CN1171999C - Nucletide specific to O-antigen of shigella dysenteriae 8 - Google Patents

Abstract

The present invention provides a specific nucleotide for the O-antigens of Shigella dysenteriae 8. The specific nucleotide is the nucleotide complete sequence of gene clusters in Shigella dysenteriae 8 for controlling the synthesis of O-antigens, such as a separated nucleotide with the total length of 9227 basic groups shown as SEQ ID NO: 1 or the nucleotide of SEQ ID NO: 1 which has one or a plurality of inserted, deleted or substituted basic groups and can simultaneously keep the functions of the separated nucleotide. The present invention also comprises the structure of O-antigen gene clusters and the oligonucleotide of glycosyltransferase genes and unit processing genes stemmed from the O-antigen gene clusters of Shigella dysenteriae 8. The present invention verifies that the oligonucleotide has high specificity to all of the O-antigen genes both of Shigella dysenteriae 8 through PCR. The present invention also discloses a method for using the oligonucleotide of the present invention to detect and identify Shigella bodyii 8 in human bodies and environment.

Description

Nucleotide to the O-antigen-specific of shigella dysenteriae 8 types

Technical field

The present invention relates to the complete nucleotide sequence of control O-antigen synthetic gene cluster in shigella dysenteriae 8 types (Shigella dysenteriae 8), particularly relate in shigella dysenteriae 8 types oligonucleotide in the control O-antigen synthetic gene cluster, can utilize these oligonucleotide of O-antigen-specific shigella dysenteriae 8 types 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 form on the glycolipid molecule lipopolysaccharide molecule [Whitfield, C. (1995) " Biosynthesis of lipopolysaccharide Oantigens ". Trends in Microbiology.3:178-185; Schnaitman, C.A.andJ.D.Klena. (1993) " Genetics of lipopolysaccharide biosynthesis inentericbacteria " .Microbiological Reviews, 57 (3): 655-682].Coding is responsible for the generally adjacent arrangement on karyomit(e) of gene of all enzyme molecules of O-antigen synthetic, form a gene cluster [Reeves, P.R., et al. (1996) " Bacterial polysaccharide synthesis and genenomenclature " Trends in Microbiology, 4:495-503].In 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-antigen loci:implicationfor Escherichia coli and Shigella relationships ". Infection andImmunity, 11:6923-6930; Lei Wang and Peter Reeves (2000) " TheEscherichia coli O111 and Salmonella enterica O35 gene clusters:geneclusters encoding the same colitose-containing O antigen are highlyconserved ". Journal of Bacteriology.182:5256-5261].The O-antigen gene bunch contains three genoids: sugared synthesis path gene, glycosyltransferase gene, oligosaccharide unit treatment gene, the required nucleoside diphosphate monose of enzymic synthesis O-antigen of wherein sugared synthesis path genes encoding; Thereby the enzyme of glycosyltransferase gene coding forwards nucleoside diphosphate monose and other molecule to and makes monose aggregate into oligosaccharide unit on the monose; The oligosaccharide unit treatment gene comprises transhipment enzyme gene and pol gene, and they transfer to the bacterium inner membrance outside with oligosaccharide unit, and repolymerization becomes polysaccharide.Glycosyltransferase gene and oligosaccharide unit treatment gene only are present in the gene cluster of carrying these genes.The difference of monose in the O-antigen, between monose between the difference of link button and the oligosaccharide unit difference of link button constituted the antigenic diversity of O-, and the composition of monose, the link button between monose and the link button between the oligosaccharide unit are by the Gene Handling in the O-antigen gene bunch, so the O-antigen gene bunch has determined O-antigenic synthetic, has also determined the antigenic diversity of O-.

Because O-antigen is extremely strong antigen, be one of important paathogenic factor of 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 ofabequose and paratose synthase genes (rfb) by polymerase chain reactionfor identification of 5.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 microbiologicalinvestigation of an outbreak of Hemolytic-Uremic Syndrome caused bydry fermented sausage contaminated with Shiga-like toxin producingEscherichia coli " .J.Clin.Microbiol.34:1622-1627] of the oligonucleotide that comes from the wbdI gene of the O-antigen-specific of E.coli O111 having been identified the toxogenic E.coli O111 of a strain, but afterwards studies show that Paton, the usefulness of A.W et.al comes from the oligonucleotide of wbdI gene and identifies that the method for the serotype of E.coli O111 has false positive results to occur.Bastin D.A.and Reeves, P.R. think, this is because the wbdI gene is sugared synthesis path gene [the Bastin D.A.andReeves of a supposition, P.R. (1995) Sequence and analysis of the O antigen gene (rfb) cluster of Escherichia coli O111. Gene 164:17-23], and have this sugar in the antigenic structure of the O-of other bacterium, so sugared synthesis path gene is not a high special for O-antigen yet

Shigellae has 46 kinds of serotypes, but have only 33 kinds of different O-antigens, intestinal bacteria have 166 kinds of different O-antigen [Reeves, P.R (1992) " Variation in O antigens; nichespecific selection and bacterial populations ". FEMSMicrobiol. Lett, 100:509-516], the two sibship is very near, and there are 12 kinds to be intestinal bacteria and the total [Ewing of Shigellae, W.H. (1986) " Edwards and Ewing ' sidentification of the Enterobacteriaceae ". Elsevier SciencePublishers, Amsterdam, The Netherlands; T.cheasty, et al. (1983) " Antigenic relationships between the enteroinvasive Escherichiacoli antigens O28ac; O112ac; O124; O136, O143, O144; O152 and andShigella O antigens " J.clin Microbiol, 17 (4): 681-684]

Summary of the invention

The Nucleotide that the purpose of this invention is to provide a kind of O-antigen-specific to shigella dysenteriae 8 types.It is the Nucleotide in the O-antigen gene bunch of shigella dysenteriae 8 types, is the special Nucleotide that comes from glycosyltransferase gene and transhipment enzyme gene and pol gene.

A time purpose of the present invention has provided the full length nucleotide sequence of the O-antigen gene bunch of shigella dysenteriae 8 types.

Another object of the present invention has provided the gene of the O-antigen gene bunch that constitutes shigella dysenteriae 8 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 orf1, orf4, orf5, orf7 gene; Sugar synthesis path gene comprises the gne gene.

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 8 types respectively comprises orf1, orf4, orf5, orf7 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 8 types; Especially the oligonucleotide of listing in the table 1, they are high specials to the O-antigen of shigella dysenteriae 8 types, and these oligonucleotide are also reconfigurable, the oligonucleotide after the combination also is a high special to the O-antigen of shigella dysenteriae 8 types.

The above-mentioned oligonucleotide that a further object of the present invention provides can be used as primer and is used for nucleic acid amplification reaction, perhaps be used for hybridization as probe, perhaps be used to make gene chip or microarray, thereby detect and identify O-antigen and the detection and evaluation shigella dysenteriae 8 types of shigella dysenteriae 8 types by these methods.

An also purpose of the present invention has provided the method for the complete sequence of the O-antigen gene bunch that separates shigella dysenteriae 8 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 to the Nucleotide of the O-antigen-specific of shigella dysenteriae 8 types, and it is the isolating Nucleotide shown in SEQ ID NO:1,9227 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 8 types, it is by 7 genomic constitutions of called after wzx, wzy, orf1, orf6, orf4, orf5, orf7, all between galF gene and gnd gene.

The Nucleotide of aforesaid O-antigen-specific to shigella dysenteriae 8 types, wherein said gene with height O-antigen-specific is: the gene of transhipment enzyme comprises the wzx gene or with wzx the gene of identity function is arranged; Pol gene wzy gene or the gene of identity function is arranged with wzy; Glycosyltransferase gene comprises orf1, orf4, orf5, orf7 gene; Wherein said gene: orf1 is the Nucleotide of 1097 to 1912 bases among the SEQ ID NO:1; Wzy is the Nucleotide of 1899 to 3125 bases among the SEQ ID NO:1; Wzx is the Nucleotide of 3180 to 4349 bases among the SEQ IDNO:1; Orf4 is the Nucleotide of 4419 to 5285 bases among the SEQID NO:1; Orf5 is the Nucleotide of 5282 to 5968 bases among the SEQ ID NO:1; Orf7 is the Nucleotide of 7004 to 7750 bases among the SEQ ID NO:1.

The Nucleotide of aforesaid O-antigen-specific to shigella dysenteriae 8 types, wherein it is to come from described wzx gene, wzy gene or glycosyltransferase gene orf1, orf4, orf5, orf7 gene; And their mixing or their reorganization.

The Nucleotide of aforesaid O-antigen-specific to shigella dysenteriae 8 types, the oligonucleotide of the wherein said orf1 of coming from gene is to being: the Nucleotide of 1103 to 1120 bases among the SEQ ID NO:1 and the Nucleotide of 1877 to 1896 bases; The Nucleotide of 1194 to 1213 bases among the SEQ ID NO:1 and the Nucleotide of 1763 to 1780 bases; The Nucleotide of 1261 to 1279 bases among the SEQ ID NO:1 and the Nucleotide of 1686 to 1704 bases; The oligonucleotide that comes from the wzy gene is to being: the Nucleotide of 1976 to 1995 bases among the SEQ ID NO:1 and the Nucleotide of 3096 to 3114 bases; The Nucleotide of 2145 to 2162 bases among the SEQ ID NO:1 and the Nucleotide of 2997 to 3014 bases; The Nucleotide of 2249 to 2268 bases among the SEQ ID NO:1 and the Nucleotide of 2834 to 2850 bases; The oligonucleotide that comes from the wzx gene is to being: the Nucleotide of 3183 to 3191 bases among the SEQ ID NO:1 and the Nucleotide of 4261 to 4278 bases; The Nucleotide of 3241 to 3257 bases among the SEQ ID NO:1 and the Nucleotide of 4091 to 4109 bases; The Nucleotide of 3301 to 3318 bases among the SEQ ID NO:1 and the Nucleotide of 3991 to 4019 bases; The oligonucleotide that comes from the orf4 gene is to being: the Nucleotide of 4441 to 4460 bases among the SEQ ID NO:1 and the Nucleotide of 5201 to 5220 bases; The Nucleotide of 4501 to 4520 bases among the SEQ ID NO:1 and the Nucleotide of 5115 to 5132 bases; The Nucleotide of 4601 to 4620 bases among the SEQ ID NO:1 and the Nucleotide of 5072 to 5088 bases; The oligonucleotide that comes from the orf5 gene is to being: the Nucleotide of 5311 to 5330 bases among the SEQ ID NO:1 and the Nucleotide of 5947 to 5965 bases; The Nucleotide of 5401 to 5420 bases among the SEQ ID NO:1 and the Nucleotide of 5895 to 5912 bases; The Nucleotide of 5501 to 5520 bases among the SEQ ID NO:1 and the Nucleotide of 5828 to 5846 bases; The oligonucleotide that comes from the orf7 gene is to being: the Nucleotide of 7059 to 7076 bases among the SEQ ID NO:1 and the Nucleotide of 7651 to 7668 bases; The Nucleotide of 7099 to 7106 bases among the SEQ ID NO:1 and the Nucleotide of 7521 to 7540 bases; The Nucleotide of 7161 to 7180 bases among the SEQ ID NO:1 and the Nucleotide of 7481 to 7500 bases.

The Nucleotide of aforesaid O-antigen-specific to shigella dysenteriae 8 types detect express the antigenic bacterium of O-, in the application of other polysaccharide antigen of the O-antigen of identifying bacterium and bacterium.

The recombinant molecule of the Nucleotide of aforesaid O-antigen-specific to shigella dysenteriae 8 types provides application in the O-antigen bacterial vaccine of expressing shigella dysenteriae 8 types in preparation.

The application of the Nucleotide of aforesaid O-antigen-specific to shigella dysenteriae 8 types is characterized in that it is used for PCR, is used for hybridization and fluoroscopic examination or makes gene chip or microarray as probe as primer, is used for bacterial detection.

The separation method of the Nucleotide of aforesaid O-antigen-specific to shigella dysenteriae 8 types is characterized in that it comprises the steps:

(1) genomic extraction: 37 ℃ of incubated overnight shigella dysenteriae 8 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 ℃ 30 minutes.Add equal-volume phenol extracting mixture, get supernatant and use isopyknic phenol again: chloroform: primary isoamyl alcohol extracting twice, get supernatant again with isopyknic ether extracting to remove remaining phenol.Supernatant rolls out DNA and washes DNA with 70% ethanol with glass yarn with 2 times of volume ethanol deposit D NA, and DNA is resuspended among the 30ul TE; Genomic dna detects by 0.4% agarose gel electrophoresis;

(2) by the O-antigen gene in pcr amplification shigella dysenteriae 8 types bunch: with the genome of shigella dysenteriae 8 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 (#1523-ATT GTGGCT GCA GGG ATC AAA GAA AT) that often is found in O-antigen gene bunch promoter region, again according to the gnd gene design downstream primer (#1524-TAG TCG CGT GNG CCT GGA TTA AGT TCG C) in O-antigen gene bunch downstream; With the Expand Long Template PCR method of BoehringerMannheim 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;

(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 1.25ul100mM 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 the 3 ' end of DNA 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; Preparation method with the electric transformed competence colibacillus cell of BiO-Rad company prepares competence bacillus coli DH 5 a cell, get after 2-3ul connects product and 50ul competence escherichia coli DH5a 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 to 6.0 milliseconds, the SOC substratum that adds 1ml after the electric shock immediately in cup makes the bacterium recovery, then bacterium is coated in and contains penbritin, on the LB solid medium of X-Gal and IPTG, 37 ℃ of incubated overnight, obtain blue white bacterium colony next day, with the white colony that obtains promptly the white clone forward on the LB solid medium that contains acillin and cultivate, from each clone, extract plasmid simultaneously, and cutting the segmental size of evaluation insertion wherein with the EcoRI enzyme, the white that obtains clone group has constituted the O-antigen gene bunch library of dysentery will Hayes 8 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 90% fraction of coverage.Residue 10% sequence is again by with the partial sequence backward sequencing, thereby obtains all sequences of O-antigen gene bunch;

(5) splicing of nucleotide sequence and analysis: the Pregap4 and the splicing of Gap4 software of the Staden package software package of publishing with Britain Camb MRC (Medical Research Council) Molecular Biology Lab and edit all sequences, thus obtain the Nucleotide full length sequence of the O-antigen gene bunch of shigella dysenteriae 8 types; The quality of sequence is mainly guaranteed by two aspects: 1) genome of shigella dysenteriae 8 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 8 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 7 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 8 types at last;

(6) screening of specific gene: at wzx, wzy, orf1, orf4, orf5, the orf7 gene design primer in the O-antigen gene of shigella dysenteriae 8 types bunch; Respectively design three pairs of primers in each gene, every pair of primer is distributed in the different places in the corresponding gene, to guarantee its specificity; Is that template is carried out PCR with these primers with the genomes of 166 strain intestinal bacteria and 43 strain Shigellaes, the correct band of any size does not all increase in other groups, promptly do not obtaining any PCR product band in the array mostly, though in the minority group, obtain PDR product band, but its size does not meet the expection size, so the O-antigen of wzx, wzy, orf1, orf4, orf5, orf7 gene pairs shigella dysenteriae 8 types all is high special.

Just, first aspect of the present invention provides the full length nucleotide sequence of the O-antigen gene bunch of shigella dysenteriae 8 types, its complete sequence shown in SEQ ID NO:1,9227 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 8 types by method of the present invention, as shown in table 3, it is altogether by 7 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 8 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 orf1, orf4, orf5, orf7 gene; Special sugared synthesis path gene in the bacterial polysaccharides antigen comprises the gne gene.Their zero positions in O-antigen gene bunch and final position and nucleotide sequence all are listed in the table 4; The invention particularly relates to glycosyltransferase gene, transhipment enzyme gene and pol gene, because sugared synthesis path gene is that the gene of synthetic nucleosides bisphosphate monose is common, common by indication to more exocellular polysaccharide now, O-antigen to bacterium is not very special, and the glycosyltransferase gene that the present invention relates to, transhipment enzyme gene and pol gene are high specials to the O-antigen of shigella dysenteriae 8 types.

The 3rd aspect of the present invention, wzx gene in the O-antigen gene bunch that comes from shigella dysenteriae 8 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, comprise orf1, orf4, orf5, the oligonucleotide of orf7 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 8 types only, and are all not obtain expecting the product of size in the pcr amplification that carries out of template other bacterium listed with table 2.In more detail, with these oligonucleotide to being that PCR that primer is done is reflected at and does not all obtain spawn in most of bacteriums.So, can determine these primers promptly the listed oligonucleotide of table 1 are high specials to the O-antigen of shigella dysenteriae 8 types.

The separation method of the Nucleotide of described O-antigen-specific to shigella dysenteriae 8 types comprises the steps: 1) genomic extraction; 2) the O-antigen gene in pcr amplification shigella dysenteriae 8 types bunch; 3) make up O-antigen gene bunch library; 4) to the cloning and sequencing in the library; 5) splicing of nucleotide sequence and analysis; 6) screening of specific gene.

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

As used herein, " oligonucleotide " mainly 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 orf1 gene (nucleotide position is 1097 to 1912 bases from SEQ ID NO:1), wzy gene (nucleotide position is 1899 to 3125 bases from SEQ ID NO:1), wzx gene (nucleotide position is 3180 to 4349 bases from SEQ ID NO:1), orf4 gene (nucleotide position is 4419 to 5285 bases from SEQ ID NO:1), orf5 gene (nucleotide position is 5282 to 5968 bases from SEQID NO:1), orf7 gene (nucleotide position is 7004 to 7750 bases from SEQID NO:1).Coming from above intragenic oligonucleotide is high specials to shigella dysenteriae 8 types.

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

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

The present invention relates to a kind of antigenic method of one or more bacterial polysaccharideses that detects in the food, these antigens can make sample can with the oligonucleotide specific hybrid of following at least one gene, these genes are: (i) gene of the encoding glycosyl transferring enzyme gene of transhipment enzyme and polysaccharase of (ii) encoding comprises the wzx gene or the gene, wzy gene of identity function is arranged or with wzy the gene of identity function is arranged with wzx.At least one oligonucleotide can be hybridized with at least one more than one such gene specific of expressing the special antigenic bacterium of O-under the situation of condition permission, and these bacteriums are shigella dysenteriae 8 types.Available PCR method detects, more can with behind the Nucleotide mark in the inventive method as probe by hybridization such as southern-blot or fluoroscopic examination, perhaps by antigen and bacterium in gene chip or the microarray assay sample.

Planner of the present invention considers following situation: when one special oligonucleotide detects when invalid, the mixture of oligonucleotide can with the target region specific hybrid with test sample.Therefore the invention provides a cover oligonucleotide and be used for detection method of the present invention.Here said oligonucleotide is meant the gene that comes from the encoding glycosyl transferring enzyme, the gene of coding transhipment enzyme and the gene of polysaccharase, comprises the wzx gene or the gene, wzy gene of identity function is arranged or with wzy the oligonucleotide of the gene of identity function is arranged with wzx.This cover oligonucleotide is special to the O-antigen of a special bacterium, and this special bacterium O-antigen is expressed by shigella dysenteriae 8 types.

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 8 types.Oligonucleotide among available the present invention is made the method test sample of primer by PCR, also can with behind the oligonucleotide molecules mark among the present invention as probe by hybridization such as southern-blot or fluoroscopic examination, perhaps by antigen and bacterium in gene chip or the microarray assay sample.

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

On the other hand, the present invention also relates to the antigenic method of one or more bacterial polysaccharideses in the sample that a kind of detection comes from patient.One or more bacterial polysaccharides antigens in the sample can make sample can with a specific hybrid in a pair of oligonucleotide in following at least one gene, these genes are: (i) gene of the encoding glycosyl transferring enzyme gene of transhipment enzyme and polysaccharase of (ii) encoding comprises the wzx gene or the gene, wzy gene of identity function is arranged or with wzy the gene of identity function is arranged with wzx.Under the situation of condition permission at least one oligonucleotide can with sample at least one express more than one such gene specific hybridization of the special antigenic bacterium of O-, these bacteriums are shigella dysenteriae 8 types.Oligonucleotide among available the present invention is made the method test sample of primer by PCR, also can will pass through hybridization as probe behind the oligonucleotide mark among the present invention, perhaps by antigen and bacterium in gene chip or the microarray assay sample.

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

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

The present invention discloses the full length sequence of the O-antigen gene bunch of shigella dysenteriae 8 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 8 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 8 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 3ul20mg/ml afterwards, 50 ℃ of incubations 2 hours add the RNase of 3ul 10mg/ml again, 65 ℃ 30 minutes.Add equal-volume phenol extracting mixture, get supernatant and use isopyknic phenol again: chloroform: primary isoamyl alcohol extracting twice, get supernatant again with isopyknic ether extracting to remove remaining phenol.Supernatant rolls out DNA and washes DNA with 70% ethanol with glass yarn with 2 times of volume ethanol deposit D NA, 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 8 types bunch:

With the genome of shigella dysenteriae 8 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 (#1523-ATT GTG GCT GCA GGG ATC AAA GAA AT) that often is found in O-antigen gene bunch promoter region, again according to the gnd gene design downstream primer (#1524-TAG TCG CGT GNG CCT GGA TTA AGT TCGC) 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 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 2.5ul dNTP (1mMdCTP subsequently, 1mMdGTP, 1mMdTTP, 10mMdATP), the T4DNA polysaccharase of 1.25ul100mM 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 the 3 ' end of DNA 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 the competent cell bacillus coli DH 5 alpha.Get a ring bacillus coli DH 5 alpha list bacterium colony in the LB of 5ml substratum, 180rpm cultivated after 10 hours, got in the LB substratum that the 2ml culture is transferred to 200ml, and 37 ℃ of 250rpm thermal agitations are cultivated OD600 about 0.5, ice bath cooling was 20 minutes then, in centrifugal 15 minutes of 4 ℃ of 4000rpm.Confide all supernatant, dispel thalline, in centrifugal 15 minutes of 4 ℃ of 4000rpm with the deionization aqua sterilisa 200ml of cold ice precooling.Deionization aqua sterilisa 100ml with cold ice precooling dispelled thalline again, in centrifugal 15 minutes of 4 ℃ of 4000rpm.With 10% glycerine suspension cell of cold ice precooling, centrifugal 10 minutes of 4 ℃ of 6000rpm abandon supernatant, precipitate 10% glycerine suspension cell with the precooling of 1ml ice at last, are competent cell.The competent cell that makes is packed as 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 α 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 8 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 90% fraction of coverage.Residue 10% sequence is again by with the partial sequence backward sequencing, thereby obtains all sequences of O-antigen gene bunch.

Embodiment 5: the splicing of nucleotide sequence and analysis:

The Pregap4 and the splicing of Gap4 software of the Staden package software package of publishing with Britain Camb MRC (Medical Research Council) Molecular Biology Lab and edit all sequences, thus the Nucleotide full length sequence (seeing sequence list) of the O-antigen gene bunch of shigella dysenteriae 8 types obtained.The quality of sequence is mainly guaranteed by two aspects: 1) genome of shigella dysenteriae 8 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 8 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 7 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 8 types at last, as shown in table 3.

By retrieving and comparing, find that it is the same that orf 1 and the glycosyltransferase gene of Escherichia coli O91 have 25% aminoacid sequence in 265 amino acid, 17% homogeny is promptly arranged, 44% similarity, the homology that height is arranged between them is described, can infer that orf 1 also is glycosyltransferase gene, called after orf 1.Orf 2 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 25% homogeny in 370 amino acid, 45% similarity, the homology that height is arranged between them is described, so name orf 2 is the wzy gene.The wzx gene of Orf3 and E.coli has 23% homogeny in 419 amino acid, 44% similarity, this wzx gene has 12 potential transmembrane domains, and the algorithm [Eisenberg by Eisenberg etc., D, Schwarz, E.etal (1984) " Analysis of membrane and surfaceprotein sequences with the hydrophobic moment plot.J.Mol.Biol.179:125-142] find that orf3 also has 12 potential transmembrane domains; and with many wzx protein similars; orf3 has about 50 amino acid whose conservative motifs at the proteic aminoterminal of wzx; so can determine orf3 is the wzx gene, called after wzx.The CpsVN gene of orf4 and Streptococcus agalactiae has 33% homogeny in 295 amino acid, 56% similarity, and the CpsVN gene is a glycosyltransferase gene; The glycosyl transferase of orf4 and Strep tococcus pneumoniae in addition, the family2 gene has 24% homogeny in 301 amino acid, and 43% similarity is so infer that orf4 is a glycosyltransferase gene, called after orf4.Orf5 and Streptococcus thermophilusEps10 gene have 32% homogeny in 246 amino acid, 52% similarity shows higher homology, this genes encoding glycosyltransferase, so infer that orf5 is a glycosyltransferase gene, called after orf5.The gne gene of orf6 and Yersinia enterocoli tica (type 0:8) has 59% homogeny in 336 amino acid, 74% similarity is so name orf6 is the gne gene.Among orf7 and the Bacillusanthracis among the gene of encoding glycosyl transferring enzyme and the Escherichia coli O157:H7 gene of encoding glycosyl transferring enzyme 42% and 36% homogeny is arranged respectively on amino acid levels, so infer that orf7 also is a glycosyltransferase gene, called after orf 7.

Infer that according to the structure of shigella dysenteriae 8 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 gne gene as GalNAc, and these genes all are found in the O-antigen gene of shigella dysenteriae 8 types bunch.

Embodiment 6: the screening of specific gene: at wzx, wzy, orf1, orf4, orf5, orf7 gene design primer in the O-antigen gene of shigella dysenteriae 8 types bunch, the position of these genes in nucleotide sequence sees Table 1.

Table 1 has been listed glycosyltransferase gene and oligosaccharide unit treatment gene and intragenic primer and PCR data in the O antigen gene bunch of shigella dysenteriae 8 types.Glycosyltransferase gene, transhipment enzyme gene and pol gene and their function corresponding and the size of the O antigen gene bunch of shigella dysenteriae 8 types in table, 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 IDNO:1.Is that template carry out PCR with listed corresponding annealing temperature in the table with the genomes of all bacterium in the table 2 with every pair of primer, has obtained corresponding PCR product, and its size is also listed in the table.

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 #101 (TTC ATC CTAAAC TCC TTA TT) and #102 (TAA TCG CAG GGG AAA GCA GG), 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 8 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, orf1, orf4, orf5, orf7 gene, each gene all has three pairs of primers detected, every pair of primer has obtained except be PCR in the 24th group after the correct band of expection size, the correct band of any size does not all increase in other groups, that is to say, not obtaining any PCR product band in the array mostly, so wzx, wzy, orf1, orf4, orf5, orf7 gene pairs shigella dysenteriae 8 types and O-antigen thereof are high specials.

At last, from shigella dysenteriae 8 types, screen gene by PCR: wzx, wzy and four glycosyltransferase genes to the O-antigen high special of shigella dysenteriae 8 types.And the oligonucleotide of these intragenic any one section 10-20nt is special to the O-antigen of shigella dysenteriae 8 types, and the primer in especially above-mentioned each gene is that oligonucleotide is high specials to detecting the back confirmation through PCR to shigella dysenteriae 8 types.These all oligonucleotide all can be used for shigella dysenteriae 8 types 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 8 types, in table, listed the structure of the O-antigen gene bunch of shigella dysenteriae 8 types, altogether by 7 genomic constitutions, each gene box indicating, and in square frame, write the title of gene, numeral be the order of the open reading frame (orf) in the O-antigen gene bunch.Two ends at O-antigen gene bunch are galF gene and gnd gene, and they do not belong to O-antigen gene bunch, and we are just with the increase full length sequence of O-antigen gene bunch of their one section sequences Design primer.

Table 4 is site plan of the gene in the O-antigen gene bunch of shigella dysenteriae 8 types, listed the accurate position of all open reading frame in complete sequence in the O-antigen gene bunch of shigella dysenteriae 8 types in the drawings, at the underscoring of the initiator codon and the terminator codon of each open reading frame.The initiator codon of open reading frame has two in Shigellae: ATG and GTG.

SEQ ID NO:1 sequence list

＜110〉Nankai University

＜120〉to the Nucleotide of the O-antigen-specific of shigella dysenteriae 8 types

＜130〉to the Nucleotide of the O-antigen-specific of shigella dysenteriae 8 types

<160>1

<170>PatentIn version 3.1

<210>1

<211>9227

<212>DNA

<213>Shigella dysenteriae

<400>1

attgtggctg cagggatcaa agaaatcctc ctggtaactc acgcgtccaa gaacgcggtc 60

gaaaaccact tcgacacctc ttatgaatta gaatctctcc ttgaactgcg cgtgaagcgc 120

caactgttgg cggaagtaca gtccatctgt ccgcctggcg tgaccattat gaacgtgcgt 180

cagggcgaac ctttaggttt aggccactcc attttatgtg cacgacccgc cattggcgac 240

aacccatttg tcgtggtact gccagacgta gtgatcgacg acgccagcgc cgacccgctg 300

cgctacaacc ttgctgccgt gattgcgcgc ttcaatgaaa cgggccgtag ccaggtgctg 360

gcaaaacgta tgccgggtga cctctctgaa tactccgtca ttcagaccaa agaaccgctg 420

gaccgtgaag gcaaagtcag ccgcattgtt gaattcatcg aaaaaccgga tcagccacaa 480

acgctggact cagacatcat ggccgttggt cgctatgtgc tttctgccga tatttggccg 540

gaacttgaac gcactcaacc tggtgcatgg gggcgtattc agctgactga tgccattgct 600

gaactggcga aaaaacagtc cgttgatgca atgctgatga ctggtgaaag ctacgactgc 660

ggtaaaaaaa tgggctatat gcaggcgttt gtgaagtatg gattgcgcaa cctgaaagaa 720

ggggcgaagt tccgtaaagg gattgagaag ctgttaagcg aataatgaaa atctgaccgg 780

atgtaacggt tgataagaaa attataacgg cagtgaggat tcgtggcgaa agtaatttgt 840

tgcgaatatt cctgccgttg ttttatataa acaatcagaa taacaacgag ttagcaatag 900

gattttagtc aaagttttcc aggattttcc ttgtttccag agcgaattgg taagacaatt 960

agcgtttgaa tttttcgagt taagcgcgag tgggtaacgc tcgtcacatc gtagacatgc 1020

atgcagtgct ctggtagctg taaagccagg ggcggtagcg ttatttatat tcataggtct 1080

aaaatagata accgagatga aaatagctat aggtatatca acattaaata atggaataaa 1140

tcaggtttgg gaaaaaataa aaaatatacc agatgaattt ttaataataa tttcacatca 1200

agtgactgat aagcaaaaac aacatgggga tatttgtaag aaaaacaatg tgataataat 1260

aacgacatac tgtaaagggt taagtaaaag cagaaatatt ttattggcta ctgcatttca 1320

aaaaagcgtt gattatatga ttataagtga tgatgatgtc gcttaccttg ttaatggact 1380

ttatgaacta aaagatagaa taatcgaaga tagaggcaag tatcattatc aaattcaaag 1440

ttgtacgcaa gacggaagac taaggaaaaa atatccgcta atgaggaaaa ggctaaatag 1500

attgtcagca tttaacattt cttcaataga aatgtgcctt aatgtgaatc aaatacagga 1560

atgtaatgtt tggtttgatg agtgtttcgg attgggggct agatataaag cgggtgaaga 1620

accgattttt gttacggatc ttatgaaaac aaaaaataat attattttta tacctgtcac 1680

aattactgtg catccaatag aaagttctgg taaaaaaatt tataatgaaa cagatgccct 1740

atcagataga agtgctattt ttgtacgctg tggtggtaga tatttagggt tactatatat 1800

cttcatattc tgggtgaaaa aatttctttt aaaaaaaaaa tgcggcagaa ttaaaaaaat 1860

agctgcatta tttattctgc taaaaggata ttcccgatat gaatatttat aaaaacagca 1920

atgaaaattc attgtcaaat aataactgta ttgtatattt tattcctttg ttattattgg 1980

caacattctt tttccctctt gttgtattga tatttattgg tgccctctcg ccattgttac 2040

acccagtatt gcgtaatttt tatttttatg cactattggt gtttattatt attttttttt 2100

ccacactaaa accatttgga gatattgcag agtatcttca tgtttaccat gagttaaatt 2160

ataatttaat tgatgttttt ggctattcaa gatttggtga tgggctggaa tttatgtttt 2220

tagccatcat gaaatttatt gggtatattt cgggtggcaa tgatgaagta tttttacttt 2280

ctacttattt tctgatcgtc ttttttttat ccaagattct taaagatgtt gataaaaaat 2340

ataaactttt tttattatcg cttttctttt ttaacttagg atttatagaa gttacatcat 2400

attttcttcgt acaggtttta tctgtggttg tttttctgta tgctataaat gagcgttcat 2460

ttaaaaaata tatctttttt ttgttgagtg ttttttttca catgtctgca gttgttaatg 2520

tttttatata tgcagtttat atgatctttg gttcaaaaaa atacccatat gcaaaaattg 2580

ttttttctat tcttattgga cttttagttg tgttttttgt ggtttataat acgccaattt 2640

attctgtgct attatcgaaa ttcacttcag tttctgggaa cgataaattc acacgtttgc 2700

cgttaaatta tattatcatt acagttgtaa atatatgctt tattataatg atagggaaga 2760

gaagtaaatg tgatgatttt aataagattt tattttgcaa ggagtgtttt ctattcttaa 2820

tattattacc tttcccagca ctttcgaata gattgggtat gattattttt ggtttttatc 2880

cctactttat cttaccctat ctaaaagctt ttgagagaaa aggtaagagt aaatatgccc 2940

tacttatttg tttatatgta gtaaatttag tgcctttttt gtatttaatg tataatgtgt 3000

ctttagggaa taacatgttt actttcctaa ataatcatcc atttactgag ggtgtgtatg 3060

gtatgattga ttacatattg gaggcaattg ataaaggtgt taactatatc aatgaaggta 3120

actagcacgt gaaaaaatac ttaagtaacc tattttggtt gttgagtgac cgagtgttca 3180

tgctggtatt tcagttgtca ctttttgctt ctatcaaaag aatctatggg ttagatatct 3240

tgggtagttg ggcaacgata atgaatatct cacaaatatt actatcgtta tttttatttg 3300

gcattgatat agtcgtagtt aaaagaattg ttgaaaatcc atcatcaacg ggcactgaga 3360

ttggatgtgc attattctta cagtttttag gactgttatt atatgcatct gcttttatta 3420

ccattgttat ttatttttac tatgatatac cgttcgcttt tatattcgtg gcgatattaa 3480

tcgttgctaa tttttttagc ttgtatgcta aagtaatatt ttttcattat tcggcattgg 3540

ttgaatcaaa atatcgtgct atcacaattc taagtagtgt ggccgtttca tatggttatc 3600

tttggtgttg tatatatttt ggttggcatg tcttttatgc ttatgttttc ttttatttaa 3660

ttcaagctct ttttagtttt actatataca aattctattt cccttattca gcaaaatgga 3720

cgattgattt agaattagtc aaaatgtatt tttttatggg gagtaaactc attgtatcaa 3780

ctattagtgt atcactattt acacagtgtg atgtgatctt attagagtct cttacaggta 3840

caaaagaggc tggggcattt agtgcagctc ttaggttatc agccatctgg tttatgtgtg 3900

gaggtttgat agcgaacgct ttttttccaa aaattgttca gcttgaaaaa ataggagagg 3960

aagaatcatt tatctttttg aaatggatat gcggagttgt aagtgtaatc tctatatatg 4020

gtgcaattat tatgatgtct ctgtcaccta taattataaa aatactatat ggcgataata 4080

tggatttatc ggcgcaagta ttaatggttc atatgtggag tggtgttttt gtttttttag 4140

gatcattttc atctaaatgg ttatttagta agaattatat taatttagaa gtgataaaaa 4200

ccattattgc tgcaattttg aatataactt tgaatatcat tgttatacca aaatatggag 4260

cggttggcgc tgcttctgta tcattgcttt cttattttat tgctaacttt ttcattttta 4320

tatttatacc aaaaactaaa aatgtttaaa atgcaattac agagtttgaa gtatattatt 4380

tttccatggc gtttgattaa tgattttgga agggttagat gtcaatttca gtaattactc 4440

ctgtattcaa tcgagctttg ttagtctatg aattatatga atccttaatg tcacaacaat 4500

cttatgattt tgaatgggtg ataattgatg atggttctac tgataattta aaagaggtta 4560

ttgacaaaat agcgtcgaca tccccattta aaatcatata tagatataag aagaacggtg 4620

gaaaacacac agccttaaat atagggatag aaatgtcatc gtttaactgg atatttattg 4680

ttgatagtga tgatatatta acgcccaacg ctattgctct agccaatgaa aaaatccagg 4740

caattgttga tgataaatgt aaagggatgg ttttcctgag ggggtataaa actacaaaag 4800

agattgtagg taaagcggaa acaattgaaa atatttctct ggaaaagttt gcaggtacaa 4860

agggagataa agcattaatc ataaaacgtg attctttact taaaaatcaa tttcctgttt 4920

ttgatggtga aaattttata actgaggcat tggtttggaa ttcaatttta gaaaatggct 4980

actttaaata tttcaatgag atcatctatt atagtgaata tttaccagga ggtttaactt 5040

ctaattatac tgatcttttg cggaaaaata tcaatggcac gatggctttt gttatcaata 5100

acttgaatct aaaaggtctt ggtattaatg tcattaaaca aaccgttttt cattttattc 5160

ccatttttaa tattagtaat ctaatagttg taaagaaaaa gacaaagttt actgttttcg 5220

ttttatttat tacatgtctt tttcttgtaa aaatcaagaa taaagttaaa ggaaaatcgt 5280

tatgataccg gcaataattc attatatttg gcttggtaaa agtgaaatcc ctaaaatata 5340

tttagattgt atggagtcgt ggaaagaaca tgctgtaaat tacgattgct atttatggaa 5400

tgaggattct tatagaaagg aatttggtca gaatgatttt gttgaagaaa tgatccaaag 5460

gaaaaagttt gcatttgctg cagatttgat ccgatgtgat gtattatatc gttttggtgg 5520

tatatatctc gatactgata tggaattagt tcgggatatt tctgcattgc gaaaaaatat 5580

tgcatttatc ggcgaagaag atattgatac gcctagttgt ggtattttgg gttgtgaacc 5640

caaattttgg cttttccaag agctaaaagc agctgtcata aaagcaaatg gtatgcaaac 5700

aattcctttt cttttaaaga atattttgga cttacatggt gtaaaaaaaa tagattcaca 5760

agatatttct actattaaag atatcacaat atactctgat aagtattttt atccatataa 5820

cccttatggt agtgccaaac gatcacaatt actttataga tatataacaa aagattgtta 5880

tgctatacat cattgggcta aaagttggaa gctttctttt ttagagagaa ttaaaagaaa 5940

aattatcatg cgatatcgta aggagtaata tgaatatcct agtaacaggt ggtgctggtt 6000

atattggttc gcatacggta cttcgcttat tagaaaatga aaatgaaatt actgttgtgg 6060

ataatttggt taattcttca tctgaggtaa ttaagcgtgt agagaatatc acgcaaaaaa 6120

gcatttgctt tatcgaaatg gatatactta acacagaatt attacacgaa gtaattatta 6180

ataaagatat tgatgcggtc attcattttg ctgggttgaa atctgtttct gagtcaatca 6240

gtaggcctct tgaatattat aagaataatg ttcagggaac aattagtgtc cttagtgcta 6300

tgataaatag caaagccaag aaaattattt ttagttcttc tgcaacggtt tatggtgagc 6360

ctgaacaaat tccattaaat gaaaaatgta aagtaggtgg tacaacaaat ccttatggca 6420

cttcgaaatt aatggcagaa caaatccttt gtgattttgc aaaagcaaat tatggttttg 6480

atattatttc attacgatac tttaatccgg taggagcaca tcccagtgga atgataggtg 6540

aggctccgaa tgggattcct aataatcttg taccatatct cactaaggtt gctataggcg 6600

aattagattc attgaaaata tttgggaatg attatcctac cagggacgga tatggagttc 6660

gggattttat tcatgttatg gatcttgccg atggacatat tgctgctttg aatgcagagt 6720

ttaaagaaaa ttctatcagg atatataatt taggtacagg taaaggatat tcagtattag 6780

agttggttga tactttcgag agaattatag ctagaaaaat aaataagtgt gttatatcaa 6840

ggcgagatgg agatatagca gaatgctggt ctgatccaat gttagccttc aatgaactcg 6900

gttggtcagc caaattcaat ttagaggata tgctacgaga ttcttggaat tggcaaataa 6960

aaaacccgaa aggctacgat caaaataaag ggaggaaata ataatggatt atcttgtgtc 7020

aattattatg cccagttata attctgaatt cacaattaag gaaagtataa aatcggtaat 7080

agaacaaaca tattctaatt gggagttgtt aattactgat gactgctcaa cagatagaac 7140

ttgtcagata gttaaagaat ttgttgagca agatgacagg ataaaacttt ttgtttcaga 7200

taaaaataaa ggcgcaggcg cagcaagaaa caactcaata aaagagtcta gtggacgatt 7260

tttggctttt ttagatagtg atgatctatg ggcacccgat aaactaaaag agcagattaa 7320

ttatatgatt atgaatggct acgcccttac ttatactgca tatagtaaaa ttgatgcata 7380

tggtaatatt aaaaaagata ttcagccccc atcaaaagtc gatttttctt ctttactgaa 7440

atccaatgtt atcggatgcc ttactgcaat ttatgacacg gaagttgtgg gtaaagtata 7500

tatgccactc atacgaaagc gtcaagatat ggcattatgg ctaataatcc tgcaaaaaat 7560

agattatgcc cactgcctaa ataaaaacct agcattttat cgcgaaggtc atttaagttt 7620

atcatcgaat aagataaaaa taataaaatc gcaatgggag ttttaccgtt attatcttgg 7680

ttttggatat gtaaaggcaa tgtattattt tctgcattat atccagcgtg cattaagaaa 7740

acacgcttaa gagtttaact ctatttgttt aattttaaaa cattgataat aattgagtct 7800

atcattcatt tattgcatgt cttattttat atctgttatc cttattaacc gtcaaatccc 7860

gcggtaaccc cctgacagga gtaaacaatg tcaaagcaac agatcggcgt agtcggtatg 7920

gcagtgatgg ggcgcaacct cgcgctcaac atcgaaagcc gtggttatac cgtctctatt 7980

ttcaaccgtt cccgtgaaaa aacggaagaa gtgattgccg aaaatccggg caagaaactg 8040

gttccttact atacggtgaa agagtttgtt gaatctctgg aaacgcctcg tcgcatcctg 8100

ttaatggtga aagcaggtgc aggcacggat gctgctattg attccctcaa gccatacctc 8160

gataaaggtg acatcatcat tgatggtggt aataccttct tccaggacac tattcgtcgt 8220

aaccgtgagc tttctgcaga aggctttaat ttcattggta ccggtgtttc cggcggtgaa 8280

gaaggtgcgc tgaaaggtcc ttccattatg cctggtgggc agaaagaagc ctatgaactg 8340

gttgctccga tcctgaccaa aatcgccgca gtggttgaag atggcgagcc atgcgttacc 8400

tatattggtg ccgatggtgc aggtcactat gtgaagatgg ttcacaacgg tattgaatac 8460

ggcgatatgc agctgattgc tgaagcctat tctctgctta aaggtggcct gaatctttcc 8520

aacgaagaac tggcgcagac gtttaccgag tggaataatg gtgaactgag cagctacctg 8580

atcgacatca ccaaagatat cttcaccaaa aaagatgaag acggtaacta cctggttgat 8640

gtgattctgg atgaagcagc aaataaaggt acgggcaaat ggaccagcca gagtgcgctg 8700

gatctcggcg aaccgctgtc gctgattacc gagtctgtgt ttgctcgtta tatctcttct 8760

ctgaaagatc agcgcgttgc cgcgtctaaa gttctctctg gcccgcaagc gcagccagct 8820

ggcgacaagg ctgagttcat cgaaaaagtt cgtcgtgctc tgtatctggg caaaatcgtt 8880

tcttacgccc aaggcttctc tcagctgcgt gcggcgtctg aagagtacaa ctgggatctg 8940

aactacggcg aaatcgcgaa gattttccgt gctggctgca tcatccgtgc gcagttcctg 9000

cagaaaatca ctgatgcata tgccgaaaat ccgcagatcg ctaatctgct gctggctccg 9060

tactttaaac aaattgccga tgattaccag caggcgctgc gtgatgtcgt tgcttatgca 9120

gtgcagaacg gtatcccggt tccgaccttc gccgctgcgg ttgcctatta tgacagctac 9180

cgtgccgctg ttctgcctgc gaacctaatc caggcccagc gcgacta 9227

Glycosyltransferase gene in the O antigen gene of table 1 shigella dysenteriae 8 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 (℃)
								Orf1	Glycosyltransferase	1092-1912	#501(1103-1120)	#502(1877-1896)	794bp	0 *	50
			#503(1194-1213)	#504(1763-1780)	587bp	0 *	52
											#505(1261-1279)	#506(1686-1704)	444bp	0 *	52
wzy	Polysaccharase	1899-3125	#507(1976-1995)	#508(3096-3114)	1139bp	0 *	53
											#509(2145-2162)	#510(2997-3014)	870bp	0 *	52
			#511(2249-2268)	#512(2834-2850)	602bp	0 *	52
								wzx	The transhipment enzyme	3180-4349	#513(3183-3191)	#514(4261-4278)	1096bp	0 *	55
			#515(3241-3257)	#516(4091-4109)	869bp	0 *	52
											#517(3301-3318)	#518(3991-4019)	719bp	0 *	50
Orf4	Glycosyltransferase	4419-5285	#519(4441-4460)	#520(5201-5220)	780bp	0 *	53
											#521(4501-4520)	#522(5115-5132)	632bp	0 *	54
			#523(4601-4620)	#524(5072-5088)	488bp	0 *	53
								Orf5	Glycosyltransferase	5282-5968	#525(5311-5330)	#526(5947-5965)	655bp	0 *	55
			#527(5401-5420)	#528(5895-5912)	512bp	0 *	52
											#529(5501-5520)	#530(5828-5846)	346bp	0 *	54
Orf7	Glycosyltransferase	7004-7750	#531(7059-7076)	#532(7651-7668)	610bp	0 *	52
											#533(7099-7106)	#534(7521-7540)	442bp	0 *	53
			#535(7161-7180)	#536(7481-7500)	340bp	0 *	54

^*In shigella dysenteriae 8 types, obtain a correct band

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

The bacterium source that contains in this group of group number

1 wild-type e. coli O1, O2, O3, O4, O10, O16, O18, O39 IMVS ^a

2 wild-type e. coli O40, O41, O48, O49, O71, O73, O88, O100 IMVS

3 wild-type e. coli O102, O109, O119, 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 IMVS

10 wild-type e. coli O62, O63, O64, O65, O66, O68, O69, O70 IMVS

11 wild-type e. coli O74, O75, O76, O77, O78, O79, O80, O81 IMVS

12 wild-type e. coli O82, O83, O84, O85, O86, O87, O89, O90 IMVS

13 wild-type e. coli O91, O92, O95, O96, O97, O98, O99, O101 IMVS

14 wild-type e. coli O112, O162, O113, O114, O115, O116, O117, O118 IMVS

15 wild-type e. coli O123, O165, O166, O167, O168, O169, O170, O171 See b

16 wild-type e. coli O172, O173, O127, O128, O129, O130, O131, O132, See c

17 wild-type e. coli O133, O134, O135, O136, O140, O141, O142, O143 IMVS

18 wild-type e. coli O144, O145, O146, O147, O148, O150, O151, O152 IMVS

19 wild-type e. coli O153,0154, O155, O156, O157, O158, O159, IMVS

20 wild-type e. coli O160, O161, O163, O8, O9, O124, O111 IMVS

21 wild-type e. coli O103, O104, O105, O106, O107, O108, O110 IMVS

22 Shigella bogdii serotypes B 4, B5, B6, B8, B9, B11, B12, B14 See d

23 Shigella bogdii serotypes B 1, B3, B7, B8, B10, B13, B15, B16, B17, B18 See d

24 shigella dysenteriae serotype D1, D2, D3, D4, D5, D6, D7, D8 See d

25 shigella dysenteriae serum D9, D10, D11, D12, D13 See d

26 shigella flexneri F6a, F1a, F1b, F2a, F2b, F3, F4a, F4b, F5 (v:7) F5 (v:4) See d

27 bacillus ceylonensis A D5, DR See d

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

b. O123 from IMVS；the rest foom Statens Serum Institut，Copenhagen，Denmark

c. 172 and 173 from Statens Serum Institut，Copenhagen，Denmark，the rest from IMVS

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

Table 3 shigella dysenteriae 8 type O antigen gene structure iron

galF

Orf1

wzy

wzx

Orf4

orf5

gne

Orf7

gnd

Table 4 dysentery Hayes bacterium 8 type O antigen gene cluster gene positions

attgtggctg cagggatcaa agaaatcctc ctggtaactc acgcgtccaa gaacgcggtc 60

gaaaaccact tcgacacctc ttatgaatta gaatctctcc ttgaactgcg cgtgaagcgc 120

caactgttgg cggaagtaca gtccatctgt ccgcctggcg tgaccattat gaacgtgcgt 180

cagggcgaac ctttaggttt aggccactcc attttatgtg cacgacccgc cattggcgac 240

aacccatttg tcgtggtact gccagacgta gtgatcgacg acgccagcgc cgacccgctg 300

cgctacaacc ttgctgccgt gattgcgcgc ttcaatgaaa cgggccgtag ccaggtgctg 360

gcaaaacgta tgccgggtga cctctctgaa tactccgtca ttcagaccaa agaaccgctg 420

gaccgtgaag gcaaagtcag ccgcattgtt gaattcatcg aaaaaccgga tcagccacaa 480

acgctggact cagacatcat ggccgttggt cgctatgtgc tttctgccga tatttggccg 540

gaacttgaac gcactcaacc tggtgcatgg gggcgtattc agctgactga tgccattgct 600

gaactggcga aaaaacagtc cgttgatgca atgctgatga ctggtgaaag ctacgactgc 660

ggtaaaaaaa tgggctatat gcaggcgttt gtgaagtatg gattgcgcaa cctgaaagaa 720

ggggcgaagt tccgtaaagg gattgagaag ctgttaagcg aa taatgaaa atctgaccgg 780

atgtaacggt tgataagaaa attataacgg cagtgaggat tcgtggcgaa agtaatttgt 840

tgcgaatatt cctgccgttg ttttatataa acaatcagaa taacaacgag ttagcaatag 900

gattttagtc aaagttttcc aggattttcc ttgtttccag agcgaattgg taagacaatt 960

agcgtttgaa tttttcgagt taagcgcgag tgggtaacgc tcgtcacatc gtagacatgc 1020

atgcagtgct ctggtagctg taaagccagg ggcggtagcg ttatttatat tcataggtct 1080

Orf1 is initial

aaaatagata accgag atga aaatagctat aggtatatca acattaaata atggaataaa 1140

tcaggtttgg gaaaaaataa aaaatatacc agatgaattt ttaataataa tttcacatca 1200

agtgactgat aagcaaaaac aacatgggga tatttgtaag aaaaacaatg tgataataat 1260

aacgacatac tgtaaagggt taagtaaaag cagaaatatt ttattggcta ctgcatttca 1320

aaaaagcgtt gattatatga ttataagtga tgatgatgtc gcttaccttg ttaatggact 1380

ttatgaacta aaagatagaa taatcgaaga tagaggcaag tatcattatc aaattcaaag 1440

ttgtacgcaa gacggaagac taaggaaaaa atatccgcta atgaggaaaa ggctaaatag 1500

attgtcagca tttaacattt cttcaataga aatgtgcctt aatgtgaatc aaatacagga 1560

atgtaatgtt tggtttgatg agtgtttcgg attgggggct agatataaag cgggtgaaga 1620

accgattttt gttacggatc ttatgaaaac aaaaaataat attattttta tacctgtcac 1680

aattactgtg catccaatag aaagttctgg taaaaaaatt tataatgaaa cagatgccct 1740

atcagataga agtgctattt ttgtacgctg tggtggtaga tatttagggt tactatatat 1800

cttcatattc tgggtgaaaa aatttctttt aaaaaaaaaa tgcggcagaa ttaaaaaaat 1860

The initial orf1 of orf2 stops

agctgcatta tttattctgc taaaaggata ttcccgat at gaatattta t aaaaacagca 1920

atgaaaattc attgtcaaat aataactgta ttgtatattt tattcctttg ttattattgg 1980

caacattctt tttccctctt gttgtattga tatttattgg tgccctctcg ccattgttac 2040

acccagtatt gcgtaatttt tatttttatg cactattggt gtttattatt attttttttt 2100

ccacactaaa accatttgga gatattgcag agtatcttca tgtttaccat gagttaaatt 2160

ataatttaat tgatgttttt ggctattcaa gatttggtga tgggctggaa tttatgtttt 2220

tagccatcat gaaatttatt gggtatattt cgggtggcaa tgatgaagta tttttacttt 2280

ctacttattt tctgatcgtc ttttttttat ccaagattct taaagatgtt gataaaaaat 2340

ataaactttt tttattatcg cttttctttt ttaacttagg atttatagaa gttacatcat 2400

attttcttcg acaggtttta tctgtggttg tttttctgta tgctataaat gagcgttcat 2460

ttaaaaaata tatctttttt ttgttgagtg ttttttttca catgtctgca gttgttaatg 2520

tttttatata tgcagtttat atgatctttg gttcaaaaaa atacccatat gcaaaaattg 2580

ttttttctat tcttattgga cttttagttg tgttttttgt ggtttataat acgccaattt 2640

attctgtgct attatcgaaa ttcacttcag tttctgggaa cgataaattc acacgtttgc 2700

cgttaaatta tattatcatt acagttgtaa atatatgctt tattataatg atagggaaga 2760

gaagtaaatg tgatgatttt aataagattt tattttgcaa ggagtgtttt ctattcttaa 2820

tattattacc tttcccagca ctttcgaata gattgggtat gattattttt ggtttttatc 2880

cctactttat cttaccctat ctaaaagctt ttgagagaaa aggtaagagt aaatatgccc 2940

tacttatttg tttatatgta gtaaatttag tgcctttttt gtatttaatg tataatgtgt 3000

ctttagggaa taacatgttt actttcctaa ataatcatcc atttactgag ggtgtgtatg 3060

gtatgattga ttacatattg gaggcaattg ataaaggtgt taactatatc aatgaaggta 3120

It is initial that orf2 stops orf3

ac tagcacgt gaaaaaatac ttaagtaacc tattttggtt gttgagtgac cgagtgttc a 3180

tgctggtatt tcagttgtca cttggtgctt ctatcaaaag aatctatggg ttagatatct 3240

tgggtagttg ggcaacgata atgaatatct cacaaatatt actatcgtta tttttatttg 3300

gcattgatat agtcgtagtt aaaagaattg ttgaaaatcc atcatcaacg ggcactgaga 3360

ttggatgtgc attattctta cagtttttag gactgttatt atatgcatct gcttttatta 3420

ccattgttat ttatttttac tatgatatac cgttcgcttt tatattcgtg gcgatattaa 3480

tcgttgctaa tttttttagc ttgtatgcta aagtaatatt ttttcattat tcggcattgg 3540

ttgaatcaaa atatcgtgct atcacaattc taagtagtgt ggccgtttca tatggttatc 3600

tttggtgttg tatatatttt ggttggcatg tcttttatgc ttatgttttc ttttatttaa 3660

ttcaagctct ttttagtttt actatataca aattctattt cccttattca gcaaaatgga 3720

cgattgattt agaattagtc aaaatgtatt tttttatggg gagtaaactc attgtatcaa 3780

ctattagtgt atcactattt acacagtgtg atgtgatctt attagagtct cttacaggta 3840

caaaagaggc tggggcattt agtgcagctc ttaggttatc agccatctgg tttatgtgtg 3900

gaggtttgat agcgaacgct ttttttccaa aaattgttca gcttgaaaaa ataggagagg 3960

aagaatcatt tatctttttg aaatggatat gcggagttgt aagtgtaatc tctatatatg 4020

gtgcaattat tatgattgct ctgtcaccta taattataaa aatactatat ggcgataata 4080

tggatttatc ggcgcaagta ttaatggttc atatgtggag tggtgttttt gtttttttag 4140

gatcattttc atctaaatgg ttatttagta agaattatat taatttagaa gtgataaaaa 4200

ccattattgc tgcaattttg aatataactt tgaatatcat tgttatacca aaatatggag 4260

cggttggcgc tgcttctgta tcattgcttt cttattttat tgctaacttt ttcattttta 4320

Orf3 stops

tatttatacc aaaaac taaa aatgtttaaa atgcaattac agagtttgaa gtatattatt 4380

Orf4 is initial

tttccatggc gtttgattaa tgattttgga agggttag at gtcaatttca gtaattactc 4440

ctgtattcaa tcgagctttg ttagtctatg aattatatga atccttaatg tcacaacaat 4500

cttatgattt tgaatgggtg ataattgatg atggttctac tgataattta aaagaggtta 4560

ttgacaaaat agcgtcgaca tccccattta aaatcatata tagatataag aagaacggtg 4620

gaaaacacac agccttaaat atagggatag aaatgtcatc gtttaactgg atatttattg 4680

ttgatagtga tgatatatta acgcccaacg ctattgctct agccaatgaa aaaatccagg 4740

caattgttga tgataaatgt aaagggatgg ttttcctgag ggggtataaa actacaaaag 4800

agattgtagg taaagcggaa acaattgaaa atatttctct ggaaaagttt gcaggtacaa 4860

agggagataa agcattaatc ataaaacgtg attctttact taaaaatcaa tttcctgttt 4920

ttgatggtga aaattttata actgaggcat tggtttggaa ttcaatttta gaaaatggct 4980

actttaaata tttcaatgag atcatctatt atagtgaata tttaccagga ggtttaactt 5040

ctaattatac tgatcttttg cggaaaaata tcaatggcac gatggctttt gttatcaata 5100

acttgaatct aaaaggtctt ggtattaatg tcattaaaca aaccgttttt cattttattc 5160

ccatttttaa tattagtaat ctaatagttg taaagaaaaa gacaaagttt actgttttcg 5220

ttttatttat tacatgtctt tttcttgtaa aaatcaagaa taaagttaaa ggaaaatcgt 5280

Orf5 is initial, and orf4 stops

tatgataccg gcaataattc attatatttg gcttggtaaa agtgaaatcc ctaaaatata 5340

tttagattgt atggagtcgt ggaaagaaca tgctgtaaat tacgattgct atttatggaa 5400

tgaggattct tatagaaagg aatttggtca gaatgatttt gttgaagaaa tgatccaaag 5460

gaaaaagttt gcatttgctg cagatttgat ccgatgtgat gtattatatc gttttggtgg 5520

tatatatctc gatactgata tggaattagt tcgggatatt tctgcattgc gaaaaaatat 5580

tgcatttatc ggcgaagaag atattgatac gcctagttgt ggtattttgg gttgtgaacc 5640

caaattttgg cttttccaag agctaaaagc agctgtcata aaagcaaatg gtatgcaaac 5700

aattcctttt cttttaaaga atattttgga cttacatggt gtaaaaaaaa tagattcaca 5760

agatatttct actattaaag atatcacaat atactctgat aagtattttt atccatataa 5820

cccttatggt agtgccaaac gatcacaatt actttataga tatataacaa aagattgtta 5880

tgctatacat cattgggcta aaagttggaa gctttctttt ttagagagaa ttaaaagaaa 5940

Orf5 stops, and orf6 is initial

aattatcatg cgatatcgta aggag taat a tgaatatcct agtaacaggt ggtgctggtt 6000

atattggttc gcatacggta cttcgcttat tagaaaatga aaatgaaatt actgttgtgg 6060

ataatttggt taattcttca tctgaggtaa ttaagcgtgt agagaatatc acgcaaaaaa 6120

gcatttgctt tatcgaaatg gatatactta acacagaatt attacacgaa gtaattatta 6180

ataaagatat tgatgcggtc attcattttg ctgggttgaa atctgtttct gagtcaatca 6240

gtaggcctct tgaatattat aagaataatg ttcagggaac aattagtgtc cttagtgcta 6300

tgataaatag caaagccaag aaaattattt ttagttcttc tgcaacggtt tatggtgagc 6360

ctgaacaaat tccattaaat gaaaaatgta aagtaggtgg tacaacaaat ccttatggca 6420

cttcgaaatt aatggcagaa caaatccttt gtgattttgc aaaagcaaat tatggttttg 6480

atattatttc attacgatac tttaatccgg taggagcaca tcccagtgga atgataggtg 6540

aggctccgaa tgggattcct aataatcttg taccatatct cactaaggtt gctataggcg 6600

aattagattc attgaaaata tttgggaatg attatcctac cagggacgga tatggagttc 6660

gggattttat tcatgttatg gatcttgccg atggacatat tgctgctttg aatgcagagt 6720

ttaaagaaaa ttctatcagg atatataatt taggtacagg taaaggatat tcagtattag 6780

agttggttga tactttcgag agaattatag ctagaaaaat aaataagtgt gttatatcaa 6840

ggcgagatgg agatatagca gaatgctggt ctgatccaat gttagccttc aatgaactcg 6900

gttggtcagc caaattcaat ttagaggata tgctacgaga ttcttggaat tggcaaataa 6960

Orf6 stops, and orf7 is initial

aaaacccgaa aggctacgat caaaataaag ggaggaaa ta ata atggatt atcttgtgtc 7020

aattattatg cccagttata attctgaatt cacaattaag gaaagtataa aatcggtaat 7080

agaacaaaca tattctaatt gggagttgtt aattactgat gactgctcaa cagatagaac 7140

ttgtcagata gttaaagaat ttgttgagca agatgacagg ataaaacttt ttgtttcaga 7200

taaaaataaa ggcgcaggcg cagcaagaaa caactcaata aaagagtcta gtggacgatt 7260

tttggctttt ttagatagtg atgatctatg ggcacccgat aaactaaaag agcagattaa 7320

ttatatgatt atgaatggct acgcccttac ttatactgca tatagtaaaa ttgatgcata 7380

tggtaatatt aaaaaagata ttcagccccc atcaaaagtc gatttttctt ctttactgaa 7440

atccaatgtt atcggatgcc ttactgcaat ttatgacacg gaagttgtgg gtaaagtata 7500

tatgccactc atacgaaagc gtcaagatat ggcattatgg ctaataatcc tgcaaaaaat 7560

agattatgcc cactgcctaa ataaaaacct agcattttat cgcgaaggtc atttaagttt 7620

atcatcgaat aagataaaaa taataaaatc gcaatgggag ttttaccgtt attatcttgg 7680

ttttggatat gtaaaggcaa tgtattattt tctgcattat atccagcgtg cattaagaaa 7740

Orf7 stops

acacgct taa gagtttaact ctatttgttt aattttaaaa cattgataat aattgagtct 7800

atcattcatt tattgcatgt cttattttat atctgttatc cttattaacc gtcaaatccc 7860

gcggtaaccc cctgacagga gtaaacaatg tcaaagcaac agatcggcgt agtcggtatg 7920

gcagtgatgg ggcgcaacct cgcgctcaac atcgaaagcc gtggttatac cgtctctatt 7980

ttcaaccgtt cccgtgaaaa aacggaagaa gtgattgccg aaaatccggg caagaaactg 8040

gttccttact atacggtgaa agagtttgtt gaatctctgg aaacgcctcg tcgcatcctg 8100

ttaatggtga aagcaggtgc aggcacggat gctgctattg attccctcaa gccatacctc 8160

gataaaggtg acatcatcat tgatggtggt aataccttct tccaggacac tattcgtcgt 8220

aaccgtgagc tttctgcaga aggctttaat ttcattggta ccggtgtttc cggcggtgaa 8280

gaaggtgcgc tgaaaggtcc ttccattatg cctggtgggc agaaagaagc ctatgaactg 8340

gttgctccga tcctgaccaa aatcgccgca gtggttgaag atggcgagcc atgcgttacc 8400

tatattggtg ccgatggtgc aggtcactat gtgaagatgg ttcacaacgg tattgaatac 8460

ggcgatatgc agctgattgc tgaagcctat tctctgctta aaggtggcct gaatctttcc 8520

aacgaagaac tggcgcagac gtttaccgag tggaataatg gtgaactgag cagctacctg 8580

atcgacatca ccaaagatat cttcaccaaa aaagatgaag acggtaacta cctggttgat 8640

gtgattctgg atgaagcagc aaataaaggt acgggcaaat ggaccagcca gagtgcgctg 8700

gatctcggcg aaccgctgtc gctgattacc gagtctgtgt ttgctcgtta tatctcttct 8760

ctgaaagatc agcgcgttgc cgcgtctaaa gttctctctg gcccgcaagc gcagccagct 8820

ggcgacaagg ctgagttcat cgaaaaagtt cgtcgtgctc tgtatctggg caaaatcgtt 8880

tcttacgccc aaggcttctc tcagctgcgt gcggcgtctg aagagtacaa ctgggatctg 8940

aactacggcg aaatcgcgaa gattttccgt gctggctgca tcatccgtgc gcagttcctg 9000

cagaaaatca ctgatgcata tgccgaaaat ccgcagatcg ctaatctgct gctggctccg 9060

tactttaaac aaattgccga tgattaccag caggcgctgc gtgatgtcgt tgcttatgca 9120

gtgcagaacg gtatcccggt tccgaccttc gccgctgcgg ttgcctatta tgacagctac 9180

cgtgccgctg ttctgcctgc gaacctaatc caggcccagc gcgacta 9227

The above only is preferred embodiment of the present invention, is not the present invention is done the restriction of any form, allly according to the technology of the present invention essence above embodiment is done any simple modification, equivalent variations and modification, still belongs to the technical solution of the present invention scope.

Claims (9)

1, a kind of Nucleotide of the O-antigen-specific to shigella dysenteriae 8 types is characterized in that it is the isolating Nucleotide shown in SEQ ID NO:1,9227 bases of total length; Perhaps described base with one or more insertions, disappearance or replacement keeps the Nucleotide of the SEQID NO:1 of described isolating functional nucleotide simultaneously.

2, according to the Nucleotide of the described O-antigen-specific to shigella dysenteriae 8 types of claim 1, it is characterized in that, it is 7 genomic constitutions by called after wzx, wzy, orf1, orf6, orf4, orf5, orf7, all between galF gene and gnd gene.

According to the Nucleotide of the described O-antigen-specific to shigella dysenteriae 8 types of claim 2, it is characterized in that 3, following gene with height O-antigen-specific is: the gene of transhipment enzyme comprises the wzx gene; Pol gene wzy gene; Glycosyltransferase gene comprises orf1, orf4, orf5, orf7 gene; Wherein said gene: orf1 is the Nucleotide of 1097 to 1912 bases among the SEQ ID NO:1; Wzy is the Nucleotide of 1899 to 3125 bases among the SEQ ID NO:1; Wzx is the Nucleotide of 3180 to 4349 bases among the SEQ ID NO:1; Orf4 is the Nucleotide of 4419 to 5285 bases among the SEQ ID NO:1; Orf5 is the Nucleotide of 5282 to 5968 bases among the SEQ ID NO:1; Orf7 is the Nucleotide of 7004 to 7750 bases among the SEQ ID NO:1.

4, according to the Nucleotide of claim 1 or 2 described O-antigen-specifics to shigella dysenteriae 8 types, it is characterized in that it is to come from described wzx gene, wzy gene or glycosyltransferase gene orf1, orf4, orf5, orf7 gene; And their mixing or their reorganization.

5, according to the Nucleotide of the described O-antigen-specific to shigella dysenteriae 8 types of claim 4, it is characterized in that the oligonucleotide of the described orf1 of coming from gene is to being: the Nucleotide of 1103 to 1120 bases among the SEQ ID NO:1 and the Nucleotide of 1877 to 1896 bases; The Nucleotide of 1194 to 1213 bases among the SEQ ID NO:1 and the Nucleotide of 1763 to 1780 bases; The Nucleotide of 1261 to 1279 bases among the SEQ ID NO:1 and the Nucleotide of 1686 to 1704 bases; The oligonucleotide that comes from the wzy gene is to being: the Nucleotide of 1976 to 1995 bases among the SEQ ID NO:1 and the Nucleotide of 3096 to 3114 bases; The Nucleotide of 2145 to 2162 bases among the SEQ ID NO:1 and the Nucleotide of 2997 to 3014 bases; The Nucleotide of 2249 to 2268 bases among the SEQ ID NO:1 and the Nucleotide of 2834 to 2850 bases; The oligonucleotide that comes from the wzx gene is to being: the Nucleotide of 3183 to 3191 bases among the SEQ ID NO:1 and the Nucleotide of 4261 to 4278 bases; The Nucleotide of 3241 to 3257 bases among the SEQ ID NO:1 and the Nucleotide of 4091 to 4109 bases; The Nucleotide of 3301 to 3318 bases among the SEQ ID NO:1 and the Nucleotide of 3991 to 4019 bases; The oligonucleotide that comes from the orf4 gene is to being: the Nucleotide of 4441 to 4460 bases among the SEQ ID NO:1 and the Nucleotide of 5201 to 5220 bases; The Nucleotide of 4501 to 4520 bases among the SEQ ID NO:1 and the Nucleotide of 5115 to 5132 bases; The Nucleotide of 4601 to 4620 bases among the SEQ ID NO:1 and the Nucleotide of 5072 to 5088 bases; The oligonucleotide that comes from the orf5 gene is to being: the Nucleotide of 5311 to 5330 bases among the SEQ ID NO:1 and the Nucleotide of 5947 to 5965 bases; The Nucleotide of 5401 to 5420 bases among the SEQ ID NO:1 and the Nucleotide of 5895 to 5912 bases; The Nucleotide of 5501 to 5520 bases among the SEQ ID NO:1 and the Nucleotide of 5828 to 5846 bases; The oligonucleotide that comes from the orf7 gene is to being: the Nucleotide of 7059 to 7076 bases among the SEQ IDNO:1 and the Nucleotide of 7651 to 7668 bases; The Nucleotide of 7099 to 7106 bases among the SEQ IDNO:1 and the Nucleotide of 7521 to 7540 bases; The Nucleotide of 7161 to 7180 bases among the SEQ IDNO:1 and the Nucleotide of 7481 to 7500 bases.

6, the Nucleotide of the described O-antigen-specific to shigella dysenteriae 8 types of claim 1 detect express the antigenic bacterium of O-, in the application of other polysaccharide antigen of the O-antigen of identifying bacterium and bacterium.

7, the recombinant molecule of the Nucleotide of the described O-antigen-specific to shigella dysenteriae 8 types of claim 1 provides application in the O-antigen bacterial vaccine of expressing shigella dysenteriae 8 types in preparation.

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

9, the separation method of the Nucleotide of the described O-antigen-specific to shigella dysenteriae 8 types of claim 1 is characterized in that it comprises the steps:

(1) genomic extraction: 37 ℃ of incubated overnight shigella dysenteriae 8 types in the LB of 5mL substratum, centrifugal collecting cell; With the pH value is 8.0 500ul 50mM Tris-HCl and 10ul 0.4M EDTA re-suspended cell, 37 ℃ of incubations 20 minutes, and 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 ℃ 30 minutes; Add equal-volume phenol extracting mixture, get supernatant and use isopyknic phenol again: chloroform: primary isoamyl alcohol extracting twice, get supernatant again with isopyknic ether extracting to remove remaining phenol; Supernatant rolls out DNA and washes DNA with 70% ethanol with glass yarn with 2 times of volume ethanol deposit D NA, and DNA is resuspended among the 30ul TE; Genomic dna detects by 0.4% agarose gel electrophoresis;

(2) by the O-antigen gene in pcr amplification shigella dysenteriae 8 types bunch: with the genome of shigella dysenteriae 8 types is that template is passed through its O-antigen gene of Long pcr amplification bunch; Be 5 '-ATT GTGGCT GCA GGG ATC AAA GAA AT-3 ' according to the JumpStart sequences Design upstream primer that often is found in O-antigen gene bunch promoter region at first, the gnd gene design downstream primer according to O-antigen gene bunch downstream is 5 '-TAG TCG CGT GNG CCT GGA TTA AGT TCG C-3 ' again; PCR method 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 use the purification kit purified pcr product;

(3) make up O-antigen gene bunch library: make up O-antigen gene bunch library with the Novagen DNaseI shot gun method that is modified, reaction system is a 300ng PCR purified product, 0.9ul 0.1M MnCl ₂, the DNaseI reaction of the 1mg/ml of 1ul dilution in 1: 2000 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: the extracting of primary isoamyl alcohol mixing solutions once, phenol: chloroform: primary isoamyl alcohol mixed volume ratio is 25: 24: 1; After using isopyknic ether extracting once again,, and wash precipitation, be resuspended at last in the 18ul water with 70% ethanol with the dehydrated alcohol deposit D NA of 2.5 times of volumes; 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: be connected 24 hours with carrier in 16 ℃ after the extracting of primary isoamyl alcohol mixing solutions and the extracting of equal-volume ether, cumulative volume is 90ul, chloroform: the mixed volume ratio of primary isoamyl alcohol is 24: 1; 10 * the buffer of 9ul and the T4DNA ligase enzyme of 25 units are wherein arranged; Be that the NaAc of 5.2 3M and the dehydrated alcohol precipitation of 2 times of volumes are connected mixture with the pH value of 1/10 volume at last, wash precipitation with 70% ethanol again, be dissolved in after the drying and obtain connecting product in the 30ul water; The preparation method of electricity consumption transformed competence colibacillus cell prepares competence escherichia coli DH5a cell, get after 2-3ul connects product and 50ul competence escherichia coli DH5a mixes, forward in the electric shock cup of 0.2cm of Bi0-Rad company and shock by electricity, voltage is 2.5 kilovolts, time is 5.0 milliseconds to 6.0 milliseconds, the SOC substratum that adds 1ml after the electric shock immediately in cup makes the bacterium recovery, then bacterium is coated in and contains penbritin, on the LB solid medium of X-Gal and IPTG, 37 ℃ of incubated overnight, obtain blue white bacterium colony next day, with the white colony that obtains promptly the white clone forward on the LB solid medium that contains acillin and cultivate, from each clone, extract plasmid simultaneously, and cutting the segmental size of evaluation insertion wherein with the EcoRI enzyme, the white that obtains clone group has constituted the O-antigen gene bunch library of dysentery will Hayes 8 types;

(4) to the cloning and sequencing in the library: from the library, select insert 120 clones of fragment more than 700bp by automatic dna sequencer to unidirectional order-checking of insertion fragment among the clone, make sequence reach 90% fraction of coverage; Residue 10% sequence is again by with the partial sequence backward sequencing, thereby obtains all sequences of O-antigen gene bunch;

(5) splicing of nucleotide sequence and analysis: with the Pregap4 of Staden package software package and the splicing of Gap4 software with edit all sequences, thereby obtain the Nucleotide full length sequence of the O-antigen gene bunch of shigella dysenteriae 8 types; The quality of sequence is mainly guaranteed by two aspects: 1, the genome of shigella dysenteriae 8 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 8 type O-antigen genes bunch, with orffinder software discovery gene, find the reading frame of 7 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 Artemis software 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 8 types at last;

(6) screening of specific gene: at wzx, wzy, orf1, orf4, orf5, the orf7 gene design primer in the O-antigen gene of shigella dysenteriae 8 types bunch; Respectively design three pairs of primers in each gene, every pair of primer is distributed in the different places in the corresponding gene, to guarantee its specificity; Is that template is carried out PCR with these primers with the genomes of 166 strain intestinal bacteria and 43 strain Shigellaes, the correct band of any size does not all increase in other groups, promptly do not obtaining any PCR product band in the array mostly, though in the minority group, obtain PDR product band, but its size does not meet the expection size, so the O-antigen of wzx, wzy, orf1, orf4, orf5, orf7 gene pairs shigella dysenteriae 8 types all is high special.