CN1252266C - nucleotide and its use - Google Patents

nucleotide and its use Download PDF

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CN1252266C
CN1252266C CNB031005373A CN03100537A CN1252266C CN 1252266 C CN1252266 C CN 1252266C CN B031005373 A CNB031005373 A CN B031005373A CN 03100537 A CN03100537 A CN 03100537A CN 1252266 C CN1252266 C CN 1252266C
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gene
nucleotide
bases
antigen
seq
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CN1438229A (en
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王磊
杨静华
冯露
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Nankai University
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Nankai University
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Abstract

The present invention provides a specific nucleotide for the O-antigens of Shigella dysenteriae 13. The specific nucleotide is the nucleotide complete sequence of gene clusters in Shigella dysenteriae 13 for controlling the synthesis of O-antigens. The present invention also comprises the structure of O-antigen gene clusters and the oligonucleotide of glycosyltransferase genes and unit processing genes (comprising wzx genes or genes with the similar functions of wzx, wzy genes or genes with the similar functions of wzy) stemmed from the O-antigen gene clusters of Shigella dysenteriae 13, and a method for obtaining bacteria O-antigen gene clusters. The present invention verifies that the oligonucleotide has high specificity to the O-antigen genes of Shigella dysenteriae 13 through PCR. The present invention also discloses a method for using the oligonucleotide of the present invention to detect and identify Shigella dysenteriae 13 in human bodies and environment.

Description

Nucleotide and application thereof to the O-antigen-specific of Shigella bogdii 13 types
Technical field
The present invention relates to the complete nucleotide sequence of control O-antigen synthetic gene cluster in Shigella bogdii 13 types (Shigella bodyii 13), particularly relate in Shigella bogdii 13 types oligonucleotide in the control O-antigen synthetic gene cluster, can utilize these oligonucleotide of O-antigen-specific Shigella bogdii 13 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: earlier by glycosyltransferase nucleoside diphosphate monose is transferred to-the individual fat molecule that is fixed on the cell inner membrance on, then in the inboard synthesis of oligose unit of inner membrance, the antigenic oligosaccharide unit of O-is transferred to the inner membrance outside by the transhipment enzyme again, then aggregate into polysaccharide by polysaccharase, be connected to again and form lipopolysaccharide molecule [Whitfield, C. (1995) " Biosynthesis of lipopolysaccharide Oantigens " .Trends in Microbiology.3:178-185 on the glycolipid molecule; Schnaitman, C.A.andJ.D.Klena. (1993) " Genetics of lipopolysaccharide biosynthesis inentericbacteria " .Microbiological Reviews, 57 (3): 655-682].Coding is responsible for the generally adjacent arrangement on karyomit(e) of gene of all enzyme molecules of O-antigen synthetic, form a gene cluster [Reeves, P.R., et al. (1996) " Bacterial polysaccharide synthesis and genenomenclature " Trends in Microbiology, 4:495-503].In Shigellae, intestinal bacteria and Salmonellas, O-antigen gene [Lei Wang.et al (2001) " Sequence analysis of four Shigella boydii O-antigen loci:implicationfor Escherichia coli and Shigella relationships " .Infection andImmunity, 11:6923-6930 bunch between galF and gnd gene; Lei Wang and Peter Reeves (2000) " 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 S.enterica major serogroups (A; B; C2; andD) ", J.Clin.Microbiol.31:2118-2123].Luk, the method for et.al is with corresponding to Salmonellas serotype E 1, D1 obtains the oligonucleotide special to the Salmonellas of different serotypes after the nucleotide sequence of the CDP-abequose in the A, the O-antigen of B and C2 and the synthetic gene of CDP-tyvelose is arranged.1996, Paton, the A.W et.al serotype [" Molecular microbiologicalinyestigation 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.colf 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, intestinal bacteria have 166 kinds of different O-antigens, the two sibship is very near, and it is the total [Ewing of intestinal bacteria and Shigellae that 12 kinds of O-antigens are arranged, W.H. (1986) " Edwards and Ewing ' s identification of theEnterobacteriaceae " .Elsevier Science Publishers, Amsterdam, TheNetherlands; T.cheasty, et al. (1983) " Antigenic relationships betweenthe enteroinvasive Escherichia coli antigensO28ac; O112ac; O124, O136, O143; O144; O152 and O164 and Shigella Oantigens " J.clin Microbiol, 17 (4): 681-684], traditional serological method can not be distinguished them.
Summary of the invention
The Nucleotide that the purpose of this invention is to provide a kind of O-antigen-specific to Shigella bogdii 13 types.It is the Nucleotide in the O-antigen gene bunch of Shigella bogdii 13 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 bogdii 13 types.
Another object of the present invention has provided the gene of the O-antigen gene bunch that constitutes Shigella bogdii 13 types: the gene of transhipment enzyme is the wzx gene or with wzx the gene of identity function is arranged; Pol gene is the wzy gene or with wzy the gene of identity function is arranged; Glycosyltransferase gene comprises orf4, orf5, orf6, wbjE, orf11, orf12 gene; Sugar synthesis path gene comprises wbjB, wbjC, wbjD.
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 bogdii 13 types respectively comprises orf4, orf5, orf6, wbjE, orf11, orf12 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 bogdii 13 types; Especially the oligonucleotide of listing in the table 1, they are high specials to the O-antigen of Shigella bogdii 13 types, and these oligonucleotide are also reconfigurable, the oligonucleotide after the combination also is a high special to the O-antigen of Shigella bogdii 13 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 bogdii 13 types of Shigella bogdii 13 types by these methods.
Also purpose of the present invention has provided the method for the complete sequence of the O-antigen gene bunch that separates Shigella bogdii 13 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 bogdii 13 types, and it is the isolating Nucleotide shown in SEQ ID NO:1,14504 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 bogdii 13 types, it is by 12 genomic constitutions, all between galF gene and gnd gene.
The Nucleotide of aforesaid O-antigen-specific to Shigella bogdii 13 types, wherein said gene is: transhipment enzyme gene comprises the wzx gene or with wzx the gene of identity function is arranged; Pol gene comprises the wzy gene or with wzy the gene of identity function is arranged; Glycosyltransferase gene comprises orf4, orf5, orf6, wbjE, orf11, orf12 gene; Wherein said gene: wzx is the Nucleotide of 1695 to 3032 bases among the SEQ ID NO:1; Wz y is the Nucleotide of 3019 to 4197 bases among the SEQ ID NO:1; Orf4 is the Nucleotide of 4184 to 4969 bases among the SEQ ID NO:1; Orf5 is the Nucleotide of 4972 to 6042 bases among the SEQ ID NO:1; Orf6 is the Nucleotide of 6138 to 7172 bases among the SEQ ID NO:1; WbjE is the Nucleotide of 10193 to 11410 bases among the SEQ ID NO:1; Orf11 is the Nucleotide of 11412 to 11930 bases among the SEQ ID NO:1; Orf12 is the Nucleotide of 11935 to 13056 bases among the SEQID NO:1.
The Nucleotide of aforesaid O-antigen-specific to Shigella bogdii 13 types wherein comes from described wzx gene, wzy gene or glycosyltransferase gene orf4, orf5, orf6, wbjE, orf11, orf12 gene; Or the oligonucleotide in the sugared synthesis path gene; And their mixing or their reorganization.
The Nucleotide of aforesaid O-antigen-specific to Shigella bogdii 13 types, the oligonucleotide of the wherein said wzx of coming from gene is to being: the Nucleotide of 2012 to 2030 bases among the SEQ ID NO:1 and the Nucleotide of 2931 to 2949 bases; The Nucleotide of 1887 to 1904 bases among the SEQ ID NO:1 and the Nucleotide of 2599 to 2616 bases; The Nucleotide of 2009 to 2026 bases among the SEQ ID NO:1 and the Nucleotide of 2493 to 2510 bases; The oligonucleotide that comes from the wzy gene is to being: the Nucleotide of 3252 to 3269 bases among the SEQ ID NO:1 and the Nucleotide of 4081 to 4099 bases; The Nucleotide of 3206 to 3224 bases among the SEQ ID NO:1 and the Nucleotide of 4160 to 4176 bases; The Nucleotide of 3565 to 3582 bases among the SEQ ID NO:1 and the Nucleotide of 4083 to 4100 bases; The oligonucleotide that comes from the orf4 gene is to being: the Nucleotide of 4399 to 4417 bases among the SEQ ID NO:1 and the Nucleotide of 4943 to 4962 bases; The Nucleotide of 4368 to 4385 bases among the SEQ ID NO:1 and the Nucleotide of 4670 to 4688 bases; The Nucleotide of 4227 to 4244 bases among the SEQ ID NO:1 and the Nucleotide of 4821 to 4840 bases; The oligonucleotide that comes from the orf5 gene is to being: the Nucleotide of 5242 to 5261 bases among the SEQ ID NO:1 and the Nucleotide of 5677 to 5694 bases; The Nucleotide of 5071 to 5088 bases among the SEQ ID NO:1 and the Nucleotide of 5774 to 5791 bases; The Nucleotide of 5600 to 5617 bases among the SEQ ID NO:1 and the Nucleotide of 5889 to 5908 bases; The oligonucleotide that comes from the orf6 gene is to being: the Nucleotide of 6089 to 6106 bases among the SEQ ID NO:1 and the Nucleotide of 6904 to 6921 bases; The Nucleotide of 6306 to 6324 bases among the SEQ ID NO:1 and the Nucleotide of 6906 to 6923 bases; The Nucleotide of 6050 to 6067 bases among the SEQ ID NO:1 and the Nucleotide of 6996 to 7013 bases; The oligonucleotide that comes from wbjE is to being: the Nucleotide of 10379 to 10397 bases among the SEQ ID NO:1 and the Nucleotide of 10953 to 10970 bases; The Nucleotide of 10448 to 10465 bases among the SEQ ID NO:1 and the Nucleotide of 10813 to 10830 bases; The Nucleotide of 10199 to 10217 bases among the SEQ ID NO:1 and the Nucleotide of 10098 to 11115 bases; The oligonucleotide that comes from the orf11 gene is to being: the Nucleotide of 11472 to 11489 bases among the SEQ ID NO:1 and the Nucleotide of 11878 to 11894 bases; The Nucleotide of 11500 to 11517 bases among the SEQ ID NO:1 and the Nucleotide of 11686 to 11703 bases; The Nucleotide of 11689 to 11708 bases among the SEQ ID NO:1 and the Nucleotide of 11906 to 11923 bases; The oligonucleotide that comes from the orf12 gene is to being: the Nucleotide of 11971 to 11990 bases among the SEQ ID NO:1 and the Nucleotide of 12872 to 12891 bases; The Nucleotide of 11973 to 11990 bases among the SEQ ID NO:1 and the Nucleotide of 12998 to 13015 bases; The Nucleotide of 12682 to 12700 bases among the SEQ ID NO:1 and the Nucleotide of 12997 to 13014 bases.
The Nucleotide of aforesaid O-antigen-specific to Shigella bogdii 13 types is detecting the application of expressing the antigenic bacterium of O-, identify other polysaccharide antigen of the O-antigen of bacterium and bacterium in diagnosis.
The recombinant molecule of the Nucleotide of aforesaid O-antigen-specific to Shigella bogdii 13 types, and can provide the O-antigen of expressing Shigella bogdii 13 types by inserting to express, and become bacterial vaccine.
The application of the Nucleotide of aforesaid O-antigen-specific to Shigella bogdii 13 types, it is used for PCR, is used for hybridization and fluoroscopic examination or is used to make gene chip or microarray as probe as primer, the bacterium in available these method human body and the environment.
The separation method of the Nucleotide of aforesaid O-antigen-specific to Shigella bogdii 13 types, comprising following step:
(1) genomic extraction: 37 ℃ of incubated overnight Shigella bogdii 13 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 liquor and use isopyknic phenol again: chloroform: primary isoamyl alcohol (25: 24: 1) extracting twice, get supernatant liquor again with isopyknic ether extracting to remove remaining phenol.Supernatant liquor rolls out DNA and washes DNA with 70% ethanol with glass yarn with 2 times of volume ethanol deposit D NA, and DNA is resuspended among the 30ul TE, and genomic dna detects by 0.4% agarose gel electrophoresis;
(2) by the O-antigen gene in pcr amplification Shigella bogdii 13 types bunch: the O-antigen gene of Shigella bogdii 13 types is bunch by the Long pcr amplification.At first according to the JumpStart sequences Design upstream primer (5 '-ATT GTG GCT GCA GGG ATCAAA GAA AT-3 ') that often is found in O-antigen gene bunch promoter region, again according to the gnd gene design downstream primer (5 '-TAGTCG CGT GNG CCT GGA TTA AGT TCG C-3 ') in O-antigen gene bunch downstream.With the Expand Long Template PCR method of Boehringer Mannheim company amplification O-antigen gene bunch, the PCR response procedures was as follows: 94 ℃ of pre-sex change 2 minutes; 94 ℃ of sex change are 10 seconds then, 60 ℃ of annealing 30 seconds, and 68 ℃ were extended 15 minutes, and carried out 30 circulations like this.At last, continue to extend 7 minutes at 68 ℃, obtain the PCR product, the agarose gel electrophoresis with 0.8% detects the size and the specificity thereof of PCR product.Merge 6 pipe 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 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 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 Bao Shi will Hayes 13 types;
(4) to the cloning and sequencing in the library: from the library, select insert 120 clones of fragment more than 700bp by Shanghai biotechnology company limited with ABI377 type automatic dna sequencer to unidirectional order-checking of insertion fragment in cloning, make sequence reach 80% fraction of coverage.Residue 20% sequence is again according to the sequences Design primer that has obtained, and direct PCR and carry out backward sequencing and survey logically to the order-checking of PCR product or to known array from the genomic dna of Shigella bogdii 13 types obtains all sequences of O-antigen gene bunch again;
(5) splicing of nucleotide sequence and analysis: the Pregap4 and the splicing of Gap4 software of the Staden package software package of publishing with Britain Camb MRC (Medical Research Council) Molecular Biology Lab and edit all sequences, thereby obtain the Nucleotide full length sequence of the O-antigen gene bunch of Shigella bogdii 13 types, the quality of sequence is mainly guaranteed by two aspects: 1) genome of Shigella bogdii 13 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 bogdii 13 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 12 openings, determine also that with the function of finding the reading frame that these are open what gene they are with the genetic comparison among the blast groupware and the GenBank, finish gene annotation with the Artemis software at Britain sanger center again, do accurate comparison between DNA and protein sequence with Clustral W software, obtain the structure of the O-antigen gene bunch of Shigella bogdii 13 types at last;
(6) screening of specific gene: at wzx, wzy, orf4, orf5, orf6, wbjE, orf11, the orf12 gene design primer in the O-antigen gene of Shigella bogdii 13 types bunch; Respectively designed 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, all primers all obtain positive findings in Shigella bogdii 13 types, 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 PCR product band, but its size does not meet the expection size, so the O-antigen of wzx, wzy, orf4, orf5, orf6, wbjE, orf11, orf12 gene pairs Shigella bogdii 13 types all is high special.
First aspect just of the present invention provides the full length nucleotide sequence of the O-antigen gene bunch of Shigella bogdii 13 types, its complete sequence shown in SEQ ID NO:1,14504 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 (listing) of the O-antigen gene bunch of Shigella bogdii 13 types by method of the present invention in Fig. 1, it is altogether by 12 genomic constitutions.Each gene box indicating in Fig. 1, 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.The initiator codon of open reading frame can be ATG, also can be GTG.Two ends at O-antigen gene bunch are galF gene and gnd gene, and these two not responsible O-of gene are antigenic synthetic, and we are just with the increase full length sequence of O-antigen gene bunch of their one section sequences Design primer.
Second aspect of the present invention provides the gene in the O-antigen gene bunch of Shigella bogdii 13 types, promptly transports enzyme gene (wzx gene or the gene of identity function arranged with wzx); Pol gene (wzy gene or the gene of identity function arranged with wzy); Glycosyltransferase gene comprises orf4, orf5, orf6, wbjE, orf11, orf12 gene; Special sugared synthesis path gene in the bacterial polysaccharides antigen comprises wbjB, wbjC, wbjD gene, and 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 bogdii 13 types.
The 3rd aspect of the present invention, wzx gene in the O-antigen gene bunch that comes from Shigella bogdii 13 types is provided or the gene, wzy gene of identity function is arranged or the gene and the glycosyltransferase gene of identity function are arranged with wzy with wzx, the oligonucleotide that comprises orf4, orf5, orf6, wbjE, orf11, orf12 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 bogdii 13 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, though in some bacterium, obtained PCR product band, but its size does not meet the expection size, this is that this problem can be avoided by being PCR with intragenic other primer because primer is attached to genomic other position and causes.So, can determine these primers promptly the listed oligonucleotide of table 1 be high special to Shigella bogdii 13 types and O-antigen thereof.
The separation method of the Nucleotide of described O-antigen-specific to Shigella bogdii 13 types comprises the steps: 1) genomic extraction; 2) the O-antigen gene in pcr amplification Shigella bogdii 13 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 wzx gene (nucleotide position is 1695 to 3032 bases from SEQ ID NO:1), wzy gene (nucleotide position is 3019 to 4197 bases from SEQ ID NO:1), orf4 gene (nucleotide position is 4184 to 4969 bases from SEQ ID NO:1), orf5 gene (nucleotide position is 4972 to 6042 bases from SEQ ID NO:1), orf6 gene (nucleotide position is 6138 to 7172 bases from SEQID NO:1), wbjE gene (nucleotide position is 10193 to 11410 bases from SEQ ID NO:1), orf11 gene (nucleotide position is 11472 to 11930 bases from SEQ ID NO:1) orf12 (nucleotide position is 11935 to 13056 bases from SEQ ID NO:1).Coming from above intragenic oligonucleotide is high special to Shigella bogdii 13 types (SEQ ID NO:1).
In addition, the antigenic gene cluster of the different O-of the coding of two genetic resemblances produces new O-antigen by gene recombination or sudden change sometimes, thereby produces new bacteria types, new mutant strain.In this environment, need filter out many specificitys that oligonucleotide is detected with raising with recombination hybridization.Therefore, the invention provides a whole set of many mixtures to oligonucleotide, they come from glycosyltransferase gene; Come from transhipment enzyme and pol gene, comprise the wzx gene or the gene, wzy gene of identity function arranged or the gene of identity function is arranged with wzy with wzx; Also come from sugared synthesis path gene.The mixture of these genes is special to a special bacterial polysaccharides antigen, is special thereby make this cover oligonucleotide to the polysaccharide antigen of this bacterium.More particularly, the mixture of these oligonucleotide is to come from glycosyltransferase gene, wzx gene or the gene, wzy gene of identity function arranged or with wzy oligonucleotide and the combination that comes from the oligonucleotide in the sugared synthesis path gene in the gene of identity function are arranged with wzx.
On the other hand, the present invention relates to the evaluation of oligonucleotide, they can be used for detecting the O-antigen of expressing the antigenic bacterium of O-and identifying bacterium in diagnosis.
The present invention relates to a kind of antigenic method of one or more bacterial polysaccharideses that detects in the food, these antigens can make sample can with the oligonucleotide specific hybrid of following at least one gene, these genes are: (i) gene of the encoding glycosyl transferring enzyme gene of transhipment enzyme and polysaccharase of (ii) encoding comprises the wzx gene or the gene, wzy gene of identity function is arranged or with wzy the gene of identity function is arranged with wzx.At least one oligonucleotide can be hybridized with at least one more than one such gene specific of expressing the special antigenic bacterium of O-under the situation of condition permission, and these bacteriums are Shigella bogdii 13 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.
The present inventor considers following situation: when one special oligonucleotide detects when invalid, the mixture of oligonucleotide can with the target region specific hybrid with test sample.Therefore the invention provides a cover oligonucleotide and be used for detection method of the present invention.Here said oligonucleotide is meant the gene that comes from the encoding glycosyl transferring enzyme, the gene of coding transhipment enzyme and the gene of polysaccharase, comprises the wzx gene or the gene, wzy gene of identity function is arranged or with wzy the oligonucleotide of the gene of identity function is arranged with wzx.This cover oligonucleotide is special to the O-antigen of a special bacterium, and this special bacterium O-antigen is expressed by Shigella bogdii 13 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 bogdii 13 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 bogdii 13 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, the inventor believes that 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 bogdii 13 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 bogdii 13 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 bogdii 13 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 liquor and use isopyknic phenol again: chloroform: primary isoamyl alcohol (25: 24: 1) extracting twice, get supernatant liquor again with isopyknic ether extracting to remove remaining phenol.Supernatant liquor rolls out DNA and washes DNA with 70% ethanol with glass yarn with 2 times of volume ethanol deposit D NA, and DNA is resuspended among the 30ul TE, and genomic dna detects by 0.4% agarose gel electrophoresis.
Embodiment 2: by the O-antigen gene in pcr amplification Shigella bogdii 13 types bunch:
The O-antigen gene of Shigella bogdii 13 types bunch is by the Long pcr amplification.At first according to the JumpStart sequences Design upstream primer (5 '-ATT GTGGCT GCA GGG ATC AAA GAA AT-3 ') that often is found in O-antigen gene bunch promoter region, again according to the gnd gene design downstream primer (5 '-TAG TCG CGT GNG CCT GGA TTA AGT TCG C-3 ') in O-antigen gene bunch downstream.With the Expand Long Template PCR method of 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.
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 liquor, dispel thalline, in centrifugal 15 minutes of 4 ℃ of 4000rpm with the deionization aqua sterilisa 200ml of cold ice precooling.Deionization aqua sterilisa 100ml with cold ice precooling dispelled thalline again, in centrifugal 15 minutes of 4 ℃ of 4000rpm.With 10% glycerine suspension cell of cold ice precooling, centrifugal 10 minutes of 4 ℃ of 6000rpm abandon supernatant liquor, precipitate 10% glycerine suspension cell with the precooling of 1ml ice at last, are competent cell.The competent cell that makes is packed as 50ul one pipe ,-70 ℃ of preservations.
Be electric transformed competence colibacillus cell at last:
Get after 2-3ul connects product and 50ul competence bacillus coli DH 5 alpha mixes, forward in the electric shock cup of 0.2cm of Bio-Rad company and shock by electricity, voltage is 2.5 kilovolts, and the time is 5.0 milliseconds-6.0 milliseconds.The SOC substratum that adds 1ml after the electric shock immediately in cup makes the bacterium recovery.Immediately bacterium is coated in 37 ℃ of inversion incubated overnight on the LB solid medium that contains penbritin, X-Gal and IPTG then, obtains blue white bacterium colony next day.With the white colony that obtains promptly the white clone forward on the LB solid medium that contains penbritin and cultivate, from each clone, extract plasmid and cut the segmental size of evaluation insertion wherein simultaneously, obtain the O-antigen gene bunch library that white clone group has constituted Shigella bogdii 13 types with the EcoRI enzyme.
Embodiment 4: to the cloning and sequencing in the library:
From the library, select insert 120 clones of fragment more than 700bp by Shanghai biotechnology company limited with ABI377 type automatic dna sequencer to unidirectional order-checking of insertion fragment in cloning, make sequence reach 80% fraction of coverage.Residue 20% sequence is again according to the sequences Design primer that has obtained, and direct PCR and carry out backward sequencing and survey logically to the order-checking of PCR product or to known array from the genomic dna of Shigella bogdii 13 types obtains all sequences of O-antigen gene bunch again.
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, thereby obtain the Nucleotide full length sequence of the O-antigen gene bunch of Shigella bogdii 13 types, the quality of sequence is mainly guaranteed by two aspects: 1) genome of Shigella bogdii 13 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 bogdii 13 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 12 openings, determine also that with the function of finding the reading frame that these are open what gene they are with the genetic comparison among the blast groupware and the GenBank, finish gene annotation with the Artemis software at Britain sanger center again, do accurate comparison between DNA and protein sequence with Clustral W software, obtain the structure of the O-antigen gene bunch of Shigella bogdii 13 types at last, as shown in table 3.
By retrieving and relatively, finding that orf1 is a transposon, it has the transposase of insertion sequence element IS200, called after orf1.The albumen that is similar to O-antigen transhipment enzyme of orf2 and Yersinia pseudotuberculosis and other bacterium has 25% homogeny in 447 amino acid whose sequences, the wzx gene with Shigella boydii has 21% homogeny in 301 amino acid whose sequences in addition.And algorithm [Eisenberg by people such as Eisenberg, D, Schwarz, E.etal (1984) .Analysisof membrane and surface protein sequences with the hydrophobic momentplot.J.Mol.Biol.179:125-142] find that orf2 has 12 potential transmembrane domains, it and many wzx protein similars, and about 50 amino acid whose conservative motifs are arranged at the proteic aminoterminal of wzx, so can determine orf2 is the wzx gene, called after wzx.The wzy gene of orf3 and Escherichia coli K12 has 20% homogeny in 391 amino acid whose sequences, in 400 amino acid whose sequences 20% homogeny is arranged with polysaccharase in the lipopolysaccharides biosynthesizing of Shewanellaoneidensis MR-1.Learn that by the Eisenberg algorithm orf3 has 10 potential transmembrane domains in addition, similar secondary structure is arranged, so determine that orf3 is the wzy gene, called after wzy to other O-antigen polysaccharase.The LgtB albumen of orf4 and Neisseria meningitidis has 24% homogeny in 174 amino acid, in 212 amino acid, 26% homogeny is arranged with the LosA albumen of Pasteurella multocida, so determine that orf4 is a glycosyltransferase, called after orf4.The tagB albumen of orf5 and Bacillussubtilis has 40% homogeny in 55 amino acid, tagB albumen is a transferring enzyme, and the transferring enzyme of orf5 and other bacterium also shows homogeny, so determine that orf5 also is a transferring enzyme, called after orf5.The glycosyltransferase WbpX of orf6 and Pseudomonas aeruginosa has 56% homogeny in 30 amino acid, in 62 amino acid, 33% homogeny is arranged with the glycosyltransferase of the supposition of Salmonella enterica subsp.Enterica, so orf6 also is a glycosyltransferase gene, called after orf6.The WbjB albumen of orf7 and Pseudomonas aeruginosa and Pasteurella multocida has 74% homogeny in 344 amino acid, in 343 amino acid, 76% homogeny is arranged with the Fnl1 albumen of Escherichia coli, these two proteic functions are the same, all with synthetic relevant [the Bernd Kneidinger et al. " Three Highly Conserved Proteins Catalyze the Conversion ofUDP-N-acetyl-D-Glucosamine to Precursors for the Biosynthesis of Oantigen in Pseudomonas aeruginosa O11 and Capsule in Staphylococcusaureus Type 5-Implications for the UDP-N-Acetyl-L-FucosamineBiosynthetic Pathway.JBC Papers in Press.Published on December 2,2002as manuscript M203867200] of L-FucAc.Synthetic L-FucAc needs three enzyme catalysiss, five steps reaction to finish, and WbjB albumen is first enzyme of catalysis L-FucAc synthetic.So can determine orf7 is the sugared synthetic gene about L-FucAc, called after wbjB.The reductase gene of orf8 and Vibrio cholerae O37 has 51% homogeny in 284 amino acid, and WbjC albumen is a reductase enzyme in the building-up reactions of L-FucAc, it is second enzyme of catalysis L-FucAc synthetic, so can determine the orf8 WbjC albumen of also encoding, be wbjC with this unnamed gene.The Fn13 albumen of orf9 and Escherichia coli has 68% homogeny in 369 aminoacid sequences, 81% similarity, in 374 aminoacid sequences, 66% homogeny is arranged with the WbjD albumen of Pseudomonasaeruginosa, 79% similarity, these two proteic functions are the same, are last enzymes of catalysis L-FucAc synthetic.So can determine the orf9 WbjD albumen of also encoding, be wbjD with this unnamed gene.The WbjE albumen of orf10 and Pseudomonasaeruginosa has 24% homogeny in 238 aminoacid sequences, WbjE albumen is the transferring enzyme of L-Fucose.In addition, the orf10 also motif " Glycos-transf-1 " with first group of glycosyltransferase of Bacillus anthracis is the same, so orf10 is a glycosyltransferase, shifts L-Fucose, is WbjE with this unnamed gene.The WbuC albumen of orf11 and Escherichia coli has 45% homogeny in 133 amino acid, WbuC albumen is a glycosyltransferase.In addition, the waaK of orf11 and Escherichia coli F632 and the RfaK gene of Escherichia coli K-12 have homogeny, and these two genes are glycosyltransferase genes, so determine that orf11 also is a glycosyltransferase, called after orf11.The acetyltransferase of orf12 and Mesorhizobium loti has 32% homogeny in 371 amino acid; in 352 amino acid, 26% homogeny is arranged with the acetyltransferase of Micromonospora megalomicea subsp nigra; orf12 is a membrane-spanning protein in addition; so determine that orf12 also is an acetyltransferase, called after orf12.
Embodiment 6: the screening of specific gene: at wzx, wzy, orf4, orf5, orf6, wbjE, orf11, orf12 gene design primer in the O-antigen gene of Shigella bogdii 13 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 bogdii 13 types.Glycosyltransferase gene, transhipment enzyme gene and pol gene and their function corresponding and the size of the O antigen gene bunch of Shigella bogdii 13 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 upstream primer (5 '-TTCATC CTA AAC TCC TTA TT-3 ') and downstream primer (5 '-TAA TCG CAG GGG AAA GCAGG-3 '), extract genome then from 166 strain intestinal bacteria, method as previously mentioned.With this to primer from the colibacillary genome of 166 strains PCR with identification of escherichia coli and detect its genomic quality.
Table 2 is 166 strain intestinal bacteria and 43 strain Shigellaes and their sources that are used to screen specific gene, and for the convenience that detects, we are divided into one group with their every 8-10 bacterium, and 27 groups altogether, all list in the table in their source.The genomic dna that contains Shigella bogdii 13 types in the 23rd group is as positive control.Do template with every group of bacterium, be PCR by following condition with every pair in the table 1 primer: 94 ℃ of pre-sex change after 2 minutes, 94 ℃ of sex change 15 seconds, annealing temperature is because of the difference different (with reference to table 1) of primer, annealing time is 50 seconds, and 72 ℃ were extended 2 minutes, and carried out 30 circulations like this.Continue to extend 10 minutes at 72 ℃ at last, reaction system is 25ul.After reaction finishes, get the 10ulPCR product and detect the fragment that amplifies by 0.8% agarose gel electrophoresis.
For wzx, wzy, orf4, orf5, orf6, wbjE, orf11, orf12 gene, each gene all has three pairs of primers detected, obtain a correct band of expection size every pair of primer only is PCR in the 23rd group after, in other groups, all do not obtained onesize specificity band.After being template PCR with the genomic dna of each bacterium in the 23rd group, find only in Shigella bogdii 13 types, to have obtained positive findings.In more detail, more than each of each gene primer is all obtained expecting the correct PCR product band of size in Shigella bogdii 13 types, all do not obtaining any PCR product band in the array mostly, though in the minority group, obtain PCR product band, but its size does not meet the expection size, be non-specific band, so wzx, wzy, orf4, orf5, orf6, wbjE, orf11, orf12 gene pairs Shigella bogdii 13 types and O-antigen thereof all are high specials.
At last, from Shigella bogdii 13 types, screen gene by PCR: wzx, wzy, orf4, orf5, orf6, wbjE, orf11, orf12 gene to the O-antigen high special of Shigella bogdii 13 types.And the oligonucleotide of these intragenic any one section 10-20nt is special to the O-antigen of Shigella bogdii 13 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 bogdii 13 types.These all oligonucleotide all can be used for Shigella bogdii 13 types in the human body and environment rapidly and accurately, and can identify its O-antigen.
Table 3 is structural tables of the O-antigen gene bunch of Shigella bogdii 13 types, has listed the structure of the O-antigen gene bunch of Shigella bogdii 13 types in table, altogether by 12 genomic constitutions.
Table 4 is location tables of the gene in the O-antigen gene bunch of Shigella bogdii 13 types, in table, listed the accurate position of all open reading frame in complete sequence in the O-antigen gene bunch of Shigella bogdii 13 types, at the underscoring of the initiator codon and the terminator codon of each open reading frame.
Sequence list
SEQUENCE LISTING
＜110〉Nankai University
＜120〉to the Nucleotide and the application thereof of the O-antigen-specific of Shigella bogdii 13 types
＜130〉to the Nucleotide and the application thereof of the O-antigen-specific of Shigella bogdii 13 types
<160>1
<170>PatentIn version 3.1
<210>1
<211>14504
<212>DNA
<213>Shigella boydii
<400>1
attgtggctg cagggatcaa agaaatcctc ctggtgactc atgcgtccaa gaacgcagtc 60
gaaaaccact tcgacacttc ttatgaacta gaatctctcc ttgaactgcg cgtaaaacgc 120
cagttgctgg cagaggttca gtccatctgc ccaccgggcg ttaccattat gaacgtgcgt 180
cagggcgaac ctttaggttt gggccactcc attctgtgtg ccagacctgc cattggcgac 240
aatccattcg ttgtggtgct gccggatgtc gttattgatg acgccagcgc cgatccgctg 300
cgttacaacc ttgccgccat gattgcgcga ttcaacgaaa cgggtcgtag ccaggttctg 360
gcaaaacgta tgccgggtga cctttctgaa tactctgtca tccagactaa agaaccgctg 420
gatcgtgaag gcaaagttag tcgcattgtt gaatttatcg aaaaacccga tcagccgcag 480
acgctggact cagacattat ggcggttggt cgctatgtgc tttctgcgga catttggcct 540
gaactggagc ggactcaacc cggtgcatgg ggacgtattc agctaactga tgcaattgct 600
gaactggcga aaaaacagtc cgttgacgca atgttaatga cgggtgacag ctacgattgc 660
ggtaagaaaa tgggctacat gcaggcattt gtgaagtacg gactacgcaa cctgaaagaa 720
ggagcgaagt tccgcaaagg gattgagaag ctgttgttag aataagatat tgcgcgacgg 780
gatagaatgt tggtgatgta acagaacggc agatgattgt ggttgaggaa gagctcactt 840
cacagtaact gccgtatttg ttgtccctaa actacctttg gggaggtggt gattaatcct 900
gtaaggatat attctgaata atgttcactt accacaagta aaaggggaca aaccgacaga 960
ggaatttaca gaagttcatc aaatatgcat tgcgttgtaa atatcacata gtctggacgc 1020
caaagtaccg cttcaggagc cagcaggata aattgggtaa agagctgtac agagccacct 1080
atattttgtg cagaataaaa gattgcgagg ttcaggagtt aaatatacaa ccagatcatg 1140
tgcatcttgt agcaatagtg cttctaaaaa tctcaatata tactctaatg gggcacctga 1200
aggtcgcagt gcaatcaggc tctacaatcg gttttcgcat atcaggaaga aactatggaa 1260
atcatttttg gccatggggc tgtgttgccg atatggtagg cgtatcagac gatatgtgat 1320
acatcaggaa aagatagaac aaacatatga acagtaaatg gtgttgctag agtagaacgc 1380
gggatatgac ggttgccccc ttatcggaga gtattctgaa aagctacctc atcaggagat 1440
gatcttttat tattaaaaac aaccactaaa attagtgatt gcatggcgtt ttttgacaaa 1500
aatgtcggat ttttccttgt tttataaagg gattgtgctc aaaatagtgg gctgaattgg 1560
aagggagggt tcctcgcaaa cgcgaaggtt ggcgggagtt ggcttgcgac acacagaatt 1620
ttgtgcagtg cactggtagc tgttgagcca ggggcggtag cgtttctgat gtgtgtgatt 1680
tttacccatt taaaatgttc agcggaaaaa ttatgaagag tgccattagg agtggtaaag 1740
agcaattttt aatgggagtc atctataaag cattatttat gttgataaca tcaataaatg 1800
ctttagtaat agtaaagcta ctgactcctg aagagttagg tatttggtat atttttatgg 1860
cgcttcaaac gcttttattt atcttaaata gcgccttgac tccgaatatt tcaagacagt 1920
acagcattgc atttacggat attggtgagg gatttgatcc ccgtcttttt cacacaaaag 1980
taattattat ttataataag ataattttag tggcaacagt catgggagtg ttttttactc 2040
tgacttatct taactatgtg cttgatatat ttaggttggc taatgtttct atattactat 2100
cttgggtaat tgtttttgct tccattttga tggaaattta ttttacatcg tatgagtgtg 2160
cattaaatgg gtttggtgaa ttccaaaaag ttaacaaagt aaattttata tcgagattga 2220
ttctaacgtt aagcattttt acactgttgt tatctgatgt gcatagtaat ggcttgctaa 2280
tattctgtgg gttatatttt tttagtaata taataaaaag ggttatggtt attaatcttt 2340
ttaatcatac ctttaaagac aaagggctaa aagaatcgcg gaaaacaata gatttttttg 2400
atagaatcag ctgtaagcta caaaaactgt cctttttatc actactttca tcaattgggg 2460
gggtcctcat tgtcagaagc ggaatgttta ttttgccctt ctatgtgcca atggaccaag 2520
tagcatctta tggattaacc tatcagcttt ttgagctcgg gtttaatgtc atatttacta 2580
tttctataat agcaactccc gcatggatct cagcctataa aaataatcca gaaggattaa 2640
aaaaatcctt cacttgggta aagtatatat ctctattgct aatgttaatt tgtgggctgt 2700
taattacatt tctgggtaat atcatattag agtttgctga tgttgaaacc agagtgcagt 2760
cagttatcct ttgtttttat ttgctggtaa tttttgtgtt gcaaattaat cacagtattt 2820
ctgggcaact cctaactatt cagaataaaa ttccttttgc ttatgcatca ttcttgagtg 2880
ggatttttac attagtgtta tcggttgttt taattaaata ttattcagtt cttggcgcta 2940
tttatgcgat attaatcagt cagctaatat ataataattg gaagtggcca tatgaagcat 3000
ataaattctt gagggtgcat ggaaatgtat gagggaaaat taaaaaacaa aatattaagt 3060
cttttcctct tatgtgaaac cttttgcttt atatatataa tatataataa ggaattagtt 3120
tccgattatt tggggagggc gataacttta agtgagttta caataagttg tatttttttt 3180
ctgctattgt tattttattt tttgttccat actttgtggg tgatttcaga aaaaataatt 3240
aaatccagga aaaagattgg tagcgtcggg aatttgaatc tttttgttct tgttcttata 3300
ttcttatatt ttatattaaa tttaaaaaca ggtattggta aagttggtca agaggataat 3360
attactttaa caggattaga taaattatta tttgcagcac caatattttt taaaataaat 3420
tttctcatgt atatttacgc tgcggggaat agaaataaga atcgtctgta ttactttaat 3480
ttgttctttt tcgcatttgc agaattaact cggggagtat cttttacaat attgttgttg 3540
ttgataatag aatacaataa gattagacgc ttcatttccc ttaggtattt tttattttca 3600
ttgctttttg gtatattgtt aattaatgtg atttacaatg tgaaatttta cgttaggatg 3660
ggggcgaaat acgagtatat tgatatctac acgtctttaa ttatgttgat gggacgtttg 3720
agtatattgt ccaatttact atttattcat gataatctct ctgatataag attatatttt 3780
tatggctcag ggtaccaagg agttgttcat gagtttttag agtcgttgac acctgtacca 3840
tcattattcg gtattaacga gaaagtgttg gagtttggta aaatattatt ctcatattcg 3900
aatgttactt ttaccagcgc aactgcagca tcgctattag gtatcattta tatttatcca 3960
aattcaattt tcaatgcgat tacaataata tgtcttagtt ggatttatat acatataatt 4020
acgggttatc ttgagtttag tcgaatgcaa cgagtagtcg cgtacttttt tgtgctgctt 4080
gcactttacc agggcttcat ggccatatta agtaattata tatatgcatt aacaatatat 4140
ttccttatag cctgtgccag aaatctttta ttgagtaaga aatatgaaac aatttagtat 4200
taaaatcttg tcattggcag atgctatcga gcgtaggagg aaagtatcta gagagtttga 4260
gctgaaatca gtactgccat tctcattttt taacggtatt tatggtaaga cactaagtaa 4320
agaaaaatta gatacaatat actcttcaga gctggcgcag aaagtactcc atcggcaact 4380
aactgcgggg gaaattggtg caacctactc acattacttg attttccgtg aggcatatga 4440
gcgtggggat gagtttgttg ttgttctgga agatgactgt tatatagata gaaattttga 4500
taccgtgtta acatcaattc ttgataagaa aaatcctaca gatgatgaaa ttatctttat 4560
tcaaagacat acgagcgaaa actcacatat aataagatct ttaggcagtg agaagataaa 4620
taaaaattat accttacatc gtatgctagg tagcgcgcag tattttgttg gtgcatacgg 4680
gtatatcgta actagagctg caatgaaaaa aatgatcgat acatacttgc caatatattg 4740
tgtatgtgat cattggtatt ttataaagaa gaaatgtcag atagaaaaat ttagtgtatt 4800
atccccagct attgttttca caaatgatga ggatgttagg aaaatagata gttttgtaga 4860
cattgaaaga agagaaatcg ctgttttcaa atcagtaggt aaaatagcaa aactaaaaat 4920
tttaattaag aaaattattt taccaatatt aaataaagac tgggaataaa agtgctgcgc 4980
tattataaag tattgactgt tctgcttaga ttaattatag cttttatatt gtaccctatt 5040
gcatatgttt ttttgaatca caaaaaatat ttcatcattg gcacacgcaa tggtgttcga 5100
gggcatgata atgctgagta ttttttgtct tattgtacaa gacacaatat taatacaaaa 5160
gtaatttata atgatgctta tcagaaagat gaattatgca aaaactctat aaaaagtata 5220
ttgtatatct tatgtgcaaa caatgtttac ataacgcact ctgaatcaga tgttttagat 5280
tatttttggc gttttattcc cggtataaag tatatattta tacagcatgg agttattgga 5340
ataaaaaaac ttccggatta tgaaaagaaa agatatcata gatatgtttc tagtagtgaa 5400
tatgaaacag aaatttttag gaatttcttc catttgaaca gtgataaaat tattaaatct 5460
ggacttccac gttttgattt ctatgactat aaccgttcaa aaaatgatga aattaaatcc 5520
tgcttaatta tgtttacctg gagaaaatcg gattctggca aggctaaatt aattaaaaaa 5580
tatactgaga tggttacttg cctgtcaaat gaagctctca ataaaattta catttgtgtt 5640
catgaagcaa acttaagtac atttaaggtc ttgcttgaag aatcgcttga attggatgat 5700
aggttttcat ttatagaaaa tgataaatta tcatgtatta ttcaaagtac agaactttta 5760
atcactgact actcgagtgt tgcatgggat tatttatatc agaataaata tattttattt 5820
tataccccag atattgatga ttataaaaat acaactggat tgtattgtga tttttccgat 5880
ttttttggcg cacatcttat gaatttcagt agaatcaatt atccagtttt ggcatggatt 5940
atgcaagaaa atgaaattaa aaataaaaac tttcttgcaa ggtataaatt ttataacatg 6000
catgcaggca tacactctga gtatatagtg aagaatagtt aaaattatga gggcgaacat 6060
tgacaactat tattgtctca gcaactgcat tggctaagag cggtgcatta acaatattaa 6120
atgagtttat tgagtatgtg tcaaatttaa aaaaatataa gtttataatt tttattcctg 6180
atagcattaa tttgcctaaa gcagttaata tcagatatat ccctgtgcct aaaaaaaatt 6240
ggctatctag gatatattgg gatagttatg gcttaagaaa atatattcga gtaaatcggc 6300
ttcaatatca ggcggtcatt tctcttcaga atacatctgt aaatgtagaa ggaaaacaga 6360
taatatatct tcaccaatct ataccattta tagattttga tattccactg acaagtttat 6420
ttaatttaaa attatggcta tacaagcgat tttattctta ttttatattt ttatttgtta 6480
acaagaatac ttcttttatt gtccaagcca aatggctgaa agagctcctt tctgataagt 6540
atcatatcga tgcaaaaaaa atatctatca taaagcccaa agcaagatat gttcctctag 6600
caactaactc tattcataat gtagccaact tatctaagca attctgtaat attatttatc 6660
cagcaacacc aatattttat aaaaatcatt gtgttattat tgatgctctt aggattttaa 6720
gagaacagcg caatattgat aacttatgtt ttaatgtcac cttttcaaaa ggtgagtatg 6780
ctaagtttga cgatttggtt aggcgttata acttgggtaa gaatattcgc tatttgggat 6840
atttgacccg agatgaatta tatcaacatt atgataatag cgcttttatg gtctacccca 6900
gttatgtgga aacctgtggg cttccgttgc tggaggctgc ttctaagcag ttgccaataa 6960
tcgctagtga tctgccttat gcatgtgaaa tgcttgaggc ttatactggc gtcgtttatg 7020
ttaagttcaa tgtctctacc gaatgggctc gagaaattga tacgatggcc cgagctggtg 7080
aaactcgaat tattccgccg ttattaaaag cggagaatga ttctgattgg aataagttag 7140
ataatattat tgaaggaaaa gatgatgttt aaaaataaaa cactactaat tacaggtgga 7200
actggatcgt ttggtaatgc tgtgctccgt cgttttttat cgacggatat tggcgaaata 7260
aggatcttta gtcgcgatga aaagaaacaa gatgatatgc gtaaaaaata ttcggatccg 7320
aagttgaagt tttatattgg cgacgtaagg gattataaca gtattttgtc tgcaactcgc 7380
ggagttgatt atatctatca tgccgcagca ttgaagcagg ttccttcctg cgagttttat 7440
ccaatggaag cagtaaaaac taacgttatt ggtacagata atgtgttaga agctgcaata 7500
gcgaataaag ttcagcgtat agtctgtttg agtacggata aagccgtata tcctatcaat 7560
gcaatgggaa catcaaaagc aatgatggaa aaagtaattg ttgcaaaatc acgcaatttg 7620
cctgaaggca taactatttg tgctacccgt tatggaaatg taatggcttc gcgtggatcg 7680
gtgataccat tatttataaa tcaaattaaa aatggtaacc ctataactat taccgatccc 7740
gatatgactc gctttatgat gacccttgat gatgccgtcg atttagtatt acatgcgttc 7800
gaatacggaa ataatggtga tattttcgta caaaaggctc ctgcggcaac aattgaaaca 7860
cttactaaag caattcaaca ggttacaaac gcacttgatc atcagataaa tattatcggg 7920
acacgtcatg gtgaaaaatt gtatgaagtt ctctgtagcc gtgaagaaat ggctgtagca 7980
gaagatcagg gaaattacta ccgtatccct gctgacaatc gagatttaaa ctatgagaaa 8040
tactttgaga agggtaacaa agatgtttcc ttactcgagg attataattc gcataatact 8100
attagacttg acgttgatgg tatggttgct ttgctgcgta aactcgaatt tatccgcaaa 8160
gtggaagctg gttatgatgc aaatccagat gagtaaagca tgaaaatact tgttataggt 8220
gctacaggaa tgctcggagg aagtttgctt cgttatttcg ctgacaagac cgagcatgat 8280
gtatatggaa ctgttcgtga tagtaacgct gaaagtaggc tagtctcgaa agctaacgcc 8340
aatataatct gcggtattga tgttcacaat gtgaataaaa tacgcagcat aatagaggaa 8400
gtaagacctg actatgtaat taattgtgtt ggtgtagtta agcagcttaa agagtctaaa 8460
tatcctattc attctattac tataaattca ttgctgcccc atcgattagc tgagatctgt 8520
tcgcataata attcgaaatt aattcatttc tctacagatt gtgtattctc tggtaataga 8580
ggtaattatt cggttaatga tgttcctgat gcctttgatc tttatggacg ttctaaatta 8640
cttggtgaag taagttatgc tccacacctt acgttaagaa catcaataat tgggcatgag 8700
caggggtcac agcatagttt aattgattgg tttttaaatc aatcaggtga ggtaaaagga 8760
tttactaaag ctgtattctc cggagttcct actgtctata tggcggagct attaaataat 8820
tatgttttta ctaatcctga tatcacaggg ttgtatcagg tgagtgttga acctattgat 8880
aagtattctc ttctttcatt agttaaagat atttatggta aagatataga tattaatgcg 8940
gatgatagtc ttgttattga cagaagtttg aactcaactg aatttaaaat tcgtacgggg 9000
atgaataatc cctcttggaa agatttgata gagaagatgt acaatgaata tagaacatat 9060
ttccaaaaag cttaaagtta ttacttttgt tggtacccgt cctgaaataa ttcgattgtc 9120
tagaataatt gctttgttgg ataaatattg tgaccatatt ttggttcata ctggacaaaa 9180
ttatgattat gagttgaatg aggttttttt ctccgattta ggaattagaa agcccgatta 9240
ctttttgaat gctgcgggta aaaatgcggc agagactatt gggcaaatta ttattaaatc 9300
tgatagtatt ctggaacaag ttaatccgca ggcactatta attttaggtg ataccaatag 9360
tgccttggtt gcaatatctg caaaaagaag aaaaatccct attttccata tggaagcagg 9420
gaatcgttgt tttgatttta gggttcctga agaaatcaat agaaaattgg ttgatcatat 9480
ttctgatatt aatctgacct atagtcatat agctcgtgat tatttactac gtgaaggcat 9540
tcccgctgat caggtgatca aaacgggatc tccgatgcgt gaagttttga attactatcg 9600
tgatgatatc caaaagtcgg ttgtacttga aacgttgaaa cttagtaata acaactattt 9660
tgttgtcagc tcccatcgcg aagagaatgt tgattcgcca gagagactta gacaactgct 9720
ggagatcctc aatgagcttg ctgaaagata taatttgcct gtcattgttt caactcatcc 9780
aagaaccaga aaaagattcg aagacttaag ttttaatgtt caccctaata tctctttcat 9840
gaagccattc ggatttattg actacatcac tttacagttg catgccagag ctgtactttc 9900
agatagtggt actattactg aagaatcttc tattcttaac ttcccagcac ttaatttacg 9960
tgaggttcat gaacgtcctg aaggtttcga agaagcctca gtgatgtttg tcggtctgaa 10020
taaagagcga gtattacagg ctcttgacat attaaatgag caaccaagat acgaatcacg 10080
cctcttgaac cttgtagagg actacaaacc cgataacgta tcagagaaag ttctcaggat 10140
tattatgagc tatactgatt ttgttaataa taaagtatgg aagaaataat cgatgcgcat 10200
tgcgttaatt tgtgatgact atctccccgg tagtactcga gttagtgcaa aaatgatgca 10260
tgaattggct cgtgaacttc tgcaaagagg tcatgagcca attgttgtga ctccggattg 10320
ttacattaag tcaatttaca aagttgagca actggatggc gttgatatct tacgttttcg 10380
taacggaaga atcaaagatg tgtccaaaat taaacgtgca atgtgtgaaa cactactctc 10440
atataatggt tggaaggcgg taaatagtta tttttccaat gatgtgattg atgctgtcat 10500
atattactca ccttctatat tctttggtcc tctggttaat aaaattaaga ggaaatggtc 10560
atgtccatct tttcttatct tacgtgacag ttttcctcag tgggtaatcg atgaaggatt 10620
aattagagag ggttctctga tatgtagata ttttagattt tttgagaaaa taaactatca 10680
agcagcagat actattggag ttatgtcgga gaggaataga gaactttttt taaatatgca 10740
tccaaatatt cagagtgttg aagtcttata taactggtgc gatactaggg cggatgcaga 10800
actggacttg gtgaataaac ctgactgtat tactaagatc gcagataaaa ttatttttct 10860
ctatggtgga aatataggca aggcacagga tatgggaaat ttaatccgtc ttgcaattgc 10920
tatgaaaacc tactcaaatg ttcatttcat ttttattggg cagggtgatg aatacgattt 10980
tgtaagtgat agtataaaaa aatattcact gagcaacgtt agcttgtttc catctgtttc 11040
acaacatgat tttaaagtta ttctcaaatt tgttcatgtt gggttattta gtttgtctaa 11100
gaaacatacc gcccataatt tccctggcaa gttacttggc tatatgaaaa atagtttacc 11160
catactaggt agtgtaaata gtggtaatga tctgtcagat gttattaata ataatgaggc 11220
gggttgtgtt tgtgacaatg gcgatgataa tgcattatta cgtgcggcaa taaggttagc 11280
aactgatcag gattatcgtt acatgtgtgg acaaaaggct aatcagctac tacatgagaa 11340
attttctgtt acttctgcta ttgacaatat aatgaagcac ttaagtaaaa aaactcagga 11400
ctataattaa catgaccttt aaattattgg actttaatct aaataatgag ttgaaagcta 11460
atgcaaagaa gagtgagcgt ttgcgatatc acttgagctt gcatggctct tataacgaac 11520
ccgttcagag aattattata tcgctaatgc gtggtacata tattcctcca cactaccatg 11580
aattcgagta tcaatgggag tattttaatg ttatatcagg taatatatgt gtgattattt 11640
ttgatcagaa tggtactgtg actgataagt ttaaattggg tcaaaatact ggtgcttacg 11700
gagttgagtt ttcttcaaac acaattcata caattctctg tgagagtgaa gatagtatta 11760
ttttagagtt aaaagaaggt cctttttttc caagtaaagc taaagttata ccgagttggg 11820
cgcctgatga gcaatattgc acatactctc gagcagaaat agtaagtgtt ttagaaaaaa 11880
tcaaaccagg ccaaaatctt cctgaagcat tattggcgca ggaacaataa tgctatggaa 11940
aaaaaacgat tttttgcact tgatagtttt cgtggtctat gcgctttgtg tgtagtcatt 12000
tttcacatga aaatattgaa tgctttttcc gagtgggatt tttttcgaaa tagtcgactg 12060
tttgttgaat ttttctttat tcttagtggg tttgttttaa cccacagcta tttaaaaaaa 12120
gaaactaatg gtttttacaa atacgctatc gctcgaacgt ttcgaatttt tccacttcac 12180
atttttatgc tttgtgtgta tgttattctt gaatctttta aattagttgc acaacagaaa 12240
ggttttgtct ttaatacacc tccttttcag ggaggaaatt cattaaaaga atttcttcct 12300
aatctgcttt taattcagtc gtggtcatcg agttttgaat acttaagctt caattatcca 12360
tcatggagta taagtgttga atattattta tattttattt tttatctggt gatatgtcca 12420
cgaacgataa ttggtagagt ttttgctggc atagtgtttt ttattatatt tctttttcaa 12480
gaatattcct cttttaagat ttttaccagc gaagtgatta gaggtggaat ttgctttggc 12540
gctggaattt tagtttatta tctttattca ttaattcatc taagtcatgc aactcgattt 12600
ttatctgtgg tttttactat actagagtct agttctcttg cattaatttg gtttgttgta 12660
agtatgactg attatgaaag tgaattattg gtggtggttg tcttttcgtc atcagttttt 12720
ctatttgcat ttgaattagg gatgatttct ttttttctga aaaaaaaacc atttcagttt 12780
tttggtaaac tatcttactc tatatatctt actcatgctt cagttctttt tgttcttata 12840
tcagcaatga tggtattaca aaagataacg ggggtggcat ttacacagca tattgataat 12900
ttacgttatc ttaattttgg aagttcattg ataaataatt ttatgatttt tgcagtgctt 12960
ggatgtgtaa tatttttttc gatgctaacc tataaattta ttgagttgcc tgcgcaaaga 13020
aagggtaaag agttatatga gcattattct aaataattgc tttaaagtta atgtgtcatt 13080
ttatttctag actatgttac tttaacaaga ctaatttaat atattgtaca cacctaatcg 13140
catcccccct gacaggagta aacaatgtca aagcaacaga tcggcgtcgt cggtatggca 13200
gtgatggggc gcaaccttgc gctcaacatc gaaagccgtg gttataccgt ctctattttc 13260
aaccgttcct gtgaaaagac ggaagaagtg attgccgaaa atccaggcaa gaaactggtt 13320
ccttactata cggtgaaaga gtttgttgaa tctcttgaaa cgcctcgtcg tatcttgtta 13380
atggtgaaag caggtgcagg cacggatgct gctattgatt cccttaagcc ataccttgaa 13440
aaaggcgaca tcatcattga tggcggtaac accttcttcc aggacaccat tcgtcgtaac 13500
cgtgagcttt ctgctgaagg ttttaacttc attggtacgg gggtttccgg tggtgaagag 13560
ggcgcgctga aaggtccttc tatcatgcct ggtggtcaga aagaagccta tgaactggtt 13620
gcaccgatcc tgaccaaaat tgctgccgta gctgaagatg gcgaaccgtg cgttacctat 13680
attggtgcag acggtgctgg tcactatgtg aagatggttc acaacggtat tgaatacggt 13740
gatatgcagt tgattgctga agcctattca ctgctgaaag gtggtctgaa tctctccaac 13800
gaagaactgg cacaaacctt tactgagtgg aataacggtg aactgagcag ctacctgatc 13860
gacatcacta aagacatttt caccaagaaa gatgaagacg gtaattatct ggttgacgtg 13920
atcctggatg aagcggctaa taaaggcacc ggtaaatgga ccagtcagag tgcgctggat 13980
ctcggcgagc cgctgtcgtt gattaccgag tctgtgtttg ctcgttatat ctcttctctg 14040
aaagagcagc gtgttgctgc atctaaagtt ctgtctggtc ctcaggcaca gccagcaggc 14100
gacaaagcgg agttcatcga gaaggttcac cgtgcgctgt atcttggcaa aatcgtttct 14160
tatgcacagg gcttctctca actgcgtgcc gcgtctgaag agtacaactg ggatctgaac 14220
tacggtgaaa tcgcgaagat tttccgtgct ggttgcatta ttcgtgcgca gttcttgcag 14280
aaaatcaccg atgcctatgc cgaaaatcca aaaatcgcta acctgctgtt agctccgtat 14340
ttcaagaaaa tcgccgatga ctaccagcag gcactgcgtg atgtcgtcgc ttatgcagtg 14400
caaaacggta tcccagttcc aaccttctct gcggcggttg cttattacga cagctatcgt 14460
gccgctgtcc tgcctgcgaa cctaatccag gcccagcgcg acta 14504
Glycosyltransferase gene in the table 1 Shigella bogdii 13 type O antigen genes bunch and oligosaccharide unit treatment gene and wherein primer and PCR data
Gene Function The base position of gene Forward primer Reverse primer PCR product length Produce the group number of correct big or small electrophoresis band The annealing temperature of PCR (℃)
wzx The transhipment enzyme 1695-3032 #59(2012-2030) #60(2931-2949) 938bp 0 * 52
#79(1887-1904) #80(2599-2616) 729bp 0 52
#83(2009-2026) #84( 2493-2510) 503bp 0 60
wzy Polysaccharase 3019-4197 #61(3252-3269) #62(4081-4099) 848bp 0 55
#85(3206-3224) #Q43(4160-4176) 955bp 0 55
#86(3565-3582) #87(4083-4100) 535bp 0 58
orf4 Glycosyltransferase 4184-4969 #273(4399-4417) #274(4943-4962) 564bp 0 50
#88(4368-4385) #89(4670-4688) 321bp 0 **** 55
#90(4227-4244) #91(4821-4840) 614bp 0 **** 55
orf5 Glycosyltransferase 4972-6042 #261(5242-5261) #262(5677-5694) 453bp 0 *** 52
#263(5071-5088) #264(5774-5791) 721bp 0 60
#265(5600-5617) #266(5889-5908) 309bp 0 56
orf6 Glycosyltransferase 6138-7172 #67(6089-6106) #68(6904-6921) 833bp 0 60
#96(6306-6324) #97(6906-6923) 617bp 0 58
#98(6050-6067) #99(6996-7013) 964bp 0 ** 60
wbjE Glycosyltransferase 10193-11410 #92(10379-10397) #93(10953-10970) 592bp 0 52
#94(10448-10465) #95(10813-10830) 356bp 0 58
#275(10199-10217) #276(10098-11115) 917bp 0 52
orf11 Glycosyltransferase 11412-11930 #267(11472-11489) #268(11878-11894) 423bp 0 52
#269(11500-11517) #270(11686-11703) 203bp 0 50
#271(11689-11708) #272(11906-11923) 235bp 0
orf12 Acetyltransferase 11935-13056 #69(11971-11990) #70(12872-12891) 921bp 0 62
#96(11973-11990) #97(12998-13015) 1043bp 0 60
#98(12682-12700) #99(12997-13014) 333bp 0 ** 58
*In one group, obtain the band of a wrong size; *In two groups, all obtain the band of a wrong size; * *In three groups, obtain the band of a wrong size; * * *In four groups, obtain the band of a wrong size
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, O164 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 from 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 is structural tables of the O-antigen gene bunch of Shigella bogdii 13 types
Table 4 is location tables of the gene in the O-antigen gene bunch of Shigella bogdii 13 types
attgtggctg cagggatcaa agaaatcctc ctggtgactc atgcgtccaa gaacgcagtc 60
gaaaaccact tcgacacttc ttatgaacta gaatctctcc ttgaactgcg cgtaaaacgc 120
cagttgctgg cagaggttca gtccatctgc ccaccgggcg ttaccattat gaacgtgcgt 180
cagggcgaac ctttaggttt gggccactcc attctgtgtg ccagacctgc cattggcgac 240
aatccattcg ttgtggtgct gccggatgtc gttattgatg acgccagcgc cgatccgctg 300
cgttacaacc ttgccgccat gattgcgcga ttcaacgaaa cgggtcgtag ccaggttctg 360
gcaaaacgta tgccgggtga cctttctgaa tactctgtca tccagactaa agaaccgctg 420
gatcgtgaag gcaaagttag tcgcattgtt gaatttatcg aaaaacccga tcagccgcag 480
acgctggact cagacattat ggcggttggt cgctatgtgc tttctgcgga catttggcct 540
gaactggagc ggactcaacc cggtgcatgg ggacgtattc agctaactga tgcaattgct 600
gaactggcga aaaaacagtc cgttgacgca atgttaatga cgggtgacag ctacgattgc 660
ggtaagaaaa tgggctacat gcaggcattt gtgaagtacg gactacgcaa cctgaaagaa 720
ggagcgaagt tccgcaaagg gattgagaag ctgttgttag aataagatat tgcgcgacgg 780
gatagaatgt tggtgatgta acagaacggc agatgattgt ggttgaggaa gagctcactt 840
cacagtaact gccgtatttg ttgtccctaa actacctttg gggaggtggt gattaatcct 900
Orf1's is initial
gtaaggatat attctgaata atgttcactt accacaagta a aaggggaca aaccgacaga 960
ggaatttaca gaagttcatc aaatatgcat tgcgttgtaa atatcacata gtctggacgc 1020
caaagtaccg cttcaggagc cagcaggata aattgggtaa agagctgtac agagccacct 1080
atattttgtg cagaataaaa gattgcgagg ttcaggagtt aaatatacaa ccagatcatg 1140
tgcatcttgt agcaatagtg cttctaaaaa tctcaatata tactctaatg gggcacctga 1200
aggtcgcagt gcaatcaggc tctacaatcg gttttcgcat atcaggaaga aactatggaa 1260
The termination of orf1
atcatttttg gccatggggc tgtgttgccg atatggtagg cgtatcagac gatatg tgat 1320
acatcaggaa aagatagaac aaacatatga acagtaaatg gtgttgctag agtagaacgc 1380
gggatatgac ggttgccccc ttatcggaga gtattctgaa aagctacctc atcaggagat 1440
gatcttttat tattaaaaac aaccactaaa attagtgatt gcatggcgtt ttttgacaaa 1500
aatgtcggat ttttccttgt tttataaagg gattgtgctc aaaatagtgg gctgaattgg 1560
aagggagggt tcctcgcaaa cgcgaaggtt ggcgggagtt ggcttgcgac acacagaatt 1620
ttgtgcagtg cactggtagc tgttgagcca ggggcggtag cgtttctgat gtgtgtgatt 1680
Orf2's is initial
tttacccatt taaa atgttc agcggaaaaa ttatgaagag tgccattagg agtggtaaag 1740
agcaattttt aatgggagtc atctataaag cattatttat gttgataaca tcaataaatg 1800
ctttagtaat agtaaagcta ctgactcctg aagagttagg tatttggtat atttttatgg 1860
cgcttcaaac gcttttattt atcttaaata gcgccttgac tccgaatatt tcaagacagt 1920
acagcattgc atttacggat attggtgagg gatttgatcc ccgtcttttt cacacaaaag 1980
taattattat ttataataag ataattttag tggcaacagt catgggagtg ttttttactc 2040
tgacttatct taactatgtg cttgatatat ttaggttggc taatgtttct atattactat 2100
cttgggtaat tgtttttgct tccattttga tggaaattta ttttacatcg tatgagtgtg 2160
cattaaatgg gtttggtgaa ttccaaaaag ttaacaaagt aaattttata tcgagattga 2220
ttctaacgtt aagcattttt acactgttgt tatctgatgt gcatagtaat ggcttgctaa 2280
tattctgtgg gttatatttt tttagtaata taataaaaag ggttatggtt attaatcttt 2340
ttaatcatac ctttaaagac aaagggctaa aagaatcgcg gaaaacaata gatttttttg 2400
atagaatcag ctgtaagcta caaaaactgt cctttttatc actactttca tcaattgggg 2460
gggtcctcat tgtcagaagc ggaatgttta ttttgccctt ctatgtgcca atggaccaag 2520
tagcatctta tggattaacc tatcagcttt ttgagctcgg gtttaatgtc atatttacta 2580
tttctataat agcaactccc gcatggatct cagcctataa aaataatcca gaaggattaa 2640
aaaaatcctt cacttgggta aagtatatat ctctattgct aatgttaatt tgtgggctgt 2700
taattacatt tctgggtaat atcatattag agtttgctga tgttgaaacc agagtgcagt 2760
cagttatcct ttgtttttat ttgctggtaa tttttgtgtt gcaaattaat cacagtattt 2820
ctgggcaact cctaactatt cagaataaaa ttccttttgc ttatgcatca ttcttgagtg 2880
ggatttttac attagtgtta tcggttgttt taattaaata ttattcagtt cttggcgcta 2940
tttatgcgat attaatcagt cagctaatat ataataattg gaagtggcca tatgaagcat 3000
The termination of the initial orf2 of orf3
ataaattctt gagggtg cat ggaaatgta t gagggaaaat taaaaaacaa aatattaagt 3060
cttttcctct tatgtgaaac cttttgcttt atatatataa tatataataa ggaattagtt 3120
tccgattatt tggggagggc gataacttta agtgagttta caataagttg tatttttttt 3180
ctgctattgt tattttattt tttgttccat actttgtggg tgatttcaga aaaaataatt 3240
aaatccagga aaaagattgg tagcgtcggg aatttgaatc tttttgttct tgttcttata 3300
ttcttatatt ttatattaaa tttaaaaaca ggtattggta aagttggtca agaggataat 3360
attactttaa caggattaga taaattatta tttgcagcac caatattttt taaaataaat 3420
tttctcatgt atatttacgc tgcggggaat agaaataaga atcgtctgta ttactttaat 3480
ttgttctttt tcgcatttgc agaattaact cggggagtat cttttacaat attgttgttg 3540
ttgataatag aatacaataa gattagacgc ttcatttccc ttaggtattt tttattttca 3600
ttgctttttg gtatattgtt aattaatgtg atttacaatg tgaaatttta cgttaggatg 3660
ggggcgaaat acgagtatat tgatatctac acgtctttaa ttatgttgat gggacgtttg 3720
agtatattgt ccaatttact atttattcat gataatctct ctgatataag attatatttt 3780
tatggctcag ggtaccaagg agttgttcat gagtttttag agtcgttgac acctgtacca 3840
tcattattcg gtattaacga gaaagtgttg gagtttggta aaatattatt ctcatattcg 3900
aatgttactt ttaccagcgc aactgcagca tcgctattag gtatcattta tatttatcca 3960
aattcaattt tcaatgcgat tacaataata tgtcttagtt ggatttatat acatataatt 4020
acgggttatc ttgagtttag tcgaatgcaa cgagtagtcg cgtacttttt tgtgctgctt 4080
gcactttacc agggcttcat ggccatatta agtaattata tatatgcatt aacaatatat 4140
The termination of the initial orf3 of orf4
ttccttatag cctgtgccag aaatctttta ttgagtaaga aat atgaaac aatt tagtat 4200
taaaatcttg tcattggcag atgctatcga gcgtaggagg aaagtatcta gagagtttga 4260
gctgaaatca gtactgccat tctcattttt taacggtatt tatggtaaga cactaagtaa 4320
agaaaaatta gatacaatat actcttcaga gctggcgcag aaagtactcc atcggcaact 4380
aactgcgggg gaaattggtg caacctactc acattacttg attttccgtg aggcatatga 4440
gcgtggggat gagtttgttg ttgttctgga agatgactgt tatatagata gaaattttga 4500
taccgtgtta acatcaattc ttgataagaa aaatcctaca gatgatgaaa ttatctttat 4560
tcaaagacat acgagcgaaa actcacatat aataagatct ttaggcagtg agaagataaa 4620
taaaaattat accttacatc gtatgctagg tagcgcgcag tattttgttg gtgcatacgg 4680
gtatatcgta actagagctg caatgaaaaa aatgatcgat acatacttgc caatatattg 4740
tgtatgtgat cattggtatt ttataaagaa gaaatgtcag atagaaaaat ttagtgtatt 4800
atccccagct attgttttca caaatgatga ggatgttagg aaaatagata gttttgtaga 4860
cattgaaaga agagaaatcg ctgttttcaa atcagtaggt aaaatagcaa aactaaaaat 4920
The termination orf5's of orf4 is initial
tttaattaag aaaattattt taccaatatt aaataaagac tgggaa taaa a gtgctgcgc 4980
tattataaag tattgactgt tctgcttaga ttaattatag cttttatatt gtaccctatt 5040
gcatatgttt ttttgaatca caaaaaatat ttcatcattg gcacacgcaa tggtgttcga 5100
gggcatgata atgctgagta ttttttgtct tattgtacaa gacacaatat taatacaaaa 5160
gtaatttata atgatgctta tcagaaagat gaattatgca aaaactctat aaaaagtata 5220
ttgtatatct tatgtgcaaa caatgtttac ataacgcact ctgaatcaga tgttttagat 5280
tatttttggc gttttattcc cggtataaag tatatattta tacagcatgg agttattgga 5340
ataaaaaaac ttccggatta tgaaaagaaa agatatcata gatatgtttc tagtagtgaa 5400
tatgaaacag aaatttttag gaatttcttc catttgaaca gtgataaaat tattaaatct 5460
ggacttccac gttttgattt ctatgactat aaccgttcaa aaaatgatga aattaaatcc 5520
tgcttaatta tgtttacctg gagaaaatcg gattctggca aggctaaatt aattaaaaaa 5580
tatactgaga tggttacttg cctgtcaaat gaagctctca ataaaattta catttgtgtt 5640
catgaagcaa acttaagtac atttaaggtc ttgcttgaag aatcgcttga attggatgat 5700
aggttttcat ttatagaaaa tgataaatta tcatgtatta ttcaaagtac agaactttta 5760
atcactgact actcgagtgt tgcatgggat tatttatatc agaataaata tattttattt 5820
tataccccag atattgatga ttataaaaat acaactggat tgtattgtga tttttccgat 5880
ttttttggcg cacatcttat gaatttcagt agaatcaatt atccagtttt ggcatggatt 5940
atgcaagaaa atgaaattaa aaataaaaac tttcttgcaa ggtataaatt ttataacatg 6000
The termination of orf5
catgcaggca tacactctga gtatatagtg aagaatagt t aaaattatga gggcgaacat 6060
tgacaactat tattgtctca gcaactgcat tggctaagag cggtgcatta acaatattaa 6120
Orf6's is initial
atgagtttat tgagt atgtg tcaaatttaa aaaaatataa gtttataatt tttattcctg 6180
atagcattaa tttgcctaaa gcagttaata tcagatatat ccctgtgcct aaaaaaaatt 6240
ggctatctag gatatattgg gatagttatg gcttaagaaa atatattcga gtaaatcggc 6300
ttcaatatca ggcggtcatt tctcttcaga atacatctgt aaatgtagaa ggaaaacaga 6360
taatatatct tcaccaatct ataccattta tagattttga tattccactg acaagtttat 6420
ttaatttaaa attatggcta tacaagcgat tttattctta ttttatattt ttatttgtta 6480
acaagaatac ttcttttatt gtccaagcca aatggctgaa agagctcctt tctgataagt 6540
atcatatcga tgcaaaaaaa atatctatca taaagcccaa agcaagatat gttcctctag 6600
caactaactc tattcataat gtagccaact tatctaagca attctgtaat attatttatc 6660
cagcaacacc aatattttat aaaaatcatt gtgttattat tgatgctctt aggattttaa 6720
gagaacagcg caatattgat aacttatgtt ttaatgtcac cttttcaaaa ggtgagtatg 6780
ctaagtttga cgatttggtt aggcgttata acttgggtaa gaatattcgc tatttgggat 6840
atttgacccg agatgaatta tatcaacatt atgataatag cgcttttatg gtctacccca 6900
gttatgtgga aacctgtggg cttccgttgc tggaggctgc ttctaagcag ttgccaataa 6960
tcgctagtga tctgccttat gcatgtgaaa tgcttgaggc ttatactggc gtcgtttatg 7020
ttaagttcaa tgtctctacc gaatgggctc gagaaattga tacgatggcc cgagctggtg 7080
aaactcgaat tattccgccg ttattaaaag cggagaatga ttctgattgg aataagttag 7140
The termination of the initial orf6 of orf7
ataatattat tgaaggaaaa g atgatgtt t aaaaataaaa cactactaat tacaggtgga 7200
actggatcgt ttggtaatgc tgtgctccgt cgttttttat cgacggatat tggcgaaata 7260
aggatcttta gtcgcgatga aaagaaacaa gatgatatgc gtaaaaaata ttcggatccg 7320
aagttgaagt tttatattgg cgacgtaagg gattataaca gtattttgtc tgcaactcgc 7380
ggagttgatt atatctatca tgccgcagca ttgaagcagg ttccttcctg cgagttttat 7440
ccaatggaag cagtaaaaac taacgttatt ggtacagata atgtgttaga agctgcaata 7500
gcgaataaag ttcagcgtat agtctgtttg agtacggata aagccgtata tcctatcaat 7560
gcaatgggaa catcaaaagc aatgatggaa aaagtaattg ttgcaaaatc acgcaatttg 7620
cctgaaggca taactatttg tgctacccgt tatggaaatg taatggcttc gcgtggatcg 7680
gtgataccat tatttataaa tcaaattaaa aatggtaacc ctataactat taccgatccc 7740
gatatgactc gctttatgat gacccttgat gatgccgtcg atttagtatt acatgcgttc 7800
gaatacggaa ataatggtga tattttcgta caaaaggctc ctgcggcaac aattgaaaca 7860
cttactaaag caattcaaca ggttacaaac gcacttgatc atcagataaa tattatcggg 7920
acacgtcatg gtgaaaaatt gtatgaagtt ctctgtagcc gtgaagaaat ggctgtagca 7980
gaagatcagg gaaattacta ccgtatccct gctgacaatc gagatttaaa ctatgagaaa 8040
tactttgaga agggtaacaa agatgtttcc ttactcgagg attataattc gcataatact 8100
attagacttg acgttgatgg tatggttgct ttgctgcgta aactcgaatt tatccgcaaa 8160
The termination orf8's of orf7 is initial
gtggaagctg gttatgatgc aaatccagat gag taaagc a tgaaaatact tgttataggt 8220
gctacaggaa tgctcggagg aagtttgctt cgttatttcg ctgacaagac cgagcatgat 8280
gtatatggaa ctgttcgtga tagtaacgct gaaagtaggc tagtctcgaa agctaacgcc 8340
aatataatct gcggtattga tgttcacaat gtgaataaaa tacgcagcat aatagaggaa 8400
gtaagacctg actatgtaat taattgtgtt ggtgtagtta agcagcttaa agagtctaaa 8460
tatcctattc attctattac tataaattca ttgctgcccc atcgattagc tgagatctgt 8520
tcgcataata attcgaaatt aattcatttc tctacagatt gtgtattctc tggtaataga 8580
ggtaattatt cggttaatga tgttcctgat gcctttgatc tttatggacg ttctaaatta 8640
cttggtgaag taagttatgc tccacacctt acgttaagaa catcaataat tgggcatgag 8700
caggggtcac agcatagttt aattgattgg tttttaaatc aatcaggtga ggtaaaagga 8760
tttactaaag ctgtattctc cggagttcct actgtctata tggcggagct attaaataat 8820
tatgttttta ctaatcctga tatcacaggg ttgtatcagg tgagtgttga acctattgat 8880
aagtattctc ttctttcatt agttaaagat atttatggta aagatataga tattaatgcg 8940
gatgatagtc ttgttattga cagaagtttg aactcaactg aatttaaaat tcgtacgggg 9000
Orf9's is initial
atgaataatc cctcttggaa agatttgata gagaagatgt aca atgaata tagaacatat 9060
The termination of orf8
ttccaaaaag ct taaagtta ttacttttgt tggtacccgt cctgaaataa ttcgattgtc 9120
tagaataatt gctttgttgg ataaatattg tgaccatatt ttggttcata ctggacaaaa 9180
ttatgattat gagttgaatg aggttttttt ctccgattta ggaattagaa agcccgatta 9240
ctttttgaat gctgcgggta aaaatgcggc agagactatt gggcaaatta ttattaaatc 9300
tgatagtatt ctggaacaag ttaatccgca ggcactatta attttaggtg ataccaatag 9360
tgccttggtt gcaatatctg caaaaagaag aaaaatccct attttccata tggaagcagg 9420
gaatcgttgt tttgatttta gggttcctga agaaatcaat agaaaattgg ttgatcatat 9480
ttctgatatt aatctgacct atagtcatat agctcgtgat tatttactac gtgaaggcat 9540
tcccgctgat caggtgatca aaacgggatc tccgatgcgt gaagttttga attactatcg 9600
tgatgatatc caaaagtcgg ttgtacttga aacgttgaaa cttagtaata acaactattt 9660
tgttgtcagc tcccatcgcg aagagaatgt tgattcgcca gagagactta gacaactgct 9720
ggagatcctc aatgagcttg ctgaaagata taatttgcct gtcattgttt caactcatcc 9780
aagaaccaga aaaagattcg aagacttaag ttttaatgtt caccctaata tctctttcat 9840
gaagccattc ggatttattg actacatcac tttacagttg catgccagag ctgtactttc 9900
agatagtggt actattactg aagaatcttc tattcttaac ttcccagcac ttaatttacg 9960
tgaggttcat gaacgtcctg aaggtttcga agaagcctca gtgatgtttg tcggtctgaa 10020
taaagagcga gtattacagg ctcttgacat attaaatgag caaccaagat acgaatcacg 10080
cctcttgaac cttgtagagg actacaaacc cgataacgta tcagagaaag ttctcaggat 10140
The termination orf10's of orf9 is initial
tattatgagc tatactgatt ttgttaataa taaagtatgg aagaaa taat cg atgcgcat 10200
tgcgttaatt tgtgatgact atctccccgg tagtactcga gttagtgcaa aaatgatgca 10260
tgaattggct cgtgaacttc tgcaaagagg tcatgagcca attgttgtga ctccggattg 10320
ttacattaag tcaatttaca aagttgagca actggatggc gttgatatct tacgttttcg 10380
taacggaaga atcaaagatg tgtccaaaat taaacgtgca atgtgtgaaa cactactctc 10440
atataatggt tggaaggcgg taaatagtta tttttccaat gatgtgattg atgctgtcat 10500
atattactca ccttctatat tctttggtcc tctggttaat aaaattaaga ggaaatggtc 10560
atgtccatct tttcttatct tacgtgacag ttttcctcag tgggtaatcg atgaaggatt 10620
aattagagag ggttctctga tatgtagata ttttagattt tttgagaaaa taaactatca 10680
agcagcagat actattggag ttatgtcgga gaggaataga gaactttttt taaatatgca 10740
tccaaatatt cagagtgttg aagtcttata taactggtgc gatactaggg cggatgcaga 10800
actggacttg gtgaataaac ctgactgtat tactaagatc gcagataaaa ttatttttct 10860
ctatggtgga aatataggca aggcacagga tatgggaaat ttaatccgtc ttgcaattgc 10920
tatgaaaacc tactcaaatg ttcatttcat ttttattggg cagggtgatg aatacgattt 10980
tgtaagtgat agtataaaaa aatattcact gagcaacgtt agcttgtttc catctgtttc 11040
acaacatgat tttaaagtta ttctcaaatt tgttcatgtt gggttattta gtttgtctaa 11100
gaaacatacc gcccataatt tccctggcaa gttacttggc tatatgaaaa atagtttacc 11160
catactaggt agtgtaaata gtggtaatga tctgtcagat gttattaata ataatgaggc 11220
gggttgtgtt tgtgacaatg gcgatgataa tgcattatta cgtgcggcaa taaggttagc 11280
aactgatcag gattatcgtt acatgtgtgg acaaaaggct aatcagctac tacatgagaa 11340
attttctgtt acttctgcta ttgacaatat aatgaagcac ttaagtaaaa aaactcagga 11400
The termination orf11's of orf10 is initial
ctataat taa c atgaccttt aaattattgg actttaatct aaataatgag ttgaaagcta 11460
atgcaaagaa gagtgagcgt ttgcgatatc acttgagctt gcatggctct tataacgaac 11520
ccgttcagag aattattata tcgctaatgc gtggtacata tattcctcca cactaccatg 11580
aattcgagta tcaatgggag tattttaatg ttatatcagg taatatatgt gtgattattt 11640
ttgatcagaa tggtactgtg actgataagt ttaaattggg tcaaaatact ggtgcttacg 11700
gagttgagtt ttcttcaaac acaattcata caattctctg tgagagtgaa gatagtatta 11760
ttttagagtt aaaagaaggt cctttttttc caagtaaagc taaagttata ccgagttggg 11820
cgcctgatga gcaatattgc acatactctc gagcagaaat agtaagtgtt ttagaaaaaa 11880
The termination orf12's of orf11 is initial
tcaaaccagg ccaaaatctt cctgaagcat tattggcgca ggaacaa taa tgct atggaa 11940
aaaaaacgat tttttgcact tgatagtttt cgtggtctat gcgctttgtg tgtagtcatt 12000
tttcacatga aaatattgaa tgctttttcc gagtgggatt tttttcgaaa tagtcgactg 12060
tttgttgaat ttttctttat tcttagtggg tttgttttaa cccacagcta tttaaaaaaa 12120
gaaactaatg gtttttacaa atacgctatc gctcgaacgt ttcgaatttt tccacttcac 12180
atttttatgc tttgtgtgta tgttattctt gaatctttta aattagttgc acaacagaaa 12240
ggttttgtct ttaatacacc tccttttcag ggaggaaatt cattaaaaga atttcttcct 12300
aatctgcttt taattcagtc gtggtcatcg agttttgaat acttaagctt caattatcca 12360
tcatggagta taagtgttga atattattta tattttattt tttatctggt gatatgtcca 12420
cgaacgataa ttggtagagt ttttgctggc atagtgtttt ttattatatt tctttttcaa 12480
gaatattcct cttttaagat ttttaccagc gaagtgatta gaggtggaat ttgctttggc 12540
gctggaattt tagtttatta tctttattca ttaattcatc taagtcatgc aactcgattt 12600
ttatctgtgg tttttactat actagagtct agttctcttg cattaatttg gtttgttgta 12660
agtatgactg attatgaaag tgaattattg gtggtggttg tcttttcgtc atcagttttt 12720
ctatttgcat ttgaattagg gatgatttct ttttttctga aaaaaaaacc atttcagttt 12780
tttggtaaac tatcttactc tatatatctt actcatgctt cagttctttt tgttcttata 12840
tcagcaatga tggtattaca aaagataacg ggggtggcat ttacacagca tattgataat 12900
ttacgttatc ttaattttgg aagttcattg ataaataatt ttatgatttt tgcagtgctt 12960
ggatgtgtaa tatttttttc gatgctaacc tataaattta ttgagttgcc tgcgcaaaga 13020
The termination of orf12
aagggtaaag agttatatga gcattattct aaa taattgc tttaaagtta atgtgtcatt 13080
ttatttctag actatgttac tttaacaaga ctaatttaat atattgtaca cacctaatcg 13140
catcccccct gacaggagta aacaatgtca aagcaacaga tcggcgtcgt cggtatggca 13200
gtgatggggc gcaaccttgc gctcaacatc gaaagccgtg gttataccgt ctctattttc 13260
aaccgttcct gtgaaaagac ggaagaagtg attgccgaaa atccaggcaa gaaactggtt 13320
ccttactata cggtgaaaga gtttgttgaa tctcttgaaa cgcctcgtcg tatcttgtta 13380
atggtgaaag caggtgcagg cacggatgct gctattgatt cccttaagcc ataccttgaa 13440
aaaggcgaca tcatcattga tggcggtaac accttcttcc aggacaccat tcgtcgtaac 13500
cgtgagcttt ctgctgaagg ttttaacttc attggtacgg gggtttccgg tggtgaagag 13560
ggcgcgctga aaggtccttc tatcatgcct ggtggtcaga aagaagccta tgaactggtt 13620
gcaccgatcc tgaccaaaat tgctgccgta gctgaagatg gcgaaccgtg cgttacctat 13680
attggtgcag acggtgctgg tcactatgtg aagatggttc acaacggtat tgaatacggt 13740
gatatgcagt tgattgctga agcctattca ctgctgaaag gtggtctgaa tctctccaac 13800
gaagaactgg cacaaacctt tactgagtgg aataacggtg aactgagcag ctacctgatc 13860
gacatcacta aagacatttt caccaagaaa gatgaagacg gtaattatct ggttgacgtg 13920
atcctggatg aagcggctaa taaaggcacc ggtaaatgga ccagtcagag tgcgctggat 13980
ctcggcgagc cgctgtcgtt gattaccgag tctgtgtttg ctcgttatat ctcttctctg 14040
aaagagcagc gtgttgctgc atctaaagtt ctgtctggtc ctcaggcaca gccagcaggc 14100
gacaaagcgg agttcatcga gaaggttcac cgtgcgctgt atcttggcaa aatcgtttct 14160
tatgcacagg gcttctctca actgcgtgcc gcgtctgaag agtacaactg ggatctgaac 14220
tacggtgaaa tcgcgaagat tttccgtgct ggttgcatta ttcgtgcgca gttcttgcag 14280
aaaatcaccg atgcctatgc cgaaaatcca aaaatcgcta acctgctgtt agctccgtat 14340
ttcaagaaaa tcgccgatga ctaccagcag gcactgcgtg atgtcgtcgc ttatgcagtg 14400
caaaacggta tcccagttcc aaccttctct gcggcggttg cttattacga cagctatcgt 14460
gccgctgtcc tgcctgcgaa cctaatccag gcccagcgcg acta 14504
The above, it only is preferred embodiment of the present invention, be not that the present invention is done any pro forma restriction, every foundation technical spirit of the present invention all still belongs in the scope of technical solution of the present invention any simple modification, equivalent variations and modification that above embodiment did.

Claims (2)

1, a kind of oligonucleotide of the O-antigen-specific to Shigella bogdii 13 types is characterized in that described oligonucleotide is to being: the Nucleotide of 2012 to 2030 bases among the SEQ ID NO:1 and the Nucleotide of 2931 to 2949 bases; The Nucleotide of 1887 to 1904 bases among the SEQ ID NO:1 and the Nucleotide of 2599 to 2616 bases; The Nucleotide of 2009 to 2026 bases among the SEQ ID NO:1 and the Nucleotide of 2493 to 2510 bases; The Nucleotide of 3252 to 3269 bases among the SEQ ID NO:1 and the Nucleotide of 4081 to 4099 bases; The Nucleotide of 3206 to 3224 bases among the SEQ ID NO:1 and the Nucleotide of 4160 to 4176 bases; The Nucleotide of 3565 to 3582 bases among the SEQ ID NO:1 and the Nucleotide of 4083 to 4100 bases; The Nucleotide of 4399 to 4417 bases among the SEQ ID NO:1 and the Nucleotide of 4943 to 4962 bases; The Nucleotide of 4368 to 4385 bases among the SEQ ID NO:1 and the Nucleotide of 4670 to 4688 bases; The Nucleotide of 4227 to 4244 bases among the SEQ ID NO:1 and the Nucleotide of 4821 to 4840 bases; The Nucleotide of 5242 to 5261 bases among the SEQ ID NO:1 and the Nucleotide of 5677 to 5694 bases; The Nucleotide of 5071 to 5088 bases among the SEQ ID NO:1 and the Nucleotide of 5774 to 5791 bases; The Nucleotide of 5600 to 5617 bases among the SEQ ID NO:1 and the Nucleotide of 5889 to 5908 bases; The Nucleotide of 6089 to 6106 bases among the SEQ ID NO:1 and the Nucleotide of 6904 to 6921 bases; The Nucleotide of 6306 to 6324 bases among the SEQ ID NO:1 and the Nucleotide of 6906 to 6923 bases; The Nucleotide of 6050 to 6067 bases among the SEQ ID NO:1 and the Nucleotide of 6996 to 7013 bases; The Nucleotide of 10379 to 10397 bases among the SEQ ID NO:1 and the Nucleotide of 10953 to 10970 bases; The Nucleotide of 10448 to 10465 bases among the SEQ ID NO:1 and the Nucleotide of 10813 to 10830 bases; The Nucleotide of 10199 to 10217 bases among the SEQ ID NO:1 and the Nucleotide of 10098 to 11115 bases; The Nucleotide of 11472 to 11489 bases among the SEQ ID NO:1 and the Nucleotide of 11878 to 11894 bases; The Nucleotide of 11500 to 11517 bases among the SEQ ID NO:1 and the Nucleotide of 11686 to 11703 bases; The Nucleotide of 11689 to 11708 bases among the SEQ ID NO:1 and the Nucleotide of 11906 to 11923 bases; The Nucleotide of 11971 to 11990 bases among the SEQ ID NO:1 and the Nucleotide of 12872 to 12891 bases; The Nucleotide of 11973 to 11990 bases among the SEQ ID NO:1 and the Nucleotide of 12998 to 13015 bases; The Nucleotide of 12682 to 12700 bases among the SEQ IDNO:1 and the Nucleotide of 12997 to 13014 bases.
2, the application of the nucleotide pair of claim 1 is characterized in that, it is used for PCR as primer, perhaps is used for hybridization and fluoroscopic examination, makes gene chip or microarray as probe, for detecting Shigella bogdii 13 types.
CNB031005373A 2003-01-20 2003-01-20 nucleotide and its use Expired - Fee Related CN1252266C (en)

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