CN1257277C - Nucleotide peculiar to 0-antigen of 0155 type bacillus coli - Google Patents

Nucleotide peculiar to 0-antigen of 0155 type bacillus coli Download PDF

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CN1257277C
CN1257277C CN 200410019042 CN200410019042A CN1257277C CN 1257277 C CN1257277 C CN 1257277C CN 200410019042 CN200410019042 CN 200410019042 CN 200410019042 A CN200410019042 A CN 200410019042A CN 1257277 C CN1257277 C CN 1257277C
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gene
antigen
nucleotide
intestinal bacteria
oligonucleotide
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CN1563057A (en
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王磊
孔庆科
郭宏杰
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Tianjin Biochip Corp
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Abstract

The present invention provides a nucleotide specific to an O-antigen of Escherichia coli O155, which is a complete nucleotide sequence of a gene cluster for controlling the synthesis of the O-antigen in Escherichia coli 0140, such as the separated nucleotide disclosed in SEQ ID NO: 1 with the full length of 12755 basic groups, or the nucleotide of SEQ ID NO: 1 comprising one or a plurality of inserted, deleted or substituted basic groups and maintaining the functions of the separated nucleotide simultaneously. The present invention also comprises an oligonucleotide of a glycosyltransferase gene and an oligosaccharide unit processing gene derived from an O-antigen gene cluster of Escherichia coli O155. In the present invention, PCR indicates that the oligonucleotide has high specificity on the O-antigen of Escherichia coli O155. The present invention also discloses a method for detecting and identifying Escherichia coli O155 in human bodies and in environments by the oligonucleotide of the present invention.

Description

Nucleotide to the O-antigen-specific of intestinal bacteria O155 type
Technical field
The present invention relates to the complete nucleotide sequence of control O-antigen synthetic gene cluster in the intestinal bacteria O155 type (Escherichia coli O155), particularly relate in the intestinal bacteria O155 type oligonucleotide in the control O-antigen synthetic gene cluster, can utilize these the oligonucleotide of the O-antigen-specific intestinal bacteria O155 type in human body and the environment and identify O-antigen in these pathogenic bacterium quickly and accurately.
Background technology
O-antigen is the O specific polysaccharide composition in the gram negative bacterium lipopolysaccharides, and it is made up of many multiple oligosaccharide unit.The antigenic building-up process of O-is studied clearlyer: by glycosyltransferase nucleoside diphosphate monose is transferred on the fat molecule that is fixed on the cell inner membrance earlier, then in the inboard synthesis of oligose unit of inner membrance, the antigenic oligosaccharide unit of O-is transferred to the inner membrance outside by the transhipment enzyme again, then aggregate into polysaccharide by polysaccharase, be connected to again and form lipopolysaccharide molecule [Whitfield, C. (1995) " Biosynthesis of lipopolysaccharide O antigens " .Trends inMicrobiology.3:178-185 on the glycolipid molecule; Schnaitman, C.A.and J.D.Klena. (1993) " Genetics oflipopolysaccharide biosynthesis in entericbacteria " .MicrobiologicalReviews, 57 (3): 655-682].Coding is responsible for the generally adjacent arrangement on karyomit(e) of gene of all enzyme molecules of O-antigen synthetic, form a gene cluster [Reeves, P.R., et al. (1981) " Bacterialpolysaccharide synthesis and gene nomenclature " Trends in Microbiology, 4:495-503].In Shigellae, intestinal bacteria and Salmonellas, O-antigen gene [Lei Wang.et al (2001) " Sequence analysis of four Shigella boydii O-antigenloci:implication for Escherichia coli and Shigella relationships " .Infection andImmunity, 11:6923-6930 bunch between galF and gnd gene; Lei Wang and Peter Reeves (2000) " The Escherichia coliO111 and Salmonella enterica O35 gene clusters:gene clusters encoding the samecolitose-containing O antigen are highly conserved " .Journal ofBacteriology.182:5256-5261].The O-antigen gene bunch contains three genoids: sugared synthesis path gene, glycosyltransferase gene, oligosaccharide unit treatment gene, the required nucleoside diphosphate monose of enzymic synthesis O-antigen of wherein sugared synthesis path genes encoding; Thereby the enzyme of glycosyltransferase gene coding forwards nucleoside diphosphate monose and other molecule to and makes monose aggregate into oligosaccharide unit on the monose; The oligosaccharide unit treatment gene comprises transhipment enzyme gene and pol gene, and they transfer to the bacterium inner membrance outside with oligosaccharide unit, and repolymerization becomes polysaccharide.Glycosyltransferase gene and oligosaccharide unit treatment gene only are present in the gene cluster of carrying these genes.The difference of monose in the O-antigen, between monose between the difference of link button and the oligosaccharide unit difference of link button constituted the antigenic diversity of O-, and the composition of monose, the link button between monose and the link button between the oligosaccharide unit are by the Gene Handling in the O-antigen gene bunch, so the O-antigen gene bunch has determined O-antigenic synthetic, has also determined the antigenic diversity of O-.
Because O-antigen is extremely strong antigen, be one of important paathogenic factor of intestinal bacteria, it has extremely strong diversity again simultaneously, and this enlightens us can study a kind of intestinal bacteria and good, highly sensitive method of the antigenic specificity of O-thereof of detecting quickly and accurately.With surperficial polysaccharide is that the serology immune response of target has been used to somatotype and the evaluation to bacterium always since the thirties in last century, is unique means of identifying pathogenic bacterium.This diagnostic method needs a large amount of antiserum(antisera)s, and the antiserum(antisera) general classes is incomplete, quantity not sufficient, and also there are some difficulties in a large amount of antiserum(antisera)s in preparation with in storing.On the other hand this method length consuming time, sensitivity is low, loss is high, poor accuracy, so, generally believe that now this traditional serology detection method will be that the modern molecular biology method replaces.1993, Luk, J.M.C et.al has identified the O-antigen [Luk of Salmonellas with the specific nucleotide sequence of Salmonellas (S.enterica) O-antigen gene bunch by PCR method, J.M.C.et.al. (1993) " Selective amplification of abequose andparatose synthase genes (rfb) by polymerase chain reaction for identification ofS.enterica major serogroups (A; B; C2; andD) ", J.Clin.Microbiol.31:2118-2123].Luk, the method for et.al is with corresponding to Salmonellas serotype E 1, D1 obtains the oligonucleotide special to the Salmonellas of different serotypes after the nucleotide sequence of the CDP-abequose in the A, the O-antigen of B and C2 and the synthetic gene of CDP-tyvelose is arranged.1981, Paton, the A.W et.al serotype [" Molecular microbiological investigation of an outbreak of Hemolytic-Uremic Syndrome caused by dry fermented sausage contaminated with Shiga-liketoxin producing Escherichia coli " .J.Clin.Microbiol.34:11552-11557] of the oligonucleotide that comes from the wbdI gene of the O-antigen-specific of E.coli O111 having been identified the toxogenic E.coli O111 of a strain, but afterwards studies show that Paton, the usefulness of A.W et.al comes from the oligonucleotide of wbdI gene and identifies that the method for the serotype of E.coli O111 has false positive results to occur.Bastin D.A.and Reeves, P.R. think, this is because the wbdI gene is sugared synthesis path gene [the Bastin D.A.andReeves of a supposition, P.R. (1995) Sequence and analysis of the O antigen gene (rfb) cluster ofEscherichia coli O111.Gene 164:17-23], and have this sugar in the antigenic structure of the O-of other bacterium, so sugared synthesis path gene is not a high special for O-antigen yet
Shigellae has 46 kinds of serotypes, but have only 33 kinds of different O-antigens, intestinal bacteria have 166 kinds of different O-antigen [Reeves, P.R (1992) " Variation in O antigens; niche specificselection and bacterial populations " .FEMS Microbiol.Lett, 100:509-516], the two sibship is very near, and there are 12 kinds to be intestinal bacteria and the total [Ewing of Shigellae, W.H. (1986) " Edwards and Ewing ' s identification ofthe Enterobacteriaceae " .Elsevier SciencePublishers, Amsterdam, The Netherlands; T.cheasty, et al. (1983) " Antigenicrelationships between the enteroinvasive Escherichia coli antigensO28ac; O112ac; O124; O136, O143, O144; O152and and Shigella O antigens " J.clinMicrobiol, 17 (4): 681-684]
Summary of the invention
The Nucleotide that the purpose of this invention is to provide a kind of O-antigen-specific to intestinal bacteria O155 type.It is the Nucleotide in the O-antigen gene bunch of intestinal bacteria O155 type, is the special Nucleotide that comes from glycosyltransferase gene and transhipment enzyme gene and pol gene.
A time purpose of the present invention has provided the full length nucleotide sequence of the O-antigen gene bunch of intestinal bacteria O155 type.
Another object of the present invention has provided the gene of the O-antigen gene bunch that constitutes intestinal bacteria O155 type: 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 orf2, orf4, orf5 gene; The acetyltransferase gene comprises the orf8 gene; The gluconic acid synthase gene comprises the ugd gene; The galactosonic acid synthase gene comprises the gla gene; The chain length controlling gene comprises the wzz gene.
Another purpose of the present invention has provided oligonucleotide, and they come from gene that intestinal bacteria O155 type comes from coding transhipment enzyme respectively is the wzx gene or with wzx the gene of identity function is arranged; The gene that comes from the coding polysaccharase is the wzy gene or with wzy the gene of identity function is arranged; They are the oligonucleotide in the said gene, and length is at 10-20nt; They are special to the O-antigen of intestinal bacteria O155 type; Especially the oligonucleotide of listing in the table 1, they are high specials to the O-antigen of intestinal bacteria O155 type, and these oligonucleotide are also reconfigurable, the oligonucleotide after the combination also is a high special to the O-antigen of intestinal bacteria O155 type.
The above-mentioned oligonucleotide that a further object of the present invention provides can be used as primer and is used for nucleic acid amplification reaction, perhaps be used for hybridization as probe, perhaps be used to make gene chip or microarray, thereby pass through O-antigen and the detection and the identification of escherichia coli O155 type of these methods detections and identification of escherichia coli O155 type.
An also purpose of the present invention has provided the method for complete sequence of the O-antigen gene bunch of separating Escherichia coli O155 type.Can obtain the complete sequence of the O-antigen gene bunch of other bacteriums according to present method operation, the complete sequence of the gene cluster of the bacterium of other polysaccharide antigens that also can obtain to encode.
The objective of the invention is to realize by following technical scheme.
The present invention is to the Nucleotide of the O-antigen-specific of intestinal bacteria O155 type, and it is the isolating Nucleotide shown in SEQ ID NO:1,12755 bases of total length; The base that perhaps has one or more insertions, disappearance or replacement keeps the Nucleotide of the SEQ ID NO:1 of described isolating functional nucleotide simultaneously.
The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O155 type, it is by orf2, wzy, orf4, orf5, wzx, orf8, ugd gla, wzz9 genomic constitution is all between galF gene and hisI gene.
The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O155 type, the gene that has high degree of specificity in the wherein said gene is: the gene of transhipment enzyme comprises the wzx gene or with wzx the gene of identity function is arranged; Pol gene comprises the wzy gene or with wzy the gene of identity function is arranged; Glycosyltransferase gene comprises orf2, orf4, orf5 gene; The acetyltransferase gene comprises the orf8 gene; The gluconic acid synthase gene comprises the ugd gene; The galactosonic acid synthase gene comprises the gla gene; The chain length controlling gene comprises the wzz gene; Wherein said gene: wzx is the Nucleotide of 6827 to 8113 bases among the SEQ ID NO:1; Wzy is the Nucleotide of 2232 to 3185 bases among the SEQ ID NO:1; Orf2 is the Nucleotide of 1213 to 2229 bases among the SEQ ID NO:1; Orf4 is 3195 to 4244 among the SEQ IDNO:1; Gene orf5 is the Nucleotide of 4358 to 5173 bases among the SEQ ID NO:1; Orf8 is the Nucleotide of 8123 to 8590 bases among the SEQ ID NO:1; Ugd is the Nucleotide of 8689 to 9855 bases among the SEQID NO:1; Gla is the Nucleotide of 9921 to 10925 bases among the SEQ ID NO:1; Wzz is the Nucleotide of 11331 to 12311 bases among the SEQ ID NO:1.
The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O155 type, wherein it also comprises and comes from described wzx gene, wzy gene and their mixing or their reorganization.
The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O155 type, the oligonucleotide that comes from the wzx gene is to being: the Nucleotide of 7226 to 7243 bases among the SEQ ID NO:1 and the Nucleotide of 8053 to 8072 bases; The Nucleotide of 7131 to 7148 bases among the SEQ ID NO:1 and the Nucleotide of 7862 to 7881 bases.The oligonucleotide that comes from the wzy gene is to being: the Nucleotide of 2295 to 2312 bases among the SEQ ID NO:1 and the Nucleotide of 2776 to 2794 bases; The Nucleotide of 2234 to 2251 bases among the SEQ ID NO:1 and the Nucleotide of 2864 to 2881 bases.
The application of the Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O155 type in detecting other polysaccharide antigen of expressing the antigenic bacterium of O-, the O-antigen of identifying bacterium and bacterium.
The recombinant molecule of the Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O155 type is providing the O-antigen of expressing intestinal bacteria O155 type by inserting to express, and the application in the preparation bacterial vaccine.
The application of the Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O155 type is characterized in that it is used for PCR, is used for hybridization and fluoroscopic examination or is used to make gene chip or microarray as probe as primer, for the application of bacterial detection.
The separation method of the Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O155 type is characterized in that it comprises the steps:
(1) genomic extraction: in substratum, cultivate intestinal bacteria O155 type, centrifugal collecting cell; The genomic dna that obtains detects by agarose gel electrophoresis;
(2) by the O-antigen gene in the pcr amplification intestinal bacteria O155 type bunch: with the genome of intestinal bacteria O155 type is that template is passed through its O-antigen gene of Long pcr amplification bunch, with the PCR product that obtains, detect the size and the specificity thereof of PCR product with agarose gel electrophoresis, merge this long PCR product, and with DNA purification kit purified pcr product;
(3) make up O-antigen gene bunch library: Long PCR purified product is used shotgun make up O-antigen gene bunch library;
(4) to the cloning and sequencing in the library: from the library, select the clone of insertion fragment more than 1kb and the insertion fragment among the clone is checked order with laboratory automatic dna sequencer commonly used, sequence reaches 100% fraction of coverage, thereby obtains all sequences of O-antigen gene bunch;
(5) splicing of nucleotide sequence and analysis: applying biological information science software splicing and edit all sequences, thus obtain the Nucleotide full length sequence of the O-antigen gene bunch of intestinal bacteria O155 type;
(6) screening of specific gene: at wzx, wzy, orf8, orf11, the orf12 gene design primer in the O-antigen gene of intestinal bacteria O155 type bunch; Respectively designed two pairs of primers in each gene, every pair of primer is distributed in the different places in the corresponding gene, to guarantee its specificity; Is that template is carried out PCR with these primers with the genomes of 166 strain intestinal bacteria and 43 strain Shigellaes, determines the antigenic high degree of specificity of O-of wzx, wzy, orf8, orf11, orf12 gene pairs intestinal bacteria O155 type;
(7) detection of primer sensitivity: cultivate intestinal bacteria O155, after the bacterial count respectively with 5 * 10 3, 5 * 10 2, 5 * 10 15 and 0 viable bacteria join in a certain amount of certain thing to be detected, sneak into the thing to be detected of bacterium and use sample as detecting, sample is added the LB substratum, getting the LB substratum that some and sample mix cross filters, filtered liquid is cultivated, carried out the PCR reaction as pcr template with oligonucleotide after the peek milliliter is handled from cultured bacterium liquid, detect its sensitivity intestinal bacteria O155.
The separation method of the Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O155 type is characterized in that it comprises the steps:
(1) genomic extraction: 37 ℃ of incubated overnight intestinal bacteria O155 types in the LB of 5mL substratum, centrifugal collecting cell.With 500ul 50mM Tris-HCl (pH8.0) and 10ul 0.4M EDTA re-suspended cell, 37 ℃ of incubations 20 minutes, the N,O-Diacetylmuramidase that adds 10ul 10mg/ml then continues insulation 20 minutes.The Proteinase K, the 15ul 10%SDS that add 3ul 20mg/ml afterwards, 50 ℃ of incubations 2 hours, the RNase that adds 3ul 10mg/ml again, 65 ℃ of incubations 30 minutes, add equal-volume phenol extracting mixture, get supernatant and use isopyknic phenol again: chloroform: primary isoamyl alcohol (25: 24: 1) mixing solutions extracting twice, get supernatant again with isopyknic ether extracting to remove remaining phenol.Supernatant rolls out DNA and washes DNA with 70% ethanol with glass yarn with 2 times of volume ethanol deposit D NA, and DNA is resuspended among the 30ul TE; Genomic dna detects by 0.4% agarose gel electrophoresis;
(2) by the O-antigen gene in the pcr amplification intestinal bacteria O155 type bunch: with the genome of intestinal bacteria O155 type is that template is passed through its O-antigen gene of Long pcr amplification bunch, at first according to the JumpStart sequences Design upstream primer (#1523-ATT GTGGCT GCA GGG ATC AAA GAA AT) that often is found in O-antigen gene bunch promoter region, again according to the gnd gene design downstream primer (#1524-TAG TCG CGT GNG CCT GGA TTA AGT TCG C) in O-antigen gene bunch downstream; With the Expand Long Template PCR method of Boehringer Mannheim company amplification O-antigen gene bunch, the PCR response procedures was as follows: 94 ℃ of pre-sex change 2 minutes; 94 ℃ of sex change are 10 seconds then, annealed 15 seconds for 60 ℃, 68 ℃ were extended 15 minutes, carry out 30 circulations like this, last, continue to extend 7 minutes at 68 ℃, obtain the PCR product, detect the size and the specificity thereof of PCR product with 0.8% agarose gel electrophoresis, merge 5 pipe 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, and enzyme is cut the dna fragmentation size is concentrated between the 1.5kb-3kb, then adds 2ul 0.1M EDTA termination reaction.Merge the same reaction system of 4 pipes, with isopyknic phenol extracting once, use isopyknic phenol: chloroform: primary isoamyl alcohol (25: 24: 1) mixing solutions extracting once, after using isopyknic ether extracting once again, dehydrated alcohol deposit D NA with 2.5 times of volumes, and wash precipitation with 70% ethanol, be resuspended at last in the 18ul water, in this mixture, add 2.5ul dNTP (1mMdCTP, 1mMdGTP, 1mMdTTP subsequently, 10mMdATP), 1.25ul the T4DNA polysaccharase of 100mM DTT and 5 units, 11 ℃ 30 minutes, enzyme is cut product mends into flush end, after 75 ℃ of termination reactions, add the Tth archaeal dna polymerase of 5 units and corresponding damping fluid thereof and system is expanded as 80ul, 70 ℃ of reactions 20 minutes make 3 of DNA ' end add the dA tail.This mixture is through the equal-volume chloroform: after primary isoamyl alcohol (24: 1) mixing solutions extracting and the extracting of equal-volume ether with 3 * 10 of Promega company -3The pGEM-T-Easy carrier connect 10 hours in 16 ℃, cumulative volume is 90ul.10 * the buffer of 9ul and the T4DNA ligase enzyme of 25 units are wherein arranged, use the dehydrated alcohol precipitation of the 3M NaAc (pH5.2) of 1/10 volume and 2 times of volumes to be connected mixture at last, wash precipitation with 70% ethanol again, be dissolved in after the drying and obtain connecting product in the 30ul water; Preparation method with the electric transformed competence colibacillus cell of BiO-Rad company prepares the competence e.colidh5, get after 2-3ul connects product and 50ul competence bacillus coli DH 5 alpha mixes, forward in the electric shock cup of 0.2cm of 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 penbritin and cultivate, from each clone, extract plasmid simultaneously, and cutting the segmental size of evaluation insertion wherein with the EcoRI enzyme, the white that obtains clone group has constituted the O-antigen gene bunch library of intestinal bacteria O155 type;
(4) to the cloning and sequencing in the library: from the library, select 81 clones of insertion fragment more than 1kb and the insertion fragment among the clone is checked order with this lab A BI3730 type automatic dna sequencer, sequence reaches 100% fraction of coverage, thereby obtains all sequences of O-antigen gene bunch;
(5) splicing of nucleotide sequence and analysis: the Pregap4 and the splicing of Gap4 software of the Staden package software package of publishing with Britain Camb MRC (Medical Research Council) Molecular Biology Lab and edit all sequences, thus obtain the Nucleotide full length sequence of the O-antigen gene bunch of intestinal bacteria O155 type; The quality of sequence is mainly guaranteed by two aspects: 1) genome of intestinal bacteria O155 type is done 5 Long PCR reactions, mix these products then to produce the library, 2) to each base, guarantee high-quality fraction of coverage more than 3, after obtaining the nucleotide sequence of intestinal bacteria O155 type O-antigen gene bunch, with American National biotechnology information science center (The National Center forBiotechnology Information, NCBI) orffinder finds gene, find the reading frame of 7 openings, determine also that with the function of finding the reading frame that these are open what gene they are with the genetic comparison among the blast groupware and the GenBank, finish gene annotation with the Artemis software at Britain sanger center again, do accurate comparison between DNA and protein sequence with Clustral W software, obtain the structure of the O-antigen gene bunch of intestinal bacteria O155 type at last;
(6) specific gene screening: at wzx, the wzy gene design primer in the O-antigen gene of dysentery intestinal bacteria O155 type bunch; Respectively designed two pairs of primers in each gene, every pair of primer is distributed in the different places in the corresponding gene, to guarantee its specificity; Is that template is carried out PCR with these primers with the genomes of 166 strain intestinal bacteria and 43 strain Shigellaes, except that a band that in the 11st group, has obtained the expection size, any product that all do not increase in other groups is so the O-antigen of wzx, wzy gene pairs intestinal bacteria O155 type all is high special.
Just, first aspect of the present invention provides the full length nucleotide sequence of the O-antigen gene bunch of intestinal bacteria O155 type, its complete sequence shown in SEQ ID NO:1,14879 bases of total length; The base that perhaps has one or more insertions, disappearance or replacement keeps the Nucleotide of the SEQ ID NO:1 of described isolating functional nucleotide simultaneously.Obtained the structure of the O-antigen gene bunch of intestinal bacteria O155 type by method of the present invention, as shown in table 3, it is altogether by orf2, wzy, and orf4, orf5, wzx, orf8, ugd, gla, wzz9 genomic constitution is all between galF gene and hisI gene.
Second aspect of the present invention provides the gene in the O-antigen gene bunch of intestinal bacteria O155 type, promptly transports enzyme gene (wzx gene or the gene of identity function arranged with wzx); Pol gene (wzy gene or the gene of identity function arranged with wzy); Glycosyltransferase gene (orf2, orf4, orf5 gene); Acetyltransferase gene (orf8 gene); Gluconic acid synthase gene (ugd gene); Galactosonic acid synthase gene (gla gene); Chain length controlling gene (wzz gene).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 transhipment enzyme gene that the present invention relates to and pol gene are high specials to the O-antigen of intestinal bacteria O155 type.
The 3rd aspect of the present invention provides the wzx gene in the O-antigen gene bunch that comes from intestinal bacteria O155 type or with wzx the gene of identity function arranged, wzy gene or the gene of identity function is arranged with wzy; Glycosyltransferase gene comprises orf2, orf4, orf5 gene; The acetyltransferase gene comprises the orf8 gene; The gluconic acid synthase gene comprises the ugd gene; The galactosonic acid synthase gene comprises the gla gene; The chain length controlling gene comprises the wzz gene, and they are any one section oligonucleotide in these genes.But, be that the oligonucleotide of listing in the table 1 is right preferentially by usefulness, in table 1, also listed these oligonucleotide to the position in O-antigen gene bunch and with these oligonucleotide to being the size of the product of the PCR reaction done of primer, the annealing temperature in these PCR reaction free lists is carried out.These primers are except that a band that has obtained the expection size in the 19th group, and any product that all do not increase in other groups is so the O-antigen of wzx, wzy gene pairs intestinal bacteria O155 type all is high special.
The separation method of the Nucleotide of described O-antigen-specific to intestinal bacteria O155 type comprises the steps: 1) genomic extraction; 2) the O-antigen gene in the pcr amplification intestinal bacteria O155 type bunch; 3) make up O-antigen gene bunch library; 4) to the cloning and sequencing in the library; 5) splicing of nucleotide sequence and analysis; 6) screening of specific gene.
Other aspects of the present invention are because disclosing of the technology of this paper is conspicuous to those skilled in the art.
As used herein, " oligonucleotide " mainly refers to derive from gene, the gene of coding transhipment enzyme and intragenic one section nucleic acid molecule of coding polysaccharase of the encoding glycosyl transferring enzyme in the O-antigen gene bunch, they can change on length, generally change in 10 to 20 Nucleotide scopes; More definite these oligonucleotide of saying are to come from wzx gene (nucleotide position is the Nucleotide of 6827 to 8113 bases from SEQ ID NO:1); Wzy gene (nucleotide position is the Nucleotide of 2232 to 3185 bases from SEQ ID NO:1); Coming from above intragenic oligonucleotide is high special to intestinal bacteria O155 type.
In addition, the antigenic gene cluster of the different O-of the coding of two genetic resemblances produces new O-antigen by gene recombination or sudden change sometimes, thereby produces new bacteria types, new mutant strain.In this environment, need filter out many specificitys that oligonucleotide is detected with raising with recombination hybridization.Therefore, the invention provides a whole set of many mixtures to oligonucleotide, they come from glycosyltransferase gene; Come from transhipment enzyme and pol gene, comprise the wzx gene or the gene, wzy gene of identity function arranged or the gene of identity function is arranged with wzy with wzx.The mixture of these genes is special to a special bacterial polysaccharides antigen, is special thereby make this cover oligonucleotide to the polysaccharide antigen of this bacterium.More particularly, the mixture of these oligonucleotide is to come from glycosyltransferase gene, wzx gene or the gene, wzy gene of identity function arranged or with wzy the combination of the oligonucleotide in the gene of identity function is arranged with wzx.
On the other hand, the present invention relates to the evaluation of oligonucleotide, they can be used for detecting the O-antigen of expressing the antigenic bacterium of O-and identifying bacterium in diagnosis.
The present invention relates to a kind of antigenic method of one or more bacterial polysaccharideses that detects in the food, these antigens can make sample can with the oligonucleotide specific hybrid of following at least one gene, these genes are: (i) gene of the encoding glycosyl transferring enzyme gene of transhipment enzyme and polysaccharase of (ii) encoding comprises the wzx gene or the gene, wzy gene of identity function is arranged or with wzy the gene of identity function is arranged with wzx.At least one oligonucleotide can be hybridized with at least one more than one such gene specific of expressing the special antigenic bacterium of O-under the situation of condition permission, and these bacteriums are intestinal bacteria O155 types.Available PCR method detects, more can with behind the Nucleotide mark in the inventive method as probe by hybridization such as southern-blot or fluoroscopic examination, perhaps by antigen and bacterium in gene chip or the microarray assay sample.
Planner of the present invention considers following situation: when one special oligonucleotide detects when invalid, the mixture of oligonucleotide can with the target region specific hybrid with test sample.Therefore the invention provides a cover oligonucleotide and be used for detection method of the present invention.Here said oligonucleotide is meant the gene that comes from the encoding glycosyl transferring enzyme, the gene of coding transhipment enzyme and the gene of polysaccharase, comprises the wzx gene or the gene, wzy gene of identity function is arranged or with wzy the oligonucleotide of the gene of identity function is arranged with wzx.This cover oligonucleotide is special to the O-antigen of a special bacterium, and this special bacterium O-antigen is expressed by intestinal bacteria O155 type.
On the other hand, the present invention relates to a kind of antigenic method of one or more bacterial polysaccharideses that detects in the movement, these antigens can make sample can with the oligonucleotide specific hybrid of following at least one gene, these genes are: (i) gene of the encoding glycosyl transferring enzyme gene of transhipment enzyme and polysaccharase of (ii) encoding comprises the wzx gene or the gene, wzy gene of identity function is arranged or with wzy the gene of identity function is arranged with wzx.At least one oligonucleotide can be expressed more than one such gene specific hybridization of the special antigenic bacterium of O-with at least one under the situation of condition permission.These bacteriums are intestinal bacteria O155 types.Oligonucleotide among available the present invention is made the method test sample of primer by PCR, also can with behind the oligonucleotide molecules mark among the present invention as probe by hybridization such as southern-blot or fluoroscopic examination, perhaps by antigen and bacterium in gene chip or the microarray assay sample.
General a pair of oligonucleotide may with same gene recombination also can with different gene recombinations, but must have in them an oligonucleotide can specific hybrid to the distinguished sequence of special antigenic type, another oligonucleotide can be hybridized in non-specific zone.Therefore, when the oligonucleotide in the special polysaccharide antigen gene cluster is reconfigured, can select specific gene mixture hybridization in a pair of oligonucleotide and the polysaccharide antigen gene cluster at least, perhaps select many mixture hybridization oligonucleotide and specific gene.Even even when all genes were all unique in the specific genes bunch, this method also can be applied to discern the nucleic acid molecule of the gene mixture in this gene cluster.Therefore the invention provides a whole set of is used to detect the many to oligonucleotide of the inventive method, many here is that the gene of the gene that comes from the encoding glycosyl transferring enzyme, coding transhipment enzyme and polysaccharase comprises the wzx gene or the gene, wzy gene of identity function arranged or with wzy the gene of identity function is arranged with wzx to oligonucleotide, this cover oligonucleotide is special to a special bacterial polysaccharides, and this cover oligonucleotide may be the Nucleotide of necessary gene during sugar synthesizes.
On the other hand, the present invention also relates to the antigenic method of one or more bacterial polysaccharideses in the sample that a kind of detection comes from patient.One or more bacterial polysaccharides antigens in the sample can make sample can with a specific hybrid in a pair of oligonucleotide in following at least one gene, these genes are: (i) gene of the encoding glycosyl transferring enzyme gene of transhipment enzyme and polysaccharase of (ii) encoding comprises the wzx gene or the gene, wzy gene of identity function is arranged or with wzy the gene of identity function is arranged with wzx.Under the situation of condition permission at least one oligonucleotide can with sample at least one express more than one such gene specific hybridization of the special antigenic bacterium of O-, these bacteriums are intestinal bacteria O155 types.Oligonucleotide among available the present invention is made the method test sample of primer by PCR, also can will pass through hybridization as probe behind the oligonucleotide mark among the present invention, perhaps by antigen and bacterium in gene chip or the microarray assay sample.
In more detail, method described above can be understood as when oligonucleotide when being used, it is not to derive from glycosyltransferase gene, wzx gene or with wzx the gene, wzy gene of identity function arranged or have on the sequence of gene of identity function with wzy that one of them oligonucleotide molecules can hybridize to one.In addition, when two oligonucleotide can both be hybridized, they may be hybridized in same gene and also may hybridize on the different genes.Also promptly, when cross reaction goes wrong, can select the mixture of oligonucleotide to detect the blended gene so that the specificity of detection to be provided.
The present inventor believes that the present invention is not necessarily limited to the above nucleotide sequence coded specific O-antigen of carrying, and is widely used in detecting all expression O-antigens and identifies the antigenic bacterium of O-.And because O-antigen is synthetic and the similarity of other polysaccharide antigens (as bacterium born of the same parents exoantigen) between synthesizing, method of the present invention and molecule also are applied to these other polysaccharide antigen.
The present invention discloses the full length sequence of the O-antigen gene bunch of intestinal bacteria O155 type first, and can from the sequence of this total length gene cluster of not cloned, produce recombinant molecule, can produce the O-antigen of expressing intestinal bacteria O155 type by inserting to express, and become useful vaccine.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should understand these embodiment only is used to the present invention is described and is not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, molecular cloning: the condition described in the laboratory manual (NewYork:Cold Spring Harbor Laboratory Press, 1989).
Embodiment 1: genomic extraction:
37 ℃ of incubated overnight intestinal bacteria O155 types in the LB of 5mL substratum, centrifugal collecting cell.With 500ul 50mM Tris-HCl (pH8.0) and 10ul 0.4M EDTA re-suspended cell, 37 ℃ of incubations 20 minutes, the N,O-Diacetylmuramidase that adds 10ul 10mg/ml then continues insulation 20 minutes.The Proteinase K, the 15ul 10%SDS that add 3ul 20mg/ml afterwards, 50 ℃ of incubations 2 hours add the RNase of 3ul 10mg/ml again, 65 ℃ of incubations 30 minutes.Add equal-volume phenol extracting mixture, get supernatant and use isopyknic phenol again: chloroform: primary isoamyl alcohol is taken out (25: 24: 1) mixing solutions and is carried twice, get supernatant again with isopyknic ether extracting to remove remaining phenol, supernatant is with 2 times of volume ethanol deposit D NA, roll out DNA and wash DNA with glass yarn, at last DNA is resuspended among the 30ul TE with 70% ethanol.Genomic dna detects by 0.4% agarose gel electrophoresis.
Embodiment 2: by the O-antigen gene in the pcr amplification intestinal bacteria O155 type bunch:
With the genome of intestinal bacteria O155 type is that template is passed through its O-antigen gene of Long pcr amplification bunch.At first according to the JumpStart sequences Design upstream primer (#1523-ATT GTG GCT GCA GGG ATC AAA GAA AT) that often is found in O-antigen gene bunch promoter region, again according to the gnd gene design downstream primer (#1524-TAG TCG CGT GNG CCTGGA TTA AGT TCG C) in O-antigen gene bunch downstream; With the Expand Long TemplatePCR method of Boehringer Mannheim company amplification O-antigen gene bunch, the PCR response procedures was as follows: 94 ℃ of pre-sex change 2 minutes; 94 ℃ of sex change are 10 seconds then, 60 ℃ of annealing 15 seconds, and 68 ℃ were extended 15 minutes, and carried out 30 circulations like this.At last, continue to extend 7 minutes at 68 ℃, obtain the PCR product, the agarose gel electrophoresis with 0.8% detects the size and the specificity thereof of PCR product.Merge 5 pipe long PCR products, and with the Wizard PCR Preps purification kit purified pcr product of Promega company.
Embodiment 3: make up O-antigen gene bunch library:
At first be the acquisition that connects product: make up O-antigen gene bunch library with the Novagen DNaseI shot gun method that is modified.Reaction system is a 300ng PCR purified product, 0.9ul 0.1M MnCl 2, 1ul1: the DNaseI of the lmg/ml of 2000 dilutions, reaction is carried out at room temperature.Enzyme is cut the dna fragmentation size is concentrated between the 1.5kb-3kb, then adds 2ul 0.1M EDTA termination reaction.Merge the same reaction system of 4 pipes, with isopyknic phenol extracting once, use isopyknic phenol: chloroform: primary isoamyl alcohol (25: 24: 1) mixing solutions extracting once, after using isopyknic ether extracting once again, dehydrated alcohol deposit D NA with 2.5 times of volumes, and wash precipitation with 70% ethanol, be resuspended at last in the 18ul water.In this mixture, add 2.5ul dNTP (1mMdCTP subsequently, 1mMdGTP, 1mMdTTP, 10mMdATP), the T4DNA polysaccharase of 1.25ul 100mM DTT and 5 units, 11 ℃ 30 minutes, enzyme is cut product mend into flush end, after 75 ℃ of termination reactions, add the Tth archaeal dna polymerase of 5 units and corresponding damping fluid thereof and system is expanded as 80ul, 70 ℃ were reacted 20 minutes, made 3 of DNA ' end add the dA tail.This mixture is through the equal-volume chloroform: after primary isoamyl alcohol (24: 1) mixing solutions extracting and the extracting of equal-volume ether with 3 * 10 of Promega company -3The pGEM-T-Easy carrier connect 10 hours in 16 ℃, cumulative volume is 90ul.10 * the buffer of 9ul and the T4DNA ligase enzyme of 25 units are wherein arranged.Use the dehydrated alcohol precipitation of the 3M NaAc (pH5.2) of 1/10 volume and 2 times of volumes to be connected mixture at last, wash precipitation with 70% ethanol again, be dissolved in after the drying and obtain connecting product in the 30ul water.
Next is the preparation of competent cell: the method that provides with reference to Bio-Rad company prepares 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 about OD6000.5, ice bath cooling was 20 minutes then, in centrifugal 15 minutes of 4 ℃ of 4000rpm.Confide all supernatant, dispel thalline, in centrifugal 15 minutes of 4 ℃ of 4000rpm with the deionization aqua sterilisa 200ml of cold ice precooling.Deionization aqua sterilisa 100ml with cold ice precooling dispelled thalline again, in centrifugal 15 minutes of 4 ℃ of 4000rpm.With 10% glycerine suspension cell of cold ice precooling, centrifugal 10 minutes of 4 ℃ of 6000rpm abandon supernatant, precipitate 10% glycerine suspension cell with the precooling of 1ml ice at last, are competent cell.The competent cell that makes is packed as 50ul one pipe ,-70 ℃ of preservations.
Be electric transformed competence colibacillus cell at last: get after 2-3ul connects product and 50ul competence bacillus coli DH 5 alpha mixes, forward in the electric shock cup of 0.2cm of Bio-Rad company and shock by electricity, voltage is 2.5 kilovolts, and the time is 5.0 milliseconds-6.0 milliseconds.The SOC substratum that adds 1ml after the electric shock immediately in cup makes the bacterium recovery.Immediately bacterium is coated in 37 ℃ of inversion incubated overnight on the LB solid medium that contains penbritin, X-Gal and IPTG then, obtains blue white bacterium colony next day.With the white colony that obtains promptly the white clone forward on the LB solid medium that contains penbritin and cultivate, from each clone, extract plasmid and cut the segmental size of evaluation insertion wherein simultaneously, obtain the O-antigen gene bunch library that white clone group has constituted intestinal bacteria O155 type with the EcoRI enzyme.
Embodiment 4: to the cloning and sequencing in the library:
From the library, select insert 81 clones of fragment more than 1kb with this lab A BI3730 type automatic dna sequencer to unidirectional order-checking of insertion fragment among the clone, make sequence reach 100% fraction of coverage, thus all sequences of acquisition O-antigen gene bunch.
Embodiment 5: the splicing of nucleotide sequence and analysis:
The Pregap4 and the splicing of Gap4 software of the Staden package software package of publishing with Britain Camb MRC (Medical Research Council) Molecular Biology Lab and edit all sequences, thus the Nucleotide full length sequence (seeing sequence list) of the O-antigen gene bunch of intestinal bacteria O155 type obtained.The quality of sequence is mainly guaranteed by two aspects: 1) genome of intestinal bacteria O155 type is done 5 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 intestinal bacteria O155 type O-antigen gene bunch, with American National biotechnology information science center (The National Center for BiotechnologyInformation, NCBI) orffinder finds gene, find the reading frame of 9 openings, determine also that with the function of finding the reading frame that these are open what gene they are with the genetic comparison among the blast groupware and the GenBank, finish gene annotation with the Artemis software at Britain sanger center again, do accurate comparison between DNA and protein sequence with ClustralW software, obtain the structure of the O-antigen gene bunch of intestinal bacteria O155 type at last, as shown in table 3.By retrieval and relatively, find orf9, ugd among the orf10, orf11 encoded protein and Escherichia coli O-antigen gene bunch, gla, the albumen of wzz genes encoding have very high consensus amino acid sequence (92-99%).We name orf9, and orf10, orf11 are ugd, glaa, and wzz, and infer the existence that gluconic acid and galactosonic acid are arranged in the O-antigen of intestinal bacteria O155.
Orf7 and orf3 are the proteic genes that there is transmembrane segment in only two codings of intestinal bacteria O155 kind.The O-antigen transferring enzyme of Orf7 encoded protein and Yersinia enterocolitica has 28% sequence identity, and it contains 12 uniform transmembrane segments by the proteic topology discovery of HMMTOP2.0 programanalysis, and this is the proteic characteristic feature of Wzx.So name orf7 is wzx.The O-antigen polysaccharase of Orf3 encoded protein and Shigella boydii has 24% consistence, 45% similarity, it contains 9 transmembrane segments by the proteic topology discovery of HMMTOP2.0 programanalysis, and hydrophilic loop (loop) in big (61 an amino acid) kytoplasm is arranged, and this is the proteic characteristic feature of Wzy.So name orf3 is wzy.
Orf2, the albumen of 4,5 four genes encodings and other known glycosyltransferases have the sequence identity of 24-58% and the sequence similarity of 52-76%.Therefore we infer this four genes encoding glycosyltransferases, and because each glycosyltransferase specificity catalysis forms a kind of disaccharide bond, so we infer that the antigenic oligosaccharide unit of O-of intestinal bacteria O155 may be made up of four monose.Because the definite function of these three genes can't be determined, so we are with these three genes temporary called after orf2, orf4 and orf5.
Embodiment 6: the screening of specific gene:
At wzx, wzy gene design primer in the O-antigen gene of intestinal bacteria O155 type bunch, the position of these genes in nucleotide sequence sees Table 1.
Embodiment 7: the detection of primer sensitivity.
Buy the live pig meat stuffing on the market, stir, be divided into the 20g portion, exist in-40 ℃ of refrigerators standby.The frozen bacterium liquid of 10 μ l intestinal bacteria O155 is inoculated in the triangular flask of 20ml LB substratum, in 37 ℃, 200 rev/mins, cultivate 12 hours to saturated, the cultured bacterium liquid that takes a morsel does 10 6With 10 7Dilution doubly, remaining bacterium liquid are put in 4 ℃ the refrigerator standby, get 50 μ l dilution bacterium liquid coating LB agar plate, and 37 degree are cultivated 12h, to being coated with plate count, calculate viable bacteria concentration in the stoste.In 5 portions of live pig meat stuffings, mix 5 * 10 respectively 3, 5 * 10 2, 5 * 10 1, 5 and 0 viable bacteria stir, and add 200ml LB substratum, and through 6 layers of filtered through gauze, filtered liquid 200 rev/mins, is cultivated 12h in 37 ℃.Get 3ml bacterium liquid in 6 from cultured bacterium liquid, centrifugal 5 minutes of 000g removes supernatant, adds 100 μ l MQ ultrapure waters and blows precipitation and mixing open, puts into 100 degree boiling water and boils 15 minutes, and lysate is in 12, and centrifugal 8 minutes of 000g gets 1 μ supernatant as pcr template.Right with 3 pairs of oligonucleotide, the Nucleotide of 7641 to 7658 bases among the SEQ ID NO:1 and the Nucleotide of 8081 to 8098 bases; The Nucleotide of 10451 to 10468 bases among the SEQ ID NO:1 and the Nucleotide of 10736 to 10753 bases; The Nucleotide of 6289 to 6308 bases among the SEQ IDNO:1 and the Nucleotide of 7198 to 7216 bases carry out the PCR reaction, and the PCR reaction system is as follows: MQ:15.7 μ l, Mg 2+: 2.5 μ l, Buffer:2.5 μ l, dNTP:1 μ l, Taq enzyme: 0.3 μ l, P1:1 μ l, P2:1 μ l, template DNA: 1 μ l.The PCR reaction conditions is: 95 ℃: 5 ', 95 ℃: 30 ", 56 ℃: 45 ", 72 ℃: 1 ', 72 ℃: 5 ', totally 30 circulations.Reaction is got 10 μ l reaction product electrophoresis after finishing, if the amplified band that conforms to the expection size is arranged, then the result is positive, if do not have, then the result is negative.Participated in 5 * 10 3, 5 * 10 2, 5 * 10 1And every part of pork filling of 5 viable bacterias all obtains positive findings in the PCR of 3 pairs of primers reaction.The pork filling that participates in 0 viable bacteria obtains negative findings in the PCR of 3 pairs of primers reaction.Illustrate that these 3 pairs of primers are 0.25 bacterium/g to the detection sensitivity of the intestinal bacteria O155 in the pork filling when using aforesaid method.
By clone and the expression in the vaccine strains of attenuation, can set up recombiant vaccine to O antigen gene bunch.O antigen is the surface antigen of topmost Gram-negative bacteria, can cause the intensive immune response, is one of best target molecule of making recombiant vaccine.Viret laboratory success in 1993 the O antigen gene of Shigellae Sonnei bunch is expressed in a strain Salmonellas Tyziai vaccine bacterium, experimentation on animals proof can cause rabbit immune response (Molecular Microbiology 1993,7:239-252).The group of China Military Medical Science Institute also similarly works being engaged in the Viret laboratory.Bunch express the O antigen gene of intestinal bacteria O111 success in 1999 in the Wang Lei laboratory in salmonella vaccine STM-1, and the bacterial strain set up of proof can cause the blood of mouse and humoral response (Microbial Pathogenesis 1999,27:55-59).So the O antigen-specific gene order of O155 of the present invention can be applied to set up recombiant vaccine.
Nucleotide sequence (shown in the SEQ IDNO:1) according to the O-antigen-specific to intestinal bacteria O155 type of the present invention, structure specific nucleic acid probe, be fixed on the carrier of chip and make biochip, after the sample that will detect is suitably handled, carry out hybridization with biochip, utilize the biochip signal analysis equipment just can obtain corresponding bacteria situation in the sample then.The DNA chip that this intestinal bacteria O antigen is identified can be directly used in clinical and other check place (as food-processing and production industry, the Micro biological Tests of animal and veterinary industry customs quarantine control etc.).This chip only need enlarge output, just can industrialization under identical condition.
Table 1 has been listed glycosyltransferase gene and oligosaccharide unit treatment gene and intragenic primer and PCR data in the O antigen gene bunch of intestinal bacteria O155 type.Glycosyltransferase gene, transhipment enzyme gene and pol gene and their function corresponding and the size of the O antigen gene bunch of intestinal bacteria O155 type in table, have been listed.In each gene, we have respectively designed two pairs of primers, and the difference that every pair of primer is distributed in the corresponding gene is local to guarantee its specificity.In table, also listed position and the size of each primer in SEQ IDNO:1.Is that template carry out PCR with listed corresponding annealing temperature in the table with the genomes of all bacterium in the table 2 with every pair of primer, has obtained corresponding PCR product, and its size is also listed in the table.
Table 2 is 166 strain intestinal bacteria and 43 strain Shigellaes and their sources that are used to screen specific gene, and for the convenience that detects, we are divided into one group with their every 8-10 bacterium, and 28 groups altogether, all list in the table in their source.
The genomic dna that contains intestinal bacteria O155 type in the 11st group is as positive control.Do template with every group of bacterium, be PCR by following condition with every pair in the table 1 primer: 95 ℃ of pre-sex change after 5 minutes, 95 ℃ of sex change 30 seconds, annealing time is 30 seconds, temperature sees Table 1,72 ℃ extended 2 minutes, carried out 25 circulations like this.Continue to extend 5 minutes at 72 ℃ at last, reaction system is 25ul.Template is dilution in 1: 20, gets 1 μ l.After reaction finishes, get the 10ulPCR product and detect the fragment that amplifies by 0.8% agarose gel electrophoresis.
For wzx, wzy gene, each gene all has two pairs of primers detected, every pair of primer has obtained except be PCR in the 11st group after the correct band of expection size, the correct band of any size does not all increase in other groups, that is to say, not obtaining any PCR product band in the array mostly, so wzx, wzy gene pairs intestinal bacteria O155 type and O-antigen thereof are high specials.
At last, from intestinal bacteria O155 type, screen gene by PCR: wzx, wzy and three glycosyltransferase genes to the O-antigen high special of intestinal bacteria O155 type.And the oligonucleotide of these intragenic any one section 10-20nt is special to the O-antigen of intestinal bacteria O155 type, and the primer in especially above-mentioned each gene is that oligonucleotide is high special to detecting the back confirmation through PCR to intestinal bacteria O155 type.These all oligonucleotide all can be used for the intestinal bacteria O155 type in the human body and environment rapidly and accurately, and can identify their O-antigen.
Table 3 is structural tables of the O-antigen gene bunch of intestinal bacteria O155 type, in table, listed the structure of the O-antigen gene bunch of intestinal bacteria O155 type, altogether by 9 genomic constitutions, each gene box indicating, and in square frame, write the title of gene, numeral be the order of the open reading frame (orf) in the O-antigen gene bunch.Two ends at O-antigen gene bunch are galF gene and hisI gene, and they do not belong to O-antigen gene bunch, and we are just with the increase full length sequence of O-antigen gene bunch of their one section sequences Design primer.
Table 4 is site plan of the gene in the O-antigen gene bunch of intestinal bacteria O155 type, listed the accurate position of all open reading frame in complete sequence in the O-antigen gene bunch of intestinal bacteria O155 type in the drawings, at the underscoring of the initiator codon and the terminator codon of each open reading frame.The initiator codon of open reading frame is ATG and GTG in intestinal bacteria.
SEQ ID NO:1 sequence (SEQUENCE LISTING)
＜110〉Nankai University
＜120〉to the Nucleotide of the O antigen-specific of intestinal bacteria O155 type
＜130〉to the Nucleotide of the O antigen-specific of intestinal bacteria O155 type
<160>1
<170>PatentIn version 3.2
<210>1
<211>12755
<212>DNA
<213>Escherichia coli
<400>1
attgtggctg cagggatcaa agaaatcctc ctggtaactc atgcgtccaa gaacgcggtc 60
gaaaaccact tcgacacctc ttatgaatta gaatctctcc ttgaacagcg cgtgaagcgt 120
caactactgg cggaagtgca atctatctgc ccgccgggcg tgactattat gaacgtgcgt 180
cagggcgaac ctttaggttt aggtcactcc attttgtgtg cacgacccgc cattggtgac 240
aatccatttg tcgtggtgct gccagacgtt gtgatcgacg acgccagcgc cgacccgcta 300
cgctacaacc ttgctgccat gattgcgcgc ttcaatgaaa cgggccgtag ccaggtgctg 360
gcaaaacgta tgccgggtga cctctctgaa tactccgtca tccagaccaa agaaccgctg 420
gaccgtgaag gcaaagtcag ccgcattgtt gaatttatcg aaaaaccgga tcagccgcag 480
acgttggact cagacatcat ggccgtaggt cgttatgtgc tttctgctga tatttggccg 540
gaacttgaac gcacgcagcc aggggcatgg ggacgtattc agctgacaga tgccatcgct 600
gaactggcga aaaaacaatc tgttgatgca atgctgatga ctggtgacag ctacgactgc 660
ggtaaaaaaa tgggctatat gcaggcgttt gtgaagtatg ggctacgtaa cctgaaagaa 720
ggggcgaagt tccgcaaagg tattgagaag ctgttaagcg aataatgaaa atctgaccga 780
atgttacggt tgataagaaa attataacgg cagtgaagat tcgtggcgag agtaatttgt 840
tgcgaatatt cctgccgtta ttttatataa acaatcagaa taacaacgag ttagcaatag 900
gattttagtc aaagttttcc aggattttcc ttgtttccag agcggattgg taagacaatt 960
agcgtttgaa tttttcgggt ttagcgcgag tgggcaacgc tcgtcacatc gtagacatgc 1020
atgcagtgct ctggtagctg taaagccagg ggcggtagcg tgaaaattaa ggatgctaat 1080
aaataattgg ttgtatgtat accacaactt attaacttat ttatgtgtaa gtctaaatag 1140
tgtggtaatt cttatcagtt ggaagatttt tacacatata aactgaaaac atcacaatat 1200
agtaaagtta aagtgggctt tattttattg tcatttttgg ttgcaacagc atattatgct 1260
acctttaaaa gaggaaacga catggggtta aatatagaat taataagtgt tgtgattact 1320
ttctttagta atgatgaatc ttcgggtaca tatttaaata atagtgtttt aagcgcatta 1380
aaccaaacat atagaaacat tgaagtcttg gtcattgatg attgctcccc aataaaagca 1440
actagctgtc ttaatggtat taatgatagt agattgtcaa ttataagact tccacaaaat 1500
ggtggaatgg ccgcagctat taatcatgga attggtctgt catctggaag ttttattgca 1560
tttcttgatt ttgatgacct ttggtacgat ttcaaactta aagagcaagt tgagttgatc 1620
aaagattata ttgataaaaa gctggctgaa cctgaaaaaa tatgttgcta ttctaaggtt 1680
ttaataaaac atgataatta cgaatatact aaaccttaca gaagtataga ggagggtgag 1740
cacgtagcag attatttgtt tgctaatgga caatttatgc aaacaagtgg gatgcttata 1800
tcaaaatcat tacttataaa agtcaaagga atggattctt ataaaagaca tacggattat 1860
cagttagtat taaaactata tcaagaaaat gctgtgttta tttattatga tcgagtttca 1920
tatatgtata aaataactga caaaaaagga gattacaaat tttcgcaaga ttggctttgt 1980
gcaaataagt ggatgatgac aaaacgttct ataagatatt tcaaaattaa tacaatatta 2040
agaagcatga ttgataatca tgcttttggt actgcgttta tatattcagt atctaacaga 2100
tttatatggt cttttggctc gtatttaata cgattttctt taaaaaaatt attacctaca 2160
cgcttattat caacgctgat taaacataaa aataaagcag cgttttctgt aaaaggaaga 2220
gagtattgaa aatgggaggc tttattacaa gattagtttt attgctgcca ttatttttat 2280
tattttattt tcctgcggac cataacaaag attattttaa ttatcaaaca gattatgact 2340
atgccatagg gcaatttgat tacctatatg agctatccgt gagatttttt agagatatat 2400
tgaactatag ttttagttca ttttgggtga ttgtgattat agtagaaatt attttcttga 2460
cgatatatta taacaaggtg aaatatatcc ttattgcata cccgggtata atttcgatga 2520
gccagttttt ttatggcact caaatacgtt atgctttggc aatgttaatt ggattagtag 2580
tattaaaaaa tagtagacgc tttattaaag tttttggagt gttaattgct tcattattcc 2640
actatggcgc cgtgatatca ttattaccat tatttatttg taggtatata aatgaaagat 2700
atttcatttt tttgtctaaa cgaaatatat tcttgattat atctgtatta acattactgt 2760
tttttttctt ctattctttc gagtcatttg tttcaatgac tcgtttttct tattatttag 2820
gtgctgaaaa attttccgat tcaaaatcga cttcatctat aatatatgcg gttgtgagtc 2880
tattgtttat tctgttttta tattcaaaaa atacagcgtt cagaaccaat ataagtaaac 2940
aatctatagt aatactgatt ttagttttga ttttttcacc tatagcagtg ctatcaggtc 3000
gcacgatgct cgtttatttt ataatggaac ctttgttaat agcatctgca ttcacctcta 3060
taaagaaaaa tagtcctgat tattttatat ctttatcgtt atctttgtct ttatttttat 3120
tctatattgc gagatgtatt tattatatcg taacggcaga tttttatttt tatgatttct 3180
tataaggaga acaaatgagg atagctcata tatataaaaa acattatgat agcggagatg 3240
gtatcactaa tgttgttgag aatttatata aacttcaaaa aaacaataat gcagatgaat 3300
atatagattt accattcatg tttgataatt gctcaaaagg taaggctcaa tatagatatt 3360
cagggtttat tgggatgcta tatcatttgt tcaaatgcaa gccaaatgta atatacttgc 3420
atggtttcta ctatttgcat tatattattc ttggtgtttg tttttatttt atcaaatcaa 3480
aagttgtttt aataccacat tgctctcttt taaatagagc aattacttat aaaaaaataa 3540
gaaagttgat atattataaa atattctcat taatttatta tgattctata atattgattc 3600
agtatttgaa ccacgaagag caaataggtt caaaaaaatt tttattctca cctcctgaat 3660
ccattatacc aaatggtgtt aactatactg aaaaacaatg tcgatctttt gattttctat 3720
ctctattcta tttagggcga tgtgatataa atcataaagg tattgacata ctatttgagt 3780
atcttactaa gattaatcaa aaaataaaat tatatggtgc agatccaaat gaaaaaataa 3840
aactaaagaa attaattcaa ttaaagaaat tagagcaaaa agttgaaata tatgatcctg 3900
tctttaatga ggataaggag agggtattct tgaataataa tatatttata ttactttcga 3960
gatatgaagg attacctatt agtgtgcttg aagcactttc ttatggatgt atttgtctag 4020
tttcatctgg aacaaatatg gctgatgaaa ttgtagctgc gaactgcgga tttaaaattg 4080
attctgtaga ggatcttatt tcagtatgga ataagctacg ggtaatgagt atatatgaat 4140
taaaagaaat gtctgaaaat tcaaaagcat taattaaaca taaatacgct tggcaaaaaa 4200
taatcatatc ttctgagatt gaattaagaa aatatgtaac ataagtcttt gtgcgtagat 4260
tttgaatgtt ctatttattc tggttagaat tttatagtca attcgatgac atatattcac 4320
acaatatttc atacagaaga ttaattagga acttaatgtg aaatttactg ttttactttc 4380
cttatataat agagagtcta aacacaactt gttcgattgt cttgaaagta ttaagtcaaa 4440
cactataaag ccagaacaga tagtcatagt ttatgatgga ccgatcagtg aagaactgga 4500
tagtattgtt actggtttta tcaaatactt accgattgag aaaataaaaa tatcagaaaa 4560
tattggacta ggcaacgcac ttaattatgg cttgaatttt tgtagacatg aaattgtttt 4620
caggatggat acagacgata aatgtaaaag cacaagattt gaacggcaac ttgaaattat 4680
aaaaataaaa aatgatatcg tgttgatggg gagtgctata gaagaattta acgaatcgat 4740
gacagtctct catggtgtaa gattttcagc cgaatatcat gatgatataa taaattatgc 4800
aaaaagaaga aacccattta atcatatgtc agtggttttt aaaaaatcaa taatacaggg 4860
gctaggaggg tatcagcatc attattttat ggaagattat aacttgtggc ttcgggttat 4920
cgctgctggg tataaaactt ataatattcc agaagtgctt gtctatgttc gaggtggaga 4980
taaaatgctt gggcgtagaa aaggaattcc ctatgtgatt agtgaggtaa aattggctaa 5040
attaaaatat aacttaaaaa ttgacagtaa tgcaggtgtt attatttctg caaccatgag 5100
gattttacct cgtttattgc ccgtcttttt gttaaaacaa atatataaag ttttaaggaa 5160
aagcaagtct tgaattctct gtgaaattta tgaatacaat ccacattgat tacatatact 5220
gaacattatc cgtttgttat aataccgcac aattctaagt caattcccct gacaggagta 5280
aacaatgtca aagcaacaga tcggcgtcgt cggtatggca gtgatggggc gcaaccttgc 5340
gcttaatatc gaaagccgtg gttataccgt ctctattttc aaccgttctc gtgaaaagac 5400
ggaagaagtg attgccgaaa atccagagaa gaaactggtt ccttactata cggtaaaaga 5460
gtttgttgaa tctctggaaa cacctcgtcg catcctgtta atggtgaaag caggtgcagg 5520
cacggatgct gctattgatt ccctcaagcc atatctcgat aaaggcgaca tcatcattga 5580
tggtggtaat accttcttcc aggacaccat tcgtcgtaac cgtgagcttt ctgcggaagg 5640
ctttaacttc attggtaccg gtgtttccgg tggtgaagag ggtgcgctga aaggtccttc 5700
cattatgccg ggtggtcaga aagaagccta tgagctggtt gctccgatcc tgaccaaaat 5760
cgctgctgtg gctgaagacg gtgagccatg cgttacctat atcggtgctg atggcgcagg 5820
tcactatgtg aagatggttc acaacggtat tgaatacggc gatatgcagt tgattgctga 5880
atcctattct ctgttgaaag gtggcctaaa cctctccaac gaagaactgg cgcagacctt 5940
taccgagtgg aataacggtg aactgagcag ctacctgatc gacatcacca aagacatctt 6000
cactaaaaaa gatgaagacg gtaattacct ggttgatgta attctggatg aagcggctaa 6060
caaaggtacc ggtaaatgga ccagccagag tgcgctggat ctcggcgaac cgctgtcgct 6120
gatcaccgag tctgtgtttg cacgttatat ctcttctttg aaagagcagc gtgttgccgc 6180
gtctaaagtt ctctctggtc cacaagcaca gccagcaggc gataaggctg agtttatcga 6240
gaaagttcgt cgtgcgctgt atctgggcaa aatcgtttct tacgctcagg gcttctctca 6300
gctgcgtgct gcgtctgaag agtacaactg ggatctgaat tacggcgaaa tcgcgaagat 6360
tttccgtgct ggctgcatca tccgtgcgca gttcctgcag aaaatcaccg atgcatatgc 6420
cgaaaatccg cagatcgcta acctgctgct ggctccgtac tttaaacaaa ttgccgatga 6480
ttaccagcag gcgctgcgcg atgtcgttgc ttatgcagta caaaacggta tcccgattcc 6540
gaccttttcc gctgcggtta cctattacga cagctaccgt gcggctgttc tgcctgcaaa 6600
cctgatccag gcacagcgtg actatttcgg tgcgcatact tataagcgca ttgataaaga 6660
aggtgtgttc cataccgaat ggctggatta atttttttct agtttttaaa gagcctgctt 6720
acgcggctct ttaatatata taaaaattat aattcagtcg attcaatcta gataaaacta 6780
ttatttttat tatttcttag ttctaagagt ctacgtgaga tattagatgt ctaactatat 6840
caaaaatgct ggttggattt tttgcgaaaa atttgtacga atagccgtag gctttatatt 6900
atttgcatta atttcaagag ttttgaatcc tggtgaattt ggcttgctat cttattatca 6960
aacaatagca accatgtttt tagcaataac caatttaggt ttcgataatg ttctgattaa 7020
cgagtttaat cagaataaaa agcataatga aatattttca acggcatttt ggtcaagaat 7080
tatagtttca gtatttgtaa taatgttatt tgctgtggga ttgtattttg atgatgtcgt 7140
gctgaagaat aaattggttt tgctgttgtg tataagtagc ttggtttttc aaagtcaaaa 7200
tacatacata ccattttttc aatctcagtt acaagcctca gttgtcacga aaattagttt 7260
ttgcgcactt attatatcct cggtatttaa agtctatttg ttgttaatca atggggatat 7320
aatgtggttt gctttttcat atagttttga ttttgctgtg tcttttttat ttttcgcttt 7380
tttttcatat aaaagaaatt atatatcaat taatccaaaa catttccggt taagtgtgct 7440
aaaacgttta ttacgctcgt cttggccaat tattgcatca tcaataatta ttgttctcta 7500
tactcgatta gatcaattaa tgattatgcg aatgttaggg gctgattcag ttgcaatatt 7560
tagtgttgct ttgaaaatat cagaggcata tttatttgtc cctgcagcat tagttacatc 7620
atattatcca ctgatttcaa agtcacctac cagagaaaat attagatttt attttgatgt 7680
tgttttttca agctctgtaa taatggctgt agtggtagct atagcgtctt attttgtaat 7740
accatatatg tttggggcta attatatcac ctcatataat atattaatta ttttgctatt 7800
tggttcaata ttttctatac tcggctcagc gtgtacaaat ttaatgatag tgtatgactt 7860
atcttattta cggttagttc gagccgttta tgggctatta ataaatttct ccctgaatat 7920
tttacttatt cctaaatatg gtattattgg tgctgcttat gcgtcttttg taagtcagat 7980
ctttgcaagt tggttaagca atagccttaa taaaaaaacg attgaatgct tcagaataca 8040
gtcaatgaca gttgtatctt tgggactaat aggatttaga caattattta tcatgctatt 8100
tagaaggggg taggcatatt atatgtttga cagttatgaa agaaagaaag gtataatgca 8160
aaggataatc ttttatttta aaacgaaatt gagaggatgt cgtgaatact attacagtct 8220
taaatattat catcagttta aaattccaat ctcgaatcat cttaccatcg gcattaagac 8280
attaggggaa aacaaaacta ctttacctca cccggtaggt atagttatag gtaagaacgt 8340
taacatcggt aaaaaatgta ttgtttatca aaatgtcact ataggggtga gtaataataa 8400
atttgaggat tatcctgtta ttggaaataa tgttactatt tatgccggag caatcataat 8460
aggtaaagta attatagggg ataatgcaat aattggtgca ggttgcatcg ttactaaaga 8520
tgtacctgaa ggaaagattg caattggttc accaatgaaa attatggata aaaaacataa 8580
taactactaa ttatctcagt gaattcttaa taattttaaa atgtgttgac tgtaatgttc 8640
gttttttgct attaatgtga aaaaacttca ttaaaaaaga atgcaaatat gaaaataaca 8700
atttcaggaa caggttatgt tggtctttca aatggtattc tgattgcgca aaatcacgaa 8760
gtggttgcac tggatatcgt tcaggccaaa gtggacatgc ttaacaagag gcagtcaccg 8820
attgttgata aggagattga agagtatctg gcgactaaag atctcaattt ccgcgctacg 8880
acagataagt atgaggcgta taaaaatgcc gattacgtta ttattgccac acctaccgat 8940
tatgatccga aaacaaatta ttttaatacc tcaagtgtgg aagcggtcat tcgtgatgtg 9000
acagaaatta atcccaacgc ggtaatgatt ataaaatcaa ctatccctgt tggttttaca 9060
gagtccatta aagaacggtt tggtattgaa aatgtgatct tttcgccgga gtttttacgc 9120
gaagggaaag cactttatga taacttgcat ccatcacgca ttgtgattgg tgagcaatcc 9180
gatcgcgctg aacgttttgc agcattatta caagaaggag ctattaagca agatattcca 9240
acgttgttta ctgactcaac ggaagctgag gcgattaaac tttttgccaa cacctatctg 9300
gcgatgcgtg tagcgtattt caatgaactt gatagttatg ctgaaagctt ggggcttaat 9360
tcacgccaga ttattgaggg cgtatgcctt gatccgcgta taggtaatca ctacaacaat 9420
ccgtcattcg gttatggtgg ttattgtctg ccaaaagata ctaagcagtt actggcaaac 9480
tatcagtcag taccgaataa cttgatctca gcaattgtcg atgccaatcg cacgcgaaaa 9540
gattttattg ccgattctat ccttgcacgt aaaccgaaag ttgttggcgt ctatcgtctg 9600
attatgaaga gtggttcgga caatttccgt gcttcatcga ttcagggtat tatgaagcga 9660
atcaaggcga aaggtgtgcc agtaatcgtc tatgagccag cgatgaaaga ggacgatttt 9720
ttccgttcgc gcgtggtacg tgatctggat gcgttcaaac aagaagctga tgttattatt 9780
tctaaccgta tgtctgccga tctggctgat gtagcagata aggtttatac gcgcgacttg 9840
tttggcaatg attaattatt ttgtttcata ctaagaaaag gccctaataa attagggcct 9900
tttcttatgg ttttgtaaaa tcatacttta tagaaattac gataccattc tacaaagttc 9960
tttaccccct ctttaactga cgtttcaggt ttgaatccta ttacgtcata tagtgcttta 10020
gtatcagcac tggtttccag tacatcaccg ggttggagag gcatcatatt tttgttggct 10080
tcaataccca gagcctcttc taaagcattg atatagtcca tcaactccac aggcgaacta 10140
ttaccaatgt tataaatacg atatggtgct gaacttgttg caggcgaccc agtttctaca 10200
gtccactgtg gatctttttc tggaataaca tcctgtaacc gaataatggc ttcggcaata 10260
tcatcaatat aggtaaagtc acgcttcatt ttgccgaagt tgtatacatc aatgctttta 10320
ccttccagca tagctttagt gaatttaaat aatgccatat ccggacgtcc ccatggacca 10380
taaaccgtaa agaaacgcag ccctgtggtc ggtaagccat acaaatgaga ataggtatgg 10440
gccatgagtt cattcgcttt tttagttgct gcataaagcg aaacaggatg atctacagag 10500
tcatctgtag agaaaggcat cttgcggttc ataccataaa cagaactgga ggaagcgtag 10560
agtagatgct gaacattatt atggcggcat ccttccagta cgttcaggaa tccaatcagg 10620
tttgcatctg catatgcatt gggattttca agggagtaac gtacaccggc ttgcgcagcg 10680
aggtttatta cgcgatcgaa ccgctcgtct gcaaacagtg tcgccatttt ctcacgatcg 10740
gccaggtcaa ttttataaaa actgaaattg tcgtgcttga gtaaatcaag tcgtgcttgt 10800
ttgaggttga catcgtaata atcatttaag ttgtcaatgc ctacaacctg atgaccagct 10860
gcaagaagcc gtttacttac atagaaaccg ataaagccag cagctcccgt aaccagaaat 10920
ttcatttata atcctcgctc aggctagaat atagccaatt ttcatctggc ataactgaaa 10980
gttaaatcat accgttagac aagaaaaaaa gataatcggt atcagttcta aacctggctg 11040
ttttttctgg taacgtgctc attttacaat caaagctgtt ctaagctgac tatacaagcc 11100
gacgtcatta tttccaaccg tatggcagaa gagcttaagg atgtggcaga taaggtctac 11160
acccgcgatc tttttggcag cgactaacat cctgttatca gggcgatttt cgccctgatt 11220
ctcttatgtt ccctttgtag taattcattc tttttatcat ttatcctata gcattcatga 11280
cgattatcgc taaattatgg cgacttaaaa ttatttcgtc cgttagggta atgatgagag 11340
tagaaaataa taatgtttct gggcaaaacc atgacccgga acagattgat ttgattgatt 11400
tactggtgca gttgtggcgc ggcaagatga caattatcat ctctgtcatt gtcgctattg 11460
ccctggctat agggtatctg gccgttgcca aagaaaagtg gacttccaca gctatcgtta 11520
ctcaacctga tgtggggcaa atcgctggtt ataccaacgc gatgaatgtg atttacggtc 11580
cggctgtacc gaaagtctcc gacattcaga cttcacttat cggacgttac agcactgcat 11640
tctcagcatt agcggaaacg ttggataatc aggaagaacc tgaaaaactg accattgagc 11700
ctactgttaa aaatcagtcc ttacctttag cggtgtctta tgttggtgaa acagctgaag 11760
gtgcgcagaa acagctggcg caatatatcc agcaagttga cgaccaggta aacgaagaac 11820
tggaaaaaga cctgaaagac aacatcgcgc tgcgtatgaa aaacttgcag gattcgctga 11880
aaacccagga agtagtcgcg caggagcaga aagatctgcg tatccgtcag attcaggaag 11940
cgttgcagta tgcgaatcag gcgcaggtga caaagccaca ggttcagcag actgaagatg 12000
tgacacaaga tacgttgttc cttttgggga gcgaagcgct ggagtcgatg attaagcatg 12060
aggcgacccg tccgttggtg ttctcatcaa actactatca gactcgtcaa aacctgctgg 12120
atatcgacaa tcttgacgtt gataaacttg atattcatgc ttaccgctat gtaatgaaac 12180
cgacgttacc tattcgtcgc gatagcccga aaaaggcaat taccttgatt ctggcggtgc 12240
tgctgggcgg catggttggc gcggggattg tgctggggcg taacgctctg cgtaattaca 12300
acgcgaagta atcttttcgg ttttaaagaa aaagggcagg gtggtgacac cttgcccgtt 12360
ttttttgccg gatgcgacaa caatatcgca tccgcttacc ccgcaactca ctgatgccgt 12420
ttacgcaggt tctcaattac cgtagttaaa tccagatcct gatcttgcaa cagcaccagc 12480
aggtgataca tcaaatcaga cgcctcgttg gtcagctcaa agcggtcatg tacagtcgcg 12540
gccagtgcgg tttccacgcc ttcttcgccc actttctgcg caatgcgttt ggtgccgctg 12600
gcatacagtt tggcggtgta ggaggtttcc gggtcggcag atttgcgttc ggcgagcagt 12660
tgttccagtt gatacaggaa cagccactgg tgagcggtgt cgccgaagca gctgctggtg 12720
cctttgtggc aggttggtcc gataggattc gccag 12755
Glycosyltransferase gene in the O antigen gene of table 1 intestinal bacteria O155 type bunch and oligosaccharide unit treatment gene and wherein Primer and PCR data
Gene Function The base position of gene Forward primer Reverse primer The length of PCR product Produce the group number of correct big or small electrophoresis band The annealing temperature of PCR (℃)
Wzx O-antigen transhipment enzyme 6827-8113 7226-7243 8053-8072 847bp 0 * 56
7131-7148 7862-7881 751bp 0 * 55
Wzy O-antigen polysaccharase 2232-3185 2295-2312 2776-2794 500bp 0 * 56
2234-2251 2864-2881 648bp 0 * 54
*Only in intestinal bacteria O155 type, obtain a correct band
Table 2166 strain intestinal bacteria and 43 strain Shigellaes and their source
Group number The bacterial strain that contains in this group The source
1, wild-type e. coli 2, wild-type e. coli 3, wild-type e. coli 4, wild-type e. coli 5, wild-type e. coli 6, wild-type e. coli 7, wild-type e. coli O1，O2，O5，O7，O12，O13，O14，O15，O16，O17，O19ab，O20， O21，O22，O23，O24，O59，O3，O11 O25，O26，O27，O28，O29，O30，O32，O31，O33，O35，O36，O37， O38，O40，O41，O42，O43，O39，O59 O44，O45，O46，O48，O49，O50，O51，O52，O54，O55，O56， O57，O58，O60，O61，O62，O64，O73 O63，O65，O66，O69，O70，O71，O74，O75，O76，O77，O78， O79，O80，O81，O82，O83，O96，O95 O84，O85，O86，O87，O88，O89，O91，O92，O98，O99，O101， O102，O103，O104，O105，O106，O100，O151 O107，O108，O109，O110，O111，O112ab，O112ac，O113， O115，O116，O118，O120，O123，O125，O126，O128 O129，O130，O131，O132，O133，O134，O135，O136，O137， O138，O139，O140，O141，O142，O143，O144，O145 IMVS a IMVS a IMVS a IMVS a IMVS a IMVS a IMVS a
8, wild-type e. coli 9, wild-type e. coli 10, wild-type e. coli 11, wild-type e. coli 12, the 8th group of bacterial strain adds the intestinal bacteria reference culture O146, O147, O148, O150, O152, O154, O156, O157, O158, O159, O160, O161, O163, O164, O165, O166 O168, O169, O170, O171, O172, O173, O155, O124 D1, D2, D3, D4, D5, D6, D7, D8, D9, D10, D11, D12 B1, B2, B3, B4, B6, B7, B8, B9, B10, B11, B12, B13, B14, B15, B16, B17, B18 F1a, F1b, F2a, F2b, F3, F4b, F5 (v:4), F5 (v:7), F6, FX becomes, FY becomes, DS, DR, O155 IMVS a b c d d d
a.Institute of Medical and Veterinary Science，Anelaide，Australia(IMVS)
b.O123 from IMVS；the rest from Statens Serum Institut，Copenhagen，Denmark
c.O172 and O173 from Statens Serum Institut，Copenhagen，Denmark，the rest from IMVS
D. China Preventive Medicial Science Institute's epidemiological study institute
Table 3 intestinal bacteria O155 type O antigen gene structure iron
Figure C20041001904200231
Table 4 intestinal bacteria O155 type O antigen gene cluster gene position
ATTGTGGCTG CAGGGATCAA AGAAATCCTC CTGGTAACTC ATGCGTCCAA GAACGCGGTC 60
GAAAACCACT TCGACACCTC TTATGAATTA GAATCTCTCC TTGAACAGCG CGTGAAGCGT 120
CAACTACTGG CGGAAGTGCA ATCTATCTGC CCGCCGGGCG TGACTATTAT GAACGTGCGT 180
CAGGGCGAAC CTTTAGGTTT AGGTCACTCC ATTTTGTGTG CACGACCCGC CATTGGTGAC 240
AATCCATTTG TCGTGGTGCT GCCAGACGTT GTGATCGACG ACGCCAGCGC CGACCCGCTA 300
CGCTACAACC TTGCTGCCAT GATTGCGCGC TTCAATGAAA CGGGCCGTAG CCAGGTGCTG 360
GCAAAACGTA TGCCGGGTGA CCTCTCTGAA TACTCCGTCA TCCAGACCAA AGAACCGCTG 420
GACCGTGAAG GCAAAGTCAG CCGCATTGTT GAATTTATCG AAAAACCGGA TCAGCCGCAG 480
ACGTTGGACT CAGACATCAT GGCCGTAGGT CGTTATGTGC TTTCTGCTGA TATTTGGCCG 540
GAACTTGAAC GCACGCAGCC AGGGGCATGG GGACGTATTC AGCTGACAGA TGCCATCGCT 600
GAACTGGCGA AAAAACAATC TGTTGATGCA ATGCTGATGA CTGGTGACAG CTACGACTGC 660
GGTAAAAAAA TGGGCTATAT GCAGGCGTTT GTGAAGTATG GGCTACGTAA CCTGAAAGAA 720
GGGGCGAAGT TCCGCAAAGG TATTGAGAAG CTGTTAAGCG AATAATGAAA ATCTGACCGA 780
ATGTTACGGT TGATAAGAAA ATTATAACGG CAGTGAAGAT TCGTGGCGAG AGTAATTTGT 840
TGCGAATATT CCTGCCGTTA TTTTATATAA ACAATCAGAA TAACAACGAG TTAGCAATAG 900
GATTTTAGTC AAAGTTTTCC AGGATTTTCC TTGTTTCCAG AGCGGATTGG TAAGACAATT 960
AGCGTTTGAA TTTTTCGGGT TTAGCGCGAG TGGGCAACGC TCGTCACATC GTAGACATGC 1020
ATGCAGTGCT CTGGTAGCTG TAAAGCCAGG GGCGGTAGCG TGAAAATTAA GGATGCTAAT 1080
AAATAATTGG TTGTATGTAT ACCACAACTT ATTAACTTAT TTATGTGTAA GTCTAAATAG 1140
TGTGGTAATT CTTATCAGTT GGAAGATTTT TACACATATA AACTGAAAAC ATCACAATAT 1200
Orf2 is initial
AGTAAAGTTA AA GTGGGCTT TATTTTATTG TCATTTTTGG TTGCAACAGC ATATTATGCT 1260
ACCTTTAAAA GAGGAAACGA CATGGGGTTA AATATAGAAT TAATAAGTGT TGTGATTACT 1320
TTCTTTAGTA ATGATGAATC TTCGGGTACA TATTTAAATA ATAGTGTTTT AAGCGCATTA 1380
AACCAAACAT ATAGAAACAT TGAAGTCTTG GTCATTGATG ATTGCTCCCC AATAAAAGCA 1440
ACTAGCTGTC TTAATGGTAT TAATGATAGT AGATTGTCAA TTATAAGACT TCCACAAAAT 1500
GGTGGAATGG CCGCAGCTAT TAATCATGGA ATTGGTCTGT CATCTGGAAG TTTTATTGCA 1560
TTTCTTGATT TTGATGACCT TTGGTACGAT TTCAAACTTA AAGAGCAAGT TGAGTTGATC 1620
AAAGATTATA TTGATAAAAA GCTGGCTGAA CCTGAAAAAA TATGTTGCTA TTCTAAGGTT 1680
TTAATAAAAC ATGATAATTA CGAATATACT AAACCTTACA GAAGTATAGA GGAGGGTGAG 1740
CACGTAGCAG ATTATTTGTT TGCTAATGGA CAATTTATGC AAACAAGTGG GATGCTTATA 1800
TCAAAATCAT TACTTATAAA AGTCAAAGGA ATGGATTCTT ATAAAAGACA TACGGATTAT 1860
CAGTTAGTAT TAAAACTATA TCAAGAAAAT GCTGTGTTTA TTTATTATGA TCGAGTTTCA 1920
TATATGTATA AAATAACTGA CAAAAAAGGA GATTACAAAT TTTCGCAAGA TTGGCTTTGT 1980
GCAAATAAGT GGATGATGAC AAAACGTTCT ATAAGATATT TCAAAATTAA TACAATATTA 2040
AGAAGCATGA TTGATAATCA TGCTTTTGGT ACTGCGTTTA TATATTCAGT ATCTAACAGA 2100
TTTATATGGT CTTTTGGCTC GTATTTAATA CGATTTTCTT TAAAAAAATT ATTACCTACA 2160
CGCTTATTAT CAACGCTGAT TAAACATAAA AATAAAGCAG CGTTTTCTGT AAAAGGAAGA 2220
It is initial that orf2 stops wzy
GAGTAT TGAA A ATGGGAGGC TTTATTACAA GATTAGTTTT ATTGCTGCCA TTATTTTTAT2280
TATTTTATTT TCCTGCGGAC CATAACAAAG ATTATTTTAA TTATCAAACA GATTATGACT 2340
ATGCCATAGG GCAATTTGAT TACCTATATG AGCTATCCGT GAGATTTTTT AGAGATATAT 2400
TGAACTATAG TTTTAGTTCA TTTTGGGTGA TTGTGATTAT AGTAGAAATT ATTTTCTTGA 2460
CGATATATTA TAACAAGGTG AAATATATCC TTATTGCATA CCCGGGTATA ATTTCGATGA 2520
GCCAGTTTTT TTATGGCACT CAAATACGTT ATGCTTTGGC AATGTTAATT GGATTAGTAG 2580
TATTAAAAAA TAGTAGACGC TTTATTAAAG TTTTTGGAGT GTTAATTGCT TCATTATTCC 2640
ACTATGGCGC CGTGATATCA TTATTACCAT TATTTATTTG TAGGTATATA AATGAAAGAT 2700
ATTTCATTTT TTTGTCTAAA CGAAATATAT TCTTGATTAT ATCTGTATTA ACATTACTGT 2760
TTTTTTTCTT CTATTCTTTC GAGTCATTTG TTTCAATGAC TCGTTTTTCT TATTATTTAG 2820
GTGCTGAAAA ATTTTCCGAT TCAAAATCGA CTTCATCTAT AATATATGCG GTTGTGAGTC 2880
TATTGTTTAT TCTGTTTTTA TATTCAAAAA ATACAGCGTT CAGAACCAAT ATAAGTAAAC 2940
AATCTATAGT AATACTGATT TTAGTTTTGA TTTTTTCACC TATAGCAGTG CTATCAGGTC 3000
GCACGATGCT CGTTTATTTT ATAATGGAAC CTTTGTTAAT AGCATCTGCA TTCACCTCTA 3060
TAAAGAAAAA TAGTCCTGAT TATTTTATAT CTTTATCGTT ATCTTTGTCT TTATTTTTAT 3120
TCTATATTGC GAGATGTATT TATTATATCG TAACGGCAGA TTTTTATTTT TATGATTTCT 3180
It is initial that wzy stops orf4
TA TAAGGAGA ACAA ATGAGG ATAGCTCATA TATATAAAAA ACATTATGAT AGCGGAGATG3240
GTATCACTAA TGTTGTTGAG AATTTATATA AACTTCAAAA AAACAATAAT GCAGATGAAT 3300
ATATAGATTT ACCATTCATG TTTGATAATT GCTCAAAAGG TAAGGCTCAA TATAGATATT 3360
CAGGGTTTAT TGGGATGCTA TATCATTTGT TCAAATGCAA GCCAAATGTA ATATACTTGC 3420
ATGGTTTCTA CTATTTGCAT TATATTATTC TTGGTGTTTG TTTTTATTTT ATCAAATCAA 3480
AAGTTGTTTT AATACCACAT TGCTCTCTTT TAAATAGAGC AATTACTTAT AAAAAAATAA 3540
GAAAGTTGAT ATATTATAAA ATATTCTCAT TAATTTATTA TGATTCTATA ATATTGATTC 3600
AGTATTTGAA CCACGAAGAG CAAATAGGTT CAAAAAAATT TTTATTCTCA CCTCCTGAAT 3660
CCATTATACC AAATGGTGTT AACTATACTG AAAAACAATG TCGATCTTTT GATTTTCTAT 3720
CTCTATTCTA TTTAGGGCGA TGTGATATAA ATCATAAAGG TATTGACATA CTATTTGAGT 3780
ATCTTACTAA GATTAATCAA AAAATAAAAT TATATGGTGC AGATCCAAAT GAAAAAATAA 3840
AACTAAAGAA ATTAATTCAA TTAAAGAAAT TAGAGCAAAA AGTTGAAATA TATGATCCTG 3900
TCTTTAATGA GGATAAGGAG AGGGTATTCT TGAATAATAA TATATTTATA TTACTTTCGA 3960
GATATGAAGG ATTACCTATT AGTGTGCTTG AAGCACTTTC TTATGGATGT ATTTGTCTAG 4020
TTTCATCTGG AACAAATATG GCTGATGAAA TTGTAGCTGC GAACTGCGGA TTTAAAATTG 4080
ATTCTGTAGA GGATCTTATT TCAGTATGGA ATAAGCTACG GGTAATGAGT ATATATGAAT 4140
TAAAAGAAAT GTCTGAAAAT TCAAAAGCAT TAATTAAACA TAAATACGCT TGGCAAAAAA 4200
TAATCATATC TTCTGAGATT GAATTAAGAA AATATGTAAC ATAAGTCTTT GTGCGTAGAT 4260
TTTGAATGTT CTATTTATTC TGGTTAGAAT TTTATAGTCA ATTCGATGAC ATATATTCAC 4320
Orf5 is initial
ACAATATTTC ATACAGAAGA TTAATTAGGA ACTTAATGTG AAATTTACTG TTTTACTTTC 4380
CTTATATAAT AGAGAGTCTA AACACAACTT GTTCGATTGT CTTGAAAGTA TTAAGTCAAA 4440
CACTATAAAG CCAGAACAGA TAGTCATAGT TTATGATGGA CCGATCAGTG AAGAACTGGA 4500
TAGTATTGTT ACTGGTTTTA TCAAATACTT ACCGATTGAG AAAATAAAAA TATCAGAAAA 4560
TATTGGACTA GGCAACGCAC TTAATTATGG CTTGAATTTT TGTAGACATG AAATTGTTTT 4620
CAGGATGGAT ACAGACGATA AATGTAAAAG CACAAGATTT GAACGGCAAC TTGAAATTAT 4680
AAAAATAAAA AATGATATCG TGTTGATGGG GAGTGCTATA GAAGAATTTA ACGAATCGAT 4740
GACAGTCTCT CATGGTGTAA GATTTTCAGC CGAATATCAT GATGATATAA TAAATTATGC 4800
AAAAAGAAGA AACCCATTTA ATCATATGTC AGTGGTTTTT AAAAAATCAA TAATACAGGG 4860
GCTAGGAGGG TATCAGCATC ATTATTTTAT GGAAGATTAT AACTTGTGGC TTCGGGTTAT 4920
CGCTGCTGGG TATAAAACTT ATAATATTCC AGAAGTGCTT GTCTATGTTC GAGGTGGAGA 4980
TAAAATGCTT GGGCGTAGAA AAGGAATTCC CTATGTGATT AGTGAGGTAA AATTGGCTAA 5040
ATTAAAATAT AACTTAAAAA TTGACAGTAA TGCAGGTGTT ATTATTTCTG CAACCATGAG 5100
GATTTTACCT CGTTTATTGC CCGTCTTTTT GTTAAAACAA ATATATAAAG TTTTAAGGAA 5160
Orf5 stops
AAGCAAGTCT TGAATTCTCT GTGAAATTTA TGAATACAAT CCACATTGAT TACATATACT 5220
GAACATTATC CGTTTGTTAT AATACCGCAC AATTCTAAGT CAATTCCCCT GACAGGAGTA 5280
Gnd is initial
AACAATGTCA AAGCAACAGA TCGGCGTCGT CGGTATGGCA GTGATGGGGC GCAACCTTGC 5340
GCTTAATATC GAAAGCCGTG GTTATACCGT CTCTATTTTC AACCGTTCTC GTGAAAAGAC 5400
GGAAGAAGTG ATTGCCGAAA ATCCAGAGAA GAAACTGGTT CCTTACTATA CGGTAAAAGA 5460
GTTTGTTGAA TCTCTGGAAA CACCTCGTCG CATCCTGTTA ATGGTGAAAG CAGGTGCAGG 5520
CACGGATGCT GCTATTGATT CCCTCAAGCC ATATCTCGAT AAAGGCGACA TCATCATTGA 5580
TGGTGGTAAT ACCTTCTTCC AGGACACCAT TCGTCGTAAC CGTGAGCTTT CTGCGGAAGG 5640
CTTTAACTTC ATTGGTACCG GTGTTTCCGG TGGTGAAGAG GGTGCGCTGA AAGGTCCTTC 5700
CATTATGCCG GGTGGTCAGA AAGAAGCCTA TGAGCTGGTT GCTCCGATCC TGACCAAAAT 5760
CGCTGCTGTG GCTGAAGACG GTGAGCCATG CGTTACCTAT ATCGGTGCTG ATGGCGCAGG 5820
TCACTATGTG AAGATGGTTC ACAACGGTAT TGAATACGGC GATATGCAGT TGATTGCTGA 5880
ATCCTATTCT CTGTTGAAAG GTGGCCTAAA CCTCTCCAAC GAAGAACTGG CGCAGACCTT 5940
TACCGAGTGG AATAACGGTG AACTGAGCAG CTACCTGATC GACATCACCA AAGACATCTT 6000
CACTAAAAAA GATGAAGACG GTAATTACCT GGTTGATGTA ATTCTGGATG AAGCGGCTAA 6060
CAAAGGTACC GGTAAATGGA CCAGCCAGAG TGCGCTGGAT CTCGGCGAAC CGCTGTCGCT 6120
GATCACCGAG TCTGTGTTTG CACGTTATAT CTCTTCTTTG AAAGAGCAGC GTGTTGCCGC 6180
GTCTAAAGTT CTCTCTGGTC CACAAGCACA GCCAGCAGGC GATAAGGCTG AGTTTATCGA 6240
GAAAGTTCGT CGTGCGCTGT ATCTGGGCAA AATCGTTTCT TACGCTCAGG GCTTCTCTCA 6300
GCTGCGTGCT GCGTCTGAAG AGTACAACTG GGATCTGAAT TACGGCGAAA TCGCGAAGAT 6360
TTTCCGTGCT GGCTGCATCA TCCGTGCGCA GTTCCTGCAG AAAATCACCG ATGCATATGC 6420
CGAAAATCCG CAGATCGCTA ACCTGCTGCT GGCTCCGTAC TTTAAACAAA TTGCCGATGA 6480
TTACCAGCAG GCGCTGCGCG ATGTCGTTGC TTATGCAGTA CAAAACGGTA TCCCGATTCC 6540
GACCTTTTCC GCTGCGGTTA CCTATTACGA CAGCTACCGT GCGGCTGTTC TGCCTGCAAA 6600
CCTGATCCAG GCACAGCGTG ACTATTTCGG TGCGCATACT TATAAGCGCA TTGATAAAGA 6660
Gnd stops
AGGTGTGTTC CATACCGAAT GGCTGGATTA ATTTTTTTCT AGTTTTTAAA GAGCCTGCTT 6720
ACGCGGCTCT TTAATATATA TAAAAATTAT AATTCAGTCG ATTCAATCTA GATAAAACTA 6780
Wzx is initial
TTATTTTTAT TATTTCTTAG TTCTAAGAGT CTACGTGAGA TATTAGATGT CTAACTATAT 6840
CAAAAATGCT GGTTGGATTT TTTGCGAAAA ATTTGTACGA ATAGCCGTAG GCTTTATATT 6900
ATTTGCATTA ATTTCAAGAG TTTTGAATCC TGGTGAATTT GGCTTGCTAT CTTATTATCA 6960
AACAATAGCA ACCATGTTTT TAGCAATAAC CAATTTAGGT TTCGATAATG TTCTGATTAA 7020
CGAGTTTAAT CAGAATAAAA AGCATAATGA AATATTTTCA ACGGCATTTT GGTCAAGAAT 7080
TATAGTTTCA GTATTTGTAA TAATGTTATT TGCTGTGGGA TTGTATTTTG ATGATGTCGT 7140
GCTGAAGAAT AAATTGGTTT TGCTGTTGTG TATAAGTAGC TTGGTTTTTC AAAGTCAAAA 7200
TACATACATA CCATTTTTTC AATCTCAGTT ACAAGCCTCA GTTGTCACGA AAATTAGTTT 7260
TTGCGCACTT ATTATATCCT CGGTATTTAA AGTCTATTTG TTGTTAATCA ATGGGGATAT 7320
AATGTGGTTT GCTTTTTCAT ATAGTTTTGA TTTTGCTGTG TCTTTTTTAT TTTTCGCTTT 7380
TTTTTCATAT AAAAGAAATT ATATATCAAT TAATCCAAAA CATTTCCGGT TAAGTGTGCT 7440
AAAACGTTTA TTACGCTCGT CTTGGCCAAT TATTGCATCA TCAATAATTA TTGTTCTCTA 7500
TACTCGATTA GATCAATTAA TGATTATGCG AATGTTAGGG GCTGATTCAG TTGCAATATT 7560
TAGTGTTGCT TTGAAAATAT CAGAGGCATA TTTATTTGTC CCTGCAGCAT TAGTTACATC 7620
ATATTATCCA CTGATTTCAA AGTCACCTAC CAGAGAAAAT ATTAGATTTT ATTTTGATGT 7680
TGTTTTTTCA AGCTCTGTAA TAATGGCTGT AGTGGTAGCT ATAGCGTCTT ATTTTGTAAT 7740
ACCATATATG TTTGGGGCTA ATTATATCAC CTCATATAAT ATATTAATTA TTTTGCTATT 7800
TGGTTCAATA TTTTCTATAC TCGGCTCAGC GTGTACAAAT TTAATGATAG TGTATGACTT 7860
ATCTTATTTA CGGTTAGTTC GAGCCGTTTA TGGGCTATTA ATAAATTTCT CCCTGAATAT 7920
TTTACTTATT CCTAAATATG GTATTATTGG TGCTGCTTAT GCGTCTTTTG TAAGTCAGAT 7980
CTTTGCAAGT TGGTTAAGCA ATAGCCTTAA TAAAAAAACG ATTGAATGCT TCAGAATACA 8040
GTCAATGACA GTTGTATCTT TGGGACTAAT AGGATTTAGA CAATTATTTA TCATGCTATT 8100
It is initial that wzx stops orf8
TAGAAGGGGG TAGGCATATT ATATGTTTGA CAGTTATGAA AGAAAGAAAG GTATAATGCA 8160
AAGGATAATC TTTTATTTTA AAACGAAATT GAGAGGATGT CGTGAATACT ATTACAGTCT 8220
TAAATATTAT CATCAGTTTA AAATTCCAAT CTCGAATCAT CTTACCATCG GCATTAAGAC 8280
ATTAGGGGAA AACAAAACTA CTTTACCTCA CCCGGTAGGT ATAGTTATAG GTAAGAACGT 8340
TAACATCGGT AAAAAATGTA TTGTTTATCA AAATGTCACT ATAGGGGTGA GTAATAATAA 8400
ATTTGAGGAT TATCCTGTTA TTGGAAATAA TGTTACTATT TATGCCGGAG CAATCATAAT 8460
AGGTAAAGTA ATTATAGGGG ATAATGCAAT AATTGGTGCA GGTTGCATCG TTACTAAAGA 8520
TGTACCTGAA GGAAAGATTG CAATTGGTTC ACCAATGAAA ATTATGGATA AAAAACATAA 8580
Orf8 stops
TAACTACTAA TTATCTCAGT GAATTCTTAA TAATTTTAAA ATGTGTTGAC TGTAATGTTC 8640
GTTTTTTGCT ATTAATGTGA AAAAACTTCA TTAAAAAAGA ATGCAAATAT GAAAATAACA 8700
ATTTCAGGAA CAGGTTATGT TGGTCTTTCA AATGGTATTC TGATTGCGCA AAATCACGAA 8760
GTGGTTGCAC TGGATATCGT TCAGGCCAAA GTGGACATGC TTAACAAGAG GCAGTCACCG 8820
ATTGTTGATA AGGAGATTGA AGAGTATCTG GCGACTAAAG ATCTCAATTT CCGCGCTACG 8880
ACAGATAAGT ATGAGGCGTA TAAAAATGCC GATTACGTTA TTATTGCCAC ACCTACCGAT 8940
TATGATCCGA AAACAAATTA TTTTAATACC TCAAGTGTGG AAGCGGTCAT TCGTGATGTG 9000
ACAGAAATTA ATCCCAACGC GGTAATGATT ATAAAATCAA CTATCCCTGT TGGTTTTACA 9060
GAGTCCATTA AAGAACGGTT TGGTATTGAA AATGTGATCT TTTCGCCGGA GTTTTTACGC 9120
GAAGGGAAAG CACTTTATGA TAACTTGCAT CCATCACGCA TTGTGATTGG TGAGCAATCC 9180
GATCGCGCTG AACGTTTTGC AGCATTATTA CAAGAAGGAG CTATTAAGCA AGATATTCCA 9240
ACGTTGTTTA CTGACTCAAC GGAAGCTGAG GCGATTAAAC TTTTTGCCAA CACCTATCTG 9300
GCGATGCGTG TAGCGTATTT CAATGAACTT GATAGTTATG CTGAAAGCTT GGGGCTTAAT 9360
TCACGCCAGA TTATTGAGGG CGTATGCCTT GATCCGCGTA TAGGTAATCA CTACAACAAT 9420
CCGTCATTCG GTTATGGTGG TTATTGTCTG CCAAAAGATA CTAAGCAGTT ACTGGCAAAC 9480
TATCAGTCAG TACCGAATAA CTTGATCTCA GCAATTGTCG ATGCCAATCG CACGCGAAAA 9540
GATTTTATTG CCGATTCTAT CCTTGCACGT AAACCGAAAG TTGTTGGCGT CTATCGTCTG 9600
ATTATGAAGA GTGGTTCGGA CAATTTCCGT GCTTCATCGA TTCAGGGTAT TATGAAGCGA 9660
ATCAAGGCGA AAGGTGTGCC AGTAATCGTC TATGAGCCAG CGATGAAAGA GGACGATTTT 9720
TTCCGTTCGC GCGTGGTACG TGATCTGGAT GCGTTCAAAC AAGAAGCTGA TGTTATTATT 9780
TCTAACCGTA TGTCTGCCGA TCTGGCTGAT GTAGCAGATA AGGTTTATAC GCGCGACTTG 9840
TTTGGCAATG ATTAATTATT TTGTTTCATA CTAAGAAAAG GCCCTAATAA ATTAGGGCCT 9900
Gla stops complement
TTTCTTATGG TTTTGTAAAA TCATACTTTA TAGAAATTAC GATACCATTC TACAAAGTTC 9960
TTTACCCCCT CTTTAACTGA CGTTTCAGGT TTGAATCCTA TTACGTCATA TAGTGCTTTA 10020
GTATCAGCAC TGGTTTCCAG TACATCACCG GGTTGGAGAG GCATCATATT TTTGTTGGCT 10080
TCAATACCCA GAGCCTCTTC TAAAGCATTG ATATAGTCCA TCAACTCCAC AGGCGAACTA 10140
TTACCAATGT TATAAATACG ATATGGTGCT GAACTTGTTG CAGGCGACCC AGTTTCTACA 10200
GTCCACTGTG GATCTTTTTC TGGAATAACA TCCTGTAACC GAATAATGGC TTCGGCAATA 10260
TCATCAATAT AGGTAAAGTC ACGCTTCATT TTGCCGAAGT TGTATACATC AATGCTTTTA 10320
CCTTCCAGCA TAGCTTTAGT GAATTTAAAT AATGCCATAT CCGGACGTCC CCATGGACCA 10380
TAAACCGTAA AGAAACGCAG CCCTGTGGTC GGTAAGCCAT ACAAATGAGA ATAGGTATGG 10440
GCCATGAGTT CATTCGCTTT TTTAGTTGCT GCATAAAGCG AAACAGGATG ATCTACAGAG 10500
TCATCTGTAG AGAAAGGCAT CTTGCGGTTC ATACCATAAA CAGAACTGGA GGAAGCGTAG 10560
AGTAGATGCT GAACATTATT ATGGCGGCAT CCTTCCAGTA CGTTCAGGAA TCCAATCAGG 10620
TTTGCATCTG CATATGCATT GGGATTTTCA AGGGAGTAAC GTACACCGGC TTGCGCAGCG 10680
AGGTTTATTA CGCGATCGAA CCGCTCGTCT GCAAACAGTG TCGCCATTTT CTCACGATCG 10740
GCCAGGTCAA TTTTATAAAA ACTGAAATTG TCGTGCTTGA GTAAATCAAG TCGTGCTTGT 10800
TTGAGGTTGA CATCGTAATA ATCATTTAAG TTGTCAATGC CTACAACCTG ATGACCAGCT 10860
GCAAGAAGCC GTTTACTTAC ATAGAAACCG ATAAAGCCAG CAGCTCCCGT AACCAGAAAT 10920
The initial complement of gla
TTCATTTATA ATCCTCGCTC AGGCTAGAAT ATAGCCAATT TTCATCTGGC ATAACTGAAA 10980
GTTAAATCAT ACCGTTAGAC AAGAAAAAAA GATAATCGGT ATCAGTTCTA AACCTGGCTG 11040
TTTTTTCTGG TAACGTGCTC ATTTTACAAT CAAAGCTGTT CTAAGCTGAC TATACAAGCC 11100
GACGTCATTA TTTCCAACCG TATGGCAGAA GAGCTTAAGG ATGTGGCAGA TAAGGTCTAC 11160
ACCCGCGATC TTTTTGGCAG CGACTAACAT CCTGTTATCA GGGCGATTTT CGCCCTGATT 11220
CTCTTATGTT CCCTTTGTAG TAATTCATTC TTTTTATCAT TTATCCTATA GCATTCATGA 11280
Wzz is initial
CGATTATCGC TAAATTATGG CGACTTAAAA TTATTTCGTC CGTTAGGGTA ATGATGAGAG 11340
TAGAAAATAA TAATGTTTCT GGGCAAAACC ATGACCCGGA ACAGATTGAT TTGATTGATT 11400
TACTGGTGCA GTTGTGGCGC GGCAAGATGA CAATTATCAT CTCTGTCATT GTCGCTATTG 11460
CCCTGGCTAT AGGGTATCTG GCCGTTGCCA AAGAAAAGTG GACTTCCACA GCTATCGTTA 11520
CTCAACCTGA TGTGGGGCAA ATCGCTGGTT ATACCAACGC GATGAATGTG ATTTACGGTC 11580
CGGCTGTACC GAAAGTCTCC GACATTCAGA CTTCACTTAT CGGACGTTAC AGCACTGCAT 11640
TCTCAGCATT AGCGGAAACG TTGGATAATC AGGAAGAACC TGAAAAACTG ACCATTGAGC 11700
CTACTGTTAA AAATCAGTCC TTACCTTTAG CGGTGTCTTA TGTTGGTGAA ACAGCTGAAG 11760
GTGCGCAGAA ACAGCTGGCG CAATATATCC AGCAAGTTGA CGACCAGGTA AACGAAGAAC 11820
TGGAAAAAGA CCTGAAAGAC AACATCGCGC TGCGTATGAA AAACTTGCAG GATTCGCTGA 11880
AAACCCAGGA AGTAGTCGCG CAGGAGCAGA AAGATCTGCG TATCCGTCAG ATTCAGGAAG 11940
CGTTGCAGTA TGCGAATCAG GCGCAGGTGA CAAAGCCACA GGTTCAGCAG ACTGAAGATG 12000
TGACACAAGA TACGTTGTTC CTTTTGGGGA GCGAAGCGCT GGAGTCGATG ATTAAGCATG 12060
AGGCGACCCG TCCGTTGGTG TTCTCATCAA ACTACTATCA GACTCGTCAA AACCTGCTGG 12120
ATATCGACAA TCTTGACGTT GATAAACTTG ATATTCATGC TTACCGCTAT GTAATGAAAC 12180
CGACGTTACC TATTCGTCGC GATAGCCCGA AAAAGGCAAT TACCTTGATT CTGGCGGTGC 12240
TGCTGGGCGG CATGGTTGGC GCGGGGATTG TGCTGGGGCG TAACGCTCTG CGTAATTACA 12300
Wzz stops
ACGCGAAGTA ATCTTTTCGG TTTTAAAGAA AAAGGGCAGG GTGGTGACAC CTTGCCCGTT 12360
HisI stops complement
TTTTTTGCCG GATGCGACAA CAATATCGCA TCCGCTTACC CCGCAACTCA CTGATGCCGT 12420
TTACGCAGGT TCTCAATTAC CGTAGTTAAA TCCAGATCCT GATCTTGCAA CAGCACCAGC 12480
AGGTGATACA TCAAATCAGA CGCCTCGTTG GTCAGCTCAA AGCGGTCATG TACAGTCGCG 12540
GCCAGTGCGG TTTCCACGCC TTCTTCGCCC ACTTTCTGCG CAATGCGTTT GGTGCCGCTG 12600
GCATACAGTT TGGCGGTGTA GGAGGTTTCC GGGTCGGCAG ATTTGCGTTC GGCGAGCAGT 12660
TGTTCCAGTT GATACAGGAA CAGCCACTGG TGAGCGGTGT CGCCGAAGCA GCTGCTGGTG 12720
CCTTTGTGGC AGGTTGGTCC GATAGGATTC GCCAG 12755
The above, instrument 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 (4)

1, a kind of oligonucleotide of the O-antigen-specific to intestinal bacteria O155 type, it is characterized in that, its wzx sequence is that the Nucleotide of 6827 to 8113 bases among the SEQ ID NO:1 or wzy sequence are the Nucleotide of 2232 to 3185 bases among the SEQ IDNO:1 or have one or more Nucleotide are inserted, lack or replaced to above-mentioned sequence, but keeps the oligonucleotide of the O-antigen function of wzx, wzy Nucleotide specific detection intestinal bacteria O155.
2, a kind of oligonucleotide of the O-antigen-specific to intestinal bacteria O155 type is characterized in that, it is that to come from the oligonucleotide of wzx gene right: the Nucleotide of 7226 to 7243 bases among the SEQ ID NO:1 and the Nucleotide of 8053 to 8072 bases; The Nucleotide of 7131 to 7148 bases among the SEQ ID NO:1 and the Nucleotide of 7862 to 7881 bases; Or the oligonucleotide that comes from the wzy gene is right: the Nucleotide of 2295 to 2312 bases among the SEQ IDNO:1 and the Nucleotide of 2776 to 2794 bases; The Nucleotide of 2234 to 2251 bases among the SEQ IDNO:1 and the Nucleotide of 2864 to 2881 bases.
3, the application of oligonucleotide in detecting the O-antigen of expressing the antigenic bacterium of O-, identifying bacterium of claim 1, any described O-antigen-specific to intestinal bacteria O155 type of 2.
4, according to the application of the oligonucleotide of claim 1, any described O-antigen-specific to intestinal bacteria O155 type of 2, it is characterized in that, it is used for PCR, is used for hybridization and fluoroscopic examination or is used to make gene chip or microarray as probe as primer, and the confession detection bodies reaches the bacterium in the environment outward.
CN 200410019042 2004-04-19 2004-04-19 Nucleotide peculiar to 0-antigen of 0155 type bacillus coli Expired - Fee Related CN1257277C (en)

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CN1257277C true CN1257277C (en) 2006-05-24

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