CN1554758A - O-antigen specific nucleotide of E.coli 074 type - Google Patents

Abstract

The present invention provides a kind of nucleotide specific to O-antigen of Escherichia coli O74, and it is the total nucleotide sequence of gene cluster of Escherichia coli O74 to control the synthesis of O-antigen, such as separated nucleotide shown in SEQ ID No. 1 with whole length of 11743 bases; nucleotide similar to that in SEQ ID No. 1 with one or several inserted, deleted or substituted bases; and oligonucleotide originated glycosyltransferase gene and oligose unit processing gene of Escherichia coli O74 antigen gene cluster. PCR proves the high specificity of the oligonucleotide on the Escherichia coli O74 antigen. The present invention also discloses the method of detecting and identifying the Escherichia coli O74 in human body and in environment with the oligonucleotide of the present invention.

Description

Nucleotide to the O-antigen-specific of intestinal bacteria O74 type

Technical field

The present invention relates to the complete nucleotide sequence of control O-antigen synthetic gene cluster in the intestinal bacteria O74 type (Escherichia coli O74), particularly relate in the intestinal bacteria O74 type oligonucleotide in the control O-antigen synthetic gene cluster, can utilize these the oligonucleotide of the O-antigen-specific intestinal bacteria O74 type in human body and the environment and identify O-antigen in these pathogenic bacterium quickly and accurately.

Background technology

O-antigen is the O specific polysaccharide composition in the gram negative bacterium lipopolysaccharides, and it is made up of many multiple oligosaccharide unit.The antigenic building-up process of O-is studied clearlyer: by glycosyltransferase nucleoside diphosphate monose is transferred on the fat molecule that is fixed on the cell inner membrance earlier, then in the inboard synthesis of oligose unit of inner membrance, the antigenic oligosaccharide unit of O-is transferred to the inner membrance outside by the transhipment enzyme again, then aggregate into polysaccharide by polysaccharase, be connected to again and form lipopolysaccharide molecule [Whitfield, C. (1995) " Biosynthesis of lipopolysaccharide O antigens " .Trends inMicrobiology.3:178-185 on the glycolipid molecule; Schnaitman, C.A.and J.D.Klena. (1993) " Genetics oflipopolysaccharide biosynthesis in entericbacteria " .MicrobiologicalReviews, 57 (3): 655-682].Coding is responsible for the generally adjacent arrangement on karyomit(e) of gene of all enzyme molecules of O-antigen synthetic, form a gene cluster [Reeves, P.R., et al. (1935) " Bacterialpolysaccharide synthesis and gene nomenclature " Trends in Microbiology, 4:495-503].In Shigellae, intestinal bacteria and Salmonellas, O-antigen gene [Lei Wang.et al (2001) " Sequence analysis of four Shigella boydii O-antigenloci:implication for Escherichia coli and Shigellla relationships " .Infection andImmunity, 11:6923-6930 bunch between galF and gnd gene; Lei Wang and Peter Reeves (2000) " The Escherichia coliO111 and Salmonella enterica O74 gene clusters:gene clusters encoding the samecolitose-containing O antigen are highly conserved " .Journal ofBacteriology.182:5256-5261].The O-antigen gene bunch contains three genoids: sugared synthesis path gene, glycosyltransferase gene, oligosaccharide unit treatment gene, the required nucleoside diphosphate monose of enzymic synthesis O-antigen of wherein sugared synthesis path genes encoding; Thereby the enzyme of glycosyltransferase gene coding forwards nucleoside diphosphate monose and other molecule to and makes monose aggregate into oligosaccharide unit on the monose; The oligosaccharide unit treatment gene comprises transhipment enzyme gene and pol gene, and they transfer to the bacterium inner membrance outside with oligosaccharide unit, and repolymerization becomes polysaccharide.Glycosyltransferase gene and oligosaccharide unit treatment gene only are present in the gene cluster of carrying these genes.The difference of monose in the O-antigen, between monose between the difference of link button and the oligosaccharide unit difference of link button constituted the antigenic diversity of O-, and the composition of monose, the link button between monose and the link button between the oligosaccharide unit are by the Gene Handling in the O-antigen gene bunch, so the O-antigen gene bunch has determined O-antigenic synthetic, has also determined the antigenic diversity of O-.

Because O-antigen is extremely strong antigen, be one of important paathogenic factor of intestinal bacteria, it has extremely strong diversity again simultaneously, and this enlightens us can study a kind of intestinal bacteria and good, highly sensitive method of the antigenic specificity of O-thereof of detecting quickly and accurately.With surperficial polysaccharide is that the serology immune response of target has been used to somatotype and the evaluation to bacterium always since the thirties in last century, is unique means of identifying pathogenic bacterium.This diagnostic method needs a large amount of antiserum(antisera)s, and the antiserum(antisera) general classes is incomplete, quantity not sufficient, and also there are some difficulties in a large amount of antiserum(antisera)s in preparation with in storing.On the other hand this method length consuming time, sensitivity is low, loss is high, poor accuracy, so, generally believe that now this traditional serology detection method will be that the modern molecular biology method replaces.1993, Luk, J.M.C et.al has identified the O-antigen [Luk of Salmonellas with the specific nucleotide sequence of Salmonellas (S.enterica) O-antigen gene bunch by PCR method, J.M.C.et.al. (1993) " Selective amplification of abequose andparatose synthase genes (rfb) by polymerase chain reaction for identification ofS.enterica major serogroups (A; B; C2; 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.Nineteen thirty-five, Paton, the A.W et.al serotype [" Molecular microbiological investigation of an outbreak of Hemolytic-Uremic Syndrome caused by dry fermented sausage contaminated with Shiga-liketoxin producing Escherichia coli " .J.Clin.Microbiol.34:1622-1627] of the oligonucleotide that comes from the wbdI gene of the O-antigen-specific of E.coli O111 having been identified the toxogenic E.coli O111 of a strain, but afterwards studies show that Paton, the usefulness of A.W et.al comes from the oligonucleotide of wbdI gene and identifies that the method for the serotype of E.coli O111 has false positive results to occur.Bastin D.A.and Reeves, P.R. think, this is because the wbdI gene is sugared synthesis path gene [the Bastin D.A.and Reeves of a supposition, P. R. (1995) Sequence and analysis of the O antigen gene (rfb) cluster of Escherichiacoli O111.Gene 164:17-23], and have this sugar in the antigenic structure of the O-of other bacterium, so sugared synthesis path gene is not a high special for O-antigen yet

Shigellae has 46 kinds of serotypes, but have only 33 kinds of different O-antigens, intestinal bacteria have 166 kinds of different O-antigen [Reeves, P.R (1992) " Variation in O antigens; niche specificselection and bacterial populations " .FEMS Microbiol.Lett, 100:509-516], the two sibship is very near, and there are 12 kinds to be intestinal bacteria and the total [Ewing of Shigellae, W.H. (1986) " Edwards and Ewing ' s identification of the Enterobacteriaceae " .Elsevier SciencePublishers, Amsterdam, The Netherlands; T.cheasty, et al. (1983) " Antigenicrelationships between the enteroinvasive Escherichia coli antigensO28ac; O112ac; O124; O136, O143, O74; O152 and and Shigellla O antigens " J.clinMicrobiol, 17 (4): 681-684]

Summary of the invention

The Nucleotide that the purpose of this invention is to provide a kind of O-antigen-specific to intestinal bacteria O74 type.It is the Nucleotide in the O-antigen gene bunch of intestinal bacteria O74 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 O74 type.

Another object of the present invention has provided the gene of the O-antigen gene bunch that constitutes intestinal bacteria O74 type: the gene of transhipment enzyme comprises the wzx gene or with wzx the gene of identity function is arranged; Pol gene comprises the wzy gene or with wzy the gene of identity function is arranged; The monose synthase gene comprises that orf1, orf2, orf3, orf4, orf5 and orf1, orf2, orf3, orf4, orf5 have the gene of identity function; Glycosyltransferase gene comprises that orf7, orf8, orf10 gene and orf7, orf8, orf10 have the gene of identity function.

Another purpose of the present invention has provided oligonucleotide, and the gene that they come from coding transhipment enzyme is the wzx gene or with wzx the gene of identity function is arranged; The gene that comes from the coding polysaccharase is the wzy gene or with wzy the gene of identity function is arranged; They are the oligonucleotide in the said gene, and length is at 10-20nt; They are special to the O-antigen of intestinal bacteria O74 type; Especially the oligonucleotide of listing in the table 1, they are high specials to the O-antigen of intestinal bacteria O74 type, and these oligonucleotide are also reconfigurable, the oligonucleotide after the combination also is a high special to the O-antigen of intestinal bacteria O74 type.

The above-mentioned oligonucleotide that a further object of the present invention provides can be used as primer and is used for nucleic acid amplification reaction, perhaps be used for hybridization as probe, perhaps be used to make gene chip or microarray, thereby pass through O-antigen and the detection and the identification of escherichia coli O74 type of these methods detections and identification of escherichia coli O74 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 O74 type.Can obtain the complete sequence of the O-antigen gene bunch of other bacteriums according to present method operation, the complete sequence of the gene cluster of the bacterium of other polysaccharide antigens that also can obtain to encode.

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

The present invention is to the Nucleotide of the O-antigen-specific of intestinal bacteria O74 type, and it is the isolating Nucleotide shown in SEQ ID NO:1,11743 bases of total length; The base that perhaps has one or more insertions, disappearance or replacement keeps the Nucleotide of the SEQ ID NO:1 of described isolating functional nucleotide simultaneously.

The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O74 type, it is by 10 genomic constitutions, all between galF gene and gnd gene.

The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O74 type, described gene is: the gene of transhipment enzyme comprises the wzx gene or with wzx the gene of identity function is arranged; Pol gene comprises the wzy gene or with wzy the gene of identity function is arranged; The monose synthase gene comprises that orf1, orf2, orf3, orf4, orf5 and orf1, orf2, orf3, orf4, orf5 have the gene of identity function; Glycosyltransferase gene comprises that orf7, orf8, orf10 gene and orf7, orf8, orf10 have the gene of identity function.Wherein said gene: wzx is the Nucleotide of 5030 to 6289 bases among the SEQ ID NO:1; Wzy is the Nucleotide of 8078 to 8253 bases among the SEQ ID NO:1; Orf1 is the Nucleotide of 1137 to 2213 bases among the SEQ ID NO:1; Orf2 is the Nucleotide of 2210 to 3073 bases among the SEQ ID NO:1; Orf3 is the Nucleotide of 3075 to 3479 bases among the SEQ ID NO:1; Orf4 is the Nucleotide of 3469 to 3927 bases among the SEQ IDNO:1; Orf5 is the Nucleotide of 3930 to 5033 bases among the SEQ ID NO:1; Orf7 is the Nucleotide of 6300 to 7241 bases among the SEQ ID NO:1; Orf8 is the Nucleotide of 7257 to 8078 bases among the SEQ ID NO:1; Orf10 is the Nucleotide of 9231 to 10313 bases among the SEQ ID NO:1.

The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O74 type, wherein it is to come from described wzx gene, wzy gene; Glycosyltransferase gene orf7, orf8, orf10 gene; And their mixing or their reorganization.

The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O74 type, the oligonucleotide of the described wzx of coming from gene is to being: the Nucleotide of 5547 to 5564 bases among the SEQ IDNO:1 and the Nucleotide of 6140 to 6157 bases; The Nucleotide of 5143 to 5160 bases among the SEQ IDNO:1 and the Nucleotide of 5414 to 5431 bases.The oligonucleotide that comes from the wzy gene is to being: the Nucleotide of 8119 to 8136 bases among the SEQ ID NO:1 and the Nucleotide of 8955 to 8972 bases; The Nucleotide of 8299 to 8316 bases among the SEQ IDNO:1 and the Nucleotide of 8563 to 8581 bases.

The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O74 type is detecting the application of expressing the antigenic bacterium of O-, identify other polysaccharide antigen of the O-antigen of bacterium and bacterium in diagnosis.

The recombinant molecule of the Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O74 type, and can provide the O-antigen of expressing intestinal bacteria O74 type by inserting to express, and become bacterial vaccine.

The application of the Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O74 type, wherein it is used for PCR, is used for hybridization and fluoroscopic examination or is used to make gene chip or microarray, the bacterium in human body and the environment as probe as primer.

The separation method of the Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O74 type is characterized in that it comprises the steps:

(1) genomic extraction: 37 ℃ of incubated overnight intestinal bacteria O74 types in the LB of 5mL substratum, centrifugal collecting cell.With 500ul 50mM Tris-HCl (pH8.0) and 10ul 0.4M EDTA re-suspended cell, 37 ℃ of incubations 20 minutes, the N,O-Diacetylmuramidase that adds 10ul 10mg/ml then continues insulation 20 minutes.The Proteinase K, the 15ul 10%SDS that add 3ul 20mg/ml afterwards, 50 ℃ of incubations 2 hours, the RNase that adds 3ul10mg/ml again, 65 ℃ of incubations 30 minutes, add equal-volume phenol extracting mixture, get supernatant and use isopyknic phenol again: chloroform: primary isoamyl alcohol (25: 24: 1) mixing solutions extracting twice, get supernatant again with isopyknic ether extracting to remove remaining phenol.Supernatant rolls out DNA and washes DNA with 70% ethanol with glass yarn with 2 times of volume ethanol deposit D NA, and DNA is resuspended among the 30ul TE; Genomic dna detects by 0.4% agarose gel electrophoresis;

(2) by the O-antigen gene in the pcr amplification intestinal bacteria O74 type bunch: with the genome of intestinal bacteria O74 type is that template is passed through its O-antigen gene of Long pcr amplification bunch, at first according to the JumpStart sequences Design upstream primer (#1523-ATT GTGGCT GCA GGG ATC AAA GAA AT) that often is found in O-antigen gene bunch promoter region, again according to the gnd gene design downstream primer (#1524-TAG TCG CGT GNG CCT GGA TTA AGT TCG C) in O-antigen gene bunch downstream; With the Expand Long Template PCR method of Boehringer Mannheim company amplification O-antigen gene bunch, the PCR response procedures was as follows: 94 ℃ of pre-sex change 2 minutes; 94 ℃ of sex change are 10 seconds then, annealed 15 seconds for 60 ℃, 68 ℃ were extended 15 minutes, carry out 30 circulations like this, last, continue to extend 7 minutes at 68 ℃, obtain the PCR product, detect the size and the specificity thereof of PCR product with 0.8% agarose gel electrophoresis, merge 5 pipe longPCR products, and with the WizardPCRPreps purification kit purified pcr product of Promega company;

(3) make up O-antigen gene bunch library: make up O-antigen gene bunch library with the Novagen DNaseI shot gun method that is modified, reaction system is a 300ng PCR purified product, 0.9ul 0.1M MnCl ₂, the DNaseI of the 1mg/ml of 1ul dilution in 1: 2000, reaction is carried out at room temperature, and enzyme is cut the dna fragmentation size is concentrated between the 1.5kb-3kb, then adds 2ul 0.1M EDTA termination reaction.Merge the same reaction system of 4 pipes, with isopyknic phenol extracting once, use isopyknic phenol: chloroform: primary isoamyl alcohol (25: 24: 1) mixing solutions extracting once, after using isopyknic ether extracting once again, dehydrated alcohol deposit D NA with 2.5 times of volumes, and wash precipitation with 70% ethanol, be resuspended at last in the 18ul water, in this mixture, add 2.5ul dNTP (1mMdCTP, 1mMdGTP, 1mMdTTP subsequently, 10mMdATP), 1.25ul the T4DNA polysaccharase of 100mMDTT and 5 units, 11 ℃ 30 minutes, enzyme is cut product mends into flush end, after 75 ℃ of termination reactions, add the Tth archaeal dna polymerase of 5 units and corresponding damping fluid thereof and system is expanded as 80ul, 70 ℃ of reactions 20 minutes make 3 of DNA ' end add the dA tail.This mixture is through the equal-volume chloroform: after primary isoamyl alcohol (24: 1) mixing solutions extracting and the extracting of equal-volume ether with 3 * 10 of Promega company ^-3The pGEM-T-Easy carrier connect 10 hours in 16 ℃, cumulative volume is 90ul.10 * the buffer of 9ul and the T4DNA ligase enzyme of 25 units are wherein arranged, use the dehydrated alcohol precipitation of the 3M NaAc (pH5.2) of 1/10 volume and 2 times of volumes to be connected mixture at last, wash precipitation with 70% ethanol again, be dissolved in after the drying and obtain connecting product in the 30ul water; Preparation method with the electric transformed competence colibacillus cell of Bi0-Rad company prepares the competence e.colidh5, get after 2-3ul connects product and 50ul competence bacillus coli DH 5 alpha mixes, forward in the electric shock cup of 0.2cm of Bi0-Rad company and shock by electricity, voltage is 2.5 kilovolts, time is 5.0 milliseconds to 6.0 milliseconds, the SOC substratum that adds 1ml after the electric shock immediately in cup makes the bacterium recovery, then bacterium is coated in and contains penbritin, on the LB solid medium of X-Gal and IPTG, 37 ℃ of incubated overnight, obtain blue white bacterium colony next day, with the white colony that obtains promptly the white clone forward on the LB solid medium that contains penbritin and cultivate, from each clone, extract plasmid simultaneously, and cutting the segmental size of evaluation insertion wherein with the EcoRI enzyme, the white that obtains clone group has constituted the O-antigen gene bunch library of intestinal bacteria O74 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 O74 type; The quality of sequence is mainly guaranteed by two aspects: 1) genome of intestinal bacteria O74 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 O74 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 10 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 O74 type at last;

(6) specific gene screening: at wzx, the wzy gene design primer in the O-antigen gene of dysentery intestinal bacteria O74 type bunch; Respectively designed two pairs of primers in each gene, every pair of primer is distributed in the different places in the corresponding gene, to guarantee its specificity; Is that template is carried out PCR with these primers with the genomes of 166 strain intestinal bacteria and 43 strain Shigellaes, except that a band that in the 13rd group, has obtained the expection size, any product that all do not increase in other groups is so the O-antigen of wzx, wzy gene pairs intestinal bacteria O74 type all is high special.

Just, first aspect of the present invention provides the full length nucleotide sequence of the O-antigen gene bunch of intestinal bacteria O74 type, its complete sequence shown in SEQ ID NO:1,11743 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 O74 type by method of the present invention, as shown in table 3, it is altogether by 11 genomic constitutions, all between galF gene and gnd gene.

Second aspect of the present invention provides the gene in the O-antigen gene bunch of intestinal bacteria O74 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); Monose synthase gene (orf1, orf2, orf3, orf4, orf5); Glycosyltransferase gene (orf7, orf8, orf10).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 O74 type.

The 3rd aspect of the present invention, wzx gene in the O-antigen gene bunch that comes from intestinal bacteria O74 type is provided or the gene, wzy gene of identity function is arranged or with wzy the gene of identity function, the oligonucleotide of gene are arranged with wzx, they are any one section oligonucleotide in these genes.But, be that the oligonucleotide of listing in the table 1 is right preferentially by usefulness, in table 1, also listed these oligonucleotide to the position in O-antigen gene bunch and with these oligonucleotide to being the size of the product of the PCR reaction done of primer, the annealing temperature in these PCR reaction free lists is carried out.These primers are except that a band that has obtained the expection size in the 13rd group, and any product that all do not increase in other groups is so the O-antigen of wzx, wzy gene pairs intestinal bacteria O74 type all is high special.

The separation method of the Nucleotide of described O-antigen-specific to intestinal bacteria O74 type comprises the steps: 1) genomic extraction; 2) the O-antigen gene in the pcr amplification intestinal bacteria O74 type bunch; 3) make up O-antigen gene bunch library; 4) to the cloning and sequencing in the library; 5) splicing of nucleotide sequence and analysis; 6) screening of specific gene.

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

As used herein, " oligonucleotide " mainly 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 5030 to 6289 bases from SEQ ID NO:1); It is high special to intestinal bacteria O74 type that wzy gene (nucleotide position is the Nucleotide of 8078 to 9253 bases from SEQ ID NO:1) comes from above intragenic oligonucleotide.

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

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

The present invention relates to a kind of antigenic method of one or more bacterial polysaccharideses that detects in the food, these antigens can make sample can with the oligonucleotide specific hybrid of following at least one gene, these genes are: (i) gene of the encoding glycosyl transferring enzyme gene of transhipment enzyme and polysaccharase of (ii) encoding comprises the wzx gene or the gene, wzy gene of identity function is arranged or with wzy the gene of identity function is arranged with wzx.At least one oligonucleotide can be hybridized with at least one more than one such gene specific of expressing the special antigenic bacterium of O-under the situation of condition permission, and these bacteriums are intestinal bacteria O74 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 O74 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 O74 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 O74 types.Oligonucleotide among available the present invention is made the method test sample of primer by PCR, also can will pass through hybridization as probe behind the oligonucleotide mark among the present invention, perhaps by antigen and bacterium in gene chip or the microarray assay sample.

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

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

The present invention discloses the full length sequence of the O-antigen gene bunch of intestinal bacteria O74 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 O74 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 O74 types in the LB of 5mL substratum, centrifugal collecting cell.With 500ul 50mM Tris-HCl (pH8.0) and 10ul 0.4M EDTA re-suspended cell, 37 ℃ of incubations 20 minutes, the N,O-Diacetylmuramidase that adds 10ul 10mg/ml then continues insulation 20 minutes.The Proteinase K, the 15ul 10%SDS that add 3ul 20mg/ml afterwards, 50 ℃ of incubations 2 hours add the RNase of 3ul 10mg/ml again, 65 ℃ of incubations 30 minutes.Add equal-volume phenol extracting mixture, get supernatant and use isopyknic phenol again: chloroform: primary isoamyl alcohol is taken out (25: 24: 1) mixing solutions and is carried twice, get supernatant again with isopyknic ether extracting to remove remaining phenol, supernatant is with 2 times of volume ethanol deposit D NA, roll out DNA and wash DNA with glass yarn, at last DNA is resuspended among the 30ul TE with 70% ethanol.Genomic dna detects by 0.4% agarose gel electrophoresis.

Embodiment 2: by the O-antigen gene in the pcr amplification intestinal bacteria O74 type bunch:

With the genome of intestinal bacteria O74 type is that template is passed through its O-antigen gene of Long pcr amplification bunch.At first according to the JumpStart sequences Design upstream primer (#1523-ATT GTG GCT GCA GGG ATC AAA GAA AT) that often is found in O-antigen gene bunch promoter region, again according to the gnd gene design downstream primer (#1524-TAG TCG CGT GNG CCT GGATTA AGT TCG C) in O-antigen gene bunch downstream; With the Expand Long Template PCR method of Boehringer Mannheim company amplification O-antigen gene bunch, the PCR response procedures was as follows: 94 ℃ of pre-sex change 2 minutes; 94 ℃ of sex change are 10 seconds then, 60 ℃ of annealing 15 seconds, and 68 ℃ were extended 15 minutes, and carried out 30 circulations like this.At last, continue to extend 7 minutes at 68 ℃, obtain the PCR product, the agarose gel electrophoresis with 0.8% detects the size and the specificity thereof of PCR product.Merge 5 pipe long PCR products, and with the Wizard PCR Preps purification kit purified pcr product of Promega company.

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

At first be the acquisition that connects product: make up O-antigen gene bunch library with the Novagen DNaseI shot gun method that is modified.Reaction system is a 300ng PCR purified product, 0.9ul 0.1M MnCl ₂, 1ul1: the DNaseI of the 1mg/ml of 2000 dilutions, reaction is carried out at room temperature.Enzyme is cut the dna fragmentation size is concentrated between the 1.5kb-3kb, then adds 2ul 0.1M EDTA termination reaction.Merge the same reaction system of 4 pipes, with isopyknic phenol extracting once, use isopyknic phenol: chloroform: primary isoamyl alcohol (25: 24: 1) mixing solutions extracting once, after using isopyknic ether extracting once again, dehydrated alcohol deposit D NA with 2.5 times of volumes, and wash precipitation with 70% ethanol, be resuspended at last in the 18ul water.In this mixture, add 2.5ul dNTP (1mMdCTP subsequently, 1mMdGTP, 1mMdTTP, 10mMdATP), the T4DNA polysaccharase of 1.25ul 100mMDTT and 5 units, 11 ℃ 30 minutes, enzyme is cut product mend into flush end, after 75 ℃ of termination reactions, add the Tth archaeal dna polymerase of 5 units and corresponding damping fluid thereof and system is expanded as 80ul, 70 ℃ were reacted 20 minutes, made 3 of DNA ' end add the dA tail.This mixture is through the equal-volume chloroform: after primary isoamyl alcohol (24: 1) mixing solutions extracting and the extracting of equal-volume ether with 3 * 10 of Promega company ^-3The pGEM-T-Easy carrier connect 10 hours in 16 ℃, cumulative volume is 90ul.10 * the buffer of 9ul and the T4DNA ligase enzyme of 25 units are wherein arranged.Use the dehydrated alcohol precipitation of the 3M NaAc (pH5.2) of 1/10 volume and 2 times of volumes to be connected mixture at last, wash precipitation with 70% ethanol again, be dissolved in after the drying and obtain connecting product in the 30ul water.

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

Be electric transformed competence colibacillus cell at last: get after 2-3ul connects product and 50ul competence bacillus coli DH 5 mixes, forward in the electric shock cup of 0.2cm of Bio-Rad company and shock by electricity, voltage is 2.5 kilovolts, and the time is 5.0 milliseconds-6.0 milliseconds.The SOC substratum that adds 1ml after the electric shock immediately in cup makes the bacterium recovery.Immediately bacterium is coated in 37 ℃ of inversion incubated overnight on the LB solid medium that contains penbritin, X-Gal and IPTG then, obtains blue white bacterium colony next day.With the white colony that obtains promptly the white clone forward on the LB solid medium that contains penbritin and cultivate, from each clone, extract plasmid and cut the segmental size of evaluation insertion wherein simultaneously, obtain the O-antigen gene bunch library that white clone group has constituted intestinal bacteria O74 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 O74 type obtained.The quality of sequence is mainly guaranteed by two aspects: 1) genome of intestinal bacteria O74 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 O74 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 10 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 O74 type at last, as shown in table 3.

By retrieving and comparing, find orf1, rmlB in 2 encoded protein and the Shigellla flexneri O-antigen gene bunch, the albumen of rmlA genes encoding has very high consensus amino acid sequence (94%, 72%),, finds orf1 by search to Pfam protein-based order sequenced data storehouse, 2 encoded protein and known RmlB, the very high (2.6 * e of the homology desired value of the consensus sequence of RmlA ^-2153.7 * e ^-108).DTDP-6-deoxy-3 among Orf3 and the Aneurinibacillus thermoaerophilus, 4-keto-hexuloseisomerase has 54% sequence homology; Acetyltransferase enzyme among Orf4 and the Campylobacter jejuni has 72% sequence homogeny; The aminotransferase of Orf5 and intestinal bacteria kind has 54% sequence homology, possible orf1, and 2,3,4,5 are responsible for synthetic a kind of rare monose, and this monose contains kharophen.

Orf6 and orf9 are the proteic genes that there is transmembrane segment in only two codings of intestinal bacteria O74 kind.The O-antigen transferring enzyme of Orf6 encoded protein and e. coli k12 has 32% sequence identity, and it contains 12 uniform transmembrane segments by the proteic topology discovery of HMMTOP2.0 programanalysis, and this is the proteic characteristic feature of Wzx.So name orf6 is wzx.The O-antigen polysaccharase of Orf9 encoded protein and Streptococcus pneumoniae has 22% consistence, 42% similarity, it contains 9 transmembrane segments by the proteic topology discovery of HMMTOP2.0 programanalysis, and hydrophilic loop (loop) in big (61 an amino acid) kytoplasm is arranged, and this is the proteic characteristic feature of Wzy.So name orf9 is wzy.

Orf7, the albumen of 8,10 three genes encodings and other known glycosyltransferases have the sequence identity of 24-36% and the sequence similarity of 44-53%.By the search to glycosyltransferase motif database among the Pfam, the homology desired value of the consensus sequence of the albumen of these three genes encodings and known glycosyltransferase family 1 and 2 is 1 * e ^-12To 5 * e ^-17, so we infer this three genes encoding glycosyltransferases, and because each glycosyltransferase specificity catalysis forms a kind of disaccharide bond, so we infer that the antigenic oligosaccharide unit of O-of intestinal bacteria O74 may be made up of four monose.Because the definite function of these three genes can't be determined, so we are with the temporary called after orf7 of these eight genes, orf8, orf9.

Embodiment 6: the screening of specific gene:

At wzx, wzy gene design primer in the O-antigen gene of intestinal bacteria O74 type bunch, the position of these genes in nucleotide sequence sees Table 1.

Table 1 has been listed glycosyltransferase gene and oligosaccharide unit treatment gene and intragenic primer and PCR data in the O antigen gene bunch of intestinal bacteria O74 type.Glycosyltransferase gene, transhipment enzyme gene and pol gene and their function corresponding and the size of the O antigen gene bunch of intestinal bacteria O74 type in table, have been listed.In each gene, we have respectively designed two pairs of primers, and the difference that every pair of primer is distributed in the corresponding gene is local to guarantee its specificity.In table, also listed position and the size of each primer in SEQ IDNO:1.Is that template carry out PCR with listed corresponding annealing temperature in the table with the genomes of all bacterium in the table 2 with every pair of primer, has obtained corresponding PCR product, and its size is also listed in the table.

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

The genomic dna that contains intestinal bacteria O74 type in the 13rd group is as positive control.Do template with every group of bacterium, be PCR by following condition with every pair in the table 1 primer: 95 ℃ of pre-sex change after 5 minutes, 95 ℃ of sex change 30 seconds, annealing time is 30 seconds, temperature sees Table 1,72 ℃ extended 2 minutes, carried out 25 circulations like this.Continue to extend 5 minutes at 72 ℃ at last, reaction system is 25ul.Template is dilution in 1: 20, gets 1 μ l.After reaction finishes, get the 10ulPCR product and detect the fragment that amplifies by 0.8% agarose gel electrophoresis.

For wzx, wzy gene, each gene all has two pairs of primers detected, every pair of primer has obtained except be PCR in the 13rd group after the correct band of expection size, the correct band of any size does not all increase in other groups, that is to say, not obtaining any PCR product band in the array mostly, so wzx, wzy gene pairs intestinal bacteria O74 type and O-antigen thereof are high specials.

At last, from intestinal bacteria O74 type, screen gene by PCR: wzx, wzy and two glycosyltransferase genes to the O-antigen high special of intestinal bacteria O74 type.And the oligonucleotide of these intragenic any one section 10-20nt is special to the O-antigen of intestinal bacteria O74 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 O74 type.These all oligonucleotide all can be used for the intestinal bacteria O74 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 O74 type, in table, listed the structure of the O-antigen gene bunch of intestinal bacteria O74 type, altogether by 10 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 O74 type, listed the accurate position of all open reading frame in complete sequence in the O-antigen gene bunch of intestinal bacteria O74 type in the drawings, at the underscoring of the initiator codon and the terminator codon of each open reading frame.The initiator codon of open reading frame has two in intestinal bacteria: ATG and GTG.

SEQUENCE?LISTING

＜110〉Nankai University

＜120〉to the Nucleotide of the O antigen-specific of intestinal bacteria O74 type

＜130〉to the Nucleotide of the O antigen-specific of intestinal bacteria O74 type

<160>1

<170>PatentIn?version?3.2

<210>1

<211>11743

<212>DNA

<213>Escherichia?coli

<400>1

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

gaaaaccact?tcgacacctc?ttatgaatta?gaatctctcc?ttgaacagcg?cgtgaagcgt 120

cagctgctgg?cggaagtgca?gtccatctgt?ccgccgggtg?tgaccattat?gaacgtgcgt 180

cagggcgaac?ctttaggttt?gggccactcc?attttatgtg?cacgacccgc?cattggtgac 240

aacccatttg?tcgtggtgct?gccagacgtt?gtgatcgacg?acgccagcgc?cgacccgctg 300

cgctacaacc?ttgctgccat?gattgcgcgc?ttcaacgaaa?caggacgcag?ccaggtgctg 360

gcaaaacgta?tgccgggtga?cctctctgaa?tactccgtca?ttcagaccaa?agaaccgctg 420

gatcgcgaag?gcaaagtcag?ccgcatcgtg?gaatttatcg?aaaaaccgga?tcagccgcag 480

acgctggact?cagacatcat?ggccgtgggt?cgctatgtgc?tttctgccga?tatttggccg 540

gaacttgaac?gcactcagcc?tggtgcatgg?gggcgtattc?agctgactga?tgccatcgct 600

gaactggcga?aaaaacagtc?cgttgatgcc?atgctgatga?caggtgacag?ctacgactgc 660

ggtaaaaaaa?tgggttatat?gcaggcattt?gtgaagtatg?gactacgcaa?cctcaaagaa 720

ggggcgaagt?tccggaaagg?gattgagaag?ctgttaagcg?aataatgaaa?atctgaccga 780

atgtaacggt?tgataagaaa?attataacgg?cagtgaagat?tcgtggcgaa?agtaatttgt 840

tgcgaatatt?cctgccgttg?ttttatataa?acaatcagaa?taacaacgag?ttagcaatag 900

gattttagtc?aaagttttcc?aggattttcc?ttgtttccag?ggcggattgg?taagacaatt 960

agcgtttgaa?tttttcgggt?taagcgcgag?tgggtaacgc?tcgtcacatc?gtagacatgc 1020

atgtagtgct?ctggtagctg?taaagccagg?ggcggtagcg?tgtattaaca?cctctatcaa 1080

tcaacctaag?agccgcttat?ttcacagcat?gctctgaagt?aatatggaat?aaataagtga 1140

agatacttgt?tactggtggc?gcaggattta?ttggttctgc?tgtagttcgt?cacattataa 1200

ataatacgca?ggatagtgtt?gttaatgtcg?ataaattaac?gtacgccgga?aacctggaat 1260

cgcttgctga?tgtttctgat?tctgaacgct?atgtttttga?acatgcggat?atttgcgatg 1320

cagctgcaat?ggcacggatt?tttgctcagc?atcagccgga?tgcagtgatg?cacttggctg 1380

ctgaaagcca?tgttgaccgt?tcaattacag?gtcctgcggc?atttattgaa?accaatattg 1440

ttggtactta?tgtccttttg?gaagccgctc?gcaattactg?gtctgctctt?gatagcgaca 1500

agaaaaatag?cttccgtttt?catcatattt?ctactgacga?agtctatggt?gatttgcctc 1560

atccagatga?agtaaataat?aacgaagaat?tacccttatt?tactgagacg?acagcttacg 1620

caccaagcag?cccttattcc?gcatcaaaag?catccagcga?tcatttagtc?cgggcgtgga 1680

aacgcaccta?tggtttaccg?accattgtga?ctaactgttc?gaataactac?ggtccttatc 1740

actttccgga?aaaattgatt?ccactagtaa?ttcttaatgc?tctggaaggt?aaggcattac 1800

ctatttatgg?caaaggggat?caaattcgtg?actggctgta?tgttgaagat?catgcgcgtg 1860

cgttatatac?cgtcgtaacc?gaaggtaaag?cgggtgaaac?ttataacatt?ggtggacaca 1920

acgaaaagaa?aaacatcgat?gtagtgctca?ctatttgtga?tttgttggat?gagattgtac 1980

cgaaagagaa?atcttaccgc?gagcaaatta?cttatgttgc?cgatcgcccg?ggacacgatc 2040

gccgttatgc?gattgatgca?gagaagatta?gccgcgaatt?gggctggaaa?ccgcaggaaa 2100

cgtttgagag?cgggattcgt?aaaacaatta?actggtattt?gaataataat?gattggtgcc 2160

ggcgagttca?agatggctct?tatcaacgtg?agcgtttggg?ggtaattaga?tgaaaggaat 2220

tattttagct?ggtggttctg?gaacaaggct?ttatccaatt?acaatgggag?tttcaaagca 2280

gctattaccc?atttatgata?aaccgatgat?ttattatcct?ttgtcagtgc?tgatgttggc 2340

aggtatacaa?gatattctta?ttattacaac?gccagaagat?cagcaggggt?tcataagatt 2400

acttgggaac?ggaagccaat?ttggtattaa?tcttaattac?gccactcaac?ggaatcctga 2460

tggtttagct?caggccttca?tcatcgggga?acaatttatt?ggtgaggatt?ctgtctgctt 2520

agttttggga?gataatattt?tttttggtca?aggatttact?ccgaaactta?gatgtgccgc 2580

tgagagggaa?ataggagcca?cgatatttgg?ttatcaggta?atggatcctg?aacgattcgg 2640

tgtggtcgag?tttgattgca?attataaagc?attaagtatt?gctgaaaaac?caaataaacc 2700

aaaatcaaac?tgggctgtaa?ctggattgta?tttttacgat?aacaatgtta?ttgatattgc 2760

aaaaaaaata?aaaccgtcgc?atcgtggtga?attagaaatc?acatctgtta?atgaggttta 2820

cttaaaaaat?aatgagttaa?ctgttgaatt?attaggtcga?gggtttgcgt?ggctggatac 2880

cggcacacat?gatagtctca?ttgaagcatc?taattttgtc?gaaaccgttc?aacgtagaca 2940

aggaatgatg?gttgcatgcc?ccgaagaaat?agcttggcgg?aatggttggt?tatctaatga 3000

agatttatat?aaattaggca?cgaaaattaa?aaagaatcta?tacggaaaat?atcttatcga 3060

tttggtgaaa?taatatgaat?gtcgagttta?ttccattaca?aaaacatggt?gatgaaagag 3120

ggatgttagt?agccctagag?caagccaaaa?acattccatt?tgaaattaaa?cgtgtttatt 3180

atatgtttgg?aactcaagga?aatgttcggc?gcgggtatca?tgcccataaa?aaaattaggc 3240

aagtagcgat?accgctaaat?ggttcatgcc?gttttcattt?agataatggt?cgtgagaaaa 3300

tagatgttgt?tttagatgat?cctgcattag?ggttgcttat?agagcctgga?gtctggcatg 3360

aaatgtacga?ttactcaaag?aactgtattc?ttctagtatt?ggcgagcgat?atttatgatg 3420

aaaatgacta?tattagaaat?tatgaggatt?ttatgaggtc?tgttaaaaat?gattcataaa 3480

ttaagcgatg?ttcaaactat?ccatataggt?aaaaaaacaa?gggtctggca?atattcagtt 3540

atattaccgg?gtgctgtaat?tggtgatgag?tgtaacatat?gtgcacatac?acttattgaa 3600

aatgatgttg?taattgggga?tagagttact?attaaatcgg?gcgtatatat?ctgggatggg 3660

gttgttattg?aagatgatgt?atttataggg?ccctgtgtaa?catttaccaa?tgataaaatc 3720

cccaggtcaa?agaaataccc?agaaaaattt?cttaagacta?ttattaaaaa?aggtgcttcc 3780

attggagcca?atgcaactat?attacctgga?ataacaattg?gtgaaaatgc?tatggttggg 3840

gctggtaccg?tagtcactaa?agatgttgca?ccttatacag?tcattgcagg?taatccaggt 3900

aaagttatta?gggatattaa?ggaataaaaa?tgatcgactt?tctcaacttg?aaaagaatta 3960

atgaaaaata?taaagcggaa?atgcaagatg?cctttaatcg?agttgtcgat?tctggctggt 4020

atattatggg?gcaagaatta?aaatattttg?aaagtgaatt?tgcacagtat?tgcggggcaa 4080

aatatgctat?aggtgtagct?aatggtttag?atgcccttta?tctcgtttta?aaagcgtgga 4140

aagaacaagg?taaattgagt?gatggcgatg?aagtgctggt?gcaggcaaac?acatatattg 4200

cctcaattct?tgctataaca?aataataatc?tcgttccggt?gttcattgaa?ccagatgaaa 4260

cgacttttaa?tttaaatccg?gcatttattc?gttcaaaaat?aacaactcgg?acaaaagtga 4320

tattgcccgt?gcatctttat?ggaaaaatat?caccaatggc?tgaaataatg?gacatcgctg 4380

aagagtacaa?gctattggtc?ctagaagatt?gtgctcaagc?tcatggtgct?gagttgaata 4440

aagtcaaagc?aggcaattgg?ggaaatgctg?ccggatttag?tttttatccg?ggtaaaaatc 4500

ttggggccat?aggtgatgcg?ggggctatta?caacatcaga?tgatgagctt?gccagtattt 4560

tgttagcatt?aagaaattat?ggttcccatg?ttaaatatga?aaatatttac?caaggtataa 4620

acagtcgctt?ggatgaattt?caggctgcag?tacttcgagt?taaattacct?aaattagata 4680

aagaaaatga?gagaaggcgt?caaatagcta?gaaagtatac?tgcagaaatt?agcaataaat 4740

acatgtcatt?accttcatac?actgacgatt?tatcacatgt?atggcatttg?tttgttgtta 4800

gaactcaatt?tagaaaagaa?ttgcagcttc?atttttcaaa?aaatgaaata?caaactatga 4860

tacattatcc?gatacctccg?cataaacaat?tagcatatac?tgctatgagt?aatatacatt 4920

tacccgtaac?cgaaagaata?cataacactg?ttatgtcatt?acctatggat?ccatcaatga 4980

ctgatgagca?cataaataaa?gttattaaag?ttgctaatga?atttaaaaaa?tgaggagact 5040

attatcagta?acaatattta?caggcctgct?gacttttatg?aaaatgtgtg?caggctttat 5100

cgttagcaag?gttgttgcaa?tatatatagg?ccctagtggg?ctggcgatgc?ttggacagat 5160

acaggatatt?gttgctcttt?tcaatgggct?ggttaactcc?ccggcaggag?ctggggttgt 5220

acgtttcact?tctcaaaatt?atgacaatgg?tgatatttct?aaatgtgtgc?catggtggcg 5280

cgcaagcatg?cgctggtgca?taatagtttc?ttctttagta?ataatttcag?ttatcccatt 5340

taataaagaa?ttatcattat?ggttatttaa?tgataattct?tattcaccag?ttatcattat 5400

aactgtttta?atactccctt?taactgcgtt?aggaacgatg?ataaattccg?ttattaacgg 5460

ccaacaaaaa?tatagagttt?ttgtttctat?tggtatgata?tccgttatca?tatcaactgc 5520

agtaatggtc?atacttataa?tgaagttcaa?ggtatttgga?gggttattag?cagtttcaat 5580

tcaatatgga?gtaattggtt?ttacttcttt?actattatgc?ttaaggtttg?catgggttag 5640

acctaattat?tggtttggta?aaacgtcgcc?agacaatatg?aagcatattt?ttcattatat 5700

gttgatggca?attacaagcg?cattttgcct?tccactatct?cttattctga?taaggaagat 5760

acttatattt?tatgttggtt?gggatttgac?tggtcaatgg?caagctgtat?ggaaaatatc 5820

tgaggcttac?ctaagcgtgg?taactcttgc?attatcaaca?tattttttac?caaaattatc 5880

taaacttaca?acatatggtg?agattaaaag?agaaataaca?tcaacggttt?tagtaataat 5940

gccattgata?gtaattatgg?gaatatttat?atatatcatg?cgtgataaaa?ttatctttct 6000

tttattcacg?aatgatttta?tagcatcaat?aagcttattc?aagtatcaat?tatgtggtga 6060

ttttattaaa?atattggctt?ggatttttgc?atatcctttg?atttcacagg?gaaaagtaaa 6120

gtggtatatt?acattagaga?tcttatcagc?cttccttttt?gttaccttga?gctttatatt 6180

tatccaaaag?cttggattgt?ctggagtatt?ttttgcatac?atagttaatt?atttatttta 6240

ttttctattt?atactgatga?actttaaaag?aatatttaag?aatatatgag?gatattattt 6300

tgcgtgaggc?tgatataatt?tctgttattg?tcttggcata?caattctgct?gatactataa 6360

catctacatt?agaaagtata?gctaaacaag?agtatggtgc?aaaaaatatt?gagttaataa 6420

taagtgatga?tgcttctcag?gatttaacaa?tatctatcat?agaaaactgg?ttagttgata 6480

actatcttaa?gttttatttt?gttaataaga?tatatcatca?taaaaatcta?gggatagtag 6540

ctaatttaga?taatgcatat?aaaaccgcaa?gcggtaaatg?gataaaggta?attgcaggtg 6600

atgacatact?aacctcgaac?tgtttatcaa?tatattctga?atttacaaaa?aaatctcaag 6660

gacgagtttt?tttatcgtat?atacagtcat?tttctcaaaa?cagcattgga?atagttaaga 6720

aggatattct?cccaccgaga?tctcaggtgg?ttctgcttaa?acatggtact?ctgcagcaac 6780

aaaaaaaaca?tttgatgaat?tttagttttt?ctgccactcc?gtcattgttt?atcgagaaaa 6840

aattattgga?tgatataggg?tatctcgata?aaaaatatcg?gctaatggaa?gattatcctc 6900

tctggcataa?aataactgat?tatggtgaaa?gaatttattt?tgttccagag?atcactgtgt 6960

tgtatagagt?gcaggaatca?gttagtaggt?caaaggagaa?gattgtaaat?ctagattttt 7020

taacagatat?tctaagattt?gaaaagatat?taatcaaaag?gttatctcca?gttagtatta 7080

cttatcatag?aagaaaacta?tggtgctatt?tgtacccatt?attaattaga?cttgttaata 7140

ataaaaaaaa?tatattttct?agaggctgta?tttttctact?aaatgtaatt?cttaaaccat 7200

tatatttgtc?aacaatagtt?agacgatatt?ttgaaaaata?aaaaggtttc?atagtgatgg 7260

ataataataa?acatacggtt?gctgtcatta?tgtcagttta?tgatggagat?gagccgtact 7320

attttgaaaa?agctattcag?agtatccaaa?aacaaaccta?ccaaagtatc?gatatatatt 7380

tatatatcga?tgcagtgcaa?aggaaagacc?ttctgaacag?gattcgacac?tataaaaaat 7440

gctctaggtt?tcatgttgta?acaggtgata?aaaaaagagg?tttagcatat?gggttaaatc 7500

gtcttttaga?caaaatccga?ggaaaaggat?atggatatat?tgccagaatg?gattctgatg 7560

atatttctac?gactgatcgc?attgaatctc?aggtgagatt?tctagaaaaa?aataaaaaca 7620

tagatgttat?aggtacaaat?tgtatagaaa?tagatgggga?tgataatgag?attttttata 7680

aaaaaatgcc?agaaactcat?catgaactaa?taaaatccat?agttaagcgc?tctccattta 7740

ttcatccttc?agttatgttt?agaggaagtg?ttgttgagca?cattagatat?aatgatagtc 7800

ttatgaatac?gcaagattat?tatttgtgga?ttgatttatt?atctaaaaaa?tataaatttc 7860

ataatatcca?acaaccatta?ttattttttc?gtgttaatga?ttcattttac?gctagacgtg 7920

ggtttggaaa?attaatcaat?gaagtgaaaa?gtagattgta?cgcgatgaag?aagttaaata 7980

aaaaaaatgt?taaaaattat?ttatacattc?tagcgttaat?atgtttacgc?ctcgcaccgg 8040

gtaatataaa?gaaatattgt?tacatgaggt?tccgataatg?aattgtatca?tgttttttta 8100

tggtgttttt?ttcgcattgc?cgttatctat?tctcaacgtg?ttaccttatg?gtatgaaaat 8160

agatgatgta?attcttttta?ttgcattaat?tgtatttcca?attttaagtt?ttaaaaaagg 8220

ttatcatgtg?acaattccat?tgtcatgttt?actatttatt?atcataactg?tgctgtttgc 8280

ctcactgtct?tttttgaagg?gaataaatag?cacgaatgta?tttttagggg?attctgcggg 8340

aactgttatt?ggtcgtataa?ttcaatctct?acttattctc?atattgatta?cttttttaaa 8400

aaacaccccg?cattatttat?taagtttttt?taaaggttat?gttatagctg?caaccttatc 8460

cctcattatt?ttttttggtt?attatgttac?gcatataaat?atatcttcat?ttactatgag 8520

aggggtatat?ttcgctaagg?atatttttca?atacaatgat?gacttaccct?ttactgttca 8580

tgtgaacaca?ttaggttctt?tttttctaat?agcgtttttt?ttattaaagt?ataacttccc 8640

taaataccaa?ttattatctt?atttgttttt?attgccttca?ctactattaa?tctccaaagg 8700

tgatatgttg?gcaatattaa?cttattttgc?ttatgtgttt?tataagagaa?ataatttaaa 8760

tgctttctta?tctctcattg?gtctattgat?ccttgtatta?ttgatgccat?atttatacaa 8820

tgtctactta?agcttatctg?attatagagt?atatgcatca?ggtagagatg?agctatatgg 8880

tgcgtcacta?tctagtataa?tagataatcc?atttgggtat?ggtctgggaa?tgcaaaataa 8940

tatactatat?tcgataacag?gcattgactt?tcctgcgcat?aatattcttt?taagtatagg 9000

gattgaactt?ggccttctat?atttattttt?gttggttgca?tatataatcg?gctggttatt 9060

attaggcaaa?gtagattata?aaatcatatt?tatttctttt?ttagtaatcg?gaatgtttgg 9120

taatgcaatg?tatttttata?agtttcattc?acttgctata?gcgctttgtt?ttctgagtgt 9180

atggcatatc?aattttaatc?acaaaactaa?acaagttgat?ttaattaata?atgaaaatac 9240

tttacattct?taataaatct?gtgctgacag?gaccaaatat?cgttgcatta?acaaacattg 9300

aacatattca?tgatcaggga?tatgatgttt?ctatttgttt?tttaaataac?ggtgacgatc 9360

taaataatat?atttcctttt?ttaaaagaaa?ttgaagtttt?ctatcttaac?ctaaaatccc 9420

ttggttttat?ttctgggcta?aaaaatttaa?attattatat?taattcaatt?aaaccaacaa 9480

taattcacag?ccattgcttt?ctacctgaca?tttataacat?atttaataag?tttttttgcg 9540

ggccaataaa?aaataagcgt?gttactacaa?ttcacaacat?acctaaagaa?gattacagat 9600

tacgatatgg?tcgtattaaa?ggaagtcttt?tgctttattt?tcataaggca?cttttccctt 9660

tttttgataa?ttgcatatgt?atctcaaaaa?ctgtacaaaa?gagtattggg?attaaaaaaa 9720

caagtgtgat?ttataatcct?gttcgtgatg?ttttttttaa?ctcaacgaaa?gatgatgtag 9780

aatgtttaac?aattgtatac?tgtggtcatt?tctctcaatt?aaaaaaccct?attacattaa 9840

ttcagttgtt?agccgattca?aaagtgaatt?ttcagttttt?aggaatcggt?gatggtcctc 9900

ttttatatca?atgtagacaa?ctggttaaaa?aagatccgcg?ttttactttt?gtcggtagag 9960

taaatcatgt?ccataaattt?tatgagaaag?caaattgttt?aatacacatt?tcacaaactg 10020

aaggtttttg?tctttcagtt?gctgaggctc?tggcttctaa?tatgtatgtc?attacaaatg 10080

aattacctgt?ttttttagaa?ctaaagcaag?ctttgagttc?agagggcttt?tacatgctta 10140

atgatataaa?taattacaat?ttagaaatgg?tattaaattc?tattaataga?aaaattgaag 10200

caggtgaaag?atgtaattct?ggtgcgtcga?atagagttaa?agctttactt?tctcctactt 10260

ttatagcaca?gcaacatatt?gtgctttatc?aaaaattgac?agtggaaaag?tgagatttca 10320

taatttttgt?actcgtatct?atattattgg?atgttgtcaa?cttgtagcgt?agtttagatt 10380

catgcccccc?tgacaggagt?aaacaatgtc?aaagcaacaa?atcggcgtcg?tcggtatggc 10440

agtgatgggg?cgcaaccttg?cgctcaacat?cgaaagccgt?ggttataccg?tctctatttt 10500

caaccgttcc?cgtgagaaaa?cggaagaagt?aattgccgaa?aatccaggca?agaaactggt 10560

tccttactat?acggtgaaag?agtttattga?atctctggaa?acgcctcgtc?gcattctgtt 10620

aatggtgaaa?gcaggtgcag?gcacggatgc?tgctattgat?tccctcaagc?catacctcga 10680

taaaggtgac?atcatcattg?atggtggtaa?caccttcttc?caggacacca?ttcgtcgtaa 10740

ccgtgagctt?tctgccgaag?gctttaactt?cattggtacc?ggtgtctccg?gtggtgaaga 10800

aggcgcgctg?aaaggtcctt?ccattatgcc?cggtgggcag?aaagaagcct?atgaactggt 10860

tgcgccgatc?ctgaccaaga?tcgccgcagt?ggctgaagac?ggtgagccat?gcgttaccta 10920

tattggtgcc?gatggcgcag?gtcactatgt?gaagatggtc?cacaacggta?ttgaatatgg 10980

cgatatgcag?ctgattgctg?aagcctattc?tctgcttaaa?ggtggcctga?acctcaccaa 11040

cgaagaactg?gcgcagacgt?ttaccgagtg?gaataacggt?gaactgagca?gctacctgat 11100

cgacatcacc?aaagatatct?tcaccaaaaa?agatgaagac?ggtaactacc?tggttgatgt 11160

gattctggat?gaagcagcaa?acaaaggtac?aggcaaatgg?accagccaga?gcgcgctgga 11220

tctcggtgaa?ccactgtcgc?ttattaccga?gtctgtgttt?gcacgttata?tctcgtctct 11280

gaaagatcag?cgcgttgccg?cgtctaaagt?tctctctggc?ccgcaagcgc?agccagctgg 11340

cgacaaggct?gagttcatcg?aaaaagttcg?ccgtgctctg?tatctgggca?aaatcgtttc 11400

ttacgcccag?ggcttctctc?agctacgcgc?cgcgtctgaa?gagtacaact?gggatctgaa 11460

ctacggcgaa?atcgcgaaga?tttttcgtgc?tggctgcatc?atccgtgcgc?agttcctgca 11520

gaaaatcacc?gatgcatatg?ccgaaaatcc?gcagatcgct?aacctgctgc?tggccccgta 11580

cttcaagcaa?attgccgatg?actatcagca?ggcgctgcgc?gatgttgtcg?cttacgcagt 11640

acagaacggt?atcccggttc?cgaccttcgc?cgctgcggtt?gcctattatg?acagctaccg 11700

cgccgctatt?cttcctgcga?acctgatcag?gccagcgcga?cta 11743

Glycosyltransferase gene in the O antigen gene of table 1 intestinal bacteria O74 type bunch and oligosaccharide unit treatment gene and wherein Primer and PCR data

Produce moving back of correct PCR

Gene base PCR product

Gene function forward primer reverse primer size electrophoresis fire temperature

The length of position

The group number of band (℃)

Wzx O-antigen transhipment enzyme 5030-6289 5547-5564 6140-6157 611bp 0 ^*493

5143-5160 5414-5431 289bp 0 ^* 48?5

Wzy O-antigen polysaccharase 8078-9253 8119-8136 8955-8972 854bp 0 ^*48.2

8299-8316 8563-8581 283bp 0 ^* 43.9

^*Only in intestinal bacteria O74 type, obtain a correct band

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

The bacterium source that contains in this group of group number

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

O21，O22，O23，O24，O59

2, wild-type e. coli O25, O26, O27, O28, O29, O30, O32, O31, O33, O35, O36, IMVS ^a

O37，O38，O40，O41，O42，O43

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

O57，O58，O60，O61，O62

4, wild-type e. coli O63, O65, O66, O69, O70, O71, O75, O76, O77, O78, O79, IMVS ^a

O80，O81，O82，O83

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

O102，O103，O104，O106

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

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

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

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

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

O159，O160，O161，O163，O164，O165，O166 b

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

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

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

B16，B17，B18

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

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

O155，O124

13, the 4th group of bacterial strain adds intestinal bacteria reference culture O74 IMVS

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

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

b.Statens?Serum?Institut，Copenhagen，Denmark

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

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

Table 3 intestinal bacteria O74 type O antigen gene structure iron

E.coli?O74?O-antigen?gene?cluster

galF rmlB rmlA orf3 orf4 orf5 wzx orf7 orf8 wzy orf10 gnd

％G+C 43％ 46％ 44％ 39％ 29％ 31％ 30％ 33％ 33％ 29％

content

Table 4 intestinal bacteria O74 type O antigen gene cluster gene position

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

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

CAGCTGCTGG?CGGAAGTGCA?GTCCATCTGT?CCGCCGGGTG?TGACCATTAT?GAACGTGCGT 180

CAGGGCGAAC?CTTTAGGTTT?GGGCCACTCC?ATTTTATGTG?CACGACCCGC?CATTGGTGAC 240

AACCCATTTG?TCGTGGTGCT?GCCAGACGTT?GTGATCGACG?ACGCCAGCGC?CGACCCGCTG 300

CGCTACAACC?TTGCTGCCAT?GATTGCGCGC?TTCAACGAAA?CAGGACGCAG?CCAGGTGCTG 360

GCAAAACGTA?TGCCGGGTGA?CCTCTCTGAA?TACTCCGTCA?TTCAGACCAA?AGAACCGCTG 420

GATCGCGAAG?GCAAAGTCAG?CCGCATCGTG?GAATTTATCG?AAAAACCGGA?TCAGCCGCAG 480

ACGCTGGACT?CAGACATCAT?GGCCGTGGGT?CGCTATGTGC?TTTCTGCCGA?TATTTGGCCG 540

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

GAACTGGCGA?AAAAACAGTC?CGTTGATGCC?ATGCTGATGA?CAGGTGACAG?CTACGACTGC 660

GGTAAAAAAA?TGGGTTATAT?GCAGGCATTT?GTGAAGTATG?GACTACGCAA?CCTCAAAGAA 720

GGGGCGAAGT?TCCGGAAAGG?GATTGAGAAG?CTGTTAAGCG?AATAATGAAA?ATCTGACCGA 780

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

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

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

AGCGTTTGAA?TTTTTCGGGT?TAAGCGCGAG?TGGGTAACGC?TCGTCACATC?GTAGACATGC 1020

ATGTAGTGCT?CTGGTAGCTG?TAAAGCCAGG?GGCGGTAGCG?TGTATTAACA?CCTCTATCAA 1080

Orf1 is initial

TCAACCTAAG?AGCCGCTTAT?TTCACAGCAT?GCTCTGAAGT?AATATGGAAT?AAATAA GTGA 1140

AGATACTTGT?TACTGGTGGC?GCAGGATTTA?TTGGTTCTGC?TGTAGTTCGT?CACATTATAA 1200

ATAATACGCA?GGATAGTGTT?GTTAATGTCG?ATAAATTAAC?GTACGCCGGA?AACCTGGAAT 1260

CGCTTGCTGA?TGTTTCTGAT?TCTGAACGCT?ATGTTTTTGA?ACATGCGGAT?ATTTGCGATG 1320

CAGCTGCAAT?GGCACGGATT?TTTGCTCAGC?ATCAGCCGGA?TGCAGTGATG?CACTTGGCTG 1380

CTGAAAGCCA?TGTTGACCGT?TCAATTACAG?GTCCTGCGGC?ATTTATTGAA?ACCAATATTG 1440

TTGGTACTTA?TGTCCTTTTG?GAAGCCGCTC?GCAATTACTG?GTCTGCTCTT?GATAGCGACA 1500

AGAAAAATAG?CTTCCGTTTT?CATCATATTT?CTACTGACGA?AGTCTATGGT?GATTTGCCTC 1560

ATCCAGATGA?AGTAAATAAT?AACGAAGAAT?TACCCTTATT?TACTGAGACG?ACAGCTTACG 1620

CACCAAGCAG?CCCTTATTCC?GCATCAAAAG?CATCCAGCGA?TCATTTAGTC?CGGGCGTGGA 1680

AACGCACCTA?TGGTTTACCG?ACCATTGTGA?CTAACTGTTC?GAATAACTAC?GGTCCTTATC 1740

ACTTTCCGGA?AAAATTGATT?CCACTAGTAA?TTCTTAATGC?TCTGGAAGGT?AAGGCATTAC 1800

CTATTTATGG?CAAAGGGGAT?CAAATTCGTG?ACTGGCTGTA?TGTTGAAGAT?CATGCGCGTG 1860

CGTTATATAC?CGTCGTAACC?GAAGGTAAAG?CGGGTGAAAC?TTATAACATT?GGTGGACACA 1920

ACGAAAAGAA?AAACATCGAT?GTAGTGCTCA?CTATTTGTGA?TTTGTTGGAT?GAGATTGTAC 1980

CGAAAGAGAA?ATCTTACCGC?GAGCAAATTA?CTTATGTTGC?CGATCGCCCG?GGACACGATC 2040

GCCGTTATGC?GATTGATGCA?GAGAAGATTA?GCCGCGAATT?GGGCTGGAAA?CCGCAGGAAA 2100

CGTTTGAGAG?CGGGATTCGT?AAAACAATTA?ACTGGTATTT?GAATAATAAT?GATTGGTGCC 2160

The initial orf1 of orf2 stops

GGCGAGTTCA?AGATGGCTCT?TATCAACGTG?AGCGTTTGGG?GGTAATTAG A? TGAAAGGAAT 2220

TATTTTAGCT?GGTGGTTCTG?GAACAAGGCT?TTATCCAATT?ACAATGGGAG?TTTCAAAGCA 2280

GCTATTACCC?ATTTATGATA?AACCGATGAT?TTATTATCCT?TTGTCAGTGC?TGATGTTGGC 2340

AGGTATACAA?GATATTCTTA?TTATTACAAC?GCCAGAAGAT?CAGCAGGGGT?TCATAAGATT 2400

ACTTGGGAAC?GGAAGCCAAT?TTGGTATTAA?TCTTAATTAC?GCCACTCAAC?GGAATCCTGA 2460

TGGTTTAGCT?CAGGCCTTCA?TCATCGGGGA?ACAATTTATT?GGTGAGGATT?CTGTCTGCTT 2520

AGTTTTGGGA?GATAATATTT?TTTTTGGTCA?AGGATTTACT?CCGAAACTTA?GATGTGCCGC 2580

TGAGAGGGAA?ATAGGAGCCA?CGATATTTGG?TTATCAGGTA?ATGGATCCTG?AACGATTCGG 2640

TGTGGTCGAG?TTTGATTGCA?ATTATAAAGC?ATTAAGTATT?GCTGAAAAAC?CAAATAAACC 2700

AAAATCAAAC?TGGGCTGTAA?CTGGATTGTA?TTTTTACGAT?AACAATGTTA?TTGATATTGC 2760

AAAAAAAATA?AAACCGTCGC?ATCGTGGTGA?ATTAGAAATC?ACATCTGTTA?ATGAGGTTTA 2820

CTTAAAAAAT?AATGAGTTAA?CTGTTGAATT?ATTAGGTCGA?GGGTTTGCGT?GGCTGGATAC 2880

CGGCACACAT?GATAGTCTCA?TTGAAGCATC?TAATTTTGTC?GAAACCGTTC?AACGTAGACA 2940

AGGAATGATG?GTTGCATGCC?CCGAAGAAAT?AGCTTGGCGG?AATGGTTGGT?TATCTAATGA 3000

AGATTTATAT?AAATTAGGCA?CGAAAATTAA?AAAGAATCTA?TACGGAAAAT?ATCTTATCGA 3060

It is initial that orf2 stops orf3

TTTGGTGAAA? TAAT ATGAAT?GTCGAGTTTA?TTCCATTACA?AAAACATGGT?GATGAAAGAG 3120

GGATGTTAGT?AGCCCTAGAG?CAAGCCAAAA?ACATTCCATT?TGAAATTAAA?CGTGTTTATT 3180

ATATGTTTGG?AACTCAAGGA?AATGTTCGGC?GCGGGTATCA?TGCCCATAAA?AAAATTAGGC 3240

AAGTAGCGAT?ACCGCTAAAT?GGTTCATGCC?GTTTTCATTT?AGATAATGGT?CGTGAGAAAA 3300

TAGATGTTGT?TTTAGATGAT?CCTGCATTAG?GGTTGCTTAT?AGAGCCTGGA?GTCTGGCATG 3360

AAATGTACGA?TTACTCAAAG?AACTGTATTC?TTCTAGTATT?GGCGAGCGAT?ATTTATGATG 3420

The initial orf3 of orf4 stops

AAAATGACTA?TATTAGAAAT?TATGAGGATT?TTATGAGGTC?TGTTAAAA AT? GATTCA TAAA 3480

TTAAGCGATG?TTCAAACTAT?CCATATAGGT?AAAAAAACAA?GGGTCTGGCA?ATATTCAGTT 3540

ATATTACCGG?GTGCTGTAAT?TGGTGATGAG?TGTAACATAT?GTGCACATAC?ACTTATTGAA 3600

AATGATGTTG?TAATTGGGGA?TAGAGTTACT?ATTAAATCGG?GCGTATATAT?CTGGGATGGG 3660

GTTGTTATTG?AAGATGATGT?ATTTATAGGG?CCCTGTGTAA?CATTTACCAA?TGATAAAATC 3720

CCCAGGTCAA?AGAAATACCC?AGAAAAATTT?CTTAAGACTA?TTATTAAAAA?AGGTGCTTCC 3780

ATTGGAGCCA?ATGCAACTAT?ATTACCTGGA?ATAACAATTG?GTGAAAATGC?TATGGTTGGG 3840

GCTGGTACCG?TAGTCACTAA?AGATGTTGCA?CCTTATACAG?TCATTGCAGG?TAATCCAGGT 3900

It is initial that orf4 stops orf5

AAAGTTATTA?GGGATATTAA?GGAA TAAAA A? TGATCGACTT?TCTCAACTTG?AAAAGAATTA3960

ATGAAAAATA?TAAAGCGGAA?ATGCAAGATG?CCTTTAATCG?AGTTGTCGAT?TCTGGCTGGT 4020

ATATTATGGG?GCAAGAATTA?AAATATTTTG?AAAGTGAATT?TGCACAGTAT?TGCGGGGCAA 4080

AATATGCTAT?AGGTGTAGCT?AATGGTTTAG?ATGCCCTTTA?TCTCGTTTTA?AAAGCGTGGA 4140

AAGAACAAGG?TAAATTGAGT?GATGGCGATG?AAGTGCTGGT?GCAGGCAAAC?ACATATATTG 4200

CCTCAATTCT?TGCTATAACA?AATAATAATC?TCGTTCCGGT?GTTCATTGAA?CCAGATGAAA 4260

CGACTTTTAA?TTTAAATCCG?GCATTTATTC?GTTCAAAAAT?AACAACTCGG?ACAAAAGTGA 4320

TATTGCCCGT?GCATCTTTAT?GGAAAAATAT?CACCAATGGC?TGAAATAATG?GACATCGCTG 4380

AAGAGTACAA?GCTATTGGTC?CTAGAAGATT?GTGCTCAAGC?TCATGGTGCT?GAGTTGAATA 4440

AAGTCAAAGC?AGGCAATTGG?GGAAATGCTG?CCGGATTTAG?TTTTTATCCG?GGTAAAAATC 4500

TTGGGGCCAT?AGGTGATGCG?GGGGCTATTA?CAACATCAGA?TGATGAGCTT?GCCAGTATTT 4560

TGTTAGCATT?AAGAAATTAT?GGTTCCCATG?TTAAATATGA?AAATATTTAC?CAAGGTATAA 4620

ACAGTCGCTT?GGATGAATTT?CAGGCTGCAG?TACTTCGAGT?TAAATTACCT?AAATTAGATA 4680

AAGAAAATGA?GAGAAGGCGT?CAAATAGCTA?GAAAGTATAC?TGCAGAAATT?AGCAATAAAT 4740

ACATGTCATT?ACCTTCATAC?ACTGACGATT?TATCACATGT?ATGGCATTTG?TTTGTTGTTA 4800

GAACTCAATT?TAGAAAAGAA?TTGCAGCTTC?ATTTTTCAAA?AAATGAAATA?CAAACTATGA 4860

TACATTATCC?GATACCTCCG?CATAAACAAT?TAGCATATAC?TGCTATGAGT?AATATACATT 4920

TACCCGTAAC?CGAAAGAATA?CATAACACTG?TTATGTCATT?ACCTATGGAT?CCATCAATGA 4980

The initial orf5 of wzx stops

CTGATGAGCA?CATAAATAAA?GTTATTAAAG?TTGCTAATGA?ATTTAAAAA A? TGAGGAGACT5040

ATTATCAGTA?ACAATATTTA?CAGGCCTGCT?GACTTTTATG?AAAATGTGTG?CAGGCTTTAT 5100

CGTTAGCAAG?GTTGTTGCAA?TATATATAGG?CCCTAGTGGG?CTGGCGATGC?TTGGACAGAT 5160

ACAGGATATT?GTTGCTCTTT?TCAATGGGCT?GGTTAACTCC?CCGGCAGGAG?CTGGGGTTGT 5220

ACGTTTCACT?TCTCAAAATT?ATGACAATGG?TGATATTTCT?AAATGTGTGC?CATGGTGGCG 5280

CGCAAGCATG?CGCTGGTGCA?TAATAGTTTC?TTCTTTAGTA?ATAATTTCAG?TTATCCCATT 5340

TAATAAAGAA?TTATCATTAT?GGTTATTTAA?TGATAATTCT?TATTCACCAG?TTATCATTAT 5400

AACTGTTTTA?ATACTCCCTT?TAACTGCGTT?AGGAACGATG?ATAAATTCCG?TTATTAACGG 5460

CCAACAAAAA?TATAGAGTTT?TTGTTTCTAT?TGGTATGATA?TCCGTTATCA?TATCAACTGC 5520

AGTAATGGTC?ATACTTATAA?TGAAGTTCAA?GGTATTTGGA?GGGTTATTAG?CAGTTTCAAT 5580

TCAATATGGA?GTAATTGGTT?TTACTTCTTT?ACTATTATGC?TTAAGGTTTG?CATGGGTTAG 5640

ACCTAATTAT?TGGTTTGGTA?AAACGTCGCC?AGACAATATG?AAGCATATTT?TTCATTATAT 5700

GTTGATGGCA?ATTACAAGCG?CATTTTGCCT?TCCACTATCT?CTTATTCTGA?TAAGGAAGAT 5760

ACTTATATTT?TATGTTGGTT?GGGATTTGAC?TGGTCAATGG?CAAGCTGTAT?GGAAAATATC 5820

TGAGGCTTAC?CTAAGCGTGG?TAACTCTTGC?ATTATCAACA?TATTTTTTAC?CAAAATTATC 5880

TAAACTTACA?ACATATGGTG?AGATTAAAAG?AGAAATAACA?TCAACGGTTT?TAGTAATAAT 5940

GCCATTGATA?GTAATTATGG?GAATATTTAT?ATATATCATG?CGTGATAAAA?TTATCTTTCT 6000

TTTATTCACG?AATGATTTTA?TAGCATCAAT?AAGCTTATTC?AAGTATCAAT?TATGTGGTGA 6060

TTTTATTAAA?ATATTGGCTT?GGATTTTTGC?ATATCCTTTG?ATTTCACAGG?GAAAAGTAAA 6120

GTGGTATATT?ACATTAGAGA?TCTTATCAGC?CTTCCTTTTT?GTTACCTTGA?GCTTTATATT 6180

TATCCAAAAG?CTTGGATTGT?CTGGAGTATT?TTTTGCATAC?ATAGTTAATT?ATTTATTTTA 6240

It is initial that wzx stops orf7

TTTTCTATTT?ATACTGATGA?ACTTTAAAAG?AATATTTAAG?AATATA TGAG?GATATTATT T6300

TGCGTGAGGC?TGATATAATT?TCTGTTATTG?TCTTGGCATA?CAATTCTGCT?GATACTATAA 6360

CATCTACATT?AGAAAGTATA?GCTAAACAAG?AGTATGGTGC?AAAAAATATT?GAGTTAATAA 6420

TAAGTGATGA?TGCTTCTCAG?GATTTAACAA?TATCTATCAT?AGAAAACTGG?TTAGTTGATA 6480

ACTATCTTAA?GTTTTATTTT?GTTAATAAGA?TATATCATCA?TAAAAATCTA?GGGATAGTAG 6540

CTAATTTAGA?TAATGCATAT?AAAACCGCAA?GCGGTAAATG?GATAAAGGTA?ATTGCAGGTG 6600

ATGACATACT?AACCTCGAAC?TGTTTATCAA?TATATTCTGA?ATTTACAAAA?AAATCTCAAG 6660

GACGAGTTTT?TTTATCGTAT?ATACAGTCAT?TTTCTCAAAA?CAGCATTGGA?ATAGTTAAGA 6720

AGGATATTCT?CCCACCGAGA?TCTCAGGTGG?TTCTGCTTAA?ACATGGTACT?CTGCAGCAAC 6780

AAAAAAAACA?TTTGATGAAT?TTTAGTTTTT?CTGCCACTCC?GTCATTGTTT?ATCGAGAAAA 6840

AATTATTGGA?TGATATAGGG?TATCTCGATA?AAAAATATCG?GCTAATGGAA?GATTATCCTC 6900

TCTGGCATAA?AATAACTGAT?TATGGTGAAA?GAATTTATTT?TGTTCCAGAG?ATCACTGTGT 6960

TGTATAGAGT?GCAGGAATCA?GTTAGTAGGT?CAAAGGAGAA?GATTGTAAAT?CTAGATTTTT 7020

TAACAGATAT?TCTAAGATTT?GAAAAGATAT?TAATCAAAAG?GTTATCTCCA?GTTAGTATTA 7080

CTTATCATAG?AAGAAAACTA?TGGTGCTATT?TGTACCCATT?ATTAATTAGA?CTTGTTAATA 7140

ATAAAAAAAA?TATATTTTCT?AGAGGCTGTA?TTTTTCTACT?AAATGTAATT?CTTAAACCAT 7200

It is initial that orf7 stops orf8

TATATTTGTC?AACAATAGTT?AGACGATATT?TTGAAAAA TA? AAAAGGTTTC?ATAGTG ATGG7260

ATAATAATAA?ACATACGGTT?GCTGTCATTA?TGTCAGTTTA?TGATGGAGAT?GAGCCGTACT 7320

ATTTTGAAAA?AGCTATTCAG?AGTATCCAAA?AACAAACCTA?CCAAAGTATC?GATATATATT 7380

TATATATCGA?TGCAGTGCAA?AGGAAAGACC?TTCTGAACAG?GATTCGACAC?TATAAAAAAT 7440

GCTCTAGGTT?TCATGTTGTA?ACAGGTGATA?AAAAAAGAGG?TTTAGCATAT?GGGTTAAATC 7500

GTCTTTTAGA?CAAAATCCGA?GGAAAAGGAT?ATGGATATAT?TGCCAGAATG?GATTCTGATG 7560

ATATTTCTAC?GACTGATCGC?ATTGAATCTC?AGGTGAGATT?TCTAGAAAAA?AATAAAAACA 7620

TAGATGTTAT?AGGTACAAAT?TGTATAGAAA?TAGATGGGGA?TGATAATGAG?ATTTTTTATA 7680

AAAAAATGCC?AGAAACTCAT?CATGAACTAA?TAAAATCCAT?AGTTAAGCGC?TCTCCATTTA 7740

TTCATCCTTC?AGTTATGTTT?AGAGGAAGTG?TTGTTGAGCA?CATTAGATAT?AATGATAGTC 7800

TTATGAATAC?GCAAGATTAT?TATTTGTGGA?TTGATTTATT?ATCTAAAAAA?TATAAATTTC 7860

ATAATATCCA?ACAACCATTA?TTATTTTTTC?GTGTTAATGA?TTCATTTTAC?GCTAGACGTG 7920

GGTTTGGAAA?ATTAATCAAT?GAAGTGAAAA?GTAGATTGTA?CGCGATGAAG?AAGTTAAATA 7980

AAAAAAATGT?TAAAAATTAT?TTATACATTC?TAGCGTTAAT?ATGTTTACGC?CTCGCACCGG 8040

It is initial that orf8 stops wzx

GTAATATAAA?GAAATATTGT?TACATGAGGT?TCCGA TAATG?AATTGTATCA?TGTTTTTTTA 8100

TGGTGTTTTT?TTCGCATTGC?CGTTATCTAT?TCTCAACGTG?TTACCTTATG?GTATGAAAAT 8160

AGATGATGTA?ATTCTTTTTA?TTGCATTAAT?TGTATTTCCA?ATTTTAAGTT?TTAAAAAAGG 8220

TTATCATGTG?ACAATTCCAT?TGTCATGTTT?ACTATTTATT?ATCATAACTG?TGCTGTTTGC 8280

CTCACTGTCT?TTTTTGAAGG?GAATAAATAG?CACGAATGTA?TTTTTAGGGG?ATTCTGCGGG 8340

AACTGTTATT?GGTCGTATAA?TTCAATCTCT?ACTTATTCTC?ATATTGATTA?CTTTTTTAAA 8400

AAACACCCCG?CATTATTTAT?TAAGTTTTTT?TAAAGGTTAT?GTTATAGCTG?CAACCTTATC 8460

CCTCATTATT?TTTTTTGGTT?ATTATGTTAC?GCATATAAAT?ATATCTTCAT?TTACTATGAG 8520

AGGGGTATAT?TTCGCTAAGG?ATATTTTTCA?ATACAATGAT?GACTTACCCT?TTACTGTTCA 8580

TGTGAACACA?TTAGGTTCTT?TTTTTCTAAT?AGCGTTTTTT?TTATTAAAGT?ATAACTTCCC 8640

TAAATACCAA?TTATTATCTT?ATTTGTTTTT?ATTGCCTTCA?CTACTATTAA?TCTCCAAAGG 8700

TGATATGTTG?GCAATATTAA?CTTATTTTGC?TTATGTGTTT?TATAAGAGAA?ATAATTTAAA 8760

TGCTTTCTTA?TCTCTCATTG?GTCTATTGAT?CCTTGTATTA?TTGATGCCAT?ATTTATACAA 8820

TGTCTACTTA?AGCTTATCTG?ATTATAGAGT?ATATGCATCA?GGTAGAGATG?AGCTATATGG 8880

TGCGTCACTA?TCTAGTATAA?TAGATAATCC?ATTTGGGTAT?GGTCTGGGAA?TGCAAAATAA 8940

TATACTATAT?TCGATAACAG?GCATTGACTT?TCCTGCGCAT?AATATTCTTT?TAAGTATAGG 9000

GATTGAACTT?GGCCTTCTAT?ATTTATTTTT?GTTGGTTGCA?TATATAATCG?GCTGGTTATT 9060

ATTAGGCAAA?GTAGATTATA?AAATCATATT?TATTTCTTTT?TTAGTAATCG?GAATGTTTGG 9120

TAATGCAATG?TATTTTTATA?AGTTTCATTC?ACTTGCTATA?GCGCTTTGTT?TTCTGAGTGT 9180

Orf10 is initial

ATGGCATATC?AATTTTAATC?ACAAAACTAA?ACAAGTTGAT?TTAATTAATA? ATGAAAATAC 9240

Wzx stops

TTTACATTCT? TAATAAATCT?GTGCTGACAG?GACCAAATAT?CGTTGCATTA?ACAAACATTG 9300

AACATATTCA?TGATCAGGGA?TATGATGTTT?CTATTTGTTT?TTTAAATAAC?GGTGACGATC 9360

TAAATAATAT?ATTTCCTTTT?TTAAAAGAAA?TTGAAGTTTT?CTATCTTAAC?CTAAAATCCC 9420

TTGGTTTTAT?TTCTGGGCTA?AAAAATTTAA?ATTATTATAT?TAATTCAATT?AAACCAACAA 9480

TAATTCACAG?CCATTGCTTT?CTACCTGACA?TTTATAACAT?ATTTAATAAG?TTTTTTTGCG 9540

GGCCAATAAA?AAATAAGCGT?GTTACTACAA?TTCACAACAT?ACCTAAAGAA?GATTACAGAT 9600

TACGATATGG?TCGTATTAAA?GGAAGTCTTT?TGCTTTATTT?TCATAAGGCA?CTTTTCCCTT 9660

TTTTTGATAA?TTGCATATGT?ATCTCAAAAA?CTGTACAAAA?GAGTATTGGG?ATTAAAAAAA 9720

CAAGTGTGAT?TTATAATCCT?GTTCGTGATG?TTTTTTTTAA?CTCAACGAAA?GATGATGTAG 9780

AATGTTTAAC?AATTGTATAC?TGTGGTCATT?TCTCTCAATT?AAAAAACCCT?ATTACATTAA 9840

TTCAGTTGTT?AGCCGATTCA?AAAGTGAATT?TTCAGTTTTT?AGGAATCGGT?GATGGTCCTC 9900

TTTTATATCA?ATGTAGACAA?CTGGTTAAAA?AAGATCCGCG?TTTTACTTTT?GTCGGTAGAG 9960

TAAATCATGT?CCATAAATTT?TATGAGAAAG?CAAATTGTTT?AATACACATT?TCACAAACTG 10020

AAGGTTTTTG?TCTTTCAGTT?GCTGAGGCTC?TGGCTTCTAA?TATGTATGTC?ATTACAAATG 10080

AATTACCTGT?TTTTTTAGAA?CTAAAGCAAG?CTTTGAGTTC?AGAGGGCTTT?TACATGCTTA 10140

ATGATATAAA?TAATTACAAT?TTAGAAATGG?TATTAAATTC?TATTAATAGA?AAAATTGAAG 10200

CAGGTGAAAG?ATGTAATTCT?GGTGCGTCGA?ATAGAGTTAA?AGCTTTACTT?TCTCCTACTT 10260

Orf10 stops

TTATAGCACA?GCAACATATT?GTGCTTTATC?AAAAATTGAC?AGTGGAAAAG? TGAGATTTCA 10320

TAATTTTTGT?ACTCGTATCT?ATATTATTGG?ATGTTGTCAA?CTTGTAGCGT?AGTTTAGATT 10380

CATGCCCCCC?TGACAGGAGT?AAACAATGTC?AAAGCAACAA?ATCGGCGTCG?TCGGTATGGC 10440

AGTGATGGGG?CGCAACCTTG?CGCTCAACAT?CGAAAGCCGT?GGTTATACCG?TCTCTATTTT 10500

CAACCGTTCC?CGTGAGAAAA?CGGAAGAAGT?AATTGCCGAA?AATCCAGGCA?AGAAACTGGT 10560

TCCTTACTAT?ACGGTGAAAG?AGTTTATTGA?ATCTCTGGAA?ACGCCTCGTC?GCATTCTGTT 10620

AATGGTGAAA?GCAGGTGCAG?GCACGGATGC?TGCTATTGAT?TCCCTCAAGC?CATACCTCGA 10680

TAAAGGTGAC?ATCATCATTG?ATGGTGGTAA?CACCTTCTTC?CAGGACACCA?TTCGTCGTAA 10740

CCGTGAGCTT?TCTGCCGAAG?GCTTTAACTT?CATTGGTACC?GGTGTCTCCG?GTGGTGAAGA 10800

AGGCGCGCTG?AAAGGTCCTT?CCATTATGCC?CGGTGGGCAG?AAAGAAGCCT?ATGAACTGGT 10860

TGCGCCGATC?CTGACCAAGA?TCGCCGCAGT?GGCTGAAGAC?GGTGAGCCAT?GCGTTACCTA 10920

TATTGGTGCC?GATGGCGCAG?GTCACTATGT?GAAGATGGTC?CACAACGGTA?TTGAATATGG 10980

CGATATGCAG?CTGATTGCTG?AAGCCTATTC?TCTGCTTAAA?GGTGGCCTGA?ACCTCACCAA 11040

CGAAGAACTG?GCGCAGACGT?TTACCGAGTG?GAATAACGGT?GAACTGAGCA?GCTACCTGAT 11100

CGACATCACC?AAAGATATCT?TCACCAAAAA?AGATGAAGAC?GGTAACTACC?TGGTTGATGT 11160

GATTCTGGAT?GAAGCAGCAA?ACAAAGGTAC?AGGCAAATGG?ACCAGCCAGA?GCGCGCTGGA 11220

TCTCGGTGAA?CCACTGTCGC?TTATTACCGA?GTCTGTGTTT?GCACGTTATA?TCTCGTCTCT 11280

GAAAGATCAG?CGCGTTGCCG?CGTCTAAAGT?TCTCTCTGGC?CCGCAAGCGC?AGCCAGCTGG 11340

CGACAAGGCT?GAGTTCATCG?AAAAAGTTCG?CCGTGCTCTG?TATCTGGGCA?AAATCGTTTC 11400

TTACGCCCAG?GGCTTCTCTC?AGCTACGCGC?CGCGTCTGAA?GAGTACAACT?GGGATCTGAA 11460

CTACGGCGAA?ATCGCGAAGA?TTTTTCGTGC?TGGCTGCATC?ATCCGTGCGC?AGTTCCTGCA 11520

GAAAATCACC?GATGCATATG?CCGAAAATCC?GCAGATCGCT?AACCTGCTGC?TGGCCCCGTA 11580

CTTCAAGCAA?ATTGCCGATG?ACTATCAGCA?GGCGCTGCGC?GATGTTGTCG?CTTACGCAGT 11640

ACAGAACGGT?ATCCCGGTTC?CGACCTTCGC?CGCTGCGGTT?GCCTATTATG?ACAGCTACCG 11700

CGCCGCTATT?CTTCCTGCGA?ACCTGATCAG?GCCAGCGCGA?CTA 11743

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

Claims (9)

1, a kind of Nucleotide of the O-antigen-specific to intestinal bacteria O74 type, it is characterized in that: it is the isolating Nucleotide shown in SEQ ID NO:1,11743 bases of total length; The base that perhaps has one or more insertions, disappearance or replacement keeps the Nucleotide of the SEQ ID NO:1 of described isolating functional nucleotide simultaneously.

2, according to the Nucleotide of the described O-antigen-specific to intestinal bacteria O74 type of claim 1, it is characterized in that: it is by 10 genomic constitutions, all between galF gene and gnd gene.

3, according to the Nucleotide of the described O-antigen-specific to intestinal bacteria O74 type of claim 2, it is characterized in that described gene is: the gene of transhipment enzyme comprises the wzx gene or with wzx the gene of identity function is arranged; Pol gene comprises the wzy gene or with wzy the gene of identity function is arranged; The monose synthase gene comprises orf1, orf2, orf3, orf4, orf5; Glycosyltransferase gene comprises orf7, orf8, orf10 gene.Wherein said gene: wzx is the Nucleotide of 5030 to 6289 bases among the SEQ ID NO:1; Wzy is the Nucleotide of 8078 to 8253 bases among the SEQ ID NO:1; Orf1 is the Nucleotide of 1137 to 2213 bases among the SEQ ID NO:1; Orf2 is the Nucleotide of 2210 to 3073 bases among the SEQ ID NO:1; Orf3 is the Nucleotide of 3075 to 3479 bases among the SEQ ID NO:1; Orf4 is the Nucleotide of 3469 to 3927 bases among the SEQ ID NO:1; Orf5 is the Nucleotide of 3930 to 5033 bases among the SEQ IDNO:1; Orf7 is the Nucleotide of 6300 to 7241 bases among the SEQ ID NO:1; Orf8 is the Nucleotide of 7257 to 8078 bases among the SEQ ID NO:1; Orf10 is the Nucleotide of 9231 to 10313 bases among the SEQ ID NO:1.

4, according to the Nucleotide of claim 1 or 2 described O-antigen-specifics to intestinal bacteria O74 type, it is characterized in that: it is to come from described wzx gene, wzy gene; The orf7 that glycosyltransferase gene comprises, orf8, orf10, and their mixing or their reorganization.

5, according to the Nucleotide of the described O-antigen high special to intestinal bacteria O74 type of claim 4, it is characterized in that the oligonucleotide of the described wzx of coming from gene is to being: the Nucleotide of 5547 to 5564 bases among the SEQ ID NO:1 and the Nucleotide of 6140 to 6157 bases; The Nucleotide of 5143 to 5160 bases among the SEQ ID NO:1 and the Nucleotide of 5414 to 5431 bases; The oligonucleotide that comes from the wzy gene is to being: the Nucleotide of 8119 to 8136 bases among the SEQ ID NO:1 and the Nucleotide of 8955 to 8972 bases; The Nucleotide of 8299 to 8316 bases among the SEQ ID NO:1 and the Nucleotide of 8563 to 8581 bases.

6, the Nucleotide of the described O-antigen-specific to intestinal bacteria O74 type of claim 1 is detecting the application of expressing the antigenic bacterium of O-, identify other polysaccharide antigen of the O-antigen of bacterium and bacterium in diagnosis.

7, the recombinant molecule of the Nucleotide of the described O-antigen-specific to intestinal bacteria O74 type of claim 1, and can provide the O-antigen of expressing intestinal bacteria O74 type by inserting to express, and become bacterial vaccine.

8, according to the application of the Nucleotide of the described O-antigen-specific to intestinal bacteria O74 type of claim 1, it is characterized in that it is used for PCR, is used for hybridization and fluoroscopic examination or is used to make gene chip or microarray, the bacterium in human body and the environment as probe as primer.

9, the separation method of the Nucleotide of the described O-antigen-specific to intestinal bacteria O74 type of claim 1 is characterized in that it comprises the steps:

(1) genomic extraction: 37 ℃ of incubated overnight intestinal bacteria O74 types in the LB of 5mL substratum, centrifugal collecting cell.With 500ul 50mM Tris-HCl (pH8.0) and 10ul 0.4M EDTA re-suspended cell, 37 ℃ of incubations 20 minutes, the N,O-Diacetylmuramidase that adds 10ul 10mg/ml then continues insulation 20 minutes, the Proteinase K, the 15ul 10%SDS that add 3ul 20mg/ml afterwards, 50 ℃ of incubations 2 hours, the RNase that adds 3ul 10mg/ml again, 65 ℃ of incubations 30 minutes; Add equal-volume phenol extracting mixture, get supernatant and use isopyknic phenol again: chloroform: twice of primary isoamyl alcohol (25: 24: 1) mixing solutions extracting, 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, DNA is resuspended among the 30ul TE with 70% ethanol; Genomic dna detects by 0.4% agarose gel electrophoresis;

(2) by the O-antigen gene in the pcr amplification intestinal bacteria O74 type bunch: with the genome of intestinal bacteria O74 type is that template is passed through its O-antigen gene of Long pcr amplification bunch, at first according to the JumpStart sequences Design upstream primer (5 '-ATT GTG GCTGCA GGG ATC AAA GAA AT-3 ') that often is found in O-antigen gene bunch promoter region, again according to the gnd gene design downstream primer (5 '-TAG TCG CGT GNG CCT GGA TTA AGT TCG C-3 ') in O-antigen gene bunch downstream; With the Expand Long Template PCR method of Boehringer Mannheim company amplification O-antigen gene bunch, the PCR response procedures was as follows: 94 ℃ of pre-sex change 2 minutes; 94 ℃ of sex change are 10 seconds then, annealed 15 seconds for 60 ℃, 68 ℃ were extended 15 minutes, carry out 30 circulations like this, last, continue to extend 7 minutes at 68 ℃, obtain the PCR product, detect the size and the specificity thereof of PCR product with 0.8% agarose gel electrophoresis, merge 5 pipe long PCR products, and with the Wizard PCR Preps purification kit purified pcr product of Promega company;

(3) make up O-antigen gene bunch library: make up O-antigen gene bunch library with the Novagen DNaseI shot gun method that is modified, reaction system is the 300ngPCR purified product, 0.9ul 0.1M MnCl ₂The DNaseI of the 1mg/ml of 1ul dilution in 1: 2000, reaction is carried out at room temperature, enzyme is cut the dna fragmentation size is concentrated between the 1.5kb-3kb, then add 2ul 0.1M EDTA termination reaction, merge the same reaction system of 4 pipes, with isopyknic phenol extracting once, use isopyknic phenol: chloroform: primary isoamyl alcohol (25: 24: 1) mixing solutions extracting once, after using isopyknic ether extracting once again, dehydrated alcohol deposit D NA with 2.5 times of volumes, and wash precipitation with 70% ethanol, be resuspended at last in the 18ul water, in this mixture, add 2.5ul dNTP (1mMdCTP subsequently, 1mMdGTP, 1mMdTTP, 10mMdATP), 1.25ul the T4DNA polysaccharase of 100mM DTT and 5 units, 11 ℃ 30 minutes, enzyme is cut product mends into flush end, after 75 ℃ of termination reactions, add the Tth archaeal dna polymerase of 5 units and corresponding damping fluid thereof and system is expanded as 80ul, 70 ℃ of reactions 20 minutes make 3 of DNA ' end add the dA tail, and this mixture is through the equal-volume chloroform: after primary isoamyl alcohol (24: 1) mixing solutions extracting and the extracting of equal-volume ether with 3 * 10 of Promega company ^-3The pGEM-T-Easy carrier connect 10 hours in 16 ℃, cumulative volume is 90ul, and the 10 * buffer of 9ul and the T4DNA ligase enzyme of 25 units are wherein arranged.Use the dehydrated alcohol precipitation of the 3M NaAc (pH5.2) of 1/10 volume and 2 times of volumes to be connected mixture at last, wash precipitation with 70% ethanol again, be dissolved in after the drying and obtain connecting product in the 30ul water; Preparation method with the electric transformed competence colibacillus cell of Bi0-Rad company prepares the competence e.colidh5, get after 2-3ul connects product and 50ul competence bacillus coli DH 5 alpha mixes, forward in the electric shock cup of 0.2cm of Bi0-Rad company and shock by electricity, voltage is 2.5 kilovolts, time is 5.0 milliseconds to 6.0 milliseconds, the SOC substratum that adds 1ml after the electric shock immediately in cup makes the bacterium recovery, then bacterium is coated in and contains penbritin, on the LB solid medium of X-Gal and IPTG, 37 ℃ of incubated overnight, obtain blue white bacterium colony next day, with the white colony that obtains promptly the white clone forward on the LB solid medium that contains acillin and cultivate, from each clone, extract plasmid simultaneously, and cutting the segmental size of evaluation insertion wherein with the EcoRI enzyme, the white that obtains clone group has constituted the O-antigen gene bunch library of intestinal bacteria O74 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 O74 type; The quality of sequence is mainly guaranteed by two aspects: 1) genome of intestinal bacteria O74 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 O74 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 10 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 O74 type at last;

(6) screening of specific gene: respectively designed two pairs of primers at wzx, wzy in the O-antigen gene of intestinal bacteria O74 type bunch 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 the 13rd group, has obtained the expection size, any product that all do not increase in other groups is so the O-antigen of wzx, wzy gene pairs intestinal bacteria O74 type all is high special.