CN1442422A - Nucleotide specific against 0-antigen of shigella dysenteria 3, colibacillus 0124 and 0164 - Google Patents

Abstract

A nucleotide specific to the O-antigen of Shigella dysenteriae 3, Escherichia coli O124 and Escherichia coil O164 is a complete nucleotide sequence of the genom for controlling the synthesis of O-antigen is shigella dysenteriae 3, such as the separated nucleotide shown by SEQ ID No.1. It has 12629 bases, or has one or more inserted, deletional or substituted bases but keeps the function of said separated neucleotide. It also includes the oligonucleotide of glycosyltransferase gene and oligose unit treating gene in O-antigen genom. A method for using said oligonucleotide to detect said three bacteria is also disclosed.

Description

Nucleotide to the O-antigen-specific of shigella dysenteriae 3 types, intestinal bacteria O124 and O164

Technical field

The present invention relates to the complete nucleotide sequence of control O-antigen synthetic gene cluster in shigella dysenteriae 3 types (Shigella dysenteriae 3), particularly relate in shigella dysenteriae 3 types oligonucleotide in the control O-antigen synthetic gene cluster, can utilize these oligonucleotide of O-antigen-specific shigella dysenteriae 3 types, intestinal bacteria O124 (Escherichia coliO124) and the intestinal bacteria O164 (Escherichia coli O164) in human body and the environment and identify O-antigen in these pathogenic bacterium quickly and accurately.

Background technology

Shigellae is the pathogenic bacterium that grow up along with the human evolution, can attack colon film epithelial cell, causes self limiting pyogenic infection focus, causes human bacillary dysentery.Human have higher susceptibility to Shigellae, only need be less than the infection that ten bacterium just can cause the people, children and adult easy infection, particularly children, easily cause acute poisoning dysentery, and the O-antigen of Shigellae is the one of the main reasons that Shigellae causes disease.

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. (1996) " Bacterialpolysaccharide synthesis and gene nomenclature " Trends in Microbiology, 4:495-503].In Shigellae, intestinal bacteria and Salmonellas, the O-antigen gene bunch between galF and gnd gene [Lei Wang.et al (2001) " Sequence analysis of four Shigella boydii O-antigenloci:implication for Escherichia coli and Shigella relationships " .Infection andImmunity, 11:6923-693 0; Lei Wang and Peter Reeves (2000) " The Escherichia coliO 111 and Salmonella enterica O35 gene clusters:gene clusters encoding the samecolitose-containing O antigen are highly conserved " .Joumal ofBacteriology.182:5256-5261].The O-antigen gene bunch contains three genoids: sugared synthesis path gene, glycosyltransferase gene, oligosaccharide unit treatment gene, the required nucleoside diphosphate monose of enzymic synthesis O-antigen of wherein sugared synthesis path genes encoding; Thereby the enzyme of glycosyltransferase gene coding forwards nucleoside diphosphate monose and other molecule to and makes monose aggregate into oligosaccharide unit on the monose; The oligosaccharide unit treatment gene comprises transhipment enzyme gene and pol gene, and they transfer to the bacterium inner membrance outside with oligosaccharide unit, and repolymerization becomes polysaccharide.Glycosyltransferase gene and oligosaccharide unit treatment gene only are present in the gene cluster of carrying these genes.The difference of monose in the O-antigen, monose asks that the difference of link button between the difference of link button and the oligosaccharide unit has 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 Shigellae, it has extremely strong diversity again simultaneously, and this enlightens us can study a kind of Shigellae 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.1996, 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 colt " .J.Clin.Microbiol.34:1622-1627] of the oligonucleotide that comes from the wbdI gene of the O-antigen-specific of E.coli O111 having been identified the toxogenic E.coli O111 of a strain, but afterwards studies show that Paton, the usefulness of A.W et.al comes from the oligonucleotide of wbdI gene and identifies that the method for the serotype of E.coli O111 has false positive results to occur.Bastin D.A.and Reeves, P.R. think, this is because the wbdI gene is sugared synthesis path gene [the Bastin D.A.andReeves of a supposition, P.R. (1995) Sequence and analysis of the O antigen gene (rfb) cluster ofEscherichia coli O111.Gene 164:17-23], and have this sugar in the antigenic structure of the O-of other bacterium, so sugared synthesis path gene is not a high special for O-antigen yet.

Shigellae has 46 kinds of serotypes, intestinal bacteria have 166 kinds of different O-antigens, 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 ofthe Enterobacteriaceae " .Elsevier SciencePublishers, Amsterdam, The Netherlands; T.cheasty, et al. (1983) " Antigenicrelationships between the enteroinvasive Escherichia coli antigensO28ac; O112ac; O124; O136; O143; O144, O152 and O164 and Shigella Oantigens " J.clin Microbiol; 17 (4): 681-684]; wherein shigella dysenteriae 3 types just have identical O-antigen [Dmitriev; B.A.; et al (1976) " Cell-walllipopolysaccharide of the ' Shigella-like ' Escherichia coliO124 " Eur.J.Biochem.64:491-498] with intestinal bacteria O124, and also the O-antigen with intestinal bacteria O136 is relevant.In addition, all known [Dmitriev, B.A., et al (1977) Carbohydr.Res, 56:207-209 of the antigenic structure of O-of shigella dysenteriae 3 types, intestinal bacteria O124 and intestinal bacteria O164; Malin Linnerborg., et al (1999) " Structural studies of the O-antigen polysaccharide from the enteroinvasiveEscherichia coli O 164 cross-reacting with Shigella dysenteriae type 3 " Eur.J.Biochem, 266:460-466], as shown below.Their O-antigenic structure is very similar as can be seen from Figure, Duos a group than intestinal bacteria O164 on last sugar of different is side chain in the O-antigenic structure of shigella dysenteriae 3 types; A β-D-Galp is arranged on the main chain in the O-antigenic structure of these external shigella dysenteriae 3 types, and a α-D-Galp is arranged on the main chain in the O-of intestinal bacteria O164 antigenic structure, traditional serological method can not be distinguished them.

The O-antigenic structure of shigella dysenteriae 3 types and intestinal bacteria O124:

→6)-β-D-Galf-(1→3)-β-D-GalpNAc-(1→3)-β-D-Galp-(1→α

4

CH3??????????????????????????↑

↑???????????????????????????1

HOOCCH-4The O-antigenic structure of)-β-D-Glcp-(1 → 6)-α-D-Glcp intestinal bacteria O164:

→6)-β-D-Galf-(1→3)-β-D-GalpNAc-(1→3)- α-D-Galp-(1→α

4

↑

1

β-D-Glcp-(1→6)-α-D-Glcp

Summary of the invention

The Nucleotide that the purpose of this invention is to provide a kind of O-antigen-specific to shigella dysenteriae 3 types, intestinal bacteria O124 and O164.It is the Nucleotide in the O-antigen gene bunch of shigella dysenteriae 3 types, is the special Nucleotide that comes from glycosyltransferase gene and transhipment enzyme gene and pol gene.

An object of the present invention is to provide the full length nucleotide sequence of the O-antigen gene bunch of shigella dysenteriae 3 types.

A time purpose of the present invention has provided the gene of the O-antigen gene bunch that constitutes shigella dysenteriae 3 types: 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 orf4, orf5, orf7, orf9 gene; Sugar synthesis path gene comprises the galf gene; The gene of two Unknown Function comprises orf2, orf3.

Another purpose of the present invention has provided oligonucleotide, and the gene that they come from encoding glycosyl transferring enzyme in the O-antigen gene bunch of shigella dysenteriae 3 types respectively comprises orf4, orf5, orf7, orf9 gene; The gene that coming from coding transhipment enzyme be the wzx gene or with wzx the gene of identity function, the gene that comes from the coding polysaccharase are arranged 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 shigella dysenteriae 3 types, intestinal bacteria O124 and O164; Especially the oligonucleotide of listing in the table 1, they are high specials to the O-antigen of shigella dysenteriae 3 types, intestinal bacteria O124 and O164, and these oligonucleotide are also reconfigurable, and the oligonucleotide after the combination also is a high special to the O-antigen of shigella dysenteriae 3 types, intestinal bacteria O124 and O164.

The above-mentioned oligonucleotide that another object of the present invention provides can be used as primer and is used for nucleic acid amplification reaction, perhaps be used for hybridization as probe, perhaps be used to make gene chip or microarray, thereby detect and identify O-antigen and detection and evaluation shigella dysenteriae 3 types, intestinal bacteria O124 and the O164 of shigella dysenteriae 3 types, intestinal bacteria O124 and O164 by these methods.

A further object of the present invention has provided the method for the complete sequence of the O-antigen gene bunch that separates shigella dysenteriae 3 types.Can obtain the complete sequence of the O-antigen gene bunch of other bacteriums according to present method operation, the complete sequence of the gene cluster of the bacterium of other polysaccharide antigens that also can obtain to encode.

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

The present invention is characterized in that to the Nucleotide of the O-antigen-specific of shigella dysenteriae 3 types, intestinal bacteria O124 and intestinal bacteria O164 it is the isolating Nucleotide shown in SEQ ID NO:1,12629 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 shigella dysenteriae 3 types, intestinal bacteria O124 and intestinal bacteria O164 is characterized in that it is by 9 genomic constitutions, all between galF gene and gnd gene.

The Nucleotide of aforesaid O-antigen-specific to shigella dysenteriae 3 types, intestinal bacteria O124 and intestinal bacteria O164 is characterized in that described gene comprises: about transhipment enzyme gene, comprise the wzx gene or with wzx the gene of identity function is arranged; Pol gene comprises the wzy gene or with wzy the gene of identity function is arranged; Glycosyltransferase gene comprises orf4, orf5, orf7, orf9 gene; Wherein said wzx gene is the Nucleotide of 1111 to 2625 bases among the SEQ ID NO:1; The orf4 gene is the Nucleotide of 5151 to 6155 bases among the SEQ ID NO:1; The orf5 gene is the Nucleotide of 6152 to 6940 bases among the SEQ ID NO:1; The wzy gene is the Nucleotide of 7014 to 8117 bases among the SEQ ID NO:1; The orf7 gene is the Nucleotide of 8146 to 9195 bases among the SEQ ID NO:1; The orf9 gene is the Nucleotide of 10435 to 11208 bases among the SEQ ID NO:1.

The Nucleotide of aforesaid O-antigen-specific to shigella dysenteriae 3 types, intestinal bacteria O124 and intestinal bacteria O164 is characterized in that it comes from described wzx gene, wzy gene or glycosyltransferase gene orf4, orf5, orf, orf9 gene; Or the oligonucleotide in the sugared synthesis path gene; And their mixing or their reorganization.

The Nucleotide of aforesaid O-antigen-specific to shigella dysenteriae 3 types, intestinal bacteria O124 and intestinal bacteria O164, it is characterized in that, the oligonucleotide of the described wzx of coming from is to being: the Nucleotide of 1221 to 1240 bases among the SEQ ID NO:1 and the Nucleotide of 2148 to 2167 bases, the Nucleotide of 1220 to 1238 bases among the SEQ ID NO:1 and the Nucleotide of 1884 to 1903 bases, the Nucleotide of 1835 to 1853 bases among the SEQ ID NO:1 and the Nucleotide of 2325 to 2345 bases; The oligonucleotide that comes from orf4 is to being: the Nucleotide of 5931 to 5909 bases among the SEQ ID NO:1 and the Nucleotide of 5824 to 5843 bases, the Nucleotide of 5377 to 5396 bases among the SEQ ID NO:1 and the Nucleotide of 6107 to 6125 bases, the Nucleotide of 5575 to 5594 bases among the SEQ ID NO:1 and the Nucleotide of 6114 to 6132 bases; The oligonucleotide that comes from orf5 is to being: the Nucleotide of 6182 to 6200 bases among the SEQ ID NO:1 and the Nucleotide of 6874 to 6895 bases, the Nucleotide of 6168 to 6186 bases among the SEQ ID NO:1 and the Nucleotide of 6824 to 6842 bases, the Nucleotide of 6172 to 6190 bases among the SEQ ID NO:1 and the Nucleotide of 6172 to 6190 bases; The oligonucleotide that comes from wzy is to being: the Nucleotide of 7969 to 7990 bases among the SEQID NO:1 and the Nucleotide of 7256 to 7276 bases, the Nucleotide of 7257 to 7277 bases among the SEQID NO:1 and the Nucleotide of 7768 to 7786 bases, the Nucleotide of 7410 to 7430 bases among the SEQID NO:1 and the Nucleotide of 7955 to 7973 bases; The oligonucleotide that comes from orf7 is to being: the Nucleotide of 8410 to 8428 bases among the SEQ ID NO:1 and the Nucleotide of 9109 to 9127 bases, the Nucleotide of 8405 to 8423 bases among the SEQ ID NO:1 and the Nucleotide of 9242 to 9260 bases, the Nucleotide of 8449 to 8467 bases among the SEQ ID NO:1 and the Nucleotide of 9040 to 9059 bases; The oligonucleotide that comes from orf9 is to being: the Nucleotide of 10450 to 10468 bases among the SEQ ID NO:1 and the Nucleotide of 11123 to 11141 bases, the Nucleotide of 10451 to 10469 bases among the SEQ ID NO:1 and the Nucleotide of 10803 to 10821 bases, the Nucleotide of 10565 to 10583 bases among the SEQ ID NO:1 and the Nucleotide of 11124 to 11142 bases; The Nucleotide of aforesaid O-antigen-specific to shigella dysenteriae 3 types, intestinal bacteria O124 and intestinal bacteria O164 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 shigella dysenteriae 3 types, intestinal bacteria O124 and intestinal bacteria O164, and can provide the O-antigen of expressing shigella dysenteriae 3 types, intestinal bacteria O124 and intestinal bacteria O164, and become bacterial vaccine by insert expressing.

The application of the Nucleotide of aforesaid O-antigen-specific to shigella dysenteriae 3 types, intestinal bacteria O124 and intestinal bacteria O164, 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, the bacterium in available these method human body and the environment.

The separation method of the Nucleotide of aforesaid O-antigen-specific to shigella dysenteriae 3 types, intestinal bacteria O124 and intestinal bacteria O164 is characterized in that, comprises the steps:

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

(2) by the O-antigen gene in pcr amplification shigella dysenteriae 3 types bunch: with the genome of shigella dysenteriae 3 types 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 GTGGCT GCA 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, 60 ℃ of annealing 30 seconds, and 68 ℃ were extended 15 minutes, and carried out 30 circulations like this.At last, continue to extend 7 minutes at 68 ℃, obtain the PCR product, the agarose gel electrophoresis with 0.8% detects the size and the specificity thereof of PCR product.Merge 6 pipe longPCR products, and with the Wizard PCR Preps purification kit purified pcr product of Promega company;

(3) make up O-antigen gene bunch library: make up O-antigen gene bunch library with the Novagen DNaseI shot gun method that is modified.Reaction system is a 300ng PCR purified product, 0.9ul 0.1M MnCl ₂, the DNaseI of the 1mg/ml of 1ul dilution in 1: 2000, reaction is carried out at room temperature.Enzyme is cut the dna fragmentation size is concentrated between the 1kb-3kb, then adds 2ul 0.1M EDTA termination reaction.Merge the same reaction system of 4 pipes, with isopyknic phenol extracting once, use isopyknic phenol: chloroform: primary isoamyl alcohol (25: 24: 1) extracting once, after using isopyknic ether extracting once again, dehydrated alcohol deposit D NA with 2.5 times of volumes, and wash precipitation with 70% ethanol, be resuspended at last in the 18ul water.In this mixture, add 2.5ul dNTP (1mMdCTP subsequently, 1mMdGTP, 1mMdTTP, 10mMdATP), the T4DNA polysaccharase of 1.25ul 100mM DTT and 5 units, 11 ℃ 30 minutes, enzyme is cut product mend into flush end, after 75 ℃ of termination reactions, add the Tth archaeal dna polymerase of 5 units and corresponding damping fluid thereof and system is expanded as 80ul, 70 ℃ were reacted 20 minutes, made 3 of DNA ' end add the dA tail.This mixture is through the equal-volume chloroform: after primary isoamyl alcohol (24: 1) mixing solutions extracting and the extracting of equal-volume ether with 3 * 10 of Promega company ^-3The pGEM-T-Easy carrier connect 24 hours in 16 ℃, cumulative volume is 90ul.10 * the buffer of 9ul and the T4DNA ligase enzyme of 25 units are wherein arranged.Use the dehydrated alcohol precipitation of the 3MNaAc (pH5.2) of 1/10 volume and 2 times of volumes to be connected mixture at last, wash precipitation with 70% ethanol again, be dissolved in after the drying and obtain connecting product in the 30ul water.Preparation method with the electric transformed competence colibacillus cell of Bio-Rad company prepares competence bacillus coli DH 5 cell, 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, time is 5.0 milliseconds-6.0 milliseconds, the SOC substratum that adds 1ml after the electric shock immediately in cup makes the bacterium recovery, then bacterium is coated in 37 ℃ of incubated overnight on the LB solid medium that contains penbritin, X-Gal and IPTG, 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, extract plasmid and cut the segmental size of evaluation insertion wherein with the EcoRI enzyme from each clone simultaneously, the white that obtains clone group has constituted the O-antigen gene bunch library of shigella dysenteriae 12 types;

(4) to the cloning and sequencing in the library: from the library, select insert 120 clones of fragment more than 700bp by Shanghai biotechnology company limited with ABI377 type automatic dna sequencer to unidirectional order-checking of insertion fragment in cloning, make sequence reach 80% fraction of coverage.Residue 20% sequence is again according to the sequences Design primer that has obtained, direct PCR and from the genomic dna of shigella dysenteriae 3 types again to the order-checking of PCR product, thus obtain all sequences of O-antigen gene bunch.In shigella dysenteriae 3 types we according to the sequences Design that has obtained two pairs of primers, as follows:

Upstream primer 5 '-TACTCTCCGCACAACGTC-3 ' and downstream primer 5 '-TGCCCAATCATCACTATCA-3 ';

Upstream primer 5 '-TAATCCCTCTGACAATAGTG-3 ' and downstream primer 5 '-GCAATGTCAGAAACTC-3 '.

(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, thereby obtain the Nucleotide full length sequence of the O-antigen gene bunch of shigella dysenteriae 3 types, the quality of sequence is mainly guaranteed by two aspects: 1) genome of shigella dysenteriae 12 types is done 6 Long PCR reactions, mix these products then to produce the library.2), guarantee high-quality fraction of coverage more than 3 to each base.After obtaining the nucleotide sequence of shigella dysenteriae 3 type O-antigen genes bunch, with American National biotechnology information science center (The National Center forBiotechnology Information, NCBI) orffinder finds gene, find the reading frame of 9 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 shigella dysenteriae 3 types at last;

(6) screening of specific gene: at wzx, wzy, orf4, orf5, orf7, the orf9 gene design primer in the O-antigen gene of shigella dysenteriae 3 types bunch.Respectively designed three pairs of primers in each gene, the difference that every pair of primer is distributed in the corresponding gene is local 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, all primers all obtain positive findings in intestinal bacteria O124 and O164, the correct band of any size does not all increase in other groups, that is to say, do not obtaining any PCR product band in the array mostly, though in the minority group, obtain PCR product band, but its size does not meet the expection size, so wzx, wzy, orf4, orf5, orf7, orf9 gene pairs shigella dysenteriae 3 types, intestinal bacteria O124 and O164 and O-antigen thereof all are high specials.

Just, first aspect of the present invention provides the full length nucleotide sequence of the O-antigen gene bunch of shigella dysenteriae 3 types, its complete sequence shown in SEQ ID NO:1,12629 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 shigella dysenteriae 3 types by method of the present invention, as described in Table 3, it is altogether by 9 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 shigella dysenteriae 3 types, promptly transports enzyme gene (wzx gene or the gene of identity function arranged with wzx); Pol gene (wzy gene or the gene of identity function arranged with wzy); Glycosyltransferase gene comprises orf4, orf5, orf7, orf9 gene; Special sugared synthesis path gene in the bacterial polysaccharides antigen comprises the glf gene; The gene of two Unknown Function comprises orf2, orf3 gene.Their zero positions in O-antigen gene bunch and final position and nucleotide sequence all are listed among Fig. 2.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 shigella dysenteriae 3 types, intestinal bacteria O124 and O164.

The 3rd aspect of the present invention, wzx gene in the O-antigen gene bunch that comes from shigella dysenteriae 3 types is provided or the gene, wzy gene of identity function is arranged or the gene and the glycosyltransferase gene of identity function are arranged with wzy with wzx, the oligonucleotide that comprises orf4, orf5, orf7, orf9 gene, 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 being to obtain expecting the product of size in the pcr amplification that carries out of template with shigella dysenteriae 3 types, intestinal bacteria O124 and O164 only, and are all not obtain expecting the product of size in the pcr amplification that carries out of template other bacterium listed with table 2.In more detail, with these oligonucleotide to being that PCR that primer is done is reflected at and does not all obtain spawn in most of bacteriums, though in some bacterium, obtained PCR product band, but its size does not meet the expection size, this is that this problem can be avoided by being PCR with intragenic other primer because primer is attached to genomic other position and causes.So, can determine these primers promptly the listed oligonucleotide of table 1 be high special to shigella dysenteriae 3 types, intestinal bacteria O124 and O164 and their O-antigen.

The separation method of the Nucleotide of described O-antigen-specific to shigella dysenteriae 3 types, intestinal bacteria O124 and intestinal bacteria O164 comprises the steps: 1) genomic extraction; 2) the O-antigen gene in pcr amplification shigella dysenteriae 3 types bunch; 3) structure in O-antigen gene bunch library; 4) to the cloning and sequencing in the library; 5) splicing of nucleotide sequence and analysis finally obtain the structure of O-antigen gene bunch; 6) screening of specific gene.

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 is meant the gene that derives from the encoding glycosyl transferring enzyme in the O-antigen gene bunch, the gene of coding transhipment enzyme and intragenic one section nucleic acid molecule of coding polysaccharase, they can change on length, generally change in 10 to 20 Nucleotide scopes.More precisely these oligonucleotide are to come from wzx gene (nucleotide position is 1111 to 2625 bases from SEQ ID NO:1), orf4 gene (nucleotide position is 5151 to 6155 bases from SEQ ID NO:1), orf5 gene (nucleotide position is 6152 to 6940 bases from SEQ ID NO:1), wzy gene (nucleotide position is 7014 to 8117 bases from SEQ ID NO:1), orf7 gene (nucleotide position is 8146 to 9195 bases from SEQID NO:1), orf9 gene (nucleotide position is 10435 to 11208 bases from SEQ ID NO:1).Coming from above intragenic oligonucleotide all is high special to shigella dysenteriae 3 types, intestinal bacteria O124 and O164.

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; Also come from sugared synthesis path gene.The mixture of these genes is special to a special bacterial polysaccharides antigen, is special thereby make this cover oligonucleotide to the polysaccharide antigen of this bacterium.More particularly, the mixture of these oligonucleotide is to come from glycosyltransferase gene, wzx gene or the gene, wzy gene of identity function arranged or with wzy oligonucleotide and the combination that comes from the oligonucleotide in the sugared synthesis path gene in the gene of identity function are 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 shigella dysenteriae 3 types, intestinal bacteria O124 and O164.Available PCR method detects, more can with behind the Nucleotide mark in the inventive method as probe by hybridization such as southern-blot or fluoroscopic examination, perhaps by antigen and bacterium in gene chip or the microarray assay sample.

The present inventor considers following situation: when one special oligonucleotide detects when invalid, the mixture of oligonucleotide can with the target region specific hybrid with test sample.Therefore the invention provides a cover oligonucleotide and be used for detection method of the present invention.Here said oligonucleotide is meant 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 shigella dysenteriae 3 types, intestinal bacteria O124 and O164.

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 shigella dysenteriae 3 types, intestinal bacteria O124 and O164.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 shigella dysenteriae 3 types, intestinal bacteria O124 and O164.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 shigella dysenteriae 3 types 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 shigella dysenteriae 3 types 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 shigella dysenteriae 3 types in the LB of 5mL substratum, centrifugal collecting cell.With 500ul 50mM Tris-HCl (pH8.0) and 10ul 0.4M EDTA re-suspended cell, 37 ℃ of incubations 20 minutes, the N,O-Diacetylmuramidase that adds 10ul 10mg/ml then continues insulation 20 minutes.The Proteinase K, the 15ul 10%SDS that add 3ul 20mg/ml afterwards, 50 ℃ of incubations 2 hours add the RNase of 3ul 10mg/ml again, 65 ℃ of incubations 30 minutes.Add equal-volume phenol extracting mixture, get supernatant liquor liquid and use isopyknic phenol again: chloroform: primary isoamyl alcohol (25: 24: 1) mixing solutions extracting twice, get supernatant liquor liquid again with isopyknic ether extracting to remove remaining phenol.Supernatant liquor liquid rolls out DNA and washes DNA with 70% ethanol with glass yarn with 2 times of volume ethanol deposit D NA, at last DNA is resuspended among the 30ul TE.Genomic dna detects by 0.4% agarose gel electrophoresis.Embodiment 2: by the O-antigen gene in pcr amplification shigella dysenteriae 3 types bunch

With the genome of shigella dysenteriae 3 types 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 GCT GCA 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 GGATTA AGT TCG C-3 ') in O-antigen gene bunch downstream.With the Expand Long TemplatePCR method of Boehringer Mannheim company amplification O-antigen gene bunch, the PCR response procedures was as follows: 94 ℃ of pre-sex change 2 minutes; 94 ℃ of sex change are 10 seconds then, 60 ℃ of annealing 30 seconds, and 68 ℃ were extended 15 minutes, and carried out 30 circulations like this.At last, continue to extend 7 minutes at 68 ℃, obtain the PCR product, the agarose gel electrophoresis with 0.8% detects the size and the specificity thereof of PCR product.Merge 6 pipe long PCR products, and with the Wizard PCR Preps purification kit purified pcr product of Promega company.Embodiment 3: make up O-antigen gene bunch library.

At first be the acquisition that connects product:

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

Next is the preparation of competent cell:

The method that provides with reference to Bio-Rad company prepares 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 about OD6000.5, ice bath cooling was 20 minutes then, in centrifugal 15 minutes of 4 ℃ of 4000rpm.Confide all supernatant liquor liquid, dispel thalline, in centrifugal 15 minutes of 4 ℃ of 4000rpm with the deionization aqua sterilisa 200ml of cold ice precooling.Deionization aqua sterilisa 100ml with cold ice precooling dispelled thalline again, in centrifugal 15 minutes of 4 ℃ of 4000rpm.With 10% glycerine suspension cell of cold ice precooling, centrifugal 10 minutes of 4 ℃ of 6000rpm abandon supernatant liquor liquid, 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 shigella dysenteriae 3 types with the EcoRI enzyme.Embodiment 4: to the cloning and sequencing in the library.

From the library, select insert 120 clones of fragment more than 700bp by Shanghai biotechnology company limited with ABI377 type automatic dna sequencer to unidirectional order-checking of insertion fragment in cloning, make sequence reach 80% fraction of coverage.Residue 20% sequence is again according to the sequences Design primer that has obtained, direct PCR and from the genomic dna of shigella dysenteriae 3 types again to the order-checking of PCR product, thus obtain all sequences of O-antigen gene bunch.Residue 20% sequence is according to the two pairs of primers of sequences Design that obtained in shigella dysenteriae 3 types, again from the genomic dna of shigella dysenteriae 3 types directly PCR and to the PCR product order-checking obtains, primer is as follows:

Upstream primer 5 '-TACTCTCCGCACAACGTC-3 ' and downstream primer 5 '-TGCCCAATCATCACTATCA-3 ';

Upstream primer 5 '-TAATCCCTCTGACAATAGTG-3 ' and downstream primer 5 '-GCAATGTCAGAAACTC-3 ' 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 shigella dysenteriae 3 types obtained.The quality of sequence is mainly guaranteed by two aspects: 1) genome of shigella dysenteriae 12 types is done 6 Long PCR reactions, mix these products then to produce the library.2), guarantee high-quality fraction of coverage more than 3 to each base.After obtaining the nucleotide sequence of shigella dysenteriae 3 type O-antigen genes bunch, with American National biotechnology information science center (The National Center for BiotechnologyInformation, NCBI) orffinder finds gene, find the reading frame of 9 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 shigella dysenteriae 3 types at last, as shown in table 3.

By retrieval and relatively, find that it is the same that orf1 and the wzx gene of E.coli O157:H7 have 17% aminoacid sequence, promptly has 17% homogeny, 42% similarity in 440 amino acid; And and the wzx gene of Bacteroides fragilis in 506 amino acid, 20% homogeny is arranged, 42% similarity, this wzx gene has 13 potential transmembrane domains, and the algorithm [Eisenberg by Eisenberg etc., D, Schwarz, E.etal (1984) " Analysis of membrane and surface proteinsequences with the hydrophobic moment plot.J.Mol.Biol.179:125-142] find that orf6 also has 13 potential transmembrane domains; and with many wzx protein similars; orf6 has about 50 amino acid whose conservative motifs at the proteic aminoterminal of wzx; so can determine orf6 is the wzx gene, called after wzx.The albumen of the hypothesis of orf2 and Sinorhizobium meliloti has 34% homogeny in 363 amino acid, 52% similarity is arranged, temporarily with orf2 called after orf2 gene.Orf3 also has 23% homogeny with the albumen of the hypothesis of Sinorhizobium meliloti in 226 amino acid, 42% similarity is temporarily with orf3 called after orf3 gene.The eps9I gene of orf4 and Streptococcus thernophilus has 30% homogeny in 239 amino acid, 54% similarity, and the eps9I gene is a glycosyltransferase gene; The CpsIaI gene of orf4 and Streptococcus agalactiae has 29% homogeny in 310 amino acid in addition, 47% similarity, the CpsIaI gene is a N-acetyl glycosyltransferase gene, so think that orf4 is a glycosyltransferase gene, called after orf4.Orf5 and colibacillary cap8J gene show higher homology, this genes encoding galactosyltransferase, so orf5 is the galactosyltransferase gene, and called after orf5.Orf6 is the albumen that a prediction has 10 transmembrane segments, the topological framework of its inner membrance has the characteristic feature of well-known O-antigen polysaccharase, in addition, the wzy gene of the coding O-antigen polysaccharase of it and E.coli has 23% homogeny, 44% similarity in 355 amino acid; In 353 amino acid 22% homogeny is arranged with the wzy gene of Streptococcus thermophilus, 44% similarity is so name orf6 is the wzy gene.Among orf7 and the Streptococcus pneumoniae among coding cpsI gene of galactosyltransferase and the Streptococcus pneumoniae cps23FU gene of coding galactosyltransferase 34.5% and 33% homogeny is arranged respectively on amino acid levels, and semi-lactosi is arranged in the O-antigenic structure of shigella dysenteriae 3 types, so determine that orf7 also is a galactosyltransferase gene, called after orf7.Orf8 has the aminoacid sequence of glf gene of O-antigen gene bunch of 63% aminoacid sequence and E.coli K12 identical, shows the homology that height is arranged between them.In addition, sugared Galf is synthesized in the enzyme catalysis of glf genes encoding, and this is the composition in the shigella dysenteriae 3 type O-antigenic structures just, so infer that orf8 is the glf gene, called after glf.The gene BL0045 of orf9 and Bifidobacterium longum and the wcfN gene of Bacteoides fragilis have 33% homogeny on amino acid levels, and these two genes are the glycosyltransferase genes in the exocellular polysaccharide gene cluster.Therefore orf9 also is a glycosyltransferase gene, called after orf9 gene.

Infer that according to the structure of shigella dysenteriae 3 types it should have four glycosyltransferase genes, shift five sugar respectively with the synthetic antigenic oligosaccharide unit of O-.Need the wzx gene that oligosaccharide unit is transferred to outside the film, the wzy gene aggregates into polysaccharide with oligosaccharide unit, special monose in the oligosaccharide unit is also synthetic by O-antigen gene bunch, and is synthetic by the glf gene as Galf, and these genes all are found in the O-antigen gene of shigella dysenteriae 3 types bunch.But in the antigenic structure of shigella dysenteriae 3 type O-, also has GalNAc.In Yersina enterocolitica O8, UDP-GalNAc is generated by the semi-lactosi mutase catalysis UDP-GlcNAc of gne genes encoding, in the O-antigen gene of shigella dysenteriae 3 types bunch, also do not find the gne gene, but Peter Reeves etc. are by having the gne gene in PCR discovery other gene clusters in some bacterium such as E.coli O124, that is to say that an O-antigen gene bunch outer gne gene transformation UDP-GlcNAc generates UDP-GalNAc and provides O-antigen to synthesize essential sugar, so we think that the gne gene may be in other gene clusters of shigella dysenteriae 3 types.Embodiment 6: the screening of specific gene.

At wzx, wzy, orf4, orf5, orf7, orf9 gene design primer in the O-antigen gene of shigella dysenteriae 3 types bunch, the position of these genes in nucleotide sequence sees Table 1.

Glycosyltransferase gene, transhipment enzyme gene and pol gene and their function corresponding and the size of the O antigen gene bunch of shigella dysenteriae 3 types in table 1, have been listed.In each gene, we have respectively designed three pairs of primers, and the difference that every pair of primer is distributed in the corresponding gene is local to guarantee its specificity.In table, also listed position and the size of each primer in SEQ ID NO:1.Is that template carry out PCR with listed corresponding annealing temperature in the table with the genomes of all bacterium in the table 2 with every pair of primer, has obtained corresponding PCR product, and its size is also listed in the table.

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

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 27 groups altogether, all list in the table in their source.

The genomic dna that contains shigella dysenteriae 3 types in the 24th 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: 94 ℃ of pre-sex change after 2 minutes, 94 ℃ of sex change 15 seconds, annealing temperature is because of the difference different (with reference to table 1) of primer, annealing time is 50 seconds, and 72 ℃ were extended 2 minutes, and carried out 30 circulations like this.Continue to extend 10 minutes at 72 ℃ at last, reaction system is 25ul.After reaction finishes, get the 10ulPCR product and detect the fragment that amplifies by 0.8% agarose gel electrophoresis.

For wzx, wzy, orf4, orf5, orf7, orf9 gene, each gene all has three pairs of primers detected, every pair of primer has obtained also all obtaining onesize specificity band the correct band of expection size in the 19th group and the 20th group except be PCR in the 24th group after.After being template PCR with the genomic dna of each bacterium in these two groups, find only in intestinal bacteria O124 and O164, to have obtained positive findings.In more detail, each of each gene all obtains expecting the correct PCR product band of size to primer more than in intestinal bacteria O124 and O164.Wzx, wzy, orf4, orf5, orf7, the orf9 gene that shigella dysenteriae 3 types are described also all is present in the O antigen gene bunch of intestinal bacteria O124 and O164.In addition, three pairs of primers that come from the orf9 gene all obtain positive findings in intestinal bacteria O136, and the primer of other genes does not all obtain positive findings in intestinal bacteria O136.Introduce as background technology, the O-antigenic structure of shigella dysenteriae 3 types and intestinal bacteria O124 is the same, very similar with the O-antigenic structure of intestinal bacteria O164, relevant with the O-antigenic structure of intestinal bacteria O136, our result has confirmed this point from another side.Except above-mentioned situation, the correct band of any size does not all increase in other groups, that is to say, do not obtaining any PCR product band in the array mostly, though in the minority group, obtain PCR product band, but its size does not meet the expection size, so wzx, wzy, orf4, orf5, orf7, orf9 gene pairs shigella dysenteriae 3 types, intestinal bacteria O124 and O164 and O-antigen thereof all are high specials.

At last, from shigella dysenteriae 3 types, screen gene by PCR: wzx, wzy and four glycosyltransferase genes to the O-antigen high special of shigella dysenteriae 3 types, intestinal bacteria O124 and O164.And the oligonucleotide of these intragenic any one section 10-20nt is special to the O-antigen of shigella dysenteriae 3 types, intestinal bacteria O124 and O164, and the primer in especially above-mentioned each gene is that oligonucleotide is high special to detecting the back confirmation through PCR to shigella dysenteriae 3 types, intestinal bacteria O124 and O164.These all oligonucleotide all can be used for shigella dysenteriae 3 types, intestinal bacteria O124 and the O164 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 shigella dysenteriae 3 types, in table, listed the structure of the O-antigen gene bunch of shigella dysenteriae 3 types, altogether by 9 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 location tables of the gene in the O-antigen gene bunch of shigella dysenteriae 3 types, in table, listed the accurate position of all open reading frame in complete sequence in the O-antigen gene bunch of shigella dysenteriae 3 types, at the underscoring of the initiator codon and the terminator codon of each open reading frame.The initiator codon of open reading frame has two in bacterium: ATG and GTG.

Sequence list

SEQUENCE LISTING＜110〉Nankai University＜120〉Nucleotide＜130 special to shigella dysenteriae 3 types, intestinal bacteria O124 and O164〉Nucleotide＜160 special to shigella dysenteriae 3 types, intestinal bacteria O124 and O164〉1＜170〉PatentIn version 3.1＜210〉1＜211〉12629＜212〉DNA＜213〉Shigella dysenteriae＜400〉1

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

gaaaaccact?tcgacacctc?ttatgaatta?gaatctctcc?ttgagcagcg?cgtgaagcgt????120

caactgttgg?cggaagtaca?atctatctgt?ccgccgggcg?tgaccattat?gaacgtgcgt????180

cagggtgaac?ctttaggttt?aggccactcc?atcttatgtg?cgcgacctgc?cattggtgac????240

aacccattta?tcgtggtact?gccagacgta?gtgatcgacg?acgccagcgc?cgacccgctg????300

cgctacaacc?ttgctgccat?gattgcgcgc?ttcaacgaaa?cgggccgtag?ccaggtgctg????360

gcaaaacgta?tgccgggtga?cctctctgaa?tactccgtca?tccagaccaa?agaaccgctg????420

gaccgtgaag?gtaaagtcag?ccgcattgtt?gaatttatcg?aaaaaccgga?tcagccgcag????480

acgctggact?cagacatcat?ggccgtaggt?cgctatgtgc?tttctgccga?tatttggccg????540

gaactagaac?gcactcagcc?tggtgcatgg?gggcgtattc?agctgactga?tgctattgcc????600

gaactggcga?aaaaacagtc?cgttgatgcc?atgctgatga?ctggagacag?ctacgactgc????660

ggtaaaaaaa?tgggctatat?gcaggcgttt?gtgaagtacg?gactgcgtaa?cctgaaagaa????720

ggggcgaagt?tccgcaaagg?tattgagagg?ttgttgaatg?attaaaaaca?tgaacaaata????780

taacggtaaa?taagagaaat?tgtaacggca?gtgaggattt?gcggcgaagc?tcaaacgtgg????840

cgaatatccc?tgtcgttatt?tattaataaa?tcatcgtact?aacaatggaa?taaaaaagtg????900ccttgtttta?agttttacag?gatattcctt?gtttctggag?gagattgata?aggcaatcag??????960catctgaatt?tatcagaact?tgtgtgtctt?gctgattaca?ttgttatatt?catgcagtgt?????1020actggtagct?gtggagccag?gggcggtagc?gtgcattaat?attcacaata?tattctttta?????1080aatgtaagca?gaattaatcg?aacctaaata?atgtcacgta?acaaaataat?cataaagaat?????1140actatgtttt?tggcaacaag?aactttagtt?tctttaagtg?tttcatttta?cacaacaaga?????1200atagttatac?aacagttggg?ggcggcagac?tatggcttat?ttaatatcat?ttatggagtt?????1260gtaacatttt?ttacattcgt?agtcaccgcg?atgaatgatt?ctgtacagcg?ttatattgca?????1320ataggtgttg?gttcacaaaa?aatatcggtt?attagagatg?ctgtaaaaaa?tagtatgttt?????1380atatttgtta?tatctgcttt?catattggca?gtatgtcttc?ttttggcaag?gggggtgatc?????1440atacataata?ttttaaatat?acctaaagat?tctattgaaa?atgcaagtgt?gttatatttg?????1500gttgcagtat?tttcaataac?catacttata?attcagacgc?cacttaatgc?gatggtgtta?????1560gcatacgaga?aaatgtcatt?ttacgcatac?atgatgatat?ttgaaatggt?agctaaaatg?????1620agtatggctt?tgttactaac?tttactggaa?aaagataagg?tcattgtata?ttcaatactt?????1680ttaggttcta?tttctttttt?taacttgttg?gtttatttat?gctactgctt?gatatgtttt?????1740aaaaaatcaa?tgtttggtgg?gagaataaaa?tttagagtgc?tgaaagaaat?ttcaactttt?????1800tcattttgga?atatatttgg?taatttctct?tacatgtgca?gagttcaggg?agtgaacatt?????1860gtaattaata?ttttttacgc?tatagccgtg?aatgcagcat?atgcgatttc?gattactgtt?????1920ttaaatgcaa?ttaatacact?tacgcaatca?ttaattacag?cattaaggcc?tcaaatattt?????1980aaatcatatg?gcgaatgtga?tttaaaaaga?tataatcatc?tagttctctt?tggttcaaaa?????2040tatacatttt?caatattgtt?tctgttaagt?agccctgtaa?ttctatgtgc?ggatgagttg?????2100ttaaagatat?ggcttgatat?tgtaccagat?tatacagttg?aatttgtgag?gttggtcatt?????2160gttgtggctt?ttattgatag?tttttcatat?agcatgattg?ctggtattca?ggctacgggt?????2220aggattaaaa?catatcaact?agttgttagt?ttaattgtcc?taattaattt?gccgttaacc?????2280tttattttgt?ttaaggcagg?aaataatgtt?ttaagtatgt?tttatccatt?tattgttact?????2340gccattatca?atcaaggttt?aaggttgtat?tttatctata?tcaatgcagg?ttttgattat?????2400aagaagtatt?tcactgttgt?gatctatcct?tgcttgttag?ctgtatgctt?atcgttagtg?????2460acagatattt?caattaaaaa?aatgcttcca?tttaacagta?ttattgatgt?tttaattgtg?????2520tgtattttta?ttttttcatt?caatacaata?attttctatt?gggttgtagt?ttctaaaaag?????2580gaaaaaaaat?ggttattaga?aaccctgaaa?agaaaagtga?aataacaata?aaagatgtta?????2640tagacaatga?actctgtact?ggttgtggag?tttgtatttc?ggaagatagt?agcaaaacct?????2700cctttatgaa?atggaattct?gaaggttttt?atgagccatg?tttctctcct?gtatcaacat?????2760tattcaatat?gcagcgtgtt?tgtcccttta?acttaagtag?ggatacatta?gtgaatgagg?????2820atgaacttgc?gcatgagttt?tttgatggta?agggatattt?agattctgag?gttgggtttt?????2880ataaaaaaat?atatgtgggt?tattctaaac?attttagaga?gacttcatcc?tccggtggta?????2940tagctacata?tgtatttgaa?caacttctta?gacgtaagta?tgttgatgct?ctgtttatag?????3000ttagggagct?tggtggttca?tatggatatc?aagtcttcga?taatccagaa?catattaagg?????3060atatgtctaa?aacaagatat?tatcctgtaa?cgctagaaaa?gttatttgat?aatatttata?????3120agcttaatgg?tagagtggcc?gtttcaggag?tggcatgttt?tattaaagcc?attaggctca?????3180aacaacatta?ccatccagaa?ctgaaagaga?aaataccatt?ccttgttggt?ataatttgtg?????3240gaggactcaa?aagtcgatat?tacactgatt?atttatcaca?gtctgctggt?tgtgtcagtg?????3300agtaccaaaa?tgctgagtat?cgagtgaaaa?agaaagatag?tcatgctctt?gactatcgtt?????3360tcacttgtgt?tgagaaatca?aataatataa?ttcattctgt?tgatatgcaa?agaatggggg?????3420atatgtgggg?ttcagggcta?tttaaggcaa?acgcatgtga?ctattgtgat?gatgttacaa?????3480ccgaactcgc?agacatatca?ttgggtgatg?cctggatttc?gccatacaat?atggatggag?????3540ctggtaataa?tgtagttgta?tgtcgctcca?ccacagctca?tgaaattatt?ttatctggga?????3600ttgagaaaaa?agacttagaa?ttaacggaac?tcgaattaga?gcagttgaaa?ttgtcacaac?????3660aaggaagttt?taaccacagg?cataaaggat?tattatatag?aataaaaaat?gcagagaaaa?????3720ataatcgctt?agtgcctgtt?aagaggaaaa?gattcttacg?ctctatatct?tttttattaa?????3780agttaattca?aaaacaacga?tcagttacaa?ggagaaaaag?catagagata?tggatggaaa?????3840ctcaaaattc?agcaactttc?gataagaaaa?tgaagggata?tttattcacg?ttgcgttggt?????3900taacagtggt?caataggaaa?ttaagtcgga?tgttcaagat?tgtttctcta?aatagaatgg?????3960gtaaaaaatg?aaagttgcaa?tattaactca?accattacat?acgaattatg?gtggcacatt?????4020acaagcctat?gcattacaaa?aagtcttgat?taatttggga?catgaaccag?agactattaa?????4080ttataggtcg?aaaattaaga?ggccactatt?tatccgtgtt?gtgttgtcaa?aaattaaaag?????4140gatagtatta?tgccgaaaaa?tcacatttga?ttttacaaca?caagatagaa?ttaatattag?????4200gacgcatcat?caatctttca?ttgatacaag?attaaactat?tcagaggaga?ttaacggtac?????4260tgaggggttg?agagattata?ttttaaaaaa?taattacggt?gctgtaataa?taggtagtga?????4320tcagacatgg?agaccaatat?attcgcctag?aatagattca?ttttttctcg?attttttaag?????4380tgatgtaaac?gatataaaaa?aaatggcata?tgcagcatct?tttggtactg?ataaatggga?????4440gttcacggag?tcacaaacta?atttatttaa?attgttactt?tcaaaatttg?actatgtttc?????4500agttagagag?tcctcaggag?tcaaattatg?tagtgaaaaa?tttggtttga?aagcagagtt????4560ggttttagat?ccgacattgc?tattgacatt?cgaagattat?actcatttat?tagacaatga????4620ctatattaat?cttcataagg?gaaaggtgtt?tagttatgta?cttgatgaga?attacgacaa????4680aaaaaactct?atagacacag?tagcaagtac?tctccgcaca?acgtcatttt?atacttaccc????4740taaaaaaatc?actaaagatg?aatatgtgat?tagaaattat?tccgagtatg?aatatccgcc????4800aatagaatat?tggatttctt?cgtttaaggc?cgctgaattt?attgtaacag?attcatttca????4860tggaacagta?ttttctatta?tttttaacaa?acccttcatc?gccattgcaa?atgaagagag????4920aggaaaggct?agatttacgt?cactattgga?aatgtttggt?cttgaaagac?gacttgtgag????4980taatttagat?gatgttgatt?taaagcttgt?aaatgaaaaa?attaactatg?gccccataaa????5040tgaaaaaatt?gcttatttta?gagaagagtg?ccttaaaaag?ttgcagttaa?tgctaataaa????5100ataatcagat?tatgtatgtc?tctctacaat?taaaatatga?atgttgatcg?atgaagaatt????5160gtaaagtatc?tgtaattatt?ccggtctata?atgcggaaaa?atatatacaa?cgttgtattt????5220taagcttatt?aaagcagaca?ttagatgatg?ttgaaattat?catcattgat?gatggttcta????5280ctgataattc?actttctata?ataaaagaaa?ctgttgcatt?gcatactgct?agtagagctc????5340gccaaaagag?aatcaatata?atctctcgag?aaaacaaagg?agttgcctat?acacgaagtc????5400agggattgag?actttcgcag?ggggaattta?tcattcattt?tgatagtgat?gattgggcaa????5460aatccaatat?gctggaggaa?atgtataaga?caattgtttc?caataatgca?gacatggtta????5520tatgcgatta?ttttttagtg?aagaataata?aagaaatact?tataaaacag?agagtggagt????5580atgagccaaa?gaaatgtatt?agatatctac?tgactggtga?gctagaaggg?tttacatgga????5640acaaactaat?tagaaaaaaa?tatatcgata?aaaataaaat?tgattttgtt?aataaaatta????5700cttatatgga?ggattttttg?ttcatattaa?gtgtgttact?acacaatcct?aaaattattt????5760ttcaggattg?tgcatattat?tattatcaaa?aaagcaatcc?taaatctcta?actagccatg????5820ctagtgtgga?taggctttct?gaaatgatta?aagccgtaag?tgaaatagaa?aagaagataa????5880ataaatataa?tttacaaaag?tatctgaata?acgagtttca?attgtttaag?ttaaaacaaa????5940aaatttggtt?tatatctata?agtaagttaa?atgttaatga?taatgtatgg?aatctctttc????6000ctgaaacaaa?tccatttata?tcaaaggtaa?atgtgctttt?ttattataaa?atagtgcttt????6060ttcttgatag?tattaaactg?cgtttttttt?caaataagat?catttatctc?attggattgg????6120tgcaggcttt?attacaagaa?aggaaagaga?agtgatacca?aaaataattc?attactgctg????6180gttcggtcgc?tctccattgt?cggaattaac?aaagcagtgc?attgcaagct?gggagaaata????6240ttgtcctgaa?tataaaatca?taagatggga?tgaaaataat?gtcgatttga?attcttgttc????6300ttttgttaga?caagcttata?aagaaaaaaa?atgggcgttc?gtttctgatt?atgtaaggct????6360taaagtggtt?aatgaatatg?gcggtatcta?tcttgataca?gatgttgaat?taataaagcc????6420attagatgat?ttgttaatat?atcctgcata?tataggcttt?gaaattaaca?aggaatggta????6480tgtaaattct?ggtttagggt?tcggttctgt?taataataat?ccggttttag?aatcattaat????6540tatggagtat?gaaaatatta?attttgtaaa?tgaggatggg?actctaaata?taacgccgtg????6600ccccataaga?gaaacaaaag?cattaacaaa?aataggttta?attcctgatg?gtcaatgtca????6660atcttttgat?aatatagtga?ttttttctgc?agattatttt?tgtcctgtat?ctataacagg????6720tgagagaaat?ttttctgata?aaacatattc?aatacatcat?tacgatgcta?gttggttttc????6780tgagcaaaag?agaaaaggcc?tgcaacgtaa?aaagagattt?ataaaactat?tcggcaacct????6840aataggcaca?tatattaata?aaccttttat?ttttgtagat?gaatgtcgcg?agttcggatt????6900agttaaagca?atcaagaata?tgcgttccaa?ttttccttaa?tttttagtgt?taaggaataa????6960gtttcgttaa?aatactttat?tcatttaatt?aataaaggtt?taatggtgat?tagatgtact????7020ttcttttgtt?cttctcaata?ttttattttg?tatacgtaat?cttaatagga?acattatgtc????7080ataactataa?agcccttttg?attttttcac?taatccctct?gacaatagtg?tcaggtataa????7140gatataatgt?cggctttgat?tttatgtcat?atgttgatta?tttcaatcaa?ctcaaatatt????7200caaatgatat?atatctagat?tctaccttta?agtatataag?ctactttacc?tattatatag????7260gaggtaacga?acagattttg?tttctgatat?atgcggtttt?ttattcggtt?gcactttatt????7320ttttaataaa?actggtattg?gaaaattata?taacggaaat?aaataatttt?tatactattg????7380gacttgtgtt?atcattttat?tcgttttatt?ttttattatc?atttaatcaa?attagggctg????7440tattatcagc?tctaatacta?tgttatgggc?tattaaaaaa?taaaatagat?ttttctttta????7500taataacaat?tacattaagt?attctttttc?actctgcagc?aatgttcatt?ttacctcttt????7560attttgtcct?aagaaaaata?aatgtaacag?ttctgttggt?gatattccct?tttttggttg????7620ttgcttcatc?ctttaatatt?ttttcagata?ttgtgagatt?cattctgaca?ttgctaaata????7680gtcgtttttt?aacatatttt?aactctgaat?attttgttcc?aaggacggga?atggaaaaaa????7740tgtatagtat?gatctcaatg?attattgttt?tgggaatggt?tgtttgtttg?tctaaattat????7800tacctaaaaa?gtttgattta?atgataaagt?ttgttattct?ttttgttcta?cttagagcga????7860tgtcaattga?tatattgatt?tttgctaggt?ttagtgattt?tcttaaacca?atggctataa????7920ttcttatatt?taccgctatt?tatttttctt?cgaataaagt?gcgacctaga?attatccttc????7980ctttttattt?aattatgaca?ttgtttatct?gtatttttaa?tataataatc?ggatcgaata????8040taactaagga?gaactattat?acttatggtt?ataacatttg?cttttttggc?aataaatgta????8100tagataaatt?ttattagtaa?ttgtcgtttg?atacttctgg?tatttatgga?aaaatatatg??????8160aataactttt?cttgtaaaga?acccccttca?tttattgaaa?ataaaattat?attatcaatt??????8220attcttcctg?tatataacgt?atcagagtat?ttaattgagt?gcctaaacag?tttgctgtta??????8280gacgttcatg?aacgttatct?taatagatgt?gaagtaattg?tggttgatga?tggttcaact??????8340gataattcat?ttgaattaat?gagagagtat?tctttgaaat?atcctgaatc?tattaagatt??????8400tattcgaaat?tcaatggtgg?tctctcagat?gcaagaaact?atggattatt?aaaatcttca??????8460ggtaaatata?tttcttttgt?tgattcagat?gatgttgtta?atagaggttt?tataaaggaa??????8520atcatcaatt?tcattgatgc?ctatgatttt?gatatattgt?catttgactt?catgaagttt??????8580tttaataata?atggtgttct?tattctttca?caaatagatg?aattctccaa?aatggcagaa??????8640agagtagatt?cggagttcta?caagtccaag?cctgtttttg?cttggaataa?agtttatcga??????8700cgttctcttt?tcgacaatga?agtatttcct?aagggatggt?attatgaaga?tgttgcatta??????8760attccgttat?tattagatag?agcaaaagtt?ttataccata?ttaatagcgt?ttgttatttt??????8820tacagacaga?gacaaggagc?aatcactttt?ttttatgaca?ataaatatct?tgatatttta??????8880aaaggagtgt?cttttttata?tgacaggtca?caatcttcat?tcattaaaac?gataattata??????8940aatcagtttt?ttactttaac?cttactatct?ttaagattgc?cgactgataa?ttatttcatg??????9000aatatgaggg?ggattattga?tacctattgt?gaaaaatttg?acttggattc?ctttgagcct??????9060aaactaatat?taaaacacat?tcctttttta?ttgcttaaaa?aattgaagtc?gctgtgtgtt??????9120tatccactgt?atttatttaa?acctgcagtt?tttttacata?aacaaattaa?acattttaga??????9180ggaaaactaa?agtgaaatat?gattatataa?ttataggtag?tggtcttttt?gggagtgtgt??????9240ttgcacatga?attagggaaa?atgaaaaaaa?aggtcaaggt?tattgagcgt?cgtgatcatg??????9300ttggtggtaa?tgtatattgt?gagaatattg?aaggaattaa?tgttcataaa?tatggcgcac??????9360atatatttca?tactaacgat?aaagaaatat?gggattatgt?aaatcaattt?gtagagttta??????9420atagatatac?aaattcacca?ttagcttttt?caaaaggtaa?aatttataat?ttacctttta??????9480acatgaatac?ctttaatcaa?ctctgggggg?ctattactcc?tcaacatgcg?aaagaaatta??????9540ttaaaaagca?aagcggtgaa?ataagtggaa?ataaaccaag?gaatcttgaa?gagcaagcta??????9600tttctttggt?gggacgagat?atttataatt?gtttgattaa?ggagtacact?gagaaacaat??????9660ggggacgacc?ttgcactgaa?ttgccatcat?ttattataaa?acgactccct?gttaggttta??????9720catacgataa?taactatttt?aatgaccagt?atcaaggaat?accagtcggt?ggttacaata??????9780aattaatcga?tgcattatta?aatggtattg?aagttcaaac?atgcactgac?tattttgaag??????9840agaagaaata?ttgggattct?attgcaaaga?aaattattta?tacagggcct?attgataagt??????9900tttattgcta?taaatttgga?gctttggatt?atcgatcatt?aaaatttgag?catgaattac??????9960tgaatgtcga?taattttcaa?gggaatgctg?ttgtaaatta?tatagacaaa?aaattcccat?????10020tcactcgtat?aatagaacat?aaattttttg?aatttggtga?acagcataat?actctcatta?????10080ctcgagagta?cccaaaagaa?ttcgaagaag?gcgatgagta?ttactatcct?gtcaatgatg?????10140aaaaaaatat?gcaaatctat?aaaatgtatt?ataatctttc?taaacaggaa?agtaatgtga?????10200tttttggtgg?acgtcttgcg?gaatataaat?attatgacat?gcatcaagtt?atcaaatcag?????10260ctttaaatgc?attccgaaaa?gaggtacgtt?taggtgaata?aagaagtaat?cgcagttatt?????10320gtaacctata?atagaaaagc?attattgctg?aaagttatag?atgctgttat?taaccaaagt?????10380tatccactaa?aaaaaatatt?gataatagat?aataatagta?ctgatggtac?ggagatgttt?????10440atttcaaata?ggctctctga?cgtggtgaag?tataaaaata?cgggggataa?tttagggggg?????10500gctggcgggt?tttatagagg?attcatagag?gcggaaaaat?atggctatga?ttatctttgg?????10560ttaatggacg?atgattgtat?gcctacagtg?aattgtcttg?aaatattaat?atcaaataac?????10620cctgatggta?ttgtccaacc?tgttagatat?aacttggatg?aaacttgtgc?agagctttct?????10680ccattaactt?atgatttatc?taacccattt?aagttaaacc?ctaaaggtac?accattaaaa?????10740aattacttaa?atacaattgg?taataaatct?gcgaaggtag?atattgaagc?tattccattt?????10800gaaggtccat?tgatttcacg?aaaggttgtt?gaaaaaatag?gctatccaga?gcctaagttt?????10860tttatttttt?gtgatgacat?tgaatacgct?attaaagcta?aaagaaaggg?gattcccatc?????10920cagtgcaatc?ttaaagccaa?ggcataccgc?cttttggtca?acaatcaagg?taatgatctt?????10980ctttcctgga?aaggatattt?tatgcttcga?aatcttttct?acctgcataa?aacgtatggg?????11040accaatttcc?ttgttagaca?aaaacctatc?gtattgggat?taggatatgc?attgtcgtgt?????11100gtattgaaat?gcaatttttc?tcaacttccc?gttatatggc?gtgctttttg?ggattcatct?????11160acgttaagaa?ataccgaaaa?attcaggccc?gaaatgagaa?gtaaataatt?tattattatg?????11220tttttattaa?ttaatgtcga?tttttactaa?aattcttaac?acgatattta?tccctgacag?????11280gagtaaacaa?tgtcaaagca?acagatcggc?gtcgtcggta?tggcagtgat?ggggcgcaac?????11340cttgcgctca?acatcgaaag?ccgtggttat?accgtctcta?ttttcaaccg?ttcccgtgaa?????11400aaaacggaag?aagtgattgc?cgaaaatcca?ggcaaaaaac?tggttcctta?ctatacggtg?????11460aaagagtttg?ttgaatctct?ggaaacgcct?cgtcgcatcc?tgttaatggt?gaaagcaggt?????11520gcaggcacgg?atgctgctat?tgattctctc?aagccatacc?tcgataaagg?tgacatcatc?????11580attgatggtg?gtaacacctt?cttccaggac?accattcgtc?gtaaccgtga?gctttctgca?????11640gaaggcttta?acttcattgg?taccggtgtt?tccggcggtg?aagagggggc?gctgaaaggg?????11700

ccgtccatca?tgcctggtgg?ccagaaagaa?gcctatgaac?tggttgcacc?gatcctgacc????11760

aaaatcgccg?cagtagctga?agacggggag?ccatgcgtta?cctatattgg?tgccgatggt????11820

gcaggtcact?atgtgaagat?ggttcacaac?ggtattgaat?acggtgatat?gcaactgatt????11880

gctgaagcct?attctctgct?taaaggtggc?ctgaacctca?ccaacgaaga?actggcgcag????11940

acctttaccg?agtggaataa?cggtgaactg?agcagctacc?tgatcgacat?cactaaagac????12000

atcttcacta?aaaaagatga?agacggtaac?tacctggttg?atgtgattct?ggatgaagca????12060

gcaaacaaag?gtaccggtaa?atggactagc?cagagcgcgc?tggatctcgg?cgaaccgctg????12120

tcgctgatta?ccgagtctgt?gtttgcacgt?tatatctctt?ctctgaaaga?tcagcgcgtt????12180

gccgcgtcta?aagttctcac?tggcccgaaa?gcacagccag?ctggcgaaaa?ggctgagttc????12240

atcgaaaaag?ttcgtcgtgc?gctgtatctg?ggcaaaatcg?tttcttacgc?tcagggcttc????12300

tctcagctac?gcgccgcgtc?tgaagagtac?aactgggatc?tgaactacgg?cgaaatcgcg????12360

aagattttcc?gtgctggttg?catcatccgt?gcgcagttcc?tgcagaaaat?caccgatgcc????12420

tatgccgaaa?atccgcagat?cgctaacctg?ctgctggctc?cgtacttcaa?gcaaattgcc????12480

gatgactacc?agcaggcgct?gcgcgatgtc?gtcgcttacg?cagtacagaa?cggtatcccg????12540

gttccgacct?tcgccgctgc?ggttgcctat?tatgacagct?accgttctgc?tgttctgcct????12600

Glycosyltransferase gene in the O antigen gene of gcgaacctaa tccaggccca gcgcgacta 12629 table 1 shigella dysenteriaes 3 types bunch and oligosaccharide unit treatment gene and wherein

Primer and PCR data

Gene	Function	The base position of gene	Forward primer	Reverse primer	The length of PCR product	Produce the group number of correct big or small electrophoresis band	The annealing temperature of PCR (℃)
								????wzx	The transhipment enzyme	????1111-2625	????#55(1221-1240)	????#56(2148-2167)	????946bp	????0 **	????50
			????#81(1220-1238)	????#82(1884-1903)	????684bp	????0	????60
											????#100(1835-1853)	????#201(2325-2345)	????511bp	????0	????58
????orf4	Glycosyltransferase	????5151-6155	????#71(5391-5409)	????#72(5824-5843)	????750bp	????0 *	????56
											????#202(5377-5396)	????#203(6107-6125)	????749bp	????0	????56
			????#204(5575-5594)	????#205(6114-6132)	????558bp	????0	????60
								????orf5	Glycosyltransferase	????6152-6940	????#73(6182-6200)	????#74(6874-6895)	????714bp	????0	????60
			????#206(6168-6186)	????#207(6824-6842)	????675bp	????0	????58
											????#208(6172-6190)	????#209(6584-6602)	????431bp	????0	????60
????wzy	Polysaccharase	????7014-8117	????#57(7969-7990)	????#58(7256-7276)	????694bp	????0	????58
											????#217(7257-7277)	????#218(7768-7786)	????530bp	????0	????60
			????#219(7410-7430)	????#220(7955-7973)	????544bp	????0	????53
								????orf7	Glycosyltransferase	????8146-9195	????#75(8410-8428)	????#76(9109-9127)	????718bp	????0	????60
			????#210(8405-8423)	????#211(9242-9260)	????856bp	????0	????56
											????#sg1(8449-8467)	????#212(9040-9059)	????980bp	????0 *	????60
????orf9	Glycosyltransferase	????10435-11208	????#77(10450-10468)	????#78(11123-11141)	????692bp	????0	????62
											????#213(10451-10469)	????#214(10803-10821)	????370bp	????0 **	????58
			????#215(10565-10583)	????#216(11124-11142)	????578bp	????0	????60

^*In one group, produce the band of a wrong size; ^*The bacterium source 1 wild-type e. coli O1 that in one group, contains in the band table 2 166 strain intestinal bacteria of two wrong sizes of generation and 43 strain Shigellaes and their this group of source group number, O2, O3, O4, O10, O16, O18, O39 IMVSa 2 wild-type e. coli O40, O41, O48, O49, O71, O73, O88, O100 IMVS 3 wild-type e. coli O102, O109, O1 19, O120, O121, O125, O126, O137 IMVS 4 wild-type e. coli O138, O139, O149, O7, O5, O6, O11, O12 IMVS 5 wild-type e. coli O13, O14, O15, O17, O19ab, O20, O21, O22 IMVS 6 wild-type e. coli O23, O24, O25, O26, O27, O28, O29, O30 IMVS 7 wild-type e. coli O32, O33, O34, O35, O36, O37, O38, O42 IMVS 8 wild-type e. coli O43, O44, O45, O46, O50, O51, O52, O53 IMVS 9 wild-type e. coli O54, O55, O56, O57, O58, O59, O60, O61 IMVS10 wild-type e. coli O62, O63, O64, O65, O66, O68, O69, O70 IMVS11 wild-type e. coli O74, O75, O76, O77, O78, O79, O80, O81 IMVS12 wild-type e. coli O82, O83, O84, O85, O86, O87, O89, O90 IMVS13 wild-type e. coli O91, O92, O95, O96, O97, O98, O99, O101 IMVS14 wild-type e. coli O112, O162, O113, O114, O115, O116, O117, O118 IMVS15 wild-type e. coli O123, O165, O166, O167, O168, O169, O170, O171 See b16 wild-type e. coli O172, O173, O127, O128, O129, O130, O131, O132, See c17 wild-type e. coli O133, O134, O135, O136, O140, O141, O142, O143 IMVS18 wild-type e. coli O144, O145, O146, O147, O148, O150, O151, O152 IMVS19 wild-type e. coli O153,0154, O155, O156, O157, O158, O159, O164 IMVS20 wild-type e. coli O160, O161, O163, O8, O9, O124, O111 IMVS21 wild-type e. coli O103, O104, O105, O106, O107, O108, O110 IMVS22 Shigella bogdii serotypes B 4, B5, B6, B8, B9, B11, B12, B14 See d23 Shigella bogdii serotypes B 1, B3, B7, B8, B10, B13, B15, B16, B17, B18 See d24 shigella dysenteriae serotype D1, D2, D3, D4, D5, D6, D7, D8 See d25 shigella dysenteriae serum D9, D10, D11, D12, D13 See d26 shigella flexneri F6a, F1a, F1b, F2a, F2b, F3, F4a, F4b, F5 (v:7) F5 (v:4) See d27 bacillus ceylonensis A D5, DR See d a. Institude of Medical and VeterinaryScience, Ane1aide, Australia b. O123 from IMVS; The rest from Statens Serum Institut, Copenhagen, Denmark c. 172 and 173 from Statens Serum Institut, Copenhagen, Denmark, epidemiological study institute of the rest from IMVS d. China Preventive Medicial Science Institute

Table 3 is structural tables of the O-antigen gene bunch of shigella dysenteriae 3 types

43O-ATTGTGGCTG CAGGGATCAA AGAAATCCTC CTGGTAACTC ACGCGTCCAA GAACGCGGTC 60GAAAACCACT TCGACACCTC TTATGAATTA GAATCTCTCC TTGAGCAGCG CGTGAAGCGT 120CAACTGTTGG CGGAAGTACA ATCTATCTGT CCGCCGGGCG TGACCATTAT GAACGTGCGT 180CAGGGTGAAC CTTTAGGTTT AGGCCACTCC ATCTTATGTG CGCGACCTGC CATTGGTGAC 240AACCCATTTA TCGTGGTACT GCCAGACGTA GTGATCGACG ACGCCAGCGC CGACCCGCTG 300CGCTACAACC TTGCTGCCAT GATTGCGCGC TTCAACGAAA CGGGCCGTAG CCAGGTGCTG 360GCAAAACGTA TGCCGGGTGA CCTCTCTGAA TACTCCGTCA TCCAGACCAA AGAACCGCTG 420GACCGTGAAG GTAAAGTCAG CCGCATTGTT GAATTTATCG AAAAACCGGA TCAGCCGCAG 480ACGCTGGACT CAGACATCAT GGCCGTAGGT CGCTATGTGC TTTCTGCCGA TATTTGGCCG 540GAACTAGAAC GCACTCAGCC TGGTGCATGG GGGCGTATTC AGCTGACTGA TGCTATTGCC 600GAACTGGCGA AAAAACAGTC CGTTGATGCC ATGCTGATGA CTGGAGACAG CTACGACTGC 660GGTAAAAAAA TGGGCTATAT GCAGGCGTTT GTGAAGTACG GACTGCGTAA CCTGAAAGAA 720GGGGCGAAGT TCCGCAAAGG TATTGAGAGG TTGTTGAATG ATTAAAAACA TGAACAAATA 780TAACGGTAAA TAAGAGAAAT TGTAACGGCA GTGAGGATTT GCGGCGAAGC TCAAACGTGG 840CGAATATCCC TGTCGTTATT TATTAATAAA TCATCGTACT AACAATGGAA TAAAAAAGTG 900CCTTGTTTTA AGTTTTACAG GATATTCCTT GTTTCTGGAG GAGATTGATA AGGCAATCAG 960CATCTGAATT TATCAGAACT TGTGTGTCTT GCTGATTACA TTGTTATATT CATGCAGTGT 1020ACTGGTAGCT GTGGAGCCAG GGGCGGTAGC GTGCATTAAT ATTCACAATA TATTCTTTTA 1080

orf1AATGTAAGCA GAATTAATCG AACCTAAATA ATGTCACGTA ACAAAATAAT CATAAAGAAT 1140ACTATGTTTT TGGCAACAAG AACTTTAGTT TCTTTAAGTG TTTCATTTTA CACAACAAGA 1200ATAGTTATAC AACAGTTGGG GGCGGCAGAC TATGGCTTAT TTAATATCAT TTATGGAGTT 1260GTAACATTTT TTACATTCGT AGTCACCGCG ATGAATGATT CTGTACAGCG TTATATTGCA 1320ATAGGTGTTG GTTCACAAAA AATATCGGTT ATTAGAGATG CTGTAAAAAA TAGTATGTTT 1380ATATTTGTTA TATCTGCTTT CATATTGGCA GTATGTCTTC TTTTGGCAAG GGGGGTGATC 1440ATACATAATA TTTTAAATAT ACCTAAAGAT TCTATTGAAA ATGCAAGTGT GTTATATTTG 1500GTTGCAGTAT TTTCAATAAC CATACTTATA ATTCAGACGC CACTTAATGC GATGGTGTTA 1560GCATACGAGA AAATGTCATT TTACGCATAC ATGATGATAT TTGAAATGGT AGCTAAAATG 1620AGTATGGCTT TGTTACTAAC TTTACTGGAA AAAGATAAGG TCATTGTATA TTCAATACTT 1680TTAGGTTCTA TTTCTTTTTT TAACTTGTTG GTTTATTTAT GCTACTGCTT GATATGTTTT 1740AAAAAATCAA TGTTTGGTGG GAGAATAAAA TTTAGAGTGC TGAAAGAAAT TTCAACTTTT 1800TCATTTTGGA ATATATTTGG TAATTTCTCT TACATGTGCA GAGTTCAGGG AGTGAACATT 1860GTAATTAATA TTTTTTACGC TATAGCCGTG AATGCAGCAT ATGCGATTTC GATTACTGTT 1920TTAAATGCAA TTAATACACT TACGCAATCA TTAATTACAG CATTAAGGCC TCAAATATTT 1980AAATCATATG GCGAATGTGA TTTAAAAAGA TATAATCATC TAGTTCTCTT TGGTTCAAAA 2040TATACATTTT CAATATTGTT TCTGTTAAGT AGCCCTGTAA TTCTATGTGC GGATGAGTTG 2100TTAAAGATAT GGCTTGATAT TGTACCAGAT TATACAGTTG AATTTGTGAG GTTGGTCATT 2160GTTGTGGCTT TTATTGATAG TTTTTCATAT AGCATGATTG CTGGTATTCA GGCTACGGGT 2220AGGATTAAAA CATATCAACT AGTTGTTAGT TTAATTGTCC TAATTAATTT GCCGTTAACC 2280TTTATTTTGT TTAAGGCAGG AAATAATGTT TTAAGTATGT TTTATCCATT TATTGTTACT 2340GCCATTATCA ATCAAGGTTT AAGGTTGTAT TTTATCTATA TCAATGCAGG TTTTGATTAT 2400AAGAAGTATT TCACTGTTGT GATCTATCCT TGCTTGTTAG CTGTATGCTT ATCGTTAGTG 2460ACAGATATTT CAATTAAAAA AATGCTTCCA TTTAACAGTA TTATTGATGT TTTAATTGTG 2520TGTATTTTTA TTTTTTCATT CAATACAATA ATTTTCTATT GGGTTGTAGT TTCTAAAAAG 2580

orf2 orf1GAAAAAAAAT GGTTATTAGA AACCCTGAAA AGAAAAGTGA AATAACAATA AAAGATGTTA 2640TAGACAATGA ACTCTGTACT GGTTGTGGAG TTTGTATTTC GGAAGATAGT AGCAAAACCT 2700CCTTTATGAA ATGGAATTCT GAAGGTTTTT ATGAGCCATG TTTCTCTCCT GTATCAACAT 2760TATTCAATAT GCAGCGTGTT TGTCCCTTTA ACTTAAGTAG GGATACATTA GTGAATGAGG 2820ATGAACTTGC GCATGAGTTT TTTGATGGTA AGGGATATTT AGATTCTGAG GTTGGGTTTT 2880ATAAAAAAAT ATATGTGGGT TATTCTAAAC ATTTTAGAGA GACTTCATCC TCCGGTGGTA 2940TAGCTACATA TGTATTTGAA CAACTTCTTA GACGTAAGTA TGTTGATGCT CTGTTTATAG 3000TTAGGGAGCT TGGTGGTTCA TATGGATATC AAGTCTTCGA TAATCCAGAA CATATTAAGG 3060ATATGTCTAA AACAAGATAT TATCCTGTAA CGCTAGAAAA GTTATTTGAT AATATTTATA 3120AGCTTAATGG TAGAGTGGCC GTTTCAGGAG TGGCATGTTT TATTAAAGCC ATTAGGCTCA 3180AACAACATTA CCATCCAGAA CTGAAAGAGA AAATACCATT CCTTGTTGGT ATAATTTGTG 3240GAGGACTCAA AAGTCGATAT TACACTGATT ATTTATCACA GTCTGCTGGT TGTGTCAGTG 3300AGTACCAAAA TGCTGAGTAT CGAGTGAAAA AGAAAGATAG TCATGCTCTT GACTATCGTT 3360TCACTTGTGT TGAGAAATCA AATAATATAA TTCATTCTGT TGATATGCAA AGAATGGGGG 3420ATATGTGGGG TTCAGGGCTA TTTAAGGCAA ACGCATGTGA CTATTGTGAT GATGTTACAA 3480CCGAACTCGC AGACATATCA TTGGGTGATG CCTGGATTTC GCCATACAAT ATGGATGGAG 3540CTGGTAATAA TGTAGTTGTA TGTCGCTCCA CCACAGCTCA TGAAATTATT TTATCTGGGA 3600TTGAGAAAAA AGACTTAGAA TTAACGGAAC TCGAATTAGA GCAGTTGAAA TTGTCACAAC 3660AAGGAAGTTT TAACCACAGG CATAAAGGAT TATTATATAG AATAAAAAAT GCAGAGAAAA 3720ATAATCGCTT AGTGCCTGTT AAGAGGAAAA GATTCTTACG CTCTATATCT TTTTTATTAA 3780AGTTAATTCA AAAACAACGA TCAGTTACAA GGAGAAAAAG CATAGAGATA TGGATGGAAA 3840CTCAAAATTC AGCAACTTTC GATAAGAAAA TGAAGGGATA TTTATTCACG TTGCGTTGGT 3900TAACAGTGGT CAATAGGAAA TTAAGTCGGA TGTTCAAGAT TGTTTCTCTA AATAGAATGG 3960 Orf3orf2GTAAAAAATG AAAGTTGCAA TATTAACTCA ACCATTACAT ACGAATTATG GTGGCACATT 4020ACAAGCCTAT GCATTACAAA AAGTCTTGAT TAATTTGGGA CATGAACCAG AGACTATTAA 4080TTATAGGTCG AAAATTAAGA GGCCACTATT TATCCGTGTT GTGTTGTCAA AAATTAAAAG 4140GATAGTATTA TGCCGAAAAA TCACATTTGA TTTTACAACA CAAGATAGAA TTAATATTAG 4200GACGCATCAT CAATCTTTCA TTGATACAAG ATTAAACTAT TCAGAGGAGA TTAACGGTAC 4260TGAGGGGTTG AGAGATTATA TTTTAAAAAA TAATTACGGT GCTGTAATAA TAGGTAGTGA 4320TCAGACATGG AGACCAATAT ATTCGCCTAG AATAGATTCA TTTTTTCTCG ATTTTTTAAG 4380TGATGTAAAC GATATAAAAA AAATGGCATA TGCAGCATCT TTTGGTACTG ATAAATGGGA 4440GTTCACGGAG TCACAAACTA ATTTATTTAA ATTGTTACTT TCAAAATTTG ACTATGTTTC 4500AGTTAGAGAG TCCTCAGGAG TCAAATTATG TAGTGAAAAA TTTGGTTTGA AAGCAGAGTT 4560GGTTTTAGAT CCGACATTGC TATTGACATT CGAAGATTAT ACTCATTTAT TAGACAATGA 4620CTATATTAAT CTTCATAAGG GAAAGGTGTT TAGTTATGTA CTTGATGAGA ATTACGACAA 4680AAAAAACTCT ATAGACACAG TAGCAAGTAC TCTCCGCACA ACGTCATTTT ATACTTACCC 4740TAAAAAAATC ACTAAAGATG AATATGTGAT TAGAAATTAT TCCGAGTATG AATATCCGCC 4800AATAGAATAT TGGATTTCTT CGTTTAAGGC CGCTGAATTT ATTGTAACAG ATTCATTTCA 4860TGGAACAGTA TTTTCTATTA TTTTTAACAA ACCCTTCATC GCCATTGCAA ATGAAGAGAG 4920AGGAAAGGCT AGATTTACGT CACTATTGGA AATGTTTGGT CTTGAAAGAC GACTTGTGAG 4980TAATTTAGAT GATGTTGATT TAAAGCTTGT AAATGAAAAA ATTAACTATG GCCCCATAAA 5040TGAAAAAATT GCTTATTTTA GAGAAGAGTG CCTTAAAAAG TTGCAGTTAA TGCTAATAAA 5100Orf3 orf4ATAATCAGAT TATGTATGTC TCTCTACAAT TAAAATATGA ATGTTGATCG ATGAAGAATT 5160GTAAAGTATC TGTAATTATT CCGGTCTATA ATGCGGAAAA ATATATACAA CGTTGTATTT 5220TAAGCTTATT AAAGCAGACA TTAGATGATG TTGAAATTAT CATCATTGAT GATGGTTCTA 5280CTGATAATTC ACTTTCTATA ATAAAAGAAA CTGTTGCATT GCATACTGCT AGTAGAGCTC 5340GCCAAAAGAG AATCAATATA ATCTCTCGAG AAAACAAAGG AGTTGCCTAT ACACGAAGTC 5400AGGGATTGAG ACTTTCGCAG GGGGAATTTA TCATTCATTT TGATAGTGAT GATTGGGCAA 5460AATCCAATAT GCTGGAGGAA ATGTATAAGA CAATTGTTTC CAATAATGCA GACATGGTTA 5520TATGCGATTA TTTTTTAGTG AAGAATAATA AAGAAATACT TATAAAACAG AGAGTGGAGT 5580ATGAGCCAAA GAAATGTATT AGATATCTAC TGACTGGTGA GCTAGAAGGG TTTACATGGA 5640ACAAACTAAT TAGAAAAAAA TATATCGATA AAAATAAAAT TGATTTTGTT AATAAAATTA 5700CTTATATGGA GGATTTTTTG TTCATATTAA GTGTGTTACT ACACAATCCT AAAATTATTT 5760TTCAGGATTG TGCATATTAT TATTATCAAA AAAGCAATCC TAAATCTCTA ACTAGCCATG 5820CTAGTGTGGA TAGGCTTTCT GAAATGATTA AAGCCGTAAG TGAAATAGAA AAGAAGATAA 5880ATAAATATAA TTTACAAAAG TATCTGAATA ACGAGTTTCA ATTGTTTAAG TTAAAACAAA 5940AAATTTGGTT TATATCTATA AGTAAGTTAA ATGTTAATGA TAATGTATGG AATCTCTTTC 6000CTGAAACAAA TCCATTTATA TCAAAGGTAA ATGTGCTTTT TTATTATAAA ATAGTGCTTT 6060TTCTTGATAG TATTAAACTG CGTTTTTTTT CAAATAAGAT CATTTATCTC ATTGGATTGG 6120

orf5 orf4TGCAGGCTTT ATTACAAGAA AGGAAAGAGA AGTGATACCA AAAATAATTC ATTACTGCTG 6180GTTCGGTCGC TCTCCATTGT CGGAATTAAC AAAGCAGTGC ATTGCAAGCT GGGAGAAATA 6240TTGTCCTGAA TATAAAATCA TAAGATGGGA TGAAAATAAT GTCGATTTGA ATTCTTGTTC 6300TTTTGTTAGA CAAGCTTATA AAGAAAAAAA ATGGGCGTTC GTTTCTGATT ATGTAAGGCT 6360TAAAGTGGTT AATGAATATG GCGGTATCTA TCTTGATACA GATGTTGAAT TAATAAAGCC 6420ATTAGATGAT TTGTTAATAT ATCCTGCATA TATAGGCTTT GAAATTAACA AGGAATGGTA 6480TGTAAATTCT GGTTTAGGGT TCGGTTCTGT TAATAATAAT CCGGTTTTAG AATCATTAAT 6540TATGGAGTAT GAAAATATTA ATTTTGTAAA TGAGGATGGG ACTCTAAATA TAACGCCGTG 6600CCCCATAAGA GAAACAAAAG CATTAACAAA AATAGGTTTA ATTCCTGATG GTCAATGTCA 6660ATCTTTTGAT AATATAGTGA TTTTTTCTGC AGATTATTTT TGTCCTGTAT CTATAACAGG 6720TGAGAGAAAT TTTTCTGATA AAACATATTC AATACATCAT TACGATGCTA GTTGGTTTTC 6780TGAGCAAAAG AGAAAAGGCC TGCAACGTAA AGAGATTT ATAAAACTAT TCGGCAACCT 6840AATAGGCACA TATATTAATA AACCTTTTAT TTTTGTAGAT GAATGTCGCG AGTTCGGATT 6900

The termination AGTTAAAGCA ATCAAGAATA TGCGTTCCAA TTTTCCTTAA TTTTTAGTGT TAAGGAATAA 6960 of orf5

orf6GTTTCGTTAA AATACTTTAT TCATTTAATT AATAAAGGTT TAATGGTGAT TAGATGTACT 7020TTCTTTTGTT CTTCTCAATA TTTTATTTTG TATACGTAAT CTTAATAGGA ACATTATGTC 7080ATAACTATAA AGCCCTTTTG ATTTTTTCAC TAATCCCTCT GACAATAGTG TCAGGTATAA 7140GATATAATGT CGGCTTTGAT TTTATGTCAT ATGTTGATTA TTTCAATCAA CTCAAATATT 7200CAAATGATAT ATATCTAGAT TCTACCTTTA AGTATATAAG CTACTTTACC TATTATATAG 7260GAGGTAACGA ACAGATTTTG TTTCTGATAT ATGCGGTTTT TTATTCGGTT GCACTTTATT 7320TTTTAATAAA ACTGGTATTG GAAAATTATA TAACGGAAAT AAATAATTTT TATACTATTG 7380GACTTGTGTT ATCATTTTAT TCGTTTTATT TTTTATTATC ATTTAATCAA ATTAGGGCTG 7440TATTATCAGC TCTAATACTA TGTTATGGGC TATTAAAAAA TAAAATAGAT TTTTCTTTTA 7500TAATAACAAT TACATTAAGT ATTCTTTTTC ACTCTGCAGC AATGTTCATT TTACCTCTTT 7560ATTTTGTCCT AAGAAAAATA AATGTAACAG TTCTGTTGGT GATATTCCCT TTTTTGGTTG 7620TTGCTTCATC CTTTAATATT TTTTCAGATA TTGTGAGATT CATTCTGACA TTGCTAAATA 7680GTCGTTTTTT AACATATTTT AACTCTGAAT ATTTTGTTCC AAGGACGGGA ATGGAAAAAA 7740TGTATAGTAT GATCTCAATG ATTATTGTTT TGGGAATGGT TGTTTGTTTG TCTAAATTAT 7800TACCTAAAAA GTTTGATTTA ATGATAAAGT TTGTTATTCT TTTTGTTCTA CTTAGAGCGA 7860TGTCAATTGA TATATTGATT TTTGCTAGGT TTAGTGATTT TCTTAAACCA ATGGCTATAA 7920TTCTTATATT TACCGCTATT TATTTTTCTT CGAATAAAGT GCGACCTAGA ATTATCCTTC 7980CTTTTTATTT AATTATGACA TTGTTTATCT GTATTTTTAA TATAATAATC GGATCGAATA 8040TAACTAAGGA GAACTATTAT ACTTATGGTT ATAACATTTG CTTTTTTGGC AATAAATGTA 8100

orf6 orf7TAGATAAATT TTATTAGTAA TTGTCGTTTG ATACTTCTGG TATTTATGGA AAAATATATG 8160AATAACTTTT CTTGTAAAGA ACCCCCTTCA TTTATTGAAA ATAAAATTAT ATTATCAATT 8220ATTCTTCCTG TATATAACGT ATCAGAGTAT TTAATTGAGT GCCTAAACAG TTTGCTGTTA 8280GACGTTCATG AACGTTATCT TAATAGATGT GAAGTAATTG TGGTTGATGA TGGTTCAACT 8340GATAATTCAT TTGAATTAAT GAGAGAGTAT TCTTTGAAAT ATCCTGAATC TATTAAGATT 8400TATTCGAAAT TCAATGGTGG TCTCTCAGAT GCAAGAAACT ATGGATTATT AAAATCTTCA 8460GGTAAATATA TTTCTTTTGT TGATTCAGAT GATGTTGTTA ATAGAGGTTT TATAAAGGAA 8520ATCATCAATT TCATTGATGC CTATGATTTT GATATATTGT CATTTGACTT CATGAAGTTT 8580TTTAATAATA ATGGTGTTCT TATTCTTTCA CAAATAGATG AATTCTCCAA AATGGCAGAA 8640AGAGTAGATT CGGAGTTCTA CAAGTCCAAG CCTGTTTTTG CTTGGAATAA AGTTTATCGA 8700CGTTCTCTTT TCGACAATGA AGTATTTCCT AAGGGATGGT ATTATGAAGA TGTTGCATTA 8760ATTCCGTTAT TATTAGATAG AGCAAAAGTT TTATACCATA TTAATAGCGT TTGTTATTTT 8820TACAGACAGA GACAAGGAGC AATCACTTTT TTTTATGACA ATAAATATCT TGATATTTTA 8880AAAGGAGTGT CTTTTTTATA TGACAGGTCA CAATCTTCAT TCATTAAAAC GATAATTATA 8940AATCAGTTTT TTACTTTAAC CTTACTATCT TTAAGATTGC CGACTGATAA TTATTTCATG 9000AATATGAGGG GGATTATTGA TACCTATTGT GAAAAATTTG ACTTGGATTC CTTTGAGCCT 9060AAACTAATAT TAAAACACAT TCCTTTTTTA TTGCTTAAAA AATTGAAGTC GCTGTGTGTT 9120TATCCACTGT ATTTATTTAA ACCTGCAGTT TTTTTACATA AACAAATTAA ACATTTTAGA 9180

The termination GGAAAACTAA AGTGAAATAT GATTATATAA TTATAGGTAG TGGTCTTTTT GGGAGTGTGT 9240 of orf7

orf8TTGCACATGA ATTAGGGAAA ATGAAAAAAA AGGTCAAGGT TATTGAGCGT CGTGATCATG 9300TTGGTGGTAA TGTATATTGT GAGAATATTG AAGGAATTAA TGTTCATAAA TATGGCGCAC 9360ATATATTTCA TACTAACGAT AAAGAAATAT GGGATTATGT AAATCAATTT GTAGAGTTTA 9420ATAGATATAC AAATTCACCA TTAGCTTTTT CAAAAGGTAA AATTTATAAT TTACCTTTTA 9480ACATGAATAC CTTTAATCAA CTCTGGGGGG CTATTACTCC TCAACATGCG AAAGAAATTA 9540TTAAAAAGCA AAGCGGTGAA ATAAGTGGAA ATAAACCAAG GAATCTTGAA GAGCAAGCTA 9600TTTCTTTGGT GGGACGAGAT ATTTATAATT GTTTGATTAA GGAGTACACT GAGAAACAAT 9660GGGGACGACC TTGCACTGAA TTGCCATCAT TTATTATAAA ACGACTCCCT GTTAGGTTTA 9720CATACGATAA TAACTATTTT AATGACCAGT ATCAAGGAAT ACCAGTCGGT GGTTACAATA 9780AATTAATCGA TGCATTATTA AATGGTATTG AAGTTCAAAC ATGCACTGAC TATTTTGAAG 9840AGAAGAAATA TTGGGATTCT ATTGCAAAGA AAATTATTTA TACAGGGCCT ATTGATAAGT 9900TTTATTGCTA TAAATTTGGA GCTTTGGATT ATCGATCATT AAAATTTGAG CATGAATTAC 9960TGAATGTCGA TAATTTTCAA GGGAATGCTG TTGTAAATTA TATAGACAAA AAATTCCCAT 10020TCACTCGTAT AATAGAACAT AAATTTTTTG AATTTGGTGA ACAGCATAAT ACTCTCATTA 10080CTCGAGAGTA CCCAAAAGAA TTCGAAGAAG GCGATGAGTA TTACTATCCT GTCAATGATG 10140AAAAAAATAT GCAAATCTAT AAAATGTATT ATAATCTTTC TAAACAGGAA AGTAATGTGA 10200TTTTTGGTGG ACGTCTTGCG GAATATAAAT ATTATGACAT GCATCAAGTT ATCAAATCAG 10260

The termination CTTTAAATGC ATTCCGAAAA GAGGTACGTT TAGGTGAATA AAGAAGTAAT CGCAGTTATT 10320GTAACCTATA ATAGAAAAGC ATTATTGCTG AAAGTTATAG ATGCTGTTAT TAACCAAAGT 10380 of orf8

orf9TATCCACTAA AAAAAATATT GATAATAGAT AATAATAGTA CTGATGGTAC GGAGATGTTT 10440ATTTCAAATA GGCTCTCTGA CGTGGTGAAG TATAAAAATA CGGGGGATAA TTTAGGGGGG 10500GCTGGCGGGT TTTATAGAGG ATTCATAGAG GCGGAAAAAT ATGGCTATGA TTATCTTTGG 10560TTAATGGACG ATGATTGTAT GCCTACAGTG AATTGTCTTG AAATATTAAT ATCAAATAAC 10620CCTGATGGTA TTGTCCAACC TGTTAGATAT AACTTGGATG AAACTTGTGC AGAGCTTTCT 10680CCATTAACTT ATGATTTATC TAACCCATTT AAGTTAAACC CTAAAGGTAC ACCATTAAAA 10740AATTACTTAA ATACAATTGG TAATAAATCT GCGAAGGTAG ATATTGAAGC TATTCCATTT 10800GAAGGTCCAT TGATTTCACG AAAGGTTGTT GAAAAAATAG GCTATCCAGA GCCTAAGTTT 10860TTTATTTTTT GTGATGACAT TGAATACGCT ATTAAAGCTA AAAGAAAGGG GATTCCCATC 10920CAGTGCAATC TTAAAGCCAA GGCATACCGC CTTTTGGTCA ACAATCAAGG TAATGATCTT 10980CTTTCCTGGA AAGGATATTT TATGCTTCGA AATCTTTTCT ACCTGCATAA AACGTATGGG 11040ACCAATTTCC TTGTTAGACA AAAACCTATC GTATTGGGAT TAGGATATGC ATTGTCGTGT 11100GTATTGAAAT GCAATTTTTC TCAACTTCCC GTTATATGGC GTGCTTTTTG GGATTCATCT 11160

orf9ACGTTAAGAA ATACCGAAAA ATTCAGGCCC GAAATGAGAA GTAAATAATT TATTATTATG 11220TTTTTATTAA TTAATGTCGA TTTTTACTAA AATTCTTAAC ACGATATTTA TCCCTGACAG 11280GAGTAAACAA TGTCAAAGCA ACAGATCGGC GTCGTCGGTA TGGCAGTGAT GGGGCGCAAC 11340CTTGCGCTCA ACATCGAAAG CCGTGGTTAT ACCGTCTCTA TTTTCAACCG TTCCCGTGAA 11400AAAACGGAAG AAGTGATTGC CGAAAATCCA GGCAAAAAAC TGGTTCCTTA CTATACGGTG 11460AAAGAGTTTG TTGAATCTCT GGAAACGCCT CGTCGCATCC TGTTAATGGT GAAAGCAGGT 11520GCAGGCACGG ATGCTGCTAT TGATTCTCTC AAGCCATACC TCGATAAAGG TGACATCATC 11580ATTGATGGTG GTAACACCTT CTTCCAGGAC ACCATTCGTC GTAACCGTGA GCTTTCTGCA 11640GAAGGCTTTA ACTTCATTGG TACCGGTGTT TCCGGCGGTG AAGAGGGGGC GCTGAAAGGG 11700CCGTCCATCA TGCCTGGTGG CCAGAAAGAA GCCTATGAAC TGGTTGCACC GATCCTGACC 11760AAAATCGCCG CAGTAGCTGA AGACGGGGAG CCATGCGTTA CCTATATTGG TGCCGATGGT 11820GCAGGTCACT ATGTGAAGAT GGTTCACAAC GGTATTGAAT ACGGTGATAT GCAACTGATT 11880GCTGAAGCCT ATTCTCTGCT TAAAGGTGGC CTGAACCTCA CCAACGAAGA ACTGGCGCAG 11940ACCTTTACCG AGTGGAATAA CGGTGAACTG AGCAGCTACC TGATCGACAT CACTAAAGAC 12000ATCTTCACTA AAAAAGATGA AGACGGTAAC TACCTGGTTG ATGTGATTCT GGATGAAGCA 12060GCAAACAAAG GTACCGGTAA ATGGACTAGC CAGAGCGCGC TGGATCTCGG CGAACCGCTG 12120TCGCTGA1TA CCGAGTCTGT GTTTGCACGT TATATCTCTT CTCTGAAAGA TCAGCGCGTT 12180GCCGCGTCTA AAGTTCTCAC TGGCCCGAAA GCACAGCCAG CTGGCGAAAA GGCTGAGTTC 12240ATCGAAAAAG TTCGTCGTGC GCTGTATCTG GGCAAAATCG TTTCTTACGC TCAGGGCTTC 12300TCTCAGCTAC GCGCCGCGTC TGAAGAGTAC AACTGGGATC TGAACTACGG CGAAATCGCG 12360AAGATTTTCC GTGCTGGTTG CATCATCCGT GCGCAGTTCC TGCAGAAAAT CACCGATGCC 12420TATGCCGAAA ATCCGCAGAT CGCTAACCTG CTGCTGGCTC CGTACTTCAA GCAAATTGCC 12480GATGACTACC AGCAGGCGCT GCGCGATGTC GTCGCTTACG CAGTACAGAA CGGTATCCCG 12540GTTCCGACCT TCGCCGCTGC GGTTGCCTAT TATGACAGCT ACCGTTCTGC TGTTCTGCCT 12600GCGAACCTAA TCCAGGCCCA GCGCGACTA 12629

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 shigella dysenteriae 3 types, intestinal bacteria O124 and intestinal bacteria O164 is characterized in that it is the isolating Nucleotide shown in SEQ ID NO:1,12629 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 shigella dysenteriae 3 types, intestinal bacteria O124 and intestinal bacteria O164 of claim 1, it is characterized in that it is by 9 genomic constitutions, all between galF gene and gnd gene.

3, according to the Nucleotide of the described O-antigen-specific to shigella dysenteriae 3 types, intestinal bacteria O124 and intestinal bacteria O164 of claim 2, it is characterized in that, described gene comprises: about transhipment enzyme gene, comprise the wzx gene or with wzx the gene of identity function is arranged; Pol gene comprises the wzy gene or with wzy the gene of identity function is arranged; Glycosyltransferase gene comprises orf4, orf5, orf7, orf9 gene; Wherein said wzx gene is the Nucleotide of 1111 to 2625 bases among the SEQ ID NO:1; The orf4 gene is the Nucleotide of 5151 to 6155 bases among the SEQ ID NO:1; The orf5 gene is the Nucleotide of 6152 to 6940 bases among the SEQ ID NO:1; The wzy gene is the Nucleotide of 7014 to 8117 bases among the SEQ ID NO:1; The orf7 gene is the Nucleotide of 8146 to 9195 bases among the SEQ ID NO:1; The orf9 gene is the Nucleotide of 10435 to 11208 bases among the SEQ ID NO:1.

4, according to the Nucleotide of claim 1 or 2 described O-antigen-specifics to shigella dysenteriae 3 types, intestinal bacteria O124 and intestinal bacteria O164, it is characterized in that it comes from described wzx gene, wzy gene or glycosyltransferase gene is orf4, orf5, orf7, orf9 gene; Or the oligonucleotide in the sugared synthesis path gene; And their mixing or their reorganization.

5, according to the Nucleotide of the described O-antigen-specific to shigella dysenteriae 3 types, intestinal bacteria O124 and intestinal bacteria O164 of claim 4, it is characterized in that, the oligonucleotide of the described wzx of coming from gene is to being: the Nucleotide of 1221 to 1240 bases among the SEQ ID NO:1 and the Nucleotide of 2148 to 2167 bases, the Nucleotide of 1220 to 1238 bases among the SEQ ID NO:1 and the Nucleotide of 1884 to 1903 bases, the Nucleotide of 1835 to 1853 bases among the SEQ ID NO:1 and the Nucleotide of 2325 to 2345 bases; The oligonucleotide that comes from the orf4 gene is to being: the Nucleotide of 5391 to 5409 bases among the SEQ ID NO:1 and the Nucleotide of 5824 to 5843 bases, the Nucleotide of 5377 to 5396 bases among the SEQ ID NO:1 and the Nucleotide of 6107 to 6125 bases, the Nucleotide of 5575 to 5594 bases among the SEQ ID NO:1 and the Nucleotide of 6114 to 6132 bases; The oligonucleotide that comes from the orf5 gene is to being: the Nucleotide of 6182 to 6200 bases among the SEQ ID NO:1 and the Nucleotide of 6874 to 6895 bases, the Nucleotide of 6168 to 6186 bases among the SEQ ID NO:1 and the Nucleotide of 6824 to 6842 bases, the Nucleotide of 6172 to 6190 bases among the SEQ ID NO:1 and the Nucleotide of 6584 to 6602 bases; The oligonucleotide that comes from the wzy gene is to being: the Nucleotide of 7969 to 7990 bases among the SEQ ID NO:1 and the Nucleotide of 7256 to 7276 bases, the Nucleotide of 7257 to 7277 bases among the SEQ ID NO:1 and the Nucleotide of 7768 to 7786 bases, the Nucleotide of 7410 to 7430 bases among the SEQ ID NO:1 and the Nucleotide of 7955 to 7973 bases; The oligonucleotide that comes from the orf7 gene is to being: the Nucleotide of 8410 to 8428 bases among the SEQ IDNO:1 and the Nucleotide of 9109 to 9127 bases, the Nucleotide of 8405 to 8423 bases among the SEQ IDNO:1 and the Nucleotide of 9242 to 9260 bases, the Nucleotide of 8449 to 8467 bases among the SEQ IDNO:1 and the Nucleotide of 9040 to 9059 bases; The oligonucleotide that comes from the orf9 gene is to being: the Nucleotide of 10450 to 10468 bases among the SEQ ID NO:1 and the Nucleotide of 11123 to 11141 bases, the Nucleotide of 10451 to 10469 bases among the SEQ ID NO:1 and the Nucleotide of 10803 to 10821 bases, the Nucleotide of 10565 to 10583 bases among the SEQ ID NO:1 and the Nucleotide of 11124 to 11142 bases.

6, the Nucleotide of the described O-antigen-specific to shigella dysenteriae 3 types, intestinal bacteria O124 and intestinal bacteria O164 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 shigella dysenteriae 3 types, intestinal bacteria O124 and intestinal bacteria O164 of claim 1, and can provide the O-antigen of expressing shigella dysenteriae 3 types, intestinal bacteria O124 and intestinal bacteria O164, and become bacterial vaccine by insert expressing.

8, the application of the Nucleotide of the described O-antigen-specific to shigella dysenteriae 3 types, intestinal bacteria O124 and intestinal bacteria O164 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 shigella dysenteriae 3 types, intestinal bacteria O124 and intestinal bacteria O164 of claim 1 is characterized in that, comprises the steps:

(1) genomic extraction: 37 ℃ of incubated overnight shigella dysenteriae 3 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 that adds 3ul 20mg/ml afterwards, 15ul 10%SDS, 50 ℃ of incubations 2 hours, the RNase that adds 3ul 10mg/ml again, 65 ℃ of incubations 30 minutes, add equal-volume phenol extracting mixture, get supernatant liquor liquid and use isopyknic phenol again: chloroform: twice of primary isoamyl alcohol (25: 24: 1) mixing solutions extracting, get supernatant liquor liquid again with isopyknic ether extracting to remove remaining phenol, supernatant liquor liquid is with 2 times of volume ethanol deposit D NA, roll out DNA and wash DNA with glass yarn with 70% ethanol, at last DNA is resuspended among the 30ul TE, genomic dna detects by 0.4% agarose gel electrophoresis;

(2) by the O-antigen gene in pcr amplification shigella dysenteriae 3 types bunch: with the genome of shigella dysenteriae 3 types 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 GTGGCT GCA 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 30 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 6 pipe long PCR products, and with the Wizard PCR Preps purification kit purified pcr product of Promega company;

(3) make up O-antigen gene bunch library: make up O-antigen gene bunch library with the Novagen DNaseI shot gun method that is modified, reaction system is a 300ng PCR purified product, 0.9ul 0.1MMnCl ₂, 1ul1: the DNaseI of the 1mg/ml of 2000 dilutions, reaction is carried out at room temperature, and enzyme is cut the dna fragmentation size is concentrated between the 1kb-3kb, then adds 2ul 0.1M EDTA termination reaction.Merge the same reaction system of 4 pipes, with isopyknic phenol extracting once, use isopyknic phenol: chloroform: primary isoamyl alcohol (25: 24: 1) mixing solutions extracting once, after using isopyknic ether extracting once again, dehydrated alcohol deposit D NA with 2.5 times of volumes, and wash precipitation with 70% ethanol, be resuspended at last in the 18ul water, in this mixture, add 2.5ul dNTP (1mMdCTP, 1mMdGTP, 1mMdTTp subsequently, 10mMdATP), 1.25ul the T4DNA polysaccharase of 100mM DTT and 5 units, 11 ℃ 30 minutes, enzyme is cut product mends into flush end, after 75 ℃ of termination reactions, add the Tth archaeal dna polymerase of 5 units and corresponding damping fluid thereof and system is expanded as 80ul, 70 ℃ of reactions 20 minutes make 3 of DNA ' end add the dA tail.This mixture is through the equal-volume chloroform: after primary isoamyl alcohol (24: 1) mixing solutions extracting and the extracting of equal-volume ether with 3 * 10 of Promega company ^-3The pGEM-T-Easy carrier connect 24 hours in 16 ℃, cumulative volume is 90ul.10 * the buffer of 9ul and the T4DNA ligase enzyme of 25 units are wherein arranged.Use the dehydrated alcohol precipitation of the 3M NaAc (pH5.2) of 1/10 volume and 2 times of volumes to be connected mixture at last, wash precipitation with 70% ethanol again, be dissolved in after the drying and obtain connecting product in the 30ul water; Preparation method with the electric transformed competence colibacillus cell of Bio-Rad company prepares competence bacillus coli DH 5 cell, 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, time is 5.0 milliseconds-6.0 milliseconds, the SOC substratum that adds 1ml after the electric shock immediately in cup makes the bacterium recovery, then bacterium is coated in and contains penbritin, 37 ℃ of incubated overnight on the LB solid medium of X-Gal and IPTG, obtain blue white bacterium colony next day, with the white colony that obtains promptly the white clone forward on the LB solid medium that contains penbritin and cultivate, extract plasmid and cut the segmental size of evaluation insertion wherein with the EcoRI enzyme from each clone simultaneously, the white that obtains clone group has constituted the O-antigen gene bunch library of shigella dysenteriae 3 types;

(4) to the cloning and sequencing in the library: from the library, select insert 120 clones of fragment more than 700bp by Shanghai biotechnology company limited with ABI377 type automatic dna sequencer to unidirectional order-checking of insertion fragment in cloning, make sequence reach 80% fraction of coverage.Residue 20% sequence is again according to the sequences Design primer that has obtained, and this primer is as follows: upstream primer 5 '-TACTCTCCGCACAACGTC-3 ' and downstream primer 5 '-TGCCCAATCATCACTATCA-3 '; Upstream primer 5 '-TAATCCCTCTGACAATAGTG-3 ' and downstream primer 5 '-GCAATGTCAGAAACTC-3 '.Direct PCR and from the genomic dna of shigella dysenteriae 3 types again to the order-checking of PCR product, thus all sequences of O-antigen gene bunch obtained.

(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, thereby obtain the Nucleotide full length sequence of the O-antigen gene bunch of shigella dysenteriae 3 types, the quality of sequence is mainly guaranteed by two aspects: 1) genome of shigella dysenteriae 3 types is done 6 LongPCR reactions, mix these products then to produce the library.2), guarantee high-quality fraction of coverage more than 3 to each base.After obtaining the nucleotide sequence of shigella dysenteriae 3 type O-antigen genes bunch, with American National biotechnology information science center (The National Center forBiotechnology Information, NCBI) orffinder finds gene, find the reading frame of 9 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 shigella dysenteriae 3 types at last;

(6) screening of specific gene: at wzx, wzy, orf4, orf5, orf7, the orf9 gene design primer in the O-antigen gene of shigella dysenteriae 3 types bunch.Respectively designed three pairs of primers in each gene, the difference that every pair of primer is distributed in the corresponding gene is local 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, all primers all obtain positive findings in intestinal bacteria O124 and O164, the correct band of any size does not all increase in other groups, that is to say, do not obtaining any PCR product band in the array mostly, though in the minority group, obtain PCR product band, but its size does not meet the expection size, so wzx, wzy, orf4, orf5, orf7, orf9 gene pairs shigella dysenteriae 3 types, intestinal bacteria O124 and O164 and O-antigen thereof all are high specials.