CN1261573C - Nucleotide with O-antigen idiocrasy against Ball's and shiga's-8 and colibacillus 0143 - Google Patents
Nucleotide with O-antigen idiocrasy against Ball's and shiga's-8 and colibacillus 0143 Download PDFInfo
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Abstract
The present invention provides a specific nucleotide to the O-antigens of Shigella boydii 8 and Escherichia coli 0143. The specific nucleotide is the nucleotide complete sequence of gene clusters in Shigella boydii 8 for controlling the synthesis of O-antigens, such as a separated nucleotide with the total length of 15124 basic groups shown as SEQ ID NO: 1 or a basic group with one or a plurality of insertion, deletion or substitution and the nucleotide of SEQ ID NO: 1 which can simultaneously keep the functions of the separated nucleotide. The present invention also comprises the oligonucleotide of glycosyltransferase genes and unit processing genes of oligosaccharide in O-antigen gene clusters stemmed from Shigella boydii 8. The PCR verifies that the specific nucleotide and the oligonucleotide have high specificity to all of the O-antigens of Shigella bodyii 8 and Escherichia coli 0143. The present invention also discloses a method for using the oligonucleotide of the present invention to detect and identify Shigella bodyii 8 and Escherichia coli 0143 in human bodies and environment.
Description
Technical field
The present invention relates to the complete nucleotide sequence of control O-antigen synthetic gene cluster in Shigella bogdii 8 types (Shigella boydii 8), particularly relate in Shigella bogdii 8 types oligonucleotide in the control O-antigen synthetic gene cluster, can utilize these oligonucleotide of O-antigen-specific Shigella bogdii 8 types and the intestinal bacteria O143 (Escherichia coliO143) in human body and the environment and identify O-antigen in these pathogenic bacterium quickly and accurately.
Background technology
Shigellae is the pathogenic bacterium that grow up along with the human evolution, can attack colon film epithelial cell, causes self limiting pyogenic infection focus, causes human bacillary dysentery.Human have higher susceptibility to Shigellae, only need be less than the infection that ten bacterium just can cause the people, children and adult easy infection, particularly children, easily cause acute poisoning dysentery, and the O-antigen of Shigellae is the one of the main reasons that Shigellae causes disease.
O-antigen is the O specific polysaccharide composition in the gram negative bacterium lipopolysaccharides, and it is made up of many multiple oligosaccharide unit.The antigenic building-up process of O-is studied clearlyer: by glycosyltransferase nucleoside diphosphate monose is transferred on the fat molecule that is fixed on the cell inner membrance earlier, then in the inboard synthesis of oligose unit of inner membrance, the antigenic oligosaccharide unit of O-is transferred to the inner membrance outside by the transhipment enzyme again, then aggregate into polysaccharide by polysaccharase, be connected to again and form lipopolysaccharide molecule [Whitfield, C. (1995) " Biosynthesis of lipopolysaccharide 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 relat ionships " .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 ant igen 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 link button between synthetic, the monose of 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 [" Molecularmicrobiological investigation of an outbreak of Hemolytic-UremicSyndrome caused by dry fermented sausage contaminated with Shiga-liketoxin producing Escherichia oli " .J.Clin.Microbiol.34:1622-1627] of having identified the toxogenic E.coli O111 of a strain at the oligonucleotide that comes from the wbdI gene of the O-antigen-specific of E.coli O111, 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 Bast in 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 that intestinal bacteria and Shigellae are total that 12 kinds of O-antigens are arranged, wherein Shigella bogdii 8 types just have identical O-antigen [Ewing with intestinal bacteria O143, W.H. (1986) " Edwardsand Ewing ' s identification of the Enterobacteriaceae " .ElsevierScience Publishers, Amsterdam, The Netherlands; T.cheasty, et al. (1983) " Antigenic relationships between the enteroinvasive Escherichiacoli antigens O28ac; O112ac; O124, O136, O143; O144; O152and O164andShigella O antigens " J.clin Microbiol, 17 (4): 681-684], traditional serotype 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 8 types and intestinal bacteria O143.It is the Nucleotide in the O-antigen gene bunch of Shigella bogdii 8 types, is the special Nucleotide that comes from glycosyltransferase gene and transhipment enzyme gene and pol gene.
A time purpose of the present invention has provided the full length nucleotide sequence of the O-antigen gene bunch of Shigella bogdii 8 types.
Another object of the present invention has provided the gene of the O-antigen gene bunch that constitutes Shigella bogdii 8 types: the gene of transhipment enzyme is the wzx gene or with wzx the gene of identity function is arranged; Pol gene is the wzy gene or with wzy the gene of identity function is arranged; Glycosyltransferase gene comprises orf3, orf6, orf7, orf10 gene.
Another purpose of the present invention has provided oligonucleotide, and the gene that they come from encoding glycosyl transferring enzyme in the O-antigen gene bunch of Shigella bogdii 8 types respectively comprises orf3, orf6, orf7, orf10 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 8 types and intestinal bacteria O143; Especially the oligonucleotide of listing in the table 1, they are high specials to the O-antigen of Shigella bogdii 8 types and intestinal bacteria O143, and these oligonucleotide are also reconfigurable, and the oligonucleotide after the combination also is a high special to the O-antigen of Shigella bogdii 8 types and intestinal bacteria O143.
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 detection and evaluation Shigella bogdii 8 types and the intestinal bacteria O143 of Shigella bogdii 8 types and intestinal bacteria O143 by these methods.
An also purpose of the present invention has provided the method for the complete sequence of the O-antigen gene bunch that separates Shigella bogdii 8 types.Can obtain the complete sequence of the O-antigen gene bunch of other bacteriums according to present method operation, the complete sequence of the gene cluster of the bacterium of other polysaccharide antigens that also can obtain to encode.
The technical scheme that the present invention is adopted for achieving the above object is:
The present invention is to the Nucleotide of the O-antigen-specific of Shigella bogdii 8 types and intestinal bacteria O143, and it is the isolating Nucleotide shown in SEQ ID NO:1,15124 bases of total length; The base that perhaps has one or more insertions, disappearance or replacement keeps the Nucleotide of the SEQ IDNO:1 of described isolating functional nucleotide simultaneously.
The Nucleotide of aforesaid O-antigen-specific to Shigella bogdii 8 types and intestinal bacteria O143, it is by 10 genomic constitutions, and they are all between galF gene and gnd gene.
The Nucleotide of aforesaid O-antigen-specific to Shigella bogdii 8 types and intestinal bacteria O143, wherein said gene is: transhipment enzyme gene comprises the wzx gene or with wzx the gene of identity function is arranged; Pol gene wzy gene or the gene of identity function is arranged with wzy; Glycosyltransferase gene comprises orf3, orf6, orf7, orf10 gene; Wherein said gene: orf3 is the Nucleotide of 3311 to 4228 bases among the SEQ ID NO:1; Wzx is the Nucleotide of 4664 to 5869 bases among the SEQ ID NO:1; Orf6 is the Nucleotide of 5851 to 6744 bases among the SEQ ID NO:1; Orf7 is the Nucleotide of 6741 to 7856 bases among the SEQ ID NO:1; Wzy is the Nucleotide of 7853 to 8935 bases among the SEQ ID NO:1; Orf10 is the Nucleotide of 12131 to 12934 bases among the SEQ ID NO:1.
The Nucleotide of aforesaid O-antigen-specific to Shigella bogdii 8 types and intestinal bacteria O143 wherein comes from described wzx gene, wzy gene or glycosyltransferase gene orf3, orf6, orf7, orf10 gene; And their mixing or their reorganization.
The Nucleotide of aforesaid O-antigen-specific to Shigella bogdii 8 types and intestinal bacteria O143, the oligonucleotide that wherein comes from the orf3 gene is to being: the Nucleotide of 3455 to 3471 bases among the SEQ ID NO:1 and the Nucleotide of 3929 to 3946 bases; The Nucleotide of 3635 to 3653 bases among the SEQ ID NO:1 and the Nucleotide of 4037 to 4056 bases; The Nucleotide of 3740 to 3759 bases among the SEQ ID NO:1 and the Nucleotide of 4201 to 4220 bases; The oligonucleotide that comes from the wzx gene is to being: the Nucleotide of 4843 to 4860 bases among the SEQ ID NO:1 and the Nucleotide of 5377 to 5394 bases; The Nucleotide of 4889 to 4906 bases among the SEQ ID NO:1 and the Nucleotide of 5461 to 5479 bases; The Nucleotide of 5391 to 5408 bases among the SEQ ID NO:1 and the Nucleotide of 5830 to 5847 bases; The oligonucleotide that comes from the orf6 gene is to being: the Nucleotide of 6032 to 6051 bases among the SEQ ID NO:1 and the Nucleotide of 6417 to 6435 bases; The Nucleotide of 6220 to 6239 bases among the SEQ ID NO:1 and the Nucleotide of 6671 to 6689 bases; The Nucleotide of 6087 to 6106 bases among the SEQ ID NO:1 and the Nucleotide of 6671 to 6689 bases; The oligonucleotide that comes from the orf7 gene is to being: the Nucleotide of 7194 to 7211 bases among the SEQ ID NO:1 and the Nucleotide of 7822 to 7839 bases; The Nucleotide of 6780 to 6797 bases among the SEQ ID NO:1 and the Nucleotide of 7222 to 7239 bases; The Nucleotide of 7010 to 7028 bases among the SEQ ID NO:1 and the Nucleotide of 7592 to 7626 bases; The oligonucleotide that comes from the wzy gene is to being: the Nucleotide of 8211 to 8228 bases among the SEQ ID NO:1 and the Nucleotide of 8844 to 8861 bases; The Nucleotide of 7892 to 7911 bases among the SEQ ID NO:1 and the Nucleotide of 8414 to 8431 bases; The Nucleotide of 8038 to 8056 bases among the SEQ ID NO:1 and the Nucleotide of 8565 to 8582 bases; The oligonucleotide that comes from the orf10 gene is to being: the Nucleotide of 12152 to 12171 bases among the SEQ ID NO:1 and the Nucleotide of 12915 to 12932 bases; The Nucleotide of 12312 to 12329 bases among the SEQ ID NO:1 and the Nucleotide of 12843 to 12860 bases; The Nucleotide of 9934 to 9952 bases among the SEQ ID NO:1 and the Nucleotide of 10513 to 10530 bases.
The Nucleotide of aforesaid O-antigen-specific to Shigella bogdii 8 types and intestinal bacteria O143 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 8 types and intestinal bacteria O143, and can provide the O-antigen of expressing Shigella bogdii 8 types and intestinal bacteria O143 by inserting to express, and become bacterial vaccine.
The application of the Nucleotide of aforesaid O-antigen-specific to Shigella bogdii 8 types and intestinal bacteria O143, wherein it is used for PCR, is used for hybridization and fluoroscopic examination or is used to make gene chip or microarray, the bacterium in human body and the environment as probe as primer.
The separation method of the Nucleotide of aforesaid O-antigen-specific to Shigella bogdii 8 types and intestinal bacteria O143 is characterized in that it comprises the steps:
(1) genomic extraction: 37 ℃ of incubated overnight Shigellaes in the LB of 5mL substratum, centrifugal collecting cell.With 500ul 50mM Tris-HCl (pH8.0) and 10ul 0.4M EDTA re-suspended cell, 37 ℃ of incubations 20 minutes, the N,O-Diacetylmuramidase that adds 10ul 10mg/ml then continues insulation 20 minutes, the Proteinase K that adds 3ul20mg/ml afterwards, 15ul 10%SDS, 50 ℃ of incubations 2 hours, the RNase that adds 3ul 10mg/ml again, 65 ℃ 30 minutes, add equal-volume phenol extracting mixture, get supernatant and use isopyknic phenol again: chloroform: twice of primary isoamyl alcohol (25: 24: 1) extracting, get supernatant again with isopyknic ether extracting to remove remaining phenol, supernatant is with 2 times of volume ethanol deposit D NA, roll out DNA and wash DNA with 70% ethanol with glass yarn, at last DNA is resuspended among the 30ul TE, genomic dna detects by 0.4% agarose gel electrophoresis;
(2) by the O-antigen gene in pcr amplification Shigella bogdii 8 types bunch: the O-antigen gene of Shigella bogdii 8 types is bunch by the Long pcr amplification, at first according to the JumpStart sequences Design upstream primer (#1523-ATT GTG GCT GCA GGG ATCAAA GAA AT) that often is found in O-antigen gene bunch promoter region, again according to the gnd gene design downstream primer (#1524-TAGTCG CGT GNG CCT GGA TTA AGT TCG C) in O-antigen gene bunch downstream.With the ExpandLong 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 fens, and carried out 30 circulations like this.At last, continue to extend 7 fens 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, use isopyknic phenol and phenol respectively: chloroform: primary isoamyl alcohol (25: 24: 1) extracting once, use twice of isopyknic ether extracting again after, with the dehydrated alcohol deposit D NA of 2.5 times of volumes, and wash precipitation with 70% ethanol, be resuspended at last in the 18ul water.In this mixture, add 2.5uldNTP (1mMdCTP subsequently, 1mMdGTP, 1mMdTTP, 10mMdATP), the T4DNA polysaccharase of 1.25ul 100mM DTT and 5 units, 11 ℃ 30 minutes, enzyme is cut product mend into flush end, after 75 ℃ of termination reactions, add the Tth archaeal dna polymerase of 5 units and corresponding damping fluid thereof and system is expanded as 80ul, 70 ℃ were reacted 20 fens, made the 3 ' end of DNA add the dA tail.This mixture is through the equal-volume chloroform: after primary isoamyl alcohol 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 8 types;
(4) to the cloning and sequencing in the library: from the library, select insert 120 clones of fragment more than 700bp by Shanghai biotechnology company limited with ABI377 type automatic dna sequencer to unidirectional order-checking of insertion fragment in cloning, make sequence reach 80% fraction of coverage.The sequence of residue 20% is again according to the sequences Design primer that has obtained, this is to primer, as follows: 5 '-TCGAGTTGGTGATATAGC-3 ' and 5 '-TTATTAATAGTCTCCGCG-3 ', direct PCR and from the genomic dna of Shigella bogdii 8 types again to the order-checking of PCR product, thus all sequences of O-antigen gene bunch obtained.
(5) splicing of nucleotide sequence and analysis: the Pregap4 and the splicing of Gap4 software of the Staden package software package of publishing with Britain Camb MRC (Medical Research Council) Molecular Biology Lab and edit all sequences, thereby obtain the Nucleotide full length sequence of the O-antigen gene bunch of Shigella bogdii 8 types, the quality of sequence is mainly guaranteed by two aspects: 1) genome of Shigella bogdii 8 types is done 6 Long PCR reactions, mix these products then to produce the library.2), guarantee high-quality fraction of coverage more than 3 to each base.After obtaining the nucleotide sequence of Shigella bogdii 8 type O-antigen genes bunch, with American National biotechnology information science center (The National Center forBiotechnology Information, NCBI) orffinder finds gene, find the reading frame of 14 openings, determine also that with the function of finding the reading frame that these are open what gene they are with the genetic comparison among the blast groupware and the GenBank, finish gene annotation with the Artemis software at Britain sanger center again, do accurate comparison between DNA and protein sequence with Clustral W software, obtain the structure of the O-antigen gene bunch of Shigella bogdii 8 types at last.
(6) screening of specific gene: at wzx, wzy, orf3, orf6, orf7, the orf10 gene design primer in the O-antigen gene of Shigella bogdii 8 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 intestinal bacteria O143, the correct band of any size does not all increase in other groups, promptly do not obtaining any PCR product band in the array mostly, though in the minority group, obtain PDR product band, but its size does not meet the expection size, so wzx, wzy, orf3, orf6, orf7, orf10 gene pairs Shigella bogdii 8 types and intestinal bacteria O143 and O-antigen thereof all are high specials.
First aspect just of the present invention provides the full length nucleotide sequence of the O-antigen gene bunch of Shigella bogdii 8 types, its complete sequence shown in SEQ ID NO:1,15124 bases of total length; The base that perhaps has one or more insertions, disappearance or replacement keeps the Nucleotide of the SEQ ID NO:1 of described isolating functional nucleotide simultaneously.Obtained the structure of the O-antigen gene bunch of Shigella bogdii 8 types by method of the present invention, as shown in table 3, it is altogether by 10 genomic constitutions, all between galF gene and gnd gene.
Second aspect of the present invention provides the gene in the O-antigen gene bunch of Shigella bogdii 8 types, promptly transports enzyme gene (wzx gene or the gene of identity function arranged with wzx); Pol gene (wzy gene or the gene of identity function arranged with wzy); Glycosyltransferase gene comprises orf3, orf6, orf7, orf10 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 8 types and intestinal bacteria O143.
The 3rd aspect of the present invention, wzx gene in the O-antigen gene bunch that comes from Shigella bogdii 8 types is provided or the gene, wzy gene of identity function is arranged or the gene and the glycosyltransferase gene of identity function are arranged with wzy with wzx, the oligonucleotide that comprises orf3, orf6, orf7, orf10 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 8 types and intestinal bacteria O143 only, and are all not obtain expecting the product of size in the pcr amplification that carries out of template all the other 165 strain intestinal bacteria listed with table 2 and 42 strain Shigellaes.In more detail, with these oligonucleotide to being that PCR that primer is done is reflected at and does not all obtain spawn in most of bacteriums.So can determine these primers promptly the listed oligonucleotide of table 1 be high special to Shigella bogdii 8 types and intestinal bacteria O143 and their O-antigen.
The separation method of the Nucleotide of the O-antigen-specific to shigella dysenteriae 8 types and intestinal bacteria O143 provided by the invention comprises the steps: (1) genomic extraction; (2) the O-antigen gene in pcr amplification Shigella bogdii 8 types bunch; (3) structure in structure O-antigen gene bunch library; (4) to the cloning and sequencing in the library: the splicing and the analysis of (5) nucleotide sequence: the screening of (6) 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 " 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 orf3 gene (nucleotide position is 3311 to 4228 bases from SEQ ID NO:1), wzx gene (nucleotide position is 4664 to 5869 bases from SEQ ID NO:1), orf6 gene (nucleotide position is 5851 to 6744 bases from SEQ ID NO:1), orf7 gene (nucleotide position is 6741 to 7856 bases from SEQ ID NO:1), wzy gene (nucleotide position is 7853 to 8935 bases from SEQ IDNO:1), orf10 gene (nucleotide position is 12131 to 12934 bases from SEQ ID NO:1).Coming from above intragenic oligonucleotide all is high special to Shigella bogdii 8 types (SEQ ID NO:1) and intestinal bacteria O143.
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 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 8 types or intestinal bacteria O143.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 8 types or intestinal bacteria O143.
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 8 types or intestinal bacteria O143.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.
Generally, 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 8 types or intestinal bacteria O143.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, or 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 8 types first, and can from the sequence of this total length gene cluster of not cloned, produce recombinant molecule, can produce the O-antigen of expressing Shigella bogdii 8 types by inserting to express, and become useful vaccine.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should understand these embodiment only is used to the present invention is described and is not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, molecular cloning: the condition described in the laboratory manual (NewYork:Cold Spring Harbor Laboratory Press, 1989).
Embodiment 1: genomic extraction
37 ℃ of incubated overnight Shigellaes in the LB of 5mL substratum, centrifugal collecting cell.With 500ul50mM 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, at last DNA is resuspended among the 30ul TE.Genomic dna detects by 0.4% agarose gel electrophoresis.
Embodiment 2: by the O-antigen gene in pcr amplification Shigella bogdii 8 types bunch
The O-antigen gene of Shigella bogdii 8 types bunch is by the Long pcr amplification.At first according to the JumpStart sequences Design upstream primer (#1523-ATTGTG GCT GCA GGG ATC AAA GAA AT) that often is found in O-antigen gene bunch promoter region, again according to the gnd gene design downstream primer (#1524-TAG TCG CGT GNG CCT GGA TTA AGT TCG C) in O-antigen gene bunch downstream.With the Expand Long Template PCR method of BoehringerMannheim company amplification O-antigen gene bunch, the PCR response procedures was as follows: 94 ℃ of pre-sex change 2 minutes; 94 ℃ of sex change are 10 seconds then, 60 ℃ of annealing 30 seconds, and 68 ℃ were extended 15 fens, and carried out 30 circulations like this.At last, continue to extend 7 fens 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, use isopyknic phenol and phenol respectively: chloroform: primary isoamyl alcohol (25: 24: 1) extracting once, use twice of isopyknic ether extracting again after, with the dehydrated alcohol deposit D NA of 2.5 times of volumes, and wash precipitation with 70% ethanol, be resuspended at last in the 18ul water.In this mixture, add 2.5uldNTP (1mMdCTP subsequently, 1mMdGTP, 1mMdTTP, 10mMdATP), the T4DNA polysaccharase of 1.25ul 100mM DTT and 5 units, 11 ℃ 30 minutes, enzyme is cut product mend into flush end, after 75 ℃ of termination reactions, add the Tth archaeal dna polymerase of 5 units and corresponding damping fluid thereof and system is expanded as 80ul, 70 ℃ were reacted 20 fens, 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-6.0ms millisecond.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 8 types with the EcoRI enzyme.
Embodiment 4: to the cloning and sequencing in the library:
From the library, select insert 120 clones of fragment more than 700bp by Shanghai biotechnology company limited with ABI377 type automatic dna sequencer to unidirectional order-checking of insertion fragment in cloning, make sequence reach 90% fraction of coverage.Residue 10% sequence is again according to the sequences Design primer that has obtained, direct PCR and from the genomic dna of Shigella bogdii 8 types again to the order-checking of PCR product, thus obtain all sequences of O-antigen gene bunch.We have designed a pair of primer in Shigella bogdii 8 types, and are as follows: 5 '-TCGAGTTGGTGATATAGC-3 ' and 5 '-TTATTAATAGTCTCCGCG-3 '
Embodiment 5: the splicing of nucleotide sequence and analysis:
The Pregap4 and the splicing of Gap4 software of the Staden package software package of publishing with Britain Camb MRC (Medical Research Council) Molecular Biology Lab and edit all sequences, thus the Nucleotide full length sequence (seeing sequence list) of the O-antigen gene bunch of Shigella bogdii 8 types obtained.The quality of sequence is mainly guaranteed by two aspects: 1) genome of Shigella bogdii 8 types is done 6 Long PCR reactions, mix these products then to produce the library.2), guarantee high-quality fraction of coverage more than 3 to each base.After obtaining the nucleotide sequence of Shigella bogdii 8 type O-antigen genes bunch, with American National biotechnology information science center (The National Center for BiotechnologyInformation, NCBI) orffinder finds gene, find the reading frame of 10 openings, determine also that with the function of finding the reading frame that these are open what gene they are with the genetic comparison among the blast groupware and the GenBank, finish gene annotation with the Artemis software at Britain sanger center again, do accurate comparison between DNA and protein sequence with Clustral W software, obtain the structure of the O-antigen gene bunch of Shigella bogdii 8 types at last, as shown in table 3.
By retrieving and comparing, the aminotransferase gene of finding orf1 and Bacillus anthracis has 27% homogeny in 370 amino acid whose sequences, 49% similarity, show very high homology is arranged between them, so can infer orf1 is aminotransferase gene, called after orf1.The phosphatase gene of orf2 and Clostridium acetobutylicum has 25% homogeny in 213 amino acid whose sequences, 46% similarity, showing has very high homology between them, are phosphatase genes so can infer orf 2, called after orf2.The glycosyltransferase gene of orf3 and Methanosarcina maze homogeny of 38% in 311 amino acid whose sequences, 55% similarity, in addition also with the EpsllQ gene of Streptococcus thermophilus 37% homogeny in 428 amino acid whose sequences, 56% similarity, EpsllQ is a glycosyltransferase gene, so infer that orf3 is the gene of an encoding glycosyl transferring enzyme, called after orf3.The acetyltransferase gene of orf4 and Clostridiumacetobutylicum has 29% homogeny in 182 amino acid whose sequences, 47% similarity shows the homology that height is arranged between them.So, can infer that orf4 is the acetyltransferase gene.The wzx gene of Orf5 and Escherichia coli K-12 has 17% homogeny in 415 amino acid, 43% similarity, in 409 amino acid, 18% homogeny is arranged with the wzx gene of Shigella bodyii, 44% similarity, and algorithm [Eisenberg by people such as Eisenberg, D, Schwarz, E.etal (1984) .Analysis of membrane and surfaceprotein sequences with the hydrophobic moment plot.J.Mol.Biol.179:125-142] find that orf5 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 orf5 is the wzx gene, called after wzx.The EpsH gene of Orf6 and Lac tobacillus delbrueckii has 28% homogeny in 268 aminoacid sequences, 49% similarity, in addition also with Streptococcus agalactiae in the cpsIbJ gene in 313 aminoacid sequences, 28% homogeny is arranged, 48% similarity, and the EpsH gene is a galactosyltransferase gene, the cpsIbJ gene also is a glycosyltransferase gene, so infer that orf6 is the gene of an encoding glycosyl transferring enzyme, called after orf6.The glycosyltransferase gene of Orf7 and Methanosarcina mazei Goel has 23% homogeny in 379 aminoacid sequences, 42% similarity, in addition also with Nostoc sp.PCC7120 in glycosyltransferase gene in 429 aminoacid sequences, 22% homogeny is arranged, 43% similarity, so infer that orf7 is the gene of an encoding glycosyl transferring enzyme, called after orf7.The wzy gene of orf8 and Escherichia coli K-12 has 19% homogeny in 388 aminoacid sequences, 41% similarity, in 447 aminoacid sequences, 20% homogeny is arranged with the O-antigen polysaccharase of Streptococcus pneumoniae, 39% similarity, learn that by the Eisenberg algorithm orf8 has 10 potential transmembrane domains in addition, to other O-antigen polysaccharase similar secondary structure is arranged, so determine that orf8 is the wzy gene, called after wzy.The gne gene of orf9 and Edwardsiella ictaluri has 70% homogeny in 338 aminoacid sequences, 80% similarity, and showing has high homology between them, so determine that orf9 is the gne gene, called after gne.The CpsVII gene of orf10 and Streptococcus agalactiaei has 35% homogeny in 322 aminoacid sequences, 54% similarity, EpsM gene with Lactobacillus delbrueckii has 29% homogeny in 336 aminoacid sequences in addition, 47% similarity, and these two genes all are glycosyltransferase genes, so infer that orf10 also is a glycosyltransferase gene, called after orf10.Embodiment 6: the screening of specific gene: at wzx, wzy, orf3, orf6, orf7, orf10 gene design primer in the O-antigen gene of Shigella bogdii 8 types bunch, the position of these genes in nucleotide sequence sees Table 1.
Table 1 has been listed glycosyltransferase gene and oligosaccharide unit treatment gene and intragenic primer and PCR data in 0 antigen gene bunch of Shigella bogdii 8 types.Glycosyltransferase gene, transhipment enzyme gene and pol gene and their function corresponding and the size of the O antigen gene bunch of Shigella bogdii 8 types in table, have been listed.In each gene, we have respectively designed three pairs of primers, and the difference that every pair of primer is distributed in the corresponding gene is local to guarantee its specificity.In table, also listed position and the size of each primer in SEQ IDNO:1.Is that template carry out PCR with listed corresponding annealing temperature in the table with the genomes of all bacterium in the table 2 with every pair of primer, has obtained corresponding PCR product, and its size is also listed in the table.Mdh (malate dehydrogenase) gene is to be present in all colibacillary genomes and a gene of high conservative, so we according to the mdh gene design primer #101 (TTC ATC CTAAAC TCC TTA TT) and #102 (TAA TCG CAG GGG AAA GCA GG), extract genome then from 166 strain intestinal bacteria, method as previously mentioned.With this to primer from the colibacillary genome of 166 strains PCR with identification of escherichia coli and detect its genomic quality.
Table 2 is 166 strain intestinal bacteria and 43 strain Shigellaes and their sources that are used to screen specific gene, and for the convenience that detects, we are divided into one group with their every 8-10 bacterium, and 27 groups altogether, all list in the table in their source.
The genomic dna that contains Shigella bogdii 8 types in the 22nd 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 fens, and carried out 30 circulations like this.Continue to extend 10 fens 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, orf3, orf6, orf7, orf10 gene, each gene all has three pairs of primers detected, every pair of primer has obtained also all obtaining onesize specificity band the correct band of expection size in the 17th group except be PCR in the 22nd group after.After being template PCR with the genomic dna of each bacterium in the 17th group, find only in intestinal bacteria O143, to have obtained positive findings.In more detail, each of each gene all obtains expecting the correct PCR product band of size to primer more than in intestinal bacteria O143.It is reported that the O-antigenic structure of Shigella bogdii 8 types and intestinal bacteria O143 is the same, our result has confirmed this point from another side.In addition, all primers are any band that all do not increase in other groups, so wzx, wzy, orf3, orf6, orf7, orf10 gene pairs Shigella bogdii 8 types and intestinal bacteria O143 and O-antigen thereof all are high specials.
At last, from Shigella bogdii 8 types, screen gene by PCR: wzx, wzy and four glycosyltransferase genes to the O-antigen high special of Shigella bogdii 8 types and intestinal bacteria O143.And the oligonucleotide of these intragenic any one section 10-20nt is special to the O-antigen of Shigella bogdii 8 types and intestinal bacteria O143, and the primer in especially above-mentioned each gene is that oligonucleotide is high special to detecting the back confirmation through PCR to Shigella bogdii 8 types and intestinal bacteria O143.These all oligonucleotide all can be used for Shigella bogdii 8 types and the intestinal bacteria O143 in the human body and environment rapidly and accurately, and can identify their O-antigen.
Table 3 is structural tables of the O-antigen gene bunch of Shigella bogdii 8 types, in table, listed the structure of the O-antigen gene bunch of Shigella bogdii 8 types, altogether by 10 genomic constitutions, each gene box indicating, and in square frame, write the title of gene, numeral be the order of the open reading frame (orf) in the O-antigen gene bunch.Two ends at O-antigen gene bunch are galF gene and gnd gene, and 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.
Table 4 is location tables of the gene in the O-antigen gene bunch of Shigella bogdii 8 types, in table, listed the accurate position of all open reading frame in complete sequence in the O-antigen gene bunch of Shigella bogdii 8 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 of the O-antigen-specific of Shigella bogdii 8 types and intestinal bacteria O143
＜130〉to the Nucleotide of the O-antigen-specific of Shigella bogdii 8 types and intestinal bacteria O143
<160>1
<170>PatentIn version 3.1
<210>1
<211>15124
<212>DNA
<213>Shigella boydii
<400>1
attgtggctg cagggatcaa agaaatcctc ctggtaactc atgcgtccaa gaacgcagtc 60
gaaaaccact tcgacacctc ttatgaatta gaatctctcc ttgaacagcg cgtgaagcgt 120
caactgctgg cggaagtaca gtccatttgc ccgccgggcg tgaccattat gaacgtgcgt 180
cagggcgaac ctttaggttt aggccactcc attttatgtg cacgacctgc tattggtgac 240
aacccatttg tcgcggtgct gccagacgtt gttatcgatg acgccagcgc cgacccgctg 300
cgctacaacc ttgctgccat gattgcgcgc ttcaacgaaa cgggccgcag ccaagtgctg 360
gcaaaacgta tgccgggtga cctctctgaa tactccgtca ttcagaccaa agagccgctg 420
gaccgcgaag gtaaagtcag ccgcattgtt gaatttatcg aaaaaccgga tcagccgcag 480
acgctggact cagacatcat ggctgtaggg cgttatgtgc tttctgccga tatttggcct 540
gaactggagc gtactcaacc tggagcatgg ggacgtattc agttgactga tgccattgct 600
gagttggcaa aaaaacaagc agttgacgca atgctgatga ctggggacag ttacgactgc 660
ggaaagaaaa tgggttatat gcaagcgttt gtgaagtatg ggctgcgtaa cctgaaggaa 720
ggcgctaagt tccgcaaagg cattgagaag ttgttaagcg agtaatgaaa atctgaccgg 780
atgtaacggt tgataagaaa attataacgg ctgtgaagat ttgcggcgaa ggtaatttgt 840
tgcgaatatt cctgccgttg ttttatataa acaatcagaa taacaaagag ttagcaatag 900
gattttcgtc aaagttttcc aggattttcc ttgtttccag agcggattgg taagacaatt 960
agcgtttgaa tttttcgggt ttagcgcgag tgggtaacgc tcgtcacatc gtaggcatgc 1020
atgcagtgct ctggtagctg taaagccagg ggcggtagcg tgcatataag agaaaacatg 1080
tggcactgat tttttactaa acaattggcg ataaatattc aataatggag attttcctta 1140
acatcaaacg ctattatttt atatattata aatgtccaaa tactcagtat atatacaaaa 1200
agagtaagat aaaacttaaa aatttaataa ggtgtctcta tatttctgac tacagacaag 1260
catggaaagt ataatatatt gaatattctg tttctgatta cttatgctct caatttgcag 1320
aagttagtga agatattcag tatgtggaat agatatatat tttttttgac acaatatgtc 1380
gattattctt cacgagatta tattaagaat agaataaaat aaatgtatgg gcttatatac 1440
gaattatgaa acataggagc aattctgaaa attatctaag ataagatatt gtgttttctt 1500
gagtatgttt aaattcttta aatgttatgc acagaacgta atggagtaaa aattgaataa 1560
gttcgtaaaa aaaatgaagc catataaact tgcatcccac gctatatggg agagtgaaaa 1620
taaagaaaca atattaaaat tagattggaa cgaatctact attgaaccgc cagaaagagt 1680
aaaaaatgca ttaatgaatt cgttggtaaa taatctaatt aggtattatc cagatgtgag 1740
caataaagat ttaataataa agatatcaaa ctatgtgggg gtgccaaagg aatatatcca 1800
gtatttttgt agttcagact cagcacacga gtctatcatt cgtacattaa ttaatgaaaa 1860
tgataaaata ttgatgttag cacctacgta tgataatttc agaacaacag gtgaagctca 1920
aggggcaaca atttgttatt caatttttga agataattac tattggtcat taaagaagtt 1980
tgaaagcgat ctcaataagt ttcaacctca aatggcctat ttatgtaacc caaataatcc 2040
tacaggtact cttattccta aaggagatat acattatctt gtagaaaaat ataaaggaat 2100
atattttatt atcgatgaag cctattatga atttgcaaaa ttaacgtgtg ctgattttgt 2160
aaactccttt aataacataa ttattacgcg aacattttcc aaagcatttt cattggcagg 2220
gatgcgaatg ggatatataa tagcgtcaca agatttgtta ggtgcaataa ataaagtcag 2280
aaatgcaaaa aatatacctc agctaagtca gattgccgct ttgaatgcat tagacgaagt 2340
tgaatatatg aataattatg ttaaggaagt taatcaggct aagcgttttt tttatgataa 2400
gttgaaaaat ataaatatag gtcaaaaaaa tattgtatat ggagagggaa actttttggt 2460
tattgagttt aaatcgatga atgataaaaa agcattcatt gaacgtttaa aatctaattt 2520
aatatttgtt cgtgatttat ctcatatgaa agaggtcgag ttatgtgtca gaattactat 2580
tggaacgaaa aaacaaatgt tgtatgtaat cagaatttta gagcagatgt atgaagttaa 2640
aaaagatagc attatttgat ctgtgtgaaa ccttagttga ttttcaaact ggcgatgggt 2700
ttatatacta tgtaatagat cgtttgggga tatgtaataa catcaaaata cagactttaa 2760
atagggtggc atcttcatta tccattttca aaatcgaaca aataataaat aaaataagat 2820
tgggggaaag tttaaggaaa agaattttat tgtatatgtt aaaaaatatt gaaagggata 2880
ttcttaatgt aatggctcgt gattacgtta atgaaattat attgcgcaga gttaatcctg 2940
ccataattaa gatattgaat aagcatttaa aagatggaga tgaggttatt attatttctg 3000
gaggctatga tatttatatt caatatttag ttaaagcatt agagattaat agctttgtat 3060
gcacaaaaat taaatttgat aatagaggca ggtgtttagg gagaattagt ggcaagaatt 3120
gcatgggata taataaagta ttaatgttaa aaggtgtttt gtcgttagat gtttctgagt 3180
atcatataac aacttactct gattctatta gcgatttacc aatattaaaa atgagcaacg 3240
ttggggtagt agtatcgaaa ggccaggcaa gagaatgggt aagacagcat aaattatttg 3300
agattctaat atgaatagaa aaaatctttc catgaaattg atgtatatat tgtataatat 3360
aaagtattta ttgaaatgct ttgtcgttct gattactcca gatagcttga ttataaaaaa 3420
gcagtttctt gaatatcagg gataccctct cggattgaac aatccgaaga cattaaatca 3480
aaagttacaa tggttgaaac taaatgacag aacaccatta catacaatat gtgctgataa 3540
aattagagtg cgagaatatg tggaaaatag gctgggaacg agagataata taattgattt 3600
gttaaaggtt tatgataccc cttttgatat tacctttgag tctttaccac agggtaagtt 3660
tgttataaaa tcgaaccact attgtggcga ttattttatt gttagagaca agaatttatt 3720
gaatattgat gaggttaagt ggaaatttat tagaacactt aattgtaata tttaccatca 3780
cggaagagaa tggcaatata aaaatattga taggcgtata attgttgaaa ggttattaga 3840
ggatgaggca gggaaagttc ctaatgatat aaaaattcat tgttttaatg gagaaccgaa 3900
atttatttat gtgtcgttag atcgtgaggg agggaattat cgttctatat atgatactag 3960
ttggaatatg ttagattttt gctgggcgag aaggggaaaa gatctttcaa aatttaatgt 4020
gaagaatatt agtaaaccaa gtcagttaga aaacgctcta aatatatcta aaaaactaag 4080
tcaaggtttc aaatatctta gggttgattt atatcttgtt caagaaaaaa tatatgtagg 4140
cgaattaaca tttcatcaag gatcggggta tgataagata actccatttg aatgggatga 4200
aaggctcgga aaggagttaa agctatgagc atattctcta gaattcaatt aatatggtat 4260
aaattatgtt ttatctgtga gtttggagta aaacgttata agcaatccca tatttatggt 4320
ccttataaga tagtagaacc atcgaatctt aaattcggta attccttttc aattaatgat 4380
tttgtgtata taaatgctag tggaggagtc attttaggcc ataatgtagt actgtctgct 4440
ggtagtaaaa ttctcagtac aggtttggta tttgaaaaga aaatctttaa atataaaaaa 4500
gataaggtag taataaaaga taatgttcag ttaggtgctg gagcaattgt tttaccggga 4560
gtggagatct gtagtaatat tattgttggt gccggagcga tagttacaaa aaacttagag 4620
gaaccaggtg tatatgttgg gcaaccggct agaaaaataa aagatgtcta gttttttaag 4680
aagtattatc tctgtaagtg ctctaaagct agtggattat attgtcccaa taataattat 4740
gccgctggtt ttaactagat ttaatttaga gggatatgga gtttattctt atgtgttaac 4800
tatatcaact tttatttcat ttatttatga tgcttgtcta aatacagtag gcgtacaaca 4860
attaaataag cttaagggta atgaaattgc tcagcagtcg tttgttattt cagcgatttt 4920
ctttaagtta gtgttttttt ttgtatgtgc gattgtgttt attataattt cagagactgc 4980
agggaagagt gattattttt gtgtcgtaat attctcgttt ggatttggca tgcttaatag 5040
ttggtactat atagcaaagg ataactttag gtttataata atagtgtcct tggtagttaa 5100
aattttagcg gttttatttg ttttatatta tgcttttgaa ttacacatgt ttatttttat 5160
tcaggcgata gcatctgctc tggttggact tattagtttg ataaaaataa agaatgataa 5220
taggttggga aaaatatcat ttagacatat taaaaattat ttgaaaaaca ttaagcatat 5280
gtacatatat agcggggtaa atgcacttat tcagcctata attaattctg ctatattggc 5340
atcaggaaat gctagcttgt tagggatata taacgtcgtt atgcgatttg tggcagtatc 5400
cgtaacattt tccaattcaa ttatcactgt cttaaataaa tgtaattcag aggtttttta 5460
ttccgtaggg cacaaaacta actatgtgga agtaaaatac agaagtcagc tactcaaatt 5520
gttgttttac agcatcgttg tttttatatg tgtggctgca atatcatatg tctctattta 5580
tatgacaaag ggagaaatat ctcgtttttt tatttttcta atattttcat ttagcctagc 5640
tgttatccca agtatactca atcaatattt aactcaatta attcatatga tacactcatc 5700
aaaaaatata gtaaaatatg agatttgttc gaatgtacta tcgttaatat ctattttttt 5760
attttcttca ttattcccat attattatat agtatatgct gtagttctat cgccaattat 5820
tattacaatt tgctgtctgt gggatttgaa atgcaaaaac ttgctgtaat catgacttac 5880
tataatgaga gttacataat actaaaaaag gcagtagaca gtactcttca ggatctggaa 5940
agattggaaa agaagaatgt gaaatataaa ttaataattg gagtcgatag gagaaatgaa 6000
gatattcctc atattgagag tttatattta aaagaagtta gtgccagaaa tgatgtgtgc 6060
gttgtttttt ttaatgagaa cgaagggttg gcggcaagtt taaataaatt aatttttggc 6120
gcgtgtaatg gctatgattt tattgccaga atggatgctg atgatattgt tattaacggg 6180
cgttttaaaa agcaattaga atatctgaat aagcattctg atgtcagttt agtcggtgga 6240
caggctgttg ttattgatga gaacgataat gttgttaggg attattttaa tatgaaccca 6300
gtagattttt cgtgggggaa tgaaataatt catccaactt ggttgttaag gtcagagctt 6360
tattataaat taggtggtta caggcgttta gatgctgctc aagattatga ttttttgtgt 6420
agggctgtat tgagtggatg tattgtaagg aatatagatg acgtggtttt attttatagg 6480
ataaggcatg gctcaattgg cagttgtaag aagtttatcc aaataaaaaa caagactatt 6540
ttatctaaat ttttcagaaa aaatgaagtt gcacctattc atttatttaa tgcaaccaac 6600
ttttcagttg agtttttttt attctctgtc attgaatcaa taaaaatcaa gcacaatatt 6660
gtggggaaat gtattgggtg gttgtctcca ctccatgcta aaaattatta tttaaattta 6720
agaaaacgaa tggggaaaaa atgagagtac ttgtttttgg ccctatgata tctggtcata 6780
ttagtaactg gattcatggt tccaaagaat atattgatta tagaattgtc acactacact 6840
atgacaaaag tatttctgag aatgttggaa taggagagat atataagata ccaagactta 6900
caaaaactaa atttgatttt atatacgcac ctgtttttct tcttttttat ttttttttat 6960
taaagcctga gttggttcat gtccattttt ttagttctta tggtttaatt tcatgtgttt 7020
tgcctagaaa agtaaagaag atactctcag tatggggaag tgacattaac aatactataa 7080
aaaagagagg gatattagga aaaatatatg taaaagcaat gaaaagtttt tgtgttgtaa 7140
attcacccgc caggcatata acaaataaaa taatatcgta tttcaatgtg aatcaggagc 7200
gaatacatac ttttcaatat ggtgtagatg tggggttctt ttcagatgaa aaaacgtcaa 7260
gcataaggga cgataaatgt ataacaataa cttctgtaag aaattgggat aatatttata 7320
acatagagtc ctttttagaa gctgttattg atatgaaaga ttatgattgt aaagggtact 7380
tttttaatat caatatacta ggccgctcat cgtcaaagac cacacatgat aagataatag 7440
aactcacaaa aaaatgtgag ggagataatt ataaagtaaa tttaataggg tttattcaaa 7500
aagaggaatt aaggtcgatt cttcataaat ctgattttgt tcttagtatt cctagcaatg 7560
atggtgctcc attatctttg atggagggcg ttttatgtaa ttgttttccg atagtatcag 7620
acatcgatgc aaacagagag ctattaggaa atcatgcaat gtatttgaat ctgcatgatg 7680
atattgacat gaaatctgtt ttttcaaggt gtgtgaaatt aatagatagt aatcaatata 7740
ggtgttcgat tgaatataac cgagatacaa taataaaaat gtttgatata aataaaaaca 7800
caaagcgtat ggttgatatt tattatcggg tcgtgtcaca aaaggaattt ttatgaaggt 7860
tttttttgat gtatttttgt tgtttttatt tgtgtttgct tttagcatcc ctgttttttt 7920
taattcaggc tatcttatat atattttttc cttgatttat ttaattacaa atatcaggat 7980
aaggactgtt ttttttagtc taataagtac aagaatattt tttattgcgt ttttgtcttt 8040
ggcctgcatg ctcttttttt cttgtatttc attggtatta aatggtacag gagatttatc 8100
tatcatgggg gttatcttta ataatttagc tgcattattc agctctgtta taattgcgtc 8160
gcatttaatc cagcataaca atgataatgt gttgaaattg ttttttctaa ttgccctctt 8220
acaatccttt tttattattc tgatgatgtt ttttcccagt ataaatgctt atattcaaag 8280
tattattaga acaaaagaag aaatagaatt tatgagtaat acatatggag gcatcagggg 8340
attaggtata tcaggttctg ttgcctttgg tcttgctata attatgtcat tattatcttt 8400
tctttcaatt gtttggttta atgaggaaga ctgtaatatt cctttctttt taaaattatt 8460
ttttatcata ctcttgctgt tcgcgtcaat ttctgctggg cgtacggctg ttttggggtt 8520
tgtcctcgga ggggtatata gttttatttg ctctgattat aaaaagaagt tttatcgggc 8580
gcttcaaatt ttgatttatg tatctctgtt tttatttatt ttttggagat tttttttaaa 8640
tgaacatgtt tatgatatct tgaaaaaata ttcggattat tcttttcaaa tgttatatta 8700
ttatttagat tcaggaaagt tatcaacaac atctacagat gaattattta atatgtattt 8760
cccgttaaca gaaaaacaga tcttcattgg ttatgggtac cataccgtta aaggatggga 8820
catattatct cgggaccgat gctggattta tgcggttagc attatttttg ggtgcggtac 8880
catcggttat tttttatttt ttgtttttta tttttttgac tatttatgct gctaatagca 8940
tcaaacctaa aatagttgca ttaatgatta caatactcag tttttctttt cattataaag 9000
gcgaggttat atttttgaat gttgcatata tgaaaataat atatatatat atattttcat 9060
ctttttatat ttgtcataaa aaaagattaa taagaagggg ttcggttaat gcatgattat 9120
tattttaatt gcaggttttt attaacaaac aacagtagaa actcgttaat tgttgacata 9180
ttatcttatt ctagatatct gtaaaacaaa ttggtttttg cctttgtttt tatgggtatt 9240
taaagactat agggtaatag cgcaacaatt taatttaagt tttttgagaa ctgtaagaat 9300
agtgaactac atagatcaca aatctataac tgaaccaggt gataatccca tgaatacaat 9360
ctataataaa taatgttttg gattagtaag tgttataaat ttacgtggaa attctatttg 9420
cgtgtttatg ttgctatttc atggaatggt tttattgcca ttgctgtagt gttattttga 9480
actaatttat ttggtgaaag attactgagt aatcttttga tactaaatcg attgtgtttt 9540
atatctttga attaaatatt tttgccaaag cactccttca ctcttatata ttcagatact 9600
ccttgtgtta taagagtaag gaatataaat tggaatatgg aaaaaggcgt gacatatttc 9660
tgttgtcttt ggttatgtac acgatgggca gagtgtaagt tttcagagat gttgcaattg 9720
tctccagata agaaaggaga gattgttatg ttgaaccaac atatcggcgt caggtgtttc 9780
gggcattacc gtgcaacaat ctatgcgatg gaatacaatg ccatgcctga tattgcaaaa 9840
ttaagaatga aaaccctgca ttttagggaa agacatggaa tcaatgcatc cgcagaaaat 9900
ttttgtgtat caatacgcgt ttaatatata ggctcggaag tgcttatctc aagtagtaaa 9960
tctttctagt atgtcttaca aaactatgac atccagaaat tttgaaagaa atatgatgaa 10020
ttagaatagg acttctcaac cttggaaaag aacagatttt tgaccgtctg aatctgtagt 10080
gtgaacagta gcatttggcc tgcccgagcg tctccactat tggaggaatg agctaggcag 10140
cacatgataa aatgtggcta atactggtgt aactgggttc ccagacttca agtccaaggt 10200
actattagcc cgctaaagac cggtgagctt atattgagct cagaatcggc gttatgttat 10260
taccgtgatt gacgaacgca gcaactacgc tttggcggtg gcggtaacgt cgcttaacag 10320
tgatgtaatc atgagtttct tctgccatca agctcggttg ttacctgttg gaatcaacca 10380
gaatataccc gattatgggg aagagttcct gggaaaattt gacaaaaagc tacaggaagc 10440
cgctatcagg caccaatagg cttatctcga tacgccgaaa atgaacacca tctgtgagcg 10500
ctttaaccga tcactgagat agccgtttat tgagtttaat gaagaattgc tctttgaaaa 10560
tctgaatatg tcaatcataa acaggctcaa tattgcatac tgtacaagaa taaaaggcct 10620
cataaattgc tcgaattact gacaccacta gaacataatt tacacgaaaa aaatgcaata 10680
tagtgttaga ctcatacaat atgtcgagat gtttagaaaa ataacgtata cttacatatt 10740
gtatttttat agtaacattg ggcttaacta ttctaaattt ttattctttc acagtttata 10800
ctatttataa tgtgtttata ttcttgtttt atatcctagg cagatagatg aaatataagg 10860
agttttatga ctattttggt tactggtggt gcaggttaca ttggttcaca tactgtatta 10920
tccttattat catacggaaa aaatgtggtg gtgttggata atctatcgaa ctcatcgtct 10980
aagtcgttag aaagggtcga aaaaatttgt ggacgtaggc ctctatttta tcatggtgat 11040
gtgcgtgata ggaaaaaact caaacatatt ttccagtatc atgatattac agatgttatt 11100
cattttgcag gattaaaagc cgtcgctgaa tctgtttcaa atccattgga atattatgat 11160
cacaatgtta acggaactat tgttttactt gaagaaatga tgcgcgctgg cgttattagt 11220
tttattttta gctcctccgc aacagtgtat ggtaagcctg aaactattcc tttgaaggaa 11280
gatagtagcg ttggcggaac aactaatcca tatggctttt caaaacttat agtagagagg 11340
gttttgaagg atatagcttt ggctgagcca acaatgcgta ttactatttt gcgttatttc 11400
aatcctgtag gagcccactc ttctggtcta ttaggtgaag atcctaatgg tgttccaaat 11460
aatttaatcc cttatattac tcaagtggct attggtaatc taaaatattt atcagtattc 11520
ggaaatgatt atcctacacc tgatggaaca ggcattagag attatattca tgttatggat 11580
ttggctgaag ggcacatagc tgctttagaa caccgtaatt ttgggggaaa ttataaagta 11640
tataatcttg gtacaggggt tggttattca gttcttgatg tgataaatac ctttaaacgt 11700
tgtactggaa ttgatatccc ttattgtttc aaacctcgtc gagttggtga tatagctgaa 11760
tgttggtctg atccatctct agcattttta gaattagggt ggtcttctcg atatgattta 11820
gatacaatga taattgattc ttggcgttgg caaaaaaaca atcccaaagg atataaataa 11880
gacgatttta atgtgctatt ttattcactg tcaatatttt tataaaacca tctttgcttt 11940
gctttgcttt gctttgcttt gctttgcttt gtagttactc tcttaattaa cggcttttta 12000
tattagggtg ggatgttaat gaaatacttg ataaatattt aatatgtgaa atttcttaat 12060
aagctaccaa ggatggatat gataaacatg atattatata atgaaactta tgtaagtttg 12120
agatgattct atgagtaccg ataaatattt agtttcagtc ataacaccca catataattc 12180
tgaaagaacg attagatgta caattgattc tgttctatct caatcatata ataatataga 12240
aatgattatt gtcgatgatt gttcgttaga taatacagtg tctatttgca gggaatatca 12300
aaaaaaagat tcaagagttg ttctcatttc aaatgagaag aattatgggg ctggtatgtc 12360
aagaaatata gcgatcagta aagctaaagg tcggtttatc gcttttttag atagtgatga 12420
tttttggcat aaagagaaaa taaaaaaaca gatagacttt atgctttcaa ataattatgc 12480
tttcacttat acctcatatc aaaaggttag ttttagcggg aaacttttat cccaaattaa 12540
tcctgtttct aaagttaatt attctgaatt attaaagact aatgtaatag gttgcttgac 12600
agctgtatat gatacttcat ctcttggtaa aatgtttatg ccaacaatca ggaaaagaca 12660
ggacatggca ctatggctga gcattttaga taaaattgat tatgcatatt gtttgaacga 12720
aatactggcc tactatcgag taggcaccga tacactctcg tcaaataaat taaaaataat 12780
attttcacaa tgggcctttt acagaaaata tttaaagttt ggtctgttta aatctcttta 12840
ttatttctcc cactatgctg ttaatgctgt tgccaaacat catataaatc cgctgtgttt 12900
gttttttaag cgccactttg cctcttggag ataattcggc aaatcttata aagaaatatt 12960
ggataatgct tttgatttgc aaatgtattt ttatatttga tgatgtcatg gatgagtttt 13020
aagtgaattt caatagaata gggaaatgtg ggtctgtgtg gacaagaagt gtgattaatg 13080
tttttgtatc agattataat ctgcaatcta tttagtaaaa ttatgtttac caagtgcctc 13140
tcctgaagaa ttatttttat aaagccttta gttatcattg aaatgaattc tgaggttaat 13200
tggtgcgaat gaacttagta ttatgataaa gattgtcttt attttactta aaagtataga 13260
atagttcatt gcattatttg tgttgtataa aacttcagag tcaggaacag tcttaagttt 13320
tattttttct aaattcattg gtggctatca tatggttatt agatgactgt gttaagatat 13380
atttcattgg ctttaaggtt ttagtacata tgacgaatag aaccaatttg aatattctgg 13440
aaacaccaac agcctcgttg tttgtcaaaa ttattttgag gttgataatt tgactctcga 13500
ttattgtaat ttataaaata taagtgagtt gaaagatata tttgaggcat ggcagttaaa 13560
tctcttatat atcgcggaga ctattaataa tagttagtct gagacactgg aaactagagt 13620
gtggagagca acgttagttt tgagtatttt ctgtaattaa tttaatataa gtttaattac 13680
atatacaatt atctgttgtt tgtcttatgc aatattttcc atatgacata tttttatatg 13740
tgtttcaata cgtttattat tctgctaatt aaataggagt gaaaatgtca aaaaaacaga 13800
ttggggttat cggtatggcg gtaatggggc gtaatcttgc actaaatatc gaaagtcacg 13860
gctatactgt ttcaattttt aaccgttctc gtgaaaaaac agaaaaaatt atagctactc 13920
attcggataa gaagttattt ccttactata caataaaaga gtttgttgaa tctctggaaa 13980
cgcctcgtcg catcctgtta atggtgaaag caggtgcagg cacggatgct gctattgatt 14040
ctctcaagcc atacctcgat aaaggtgaca tcatcattga tggtggtaac accttcttcc 14100
gagacaccat tcgtcgtaac cgtgagctgt ctgcagaagg ctttaacttt atcggtagcg 14160
gtgtttctgg tggtgaagaa ggtgcgctga aaggaccttc catcatgcca ggtggccaga 14220
aagaagccta tgaactggtc gcaccgatcc tgaccaaaat cgccgcagtg gctgaagatg 14280
gggagccatg tgttacctat attggtgccg atggcgcagg tcactatgtg aagatggttc 14340
acaacggtat tgaatacgga gatatgcagc tgattgctga agcctattct ctgcttaaag 14400
gtggcctgaa cctcaccaac gaagaactgg cgcagacctt taccgagtgg aataatggtg 14460
aactaagcag ctacctgatc gacatcacca aagacaactt cactaaaaaa gatgaagatg 14520
gtaactacct ggttgatgtg attctggatg aagcggctaa caaaggtacc ggtaaatgga 14580
ccagccaaag cgcgctagat ctcggtgaac cgctgtcgct cattaccgag tctgtgtttg 14640
cacgatacat ctcttctctg aaagagcagc gtgttgccgc gtccaaagta ctgtctggtc 14700
cgcaagcgca gccagcaggc gacaaggctg agttcatcga aaaagttcgt cgtgctctgt 14760
atctgggcaa aatcgtttct tacgcccagg gcttctctca gttgcgtgct gcgtctgaag 14820
agtacaactg ggatctgaac tatggcgaaa tcgcgaagat tttccgtgct ggctgcatta 14880
tccgtgcgca gttcctgcag aaaatcaccg atgcttatgc cgaaaatccg cagatcgcta 14940
acctgctgct ggctccgtat ttcaagcaaa ttgccgatga ctaccagcag gcgctgcgcg 15000
atgtcgtcgc ttatgcggta cagaacggta tcccggttcc gaccttcgcc gctgcggttg 15060
cctattatga cagctaccgc gccgctgttc ttcctgcgaa cctaatccag gcccagcgcg 15120
acta 15124
Glycosyltransferase gene and oligosaccharide unit treatment gene in the table 1 Shigella bogdii 8 type O antigen genes bunch
And wherein primer and PCR data
Table 2166 strain intestinal bacteria and 43 strain Shigellaes and their source
| 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 (℃) |
| Orf3 | Glycosyltransferase | 3311-4228 | #301(3455-3471) | #302(3929-3946) | 492bp | 0 | 50 |
| #303(3635-3653) | #304(4037-4056) | 423bp | 0 | 54 | |||
| #305(3740-3759) | #306(4201-4220) | 481bp | 0 | 54 | |||
| wzx | The transhipment enzyme | 4664-5869 | #307(4843-4860) | #308(5377-5394) | 552bp | 0 | 50 |
| #309(4889-4906) | #310(5461-5479) | 591bp | 0 | 53 | |||
| #311(5391-5408) | #312(5830-5847) | 457bp | 0 | 55 | |||
| 0rf6 | Glycosyltransferase | 5851-6744 | #313(6032-6051) | #314(6417-6435) | 404bp | 0 | 50 |
| #315(6220-6239) | #316(6671-6689) | 470bp | 0 | 54 | |||
| #317(6087-6106) | #318(6671-6689) | 611bp | 0 | 55 | |||
| Orf7 | Glycosyltransferase | 6741-7856 | #319(7194-7211) | #320(7822-7839) | 648bp | 0 | 50 |
| #321(6780-6797) | #322(7222-7239) | 460bp | 0 | 54 | |||
| #323(7010-7028) | #324(7592-7626) | 617bp | 0 | 50 | |||
| wzy | Polysaccharase | 7853-8935 | #325(8211-8228) | #326(8844-8861) | 651bp | 0 | 52 |
| #327(7892-7911) | #328(8414-8431) | 540bp | 0 | 54 | |||
| #329(8038-8056) | #330(8565-8582) | 545bp | 0 | 54 | |||
| orf10 | Glycosyltransferase | 12131-12934 | #331(12152-12171) | #332(12915-12932) | 781bp | 0 | 55 |
| #333(12312-12329) | #334(12843-12860) | 549bp | 0 | 55 | |||
| #335(9934-9952) | #336(10513-10530) | 597bp | 0 | 55 |
| Group number | The bacterial strain that contains in this group | The source |
| 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 | Wild-type e. coli O1, O2, O3, O4, O10, O16, O18, O39 wild-type e. coli O40, O41, O48, O49, O71, O73, O88, O100 wild-type e. coli O102, O109, O119, O120, O121, O125, O126, O137 wild-type e. coli O138, O139, O149, O7, O5, O6, O11, O12 wild-type e. coli O13, O14, O15, O17, O19ab, O20, O21, O22 wild-type e. coli O23, O24, O25, O26, O27, O28, O29, O30 wild-type e. coli O32, O33, O34, O35, O36, O37, O38, O42 wild-type e. coli O43, O44, O45, O46, O50, O51, O52, O53 wild-type e. coli O54, O55, O56, O57, O58, O59, O60, O61 wild-type e. coli O62, O63, O64, O65, O66, O68, O69, O70 wild-type e. coli O74, O75, O76, O77, O78, O79, O80, O81 wild-type e. coli O82, O83, O84, O85, O86, O87, O89, O90 wild-type e. coli O91, O92, O95, O96, O97, O98, O99, O101 wild-type e. coli O112, O162, O113, O114, O115, O116, O117, O118 wild-type e. coli O123, O165, O166, O167, O168, O169, O170, O171 wild-type e. coli O172, O173, O127, O128, O129, O130, O131, O132 | IMVS a IMVS IMVS IMVS IMVS IMVS IMVS IMVS IMVS IMVS IMVS IMVS IMVS IMVS See b See c |
| 17 18 19 20 21 22 23 24 25 26 27 | Wild-type e. coli O133, O134, O135, O136, O140, O141, O142, O143 wild-type e. coli O144, O145, O146, O147, O148, O150, O151, O152 wild-type e. coli O153,0154, O155, O156, O157, O158, O159, O164 wild-type e. coli O160, O161, O163, O8, O9, O124, O111 wild-type e. coli O103, O104, O105, O106, O107, O108, O110 Shigella bogdii serotypes B 4, B5, B6, B8, B9, B11, B12, B14 Shigella bogdii serotypes B 1, B3, B7, B8, B10, B13, B15, B16, B17, B18 shigella dysenteriae serotype D1, D2, D3, D4, D5, D6, D7, D8 shigella dysenteriae serum D9, D10, D11, D12, D13 shigella flexneri F6a, F1a, F1b, F2a, F2b, F3, F4a, F4b, F5 (v:7) F5 (v:4) bacillus ceylonensis A D5, DR | IMVS IMVS IMVS IMVS IMVS See d See d See d See d See d 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 8 types
Table 4 is location tables of the gene in the O-antigen gene bunch of Shigella bogdii 8 types
attgtggctg cagggatcaa agaaatcctc ctggtaactc atgcgtccaa gaacgcagtc 60
gaaaaccact tcgacacctc ttatgaatta gaatctctcc ttgaacagcg cgtgaagcgt 120
caactgctgg cggaagtaca gtccatttgc ccgccgggcg tgaccattat gaacgtgcgt 180
cagggcgaac ctttaggttt aggccactcc attttatgtg cacgacctgc tattggtgac 240
aacccatttg tcgcggtgct gccagacgtt gttatcgatg acgccagcgc cgacccgctg 300
cgctacaacc ttgctgccat gattgcgcgc ttcaacgaaa cgggccgcag ccaagtgctg 360
gcaaaacgta tgccgggtga cctctctgaa tactccgtca ttcagaccaa agagccgctg 420
gaccgcgaag gtaaagtcag ccgcattgtt gaatttatcg aaaaaccgga tcagccgcag 480
acgctggact cagacatcat ggctgtaggg cgttatgtgc tttctgccga tatttggcct 540
gaactggagc gtactcaacc tggagcatgg ggacgtattc agttgactga tgccattgct 600
gagttggcaa aaaaacaagc agttgacgca atgctgatga ctggggacag ttacgactgc 660
ggaaagaaaa tgggttatat gcaagcgttt gtgaagtatg ggctgcgtaa cctgaaggaa 720
ggcgctaagt tccgcaaagg cattgagaag ttgttaagcg agtaatgaaa atctgaccgg 780
atgtaacggt tgataagaaa attataacgg ctgtgaagat ttgcggcgaa ggtaatttgt 840
tgcgaatatt cctgccgttg ttttatataa acaatcagaa taacaaagag ttagcaatag 900
gattttcgtc aaagttttcc aggattttcc ttgtttccag agcggattgg taagacaatt 960
agcgtttgaa tttttcgggt ttagcgcgag tgggtaacgc tcgtcacatc gtaggcatgc 1020
atgcagtgct ctggtagctg taaagccagg ggcggtagcg tgcatataag agaaaacatg 1080
tggcactgat tttttactaa acaattggcg ataaatattc aataatggag attttcctta 1140
acatcaaacg ctattatttt atatattata aatgtccaaa tactcagtat atatacaaaa 1200
agagtaagat aaaacttaaa aatttaataa ggtgtctcta tatttctgac tacagacaag 1260
catggaaagt ataatatatt gaatattctg tttctgatta cttatgctct caatttgcag 1320
aagttagtga agatattcag tatgtggaat agatatatat tttttttgac acaatatgtc 1380
gattattctt cacgagatta tattaagaat agaataaaat aaatgtatgg gcttatatac 1440
gaattatgaa acataggagc aattctgaaa attatctaag ataagatatt gtgttttctt 1500
gagtatgttt aaattcttta aatgttatgc acagaacgta atggagtaaa aattgaataa 1560
Orf1's is initial
gttcgtaaaa aaa
atgaagc catataaact tgcatcccac gctatatggg agagtgaaaa 1620
taaagaaaca atattaaaat tagattggaa cgaatctact attgaaccgc cagaaagagt 1680
aaaaaatgca ttaatgaatt cgttggtaaa taatctaatt aggtattatc cagatgtgag 1740
caataaagat ttaataataa agatatcaaa ctatgtgggg gtgccaaagg aatatatcca 1800
gtatttttgt agttcagact cagcacacga gtctatcatt cgtacattaa ttaatgaaaa 1860
tgataaaata ttgatgttag cacctacgta tgataatttc agaacaacag gtgaagctca 1920
aggggcaaca atttgttatt caatttttga agataattac tattggtcat taaagaagtt 1980
tgaaagcgat ctcaataagt ttcaacctca aatggcctat ttatgtaacc caaataatcc 2040
tacaggtact cttattccta aaggagatat acattatctt gtagaaaaat ataaaggaat 2100
atattttatt atcgatgaag cctattatga atttgcaaaa ttaacgtgtg ctgattttgt 2160
aaactccttt aataacataa ttattacgcg aacattttcc aaagcatttt cattggcagg 2220
gatgcgaatg ggatatataa tagcgtcaca agatttgtta ggtgcaataa ataaagtcag 2280
aaatgcaaaa aatatacctc agctaagtca gattgccgct ttgaatgcat tagacgaagt 2340
tgaatatatg aataattatg ttaaggaagt taatcaggct aagcgttttt tttatgataa 2400
gttgaaaaat ataaatatag gtcaaaaaaa tattgtatat ggagagggaa actttttggt 2460
tattgagttt aaatcgatga atgataaaaa agcattcatt gaacgtttaa aatctaattt 2520
aatatttgtt cgtgatttat ctcatatgaa agaggtcgag ttatgtgtca gaattactat 2580
Orf2's is initial
tggaacgaaa aaacaaatgt tgtatgtaat cagaatttta gagcagatgt
atgaagttaa 2640
The termination of orf1
aaaagatagc attatt
tgat ctgtgtgaaa ccttagttga ttttcaaact ggcgatgggt 2700
ttatatacta tgtaatagat cgtttgggga tatgtaataa catcaaaata cagactttaa 2760
atagggtggc atcttcatta tccattttca aaatcgaaca aataataaat aaaataagat 2820
tgggggaaag tttaaggaaa agaattttat tgtatatgtt aaaaaatatt gaaagggata 2880
ttcttaatgt aatggctcgt gattacgtta atgaaattat attgcgcaga gttaatcctg 2940
ccataattaa gatattgaat aagcatttaa aagatggaga tgaggttatt attatttctg 3000
gaggctatga tatttatatt caatatttag ttaaagcatt agagattaat agctttgtat 3060
gcacaaaaat taaatttgat aatagaggca ggtgtttagg gagaattagt ggcaagaatt 3120
gcatgggata taataaagta ttaatgttaa aaggtgtttt gtcgttagat gtttctgagt 3180
atcatataac aacttactct gattctatta gcgatttacc aatattaaaa atgagcaacg 3240
ttggggtagt agtatcgaaa ggccaggcaa gagaatgggt aagacagcat aaattatttg 3300
Orf3's is initial, the termination of orf2
agattctaat
atgaatagaa aaaatctttc catgaaattg atgtatatat tgtataatat 3360
aaagtattta ttgaaatgct ttgtcgttct gattactcca gatagcttga ttataaaaaa 3420
gcagtttctt gaatatcagg gataccctct cggattgaac aatccgaaga cattaaatca 3480
aaagttacaa tggttgaaac taaatgacag aacaccatta catacaatat gtgctgataa 3540
aattagagtg cgagaatatg tggaaaatag gctgggaacg agagataata taattgattt 3600
gttaaaggtt tatgataccc cttttgatat tacctttgag tctttaccac agggtaagtt 3660
tgttataaaa tcgaaccact attgtggcga ttattttatt gttagagaca agaatttatt 3720
gaatattgat gaggttaagt ggaaatttat tagaacactt aattgtaata tttaccatca 3780
cggaagagaa tggcaatata aaaatattga taggcgtata attgttgaaa ggttattaga 3840
ggatgaggca gggaaagttc ctaatgatat aaaaattcat tgttttaatg gagaaccgaa 3900
atttatttat gtgtcgttag atcgtgaggg agggaattat cgttctatat atgatactag 3960
ttggaatatg ttagattttt gctgggcgag aaggggaaaa gatctttcaa aatttaatgt 4020
gaagaatatt agtaaaccaa gtcagttaga aaacgctcta aatatatcta aaaaactaag 4080
tcaaggtttc aaatatctta gggttgattt atatcttgtt caagaaaaaa tatatgtagg 4140
cgaattaaca tttcatcaag gatcggggta tgataagata actccatttg aatgggatga 4200
Orf4's is initial, the termination of orf3
aaggctcgga aaggagttaa agct
atgagc atattctcta gaattcaatt aatatggtat 4260
aaattatgtt ttatctgtga gtttggagta aaacgttata agcaatccca tatttatggt 4320
ccttataaga tagtagaacc atcgaatctt aaattcggta attccttttc aattaatgat 4380
tttgtgtata taaatgctag tggaggagtc attttaggcc ataatgtagt actgtctgct 4440
ggtagtaaaa ttctcagtac aggtttggta tttgaaaaga aaatctttaa atataaaaaa 4500
gataaggtag taataaaaga taatgttcag ttaggtgctg gagcaattgt tttaccggga 4560
gtggagatct gtagtaatat tattgttggt gccggagcga tagttacaaa aaacttagag 4620
Orf5's is initial, the termination of orf4
gaaccaggtg tatatgttgg gcaaccggct agaaaaataa aag
atgtc
ta gttttttaag 4680
aagtattatc tctgtaagtg ctctaaagct agtggattat attgtcccaa taataattat 4740
gccgctggtt ttaactagat ttaatttaga gggatatgga gtttattctt atgtgttaac 4800
tatatcaact tttatttcat ttatttatga tgcttgtcta aatacagtag gcgtacaaca 4860
attaaataag cttaagggta atgaaattgc tcagcagtcg tttgttattt cagcgatttt 4920
ctttaagtta gtgttttttt ttgtatgtgc gattgtgttt attataattt cagagactgc 4980
agggaagagt gattattttt gtgtcgtaat attctcgttt ggatttggca tgcttaatag 5040
ttggtactat atagcaaagg ataactttag gtttataata atagtgtcct tggtagttaa 5100
aattttagcg gttttatttg ttttatatta tgcttttgaa ttacacatgt ttatttttat 5160
tcaggcgata gcatctgctc tggttggact tattagtttg ataaaaataa agaatgataa 5220
taggttggga aaaatatcat ttagacatat taaaaattat ttgaaaaaca ttaagcatat 5280
gtacatatat agcggggtaa atgcacttat tcagcctata attaattctg ctatattggc 5340
atcaggaaat gctagcttgt tagggatata taacgtcgtt atgcgatttg tggcagtatc 5400
cgtaacattt tccaattcaa ttatcactgt cttaaataaa tgtaattcag aggtttttta 5460
ttccgtaggg cacaaaacta actatgtgga agtaaaatac agaagtcagc tactcaaatt 5520
gttgttttac agcatcgttg tttttatatg tgtggctgca atatcatatg tctctattta 5580
tatgacaaag ggagaaatat ctcgtttttt tatttttcta atattttcat ttagcctagc 5640
tgttatccca agtatactca atcaatattt aactcaatta attcatatga tacactcatc 5700
aaaaaatata gtaaaatatg agatttgttc gaatgtacta tcgttaatat ctattttttt 5760
attttcttca ttattcccat attattatat agtatatgct gtagttctat cgccaattat 5820
The termination of the initial orf5 of orf6
tattacaatt tgctgtctgt gggatttgaa
atgcaaaaac ttgctg
taat catgacttac 5880
tataatgaga gttacataat actaaaaaag gcagtagaca gtactcttca ggatctggaa 5940
agattggaaa agaagaatgt gaaatataaa ttaataattg gagtcgatag gagaaatgaa 6000
gatattcctc atattgagag tttatattta aaagaagtta gtgccagaaa tgatgtgtgc 6060
gttgtttttt ttaatgagaa cgaagggttg gcggcaagtt taaataaatt aatttttggc 6120
gcgtgtaatg gctatgattt tattgccaga atggatgctg atgatattgt tattaacggg 6180
cgttttaaaa agcaattaga atatctgaat aagcattctg atgtcagttt agtcggtgga 6240
caggctgttg ttattgatga gaacgataat gttgttaggg attattttaa tatgaaccca 6300
gtagattttt cgtgggggaa tgaaataatt catccaactt ggttgttaag gtcagagctt 6360
tattataaat taggtggtta caggcgttta gatgctgctc aagattatga ttttttgtgt 6420
agggctgtat tgagtggatg tattgtaagg aatatagatg acgtggtttt attttatagg 6480
ataaggcatg gctcaattgg cagttgtaag aagtttatcc aaataaaaaa caagactatt 6540
ttatctaaat ttttcagaaa aaatgaagtt gcacctattc atttatttaa tgcaaccaac 6600
ttttcagttg agtttttttt attctctgtc attgaatcaa taaaaatcaa gcacaatatt 6660
gtggggaaat gtattgggtg gttgtctcca ctccatgcta aaaattatta tttaaattta 6720
Orf7's is initial, the termination of orf6
agaaaacgaa tggggaaaaa
atgagagtac ttgtttttgg ccctatgata tctggtcata 6780
ttagtaactg gattcatggt tccaaagaat atattgatta tagaattgtc acactacact 6840
atgacaaaag tatttctgag aatgttggaa taggagagat atataagata ccaagactta 6900
caaaaactaa atttgatttt atatacgcac ctgtttttct tcttttttat ttttttttat 6960
taaagcctga gttggttcat gtccattttt ttagttctta tggtttaatt tcatgtgttt 7020
tgcctagaaa agtaaagaag atactctcag tatggggaag tgacattaac aatactataa 7080
aaaagagagg gatattagga aaaatatatg taaaagcaat gaaaagtttt tgtgttgtaa 7140
attcacccgc caggcatata acaaataaaa taatatcgta tttcaatgtg aatcaggagc 7200
gaatacatac ttttcaatat ggtgtagatg tggggttctt ttcagatgaa aaaacgtcaa 7260
gcataaggga cgataaatgt ataacaataa cttctgtaag aaattgggat aatatttata 7320
acatagagtc ctttttagaa gctgttattg atatgaaaga ttatgattgt aaagggtact 7380
tttttaatat caatatacta ggccgctcat cgtcaaagac cacacatgat aagataatag 7440
aactcacaaa aaaatgtgag ggagataatt ataaagtaaa tttaataggg tttattcaaa 7500
aagaggaatt aaggtcgatt cttcataaat ctgattttgt tcttagtatt cctagcaatg 7560
atggtgctcc attatctttg atggagggcg ttttatgtaa ttgttttccg atagtatcag 7620
acatcgatgc aaacagagag ctattaggaa atcatgcaat gtatttgaat ctgcatgatg 7680
atattgacat gaaatctgtt ttttcaaggt gtgtgaaatt aatagatagt aatcaatata 7740
ggtgttcgat tgaatataac cgagatacaa taataaaaat gtttgatata aataaaaaca 7800
Orf8's is initial, the termination of orf7
caaagcgtat ggttgatatt tattatcggg tcgtgtcaca aaaggaattt tt
atgaaggt 7860
tttttttgat gtatttttgt tgtttttatt tgtgtttgct tttagcatcc ctgttttttt 7920
taattcaggc tatcttatat atattttttc cttgatttat ttaattacaa atatcaggat 7980
aaggactgtt ttttttagtc taataagtac aagaatattt tttattgcgt ttttgtcttt 8040
ggcctgcatg ctcttttttt cttgtatttc attggtatta aatggtacag gagatttatc 8100
tatcatgggg gttatcttta ataatttagc tgcattattc agctctgtta taattgcgtc 8160
gcatttaatc cagcataaca atgataatgt gttgaaattg ttttttctaa ttgccctctt 8220
acaatccttt tttattattc tgatgatgtt ttttcccagt ataaatgctt atattcaaag 8280
tattattaga acaaaagaag aaatagaatt tatgagtaat acatatggag gcatcagggg 8340
attaggtata tcaggttctg ttgcctttgg tcttgctata attatgtcat tattatcttt 8400
tctttcaatt gtttggttta atgaggaaga ctgtaatatt cctttctttt taaaattatt 8460
ttttatcata ctcttgctgt tcgcgtcaat ttctgctggg cgtacggctg ttttggggtt 8520
tgtcctcgga ggggtatata gttttatttg ctctgattat aaaaagaagt tttatcgggc 8580
gcttcaaatt ttgatttatg tatctctgtt tttatttatt ttttggagat tttttttaaa 8640
tgaacatgtt tatgatatct tgaaaaaata ttcggattat tcttttcaaa tgttatatta 8700
ttatttagat tcaggaaagt tatcaacaac atctacagat gaattattta atatgtattt 8760
cccgttaaca gaaaaacaga tcttcattgg ttatgggtac cataccgtta aaggatggga 8820
catattatct cgggaccgat gctggattta tgcggttagc attatttttg ggtgcggtac 8880
The termination of orf8
catcggttat tttttatttt ttgtttttta tttttttgac tatttatgct gc
taatagca 8940
tcaaacctaa aatagttgca ttaatgatta caatactcag tttttctttt cattataaag 9000
gcgaggttat atttttgaat gttgcatata tgaaaataat atatatatat atattttcat 9060
ctttttatat ttgtcataaa aaaagattaa taagaagggg ttcggttaat gcatgattat 9120
tattttaatt gcaggttttt attaacaaac aacagtagaa actcgttaat tgttgacata 9180
ttatcttatt ctagatatct gtaaaacaaa ttggtttttg cctttgtttt tatgggtatt 9240
taaagactat agggtaatag cgcaacaatt taatttaagt tttttgagaa ctgtaagaat 9300
agtgaactac atagatcaca aatctataac tgaaccaggt gataatccca tgaatacaat 9360
ctataataaa taatgttttg gattagtaag tgttataaat ttacgtggaa attctatttg 9420
cgtgtttatg ttgctatttc atggaatggt tttattgcca ttgctgtagt gttattttga 9480
actaatttat ttggtgaaag attactgagt aatcttttga tactaaatcg attgtgtttt 9540
atatctttga attaaatatt tttgccaaag cactccttca ctcttatata ttcagatact 9600
ccttgtgtta taagagtaag gaatataaat tggaatatgg aaaaaggcgt gacatatttc 9660
tgttgtcttt ggttatgtac acgatgggca gagtgtaagt tttcagagat gttgcaattg 9720
tctccagata agaaaggaga gattgttatg ttgaaccaac atatcggcgt caggtgtttc 9780
gggcattacc gtgcaacaat ctatgcgatg gaatacaatg ccatgcctga tattgcaaaa 9840
ttaagaatga aaaccctgca ttttagggaa agacatggaa tcaatgcatc cgcagaaaat 9900
ttttgtgtat caatacgcgt ttaatatata ggctcggaag tgcttatctc aagtagtaaa 9960
tctttctagt atgtcttaca aaactatgac atccagaaat tttgaaagaa atatgatgaa 10020
ttagaatagg acttctcaac cttggaaaag aacagatttt tgaccgtctg aatctgtagt 10080
gtgaacagta gcatttggcc tgcccgagcg tctccactat tggaggaatg agctaggcag 10140
cacatgataa aatgtggcta atactggtgt aactgggttc ccagacttca agtccaaggt 10200
actattagcc cgctaaagac cggtgagctt atattgagct cagaatcggc gttatgttat 10260
taccgtgatt gacgaacgca gcaactacgc tttggcggtg gcggtaacgt cgcttaacag 10320
tgatgtaatc atgagtttct tctgccatca agctcggttg ttacctgttg gaatcaacca 10380
gaatataccc gattatgggg aagagttcct gggaaaattt gacaaaaagc tacaggaagc 10440
cgctatcagg caccaatagg cttatctcga tacgccgaaa atgaacacca tctgtgagcg 10500
ctttaaccga tcactgagat agccgtttat tgagtttaat gaagaattgc tctttgaaaa 10560
tctgaatatg tcaatcataa acaggctcaa tattgcatac tgtacaagaa taaaaggcct 10620
cataaattgc tcgaattact gacaccacta gaacataatt tacacgaaaa aaatgcaata 10680
tagtgttaga ctcatacaat atgtcgagat gtttagaaaa ataacgtata cttacatatt 10740
gtatttttat agtaacattg ggcttaacta ttctaaattt ttattctttc acagtttata 10800
ctatttataa tgtgtttata ttcttgtttt atatcctagg cagatagatg aaatataagg 10860
Orf9's is initial
agtttt
atga ctattttggt tactggtggt gcaggttaca ttggttcaca tactgtatta 10920
tccttattat catacggaaa aaatgtggtg gtgttggata atctatcgaa ctcatcgtct 10980
aagtcgttag aaagggtcga aaaaatttgt ggacgtaggc ctctatttta tcatggtgat 11040
gtgcgtgata ggaaaaaact caaacatatt ttccagtatc atgatattac agatgttatt 11100
cattttgcag gattaaaagc cgtcgctgaa tctgtttcaa atccattgga atattatgat 11160
cacaatgtta acggaactat tgttttactt gaagaaatga tgcgcgctgg cgttattagt 11220
tttattttta gctcctccgc aacagtgtat ggtaagcctg aaactattcc tttgaaggaa 11280
gatagtagcg ttggcggaac aactaatcca tatggctttt caaaacttat agtagagagg 11340
gttttgaagg atatagcttt ggctgagcca acaatgcgta ttactatttt gcgttatttc 11400
aatcctgtag gagcccactc ttctggtcta ttaggtgaag atcctaatgg tgttccaaat 11460
aatttaatcc cttatattac tcaagtggct attggtaatc taaaatattt atcagtattc 11520
ggaaatgatt atcctacacc tgatggaaca ggcattagag attatattca tgttatggat 11580
ttggctgaag ggcacatagc tgctttagaa caccgtaatt ttgggggaaa ttataaagta 11640
tataatcttg gtacaggggt tggttattca gttcttgatg tgataaatac ctttaaacgt 11700
tgtactggaa ttgatatccc ttattgtttc aaacctcgtc gagttggtga tatagctgaa 11760
tgttggtctg atccatctct agcattttta gaattagggt ggtcttctcg atatgattta 11820
The termination of orf9
gatacaatga taattgattc ttggcgttgg caaaaaaaca atcccaaagg atataaa
taa 11880
gacgatttta atgtgctatt ttattcactg tcaatatttt tataaaacca tctttgcttt 11940
gctttgcttt gctttgcttt gctttgcttt gtagttactc tcttaattaa cggcttttta 12000
tattagggtg ggatgttaat gaaatacttg ataaatattt aatatgtgaa atttcttaat 12060
aagctaccaa ggatggatat gataaacatg atattatata atgaaactta tgtaagtttg 12120
Orf10's is initial
agatgattct
atgagtaccg ataaatattt agtttcagtc ataacaccca catataattc 12180
tgaaagaacg attagatgta caattgattc tgttctatct caatcatata ataatataga 12240
aatgattatt gtcgatgatt gttcgttaga taatacagtg tctatttgca gggaatatca 12300
aaaaaaagat tcaagagttg ttctcatttc aaatgagaag aattatgggg ctggtatgtc 12360
aagaaatata gcgatcagta aagctaaagg tcggtttatc gcttttttag atagtgatga 12420
tttttggcat aaagagaaaa taaaaaaaca gatagacttt atgctttcaa ataattatgc 12480
tttcacttat acctcatatc aaaaggttag ttttagcggg aaacttttat cccaaattaa 12540
tcctgtttct aaagttaatt attctgaatt attaaagact aatgtaatag gttgcttgac 12600
agctgtatat gatacttcat ctcttggtaa aatgtttatg ccaacaatca ggaaaagaca 12660
ggacatggca ctatggctga gcattttaga taaaattgat tatgcatatt gtttgaacga 12720
aatactggcc tactatcgag taggcaccga tacactctcg tcaaataaat taaaaataat 12780
attttcacaa tgggcctttt acagaaaata tttaaagttt ggtctgttta aatctcttta 12840
ttatttctcc cactatgctg ttaatgctgt tgccaaacat catataaatc cgctgtgttt 12900
The termination of orf10
gttttttaag cgccactttg cctcttggag a
taattcggc aaatcttata aagaaatatt 12960
ggataatgct tttgatttgc aaatgtattt ttatatttga tgatgtcatg gatgagtttt 13020
aagtgaattt caatagaata gggaaatgtg ggtctgtgtg gacaagaagt gtgattaatg 13080
tttttgtatc agattataat ctgcaatcta tttagtaaaa ttatgtttac caagtgcctc 13140
tcctgaagaa ttatttttat aaagccttta gttatcattg aaatgaattc tgaggttaat 13200
tggtgcgaat gaacttagta ttatgataaa gattgtcttt attttactta aaagtataga 13260
atagttcatt gcattatttg tgttgtataa aacttcagag tcaggaacag tcttaagttt 13320
tattttttct aaattcattg gtggctatca tatggttatt agatgactgt gttaagatat 13380
atttcattgg ctttaaggtt ttagtacata tgacgaatag aaccaatttg aatattctgg 13440
aaacaccaac agcctcgttg tttgtcaaaa ttattttgag gttgataatt tgactctcga 13500
ttattgtaat ttataaaata taagtgagtt gaaagatata tttgaggcat ggcagttaaa 13560
tctcttatat atcgcggaga ctattaataa tagttagtct gagacactgg aaactagagt 13620
gtggagagca acgttagttt tgagtatttt ctgtaattaa tttaatataa gtttaattac 13680
atatacaatt atctgttgtt tgtcttatgc aatattttcc atatgacata tttttatatg 13740
tgtttcaata cgtttattat tctgctaatt aaataggagt gaaaatgtca aaaaaacaga 13800
ttggggttat cggtatggcg gtaatggggc gtaatcttgc actaaatatc gaaagtcacg 13860
gctatactgt ttcaattttt aaccgttctc gtgaaaaaac agaaaaaatt atagctactc 13920
attcggataa gaagttattt ccttactata caataaaaga gtttgttgaa tctctggaaa 13980
cgcctcgtcg catcctgtta atggtgaaag caggtgcagg cacggatgct gctattgatt 14040
ctctcaagcc atacctcgat aaaggtgaca tcatcattga tggtggtaac accttcttcc 14100
gagacaccat tcgtcgtaac cgtgagctgt ctgcagaagg ctttaacttt atcggtagcg 14160
gtgtttctgg tggtgaagaa ggtgcgctga aaggaccttc catcatgcca ggtggccaga 14220
aagaagccta tgaactggtc gcaccgatcc tgaccaaaat cgccgcagtg gctgaagatg 14280
gggagccatg tgttacctat attggtgccg atggcgcagg tcactatgtg aagatggttc 14340
acaacggtat tgaatacgga gatatgcagc tgattgctga agcctattct ctgcttaaag 14400
gtggcctgaa cctcaccaac gaagaactgg cgcagacctt taccgagtgg aataatggtg 14460
aactaagcag ctacctgatc gacatcacca aagacaactt cactaaaaaa gatgaagatg 14520
gtaactacct ggttgatgtg attctggatg aagcggctaa caaaggtacc ggtaaatgga 14580
ccagccaaag cgcgctagat ctcggtgaac cgctgtcgct cattaccgag tctgtgtttg 14640
cacgatacat ctcttctctg aaagagcagc gtgttgccgc gtccaaagta ctgtctggtc 14700
cgcaagcgca gccagcaggc gacaaggctg agttcatcga aaaagttcgt cgtgctctgt 14760
atctgggcaa aatcgtttct tacgcccagg gcttctctca gttgcgtgct gcgtctgaag 14820
agtacaactg ggatctgaac tatggcgaaa tcgcgaagat tttccgtgct ggctgcatta 14880
tccgtgcgca gttcctgcag aaaatcaccg atgcttatgc cgaaaatccg cagatcgcta 14940
acctgctgct ggctccgtat ttcaagcaaa ttgccgatga ctaccagcag gcgctgcgcg 15000
atgtcgtcgc ttatgcggta cagaacggta tcccggttcc gaccttcgcc gctgcggttg 15060
cctattatga cagctaccgc gccgctgttc ttcctgcgaa cctaatccag gcccagcgcg 15120
acta 15124
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 8 types and intestinal bacteria O143 is characterized in that described oligonucleotide is to being: the Nucleotide of 3455 to 3471 bases among the SEQ ID NO:1 and the Nucleotide of 3929 to 3946 bases; The Nucleotide of 3635 to 3653 bases among the SEQ ID NO:1 and the Nucleotide of 4037 to 4056 bases; The Nucleotide of 3740 to 3759 bases among the SEQ ID NO:1 and the Nucleotide of 4201 to 4220 bases; The Nucleotide of 4843 to 4860 bases among the SEQ ID NO:1 and the Nucleotide of 5377 to 5394 bases; The Nucleotide of 4889 to 4906 bases among the SEQ ID NO:1 and the Nucleotide of 5461 to 5479 bases; The Nucleotide of 5391 to 5408 bases among the SEQ ID NO:1 and the Nucleotide of 5830 to 5847 bases; The Nucleotide of 6032 to 6051 bases among the SEQ ID NO:1 and the Nucleotide of 6417 to 6435 bases; The Nucleotide of 6220 to 6239 bases among the SEQ ID NO:1 and the Nucleotide of 6671 to 6689 bases; The Nucleotide of 6087 to 6106 bases among the SEQ ID NO:1 and the Nucleotide of 6671 to 6689 bases; The Nucleotide of 7194 to 7211 bases among the SEQ ID NO:1 and the Nucleotide of 7822 to 7839 bases; The Nucleotide of 6780 to 6797 bases among the SEQ ID NO:1 and the Nucleotide of 7222 to 7239 bases; The Nucleotide of 7010 to 7028 bases among the SEQ ID NO:1 and the Nucleotide of 7592 to 7626 bases; The Nucleotide of 8211 to 8228 bases among the SEQ ID NO:1 and the Nucleotide of 8844 to 8861 bases; The Nucleotide of 7892 to 7911 bases among the SEQ ID NO:1 and the Nucleotide of 8414 to 8431 bases; The Nucleotide of 8038 to 8056 bases among the SEQ ID NO:1 and the Nucleotide of 8565 to 8582 bases; The Nucleotide of 12152 to 12171 bases among the SEQ ID NO:1 and the Nucleotide of 12915 to 12932 bases; The Nucleotide of 12312 to 12329 bases among the SEQ ID NO:1 and the Nucleotide of 12843 to 12860 bases; The Nucleotide of 9934 to 9952 bases among the SEQ ID NO:1 and the Nucleotide of 10513 to 10530 bases.
2, the application of the described Nucleotide 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 confession detection Shigella bogdii 8 types and intestinal bacteria O143 type as probe.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB031003567A CN1261573C (en) | 2003-01-20 | 2003-01-20 | Nucleotide with O-antigen idiocrasy against Ball's and shiga's-8 and colibacillus 0143 |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB031003567A CN1261573C (en) | 2003-01-20 | 2003-01-20 | Nucleotide with O-antigen idiocrasy against Ball's and shiga's-8 and colibacillus 0143 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1439645A CN1439645A (en) | 2003-09-03 |
| CN1261573C true CN1261573C (en) | 2006-06-28 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB031003567A Expired - Fee Related CN1261573C (en) | 2003-01-20 | 2003-01-20 | Nucleotide with O-antigen idiocrasy against Ball's and shiga's-8 and colibacillus 0143 |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1253571C (en) * | 2004-03-12 | 2006-04-26 | 天津生物芯片技术有限责任公司 | Nucleotide peculiar to 0-antigen of 0114 type bacillus coil |
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| CN1439645A (en) | 2003-09-03 |
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