CN1300319C - Nucleotide specific to 0174 type O antigen of bacillus coli - Google Patents

Abstract

The present invention provides a specific nucleotide for the O-antigens of Escherichia coli O174. The specific nucleotide is the nucleotide complete sequence of gene clusters in Escherichia coli O174 for controlling the synthesis of O-antigens, such as a separated nucleotide with the total length of 5668 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 present invention also comprises the oligonucleotide of glycosyltransferase genes and unit processing genes stemmed from the O-antigen gene clusters of Escherichia coli O174. The present invention verifies that the oligonucleotide has high specificity to all of the O-antigen genes of Escherichia coli O174 through PCR. The present invention also discloses a method for using the oligonucleotide of the present invention to detect and identify Escherichia coli O174.

Description

Nucleotide to the O-antigen-specific of intestinal bacteria O174 type

Technical field

The present invention relates to the complete nucleotide sequence of control O-antigen synthetic gene cluster in the intestinal bacteria O174 type (Escherichia coli O174), particularly relate in the intestinal bacteria O174 type oligonucleotide in the control O-antigen synthetic gene cluster, can utilize these the oligonucleotide of the O-antigen-specific intestinal bacteria O174 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 Oantigens " .Trends in Microbiology.3:178-185 on the glycolipid molecule; Schnaitman, C.A.andJ.D.Klena. (1993) " Genetics of lipopolysaccharide biosynthesis inentericbacteria " .Microbiological Reviews, 57 (3): 655-682].Coding is responsible for the generally adjacent arrangement on karyomit(e) of gene of all enzyme molecules of O-antigen synthetic, form a gene cluster [Reeves, P.R., et al. (1996) " Bacterial polysaccharide synthesis and genenomenclature " Trends in Microbiology, 4:495-503].In Shigellae, intestinal bacteria and Salmonellas, O-antigen gene [Lei Wang.et al (2001) " Sequence analysis of four Shigella boydii O-antigen loci:implicationfor Escherichia coli and Shigella relationships " .Infection andImmunity, 11:6923-6930 bunch between galF and gnd gene; Lei Wang and Peter Reeves (2000) " TheEscherichia coli O111 and Salmonella enterica O35 gene clusters:geneclusters encoding the same colitose-containing O antigen are highlyconserved " .Journal of Bacteriology.182:5256-5261].The O-antigen gene bunch contains three genoids: sugared synthesis path gene, glycosyltransferase gene, oligosaccharide unit treatment gene, the required nucleoside diphosphate monose of enzymic synthesis O-antigen of wherein sugared synthesis path genes encoding; Thereby the enzyme of glycosyltransferase gene coding forwards nucleoside diphosphate monose and other molecule to and makes monose aggregate into oligosaccharide unit on the monose; The oligosaccharide unit treatment gene comprises transhipment enzyme gene and pol gene, and they transfer to the bacterium inner membrance outside with oligosaccharide unit, and repolymerization becomes polysaccharide.Glycosyltransferase gene and oligosaccharide unit treatment gene only are present in the gene cluster of carrying these genes.The difference of monose in the O-antigen, between monose between the difference of link button and the oligosaccharide unit difference of link button constituted the antigenic diversity of O-, and the composition of monose, the link button between monose and the link button between the oligosaccharide unit are by the Gene Handling in the O-antigen gene bunch, so the O-antigen gene bunch has determined O-antigenic synthetic, has also determined the antigenic diversity of O-.

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

Shigellae has 46 kinds of serotypes, but have only 33 kinds of different O-antigens, intestinal bacteria have 166 kinds of different O-antigen [Reeves, P.R (1992) " Variation in O antigens; nichespecific selection and bacterial populations " .FEMSMicrobiol.Lett, 100:509-516], the two sibship is very near, and 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 SciencePublishers, Amsterdam, The Netherlands; T.cheasty, et al. (1983) " Antigenic relationships between the enteroinvasive Escherichiacoli 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 O174 type.It is the Nucleotide in the O-antigen gene bunch of intestinal bacteria O174 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 O174 type.

Another object of the present invention has provided the gene of the O-antigen gene bunch that constitutes intestinal bacteria O174 type: 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.

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 O174 type respectively comprises orf1, orf4, orf5 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 O174 type; Especially the oligonucleotide of listing in the table 1, they are high specials to the O-antigen of intestinal bacteria O174 type, and these oligonucleotide are also reconfigurable, the oligonucleotide after the combination also is a high special to the O-antigen of intestinal bacteria O174 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 O174 type of these methods detections and identification of escherichia coli O174 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 O174 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 characterized in that the Nucleotide of the O-antigen-specific of intestinal bacteria O174 type: it is the isolating Nucleotide shown in SEQ ID NO:1,5668 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.

The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O174 type, comprising called after orf1, wzx, wzy, orf4,5 genomic constitutions of orf5 are all between galF gene and gnd gene.

The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O174 type, 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 orf1, orf4, orf5 gene; Wherein said gene: wzx is the Nucleotide of 805 to 2076 bases among the SEQ ID NO:1; Wzy is the Nucleotide of 2057 to 3196 bases among the SEQ ID NO:1.

The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O174 type wherein also comprises coming from described wzx gene, wzy gene or glycosyltransferase gene orf1, orf4, orf5 gene and their mixing or their reorganization.

The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O174 type is characterized in that the oligonucleotide that comes from the wzx gene is to being: the Nucleotide of 891 to 908 bases among the SEQ ID NO:1 and the Nucleotide of 1529 to 1546 bases; The Nucleotide of 1479 to 1498 bases among the SEQ ID NO:1 and the Nucleotide of 1861 to 1878 bases; The oligonucleotide that comes from the wzy gene is to being: the Nucleotide of 2421 to 2439 bases among the SEQ ID NO:1 and the Nucleotide of 3160 to 3179 bases; The Nucleotide of 2512 to 2529 bases among the SEQ ID NO:1 and the Nucleotide of 2944 to 2961 bases

The application of the Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O174 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 O174 type is providing the O-antigen of expressing intestinal bacteria O174 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 O174 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 O174 type is characterized in that it comprises the steps:

(1) genomic extraction: in substratum, cultivate intestinal bacteria O174 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 O174 type bunch: with the genome of intestinal bacteria O174 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 O174 type:

(6) screening of specific gene: at wzx, wzy, the gene design primer in the O-antigen gene of intestinal bacteria O174 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 former high degree of specificity of O-of wzx, wzy gene pairs intestinal bacteria O174 type;

(7) detection of primer sensitivity: cultivate intestinal bacteria O174, 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 O174.

The separation method of the Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O174 type is characterized in that it comprises the steps:

(1) genomic extraction: 37 ℃ of incubated overnight intestinal bacteria O174 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 3ul10mg/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 O174 type bunch: with the genome of intestinal bacteria O174 type is that template is passed through its O-antigen gene of Long pcr amplification bunch, at first according to the jumpstart sequences Design upstream primer (wl-10985-ATTGGTAGCTGTAAGCCAAGGGCGGTAGCGT-3) that often is found in O-antigen gene bunch promoter region, again according to the gnd gene design downstream primer (wl-22115-CACTGCCATACCGACGACGCCGATCTGTTGCTTGG-3) in O-antigen gene bunch downstream; With the Expand Long Template PCR method of Boehringer Mannheim company amplification O-antigen gene bunch, the PCR response procedures was as follows: 94 ℃ of pre-sex change 2 minutes; 94 ℃ of sex change are 10 seconds then, 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 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 80ul, 70 ℃ of reactions 20 minutes make the 3 ' end of DNA 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 BiO-Rad company prepares the competence e.colidh5, get after 2-3ul connects product and 50ul competence bacillus coli DH 5 alpha mixes, forward in the electric shock cup of 0.2cm of BiO-Rad company and shock by electricity, voltage is 2.5 kilovolts, time is 5.0 milliseconds 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 O174 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 O174 type; The quality of sequence is mainly guaranteed by two aspects: 1) genome of intestinal bacteria O174 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 O174 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 5 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 O174 type at last;

(6) specific gene screening: at wzx, the wzy gene design primer in the O-antigen gene of dysentery intestinal bacteria O174 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 intestinal bacteria O174 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 O174 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 O174 is inoculated in the triangular flask of 20ml LB substratum, in 37 ℃, 200 rev/mins, cultivate 12 hours to saturated, the cultured bacterium liquid that takes a morsel does 10 ⁶With 10 ⁷Dilution doubly, remaining bacterium liquid are put in 4 ℃ the refrigerator standby, get 50 μ l dilution bacterium liquid coating LB agar plate, and 37 degree are cultivated 12h, to being coated with plate count, calculate viable bacteria concentration in the stoste.In 5 portions of live pig meat stuffings, mix 5 * 10 respectively ³, 5 * 10 ², 5 * 10 ¹, 5 and 0 viable bacteria stir, and add 200ml LB substratum, and through 6 layers of filtered through gauze, filtered liquid 200 rev/mins, is cultivated 12h in 37 ℃.Get 3ml bacterium liquid in 6 from cultured bacterium liquid, centrifugal 5 minutes of 000g removes supernatant, adds 100 μ l MQ ultrapure waters and blows precipitation and mixing open, puts into 100 degree boiling water and boils 15 minutes, and lysate is in 12, and centrifugal 8 minutes of 000g gets 1 μ supernatant as pcr template.The Nucleotide of 891 to 908 bases among the SEQ ID NO:1 and the Nucleotide of 1529 to 1546 bases; The Nucleotide of 1479 to 1498 bases among the SEQ ID NO:1 and the Nucleotide of 1861 to 1878 bases; The Nucleotide of 2421 to 2439 bases among the SEQ ID NO:1 and the Nucleotide of 3160 to 3179 bases; The Nucleotide of 2512 to 2529 bases among the SEQ ID NO:1 and the Nucleotide of 2944 to 2961 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 O174 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 O174 type, its complete sequence shown in SEQ ID NO:1,5668 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 O174 type by method of the present invention, as shown in table 3, it comprises called after orf1, wzx, wzy, orf4, orf5,5 genomic constitutions, all between jumpstart sequence and gnd gene.

Second aspect of the present invention provides the gene in the O-antigen gene bunch of intestinal bacteria O174 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.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 O174 type.

The 3rd aspect of the present invention, wzx gene in the O-antigen gene bunch that comes from intestinal bacteria O174 type is provided or the gene, wzy gene of identity function is arranged or with wzy the gene of identity function is arranged with wzx, 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 wzx, wzy gene pairs intestinal bacteria O174 type all is high special.

The separation method of the Nucleotide of described O-antigen-specific to intestinal bacteria O174 type comprises the steps: 1) genomic extraction; 2) the O-antigen gene in the pcr amplification intestinal bacteria O174 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 the oligonucleotide of wzx gene to being: the Nucleotide of 891 to 908 bases among the SEQ ID NO:1 and the Nucleotide of 1529 to 1546 bases; The Nucleotide of 1479 to 1498 bases among the SEQ ID NO:1 and the Nucleotide of 1861 to 1878 bases; The oligonucleotide that comes from the wzy gene is to being: the Nucleotide of 2421 to 2439 bases among the SEQ IDNO:1 and the Nucleotide of 3160 to 3179 bases; The Nucleotide of 2512 to 2529 bases among the SEQ IDNO:1 and the Nucleotide of 2944 to 2961 bases.Coming from above intragenic oligonucleotide is high special to intestinal bacteria O174 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 O174 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 O174 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 O174 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 O174 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 O174 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 O174 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 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 O174 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 O174 type bunch:

With the genome of intestinal bacteria O174 type is that template is passed through its O-antigen gene of Long pcr amplification bunch.At first according to the jumpstart sequences Design upstream primer (wl-1098 that often is found in O-antigen gene bunch promoter region, 5-AITGGTAGCTGTAAGCCAAGGGCGGTAGCGT-3), again according to the gnd gene design downstream primer in O-antigen gene bunch downstream (wl-2211,5-CACTGCCATACCGACGACGCCGATCTGTTGCTTGG-3); 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, the DNaseI of the 1mg/ml of 1ul dilution in 1: 2000, reaction is carried out at room temperature.Enzyme is cut the dna fragmentation size is concentrated between the 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 1.25ul 100mM DTT and 5 units, 11 ℃ 30 minutes, enzyme is cut product mend into flush end, after 75 ℃ of termination reactions, add the Tth archaeal dna polymerase of 5 units and corresponding damping fluid thereof and system is expanded as 80ul, 70 ℃ were reacted 20 minutes, made the 3 ' end of DNA 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 the single bacterium colony of a ring bacillus coli DH 5 Q 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 bacillus coli DH 5 alpha mixes, forward in the electric shock cup of 0.2cm of Bio-Rad company and shock by electricity, voltage is 2.5 kilovolts, and the time is 5.0 milliseconds-6.0 milliseconds.The SOC substratum that adds 1ml after the electric shock immediately in cup makes the bacterium recovery.Immediately bacterium is coated in 37 ℃ of inversion incubated overnight on the LB solid medium that contains penbritin, X-Gal and IPTG then, obtains blue white bacterium colony next day.With the white colony that obtains promptly the white clone forward on the LB solid medium that contains penbritin and cultivate, from each clone, extract plasmid and cut the segmental size of evaluation insertion wherein simultaneously, obtain the O-antigen gene bunch library that white clone group has constituted intestinal bacteria O174 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 O174 type obtained.The quality of sequence is mainly guaranteed by two aspects: 1) genome of intestinal bacteria O174 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 O174 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 5 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 O174 type at last, as shown in table 3.

By retrieving and relatively, finding that orf3 and orf2 are the proteic genes that there is transmembrane segment in only two codings among the intestinal bacteria O174.The O-antigen transferring enzyme of orf2 encoded protein and Yersinia enterocolitica has 27% sequence identity, 49% similarity, 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 orf2 is wzx.The O-antigen polysaccharase of orf3 encoded protein and Bacillus cereus ATCC10987 has 24% consistence, 46% similarity, it contains 12 transmembrane segments by the proteic topology discovery 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 orf3 is wzy.

Orf1, orf4, the albumen of three genes encodings of orf5 and other known glycosyltransferases have the sequence identity of 25-38% and the sequence similarity of 50-57%.By search to glycosyltransferase motif database among the Pfam, the homology desired value of the consensus sequence of the albumen of these three genes encodings and known glycosyltransferase family 1 and 2 is very high, therefore we infer this four 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 O174 may be made up of four monose.Because the definite function of these three genes can't be determined, so we are with the temporary called after orf1 of these three genes, orf4, orf5.

Embodiment 6: the screening of specific gene.

Wzx, wzy gene design primer in the O-antigen gene of intestinal bacteria O174 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 intestinal bacteria O174 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 O174 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 O174 is inoculated in the triangular flask of 20ml LB substratum, in 37 ℃, 200 rev/mins, cultivate 12 hours to saturated, the cultured bacterium liquid that takes a morsel does 10 ⁶With 10 ⁷Dilution doubly, remaining bacterium liquid are put in 4 ℃ the refrigerator standby, get 50 μ l dilution bacterium liquid coating LB agar plate, and 37 degree are cultivated 12h, to being coated with plate count, calculate viable bacteria concentration in the stoste.In 5 portions of live pig meat stuffings, mix 5 * 10 respectively ³, 5 * 10 ², 5 * 10 ¹, 5 and 0 viable bacteria stir, and add 200ml LB substratum, and through 6 layers of filtered through gauze, filtered liquid 200 rev/mins, is cultivated 12h in 37 ℃.Get 3ml bacterium liquid in 6 from cultured bacterium liquid, centrifugal 5 minutes of 000g removes supernatant, adds 100 μ l MQ ultrapure waters and blows precipitation and mixing open, puts into 100 degree boiling water and boils 15 minutes, and lysate is in 12, and centrifugal 8 minutes of 000g gets 1 μ supernatant as pcr template.Right with 4 pairs of oligonucleotide, the Nucleotide of 891 to 908 bases among the SEQ ID NO:1 and the Nucleotide of 1529 to 1546 bases; The Nucleotide of 1479 to 1498 bases among the SEQ ID NO:1 and the Nucleotide of 1861 to 1878 bases; The Nucleotide of 2421 to 2439 bases among the SEQ ID NO:1 and the Nucleotide of 3160 to 3179 bases; The Nucleotide of 2512 to 2529 bases among the SEQ ID NO:1 and the Nucleotide of 2944 to 2961 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 O174 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 O174 of the present invention can be applied to set up recombiant vaccine.

When molecular probe nucleotide sequence and target DNA sequence homology greater than 85% the time, can accurately aim sequence be hybridized out.The homology that requires both in the Southern of low preciseness hybridization is greater than 65% (" molecular cloning experiment guide " third edition, the 509th page, low preciseness hybridization).The homology search of specific nucleotide sequence among the present invention in Genebank shows does not have homology to exist greater than other genes of 65%.So in hybrid experiment, the specific nucleotide sequence among the present invention can only draw positive findings to the purpose bacterium as molecular probe.The Southern hybrid method is not strict with for the length of molecular probe, can use hybridization to whole sequence from 20bp or above oligonucleotide in the specific nucleotide sequence among the present invention.In a Southern experiment, utilize the relevant specific gene (more than the 1000bp) of Salmonellas to do molecular probe, success tell this bacterium (Liu D, Verma NK, RomanaLK, Reeves PR., 1991 Relationships among the rfb regions of Salmonellaserovars A, B, and D.J Bacteriol.173 (15): 4814-4819.), the experiment of a lot of this areas shows that all the gene order about 1000-2000bp can be used as molecular probe.Gene chip is the same with Southern hybridization ratio juris, also similar in the requirement of selecting molecular probe for use, so specific nucleotide sequence among the present invention and oligonucleotide fragment wherein can detect this purpose bacterium as molecular probe in hybridization, comprise the ordinary method of multiple hybridization such as Southern, gene chip.

Nucleotide sequence (shown in the SEQ IDNO:1) according to the O-antigen-specific to intestinal bacteria O174 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 O174 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 O174 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 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 intestinal bacteria O174 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 wzx, wzy gene, each gene all has two pairs of primers detected, every pair of primer has obtained except be PCR in the 12nd 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 O174 type and O-antigen thereof are high specials.

At last, from intestinal bacteria O174 type, screen gene by PCR: three glycosyltransferase genes to the O-antigen high special of intestinal bacteria O174 type.And the oligonucleotide of these intragenic any one section 10-20nt is special to the O-antigen of intestinal bacteria O174 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 O174 type.These all oligonucleotide all can be used for the intestinal bacteria O174 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 O174 type, in table, listed the structure of the O-antigen gene bunch of intestinal bacteria O174 type, altogether by 5 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 jumpstart sequence 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 O174 type, listed the accurate position of all open reading frame in complete sequence in the O-antigen gene bunch of intestinal bacteria O174 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 O174 type

＜130〉to the Nucleotide of the O antigen-specific of intestinal bacteria O174 type

<160>1

<170>PatentIn version 3.2

<210>1

<211>5668

<212>DNA

<213>Escherichia coli

<400>1

ccttttgcat tggtagctgt aagccaaggg cggtagcgta aacatttgag aggaattaat 60

gagtacagtt acactcgtta taactagttg cggtagattt gaattattag aaaaaacaat 120

atcttctctt gtaaataggt acccatttac agagaagatc ataattgagg attctggtaa 180

tgtaaaagtt ataaacaaaa taaaagaaaa atatgatagg gattttacaa tcttaataaa 240

tgaaaaaaac attggacaaa ttaaaagcat cgataaggca tatagtttag tcactaccga 300

atatatattc cattgtgaag atgattggca tttctataga gacggattca tagaagattc 360

attagatata ttgaaagagt atagacacat ttcaatggta tcacttagag attggctaaa 420

tgatgtatcg atcaattgtc atatggaaag atccacgctc ttacaaacgg gaaatggaac 480

aagatttttc atgttaaaac ccaaaaatgg tgatggctgg ggagggtatt cgttcaaccc 540

ggggctgaga cgacttcaag attataaaga aataatcgga caattttcaa aagtagggca 600

tgaaaaaaat ataagtttat attttttaga aaaaggaatg aatatggcgc tattagaatc 660

ttcagctgtt gagcacatag gttggaatca ccatatattg agtaaaaata atcctcaaga 720

aagattttat ttgcttaaaa aatatttacc aaaggaagta actaatgtgc tcaagataat 780

ttatagaaaa attgctgggt gattatgaaa tatcttttta tttggtctct tactcccaaa 840

ttatatgaat taagccttaa tctttctatc gggatagctc ttataaatta tctaggacct 900

gtagaacaag gtaaaatagg ttttttaatt aatttatgca cattaatgag ttttcttacc 960

acgcttggaa ttgggcctgt atattctaat tttgttagtc gttcaaataa tgaaagatta 1020

atatccaata aatttaagga gaatgttaca ttaaggtttt taggttatat tttgtttctt 1080

ctggtttcac ttttatttct attcttcttt aaaagggggt tgatatattt atcaatacca 1140

tttttaatag ggaaattatt tttcagtttt gatatatatt ataacctcat tgaaggtaag 1200

gctggattta aaaattatgc aatttcaaaa tttatatctt tgacatgtat aaatgtattt 1260

cggttatatt gtatatatgc acaattagat atatattggg ttgcgttttc attctttttg 1320

actgatttct tgacattttt tgtgtatttt ttgttatttg acaaatttaa gtatttcggt 1380

tttaaatttg attacaaaaa atcattaatt atatttaaga taaactataa gttagcatta 1440

tctacaatag ttgtaagtct ctttactcag ctggatatag taatgatcgg aactatgctt 1500

ggagataaag ctgcaggtga gtattatgca tcaactagac tcgctactcc tttagttttt 1560

atatcaacaa ttattataag cacttttttt tcaaaacttt ctagaaactg ggttgttaat 1620

aaaaaagaat attatgaact tttagctttc attagtggaa gtataatttt ttcgtattca 1680

gcaattgtac tcattatttt tatatttgga aacgatatat ttttcttgtt tttcagttca 1740

gaatataaag cctcttatga tttattctta atccatattg taggtttgat ttttgtactt 1800

ttaggcccgc ttactgggaa gcatttaata attaaaaaag attatggggc agaacttagc 1860

aaaaccttat tggcagcaat tgttaatata gccttaactt ccattgcagt gttaaaatat 1920

gaaaatctta acttagtggc cgttagtaca ttaataagtt atatgattgc taattttggt 1980

tactttatag taaaaaaaga ttggctatta atcaaagcta tattgaatgg tgttaatccc 2040

ctttttttgg tgagatatgc taaaaagata ctctaacctt atattttctt ttgttattgc 2100

gttttccttt ttttcatacg taatgaatcc cagtttaaaa tttatatata aatcaccagt 2160

aataaatttt atcccttttt tgttatgttg tttgataaca atcatttttc ttacagagaa 2220

aaaaatcaag cttaactatt atgaattatt ttcaattctt tatgttgggt tcttttttat 2280

acttcaatat acctatttta tacaatcgac agattcaata gaaactttac tacgattaat 2340

gtcggtaaat ttttcattct tatttggatt actattaggc tggtgtttta aaagggagtc 2400

aattgagaag ctttttatac tatgggtcct attactttct attgctaata tctgcggtgt 2460

aatcaattat tctgatgggg tggaatttaa tcatttgaac tttacattac ctctagggac 2520

tgttgttaca tggttaattt ttaaagcatt tatgaaagat actgataggt acatattcac 2580

attatctgtt atattatttt tattttttaa tataatattt gccggaagtc ggactgctat 2640

ttttctacca atcttagtaa gtttatttat tatggtgata tttagaaaat atgtgtctat 2700

aaagaaaaca gtgatctcat tcattatatt gataacaata actataatat cattgcctta 2760

tattttaagt aacttgaatg cttatttttt aagcaaggtt caaaatatgg ctgatatcag 2820

tgaggatagt cgttataatt tatatttgaa atgttttaat atgcttttag agcatcctct 2880

tggaataggg tatggcaatt ataaatattt tatcactgaa ccttatcctc ataatattct 2940

tttagaaatt gggctgaata gtggggtttt aggatgtttt gtgtttataa cctatgtgat 3000

gatcaccaca atagtcataa taaagaaaac aaaatcctgc tataatgaaa aaaatctatt 3060

tgttttgact atatttttat attcactttt tagctggatg ttttcaaacg attttgcaag 3120

tagcagtgtt gtttttttct tattgggtgt gctaggtcac atagcctcta gtaataatga 3180

aaaagagatt aaataaatat gttttctata gtaattacaa ctaaagaccg ggcttattat 3240

ttgaaaagag cagttgacag catattaaaa tcaactattt ctccaaaaga tatcgtaata 3300

attaatgatg gtgggaaggc tatatgtgaa aatgattttc cacctatgaa acatattaca 3360

ttaagtatag ttaataatcg tttttcaatg ggagctaact attcaagaaa tcaaggaatt 3420

gataaagcct taactaatta tgtattttta cttgatgatg atgatgcctt tactccaact 3480

acctttgaaa atagaattaa tataattaaa tcttcagtgg atattggggt cgtttttact 3540

ggaattaata tagtatcatc caaaaatttg gataaagtaa ttagaaaatc aaaaaacatc 3600

aatgaccaaa tcactacgca tcagcttcta acagaaggca atgttatcgg atctacatca 3660

cgtgcattaa ttcgtaaaga tttattctgt gaagcgggaa gatttgatcc tcaattaagc 3720

tgtcttcagg attatgattt atggattaga atgtcattgg tatcaagaat tgtcaatgat 3780

cacaaatacg gagtttatta tacaatacac gacaatgggc gacaaattag tactaattat 3840

gaaaaatata tgcaagtagg aaaattgtta ataaataaat atgattctct tcttaataaa 3900

aatacaatta atgcttttaa gtctaatatt tacttaagag ttgcaatatc tgcctctgct 3960

tcatcaaata aagcaagatt aaaatattct tttatgtcat taaaatatag gcttaatatt 4020

aaagctttag ctttgttcct attccctagc tctgttctta aaagatttta taattatgta 4080

taagtttgaa tgagtaattg caatattatt acgttttctg tatttaaact caagattttt 4140

tcttttcacc tttctttttg ctaggcactt gttgaatagt agatggatgt gtaatacaat 4200

aatggaagat gtcttagtga tttatacatt tcaataaact ttcaaaaaaa gttaattata 4260

acgcatatat ttaaatgatt tatatttaat gtttgttagt cagataaata ttttattgtc 4320

tctatagcta taggcttttg ttacttttaa ataaagaaga tatgtgaaaa ataaacaatt 4380

ttaatgaaga gtggagccat gtgattcatg tgttctaatt tttatcttaa taacaatgat 4440

gtacaatggc atggttacaa tactatttaa tatggtaggg gtagtatctt tgaagtagtt 4500

tatattaaaa actatgttcg taaattatat atgttccgac aatatataaa gattttagat 4560

agagggatta tctagtaata caatgcgtta gataaaataa atccgtatct ctattactaa 4620

aacaattaaa tgttttatta aagacaagga tattaagagg gcatattaat gaaaggtttt 4680

tcggtactta tgtctgttta taaaggtgaa aaggaaaact atcttgatga gtgttttaaa 4740

agcctgcatg aacagacaat taaagctaat gaaatcattt tggttattga cggcccagtt 4800

tccaaagaat tatatcaggt tatagataaa tggggggagg ttctgcccat tataaaagtt 4860

aaacttgata aaaatgtagg gcttggacaa gctttaaata taggccttaa gcactgtaat 4920

tttgagttgg ttgcacgaat ggatactgat gattattgtg taaaggatag atttttatta 4980

cagatgaaat tcttcgaaga acacaatgat ataatggtct tggggggcga gatagaggaa 5040

tacgaccaat ccttgagtat tccattaggc aaaagaacaa cagcattatc tcacgaagaa 5100

ataattgaac ttgcaaaaaa aagaaatcct ctcaaccata tgacagtcat gtataaaaaa 5160

agctttatct taagtgttgg aggttatcaa catcatttat atatggaaga ttataatctt 5220

tggttgagag tattagcttc tggtggttgt atttgcaact tacctaaggt attagtgcat 5280

gtaagagctg gggaagaaat gatcaaaaga cgaaaaggct ggatatatat caagagtgaa 5340

atacagctag cacgtttaaa aagtaagtta aatataacct gtttctggaa taactactat 5400

acaatgacac ttaggatcct tgccagacta atgccgacgc cacttctaaa attcgtatat 5460

tctaaactta gaacgtctaa attagcttga tgttgacatt tcatttgtgg ggagggtgaa 5520

ttaattcatt taaaatgata aaacattgcg ccaaatgcca ataaaaagtt atatttttac 5580

acggaatctg aagtaacctt tatagttcat cccctgacag gagtaaacaa tgtccaagca 5640

acaga tcggc gtcgtcggta tggcagtg 5668

Glycosyltransferase gene in the O antigen gene of table 1 intestinal bacteria O174 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 (℃)
								wzx	The transhipment enzyme	805-2076	891-908	1529-1546	656bp	0 a	50
			1479-1498	1861-1878	928bp	0 *	56
								wzy	Polysaccharase	2057-3196	2421-2439	3160-3179	400bp	0 b	56
			2512-2529	2944-2961	450bp	0 *	60

^*Only in intestinal bacteria O174 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 10, wild-type e. coli 11, wild-type e. coli 12, the 9th group of bacterial strain adds the intestinal bacteria reference culture	O1，O2，O5，O7，O12，O13，O14，O15，O16，O17，O19ab，O20， O21，O22，O23，O24，O59，O3，O11 O25，O26，O27，O28，O174，O30，O32，O31，O33，O35，O36，O37， O38，O40，O41，O42，O43，O39，O59 O44，O45，O46，O48，O49，O50，O51，O52，O54，O55，O56， O57，O58，O60，O61，O62，O64，O73 O63，O65，O66，O69，O70，O71，O74，O75，O76，O77，O78， O79，O80，O81，O82，O83，O96，O95 O84，O85，O86，O87，O88，O89，O91，O92，O98，O99，O101， O102，O103，O104，O105，O106，O100，O151 O107，O108，O109，O110，O111，O112ab，O112ac，O113， O115，O116，O118，O120，O123，O125，O126，O128 O129，O130，O131，O132，O133，O134，O135，O136，O137， O138，O139，O140，O141，O142，O143，O144，O145 O146，O147，O148，O150，O152，O154，O156，O157，O158， O159，O160，O161，O163，O164，O165，O166 O168，O169，O170，O171，O172，O173，O155，O124 D1，D2，D3，D4，D5，D6，D7，D8，D9，D10，D11，D12 B1，B2，B3，B4，B6，B7，B8，B9，B10，B11，B12，B13，B14，B15， B16，B17，B18 F1a，F1b，F2a，F2b，F3，F4b，F5(v：4)，F5(v：7)，F6，F X becomes，F Y becomes，DS，DR O174	IMVS a IMVS a IMVS a IMVS a IMVS a IMVS a IMVS a IMVS a b c d d d IMVS

^*For the convenience that detects, we are divided into one group with every 13-19 bacterium, 12 groups altogether

a.Institute of Medical and Veterinary Science(IMVS)，Anelaide，Australia

b.Statens Serum Institut，Copenbagen，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 O174 type O antigen gene structure iron

Table 4 intestinal bacteria O174 type O antigen gene cluster gene position

Orf1's is initial

CCTTTTGCAT TGGTAGCTGT AAGCCAAGGG CGGTAGCGTA AACATTTGAG AGGAATTA AT 60

GAGTACAGTT ACACTCGTTA TAACTAGTTG CGGTAGATTT GAATTATTAG AAAAAACAAT 120

ATCTTCTCTT GTAAATAGGT ACCCATTTAC AGAGAAGATC ATAATTGAGG ATTCTGGTAA 180

TGTAAAAGTT ATAAACAAAA TAAAAGAAAA ATATGATAGG GATTTTACAA TCTTAATAAA 240

TGAAAAAAAC ATTGGACAAA TTAAAAGCAT CGATAAGGCA TATAGTTTAG TCACTACCGA 300

ATATATATTC CATTGTGAAG ATGATTGGCA TTTCTATAGA GACGGATTCA TAGAAGATTC 360

ATTAGATATA TTGAAAGAGT ATAGACACAT TTCAATGGTA TCACTTAGAG ATTGGCTAAA 420

TGATGTATCG ATCAATTGTC ATATGGAAAG ATCCACGCTC TTACAAACGG GAAATGGAAC 480

AAGATTTTTC ATGTTAAAAC CCAAAAATGG TGATGGCTGG GGAGGGTATT CGTTCAACCC 540

GGGGCTGAGA CGACTTCAAG ATTATAAAGA AATAATCGGA CAATTTTCAA AAGTAGGGCA 600

TGAAAAAAAT ATAAGTTTAT ATTTTTTAGA AAAAGGAATG AATATGGCGC TATTAGAATC 660

TTCAGCTGTT GAGCACATAG GTTGGAATCA CCATATATTG AGTAAAAATA ATCCTCAAGA 720

AAGATTTTAT TTGCTTAAAA AATATTTACC AAAGGAAGTA ACTAATGTGC TCAAGATAAT 780

The termination wzx's of orf1 is initial

TTATAGAAAA ATTGCTGGG T GATT ATGAAA TATCTTTTTA TTTGGTCTCT TACTCCCAAA 840

TTATATGAAT TAAGCCTTAA TCTTTCTATC GGGATAGCTC TTATAAATTA TCTAGGACCT 900

GTAGAACAAG GTAAAATAGG TTTTTTAATT AATTTATGCA CATTAATGAG TTTTCTTACC 960

ACGCTTGGAA TTGGGCCTGT ATATTCTAAT TTTGTTAGTC GTTCAAATAA TGAAAGATTA 1020

ATATCCAATA AATTTAAGGA GAATGTTACA TTAAGGTTTT TAGGTTATAT TTTGTTTCTT 1080

CTGGTTTCAC TTTTATTTCT ATTCTTCTTT AAAAGGGGGT TGATATATTT ATCAATACCA 1140

TTTTTAATAG GGAAATTATT TTTCAGTTTT GATATATATT ATAACCTCAT TGAAGGTAAG 1200

GCTGGATTTA AAAATTATGC AATTTCAAAA TTTATATCTT TGACATGTAT AAATGTATTT 1260

CGGTTATATT GTATATATGC ACAATTAGAT ATATATTGGG TTGCGTTTTC ATTCTTTTTG 1320

ACTGATTTCT TGACATTTTT TGTGTATTTT TTGTTATTTG ACAAATTTAA GTATTTCGGT 1380

TTTAAATTTG ATTACAAAAA ATCATTAATT ATATTTAAGA TAAACTATAA GTTAGCATTA 1440

TCTACAATAG TTGTAAGTCT CTTTACTCAG CTGGATATAG TAATGATCGG AACTATGCTT 1500

GGAGATAAAG CTGCAGGTGA GTATTATGCA TCAACTAGAC TCGCTACTCC TTTAGTTTTT 1560

ATATCAACAA TTATTATAAG CACTTTTTTT TCAAAACTTT CTAGAAACTG GGTTGTTAAT 1620

AAAAAAGAAT ATTATGAACT TTTAGCTTTC ATTAGTGGAA GTATAATTTT TTCGTATTCA 1680

GCAATTGTAC TCATTATTTT TATATTTGGA AACGATATAT TTTTCTTGTT TTTCAGTTCA 1740

GAATATAAAG CCTCTTATGA TTTATTGTTA ATCCATATTG TAGGTTTGAT TTTTGTACTT 1800

TTAGGCCCGC TTACTGGGAA GCATTTAATA ATTAAAAAAG ATTATGGGGC AGAACTTAGC 1860

AAAACCTTAT TGGCAGCAAT TGTTAATATA GCCTTAACTT CCATTGCAGT GTTAAAATAT 1920

GAAAATCTTA ACTTAGTGGC CGTTAGTACA TTAATAAGTT ATATGATTGC TAATTTTGGT 1980

TACTTTATAG TAAAAAAAGA TTGGCTATTA ATCAAAGCTA TATTGAATGG TGTTAATCCC 2040

The termination of the initial wzx of wzy

CTTTTTTTGG TGAGAT ATGC TAAAAAGATA CTC TAACCTT ATATTTTCTT TTGTTATTGC 2100

GTTTTCCTTT TTTTCATACG TAATGAATCC CAGTTTAAAA TTTATATATA AATCACCAGT 2160

AATAAATTTT ATCCCTTTTT TGTTATGTTG TTTGATAACA ATCATTTTTC TTACAGAGAA 2220

AAAAATCAAG CTTAACTATT ATGAATTATT TTCAATTCTT TATGTTGGGT TCTTTTTTAT 2280

ACTTCAATAT ACCTATTTTA TACAATCGAC AGATTCAATA GAAACTTTAC TACGATTAAT 2340

GTCGGTAAAT TTTTCATTCT TATTTGGATT ACTATTAGGC TGGTGTTTTA AAAGGGAGTC 2400

AATTGAGAAG CTTTTTATAC TATGGGTCCT ATTACTTTCT ATTGCTAATA TCTGCGGTGT 2460

AATCAATTAT TCTGATGGGG TGGAATTTAA TCATTTGAAC TTTACATTAC CTCTAGGGAC 2520

TGTTGTTACA TGGTTAATTT TTAAAGCATT TATGAAAGAT ACTGATAGGT ACATATTCAC 2580

ATTATCTGTT ATATTATTTT TATTTTTTAA TATAATATTT GCCGGAAGTC GGACTGCTAT 2640

TTTTCTACCA ATCTTAGTAA GTTTATTTAT TATGGTGATA TTTAGAAAAT ATGTGTCTAT 2700

AAAGAAAACA GTGATCTCAT TCATTATATT GATAACAATA ACTATAATAT CATTGCCTTA 2760

TATTTTAAGT AACTTGAATG CTTATTTTTT AAGCAAGGTT CAAAATATGG CTGATATCAG 2820

TGAGGATAGT CGTTATAATT TATATTTGAA ATGTTTTAAT ATGCTTTTAG AGCATCCTCT 2880

TGGAATAGGG TATGGCAATT ATAAATATTT TATCACTGAA CCTTATCCTC ATAATATTCT 2940

TTTAGAAATT GGGCTGAATA GTGGGGTTTT AGGATGTTTT GTGTTTATAA CCTATGTGAT 3000

GATCACCACA ATAGTCATAA TAAAGAAAAC AAAATCCTGC TATAATGAAA AAAATCTATT 3060

TGTTTTGACT ATATTTTTAT ATTCACTTTT TAGCTGGATG TTTTCAAACG ATTTTGCAAG 3120

TAGCAGTGTT GTTTTTTTCT TATTGGGTGT GCTAGGTCAC ATAGCCTCTA GTAATAATGA 3180

Wzy stops the initial of orf4

AAAAGAGATT AAA TAAAT AT GTTTTCTATA GTAATTACAA CTAAAGACCG GGCTTATTAT 3240

TTGAAAAGAG CAGTTGACAG CATATTAAAA TCAACTATTT CTCCAAAAGA TATCGTAATA 3300

ATTAATGATG GTGGGAAGGC TATATGTGAA AATGATTTTC CACCTATGAA ACATATTACA 3360

TTAAGTATAG TTAATAATCG TTTTTCAATG GGAGCTAACT ATTCAAGAAA TCAAGGAATT 3420

GATAAAGCCT TAACTAATTA TGTATTTTTA CTTGATGATG ATGATGCCTT TACTCCAACT 3480

ACCTTTGAAA ATAGAATTAA TATAATTAAA TCTTCAGTGG ATATTGGGGT CGTTTTTACT 3540

GGAATTAATA TAGTATCATC CAAAAATTTG GATAAAGTAA TTAGAAAATC AAAAAACATC 3600

AATGACCAAA TCACTACGCA TCAGCTTCTA ACAGAAGGCA ATGTTATCGG ATCTACATCA 3660

CGTGCATTAA TTCGTAAAGA TTTATTCTGT GAAGCGGGAA GATTTGATCC TCAATTAAGC 3720

TGTCTTCAGG ATTATGATTT ATGGATTAGA ATGTCATTGG TATCAAGAAT TGTCAATGAT 3780

CACAAATACG GAGTTTATTA TACAATACAC GACAATGGGC GACAAATTAG TACTAATTAT 3840

GAAAAATATA TGCAAGTAGG AAAATTGTTA ATAAATAAAT ATGATTCTCT TCTTAATAAA 3900

AATACAATTA ATGCTTTTAA GTCTAATATT TACTTAAGAG TTGCAATATC TGCCTCTGCT 3960

TCATCAAATA AAGCAAGATT AAAATATTCT TTTATGTCAT TAAAATATAG GCTTAATATT 4020

AAAGCTTTAG CTTTGTTCCT ATTCCCTAGC TCTGTTCTTA AAAGATTTTA TAATTATGTA 4080

The termination of orf4

TAAGTTTGAA TGAGTAATTG CAATATTATT ACGTTTTCTG TATTTAAACT CAAGATTTTT 4140

TCTTTTCACC TTTCTTTTTG CTAGGCACTT GTTGAATAGT AGATGGATGT GTAATACAAT 4200

AATGGAAGAT GTCTTAGTGA TTTATACATT TCAATAAACT TTCAAAAAAA GTTAATTATA 4260

ACGCATATAT TTAAATGATT TATATTTAAT GTTTGTTAGT CAGATAAATA TTTTATTGTC 4320

TCTATAGCTA TAGGCTTTTG TTACTTTTAA ATAAAGAAGA TATGTGAAAA ATAAACAATT 4380

TTAATGAAGA GTGGAGCCAT GTGATTCATG TGTTCTAATT TTTATCTTAA TAACAATGAT 4440

GTACAATGGC ATGGTTACAA TACTATTTAA TATGGTAGGG GTAGTATCTT TGAAGTAGTT 4500

TATATTAAAA ACTATGTTCG TAAATTATAT ATGTTCCGAC AATATATAAA GATTTTAGAT 4560

AGAGGGATTA TCTAGTAATA CAATGCGTTA GATAAAATAA ATCCGTATCT CTATTACTAA 4620

Orf5's is initial

AACAATTAAA TGTTTTATTA AAGACAAGGA TATTAAGAGG GCATATTA AT GAAAGGTTTT 4680

TCGGTACTTA TGTCTGTTTA TAAAGGTGAA AAGGAAAACT ATCTTGATGA GTGTTTTAAA 4740

AGCCTGCATG AACAGACAAT TAAAGCTAAT GAAATCATTT TGGTTATTGA CGGCCCAGTT 4800

TCCAAAGAAT TATATCAGGT TATAGATAAA TGGGGGGAGG TTCTGCCCAT TATAAAAGTT 4860

AAACTTGATA AAAATGTAGG GCTTGGACAA GCTTTAAATA TAGGCCTTAA GCACTGTAAT 4920

TTTGAGTTGG TTGCACGAAT GGATACTGAT GATTATTGTG TAAAGGATAG ATTTTTATTA 4980

CAGATGAAAT TCTTCGAAGA ACACAATGAT ATAATGGTCT TGGGGGGCGA GATAGAGGAA 5040

TACGACCAAT CCTTGAGTAT TCCATTAGGC AAAAGAACAA CAGCATTATC TCACGAAGAA 5100

ATAATTGAAC TTGCAAAAAA AAGAAATCCT CTCAACCATA TGACAGTCAT GTATAAAAAA 5160

AGCTTTATCT TAAGTGTTGG AGGTTATCAA CATCATTTAT ATATGGAAGA TTATAATCTT 5220

TGGTTGAGAG TATTAGCTTC TGGTGGTTGT ATTTGCAACT TACCTAAGGT ATTAGTGCAT 5280

GTAAGAGCTG GGGAAGAAAT GATCAAAAGA CGAAAAGGCT GGATATATAT CAAGAGTGAA 5340

ATACAGCTAG CACGTTTAAA AAGTAAGTTA AATATAACCT GTTTCTGGAA TAACTACTAT 5400

ACAATGACAC TTAGGATCCT TGCCAGACTA ATGCCGACGC CACTTCTAAA ATTCGTATAT 5460

The termination of orf5

TCTAAACTTA GAACGTCTAA ATTAGCT TGA TGTTGACATT TCATTTGTGG GGAGGGTGAA 5520

TTAATTCATT TAAAATGATA AAACATTGCG CCAAATGCCA ATAAAAAGTT ATATTTTTAC 5580

ACGGAATCTG AAGTAACCTT TATAGTTCAT CCCCTGACAG GAGTAAACAA TGTCCAAGCA 5640

ACAGATCGGC GTCGTCGGTA TGGCAGTG 5668

The invention has the beneficial effects as follows: the special molecular marker (molecular probe) that the objective of the invention is to seek and use this bacterium, it is specific nucleotide sequence, therefore technical scheme of the present invention has uniqueness, the difference of itself and conventional study method is, search out and have specific nucleotide sequence, and guarantee the specificity of molecule marker by reliable experimental, can utilize and well known to a person skilled in the art mature technology (as PCR or gene chip etc.), use the technology of this marker bacterial detection, make all those skilled in the art can be, realize purpose of the present invention easily and obtain the result of use of expection according to technology contents provided by the invention.Already provided experimental data among the present invention, fully proved the specificity of this nucleotide sequence, and can be applied to the detection of this bacterium, use modern molecular biology method for determining bacteria of the present invention with respect to traditional serology immune response, have fast, accurately, advantage cheaply.The present invention order-checking is also inferred the function of each gene with information biology software, be in order to seek specificity Nucleotide, the gene that some specific functions are arranged in the bacterium is a high special, gene that utilizes above method to infer these specific functions and their position, utilize experiment to prove then, the function of these genes that will be inferred, remove to search out better faster specificity Nucleotide as a kind of road sign, like this, can reduce the blindness of research experiment, accelerate the progress of experiment, reduce experiment fees.

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 (3)

1, a kind of oligonucleotide of the O-antigen-specific to intestinal bacteria O174 type is characterized in that it is the Nucleotide that is selected from 891 to 908 bases among the SEQ ID NO:1; The Nucleotide of 1529 to 1546 bases among the SEQ IDNO:1; The Nucleotide of 1479 to 1498 bases among the SEQ ID NO:1; The Nucleotide of 1861 to 1878 bases among the SEQ ID NO:1; The Nucleotide of 2421 to 2439 bases among the SEQID NO:1; The Nucleotide of 3160 to 3179 bases among the SEQ ID NO:1; The Nucleotide of 2512 to 2529 bases among the SEQ ID NO:1; The Nucleotide of 2944 to 2961 bases among the SEQ ID NO:1.

2, a kind of oligonucleotide of the O-antigen-specific to intestinal bacteria O174 type is right, it is characterized in that it is to be selected from the Nucleotide of 891 to 908 bases among the SEQ ID NO:1 and the Nucleotide of 1529 to 1546 bases; The Nucleotide of 1479 to 1498 bases among the SEQ ID NO:1 and the Nucleotide of 1861 to 1878 bases; The Nucleotide of 2421 to 2439 bases among the SEQ ID NO:1 and the Nucleotide of 3160 to 3179 bases; The Nucleotide of 2512 to 2529 bases among the SEQ ID NO:1 and the Nucleotide of 2944 to 2961 bases.

3, the right application of the oligonucleotide of the oligonucleotide of claim 1 or claim 2, it is characterized in that, it is used for PCR as primer, perhaps is used for hybridization and fluoroscopic examination, manufacturing gene chip or microarray as probe, for detecting intestinal bacteria O174 type.