CN1570114A - Nucleotide specific for bacillus coli O167 type and Sh.boydii 3 type O-antigen - Google Patents

Abstract

The invention provides an O-antigen specific nucleotide to Escherichia coli O167 and Shigella boydii3, which is the whole sequence of gene cluster controlling O-antigen synthesis in Escherichia coli O167 and Shigella boydii3, such as separating nucleotide sequence of SEQ ID NO:1 , Escherichia coli O167 being 12864 base, Shigella boydii3 being 12199 base, or one or several base substituted, defected or inserted SEQ ID NO:1 and at same time have same function with SEQ ID NO:1, also comprise oligonucleotide of glycosyltransferase gene and oligosaccharide disposing gene derived from O-antigen gene cluster of Escherichia coli O167 and Shigella boydii3. It is validated that oligonucleotide is highly specific to Escherichia coli O167 and Shigella boydii3 O-antigen. The invention also discloses Escherichia coli O167 and Shigella boydii3 detection and identification method using inventive oligonucleotide.

Description

Nucleotide to the O-antigen-specific of intestinal bacteria O167 type and Shigella bogdii 3 types

Technical field

The present invention relates to the complete nucleotide sequence of control O-antigen synthetic gene cluster in intestinal bacteria O167 type (Escherichia coli O167) and Shigella bogdii 3 types (Shigella boydii 3), particularly relate in intestinal bacteria O167 type and Shigella bogdii 3 types oligonucleotide in the control O-antigen synthetic gene cluster, can utilize these oligonucleotide of O-antigen-specific intestinal bacteria O167 type and Shigella bogdii 3 types and identify O-antigen in these pathogenic bacterium in human body and the environment quickly and accurately.

Background technology

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

Because O-antigen is extremely strong antigen, be one of important paathogenic factor of 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 of abequose and paratosesynthase genes (rfb) by polymerase chain reaction for identification of S.enterica majorserogroups (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-Uremic Syndrome caused by dryfermented sausage contaminated with Shiga-like toxin producing Escherichiacoli " .J.Clin.Microbiol.34:1622-1627] of the oligonucleotide that comes from the wbdI gene of the O-antigen-specific of E.coli O111 having been identified the toxogenic E.coli O111 of a strain, but afterwards studies show that Paton, the usefulness of A.W et.al comes from the oligonucleotide of wbdI gene and identifies that the method for the serotype of E.coli O111 has false positive results to occur.BastinD.A.and Reeves, P.R. think, this is because the wbdI gene is sugared synthesis path gene [the BastinD.A.and Reeves of a supposition, P.R. (1995) Sequence and analysis of the O antigen gene (rfb) cluster ofEscherichia coli O111.Gene 164:17-23], and in the antigenic structure of the O-of other bacterium, also has this sugar, so sugared synthesis path gene is not that the Shigellae of high special has 46 kinds of serotypes for O-antigen, but have only 33 kinds of different O-antigens, intestinal bacteria have 166 kinds of different O-antigen [Reeves, P.R (1992) " Variation in O antigens; niche specific selection and bacterialpopulations " .FEMS Microbiol.Lett, 100:509-516], the two sibship is very near, and there are 12 kinds to be intestinal bacteria and the total [Ewing of Shigellae, W.H. (1986) " Edwards and Ewing ' sidentification of the Enterobacteriaceae " .Elsevier Science Publishers, Amsterdam, TheNetherlands; T.cheasty, et al. (1983) " Antigenic relationships between the enteroinvasiveEscherichia coli antigens O28ac; O112ac; O124; O136, O143, O144; O152 and Shigella Oantigens " J.clin Microbiol, 17 (4): 681-684]

Summary of the invention

The Nucleotide that the purpose of this invention is to provide a kind of O-antigen-specific to intestinal bacteria O167 type and Shigella bogdii 3 types.It is the Nucleotide in the O-antigen gene bunch of intestinal bacteria O167 type and Shigella bogdii 3 types, is the special Nucleotide that comes from glycosyltransferase gene and transhipment enzyme gene and pol gene.

A time purpose of the present invention has provided the full length nucleotide sequence of the O-antigen gene bunch of intestinal bacteria O167 type and Shigella bogdii 3 types.

Another object of the present invention has provided the gene of the O-antigen gene bunch that constitutes intestinal bacteria O167 type and Shigella bogdii 3 types: the gene of transhipment enzyme is the wzx gene or with wzx the gene of identity function is arranged; Pol gene is the wzy gene or with wzy the gene of identity function is arranged; Glycosyltransferase gene comprises orf3, orf4, orf5, orf7, orf8, orf9 gene.

Another purpose of the present invention has provided oligonucleotide, and the gene that they come from coding transhipment enzyme respectively is the wzx gene or with wzx the gene of identity function is arranged; The gene that comes from the coding polysaccharase is the wzy gene or with wzy the gene (table 1) of identity function is arranged; They are the oligonucleotide in the said gene, and length is at 10-20nt; They are high specials to the O-antigen of intestinal bacteria O167 type and Shigella bogdii 3 types; And these oligonucleotide are also reconfigurable, and the oligonucleotide after the combination also is a high special to the O-antigen of intestinal bacteria O167 type and Shigella bogdii 3 types.

The above-mentioned oligonucleotide that a further object of the present invention provides can be used as primer and is used for nucleic acid amplification reaction, perhaps be used for hybridization as probe, perhaps be used to make gene chip or microarray, thereby pass through O-antigen and detection and the identification of escherichia coli O167 type and Shigella bogdii 3 types of these methods detections and identification of escherichia coli O167 type and Shigella bogdii 3 types.

A present invention also purpose has provided the method for complete sequence of the O-antigen gene bunch of separating Escherichia coli O167 type and Shigella bogdii 3 types.Can obtain the complete sequence of the O-antigen gene bunch of other bacteriums according to present method operation, the complete sequence of the gene cluster of the bacterium of other polysaccharide antigens that also can obtain to encode.

The objective of the invention is to realize by following technical scheme.

The present invention is to the Nucleotide of the O-antigen-specific of intestinal bacteria O167 type and Shigella bogdii 3 types, it is characterized in that: it is the isolating Nucleotide shown in SEQ ID NO:1,12864 bases of intestinal bacteria O167 type total length, 12199 bases of Shigella bogdii 3 types; Perhaps described base with one or more insertions, disappearance or replacement keeps the Nucleotide of the SEQ IDNO:1 of described isolating functional nucleotide simultaneously.

The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O167 type and Shigella bogdii 3 types, comprising called after wzx, glf, orf3, orf4, orf5, wzy, orf7, orf8,9 genomic constitutions of orf9 are all between galF gene and gnd gene.

The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O167 type and Shigella bogdii 3 types, the gene that has high degree of specificity in the wherein said gene is: transhipment enzyme gene, it comprises the wzx gene; Pol gene, it comprises the wzy gene; Glycosyltransferase gene, it comprises orf3, orf4, orf5, orf7, orf8, orf9 gene; Wherein said gene: wzx is the Nucleotide of 1774 to 3039 bases among the SEQ ID NO:1; Wzy is the Nucleotide of 7165 to 8415 bases among the SEQ ID NO:1; Orf3 is the Nucleotide of 4153 to 5355 bases among the SEQ IDNO:1; Orf4 is the Nucleotide of 5355 to 6209 bases among the SEQ ID NO:1; Orf5 is the Nucleotide of 6209 to 7117 bases among the SEQ ID NO:1; Orf7 is 8415 to 9506 nucleotide bases among the SEQ ID NO:1; Orf8 is the Nucleotide of 9503 to 10462 bases among the SEQ ID NO:1; Orf9 is 10570 to 11472 nucleotide bases among the SEQ ID NO:1.

The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O167 type and Shigella bogdii 3 types wherein also comprises coming from described wzx gene, wzy gene and their mixing or their reorganization.

The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O167 type and Shigella bogdii 3 types, it is characterized in that the oligonucleotide that wherein comes from the wzx gene is to being: the Nucleotide of 2281 to 2300 bases among the SEQ ID NO:1 and the Nucleotide of 2925 to 2944 bases; The Nucleotide of 1973 to 1992 bases among the SEQ ID NO:1 and 2863 to 2882 nucleotide bases; The oligonucleotide that comes from the wzy gene is to being: the Nucleotide of 7318 to 7337 bases and 8047 to 8066 nucleotide bases among the SEQ ID NO:1; The Nucleotide of 7667 to 7686 bases and 8168 to 8184 nucleotide bases among the SEQ ID NO:1.

The application of the Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O167 type and Shigella bogdii 3 types in detecting other polysaccharide antigen of expressing the antigenic bacterium of O-, the O-antigen of identifying bacterium and bacterium.

The recombinant molecule of the Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O167 type and Shigella bogdii 3 types, providing the O-antigen of expressing intestinal bacteria O167 type and Shigella bogdii 3 types by inserting to express, and the application in the preparation bacterial vaccine.

The application of the Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O167 type and Shigella bogdii 3 types, it is characterized in that, it is used for PCR, is used for hybridization and fluoroscopic examination or is used to make gene chip or microarray as probe as primer, for the application of bacterial detection.

The separation method of the Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O167 type and Shigella bogdii 3 types is characterized in that it comprises the steps:

(1) genomic extraction: in substratum, cultivate intestinal bacteria O167 type or Shigella bogdii 3 types, centrifugal collecting cell; The genomic dna that obtains detects by agarose gel electrophoresis;

(2) by the O-antigen gene in pcr amplification intestinal bacteria O167 type or Shigella bogdii 3 types bunch: with intestinal bacteria O167 type or Shigella bogdii 3 type genomes 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 O167 type or Shigella bogdii 3 types:

(6) screening of specific gene: at wzx, the wzy gene design primer in the O-antigen gene of intestinal bacteria O167 type or Shigella bogdii 3 types bunch; Respectively designed two pairs of primers in each gene, every pair of primer is distributed in the different places in the corresponding gene, to guarantee its specificity; Is that template is carried out PCR with these primers with the genomes of 166 strain intestinal bacteria and 43 strain Shigellaes, determines the antigenic high degree of specificity of O-of wzx, wzy gene pairs intestinal bacteria O167 type or Shigella bogdii 3 types;

(7) detection of primer sensitivity: cultivate intestinal bacteria O167, 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 O167.

The separation method of the Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O167 type or Shigella bogdii 3 types is characterized in that it comprises the steps:

(1) genomic extraction: 37 ℃ of incubated overnight intestinal bacteria O167 types or Shigella bogdii 3 types in the LB of 5mL substratum, centrifugal collecting cell.With 500 μ l 50mM Tris-HCl (pH8.0) and 10 μ l0.4M EDTA re-suspended cells, 37 ℃ of incubations 20 minutes, the N,O-Diacetylmuramidase that adds 10 μ l 10mg/mL then continues insulation 20 minutes.The Proteinase K, the 15 μ l 10%SDS that add 3 μ l 20mg/mL afterwards, 50 ℃ of incubations 2 hours, the RNase that adds 3 μ l 10mg/mL again, 65 ℃ of incubations 30 minutes, add equal-volume phenol extracting mixture, get supernatant and use isopyknic phenol again: chloroform: primary isoamyl alcohol (25: 24: 1) mixing solutions extracting twice, get supernatant again with isopyknic ether extracting to remove remaining phenol.Supernatant rolls out DNA and washes DNA with 70% ethanol with glass yarn with 2 times of volume ethanol deposit D NA, and DNA is resuspended among the 30 μ l TE; Genomic dna detects by 0.4% agarose gel electrophoresis;

(2) by the O-antigen gene in pcr amplification intestinal bacteria O167 type or Shigella bogdii 3 types bunch: with the genome of intestinal bacteria O167 type or Shigella bogdii 3 types is that template is passed through its O-antigen gene of Long pcr amplification bunch, at first according to the galF sequences Design upstream primer (#1523-ATT GTG GCT GCA GGG ATC AAA GAA AT) that often is found in O-antigen gene bunch promoter region, again according to the gnd gene design downstream primer (#1524-TAG TCG CGTGNG CCT GGA TTA AGT TCG C) in O-antigen gene bunch downstream; With the ExpandLong Template PCR method of Boehringer Mannheim company amplification O-antigen gene bunch, the PCR response procedures was as follows: 94 ℃ of pre-sex change 2 minutes; 94 ℃ of sex change are 10 seconds then, Annealed 15 seconds, 68 ℃ were extended 15 minutes, carry 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 5 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, 0.9 μ l 0.1M MnCl ₂, the DNaseI of the 1mg/mL of 1 μ l dilution in 1: 2000, reaction is carried out at room temperature, and enzyme is cut the dna fragmentation size is concentrated between the 1.5kb-3kb, then adds 2 μ l 0.1M EDTA termination reactions.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 18 μ l water, in this mixture, add 2.5 μ l dNTP (1mMdCTP, 1mMdGTP, 1mMdTTP subsequently, 10mMdATP), 1.25 the T4DNA polysaccharase of μ l 100mM DTT and 5 units, 11 ℃ 30 minutes, enzyme is cut product mends 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 80 μ l, 70 ℃ of reactions 20 minutes make 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 10 hours in 16 ℃, cumulative volume is 90 μ l.10 * the buffer of 9 μ l and the T4DNA ligase enzyme of 25 units are wherein arranged, use the 3M NaAc (pH5.2) of 1/10 volume and the dehydrated alcohol precipitation of 2 times of volumes to be connected mixture at last, wash precipitation with 70% ethanol again, be dissolved in after the drying in the 30 μ l water and obtain connecting product; Preparation method with the electric transformed competence colibacillus cell of Bio-Rad company prepares the competence e.colidh5, get after 2-3 μ l connects product and 50 μ l competence bacillus coli DH 5 alphas mix, forward in the electric shock cup of 0.2cm of Bio-Rad company and shock by electricity, voltage is 2.5 kilovolts, time is 5.0 milliseconds to 6.0 milliseconds, the SOC substratum that adds 1mL after the electric shock immediately in cup makes the bacterium recovery, then bacterium is coated in and contains penbritin, on the LB solid medium of X-Gal and IPTG, 37 ℃ of incubated overnight, obtain blue white bacterium colony next day, with the white colony that obtains promptly the white clone forward on the LB solid medium that contains penbritin and cultivate, from each clone, extract plasmid simultaneously, and cutting the segmental size of evaluation insertion wherein with the EcoRI enzyme, the white that obtains clone group has constituted the O-antigen gene bunch library of intestinal bacteria O167 type or Shigella bogdii 3 types;

(4) to the cloning and sequencing in the library: from the library, select 96 clones of insertion fragment more than 1kb and the insertion fragment among the clone is checked order with this lab A BI3730 type automatic dna sequencer, sequence reaches 100% fraction of coverage, thereby obtains 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 Research Council) Molecular Biology Lab and edit all sequences, thus obtain the Nucleotide full length sequence of the O-antigen gene bunch of intestinal bacteria O167 type or Shigella bogdii 3 types; The quality of sequence is mainly guaranteed by two aspects: 1) genome of intestinal bacteria O167 type or Shigella bogdii 3 types is done 5 Long PCR reactions, mix these products then to produce the library, 2) to each base, guarantee high-quality fraction of coverage more than 3, after obtaining the nucleotide sequence of intestinal bacteria O167 type and Shigella bogdii 3 type O-antigen genes bunch, with American National biotechnology information science center (The National Center for BiotechnologyInformation, NCBI) Orffinder finds gene, find the reading frame of 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 ClustralW software, obtain the structure of the O-antigen gene bunch of intestinal bacteria O167 type and Shigella bogdii 3 types at last;

(6) specific gene screening: at wzx, the wzy gene design primer in the O-antigen gene of intestinal bacteria O167 type and Shigella bogdii 3 types bunch; Respectively designed two pairs of primers in each gene, every pair of primer is distributed in the different places in the corresponding gene, to guarantee its specificity; Is that template is carried out PCR with these primers with the genomes of 166 strain intestinal bacteria and 43 strain Shigellaes, except that a band that in containing intestinal bacteria O167 group, has obtained the expection size, the correct product of the expection clip size that all do not increase in other groups is so the O-antigen of wzx, wzy gene pairs intestinal bacteria O167 type and Shigella bogdii 3 types all is high special.

(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 O167 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 l supernatant as pcr template.Right with 4 pairs of oligonucleotide, the Nucleotide of 2281 to 2300 bases among the SEQ ID NO:1 and the Nucleotide of 2925 to 2944 bases; The Nucleotide of 1973 to 1992 bases among the SEQ ID NO:1 and the Nucleotide of 2863 to 2882 bases; The Nucleotide of 7318 to 7337 bases among the SEQ ID NO:1 and the Nucleotide of 8047 to 8066 bases; The Nucleotide of 7667 to 7686 bases among the SEQ ID NO:1 and the Nucleotide of 8168 to 8184 bases carry out the PCR reaction, and 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 O167 in the pork filling when using aforesaid method.

Just, first aspect of the present invention, the full length nucleotide sequence of the O-antigen gene bunch of intestinal bacteria O167 type and Shigella bogdii 3 types is provided, its complete sequence is shown in SEQ ID NO:1,12864 bases of intestinal bacteria O167 type total length, 12199 bases of Shigella bogdii 3 types; 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 O167 type and Shigella bogdii 3 types by method of the present invention, as shown in table 3, it comprises called after wzx, glf, orf3, orf4, orf5, wzy, orf7, orf8,9 genomic constitutions of orf9 are all between galF gene and gnd gene.

Second aspect of the present invention provides the gene in the O-antigen gene bunch of intestinal bacteria O167 type and Shigella bogdii 3 types, promptly transports enzyme gene (wzx gene or the gene of identity function arranged with wzx); Pol gene (wzy gene or the gene of identity function arranged with wzy); Glycosyltransferase gene (orf3, orf4, orf5, orf7, orf8, orf9 gene).Their zero positions in O-antigen gene bunch and final position and nucleotide sequence all are listed in the table 4; The invention particularly relates to glycosyltransferase gene, transhipment enzyme gene and pol gene, because sugared synthesis path gene is that the gene of synthetic nucleosides bisphosphate monose is common, common by indication to more exocellular polysaccharide now, O-antigen to bacterium is not very special, and the glycosyltransferase gene that the present invention relates to, transhipment enzyme gene and pol gene are high specials to the O-antigen of intestinal bacteria O167 type and Shigella bogdii 3 types.

The 3rd aspect of the present invention, wzx gene in the O-antigen gene bunch that comes from intestinal bacteria O167 type and Shigella bogdii 3 types is provided or the gene of identity function and wzy gene is arranged or with wzy the oligonucleotide (table 1) of the gene of identity function is arranged with wzx, they are any one section oligonucleotide in these genes.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 except that a band that has obtained the expection size in the 13rd group, and any product that all do not increase in other groups is so the O-antigen of wzx, wzy gene pairs intestinal bacteria O167 type and Shigella bogdii 3 types all is high special.

The separation method of the Nucleotide of described O-antigen-specific to intestinal bacteria O167 type and Shigella bogdii 3 types comprises the steps: 1) genomic extraction; 2) the O-antigen gene in pcr amplification intestinal bacteria O167 type or Shigella bogdii 3 types bunch; 3) make up O-antigen gene bunch library; 4) to the cloning and sequencing in the library; 5) splicing of nucleotide sequence and analysis; 6) screening of specific gene; 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 used herein, " oligonucleotide " mainly refer to derive from the gene of the coding transhipment enzyme in the O-antigen gene bunch and intragenic one section nucleic acid molecule of coding polysaccharase, and they can change on length, generally change in 10 to 20 Nucleotide scopes; More definite these oligonucleotide of saying are to come from wzx gene (nucleotide position is the Nucleotide of 1774 to 3039 bases from SEQ ID NO:1), wzy gene (nucleotide position is the Nucleotide of 7165 to 8415 bases from SEQ ID NO:1); Coming from above intragenic oligonucleotide is high specials to intestinal bacteria O167 type and Shigella bogdii 3 types.

In addition, the antigenic gene cluster of the different O-of the coding of two genetic resemblances produces new O-antigen by gene recombination or sudden change sometimes, thereby produces new bacteria types, new mutant strain.In this environment, need filter out many specificitys that oligonucleotide is detected with raising with recombination hybridization.Therefore, the invention provides a whole set of many mixtures to oligonucleotide, they come from transhipment enzyme and pol gene, comprise the wzx gene or the gene, wzy gene of identity function are arranged or with wzy the gene of identity function is arranged with wzx.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 the wzx gene or the gene, wzy gene of identity function arranged or with wzy the combination of the oligonucleotide in the gene of identity function is arranged with wzx.

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

The present invention relates to a kind of antigenic method of one or more bacterial polysaccharideses that detects in the food, these antigens can make sample can with the oligonucleotide specific hybrid of following at least one gene, these genes are: (i) gene of encoding glycosyl transferring enzyme; The (ii) gene of coding transhipment enzyme and polysaccharase comprises the wzx gene or the gene, wzy gene of identity function is arranged or with wzy the gene of identity function is arranged with wzx.At least one oligonucleotide can be hybridized with at least one more than one such gene specific of expressing the special antigenic bacterium of O-under the situation of condition permission, and these bacteriums are intestinal bacteria O167 type or Shigella bogdii 3 types.Available PCR method detects, more can with behind the Nucleotide mark in the inventive method as probe by hybridization such as Southern-blot or fluoroscopic examination, perhaps by antigen and bacterium in gene chip or the microarray assay sample.

Planner of the present invention considers following situation: when one special oligonucleotide detects when invalid, the mixture of oligonucleotide can with the target region specific hybrid with test sample.Therefore the invention provides a cover oligonucleotide and be used for detection method of the present invention.Here said oligonucleotide is meant the gene that coming from coding transhipment enzyme and the gene of polysaccharase, comprises the wzx gene or the gene, wzy gene of identity function is arranged or with wzy the oligonucleotide of the gene of identity function is arranged with wzx.This cover oligonucleotide is special to the O-antigen of a special bacterium, and this special bacterium O-antigen is expressed by intestinal bacteria O167 type or Shigella bogdii 3 types.

On the other hand, the present invention relates to a kind of antigenic method of one or more bacterial polysaccharideses that detects in the movement, these antigens can make sample can with the oligonucleotide specific hybrid of following at least one gene, these genes are: (i) gene of encoding glycosyl transferring enzyme; The (ii) gene of coding transhipment enzyme and polysaccharase comprises the wzx gene or the gene, wzy gene of identity function is arranged or with wzy the gene of identity function is arranged with wzx.At least one oligonucleotide can be expressed more than one such gene specific hybridization of the special antigenic bacterium of O-with at least one under the situation of condition permission.These bacteriums are intestinal bacteria O167 type or Shigella bogdii 3 types.Available oligonucleotide of the present invention is made the method test sample of primer by PCR, also can with behind the oligonucleotide molecules mark of the present invention as probe by hybridization such as Southern-blot or fluoroscopic examination, or by antigen and bacterium in gene chip or the microarray assay sample.

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

On the other hand, the present invention also relates to the antigenic method of one or more bacterial polysaccharideses in the sample that a kind of detection comes from patient.One or more bacterial polysaccharides antigens in the sample can make sample can with a specific hybrid in a pair of oligonucleotide in following at least one gene, these genes are: (i) gene of encoding glycosyl transferring enzyme; The (ii) gene of coding transhipment enzyme and polysaccharase comprises the wzx gene or the gene, wzy gene of identity function is arranged or with wzy the gene of identity function is arranged with wzx.Under the situation of condition permission at least one oligonucleotide can with sample at least one express more than one such gene specific hybridization of the special antigenic bacterium of O-, these bacteriums are intestinal bacteria O167 type or Shigella bogdii 3 types.Oligonucleotide among available the present invention is made the method test sample of primer by PCR, also can will pass through hybridization as probe behind the oligonucleotide mark among the present invention, perhaps by antigen and bacterium in gene chip or the microarray assay sample.

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

The present inventor believes that the present invention is not necessarily limited to the above nucleotide sequence coded specific O-antigen of carrying, and is widely used in detecting all expression O-antigens and identifies the antigenic bacterium of O-.And because O-antigen is synthetic and the similarity of other polysaccharide antigens (as bacterium born of the same parents exoantigen) between synthesizing, 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 O167 type and Shigella bogdii 3 types first, and can from the sequence of this total length gene cluster of not cloned, produce recombinant molecule, can produce the O-antigen of expressing intestinal bacteria O167 type and Shigella bogdii 3 types by inserting to express, and become useful vaccine.

Embodiment

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

Embodiment 1: genomic extraction:

37 ℃ of incubated overnight intestinal bacteria O167 types or Shigella bogdii 3 types in the LB of 5mL substratum, centrifugal collecting cell.With 500 μ l 50mM Tris-HCl (pH8.0) and 10 μ l 0.4M EDTA re-suspended cells, 37 ℃ of incubations 20 minutes, the N,O-Diacetylmuramidase that adds 10 μ l 10mg/mL then continues insulation 20 minutes.The Proteinase K, the 15 μ l 10%SDS that add 3 μ l 20mg/mL afterwards, 50 ℃ of incubations 2 hours add the RNase of 3 μ l10mg/mL again, 65 ℃ of incubations 30 minutes.Add equal-volume phenol extracting mixture, get supernatant and use isopyknic phenol again: chloroform: primary isoamyl alcohol is taken out (25: 24: 1) mixing solutions and is carried twice, get supernatant again with isopyknic ether extracting to remove remaining phenol, supernatant is with 2 times of volume ethanol deposit D NA, roll out DNA and wash DNA with glass yarn, at last DNA is resuspended among the 30 μ l TE with 70% ethanol.Genomic dna detects by 0.4% agarose gel electrophoresis.

Embodiment 2: by the O-antigen gene in pcr amplification intestinal bacteria O167 type or Shigella bogdii 3 types bunch:

With the genome of intestinal bacteria O167 type or Shigella bogdii 3 types is that template is passed through its O-antigen gene of Long pcr amplification bunch.At first according to the galF sequences Design upstream primer (#1523-ATT GTG GCT GCA GGG ATC AAA GAA AT) that often is found in O-antigen gene bunch promoter region, again according to the gnd gene design downstream primer (#1524-TAG TCG CGTGNG CCT GGA TTA AGT TCG C) in O-antigen gene bunch downstream; With the ExpandLong Template PCR method of Boehringer Mannheim company amplification O-antigen gene bunch, the PCR response procedures was as follows: 94 ℃ of pre-sex change 2 minutes; 94 ℃ of sex change are 10 seconds then, Annealed 15 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 5 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, 0.9 μ l 0.1M MnCl ₂, 1 μ l1: the DNaseI of the 1mg/mL of 2000 dilutions, reaction is carried out at room temperature.Enzyme is cut the dna fragmentation size is concentrated between the 1.5kb-3kb, then adds 2 μ l 0.1M EDTA termination reactions.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 18 μ l water.In this mixture, add 2.5 μ l dNTP (1mMdCTP subsequently, 1mMdGTP, 1mMdTTP, 10mMdATP), the T4DNA polysaccharase of 1.25 μ l 100mM DTT and 5 units, 11 ℃ 30 minutes, enzyme is cut product mend into flush end, after 75 ℃ of termination reactions, add the Tth archaeal dna polymerase of 5 units and corresponding damping fluid thereof and system is expanded as 80 μ l, 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 10 hours in 16 ℃, cumulative volume is 90 μ l.10 * the buffer of 9 μ l 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 in the 30 μ l water and obtain connecting product.

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, 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, precipitate 10% glycerine suspension cell with the precooling of 1mL ice at last, are competent cell.The competent cell that makes is packed as 50 μ l, one pipe ,-70 ℃ of preservations.

Be electric transformed competence colibacillus cell at last: get after 2-3 μ l connects product and 50 μ l competence bacillus coli DH 5 alphas mix, 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 O167 type or Shigella bogdii 3 types with the EcoRI enzyme.

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

From the library, select insert 96 clones of fragment more than 1kb with this lab A BI3730 type automatic dna sequencer to unidirectional order-checking of insertion fragment among the clone, make sequence reach 100% fraction of coverage, thus all sequences of acquisition O-antigen gene bunch.

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 O167 type and Shigella bogdii 3 types obtained.The quality of sequence is mainly guaranteed by two aspects: 1) genome of intestinal bacteria O167 type and Shigella bogdii 3 types is done 5 Long PCR reactions, mix these products then to produce the library.2), guarantee high-quality fraction of coverage more than 3 to each base.After obtaining the nucleotide sequence of intestinal bacteria O167 type and Shigella bogdii 3 type O-antigen genes bunch, with American National biotechnology information science center (The National Center for Biotechnology 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 O167 type and Shigella bogdii 3 types at last, as shown in table 3.

By retrieving and comparing, the aminoacid sequence of finding the UDP-galactopyranose mutase (AAC75097) that encodes among orf2 encoded protein and the Escherichia coli K12 has 72% consistence and 85% similarity, by search, find that the homology desired value of the consensus sequence of orf2 encoded protein and known UDP-galactopyranose mutase is 8.4e to Pfam protein-based order sequenced data storehouse ^-138Therefore we can determine that this gene is glf.

Orf1 and orf6 are the proteic genes that there is transmembrane segment in only two codings among the intestinal bacteria O167.The aminoacid sequence that is speculated as O-antigen transferring enzyme (AAC75098) of orf1 encoded protein and Escherichia coli has 37% consistence and 61% similarity, it contains 11 uniform transmembrane segments by the proteic topology discovery of HMMTOP2.0 programanalysis, and this is the proteic characteristic feature of Wzx.So name orf1 is wzx.The O-antigen polysaccharase (CAD64465) of orf6 encoded protein and Lactobacillus plantarum WCFS1 has 25% consistence and 43% similarity, it contains 11 transmembrane segments by the proteic topology discovery of HMMTOP2.0 programanalysis, and hydrophilic loop (loop) in the big kytoplasm is arranged, and this is a Wzy albumen characteristic feature.So name orf6 is wzy.Orf3, orf4, orf5, orf7, orf8, the albumen of six genes encodings of orf9 and other known glycosyltransferases have the sequence identity of 25-39% and the sequence similarity of 49-59%.By the search to glycosyltransferase motif database among the Pfam, the homology desired value of the albumen of these six genes encodings and the consensus sequence of known glycosyltransferase is very high, so we infer this six genes encoding glycosyltransferases.Because the definite function of these six genes can't be determined, so we are with these six genes temporary called after orf3, orf4, orf5, orf7, orf8 and orf9.

Embodiment 6: the screening of specific gene.

Wzx, wzy gene design primer in the O-antigen gene of intestinal bacteria O167 type and Shigella bogdii 3 types bunch have respectively designed two pairs of primers in each gene, every pair of primer is distributed in different local 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, except that a band that in containing intestinal bacteria O167 type and Shigella bogdii 3 type groups, has obtained the expection size, the correct product of the expection clip size that all do not increase in other groups is so the position in the nucleotide sequence of wzx, wzy gene pairs intestinal bacteria O167 type and Shigella bogdii 3 types sees Table 1.

Embodiment 7: the detection of primer sensitivity.O-antigen all is high special; The live pig meat stuffing of these genes on nuclear purchase market stirs, and is divided into the 20g portion, exists in-40 ℃ of refrigerators standby.The frozen bacterium liquid of 10 μ l intestinal bacteria O167 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 l supernatant as pcr template.Right with 4 pairs of oligonucleotide, the Nucleotide of 2281 to 2300 bases among the SEQ ID NO:1 and the Nucleotide of 2925 to 2944 bases; The Nucleotide of 1973 to 1992 bases among the SEQ ID NO:1 and the Nucleotide of 2863 to 2882 bases; The Nucleotide of 7318 to 7337 bases among the SEQ ID NO:1 and the Nucleotide of 8047 to 8066 bases; The Nucleotide of 7667 to 7686 bases among the SEQ ID NO:1 and the Nucleotide of 8168 to 8184 bases carry out the PCR reaction, and 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 O167 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 intestinal bacteria O167 of the present invention can be applied to set up recombiant vaccine.

Nucleotide sequence (shown in the SEQ ID NO:1) according to the O-antigen-specific to intestinal bacteria O167 type and Shigella bogdii 3 types of the present invention, structure specific nucleic acid probe, be fixed on the carrier of chip and make biochip, after the sample that will detect is suitably handled, carry out hybridization with biochip, utilize the biochip signal analysis equipment just can obtain corresponding bacteria situation in the sample then.The DNA chip that this intestinal bacteria O-antigen is identified can be directly used in clinical and other check place (as food-processing and production industry, the Micro biological Tests of animal and veterinary industry customs quarantine control etc.).This chip only need enlarge output, just can industrialization under identical condition.

Table 1 has been listed transhipment enzyme gene and pol gene and intragenic primer and PCR data in the O-antigen gene bunch of intestinal bacteria O167 type and Shigella bogdii 3 types.Transhipment enzyme gene and pol gene and their function corresponding and the size of the O-antigen gene bunch of intestinal bacteria O167 type and Shigella bogdii 3 types in table, 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.

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

The genomic dna that contains Shigella bogdii 3 types in the 13rd group is as positive control; The genomic dna that contains intestinal bacteria O167 type in the 14th group is as positive control.Do template with every group of bacterium, be PCR by following condition with every pair in the table 1 primer: 95 ℃ of pre-sex change after 5 minutes, 95 ℃ of sex change 30 seconds, annealing time is 30 seconds, temperature sees Table 1,72 ℃ extended 2 minutes, carried out 25 circulations like this.Continue to extend 5 minutes at 72 ℃ at last, reaction system is 25 μ l.Template is dilution in 1: 20, gets 1 μ l.After reaction finishes, get 10 μ l PCR products 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, every pair of primer has obtained except be PCR in the 13rd and 14 group after the correct band of expection size, the correct band of any size does not all increase in other groups, that is to say, not obtaining any PCR product band in the array mostly, so wzx, wzy gene pairs intestinal bacteria O167 type and Shigella bogdii 3 types and O-antigen thereof are high specials.And the oligonucleotide of these intragenic any one section 10-20nt is special to the O-antigen of intestinal bacteria O167 type and Shigella bogdii 3 types, and the primer in especially above-mentioned each gene is that oligonucleotide is high specials to detecting the back confirmation through PCR to intestinal bacteria O167 type and Shigella bogdii 3 types.These all oligonucleotide all can be used for intestinal bacteria O167 type and Shigella bogdii 3 types in the human body and environment rapidly and accurately, and can identify their O-antigen.

Table 3 is structural tables of the O-antigen gene bunch of intestinal bacteria O167 type and Shigella bogdii 3 types, in table, listed the structure of the O-antigen gene bunch of intestinal bacteria O167 type and Shigella bogdii 3 types, altogether by 9 genomic constitutions, each gene box indicating, and in square frame, write the title of gene, numeral be the order of the open reading frame (orf) in the O-antigen gene bunch.Two ends at O-antigen gene bunch are galF gene and gnd gene, and 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 site plan of the gene in the O-antigen gene bunch of intestinal bacteria O167 type and Shigella bogdii 3 types, listed the accurate position of all open reading frame in complete sequence in the O-antigen gene bunch of intestinal bacteria O167 type and Shigella bogdii 3 types in the drawings, 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 intestinal bacteria: 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 O167 type and Shigella bogdii 3 types

＜130〉to the Nucleotide of the O-antigen-specific of intestinal bacteria O167 type and Shigella bogdii 3 types

<160>1

<170>PatentIn?version?3.2

<210>1

<211>12864

<212>DNA

<213>Escherichia?coli

<400>1

attgtggctg?cagggatcaa?agaaatcctc?ctggtaactc?acgcgtccaa?gaacgcggtc???????????60

gaaaaccact?tcgacacctc? ?gaatctctcc?ttgaacagcg?cgtgaagcgt??????120

caactactgg?cggaagtgca?gtccatctgt?ccgccgggcg?tgaccattat?gaacgtgcgt??????????180

cagggcgaac?ctttaggttt?gggccactcc?attttgtgtg?cacgacctgc?cattggtgac??????????240

aacccatttg?tcgtggtgct?gccagacgtt?gtgatcgatg?acgccagcgc?cgacccgctg??????????300

cgctacaatc?ttgctgccat?gattgcgcgc?ttcaacgaaa?cgggccgcag?ccaggtgctg??????????360

gcaaaacgta?tgccgggtga?cctctctgaa?tactccgtca?ttcagaccaa?agaaccgctg??????????420

gatcgtgaag?gtaaagtcag?ccgcattgtt?gaatttatcg?aaaaaccgga?tcagccgcag??????????480

acgctggact?cagatattat?ggccgttggt?cgctatgtgc?tttctgccga?tatttggccg??????????540

gaacttgaac?gtactcagcc?tggtgcatgg?gggcgtattc?agctgactga?tgccattgcc??????????600

gaactggcga?aaaaacagtc?cgttgatgca?atgctgatga?ctggtgacag?ctacgactgc??????????660

ggtaaaaaaa?tgggctatat?gcaggcgttt?gtgaagtatg?gactgcgcaa?cctgaaagaa??????????720

ggggcgaagt?tccgtaaagg?tattgaaaag?ctgttaagcg?aataatgaaa?atctgaccga??????????780

atgtaacggt?tgataagaaa?attataacgg?cggtgaagat?tcgtggcgaa?agtaatttgt??????????840

tgcgaatatt?cctgccgtta?ttttatataa?acaatcagaa?taacaacgag?ttagcaatag??????????900

gattttagtc?aaagttttcc?aggattttcc?ttgtttccgt?agctgattgg?taagacaatt??????????960

agtgtttgaa? ?ttagcgcgag?tgggtaacgc?tcgtcacatc?gtaggcatgc?????1020

atgcagtgct?ctggtagctg?taaagccagg?ggcggtagcg?tgtctagggc?agggaaagat?????????1080

?tgcttttttg?tactaaacaa?tttgcatttt?atgttcaata?attgagacat??????1140

tccttattac?ctaaaactgt?ttttcattgc?ttatacatga?gcaaatactc?cttacataat?????????1200

taaggagaac?aaaattgaac?ttaaaaaatt?gatggaacat?atttctatta?tcctcgatta?????????1260

tagacaagct?aggaaagtga?aacataaatt?gtttgatatt?ctactgttga?ctatttgcgc????????1320

acttatttct?ggtgcagaag?gctgaaaaga?tatagatggt?ttttgggaaa?cgcatcttga????????1380

tttttttaac?

ttttgaaaat?ggtattccta?ttcactatac?cattgccaga????1440

?gtatcagttc?tacaaaattt?catgagtgtt?ttattaactg?gatgcgtgat?????1500

tgtcattctt?aagatgataa?agacgtcatt?gcaattgatg?gaaaaacgct?ccggcattct????????1560

tatgacaaga?gtcgtcgcag?gagagcgatt?catgtcattg?gtgcattctc?aactatgcac????????1620

agtctggtca?tcggacagat?caagacggat?gaacagaaac?tacctagcgt?cagtccttgc????????1680

ggggagcggg?ctttcgtaat?cttgcctgta?gcgttatttg?ttttcgagaa?taaatatata????????1740

atactccatg?tggatattta?gttaaaagag?gatgtggtga?aaacagattt?gactgtattt????????1800

aagaatatta?tatcgctatt?tagcattcgt?tttgctggct?acataatacc?tttaatcacg????????1860

ttgccgtatc?ttgtcaaggt?acttcaacct?gttggatttg?gatatttatc?atttggtatt????????1920

gctgttttac?aatattgtac?gctagttgtt?aattatgggt?ttgatctttc?agcaacaaga????????1980

aaaattgcac?agaatagaga?aaatgtgcat?tacatcagcg?atattttttg?gaatattctt????????2040

tttttgcggt?tgtttatttc?tcttgtaggt?tttttagtat?tagtggtttt?tatccattta????????2100

tttcctgatt?tgtatcgaat?taaatggatt?ttgctttggg?ggtatttgtc?tgttttgggg????????2160

acagctctct?ttccaatgtg?gttatttcag?gggaaagaac?agcttggacg?aatatcagca????????2220

gctcgtatat?ttttacagat?ttcttgcgta?cctttatttt?ttgtttttgt?aaaaaacact????????2280

caagatgatt?gggtcgcatc?actaatatat?gctctaccgc?agttgttaat?atcagtagtg????????2340

gcagtatata?tcattcataa?aaggaagtgg?attgtattaa?aaaaaataaa?acttgcaaca????????2400

atcaagaaag?aattaataga?tggttggcat?ctttttatat?ctacggcagc?aattagtctc????????2460

tatacaacaa?ccattactgt?agtcttaggg? gtccctatag?tgttgccata?????2520

tatacttctg?caaataaaat?attacaggcg?gctcaggggc?tttatcaacc?aatatcctct????????2580

gcattttacc?cgcgaataaa? aaagaaaata?aacaattagc?atttaaaact?????2640

ataagaaaac?tatttaaaat?acagtttgtt?attactattt?gcataagcgt?tgggttgttt????????2700

ttgttttcta?atattgttgt?tgatatttta?tacggtgatg?attatcgaaa?tgcggcatta????????2760

attcttcagc?tattatcacc?tattcctata?gttgtgggat?gtagtaacat?ttttggaata????????2820

caaatattat?taacatgtgg?ttataaaaag?gagttttcca?ctattttact?gatctcaggg????????2880

cttattaata?tgattttgat?atttcctctt?tgctataagt?ttaacgagtt?tggagcagca????????2940

ttttctgttt?taattacaga?gatatttgtt?actgtttcta?tgctgtttgt?ggttattctg????????3000

aaaaaaatac?cggtttggga?gagtaagcgt?aatgtatgat?tatttgattg?tcggcagcgg????????3060

gctttttggc?gcggtttgtg?caaatgagct?taaaaaaaga?aatgtcaaaa? ????3120

agataaacgc?aaccatgtag?gagggaatgt?ttataccgaa?aacattaatg?gaattcaagt????????3180

gcataaatat?ggtgcacata?tatttcatac?taatgataag?tatatttggg?agtatgtaaa????????3240

taaacttgtt?gagtttaatc?gttttacaaa?ttcacctcta?gcttatttta?aaggggaact????????3300

ttttaattta?ccatttaaca?tgaatacttt?ttatcagttg?tggggagtga?aaaaaccttc????????3360

agaagctgcc?gctattatag?

?aaaagtaatg?ctaaaaaaag?aacctacaaa????3420

tttggaagag?caggctattt?cactagttgg?agtagatatc?tatgagaaat?tgattaaggg????????3480

atataccgaa?aaacagtggg?gacgtagttg?taaagagtta?ccatccttca?ttattcgacg????????3540

actaccagta?agatttacat?ttgacaataa?ttatttttct?gacaaatatc?agggtattcc????????3600

tgttggggga?tatacgaatt?taattgaaaa?aatgcttgag?ggagttgagg?ttcatttagg????????3660

cattgactat?cttaaagaac?gcaagaaata?ttcgaaaatt?gcaaaaaaaa?tcatatatac????????3720

aggaccgatt?gatgagtatt?ttgataatgt?gctaggacag?ttggagtata?ggtcattaaa????????3780

atttcaaaca?gaaaaaatgg?atattcctaa?ttatcagggg?aatgcagtgg?ttaattacac????????3840

tgaaaaagaa?ataccattta?cgcgaattat?agaacataag?cattttgatt?ttgttgaatc????????3900

taattttacc?ataattacta?aagaataccc?taaagagtgg?agtaatggtg?atgagccata????????3960

ctaccctatc?aatgatgaga?aaaacatggc?tttatataga?aaatataaat?ctttggctga????????4020

tgaggaaaat?gtgatttttg?gtgggcgttt?ggctgaatat?aaatattatg?acatgcacca????????4080

agttattcgg?tctgccttaa?actgtgtgaa?gcatatttaa?ttattaagga?ggggggaggg????????4140

aggcatattt?ttatgaagct?atattatatc?tgttcggatt?tttatccttc?tggcactggt????????4200

tttgcaatat?cgtttacgaa?ttttatacag?tttatattag?attcaaataa?attcgagcat????????4260

atcacgatac?taactacaaa?ttctaaagca?tctttgcctg?aaaatttcaa?cgaacgtgtt????????4320

cttttgatgt?cagaggtgag?ttgaagttag?ctaggtactt?tagatcatct??????4380

attcttcttg?acttgtattc?aatatttaaa?tatagacgca?ttctgcatat?gttaaaatgt????????4440

tgttgtatta?agtctgatga?tgtattcttt?tatgaggagt?tttattttgg?caatctttat????????4500

aaatatttga?aaagacgctt?tccttgtaat?aagcatataa?tacgagtgca?tgggacaatg????????4560

ccagagtttg?tgacgtttga?tcgttcaaat?agatatagaa?aaatattatt?taaacaagct????????4620

ttagagctaa?ataacaatag?agaaaataaa?ataacaattg?caacgacaac?atctttttat????????4680

attgatttca?ttaatactca?tatacttaaa?aatcagtatg?gaacaatatg?taatgtagat????????4740

tatatttttc?taccaaatag?tatatatagt?aataaaaata?ttccgcagac?acaaaagtca????????4800

gaaaatatgt?ccaattcttt?aactttattg?cagttgggga?gaatggataa?aggtgggtac????????4860

ttccaaaaag?ggtttgatga?tacgattaaa?gcattaaatt?atattaattc?agatgttttt????????4920

ttatcccatc?ggatacgtct?tgttactatt?ggtagtggtg?agaaaaaaaa?atattttaaa????????4980

gataaaatga?gggatctaaa?aaacatagca?tttgaacatt?atgaaaatat?aaataatgaa????????5040

gctgttaatg?acttaataat?gcaggctgat?gttatactct?taccatcaag?atgtgaaggt????????5100

atgtcaatgt?ttgcagtcga?agctatttcg?ctaggaaaac?caataattac?gacaagaaat????????5160

actggagttg?atgatatatg?tattgaagga?gttaatagtc?tgaaatttga?tatgtttaat????????5220

tatgttgagt?atgcacaagc?tatcgaaaaa?ataatcaaag?aaccgcattt?gattagacaa????????5280

atgggatata?atgcttttgc?tgtttcaaaa?gaaaatgaaa?agaaattaaa?agcaaatata????????5340

gaggcctttt?tgtgatgatt?tatagatata?tattaaatct?atttcctgac?actctattta????????5400

ttagattgcg?atattttaaa?cgatttaagc?gtattttaca?cttgagaaaa?ccgaagcgat????????5460

ttactgagaa?aattatgtgg?tataaacttt?tttatcatga?taatttgatg?actatctgcg????????5520

cagataagta?tcgtgtaaga?gaatatataa?aggaaaaggg?gttcggtgat?atattggttc????????5580

cattgtttaa?agtatataat?actccagatg?aaataaaaat?tgatgaattg?ccttgtcaat????????5640

ttattttaaa?agggaataat?ggaagtgaga?ctaatttagt?tgttagggat?aagagtaaaa????????5700

taaccgagaa?gtatataagg?catatagttt?ctggatggat?ggctaataac?caagttatta????????5760

ctttggggaa?agagtggtgt?tatcagaata?ttgaatttaa?gataactgtg?gagcagctat????????5820

tagaatcaga?aggagaggat?ggcattgatg?actataaatt?tctttgtttt?aatggtcgcg????????5880

ttcattatat?atgggttgat?aaaaatcgat?ttacaagcca?caccagaact?ttttttgatc????????5940

gagactggaa?tgtgataaat?

atcatccagc?tgctagtttt?tctatagata?????6000

agccttgggg?? atgctacata?tcgctgaaga?tataggtaaa?gatttcccat????6060

ttgctagagt?tgatttttat?agtgtaaaca?aaaaggttta?ttttggtgag?ataacttttt????????6120

atccatggag?tggcatggtg?cagtttcaac?cagatagttt?tgattttgat?atgggtgata????????6180

aatttattct?gccaaaagag?atgttataat?gagtaagttg?tccgtaatta?tgtctgtata????????6240

ttcagagtcc?gaaaaaatgc?taagacaatc?tattgattct?gtattgaatc?agtcatttag????????6300

ggattttgaa?tttatcatag?ttaatgataa?cccagagaga?atcgagataa?tacagttgct????????6360

tcgttattat?atggaaaaag?ataatagggt?taaggtattt?tcaaatgaga?tcaataaagg????????6420

gttgacaaag?agtcttaata?ttgcagcaaa?aaaaagcaac?agtaaatata?ttgccagaat????????6480

ggacgctgat?gatatttctg?atattacgag?gtttgataga?cagttagata?tacttgagaa????????6540

aaatgatgat?atagatatat?gtggtagtaa?tgttaaggta?attaatgtcc?atgataatgt????????6600

tacagggcac?tataggtatc?ctgaaagatc?ttcaaaaata?aaggggaagt?tattctttcg????????6660

tgattgtctt?tgccatccag?ccacaatgtt?tcggcgttcg?tttttcattg?aggtcggcgg????????6720

atatagtgag?gattttaaaa?aagctcaaga?ctatgaactt?tggattaagg?

????6780

tgataaaggg?atatataata?ttcagactcc?cttattatac?tatcgtatgc?attctgaaaa????????6840

tatttcgaca?aaaaatattg?atgaacaaca?ttattattca?ataaaggcaa?aagtactact????????6900

gtattcattt?tttatggatc?agcagcaatc?attgaaaatc?atagattata?tagagggaca????????6960

gcaaggcaat?gttcatatta?tacgtgtgtt

?tttgttttag?ctgggcgatt?????7020

aaataaaaaa?attaatggtt?tttcatatat?atatttcttc?tttttattgg?tgaaaaggtt????????7080

gatttacata?aaaattataa?gacgtgctat?tttttagtag?agttaaaatc?cttttattat????????7140

atcctccttg?caatgagtta?ttaaatgaat?aacactttac?gacagtatag?tttagtgcaa????????7200

gtctttataa?caatgtactg?cctctttccc?ttctattttc?ataacatata? ????7260

actttttctt?tatattcatt?agcactaagc?tttgtagttt?atacgttttt?ctataaagtt????????7320

aggtttgttt?caaggtcagg?ggcaattaca?atccttgcta?gcacgacaac?atttttgatt????????7380

tttttattta?tgagcatgct?cattaatttg?tcgagcgata?tgacctttgt?ttatgaacaa????????7440

gtttttggtt?tttctaagca?gttactatta?gcattagttc?ctttttgtct?ctatttttcg????????7500

?atgatattaa?ttacgcagta?cacagtattt?tattgttgtt?tattagagcc????7560

ataagcttgt?atgtttttat?ttctgttata?tttctgatac?ctgaagttag?aaattattgg????????7620

attgatatta?tatattttag?tagtgatcaa?aataaggaat?taataaattc?tgcttcatac????????7680

tatacgcgct?ttggtttgca?aggattttct?ggattcagac?atgctattct?atgcgcgatt????????7740

gctctaattt?ttagtatata?tttatttatg?tgtggttata?aaaaaataaa?aaattattgg????????7800

atgagtgtgt?ttcttgtttg?tattgggtgt?ttcctatacg?gccgtattgc?tatagctgtg????????7860

atactatgta?taactttaat?atcattattt?tatttattat?gtaagtttga?aaaactttat????????7920

tttatattta?ttgtttgctt?atgcgtgggg?acgattgcat?gtgtagtata?tatgaatatt????????7980

gattatattc?aggatagtta?ttcattagct?tggattttcg?aaccattaat?aaattttatt????????8040

aatgaaggtc?agtttacaag?tcattcgaca?aatgatttat?ccagcatgta?tataatgccg????????8100

aatgaaaata?caatgttgtt?aggtgatggg?aaatatacta?atcatgatgg?aagttattat????????8160

atgcatacgg?atgttggatt?tttaaggcct?gttttttttg?gaggtatttt?ttttgttttc????????8220

ttttactata?tgatgttaat?attgccactt?atattaatta?ttaagaattc?tgggcttgaa??????????8280

cgaagaaata?tattaacatt?gtttttggct?ttggctttga?ttgtttttga?gttgaaggga??????????8340

gagtcattta?tatcattttc?

?ttttctttca?tactcattcc?tattatttgt?????8400

aaaaggttaa?attgatgaga?atatcattag?aagagaattc?agtagttttt?attctatatc??????????8460

aatttcatat?tggcggtgtc?gaaa8tgcac?taataaatct?gcttgagaat?attaaaaaga??????????8520

agagagtata?tttgatcttt?gcaatgaata?agatcaacaa?tgaactccta?aaaaaaattc??????????8580

ctcataacgt?caaaataatt?ggactaggaa?taaataataa?aaatatatgt?catttaattg??????????8640

gttatacacg?tagattaagc?aaacgatatg?attttagtag?aacacacatt?gttaattttt??????????8700

ctgataccct?atcaactcta?ataattcaat?attctttgaa?tgcaaaatta?aagacatcgt??????????8760

gggttcattg?caatccatta?actttgataa?actctcgttc?atttcagtta?tataagtatt??????????8820

tattagttag?aaatgataaa?atagtttgtt?tgtgtgaatc?acagaaaaat?ctcttaataa??????????8880

aggtgatgcc?caaagcggaa?aaaaaagtca?ccattatata?taattgtgtt?gatgctgata??????????8940

tcctttcaaa?aggagctcaa?gaaaaagttg?acgttgatta?taaatatata?ataaaagttg??????????9000

caagatttga?tgaaagaaca?aaagatttca?ttactcttat?agatgcatat?tgtaaactac??????????9060

ctaaagaaac?acaagatacg?tataaacttg?tattattagg?tgatggacct?gatctcgaaa??????????9120

atgttagaaa?ttatacaaga?aaaaaaaaca?acatgcaaaa?catatttttt?ccgggtaaag??????????9180

atgtcaatcc?ttttaaatgg?attaaacatt?cgcagttctt?ggtgcactcc?tcaacatcag??????????9240

agggcttttc?tcttgttctg?ttggaggcca?tgttattagg?taaagttgtg?attgcatctg??????????9300

attgtaattg?tggtccttct?gatatactgg?ataatgggaa?tgcaggattc?ctattcaatg??????????9360

ttggtgatgt?tgatagctta?acctgtatta?tgcaaaattt?attagttaat?caagacatca??????????9420

tcaatatata?taccaatcgc?tctcttgtta?gaggcgttga?attacagttg?aaagcaaata??????????9480

ggcaggttaa?ggagtatttt?ttgtgagtag?tgtatctgta?tgcattgtga?cttttaatcg??????????9540

taaaaatgac?ctgttgagat?gtctagataa?tgttcttaaa?cagtcacacg?ttgtcagtaa??????????9600

tatccttata?tatgataatg?catctactga?tgggaccact?cggtttctgc?ttagtaaata??????????9660

ttgcgctata?ttatctccat?caaatattcc?tcaaaagatt?gcaaccctaa?atgacataaa??????????9720

tatctggctt?gttacagcga?cagagaactc?tggaggtgct?ggcgggtttc?attattctgt??????????9780

?cgtgaaatat?tcgatactga?ttattattgg?ttaatggatg?acgatggtta??????9840

cccttctaaa?gattgtttaa?atattcttat?gcaatatgca?gagacgcata?attctgatta??????????9900

?gttagtatag?atattgataa?taatgaacag?ctatcttggc?caacacgaaa??????9960

aaaaaaccat?gcaaaaactg?aatgttataa?ggaattatat?gagtcgtggg?gagatgttat?????????10020

ggattatgtt?acacctttta?atggttctct?cttaagtaaa?aaatgtattg?atagtgttgg?????????10080

gtatgtgaat?aaagattttt?ttatttgggg?ggatgagtat?gaacattact?ggagatgcaa?????????10140

?attcatcctg?taacaattat?gaatgcaaaa?ttctatcacc?cgtcacaaaa??????10200

gttacctctt?cagccatgtt?tttttggatt?gattaaatta?ccctatgtcg?attctccatt?????????10260

acgtatggta?tgcctagttc?gaaattatac?atatatttat?aaaaaatatg?actcaaaatt?????????10320

aaaaataatt?ctcaagctta?tatcttatag?ttggtttttc?ttaataacaa?gaaagcttga?????????10380

?tttgtcttat?acttaaaaag?tgttcttgat?ggattaagaa?atgattttac??????10440

aagacataga?gattttttat?

?gatacatttc?ataaaaacat?aatgataatg?????10500

gataatcaat?ttgcatctta?attattttta?ctaccttccc?tgctcgataa?atatgtttat?????????10560

agggttaata?tgactgttat?tgctatagtt?gtaacattta?atcgctgtgc?gcttttgaaa?????????10620

aaagttttgc?attcgttgct?ttctcaatca?attgcattaa?ataaaataat?aatcattgat?????????10680

aatgatagta?atgatgatac?agctaaagtc?gtgcatgatt?tttctgaagt?ggatgatatt?????????10740

ttttattatt?ataacacagg?agataatctg?ggaggggctg?gagggtttta?tcaggggttt?????????10800

aaaatcgcag? ?ttatgactat?ctttggttaa?tggatgatga?ccttctacct?????10860

gagcctgatt?gcctagagaa?attaatccaa?gataggccag?aaggaattgt?tcagccggta?????????10920

agatacgatc?ttgatggtgc?ttgtgctgaa?atatcacctg?ttgagtataa?tcttcaaaaa?????????10980

atattttgca?ggaatcctaa?aaccaaaaca?gtaaaagaag?tgatttcaac?agttatttct?????????11040

gataattgtc?gggaaatcga?tattgcaggt?gttccttttg?aaggtccttt?aatttcaaag?????????11100

tcggtagtta?ataaagtggg?gtatccaaat?cctgattttt?ttatttttaa?tgatgatctt?????????11160

gattattcgt?taaggacgag?aagtaaaggt?ttctctataa?agtgtatagt?cgacgccagg?????????11220

gctacaaggc?ttttaaaaaa?taatcaaaaa?aatgatttga?aaagctggaa?aggatatttc?????????11280

atgttgagaa?atcattatta?tatcctacga?aactatggtg?aaaataagct?cgttaagaat?????????11340

cgagtctatc?tcatcatgtt?ttattatttt?ttgaagtcag?ttttttcttt?tgattataag?????????11400

ttcgcaaaag?tggttatttt?ttcttttaaa?gattcatttt?cgcttaaaaa?tagcaaaaga?????????11460

tttcgaccat?agtttttatt?tttggataaa?cgctgttatc?acaactgatc?ggagtaattc?????????11520

ttaaatgtca?aagcaacaga?tcggcgtcgt?cggtatggca?gtgatggggc?gcaaccttgc?????????11580

gctcaacatc?gaaagccgtg?gttataccgt?ctctattttc?aaccgttccc?gtgaaaagac?????????11640

ggaagaagtg?attgccgaaa?atccaggcaa?gaaactggtt?ccttactata?cggtgaaaga?????????11700

gtttgttgaa?tctctggaaa?cgcctcgtcg?catcctgtta?atggtgaaag?caggtgcagg???11760

cacggatgct?gctattgatt?ccctcaagcc?atacctcgat?aaaggtgaca?tcatcattga???11820

tggtggtaat?accttcttcc?aggacaccat?tcgtcgtaac?cgtgagcttt?ctgcagaagg???11880

ctttaacttc?atcggtaccg?gtgtttccgg?tggtgaagag?ggcgcgctga?aaggtccttc???11940

cattatgcct?ggtggccaga?aagaagccta?tgaactggtt?gctccggtcc?tgaccaaaat???12000

cgccgcagta?gctgaagacg?gtgagccatg?cgttacctat?attggtgccg?atggcgcagg???12060

tcactatgtg?aagatggttc?ataacggtat?tgaatacggt?gatatgcagc?tgattgctga???12120

agcctattct?ctgcttaaag?gtggcctgaa?cctcaccaac?gaagaattgg?cgcagacctt???12180

taccgagtgg?aataacggtg?aactgagcag?ctacctgatc?gacatcacca?aagatatctt???12240

caccaaaaaa?gatgaagacg?gtaactacct?ggttgatgtg?atcctggatg?aagctgctaa???12300

caaaggtacc?ggtaaatgga?ccagccagag?cgcgctggat?ctcggcgaac?cgctgtcgct???12360

gattaccgag?tctgtgtttg?cacgttatat?ctcttctctg?aaagatcagc?gtgttgccgc???12420

atctaaagtt?ctctctggcc?cgcaagcgca?gccagctggc?gacaaaggtg?agttcatcga???12480

aaaagttcgt?cgtgctctgt?atctgggcaa?aatcgtttct?tacgcgcagg?gcttctctca???12540

gctgcgtgct?gcgtctgaag?agtacaactg?gaatctgaac?tacggcgaaa?tcgcgaagat???12600

tttccgtgct?ggctgcatca?tccgtgcgca?gttcctgcag?aaaatcaccg?atgcatatac???12660

agaaaatccg?cagatcgcta?acctgctgct?ggctccgtac?ttcaagcaaa?tcgccgatga???12720

ctaccagcag?gcgctgcgtg?atgtcgttgc?ttatgcggta?cagaacggta?tcccggttcc???12780

gaccttcgcc?gctgcggttg?cctattatga?cagctaccgt?gccgctgttc?tgcctgcgaa???12840

cttgatccag?gcacagcgtg?acta??????????????????????????????????????????12864

It in the sequence list nucleotide sequence of the O antigen gene bunch of intestinal bacteria O167, it is the same that preceding 12199 nucleotides sequence of the nucleotide sequence of the O antigen gene of Shigella bogdii 3 types bunch and the O antigen gene of intestinal bacteria O167 bunch shows 99.78%, different base box indicating is the Nucleotide of Shigella bogdii 3 types above this base; And 665 bases of the O antigen gene bunch back of intestinal bacteria O167 and O antigen gene are irrelevant, so not survey in Shigella bogdii 3 types.

Oligosaccharide unit treatment gene in the O-antigen gene of table 1 intestinal bacteria O167 type and Shigella bogdii 3 types bunch and wherein Primer and PCR data

Gene	Function	The base position of gene	Forward primer	Reverse primer	The length of PCR product	Produce the group number of correct big or small electrophoresis band	The annealing temperature of PCR (℃)
								??wzx	The transhipment enzyme	???1774-3039	??2281-2300	??2925-2944	??664bp	???0 *	???65
			??1973-1992	??2863-2882	??910bp	???0 *	???63.6
								??wzy	Polysaccharase	???7165-8415	??7318-7337	??8047-8066	??749bp	???0 *	???65
			??7667-7686	??8168-8184	??518bp	???0 *	???60.9

^*Only in intestinal bacteria O167 type and Shigella bogdii 3 types, obtain a correct band

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

The bacterium source that contains in this group of group number

1, wild-type e. coli O1, O2, O5, O7, O8, O9, O12, O13, O14, O15, O16, O17, O18, O19ab, IMVS ^a

O20，O21，O22，O23，O24

2, wild-type e. coli O4, O10, O25, O26, O27, O28, O29, O30, O32, O33, O34, O35, IMVS ^a

O36，O37，O38，O40，O41，O42，O43

3, wild-type e. coli O6, O44, O45, O46, O48, O49, O50, O51, O52, O54, O55, O56, IMVS ^a

O57，O58，O60，O61，O62，O53

4, wild-type e. coli O63, O65, O66, O69, O70, O71, O74, O75, O76, O77, O78, IMVS ^a

O79，O80，O81，O82，O83，O68

5, wild-type e. coli O84, O85, O86, O87, O88, O89, O90, O91, O92, O98, O99, IMVS ^a

O101，O102，O103，O104，O105，O106，O97

6, wild-type e. coli O107, O108, O109, O110, O111, O11ab, O112ac, O113, IMVS ^a

O115，O116，O118，O120，O123，O125，O126，O128，O117

7, wild-type e. coli O129, O130, O131, O132, O133, O134, O135, O136, O137, IMVS ^a

O138，O139，O141，O142，O143，O144，O145，O140

8, wild-type e. coli O146 O147, O148, O150, O152, O154, O156, O157, O158, IMVS ^a

O159，O160，O161，O163，O164，O165，O166，O153?????????????????????????b

9, wild-type e. coli O168, O169, O170, O171, O172, O173, c

Shigella dysenteriae D1, D2, D3, D4, D5 D6, D7, D8, D9, D10, D11, D12, D13 d

10, Shigella bogdii B1, B2, B3, B4, B6, B7, B8, B9, B10, B11, B12, B13, B14, B15, d

B16，B17，B18

11, shigella flexneri F1a, F1b, F2a, F2b, F3, F4a, F4b, F5 (v:4), F5 (v:7), F6, d

DS，DR

12, wild-type e. coli O3, O11, O39, O59, O64, O73, O96, O95, O100, O114, O151, O155, IMVS ^a

O124，O162，O121，O127，O149，O119

13, the 10th group of bacterial strain adds Shigella bogdii bacterium reference culture 3 type IMVS ^a

14, the 12nd group of bacterial strain adds intestinal bacteria reference culture O167

a.?Institute?of?MediCxcal?and?Veterinary?Science(IMVS)，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 intestinal bacteria O167 type and Shigella bogdii 3 type O-antigen gene structure iron

Escherichia?coli?O167?O?antigen?gene?cluster

orf#??galF??H-repeat???wzx??????glf????orf3??????orf4?????orf5??????wzy??????orf7??????orf8?????orf9???gnd

G+C％????????????????????32.1?????32.6???28.8??????31.5?????30.0??????28.3??????30.0??????31.7?????32.3

Table 4 intestinal bacteria O167 type and Shigella bogdii 3 type O-antigen gene cluster gene positions

ATTGTGGCTG?CAGGGATCAA?AGAAATCCTC?CTGGTAACTC?ACGCGTCCAA?GAACGCGGTC???????????60

GAAAACCACT?TCGACACCTC?

?GAATCTCTCC?TTGAACAGCG?CGTGAAGCGT??????120

CAACTACTGG?CGGAAGTGCA?GTCCATCTGT?CCGCCGGGCG?TGACCATTAT?GAACGTGCGT???????????180

CAGGGCGAAC?CTTTAGGTTT?GGGCCACTCC?ATTTTGTGTG?CACGACCTGC?CATTGGTGAC???????????240

AACCCATTTG?TCGTGGTGCT?GCCAGACGTT?GTGATCGATG?ACGCCAGCGC?CGACCCGCTG???????????300

CGCTACAATC?TTGCTGCCAT?GATTGCGCGC?TTCAACGAAA?CGGGCCGCAG?CCAGGTGCTG???????????360

GCAAAACGTA?TGCCGGGTGA?CCTCTCTGAA?TACTCCGTCA?TTCAGACCAA?AGAACCGCTG???????????420

GATCGTGAAG?GTAAAGTCAG?CCGCATTGTT?GAATTTATCG?AAAAACCGGA?TCAGCCGCAG???????????480

ACGCTGGACT?CAGATATTAT?GGCCGTTGGT?CGCTATGTGC?TTTCTGCCGA?TATTTGGCCG???????????540

GAACTTGAAC?GTACTCAGCC?TGGTGCATGG?GGGCGTATTC?AGCTGACTGA?TGCCATTGCC???????????600

GAACTGGCGA?AAAAACAGTC?CGTTGATGCA?ATGCTGATGA?CTGGTGACAG?CTACGACTGC???????????660

GGTAAAAAAA?TGGGCTATAT?GCAGGCGTTT?GTGAAGTATG?GACTGCGCAA?CCTGAAAGAA???????????720

GGGGCGAAGT?TCCGTAAAGG?TATTGAAAAG?CTGTTAAGCG?AATAATGAAA?ATCTGACCGA???????????780

ATGTAACGGT?TGATAAGAAA?ATTATAACGG?CGGTGAAGAT?TCGTGGCGAA?AGTAATTTGT???????????840

TGCGAATATT?CCTGCCGTTA?TTTTATATAA?ACAATCAGAA?TAACAACGAG?TTAGCAATAG???????????900

GATTTTAGTC?AAAGTTTTCC?AGGATTTTCC?TTGTTTCCGT?AGCTGATTGG?TAAGACAATT???????????960

AGTGTTTGAA?

?TTAGCGCGAG?TGGGTAACGC?TCGTCACATC?GTAGGCATGC?????1020

ATGCAGTGCT?CTGGTAGCTG?TAAAGCCAGG?GGCGGTAGCG?TGTCTAGGGC?AGGGAAAGAT??????????1080

?TGCTTTTTTG?TACTAAACAA?TTTGCATTTT?ATGTTCAATA?ATTGAGACAT???????1140

TCCTTATTAC?CTAAAACTGT?TTTTCATTGC?TTATACATGA?GCAAATACTC?CTTACATAAT??????????1200

TAAGGAGAAC?AAAATTGAAC?TTAAAAAATT?GATGGAACAT?ATTTCTATTA?TCCTCGATTA??????????1260

TAGACAAGCT?AGGAAAGTGA?AACATAAATT?GTTTGATATT?CTACTGTTGA?CTATTTGCGC??????????1320

ACTTATTTCT?GGTGCAGAAG?GCTGAAAAGA?TATAGATGGT?TTTTGGGAAA?CGCATCTTGA??????????1380

TTTTTTTAAC? ?TTTTGAAAAT?GGTATTCCTA?TTCACTATAC?CATTGCCAGA?????1440

?GTATCAGTTC?TACAAAATTT?CATGAGTGTT?TTATTAACTG?GATGCGTGAT???????1500

TGTCATTCTT?AAGATGATAA?AGACGTCATT?GCAATTGATG?GAAAAACGCT?CCGGCATTCT??????????1560

TATGACAAGA?GTCGTCGCAG?GAGAGCGATT?CATGTCATTG?GTGCATTCTC?AACTATGCAC??????????1620

AGTCTGGTCA?TCGGACAGAT?CAAGACGGAT?GAACAGAAAC?TACCTAGCGT?CAGTCCTTGC??????????1680

GGGGAGCGGG?CTTTCGTAAT?CTTGCCTGTA?GCGTTATTTG?TTTTCGAGAA?TAAATATATA??????????1740

Orf1's is initial

ATACTCCATG?TGGATATTTA?GTTAAAAGAG?GAT GTGGTGA?AAACAGATTT?GACTGTATTT????1800

AAGAATATTA?TATCGCTATT?TAGCATTCGT?TTTGCTGGCT?ACATAATACC?TTTAATCACG??????1860

TTGCCGTATC?TTGTCAAGGT?ACTTCAACCT?GTTGGATTTG?GATATTTATC?ATTTGGTATT??????1920

GCTGTTTTAC?AATATTGTAC?GCTAGTTGTT?AATTATGGGT?TTGATCTTTC?AGCAACAAGA??????1980

AAAATTGCAC?AGAATAGAGA?AAATGTGCAT?TACATCAGCG?ATATTTTTTG?GAATATTCTT??????2040

TTTTTGCGGT?TGTTTATTTC?TCTTGTAGGT?TTTTTAGTAT?TAGTGGTTTT?TATCCATTTA??????2100

TTTCCTGATT?TGTATCGAAT?TAAATGGATT?TTGCTTTGGG?GGTATTTGTC?TGTTTTGGGG??????2160

ACAGCTCTCT?TTCCAATGTG?GTTATTTCAG?GGGAAAGAAC?AGCTTGGACG?AATATCAGCA??????2220

GCTCGTATAT?TTTTACAGAT?TTCTTGCGTA?CCTTTATTTT?TTGTTTTTGT?AAAAAACACT??????2280

CAAGATGATT?GGGTCGCATC?ACTAATATAT?GCTCTACCGC?AGTTGTTAAT?ATCAGTAGTG??????2340

GCAGTATATA?TCATTCATAA?AAGGAAGTGG?ATTGTATTAA?AAAAAATAAA?ACTTGCAACA??????2400

ATCAAGAAAG?AATTAATAGA?TGGTTGGCAT?CTTTTTATAT?CTACGGCAGC?AATTAGTCTC??????2460

TATACAACAA?CCATTACTGT?AGTCTTAGGG? ?GTCCCTATAG?TGTTGCCATA??2520

TATACTTCTG?CAAATAAAAT?ATTACAGGCG?GCTCAGGGGC?TTTATCAACC?AATATCCTCT??????2580

GCATTTTACC?CGCGAATAAA?

?AAAGAAAATA?AACAATTAGC?ATTTAAAACT?2640

ATAAGAAAAC?TATTTAAAAT?ACAGTTTGTT?ATTACTATTT?GCATAAGCGT?TGGGTTGTTT??????2700

TTGTTTTCTA?ATATTGTTGT?TGATATTTTA?TACGGTGATG?ATTATCGAAA?TGCGGCATTA??????2760

ATTCTTCAGC?TATTATCACC?TATTCCTATA?GTTGTGGGAT?GTAGTAACAT?TTTTGGAATA??????2820

CAAATATTAT?TAACATGTGG?TTATAAAAAG?GAGTTTTCCA?CTATTTTACT?GATCTCAGGG??????2880

CTTATTAATA?TGATTTTGAT?ATTTCCTCTT?TGCTATAAGT?TTAACGAGTT?TGGAGCAGCA??????2940

TTTTCTGTTT?TAATTACAGA?GATATTTGTT?ACTGTTTCTA?TGCTGTTTGT?GGTTATTCTG??????3000

The termination of the initial orf1 of orf2

AAAAAAATAC?CGGTTTGGGA?GAGTAAGCGT?A ATGTA TGAT?TATTTGATTG?TCGGCAGCGG??3060

GCTTTTTGGC?GCGGTTTGTG?CAAATGAGCT?TAAAAAAAGA?AATGTCAAAA?

?3120

AGATAAAGGC?AACCATGTAG?GAGGGAATGT?TTATACCGAA?AACATTAATG?GAATTCAAGT??????3180

GCATAAATAT?GGTGCACATA?TATTTCATAC?TAATGATAAG?TATATTTGGG?AGTATGTAAA??????3240

TAAACTTGTT?GAGTTTAATC?GTTTTACAAA?TTCACCTCTA?GCTTATTTTA?AAGGGGAACT??????3300

TTTTAATTTA?CCATTTAACA?TGAATACTTT?TTATCAGTTG?TGGGGAGTGA?AAAAACCTTC??????3360

AGAAGCTGCC?GCTATTATAG? ?AAAAGTAATG?CTAAAAAAAG?AACCTACAAA?3420

TTTGGAAGAG?CAGGCTATTT?CACTAGTTGG?AGTAGATATC?TATGAGAAAT?TGATTAAGGG??????3480

ATATACCGAA?AAACAGTGGG?GACGTAGTTG?TAAAGAGTTA?CCATCCTTCA?TTATTCGACG??????3540

ACTACCAGTA?AGATTTACAT?TTGACAATAA?TTATTTTTCT?GACAAATATC?AGGGTATTCC??????3600

TGTTGGGGGA?TATACGAATT?TAATTGAAAA?AATGCTTGAG?GGAGTTGAGG?TTCATTTAGG??????3660

CATTGACTAT?CTTAAAGAAC?GCAAGAAATA?TTCGAAAATT?GCAAAAAAAA?TCATATATAC??????3720

AGGACCGATT?GATGAGTATT?TTGATAATGT?GCTAGGACAG?TTGGAGTATA?GGTCATTAAA??????3780

ATTTCAAACA?GAAAAAATGG?ATATTCCTAA?TTATCAGGGG?AATGCAGTGG?TTAATTACAC??????3840

TGAAAAAGAA?ATACCATTTA?CGCGAATTAT?AGAACATAAG?CATTTTGATT?TTGTTGAATC??????3900

TAATTTTACC?ATAATTACTA?AAGAATACCC?TAAAGAGTGG?AGTAATGGTG?ATGAGCCATA??????3960

CTACCCTATC?AATGATGAGA?AAAACATGGC?TTTATATAGA?AAATATAAAT?CTTTGGCTGA??????4020

TGAGGAAAAT?GTGATTTTTG?GTGGGCGTTT?GGCTGAATAT?AAATATTATG?ACATGCACCA??????4080

The termination of orf2

AGTTATTCGG?TCTGCCTTAA?ACTGTGTGAA?GCATATT TAA?TTATTAAGGA?GGGGGGAGGG????4140

Orf3's is initial

AGGCATATTT?TT ATGAAGCT?ATATTATATC?TGTTCGGATT?TTTATCCTTC?TGGCACTGGT????4200

TTTGCAATAT?CGTTTACGAA?TTTTATACAG?TTTATATTAG?ATTCAAATAA?ATTCGAGCAT??????4260

ATCACGATAC?TAACTACAAA?TTCTAAAGCA?TCTTTGCCTG?AAAATTTCAA?CGAACGTGTT??????4320

?CTTTTGATGT?CAGAGGTGAG?TTGAAGTTAG?CTAGGTACTT?TAGATCATCT4380

ATTCTTCTTG?ACTTGTATTC?AATATTTAAA?TATAGACGCA?TTCTGCATAT?GTTAAAATGT??????4440

TGTTGTATTA?AGTCTGATGA?TGTATTCTTT?TATGAGGAGT?TTTATTTTGG?CAATCTTTAT??????4500

AAATATTTGA?AAAGACGCTT?TCCTTGTAAT?AAGCATATAA?TACGAGTGCA?TGGGACAATG??????4560

CCAGAGTTTG?TGACGTTTGA?TCGTTCAAAT?AGATATAGAA?AAATATTATT?TAAACAAGCT??????4620

TTAGAGCTAA?AAAACAATAG?AGAAAATAAA?ATAACAATTG?CAACGACAAC?ATCTTTTTAT??????4680

ATTGATTTCA?TTAATACTCA?TATACTTAAA?AATCAGTATG?GAACAATATG?TAATGTAGAT??????4740

TATATTTTTC?TACCAAATAG?TATATATAGT?AATAAAAATA?TTCCGCAGAC?ACAAAAGTCA????????4800

GAAAATATGT?CCAATTCTTT?AACTTTATTG?CAGTTGGGGA?GAATGGATAA?AGGTGGGTAC????????4860

TTCCAAAAAG?GGTTTGATGA?TACGATTAAA?GCATTAAATT?ATATTAATTC?AGATGTTTTT????????4920

TTATCCCATC?GGATACGTCT?TGTTACTATT?GGTAGTGGTG?AGAAAAAAAA?ATATTTTAAA????????4980

GATAAAATGA?GGGATCTAAA?AAACATAGCA?TTTGAACATT?ATGAAAATAT?AAATAATGAA????????5040

GCTGTTAATG?ACTTAATAAT?GCAGGCTGAT?GTTATACTCT?TACCATCAAG?ATGTGAAGGT????????5100

ATGTCAATGT?TTGCAGTCGA?AGCTATTTCG?CTAGGAAAAC?CAATAATTAC?GACAAGAAAT????????5160

ACTGGAGTTG?ATGATATATG?TATTGAAGGA?GTTAATAGTC?TGAAATTTGA?TATGTTTAAT????????5220

TATGTTGAGT?ATGCACAAGC?TATCGAAAAA?ATAATCAAAG?AACCGCATTT?GATTAGACAA????????5280

ATGGGATATA?ATGCTTTTGC?TGTTTCAAAA?GAAAATGAAA?AGAAATTAAA?AGCAAATATA????????5340

The termination orf4's of orf3 is initial

GAGGCCTTTT?TG TGATGATT?TATAGATATA?TATTAAATCT?ATTTCCTGAC?ACTCTATTTA??????5400

TTAGATTGCG?ATATTTTAAA?CGATTTAAGC?GTATTTTACA?CTTGAGAAAA?CCGAAGCGAT????????5460

TTACTGAGAA?AATTATGTGG?TATAAACTTT?TTTATCATGA?TAATTTGATG?ACTATCTGCG????????5520

CAGATAAGTA?TCGTGTAAGA?GAATATATAA?AGGAAAAGGG?GTTCGGTGAT?ATATTGGTTC????????5580

CATTGTTTAA?AGTATATAAT?ACTCCAGATG?AAATAAAAAT?TGATGAATTG?CCTTGTCAAT????????5640

TTATTTTAAA?AGGGAATAAT?GGAAGTGAGA?CTAATTTAGT?TGTTAGGGAT?AAGAGTAAAA????????5700

TAACCGAGAA?GTATATAAGG?CATATAGTTT?CTGGATGGAT?GGCTAATAAC?CAAGTTATTA????????5760

CTTTGGGGAA?AGAGTGGTGT?TATCAGAATA?TTGAATTTAA?GATAACTGTG?GAGCAGCTAT????????5820

TAGAATCAGA?AGGAGAGGAT?GGCATTGATG?ACTATAAATT?TCTTTGTTTT?AATGGTCGCG????????5880

TTCATTATAT?ATGGGTTGAT?AAAAATCGAT?TTACAAGCCA?CACCAGAACT?TTTTTTGATC????????5940

GAGACTGGAA?TGTGATAAAT?

?ATCATCCAGC?TGCTAGTTTT?TCTATAGATA???6000

AGCCTTGGGG?

?ATGCTACATA?TCGCTGAAGA?TATAGGTAAA?GATTTCCCAT????6060

TTGCTAGAGT?TGATTTTTAT?AGTGTAAACA?AAAAGGTTTA?TTTTGGTGAG?ATAACTTTTT????????6120

ATCCATGGAG?TGGCATGGTG?CAGTTTCAAC?CAGATAGTTT?TGATTTTGAT?ATGGGTGATA????????6180

The termination orf5's of orf4 is initial

AATTTATTCT?GCCAAAAGAG?ATGTTA TAAT?GAGTAAGTTG?TCCGTAATTA?TGTCTGTATA??????6240

TTCAGAGTCC?GAAAAAATGC?TAAGACAATC?TATTGATTCT?GTATTGAATC?AGTCATTTAG????????6300

GGATTTTGAA?TTTATCATAG?TTAATGATAA?CCCAGAGAGA?ATCGAGATAA?TACAGTTGCT????????6360

TCGTTATTAT?ATGGAAAAAG?ATAATAGGGT?TAAGGTATTT?TCAAATGAGA?TCAATAAAGG????????6420

GTTGACAAAG?AGTCTTAATA?TTGCAGCAAA?AAAAAGCAAC?AGTAAATATA?TTGCCAGAAT????????6480

GGACGCTGAT?GATATTTCTG?ATATTACGAG?GTTTGATAGA?CAGTTAGATA?TACTTGAGAA????????6540

AAATGATGAT?ATAGATATAT?GTGGTAGTAA?TGTTAAGGTA?ATTAATGTCC?ATGATAATGT????????6600

TACAGGGCAC?TATAGGTATC?CTGAAAGATC?TTCAAAAATA?AAGGGGAAGT?TATTCTTTCG????????6660

TGATTGTCTT?TGCCATCCAG?CCACAATGTT?TCGGCGTTCG?TTTTTCATTG?AGGTCGGCGG????????6720

ATATAGTGAG?GATTTTAAAA?AAGCTCAAGA?CTATGAACTT?TGGATTAAGG? ???6780

TGATAAAGGG?ATATATAATA?TTCAGACTCC?CTTATTATAC?TATCGTATGC?ATTCTGAAAA????????6840

TATTTCGACA?AAAAATATTG?ATGAACAACA?TTATTATTCA?ATAAAGGCAA?AAGTACTACT????????6900

GTATTCATTT?TTTATGGATC?AGCAGCAATC?ATTGAAAATC?ATAGATTATA?TAGAGGGACA????????6960

GCAAGGCAAT?GTTCATATTA?TACGTGTGTT? ?TTTGTTTTAG?CTGGGCGATT????7020

AAATAAAAAA?ATTAATGGTT?TTTCATATAT?ATATTTCTTC?TTTTTATTGG?TGAAAAGGTT????????7080

The termination of orf5

GATTTACATA?AAAATTATAA?GACGTGCTAT?TTTT TAGTAG?AGTTAAAATC?CTTTTATTAT??????7140

Orf6's is initial

ATCCTCCTTG?CAATGAGTTA?TTAA ATGAAT?AACACTTTAC?GACAGTATAG?TTTAGTGCAA??????7200

GTCTTTATAA?CAATGTACTG?CCTCTTTCCC?TTCTATTTTC?ATAACATATA?

????7260

ACTTTTTCTT?TATATTCATT?AGCACTAAGC?TTTGTAGTTT?ATACGTTTTT?CTATAAAGTT????????7320

AGGTTTGTTT?CAAGGTCAGG?GGCAATTACA?ATCCTTGCTA?GCACGACAAC?ATTTTTGATT????????7380

TTTTTATTTA?TGAGCATGCT?CATTAATTTG?TCGAGCGATA?TGACCTTTGT?TTATGAACAA????????7440

GTTTTTGGTT?TTTCTAAGCA?GTTACTATTA?GCATTAGTTC?CTTTTTGTCT?CTATTTTTCG????????7500

?ATGATATTAA?TTACGCAGTA?CACAGTATTT?TATTGTTGTT?TATTAGAGCC?????7560

ATAAGCTTGT?ATGTTTTTAT?TTCTGTTATA?TTTCTGATAC?CTGAAGTTAG?AAATTATTGG????????7620

ATTGATATTA?TATATTTTAG?TAGTGATCAA?AATAAGGAAT?TAATAAATTC?TGCTTCATAC????????7680

TATACGCGCT?TTGGTTTGCA?GGGATTTTCT?GGATTCAGAC?ATGCTATTCT?ATGCGCGATT????????7740

GCTCTAATTT?TTAGTATATA?TTTATTTATG?TGTGGTTATA?AAAAAATAAA?AAATTATTGG????????7800

ATGAGTGTGT?TTCTTGTTTG?TATTGGGTGT?TTCCTATACG?GCCGTATTGC?TATAGCTGTG????????7860

ATACTATGTA?TAACTTTAAT?ATCATTATTT?TATTTATTAT?GTAAGTTTGA?AAAACTTTAT????????7920

TTTATATTTA?TTGTTTGCTT?ATGCGTGGGG?ACGATTGCAT?GTGTAGTATA?TATGAATATT????????7980

GATTATATTC?AGGATAGTTA?TTCATTAGCT?TGGATTTTCG?AACCATTAAT?AAATTTTATT????????8040

AATGAAGGTC?AGTTTACAAG?TCATTCGACA?AATGATTTAT?CCAGCATGTA?TATAATGCCG????????8100

AATGAAAATA?CAATGTTGTT?AGGTGATGGG?AAATATACTA?ATCATGATGG?AAGTTATTAT????????8160

ATGCATACGG?ATGTTGGATT?TTTAAGGCCT?GTTTTTTTTG?GAGGTATTTT?TTTTGTTTTC????????8220

TTTTACTATA?TGATGTTAAT?ATTGCCACTT?ATATTAATTA?TTAAGAATTC?TGGGCTTGAA????????8280

CGAAGAAATA?TATTAACATT?GTTTTTGGCT?TTGGCTTTGA?TTGTTTTTGA?GTTGAAGGGA????????8340

GAGTCATTTA?TATCATTTTC?

?TTTTCTTTCA?TACTCATTCC?TATTATTTGT???8400

The termination orf7's of orf6 is initial

AAAAGGTTAA?AT TGATGAGA?ATATCATTAG?AAGAGAATTC?AGTAGTTTTT?ATTCTATATC??????8460

AATTTCATAT?TGGCGGTGTC?GAAAGTGCAC?TAATAAATCT?GCTTGAGAAT?ATTAAAAAGA????????8520

AGAGAGTATA?TTTGATCTTT?GCAATGAATA?AGATCAACAA?TGAACTCCTA?AAAAAAATTC????????8580

CTCATAACGT?CAAAATAATT?GGACTAGGAA?TAAATAATAA?AAATATATGT?CATTTAATTG????????8640

GTTATACACG?TAGATTAAGC?AAACGATATG?ATTTTAGTAG?AACACACATT?GTTAATTTTT????????8700

CTGATACCCT?ATCAACTCTA?ATAATTCAAT?ATTCTTTGAA?TGCAAAATTA?AAGACATCGT????????8760

GGGTTCATTG?CAATCCATTA?ACTTTGATAA?ACTCTCGTTC?ATTTCAGTTA?TATAAGTATT????????8820

TATTAGTTAG?AAATGATAAA?ATAGTTTGTT?TGTGTGAATC?ACAGAAAAAT?CTCTTAATAA????????8880

AGGTGATGCC?CAAAGCGGAA?AAAAAAGTCA?CCATTATATA?TAATTGTGTT?GATGCTGATA????????8940

TCCTTTCAAA?AGGAGCTCAA?GAAAAAGTTG?ACGTTGATTA?TAAATATATA?ATAAAAGTTG????????9000

CAAGATTTGA?TGAAAGAACA?AAAGATTTCA?TTACTCTTAT?AGATGCATAT?TGTAAACTAC????????9060

CTAAAGAAAC?ACAAGATACG?TATAAACTTG?TATTATTAGG?TGATGGACCT?GATCTCGAAA????????9120

ATGTTAGAAA?TTATACAAGA?AAAAAAAACA?ACATGCAAAA?CATATTTTTT?CCGGGTAAAG????????9180

ATGTCAATCC?TTTTAAATGG?ATTAAACATT?CGCAGTTCTT?GGTGCACTCC?TCAACATCAG????????9240

AGGGCTTTTC?TCTTGTTCTG?TTGGAGGCCA?TGTTATTAGG?TAAAGTTGTG?ATTGCATCTG????????9300

ATTGTAATTG?TGGTCCTTCT?GATATACTGG?ATAATGGGAA?TGCAGGATTC?CTATTCAATG????????9360

TTGGTGATGT?TGATAGCTTA?ACCTGTATTA?TGCAAAATTT?ATTAGTTAAT?CAAGACATCA????????9420

TCAATATATA?TACCAATCGC?TCTCTTGTTA?GAGGCGTTGA?ATTACAGTTG?AAAGCAAATA????????9480

The termination of the initial orf7 of orf8

GGCAGGTTAA?GGAGTATTTT?T TGTGAGTAG?TGTATCTGTA?TGCATTGTGA?CTTTTAATCG??????9540

TAAAAATGAC?CTGTTGAGAT?GTCTAGATAA?TGTTCTTAAA?CAGTCACACG?TTGTCAGTAA????????9600

TATCCTTATA?TATGATAATG?CATCTACTGA?TGGGACCACT?CGGTTTCTGC?TTAGTAAATA????????9660

TTGCGCTATA?TTATCTCCAT?CAAATATTCC?TCAAAAGATT?GCAACCCTAA?ATGACATAAA????????9720

TATCTGGCTT?GTTACAGCGA?CAGAGAACTC?TGGAGGTGCT?GGCGGGTTTC?ATTATTCTGT????????9780

?CGTGAAATAT?TCGATACTGA?TTATTATTGG?TTAATGGATG?ACGATGGTTA?????9840

CCCTTCTAAA?GATTGTTTAA?ATATTCTTAT?GCAATATGCA?GAGACGCATA?ATTCTGATTA????????9900

?GTTAGTATAG?ATATTGATAA?TAATGAACAG?CTATCTTGGC?CAACACGAAA????9960

AAAAAACCAT?GCAAAAACTG?AATGTTATAA?GGAATTATAT?GAGTCGTGGG?GAGATGTTAT???????10020

GGATTATGTT?ACACCTTTTA?ATGGTTCTCT?CTTAAGTAAA?AAATGTATTG?ATAGTGTTGG???????10080

GTATGTGAAT?AAAGATTTTT?TTATTTGGGG?GGATGAGTAT?GAACATTACT?GGAGATGCAA???????10140

?ATTCATCCTG?TAACAATTAT?GAATGCAAAA?TTCTATCACC?CGTCACAAAA???10200

GTTACCTCTT?CAGCCATGTT?TTTTTGGATT?GATTAAATTA?CCCTATGTCG?ATTCTCCATT???????10260

ACGTATGGTA?TGCCTAGTTC?GAAATTATAC?ATATATTTAT?AAAAAATATG?ACTCAAAATT???????10320

AAAAATAATT?CTCAAGCTTA?TATCTTATAG?TTGGTTTTTC?TTAATAACAA?GAAAGCTTGA???????10380

?TTTGTCTTAT?ACTTAAAAAG?TGTTCTTGAT?GGATTAAGAA?ATGATTTTAC????10440

The termination G of orf8

AAGACATAGA?GATTTTTTA T ?GATACATTTC?ATAAAAACAT?AATGATAATG??10500

GATAATCAAT?TTGCATCTTA?ATTATTTTTA?CTACCTTCCC?TGCTCGATAA?ATATGTTTAT???????10560

AGGGTTAATA?TGACTGTTAT?TGCTATACTT?GTAACATTTA?ATCGCTGTGC?GCTTTTGAAA???????10620

AAAGTTTTGC?ATTCGTTGCT?TTCTCAATCA?ATTGCATTAA?ATAAAATAAT?AATCATTGAT???????10680

AATGATAGTA?ATGATGATAC?AGCTAAAGTC?GTGCATGATT?TTTCTGAAGT?GGATGATATT???????10740

TTTTATTATT?ATAACACAGG?AGATAATCTG?GGAGGGGCTG?GAGGGTTTTA?TCAGGGGTTT???????10800

AAAATCGCAG? ?TTATGACTAT?CTTTGGTTAA?TGGATGATGA?CCTTCTACCT??10860

GAGCCTGATT?GCCTAGAGAA?ATTAATCCAA?GATAGGCCAG?AAGGAATTGT?TCAGCCGGTA???????10920

AGATACGATC?TTGATGGTGC?TTGTGCTGAA?ATATCACCTG?TTGAGTATAA?TCTTCAAAAA???????10980

ATATTTTGCA?GGAATCCTAA?AACCAAAACA?GTAAAAGAAG?TGATTTCAAC?AGTTATTTCT???????11040

GATAATTGTC?GGGAAATCGA?TATTGCAGGT?GTTCCTTTTG?AAGGTCCTTT?AATTTCAAAG???????11100

TCGGTAGTTA?ATAAAGTGGG?GTATCCAAAT?CCTGATTTTT?TTATTTTTAA?TGATGATCTT???????11160

GATTATTCGT?TAAGGACGAG?AAGTAAACGT?TTCTCTATAA?AGTGTATAGT?CGACGCCAGG???????11220

GCTACAAGGC?TTTTAAAAAA?TAATCAAAAA?AATGATTTGA?AAAGCTGGAA?AGGATATTTC???????11280

ATGTTGAGAA?ATCATTATTA?TATCCTACGA?AACTATGGTG?AAAATAAGCT?CGTTAAGAAT???????11340

CGAGTCTATC?TCATCATGTT?TTATTATTTT?TTGAAGTCAG?TTTTTTCTTT?TGATTATAAG???????11400

TTCGCAAAAG?TGGTTATTTT?TTCTTTTAAA?GATTCATTTT?CGCTTAAAAA?TAGCAAAAGA???????11460

TTTCGACCAT?AGTTTTTATT?TTTGGATAAA?CGCTGTTATC?ACAACTCATC?GGAGTAATTC???????11520

TTAAATGTCA?AAGCAACAGA?TCGGCGTCGT?CGGTATGGCA?GTGATGGGGC?GCAACCTTGC???????11580

GCTCAACATC?GAAAGCCGTG?GTTATACCGT?CTCTATTTTC?AACCGTTCCC?GTGAAAAGAC???????11640

GGAAGAAGTG?ATTGCCGAAA?ATCCAGGCAA?GAAACTGGTT?CCTTACTATA?CGGTGAAAGA???????11700

GTTTGTTGAA?TCTCTGGAAA?CGCCTCGTCG?CATCCTGTTA?ATGGTGAAAG?CAGGTGCAGG???????11760

CACGGATGCT?GCTATTGATT?CCCTCAAGCC?ATACCTCGAT?AAAGGTGACA?TCATCATTGA???????11820

TGGTGGTAAT?ACCTTCTTCC?AGGACACCAT?TCGTCGTAAC?CGTGAGCTTT?CTGCAGAAGG???????11880

CTTTAACTTC?ATCGGTACCG?GTGTTTCCGG?TGGTGAAGAG?GGCGCGCTGA?AAGGTCCTTC???????11940

CATTATGCCT?GGTGGCCAGA?AAGAAGCCTA?TGAACTGGTT?GCTCCGGTCC?TGACCAAAAT???????12000

CGCCGCAGTA?GCTGAAGACG?GTGAGCCATG?CGTTACCTAT?ATTGGTGCCG?ATGGCGCAGG???????12060

TCACTATGTG?AAGATGGTTC?ATAACGGTAT?TGAATACGGT?GATATGCAGC?TGATTGCTGA???????12120

AGCCTATTCT?CTGCTTAAAG?GTGGCCTGAA?CCTCACCAAC?GAAGAATTGG?CGCAGACCTT???????12180

TACCGAGTGG?AATAACGGTG?AACTGAGCAG?CTACCTGATC?GACATCACCA?AAGATATCTT???????12240

CACCAAAAAA?GATGAAGACG?GTAACTACCT?GGTTGATGTG?ATCCTGGATG?AAGCTGCTAA???????12300

CAAAGGTACC?GGTAAATGGA?CCAGCCAGAG?CGCGCTGGAT?CTCGGCGAAC?CGCTGTCGCT???????12360

GATTACCGAG?TCTGTGTTTG?CACGTTATAT?CTCTTCTCTG?AAAGATCAGC?GTGTTGCCGC???????12420

ATCTAAAGTT?CTCTCTGGCC?CGCAAGCGCA?GCCAGCTGGC?GACAAAGGTG?AGTTCATCGA???????12480

AAAAGTTCGT?CGTGCTCTGT?ATCTGGGCAA?AATCGTTTCT?TACGCGCAGG?GCTTCTCTCA???????12540

GCTGCGTGCT?GCGTCTGAAG?AGTACAACTG?GAATCTGAAC?TACGGCGAAA?TCGCGAAGAT???????12600

TTTCCGTGCT?GGCTGCATCA?TCCGTGCGCA?GTTCCTGCAG?AAAATCACCG?ATGCATATAC???????12660

AGAAAATCCG?CAGATCGCTA?ACCTGCTGCT?GGCTCCGTAC?TTCAAGCAAA?TCGCCGATGA???????12720

CTACCAGCAG?GCGCTGCGTG?ATGTCGTTGC?TTATGCGGTA?CAGAACGGTA?TCCCGGTTCC???????12780

GACCTTCGCC?GCTGCGGTTG?CCTATTATGA?CAGCTACCGT?GCCGCTGTTC?TGCCTGCGAA???????12840

CTTGATCCAG?GCACAGCGTG?ACTA??????????????????????????????????????????????12864

It in the sequence list nucleotide sequence of the O antigen gene bunch of intestinal bacteria O167, it is the same that preceding 12199 nucleotides sequence of the nucleotide sequence of the O antigen gene of Shigella bogdii 3 types bunch and the O antigen gene of intestinal bacteria O167 bunch shows 99.78%, different base box indicating is the Nucleotide of Shigella bogdii 3 types above this base; And 665 bases of the O antigen gene bunch back of intestinal bacteria O167 and O antigen gene are irrelevant, so not survey in Shigella bogdii 3 types.The initial sum terminated position of each gene of the O antigen gene of intestinal bacteria O167 and Shigella bogdii 3 types bunch all is the same as can be known from Table 4.

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

Claims (10)

1, a kind of Nucleotide of the O-antigen-specific to intestinal bacteria O167 type and Shigella bogdii 3 types, it is characterized in that: it is the isolating Nucleotide shown in SEQ ID NO:1,12864 bases of total length; Perhaps described base with one or more insertions, disappearance or replacement keeps the Nucleotide of the SEQ ID NO:1 of described isolating functional nucleotide simultaneously.

2, according to the Nucleotide of the described O-antigen-specific to intestinal bacteria O167 type and Shigella bogdii 3 types of claim 1, it is characterized in that: it comprises called after wzx, glf, orf3, orf4, orf5, wzy, orf7, orf8,9 genomic constitutions of orf9 are all between galF gene and gnd gene.

3, according to the Nucleotide of the described O-antigen-specific to intestinal bacteria O167 type and Shigella bogdii 3 types of claim 2, it is characterized in that the gene that has high degree of specificity in the described gene is: transhipment enzyme gene, it comprises the wzx gene; Pol gene, it comprises the wzy gene; Glycosyltransferase gene, it comprises orf3, orf4, orf5, orf7, orf8, orf9 gene; Wherein said gene: wzx is the Nucleotide of 1774 to 3039 bases among the SEQ IDNO:1; Wzy is the Nucleotide of 7165 to 8415 bases among the SEQ ID NO:1; Orf3 is the Nucleotide of 4153 to 5355 bases among the SEQ ID NO:1; Orf4 is the Nucleotide of 5355 to 6209 bases among the SEQ IDNO:1; Orf5 is the Nucleotide of 6209 to 7117 bases among the SEQ ID NO:1; Orf7 is the Nucleotide of 8415 to 9506 bases among the SEQ ID NO:1; Orf8 is the Nucleotide of 9503 to 10462 bases among the SEQ ID NO:1; Orf9 is the Nucleotide of 10570 to 11472 bases among the SEQ ID NO:1.

4, according to the Nucleotide of claim 1 or 2 described O-antigen-specifics to intestinal bacteria O167 type and Shigella bogdii 3 types, it is characterized in that: it also comprises and comes from described wzx gene, wzy gene and their mixing or their reorganization.

5, according to the Nucleotide of the described O-antigen-specific to intestinal bacteria O167 type and Shigella bogdii 3 types of claim 4, it is characterized in that the oligonucleotide that wherein comes from the wzx gene is to being: the Nucleotide of 2281 to 2300 bases among the SEQ IDNO:1 and the Nucleotide of 2925 to 2944 bases; The Nucleotide of 1973 to 1992 bases among the SEQ IDNO:1 and the Nucleotide of 2863 to 2882 bases; The oligonucleotide that comes from the wzy gene is to being: the Nucleotide of 7318 to 7337 bases among the SEQ ID NO:1 and the Nucleotide of 8047 to 8066 bases; The Nucleotide of 7667 to 7686 bases among the SEQ ID NO:1 and the Nucleotide of 8168 to 8184 bases.

6, the application of Nucleotide in detecting other polysaccharide antigen of expressing the antigenic bacterium of O-, the O-antigen of identifying bacterium and bacterium of the described O-antigen-specific to intestinal bacteria O167 type and Shigella bogdii 3 types of claim 1.

7, the recombinant molecule of the Nucleotide of the described O-antigen-specific to intestinal bacteria O167 type and Shigella bogdii 3 types of claim 1, providing the O-antigen of expressing intestinal bacteria O167 type and Shigella bogdii 3 types by inserting to express, and the application in the preparation bacterial vaccine.

8, according to the application of the Nucleotide of the described O-antigen-specific to intestinal bacteria O167 type and Shigella bogdii 3 types of claim 1, it is characterized in that, it is used for PCR, is used for hybridization and fluoroscopic examination or is used to make gene chip or microarray as probe as primer, for bacterial detection.

9, the separation method of the Nucleotide of the described O-antigen-specific to intestinal bacteria O167 type and Shigella bogdii 3 types of claim 1 is characterized in that it comprises the steps:

(1) genomic extraction: in substratum, cultivate intestinal bacteria O167 type or Shigella bogdii 3 types, centrifugal collecting cell; The genomic dna that obtains detects by agarose gel electrophoresis;

(2) by the O-antigen gene in pcr amplification intestinal bacteria O167 type or Shigella bogdii 3 types bunch: with the genome of intestinal bacteria O167 type or Shigella bogdii 3 types is template by increase its O-antigen gene bunch of LongPCR, 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 O167 type or Shigella bogdii 3 types;

(6) screening of specific gene: at wzx, the wzy gene design primer in the O-antigen gene of intestinal bacteria O167 type and Shigella bogdii 3 types bunch; Respectively designed two pairs of primers in each gene, every pair of primer is distributed in the different places in the corresponding gene, to guarantee its specificity; Is that template is carried out PCR with these primers with the genomes of 166 strain intestinal bacteria and 43 strain Shigellaes, determines the antigenic high degree of specificity of O-of wzx, wzy gene pairs intestinal bacteria O167 type and Shigella bogdii 3 types;

(7) detection of primer sensitivity: cultivate intestinal bacteria O167, 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 O167.

10, the separation method of the Nucleotide of the O-antigen-specific to intestinal bacteria O167 type and Shigella bogdii 3 types according to claim 9 is characterized in that it comprises the steps:

(1) genomic extraction: 37 ℃ of incubated overnight intestinal bacteria O167 types or Shigella bogdii 3 types in the LB of 5mL substratum, centrifugal collecting cell; With the pH value is 8.0 500 μ l 50mM Tris-HCl and 10 μ l 0.4MEDTA re-suspended cells, 37 ℃ of incubations 20 minutes, and the N,O-Diacetylmuramidase that adds 10 μ l 10mg/mL then continues insulation 20 minutes; The Proteinase K, the 15 μ l10%SDS that add 3 μ l 20mg/mL afterwards, 50 ℃ of incubations 2 hours, the RNase that adds 3 μ l 10mg/mL again, 65 ℃ of incubations 30 minutes, add equal-volume phenol extracting mixture, get supernatant and use isopyknic phenol again: chloroform: primary isoamyl alcohol mixing solutions extracting twice, get supernatant again with isopyknic ether extracting to remove remaining phenol, phenol: chloroform: the mixed volume ratio of primary isoamyl alcohol is 25: 24: 1; Supernatant rolls out DNA and washes DNA with 70% ethanol with glass yarn with 2 times of volume ethanol deposit D NA, and DNA is resuspended among the 30 μ l TE; Genomic dna detects by 0.4% agarose gel electrophoresis;

(2) by the O-antigen gene in pcr amplification intestinal bacteria O167 type or Shigella bogdii 3 types bunch: with the genome of intestinal bacteria O167 type or Shigella bogdii 3 types is that template is passed through its O-antigen gene of Long pcr amplification bunch, be #1523-ATT GTG GCT GCA GGG ATC AAA GAA AT according to the galF sequences Design upstream primer that often is found in O-antigen gene bunch promoter region at first, the gnd gene design downstream primer according to O-antigen gene bunch downstream is #1524-TAG TCG CGTGNG CCT GGA TTA AGT TCG C again; With the Expand LongTemplate 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, 55 annealing 15 seconds, 68 ℃ were extended 15 minutes, carry out 30 circulations like this, last, continue to extend 7 minutes at 68 ℃, obtain the PCR product, detect the size and the specificity thereof of PCR product with 0.8% agarose gel electrophoresis, merge 5 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 the 300ngPCR purified product, 0.9 μ l 0.1M MnCl ₂, the DNaseI of the 1mg/mL of 1 μ l dilution in 1: 2000, reaction is carried out at room temperature, and enzyme is cut the dna fragmentation size is concentrated between the 1.5kb-3kb, then adds 2 μ l 0.1M EDTA termination reactions; Merge the same reaction system of 4 pipes, with isopyknic phenol extracting once, use isopyknic phenol: chloroform: the mixing solutions extracting of primary isoamyl alcohol once, phenol: chloroform: the mixed volume ratio of primary isoamyl alcohol is after using isopyknic ether extracting once again at 25: 24: 1, dehydrated alcohol deposit D NA with 2.5 times of volumes, and wash precipitation with 70% ethanol, be resuspended at last in the 18 μ l water, in this mixture, add 2.5 μ ldNTP (1mMdCTP, 1mMdGTP, 1mMdTTP subsequently, 10mMdATP), 1.25 the T4DNA polysaccharase of μ l 100mMDTT and 5 units, 11 ℃ 30 minutes, enzyme is cut product mends 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 80 μ l, 70 ℃ of reactions 20 minutes make 3 of DNA ' end add the dA tail; This mixture is through the equal-volume chloroform: after the mixing solutions extracting of primary isoamyl alcohol and the extracting of equal-volume ether with 3 * 10 of Promega company ^-3The pGEM-T-Easy carrier connect 10 hours in 16 ℃, cumulative volume is 90 μ l, chloroform: the mixed volume ratio of primary isoamyl alcohol is 24: 1; 10 * the buffer of 9 μ l and the T4DNA ligase enzyme of 25 units are wherein arranged, be that 5.2 the 3M NaAc and the dehydrated alcohol precipitation of 2 times of volumes are connected mixture with the pH value of 1/10 volume at last, wash precipitation with 70% ethanol again, be dissolved in after the drying in the 30 μ l water and obtain connecting product; Preparation method with the electric transformed competence colibacillus cell of Bio-Rad company prepares the competence e.colidh5, get after 2-3 μ l connects product and 50 μ l competence bacillus coli DH 5 alphas mix, forward in the electric shock cup of 0.2cm of Bio-Rad company and shock by electricity, voltage is 2.5 kilovolts, time is 5.0 milliseconds to 6.0 milliseconds, the SOC substratum that adds 1mL after the electric shock immediately in cup makes the bacterium recovery, then bacterium is coated in and contains penbritin, on the LB solid medium of X-Gal and IPTG, 37 ℃ of incubated overnight, obtain blue white bacterium colony next day, with the white colony that obtains promptly the white clone forward on the LB solid medium that contains penbritin and cultivate, from each clone, extract plasmid simultaneously, and cutting the segmental size of evaluation insertion wherein with the EcoRI enzyme, the white that obtains clone group has constituted the O-antigen gene bunch library of intestinal bacteria O167 type or Shigella bogdii 3 types;

(4) to the cloning and sequencing in the library: from the library, select 96 clones of insertion fragment more than 1kb and the insertion fragment among the clone is checked order with this lab A BI3730 type automatic dna sequencer, sequence reaches 100% fraction of coverage, thereby obtains 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 Molecular Biology Lab and edit all sequences, thus obtain the Nucleotide full length sequence of the O-antigen gene bunch of intestinal bacteria O167 type or Shigella bogdii 3 types; The quality of sequence is mainly guaranteed by two aspects: 1) genome of intestinal bacteria O167 type or Shigella bogdii 3 types is done 5 Long PCR reactions, mix these products then to produce the library, 2) to each base, guarantee high-quality fraction of coverage more than 3, after obtaining the nucleotide sequence of intestinal bacteria O167 type or Shigella bogdii 3 type O-antigen genes bunch, Orffmder with American National biotechnology information science center 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 O167 type or Shigella bogdii 3 types at last;

(6) specific gene screening: at wzx, the wzy gene design primer in the O-antigen gene of intestinal bacteria O167 type and Shigella bogdii 3 types bunch; Respectively designed two pairs of primers in each gene, every pair of primer is distributed in the different places in the corresponding gene, to guarantee its specificity; Is that template is carried out PCR with these primers with the genomes of 166 strain intestinal bacteria and 43 strain Shigellaes, except that a band that in containing intestinal bacteria O167 group, has obtained the expection size, the correct product of the expection clip size that all do not increase in other groups is so the O-antigen of wzx, wzy gene pairs intestinal bacteria O167 type and Shigella bogdii 3 types all is high special;

(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 O167 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 l supernatant as pcr template; To carrying out the PCR reaction, the PCR reaction system is as follows: MQ:15.7 μ l, Mg with oligonucleotide 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 primer reaction; The pork filling that participates in 0 viable bacteria obtains negative findings in the PCR of primer reaction; Illustrate that primer is 0.25 bacterium/g to the detection sensitivity of the intestinal bacteria O167 in the pork filling when using aforesaid method.