CN1563059A

CN1563059A - Nucleotide peculiar to 0-antigen of 0163 type bacillus coli

Info

Publication number: CN1563059A
Application number: CN 200410019044
Authority: CN
Inventors: 王磊; 杨静华; 冯露
Original assignee: Tianjin Biochip Corp
Current assignee: Tianjin Biochip Corp
Priority date: 2004-04-19
Filing date: 2004-04-19
Publication date: 2005-01-12
Anticipated expiration: 2024-04-19
Also published as: CN1257279C

Abstract

The invention provides a nucleotide which is specific to O-antigen of Escherichia coli O163, it is a nucleotide complete sequence of gene cluster for controlling synthesis of O-antigenin Escherichia coli O163, as the separated nucleotide shown by SEQ ID No:1, its total length has 15433 bases; or the nucleotide of SEQ ID No:1, which has one or several inserted, deleted or substituted bases, and retains the function of the described separated nucleotide at the same time; it also includes the oligonucleotide of the oligosaccharide unit treatment gene (including wzx gene or gene whose function is similar to that of wzx and wzy gene or gene whose function is similar to that of wzy) originated from O-antigen gene cluster of Escherichia coli O163. Said invention utilizes PCR to verify that the oligonucleotide has high specificity to O-antigen of Escherichia coli O163, and said invention also discloses the method for detecting and identifying Escherichia coli O163 in human body.

Description

Nucleotide to the O-antigen-specific of intestinal bacteria O163

Technical field

The present invention relates to the complete nucleotide sequence of control O-antigen synthetic gene cluster among the intestinal bacteria O163 (Escherichia coli O163), particularly relate among the intestinal bacteria O163 oligonucleotide in the control O-antigen synthetic gene cluster, can utilize these the oligonucleotide of the O-antigen-specific intestinal bacteria O163 in human body and the environment and identify O-antigen in these pathogenic bacterium quickly and accurately.

Background technology

The lipopolysaccharides that is positioned at the intestinal bacteria surface is the morbific inducements of intestinal bacteria, and O-antigen is lipopolysaccharides outermost layer structure, is the target of immune system recognition and the site of phage absorption.The antigenic disappearance of O-can cause the serum sensitivity of many pathogenic agent, perhaps seriously undermines the virulence [Frank etal (1987) " The function of antibody and complement in the lysis ofbacteria " .Rev Infect Dis 177:1750-1753.Pluschke G et al " Role of thecapsule and the O-antigen in resistance of O18:K1 Escherichia coli tocomplement-mediated king " .J Bacteriol 42:907-913] of pathogenic agent.Intestinal bacteria are kinds, and the bacterial strain in planting is generally identified by O-antigen and H-antigen (sometimes by K-antigen).Wherein O-antigen has the height diversity, intestinal bacteria have 166 kinds of different O-antigens, the antigenic variation of O-may be colibacillary origin and keep its multifarious major cause [Reeves, P.R (1992) " Variation in antigens; niche specific selection and bacterialpopulations " .FEMS Microbiol.Lett, 100:509-516].

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 o-antigen transhipment enzyme again, then aggregate into polysaccharide by polysaccharase, be connected to again and form lipopolysaccharide molecule [Whitfield, C. (1995) " Biosynthesis of lipopolysaccharide Oantigens " .Trends in Microbiology.3:178-185 on the glycolipid molecule; Schnaitman, C.A.andJ.D.Klena. (1993) " Genetics of lipopolysaccharide biosynthesis inentericbacteria " .Microbiological Reviews, 57 (3): 655-682].Coding is responsible for the generally adjacent arrangement on karyomit(e) of gene of all enzyme molecules of O-antigen synthetic, form a gene cluster [Reeves, P.R., et al. (1996) " Bacterial polysaccharide synthesis and genenomenclature " Trends in Microbiology, 4:495-503].In intestinal bacteria, Shigellae and Salmonellas, O-antigen gene [Lei Wang.et al (2001) " Sequence analysis of four Shigella boydii O-antigen loci:implicationfor Escherichia coli and Shigella relationships " .Infection andImmunity, 11:6923-6930 bunch between galF and gnd gene; Lei Wang and Peter Reeves (2000) " The Escherichiacoli O111 and Salmonella enterica O35 gene clusters:gene clusters encodingthe same colitose-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 o-antigen transhipment enzyme gene and pol gene, and they transfer to the bacterium inner membrance outside with oligosaccharide unit, and repolymerization becomes polysaccharide.Glycosyltransferase gene and oligosaccharide unit treatment gene only are present in the gene cluster of carrying these genes.The difference of monose in the O-antigen, between monose between the difference of link button and the oligosaccharide unit difference of link button constituted the antigenic diversity of O-, and the composition of monose, the link button between monose and the link button between the oligosaccharide unit are by the Gene Handling in the O-antigen gene bunch, so the O-antigen gene bunch has determined O-antigenic synthetic, has also determined the antigenic diversity of O-.

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

Summary of the invention

The Nucleotide that the purpose of this invention is to provide a kind of O-antigen-specific to intestinal bacteria O163.It is the Nucleotide in the O-antigen gene bunch of intestinal bacteria O163, is the special Nucleotide that comes from o-antigen transhipment enzyme gene, pol gene and glycosyltransferase gene.

An object of the present invention is to provide the full length nucleotide sequence of the O-antigen gene bunch of intestinal bacteria O163.

A time purpose of the present invention has provided the gene of the O-antigen gene bunch that constitutes intestinal bacteria O163: transhipment enzyme gene is the wzx gene or with wzx the gene of identity function is arranged; Pol gene is the wzy gene or with wzy the gene of identity function is arranged; Glycosyltransferase gene comprises orf5, orf6, orf10, orf11 gene; Sugar synthesis path gene comprises manC, manB, fnlA, qnlA, qnlB.Their zero positions in O-antigen gene bunch and final position and nucleotide sequence all are listed in the table 4.

Another purpose of the present invention has provided oligonucleotide, and they come from the gene of coding transhipment enzyme in the O-antigen gene bunch of intestinal bacteria O163 respectively, comprises the wzx gene or with wzx the gene of identity function is arranged; Come from the gene of coding polysaccharase, comprise the wzy gene or the gene of identity function is arranged with wzy; Come from the gene of encoding glycosyl transferring enzyme, comprise orf5, orf6, orf10 gene.They are the oligonucleotide in the said gene, and length is at 10-20nt; They are special to the O-antigen of intestinal bacteria O163; The oligonucleotide of the gene of especially listing in the table 1 that comes from coding transhipment enzyme and the gene of polysaccharase, they are high specials to the O-antigen of intestinal bacteria O163, and these oligonucleotide are also reconfigurable, and the oligonucleotide after the combination also is a high special to the O-antigen of intestinal bacteria O163.

The above-mentioned oligonucleotide that another 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 detect O-antigen and detection and identification of escherichia coli O163 with identification of escherichia coli O163 by these methods.

A further object of the present invention has provided the method for complete sequence of the O-antigen gene bunch of separating Escherichia coli O163.Can obtain the complete sequence of the O-antigen gene bunch of other bacteriums according to present method operation, the complete sequence of the gene cluster of the bacterium of other polysaccharide antigens that also can obtain to encode.

The objective of the invention is to realize by following technical scheme.

The present invention is characterized in that to the Nucleotide of the O-antigen-specific of intestinal bacteria O163 it is the isolating Nucleotide shown in SEQ ID NO:1,15433 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 O163, it is characterized in that, it comprises 11 genomic constitutions of called after manC, manB, wzx, wzy, orf5, orf6, fnlA, qnlA, qnlB, orf10, orf11, all between galF gene and gnd gene.

The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O163 is characterized in that the gene that has high degree of specificity in the described gene comprises: transhipment enzyme gene 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; Glycosyltransferase gene comprises orf5, orf6, orf10 gene.Wherein said transhipment enzyme gene is the Nucleotide of 4349 to 5602 bases among the SEQID NO:1; Described pol gene is the Nucleotide of 5599 to 6834 bases among the SEQ ID NO:1; Described orf5 gene is the Nucleotide of 6852 to 7586 bases among the SEQ ID NO:1; The orf6 gene is the Nucleotide of 7880 to 8983 bases among the SEQ ID NO:1; The Orf10 gene is the Nucleotide of 12196 to 13389 bases among the SEQ ID NO:1.

The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O163 is characterized in that it comes from the oligonucleotide of described wzx gene or wzy gene or glycosyltransferase gene; And their mixing or their reorganization.

The Nucleotide of aforesaid O-antigen high special to intestinal bacteria O163, it is characterized in that, the oligonucleotide of the described wzx of coming from gene is to being: the Nucleotide of 4500 to 4518 bases among the SEQ ID NO:1 and the Nucleotide of 5425 to 5442 bases, the Nucleotide of 5100 to 5117 bases among the SEQ ID NO:1 and the Nucleotide of 5503 to 5520 bases; The oligonucleotide of the described wzy of coming from gene is to being: the Nucleotide of 6102 to 6119 bases among the SEQ ID NO:1 and the Nucleotide of 6738 to 6755 bases, the Nucleotide of 6223 to 6240 bases among the SEQ ID NO:1 and the Nucleotide of 6784 to 6801 bases.

The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O163 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 O163 is providing the O-antigen of expressing intestinal bacteria O163 by inserting to express, and the application in the preparation bacterial vaccine.

The application of the Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O163 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, bacterial detection as probe as primer.

The separation method of the Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O163 is characterized in that, comprises the steps:

(1) genomic extraction: in substratum, cultivate intestinal bacteria O163, centrifugal collecting cell; The genomic dna that obtains detects by agarose gel electrophoresis;

(2) by the O-antigen gene among the pcr amplification intestinal bacteria O163 bunch: with the genome of intestinal bacteria O163 is that template is passed through its O-antigen gene of Long pcr amplification bunch; The PCR product detects the PCR product with agarose gel electrophoresis size and specificity thereof will be obtained; Merge long PCR product, and with DNA purification kit purified pcr product;

(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;

(4) to the cloning and sequencing in the library: from the library, select insert fragment more than 1000bp with laboratory automatic dna sequencer commonly used to unidirectional order-checking of insertion fragment among the clone, make sequence reach 80% fraction of coverage, thereby obtain all sequences of O-antigen gene bunch.

(5) splicing of nucleotide sequence and analysis: with information biology software splicing with edit all sequences, thereby obtain the Nucleotide full length sequence of the O-antigen gene bunch of intestinal bacteria O163;

(6) specific gene screening: at wzx and wzy gene design primer in the O-antigen gene of intestinal bacteria O163 bunch; Respectively design two pairs of primers in each gene, every pair of primer is distributed in the corresponding gene different local to guarantee its specificity; Is that template is carried out PCR with these primers with the intestinal bacteria and the 43 strain Shigellae genomes of 166 kinds of serotypes, determines that wzx, wzy gene pairs intestinal bacteria O163 and O-antigen thereof all are high specials;

(7) detection of primer sensitivity: cultivate intestinal bacteria O163, after the bacterial count respectively with 5 * 10 ³, 5 * 10 ², 5 * 10 ¹5 and 0 viable bacteria join in a certain amount of certain thing to be detected, sneak into the thing to be detected of bacterium and use sample as detecting, sample is added the LB substratum, getting the LB substratum that some and sample mix cross filters, filtered liquid is cultivated, carried out the PCR reaction as pcr template with oligonucleotide after the peek milliliter is handled from cultured bacterium liquid, detect its sensitivity intestinal bacteria O163.

(1) genomic extraction: 37 ℃ of incubated overnight intestinal bacteria O163 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, use isopyknic phenol again: chloroform: twice of the solution extracting of primary isoamyl alcohol (25: 24: 1), 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 70% ethanol with glass yarn, at last DNA is resuspended among the 30ul TE, genomic dna detects by 0.4% agarose gel electrophoresis;

(2) by the O-antigen gene among the pcr amplification intestinal bacteria O163 bunch: with the genome of intestinal bacteria O163 is that template is passed through its O-antigen gene of Long pcr amplification bunch; At first according to the galF gene design upstream primer (5 '-ATT GTG GCT GCA GGG ATCAAA GAA ATC-3 ') that often is found in O-antigen gene bunch upstream, again according to the gnd gene design downstream primer (5 '-TAGTCG CGC TGN GCC TGG ATT AAG TTC GC-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 is as follows: 94 ℃ of pre-sex change 2 minutes, 94 ℃ of sex change were 10 seconds then, 60 ℃ of annealing 30 seconds, 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 6 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; Enzyme is cut the dna fragmentation size is concentrated between the 1kb-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) solution 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 ℃ were reacted 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) solution extracting and the extracting of equal-volume ether with 3 * 10 of Promega company ^-3The pGEM-T-Easy carrier connect 24 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 3MNaAc (pH5.2) of 1/10 volume and 2 times of volumes to be connected mixture at last, wash precipitation with 70% ethanol again, be dissolved in after the drying and obtain connecting product in the 30ul water; Preparation method with the electric transformed competence colibacillus cell of Bio-Rad company prepares the competence e.colidh5, get after 2-3ul connects product and 50ul competence bacillus coli DH 5 alpha mixes, forward in the electric shock cup of 0.2cm of Bio-Rad company and shock by electricity, voltage is 2.5 kilovolts, time is 5.0 milliseconds-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, 37 ℃ of incubated overnight on the LB solid medium of X-Gal and IPTG, 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, extract plasmid and cut the segmental size of evaluation insertion wherein with the EcoRI enzyme from each clone simultaneously, the white that obtains clone group has constituted the O-antigen gene bunch library of intestinal bacteria O163;

(4) to the cloning and sequencing in the library: from the library, select insert 100 clones of fragment more than 1000bp by Shanghai biotechnology company limited with ABI377 type automatic dna sequencer to unidirectional order-checking of insertion fragment in cloning, make sequence reach 80% fraction of coverage, carry out backward sequencing by the sequence that will interrelate again and survey logically obtaining remaining 20% sequence, thereby obtain 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 ResearchCouncil) Molecular Biology Lab and edit all sequences, thereby obtain the Nucleotide full length sequence of the O-antigen gene bunch of intestinal bacteria O163, the quality of sequence is mainly guaranteed by two aspects: 1) genome of intestinal bacteria O163 is done 6 Long PCR reactions, mix these products then to produce the library.2), guarantee high-quality fraction of coverage more than 3 to each base; Behind the nucleotide sequence of the O-antigen gene that obtains intestinal bacteria O163 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 O163 at last;

(6) specific gene screening: at wzx and wzy gene design primer in the O-antigen gene of intestinal bacteria O163 bunch; Respectively design two pairs of primers in each gene, every pair of primer is distributed in the corresponding gene different local to guarantee its specificity; Is that template is carried out PCR with these primers with the intestinal bacteria and the 43 strain Shigellae genomes of 166 kinds of serotypes, all primers obtain positive findings in intestinal bacteria O163, the correct band of any size does not increase in other groups, just, do not obtaining any PCR product band in the array mostly, though obtain PCR product band in the minority group, its size does not meet the expection size, so wzx, wzy gene pairs intestinal bacteria O163 and O-antigen thereof all are high specials.

(7) detection of primer sensitivity: the frozen bacterium liquid of intestinal bacteria O163 is inoculated in the triangular flask of LB substratum, 30 ℃ of-40 ℃ of cultivations, 180 to 250 rev/mins, cultivate a few hours to saturated, get cultured bacterium liquid dilution, get dilution bacterium liquid coating LB agar plate, 30 ℃ to 40 ℃, cultivate a few hours counting, calculate viable bacteria concentration in the stoste; In being the live pig meat stuffing of 20g, 5 parts of weight mix 5 * 10 respectively ³, 5 * 10 ², 5 * 10 ¹, 5 and 0 viable bacteria stir, and add the LB substratum, filter, and filtered liquid 180 to 250 rev/mins, is cultivated a few hours in 30 ℃ of-40 ℃ of cultivations; Peek ml is in 6 from cultured bacterium liquid, and centrifugal several minutes of 000g removes supernatant, adds the MQ ultrapure water and blows precipitation and mixing open, puts into 100 ℃ of boiling water and boils several minutes, and lysate is in 12, and centrifugal several minutes of 000g gets supernatant as pcr template; Right with 4 pairs of oligonucleotide, the Nucleotide of 4500 to 4518 bases among the SEQ ID NO:1 and the Nucleotide of 5425 to 5442 bases; The Nucleotide of 5100 to 5117 bases among the SEQ ID NO:1 and the Nucleotide of 5503 to 5520 bases; The Nucleotide of 6102 to 6119 bases among the SEQ ID NO:1 and the Nucleotide of 6738 to 6755 bases; The Nucleotide of 6223 to 6240 bases among the SEQ ID NO:1 and the Nucleotide of 6784 to 6801 bases carry out the PCR reaction, and the PCR reaction system is as follows: MQ:15.7 μ l, Mg ²⁺: 2.5 μ l, Buffer:2.5 μ l, dNTP:1 μ l, Taq enzyme: 0.3 μ l, P1:1 μ l, P2:1 μ l, template DNA: 1 μ l.The PCR reaction conditions is: 95 ℃: 5 ', 95 ℃: 30 ", 56 ℃: 45 ", 72 ℃: 1 ', 72 ℃: 5 ', totally 30 circulations; Reaction is got 10 μ l reaction product electrophoresis after finishing, if the amplified band that conforms to the expection size is arranged, then the result is positive, if do not have, then the result is negative; Participated in 5 * 10 ³, 5 * 10 ², 5 * 10 ¹And every part of pork filling of 5 viable bacterias all obtains positive findings in the PCR of 4 pairs of primers reaction; The pork filling that participates in 0 viable bacteria obtains negative findings in the PCR of 4 pairs of primers reaction; Illustrate that these 4 pairs of primers are 0.25 bacterium/g to the detection sensitivity of the intestinal bacteria O163 in the pork filling when using aforesaid method.

Just, first aspect of the present invention provides the full length nucleotide sequence of the O-antigen gene bunch of intestinal bacteria O163, its complete sequence shown in SEQ ID NO:1,15433 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 O163 by method of the present invention, as described in Table 3, its 11 genes are all between galF gene and gnd gene.

Second aspect of the present invention provides the gene in the O-antigen gene bunch of intestinal bacteria O163, promptly transports enzyme gene (wzx gene or the gene of identity function arranged with wzx); Pol gene (wzy gene or the gene of identity function arranged with wzy); Glycosyltransferase gene comprises orf5, orf6, orf10 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 O-antigen 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 special, and the o-antigen that the present invention relates to transhipment enzyme gene, pol gene and glycosyltransferase gene are special to the O-antigen of intestinal bacteria O163.

The 3rd aspect of the present invention, wzy gene in the O-antigen gene bunch that comes from intestinal bacteria O163 is provided or the gene of identity function and wzx gene is arranged or with wzx the oligonucleotide of gene of identity function and the oligonucleotide that glycosyltransferase gene comprises orf5, orf6, orf10 gene are arranged with wzy, they are any one section oligonucleotide in these genes.But, be to list in the wzx gene in the O-antigen gene bunch that comes from intestinal bacteria O163 in the table 1 or the gene, wzy gene of identity function arranged or have the oligonucleotide of gene of identity function right preferentially with wzy with wzx 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 being to obtain expecting the product of size in the pcr amplification that carries out of template with intestinal bacteria O163 only, and are all not obtain expecting the product of size in the pcr amplification that carries out of template other bacterium listed with table 2.In more detail, with these oligonucleotide to being that PCR that primer is done is reflected at and does not all obtain spawn in most of bacteriums, so, can determine these primers promptly the listed oligonucleotide of table 1 be high special to intestinal bacteria O163 and their O-antigen.

The separation method of the Nucleotide of described O-antigen-specific to intestinal bacteria O163 comprises the steps: 1) genomic extraction; 2) the O-antigen gene among the pcr amplification intestinal bacteria O163 bunch; 3) structure in O-antigen gene bunch library; 4) to the cloning and sequencing in the library; 5) splicing of nucleotide sequence and analysis finally obtain the structure of O-antigen gene bunch; 6) screening of specific gene; 7) primer sensitivity detects.

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

As described herein, " oligonucleotide " mainly is meant gene, the gene of coding polysaccharase and one section nucleic acid molecule in the encoding glycosyl transferase gene of the coding transhipment enzyme that derives from the O-antigen gene bunch, they can change on length, generally change in 10 to 20 Nucleotide scopes.Especially come from wzx gene (nucleotide position is 4349 to 5602 bases from SEQ ID NO:1), the oligonucleotide in the wzy gene (nucleotide position is 5599 to 6834 bases from SEQ ID NO:1) all is a high special to intestinal bacteria O163.

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 transhipment enzyme gene, comprise the wzx gene or with wzx the gene of identity function are arranged; Come from pol gene, comprise the wzy gene or the gene of identity function is arranged with wzy; Come from glycosyltransferase gene, comprise orf5, orf6, orf10 gene.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 transhipment enzyme gene, come from pol gene and come from the combination of the oligonucleotide in the glycosyltransferase gene.

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) coding transhipment enzyme gene, comprise the wzx gene or (ii) the encode gene of polysaccharase of the gene of identity function is arranged, comprise the wzy gene or the gene of identity function is arranged with wzy with wzx.(iii) the encoding glycosyl transferase gene comprises orf5, orf6, orf10 gene.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 O163.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.

The present inventor 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 and comes from that coding transhipment enzyme gene comprises the wzx gene or have the gene of gene, the coding polysaccharase of identity function to comprise the wzy gene with wzx or with wzy the oligonucleotide of gene of identity function and the oligonucleotide that the encoding glycosyl transferase gene comprises orf5, orf6, orf10 gene are arranged.This cover oligonucleotide is special to the O-antigen of a special bacterium, and this special bacterium O-antigen is expressed by intestinal bacteria O163.

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 coding transhipment enzyme, comprise the wzx gene or (ii) the encode gene of polysaccharase of the gene of identity function is arranged with wzx, the gene that comprises the wzy gene or identity function arranged with wzy is the encoding glycosyl transferase gene (iii), comprises orf5, orf6, orf10 gene.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 O163.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 and be used to detect the many to oligonucleotide of the inventive method, many here is that the gene that comes from coding transhipment enzyme comprises the wzx gene or with wzx the gene of identity function arranged to oligonucleotide; The gene that comes from the coding polysaccharase comprises the wzy gene or with wzy the gene of identity function is arranged; The gene that comes from the encoding glycosyl transferring enzyme comprises orf5, orf6, orf10 gene.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 coding transhipment enzyme, comprise the wzx gene or (ii) the encode gene of polysaccharase of the gene of identity function is arranged with wzx, the gene that comprises the wzy gene or identity function arranged with wzy is the encoding glycosyl transferase gene (iii), comprises orf5, orf6, orf10 gene.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 O163.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 the gene of identity function and wzy gene are arranged or have the gene of identity function and glycosyltransferase gene to comprise on the sequence of orf5, orf6, orf10 gene with wzy with wzx 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-.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 O163 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 O163 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 O163 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, use isopyknic phenol again: chloroform: primary isoamyl alcohol (25: 24: 1) solution 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, at last DNA is resuspended among the 30ul TE.Genomic dna detects by 0.4% agarose gel electrophoresis.

Embodiment 2: by the O-antigen gene among the pcr amplification intestinal bacteria O163 bunch

With the genome of intestinal bacteria O163 is that template is passed through its O-antigen gene of Long pcr amplification bunch.At first according to the galF gene design upstream primer (5 '-ATTGTG GCT GCA GGG ATC AAA GAA ATC-3 ') that often is found in O-antigen gene bunch upstream, again according to the gnd gene design downstream primer (5 '-TAG TCG CGC TGN GCC TGG ATT AAG TTC GC-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, 61 ℃ of annealing 30 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 6 pipe long PCR products, and with the Wizard PCR Preps purification kit purified pcr product of Promega company.

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

At first be the acquisition that connects product:

Make up O-antigen gene bunch library with the Novagen DNaseI shot gun method that is modified.Reaction system is a 300ng PCR purified product, 0.9ul 0.1M MnCl ₂, the DNaseI of the 1mg/ml of 1ul dilution in 1: 2000, reaction is carried out at room temperature.Enzyme is cut the dna fragmentation size is concentrated between the 1kb-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 ℃ were reacted 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 24 hours in 16 ℃, cumulative volume is 90ul.10 * the buffer of 9ul and the T4DNA ligase enzyme of 25 units are wherein arranged.Use the dehydrated alcohol precipitation of the 3M NaAc (pH5.2) of 1/10 volume and 2 times of volumes to be connected mixture at last, wash precipitation with 70% ethanol again, be dissolved in after the drying and obtain connecting product in the 30ul water.

Next is the preparation of competent cell:

The method that provides with reference to Bio-Rad company prepares the competent cell bacillus coli DH 5 alpha.Get a ring bacillus coli DH 5 alpha list bacterium colony in the LB of 5ml substratum, 180rpm cultivated after 10 hours, got in the LB substratum that the 2ml culture is transferred to 200ml, and 37 ℃ of 250rpm thermal agitations are cultivated OD600 about 0.5, ice bath cooling was 20 minutes then, in centrifugal 15 minutes of 4 ℃ of 4000rpm.Confide all supernatant 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 alpha mixes, forward in the electric shock cup of 0.2cm of Bio-Rad company and shock by electricity, voltage is 2.5 kilovolts, and the time is 5.0 milliseconds-6.0 milliseconds.The SOC substratum that adds 1ml after the electric shock immediately in cup makes the bacterium recovery.Immediately bacterium is coated in 37 ℃ of inversion incubated overnight on the LB solid medium that contains penbritin, X-Gal and IPTG then, obtains blue white bacterium colony next day.With the white colony that obtains promptly the white clone forward on the LB solid medium that contains penbritin and cultivate, from each clone, extract plasmid and cut the segmental size of evaluation insertion wherein simultaneously, obtain the O-antigen gene bunch library that white clone group has constituted intestinal bacteria O163 with the EcoRI enzyme.

Embodiment 4: to the cloning and sequencing in the library.

From the library, select insert 100 clones of fragment more than 1000bp by Shanghai biotechnology company limited with ABI377 type automatic dna sequencer to unidirectional order-checking of insertion fragment in cloning, make sequence reach 80% fraction of coverage.Residue 20% sequence is surveyed logical obtaining by backward sequencing and with some sequence again, obtains all sequences of O-antigen gene bunch at last.

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 O163 obtained.The quality of sequence is mainly guaranteed by two aspects: 1) genome of intestinal bacteria O163 is done 6 LongPCR reactions, mix these products then to produce the library.2), guarantee high-quality fraction of coverage more than 3 to each base.Behind the nucleotide sequence of the O-antigen gene that obtains intestinal bacteria O163 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 Clustral W software, obtain the structure of the O-antigen gene bunch of intestinal bacteria O163 at last, as shown in table 3.

By retrieving and relatively, finding that the mannose-1-phosphate guanyltransferase of orf1 and Shigella flexneri 2a str.301 has 78% homogeny, 88% similarity in 478 amino acid.Mannose-1-phosphate guanyltransferase is by the manC genes encoding, and the homogeny of height shows that orf1 also is the manC gene, called after manC.The phosphomannomutase of Orf2 and Escherichia coli O157:H7 has 85% homogeny in 454 amino acid, 93% similarity.Phosphomannomutase is by the manB genes encoding, and the homogeny of height shows that orf2 also is the manB gene, called after manB.The O-antigen transhipment enzyme Wzx of Orf3 and Escherichia coli has 24% homogeny, 46% similarity in 412 amino acid whose sequences.And find that by people's such as Eisenberg algorithm orf5 has 12 potential transmembrane domains, the proteic aminoterminal of Wzx has about 40 amino acid whose conservative motifs, is the wzx gene so can determine orf3, called after wzx.The O-antigen polysaccharase of Orf5 and Yersinia pseudotuberculosis has 31% homogeny, 55% similarity in 402 amino acid whose sequences.And algorithm [Eisenberg by people such as Eisenberg, D, Schwarz, E.etal (1984) .Analysis of membrane and surfaceprotein sequences with the hydrophobic moment plot.J.Mol.Biol.179:125-142] find that orf5 has 9 potential transmembrane domains, it has similar secondary structure to many Wzy albumen, a big loop is arranged, feature with typical O-antigen polysaccharase, so determine that orf5 is the wzy gene, called after wzy.The glycosyltransferase of Orf5 and Thermoanaerobacter tengcongensis has 24% homogeny in 153 amino acid, 49% similarity, in genbank, seek conservative functional domain, find that the Evalue of the conservative functional domain PF00535 of orf5 and glycosyltransferase family is 2 * e ^-5, infer that orf5 also is a glycosyltransferase, with the temporary called after orf5 of orf5.The glycosyltransferase of Orf6 and Nostoc sp.PCC 7120 has 26% homogeny in 289 amino acid, 47% similarity, in genbank, seek conservative functional domain, find that the E value of the conservative functional domain PF00534 of Orf6 and glycosyltransferase family 1 is 3.7 * e ^-7, infer that orf6 also is a glycosyltransferase, temporarily called after orf6.The UDP-D-GlcNAc 4 of Orf7 and Vibrio cholerae O37,6-dehydratase/5-epimerase/3-epimerase has 72% homogeny in 343 amino acid, 85% similarity.UDP-D-GlcNAc 4, and 6-dehydratase/5-epimerase/3-epimerase is by the fnlA genes encoding, and the homogeny of height shows that orf1 also is the fnlA gene, called after fnlA.The dTDP-4-dehydrorhamnosereductase of Orf8 and Vibrio cholerae O37 has 54% homogeny in 285 amino acid, 68% similarity.DTDP-4-dehydrorhamnose reductase is by the qnlA genes encoding, and the homogeny of height shows that orf1 also is the qnlA gene, called after qnlA.The UDP-N-acetylglucosamine 2-epimerase of Orf9 and Vibrio cholerae O37 has 61% homogeny in 376 amino acid, 79% similarity.UDP-N-acetylglu-cosamine 2-epimerase is by the qnlB genes encoding, and the homogeny of height shows that orf1 also is the qnlB gene, called after qnlB.The glycosyltransferase of Orf10 and Escherichia coli has 53% homogeny in 379 amino acid, 68% similarity, in genbank, seek conservative functional domain, find that the Evalue of the conservative functional domain PF00534 of orf9 and glycosyltransferase family 1 is 3.5 * e ^-5, therefore infer that orf10 also is a glycosyltransferase, temporarily called after orf10.The WbuC protein of Orf11 and Escherichia coli has 39% homogeny in 127 amino acid whose sequences, 62% similarity, called after orf11 temporarily.

Embodiment 6: the screening of specific gene

At wzx, wzy gene design primer in the O-antigen gene of intestinal bacteria O163 bunch, the position of these genes in nucleotide sequence sees Table 1.

Transhipment enzyme gene, pol gene and their function corresponding and the size of the O antigen gene bunch of intestinal bacteria O163 in table 1, have been listed.In each gene, we have respectively designed two pairs of primers, and the difference that every pair of primer is distributed in the corresponding gene is local to guarantee its specificity.In table, also listed position and the size of each primer in SEQ ID NO:1.Is that template carry out PCR with listed corresponding annealing temperature in the table with the genomes of all bacterium in the table 2 with every pair of primer, has obtained corresponding PCR product, and its size is also listed in the table.

Mdh (malate dehydrogenase) gene is to be present in all colibacillary genomes and a gene of high conservative, so we according to the mdh gene design primer (5 '-TTC ATC CTA AACTCC TTA TT-3 ') and (5 '-TAA TCG CAG GGG AAA GCA GG-3 '), extract genome then from the intestinal bacteria of 166 kinds of serotypes, method as previously mentioned.With this to primer from the colibacillary genome of 166 kinds of serotypes PCR with identification of escherichia coli and detect its genomic quality.

Table 2 is intestinal bacteria and 43 strain Shigellaes and their sources that are used to screen 166 kinds of serotypes of specific gene, and for the convenience that detects, we are divided into one group, 13 groups altogether with their every 12-19 bacterium.All list in the table in their source.

In the 8th group, contain the genomic dna of intestinal bacteria O163 as positive control.In the 13rd group is the genomic dna that does not contain intestinal bacteria O163, as negative 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 2 minutes, 95 ℃ of sex change 15 seconds, annealing temperature is because of the difference different (with reference to table 1) of primer, annealing time is 50 seconds, and 72 ℃ were extended 2 minutes, and carried out 30 circulations like this.Continue to extend 10 minutes at 72 ℃ at last, reaction system is 25ul.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, and every pair of primer has obtained except be PCR in the 3rd group after the correct band of expection size, and the correct band of any size does not all increase in other groups.So wzx, wzy gene pairs intestinal bacteria O163 and O-antigen thereof all are high specials.

At last, from intestinal bacteria O163, screen gene by PCR: wzx, wzy gene to the O-antigen high special of intestinal bacteria O163.And the oligonucleotide of these intragenic any one section 10-20nt is special to the O-antigen of intestinal bacteria O163, 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 O163.These all oligonucleotide all can be used for the intestinal bacteria O163 in the human body and environment rapidly and accurately, and can identify their O-antigen.

By clone and the expression in the vaccine strains of attenuation, can set up recombiant vaccine to O antigen gene bunch.O antigen is the surface antigen of topmost Gram-negative bacteria, can cause the intensive immune response, is one of best target molecule of making recombiant vaccine.Viret laboratory success in 1993 the O antigen gene of Shigellae Sonnei bunch is expressed in a strain Salmonellas Tyziai vaccine bacterium, experimentation on animals proof can cause rabbit immune response (Molecular Microbiology1993,7:239-252).The group of China Military Medical Science Institute also similarly works being engaged in the Viret laboratory.Bunch express the O antigen gene of intestinal bacteria O111 success in 1999 in the Wang Lei laboratory in salmonella vaccine STM-1, and the bacterial strain set up of proof can cause the blood of mouse and humoral response (Microbial Pathogenesis 1999,27:55-59).So the O antigen-specific gene order of O163 of the present invention can be applied to set up recombiant vaccine.

Nucleotide sequence (shown in the SEQ IDNO:1) according to the O-antigen-specific to intestinal bacteria O163 type of the present invention, structure specific nucleic acid probe, be fixed on the carrier of chip and make biochip, after the sample that will detect is suitably handled, carry out hybridization with biochip, utilize the biochip signal analysis equipment just can obtain corresponding bacteria situation in the sample then.The DNA chip that this intestinal bacteria O antigen is identified can be directly used in clinical and other check place (as food-processing and production industry, the Micro biological Tests of animal and veterinary industry customs quarantine control etc.).This chip only need enlarge output, just can industrialization under identical condition.

Table 3 is structural tables of the O-antigen gene bunch of intestinal bacteria O163, has listed the structure of the O-antigen gene bunch of intestinal bacteria O163 in table, altogether by 11 genomic constitutions, and each gene box indicating, and in square frame, write the title of gene.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 location tables of the gene in the O-antigen gene bunch of intestinal bacteria O163, in table, listed the accurate position of all open reading frame in complete sequence in the O-antigen gene bunch of intestinal bacteria O163, 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 bacterium: ATG and GTG.

Sequence list

SEQUENCE?LISTING

＜110〉Tianjin Biochip Technology Co., Ltd

＜120〉to the Nucleotide of the O-antigen-specific of intestinal bacteria O163

<160>1

<170>PatentIn?version?3.1

<210>1

<211>15433

<212>DNA

<213>Escherichia?coli

<400>1

attgtggctg?cagggatcaa?agaaatcctc?ctggtaactc?acgcgtccaa?gaacgcggtc 60

gaaaaccact?tcgacacctc?ttatgaatta?gaatctctcc?ttgaactgcg?cgtgaagcgt 120

caactgctgg?cggaagtaca?gtccatttgc?ccgccgggcg?tgaccatcat?gaacgtgcgt 180

cagggcgaac?ttttaggttt?gggccactcc?atcttgtgtg?cacgacctgc?cattggtgac 240

aacccatttg?tcgtggtact?gccagacgtt?gtaatcgacg?acgccagcgc?tgacccgctg 300

cgctataacc?ttgctgccat?gattgcgcgc?ttcaatgaaa?cgggacgcag?ccaggtgctg 360

gcaaaacgta?tgccgggcga?tctctctgaa?tactccgtca?ttcagaccaa?agaaccgctg 420

gatcgtgaag?gtaaagtcag?ccgcattgtt?gaattcatag?aaaaaccgga?tcagccacag 480

acgctggact?cagacattat?ggccgttggt?cgctatgtgc?tttctgccga?tatttggccg 540

gaactggaac?gcactcagcc?aggtgcatgg?ggacgtatcc?aactgactga?tgccattgcc 600

gaactggcga?aaaaacagtc?cgttgatgcc?atgctgatga?ctggtgacag?ctacgactgc 660

ggtaaaaaaa?tgggctatat?gcaggcgttc?gtgaagtatg?gcctacgcaa?cctgaaagaa 720

ggggcgaagt?tccgtaaagg?gattgagaag?ctgttaagcg?aataatgaaa?atctgaccga 780

atgtaacggt?tgataagaaa?attataacgg?cggtgagatt?cgtggcgaaa?gtaatttgtt 840

tcgaatattc?ctgccgttat?tttatataac?aatcagaaca?acaacgagtt?agcaatagga 900

ttttagtcaa?agttttctag?gatttccctt?gtttccagag?cggattggta?gacattagcg 960

ttgaattttc?cgggtttagc?gcgaggtagg?taacgctcgt?cacatcgtag?acatgtatgc 1020

agtgccctgg?tagctgtaaa?gccaggggcg?gtagcgtgca?ttaatacctc?tattaatcaa 1080

actgagagcc?gcttatttca?cagcatgctc?tgaagtaata?tggaataaat?taagtgaaaa 1140

tacttgttac?tggtggcgca?ggatttattg?gttctgctgt?agttcgtcac?attataaata 1200

atacgcagga?tagtgttgtt?aatgtcgata?aattaacgta?cgccggaaac?ctggaatcac 1260

ttgcagatgt?ttctgattct?gaacgctatg?tttttgaaca?tgcggatatt?tgtgatgcag 1320

ctgcaatggc?acggattttt?gctcagcatc?agccggatgc?agtgatgcac?ctggctgctg 1380

aaagccatgt?tgaccgttca?attacaggcc?ctgcggcatt?tattgaaacc?aatattgttg 1440

gtacttatgt?ccttttggaa?gctgctcgga?attactggtc?tgctcttgat?ggcgacaaga 1500

aaaatagctt?ccgttttcat?catatttcta?ctgacgaagt?ctatggtgat?ttgcctcatc 1560

cagatgaagt?aaataataca?gaagaattac?ccttatttac?tgagacaaca?gcttacgcac 1620

caagcagccc?ttattctgca?tcaaaagctt?ccagcgatca?tttagtccgt?gcgtggaaac 1680

gtacctatgg?tttaccgacc?attgtgacta?attgctctaa?caattatggt?ccttatcatt 1740

tcccggaaaa?attgattcca?ttggttattc?tcaatgctct?ggaaggtaaa?ggattaccta 1800

tttatggtaa?aggggatcaa?attcgcgact?ggctgtatgt?tgaagatcat?gcgcgtgcgt 1860

tatataccgt?cgtaaccgaa?ggtaaagcgg?gtgaaactta?taacattggt?ggacacaacg 1920

aaaagaaaaa?catcgatgta?gtgctcacta?tttgtgattt?gttggatgag?attgtaccga 1980

aagagaaatc?ttatcgtgag?caaatcactt?atgttgccga?tcgtccggga?cacgatcgcc 2040

gttatgcgat?tgatgctgag?aagattggta?gcgaattggg?atggaaacca?caggaaacgt 2100

ttgagagcgg?gattcgtaaa?acggtggaat?ggtacctggc?taatgcaaaa?tgggttgaga 2160

atgtgaaaag?tggtgcctat?caatcgtgga?ttgaacagaa?ctatgagggc?cgcaagtaat 2220

gaatatcctc?cttttcggca?aaacagggca?ggtaggttgg?gaactacagc?gttctcttgc 2280

tcctctgggt?aacttgattg?ctcttgatgt?tcactccact?gattattgtg?gcgatttcag 2340

caacccagaa?ggtgtggctg?aaaccgtcaa?aaaaattcgc?ccagatgtta?ttgttaatgc 2400

tgcggctcat?accgcagtag?ataaagctga?gtcagaaccc?gaatttgcac?aattactcaa 2460

tgcgaccagc?gttgaatcaa?ttgcaaaagc?ggctaatgaa?gttggggcct?gggtaattca 2520

tttctcaact?gactacgtat?tccctggaaa?tggcgacact?ccatggctgg?agactgatgc 2580

aaccgcaccg?ctaaatgttt?acggtgaaac?caagttagct?ggagaaaaag?cattacaaga 2640

gcattgtgcg?aagtacctta?ttttccggac?cagctgggtc?tatgcaggta?aaggaaataa 2700

cttcgccaaa?acaatgttgc?gtctggcaaa?agagcgtgaa?gaattagcgg?ttattaacga 2760

tcagtttggt?gcgccaacag?gtgctgaact?gctggctgat?tgtacagcac?atgccattcg 2820

tgtcgcactg?aataaaccgg?atgtcgcagg?cttgtaccat?ttggtagcca?gtggtgccac 2880

aacctggtac?gattatgctg?cgctggtttt?tgaagaggcg?cgcaaagcag?gcattcccct 2940

tgcactcaac?aagctcaacg?cagtaccaac?aactgcctat?cctacaccag?ctcgtcgtcc 3000

acataactct?cgccttaata?cagaaaaatt?tcagcagaac?tttgcgcttg?tcttgcctga 3060

ctggcaggtt?ggcgtgaaac?gaatgctcaa?cgaattattt?acgactacag?caatttaata 3120

gtttttgtat?cttgttcgtg?atggtggagc?aagatgaatt?aaaaggaatg?atgaaatgaa 3180

aacgcgtaaa?ggtattattt?tagcgggtgg?ttctggtact?cgtctttatc?ctgtaactat 3240

ggctgtcagt?aaacagctgt?taccgattta?tgataaaccg?atgatctatt?acccgctctc 3300

tacactgatg?ttggcgggta?ttcgcgatat?tttgattatc?agtacgccac?aggatactcc 3360

tcgttttcaa?caactgctgg?gtgacgggag?ccagtggggc?ctgaatcttc?agtacaaagt 3420

tcaaccgagt?ccggatggtc?ttgcgcaggc?gtttattatc?ggtgaagagt?ttatcggtgg 3480

tgatgattgt?gctttggttc?taggtgataa?tatcttttac?ggtcacgatc?tgccgaagtt 3540

aatggatgtc?gctgttaaca?aagaaagtgg?tgcaacggta?tttgcttatc?acgtaaatga 3600

tcctgaacgc?tacggtgtcg?ttgagtttga?taaaagcggt?acggcaatta?gcctggaaga 3660

aaaaccgcta?cagccaaaaa?gtaattatgc?ggtaactggg?ctttattttt?atgataacga 3720

tgttgtcgaa?atgtcgaaaa?atcttaagcc?ttctgcccgt?ggtgaactgg?aaattaccga 3780

tattaaccgt?atttatatgg?aacaggggcg?tttatccgtt?gccatgatgg?ggcgtggtta 3840

tgcatggctg?gatacgggga?cgcatcaaag?tcttattgag?gcaagcaact?ttattgcaac 3900

aattgaagag?cgtcaggggc?tgaaagtttc?ctgcccggaa?gaaattgctt?accgtaaagg 3960

gtttattgat?gctgagcaag?taaaagtgtt?agtacaacct?ctgaaaaaaa?atgcttatgg 4020

tcagtaccta?ctaaaaatga?ttgaaggtta?ttaataaaat?gaacgtaatt?aaaactgaaa 4080

ttcctgatgt?attaattttt?gaaccgaaag?tttttggtga?tgagcgtggt?ttctttatgg 4140

aaagctttaa?tcagaaagtt?ttcgaagagg?ctgtagggcg?taaggttgaa?tttgttcagg 4200

ataaccattc?taagtctaat?aaaggcgttt?tacgcgggct?ccattaccag?ttagaacctt 4260

atgcgcaagg?caaacttgtg?cgctgtgttg?ttggtgaggt?ctttgatgtt?gcggttgaca 4320

ttcgaaaatc?gtcacctacg?tttggcaagt?gggttggtgt?gaatttgtcg?gcggcgaata 4380

agcgccaatt?atggataccg?gaaggatttg?ctcatggctt?tttaagccta?acagataaca 4440

ctgaattttt?gtataaaact?aataacttct?ataatagaaa?ttatgaaaga?tgtttaaaat 4500

ttgatgatat?agaattaagc?atcaaatggc?caattggcga?cgaaactgag?ttgatcgttt 4560

cagaaaaaga?tatgttaggc?aatattttta?gtaaattata?gtttttatca?tagcggtatt 4620

taagtttaat?acttaccgct?atgattttta?catctttttt?gtaaaggtgc?ttaaatgcgc 4680

gctagtccat?tttttcggtc?agttaaatat?ggtattgtat?tttctatagt?aataatgata 4740

cttactttta?ttgtaaggca?gtgtattatt?aattcgtttg?gtgaaaattt?aacaggttat 4800

tatttgttaa?taaaccaatt?agtcggttat?ctaaatttag?ctgaattggg?tttaactacg 4860

gcatcagtat?atttattgtt?caagccgttg?aattctgaaa?ataaatatga?cattgttggt 4920

tatttttata?ttattcacag?aatatttaaa?tttattgcat?cagcaatatt?atttattggg 4980

ctaacactct?cttttattat?gccctattta?gtcaaagata?atataaattg?gcatgatatt 5040

tacataccat?gggtttttta?tgtcattgca?acagcattgt?cgtattttta?tagtgcagaa 5100

actgttttgt?taacagcaag?tgaaaaacta?tatattgttc?ggattgtgaa?tggtttggct 5160

cgagttatta?cttttttaat?acaaatagtt?gtgcttaaaa?atggtggtgg?ttttattatt 5220

tttagtgcgt?tagagatttt?atatgcgcta?attcaatata?tactatttag?atattatgtt 5280

ctaaaactgt?acggggcata?tttatatagt?aagataacac?tatcaaaaga?acttccgcta 5340

ataaagcaaa?caataataaa?agagataaaa?aagacattta?tccacaagtt?atcaggtgtt 5400

atgatcttta?atacagacta?tataattatt?agcattttta?taggattatc?tacaataacg 5460

atatattcat?cctatttaat?gttcatacaa?gcttcagcta?taataattgg?aacaattgta 5520

agtccattgg?gggcatacat?tggaaatttt?ttacatagaa?ccgacagtaa?tctaacgtat 5580

ttaaaattca?attcaataaa?tacgattttt?ttcttattgg?catcgattgg?ttgtattata 5640

tacaacaata?tctcaactcc?ttttgtacaa?ctgtggatgg?gcaaagatat?cattttttct 5700

caagatattg?tggcgttgct?agcggttaac?tttttttgtt?tagtagcgag?gagcgctgta 5760

gatattttta?aggtcgcata?tggatatatg?tctgatattc?atttaccaat?aattgaaggt 5820

actttaaatt?taattatatc?tttatctttg?gttaattttt?atggtgtcaa?aggtgtcatt 5880

tatggaacta?tatgttcaaa?tgttatcatt?atcatgctgg?cgaggccctg?gtatttatat 5940

aaagaggcct?tcaatttatg?cactatagat?ttcataaaag?atcatattca?attatgggta 6000

atatcattaa?tgattctcat?cactttgtta?agtggtcgag?ttaatgatat?gtatacactt 6060

ttcgagaaat?acttaatgaa?taattgggat?gttagcaata?agctaattga?acaacttctc 6120

acacatagtc?tctttttgat?tttatcaaca?atatttgtag?taattatcta?tattatagtc 6180

acccctaaaa?attgccttgc?ttcagtaaaa?atattaacca?ataaagggtt?ataatgaatc 6240

caatacctat?tttcataatg?actagaaatg?atggtgttta?tctgcaggac?tgtttgaact 6300

caatccttaa?aaataccgta?tacccattta?atctgtatgt?tattgataat?aactcttcag 6360

ataaattaca?tttaaaaatt?cttgaggaat?ataaaactcg?gcagaatgtg?aatgtaataa 6420

gaaataagag?taacttatgg?gttttaggtc?taaataagca?tcttgagaaa?gttaaaaagg 6480

aaagtgattc?ttcttatttt?gtgcttactg?acggtgatat?tatttttcct?gagcctggaa 6540

taaatggatg?ttggttgact?caattagtaa?tatatatgga?cacgtataaa?tgcattggta 6600

aaataggtat?gtcactaaat?tgggattcga?taagggatga?cccattctat?gaagaaattt 6660

atgagcaaga?aaaaaaatta?tataataaca?ataaaaaaat?agaaaatctt?tatatatcac 6720

cagtagatac?aaccgcagct?atatatcgct?gggattggtc?tatcgaagga?tataaatttt 6780

atcctgatca?tatacgctat?cttagaccgg?agctatactc?atgtaggact?ccaaaagaat 6840

ttaatgcaaa?acatttaggt?tggttagttt?ataaaaataa?taatggccaa?gcaattaaaa 6900

aactagatga?gaaaattaaa?tgtttcacct?tagttggagc?tgatataaaa?aaaacacaac 6960

taaaaaaggc?tagcctgaag?gtgagaatgt?ttaattcttt?atgtggaaaa?cccataaaat 7020

atttttggag?tatgcgacgt?attttatata?ctatattcta?tacccttaaa?aaaggaattt 7080

ggctttatga?taatcattga?gaaaacaaga?tgataccata?ttttttaatg?ttcgcatgcc 7140

ttgtgatctt?ttctttattt?aaaggtaatg?acagaagagt?attaaatgct?tctatgatca 7200

tccttggcat?tttttgcgcc?atgaggggaa?acaatgttga?tagagactat?gagacgtata 7260

taagtattta?tacatatatt?atagaaggtt?atagctatgc?tattgagcca?acctttcatc 7320

tattatcaat?aatttcaaat?acccttacgg?gtacaccatt?tcttatattt?tgcgtttatg 7380

caggacttgc?tatttatttt?aaaataaaat?ttattgaata?ttggtcgcct?tatttattgc 7440

tttctgtatt?gctctatttt?tccaatgtgt?atttactaca?tgagatgact?caaattagga 7500

ttgggttagc?aagtgccatt?ggttttttta?gtttgaaata?tttaataacc?gcagaaaaga 7560

aaaaatattt?tatttgggtg?ttcatcgcta?tgactttcca?tttttcaatg?gctgtatttt 7620

ttttgttgcc?attgttaaat?tcaaagcgat?tgtctggcag?gtatgtcacc?atatattgga 7680

taagtattat?tttactttac?tttttatctt?attataaaat?tgatttaagt?gtctttttaa 7740

aatattttga?tataggcata?ataaattcaa?aatatgattt?gtatcgagag?caaggggaaa 7800

ataatgatac?tagtgtgaat?atttattctg?cactgcaata?ttttcatttg?tttgtgattt 7860

ttctaagtat?ggtctatcgc?gactcgttca?gatatgatga?aaagtatata?attatgttaa 7920

aaatttattc?attaggacct?ttatcattat?tagtgttttc?aaatgttcct?ggtttttcat 7980

tacgtttatc?tgaattgttt?aatgtagggg?aaatagtttt?attaccaatg?ctcgttagtc 8040

acattaaaca?aattaagcta?gcctacctgt?cagtaatatt?gatttctctt?tgtttgttgc 8100

ttattaattt?gtattatttg?tccttgttga?aggaatattc?aatctgatgc?ttattttacc 8160

tatagttgtt?atttataaat?gcaaactcga?aaattctgca?agtttaaata?cattattaaa 8220

atgtgaatca?aatgaattta?taaaggaaat?tttcgtatat?aataattctc?ctgatgaaat 8280

atttataccg?gattattata?tgggtgttaa?agtttataag?atagatgatt?ataataactc 8340

tggtgttagt?agagcttata?ataaaggttt?aaaacttgct?tcaaatctaa?attataagta 8400

tgttcttctt?ttagaccaag?atacatatat?tcctaaggac?tttttgaaaa?tatgtaatga 8460

aactatatca?cagcatttaa?atataaaact?tttctgtcct?atattaaaaa?caaaacgtgg 8520

tgtcatttgt?tctccattgt?catataagta?tcatagagga?tttcatgtta?agaatattgt 8580

cgctggggaa?aagggattaa?gcaaattatc?tcctataaat?agcggaatga?tattagatgt 8640

tgaagccgct?cttaagtgtg?gtggatataa?tgaaaatgtt?tttctagatt?ttagtgattt 8700

tcaatttatt?gagagattta?agaaaaaaaa?tcattcattc?tatgttttac?caatagtatt 8760

aacgcaagat?ttttcaggag?aagaaactga?taaagaaaaa?acactaaggc?gttttcatat 8820

ttattgtata?tgtgctaaaa?actgtactaa?aaaaaatttt?acagatcaat?ttatctattt 8880

ttttatggtt?ttactcagag?caatgaagtt?gacttttaaa?atcaaaacta?taaacccatt 8940

aatcactttt?tatacaagat?atctccgggg?gtaagatgat?ttctgtatgc?attccgactt 9000

ataatggtga?gaattatata?aaacaacaaa?ttctcagcat?acttaatgag?ttgggtgaag 9060

atgatgagat?tgttatttct?gatgatggtt?caacagatat?gacattagca?ataatagaag 9120

aaataaatga?taaacgaatt?attgtaatta?aaaatgagga?aaacattcaa?tgcgaatcct 9180

acaataaccg?aactgaaaaa?ttgctaaaaa?aagtatcatt?gaatctgcaa?aatgccttat 9240

tacattgtaa?aggagattat?atatatttag?ctgaccaaga?tgatatatgg?aagaaagggc 9300

ggattactaa?tacattacct?ttattagcag?agaataagcc?tattttggtt?gtgtgtgatt 9360

gttgtgttgt?tgatgaaaat?aatactataa?cgcagaggtc?atattttgat?tacgttaccc 9420

cttcgcaaaa?tttatggaga?acagtaatta?agagttcctt?tcatgggtgc?tgtatgtgtt 9480

ttaatagaat?tttattagca?aaagcatttc?cgtttcctca?ctatagtttg?gggcatgatt 9540

tatggattgg?tttaattgca?ataaaatttg?gtctcgtata?tttttgtcca?gaagttttag 9600

tggagtatag?gcgacattcg?acaactgtta?cgtctacagg?tagtaaaagt?aagaatagtt 9660

ttggatttaa?aatacgttac?agaattatgt?tattaattga?atatttgaaa?ctatataaaa 9720

gaaaatgaaa?aaaattgcac?atgttttagt?tcttcccaaa?atggcaggtt?ctcaaaaatt 9780

ttgccatatg?cttctctcaa?aaatagaggg?gtatgaaaaa?tttgttcttg?tctctgaatg 9840

tgaggatgtc?gatttagaac?aacgagctga?gtttataaaa?aattttgaat?caattaatgt 9900

caatattatt?tggtgtaaga?accttaagag?aaatattggt?aaatctgatg?taaaagcttt 9960

tgttgagtta?tataatattt?ttaaacaata?tgattttgat?atagttcata?ctaattcgac 10020

taagcctggc?attctggcta?ggattgccgc?taaaatggcg?ggtgtaaaaa?aaataataca 10080

tacagtacac?ggcatttctt?tttatagagg?acaaccttat?atcaaaaggt?taatatactg 10140

gataatagaa?gtttttgcac?ttcagtttgg?tgatcttaac?atatgtgtca?ataaattcta 10200

tcagaaatat?tataaaattt?ttttttggaa?gaaaagtatt?accatttata?atggctatgg 10260

tttttccgaa?attaaaaaaa?taaacaaaat?ggcaaaagac?agtaatgaaa?aaagattttt 10320

gtttgtagga?cggctagatg?agcaaaaaga?tccgctcaca?cttatcaaag?cttttgaact 10380

tattgaaaaa?aaatatccta?atgtatattt?agatattgtt?ggcgatggtg?aattaaaagg 10440

gcattgcgaa?gaattggtgg?aaaaattaaa?aattcaagat?aaggtgatat?ttcatggttg 10500

ggtagaaaaa?ccatactctt?tttacttaaa?ttgcaatgta?tttatttgcc?cgtcaaaata 10560

tgaggcattt?ggttttattt?tcgttgaagc?tgcttatttt?aaaaaaccaa?taataactac 10620

taatgtagaa?ggtattccgg?aggtcgtggt?agattcgaaa?atgggctatt?tgattaagcc 10680

gggaaatttc?ctagatttat?caaagagaat?gacaaattta?ctagagtcta?attctttatg 10740

tgtagctatg?ggtgaatatg?gtcataaata?tgttacaagt?aatttcgata?ttgagaaatg 10800

tatcgaaaaa?tatcaggctg?tatatgatga?cctttgaagg?cttttggtat?gatgaaagta 10860

gtgtttaaaa?tttatattcc?ttatttgatt?gtaatgattg?ctgtgtttgt?catgctaacg 10920

tatacagaca?tggtaagtaa?attttctagt?attgctttag?agtacttctg?tatatttaca 10980

tccctttttg?tttttaaaaa?aatttataga?ggaataattt?ggactttagt?cgtagggata 11040

cttggagctc?agatatcatc?tttatataca?agtggaaatt?atgtcatacc?tcttactctc 11100

tcaaacgttg?gtgagtataa?tgcgttaggt?tttgagcttt?tatttaagtt?gttatgtata 11160

tcactcttat?ttttatgtgt?ggcacaaatt?atctttagtc?ctgtttttac?ttatgatatt 11220

ccacggcgga?aaacattttt?attagctctt?cttttcttgc?ctctaataaa?tggtccttta 11280

gttaagttca?cagagactct?ttatttttac?tataagcaag?tgactttttc?ccctgcgtat 11340

aattatcctg?caattgcaaa?aaaattttta?aaaacagata?tttggcatga?tgagtcactt 11400

cttttgaaaa?ataaaaaacc?taatgtgata?gtaattttta?ctgaaggaat?gtcttttaat 11460

gtaattgata?gcgttaataa?tttaggcctc?ggagtaactc?caaagctcga?tgaaatcatg 11520

aagaaatcat?ttttctttat?aaattattat?aatcatacag?cagctacttt?tcgagggtta 11580

agagggcaat?taacttctgc?ttatcaattt?aaagatggtg?taggtgcaaa?tggtgatggt 11640

ttttttgaaa?taacaaatca?aaaagttaaa?tcaatatata?ataaaagatt?agtctcttta 11700

ccggagatat?tgaattcaaa?tggttataag?acgatatttc?tttcttcaac?agaaaaaact 11760

agcactttga?atgcgatgct?gaaaacctta?tcattcaatg?aagttttggg?aatgggagat 11820

tttgattttt?atcagaatga?ccgtatgtca?gataagcaaa?ctttcatagc?gcttaaggaa 11880

gttgttgaaa?gaaacaagaa?taataaattt?ttcattggag?tttacccatc?tggaactcat 11940

catggtttag?atagccctga?tttgcgtttt?agagatggtt?caaattctta?ttataacaaa 12000

ttttataatt?ttgatcatca?agttggcaag?ttcatagatt?atcttacgtc?cacaggtctt 12060

ataaataata?ccttggtggt?tattacggct?gatcattcaa?ctttccctac?accacaattt 12120

aataaatctt?ttagttcaaa?ttctgattat?tttgttgatg?caataccttt?aataattttg 12180

gggcaggaaa?tagagtctaa?aaaaaataat?gcgcatggga?agaatagttt?agcattggca 12240

ccaacgatac?taaatttatt?aaatattaac?cattatccta?atttcttttt?aggctgctcg 12300

ctcttggatg?taaaatgcca?aagtactttt?agtcatatat?cggcaattgg?aaattctttt 12360

tttaaaaccg?gcgataaaaa?gtgttcttca?gatgattata?acgttaaaaa?attagataat 12420

tctagcgata?taattaattt?ctataatgtt?agtggttaaa?gtataaatac?agcttatagt 12480

tgactgatat?atatggttaa?tgtttttata?gtgaatattt?tttcaagccg?cacaccctcg 12540

cggtgaccac?cccctgacag?gagtaaacaa?tgtcaaagca?acagatcggc?gtcgtcggta 12600

tggcagtgat?ggggcgcaac?cttgcgctca?acatcgaaag?ccgtggttat?accgtctcta 12660

ttttcaaccg?ttcccgtgag?aagacggaag?aagtgattgc?cgaaaatcca?ggcaagaaat 12720

tggttcctta?ctttacggtg?aaagagtttg?ttgaatctct?ggaaacgcct?cgtcgcatcc 12780

tgttaatggt?gaaagcaggt?gcaggcacgg?atgctgctat?tgattctctc?aagccatacc 12840

tcgataaagg?tgacatcatc?attgatggtg?gtaatacctt?cttccaggac?accattcgtc 12900

gtaatcgtga?gctttctgcc?gaaggcttta?acttcattgg?taccggtgtc?tccggtggtg 12960

aagaaggcgc?gctgaaaggt?ccttccatta?tgcctggtgg?gcagaaagaa?gcctatgaac 13020

tggttgcacc?gatcctgacc?aaaatcgccg?cagtggctga?agacggtgag?ccatgcgtta 13080

cctatatcgg?tgccgatggc?gcaggccatt?atgtgaagat?ggttcacaac?ggtattgaat 13140

acggagatat?gcagctgatt?gctgaagcct?attctctgct?taaaggtggc?ctgaatcttt 13200

ccaacgaaga?actggcgcag?acctttaccg?agtgggataa?cggtgaactg?agcagctacc 13260

tgatcgacat?caccaaagac?atcttcacta?aaaaagatga?agacggtaac?tacctggttg 13320

atgtgattct?ggatgaagcg?gct 15433

Wzx gene, wzy gene and wherein primer and PCR data in the O antigen gene of table 1 intestinal bacteria 0163 bunch

Produce positive PCR's

The forward primer position-reversed primer location PCR product of base gene is size annealing temperature really

Function

Because of base position length electrophoresis band width

The group number (℃)

Wzx O-antigen 4349-5602 4500-4518 5425-5442 1093bp 0 58

The transhipment enzyme

5100-5117 5503-5520 420bp 0 58

Wzy O-antigen 5599-6834 6102-6119 6738-6755 653bp 0 58

Polysaccharase

6223-6240 6784-6801 578bp 0 56

The intestinal bacteria of 166 kinds of serotypes of table 2 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, O8, O9, O12, O13, O14, O15, O16, O17, O18, IMVS ^a

O19ab，O20，O21，O22，O23，O24

2, wild-type e. coli O4, O10, O25, O26, O27, O28, O29, O30, O32, O33, O34, O35, IMVS ^a

O36，O37，O38，O40，O41，O42，O43

3, wild-type e. coli O6, O44, O45, O46, O48, O49, O50, O51, O52, O54, O55, O56, IMVS ^a

O57，O58，O60，O61，O62，O53

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

O79，O80，O81，O82，O83，O68

5, wild-type e. coli O84, O85, O86, O87, O88, O89, O90, O91, O92, O98, O99, IMVS ^a

O101，O102，O103，O104，O105，O106，O97，

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

O115，O116，O118，O120，O123，O125，O126，O128，O117

7, wild-type e. coli O129, O130, O131, O132, O133, O134, O135, O51, O137, IMVS ^a

O138，O139，O141，O142，O143，O144，O145，O140

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

O159，O160，O161，O163，O164，O165，O166，O153 b

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

Shigella dysenteriae D1, D2, D3, D4, D5, D6, D7, D8, D9, D10, D11, D12, D13 d

10, Shigella bogdii B1, B2, B3, B4, B6, B7, B8, B9, B10, B11, B12, B13, B14, B15, d

B16，B17，B18

11, shigella flexneri F1a, F1b, F2a, F2b, F3, F4a, F4b, F5 (v:4), F5 (v:7), F6, d

DS，DR

12, wild-type e. coli O3, O11, O39, O59, O64, O73, O96, O95, O100, O114, O151, O155, IMVS ^a

O124，O167，O162，O121，O127，O149，O119

13, wild-type e. coli is removed the 8th group of bacterium of intestinal bacteria O163

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

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 is structural tables of the O-antigen gene bunch of intestinal bacteria O163

E.coli?O163?O-antigen?gene?cluster

#orf?galF manC manB wzx wzy orf5 orf6 fnlA qnlA qnlB orf10 orf11 gnd

Table 4 is location tables of the gene in the O-antigen gene bunch of intestinal bacteria O163

ATTGTGGCTG?CAGGGATCAA?AGAAATCCTC?CTGGTAACTC?ACGCGTCCAA?GAACGCGGTC 60

GAAAACCACT?TCGACACCTC?TTATGAATTA?GAATCTCTCC?TTGAACAGCG?CGTGAAGCGT 120

CAACTGCTGG?CGGAAGTACA?GTCCATTTGC?CCGCCGGGCG?TGACCATTAT?GAACGTGCGT 180

CAGGGCGAAC?CTTTAGGTTT?GGGTCACTCC?ATTTTATGTG?CACGACCCGC?CATTGGTGAC 240

AATCCATTTG?TCGTGGTGCT?GCCGGACGTT?GTGATCGACG?ACGCCAGCGC?CGACCCGCTG 300

CGCTACAACC?TTGCAGCCAT?GATTGCGCGC?TTCAACGAAA?CGGGCCGCAG?TCAGGTGCTG 360

GCAAAACGTA?TGCCGGGCGA?TCTCTCTGAA?TACTCCGTCA?TTCAGACAAA?AGAACCACTG 420

GACCGTGAAG?GTAAAGTCAG?CCGCATTGTT?GAATTTATCG?AAAAACCGGA?TCAGCCGCAG 480

ACGCTGGACT?CAGACATCAT?GGCCGTTGGT?CGCTATGTGC?TTTCTGCCGA?TATTTGGCCG 540

GAACTTGAAC?GCACTCAGCC?TGGTGCATGG?GGGCGTATTC?AGCTGACTGA?TGCCATCGCT 600

GAACTGGCGA?AAAAACAGGC?TGTTGATGCA?ATGCTGATGA?CAGGCGACAG?CTACGACTGC 660

GGTAAAAAAA?TGGGCTATAT?GCAGGCGTTT?GTGAAGTATG?GACTGCGTAA?CCTAAAAGAA 720

GGGGCGAAGT?TCCGTAAAGG?GATTGAGAAG?CTGTTAAGCG?AATAATGAAA?ATCTGACCGG 780

ATGTAACGGT?TGATAAGAAA?ATTATAACGG?CAGTGAAGAT?TCGTGGCGAA?AGTAATTTGT 840

TGCGAATATT?CCTGCCGTTG?TTTTATATAA?ACAATCAGAA?TAACAACGAG?TTAGCAATAG 900

GATTTTAGTC?AAAGTTTTCC?AGGATTTTCC?TTGTTTCCAG?GGCGGATTGG?TAAGACAATT 960

AGCGTTTGAA?TTTTTCGGGT?TAAGCGCGAG?TGGGTAACGC?TCGCTACATC?GTAGGCATGC 1020

ATGCAGTGCT?CTGGTAGCTG?TAAAGCCAGG?GGCGGTAGCG?TGTCTGATGA?ATAAATCGAT 1080

TTGTTCAATA?TAGTAATCTA?CTAGAAAAAC?TTTTCATCCC?TCATAATTAT?CGATGTCAAT 1140

ATTTGAAATA?TAATTAGCAA?CCAACAGTTG?ATTAAAATAG?TTCAATGTTT?CTGCATAATA 1200

ATTATATTGA?TGGTAAAAAA?TTAAACATAA?CGAACCCTAT?TGTTTTATGC?GTTTATTTAT 1260

AACGTAAGGC?CCATTTAATA?GCTCTGTAAA?TAATGTAATT?CACTTAAATT?TTCTTTGTTT 1320

TACCAAATAC?CCTTTGGTTT?AACCGAATTT?TATATTTAAC?CTTATCTTCA?AGGTATGAGT 1380

Orf1's is initial

TATAGCTCTG?GGCCTTAGAA?TGAGTTAAT A?TGATGAATCA?CGAAAAAATT?TTCCCAATAA 1440

TCATGGCTGG?TGGGGCAGGT?AGTCGTCTGT?GGCCACTGTC?CCGCGAGCTT?TATCCAAAAC 1500

AATTCATTCG?CCTTAAAGGC?GAACTCACCA?TGTTACAGGC?AACTCTAAGC?CGTTTGAATA 1560

CTTTGATGTG?TAATGATCCG?ATTATTATAT?GCAACGAACA?GCACCGCTTT?ATTGTTGCAG 1620

AGCAGGTGCG?CCAGCTGAAC?AAACGGACCG?AAAATATCAT?TTTGGAGCCT?GTCGGTCGTA 1680

ACACCGCTCC?GGCAGTTGCC?CTCGCGGCGC?TGGCGGCAAA?ACGCAGTCGC?CCGGACTGTG 1740

ATCCGTTGAT?GCTGGTTCTG?GCTGCTGACC?ATGTTATTCA?GAACGAAGAT?GCATTTCGCG 1800

AAGCGGTAAA?GGTGGCTATC?CCTTACGCGG?AAAACGGCAA?GTTGGTGACT?TTCGGCATCG 1860

TGCCTGATAC?GCCGGAAACG?GGATATGGCT?ACATTCGTCG?TGGTGAGGCT?CTTTTTAGCG 1920

TAGATGAGTT?CAAAGCCTTT?GCTGTAAAAA?CATTTATAGA?AAAGCCTAAT?CTTGAAACTG 1980

CGATCGATTA?CGTCTCAAGT?GGTGAGTATT?ACTGGAACAG?TGGTATGTTC?ATGTTCCGTG 2040

CAGGTCGCTA?CCTGGAAGAA?CTTGAAAAAT?ACCGTCCGGA?TATCTTAAGT?GCCTGCGAAA 2100

AATCGATGGC?AGTGGTTGAT?CTTGATCTCG?ATTTTATCCG?CGTGGATGAA?GAAGCCTTTC 2160

TCTGCTGTCC?GGAAGAGTCC?ATTGACTATG?CAGTGATGGA?ACATACGGCG?GATGCCGTGG 2220

TGGTGCCAAT?GGATGCGGGC?TGGAGTGATG?TCGGTTCATG?GTCTTCTTTA?TGGGAAGTTA 2280

GCGATAAAAC?TCGTGAGGGA?AATGTTTGCA?CTGGAGATGT?GATTTCATTG?AACTCTGGCA 2340

ATAATCTTAT?TTTCTCCGAT?ACAAGCTTAA?TTGCCACAGT?TGGTGTGCAT?AATCTAATTA 2400

TAGTACAGAC?TAAAGACGCG?GTACTGATTG?CTGACCGTGC?CGCTGTGCAA?GACGTTAAAA 2460

AAGTGGTGGA?GAAGATCAAG?ACTGATGGTC?GCCATGAACA?CCATATTCAT?CGTGAAGTCT 2520

ACCGTCCATG?GGGTAAATAT?GACTCCATCG?ACACCGGTGA?GCGTTACCAG?GTGAAACGCA 2580

TCACAGTGAA?TCCGGGTGAA?GGAATGTCGG?TGCAGATGCA?CCATCACCGT?GCCAAACACT 2640

GGGTCGTGGT?GGCTGGTACC?GCCAAAGTGA?CCATCAATGG?TGAAGCGAAG?CTGCTCGGTG 2700

AAAACGAGTC?CATTTATATT?CCGCTGGGTG?CGACGCATTG?CCTGGAAAAC?CCGGGGAAAA 2760

TTCCGCTCGA?CTTAATTGAG?GTGCGCTCTG?GCTCCTATCT?GGAAGAGGAC?GACATCATCC 2820

The termination of orf1

GCTTCCAGGA?TCGCTACGGG?CGGGTG TAGC?ACTCCGCATC?ATGCCCAGTG?GCGCTGAGCT 2880

TGCTGGATCT?ACGAATGAAA?AAATAGATTT?GACCATTCAA?GGGTCAGGAC?TAATTTTGCC 2940

Orf2's is initial

TAAAAAGGAG?CCATC ATGGA?AAAATTAACC?TGTTTTAAAG?CTTACGATGT?TCGCGGCAAG 3000

CTAGGTGAGG?AGCTGAATGA?AGACATTGCG?TGGCGCATCG?GCCGCGCGTA?TGGCGAATAT 3060

TTTAAGCCAC?GAACTATCGT?CTTAGGCGGC?GATGTGCGTC?TGTCCAGCAA?AGCCCTGAAG 3120

CTGGCGTTGG?CGAAAGGACT?GCAGGACGCT?GGTGTTGATG?TGCTTGATAT?CGGCCTTTCC 3180

GGTACCGAAG?AGATCTATTT?TGCAACTTTC?CATTTGGGCG?TGGACGGTGG?TATTGAAGTG 3240

ACAGCTAGCC?ATAACCCGGT?GGACTATAAC?GGTATGAAGA?TTGTGCGCGA?AGGTGCACGT 3300

CCTATCAGCG?GTGATACCGG?CCTGCGCGAT?GTGCAGCGCT?TGGCTGAGGC?CAACGACTTC 3360

CCGGCGGTGA?ATGAAGCCAA?ACGCGGTAGT?TATAAGCAAA?TCAACTTGCA?AAAAGAGTAC 3420

ATCGACCACT?TGCTAGGCTA?TGTCAACGTG?GCAAACTTCA?AACCACTCAC?TCTTGTAATC 3480

AATTCCGGAA?ATGGAGCGGC?AGGACCGGTT?GTCGATGCAC?TTGAAGCTCG?CTTTAAGGCG 3540

CTGAACGTGC?CTATAAAATT?CGTTAAAGTG?AACAATACGC?CTGACGGCAA?CTTCCCGAAC 3600

GGTATTCCTA?ACCCGTTGTT?GCCAGAGTGC?CGTTCTGATA?CAAGCAATGC?GGTGATTGAG 3660

CTCGGTGCAG?ACATGGGAAT?TGCCTTTGAC?GGTGATTTCG?ACCGCTGCTT?CCTGTTCGAC 3720

GAAAAAGGAC?AGTTTATCGA?GGGTTACTAT?ATAGTTGGTC?TGCTGGCGGA?AGCCTTTCTT 3780

GAAAAACACC?CTGGTGCAAA?AATTATCCAT?GATCCGCGTC?TCTCCTGGAA?CACTGTTGAT 3840

GTGGTGACTG?CCGCAGGTGG?CACGCCGGTA?ATGTCGAAAA?CAGGACACGC?CTTTATTAAA 3900

GAACGTATGC?GCAAGGAAGA?CGCCATCTAC?GGTGGCGAAA?TGAGCGCGCA?CCATTACTTC 3960

CGTGATTTCG?CTTACTGTGA?TAGCGGCATG?ATCCCATGGT?TGCTGGTGAC?GGAGATGCTG 4020

TGTCTGAAGG?GCAAGTCTCT?GGGCGAGATG?GTTCGCGACC?GCATGACTGC?CTATCCTGCA 4080

AGCGGTGAAA?TCAACAGCAC?CTTGGCACAC?CCCGCTGAGG?CGATAGCGCG?GGTGGAGCAG 4140

CACTTTGCGA?AAGAGGCACT?GGAGGTGGAT?CGCACCGATG?GTCTTAGCAT?GTCGTTTGAT 4200

GAATGGCGCT?TCAATCTGCG?TTCGTCGAAT?ACCGAGCCGG?TGGTACGTTT?GAACGTCGAA 4260

TCCCGGTCTA?ATATCGAGCT?AATGGAAATA?AAAACTAAAG?AGATAATGGC?TCTATTAGAT 4320

The termination of the initial Orf2 of Orf3

AATAATAATT?CAGGGGGCCG?AGCTGAGA AT?GTT TAGTCAG?CTAATTCAGC?AGTTGAGCTC 4380

GTTTGTAGCG?AGGATATTAG?TATCATTAAT?TTCTCTTCTT?CTCAGCGTGA?TAATTGTACG 4440

GTTATATACG?AAAGATATAG?TTGGTGATTT?TTTTTTGCTA?ACCAGTTATT?CGACTGTTTT 4500

GGCCCAAGTG?TTCTTGTTTG?GAATGGCACC?TGTTTTAAAC?ATTATGTCAT?CCAAGGGGGT 4560

TCAGTTAAAT?AAAATAGTAA?TGGAGATAGC?CAAGAAAAAT?GTAGTATCTA?TGCTATGTAT 4620

GTTTTTATGT?GGCTTAATTA?TACTGGCTTT?TTTTGCTAAA?GTTGATTATT?CGAGTTTAAT 4680

TGTGCTTTCA?CTTCTTTCAG?GAGTTCTATT?AATTATTAGT?GAACTAATGA?AGGGACAAGG 4740

TATGTATATT?CTCTCCCAAA?TCTTCAATGG?AGGTTTGAAT?AACATTCTCT?TTCTTATTCT 4800

TATAGTCGTT?TTTATACCAA?ATAAAGAAGA?AACAGATCTC?GAGGATATTA?TTTATTTTTA 4860

TGCGTTAGCA?TATATGTGTG?TTATTGTAGG?TGGTTCTATA?TACTTGCGCA?TTAAATATAA 4920

GGTAAGTAAG?AGTAAAGGAT?TAATTGATGA?TGTTATTACG?TATTCAATTA?TTTTACCGAT 4980

ATTCATATCA?ACTGTTATTG?TATATATGTT?CTCACAAATA?GACCTGTGGT?TTGTATCTCG 5040

TTTATTTGAT?AATGATGTAG?TCGCTCAGTA?TGGATTAGCA?ATTAGATTGA?CGGCAGTATT 5100

GAGTTTTTCG?ACTTTATCAG?TTAGAGCTAT?AGCAGCAGCA?AGAATCCCAA?AATTAATGAA 5160

CGACATTCAG?GCGTTGCAAA?AAGATGTATC?CCTCAGTTGT?TTATTTTCAT?TTTTAGTGAG 5220

TTTAGTGATT?TTAAGTGGGA?TAGCATTATT?TGGCAAATTA?TTTATTGGCC?TTGTTTATGG 5280

CAACAGCTAT?GAATTAACTT?GGTATGTTCT?ACTAATTTTC?TCATTTGGAC?AGATGATTAA 5340

TGCTGCAACT?GGTCCCTGCG?ATTATTTACT?AAGTCATACA?GGGCATGGAA?GATACCTTAT 5400

GATAATCTCA?CTTGTTTCAT?TCTTATTTTT?AATGACTCTA?TTGGCAATAA?TAACAATAAG 5460

ATTCAACAGT?GTGTTTTTAT?TCTGTGGCGC?TGTATCATTT?ACAATCTGCT?TGCAAAATCT 5520

GATTATAATG?TACGTAGCAT?TTAAAAAAAC?GGGTGTACTT?TCACTACCCT?TAATAAATTT 5580

The termination of the initial Orf3 of orf4

TAAAAGAGCA?TATGTATC AT?GACAAGGAAA?ATGAATACTG?CAGTTTTATC?ATTCATGATG 5640

TTATATATTC?TTACTAGTCT?TATTTCAGTT?TATCAAACAT?TGAATCAAGA?TTTATTTCCT 5700

GGTGAACTTC?ATAGATATGT?AAGAACTATT?GCTGATGATG?AGGTTGTGAT?TATAACAACT 5760

CTGAATATTA?TATCCTTCAT?AATATGTTAT?ATAACATTTT?TGTTATTTCA?AAAATTTAGG 5820

TTCAGGCTGA?ACAAAAACTA?TCATTATGAA?TTGAATGATA?AGCGTATCCA?TTTCATAATT 5880

TTCGTAGTGT?TAATATCTCA?ACTTATTTTT?CTCTACTACA?CAGGTATTGG?TAAAGTTACT 5940

GTTAATGTCG?AACAGCGTAG?TACTTCATCA?TTTAGCGCTT?TTTTTGCAAT?CTTGAAGCCA 6000

GAACCTTTTA?TTTTTTTCTA?TTTTTTATAT?TATAGATTGA?AACCCGGATT?TACTTTTATT 6060

AAAAATAAGA?TTTTTATTTT?TAATATGTTT?TTGTTTATTG?TTTTTAAATT?ATTTCAAGGG 6120

TGGACAAGTT?TTTTGTTAAT?ACTTTTCTTT?CTTGAAGTTC?ATGCCAGAGT?ACAATTTACT 6180

AAAAACAGGT?TGAGATATGT?ACTCTTTATA?CCTTTAATTA?TTATTTTTTT?TGGTGGCTGG 6240

GTTTATCAGT?ATGCTTATGT?ATTAAAGAAT?GAAATTAGGG?GGGCGAAAGT?TGAAAGTATT 6300

ACGTACTTGC?AGGGAGTGGA?ACATCTTGCA?TCACGTTTAT?CGATGAATCC?TAACTCGCTT 6360

GGGGCTTATC?AAAATTATTC?TAAAGTTATA?AACTTATATC?AGCAAGAAGG?TCTTGCTCTC 6420

AAAGAATCAA?AAGCATTTCT?TCGACCTATT?ACACCAGGTG?GTTTGGTGGA?TAAAAATTTC 6480

AGAAATATAA?ATAATAATGT?AATGACATCA?TATACACCGG?ATCTAAATCA?ATTTACGAGT 6540

TCAGATTTTG?GCATTGTAAT?GTACTATTCA?ATATTGTTTA?ATGCAAGTCT?ACCTGATTTT 6600

GTATTAAGTA?TAATAATGAC?TGTTTTACTG?TTAGTAATTG?CTAAAATGTT?TTTTGATTCA 6660

ATTAGTAGTA?TTCCTGGACA?GTGCGATATT?TTATTCTTTT?TTGTATTGTT?CTATACCTTT 6720

TATACTGTTA?GTTTAGAAAA?TGTTTTTGGG?CAAAACTTTA?TTCCTTATAT?CTTTAGTTTT 6780

The termination of Orf4

GTAATATATT?ATTTTTTAGG?TGGAATAAAG?AAAGTACACT?TTGTAGAAAA?T TAATTTGAG 6840

Orf5's is initial

ATTGTGTGAT?T ATGATAGTT?AAAATGTGGC?CTCTTCAAAA?AGAATCAACG?GCAAATCAAT 6900

ATACTTCGTT?GATTCTACAT?GATGTGATTG?AATATAAACC?TCACAAATGG?AATCTGAAAA 6960

ATGTGTTGGA?TATGAGGTTC?GATATTTTCC?ATATGCATTG?GCCTGAAACT?TATTTAAACT 7020

TACCTAAACG?GTATCAACAG?TTAATTGGGA?CTCTGTGTGT?TGTATTGTTT?TTAGTTGTAT 7080

GCAAGCTTTT?TTCTAAAAAA?ATCGTATGGA?CCGTTCATAA?TTTCAAGCCT?CATGAAAATC 7140

ACAATAATAT?TAAGATATCA?GAATTTTTTC?GTACATTCCT?TGTTCGAATG?GTAGATAAAT 7200

TTATTTTTTT?AACAGAAGTT?AGCAAAGATG?AATTTCTGAA?AATTTACAAT?ATTGATAATT 7260

CAAGATTAAC?TGTGATACAT?CACCCACTGT?ATCAAGTAAA?TGAGTGTTGC?CATGATATTT 7320

CAGCAGCTAA?TCATTCATCA?ATTAATAATA?AATATCTTTT?TTTCGGATTA?ATTAGGCCTT 7380

ATAAAAACAT?AGAGTTATTA?ATGAATGAAT?TTATTAAATA?TGATTCTGAT?TCGGTATTGA 7440

TAATTATGGG?AGGGTGCTCC?AACACATTAG?CTACTGAATT?AAGGAACATG?AAGAAAAAAA 7500

TTGATTTTAA?AGAACGCATC?AAATTTAAAT?TTGGGTACTA?TAATGAACAA?CAACTTCAGC 7560

The termination of Orf5

AGAGCTCAAT?CAATGTAAAG?GTG TAATAAT?ACCATACTCA?GACATAATTG?AATTTCTGGT 7620

GTGCTATATC?GTGCAATAAC?TGCGGGTGTA?AATGTGTTAC?TGCCTCGTAT?AAGATATACA 7680

GAAGAATTGG?TCAATTCTCT?TAAATATCAA?GGTGCTGTAT?TTTGTGACCC?TCCACTTGAT 7740

ACGAATGATA?TAGCGACGTT?TAATGGCATA?CCCTCAGTAA?ATCATAATAA?TTTTGATGTG 7800

GATACATATA?ACACATATAT?AAAAAATGCA?CATTATGAAC?TTTATCAAAC?CATAATTTAA 7860

Orf6's is initial

GACTTAAATT?AAGCGTAAA A?TGAAGAATAT?ATTAATAGTT?TGTGATTATT?TTTATCCCGC 7920

TTTCAAAGCG?GGAGGGCCTA?TTAAAAGTCT?CGGAAATATA?GCCAAAACTT?TCAATGACAG 7980

AAATTACATA?ACCGTATTGA?CCTACAATCA?GGATATTGAT?GGTCAGATCA?TAAATAAATC 8040

TGGCACTGAA?ATTTTTGAAA?AAAATATTGT?CGTTTATTAT?GCTCAAAACC?TGCTGAATTT 8100

TGTTAAATTG?TATTTAAAAC?TTTACAAAAC?AGCTGATATA?ATCTATTTAA?ATAGTTTTTT 8160

TTCGAGTAGA?ACTACGTTTC?CTGCATTAAT?GCTTAAGAAG?ACTAGCGCGA?AGGTTTTGCT 8220

GGCCCCTCGT?GGTGAACTAA?CATTAGGTGC?ATTGACGTTA?AAACCATTAA?AAAAGAAAAT 8280

ATATATAAAA?TTATTTAATA?TTTTCGCTAA?CAAAAATGAA?ATTTATTTTC?AATTTACATC 8340

TGAAGAAGAA?AAGAATGAAT?CATTGATATC?CTTAAAAAAA?GGATTTTCAT?ATACTGTCAT 8400

TCCTAATATG?CATGATCCGA?TTCCACCTTA?TATATATAAG?CATAAGAAGG?AAGGTGAAAT 8460

CCATATTTCA?TTTATTTCGC?GTATTTCCCC?CAAAAAAAAT?CTCAAAGCAG?TGCTATTGTC 8520

TCTTTTAAAT?ATAGCCCAAG?GCGACATACT?CCTTAGTATT?GCAGGAAATA?TCGAGGATGA 8580

AAAATATTGG?GCGGAATGTA?TGTTGATAAT?AAATAAATTA?CCACAGAATG?TTAAAGTAAA 8640

TTATCTCGGT?GGAATCAAGC?CTACTCAGGT?AATTGATTTA?CTTAAGAGCT?CGCATTTGTT 8700

TTTCTTGCCA?ACATTAAATG?AGAACTATGG?GCATGCTATT?GTTGAGTCCA?TGATAAACTC 8760

AAATGTTGTG?CTAATATCCG?ATCAAACACC?ATGGTCCGAT?GTGCAATGGA?ATGGCGGTTA 8820

TGTTGCAGGT?TATAATGATA?CTGATACATT?CAAGAAGTGT?ATAACCGAAT?GTATGAAGTT 8880

CGATGAGTCG?CAATTTAATA?ATAAATCACG?ACAAGTCTAT?GACTATGCAC?GAAATGCTTT 8940

The termination of Orf6

AACTAAACAT?GAATATAAAA?CATCAACTTT?ATTCGATATT? TAGATGTAAA?TTTATGCTTT 9000

TTCATTTTAC?TTAAGTTTCT?ATGTAAACAG?TTATTCCCAA?TTAACGGTGT?AGTATTATTT 9060

AACATCTTTT?ATTTAAAATT?TTCAATGTGG?TAATGAATTG?AGTTTTATTG?CTCGCTTTTC 9120

Orf7's is initial

TTCATATAAA?AACACTTTAC?GTGTTTGCTC?ACTCTAGAGG?AATTAGT ATG?TTTAAAGATA 9180

AGGTTTTACT?TATTACAGGT?GGCACAGGTT?CATTTGGTAA?TGCTGTATTA?AAGCGTTTTT 9240

TAGATACGAA?TATCAAAGAA?ATTCGCATTT?TTTCGAGAGA?TGAGAAAAAA?CAAGATGATA 9300

TGCGAAAGAA?ATATAGTAAC?AATAAACTCA?AGTTTTATAT?CGGTGACGTA?AGGGATTATT 9360

CGAGTGTGTT?AAATGCATCT?CGAGGAGTTG?ATTTTATTTA?TCATGCGGCT?GCATTAAAAC 9420

AGGTTCCTTC?TTGTGAATTT?TATCCAATGG?AAGCAGTAAA?AACTAATATT?ATTGGCACTG 9480

ATAATGTTCT?GGAAGCAGCT?ATTGCCAATA?AAGTGTCAAG?AGTAGTGTGT?TTGAGTACTG 9540

ATAAAGCAGT?ATACCCAATC?AATGCTATGG?GAACATCTAA?AGCAATGATG?GAAAAGGTCA 9600

TAGTTGCTAA?ATCTCGTAAT?GTTCCTGATG?AAATGGTTAT?ATGTGCAACA?CGTTATGGGA 9660

ATGTTATGGC?TTCAAGAGGT?TCTGTTATTC?CCCTTTTTGT?TAATCAGATA?TTAACTGGTA 9720

AACCGATTAC?AATCACCGAT?CCAAATATGA?CTCGTTTCAT?GATGACATTA?GATGATGCAG 9780

TTGATTTGGT?TGTACATGCT?TTTAAACACG?GACAAAATGG?AGATATTTTT?GTACAGAAAT 9840

CTCCTGCTGC?TACCATTGAA?GTTTTGACTC?ATGCGATTCT?AGAAATTATG?AAATCGGCTG 9900

ATCACACTGT?TAATATTATC?GGTACACGTC?ATGGCGAAAA?ACTATATGAG?GTTTTATGTA 9960

GCAGAGAAGA?GATGTTGGTA?GCCGAAGATC?AAGGTAACTA?TTATAGAATA?CCGTGCGATA 10020

AGCGAGATTT?GAATTATGAA?AAATATTTTG?AGAAAGGCAA?CAAAAAAGTT?GAAACAATAG 10080

ATGATTATAA?TTCACATAAT?ACATATCGTT?TAAATACTCA?AGAGATGGTT?TCATTACTAC 10140

The initial Orf7 of orf8 stops

GTAAACTTAC?CTATATCAAT?AAAATTGAAG?CTGGTGAAAA?GGCTGATCCT?G ATGAA TAAA?10200

AGAATATTGA?TTGTTGGTGC?TAATGGCATG?CTGGGCAGCA?GTCTGCTACG?ATATTTTACC 10260

CAATATAGTA?GATATGATGT?TTTGGGTACT?GTACGTAGTA?ACAGTGCAAA?GGCAAAACTA 10320

TCTAAACAAG?GTTTTGATAA?TATCATATCT?CAAGTTGATG?TGCTTGAATA?TGAAACAATC 10380

GAGAATGTTA?TTTCAGACTG?GAAACCAGAT?TTTTTATTTA?ATTGTGTAGG?TATTATCAAA 10440

CAGCTTGACG?CTGCGAAGAA?TAACATACTT?TCATTATCAA?TCAATTCTTT?GTTGCCACAC 10500

CAACTTGCTA?AAACATGTAC?TCGTAATGAA?ACTAAACTAA?TACATTTTTC?TACTGATTGC 10560

GTATTTAGTG?GTAAGCAAGG?TAATTATTGT?GAAGAAAGCT?TTCCAGATGC?TTACGATCTA 10620

TATGGAAAAT?CAAAGCAACT?TGGTGAAGTG?GTTTATGACG?GTCATTTAAC?TTTACGGACT 10680

TCAATTATTG?GGCATGAAAT?CTCAAGCCAG?CACAGTTTGA?TCGATTGGTT?TTTATCTCAA 10740

AAAAAATATA?TTTATGGATA?CTCAAAAGCT?ATTTTTTCTG?GAATGCCAAC?AGTATATGTA 10800

GCCGAGATTA?TTCACGACTA?TATTCTTCCA?AATCCAAACT?GTTGTGGGCT?AAGACACCTT 10860

AGTGTTGATC?CTATTGATAA?ATTCAGCTTA?TTGAAGCTAG?TAAAAAAGCA?ATACTCTCAC 10920

AATATTGAGA?TTATAGAATC?AAGTGATTTA?GAGATTGATC?GTAGTCTTGA?TTCATTTCTA 10980

Orf9's is initial

TTACGCCAAG?AAGTGGGGTT?TTATCCAGCA?AGTTGGCCTA?AACTGATTGA?GAAAATGA AT 11040

The termination of Orf8

GATGAATACA?ATAAATTCTT?CCGT TAAAAA?AATGAAAGTT?GTTACTGTTG?TGGGTACCAG 11100

ACCGGAGATA?ATTCGTTTAT?CGAGAGTAAT?GGCTGTTCTT?GATCAGTACA?CAGAACATAC 11160

CATCATTCAC?ACTGGACAGA?ACTATGATTA?TGAACTTAAT?GAGGTTTTCT?TCAAGGAGTT 11220

AGAGATCAGA?AAACCAGATT?ACTTCCTTAA?TGCAGCAGGT?TCTAACCCAG?CTGAAACTAT 11280

TGGTAATATA?ATTATTGAAG?CAGACAAAAT?TTTTGACATA?ATTTCTCCAG?AGGCTCTTTT 11340

AATACTTGGG?GATACAAATA?GTGCACTAGT?TTCTATTGCA?GCTAAAAGAC?GTAAAATTCC 11400

AATATTTCAT?ATGGAAGCAG?GAAACAGATG?TTTTGATTAT?CGAGTTCCTG?AGGAAATTAA 11460

TCGAAAGATT?GTGGATCATA?TTGCCGATAT?AAACCTTACT?TACAGCGAAA?TTGCTCGTGA 11520

GTACTTGTTG?CGTGAAGGGA?TTCCTGCAGA?TCAGATTATC?AAAACAGGTA?GCCCAATGCG 11580

TGAGGTTTTA?AATTATTATA?ATGGAGAGAT?AAATAAATCT?AAAGCACTCG?AAAAGTTGAA 11640

TTTAACTCCT?AATGAGTATT?TTCTGGTCAG?CGCACATCGA?GAAGAGAATG?TAGATTCTCC 11700

TGAAAAATTA?AAAATTTTTA?TTAACATTCT?TAATAAAATA?TCGGATAAAT?ATGATTTGCC 11760

AATAATAGTA?TCTACGCACC?CACGAACCCG?TAATCGAATG?GATAAGCTTA?ATGTAGAAAT 11820

AAGCAATAAT?GTTTATTTTA?TGAAACCATT?CGGTTTCTTA?GATTATGTTT?CTTTGCAAAA 11880

GAATGCTCGC?GTAGTTCTGT?CTGACAGCGG?TACAATAACG?GAAGAATCTT?CGATTTTAAA 11940

TTTTCCCGCT?CTTAATTTAC?GTGAAGTTCA?TGAACGTCCG?GAAGGTTTCG?AAGAAGCTGC 12000

CGTAATGTTC?GTTGGGCTGG?ATGCTGTTCG?TATTTTTCAA?GGTATTGAAA?TTTTGGATCA 12060

ACAGCAGAGA?GGACCGGATA?ATCGAGATTT?GTATTTGGTC?AATGATTATT?CGGTTGACAA 12120

TGTATCGCTA?AAAGTCCTTA?GAATCATAAT?GAGCTATACT?AATTTTGTTA?ACCAAAAGGT 12180

The termination orf10's of Orf9 is initial

TTGGAAAAAG?CAA TAATGCG?AATAGCGTTA?ATCTGTGATG?ATTATCTGCC?TGATAGCACA 12240

CGTGTTAGTG?CAAAAATGAT?GCATGAATTG?GCTTGTGGAC?TTTTAAGAGA?AGGCCATGAG 12300

CCGGTTGTAT?TTTGCCCTAA?TGGTGGTGTT?GGTACTGAAT?TAAAAGTAAT?TTATTTGGAT 12360

GGAATATGTA?TTTATAAATA?TCCAAATGGA?CAAACAAAAG?ATATATCAAA?AATTAAGCGT 12420

GCATTTAACG?AAACTATGCT?TTCATTCAAT?GCATGGCATT?ACCTTTCAAG?CGAAATTAAA 12480

CGTAAAAAAA?TTGATGGTGT?TATTTATTAT?TCACCATCAA?TTTTCTTTGG?GGCTTTTGTA 12540

AAAAAAATTA?AAAAATATTG?GAAATGTAAA?TCATATCTAA?TTTTACGTGA?TTCATTTCCC 12600

CAATGGTTAG?TTGACCAAAG?GATAATTCGT?AGAAATGGTA?TGCTTGAGAA?ATATTTTCGT 12660

TTTTTTGAAA?GAAGAATTA TACTGCAGCC?AATTATATTG?GTGTAATGTC?ATCTAAAAAT 12720

AAAGAACTTT?TTACATTACA?GTATCCCCAT?TACAAAAACG?TAGATGTCTT?ATATAATTGG 12780

GCAGATCTTA?ATAGCACTCA?AGATGTTTCA?CCAAGTGATA?TTTTAGAAAG?GCTATCATTG 12840

GCTGATAAAG?TTATATTTTT?CTATGGAGGA?AATATAGGTC?ATGCTCAAGA?CATGGCAAAT 12900

TTAATGAGAT?TGGCAATAGG?TATGCGTACT?ATTAATTACG?TTCATTTTCT?TTTTGTTGGA 12960

CAAGGGGATG?AGGTCGAACT?TGTTAAAAAA?ACAATTGCCG?AAAATGCACT?TGAGAACTGT 13020

ACATACTTAC?CTGCAATTAG?TCAGTCGGAA?TATAAATCAA?TTTTAAAGGT?TGTTCATGTA 13080

GGGTTGTTTA?GCCTTGCAAA?GAATCATTCT?GTGCATAATT?TTCCGGGCAA?ACTTCTAGGA 13140

TATATGGCAA?ATAAATTACC?AATTTTAGGT?AGTGTGAATG?AAGGTAATGA?TCTTATGCAA 13200

GTGCTCATTG?GCGCAAAGGC?TGGATATGTG?CACGTTAATG?GATATGATGC?AGAATTATTG 13260

CAATCAGCTA?TAAAATTAGC?TACGGAAGAG?AAATTAAGGA?ATGAATTAGG?AGAAAATGGT 13320

CATTCACTTT?TAGAGAAGAC?CTTTTCTACG?CAATCTGCTG?TCGAGAACAT?TTTGTTACGG 13380

The termination orf11's of orf10 is initial

CTTAAG TGAA?TTATTAAATG?TAGGAATTGA?T ATGTCGGTT?ACTGTATTTT?ATAAAGAGCA?13440

GTTAAATGAA?TTGTCAAATC?TTGCAAGTAA?TAACCCTCGG?CTAAGGGCTC?ATTTAAACTT 13500

ACATAAGAGT?TATCAAGATA?AGATTCAGAG?GATTTTAATT?TCCTTAAAGA?GGGGAACATA 13560

CATACCGCCA?CATTATCATA?AATATGATCA?TCAGTGGGGA?ACATTTCAAG?TGATCGAGGG 13620

TATAGTCGAT?TTATATATAT?TTAATAATTC?AGGTACTGTC?TTAGATATTA?TTAATTTGGG 13680

AGATGTAAAC?GGTGCTTTAT?TTGCACAAAT?AGAACCCTAT?ACCATACATA?CACTTGTCTG 13740

CCGTTCTCCT?TATGCTTCTG?TAATCGAAAT?TAAAGAAGGG?CCTTTTGTTG?AGGCAGAGGC 13800

TAAAATTGTT?CCCTCTTGGG?TGTATCCGGA?AGGCTGTAGA?GAGTACTTAC?GTTCTGATAT 13860

The termination of orf11

CGTATCAATG?CTTGAAACTC?TCAAGATTAA?TGAAGTATAT?AAGCTT TAAC?TGATTAAGGA 13920

TAATATGGAA?TCAATTTAAT?TAAATCAATC?ACCACCTCCC?TAGGATGTGG?TTTAGGAATA 13980

ACAATTATAA?AATTTCTTAA?TGTTGAAAGA?GGCTGCAGTT?TCCCTCTATT?TTTTTATTAG 14040

GACCTGAGTT?AGCATCAAAG?TTACATTCAA?GCCGCATACA?TCGCGGTGAA?CACCCCCTGA 14100

CAGGAGTAAA?CAATGTCAAA?GCAACAGATC?GGCGTCGTCG?GTATGGCAGT?GATGGGGCGC 14160

AACCTTGCGC?TCAACATCGA?AAGCCGTGGT?TATACCGTCT?CTATTTTCAA?CCGTTCCCGT 14220

GAAAAGACGG?AAGAAGTGAT?TGCCGAAAAT?CCAGGCAAGA?AACTGGTTCC?TTACTATACG 14280

GTGAAAGAGT?TTGTTGAATC?TCTGGAAACG?CCTCGTCGCA?TCCTGTTAAT?GGTGAAAGCA 14340

GGTGCAGGCA?CGGATGCTGC?TATTGATTCT?CTCAAGCCAT?ACCTCGATAA?AGGTGACATC 14400

ATCATTGATG?GCGGTAACAC?CTTCTTCCAG?GATACCATCC?GTCGTAACCG?TGAGCTTTCT 14460

GCCGAAGGCT?TTAACTTCAT?TGGTACCGGT?GTTTCCGGTG?GTGAACAAGG?TGCGCTGAAA 14520

GGTCCTTCCA?TTATGCCTGG?TGGGCAGAAA?GAAGCCTATG?AACTGGTTGC?ACCGATCCTG 14580

ACCAAAATCG?CCGCAGTGGC?TGAAGATGGC?GAACCGTGTG?TTACCTATAT?TGGTGCCGAT 14640

GGCGCGGGTC?ACTATGTAAA?AATGGTTCAC?AACGGTATTG?AATACGGTGA?TATGCAGCTG 14700

ATTGCTGAAG?CCTACTCTTT?GCTTAAAGGT?GGCTTGAACC?TTTCCAACGA?AGAACTGGCG 14760

CAGACCTTTA?CTGAGTGGAA?TAACGGTGAA?CTGAGCAGCT?ACCTGATTGA?CATCACAAAA 14820

GACATCTTCA?CTAAAAAAGA?TGAAGACGGT?AACTACCTGG?TTGATGTGAT?TCTGGATGAA 14880

GCGGCTAACA?AAGGTACCGG?TAAATGGACC?AGCCAGAGCG?CGCTGGATCT?CGGTGAACCG 14940

CTGTCGCTGA?TTACCGAGTC?TGTGTTTGCA?CGTTATATCT?CTTCTCTGAA?AGATCAGCGT 15000

GTTGCCGCAT?CTAAAGTTCT?CTCTGGCCCG?CAAGCGCAGC?CAGCTGGCGA?CAAGGCTGAG 15060

TTCATCGAAA?AAGTTCGTCG?TGCGCTGTAT?CTGGGCAAAA?TCGTTTCTTA?CGCTCAGGGC 15120

TTCTCTCAGC?TACGCGCCGC?GTCTGAAGAG?TACAACTGGG?ATCTGAAATA?CGGCGAAATC 15180

GCGAAGATTT?TCCGTGCTGG?CTGCATCATC?CGTGCGCAGT?TCCTGCAGAA?AATCACCGAT 15240

GCTTATGCCG?AAAATCCGCA?GATCGCTAAC?CTGCTGCTGG?CTCCGTACTT?CAAGCAAATT 15300

GCCGATGACT?ATCAGCAGGC?GCTGCGTGAT?GTCGTTGCTT?ATGCAGTACA?GAACGGTATC 15360

CCGGTTCCGA?CCTTCGCCGC?TGCGGTTGCC?TATTATGACA?GCTACCGTAA?CGGCTGTTCT 15420

GCCTGCGAAC?CTA 15433

Only being preferred embodiment of the present invention below, is not that the present invention is imposed any restrictions, all according to the technology of the present invention essence to above embodiment make an amendment, equivalent variations and modification, all belong in the scope of technical solution of the present invention.

Claims

1, a kind of Nucleotide of the O-antigen-specific to intestinal bacteria O163 is characterized in that it is the isolating Nucleotide shown in SEQ ID NO:1,15433 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.

2, according to the Nucleotide of the described O-antigen-specific to intestinal bacteria O163 of claim 1, it is characterized in that, it comprises 11 genomic constitutions of called after manC, manB, wzx, wzy, orf5, orf6, fnlA, qnlA, qnlB, orf10, orf11, all between galF gene and gnd gene.

3, according to the Nucleotide of the described O-antigen-specific to intestinal bacteria O163 of claim 2, it is characterized in that the gene that has high degree of specificity in the described gene comprises: transhipment enzyme gene 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; Glycosyltransferase gene comprises orf5, orf6, orf10 gene.Wherein said transhipment enzyme gene is the Nucleotide of 4349 to 5602 bases among the SEQ ID NO:1; Described pol gene is the Nucleotide of 5599 to 6834 bases among the SEQ ID NO:1; Described orf5 gene is the Nucleotide of 6852 to 7586 bases among the SEQ ID NO:1; The orf6 gene is the Nucleotide of 7880 to 8983 bases among the SEQ ID NO:1; The orf10 gene is the Nucleotide of 12196 to 13389 bases among the SEQ ID NO:1.

4, according to the Nucleotide of claim 1 or 2 described O-antigen-specifics to intestinal bacteria O163, it is characterized in that, it also comprises oligonucleotide or the glycosyltransferase gene that comes from described wzx gene or the wzy gene, and their mixing or their reorganization.

5, according to the Nucleotide of the described O-antigen high special to intestinal bacteria O163 of claim 4, it is characterized in that the oligonucleotide of the described wzx of coming from gene is to being:

The Nucleotide of 4500 to 4518 bases and 5425 to 5442 nucleotide bases among the SEQ ID NO:1,

The Nucleotide of 5100 to 5117 bases and 5503 to 5520 nucleotide bases among the SEQ ID NO:1;

The oligonucleotide of the described wzy of coming from gene is to being:

The Nucleotide of 6102 to 6119 bases and 6738 to 6755 nucleotide bases among the SEQ ID NO:1,

The Nucleotide of 6223 to 6240 bases and 6784 to 6801 nucleotide bases among the SEQ ID NO:1.

6, the Nucleotide of the described O-antigen-specific to intestinal bacteria O163 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 O163 of claim 1 is providing the O-antigen of expressing intestinal bacteria O163 by inserting to express, and the application in the preparation bacterial vaccine.

8, the application of the Nucleotide of the described O-antigen-specific to intestinal bacteria O163 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, bacterial detection as probe as primer.

9, the separation method of the Nucleotide of the described O-antigen-specific to intestinal bacteria O163 of claim 1 is characterized in that, comprises the steps:

10, the separation and the authentication method of the Nucleotide of the described O-antigen-specific to intestinal bacteria O163 of claim 9 is characterized in that, comprise the steps:

(7) detection of primer sensitivity: the frozen bacterium liquid of intestinal bacteria O163 is inoculated in the triangular flask of LB substratum, 30 ℃ of-40 ℃ of cultivations, 180 to 250 rev/mins, cultivate a few hours to saturated, get cultured bacterium liquid dilution, get dilution bacterium liquid coating LB agar plate, 30 ℃ to 40 ℃, cultivate a few hours counting, calculate viable bacteria concentration in the stoste; In being the live pig meat stuffing of 20g, 5 parts of weight mix 5 * 10 respectively ³, 5 * 10 ², 5 * 10 ¹, 5 and 0 viable bacteria stir, and add the LB substratum, filter, and filtered liquid 180 to 250 rev/mins, is cultivated a few hours in 30 ℃ of-40 ℃ of cultivations; Peek ml is in 6 from cultured bacterium liquid, and centrifugal several minutes of 000g removes supernatant, adds the MQ ultrapure water and blows precipitation and mixing open, puts into 100 ℃ of boiling water and boils several minutes, and lysate is in 12, and centrifugal several minutes of 000g gets supernatant as pcr template; To carrying out the PCR reaction, the PCR reaction system is as follows: MQ:15.7 μ l, Mg with 4 pairs of oligonucleotide ²⁺: 2.5 μ l, Buffer:2.5 μ l, dNTP:1 μ l, Taq enzyme: 0.3 μ l, P1:1 μ l, P2:1 μ l, template DNA: 1 μ l.The PCR reaction conditions is: 95 ℃: 5 ', 95 ℃: 30 ", 56 ℃: 45 ", 72 ℃: 1 ', 72 ℃: 5 ', totally 30 circulations; Reaction is got 10 μ l reaction product electrophoresis after finishing, if the amplified band that conforms to the expection size is arranged, then the result is positive, if do not have, then the result is negative; Participated in 5 * 10 ³, 5 * 10 ², 5 * 10 ¹And every part of pork filling of 5 viable bacterias all obtains positive findings in the PCR of 4 pairs of primers reaction; The pork filling that participates in 0 viable bacteria obtains negative findings in the PCR of 4 pairs of primers reaction; Illustrate that these 4 pairs of primers are 0.25 bacterium/g to the detection sensitivity of the intestinal bacteria O163 in the pork filling when using aforesaid method.