273 to 4290 bases among the SEQ ID NO:1 and the Nucleotide of 5050 to 5072 bases; The Nucleotide of 4802 to 4819 bases among the SEQ ID NO:1 and the Nucleotide of 5286 to 5303 bases; The Nucleotide of 6253 to 6270 bases among the SEQ ID NO:1 and the Nucleotide of 6705 to 6722 bases; Or the Nucleotide of the Nucleotide of 6155 to 6172 bases among the SEQ ID NO:1 and 6627 to 6644 bases.

2, the right application of the described oligonucleotide of claim 1 is characterized in that it is used for PCR as primer, perhaps is used for hybridization and fluoroscopic examination as probe or is used to make gene chip or microarray confession detection Shigella bogdii 2 types.