CN1261574C - Nucleotide peculiar to 0-antigen of 29 type bacillus coli - Google Patents
Nucleotide peculiar to 0-antigen of 29 type bacillus coli Download PDFInfo
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- CN1261574C CN1261574C CN 200410019023 CN200410019023A CN1261574C CN 1261574 C CN1261574 C CN 1261574C CN 200410019023 CN200410019023 CN 200410019023 CN 200410019023 A CN200410019023 A CN 200410019023A CN 1261574 C CN1261574 C CN 1261574C
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Abstract
The present invention provides a specific nucleotide for an O-antigen of Escherichia coli O29. The specific nucleotide is the nucleotide complete sequence of gene clusters in Escherichia coli O29 for controlling the synthesis of an O-antigen, such as a separated nucleotide with the total length of 14250 basic groups shown as SEQ ID NO: 1 or the nucleotide of SEQ ID NO: 1 which has one or a plurality of inserted, deleted or substituted basic groups and can simultaneously keep the functions of the separated nucleotide. The specific nucleotide also comprises the oligonucleotides of glycosyl transferase genes and oligosaccharide unit processing genes in the O-antigen gene clusters of Escherichia coli O29. The present invention verifies that the oligonucleotide has high specificity to all of the O-antigen genes of Escherichia coli O29 through PCR. The present invention also discloses a method for detecting and identifying Escherichia coli O29 by the oligonucleotide of the present invention.
Description
Technical field
The present invention relates to the complete nucleotide sequence of control O-antigen synthetic gene cluster in intestinal bacteria 29 types (Escherichia coli O29), particularly relate in intestinal bacteria 29 types oligonucleotide in the control O-antigen synthetic gene cluster, can utilize these oligonucleotide of O-antigen-specific intestinal bacteria 29 types 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. (1996) " 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, l1:6923-6930 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 sanecolitose-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 tool O-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 identificaion ofS.enterica maior serogroups (A; B; C2; andD) ", J.Clin.Microbiol.31:2118-2123].Luk, the method for et.al is with corresponding to Salmonellas serotype E 1, D1 obtains the oligonucleotide special to the Salmonellas of different serotypes after the nucleotide sequence of the CDP-abequose in the A, the O-antigen of B and C2 and the synthetic gene of CDP-tyvelose is arranged.1996, Paton, the A.W et.al serotype [" Molecular microbiological investigation of an outbreak of Hemolytic-Uremic Syndrome caused by dry fermented sausage contaminated with Shiga-liketoxin producing Escherichia coli " .J.Clin.Microbiol.34:1622-1627] of the oligonucleotide that comes from the wbdI gene of the O-antigen-specific of E.coli O111 having been identified the toxogenic E.coli O111 of a strain, but afterwards studies show that Paton, the usefulness of A.W et.al comes from the oligonucleotide of wbdI gene and identifies that the method for the serotype of E.coli O111 has false positive results to occur.Bastin D.A.and Reeves, P.R. think, this is because the wbdI gene is sugared synthesis path gene [the Bastin D.A.andReeves of a supposition, P.R. (1995) Sequence and analysis of the O antigen gene (rfb) cluster 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 of theEnterobacteriaceae " .Elsevier Science Publishers, Amsterdam, TheNetherlands; T.cheasty, et al. (1983) " Antigenic relationships between theenteroinvasive Escherichia coli antigensO28ac; O112ac; O124; O136, O143, O144; O152 and 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 29 types.It is the Nucleotide in the O-antigen gene bunch of intestinal bacteria 29 types, is the special Nucleotide that comes from glycosyltransferase gene and transhipment enzyme gene and pol gene.
A time purpose of the present invention has provided the full length nucleotide sequence of the O-antigen gene bunch of intestinal bacteria 29 types.
Another object of the present invention has provided the gene of the O-antigen gene bunch that constitutes intestinal bacteria 29 types: the gene of transhipment enzyme is the wzx gene or with wzx the gene of identity function is arranged; Pol gene is the wzy gene or with wzy the gene of identity function is arranged.
Another purpose of the present invention has provided oligonucleotide, and the gene that they come from encoding glycosyl transferring enzyme in the O-antigen gene bunch of intestinal bacteria 29 types respectively comprises orf8, orf11, orf12 gene; The gene that coming from coding transhipment enzyme 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 29 types; Especially the oligonucleotide of listing in the table 1, they are high specials to the O-antigen of intestinal bacteria 29 types, and these oligonucleotide are also reconfigurable, the oligonucleotide after the combination also is a high special to the O-antigen of intestinal bacteria 29 types.
The above-mentioned oligonucleotide that a further object of the present invention provides can be used as primer and is used for nucleic acid amplification reaction, perhaps be used for hybridization as probe, or be used to make gene chip or microarray, thereby pass through O-antigen and the detection and identification of escherichia coli 29 types of these methods detections and identification of escherichia coli 29 types.
An also purpose of the present invention has provided the method for complete sequence of the O-antigen gene bunch of separating Escherichia coli 29 types.Can obtain the complete sequence of the O-antigen gene bunch of other bacteriums according to present method operation, the complete sequence of the gene cluster of the bacterium of other polysaccharide antigens that also can obtain to encode.
The objective of the invention is to realize by following technical scheme.
The present invention is characterized in that the Nucleotide of the O-antigen-specific of intestinal bacteria 29 types: it is the isolating Nucleotide shown in SEQID NO:1,14250 bases of total length; Perhaps described base with 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 29 types, comprising called after orf1, orf2, orf3, orf4, wzx, wzy, orf7, fnlA, fnlB, fnlC, wbuB, 12 genomic constitutions of wbuC are all between galF gene and gnd gene.
The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria 29 types, the gene that has high degree of specificity in the wherein said gene is: transhipment enzyme gene, it comprises the wzx gene; Pol gene, it comprises the wzy gene; Glycosyltransferase gene, it comprises orf2, orf3, orf7, the orfl1 gene; Wherein said gene: wzx is the Nucleotide of 4778 to 6178 bases among the SEQ ID NO:1; Wzy is the Nucleotide of 6175 to 7260 bases among the SEQ ID NO:1.
The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria 29 types wherein also comprises coming from described wzx gene, wzy gene or glycosyltransferase gene orf2, orf3, orf7, orf11 gene and their mixing or their reorganization.
The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria 29 types is characterized in that the oligonucleotide that comes from the wzx gene is to being: the Nucleotide of 4891 to 4900 bases among the SEQ ID NO:1 and the Nucleotide of 5571 to 5589 bases; The Nucleotide of 4858 to 4876 bases among the SEQ IDNO:1 and the Nucleotide of 5551 to 5569 bases; The oligonucleotide that comes from the wzy gene is to being: the Nucleotide of 6575 to 6593 bases among the SEQ ID NO:1 and the Nucleotide of 7201 to 7221 bases; The Nucleotide of 6455 to 6473 bases among the SEQ ID NO:1 and the Nucleotide of 7060 to 7078 bases.
The application of the Nucleotide of aforesaid O-antigen-specific to intestinal bacteria 29 types 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 29 types is providing the O-antigen of expressing intestinal bacteria 29 types 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 29 types 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 29 types is characterized in that it comprises the steps:
(1) genomic extraction: in substratum, cultivate intestinal bacteria 29 types, centrifugal collecting cell; The genomic dna that obtains detects by agarose gel electrophoresis;
(2) by the O-antigen gene in pcr amplification intestinal bacteria 29 types bunch: with the genome of intestinal bacteria 29 types 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 29 types;
(6) screening of specific gene: at wzx, wzy, the gene design primer in the O-antigen gene of intestinal bacteria 29 types 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 gene pairs intestinal bacteria 29 types;
(7) detection of primer sensitivity: cultivate intestinal bacteria O29, 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 O29.
The separation method of the Nucleotide of aforesaid O-antigen-specific to intestinal bacteria 29 types is characterized in that it comprises the steps:
(1) genomic extraction: 37 ℃ of incubated overnight intestinal bacteria 29 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 3ul10mg/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 pcr amplification intestinal bacteria 29 types bunch: with the genome of intestinal bacteria 29 types is that template is passed through its O-antigen gene of Long pcr amplification bunch, at first according to the galF sequences Design upstream primer (#1523-ATT GTG GCTGCA 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 100mMDTT 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 lml 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 29 types;
(4) to the cloning and sequencing in the library: from the library, select 96 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 29 types; The quality of sequence is mainly guaranteed by two aspects: 1) genome of intestinal bacteria 29 types 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 29 type O-antigen genes bunch, with American National biotechnology information science center (The National Center for BiotechnologyInformation, NCBI) orffinder finds gene, find the reading frame of 12 openings, determine also that with the function of finding the reading frame that these are open what gene they are with the genetic comparison among the blast groupware and the GenBank, finish gene annotation with the Artemis software at Britain sanger center again, do accurate comparison between DNA and protein sequence with ClustralW software, obtain the structure of the O-antigen gene bunch of intestinal bacteria 29 types at last;
(6) specific gene screening: at wzx, wzy, the gene gene design primer in the O-antigen gene of dysentery intestinal bacteria 29 types 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 containing intestinal bacteria O114 group, has obtained the expection size, the correct product of the expection clip size that all do not increase in other groups is so the O-antigen of wzx, wzy gene pairs intestinal bacteria 29 types all is high special.
(7) 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 O29 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 O29 in the pork filling when using aforesaid method.
Just, first aspect of the present invention provides the full length nucleotide sequence of the O-antigen gene bunch of intestinal bacteria 29 types, its complete sequence shown in SEQ ID NO:1,14250 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 29 types by method of the present invention, as shown in table 3, it comprises called after orf1, orf2, orf3, orf4, wzx, wzy, orf7, fnlA, fnlB, fnlC, wbuB, 12 genomic constitutions of wbuC are all between galF gene and gnd gene.
Second aspect of the present invention provides the gene in the O-antigen gene bunch of intestinal bacteria 29 types, promptly transports enzyme gene (wzx gene or the gene of identity function arranged with wzx); Pol gene (wzy gene or the gene of identity function arranged with wzy); Glycosyltransferase gene.Their zero positions in O-antigen gene bunch and final position and nucleotide sequence all are listed in the table 4; The invention particularly relates to glycosyltransferase gene, transhipment enzyme gene and pol gene, because sugared synthesis path gene is that the gene of synthetic nucleosides bisphosphate monose is common, common by indication to more exocellular polysaccharide now, O-antigen to bacterium is not very special, and the glycosyltransferase gene that the present invention relates to, transhipment enzyme gene and pol gene are high specials to the O-antigen of intestinal bacteria 29 types.
The 3rd aspect of the present invention provides the wzx gene in the O-antigen gene bunch that comes from intestinal bacteria 29 types or the gene, wzy gene of identity function arranged or with wzy the gene of identity function is arranged with wzx, 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 13rd group, and any product that all do not increase in other groups is so the O-antigen of wzx, wzy gene pairs intestinal bacteria 29 types all is high special.
The separation method of the Nucleotide of described O-antigen-specific to intestinal bacteria 29 types comprises the steps: 1) genomic extraction; 2) the O-antigen gene in pcr amplification intestinal bacteria 29 types bunch; 3) make up O-antigen gene bunch library; 4) to the cloning and sequencing in the library; 5) splicing of nucleotide sequence and analysis; 6) screening of specific gene; 7) detection of primer sensitivity.
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 the oligonucleotide of wzx gene to being: the Nucleotide of 4891 to 4900 bases among the SEQ ID NO:1 and the Nucleotide of 5571 to 5589 bases; The Nucleotide of 4858 to 4876 bases among the SEQ ID NO:1 and the Nucleotide of 5551 to 5569 bases; The oligonucleotide that comes from the wzy gene is to being: the Nucleotide of 6575 to 6593 bases among the SEQ ID NO:1 and the Nucleotide of 7201 to 7221 bases; The Nucleotide of 6455 to 6473 bases among the SEQ ID NO:1 and the Nucleotide of 7060 to 7078 bases.Coming from above intragenic oligonucleotide is high specials to intestinal bacteria 29 types.
In addition, the antigenic gene cluster of the different O-of the coding of two genetic resemblances produces new O-antigen by gene recombination or sudden change sometimes, thereby produces new bacteria types, new mutant strain.In this environment, need filter out many specificitys that oligonucleotide is detected with raising with recombination hybridization.Therefore, the invention provides a whole set of many mixtures to oligonucleotide, they come from glycosyltransferase gene; Come from transhipment enzyme and pol gene, comprise the wzx gene or the gene, wzy gene of identity function arranged or the gene of identity function is arranged with wzy with wzx.The mixture of these genes is special to a special bacterial polysaccharides antigen, is special thereby make this cover oligonucleotide to the polysaccharide antigen of this bacterium.More particularly, the mixture of these oligonucleotide is to come from glycosyltransferase gene, wzx gene or the gene, wzy gene of identity function arranged or with wzy the combination of the oligonucleotide in the gene of identity function is arranged with wzx.
On the other hand, the present invention relates to the evaluation of oligonucleotide, they can be used for detecting the O-antigen of expressing the antigenic bacterium of O-and identifying bacterium in diagnosis.
The present invention relates to a kind of antigenic method of one or more bacterial polysaccharideses that detects in the food, these antigens can make sample can with the oligonucleotide specific hybrid of following at least one gene, these genes are: (i) gene of the encoding glycosyl transferring enzyme gene of transhipment enzyme and polysaccharase of (ii) encoding comprises the wzx gene or the gene, wzy gene of identity function is arranged or with wzy the gene of identity function is arranged with wzx.At least one oligonucleotide can be hybridized with at least one more than one such gene specific of expressing the special antigenic bacterium of O-under the situation of condition permission, and these bacteriums are intestinal bacteria 29 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 29 types.
On the other hand, the present invention relates to a kind of antigenic method of one or more bacterial polysaccharideses that detects in the movement, these antigens can make sample can with the oligonucleotide specific hybrid of following at least one gene, these genes are: (i) gene of the encoding glycosyl transferring enzyme gene of transhipment enzyme and polysaccharase of (ii) encoding comprises the wzx gene or the gene, wzy gene of identity function is arranged or with wzy the gene of identity function is arranged with wzx.At least one oligonucleotide can be expressed more than one such gene specific hybridization of the special antigenic bacterium of O-with at least one under the situation of condition permission.These bacteriums are intestinal bacteria 29 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 29 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 29 types first, and can from the sequence of this total length gene cluster of not cloned, produce recombinant molecule, can produce the O-antigen of expressing intestinal bacteria 29 types by inserting to express, and become useful vaccine.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should understand these embodiment only is used to the present invention is described and is not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, molecular cloning: the condition described in the laboratory manual (NewYork:Cold Spring Harbor Laboratory Press, 1989).
Embodiment 1: genomic extraction:
37 ℃ of incubated overnight intestinal bacteria 29 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 pcr amplification intestinal bacteria 29 types bunch:
With the genome of intestinal bacteria 29 types is that template is passed through its O-antigen gene of Long pcr amplification bunch.At first according to the galF sequences Design upstream primer (#1523-ATT GTG GCT GCA GGG ATC AAA GAA AT) that often is found in O-antigen gene bunch promoter region, again according to the gnd gene design downstream primer (#1524-TAG TCG CGT GNG CCT GGA TTA AGTTCG 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, 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 PCRPreps 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 1mg/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 100mMDTT 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 escherichia coli DH5a.Get a ring bacillus coli DH 5 alpha list bacterium colony in the LB of 5ml substratum, 180rpm cultivated after 10 hours, got in the LB substratum that the 2ml culture is transferred to 200ml, and 37 ℃ of 250rpm thermal agitations are cultivated OD600 about 0.5, ice bath cooling was 20 minutes then, in centrifugal 15 minutes of 4 ℃ of 4000rpm.Confide all supernatant, dispel thalline, in centrifugal 15 minutes of 4 ℃ of 4000rpm with the deionization aqua sterilisa 200ml of cold ice precooling.Deionization aqua sterilisa 100ml with cold ice precooling dispelled thalline again, in centrifugal 15 minutes of 4 ℃ of 4000rpm.With 10% glycerine suspension cell of cold ice precooling, centrifugal 10 minutes of 4 ℃ of 6000rpm abandon supernatant, precipitate 10% glycerine suspension cell with the precooling of 1ml ice at last, are competent cell.The competent cell that makes is packed as 50ul one pipe ,-70 ℃ of preservations.
Be electric transformed competence colibacillus cell at last: get after 2-3ul connects product and 50ul competence bacillus coli DH 5 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 constitutes fourth intestinal bacteria 29 types with the EcoRI enzyme.
Embodiment 4: to the cloning and sequencing in the library:
From the library, select insert 96 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 29 types obtained.The quality of sequence is mainly guaranteed by two aspects: 1) genome of intestinal bacteria 29 types 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 29 type O-antigen genes bunch, with American National biotechnology information science center (The National Center for Biotechnology Information, NCBI) orffinder finds gene, find the reading frame of 12 openings, determine also that with the function of finding the reading frame that these are open what gene they are with the genetic comparison among the blast groupware and the GenBank, finish gene annotation with the Artemis software at Britain sanger center again, do accurate comparison between DNA and protein sequence with Clustral W software, obtain the structure of the O-antigen gene bunch of intestinal bacteria 29 types at last, as shown in table 3.
By retrieving and comparing, find that the glycerol-3-phosphatecytidyltransferase Escherichia coli that encodes in orf1 encoded protein and the intestinal bacteria O-antigen gene bunch has very high consensus amino acid sequence (63%), by the search to Pfam protein-based order sequenced data storehouse, the homology desired value of the consensus sequence of discovery orf1 encoded protein and known Cytidylyltransferase is very high.Because the definite function of this gene can't be determined, so we are with the temporary called after orf1 of this gene.The Fnl1 of coding has very high consensus amino acid sequence (89%) in Orf8 encoded protein and the intestinal bacteria O-antigen gene bunch, the Fnl2 of coding has very high consensus amino acid sequence (71%) in Orf9 encoded protein and the intestinal bacteria O-antigen gene bunch, the Fnl3 of coding has very high consensus amino acid sequence (89%) in Orf10 encoded protein and the intestinal bacteria O-antigen gene bunch) by search to Pfam protein-based order sequenced data storehouse, find orf8, the homology desired value of the consensus sequence of the synthetic enzyme of 9,10 encoded protein and known Fuc2NAc is very high.Therefore we are with the temporary called after fnlA of this gene, B, C.
Orf5 and orf6 are the proteic genes that there is transmembrane segment in only two codings of intestinal bacteria O29 kind.The O-antigen transferring enzyme of Orf5 encoded protein and Streptococcus thermophilus has 23% sequence identity, 43% similarity, it contains 10 uniform transmembrane segments by the proteic topology discovery of HMMTOP2.0 programanalysis, and this is the proteic characteristic feature of Wzx.So name orf5 is wzx.The O-antigen polysaccharase of Orf6 encoded protein and Sa/monella enterica has 21% consistence, 43% similarity, it contains 10 transmembrane segments by the proteic topology discovery of HMMTOP2.0 programanalysis, and hydrophilic loop (loop) in the big kytoplasm is arranged, and this is the proteic characteristic feature of Wzy.So name orf6 is wzy.
Orf2, orf3, orf4, orf7, the albumen of five genes encodings of orf11 and other known glycosyltransferases have the sequence identity of 25-38% and the sequence similarity of 50-57%.By search to glycosyltransferase motif database among the Pfam, the homology desired value of the consensus sequence of the albumen of these five genes encodings and known glycosyltransferase family 1 and 2 is very high, therefore we infer this five 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 O29 may be made up of six monose.Because the definite function of these five genes can't be determined, so we are with the temporary called after Orf2 of these five genes, orf3, orf4, orf7, orf11.
WbuC among orf12 and the intestinal bacteria O26 has 69% sequence homology, because the WbuC conjecture is the gene that does not have function, so our supposition may be for there not being the gene of function, with its called after orf12 yet.
Embodiment 6: the screening of specific gene.
Wzx, wzy gene design primer in the O-antigen gene of intestinal bacteria 29 types bunch have respectively designed two pairs of primers in each gene, every pair of primer is distributed in different local 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 containing intestinal bacteria O29 group, has obtained the expection size, the correct product of the expection clip size that all do not increase in other groups is so the O-antigen of wzx, wzy gene pairs intestinal bacteria 29 types all is high special; The position of these genes in nucleotide sequence is shown in 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 O29 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 O29 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 O114 of the present invention can be applied to set up recombiant vaccine.
Nucleotide sequence (shown in the SEQ ID NO:1) according to the O-antigen-specific to intestinal bacteria 29 types 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 29 types.Glycosyltransferase gene, transhipment enzyme gene and pol gene and their function corresponding and the size of the O antigen gene bunch of intestinal bacteria 29 types 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 ID NO:1.Is that template carry out PCR with listed corresponding annealing temperature in the table with the genomes of all bacterium in the table 2 with every pair of primer, has obtained corresponding PCR product, and its size is also listed in the table.
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 12-19 bacterium, and 12 groups altogether, all list in the table in their source.
The genomic dna that contains intestinal bacteria 29 types in the 13rd 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 12nd 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 29 types and O-antigen thereof are high specials.
At last, from intestinal bacteria 29 types, screen gene by PCR: three glycosyltransferase genes to the O-antigen high special of intestinal bacteria 29 types.And the oligonucleotide of these intragenic any one section 10-20nt is special to the O-antigen of intestinal bacteria 29 types, and the primer in especially above-mentioned each gene is that oligonucleotide is high specials to detecting the back confirmation through PCR to intestinal bacteria 29 types.These all oligonucleotide all can be used for intestinal bacteria 29 types in the human body and environment rapidly and accurately, and can identify their O-antigen.
Table 3 is structural tables of the O-antigen gene bunch of intestinal bacteria 29 types, in table, listed the structure of the O-antigen gene bunch of intestinal bacteria 29 types, altogether by 12 genomic constitutions, each gene box indicating, and in square frame, write the title of gene, numeral be the order of the open reading frame (orf) in the O-antigen gene bunch.Two ends at O-antigen gene bunch are galF gene and gnd gene, and they do not belong to O-antigen gene bunch, and we are just with the increase full length sequence of O-antigen gene bunch of their one section sequences Design primer.
Table 4 is site plan of the gene in the O-antigen gene bunch of intestinal bacteria 29 types, listed the accurate position of all open reading frame in complete sequence in the O-antigen gene bunch of intestinal bacteria 29 types in the drawings, at the underscoring of the initiator codon and the terminator codon of each open reading frame.The initiator codon of open reading frame has two in intestinal bacteria: ATG and GTG.
SEQ ID NO:1 sequence (SEQUENCE LISTING)
＜110〉Tianjin Bioarray Solutions Ltd.
＜120〉to the Nucleotide of the O antigen-specific of intestinal bacteria 29 types
＜130〉to the Nucleotide of the O antigen-specific of intestinal bacteria 29 types
<160>1
<170>PatentIn version 3.2
<210>1
<211>14250
<212>DNA
<213>Escherichia coli
<400>1
attgtggctg cagggatcaa agaaattgtt ctggttacgc actcgtccaa gaatgcggtc 60
gaaaaccact tcgacacctc ttacgaactc gaagcgctgc tggagcagcg tgttaaacgt 120
caactgctgg cggaagtgca gtccatttgt cctcctggcg tgaccatcat gaacgtgcgt 180
caggcgcagc cgctgggcct gggccactcc atcctgtgtg ctcgccctgt cgtgggcgac 240
aacccgttta tcgtggtcct gccggatatc atcatcgata ccgcttctgc ggatccgctg 300
cgctataacc tggcggcgat ggtggcgcgt ttcaacgaaa caggccgcag ccaggtgctg 360
gcgaaacgca tgaaaggcga tctgtccgag tactctatta tccagactaa agaagcgctg 420
gagacagaag ggcaggtgag ccgcatcgtt gagttcatcg aaaaaccgga tcagccgcag 480
acgctggatt ctgacctgat ggcggttggt cgttatgtcc tgaacgcgga tatctgggcc 540
gagctggaaa aaaccgagcc aggcgcctgg gatcgtatcc agttaaccga tgcgatcgcc 600
gagctggcga aaaagcagtc tgttgacgcg atgctgatga ccggtgagag ctacgactgc 660
ggtaagaagc tgggctacat gcaggcgttt gtgaactatg ggctgcggaa cctgaaggaa 720
ggggcgaagt tcagaagccg gattgagaag ttgttagcta acgactgatt ttatcttagt 780
tgatttgcac aagcggcaat catcatttgg ggatgttgaa acataaccaa aatggtagct 840
gccgtttttt gttattaatt ctaatgataa catggattta tctgatttaa atctggtcag 900
atttgtaacg atttgtgctt gtttctgagg tcgtttagga cgacaattag cagagttgta 960
atgcagttgt ctagtggcag ttagtgtccg atgacagaca acagaaaatg aaagaatcat 1020
gcatcattca gtgcactggt agctgttgag ccaggggcgg tagcatattt aattgtgaga 1080
gaacaatgaa gagaattatt acgtttggta catttgatgt ttttcatgtt ggtcatgtta 1140
atatccttga gcgaactgct tcacttggcg actatcttat tgtaggtgta agttcagata 1200
aattgaattt caataaaaaa ggtcgttacc ccatttataa tcaagaagac cgctgtcgta 1260
taataaattc tcttagagtt gtgaatgatg tatttattga agaatcttta gagcaaaaaa 1320
aagagtatat aatccaatac gaagctgata ttttagttat gggtgatgat tgggctggcc 1380
gatttgattg ggtaaatgac atttgtgatg ttatttattt accaagaaca ccatcagtat 1440
ctaccacaga aattattgaa gttgtgaaga ccctcagatg acgaatttca aaaaaatagc 1500
aagaaaaata attagtaaaa gcatttcatt gttaaattat tttatcccca agaaaaaaaa 1560
tagaattttt ttcaaaagca aacctgacta ctcaggtaat tgtaaggcac taagcgatta 1620
tattatagag aaaaaacttc catatcatat cgtttggtct gttaaaaaag aaattaatca 1680
aaaaggaatt actgtagtaa gagctggttc attgaaagaa tttttctatt actttacaag 1740
taaatatgtt attactacgc ataatgaaat gataggtcca attgcaacaa atcaaaaata 1800
cataagcttg tggcatggaa tgccttttaa gaaaatttgt tatcttggag agaatgatca 1860
tcaaggaatg atagattatt cagccattag gattgccact tccgaagtaa tgcgttctat 1920
aatttcagca agctttcgtg aaaaagccaa taatgtatat attacaggcc agcctcgtaa 1980
tgattttttg ttcaaaccaa ttagtctgac agatattggt attaaatcta taaagaacaa 2040
aaaaattgta atgtttgctc caacatttcg tatgaataat gaagatataa gatattctga 2100
tggagcggaa attattgata ataattttct tcgagtaaat gatttctgca tggaagagat 2160
agattattat cttgaacaaa gcaatttaca tttgatatta aaattgcatc catacgagga 2220
ggaatatttc cgcgggatcg caacactaag ttcgaatata actattataa gttctgatga 2280
actcacgcaa aaaaatatcg atttgaatca gttgctttcc ttggtcgata ttttaataac 2340
agattactca tcaatttatt tagattactt aattctaaat aaacctctaa tttttttagt 2400
tcctgatgta gatgcttata gttctgcacg ggtgggttt actttagaac cttttgattt 2460
ttggacgcca ggagataagg ttagctgtca aaggtcatta ttgaattcaa taaataaaat 2520
aatcactgga aatgatgagt atgctgaaaa acgtaaccaa ataaatctca taataaataa 2580
atactctgat gctaataata gccagagagt cattgaattg atgaagagtt tatcatgaaa 2640
atatacattt tcttaggaga tttaagttcg aaaggtggta ttgagcgtgt ttcagtagca 2700
ttagccaatg gattggctaa attttatgat gtcactatca ttagcttata tcgtgcaact 2760
agaaatttat catttgtccc ggatgaaaag gtcaatgtga tctatttgta cgatgaattt 2820
gaaaagagta tgtataatcg taatctggga gctatcatag ggttaaagtt tgatttctta 2880
tatattataa aaaagttgaa gcaacttaaa agattaaatt tagaactaaa taaaaatgat 2940
gtgcttattt caagcgatat taaaatgtct ctgctattat ttttctatgc aaaaaaaagc 3000
aaaattatag ctattgagca ttttgaacat gatgttggta atttagtgtt gagaaaaatt 3060
agaagcgcac tatatcctaa attatcagca gttgtatcgc aaactggcga agatcaaatt 3120
aagtatttgc aatggttacc tagaaagaaa cataaaatca ttccaaatat tattagcttc 3180
gaagcgaccg atataccgca aaataaaata gaacaaaaaa atgtacttgc tgtgggacga 3240
ttaactcatc aaaagggttt tgatttactt ctacaagctt gggcagacgc aaatactcat 3300
gattggcgct taaagatcat tggagacgga gaagagctga accatttaaa ttctctaatt 3360
accgagttaa atatctctaa cgctgaaatt atcccttttc aaaaggatat tcaaaggcat 3420
tattcttctg caggaatatt cgtactttct tctcgctttg agggtttggg tatggtgctt 3480
ttagaagctc ttagcagcgg cttggcgtgt attagttttg attgtccagc tggtcctaaa 3540
agtataatct caagtgataa tggggtgtta gttccaactg gtgacactat aaaattatca 3600
caagctattt ctttcttgat aaataacgaa gaagaaagaa aacggttaca aaacaaagct 3660
gctgcttctg ttgaaaaatt caaagaatca aacgtaattg caaaatggcg agagttattg 3720
aatgaaattt catagaaaac aatatgatgc agtatttctt acaaacgtac ctgctttcta 3780
taaagtaaat ttgttcaacg aaattcaaaa aaataaagat atttttgtta tattcgtttc 3840
agataagtcg aaaattagga atgaagattt ttattcatac gactttgcgt ttgaccactt 3900
ttttcttaat gaagagaact ttgaagaacg aagtaaaata aaaacactat tcaatttact 3960
tagagtgtta cgaacgattt cgtacaaaaa aattatatat tcaggatggg aagttaaaga 4020
agtaactcta gctgcattat ttaataagcg ggaaaaaaat gcaatagtga tcgaaagtag 4080
cattattgaa acgaagaaaa ctggtttgac atggcttcta aaaaaaatag ctatcggtcg 4140
tatgtcgctt gcttacccat cagggctatt gcaaaaagca atattggaga cattctcttt 4200
taaaggaaaa actgttatta cccatggcgt tggtatctct aatttaaaag agaccagttt 4260
tcataataaa aaaacatgta caaggaataa tcctcttcgt ttcatctatg ttggaaggat 4320
ttcatcggaa aaaaatatag attttatggt gaaagtattt aaaactcttc cttacgaatt 4380
gatactaatt ggagatggtc cattaaaaaa acagtttgac gataaaacct atagcaatat 4440
cagattttta ggctatattg acaataaaaa attatcaaaa gaactactta aaagtgattg 4500
ttttattctt ccgtcattat ctgaaccgtg gggattagta gtagaggagg ctttgacatt 4560
agggcttccg gttatagtca gtaaccatgt gggctgtcat agtgatttag tcaatgatag 4620
aaatggcatt atattcgatg tgaacgatac acaatccttt attgacgcat tgtcgaaaat 4680
ggaaaaaaat tatgaacgct ttgcccgtgg tgccagcgaa ttccatgcta gtgaaatagc 4740
aaaagcacag atcgatgctt atgtaggtag catctgatga gcaacaaaaa caaaacattt 4800
aaagatttta taaattattt tttaggcgat ctttttgtca aaggatttat gtttatctcg 4860
ctacctctac tttcgagaat aatgtcgcca gaagattatg ggcggatgtc ccttataaat 4920
gcagctgtta tgattttata tgtttttata agtttaaatc tccaaaatgc tgttatcaac 4980
gcttacatga aaaatgaagt tgattttcca gtatatctag gtagtgtatt atggggattg 5040
accgccgcgc aagtattgtt agtcgcttta agtatatatt ttgctgttcc gcttggaatg 5100
ttattgagta ttagcaaata tgatgtttat tgggttgtcg ccatttgtat tttgctaagc 5160
tatatatata tatatactag ctatctacag ggtgcacggc tcagttctag ctttgtcaaa 5220
ctgaatatat ttagtaagat atcagaagtt gttgtcattt ttgtctttgc ttggttttta 5280
actcaagata aataccttgc taaggtatat gctcagttag tgattagctt tattttactg 5340
acatatgtgt tgaagaaact aaaaaagata gctgtattca aatttaatgt acgatatttt 5400
atatcagcac ttgcatttag ttctccatta attatacatg ttctttcaaa tgctttgctt 5460
tcacaagttg accgattgtt tatcgcaaag atgttgggag aggggcaggc tggtatatat 5520
tctttcgcat ataatatcgg aatgtgtata ttagtggttg ttatggcctg gaactcttcg 5580
tggcaaccta aattatataa gcttattgat tcgaaggata atggtaaaat aattcgaatt 5640
gtcgatgtaa gttccttatt attattaata gtatcatttt tatctattct tttttcaaaa 5700
cagatggttg aagtccttgc ggacaatcgc tatagggaaa gtatatccgt cgttcctgtc 5760
atattgattg gtaactctct gattcatatt tatttaagct atgttaactt tactttctat 5820
aagaaaaaaa caatatatgt ttcaattggt acattgcttg cggtagcgat aaatattgct 5880
ttgaattata tactcatacc aatatacggt atccacggag cagcatgggc tacagtaata 5940
gcttatttta tgctggcatt tttccattat ctcatagcaa caataatgtt aaaggcaaac 6000
ccactctcat tgtttctgtt attatggtat tcagctttgt tattggcttc gtatttctta 6060
gtaatatacc ttgactcttt gtctctttgg atctctttat caataaaggc aatgatcatt 6120
tttattatcc tcatcatcct tatgaaaaca aaaatctata atgaattaaa ggaatgacag 6180
tgtcgatata tcttctgctt ctagctttcg tatttctgct tgcaatgtca gattttttta 6240
tcattgctga tgccagaaat cgttttttac tttatataat actattttcg ctattagtaa 6300
cctttatagg tttaagatat caaactggac ttgattggtt attttataac aatctattta 6360
atggagaggg tttttcatta gccattgagc ctggatatta ttttttctcg tatgtttcat 6420
cttttttaat ggggtattgg atttatcagg cattaataac cgccgttcta ataatatgtt 6480
taaaaacatt ctttgaaaaa aacactaaaa actatctgtt ttgcataggt tttttcttct 6540
tatatcaatt tattttcgtg acggaagcaa tacggcaaat aatcgctctg tcaattattt 6600
tggttgcata taaaaagttt tatgatggta agaaattgca atttcacatg cttaccattt 6660
tggcttgttc attccatatt tctgctgtaa ttgtctttat tttgattccg tttttaaaac 6720
gcagaaatat atatatatta aaaatactga caatcgttgg tttagttttg gcaattttta 6780
gtgtttatcc tgttgactac ttaatacagt tactgtcatt gctccctgct ggtggttata 6840
tagaaaagat aagatggtat agtcaggatg attatgctgg atcagtactt acgttttcat 6900
tagtattcaa agtttttgtt gtgcttttat tcgattacag atttaaatca ataaaatcac 6960
atggtcaaag tcttattaat gcgagggcat atgattttat ttatacttcg gtttatttaa 7020
tgatattcat ggacgtttat cttggtaggt ttggtactat tagcaccagg cttgacgtat 7080
actttatacc atgtttttta atagctctta atcatttaat aaatgaacac aaacaaggtg 7140
tgagtcgttt catattcttc ttcgttgtca tggtttactt tactataaat tatctcagta 7200
ttatgaacgg atattacttc gagaaatttt acagtccctt atcaaaatta tataactgaa 7260
tttttaaatc cgggcagtta tagtgataga gggtgggatg ttagatatta tttcagcaat 7320
aaggaattat tgcagtgaat cttttgatta atgctagtaa tctgtatgtt ggtggaggag 7380
tccaggtagc aatttctgta ctggaagagt tatctgattc atcattttct tttattgcgg 7440
ttgtttcacc agttgtttat tcgcagttaa gcgatgatgc agcatcatgt tgtatagtga 7500
ttgaatcttc tccatcaaaa ttgttgaatt ttaaagttag aaggcaactc gacgatatag 7560
ttaaaaaaaa tgatatttct gtagttttca caatcttcgg tcctagctat tggtctccga 7620
aaaatgttaa acatgctatt ggttttgcgc tcccttggtt gatttatgat attgaatata 7680
tatttaaaaa attgacttta aaagctaaat tgaaattttg cattttaaaa ttattacagc 7740
catattactt caagaaaaat gccgacttaa tttttactga aacagatgat gtcaacttgc 7800
gggtaacaaa acttcttaac tttgaaaaag aacaagtcta tactgtttca aacacactta 7860
atggtttatt aaaaaattca aactgttatg attacagcat tttggataga ttacctacga 7920
aagaaccaaa tgatatttgg ttggtaacta tttctcataa ttaccctcat aagaacttag 7980
aagtcattaa agaattagta acagtattgc caccttgtta taagtttatt ttaacagttt 8040
caagtgattt tttgcagcta gtcccaaaag agcatagaga gcgagtcatc accataggta 8100
atgcaacact tagtcaatgt gctccgttgt atgaagtttg tgatggatta ttcatgccaa 8160
cacttttaga atgctttagt gcttcttact tagaagcaat gtacatgaaa aaaataatct 8220
tcacttctga tctgcctttt gctcacactg tttgcaaaga tgcagcattt tactttgctc 8280
ctcatgatgt tgaaaatatt aggagtacac ttgttaatgg ctttcaaaac aaagaaattc 8340
ttaaccataa attaaatgaa ggttcgaaaa tttatgaaag ttttccttct gcgaaagctc 8400
gagcattgca gtatatagat ataataaaat ccaacttggt ataaattgta gattttgagg 8460
tgttaaaaat gtttaaagat aaggttttac ttattactgg cggaacaggg tctttcggta 8620
acgctgtact taatcgtttt cttgaaactg atattaaaga aataaggatt ttttctcgcg 8580
atgaaaaaaa acaagacgat atgcggaaaa aatataataa ttctaagtta aaattctaca 8640
ttggggatgt aagggattat tccagcattc taagcgccgc acgtggcgtc gactttatat 8700
accatgctgc tgcattaaaa caagttccgt catgtgaatt tcatcctctg gaagcagtga 8760
aaactaacgt attaggtaca gagaatgtgc ttgaagcagc aattgcaaac caggttaaac 8820
gtgtcgtttg cttaagcacg gataaggcag tatatccgat taatgccatg ggtatttcca 8880
aagcaatgat ggaaaaagtg atggtcgcaa aatcacgtaa tgttaatagc gacaaaactg 8940
tgatttgcgg tactcgctac ggcaacgtga tggcttcacg tggttccgtt attcccttat 9000
tcgttgatct gatcaaagcg ggcaaagcgc tgacgatcac tgacccaaat atgacccgtt 9060
ttatgatgac gcttgaagat gctgtcgatc tggtcctata tgcgtttgaa catggtaata 9120
atggcgatat atttgtccaa aaagcgcctg ctgcaacgat tgaaacatta gccattgctc 9180
tcaaagaact tcttaatgtt gagcaacatc ctgttaatgt tatcggaacg cgccatggtg 9240
agaaacttta tgaagcgctg cttagccgtg aagagatgat tgccgccatt gacatggggg 9300
attactaccg tgttccacca gacctgcgcg atcttaacta tggaaaatac gtagaacaag 9360
gtgatagccg aatctcagca gtagaggatt ataactctca caatacacag cgactggatg 9420
ttgaagggat gaaaacgctt cttttgaaat taccttttat tcgtgcactg cgtgcgggtg 9480
aacattacga tctggatgcc taatatgaaa atcctgatta ctggagctga tggttttatt 9540
ggacgtaacc tgtgcttacg ccttcaggaa gcaggctact gtgaccttgt taagattgac 9600
cgcggttcaa gtgcggctga tctggaaact ggccttcaag atgctgattt tgtctatcat 9660
ctcgcaggta tcaatcgacc taaaaacgtt gatgaatttg ccgaggggaa tagcaatctg 9720
actcaacaga ttgttgatta tcttttagcc aagcataaaa gcatacctat tatgatcagt 9780
tcttccattc aggctgaact ggttaatgct tatggtcaaa gtaaagctgc agcagaaaaa 9840
catattgaac gctatgcagc tgaaagcggt gcagcttatt ttatttatcg ttatccgaat 9900
gtttttggta agtggtgtaa gcctaattat aattcgttcg tggcgacctt ttgccataat 9960
attgccaaca atatcgatat cacgatcaat gactccttcg cgcctgttaa tcttgtttat 10020
attgatgatg tctgttctga tgcgataaag ctcttgtctg gaaaggttga aagcgggtac 10080
aaaactgtta agccagtata ttcgacgaca gtaggtgagg tggcggaatt actttatcgc 10140
ttcaaagaaa gccgttccac tcttgtcacc gaggctgtag gaacagggtt catccgcgcg 10200
ctgtattcga cgtggttaag ttatctccca gccgatatgt ttgcgtattc agttccctct 10260
tacggagacg cccgaggggt tttttgtgaa atgttaaaaa ccccttcagc ggggcagttt 10320
tcatttttta cagcgcatcc cggcattaca cgtgggggtc attatcatca caccaaaaat 10380
gagaagttcc tggtcattcg cggccaggca tgctttaggt ttgaacatgt gattaccggt 10440
gagcgatatg agatgaatgt ttcctcagat gagttcaaaa tcgttgagac agtccccggc 10500
tggacacatg atgttacaaa tattggagcg gatgaattga tagtcatgct gtgggcaaac 10560
gaaattttca gtcgcgatga gcctgatact attgcgagac ctctgtaatg aaaaaactaa 10620
aaattatgtc tgttgttggt acgcgtcccg agattatccg tctgtcacgc gttctcgcta 10680
agcttgatga acactgcgag catattcttg tccatactgg tcaaaactat gattatgagt 10740
taaacgaagt attctttaat gaccttggtg tacgaaaacc cgattacttt ttaaatgctg 10800
ctgggaaaaa tgctgcagaa accatcggtc aggttatcat caaagttgat gaggtacttg 10860
aatccgaaaa gcctgaagca atgttggtgc tgggtgatac aaactcctgt atttctgcaa 10920
ttccagccaa acgccgtaaa gtgcctatct tccatatgga agctggcaat cgttgtttcg 10980
atcagcgcgt cccggaagag accaatcggc gcattgtaga ccataccgct gacatcaata 11040
tgacctacag cgatattgca cgtgaatacc ttcttgctga aggcctccca gcggaccgaa 11100
ttattaaaac cggtagccca atgtttgagg tacttacgta ttatatgcct caaatcgata 11160
attcggatgt actgtcgcgt ctgaacctgc gttcaggcga atttttcgtc gtcagcgcgc 11220
atcgtgaaga gaatgttgat tctcccaaac agctagtcaa gcttgcgact attctcaata 11280
ctattgctga aaaatatgat ttgccggtta ttgtatccac tcatccgcgg acacgtaatc 11340
gtattaacga gcaagggatt gaattccatc caaatattaa tctactgaaa ccgttaggtt 11400
tccatgatta caaccatttg cagaaaaatt cacgtgctgt gctgtctgac agcggtacga 11460
taactgaaga atcttccatt atgaatttcc cggcggtaaa tattcgggaa gcacatgagc 11520
gtccagaggg ctttgaagaa gcctctgtca tgatggtggg cctggattgt gaacgtgttc 11580
tacaggcact ggatattctg gcaacacagc cccgcggcga aacccgtctt ttacgacaag 11640
taagtgacta cagcatgcct aatgtgtcag ataaagtcgt cagaatcgtt cattcttata 11700
ctgattatgt caagcgagtc gtctggaaag aatactgatg aaacttgctt taatcataga 11760
tgattacctg cccaatagta cgcgagttgg tgcaaaaatg tttcatgaat tggctcagga 11820
atttattcgc cgtgggcatg atgttacggt aattacacct gacatctgtc ttcaggatga 11880
tgtgtccttt agcaccttcc agggggtcaa gacatggcgt ttcaaaagtg ggcctctcaa 11940
ggatgtgagc aaaattcaac gagccatcaa tgaaacactt ctatcctatc gtgcctggaa 12000
ttccattaaa agccagataa aaaaagagac ttttgatggg gtagtttatt actcaccctc 12060
cattttctgg gggcacttag tcaagaaaat taaatctcgt tgccagtgtc cggcttacct 12120
gattttgagg gatatgttcc cgcaatgggt gatagacgct ggcatgttaa aagccggttc 12180
cctcatcgaa cgctatttcc gtgttttcga aagatcatct tatcgccagg caaaccgtat 12240
cgggctgatg tcagataaga atcttgaggt ctttcgggtt aacaacaaag gttatccttg 12300
tgaggttttg cgtaactggg cttctctcac gccgacggta ccaccccagg gttatatccc 12360
attgcgtcag cgtcttggcc ttgatgataa agttattttc ttctatggag ggaatatcgg 12420
tcatgcgcag gatatgggga atctgatgcg tcttgctcga aaaatggctg agcatccgca 12480
agcccatttt ttatttatcg ggcaggggga tgaagttgaa ttaatcaatt ctttggctgc 12540
cgagtggtca ttgcccaact ttacgtattt ggcctctgtc aatcaggatg aatttaaatt 12600
tatcctctcg gaaatggata taggcttgtt ttcactttcc gccagacatt cttcgcatag 12660
| E.coli | Gene | Gene Base positions of genes | Base positions of forward /reverse primers | Annealing temp (℃)of PCR |
| O29 | wzx wzy | 4777/6177 6174/7259 | 4891-4900/5571-5589 4858-4876/5551-5569 6575-6593/7201-7221 6455-6473/7060-7078 | 58 60 58 58 |
tttcccgggt aaattgctag ggtatatggt tcagtcctta cctatactag gcagtgtgaa 12720
tgctggtaac gatttgcttg atgtcgtcaa tcaaaataat gccggattaa ttcatatcaa 12780
tggtgaggat gacaagcttc atgagtccgc actgttaatg cttaaggatg ttgctgcgcg 12840
acgtcaattc ggcttaggcg cgaatgcatt attgagagaa cagttctccg ttgagtctgc 12900
ggcacagact atagaaatga ggttagaggc atgtaatgcg actcattgat actcaccaac 12960
ttgaagcttt atacgaacaa gccggaaaat ctgcacgctt gcgcgctcat ctcttattgc 13020
acaattcgca ccgagagaaa gtgcaacgtc tgctcattgc cttggttcag ggcagctatg 13080
tcgatccgca tttccatgaa cttcctcatc aatgggagat gtttgtcgtt atgcaggggc 13140
aggttcaagt ctgtttgtac ggcaaagatg gcgaaatcat taatcaattt gtcgctggag 13200
agaatactgc aataagcgta gtcgagtttt ctccgggtga tatacacagt gtcgaatgtc 13260
tctctccgag agcattaatg atggaagtga aagaagggcc ttttgatcct tcctttgcca 13320
aggcgttcat ctaacgcccc tctgaatcgc atcttccgct atctactcag gctcatcctg 13380
agttaacatc taagccacat ttcaagccgc gcacagtcgc ggcgaccaca cctgacagga 13440
gtatgtaatg tccaagcaac agatcggcgt tgtcggtatg gcagtgatgg ggcgcaacct 13500
ggcgctcaac atcgaaagcc gtggttatac cgtctccgtt ttcaaccgct cccgtgataa 13560
gaccgaagaa gtcatcgctg agaatccggg taagaaactg gttcctttct atacggttaa 13620
agagtttgtt gaatctctgg aaaggcctcg tcgtatcctg ttaatggtga aagcgggcgc 13680
aggtaccgat gcagccatcg attccctgaa accttatctg gacaaaggcg acatcatcat 13740
tgatggtggt aacaccttct tccaggacac cattcgtcgt aaccgtgaac tctctgctga 13800
aggtttcaac ttcatcggta ccggtgtttc cggtggtgaa gagggcgcgc tgaaaggccc 13860
atctatcatg cctggcggcc agaaagaagc gtacgagctt gttgcgccaa tcctgaccaa 13920
aatcgctgcg gttgcggaag atggtgagcc gtgtgtgacc tatatcggtc cggacggtgc 13980
aggccactat gttaagatgg ttcacaacgg catcgaatac ggcgatatgc agctgattgc 14040
tgaagcctac tctctgctga aggggggcct gaacctttcc aacgaagaac tagcagagac 14100
cttcactgag tggaacaaag gcgagctgaa cagctatctg atcgacatca ccaaatatat 14160
cttcacgaag agagatgaag agggtaaata cctgggtcga tgtgattcct tgacgaagcc 14220
gccgaacaag ggtaccggca aatgggacca 14250
Glycosyltransferase gene in the O antigen gene of table 1 intestinal bacteria 29 types bunch and oligosaccharide unit treatment gene and drawing wherein
Thing and PCR data
*Only in intestinal bacteria 29 types, obtain a correct band
aIn 12 groups shown in the table 2, there are 3 groups to obtain the band of positional fault
bIn 12 groups shown in the table 2,12 groups have all obtained the band of positional fault
Table 2 166 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 | O1，O2，O5，O7，O12，O13，O14，O15，O16，O17，O19ab，O20， O21，O22，O23，O24，O59，O3，O11 | IMVS a |
| 2, wild-type e. coli | O25，O26，O27，O28，O29，O30，O32，O31，O33，O35，O36，O37， O38，O40，O41，O42，O43，O39，O59 | IMVS a |
| 3, wild-type e. coli | O44，O45，O46，O48，O49，O50，O51，O52，O54，O55，O56， | IMVS a |
| O57，O58，O60，O61，O62，O64，O73 | ||
| 4, wild-type e. coli | O63，O65，O66，O69，O70，O71，O74，O75，O76，O77，O78， O79，O80，O81，O82，O83，O96，O95 | MVS a |
| 5, wild-type e. coli | O84，O85，O86，O87，O88，O89，O91，O92，O98，O99，O101， O102，O103，O104，O105，O106，O100，O151 | MVS a |
| 6, wild-type e. coli | O107，O108，O109，O110，O111，O112ab，O112ac，O113， O115，O116，O118，O120，O123，O125，O126，O128 | IMVS a |
| 7, wild-type e. coli | O129，O130，O131，O132，O133，O134，O135，O136，O137， O138，O139，O140，O141，O142，O143，O144，O145 | IMVS a |
| 8, wild-type e. coli | O146，O147，O148，O150，O152，O154，O156，O157，O158， O159，O160，O161，O163，O164，O165，O166 | IMVS a b |
| 9, wild-type e. coli | O168，O169，O170，O171，O172，O173，O155，O124 D1，D2，D3，D4，D5，D6，D7，D8，D9，D10，D11，D12 | c d |
| 10, wild-type e. coli | B1，B2，B3，B4，B6，B7，B8，B9，B10，B11，B12，B13，B14，B15， B16，B17，B18 | d |
| 11, wild-type e. coli | F1a，F1b，F2a，F2b，F3，F4b，F5(v：4)，F5(v：7)，F6，F X becomes，F Y becomes，DS，DR， | d |
| 12, the 9th group of bacterial strain adds the intestinal bacteria reference culture | O29 | IMVS |
*For the convenience that detects, we are divided into one group with every 13-19 bacterium, 12 groups altogether
a.Institute of Medical and Veterinary Science(IMVS)，Anelaide，Australia
b.Statens Serum Institut，Copenhagen，Denmark
C.O172 and O173 come from statens Serum Institut, Copenhagen, and Denmark, all the other come from IMVS
D. China Preventive Medicial Science Institute's epidemiological study institute
Table 3 intestinal bacteria 29 type O antigen gene structure iron
orf1orf2orf3orf4wzxwzyorf7fnlAfnlBfnlCwbuBwbuC
Table 4 intestinal bacteria 29 type O antigen gene cluster gene positions
ATTGTGGCTG CAGGGATCAA AGAAATTGTT CTGGTTACGC ACTCGTCCAA GAATGCGGTC 60
GAAAACCACT TCGACACCTC TTACGAACTC GAAGCGCTGC TGGAGCAGCG TGTTAAACGT 120
CAACTGCTGG CGGAAGTGCA GTCCATTTGT CCTCCTGGCG TGACCATCAT GAACGTGCGT 180
CAGGCGCAGC CGCTGGGCCT GGGCCACTCC ATCCTGTGTG CTCGCCCTGT CGTGGGCGAC 240
AACCCGTTTA TCGTGGTCCT GCCGGATATC ATCATCGATA CCGCTTCTGC GGATCCGCTG 300
CGCTATAACC TGGCGGCGAT GGTGGCGCGT TTCAACGAAA CAGGCCGCAG CCAGGTGCTG 360
GCGAAACGCA TGAAAGGCGA TCTGTCCGAG TACTCTATTA TCCAGACTAA AGAAGCGCTG 420
GAGACAGAAG GGCAGGTGAG CCGCATCGTT GAGTTCATCG AAAAACCGGA TCAGCCGCAG 480
ACGCTGGATT CTGACCTGAT GGCGGTTGGT CGTTATGTCC TGAACGCGGA TATCTGGGCC 540
GAGCTGGAAA AAACCGAGCC AGGCGCCTGG GATCGTATCC AGTTAACCGA TGCGATCGCC 600
GAGCTGGCGA AAAAGCAGTC TGTTGACGCG ATGCTGATGA CCGGTGAGAG CTACGACTGC 660
GGTAAGAAGC TGGGCTACAT GCAGGCGTTT GTGAACTATG GGCTGCGGAA CCTGAAGGAA 720
GGGGCGAAGT TCAGAAGCCG GATTGAGAAG TTGTTAGCTA ACGACTGATT TTATCTTAGT 780
TGATTTGCAC AAGCGGCAAT CATCATTTGG GGATGTTGAA ACATAACCAA AATGGTAGCT 840
GCCGTTTTTT GTTATTAATT CTAATGATAA CATGGATTTA TCTGATTTAA ATCTGGTCAG 900
ATTTGTAACG ATTTGTGCTT GTTTCTGAGG TCGTTTAGGA CGACAATTAG CAGAGTTGTA 960
ATGCAGTTGT CTAGTGGCAG TTAGTGTCCG ATGACAGACA ACAGAAAATG AAAGAATCAT 1020
GCATCATTCA GTGCACTGGT AGCTGTTGAG CCAGGGGCGG TAGCATATTT AATTGTGAGA 1080
Orf1 is initial
GAACA
ATGAA GAGAATTATT ACGTTTGGTA CATTTGATGT TTTTCATGTT GGTCATGTTA 1140
ATATCCTTGA GCGAACTGCT TCACTTGGCG ACTATCTTAT TGTAGGTGTA AGTTCAGATA 1200
AATTGAATTT CAATAAAAAA GGTCGTTACC CCATTTATAA TCAAGAAGAC CGCTGTCGTA 1260
TAATAAATTC TCTTAGAGTT GTGAATGATG TATTTATTGA AGAATCTTTA GAGCAAAAAA 1320
AAGAGTATAT AATCCAATAC GAAGCTGATA TTTTAGTTAT GGGTGATGAT TGGGCTGGCC 1380
GATTTGATTG GGTAAATGAC ATTTGTGATG TTATTTATTT ACCAAGAACA CCATCAGTAT 1440
Orf1 stops orf2 to begin
CTACCACAGA AATTATTGAA GTTGTGAAGA CCCTCAG
ATG ACGAATTTCA AAAAAATAGC 1500
AAGAAAAATA ATTAGTAAAA GCATTTCATT GTTAAATTAT TTTATCCCCA AGAAAAAAAA 1560
TAGAATTTTT TTCAAAAGCA AACCTGACTA CTCAGGTAAT TGTAAGGCAC TAAGCGATTA 1620
TATTATAGAG AAAAAACTTC CATATCATAT CGTTTGGTCT GTTAAAAAAG AAATTAATCA 1680
AAAAGGAATT ACTGTAGTAA GAGCTGGTTC ATTGAAAGAA TTTTTCTATT ACTTTACAAG 1740
TAAATATGTT ATTACTACGC ATAATGAAAT GATAGGTCCA ATTGCAACAA ATCAAAAATA 1800
CATAAGCTTG TGGCATGGAA TGCCTTTTAA GAAAATTTGT TATCTTGGAG AGAATGATCA 1860
TCAAGGAATG ATAGATTATT CAGCCATTAG GATTGCCACT TCCGAAGTAA TGCGTTCTAT 1920
AATTTCAGCA AGCTTTCGTG AAAAAGCCAA TAATGTATAT ATTACAGGCC AGCCTCGTAA 1980
TGATTTTTTG TTCAAACCAA TTAGTCTGAC AGATATTGGT ATTAAATCTA TAAAGAACAA 2040
AAAAATTGTA ATGTTTGCTC CAACATTTCG TATGAATAAT GAAGATATAA GATATTCTGA 2100
TGGAGCGGAA ATTATTGATA ATAATTTTCT TCGAGTAAAT GATTTCTGCA TGGAAGAGAT 2160
AGATTATTAT CTTGAACAAA GCAATTTACA TTTGATATTA AAATTGCATC CATACGAGGA 2220
GGAATATTTC CGCGGGATCG CAACACTAAG TTCGAATATA ACTATTATAA GTTCTGATGA 2280
ACTCACGCAA AAAAATATCG ATTTGAATCA GTTGCTTTCC TTGGTCGATA TTTTAATAAC 2340
AGATTACTCA TCAATTTATT TAGATTACTT AATTCTAAAT AAACCTCTAA TTTTTTTAGT 2400
TCCTGATGTA GATGCTTATA GTTCTGCACG CGGTGGGTTT ACTTTAGAAC CTTTTGATTT 2460
TTGGACGCCA GGAGATAAGG TTAGCTGTCA AAGGTCATTA TTGAATTCAA TAAATAAAAT 2520
AATCACTGGA AATGATGAGT ATGCTGAAAA ACGTAACCAA ATAAATCTCA TAATAAATAA 2580
The initial orf2 of orf3 stops
ATACTCTGAT GCTAATAATA GCCAGAGAGT CATTGAATTG ATGAAGAGTT TATC
ATGAAA 2640
ATATACATTT TCTTAGGAGA TTTAAGTTCG AAAGGTGGTA TTGAGCGTGT TTCAGTAGCA 2700
TTAGCCAATG GATTGGCTAA ATTTTATGAT GTCACTATCA TTAGCTTATA TCGTGCAACT 2760
AGAAATTTAT CATTTGTCCC GGATGAAAAG GTCAATGTGA TCTATTTGTA CGATGAATTT 2820
GAAAAGAGTA TGTATAATCG TAATCTGGGA GCTATCATAG GGTTAAAGTT TGATTTCTTA 2880
TATATTATAA AAAAGTTGAA GCAACTTAAA AGATTAAATT TAGAACTAAA TAAAAATGAT 2940
GTGCTTATTT CAAGCGATAT TAAAATGTCT CTGCTATTAT TTTTCTATGC AAAAAAAAGC 3000
AAAATTATAG CTATTGAGCA TTTTGAACAT GATGTTGGTA ATTTAGTGTT GAGAAAAATT 3060
AGAAGCGCAC TATATCCTAA ATTATCAGCA GTTGTATCGC AAACTGGCGA AGATCAAATT 3120
AAGTATTTGC AATGGTTACC TAGAAAGAAA CATAAAATCA TTCCAAATAT TATTAGCTTC 3180
GAAGCGACCG ATATACCGCA AAATAAAATA GAACAAAAAA ATGTACTTGC TGTGGGACGA 3240
TTAACTCATC AAAAGGGTTT TGATTTACTT CTACAAGCTT GGGCAGACGC AAATACTCAT 3300
GATTGGCGCT TAAAGATCAT TGGAGACGGA GAAGAGCTGA ACCATTTAAA TTCTCTAATT 3360
ACCGAGTTAA ATATCTCTAA CGCTGAAATT ATCCCTTTTC AAAAGGATAT TCAAAGGCAT 3420
TATTCTTCTG CAGGAATATT CGTACTTTCT TCTCGCTTTG AGGGTTTGGG TATGGTGCTT 3480
TTAGAAGCTC TTAGCAGCGG CTTGGCGTGT ATTAGTTTTG ATTGTCCAGC TGGTCCTAAA 3540
AGTATAATCT CAAGTGATAA TGGGGTGTTA GTTCCAACTG GTGACACTAT AAAATTATCA 3600
CAAGCTATTT CTTTCTTGAT AAATAACGAA GAAGAAAGAA AACGGTTACA AAACAAAGCT 3660
GCTGCTTCTG TTGAAAAATT CAAAGAATCA AACGTAATTG CAAAATGGCG AGAGTTATTG 3720
The initial orf3 of orf4 stops
A
ATGAAATTT CA
TAGAAAAC AATATGATGC AGTATTTCTT ACAAACGTAC CTGCTTTCTA3780
TAAAGTAAAT TTGTTCAACG AAATTCAAAA AAATAAAGAT ATTTTTGTTA TATTCGTTTC 3840
AGATAAGTCG AAAATTAGGA ATGAAGATTT TTATTCATAC GACTTTGCGT TTGACCACTT 3900
TTTTCTTAAT GAAGAGAACT TTGAAGAACG AAGTAAAATA AAAACACTAT TCAATTTACT 3960
TAGAGTGTTA CGAACGATTT CGTACAAAAA AATTATATAT TCAGGATGGG AAGTTAAAGA 4020
AGTAACTCTA GCTGCATTAT TTAATAAGCG GGAAAAAAAT GCAATAGTGA TCGAAAGTAG 4080
CATTATTGAA ACGAAGAAAA CTGGTTTGAC ATGGCTTCTA AAAAAAATAG CTATCGGTCG 4140
TATGTCGCTT GCTTACCCAT CAGGGCTATT GCAAAAAGCA ATATTGGAGA CATTCTCTTT 4200
TAAAGGAAAA ACTGTTATTA CCCATGGCGT TGGTATCTCT AATTTAAAAG AGACCAGTTT 4260
TCATAATAAA AAAACATGTA CAAGGAATAA TCCTCTTCGT TTCATCTATG TTGGAAGGAT 4320
TTCATCGGAA AAAAATATAG ATTTTATGGT GAAAGTATTT AAAACTCTTC CTTACGAATT 4380
GATACTAATT GGAGATGGTC CATTAAAAAA ACAGTTTGAC GATAAAACCT ATAGCAATAT 4440
CAGATTTTTA GGCTATATTG ACAATAAAAA ATTATCAAAA GAACTACTTA AAAGTGATTG 4500
TTTTATTCTT CCGTCATTAT CTGAACCGTG GGGATTAGTA GTAGAGGAGG CTTTGACATT 4560
AGGGCTTCCG GTTATAGTCA GTAACCATGT GGGCTGTCAT AGTGATTTAG TCAATGATAG 4620
AAATGGCATT ATATTCGATG TGAACGATAC ACAATCCTTT ATTGACGCAT TGTCGAAAAT 4680
GGAAAAAAAT TATGAACGCT TTGCCCGTGG TGCCAGCGAA TTCCATGCTA GTGAAATAGC 4740
It is initial that orf4 stops orf5
AAAAGCACAG ATCGATGCTT ATGTAGGTAG CATC
TGATGA GCAACAAAAA CAAAACATTT 4800
AAAGATTTTA TAAATTATTT TTTAGGCGAT CTTTTTGTCA AAGGATTTAT GTTTATCTCG 4860
CTACCTCTAC TTTCGAGAAT AATGTCGCCA GAAGATTATG GGCGGATGTC CCTTATAAAT 4920
GCAGCTGTTA TGATTTTATA TGTTTTTATA AGTTTAAATC TCCAAAATGC TGTTATCAAC 4980
GCTTACATGA AAAATGAAGT TGATTTTCCA GTATATCTAG GTAGTGTATT ATGGGGATTG 5040
ACCGCCGCGC AAGTATTGTT AGTCGCTTTA AGTATATATT TTGCTGTTCC GCTTGGAATG 5100
TTATTGAGTA TTAGCAAATA TGATGTTTAT TGGGTTGTCG CCATTTGTAT TTTGCTAAGC 5160
TATATATATA TATATACTAG CTATCTACAG GGTGCACGGC TCAGTTCTAG CTTTGTCAAA 5220
CTGAATATAT TTAGTAAGAT ATCAGAAGTT GTTGTCATTT TTGTCTTTGC TTGGTTTTTA 5280
ACTCAAGATA AATACCTTGC TAAGGTATAT GCTCAGTTAG TGATTAGCTT TATTTTACTG 5340
ACATATGTGT TGAAGAAACT AAAAAAGATA GCTGTATTCA AATTTAATGT ACGATATTTT 5400
ATATCAGCAC TTGCATTTAG TTCTCCATTA ATTATACATG TTCTTTCAAA TGCTTTGCTT 5460
TCACAAGTTG ACCGATTGTT TATCGCAAAG ATGTTGGGAG AGGGGCAGGC TGGTATATAT 5520
TCTTTCGCAT ATAATATCGG AATGTGTATA TTAGTGGTTG TTATGGCCTG GAACTCTTCG 5580
TGGCAACCTA AATTATATAA GCTTATTGAT TCGAAGGATA ATGGTAAAAT AATTCGAATT 5640
GTCGATGTAA GTTCCTTATT ATTATTAATA GTATCATTTT TATCTATTCT TTTTTCAAAA 5700
CAGATGGTTG AAGTCCTTGC GGACAATCGC TATAGGGAAA GTATATCCGT CGTTCCTGTC 5760
ATATTGATTG GTAACTCTCT GATTCATATT TATTTAAGCT ATGTTAACTT TACTTTCTAT 5820
AAGAAAAAAA CAATATATGT TTCAATTGGT ACATTGCTTG CGGTAGCGAT AAATATTGCT 5880
TTGAATTATA TACTCATACC AATATACGGT ATCCACGGAG CAGCATGGGC TACAGTAATA 5940
GCTTATTTTA TGCTGGCATT TTTCCATTAT CTCATAGCAA CAATAATGTT AAAGGCAAAC 6000
CCACTCTCAT TGTTTCTGTT ATTATGGTAT TCAGCTTTGT TATTGGCTTC GTATTTCTTA 6060
GTAATATACC TTGACTCTTT GTCTCTTTGG ATCTCTTTAT CAATAAAGGC AATGATCATT 6120
It is initial that orf5 stops orf6
TTTATTATCC TCATCATCCT TATGAAAACA AAAATCTATA ATGAAT
TAAA GGA
ATGACAG6180
TGTCGATATA TCTTCTGCTT CTAGCTTTCG TATTTCTGCT TGCAATGTCA GATTTTTTTA 6240
TCATTGCTGA TGCCAGAAAT CGTTTTTTAC TTTATATAAT ACTATTTTCG CTATTAGTAA 6300
CCTTTATAGG TTTAAGATAT CAAACTGGAC TTGATTGGTT ATTTTATAAC AATCTATTTA 6360
ATGGAGAGGG TTTTTCATTA GCCATTGAGC CTGGATATTA TTTTTTCTCG TATGTTTCAT 6420
CTTTTTTAAT GGGGTATTGG ATTTATCAGG CATTAATAAC CGCCGTTCTA ATAATATGTT 6480
TAAAAACATT CTTTGAAAAA AACACTAAAA ACTATCTGTT TTGCATAGGT TTTTTCTTCT 6540
TATATCAATT TATTTTCGTG ACGGAAGCAA TACGGCAAAT AATCGCTCTG TCAATTATTT 6600
TGGTTGCATA TAAAAAGTTT TATGATGGTA AGAAATTGCA ATTTCACATG CTTACCATTT 6660
TGGCTTGTTC ATTCCATATT TCTGCTGTAA TTGTCTTTAT TTTGATTCCG TTTTTAAAAC 6720
GCAGAAATAT ATATATATTA AAAATACTGA CAATCGTTGG TTTAGTTTTG GCAATTTTTA 6780
GTGTTTATCC TGTTGACTAC TTAATACAGT TACTGTCATT GCTCCCTGCT GGTGGTTATA 6840
TAGAAAAGAT AAGATGGTAT AGTCAGGATG ATTATGCTGG ATCAGTACTT ACGTTTTCAT 6900
TAGTATTCAA AGTTTTTGTT GTGCTTTTAT TCGATTACAG ATTTAAATCA ATAAAATCAC 6960
ATGGTCAAAG TCTTATTAAT GCGAGGGCAT ATGATTTTAT TTATACTTCG GTTTATTTAA 7020
TGATATTCAT GGACGTTTAT CTTGGTAGGT TTGGTACTAT TAGCACCAGG CTTGACGTAT 7080
ACTTTATACC ATGTTTTTTA ATAGCTCTTA ATCATTTAAT AAATGAACAC AAACAAGGTG 7140
TGAGTCGTTT CATATTCTTC TTCGTTGTCA TGGTTTACTT TACTATAAAT TATCTCAGTA 7200
Orf6 stops
TTATGAACGG ATATTACTTC GAGAAATTTT ACAGTCCCTT ATCAAAATTA TATAAC
TGAA 7260
TTTTTAAATC CGGGCAGTTA TAGTGATAGA GGGTGGGATG TTAGATATTA TTTCAGCAAT 7320
Orf7 stops
AAGGAATTAT TGC
AGTGAAT CTTTTGATTA ATGCTAGTAA TCTGTATGTT GGTGGAGGAG 7380
TCCAGGTAGC AATTTCTGTA CTGGAAGAGT TATCTGATTC ATCATTTTCT TTTATTGCGG 7440
TTGTTTCACC AGTTGTTTAT TCGCAGTTAA GCGATGATGC AGCATCATGT TGTATAGTGA 7500
TTGAATCTTC TCCATCAAAA TTGTTGAATT TTAAAGTTAG AAGGCAACTC GACGATATAG 7560
TTAAAAAAAA TGATATTTCT GTAGTTTTCA CAATCTTCGG TCCTAGCTAT TGGTCTCCGA 7620
AAAATGTTAA ACATGCTATT GGTTTTGCGC TCCCTTGGTT GATTTATGAT ATTGAATATA 7680
TATTTAAAAA ATTGACTTTA AAAGCTAAAT TGAAATTTTG CATTTTAAAA TTATTACAGC 7740
CATATTACTT CAAGAAAAAT GCCGACTTAA TTTTTACTGA AACAGATGAT GTCAACTTGC 7800
GGGTAACAAA ACTTCTTAAC TTTGAAAAAG AACAAGTCTA TACTGTTTCA AACACACTTA 7860
ATGGTTTATT AAAAAATTCA AACTGTTATG ATTACAGCAT TTTGGATAGA TTACCTACGA 7920
AAGAACCAAA TGATATTTGG TTGGTAACTA TTTCTCATAA TTACCCTCAT AAGAACTTAG 7980
AAGTCATTAA AGAATTAGTA ACAGTATTGC CACCTTGTTA TAAGTTTATT TTAACAGTTT 8040
CAAGTGATTT TTTGCAGCTA GTCCCAAAAG AGCATAGAGA GCGAGTCATC ACCATAGGTA 8100
ATGCAACACT TAGTCAATGT GCTCCGTTGT ATGAAGTTTG TGATGGATTA TTCATGCCAA 8160
CACTTTTAGA ATGCTTTAGT GCTTCTTACT TAGAAGCAAT GTACATGAAA AAAATAATCT 8220
TCACTTCTGA TCTGCCTTTT GCTCACACTG TTTGCAAAGA TGCAGCATTT TACTTTGCTC 8280
CTCATGATGT TGAAAATATT AGGAGTACAC TTGTTAATGG CTTTCAAAAC AAAGAAATTC 8340
TTAACCATAA ATTAAATGAA GGTTCGAAAA TTTATGAAAG TTTTCCTTCT GCGAAAGCTC 8400
Orf7 stops
GAGCATTGCA GTATATAGAT ATAATAAAAT CCAACTTGGT A
TAAATTGTA GATTTTGAGG 8460
Orf8 is initial
TGTTAAAA
AT GTTTAAAGAT AAGGTTTTAC TTATTACTGG CGGAACAGGG TCTTTCGGTA 8520
ACGCTGTACT TAATCGTTTT CTTGAAACTG ATATTAAAGA AATAAGGATT TTTTCTCGCG 8580
ATGAAAAAAA ACAAGACGAT ATGCGGAAAA AATATAATAA TTCTAAGTTA AAATTCTACA 8640
TTGGGGATGT AAGGGATTAT TCCAGCATTC TAAGCGCCGC ACGTGGCGTC GACTTTATAT 8700
ACCATGCTGC TGCATTAAAA CAAGTTCCGT CATGTGAATT TCATCCTCTG GAAGCAGTGA 8760
AAACTAACGT ATTAGGTACA GAGAATGTGC TTGAAGCAGC AATTGCAAAC CAGGTTAAAC 8820
GTGTCGTTTG CTTAAGCACG GATAAGGCAG TATATCCGAT TAATGCCATG GGTATTTCCA 8880
AAGCAATGAT GGAAAAAGTG ATGGTCGCAA AATCACGTAA TGTTAATAGC GACAAAACTG 8940
TGATTTGCGG TACTCGCTAC GGCAACGTGA TGGCTTCACG TGGTTCCGTT ATTCCCTTAT 9000
TCGTTGATCT GATCAAAGCG GGCAAAGCGC TGACGATCAC TGACCCAAAT ATGACCCGTT 9060
TTATGATGAC GCTTGAAGAT GCTGTCGATC TGGTCCTATA TGCGTTTGAA CATGGTAATA 9120
ATGGCGATAT ATTTGTCCAA AAAGCGCCTG CTGCAACGAT TGAAACATTA GCCATTGCTC 9130
TCAAAGAACT TCTTAATGTT GAGCAACATC CTGTTAATGT TATCGGAACG CGCCATGGTG 9240
AGAAACTTTA TGAAGCGCTG CTTAGCCGTG AAGAGATGAT TGCCGCCATT GACATGGGGG 9300
ATTACTACCG TGTTCCACCA GACCTGCGCG ATCTTAACTA TGGAAAATAC GTAGAACAAG 9360
GTGATAGCCG AATCTCAGCA GTAGAGGATT ATAACTCTCA CAATACACAG CGACTGGATG 9420
TTGAAGGGAT GAAAACGCTT CTTTTGAAAT TACCTTTTAT TCGTGCACTG CGTGCGGGTG 9480
It is initial that orf8 stops orf9
AACATTACGA TCTGGATGCC
TAAT
ATGAAA ATCCTGATTA CTGGAGCTGA TGGTTTTATT 9540
GGACGTAACC TGTGCTTACG CCTTCAGGAA GCAGGCTACT GTGACCTTGT TAAGATTGAC 9600
CGCGGTTCAA GTGCGGCTGA TCTGGAAACT GGCCTTCAAG ATGCTGATTT TGTCTATCAT 9660
CTCGCAGGTA TCAATCGACC TAAAAACGTT GATGAATTTG CCGAGGGGAA TAGCAATCTG 9720
ACTCAACAGA TTGTTGATTA TCTTTTAGCC AAGCATAAAA GCATACCTAT TATGATCAGT 9780
TCTTCCATTC AGGCTGAACT GGTTAATGCT TATGGTCAAA GTAAAGCTGC AGCAGAAAAA 9840
CATATTGAAC GCTATGCAGC TGAAAGCGGT GCAGCTTATT TTATTTATCG TTATCCGAAT 9900
GTTTTTGGTA AGTGGTGTAA GCCTAATTAT AATTCGTTCG TGGCGACCTT TTGCCATAAT 9960
ATTGCCAACA ATATCGATAT CACGATCAAT GACTCCTTCG CGCCTGTTAA TCTTGTTTAT 10020
ATTGATGATG TCTGTTCTGA TGCGATAAAG CTCTTGTCTG GAAAGGTTGA AAGCGGGTAC 10080
AAAACTGTTA AGCCAGTATA TTCGACGACA GTAGGTGAGG TGGCGGAATT ACTTTATCGC 10140
TTCAAAGAAA GCCGTTCCAC TCTTGTCACC GAGGCTGTAG GAACAGGGTT CATCCGCGCG 10200
CTGTATTCGA CGTGGTTAAG TTATCTCCCA GCCGATATGT TTGCGTATTC AGTTCCCTCT 10260
TACGGAGACG CCCGAGGGGT TTTTTGTGAA ATGTTAAAAA CCCCTTCAGC GGGGCAGTTT 10320
TCATTTTTTA CAGCGCATCC CGGCATTACA CGTGGGGGTC ATTATCATCA CACCAAAAAT 10380
GAGAAGTTCC TGGTCATTCG CGGCCAGGCA TGCTTTAGGT TTGAACATGT GATTACCGGT 10440
GAGCGATATG AGATGAATGT TTCCTCAGAT GAGTTCAAAA TCGTTGAGAC AGTCCCCGGC 10500
TGGACACATG ATGTTACAAA TATTGGAGCG GATGAATTGA TAGTCATGCT GTGGGCAAAC 10560
It is initial that orf9 stops orf10
GAAATTTTCA GTCGCGATGA GCCTGATACT ATTGCGAGAC CTCTG
TAATG AAAAAACTAA 10620
AAATTATGTC TGTTGTTGGT ACGCGTCCCG AGATTATCCG TCTGTCACGC GTTCTCGCTA 10680
AGCTTGATGA ACACTGCGAG CATATTCTTG TCCATACTGG TCAAAACTAT GATTATGAGT 10740
TAAACGAAGT ATTCTTTAAT GACCTTGGTG TACGAAAACC CGATTACTTT TTAAATGCTG 10800
CTGGGAAAAA TGCTGCAGAA ACCATCGGTC AGGTTATCAT CAAAGTTGAT GAGGTACTTG 10860
AATCCGAAAA GCCTGAAGCA ATGTTGGTGC TGGGTGATAC AAACTCCTGT ATTTCTGCAA 10920
TTCCAGCCAA ACGCCGTAAA GTGCCTATCT TCCATATGGA AGCTGGCAAT CGTTGTTTCG 10980
ATCAGCGCGT CCCGGAAGAG ACCAATCGGC GCATTGTAGA CCATACCGCT GACATCAATA 11040
TGACCTACAG CGATATTGCA CGTGAATACC TTCTTGCTGA AGGCCTCCCA GCGGACCGAA 11100
TTATTAAAAC CGGTAGCCCA ATGTTTGAGG TACTTACGTA TTATATGCCT CAAATCGATA 11160
ATTCGGATGT ACTGTCGCGT CTGAACCTGC GTTCAGGCGA ATTTTTCGTC GTCAGCGCGC 11220
ATCGTGAAGA GAATGTTGAT TCTCCCAAAC AGCTAGTCAA GCTTGCGACT ATTCTCAATA 11280
CTATTGCTGA AAAATATGAT TTGCCGGTTA TTGTATCCAC TCATCCGCGG ACACGTAATC 11340
GTATTAACGA GCAAGGGATT GAATTCCATC CAAATATTAA TCTACTGAAA CCGTTAGGTT 11400
TCCATGATTA CAACCATTTG CAGAAAAATT CACGTGCTGT GCTGTCTGAC AGCGGTACGA 11460
TAACTGAAGA ATCTTCCATT ATGAATTTCC CGGCGGTAAA TATTCGGGAA GCACATGAGC 11520
GTCCAGAGGG CTTTGAAGAA GCCTCTGTCA TGATGGTGGG CCTGGATTGT GAACGTGTTC 11580
TACAGGCACT GGATATTCTG GCAACACAGC CCCGCGGCGA AACCCGTCTT TTACGACAAG 11640
TAAGTGACTA CAGCATGCCT AATGTGTCAG ATAAAGTCGT CAGAATCGTT CATTCTTATA 11700
It is initial that orf10 stops orf11
CTGATTATGT CAAGCGAGTC GTCTGGAAAG AATAC
TGATG AAACTTGCTT TAATCATAGA 11760
TGATTACCTG CCCAATAGTA CGCGAGTTGG TGCAAAAATG TTTCATGAAT TGGCTCAGGA 11820
ATTTATTCGC CGTGGGCATG ATGTTACGGT AATTACACCT GACATCTGTC TTCAGGATGA 11880
TGTGTCCTTT AGCACCTTCC AGGGGGTCAA GACATGGCGT TTCAAAAGTG GGCCTCTCAA 11940
GGATGTGAGC AAAATTCAAC GAGCCATCAA TGAAACACTT CTATCCTATC GTGCCTGGAA 12000
TTCCATTAAA AGCCAGATAA AAAAAGAGAC TTTTGATGGG GTAGTTTATT ACTCACCCTC 12060
CATTTTCTGG GGGCACTTAG TCAAGAAAAT TAAATCTCGT TGCCAGTGTC CGGCTTACCT 12120
GATTTTGAGG GATATGTTCC CGCAATGGGT GATAGACGCT GGCATGTTAA AAGCCGGTTC 12180
CCTCATCGAA CGCTATTTCC GTGTTTTCGA AAGATCATCT TATCGCCAGG CAAACCGTAT 12240
CGGGCTGATG TCAGATAAGA ATCTTGAGGT CTTTCGGGTT AACAACAAAG GTTATCCTTG 12300
TGAGGTTTTG CGTAACTGGG CTTCTCTCAC GCCGACGGTA CCACCCCAGG GTTATATCCC 12360
ATTGCGTCAG CGTCTTGGCC TTGATGATAA AGTTATTTTC TTCTATGGAG GGAATATCGG 12420
TCATGCGCAG GATATGGGGA ATCTGATGCG TCTTGCTCGA AAAATGGCTG AGCATCCGCA 12480
AGCCCATTTT TTATTTATCG GGCAGGGGGA TGAAGTTGAA TTAATCAATT CTTTGGCTGC 12540
CGAGTGGTCA TTGCCCAACT TTACGTATTT GGCCTCTGTC AATCAGGATG AATTTAAATT 12600
TATCCTCTCG GAAATGGATA TAGGCTTGTT TTCACTTTCC GCCAGACATT CTTCGCATAA 12660
TTTCCCGGGT AAATTGCTAG GGTATATGGT TCAGTCCTTA CCTATACTAG GCAGTGTGAA 12720
TGCTGGTAAC GATTTGCTTG ATGTCGTCAA TCAAAATAAT GCCGGATTAA TTCATATCAA 12780
TGGTGAGGAT GACAAGCTTC ATGAGTCCGC ACTGTTAATG CTTAAGGATG TTGCTGCGCG 12840
ACGTCAATTC GGCTTAGGCG CGAATGCATT ATTGAGAGAA CAGTTCTCCG TTGAGTCTGC 12900
The initial orf11 of orf12 stops
GGCACAGACT ATAGAAATGA GGTTAGAGGC ATGTA
ATGCG ACTCAT
TGAT ACTCACCAAC12960
TTGAAGCTTT ATACGAACAA GCCGGAAAAT CTGCACGCTT GCGCGCTCAT CTCTTATTGC 13020
ACAATTCGCA CCGAGAGAAA GTGCAACGTC TGCTCATTGC CTTGGTTCAG GGCAGCTATG 13080
TCGATCCGCA TTTCCATGAA CTTCCTCATC AATGGGAGAT GTTTGTCGTT ATGCAGGGGC 13140
AGGTTCAAGT CTGTTTGTAC GGCAAAGATG GCGAAATCAT TAATCAATTT GTCGCTGGAG 13200
AGAATACTGC AATAAGCGTA GTCGAGTTTT CTCCGGGTGA TATACACAGT GTCGAATGTC 13260
TCTCTCCGAG AGCATTAATG ATGGAAGTGA AAGAAGGGCC TTTTGATCCT TCCTTTGCCA 13320
Orf12 stops
AGGCGTTCAT C
TAACGCCCC TCTGAATCGC ATCTTCCGCT ATCTACTCAG GCTCATCCTG 13380
AGTTAACATC TAAGCCACAT TTCAAGCCGC GCACAGTCGC GGCGACCACA CCTGACAGGA 13440
GTATGTAATG TCCAAGCAAC AGATCGGCGT TGTCGGTATG GCAGTGATGG GGCGCAACCT 13500
GGCGCTCAAC ATCGAAAGCC GTGGTTATAC CGTCTCCGTT TTCAACCGCT CCCGTGATAA 13560
GACCGAAGAA GTCATCGCTG AGAATCCGGG TAAGAAACTG GTTCCTTTCT ATACGGTTAA 13620
AGAGTTTGTT GAATCTCTGG AAAGGCCTCG TCGTATCCTG TTAATGGTGA AAGCGGGCGC 13680
AGGTACCGAT GCAGCCATCG ATTCCCTGAA ACCTTATCTG GACAAAGGCG ACATCATCAT 13740
TGATGGTGGT AACACCTTCT TCCAGGACAC CATTCGTCGT AACCGTGAAC TCTCTGCTGA 13800
AGGTTTCAAC TTCATCGGTA CCGGTGTTTC CGGTGGTGAA GAGGGCGCGC TGAAAGGCCC 13860
ATCTATCATG CCTGGCGGCC AGAAAGAAGC GTACGAGCTT GTTGCGCCAA TCCTGACCAA 13920
AATCGCTGCG GTTGCGGAAG ATGGTGAGCC GTGTGTGACC TATATCGGTC CGGACGGTGC 13980
AGGCCACTAT GTTAAGATGG TTCACAACGG CATCGAATAC GGCGATATGC AGCTGATTGC 14040
TGAAGCCTAC TCTCTGCTGA AGGGGGGCCT GAACCTTTCC AACGAAGAAC TAGCAGAGAC 14100
CTTCACTGAG TGGAACAAAG GCGAGCTGAA CAGCTATCTG ATCGACATCA CCAAATATAT 14160
CTTCACGAAG AGAGATGAAG AGGGTAAATA CCTGGGTCGA TGTGATTCCT TGACGAAGCC 14220
GCCGAACAAG GGTACCGGCA AATGGGACCA 14250
The above, it only is preferred embodiment of the present invention, be not that the present invention is done any pro forma restriction, every foundation technical spirit of the present invention all still belongs in the scope of technical solution of the present invention any simple modification, equivalent variations and modification that above embodiment did.
Claims (4)
1, a kind of oligonucleotide of the O-antigen-specific to intestinal bacteria 29 types, it is characterized in that: its wzx sequence is the Nucleotide of 4778 to 6178 bases among the SEQ ID NO:1 or Nucleotide that the wzy sequence is 6175 to 7260 bases among the SEQ IDNO:1 or describedly has above-mentioned sequence and insert, lack or replace one or more Nucleotide, but keeps the oligonucleotide of the O-antigen function of wzx, wzy Nucleotide specific detection intestinal bacteria 29 types.
2, a kind of oligonucleotide of the O-antigen-specific to intestinal bacteria 29 types, it is characterized in that, it is that to come from the oligonucleotide of wzx gene right: the Nucleotide of 4891 to 4900 bases among the SEQ ID NO:1 and the Nucleotide of 5571 to 5589 bases, the Nucleotide of 4858 to 4876 bases among the SEQ ID NO:1 and the Nucleotide of 5551 to 5569 bases; Or the oligonucleotide that comes from the wzy gene is right: the Nucleotide of 6575 to 6593 bases among the SEQ IDNO:1 and the Nucleotide of 7201 to 7221 bases, the Nucleotide of 6455 to 6473 bases among the SEQ IDNO:1 and the Nucleotide of 7060 to 7078 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 29 types of 2.
4, according to the application of the oligonucleotide of claim 1, any described O-antigen-specific to intestinal bacteria 29 types 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.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410019023 CN1261574C (en) | 2004-04-19 | 2004-04-19 | Nucleotide peculiar to 0-antigen of 29 type bacillus coli |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410019023 CN1261574C (en) | 2004-04-19 | 2004-04-19 | Nucleotide peculiar to 0-antigen of 29 type bacillus coli |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1563038A CN1563038A (en) | 2005-01-12 |
| CN1261574C true CN1261574C (en) | 2006-06-28 |
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