CN1256437C - Nucleotide peculiar to 0-antigen of 12 type Baoshi Sh.dysenterae - Google Patents

Abstract

The present invention provides a specific nucleotide for an O-antigen of Shigella bodyii 12. The specific nucleotide is the nucleotide complete sequence of gene clusters in Shigella bodyii 12 for controlling the synthesis of an O-antigen, such as a separated nucleotide with the total length of 15278 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 oligonucleotides of oligosaccharide unit processing genes such as wzx genes or genes with the similar functions of wzx and wzy genes or genes with the similar functions of wzy in the O-antigen gene clusters of Shigella bodyii 12. The present invention verifies that the oligonucleotide has high specificity to all of the O-antigen genes of Shigella bodyii 12 through PCR. The present invention also discloses a method for detecting and identifying Shigella bodyii 12 in human bodies and environment by the oligonucleotide of the present invention.

Description

Nucleotide to the O-antigen-specific of Shigella bogdii 12 types

Technical field

The present invention relates to the complete nucleotide sequence of control 0-antigen synthetic gene cluster in Shigella bogdii 12 types (Shigella bodyii 12), particularly relate in Shigella bogdii 12 types oligonucleotide in the control O-antigen synthetic gene cluster, can utilize these oligonucleotide of O-antigen-specific Shigella bogdii 12 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:K1 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 " .Irends 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 sane 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 0-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 syntha se 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-UremicSvndrome 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 12 types.It is the Nucleotide in the O-antigen gene bunch of Shigella bogdii 12 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 12 types.

A time purpose of the present invention has provided the gene of the O-antigen gene bunch that constitutes Shigella bogdii 12 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, manB, manC.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 12 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 12 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 12 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 12 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 12 types of Shigella bogdii 12 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 12 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 12 types it is the isolating Nucleotide shown in SEQ ID NO:1,15278 bases of total length; The base that perhaps has one or more insertions, disappearance or replacement keeps the Nucleotide of the SEQ IDNO:1 of described isolating functional nucleotide simultaneously.

The Nucleotide of aforesaid O-antigen-specific to Shigella bogdii 12 types, it is characterized in that, comprising 12 genome Chengdu of called after rmlB, rmlD, IS630, rmlA, rmlC, orf5, wzx, wzy, orf8, orf9, orf10, manC, manB between galF gene and gnd gene.

The Nucleotide of aforesaid 0-antigen-specific to Shigella bogdii 12 types is characterized in that the gene that has high degree of specificity in the 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 6246 to 7580 bases among the SEQ ID NO:1; Pol gene is the Nucleotide of 7561 to 8700 bases among the SEQ ID NO:1.Orf5 is the Nucleotide of 5179 to 6270 bases among the SEQ ID NO:1; Orf8 is the Nucleotide of 8717 to 9538 bases among the SEQ ID NO:1; Orf9 is the Nucleotide of 9535 to 10653 bases among the SEQ ID NO:1; Orf10 is the Nucleotide of 10650 to 11471 bases among the SEQ ID NO:1.

The Nucleotide of aforesaid O-antigen-specific to Shigella bogdii 12 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 12 types, the oligonucleotide that it is characterized in that the described wzx of coming from gene is to being: the Nucleotide of 6435 to 6452 bases among the SEQ ID NO:1 and the Nucleotide of 7094 to 7111 bases, the Nucleotide of 6709 to 6726 bases among the SEQ ID NO:1 and the Nucleotide of 7280 to 7297 bases; The oligonucleotide that comes from the wzy gene is to being: the Nucleotide of 7679 to 7696 bases among the SEQ ID NO:1 and the Nucleotide of 8511 to 8528 bases, the Nucleotide of 8270 to 8287 bases among the SEQ ID NO:1 and the Nucleotide of 8667 to 8684 bases.

The application of the Nucleotide of aforesaid O-antigen-specific to Shigella bogdii 12 types in detecting 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 Shigella bogdii 12 types is providing the O-antigen of expressing Shigella bogdii 12 types by inserting to express, and the application in the preparation bacterial vaccine.

The application of the Nucleotide of aforesaid O-antigen-specific to Shigella bogdii 12 types 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 Shigella bogdii 12 is characterized in that, comprises the steps:

(1) genomic extraction: in substratum, cultivate Shigella bogdii 12 types, centrifugal collecting cell; The genomic dna that obtains detects by agarose gel electrophoresis;

(2) by the O-antigen gene in pcr amplification Shigella bogdii 12 types bunch: with the genome of Shigella bogdii 12 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 Shigella bogdii 12 types;

(6) screening of specific gene: at wzx, the wzy gene design primer in the O-antigen gene of Shigella bogdii 12 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 Shigella bogdii 12 types;

(7) detection of primer sensitivity: cultivate Shigella bogdii 12, 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 Shigella bogdii 12.

The separation method of the Nucleotide of aforesaid O-antigen-specific to Shigella bogdii 12 types is characterized in that, comprises the steps:

(1) genomic extraction: 37 ℃ of incubated overnight Shigella bogdii 12 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 12 types bunch: with the genome of Shigella bogdii 12 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 GGGATC 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 ATT AAG 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 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 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 Shigella bogdii 12 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 ABI 377 type automatic dna sequencers 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 12 types, the quality of sequence is mainly guaranteed by two aspects: 1) genome of Shigella bogdii 12 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 12 types bunch, with American National biotechnology information science center (The NationalCenter for Biotechnology Information, NCBI) orffinder finds gene, find 8 open reading frames, 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 12 types at last;

(6) screening of specific gene: at wzx, the wzy gene design primer in the O-antigen gene of Shigella bogdii 12 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 Shigella bogdii 12 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 Shigella bogdii 12 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 Shigella bogdiis, 12 types is inoculated in the triangular flask of 20nl 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 l 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 6435 to 6452 bases among the SEQ ID NO:1 and the Nucleotide of 7094 to 7111 bases, the Nucleotide of 6709 to 6726 bases among the SEQ ID NO:1 and the Nucleotide of 7280 to 7297 bases, the Nucleotide of 7679 to 7696 bases among the SEQ ID NO:1 and the Nucleotide of 8511 to 8528 bases, the Nucleotide of 8270 to 8287 bases among the SEQ ID NO:1 and the Nucleotide of 8667 to 8684 bases, carry out the PCR reaction, the PCR reaction system is as follows: MQ:15.7 μ l, Mg2+:2.5 μ l, Buffer:2.5 μ l, dNTP:1 μ l, Taq enzyme: 0.31, 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 12 types of the Shigella bogdii in the pork filling when using aforesaid method.

Just, first aspect of the present invention provides the full length nucleotide sequence of the O-antigen gene bunch of Shigella bogdii 12 types, its complete sequence shown in SEQ ID NO:1,15278 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 12 types by method of the present invention, as described in Table 3, it comprises called after rmlB, rmlD, IS630, rmlA, rmlC, orf5, wzx, wzy, orf8, orf9, orf10, manC, 12 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 Shigella bogdii 12 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, orf9orf10; Special sugared synthesis path gene in the bacterial polysaccharides antigen comprises rmlB, rmlD, rmlA, rmlC, manB, manC 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, pol gene and glycosyltransferase 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 12 types.

The 3rd aspect of the present invention, wzy gene in the O-antigen gene bunch that comes from Shigella bogdii 12 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 12 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 1 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 12 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 12 types and their O-antigen.

The separation method of the Nucleotide of described O-antigen-specific to Shigella bogdii 12 types comprises the steps: 1) genomic extraction; 2) the O-antigen gene in pcr amplification Shigella bogdii 12 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 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 6246 to 7580 bases from SEQ ID NO:1), the oligonucleotide of wzy gene (nucleotide position is 7561 to 8700 bases from SEQ ID NO:1) all is a high special to Shigella bogdii 12 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 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 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 12 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, orfl0 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 12 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 12 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 12 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, or 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 12 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 12 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 12 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 12 types bunch

With the genome of Shigella bogdii 12 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 ATT AAG 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-inferior, 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 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 Shigella bogdii 12 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 12 types obtained.The quality of sequence is mainly guaranteed by two aspects: 1) genome of Shigella bogdii 12 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 12 types bunch, with American National biotechnology information science center (The National Center forBiotechnology Information, NCBI) orffinder finds gene, find the reading frame of 12 openings, with the genetic comparison among the blast groupware and the GenBank to send out. the function of existing these open reading frames also determines that what gene they are, 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 12 types at last, as shown in table 3.

By retrieving and relatively, finding the dTDP-D-glucose-4 of orf1 and Shigella boydii, the 6-desaturase has 98% homogeny in 361 amino acid, 99% 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 and Shigella boydii-4-rhamnosyl reductase enzyme has 96% homogeny, 97% 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 and Shigella boydii has 96% homogeny, 99% similarity in 292 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 and Shigella boydii, the 5-mutase has 85% homogeny in 180 amino acid, 88% 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.Seek conservative functional domain and find that Orf5 is similar to the glycosyltransferase family of pfam00534 in Genbank, the E value is 1.8 * e ^-19Comparison shows that by blast the glycosyltransferase B of seminose of orf5 and Campylobacter fetus has 31% homogeny respectively in 236 amino acid, 51% similarity, the homology that has between them is described, therefore infer that orf5 is a glycosyltransferase gene, temporarily called after orf5.The O-antigen transhipment enzyme of 0rf6 and Chromobacterium violaceum ATCC 12472 has 27% homogeny, 49% similarity in 386 amino acid whose sequences.And algorithm [Eisenberg by people such as Eisenberg, D, Schwarz, E.et al (1984) .Analysis of membrane and surfaceprotein sequences with the hydrophobic moment plot.J.Mol.Biol.179:125-142] find that orf6 has 10 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 orf6 is the wzx gene, called after wzx.Blast comparison shows that orf7 and many Wzy albumen with, for example and Salmonella enterica in 399 amino acid, 29% homogeny is arranged, 51% similarity.Learn that by the Eisenberg algorithm orf7 has 10 potential transmembrane domains in addition, similar secondary structure is arranged, have the feature of typical O-antigen polysaccharase, so determine that orf7 is the wzy gene, called after wzy to other O-antigen polysaccharase.The WbbB albumen of Orf8 and Escherichia coli O7 has 43% homogeny in 197 amino acid, 63% similarity, WbbB albumen are glycosyltransferases, and rhamnosyl is transferred on the seminose, infer that orf8 is a glycosyltransferase gene, temporarily called after orf8.Seek conservative functional domain and find that Orf9 is similar to the glycosyltransferase family of pfam00534 in Genbank, the E value is 2.7 * e ^-22And the WbbC albumen of orf9 and Escherichia coli 07 has 53% homogeny respectively in 345 amino acid, 71% similarity, and illustrating has higher homology between them, therefore infer that orf9 also is a glycosyltransferase gene, temporarily called after orf9.Seek conservative functional domain and find that Orf10 is similar to the glycosyltransferase family of pfam00534 in Genbank, the E value is 5.3 * e ^-23And the WbbD albumen of orf10 and Escherichia coli 07 has 55% homogeny respectively in 272 amino acid, 73% similarity, and illustrating has higher homology between them, therefore infer that orf10 also is a glycosyltransferase gene, temporarily called after orf10.Relatively find by blast, two synthase genes of the synthetic seminose of orf11 and orf12 and many bacterium have very high homogeny (identity), wherein: the ManC of orf11 and Shigella bogdii has 58% homogeny respectively in 475 amino acid, 76% similarity; The ManB of Orf12 and Shigella bogdii has 98% homogeny in 453 amino acid, 98% similarity; Illustrate that they all have the homology of height, can determine that orf11, orf12 are respectively manC, the manB gene of synthetic seminose, therefore difference called after manC, manB.

Embodiment 6: the screening of specific gene

At wzx, wzy gene design primer in the O-antigen gene of Shigella bogdii 12 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 12 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 12-19 bacterium, and 13 groups altogether, all list in the table in their source.

The genomic dna that contains Shigella bogdii 12 types in the 10th group is as positive control.In the 13rd group is the genomic dna that does not contain Shigella bogdii 12 types, as negative 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 10th 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 12 types and 0-antigen thereof all are high specials.

At last, from Shigella bogdii 12 types, screen gene by PCR: wzx, wzy gene to the O-antigen high special of Shigella bogdii 12 types.And the oligonucleotide of these intragenic any one section 10-20nt is special to the O-antigen of Shigella bogdii 12 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 12 types.These all oligonucleotide all can be used for Shigella bogdii 12 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 Shigella bogdiis, 12 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 LR 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 6435 to 6452 bases among the SEQ ID NO:1 and the Nucleotide of 7094 to 7111 bases, the Nucleotide of 6709 to 6726 bases among the SEQ ID NO:1 and the Nucleotide of 7280 to 7297 bases, the Nucleotide of 7679 to 7696 bases among the SEQ ID NO:1 and the Nucleotide of 8511 to 8528 bases, the Nucleotide of 8270 to 8287 bases among the SEQ ID NO:1 and the Nucleotide of 8667 to 8684 bases, carry out the PCR reaction, the PCR reaction system is as follows: MQ:5.7 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 12 types of the Shigella bogdii in the pork filling 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 B12 of the present invention can be applied to set up recombiant vaccine.

Nucleotide sequence (shown in the SEQ IDNO:1) according to the O-antigen-specific to Shigella bogdii 12 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 Shigella bogdii 12 types, has listed the structure of the O-antigen gene bunch of Shigella bogdii 12 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 12 types, in table, listed the accurate position of all open reading frame in complete sequence in the O-antigen gene bunch of Shigella bogdii 12 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

SEQ ID NO:1 sequence (SEQUENCE LISTING)

＜110〉Tianjin Biochip Technology Co., Ltd

＜120〉to the Nucleotide of the O-antigen-specific of Shigella bogdii 12 types

<160>1

<170>PatentIn version 3.1

<210>1

<211>15278

<212>DNA

<213>Shigella boydii

<400>1

atccaactaa ctgatgctat tgccgaactg gcgaaaaaac agtccgttga tgccatgctg 60

atgaccggcg acagttacga ctgcggtaaa aaaatgggct atatgcaggc gttcgtgaag 120

tatggattac gcaacctgaa agaaggggcg aagttccgca aaggcattga gaagttgtta 180

agcgaataat gaaaatctga ccggatgtaa cggttgataa gaaaattata acggcagtga 240

agattcgtag caaaagtaat ttgttgcgaa tattcctgcc gttgttttat ataaatattc 300

agaataacaa cgagttagca ataggatttt agtcaaagtt ttccaggatt ttccttgttt 360

ccagagcgga ttggtaagac aattagcgtt tgaatttttc gagctaagcg cgagtgggga 420

acgctcgcta catcgtaggc atgcagtgtt ctggtagctg taaagccagg ggcggtagog 480

tgcattaata cctctattaa tcaaactgag agccgctaat ttcacagcat gctctgaagt 540

aatatggaat aaattaagtg aaaatacttg ttactggtgg cgcaggattt attggttctg 600

ctgtagttcg tcacattata aataatacgc aggatagtgt tgttaatgtc gataaattaa 660

cgtacgccgg aaacctggaa tcacttgctg atgtttctga ttctgaacgc tatgtttttg 720

aacatacaga tatttgcgat gcagctgcaa tggcacggat ttttgctcag catcagccgg 780

atgcagtgat gcacctggct gctgaaagcc atgttgaccg ttcaattaca ggtcctgcgg 840

catttattga aaccaatatt gttggaactt atgtcctttt agaagccgct cgcaattact 900

ggtctgctct tgatagcgac aagaaaaata gcttccgttt tcatcatatt tctactgacg 960

aagtctatgg tgatttgcct catcctgacg aggtaaataa tacagaagaa ctacccttat 1020

ttactgagac gacagcttac gcgccaagca gcccttattc cgcatccaaa gcatccagcg 1080

atcatttagt ccgcgcgtgg aaacgtacct atggtttacc gaccattgtg actaattgct 1140

ctaacaatta tggtccttat catttcccgg aaaagcttat tccattggtt attcttaatg 1200

ctctggaaga taaagcatta cctatttatg gtaaagggga tcaaattcgc gattggctgt 1260

atgttgaaga tcatgcgcgt gcgttatata ccgtcgtaac cgaaggtaaa gcgggtgaaa 1320

cttataacat tggtgggcac aacgaaaaga aaaacataga tgtagtgctc actatttgtg 1380

atttgctgga tgagattgta ccgaaagaga aatcttatcg tgagcaaatc acttatgttg 1440

ccgatcgtcc gggacacgat cgccgttatg ccattgatgc tgataagatt ggtcgtgaat 1500

tgggatggaa accacaggaa acgtttgaga gcggcattcg taaaacggtg gaatggtacc 1560

tgtccaatac aaaatgggtt gataatgtga aaagtggtgc ctatcaatcg tggattgaac 1620

agaactatga gggccgcaag taatgaatat cctccttttc ggcaaaacag ggcaggtagg 1680

ttgggaacta cagcgtgccc tggctcctct gggtaatttg attgctcttg atgttcactc 1740

cactgattat tgtggtgatt ttagtaatcc tgaaggtgta ggtgaaaccg taagaagcat 1800

tcggcctgat attattgtca acgcagccgc tcacaccgca gttgacaaag cagaatcaga 1860

accggagttt gcacaattac ttaacgcgac aagtgtcgaa gcgattgcga aagcagccaa 1920

tgaagtcggt gcctgggtta ttcactactc tactgactat gtatttccgg gaaccggtga 1980

aataccatgg caggagtcgg atgcaaccgc accgctaaat gtttacggtg aaaccaagtt 2040

agccggagaa aaagcattac aagagcattg tgtgaagcac cttattttcc ggaccagctg 2100

ggtctatgca ggtaaaggaa ataacttcgc caaaacgatg ttacgactgg caaaagagcg 2160

tgaagaattg gccgttatta acgatcagtt tggtgcgcca acaggtgctg agctgctggc 2220

tgattgtaca gcacatgcca ttcgtgtcgc actgaataaa ccggaagtcg caggcttgta 2280

ccatttggta gccagtggta ccacaacctg gcacgattat gctgcgctgg tttttgaaga 2340

ggcgtgcaaa gcaggcattc ccctcgcact caacaagctc aacgctgtac caacaacagc 2400

ctatcctaca ccagcacgtc gtccacataa ctctcgtctt aatacagaaa aatttcagca 2460

gaattttgcg cttgtattgc cagactggca ggttggcgtg aaacgcatgc tcaacgaatt 2520

atttacgact accgcaattt aatagttttt gcatcttgtt cgtgatggtg gagcaagatg 2580

aattaaatag ctgcgcttaa taccgctaca cttttgccag tccatgtttg cctccgggga 2640

atgggctgac ggtttccata aaatggcgaa cttttttcaa cagttgccac attgagcggc 2700

actgatgatt acgcgttatt gtgtcgtgaa gtgcctgcca tagccgttca acatgattca 2760

cccatggcga gtaaaccggc tgataaatga ccctgaactt cgggttctcc ttcagccagc 2820

tctgtgtttc ccggtttttg tggataatgt agttgtccac gatcagcgtg atggttttcg 2880

cccgacggta tgtcgcttta agccgcttca gcaggctgat gaacagcgcc gaacttttgc 2940

tgttgccgcc cacatagctg actttacctg tcccgctgtg cagcgctccg gccagataat 3000

atttttcatt ctgtcccggc gtgaccaccc gtttttgctg tccgcgcagt tgccagtctg 3060

caccgatttt gggattaaga tggatatcca cttcatcttc ataaaagacc ggatgttctg 3120

cgctgcattc gtccagtgct ttatggattg ctgccatctt ttcatcttta tgcgggtcac 3180

ggatacgcag agttggcgca gcccttcgcc acacaatccc cgcagacggc aaccagcggc 3240

gaacggttcc ggcatttaac tggcaaccgg ttatctcatt gatttttatt gccagcagtt 3300

ctgtactcca gcgtgaacgc tggtagccaa agtcgccggg agaatgtttt accagctcac 3360

gtaacagtgt gcagatatgc tcaaacggcc agcgacgggc acgcccggca ggtaatgatt 3420

tcagtccctc aacacccgac tgcgtgaacc agttaatcca gcgtccaaca gaggagcggg 3480

cgcagcagag cgttctggca acgtcgctga cacggtcgcc ccggtgcagc atcagcatgg 3540

cagtcagtct gcgggcataa tttctatcgt gtgttttatg gatggctttc tgcatcaggc 3600

gtcgttcgtc acgggaaatt ggtgctatga tcggcattgc tcagtccggt tggtgatttg 3660

ttttgatttg gcgattgatc agatcgcaca atccgggcta agttccctca aagtgatcta 3720

ctattccgcg cagctattta aaaggaatga tgaaatgaaa acgcgtaaag gtattatttt 3780

agcgggggga tctggtactc gtctttatcc tgtcactatg gctgtcagta aacagctgtt 3840

accgatttat gataaaccga tgatttatta tccgctatct acactgatgt tagcgggtat 3900

tcgcgatatt ctgattatca gtacgccaca ggatactcct cgttttcaac aactgctggg 3960

tgatggtagc cagtggggcc ttaatcttca gtataaagta caacctagtc cagatggttt 4020

ggcacaagct ttcattattg gagaagattt tattggtagg gatgattgcg cattaattct 4080

aggtgataat atcttttacg gtcatgacct accaaaatta atggatacag ctgttaatag 4140

agagagtggt gcaacggtat ttgcctatca cgttaatgat ccagaacgtt acggtgtggt 4200

ggaatttgat aaaaacggca cggcaataag cctggaagaa aaaccgcttc aaccgaaaag 4260

caactatgca gttacagggc tttatttcta tgataatgac gtcgtggaaa tggcgaaaaa 4320

tcttaagcct tctgcccgtg gtgaactgga aattaccgat attaatcgta tctatatgga 4380

acgggggcgt ttgtctgttg ccatgatggg ccgtggttat gcatggctgg acacggggac 4440

acatcaaagc ctgattgagg caagcaactt catcgcaaca attgaagagc gtcaggggtt 4500

aaaagtttcc tgcccggaag aaattgctta ccgtaaaggg tttattgatt ctgagcaggt 4560

gaaagtatta gctgatccgc tgaaaaaaaa tgcttatggt cagtatctgc ttaaaatgat 4620

taaaggttat taataaaatg aacgtaatta aaacagaaat tccagatgta ctgatttttg 4680

aaccgaaagt ttttggtgat gagcgtggtt tcttttttga gagctttaac cagaaggttt 4740

ttgaggaagc tgtaggccgc aaagttgaat ttgtccagga taaccattcg aagtcttgta 4800

aaggtgtttt acgcggactg cattatcaat tagaacctta tgcgcaaggg aaattggtgc 4860

gctgcgttgt tggtgaagtt tttgacgtag cggttgatat tcgtaaatcg tcacctactt 4920

ttggcaagtg ggttggggtg aatttatctg ctgagaataa gcgccagttg tggatcccgg 4980

aggggtttgc tcatggtttt ttggtgctga gtgagacggc ggagtttgtt tataagacga 5040

ctcagtatta tcatcctgaa tgtgaacgag ggatcagatg ggatgatgaa aatatagggg 5100

tagattggcc tctgtctaca gatttaatac tatcttttaa agataccaag catccaaaat 5160

tcaacggtat tttataaaat gaaaaattat agtgaattat taataatcca tgaatatgga 5220

gaaccatctc attaccaagg aataatagag attgctcagg agagaagaat taaggtccgt 5280

tttttagaat tcagtatttt acataatatt tatgctgcaa ttaaaaaaaa gaaaataaat 5340

gccgttataa aggcgatcaa agattatttg atattgatga tatactcatt ctttccaaga 5400

atgaataatg gaaagttggt tgttataggc atggccccgt tagataaata tgttttttta 5460

ataaataaat tattgaaggg acaagattat atttatcaca catcatggct ttattgggat 5520

ggcagtgact atcctaaaaa aacatataaa aatcctgagg tattaattga tgagtggaag 5580

acttttttaa ataatgctaa agcggttgca tgtgtaacac ctgattctgc gcatagttta 5640

atttcaaact ttaatatgaa agataaaaca ttcattgttt atcattcgta tgactcaaat 5700

attttctatc cttcgccaag gcaagaacga aaaaaaacat taacgatatg ttacttaggg 5760

cgacttgaaa aaaacaaagg aattgaaaat cttatttata ttattgaaaa taatgataat 5820

gttaatttta aaataatggg cagcggaagt tactcaagca aaataactga gttaagcagg 5880

tcaaagacta atgttgaata cttagggtac ctatcggata aaaaattaat agcagaacaa 5940

ttgcggcaaa ctgatattat attacttcca tcaattaaaa ttaacgggtg ggaagaatta 6000

tttggaatcg cattaataga gtcaatggct tgtggggtag tacctattac tactgatcac 6060

aaaggtccta ggaccatact aaagaataaa ttattagcag agctcatttt cagtgagaat 6120

gaatttgttg agtcggcaca gaagatcatc acagatttaa ataatgatga atttttatta 6180

caaaagtaca aggattgtgc gattgctgag gcaaagtttt atgaaaagaa aaatatatca 6240

aataaatgga gggaagttat tcacaaataa tttatgacaa catttttaat gtctatcttt 6300

atgagttgaa aataatggcc ataattaatg gtgtttttaa aaatattgct ttgcttattt 6360

ctggaaccgc agtagctcaa ttaattaatt tttgttttat tcctttgatt acaagattgt 6420

atggtcctga gtcttatgga atgatgggaa gcttaatggc gtttattaac attatgattc 6480

cgatagcttc attaagtttt ccaattgcaa ttgtgttacc aaagtctgat aaaaccgcta 6540

tttatattgc ttaccttagc atttgtatag gtttggtggg tgcaggagtt ctattaatcg 6600

taataggggg attggaatat ttagatggaa gtttcagtca ataccctgaa tggtatatat 6660

tactattacc atttatcatg acattcctac ctctacagca aagtggacag caatggctaa 6720

taaggaaaaa acagtttaaa gaaatagcaa aagtatctgt cagtcaagca ttaattatga 6780

atggtttgaa ggtaatcggg ggagggattt taccaacaca aatcatgata atatatacaa 6840

caggtgcagg agtaattttt caatgcttac agttctttta tcgatcttat aagaaagggc 6900

ttggaattcc tttatcaaaa ataaaaaaga attattttat aagggttgcg cgcggctatt 6960

atgattttcc gctatataga actccgcaag tattaatcaa tgcgttgtct caaaatttgc 7020

cgatattttt gttggggtat tattttgatt ctaaaatagt aggcttttat gtgcttgctc 7080

aaacgttact aactgctcca gtgactttgt taagcacttc gataagtaat gtatattatc 7140

ctacaatcgc agagaaattt acccaaaaaa aacctattta taatttctta agaaaaacaa 7200

tttggggact gtttattgta tccttgttta tctatttgcc atgtatattg ttatctggca 7260

ttgtatttaa aattattttt ggaggtgggt ggattacgtc tagcattttt tttcaatgga 7320

tggctattta ttgcattttt tggttaagtg ctagacccgc gatggatact ataccgccat 7380

tgaaaattca aaactacttt cttctatatg aagttatttc ttttttattg aggattttaa 7440

gcctaatgtt aggctattat ttatataaag atgcgataat ttgtattatt atgttttcat 7500

caaccaatgc agtcacatat gctgtcttga tagtttatat tttatttcag gcaaaagtaa 7560

atgatagaaa atatacataa ttcactacaa gagttaaaga gtgatcatat taataaatat 7620

aatggctcaa acttttatta ctatgtctca gtatttggtc tattacttat ctcatttcga 7680

cttcagatac cctcacttat atttattata ggtgcgatta ttataagagg tactactttt 7740

aagcaaaata tatctggttg gctttggttc ggaagtatta cttttttttt tggaatatat 7800

ctttttttgg ggttggataa ttatgaaaga ggtgctgata agtttttatc acgaacttta 7860

gtaatatatc tttatttcgt ttttttttac gggttgggat gtgttagaac tcaaaataga 7920

gaacgactct tatctttttt ttcattattt agcgtgattt atgtctatgc tgtctgtatc 7980

tatagtaaga ttagcggtta tccaggatat aatggaatat atgatgtgtt caatgggaat 8040

gatgaaaatt ctccattata tgcattacag ttgattctat ttacaatttt atatcttcac 8100

ctcaatgcag ggaaaataaa taaaatatgg gttgggttgt taattatcac atctttttat 8160

tttagtgtta tttatcttgg ctctcgagca tccttcttat tattaatttt ttatttcatt 8220

tttgtgatct ttaaacataa aactaacttg ttaatatctt tcattatttt tatcccgttc 8280

atagttacat tatttattta tataaatgac tttaattttt taaattattt agattttgga 8340

gggtttgaaa atcgaggact aagcagtcct cgatttagta tgcttgaata tggtttaaag 8400

aatttcatga attatccatg gggaggttta attgttcatg gtgaaggtta tgatggtatc 8460

tggctccata atatgttatt agatatagtt agggtatcag gttactatac tatgtttgta 8520

tggctgttca ttttattaat ggcaggttta gttattctga aacgtgaaaa attttatttt 8580

ccaatatttc taattctaaa tatagcttta atgcaagatt tagcatttga tggttttttt 8640

aacataatgg ctctggagtt ttttttattg ggtgtagctg tacctggttt aagaaaataa 8700

aggaaaataa aatataatga atatgtatat tgtggtttgt gtatataccc tgcccccagc 8760

gtatataaaa aaatggttta agttacattt gcataatact aaatataaac ttattattgt 8820

cgataacaac ttaagaagac aaataacaga tccaactgtt attattggca caaatactct 8880

taatgaattc tcagcataca atgaagggct tcagctacta aaaaaagaat ttgaagatga 8940

atatgacata atattgatgt tgaatgatac tttgttcact aggcataatg cgaaattttt 9000

cttaaaacat ttattaaagt ataaaaatac agttgctcgc ttatcaatcc cggctatcgg 9060

tggaagaata gatccatata ataatatctg ttatagaaac ccatggagca atgacattgg 9120

ttatatatct tcattttgta taataatgaa taaaccagca agagatctat atttgaagtt 9180

actatctgat atatccccaa cttttccatt tgttgattca gttactgagc ttttcaactg 9240

gagtactcat atagacagaa gatttaaaga atttgtgatc agtcatctga tagatacaga 9300

cacagctact gtgtggtacc aatccaagaa taatattaaa aatatagaaa gattgaatgt 9360

taaaggtaag tgtgtttttc ttgaacatta tgtatcagga aatatttcta aacatggagt 9420

attggtttca atctttccca cgtggaagca gaaaactcag cattttattt atgagcaaat 9480

cgcaaaaatg gaaagaaaat tattatcgat actaaatttt aaagttggtt caaaatgaaa 9540

aaaagaattt taataaatct attgagtttt tctgaagaaa aatattacgg tgtgggagtt 9600

tatttcagag atgttgctat taaaagtatt aacagggtta ttgaagaact agaatgcgaa 9660

atatgtattt tatatttggc acatattccg atacaaaaga tgtttacttt ttcaaaaaat 9720

gaaaacataa aatatatacc tgtaaagaaa attaatagca aactcacaag agttttctac 9780

gaacaaataa tgttaccttt gtatcataga gattatgatg tgatttatag tcctaataat 9840

ataaatccat taatattagc tttttcaaaa aaatctataa taactatcca cgatttgcta 9900

ccattcaaga gaaagaatag atttagttta ttgcagagac aatatttgaa actatttact 9960

tatctatctg caaagtttgc atataaaata ataactgttt ctaattattc caagatggag 10020

attatgcgtc ttcttggtac cagtggtgat aaagttatag tgacttataa tacgttacca 10080

aatatacata tagagaatcc atcaattaat tgttggatta attgtaaata ctttttttct 10140

gttggagcat tgcaatctga taaacagtat gatttgatga ttaaagcttt ctataaattt 10200

attcaaatgg atgagcagta taaagattac aaattgctta ttgctggtgg ggatttagga 10260

gcaaaaaaat ccttatgtaa cctaattgat gagtttaatt taaccggcaa ggttattctc 10320

ttagggtata tttcagaact cgaaaaaaat tcactatata gttcatgtac tgctagcctg 10380

ttactaggaa aaagtgaggg attcgggatt cctgtgatcg aaagtatgtt ccacaacaaa 10440

cctgtaattg tttcagatct gggggcatta cctgaaatag ttggtgatgc tggaatcgtt 10500

gttaactcga atctaaatga tttaacaaat gctatggtaa atgtatgtaa tttgaacctg 10560

tcagagaaag tatttaagca tgagttagat aggtttgaaa gtgaaaagca gattgaatgt 10620

ctgacttctg taattaaaaa ggctatttca tgagtgaaaa ttcaatgttc ttttctcttt 10680

tgatggctgt ccgtaagaaa gatgatccaa cctttttgtt tgaggcactt aaatcaattg 10740

caaataatag tctacaacct acagaggtaa tcttggtcga agatggacct attactcggg 10800

aattaagaaa agttattaac ggttggtcaa atgttttaaa tattaaaagt attggtttag 10860

agagaaactc tggattaggt gctgctctta atgaagggct taaatattgt aaatatgaag 10920

tgattgtgcg agccgatgct gatgatatta accgaccaaa tagatttcag tgccaactaa 10980

attatttatt gcaaaatcct gatattgata tattaagttc gtgggtcgag gaattctcag 11040

tgatacctgg tgataaaact aaagtgagaa aagttcctcc tcaacaatca ttagacattt 11100

tttcaaagaa aagatgtcca tttaatcatc cagctgtatg ttttagaaaa aaaactatac 11160

tgtcactcgg tggctatggt aatgagcatt tatatgagga ttactcatta tggatgaaaa 11220

tgcttaattc cggatgcaaa ggggacaata tacaagaaat attagttgat atgcgttttt 11280

ctgatgaggc attgaaacgc aggggaggga taaaatatgc tttatcagaa ttaagaactc 11340

aatggaattt ttatgttttg aattatattt ctcttccaag gtttgtttca aatgctatat 11400

gtcgtaccag tgtcagaatc attccaaata aaattcgtgt gagtttatac aaaatctttc 11460

ttcggagcta actatgaaag agaaacaaat tatcccagta attttatgtg gtggaaatgg 11520

tactagactc tggccattgt ctagggaaca ataccctaag caatttcttt ctttgcaagg 11580

gaatgtgtca atgttacagg ccactatttc aagattagct aatctaaagt gctccgaacc 11640

gatagtcgtt tgtaatgata agcatcgctt tatcgttgca gaacaactaa aaaaaatcgg 11700

taaattatcg aataatattg tactggagcc tgaaggtaag aatactgcgc cagcaattgc 11760

attgtctgcg tttatcgctg agaaaattac aaatatatca tcagctatat tgttagttct 11820

ggctgcagat catattatag agaatgaaag cgtttttatt gagactataa aaaaaaccat 11880

ttcgttagct caattaggaa agctggttac tttcggaatt acccctagat atgcagaaac 11940

gggttacggc tatattcata gaggtaataa agtaaacaat aatgacgtta tcgcttatca 12000

agtgtctaat tttgttgaaa aaccaaatct aactttggca cgaaaatatt tgaaaaatgg 12060

agagtattat ttgaatagcg gtatgttttt atttcgcgta gatatttatt taaaggaatt 12120

gaagaaatat agaccagaaa tatatcggat ttgtttgagt tctgtaaaaa atatagggca 12180

tgatttggat tttattagga ttaatgcatc tcaatttaat aaaagcccat cggagtctat 12240

cgattgtgca gtcatggaaa atacaagtga tgcagttgtc gttcctatgg acgttggatg 12300

gagtgatgtc ggatcatggg aatcattatg ggatgttagc agcaaggatg aacatgggaa 12360

ttttagcaaa ggaaacgtta ttagttttga tgctcatgat aattatatat atgctgaatc 12420

atcgctggta acgacactcg gaatcgaaaa cttaattgtt attcaaacga aagacgcttt 12480

gcttgttgct gataaaaaaa aatctcaaga agtaaagcta attgttgata aacttaaaaa 12540

tgaaaatctg aaagagtatt ctttacatcg agaagttcat agaccatggg gtaaatataa 12600

ttatctttgt gaaggcgtag gataccaagt taagcatata ttagttaatc cgggagagtc 12660

actgtcactt caaatgcatt attatagagt agagcattgg attgtcattt ctggaacagc 12720

taaagtccaa ataaactcag aagaaaaaat tattagtgaa aatgaatcaa tatttattcc 12780

atcgggagtt aaacatagtt tggaaaattt tggggaagta ccattaataa taatcgaagt 12840

aagaactggc gagtattttg gagaagatga tgttattaga tttgataatt atcatagttt 12900

agataaatag tggttactta agaatgaata tgcttacttg ttttaaagcc tatgatattc 12960

gcggaaaatt aggcgaagaa ctgaatgaag atatcgcctg gcgtattggg cgtgcctatg 13020

gcgaatttct caaaccgaaa accattgttt taggcggtga tgtccgtctc accagcgaaa 13080

ccttaaaact ggcgctggcg aaagggttac aggatgcggg cgtcgacgtg ctggatatcg 13140

gcatgtccgg caccgaagag atctatttcg ccacgttcca tctcggtgtg gatggcggca 13200

tcgaagttac cgccagccat aacccgatgg gttataacgg catgaagctg gtgcgcgagg 13260

gggctcgccc gatcagcggt gataccggac tgcgcgacgt ccagcgtctg gcagaagcca 13320

acgactttcc tcccgttgat gaaaccaaac gcggtcgcta tcaacaaatc aatctgcgtg 13380

gcgcttacgt tgatcacctg ttcggttata tcaatgtcaa aaacctcacg ccgctcaagc 13440

tggtgatcaa ctccgggaac ggcgcagcgg gtccggtggt ggacgctatc gaagcccgct 13500

ttaaagccct cggcgcacct gtggaattga acaaagtgca caacacgccg gacggcaatt 13560

tccccaacgg tattcctaac ccgctgcagc cggaatgtcg cgacgacacc cgcaatgcgg 13620

tcatcaaaca cggcgcggat atgggcattg cctttgatgg cgattttgac cgctgtttcc 13680

tgtttgacga aaaagggcag tttatcgagg gctattacat tgtcggcctg ctggcagaag 13740

cgttcctcga aaaaaatccc ggcgcgaaga tcatccacga tccgcgtctc tcctggaata 13800

ccgttgatgt ggtgaccgcc gcgggcggca ctccggtgat gtcgaaaacc ggacacgcct 13860

ttatcaaaga acgtatgcgc aaggaggacg ccatctacgg tggcgaaatg agcgcccacc 13920

attacttccg tgatttcgct tactgcgaca gcggcatgat cccgtggctg ctggtcgccg 13980

aactggtgtg cctgaaagag aaaacgctgg gcgaactggt acgcgaccgg atggcggcgt 14040

ttccggcaag cggtgagatc aacagcaaac tggcgcaacc cgttgaggcg attagccgct 14100

tggaacagca ttttagccgt gaggcgctgg cggtggatcg caccgatggc atcagcatga 14160

cctttgccga ctggcgcttt aacctgcgca cctccaatac cgaaccggtg gtgcggttga 14220

atgtggaatc gcgcggtgat gtgccgctga tggaagaaaa aacaaaactt atccttgagt 14280

tactgagtaa gtaatccagt aatttcatat aaatgggttt taaaagacgg aaaagatgag 14340

atatccagtg tggtatagcc aaggtaatgc tattcagcat ctctatgagt gagttaacat 14400

ctataccaca tttaagccgc acactcggcg gtgaccaccc cctgacagga gtaaacaatg 14460

tcaaagcaac agatcggcgt cgtcggtatg gcagtgatgg ggcgcaacct tgcgctcaat 14520

atcgaaagcc gtggttatac cgtctctatt ttcaaccgtt cccgtgagaa aacggaagaa 14580

gtgattgccg aaaatccagg caagaaactg gttccttact acacggtgaa agagtttgtt 14640

gaatctctgg aaacgcctcg tcgcatcctg ttaatggtga aagcaggtgc aggcacggat 14700

gctgctattg attctctcaa gccatacctc gataaaggtg acatcatcat tgatggtggt 14760

aacaccttct tccaggacac cattcgtcgt aaccgtgagc tgtctgccga aggttttaac 14820

ttcattggta ccggtgtttc cggtggtgag gagggcgcac taaaaggtcc ttccattatg 14880

cctggtggcc agaaagaagc ctatgaactg gttgctccga tcctaaccaa aatcgccgca 14940

gtggctgaag acggtgagcc atgcgttacc tatattggtg ccgatggtgc aggtcactat 15000

gtgaagatgg ttcacaacgg tattgaatac ggcgatatgc agctgattgc tgaagcctat 15060

tctctgctta aaggtggcct gaacctcacc aacgaagaac tggcgcagac ctttaccgag 15120

tggaataacg gtgaactgag cagctacctg atcgacatca ccaaagatat cttcaccaaa 15180

aaagatgaag agggttacta tctggttgat gtgattctgg atgaagcggc taacaaaggt 15240

accggttaat ggaccagcca gagtgcgctg gatctcgg 15278

Wzx gene, wzy gene and wherein primer and PCR data in the O antigen gene of table 1 Shigella bogdii 12 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	6246-7580 7561-8700	6435-6452 6709-6726 7679-7696 8270-8287	7094-7111 7280-7297 8511-8528 8667-8684	677bp 589bp 850bp 415bp	0 0 0 0	56 56 58 58

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, Shigella bogdii	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, B12, 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, O136, 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 removes the 10th group of bacterium of B12	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 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 Shigella bogdii 12 types

galF rmlB rmlD IS630 rmlArmlC orf5 wzx wzy orf8 orf9 orf10 manC manB gnd

1kb

Table 4 is location tables of the gene in the O-antigen gene bunch of Shigella bogdii 12 types

ATCCAACTAA CTGATGCTAT TGCCGAACTG GCGAAAAAAC AGTCCGTTGA TGCCATGCTG 60

ATGACCGGCG ACAGTTACGA CTGCGGTAAA AAAATGGGCT ATATGCAGGC GTTCGTGAAG 120

TATGGATTAC GCAACCTGAA AGAAGGGGCG AAGTTCCGCA AAGGCATTGA GAAGTTGTTA 180

AGCGAATAAT GAAAATCTGA CCGGATGTAA CGGTTGATAA GAAAATTATA ACGGCAGTGA 24O

AGATTCGTAG CAAAAGTAAT TTGTTGCGAA TATTCCTGCC GTTGTTTTAT ATAAATATTC 300

AGAATAACAA CGAGTTAGCA ATAGGATTTT AGTCAAAGTT TTCCAGGATT TTCCTTGTTT 360

CCAGAGCGGA TTGGTAAGAC AATTAGCGTT TGAATTTTTC GAGCTAAGCG CGAGTGGGGA 420

ACGCTCGCTA CATCGTAGGC ATGCAGTGTT CTGGTAGCTG TAAAGCCAGG GGCGGTAGCG 480

TGCATTAATA CCTCTATTAA TCAAACTGAG AGCCGCTAAT TTCACAGCAT GCTCTGAAGT 540

Orf7 is initial

AATATGGAATAAATTAA GTG AAAATACTTG TTACTGGTGG CGCAGGATTT ATTGGTTCTG 600

CTGTAGTTCG TCACATTATA AATAATACGC AGGATAGTGT TGTTAATGTC GATAAATTAA 660

CGTACGCCGG AAACCTGGAA TCACTTGCTG ATGTTTCTGA TTCTGAACGC TATGTTTTTG 720

AACATACAGA TATTTGCGAT GCAGCTGCAA TGGCACGGAT TTTTGCTCAG CATCAGCCGG 780

ATGCAGTGAT GCACCTGGCT GCTGAAAGCC ATGTTGACCG TTCAATTACA GGTCCTGCGG 840

CATTTATTGA AACCAATATT GTTGGAACTT ATGTCCTTTT AGAAGCCGCT CGCAATTACT 900

GGTCTGCTCT TGATAGCGAC AAGAAAAATA GCTTCCGTTT TCATCATATT TCTACTGACG 960

AAGTCTATGG TGATTTGCCT CATCCTGACG AGGTAAATAA TACAGAAGAA CTACCCTTAT 1020

TTACTGAGAC GACAGCTTAC GCGCCAAGCA GCCCTTATTC CGCATCCAAA GCATCCAGCG 1080

ATCATTTAGT CCGCGCGTGG AAACGTACCT ATGGTTTACC GACCATTGTG ACTAATTGCT 1140

CTAACAATTA TGGTCCTTAT CATTTCCCGG AAAAGCTTAT TCCATTGGTT ATTCTTAATG 1200

CTCTGGAAGA TAAAGCATTA CCTATTTATG GTAAAGGGGA TCAAATTCGC GATTGGCTGT 1260

ATGTTGAAGA TCATGCGCGT GCGTTATATA CCGTCGTAAC CGAAGGTAAA GCGGGTGAAA 1320

CTTATAACAT TGGTGGGCAC AACGAAAAGA AAAACATAGA TGTAGTGCTC ACTATTTGTG 1380

ATTTGCTGGA TGAGATTGTA CCGAAAGAGA AATCTTATCG TGAGCAAATC ACTTATGTTG 1440

CCGATCGTCC GGGACACGAT CGCCGTTATG CCATTGATGC TGATAAGATT GGTCGTGAAT 1500

TGGGATGGAA ACCACAGGAA ACGTTTGAGA GCGGCATTCG TAAAACGGTG GAATGGTACC 1560

TGTCCAATAC AAAATGGGTT GATAATGTGA AAAGTGGTGC CTATCAATCG TGGATTGAAC 1620

It is initial that Orf1 stops Orf2

AGAACTATGA GGGCCGCAAG TAATGAATAT CCTCCTTTTC GGCAAAACAG GGCAGGTAGG 1680

TTGGGAACTA CAGCGTGCCC TGGCTCCTCT GGGTAATTTG ATTGCTCTTG ATGTTCACTC 1740

CACTGATTAT TGTGGTGATT TTAGTAATCC TGAAGGTGTA GGTGAAACCG TAAGAAGCAT 1800

TCGGCCTGAT ATTATTGTCA ACGCAGCCGC TCACACCGCA GTTGACAAAG CAGAATCAGA 1860

ACCGGAGTTT GCACAATTAC TTAACGCGAC AAGTGTCGAA GCGATTGCGA AAGCAGCCAA 1920

TGAAGTCGGT GCCTGGGTTA TTCACTACTC TACTGACTAT GTATTTCCGG GAACCGGTGA 1980

AATACCATGG CAGGAGTCGG ATGCAACCGC ACCGCTAAAT GTTTACGGTG AAACCAAGTT 2040

AGCCGGAGAA AAAGCATTAC AAGAGCATTG TGTGAAGCAC CTTATTTTCC GGACCAGCTG 2100

GGTCTATGCA GGTAAAGGAA ATAACTTCGC CAAAACGATG TTACGACTGG CAAAAGAGCG 2160

TGAAGAATTG GCCGTTATTA ACGATCAGTT TGGTGCGCCA ACAGGTGCTG AGCTGCTGGC 2220

TGATTGTACA GCACATGCCA TTCGTGTCGC ACTGAATAAA CCGGAAGTCG CAGGCTTGTA 2280

CCATTTGGTA GCCAGTGGTA CCACAACCTG GCACGATTAT GCTGCGCTGG TTTTTGAAGA 2340

GGCGTGCAAA GCAGGCATTC CCCTCGCACT CAACAAGCTC AACGCTGTAC CAACAACAGC 2400

CTATCCTACA CCAGCACGTC GTCCACATAA CTCTCGTCTT AATACAGAAA AATTTCAGCA 2460

AATTTTGCG CTTGTATTGC CAGACTGGCA GGTTGGCGTG AAACGCATGC TCAACGAATT 2520

Orf2 stops

ATTTACGACT ACCGCAATTTAA TAGTTTTT GCATCTTGTT CGTGATGGTG GAGCAAGATG 2580

AATTAAATAG CTGCGCTTAA TACCGCTACA CTTTTGCCAG TCCATGTTTG CCTCCGGGGA 2640

ATGGGCTGAC GGTTTCCATA AAATGGCGAA CTTTTTTCAA CAGTTGCCAC ATTGAGCGGC 2700

ACTGATGATT ACGCGTTATT GTGTCGTGAA GTGCCTGCCA TAGCCGTTCA ACATGATTCA 2760

CCCATGGCGA GTAAACCGGC TGATAAATGA CCCTGAACTT CGGGTTCTCC TTCAGCCAGC 2820

TCTGTGTTTC CCGGTTTTTG TGGATAATGT AGTTGTCCAC GATCAGCGTG ATGGTTTTCG 2880

CCCGACGGTA TGTCGCTTTA AGCCGCTTCA GCAGGCTGAT GAACAGCGCC GAACTTTTGC 2940

TGTTGCCGCC CACATAGCTG ACTTTACCTG TCCCGCTGTG CAGCGCTCCG GCCAGATAAT 3000

ATTTTTCATT CTGTCCCGGC GTGACCACCC GTTTTTGCTG TCCGCGCAGT TGCCAGTCTG 3060

CACCGATTTT GGGATTAAGA TGGATATCCA CTTCATCTTC ATAAAAGACC GGATGTTCTG 3120

CGCTGCATTC GTCCAGTGCT TTATGGATTG CTGCCATCTT TTCATCTTTA TGCGGGTCAC 3180

GGATACGCAG AGTTGGCGCA GCCCTTCGCC ACACAATCCC CGCAGACGGC AACCAGCGGC 3240

GAACGGTTCC GGCATTTAAC TGGCAACCGG TTATCTCATT GATTTTTATT GCCAGCAGTT 3300

CTGTACTCCA GCGTGAACGC TGGTAGCCAA AGTCGCCGGG AGAATGTTTT ACCAGCTCAC 3360

GTAACAGTGT GCAGATATGC TCAAACGGCC AGCGACGGGC ACGCCCGGCA GGTAATGATT 3420

TCAGTCCCTC AACACCCGAC TGCGTGAACC AGTTAATCCA GCGTCCAACA GAGGAGCGGG 3480

CGCAGCAGAG CGTTCTGGCA ACGTCGCTGA CACGGTCGCC CCGGTGCAGC ATCAGCATGG 3540

CAGTCAGTCT GCGGGCATAA TTTCTATCGT GTGTTTTATG GATGGCTTTC TGCATCAGGC 3600

GTCGTTCGTC ACGGGAAATT GGTGCTATGA TCGGCATTGC TCAGTCCGGT TGGTGATTTG 3660

TTTTGATTTG GCGATTGATC AGATCGCACA ATCCGGGCTA AGTTCCCTCA AAGTGATCTA 3720

Orf3 is initial

CTATTCCGCG CAGCTATTTA AAAGGAATGA TGAA ATGAAA ACGCGTAAAG GTATTATTTT 3780

AGCGGGGGGA TCTGGTACTC GTCTTTATCC TGTCACTATG GCTGTCAGTA AACAGCTGTT 3840

ACCGATTTAT GATAAACCGA TGATTTATTA TCCGCTATCT ACACTGATGT TAGCGGGTAT 3900

TCGCGATATT CTGATTATCA GTACGCCACA GGATACTCCT CGTTTTCAAC AACTGCTGGG 3960

TGATGGTAGC CAGTGGGGCC TTAATCTTCA GTATAAAGTA CAACCTAGTC CAGATGGTTT 4020

GGCACAAGCT TTCATTATTG GAGAAGATTT TATTGGTAGG GATGATTGCG CATTAATTCT 4080

AGGTGATAAT ATCTTTTACG GTCATGACCT ACCAAAATTA ATGGATACAG CTGTTAATAG 4140

AGAGAGTGGT GCAACGGTAT TTGCCTATCA CGTTAATGAT CCAGAACGTT ACGGTGTGGT 4200

GGAATTTGAT AAAAACGGCA CGGCAATAAG CCTGGAAGAA AAACCGCTTC AACCGAAAAG 4260

CAACTATGCA GTTACAGGGC TTTATTTCTA TGATAATGAC GTCGTGGAAA TGGCGAAAAA 4320

TCTTAAGCCT TCTGCCCGTG GTGAACTGGA AATTACCGAT ATTAATCGTA TCTATATGGA 4380

ACGGGGGCGT TTGTCTGTTG CCATGATGGG CCGTGGTTAT GCATGGCTGG ACACGGGGAC 4440

ACATCAAAGC CTGATTGAGG CAAGCAACTT CATCGCAACA ATTGAAGAGC GTCAGGGGTT 4500

AAAAGTTTCC TGCCCGGAAG AAATTGCTTA CCGTAAAGGG TTTATTGATT CTGAGCAGGT 4560

GAAAGTATTA GCTGATCCGC TGAAAAAAAA TGCTTATGGT CAGTATCTGC TTAAAATGAT 4620

It is initial that Orf3 stops Orf4

TAAAGGTTAT TAATAAA ATG AACGTAATTA AAACAGAAAT TCCAGATGTA CTGATTTTTG 4680

AACCGAAAGT TTTTGGTGAT GAGCGTGGTT TCTTTTTTGA GAGCTTTAAC CAGAAGGTTT 4740

TTGAGGAAGC TGTAGGCCGC AAAGTTGAAT TTGTCCAGGA TAACCATTCG AAGTCTTGTA 4800

AAGGTGTTTT ACGCGGACTG CATTATCAAT TAGAACCTTA TGCGCAAGGG AAATTGGTGC 4860

GCTGCGTTGT TGGTGAAGTT TTTGACGTAG CGGTTGATAT TCGTAAATCG TCACCTACTT 4920

TTGGCAAGTG GGTTGGGGTG AATTTATCTG CTGAGAATAA GCGCCAGTTG TGGATCCCGG 4980

AGGGGTTTGC TCATGGTTTT TTGGTGCTGA GTGAGACGGC GGAGTTTGTT TATAAGACGA 5040

CTCAGTATTA TCATCCTGAA TGTGAACGAG GGATCAGATG GGATGATGAA AATATAGGGG 5100

TAGATTGGCC TCTGTCTACA GATTTAATAC TATCTTTTAA AGATACCAAG CATCCAAAAT 5160

It is initial that Orf4 stops Orf5

TCAACGGTATTTTA TAAA AT GAAAAATTAT AGTGAATTAT TAATAATCCA TGAATATGGA 5220

GAACCATCTC ATTACCAAGG AATAATAGAG ATTGCTCAGG AGAGAAGAAT TAAGGTCCGT 5280

TTTTTAGAAT TCAGTATTTT ACATAATATT TATGCTGCAA TTAAAAAAAA GAAAATAAAT 5340

GCCGTTATAA AGGCGATCAA AGATTATTTG ATATTGATGA TATACTCATT CTTTCCAAGA 5400

ATGAATAATG GAAAGTTGGT TGTTATAGGC ATGGCCCCGT TAGATAAATA TGTTTTTTTA 5460

ATAAATAAAT TATTGAAGGG ACAAGATTAT ATTTATCACA CATCATGGCT TTATTGGGAT 5520

GGCAGTGACT ATCCTAAAAA AACATATAAA AATCCTGAGG TATTAATTGA TGAGTGGAAG 5580

ACTTTTTTAA ATAATGCTAA AGCGGTTGCA TGTGTAACAC CTGATTCTGC GCATAGTTTA 5640

ATTTCAAACT TTAATATGAA AGATAAAACA TTCATTGTTT ATCATTCGTA TGACTCAAAT 5700

ATTTTCTATC CTTCGCCAAG GCAAGAACGA AAAAAAACAT TAACGATATG TTACTTAGGG 5760

CGACTTGAAA AAAACAAAGG AATTGAAAAT CTTATTTATA TTATTGAAAA TAATGATAAT 5820

GTTAATTTTA AAATAATGGG CAGCGGAAGT TACTCAAGCA AAATAACTGA GTTAAGCAGG 5880

TCAAAGACTA ATGTTGAATA CTTAGGGTAC CTATCGGATA AAAAATTAAT AGCAGAACAA 5940

TTGCGGCAAA CTGATATTAT ATTACTTCCA TCAATTAAAA TTAACGGGTG GGAAGAATTA 6000

TTTGGAATCG CATTAATAGA GTCAATGGCT TGTGGGGTAG TACCTATTAC TACTGATCAC 6060

AAAGGTCCTA GGACCATACT AAAGAATAAA TTATTAGCAG AGCTCATTTT CAGTGAGAAT 6120

GAATTTGTTG AGTCGGCACA GAAGATCATC ACAGATTTAA ATAATGATGA ATTTTTATTA 6180

CAAAAGTACA AGGATTGTGC GATTGCTGAG GCAAAGTTTT ATGAAAAGAA AAATATATCA 6240

The initial Orf5 of Orf6 stops

AATAA ATGGA GGGAAGTTATTCACAAA TAA TTTATGACAA CATTTTTAAT GTCTATCTTT 6300

ATGAGTTGAA AATAATGGCC ATAATTAATG GTGTTTTTAA AAATATTGCT TTGCTTATTT 6360

CTGGAACCGC AGTAGCTCAA TTAATTAATT TTTGTTTTAT TCCTTTGATT ACAAGATTGT 6420

ATGGTCCTGA GTCTTATGGA ATGATGGGAA GCTTAATGGC GTTTATTAAC ATTATGATTC 6480

CGATAGCTTC ATTAAGTTTT CCAATTGCAA TTGTGTTACC AAAGTCTGAT AAAACCGCTA 6540

TTTATATTGC TTACCTTAGC ATTTGTATAG GTTTGGTGGG TGCAGGAGTT CTATTAATCG 6600

TAATAGGGGG ATTGGAATAT TTAGATGGAA GTTTCAGTCA ATACCCTGAA TGGTATATAT 6660

TACTATTACC ATTTATCATG ACATTCCTAC CTCTACAGCA AAGTGGACAG CAATGGCTAA 6720

TAAGGAAAAA ACAGTTTAAA GAAATAGCAA AAGTATCTGT CAGTCAAGCA TTAATTATGA 6780

ATGGTTTGAA GGTAATCGGG GGAGGGATTT TACCAACACA AATCATGATA ATATATACAA 6840

CAGGTGCAGG AGTAATTTTT CAATGCTTAC AGTTCTTTTA TCGATCTTAT AAGAAAGGGC 6900

TTGGAATTCC TTTATCAAAA ATAAAAAAGA ATTATTTTAT AAGGGTTGCG CGCGGCTATT 6960

ATGATTTTCC GCTATATAGA ACTCCGCAAG TATTAATCAA TGCGTTGTCT CAAAATTTGC 7020

CGATATTTTT GTTGGGGTAT TATTTTGATT CTAAAATAGT AGGCTTTTAT GTGCTTGCTC 7080

AAACGTTACT AACTGCTCCA GTGACTTTGT TAAGCACTTC GATAAGTAAT GTATATTATC 7140

CTACAATCGC AGAGAAATTT ACCCAAAAAA AACCTATTTA TAATTTCTTA AGAAAAACAA 7200

TTTGGGGACT GTTTATTGTA TCCTTGTTTA TCTATTTGCC ATGTATATTG TTATCTGGCA 7260

TTGTATTTAA AATTATTTTT GGAGGTGGGT GGATTACGTC TAGCATTTTT TTTCAATGGA 7320

TGGCTATTTA TTGCATTTTT TGGTTAAGTG CTAGACCCGC GATGGATACT ATACCGCCAT 7380

TGAAAATTCA AAACTACTTT CTTCTATATG AAGTTATTTC TTTTTTATTG AGGATTTTAA 7440

GCCTAATGTT AGGCTATTAT TTATATAAAG ATGCGATAAT TTGTATTATT ATGTTTTCAT 7500

CAACCAATGC AGTCACATAT GCTGTCTTGA TAGTTTATAT TTTATTTCAG GCAAAAGTAA 7560

The initial Orf6 of Orf7 stops

ATGATAGAAA ATATACA TAA TTCACTACAA GAGTTAAAGA GTGATCATAT TAATAAATAT 7620

AATGGCTCAA ACTTTTATTA CTATGTCTCA GTATTTGGTC TATTACTTAT CTCATTTCGA 7680

CTTCAGATAC CCTCACTTAT ATTTATTATA GGTGCGATTA TTATAAGAGG TACTACTTTT 7740

AAGCAAAATA TATCTGGTTG GCTTTGGTTC GGAAGTATTA CTTTTTTTTT TGGAATATAT 7800

CTTTTTTTGG GGTTGGATAA TTATGAAAGA GGTGCTGATA AGTTTTTATC ACGAACTTTA 7860

GTAATATATC TTTATTTCGT TTTTTTTTAC GGGTTGGGAT GTGTTAGAAC TCAAAATAGA 7920

GAACGACTCT TATCTTTTTT TTCATTATTT AGCGTGATTT ATGTCTATGC TGTCTGTATC 7980

TATAGTAAGA TTAGCGGTTA TCCAGGATAT AATGGAATAT ATGATGTGTT CAATGGGAAT 8040

GATGAAAATT CTCCATTATA TGCATTACAG TTGATTCTAT TTACAATTTT ATATCTTCAC 8100

CTCAATGCAG GGAAAATAAA TAAAATATGG GTTGGGTTGT TAATTATCAC ATCTTTTTAT 8160

TTTAGTGTTA TTTATCTTGG CTCTCGAGCA TCCTTCTTAT TATTAATTTT TTATTTCATT 8220

TTTGTGATCT TTAAACATAA AACTAACTTG TTAATATCTT TCATTATTTT TATCCCGTTC 8280

ATAGTTACAT TATTTATTTA TATAAATGAC TTTAATTTTT TAAATTATTT AGATTTTGGA 8340

GGGTTTGAAA ATCGAGGACT AAGCAGTCCT CGATTTAGTA TGCTTGAATA TGGTTTAAAG 8400

AATTTCATGA ATTATCCATG GGGAGGTTTA ATTGTTCATG GTGAAGGTTA TGATGGTATC 8460

TGGCTCCATA ATATGTTATT AGATATAGTT AGGGTATCAG GTTACTATAC TATGTTTGTA 8520

TGGCTGTTCA TTTTATTAAT GGCAGGTTTA GTTATTCTGA AACGTGAAAA ATTTTATTTT 8580

CCAATATTTC TAATTCTAAA TATAGCTTTA ATGCAAGATT TAGCATTTGA TGGTTTTTTT 8640

Orf7 stops

AACATAATGG CTCTGGAGTT TTTTTTATTG GGTGTAGCTG TACCTGGTTTAAGAAAA TAA 8700

Orf8 is initial

AGGAAAATAA AATATA ATGA ATATGTATAT TGTGGTTTGT GTATATACCC TGCCCCCAGC 8760

GTATATAAAA AAATGGTTTA AGTTACATTT GCATAATACT AAATATAAAC TTATTATTGT 8820

CGATAACAAC TTAAGAAGAC AAATAACAGA TCCAACTGTT ATTATTGGCA CAAATACTCT 8880

TAATGAATTC TCAGCATACA ATGAAGGGCT TCAGCTACTA AAAAAAGAAT TTGAAGATGA 8940

ATATGACATA ATATTGATGT TGAATGATAC TTTGTTCACT AGGCATAATG CGAAATTTTT 9000

CTTAAAACAT TTATTAAAGT ATAAAAATAC AGTTGCTCGC TTATCAATCC CGGCTATCGG 9060

TGGAAGAATA GATCCATATA ATAATATCTG TTATAGAAAC CCATGGAGCA ATGACATTGG 9120

TTATATATCT TCATTTTGTA TAATAATGAA TAAACCAGCA AGAGATCTAT ATTTGAAGTT 9180

ACTATCTGAT ATATCCCCAA CTTTTCCATT TGTTGATTCA GTTACTGAGC TTTTCAACTG 9240

GAGTACTCAT ATAGACAGAA GATTTAAAGA ATTTGTGATC AGTCATCTGA TAGATACAGA 9300

CACAGCTACT GTGTGGTACC AATCCAAGAA TAATATTAAA AATATAGAAA GATTGAATGT 9360

TAAAGGTAAG TGTGTTTTTC TTGAACATTA TGTATCAGGA AATATTTCTA AACATGGAGT 9420

ATTGGTTTCA ATCTTTCCCA CGTGGAAGCA GAAAACTCAG CATTTTATTT ATGAGCAAAT 9480

The initial Orf8 of Orf9 stops

CGCAAAAATG GAAAGAAAAT TATTATCGAT ACTAAATTTT AAAGTTGGTT CAAA ATGAAA 9540

AAAAGAATTT TAATAAATCT ATTGAGTTTT TCTGAAGAAA AATATTACGG TGTGGGAGTT 9600

TATTTCAGAG ATGTTGCTAT TAAAAGTATT AACAGGGTTA TTGAAGAACT AGAATGCGAA 9660

ATATGTAATT ATATTTGGC ACATATTCCG ATACAAAAGA TGTTTACTTT TTCAAAAAAT 9720

GAAAACATAA AATATATACC TGTAAAGAAA ATTAATAGCA AACTCACAAG AGTTTTCTAC 9780

GAACAAATAA TGTTACCTTT GTATCATAGA GATTATGATG TGATTTATAG TCCTAATAAT 9840

ATAAATCCAT TAATATTAGC TTTTTCAAAA AAATCTATAA TAACTATCCA CGATTTGCTA 9900

CCATTCAAGA GAAAGAATAG ATTTAGTTTA TTGCAGAGAC AATATTTGAA ACTATTTACT 9960

TATCTATCTG CAAAGTTTGC ATATAAAATA ATAACTGTTT CTAATTATTC CAAGATGGAG 10020

ATTATGCGTC TTCTTGGTAC CAGTGGTGAT AAAGTTATAG TGACTTATAA TACGTTACCA 10080

AATATACATA TAGAGAATCC ATCAATTAAT TGTTGGATTA ATTGTAAATA CTTTTTTTCT 10140

GTTGGAGCAT TGCAATCTGA TAAACAGTAT GATTTGATGA TTAAAGCTTT CTATAAATTT 10200

ATTCAAATGG ATGAGCAGTA TAAAGATTAC AAATTGCTTA TTGCTGGTGG GGATTTAGGA 10260

GCAAAAAAAT CCTTATGTAA CCTAATTGAT GAGTTTAATT TAACCGGCAA GGTTATTCTC 10320

TTAGGGTATA TTTCAGAACT CGAAAAAAAT TCACTATATA GTTCATGTAC TGCTAGCCTG 10380

TTACTAGGAA AAAGTGAGGG ATTCGGGATT CCTGTGATCG AAAGTATGTT CCACAACAAA 10440

CCTGTAATTG TTTCAGATCT GGGGGCATTA CCTGAAATAG TTGGTGATGC TGGAATCGTT 10500

GTTAACTCGA ATCTAAATGA TTTAACAAAT GCTATGGTAA ATGTATGTAA TTTGAACCTG 10560

TCAGAGAAAG TATTTAAGCA TGAGTTAGAT AGGTTTGAAA GTGAAAAGCA GATTGAATGT 10620

The initial Orf9 of Orf10 stops

CTGACTTCTG TAATTAAAAA GGCTATTTC A TGAGTGAAAA TTCAATGTTC TTTTCTCTTT 10680

TGATGGCTGT CCGTAAGAAA GATGATCCAA CCTTTTTGTT TGAGGCACTT AAATCAATTG 10740

CAAATAATAG TCTACAACCT ACAGAGGTAA TCTTGGTCGA AGATGGACCT ATTACTCGGG 10800

AATTAAGAAA AGTTATTAAC GGTTGGTCAA ATGTTTTAAA TATTAAAAGT ATTGGTTTAG 10860

AGAGAAACTC TGGATTAGGT GCTGCTCTTA ATGAAGGGCT TAAATATTGT AAATATGAAG 10920

TGATTGTGCG AGCCGATGCT GATGATATTA ACCGACCAAA TAGATTTCAG TGCCAACTAA 10980

ATTATTTATT GCAAAATCCT GATATTGATA TATTAAGTTC GTGGGTCGAG GAATTCTCAG 11040

TGATACCTGG TGATAAAACT AAAGTGAGAA AAGTTCCTCC TCAACAATCA TTAGACATTT 11100

TTTCAAAGAA AAGATGTCCA TTTAATCATC CAGCTGTATG TTTTAGAAAA AAAACTATAC 11160

TGTCACTCGG TGGCTATGGT AATGAGCATT TATATGAGGA TTACTCATTA TGGATGAAAA 11220

TGCTTAATTC CGGATGCAAA GGGGACAATA TACAAGAAAT ATTAGTTGAT ATGCGTTTTT 11280

CTGATGAGGC ATTGAAACGC AGGGGAGGGA TAAAATATGC TTTATCAGAA TTAAGAACTC 11340

AATGGAATTT TTATGTTTTG AATTATATTT CTCTTCCAAG GTTTGTTTCA AATGCTATAT 11400

GTCGTACCAG TGTCAGAATC ATTCCAAATA AAATTCGTGT GAGTTTATAC AAAATCTTTC 11460

It is initial that Orf10 stops Orf11

TTCGGAGC TA ACT ATGAAAGAGAAACAAAT TATCCCAGTA ATTTTATGTG GTGGAAATGG 11520

TACTAGACTC TGGCCATTGT CTAGGGAACA ATACCCTAAG CAATTTCTTT CTTTGCAAGG 11580

GAATGTGTCA ATGTTACAGG CCACTATTTC AAGATTAGCT AATCTAAAGT GCTCCGAACC 11640

GATAGTCGTT TGTAATGATA AGCATCGCTT TATCGTTGCA GAACAACTAA AAAAAATCGG 11700

TAAATTATCG AATAATATTG TACTGGAGCC TGAAGGTAAG AATACTGCGC CAGCAATTGC 11760

ATTGTCTGCG TTTATCGCTG AGAAAATTAC AAATATATCA TCAGCTATAT TGTTAGTTCT 11820

GGCTGCAGAT CATATTATAG AGAATGAAAG CGTTTTTATT GAGACTATAA AAAAAACCAT 11880

TTCGTTAGCT CAATTAGGAA AGCTGGTTAC TTTCGGAATT ACCcCTAGAT ATGCAGAAAC 11940

GGGTTACGGC TATATTCATA GAGGTAATAA AGTAAACAAT AATGACGTTA TCGCTTATCA 12000

AGTGTCTAAT TTTGTTGAAA AACCAAATCT AACTTTGGCA CGAAAATATT TGAAAAATGG 12060

AGAGTATTAT TTGAATAGCG GTATGTTTTT ATTTCGCGTA GATATTTATT TAAAGGAATT 12120

GAAGAAATAT AGACCAGAAA TATATCGGAT TTGTTTGAGT TCTGTAAAAA ATATAGGGCA 12180

TGATTTGGAT TTTATTAGGA TTAATGCATC TCAATTTAAT AAAAGCCCAT CGGAGTCTAT 12240

CGATTGTGCA GTCATGGAAA ATACAAGTGA TGCAGTTGTC GTTCCTATGG ACGTTGGATG 12300

GAGTGATGTC GGATCATGGG AATCATTATG GGATGTTAGC AGCAAGGATG AACATGGGAA 12360

TTTTAGCAAA GGAAACGTTA TTAGTTTTGA TGCTCATGAT AATTATATAT ATGCTGAATC 12420

ATCGCTGGTA ACGACACTCG GAATCGAAAA CTTAATTGTT ATTCAAACGA AAGACGCTTT 12480

GCTTGTTGCT GATAAAAAAA AATCTCAAGA AGTAAAGCTA ATTGTTGATA AACTTAAAAA 12540

TGAAAATCTG AAAGAGTATT CTTTACATCG AGAAGTTCAT AGACCATGGG GTAAATATAA 12600

TTATCTTTGT GAAGGCGTAG GATACCAAGT TAAGCATATA TTAGTTAATC CGGGAGAGTC 12660

ACTGTCACTT CAAATGCATT ATTATAGAGT AGAGCATTGG ATTGTCATTT CTGGAACAGC 12720

TAAAGTCCAA ATAAACTCAG AAGAAAAAAT TATTAGTGAA AATGAATCAA TATTTATTCC 12780

ATCGGGAGTT AAACATAGTT TGGAAAATTT TGGGGAAGTA CCATTAATAA TAATCGAAGT 12840

AAGAACTGGC GAGTATTTTG GAGAAGATGA TGTTATTAGA TTTGATAATT ATCATAGTTT 12900

It is initial that Orf11 stops Orf12

AGATAAA TAGTGGTTACTTA AGAATGAAT A TGCTTACTTG TTTTAAAGCC TATGATATTC 12960

GCGGAAAATT AGGCGAAGAA CTGAATGAAG ATATCGCCTG GCGTATTGGG CGTGCCTATG 13020

GCGAATTTCT CAAACCGAAA ACCATTGTTT TAGGCGGTGA TGTCCGTCTC ACCAGCGAAA 13080

CCTTAAAACT GGCGCTGGCG AAAGGGTTAC AGGATGCGGG CGTCGACGTG CTGGATATCG 13140

GCATGTCCGG CACCGAAGAG ATCTATTTCG CCACGTTCCA TCTCGGTGTG GATGGCGGCA 13200

TCGAAGTTAC CGCCAGCCAT AACCCGATGG GTTATAACGG CATGAAGCTG GTGCGCGAGG 13260

GGGCTCGCCC GATCAGCGGT GATACCGGAC TGCGCGACGT CCAGCGTCTG GCAGAAGCCA 13320

ACGACTTTCC TCCCGTTGAT GAAACCAAAC GCGGTCGCTA TCAACAAATC AATCTGCGTG 13380

GCGCTTACGT TGATCACCTG TTCGGTTATA TCAATGTCAA AAACCTCACG CCGCTCAAGC 13440

TGGTGATCAA CTCCGGGAAC GGCGCAGCGG GTCCGGTGGT GGACGCTATC GAAGCCCGCT 13500

TTAAAGCCCT CGGCGCACCT GTGGAATTGA ACAAAGTGCA CAACACGCCG GACGGCAATT 13560

TCCCCAACGG TATTCCTAAC CCGCTGCAGC CGGAATGTCG CGACGACACC CGCAATGCGG 13620

TCATCAAACA CGGCGCGGAT ATGGGCATTG CCTTTGATGG CGATTTTGAC CGCTGTTTCC 13680

TGTTTGACGA AAAAGGGCAG TTTATCGAGG GCTATTACAT TGTCGGCCTG CTGGCAGAAG 13740

CGTTCCTCGA AAAAAATCCC GGCGCGAAGA TCATCCACGA TCCGCGTCTC TCCTGGAATA 13800

CCGTTGATGT GGTGACCGCC GCGGGCGGCA CTCCGGTGAT GTCGAAAACC GGACACGCCT 13860

TTATCAAAGA ACGTATGCGC AAGGAGGACG CCATCTACGG TGGCGAAATG AGCGCCCACC 13920

ATTACTTCCG TGATTTCGCT TACTGCGACA GCGGCATGAT CCCGTGGCTG CTGGTCGCCG 13980

AACTGGTGTG CCTGAAAGAG AAAACGCTGG GCGAACTGGT ACGCGACCGG ATGGCGGCGT 14040

TTCCGGCAAG CGGTGAGATC AACAGCAAAC TGGCGCAACC CGTTGAGGCG ATTAGCCGCT 14100

TGGAACAGCA TTTTAGCCGT GAGGCGCTGG CGGTGGATCG CACCGATGGC ATCAGCATGA 14160

CCTTTGCCGA CTGGCGCTTT AACCTGCGCA CCTCCAATAC CGAACCGGTG GTGCGGTTGA 14220

ATGTGGAATC GCGCGGTGAT GTGCCGCTGA TGGAAGAAAA AACAAAACTT ATCCTTGAGT 14280

Orf12 stops

TACTGAGTAAG TAATCCAGT AATTTCATAT AAATGGGTTT TAAAAGACGG AAAAGATGAG 14340

ATATCCAGTG TGGTATAGCC AAGGTAATGC TATTCAGCAT CTCTATGAGT GAGTTAACAT 14400

CTATACCACA TTTAAGCCGC ACACTCGGCG GTGACCACCC CCTGACAGGA GTAAACAATG 14460

TCAAAGCAAC AGATCGGCGT CGTCGGTATG GCAGTGATGG GGCGCAACCT TGCGCTCAAT 14520

ATCGAAAGCC GTGGTTATAC CGTCTCTATT TTCAACCGTT CCCGTGAGAA AACGGAAGAA 14580

GTGATTGCCG AAAATCCAGG CAAGAAACTG GTTCCTTACT ACACGGTGAA AGAGTTTGTT 14640

GAATCTCTGG AAACGCCTCG TCGCATCCTG TTAATGGTGA AAGCAGGTGC AGGCACGGAT 14700

GCTGCTATTG ATTCTCTCAA GCCATACCTC GATAAAGGTG ACATCATCAT TGATGGTGGT 14760

AACACCTTCT TCCAGGACAC CATTCGTCGT AACCGTGAGC TGTCTGCCGA AGGTTTTAAC 14820

TTCATTGGTA CCGGTGTTTC CGGTGGTGAG GAGGGCGCAC TAAAAGGTCC TTCCATTATG 14880

CCTGGTGGCC AGAAAGAAGC CTATGAACTG GTTGCTCCGA TCCTAACCAA AATCGCCGCA 14940

GTGGCTGAAG ACGGTGAGCC ATGCGTTACC TATATTGGTG CCGATGGTGC AGGTCACTAT 15000

GTGAAGATGG TTCACAACGG TATTGAATAC GGCGATATGC AGCTGATTGC TGAAGCCTAT 15060

TCTCTGCTTA AAGGTGGCCT GAACCTCACC AACGAAGAAC TGGCGCAGAC CTTTACCGAG 15120

TGGAATAACG GTGAACTGAG CAGCTACCTG ATCGACATCA CCAAAGATAT CTTCACCAAA 15180

AAAGATGAAG AGGGTTACTA TCTGGTTGAT GTGATTCTGG ATGAAGCGGC TAACAAAGGT 15240

ACCGGTTAAT GGACCAGCCA GAGTGCGCTG GATCTCGG 15278

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 (4)

1, a kind of oligonucleotide of the O-antigen-specific to Shigella bogdii 12 types, it is characterized in that: its wzx sequence is that the Nucleotide or the wzy sequence of 6246 to 7580 bases among the SEQ ID NO:1 is the Nucleotide of 7561 to 8700 bases among the SEQID NO:1 or has above-mentioned sequence insertion, lack or replace one or more Nucleotide, but keeps wzx, wzy Nucleotide to detect the oligonucleotide of the O-antigen function of Shigella bogdii 12 types.

2, a kind of oligonucleotide of the O-antigen-specific to Shigella bogdii 12 types, it is characterized in that, it is that to come from the oligonucleotide of wzx gene right: the Nucleotide of 6435 to 6452 bases among the SEQ ID NO:1 and the Nucleotide of 7094 to 7111 bases, the Nucleotide of 6709 to 6726 bases among the SEQ ID NO:1 and the Nucleotide of 7280 to 7297 bases; Or the oligonucleotide that comes from the wzy gene is right: the Nucleotide of 7679 to 7696 bases among the SEQ ID NO:1 and the Nucleotide of 8511 to 8528 bases, the Nucleotide of 8270 to 8287 bases among the SEQ ID NO:1 and the Nucleotide of 8667 to 8684 bases.

3, the application of oligonucleotide in detecting the O-antigen of expressing the antigenic bacterium of O-, identifying bacterium of claim 1, any described O-antigen-specific to Shigella bogdii 12 types of 2.

4, according to the application of the oligonucleotide of claim 1, any described O-antigen-specific to Shigella bogdii 12 types of 2, 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, and the confession detection bodies reaches the bacterium in the environment outward.