CN1257275C - Nucleotide peculiar to 0-antigen of 0136 type bacillus coli - Google Patents

Abstract

The present invention provides a specific nucleotide for an O-antigen of Escherichia coli O136. The specific nucleotide is the nucleotide complete sequence of gene clusters in Escherichia coli O136 for controlling the synthesis of an O-antigen, such as a separated nucleotide with the total length of 11895 basic groups shown as SEQ ID NO: 1 or the nucleotide of SEQ ID NO: 1 which has one or a plurality of inserted, deleted or substituted basic groups and can simultaneously keep the functions of the separated nucleotide. The specific nucleotide also comprises the oligonucleotide from oligosaccharide unit processing genes (comprising wzx genes or genes with the similar functions of wzx and wzy genes or genes with the similar functions of wzy) in the O-antigen gene clusters of Escherichia coli O136. The present invention verifies that the oligonucleotide has high specificity to all of the O-antigen genes of Escherichia coli O136 through PCR. The present invention also discloses a method for detecting and identifying Escherichia coli O136 in human bodies and environment by the oligonucleotide of the present invention.

Description

Nucleotide to the O-antigen-specific of intestinal bacteria O136

Technical field

The present invention relates to the complete nucleotide sequence of control O-antigen synthetic gene cluster among the intestinal bacteria O136 (Escherichia coli O136), particularly relate among the intestinal bacteria O136 oligonucleotide in the control O-antigen synthetic gene cluster, can utilize these the oligonucleotide of the O-antigen-specific intestinal bacteria O136 in human body and the environment and identify O-antigen in these pathogenic bacterium quickly and accurately.

Background technology

Intestinal bacteria O136 is important pathogenic bacterium, it belongs to STEC (Shiga toxin-producingEscherichia coli), promptly produce the intestinal bacteria of Shiga toxin, it produces Stx2 and Stx1 toxin [Steven P et al (2001) " Virulence Properties and Serotypes of ShigaToxin-Producing Escherichia coli from Healthy AustralianSlaughter-Age Sheep " .Journal of Clinical Microbiology, Vol.39, No.5,2017-2021]; It also belongs to EIEC (enteroinvasive Escherichia coli) is enteroinvasive E.Coli, produce CNF1 and CNF2 toxin [Blanco Metal (1993) " Toxicproperties of enteroinvasive Escherichia coli " Microbiologia.9 (2): 149-52], cause the diarrhoea of people and livestock, therefore the detection to intestinal bacteria O136 is important.Simultaneously, intestinal bacteria O136 and pathogenic bacterium shigella dysenteriae 3 types and Shigella bogdii 1 type all have serological cross reaction, with their difficulty [Cheasty T relatively of serological method difference, Rowe B " Antigenic relationships between the enteroinvasive Escherichia coliO antigens O28ac; O112ac; O124; O136; O143; O144, O152, and O164 andShigella O antigens " J Clin Microbiol.1983 Apr; 17 (4): 681-4], so need the method that can detect intestinal bacteria O136 quickly and accurately.

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

O-antigen is the O specific polysaccharide composition in the gram negative bacterium lipopolysaccharides, and it is made up of many multiple oligosaccharide unit.The antigenic building-up process of O-is studied clearlyer: by glycosyltransferase nucleoside diphosphate monose is transferred on the fat molecule that is fixed on the cell inner membrance earlier, then in the inboard synthesis of oligose unit of inner membrance, the antigenic oligosaccharide unit of O-is transferred to the inner membrance outside by O-antigen transhipment enzyme again, then aggregate into polysaccharide by polysaccharase, be connected to again and form lipopolysaccharide molecule [Whitfield, C. (1995) " Biosynthesis of lipopolysaccharide Oantigens " .Trends in Microbiology.3:178-185 on the glycolipid molecule; Schnaitman, C.A.andJ.D.Klena. (1993) " Genetics of lipopolysaccharide biosynthesis inentericbacteria " .Mcrobiological Reviews, 57 (3): 655-682].Coding is responsible for the generally adjacent arrangement on karyomit(e) of gene of all enzyme molecules of O-antigen synthetic, form a gene cluster [Reeves, P.R., et al. (1996) " Bacterial polysaccharide synthesis and genenomenclature " Trends in Microbiology, 4:495-503].In intestinal bacteria, Shigellae and Salmonellas, O-antigen gene [Lei Wang.et al (2001) " Sequence analysis of four Shigella boydii O-antigen loci:implicationfor Escherichia coli and Shigella relationships " .Infection andImmunity, 11:6923-6930 bunch between galF and gnd gene; Lei Wang and Peter Reeves (2000) " The Escherichiacoli O111 and Salmonella enterica O35 gene clusters:gene clusters encodingthe same colitose-containing O antigen are highly conserved " .Journal ofBacteriology.182:5256-5261].The O-antigen gene bunch contains three genoids: sugared synthesis path gene, glycosyltransferase gene, oligosaccharide unit treatment gene, the required nucleoside diphosphate monose of enzymic synthesis O-antigen of wherein sugared synthesis path genes encoding; Thereby the enzyme of glycosyltransferase gene coding forwards nucleoside diphosphate monose and other molecule to and makes monose aggregate into oligosaccharide unit on the monose; The oligosaccharide unit treatment gene comprises o-antigen transhipment enzyme gene and pol gene, and they transfer to the bacterium inner membrance outside with oligosaccharide unit, and repolymerization becomes polysaccharide.Glycosyltransferase gene and oligosaccharide unit treatment gene only are present in the gene cluster of carrying these genes.The difference of monose in the O-antigen, between monose between the difference of link button and the oligosaccharide unit difference of link button constituted the antigenic diversity of O-, and the composition of monose, the link button between monose and the link button between the oligosaccharide unit are by the Gene Handling in the O-antigen gene bunch, so the O-antigen gene bunch has determined O-antigenic synthetic, has also determined the antigenic diversity of O-.

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

The antigenic structure of the O-of intestinal bacteria O136 is known, and is as follows.The repeating unit that it is made up of 3 sugar, wherein, Non pA is the sugar [Mikael Staaf et al (1999) " Structuredetermination of the O-antigenic polysaccharide from the enteroinvasiveEscherichia coliO136 " .European Journal of Biochemistry, Volume 263 Issue 3 Page656] of an important the unknown.

→4)-β-Non pA-(2→4)-β-D-Gal p-(1→4)-β-D-Glc pNAc-(1→

Summary of the invention

The Nucleotide that the purpose of this invention is to provide a kind of O-antigen-specific to intestinal bacteria O136.It is the Nucleotide in the O-antigen gene bunch of intestinal bacteria O136, is the special Nucleotide that comes from o-antigen transhipment enzyme gene, pol gene and glycosyltransferase gene.

An object of the present invention is to provide the full length nucleotide sequence of the O-antigen gene bunch of intestinal bacteria O136.

A time purpose of the present invention has provided the gene of the O-antigen gene bunch that constitutes intestinal bacteria O136: transhipment enzyme gene is the wzx gene or with wzx the gene of identity function is arranged; Pol gene is the wzy gene or with wzy the gene of identity function is arranged; Glycosyltransferase gene comprises orf2, orf4, orf7, orf9 gene; Sugar synthesis path gene comprises orf1, NeuA, NeuB.Their zero positions in O-antigen gene bunch and final position and nucleotide sequence all are listed in the table 4.

Another purpose of the present invention has provided oligonucleotide, and they come from the gene of coding transhipment enzyme in the O-antigen gene bunch of intestinal bacteria O136 respectively, comprises the wzx gene or with wzx the gene of identity function is arranged; Come from the gene of coding polysaccharase, comprise the wzy gene or the gene of identity function is arranged with wzy; Come from the gene of encoding glycosyl transferring enzyme, comprise orf2, orf4, orf7, orf9 gene.They are the oligonucleotide in the said gene, and length is at 10-20nt; They are special to the O-antigen of intestinal bacteria O136; The oligonucleotide of the gene of especially listing in the table 1 that comes from coding transhipment enzyme and the gene of polysaccharase, they are high specials to the O-antigen of intestinal bacteria O136, and these oligonucleotide are also reconfigurable, and the oligonucleotide after the combination also is a high special to the O-antigen of intestinal bacteria O136.

The above-mentioned oligonucleotide that another object of the present invention provides can be used as primer and is used for nucleic acid amplification reaction, perhaps be used for hybridization as probe, perhaps be used to make gene chip or microarray, thereby detect O-antigen and detection and identification of escherichia coli O136 with identification of escherichia coli O136 by these methods.

A further object of the present invention has provided the method for complete sequence of the O-antigen gene bunch of separating Escherichia coli O136.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 intestinal bacteria O136 it is the isolating Nucleotide shown in SEQ ID NO:1,11895 bases of total length; The base that perhaps has one or more insertions, disappearance or replacement keeps the Nucleotide of the SEQ ID NO:1 of described isolating functional nucleotide simultaneously.

The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O136 is characterized in that it comprises called after orf1, orf2, and neuA, orf4, neuB, wzx, orf7, wzy, 9 genomic constitutions of orf9 are all between galF gene and gnd gene.

The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O136 is characterized in that described gene comprises: transhipment enzyme gene is the wzx gene or with wzx the gene of identity function is arranged; Pol gene is the wzy gene or with wzy the gene of identity function is arranged; Glycosyltransferase gene comprises orf2, orf4, orf7, orf9 gene.Wherein said transhipment enzyme gene is the Nucleotide of 6609 to 7847 bases among the SEQ ID NO:1; Described pol gene is the Nucleotide of 8933 to 10165 bases among the SEQ ID NO:1; Described orf2 gene is the Nucleotide of 2134 to 3288 bases among the SEQ ID NO:1; The orf4 gene is the Nucleotide of 3973 to 5511 bases among the SEQ ID NO:1; The orf7 gene is the Nucleotide of 7895 to 8923 bases among the SEQ ID NO:1; Orf9 is the Nucleotide of 10152 to 10892 bases among the SEQ ID NO:1.

The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O136 is characterized in that it comes from the oligonucleotide of described wzx gene or wzy gene or glycosyltransferase gene; And their mixing or their reorganization.

The Nucleotide of aforesaid O-antigen high special to intestinal bacteria O136, it is characterized in that, the oligonucleotide of the described wzx of coming from gene is to being: the Nucleotide of 6620 to 6637 bases among the SEQ ID NO:1 and the Nucleotide of 7254 to 7261 bases, the Nucleotide of 6597 to 6614 bases among the SEQ ID NO:1 and the Nucleotide of 7048 to 7065 bases; The oligonucleotide of the described wzy of coming from gene is to being: the Nucleotide of 9525 to 9542 bases among the SEQ ID NO:1 and the Nucleotide of 10067 to 10084 bases, the Nucleotide of 8973 to 8992 bases among the SEQ ID NO:1 and the Nucleotide of 9885 to 9902 bases.

The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O136 is in the application that detects other polysaccharide antigen of expressing the antigenic bacterium of O-, the O-antigen of identifying bacterium and bacterium.

The recombinant molecule of the Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O136, and can provide the O-antigen of expressing intestinal bacteria O136 by inserting to express, and the application in the preparation bacterial vaccine.

The application of the Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O136 is characterized in that it is used for PCR, is used for hybridization and fluoroscopic examination or is used to make gene chip or microarray as probe as primer, for the application of bacterial detection.

The separation method of the Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O136 is characterized in that, comprises the steps:

(1) genomic extraction: in substratum, cultivate intestinal bacteria O136 type, centrifugal collecting cell; The genomic dna that obtains detects by agarose gel electrophoresis;

(2) by the O-antigen gene in the pcr amplification intestinal bacteria O136 type bunch: with the genome of intestinal bacteria O136 type is that template is passed through its O-antigen gene of Long pcr amplification bunch, with the PCR product that obtains, detect the size and the specificity thereof of PCR product with agarose gel electrophoresis, merge this long PCR product, and with DNA purification kit purified pcr product;

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

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

(5) splicing of nucleotide sequence and analysis: applying biological information science software splicing and edit all sequences, thus obtain the Nucleotide full length sequence of the O-antigen gene bunch of intestinal bacteria O136 type;

(6) screening of specific gene: at wzx, the wzy gene design primer in the O-antigen gene of intestinal bacteria O136 type bunch; Respectively designed two pairs of primers in each gene, every pair of primer is distributed in the different places in the corresponding gene, to guarantee its specificity; Is that template is carried out PCR with these primers with the genomes of 166 strain intestinal bacteria and 43 strain Shigellaes, determines the antigenic high degree of specificity of O-of wzx, wzy gene pairs intestinal bacteria O136 type;

(7) detection of primer sensitivity: cultivate intestinal bacteria O136, after the bacterial count respectively with 5 * 10 ³, 5 * 10 ², 5 * 10 ¹5 and 0 viable bacteria join in a certain amount of certain thing to be detected, sneak into the thing to be detected of bacterium and use sample as detecting, sample is added the LB substratum, getting the LB substratum that some and sample mix cross filters, filtered liquid is cultivated, carried out the PCR reaction as pcr template with oligonucleotide after the peek milliliter is handled from cultured bacterium liquid, detect its sensitivity intestinal bacteria O136.

The separation method of the Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O136 is characterized in that, comprises the steps:

(1) genomic extraction: 37 ℃ of incubated overnight intestinal bacteria O136 in the LB of 5mL substratum, centrifugal collecting cell.With 500ul 50mM Tris-HCl (pH8.0) and 10ul 0.4M EDTA re-suspended cell, 37 ℃ of incubations 20 minutes, the N,O-Diacetylmuramidase that adds 10ul 10mg/ml then continues insulation 20 minutes.The Proteinase K, the 15ul 10%SDS that add 3ul 20mg/ml afterwards, 50 ℃ of incubations 2 hours add the RNase of 3ul 10mg/ml again, 65 ℃ of incubations 30 minutes.Add equal-volume phenol extracting mixture, get supernatant liquor and use isopyknic phenol again: chloroform: twice of primary isoamyl alcohol (25: 24: 1) mixing solutions extracting, get supernatant liquor, again with isopyknic ether extracting to remove remaining phenol, supernatant liquor is with 2 times of volume ethanol deposit D NA, roll out DNA and wash DNA with 70% ethanol with glass yarn, at last DNA is resuspended among the 30ul TE, genomic dna detects by 0.4% agarose gel electrophoresis;

(2) by the O-antigen gene among the pcr amplification intestinal bacteria O136 bunch: with the genome of intestinal bacteria O136 is that template is passed through its O-antigen gene of Long pcr amplification bunch; At first according to the galF gene design upstream primer (5 '-ATT GTG GCT GCA GGG ATCAAA GAA ATC-3 ') that often is found in O-antigen gene bunch upstream, again according to the gnd gene design downstream primer (5 '-TAGTCG CGC TGN GCC TGG ATT AAG TTC GC-3 ') in O-antigen gene bunch downstream.With the Expand Long Template PCR method of Boehr inger Mannheim company amplification O-antigen gene bunch, the PCR response procedures is as follows: 94 ℃ of pre-sex change 2 minutes, 94 ℃ of sex change were 10 seconds then, 60 ℃ of annealing 30 seconds, 68 ℃ were extended 15 minutes, and carried out 30 circulations like this; At last, continue to extend 7 minutes at 68 ℃, obtain the PCR product, the agarose gel electrophoresis with 0.8% detects the size and the specificity thereof of PCR product; Merge 6 pipe long PCR products, and with the Wizard PCR Preps purification kit purified pcr product of Promega company;

(3) make up O-antigen gene bunch library: make up O-antigen gene bunch library with the Novagen DNaseI shot gun method that is modified.Reaction system is a 300ng PCR purified product, 0.9ul 0.1M MnCl2, and the DNaseI of the 1mg/ml of 1ul dilution in 1: 2000, reaction is carried out at room temperature; Enzyme is cut the dna fragmentation size is concentrated between the 1kb-3kb, then adds 2ul 0.1M EDTA termination reaction; Merge the same reaction system of 4 pipes, with isopyknic phenol extracting once, use isopyknic phenol: chloroform: primary isoamyl alcohol (25: 24: 1) mixing solutions extracting once, after using isopyknic ether extracting once again, dehydrated alcohol deposit D NA with 2.5 times of volumes, and wash precipitation with 70% ethanol, be resuspended at last in the 18ul water; In this mixture, add 2.5ul dNTP (1mMdCTP subsequently, 1mMdGTP, 1mMdTTP, 10mMdATP), 1.25ul the T4DNA polysaccharase of 100mM DTT and 5 units, 11 ℃ were reacted 30 minutes, enzyme is cut product mend into flush end, after 75 ℃ of termination reactions, add the Tth archaeal dna polymerase of 5 units and corresponding damping fluid thereof and system is expanded as 80ul, 70 ℃ of reactions 20 minutes make 3 of DNA ' end add the dA tail, and this mixture is through the equal-volume chloroform: after primary isoamyl alcohol (24: 1) mixing solutions extracting and the extracting of equal-volume ether with 3 * 10 of Promega company ^-3The pGEM-T-Easy carrier connect 24 hours in 16 ℃, cumulative volume is 90ul, and the 10 * buffer of 9ul and the T4DNA ligase enzyme of 25 units are wherein arranged; Use the dehydrated alcohol precipitation of the 3M NaAc (pH5.2) of 1/10 volume and 2 times of volumes to be connected mixture at last, wash precipitation with 70% ethanol again, be dissolved in after the drying and obtain connecting product in the 30ul water.Preparation method with the electric transformed competence colibacillus cell of Bio-Rad company prepares the competence e.colidh5, get after 2-3ul connects product and 50ul competence bacillus coli DH 5 alpha mixes, forward in the electric shock cup of 0.2cm of Bio-Rad company and shock by electricity, voltage is 2.5 kilovolts, time is 5.0 milliseconds-6.0 milliseconds, the SOC substratum that adds 1ml after the electric shock immediately in cup makes the bacterium recovery, then bacterium is coated in and contains penbritin, 37 ℃ of incubated overnight on the LB solid medium of X-Gal and IPTG, obtain blue white bacterium colony next day, with the white colony that obtains promptly the white clone forward on the LB solid medium that contains penbritin and cultivate, extract plasmid and cut the segmental size of evaluation insertion wherein with the EcoRI enzyme from each clone simultaneously, the white that obtains clone group has constituted the O-antigen gene bunch library of intestinal bacteria O136;

(4) to the cloning and sequencing in the library: from the library, select insert 100 clones of fragment more than 1000bp by Shanghai biotechnology company limited with ABI377 type automatic dna sequencer to unidirectional order-checking of insertion fragment in cloning, make sequence reach 80% fraction of coverage, carry out backward sequencing by the sequence that will interrelate again and survey logically obtaining remaining 20% sequence, thereby obtain all sequences of O-antigen gene bunch;

(5) splicing of nucleotide sequence and analysis: the Pregap4 and the splicing of Gap4 software of the Staden package software package of publishing with Britain Camb MRC (Medical ResearchCouncil) Molecular Biology Lab and edit all sequences, thereby obtain the Nucleotide full length sequence of the O-antigen gene bunch of intestinal bacteria O136, the quality of sequence is mainly guaranteed by two aspects: 1) genome of intestinal bacteria O136 is done 6 Long PCR reactions, mix these products then to produce the library.2), guarantee high-quality fraction of coverage more than 3 to each base; Behind the nucleotide sequence of the O-antigen gene that obtains intestinal bacteria O136 bunch, with American National biotechnology information science center (The National Center forBiotechnology Information, NCBI) orffinder finds gene, find the reading frame of 9 openings, determine also that with the function of finding the reading frame that these are open what gene they are with the genetic comparison among the blast groupware and the GenBank, finish gene annotation with the Artemis software at Britain sanger center again, do accurate comparison between DNA and protein sequence with Clustral W software, obtain the structure of the O-antigen gene bunch of intestinal bacteria O136 at last;

(6) screening of specific gene: at wzx, wzy, the orf8 gene design primer in the O-antigen gene of intestinal bacteria O136 bunch; Respectively designed two pairs of primers in each gene, the difference that every pair of primer is distributed in the corresponding gene is local to guarantee its specificity; Is that template is carried out PCR with these primers with the intestinal bacteria of 166 kinds of serotypes and the genome of 43 strain Shigellaes, all primers all obtain positive findings in intestinal bacteria O136, the correct band of any size does not all increase in other groups, that is to say, do not obtaining any PCR product band in the array mostly, though obtain PCR product band in the minority group, its size does not meet the expection size, so wzx, wzy gene pairs intestinal bacteria O136 and O-antigen thereof all are high specials.

(7) detection of primer sensitivity: buy the live pig meat stuffing on the market, stir, be divided into the 20g portion, exist in-40 ℃ of refrigerators standby.The frozen bacterium liquid of 10 μ l intestinal bacteria O136 is inoculated in the triangular flask of 20ml LB substratum, in 37 ℃, 200 rev/mins, cultivate 12 hours to saturated, the cultured bacterium liquid that takes a morsel does 10 ⁶With 10 ⁷Dilution doubly, remaining bacterium liquid are put in 4 ℃ the refrigerator standby, get 50 μ l dilution bacterium liquid coating LB agar plate, and 37 degree are cultivated 12h, to being coated with plate count, calculate viable bacteria concentration in the stoste.In 5 portions of live pig meat stuffings, mix 5 * 10 respectively ³, 5 * 10 ², 5 * 10 ¹, 5 and 0 viable bacteria stir, and add 200ml LB substratum, and through 6 layers of filtered through gauze, filtered liquid 200 rev/mins, is cultivated 12h in 37 ℃.Get 3ml bacterium liquid in 6 from cultured bacterium liquid, centrifugal 5 minutes of 000g removes supernatant, adds 100 μ l MQ ultrapure waters and blows precipitation and mixing open, puts into 100 degree boiling water and boils 15 minutes, and lysate is in 12, and centrifugal 8 minutes of 000g gets 1 μ supernatant as pcr template.With 4 pairs of oligonucleotide to the Nucleotide of 6620 to 6637 bases among the SEQ ID NO:1 and the Nucleotide of 7254 to 7261 bases, the Nucleotide of 6597 to 6614 bases among the SEQ ID NO:1 and the Nucleotide of 7048 to 7065 bases, the Nucleotide of 9525 to 9542 bases among the SEQ ID NO:1 and the Nucleotide of 10067 to 10084 bases, the Nucleotide of 8973 to 8992 bases among the SEQ ID NO:1 and the Nucleotide of 9885 to 9902 bases carry out the PCR reaction, the PCR reaction system is as follows: MQ:15.7 μ l, Mg ²⁺: 2.5 μ l, Buffer:2.5 μ l, dNTP:1 μ l, Taq enzyme: 0.3 μ l, P1:1 μ l, P2:1 μ l, template DNA: 1 μ l.The PCR reaction conditions is: 95 ℃: 5 ', 95 ℃: 30 ", 56 ℃: 45 ", 72 ℃: 1 ', 72 ℃: 5 ', totally 30 circulations.Reaction is got 10 μ l reaction product electrophoresis after finishing, if the amplified band that conforms to the expection size is arranged, then the result is positive, if do not have, then the result is negative.Participated in 5 * 10 ³, 5 * 10 ², 5 * 10 ¹And every part of pork filling of 5 viable bacterias all obtains positive findings in the PCR of 4 pairs of primers reaction.The pork filling that participates in 0 viable bacteria obtains negative findings in the PCR of 4 pairs of primers reaction.Illustrate that these 4 pairs of primers are 0.25 bacterium/g to the detection sensitivity of the intestinal bacteria O136 in the pork filling when using aforesaid method.

Just, first aspect of the present invention provides the full length nucleotide sequence of the O-antigen gene bunch of intestinal bacteria O136, its complete sequence shown in SEQ ID NO:1,11895 bases of total length; The base that perhaps has one or more insertions, disappearance or replacement keeps the Nucleotide of the SEQ ID NO:1 of described isolating functional nucleotide simultaneously.Obtained the structure of the O-antigen gene bunch of intestinal bacteria O136 by method of the present invention, as described in Table 3, it comprises called after orf1, orf2, neuA, orf4, neuB, wzx, orf7, wzy, 9 genome Chengdu of orf9 are between galF gene and gnd gene.

Second aspect of the present invention provides the gene in the O-antigen gene bunch of intestinal bacteria O136, 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 orf2, orf4, orf7, orf9 gene.Special sugared synthesis path gene in the bacterial polysaccharides antigen comprises orf1, NeuA, NeuB gene.Their zero positions in O-antigen gene bunch and final position and nucleotide sequence all are listed in the table 4.The invention particularly relates to o-antigen transhipment enzyme gene and pol gene, because sugared synthesis path gene is that the gene of synthetic nucleosides bisphosphate monose is common, common by indication to more exocellular polysaccharide now, O-antigen to bacterium is not special, and the o-antigen that the present invention relates to transhipment enzyme gene, pol gene and glycosyltransferase gene are special to the O-antigen of intestinal bacteria O136.

The 3rd aspect of the present invention, wzy gene in the O-antigen gene bunch that comes from intestinal bacteria O136 is provided or the gene of identity function and wzx gene is arranged or with wzx the oligonucleotide of gene of identity function and the oligonucleotide that glycosyltransferase gene comprises orr2, orf4, orff7, orf9 gene are arranged with wzy, they are any one section oligonucleotide in these genes.But, be to list in the wzy gene in the O-antigen gene bunch that comes from intestinal bacteria O136 in the table 1 or the gene, wzx gene of identity function arranged or have the oligonucleotide of gene of identity function right preferentially with wzx with wzy by usefulness.In table 1, also listed these oligonucleotide to the position in O-antigen gene bunch and with these oligonucleotide to being the size of the product of the PCR reaction done of primer, the annealing temperature in these PCR reaction free lists is carried out.These primers are being to obtain expecting the product of size in the pcr amplification that carries out of template with intestinal bacteria O136 only, and are all not obtain expecting the product of size in the pcr amplification that carries out of template other bacterium listed with table 2.In more detail, with these oligonucleotide to being that PCR that primer is done is reflected at and does not all obtain spawn in most of bacteriums, though in some bacterium, obtained PCR product band, but its size does not meet the expection size, this is that this problem can be avoided by being PCR with intragenic other primer because primer is attached to genomic other position and causes.So, can determine these primers promptly the listed oligonucleotide of table 1 be high special to intestinal bacteria O136 and their O-antigen.

The separation and the authentication method of the Nucleotide of described O-antigen-specific to intestinal bacteria O136 comprise the steps: 1) genomic extraction; 2) the O-antigen gene among the pcr amplification intestinal bacteria O136 bunch; 3) structure in O-antigen gene bunch library; 4) to the cloning and sequencing in the library; 5) splicing of nucleotide sequence and analysis finally obtain the structure of O-antigen gene bunch; 6) screening of specific gene; 7) detection of primer sensitivity.

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

As described herein, " oligonucleotide " mainly is meant gene, the gene of coding polysaccharase and one section nucleic acid molecule in the encoding glycosyl transferase gene of the coding transhipment enzyme that derives from the O-antigen gene bunch, they can change on length, generally change in 10 to 20 Nucleotide scopes.Especially come from wzx gene (nucleotide position is 6609 to 7847 bases from SEQ ID NO:1), the oligonucleotide in the wzy gene (nucleotide position is 8933 to 10165 bases from SEQ ID NO:1) all is a high special to intestinal bacteria O136.

In addition, the antigenic gene cluster of the different O-of the coding of two genetic resemblances produces new O-antigen by gene recombination or sudden change sometimes, thereby produces new bacteria types, new mutant strain.In this environment, need filter out many specificitys that oligonucleotide is detected with raising with recombination hybridization.Therefore, the invention provides a whole set of many mixtures to oligonucleotide, they come from transhipment enzyme gene, comprise the wzx gene or with wzx the gene of identity function are arranged; Come from pol gene, comprise the wzy gene or the gene of identity function is arranged with wzy; Come from glycosyltransferase gene, comprise orf2, orf4, orf7, orf9 gene.The mixture of these genes is special to a special bacterial polysaccharides antigen, is special thereby make this cover oligonucleotide to the polysaccharide antigen of this bacterium.More particularly, the mixture of these oligonucleotide is to come from transhipment enzyme gene, come from pol gene and come from the combination of the oligonucleotide in the glycosyltransferase gene.

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

The present invention relates to a kind of antigenic method of one or more bacterial polysaccharideses that detects in the food, these antigens can make sample can with the oligonucleotide specific hybrid of following at least one gene, these genes are: (i) coding transhipment enzyme gene, comprise the wzx gene or (ii) the encode gene of polysaccharase of the gene of identity function is arranged, comprise the wzy gene or the gene of identity function is arranged with wzy with wzx.(iii) the encoding glycosyl transferase gene comprises orf2, orf4, orf7, orf9 gene.At least one oligonucleotide can be hybridized with at least one more than one such gene specific of expressing the special antigenic bacterium of O-under the situation of condition permission, and these bacteriums are intestinal bacteria O136.Available PCR method detects, more can with behind the Nucleotide mark in the inventive method as probe by hybridization such as southern-blot or fluoroscopic examination, perhaps by antigen and bacterium in gene chip or the microarray assay sample.

The present inventor considers following situation: when one special oligonucleotide detects when invalid, the mixture of oligonucleotide can with the target region specific hybrid with test sample.Therefore the invention provides a cover oligonucleotide and be used for detection method of the present invention.Here said oligonucleotide is meant and comes from that coding transhipment enzyme gene comprises the wzx gene or have the gene of gene, the coding polysaccharase of identity function to comprise the wzy gene with wzx or with wzy the oligonucleotide of gene of identity function and the oligonucleotide that the encoding glycosyl transferase gene comprises orf2, orf4, orf7, orf9 gene are arranged.This cover oligonucleotide is special to the O-antigen of a special bacterium, and this special bacterium O-antigen is expressed by intestinal bacteria O136.

On the other hand, the present invention relates to a kind of antigenic method of one or more bacterial polysaccharideses that detects in the movement, these antigens can make sample can with the oligonucleotide specific hybrid of following at least one gene, these genes are: (i) gene of coding transhipment enzyme, comprise the wzx gene or (ii) the encode gene of polysaccharase of the gene of identity function is arranged with wzx, the gene that comprises the wzy gene or identity function arranged with wzy is the encoding glycosyl transferase gene (iii), comprises orf2, orf4, orf7, orf9 gene.At least one oligonucleotide can be expressed more than one such gene specific hybridization of the special antigenic bacterium of O-with at least one under the situation of condition permission.These bacteriums are intestinal bacteria O136.Oligonucleotide among available the present invention is made the method test sample of primer by PCR, also can with behind the oligonucleotide molecules mark among the present invention as probe by hybridization such as southern-blot or fluoroscopic examination, perhaps by antigen and bacterium in gene chip or the microarray assay sample.

General a pair of oligonucleotide may with same gene recombination also can with different gene recombinations, but must have in them an oligonucleotide can specific hybrid to the distinguished sequence of special antigenic type, another oligonucleotide can be hybridized in non-specific zone.Therefore, when the oligonucleotide in the special polysaccharide antigen gene cluster is reconfigured, can select specific gene mixture hybridization in a pair of oligonucleotide and the polysaccharide antigen gene cluster at least, perhaps select many mixture hybridization oligonucleotide and specific gene.Even even when all genes were all unique in the specific genes bunch, this method also can be applied to discern the nucleic acid molecule of the gene mixture in this gene cluster.Therefore the invention provides a whole set of and be used to detect the many to oligonucleotide of the inventive method, many here is that the gene that comes from coding transhipment enzyme comprises the wzx gene or with wzx the gene of identity function arranged to oligonucleotide; The gene that comes from the coding polysaccharase comprises the wzy gene or with wzy the gene of identity function is arranged; The gene that comes from the encoding glycosyl transferring enzyme comprises orf2, orf4, orf7, orf9 gene.This cover oligonucleotide is special to a special bacterial polysaccharides, and this cover oligonucleotide may be the Nucleotide of necessary gene during sugar synthesizes.

On the other hand, the present invention also relates to the antigenic method of one or more bacterial polysaccharideses in the sample that a kind of detection comes from patient.One or more bacterial polysaccharides antigens in the sample can make sample can with a specific hybrid in a pair of oligonucleotide in following at least one gene, these genes are: (i) gene of coding transhipment enzyme, comprise the wzx gene or (ii) the encode gene of polysaccharase of the gene of identity function is arranged with wzx, the gene that comprises the wzy gene or identity function arranged with wzy is the encoding glycosyl transferase gene (iii), comprises orf2, orf4, orf7, orf9 gene.Under the situation of condition permission at least one oligonucleotide can with sample at least one express more than one such gene specific hybridization of the special antigenic bacterium of O-, these bacteriums are intestinal bacteria O136.Oligonucleotide among available the present invention is made the method test sample of primer by PCR, also can will pass through hybridization as probe behind the oligonucleotide mark among the present invention, perhaps by antigen and bacterium in gene chip or the microarray assay sample.

In more detail, method described above can be understood as when oligonucleotide when being used, it is not to derive from the wzx gene or the gene of identity function and wzy gene are arranged or have the gene of identity function and glycosyltransferase gene to comprise on the sequence of orf2, orf4, orf7, orf9 gene with wzy with wzx that one of them oligonucleotide molecules can hybridize to one.In addition, when two oligonucleotide can both be hybridized, they may be hybridized in same gene and also may hybridize on the different genes.Also promptly, when cross reaction goes wrong, can select oligonucleotide mixture to detect the blended gene so that the specificity of detection to be provided.

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

The present invention discloses the full length sequence of the O-antigen gene bunch of intestinal bacteria O136 first, and can from the sequence of this total length gene cluster of not cloned, produce recombinant molecule, can produce the O-antigen of expressing intestinal bacteria O136 by inserting to express, and become useful vaccine.

Embodiment:

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

Embodiment 1: genomic extraction.

37 ℃ of incubated overnight intestinal bacteria O136 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 10ul10mg/ml then continues insulation 20 minutes.The Proteinase K, the 15ul 10%SDS that add 3ul 20mg/ml afterwards, 50 ℃ of incubations 2 hours add the RNase of 3ul 10mg/ml again, 65 ℃ of incubations 30 minutes.Add equal-volume phenol extracting mixture, get supernatant liquor, use isopyknic phenol again: chloroform: primary isoamyl alcohol (25: 24: 1) solution extracting twice, get supernatant liquor, again with isopyknic ether extracting to remove remaining phenol.Supernatant liquor rolls out DNA and washes DNA with 70% ethanol with glass yarn with 2 times of volume ethanol deposit D NA, at last DNA is resuspended among the 30ul TE.Genomic dna detects by 0.4% agarose gel electrophoresis.

Embodiment 2: by the O-antigen gene among the pcr amplification intestinal bacteria O136 bunch

With the genome of intestinal bacteria O136 is that template is passed through its O-antigen gene of Long pcr amplification bunch.At first according to the galF gene design upstream primer (5 '-ATTGTG GCT GCA GGG ATC AAA GAA ATC-3 ') that often is found in O-antigen gene bunch upstream, again according to the gnd gene design downstream primer (5 '-TAG TCG CGC TGN GCC TGG ATT AAG TTC GC-3 ') in O-antigen gene bunch downstream.With the Expand Long Template PCR method of Boehringer Mannheim company amplification O-antigen gene bunch, the PCR response procedures was as follows: 94 ℃ of pre-sex change 2 minutes; 94 ℃ of sex change are 10 seconds then, 61 ℃ of annealing 30 seconds, and 68 ℃ were extended 15 minutes, and carried out 30 circulations like this; At last, continue to extend 7 minutes at 68 ℃, obtain the PCR product, the agarose gel electrophoresis with 0.8% detects the size and the specificity thereof of PCR product.Merge 6 pipe long PCR products, and with the Wizard PCR Preps purification kit purified pcr product of Promega company.

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

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

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

Be electric transformed competence colibacillus cell at last: get after 2-3ul connects product and 50ul competence bacillus coli DH 5 alpha mixes, forward in the electric shock cup of 0.2cm of Bio-Rad company and shock by electricity, voltage is 2.5 kilovolts, and the time is 5.0 milliseconds-6.0 milliseconds.The SOC substratum that adds 1ml after the electric shock immediately in cup makes the bacterium recovery.Immediately bacterium is coated in 37 ℃ of inversion incubated overnight on the LB solid medium that contains penbritin, X-Gal and IPTG then, obtains blue white bacterium colony next day.With the white colony that obtains promptly the white clone forward on the LB solid medium that contains penbritin and cultivate, from each clone, extract plasmid and cut the segmental size of evaluation insertion wherein simultaneously, obtain the O-antigen gene bunch library that white clone group has constituted intestinal bacteria O136 with the EcoRI enzyme.

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

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

Embodiment 5: the splicing of nucleotide sequence and analysis.

The Pregap4 and the splicing of Gap4 software of the Staden package software package of publishing with Britain Camb MRC (Medical Research Council) Molecular Biology Lab and edit all sequences, thus the Nucleotide full length sequence (seeing sequence list) of the O-antigen gene bunch of intestinal bacteria O136 obtained.The quality of sequence is mainly guaranteed by two aspects: 1) genome of intestinal bacteria O136 is done 6 LongPCR reactions, mix these products then to produce the library.2), guarantee high-quality fraction of coverage more than 3 to each base.Behind the nucleotide sequence of the O-antigen gene that obtains intestinal bacteria O136 bunch, with American National biotechnology information science center (The National Center for BiotechnologyInformation, NCBI) orffinder finds gene, find the reading frame of 9 openings, determine also that with the function of finding the reading frame that these are open what gene they are with the genetic comparison among the blast groupware and the GenBank, finish gene annotation with the Artemis software at Britain sanger center again, do accurate comparison between DNA and protein sequence with Clustral W software, obtain the structure of the O-antigen gene bunch of intestinal bacteria O136 at last, as shown in table 3.

By retrieving and relatively, finding that the flaA1 albumen of orf1 and Helicobacter pylori 26695 has 57% homogeny, 73% similarity in 325 amino acid; FlaA1 albumen is the mutase in the biosynthesizing of polysaccharide, so orf1 also is a mutase, and called after orf1 temporarily.The transaminase of the supposition of Orf2 and Campylobacter jejuni subsp.jejuni NCTC 11168 has 40% homogeny in 372 amino acid, 59% similarity, therefore infer that orf2 also is the gene of a transaminase, temporarily called after orf2.The NeuA of orf3 and Aeromonas punctata has 59% homogeny in 225 amino acid, 76% similarity, and higher homogeny shows the orf3 NeuA that also encodes, therefore with orf3 called after neuA.The acetyltransferase of Orf4 and Bacteroidesthetaiotaomicron VPI-5482 has 35% homogeny in 89 amino acid; 50% similarity; illustrating has higher homology between them, therefore infer that orf4 also is an acetyltransferase gene, temporarily called after orf4.The NeuB of Orf5 and Aeromonas punctata has 76% homogeny in 345 amino acid, 84% similarity, and the homogeny of height shows the orf5 NeuB that also encodes, therefore with orf5 called after neuB.In genbank, seek conservative functional domain, find that the O-antigen transhipment enzyme of orf6 and Pseudomonas aeruginosa has 34% homogeny, 56% similarity in 386 amino acid whose sequences.And algorithm [Eisenberg by people such as Eisenberg, D, Schwarz, E.etal (1984) .Analysis of membrane and surfaceprotein sequences with the hydrophobic moment plot.J.Mol.Biol.179:125-142] find that orf6 has 12 potential transmembrane domains, it and many Wzx protein similars, and have-individual about 40 amino acid whose conservative motifs at the proteic aminoterminal of Wzx, so can determine orf6 is the wzx gene, called after wzx.The structure of intestinal bacteria O136 is known, knows its antigenic synthetic also glycosyltransferase of needs of O-according to its structure, so infers that orf7 also is a glycosyltransferase gene, temporarily called after orf7.Blast comparison shows that orf8 and many Wzy protein similars, for example and Vibro cholerae in 227 amino acid, 23% homogeny is arranged, 41% similarity.Learn that by the Eisenberg algorithm orf8 has 11 potential transmembrane domains in addition, to other O one antigen polysaccharase similar secondary structure is arranged, a big loop is arranged, have the feature of typical O-antigen polysaccharase, so determine that orf8 is the wzy gene, called after wzy.Seek conservative functional domain in genbank, find that orf9 is similar to the WecB of glycosyltransferase family of PF03808, the E value is 7 * e ^-8, find relatively that by carrying out blast the glycosyltransferase of orf9 and Vibro cholerae has 23% homogeny respectively in 392 amino acid, 57% similarity, illustrating has higher homology between them, therefore infer that orf9 also is a glycosyltransferase gene, temporarily called after orf9.

Embodiment 6: the screening of specific gene

At wzx, wzy gene design primer in the O-antigen gene of intestinal bacteria O136 bunch, the position of these genes in nucleotide sequence sees Table 1.

Transhipment enzyme gene, pol gene and their function corresponding and the size of the O antigen gene bunch of intestinal bacteria O136 in table 1, have been listed.In each gene, we have respectively designed two pairs of primers, and the difference that every pair of primer is distributed in the corresponding gene is local to guarantee its specificity.In table, also listed position and the size of each primer in SEQ ID NO:1.Is that template carry out PCR with listed corresponding annealing temperature in the table with the genomes of all bacterium in the table 2 with every pair of primer, has obtained corresponding PCR product, and its size is also listed in the table.

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

Table 2 is intestinal bacteria and 43 strain Shigellaes and their sources that are used to screen 166 kinds of serotypes of specific gene, and for the convenience that detects, we are divided into one group, 13 groups altogether with their every 12-19 bacterium.All list in the table in their source.

In the 7th group, contain the genomic dna of intestinal bacteria O136 as positive control.In the 13rd group is the genomic dna that does not contain intestinal bacteria O136, as negative control.Do template with every group of bacterium, be PCR by following condition with every pair in the table 1 primer: 95 ℃ of pre-sex change after 2 minutes, 95 ℃ of sex change 15 seconds, annealing temperature is because of the difference different (with reference to table 1) of primer, annealing time is 50 seconds, and 72 ℃ were extended 2 minutes, and carried out 30 circulations like this.Continue to extend 10 minutes at 72 ℃ at last, reaction system is 25ul.After reaction finishes, get the 10ulPCR product and detect the fragment that amplifies by 0.8% agarose gel electrophoresis.

For wzx, wzy gene, each gene all has two pairs of primers detected, and every pair of primer has obtained except be PCR in the 7th group after the correct band of expection size, and the correct band of any size does not all increase in other groups.Obtain the PCR product band of a 2kb according to a pair of primer of wzy gene design in other group, size is incorrect, is a nonspecific product band, so wzx, wzy gene pairs intestinal bacteria O136 and O-antigen thereof all are high specials.

At last, from intestinal bacteria O136, screen gene by PCR: wzx, wzy gene to the O-antigen high special of intestinal bacteria O136.And the oligonucleotide of these intragenic any one section 10-20nt is special to the O-antigen of intestinal bacteria O136, and the primer in especially above-mentioned each gene is that oligonucleotide is high special to detecting the back confirmation through PCR to intestinal bacteria O136.These all oligonucleotide all can be used for the intestinal bacteria O136 in the human body and environment rapidly and accurately, and can identify their O-antigen.

(7) detection of primer sensitivity: buy the live pig meat stuffing on the market, stir, be divided into the 20g portion, exist in-40 ℃ of refrigerators standby.The frozen bacterium liquid of 10 μ l intestinal bacteria O136 is inoculated in the triangular flask of 20ml LB substratum, in 37 ℃, 200 rev/mins, cultivate 12 hours to saturated, the cultured bacterium liquid that takes a morsel does 10 ⁶With 10 ⁷Dilution doubly, remaining bacterium liquid are put in 4 ℃ the refrigerator standby, get 50 μ l dilution bacterium liquid coating LB agar plate, and 37 degree are cultivated 12h, to being coated with plate count, calculate viable bacteria concentration in the stoste.In 5 portions of live pig meat stuffings, mix 5 * 10 respectively ³, 5 * 10 ², 5 * 10 ¹, 5 and 0 viable bacteria stir, and add 200ml LB substratum, and through 6 layers of filtered through gauze, filtered liquid 200 rev/mins, is cultivated 12h in 37 ℃.Get 3ml bacterium liquid in 6 from cultured bacterium liquid, centrifugal 5 minutes of 000g removes supernatant, adds 100 μ l MQ ultrapure waters and blows precipitation and mixing open, puts into 100 degree boiling water and boils 15 minutes, and lysate is in 12, and centrifugal 8 minutes of 000g gets 1 μ supernatant as pcr template.Right with four pairs of oligonucleotide, the Nucleotide of 6620 to 6637 bases among the SEQ ID NO:1 and the Nucleotide of 7254 to 7261 bases, the Nucleotide of 6597 to 6614 bases among the SEQ ID NO:1 and the Nucleotide of 7048 to 7065 bases, the Nucleotide of 9525 to 9542 bases among the SEQ ID NO:1 and the Nucleotide of 10067 to 10084 bases, the Nucleotide of 8973 to 8992 bases among the SEQ ID NO:1 and the Nucleotide of 9885 to 9902 bases carry out the PCR reaction, the PCR reaction system is as follows: MQ:15.7 μ l, Mg ²⁺: 2.5 μ l, Buffer:2.5 μ l, dNTP:1 μ l, Taq enzyme: 0.3 μ l, P1:1 μ l, P2:1 μ l, template DNA: 1 μ l.The PCR reaction conditions is: 95 ℃: 5 ', 95 ℃: 30 ", 56 ℃: 45 ", 72 ℃: 1 ', 72 ℃: 5 ', totally 30 circulations.Reaction is got 10 μ l reaction product electrophoresis after finishing, if the amplified band that conforms to the expection size is arranged, then the result is positive, if do not have, then the result is negative.Participated in 5 * 10 ³, 5 * 10 ², 5 * 10 ¹And every part of pork filling of 5 viable bacterias all obtains positive findings in the PCR of 4 pairs of primers reaction.The pork filling that participates in 0 viable bacteria obtains negative findings in the PCR of 4 pairs of primers reaction.Illustrate that these 4 pairs of primers are 0.25 bacterium/g to the detection sensitivity of the intestinal bacteria O136 in the pork filling when using aforesaid method.

By clone and the expression in the vaccine strains of attenuation, can set up recombiant vaccine to O antigen gene bunch.O antigen is the surface antigen of topmost Gram-negative bacteria, can cause the intensive immune response, is one of best target molecule of making recombiant vaccine.Viret laboratory success in 1993 the O antigen gene of Shigellae Sonnei bunch is expressed in a strain Salmonellas Tyziai vaccine bacterium, experimentation on animals proof can cause rabbit immune response (Molecular Microbiology1993,7:239-252).The group of China Military Medical Science Institute also similarly works being engaged in the Viret laboratory.Bunch express the O antigen gene of intestinal bacteria O111 success in 1999 in the Wang Lei laboratory in salmonella vaccine STM-1, and the bacterial strain set up of proof can cause the blood of mouse and humoral response (Microbial Pathogenesis 1999,27:55-59).So the O antigen-specific gene order of O136 of the present invention can be applied to set up recombiant vaccine.

Nucleotide sequence (shown in the SEQ IDNO:1) according to the O-antigen-specific to intestinal bacteria O136 type of the present invention, structure specific nucleic acid probe, be fixed on the carrier of chip and make biochip, after the sample that will detect is suitably handled, carry out hybridization with biochip, utilize the biochip signal analysis equipment just can obtain corresponding bacteria situation in the sample then.The DNA chip that this intestinal bacteria O antigen is identified can be directly used in clinical and other check place (as food-processing and production industry, the Micro biological Tests of animal and veterinary industry customs quarantine control etc.).This chip only need enlarge output, just can industrialization under identical condition.

Table 3 is structural tables of the O-antigen gene bunch of intestinal bacteria O136, has listed the structure of the O-antigen gene bunch of intestinal bacteria O136 in table, altogether by 9 genomic constitutions, and each gene box indicating, and in square frame, write the title of gene.Two ends at O-antigen gene bunch are galF gene and gnd gene, and they do not belong to O-antigen gene bunch, and we are just with the increase full length sequence of O-antigen gene bunch of their one section sequences Design primer.

Table 4 is location tables of the gene in the O-antigen gene bunch of intestinal bacteria O136, in table, listed the accurate position of all open reading frame in complete sequence in the O-antigen gene bunch of intestinal bacteria O136, at the underscoring of the initiator codon and the terminator codon of each open reading frame.The initiator codon of open reading frame has two in bacterium: ATG and GTG.

SEQ ID NO:1 sequence (SEQUENCE LISTING)

＜110〉Tianjin Biochip Technology Co., Ltd

＜120〉to the Nucleotide of the O-antigen-specific of intestinal bacteria O136

<160>1

<170>PatentIn version 3.1

<210>1

<211>11895

<212>DNA

<213>Escherichia coli

<400>1

attgtggctg cagggatcaa agaaatcctc ctggtaactc acgcgtccaa gaacgcggtc 60

gaaaaccact tcgacacctc ttatgaatta gaatctcttc ttgagcagcg cgtgaagcgt 120

caactgcttg cggaagtgca gtccatctgt ccaccgggcg tgaccattat gaacgtgcgt 180

cagggcgaac ctttaggttt aggccactcc attttgtgtg cacgacccgc cattggtgac 240

aacccatttg tcgtggtgct gccagacgta gtgatcgacg acgccagcgc cgacccgctg 300

cgctacaacc ttgctgccat gattgcgcgc ttcaatgaaa cgggccgtag ccaggtgctg 360

gcaaaacgta tgccgggtga cctctctgaa tactccgtca tccagaccaa agaaccgctg 420

gatcgtgaag gtaaagtcag ccgcattgtt gaatttatcg aaaaaccgga tcagccgcag 480

acgttggact cagacatcat ggccgtaggt cgttatgtgc tttctgccga tatttggccg 540

gaacttgaac gcacgcagcc aggggcatgg ggacgtattc agctgactga tgctatcgct 600

gaactggcga aaaaacaatc cgttgatgcc atgctgatga caggtgacag ctacgactgc 660

ggtaaaaaaa tgggttatat gcaggcattt gtgaagtatg gactacgcaa tctgaaagaa 720

ggggcgaagt tccgtaaagg gattgagaag ctgttaagcg aataatgaaa atctgaccgt 780

atgtaacggt tgataagaaa attataacgg cagtgaagat ttgtggcgaa agtaatttgt 840

tgggaatttt cctgccgttg ttttatataa acaatcagga taacaacgag ttagcaatag 900

gattttagtc aaagttttcc aggattttcc ttgtttccag agcggattgg taagacaatt 960

agcgtttgaa tttttcgggt ttagcgcgag tgggtaacgc tcgctacatc gtaggcatgc 1020

atgcagtgct ctggtagctg taaagccagg ggcggtagcg tgcataaact tagggaagtt 1080

gttacgaatt ataaagatcg cagcaatttc gtattgtgtc ttatctggtg aaaaaatgtt 1140

tgataataaa acgattcttg taaccggcgg taccggttca tttggcaata agttcgttcg 1200

catgacatta gaaaactata atcctaaaaa gattattatt tattctcgtg atgaaatgaa 1260

acagtgggaa atggcaaaaa aattcaagga tgaaaatcgg atccgcttct ttattggtga 1320

tgttcgtgat aaagatcgtc tttatcgcgc attggatggc gttgattttg ttgttcatgc 1380

tgcagcaaca aaaatcgtcc ctacagccga gtataatccc tttgaatgtg taaaaacgaa 1440

tatcaatggt gctatgaacg tgattgatgc ttgtattgac aaaggcatcg aacgcgtagt 1500

tgcgctttct accgataaag caagtagccc tgcgaatctg tatggtgcaa ctaagctagc 1560

atctgataaa ctttttgttg caggtaactc ctattctggc gcgacaaaaa cccgttttgc 1620

tgttgttcgc tatggaaatg ttatggggtc tcgtggctca gtaataccat tcttcttatc 1680

tattaaggag aacggtgaac tgccgataac tgatgagcgt atgacacgct ttatgattac 1740

tctggagcag ggagttgaat tggtttggca tgcgtttaaa gatatggtcg gtggtgaagt 1800

ttatgtaaaa aaaatacctt ctatgaaagt cactgatata gctacagctg ttgcacctaa 1860

tgctaaacaa aaaataatcg gcattcgacc tggcgagaaa ctccacgaac aaatgatcag 1920

cgctgaagat tcttattaca cctatgaata tccagagcat tttaaaattc ttccagctat 1980

tcacaactgg tgtaactcac ctgaaagaat taaagatggc aaaaaagtac cagaaggttt 2040

cgtttatgaa agtgatagta acgcagaatg gatgagcatt gaagaacttc gtcaatggat 2100

cgacgataat cgtgaaaaag taggtaacat ctaatgaaat ttattcctta cggacggcag 2160

gatatttctg acgaagatat aagtgctgta gtggatgtac ttaagtccga gttcttgact 2220

caaggaccat atgttcctaa gtttgagaaa acaatagcta attatgtgaa tgttaagcat 2280

gctgttgcag taaatagtgc gacgtcagca ctccatattg cttgtctggc actggggatg 2340

aaaaaaggcg attggctttg gacatcacca aatacttttg ttgcttctgc gaactgtgct 2400

ctttattgtg gcgctaatgt cagttttgtt gatatcgatg cgcgaacata taatatgagc 2460

gttagtgcct tagaagcgaa actcatgtct gcgaaaaata atggaacttt acctaaagtt 2520

gttgttcctg tcgcatttgc aggacagtca tgtgaaatgg aggcaatata taaactctca 2580

aaagagtatg gattttcaat tatcgaagat gcttcacatg caattggcgg aagttatcag 2640

ggagaaaaaa taggtaattc gcactttgca gatattacta tattgagctt tcatcctgtt 2700

aaaattatca caactgcaga aggggggatg gctctaacca ataacgatag tttggcggaa 2760

aaaatgcagc tattccgtag ccatggaatt acccgtgata ttaatcatat gacaaaagta 2820

agtgaaggcg attggtatta tcaacaaatt gatctcggtc tgaattacag aatgaccgaa 2880

attcaggcag cgttgggtat gagtcagttg aaacgtattg atggctttgt aacacgccgt 2940

catgaattgg ctgagcgtta caaaaaagct cttgaaggta ttcctatttc tctgccattc 3000

caggccgaag gcggctacag tgcttttcac ctttatccta taactgttaa aaattccaaa 3060

ctgcggaaat cactatttga ttatctacgg aataaaaata tcggggtcaa tgtccattat 3120

attccagttc atactcagcc gtattatgaa aagttagggc ataaagtcgg ggattatcct 3180

gttgctgagg actactactc ccgggccctc agtataccga tgtattctgc tttaacaaac 3240

gaagatcagg attatgtaat ccaatgcatt cgggagtttt ttaagtgaac gtggcaatca 3300

ttccagctcg cggtggcagt aaacgtatcc cgcaaaaaaa tatcaaaatg ttttgtggga 3360

aaccaatgat tgcttggtcg attaatgccg ctcgaaagag cggcgtgttt gatcgaatta 3420

ttgtttcgac ggatgatgca gaaattgcgg ctgtagcgag aaaatatggc gctgaagttc 3480

cgtttacgcg cccagaagaa ctttcaaatg attttgctgc aacaattcct gtcatacgac 3540

acgctgttga atggcttgtt aataatgggt gtatagcaga ttttgtgtgc tgtatatatg 3600

caactgcacc atttattcgt tctgaagata ttattcgtgg gttgacaata attagggaac 3660

aacaggcaga ctatgctttt actgttactc ggtaccctta tccgatccaa cgcgcactca 3720

aaatagggca tgaaaaccag attagtatgt tttctcctga aatgttccat gttcgttctc 3780

aggatctcga agaatcttgg catgatgccg gacagtttta ctggggaaca gtatctgcat 3840

ggttgcaaga aaagccaata tttagcgcta actcctattc aattgttcta ccgcgtgagc 3900

gtgtccaaga tattgatact ccagaagact ggcgagtagc tgagtggtta tataaaacca 3960

tggagtttaa aaatgaaagt gtttctgcgg gttgattcgt ctctatctat cggctccggt 4020

catattatta gatgtttaaa tttagcaata gcactaagga acgtaggggc tgaatgtatt 4080

tttatatcta aaaaacatcg tggcaatatt ttgtttaaga tcgaacaggc cgaattttta 4140

taccaagtga ttccgacacc tgaagagtat gatatctatg taagcgaaga aaaatattgg 4200

ctcaatggta gtcaaaatga tgatgctttg caattcagtt tactgataaa aaaacaatgt 4260

gaaaatccag atattattat cgttgatcat tattcactcg attatgaatg ggaattaata 4320

attaagcgta atttccctga agctaaactt atagttatcg atgatctttg taatcgcccc 4380

cactgttgtg atctattgat agatcagaca tatttacgtc atgaaaaaga atatacatgc 4440

ttaaacattt ggggagggaa gatattaacg gggcccaagt atgcactact agatcctgtt 4500

ttttcaaaat taagggagca gtcaataaat cgtaaaactc agctagaatt tcctcaaagg 4560

ctaatgataa caatgggagg agttgatgta aataatataa ctgggaaagt cttgcgttac 4620

attgaaaata aaaacctaaa aagcatcgaa aaaattactg ttatactcgg tagcgcgtgt 4680

ccgcatcggg aggaaattga ggcattggtt gctgattcca aatatcctat aaacatatta 4740

accaatgttg aaaacatggc tgaacttatg ctagaacatg attttgcgat tggtgcaatg 4800

ggcgggacaa catgggaacg ttgtgtaatg gctttacctg cagtgaatat cgcaattgcc 4860

aataaccaga gcacgattgc cacgaatttt tctaaagctg gcgctattgt tttacattca 4920

gataatttta ctaagaatga tttttataat gcttttaatc ggttgataac tgattatcat 4980

caacagcgtg atcttgtaat gagtatatgt gatggtcaag gactcatcag agatatacag 5040

gaaattattc cttgcttttc tacagatggc ataaatgtga cgcttagatt tgcaacgttg 5100

gatgacatta attttgttta ccaacttcag tgtgagccac aaactcgaaa gtttgcacga 5160

aacccaaata ttccttcata tgatagtcac acagaatgga tgcgtcgtaa attagttgaa 5220

caaaatagtt ttttctatat cattgagcat ataggagcgt gtggagtatt gcgtttagat 5280

ccaatagaac atgagttagc acaatacgaa atttcgatat ttttgacgac agctagtatg 5340

gggaaaggaa ttgcaaaagc ggctatcaga cgtgcaacta tgttacataa agatacagta 5400

atactggcaa cggtactgta cgaaaatttt gcttctcatc gactttttga acaaattggt 5460

ttcaataaga tctcttcatg tgaatatatt aatagggggg ggaatgagta aatatataac 5520

gattgatggt cggaaaattg gtaaagaata ctcaccatat gtcattgctg aattgtccgc 5580

aaatcacaat ggtgatataa atcgtgcttt caaaattatg gaggctgcta aattagccgg 5640

cgctgatgca ataaaattac agacatatcg tgctgataca attactatcg attgtaactc 5700

tgaaggtttt caaattcatg gtggcctatg ggatgggcaa actttattta atttatataa 5760

aggtgctcaa atgccctggg aatggcataa accattattt gaaaaagcga aagaactagg 5820

tattaccatt ttcagtagtc catttgactt tactgcagtt gatctactag aggaattagg 5880

tgcacccgca tataagattg cttcatttga agcaattgac atacctctga tcaaatatgt 5940

agccagaaca ggtaaaccga tgattatctc tacaggtatg gctaatgaac aagaaattca 6000

ggaagcaatt gatgccgcga aagatggggg atgtaaagag cttgttgtgt tgcattgtgt 6060

tagcggatat ccagcaccag cggaagatta taatttagta acaatacttg atatggctga 6120

gagatacggt gtgattactg ggttatccga tcacacaata gataatacca cggctattgc 6180

atccgtctca cttggagcta atgtaattga aaaacatgtt acactggaca gaaaaggagg 6240

tggacctgat gatagttttt ctctggaacc tagtgagcta agagcacttt gcaaagacgt 6300

taagacagct tggcgagcat taggaaaaat cagttataac cataaggaaa gtgaaaaagg 6360

gaatgtcaaa ttcagacgat cactttatgt tgtaaaagat gttgcaaaag gtgaggaaat 6420

aacaacgcaa aatgtgcgta gtattcgccc tggttttggt ttagctccaa aacatttaga 6480

gcatgtgtta ggaaaaaaat tcttgacaga tctacctgca ggcacagctt taactttcga 6540

tattatcgaa taatatagta ccctcttaac aaagagggtg aatcaagctc attatataaa 6600

tcgatgatat ggaatgtaga acagtaataa tgttgctcct cagaggggca acactcgcaa 6660

gtaagttttt acttgttatt tttttagcta agtttgccac gtacaaaaat ttagccgact 6720

atacaataat tgcagtcact attagttatt tgttattttt tttggggttt gatttttata 6780

catactcaac tagggaaatt ataaaaaaag gctttaccaa aagtggtgaa cttttatgta 6840

accagttgta tttatacata tcaatgtatt tgatattaat tccaatagta tacgggttaa 6900

attatttttc tgttctaagt gttggtgttt tgttttattt tgttgttata acagaacatt 6960

tcacacaaga atgcatgaga atcattatta taaataataa acctgtaaaa gcaaattttc 7020

aatttttttt acgctcttca ttttggatat atatatatat agcatattgt tattggaaag 7080

ccacttattc attggatatt ttgcttttat tctggttgat atcgaatgtg tgctctatag 7140

tttattctgt cagtgaattt aaaatgatta attataaaga tgataaaata tatagagttg 7200

atttcaaatg gatcaagaag ggggtggcaa ttgcaatacc attactcgtt accacattaa 7260

tgctgagggg gggatatgtt acggacaggt atattttaaa atatctctca tctactgaag 7320

tattagctgt ttactcattt tatagtaaca tgtctaatgc tttgattgca ttcattgatg 7380

ctgctgtaat aatgatattt taccccaaag tcatttctgc ttataatgaa ctaaatatag 7440

aggaatataa taaaacattg gtgttattta agcgcaatgt tgtaaaggtc ggttgctgtt 7500

ccttttttat tttgagtgta gctgtaccag taatatgttt atttttgaat aaatcagaat 7560

ttattaattc tattattatg ttttatatat tattaacttc agcatttata tattcattta 7620

gtctagtata tcattatgaa ttatatgcca gacataaaga ttttatatta tttaagtgta 7680

catttttttc atttctgctt actgtcatag ggcaatatgt ttttgcttct ttatttggtg 7740

gcataggcat ggcgatttca attcttttat tttcattaat gttattatcc acaaagtatt 7800

tatctattat caatattaaa aaaaatgagt gttttggcaa ctcatagtag ctaaactaga 7860

atcaacaagt aaatatcact ggtttggagt aataagtgaa tctttatatt attcaaaatg 7920

atacgcattt caaaaatttt attgaattgg ctactgaagg cgattttttt atagatcttg 7980

caaggtttac aggtcaggat atcaattacg aattatttta taaaaaagga gtgtcagaaa 8040

ttaatttagg gttacatgat gctaattttt atttgtttag ttcttatcag aaaattaaat 8100

tttgtttcaa ttttgagatg attgtaaaaa aaataatatc tgcttaccgc tttgaaaaaa 8160

tcataacagg gaatgatggt gcattacaaa aaataataat aaaacaagca ttaaagaata 8220

atagcaaagg aagagtagaa atgtggttgg atggtttaat ttcattgaat aaaaacagag 8280

taattaattc agcaaaagta tttatgtcgt atatcgctga caagattggt ttgtcttctt 8340

atgtaccttc agttataggg acaagtagtt acgttgatac attgtatgta atggatcaat 8400

ctgtaataaa agagtataat tttctaatga ttaaaccaaa agttaaaaaa attgaaaaga 8460

gattgtttcc tcggcataaa aaattaatat ctttggcaca taatcatgag agaagagctc 8520

aggtctgtaa agtattgtat ttaacaagtg catggtcatt tcatggacat aataaatgtc 8580

aaaatattca gtatcaccag attttaaatt tgttatctaa tttcgaaggt aataaagata 8640

ttgactttca aattcgaatc catccaagag acattataac aaactattct gaaattccgg 8700

tgggggttat ttctagagta aaatcatttg aggaagatat tttatcagct agcattatta 8760

tttctgcaag atctacaggg cttttcgagg ctgacatgat agggaagaaa gtgatagtat 8820

atgacgaagg ttttgaaggg gattttatga atgaatattt tgatacactt cccagatgtg 8880

tcgatttgaa tgaaatgaat tcatttatta gtcaacaagg ttaatttata taatgtcttc 8940

tacacaagtt ttttcattgc tactggggtt ttatttagta tcagtaaatc ttttaattat 9000

cccattagat tatttcaacg tagaacgctc aattctgatt attccatatg tatttatatt 9060

ttctctgagt ttgttttttt ttgcgttagc aactattggt gatttttata aatcgccaaa 9120

acaaattttt tatgtgcaag tttgttttgt ttttgtgata ataattccat catgtttgag 9180

ttttatacaa aatacattat tagaagtagt gcctagcatg atgaggtaca tgtcatattt 9240

attcacattt gtatttgtct actatttctc tgccaaaaat tggtttactg ttaaaaattt 9300

agatgtgtcc atcaaattta tatcgattat cttgttcatt ttttcaatac ttcaaatagt 9360

aaaaggcgag atgatatata tgaacggggc ttatcgtttg tcaagtattt atgggacaac 9420

gcctgctggt tttgcgttga ttactctcgt cttttctgtt tatttctata gtcaattgca 9480

cgtttcggat agcggtaagt ttaaaaaaat atttccattt gcttttttta tgattaatgt 9540

atctatgatg gcgttgacgc aaagccgaca atcaataatg acattgttat tgcttatttg 9600

tatatttcat tttgttaggg caaagaatat atttaagttt ttttcgctta tatttatttt 9660

attgttaatc tatgtatttt atttgttggt tgttaatacg gacttttttc cacgattaac 9720

tcaaatgctt tttaattact catcagattc atcaacacat acacgtattt ctattattac 9780

tcaatcgtat gaacatcttg atgttaatga tttaatttat gggattggtt taggcggatt 9840

taatcacttc tactacaaga ttagtggtga aataggtgtt gcagcccata atgatttttt 9900

cctttttttt gttgaaggtg gcgtaatagc tttgttcttt tattgccttt ttttatgcgg 9960

tggagtgtca ttttgggggc gggcgattaa acagcaaggt gatagttatt ttacaccgtt 10020

gcttgttttt tgtgctttat acgtattttc ttttttgaat aatccttatt attacccaca 10080

agtgcaagtt gttgcttctg cattgatggg cgtgtatttg tcagaatata taaagaaaca 10140

aaaggtaaat tatgttttgc catgaacttg aaaacaaaat gaaatctcag gaattttttg 10200

tcgaaactct caaaaatagc cagcttaata aaaccagaat tagttttctt aacccctatt 10260

catacccttt aatttcaaga aataaaaaaa tcatcaaggg ttttgattat tggtttgcag 10320

atggtttaac actatgtatt gttactaatt tgtttagaga gaagtctgat ataataaaaa 10380

gaggcagttt tgatttttca tcactggcgg gaatatgttt cgattattta caaactcatt 10440

ctctttctgt tgctattatt ggtggaagta atgatgagat tcatcaagca tgtgaatatt 10500

tttccattcg atatccatcc ttaaaaattt gttttaagca tcatgggtat atagatatag 10560

acaagtgtga tcatataata aatgagctta aaaaatctga agtgcaagca ttaattgtag 10620

gaatgggaac acctaagcaa gaagagttta tattaaaggt tgaaaaacat ataataagtt 10680

gcaagctttt tttaacttgt ggaggtttta tttcacaaac agcgagtagc ggtgattatt 10740

atcacccatt aatcaaaaga ttaggtcttc gttggctaca gcgtgcagtg ctttataagc 10800

atgttaggcg caggctttta ctcaattatc cctgttttta ttttagatat atatgtagtg 10860

caatattaaa tatcttatat gctaaaaaat agcaggataa ttatcaatgt agtatgctaa 10920

aattgaccaa tatcgtgaag ttatcaaatg ttatatcaga taacatgaaa caatatacga 10980

accgctacac gcagtaaccc ctgacaggag taaacaatgt caaagcaaca gatcggcgtc 11040

gtcggtatgg cagtgatggg gcgcaacctt gcgctcaaca tcgaaagccg tggttatacc 11100

gtctctattt tcaaccgttc ccgtgaaaag accgaagaag tgattgccga aaatccaggc 11160

aagaaactgg ttccttacta tacggtgaaa gagtttgttg aatctctgga aacgcctcgt 11220

cgcatcctgt taatggtgaa agcaggtgca ggcacggatg ctgctattga ttctctcaag 11280

ccatacctcg ataaaggtga catcatcatt gatggtggta ataccttctt cctggacacc 11340

attcgccgta atcgtgagct ttctgccgaa ggctttaact tcattggtac cggtgtttcc 11400

ggtggtgaag aaggtgcgct gaaaggtcct tccattatgc ctggtggtca gaaagaagcc 11460

tatgaactgg ttgcaccgat cctgaccaaa atcgccgcag tggctgaaga tggcgaaccg 11520

tgcgttacct atattggtgc cgatggtgca ggtcattacg tgaagatggt tcacaacggt 11580

attgaatacg gcgatatgca gctgattgct gaagcctatt ctctgcttaa aggtggcctg 11640

aatctttcca acgaagaact agcgcagacg tttaccgagt ggaataacgg tgaactgagc 11700

agctacctga tcgacatcac caaagatatc ttcaccaaaa aagatgaaga cggtaactac 11760

ctggttgatg tgattctgga tgaagcggct aacaaaggta ccggtaaatg gaccagccag 11820

agtgcgctgg atctcggcga accgctgtcg ctgattaccg agtctgtgtt tgcacgttat 11880

atctcttctc tgaaa 11895

Wzx gene, wzy gene and wherein primer and PCR data in the O antigen gene of table 1 intestinal bacteria O136 bunch

Gene	Function	The base position of gene	The forward primer position	The reverse primer position	PCR product length	Produce the group number of correct big or small electrophoresis band	The annealing temperature of PCR (℃)
								Wzx Wzy	O-antigen transhipment enzyme O-antigen polysaccharase	6609-7847 8933-10165	6620-6637 6597-6614 9525-9542 8973-8992	7254-7261 7048-7065 10067-10084 9885-9902	642bp 462bp 560bp 930bp	0 0 0 0 *	58 58 62 60

* in other six groups, produce the band of a wrong size

The intestinal bacteria of 166 kinds of serotypes of table 2 and 43 strain Shigellaes and their source

Group number	The bacterial strain that contains in this group	The source
			1, wild-type e. coli 2, wild-type e. coli 3, wild-type e. coli 4, wild-type e. coli 5, wild-type e. coli 6, wild-type e. coli 7, wild-type e. coli 8, wild-type e. coli 9, wild-type e. coli shigella dysenteriae 10, Shigella bogdii 11, shigella flexneri	O1，O2，O5，O7，O8，O9，O12，O13，O14，O15，O16，O17，O18， O19ab，O20，O21，O22，O23，O24 O4，O10，O25，O26，O27，O28，O29，O30，O32，O33，O34，O35， O136，O37，O38，O40，O41，O42，O43 O6，O44，O45，O46，O48，O49，O50，O51，O52，O54，O55，O56， O57，O58，O60，O61，O62，O53 O63，O65，O66，O69，O70，O71，O74，O75，O76，O77，O78， O79，O80，O81，O82，O83，O68 O84，O85，O86，O87，O88，O89，O90，O91，O92，O98，O99， O101，O102，O103，O104，O105，O106，O97， O107，O108，O109，O110，O111，O112ab，O112ac，O113， O115，O116，O118，O120，O123，O125，O126，O128，O117 O129，O130，O131，O132，O133，O134，O135，O136，O137， O138，O139，O141，O142，O143，O144，O145，O140 O146，O147，O148，O150，O152，O154，O156，O157，O158， O159，O160，O161，O163，O164，O165，O166，O153 O168，O169，O170，O171，O172，O173， D1，D2，D3，D4，D5，D6，D7，D8，D9，D10，D11，D12，D13 B1，B2，B3，B4，B6，B7，B8，B9，B10，B11，B12，B13，B14，B15， B16，B17，B18 F1a，F1b，F2a，F2b，F3，F4a，F4b，F5(v：4)，F5(v：7)，F6，	IMVS a IMVS a IMVS a IMVS a IMVS a IMVS a IMVS d IMVS d b d d d

12, wild-type e. coli 13, wild-type e. coli

DS, DR O3, O11, O39, O59, O64, O73, O96, O95, O100, O114, O151, O155, O124, O167, O162, O121, O127, O149, O119 removes the 7th group of bacterium of intestinal bacteria O136

IMVS a

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

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

b.Statens Serum Institut，Copenhagen，Denmark

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

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

Table 3 is structural tables of the O-antigen gene bunch of intestinal bacteria O136

Table 4 is location tables of the gene in the O-antigen gene bunch of intestinal bacteria O136

ATTGTGGCTG CAGGGATCAA AGAAATCCTC CTGGTAACTC ACGCGTCCAA GAACGCGGTC 60

GAAAACCACT TCGACACCTC TTATGAATTA GAATCTCTTC TTGAGCAGCG CGTGAAGCGT 120

CAACTGCTTG CGGAAGTGCA GTCCATCTGT CCACCGGGCG TGACCATTAT GAACGTGCGT 180

CAGGGCGAAC CTTTAGGTTT AGGCCACTCC ATTTTGTGTG CACGACCCGC CATTGGTGAC 240

AACCCATTTG TCGTGGTGCT GCCAGACGTA GTGATCGACG ACGCCAGCGC CGACCCGCTG 300

CGCTACAACC TTGCTGCCAT GATTGCGCGC TTCAATGAAA CGGGCCGTAG CCAGGTGCTG 360

GCAAAACGTA TGCCGGGTGA CCTCTCTGAA TACTCCGTCA TCCAGACCAA AGAACCGCTG 420

GATCGTGAAG GTAAAGTCAG CCGCATTGTT GAATTTATCG AAAAACCGGA TCAGCCGCAG 480

ACGTTGGACT CAGACATCAT GGCCGTAGGT CGTTATGTGC TTTCTGCCGA TATTTGGCCG 540

GAACTTGAAC GCACGCAGCC AGGGGCATGG GGACGTATTC AGCTGACTGA TGCTATCGCT 600

GAACTGGCGA AAAAACAATC CGTTGATGCC ATGCTGATGA CAGGTGACAG CTACGACTGC 660

GGTAAAAAAA TGGGTTATAT GCAGGCATTT GTGAAGTATG GACTACGCAA TCTGAAAGAA 720

GGGGCGAAGT TCCGTAAAGG GATTGAGAAG CTGTTAAGCG AATAATGAAA ATCTGACCGT 780

ATGTAACGGT TGATAAGAAA ATTATAACGG CAGTGAAGAT TTGTGGCGAA AGTAATTTGT 840

TGCGAATTTT CCTGCCGTTG TTTTATATAA ACAATCAGGA TAACAACGAG TTAGCAATAG 900

GATTTTAGTC AAAGTTTTCC AGGATTTTCC TTGTTTCCAG AGCGGATTGG TAAGACAATT 960

AGCGTTTGAA TTTTTCGGGT TTAGCGCGAG TGGGTAACGC TCGCTACATC GTAGGCATGC 1020

ATGCAGTGCT CTGGTAGCTG TAAAGCCAGG GGCGGTAGCG TGCATAAACT TAGGGAAGTT 1080

Orf1's is initial

GTTACGAATT ATAAAGATCG CAGCAATTTC GTATTGTGTC TTATCTGGTG AAAAA ATGTT 1140

TGATAATAAA ACGATTCTTG TAACCGGCGG TACCGGTTCA TTTGGCAATA AGTTCGTTCG 1200

CATGACATTA GAAAACTATA ATCCTAAAAA GATTATTATT TATTCTCGTG ATGAAATGAA 1260

ACAGTGGGAA ATGGCAAAAA AATTCAAGGA TGAAAATCGG ATCCGCTTCT TTATTGGTGA 1320

TGTTCGTGAT AAAGATCGTC TTTATCGCGC ATTGGATGGC GTTGATTTTG TTGTTCATGC 1380

TGCAGCAACA AAAATCGTCC CTACAGCCGA GTATAATCCC TTTGAATGTG TAAAAACGAA 1440

TATCAATGGT GCTATGAACG TGATTGATGC TTGTATTGAC AAAGGCATCG AACGCGTAGT 1500

TGCGCTTTCT ACCGATAAAG CAAGTAGCCC TGCGAATCTG TATGGTGCAA CTAAGCTAGC 1560

ATCTGATAAA CTTTTTGTTG CAGGTAACTC CTATTCTGGC GCGACAAAAA CCCGTTTTGC 1620

TGTTGTTCGC TATGGAAATG TTATGGGGTC TCGTGGCTCA GTAATACCAT TCTTCTTATC 1680

TATTAAGGAG AACGGTGAAC TGCCGATAAC TGATGAGCGT ATGACACGCT TTATGATTAC 1740

TCTGGAGCAG GGAGTTGAAT TGGTTTGGCA TGCGTTTAAA GATATGGTCG GTGGTGAAGT 1800

TTATGTAAAA AAAATACCTT CTATGAAAGT CACTGATATA GCTACAGCTG TTGCACCTAA 1860

TGCTAAACAA AAAATAATCG GCATTCGACC TGGCGAGAAA CTCCACGAAC AAATGATCAG 1920

CGCTGAAGAT TCTTATTACA CCTATGAATA TCCAGAGCAT TTTAAAATTC TTCCAGCTAT 1980

TCACAACTGG TGTAACTCAC CTGAAAGAAT TAAAGATGGC AAAAAAGTAC CAGAAGGTTT 2040

CGTTTATGAA AGTGATAGTA ACGCAGAATG GATGAGCATT GAAGAACTTC GTCAATGGAT 2100

The termination Orf2's of Orf1 is initial

CGACGATAAT CGTGAAAAAG TAGGTAACAT C TAATGAAAT TTATTCCTTA CGGACGGCAG 2160

GATATTTCTG ACGAAGATAT AAGTGCTGTA GTGGATGTAC TTAAGTCCGA GTTCTTGACT 2220

CAAGGACCAT ATGTTCCTAA GTTTGAGAAA ACAATAGCTA ATTATGTGAA TGTTAAGCAT 2280

GCTGTTGCAG TAAATAGTGC GACGTCAGCA CTCCATATTG CTTGTCTGGC ACTGGGGATG 2340

AAAAAAGGCG ATTGGCTTTG GACATCACCA AATACTTTTG TTGCTTCTGC GAACTGTGCT 2400

CTTTATTGTG GCGCTAATGT CAGTTTTGTT GATATCGATG CGCGAACATA TAATATGAGC 2460

GTTAGTGCCT TAGAAGCGAA ACTCATGTCT GCGAAAAATA ATGGAACTTT ACCTAAAGTT 2520

GTTGTTCCTG TCGCATTTGC AGGACAGTCA TGTGAAATGG AGGCAATATA TAAACTCTCA 2580

AAAGAGTATG GATTTTCAAT TATCGAAGAT GCTTCACATG CAATTGGCGG AAGTTATCAG 2640

GGAGAAAAAA TAGGTAATTC GCACTTTGCA GATATTACTA TATTGAGCTT TCATCCTGTT 2700

AAAATTATCA CAACTGCAGA AGGGGGGATG GCTCTAACCA ATAACGATAG TTTGGCGGAA 2760

AAAATGCAGC TATTCCGTAG CCATGGAATT ACCCGTGATA TTAATCATAT GACAAAAGTA 2820

AGTGAAGGCG ATTGGTATTA TCAACAAATT GATCTCGGTC TGAATTACAG AATGACCGAA 2880

ATTCAGGCAG CGTTGGGTAT GAGTCAGTTG AAACGTATTG ATGGCTTTGT AACACGCCGT 2940

CATGAATTGG CTGAGCGTTA CAAAAAAGCT CTTGAAGGTA TTCCTATTTC TCTGCCATTC 3000

CAGGCCGAAG GCGGCTACAG TGCTTTTCAC CTTTATCCTA TAACTGTTAA AAATTCCAAA 3060

CTGCGGAAAT CACTATTTGA TTATCTACGG AATAAAAATA TCGGGGTCAA TGTCCATTAT 3120

ATTCCAGTTC ATACTCAGCC GTATTATGAA AAGTTAGGGC ATAAAGTCGG GGATTATCCT 3180

GTTGCTGAGG ACTACTACTC CCGGGCCCTC AGTATACCGA TGTATTCTGC TTTAACAAAC 3240

The termination of Orf2

GAAGATCAGG ATTATGTAAT CCAATGCATT CGGGAGTTTT TTAAG TGAAC GTGGCAATCA 3300

Orf3's is initial

TTCCAGCTCG CGGTGGCAGT AAACGTATCC CGCAAAAAAA TATCAAA ATG TTTTGTGGGA 3360

AACCAATGAT TGCTTGGTCG ATTAATGCCG CTCGAAAGAG CGGCGTGTTT GATCGAATTA 3420

TTGTTTCGAC GGATGATGCA GAAATTGCGG CTGTAGCGAG AAAATATGGC GCTGAAGTTC 3480

CGTTTACGCG CCCAGAAGAA CTTTCAAATG ATTTTGCTGC AACAATTCCT GTCATACGAC 3540

ACGCTGTTGA ATGGCTTGTT AATAATGGGT GTATAGCAGA TTTTGTGTGC TGTATATATG 3600

CAACTGCACC ATTTATTCGT TCTGAAGATA TTATTCGTGG GTTGACAATA ATTAGGGAAC 3660

AACAGGCAGA CTATGCTTTT ACTGTTACTC GGTACCCTTA TCCGATCCAA CGCGCACTCA 3720

AAATAGGGCA TGAAAACCAG ATTAGTATGT TTTCTCCTGA AATGTTCCAT GTTCGTTCTC 3780

AGGATCTCGA AGAATCTTGG CATGATGCCG GACAGTTTTA CTGGGGAACA GTATCTGCAT 3840

GGTTGCAAGA AAAGCCAATA TTTAGCGCTA ACTCCTATTC AATTGTTCTA CCGCGTGAGC 3900

GTGTCCAAGA TATTGATACT CCAGAAGACT GGCGAGTAGC TGAGTGGTTA TATAAAACCA 3960

The termination of the initial Orf3 of Orf4

TGGAGTTTAA AA ATGAAAGT GTTTCTGCGG GT TGATTCGT CTCTATCTAT CGGCTCCGGT4020

CATATTATTA GATGTTTAAA TTTAGCAATA GCACTAAGGA ACGTAGGGGC TGAATGTATT 4080

TTTATATCTA AAAAACATCG TGGCAATATT TTGTTTAAGA TCGAACAGGC CGAATTTTTA 4140

TACCAAGTGA TTCCGACACC TGAAGAGTAT GATATCTATG TAAGCGAAGA AAAATATTGG 4200

CTCAATGGTA GTCAAAATGA TGATGCTTTG CAATTCAGTT TACTGATAAA AAAACAATGT 4260

GAAAATCCAG ATATTATTAT CGTTGATCAT TATTCACTCG ATTATGAATG GGAATTAATA 4320

ATTAAGCGTA ATTTCCCTGA AGCTAAACTT ATAGTTATCG ATGATCTTTG TAATCGCCCC 4380

CACTGTTGTG ATCTATTGAT AGATCAGACA TATTTACGTC ATGAAAAAGA ATATACATGC 4440

TTAAACATTT GGGGAGGGAA GATATTAACG GGGCCCAAGT ATGCACTACT AGATCCTGTT 4500

TTTTCAAAAT TAAGGGAGCA GTCAATAAAT CGTAAAACTC AGCTAGAATT TCCTCAAAGG 4560

CTAATGATAA CAATGGGAGG AGTTGATGTA AATAATATAA CTGGGAAAGT CTTGCGTTAC 4620

ATTGAAAATA AAAACCTAAA AAGCATCGAA AAAATTACTG TTATACTCGG TAGCGCGTGT 4680

CCGCATCGGG AGGAAATTGA GGCATTGGTT GCTGATTCCA AATATCCTAT AAACATATTA 4740

ACCAATGTTG AAAACATGGC TGAACTTATG CTAGAACATG ATTTTGCGAT TGGTGCAATG 4800

GGCGGGACAA CATGGGAACG TTGTGTAATG GCTTTACCTG CAGTGAATAT CGCAATTGCC 4860

AATAACCAGA GCACGATTGC CACGAATTTT TCTAAAGCTG GCGCTATTGT TTTACATTCA 4920

GATAATTTTA CTAAGAATGA TTTTTATAAT GCTTTTAATC GGTTGATAAC TGATTATCAT 4980

CAACAGCGTG ATCTTGTAAT GAGTATATGT GATGGTCAAG GACTCATCAG AGATATACAG 5040

GAAATTATTC CTTGCTTTTC TACAGATGGC ATAAATGTGA CGCTTAGATT TGCAACGTTG 5100

GATGACATTA ATTTTGTTTA CCAACTTCAG TGTGAGCCAC AAACTCGAAA GTTTGCACGA 5160

AACCCAAATA TTCCTTCATA TGATAGTCAC ACAGAATGGA TGCGTCGTAA ATTAGTTGAA 5220

CAAAATAGTT TTTTCTATAT CATTGAGCAT ATAGGAGCGT GTGGAGTATT GCGTTTAGAT 5280

CCAATAGAAC ATGAGTTAGC ACAATACGAA ATTTCGATAT TTTTGACGAC AGCTAGTATG 5340

GGGAAAGGAA TTGCAAAAGC GGCTATCAGA CGTGCAACTA TGTTACATAA AGATACAGTA 5400

ATACTGGCAA CGGTACTGTA CGAAAATTTT GCTTCTCATC GACTTTTTGA ACAAATTGGT 5460

The termination of the initial Orf4 of Orf5

TTCAATAAGA TCTCTTCATG TGAATATATT AATAGGGGGG GGA ATGAG TA AATATATAAC5520

GATTGATGGT CGGAAAATTG GTAAAGAATA CTCACCATAT GTCATTGCTG AATTGTCCGC 5580

AAATCACAAT GGTGATATAA ATCGTGCTTT CAAAATTATG GAGGCTGCTA AATTAGCCGG 5640

CGCTGATGCA ATAAAATTAC AGACATATCG TGCTGATACA ATTACTATCG ATTGTAACTC 5700

TGAAGGTTTT CAAATTCATG GTGGCCTATG GGATGGGCAA ACTTTATTTA ATTTATATAA 5760

AGGTGCTCAA ATGCCCTGGG AATGGCATAA ACCATTATTT GAAAAAGCGA AAGAACTAGG 5820

TATTACCATT TTCAGTAGTC CATTTGACTT TACTGCAGTT GATCTACTAG AGGAATTAGG 5880

TGCACCCGCA TATAAGATTG CTTCATTTGA AGCAATTGAC ATACCTCTGA TCAAATATGT 5940

AGCCAGAACA GGTAAACCGA TGATTATCTC TACAGGTATG GCTAATGAAC AAGAAATTCA 6000

GGAAGCAATT GATGCCGCGA AAGATGGGGG ATGTAAAGAG CTTGTTGTGT TGCATTGTGT 6060

TAGCGGATAT CCAGCACCAG CGGAAGATTA TAATTTAGTA ACAATACTTG ATATGGCTGA 6120

GAGATACGGT GTGATTACTG GGTTATCCGA TCACACAATA GATAATACCA CGGCTATTGC 6180

ATCCGTCTCA CTTGGAGCTA ATGTAATTGA AAAACATGTT ACACTGGACA GAAAAGGAGG 6240

TGGACCTGAT GATAGTTTTT CTCTGGAACC TAGTGAGCTA AGAGCACTTT GCAAAGACGT 6300

TAAGACAGCT TGGCGAGCAT TAGGAAAAAT CAGTTATAAC CATAAGGAAA GTGAAAAAGG 6360

GAATGTCAAA TTCAGACGAT CACTTTATGT TGTAAAAGAT GTTGCAAAAG GTGAGGAAAT 6420

AACAACGCAA AATGTGCGTA GTATTCGCCC TGGTTTTGGT TTAGCTCCAA AACATTTAGA 6480

GCATGTGTTA GGAAAAAAAT TCTTGACAGA TCTACCTGCA GGCACAGCTT TAACTTTCGA 6540

The termination of Orf5

TATTATCGAA TAATATAGTA CCCTCTTAAC AAAGAGGGTG AATCAAGCTC ATTATATAAA 6600

Orf6's is initial

TCGATGATAT GGAATGTAGA ACAGTAATAA TGTTGCTCCT CAGAGGGGCA ACACTCGCAA 6660

GTAAGTTTTT ACTTGTTATT TTTTTAGCTA AGTTTGCCAC GTACAAAAAT TTAGCCGACT 6720

ATACAATAAT TGCAGTCACT ATTAGTTATT TGTTATTTTT TTTGGGGTTT GATTTTTATA 6780

CATACTCAAC TAGGGAAATT ATAAAAAAAG GCTTTACCAA AAGTGGTGAA CTTTTATGTA 6840

ACCAGTTGTA TTTATACATA TCAATGTATT TGATATTAAT TCCAATAGTA TACGGGTTAA 6900

ATTATTTTTC TGTTCTAAGT GTTGGTGTTT TGTTTTATTT TGTTGTTATA ACAGAACATT 6960

TCACACAAGA ATGCATGAGA ATCATTATTA TAAATAATAA ACCTGTAAAA GCAAATTTTC 7020

AATTTTTTTT ACGCTCTTCA TTTTGGATAT ATATATATAT AGCATATTGT TATTGGAAAG 7080

CCACTTATTC ATTGGATATT TTGCTTTTAT TCTGGTTGAT ATCGAATGTG TGCTCTATAG 7140

TTTATTCTGT CAGTGAATTT AAAATGATTA ATTATAAAGA TGATAAAATA TATAGAGTTG 7200

ATTTCAAATG GATCAAGAAG GGGGTGGCAA TTGCAATACC ATTACTCGTT ACCACATTAA 7260

TGCTGAGGGG GGGATATGTT ACGGACAGGT ATATTTTAAA ATATCTCTCA TCTACTGAAG 7320

TATTAGCTGT TTACTCATTT TATAGTAACA TGTCTAATGC TTTGATTGCA TTCATTGATG 7380

CTGCTGTAAT AATGATATTT TACCCCAAAG TCATTTCTGC TTATAATGAA CTAAATATAG 7440

AGGAATATAA TAAAACATTG GTGTTATTTA AGCGCAATGT TGTAAAGGTC GGTTGCTGTT 7500

CCTTTTTTAT TTTGAGTGTA GCTGTACCAG TAATATGTTT ATTTTTGAAT AAATCAGAAT 7560

TTATTAATTC TATTATTATG TTTTATATAT TATTAACTTC AGCATTTATA TATTCATTTA 7620

GTCTAGTATA TCATTATGAA TTATATGCCA GACATAAAGA TTTTATATTA TTTAAGTGTA 7680

CATTTTTTTC ATTTCTGCTT ACTGTCATAG GGCAATATGT TTTTGCTTCT TTATTTGGTG 7740

GCATAGGCAT GGCGATTTCA ATTCTTTTAT TTTCATTAAT GTTATTATCC ACAAAGTATT 7800

The termination of Orf6

TATCTATTAT CAATATTAAA AAAAATGAGT GTTTTGGCAA CTCATAGTAG CTAAACTAGA 7860

ATCAACAAGT AAATATCACT GGTTTGGAGT AATAAGTGAA TCTTTATATT ATTCAAAATG 7920

ATACGCATTT CAAAAATTTT ATTGAATTGG CTACTGAAGG CGATTTTTTT ATAGATCTTG 7980

CAAGGTTTAC AGGTCAGGAT ATCAATTACG AATTATTTTA TAAAAAAGGA GTGTCAGAAA 8040

TTAATTTAGG GTTACATGAT GCTAATTTTT ATTTGTTTAG TTCTTATCAG AAAATTAAAT 8100

Orf7's is initial

TTTGTTTCAA TTTTGAGATG ATTGTAAAAA AAATAATATC TGCTTACCGC TTTGAAAAAA 8160

TCATAACAGG GAATGATGGT GCATTACAAA AAATAATAAT AAAACAAGCA TTAAAGAATA 8220

ATAGCAAAGG AAGAGTAGAA ATGTGGTTGG ATGGTTTAAT TTCATTGAAT AAAAACAGAG 8280

TAATTAATTC AGCAAAAGTA TTTATGTCGT ATATCGCTGA CAAGATTGGT TTGTCTTCTT 8340

ATGTACCTTC AGTTATAGGG ACAAGTAGTT ACGTTGATAC ATTGTATGTA ATGGATCAAT 8400

CTGTAATAAA AGAGTATAAT TTTCTAATGA TTAAACCAAA AGTTAAAAAA ATTGAAAAGA 8460

GATTGTTTCC TCGGCATAAA AAATTAATAT CTTTGGCACA TAATCATGAG AGAAGAGCTC 8520

AGGTCTGTAA AGTATTGTAT TTAACAAGTG CATGGTCATT TCATGGACAT AATAAATGTC 8580

AAAATATTCA GTATCACCAG ATTTTAAATT TGTTATCTAA TTTCGAAGGT AATAAAGATA 8640

TTGACTTTCA AATTCGAATC CATCCAAGAG ACATTATAAC AAACTATTCT GAAATTCCGG 8700

TGGGGGTTAT TTCTAGAGTA AAATCATTTG AGGAAGATAT TTTATCAGCT AGCATTATTA 8760

TTTCTGCAAG ATCTACAGGG CTTTTCGAGG CTGACATGAT AGGGAAGAAA GTGATAGTAT 8820

ATGACGAAGG TTTTGAAGGG GATTTTATGA ATGAATATTT TGATACACTT CCCAGATGTG 8880

The termination Orf8's of Orf7 is initial

TCGATTTGAA TGAAATGAAT TCATTTATTA GTCAACAAGG TTAATTTATA TAATGTCTTC 8940

TACACAAGTT TTTTCATTGC TACTGGGGTT TTATTTAGTA TCAGTAAATC TTTTAATTAT 9000

CCCATTAGAT TATTTCAACG TAGAACGCTC AATTCTGATT ATTCCATATG TATTTATATT 9060

TTCTCTGAGT TTGTTTTTTT TTGCGTTAGC AACTATTGGT GATTTTTATA AATCGCCAAA 9120

ACAAATTTTT TATGTGCAAG TTTGTTTTGT TTTTGTGATA ATAATTCCAT CATGTTTGAG 9180

TTTTATACAA AATACATTAT TAGAAGTAGT GCCTAGCATG ATGAGGTACA TGTCATATTT 9240

ATTCACATTT GTATTTGTCT ACTATTTCTC TGCCAAAAAT TGGTTTACTG TTAAAAATTT 9300

AGATGTGTCC ATCAAATTTA TATCGATTAT CTTGTTCATT TTTTCAATAC TTCAAATAGT 9360

AAAAGGCGAG ATGATATATA TGAACGGGGC TTATCGTTTG TCAAGTATTT ATGGGACAAC 9420

GCCTGCTGGT TTTGCGTTGA TTACTCTCGT CTTTTCTGTT TATTTCTATA GTCAATTGCA 9480

CGTTTCGGAT AGCGGTAAGT TTAAAAAAAT ATTTCCATTT GCTTTTTTTA TGATTAATGT 9540

ATCTATGATG GCGTTGACGC AAAGCCGACA ATCAATAATG ACATTGTTAT TGCTTATTTG 9600

TATATTTCAT TTTGTTAGGG CAAAGAATAT ATTTAAGTTT TTTTCGCTTA TATTTATTTT 9660

ATTGTTAATC TATGTATTTT ATTTGTTGGT TGTTAATACG GACTTTTTTC CACGATTAAC 9720

TCAAATGCTT TTTAATTACT CATCAGATTC ATCAACACAT ACACGTATTT CTATTATTAC 9780

TCAATCGTAT GAACATCTTG ATGTTAATGA TTTAATTTAT GGGATTGGTT TAGGCGGATT 9840

TAATCACTTC TACTACAAGA TTAGTGGTGA AATAGGTGTT GCAGCCCATA ATGATTTTTT 9900

CCTTTTTTTT GTTGAAGGTG GCGTAATAGC TTTGTTCTTT TATTGCCTTT TTTTATGCGG 9960

TGGAGTGTCA TTTTGGGGGC GGGCGATTAA ACAGCAAGGT GATAGTTATT TTACACCGTT 10020

GCTTGTTTTT TGTGCTTTAT ACGTATTTTC TTTTTTGAAT AATCCTTATT ATTACCCACA 10080

AGTGCAAGTT GTTGCTTCTG CATTGATGGG CGTGTATTTG TCAGAATATA TAAAGAAACA 10140

The termination of the initial Orf8 of Orf9

AAAGGTAAAT TATGTTTTGC CATGAACTTG AAAACAAAAT GAAATCTCAG GAATTTTTTG 10200

TCGAAACTCT CAAAAATAGC CAGCTTAATA AAACCAGAAT TAGTTTTCTT AACCCCTATT 10260

CATACCCTTT AATTTCAAGA AATAAAAAAA TCATCAAGGG TTTTGATTAT TGGTTTGCAG 10320

ATGGTTTAAC ACTATGTATT GTTACTAATT TGTTTAGAGA GAAGTCTGAT ATAATAAAAA 10380

GAGGCAGTTT TGATTTTTCA TCACTGGCGG GAATATGTTT CGATTATTTA CAAACTCATT 10440

CTCTTTCTGT TGCTATTATT GGTGGAAGTA ATGATGAGAT TCATCAAGCA TGTGAATATT 10500

TTTCCATTCG ATATCCATCC TTAAAAATTT GTTTTAAGCA TCATGGGTAT ATAGATATAG 10560

ACAAGTGTGA TCATATAATA AATGAGCTTA AAAAATCTGA AGTGCAAGCA TTAATTGTAG 10620

GAATGGGAAC ACCTAAGCAA GAAGAGTTTA TATTAAAGGT TGAAAAACAT ATAATAAGTT 10680

GCAAGCTTTT TTTAACTTGT GGAGGTTTTA TTTCACAAAC AGCGAGTAGC GGTGATTATT 10740

ATCACCCATT AATCAAAAGA TTAGGTCTTC GTTGGCTACA GCGTGCAGTG CTTTATAAGC 10800

ATGTTAGGCG CAGGCTTTTA CTCAATTATC CCTGTTTTTA TTTTAGATAT ATATGTAGTG 10860

The termination of Orf9

CAATATTAAA TATCTTATAT GCTAAAAAAT AGCAGGATAA TTATCAATGT AGTATGCTAA 10920

AATTGACCAA TATCGTGAAG TTATCAAATG TTATATCAGA TAACATGAAA CAATATACGA 10980

ACCGCTACAC GCAGTAACCC CTGACAGGAG TAAACAATGT CAAAGCAACA GATCGGCGTC 11040

GTCGGTATGG CAGTGATGGG GCGCAACCTT GCGCTCAACA TCGAAAGCCG TGGTTATACC 11100

GTCTCTATTT TCAACCGTTC CCGTGAAAAG ACCGAAGAAG TGATTGCCGA AAATCCAGGC 11160

AAGAAACTGG TTCCTTACTA TACGGTGAAA GAGTTTGTTG AATCTCTGGA AACGCCTCGT 11220

CGCATCCTGT TAATGGTGAA AGCAGGTGCA GGCACGGATG CTGCTATTGA TTCTCTCAAG 11280

CCATACCTCG ATAAAGGTGA CATCATCATT GATGGTGGTA ATACCTTCTT CCTGGACACC 11340

ATTCGCCGTA ATCGTGAGCT TTCTGCCGAA GGCTTTAACT TCATTGGTAC CGGTGTTTCC 11400

GGTGGTGAAG AAGGTGCGCT GAAAGGTCCT TCCATTATGC CTGGTGGTCA GAAAGAAGCC 11460

TATGAACTGG TTGCACCGAT CCTGACCAAA ATCGCCGCAG TGGCTGAAGA TGGCGAACCG 11520

TGCGTTACCT ATATTGGTGC CGATGGTGCA GGTCATTACG TGAAGATGGT TCACAACGGT 11580

ATTGAATACG GCGATATGCA GCTGATTGCT GAAGCCTATT CTCTGCTTAA AGGTGGCCTG 11640

AATCTTTCCA ACGAAGAACT AGCGCAGACG TTTACCGAGT GGAATAACGG TGAACTGAGC 11700

AGCTACCTGA TCGACATCAC CAAAGATATC TTCACCAAAA AAGATGAAGA CGGTAACTAC 11760

CTGGTTGATG TGATTCTGGA TGAAGCGGCT AACAAAGGTA CCGGTAAATG GACCAGCCAG 11820

AGTGCGCTGG ATCTCGGCGA ACCGCTGTCG CTGATTACCG AGTCTGTGTT TGCACGTTAT 11880

ATCTCTTCTC TGAAA 11895

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

Claims (4)

1, a kind of oligonucleotide of the O-antigen-specific to intestinal bacteria O136, it is characterized in that, its wzx sequence is that the Nucleotide or the wzy sequence of 6609 to 7847 bases among the SEQ ID NO:1 is the Nucleotide of 8933 to 10165 bases among the SEQ ID NO:1 or has above-mentioned sequence insertion, lack or replace one or more Nucleotide, but keeps the oligonucleotide of the O-antigen function of wzx, wzy Nucleotide specific detection intestinal bacteria O136.

2, a kind of oligonucleotide of the O-antigen high special to intestinal bacteria O136, it is characterized in that, it is that to come from the oligonucleotide of wzx gene right: the Nucleotide of 6620 to 6637 bases among the SEQ ID NO:1 and the Nucleotide of 7254 to 7261 bases, the Nucleotide of 6597 to 6614 bases among the SEQ ID NO:1 and the Nucleotide of 7048 to 7065 bases; Or the oligonucleotide that comes from the wzy gene is right: the Nucleotide of 9525 to 9542 bases among the SEQ ID NO:1 and the Nucleotide of 10067 to 10084 bases, the Nucleotide of 8973 to 8992 bases among the SEQ ID NO:1 and the Nucleotide of 9885 to 9902 bases.

3, the application of oligonucleotide in detecting the O-antigen of expressing the antigenic bacterium of O-, identifying bacterium of claim 1, any described O-antigen-specific to intestinal bacteria O136 type of 2.

4, the application of the oligonucleotide of claim 1, any described O-antigen-specific to intestinal bacteria O136 type of 2, it is characterized in that, it is used for PCR, is used for hybridization and fluoroscopic examination or is used to make gene chip or microarray as probe as primer, and the confession detection bodies reaches the bacterium in the environment outward.