CN1313477C - Nucleotide specific to O-antigen of 025 type b acillus coli - Google Patents

Abstract

The present invention provides a specific nucleotide for the O-antigens of Escherichia coli O25. The specific nucleotide is the nucleotide complete sequence of gene clusters in escherichia coli for controlling the synthesis of O-antigens, such as a separated nucleotide with the total length of 17566 basic groups shown as SEQ ID NO: 1 or the nucleotide of SEQ ID NO: 1 which has one or a plurality of inserted, deleted or substituted basic groups and can simultaneously keep the functions of the separated nucleotide. The present invention also comprises the oligonucleotide of glycosyltransferase genes and unit processing genes stemmed from the O-antigen gene clusters of Escherichia coli O25. The present invention verifies that the oligonucleotide has high specificity to all of the O-antigen genes of Escherichia coli O25 PCR. The present invention also discloses a method for using the oligonucleotide of the present invention to detect and identify Escherichia coli O25.

Description

Nucleotide to the O-antigen-specific of intestinal bacteria O25 type

Technical field

The present invention relates to the complete nucleotide sequence of control O-antigen synthetic gene cluster in the intestinal bacteria O25 type (Escherichia coli O25), particularly relate in the intestinal bacteria O25 type oligonucleotide in the control O-antigen synthetic gene cluster, can utilize these the oligonucleotide of the O-antigen-specific intestinal bacteria O25 type in human body and the environment and identify O-antigen in these pathogenic bacterium quickly and accurately.

Background technology

O-antigen is the O specific polysaccharide composition in the gram negative bacterium lipopolysaccharides, and it is made up of many multiple oligosaccharide unit.The antigenic building-up process of O-is studied clearlyer: by glycosyltransferase nucleoside diphosphate monose is transferred on the fat molecule that is fixed on the cell inner membrance earlier, then in the inboard synthesis of oligose unit of inner membrance, the antigenic oligosaccharide unit of O-is transferred to the inner membrance outside by the transhipment enzyme again, then aggregate into polysaccharide by polysaccharase, be connected to again and form lipopolysaccharide molecule [Whitfield, C. (1995) " Biosynthesis of lipopolysaccharide O antigens " .Trends inMicrobiology.3:178-185 on the glycolipid molecule; Schnaitman, C.A.and J.D.Klena. (1993) " Genetics of lipopolysaccharidebiosynthesis in entericbacteria " .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 gene nomenclature " Trends inMicrobiology, 4:495-503].In Shigellae, intestinal bacteria and Salmonellas, O-antigen gene [Lei Wang.et al (2001) " Sequence analysis of four shigella boydiiO-antigen loci:implication for Escherichia coli and Shigella relationships " .Infection andImmunity, 11:6923-6930 bunch between galF and gnd gene; Lei Wang and Peter Reeves (2000) " The Escherichia coli O111 andSalmonella enterica O35 gene clusters:gene clusters encoding the same colitose-containing Oantigen are highly conserved " .Journal of Bacteriology.182:5256-5261].The O-antigen gene bunch contains three genoids: sugared synthesis path gene, glycosyltransferase gene, oligosaccharide unit treatment gene, the required nucleoside diphosphate monose of enzymic synthesis O-antigen of wherein sugared synthesis path genes encoding; Thereby the enzyme of glycosyltransferase gene coding forwards nucleoside diphosphate monose and other molecule to and makes monose aggregate into oligosaccharide unit on the monose; The oligosaccharide unit treatment gene comprises transhipment enzyme gene and pol gene, and they transfer to the bacterium inner membrance outside with oligosaccharide unit, and repolymerization becomes polysaccharide.Glycosyltransferase gene and oligosaccharide unit treatment gene only are present in the gene cluster of carrying these genes.The difference of monose in the O-antigen, between monose between the difference of link button and the oligosaccharide unit difference of link button constituted the antigenic diversity of O-, and the composition of monose, the link button between monose and the link button between the oligosaccharide unit are by the Gene Handling in the O-antigen gene bunch, so the O-antigen gene bunch has determined O-antigenic synthetic, has also determined the antigenic diversity of O-.

Because O-antigen is extremely strong antigen, be one of important paathogenic factor of intestinal bacteria, it has extremely strong diversity again simultaneously, and this enlightens us can study a kind of intestinal bacteria and good, highly sensitive method of the antigenic specificity of O-thereof of detecting quickly and accurately.With surperficial polysaccharide is that the serology immune response of target has been used to somatotype and the evaluation to bacterium always since the thirties in last century, is unique means of identifying pathogenic bacterium.This diagnostic method needs a large amount of antiserum(antisera)s, and the antiserum(antisera) general classes is incomplete, quantity not sufficient, and also there are some difficulties in a large amount of antiserum(antisera)s in preparation with in storing.On the other hand this method length consuming time, sensitivity is low, loss is high, poor accuracy, so, generally believe that now this traditional serology detection method will be that the modern molecular biology method replaces.1993, Luk, J.M.C et.al has identified the O-antigen [Luk of Salmonellas with the specific nucleotide sequence of Salmonellas (S.enterica) O-antigen gene bunch by PCR method, J.M.C.et.al. (1993) " Selective amplification of abequose and paratosesynthase genes (rfb) by polymerase chain reaction for identification of S.enterica majorserogroups (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-Uremic Syndrome caused by dryfermented sausage contaminated with Shiga-like toxin producing Escherichiacoli " .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.and Reeves 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 in the antigenic structure of the O-of other bacterium, also has this sugar, so sugared synthesis path gene is not that the Shigellae of high special has 46 kinds of serotypes for O-antigen, but have only 33 kinds of different O-antigens, intestinal bacteria have 166 kinds of different O-antigen [Reeves, P.R (1992) " Variation in O antigens; niche specific selection and bacterialpopulations " .FEMS Microbiol.Lett, 100:509-516], the two sibship is very near, and there are 12 kinds to be intestinal bacteria and the total [Ewing of Shigellae, W.H. (1986) " Edwards and Ewing ' sidentification of the Enterobacteriaceae " .Elsevier Science Publishers, Amsterdam, TheNetherlands; T.cheasty, et al. (1983) " Antigenic relationships between the enteroinvasiveEscherichia coli antigens O28ac; O112ac; O124; O136, O143, O144; O152 and and Shigella Oantigens " J.clin Microbiol, 17 (4): 681-684].

Summary of the invention

The Nucleotide that the purpose of this invention is to provide a kind of O-antigen-specific to intestinal bacteria O25 type.It is the Nucleotide in the O-antigen gene bunch of intestinal bacteria O25 type, is the special Nucleotide that comes from glycosyltransferase gene and transhipment enzyme gene and pol gene.

A time purpose of the present invention has provided the full length nucleotide sequence of the O-antigen gene bunch of intestinal bacteria O25 type.

Another object of the present invention has provided the gene of the O-antigen gene bunch that constitutes intestinal bacteria O25 type: the gene of transhipment enzyme is the wzx gene or with wzx the gene of identity function is arranged; Pol gene is the wzy gene or with wzy the gene of identity function is arranged; Glycosyltransferase gene comprises orf6, orf7, orf9, orf13 gene.

Another purpose of the present invention has provided oligonucleotide, and the gene that they come from coding transhipment enzyme in the O-antigen gene bunch of intestinal bacteria O25 type respectively is the wzx gene or with wzx the gene of identity function is arranged; The gene that comes from the coding polysaccharase is the wzy gene or with wzy the gene (table 1) of identity function is arranged; They are the oligonucleotide in the said gene, and length is at 10-25nt; They are high specials to the O-antigen of intestinal bacteria O25 type; And these oligonucleotide are also reconfigurable, and the oligonucleotide after the combination also is a high special to the O-antigen of intestinal bacteria O25 type.

A further object of the present invention provides above-mentioned oligonucleotide and can be used as primer and be used for nucleic acid amplification reaction, perhaps be used for hybridization as probe, perhaps be used to make gene chip or microarray, thereby pass through O-antigen and the detection and the identification of escherichia coli O25 type of these methods detections and identification of escherichia coli O25 type.

An also purpose of the present invention has provided the method for complete sequence of the O-antigen gene bunch of separating Escherichia coli O25 type.Can obtain the complete sequence of the O-antigen gene bunch of other bacteriums according to present method operation, the complete sequence of the gene cluster of the bacterium of other polysaccharide antigens that also can obtain to encode.

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

The present invention is characterized in that the Nucleotide of the O-antigen-specific of intestinal bacteria O25 type: it is the isolating Nucleotide shown in SEQ ID NO:1,17566 bases of total length; Perhaps described base with one or more insertions, disappearance or replacement keeps the Nucleotide of the SEQ IDNO:1 of described isolating functional nucleotide simultaneously.

The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O25 type is comprising called after rmlB, rmlD, rmlA, rmlC, wzx, orf6, orf7, wzy, orf9, fnl1, fnl2, fnl3, orf13,14 genomic constitutions of orf14 are all between galF gene and gnd gene.

The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O25 type, the gene that has high degree of specificity in the wherein said gene is: transhipment enzyme gene, it comprises the wzx gene; Pol gene, it comprises the wzy gene; Glycosyltransferase gene, it comprises orf6, orf7, orf9, orf13 gene; Wherein said gene: wzx is the Nucleotide of 4604 to 5863 bases among the SEQ ID NO:1; Wzy is the Nucleotide of 8039 to 9100 bases among the SEQ IDNO:1; Orf6 is the Nucleotide of 5868 to 6833 bases among the SEQ ID NO:1; Orf7 is the Nucleotide of 6919 to 8067 bases among the SEQ ID NO:1; Orf9 is the Nucleotide of 10127 to 11230 bases among the SEQ ID NO:1; Orf13 is the Nucleotide of 14508 to 15716 bases among the SEQ ID NO:1.

The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O25 type wherein also comprises coming from described wzx gene, wzy gene and their mixing or their reorganization.

The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O25 type is characterized in that the oligonucleotide that wherein comes from the wzx gene is to being: the Nucleotide of 4946 to 4965 bases among the SEQ ID NO:1 and the Nucleotide of 5329 to 5348 bases; The Nucleotide of 5327 to 5346 bases among the SEQ ID NO:1 and the Nucleotide of 5778 to 5797 bases.The oligonucleotide that comes from the wzy gene is to being: the Nucleotide of 8622 to 8641 bases among the SEQ IDNO:1 and the Nucleotide of 8973 to 8980 bases; The Nucleotide of 8349 to 8366 bases among the SEQ IDNO:1 and the Nucleotide of 8993 to 9009 bases.

The application of the Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O25 type in detecting other polysaccharide antigen of expressing the antigenic bacterium of O-, the O-antigen of identifying bacterium and bacterium.

The recombinant molecule of the Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O25 type is providing the O-antigen of expressing intestinal bacteria O25 type by inserting to express, and the application in the preparation bacterial vaccine.

The application of the Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O25 type is characterized in that it is used for PCR, is used for hybridization and fluoroscopic examination or is used to make gene chip or microarray as probe as primer, for the application of bacterial detection.

The separation method of the Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O25 type is characterized in that it comprises the steps:

(1) genomic extraction: in substratum, cultivate intestinal bacteria O25 type, centrifugal collecting cell; The genomic dna that obtains detects by agarose gel electrophoresis;

(2) by the O-antigen gene in the pcr amplification intestinal bacteria O25 type bunch: with the genome of intestinal bacteria O25 type is that template is passed through its O-antigen gene of Long pcr amplification bunch, with the PCR product that obtains, detect the size and the specificity thereof of PCR product with agarose gel electrophoresis, merge this long PCR product, and with DNA purification kit purified pcr product;

(3) make up O-antigen gene bunch library: Long PCR purified product is used shotgun make up O-antigen gene bunch library;

(4) to the cloning and sequencing in the library: from the library, select the clone of insertion fragment more than 1kb and the insertion fragment among the clone is checked order with laboratory automatic dna sequencer commonly used, sequence reaches 100% fraction of coverage, thereby obtains all sequences of O-antigen gene bunch;

(5) splicing of nucleotide sequence and analysis: applying biological information science software splicing and edit all sequences, thus obtain the Nucleotide full length sequence of the O-antigen gene bunch of intestinal bacteria O25 type;

(6) screening of specific gene: at wzx, the wzy gene design primer in the O-antigen gene of intestinal bacteria O25 type bunch; Respectively designed two pairs of primers in each gene, every pair of primer is distributed in the different places in the corresponding gene, to guarantee its specificity; Is that template is carried out PCR with these primers with the genomes of 166 strain intestinal bacteria and 43 strain Shigellaes, determines the antigenic high degree of specificity of O-of wzx, wzy gene pairs intestinal bacteria O25 type;

(7) detection of primer sensitivity: cultivate intestinal bacteria O25, 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 O25.

The separation method of the Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O25 type is characterized in that it comprises the steps:

(1) genomic extraction: 37 ℃ of incubated overnight intestinal bacteria O25 types in the LB of 5mL substratum, centrifugal collecting cell.With 500 μ l 50mM Tris-HCl (pH8.0) and 10 μ l 0.4M EDTA re-suspended cells, 37 ℃ of incubations 20 minutes, the N,O-Diacetylmuramidase that adds 10 μ l 10mg/mL then continues insulation 20 minutes.The Proteinase K, the 15 μ l 10%SDS that add 3 μ l 20mg/mL afterwards, 50 ℃ of incubations 2 hours, the RNase that adds 3 μ l 10mg/mL again, 65 ℃ of incubations 30 minutes, add equal-volume phenol extracting mixture, get supernatant and use isopyknic phenol again: chloroform: primary isoamyl alcohol (25: 24: 1) mixing solutions extracting twice, get supernatant again with isopyknic ether extracting to remove remaining phenol.Supernatant rolls out DNA and washes DNA with 70% ethanol with glass yarn with 2 times of volume ethanol deposit D NA, and DNA is resuspended among the 30 μ l TE; Genomic dna detects by 0.4% agarose gel electrophoresis;

(2) by the O-antigen gene in the pcr amplification intestinal bacteria O25 type bunch: with the genome of intestinal bacteria O25 type 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 (#1523-ATT GTG GCTGCA GGG ATC AAA GAA AT) that often is found in O-antigen gene bunch promoter region, again according to the gnd gene design downstream primer (#1524-TAG TCG CGT GNG CCT GGA TTA AGT TCG C) in O-antigen gene bunch downstream; With the Expand Long Template PCR method of Boehringer Mannheim company amplification O-antigen gene bunch, the PCR response procedures was as follows: 94 ℃ of pre-sex change 2 minutes; 94 ℃ of sex change are 10 seconds then, 55 annealing 15 seconds, 68 ℃ were extended 15 minutes, carry out 30 circulations like this, last, continue to extend 7 minutes at 68 ℃, obtain the PCR product, detect the size and the specificity thereof of PCR product with 0.8% agarose gel electrophoresis, merge 5 pipe long PCR products, and with the Wizard PCR Preps purification kit purified pcr product of Promega company;

(3) make up O-antigen gene bunch library: make up O-antigen gene bunch library with the Novagen DNaseI shot gun method that is modified, reaction system is a 300ng PCR purified product, 0.9 μ l 0.1MMnCl ₂, the DNaseI of the 1mg/mL of 1 μ l dilution in 1: 2000, reaction is carried out at room temperature, and enzyme is cut the dna fragmentation size is concentrated between the 1.5kb-3kb, then adds 2 μ l 0.1M EDTA termination reactions.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 18 μ l water, in this mixture, add 2.5 μ l dNTP (1mMdCTP, 1mMdGTP, 1mMdTTP subsequently, 10mMdATP), 1.25 the T4DNA polysaccharase of μ l 100mM DTT and 5 units, 11 ℃ 30 minutes, enzyme is cut product mends into flush end, after 75 ℃ of termination reactions, add the Tth archaeal dna polymerase of 5 units and corresponding damping fluid thereof and system is expanded as 80 μ l, 70 ℃ of reactions 20 minutes make 3 of DNA ' end add the dA tail.This mixture is through the equal-volume chloroform: after primary isoamyl alcohol (24: 1) mixing solutions extracting and the extracting of equal-volume ether with 3 * 10 of Promega company ^-3The pGEM-T-Easy carrier connect 10 hours in 16 ℃, cumulative volume is 90 μ l.10 * the buffer of 9 μ l and the T4DNA ligase enzyme of 25 units are wherein arranged, use the 3M NaAc (pH5.2) of 1/10 volume and the dehydrated alcohol precipitation of 2 times of volumes to be connected mixture at last, wash precipitation with 70% ethanol again, be dissolved in after the drying in the 30 μ l water and obtain connecting product; Preparation method with the electric transformed competence colibacillus cell of Bio-Rad company prepares the competence e.colidh5, get after 2-3 μ l connects product and 50 μ l competence bacillus coli DH 5 alphas mix, forward in the electric shock cup of 0.2cm of Bio-Rad company and shock by electricity, voltage is 2.5 kilovolts, time is 5.0 milliseconds to 6.0 milliseconds, the SOC substratum that adds 1mL after the electric shock immediately in cup makes the bacterium recovery, then bacterium is coated in and contains penbritin, on the LB solid medium of X-Gal and IPTG, 37 ℃ of incubated overnight, obtain blue white bacterium colony next day, with the white colony that obtains promptly the white clone forward on the LB solid medium that contains penbritin and cultivate, from each clone, extract plasmid simultaneously, and cutting the segmental size of evaluation insertion wherein with the EcoRI enzyme, the white that obtains clone group has constituted the O-antigen gene bunch library of intestinal bacteria O25 type;

(4) to the cloning and sequencing in the library: from the library, select 96 clones of insertion fragment more than 1kb and the insertion fragment among the clone is checked order with this lab A BI3730 type automatic dna sequencer, sequence reaches 100% fraction of coverage, thereby obtains all sequences of O-antigen gene bunch;

(5) splicing of nucleotide sequence and analysis: the Pregap4 and the splicing of Gap4 software of the Staden package software package of publishing with Britain Camb MRC (Medical Research Council) Molecular Biology Lab and edit all sequences, thus obtain the Nucleotide full length sequence of the O-antigen gene bunch of intestinal bacteria O25 type; The quality of sequence is mainly guaranteed by two aspects: 1) genome of intestinal bacteria O25 type is done 5 Long PCR reactions, mix these products then to produce the library, 2) to each base, guarantee high-quality fraction of coverage more than 3, after obtaining the nucleotide sequence of intestinal bacteria O25 type O-antigen gene bunch, with American National biotechnology information science center (The National Center forBiotechnology Information, NCBI) Orffinder finds gene, find the reading frame of 14 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 O25 type at last;

(6) specific gene screening: at wzx, the wzy gene design primer in the O-antigen gene of dysentery intestinal bacteria O25 type bunch; Respectively designed two pairs of primers in each gene, every pair of primer is distributed in the different places in the corresponding gene, to guarantee its specificity; Is that template is carried out PCR with these primers with the genomes of 166 strain intestinal bacteria and 43 strain Shigellaes, except that a band that in containing intestinal bacteria O25 group, has obtained the expection size, the correct product of the expection clip size that all do not increase in other groups is so the O-antigen of wzx, wzy gene pairs intestinal bacteria O25 type all is high special.

(7) detection of primer sensitivity: buy the live pig meat stuffing on the market, stir, be divided into the 20g portion, exist in-40 ℃ of refrigerators standby.The frozen bacterium liquid of 10 μ l intestinal bacteria O25 is inoculated in the triangular flask of 20mL LB substratum, in 37 ℃, 200 rev/mins, cultivate 12 hours to saturated, the cultured bacterium liquid that takes a morsel does 10 ⁶With 10 ⁷Dilution doubly, remaining bacterium liquid are put in 4 ℃ the refrigerator standby, get 50 μ l dilution bacterium liquid coating LB agar plate, and 37 degree are cultivated 12h, to being coated with plate count, calculate viable bacteria concentration in the stoste.In 5 portions of live pig meat stuffings, mix 5 * 10 respectively ³, 5 * 10 ², 5 * 10 ¹, 5 and 0 viable bacteria stir, and add 200mL LB substratum, and through 6 layers of filtered through gauze, filtered liquid 200 rev/mins, is cultivated 12h in 37 ℃.Get 3mL bacterium liquid in 6 from cultured bacterium liquid, centrifugal 5 minutes of 000g removes supernatant, adds 100 μ lMQ ultrapure waters and blows precipitation and mixing open, puts into 100 degree boiling water and boils 15 minutes, and lysate is in 12, and centrifugal 8 minutes of 000g gets 1 μ supernatant as pcr template.Right with 4 pairs of oligonucleotide, the Nucleotide of 4946 to 4965 bases among the SEQ ID NO:1 and the Nucleotide of 5329 to 5348 bases; The Nucleotide of 5327 to 5346 bases among the SEQ ID NO:1 and the Nucleotide of 5778 to 5797 bases; The Nucleotide of 8622 to 8641 bases among the SEQ ID NO:1 and the Nucleotide of 8973 to 8980 bases; The Nucleotide of 8349 to 8366 bases among the SEQ ID NO:1 and the Nucleotide of 8993 to 9009 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 O25 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 O25 type, its complete sequence shown in SEQ ID NO:1,17566 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 O25 type by method of the present invention, as shown in table 3, it comprises called after rmlB, rmlD, rmlA, rmlC, wzx, orf6, orf7, wzy, orf9, fnl1, fnl2, fnl3, orf13,14 genomic constitutions of orf14 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 O25 type, promptly transports enzyme gene (wzx gene or the gene of identity function arranged with wzx); Pol gene (wzy gene or the gene of identity function arranged with wzy); Glycosyltransferase gene (orf6, orf7, orf9, orf13 gene).Their zero positions in O-antigen gene bunch and final position and nucleotide sequence all are listed in the table 4; The invention particularly relates to glycosyltransferase gene, transhipment enzyme gene and pol gene, because sugared synthesis path gene is that the gene of synthetic nucleosides bisphosphate monose is common, common by indication to more exocellular polysaccharide now, O-antigen to bacterium is not very special, and the glycosyltransferase gene that the present invention relates to, transhipment enzyme gene and pol gene are high specials to the O-antigen of intestinal bacteria O25 type.

The 3rd aspect of the present invention, wzx gene in the O-antigen gene bunch that comes from intestinal bacteria O25 type is provided or the gene of identity function and wzy gene is arranged or with wzy the oligonucleotide (table 1) of the gene of identity function is arranged with wzx, they are any one section oligonucleotide in these genes.In table 1, also listed these oligonucleotide to the position in O-antigen gene bunch and with these oligonucleotide to being the size of the product of the PCR reaction done of primer, the annealing temperature in these PCR reaction free lists is carried out.These primers are except that a band that has obtained the expection size in the 2nd group, and any product that all do not increase in other groups is so the O-antigen of wzx, wzy gene pairs intestinal bacteria O25 type all is high special.

The separation method of the Nucleotide of described O-antigen-specific to intestinal bacteria O25 type comprises the steps: 1) genomic extraction; 2) the O-antigen gene in the pcr amplification intestinal bacteria O25 type bunch; 3) make up O-antigen gene bunch library; 4) to the cloning and sequencing in the library; 5) splicing of nucleotide sequence and analysis; 6) screening of specific gene; 7) detection of primer sensitivity.

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

As used herein, " oligonucleotide " mainly refer to derive from the gene of the coding transhipment enzyme in the O-antigen gene bunch and intragenic one section nucleic acid molecule of coding polysaccharase, and they can change on length, generally change in 10 to 20 Nucleotide scopes; More definite these oligonucleotide of saying are to come from wzx gene (nucleotide position is the Nucleotide of 4604 to 5863 bases from SEQ ID NO:1); Wzy gene (nucleotide position is the Nucleotide of 8039 to 9100 bases from SEQ ID NO:1); Coming from above intragenic oligonucleotide is high special to intestinal bacteria O25 type.

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

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

The present invention relates to a kind of antigenic method of one or more bacterial polysaccharideses that detects in the food, these antigens can make sample can with the oligonucleotide specific hybrid of following at least one gene, these genes are: (i) gene of the encoding glycosyl transferring enzyme gene of transhipment enzyme and polysaccharase of (ii) encoding comprises the wzx gene or the gene, wzy gene of identity function is arranged or with wzy the gene of identity function is arranged with wzx.At least one oligonucleotide can be hybridized with at least one more than one such gene specific of expressing the special antigenic bacterium of O-under the situation of condition permission, and these bacteriums are intestinal bacteria O25 types.Available PCR method detects, more can with behind the Nucleotide mark in the inventive method as probe by hybridization such as Southern-blot or fluoroscopic examination, perhaps by antigen and bacterium in gene chip or the microarray assay sample.

Planner of the present invention considers following situation: when one special oligonucleotide detects when invalid, the mixture of oligonucleotide can with the target region specific hybrid with test sample.Therefore the invention provides a cover oligonucleotide and be used for detection method of the present invention.Here said oligonucleotide is meant the gene that comes from the encoding glycosyl transferring enzyme, the gene of coding transhipment enzyme and the gene of polysaccharase, comprises the wzx gene or the gene, wzy gene of identity function is arranged or with wzy the oligonucleotide of the gene of identity function is arranged with wzx.This cover oligonucleotide is special to the O-antigen of a special bacterium, and this special bacterium O-antigen is expressed by intestinal bacteria O25 type.

On the other hand, the present invention relates to a kind of antigenic method of one or more bacterial polysaccharideses that detects in the movement, these antigens can make sample can with the oligonucleotide specific hybrid of following at least one gene, these genes are: (i) gene of the encoding glycosyl transferring enzyme gene of transhipment enzyme and polysaccharase of (ii) encoding comprises the wzx gene or the gene, wzy gene of identity function is arranged or with wzy the gene of identity function is arranged with wzx.At least one oligonucleotide can be expressed more than one such gene specific hybridization of the special antigenic bacterium of O-with at least one under the situation of condition permission.These bacteriums are intestinal bacteria O25 types.Oligonucleotide among available the present invention is made the method test sample of primer by PCR, also can with behind the oligonucleotide molecules mark among the present invention as probe by hybridization such as Southern-blot or fluoroscopic examination, perhaps by antigen and bacterium in gene chip or the microarray assay sample.

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

On the other hand, the present invention also relates to the antigenic method of one or more bacterial polysaccharideses in the sample that a kind of detection comes from patient.One or more bacterial polysaccharides antigens in the sample can make sample can with a specific hybrid in a pair of oligonucleotide in following at least one gene, these genes are: (i) gene of the encoding glycosyl transferring enzyme gene of transhipment enzyme and polysaccharase of (ii) encoding comprises the wzx gene or the gene, wzy gene of identity function is arranged or with wzy the gene of identity function is arranged with wzx.Under the situation of condition permission at least one oligonucleotide can with sample at least one express more than one such gene specific hybridization of the special antigenic bacterium of O-, these bacteriums are intestinal bacteria O25 types.Oligonucleotide among available the present invention is made the method test sample of primer by PCR, also can will pass through hybridization as probe behind the oligonucleotide mark among the present invention, perhaps by antigen and bacterium in gene chip or the microarray assay sample.

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

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

The present invention discloses the full length sequence of the O-antigen gene bunch of intestinal bacteria O25 type first, and can from the sequence of this total length gene cluster of not cloned, produce recombinant molecule, can produce the O-antigen of expressing intestinal bacteria O25 type by inserting to express, and become useful vaccine.

Embodiment

Below in conjunction with specific embodiment, further set forth the present invention.Should understand these embodiment only is used to the present invention is described and is not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, molecular cloning: the condition described in the laboratory manual (NewYork:Cold Spring Harbor Laboratory Press, 1989).

Embodiment 1: genomic extraction:

37 ℃ of incubated overnight intestinal bacteria O25 types in the LB of 5mL substratum, centrifugal collecting cell.With 500 μ l 50mM Tris-HCl (pH8.0) and 10 μ l 0.4M EDTA re-suspended cells, 37 ℃ of incubations 20 minutes, the N,O-Diacetylmuramidase that adds 10 μ l 10mg/mL then continues insulation 20 minutes.The Proteinase K, the 15 μ l 10%SDS that add 3 μ l 20mg/mL afterwards, 50 ℃ of incubations 2 hours add the RNase of 3 μ l 10mg/mL again, 65 ℃ of incubations 30 minutes.Add equal-volume phenol extracting mixture, get supernatant and use isopyknic phenol again: chloroform: primary isoamyl alcohol is taken out (25: 24: 1) mixing solutions and is carried twice, get supernatant again with isopyknic ether extracting to remove remaining phenol, supernatant is with 2 times of volume ethanol deposit D NA, roll out DNA and wash DNA with glass yarn, at last DNA is resuspended among the 30 μ l TE with 70% ethanol.Genomic dna detects by 0.4% agarose gel electrophoresis.

Embodiment 2: by the O-antigen gene in the pcr amplification intestinal bacteria O25 type bunch:

With the genome of intestinal bacteria O25 type 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 (#1523-ATT GTG GCT GCA GGG ATC AAA GAA AT) that often is found in O-antigen gene bunch promoter region, again according to the gnd gene design downstream primer (#1524-TAG TCG CGT GNG CCT GGA TTAAGT TCG C) in O-antigen gene bunch downstream; With the Expand Long Template PCR method of Boehringer Mannheim company amplification O-antigen gene bunch, the PCR response procedures was as follows: 94 ℃ of pre-sex change 2 minutes; 94 ℃ of sex change are 10 seconds then, 55 annealing 15 seconds, and 68 ℃ were extended 15 minutes, and carried out 30 circulations like this.At last, continue to extend 7 minutes at 68 ℃, obtain the PCR product, the agarose gel electrophoresis with 0.8% detects the size and the specificity thereof of PCR product.Merge 5 pipe long PCR products, and with the WizardPCR 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, 0.9 μ l 0.1M MnCl ₂, 1 μ l1: the DNaseI of the 1mg/mL of 2000 dilutions, reaction is carried out at room temperature.Enzyme is cut the dna fragmentation size is concentrated between the 1.5kb-3kb, then adds 2 μ l 0.1M EDTA termination reactions.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 18 μ l water.In this mixture, add 2.5 μ l dNTP (1mMdCTP subsequently, 1mMdGTP, 1mMdTTP, 10mMdATP), the T4DNA polysaccharase of 1.25 μ l 100mM DTT and 5 units, 11 ℃ 30 minutes, enzyme is cut product mend into flush end, after 75 ℃ of termination reactions, add the Tth archaeal dna polymerase of 5 units and corresponding damping fluid thereof and system is expanded as 80 μ l, 70 ℃ were reacted 20 minutes, made 3 of DNA ' end add the dA tail.This mixture is through the equal-volume chloroform: after primary isoamyl alcohol (24: 1) mixing solutions extracting and the extracting of equal-volume ether with 3 * 10 of Promega company ^-3The pGEM-T-Easy carrier connect 10 hours in 16 ℃, cumulative volume is 90 μ l.10 * the buffer of 9 μ l 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 in the 30 μ l water and obtain connecting product.

Next is the preparation of competent cell: the method that provides with reference to Bio-Rad company prepares the competent cell bacillus coli DH 5 alpha.Get a ring bacillus coli DH 5 alpha list bacterium colony in the LB of 5mL substratum, 180rpm cultivated after 10 hours, got in the LB substratum that the 2mL culture is transferred to 200mL, and 37 ℃ of 250rpm thermal agitations are cultivated OD600 about 0.5, ice bath cooling was 20 minutes then, in centrifugal 15 minutes of 4 ℃ of 4000rpm.Confide all supernatant, dispel thalline, in centrifugal 15 minutes of 4 ℃ of 4000rpm with the deionization aqua sterilisa 200mL of cold ice precooling.Deionization aqua sterilisa 100mL with cold ice precooling dispelled thalline again, in centrifugal 15 minutes of 4 ℃ of 4000rpm.With 10% glycerine suspension cell of cold ice precooling, centrifugal 10 minutes of 4 ℃ of 6000rpm abandon supernatant, precipitate 10% glycerine suspension cell with the precooling of 1mL ice at last, are competent cell.The competent cell that makes is packed as 50 μ l, one pipe ,-70 ℃ of preservations.

Be electric transformed competence colibacillus cell at last: get after 2-3 μ l connects product and 50 μ l competence bacillus coli DH 5 alphas mix, 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 O25 type with the EcoRI enzyme.

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

From the library, select insert 96 clones of fragment more than 1kb with this lab A BI3730 type automatic dna sequencer to unidirectional order-checking of insertion fragment among the clone, make sequence reach 100% fraction of coverage, thus all sequences of acquisition O-antigen gene bunch.

Embodiment 5: the splicing of nucleotide sequence and analysis:

The Pregap4 and the splicing of Gap4 software of the Staden package software package of publishing with Britain Camb MRC (Medical Research Council) Molecular Biology Lab and edit all sequences, thus the Nucleotide full length sequence (seeing sequence list) of the O-antigen gene bunch of intestinal bacteria O25 type obtained.The quality of sequence is mainly guaranteed by two aspects: 1) genome of intestinal bacteria O25 type is done 5 LongPCR reactions, mix these products then to produce the library.2), guarantee high-quality fraction of coverage more than 3 to each base.After obtaining the nucleotide sequence of intestinal bacteria O25 type O-antigen gene bunch, with American National biotechnology information science center (The National Center for BiotechnologyInformation, NCBI) Orffinder finds gene, find the reading frame of 14 openings, determine also that with the function of finding the reading frame that these are open what gene they are with the genetic comparison among the Blast groupware and the GenBank, finish gene annotation with the Artemis software at Britain Sanger center again, do accurate comparison between DNA and protein sequence with ClustralW software, obtain the structure of the O-antigen gene bunch of intestinal bacteria O25 type at last, as shown in table 3.

By retrieving and comparing, find the DTDP-glucose 4 of the rmlB genes encoding in orf1 encoded protein and Escherichia coli K12 (AAC75102) the O-antigen, 6-dehydratase has 97% consistence and 98% similarity in 361 amino acid whose sequences, show the homology that height is arranged between them, therefore, be rmlB with the temporary transient called after of orf1.The dTDP-6-deoxy-L-mannose-dehydrogenase of the rmlD genes encoding in orf2 encoded protein and Escherichia coliK12 (AAC75101) the O-antigen has 97% consistence and 98% similarity in 299 amino acid whose sequences, show the homology that height is arranged between them, therefore, be rmlD with the temporary transient called after of orf2.The glucose-1-phosphate thymidylyltransferase of the rmla genes encoding in orf3 encoded protein and Escherichia coli K12 (AAC75100) the O-antigen has 93% consistence and 96% similarity in 290 amino acid whose sequences, show the homology that height is arranged between them, therefore, be rmlA with the temporary transient called after of orf3.The dTDP-6-deoxy-D-glucose-3 of the rmlC genes encoding in orf4 encoded protein and Escherichia coli K12 (AAC75099) the O-antigen, 5epimerase has 67% consistence and 76% similarity in 169 amino acid whose sequences, show the homology that height is arranged between them, therefore, be rmlC with the temporary transient called after of orf4.The FucNAc synthetase of the fnl1 genes encoding in orf10 encoded protein and Escherichia coli (AAN60461) the O-antigen has 94% consistence and 97% similarity in 344 amino acid whose sequences, show the homology that height is arranged between them, therefore, be fnl1 with the temporary transient called after of orf10.The FucNAc synthetase of the fnl2 genes encoding in orf11 encoded protein and Escherichia coli (AAN60462) the O-antigen has 95% consistence and 97% similarity in 374 amino acid whose sequences, show the homology that height is arranged between them, therefore, be fnl2 with the temporary transient called after of orf11.The FucNAc synthetase of the fnl3 genes encoding in orf12 encoded protein and Escherichia coli (AAN60463) the O-antigen has 98% consistence and 99% similarity in 370 amino acid whose sequences, show the homology that height is arranged between them, therefore, be fnl3 with the temporary transient called after of orf12.WbuC protein in orf14 encoded protein and Escherichia coli (AAN60465) the O-antigen has 94% consistence and 98% similarity, show the homology that height is arranged between them, but the definite function of this open reading frame is also unclear, therefore, be orf14 with the temporary transient called after of orf14.

Orf5 and orf8 are the proteic genes that there is transmembrane segment in only two codings of intestinal bacteria O25 kind.The O-antigen of orf5 encoded protein and Shigella flexneri 2a str.301 (AAN43639) transhipment enzyme has 43% sequence identity and 65% similarity, it contains 12 uniform transmembrane segments by the proteic topology discovery of HMMTOP2.0 programanalysis, and this is the proteic characteristic feature of Wzx.So name orf5 is wzx.The O-antigen polysaccharase of orf8 encoded protein and Aeromonas hydrophila (AAM22551) has 29% consistence, 46% similarity, it contains 9 transmembrane segments by the proteic topology discovery of HMMTOP2.0 programanalysis, and hydrophilic loop (loop) in the big kytoplasm is arranged, and this is the proteic characteristic feature of Wzy.So name orf8 is wzy.

Albumen and other known glycosyltransferases of orf6, orf7, orf9, four genes encodings of orf13 have the sequence identity of 32-96% and the sequence similarity of 54-97%.By search to glycosyltransferase motif database among the Pfam, the homology desired value of the consensus sequence of the albumen of these four genes encodings and known glycosyltransferase family 1 and 2 is very high, therefore we infer this four genes encoding glycosyltransferases, and because each glycosyltransferase specificity catalysis forms a kind of disaccharide bond, this is consistent with the monose quantity of oligosaccharide unit in the intestinal bacteria O18 type O-antigen.Because the definite function of these four genes can't be determined, so we are with the temporary called after orf6 of these four genes, orf7, orf9, orf13.

Embodiment 6: the screening of specific gene.

Wzx, wzy gene design primer in the O-antigen gene of intestinal bacteria O25 type bunch have respectively designed two pairs of primers in each gene, every pair of primer is distributed in different local in the corresponding gene, to guarantee its specificity; Is that template is carried out PCR with these primers with the genomes of 166 strain intestinal bacteria and 43 strain Shigellaes, except that a band that in containing intestinal bacteria O25 group, has obtained the expection size, the correct product of the expection clip size that all do not increase in other groups is so the O-antigen of wzx, wzy gene pairs intestinal bacteria O25 type all is high special; The position of these genes in nucleotide sequence sees Table 1.

Embodiment 7: the detection of primer sensitivity.

Buy the live pig meat stuffing on the market, stir, be divided into the 20g portion, exist in-40 ℃ of refrigerators standby.The frozen bacterium liquid of 10 μ l intestinal bacteria O25 is inoculated in the triangular flask of 20mL LB substratum, in 37 ℃, 200 rev/mins, cultivate 12 hours to saturated, the cultured bacterium liquid that takes a morsel is done 106 and 107 times dilution, remaining bacterium liquid is put in 4 ℃ the refrigerator standby, get 50 μ l dilution bacterium liquid coating LB agar plate, 37 degree are cultivated 12h, to being coated with plate count, calculate viable bacteria concentration in the stoste.In 5 portions of live pig meat stuffings, mix 5 * 10 respectively ³, 5 * 10 ², 5 * 10 ¹, 5 and 0 viable bacteria stir, and add 200mL LB substratum, and through 6 layers of filtered through gauze, filtered liquid 200 rev/mins, is cultivated 12h in 37 ℃.Get 3mL bacterium liquid in 6 from cultured bacterium liquid, centrifugal 5 minutes of 000g removes supernatant, adds 100 μ l MQ ultrapure waters and blows precipitation and mixing open, puts into 100 degree boiling water and boils 15 minutes, and lysate is in 12, and centrifugal 8 minutes of 000g gets 1 μ supernatant as pcr template.Right with 4 pairs of oligonucleotide, the Nucleotide of 4946 to 4965 bases among the SEQ ID NO:1 and the Nucleotide of 5329 to 5348 bases; The Nucleotide of 5327 to 5346 bases among the SEQ ID NO:1 and the Nucleotide of 5778 to 5797 bases; The Nucleotide of 8622 to 8641 bases among the SEQ ID NO:1 and the Nucleotide of 8973 to 8980 bases; The Nucleotide of 8349 to 8366 bases among the SEQ ID NO:1 and the Nucleotide of 8993 to 9009 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 ", 55 ℃: 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 O25 in the pork filling when using aforesaid method.

By clone and the expression in the vaccine strains of attenuation, can set up recombiant vaccine to O-antigen gene bunch.O-antigen is the surface antigen of topmost Gram-negative bacteria, can cause the intensive immune response, is one of best target molecule of making recombiant vaccine.Viret laboratory success in 1993 the O-antigen gene of Shigellae Sonnei bunch is expressed in a strain Salmonellas Tyziai vaccine bacterium, experimentation on animals proof can cause rabbit immune response (Molecular Microbiology1993,7:239-252).The group of China Military Medical Science Institute also similarly works being engaged in the Viret laboratory.Bunch express the O-antigen gene of intestinal bacteria O111 success in 1999 in the Wang Lei laboratory in salmonella vaccine STM-1, and the bacterial strain set up of proof can cause the blood of mouse and humoral response (Microbial Pathogenesis 1999,27:55-59).So the O-antigen-specific gene order of intestinal bacteria O25 of the present invention can be applied to set up recombiant vaccine.

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

Nucleotide sequence (shown in the SEQ IDNO:1) according to the O-antigen-specific to intestinal bacteria O25 type of the present invention, structure specific nucleic acid probe, be fixed on the carrier of chip and make biochip, after the sample that will detect is suitably handled, carry out hybridization with biochip, utilize the biochip signal analysis equipment just can obtain corresponding bacteria situation in the sample then.The DNA chip that this intestinal bacteria O-antigen is identified can be directly used in clinical and other check place (as food-processing and production industry, the Micro biological Tests of animal and veterinary industry customs quarantine control etc.).This chip only need enlarge output, just can industrialization under identical condition.

Table 1 has been listed transhipment enzyme gene and pol gene and intragenic primer and PCR data in the O-antigen gene bunch of intestinal bacteria O25 type.Transhipment enzyme gene and pol gene and their function corresponding and the size of the O-antigen gene bunch of intestinal bacteria O25 type in table, have been listed.In each gene, we have respectively designed two pairs of primers, and the difference that every pair of primer is distributed in the corresponding gene is local to guarantee its specificity.In table, also listed position and the size of each primer in SEQ ID NO:1.Is that template carry out PCR with listed corresponding annealing temperature in the table with the genomes of all bacterium in the table 2 with every pair of primer, has obtained corresponding PCR product, and its size is also listed in the table.

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

The genomic dna that contains intestinal bacteria O25 type in the 2nd group is as positive control.Do template with every group of bacterium, be PCR by following condition with every pair in the table 1 primer: 95 ℃ of pre-sex change after 5 minutes, 95 ℃ of sex change 30 seconds, annealing time is 30 seconds, temperature sees Table 1,72 ℃ extended 2 minutes, carried out 25 circulations like this.Continue to extend 5 minutes at 72 ℃ at last, reaction system is 25 μ l.Template is dilution in 1: 20, gets 1 μ l.After reaction finishes, get 10 μ lPCR products and detect the fragment that amplifies by 0.8% agarose gel electrophoresis.

For wzx, wzy gene, each gene all has two pairs of primers detected, every pair of primer has obtained except be PCR in the 5th group after the correct band of expection size, the correct band of any size does not all increase in other groups, that is to say, not obtaining any PCR product band in the array mostly, so wzx, wzy gene pairs intestinal bacteria O25 type and O-antigen thereof are high specials.And the oligonucleotide of these intragenic any one section 10-25nt is special to the O-antigen of intestinal bacteria O25 type, and the primer in especially above-mentioned each gene is that oligonucleotide is high special to detecting the back confirmation through PCR to intestinal bacteria O25 type.These all oligonucleotide all can be used for the intestinal bacteria O25 type in the human body and environment rapidly and accurately, and can identify their O-antigen.

Table 3 is structural tables of the O-antigen gene bunch of intestinal bacteria O25 type, in table, listed the structure of the O-antigen gene bunch of intestinal bacteria O25 type, altogether by 14 genomic constitutions, each gene box indicating, and in square frame, write the title of gene, numeral be the order of the open reading frame (orf) in the O-antigen gene bunch.Two ends at O-antigen gene bunch are galF gene and gnd gene, and they do not belong to O-antigen gene bunch, and we are just with the increase full length sequence of O-antigen gene bunch of their one section sequences Design primer.

Table 4 is site plan of the gene in the O-antigen gene bunch of intestinal bacteria O25 type, listed the accurate position of all open reading frame in complete sequence in the O-antigen gene bunch of intestinal bacteria O25 type in the drawings, at the underscoring of the initiator codon and the terminator codon of each open reading frame.The initiator codon of open reading frame has two in intestinal bacteria: ATG and GTG.

SEQ ID NO:1 sequence (SEQUENCE LISTING)

SEQUENCE LISTING

＜110〉Tianjin Biochip Technology Co., Ltd

＜120〉to the Nucleotide of the O-antigen-specific of intestinal bacteria O25 type

＜130〉to the Nucleotide of the O-antigen-specific of intestinal bacteria O25 type

<160>1

<170>PatentIn version 3.2

<210>1

<211>17566

<212>DNA

<213>Escherichia coli

<400>1

attgtggctg cagggatcaa agaaatcctc ctggtaactc acgcgtccaa gaacgcggtc 60

gaaaaccact tcgacacctc ttatgaatta gaatctctcc ttgaacagcg cgtgaagcgt 120

caactgctgg cggaagtgca atctatctgc ccgccgggcg tgactattat gaacgtgcgt 180

cagggcgaac ctttaggttt aggccactcc attttgtgtg cgcgacctgc cattggtgac 240

aacccgtttg tcgtggtact gccagacgtt gtgatcgacg atgccagcgc cgacccgcta 300

cgttacaacc ttgctgccat gattgcgcgc ttcaacgaaa cgggccgcag ccaggtgctg 360

gcaaaacgta tgccgggtga cctctctgaa tactcggtca tccagaccaa agaaccgctg 420

gatcgcgaag gtaaagtcag ccgcattgtt gaatttatcg aaaaaccgga tcagccgcag 480

acgctggact cagacatcat ggccgttggt cgctatgtgc tttctgccga tatttggccg 540

gaacttgaac gcacgcagcc tggtgcatgg ggacgtattc agctgactga tgccattgct 600

gaactggcga aaaaacagtc cgttgatgcc atgctgatga ccggcgacag ctacgactgc 660

ggtaaaaaaa tgggctatat gcaggcgttt gtgaagtatg gactgcgtaa cctaaaagaa 720

ggggcgaagt tccgtaaagg gattgagaag ctgttaagcg aataatgaaa atctgaccgg 780

atgtaacggt tgataagaaa attataacgg cagtgaagat tcgtggcgaa agtaatttgc 840

tgcgaatatt cctgccgttg ttttatataa acaatcagaa taacaacgag ttagcaatag 900

gattttagtc aaagttttcc aggattttcc ttgtttccag agcggattgg taagacaatt 960

agcgtttgaa tttttcgggt ttagcgcgag tgggtaacgc tcgtcacatc gtagacatgc 1020

atgcagtgct ctggtagctg taaagccagg ggcggtagcg tgcgttaala cctctattaa 1080

tcaaactaag agccgctaat ttcacagcat gctctgaagt aatatggaat aaaaaagtga 1140

agatacttgt tactggtggc gcaggattta ttggttctgc tgtagttcgt cacattataa 1200

ataatacgca ggatagtgtt gttaatgtcg ataaattaac gtacgccgga aacctggaat 1260

cacttgctga tgtttctgat tctgaacgct atgtttttga acatgcggat atttgcgatg 1320

ctgctgcaat ggcgcggatt tttgctcagc atcagccgga tgcagtgatg cacctggctg 1380

ctgaaagcca tgtggatcgt tctatcactg gccctgcggc atttattgaa accaatattg 1440

ttggtactta tgtcctttta gaagccgctc gcaattactg gtctgctctt gatagcgaca 1500

agaaaaatag cttccgtttt catcatattt ctactgacga agtctatggt gatttgcctc 1560

atcctgacga agtaaataat aaagaaaaat tacccttatt tattgagaca acagcttacg 1620

caccaagcag cccttattcc gcatccaaag catccagcga tcatttagtc cgcgcgtgga 1680

aacgtaccta tggtttaccg accattgtga ctaactgttc gaataactac ggtccttatc 1740

actttccgga aaaattgatt ccactagtaa ttcttaatgc tctggaaggt aaggcattac 1800

ctatttatgg caaaggggat caaattcgtg attggctgta tgttgaagat catgcgcgtg 1860

cattatatac cgtcgtaacc gaaggtaaag cgggtgaaac ttataacatt ggtgggcaca 1920

acgaaaagaa aaacattgat gtagtgctca ctatttgtaa tttgctggat gagattgtac 1980

cgaaagagaa atcttatcgt gagcaaatca cttatgtcgc cgatcgtccg ggacacgatc 2040

gccgttatgc cattgatgct gagaagattg gtcgcgaatt gggatggaaa ccacaggaaa 2100

cgtttgagag cgggattcgg aagacagtgg aatggtacct gtccaataca aaatgggttg 2160

ataatgtaaa aagtggtgcc tatcaatcgt ggattgaaca gaactatgag ggccgccagt 2220

aatgaatatc ctcctttttg gcaaaacagg gcaggtaggt tgggaactac agcgtgctct 2280

ggcacctttg ggtaatttga ttgctcttga tgttcactcc actgattact gtggtgattt 2340

tagtaatcct gaaggtgtag ctgaaaccgt aagaagcatt cggcctgata ttattgtcaa 2400

cgcagccgct cacaccgcag tagacaaagc agaatcagaa ccggagtttg cacaattact 2460

taacgcaaca agtgtcgaag cgattgcgaa agcagcaaat gaagttggag cttgggttat 2520

acactactcc accgattatg ttttccctgg cagtggtgac aggccatggc tggaaacgga 2580

tgcaacagcg ccgctaaatg tttacggtga aactaagcta gctggggaaa aagcgttaca 2640

agaacattgc gcaaagcatc ttattttccg taccagctgg gtatacgctg gtaaaggaaa 2700

taattttgcc aaaacgatgt tgcgtctggc aaaagaacgc gaagagttgg ctgtgataaa 2760

cgatcaattt ggcgcaccaa cgggtgctga gcttctggcc gattgtacag cacatgccat 2820

tcgtgtcgca ctgaataaac cggatgtcgc aggcttgtac catctggtag ctagtggtac 2880

cacgacctgg tacgattatg ctgcgctggt ttttgaagag gcgcgcaaag cagatattcc 2940

cctcgcactc aacaaactca atgcggtacc aacgacagcc tatcctacac cagcttgtcg 3000

tccacataac tctcgactta atacagaaaa atttcagcag aattttgcgc ttgtcttgcc 3060

tgactggcag gttggtgtga aacgcatgct caacgaatta tttacgacta cagcaattta 3120

atagtttttg catcttgttc gtgatgatgg agcaagatga attaaaagga atgatgaaat 3180

gaaaacgcgt aagggtatta ttttagcggg tggttctggt actcgtcttt atcctgtgac 3240

tatggcagtc agtaaacagc tattaccgat ttatgataaa ccgatgatct attacccgct 3300

ctctacactg atgttggcgg gtattcgcga tattttgatt attagtacgc cacaggatac 3360

tcctcgtttt caacaactgc tgggtgacgg tagccagtgg gggctaaatc ttcagtacaa 3420

agtgcaactg actccagatg ggcttgcgca ggcatttatt atcggtgaag agtttattgg 3480

tggtgatgat tgtgctttga ttcttggtga taatatcttt tacggtcacg atctgccgag 3540

gttaatggat gccgctgtta acaaagaaag tggtgcaacg gtatttgcct atcacgtaaa 3600

tgatcctgaa cgctatggtg tcgttgagtt tgataaaaac ggtactgcaa tcagcttgga 3660

agaaaaaccg ttacaaccaa aaagtaatta tgcggtaacc gggctttatt tctatgataa 3720

cgacgttgtg gaaatggcga aaaaccttaa gccttctgcc cgtggtgaac tggaaattac 3780

cgatattaac cgtgtctata tggaacaggg gcgtttatct gttgccatga tgggccgtgg 3840

ttatgcatgg ttagacacgg ggacacatca aagtctgatt gaagcaagta acttcattgc 3900

aacaattgaa gagcgacaag gtttaaaggt atcttgcccg gaagaaattg cttatcgtaa 3960

aggctttatt gacgctgagc aggttaatgt attagcagaa ccactaaaga aaaatgctta 4020

tggtcagtat ctgctaaaaa tgattaaagg ttactaaaaa tgaatgtaat taaaactgaa 4080

attcctgatg tattaatttt ggaaccggaa gtttttggtg atgagcgcgg tttttttatg 4140

gaaagcttta atcagaaagt tttcgaagag gctgtagggc gtaaggttga atttgttcag 4200

gataaccatt ctaaatcaat taagggtgta ttacgcggat tgcactatca gctggaacct 4260

tatgctcaag gtaaattagt tcgttgtgtg gtcggtgagg tttttgatgt agcagttgat 4320

attcgtaaat cgtcaggtac atttgggaaa tgggttgggg tgaatttgtc tgctgagaac 4380

aagcgtcagt tgtggatacc tgaaggattt gctcatggat ttttggtact tagtgattta 4440

gcagaagttt tatataaaac gaatcaatat tatgctccat cacatgaacg aaatattatc 4500

tggaatgacc ccttgcttaa tattaaatgg ccaaggacag cactgattac tctgtctgat 4560

aaggatgcaa atggggaaaa attagaacta agtgagtatt gaaatgtctc tcttaaaaca 4620

tagtatatgg aatgttgcgg gctactttat accaacatta attgcaatcc cagcgtttgg 4680

attaattgca agggaaattg gtgtagaact atttggtttg tatactttat caatgatttt 4740

tatagggtat gctagtatat ttgatgctgg gttaacaaga gctgtagtac gcgaaatagc 4800

attactaaaa aacagactgg acgattgtaa tacgataata gtaacttcta ttattgctgt 4860

ggtattttta gggggtatcg gaggcggggg agtgtttctg cttaaagacc atattattga 4920

actgttaaat atgtcaccaa tatactacgt cgatgcgata aagtctctaa tattattatc 4980

atctctgata cctgtattct tagtcacgca aatactatta gcagagcttg aggggcggga 5040

atattttgga atcctaaata tacaaaaaag tttagggaat tctttaattg cagggttgcc 5100

tgcattattt gttttaatta atcaaacact tttttctgca attattggtg tagctattgc 5160

aagagttata tgcttgtggt taagctacat tatgagcagg aaaagaataa ctatcgatat 5220

ttcatttttt tcaataactg ttttaaaacg gttatttaga tatggcgggt gggtaactat 5280

aagtaacata atatctccta tattagcgag tatggataga tttattctat ctcatatcca 5340

gggagcatca aaaatatcat tctatacagt ccctaatgag ctggtaacta ggcttggaat 5400

agttccaggc tctcttggga aagctgtttt tccaaaatta agccatgcaa gtagttttac 5460

agcgtcatat gcagaacaaa aaaaagcgta tatattaatg actgtcattg tattgccttt 5520

ggttttattt atatattact acgcaaagtt tattttaaca ttgtggatgg gggctgagta 5580

tgcagggatt tcagtcgaaa tattacggat tatgcttata gggtatatct ttaactgtta 5640

ttcacaaatc tcttttgcca atatacaggc atttggaaaa gcaaaataca ctgcatacat 5700

ccatatgatg gaatttattc cttatttgat aatgttatat ataatttcaa aggaatgtgg 5760

ggttattggt gtggcgtggt tatggacaat tcgagtgata attgattttt tgatgctttt 5820

ttatatgagt tatcgttgta ataatcttat gaaaaaaggg tagcctgatg atatatattg 5880

tagtgttaaa ttggaatggg gctatagata ccattaattg tgttaaaagt ttaatgaatt 5940

taaattatga tgattataaa attatcgttg ttgataactg ttctacggat aactcatatg 6000

attctataaa agaaaatctt aatgcattat atattactgg taaaagtttc attgaggtga 6060

agtatgagga tagaagtaaa tatcaaacat tagaaaacga taaaatcata ttaatacaat 6120

ctccgaaaaa taatgggtac gcaagtggta ataacattgg aatagagctc gcacttaatc 6180

aggaggatat gaaatacgtc tgggttctga ataatgatac tgaagtggat aaagaggctt 6240

taactcattt aattagtaaa tgtgattcag ataaaaatat agggatttgc ggttctcgtt 6300

tagtctattt taccgataga gagatgcagc aaggactggg tggggtacat aacagatggt 6360

tatgcactac aaaaaattat gagatgggaa gattagtttc caaaaaatat gatgatgaag 6420

tcattagcaa taatatagat tatataattg gcgcatcaat gtttttctct agagaatgtt 6480

tggaaacagt tggattgatg aatgaagaat attttttata ctatgaagag ttagatattt 6540

gcctcagagc aaaagcaaag aactttaaat taggtatttg ctcagatagt ttggtttatc 6600

ataagatagg tgcaagtact gatgggggaa agagtatgat agctgacctt tgttcaataa 6660

aaaataggct ggccattaca gaaaagtttt atccacaata ttattggacg gtatggttgt 6720

cactctttgt tgtagcattt aaccgtgcta gaagaggtga gtttaataag atgaaaagat 6780

gtttgaatgt tatgtttaac ttcaaacgaa ataaaggtag caaatgccat tagaatatgc 6840

atttaatgat ggtgttatta aataaatgta tagtttgata tgtacctgca ggcggccgcg 6900

aattcactag ggtatttaat gaaagtggct tttttatctg cttatgatcc actatctaca 6960

tccagttggt cgggcacacc ttattatatg ctaaaggcat tatcgaagag aaatatttcc 7020

attgaaatat taggaccggt aaatagctat atggtataca tgttaaaagc atataaatta 7080

atattaaggt gtttcggaaa ggaatatgat tatagtcgtt cgaagttgct ttccaagtat 7140

tacggtagaa tatttgagag aaaattaaaa aaaattgatg gtttggattt tattattgca 7200

cctgcaggtt cttcacaaat tgctttttta gaaacaaata taccaataat atatctatcg 7260

gatacaacat atgatcaatt aaaaaactat tatccgaatt taaataaaaa aacaattata 7320

aatgatgagg atgcaagttt aatcgaacgc aaggctattg aaaaagcaac agtcgtatct 7380

ttcccatcta aatgggcaat ggatttttgc aaggactatt acagattgga ttctgataaa 7440

ttagttgaaa taccatgggg ggctaattta tttgaagata ttcattttgc taataaaact 7500

ataattcaaa agattagtta tacttgtctt ttcttggggg tcgattggga aagaaaaggt 7560

ggtaaaacag cattgaaagc aattgaatat gtaagagagt tatatgggat cgatgttaga 7620

ctaaaaattt gtggatgtac tccgaatcaa gagattttac ctgcttgggt tgaattaatt 7680

gataaaataa ataaaaataa cgttgaagaa tatcagaaat tcatcgatgt gttatctaac 7740

gctgatatac ttcttttacc aaccattgct gaatgttacg gaatggtatt ttgtgaagct 7800

gcagcttatg gattgccagt tgtcgccaca gatacaggtg gaattagttc tatagttatc 7860

aacgaaagga cggggatatt aattaaagac tcgtcagact ataagcactt tggaaatgca 7920

attcataaaa taattagctc cgtagagact tatcaaaact actcccaaaa cgcaagagtt 7980

agatataata aaatattgca ttgggacaat tgggcaaaaa agataattga gattatgtat 8040

gagcataaga atagaagaac caaatagcac aaaaagaatt atatttttat ttttactttt 8100

ccttgttttt cctgattttt tgttttatac attagggatt gataatttta gcatttcaac 8160

gataatctcc attatattgc tttttgtttt tttaagggct aaaaacactt gcaaagataa 8220

tttgctaata atagtagtat tattcatatt gttgtgtttt aactgtctgt taagtatgct 8280

atttaatatt gaacaggttt tatcatttaa aattgtactt tcaatgtata gcatcttaat 8340

aatggcatac gtctcctctt gttatgcaca gactttgtgg ttatgttctg aagaaatact 8400

taagagatcc gtcttttatt tgttcgcatt tctttgcctt attggcatta taagtattct 8460

tttacagaaa actgagatta tacatgataa aagtatgatt ctttttcctg aaccatcagc 8520

atttgcattg gttttcatac ctatcttttc attttgttta tactatacaa gggggggggg 8580

ggtactattg ctctatatat tatctttggg tattgcgtta ggtatccaga acttaacgat 8640

gttagtaggc attgtgatta gtgtttttgt gatgaaaaaa ataacaataa ggcaaactat 8700

tgttatattt ttaggggcat ggattttttc catgatatta agtgatttag acatttctta 8760

ctatacatcg cggcttgatt ttaaaaatac tacgaactta tcagtgcttg tatatctttc 8820

aggaattgaa agagctttct tgaattttat tacaagttat ggtcttggta ttggttttca 8880

acaaatgggc gtgaatgggg aggtaggagt gtatcaacaa attttagctg atcttgatgc 8940

ccctatgtta aatatatacg atggctcatt tatttcttcg aagttaatat ctgagtttgg 9000

gtttattggt gcaataatgt gcattttcta tctttttatt tttttcgatt ttatctgcgt 9060

ttcaaaaaaa ataagagata tccaccgcag tatattttag catatagttt ctatatgtgt 9120

ttcttcatcc ccctttttat acgtggtgct ggctatataa atccctatgt gtttatgtta 9180

ttttcatcaa tatttttatg caaatatcat gctaaaatta tcttgatgaa atctaatgtc 9240

aaaatggcta tataatagta gattatgtta tcattaatac gtaaataaca tatattctaa 9300

ccagggcata aataatgtgc ataaaaaaat taagttaatt aaacgatatg gcctttatgg 9360

tggtcttagg cttcttaaag attattctta acaaaagttt tattttgttc aaatgttagg 9420

gttattagat tttcatgtta tcttagaaaa gatggaagtg ttagttttgg aaaagggttt 9480

acatcaggtg tagtattagg aggtgataca tttatggatg ctatagtgtc tattggagaa 9540

aatgttcaaa ttaatgatta tgttccacat cgcggctatt aataatgcca ttattggtag 9600

agatacatta atagcaagta aagtatttat tagtgatcat aatcatggta ttttttctaa 9660

atctgatatc catagttcac caactattat tccttcatct aggtctcttg aatctgcacc 9720

tgtgtatatt gaagagcgtg tgtggattgg tgaaaacgtg acaatattac caggtgcgtg 9780

tataggtaat ggtgtagtta ttggcgcaaa tagtgttgtt cgtggtgaga ttcttaataa 9840

tgtgatctga gttgaccccc gaaaacacca gacagtagat gtatcttaaa ataagaggta 9900

ggcttggaca tatatctaac ttaagataca atccctgtct ggaaataaga gggcaagaac 9960

agaaaatgtt tcgatttggg aaagatagga tatggcttct tatgtagtat ataatatcct 10020

gcattcattc ggataacttc ctatggaagt gtactttgct ctgtctgttt tcatttgttg 10080

aaaatttatg ttaataagaa gctttagata accacttagg aactgtatgt ttgatttgtc 10140

caaaagttat attgttgtaa gtgcgacggc gctggcctcc ggaggtgcat taactatatt 10200

aaagcaattt ataaaacatg catcacaaaa ttcaaatgac tatattatgt ttgtatctgc 10260

gggattagag ttgccggtct gtgataacat catttacata gaaaacacac caaaaggatg 10320

gttgaaaaga atatattggg attggttcgg ttgtcgaaag tttatctctg aacataagat 10380

taacgttaag aaagtaattt ctctacaaaa ttccagtttg aatgttcctt atgagcagat 10440

tatttacttg caccagccaa ttcctttcag taaagttgat tcttttttaa atgatatcac l0500

gttcgataac gtaaaacttt ttttatataa aaatttttat tcctatttta tatttaaata 10560

tgtgaatgca aatacaacca tcgtagtgca aacgaattgg atgaaaaaag gagtgttgga 10620

tcaatgtgat aagattagtt ccgaaagggt ccttgttata aaacctgata tcaaaacatt 10680

taataatact aaatttgatg tagataagga tgtatctgtt aaaacactct tatatccagc 10740

gacaccactt acctataaaa atcatttggt cattctgaag gcgttggtta ttttaaagca 10800

aaagtatttt atagatgatc tgaaattcca agtgactttt gaaaagaata ggtacaaaaa 10860

ttttgataag tttgtgcaat taaataacct aagcaaaaac attgattatc tcggtgttct 10920

ttcatactcg aaattgcaaa aaaaatatat ggcggcatct tttattgttt ttcctagcta l0980

tatagaatca tatgggttac cactcatcga agctgctagt ttaggaaaaa aaatcattag 11040

tagtgatctt ccttatgccc gtgatgtttt aaagaattat agcggcgtag attttgtaat 11100

ttacaatgat gaagatggct gggctagggc gttgtttaat gttttaaatg gcaattcgaa 11160

gctcaatttt aggccttatg aaaaagatag tcgttcatct tggccacagt tcttctctat 11220

tttaaaataa ggtgtattat gtttaatggt aaaatattgt taattactgg tggtacgggg 11280

tctttcggta atgctgttct aagacgtttt cttgatacag atatcaaaga aatacgtatt 11340

ttttcccggg atgaaaaaaa acaagatgac atgaggaaaa aatataataa tcagaagctt 11400

aagttctata taggtgatgt tcgcgattat tcgagtattc tcaatgcttc tcgaggtgtt 11460

gattttattt atcatgctgc agccctgaag caagtgcctt cctgcgaatt ccaccccatg 11520

gaagctgtaa aaacgaatat tttaggtact gaaaacgttc tagaagctgc aatagctaat 11580

cgcgttaggc gaattgtatg tctgagtaca gataaagccg tatatcctat taatgcaatg 11640

ggcatatcta aagcaatgat ggaaaaagtc attgttgcaa aatcacgtaa tcttgatagt 11700

tcaaaaacag ttatctgcgg aacacgttat gggaatgtaa tggcttcacg tggatcggtc 11760

atcccattgt ttgttgatct aatcaaatct ggtaaaccat tgaccattac cgatcccaat 11820

atgactcgtt tcatgatgac gcttgaggat gctgttgatc tggtccttta tgccttcgag 11880

catggaaata acggtgatat tttcgttcag aaagctcctg cggcaacaat tcaaacatta 11940

gccattgcac ttaaggaatt gctaaatgcc catgaacatc caatcaatat tattggaact 12000

cgacacgggg aaaaacttta cgaagcgtta ttgagccgag aggaaatgat agcagcggaa 12060

gatatgggtg attattatcg tgttccacca gatctccgcg atttgaacta tggtaaatat 12120

gtggaacatg gtgaccgtcg tatctcggaa gtggaagatt ataactctca taatactgag 12180

agattagatg ttgagggtat gaaaaaatta ctgctaaaac ttccttttat ccgggcaatt 12240

cgttctggtg aagattatga gttggattca taatatgaaa attttagtta ctggtgcttc 12300

agggtttatc ggccgtaatt tggttttccg ccttaaggag gctggttata acgaacttat 12360

tacgatagat cgtaactctt ctttggcgga tttagagcag ggacttaagc aggcagattt 12420

catttttcac cttgcaggag taaatcgtcc tgtgaaggag agtgaatttg gagagggaaa 12480

tagcaacgta actcaacaga ttgttgatgt tctgaaaaaa aataataaaa atactcctat 12540

catgctgagt tcttccatcc aggctgaatg tgataacgct tatggaaaga gtaaagcgac 12600

tgcggagaaa atcattcagc agtatgggga aacgacaaat gccaaatatt atatttatcg 12660

cttgccgaat gtattcggta agtggtgtcg cccaaattat aactccttta tagcaacttt 12720

ctgccatcgc attgcaaatg atgaaactat tacaattaat gatccttcag cagttgttga 12780

tctggtgtat atagatgact tttgttctga catattaaag ctattggaag gagcgaacga 12840

aactggttac aggacattcg gtccaattta ttctgttact gttggtgaag tggcgcaatt 12900

aatttaccga tttaaagaaa gtcgccaaac attaatcacc gaagatgtag gtaatggatt 12960

tacacgtgca ttgtactcaa catggttaag ttatctgtct cctgaacagt ttgcgtatac 13020

ggttccttct tatagtgatg atagaggggt attctgtgaa gtattgaaaa cgaaaaacgc 13080

gggccagttt tcgttcttta ctgcgcatcc aggaattact cgggggggac attatcatca 13140

ttccaaaaat gagaaattta ttgtcatccg aggaagtgct tgtttcaaat ttgaaaatat 13200

tgtcacgggt gagcgatatg aatttaatgt ctcctcagat gattttaaaa ttgttgaaac 13260

agttccgggg tggacgcatg acatcactaa taatggctcg gatgagctag ttgttatgct 13320

ttgggcaaat gaaatattta atcgttctga accagatact atagcgagag ttttatcgtg 13380

aaaaaattga aagtcatgtc ggttgttggg actcgtccag aaattattcg actctcgcgt 13440

gtccttgcaa aattagatga atattgtgac caccttatta ttcatactgg ccaaaactac 13500

gattatgaat tgaatgaagt ttttttcaaa gatttgggtg ttcgcaaacc agattatttt 13560

cttaatgccg caggtaaaaa tgcagcagag actattggac aagttatcat taaagttgat 13620

gatgtccttg aacaggaaaa accagaagct atgttagttc ttggcgatac taactcctgt 13680

atttcagcaa taccagcaaa gcgtcgaaaa attccgatct tccatatgga agcggggaat 13740

cgttgttttg accaacgcgt accggaagaa actaacagaa aaatagttga ccacactgct 13800

gatatcaata tgacatatag cgatatcgct cgtgaatatc ttctggctga aggtgtacca 13860

gccgatagaa ttattaaaac cggtagccca atgtttgaag tactcactca ttatatgccg 13920

cagattgatg gttccgatgt actttctcgc ctgaatttaa cacctgggaa tttctttgtg 13980

gtaagtgccc acagagaaga aaatgttgat acccctaaac agcttgcgaa actggcgaat 14040

atacttaata ccgtggctga aaaatatgat gtcccggtag ttgtttctac tcatcctcgc 14100

actcgtaacc gcatcaatga aaacggtatt caattccata aaaatatctt gcttcttaag 14160

ccattaggat ttcacgatta caaccatctg caaaaaaatg cacgtgctgt tttatcggat 14220

agtgggacta ttacagaaga gtcctccatt atgaacttcc ctgcactcaa tatacgagaa 14280

gcgcacgaac gcccggaagg cttcgaagaa ggggcagtaa tgatggtcgg tcttgaatct 14340

gagcgcgttt tacaggcatt agaaattatt gcaacacagc ctcgtggaga agtacgctta 14400

cttcgtcagg tcagtgacta tagcatgccc aatgtttcag ataaagttgt gcgtattatc 14460

cattcatata ctgactacgt taaacgggtt gtctggaaac aatactaatg aaacttgcat 14520

taatcattga tgattatttg ccccatagca cacgtgttgg ggctaaaatg tttcatgagt 14580

taggccttga attgttgagc agaggccatg atgtaactgt aattacgcct gacatcacat 14640

tacaagcaat ctattctgtt agtatgattg atggtataaa ggtttggcgt ttcaaaagtg 14700

gacctttaaa ggatataggt aaggctaagc gtgccataaa tgaaactctt ttatcttttc 14760

gtgcatggcg tgcattaaag cacctcattc aacatgatac atttgatggt atcgtttatt 14820

attccccctc tattttttgg ggagacttgg ttaaaaaaat aaaacagcga tgccagtgcc 14880

caagctacct ggtcctaagg gatatgtttc cacagtgggt cattgatgca ggtatgttga 14940

aagccggttc gccaattgaa aaatatttca ggtattttga aaaaaaatca tatcagcagg 15000

ctgaccggat aggggtaatg tctgataaga atcttgagat atttcgtcag accaataaag 15060

gttatccgtg tgaagtttta cgtaattggg cctcaatgac tcctgtgtct gccagcgatg 15120

attatcattc acttcgtcaa aaatacgatc taaaagataa agtcattttt ttctatggcg 15180

gtaatattgg gcatgctcag gatatggcga acttaatgcg ccttgcgcgt aatatgatgc 15240

gttatcatga tgctcatttc ctgtttatag ggcagggtga tgaagttgac ctgataaaat 15300

ctcttgctgc agaatggaat ttaactaatt tcactcacct accttcagtg aaccaggaag 15360

agtttaaatt aattttatct gaagttgatg tcggcctgtt ctccctttca tctcgccatt 15420

cttcacataa tttccctggg aaattactag ggtatatggt tcaatcaatc ccgatccttg 15480

ggagtgtgaa tggcggcaat gatttaatgg atgtaattaa taagcacagg gccggtttca 15540

ttcatgttaa tggtgaagat gataaactgt tcgaatctgc acaattgctt cttgcagatt 15600

cggctttaag aaagcagtta ggtcagaacg ctaatgtgtt gttaaaatct caattttcgg 15660

ttgaatcggc ggcacatact atcgaagtcc gactggaggc aggagaatgc gtttagttga 15720

tgacaatatt ctggatgagc tttttcgcac agcagtaaat tctgaacgtt tgcgcgctca 15780

ttatttattg cacgcatctc atcaggagaa ggttcaacgt ttacttattg catttgtacg 15840

cgacagctat gttgagcccc attggcatga gttaccgcat cagtgggaaa tgtttgtcgt 15900

catgcaaggg caattagaag tttgtttgta tgagaaaaat ggtgagatac aaaaacagtt 15960

tattgttgga gacggtactg gaataagcgt catggaattt tccccaggtg atatacatag 16020

tgtcaaatgc ctgtcaccga atgcccttat gttggagata aaggaaggac catttgaccc 16080

actgaaagct aaggcttttt ctaagtggtt atagggcgat acaccaccct ttattcttct 16140

atcttattct atacatgctg ggttaccatc ttagcttctt caagccgcgc aaccccgcgg 16200

tgaacacccc ctgacaggag taaacaatgt caaagcaaca gatcggcgtc gtcggtatgg 16260

cagtgatggg gcgcaacctt gcgctcaaca tcgaaagccg tggttatacc gtctctattt 16320

tcaaccgttc ccgtgaaaag acggaagaag tgattgccga aaatccaggc aagaaactgg 16380

ttccttacta tacggtgaaa gagtttgttg aatctctgga aacgcctcgt cgcatcctgt 16440

taatggtgaa agcaggtgca ggcacggatg ctgctattga ttctctcaag ccatacctcg 16500

ataaaggtga catcatcatt gatggtggta acaccttctt ccaggacaca attcgtcgta 16560

accgtgagct gtctgcagaa ggttttaact ttatcggtac cggtgtttcc ggtggtgaag 16620

aaggtgcgct gaaaggacct tccatcatgc ctggtgggca gaaagaagcc tatgaactgg 16680

tcgctccgat cctgaccaaa atcgccgccg ttgctgaaga tggcgaaccg tgcgttacct 16740

atattggtgc cgatggcgca ggtcactatg tgaagatggt tcacaacggt attgaatacg 16800

gtgatatgca gctgattgct gaagcctatt ctctgcttaa aggtggcatg aatctctcta 16860

acgaagaact ggcgcagacg tttaccgagt ggaataacgg tgaactaagc agctacctga 16920

tcgacatcac caaagacatc ttcactaaaa aagatgaaga cggtaactac ctggttgatg 16980

tgatcctgga tgaagcggct aacaaaggta ccggtaaatg gaccagccag agcgcgctgg 17040

atctcggcga accgctgtcg ctgattaccg agtctgtgtt tgcacgttat atctcttctc 17100

tgaaagagca gcgtgttgcc gcatctaaag ttctctctgg cccgcaagcg cagccagctg 17160

gcgacaaagg tgagttcatc gaaaaagttc gccgtgcgct gtatcttggc aaaatcgttt 17220

cttacgctca gggcttctct cagctgcgtg cagcgtctga agagtacaac tgggatctga 17280

actacggcga aatcgcgaag attttccgtg ctggttgcat cattcgtgcg cagttcttgc 17340

agaaaatcac cgatgcttat gccgaaaatc cgcagatcgc taacctgctg ctggctccgt 17400

acttcaagca aattgccgat gactaccagc aggcgctgcg tgatgtcgt tgcttatgcag 17460

tacagaacgg tatcccggtt ccgaccttcg ctgctgcggt tgcctattac gatagctacc 17520

gtgccgctgt tctgcctgcg aacataatcc aggcgcagcg tgacta 17566

Oligosaccharide unit treatment gene in the O-antigen gene of table 1 intestinal bacteria O25 type bunch and primer and PCR data wherein

Gene	Function	The base position of gene	Forward primer	Reverse primer	The length of PCR product	Produce the group number of correct big or small electrophoresis band	The annealing temperature of PCR (℃)
								wzx	The transhipment enzyme	4604-5863	4946-4965	5329-5348	403	0 *	60
			5327-5346	5778-5797	471	0 *	60
								wzy	Polysaccharase	8039-9100	8622-8641	8973-8980	359	0 *	55
			8349-8366	8993-9009	661	0 *	55

^*Only in intestinal bacteria O25 type, obtain a correct band

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

The bacterium source that contains in this group of group number

1, wild-type e. coli O1, O2, O5, O7, 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，O126，O128，O117

7, wild-type e. coli O129, O130, O131, O132, O133, O134, O135, O136, 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, the 2nd group of bacterial strain removes intestinal bacteria reference culture O25 IMVS ^a

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 intestinal bacteria O25 type O antigen gene structure iron

#orf galF rmlB rmlD rmlA rmlC wzx orf8 orf7 wzy orf9 fnl1 fnl2 fnl3 orf13 orf14 gnd

G+C％ 43.0 46.8 42.1 37.4 30.7 31.2 31.8 29.9 31.7 38.6 38.6 40.9 39.8 41.2

content

Table 4 intestinal bacteria O25 type O-antigen gene cluster gene position

attgtggctg cagggatcaa agaaatcctc ctggtaactc acgcgtccaa gaacgcggtc 60

gaaaaccact tcgacacctc ttatgaatta gaatctctcc ttgaacagcg cgtgaagcgt 120

caactgctgg cggaagtgca atctatctgc ccgccgggcg tgactattat gaacgtgcgt 180

cagggcgaac ctttaggttt aggccactcc attttgtgtg cgcgacctgc cattggtgac 240

aacccgtttg tcgtggtact gccagacgtt gtgatcgacg atgccagcgc cgacccgcta 300

cgttacaacc ttgctgccat gattgcgcgc ttcaacgaaa cgggccgcag ccaggtgctg 360

gcaaaacgta tgccgggtga cctctctgaa tactcggtca tccagaccaa agaaccgctg 420

gatcgcgaag gtaaagtcag ccgcattgtt gaatttatcg aaaaaccgga tcagccgcag 480

acgctggact cagacatcat ggccgttggt cgctatgtgc tttctgccga tatttggccg 540

gaacttgaac gcacgcagcc tggtgcatgg ggacgtattc agctgactga tgccattgct 600

gaactggcga aaaaacagtc cgttgatgcc atgctgatga ccggcgacag ctacgactgc 660

ggtaaaaaaa tgggctatat gcaggcgttt gtgaagtatg gactgcgtaa cctaaaagaa 720

ggggcgaagt tccgtaaagg gattgagaag ctgttaagcg aataatgaaa atctgaccgg 780

atgtaacggt tgataagaaa attataacgg cagtgaagat tcgtggcgaa agtaatttgc 840

tgcgaatatt cctgccgttg ttttatataa acaatcagaa taacaacgag ttagcaatag 900

gattttagtc aaagttttcc aggattttcc ttgtttccag agcggattgg taagacaatt 960

agcgtttgaa tttttcgggt ttagcgcgag tgggtaacgc tcgtcacatc gtagacatgc 1020

atgcagtgct ctggtagctg taaagccagg ggcggtagcg tgcgttaata cctctattaa 1080

Orf1's is initial

tcaaactaag agccgctaat ttcacagcat gctctgaagt aatatggaat aaaaaa gtga 1140

agatacttgt tactggtggc gcaggattta ttggttctgc tgtagttcgt cacattataa 1200

ataatacgca ggatagtgtt gttaatgtcg ataaattaac gtacgccgga aacctggaat 1260

cacttgctga tgtttctgat tctgaacgct atgtttttga acatgcggat atttgcgatg 1320

ctgctgcaat ggcgcggatt tttgctcagc atcagccgga tgcagtgatg cacctggctg 1380

ctgaaagcca tgtggatcgt tctatcactg gccctgcggc atttattgaa accaatattg 1440

ttggtactta tgtcctttta gaagccgctc gcaattactg gtctgctctt gatagcgaca 1500

agaaaaatag cttccgtttt catcatattt ctactgacga agtctatggt gatttgcctc 1560

atcctgacga agtaaataat aaagaaaaat tacccttatt tattgagaca acagcttacg 1620

caccaagcag cccttattcc gcatccaaag catccagcga tcatttagtc cgcgcgtgga 1680

aacgtaccta tggtttaccg accattgtga ctaactgttc gaataactac ggtccttatc 1740

actttccgga aaaattgatt ccactagtaa ttcttaatgc tctggaaggt aaggcattac 1800

ctatttatgg caaaggggat caaattcgtg attggctgta tgttgaagat catgcgcgtg 1860

cattatatac cgtcgtaacc gaaggtaaag cgggtgaaac ttataacatt ggtgggcaca 1920

acgaaaagaa aaacattgat gtagtgctca ctatttgtaa tttgctggat gagattgtac 1980

cgaaagagaa atcttatcgt gagcaaatca cttatgtcgc cgatcgtccg ggacacgatc 2040

gccgttatgc cattgatgct gagaagattg gtcgcgaatt gggatggaaa ccacaggaaa 2100

cgtttgagag cgggattcgg aagacagtgg aatggtacct gtccaataca aaatgggttg 2160

ataatgtaaa aagtggtgcc tatcaatcgt ggattgaaca gaactatgag ggccgccag t 2220

The termination orf2's of orf1 is initial

aatgaatatc ctcctttttg gcaaaacagg gcaggtaggt tgggaactac agcgtgctct 2280

ggcacctttg ggtaatttga ttgctcttga tgttcactcc actgattact gtggtgattt 2340

tagtaatcct gaaggtgtag ctgaaaccgt aagaagcatt cggcctgata ttattgtcaa 2400

cgcagccgct cacaccgcag tagacaaagc agaatcagaa ccggagtttg cacaattact 2460

taacgcaaca agtgtcgaag cgattgcgaa agcagcaaat gaagttggag cttgggttat 2520

acactactcc accgattatg ttttccctgg cagtggtgac aggccatggc tggaaacgga 2580

tgcaacagcg ccgctaaatg tttacggtga aactaagcta gctggggaaa aagcgttaca 2640

agaacattgc gcaaagcatc ttattttccg taccagctgg gtatacgctg gtaaaggaaa 2700

taattttgcc aaaacgatgt tgcgtctggc aaaagaacgc gaagagttgg ctgtgataaa 2760

cgatcaattt ggcgcaccaa cgggtgctga gcttctggcc gattgtacag cacatgccat 2820

tcgtgtcgca ctgaataaac cggatgtcgc aggcttgtac catctggtag ctagtggtac 2880

cacgacctgg tacgattatg ctgcgctggt ttttgaagag gcgcgcaaag cagatattcc 2940

cctcgcactc aacaaactca atgcggtacc aacgacagcc tatcctacac cagcttgtcg 3000

tccacataac tctcgactta atacagaaaa atttcagcag aattttgcgc ttgtcttgcc 3060

The termination of orf2

tgactggcag gttggtgtga aacgcatgct caacgaatta tttacgacta cagcaatt ta 3120

Orf3's is initial

atagtttttg catcttgttc gtgatgatgg agcaagatga attaaaagga atgatgaa at 3180

gaaaacgcgt aagggtatta ttttagcggg tggttctggt actcgtcttt atcctgtgac 3240

tatggcagtc agtaaacagc tattaccgat ttatgataaa ccgatgatct attacccgct 3300

ctctacactg atgttggcgg gtattcgcga tattttgatt attagtacgc cacaggatac 3360

tcctcgtttt caacaactgc tgggtgacgg tagccagtgg gggctaaatc ttcagtacaa 3420

agtgcaactg actccagatg ggcttgcgca ggcatttatt atcggtgaag agtttattgg 3480

tggtgatgat tgtgctttga ttcttggtga taatatcttt tacggtcacg atctgccgag 3540

gttaatggat gccgctgtta acaaagaaag tggtgcaacg gtatttgcct atcacgtaaa 3600

tgatcctgaa cgctatggtg tcgttgagtt tgataaaaac ggtactgcaa tcagcttgga 3660

agaaaaaccg ttacaaccaa aaagtaatta tgcggtaacc gggctttatt tctatgataa 3720

cgacgttgtg gaaatggcga aaaaccttaa gccttctgcc cgtggtgaac tggaaattac 3780

cgatattaac cgtgtctata tggaacaggg gcgtttatct gttgccatga tgggccgtgg 3840

ttatgcatgg ttagacacgg ggacacatca aagtctgatt gaagcaagta acttcattgc 3900

aacaattgaa gagcgacaag gtttaaaggt atcttgcccg gaagaaattg cttatcgtaa 3960

aggctttatt gacgctgagc aggttaatgt attagcagaa ccactaaaga aaaatgctta 4020

The termination orf4's of orf3 is initial

tggtcagtat ctgctaaaaa tgattaaagg ttac taaaa a tgaatgtaat taaaactgaa 4080

attcctgatg tattaatttt ggaaccggaa gtttttggtg atgagcgcgg tttttttatg 4140

gaaagcttta atcagaaagt tttcgaagag gctgtagggc gtaaggttga atttgttcag 4200

gataaccatt ctaaatcaat taagggtgta ttacgcggat tgcactatca gctggaacct 4260

tatgctcaag gtaaattagt tcgttgtgtg gtcggtgagg tttttgatgt agcagttgat 4320

attcgtaaat cgtcaggtac atttgggaaa tgggttgggg tgaatttgtc tgctgagaac 4380

aagcgtcagt tgtggatacc tgaaggattt gctcatggat ttttggtact tagtgattta 4440

gcagaagttt tatataaaac gaatcaatat tatgctccat cacatgaacg aaatattatc 4500

tggaatgacc ccttgcttaa tattaaatgg ccaaggacag cactgattac tctgtctgat 4560

The termination orf5's of orf4 is initial

aaggatgcaa atggggaaaa attagaacta agtgagtat t gaa atgtctc tcttaaaaca 4620

tagtatatgg aatgttgcgg gctactttat accaacatta attgcaatcc cagcgtttgg 4680

attaattgca agggaaattg gtgtagaact atttggtttg tatactttat caatgatttt 4740

tatagggtat gctagtatat ttgatgctgg gttaacaaga gctgtagtac gcgaaatagc 4800

attactaaaa aacagactgg acgattgtaa tacgataata gtaacttcta ttattgctgt 4860

ggtattttta gggggtatcg gaggcggggg agtgtttctg cttaaagacc atattattga 4920

actgttaaat atgtcaccaa tatactacgt cgatgcgata aagtctctaa tattattatc 4980

atctctgata cctgtattct tagtcacgca aatactatta gcagagcttg aggggcggga 5040

atattttgga atcctaaata tacaaaaaag tttagggaat tctttaattg cagggttgcc 5100

tgcattattt gttttaatta atcaaacact tttttctgca attattggtg tagctattgc 5160

aagagttata tgcttgtggt taagctacat tatgagcagg aaaagaataa ctatcgatat 5220

ttcatttttt tcaataactg ttttaaaacg gttatttaga tatggcgggt gggtaactat 5280

aagtaacata atatctccta tattagcgag tatggataga tttattctat ctcatatcca 5340

gggagcatca aaaatatcat tctatacagt ccctaatgag ctggtaacta ggcttggaat 5400

agttccaggc tctcttggga aagctgtttt tccaaaatta agccatgcaa gtagttttac 5460

agcgtcatat gcagaacaaa aaaaagcgta tatattaatg actgtcattg tattgccttt 5520

ggttttattt atatattact acgcaaagtt tattttaaca ttgtggatgg gggctgagta 5580

tgcagggatt tcagtcgaaa tattacggat tatgcttata gggtatatct ttaactgtta 5640

ttcacaaatc tcttttgcca atatacaggc atttggaaaa gcaaaataca ctgcatacat 5700

ccatatgatg gaatttattc cttatttgat aatgttatat ataatttcaa aggaatgtgg 5760

ggttattggt gtggcgtggt tatggacaat tcgagtgata attgattttt tgatgctttt 5820

The termination orf6's of orf5 is initial

ttatatgagt tatcgttgta ataatcttat gaaaaaaggg tagcct gatg atatatattg 5880

tagtgttaaa ttggaatggg gctatagata ccattaattg tgttaaaagt ttaatgaatt 5940

taaattatga tgattataaa attatcgttg ttgataactg ttctacggat aactcatatg 6000

attctataaa agaaaatctt aatgcattat atattactgg taaaagtttc attgaggtga 6060

agtatgagga tagaagtaaa tatcaaacat tagaaaacga taaaatcata ttaatacaat 6120

ctccgaaaaa taatgggtac gcaagtggta ataacattgg aatagagctc gcacttaatc 6180

aggaggatat gaaatacgtc tgggttctga ataatgatac tgaagtggat aaagaggctt 6240

taactcattt aattagtaaa tgtgattcag ataaaaatat agggatttgc ggttctcgtt 6300

tagtctattt taccgataga gagatgcagc aaggactggg tggggtacat aacagatggt 6360

tatgcactac aaaaaattat gagatgggaa gattagtttc caaaaaatat gatgatgaag 6420

tcattagcaa taatatagat tatataattg gcgcatcaat gtttttctct agagaatgtt 6480

tggaaacagt tggattgatg aatgaagaat attttttata ctatgaagag ttagatattt 6540

gcctcagagc aaaagcaaag aactttaaat taggtatttg ctcagatagt ttggtttatc 6600

ataagatagg tgcaagtact gatgggggaa agagtatgat agctgacctt tgttcaataa 6660

aaaataggct ggccattaca gaaaagtttt atccacaata ttattggacg gtatggttgt 6720

cactctttgt tgtagcattt aaccgtgcta gaagaggtga gtttaataag atgaaaagat 6780

The termination of orf6

gtttgaatgt tatgtttaac ttcaaacgaa ataaaggtag caaatgccat tagaatatgc 6840

atttaatgat ggtgttatta aataaatgta tagtttgata tgtacctgca ggcggccgcg 6900

Orf7's is initial

aattcactag ggtattta at gaaagtggct tttttatctg cttatgatcc actatctaca 6960

tccagttggt cgggcacacc ttattatatg ctaaaggcat tatcgaagag aaatatttcc 7020

attgaaatat taggaccggt aaatagctat atggtataca tgttaaaagc atataaatta 7080

atattaaggt gtttcggaaa ggaatatgat tatagtcgtt cgaagttgct ttccaagtat 7140

tacggtagaa tatttgagag aaaattaaaa aaaattgatg gtttggattt tattattgca 7200

cctgcaggtt cttcacaaat tgctttttta gaaacaaata taccaataat atatctatcg 7260

gatacaacat atgatcaatt aaaaaactat tatccgaatt taaataaaaa aacaattata 7320

aatgatgagg atgcaagttt aatcgaacgc aaggctattg aaaaagcaac agtcgtatct 7380

ttcccatcta aatgggcaat ggatttttgc aaggactatt acagattgga ttctgataaa 7440

ttagttgaaa taccatgggg ggctaattta tttgaagata ttcattttgc taataaaact 7500

ataattcaaa agattagtta tacttgtctt ttcttggggg tcgattggga aagaaaaggt 7560

ggtaaaacag cattgaaagc aattgaatat gtaagagagt tatatgggat cgatgttaga 7620

ctaaaaattt gtggatgtac tccgaatcaa gagattttac ctgcttgggt tgaattaatt 7680

gataaaataa ataaaaataa cgttgaagaa tatcagaaat tcatcgatgt gttatctaac 7740

gctgatatac ttcttttacc aaccattgct gaatgttacg gaatggtatt ttgtgaagct 7800

gcagcttatg gattgccagt tgtcgccaca gatacaggtg gaattagttc tatagttatc 7860

aacgaaagga cggggatatt aattaaagac tcgtcagact ataagcactt tggaaatgca 7920

attcataaaa taattagctc cgtagagact tatcaaaact actcccaaaa cgcaagagtt 7980

Orf8's is initial

agatataata aaatattgca ttgggacaat tgggcaaaaa agataattga gattatgt at 8040

The termination of orf7

gagcataaga atagaagaac caaa tagcac aaaaagaatt atatttttat ttttactttt 8100

ccttgttttt cctgattttt tgttttatac attagggatt gataatttta gcatttcaac 8160

gataatctcc attatattgc tttttgtttt tttaagggct aaaaacactt gcaaagataa 8220

tttgctaata atagtagtat tattcatatt gttgtgtttt aactgtctgt taagtatgct 8280

atttaatatt gaacaggttt tatcatttaa aattgtactt tcaatgtata gcatcttaat 8340

aatggcatac gtctcctctt gttatgcaca gactttgtgg ttatgttctg aagaaatact 8400

taagagatcc gtcttttatt tgttcgcatt tctttgcctt attggcatta taagtattct 8460

tttacagaaa actgagatta tacatgataa aagtatgatt ctttttcctg aaccatcagc 8520

atttgcattg gttttcatac ctatcttttc attttgttta tactatacaa gggggggggg 8580

ggtactattg ctctatatat tatctttggg tattgcgtta ggtatccaga acttaacgat 8640

gttagtaggc attgtgatta gtgtttttgt gatgaaaaaa ataacaataa ggcaaactat 8700

tgttatattt ttaggggcat ggattttttc catgatatta agtgatttag acatttctta 8760

ctatacatcg cggcttgatt ttaaaaatac tacgaactta tcagtgcttg tatatctttc 8820

aggaattgaa agagctttct tgaattttat tacaagttat ggtcttggta ttggttttca 8880

acaaatgggc gtgaatgggg aggtaggagt gtatcaacaa attttagctg atcttgatgc 8940

ccctatgtta aatatatacg atggctcatt tatttcttcg aagttaatat ctgagtttgg 9000

gtttattggt gcaataatgt gcattttcta tctttttatt tttttcgatt ttatctgcgt 9060

The termination of orf8

ttcaaaaaaa ataagagata tccaccgcag tatattt tag catatagttt ctatatgtgt 9120

ttcttcatcc ccctttttat acgtggtgct ggctatataa atccctatgt gtttatgtta 9180

ttttcatcaa tatttttatg caaatatcat gctaaaatta tcttgatgaa atctaatgtc 9240

aaaatggcta tataatagta gattatgtta tcattaatac gtaaataaca tatattctaa 9300

ccagggcata aataatgtgc ataaaaaaat taagttaatt aaacgatatg gcctttatgg 9360

tggtcttagg cttcttaaag attattctta acaaaagttt tattttgttc aaatgttagg 9420

gttattagat tttcatgtta tcttagaaaa gatggaagtg ttagttttgg aaaagggttt 9480

acatcaggtg tagtattagg aggtgataca tttatggatg ctatagtgtc tattggagaa 9540

aatgttcaaa ttaatgatta tgttccacat cgcggctatt aataatgcca ttattggtag 9600

agatacatta atagcaagta aagtatttat tagtgatcat aatcatggta ttttttctaa 9660

atctgatatc catagttcac caactattat tccttcatct aggtctcttg aatctgcacc 9720

tgtgtatatt gaagagcgtg tgtggattgg tgaaaacgtg acaatattac caggtgcgtg 9780

tataggtaat ggtgtagtta ttggcgcaaa tagtgttgtt cgtggtgaga ttcttaataa 9840

tgtgatctga gttgaccccc gaaaacacca gacagtagat gtatcttaaa ataagaggta 9900

ggcttggaca tatatctaac ttaagataca atccctgtct ggaaataaga gggcaagaac 9960

agaaaatgtt tcgatttggg aaagatagga tatggcttct tatgtagtat ataatatcct 10020

gcattcattc ggataacttc ctatggaagt gtactttgct ctgtctgttt tcatttgttg 10080

Orf9's is initial

aaaatttatg ttaataagaa gctttagata accacttagg aactgt atgt ttgatttgtc 10140

caaaagttat attgttgtaa gtgcgacggc gctggcctcc ggaggtgcat taactatatt 10200

aaagcaattt ataaaacatg catcacaaaa ttcaaatgac tatattatgt ttgtatctgc 10260

gggattagag ttgccggtct gtgataacat catttacata gaaaacacac caaaaggatg 10320

gttgaaaaga atatattggg attggttcgg ttgtcgaaag tttatctctg aacataagat 10380

taacgttaag aaagtaattt ctctacaaaa ttccagtttg aatgttcctt atgagcagat 10440

tatttacttg caccagccaa ttcctttcag taaagttgat tcttttttaa atgatatcac 10500

gttcgataac gtaaaacttt ttttatataa aaatttttat tcctatttta tatttaaata 10560

tgtgaatgca aatacaacca tcgtagtgca aacgaattgg atgaaaaaag gagtgttgga 10620

tcaatgtgat aagattagtt ccgaaagggt ccttgttata aaacctgata tcaaaacatt 10680

taataatact aaatttgatg tagataagga tgtatctgtt aaaacactct tatatccagc 10740

gacaccactt acctataaaa atcatttggt cattctgaag gcgttggtta ttttaaagca 10800

aaagtatttt atagatgatc tgaaattcca agtgactttt gaaaagaata ggtacaaaaa 10860

ttttgataag tttgtgcaat taaataacct aagcaaaaac attgattatc tcggtgttct 10920

ttcatactcg aaattgcaaa aaaaatatat ggcggcatct tttattgttt ttcctagcta 10980

tatagaatca tatgggttac cactcatcga agctgctagt ttaggaaaaa aaatcattag 11040

tagtgatctt ccttatgccc gtgatgtttt aaagaattat agcggcgtag attttgtaat 11100

ttacaatgat gaagatggct gggctagggc gttgtttaat gttttaaatg gcaattcgaa 11160

gctcaatttt aggccttatg aaaaagatag tcgttcatct tggccacagt tcttctctat 11220

The termination orf10's of orf9 is initial

tttaaaa taa ggtgtatt at gtttaatggt aaaatattgt taattactgg tggtacgggg 11280

tctttcggta atgctgttct aagacgtttt cttgatacag atatcaaaga aatacgtatt 11340

ttttcccggg atgaaaaaaa acaagatgac atgaggaaaa aatataataa tcagaagctt 11400

aagttctata taggtgatgt tcgcgattat tcgagtattc tcaatgcttc tcgaggtgtt 11460

gattttattt atcatgctgc agccctgaag caagtgcctt cctgcgaatt ccaccccatg 11520

gaagctgtaa aaacgaatat tttaggtact gaaaacgttc tagaagctgc aatagctaat 11580

cgcgttaggc gaattgtatg tctgagtaca gataaagccg tatatcctat taatgcaatg 11640

ggcatatcta aagcaatgat ggaaaaagtc attgttgcaa aatcacgtaa tcttgatagt 11700

tcaaaaacag ttatctgcgg aacacgttat gggaatgtaa tggcttcacg tggatcggtc 11760

atcccattgt ttgttgatct aatcaaatct ggtaaaccat tgaccattac cgatcccaat 11820

atgactcgtt tcatgatgac gcttgaggat gctgttgatc tggtccttta tgccttcgag 11880

catggaaata acggtgatat tttcgttcag aaagctcctg cggcaacaat tcaaacatta 11940

gccattgcac ttaaggaatt gctaaatgcc catgaacatc caatcaatat tattggaact 12000

cgacacgggg aaaaacttta cgaagcgtta ttgagccgag aggaaatgat agcagcggaa 12060

gatatgggtg attattatcg tgttccacca gatctccgcg atttgaacta tggtaaatat 12120

gtggaacatg gtgaccgtcg tatctcggaa gtggaagatt ataactctca taatactgag 12180

agattagatg ttgagggtat gaaaaaatta ctgctaaaac ttccttttat ccgggcaatt 12240

The termination of the initial orf10 of orf11

cgttctggtg aagatt atga gttggattca taatatgaaa attttagtta ctggtgcttc 12300

agggtttatc ggccgtaatt tggttttccg ccttaaggag gctggttata acgaacttat 12360

tacgatagat cgtaactctt ctttggcgga tttagagcag ggacttaagc aggcagattt 12420

catttttcac cttgcaggag taaatcgtcc tgtgaaggag agtgaatttg gagagggaaa 12480

tagcaacgta actcaacaga ttgttgatgt tctgaaaaaa aataataaaa atactcctat 12540

catgctgagt tcttccatcc aggctgaatg tgataacgct tatggaaaga gtaaagcgac 12600

tgcggagaaa atcattcagc agtatgggga aacgacaaat gccaaatatt atatttatcg 12660

cttgccgaat gtattcggta agtggtgtcg cccaaattat aactccttta tagcaacttt 12720

ctgccatcgc attgcaaatg atgaaactat tacaattaat gatccttcag cagttgttga 12780

tctggtgtat atagatgact tttgttctga catattaaag ctattggaag gagcgaacga 12840

aactggttac aggacattcg gtccaattta ttctgttact gttggtgaag tggcgcaatt 12900

aatttaccga tttaaagaaa gtcgccaaac attaatcacc gaagatgtag gtaatggatt 12960

tacacgtgca ttgtactcaa catggttaag ttatctgtct cctgaacagt ttgcgtatac 13020

ggttccttct tatagtgatg atagaggggt attctgtgaa gtattgaaaa cgaaaaacgc 13080

gggccagttt tcgttcttta ctgcgcatcc aggaattact cgggggggac attatcatca 13140

ttccaaaaat gagaaattta ttgtcatccg aggaagtgct tgtttcaaat ttgaaaatat 13200

tgtcacgggt gagcgatatg aatttaatgt ctcctcagat gattttaaaa ttgttgaaac 13260

agttccgggg tggacgcatg acatcactaa taatggctcg gatgagctag ttgttatgct 13320

The termination of orf11

ttgggcaaat gaaatattta atcgttctga accagatact atagcgagag ttttatcg tg 13380

Orf12's is initial

aaaaaattga aagtc atgtc ggttgttggg actcgtccag aaattattcg actctcgcgt 13440

gtccttgcaa aattagatga atattgtgac caccttatta ttcatactgg ccaaaactac 13500

gattatgaat tgaatgaagt ttttttcaaa gatttgggtg ttcgcaaacc agattatttt 13560

cttaatgccg caggtaaaaa tgcagcagag actattggac aagttatcat taaagttgat 13620

gatgtccttg aacaggaaaa accagaagct atgttagttc ttggcgatac taactcctgt 13680

atttcagcaa taccagcaaa gcgtcgaaaa attccgatct tccatatgga agcggggaat 13740

cgttgttttg accaacgcgt accggaagaa actaacagaa aaatagttga ccacactgct 13800

gatatcaata tgacatatag cgatatcgct cgtgaatatc ttctggctga aggtgtacca 13860

gccgatagaa ttattaaaac cggtagccca atgtttgaag tactcactca ttatatgccg 13920

cagattgatg gttccgatgt actttctcgc ctgaatttaa cacctgggaa tttctttgtg 13980

gtaagtgccc acagagaaga aaatgttgat acccctaaac agcttgcgaa actggcgaat 14040

atacttaata ccgtggctga aaaatatgat gtcccggtag ttgtttctac tcatcctcgc 14100

actcgtaacc gcatcaatga aaacggtatt caattccata aaaatatctt gcttcttaag 14160

ccattaggat ttcacgatta caaccatctg caaaaaaatg cacgtgctgt tttatcggat 14220

agtgggacta ttacagaaga gtcctccatt atgaacttcc ctgcactcaa tatacgagaa 14280

gcgcacgaac gcccggaagg cttcgaagaa ggggcagtaa tgatggtcgg tcttgaatct 14340

gagcgcgttt tacaggcatt agaaattatt gcaacacagc ctcgtggaga agtacgctta 14400

cttcgtcagg tcagtgacta tagcatgccc aatgtttcag ataaagttgt gcgtattatc 14460

The termination orf13's of orf12 is initial

cattcatata ctgactacgt taaacgggtt gtctggaaac aatac taatg aaacttgcat 14520

taatcattga tgattatttg ccccatagca cacgtgttgg ggctaaaatg tttcatgagt 14580

taggccttga attgttgagc agaggccatg atgtaactgt aattacgcct gacatcacat 14640

tacaagcaat ctattctgtt agtatgattg atggtataaa ggtttggcgt ttcaaaagtg 14700

gacctttaaa ggatataggt aaggctaagc gtgccataaa tgaaactctt ttatcttttc 14760

gtgcatggcg tgcattaaag cacctcattc aacatgatac atttgatggt atcgtttatt 14820

attccccctc tattttttgg ggagacttgg ttaaaaaaat aaaacagcga tgccagtgcc 14880

caagctacct ggtcctaagg gatatgtttc cacagtgggt cattgatgca ggtatgttga 14940

aagccggttc gccaattgaa aaatatttca ggtattttga aaaaaaatca tatcagcagg 15000

ctgaccggat aggggtaatg tctgataaga atcttgagat atttcgtcag accaataaag 15060

gttatccgtg tgaagtttta cgtaattggg cctcaatgac tcctgtgtct gccagcgatg 15120

attatcattc acttcgtcaa aaatacgatc taaaagataa agtcattttt ttctatggcg 15180

gtaatattgg gcatgctcag gatatggcga acttaatgcg ccttgcgcgt aatatgatgc 15240

gttatcatga tgctcatttc ctgtttatag ggcagggtga tgaagttgac ctgataaaat 15300

ctcttgctgc agaatggaat ttaactaatt tcactcacct accttcagtg aaccaggaag 15360

agtttaaatt aattttatct gaagttgatg tcggcctgtt ctccctttca tctcgccatt 15420

cttcacataa tttccctggg aaattactag ggtatatggt tcaatcaatc ccgatccttg 15480

ggagtgtgaa tggcggcaat gatttaatgg atgtaattaa taagcacagg gccggtttca 15540

ttcatgttaa tggtgaagat gataaactgt tcgaatctgc acaattgctt cttgcagatt 15600

cggctttaag aaagcagtta ggtcagaacg ctaatgtgtt gttaaaatct caattttcgg 15660

The termination of the initial orf13 of orf14

ttgaatcggc ggcacatact atcgaagtcc gactggaggc aggaga atgc gtt tagttga 15720

tgacaatatt ctggatgagc tttttcgcac agcagtaaat tctgaacgtt tgcgcgctca 15780

ttatttattg cacgcatctc atcaggagaa ggttcaacgt ttacttattg catttgtacg 15840

cgacagctat gttgagcccc attggcatga gttaccgcat cagtgggaaa tgtttgtcgt 15900

catgcaaggg caattagaag tttgtttgta tgagaaaaat ggtgagatac aaaaacagtt 15960

tattgttgga gacggtactg gaataagcgt catggaattt tccccaggtg atatacatag 16020

tgtcaaatgc ctgtcaccga atgcccttat gttggagata aaggaaggac catttgaccc 16080

The termination of orf14

actgaaagct aaggcttttt ctaagtggtt a tagggcgat acaccaccct ttattcttct 16140

atcttattct atacatgctg ggttaccatc ttagcttctt caagccgcgc aaccccgcgg 16200

tgaacacccc ctgacaggag taaacaatgt caaagcaaca gatcggcgtc gtcggtatgg 16260

cagtgatggg gcgcaacctt gcgctcaaca tcgaaagccg tggttatacc gtctctattt 16320

tcaaccgttc ccgtgaaaag acggaagaag tgattgccga aaatccaggc aagaaactgg 16380

ttccttacta tacggtgaaa gagtttgttg aatctctgga aacgcctcgt cgcatcctgt 16440

taatggtgaa agcaggtgca ggcacggatg ctgctattga ttctctcaag ccatacctcg 16500

ataaaggtga catcatcatt gatggtggta acaccttctt ccaggacaca attcgtcgta 16560

accgtgagct gtctgcagaa ggttttaact ttatcggtac cggtgtttcc ggtggtgaag 16620

aaggtgcgct gaaaggacct tccatcatgc ctggtgggca gaaagaagcc tatgaactgg 16680

tcgctccgat cctgaccaaa atcgccgccg ttgctgaaga tggcgaaccg tgcgttacct 16740

atattggtgc cgatggcgca ggtcactatg tgaagatggt tcacaacggt attgaatacg 16800

gtgatatgca gctgattgct gaagcctatt ctctgcttaa aggtggcatg aatctctcta 16860

acgaagaact ggcgcagacg tttaccgagt ggaataacgg tgaactaagc agctacctga 16920

tcgacatcac caaagacatc ttcactaaaa aagatgaaga cggtaactac ctggttgatg 16980

tgatcctgga tgaagcggct aacaaaggta ccggtaaatg gaccagccag agcgcgctgg 17040

atctcggcga accgctgtcg ctgattaccg agtctgtgtt tgcacgttat atctcttctc 17100

tgaaagagca gcgtgttgcc gcatctaaag ttctctctgg cccgcaagcg cagccagctg 17160

gcgacaaagg tgagttcatc gaaaaagttc gccgtgcgct gtatcttggc aaaatcgttt 17220

cttacgctca gggcttctct cagctgcgtg cagcgtctga agagtacaac tgggatctga 17280

actacggcga aatcgcgaag attttccgtg ctggttgcat cattcgtgcg cagttcttgc 17340

agaaaatcac cgatgcttat gccgaaaatc cgcagatcgc taacctgctg ctggctccgt 17400

acttcaagca aattgccgat gactaccagc aggcgctgcg tgatgtcgtt gcttatgcag 17460

tacagaacgg tatcccggtt ccgaccttcg ctgctgcggt tgcctattac gatagctacc 17520

gtgccgctgt tctgcctgcg aacataatcc aggcgcagcg tgacta 17566

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

The above, it only is preferred embodiment of the present invention, be not that the present invention is done any pro forma restriction, every foundation technical spirit of the present invention all still belongs in the scope of technical solution of the present invention any simple modification, equivalent variations and modification that above embodiment did.

Claims (10)

1, a kind of Nucleotide of the O-antigen-specific to intestinal bacteria O25 type, it is characterized in that: it is the isolating Nucleotide shown in SEQ ID NO:1,17566 bases of total length.

2, according to the Nucleotide of the described O-antigen-specific to intestinal bacteria O25 type of claim 1, it is characterized in that: it comprises called after rmlB, rmlD, rmlA, rmlC, wzx, orf6, orf7, wzy, orf9, fnl1, fnl2, fnl3, orfl3,14 genomic constitutions of orf14 are all between galF gene and gnd gene.

3, according to the Nucleotide of the described O-antigen-specific to intestinal bacteria O25 type of claim 2, it is characterized in that the gene that has high degree of specificity in the described gene is: transhipment enzyme gene, it comprises the wzx gene; Pol gene, it comprises the wzy gene; Glycosyltransferase gene, it comprises orf6, orf7, orf9, orf13 gene; Wherein said gene: wzx is the Nucleotide of 4604 to 5863 bases among the SEQ ID NO:1; Wzy is the Nucleotide of 8039 to 9100 bases among the SEQ ID NO:1; Orf6 is the Nucleotide of 5868 to 6833 bases among the SEQ IDNO:1; Orf7 is the Nucleotide of 6919 to 8067 bases among the SEQ ID NO:1; Orf9 is the Nucleotide of 10127 to 11230 bases among the SEQ ID NO:1; Orf13 is the Nucleotide of 14508 to 15716 bases among the SEQ ID NO:1.

4, according to the Nucleotide of claim 1 or 2 described O-antigen-specifics to intestinal bacteria O25 type, it is characterized in that: it also comprises and comes from described wzx gene, wzy gene and their mixing or their reorganization.

5, according to the Nucleotide of the described O-antigen-specific to intestinal bacteria O25 type of claim 4, it is characterized in that the oligonucleotide that wherein comes from the wzx gene is to being: the Nucleotide of 4946 to 4965 bases among the SEQ ID NO:1 and the Nucleotide of 5329 to 5348 bases; The Nucleotide of 5327 to 5346 bases among the SEQ ID NO:1 and the Nucleotide of 5778 to 5797 bases.The oligonucleotide that comes from the wzy gene is to being: the Nucleotide of 8622 to 8641 bases among the SEQ ID NO:1 and the Nucleotide of 8973 to 8980 bases; The Nucleotide of 8349 to 8366 bases among the SEQ ID NO:1 and the Nucleotide of 8993 to 9009 bases.

6, the application of Nucleotide in detecting other polysaccharide antigen of expressing the antigenic bacterium of O-, the O-antigen of identifying bacterium and bacterium of the described O-antigen-specific to intestinal bacteria O25 type of claim 1.

7, the recombinant molecule of the Nucleotide of the described O-antigen-specific to intestinal bacteria O25 type of claim 1 is providing the O-antigen of expressing intestinal bacteria O25 type by inserting to express, and the application in the preparation bacterial vaccine.

8, according to the application of the Nucleotide of the described O-antigen-specific to intestinal bacteria O25 type of claim 1, it is characterized in that, it is used for PCR, is used for hybridization and fluoroscopic examination or is used to make gene chip or microarray as probe as primer, for the application of bacterial detection.

9, the separation method of the Nucleotide of the described O-antigen-specific to intestinal bacteria O25 type of claim 1 is characterized in that it comprises the steps:

(1) genomic extraction: in substratum, cultivate intestinal bacteria O25 type, centrifugal collecting cell; The genomic dna that obtains detects by agarose gel electrophoresis;

(2) by the O-antigen gene in the pcr amplification intestinal bacteria O25 type bunch: with the genome of intestinal bacteria O25 type is that template is passed through its O-antigen gene of Long pcr amplification bunch, with the PCR product that obtains, detect the size and the specificity thereof of PCR product with agarose gel electrophoresis, merge this long PCR product, and with DNA purification kit purified pcr product;

(3) make up O-antigen gene bunch library: Long PCR purified product is used shotgun make up O-antigen gene bunch library;

(4) to the cloning and sequencing in the library: from the library, select the clone of insertion fragment more than 1kb and the insertion fragment among the clone is checked order with laboratory automatic dna sequencer commonly used, sequence reaches 100% fraction of coverage, thereby obtains all sequences of O-antigen gene bunch;

(5) splicing of nucleotide sequence and analysis: applying biological information science software splicing and edit all sequences, thus obtain the Nucleotide full length sequence of the O-antigen gene bunch of intestinal bacteria O25 type;

(6) screening of specific gene: at wzx, the wzy gene design primer in the O-antigen gene of intestinal bacteria O25 type bunch; Respectively designed two pairs of primers in each gene, every pair of primer is distributed in the different places in the corresponding gene, to guarantee its specificity; Is that template is carried out PCR with these primers with the genomes of 166 strain intestinal bacteria and 43 strain Shigellaes, determines the antigenic high degree of specificity of O-of wzx, wzy gene pairs intestinal bacteria O25 type;

(7) detection of primer sensitivity: cultivate intestinal bacteria O25, 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 O25.

10, the separation method of the Nucleotide of the O-antigen-specific to intestinal bacteria O25 type according to claim 9 is characterized in that it comprises the steps:

(1) genomic extraction: 37 ℃ of incubated overnight intestinal bacteria O25 types in the LB of 5mL substratum, centrifugal collecting cell; With the pH value is 8.0 500 l 50mM Tris-HCl and 10 l 0.4M EDTA re-suspended cells, 37 ℃ of incubations 20 minutes, and the N,O-Diacetylmuramidase that adds 10 l 10mg/mL then continues insulation 20 minutes; The Proteinase K, the 15 l 10%SDS that add 3 l 20mg/mL afterwards, 50 ℃ of incubations 2 hours, the RNase that adds 3 l 10mg/mL again, 65 ℃ of incubations 30 minutes, add equal-volume phenol extracting mixture, get supernatant and use isopyknic phenol again: chloroform: primary isoamyl alcohol mixing solutions extracting twice, get supernatant again with isopyknic ether extracting to remove remaining phenol, phenol: chloroform: the mixed volume ratio of primary isoamyl alcohol is 25: 24: 1; Supernatant rolls out DNA and washes DNA with 70% ethanol with glass yarn with 2 times of volume ethanol deposit D NA, and DNA is resuspended among the 30 l TE; Genomic dna detects by 0.4% agarose gel electrophoresis;

(2) by the O-antigen gene in the pcr amplification intestinal bacteria O25 type bunch: with the genome of intestinal bacteria O25 type is that template is passed through its O-antigen gene of Long pcr amplification bunch, be #1523-ATT GTG GCTGCA GGG ATC AAA GAA AT according to the galF gene design upstream primer that often is found in O-antigen gene bunch promoter region at first, the gnd gene design downstream primer according to O-antigen gene bunch downstream is #1524-TAG TCG CGT GNG CCT GGA TTA AGT TCG C again; 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, 55 annealing 15 seconds, 68 ℃ were extended 15 minutes, carry out 30 circulations like this, last, continue to extend 7 minutes at 68 ℃, obtain the PCR product, detect the size and the specificity thereof of PCR product with 0.8% agarose gel electrophoresis, merge 5 pipe long PCR products, and with the Wizard PCR Preps purification kit purified pcr product of Promega company;

(3) make up O-antigen gene bunch library: make up O-antigen gene bunch library with the Novagen DNaseI shot gun method that is modified, reaction system is a 300ng PCR purified product, 0.9 l 0.1M MnCl ₂, the DNaseI of the 1mg/mL of 1 l dilution in 1: 2000, reaction is carried out at room temperature, and enzyme is cut the dna fragmentation size is concentrated between the 1.5kb-3kb, then adds 2 l 0.1M EDTA termination reactions; Merge the same reaction system of 4 pipes, with isopyknic phenol extracting once, use isopyknic phenol: chloroform: the mixing solutions extracting of primary isoamyl alcohol once, phenol: chloroform: the mixed volume ratio of primary isoamyl alcohol is after using isopyknic ether extracting once again at 25: 24: 1, dehydrated alcohol deposit D NA with 2.5 times of volumes, and wash precipitation with 70% ethanol, be resuspended at last in the 18 l water, in this mixture, add 2.5 ldNTP (1mMdCTP, 1mMdGTP, 1mMdTTP subsequently, 10mMdATP), 1.25 the T4DNA polysaccharase of l 100mM DTT and 5 units, 11 ℃ 30 minutes, enzyme is cut product mends into flush end, after 75 ℃ of termination reactions, add the Tth archaeal dna polymerase of 5 units and corresponding damping fluid thereof and system is expanded as 80 l, 70 ℃ of reactions 20 minutes make 3 of DNA ' end add the dA tail; This mixture is through the equal-volume chloroform: after the mixing solutions extracting of primary isoamyl alcohol and the extracting of equal-volume ether with 3 * 10 of Promega company ^-3The pGEM-T-Easy carrier connect 10 hours in 16 ℃, cumulative volume is 90 l, chloroform: the mixed volume ratio of primary isoamyl alcohol is 24: 1; 10 * the buffer of 9 l and the T4DNA ligase enzyme of 25 units are wherein arranged, be that 5.2 the 3M NaAc and the dehydrated alcohol precipitation of 2 times of volumes are connected mixture with the pH value of 1/10 volume at last, wash precipitation with 70% ethanol again, be dissolved in after the drying in the 30 l water and obtain connecting product; Preparation method with the electric transformed competence colibacillus cell of Bio-Rad company prepares the competence e.colidh5, get after 2-3 l connects product and 50 l competence bacillus coli DH 5 alphas mix, forward in the electric shock cup of 0.2cm of Bio-Rad company and shock by electricity, voltage is 2.5 dried volts, time is 5.0 milliseconds to 6.0 milliseconds, the SOC substratum that adds 1mL after the electric shock immediately in cup makes the bacterium recovery, then bacterium is coated in and contains penbritin, on the LB solid medium of X-Gal and IPTG, 37 ℃ of incubated overnight, obtain blue white bacterium colony next day, with the white colony that obtains promptly the white clone forward on the LB solid medium that contains penbritin and cultivate, from each clone, extract plasmid simultaneously, and cutting the segmental size of evaluation insertion wherein with the EcoRI enzyme, the white that obtains clone group has constituted the O-antigen gene bunch library of intestinal bacteria O25 type;

(4) to the cloning and sequencing in the library: from the library, select 96 clones of insertion fragment more than 1kb and the insertion fragment among the clone is checked order with this lab A BI3730 type automatic dna sequencer, sequence reaches 100% fraction of coverage, thereby obtains all sequences of O-antigen gene bunch;

(5) splicing of nucleotide sequence and analysis: the Pregap4 and the splicing of Gap4 software of the Staden package software package of publishing with Britain Camb MRC Molecular Biology Lab and edit all sequences, thus obtain the Nucleotide full length sequence of the O-antigen gene bunch of intestinal bacteria O25 type; The quality of sequence is mainly guaranteed by two aspects: 1) genome of intestinal bacteria O25 type is done 5 LongPCR reactions, mix these products then to produce the library, 2) to each base, guarantee high-quality fraction of coverage more than 3, after obtaining the nucleotide sequence of intestinal bacteria O25 type O-antigen gene bunch, Orffinder with American National biotechnology information science center finds gene, find the reading frame of 14 openings, determine also that with the function of finding the reading frame that these are open what gene they are with the genetic comparison among the Blast groupware and the GenBank, finish gene annotation with the Artemis software at Britain Sanger center again, do accurate comparison between DNA and protein sequence with ClustralW software, obtain the structure of the O-antigen gene bunch of intestinal bacteria O25 type at last;

(6) specific gene screening: at wzx, the wzy gene design primer in the O-antigen gene of intestinal bacteria O25 type bunch; Respectively designed two pairs of primers in each gene, every pair of primer is distributed in the different places in the corresponding gene, to guarantee its specificity; Is that template is carried out PCR with these primers with the genomes of 166 strain intestinal bacteria and 43 strain Shigellaes, except that a band that in containing intestinal bacteria O25 group, has obtained the expection size, the correct product of the expection clip size that in other groups, all do not increase, so wzx, the O-antigen of wzy gene pairs intestinal bacteria O25 type all is high special.

(7) detection of primer sensitivity: buy the live pig meat stuffing on the market, stir, be divided into the 20g portion, exist in-40 ℃ of refrigerators standby; The frozen bacterium liquid of 10 μ l intestinal bacteria O25 is inoculated in the triangular flask of 20mL LB substratum, in 37 ℃, 200 rev/mins, cultivate 12 hours to saturated, the cultured bacterium liquid that takes a morsel does 10 ⁶With 10 ⁷Dilution doubly, remaining bacterium liquid are put in 4 ℃ the refrigerator standby, get 50 μ l dilution bacterium liquid coating LB agar plate, and 37 degree are cultivated 12h, to being coated with plate count, calculate viable bacteria concentration in the stoste.In 5 portions of live pig meat stuffings, mix 5 * 10 respectively ³, 5 * 10 ², 5 * 10 ¹, 5 and 0 viable bacteria stir, and add 200mL LB substratum, and through 6 layers of filtered through gauze, filtered liquid 200 rev/mins, is cultivated 12h in 37 ℃.Get 3mL bacterium liquid in 6 from cultured bacterium liquid, centrifugal 5 minutes of 000g removes supernatant, adds 100 μ l MQ ultrapure waters and blows precipitation and mixing open, puts into 100 degree boiling water and boils 15 minutes, and lysate is in 12, and centrifugal 8 minutes of 000g gets 1 μ supernatant as pcr template; Right with 4 pairs of oligonucleotide, the Nucleotide of 4946 to 4965 bases among the SEQ ID NO:1 and the Nucleotide of 5329 to 5348 bases; The Nucleotide of 5327 to 5346 bases among the SEQ ID NO:1 and the Nucleotide of 5778 to 5797 bases; The Nucleotide of 8622 to 8641 bases among the SEQ ID NO:1 and the Nucleotide of 8973 to 8980 bases; The Nucleotide of 8349 to 8366 bases among the SEQ ID NO:1 and the Nucleotide of 8993 to 9009 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 ", 55 ℃: 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 O25 in the pork filling when using aforesaid method.