CN1324133C - O-antigen specific nucleotide of E.coli 071 type - Google Patents

Abstract

The present invention provides a nucleotide specific to an O-antigen of Escherichia coli O71, which is a complete nucleotide sequence of a gene cluster for controlling the synthesis of the O-antigen in Escherichia coli O71, such as the separated nucleotide disclosed in SEQ ID NO: 1 with the full length of 13743 basic groups, or the nucleotide of SEQ ID NO: 1 comprising one or a plurality of inserted, deleted or substituted basic groups and maintaining the functions of the separated nucleotide simultaneously. The present invention also comprises an oligonucleotide of a glycosyltransferase gene and an oligosaccharide unit processing gene derived from an O-antigen gene cluster of Escherichia coli O71. In the present invention, PCR indicates that the oligonucleotide has high specificity on the O-antigen of Escherichia coli O71. The present invention also discloses a method for detecting and identifying Escherichia coli O71 in human bodies and in environments by the oligonucleotide of the present invention.

Description

Nucleotide to the O-antigen-specific of intestinal bacteria O71 type

Technical field

The present invention relates to the complete nucleotide sequence of control O-antigen synthetic gene cluster in the intestinal bacteria O71 type (Escherichia coli O71), particularly relate in the intestinal bacteria O71 type oligonucleotide in the control O-antigen synthetic gene cluster, can utilize these the oligonucleotide of the O-antigen-specific intestinal bacteria O71 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. (1935) " 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 O71 gene clusters:gene clusters encoding the samecolitose-containing O antigen are highly conserved " .Journal ofBacteriology.182:5256-5261].The O-antigen gene bunch contains three genoids: sugared synthesis path gene, glycosyltransferase gene, oligosaccharide unit treatment gene, the required nucleoside diphosphate monose of enzymic synthesis O-antigen of wherein sugared synthesis path genes encoding; Thereby the enzyme of glycosyltransferase gene coding forwards nucleoside diphosphate monose and other molecule to and makes monose aggregate into oligosaccharide unit on the monose; The oligosaccharide unit treatment gene comprises transhipment enzyme gene and pol gene, and they transfer to the bacterium inner membrance outside with oligosaccharide unit, and repolymerization becomes polysaccharide.Glycosyltransferase gene and oligosaccharide unit treatment gene only are present in the gene cluster of carrying these genes.The difference of monose in the O-antigen, between monose between the difference of link button and the oligosaccharide unit difference of link button constituted the antigenic diversity of O-, and the composition of monose, the link button between monose and the link button between the oligosaccharide unit are by the Gene Handling in the O-antigen gene bunch, so the O-antigen gene bunch has determined O-antigenic synthetic, has also determined the antigenic diversity of O-.

Because O-antigen is extremely strong antigen, be one of important paathogenic factor of intestinal bacteria, it has extremely strong diversity again simultaneously, and this enlightens us can study a kind of intestinal bacteria and good, highly sensitive method of the antigenic specificity of O-thereof of detecting quickly and accurately.With surperficial polysaccharide is that the serology immune response of target has been used to somatotype and the evaluation to bacterium always since the thirties in last century, is unique means of identifying pathogenic bacterium.This diagnostic method needs a large amount of antiserum(antisera)s, and the antiserum(antisera) general classes is incomplete, quantity not sufficient, and also there are some difficulties in a large amount of antiserum(antisera)s in preparation with in storing.On the other hand this method length consuming time, sensitivity is low, loss is high, poor accuracy, so, generally believe that now this traditional serology detection method will be that the modern molecular biology method replaces.1993, Luk, J.M.C et.al has identified the O-antigen [Luk of Salmonellas with the specific nucleotide sequence of Salmonellas (S.enterica) O-antigen gene bunch by PCR method, J.M.C.et.al. (1993) " Selective amplification of abequose andparatose synthase genes (rfb) by polymerase chain reaction for identification ofS.enterica major serogroups (A; B; C2; and D) ", 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.Nineteen thirty-five, 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 O71 type.It is the Nucleotide in the O-antigen gene bunch of intestinal bacteria O71 type, is the special Nucleotide that comes from glycosyltransferase gene and transhipment enzyme gene and pol gene.

A time purpose of the present invention has provided the full length nucleotide sequence of the O-antigen gene bunch of intestinal bacteria O71 type.

Another object of the present invention has provided the gene of the O-antigen gene bunch that constitutes intestinal bacteria O71 type: the gene of transhipment enzyme is the wzx gene or with wzx the gene of identity function is arranged; Pol gene is the wzy gene or with wzy the gene of identity function is arranged; The rhamnosyl synthase gene comprises the rmlBDAC gene or with rmlBDAC the gene of identity function is arranged; A kind of rare monose synthase gene comprises orf6 and orf7 or with orf6 and orf7 the gene of identity function is arranged; Acetyltransferase gene orf12 or the gene of identity function is arranged with acetyltransferase; Glycosyltransferase gene comprises orf4, orf11 gene; The gene orf9 of a unknown function.

Another purpose of the present invention has provided oligonucleotide, the gene that they come from coding transhipment enzyme respectively be the wzx gene or with wzx have identity function gene they are oligonucleotide in the said gene, length is at 10-20nt; They are special to the O-antigen of intestinal bacteria O71 type; Especially the oligonucleotide of listing in the table 1, they are high specials to the O-antigen of intestinal bacteria O71 type, and these oligonucleotide are also reconfigurable, the oligonucleotide after the combination also is a high special to the O-antigen of intestinal bacteria O71 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 O71 type of these methods detections and identification of escherichia coli O71 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 O71 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 O71 type, and it is the isolating Nucleotide shown in SEQ ID NO:1,13743 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 O71 type, it is by 12 genomic constitutions, all between galF gene and gnd gene.

The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O71 type, wherein said gene is: wzx is the Nucleotide of 6987 to 8261 bases among the SEQ ID NO:1; Wzy is the Nucleotide of 9666 to 10955 bases among the SEQ ID NO:1; RmlBDAC is respectively the Nucleotide of 1137 to 2222,2222 to 3121,3179 to 4051,4925 to 5473 bases among the SEQ ID NO:1; Orf4 is the Nucleotide of 4078 to 4887 bases among the SEQ ID NO:1; Orf6 is the Nucleotide of 5466 to 5864 bases among the SEQ ID NO:1; Orf7 is the Nucleotide of 5884 to 6990 bases among the SEQ ID NO:1; Orf9 is the Nucleotide of 8248 to 9666 bases among the SEQ ID NO:1; Orf11 is the Nucleotide of 10948 to 11775 bases among the SEQ ID NO:1; Orf712 is the Nucleotide of 11778 to 12248 bases among the SEQ ID NO:1.

The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O71 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 O71 type, the oligonucleotide of the wherein said wzx of coming from gene is to being: the Nucleotide of 7515 to 7533 bases among the SEQ ID NO:1 and the Nucleotide of 7839 to 7859 bases; The Nucleotide of 7139 to 7157 bases among the SEQ ID NO:1 and the Nucleotide of 8051 to 8069 bases.The oligonucleotide that comes from the wzy gene is to being: the Nucleotide of 10332 to 10350 bases among the SEQ ID NO:1 and the Nucleotide of 10581 to 10609 bases; The Nucleotide of 9981 to 9999 bases among the SEQ ID NO:1 and the Nucleotide of 10670 to 10688 bases.

The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O71 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 O71 type, and can provide the O-antigen of expressing intestinal bacteria O71 type by inserting to express, and become bacterial vaccine.

The application of the Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O71 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 O71 type is characterized in that it comprises the steps:

(1) genomic extraction: 37 ℃ of incubated overnight intestinal bacteria O71 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 O71 type bunch: with the genome of intestinal bacteria O71 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 long PCR products, and with the Wizard PCR Preps purification kit purified pcr product of Promega company;

(3) make up O-antigen gene bunch library: make up O-antigen gene bunch library with the Novagen DNaseI shot gun method that is modified, reaction system is a 300ng PCR purified product, 0.9ul 0.1M MnCl ₂, the DNaseI of the 1mg/ml of 1ul dilution in 1: 2000, reaction is carried out at room temperature, and enzyme is cut the dna fragmentation size is concentrated between the 1.5kb-3kb, then adds 2ul 0.1M EDTA termination reaction.Merge the same reaction system of 4 pipes, with isopyknic phenol extracting once, use isopyknic phenol: chloroform: primary isoamyl alcohol (25: 24: 1) mixing solutions extracting once, after using isopyknic ether extracting once again, dehydrated alcohol deposit D NA with 2.5 times of volumes, and wash precipitation with 70% ethanol, be resuspended at last in the 18ul water, in this mixture, add 2.5ul dNTP (1mMdCTP, 1mMdGTP, 1mMdTTP subsequently, 10mMdATP), 1.25ul the T4DNA polysaccharase of 100mM DTT and 5 units, 11 ℃ 30 minutes, enzyme is cut product mends into flush end, after 75 ℃ of termination reactions, add the Tth archaeal dna polymerase of 5 units and corresponding damping fluid thereof and system is expanded as 80ul, 70 ℃ of reactions 20 minutes make 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 O71 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 O71 type; The quality of sequence is mainly guaranteed by two aspects: 1) genome of intestinal bacteria O71 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 O71 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 12 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 O71 type at last;

(6) specific gene screening: at wzx, the wzy gene design primer in the O-antigen gene of dysentery intestinal bacteria O71 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 O71 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 O71 type, its complete sequence shown in SEQ ID NO:1,13743 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 O71 type by method of the present invention, as shown in table 3, it is altogether by 12 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 O71 type, promptly transports enzyme gene (wzx gene or the gene of identity function arranged with wzx); Pol gene (wzy gene or the gene of identity function arranged with wzy); Rhamnosyl synthase gene (rmlBDAC gene or the gene of identity function arranged with rmlBDAC); A kind of monose synthase gene (7 genes have the gene of identity function for orf6,7 genes and orf6); A kind of unknown function gene; Glycosyltransferase gene (orf4, orf11 gene); Special sugared synthesis path gene in the bacterial polysaccharides antigen comprises orf6,7,12 genes.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 O71 type.

The 3rd aspect of the present invention, wzx gene in the O-antigen gene bunch that comes from intestinal bacteria O71 type is provided or the gene, wzy gene of identity function is arranged or with wzy the gene oligonucleotide of identity function is arranged with wzx, rhamnosyl is synthetic not to have gene (rmlBDAC gene or the gene of identity function arranged with rmlBDAC); A kind of monose synthase gene (7 genes have the gene of identity function for orf6,7 genes and orf6); A kind of unknown function gene; Glycosyltransferase gene (orf4, orf11 gene); Special sugared synthesis path gene in the bacterial polysaccharides antigen comprises orf6, and they are any one section oligonucleotide in these genes for 7,12 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 O71 type all is high special.

The separation method of the Nucleotide of described O-antigen-specific to intestinal bacteria O71 type comprises the steps: 1) genomic extraction; 2) the O-antigen gene in the pcr amplification intestinal bacteria O71 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 6987 to 8261 bases among the SEQ ID NO:1); It is high special to intestinal bacteria O71 type that wzy gene (nucleotide position is to be the Nucleotide of 9666 to 10955 bases the SEQ ID NO:1 from wzy) comes from above intragenic oligonucleotide.

In addition, the antigenic gene cluster of the different O-of the coding of two genetic resemblances produces new O-antigen by gene recombination or sudden change sometimes, thereby produces new bacteria types, new mutant strain.In this environment, need filter out many specificitys that oligonucleotide is detected with raising with recombination hybridization.Therefore, the invention provides a whole set of many mixtures to oligonucleotide, they come from glycosyltransferase gene; Come from transhipment enzyme and pol gene, comprise the wzx gene or the gene, wzy gene of identity function arranged or the gene of identity function is arranged with wzy with wzx.The mixture of these genes is special to a special bacterial polysaccharides antigen, is special thereby make this cover oligonucleotide to the polysaccharide antigen of this bacterium.More particularly, the mixture of these oligonucleotide is to come from glycosyltransferase gene, wzx gene or the gene, wzy gene of identity function arranged or with wzy the combination of the oligonucleotide in the gene of identity function is arranged with wzx.

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

The present invention relates to a kind of antigenic method of one or more bacterial polysaccharideses that detects in the food, these antigens can make sample can with the oligonucleotide specific hybrid of following at least one gene, these genes are: (i) gene of the encoding glycosyl transferring enzyme gene of transhipment enzyme and polysaccharase of (ii) encoding comprises the wzx gene or the gene, wzy gene of identity function is arranged or with wzy the gene of identity function is arranged with wzx.At least one oligonucleotide can be hybridized with at least one more than one such gene specific of expressing the special antigenic bacterium of O-under the situation of condition permission, and these bacteriums are intestinal bacteria O71 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 O71 type.

On the other hand, the present invention relates to a kind of antigenic method of one or more bacterial polysaccharideses that detects in the movement, these antigens can make sample can with the oligonucleotide specific hybrid of following at least one gene, these genes are: (i) gene of the encoding glycosyl transferring enzyme gene of transhipment enzyme and polysaccharase of (ii) encoding comprises the wzx gene or the gene, wzy gene of identity function is arranged or with wzy the gene of identity function is arranged with wzx.At least one oligonucleotide can be expressed more than one such gene specific hybridization of the special antigenic bacterium of O-with at least one under the situation of condition permission.These bacteriums are intestinal bacteria O71 types.Oligonucleotide among available the present invention is made the method test sample of primer by PCR, also can with behind the oligonucleotide molecules mark among the present invention as probe by hybridization such as southern-blot or fluoroscopic examination, perhaps by antigen and bacterium in gene chip or the microarray assay sample.

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

On the other hand, the present invention also relates to the antigenic method of one or more bacterial polysaccharideses in the sample that a kind of detection comes from patient.One or more bacterial polysaccharides antigens in the sample can make sample can with a specific hybrid in a pair of oligonucleotide in following at least one gene, these genes are: (i) gene of the encoding glycosyl transferring enzyme gene of transhipment enzyme and polysaccharase of (ii) encoding comprises the wzx gene or the gene, wzy gene of identity function is arranged or with wzy the gene of identity function is arranged with wzx.Under the situation of condition permission at least one oligonucleotide can with sample at least one express more than one such gene specific hybridization of the special antigenic bacterium of O-, these bacteriums are intestinal bacteria O71 types.Oligonucleotide among available the present invention is made the method test sample of primer by PCR, also can will pass through hybridization as probe behind the oligonucleotide mark among the present invention, perhaps by antigen and bacterium in gene chip or the microarray assay sample.

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

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

The present invention discloses the full length sequence of the O-antigen gene bunch of intestinal bacteria O71 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 O71 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 O71 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 O71 type bunch:

With the genome of intestinal bacteria O71 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 CCT GGATTA AGT TCG C) in O-antigen gene bunch downstream; With the Expand Long Template PCR method of Boehringer Mannheim company amplification O-antigen gene bunch, the PCR response procedures was as follows: 94 ℃ of pre-sex change 2 minutes; 94 ℃ of sex change are 10 seconds then, 60 ℃ of annealing 15 seconds, and 68 ℃ were extended 15 minutes, and carried out 30 circulations like this.At last, continue to extend 7 minutes at 68 ℃, obtain the PCR product, the agarose gel electrophoresis with 0.8% detects the size and the specificity thereof of PCR product.Merge 5 pipe long PCR products, and with the Wizard PCR Preps purification kit purified pcr product of Promega company.

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

At first be the acquisition that connects product: make up O-antigen gene bunch library with the Novagen DNaseI shot gun method that is modified.Reaction system is a 300ng PCR purified product, 0.9ul 0.1M MnCl ₂, 1ul1: the DNaseI of the 1mg/ml of 2000 dilutions, reaction is carried out at room temperature.Enzyme is cut the dna fragmentation size is concentrated between the 1.5kb-3kb, then adds 2ul 0.1M EDTA termination reaction.Merge the same reaction system of 4 pipes, with isopyknic phenol extracting once, use isopyknic phenol: chloroform: primary isoamyl alcohol (25: 24: 1) mixing solutions extracting once, after using isopyknic ether extracting once again, dehydrated alcohol deposit D NA with 2.5 times of volumes, and wash precipitation with 70% ethanol, be resuspended at last in the 18ul water.In this mixture, add 2.5ul dNTP (1mMdCTP subsequently, 1mMdGTP, 1mMdTTP, 10mMdATP), the T4DNA polysaccharase of 1.25ul 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 O71 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 O71 type obtained.The quality of sequence is mainly guaranteed by two aspects: 1) genome of intestinal bacteria O71 type is done 5 LongPCR reactions, mix these products then to produce the library.2), guarantee high-quality fraction of coverage more than 3 to each base.After obtaining the nucleotide sequence of intestinal bacteria O71 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 12 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 O71 type at last, as shown in table 3.

By retrieving and comparing, find orf1,2, the albumen of rmlBDA genes encoding has very high consensus amino acid sequence in 3 encoded protein and the Shigella boydi antigen gene bunch, by search to Pfam protein-based order sequenced data storehouse, find orf1, the homology desired value of the consensus sequence of 2,3 encoded protein and known rmlBDA is very high.RmlBDA is responsible for a kind of rare monose in the synthetic rhamnosyl O-antigen.RmlC of Orf5 and orf4 encoded protein and Salmonellas and glycosyltransferase gene encoded protein have higher similarity.Orf6, the albumen of the genes encoding of synthetic a kind of monose has very high consensus amino acid sequence among 7 encoded protein and the Aneurinibacillus thermoaerophilus, orf6,7 also should synthesize a kind of monose, called after orf6,7.The gene of coding acetyltransferase has 63% homogeny among orf12 encoded protein and the Listonella anguillarum, infers that orf12 is the acetyltransferase gene, called after orf12.

Orf8 and orf10 are the proteic genes that there is transmembrane segment in only two codings of intestinal bacteria O71 kind.Orf1 encoded protein and colibacillary O-antigen transferring enzyme have 28% 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 orf8 is wzx.Orf10 encoded protein and Streptococcus pneumoniae O-antigen polysaccharase have 20% consistence, 42% 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 orf10 is wzy.

Orf4, the albumen of 11 two genes encodings and other known glycosyltransferases have the sequence identity of 24-36% and the sequence similarity of 44-53%.By the search to glycosyltransferase motif database among the Pfam, the homology desired value of the consensus sequence of the albumen of these two genes encodings and known glycosyltransferase family 1 and 2 is 1 * e ^-12To 5 * e ^-17, so we infer this two 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 O71 may be made up of three monose.Because the definite function of these three genes can't be determined, so we are with the temporary called after orf4 of these four genes, orf11.The gene orf9 of a unknown function.

Embodiment 6: the screening of specific gene:

At wzx, wzy gene design primer in the O-antigen gene of intestinal bacteria O71 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 O71 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 O71 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 O71 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 O71 type and O-antigen thereof are high specials.

At last, from intestinal bacteria O71 type, screen gene by PCR: wzx, wzy gene to the O-antigen high special of intestinal bacteria O71 type.And the oligonucleotide of these intragenic any one section 10-20nt is special to the O-antigen of intestinal bacteria O71 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 O71 type.These all oligonucleotide all can be used for the intestinal bacteria O71 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 O71 type, in table, listed the structure of the O-antigen gene bunch of intestinal bacteria O71 type, altogether by 12 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 O71 type, listed the accurate position of all open reading frame in complete sequence in the O-antigen gene bunch of intestinal bacteria O71 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 O71 type

＜130〉to the Nucleotide of the O antigen-specific of intestinal bacteria O71 type

<160>1

<170>PatentIn version 3.2

<210>1

<211>13743

<212>DNA

<213>Escherichia coli

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attgtggctg cagggatcaa agaaatcctc ctggtaactc acgcgtccaa gaacgcggtc 60

gaaaaccact tcgacacctc atatgaatta gaatctctcc ttgagcagcg cgtgaagcgt 120

caactgcttg cggaagtgca gtccatctgt ccaccgggcg tgaccattat gaacgtgcgt 180

cagggcgaac ctttaggttt aggccactcc attttgtgtg cacgacccgc cattggtgac 240

aacccatttg tcgtggtgct gccagacgtt gtgatcgacg atgccagcgc cgacccgcta 300

cgctacaacc ttgctgccat gattgcgcgc ttcaatgaaa cgggccgtag ccaagtgctg 360

gcaaaacgta tgccgggtga cctctctgaa tactccgtca tccagaccaa agaaccgctg 420

gaccgtgaag gcaaagtcag ccgcattgtt gaattcatcg aaaaaccgga tcagccacac 480

acgctggact cagacatcat ggccgtgggt cgctatgtgc tttctgccga tatttggccg 540

gaacttgaac gcactcagcc tggtgcatgg gggcgtattc agctgactga tgccattgcc 600

gaactggcga aaaaacagtc cgttgatgcc atgctgatga caggtgacag ctacgactgc 660

ggtaaaaaaa tggggtatat gcaggcattt gtgaagtatg gactacgcaa cctcaaagaa 720

ggggcgaagt tccgtaaagg gattgagaag ctgttaagcg aataatgaaa atctgaccgg 780

atgtaacggt tgataagaaa attataacgg cagtgaagat tcgtggcgaa agtaatttgt 840

tgcgaatatt cctgccgtta ttttatataa acaatcagaa taacaacgag ttagcaatag 900

gattttagtc aaagttttcc aggattttcc ttgtttccag agcggattgg taagacaatt 960

agcgtttgaa tttttcgggt ttagcgcgag tgggcaacgc tcgtcacatc gtagacatgc 1020

atgcagtgct ctggtagctg taaagccagg ggcggtagcg tgcattaata cctctcttaa 1080

tcaaactaag agccgctaat ttcacagcat gctctgaagt aatatggaat aaaaaagtga 1140

agatacttgt tactggtggc gcaggattta ttggttctgc tgtagttcgt cacattataa 1200

ataatacgca ggatagtgtt gttaatgtcg ataaattaac gtacgccgga aacctggaat 1260

cacttgctga tgtttctgat tctgaacgct atgtttttga acatgcggat atttgcgatg 1320

ctgctgcaat ggcgcggatt tttgctcagc atcagccgga tgcagtgatg cacctggctg 1380

ctgaaagcca tgttgaccgt tcaattacag gtcctgcggc atttattgaa accaatattg 1440

ttggtactta tgtccttttg gaagccgctc gcaattactg gtctgctctt gatagcgaca 1500

agaaaaatag cttccgtttt catcatattt ctactgacga agtctatggc gatctgcctc 1560

atcctgacga agtaaataat aaagaagaat tacccctctt tactgagacg acagcttacg 1620

cgccaagcag cccatattct gcttctaaag catccagcga tcatttagtc cgcgcgtgga 1680

aacgtaccta tggtttaccg accattgtga ctaactgttc gaataactac ggcccttatc 1740

actttccgga aaaattgatt ccgttggtaa ttcttaatgc tctggaaggt aaggcattac 1800

ctatttatgg caaaggggat caaattcgtg actggctgta tgttgaagat catgcgcgtg 1860

cgttatatac cgtcgtaacc gaaggtaaag cgggtggaac ttataacatt ggtggacaca 1920

acgaaaagaa aaacatcgat gtagtgctca ctatttgtga tttgttggat gagattgtac 1980

cgaaagagaa atcttaccgc gagcaaatca cttatgttgc cgatcgtccg ggacacgatc 2040

gccgttatgc cattgatgct gagaagactg gtcgcgaatt gggatggaaa ccacaggaaa 2100

cgtttgagag cgggattcgg aagacagtgg aatggtacct gtccaataca aaatgggttg 2160

ttaatgtgaa aagtggtgcc tatcaatcgt ggattgaaca gaactatgag ggccgccagt 2220

aatgaatatt ctccttttcg gcaaaacagg gcaggtaggt tgggaactac agcgtgctct 2280

ggcacctttg ggtaatttga ttgctcttga tgttcactcc actgattatt gtggtgattt 2340

tagtaatcct gaaggtgtgg ctgaaaccgt caaaaaaatt cgccctgatg ttattgttaa 2400

tgctgcggct catactgcag tagataagct gagtcagaac ccgaatttgc acaattactc 2460

aatgcgacca gcgttgaatc aattgcaaaa gcggccaatg aaagttgggg cttgggtaat 2520

tcattactca actgactacg tatttccggg aaccggtgaa ataccatggc tggagacgga 2580

tgcaaccgca ccgctaaatg tttacggtga aaccaagtta gccggagaaa aagcgttaca 2640

ggaaaattgc gcgaagcatc ttattttccg taccagctgg gtatacgcag gtaaaggaaa 2700

taacttcgcc aaaacgatgt tgcgtctggc aaaagagcgc gaagaactgg ctgtgattaa 2760

tgatcaattt ggtgcgccaa caggtgctga gctgctggca gattgtacgg cacatgctat 2820

tcgtgtggca ctgaataaac cagaagtcgc aggcttgtat catctggtag ccagtggtac 2880

cacaacttgg cacgattatg ctgcgctggt ttttgaagag gcgcgcaaag caggcattcc 2940

ccttgcactc aacaagctca acgcaatatc aacaacagcc tatcctacac cagctcgtcg 3000

cccacataac tctcgcctta atacagaaaa atttcagcag aactttgcgc ttgtcttgcc 3060

tgactggcag gttggcgtga aacgaatgct caacgaatta tttacgacta cagcaattta 3120

attgtttttg catcttgttc gtgatgatgg tgcaagatga attaaaagga atgatgaaat 3180

gaaaacgcgt aaaggtatta ttttagcggg tggttctggt actcgtcttt atcctgtgac 3240

tatggccgtg agtaaacaac ttctgcctat ttatgataag cctatgattt attacccgct 3300

ctcaaccctg atgctggcgg gtattcgtga tattttgatt attagtacgc cacaagacac 3360

accacgtttt gagcaattac tgggcgacgg tagccagtgg gggcttaatc ttcagtacaa 3420

agtgcaacct agtccagatg gtctggcaca agctttcatt attggagaag attttattgg 3480

tggtgatgat tgtgcgttgg tcttgggtga taatatcttt tacggtcatg acctaccaaa 3540

attaatggat acagctgtta atagagagag tggcgcaacg gtatttgcct atcacgttaa 3600

tgatccagaa cgttatggtg tggtggagtt tgataaaaac ggcacggcaa taagcttgga 3660

agaaaaaccg cttcaaccga aaagcaatta tgcagttaca ggactttatt tctatgataa 3720

tgatgtcgtg gaaatggcaa aaaaactgaa accatctgcc cgtggcgaag tcgaaatcac 3780

agatattaac cgtatttata tggaacaggg gcgtttgtct gtagccatga tggggcgtgg 3840

ttatgcttgg ttggacacgg gtacacatca aagtcttatt gaggcaagta acttcattgc 3900

aacaatagaa gaacgccagg ggctgaaagt ttcctgtcca gaagagattg cttaccgtaa 3960

agggtttatt ggaaaagagc aactagagct attagctaag tcgcttatga aaaatcaata 4020

tggaaaatat cttcttaaaa tgatcgggta aataataata ctttttcagg aaatgaaatg 4080

aatttcagtg tattgatgtc tctttatatt aaagagaagc ccgaatatct acatgaatgc 4140

ttaaaaagtc tatatgaaca aacaaagcaa gctgatgaga tagtgcttgt ctatgatgga 4200

ccaattacaa acgaattaga taaagtagtt aattattggt taaatagttt gccaataaag 4260

ataataaaat taagcaaaaa tgtcggtttg ggtcaggccc tgaataaggg gttagctcaa 4320

tgtgaaaatg attatgtcgc tagaatggat acggatgata tatgccatcc caagagattt 4380

gaaattcaaa ttgatttttt aaataaaaac aagaatataa gtgttgttgg ttcatatata 4440

gaggagtttt cagaaactat agaaaacagg ttgtcgcaaa aaattgtacc gacaacgcac 4500

aatgagataa tgaaaaatat aggaaaacga aatccattta accacatgac agttgtgttc 4560

aataagagtg aaataataaa ggtgggtgga tatgaacatc attatttaat ggaagattat 4620

aatttatggt tgcggctttt aaaaaataat attcaggcag ccaatttgcc tttaacatta 4680

gtttatgcca gaggtgggga tggcatgatt tataggaggt ccggtttacg ttatattaaa 4740

agtgaatata aattgtataa gttaaaacaa agattaggat atcaaaatta tattatatcc 4800

tcattggtgt tttttttaag gagtgtacca agggtttttc ctccttacat tttaaaagca 4860

atttatagat tcttgcgaaa gaaataacag tataaaaatg cattatatta tttagagggg 4920

gcttatgaaa tattcaagaa caacaataca agatgctata gtgattacac ctgaagtatt 4980

tgctgattct agggggtatt tttttgaaag ctttaattat cgggattttc aaaggattat 5040

taatcgagag atagtttttt gtcaagataa ccaatcatta tcgcataagg gagttgtcag 5100

gggactacat tttcaagtaa atccaaagga acaggcaaaa ttggtaagat gtataaatgg 5160

ttcggtgtat gatgtgattg tagacttgcg caaaaactcc ccttcttata agtgttggtt 5220

cggaatttta ttatcgtctg ataataaaaa acagttatgg attccagagg gatgtgctca 5280

tggttttatc tcattagagg acaatacgga attattatat aagactactg aattttattc 5340

tcctgaccat gaacgctgca tcatttggaa tgatcctgac ttaggaataa gttggccaga 5400

atatagctat ataatttccg ataaagataa gaatggaatt tccttcgctg aatgggagag 5460

cttaggtgat taaggtgaaa ttaattccac ttcaaacgca tggtgacgat agaggttcgc 5520

ttgttgcttt agaagagggg tataatatac catttcaaat taaacgcgtt tattatattt 5580

ttaacacaaa gaaaggagtc atgagaggtt ttcatgcaca taaaaactta aaacaggttg 5640

caatagctgt tagaggaagt tgtaagttta aattagacaa tggtaccgat agtgttgagg 5700

ttctactgaa tgatcctgcg caaggtctat tgatagaatc atttatttgg agagaaatgt 5760

atgacttttc cgaggactgc gtattaatgg ttttagccga ctctcattac gatgagaacg 5820

attacgttag agactacaat gctttcttaa atctggtggc ttaataataa taaggataca 5880

ataatggtta atttcttaag tttaaaaaat ataaactcac gctataatga ggaattaaaa 5940

aaagcatgtg caagagttat tgactctggc tggtatgtaa tgggggagga attacaccag 6000

ttcgaaaacg agttcgctaa atattgtggt gtaaaaggtt ctctgggggt ggcgaatggg 6060

ctagatgctt tgattctttc attaagagct tggcgtgaaa tggggaaaat taagccgggt 6120

gatgaggtac ttgttccagc taatacatat atagcttcaa ttattgcaat tacagaaaat 6180

aatctggtgc ctgtatttgt tgagccggat tataatactt ataatttaga tccagataaa 6240

attgaatctg cgattactga aaaaacaaaa gtgattttac ctgttcattt gtatggacga 6300

atttgtccta tggaagaaat attatcattg gcgaaaaaat ataatcttct tgtgttagag 6360

gattgtgctc aggcgcatgg tgcatctctt aatggcagaa aaacaggggg gtggggagat 6420

gcgggggctt ttagttttta tccaggtaaa aatttggggg cccttggtga ttctggggct 6480

attactagca atgatttaga ttttctgaat gtaattcaag ctttaagaaa ttatgggtca 6540

caccaaaaat atgtaaatca atatattggt gtgaatagtc gattagatga aattcaagca 6600

gctatgctaa gggttaaatt aaaatacctg gatagtgata acaaaagaag gaaagaaatt 6660

gctcaatatt acattgattc aataataaat cctttagtcc aattgccgaa atcatgcagt 6720

aattatgatg aaaatgtttg gcatttattt gttgttaaaa ctcgatatag agagtatctt 6780

caaaaattcc tgagtaaaca gagtattcaa acattaattc actatccaca ccctccacat 6840

aaacaaatag cttataagca gtttaattcc atttctttgc cattgacaga aaaaatccat 6900

aatgaggttt tgtctttgcc aatggatcca actctttctg aagaggagat acaaaaggtt 6960

gtcaatgcgg taaatggttt tactctatga gttcattttt taagatatca ttctttagtg 7020

gttgtttgac attattcaag atgttaatgg gctttttaat tgctaaagta atcgctattt 7080

atacagggcc atctggtatg gccttacttg gtcagcttca aagtttagtc acaggtatta 7140

atgggatcgt aaatgcacct gtaggaaatg gtatcgttaa atatactgca gagtttaaac 7200

tagaaggtgc agaatgttgc tctagatggt ggaaaccagc aatcggcttt tcattccttt 7260

tttgtttggt tttatcgata gtttttatac catttgcaaa taaaatatca tttattttat 7320

ttggctctta tgaatatgat tatgtggtta ttatagcagt atgtaactta ccttttgcag 7380

ctttcggtac tatgataatg tctattgtca atggaatggg gaagtataga ctttatatat 7440

tatatgggtt tatttctaat cttataacgt cgataataat gttacttatg ctaataaaat 7500

atggtattgt tggtgcatta ttagccactt caatacaata tggtcttatt ggtataattt 7560

tgatatgttt gaattataat caggaatggt tcaaacataa ttatttttat ccctacgata 7620

gtatttttaa ttcagaagca aaggatattt tagcttatat attgatggct ataacaagtg 7680

caattagctt acccttgact ttaatcgtaa ttagaaaaat tctcctaaat atgacggata 7740

taactaccgt tggacaatgg caggcggtat ggaaaatatc tgaagtatat ataggagtcg 7800

tgactattgc acttagtact tattttttgc ctaagttgtc tgctctaaat gatacggcta 7860

aaataaaaaa tgaaattggt attgtcctta aatatactat tccatttgtt tgtgttgccg 7920

cggttctcat tttcgctttt cgcgatctga taataacgct cttattcacg aatgagtttt 7980

atcctgcacg tgatcttttt tttattcaac ttgtaggtga tataattaaa ataataagtt 8040

ggatttatgc attcactatg ttggcttcaa aagcaactag gtggtatgtt ggatctgaat 8100

tatttttttg tatttcatgg tgtttaatta gttttgttct tgttaatata tataaggcgc 8160

aaggaattaa tatagcatat tgcattagct atgttatgta ttttttaata atttatctca 8220

acctaaataa gattataaag aaaaagaatg aacctcattg aaattattaa actccaaatt 8280

atcaaaactt ctgatattaa taagaaaata agactgctca aggagttggg gggaatgcta 8340

tgggccaata acgttggcat atacaactta aattcacttg aaagggcagt tatcgaagat 8400

gtagcaagag attatacgcc acaaattggt agtcaatata aaaagaaaaa aaataatgta 8460

tttgtgatta ctataggcta catgtctggt gggcatacaa ggttaatgga aaatcttgca 8520

aatatgctag ataataaatg tgaccttata attactggaa acgcatctgt tacgctaaaa 8580

aaaaggcttg aaaaatattt caatgaaatt tttgaaattc ctgtttacaa tgacactaac 8640

attaatgaac tttataagtt aagcgctaat cttgagagat acgaaaatat aatacttaat 8700

atacaccctg atgatattgc gagcgttatt gcagctggtc ttgcaaaaaa aaatataaac 8760

acatacgttc actttgttaa tcatgcagat catgttttta gcttcggtcc gaccgtagca 8820

gatgtctggt atcaaattag ccaattcggg gctaaacttg atcaattaag agagctaagt 8880

ggtaaaacta cattcttggg gataccgatt gcgataaatg atgatcattt atctaataat 8940

atgatttcca ctaatgatga agtgaaatgt attatcactg ctggctctgc aaataaatac 9000

aagccgcata aaggctattc tctcataaaa aaaattcctg aaatattata tcaatttcca 9060

gaagcatcaa taatagtagt aggtactaac atcctatttt gctattggtg gtggggaact 9120

tttttaaaat tcagaaagcg tattaaattc tataaagcac ttaatcattc tgaatatctt 9180

ttacttatgc aaaatgctga tatttatttg gatagttatc ctattcccgg ggggacggca 9240

tttgttgagc agtcctttct gggtaaaaaa tgtgccgggt atatttcacc gcaacaaggg 9300

tatacaccac ttgaaaacat aaaatgcgac atcacaagtt ctgagttaaa caaaataaat 9360

aacgttgata taattgagaa aataaaatat gttcatcctt ttgataatgt aaaaaataga 9420

cttttaagcg ccatcaacaa acaagtgatg actactataa actgggatta ttattacaaa 9480

tggactggtg acattaactt ttttaaactc gaaaaaataa gagaaattcc attatattta 9540

ttgctggaat gtgtgaaaga aaattctagc ttaaaatgta taatatcaca atataaattt 9600

ataactatta tttctttaat taaaaatttt atcaggctct atgtcagtaa actaaagggg 9660

aaataatgga atatactatt attattattt tttttgttct gttgtatttt acatctaaag 9720

ttacgtggcc ctttagtgga aaaaatgcac atccaattaa ctttgtttta ttcacgcaaa 9780

tatttatatt aacgctgcca ggtgttctta ttttaacatt ttttaaagag tgcgcgggat 9840

ctataacatg tgagataata gctgaaaaca tcaaaataaa cgtgatgttg gattatttta 9900

ctgtgctctg ttttatcctt ttgggggtgc tttcttcatt tttcttgtta aaaggtaaaa 9960

ctgattttcg gccagcatcg aatgcccata gacgatatct aaatacgtgc tttatattaa 10020

ctctacttat ttttttggta aaaattattt ctgttaatca agttcctcta tttcttacat 10080

tacttggtca taaagatgct gcagagattc aaaaagctga gatccttaga ggggaagatg 10140

ggttttcatt tccggtaata aattttctaa taaaatattt tcctttgtat ttttattaca 10200

gtacgttgat tggtttgttt aaacgcaaag ttactatagt ttactttgta atggcttttc 10260

ttgtcacgtc tgtgacaatg ctttacgatt tacaaaaagg tccggttgtt attatgatta 10320

ttggctcatt ttggctttat tgggcttata ccggaaaagc aaaaatgata gtagtaggtg 10380

ggatattcag tttattttta gtaggtgttc tattttatat ctccttcgat tttggtgatg 10440

atacccaata ttttattacc gctgtgttaa ataggacctt cattgctcag aatgatggaa 10500

tgtattgggt atatcaatat tataatatat taccaaataa agaactgtat tctttatggg 10560

gggtcccact tgcacaacag tttggtatcc cgcagattga cccattaagt gatattattg 10620

gtgttgtttt tcctcaagct aaggatagtt gggttaatac aacgacattt ttgttaggtg 10680

aggcaaaggc aattttcggt gactatagtt ccattatctc atctttagta gtatttatta 10740

atgtgtttgt tgtatgtgtg ctatcaacac ttttagtaag agtatcaaaa aatatttttt 10800

atcccgcaat attcgttatg attcaaacta taccatttgc aaacaatttg actgacttat 10860

tatatggtag atttattttt ggttttttag tctttatgtt atttccagtg cttatatgtt 10920

ttttgtgtta tggaaaatta aaaaatgatg aatagaaaag tgcttattat aatggttacc 10980

tataatcctg attggagtaa ggtaattaat aaagtgaagt ctttttgtgt tgcaggaaca 11040

gtttttgttt ctgataatag cgattctgat tcctcatcat tgataaacta tgaaaatgtt 11100

atatacagat ataatcatgg taatgtaggc atagcaaaag ctcagaattt aggtattgaa 11160

tatgcgatgc agtataatta tgagtttgta gcttttgttg atgatgatag taatctgagc 11220

gggggtaaaa ttacacagtt aacaaataat ttatgtaaat taggcagtct tggtgaaaat 11280

attgcagcat tttgcgctta ccctagtgaa agaggtggtt ttgacaagac aaaaaaaact 11340

tcgcttggta gacaattatt gttaactaag aatttaatga gttcaggatc tttaacaaag 11400

gttgatgtat ttaaaaagat aggtttattt gatgaagatt tatttattga ctatgtggat 11460

tatgaatggg gctggagagc actggaaaaa aattttttaa tcgtaattga tacatcagta 11520

gaatttgaac attcacttgg tcaggggcgc tcaaaaattg gattagggat cccctctcct 11580

atccgacatt tttatcaaac tagaaatctt ttatggatat caaggctaaa ttatgtacca 11640

atatattgga aacttaaaca gtttttactt cttccaattc gctatttcta ttttggtttt 11700

ttttataatc actctaaatt acggcgaacc cattttctca aagggtttgc cgcagggtta 11760

aaatttaaat catagtttta tgagataata tcatcaacgg gacgaataat tttagctgga 11820

ttgccagcaa caattacatt atccggaata tccttcgtaa ctacggagcc tgcgccaata 11880

attgaatttt caccgatgat aatacccggc aaaattgttg cgttcgcacc aatagaagca 11940

ttctttttaa tatgtatcgt tggataataa tctgggtatt ttttagatct tggataaata 12000

tcatttgtaa atgtaacatt tggacctata aaaacattat cttcaattat gactccatcc 12060

catatataga ctccagattt tatagtgaca ttatcgccaa taataacttt attttcgatt 12120

aaagtatgtg cgcaaatatt gcagttatta ccaattacag cgccttcaaa aataatgctg 12180

tattgccaaa tggttgtatt tagaccgatt tttacagttt ttacatcact taatggatga 12240

attttcataa attctaccct tcatttttaa aacaataata ttgtaaatga tatcatttag 12300

tattaatgat atcattagac gcctgaatag taaaaagttt tccctttggt tataaagtca 12360

aaagttaagt agcgtaaatc ggtattctga caggagtaaa acatgtcaaa gcaacagatc 12420

ggcgtcgtcg gtatggcagt gatggggcgc aaccttgcgc tcaacatcga aagccgtggt 12480

tataccgtct ctattttcaa ccgttcccgt gaaaagacgg aagaagtgat tgccgaaaat 12540

ccaggcaaga aactggttcc ttactatacg gtgaaagagt ttgttgaatc tctggaaacg 12600

cctcgtcgca tcctgttaat ggtgaaagca ggtgcaggca cggatgctgc tattgattcc 12660

ctcaagccgt acctcgataa aggtgacatc atcattgatg gtggtaacac cttcttccag 12720

gacaccattc gtcgtaaccg tgagctttct tcagaaggct ttaacttcat cggtaccggt 12780

gtttccggcg gagaagaggg ggcgctgaaa gggccgtcca tcatgcctgg tggccagaaa 12840

gaagcctatg aactggttgc gccgatcctg accaaaatcg ccgcagtagc tgaagacggt 12900

gagccatgcg ttacctatat tggtgccgat ggcgcaggtc actatgtgaa gatggttcac 12960

aacggtattg aatacggaga tatgcaactg attgctgaag cctactctct gcttaaaggt 13020

ggcctgaacc tcaccaacga agaactggcg cagacgttta ccgagtggaa taacggtgaa 13080

ctgagcagct acctgatcga catcaccaaa gacatcttca ctaaaaaaga tgaagacggt 13140

aactacctgg ttgatgtgat cctggatgaa gcggctaaca aaggtaccgg taaatggacc 13200

agccagagcg cgcttgatct cggcgaaccg ctgtcgctga ttaccgagtc tgtgtttgca 13260

cgatacatct cttctctgaa agagcagcgt gttgccgcat ctaaagttct ttctggtccg 13320

caagcgcagc ctgctggcga caaaggtgaa ttcatcgaaa aagttcgccg tgcgctgtat 13380

ctgggtaaaa tcgtttctta cgctcagggc ttctctcagc tacgcgctgc gtctgaagag 13440

tacaactggg atctgaacta cggtgaaatc gcgaagattt tccgtgctgg ctgcatcatc 13500

cgtgcgcagt tcctgcagaa aatcaccgat gcttatgccg aaaatccgca gatcgctaac 13560

ctgttgctgg ctccttactt caagcaaatt gccgatgact accagcaggc gctgcgcgat 13620

gtcgtcgctt acgcagtaca gaacggtatc ccggtgccga ccttcgccgc tgctgttgcc 13680

tattacgaca gctaccgcgc cgctgttctg cctgcgaacc taattcaggc acagcgcgac 13740

taa 13743

Glycosyltransferase gene in the O antigen gene of table 1 intestinal bacteria O71 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 Wzy	O-antigen transhipment enzyme O-antigen polysaccharase	6987-8261 9666-10955	7515-7533 7139-7157 10332-10350 9981-9999	7839-7859 8051-8069 10581-10609 10670-10688	345bp 931bp 278bp 708bp	0 * 0 * 0 * 0 *	56 55 56 54

^*Only in intestinal bacteria O71 type, obtain a correct band

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

Group number	The bacterial strain that contains in this group	The source
			1, wild-type e. coli 2, wild-type e. coli 3, wild-type e. coli 4, wild-type e. coli 5, wild-type e. coli 6, wild-type e. coli 7, wild-type e. coli 8, wild-type e. coli 9, wild-type e. coli Shigellae 10, wild-type Shigellae 11, wild-type Shigellae 12, wild-type e. coli 13, the 4th group of bacterial strain adds intestinal bacteria reference culture O71	O1，O2，O5，O7，O12，O13，O14，O15，O16，O17，O19ab，O20， O21，O22，O23，O24，O59 O25，O26，O27，O28，O29，O30，O32，O33，O35，O36，O37， O38，O40，O41，O42，O43 O44，O45，O46，O48，O49，O50，O51，O52，O54，O55，O56， O57，O58，O60，O61，O62 O63，O65，O66，O69，O70，O72，O74，O75，O76，O77，O78， O79，O80，O81，O82，O83 O84，O85，O86，O87，O88，O89，O91，O92，O98，O99，O101， O102，O103，O104，O106 O107，O108，O109，O110，O111，O112ab，O112ac，O113， O115，O116，O118，O120，O123，O125，O126，O128 O129，O130，O131，O132，O133，O134，O135，O136，O137， O138，O139，O140，O141，O142，O143，O144，O145 O146，O147，O148，O150，O152，O154，O156，O157，O158， O159，O160，O161，O163，O164，O165，O166 O168，O169，O170，O171，O172，O173， D1，D2，D3，D4，D5，D6，D7，D8，D9，D10，D11，D12 B1，B2，B3，B4，B6，B7，B8，B9，B10，B11，B12，B13，B14，B15， B16，B17，B18 F1a，F1b，F2a，F2b，F3，F4b，F5(v：4)，F5(v：7)，F6，F X becomes，F Y becomes，DS，DR， O3，O11，O39，O59，O64，O73，O96，O95，O100，O114，O151， O155，O124	IMVS a IMVS a IMVS a IMVS a IMVS a IMVS a IMVS a IMVS a b c d d d IMVS a 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 O71 type O antigen gene structure iron

orf# galF rmlB rmlD rmlA orf4rmlC orf6 orf7 wzx orf9 wzy orf11 orf12 gnd

G+C％content 44 46 42 31 34 36 35 30 31 31 32 32

E.coli O71 O-antigen gene cluster

Table 4 intestinal bacteria O71 type O antigen gene cluster gene position

ATTGTGGCTG CAGGGATCAA AGAAATCCTC CTGGTAACTC ACGCGTCCAA GAACGCGGTC 60

GAAAACCACT TCGACACCTC ATATGAATTA GAATCTCTCC TTGAGCAGCG CGTGAAGCGT 120

CAACTGCTTG CGGAAGTGCA GTCCATCTGT CCACCGGGCG TGACCATTAT GAACGTGCGT 180

CAGGGCGAAC CTTTAGGTTT AGGCCACTCC ATTTTGTGTG CACGACCCGC CATTGGTGAC 240

AACCCATTTG TCGTGGTGCT GCCAGACGTT GTGATCGACG ATGCCAGCGC CGACCCGCTA 300

CGCTACAACC TTGCTGCCAT GATTGCGCGC TTCAATGAAA CGGGCCGTAG CCAAGTGCTG 360

GCAAAACGTA TGCCGGGTGA CCTCTCTGAA TACTCCGTCA TCCAGACCAA AGAACCGCTG 420

GACCGTGAAG GCAAAGTCAG CCGCATTGTT GAATTCATCG AAAAACCGGA TCAGCCACAC 480

ACGCTGGACT CAGACATCAT GGCCGTGGGT CGCTATGTGC TTTCTGCCGA TATTTGGCCG 540

GAACTTGAAC GCACTCAGCC TGGTGCATGG GGGCGTATTC AGCTGACTGA TGCCATTGCC 600

GAACTGGCGA AAAAACAGTC CGTTGATGCC ATGCTGATGA CAGGTGACAG CTACGACTGC 660

GGTAAAAAAA TGGGGTATAT GCAGGCATTT GTGAAGTATG GACTACGCAA CCTCAAAGAA 720

GGGGCGAAGT TCCGTAAAGG GATTGAGAAG CTGTTAAGCG AATAATGAAA ATCTGACCGG 780

ATGTAACGGT TGATAAGAAA ATTATAACGG CAGTGAAGAT TCGTGGCGAA AGTAATTTGT 840

TGCGAATATT CCTGCCGTTA TTTTATATAA ACAATCAGAA TAACAACGAG TTAGCAATAG 900

GATTTTAGTC AAAGTTTTCC AGGATTTTCC TTGTTTCCAG AGCGGATTGG TAAGACAATT 960

AGCGTTTGAA TTTTTCGGGT TTAGCGCGAG TGGGCAACGC TCGTCACATC GTAGACATGC 1020

ATGCAGTGCT CTGGTAGCTG TAAAGCCAGG GGCGGTAGCG TGCATTAATA CCTCTCTTAA 1080

Orf1's is initial

TCAAACTAAG AGCCGCTAAT TTCACAGCAT GCTCTGAAGT AATATGGAAT AAAAAA GTGA 1140

AGATACTTGT TACTGGTGGC GCAGGATTTA TTGGTTCTGC TGTAGTTCGT CACATTATAA 1200

ATAATACGCA GGATAGTGTT GTTAATGTCG ATAAATTAAC GTACGCCGGA AACCTGGAAT 1260

CACTTGCTGA TGTTTCTGAT TCTGAACGCT ATGTTTTTGA ACATGCGGAT ATTTGCGATG 1320

CTGCTGCAAT GGCGCGGATT TTTGCTCAGC ATCAGCCGGA TGCAGTGATG CACCTGGCTG 1380

CTGAAAGCCA TGTTGACCGT TCAATTACAG GTCCTGCGGC ATTTATTGAA ACCAATATTG 1440

TTGGTACTTA TGTCCTTTTG GAAGCCGCTC GCAATTACTG GTCTGCTCTT GATAGCGACA 1500

AGAAAAATAG CTTCCGTTTT CATCATATTT CTACTGACGA AGTCTATGGC GATCTGCCTC 1560

ATCCTGACGA AGTAAATAAT AAAGAAGAAT TACCCCTCTT TACTGAGACG ACAGCTTACG 1620

CGCCAAGCAG CCCATATTCT GCTTCTAAAG CATCCAGCGA TCATTTAGTC CGCGCGTGGA 1680

AACGTACCTA TGGTTTACCG ACCATTGTGA CTAACTGTTC GAATAACTAC GGCCCTTATC 1740

ACTTTCCGGA AAAATTGATT CCGTTGGTAA TTCTTAATGC TCTGGAAGGT AAGGCATTAC 1800

CTATTTATGG CAAAGGGGAT CAAATTCGTG ACTGGCTGTA TGTTGAAGAT CATGCGCGTG 1860

CGTTATATAC CGTCGTAACC GAAGGTAAAG CGGGTGGAAC TTATAACATT GGTGGACACA 1920

ACGAAAAGAA AAACATCGAT GTAGTGCTCA CTATTTGTGA TTTGTTGGAT GAGATTGTAC 1980

CGAAAGAGAA ATCTTACCGC GAGCAAATCA CTTATGTTGC CGATCGTCCG GGACACGATC 2040

GCCGTTATGC CATTGATGCT GAGAAGACTG GTCGCGAATT GGGATGGAAA CCACAGGAAA 2100

CGTTTGAGAG CGGGATTCGG AAGACAGTGG AATGGTACCT GTCCAATACA AAATGGGTTG 2160

The termination of orf1

TTAATGTGAA AAGTGGTGCC TATCAATCGT GGATTGAACA GAACTATGAG GGCCGCCAG T2220 orf2's is initial

AATGAATATT CTCCTTTTCG GCAAAACAGG GCAGGTAGGT TGGGAACTAC AGCGTGCTCT 2280

GGCACCTTTG GGTAATTTGA TTGCTCTTGA TGTTCACTCC ACTGATTATT GTGGTGATTT 2340

TAGTAATCCT GAAGGTGTGG CTGAAACCGT CAAAAAAATT CGCCCTGATG TTATTGTTAA 2400

TGCTGCGGCT CATACTGCAG TAGATAAGCT GAGTCAGAAC CCGAATTTGC ACAATTACTC 2460

AATGCGACCA GCGTTGAATC AATTGCAAAA GCGGCCAATG AAAGTTGGGG CTTGGGTAAT 2520

TCATTACTCA ACTGACTACG TATTTCCGGG AACCGGTGAA ATACCATGGC TGGAGACGGA 2580

TGCAACCGCA CCGCTAAATG TTTACGGTGA AACCAAGTTA GCCGGAGAAA AAGCGTTACA 2640

GGAAAATTGC GCGAAGCATC TTATTTTCCG TACCAGCTGG GTATACGCAG GTAAAGGAAA 2700

TAACTTCGCC AAAACGATGT TGCGTCTGGC AAAAGAGCGC GAAGAACTGG CTGTGATTAA 2760

TGATCAATTT GGTGCGCCAA CAGGTGCTGA GCTGCTGGCA GATTGTACGG CACATGCTAT 2820

TCGTGTGGCA CTGAATAAAC CAGAAGTCGC AGGCTTGTAT CATCTGGTAG CCAGTGGTAC 2880

CACAACTTGG CACGATTATG CTGCGCTGGT TTTTGAAGAG GCGCGCAAAG CAGGCATTCC 2940

CCTTGCACTC AACAAGCTCA ACGCAATATC AACAACAGCC TATCCTACAC CAGCTCGTCG 3000

CCCACATAAC TCTCGCCTTA ATACAGAAAA ATTTCAGCAG AACTTTGCGC TTGTCTTGCC 3060

The termination of orf2

TGACTGGCAG GTTGGCGTGA AACGAATGCT CAACGAATTA TTTACGACTA CAGCAATT TA 3120

Orf3's is initial

ATTGTTTTTG CATCTTGTTC GTGATGATGG TGCAAGATGA ATTAAAAGGA ATGATGAA AT 3180

GAAAACGCGT AAAGGTATTA TTTTAGCGGG TGGTTCTGGT ACTCGTCTTT ATCCTGTGAC 3240

TATGGCCGTG AGTAAACAAC TTCTGCCTAT TTATGATAAG CCTATGATTT ATTACCCGCT 3300

CTCAACCCTG ATGCTGGCGG GTATTCGTGA TATTTTGATT ATTAGTACGC CACAAGACAC 3360

ACCACGTTTT GAGCAATTAC TGGGCGACGG TAGCCAGTGG GGGCTTAATC TTCAGTACAA 3420

AGTGCAACCT AGTCCAGATG GTCTGGCACA AGCTTTCATT ATTGGAGAAG ATTTTATTGG 3480

TGGTGATGAT TGTGCGTTGG TCTTGGGTGA TAATATCTTT TACGGTCATG ACCTACCAAA 3540

ATTAATGGAT ACAGCTGTTA ATAGAGAGAG TGGCGCAACG GTATTTGCCT ATCACGTTAA 3600

TGATCCAGAA CGTTATGGTG TGGTGGAGTT TGATAAAAAC GGCACGGCAA TAAGCTTGGA 3660

AGAAAAACCG CTTCAACCGA AAAGCAATTA TGCAGTTACA GGACTTTATT TCTATGATAA 3720

TGATGTCGTG GAAATGGCAA AAAAACTGAA ACCATCTGCC CGTGGCGAAG TCGAAATCAC 3780

AGATATTAAC CGTATTTATA TGGAACAGGG GCGTTTGTCT GTAGCCATGA TGGGGCGTGG 3840

TTATGCTTGG TTGGACACGG GTACACATCA AAGTCTTATT GAGGCAAGTA ACTTCATTGC 3900

AACAATAGAA GAACGCCAGG GGCTGAAAGT TTCCTGTCCA GAAGAGATTG CTTACCGTAA 3960

AGGGTTTATT GGAAAAGAGC AACTAGAGCT ATTAGCTAAG TCGCTTATGA AAAATCAATA 4020

The termination orf4's of orf3 is initial

TGGAAAATAT CTTCTTAAAA TGATCGGG TA AATAATAATA CTTTTTCAGG AAATGAA ATG 4080

AATTTCAGTG TATTGATGTC TCTTTATATT AAAGAGAAGC CCGAATATCT ACATGAATGC 4140

TTAAAAAGTC TATATGAACA AACAAAGCAA GCTGATGAGA TAGTGCTTGT CTATGATGGA 4200

CCAATTACAA ACGAATTAGA TAAAGTAGTT AATTATTGGT TAAATAGTTT GCCAATAAAG 4260

ATAATAAAAT TAAGCAAAAA TGTCGGTTTG GGTCAGGCCC TGAATAAGGG GTTAGCTCAA 4320

TGTGAAAATG ATTATGTCGC TAGAATGGAT ACGGATGATA TATGCCATCC CAAGAGATTT 4380

GAAATTCAAA TTGATTTTTT AAATAAAAAC AAGAATATAA GTGTTGTTGG TTCATATATA 4440

GAGGAGTTTT CAGAAACTAT AGAAAACAGG TTGTCGCAAA AAATTGTACC GACAACGCAC 4500

AATGAGATAA TGAAAAATAT AGGAAAACGA AATCCATTTA ACCACATGAC AGTTGTGTTC 4560

AATAAGAGTG AAATAATAAA GGTGGGTGGA TATGAACATC ATTATTTAAT GGAAGATTAT 4620

AATTTATGGT TGCGGCTTTT AAAAAATAAT ATTCAGGCAG CCAATTTGCC TTTAACATTA 4680

GTTTATGCCA GAGGTGGGGA TGGCATGATT TATAGGAGGT CCGGTTTACG TTATATTAAA 4740

AGTGAATATA AATTGTATAA GTTAAAACAA AGATTAGGAT ATCAAAATTA TATTATATCC 4800

TCATTGGTGT TTTTTTTAAG GAGTGTACCA AGGGTTTTTC CTCCTTACAT TTTAAAAGCA 4860

The termination of orf4

ATTTATAGAT TCTTGCGAAA GAAA TAACAG TATAAAAATG CATTATATTA TTTAGAGGGG 4920

Orf5's is initial

GCTT ATGAAA TATTCAAGAA CAACAATACA AGATGCTATA GTGATTACAC CTGAAGTATT 4980

TGCTGATTCT AGGGGGTATT TTTTTGAAAG CTTTAATTAT CGGGATTTTC AAAGGATTAT 5040

TAATCGAGAG ATAGTTTTTT GTCAAGATAA CCAATCATTA TCGCATAAGG GAGTTGTCAG 5100

GGGACTACAT TTTCAAGTAA ATCCAAAGGA ACAGGCAAAA TTGGTAAGAT GTATAAATGG 5160

TTCGGTGTAT GATGTGATTG TAGACTTGCG CAAAAACTCC CCTTCTTATA AGTGTTGGTT 5220

CGGAATTTTA TTATCGTCTG ATAATAAAAA ACAGTTATGG ATTCCAGAGG GATGTGCTCA 5280

TGGTTTTATC TCATTAGAGG ACAATACGGA ATTATTATAT AAGACTACTG AATTTTATTC 5340

TCCTGACCAT GAACGCTGCA TCATTTGGAA TGATCCTGAC TTAGGAATAA GTTGGCCAGA 5400

ATATAGCTAT ATAATTTCCG ATAAAGATAA GAATGGAATT TCCTTCGCTG AATGGGAGAG 5460

The termination of the initial orf5 of orf6

CTTAG GTGAT TAAGGTGAAA TTAATTCCAC TTCAAACGCA TGGTGACGAT AGAGGTTCGC 5520

TTGTTGCTTT AGAAGAGGGG TATAATATAC CATTTCAAAT TAAACGCGTT TATTATATTT 5580

TTAACACAAA GAAAGGAGTC ATGAGAGGTT TTCATGCACA TAAAAACTTA AAACAGGTTG 5640

CAATAGCTGT TAGAGGAAGT TGTAAGTTTA AATTAGACAA TGGTACCGAT AGTGTTGAGG 5700

TTCTACTGAA TGATCCTGCG CAAGGTCTAT TGATAGAATC ATTTATTTGG AGAGAAATGT 5760

ATGACTTTTC CGAGGACTGC GTATTAATGG TTTTAGCCGA CTCTCATTAC GATGAGAACG 5820

The termination of orf6

ATTACGTTAG AGACTACAAT GCTTTCTTAA ATCTGGTGGC T TAATAATAA TAAGGATACA 5880

Orf7's is initial

ATA ATGTTA ATTTCTTAAG TTTAAAAAAT ATAAACTCAC GCTATAATGA GGAATTAAAA 5940

AAAGCATGTG CAAGAGTTAT TGACTCTGGC TGGTATGTAA TGGGGGAGGA ATTACACCAG 6000

TTCGAAAACG AGTTCGCTAA ATATTGTGGT GTAAAAGGTT CTCTGGGGGT GGCGAATGGG 6060

CTAGATGCTT TGATTCTTTC ATTAAGAGCT TGGCGTGAAA TGGGGAAAAT TAAGCCGGGT 6120

GATGAGGTAC TTGTTCCAGC TAATACATAT ATAGCTTCAA TTATTGCAAT TACAGAAAAT 6180

AATCTGGTGC CTGTATTTGT TGAGCCGGAT TATAATACTT ATAATTTAGA TCCAGATAAA 6240

ATTGAATCTG CGATTACTGA AAAAACAAAA GTGATTTTAC CTGTTCATTT GTATGGACGA 6300

ATTTGTCCTA TGGAAGAAAT ATTATCATTG GCGAAAAAAT ATAATCTTCT TGTGTTAGAG 6360

GATTGTGCTC AGGCGCATGG TGCATCTCTT AATGGCAGAA AAACAGGGGG GTGGGGAGAT 6420

GCGGGGGCTT TTAGTTTTTA TCCAGGTAAA AATTTGGGGG CCCTTGGTGA TTCTGGGGCT 6480

ATTACTAGCA ATGATTTAGA TTTTCTGAAT GTAATTCAAG CTTTAAGAAA TTATGGGTCA 6540

CACCAAAAAT ATGTAAATCA ATATATTGGT GTGAATAGTC GATTAGATGA AATTCAAGCA 6600

GCTATGCTAA GGGTTAAATT AAAATACCTG GATAGTGATA ACAAAAGAAG GAAAGAAATT 6660

GCTCAATATT ACATTGATTC AATAATAAAT CCTTTAGTCC AATTGCCGAA ATCATGCAGT 6720

AATTATGATG AAAATGTTTG GCATTTATTT GTTGTTAAAA CTCGATATAG AGAGTATCTT 6780

CAAAAATTCC TGAGTAAACA GAGTATTCAA ACATTAATTC ACTATCCACA CCCTCCACAT 6840

AAACAAATAG CTTATAAGCA GTTTAATTCC ATTTCTTTGC CATTGACAGA AAAAATCCAT 6900

AATGAGGTTT TGTCTTTGCC AATGGATCCA ACTCTTTCTG AAGAGGAGAT ACAAAAGGTT 6960

The termination of the initial orf7 of orf8

GTCAATGCGG TAAATGGTTT TACTCT ATGA GTTCATTTTT TAAGATATCA TTCTTTAGTG 7020

GTTGTTTGAC ATTATTCAAG ATGTTAATGG GCTTTTTAAT TGCTAAAGTA ATCGCTATTT 7080

ATACAGGGCC ATCTGGTATG GCCTTACTTG GTCAGCTTCA AAGTTTAGTC ACAGGTATTA 7140

ATGGGATCGT AAATGCACCT GTAGGAAATG GTATCGTTAA ATATACTGCA GAGTTTAAAC 7200

TAGAAGGTGC AGAATGTTGC TCTAGATGGT GGAAACCAGC AATCGGCTTT TCATTCCTTT 7260

TTTGTTTGGT TTTATCGATA GTTTTTATAC CATTTGCAAA TAAAATATCA TTTATTTTAT 7320

TTGGCTCTTA TGAATATGAT TATGTGGTTA TTATAGCAGT ATGTAACTTA CCTTTTGCAG 7380

CTTTCGGTAC TATGATAATG TCTATTGTCA ATGGAATGGG GAAGTATAGA CTTTATATAT 7440

TATATGGGTT TATTTCTAAT CTTATAACGT CGATAATAAT GTTACTTATG CTAATAAAAT 7500

ATGGTATTGT TGGTGCATTA TTAGCCACTT CAATACAATA TGGTCTTATT GGTATAATTT 7560

TGATATGTTT GAATTATAAT CAGGAATGGT TCAAACATAA TTATTTTTAT CCCTACGATA 7620

GTATTTTTAA TTCAGAAGCA AAGGATATTT TAGCTTATAT ATTGATGGCT ATAACAAGTG 7680

CAATTAGCTT ACCCTTGACT TTAATCGTAA TTAGAAAAAT TCTCCTAAAT ATGACGGATA 7740

TAACTACCGT TGGACAATGG CAGGCGGTAT GGAAAATATC TGAAGTATAT ATAGGAGTCG 7800

TGACTATTGC ACTTAGTACT TATTTTTTGC CTAAGTTGTC TGCTCTAAAT GATACGGCTA 7860

AAATAAAAAA TGAAATTGGT ATTGTCCTTA AATATACTAT TCCATTTGTT TGTGTTGCCG 7920

CGGTTCTCAT TTTCGCTTTT CGCGATCTGA TAATAACGCT CTTATTCACG AATGAGTTTT 7980

ATCCTGCACG TGATCTTTTT TTTATTCAAC TTGTAGGTGA TATAATTAAA ATAATAAGTT 8040

GGATTTATGC ATTCACTATG TTGGCTTCAA AAGCAACTAG GTGGTATGTT GGATCTGAAT 8100

TATTTTTTTG TATTTCATGG TGTTTAATTA GTTTTGTTCT TGTTAATATA TATAAGGCGC 8160

AAGGAATTAA TATAGCATAT TGCATTAGCT ATGTTATGTA TTTTTTAATA ATTTATCTCA 8220

The termination of the initial orf8 of orf9

ACCTAAATAA GATTATAAAG AAAAAGA ATG AACCTCAT TG AAATTATTAA ACTCCAAATT 8280

ATCAAAACTT CTGATATTAA TAAGAAAATA AGACTGCTCA AGGAGTTGGG GGGAATGCTA 8340

TGGGCCAATA ACGTTGGCAT ATACAACTTA AATTCACTTG AAAGGGCAGT TATCGAAGAT 8400

GTAGCAAGAG ATTATACGCC ACAAATTGGT AGTCAATATA AAAAGAAAAA AAATAATGTA 8460

TTTGTGATTA CTATAGGCTA CATGTCTGGT GGGCATACAA GGTTAATGGA AAATCTTGCA 8520

AATATGCTAG ATAATAAATG TGACCTTATA ATTACTGGAA ACGCATCTGT TACGCTAAAA 8580

AAAAGGCTTG AAAAATATTT CAATGAAATT TTTGAAATTC CTGTTTACAA TGACACTAAC 8640

ATTAATGAAC TTTATAAGTT AAGCGCTAAT CTTGAGAGAT ACGAAAATAT AATACTTAAT 8700

ATACACCCTG ATGATATTGC GAGCGTTATT GCAGCTGGTC TTGCAAAAAA AAATATAAAC 8760

ACATACGTTC ACTTTGTTAA TCATGCAGAT CATGTTTTTA GCTTCGGTCC GACCGTAGCA 8820

GATGTCTGGT ATCAAATTAG CCAATTCGGG GCTAAACTTG ATCAATTAAG AGAGCTAAGT 8880

GGTAAAACTA CATTCTTGGG GATACCGATT GCGATAAATG ATGATCATTT ATCTAATAAT 8940

ATGATTTCCA CTAATGATGA AGTGAAATGT ATTATCACTG CTGGCTCTGC AAATAAATAC 9000

AAGCCGCATA AAGGCTATTC TCTCATAAAA AAAATTCCTG AAATATTATA TCAATTTCCA 9060

GAAGCATCAA TAATAGTAGT AGGTACTAAC ATCCTATTTT GCTATTGGTG GTGGGGAACT 9120

TTTTTAAAAT TCAGAAAGCG TATTAAATTC TATAAAGCAC TTAATCATTC TGAATATCTT 9180

TTACTTATGC AAAATGCTGA TATTTATTTG GATAGTTATC CTATTCCCGG GGGGACGGCA 9240

TTTGTTGAGC AGTCCTTTCT GGGTAAAAAA TGTGCCGGGT ATATTTCACC GCAACAAGGG 9300

TATACACCAC TTGAAAACAT AAAATGCGAC ATCACAAGTT CTGAGTTAAA CAAAATAAAT 9360

AACGTTGATA TAATTGAGAA AATAAAATAT GTTCATCCTT TTGATAATGT AAAAAATAGA 9420

CTTTTAAGCG CCATCAACAA ACAAGTGATG ACTACTATAA ACTGGGATTA TTATTACAAA 9480

TGGACTGGTG ACATTAACTT TTTTAAACTC GAAAAAATAA GAGAAATTCC ATTATATTTA 9540

TTGCTGGAAT GTGTGAAAGA AAATTCTAGC TTAAAATGTA TAATATCACA ATATAAATTT 9600

The termination orf10's of ATAACTATTA TTTCTTTAAT TAAAAATTTT ATCAGGCTCT ATGTCAGTAA ACTAAAGGGG 9660 orf9 is initial

AAA TAATGGA ATATACTATT ATTATTATTT TTTTTGTTCT GTTGTATTTT ACATCTAAAG 9720

TTACGTGGCC CTTTAGTGGA AAAAATGCAC ATCCAATTAA CTTTGTTTTA TTCACGCAAA 9780

TATTTATATT AACGCTGCCA GGTGTTCTTA TTTTAACATT TTTTAAAGAG TGCGCGGGAT 9840

CTATAACATG TGAGATAATA GCTGAAAACA TCAAAATAAA CGTGATGTTG GATTATTTTA 9900

CTGTGCTCTG TTTTATCCTT TTGGGGGTGC TTTCTTCATT TTTCTTGTTA AAAGGTAAAA 9960

CTGATTTTCG GCCAGCATCG AATGCCCATA GACGATATCT AAATACGTGC TTTATATTAA 10020

CTCTACTTAT TTTTTTGGTA AAAATTATTT CTGTTAATCA AGTTCCTCTA TTTCTTACAT 10080

TACTTGGTCA TAAAGATGCT GCAGAGATTC AAAAAGCTGA GATCCTTAGA GGGGAAGATG 10140

GGTTTTCATT TCCGGTAATA AATTTTCTAA TAAAATATTT TCCTTTGTAT TTTTATTACA 10200

GTACGTTGAT TGGTTTGTTT AAACGCAAAG TTACTATAGT TTACTTTGTA ATGGCTTTTC 10260

TTGTCACGTC TGTGACAATG CTTTACGATT TACAAAAAGG TCCGGTTGTT ATTATGATTA 10320

TTGGCTCATT TTGGCTTTAT TGGGCTTATA CCGGAAAAGC AAAAATGATA GTAGTAGGTG 10380

GGATATTCAG TTTATTTTTA GTAGGTGTTC TATTTTATAT CTCCTTCGAT TTTGGTGATG 10440

ATACCCAATA TTTTATTACC GCTGTGTTAA ATAGGACCTT CATTGCTCAG AATGATGGAA 10500

TGTATTGGGT ATATCAATAT TATAATATAT TACCAAATAA AGAACTGTAT TCTTTATGGG 10560

GGGTCCCACT TGCACAACAG TTTGGTATCC CGCAGATTGA CCCATTAAGT GATATTATTG 10620

GTGTTGTTTT TCCTCAAGCT AAGGATAGTT GGGTTAATAC AACGACATTT TTGTTAGGTG 10680

AGGCAAAGGC AATTTTCGGT GACTATAGTT CCATTATCTC ATCTTTAGTA GTATTTATTA 10740

ATGTGTTTGT TGTATGTGTG CTATCAACAC TTTTAGTAAG AGTATCAAAA AATATTTTTT 10800

ATCCCGCAAT ATTCGTTATG ATTCAAACTA TACCATTTGC AAACAATTTG ACTGACTTAT 10860

TATATGGTAG ATTTATTTTT GGTTTTTTAG TCTTTATGTT ATTTCCAGTG CTTATATGTT 10920

The termination of the initial orf10 of orf11

TTTTGTGTTA TGGAAAATTA AAAAATG ATG AA TAGAAAAG TGCTTATTAT AATGGTTACC 10980

TATAATCCTG ATTGGAGTAA GGTAATTAAT AAAGTGAAGT CTTTTTGTGT TGCAGGAACA 11040

GTTTTTGTTT CTGATAATAG CGATTCTGAT TCCTCATCAT TGATAAACTA TGAAAATGTT 11100

ATATACAGAT ATAATCATGG TAATGTAGGC ATAGCAAAAG CTCAGAATTT AGGTATTGAA 11160

TATGCGATGC AGTATAATTA TGAGTTTGTA GCTTTTGTTG ATGATGATAG TAATCTGAGC 11220

GGGGGTAAAA TTACACAGTT AACAAATAAT TTATGTAAAT TAGGCAGTCT TGGTGAAAAT 11280

ATTGCAGCAT TTTGCGCTTA CCCTAGTGAA AGAGGTGGTT TTGACAAGAC AAAAAAAACT 11340

TCGCTTGGTA GACAATTATT GTTAACTAAG AATTTAATGA GTTCAGGATC TTTAACAAAG 11400

GTTGATGTAT TTAAAAAGAT AGGTTTATTT GATGAAGATT TATTTATTGA CTATGTGGAT 11460

TATGAATGGG GCTGGAGAGC ACTGGAAAAA AATTTTTTAA TCGTAATTGA TACATCAGTA 11520

GAATTTGAAC ATTCACTTGG TCAGGGGCGC TCAAAAATTG GATTAGGGAT CCCCTCTCCT 11580

ATCCGACATT TTTATCAAAC TAGAAATCTT TTATGGATAT CAAGGCTAAA TTATGTACCA 11640

ATATATTGGA AACTTAAACA GTTTTTACTT CTTCCAATTC GCTATTTCTA TTTTGGTTTT 11700

TTTTATAATC ACTCTAAATT ACGGCGAACC CATTTTCTCA AAGGGTTTGC CGCAGGGTTA 11760

The termination of the termination orf12 of orf11

AAATTTAAAT CA TAGTT TTA TGAGATAATA TCATCAACGG GACGAATAAT TTTAGCTGGA 11820

TTGCCAGCAA CAATTACATT ATCCGGAATA TCCTTCGTAA CTACGGAGCC TGCGCCAATA 11880

ATTGAATTTT CACCGATGAT AATACCCGGC AAAATTGTTG CGTTCGCACC AATAGAAGCA 11940

TTCTTTTTAA TATGTATCGT TGGATAATAA TCTGGGTATT TTTTAGATCT TGGATAAATA 12000

TCATTTGTAA ATGTAACATT TGGACCTATA AAAACATTAT CTTCAATTAT GACTCCATCC 12060

CATATATAGA CTCCAGATTT TATAGTGACA TTATCGCCAA TAATAACTTT ATTTTCGATT 12120

AAAGTATGTG CGCAAATATT GCAGTTATTA CCAATTACAG CGCCTTCAAA AATAATGCTG 12180

TATTGCCAAA TGGTTGTATT TAGACCGATT TTTACAGTTT TTACATCACT TAATGGATGA 12240

Orf12's is initial

ATTTT CATAA ATTCTACCCT TCATTTTTAA AACAATAATA TTGTAAATGA TATCATTTAG 12300

TATTAATGAT ATCATTAGAC GCCTGAATAG TAAAAAGTTT TCCCTTTGGT TATAAAGTCA 12360

AAAGTTAAGT AGCGTAAATC GGTATTCTGA CAGGAGTAAA ACATGTCAAA GCAACAGATC 12420

GGCGTCGTCG GTATGGCAGT GATGGGGCGC AACCTTGCGC TCAACATCGA AAGCCGTGGT 12480

TATACCGTCT CTATTTTCAA CCGTTCCCGT GAAAAGACGG AAGAAGTGAT TGCCGAAAAT 12540

CCAGGCAAGA AACTGGTTCC TTACTATACG GTGAAAGAGT TTGTTGAATC TCTGGAAACG 12600

CCTCGTCGCA TCCTGTTAAT GGTGAAAGCA GGTGCAGGCA CGGATGCTGC TATTGATTCC 12660

CTCAAGCCGT ACCTCGATAA AGGTGACATC ATCATTGATG GTGGTAACAC CTTCTTCCAG 12720

GACACCATTC GTCGTAACCG TGAGCTTTCT TCAGAAGGCT TTAACTTCAT CGGTACCGGT 12780

GTTTCCGGCG GAGAAGAGGG GGCGCTGAAA GGGCCGTCCA TCATGCCTGG TGGCCAGAAA 12840

GAAGCCTATG AACTGGTTGC GCCGATCCTG ACCAAAATCG CCGCAGTAGC TGAAGACGGT 12900

GAGCCATGCG TTACCTATAT TGGTGCCGAT GGCGCAGGTC ACTATGTGAA GATGGTTCAC 12960

AACGGTATTG AATACGGAGA TATGCAACTG ATTGCTGAAG CCTACTCTCT GCTTAAAGGT 13020

GGCCTGAACC TCACCAACGA AGAACTGGCG CAGACGTTTA CCGAGTGGAA TAACGGTGAA 13080

CTGAGCAGCT ACCTGATCGA CATCACCAAA GACATCTTCA CTAAAAAAGA TGAAGACGGT 13140

AACTACCTGG TTGATGTGAT CCTGGATGAA GCGGCTAACA AAGGTACCGG TAAATGGACC 13200

AGCCAGAGCG CGCTTGATCT CGGCGAACCG CTGTCGCTGA TTACCGAGTC TGTGTTTGCA 13260

CGATACATCT CTTCTCTGAA AGAGCAGCGT GTTGCCGCAT CTAAAGTTCT TTCTGGTCCG 13320

CAAGCGCAGC CTGCTGGCGA CAAAGGTGAA TTCATCGAAA AAGTTCGCCG TGCGCTGTAT 13380

CTGGGTAAAA TCGTTTCTTA CGCTCAGGGC TTCTCTCAGC TACGCGCTGC GTCTGAAGAG 13440

TACAACTGGG ATCTGAACTA CGGTGAAATC GCGAAGATTT TCCGTGCTGG CTGCATCATC 13500

CGTGCGCAGT TCCTGCAGAA AATCACCGAT GCTTATGCCG AAAATCCGCA GATCGCTAAC 13560

CTGTTGCTGG CTCCTTACTT CAAGCAAATT GCCGATGACT ACCAGCAGGC GCTGCGCGAT 13620

GTCGTCGCTT ACGCAGTACA GAACGGTATC CCGGTGCCGA CCTTCGCCGC TGCTGTTGCC 13680

TATTACGACA GCTACCGCGC CGCTGTTCTG CCTGCGAACC TAATTCAGGC ACAGCGCGAC 13740

TAA 13743

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 (2)

1, a kind of primer of the O-antigen-specific to intestinal bacteria O71 type is right, it is characterized in that described primer is to being selected from: the forward primer of 10332 to 10350 bases among the forward primer of 7139 to 7157 bases among the forward primer of 7515 to 7533 bases among the SEQ ID NO:1 and the reverse primer of 7839 to 7859 bases, the SEQ ID NO:1 and the reverse primer of 8051 to 8069 bases, the SEQ ID NO:1 and the reverse primer of 10581 to 10609 bases or the forward primer of 9981 to 9999 bases among the SEQ ID NO:1 and the reverse primer of 10670 to 10688 bases.

2, the described primer of claim 1 is to the application in the intestinal bacteria O71 type in testing environment.