CN1274831C - Nucleotide specific to O-antigen of shigella dysenteriae I and Bacillus coli 0121 - Google Patents

Abstract

The present invention provides a specific nucleotide for the O-antigens of Shigella dysenteriae 7 and Escherichia coli O121. The specific nucleotide is the nucleotide complete sequence of gene clusters in Shigella dysenteriae 7 for controlling the synthesis of O-antigens, such as a separated nucleotide with the total length of 17433 basic groups shown as SEQ ID NO: 1 or the nucleotide having one or a plurality of inserted, deleted or substituted basic groups and simultaneously keeping the functions of the separated nucleotide of SEQ ID NO: 1. The present invention also comprises the oligonucleotide of glycosyltransferase genes and oligonucleotide unit processing genes in O-antigen gene clusters stemmed from Shigella dysenteriae 7, and a method for obtaining bacteria O-antigen gene clusters. The present invention verifies that the specific nucleotide and the oligonucleotide have high specificity to all of the O-antigens of Shigella dysenteriae 7 and Escherichia coli O121 through PCR. The present invention also discloses a method for using the oligonucleotide of the present invention to detect and identify Shigella dysenteriae 7 and Escherichia coli O121 in human bodies and environment.

Description

Nucleotide to the O-antigen-specific of shigella dysenteriae 7 types and intestinal bacteria O121

Technical field

The present invention relates to the complete nucleotide sequence of control O-antigen synthetic gene cluster in shigella dysenteriae 7 types (Shigella dysenteriae 7), particularly relate in shigella dysenteriae 7 types oligonucleotide in the control O-antigen synthetic gene cluster, can utilize these oligonucleotide of O-antigen-specific shigella dysenteriae 7 types and the intestinal bacteria O121 (Escherichiacoli O121) 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.

O-antigen is the O specific polysaccharide composition in the gram negative bacterium lipopolysaccharides, and it is made up of many multiple oligosaccharide unit.The antigenic building-up process of O-is studied clearlyer: by glycosyltransferase nucleoside diphosphate monose is transferred on the fat molecule that is fixed on the cell inner membrance earlier, then in the inboard synthesis of oligose unit of inner membrance, the antigenic oligosaccharide unit of O-is transferred to the inner membrance outside by the transhipment enzyme again, then aggregate into polysaccharide by polysaccharase, be connected to again and form lipopolysaccharide molecule [Whitfield, C. (1995) " Biosynthesis of lipopolysaccharide Oantigens " .Trends in Microbiology.3:178-185 on the glycolipid molecule; Schnaitman, C.A.andJ.D.Klena. (1993) " Genetics of lipopolysaccharide biosynthesis inentericbacteria " .Microbiological Reviews, 57 (3): 655-682].Coding is responsible for the generally adjacent arrangement on karyomit(e) of gene of all enzyme molecules of O-antigen synthetic, form a gene cluster [Reeves, P.R., et al. (1996) " Bacterial polysaccharide synthesis and genenomenclature " Trends in Microbiology, 4:495-503].In Shigellae, intestinal bacteria 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) " TheEscherichia coli O111 and Salmonella enterica O35 gene clusters:geneclusters encoding the same colitose-containing O antigen are highlyconserved " .Journal of Bacteriology.182:5256-5261].The O-antigen gene bunch contains three genoids: sugared synthesis path gene, glycosyltransferase gene, oligosaccharide unit treatment gene.The required nucleoside diphosphate monose of enzymic synthesis O-antigen of wherein sugared synthesis path genes encoding; Thereby the enzyme of glycosyltransferase gene coding forwards nucleoside diphosphate monose and other molecule to and makes monose aggregate into oligosaccharide unit on the monose; The oligosaccharide unit treatment gene comprises transhipment enzyme gene and pol gene, and they transfer to the bacterium inner membrance outside with oligosaccharide unit, and repolymerization becomes polysaccharide.Glycosyltransferase gene and oligosaccharide unit treatment gene only are present in the gene cluster of carrying these genes.The difference of monose in the O-antigen, between monose between the difference of link button and the oligosaccharide unit difference of link button constituted the antigenic diversity of O-, and the composition of monose, the link button between monose and the link button between the oligosaccharide unit are by the Gene Handling in the O-antigen gene bunch, so the O-antigen gene bunch has determined O-antigenic synthetic, has also determined the antigenic diversity of O-.

Because O-antigen is extremely strong antigen, be one of important paathogenic factor of Shigellae, it has extremely strong diversity again simultaneously, and this enlightens us can study a kind of Shigellae and good, highly sensitive method of the antigenic specificity of O-thereof of detecting quickly and accurately.With surperficial polysaccharide is that the serology immune response of target has been used to somatotype and the evaluation to bacterium always since the thirties in last century, is unique means of identifying pathogenic bacterium.This diagnostic method needs a large amount of antiserum(antisera)s, and the antiserum(antisera) general classes is incomplete, quantity not sufficient, and also there are some difficulties in a large amount of antiserum(antisera)s in preparation with in storing.On the other hand this method length consuming time, sensitivity is low, loss is high, poor accuracy, so, generally believe that now this traditional serology detection method will be that the modern molecular biology method replaces.1993, Luk, J.M.C et.al has identified the O-antigen [Luk of Salmonellas with the specific nucleotide sequence of Salmonellas (S.enterica) O-antigen gene bunch by PCR method, J.M.C.et.al. (1993) " Selective amplification ofabequose and paratose synthase genes (rfb) by polymerase chain reactionfor identification of S.enterica major serogroups (A; B; C2; andD) ", J.Clin.Microbiol.31:2118-2123].Luk, the method for et.al is with corresponding to Salmonellas serotype E 1, D1 obtains the oligonucleotide special to the Salmonellas of different serotypes after the nucleotide sequence of the CDP-abequose in the A, the O-antigen of B and C2 and the synthetic gene of CDP-tyvelose is arranged.1996, Paton, the A.W et.al serotype [" Molecularmicrobiological investigation of an outbreak of Hemolytic-UremicSyndrome caused by dry fermented sausage contaminatedwith Shiga-like toxin producing Escherichiacoli " .J.Clin.Microbiol.34:1622-1627] of having identified the toxogenic E.coli O111 of a strain at the oligonucleotide that comes from the wbdI gene of the O-antigen-specific of E.coli O111, but afterwards studies show that Paton, the usefulness of A.Wet.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.and Reeves of a supposition, P.R. (1995) Sequence andanalysis of the O antigen gene (rfb) cluster of Escherichia coliO111.Gene 164:17-23], and have this sugar in the antigenic structure of the O-of other bacterium, so sugared synthesis path gene is not a high special for O-antigen yet.

Shigellae has 46 kinds of serotypes, but have only 33 kinds of different O-antigens, intestinal bacteria have 166 kinds of different O-antigen [Reeves, P.R (1992) " Variation in O antigens; nichespecific selection and bacterial populations " .FEMSMicrobiol.Lett, 100:509-516], the two sibship is very near, and it is that intestinal bacteria and Shigellae are total that 12 kinds of O-antigens are arranged, wherein shigella dysenteriae 7 types just have identical O-antigen [Ewing with intestinal bacteria O121, W.H. (1986) " Edwards and Ewing ' s identificationof the Enterobacteriaceae " .Elsevier SciencePublishers, Amsterdam, The Netherlands; T.cheasty, et al. (1983) " Antigenic relationships between the enteroinvasive Escherichiacoli antigens O28ac; O112ac; O124, O136, O143; O144; O152 and O164 andShigella O antigens " J.clin Microbiol, 17 (4): 681-684], traditional serotype method can not be distinguished them.

Summary of the invention

The Nucleotide that the purpose of this invention is to provide a kind of O-antigen-specific to shigella dysenteriae 7 types and intestinal bacteria O121, it is the Nucleotide in the O-antigen gene bunch of shigella dysenteriae 7 types, is the special Nucleotide that comes from glycosyltransferase gene and transhipment enzyme gene and pol gene.

A time purpose of the present invention has provided the full length nucleotide sequence of the O-antigen gene bunch of shigella dysenteriae 7 types.

Another object of the present invention has provided the gene of the O-antigen gene bunch that constitutes shigella dysenteriae 7 types: the gene of transhipment enzyme is the wzx gene or with wzx the gene of identity function is arranged; Pol gene is the wzy gene or with wzy the gene of identity function is arranged; Glycosyltransferase gene comprises orf9, orf13, orf14 gene; Sugar synthesis path gene comprises orf1, orf2, rmlB, rmlA, vioA, orf6, orf7, orf10, orf12.

Another purpose of the present invention has provided oligonucleotide, and the gene that they come from encoding glycosyl transferring enzyme in the O-antigen gene bunch of shigella dysenteriae 7 types respectively comprises orf9, orf13, orf14 gene; The gene that coming from coding transhipment enzyme be the wzx gene or with wzx the gene of identity function, the gene that comes from the coding polysaccharase are arranged is the wzy gene or with wzy the gene of identity function is arranged; They are the oligonucleotide in the said gene, and length is at 10-20nt; They are special to the O-antigen of shigella dysenteriae 7 types and intestinal bacteria O121; Especially the oligonucleotide of listing in the table 1, they are high specials to the O-antigen of shigella dysenteriae 7 types and intestinal bacteria O121, and these oligonucleotide are also reconfigurable, and the oligonucleotide after the combination also is a high special to the O-antigen of shigella dysenteriae 7 types and intestinal bacteria O121.

The above-mentioned oligonucleotide that a further object of the present invention provides can be used as primer and is used for nucleic acid amplification reaction, perhaps be used for hybridization as probe, perhaps be used to make gene chip or microarray, thereby detect and identify O-antigen and detection and evaluation shigella dysenteriae 7 types and the intestinal bacteria O121 of shigella dysenteriae 7 types and intestinal bacteria O121 by these methods.

An also purpose of the present invention has provided the method for the complete sequence of the O-antigen gene bunch that separates shigella dysenteriae 7 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 dysenteriae 7 types and intestinal bacteria O121 it is the isolating Nucleotide shown in SEQ ID NO:1,17433 bases of total length; The base that perhaps has one or more insertions, disappearance or replacement keeps the Nucleotide of the SEQ ID NO:1 of described isolating functional nucleotide simultaneously.

The Nucleotide of aforesaid O-antigen-specific to shigella dysenteriae 7 types and intestinal bacteria O121 is characterized in that it is by 14 genomic constitutions, all between galF gene and gnd gene.

The Nucleotide of aforesaid O-antigen-specific to shigella dysenteriae 7 types and intestinal bacteria O121 is characterized in that described gene comprises: about transhipment enzyme gene, comprise 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 orf9, orf13, orf14 gene; Wherein, described wzx gene is the Nucleotide of 7800 to 9194 bases among the SEQID NO:1; The orf9 gene is the Nucleotide of 9200 to 10090 bases among the SEQ ID NO:1; The wzy gene is the Nucleotide of 10646 to 11836 bases among the SEQ ID NO:1; The orf13 gene is the Nucleotide of 13691 to 14848 bases among the SEQ ID NO:1; The orf14 gene is the Nucleotide of 14849 to 15973 bases among the SEQ ID NO:1.

The Nucleotide of aforesaid O-antigen-specific to shigella dysenteriae 7 types and intestinal bacteria O121 is characterized in that it is to come from described wzx gene, wzy gene or glycosyltransferase gene orf9, orf13, orf14 gene; Or the oligonucleotide in the sugared synthesis path gene; And their mixing or their reorganization.

The Nucleotide of aforesaid O-antigen-specific to shigella dysenteriae 7 types and intestinal bacteria O121, it is characterized in that the oligonucleotide of the described orf9 of coming from gene is to being: the Nucleotide of 9336 to 9353 bases among the SEQ ID NO:1 and the Nucleotide of 11117 to 10034 bases; The Nucleotide of 9448 to 9465 bases among the SEQ ID NO:1 and the Nucleotide of 9864 to 9881 bases; The Nucleotide of 9274 to 9292 bases among the SEQ ID NO:1 and the Nucleotide of 9746 to 9763 bases; The oligonucleotide that comes from the wzy gene is to being: the Nucleotide of 11120 to 11137 bases among the SEQ ID NO:1 and the Nucleotide of 11366 to 11383 bases; The Nucleotide of 10957 to 10976 bases among the SEQ ID NO:1 and the Nucleotide of 11547 to 11564 bases; The Nucleotide of 10707 to 10725 bases among the SEQ ID NO:1 and the Nucleotide of 11767 to 11785 bases; The oligonucleotide that comes from the orf13 gene is to being: the Nucleotide of 14084 to 14101 bases among the SEQ ID NO:1 and the Nucleotide of 14420 to 14437 bases; The Nucleotide of 13881 to 13899 bases among the SEQ ID NO:1 and the Nucleotide of 14761 to 14798 bases; The Nucleotide of 13975 to 13992 bases among the SEQ ID NO:1 and the Nucleotide of 14361 to 14379 bases; The oligonucleotide that comes from the orf14 gene is to being: the Nucleotide of 15225 to 15244 bases among the SEQ ID NO:1 and the Nucleotide of 15765 to 15782 bases; The Nucleotide of 14975 to 14993 bases among the SEQ ID NO:1 and the Nucleotide of 15911 to 14930 bases; The Nucleotide of 15011 to 15030 bases among the SEQ ID NO:1 and the Nucleotide of 15688 to 15706 bases;

The Nucleotide of aforesaid O-antigen-specific to shigella dysenteriae 7 types and intestinal bacteria O121 is detecting the application of expressing the antigenic bacterium of O-, identify other polysaccharide antigen of the O-antigen of bacterium and bacterium in diagnosis.

The recombinant molecule of the Nucleotide of aforesaid O-antigen-specific to shigella dysenteriae 7 types and intestinal bacteria O121, and can provide the O-antigen of expressing shigella dysenteriae 7 types and intestinal bacteria O121 by inserting to express, and become bacterial vaccine.

The application of the Nucleotide of aforesaid O-antigen-specific to shigella dysenteriae 7 types and intestinal bacteria O121, 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, the bacterium in human body and the environment as probe as primer.

The separation method of the Nucleotide of aforesaid O-antigen-specific to shigella dysenteriae 7 types and intestinal bacteria O121 is characterized in that it comprises the steps:

(1) genomic extraction: 37 ℃ of incubated overnight Shigellaes 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 that adds 3ul20mg/ml afterwards, 15ul 10%SDS, 50 ℃ of incubations 2 hours, the RNase that adds 3ul 10mg/ml again, 65 ℃ 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) 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 dysenteriae 7 types bunch: the O-antigen gene of shigella dysenteriae 7 types is bunch by the Long pcr amplification, at first according to the JumpStart sequences Design upstream primer (#1523-ATT GTG GCT GCA GGG ATCAAA GAA AT) that often is found in O-antigen gene bunch promoter region, again according to the gnd gene design downstream primer (#1524-TAGTCG CGT GNG CCT GGA TTA AGT TCG C) in O-antigen gene bunch downstream.With the ExpandLong 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, 60 ℃ of annealing 30 seconds, and 68 ℃ were extended 15 fens, and carried out 30 circulations like this.At last, continue to extend 7 fens 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, use isopyknic phenol and phenol respectively: chloroform: primary isoamyl alcohol (25: 24: 1) extracting once, use twice of isopyknic ether extracting again after, with the dehydrated alcohol deposit D NA of 2.5 times of volumes, and wash precipitation with 70% ethanol, be resuspended at last in the 18ul water.In this mixture, add 2.5uldNTP (1mMdCTP subsequently, 1mMdGTP, 1mMdTTP, 10mMdATP), the T4DNA polysaccharase of 1.25ul 100mM DTT and 5 units, 11 ℃ 30 minutes, enzyme is cut product mend into flush end, after 75 ℃ of termination reactions, add the Tth archaeal dna polymerase of 5 units and corresponding damping fluid thereof and system is expanded as 80ul, 70 ℃ were reacted 20 fens, made the 3 ' end of DNA add the dA tail.This mixture is through the equal-volume chloroform: after primary isoamyl alcohol (24: 1) 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; Preparation method with the electric transformed competence colibacillus cell of BiO-Rad company prepares competence escherichia coli DH5a cell, get after 2-3ul connects product and 50ul competence escherichia coli DH5a 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 to 6.0 milliseconds, the SOC substratum that adds 1ml after the electric shock immediately in cup makes the bacterium recovery, then bacterium is coated in and contains penbritin, on the LB solid medium of X-Gal and IPTG, 37 ℃ of incubated overnight, obtain blue white bacterium colony next day, with the white colony that obtains promptly the white clone forward on the LB solid medium that contains acillin and cultivate, from each clone, extract plasmid simultaneously, and cutting the segmental size of evaluation insertion wherein with the EcoRI enzyme, the white that obtains clone group has constituted the O-antigen gene bunch library of dysentery will Hayes 7 types;

(4) to the cloning and sequencing in the library: from the library, select insert 120 clones of fragment more than 700bp 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.According to the sequences Design primer that has obtained, this primer is as follows again for the sequence of residue 20%:

Upstream primer 5 '-GCATGGGTTACTGTACTAGC-3 ' and downstream primer 3 '-AATGGCATCAATACCCGC-5 ' reaches

Upstream primer 5 '-TGGCGGTATTGAGAGAGT-3 ' and downstream primer 3 '-TTACAGGCTACTTCTCTTC-5 '

Direct PCR and from the genomic dna of shigella dysenteriae 7 types again to the order-checking of PCR product, thus all sequences of O-antigen gene bunch obtained.

(5) splicing of nucleotide sequence and analysis: the Pregap4 and the splicing of Gap4 software of the Staden package software package of publishing with Britain Camb MRC (Medical Research Council) Molecular Biology Lab and edit all sequences, thereby obtain the Nucleotide full length sequence of the O-antigen gene bunch of shigella dysenteriae 7 types, the quality of sequence is mainly guaranteed by two aspects: 1) genome of shigella dysenteriae 7 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.After obtaining the nucleotide sequence of shigella dysenteriae 7 type O-antigen genes bunch, with American National biotechnology information science center (The National Center forBiotechnology Information, NCBI) orffinder finds gene, find the reading frame of 14 openings, determine also that with the function of finding the reading frame that these are open what gene they are with the genetic comparison among the blast groupware and the GenBank, finish gene annotation with the Artemis software at Britain sanger center again, do accurate comparison between DNA and protein sequence with Clustral W software, obtain the structure of the O-antigen gene bunch of shigella dysenteriae 7 types at last, as shown in table 3.

(6) screening of specific gene: at wzx, wzy, orf9, orf13, the orf14 gene design primer in the O-antigen gene of shigella dysenteriae 7 types bunch; Respectively designed three 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, all primers all obtain positive findings in shigella dysenteriae 7 types and intestinal bacteria O121, except that wzx, the correct band of any size does not all increase in other groups, promptly do not obtaining any PCR product band in the array mostly, though in the minority group, obtain PDR product band, but its size does not meet the expection size, in 1 strain intestinal bacteria, can obtain the specific amplification of wzx, begin at the colibacillary special row research of this strain.So wzy, orf9, orf13, orf14 gene pairs shigella dysenteriae 7 types and intestinal bacteria O121 and O-antigen thereof all are high specials.

Just, first aspect of the present invention provides the full length nucleotide sequence of the O-antigen gene bunch of shigella dysenteriae 7 types, its complete sequence shown in SEQ ID NO:1,17433 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 (listing) of the O-antigen gene bunch of shigella dysenteriae 7 types by method of the present invention in table 3, it is altogether by 11 genomic constitutions, all between galF gene and gnd gene.

Second aspect of the present invention provides the gene in the O-antigen gene bunch of shigella dysenteriae 7 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 orf9, orf13, orf14 gene; Special sugared synthesis path gene in the bacterial polysaccharides antigen comprises orf1, orf2, and rmlB, rmlA, vioA, orf6, orf7, orf10, the orf12 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 glycosyltransferase gene, transhipment enzyme gene and pol gene, because sugared synthesis path gene is that the gene of synthetic nucleosides bisphosphate monose is common, common by indication to more exocellular polysaccharide now, O-antigen to bacterium is not very special, and the glycosyltransferase gene that the present invention relates to, transhipment enzyme gene and pol gene are high specials to the O-antigen of shigella dysenteriae 7 types and intestinal bacteria O121.

The 3rd aspect of the present invention, wzx gene in the O-antigen gene bunch that comes from shigella dysenteriae 7 types is provided or the gene, wzy gene of identity function is arranged or the gene and the glycosyltransferase gene of identity function are arranged with wzy with wzx, the oligonucleotide that comprises orf9, orf13, orf14 gene, they are any one section oligonucleotide in these genes.But, be that the oligonucleotide of listing in the table 1 is right preferentially by usefulness, in table 1, also listed these oligonucleotide to the position in O-antigen gene bunch and with these oligonucleotide to being the size of the product of the PCR reaction done of primer, the annealing temperature in these PCR reaction free lists is carried out.These primers are being to obtain expecting the product of size in the pcr amplification that carries out of template with shigella dysenteriae 7 types and intestinal bacteria O121 only, and are all not obtain expecting the product of size in the pcr amplification that carries out of template the 166 strain intestinal bacteria listed with table 2 and 43 strain Shigellaes.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 dysenteriae 7 types and intestinal bacteria O121 and their O-antigen.

The separation method of the Nucleotide of the O-antigen-specific to shigella dysenteriae 7 types and intestinal bacteria O121 provided by the invention comprises the steps: (1) genomic extraction; (2) by the O-antigen gene in the pcr amplification shigella boydii 7 bunch; (3) make up O-antigen gene bunch library; (4) to the cloning and sequencing in the library; (5) splicing of nucleotide sequence and analysis; (6) screening of specific gene.

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

As used herein, " oligonucleotide " is meant the gene that derives from the encoding glycosyl transferring enzyme in the O-antigen gene bunch, the gene of coding transhipment enzyme and intragenic one section nucleic acid molecule of coding polysaccharase, they can change on length, generally change in 10 to 20 Nucleotide scopes.More precisely these oligonucleotide are to come from wzx gene (nucleotide position is 7800 to 9194 bases from SEQ ID NO:1), orf9 gene (nucleotide position is 9200 to 10090 bases from SEQ ID NO:1), wzy gene (nucleotide position is 10646 to 11836 bases from SEQ ID NO:1), orf13 gene (nucleotide position is 13691 to 14848 bases from SEQ ID NO:1), orf14 gene (nucleotide position is 14849 to 15973 bases from SEQID NO:1).Coming from above intragenic oligonucleotide all is high special to shigella dysenteriae 7 types (SEQ ID NO:1) and intestinal bacteria O121.

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

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

The present invention relates to a kind of antigenic method of one or more bacterial polysaccharideses that detects in the food, these antigens can make sample can with the oligonucleotide specific hybrid of following at least one gene, these genes are: (i) gene of the encoding glycosyl transferring enzyme gene of transhipment enzyme and polysaccharase of (ii) encoding comprises the wzx gene or the gene, wzy gene of identity function is arranged or with wzy the gene of identity function is arranged with wzx.At least one oligonucleotide can be hybridized with at least one more than one such gene specific of expressing the special antigenic bacterium of O-under the situation of condition permission, and these bacteriums are shigella dysenteriae 7 types or intestinal bacteria O121.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, or 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 the gene that comes from the encoding glycosyl transferring enzyme, the gene of coding transhipment enzyme and the gene of polysaccharase, comprises the wzx gene or the gene, wzy gene of identity function is arranged or with wzy the oligonucleotide of the gene of identity function is arranged with wzx.This cover oligonucleotide is special to the O-antigen of a special bacterium, and this special bacterium O-antigen is expressed by shigella dysenteriae 7 types or intestinal bacteria O121.

On the other hand, the present invention relates to a kind of antigenic method of one or more bacterial polysaccharideses that detects in the movement, these antigens can make sample can with the oligonucleotide specific hybrid of following at least one gene, these genes are: (i) gene of the encoding glycosyl transferring enzyme gene of transhipment enzyme and polysaccharase of (ii) encoding comprises the wzx gene or the gene, wzy gene of identity function is arranged or with wzy the gene of identity function is arranged with wzx.At least one oligonucleotide can be expressed more than one such gene specific hybridization of the special antigenic bacterium of O-with at least one under the situation of condition permission.These bacteriums are shigella dysenteriae 7 types or intestinal bacteria O121.Oligonucleotide among available the present invention is made the method test sample of primer by PCR, also can with behind the oligonucleotide molecules mark among the present invention as probe by hybridization such as southern-blot or fluoroscopic examination, perhaps by antigen and bacterium in gene chip or the microarray assay sample.

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

On the other hand, the present invention also relates to the antigenic method of one or more bacterial polysaccharideses in the sample that a kind of detection comes from patient.One or more bacterial polysaccharides antigens in the sample can make sample can with a specific hybrid in a pair of oligonucleotide in following at least one gene, these genes are: (i) gene of the encoding glycosyl transferring enzyme gene of transhipment enzyme and polysaccharase of (ii) encoding comprises the wzx gene or the gene, wzy gene of identity function is arranged or with wzy the gene of identity function is arranged with wzx.Under the situation of condition permission at least one oligonucleotide can with sample at least one express more than one such gene specific hybridization of the special antigenic bacterium of O-, these bacteriums are shigella dysenteriae 7 types or intestinal bacteria O121.Oligonucleotide among available the present invention is made the method test sample of primer by PCR, also can pass through hybridization as probe behind the oligonucleotide mark among the present invention, or passes through the antigen and the bacterium of gene chip or microarray assay sample.

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

The present inventor believes that the present invention is not necessarily limited to the above nucleotide sequence coded specific O-antigen of carrying, and is widely used in detecting all expression O-antigens and identifies the antigenic bacterium of O-.And because O-antigen is synthetic and the similarity of other polysaccharide antigens (as bacterium born of the same parents exoantigen) between synthesizing, the inventor believes that 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 dysenteriae 7 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 dysenteriae 7 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 Shigellaes in the LB of 5mL substratum, centrifugal collecting cell.With 500ul50mM 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 ℃ 30 minutes.Add equal-volume phenol extracting mixture, get supernatant liquor and use isopyknic phenol again: chloroform: primary isoamyl alcohol (25: 24: 1) 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 dysenteriae 7 types bunch.

The O-antigen gene of shigella dysenteriae 7 types bunch is by the Long pcr amplification.At first according to the JumpStart sequences Design upstream primer (#1523-ATTGTG GCT GCA GGG ATC AAA GAA AT) that often is found in O-antigen gene bunch promoter region, again according to the gnd gene design downstream primer (#1524-TAG TCG CGT GNG CCT GGA TTA AGT TCG C) in O-antigen gene bunch downstream.With the Expand Long Template PCR method of BoehringerMannheim company amplification O-antigen gene bunch, the PCR response procedures was as follows: 94 ℃ of pre-sex change 2 minutes; 94 ℃ of sex change are 10 seconds then, 60 ℃ of annealing 30 seconds, and 68 ℃ were extended 15 fens, and carried out 30 circulations like this.At last, continue to extend 7 fens 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, use isopyknic phenol and phenol respectively: chloroform: primary isoamyl alcohol (25: 24: 1) extracting once, use twice of isopyknic ether extracting again after, with the dehydrated alcohol deposit D NA of 2.5 times of volumes, and wash precipitation with 70% ethanol, be resuspended at last in the 18ul water.In this mixture, add 2.5uldNTP (1mMdCTP subsequently, 1mMdGTP, 1mMdTTP, 10mMdATP), the T4DNA polysaccharase of 1.25ul 100mM DTT and 5 units, 11 ℃ 30 minutes, enzyme is cut product mend into flush end, after 75 ℃ of termination reactions, add the Tth archaeal dna polymerase of 5 units and corresponding damping fluid thereof and system is expanded as 80ul, 70 ℃ were reacted 20 fens, made the 3 ' end of DNA add the dA tail.This mixture is through the equal-volume chloroform: after primary isoamyl alcohol 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-6.0ms millisecond.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, also cutting evaluation with the EcoRI enzyme wherein inserts segmental size to extract plasmid simultaneously from each clone, and the white clone group who obtains has constituted the O-antigen gene bunch library of shigella dysenteriae 7 types.

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

From the library, select insert 120 clones of fragment more than 700bp 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 again according to the sequences Design primer that has obtained, direct PCR and from the genomic dna of shigella dysenteriae 7 types again to the order-checking of PCR product, thus obtain all sequences of O-antigen gene bunch.We have designed two pairs of primers in shigella dysenteriae 7 types, and are as follows:

Upstream primer 5 '-GCATGGGTTACTGTACTAGC-3 ' and downstream primer 3 '-AATGGCATCAATACCCGC-5 '

And upstream primer 5 '-TGGCGGTATTGAGAGAGT-3 ' and downstream primer 3 '-TTACAGGCTACTTCTCTTC-5 '

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 dysenteriae 7 types obtained.The quality of sequence is mainly guaranteed by two aspects: 1) genome of shigella dysenteriae 7 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.After obtaining the nucleotide sequence of shigella dysenteriae 7 type O-antigen genes bunch, with American National biotechnology information science center (The National Center for BiotechnologyInformation, NCBI) orffinder finds gene, find the reading frame of 14 openings, determine also that with the function of finding the reading frame that these are open what gene they are with the genetic comparison among the blast groupware and the GenBank, finish gene annotation with the Artemis software at Britain sanger center again, do accurate comparison between DNA and protein sequence with Clustral W software, obtain the structure of the O-antigen gene bunch of shigella dysenteriae 7 types at last, as shown in table 3.

By retrieving and relatively, finding that orf 1 and the synthetic enzyme ORF1S gene of Shigella sonnei all have 71% homogeny in 426 amino acid whose sequences, show the homology that height is arranged between them.So can determine orf 1 is synthase gene.Orf 2 has 70% homogeny with the synthetic enzyme ORF2S gene of Shigella sonnei in 339 amino acid whose sequences, show the homology that height is arranged between them.So can determine orf 2 is synthase genes.Orf 3 has 71% homogeny with the rmlB gene of Salmonella enterica in 360 amino acid whose sequences, show the homology that height is arranged between them.So, can determine that orf 3 is rmlB genes.Orf 4 has 72% homogeny with the rmlA gene of Aeromonas hydrophila in 287 amino acid whose sequences, show the homology that height is also arranged between them.So, can determine that orf 4 is rmlA genes.Orf8 and colibacillary wzx gene have 21% homogeny in 307 aminoacid sequences, and the algorithm by people such as Eisenberg

[Eisenberg, D, Schwarz, E.et al (1984) .Analysis of membrane andsurface protein sequences with the hydrophobic momentplot.J.Mol.Biol.179:125-142] find that orf8 has 12 potential transmembrane domains, it and many wzx protein similars, and about 50 amino acid whose conservative motifs are arranged at the proteic aminoterminal of wzx, and be the wzx gene so can determine orf8, called after wzx.The glycosyltansferase gene of Orf9 and Clostridiumacetobutylicum has 27% homogeny in 238 amino acid, be a glycosyltransferase gene, called after orf9.The acetyltransferase gene of Orf10 and Thermotoga maritima has 39% homogeny in 171 aminoacid sequences, 59% similarity is so determine that orf10 is an acetyltransferase gene, called after orf10.Orf11 and colibacillary wzy gene have 22% homogeny in 366 aminoacid sequences, learn that by the Eisenberg algorithm orf11 has 10 potential transmembrane domains in addition, to other O-antigen polysaccharase similar secondary structure is arranged, so determine that orf11 is exactly the wzy gene, called after wzy.The synthetic enzyme wbpS gene of Orf12 and Pseudomonas aeruginosa has 56% homogeny in 628 aminoacid sequences, 72% similarity is so determine that orf12 is a synthetic enzyme enzyme gene, called after orf12.The glycosyltansferase gene of Orf13 and Pseudomonasaeruginosa has 46% homogeny in 373 aminoacid sequences, 68% similarity is a glycosyltransferase gene so can determine orf13, called after orf13.The glycosyltansferase gene of Orf14 and Pseudomonas aeruginosa has 54% homogeny in 372 aminoacid sequences, 71% similarity is a glycosyltransferase gene so can determine orf14, called after orf14.

Embodiment 6: the screening of specific gene.At wzx, wzy, orf9, orf13, orf14 gene design primer in the O-antigen gene of shigella dysenteriae 7 types bunch, the position of these genes in nucleotide sequence sees Table 1.

Table 1 has been listed glycosyltransferase gene and oligosaccharide unit treatment gene and intragenic primer and PCR data in the O antigen gene bunch of shigella dysenteriae 7 types.Glycosyltransferase gene, transhipment enzyme gene and pol gene and their function corresponding and the size of the O antigen gene bunch of shigella dysenteriae 7 types in table, have been listed.In each gene, we have respectively designed three pairs of primers, and the difference that every pair of primer is distributed in the corresponding gene is local to guarantee its specificity.In table, also listed position and the size of each primer in SEQ IDNO:1.Is that template carry out PCR with listed corresponding annealing temperature in the table with the genomes of all bacterium in the table 2 with every pair of primer, has obtained corresponding PCR product, and its size is also listed in the table.

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 #101 (TTC ATC CTAAAC TCC TTA TT) and #102 (TAA TCG CAG GGG AAA GCA GG), extract genome then from 166 strain intestinal bacteria, method as previously mentioned.With this to primer from the colibacillary genome of 166 strains PCR with identification of escherichia coli and detect its genome quality.

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

The genomic dna that contains shigella dysenteriae 7 types in the 25th group is as positive control.Do template with every group of bacterium, be PCR by following condition with every pair in the table 1 primer: 94 ℃ of pre-sex change after 2 minutes, 94 ℃ of sex change 15 seconds, annealing temperature is because of the difference different (with reference to table 1) of primer, annealing time is 50 seconds, and 72 ℃ were extended 2 fens, and carried out 30 circulations like this.Continue to extend 10 fens at 72 ℃ at last, reaction system is 25ul.After reaction finishes, get the 10ulPCR product and detect the fragment that amplifies by 0.8% agarose gel electrophoresis.

For wzx, wzy, orf9, orf13, orf14 gene, each gene all has 3 pairs of primers detected, every pair of primer has obtained also all obtaining onesize specificity band the correct band of expection size in the 3rd group except be PCR in the 25th group after.After being template PCR with the genomic dna of each bacterium in the 3rd group, find only in intestinal bacteria O121, to have obtained positive findings.In more detail, each of each gene all obtains expecting the correct PCR product band of size to primer more than in intestinal bacteria O121.It is reported that the O-antigenic structure of shigella dysenteriae 7 types and intestinal bacteria O121 is the same, our result has confirmed this point from another side.In addition, all primers are the correct band of any size that all do not 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, orf9, orf13, orf14 gene pairs shigella dysenteriae 7 types and intestinal bacteria O121 and O-antigen thereof all are high specials.

At last, from shigella dysenteriae 7 types, screen gene by PCR: wzy and four glycosyltransferase genes to the O-antigen high special of shigella dysenteriae 7 types and intestinal bacteria O121.And the oligonucleotide of these intragenic any one section 10-20nt is special to the O-antigen of shigella dysenteriae 7 types and intestinal bacteria O121, and the primer in especially above-mentioned each gene is that oligonucleotide is high special to detecting the back confirmation through PCR to shigella dysenteriae 7 types and intestinal bacteria O121.These all oligonucleotide all can be used for shigella dysenteriae 7 types and the intestinal bacteria O121 in the human body and environment rapidly and accurately, and can identify their O-antigen.

Table 3 is structural tables of the O-antigen gene bunch of shigella dysenteriae 7 types, in table, listed the structure of the O-antigen gene bunch of shigella dysenteriae 7 types, altogether by 14 genomic constitutions, each gene box indicating, and in square frame, write the title of gene, numeral be the order of the open reading frame (orf) in the O-antigen gene bunch.Two ends at O-antigen gene bunch are galF gene and gnd gene, and these two not responsible O-of gene are antigenic synthetic, 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 dysenteriae 7 types, in table, listed the accurate position of all open reading frame in complete sequence in the O-antigen gene bunch of shigella dysenteriae 7 types, at the underscoring of the initiator codon and the terminator codon of each open reading frame.

Sequence list

SEQUENCE LISTING

＜110〉Nankai University

＜120〉to the Nucleotide of the O-antigen-specific of shigella dysenteriae 7 types and intestinal bacteria O121

＜130〉to the Nucleotide of the O-antigen-specific of shigella dysenteriae 7 types and intestinal bacteria O121

<160>1

<170>PatentIn version 3.1

<210>1

<211>17433

<212>DNA

<213>Shigella dysenteriae

<400>1

ctcctggtaa ctcacgcgtc caagaacgcg gtcgaaaacc acttcgacac ctcttatgaa 60

ttagaatctc tccttgaact gcgcgtgaag cgtcaactgc tggcggaagt acagtccatt 120

tgcccgccgg gcgtgacaat tatgaacgtg cgtcagggtg aacctttagg tttgggccac 180

tccattttat gtgcacgacc tgccattggt gacaatccat ttgtcgtggt gctgccagac 240

gttgtgatcg acgacgccag cgccgacccg ctgcgctaca accttgctgc catgattgcg 300

cgcttcaacg aaacgggccg cagccaggtg ctggcaaaac gtatgccggg tgacctctct 360

gaatactcct tcatccagac caaagaacct ctggatcgcg aaggtaaagt cagccgcatt 420

gttgaattca tcgaaaaacc ggatcagcca caaacgctgg aatcagacat catggccgtg 480

ggccgttatg tgctttctgc cgatatttgg ccggaacttg aacgcactca gccaggtgca 540

tggggacgta ttcagctgac tgatgccatt gccgaactgg cgaaaaaaca gtctgttgac 600

gccatgctga tgactggtga cagctacgac tgtggtaaaa aaatgggtta tatgcaggcg 660

tttgtgaagt atggactacg caacctgaaa gaaggagcga agttccgcaa aggtattgag 720

aaattgctta gcgagtaagt ttaaaaaata gacgccctta tagggcgtaa taacaaataa 780

cggtagtcaa cattcgacgc ggtgatgcag atatgcccgg aatgctgata ccgttttttc 840

attctaaaaa actcatcatt tcattgagtt aactacaaaa tttagcactg ttttttataa 900

tgtttcttct tgtttctggc atcaattggt aagataatta gtgtttgagt ttagaggctt 960

tgcggcagag aagcggagct taacacgtct gtgagagtac gcagtgcact ggtagctgta 1020

aagccagtgg cggtagcgtg tttaaataaa tacattagta ctactacata ttacatcatt 1080

gtaggctatt taagcgctac atgttaagcg acagcgctag caatcaaatc ttttaaagtt 1140

acttctcagg aatagtaaaa tgagctttaa tcttgaaaat tataaaatag gaattattgg 1200

gctgggttac gttggcttac ctttagctgt tgaatttggc aaaaaaataa agactcttgg 1260

ttttgatatc aacgagaaac ggataaaaga attatcagca ggtaatgact ttacgcttga 1320

atgtagttct gatgaactgg ctcaagctta ctatcttcaa tattcatgtg attataaaga 1380

attagcgact tgcaatgtat tcattgtgac ggtacctact ccaattgatg aatataaaca 1440

acctgattta acacctcttg ttaaagcctc ggaaacactc ggaaaaataa tcaaaaaaga 1500

tgatattatt atctacgaat caactgttta tccaggtgca acagaagaag tatgtatccc 1560

agtaattgag aagatctcag gtctggtatt taatatagat ttttttgccg gttacagccc 1620

ggaacgcatt aatccgggtg acaaagagca ccgcgtgaca acgatcaaaa aagtaacttc 1680

tggttcaacg cctgaaattg ctgagattgt cgataaatta tatggaacca tcatcaccgc 1740

tggaacgcat aaagcatctt cgatacgtgt ggcggaagcg gctaaagtga ttgagaacac 1800

ccagcgtgat ttgaatattg ctctgattaa cgaacttgca atgattttca ataagctggg 1860

gattgatacg gaggaagtat taaaagccgc aggcactaaa tggaacttct taccattcag 1920

accaggctta gttggggggc attgcatcgg agttgatccc tactatttga ctcataaagc 1980

gcaagcgata ggttatcatc cagaagtcat cctggcgggg cgtagaatta atgatggaat 2040

gagtaaatat gtagcatcac agttcattaa agccatgctc aagaaaaaat tccaaattga 2100

aggtgctaat gtcttaatta tggggcttac ctttaaagaa aactgtccgg atcttcgtaa 2160

tacaaaagtt gtagatatct tggctgaact gaaagaatac aacgtgaacg tggatattta 2220

tgatccatgg gttgatgttg aagaagccta ttatgagtat gggattaatg tattaaacac 2280

attacctgat aagaaatatg atggagtgat ctttgcagtt gctcactctc aatttaaaaa 2340

tatgagtaaa gaagaagtcc atggaataat tagaaatgat ggtgttattt atgatttgaa 2400

atacatcatt gaccctaact tagtagatat tcgtctttaa tcaatacttg atgcaatgtg 2460

tcagagagga tgcctaatga aatataaaaa aatagaagac gctttgtctt ctaaatctga 2520

aacatggttg attacaggtg ttgcgggatt tatcggttct aacctattag aggccttact 2580

aaaacttaat caaaaagtag ttggcttaga taattttgca actggacatc aagcaaacct 2640

tgatgaggtt aaagagtctg tttcagctga acaatggtct gcttttacct ttgtggaagg 2700

agacatatgt gagccagaga cgtgtgtgga ggttgttaaa ggagttgatc atgtcctaca 2760

tcaggctgca ttaggttctg tgcctcgttc aattaaagat ccaattacta caaataatac 2820

caatatatcg ggtttcatta atatgttggt tgccgccaaa gatgccaatg tcagaagttt 2880

cacttacgct gcaagtagct caacgtatgg tgatcatcct gcattaccta aaattgaaga 2940

aaatataggt aatccgctat ctccatacgc agtgacaaag tatgtaaatg aattatatgc 3000

gcaagtattt gcaagaacct acggattcaa atcaattgga ttaagatact ttaatgtatt 3060

tggaaaacgc caggatccta atggggcata tgctgctgtg ataccgaagt ggactgctgc 3120

aatgatcaat gatgaaccgc tgtatatcaa tggtgatggc gaaacaagta gagacttttg 3180

ttttattgag aatgtcgtgc aaatgaatat tcttgctgca cagtctgatg cgtctgcaag 3240

agatcaggtt tataacgttg cagttggtga ccgtactacg cttaaacaat tattcactgt 3300

attaaagaat gcacttaatg aaaatggtgt ttcgtataac aaagaacctg aatataaaga 3360

ttttcgtgca ggtgatgttc gccattcgca agctgatatc actaaagcca aaactagact 3420

tggatacgag ccacagttca gtatatcgga gggcatcctc aaagcgatgc cgtggtatgt 3480

aaactctctg ggcaacaaaa aataaccatg aaaatcctta ttacaggtgg tgctggtttt 3540

attgggtcag ctgttattag atacatcatt aatcatacta atgacagtgt cgtaaatgtc 3600

gataaattga cttacgcagg aaaccttcaa tcacttaaag atatagaaaa aaatacgcgc 3660

tataaatttg agtgcgcaga tatctgcgat agcgcaaaga tgcaagagat atttaccaat 3720

catcaaccag atgctgttat gcatttggcc gctgagagtc atgttgatcg gtcaattaat 3780

ggcccgtttg agtttataca gaccaatatc ataggcacct ttatcttact tgaaatagcc 3840

aagtcttact ggctgcaact tagtgaaagc aaaaaaaaga agtttatttt tcatcatgtt 3900

tcgacagatg aagtgtatgg tgatctagct catccagatg tactggatga aggttcacca 3960

ttgcctttat ttactgaaaa aaatggctac gcgccaagca gtccttattc agcctccaaa 4020

gcatctagcg atcatttagt tcgtgcttgg tacagaactt gggggcttcc tgtaatagta 4080

acgaattgtt ccaataatta tggtccctat catttccccg aaaaattaat cccattgacg 4140

atccttaatg cgctgcaagg tcagccattg ccagtttatg gcacagggga tcaaatccgg 4200

gattggttat atgttgaaga ccatgctagg gcacttcatc ttgtcatcag cagtaaacgt 4260

gttggagaaa catataacat aggtggctat aatgaaatga agaatattga tgtcgttcag 4320

aaggtctgcg aaatattaga tgaaatccaa ccattgagtg aaggatctta caaaaaactg 4380

attaattttg taaaagacag accggggcat gaccgtcggt atgccattga tgctaccaaa 4440

attacagagc atttaggttg gcaaccaatt gagacatttg agtctggtat tagaaaaaca 4500

gttcattggt atctacaaaa taaagcttgg gttgagtcag ttaaaaatgg aaattataaa 4560

aaatggctta ctcagcagta tggagaaatg aaatgaaagg aataatcctc gccggtggct 4620

ctggtacaag actttatccc atcacgaagg gaatatcaaa acagttgatg cctgtttatg 4680

ataaacctat gatctattat ccactttctg tattaatgct agcaggtatc aaagatatat 4740

taatcataac aacgcctgaa gatctgaata attttcagcg attactcggc gatggtaaac 4800

gttttggtat taatctctca tttgaggtgc agttaaaacc tgaagggtta gcacaggctt 4860

tcattattgg gggggaattt ataggcaatg atgcggtctg tttggcactt ggagataata 4920

ttttttatgg acaaaacttt agtccaaaac tcaaagaggc tgcacaatta attgatggtg 4980

caaccgtatt tggatatcag gtaaaagatc cagaacgatt tggtatcgta gagtttgacg 5040

ctaataaaaa agcactgtct atagaagaaa aaccggcaaa accaaaatca aattatgcag 5100

ttacaggtct ttacttttat gataataacg tagttaacat tgccaaaaca attaaacctt 5160

cagagcgcgg cgagttagag attaccagta taaacgaagt ctatcttcgc aatgggaaat 5220

tgaatgttga gttgttgggg cgaggcttta cctggcttga taccggaaca catcaaagca 5280

tgttggaagc atctcatttt gtggaaacta tcgaacaaca tcaaggtttc aaaatcgcat 5340

gtctagaaga aatcgccttg tacaatggat ggctaaccaa agatgaagta ttcaaaatag 5400

gtaatgagta caaaaaaaat ggttacggtc aatatcttct ctctttggtt aaggacaaat 5460

aatggaaaag ccaatctttg taacgcaacc taatttacca ccgctagagg agtttatacc 5520

atatctggaa atcatttggc agaataagca atttacaaat aatggtccaa tgcatcaaaa 5580

attagaaaaa aaattatgcg agtttcttgg tgttgaatac attagtctat ttaataatgg 5640

gactattgcg cttataaccg cagtacaggc tttgggtgtt aaaggcgaag taattaccac 5700

accatattcc tttgtagcaa ctgcacactc cttggtctta aatgggctta aacctgtttt 5760

tgtcgatatt gatcccaaaa ccttaaatat cgatccgaga agaattgaag aggcgattac 5820

ccctgaaacg caggcaataa tgccggtgca ctgctatggg aatccttgcg atacacaagc 5880

tattgctgat attgcgcaaa aatataattt aaaggtcatt tatgatgctg cgcatgcctt 5940

tggcgttgaa gatgatgatg gaagtgttct tcgccatgga gatctaagtg tcttaagttt 6000

ccatgcaact aaagtgttca gcacttttga aggcggagct attgtgtgta atagtaaaga 6060

aatgaaagaa aaaattgata gactaaaaaa ctttggttat atcgatgaaa ctaacatcaa 6120

tatcattggc tctaatggaa aaatgagcga agttaatgct gctttcggct tgctacaatt 6180

agaacatatg gatacttttc tacgtggtcg aatgaatgct gacatgtttt atcggcagaa 6240

acttaaagat atcactggta taagcatagt aattcccagc ggccagaaaa tatcgaattt 6300

ttcatatttc cctatattgg ttgaatctga ttttccgtta tctcgtgatg aactatttaa 6360

ttatctgaag aaccagaata tttttgcaag acgttatttt tatcctgtta taccagattt 6420

tcaagcgtat ttgaatgtag gtgaagtctg tgatgtcaag aatgcccgtg aaatagcctc 6480

gaaagtgctt tgcctaccga tgcatgcaga attgagctct gatatcttag aatatattgt 6540

aagtacgatt agggagatta aatgattgcc ataatgcagc cctatttatt tccatattta 6600

gggtatttcc aattgatcac ggcatcagat acattcgtgc tttttgatga tgttaattat 6660

ataaagaaag gatatatcaa cagaaacaac attttgctaa atggatctgc atataggttt 6720

acggttcctg tgcaaaatat atcacagaat aaaaaaatta atgagcatta tttttctgat 6780

gacactgata atctattaaa aacctttaac atggcatatg caaaagctcc atactatatg 6840

gatatatatc ctattataga gagagcttta aaaggaacag agagaaaggt tagttctgtt 6900

tgtttcaatg ctattaacga tatactggat tatctcggga tagaaaaaaa tattgtcttc 6960

tcaagtaata ttgattatga cagatgtgct agtgcaactg ataaactaat tgggataaca 7020

aaagcattag aaagcaatta ctatattaat gcaagtggtg gacaaaatct ctacgattac 7080

gattatttta aaagcaaaaa tattacgctc gcgtttattc aaatggatga tattgagtat 7140

acacagaatg gaaagaatgc tgagtttatt cctaacttgt caatattaga tgtcttaatg 7200

tggtcttctc caaaaaatgt gttggaattg ctaaataaat atactctgat aacgagcggg 7260

cataattatg gcaaatgata cgttgatttt tttaagtaaa gttatccaag atgatctaaa 7320

cctgaaatgt tcgattctta ataactatca caagatggat gactacattc aaaaattaat 7380

cgaacatgct acaataattt caatcagaga gaatggtcaa gtaattggtg ctatctgttt 7440

ctattgcaat aatcaaaaag atctaattgc gtatgtaagt atgattgcag ttcatattga 7500

tcatagaggg aaaggaatcg gcgacgagct tctaagtttt gcaattgcta attgtagaag 7560

aaaggggttc gaactttgca agttagaagt caacaaaaga aatgttagag cattaaccct 7620

ttacagtaaa aatgattttt caatattatc agaatctgag aatacttatt tgatgcagaa 7680

aaaattataa ataggctttg aatattgtgt ctttaaaaaa aaatattatc attagctatg 7740

ctgcacaaat atatgtcagt gctgtcggga ttttaatact ccctctgtac atcaagtaca 7800

tgggagcaga gtcatatgga ttaattggct tttttactat gcttcaggcg ctgtttggtc 7860

tcttagactt agggctaact ccaacaattg gtcgtgaaac agctcgctat catggcggga 7920

caatgacagt gctggactac agaaaattgc taagaactct tcatattttg ttcttcacta 7980

tcgctactat tggtggagta tttctatttt tcttagcacc catcatttcg gttcattggt 8040

taaaggttac aacaatatca attgacaccg taaattactg tgtaaagata atggcattct 8100

ctatagcatt acgatggata ggtgggttat ataggggaat cattagcggt agcgaaaggt 8160

tagactggct aagctattta aatattttta taacaacgct tagattcatc attatattcc 8220

ctgtcatgaa agtaaaaggt tatactcctt tcgtattttt cacatatcaa ttaatcgttg 8280

cgatcattga gtatttgtct ttattatgtt tgtctacttt attaactcct aaattgaaat 8340

cagcaaacga gaaaattggt ttttcatttt cgcctattat gtcactgatg cgcttttcct 8400

taacagttgc ttttacttcc tctatttgga tattgattac tcagagtgat aaactcatct 8460

tgtcgggtat attggaatta agtgaatacg ggcatttcac actttctgtg ttgttggcta 8520

gtggcattct gatgattaat attccggtta caaactctat attaccgagg cttgcgcgtt 8580

tacatgctga aaataagcat gatgaacttc tccgtattta taaaaatacg actatgttag 8640

tatgcatctt gggagtaagc gcatcgctag ttgttgctat ctatgcagaa ccattgcttc 8700

ttatctggac tggtgatgca gaagtttcag caagtgcatc gcctattctc tcattgtatg 8760

ccattggaaa tggtttactg gcagtatcag ctttcccata ttatctacaa tatgctcaag 8820

ggcggctaaa gtatcacttc tggggaaata ttgtaatgtt cttcatgcta atccccacaa 8880

ttattgtact tgctcgtaat tttggtggag ttggcgcggg ttatgcctgg ttaatagtta 8940

atgtctttta tctttttgta tggactgcat tggttcatca taaattaata cccggattac 9000

atctctcttg gttaatgagt atcggtatga taacaattcc taccggtatt attgtatttt 9060

ttctctcctt tattgtccga tttagcggta atagattgca tgacataatt gtgctgggct 9120

taatatcaat tttggctctg atggtttcaa tagtatcatg ctggctaatc aaaagaatga 9180

atctcggggt gtaaaataaa tggaaggaac agttcctaag gtctcagtct gtgtgataac 9240

atataaccag gcgaaatata taaagcaatg catagaaagc ctaatcactc aggactgtga 9300

tttcgattat gaaataattg tgagcgatga ttgttcgacg gataatacac gagaaatttt 9360

agaacactta tatcatcaat atccagaaaa gatacgtata tttatacatg aaaagaatct 9420

tggggttact aaaaattatc ttttcctgca tgaacaggcg caaggcgagt atattgcaca 9480

tgttgatggt gatgactact atttttcaaa taaattaagt ttacaagcac gatatcttga 9540

tgaaaataaa gagtgtaata ttgtttggca tcctatgttg ttagataata attcacgagt 9600

atttaatggg tatcagcaga gtggaactag ttttgccgat ttaaaattta ctcaaggtga 9660

cataattcaa tatatttctg tcggtaaaaa cagctcaaag atgtatcgaa aaaccgtacg 9720

agatattgat atacctgcat tcgagctcgt tgactacctc gttaatgttg aacaagttca 9780

gaatggctat ggtggatacg cttcgaatga acctctggga gtatatcgag taggtgtagg 9840

aatttcatct tctggagaca aaactcgcat tgctcttcgg gatacttttc tttatctatt 9900

aaaaaagtat ccaaaataca gattagaaat aaatacagcg gccttaacat attttatccg 9960

cgatatcttt gcaagaagaa aatctgcaaa gattttttta catgtatgga taaaaacgtt 10020

tcatcccttt tcagtaatta aattacttaa ggggatgagt acaattaaaa aattaaaata 10080

tagagcataa gtatgattaa aatcgttaac gaggacttac atgcttttac taaaaatggc 10140

agtttcgcta gtaaagttaa atgcacaatt ataggccaca cttttcatct agtgctcatg 10200

atacgcttag ggcagtttct atcaaagata cctgtgatcg gcgcattttt tagattggtc 10260

attgaatatt ccataagaat catattttct agtgatataa gtctaagggc aaggattggt 10320

ggtggattag taataatgca tggccatgat atcgttattg gaagagatgt tgtcataggc 10380

agaaattgta aaattctgaa tggcgtcact ttaggtaata aggacacgga atcaacagaa 10440

aaccagcaac cagtagtggg tgataatgtg ataattggta ctggagctaa aattctgggg 10500

aaagtagtta taggaaataa tgttaaaatt ggagctaata gtgtagttat ttctgacatt 10560

gctccagatt ctgttgctgt tggtatacct gcaagggtta ctaaaggtat ctaataggtt 10620

aagataagta acgaagagat tcatcatgat aaatctaaat ccagtagcta tcctaacctt 10680

actttattta tctatcaatt ttttatcaat gttgatagga tgttctagtg gtgaaataca 10740

agtagaaaca tcaattttta gagttagtga agaaagtcta atttattcat ttttacttca 10800

agctatctgt cttatttttt tatattacat ttataaatat tttacaaata gaatctcata 10860

tcccccatta acatttaaag caaagtgggg aagggcgtta cttatcattc aaatcgcatt 10920

tattattttt aacacacaaa tgggcgttaa tacagccggt agtgttgaaa ggatagaagg 10980

acaatcatta agcaactatc tttttataat tttacaaccc gatattctag tagccgttat 11040

ttcagtatgc ttaaattcag gtttcctatt ttggactaat atcttagtat acctgctatc 11100

catgttcctt agaggttgga tgggtggaac ctttgtcata ctctttttga tattatcacg 11160

ttatcaaaat ttaaggatct cactaaaaac ttttttggta tccttatgtt ctctattgct 11220

tcttttctcc attttgcctg cactcattga agcgaaatgg gcaatgagga ccggtatatc 11280

tctatcagta tttataagca atatgagttc atatgttact ccagagaatt attatgctgg 11340

cataaactat ctgttaaata gatttcaaca tgtcggccat ttagcgttga tatatgaaaa 11400

tgcagatgat atatttaaaa aatataacgc aggttatttt agctcatatt atatggatgg 11460

cattcctcag tatcttcttg ttaagatgta taacttagat atgtataagc tcagctttta 11520

tcttgttcaa tatttctttg atataacaga accgacttgg aatattaaca cgggcgtggt 11580

tggatggctt tatatattaa ggtatgaatc catacttttt gcgttttata taatgttatt 11640

gttgctggtt ccttattatg tagttagtag atttgctggt aagagaatgt tgagtgtttt 11700

ggcttgcttt tctattattt atttatttca cggatggttg ggagcctatg ttaacttagc 11760

attttacgca tgtataataa gtctgcttgc aaatataaga ttatacagaa ctgtatatat 11820

tccatgtgag aaataggatt gttatgtgtg gattagctgg tttccttgaa tcaaatttaa 11880

atgataataa tagtgctaaa gttttacatt cgatgggtga atccatatac cgaaggggac 11940

cagatggtac aggaatatgg tttgaaaaca ttgagaataa aataaatgtt ggttttgttc 12000

atactcgatt agctattatt gatctctcag acgccggtca acagccgatg cattctcact 12060

gcggtcgtta tgtaattgca ttcaatggtg aaatatataa tcatcttgaa ctaagatatc 12120

tattagaaag taataatcat atttcctggc gtggccattc tgatacggaa acgcttttgg 12180

catgctttgg tatatggggt attgataaga cattgcaggc atgtgttgga atgtttgcaa 12240

tagccttatg ggacaaaaaa gaacatcggt taacattagc acgtgataga gttggtgaaa 12300

aaccgttgta ctggggatgg caagatgatg cccttctttt tggctcagaa ttaaaagcgt 12360

tgaaaactca tccggcattt agtgcagaaa taaacagaga cgcactttgc ttgctattaa 12420

ggcataacta tattcctgca ccacatacaa tatatcgagg gatccaaaaa ctacaaccgg 12480

gccactatct aagtgttgag ttatcaaatg atgaaagaaa ggagacactt catcagtact 12540

ggtcatatga gaaagtagta caagaaggat tagtgaatcc tttcatggga tcacctatac 12600

aagcagtaga acatcttgag tctttaatcg ctcaaagtat taatggacag ttaatttctg 12660

atgtccctct tggcgctttt cttagtggtg gaattgatag tagtctgatt gtgtctatca 12720

tgcagtcttt atcatcatct ccagtgaaaa cattctctat aggatttgat gataaaaaat 12780

atgatgaggc tatctttgct gttgaagttg cccgtcattt aggtaccgat cacaccgaaa 12840

tgtacgtaaa tgacaaagat atacaggatg ttattccact tttgcctgaa gtgtattgtg 12900

aaccttttgc tgatagttct cagttgccaa cttttttggt cagtaaaatg gcacagcaac 12960

atgttaccgt cgcgttgagt ggtgatgcag gtgatgagtt atttggcgga tatacgccat 13020

atagctttac gccaaagtat tggaattatg cgcgaaatat accattgcaa attagaaaat 13080

ttgcttctag gtatattgac aaattgccag ttaatccgaa gtttaaaaaa ttaatagagt 13140

gtatggcagt tcagaatccg cagctttttt acaggaatat cattagccat tggcttaatc 13200

ctgaagagtt agtcaaaaac tcgagtgagc catcaactgc atttagttca tctactacgt 13260

tccctgtaaa tggcggatat gttgactgga tgatgtcggt agatgctcaa gtctatatga 13320

cagatgatat tttagtcaaa gttgacaggg ctgctatgta taatagcctc gaaacccgag 13380

tgccattatt ggaccaccgt attatcgaat ttgcttggca gttaccggtt tctttaaaga 13440

ttaataatgg tgtaggtaaa tggcctctgc gagaaatact ctataaacga gtaccgaaat 13500

caatgattga acggccgaaa aaaggattct ctgtaccgct atcatcgtgg ttacgcggac 13560

ctttgcgtga ttgggcggaa gagttgattg cttataaacg tctcgcagat gaaggatatt 13620

ttaatgttgc tgctgtgaga agttgctgga atctgcatat ccagggacag gctgactatt 13680

caagaaaatt atggagcata cttatgtttc aatcatggtt agaaaaccag aaatgaaagt 13740

cgttcatatt ataatcgatt tgaatgttgg tggggcggag ctcatgcttc agcgtcttat 13800

taagcatacc gctgataata atgttgaaca tgttgtcatc tctcttactg atattggcac 13860

actcggtgag gaaattctga agagtggggt agatgtaaag tgcttaaaca ttaattcccc 13920

aattaagtat gccaccagtt tattgaagct ttataagcta tttactaaaa ttaagcctga 13980

tgttgttcat acctggatgt atcacagtga tcttattggt ggtatctctg cgaaattagc 14040

cggcgttaaa aaaataattt ggtgtgttag aagtactgac attagtaaag gaggaaacaa 14100

gcttacgctt ctgataagat ggttatgcgc taaaatatca tcgtttattc ctgatactat 14160

cgtttatgct gcaaacgctt cgaaggaagt tcatgaacag tgcggttatg ataagaataa 14220

atcgttggtc attgccaatg gatttgattt aactaaacta tcgccagata aattttccag 14280

aagcgattta agaaaagaaa tcggcttagc agaaaatgat gttgtggtat gttcagttgg 14340

tcgctatagt ccagtaaaag atcactcaac ctttatttca gcagctcttc aacttgcaga 14400

agaatttagt aatgtacgct tcttattagt cggtcgtggc ttgaccaccc agaataaaat 14460

aataatgaca cagcttagtg taagctgtca ctcagataaa tttattcttc ttggtgagcg 14520

tagcgatgtt cctgcatgtt taaatgcttc tgatattttt tgccttcatt cagtaacaga 14580

aggcttccct aatgtcttag gagaagcaat ggcaatggga attccttccg tgacttcaaa 14640

cgttggtgat gccgcatttt tacttgataa ggttgatttt gttgttccag ccggaaactc 14700

gtatttatta gcacagaaac tgcgggaagt tattttgtta agtcgaaaaa accgcactaa 14760

gttaggtcgt atattaaggc tgcgaattga gaatgagttt tctatggata ctgtagccaa 14820

taaatattta tccttatata aaaattaaat gaaaaagatt gtttttataa tcaataatgt 14880

cgatttctta atatctcatc gattacctat cttactcgag gctcagaaaa atgggtttca 14940

ggttcatgtg attgctccta attccaggaa taatgagcta ctaaagaagc ataaaataat 15000

gggtcatgat ctctttcttt ccagaggagg taataaccct ttttatgatt tatttatttt 15060

gcttcagctt acaaaaattc tgaaatttct caagcccgac cttgtccatc ttgtcacgat 15120

taaaccaaca ctttatggtg ggattgctgc cagaatcgcc aaagtgcctc atgtcgttgc 15180

agctgtctct gggctgggaa ccgtattctt gagcagagga atcatcagtg gcttacgtcg 15240

cttactggtt acaaccttgt atcactccgc tctgaaacat aaaagaatac gcgttatctt 15300

tcagaaccct gatgatcgtg agttacttgt gagcgcaggc atattaaagg tttccaattc 15360

ctgtctgatc agaggttcgg gggttgattt aagagagtat ccttatcttc ctgaaaaagt 15420

tcacggtaag actgtagtaa tggcctccag gctactaagg gataaagggg tttatgagtt 15480

tatcgaagcc gctcgcttac ttaaacaaag gaatgtagag gctgatatta gaatcattgg 15540

ttctccagat acctgtaatc cgacaagtat cactgaggct gaaataagca aatgggcatc 15600

agaaaatatt gctgaattct gtggatttag aagtgacatt gctaagcagt actctaatgc 15660

taatgtaata tgcttgccct catatcgaga agggttacct aaatgtctgg ttgaagccgc 15720

agcgtgtggt agggcagttg ttacaactga tgtaccaggt tgcagggatg cgatagtggc 15780

aaatgttact gggatgttag tcgcagttcg ggatcctgta tcgttagcag atgcgattga 15840

gtttctgcta aaaaatccag atgaaagaat aaaaatgggg aaggcgggga gattgttagc 15900

tgaaaatgag tactcaatcg aacatatagt gaatcagcac ttatccattt acaatgactt 15960

aattcactcc tgacgcttag tgctggtgcc atttatgatc attctttttg catcctattt 16020

ggcacttgat gctatccatc gcgccctaac ctaagttaac atcttcaata cattcaagcc 16080

gcgcatacgt cgcgtggtga ccacacctga caggagtatg taatgtcaaa gcaacagatc 16140

ggcgtcgtcg gtatggcagt gatggggcgc aaccttgcgc tcaacatcga aagccgtggt 16200

tataccgtct ctattttcaa ccgttcccgt gagaaaacgg aagaagtaat tgccgaaaac 16260

ccaggcaaga aactggttcc ttactatacg gtgaaagagt ttgttgaatc tctggaaacg 16320

cctcgtcgca tcctgttaat ggtgaaagca ggtgcaggca cggatgctgc tattgattcc 16380

ctcaaaccat atctcgataa aggtgacatc atcattgatg gcggtaacac cttcttccag 16440

gacaccattc gtcgtaaccg tgagctttct gccgaaggct ttaacttcat tggtaccggt 16500

gtctccggtg gtgaagaagg cgcgctgaaa ggtccttcca ttatgcctgg tgggcagaaa 16560

gaagcctatg aactggttgc accgatcctg accaaaatcg ccgcagtggc tgaagacggt 16620

gagccatgcg ttacctatat tggtgccgat ggcgcaggtc actatgtgaa gatggttcac 16680

aacggtattg aatacggcga tatgcagctg attgctgaag cctattctct gcttaaaggt 16740

ggcctgaatc tcaccaacga agaattggcg cagaccttta ccgagtggaa taacggtgaa 16800

ctgagcagct acctgatcga catcaccaaa gacattttca ctaaaaaaga tgaagacggt 16860

aactacctgg ttgatgtgat tctggatgaa gcagcaaaca aaggtaccgg taaatggact 16920

agccagagcg cgctggatct cggcgaaccg ctgtcgctga ttacagagtc tgtgtttgca 16980

cgttatatct cttctctgaa agagcagcgc gttgccgcgt ctaacgtact gactggtccg 17040

caagcgcagc cagcaggcga caaggctgag tttatcgaaa aagttcgccg tgcgctgtat 17100

ctgggcaaaa tcgtttctta cgctcagggc ttttctcagc tgcgtgctgc gtctgaagag 17160

tacaactggg acctgaatta cggcgaaatc gcgaagattt tccgtgctgg ctgcatcatt 17220

cgtgcgcagt tcctgcagaa aattaccgat gcttatgccg aaaatccgca gatcgctaac 17280

ctgctgctgg ctccgtactt caagcaaatt gccgatgatt accagcaggc gctgcgtgat 17340

gtcgttgctt atgcggtaca gaacggtatc ccggttccga ccttcgctgc tgcggttgcc 17400

tattacgata gctaccgtgc cgctgttctg cct 17433

Glycosyltransferase gene in the table 1 shigella dysenteriae 7 type O antigen genes bunch and oligosaccharide unit treatment gene and wherein primer and PCR data

Gene	Function	The base position of gene	Forward primer	Reverse primer	PCR product length	Produce the group number of correct big or small electrophoresis band	The annealing temperature of PCR (℃)
								Wzx	The transhipment enzyme	7800 to 9194	#45 (7871 to 7888)	#46 (8697 to 8715)	845bp	0 *	60
			#135 (8152 to 8169)	#136 (9067 to 9086)	935bp	0 *	60
											#137 (8597 to 8614)	#138 (9102 to 9119)	523bp	0 *	60
Orf9	Glycosyltransferase	9200 to 10090	#47 (9336 to 9353)	#48 (11117 to 10034)	699bp	0	60
											#139 (9448 to 9465)	#140 (9864 to 9881)	434bp	0 **	60
			#141 (9274 to 9292)	#142 (9746 to 9763)	490bp	0	58
								Wzy	Polysaccharase	10646 to 11836	#49 (11120 to 11137)	#50 (11366 to 11383)	264bp	0	50
			#143 (10957 to 10976)	#144 (11547 to 11564)	608bp	0	55
											#145 (10707 to 10725)	#146 (11767 to 11785)	1079bp	0	50
Orf13	Glycosyltransferase	13691 to 14848	#115 (14084 to 14101)	#116 (14420 to 14437)	354bp	0	45
											#401 (13881 to 13899)	#402 (14761 to 14798)	918bp	0	48 ***
			#403 (13975 to 13992)	#404 (14361 to 14379)	405bp	0	48 ***
								Orf14	Glycosyltransferase	14849 to 15973	#117 (15225 to 15244)	#118 (15765 to 15782)	528bp	0	58
			#405 (14975 to 14993)	#406 (15911 to 14930)	956bp	0	48 ***
											#407 (15011 to 15030)	#408 (15688 to 15706)	696bp	0	48 ***

* in strain intestinal bacteria, obtain the band of 1 correct size

* obtains the band of 3 wrong sizes in all groups

* * obtains the band of 1 wrong size in all groups

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

Group number	The bacterial strain that contains in this group	The source
			1 2 3 4 5 6 7	Wild-type e. coli O1, O2, O3, O10, O16, O18, O39 wild-type e. coli O40, O41, O48, O49, O71, O73, O88, O100 wild-type e. coli O102, O109, O119, O120, O121, O125, O126, O137 wild-type e. coli O138, O139, O149, O7, O5, O6, O11, O12 wild-type e. coli O13, O14, O15, O17, O19ab, O20, O21, O22 wild-type e. coli O23, O24, O25, O26, O27, O28, O29, O30 wild-type e. coli O32, O33, O34, O35, O36, O37, O38, O42	IMVS a IMVS IMVS IMVS IMVS IMVS IMVS

8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27	Wild-type e. coli O43, O44, O45, O46, O50, O51, O52, O53 wild-type e. coli O54, O55, O56, O57, O58, O59, O60, O61 wild-type e. coli O62, O63, O64, O65, O66, O68, O69, O70 wild-type e. coli O74, O75, O76, O77, O78, O79, O80, O81 wild-type e. coli O82, O83, O84, O85, O86, O87, O89, O90 wild-type e. coli O91, O92, O95, O96, O97, O98, O99, O101 wild-type e. coli O112, O162, O113, O114, O115, O116, O117, O118 wild-type e. coli O123, O165, O166, O167, O168, O169, O170, O171 wild-type e. coli O172, O173, O127, O128, O129, O130, O131, O132, wild-type e. coli O133, O134, O135, O136, O140, O141, O142, O143 wild-type e. coli O144, O145, O146, O147, O148, O150, O151, O152 wild-type e. coli O153, O154, O155, O156, O157, O158, O159, O164 wild-type e. coli O160, O161, O163, O8, O9, O124, O111 wild-type e. coli O103, O104, O105, O106, O107, O108, O110 Shigella bogdii serotypes B 4, B5, B6, B8, B9, B11, B12, B14 Shigella bogdii serotypes B 1, B3, B7, B8, B10, B13, B15, B16, B17, B18 shigella dysenteriae serotype D1, D2, D3, D4, D5, D6, D8 shigella dysenteriae serum D9, D10, D11, D12, D13 shigella flexneri F6a, F1a, F1b, F2a, F2b, F3, F4a, F4b, F5 (v:7) F5 (v:4) bacillus ceylonensis A D5, DR	IMVS IMVS IMVS IMVS IMVS IMVS IMVS See b See c IMVS IMVS IMVS IMVS IMVS See d See d See d See d See d See d
			A.Institude of Medical and Veterinary Science, Anelaide, Australia b.O123 from IMVS; The rest from Statens Serum Institut, Copenhagen, Denmark is and 173 from Statens Serum Institut c.172, Copenhagen, Denmark, epidemiological study institute of the rest from IMVS d. China Preventive Medicial Science Institute

Table 3 is structural tables of the O-antigen gene bunch of shigella dysenteriae 7 types

Table 4 is location tables of the gene in the O-antigen gene bunch of shigella dysenteriae 7 types

ctcctggtaa ctcacgcgtc caagaacgcg gtcgaaaacc acttcgacac ctcttatgaa 60

ttagaatctc tccttgaact gcgcgtgaag cgtcaactgc tggcggaagt acagtccatt 120

tgcccgccgg gcgtgacaat tatgaacgtg cgtcagggtg aacctttagg tttgggccac 180

tccattttat gtgcacgacc tgccattggt gacaatccat ttgtcgtggt gctgccagac 240

gttgtgatcg acgacgccag cgccgacccg ctgcgctaca accttgctgc catgattgcg 300

cgcttcaacg aaacgggccg cagccaggtg ctggcaaaac gtatgccggg tgacctctct 360

gaatactcct tcatccagac caaagaacct ctggatcgcg aaggtaaagt cagccgcatt 420

gttgaattca tcgaaaaacc ggatcagcca caaacgctgg aatcagacat catggccgtg 480

ggccgttatg tgctttctgc cgatatttgg ccggaacttg aacgcactca gccaggtgca 540

tggggacgta ttcagctgac tgatgccatt gccgaactgg cgaaaaaaca gtctgttgac 600

gccatgctga tgactggtga cagctacgac tgtggtaaaa aaatgggtta tatgcaggcg 660

tttgtgaagt atggactacg caacctgaaa gaaggagcga agttccgcaa aggtattgag 720

aaattgctta gcgagtaagt ttaaaaaata gacgccctta tagggcgtaa taacaaataa 780

cggtagtcaa cattcgacgc ggtgatgcag atatgcccgg aatgctgata ccgttttttc 840

attctaaaaa actcatcatt tcattgagtt aactacaaaa tttagcactg ttttttataa 900

tgtttcttct tgtttctggc atcaattggt aagataatta gtgtttgagt ttagaggctt 960

tgcggcagag aagcggagct taacacgtct gtgagagtac gcagtgcact ggtagctgta 1020

aagccagtgg cggtagcgtg tttaaataaa tacattagta ctactacata ttacatcatt 1080

gtaggctatt taagcgctac atgttaagcg acagcgctag caatcaaatc ttttaaagtt 1140

Orf1's is initial

acttctcagg aatagtaaa a tgagctttaa tcttgaaaat tataaaatag gaattattgg 1200

gctgggttac gttggcttac ctttagctgt tgaatttggc aaaaaaataa agactcttgg 1260

ttttgatatc aacgagaaac ggataaaaga attatcagca ggtaatgact ttacgcttga 1320

atgtagttct gatgaactgg ctcaagctta ctatcttcaa tattcatgtg attataaaga 1380

attagcgact tgcaatgtat tcattgtgac ggtacctact ccaattgatg aatataaaca 1440

acctgattta acacctcttg ttaaagcctc ggaaacactc ggaaaaataa tcaaaaaaga 1500

tgatattatt atctacgaat caactgttta tccaggtgca acagaagaag tatgtatccc 1560

agtaattgag aagatctcag gtctggtatt taatatagat ttttttgccg gttacagccc 1620

ggaacgcatt aatccgggtg acaaagagca ccgcgtgaca acgatcaaaa aagtaacttc 1680

tggttcaacg cctgaaattg ctgagattgt cgataaatta tatggaacca tcatcaccgc 1740

tggaacgcat aaagcatctt cgatacgtgt ggcggaagcg gctaaagtga ttgagaacac 1800

ccagcgtgat ttgaatattg ctctgattaa cgaacttgca atgattttca ataagctggg 1860

gattgatacg gaggaagtat taaaagccgc aggcactaaa tggaacttct taccattcag 1920

accaggctta gttggggggc attgcatcgg agttgatccc tactatttga ctcataaagc 1980

gcaagcgata ggttatcatc cagaagtcat cctggcgggg cgtagaatta atgatggaat 2040

gagtaaatat gtagcatcac agttcattaa agccatgctc aagaaaaaat tccaaattga 2100

aggtgctaat gtcttaatta tggggcttac ctttaaagaa aactgtccgg atcttcgtaa 2160

tacaaaagtt gtagatatct tggctgaact gaaagaatac aacgtgaacg tggatattta 2220

tgatccatgg gttgatgttg aagaagccta ttatgagtat gggattaatg tattaaacac 2280

attacctgat aagaaatatg atggagtgat ctttgcagtt gctcactctc aatttaaaaa 2340

tatgagtaaa gaagaagtcc atggaataat tagaaatgat ggtgttattt atgatttgaa 2400

The termination orf2's of orf1 is initial

atacatcatt gaccctaact tagtagatat tcgtctt taa tcaatacttg atgcaatgtg 2460

tcagagagga tgcctaatga aatataaaaa aatagaagac gctttgtctt ctaaatctga 2520

aacatggttg attacaggtg ttgcgggatt tatcggttct aacctattag aggccttact 2580

aaaacttaat caaaaagtag ttggcttaga taattttgca actggacatc aagcaaacct 2640

tgatgaggtt aaagagtctg tttcagctga acaatggtct gcttttacct ttgtggaagg 2700

agacatatgt gagccagaga cgtgtgtgga ggttgttaaa ggagttgatc atgtcctaca 2760

tcaggctgca ttaggttctg tgcctcgttc aattaaagat ccaattacta caaataatac 2820

caatatatcg ggtttcatta atatgttggt tgccgccaaa gatgccaatg tcagaagttt 2880

cacttacgct gcaagtagct caacgtatgg tgatcatcct gcattaccta aaattgaaga 2940

aaatataggt aatccgctat ctccatacgc agtgacaaag tatgtaaatg aattatatgc 3000

gcaagtattt gcaagaacct acggattcaa atcaattgga ttaagatact ttaatgtatt 3060

tggaaaacgc caggatccta atggggcata tgctgctgtg ataccgaagt ggactgctgc 3120

aatgatcaat gatgaaccgc tgtatatcaa tggtgatggc gaaacaagta gagacttttg 3180

ttttattgag aatgtcgtgc aaatgaatat tcttgctgca cagtctgatg cgtctgcaag 3240

agatcaggtt tataacgttg cagttggtga ccgtactacg cttaaacaat tattcactgt 3300

attaaagaat gcacttaatg aaaatggtgt ttcgtataac aaagaacctg aatataaaga 3360

ttttcgtgca ggtgatgttc gccattcgca agctgatatc actaaagcca aaactagact 3420

tggatacgag ccacagttca gtatatcgga gggcatcctc aaagcgatgc cgtggtatgt 3480

The termination orf3's of orf2 is initial

aaactctctg ggcaacaaaa aa taacc atg aaaatcctta ttacaggtgg tgctggtttt 3540

attgggtcag ctgttattag atacatcatt aatcatacta atgacagtgt cgtaaatgtc 3600

gataaattga cttacgcagg aaaccttcaa tcacttaaag atatagaaaa aaatacgcgc 3660

tataaatttg agtgcgcaga tatctgcgat agcgcaaaga tgcaagagat atttaccaat 3720

catcaaccag atgctgttat gcatttggcc gctgagagtc atgttgatcg gtcaattaat 3780

ggcccgtttg agtttataca gaccaatatc ataggcacct ttatcttact tgaaatagcc 3840

aagtcttact ggctgcaact tagtgaaagc aaaaaaaaga agtttatttt tcatcatgtt 3900

tcgacagatg aagtgtatgg tgatctagct catccagatg tactggatga aggttcacca 3960

ttgcctttat ttactgaaaa aaatggctac gcgccaagca gtccttattc agcctccaaa 4020

gcatctagcg atcatttagt tcgtgcttgg tacagaactt gggggcttcc tgtaatagta 4080

acgaattgtt ccaataatta tggtccctat catttccccg aaaaattaat cccattgacg 4140

atccttaatg cgctgcaagg tcagccattg ccagtttatg gcacagggga tcaaatccgg 4200

gattggttat atgttgaaga ccatgctagg gcacttcatc ttgtcatcag cagtaaacgt 4260

gttggagaaa catataacat aggtggctat aatgaaatga agaatattga tgtcgttcag 4320

aaggtctgcg aaatattaga tgaaatccaa ccattgagtg aaggatctta caaaaaactg 4380

attaattttg taaaagacag accggggcat gaccgtcggt atgccattga tgctaccaaa 4440

attacagagc atttaggttg gcaaccaatt gagacatttg agtctggtat tagaaaaaca 4500

gttcattggt atctacaaaa taaagcttgg gttgagtcag ttaaaaatgg aaattataaa 4560

The termination of the initial orf3 of orf4

aa atggctta ctcagcagta tggagaaatg aaa tgaaagg aataatcctc gccggtggct 4620

ctggtacaag actttatccc atcacgaagg gaatatcaaa acagttgatg cctgtttatg 4680

ataaacctat gatctattat ccactttctg tattaatgct agcaggtatc aaagatatat 4740

taatcataac aacgcctgaa gatctgaata attttcagcg attactcggc gatggtaaac 4800

gttttggtat taatctctca tttgaggtgc agttaaaacc tgaagggtta gcacaggctt 4860

tcattattgg gggggaattt ataggcaatg atgcggtctg tttggcactt ggagataata 4920

ttttttatgg acaaaacttt agtccaaaac tcaaagaggc tgcacaatta attgatggtg 4980

caaccgtatt tggatatcag gtaaaagatc cagaacgatt tggtatcgta gagtttgacg 5040

ctaataaaaa agcactgtct atagaagaaa aaccggcaaa accaaaatca aattatgcag 5100

ttacaggtct ttacttttat gataataacg tagttaacat tgccaaaaca attaaacctt 5160

cagagcgcgg cgagttagag attaccagta taaacgaagt ctatcttcgc aatgggaaat 5220

tgaatgttga gttgttgggg cgaggcttta cctggcttga taccggaaca catcaaagca 5280

tgttggaagc atctcatttt gtggaaacta tcgaacaaca tcaaggtttc aaaatcgcat 5340

gtctagaaga aatcgccttg tacaatggat ggctaaccaa agatgaagta ttcaaaatag 5400

The termination of orf4

gtaatgagta caaaaaaaat ggttacggtc aatatcttct ctctttggtt aaggacaaa t 5460

Orf5's is initial

aatggaaaag ccaatctttg taacgcaacc taatttacca ccgctagagg agtttatacc 5520

atatctggaa atcatttggc agaataagca atttacaaat aatggtccaa tgcatcaaaa 5580

attagaaaaa aaattatgcg agtttcttgg tgttgaatac attagtctat ttaataatgg 5640

gactattgcg cttataaccg cagtacaggc tttgggtgtt aaaggcgaag taattaccac 5700

accatattcc tttgtagcaa ctgcacactc cttggtctta aatgggctta aacctgtttt 5760

tgtcgatatt gatcccaaaa ccttaaatat cgatccgaga agaattgaag aggcgattac 5820

ccctgaaacg caggcaataa tgccggtgca ctgctatggg aatccttgcg atacacaagc 5880

tattgctgat attgcgcaaa aatataattt aaaggtcatt tatgatgctg cgcatgcctt 5940

tggcgttgaa gatgatgatg gaagtgttct tcgccatgga gatctaagtg tcttaagttt 6000

ccatgcaact aaagtgttca gcacttttga aggcggagct attgtgtgta atagtaaaga 6060

aatgaaagaa aaaattgata gactaaaaaa ctttggttat atcgatgaaa ctaacatcaa 6120

tatcattggc tctaatggaa aaatgagcga agttaatgct gctttcggct tgctacaatt 6180

agaacatatg gatacttttc tacgtggtcg aatgaatgct gacatgtttt atcggcagaa 6240

acttaaagat atcactggta taagcatagt aattcccagc ggccagaaaa tatcgaattt 6300

ttcatatttc cctatattgg ttgaatctga ttttccgtta tctcgtgatg aactatttaa 6360

ttatctgaag aaccagaata tttttgcaag acgttatttt tatcctgtta taccagattt 6420

tcaagcgtat ttgaatgtag gtgaagtctg tgatgtcaag aatgcccgtg aaatagcctc 6480

gaaagtgctt tgcctaccga tgcatgcaga attgagctct gatatcttag aatatattgt 6540

The termination of the initial orf5 of orf6

aagtacgatt agggagatta a atgattgcc ataatgcagc cctatttatt tccatattta 6600

gggtatttcc aattgatcac ggcatcagat acattcgtgc tttttgatga tgttaattat 6660

ataaagaaag gatatatcaa cagaaacaac attttgctaa atggatctgc atataggttt 6720

acggttcctg tgcaaaatat atcacagaat aaaaaaatta atgagcatta tttttctgat 6780

gacactgata atctattaaa aacctttaac atggcatatg caaaagctcc atactatatg 6840

gatatatatc ctattataga gagagcttta aaaggaacag agagaaaggt tagttctgtt 6900

tgtttcaatg ctattaacga tatactggat tatctcggga tagaaaaaaa tattgtcttc 6960

tcaagtaata ttgattatga cagatgtgct agtgcaactg ataaactaat tgggataaca 7020

aaagcattag aaagcaatta ctatattaat gcaagtggtg gacaaaatct ctacgattac 7080

gattatttta aaagcaaaaa tattacgctc gcgtttattc aaatggatga tattgagtat 7140

acacagaatg gaaagaatgc tgagtttatt cctaacttgt caatattaga tgtcttaatg 7200

tggtcttctc caaaaaatgt gttggaattg ctaaataaat atactctgat aacgagcggg 7260

The termination of the initial orf6 of orf7

cataatt atg gcaaa tgata cgttgatttt tttaagtaaa gttatccaag atgatctaaa 7320

cctgaaatgt tcgattctta ataactatca caagatggat gactacattc aaaaattaat 7380

cgaacatgct acaataattt caatcagaga gaatggtcaa gtaattggtg ctatctgttt 7440

ctattgcaat aatcaaaaag atctaattgc gtatgtaagt atgattgcag ttcatattga 7500

tcatagaggg aaaggaatcg gcgacgagct tctaagtttt gcaattgcta attgtagaag 7560

aaaggggttc gaactttgca agttagaagt caacaaaaga aatgttagag cattaaccct 7620

ttacagtaaa aatgattttt caatattatc agaatctgag aatacttatt tgatgcagaa 7680

The termination of orf7

aaaatta taa ataggctttg aatattgtgt ctttaaaaaa aaatattatc attagctatg 7740

Orf8's is initial

ctgcacaaat atatgtcagt gctgtcggga ttttaatact ccctctgtac atcaagtac a 7800

tgggagcaga gtcatatgga ttaattggct tttttactat gcttcaggcg ctgtttggtc 7860

tcttagactt agggctaact ccaacaattg gtcgtgaaac agctcgctat catggcggga 7920

caatgacagt gctggactac agaaaattgc taagaactct tcatattttg ttcttcacta 7980

tcgctactat tggtggagta tttctatttt tcttagcacc catcatttcg gttcattggt 8040

taaaggttac aacaatatca attgacaccg taaattactg tgtaaagata atggcattct 8100

ctatagcatt acgatggata ggtgggttat ataggggaat cattagcggt agcgaaaggt 8160

tagactggct aagctattta aatattttta taacaacgct tagattcatc attatattcc 8220

ctgtcatgaa agtaaaaggt tatactcctt tcgtattttt cacatatcaa ttaatcgttg 8280

cgatcattga gtatttgtct ttattatgtt tgtctacttt attaactcct aaattgaaat 8340

cagcaaacga gaaaattggt ttttcatttt cgcctattat gtcactgatg cgcttttcct 8400

taacagttgc ttttacttcc tctatttgga tattgattac tcagagtgat aaactcatct 8460

tgtcgggtat attggaatta agtgaatacg ggcatttcac actttctgtg ttgttggcta 8520

gtggcattct gatgattaat attccggtta caaactctat attaccgagg cttgcgcgtt 8580

tacatgctga aaataagcat gatgaacttc tccgtattta taaaaatacg actatgttag 8640

tatgcatctt gggagtaagc gcatcgctag ttgttgctat ctatgcagaa ccattgcttc 8700

ttatctggac tggtgatgca gaagtttcag caagtgcatc gcctattctc tcattgtatg 8760

ccattggaaa tggtttactg gcagtatcag ctttcccata ttatctacaa tatgctcaag 8820

ggcggctaaa gtatcacttc tggggaaata ttgtaatgtt cttcatgcta atccccacaa 8880

ttattgtact tgctcgtaat tttggtggag ttggcgcggg ttatgcctgg ttaatagtta 8940

atgtctttta tctttttgta tggactgcat tggttcatca taaattaata cccggattac 9000

atctctcttg gttaatgagt atcggtatga taacaattcc taccggtatt attgtatttt 9060

ttctctcctt tattgtccga tttagcggta atagattgca tgacataatt gtgctgggct 9120

taatatcaat tttggctctg atggtttcaa tagtatcatg ctggctaatc aaaagaatga 9180

The termination orf9's of orf8 is initial

atctcggggt g taaaataa a tggaaggaac agttcctaag gtctcagtct gtgtgataac 9240

atataaccag gcgaaatata taaagcaatg catagaaagc ctaatcactc aggactgtga 9300

tttcgattat gaaataattg tgagcgatga ttgttcgacg gataatacac gagaaatttt 9360

agaacactta tatcatcaat atccagaaaa gatacgtata tttatacatg aaaagaatct 9420

tggggttact aaaaattatc ttttcctgca tgaacaggcg caaggcgagt atattgcaca 9480

tgttgatggt gatgactact atttttcaaa taaattaagt ttacaagcac gatatcttga 9540

tgaaaataaa gagtgtaata ttgtttggca tcctatgttg ttagataata attcacgagt 9600

atttaatggg tatcagcaga gtggaactag ttttgccgat ttaaaattta ctcaaggtga 9660

cataattcaa tatatttctg tcggtaaaaa cagctcaaag atgtatcgaa aaaccgtacg 9720

agatattgat atacctgcat tcgagctcgt tgactacctc gttaatgttg aacaagttca 9780

gaatggctat ggtggatacg cttcgaatga acctctggga gtatatcgag taggtgtagg 9840

aatttcatct tctggagaca aaactcgcat tgctcttcgg gatacttttc tttatctatt 9900

aaaaaagtat ccaaaataca gattagaaat aaatacagcg gccttaacat attttatccg 9960

cgatatcttt gcaagaagaa aatctgcaaa gattttttta catgtatgga taaaaacgtt 10020

tcatcccttt tcagtaatta aattacttaa ggggatgagt acaattaaaa aattaaaata 10080

The termination orf10's of orf9 is initial

tagagca taa gt atgattaa aatcgttaac gaggacttac atgcttttac taaaaatggc 10140

agtttcgcta gtaaagttaa atgcacaatt ataggccaca cttttcatct agtgctcatg 10200

atacgcttag ggcagtttct atcaaagata cctgtgatcg gcgcattttt tagattggtc 10260

attgaatatt ccataagaat catattttct agtgatataa gtctaagggc aaggattggt 10320

ggtggattag taataatgca tggccatgat atcgttattg gaagagatgt tgtcataggc 10380

agaaattgta aaattctgaa tggcgtcact ttaggtaata aggacacgga atcaacagaa 10440

aaccagcaac cagtagtggg tgataatgtg ataattggta ctggagctaa aattctgggg 10500

aaagtagtta taggaaataa tgttaaaatt ggagctaata gtgtagttat ttctgacatt 10560

The termination of orf10

gctccagatt ctgttgctgt tggtatacct gcaagggtta ctaaaggtat c taataggtt 10620

Orf11's is initial

aagataagta acgaagagat tcatc atgat aaatctaaat ccagtagcta tcctaacctt 10680

actttattta tctatcaatt ttttatcaat gttgatagga tgttctagtg gtgaaataca 10740

agtagaaaca tcaattttta gagttagtga agaaagtcta atttattcat ttttacttca 10800

agctatctgt cttatttttt tatattacat ttataaatat tttacaaata gaatctcata 10860

tcccccatta acatttaaag caaagtgggg aagggcgtta cttatcattc aaatcgcatt 10920

tattattttt aacacacaaa tgggcgttaa tacagccggt agtgttgaaa ggatagaagg 10980

acaatcatta agcaactatc tttttataat tttacaaccc gatattctag tagccgttat 11040

ttcagtatgc ttaaattcag gtttcctatt ttggactaat atcttagtat acctgctatc 11100

catgttcctt agaggttgga tgggtggaac ctttgtcata ctctttttga tattatcacg 11160

ttatcaaaat ttaaggatct cactaaaaac ttttttggta tccttatgtt ctctattgct 11220

tcttttctcc attttgcctg cactcattga agcgaaatgg gcaatgagga ccggtatatc 11280

tctatcagta tttataagca atatgagttc atatgttact ccagagaatt attatgctgg 11340

cataaactat ctgttaaata gatttcaaca tgtcggccat ttagcgttga tatatgaaaa 11400

tgcagatgat atatttaaaa aatataacgc aggttatttt agctcatatt atatggatgg 11460

cattcctcag tatcttcttg ttaagatgta taacttagat atgtataagc tcagctttta 11520

tcttgttcaa tatttctttg atataacaga accgacttgg aatattaaca cgggcgtggt 11580

tggatggctt tatatattaa ggtatgaatc catacttttt gcgttttata taatgttatt 11640

gttgctggtt ccttattatg tagttagtag atttgctggt aagagaatgt tgagtgtttt 11700

ggcttgcttt tctattattt atttatttca cggatggttg ggagcctatg ttaacttagc 11760

attttacgca tgtataataa gtctgcttgc aaatataaga ttatacagaa ctgtatatat 11820

The termination orf12's of orf11 is initial

tccatgtgag aaa taggatt gtt atgtgtg gattagctgg tttccttgaa tcaaatttaa 11880

atgataataa tagtgctaaa gttttacatt cgatgggtga atccatatac cgaaggggac 11940

cagatggtac aggaatatgg tttgaaaaca ttgagaataa aataaatgtt ggttttgttc 12000

atactcgatt agctattatt gatctctcag acgccggtca acagccgatg cattctcact 12060

gcggtcgtta tgtaattgca ttcaatggtg aaatatataa tcatcttgaa ctaagatatc 12120

tattagaaag taataatcat atttcctggc gtggccattc tgatacggaa acgcttttgg 12180

catgctttgg tatatggggt attgataaga cattgcaggc atgtgttgga atgtttgcaa 12240

tagccttatg ggacaaaaaa gaacatcggt taacattagc acgtgataga gttggtgaaa 12300

aaccgttgta ctggggatgg caagatgatg cccttctttt tggctcagaa ttaaaagcgt 12360

tgaaaactca tccggcattt agtgcagaaa taaacagaga cgcactttgc ttgctattaa 12420

ggcataacta tattcctgca ccacatacaa tatatcgagg gatccaaaaa ctacaaccgg 12480

gccactatct aagtgttgag ttatcaaatg atgaaagaaa ggagacactt catcagtact 12540

ggtcatatga gaaagtagta caagaaggat tagtgaatcc tttcatggga tcacctatac 12600

aagcagtaga acatcttgag tctttaatcg ctcaaagtat taatggacag ttaatttctg 12660

atgtccctct tggcgctttt cttagtggtg gaattgatag tagtctgatt gtgtctatca 12720

tgcagtcttt atcatcatct ccagtgaaaa cattctctat aggatttgat gataaaaaat 12780

atgatgaggc tatctttgct gttgaagttg cccgtcattt aggtaccgat cacaccgaaa 12840

tgtacgtaaa tgacaaagat atacaggatg ttattccact tttgcctgaa gtgtattgtg 12900

aaccttttgc tgatagttct cagttgccaa cttttttggt cagtaaaatg gcacagcaac 12960

atgttaccgt cgcgttgagt ggtgatgcag gtgatgagtt atttggcgga tatacgccat 13020

atagctttac gccaaagtat tggaattatg cgcgaaatat accattgcaa attagaaaat 13080

ttgcttctag gtatattgac aaattgccag ttaatccgaa gtttaaaaaa ttaatagagt 13140

gtatggcagt tcagaatccg cagctttttt acaggaatat cattagccat tggcttaatc 13200

ctgaagagtt agtcaaaaac tcgagtgagc catcaactgc atttagttca tctactacgt 13260

tccctgtaaa tggcggatat gttgactgga tgatgtcggt agatgctcaa gtctatatga 13320

cagatgatat tttagtcaaa gttgacaggg ctgctatgta taatagcctc gaaacccgag 13380

tgccattatt ggaccaccgt attatcgaat ttgcttggca gttaccggtt tctttaaaga 13440

ttaataatgg tgtaggtaaa tggcctctgc gagaaatact ctataaacga gtaccgaaat 13500

caatgattga acggccgaaa aaaggattct ctgtaccgct atcatcgtgg ttacgcggac 13560

ctttgcgtga ttgggcggaa gagttgattg cttataaacg tctcgcagat gaaggatatt 13620

ttaatgttgc tgctgtgaga agttgctgga atctgcatat ccagggacag gctgactatt 13680

The termination of the initial orf12 of orf13

caagaaaatt atggagcata cttatgtttc aatcatggtt agaaaaccag aaa tgaaagt 13740

cgttcatatt ataatcgatt tgaatgttgg tggggcggag ctcatgcttc agcgtcttat 13800

taagcatacc gctgataata atgttgaaca tgttgtcatc tctcttactg atattggcac 13860

actcggtgag gaaattctga agagtggggt agatgtaaag tgcttaaaca ttaattcccc 13920

aattaagtat gccaccagtt tattgaagct ttataagcta tttactaaaa ttaagcctga 13980

tgttgttcat acctggatgt atcacagtga tcttattggt ggtatctctg cgaaattagc 14040

cggcgttaaa aaadtaattt ggtgtgttag aagtactgac attagtaaag gaggaaacaa 14100

gcttacgctt ctgataagat ggttatgcgc taaaatatca tcgtttattc ctgatactat 14160

cgtttatgct gcaaacgctt cgaaggaagt tcatgaacag tgcggttatg ataagaataa 14220

atcgttggtc attgccaatg gatttgattt aactaaacta tcgccagata aattttccag 14280

aagcgattta agaaaagaaa tcggcttagc agaaaatgat gttgtggtat gttcagttgg 14340

tcgctatagt ccagtaaaag atcactcaac ctttatttca gcagctcttc aacttgcaga 14400

agaatttagt aatgtacgct tcttattagt cggtcgtggc ttgaccaccc agaataaaat 14460

aataatgaca cagcttagtg taagctgtca ctcagataaa tttattcttc ttggtgagcg 14520

tagcgatgtt cctgcatgtt taaatgcttc tgatattttt tgccttcatt cagtaacaga 14580

aggcttccct aatgtcttag gagaagcaat ggcaatggga attccttccg tgacttcaaa 14640

cgttggtgat gccgcatttt tacttgataa ggttgatttt gttgttccag ccggaaactc 14700

gtatttatta gcacagaaac tgcgggaagt tattttgtta agtcgaaaaa accgcactaa 14760

gttaggtcgt atattaaggc tgcgaattga gaatgagttt tctatggata ctgtagccaa 14820

The termination orf14's of orf13 is initial

taaatattta tccttatata aaaat taaat gaaaaagatt gtttttataa tcaataatgt 14880

cgatttctta atatctcatc gattacctat cttactcgag gctcagaaaa atgggtttca 14940

ggttcatgtg attgctccta attccaggaa taatgagcta ctaaagaagc ataaaataat 15000

gggtcatgat ctctttcttt ccagaggagg taataaccct ttttatgatt tatttatttt 15060

gcttcagctt acaaaaattc tgaaatttct caagcccgac cttgtccatc ttgtcacgat 15120

taaaccaaca ctttatggtg ggattgctgc cagaatcgcc aaagtgcctc atgtcgttgc 15180

agctgtctct gggctgggaa ccgtattctt gagcagagga atcatcagtg gcttacgtcg 15240

cttactggtt acaaccttgt atcactccgc tctgaaacat aaaagaatac gcgttatctt 15300

tcagaaccct gatgatcgtg agttacttgt gagcgcaggc atattaaagg tttccaattc 15360

ctgtctgatc agaggttcgg gggttgattt aagagagtat ccttatcttc ctgaaaaagt 15420

tcacggtaag actgtagtaa tggcctccag gctactaagg gataaagggg tttatgagtt 15480

tatcgaagcc gctcgcttac ttaaacaaag gaatgtagag gctgatatta gaatcattgg 15540

ttctccagat acctgtaatc cgacaagtat cactgaggct gaaataagca aatgggcatc 15600

agaaaatatt gctgaattct gtggatttag aagtgacatt gctaagcagt actctaatgc 15660

taatgtaata tgcttgccct catatcgaga agggttacct aaatgtctgg ttgaagccgc 15720

agcgtgtggt agggcagttg ttacaactga tgtaccaggt tgcagggatg cgatagtggc 15780

aaatgttact gggatgttag tcgcagttcg ggatcctgta tcgttagcag atgcgattga 15840

gtttctgcta aaaaatccag atgaaagaat aaaaatgggg aaggcgggga gattgttagc 15900

tgaaaatgag tactcaatcg aacatatagt gaatcagcac ttatccattt acaatgactt 15960

The termination of orf14

aattcactcc tgacgcttag tgctggtgcc atttatgatc attctttttg catcctattt 16020

ggcacttgat gctatccatc gcgccctaac ctaagttaac atcttcaata cattcaagcc 16080

gcgcatacgt cgcgtggtga ccacacctga caggagtatg taatgtcaaa gcaacagatc 16140

ggcgtcgtcg gtatggcagt gatggggcgc aaccttgcgc tcaacatcga aagccgtggt 16200

tataccgtct ctattttcaa ccgttcccgt gagaaaacgg aagaagtaat tgccgaaaac 16260

ccaggcaaga aactggttcc ttactatacg gtgaaagagt ttgttgaatc tctggaaacg 16320

cctcgtcgca tcctgttaat ggtgaaagca ggtgcaggca cggatgctgc tattgattcc 16380

ctcaaaccat atctcgataa aggtgacatc atcattgatg gcggtaacac cttcttccag 16440

gacaccattc gtcgtaaccg tgagctttct gccgaaggct ttaacttcat tggtaccggt 16500

gtctccggtg gtgaagaagg cgcgctgaaa ggtccttcca ttatgcctgg tgggcagaaa 16560

gaagcctatg aactggttgc accgatcctg accaaaatcg ccgcagtggc tgaagacggt 16620

gagccatgcg ttacctatat tggtgccgat ggcgcaggtc actatgtgaa gatggttcac 16680

aacggtattg aatacggcga tatgcagctg attgctgaag cctattctct gcttaaaggt 16740

ggcctgaatc tcaccaacga agaattggcg cagaccttta ccgagtggaa taacggtgaa 16800

ctgagcagct acctgatcga catcaccaaa gacattttca ctaaaaaaga tgaagacggt 16860

aactacctgg ttgatgtgat tctggatgaa gcagcaaaca aaggtaccgg taaatggact 16920

agccagagcg cgctggatct cggcgaaccg ctgtcgctga ttacagagtc tgtgtttgca 16980

cgttatatct cttctctgaa agagcagcgc gttgccgcgt ctaacgtact gactggtccg 17040

caagcgcagc cagcaggcga caaggctgag tttatcgaaa aagttcgccg tgcgctgtat 17100

ctgggcaaaa tcgtttctta cgctcagggc ttttctcagc tgcgtgctgc gtctgaagag 17160

tacaactggg acctgaatta cggcgaaatc gcgaagattt tccgtgctgg ctgcatcatt 17220

cgtgcgcagt tcctgcagaa aattaccgat gcttatgccg aaaatccgca gatcgctaac 17280

ctgctgctgg ctccgtactt caagcaaatt gccgatgatt accagcaggc gctgcgtgat 17340

gtcgttgctt atgcggtaca gaacggtatc ccggttccga ccttcgctgc tgcggttgcc 17400

tattacgata gctaccgtgc cgctgttctg cct 17433

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

Claims (2)

1, a kind of oligonucleotide of the O-antigen-specific to shigella dysenteriae 7 types and intestinal bacteria O121, it is characterized in that, described oligonucleotide is to being: the Nucleotide of 7871 to 7888 bases among the SEQ ID NO:1 and the Nucleotide of 8697 to 8715 bases, the Nucleotide of 8152 to 8169 bases among the SEQ ID NO:1 and the Nucleotide of 9067 to 9086 bases, the Nucleotide of 8597 to 8614 bases among the SEQ ID NO:1 and the Nucleotide of 9102 to 9119 bases; The Nucleotide of 9336 to 9353 bases among the SEQ ID NO:1 and the Nucleotide of 11117 to 10034 bases, the Nucleotide of 9448 to 9465 bases among the SEQ ID NO:1 and the Nucleotide of 9864 to 9881 bases, the Nucleotide of 9274 to 9292 bases among the SEQ ID NO:1 and the Nucleotide of 9746 to 9763 bases; The Nucleotide of 11120 to 11137 bases among the SEQ ID NO:1 and the Nucleotide of 11366 to 11383 bases, the Nucleotide of 10957 to 10976 bases among the SEQ ID NO:1 and the Nucleotide of 11547 to 11564 bases, the Nucleotide of 10707 to 10725 bases among the SEQ ID NO:1 and the Nucleotide of 11767 to 11785 bases; The Nucleotide of 14084 to 14101 bases among the SEQ ID NO:1 and the Nucleotide of 14420 to 14437 bases, the Nucleotide of 13881 to 13889 bases among the SEQ ID NO:1 and the Nucleotide of 14761 to 14798 bases, the Nucleotide of 15225 to 15244 bases among the Nucleotide of 13975 to 13992 bases among the SEQ IDNO:1 and the Nucleotide of 14361 to 14379 bases or the SEQID NO:1 and the Nucleotide of 15765 to 15782 bases, the Nucleotide of 14975 to 14993 bases among the SEQ ID NO:1 and the Nucleotide of 15911 to 14930 bases, the Nucleotide of 15011 to 15030 bases among the SEQ ID NO:1 and the Nucleotide of 15688 to 15706 bases.

2, the right application of the described oligonucleotide of claim 1 is characterized in that it is used for PCR as primer, perhaps is used for hybridization and fluoroscopic examination, makes gene chip or microarray confession detection shigella dysenteriae 7 types and intestinal bacteria O121 type as probe.