CN1256435C - Nucleotide peculiar to 0-antigen of 110 type bacillus coli - Google Patents

Abstract

The present invention provides a specific nucleotide for an O-antigen of Escherichia coli O110. The specific nucleotide is the nucleotide complete sequence of gene clusters in Escherichia coli O110 for controlling the synthesis of an O-antigen, such as a separated nucleotide with the total length of 9800 basic groups shown as SEQ ID NO: 1 or the nucleotide of SEQ ID NO: 1 which has one or a plurality of inserted, deleted or substituted basic groups and can simultaneously keep the functions of the separated nucleotide. The specific nucleotide also comprises the oligonucleotide from glycosyltransferase genes and oligosaccharide unit processing genes in the O-antigen gene clusters of Escherichia coli O110. The present invention verifies that the oligonucleotide has high specificity to all of the O-antigen genes of Escherichia coli O110 through PCR. The present invention also discloses a method for detecting and identifying Escherichia coli O110 in the human body and the environment by the oligonucleotide of the present invention.

Description

Nucleotide to the O-antigen-specific of intestinal bacteria O110 type

Technical field

The present invention relates to the complete nucleotide sequence of control O-antigen synthetic gene cluster in the intestinal bacteria O110 type (Escherichia coli O110), particularly relate in the intestinal bacteria O110 type oligonucleotide in the control O-antigen synthetic gene cluster, can utilize these the oligonucleotide of the O-antigen-specific intestinal bacteria O110 type in human body and the environment and identify O-antigen in these pathogenic bacterium 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 oflipopolysaccharide biosynthesis in entericbacteria " .MicrobiologicalReviews, 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) " Bacterialpolysaccharide synthesis and gene nomenclature " Trends in Microbiology, 4:495-503].In Shigellae, intestinal bacteria and Salmonellas, O-antigen gene [Lei Wang.et al (2001) " Sequence analysis of four Shigella boydii O-antigenloci: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 coliO111 and Salmonella enterica O35 gene clusters:gene clusters encoding the samecolitose-containing O antigen are highly conserved " .Journal ofBacteriology.182:5256-5261].The O-antigen gene bunch contains three genoids: sugared synthesis path gene, glycosyltransferase gene, oligosaccharide unit treatment gene, the required nucleoside diphosphate monose of enzymic synthesis O-antigen of wherein sugared synthesis path genes encoding; Thereby the enzyme of glycosyltransferase gene coding forwards nucleoside diphosphate monose and other molecule to and makes monose aggregate into oligosaccharide unit on the monose; The oligosaccharide unit treatment gene comprises 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 andparatose synthase genes (rfb) by polymerase chain reaction for identification ofSenterica major serogroups (A; B; C2; andD) ", J.Clin.Microbiol.31:2118-2123].Luk, the method for et.al is with corresponding to Salmonellas serotype E 1, D1 obtains the oligonucleotide special to the Salmonellas of different serotypes after the nucleotide sequence of the CDP-abequose in the A, the O-antigen of B and C2 and the synthetic gene of CDP-tyvelose is arranged.1996, Paton, the A.W et.al serotype [" Molecular microbiological investigation of an outbreak of Hemolytic-Uremic Syndrome caused by dry fermented sausage contaminated with Shiga-liketoxin producing Escherichia coli " .J.Clin.Microbiol.34:1622-1627] of the oligonucleotide that comes from the wbdI gene of the O-antigen-specific of E.coli O111 having been identified the toxogenic E.coli O111 of a strain, but afterwards studies show that Paton, the usefulness of A.W et.al comes from the oligonucleotide of wbdI gene and identifies that the method for the serotype of E.coli O111 has false positive results to occur.Bastin D.A.and Reeves, P.R. think, this is because the wbdI gene is sugared synthesis path gene [the Bastin D.A.andReeves of a supposition, P.R. (1995) Sequence and analysis of the O antigen gene (rfb) cluster ofEscherichia coli O111.Gene 164:17-23], and have this sugar in the antigenic structure of the O-of other bacterium, so sugared synthesis path gene is not a high special for O-antigen yet

Shigellae has 46 kinds of serotypes, but have only 33 kinds of different O-antigens, intestinal bacteria have 166 kinds of different O-antigen [Reeves, P.R (1992) " Variation in O antigens; niche specificselection and bacterial populations " .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 ofthe Enterobacteriaceae " .Elsevier SciencePublishers, Amsterdam, The Netherlands; T.cheasty, et al. (1983) " Antigenicrelationships between the enteroinvasive Escherichia coli antigensO28ac; O112ac; O124; O136, O143, O144; O152and and Shigella O antigens " J.clinMicrobiol, 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 O110 type.It is the Nucleotide in the O-antigen gene bunch of intestinal bacteria O110 type, 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 O110 type.

The present invention another purpose has provided the gene of the O-antigen gene bunch that constitutes intestinal bacteria O110 type: transhipment enzyme gene is the wzx gene or with wzx the gene of identity function is arranged; Pol gene is the wzy gene or with wzy the gene of identity function is arranged; Glycosyltransferase gene comprises orf1, orf2, orf7 gene.

Another purpose of the present invention has provided oligonucleotide, and the gene that they come from encoding glycosyl transferring enzyme in the O-antigen gene bunch of intestinal bacteria O110 type respectively comprises orf1, orf2, orf7 gene; The gene that coming from coding transhipment enzyme 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 of identity function is arranged; They are the oligonucleotide in the said gene, and length is at 10-20nt; They are special to the O-antigen of intestinal bacteria O110 type; Especially the oligonucleotide of listing in the table 1, they are high specials to the O-antigen of intestinal bacteria O110 type, and these oligonucleotide are also reconfigurable, the oligonucleotide after the combination also is a high special to the O-antigen of intestinal bacteria O110 type.

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 the detection and the identification of escherichia coli O110 type of these methods detections and identification of escherichia coli O110 type.

An also purpose of the present invention has provided the method for complete sequence of the O-antigen gene bunch of separating Escherichia coli O110 type.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 O110 type, and it is the isolating Nucleotide shown in SEQ ID NO:1,9800 bases of total length; The base that perhaps has one or more insertions, disappearance or replacement keeps the Nucleotide of the SEQ ID NO:1 of described isolating functional nucleotide simultaneously.

The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O110 type, comprising called after orf1, orf2, orf3, wzx, orf5, wzy, 7 genomic constitutions of orf7 are all between galF gene and gnd gene.

The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O110 type, the gene that has high degree of specificity in the wherein said gene is: the gene of transhipment enzyme comprises the wzx gene or with wzx the gene of identity function is arranged; Pol gene wzy gene or the gene of identity function is arranged with wzy; Glycosyltransferase gene comprises orf1, orf2, orf7 gene; Wherein said gene: wzx is the Nucleotide of 4245 to 5495 bases among the SEQ ID NO:1; Wzy is the Nucleotide of 6617 among the SEQIDNO:1 to 7825 bases; Orf1 is the Nucleotide of 1113 to 2204 bases among the SEQ ID NO:1; Orf2 is the Nucleotide of 2191 to 3267 bases among the SEQ ID NO:1; Orf7 is the Nucleotide of 7803 to 8966 bases among the SEQ ID NO:1.

The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O110 type, wherein it also comprises and comes from described wzx gene, wzy gene or glycosyltransferase gene orf1, orf2, orf7 gene; And their mixing or their reorganization.

The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O110 type, the oligonucleotide of the wherein said orf1 of coming from gene is to being: the Nucleotide of 1884 to 1901 bases among the SEQ ID NO:1 and the Nucleotide of 2150 to 2167 bases; The Nucleotide of 1514 to 1531 bases among the SEQ ID NO:1 and the Nucleotide of 1974 to 1991 bases.The oligonucleotide that comes from the orf2 gene is to being: the Nucleotide of 2638 to 2655 bases among the SEQ ID NO:1 and the Nucleotide of 3071 to 3088 bases; The Nucleotide of 2191 to 2208 bases among the SEQ ID NO:1 and the Nucleotide of 2382 to 2399 bases.The oligonucleotide that comes from the orf7 gene is to being: the Nucleotide of 7871 to 7888 bases among the SEQ ID NO:1 and the Nucleotide of 8140 to 8157 bases; The Nucleotide of 8444 to 8461 bases among the SEQ ID NO:1 and the Nucleotide of 8896 to 8913 bases.

The application of the Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O110 type 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 O110 type is providing the O-antigen of expressing intestinal bacteria O110 type 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 O110 type 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 O110 type is characterized in that it comprises the steps:

(1) genomic extraction: in substratum, cultivate intestinal bacteria O110 type, centrifugal collecting cell; The genomic dna that obtains detects by agarose gel electrophoresis;

(2) by the O-antigen gene in the pcr amplification intestinal bacteria O110 type bunch: with the genome of intestinal bacteria O110 type is that template is passed through its O-antigen gene of Long pcr amplification bunch, with the PCR product that obtains, detect the size and the specificity thereof of PCR product with agarose gel electrophoresis, merge this long PCR product, and with DNA purification kit purified pcr product;

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

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

(5) splicing of nucleotide sequence and analysis: applying biological information science software splicing and edit all sequences, thus obtain the Nucleotide full length sequence of the O-antigen gene bunch of intestinal bacteria O110 type;

(6) screening of specific gene: at orf1, orf2, the orf7 gene design primer in the O-antigen gene of intestinal bacteria O110 type bunch; Respectively designed two pairs of primers in each gene, every pair of primer is distributed in the different places in the corresponding gene, to guarantee its specificity; Is that template is carried out PCR with these primers with the genomes of 166 strain intestinal bacteria and 43 strain Shigellaes, determines the antigenic high degree of specificity of O-of orf1, orf2, orf7 gene pairs intestinal bacteria O110 type;

(7) detection of primer sensitivity: cultivate intestinal bacteria O110, 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 O110.

The separation method of the Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O110 type is characterized in that it comprises the steps:

(1) genomic extraction: 37 ℃ of incubated overnight intestinal bacteria O110 types in the LB of 5mL substratum, centrifugal collecting cell.With 500ul 50mM Tris-HCl (pH8.0) and 10ul 0.4M EDTA re-suspended cell, 37 ℃ of incubations 20 minutes, the N,O-Diacetylmuramidase that adds 10ul 10mg/ml then continues insulation 20 minutes.The Proteinase K, the 15ul 10%SDS that add 3ul 20mg/ml afterwards, 50 ℃ of incubations 2 hours, the RNase that adds 3ul 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 30ul TE; Genomic dna detects by 0.4% agarose gel electrophoresis;

(2) by the O-antigen gene in the pcr amplification intestinal bacteria O110 type bunch: with the genome of intestinal bacteria O110 type 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 GCTGCA GGG ATC AAA GAA AT) that often is found in O-antigen gene bunch promoter region, again according to the gnd gene design downstream primer (#1524-TAG TCG CGT GNG CCT GGA TTA AGT TCG C) in O-antigen gene bunch downstream; With the Expand Long Template PCR method of 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 for 60 ℃, 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 a 300ng PCR purified product, 0.9ul 0.1M MnCl ₂, the DNaseI of the 1mg/ml of 1ul dilution in 1: 2000, reaction is carried out at room temperature, and enzyme is cut the dna fragmentation size is concentrated between the 1.5kb-3kb, then adds 2ul 0.1M EDTA termination reaction.Merge the same reaction system of 4 pipes, with isopyknic phenol extracting once, use isopyknic phenol: chloroform: primary isoamyl alcohol (25: 24: 1) mixing solutions extracting once, after using isopyknic ether extracting once again, dehydrated alcohol deposit D NA with 2.5 times of volumes, and wash precipitation with 70% ethanol, be resuspended at last in the 18ul water, in this mixture, add 2.5ul dNTP (1mMdCTP, 1mMdGTP, 1mMdTTP subsequently, 10mMdATP), 1.25ul the T4DNA polysaccharase of 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 80ul, 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 90ul.10 * the buffer of 9ul and the T4DNA ligase enzyme of 25 units are wherein arranged, use the dehydrated alcohol precipitation of the 3M NaAc (pH5.2) of 1/10 volume and 2 times of volumes to be connected mixture at last, wash precipitation with 70% ethanol again, be dissolved in after the drying and obtain connecting product in the 30ul water; Preparation method with the electric transformed competence colibacillus cell of Bi0-Rad company prepares the competence e.colidh5, get after 2-3ul connects product and 50ul competence bacillus coli DH 5 alpha mixes, forward in the electric shock cup of 0.2cm of Bi0-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 O110 type;

(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 O110 type; The quality of sequence is mainly guaranteed by two aspects: 1) genome of intestinal bacteria O110 type 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 O110 type O-antigen gene bunch, with American National biotechnology information science center (The National Center forBiotechnology Information, NCBI) orffinder finds gene, find the reading frame of 7 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 O110 type at last;

(6) screening of specific gene: at orf1, orf2, the orf7 gene design primer in the O-antigen gene of intestinal bacteria O110 type bunch; Respectively designed two pairs of primers in each gene, every pair of primer is distributed in the different places in the corresponding gene, to guarantee its specificity; Is that template is carried out PCR with these primers with the genomes of 166 strain intestinal bacteria and 43 strain Shigellaes, except that a band that in containing the 13rd group of intestinal bacteria O110, 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 O110 type 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 O110 is inoculated in the triangular flask of 20mlLB 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 200 ml LB substratum, and through 6 layers of filtered through gauze, filtered liquid 200 rev/mins, is cultivated 12h in 37 ℃.Get 3ml bacterium liquid in 6 from cultured bacterium liquid, centrifugal 5 minutes of 000g removes supernatant, adds 100 μ l MQ ultrapure waters and blows precipitation and mixing open, puts into 100 degree boiling water and boils 15 minutes, and lysate is in 12, and centrifugal 8 minutes of 000g gets 1 μ supernatant as pcr template.Right with 6 pairs of oligonucleotide, the Nucleotide of 1884 to 1901 bases among the SEQ ID NO:1 and the Nucleotide of 2150 to 2167 bases; The Nucleotide of 1514 to 1531 bases among the SEQ ID NO:1 and the Nucleotide of 1974 to 1991 bases.The Nucleotide of 2638 to 2655 bases among the SEQ ID NO:1 and the Nucleotide of 3071 to 3088 bases; The Nucleotide of 2191 to 2208 bases among the SEQ IDNO:1 and the Nucleotide of 2382 to 2399 bases.The Nucleotide of 7871 to 7888 bases among the SEQ ID NO:1 and the Nucleotide of 8140 to 8157 bases; The Nucleotide of 8444 to 8461 bases among the SEQ ID NO:1 and the Nucleotide of 8896 to 8913 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 6 pairs of primers reaction.The pork filling that participates in 0 viable bacteria obtains negative findings in the PCR of 6 pairs of primers reaction.Illustrate that these 6 pairs of primers are 0.25 bacterium/g to the detection sensitivity of the intestinal bacteria O110 in the pork filling when using aforesaid method.

Just, first aspect of the present invention provides the full length nucleotide sequence of the O-antigen gene bunch of intestinal bacteria O110 type, its complete sequence shown in SEQ ID NO:1,9800 bases of total length; The base that perhaps has one or more insertions, disappearance or replacement keeps the Nucleotide of the SEQ ID NO:1 of described isolating functional nucleotide simultaneously.Obtained the structure of the O-antigen gene bunch of intestinal bacteria O110 type by method of the present invention, as shown in table 3, it comprises called after orf1, orf2, and orf3, wzx, orf5, wzy, 7 genomic constitutions of orf7 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 O110 type, 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 (orf1, orf2, orf7 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 intestinal bacteria O110 type O-antigen.

The 3rd aspect of the present invention, wzx gene in the O-antigen gene bunch that comes from intestinal bacteria O110 type is provided or the gene, wzy gene of identity function is arranged or the gene and the glycosyltransferase gene of identity function are arranged with wzy with wzx, the oligonucleotide that comprises orf1, orf2, orf7 gene, they are any one section oligonucleotide in these genes.But, be that the oligonucleotide of listing in the table 1 is right preferentially by usefulness, in table 1, also listed these oligonucleotide to the position in O-antigen gene bunch and with these oligonucleotide to being the size of the product of the PCR reaction done of primer, the annealing temperature in these PCR reaction free lists is carried out.These primers are 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 orf1, orf2, orf7 gene pairs intestinal bacteria O110 type all is high special.

The separation method of the Nucleotide of described O-antigen-specific to intestinal bacteria O110 type comprises the steps: 1) genomic extraction; 2) the O-antigen gene in the pcr amplification intestinal bacteria O110 type 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 refers to derive from gene, the gene of coding transhipment enzyme and intragenic one section nucleic acid molecule of coding polysaccharase of the encoding glycosyl transferring enzyme in the O-antigen gene bunch, they can change on length, generally change in 10 to 20 Nucleotide scopes; More definite these oligonucleotide of saying are to come from orf1 gene (nucleotide position is the Nucleotide of 1113 to 2204 bases from SEQ ID NO:1); Orf2 gene (nucleotide position is the Nucleotide of 2191 to 3267 bases from SEQ ID NO:1); Orf7 gene (nucleotide position is the Nucleotide of 7803 to 8966 bases from SEQ ID NO:1). coming from the interior oligonucleotide of above gene is high special to intestinal bacteria O110 type.

In addition, the antigenic gene cluster of the different O-of the coding of two genetic resemblances produces new O-antigen by gene recombination or sudden change sometimes, thereby produces new bacteria types, new mutant strain.In this environment, need filter out many specificitys that oligonucleotide is detected with raising with recombination hybridization.Therefore, the invention provides a whole set of many mixtures to oligonucleotide, they come from glycosyltransferase gene; Come from transhipment enzyme and pol gene, comprise the wzx gene or the gene, wzy gene of identity function arranged or the gene of identity function is arranged with wzy with wzx.The mixture of these genes is special to a special bacterial polysaccharides antigen, is special thereby make this cover oligonucleotide to the polysaccharide antigen of this bacterium.More particularly, the mixture of these oligonucleotide is to come from glycosyltransferase gene, wzx gene or the gene, wzy 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 the encoding glycosyl transferring enzyme gene of transhipment enzyme and polysaccharase of (ii) encoding comprises the wzx gene or the gene, wzy gene of identity function is arranged or with wzy the gene of identity function is arranged with wzx.At least one oligonucleotide can be hybridized with at least one more than one such gene specific of expressing the special antigenic bacterium of O-under the situation of condition permission, and these bacteriums are intestinal bacteria O110 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 comes from the encoding glycosyl transferring enzyme, the gene of coding transhipment enzyme and the gene of polysaccharase, comprises the wzx gene or the gene, wzy gene of identity function is arranged or with wzy the oligonucleotide of the gene of identity function is arranged with wzx.This cover oligonucleotide is special to the O-antigen of a special bacterium, and this special bacterium O-antigen is expressed by intestinal bacteria O110 type.

On the other hand, the present invention relates to a kind of antigenic method of one or more bacterial polysaccharideses that detects in the movement, these antigens can make sample can with the oligonucleotide specific hybrid of following at least one gene, these genes are: (i) gene of the encoding glycosyl transferring enzyme gene of transhipment enzyme and polysaccharase of (ii) encoding comprises the wzx gene or the gene, wzy gene of identity function is arranged or with wzy the gene of identity function is arranged with wzx.At least one oligonucleotide can be expressed more than one such gene specific hybridization of the special antigenic bacterium of O-with at least one under the situation of condition permission.These bacteriums are intestinal bacteria O110 types.Oligonucleotide among available the present invention is made the method test sample of primer by PCR, also can with behind the oligonucleotide molecules mark among the present invention as probe by hybridization such as southern-blot or fluoroscopic examination, perhaps by antigen and bacterium in gene chip or the microarray assay sample.

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

On the other hand, the present invention also relates to the antigenic method of one or more bacterial polysaccharideses in the sample that a kind of detection comes from patient.One or more bacterial polysaccharides antigens in the sample can make sample can with a specific hybrid in a pair of oligonucleotide in following at least one gene, these genes are: (i) gene of the encoding glycosyl transferring enzyme gene of transhipment enzyme and polysaccharase of (ii) encoding comprises the wzx gene or the gene, wzy gene of identity function is arranged or with wzy the gene of identity function is arranged with wzx.Under the situation of condition permission at least one oligonucleotide can with sample at least one express more than one such gene specific hybridization of the special antigenic bacterium of O-, these bacteriums are intestinal bacteria O110 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 glycosyltransferase gene, wzx gene or with wzx the gene, wzy gene of identity function arranged or have on the sequence of gene of identity function with wzy that one of them oligonucleotide molecules can hybridize to one.In addition, when two oligonucleotide can both be hybridized, they may be hybridized in same gene and also may hybridize on the different genes.Also promptly, when cross reaction goes wrong, can select the mixture of oligonucleotide to detect the blended gene so that the specificity of detection to be provided.

The present inventor believes that the present invention is not necessarily limited to the above nucleotide sequence coded specific O-antigen of carrying, and is widely used in detecting all expression O-antigens and identifies the antigenic bacterium of O-.And because O-antigen is synthetic and the similarity of other polysaccharide antigens (as bacterium born of the same parents exoantigen) between synthesizing, 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 O110 type 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 O110 type 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 instrument 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 O110 types in the LB of 5mL substratum, centrifugal collecting cell.With 500ul 50mM Tris-HCl (pH8.0) and 10ul 0.4M EDTA re-suspended cell, 37 ℃ of incubations 20 minutes, the N,O-Diacetylmuramidase that adds 10ul 10mg/ml then continues insulation 20 minutes.The Proteinase K, the 15ul 10%SDS that add 3ul 20mg/ml afterwards, 50 ℃ of incubations 2 hours add the RNase of 3ul 10mg/ml again, 65 ℃ of incubations 30 minutes.Add equal-volume phenol extracting mixture, get supernatant 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 30ul TE with 70% ethanol.Genomic dna detects by 0.4% agarose gel electrophoresis.

Embodiment 2: by the O-antigen gene in the pcr amplification intestinal bacteria O110 type bunch:

With the genome of intestinal bacteria O110 type 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 CGT GNG CCT GGATTA AGT TCG C) in O-antigen gene bunch downstream; With the Expand Long Template PCR method of Boehringer Mannheim company amplification O-antigen gene bunch, the PCR response procedures was as follows: 94 ℃ of pre-sex change 2 minutes; 94 ℃ of sex change are 10 seconds then, 60 ℃ of annealing 15 seconds, and 68 ℃ were extended 15 minutes, and carried out 30 circulations like this.At last, continue to extend 7 minutes at 68 ℃, obtain the PCR product, the agarose gel electrophoresis with 0.8% detects the size and the specificity thereof of PCR product.Merge 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, 0.9ul 0.1M MnCl ₂, 1ul1: 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 2ul 0.1M EDTA termination reaction.Merge the same reaction system of 4 pipes, with isopyknic phenol extracting once, use isopyknic phenol: chloroform: primary isoamyl alcohol (25: 24: 1) mixing solutions extracting once, after using isopyknic ether extracting once again, dehydrated alcohol deposit D NA with 2.5 times of volumes, and wash precipitation with 70% ethanol, be resuspended at last in the 18ul water.In this mixture, add 2.5ul dNTP (1mMdCTP subsequently, 1mMdGTP, 1mMdTTP, 10mMdATP), the T4DNA polysaccharase of 1.25 ul 100mM DTT and 5 units, 11 ℃ 30 minutes, enzyme is cut product mend into flush end, after 75 ℃ of termination reactions, add the Tth archaeal dna polymerase of 5 units and corresponding damping fluid thereof and system is expanded as 80ul, 70 ℃ were reacted 20 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 90ul.10 * the buffer of 9ul and the T4DNA ligase enzyme of 25 units are wherein arranged.Use the dehydrated alcohol precipitation of the 3M NaAc (pH5.2) of 1/10 volume and 2 times of volumes to be connected mixture at last, wash precipitation with 70% ethanol again, be dissolved in after the drying and obtain connecting product in the 30ul water.

Next is the preparation of competent cell: the method that provides with reference to Bio-Rad company prepares the competent cell bacillus coli DH 5 alpha.Get a ring bacillus coli DH 5 alpha list bacterium colony in the LB of 5ml substratum, 180rpm cultivated after 10 hours, got in the LB substratum that the 2ml culture is transferred to 200ml, and 37 ℃ of 250rpm thermal agitations are cultivated OD600 about 0.5, ice bath cooling was 20 minutes then, in centrifugal 15 minutes of 4 ℃ of 4000rpm.Confide all supernatant, 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 50ul one pipe ,-70 ℃ of preservations.

Be electric transformed competence colibacillus cell at last: get after 2-3ul connects product and 50ul competence escherichia coli DH5a mixes, forward in the electric shock cup of 0.2cm of Bio-Rad company and shock by electricity, voltage is 2.5 kilovolts, 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 O110 type 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 O110 type obtained.The quality of sequence is mainly guaranteed by two aspects: 1) genome of intestinal bacteria O110 type 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 O110 type O-antigen gene bunch, with American National biotechnology information science center (The National Center for BiotechnologyInformation, NCBI) orffinder finds gene, find the reading frame of 7 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 O110 type at last, as shown in table 3.

By retrieving and relatively, finding that orf4 and orf6 are the proteic genes that there is transmembrane segment in only two codings of intestinal bacteria O110 kind.The O-antigen transferring enzyme of orf4 encoded protein and Methanosarcina mazei Goel has 25% sequence identity, it contains 12 uniform transmembrane segments by the proteic topology discovery of HMMTOP2.0 programanalysis, and this is the proteic characteristic feature of Wzx.So name orf4 is wzx.The O-antigen polysaccharase of orf6 encoded protein and intestinal bacteria O6 has 20% consistence, 44% similarity, contain 9 transmembrane segments by the proteic topology discovery tool of HMMTOP2.0 programanalysis, and hydrophilic loop (loop) in the big kytoplasm is arranged, and this is the proteic characteristic feature of Wzy.So name orf6 is wzy.

Orf1, the albumen of 2,7 three genes encodings and other known glycosyltransferases have the sequence identity of 26-42% and the sequence similarity of 47-60%.Therefore we infer this three genes encoding glycosyltransferases, and because each glycosyltransferase specificity catalysis forms a kind of disaccharide bond, so we infer that the antigenic oligosaccharide unit of O-of intestinal bacteria O110 may be made up of four monose.Because the definite function of these three genes can't be determined, so we are with these three genes temporary called after orf1, orf2 and orf7.

The albumen of polyglutamatesynthase capA genes encoding has 28% consensus amino acid sequence in orf5 encoded protein and the Myxococcus xanthus O-antigen gene bunch, because the definite function of this gene can't be determined, so we are with the temporary called after orf5 of this gene.The definite function of orf3 gene can't be determined, so we are with the temporary called after orf3 of this gene.

Embodiment 6: the screening of specific gene:

Orf1, orf2, orf7 gene design primer in the O-antigen gene of intestinal bacteria O110 type 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 the 13rd group of intestinal bacteria O110, 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 orf1, orf2, orf7 gene pairs intestinal bacteria O110 type all is high special; The position of these genes in nucleotide sequence sees Table 1.

Embodiment 7: the detection of primer sensitivity.

Buy the live pig meat stuffing on the market, stir, be divided into the 20g portion, exist in-40 ℃ of refrigerators standby.The frozen bacterium liquid of 10 μ l intestinal bacteria O110 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 200 ml LB substratum, and through 6 layers of filtered through gauze, filtered liquid 200 rev/mins, is cultivated 12h in 37 ℃.Get 3ml bacterium liquid in 6 from cultured bacterium liquid, centrifugal 5 minutes of 000g removes supernatant, adds 100 μ l MQ ultrapure waters and blows precipitation and mixing open, puts into 100 degree boiling water and boils 15 minutes, and lysate is in 12, and centrifugal 8 minutes of 000g gets 1 μ supernatant as pcr template.Right with 6 pairs of oligonucleotide, the Nucleotide of 1884 to 1901 bases among the SEQ ID NO:1 and the Nucleotide of 2150 to 2167 bases; The Nucleotide of 1514 to 1531 bases among the SEQ ID NO:1 and the Nucleotide of 1974 to 1991 bases.The Nucleotide of 2638 to 2655 bases among the SEQ ID NO:1 and the Nucleotide of 3071 to 3088 bases; The Nucleotide of 2191 to 2208 bases among the SEQ ID NO:1 and the Nucleotide of 2382 to 2399 bases.The Nucleotide of 7871 to 7888 bases among the SEQ ID NO:1 and the Nucleotide of 8140 to 8157 bases; The Nucleotide of 8444 to 8461 bases among the SEQ ID NO:1 and the Nucleotide of 8896 to 8913 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 3 pairs of primers reaction.The pork filling that participates in 0 viable bacteria obtains negative findings in the PCR of 3 pairs of primers reaction.Illustrate that these 3 pairs of primers are 0.25 bacterium/g to the detection sensitivity of the intestinal bacteria O114 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 Microbiology 1993,7:239-252).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 O110 of the present invention can be applied to set up recombiant vaccine.

Nucleotide sequence (shown in the SEQ IDNO:1) according to the O-antigen-specific to intestinal bacteria O110 type of the present invention, structure specific nucleic acid probe, be fixed on the carrier of chip and make biochip, after the sample that will detect is suitably handled, carry out hybridization with biochip, utilize the biochip signal analysis equipment just can obtain corresponding bacteria situation in the sample then.The DNA chip that this intestinal bacteria O antigen is identified can be directly used in clinical and other check place (as food-processing and production industry, the Micro biological Tests of animal and veterinary industry customs quarantine control etc.).This chip only need enlarge output, just can industrialization under identical condition.

Table 1 has been listed glycosyltransferase gene and oligosaccharide unit treatment gene and intragenic primer and PCR data in the O antigen gene bunch of intestinal bacteria O110 type.Glycosyltransferase gene, transhipment enzyme gene and pol gene and their function corresponding and the size of the O antigen gene bunch of intestinal bacteria O110 type 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 IDNO:1.Is that template carry out PCR with listed corresponding annealing temperature in the table with the genomes of all bacterium in the table 2 with every pair of primer, has obtained corresponding PCR product, and its size is also listed in the table.

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 intestinal bacteria O110 type in the 13rd 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 25ul.Template is dilution in 1: 20, gets 1 μ l.After reaction finishes, get the 10ulPCR product and detect the fragment that amplifies by 0.8% agarose gel electrophoresis.

For orf1, orf2, orf7 gene, each gene all has two pairs of primers detected, every pair of primer has obtained except be PCR in the 28th 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 orf1, orf2, orf7 gene pairs intestinal bacteria O110 type and O-antigen thereof are high specials.

At last, from intestinal bacteria O110 type, screen gene by PCR: three glycosyltransferase genes (orf1, orf2, orf7 gene) to the O-antigen high special of intestinal bacteria O110 type.And the oligonucleotide of these intragenic any one section 10-20nt is special to the O-antigen of intestinal bacteria O110 type, and the primer in especially above-mentioned each gene is that oligonucleotide is high special to detecting the back confirmation through PCR to intestinal bacteria O110 type.These all oligonucleotide all can be used for the intestinal bacteria O110 type 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 O110 type, in table, listed the structure of the O-antigen gene bunch of intestinal bacteria O110 type, altogether by 7 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 O110 type, listed the accurate position of all open reading frame in complete sequence in the O-antigen gene bunch of intestinal bacteria O110 type 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 O110 type

＜130〉to the Nucleotide of the O antigen-specific of intestinal bacteria O110 type

<160>1

<170>PatentIn version 3.2

<210>1

<211>9800

<212>DNA

<213>Escherichia coli

<400>1

attgtggctg cagggatcaa agaaatcctc ctggtaactc acgcgtccaa gaacgcggtc 60

gaaaaccact tcgacacctc ttatgaatta gaatctctcc ttgaacagcg cgtgaagcgt 120

cagctgctgg cggaagtgca gtccatctgt ccgccgggtg tgaccattat gaacgtgcgt 180

cagggcgaac ctttaggttt gggccactcc attttatgtg cacgacccgc cattggtgac 240

aacccatttg tcgtggtgct gccagacgtt gtgatcgacg acgccagcgc cgacccgctg 300

cgctacaacc ttgctgccat gattgcgcgc ttcaacgaaa caggacgcag ccaggtgctg 360

gcaaaacgta tgccgggtga cctctctgaa tactccgtca ttcagaccaa agaaccgctg 420

gatcgcgaag gcaaagtcag ccgcatcgtg gaatttatcg aaaaaccgga tcagccgcag 480

acgctggact cagacatcat ggccgtgggt cgctatgtgc tttctgccga tatttggccg 540

gaacttgaac gcactcagcc tggtgcatgg gggcgtattc agctgactga tgccatcgct 600

gaactggcga aaaaacagtc cgttgatgcc atgctgatga caggtgacag ctacgactgc 660

ggtaaaaaaa tgggttatat gcaggcattt gtgaagtatg gactgcgcaa cctgaaagaa 720

ggggcgaagt tccgtaaagg gattgagaag ctgttaagcg aataatgaaa atctgaccgg 780

atgtaacggt tgataagaaa attataacgg cagtgaagat tcgtggcgaa agtaatttgt 840

tgcgaatatt cctgccgttg ttttatataa acaatcagaa taacaacgag ttagcaatag 900

gattttagtc aaagttttcc agggttttcc ttgtttccag agcggattgg taagacaatt 960

agcgtttgaa tttttcgggt ttagcgcgag tgggtaacgc tcgctacatc gtaggcatgc 1020

atgcagtgct ctggtagctg taaagccagg ggcggtagcg tggttgttgt ggaattaatt 1080

aatagcgagt ttgtgtttga gattttatta aaatgaaaaa aattctacat atacaattga 1140

tgccgattct tagtggtgcg caaaaggttt gcttggatga aattgagaat ttaggaaaga 1200

actatgaaca atggttgctt tgctcgggtg ttggaccttt tactaaaaag gcatctgaat 1260

ttggagtcaa acatatagta gtagataatc ttaaaagaga gatatcgatt agagatgatt 1320

tattggctct ttttaagata tatcaaaaca ttaaaaacaa gaagtttgat attgttcata 1380

ctcactcttc taagactggt tttttaggta gggtcgcagc gaggtgtgcc ggtgtaaaca 1440

ccatagtaca tactgtacat ggttttgcat ttccctcaac ggaaaatata ctcttaaaaa 1500

taatatatta tgtgatggag gttatagcag ctttatgtac tgatgcttta attgttatga 1560

ataaaagtga ttacgaatta gcaaaaaaat atttaccaat atcgaataat aaaatatatc 1620

ttcttaataa cggtgtaaaa ctagaaaaat ttaagtgtaa gggagataat tacacaaata 1680

atgacacaat taaaatcgtg atggttgggc gtttatgtga gcaaaaaaac ccattgttat 1740

tactgaatgc attttgttct ctgcaggata attatcaatt gtattatata ggtgatggac 1800

cattacgaac tgaaatagaa aaagaaattt taaataaaaa attaaatgag agagtcaaaa 1860

tattggggtg ggtagataat gtacccgaag tattgccatc ttttgatatt tttgttttgc 1920

catcaagatg ggaaggtatg ccattggcaa tgcttgaagc tatggcatca ggcttgcctg 1980

tgatttcgtc agatattcca tctaatagat ttttaattga aaatactaat ggtgagttat 2040

ttaaaagtga agatgctatc tctttaaaag aaaaaataga atatttgggg agagatccca 2100

agaaaagagc gttgatttcc caagatgcct ataaaaaaat aaaagccaat ttcagtttgg 2160

acgcaagaat atgctctttg aggagaatat atgaggagca ataaagtcat tttgtttatt 2220

gattcattta agaaaggggg ggctgaacgt gtttgttcaa attatgcatc aatacttttt 2280

aatcataatg ttgatgtaga aatatggact tataatatag gtgacaaaaa aatgctgcag 2340

gatatatgcg gcaaaataaa agttcatcaa tttaattcta tgaatgggac aagatgttta 2400

ttaaaaataa taaataaatt tcgtaaggaa actaatgcaa tatttatcgc atttaatcat 2460

caaatagcac ttttattaat tatgtcaagg tatgtaacgg ggaaaaaaaa tattatcata 2520

gctcgtaatg taaattactt aagtaaagat ctttataaag atacttcgtt aaaaactaag 2580

cttgtgtctt ttttgatgac caaactgtac agtaagtgcg atcgttttat tgctcaatgc 2640

gaggcaatga aacaagatat gattaattct tttaatatta ccccatcaaa aataaaagta 2700

atatataatc ctttatctcc tcaaattaaa aaacaaagag tttctcaaga gtgcgaaata 2760

aatacggata tactatttgt cggtcgactt gaaaagcaaa aaggaattga taaattagta 2820

aggatcatta actatgtact tgatatcgaa aaagatgtga cgataaaaat aataggaaat 2880

ggttcgttag tatcgctttt aaaagaaata aaacgtagac cgggaactga aataattcat 2940

ttgcctgaag ttgataatat ggcagagcat taccaaaatt cgcgaatgct tattttaacg 3000

tcaaattatg aagggtttcc taatgtatta atcgaagctt taggaagtgg tatacctgtt 3060

atcagttttg attgtccctc tgggccagcg gagattatag atgatggtaa gaatggtttt 3120

ttaattcctg ttgatgatta tgattattat gttgatagaa tttttttcct tttaaaaaat 3180

aatctgaata gttatatccc agaaaagatt gagaatggtg aaaaagattt gttaagttta 3240

tataatgagg ttagcaatga aaattaatat tattactgtg gatggatatt ttggacaaag 3300

attaccgaca tcaaaaagcc tggaactaca atctttagag caacacttga atttatatgg 3360

agtaaaatgt gaattcattg atattcaaga tgttcagaat ttacgtttag atagtgatac 3420

tcgctgtatt ataggtagtc atcaaaatcc gggtgtaaaa aaatatatag atgacgtaat 3480

atctataaaa tttgaaaatt atagagacag tttaatacca agtgtgcgaa atatattagc 3540

gcatgaaaac aaaggtgtgc aagctattat aatgaaagaa tgccctgatt tatttgttca 3600

acaaaattat tattatcgtg atgacataga aataaataaa gtaaaagtac tgaaacggac 3660

gtatggagca gggagttttg gggttgcatt agctacagat aataaaactt ttttaaaaca 3720

aaaaaagaaa atgttttatt ctgatttgaa atttgatgat attaagtttt atatatttga 3780

atgtttaaag aaatatatat ttccctcaaa acttagtaaa gaacacacta tttattttca 3840

aagatacaat ccttacgttt tacaagattt tattcctgat ttgaatagtg attataaggt 3900

gttagtattt ggcaataaat attatgttct taaaaggaat gttcgggata atgactttcg 3960

tgcaagtgga agtggaaaat ttgtatttga ggatgtttct gaaaagtttc tctcatttgc 4020

cagaaaagta atagaatttt tgcacactcc atatgcatct cttgatatag tagaaagggg 4080

ggatggattt ggctgtattg aatttcagtg cgtacatttt ggaccatata ctcaacttaa 4140

tgcggagtat tattatgaat ttaaaaatgg taagtggatt agagaaaaaa ataattatac 4200

tttagagcag gtctatgctg aagcattagt ggatttttta aaaaatgaat aattttataa 4260

caaatatcct taaattaata tctggtactg ctggtgggca acttatcgca ttgttattga 4320

tgcctattgt aacaaggctg gttacagtag atgagatggg gaagtattca cttgtactgg 4380

ctataattat ggtgctaagt agtgttgcaa gcatgaaact tgagcttgcc gttatgactt 4440

tatcatcaaa gacgcttgtc attcatacat tgtctgcttt ttgtaatgtc aactttataa 4500

ttggtattct ggtacttatt acaggtattt tgttgcattt ttccattgag cattttccat 4560

tgacagcggt tgatgtagtc cttaccgcgt tagctttgta tttcttttct cttgcacaat 4620

ttgtgtcatc ctattattca gcacttgatg agttcaaaaa aagcgcaata aacagatttg 4680

ccaaacctgt attagctgtt gtttttcaga tagtattagt aaatttagga tttggcgaaa 4740

atggcttatt attaggattt gttataggtt atatactaag ttgttttata tttgattcta 4800

aattcatatt gatgacgatg tgtcatattg attttaggtt cgtgaaagag tcttacaaac 4860

agtttaaatc atattttatt tttcaggcac cagctggtgt tattaattca ttatcacaaa 4920

atgtcgcaaa ttttatgcta atcggtatga taggagccta ttcgcttggt atttattcac 4980

tttcgtttcg catgttacaa gcacctattt ccttggtaag tgtttctgtg cgcgatgcat 5040

atttctcacg tgcaaaatcg atatatgaga aagaaccatc gaaacttgca aatgacatgt 5100

tatatgttat tattactttg acaatattat cggggttggt ctccgcgatt attttttcat 5160

atcttccttt tattttcgca caactatttg gaaattcttg gagagagtca ggtgtcgtgg 5220

catcatacct cctcccttgg tttgttctgt tattttccaa tcccccagct tcagtagttg 5280

ctaatataat aaacatgcag aggtttatat ttatatttga gatcgcaaat ctaattgttc 5340

gggtattaac attaacattt tcttggtggt tttataaaga ttttaaactg ttaatagttc 5400

attttagttt tgccggagta attatgaatt tgctatatat tctgatggtt tataaaatgg 5460

ctattgaaaa atgcaaggta caacatgcaa aataacaaaa tcattattgg tggtgatgtt 5520

tcttttgatc aagttaaagt tgacgagtac gcattaagtg gattgagtaa attgtcttca 5580

tttctcggtg aggcggaata ttctattgta aatttagaat gtcctattac caatagccga 5640

gaaaaaatta acaagattgg tcccaatata aagtgtggga ctaatgatgg aacgaaatta 5700

ttatcagcat tgaacttgac tcatgtaacc cttgcaaata atcatattat ggattatgga 5760

tatgaggggt tgaaagatac aatagaacta tgtaattcat taaatattga aacttttggg 5820

gctggaatta actcagagca ggctagcgca ataccagcga taatattatc caatgaaaaa 5880

aagattgcta taataaatgc ttgcgaaaaa gaattttgta tttcagatct aaataagcct 5940

ggagctgcgc attgtaatgt aatcgatatg tatcaccgta ttacagaagc taagaaaaat 6000

aatgatgagg tagtagttat tttacacggt ggacatgagc attataaata tgttagtcca 6060

gagtatcaaa agaaacttcg tttttttgct gagttgggcg tgtcggctat tatatctcat 6120

catacacact ttgcctctgg atatgaaata tgggcaggat gtcctatatt ttatagcttg 6180

ggtaatttga ttttttctcg cgaaaattat aaagattatt ggtaccatgg ataccttgta 6240

actttagatt tttctgaaga aaaaatatca tttgaactta ctccatacag gcaaggtaaa 6300

aaaaataatc tatttgagtt tatagctggg gatgaaaaag aaaaatattt aaaagatata 6360

catgaaataa ataatgtttt gcaaaataga gagttattaa tggatttatg gacgaaatat 6420

gttcataaaa atgaatatgc ttatttttgt tgggttaatg gtttaaatcg tttgcagcgc 6480

atgattttga aaaaaattgg aattattaga tttattaaat ttggactcag taatagtaaa 6540

aaacagctaa gactttggca attgattaat tgtgagtcac ataaagaaat gacggaaaaa 6600

gtgttagagg tagtgaatga tgacatcaga aggaactaaa actccacttt tttcgtatat 6660

gctttccttt tcattgttat ttataatgtc ttgtgaaagt tttaatgaaa caagatatct 6720

tcccgtattt tttcttttgt ttatcactgc attttattac attttcttta taaggaagat 6780

ctctgtatct cttttatatt ccttcggttt agtattgtct ctggtcatta ttttgggagc 6840

gttgtcttca ttgccagtat tgtcgtataa acaaaatttg gtcaattatg gactgtcgga 6900

tttttatata tctattgttt ttaagttgct ggtagcattt tttatattgg ctctctgtcc 6960

aaataattta acattaataa acatcatcaa aggggttttg attgttcatt taagtgtttt 7020

ttatgtacag gcggctattg tctacttaac aggttactat atagatttgt tatatcctgt 7080

tacgggagag ttacaacgtt atgaaagtct tttctttttg ccttggatag gagcggttta 7140

tagacctacg gggttgtatg ttgaaccctc aactttttct tcttttattt tatggttgct 7200

ttcaataaaa attttatgcc agaaaaattt ttgcagattt gatatatttg tgatcgtttc 7260

atcaatgctc tcgctatcat tggctgcaat tgtttatggt gcaatgttcc ttttacttgc 7320

tttactatgt tctggttcga ttaaactaat taaaaaaatt caatttgttt tattgttact 7380

tcctgctcca tttgtatttt atgaaatttt caaagctcgc cttgaggcat taggaggaag 7440

tggtgaaagt ttgagggaaa atttaaataa attggttttt tctcaagatc cattaacatt 7500

gctatttggt aatggtttac tgggattacc ggccgaggta agtgacttaa attcaggtgc 7560

tgaactctgg cgtttaggta tcgctgctct gaatgataac gggctttggt tgttttttat 7620

cataaaagta ggattggttg gtttatttat actaataatt ataatgttac tcattacacg 7680

aaaaaaaata gattttatag tgttcgtcat attattactg acgaaggtta ctttttttag 7740

ctttatattt gttttttatt ttttgacaat ggtttattta ttattgaagc gcggttctaa 7800

ctatgaaaaa aatactattt attgaatccc ataacggtaa aatatccggc gcacagaagg 7860

tgacattaaa tgtggctaaa cttctgtctg aaaggggtga tgatttaaca gttatgtatc 7920

cagggagcta tttagacgaa ttggctttga aatacaaatc ttatgcaaaa gctattaaat 7980

gctacaaaat gccaagtagt ttaggtagtg gaggatttga tgggttaaat gtgttgcaaa 8040

aaatacaagt aatcattgtt tcttttttga aaatgatttt tttttatttt agtgtttcct 8100

ggtattcaag gaagaataaa tttgattaca tttatgcata tgacccaaga ggactatttc 8160

tagcattgcc tgtagcagtg ctctctaaaa aaaagattat atggcatctc catggaaaat 8220

ttttatatgg tgacttcttt tttaaaatta taaatttttt ttgctataga gtcttagtcc 8280

catctcacgc tattaaagaa gcattaccta attcgaaaaa aattaatgtt ctttataatg 8340

gttttgaatt tagagaaatg ggtaatgttc catataaaaa tagtaatgtg cttaagattt 8400

tatttgtcgg ggtttttaca ccgcaaaagg gagtgcattt acttttggag gcactttcac 8460

tttataaaaa taaaaataaa aaaattgaat taacccttgt aggggatata ttagatgatg 8520

attggaaatg gtacgctgat ttgctaaatg aaaaaattaa acttttgcct gaaaatattt 8580

ctatatgtac accaggctgg gtaaaagatg tctctttgta tttaaaattg ggtgattatt 8640

tgatatttcc ttctcaacat tcatgtgaat tgttaataaa caatgtttct cgtaagttct 8700

atggcagtga agcattacca accgtgttaa ttgaagctca agccatgaat ttaccagtaa 8760

ttgcaaatga tgtgcctggg gtcagagaaa ttgtacaagt gaatggaggg ttcgtggtta 8820

atatgaatag tgttgatgat atactattta ttattgaaag gattgctaaa gagccaagaa 8880

atatgtttac tgtcaaatca gaggaaacca gacgtaagtt ttcattagat gctatgcgaa 8940

agcaattata ttgtttaata ccatgaaata tcataccata tccatatatg gtaattttac 9000

tctaacatac ttctaatcag caaaagccgt gacaacattc acggtaatcc ctgcagacag 9060

gagtaaacaa tgtcaaagca acagatcggc gtcgtcggta tggccgtgat ggggcgcaac 9120

cttgcgctca acatcgaaag ccgtggttat accgtctcta ttttcaaccg ttcccgtgaa 9180

aagacggaag aagtgattgc cgaaaatcca ggcaagaaac tggttcctta ctatacggtg 9240

aaagagtttg ttgaatctct ggaaacgcct cgtcgcatcc tgttaatggt gaaagcaggt 9300

gcaggcacgg atgctgctat tgattccctc aagccatacc tcgataaagg tgacatcatt 9360

attgatggtg gtaatacctt cttcaaggac actattcgtc gtaatcgtga gctatctgca 9420

gaaggcttta acttcattgg taccggtgtt tccggaggcg aagaaggcgc gttgaaaggt 9480

ccttccatta tgcctggtgg ccagaaagaa gcctatgaac tggttgcacc aatcctgacc 9540

aaaatcgccg cagtagctga agacggtgag ccatgcgtta cctatattgg ggccgatggt 9600

gcaggtcact atgtgaagat ggttcacaac ggtattgaat acggcgatat gcagctgatt 9660

gctgaagcct attctctgct taaagatggc ctgaacctca ccaacgaaga actggcacag 9720

acctttaccg agtggaataa cggtgaactg agcagctacc tgatcgacat caccaaagat 9780

atcttcacca aaaaagatga 9800

Glycosyltransferase gene in the O antigen gene of table 1 intestinal bacteria O110 type bunch and oligosaccharide unit treatment gene 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 (℃)
								Orf1	Glycosyltransferase	1113-2204	1884-1901	2150-2167	284bp	0 *	56
			1514-1531	1974-1991	478bp	0 *	56
								Orf2	Glycosyltransferase	2191-3267	2638-2655	3071-3088	451bp	0 *	56
			2191-2208	2382-2399	209bp	0 *	56
								Orf7	Glycosyltransferase	7803-8966	7871-7888	8140-8157	287bp	0 *	56
			8444-8461	8896-8913	470bp	0 *	56

^*Only in intestinal bacteria O110 type, obtain a correct band

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

Group number	The bacterial strain that contains in this group	The source
			1, wild-type e. coli 2, wild-type e. coli 3, wild-type e. coli 4, wild-type e. coli 5, wild-type e. coli 6, wild-type e. coli 7, wild-type e. coli 8, wild-type e. coli 9, wild-type e. coli shigella dysenteriae 10, Shigella bogdii 11, shigella flexneri 12, wild-type e. coli	O1，O2，O5，O7，O8，O9，O12，O13，O14，O15，O16，O17，O18， O19ab，O20，O21，O22，O23，O24 O4，O10，O25，O26，O27，O28，O29，O30，O32，O33，O34，O35， O36，O37，O38，O40，O41，O42，O43 O6，O44，O45，O46，O48，O49，O50，O51，O52，O54，O55，O56， O57，O58，O60，O61，O62，O53 O63，O65，O66，O69，O70，O71，O74，O75，O76，O77，O78， O79，O80，O81，O82，O83，O68 O84，O85，O86，O87，O88，O89，O90，O91，O92，O98，O99， O101，O102，O103，O104，O105，O106，O97 O107，O108，O109，O111，O112ab，O112ac，O113， O115，O116，O118，O120，O123，O125，O126，O128，O117 O129，O130，O131，O132，O133，O134，O135，O136，O137， O138，O139，O141，O142，O143，O144，O145，O140 O146，O147，O148，O150，O152，O154，O156，O157，O158， O159，O160，O161，O163，O164，O165，O166，O153 O168，O169，O170，O171，O172，O173， D1，D2，D3，D4，D5，D6，D7，D8，D9，D10，D11，D12，D13 B1，B2，B3，B4，B6，B7，B8，B9，B10，B11，B12，B13，B14，B15， B16，B17，B18 F1a，F1b，F2a，F2b，F3，F4a，F4b，F5(v：4)，F5(v：7)，F6， DS，DR O3，O11，O39，O59，O64，O73，O96，O95，O114，O151，O155， O124，O167，O162，O121，O127，O149，O119	IMVS a IMVS a IMVS a IMVS a IMVS a IMVS a IMVS a IMVS a b c d d d IMVS a

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

O110

IMVS a

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

a.Institute of Medica1 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 O11O type O antigen gene structure iron

E.coli O110 O-antigen gene cluster

#orf galF orf1 orf2 orf3 wzx orfs wzy orf7 gnd

％G+C 40.0 29.0 28.8 33.1 31.2 30.9 31.3

content

Table 4 intestinal bacteria O110 type O antigen gene cluster gene position

ATTGTGGCTG CAGGGATCAA AGAAATCCTC CTGGTAACTC ACGCGTCCAA GAACGCGGTC 60

GAAAACCACT TCGACACCTC TTATGAATTA GAATCTCTCC TTGAACAGCG CGTGAAGCGT 120

CAGCTGCTGG CGGAAGTGCA GTCCATCTGT CCGCCGGGTG TGACCATTAT GAACGTGCGT 180

CAGGGCGAAC CTTTAGGTTT GGGCCACTCC ATTTTATGTG CACGACCCGC CATTGGTGAC 240

AACCCATTTG TCGTGGTGCT GCCAGACGTT GTGATCGACG ACGCCAGCGC CGACCCGCTG 300

CGCTACAACC TTGCTGCCAT GATTGCGCGC TTCAACGAAA CAGGACGCAG CCAGGTGCTG 360

GCAAAACGTA TGCCGGGTGA CCTCTCTGAA TACTCCGTCA TTCAGACCAA AGAACCGCTG 420

GATCGCGAAG GCAAAGTCAG CCGCATCGTG GAATTTATCG AAAAACCGGA TCAGCCGCAG 480

ACGCTGGACT CAGACATCAT GGCCGTGGGT CGCTATGTGC TTTCTGCCGA TATTTGGCCG 540

GAACTTGAAC GCACTCAGCC TGGTGCATGG GGGCGTATTC AGCTGACTGA TGCCATCGCT 600

GAACTGGCGA AAAAACAGTC CGTTGATGCC ATGCTGATGA CAGGTGACAG CTACGACTGC 660

GGTAAAAAAA TGGGTTATAT GCAGGCATTT GTGAAGTATG GACTGCGCAA CCTGAAAGAA 720

GGGGCGAAGT TCCGTAAAGG GATTGAGAAG CTGTTAAGCG AATAATGAAA ATCTGACCGG 780

ATGTAACGGT TGATAAGAAA ATTATAACGG CAGTGAAGAT TCGTGGCGAA AGTAATTTGT 840

TGCGAATATT CCTGCCGTTG TTTTATATAA ACAATCAGAA TAACAACGAG TTAGCAATAC 900

GATTTTAGTC AAAGTTTTCC AGGGTTTTCC TTGTTTCCAG AGCGGATTGG TAAGACAATT 960

AGCGTTTGAA TTTTTCGGGT TTAGCGCGAG TGGGTAACGC TCGCTACATC GTAGGCATGC 1020

ATGCAGTGCT CTGGTAGCTG TAAAGCCAGG GGCGGTAGCG TGGTTGTTGT GGAATTAATT 1080

Orf1's is initial

AATAGCGAGT TTGTGTTTGA GATTTTATTA AA ATGAAAAA AATTCTACAT ATACAATTGA 1140

TGCCGATTCT TAGTGGTGCG CAAAAGGTTT GCTTGGATGA AATTGAGAAT TTAGGAAAGA 1200

ACTATGAACA ATGGTTGCTT TGCTCGGGTG TTGGACCTTT TACTAAAAAG GCATCTGAAT 1260

TTGGAGTCAA ACATATAGTA GTAGATAATC TTAAAAGAGA GATATCGATT AGAGATGATT 1320

TATTGGCTCT TTTTAAGATA TATCAAAACA TTAAAAACAA GAAGTTTGAT ATTGTTCATA 1380

CTCACTCTTC TAAGACTGGT TTTTTAGGTA GGGTCGCAGC GAGGTGTGCC GGTGTAAACA 1440

CCATAGTACA TACTGTACAT GGTTTTGCAT TTCCCTCAAC GGAAAATATA CTCTTAAAAA 1500

TAATATATTA TGTGATGGAG GTTATAGCAG CTTTATGTAC TGATGCTTTA ATTGTTATGA 1560

ATAAAAGTGA TTACGAATTA GCAAAAAAAT ATTTACCAAT ATCGAATAAT AAAATATATC 1620

TTCTTAATAA CGGTGTAAAA CTAGAAAAAT TTAAGTGTAA GGGAGATAAT TACACAAATA 1680

ATGACACAAT TAAAATCGTG ATGGTTGGGC GTTTATGTGA GCAAAAAAAC CCATTGTTAT 1740

TACTGAATGC ATTTTGTTCT CTGCAGGATA ATTATCAATT GTATTATATA GGTGATGGAC 1800

CATTACGAAC TGAAATAGAA AAAGAAATTT TAAATAAAAA ATTAAATGAG AGAGTCAAAA 1860

TATTGGGGTG GGTAGATAAT GTACCCGAAG TATTGCCATC TTTTGATATT TTTGTTTTGC 1920

CATCAAGATG GGAAGGTATG CCATTGGCAA TGCTTGAAGC TATGGCATCA GGCTTGCCTG 1980

TGATTTCGTC AGATATTCCA TCTAATAGAT TTTTAATTGA AAATACTAAT GGTGAGTTAT 2040

TTAAAAGTGA AGATGCTATC TCTTTAAAAG AAAAAATAGA ATATTTGGGG AGAGATCCCA 2100

AGAAAAGAGC GTTGATTTCC CAAGATGCCT ATAAAAAAAT AAAAGCCAAT TTCAGTTTGG 2160

The termination of the initial orfl of orf2

ACGCAAGAAT ATGCTCTTTG AGGAGAAT AT ATGAGGAGCA ATA AAGTCAT TTTGTTTATT2220

GATTCATTTA AGAAAGGGGG GGCTGAACGT GTTTGTTCAA ATTATGCATC AATACTTTTT 2280

AATCATAATG TTGATGTAGA AATATGGACT TATAATATAG GTGACAAAAA AATGCTGCAG 2340

GATATATGCG GCAAAATAAA AGTTCATCAA TTTAATTCTA TGAATGGGAC AAGATGTTTA 2400

TTAAAAATAA TAAATAAATT TCGTAAGGAA ACTAATGCAA TATTTATCGC ATTTAATCAT 2460

CAAATAGCAC TTTTATTAAT TATGTCAAGG TATGTAACGG GGAAAAAAAA TATTATCATA 2520

GCTCGTAATG TAAATTACTT AAGTAAAGAT CTTTATAAAG ATACTTCGTT AAAAACTAAG 2580

CTTGTGTCTT TTTTGATGAC CAAACTGTAC AGTAAGTGCG ATCGTTTTAT TGCTCAATGC 2640

GAGGCAATGA AACAAGATAT GATTAATTCT TTTAATATTA CCCCATCAAA AATAAAAGTA 2700

ATATATAATC CTTTATCTCC TCAAATTAAA AAACAAAGAG TTTCTCAAGA GTGCGAAATA 2760

AATACGGATA TACTATTTGT CGGTCGACTT GAAAAGCAAA AAGGAATTGA TAAATTAGTA 2820

AGGATCATTA ACTATGTACT TGATATCGAA AAAGATGTGA CGATAAAAAT AATAGGAAAT 2880

GGTTCGTTAG TATCGCTTTT AAAAGAAATA AAACGTAGAC CGGGAACTGA AATAATTCAT 2940

TTGCCTGAAG TTGATAATAT GGCAGAGCAT TACCAAAATT CGCGAATGCT TATTTTAACG 3000

TCAAATTATG AAGGGTTTCC TAATGTATTA ATCGAAGCTT TAGGAAGTGG TATACCTGTT 3060

ATCAGTTTTG ATTGTCCCTC TGGGCCAGCG GAGATTATAG ATGATGGTAA GAATGGTTTT 3120

TTAATTCCTG TTGATGATTA TGATTATTAT GTTGATAGAA TTTTTTTCCT TTTAAAAAAT 3180

AATCTGAATA GTTATATCCC AGAAAAGATT GAGAATGGTG AAAAAGATTT GTTAAGTTTA 3240

The termination of the initial orf2 of orf3

TATAATGAGG TTAGCA ATGA AAAT TAATAT TATTACTGTG GATGGATATT TTGGACAAAG3300

ATTACCGACA TCAAAAAGCC TGGAACTACA ATCTTTAGAG CAACACTTGA ATTTATATGG 3360

AGTAAAATGT GAATTCATTG ATATTCAAGA TGTTCAGAAT TTACGTTTAG ATAGTGATAC 3420

TCGCTGTATT ATAGGTAGTC ATCAAAATCC GGGTGTAAAA AAATATATAG ATGACGTAAT 3480

ATCTATAAAA TTTGAAATTT ATAGAGACAG TTTAATACCA AGTGTGCGAA ATATATTAGC 3540

GCATGAAAAC AAAGGTGTGC AAGCTATTAT AATGAAAGAA TGCCCTGATT TATTTGTTCA 3600

ACAAAATTAT TATTATCGTG ATGACATAGA AATAAATAAA GTAAAAGTAC TGAAACGGAC 3660

GTATGGAGCA GGGAGTTTTG GGGTTGCATT AGCTACAGAT AATAAAACTT TTTTAAAACA 3720

AAAAAAGAAA ATGTTTTATT CTGATTTGAA ATTTGATGAT ATTAAGTTTT ATATATTTGA 3780

ATGTTTAAAG AAATATATAT TTCCCTCAAA ACTTAGTAAA GAACACACTA TTTATTTTCA 3840

AAGATACAAT CCTTACGTTT TACAAGATTT TATTCCTGAT TTGAATAGTG ATTATAACGT 3900

GTTAGTATTT GGCAATAAAT ATTATGTTCT TAAAAGGAAT GTTCGGGATA ATGACTTTCG 3960

TGCAAGTGGA AGTGGAAAAT TTGTATTTGA GGATGTTTCT GAAAAGTTTC TCTCATTTGC 4020

CAGAAAAGTA ATAGAATTTT TGCACACTCC ATATGCATCT CTTGATATAG TAGAAAGGGG 4080

GGATGGATTT GGCTGTATTG AATTTCAGTG CGTACATTTT GGACCATATA CTCAACTTAA 4140

TGCGGAGTAT TATTATGAAT TTAAAAATGG TAAGTGGATT AGAGAAAAAA ATAATTATAC 4200

The termination of the initial orf3 of orf4

TTTAGAGCAG GTCTATGCTG AAGCATTAGT GGATTTTTTA AAAA ATGAA T AATTTTATAA4260

CAAATATCCT TAAATTAATA TCTGGTACTG CTGGTGGGCA ACTTATCGCA TTGTTATTGA 4320

TGCCTATTGT AACAAGGCTG GTTACAGTAG ATGAGATGGG GAAGTATTCA CTTGTACTGG 4380

CTATAATTAT GGTGCTAAGT AGTGTTGCAA GCATGAAACT TGAGCTTGCC GTTATGACTT 4440

TATCATCAAA GACGCTTGTC ATTCATACAT TGTCTGCTTT TTGTAATGTC AACTTTATAA 4500

TTGGTATTCT GGTACTTATT ACAGGTATTT TGTTGCATTT TTCCATTGAG CATTTTCCAT 4560

TGACAGCGGT TGATGTAGTC CTTACCGCGT TAGCTTTGTA TTTCTTTTCT CTTGCACAAT 4620

TTGTGTCATC CTATTATTCA GCACTTGATG AGTTCAAAAA AAGCGCAATA AACAGATTTG 4680

CCAAACCTGT ATTAGCTGTT GTTTTTCAGA TAGTATTAGT AAATTTAGGA TTTGGCGAAA 4740

ATGGCTTATT ATTAGGATTT GTTATAGGTT ATATACTAAG TTGTTTTATA TTTGATTCTA 4800

AATTCATATT GATGACGATG TGTCATATTG ATTTTAGGTT CGTGAAAGAG TCTTACAAAC 4860

AGTTTAAATC ATATTTTATT TTTCAGGCAC CAGCTGGTGT TATTAATTCA TTATCACAAA 4920

ATGTCGCAAA TTTTATGCTA ATCGGTATGA TAGGAGCCTA TTCGCTTGGT ATTTATTCAC 4980

TTTCGTTTCG CATGTTACAA GCACCTATTT CCTTGGTAAG TGTTTCTGTG CGCGATGCAT 5040

ATTTCTCACG TGCAAAATCG ATATATGAGA AAGAACCATC GAAACTTGCA AATGACATGT 5100

TATATGTTAT TATTACTTTG ACAATATTAT CGGGGTTGGT CTCCGCGATT ATTTTTTCAT 5160

ATCTTCCTTT TATTTTCGCA CAACTATTTG GAAATTCTTG GAGAGAGTCA GGTGTCGTGG 5220

CATCATACCT CCTCCCTTGG TTTGTTCTGT TATTTTCCAA TCCCCCAGCT TCAGTAGTTC 5280

CTAATATAAT AAACATGCAG AGGTTTATAT TTATATTTGA GATCGCAAAT CTAATTGTTC 5340

GGGTATTAAC ATTAACATTT TCTTGGTGGT TTTATAAAGA TTTTAAACTG TTAATAGTTC 5400

ATTTTAGTTT TGCCGGAGTA ATTATGAATT TGCTATATAT TCTGATGGTT TATAAAATGG 5460

The termination of the initial orf4 of orf5

CTATTGAAAA ATGCAAGGTA CAAC ATGCAA AA TAACAAAA TCATTATTGG TGGTGATGTT5520

TCTTTTGATC AAGTTAAAGT TGACGAGTAC GCATTAAGTG GATTGAGTAA ATTGTCTTCA 5580

TTTCTCGGTG AGGCGGAATA TTCTATTGTA AATTTAGAAT GTCCTATTAC CAATAGCCGA 5640

GAAAAAATTA ACAAGATTGG TCCCAATATA AAGTGTGGGA CTAATGATGG AACGAAATTA 5700

TTATCAGCAT TGAACTTGAC TCATGTAACC CTTGCAAATA ATCATATTAT GGATTATGGA 5760

TATGAGGGGT TGAAAGATAC AATAGAACTA TGTAATTCAT TAAATATTGA AACTTTTGGG 5820

GCTGGAATTA ACTCAGAGCA GGCTAGCGCA ATACCAGCGA TAATATTATC CAATGAAAAA 5880

AAGATTGCTA TAATAAATGC TTGCGAAAAA GAATTTTGTA TTTCAGATCT AAATAAGCCT 5940

GGAGCTGCGC ATTGTAATGT AATCGATATG TATCACCGTA TTACAGAAGC TAAGAAAAAT 6000

AATGATGAGG TAGTAGTTAT TTTACACGGT GGACATGAGC ATTATAAATA TGTTAGTCCA 6060

GAGTATCAAA AGAAACTTCG TTTTTTTGCT GAGTTGGGCG TGTCGGCTAT TATATCTCAT 6120

CATACACACT TTGCCTCTGG ATATGAAATA TGGGCAGGAT GTCCTATATT TTATAGCTTG 6180

GGTAATTTGA TTTTTTCTCG CGAAAATTAT AAAGATTATT GGTACCATGG ATACCTTGTA 6240

ACTTTAGATT TTTCTGAAGA AAAAATATCA TTTGAACTTA CTCCATACAG GCAAGGTAAA 6300

AAAAATAATC TATTTGAGTT TATAGCTGGG GATGAAAAAG AAAAATATTT AAAAGATATA 6360

CATGAAATAA ATAATGTTTT GCAAAATAGA GAGTTATTAA TGGATTTATG GACGAAATAT 6420

GTTCATAAAA ATGAATATGC TTATTTTTGT TGGGTTAATG GTTTAAATCG TTTGCAGCGC 6480

ATGATTTTGA AAAAAATTGG AATTATTAGA TTTATTAAAT TTGGACTCAG TAATAGTAAA 6540

AAACAGCTAA GACTTTGGCA ATTGATTAAT TGTGAGTCAC ATAAAGAAAT GACGGAAAAA 6600

The termination of the initial orf5 of orf6

GTGTTAGAGG TAGTGA ATGA TGACATCAGA AGGAAC TAAA ACTCCACTTT TTTCGTATAT6660

GCTTTCCTTT TCATTGTTAT TTATAATGTC TTGTGAAAGT TTTAATGAAA CAAGATATCT 6720

TCCCGTATTT TTTCTTTTGT TTATCACTGC ATTTTATTAC ATTTTCTTTA TAAGGAAGAT 6780

CTCTGTATCT CTTTTATATT CCTTCGGTTT AGTATTGTCT CTGGTCATTA TTTTGGGAGC 6840

GTTGTCTTCA TTGCCAGTAT TGTCGTATAA ACAAAATTTG GTCAATTATG GACTGTCGGA 6900

TTTTTATATA TCTATTGTTT TTAAGTTGCT GGTAGCATTT TTTATATTGG CTCTCTGTCC 6960

AAATAATTTA ACATTAATAA ACATCATCAA AGGGGTTTTG ATTGTTCATT TAAGTCTTTT 7020

TTATGTACAG GCGGCTATTG TCTACTTAAC AGGTTACTAT ATAGATTTGT TATATCCTGT 7080

TACGGGAGAG TTACAACGTT ATGAAAGTCT TTTCTTTTTG CCTTGGATAG GAGCGGTTTA 7140

TAGACCTACG GGGTTGTATG TTGAACCCTC AACTTTTTCT TCTTTTATTT TATGGTTGCT 7200

TTCAATAAAA ATTTTATGCC AGAAAAATTT TTGCAGATTT GATATATTTG TGATCGTTTC 7260

ATCAATGCTC TCGCTATCAT TGGCTGCAAT TGTTTATGGT GCAATGTTCC TTTTACTTGC 7320

TTTACTATGT TCTGGTTCGA TTAAACTAAT TAAAAAAATT CAATTTGTTT TATTGTTACT 7380

TCCTGCTCCA TTTGTATTTT ATGAAATTTT CAAAGCTCGC CTTGAGGCAT TAGGAGGAAG 7440

TGGTGAAAGT TTGAGGGAAA ATTTAAATAA ATTGGTTTTT TCTCAAGATC CATTAACATT 7500

GCTATTTGGT AATGGTTTAC TGGGATTACC GGCCGAGGTA AGTGACTTAA ATTCAGGTGC 7560

TGAACTCTGG CGTTTAGGTA TCGCTGCTCT GAATGATAAC GGGCTTTGGT TGTTTTTTAT 7620

CATAAAAGTA GGATTGGTTG GTTTATTTAT ACTAATAATT ATAATGTTAC TCATTACACG 7680

AAAAAAAATA GATTTTATAG TGTTCGTCAT ATTATTACTG ACGAAGGTTA CTTTTTTTAG 7740

CTTTATATTT GTTTTTTATT TTTTGACAAT GGTTTATTTA TTATTGAAGC GCGGTTCTAA 7800

The termination of the initial orf6 of orf7

CT ATGAAAAA AATACTATTT AT TGAATCCC ATAACGGTAA AATATCCGGC GCACAGAAGG7860

TGACATTAAA TGTGGCTAAA CTTCTGTCTG AAAGGGGTGA TGATTTAACA GTTATGTATC 7920

CAGGGAGCTA TTTAGACGAA TTGGCTTTGA AATACAAATC TTATGCAAAA GCTATTAAAT 7980

GCTACAAAAT GCCAAGTAGT TTAGGTAGTG GAGGATTTGA TGGGTTAAAT GTGTTGCAAA 8040

AAATACAAGT AATCATTGTT TCTTTTTTGA AAATGATTTT TTTTTATTTT AGTGTTTCCT 8100

GGTATTCAAG GAAGAATAAA TTTGATTACA TTTATGCATA TGACCCAAGA GGACTATTTC 8160

TAGCATTGCC TGTAGCAGTG CTCTCTAAAA AAAAGATTAT ATGGCATCTC CATGGAAAAT 8220

TTTTATATGG TGACTTCTTT TTTAAAATTA TAAATTTTTT TTGCTATAGA GTCTTAGTCC 8280

CATCTCACGC TATTAAAGAA GCATTACCTA ATTCGAAAAA AATTAATGTT CTTTATAATC 8340

GTTTTGAATT TAGAGAAATG GGTAATGTTC CATATAAAAA TAGTAATGTG CTTAAGATTT 8400

TATTTGTCGG GGTTTTTACA CCGCAAAAGG GAGTGCATTT ACTTTTGGAG GCACTTTCAC 8460

TTTATAAAAA TAAAAATAAA AAAATTGAAT TAACCCTTGT AGGGGATATA TTAGATGATG 8520

ATTGGAAATG GTACGCTGAT TTGCTAAATG AAAAAATTAA ACTTTTGCCT GAAAATATTT 8580

CTATATGTAC ACCAGGCTGG GTAAAAGATG TCTCTTTGTA TTTAAAATTG GGTGATTATT 8640

TGATATTTCC TTCTCAACAT TCATGTGAAT TGTTAATAAA CAATGTTTCT CGTAAGTTCT 8700

ATGGCAGTGA AGCATTACCA ACCGTGTTAA TTGAAGCTCA AGCCATGAAT TTACCAGTAA 8760

TTGCAAATGA TGTGCCTGGG GTCAGAGAAA TTGTACAAGT GAATGGAGGG TTCGTGGTTA 8820

ATATGAATAG TGTTGATGAT ATACTATTTA TTATTGAAAG GATTGCTAAA GAGCCAAGAA 8880

ATATGTTTAC TGTCAAATCA GAGGAAACCA GACGTAAGTT TTCATTAGAT GCTATGCGAA 8940

The termination of orf7

AGCAATTATA TTGTTTAATA CCA TGAAATA TCATACCATA TCCATATATG GTAATTTTAC 9000

TCTAACATAC TTCTAATCAG CAAAAGCCGT GACAACATTC ACGGTAATCC CTGCAGACAG 9060

GAGTAAACAA TGTCAAAGCA ACAGATCGGC GTCGTCGGTA TGGCCGTGAT GGGGCGCAAC 9120

CTTGCGCTCA ACATCGAAAG CCGTGGTTAT ACCGTCTCTA TTTTCAACCG TTCCCGTGAA 9180

AAGACGGAAG AAGTGATTGC CGAAAATCCA GGCAAGAAAC TGGTTCCTTA CTATACGGTG 9240

AAAGAGTTTG TTGAATCTCT GGAAACGCCT CGTCGCATCC TGTTAATGGT GAAAGCAGGT 9300

GCAGGCACGG ATGCTGCTAT TGATTCCCTC AAGCCATACC TCGATAAAGG TGACATCATT 9360

ATTGATGGTG GTAATACCTT CTTCAAGGAC ACTATTCGTC GTAATCGTGA GCTATCTGCA 9420

GAAGGCTTTA ACTTCATTGG TACCGGTGTT TCCGGAGGCG AAGAAGGCGC GTTGAAAGGT 9480

CCTTCCATTA TGCCTGGTGG CCAGAAAGAA GCCTATGAAC TGGTTGCACC AATCCTCACC 9540

AAAATCGCCG CAGTAGCTGA AGACGGTGAG CCATGCGTTA CCTATATTGG GGCCGATGGT 9600

GCAGGTCACT ATGTGAAGAT GGTTCACAAC GGTATTGAAT ACGGCGATAT GCAGCTGATT 9660

GCTGAAGCCT ATTCTCTGCT TAAAGATGGC CTGAACCTCA CCAACGAAGA ACTGGCACAG 9720

ACCTTTACCG AGTGGAATAA CGGTGAACTG AGCAGCTACC TGATCGACAT CACCAAAGAT 9780

ATCTTCACCA AAAAGATGA 9800

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 (4)

1, a kind of oligonucleotide of the O-antigen-specific to intestinal bacteria O110 type, it is characterized in that: its orf1 sequence is that Nucleotide, the orf2 sequence of 1113 to 2204 bases among the SEQ ID NO:1 is that Nucleotide, the orf7 sequence of 2191 to 3267 bases among the SEQID NO:1 is the Nucleotide of 7803 to 8966 bases among the SEQ ID NO:1 or has above-mentioned sequence and insert, lack or replace one or more Nucleotide, but keeps the oligonucleotide of the O-antigen function of wzx, wzy, orf1, orf2, orf7 Nucleotide specific detection intestinal bacteria O110 type.

2, a kind of oligonucleotide of the O-antigen-specific to intestinal bacteria O110 type, it is characterized in that, it is that to come from the oligonucleotide of orf1 gene right: the Nucleotide of 1884 to 1901 bases among the SEQ ID NO:1 and the Nucleotide of 2150 to 2167 bases, the Nucleotide of 1514 to 1531 bases among the SEQID NO:1 and the Nucleotide of 1974 to 1991 bases; The oligonucleotide that comes from the orf2 gene is right: the Nucleotide of 2638 to 2655 bases among the SEQ ID NO:1 and the Nucleotide of 3071 to 3088 bases, the Nucleotide of 2191 to 2208 bases among the SEQ ID NO:1 and the Nucleotide of 2382 to 2399 bases; The oligonucleotide that comes from the orf7 gene is right: the Nucleotide of 7871 to 7888 bases among the SEQ ID NO:1 and the Nucleotide of 8140 to 8157 bases, the Nucleotide of 8444 to 8461 bases among the SEQ ID NO:1 and the Nucleotide of 8896 to 8913 bases.

3, the application of oligonucleotide in detecting the O-antigen of expressing the antigenic bacterium of O-, identifying bacterium of claim 1, any described O-antigen-specific to intestinal bacteria O110 type of 2.

4, according to the application of the oligonucleotide of claim 1, any described O-antigen-specific to intestinal bacteria O110 type of 2, it is characterized in that, it is used for PCR, is used for hybridization and fluoroscopic examination or is used to make gene chip or microarray as probe as primer, and the confession detection bodies reaches the bacterium in the environment outward.