CN1554761A - O-antigen specific nucleotide of E.coli 0142 type - Google Patents

Abstract

The present invention provides a kind of nucleotide specific to O-antigen of Escherichia coli O142, and it is the total nucleotide sequence of gene cluster of Escherichia coli O142 to control the synthesis of O-antigen, such as separated nucleotide shown in SEQ ID No. 1 with whole length of 13584 bases; nucleotide similar to that in SEQ ID No. 1 with one or several inserted, deleted or substituted bases; and oligonucleotide originated glycosyltransferase gene and oligose unit processing gene of Escherichia coli O142 antigen gene cluster. PCR proves the high specificity of the oligonucleotide on the Escherichia coli O142 antigen. The present invention also discloses the method of detecting and identifying the Escherichia coli O142 in human body and in environment with the oligonucleotide of the present invention.

Description

Nucleotide to the O-antigen-specific of intestinal bacteria O142 type

Technical field

The present invention relates to the complete nucleotide sequence of control O-antigen synthetic gene cluster in the intestinal bacteria O142 type (Escherichia coli O142), particularly relate in the intestinal bacteria O142 type oligonucleotide in the control O-antigen synthetic gene cluster, can utilize these the oligonucleotide of the O-antigen-specific intestinal bacteria O142 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. (19142) " 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.19142, 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; O152 and 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 O142 type.It is the Nucleotide in the O-antigen gene bunch of intestinal bacteria O142 type, is the special Nucleotide that comes from glycosyl transport 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 O142 type.

Another object of the present invention has provided the gene of the O-antigen gene bunch that constitutes intestinal bacteria O142 type: the gene of transhipment enzyme comprises the wzx gene or with wzx the gene of identity function is arranged; Pol gene comprises the wzy gene or with wzy the gene of identity function is arranged; Mutase gene comprises the gne gene; Glycosyltransferase gene comprises orf1, orf8, orf9, orf10 gene; The FscM gene comprises rmlB, rmlD, rmlA, rmlC gene.

Another purpose of the present invention has provided oligonucleotide, and the gene that they come from intestinal bacteria O142 type coding transhipment enzyme respectively is the wzx gene or with wzx the gene of identity function is arranged; The gene that comes from the coding polysaccharase is the wzy gene or with wzy the gene 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 O142 type; Especially the oligonucleotide of listing in the table 1, they are high specials to the O-antigen of intestinal bacteria O142 type, and these oligonucleotide are also reconfigurable, the oligonucleotide after the combination also is a high special to the O-antigen of intestinal bacteria O142 type.

The above-mentioned oligonucleotide that a further object of the present invention provides can be used as primer and is used for nucleic acid amplification reaction, perhaps be used for hybridization as probe, perhaps be used to make gene chip or microarray, thereby pass through O-antigen and the detection and the identification of escherichia coli O142 type of these methods detections and identification of escherichia coli O142 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 O142 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 O142 type, and it is the isolating Nucleotide shown in SEQ ID NO:1,13584 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 O142 type, it is by 11 genomic constitutions, all between galF gene and gnd gene.

The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O142 type, 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 comprises the wzy gene or with wzy the gene of identity function is arranged; Mutase gene comprises the gne gene; Glycosyltransferase gene comprises orf1, orf8, orf9, orf10 gene; The FscM gene comprises rmlB, rmlD, rmlA, rmlC gene; Wherein said gene: wzx is the Nucleotide of 5649 to 6893 bases among the SEQ ID NO:1; Wzy is the Nucleotide of 6945 to 8126 bases among the SEQ ID NO:1; Gne is the Nucleotide of 11163 to 12182 bases among the SEQ ID NO:1; Orf1 is the Nucleotide of 1140 to 2156 bases among the SEQ ID NO:1; Orf8 is the Nucleotide of 8108 to 9043 bases among the SEQ ID NO:1; Orf9 is the Nucleotide of 9040 to 9828 bases among the SEQ ID NO:1; Orf10 is the Nucleotide of 9825 to 10919 bases among the SEQ ID NO:1; RmlB is the Nucleotide of 2176 to 3261 bases among the SEQ ID NO:1; RmlD is the Nucleotide of 3261 to 4160 bases among the SEQ ID NO:1; RmlA is the Nucleotide of 4212 to 5090 bases among the SEQ ID NO:1; RmlC is the Nucleotide of 5095 to 5652 bases among the SEQID NO:1.

The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O142 type, wherein it is to come from described wzx gene, wzy gene; And their mixing or their reorganization.

The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O142 type, the oligonucleotide of the wherein said wzx of coming from gene is to being: the Nucleotide of 5901 to 5920 bases among the SEQ ID NO:1 and the Nucleotide of 6155 to 6173 bases; The Nucleotide of 6496 to 6513 bases among the SEQ ID NO:1 and the Nucleotide of 6754 to 6771 bases.The oligonucleotide that comes from the wzy gene is to being: the Nucleotide of 7463 to 7480 bases among the SEQ ID NO:1 and the Nucleotide of 7902 to 7919 bases; The Nucleotide of 6966 to 6983 bases among the SEQ ID NO:1 and the Nucleotide of 7288 to 7306 bases.

The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O142 type is detecting the application of expressing the antigenic bacterium of O-, identify other polysaccharide antigen of the O-antigen of bacterium and bacterium in diagnosis.

The recombinant molecule of the Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O142 type, and can provide the O-antigen of expressing intestinal bacteria O142 type by inserting to express, and become bacterial vaccine.

The application of the Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O142 type, wherein it is used for PCR, is used for hybridization and fluoroscopic examination or is used to make gene chip or microarray, the bacterium in human body and the environment as probe as primer.

The separation method of the Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O142 type is characterized in that it comprises the steps:

(1) genomic extraction: 37 ℃ of incubated overnight intestinal bacteria O142 types in the LB of 5mL substratum, centrifugal collecting cell.With 500ul 50mM Tris-HCl (pH8.0) and 10ul 0.4M EDTA re-suspended cell, 37 ℃ of incubations 20 minutes, the N,O-Diacetylmuramidase that adds 10ul 10mg/ml then continues insulation 20 minutes.The Proteinase K, the 15ul 10%SDS that add 3ul 20mg/ml afterwards, 50 ℃ of incubations 2 hours, the RNase that adds 3ul 10mg/ml again, 65 ℃ of incubations 30 minutes, add equal-volume phenol extracting mixture, get supernatant liquor and use isopyknic phenol again: chloroform: primary isoamyl alcohol (25: 24: 1) mixing solutions extracting twice, get supernatant liquor again with isopyknic ether extracting to remove remaining phenol.Supernatant liquor rolls out DNA and washes DNA with 70% ethanol with glass yarn with 2 times of volume ethanol deposit D NA, 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 O142 type bunch: with the genome of intestinal bacteria O142 type is that template is passed through its O-antigen gene of Long pcr amplification bunch, at first according to the JumpStart sequences Design upstream primer (#1523-ATT GTGGCT 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 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 longPCR 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 O142 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 O142 type; The quality of sequence is mainly guaranteed by two aspects: 1) genome of intestinal bacteria O142 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 O142 type O-antigen gene bunch, with American National biotechnology information science center (The National Center forBiotechnology Information, NCBI) orffinder finds gene, find the reading frame of 11 openings, determine also that with the function of finding the reading frame that these are open what gene they are with the genetic comparison among the blast groupware and the GenBank, finish gene annotation with the Artemis software at Britain sanger center again, do accurate comparison between DNA and protein sequence with Clustral W software, obtain the structure of the O-antigen gene bunch of intestinal bacteria O142 type at last;

(6) specific gene screening: at wzx, the wzy gene design primer in the O-antigen gene of dysentery intestinal bacteria O142 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 the 13rd group, has obtained the expection size, any product that all do not increase in other groups is so the O-antigen of wzx, wzy gene pairs intestinal bacteria O142 type all is high special.

Just, first aspect of the present invention provides the full length nucleotide sequence of the O-antigen gene bunch of intestinal bacteria O142 type, its complete sequence shown in SEQ ID NO:1,13584 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 O142 type by method of the present invention, as shown in table 3, it is altogether by 11 genomic constitutions, 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 O142 type, promptly transports the gene of enzyme, comprises the wzx gene or with wzx the gene of identity function arranged; Pol gene comprises the wzy gene or with wzy the gene of identity function is arranged; Glycosyltransferase gene comprises orf1, orf8, orf9, orf10 gene; The FscM gene comprises rmlB, rmlD, rmlA, rmlC gene; Special sugared synthesis path gene in the bacterial polysaccharides antigen comprises the gne gene.Their zero positions in O-antigen gene bunch and final position and nucleotide sequence all are listed in the table 4; The invention particularly relates to glycosyltransferase gene, transhipment enzyme gene and pol gene, because sugared synthesis path gene is that the gene of synthetic nucleosides bisphosphate monose is common, common by indication to more exocellular polysaccharide now, O-antigen to bacterium is not very special, and the glycosyltransferase gene that the present invention relates to, transhipment enzyme gene and pol gene are high specials to the O-antigen of intestinal bacteria O142 type.

The 3rd aspect of the present invention, wzx gene in the O-antigen gene bunch that comes from intestinal bacteria O142 type is provided 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, 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 O142 type all is high special.

The separation method of the Nucleotide of described O-antigen-specific to intestinal bacteria O142 type comprises the steps: 1) genomic extraction; 2) the O-antigen gene in the pcr amplification intestinal bacteria O142 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.

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

As used herein, " oligonucleotide " mainly refers to derive from gene, the gene of coding transhipment enzyme and intragenic one section nucleic acid molecule of coding polysaccharase of the encoding glycosyl transferring enzyme in the O-antigen gene bunch, they can change on length, generally change in 10 to 20 Nucleotide scopes; More definite these oligonucleotide of saying are to come from wzx gene (nucleotide position is the Nucleotide of 5649 to 6893 bases from SEQ ID NO:1); Wzy gene (nucleotide position is the Nucleotide of 6945 to 8126 bases from SEQ ID NO:1); Coming from above intragenic oligonucleotide is high special to intestinal bacteria O142 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: the gene of coding transhipment enzyme and polysaccharase comprises the wzx gene or the gene, wzy gene of identity function is arranged or with wzy the gene of identity function is arranged with wzx.At least one oligonucleotide can be hybridized with at least one more than one such gene specific of expressing the special antigenic bacterium of O-under the situation of condition permission, and these bacteriums are intestinal bacteria O142 types.Available PCR method detects, more can with behind the Nucleotide mark in the inventive method as probe by hybridization such as southern-blot or fluoroscopic examination, perhaps by antigen and bacterium in gene chip or the microarray assay sample.

Planner of the present invention considers following situation: when one special oligonucleotide detects when invalid, the mixture of oligonucleotide can with the target region specific hybrid with test sample.Therefore the invention provides a cover oligonucleotide and be used for detection method of the present invention.Here said oligonucleotide is meant the gene that comes from the encoding glycosyl transferring enzyme, the gene of coding transhipment enzyme and the gene of polysaccharase, comprises the wzx gene or the gene, wzy gene of identity function is arranged or with wzy the oligonucleotide of the gene of identity function is arranged with wzx.This cover oligonucleotide is special to the O-antigen of a special bacterium, and this special bacterium O-antigen is expressed by intestinal bacteria O142 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: the gene of coding transhipment enzyme and polysaccharase comprises the wzx gene or the gene, wzy gene of identity function is arranged or with wzy the gene of identity function is arranged with wzx.At least one oligonucleotide can be expressed more than one such gene specific hybridization of the special antigenic bacterium of O-with at least one under the situation of condition permission.These bacteriums are intestinal bacteria O142 types.Oligonucleotide among available the present invention is made the method test sample of primer by PCR, also can with behind the oligonucleotide molecules mark among the present invention as probe by hybridization such as southern-blot or fluoroscopic examination, perhaps by antigen and bacterium in gene chip or the microarray assay sample.

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

On the other hand, the present invention also relates to the antigenic method of one or more bacterial polysaccharideses in the sample that a kind of detection comes from patient.One or more bacterial polysaccharides antigens in the sample can make sample can with a specific hybrid in a pair of oligonucleotide in following at least one gene, these genes are: the gene of coding transhipment enzyme and polysaccharase comprises the wzx gene or the gene, wzy gene of identity function is arranged or with wzy the gene of identity function is arranged with wzx.Under the situation of condition permission at least one oligonucleotide can with sample at least one express more than one such gene specific hybridization of the special antigenic bacterium of O-, these bacteriums are intestinal bacteria O142 types.Oligonucleotide among available the present invention is made the method test sample of primer by PCR, also can will pass through hybridization as probe behind the oligonucleotide mark among the present invention, perhaps by antigen and bacterium in gene chip or the microarray assay sample.

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

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

The present invention discloses the full length sequence of the O-antigen gene bunch of intestinal bacteria O142 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 O142 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 O142 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 liquor and use isopyknic phenol again: chloroform: primary isoamyl alcohol is taken out (25: 24: 1) mixing solutions and is carried twice, get supernatant liquor again with isopyknic ether extracting to remove remaining phenol, supernatant liquor is with 2 times of volume ethanol deposit D NA, roll out DNA and wash DNA with 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 O142 type bunch:

With the genome of intestinal bacteria O142 type is that template is passed through its O-antigen gene of Long pcr amplification bunch.At first according to the JumpStart sequences Design upstream primer (#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 CCTGGA TTA AGT TCG C) in O-antigen gene bunch downstream; With the Expand Long TemplatePCR method of Boehringer Mannheim company amplification O-antigen gene bunch, the PCR response procedures was as follows: 94 ℃ of pre-sex change 2 minutes; 94 ℃ of sex change are 10 seconds then, 60 ℃ of annealing 15 seconds, and 68 ℃ were extended 15 minutes, and carried out 30 circulations like this.At last, continue to extend 7 minutes at 68 ℃, obtain the PCR product, the agarose gel electrophoresis with 0.8% detects the size and the specificity thereof of PCR product.Merge 5 pipe long PCR products, and with the Wizard PCR Preps purification kit purified pcr product of Promega company.

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

At first be the acquisition that connects product: make up O-antigen gene bunch library with the Novagen DNaseI shot gun method that is modified.Reaction system is a 300ng PCR purified product, 0.9ul 0.1M MnCl ₂, 1ul1: the DNaseI of the 1mg/ml of 2000 dilutions, reaction is carried out at room temperature.Enzyme is cut the dna fragmentation size is concentrated between the 1.5kb-3kb, then adds 2ul 0.1M EDTA termination reaction.Merge the same reaction system of 4 pipes, with isopyknic phenol extracting once, use isopyknic phenol: chloroform: primary isoamyl alcohol (25: 24: 1) mixing solutions extracting once, after using isopyknic ether extracting once again, dehydrated alcohol deposit D NA with 2.5 times of volumes, and wash precipitation with 70% ethanol, be resuspended at last in the 18ul water.In this mixture, add 2.5ul dNTP (1mMdCTP subsequently, 1mMdGTP, 1mMdTTP, 10mMdATP), the T4DNA polysaccharase of 1.25ul 100mM DTT and 5 units, 11 ℃ 30 minutes, enzyme is cut product mend into flush end, after 75 ℃ of termination reactions, add the Tth archaeal dna polymerase of 5 units and corresponding damping fluid thereof and system is expanded as 80ul, 70 ℃ were reacted 20 minutes, made 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 competent cell bacillus coli DH 5.Get the single bacterium colony of a ring bacillus coli DH 5 in the LB of 5ml substratum, 180rpm cultivated after 10 hours, got in the LB substratum that the 2ml culture is transferred to 200ml, and 37 ℃ of 250rpm thermal agitations are cultivated OD600 about 0.5, ice bath cooling was 20 minutes then, in centrifugal 15 minutes of 4 ℃ of 4000rpm.Confide all supernatant liquor, dispel thalline, in centrifugal 15 minutes of 4 ℃ of 4000rpm with the deionization aqua sterilisa 200ml of cold ice precooling.Deionization aqua sterilisa 100ml with cold ice precooling dispelled thalline again, in centrifugal 15 minutes of 4 ℃ of 4000rpm.With 10% glycerine suspension cell of cold ice precooling, centrifugal 10 minutes of 4 ℃ of 6000rpm abandon supernatant liquor, precipitate 10% glycerine suspension cell with the precooling of 1ml ice at last, are competent cell.The competent cell that makes is packed as 50ul one pipe ,-70 ℃ of preservations.

Be electric transformed competence colibacillus cell at last: get after 2-3ul connects product and 50ul competence bacillus coli DH 5 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 O142 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 O142 type obtained.The quality of sequence is mainly guaranteed by two aspects: 1) genome of intestinal bacteria O142 type is done 5 Long PCR reactions, mix these products then to produce the library.2), guarantee high-quality fraction of coverage more than 3 to each base.After obtaining the nucleotide sequence of intestinal bacteria O142 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 11 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 O142 type at last, as shown in table 3.

By retrieving and comparing, find that orf2, orf3, orf4, orf5 encoded protein and rmlB, rmlD, rmlA, rmlC gene coded protein have the sequence identity of 74-92%, the similarity of 83-96% is rmlB, rmlD, rmlA, rmlC so name orf2, orf3, orf4, orf5.We think the existence of rhamnosyl in the O antigen of intestinal bacteria O154; Orf5 encoded protein and Yersinia gne encoded protein have 61% homogeny, and so 78% similarity is orf5 called after gne

Orf6 and orf7 are the proteic genes that there is transmembrane segment in only two codings of intestinal bacteria O142 kind.The O-antigen transferring enzyme of Orf6 encoded protein and Shigella flexneri 2a has 49% sequence identity, and it contains 12 uniform transmembrane segments by the proteic topology discovery of HMMTOP2.0 programanalysis, and this is the proteic characteristic feature of Wzx.So name orf6 is wzx.Orf7 encoded protein and colibacillary O-antigen polysaccharase have 24% consistence, 48% similarity, it contains 9 transmembrane segments by the proteic topology discovery of HMMTOP2.0 programanalysis, and hydrophilic loop (loop) in big (61 an amino acid) kytoplasm is arranged, and this is the proteic characteristic feature of Wzy.So name orf7 is wzy.

The albumen of orf1,8,9,10 4 genes encodings and other known glycosyltransferases have the sequence identity of 34-47% and the sequence similarity of 54-68%.By the 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 7.7 * e ^-22To 1.1 * e ^-45, 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 O142 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, orf8, orf9 and orf10.

Embodiment 6: the screening of specific gene:

At wzx, wzy gene design primer in the O-antigen gene of intestinal bacteria O142 type bunch, the position of these genes in nucleotide sequence sees Table 1.

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 O142 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 O142 type in table, have been listed.In each gene, we have respectively designed two pairs of primers, and the difference that every pair of primer is distributed in the corresponding gene is local to guarantee its specificity.In table, also listed position and the size of each primer in SEQ IDNO:1.Is that template carry out PCR with listed corresponding annealing temperature in the table with the genomes of all bacterium in the table 2 with every pair of primer, has obtained corresponding PCR product, and its size is also listed in the table.

Table 2 is 166 strain intestinal bacteria and 43 strain Shigellaes and their sources that are used to screen specific gene, and for the convenience that detects, we are divided into one group with their every 8-10 bacterium, and 13 groups altogether, all list in the table in their source.

The genomic dna that contains intestinal bacteria O142 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 O142 type and O-antigen thereof are high specials.

At last, from intestinal bacteria O142 type, screen gene by PCR: wzx, wzy gene to the O-antigen high special of intestinal bacteria O142 type.And the oligonucleotide of these intragenic any one section 10-20nt is special to the O-antigen of intestinal bacteria O142 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 O142 type.These all oligonucleotide all can be used for the intestinal bacteria O142 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 O142 type, in table, listed the structure of the O-antigen gene bunch of intestinal bacteria O142 type, altogether by 11 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 O142 type, listed the accurate position of all open reading frame in complete sequence in the O-antigen gene bunch of intestinal bacteria O142 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.

SEQUENCE?LISTING

＜110〉Nankai University

＜120〉to the Nucleotide of the O antigen-specific of intestinal bacteria O142 type

＜130〉to the Nucleotide of the O antigen-specific of intestinal bacteria O142 type

<160>1

<170>PatentIn?version?3.2

<210>1

<211>13584

<212>DNA

<213>Escherichia?coli

<400>1

attgtggctg?cagggatcaa?agaaatcctc?ctggtaactc?acgcgtccaa?gaacgcagtc?????60

gaaaaccact?tcgacacctc?ttatgaatta?gaatctctcc?ttgaactgcg?cgtgaagcgc????120

caactgctgg?cggaagtaca?gtccatctgt?ccgccgggcg?tgaccattat?gaacgtgcgt????180

cagggcgaac?ctttaggttt?aggccactcc?attttatgtg?cacgacctgc?cattggtgac????240

aacccatttg?tcgtggtgct?gccagacgtt?gttatcgatg?acgccagcgc?cgacccgctg????300

cgctacaacc?ttgctgccat?gattgcgcgc?ttcaatgaaa?cgggccgtag?ccaagtgctg????360

gcaaaacgta?tgccgggtga?cctctctgaa?tactccgtca?ttcaaaccaa?agagccgctg????420

gatcgggaag?gtaaagtcag?ccgcattgtt?gaattcatcg?aaaagccgga?tcagccgcag????480

acgttggact?cagacatcat?ggccgtgggt?cgctatgtgc?tttctgccga?tatttggccg????540

gaacttgaac?gcactcagcc?tggtgcatgg?gggcgtattc?aactgactga?tgccatcgct????600

gaactggcga?aaaaacagtc?cgttgatgcc?atgctgatga?ccggtgacag?ctacgactgc????660

ggtaaaaaaa?tgggctatat?gcaggcgttc?gtgaagtatg?gattacgcaa?cctgaaagaa????720

ggggcgaaat?tccgtaaagg?gattgagaag?ttgctaagcg?agtgagctta?aaaaatcatg????780

ttctttatga?gcatagaact?gtattggttg?ataatcattc?gaagaagtta?ggtcaatact????840

tcgaaaatat?tgctgccgtt?ttgttttaga?ggaactttgt?taaagtttat?agagttatat????900

attaaggtca?gacctacatt?ttataaaatt?tactcttgtt?tcttacatca?ataggtaaga????960

caattggtct?ttaactttcg?ggactttgcg?cactaggcaa?agttagatat?acctatggta???1020

gtatgtagcg?cactggttgc?tgttaagcca?ggggcggtag?cgtgcttaaa?taatagcaac???1080

tctattttag?ataattgaac?gattatgtaa?aatacagacc?aacataaatt?aacatcgata???1140

tgagcaacat?agcatttatt?gtgacaaaat?ctgagattgg?tggtgcacaa?acttggacta???1200

acgatatgat?ggaattagtc?agaaatggta?atactgtgca?tttgattacc?tctgaaattg???1260

gatggctgac?tgaacaaaat?aattacgata?actgtttttt?attaccacag?ctaaagaaaa???1320

aatttagctt?ttggggttac?ttgtctctcc?ttaaatatat?taaaaaagaa?aaaataaatg???1380

taatggtggc?gagttctgct?aatgcaggta?ttttttcaag?attatgtaaa?ctatttcata???1440

gatttaaatg?catttatgta?tcgcatggtt?ggtcctgtat?ttataatgga?ggtgctttta???1500

aacgaatatt?tattaaggta?gagaaatact?tgtcgtattt?aacagatgtt?gtttggtgtg???1560

tatcttatag?tgatgaaata?agtgcgaaag?aaaaaatagg?cataaaaaga?aaaaagatta???1620

aaatggtaca?taactctgta?tcaggtatac?ccaaaaagtt?acatgcagga?gaaaagaaaa???1680

aagtaatttt?tgtaggtcgc?cttactcatc?ctaaacgtcc?tgaattattg?gctcgagtaa???1740

tttctaaaaa?tccagatata?taccttgata?tagcgggagg?aggtgagtat?ctggataagc???1800

taaaagaaga?gtttgaaagt?tataagaata?ttaatttcct?tggggaagtc?aactctttta???1860

atcagtatct?taattatgat?atttttgccc?taatttcaga?tagtgaagga?cttcccatgt???1920

ctgcattaga?ggcccataca?gcgggaattc?cactcttgat?gagtgatgtt?gggggatgta???1980

aagagttggt?gagtggtaat?ggcttgctgg?tttctaatga?tgaaaaagat?attgaaagaa???2040

agctccgaaa?aattattgac?gaatatgatt?gttatttttt?atgttcacaa?gctgataaaa???2100

ataaatttga?gttcaaaaac?tatattgaaa?aatataaaga?aataatatta?acgtgatatt???2160

tttagtgagt?ttaaaatgaa?aatatttgtt?actggtggag?cgggttttat?tggatctgct???2220

gttgtacgtt?atattataaa?taatacacaa?gactgtgttg?tcaatattga?taaattaacg???2280

tatgcaggta?acctcgaatc?actgagcgtt?gtcgcaaata?atgaacgcta?ttcttttgag???2340

catgcagata?tatgtgactt?agaagctatg?gtgcgaatct?tcaatgaata?tcaacctgat???2400

gcagttatgc?atctcgctgc?tgagagccat?gtagatcgat?ccatctctgg?ccccgcagcc???2460

ttcatagaaa?ctaatattct?ggggacttac?gtccttctgg?aagctgcacg?ccagtattgg???2520

tgcacactca?atgatgtgaa?aaaagagaaa?ttccgttttc?atcatatttc?aactgacgaa???2580

gtctatggcg?atctacccca?tccggacgaa?gttgagcagg?ggacacctct?tcctcttttc???2640

acagagaaaa?cagcttatgc?gccaagcagt?ccttattctg?cttctaaagc?ctccagcgat???2700

catctggttc?gcgcatggct?gcgtacctat?ggtttaccaa?ccattatcac?caactgctca???2760

aataattacg?gtccatatca?atttccggaa?aaactgattc?ccttggttat?cctaaatgcg???2820

ctcgcaggta?aatcattacc?gatttatggc?aaaggcgatc?agattcgcga?ctggctttat???2880

gtcgaggacc?atgcttgcgc?actttattcc?gtgctgacaa?aaggtaagcc?tggagaaacc????2940

tataatcttg?gcgggtacaa?cgagaagaaa?aatctcgacg?taattcataa?tatttgtgat????3000

ctgctcgatg?agatcatacc?caaagagaca?tcatatcgtg?aacaaattac?ttatgtggct????3060

gacagacccg?gacatgatcg?tcgttatgct?attgatgcag?ataaaattat?gcgtgagtta????3120

ggatggaaac?ctgtggaaac?attcgaaagc?gggattcgta?aaaccgttga?gtggtatctg????3180

gcaaatactc?tttgggtaga?aaacatcaaa?agcgggacat?atcaaaggtg?gatcgagcag????3240

aactacaggg?agcgccagta?atgaatattc?tgttgtttgg?taaaaatgga?caggtaggat????3300

gggaactaca?acgttcgctc?gctcctcttg?gaaatcttat?agcgctggat?gttcattcaa????3360

cagattactg?tggtgatttt?agtaatcccc?aaggtgtggc?tgaaaccatc?cgttgtgttc????3420

gcccggatgt?aattgtcaat?gctgccgccc?atacggccgt?cgataaagca?gaaagtgaga????3480

ttgattatgc?gctattactc?aatgcgatta?gcgtagaagc?gctcgctaaa?gctgccgatg????3540

aaattggtgc?atgggttgtg?cattattcga?cagattacgt?tttccctgga?acaggagaaa????3600

cggcatggct?tgaaactgac?gagacagcac?cgctgaacgt?ttatggtgaa?acgaaacttg????3660

cgggcgaaaa?agctcttcag?gtaaattgca?gtaagcatct?aatttttcgt?actagctggg????3720

tttatgcagt?taacgggaat?aatttcgcga?agacaatgtt?gcgtcttgca?caagaacatc????3780

aaatgttgtc?cgttataaac?gatcagtttg?gcgcaccgac?gggagcggaa?ttgctagcag????3840

attgtacggc?acatgctatt?cggattgcat?tgaataagcc?agaggttgca?ggtctttatc????3900

atctggttgc?gagtggaacg?acaacttggt?atgaatatgc?atccttagtt?tttgaagaag????3960

cacgtaaatt?cggtatcgaa?ttggccgtaa?ctcatgttaa?tggtgtgcca?acaagcactt????4020

atgctactcc?agcacggcgt?ccaattaatt?cccggttaaa?tactgataag?tttcaaaata????4080

aatttggatt?agtattgcca?aattgggaaa?taggtgtcaa?acgtatgttg?actgagattt????4140

ttacattaac?gacaatctga?ttttaatttt?tatgtcagcc?ttcgagtatt?ttgcgtttaa????4200

gagaaattaa?catgaaaaca?cgtaaaggga?ttattctggc?cggtggttcc?ggtacacgtc????4260

tgtatccagt?gactatggct?gttagtaaac?aattgttgcc?tatttatgat?aagccgatga????4320

tttattatcc?actctcgacg?ctaatgctgg?cggggattcg?ggatatactt?attattagca????4380

ccccacagga?tacaccccgg?tttgaacaat?tgctcggaga?cggaactcaa?tggggattga????4440

atcttcaata?tcaagtacag?ccaaggccgg?atggtttggc?gcaagcattt?attattggtg????4500

aagagttcat?tggtaaagat?gattgtgctc?tagtattggg?cgataatatt?ttctatggac????4560

atgatttacc?aaaactcact?gatatcgcag?ttaataaaaa?aagtggtgca?actgtttttg????4620

catatcacgt?taatgatccg?gaacgttatg?gcgttattga?gtttgataaa?gatggtacag????4680

cgatatcttt?agaagaaaaa?ccgctagtgc?caaaaagtaa?ttatgctgta?actgggcttt????4740

atttctatga?caacagtgtg?attgaaatgg?ctaaaaatct?caagccttcc?gtgcgtggag????4800

agctggaaat?caccgatatc?aaccgtattt?atatggagca?gggaaaactt?tgtgtcgcca????4860

tgatggggag?gggctatgct?tggctggata?cgggaacaca?tcaaagcctg?atagaagcga????4920

gcaactttat?tgctactatt?gaagagcgtc?agggattgaa?agtttcttgt?ccggaagaga????4980

tagcgtttcg?caagggtttt?attgatgctg?agcaggttaa?aaaactagca?gcaccgttat????5040

caaagaatgc?ctatggacaa?tatctcctta?agatgattaa?tggtgattaa?taaaatgaat????5100

gtaattaaaa?cagaaatccc?tgatgtactc?atttttgagc?caaaagtttt?ttgcgatgct????5160

cgtggttttt?tctttgagag?ttttaatctg?aaaatatttg?cagaggccgt?tggtagaaac????5220

gttgaatttg?ttcaggacaa?tcattcaaaa?tccaaaaaag?gggttttacg?aggtcttcac????5280

tatcaggtag?ccccgtttgc?tcagggaaaa?ttagtgagat?gcattgctgg?tgaagtatat????5340

gatgtcgctg?ttgatctacg?taaatcatct?ccgacctttg?ctaaatgggt?tggtgtaaat????5400

ctttcggcta?agaataaacg?ccaattgtgg?attcctgaag?gattcgcgca?tggttttatg????5460

gtgttgagtg?atgaagctga?atttgtatac?aaaactacga?attattatag?ccccaaatca????5520

gaacgttcaa?taaactatgc?cgactcacag?ataaatatta?agtggccgtc?aagttttaat????5580

ttgaaacttt?cacggaaaga?cgaaattgca?cctcagttgg?ctattatttt?agaaaatgag????5640

ctttttgaat?gagcattata?aaaaacagtc?tttggaatgt?tgtcgggtat?attgtccctg????5700

ctattgtcac?gatccctgcg?ttgggaatat?taggtcgaat?cttaggtgca?gaaaccttcg????5760

gtgtatttac?tcttgcgcta?gcaatagttg?ggtacgctag?tatttttgat?gtaggcttat????5820

caagagctgt?tattcgagaa?atagcgttat?ttcgagatga?tcaagaagaa?aaaagaagaa????5880

taatattcac?agcatcatta?ttagttacag?taatgggagt?tactgctgca?ttagtattgt????5940

atattgcaag?tgatgtaata?gctaatttgt?tgaagatcag?ctctgagtta?catttgagtg????6000

ttgttaattc?tctacatata?ctctcactct?ccatccccgt?ttatttggta?acacaaatat????6060

ggcttgcaat?tctagaggga?gaagagaagt?ttggtctatt?aaatatttat?aaatctatta????6120

ctggttctct?aatttcacta?cttccggtta?tttgtatctt?tatttctccc?tccattgagt????6180

atgctattat?tggcttggtt?gtatctcgat?tggtttgtat?gttgtttgct?ttctttttat????6240

gtaaaagaat?aatagtggag?tcatatttcg?agtttagcaa?actaacattg?aaacgaatgc????6300

taatgtttgg?gggatggata?acagtaagca?atattataag?tccgttaatg?gcctattttg????6360

accgatttat?tgtttctaat?caattaggtg?cagcagtcgt?tgccttttat?acagctcctt????6420

ctgaaattat?tgcaaggtta?ggtattgttc?ccggtgcttt?tgcccgcgct?atttttcctc????6480

gtttgagttg?ttcaaatgat?gtccacgata?gaaaaaagaa?caaaaaaata?gtatcattac????6540

ttcttttcct?gataacagtt?ccagtattta?ttgtaggact?tttggccagt?aataaattta????6600

tggttttgtg?gatgggacct?gaatttgcag?gtacttcagc?taatatattg?gttattcttc????6660

ttctcggttt?tgttttcaat?tcattagcac?aggtcccttt?tgctagtata?cagtcacgtg????6720

gttatgctaa?aatcactgca?tatatacata?tggtggagct?aatcccttat?ttgatggctc????6780

tgttttattt?tattaataac?tatggaatta?ttggtgccgc?gtatgcatgg?agtataagag????6840

tgactattga?ttatatatta?ttggcatttt?ttgacagatg?ctttgataag?tagcataaga????6900

aaattttaat?aatcgctatc?aaagaacgga?ggaaatgacc?atttatgtta?tatgttctca????6960

cttttgcgct?cattacatca?ttcgggcttt?ttattgcact?ttatctggtc?aaatgtaact????7020

ttacatcacc?tctgtcttta?cattgtttcg?catggttttt?cgttagtagc?actggattgt????7080

ttgcttatga?tgagtttatt?gattttcccg?agataagctt?ctatgcagta?atgatttggt????7140

acctgattgt?atattttatt?ttaataacag?gtgagttaat?atccttaaat?ataaaaagtg????7200

tgaactattt?taaaaataag?gaatatatat?gtggaagata?ttggatcata?gtaataccat????7260

tgtctgctta?tacaatttac?gaaatttata?gagtgggaaa?tactgggcct?gcatcatttt????7320

tccttaattt?acgtctcgca?aacattattg?atgattacga?aggtgaaaag?ttcacattga????7380

tgacggctat?atatccagtc?ttgatagcga?tgttttccat?cgtatgcata?tcttgttcat????7440

caaagaagaa?taagtacgca?ttatggctat?ggtctattct?attttgtatt?ggcactatgg????7500

gtaaatttgc?tgtaataacc?ccaattttaa?ttttctatat?aatccgtgag?ttgacgggag????7560

gcttaaataa?aaagaggatg?gtttttattg?ttccaggtgt?aatctctgct?attcttttta????7620

tgcatttcat?acgaatgtct?agtggagata?gtactacaat?aagttctgtt?ctaggggtat????7680

atatttactc?gccactactt?gcattgagta?agttacctga?actaaatata?aatggtgaat????7740

ctggtgaata?tacattcaga?tttttgattg?caattctata?taaagtaggc?ctgtcctcaa????7800

atgagccggt?taaaactatt?ttagattacg?tcaacgtgcc?cgttccaaca?aatgttttta????7860

cggtgatgca?accattctac?caagattatt?ctttatttgg?agtagcattt?ggagcaatat????7920

tttatgggat?tatatattct?tcaatatatc?ttctggcaaa?aaaaggaaat?ccagttgccc????7980

tgcttattta?tgccgttctt?gcaattagcc?tatttacttc?tttttttgca?gaaactttaa????8040

taactaacct?tgcaggtaat?ataaaggtag?taatttgcat?ataccttcta?tggagattta????8100

cagtaagatg?caagataaaa?ccgtaacgat?attaatggca?acttataatg?gaagtgcttt????8160

tatagagaac?cagattcttt?cattacagca?acaaaaatat?aaagattgga?tactctatat????8220

tcatgatgat?ggttcctctg?atgatacctt?agatattata?aagagaattc?agcttacaga????8280

acctcgtatt?aatcttattg?aggatgggct?tacgagactt?ggtgcaggga?aaaattttct????8340

ttcattagta?aaatattcag?ctacaaatta?tactatattt?tgcgatcaag?atgatatttg????8400

gctcgaaaac?aaattaagcg?agatgattgt?tttcgcagat?agtaaagggc?tggctagtag?????8460

taagttacca?tctatgattt?atgcagatgg?gtatgcattt?gacgatagca?caggtgagat?????8520

tgatttttgt?ggtatatcac?ataatcatgc?tacgagattg?aaagatttct?tatttttcaa?????8580

tgctggctat?caagggtgtt?caatattatt?taataaagca?atggttgaca?ttgcagcaaa?????8640

ttaccatggt?tatgtccatt?tacatgatga?tgtcgtaagt?ctgatcgccc?attctttggg?????8700

gaatgtgtat?tttttaccaa?aaaaattgat?gttatatcgc?caacacttag?gggcagtaac?????8760

tggccaaaag?aagtttaata?acagattcat?ttcaatgctg?acatccaaag?ttaattatct?????8820

tttgtcaaga?gagcattttt?tagttaagag?atctttttac?gataattacc?atcatctttt?????8880

aacggctgag?attaaaaatg?actttgaagt?cttttttaaa?ttttgccaaa?caaaaaacaa?????8940

actttcacag?ttagtgcttt?tattgaagca?tgattttcgc?ttaaataaca?gcagattaaa?????9000

gctattgtta?aaatgcatag?taagaaggac?atttagtcaa?tgatctccgt?cttaacacct?????9060

acttataatc?gtgcgtatac?cttgaagcga?ttatacgagt?cgttaatttg?tcaaacgact?????9120

aaatcctttg?aatgggttgt?ggttaatgat?ggtagtaacg?atgagactga?gtcaataata?????9180

aaagagtttc?agcaacaaaa?tattattaaa?attatttatt?acaaacagga?aaataaaggt?????9240

aaaactcagg?ctcttaatgc?aggtatacaa?ttatgtgcag?gaagtgatat?tttaatcgtt?????9300

gatagcgatg?atctattgac?gtcggatgct?atagcttgca?ttgaagctag?tttgactcag?????9360

gagaagatat?ataataaaaa?aatatcgggg?gtggcatttc?gtaaagcata?tctggatgaa?????9420

accataattg?gtactgtttt?tgatgatagt?gttgatagtt?tttgttatct?ttctgcaact?????9480

gacgctgggc?atcttttcaa?aggcgatctt?gcatattgct?ttaagaaaga?aatgctgcaa?????9540

atgtttcctt?ttccatactt?tcataacgaa?aagtttgttc?ctgaattata?catttggaac?????9600

aagattacag?atcacgcatt?agtaaaattt?cataaaaaaa?aagcagtata?cctatgtgag?????9660

tatctcgaag?atgggttatc?taaaaacttc?aaaacacaat?tattaaaaaa?tcccaaaggg?????9720

tttagtatct?attatattga?tcagtttaag?cgtgaaacca?actacattcg?aaaattaaaa?????9780

atgttgattc?gttattttca?gtgtaaatta?tatgagctaa?aaaaatgaat?atagtgtttg?????9840

tgattactgg?attgggactt?gggggggctg?aaaagcaagt?atgcttattg?gctgatcgac?????9900

tggctgaggc?agagcatcaa?atatcgatag?tgtcccttac?aggaggatgt?gttgttaagc?????9960

cagataataa?gcatattgaa?atttaccacc?ttaatatgga?taaaagccta?ataagtttct????10020

ttttaggtgt?tataaaatta?cgtaatatta?tttctactat?taagcctgat?attgtgcata????10080

gccatatgtt?ccatgcaaat?atcatgtcta?gattaagtaa?gttactattg?ccttattccc????10140

ataagctcat?atgtacagca?catagcagat?atgagggagg?gaggcttcgt?atgctttgtt????10200

atcgtttaac?agattatttc?agtgatataa?atacaaacgt?tagtaaagag?gcgctcgatg????10260

aatatgttaa?taataaatat?ttctcagcga?ctaaatctat?tgttgtttat?aatggcgttg????10320

atacagaaaa?atttaatttc?agtattgata?atcgtattta?tataagagag?caattgtcta????10380

ttaataacca?tgataggttg?attctcagtg?ttggcagatt?aaaccctgct?aaagattatc????10440

ctaatctgct?agcagcattc?atgcttttac?ctgagcacta?taagcttgtt?atcattggtg????10500

agggagacgt?tcgttcacag?attgaacaga?taattaaaga?tcacaagtta?gggacgcgtg????10560

ttcagttact?tggtagtgta?aataatgtca?atgattatta?ttctgcttgt?gatttatttg????10620

tcttatcttc?ggcttgggaa?ggttttagct?tagttgttat?cgaggccatg?gcatgtcaac????10680

gcatcgctgt?atgtactgat?gcaggtggag?taaaagaagc?ttttactgat?cgtcattaca????10740

ttgtaccaac?ttcaaatgcc?gcagctttag?ctcgcaaaat?tattgaagtt?gatatgttaa????10800

ctattgatcg?taaaaatgaa?atacaaaatg?ataataggaa?taatgttgtt?aacaagtttt????10860

caattaatgc?tattgttaat?cattggttaa?caatctataa?aaatataact?atgcagtaga????10920

taaatctatt?acataatgat?gaattaaaaa?caatattaat?ttttagctgt?tttttataat????10980

acctagttca?atatggatta?aattattttg?ttttttaatt?tgtaatctgg?aataggttat????11040

tatatttact?agattgaatg?tagttcaatg?gatagattat?cacgttaaca?tttaattgaa????11100

gtataagttg?tgtggcgttg?tgttgcttta?aattcttaat?aaataattaa?ggttggtgat????11160

taatgactat?tttagtaacg?ggcggtgctg?gctatatagg?ttcgcataca?gtggtgagac????11220

tgcttgaaaa?gggtaaagaa?attgttattc?ttgatgattt?tactaattca?tttcctgaaa????11280

cattgaatag?aataaaaata?attaccggtg?ttaaaccttt?tttttacgaa?ggttctgtcc????11340

ttgataggaa?tttattgaaa?aaaatctttg?tcgaaaataa?catcaccgat?gttattcatt????11400

ttgcagggct?caaatcagtt?ggtgaatccg?tatcgtcccc?tcttaagtat?tatgaagtca????11460

atatagcagg?aagtttgcat?ctagttgaag?agatgattac?gcacaatata?agcaatttta????11520

tttttagctc?ttctgcaaca?gtatatggtg?aaccagaaac?tattccattg?acggagtcct????11580

ctcgcattgg?tggcactaca?aacccttatg?gtacatctaa?gcttatggtt?gagaaaatac????11640

ttgaggatgt?tactcgttct?aatcctgagt?ttagaaccac?aattttacga?tattttaatc????11700

ccgttggtgc?tcatccttct?ggtgatatgg?gcgaagatcc?gaatggtatt?ccaaataatc????11760

tcatgcctta?tatctgtcag?gttgctattg?gtaagtataa?gcaagtttca?gtatacggaa????11820

gcgattatcc?aacaaaggat?ggtactggag?tccgtgactt?tatacatgta?atggatcttg????11880

ctgaaggtca?tgttgctgct?ttagagcaca?gaaataaggg?accaaatcat?aaagtttaca????11940

acttgggcac?aggcactggt?tattctgttt?tggaactcct?gacagctttt?gaaagagtaa????12000

cttctcgtaa?agtaccttac?gttttaagtg?aaagacgccc?tggagatatc?gccgaatgtt????12060

ggtctaatcc?ttcgaaggcg?tatgcggaac?ttggatggaa?agcgaagcgc?ggactggaag????12120

acatggttcg?agatgcctgg?aattggcaac?aaaagaatcc?aaacggttat?aaaaaagaat????12180

gaatgatcaa?agaaattttt?gtcaactgca?aaaattaccg?aatttatgta?tcctgagtta????12240

acatagcact?aacattgaat?tgcgttatgt?ttcccagtat?cacctctgac?aggagtaaac????12300

aatgtcaaag?caacagatcg?gcgtcgtcgg?tatggcagtg?atggggcgca?accttgcgct????12360

caacatcgaa?agccgtggtt?ataccgtctc?tattttcaac?cgttcccgtg?aaaagacgga????12420

agaagtgatt?gccgaaaatc?cgggcaagaa?actggttcct?tactatacgg?tgaaagagtt????12480

tgttgaatct?ctggaaacgc?ctcgtcgcat?cctgttaatg?gtgaaagcag?gtgcaggcac????12540

ggatgctgct?attgattctc?tcaagccata?cctcgataaa?ggcgacatca?tcattgatgg????12600

cggtaatacc?ttcttccagg?acaccattcg?tcgtaaccgt?gagctttctg?cagaaggctt????12660

taacttcatc?ggtaccggtg?tttccggcgg?tgaagaaggt?gcgctgaaag?gtccttccat????12720

tatgcctggc?ggacagaaag?aagcctatga?actggttgcg?ccgatcctga?ccaagatcgc????12780

cgccgttgct?gaagatggcg?aaccgtgcgt?tacatatatt?ggtgccgatg?gcgcgggtca????12840

ctatgtaaaa?atggttcaca?acggtattga?atacggtgat?atgcaactga?ttgctgaagc????12900

ctattctctc?ctgaaaggcg?gcctgaatct?ctctaacgaa?gaactggcac?agacctttac????12960

cgagtggaat?aacggtgaac?tgagcagcta?cctgatcgac?atcaccaaag?acatcttcac????13020

caaaaaagat?gaagacggta?actacctggt?tgatgtgatt?ctggatgaag?cagcaaacaa????13080

aggtaccggt?aaatggacca?gccagagtgc?gctggatctc?ggcgaaccgc?tgtcgctgat????13140

taccgagtct?gtgtttgcac?gttatatctc?ttctctgaaa?gatcagcgtg?ttgccgcatc????13200

taaagttctc?tctggcccgc?aagcacagcc?agcaggcgag?aaggctgagt?tcatcgaaaa????13260

agttcgccgt?gcgctgtatc?tgggtaaaat?cgtttcttac?gctcagggct?tctctcagct????13320

gcgtgctgcg?tctgaagagt?ataactggga?tctgaactac?ggcgaaatca?cgaagatttt????13380

ccgtgctggc?tgcatcatcc?gtgcgcagtt?cctgcagaaa?atcaccgatg?cttatgccga????13440

aaatccgcag?atcgctaacc?tgctgctggc?tccgtacttc?aagcaaattg?ccgatgacta????13500

ccagcaggcg?ctgcgtgatg?tcgttgctta?tgcagtacag?aacggtatcc?cggttccgac????13560

cttctctgct?tcggttgcct?atta???????????????????????????????????????????13584

Glycosyltransferase gene in the O antigen gene of table 1 intestinal bacteria O142 type bunch and oligosaccharide unit treatment gene and wherein Primer and PCR data

Gene	Function	The base position of gene	Forward primer	Reverse primer	The length of PCR product	Produce the group number of correct big or small electrophoresis band	The annealing temperature of PCR (℃)
								????Wzx	O-antigen transhipment enzyme	5649-6893	????5901-5920	????6155-6173	????273bp	????0 *	????56
????6496-6513	????6754-6771	????276bp	????0 *	????54
					????Wzy	O-antigen polysaccharase	6945-8126				????7463-7480	????7902-7919	????457bp	????0 *	????56
????6966-6983	????7288-7306	????341bp	????0 *	????60

^*Only in intestinal bacteria O142 type, obtain a correct band

Table 2166 strain intestinal bacteria and 43 strain Shigellaes and their source

The bacterium source that contains in this group of group number

1, wild-type e. coli O1, O2, O5, O7, O12, O13, O14, O15, O16, O17, O19ab, O20, IMVS ^a

O21，O22，O23，O24，O59

2, wild-type e. coli O25, O26, O27, O28, O29, O30, O32, O33, O35, O36, O37, IMVS ^a

O38，O40，O41，O42，O43

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

O57，O58，O60，O61，O62

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

O79，O80，O81，O82，O83

5, wild-type e. coli O84, O85, O86, O87, O88, O89, O91, O92, O98, O99, O101, IMVS ^a

O102，O103，O104，O106

6, wild-type e. coli O107, O108, O109, O110, O111, O112ab, O112ac, O113, IMVS ^a

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

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

O138，O139，O140，O141，O143，O144，O145

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

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

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

With Shigellae D1, D2, D3, D4, D5, D6, D7, D8, D9, D10, D11, D12 d

10, wild-type Shigellae B1, B2, B3, B4, B6, B7, B8, B9, B10, B11, B12, B13, B14, B15, d

B16，B17，B18

11, wild-type Shigellae F1a, F1b, F2a, F2b, F3, F4b, F5 (v:4), F5 (v:7), F6, F _{X becomes}, F _{Y becomes}, DS, DR, d

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

O155，O124

13, the 7th group of bacterial strain adds intestinal bacteria reference culture O142 IMVS

^*For the convenience that detects, we are divided into one group with every 13-17 bacterium, 13 groups altogether

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

b.Statens?Serum?Institut，Copenhagen，Denmark

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

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

Table 3 intestinal bacteria O142 type O antigen gene structure iron

orf#???galF??orf1???rmlB???rmlD???rmlA??rmlC???wzx????wzy???orf8??orf9????orf10????gne????gnd

G+C??????????32?????43?????43?????41????37?????35?????33????32????32??????34???????39

Table 4 intestinal bacteria O142 type O antigen gene cluster gene position

ATTGTGGCTG?CAGGGATCAA?AGAAATCCTC?CTGGTAACTC?ACGCGTCCAA?GAACGCAGTC??????60

GAAAACCACT?TCGACACCTC?TTATGAATTA?GAATCTCTCC?TTGAACTGCG?CGTGAAGCGC?????120

CAACTGCTGG?CGGAAGTACA?GTCCATCTGT?CCGCCGGGCG?TGACCATTAT?GAACGTGCGT?????180

CAGGGCGAAC?CTTTAGGTTT?AGGCCACTCC?ATTTTATGTG?CACGACCTGC?CATTGGTGAC?????240

AACCCATTTG?TCGTGGTGCT?GCCAGACGTT?GTTATCGATG?ACGCCAGCGC?CGACCCGCTG?????300

CGCTACAACC?TTGCTGCCAT?GATTGCGCGC?TTCAATGAAA?CGGGCCGTAG?CCAAGTGCTG?????360

GCAAAACGTA?TGCCGGGTGA?CCTCTCTGAA?TACTCCGTCA?TTCAAACCAA?AGAGCCGCTG?????420

GATCGGGAAG?GTAAAGTCAG?CCGCATTGTT?GAATTCATCG?AAAAGCCGGA?TCAGCCGCAG?????480

ACGTTGGACT?CAGACATCAT?GGCCGTGGGT?CGCTATGTGC?TTTCTGCCGA?TATTTGGCCG?????540

GAACTTGAAC?GCACTCAGCC?TGGTGCATGG?GGGCGTATTC?AACTGACTGA?TGCCATCGCT?????600

GAACTGGCGA?AAAAACAGTC?CGTTGATGCC?ATGCTGATGA?CCGGTGACAG?CTACGACTGC?????660

GGTAAAAAAA?TGGGCTATAT?GCAGGCGTTC?GTGAAGTATG?GATTACGCAA?CCTGAAAGAA?????720

GGGGCGAAAT?TCCGTAAAGG?GATTGAGAAG?TTGCTAAGCG?AGTGAGCTTA?AAAAATCATG?????780

TTCTTTATGA?GCATAGAACT?GTATTGGTTG?ATAATCATTC?GAAGAAGTTA?GGTCAATACT?????840

TCGAAAATAT?TGCTGCCGTT?TTGTTTTAGA?GGAACTTTGT?TAAAGTTTAT?AGAGTTATAT?????900

ATTAAGGTCA?GACCTACATT?TTATAAAATT?TACTCTTGTT?TCTTACATCA?ATAGGTAAGA?????960

CAATTGGTCT?TTAACTTTCG?GGACTTTGCG?CACTAGGCAA?AGTTAGATAT?ACCTATGGTA????1020

GTATGTAGCG?CACTGGTTGC?TGTTAAGCCA?GGGGCGGTAG?CGTGCTTAAA?TAATAGCAAC????1080

Orf1's is initial

TCTATTTTAG?ATAATTGAAC?GATTATGTAA?AATACAGACC?AACATAAATT?AACATCGATA????1140

TGAGCAACAT?AGCATTTATT?GTGACAAAAT?CTGAGATTGG?TGGTGCACAA?ACTTGGACTA???1200

ACGATATGAT?GGAATTAGTC?AGAAATGGTA?ATACTGTGCA?TTTGATTACC?TCTGAAATTG????1260

GATGGCTGAC?TGAACAAAAT?AATTACGATA?ACTGTTTTTT?ATTACCACAG?CTAAAGAAAA????1320

AATTTAGCTT?TTGGGGTTAC?TTGTCTCTCC?TTAAATATAT?TAAAAAAGAA?AAAATAAATG????1380

TAATGGTGGC?GAGTTCTGCT?AATGCAGGTA?TTTTTTCAAG?ATTATGTAAA?CTATTTCATA????1440

GATTTAAATG?CATTTATGTA?TCGCATGGTT?GGTCCTGTAT?TTATAATGGA?GGTGCTTTTA????1500

AACGAATATT?TATTAAGGTA?GAGAAATACT?TGTCGTATTT?AACAGATGTT?GTTTGGTGTG????1560

TATCTTATAG?TGATGAAATA?AGTGCGAAAG?AAAAAATAGG?CATAAAAAGA?AAAAAGATTA????1620

AAATGGTACA?TAACTCTGTA?TCAGGTATAC?CCAAAAAGTT?ACATGCAGGA?GAAAAGAAAA????1680

AAGTAATTTT?TGTAGGTCGC?CTTACTCATC?CTAAACGTCC?TGAATTATTG?GCTCGAGTAA????1740

TTTCTAAAAA?TCCAGATATA?TACCTTGATA?TAGCGGGAGG?AGGTGAGTAT?CTGGATAAGC????1800

TAAAAGAAGA?GTTTGAAAGT?TATAAGAATA?TTAATTTCCT?TGGGGAAGTC?AACTCTTTTA????1860

ATCAGTATCT?TAATTATGAT?ATTTTTGCCC?TAATTTCAGA?TAGTGAAGGA?CTTCCCATGT????1920

CTGCATTAGA?GGCCCATACA?GCGGGAATTC?CACTCTTGAT?GAGTGATGTT?GGGGGATGTA????1980

AAGAGTTGGT?GAGTGGTAAT?GGCTTGCTGG?TTTCTAATGA?TGAAAAAGAT?ATTGAAAGAA????2040

AGCTCCGAAA?AATTATTGAC?GAATATGATT?GTTATTTTTT?ATGTTCACAA?GCTGATAAAA????2100

The termination of orf1

ATAAATTTGA?GTTCAAAAAC?TATATTGAAA?AATATAAAGA?AATAATATTA?ACG TGATATT??2160

RmlB's is initial

TTTAGTGAGT?TTAAA ATGAA?AATATTTGTT?ACTGGTGGAG?CGGGTTTTAT?TGGATCTGCT??2220

GTTGTACGTT?ATATTATAAA?TAATACACAA?GACTGTGTTG?TCAATATTGA?TAAATTAACG????2280

TATGCAGGTA?ACCTCGAATC?ACTGAGCGTT?GTCGCAAATA?ATGAACGCTA?TTCTTTTGAG????2340

CATGCAGATA?TATGTGACTT?AGAAGCTATG?GTGCGAATCT?TCAATGAATA?TCAACCTGAT????2400

GCAGTTATGC?ATCTCGCTGC?TGAGAGCCAT?GTAGATCGAT?CCATCTCTGG?CCCCGCAGCC????2460

TTCATAGAAA?CTAATATTCT?GGGGACTTAC?GTCCTTCTGG?AAGCTGCACG?CCAGTATTGG????2520

TGCACACTCA?ATGATGTGAA?AAAAGAGAAA?TTCCGTTTTC?ATCATATTTC?AACTGACGAA????2580

GTCTATGGCG?ATCTACCCCA?TCCGGACGAA?GTTGAGCAGG?GGACACCTCT?TCCTCTTTTC????2640

ACAGAGAAAA?CAGCTTATGC?GCCAAGCAGT?CCTTATTCTG?CTTCTAAAGC?CTCCAGCGAT????2700

CATCTGGTTC?GCGCATGGCT?GCGTACCTAT?GGTTTACCAA?CCATTATCAC?CAACTGCTCA????2760

AATAATTACG?GTCCATATCA?ATTTCCGGAA?AAACTGATTC?CCTTGGTTAT?CCTAAATGCG????2820

CTCGCAGGTA?AATCATTACC?GATTTATGGC?AAAGGCGATC?AGATTCGCGA?CTGGCTTTAT????2880

GTCGAGGACC?ATGCTTGCGC?ACTTTATTCC?GTGCTGACAA?AAGGTAAGCC?TGGAGAAACC????2940

TATAATCTTG?GCGGGTACAA?CGAGAAGAAA?AATCTCGACG?TAATTCATAA?TATTTGTGAT????3000

CTGCTCGATG?AGATCATACC?CAAAGAGACA?TCATATCGTG?AACAAATTAC?TTATGTGGCT????3060

GACAGACCCG?GACATGATCG?TCGTTATGCT?ATTGATGCAG?ATAAAATTAT?GCGTGAGTTA????3120

GGATGGAAAC?CTGTGGAAAC?ATTCGAAAGC?GGGATTCGTA?AAACCGTTGA?GTGGTATCTG????3180

GCAAATACTC?TTTGGGTAGA?AAACATCAAA?AGCGGGACAT?ATCAAAGGTG?GATCGAGCAG????3240

The termination rmlD's of rmlB is initial

AACTACAGGG?AGCGCCAG TA?ATGAATATTC?TGTTGTTTGG?TAAAAATGGA?CAGGTAGGAT??3300

GGGAACTACA?ACGTTCGCTC?GCTCCTCTTG?GAAATCTTAT?AGCGCTGGAT?GTTCATTCAA????3360

CAGATTACTG?TGGTGATTTT?AGTAATCCCC?AAGGTGTGGC?TGAAACCATC?CGTTGTGTTC????3420

GCCCGGATGT?AATTGTCAAT?GCTGCCGCCC?ATACGGCCGT?CGATAAAGCA?GAAAGTGAGA????3480

TTGATTATGC?GCTATTACTC?AATGCGATTA?GCGTAGAAGC?GCTCGCTAAA?GCTGCCGATG????3540

AAATTGGTGC?ATGGGTTGTG?CATTATTCGA?CAGATTACGT?TTTCCCTGGA?ACAGGAGAAA????3600

CGGCATGGCT?TGAAACTGAC?GAGACAGCAC?CGCTGAACGT?TTATGGTGAA?ACGAAACTTG????3660

CGGGCGAAAA?AGCTCTTCAG?GTAAATTGCA?GTAAGCATCT?AATTTTTCGT?ACTAGCTGGG????3720

TTTATGCAGT?TAACGGGAAT?AATTTCGCGA?AGACAATGTT?GCGTCTTGCA?CAAGAACATC????3780

AAATGTTGTC?CGTTATAAAC?GATCAGTTTG?GCGCACCGAC?GGGAGCGGAA?TTGCTAGCAG????3840

ATTGTACGGC?ACATGCTATT?CGGATTGCAT?TGAATAAGCC?AGAGGTTGCA?GGTCTTTATC????3900

ATCTGGTTGC?GAGTGGAACG?ACAACTTGGT?ATGAATATGC?ATCCTTAGTT?TTTGAAGAAG????3960

CACGTAAATT?CGGTATCGAA?TTGGCCGTAA?CTCATGTTAA?TGGTGTGCCA?ACAAGCACTT????4020

ATGCTACTCC?AGCACGGCGT?CCAATTAATT?CCCGGTTAAA?TACTGATAAG?TTTCAAAATA????4080

AATTTGGATT?AGTATTGCCA?AATTGGGAAA?TAGGTGTCAA?ACGTATGTTG?ACTGAGATTT????4140

The termination of rmlD

TTACATTAAC?GACAATC TGA?TTTTAATTTT?TATGTCAGCC?TTCGAGTATT?TTGCGTTTAA??4200

RmlA's is initial

GAGAAATTAA?C ATGAAAACA?CGTAAAGGGA?TTATTCTGGC?CGGTGGTTCC?GGTACACGTC??4260

TGTATCCAGT?GACTATGGCT?GTTAGTAAAC?AATTGTTGCC?TATTTATGAT?AAGCCGATGA????4320

TTTATTATCC?ACTCTCGACG?CTAATGCTGG?CGGGGATTCG?GGATATACTT?ATTATTAGCA????4380

CCCCACAGGA?TACACCCCGG?TTTGAACAAT?TGCTCGGAGA?CGGAACTCAA?TGGGGATTGA????4440

ATCTTCAATA?TCAAGTACAG?CCAAGGCCGG?ATGGTTTGGC?GCAAGCATTT?ATTATTGGTG????4500

AAGAGTTCAT?TGGTAAAGAT?GATTGTGCTC?TAGTATTGGG?CGATAATATT?TTCTATGGAC????4560

ATGATTTACC?AAAACTCACT?GATATCGCAG?TTAATAAAAA?AAGTGGTGCA?ACTGTTTTTG?????4620

CATATCACGT?TAATGATCCG?GAACGTTATG?GCGTTATTGA?GTTTGATAAA?GATGGTACAG?????4680

CGATATCTTT?AGAAGAAAAA?CCGCTAGTGC?CAAAAAGTAA?TTATGCTGTA?ACTGGGCTTT?????4740

ATTTCTATGA?CAACAGTGTG?ATTGAAATGG?CTAAAAATCT?CAAGCCTTCC?GTGCGTGGAG?????4800

AGCTGGAAAT?CACCGATATC?AACCGTATTT?ATATGGAGCA?GGGAAAACTT?TGTGTCGCCA?????4860

TGATGGGGAG?GGGCTATGCT?TGGCTGGATA?CGGGAACACA?TCAAAGCCTG?ATAGAAGCGA?????4920

GCAACTTTAT?TGCTACTATT?GAAGAGCGTC?AGGGATTGAA?AGTTTCTTGT?CCGGAAGAGA?????4980

TAGCGTTTCG?CAAGGGTTTT?ATTGATGCTG?AGCAGGTTAA?AAAACTAGCA?GCACCGTTAT?????5040

The termination rmlC's of rmlA is initial

CAAAGAATGC?CTATGGACAA?TATCTCCTTA?AGATGATTAA?TGGTGAT TAA?TAAA ATGAAT?5100

GTAATTAAAA?CAGAAATCCC?TGATGTACTC?ATTTTTGAGC?CAAAAGTTTT?TTGCGATGCT?????5160

CGTGGTTTTT?TCTTTGAGAG?TTTTAATCTG?AAAATATTTG?CAGAGGCCGT?TGGTAGAAAC?????5220

GTTGAATTTG?TTCAGGACAA?TCATTCAAAA?TCCAAAAAAG?GGGTTTTACG?AGGTCTTCAC?????5280

TATCAGGTAG?CCCCGTTTGC?TCAGGGAAAA?TTAGTGAGAT?GCATTGCTGG?TGAAGTATAT?????5340

GATGTCGCTG?TTGATCTACG?TAAATCATCT?CCGACCTTTG?CTAAATGGGT?TGGTGTAAAT?????5400

CTTTCGGCTA?AGAATAAACG?CCAATTGTGG?ATTCCTGAAG?GATTCGCGCA?TGGTTTTATG?????5460

GTGTTGAGTG?ATGAAGCTGA?ATTTGTATAC?AAAACTACGA?ATTATTATAG?CCCCAAATCA?????5520

GAACGTTCAA?TAAACTATGC?CGACTCACAG?ATAAATATTA?AGTGGCCGTC?AAGTTTTAAT?????5580

TTGAAACTTT?CACGGAAAGA?CGAAATTGCA?CCTCAGTTGG?CTATTATTTT?AGAAAATGAG?????5640

The termination of the initial rmlC of wzx

CTTTTTGA AT? GAGCATTATA?AAAAACAGTC?TTTGGAATGT?TGTCGGGTAT?ATTGTCCCTG???5700

CTATTGTCAC?GATCCCTGCG?TTGGGAATAT?TAGGTCGAAT?CTTAGGTGCA?GAAACCTTCG?????5760

GTGTATTTAC?TCTTGCGCTA?GCAATAGTTG?GGTACGCTAG?TATTTTTGAT?GTAGGCTTAT?????5820

CAAGAGCTGT?TATTCGAGAA?ATAGCGTTAT?TTCGAGATGA?TCAAGAAGAA?AAAAGAAGAA?????5880

TAATATTCAC?AGCATCATTA?TTAGTTACAG?TAATGGGAGT?TACTGCTGCA?TTAGTATTGT?????5940

ATATTGCAAG?TGATGTAATA?GCTAATTTGT?TGAAGATCAG?CTCTGAGTTA?CATTTGAGTG?????6000

TTGTTAATTC?TCTACATATA?CTCTCACTCT?CCATCCCCGT?TTATTTGGTA?ACACAAATAT?????6060

GGCTTGCAAT?TCTAGAGGGA?GAAGAGAAGT?TTGGTCTATT?AAATATTTAT?AAATCTATTA?????6120

CTGGTTCTCT?AATTTCACTA?CTTCCGGTTA?TTTGTATCTT?TATTTCTCCC?TCCATTGAGT?????6180

ATGCTATTAT?TGGCTTGGTT?GTATCTCGAT?TGGTTTGTAT?GTTGTTTGCT?TTCTTTTTAT?????6240

GTAAAAGAAT?AATAGTGGAG?TCATATTTCG?AGTTTAGCAA?ACTAACATTG?AAACGAATGC?????6300

TAATGTTTGG?GGGATGGATA?ACAGTAAGCA?ATATTATAAG?TCCGTTAATG?GCCTATTTTG?????6360

ACCGATTTAT?TGTTTCTAAT?CAATTAGGTG?CAGCAGTCGT?TGCCTTTTAT?ACAGCTCCTT?????6420

CTGAAATTAT?TGCAAGGTTA?GGTATTGTTC?CCGGTGCTTT?TGCCCGCGCT?ATTTTTCCTC?????6480

GTTTGAGTTG?TTCAAATGAT?GTCCACGATA?GAAAAAAGAA?CAAAAAAATA?GTATCATTAC?????6540

TTCTTTTCCT?GATAACAGTT?CCAGTATTTA?TTGTAGGACT?TTTGGCCAGT?AATAAATTTA?????6600

TGGTTTTGTG?GATGGGACCT?GAATTTGCAG?GTACTTCAGC?TAATATATTG?GTTATTCTTC?????6660

TTCTCGGTTT?TGTTTTCAAT?TCATTAGCAC?AGGTCCCTTT?TGCTAGTATA?CAGTCACGTG?????6720

GTTATGCTAA?AATCACTGCA?TATATACATA?TGGTGGAGCT?AATCCCTTAT?TTGATGGCTC?????6780

TGTTTTATTT?TATTAATAAC?TATGGAATTA?TTGGTGCCGC?GTATGCATGG?AGTATAAGAG?????6840

The termination of wzx

TGACTATTGA?TTATATATTA?TTGGCATTTT?TTGACAGATG?CTTTGATAAG? TAGCATAAGA???6900

Wzy's is initial

AAATTTTAAT?AATCGCTATC?AAAGAACGGA?GGAAATGACC?ATTT ATGTTA?TATGTTCTCA???6960

CTTTTGCGCT?CATTACATCA?TTCGGGCTTT?TTATTGCACT?TTATCTGGTC?AAATGTAACT?????7020

TTACATCACC?TCTGTCTTTA?CATTGTTTCG?CATGGTTTTT?CGTTAGTAGC?ACTGGATTGT?????7080

TTGCTTATGA?TGAGTTTATT?GATTTTCCCG?AGATAAGCTT?CTATGCAGTA?ATGATTTGGT?????7140

ACCTGATTGT?ATATTTTATT?TTAATAACAG?GTGAGTTAAT?ATCCTTAAAT?ATAAAAAGTG?????7200

TGAACTATTT?TAAAAATAAG?GAATATATAT?GTGGAAGATA?TTGGATCATA?GTAATACCAT?????7260

TGTCTGCTTA?TACAATTTAC?GAAATTTATA?GAGTGGGAAA?TACTGGGCCT?GCATCATTTT?????7320

TCCTTAATTT?ACGTCTCGCA?AACATTATTG?ATGATTACGA?AGGTGAAAAG?TTCACATTGA?????7380

TGACGGCTAT?ATATCCAGTC?TTGATAGCGA?TGTTTTCCAT?CGTATGCATA?TCTTGTTCAT?????7440

CAAAGAAGAA?TAAGTACGCA?TTATGGCTAT?GGTCTATTCT?ATTTTGTATT?GGCACTATGG?????7500

GTAAATTTGC?TGTAATAACC?CCAATTTTAA?TTTTCTATAT?AATCCGTGAG?TTGACGGGAG?????7560

GCTTAAATAA?AAAGAGGATG?GTTTTTATTG?TTCCAGGTGT?AATCTCTGCT?ATTCTTTTTA?????7620

TGCATTTCAT?ACGAATGTCT?AGTGGAGATA?GTACTACAAT?AAGTTCTGTT?CTAGGGGTAT?????7680

ATATTTACTC?GCCACTACTT?GCATTGAGTA?AGTTACCTGA?ACTAAATATA?AATGGTGAAT?????7740

CTGGTGAATA?TACATTCAGA?TTTTTGATTG?CAATTCTATA?TAAAGTAGGC?CTGTCCTCAA?????7800

ATGAGCCGGT?TAAAACTATT?TTAGATTACG?TCAACGTGCC?CGTTCCAACA?AATGTTTTTA?????7860

CGGTGATGCA?ACCATTCTAC?CAAGATTATT?CTTTATTTGG?AGTAGCATTT?GGAGCAATAT?????7920

TTTATGGGAT?TATATATTCT?TCAATATATC?TTCTGGCAAA?AAAAGGAAAT?CCAGTTGCCC?????7980

TGCTTATTTA?TGCCGTTCTT?GCAATTAGCC?TATTTACTTC?TTTTTTTGCA?GAAACTTTAA?????8040

TAACTAACCT?TGCAGGTAAT?ATAAAGGTAG?TAATTTGCAT?ATACCTTCTA?TGGAGATTTA?????8100

The termination of the initial wzy of orf8

CAGTAAG ATG?CAAGATAAAA?CCG TAACGAT?ATTAATGGCA?ACTTATAATG?GAAGTGCTTT?8160

TATAGAGAAC?CAGATTCTTT?CATTACAGCA?ACAAAAATAT?AAAGATTGGA?TACTCTATAT?????8220

TCATGATGAT?GGTTCCTCTG?ATGATACCTT?AGATATTATA?AAGAGAATTC?AGCTTACAGA?????8280

ACCTCGTATT?AATCTTATTG?AGGATGGGCT?TACGAGACTT?GGTGCAGGGA?AAAATTTTCT?????8340

TTCATTAGTA?AAATATTCAG?CTACAAATTA?TACTATATTT?TGCGATCAAG?ATGATATTTG?????8400

GCTCGAAAAC?AAATTAAGCG?AGATGATTGT?TTTCGCAGAT?AGTAAAGGGC?TGGCTAGTAG?????8460

TAAGTTACCA?TCTATGATTT?ATGCAGATGG?GTATGCATTT?GACGATAGCA?CAGGTGAGAT?????8520

TGATTTTTGT?GGTATATCAC?ATAATCATGC?TACGAGATTG?AAAGATTTCT?TATTTTTCAA?????8580

TGCTGGCTAT?CAAGGGTGTT?CAATATTATT?TAATAAAGCA?ATGGTTGACA?TTGCAGCAAA?????8640

TTACCATGGT?TATGTCCATT?TACATGATGA?TGTCGTAAGT?CTGATCGCCC?ATTCTTTGGG?????8700

GAATGTGTAT?TTTTTACCAA?AAAAATTGAT?GTTATATCGC?CAACACTTAG?GGGCAGTAAC?????8760

TGGCCAAAAG?AAGTTTAATA?ACAGATTCAT?TTCAATGCTG?ACATCCAAAG?TTAATTATCT?????8820

TTTGTCAAGA?GAGCATTTTT?TAGTTAAGAG?ATCTTTTTAC?GATAATTACC?ATCATCTTTT?????8880

AACGGCTGAG?ATTAAAAATG?ACTTTGAAGT?CTTTTTTAAA?TTTTGCCAAA?CAAAAAACAA?????8940

ACTTTCACAG?TTAGTGCTTT?TATTGAAGCA?TGATTTTCGC?TTAAATAACA?GCAGATTAAA?????9000

The termination of the initial orf8 of orf9

GCTATTGTTA?AAATGCATAG?TAAGAAGGAC?ATTTAGTCA A?TGATCTCCGT?CTTAACACCT???9060

ACTTATAATC?GTGCGTATAC?CTTGAAGCGA?TTATACGAGT?CGTTAATTTG?TCAAACGACT?????9120

AAATCCTTTG?AATGGGTTGT?GGTTAATGAT?GGTAGTAACG?ATGAGACTGA?GTCAATAATA?????9180

AAAGAGTTTC?AGCAACAAAA?TATTATTAAA?ATTATTTATT?ACAAACAGGA?AAATAAAGGT?????9240

AAAACTCAGG?CTCTTAATGC?AGGTATACAA?TTATGTGCAG?GAAGTGATAT?TTTAATCGTT?????9300

GATAGCGATG?ATCTATTGAC?GTCGGATGCT?ATAGCTTGCA?TTGAAGCTAG?TTTGACTCAG?????9360

GAGAAGATAT?ATAATAAAAA?AATATCGGGG?GTGGCATTTC?GTAAAGCATA?TCTGGATGAA?????9420

ACCATAATTG?GTACTGTTTT?TGATGATAGT?GTTGATAGTT?TTTGTTATCT?TTCTGCAACT?????9480

GACGCTGGGC?ATCTTTTCAA?AGGCGATCTT?GCATATTGCT?TTAAGAAAGA?AATGCTGCAA?????9540

ATGTTTCCTT?TTCCATACTT?TCATAACGAA?AAGTTTGTTC?CTGAATTATA?CATTTGGAAC?????9600

AAGATTACAG?ATCACGCATT?AGTAAAATTT?CATAAAAAAA?AAGCAGTATA?CCTATGTGAG?????9660

TATCTCGAAG?ATGGGTTATC?TAAAAACTTC?AAAACACAAT?TATTAAAAAA?TCCCAAAGGG?????9720

TTTAGTATCT?ATTATATTGA?TCAGTTTAAG?CGTGAAACCA?ACTACATTCG?AAAATTAAAA?????9780

The termination of the initial orf9 of orf10

ATGTTGATTC?GTTATTTTCA?GTGTAAATTA?TATGAGCTAA?AAAA ATGAAT?ATAGTGTTTG???9840

TGATTACTGG?ATTGGGACTT?GGGGGGGCTG?AAAAGCAAGT?ATGCTTATTG?GCTGATCGAC?????9900

TGGCTGAGGC?AGAGCATCAA?ATATCGATAG?TGTCCCTTAC?AGGAGGATGT?GTTGTTAAGC?????9960

CAGATAATAA?GCATATTGAA?ATTTACCACC?TTAATATGGA?TAAAAGCCTA?ATAAGTTTCT????10020

TTTTAGGTGT?TATAAAATTA?CGTAATATTA?TTTCTACTAT?TAAGCCTGAT?ATTGTGCATA????10080

GCCATATGTT?CCATGCAAAT?ATCATGTCTA?GATTAAGTAA?GTTACTATTG?CCTTATTCCC????10140

ATAAGCTCAT?ATGTACAGCA?CATAGCAGAT?ATGAGGGAGG?GAGGCTTCGT?ATGCTTTGTT????10200

ATCGTTTAAC?AGATTATTTC?AGTGATATAA?ATACAAACGT?TAGTAAAGAG?GCGCTCGATG????10260

AATATGTTAA?TAATAAATAT?TTCTCAGCGA?CTAAATCTAT?TGTTGTTTAT?AATGGCGTTG????10320

ATACAGAAAA?ATTTAATTTC?AGTATTGATA?ATCGTATTTA?TATAAGAGAG?CAATTGTCTA????10380

TTAATAACCA?TGATAGGTTG?ATTCTCAGTG?TTGGCAGATT?AAACCCTGCT?AAAGATTATC????10440

CTAATCTGCT?AGCAGCATTC?ATGCTTTTAC?CTGAGCACTA?TAAGCTTGTT?ATCATTGGTG????10500

AGGGAGACGT?TCGTTCACAG?ATTGAACAGA?TAATTAAAGA?TCACAAGTTA?GGGACGCGTG????10560

TTCAGTTACT?TGGTAGTGTA?AATAATGTCA?ATGATTATTA?TTCTGCTTGT?GATTTATTTG????10620

TCTTATCTTC?GGCTTGGGAA?GGTTTTAGCT?TAGTTGTTAT?CGAGGCCATG?GCATGTCAAC????10680

GCATCGCTGT?ATGTACTGAT?GCAGGTGGAG?TAAAAGAAGC?TTTTACTGAT?CGTCATTACA????10740

TTGTACCAAC?TTCAAATGCC?GCAGCTTTAG?CTCGCAAAAT?TATTGAAGTT?GATATGTTAA????10800

CTATTGATCG?TAAAAATGAA?ATACAAAATG?ATAATAGGAA?TAATGTTGTT?AACAAGTTTT????10860

The termination of orf10

CAATTAATGC?TATTGTTAAT?CATTGGTTAA?CAATCTATAA?AAATATAACT?ATGCAG TAGA??10920

TAAATCTATT?ACATAATGAT?GAATTAAAAA?CAATATTAAT?TTTTAGCTGT?TTTTTATAAT????10980

ACCTAGTTCA?ATATGGATTA?AATTATTTTG?TTTTTTAATT?TGTAATCTGG?AATAGGTTAT????11040

TATATTTACT?AGATTGAATG?TAGTTCAATG?GATAGATTAT?CACGTTAACA?TTTAATTGAA????11100

GTATAAGTTG?TGTGGCGTTG?TGTTGCTTTA?AATTCTTAAT?AAATAATTAA?GGTTGGTGAT????11160

Gne's is initial

TA ATGACTAT?TTTAGTAACG?GGCGGTGCTG?GCTATATAGG?TTCGCATACA?GTGGTGAGAC??11220

TGCTTGAAAA?GGGTAAAGAA?ATTGTTATTC?TTGATGATTT?TACTAATTCA?TTTCCTGAAA????11280

CATTGAATAG?AATAAAAATA?ATTACCGGTG?TTAAACCTTT?TTTTTACGAA?GGTTCTGTCC????11340

TTGATAGGAA?TTTATTGAAA?AAAATCTTTG?TCGAAAATAA?CATCACCGAT?GTTATTCATT????11400

TTGCAGGGCT?CAAATCAGTT?GGTGAATCCG?TATCGTCCCC?TCTTAAGTAT?TATGAAGTCA????11460

ATATAGCAGG?AAGTTTGCAT?CTAGTTGAAG?AGATGATTAC?GCACAATATA?AGCAATTTTA????11520

TTTTTAGCTC?TTCTGCAACA?GTATATGGTG?AACCAGAAAC?TATTCCATTG?ACGGAGTCCT????11580

CTCGCATTGG?TGGCACTACA?AACCCTTATG?GTACATCTAA?GCTTATGGTT?GAGAAAATAC????11640

TTGAGGATGT?TACTCGTTCT?AATCCTGAGT?TTAGAACCAC?AATTTTACGA?TATTTTAATC????11700

CCGTTGGTGC?TCATCCTTCT?GGTGATATGG?GCGAAGATCC?GAATGGTATT?CCAAATAATC????11760

TCATGCCTTA?TATCTGTCAG?GTTGCTATTG?GTAAGTATAA?GCAAGTTTCA?GTATACGGAA????11820

GCGATTATCC?AACAAAGGAT?GGTACTGGAG?TCCGTGACTT?TATACATGTA?ATGGATCTTG????11880

CTGAAGGTCA?TGTTGCTGCT?TTAGAGCACA?GAAATAAGGG?ACCAAATCAT?AAAGTTTACA????11940

ACTTGGGCAC?AGGCACTGGT?TATTCTGTTT?TGGAACTCCT?GACAGCTTTT?GAAAGAGTAA????12000

CTTCTCGTAA?AGTACCTTAC?GTTTTAAGTG?AAAGACGCCC?TGGAGATATC?GCCGAATGTT????12060

GGTCTAATCC?TTCGAAGGCG?TATGCGGAAC?TTGGATGGAA?AGCGAAGCGC?GGACTGGAAG????12120

The termination of gne

ACATGGTTCG?AGATGCCTGG?AATTGGCAAC?AAAAGAATCC?AAACGGTTAT?AAAAAAGAA T???12180

GAATGATCAA?AGAAATTTTT?GTCAACTGCA?AAAATTACCG?AATTTATGTA?TCCTGAGTTA???12240

ACATAGCACT?AACATTGAAT?TGCGTTATGT?TTCCCAGTAT?CACCTCTGAC?AGGAGTAAAC????12300

AATGTCAAAG?CAACAGATCG?GCGTCGTCGG?TATGGCAGTG?ATGGGGCGCA?ACCTTGCGCT????12360

CAACATCGAA?AGCCGTGGTT?ATACCGTCTC?TATTTTCAAC?CGTTCCCGTG?AAAAGACGGA????12420

AGAAGTGATT?GCCGAAAATC?CGGGCAAGAA?ACTGGTTCCT?TACTATACGG?TGAAAGAGTT????12480

TGTTGAATCT?CTGGAAACGC?CTCGTCGCAT?CCTGTTAATG?GTGAAAGCAG?GTGCAGGCAC????12540

GGATGCTGCT?ATTGATTCTC?TCAAGCCATA?CCTCGATAAA?GGCGACATCA?TCATTGATGG????12600

CGGTAATACC?TTCTTCCAGG?ACACCATTCG?TCGTAACCGT?GAGCTTTCTG?CAGAAGGCTT????12660

TAACTTCATC?GGTACCGGTG?TTTCCGGCGG?TGAAGAAGGT?GCGCTGAAAG?GTCCTTCCAT????12720

TATGCCTGGC?GGACAGAAAG?AAGCCTATGA?ACTGGTTGCG?CCGATCCTGA?CCAAGATCGC????12780

CGCCGTTGCT?GAAGATGGCG?AACCGTGCGT?TACATATATT?GGTGCCGATG?GCGCGGGTCA????12840

CTATGTAAAA?ATGGTTCACA?ACGGTATTGA?ATACGGTGAT?ATGCAACTGA?TTGCTGAAGC????12900

CTATTCTCTC?CTGAAAGGCG?GCCTGAATCT?CTCTAACGAA?GAACTGGCAC?AGACCTTTAC????12960

CGAGTGGAAT?AACGGTGAAC?TGAGCAGCTA?CCTGATCGAC?ATCACCAAAG?ACATCTTCAC????13020

CAAAAAAGAT?GAAGACGGTA?ACTACCTGGT?TGATGTGATT?CTGGATGAAG?CAGCAAACAA????13080

AGGTACCGGT?AAATGGACCA?GCCAGAGTGC?GCTGGATCTC?GGCGAACCGC?TGTCGCTGAT????13140

TACCGAGTCT?GTGTTTGCAC?GTTATATCTC?TTCTCTGAAA?GATCAGCGTG?TTGCCGCATC????13200

TAAAGTTCTC?TCTGGCCCGC?AAGCACAGCC?AGCAGGCGAG?AAGGCTGAGT?TCATCGAAAA????13260

AGTTCGCCGT?GCGCTGTATC?TGGGTAAAAT?CGTTTCTTAC?GCTCAGGGCT?TCTCTCAGCT????13320

GCGTGCTGCG?TCTGAAGAGT?ATAACTGGGA?TCTGAACTAC?GGCGAAATCA?CGAAGATTTT????13380

CCGTGCTGGC?TGCATCATCC?GTGCGCAGTT?CCTGCAGAAA?ATCACCGATG?CTTATGCCGA????13440

AAATCCGCAG?ATCGCTAACC?TGCTGCTGGC?TCCGTACTTC?AAGCAAATTG?CCGATGACTA????13500

CCAGCAGGCG?CTGCGTGATG?TCGTTGCTTA?TGCAGTACAG?AACGGTATCC?CGGTTCCGAC????13560

CTTCTCTGCT?TCGGTTGCCT?ATTA???????????????????????????????????????????13584

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 (9)

1, a kind of Nucleotide of the O-antigen-specific to intestinal bacteria O142 type, it is characterized in that: it is the isolating Nucleotide shown in SEQ ID NO:1,13584 bases of total length; The base that perhaps has one or more insertions, disappearance or replacement keeps the Nucleotide of the SEQ IDNO:1 of described isolating functional nucleotide simultaneously.

2, according to the Nucleotide of the described O-antigen-specific to intestinal bacteria O142 type of claim 1, it is characterized in that: it is by 11 genomic constitutions, all between galF gene and gnd gene.

3, according to the Nucleotide of the described O-antigen-specific to intestinal bacteria O142 type of claim 2, it is characterized in that described gene is: the gene of transhipment enzyme comprises the wzx gene or with wzx the gene of identity function is arranged; Pol gene comprises the wzy gene or with wzy the gene of identity function is arranged; Mutase gene comprises the gne gene; Glycosyltransferase gene comprises orf1, orf8, orf9, orf10 gene; The FscM gene comprises rmlB, rmlD, rmlA, rmlC gene; Wherein said gene: wzx is the Nucleotide of 5649 to 6893 bases among the SEQ ID NO:1; Wzy is the Nucleotide of 6945 to 8126 bases among the SEQ ID NO:1; Gne is the Nucleotide of 11163 to 12182 bases among the SEQ ID NO:1; Orf1 is the Nucleotide of 1140 to 2156 bases among the SEQ ID NO:1; Orf8 is the Nucleotide of 8108 to 9043 bases among the SEQ IDNO:1; Orf9 is the Nucleotide of 9040 to 9828 bases among the SEQ ID NO:1; Orf10 is the Nucleotide of 9825 to 10919 bases among the SEQ ID NO:1; RmlB is the Nucleotide of 2176 to 3261 bases among the SEQ ID NO:1; RmlD is the Nucleotide of 3261 to 4160 bases among the SEQ ID NO:1; RmlA is the Nucleotide of 4212 to 5090 bases among the SEQ ID NO:1; RmlC is the Nucleotide of 5095 to 5652 bases among the SEQ ID NO:1.

4, according to the Nucleotide of claim 1 or 2 described O-antigen-specifics to intestinal bacteria O142 type, it is characterized in that: it is to come from described wzx gene, wzy gene; The orf1 that glycosyltransferase gene comprises, orf8, orf9, orf10 gene; And their mixing or their reorganization.

5, according to the Nucleotide of the described O-antigen high special to intestinal bacteria O142 type of claim 4, it is characterized in that the oligonucleotide of the described wzx of coming from gene is to being: the Nucleotide of 5901 to 5920 bases among the SEQ ID NO:1 and the Nucleotide of 6155 to 6173 bases; The Nucleotide of 6496 to 6513 bases among the SEQ ID NO:1 and the Nucleotide of 6754 to 6771 bases; The oligonucleotide that comes from the wzy gene is to being: the Nucleotide of 7463 to 7480 bases among the SEQ ID NO:1 and the Nucleotide of 7902 to 7919 bases; The Nucleotide of 6966 to 6983 bases among the SEQ ID NO:1 and the Nucleotide of 7288 to 7306 bases.

6, the Nucleotide of the described O-antigen-specific to intestinal bacteria O142 type of claim 1 is detecting the application of expressing the antigenic bacterium of O-, identify other polysaccharide antigen of the O-antigen of bacterium and bacterium in diagnosis.

7, the recombinant molecule of the Nucleotide of the described O-antigen-specific to intestinal bacteria O142 type of claim 1, and can provide the O-antigen of expressing intestinal bacteria O142 type by inserting to express, and become bacterial vaccine.

8, according to the application of the Nucleotide of the described O-antigen-specific to intestinal bacteria O142 type of claim 1, it is characterized in that it is used for PCR, is used for hybridization and fluoroscopic examination or is used to make gene chip or microarray, the bacterium in human body and the environment as probe as primer.

9, the separation method of the Nucleotide of the described O-antigen-specific to intestinal bacteria O142 type of claim 1 is characterized in that it comprises the steps:

(1) genomic extraction: 37 ℃ of incubated overnight intestinal bacteria O142 types in the LB of 5mL substratum, centrifugal collecting cell.With 500ul 50mM Tris-HCl (pH8.0) and 10ul 0.4M EDTA re-suspended cell, 37 ℃ of incubations 20 minutes, the N,O-Diacetylmuramidase that adds 10ul 10mg/ml then continues insulation 20 minutes, the Proteinase K, the 15ul 10%SDS that add 3ul 20mg/ml afterwards, 50 ℃ of incubations 2 hours, the RNase that adds 3ul 10mg/ml again, 65 ℃ of incubations 30 minutes.Add equal-volume phenol extracting mixture, get supernatant liquor and use isopyknic phenol again: chloroform: twice of primary isoamyl alcohol (25: 24: 1) mixing solutions extracting, get supernatant liquor again with isopyknic ether extracting to remove remaining phenol, supernatant liquor is with 2 times of volume ethanol deposit D NA, roll out DNA and wash DNA with glass yarn, DNA is resuspended among the 30ul TE with 70% ethanol; Genomic dna detects by 0.4% agarose gel electrophoresis;

(2) by the O-antigen gene in the pcr amplification intestinal bacteria O142 type bunch: with the genome of intestinal bacteria O142 type is that template is passed through its O-antigen gene of Long pcr amplification bunch, at first according to the JumpStart sequences Design upstream primer (5 '-ATT GTG GCTGCA GGG ATC AAA GAA AT-3 ') that often is found in O-antigen gene bunch promoter region, again according to the gnd gene design downstream primer (5 '-TAG TCG CGT GNG CCT GGA TTA AGT TCG C-3 ') in O-antigen gene bunch downstream; With the Expand Long Template PCR method of Boehringer Mannheim company amplification O-antigen gene bunch, the PCR response procedures was as follows: 94 ℃ of pre-sex change 2 minutes; 94 ℃ of sex change are 10 seconds then, annealed 15 seconds for 60 ℃, 68 ℃ were extended 15 minutes, carry out 30 circulations like this, last, continue to extend 7 minutes at 68 ℃, obtain the PCR product, detect the size and the specificity thereof of PCR product with 0.8% agarose gel electrophoresis, merge 5 pipe long PCR products, and with the Wizard PCR Preps purification kit purified pcr product of Promega company;

(3) make up O-antigen gene bunch library: make up O-antigen gene bunch library with the Novagen DNaseI shot gun method that is modified, reaction system is a 300ng PCR purified product, 0.9ul 0.1M MnCl ₂The DNaseI of the 1mg/ml of 1ul dilution in 1: 2000, reaction is carried out at room temperature, enzyme is cut the dna fragmentation size is concentrated between the 1.5kb-3kb, then add 2ul 0.1M EDTA termination reaction, merge the same reaction system of 4 pipes, with isopyknic phenol extracting once, use isopyknic phenol: chloroform: primary isoamyl alcohol (25: 24: 1) mixing solutions extracting once, after using isopyknic ether extracting once again, dehydrated alcohol deposit D NA with 2.5 times of volumes, and wash precipitation with 70% ethanol, be resuspended at last in the 18ul water, in this mixture, add 2.5ul dNTP (1mMdCTP subsequently, 1mMdGTP, 1mMdTTP, 10mMdATP), 1.25ul the T4DNA polysaccharase of 100mM DTT and 5 units, 11 ℃ 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, and this mixture is through the equal-volume chloroform: after primary isoamyl alcohol (24: 1) mixing solutions extracting and the extracting of equal-volume ether with 3 * 10 of Promega company ^-3The pGEM-T-Easy carrier connect 10 hours in 16 ℃, cumulative volume is 90ul, and the 10 * buffer of 9ul and the T4DNA ligase enzyme of 25 units are wherein arranged.Use the dehydrated alcohol precipitation of the 3M NaAc (pH5.2) of 1/10 volume and 2 times of volumes to be connected mixture at last, wash precipitation with 70% ethanol again, be dissolved in after the drying and obtain connecting product in the 30ul water; Preparation method with the electric transformed competence colibacillus cell of 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 acillin 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 O142 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 O142 type; The quality of sequence is mainly guaranteed by two aspects: 1) genome of intestinal bacteria O142 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 O142 type O-antigen gene bunch, with American National biotechnology information science center (The National Center forBiotechnology Information, NCBI) orffinder finds gene, find the reading frame of 11 openings, determine also that with the function of finding the reading frame that these are open what gene they are with the genetic comparison among the blast groupware and the GenBank, finish gene annotation with the Artemis software at Britain sanger center again, do accurate comparison between DNA and protein sequence with Clustral W software, obtain the structure of the O-antigen gene bunch of intestinal bacteria O142 type at last;

(6) screening of specific gene: at wzx, the wzy gene design primer in the O-antigen gene of intestinal bacteria O142 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 the 13rd group, has obtained the expection size, any product that all do not increase in other groups is so the O-antigen of wzx, wzy gene pairs intestinal bacteria O142 type all is high special.