CN100345967C - Nucleotide peculiar to 0-antigen of 03 type bacillus coli - Google Patents

Nucleotide peculiar to 0-antigen of 03 type bacillus coli Download PDF

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CN100345967C
CN100345967C CNB2004100190178A CN200410019017A CN100345967C CN 100345967 C CN100345967 C CN 100345967C CN B2004100190178 A CNB2004100190178 A CN B2004100190178A CN 200410019017 A CN200410019017 A CN 200410019017A CN 100345967 C CN100345967 C CN 100345967C
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gene
antigen
nucleotide
intestinal bacteria
oligonucleotide
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CN1563032A (en
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王磊
陶江
冯露
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Tianjin Biochip Corp
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Tianjin Biochip Corp
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Abstract

The present invention provides a specific nucleotide for an O-antigen of Escherichia coli O3. The specific nucleotide is the nucleotide complete sequence of gene clusters in Escherichia coli O3 for controlling the synthesis of an O-antigen, such as a separated nucleotide with the total length of 14049 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 oligonucleotides of glycosyl transferase genes and oligosaccharide unit processing genes in the O-antigen gene clusters of Escherichia coli O3. The present invention verifies that the oligonucleotide has high specificity to all of the O-antigen genes of Escherichia coli O3 through PCR. The present invention also discloses a method for detecting and identifying Escherichia coli O3 in the human body and the environment by the oligonucleotide of the present invention.

Description

Nucleotide to the O-antigen-specific of intestinal bacteria O3 type
Technical field
The present invention relates to the complete nucleotide sequence of control O-antigen synthetic gene cluster in the intestinal bacteria O3 type (Escherichia coli O3), particularly relate in the intestinal bacteria O3 type oligonucleotide in the control O-antigen synthetic gene cluster, can utilize these the oligonucleotide of the O-antigen-specific intestinal bacteria O3 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 " .Joumal 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 ofS.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 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 ' s identification of the 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 O3 type.It is the Nucleotide in the O-antigen gene bunch of intestinal bacteria O3 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 O3 type.
Another object of the present invention has provided the gene of the O-antigen gene bunch that constitutes intestinal bacteria O3 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 coding transhipment enzyme in the O-antigen gene bunch of intestinal bacteria O3 type respectively is the wzx gene or with wzx the gene of identity function is arranged; The gene that comes from the coding polysaccharase is the wzy gene or with wzy the gene 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 O3 type; Especially the oligonucleotide of listing in the table 1, they are high specials to the O-antigen of intestinal bacteria O3 type, and these oligonucleotide are also reconfigurable, the oligonucleotide after the combination also is a high special to the O-antigen of intestinal bacteria O3 type.
The above-mentioned oligonucleotide that still 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 O3 type of these methods detections and identification of escherichia coli O3 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 O3 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 O3 type, and it is the isolating Nucleotide shown in SEQ ID NO:1,14049 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 O3 type, it is by called after orf1, wzx, orf3, wzy, fnlA, qnlA, orf7, orf8,9 genomic constitutions of orf9 are all between galF gene and gnd gene.
The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O3 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; Wherein said gene: wzx is the Nucleotide of 4566 to 6059 bases among the SEQ ID NO:1; Wzy is the Nucleotide of 7207 to 8292 bases among the SEQ ID NO:1; Orf1 is the Nucleotide of 2762 to 3700 bases among the SEQ ID NO:1; Orf3 is the Nucleotide of 6041 to 7210 bases among the SEQ ID NO:1; Orf8 is the Nucleotide of 10686 to 11483 bases among the SEQ ID NO:1; Orf9 is the Nucleotide of 11486 to 12604 bases among the SEQ ID NO:1.
The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O3 type, wherein it also comprises and comes from described wzx gene, wzy gene; And their mixing or their reorganization.
The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O3 type, the oligonucleotide of the wherein said wzx of coming from gene is to being: the Nucleotide of 5099 to 5114 bases among the SEQ ID NO:1 and the Nucleotide of 5575 to 5590 bases; The Nucleotide of 4897 to 4913 bases among the SEQ ID NO:1 and the Nucleotide of 5669 to 5684 bases.The oligonucleotide that comes from the wzy gene is to being: the Nucleotide of 7388 to 7403 bases among the SEQ ID NO:1 and the Nucleotide of 7921 to 7936 bases; The Nucleotide of 7529 to 7545 bases among the SEQ ID NO:1 and the Nucleotide of 8130 to 8145 bases.
The application of the Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O3 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 O3 type is providing the O-antigen of expressing intestinal bacteria O3 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 O3 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 O3 type is characterized in that it comprises the steps:
(1) genomic extraction: in substratum, cultivate intestinal bacteria O3 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 O3 type bunch: with the genome of intestinal bacteria O3 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 O3 type;
(6) screening of specific gene: at wzx, the wzy gene design primer in the O-antigen gene of intestinal bacteria O3 type bunch; Respectively designed two pairs of primers in each gene, every pair of primer is distributed in the different places in the corresponding gene, to guarantee its specificity; Is that template is carried out PCR with these primers with the genomes of 166 strain intestinal bacteria and 43 strain Shigellaes, determines the antigenic high degree of specificity of O-of wzx, wzy gene pairs intestinal bacteria O3 type;
(7) detection of primer sensitivity: cultivate intestinal bacteria O3, 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 O3.
The separation method of the Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O3 type is characterized in that it comprises the steps:
(1) genomic extraction: 37 ℃ of incubated overnight intestinal bacteria O3 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 O3 type bunch: with the genome of intestinal bacteria O3 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 O3 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 O3 type; The quality of sequence is mainly guaranteed by two aspects: 1) genome of intestinal bacteria O3 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 O3 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 9 openings, determine also that with the function of finding the reading frame that these are open what gene they are with the genetic comparison among the blast groupware and the GenBank, finish gene annotation with the Artemis software at Britain sanger center again, do accurate comparison between DNA and protein sequence with ClustralW software, obtain the structure of the O-antigen gene bunch of intestinal bacteria O3 type at last;
(6) screening of specific gene: at wzx, the wzy gene design primer in the O-antigen gene of intestinal bacteria O3 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 O3, 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 O3 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 O3 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 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 5099 to 5114 bases among the SEQ ID NO:1 and the Nucleotide of 5575 to 5590 bases; The Nucleotide of 4897 to 4913 bases among the SEQ ID NO:1 and the Nucleotide of 5669 to 5684 bases.The Nucleotide of 7388 to 7403 bases among the SEQ ID NO:1 and the Nucleotide of 7921 to 7936 bases; The Nucleotide of 7529 to 7545 bases among the SEQ ID NO:1 and the Nucleotide of 8130 to 8145 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 O3 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 O3 type, its complete sequence shown in SEQ ID NO:1,14049 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 O3 type by method of the present invention, as shown in table 3, it is altogether by called after orf1 respectively, wzx, orf3, wzy, fnlA, qnlA, orf7, orf8,9 genomic constitutions of orf9 are all between galF gene and gnd gene.
Second aspect of the present invention provides the gene in the O-antigen gene bunch of intestinal bacteria O3 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).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 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 O3 type.
The 3rd aspect of the present invention provides the wzx gene in the O-antigen gene bunch that comes from intestinal bacteria O3 type or the gene, wzy gene of identity function arranged or with wzy the gene of identity function is arranged with wzx, and 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 O3 type all is high special.
The separation method of the Nucleotide of described O-antigen-specific to intestinal bacteria O3 type comprises the steps: 1) genomic extraction; 2) the O-antigen gene in the pcr amplification intestinal bacteria O3 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 refer to derive from the gene of the coding transhipment enzyme in the O-antigen gene bunch and intragenic one section nucleic acid molecule of coding polysaccharase, and they can change on length, generally change in 10 to 20 Nucleotide scopes; More definite these oligonucleotide of saying are to come from wzx gene (nucleotide position is the Nucleotide of 4566 to 6059 bases from SEQ ID NO:1), wzy gene (nucleotide position is the Nucleotide of 7207 to 8292 bases from SEQ ID NO:1).Coming from above intragenic oligonucleotide is high special to intestinal bacteria O3 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 O3 types.Available PCR method detects, more can with behind the Nucleotide mark in the inventive method as probe by hybridization such as southern-blot or fluoroscopic examination, perhaps by antigen and bacterium in gene chip or the microarray assay sample.
Planner of the present invention considers following situation: when one special oligonucleotide detects when invalid, the mixture of oligonucleotide can with the target region specific hybrid with test sample.Therefore the invention provides a cover oligonucleotide and be used for detection method of the present invention.Here said oligonucleotide is meant the gene that coming from coding transhipment enzyme and the gene of polysaccharase, comprises the wzx gene or the gene, wzy gene of identity function is arranged or with wzy the oligonucleotide of the gene of identity function is arranged with wzx.This cover oligonucleotide is special to the O-antigen of a special bacterium, and this special bacterium O-antigen is expressed by intestinal bacteria O3 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 O3 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 that comes from coding transhipment enzyme and polysaccharase comprises the wzx gene or the gene, wzy gene of identity function arranged or with wzy the gene of identity function is arranged with wzx to oligonucleotide, this cover oligonucleotide is special to a special bacterial polysaccharides, and this cover oligonucleotide may be the Nucleotide of necessary gene during sugar synthesizes.
On the other hand, the present invention also relates to the antigenic method of one or more bacterial polysaccharideses in the sample that a kind of detection comes from patient.One or more bacterial polysaccharides antigens in the sample can make sample can with a specific hybrid in a pair of oligonucleotide in following at least one gene, these genes are: (i) gene of 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 O3 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 O3 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 O3 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 O3 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 O3 type bunch:
With the genome of intestinal bacteria O3 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 GGA TTA AGTTCG 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 PCRPreps 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 1.25ul 100mMDTT 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 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 O3 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 O3 type obtained.The quality of sequence is mainly guaranteed by two aspects: 1) genome of intestinal bacteria O3 type is done 5 LongPCR 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 O3 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 9 openings, determine also that with the function of finding the reading frame that these are open what gene they are with the genetic comparison among the blast groupware and the GenBank, finish gene annotation with the Artemis software at Britain sanger center again, do accurate comparison between DNA and protein sequence with ClustralW software, obtain the structure of the O-antigen gene bunch of intestinal bacteria O3 type at last, as shown in table 3.
By retrieving and relatively, finding that orf2 and orf4 are the proteic genes that there is transmembrane segment in only two codings of intestinal bacteria O3 kind.The O-antigen transferring enzyme of orf2 encoded protein and intestinal bacteria (AAL67558) has 22% sequence identity and 45% similarity, it contains 14 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 orf4 encoded protein and intestinal bacteria (AAL27339) has 25% consistence, 46% similarity, it contains 11 transmembrane segments by the proteic topology discovery of HMMTOP2.0 programanalysis, and hydrophilic loop (loop) in the big kytoplasm is arranged, and this is the proteic characteristic feature of Wzy.So name orf4 is wzy.
Orf 1,3, and the albumen of 8,9 four genes encodings and other known glycosyltransferases have the sequence identity of 28-48% and the sequence similarity of 51-65%.By search to glycosyltransferase motif database among the Pfam, the homology desired value of the consensus sequence of the albumen of these four genes encodings and known glycosyltransferase family 1 and 2 is 6 * E-37To 1.2 * E-9, so 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 O3 may be made up of five monose.Because the definite function of these four genes can't be determined, so we are with these four genes temporary called after orf1, orf3, orf8 and orf9.
The FnlA albumen of fnlA genes encoding has 65% consensus amino acid sequence and 80% similarity in the albumen of orf5 genes encoding and the Vibrio cholerae O37 O-antigen gene bunch, so we are fnlA with the orf5 unnamed gene.The QnlA albumen of qnlA genes encoding has 47% consensus amino acid sequence and 65% similarity in the albumen of orf6 genes encoding and the Vibrio cholerae O37 O-antigen gene bunch, so we are qnlA with the orf6 unnamed gene.A kind of hypothetical protein of coding has 37% consensus amino acid sequence in the albumen of orf7 genes encoding and the Pseudomonasaeruginosa PAO1O-antigen gene bunch, owing to wouldn't know its function, so we are with the temporary called after orf7 of orf7 gene.
Embodiment 6: the screening of specific gene:
Wzx, wzy gene design primer in the O-antigen gene of intestinal bacteria O3 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 O3 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 O3 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 O3 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 5099 to 5114 bases among the SEQ ID NO:1 and the Nucleotide of 5575 to 5590 bases; The Nucleotide of 4897 to 4913 bases among the SEQ ID NO:1 and the Nucleotide of 5669 to 5684 bases.The Nucleotide of 7388 to 7403 bases among the SEQ ID NO:1 and the Nucleotide of 7921 to 7936 bases; The Nucleotide of 7529 to 7545 bases among the SEQ ID NO:1 and the Nucleotide of 8130 to 8145 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 O3 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 O3 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 O3 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 O3 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 O3 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 O3 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 13rd 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 O3 type and O-antigen thereof are high specials.
At last, from intestinal bacteria O3 type, screen gene by PCR: wzx, wzy to the O-antigen high special of intestinal bacteria O3 type.And the oligonucleotide of these intragenic any one section 10-20nt is special to the O-antigen of intestinal bacteria O3 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 O3 type.These all oligonucleotide all can be used for the intestinal bacteria O3 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 O3 type, in table, listed the structure of the O-antigen gene bunch of intestinal bacteria O3 type, altogether by 9 genomic constitutions, each gene box indicating, and in square frame, write the title of gene, numeral be the order of the open reading frame (orf) in the O-antigen gene bunch.Two ends at O-antigen gene bunch are galF gene and gnd gene, and they do not belong to O-antigen gene bunch, and we are just with the increase full length sequence of O-antigen gene bunch of their one section sequences Design primer.
Table 4 is site plan of the gene in the O-antigen gene bunch of intestinal bacteria O3 type, listed the accurate position of all open reading frame in complete sequence in the O-antigen gene bunch of intestinal bacteria O3 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 0 antigen-specific of intestinal bacteria O3 type
＜130〉to the Nucleotide of the O antigen-specific of intestinal bacteria O3 type
<160>1
<170>PatentIn?version?3.2
<210>1
<211>14049
<212>DNA
<213>Escherichia?coli
<400>1
attgtggctg?cagggatcaa?agaaatcctc?ctggtaactc?acgcgtccaa?gaacgcggtc 60
gaaaaccact?tcgacacctc?ttatgaatta?gaatctctcc?ttgaacagcg?cgtgaagcgt 120
caactgctgg?cggaagtaca?atctatctgt?ccgccgggcg?tgaccattat?gaacgtgcgt 180
cagggtgaac?ctttaggttt?aggccactcc?attttgtgtg?cacgccccgc?cattggcgac 240
aacccatttg?tcgtggtgct?gccggacgtt?gttatcgatg?atgccagcgc?cgacccgttg 300
cgttacaacc?ttgctgccat?gattgcgcgc?ttcaatgaaa?cgggacgcag?tcaggtgctg 360
gcaaaacgta?tgccgggtga?cctctctgaa?tactccgtca?tccagaccaa?agaaccactg 420
gatcgcgaag?gtaaagtcag?ccgcattgtt?gaatttatcg?aaaaaccgga?tcagccgcag 480
acgttggact?cagacatcat?ggccgttggt?cgctatgtgc?tttctgccga?tatttggccg 540
gaacttgaac?gcacgcagcc?aggtgcatgg?gggcgtattc?agctgactga?tgccatcgct 600
gaactggcga?aaaaacagtc?cgttgacgcc?atgctgatga?ctggggacag?ttacgactgc 660
ggtaaaaaaa?tgggctatat?gcaggcgttc?gtgaagtatg?gactacgcaa?cctgaaagaa 720
ggggcgaagt?tccgtaaagg?gattgagaag?ctgttaagcg?aataatgaaa?atctgaccgg 780
atgtaacggt?tgataagaaa?attataacgg?cagtgaagat?tcgtggcgaa?agtaatttgt 840
tgcgaatttt?cctgccgttg?ttttatataa?acaatcagaa?taacaacgag?ttagcaatag 900
gattttagtc?aaagttttcc?aggattttcc?ttgtttccag?agcggattgg?taagacaatt 960
agcgtttgaa?tttttcgggt?ttagcgcgag?tgggtaacgc?tcgctacatc?gtaggcatgc 1020
atgcagtgct?ctggtagctg?taaagccagg?ggcggtagcg?tatctagggg?aagatcatcg 1080
tgtgcggtaa?gcacaaaaat?gcaaaaagga?acgttttgca?tgtaataagc?ataagcttgt 1140
aaagagatca?gggaacaggg?aaagatcaca?taacgccggt?tttttgtact?aaataattcg 1200
cactaaatgt?tcaataattg?agacattcct?tattgtctaa?aactgttttt?cacagcttat 1260
atatgatcaa?atactcctta?cataaataag?gagaacaaaa?tggaacttaa?aaaactgatg 1320
gaacatattt?ctattatccc?cgattacaga?caagcctgga?aagtagaata?taaattttca 1380
gatactctac?tattgactat?ttgtgctgtc?atttctggtg?cagaaggttg?ggaagatata 1440
gaggattttg?gtgaaacgca?tctcgatttt?ttgaagcaat?atggtgattt?tgaaaatggt 1500
attcctgtcc?atgataccat?tgccggagtt?gtatcctgta?ttagtcctgc?aaaatttcac 1560
gagtgcttta?ttaactggat?gcgtgattgc?cattgttcag?attataaaga?cgtcattgca 1620
attgatggga?aaacgctccg?gcactcttat?gataagagtc?gtcgcagggg?agcgattcat 1680
gtcattagtg?tgtcctcaac?aatgcacagt?ctggttctcg?gacagatcaa?gacggatgaa 1740
aaatctaatg?agattacagc?tattcctgaa?cttcttaaca?tgatggatat?taaaggaaaa 1800
atcatcacaa?cggatgcgat?ggggtgccag?aaagatattg?cagaaaaaat?acaaaaacag 1860
ggaggtgatt?atttattcgc?tgtaaaggga?aatcaggggc?gactaaataa?agccttcgag 1920
gaaaaatttc?cgctgaaaga?attaaataat?ccaaagtatg?acagttacgc?aattagtgaa 1980
aagaatcacg?gcagagaaga?aatccgtctt?catattgttt?gcgatgtccc?tgatgaactt 2040
attgatttca?cgtttgaatg?gaaaggactg?aaaaaaatta?tgcgtggcag?tctcctttcg 2100
gtcaataata?gcagaacaaa?agaaagagcc?agaaatgacg?gtcagatatt?atatcagttc 2160
tgctgattta?accgctgaga?agtttgcgac?agcgattcga?aatcactggc?acgtggagaa 2220
taagttgctc?tggcgtctgg?atgtagtaat?gaatgaaaga?cgactgcaaa?ataggaagag 2280
gaaacgttca?gggataagac?atattgcaat?taatattttg?acgaaagata?aggtattcaa 2340
gcacggttaa?gacgttcgat?gaagacagcg?actacctgcg?tcagtctttg?tgaggagcgg 2400
actctagtaa?tccaactttg?ttgttaggtt?ttactcgcgg?aataatcatg?ggcgaatttt 2460
attaataatg?tatattttcc?taattaaaaa?catgttaaga?aaagaaacaa?aactttcaag 2520
ctaggttact?tatgatatga?agtcaacaca?ttctaaaatt?aaaattgcac?atgttcaatt 2580
gatgccacta?ctgagtggtg?ttcagcgagt?atcgttacaa?gaattcgaat?tattaccaaa 2640
tgaacagttc?gacattaatt?taatttgcaa?agagagtgga?ccataaactg?attatttaga 2700
tgatagtgta?agggcttttt?ttgtcccgac?attatgtaga?aatatatcgc?tcataaagga 2760
tatgaaatca?ttaatatctt?tatataaatt?attgaaaaaa?gaaaaatatg?atatcgtcca 2820
tacacactcc?tctaaaactg?gtattctagg?gcggatagct?gcgcggctgg?caggagttcc 2880
ttgtgtagta?catactgtac?atggctttgc?ctttgaaagc?acaaaaagaa?aatcagtaaa 2940
gctcgtttat?aaatggctgg?aaatatttgc?agccaaatgc?actacccgat?taatttgtct 3000
tcataatgaa?gataaagaaa?tatgtataaa?agaactatat?gttgatccaa?tgaaaatttc 3060
tgtaattcca?aatggagttg?atttggaaaa?attcgctcca?gcaattaata?aaggagactt 3120
aaaagaaaaa?atattagggt?taaaaagaaa?tagctttgta?tttaccatgg?tgggacggtt 3180
gtggccacaa?aaaaatccgt?tatattttgc?tgaagcagca?aagtatatta?tagaaaataa 3240
cttaatacct?gattcagttt?ttgtcatagt?tggtgatggg?gagttgatga?atgatttaaa 3300
atacaattat?caaacagata?tgaatttaaa?aaaaaggtta?ttacttttag?gctggcgaaa 3360
tgatatccca?aacatattaa?aagcaagtga?cgtttttgtt?cttccttctt?tatgggaagg 3420
aatgcctctt?gccatattag?aagctcaatc?tacagggtta?ccatgtatcg?tttctaatat 3480
caatggtaat?aattgtttgg?ttaagaatga?atttgatgga?ttcttaattg?aacttaatga 3540
tattgatagc?tttataaatg?cgttagttag?agtgacagac?gataaagtct?taaacattat 3600
gtcaaaaaat?tgccgtaata?aaattgttaa?tggatttaat?atagttaaaa?gagtggataa 3660
aattaaagat?ctatatattg?atatggttgt?taataaatga?ttgtgtgctt?tttgaatgtt 3720
tatatcaata?tcgcactgta?tattttattt?tatgtagatt?tattatgaat?ataaaggcat 3780
ggtgatttac?atcattattt?atatacagac?gctgttattg?gtatagagaa?atatcatata 3840
acattagttt?tcgtacaaaa?tgtaaaaaat?gaaagattta?ttactaagta?agactatttt 3900
tgataaccta?tatattatca?aatactctgt?acataaagga?gggaaatgga?acatgaaaaa 3960
taatagtcgt?atttctgtta?accaatttca?aacaacctgg?gaaagtggaa?cgtaaattat 4020
tagatattta?ctactgatta?tttgctcctg?cttcgtcatt?tctgacgcag?aaggatggga 4080
gtatacaaag?gatgtttgtg?agacgtgtgt?cgatttcttg?aagcaatacg?atgatttggg 4140
acatggtact?cctgtttata?ataccatagc?tgagttctat?tccgtatcag?tcctattaac 4200
tggttacgtg?atgttattct?tcagattata?aagacatcat?tgcagtaggt?ggaacttaat 4260
agcggagcag?aataaagagt?ttaaaatgat?attcaatatt?gtatcagttc?tgctggttgt 4320
ttaaattctg?agaagtccgc?gatcgtgatt?tgaaactgct?agaatgttga?gaataatcag 4380
cgctggtgtc?tggatgtagt?aatgaatgaa?gagaagtgct?caataagcaa?agcagctatg 4440
gacagtgatt?agcccgcatc?agttcttgca?ggggtgtgtt?tttattatct?ttccctgatt 4500
attgggtgac?tgtgtgactt?atacattctc?ttattttgag?tgtgatggta?aaaatgtttg 4560
actctatgca?tgttcgggtt?gctgtaaatt?ttgcatcaaa?gataacattt?gctttgatat 4620
ctataattac?aattccagtt?ataaaatcaa?aattaggagt?aaatgcttat?ggtgtcctaa 4680
cattctttct?atcaatccag?gtagttatat?tactgcttga?ggggggaatg?agtgccacaa 4740
tggctagagg?actattgaat?aaaaaatata?agatatacaa?tataagtaaa?gcagagtacc 4800
aaagtgttta?ttttatcttt?tttatattag?tgtcattatt?tgtattttca?atttttattg 4860
tctttaatca?taatatttcc?acattgattt?acaaccttga?tgatagggaa?gtctatagtt 4920
ttattagtgc?tgattatgtt?attttgctgg?ctggtgcaat?gattgccttg?cagttttata 4980
tcatatatta?tgaagcatta?tttgtggctt?atgaaaaaca?agtctcttat?ggaattcata 5040
taatagtatt?taatgtaatc?aaaaccataa?tatcattgtt?cttgttgatt?gtctttaatc 5100
ttgggttgga?tgtttatttt?ttagttcatg?tattttgtac?ctttatactt?attattttat 5160
tgcacattaa?ggcaataaaa?aacggattat?tagaaataac?aacattgcga?gtgaaaaaaa 5220
tatacacaaa?aatatcattt?atgaaagaag?aatttcattt?ttctaaaaat?atattatgcg 5280
tgtccattct?ttctgcaatt?gcattccaag?ctgacagact?atttattaca?aaatatatgg 5340
gtattagtgc?tgttggaatg?tatggtattg?cttatacatt?atcaacagta?ccaactttat 5400
ttacatcatc?attgtataat?gttatctatc?caagacttat?tattttaaca?agagagcaaa 5460
gcagcagtgc?gatttcattt?tttgaattag?tatcgaaatt?aatatatatt?atcataatac 5520
ccacttgtgt?gattatttcg?cttaattcga?cccaaatcct?aaatatttgg?ataggaaagc 5580
acgaaggaat?aatatcattg?ctgttaagta?ttctaataat?agcgacgtta?tttcaggggt 5640
tgcaggtgat?tccttttgca?ctacagatgg?ctcaagaacg?actaaaattt?acagttttca 5700
tgaatgctat?atttgtgccc?agcctaatat?tggcatatta?tttggttagt?attcataaaa 5760
ttctgttata?catctccata?gtgtggtcag?tatataatat?aataactttt?ttctctttaa 5820
ctatttattt?atatgctaaa?aataaaatgt?atacaagact?acttatttta?tattttaata 5880
tatgcttgtg?tgctgtcgta?acagtagtta?tatgtagcat?ttttaagaat?caagaatctc 5940
aaatattctt?gtctctagtt?aatatttctc?ttcagttttt?tatgtcgatg?agtttgtgca 6000
taatactgtt?gttctggaaa?actatatatg?gagcattgag?atgtgtttgt?tgtcggtagt 6060
aattgcgact?cataatcgcg?cgggatatgc?taaaaaaagc?atagaaagta?ttctttcaat 6120
tgatgttgaa?gaatttcaac?taattattca?tgatacaagc?gataataatg?tacttgagga 6180
attttgcgct?gaaatccagg?atgcgagatt?aaaatatatc?cattgcaatg?aattattgtc 6240
gatgacagaa?aattttaata?gagcattaga?atatgttcag?ggagaatata?ctataatgat 6300
aggagatgat?gatacgattt?tatcgactgc?gataaaatat?tgtagatatg?cgaaagaaaa 6360
aaaaattgat?gtaatctcgc?agaagatatc?tgcagagtat?tgttggccgg?attttagatc 6420
taagatctca?ggggatttta?acgcatcgtc?tctgatagtg?aacaaattta?caagtaacca 6480
agttacttat?gaggggcgta?aacaatacta?tgaagcagtt?aaaaactgtt?ttcaaggtac 6540
tgcaagcctc?cccaagttat?atcatggtat?tgttaaaact?tctttattgt?taaagataaa 6600
agaaaataca?ggtacttact?ttcatggggt?tagccctgat?atttcaattg?cattgtcgtt 6660
agcgtataac?tgcgaacgat?atattgaagt?ggattatcca?attacccttc?caggatcttc 6720
ggggggaagt?aacgcaggta?gaagtgcaat?gcgtactcat?gttggtaaat?tagacgacga 6780
tccacatatg?aaaagattta?aaagttatgt?ctggccaaac?ttattgccaa?gatatttcag 6840
tgtagagact?gtgtgggctc?aggcaggctt?agtaacaatc?caaaaaatag?ataataataa 6900
aggatttaat?tttattaagt?tctatgcatt?atgtttgatt?aaacatcgac?cctataaaaa 6960
atttattatt?gatgagataa?aaaaatacaa?gaaagtaaaa?ggagggttta?attgtataat 7020
ttatacaaaa?attatttatc?acatgtctta?tttttatatt?aatggaatat?caaatctttc 7080
taaaaaaatt?ataaaatatg?tcttcaaaaa?acttaagtct?caagagaagc?aaaaaaatat 7140
aaaagcaaat?gatatttttg?aagccaggat?gataattgaa?aaggaattat?caaaggaaac 7200
atttgaatga?ttatattttc?tacttttcta?cttttctact?tttttatttt?tctttcattg 7260
cttcctgttt?taattaaaga?gggagctaga?gggaatggca?aatattttta?tttctttgat 7320
ttatatattt?acttagtatt?agctagtctt?attatttata?taatttgtac?acgagatatt 7380
ttttctgact?ccgatacaga?acgttatctt?agtatattag?aatattatag?aataaatggc 7440
ttttatcttt?ttgataagaa?ctatattttt?gctgtgtata?catatttgta?ctctctatat 7500
atagatggtt?actatatgta?tatgataatc?ccacttatta?ccctgctata?ttcatattgt 7560
aagttagtaa?aaccactaaa?atatcctgtt?tatagttttt?ctttagtatt?aatagcaaca 7620
tttcctgttt?tttggcaatt?gagcgcaagc?gcatttaaac?aatgcattgc?aatatctttt 7680
attcttttag?cattagtgca?tattatacaa?acaaaatata?aacaagcggt?gcttttatct 7740
cttatagcta?gttccttcca?tttcacagca?ttaacgtttt?ttatattttc?gttgtttttc 7800
tatattagaa?aggaaagggt?cagtttaaat?ttattttttt?taatttggtt?ttttagtgtc 7860
atttttagca?taagcggatt?aaattcgtat?attccttcaa?ttataccttt?ttatgattta 7920
gtgggtgaaa?gtaaggtcaa?tatttatctt?gaatcaacaa?ataatgcaac?tggttttcgt 7980
gttgatttct?ttttgttttc?actagcccct?gtgttaatta?tatttattaa?cagaacatta 8040
gtaataaaag?atcgctatat?taaaatgatt?gcattaagtt?atttatattt?caattctata 8100
tataatataa?tgtcattcat?cccattttca?gatagagttg?ccgcatattc?atggtgtctt 8160
tcccccatta?ttctattatg?gagtgctctt?aagtgtagat?catcaattta?tatgttaagt 8220
tctataggga?tcgcgttatt?aaatcctttg?atttttatgt?actataattc?aatgtttatt 8280
tttttaaagt?gaggatatat?gtttaacggt?aaaaagattt?taatcactgg?tggtactggg 8340
tcatttggta?atgcagtgtt?acgtcggttt?cttgagacag?atattcaaga?ggttattatt 8400
ttcagtagag?atgaaaaaaa?acaagatgag?atgagaacgc?tatatcgcaa?tgataaaatg 8460
aaatttatta?taggtgatat?tagagattat?acatctgttt?tagctgcaac?tagtgacgtt 8520
gactatatat?tccatgctgc?ggctttaaaa?caggtgcctt?catgtgaatt?ttatccatgg 8580
gaagcagtta?aaacgaatat?aattggtaca?gaaaatttat?taaatgctgc?aaaacaaaat 8640
aaagttaaaa?aaacaatttg?tttgagcact?gataaagctg?tgtatccaat?taacgcaatg 8700
ggaatttcga?aggctatgat?ggaaaaagtg?atgacagcaa?aggctagaac?gttcaacaat 8760
gaagattcag?ttgtttgcgg?aactcgttat?ggcaatgtta?tggcatcacg?cggctctgtt 8820
atccctctgt?tcattcagca?gattcaaaca?aatcaaccat?taactataac?agaccctgaa 8880
atgactagat?tcatgatgac?gttagatgat?gctgtagatc?tcgtattatt?tgcatttaat 8940
aacgggcaaa?acggtgacat?ttttgttcag?aaagcgcctg?ccgcgacaat?tgctgtgctt 9000
gctaaagcat?tagttgaatt?gatgggggtg?ccaaaccatc?ctattaaagt?agttggttca 9060
cgtcatgggg?aaaagatata?tgaaacctta?cttagccgtg?aagaaatggc?aaaagcgatt 9120
gatcatggag?attattatca?gattcaacca?gataatcgtg?atctgaatta?tgacaaatat 9180
ttaagcgaag?ggagtaaaaa?aattgagctg?tttgatgatt?acaattccca?taatacatat 9240
agactcaatg?ttgatgaaat?gaaaaaatta?ttgttaaaac?tcaatatcat?tcaaaatgtg 9300
ttaaatagtt?aaggataatt?tataatgcaa?aaaatattga?tcttaggtgt?gagtggtatg 9360
cttgggcata?ccctctttcg?ttttttatca?tctcagcagg?atcttttagt?aactggtaca 9420
gtacgccata?ttacgcaaga?aataaaatta?gtatcatcta?agaatatagt?cctgatgaga 9480
gatgtcttta?acaaagaaaa?attagaaaac?attataaatt?cgaatgatgt?tattataaat 9540
tgcattggcg?caattaagca?gaaatatgat?aataaaaaca?cagattatat?tctattaaat 9600
tcgtactttc?cacatcttct?taatgaaatt?tgtatagaat?ctaataaaag?actaattcat 9660
ttctcaacag?attgtatttt?taacggtgag?aaaggaaatt?atgatgatgg?tagtttatca 9720
aatgttatag?atatgtatgg?taaatctaaa?tatttaggtg?aggtttatgg?tgataaaaca 9780
cttactttaa?ggacatcatt?aattgggcat?gaattaaaat?catcgtatag?tttgattgat 9840
tggtttttga?actcaaactc?aactgttaat?ggttatacaa?aagctatttt?cagtggattg 9900
ccaaccatag?aaattgcgca?ttttttatat?gagcatgttt?tacaatccca?gttgcatggt 9960
atttataatt?tatcagcagc?accaatttca?aaattcgatt?tattgacttt?agtcaatgaa 10020
gtatacaaac?ttaataaaac?catcgttcct?aaaagtgact?ttgtgattga?tcgttctctt 10080
gactctcata?ggttgcgttc?attaactaag?tatacacctc?ctgactggaa?tactcttgtt 10140
gtaaagatgt?atttagatta?cttaagttta?ggagcattgc?ttaatgaaag?ataacttatc 10200
tttttctaaa?ctgatttctg?aagctgagat?taactccaga?aatcgttctc?accttaattt 10260
gcatgaatct?ttttctgatc?ctatacagaa?aactttgatt?tgctgttgtt?cgaatacgta 10320
tattcctcct?cactatcata?aatttgccca?tcaaagtgaa?ttgtttatta?gattaaaagg 10380
atgttttagt?ctattaattt?tttctgttga?tggagtgctt?acccgtcgta?taactataga 10440
tgaggagaat?cctctttatg?agataaaacc?aggtgttatt?catacggtgg?tagctttaga 10500
cagacataac?attttacttg?aaattaagag?gggaccgtat?gtcgaatctg?aagcgaaaaa 10560
ttttcctgat?tgggtaacat?tagaaaatag?cgataatcaa?cttgatttag?agcgattgcg 10620
aacattgaag?gtcggtgata?agttatcata?acaaggctgt?tataaattta?ttttaggtga 10680
tatctatgct?tccattaata?tctattatta?cgccgacata?taatagagca?tatactctag 10740
atcggttata?taattcttta?gtaacacaag?actctgaaaa?attcgaatgg?ataataatcg 10800
acgatggtag?tacagatgaa?acacggaaga?aatgtttagg?ttatataaat?gattccattt 10860
ttaaaataca?atattacaaa?caaaataata?gtggtaaacc?cgcagctatc?aatttaggag 10920
taaccaaagc?tgttggcgat?tatattttta?ttgtagatag?tgatgatgct?ttaacattaa 10980
atgcaattgg?tactatttat?gactgtattg?taaaatacac?agtcgcatct?gactttgaat 11040
tttccggctt?aggatttaga?aaatcttttt?ttgatgggaa?aatgctcggc?aaacatattg 11100
agcggaatac?gaattcacct?atgctgttac?atagcacaga?ttgtaataat?ttatttaaag 11160
tcgatttagc?atattgtttt?aaacgagagt?tgatgattaa?atatcctttc?ccaaagttta 11220
aagatgaaaa?atttgttcca?gaactgttta?tatggaataa?aattacagat?gttgccaaaa 11280
taatattttt?tcctaatgtt?gttgtatatt?attgtgagta?tctggctgat?ggacttacaa 11340
ataattttaa?aaaacaactt?cgacataatc?caattggctt?tgcagtatat?tataaagatc 11400
agttccatag?agaacattct?atatggggga?aaataaaaat?ggcaataaga?tatatccaat 11460
gtattatata?taaattcata?tagtcatgca?gggttcatcc?attcatttaa?ttagttgcta 11520
tgatttttat?atgaaaatat?tacatgttgt?accacagtta?aagcatggtg?gtgtcgaaac 11580
tgctgttttt?aattctattg?aaaggataag?aaagagtgga?tatgatttta?atgttttgac 11640
tattgaaaga?ggaaactgct?ttaacaattc?atatgtaaaa?gtagaacact?gcttaaattc 11700
ttctgtatac?aatccagttt?gtttttataa?atttattagg?tttattcata?aagaaaaacc 11760
tgatttgata?gtttattctt?tatggaaatc?tgcattgtta?ggaatttcat?tgtgtctttt 11820
aaaacctttt?ctcaaaagta?agcctaaaac?cgcgttaatt?atacataata?ctaggtttgc 11880
acattttgtt?gattacataa?taacaaaact?ttcacttaaa?ataaccgact?ctatatattt 11940
tgattcggaa?aaatcaaaag?cttactttct?gaatagagat?tatgcaacag?gcgaggttat 12000
tagttttatc?gataaaaaga?tggtggcaaa?aaaatttaat?aatatcaatg?gtgagattaa 12060
gtttgtattt?attggtaggt?tgcactctca?aaaaggggtt?gatagggcat?taaaatatat 12120
acatcttttt?aaaaagcaac?atgaagaaat?acgatttgat?atatatggtc?cagacgaagg 12180
agaattgaat?aatattataa?ataaggtaaa?taaattagga?ttgcaggata?aagttgtcta 12240
taaaggtgtt?gtcgataata?acaaagttaa?tgatatatta?acccaatatg?acttttatct 12300
tcaattcagc?cattttgaag?gaatggcaat?gtctgttgtt?gatgcgatgc?aggttggggt 12360
tattccaatc?gttaataatg?ttggtgagat?accaaattac?gtaaaaaaca?atattaacgg 12420
cattatctta?gatgataaaa?agcttggtga?tgaaaactat?cttaatttaa?atatatcatg 12480
cttattaaat?atcataaaga?atgatggtgg?atatttttta?tataataacg?cgataaagac 12540
atttcaggat?aataatactt?atgtagatga?ctatataaag?aaaataataa?atgctaacaa 12600
ctgatatcat?gtgttaaaat?ggtttatata?gctcctacta?aataatttgg?ctttttcgta 12660
atggctgtgt?agccgcaatc?tcgcagtaac?cccctgacag?gagtaaacaa?tgtcaaagca 12720
acagatcggc?gtcgtcggta?tggcagtgat?gggacgcaac?cttgcgctca?acatcgaaag 12780
ccgtggttat?accgtctcta?ttttcaaccg?ttcccgtgaa?aagacggaag?aagtgattgc 12840
cgaaaatcca?ggcaaaaaac?tggttcctta?ctatacggtg?aaagagtttg?ttgaatctct 12900
ggaaacgcct?cgtcgcatcc?tgttaatggt?gaaagcaggt?gcaggcacgg?atgctgctat 12960
tgattccctc?aagccatacc?tcgataaagg?tgacatcatc?attgatggtg?gtaacacctt 13020
cttccaggac?accattcgtc?gtaaccgtga?gctttctgca?gaaggcttta?actttatcgg 13080
taccggtgtt?tccggtggtg?aagaaggtgc?gctgaaaggt?ccttccatta?tgcctggcgg 13140
acagaaagaa?gcctataaac?tggttgcacc?gatcctgacc?aaaatcgccg?cagtggctga 13200
agacggggag?ccatgcgtta?cctatattgg?tgccgatggc?gcaggtcact?atgttaagat 13260
ggttcacaac?ggtattgaat?acggtgatat?gcagctgatt?gctgaagcct?attctctgct 13320
taaaggcggc?ctgaatctct?ctaacgaaga?actggcacag?acctttaccg?agtggaataa 13380
cggtgaactg?agcagctacc?tgatcgacat?caccaaagac?atcttcacta?aaaaagatga 13440
agacggtaac?tacctggttg?atgtgatcct?ggatgaagcg?gctaacaaag?gtaccggtaa 13500
atggaccagc?cagagcgcgc?tggatctcgg?agaaccgctg?tcgctgatta?ccgagtctgt 13560
gtttgcacgt?tatatctctt?ctctgaaaga?tcagcgcgtt?gccgcatcta?aagttctctc 13620
tggtccgcaa?gcgcagccag?ctggcgacaa?ggctgagttc?atcgaaaaag?ttcgtcgtgc 13680
gctgtatctg?ggcaaaatcg?tttcttacgc?tcagggcttc?tctcagttgc?gtgctgcgtc 13740
tgaagagtac?aactgggatc?tgaactacgg?cgaaatcgcg?aagattttcc?gtgctggctg 13800
catcatccgt?gcgcagttcc?tgcagaaaat?caccgatgca?tatgccgaaa?atccgcagat 13860
cgctaacctg?ctgctggccc?cgtacttcaa?gcaaattgcc?gatgactacc?agcaggcgct 13920
gcgcgatgtc?gtcgcttatg?cagtacagaa?cggtatcccg?gttccgacct?tcgccgctgc 13980
ggttgcctat?tacgatagct?accgtgccgc?tgttctgcct?gcgaacttaa?tccaggccca 14040
gcgcgacta 14049
Oligosaccharide unit treatment gene in the O antigen gene cluster of table 1 Escherichia coli O3 type 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 O-antigen transhipment enzyme   4566-6059   5099-5114   5575-5590   491bp   0 *   55.3
  4897-4913   5669-5684   787bp   0 *   55.3
  Wzx O-antigen polymerase   7207-8292   7388-7403   7921-7936   549bp   0 *   58.1
  7529-7545   8130-8145   617bp   0 *   58.1
*Only in Escherichia coli O3 type, obtain a correct band
Table 2 166 strain Escherichia coli and 43 strain Shigellas and their source
The bacterium source that contains in this group of group number
1, wild-type e. coli O1, O2, O5, O7, O8, O9, O12, O13, O14, O15, O16, O17, O18, IMVSa
                    O19ab，O20，O21，O22，O23，O24，
2, wild-type e. coli O4, O10, O25, O26, O27, O28, O29, O30, O32, O33, O34, O35, IMVSa
                    O36，O37，O38，O40，O41，O42，O43
3, wild-type e. coli O6, O44, O45, O46, O48, O49, O50, O51, O52, O54, O55, O56, IMVSa
                    O57，O58，O60，O61，O62，O53
4, wild-type e. coli O63, O65, O66, O69, O70, O71, O74, O75, O76, O77, O78, IMVSa
                    O79，O80，O81，O82，O83，O68
5, wild-type e. coli O84, O85, O86, O87, O88, O89, O90, O91, O92, O98, O99, IMVSa
                    O101，O102，O103，O104，O105，O106，O97
6, wild-type e. coli O107, O108, O109, O110, O111, O112ab, O112ac, O113, IMVSa
                    O115，O116，O118，O120，O123，O125，O126，O128，O117
7, wild-type e. coli O129, O130, O131, O132, O133, O134, O135, O136, O137, IMVSa
                    O138，O139，O141，O142，O143，O144，O145，O140
8, wild-type e. coli O146, O147, O148, O150, O152, O154, O156, O157, O158, IMVSa
                    O159，O160，O161，O163，O164，O165，O166，O153                 b
9, wild-type e. coli O168, O169, O170, O171, O172, O173, c
Shigella dysenteriae D1, D2, D3, D4, D5, D6, D7, D8, D9, D10, D11, D12, D13 d
10, Shigella bogdii B1, B2, B3, B4, B6, B7, B8, B9, B10, B11, B12, B13, B14, B15, d
                    B16，B17，B18
11, shigella flexneri F1a, F1b, F2a, F2b, F3, F4a, F4b, F5 (v:4), F5 (v:7), F6, d
                    DS，DR
12, wild-type e. coli O11, O39, O59, O64, O73, O96, O95, O100, O114, O151, O155, IMVSa
                    O124，O167，O162，O121，O127，O149，O119
13, the 12nd group of bacterial strain adds Escherichia coli reference culture O3 IMVSa
*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，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 O3 type O antigen gene structure iron
E.coli?O3?O-antigen?gene?cluster
Figure C20041001901700271
#orf?galF orf1 wzx orf3 wzy fnlA qnlA orf7 orf8 orf9 gnd
G+C％ 30.5 28.5 31.3 26.3 34.6 29.8 33.3 29.1 28.0
content
Table 4 intestinal bacteria O3 type O antigen gene cluster gene position
ATTGTGGCTG?CAGGGATCAA?AGAAATCCTC?CTGGTAACTC?ACGCGTCCAA?GAACGCGGTC 60
GAAAACCACT?TCGACACCTC?TTATGAATTA?GAATCTCTCC?TTGAACAGCG?CGTGAAGCGT 120
CAACTGCTGG?CGGAAGTACA?ATCTATCTGT?CCGCCGGGCG?TGACCATTAT?GAACGTGCGT 180
CAGGGTGAAC?CTTTAGGTTT?AGGCCACTCC?ATTTTGTGTG?CACGCCCCGC?CATTGGCGAC 240
AACCCATTTG?TCGTGGTGCT?GCCGGACGTT?GTTATCGATG?ATGCCAGCGC?CGACCCGTTG 300
CGTTACAACC?TTGCTGCCAT?GATTGCGCGC?TTCAATGAAA?CGGGACGCAG?TCAGGTGCTG 360
GCAAAACGTA?TGCCGGGTGA?CCTCTCTGAA?TACTCCGTCA?TCCAGACCAA?AGAACCACTG 420
GATCGCGAAG?GTAAAGTCAG?CCGCATTGTT?GAATTTATCG?AAAAACCGGA?TCAGCCGCAG 480
ACGTTGGACT?CAGACATCAT?GGCCGTTGGT?CGCTATGTGC?TTTCTGCCGA?TATTTGGCCG 540
GAACTTGAAC?GCACGCAGCC?AGGTGCATGG?GGGCGTATTC?AGCTGACTGA?TGCCATCGCT 600
GAACTGGCGA?AAAAACAGTC?CGTTGACGCC?ATGCTGATGA?CTGGGGACAG?TTACGACTGC 660
GGTAAAAAAA?TGGGCTATAT?GCAGGCGTTC?GTGAAGTATG?GACTACGCAA?CCTGAAAGAA 720
GGGGCGAAGT?TCCGTAAAGG?GATTGAGAAG?CTGTTAAGCG?AATAATGAAA?ATCTGACCGG 780
ATGTAACGGT?TGATAAGAAA?ATTATAACGG?CAGTGAAGAT?TCGTGGCGAA?AGTAATTTGT 840
TGCGAATTTT?CCTGCCGTTG?TTTTATATAA?ACAATCAGAA?TAACAACGAG?TTAGCAATAG 900
GATTTTAGTC?AAAGTTTTCC?AGGATTTTCC?TTGTTTCCAG?AGCGGATTGG?TAAGACAATT 960
AGCGTTTGAA?TTTTTCGGGT?TTAGCGCGAG?TGGGTAACGC?TCGCTACATC?GTAGGCATGC 1020
ATGCAGTGCT?CTGGTAGCTG?TAAAGCCAGG?GGCGGTAGCG?TATCTAGGGG?AAGATCATCG 1080
TGTGCGGTAA?GCACAAAAAT?GCAAAAAGGA?ACGTTTTGCA?TGTAATAAGC?ATAAGCTTGT 1140
AAAGAGATCA?GGGAACAGGG?AAAGATCACA?TAACGCCGGT?TTTTTGTACT?AAATAATTCG 1200
CACTAAATGT?TCAATAATTG?AGACATTCCT?TATTGTCTAA?AACTGTTTTT?CACAGCTTAT 1260
ATATGATCAA?ATACTCCTTA?CATAAATAAG?GAGAACAAAA?TGGAACTTAA?AAAACTGATG 1320
GAACATATTT?CTATTATCCC?CGATTACAGA?CAAGCCTGGA?AAGTAGAATA?TAAATTTTCA 1380
GATACTCTAC?TATTGACTAT?TTGTGCTGTC?ATTTCTGGTG?CAGAAGGTTG?GGAAGATATA 1440
GAGGATTTTG?GTGAAACGCA?TCTCGATTTT?TTGAAGCAAT?ATGGTGATTT?TGAAAATGGT 1500
ATTCCTGTCC?ATGATACCAT?TGCCGGAGTT?GTATCCTGTA?TTAGTCCTGC?AAAATTTCAC 1560
GAGTGCTTTA?TTAACTGGAT?GCGTGATTGC?CATTGTTCAG?ATTATAAAGA?CGTCATTGCA 1620
ATTGATGGGA?AAACGCTCCG?GCACTCTTAT?GATAAGAGTC?GTCGCAGGGG?AGCGATTCAT 1680
GTCATTAGTG?TGTCCTCAAC?AATGCACAGT?CTGGTTCTCG?GACAGATCAA?GACGGATGAA 1740
AAATCTAATG?AGATTACAGC?TATTCCTGAA?CTTCTTAACA?TGATGGATAT?TAAAGGAAAA 1800
ATCATCACAA?CGGATGCGAT?GGGGTGCCAG?AAAGATATTG?CAGAAAAAAT?ACAAAAACAG 1860
GGAGGTGATT?ATTTATTCGC?TGTAAAGGGA?AATCAGGGGC?GACTAAATAA?AGCCTTCGAG 1920
GAAAAATTTC?CGCTGAAAGA?ATTAAATAAT?CCAAAGTATG?ACAGTTACGC?AATTAGTGAA 1980
AAGAATCACG?GCAGAGAAGA?AATCCGTCTT?CATATTGTTT?GCGATGTCCC?TGATGAACTT 2040
ATTGATTTCA?CGTTTGAATG?GAAAGGACTG?AAAAAAATTA?TGCGTGGCAG?TCTCCTTTCG 2100
GTCAATAATA?GCAGAACAAA?AGAAAGAGCC?AGAAATGACG?GTCAGATATT?ATATCAGTTC 2160
TGCTGATTTA?ACCGCTGAGA?AGTTTGCGAC?AGCGATTCGA?AATCACTGGC?ACGTGGAGAA 2220
TAAGTTGCTC?TGGCGTCTGG?ATGTAGTAAT?GAATGAAAGA?CGACTGCAAA?ATAGGAAGAG 2280
GAAACGTTCA?GGGATAAGAC?ATATTGCAAT?TAATATTTTG?ACGAAAGATA?AGGTATTCAA 2340
GCACGGTTAA?GACGTTCGAT?GAAGACAGCG?ACTACCTGCG?TCAGTCTTTG?TGAGGAGCGG 2400
ACTCTAGTAA?TCCAACTTTG?TTGTTAGGTT?TTACTCGCGG?AATAATCATG?GGCGAATTTT 2460
ATTAATAATG?TATATTTTCC?TAATTAAAAA?CATGTTAAGA?AAAGAAACAA?AACTTTCAAG 2520
CTAGGTTACT?TATGATATGA?AGTCAACACA?TTCTAAAATT?AAAATTGCAC?ATGTTCAATT 2580
GATGCCACTA?CTGAGTGGTG?TTCAGCGAGT?ATCGTTACAA?GAATTCGAAT?TATTACCAAA 2640
TGAACAGTTC?GACATTAATT?TAATTTGCAA?AGAGAGTGGA?CCATAAACTG?ATTATTTAGA 2700
TGATAGTGTA?AGGGCTTTTT?TTGTCCCGAC?ATTATGTAGA?AATATATCGC?TCATAAAGGA 2760
Orf1's is initial
T ATGAAATCA?TTAATATCTT?TATATAAATT?ATTGAAAAAA?GAAAAATATG?ATATCGTCCA 2820
TACACACTCC?TCTAAAACTG?GTATTCTAGG?GCGGATAGCT?GCGCGGCTGG?CAGGAGTTCC 2880
TTGTGTAGTA?CATACTGTAC?ATGGCTTTGC?CTTTGAAAGC?ACAAAAAGAA?AATCAGTAAA 2940
GCTCGTTTAT?AAATGGCTGG?AAATATTTGC?AGCCAAATGC?ACTACCCGAT?TAATTTGTCT 3000
TCATAATGAA?GATAAAGAAA?TATGTATAAA?AGAACTATAT?GTTGATCCAA?TGAAAATTTC 3060
TGTAATTCCA?AATGGAGTTG?ATTTGGAAAA?ATTCGCTCCA?GCAATTAATA?AAGGAGACTT 3120
AAAAGAAAAA?ATATTAGGGT?TAAAAAGAAA?TAGCTTTGTA?TTTACCATGG?TGGGACGGTT 3180
GTGGCCACAA?AAAAATCCGT?TATATTTTGC?TGAAGCAGCA?AAGTATATTA?TAGAAAATAA 3240
CTTAATACCT?GATTCAGTTT?TTGTCATAGT?TGGTGATGGG?GAGTTGATGA?ATGATTTAAA 3300
ATACAATTAT?CAAACAGATA?TGAATTTAAA?AAAAAGGTTA?TTACTTTTAG?GCTGGCGAAA 3360
TGATATCCCA?AACATATTAA?AAGCAAGTGA?CGTTTTTGTT?CTTCCTTCTT?TATGGGAAGG 3420
AATGCCTCTT?GCCATATTAG?AAGCTCAATC?TACAGGGTTA?CCATGTATCG?TTTCTAATAT 3480
CAATGGTAAT?AATTGTTTGG?TTAAGAATGA?ATTTGATGGA?TTCTTAATTG?AACTTAATGA 3540
TATTGATAGC?TTTATAAATG?CGTTAGTTAG?AGTGACAGAC?GATAAAGTCT?TAAACATTAT 3600
GTCAAAAAAT?TGCCGTAATA?AAATTGTTAA?TGGATTTAAT?ATAGTTAAAA?GAGTGGATAA 3660
The termination of orf1
AATTAAAGAT?CTATATATTG?ATATGGTTGT?TAATAAA TGA?TTGTGTGCTT?TTTGAATGTT 3720
TATATCAATA?TCGCACTGTA?TATTTTATTT?TATGTAGATT?TATTATGAAT?ATAAAGGCAT 3780
GGTGATTTAC?ATCATTATTT?ATATACAGAC?GCTGTTATTG?GTATAGAGAA?ATATCATATA 3840
ACATTAGTTT?TCGTACAAAA?TGTAAAAAAT?GAAAGATTTA?TTACTAAGTA?AGACTATTTT 3900
TGATAACCTA?TATATTATCA?AATACTCTGT?ACATAAAGGA?GGGAAATGGA?ACATGAAAAA 3960
TAATAGTCGT?ATTTCTGTTA?ACCAATTTCA?AACAACCTGG?GAAAGTGGAA?CGTAAATTAT 4020
TAGATATTTA?CTACTGATTA?TTTGCTCCTG?CTTCGTCATT?TCTGACGCAG?AAGGATGGGA 4080
GTATACAAAG?GATGTTTGTG?AGACGTGTGT?CGATTTCTTG?AAGCAATACG?ATGATTTGGG 4140
ACATGGTACT?CCTGTTTATA?ATACCATAGC?TGAGTTCTAT?TCCGTATCAG?TCCTATTAAC 4200
TGGTTACGTG?ATGTTATTCT?TCAGATTATA?AAGACATCAT?TGCAGTAGGT?GGAACTTAAT 4260
AGCGGAGCAG?AATAAAGAGT?TTAAAATGAT?ATTCAATATT?GTATCAGTTC?TGCTGGTTGT 4320
TTAAATTCTG?AGAAGTCCGC?GATCGTGATT?TGAAACTGCT?AGAATGTTGA?GAATAATCAG 4380
CGCTGGTGTC?TGGATGTAGT?AATGAATGAA?GAGAAGTGCT?CAATAAGCAA?AGCAGCTATG 4440
GACAGTGATT?AGCCCGCATC?AGTTCTTGCA?GGGGTGTGTT?TTTATTATCT?TTCCCTGATT 4500
ATTGGGTGAC?TGTGTGACTT?ATACATTCTC?TTATTTTGAG?TGTGATGGTA?AAAATGTTTG 4560
Orf2's is initial
ACTCT ATGCA?TGTTCGGGTT?GCTGTAAATT?TTGCATCAAA?GATAACATTT?GCTTTGATAT 4620
CTATAATTAC?AATTCCAGTT?ATAAA?TCAA?AATTAGGAGT?AAATGCTTAT?GGTGTCCTAA 4680
CATTCTTTCT?ATCAATCCAG?GTAGTTATAT?TACTGCTTGA?GGGGGGAATG?AGTGCCACAA 4740
TGGCTAGAGG?ACTATTGAAT?AAAAAATATA?AGATATACAA?TATAAGTAAA?GCAGAGTACC 4800
AAAGTGTTTA?TTTTATCTTT?TTTATATTAG?TGTCATTATT?TGTATTTTCA?ATTTTTATTG 4860
TCTTTAATCA?TAATATTTCC?ACATTGATTT?ACAACCTTGA?TGATAGGGAA?GTCTATAGTT 4920
TTATTAGTGC?TGATTATGTT?ATTTTGCTGG?CTGGTGCAAT?GATTGCCTTG?CAGTTTTATA 4980
TCATATATTA?TGAAGCATTA?TTTGTGGCTT?ATGAAAAACA?AGTCTCTTAT?GGAATTCATA 5040
TAATAGTATT?TAATGTAATC?AAAACCATAA?TATCATTGTT?CTTGTTGATT?GTCTTTAATC 5100
TTGGGTTGGA?TGTTTATTTT?TTAGTTCATG?TATTTTGTAC?CTTTATACTT?ATTATTTTAT 5160
TGCACATTAA?GGCAATAAAA?AACGGATTAT?TAGAAATAAC?AACATTGCGA?GTGAAAAAAA 5220
TATACACAAA?AATATCATTT?ATGAAAGAAG?AATTTCATTT?TTCTAAAAAT?ATATTATGCG 5280
TGTCCATTCT?TTCTGCAATT?GCATTCCAAG?CTGACAGACT?ATTTATTACA?AAATATATGG 5340
GTATTAGTGC?TGTTGGAATG?TATGGTATTG?CTTATACATT?ATCAACAGTA?CCAACTTTAT 5400
TTACATCATC?ATTGTATAAT?GTTATCTATC?CAAGACTTAT?TATTTTAACA?AGAGAGCAAA 5460
GCAGCAGTGC?GATTTCATTT?TTTGAATTAG?TATCGAAATT?AATATATATT?ATCATAATAC 5520
CCACTTGTGT?GATTATTTCG?CTTAATTCGA?CCCAAATCCT?AAATATTTGG?ATAGGAAAGC 5580
ACGAAGGAAT?AATATCATTG?CTGTTAAGTA?TTCTAATAAT?AGCGACGTTA?TTTCAGGGGT 5640
TGCAGGTGAT?TCCTTTTGCA?CTACAGATGG?CTCAAGAACG?ACTAAAATTT?ACAGTTTTCA 5700
TGAATGCTAT?ATTTGTGCCC?AGCCTAATAT?TGGCATATTA?TTTGGTTAGT?ATTCATAAAA 5760
TTCTGTTATA?CATCTCCATA?GTGTGGTCAG?TATATAATAT?AATAACTTTT?TTCTCTTTAA 5820
CTATTTATTT?ATATGCTAAA?AATAAAATGT?ATACAAGACT?ACTTATTTTA?TATTTTAATA 5880
TATGCTTGTG?TGCTGTCGTA?ACAGTAGTTA?TATGTAGCAT?TTTTAAGAAT?CAAGAATCTC 5940
AAATATTCTT?GTCTCTAGTT?AATATTTCTC?TTCAGTTTTT?TATGTCGATG?AGTTTGTGCA 6000
The termination of the initial orf2 of orf3
TAATACTGTT?GTTCTGGAAA?ACTATATATG?GAGCATTGAG? ATGTGTTTGT?TGTCGG TAGT6060
AATTGCGACT?CATAATCGCG?CGGGATATGC?TAAAAAAAGC?ATAGAAAGTA?TTCTTTCAAT 6120
TGATGTTGAA?GAATTTCAAC?TAATTATTCA?TGATACAAGC?GATAATAATG?TACTTGAGGA 6180
ATTTTGCGCT?GAAATCCAGG?ATGCGAGATT?AAAATATATC?CATTGCAATG?AATTATTGTC 6240
GATGACAGAA?AATTTTAATA?GAGCATTAGA?ATATGTTCAG?GGAGAATATA?CTATAATGAT 6300
AGGAGATGAT?GATACGATTT?TATCGACTGC?GATAAA?TAT?TGTAGATATG?CGAAAGAAAA 6360
AAAAATTGAT?GTAATCTCGC?AGAAGATATC?TGCAGAGTAT?TGTTGGCCGG?ATTTTAGATC 6420
TAAGATCTCA?GGGGATTTTA?ACGCATCGTC?TCTGATAGTG?AACAAATTTA?CAAGTAACCA 6480
AGTTACTTAT?GAGGGGCGTA?AACAATACTA?TGAAGCAGTT?AAAAACTGTT?TTCAAGGTAC 6540
TGCAAGCCTC?CCCAAGTTAT?ATCATGGTAT?TGTTAAAACT?TCTTTATTGT?TAAAGATAAA 6600
AG?AAATACA?GGTACTTACT?TTCATGGGGT?TAGCCCTGAT?ATTTCAATTG?CATTGTCGTT 6660
AGCGTATAAC?TGCGAACGAT?ATATTGAAGT?GGATTATCCA?ATTACCCTTC?CAGGATCTTC 6720
GGGGGGAAGT?AACGCAGGTA?GAAGTGCAAT?GCGTACTCAT?GTTGGTAAAT?TAGACGACGA 6780
TCCACATATG?AAAAGATTTA?AAAGTTATGT?CTGGCCAAAC?TTATTGCCAA?GATATTTCAG 6840
TGTAGAGACT?GTGTGGGCTC?AGGCAGGCTT?AGTAACAATC?CAAAAAATAG?ATAATAATAA 6900
AGGATTTAAT?TTTATTAAGT?TCTATGCATT?ATGTTTGATT?AAACATCGAC?CCTATAAAAA 6960
ATTTATTATT?GATGAGATAA?AAAAATACAA?GAAAGTAAAA?GGAGGGTTTA?ATTGTATAAT 7020
TTATACAAAA?ATTATTTATC?ACATGTCTTA?TTTTTATATT?AATGGAATAT?CAAATCTTTC 7080
TAAAAAAATT?ATAAAATATG?TCTTCAAAAA?ACTTAAGTCT?CAAGAGAAGC?AAAAAAATAT 7140
AAAAGCAAAT?GATATTTTTG?AAGCCAGGAT?GATAATTGAA?AAGGAATTAT?CAAAGGAAAC 7200
The termination of the initial orf3 of orf4
ATTTGA ATGA?TTATATTTTC?TACTTTTCTA?CTTTTCTACT?TTTTTATTTT?TCTTTCATTG 7260
CTTCCTGTTT?TAATTAAAGA?GGGAGCTAGA?GGGAATGGCA?AATATTTTTA?TTTCTTTGAT 7320
TTATATATTT?ACTTAGTATT?AGCTAGTCTT?ATTATTTATA?TAATTTGTAC?ACGAGATATT 7380
TTTTCTGACT?CCGATACAGA?ACGTTATCTT?AGTATATTAG?AATATTATAG?AATAAATGGC 7440
TTTTATCTTT?TTGATAAGAA?CTATATTTTT?GCTGTGTATA?CATATTTGTA?CTCTCTATAT 7500
ATAGATGGTT?ACTATATGTA?TATGATAATC?CCACTTATTA?CCCTGCTATA?TTCATATTGT 7560
AAGTTAGTAA?AACCACTAAA?ATATCCTGTT?TATAGTTTTT?CTTTAGTATT?AATAGCAACA 7620
TTTCCTGTTT?TTTGGCAATT?GAGCGCAAGC?GCATTTAAAC?AATGCATTGC?AATATCTTTT 7680
ATTCTTTTAG?CATTAGTGCA?TATTATACAA?ACAAAATATA?AACAAGCGGT?GCTTTTATCT 7740
CTTATAGCTA?GTTCCTTCCA?TTTCACAGCA?TTAACGTTTT?TTATATTTTC?GTTGTTTTTC 7800
TATATTAGAA?AGGAAAGGGT?CAGTTTAAAT?TTATTTTTTT?TAATTTGGTT?TTTTAGTGTC 7860
ATTTTTAGCA?TAAGCGGATT?AAATTCGTAT?ATTCCTTCAA?TTATACCTTT?TTATGATTTA 7920
GTGGGTGAAA?GTAAGGTCAA?TATTTATCTT?GAATCAACAA?ATAATGCAAC?TGGTTTTCGT 7980
GTTGATTTCT?TTTTGTTTTC?ACTAGCCCCT?GTGTTAATTA?TATTTATTAA?CAGAACATTA 8040
GTAATAAAAG?ATCGCTATAT?TAAAATGATT?GCATTAAGTT?ATTTATATTT?CAATTCTATA 8100
TATAATATAA?TGTCATTCAT?CCCATTTTCA?GATAGAGTTG?CCGCATATTC?ATGGTGTCTT 8160
TCCCCCATTA?TTCTATTATG?GAGTGCTCTT?AAGTGTAGAT?CATCAATTTA?TATGTTAAGT 8220
TCTATAGGGA?TCGCGTTATT?AAATCCTTTG?ATTTTTATGT?ACTATAATTC?AATGTTTATT 8280
The termination orf5's of orf4 is initial
TTTTTAAAG T?GAGGATAT AT?GTTTAACGGT?AAAAAGATTT?TAATCACTGG?TGGTACTGGG8340
TCATTTGGTA?ATGCAGTGTT?ACGTCGGTTT?CTTGAGACAG?ATATTCAAGA?GGTTATTATT 8400
TTCAGTAGAG?ATGAAAAAAA?ACAAGATGAG?ATGAGAACGC?TATATCGCAA?TGATAAAATG 8460
AAATTTATTA?TAGGTGATAT?TAGAGATTAT?ACATCTGTTT?TAGCTGCAAC?TAGTGACGTT 8520
GACTATATAT?TCCATGCTGC?GGCTTTAAAA?CAGGTGCCTT?CATGTGAATT?TTATCCATGG 8580
GAAGCAGTTA?AAACGAATAT?AATTGGTACA?GAAAATTTAT?TAAATGCTGC?AAAACAAAAT 8640
AAAGTTAAAA?AAACAATTTG?TTTGAGCACT?GATAAAGCTG?TGTATCCAAT?TAACGCAATG 8700
GGAATTTCGA?AGGCTATGAT?GGAAAA?GTG?ATGACAGCAA?AGGCTAGAAC?GTTCAACAAT 8760
GAAGATTCAG?TTGTTTGCGG?AACTCGTTAT?GGCAATGTTA?TGGCATCACG?CGGCTCTGTT 8820
ATCCCTCTGT?TCATTCAGCA?GATTCAAACA?AATCAACCAT?TAACTATAAC?AGACCCTGAA 8880
ATGACTAGAT?TCATGATGAC?GTTAGATGAT?GCTGTAGATC?TCGTATTATT?TGCATTTAAT 8940
AACGGGCAAA?ACGGTGACAT?TTTTGTTCAG?AAAGCGCCTG?CCGCGACAAT?TGCTGTGCTT 9000
GCTAAAGCAT?TAGTTGAATT?GATGGGGGTG?CCAAACCATC?CTATTAAAGT?AGTTGGTTCA 9060
CGTCATGGGG?AAAAGATATA?TGAAACCTTA?CTTAGCCGTG?AAGAAATGGC?AAAAGCGATT 9120
GATCATGGAG?ATTATTATCA?GATTCAACCA?GATAATCGTG?ATCTGAATTA?TGACAAATAT 9180
TTAAGCGAAG?GGAGTAAAAA?AATTGAGCTG?TTTGATGATT?ACAATTCCCA?TAATACATAT 9240
AGACTCAATG?TTGATGAAAT?GAAAAAATTA?TTGTTAAAAC?TCAATATCAT?TCAAAATGTG 9300
The termination orf6's of orf5 is initial
TTAAATAGT T?AAGGATAATT?TATA ATGCAA?AAAATATTGA?TCTTAGGTGT?GAGTGGTATG9360
CTTGGGCATA?CCCTCTTTCG?TTTTTTATCA?TCTCAGCAGG?ATCTTTTAGT?AACTGGTACA 9420
GTACGCCATA?TTACGCAAGA?AATAAAATTA?GTATCATCTA?AGAATATAGT?CCTGATGAGA 9480
GATGTCTTTA?ACAAAGAAAA?ATTAGAAAAC?ATTATAAATT?CGAATGATGT?TATTATAAAT 9540
TGCATTGGCG?CAATTAAGCA?GAAATATGAT?AATAAAAACA?CAGATTATAT?TCTATTAAAT 9600
TCGTACTTTC?CACATCTTCT?TAATGAAATT?TGTATAGAAT?CTAATAAAAG?ACTAATTCAT 9660
TTCTCAACAG?ATTGTATTTT?TAACGGTGAG?AAAGGAAATT?ATGATGATGG?TAGTTTATCA 9720
AATGTTATAG?ATATGTATGG?TAAATCTAAA?TATTTAGGTG?AGGTTTATGG?TGATAAAACA 9780
CTTACTTTAA?GGACATCATT?AATTGGGCAT?GAATT?AAAT?CATCGTATAG?TTTGATTGAT 9840
TGGTTTTTGA?ACTCAAACTC?AACTGTTAAT?GGTTATACAA?AAGCTATTTT?CAGTGGATTG 9900
CCAACCATAG?AAATTGCGCA?TTTTTTATAT?GAGCATGTTT?TACAATCCCA?GTTGCATGGT 9960
ATTTATAATT?TATCAGCAGC?ACCAATTTCA?AAATTCGATT?TATTGACTTT?AGTCAATGAA 10020
GTATACAAAC?TTAATAAAAC?CATCGTTCCT?AAAAGTGACT?TTGTGATTGA?TCGTTCTCTT 10080
GACTCTCATA?GGTTGCGTTC?ATTAACTAAG?TATACACCTC?CTGACTGGAA?TACTCTTGTT 10140
The termination of the initial orf6 of orf7
GTAAAGATGT?ATTTAGATTA?CTTAAGTTTA?GGAGCATTGC?TTA ATGAAAG?A TAACTTATC10200
TTTTTCTAAA?CTGATTTCTG?AAGCTGAGAT?TAACTCCAGA?AATCGTTCTC?ACCTTAATTT 10260
GCATGAATCT?TTTTCTGATC?CTATACAGAA?AACTTTGATT?TGCTGTTGTT?CGAATACGTA 10320
TATTCCTCCT?CACTATCATA?AATTTGCCCA?TCAAAGTGAA?TTGTTTATTA?GATTAAAAGG 10380
ATGTTTTAGT?CTATTAATTT?TTTCTGTTGA?TGGAGTGCTT?ACCCGTCGTA?TAACTATAGA 10440
TGAGGAGAAT?CCTCTTTATG?AGATAAAACC?AGGTGTTATT?CATACGGTGG?TAGCTTTAGA 10500
CAGACATAAC?ATTTTACTTG?AAATTAAGAG?GGGACCGTAT?GTCGAATCTG?AAGCGAAAAA 10560
TTTTCCTGAT?TGGGTAACAT?TAG?AAATAG?CGATAATCAA?CTTGATTTAG?AGCGATTGCG 10620
The termination of orf7
AACATTGAAG?GTCGGTGATA?AGTTATCA TA?ACAAGGCTGT?TATAAATTTA?TTTTAGGTGA 10680
Orf8's is initial
TATCT ATGCT?TCCATTAATA?TCTATTATTA?CGCCGACATA?TAATAGAGCA?TATACTCTAG 10740
ATCGGTTATA?TAATTCTTTA?GTAACACAAG?ACTCTGAAAA?ATTCGAATGG?ATAATAATCG 10800
ACGATGGTAG?TACAGATGAA?ACACGGAAGA?AATGTTTAGG?TTATATAAAT?GATTCCATTT 10860
TTAAAATACA?ATATTACAAA?CAAAATAATA?GTGGTAAACC?CGCAGCTATC?AATTTAGGAG 10920
TAACCAAAGC?TGTTGGCGAT?TATATTTTTA?TTGTAGATAG?TGATGATGCT?TTAACATTAA 10980
ATGCAATTGG?TACTATTTAT?GACTGTATTG?TAAAATACAC?AGTCGCATCT?GACTTTGAAT 11040
TTTCCGGCTT?AGGATTTAGA?AAATCTTTTT?TTGATGGGAA?AATGCTCGGC?AAACATATTG 11100
AGCGGAATAC?GAATTCACCT?ATGCTGTTAC?ATAGCACAGA?TTGTAATAAT?TTATTTAAAG 11160
TCGATTTAGC?ATATTGTTTT?AAACGAGAGT?TGATGATTAA?ATATCCTTTC?CCAAAGTTTA 11220
AAGATGAAAA?ATTTGTTCCA?GAACTGTTTA?TATGGAATAA?AATTACAGAT?GTTGCCAAAA 11280
TAATATTTTT?TCCTAATGTT?GTTGTATATT?ATTGTGAGTA?TCTGGCTGAT?GGACTTACAA 11340
ATAATTTTAA?AAAACAACTT?CGACATAATC?CAATTGGCTT?TGCAGTATAT?TATAAAGATC 11400
AGTTCCATAG?AGAACATTCT?ATATGGGGGA?AAATAAAAAT?GGCAATAAGA?TATATCCAAT 11460
The termination orf9's of orf8 is initial
GTATTATATA?TAAATTCATA? TAGTC ATGCA?GGGTTCATCC?ATTCATTTAA?TTAGTTGCTA11520
TGATTTTTAT?ATGAAAATAT?TACATGTTGT?ACCACAGTTA?AAGCATGGTG?GTGTCGAAAC 11580
TGCTGTTTTT?AATTCTATTG?AAAGGATAAG?AAAGAGTGGA?TATGATTTTA?ATGTTTTGAC 11640
TATTGAAAGA?GGAAACTGCT?TTAACAATTC?ATATGTAAA GTAGAACACT?GCTTAAATTC 11700
TTCTGTATAC?AATCCAGTTT?GTTTTTATAA?ATTTATTAGG?TTTATTCATA?AAGAAAAACC 11760
TGATTTGATA?GTTTATTCTT?TATGGAAATC?TGCATTGTTA?GGAATTTCAT?TGTGTCTTTT 11820
AAAACCTTTT?CTCAAAAGTA?AGCCTAAAAC?CGCGTTAATT?ATACATAATA?CTAGGTTTGC 11880
ACATTTTGTT?GATTACATAA?TAACAAAACT?TTCACTTAAA?ATAACCGACT?CTATATATTT 11940
TGATTCGGAA?AAATCAAAAG?CTTACTTTCT?GAATAGAGAT?TATGCAACAG?GCGAGGTTAT 12000
TAGTTTTATC?GATAAAAAGA?TGGTGGCAAA?AAAATTTAAT?AATATCAATG?GTGAGATTAA 12060
GTTTGTATTT?ATTGGTAGGT?TGCACTCTCA?AAAAGGGGTT?GATAGGGCAT?TAAAATATAT 12120
ACATCTTTTT?AAAAAGCAAC?ATGAAGAAAT?ACGATTTGAT?ATATATGGTC?CAGACGAAGG 12180
AGAATTGAAT?AATATTATAA?ATAAGGTAAA?TAAATTAGGA?TTGCAGGATA?AAGTTGTCTA 12240
TAAAGGTGTT?GTCGATAATA?ACAAAGTTAA?TGATATATTA?ACCCAATATG?ACTTTTATCT 12300
TCAATTCAGC?CATTTTGAAG?GAATGGCAAT?GTCTGTTGTT?GATGCGATGC?AGGTTGGGGT 12360
TATTCCAATC?GTTAATAATG?TTGGTGAGAT?ACCAAATTAC?GTAAAAAACA?ATATTAACGG 12420
CATTATCTTA?GATGATAAAA?AGCTTGGTGA?TGAAAACTAT?CTTAATTTAA?ATATATCATG 12480
CTTATTAAAT?ATCATAAAGA?ATGATGGTGG?ATATTTTTTA?TATAATAACG?CGATAAAGAC 12540
ATTTCAGGAT?AATAATACTT?ATGTAGATGA?CTATATAAAG?AAAATAATAA?ATGCTAACAA 12600
The termination of orf9
C TGATATCAT?GTGTTAAAAT?GGTTTATATA?GCTCCTACTA?AATAATTTGG?CTTTTTCGTA 12660
ATGGCTGTGT?AGCCGCAATC?TCGCAGTAAC?CCCCTGACAG?GAGTAAACAA?TGTCAAAGCA 12720
ACAGATCGGC?GTCGTCGGTA?TGGCAGTGAT?GGGACGCAAC?CTTGCGCTCA?ACATCGAAAG 12780
CCGTGGTTAT?ACCGTCTCTA?TTTTCAACCG?TTCCCGTGAA?AAGACGGAAG?AAGTGATTGC 12840
CGAAAATCCA?GGCAAAAAAC?TGGTTCCTTA?CTATACGGTG?AAAGAGTTTG?TTGAATCTCT 12900
GGAAACGCCT?CGTCGCATCC?TGTTAATGGT?GAAAGCAGGT?GCAGGCACGG?ATGCTGCTAT 12960
TGATTCCCTC?AAGCCATACC?TCGATAAAGG?TGACATCATC?ATTGATGGTG?GTAACACCTT 13020
CTTCCAGGAC?ACCATTCGTC?GTAACCGTGA?GCTTTCTGCA?GAAGGCTTTA?ACTTTATCGG 13080
TACCGGTGTT?TCCGGTGGTG?AAGAAGGTGC?GCTGAAAGGT?CCTTCCATTA?TGCCTGGCGG 13140
ACAGAAAGAA?GCCTATAAAC?TGGTTGCACC?GATCCTGACC?AAAATCGCCG?CAGTGGCTGA 13200
AGACGGGGAG?CCATGCGTTA?CCTATATTGG?TGCCGATGGC?GCAGGTCACT?ATGTTAAGAT 13260
GGTTCACAAC?GGTATTGAAT?ACGGTGATAT?GCAGCTGATT?GCTGAAGCCT?ATTCTCTGCT 13320
TAAAGGCGGC?CTGAATCTCT?CTAACGAAGA?ACTGGCACAG?ACCTTTACCG?AGTGGAATAA 13380
CGGTGAACTG?AGCAGCTACC?TGATCGACAT?CACCAAAGAC?ATCTTCACTA?AAAAAGATGA 13440
AGACGGTAAC?TACCTGGTTG?ATGTGATCCT?GGATGAAGCG?GCTAACAAAG?GTACCGGTAA 13500
ATGGACCAGC?CAGAGCGCGC?TGGATCTCGG?AGAACCGCTG?TCGCTGATTA?CCGAGTCTGT 13560
GTTTGCACGT?TATATCTCTT?CTCTGAAAGA?TCAGCGCGTT?GCCGCATCTA?AAGTTCTCTC 13620
TGGTCCGCAA?GCGCAGCCAG?CTGGCGACAA?GGCTGAGTTC?ATCGAAAAAG?TTCGTCGTGC 13680
GCTGTATCTG?GGCAAAATCG?TTTCTTACGC?TCAGGGCTTC?TCTCAGTTGC?GTGCTGCGTC 13740
TGAAGAGTAC?AACTGGGATC?TGAACTACGG?CGAAATCGCG?AAGATTTTCC?GTGCTGGCTG 13800
CATCATCCGT?GCGCAGTTCC?TGCAGAAAAT?CACCGATGCA?TATGCCGAAA?ATCCGCAGAT 13860
CGCTAACCTG?CTGCTGGCCC?CGTACTTCAA?GCAAATTGCC?GATGACTACC?AGCAGGCGCT 13920
GCGCGATGTC?GTCGCTTATG?CAGTACAGAA?CGGTATCCCG?GTTCCGACCT?TCGCCGCTGC 13980
GGTTGCCTAT?TACGATAGCT?ACCGTGCCGC?TGTTCTGCCT?GCGAACTTAA?TCCAGGCCCA 14040
GCGCGACTA 14049
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 O3 type, it is characterized in that: its wzx sequence is that the Nucleotide or the wzy of 4566 to 6059 bases among the SEQ ID NO:1 is that sequence is the Nucleotide of 7207 to 8292 bases among the SEQ IDNO:1 or has above-mentioned sequence insertion, lacks or replace one or more Nucleotide, but keeps the oligonucleotide of the O-antigen function of wzx, wzy Nucleotide specific detection intestinal bacteria O3 type.
2, a kind of oligonucleotide of the O-antigen-specific to intestinal bacteria O3 type, it is characterized in that, it is that to come from the oligonucleotide of wzx gene right: the Nucleotide of 5099 to 5114 bases among the SEQ ID NO:1 and the Nucleotide of 5575 to 5590 bases, the Nucleotide of 4897 to 4913 bases among the SEQ ID NO:1 and the Nucleotide of 5669 to 5684 bases; Or the oligonucleotide that comes from the wzy gene is right: the Nucleotide of 7388 to 7403 bases among the SEQ IDNO:1 and the Nucleotide of 7921 to 7936 bases, the Nucleotide of 7529 to 7545 bases among the SEQ IDNO:1 and the Nucleotide of 8130 to 8145 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 O3 type of 2.
4, according to the application of the oligonucleotide of claim 1, any described O-antigen-specific to intestinal bacteria O3 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.
CNB2004100190178A 2004-04-19 2004-04-19 Nucleotide peculiar to 0-antigen of 03 type bacillus coli Expired - Fee Related CN100345967C (en)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998050531A1 (en) * 1997-05-01 1998-11-12 The University Of Sydney Nucleic acid molecules specific for bacterial antigens and uses thereof
CN1442423A (en) * 2003-04-15 2003-09-17 南开大学 Nucleotide specific against 0-antigen of colibacillus 0107
CN1442422A (en) * 2003-04-15 2003-09-17 南开大学 Nucleotide specific against 0-antigen of shigella dysenteria 3, colibacillus 0124 and 0164
CN1442424A (en) * 2003-04-15 2003-09-17 南开大学 Nucleotide specific against 0-antigen of colibacillus 0172
CN1442421A (en) * 2003-04-15 2003-09-17 南开大学 Nucleotide specific against o-antigen of colibacillus 0150

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998050531A1 (en) * 1997-05-01 1998-11-12 The University Of Sydney Nucleic acid molecules specific for bacterial antigens and uses thereof
CN1442423A (en) * 2003-04-15 2003-09-17 南开大学 Nucleotide specific against 0-antigen of colibacillus 0107
CN1442422A (en) * 2003-04-15 2003-09-17 南开大学 Nucleotide specific against 0-antigen of shigella dysenteria 3, colibacillus 0124 and 0164
CN1442424A (en) * 2003-04-15 2003-09-17 南开大学 Nucleotide specific against 0-antigen of colibacillus 0172
CN1442421A (en) * 2003-04-15 2003-09-17 南开大学 Nucleotide specific against o-antigen of colibacillus 0150

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
sequence and analysis of the O antigen gene(rfb) cluster ofescherichia coil O111. DAVIDA. BASTIN ET AL,GENE,Vol.164 1995 *

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