CN1316026C - Nucleotide specific for escherichia coli 0134 O-antigen - Google Patents
Nucleotide specific for escherichia coli 0134 O-antigen Download PDFInfo
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- CN1316026C CN1316026C CNB2004100941001A CN200410094100A CN1316026C CN 1316026 C CN1316026 C CN 1316026C CN B2004100941001 A CNB2004100941001 A CN B2004100941001A CN 200410094100 A CN200410094100 A CN 200410094100A CN 1316026 C CN1316026 C CN 1316026C
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Abstract
The present invention provides a specific nucleotide for an O-antigen of Escherichia coli O134. The specific nucleotide is the nucleotide complete sequence of gene clusters in Escherichia coli O134 for controlling the synthesis of an O-antigen, such as a separated nucleotide with the total length of 7530 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 oligonucleotide from oligosaccharide unit processing genes in the O-antigen gene clusters of Escherichia coli O134. The present invention verifies that the oligonucleotide has high specificity to all of the O-antigen genes of Escherichia coli O134 through PCR. The present invention also discloses a method for detecting and identifying Escherichia coli O134 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 the intestinal bacteria O134 type (Escherichia coli O134), particularly relate in the intestinal bacteria O134 type oligonucleotide in the control O-antigen synthetic gene cluster, can utilize these the oligonucleotide of the O-antigen-specific intestinal bacteria O134 type in human body and the environment and identify O-antigen in these pathogenic bacterium quickly and accurately.
Background technology
O-antigen is the O specific polysaccharide composition in the gram negative bacterium lipopolysaccharides, and it is made up of many multiple oligosaccharide unit.The antigenic building-up process of O-is studied clearlyer: by glycosyltransferase nucleoside diphosphate monose is transferred on the fat molecule that is fixed on the cell inner membrance earlier, then in the inboard synthesis of oligose unit of inner membrance, the antigenic oligosaccharide unit of O-is transferred to the inner membrance outside by the transhipment enzyme again, then aggregate into polysaccharide by polysaccharase, be connected to again on the glycolipid molecule and form lipopolysaccharide molecule [Whitfield, C. (1995) " Biosynthesis of lipopolysaccharide O antigens ", Trends inMicrobiology.3:178-185; Schnaitman, C.A.and J.D.Klena. (1993) " Genetics of lipopolysaccharidebiosynthesis in entericbacteria " .Microbiological Reviews, 57 (3): 655-682].Coding is responsible for the generally adjacent arrangement on karyomit(e) of gene of all enzyme molecules of O-antigen synthetic, form a gene cluster [Reeves, P.R., etal. (1996) " Bacterial polysaccharide 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-antigen loci:implication forEscherichia coli and Shigella relationships " .Infection and Immunity, 11:6923-6930 bunch between galF and gnd gene; Lei Wang andPeter Reeves (2000) " The Escherichia coli O111 and Salmonella enterica O35 gene clusters:geneclusters encoding the same colitose-containing O antigen are highly conserved " .Journal ofBacteriology.182:5256-5261].The O-antigen gene bunch contains three genoids: sugared synthesis path gene, glycosyltransferase gene, oligosaccharide unit treatment gene, the required nucleoside diphosphate monose of enzymic synthesis O-antigen of wherein sugared synthesis path genes encoding; Thereby the enzyme of glycosyltransferase gene coding forwards nucleoside diphosphate monose and other molecule to and makes monose aggregate into oligosaccharide unit on the monose; The oligosaccharide unit treatment gene comprises transhipment enzyme gene and pol gene, and they transfer to the bacterium inner membrance outside with oligosaccharide unit, and repolymerization becomes polysaccharide.Glycosyltransferase gene and oligosaccharide unit treatment gene only are present in the gene cluster of carrying these genes.The difference of monose in the O-antigen, between monose between the difference of link button and the oligosaccharide unit difference of link button constituted the antigenic diversity of O-, and the composition of monose, the link button between monose and the link button between the oligosaccharide unit are by the Gene Handling in the O-antigen gene bunch, so the O-antigen gene bunch has determined O-antigenic synthetic, has also determined the antigenic diversity of O-.
Because O-antigen is extremely strong antigen, be one of important paathogenic factor of intestinal bacteria, it has extremely strong diversity again simultaneously, and this enlightens us can study a kind of intestinal bacteria and good, highly sensitive method of the antigenic specificity of O-thereof of detecting quickly and accurately.With surperficial polysaccharide is that the serology immune response of target has been used to somatotype and the evaluation to bacterium always since the thirties in last century, is unique means of identifying pathogenic bacterium.This diagnostic method needs a large amount of antiserum(antisera)s, and the antiserum(antisera) general classes is incomplete, quantity not sufficient, and also there are some difficulties in a large amount of antiserum(antisera)s in preparation with in storing.On the other hand this method length consuming time, sensitivity is low, loss is high, poor accuracy, so, generally believe that now this traditional serology detection method will be that the modern molecular biology method replaces.1993, Luk, J.M.C et.al has identified the O-antigen [Luk of Salmonellas with the specific nucleotide sequence of Salmonellas (S.enterica) O-antigen gene bunch by PCR method, J.M.C.et.al. (1993) " Selective amplification of abequose and paratosesynthase genes (rfb) by polymerase chain reaction for identification of S.enterica majorserogroups (A; B; C2; andD) ", J.Clin.Microbiol.31:2118-2123].Luk, the method for et.al is with corresponding to Salmonellas serotype E 1, D1 obtains the oligonucleotide special to the Salmonellas of different serotypes after the nucleotide sequence of the CDP-abequose in the A, the O-antigen of B and C2 and the synthetic gene of CDP-tyvelose is arranged.1996, Paton, the A.W et.al serotype [" Molecularmicrobiological investigation of an outbreak of Hemolytic-Uremic Syndrome caused by dryfermented sausage contaminated with Shiga-like toxin producing Escherichiacoli " .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.and Reeves of a supposition, P.R. (1995) Sequence and analysis of the O antigen gene (rfb) cluster of Escherichia coli O111.Gene164:17-23], and in the antigenic structure of the O-of other bacterium, also has this sugar, so sugared synthesis path gene is not that the Shigellae of high special has 46 kinds of serotypes for O-antigen, 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 specific selection andbacterial 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 andEwing ' s identification of the Enterobacteriaceae " .Elsevier Science Publishers, Amsterdam, TheNetherlands; T.cheasty, et al. (1983) " Antigenic relationships between the enteroinvasiveEscherichia coli antigens O28ac; O112ac; O124; O136, O143, O144; O152 and and Shigella Oantigens " J.clin Microbiol, 17 (4): 681-684].
Summary of the invention
The Nucleotide that the purpose of this invention is to provide a kind of O-antigen-specific to intestinal bacteria O134 type.It is the Nucleotide in the O-antigen gene bunch of intestinal bacteria O134 type, is the special Nucleotide that comes from glycosyltransferase gene and transhipment enzyme gene and pol gene.
A time purpose of the present invention has provided the full length nucleotide sequence of the O-antigen gene bunch of intestinal bacteria O134 type.
Another object of the present invention has provided the gene of the O-antigen gene bunch that constitutes intestinal bacteria O134 type: the gene of transhipment enzyme is the wzx gene or with wzx the gene of identity function is arranged; Pol gene is the wzy gene or with wzy the gene of identity function is arranged; Glycosyltransferase gene comprises orf1, orf3, orf5, orf6 gene.
Another purpose of the present invention has provided oligonucleotide, and the gene that they come from coding transhipment enzyme in the O-antigen gene bunch of intestinal bacteria O134 type respectively is the wzx gene or with wzx the gene of identity function is arranged; The gene that comes from the coding polysaccharase is the wzy gene or with wzy the gene (table 1) of identity function is arranged; They are the oligonucleotide in the said gene, and length is at 10-25nt; They are high specials to the O-antigen of intestinal bacteria O134 type; And these oligonucleotide are also reconfigurable, and the oligonucleotide after the combination also is a high special to the O-antigen of intestinal bacteria O134 type.
The above-mentioned oligonucleotide that a further object of the present invention provides can be used as primer and is used for nucleic acid amplification reaction, perhaps be used for hybridization as probe, perhaps be used to make gene chip or microarray, thereby pass through O-antigen and the detection and the identification of escherichia coli O134 type of these methods detections and identification of escherichia coli O134 type.
An also purpose of the present invention has provided the method for complete sequence of the O-antigen gene bunch of separating Escherichia coli O134 type.Can obtain the complete sequence of the O-antigen gene bunch of other bacteriums according to present method operation, the complete sequence of the gene cluster of the bacterium of other polysaccharide antigens that also can obtain to encode.
The objective of the invention is to realize by following technical scheme.
The present invention is characterized in that the Nucleotide of the O-antigen-specific of intestinal bacteria O134 type: it is the isolating Nucleotide shown in SEQ ID NO:1,7530 bases of total length; Perhaps described base with one or more insertions, disappearance or replacement keeps the Nucleotide of the SEQ IDNO:1 of described isolating functional nucleotide simultaneously.
The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O134 type, comprising called after orf1, wzx, orf3, wzy, orf5,6 genomic constitutions of orf6 all are positioned between galF gene and gnd gene.
The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O134 type, 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 orf1, orf3, orf5, orf6 gene; Wherein said gene: wzx is the Nucleotide of 1920 to 3221 bases among the SEQ ID NO:1; Wzy is the Nucleotide of 4125 to 5264 bases among the SEQ IDNO:1; Orf1 is the Nucleotide of 1073 to 1918 bases among the SEQ ID NO:1; Orf3 is that the Nucleotide orf5 of 3223 to 4128 bases among the SEQ ID NO:1 is the Nucleotide of 5221 to 6054 bases among the SEQ ID NO:1; Orf6 is the Nucleotide of 6057 to 6785 bases among the SEQ ID NO:1.
The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O134 type wherein also comprises coming from described wzx gene, wzy gene and their mixing or their reorganization.
The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O134 type is characterized in that the oligonucleotide that wherein comes from the wzx gene is to being: the Nucleotide of 2146 to 2164 bases among the SEQ ID NO:1 and the Nucleotide of 2907 to 2924 bases; The Nucleotide of 2709 to 2726 bases among the SEQ ID NO:1 and the Nucleotide of 3169 to 3190 bases.The oligonucleotide that comes from the wzy gene is to being: the Nucleotide of 4558 to 4577 bases among the SEQ IDNO:1 and the Nucleotide of 5003 to 5023 bases; The Nucleotide of 4712 to 4732 bases among the SEQ IDNO:1 and the Nucleotide of 5120 to 5137 bases.
The application of the Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O134 type in detecting other polysaccharide antigen of expressing the antigenic bacterium of O-, the O-antigen of identifying bacterium and bacterium.
The recombinant molecule of the Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O134 type is providing the O-antigen of expressing intestinal bacteria O134 type and the application in the preparation bacterial vaccine by inserting to express.
The application of the Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O134 type is characterized in that it is used for PCR, is used for hybridization and fluoroscopic examination or is used to make gene chip or microarray as probe as primer, for the application of bacterial detection.
The separation method of the Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O134 type is characterized in that it comprises the steps:
(1) genomic extraction: in substratum, cultivate intestinal bacteria O134 type, centrifugal collecting cell; The genomic dna that obtains detects by agarose gel electrophoresis;
(2) by the O-antigen gene in the pcr amplification intestinal bacteria O134 type bunch: with the genome of intestinal bacteria O134 type is template by increase its O-antigen gene bunch of LongPCR, 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 O134 type;
(6) screening of specific gene: at wzx, the wzy gene design primer in the O-antigen gene of intestinal bacteria O134 type bunch; Respectively designed two pairs of primers in each gene, every pair of primer is distributed in the different places in the corresponding gene, to guarantee its specificity; Is that template is carried out PCR with these primers with the genomes of 166 strain intestinal bacteria and 43 strain Shigellaes, determines the antigenic high degree of specificity of O-of wzx, wzy gene pairs intestinal bacteria O134 type;
(7) detection of primer sensitivity: cultivate intestinal bacteria O134, 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 O134.
The separation method of the Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O134 type is characterized in that it comprises the steps:
(1) genomic extraction: 37 ℃ of incubated overnight intestinal bacteria O134 types in the LB of 5mL substratum, centrifugal collecting cell.With 500 μ l 50mM Tris-HCl (pH8.0) and 10 μ l 0.4M EDTA re-suspended cells, 37 ℃ of incubations 20 minutes, the N,O-Diacetylmuramidase that adds 10 μ l 10mg/mL then continues insulation 20 minutes.The Proteinase K, the 15 μ l 10%SDS that add 3 μ l 20mg/mL afterwards, 50 ℃ of incubations 2 hours, the RNase that adds 3 μ l 10mg/mL again, 65 ℃ of incubations 30 minutes, add equal-volume phenol extracting mixture, get supernatant and use isopyknic phenol again: chloroform: primary isoamyl alcohol (25: 24: 1) mixing solutions extracting twice, get supernatant again with isopyknic ether extracting to remove remaining phenol.Supernatant rolls out DNA and washes DNA with 70% ethanol with glass yarn with 2 times of volume ethanol deposit D NA, and DNA is resuspended among the 30 μ l TE; Genomic dna detects by 0.4% agarose gel electrophoresis;
(2) by the O-antigen gene in the pcr amplification intestinal bacteria O134 type bunch: with the genome of intestinal bacteria O134 type is that template is passed through its O-antigen gene of Long pcr amplification bunch, at first according to the galF gene 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, 55 annealing 15 seconds, 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, 0.9 μ l 0.1MMnCl
2, the DNaseI of the 1mg/mL of 1 μ l 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 2 μ l 0.1M EDTA termination reactions.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 18 μ l water, in this mixture, add 2.5 μ l dNTP (1mMdCTP, 1mMdGTP, 1mMdTTP subsequently, 10mMdATP), 1.25 the T4DNA polysaccharase of μ l 100mM DTT and 5 units, 11 ℃ 30 minutes, enzyme is cut product mends into flush end, after 75 ℃ of termination reactions, add the TthDNA polysaccharase of 5 units and corresponding damping fluid thereof and system is expanded as 80 μ l, 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 90 μ l.10 * the buffer of 9 μ l and the T4DNA ligase enzyme of 25 units are wherein arranged, use the 3M NaAc (pH5.2) of 1/10 volume and the dehydrated alcohol precipitation of 2 times of volumes to be connected mixture at last, wash precipitation with 70% ethanol again, be dissolved in after the drying in the 30 μ l water and obtain connecting product; Preparation method with the electric transformed competence colibacillus cell of Bio-Rad company prepares the competence e.colidh5, get after 2-3 μ l connects product and 50 μ l competence bacillus coli DH 5 alphas mix, forward in the electric shock cup of 0.2cm of Bio-Rad company and shock by electricity, voltage is 2.5 kilovolts, time is 5.0 milliseconds to 6.0 milliseconds, the SOC substratum that adds 1mL after the electric shock immediately in cup makes the bacterium recovery, then bacterium is coated in and contains penbritin, on the LB solid medium of X-Gal and IPTG, 37 ℃ of incubated overnight, obtain blue white bacterium colony next day, with the white colony that obtains promptly the white clone forward on the LB solid medium that contains penbritin and cultivate, from each clone, extract plasmid simultaneously, and cutting the segmental size of evaluation insertion wherein with the EcoRI enzyme, the white that obtains clone group has constituted the O-antigen gene bunch library of intestinal bacteria O134 type;
(4) to the cloning and sequencing in the library: from the library, select 95 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 O134 type; The quality of sequence is mainly guaranteed by two aspects: 1) genome of intestinal bacteria O134 type is done 5 Long PCR reactions, mix these products then to produce the library, 2) to each base, guarantee high-quality fraction of coverage more than 3, after obtaining the nucleotide sequence of intestinal bacteria O134 type O-antigen gene bunch, with American National biotechnology information science center (The National Center forBiotechnology Information, NCBI) Orffinder finds gene, find the reading frame of 6 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 O134 type at last;
(6) specific gene screening: at wzx, the wzy gene design primer in the O-antigen gene of dysentery intestinal bacteria O134 type bunch; Respectively designed two pairs of primers in each gene, every pair of primer is distributed in the different places in the corresponding gene, to guarantee its specificity; Is that template is carried out PCR with these primers with the genomes of 166 strain intestinal bacteria and 43 strain Shigellaes, except that a band that in containing intestinal bacteria O134 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 O134 type 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 O134 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 lMQ 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 4 pairs of oligonucleotide, the Nucleotide of 2146 to 2164 bases among the SEQ ID NO:1 and the Nucleotide of 2907 to 2924 bases; The Nucleotide of 2709 to 2726 bases among the SEQ ID NO:1 and the Nucleotide of 3169 to 3190 bases; The Nucleotide of 4558 to 4577 bases among the SEQ ID NO:1 and the Nucleotide of 5003 to 5023 bases; The Nucleotide of 4712 to 4732 bases among the SEQ ID NO:1 and the Nucleotide of 5120 to 5137 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 4 pairs of primers reaction.The pork filling that participates in 0 viable bacteria obtains negative findings in the PCR of 4 pairs of primers reaction.Illustrate that these 4 pairs of primers are 0.25 bacterium/g to the detection sensitivity of the intestinal bacteria O134 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 O134 type, its complete sequence shown in SEQ ID NO:1,7530 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 O134 type by method of the present invention, as shown in table 3, it comprises called after orf1, wzx, and orf3, wzy, orf5,6 genomic constitutions of orf6 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 O134 type, promptly transports enzyme gene (wzx gene or the gene of identity function arranged with wzx); Pol gene (wzy gene or the gene of identity function arranged with wzy); Glycosyltransferase gene (orf2, orf3, orf6, orf11 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 O134 type.
The 3rd aspect of the present invention, wzx gene in the O-antigen gene bunch that comes from intestinal bacteria O134 type is provided or the gene of identity function and wzy gene is arranged or with wzy the oligonucleotide (table 1) of the gene of identity function is arranged with wzx, they are any one section oligonucleotide in these genes.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 7th group, and any product that all do not increase in other groups is so the O-antigen of wzx, wzy gene pairs intestinal bacteria O134 type all is high special.
The separation method of the Nucleotide of described O-antigen-specific to intestinal bacteria O134 type comprises the steps: 1) genomic extraction; 2) the O-antigen gene in the pcr amplification intestinal bacteria O134 type bunch; 3) make up O-antigen gene bunch library; 4) to the cloning and sequencing in the library; 5) splicing of nucleotide sequence and analysis; 6) screening of specific gene; 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 refer to derive from the gene of the coding transhipment enzyme in the O-antigen gene bunch and intragenic one section nucleic acid molecule of coding polysaccharase, and they can change on length, generally change in 10 to 20 Nucleotide scopes; More definite these oligonucleotide of saying are to come from wzx gene (nucleotide position is the Nucleotide of 1920 to 3221 bases from SEQ ID NO:1); Wzy gene (nucleotide position is the Nucleotide of 4125 to 5264 bases from SEQ ID NO:1); Coming from above intragenic oligonucleotide is high special to intestinal bacteria O134 type.
In addition, the antigenic gene cluster of the different O-of the coding of two genetic resemblances produces new O-antigen by gene recombination or sudden change sometimes, thereby produces new bacteria types, new mutant strain.In this environment, need filter out many specificitys that oligonucleotide is detected with raising with recombination hybridization.Therefore, the invention provides a whole set of many mixtures to oligonucleotide, they come from transhipment enzyme and pol gene, comprise the wzx gene or the gene, wzy gene of identity function are arranged or with wzy the gene of identity function is arranged 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 the 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 O134 types.Available PCR method detects, more can with behind the Nucleotide mark in the inventive method as probe by hybridization such as Southem-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 O134 type.
On the other hand, the present invention relates to a kind of antigenic method of one or more bacterial polysaccharideses that detects in the movement, these antigens can make sample can with the oligonucleotide specific hybrid of following at least one gene, these genes are: (i) gene of the encoding glycosyl transferring enzyme gene of transhipment enzyme and polysaccharase of (ii) encoding comprises the wzx gene or the gene, wzy gene of identity function is arranged or with wzy the gene of identity function is arranged with wzx.At least one oligonucleotide can be expressed more than one such gene specific hybridization of the special antigenic bacterium of O-with at least one under the situation of condition permission.These bacteriums are intestinal bacteria O134 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 O134 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 the 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 O134 type first, and can from the sequence of this total length gene cluster of not cloned, produce recombinant molecule, can produce the O-antigen of expressing intestinal bacteria O134 type by inserting to express, and become useful vaccine.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should understand these embodiment only is used to the present invention is described and is not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, molecular cloning: the condition described in the laboratory manual (NewYork:Cold Spring Harbor Laboratory Press, 1989).
Embodiment 1: genomic extraction:
37 ℃ of incubated overnight intestinal bacteria O134 types in the LB of 5mL substratum, centrifugal collecting cell.With 500 μ l 50mM Tris-HCl (pH8.0) and 10 μ l 0.4M EDTA re-suspended cells, 37 ℃ of incubations 20 minutes, the N,O-Diacetylmuramidase that adds 10 μ l 10mg/mL then continues insulation 20 minutes.The Proteinase K, the 15 μ l 10%SDS that add 3 μ l 20mg/mL afterwards, 50 ℃ of incubations 2 hours add the RNase of 3 μ l 100mg/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 30 μ l TE with 70% ethanol.Genomic dna detects by agarose gel electrophoresis O.4%.
Embodiment 2: by the O-antigen gene in the pcr amplification intestinal bacteria O134 type bunch:
With the genome of intestinal bacteria O134 type is that template is passed through its O-antigen gene of Long pcr amplification bunch.At first according to the galF gene 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 TTAAGT 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, 55 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 longPCR products, and with the WizardPCR Preps purification kit purified pcr product of Promega company.
Embodiment 3: make up O-antigen gene bunch library:
At first be the acquisition that connects product: make up O-antigen gene bunch library with the Novagen DNaseI shot gun method that is modified.Reaction system is a 300ng PCR purified product, 0.9 μ l 0.1M MnCl
2, 1 μ l1: 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 2 μ l 0.1M EDTA termination reactions.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 18 μ l water.In this mixture, add 2.5 μ l dNTP (1mMdCTP subsequently, 1mMdGTP, 1mMdTTP, 10mMdATP), the T4DNA polysaccharase of 1.25 μ l 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 80 μ l, 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 90 μ l.10 * the buffer of 9 μ l 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 in the 30 μ l water and obtain connecting product.
Next is the preparation of competent cell: the method that provides with reference to Bio-Rad company prepares the competent cell bacillus coli DH 5 alpha.Get a ring bacillus coli DH 5 alpha list bacterium colony in the LB of 5mL substratum, 180rpm cultivated after 10 hours, got in the LB substratum that the 2mL culture is transferred to 200mL, and 37 ℃ of 250rpm thermal agitations are cultivated OD600 about 0.5, ice bath cooling was 20 minutes then, in centrifugal 15 minutes of 4 ℃ of 4000rpm.Confide all supernatant, dispel thalline, in centrifugal 15 minutes of 4 ℃ of 4000rpm with the deionization aqua sterilisa 200mL of cold ice precooling.Deionization aqua sterilisa 100mL with cold ice precooling dispelled thalline again, in centrifugal 15 minutes of 4 ℃ of 4000rpm.With 10% glycerine suspension cell of cold ice precooling, centrifugal 10 minutes of 4 ℃ of 6000rpm abandon supernatant, precipitate 10% glycerine suspension cell with the precooling of 1mL ice at last, are competent cell.The competent cell that makes is packed as 50 μ l, one pipe ,-70 ℃ of preservations.
Be electric transformed competence colibacillus cell at last: get after 2-3 μ l connects product and 50 μ l competence bacillus coli DH 5 alphas mix, forward in the electric shock cup of 0.2cm of Bio-Rad company and shock by electricity, voltage is 2.5 kilovolts, and the time is 5.0 milliseconds-6.0 milliseconds.The SOC substratum that adds 1mL after the electric shock immediately in cup makes the bacterium recovery.Immediately bacterium is coated in 37 ℃ of inversion incubated overnight on the LB solid medium that contains penbritin, X-Gal and IPTG then, obtains blue white bacterium colony next day.With the white colony that obtains promptly the white clone forward on the LB solid medium that contains penbritin and cultivate, from each clone, extract plasmid and cut the segmental size of evaluation insertion wherein simultaneously, obtain the O-antigen gene bunch library that white clone group has constituted intestinal bacteria O134 type with the EcoRI enzyme.
Embodiment 4: to the cloning and sequencing in the library:
From the library, select insert 95 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 O134 type obtained.The quality of sequence is mainly guaranteed by two aspects: 1) genome of intestinal bacteria O134 type is done 5 Long PCR reactions, mix these products then to produce the library.2), guarantee high-quality fraction of coverage more than 3 to each base.After obtaining the nucleotide sequence of intestinal bacteria O134 type O-antigen gene bunch, with American National biotechnology information science center (The National Center for BiotechnologyInformation, NCBI) Orffinder finds gene, find the reading frame of 6 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 O134 type at last, as shown in table 3.
Orf2 and orf4 are the proteic genes that there is transmembrane segment in only two codings of intestinal bacteria O134 kind.The O-antigen transhipment enzyme of orf2 encoded protein and intestinal bacteria (gb|AAD21572.1|/419) has 27% sequence identity, 47% 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 orf2 is wzx.The polysaccharase of Orf4 encoded protein and streptococcus pneumoniae (gb|AAK20705) coding has 24% consistence, 47% 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 orf4 is wzy.
Albumen and other known glycosyltransferases of orf1, orf3, orf5, four genes encodings of orf6 have the sequence identity of 26-48% and the sequence similarity of 45-68%.By search to glycosyltransferase motif database among the Pfam, the homology desired value of the consensus sequence of the albumen of these four genes encodings and known glycosyltransferase family 1 and 2 is very high, therefore we infer this four genes encoding glycosyltransferases, and because each glycosyltransferase specificity catalysis forms a kind of disaccharide bond, so we infer that the antigenic oligosaccharide unit of O-of intestinal bacteria O134 may be made up of five monose.Because the definite function of these four genes can't be determined, so we are with the temporary called after orf1 of these four genes, orf3, orf5, orf6.
Embodiment 6: the screening of specific gene.
Wzx, wzy gene design primer in the O-antigen gene of intestinal bacteria O134 type 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 O134 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 O134 type all is high special; The position of these genes in nucleotide sequence sees Table 1.
Embodiment 7: the detection of primer sensitivity.
Buy the live pig meat stuffing on the market, stir, be divided into the 20g portion, exist in-40 ℃ of refrigerators standby.The frozen bacterium liquid of 10 μ l intestinal bacteria O134 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 4 pairs of oligonucleotide, the Nucleotide of 2146 to 2164 bases among the SEQ ID NO:1 and the Nucleotide of 2907 to 2924 bases; The Nucleotide of 2709 to 2726 bases among the SEQ ID NO:1 and the Nucleotide of 3169 to 3190 bases; The Nucleotide of 4558 to 4577 bases among the SEQ ID NO:1 and the Nucleotide of 5003 to 5023 bases; The Nucleotide of 4712 to 4732 bases among the SEQ ID NO:1 and the Nucleotide of 5120 to 5137 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 ", 55 ℃: 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 4 pairs of primers reaction.The pork filling that participates in 0 viable bacteria obtains negative findings in the PCR of 4 pairs of primers reaction.Illustrate that these 4 pairs of primers are 0.25 bacterium/g to the detection sensitivity of the intestinal bacteria O134 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 Microbiology1993,7:239-252).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 intestinal bacteria O134 of the present invention can be applied to set up recombiant vaccine.
When molecular probe nucleotide sequence and target DNA sequence homology greater than 85% the time, can accurately aim sequence be hybridized out.The homology that requires both in the Southern of low preciseness hybridization is greater than 65% (" molecular cloning experiment guide " third edition, the 509th page, low preciseness hybridization).The homology search of specific nucleotide sequence among the present invention in Genebank shows does not have homology to exist greater than other genes of 65%.So in hybrid experiment, the specific nucleotide sequence among the present invention can only draw positive findings to the purpose bacterium as molecular probe.The Southern hybrid method is not strict with for the length of molecular probe, can use hybridization to whole sequence from 20bp or above oligonucleotide in the specific nucleotide sequence among the present invention.In a Southern experiment, utilize the relevant specific gene (more than the 1000bp) of Salmonellas to do molecular probe, success tell this bacterium (Liu D, VermaNK, Romana LK, Reeves PR., 1991 Relationships among the rfb regions ofSalmonella serovars A, B, and D.J Bacteriol.173 (15): 4814-4819.), the experiment of a lot of this areas shows that all the gene order about 1000-2000bp can be used as molecular probe.Gene chip is the same with Southern hybridization ratio juris, also similar in the requirement of selecting molecular probe for use, so specific nucleotide sequence among the present invention and oligonucleotide fragment wherein can detect this purpose bacterium as molecular probe in hybridization, comprise the ordinary method of multiple hybridization such as Southern, gene chip.
Nucleotide sequence (shown in the SEQ IDNO:1) according to the O-antigen-specific to intestinal bacteria O134 type of the present invention, structure specific nucleic acid probe, be fixed on the carrier of chip and make biochip, after the sample that will detect is suitably handled, carry out hybridization with biochip, utilize the biochip signal analysis equipment just can obtain corresponding bacteria situation in the sample then.The DNA chip that this intestinal bacteria O-antigen is identified can be directly used in clinical and other check place (as food-processing and production industry, the Micro biological Tests of animal and veterinary industry customs quarantine control etc.).This chip only need enlarge output, just can industrialization under identical condition.
Table 1 has been listed transhipment enzyme gene and pol gene and intragenic primer and PCR data in the O-antigen gene bunch of intestinal bacteria O134 type.Transhipment enzyme gene and pol gene and their function corresponding and the size of the O-antigen gene bunch of intestinal bacteria O134 type in table, have been listed.In each gene, we have respectively designed two pairs of primers, and the difference that every pair of primer is distributed in the corresponding gene is local to guarantee its specificity.In table, also listed position and the size of each primer in SEQ 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 O134 type in the 7th 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 25 μ l.Template is dilution in 1: 20, gets 1 μ l.After reaction finishes, get 10 μ lPCR products 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 5th 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 O134 type and O-antigen thereof are high specials.And the oligonucleotide of these intragenic any one section 10-25nt is special to the O-antigen of intestinal bacteria O134 type, and the primer in especially above-mentioned each gene is that oligonucleotide is high special to detecting the back confirmation through PCR to intestinal bacteria O134 type.These all oligonucleotide all can be used for the intestinal bacteria O134 type in the human body and environment rapidly and accurately, and can identify their O-antigen.
Table 3 is structural tables of the O-antigen gene bunch of intestinal bacteria O134 type, in table, listed the structure of the O-antigen gene bunch of intestinal bacteria O134 type, altogether by 6 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 O134 type, listed the accurate position of all open reading frame in complete sequence in the O-antigen gene bunch of intestinal bacteria O134 type in the drawings, at the underscoring of the initiator codon and the terminator codon of each open reading frame.The initiator codon of open reading frame has two in intestinal bacteria: ATG and GTG.
SEQ ID NO:1 sequence (SEQUENCE LISTING)
SEQUENCE LISTING
＜110〉Tianjin Biochip Technology Co., Ltd
＜120〉to the Nucleotide of the O-antigen-specific of intestinal bacteria O134 type
＜130〉to the Nucleotide of the O-antigen-specific of intestinal bacteria O134 type
<160>1
<170>PatentIn version3.2
<210>1
<211>7530
<212>DNA
<213>Escherichia coli
<400>1
attgtggctg cagggatcaa agaaatcctc ctggtaactc atgcgtccaa gaacgcggtc 60
gaaaaccact tcgacacctc ttatgaatta gaatctctcc ttgaacagcg cgtgaagcgt 120
caactactgg cggaagtaca atctatctgt ccgccgggcg tgaccattat gaacgtgcgt 180
cagggtgaac ctttaggttt aggccactcc attttatgtg cgcgacctgc cattggtgac 240
aatccatttg tcgtggtgct gccagacgtt gtgatcgacg acgccagcgc cgacccgcta 300
cgctacaacc ttgctgccat gattgcgcgc ttcaatgaaa cgggccgtag ccaggtgctg 360
gcaaaacgta tgccgggtga gctctctgaa tactcagtca ttcagacaaa agaaccactg 420
gatcgtgaag gtaaagtcag ccgcattgtt gaattcatcg aaaagccgga tcagccgcag 480
acgctggact cagacatcat ggccgttggt cgctatgtgc tttctgccga tatttggccg 540
gaacttgaac gtactcagcc tggtgcatgg ggacgtattc agctgactga tgccattgcc 600
gagctggcga aaaaacagtc cgttgatgcc atgctgatga ccggcgacag ctacgactgc 660
ggtaaaaaaa tgggttatat gcaggcgttt gtgaagtatg gactacgcaa cctgaaagaa 720
ggggcgaagt tccgtaaagg gattgagaag ctgttaagcg aataatgaaa atctaaccga 780
atgtaacggt tgacaagaaa attataacgg cggtgaagat tcgtggcgaa agtaatttgt 840
tgcgaatatt cctgccgtag ttttatataa acaatcagag taacaacgag ttagcaatag 900
gattttaatc aaagttttcc aggattttcc ttgtttccag agctgattgg taagacaatt 960
agcgtttgaa tttttcgggt ttagcgcgag tgggtaatgc tcgtcacatc gtaggcatgc 1020
atgcagtgct ctggtagctg taaagccagg ggcggtagct tggtaaaaaa tcatgataaa 1080
tgtaagaaaa atagcaaaat taattattgg cgataatttg tattggaagt tgaagttttt 1140
ttatgccact aaaaaatggc ctaatattta tgatccaact ttttttaatg aaaaagttcg 1200
ctggcgaatg ctgcacgacc aaaattatct ttatgttaaa tgcgctgata agtatgaggt 1260
caggaactat attgagaaaa aaattggcag tgaatattta gttccgcttc gttatatgtt 1320
tactaataat gatgatatta ctgataaatg tattctgagt aatagtgtag caaagtctaa 1380
ccatggtgca ggtatgatag agttcattcc tgagacgtat tcattaagcg agttgaagaa 1440
tacttttgga aaatggctaa atcttgacta tacctccgag tccaatgaaa aacattatag 1500
taggattccg agaaaaattc ttattgaaga gtctttgtgt aaggagggaa aacctcctgt 1560
cgattataaa tttcatacat ttaaacaagc agatggttca tttcgttttg ttcttcagtt 1620
ggtaaatggt cggtttcata atgaatcaag aggttattat ctggaggatc ttaataaatg 1680
catatggtat catggtgaag gttttcattc tattccacaa gagcatataa aacctttaaa 1740
aaaggctatg gaattgtcat caattctatg tgaagatttt aattacgtac gagttgattg 1800
gtatatctat gataataagc tttattttgg tgaattaacc tttacacctg gtgctggaag 1860
ttcatatgaa tttggtaatg agcttgaaaa attaatggcc aaatattggc agttgtaaaa 1920
tgaaaataat atataacaca ctttggatga tatttgaaaa attagtatta tccctatctt 1980
tattaatcat taatgcctat gttgcgagat atttagggcc tcagcaatat ggggttatag 2040
cctattctat tacactgctc agtatttctg tggttattag taattttggc actaacacat 2100
taattttcca aaaaataagc aaaaatcaca gtgttgcaaa aatcattata ccgccgatac 2160
agatattgaa aacaatatta tacataatat gtatacttat tatgtttttc tgtttatata 2220
tttttagtta tgattttgat ccttatctga catttggttt agctgtagca ttttactttc 2280
aagctattga tagctataca tggtataatg atgcaaaatt gaaatcaaag tacaatagtc 2340
ttattaatac aatagcctta ataggtgcta ttataagtcg ttactatttg gtaaaactat 2400
cgatggattt aaaatggttt gctattcctt atgtatgcaa ttacgggata tcttttttac 2460
tgaaaagata tttttatatt aaagaagaaa aaactaaaag catcaaatca tttatatttg 2520
aaaaaaaaga tcatacacta aaagttatat ctttctttat aatttcaggt gcgccattat 2580
ttttaagtga aatatcaatt cttatttata caagacttac aaacttatat ctaggacaaa 2640
ttaaagatat tgatgcggtc ggcatattca atgcatctta tgttttagct actgcctgga 2700
cagtcgttcc tttggcattt gttacgagtg tctttactat aatctataaa gaaaaggata 2760
tttctaacag ggctttaatt ggaacattct tgactttgtt gatgttgttt tttggttttt 2820
tacttgtcat attgtgttat tttttcaaac aacagattat cagtattgta tatggctccg 2880
gtttcgagtt atcttcttct atattacctg tgcttattct tggctcagtt ttctctcttt 2940
taggtgttat ttcctatcgg atgataatat caatgcatgg gtacaaatat atagcaaaaa 3000
agatggttat tatgggggtg ataagtattc cattaaatta ttatcttatt aaatcgttag 3060
gtattattgg tgcagcttat gctacagtta gtattgaact tatatcagcc acattggcga 3120
attatttttt taaaggtggg gtgattgcaa aaatgcattt aaaaatgata acgtctccct 3180
acagtgtatc tatgcaagct ataactaaaa tactgaaata aaatgaaaga tcaagcatta 3240
gtctctatta ttattccaac atatgggcgg tgtgacactt taaatcttgc tgtccaaagt 3300
gtactaaaac aggattataa aaacatagaa gtattggtta ttgatgataa tgataatcca 3360
ataatgtcta agaaagttcg tgccaatcta tcggaatatc aatccgatcc cagattaaag 3420
tattttagtg atggggttaa tagaggtgga gcaggggcaa gaaataaagg aattgaattg 3480
tcaataggtt attatattac ttttcttgat gacgatgatt tgtttttaga aaataaaata 3540
tcaaagcaaa ttgacttttt agaaaaagaa caacttgatg tatgcctttg tgatatgtat 3600
tttaaacaag gagaccgttt tataaaaaaa tcaaattgct atgcaaatgg aacaacatta 3660
aaagaattta tactcaaggg aaattgttac acgccaatga tcatgtgtaa acgacatgtt 3720
ctaattgata ttggtggatt cgttttaact cctcggtttc aagatcactt attaatgatt 3780
aggattcttt ctaatacttc gaaagtgggg catttatctg agcaattatt tgttcataat 3840
aaccataatg gtgaaaggat tacatatagt aataaatctg ctgaagcata tatattacgc 3900
gaaaagatgg agcgagaata tatttattta ttaaattcaa atgaacgtaa aacattcgaa 3960
ttcaagaagg cacttataag gatgaaaata tatagattaa aaaatgaaca tatgaagtta 4020
cttaaaataa cattatatgc catgaaaaac ctcactaact tttcagatgt ttacgcatta 4080
ggaaaaactt taataagagt gatgtttttt gggagtaaaa atgtgtgaaa acaaataaca 4140
aaaaaataaa agcattgatt ttttttatat caatatttct ctactttttc aaaagtgttt 4200
ttatttcatt agagcttcct gaggatttaa tagggggagt atcaaatatc ataaatgcac 4260
taatattggc tatattatct ccagtcattc tactgtcaaa taaaaaacaa tgtgcattta 4320
taattttttt aattatattg tttgtttata atgccatttt ttataataac atctatatgt 4380
ttggttttat aattattgca tgcatgttga tactaagtaa cggaatatat tggagggata 4440
ttatcagata cttgtcactg gcccattttt tggtgtttct ttttatcatt cctttagttt 4500
tttttgcaga aaaatactct tttattgatg atagatttgg agttagatat acatttggct 4560
tccataatcc gaatacattt agtcaatatt taatttcttt atatgtagtg ttcttgttgt 4620
tatttgttga agttatcaaa aaaaaatctc tgcaattttt gactgttata atattgactc 4680
tatttatata tggggtgata tccttaactg gttcaaggac gggaatgata ttaactatta 4740
ttaccggctt cggtttttta gcatgtacac tgtctaaaac gaatgataaa aaaagaagaa 4800
agttcgtata tttatacata ctaggtgcag gaatattatg ttttatacaa tattatcttg 4860
ccttaacata taatcattca gagttctcaa aaaacataaa tacaattctt tctggtagaa 4920
tatggttttc ttcacaatta actagccaat tatggcctgt cccatatttc catggtgtga 4980
atattaatga ttatttgcca attgatttct tctacgtggc ttatttttac aatcttggga 5040
tttttattgg ttcttggttt atatacctct ttataagaaa aatgtttgtg caaacatata 5100
ctcctgttat ggtaattgcg ctttggttat cgcttgcaat aacggtcact gaaaattatt 5160
atgcaattcc gttgtataat attagtttat ttattgtttt ttcctcaaga tatgtgatta 5220
atgaaaatga aaacagctgt acttatccaa gctcacaaaa atgaaaatta tattagagaa 5280
ttggcgttaa acaatccatc agttcgtttt tatgtacata tggatgccaa gtatccaaat 5340
aaaatccaat ggattaaaaa tgaatgtatt gataacatct atttaattga aaatccagtt 5400
tcggtttact gggggggaag tagtcagatt tttgcaactc tattattgat gaagaaggca 5460
tacagtgata agagaaataa gttttttcat ctcgtaagtt cagaatgcgt tccattaaag 5520
tcatttgttg aaattgaaaa cgaatggagc atgaatgaaa actgtcaatt tattgagtcg 5580
catagagata aaaataatga gtggagacta aaggttcggg tccctcactc aaatacgaag 5640
tatcttagga cttttttagg acgatgtgcc aataaactct ttaaagtaac tactttttta 5700
tgggatagtg taggtttaaa tcccgaaaac tatttttttg gttcccaatg gttttcggta 5760
actagaaatt ttgttgagca agtaatagat gaagataatg aagatttttt taaaacattc 5820
cataatataa cttgtgcaga tgagcatgct tttgctattt ttgcacgaac aaaatattct 5880
aacccaaatg atattcaaga taacaataaa agattcatta agtttttagg taagtcaagt 5940
ccagaatatt taagtttaga tcaagtgggg cagattaaga atcttaactc gtattggttt 6000
tgcaggaagg tgagatgtaa tgatttatta aaaataataa ggcaggagaa gtgattatgc 6060
ttttttctat cattatgccg gcatataatt cacaggcaac aataaaagaa agtatagcat 6120
ccgtgcttaa tcagacatat caaaattttg aattaattat aattaatgat aattcaagtg 6180
acgcaacttt atcgataata acaaatttct gtcatgatag aaggattctt gtattaaata 6240
atgaggaaaa tatgggagtg gcacactctc gtaacagagg attggaaatg gcatctggag 6300
aaataattgc ctttctggat agtgacgata tatggtaccc caataaattg gaagagcaat 6360
ataattgttt tctttctggt cacaaaattg tctgctctta ttatgatgta attgactctg 6420
aagggaatat tgttggaaca agaaatgcac ctacactcgt tacatttgaa aaaatgctta 6480
aaagtaattt tataggtaac ttgactggtg catatgcctc ctcttttttt ggaaaatgtt 6540
atcagagaaa tattgggcat gaggattata ttatgtggtt ggaattggta aaaaaacaac 6600
ctgcttattg cattaaaaat aaactagccg cataccgaat ttcaaataaa tctctttctt 6660
ctaataaaat gaaagtggta atttggcaat ggaaaatata tcgtaaagca ttaggtatga 6720
atattatcaa gtctttatat tacttcttga attatatatg ttttgcgata aaaaaaagaa 6780
actaatattc aataagtttt aatttttttt tcattcaatt tcagtaataa ttgtagttat 6840
ttgcgtatag ttatacccta accgaacata cccgcagaca acaccccctg acaggagtaa 6900
acaatgtcaa agcaacagat cggcgtcgtc ggtatggcag tgatggggcg caaccttgcg 6960
ctcaatatcg aaagccgtgg ttataccgtc tctattttca accgttcccg tgaaaagacg 7020
gaagaagtaa ttgctgaaaa tccaggcaag aaactggttc cttactatac ggtgaaagag 7080
tttgttgaat ctctggaaac gcctcgtcgc atcctgttaa tggtgaaagc aggtgcaggc 7140
acggatgctg ctattgattc cctcaagcca tacctcgata aaggtgacat catcattgat 7200
ggtggtaata ccttcttcca ggacaccatt cgtcgtaatc gtgagctttc tgcagaaggc 7260
tttaacttca ttggtaccgg tgtttccggc ggtgaagaag gtgcgctgaa aggtccttcc 7320
attatgcctg gtgggcagaa agaagcctat gaactggttg caccgatcct gaccaaaatc 7380
gccgcagtgg ctgaagacgg cgaaccgtgc gttacctata ttggtgccga tggcgcaggt 7440
cactatgtga agatggttca caacggtatt gaatacggtg atatgcagct gattgctgaa 7500
gcctattctc tgctaaaagg tggcctgaac 7530
Oligosaccharide unit treatment gene in the O-antigen gene of table 1 intestinal bacteria O134 type bunch and primer and PCR data wherein
| Gene | Function | The base position of gene | Forward primer | Reverse primer | The length of PCR product | Produce the group number of correct big or small electrophoresis band | The annealing temperature of PCR (℃) |
| wzx | The transhipment enzyme | 1920-3221 | 2146-2164 | 2907-2924 | 779 | 0 * | 60 |
| 2709-2726 | 3169-3190 | 482 | 0 * | 60 | |||
| wzy | Polysaccharase | 4125-5264 | 4558-4577 | 5003-5023 | 466 | 0 * | 60 |
| 4712-4732 | 5120-5137 | 426 | 0 * | 55 |
*Only in intestinal bacteria O134 type, obtain a correct band
Table 2 166 strain intestinal bacteria and 43 strain Shigellaes and their source
| Group number 1, wild-type e. coli 2, wild-type e. coli | The bacterial strain O1 that contains in this group, O2, O5, O7, O8, O9, O12, O13, O14, O15, O16, O17, O18, O19ab, O20, O21, O22, O23, O24 O4, O10, O25, O26, O27, O28, O29, O30, O32, O33, O34, O35, O36, O37, O38, O40, O41, O42, O43 | Source 1MVS a1MVs a |
| 3, wild-type e. coli 4, wild-type e. coli 5, wild-type e. coli 6, wild-type e. coli 7, wild-type e. coli 8, wild-type e. coli 9, wild-type e. coli shigella dysenteriae 10, Shigella bogdii 11, shigella flexneri 12, wild-type e. coli 13, the 7th group of bacterial strain removes the intestinal bacteria reference culture | O6，O44，O45，O46，O48，O49，O50，O51，O52，O54，O55，O56， O57，O58，O60，O61，O62，O53 O63，O65，O66，O69，O70，O71，O74，O75，O76，O77，O78， O79，O80，O81，O82，O83，O68 O84，O85，O86，O87，O88，O89，O90，O91，O92，O98，O99， O101，O102，O103，O104，O105，O106，O97 O107，O108，O109，O110，O111，O112ab，O112ac,O113， O115，O116，O118，O120，O123，O126，O128，O117 O129，O130，O131，O132，O133，O134，O135，O136，O137， O138，O139，O141，O142，O143，O144，O145，O140 O146，O147，O148，O150，O152，O154，O156，O157，O158， O159，O160，O161，O163，O164，O165，O166，O153 O168，O169，O170，O171，O172，O173， D1，D2，D3，D4，D5，D6，D7，D8，D9，D10，D11，D12，D13 B1，B2，B3，B4，B6，B7，B8，B9，B10，B11，B12，B13，B14，B15， B16，B17，B18 F1a，F1b，F2a，F2b，F3，F4a，F4b，F5(v:4)，F5(v:7)，F6， DS，DR O3，O11，O39，O59，O64，O73，O96，O95，O100，O114，O151，O155， O124，O167，O162，O121，O127，O149，O119 O134 | IMVS a IMVS a IMVS a IMVS a IMVS a IMVS a b c d d d IMVS a IMVS a |
For the convenience that detects, every 12-19 bacterium is divided into one group, and 12 groups altogether, the 13rd group as negative control
a.Institude of Medical and Veterinary Science，Anelaide，Australia
b.Sratens 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 O134 type O antigen gene structure iron
#orf galF orf1 orf2 orf3 orf4 orf5 orf6 gnd
％G+C 51.9 31.8 28.7 29.7 27.6 31.4 30.5 49.0
content
Table 4 intestinal bacteria O134 type O-antigen gene cluster gene position
attgtggctg cagggatcaa agaaatcctc ctggtaactc atgcgtccaa gaacgcggtc 60
gaaaaccact tcgacacctc ttatgaatta gaatctctcc ttgaacagcg cgtgaagcgt 120
caactactgg cggaagtaca atctatctgt ccgccgggcg tgaccattat gaacgtgcgt 180
cagggtgaac ctttaggttt aggccactcc attttatgtg cgcgacctgc cattggtgac 240
aatccatttg tcgtggtgct gccagacgtt gtgatcgacg acgccagcgc cgacccgcta 300
cgctacaacc ttgctgccat gattgcgcgc ttcaatgaaa cgggccgtag ccaggtgctg 360
gcaaaacgta tgccgggtga gctctctgaa tactcagtca ttcagacaaa agaaccactg 420
gatcgtgaag gtaaagtcag ccgcattgtt gaattcatcg aaaagccgga tcagccgcag 480
acgctggact cagacatcat ggccgttggt cgctatgtgc tttctgccga tatttggccg 540
gaacttgaac gtactcagcc tggtgcatgg ggacgtattc agctgactga tgccattgcc 600
gagctggcga aaaaacagtc cgttgatgcc atgctgatga ccggcgacag ctacgactgc 660
ggtaaaaaaa tgggttatat gcaggcgttt gtgaagtatg gactacgcaa cctgaaagaa 720
ggggcgaagt tccgtaaagg gattgagaag ctgttaagcg aataatgaaa atctaaccga 780
atgtaacggt tgacaagaaa attataacgg cggtgaagat tcgtggcgaa agtaatttgt 840
tgcgaatatt cctgccgtag ttttatataa acaatcagag taacaacgag ttagcaatag 900
gattttaatc aaagttttcc aggattttcc ttgtttccag agctgattgg taagacaatt 960
agcgtttgaa tttttcgggt ttagcgcgag tgggtaatgc tcgtcacatc gtaggcatgc 1020
Orf1's is initial
atgcagtgct ctggtagctg taaagccagg ggcggtagct tggtaaaaaa tc
atgataaa 1080
tgtaagaaaa atagcaaaat taattattgg cgataatttg tattggaagt tgaagttttt 1140
ttatgccact aaaaaatggc ctaatattta tgatccaact ttttttaatg aaaaagttcg 1200
ctggcgaatg ctgcacgacc aaaattatct ttatgttaaa tgcgctgata agtatgaggt 1260
caggaactat attgagaaaa aaattggcag tgaatattta gttccgcttc gttatatgtt 1320
tactaataat gatgatatta ctgataaatg tattctgagt aatagtgtag caaagtctaa 1380
ccatggtgca ggtatgatag agttcattcc tgagacgtat tcattaagcg agttgaagaa 1440
tacttttgga aaatggctaa atcttgacta tacctccgag tccaatgaaa aacattatag 1500
taggattccg agaaaaattc ttattgaaga gtctttgtgt aaggagggaa aacctcctgt 1560
cgattataaa tttcatacat ttaaacaagc agatggttca tttcgttttg ttcttcagtt 1620
ggtaaatggt cggtttcata atgaatcaag aggttattat ctggaggatc ttaataaatg 1680
catatggtat catggtgaag gttttcattc tattccacaa gagcatataa aacctttaaa 1740
aaaggctatg gaattgtcat caattctatg tgaagatttt aattacgtac gagttgattg 1800
gtatatctat gataataagc tttattttgg tgaattaacc tttacacctg gtgctggaag 1860
The termination of orf1
ttcatatgaa tttggtaatg agcttgaaaa attaatggcc aaatattggc agttg
taaaa 1920
Orf2's is initial
tgaaaataat atataacaca ctttggatga tatttgaaaa attagtatta tccctatctt 1980
tattaatcat taatgcctat gttgcgagat atttagggcc tcagcaatat ggggttatag 2040
cctattctat tacactgctc agtatttctg tggttattag taattttggc actaacacat 2100
taattttcca aaaaataagc aaaaatcaca gtgttgcaaa aatcattata ccgccgatac 2160
agatattgaa aacaatatta tacataatat gtatacttat tatgtttttc tgtttatata 2220
tttttagtta tgattttgat ccttatctga catttggttt agctgtagca ttttactttc 2280
aagctattga tagctataca tggtataatg atgcaaaatt gaaatcaaag tacaatagtc 2340
ttattaatac aatagcctta ataggtgcta ttataagtcg ttactatttg gtaaaactat 2400
cgatggattt aaaatggttt gctattcctt atgtatgcaa ttacgggata tcttttttac 2460
tgaaaagata tttttatatt aaagaagaaa aaactaaaag catcaaatca tttatatttg 2520
aaaaaaaaga tcatacacta aaagttatat ctttctttat aatttcaggt gcgccattat 2580
ttttaagtga aatatcaatt cttatttata caagacttac aaacttatat ctaggacaaa 2640
ttaaagatat tgatgcggtc ggcatattca atgcatctta tgttttagct actgcctgga 2700
cagtcgttcc tttggcattt gttacgagtg tctttactat aatctataaa gaaaaggata 2760
tttctaacag ggctttaatt ggaacattct tgactttgtt gatgttgttt tttggttttt 2820
tacttgtcat attgtgttat tttttcaaac aacagattat cagtattgta tatggctccg 2880
gtttcgagtt atcttcttct atattacctg tgcttattct tggctcagtt ttctctcttt 2940
taggtgttat ttcctatcgg atgataatat caatgcatgg gtacaaatat atagcaaaaa 3000
agatggttat tatgggggtg ataagtattc cattaaatta ttatcttatt aaatcgttag 3060
gtattattgg tgcagcttat gctacagtta gtattgaact tatatcagcc acattggcga 3120
attatttttt taaaggtggg gtgattgcaa aaatgcattt aaaaatgata acgtctccct 3180
The termination orf3's of orf2 is initial
acagtgtatc tatgcaagct ataactaaaa tactgaaa
ta aaatgaaaga tcaagcatta 3240
gtctctatta ttattccaac atatgggcgg tgtgacactt taaatcttgc tgtccaaagt 3300
gtactaaaac aggattataa aaacatagaa gtattggtta ttgatgataa tgataatcca 3360
ataatgtcta agaaagttcg tgccaatcta tcggaatatc aatccgatcc cagattaaag 3420
tattttagtg atggggttaa tagaggtgga gcaggggcaa gaaataaagg aattgaattg 3480
tcaataggtt attatattac ttttcttgat gacgatgatt tgtttttaga aaataaaata 3540
tcaaagcaaa ttgacttttt agaaaaagaa caacttgatg tatgcctttg tgatatgtat 3600
tttaaacaag gagaccgttt tataaaaaaa tcaaattgct atgcaaatgg aacaacatta 3660
aaagaattta tactcaaggg aaattgttac acgccaatga tcatgtgtaa acgacatgtt 3720
ctaattgata ttggtggatt cgttttaact cctcggtttc aagatcactt attaatgatt 3780
aggattcttt ctaatacttc gaaagtgggg catttatctg agcaattatt tgttcataat 3840
aaccataatg gtgaaaggat tacatatagt aataaatctg ctgaagcata tatattacgc 3900
gaaaagatgg agcgagaata tatttattta ttaaattcaa atgaacgtaa aacattcgaa 3960
ttcaagaagg cacttataag gatgaaaata tatagattaa aaaatgaaca tatgaagtta 4020
cttaaaataa cattatatgc catgaaaaac ctcactaact tttcagatgt ttacgcatta 4080
The termination of the initial orf3 of orf4
ggaaaaactt taataagagt gatgtttttt gggagtaaaa atgt
gtgaaa acaaataaca 4140
aaaaaataaa agcattgatt ttttttatat caatatttct ctactttttc aaaagtgttt 4200
ttatttcatt agagcttcct gaggatttaa tagggggagt atcaaatatc ataaatgcac 4260
taatattggc tatattatct ccagtcattc tactgtcaaa taaaaaacaa tgtgcattta 4320
taattttttt aattatattg tttgtttata atgccatttt ttataataac atctatatgt 4380
ttggttttat aattattgca tgcatgttga tactaagtaa cggaatatat tggagggata 4440
ttatcagata cttgtcactg gcccattttt tggtgtttct ttttatcatt cctttagttt 4500
tttttgcaga aaaatactct tttattgatg atagatttgg agttagatat acatttggct 4560
tccataatcc gaatacattt agtcaatatt taatttcttt atatgtagtg ttcttgttgt 4620
tatttgttga agttatcaaa aaaaaatctc tgcaattttt gactgttata atattgactc 4680
tatttatata tggggtgata tccttaactg gttcaaggac gggaatgata ttaactatta 4740
ttaccggctt cggtttttta gcatgtacac tgtctaaaac gaatgataaa aaaagaagaa 4800
agttcgtata tttatacata ctaggtgcag gaatattatg ttttatacaa tattatcttg 4860
ccttaacata taatcattca gagttctcaa aaaacataaa tacaattctt tctggtagaa 4920
tatggttttc ttcacaatta actagccaat tatggcctgt cccatatttc catggtgtga 4980
atattaatga ttatttgcca attgatttct tctacgtggc ttatttttac aatcttggga 5040
tttttattgg ttcttggttt atatacctct ttataagaaa aatgtttgtg caaacatata 5100
ctcctgttat ggtaattgcg ctttggttat cgcttgcaat aacggtcact gaaaattatt 5160
atgcaattcc gttgtataat attagtttat ttattgtttt ttcctcaaga tatgtgatta 5220
The termination of the initial orf4 of orf5
atgaaaatga aaacagctgt acttatccaa gctcacaaaa a
tgaaaatta tattagagaa 5280
ttggcgttaa acaatccatc agttcgtttt tatgtacata tggatgccaa gtatccaaat 5340
aaaatccaat ggattaaaaa tgaatgtatt gataacatct atttaattga aaatccagtt 5400
tcggtttact gggggggaag tagtcagatt tttgcaactc tattattgat gaagaaggca 5460
tacagtgata agagaaataa gttttttcat ctcgtaagtt cagaatgcgt tccattaaag 5520
tcatttgttg aaattgaaaa cgaatggagc atgaatgaaa actgtcaatt tattgagtcg 5580
catagagata aaaataatga gtggagacta aaggttcggg tccctcactc aaatacgaag 5640
tatcttagga cttttttagg acgatgtgcc aataaactct ttaaagtaac tactttttta 5700
tgggatagtg taggtttaaa tcccgaaaac tatttttttg gttcccaatg gttttcggta 5760
actagaaatt ttgttgagca agtaatagat gaagataatg aagatttttt taaaacattc 5820
cataatataa cttgtgcaga tgagcatgct tttgctattt ttgcacgaac aaaatattct 5880
aacccaaatg atattcaaga taacaataaa agattcatta agtttttagg taagtcaagt 5940
ccagaatatt taagtttaga tcaagtgggg cagattaaga atcttaactc gtattggttt 6000
The termination orf6's of orf5 is initial
tgcaggaagg tgagatgtaa tgatttatta aaaataataa ggcaggagaa g
tgatt
atgc 6060
ttttttctat cattatgccg gcatataatt cacaggcaac aataaaagaa agtatagcat 6120
ccgtgcttaa tcagacatat caaaattttg aattaattat aattaatgat aattcaagtg 6180
acgcaacttt atcgataata acaaatttct gtcatgatag aaggattctt gtattaaata 6240
atgaggaaaa tatgggagtg gcacactctc gtaacagagg attggaaatg gcatctggag 6300
aaataattgc ctttctggat agtgacgata tatggtaccc caataaattg gaagagcaat 6360
ataattgttt tctttctggt cacaaaattg tctgctctta ttatgatgta attgactctg 6420
aagggaatat tgttggaaca agaaatgcac ctacactcgt tacatttgaa aaaatgctta 6480
aaagtaattt tataggtaac ttgactggtg catatgcctc ctcttttttt ggaaaatgtt 6540
atcagagaaa tattgggcat gaggattata ttatgtggtt ggaattggta aaaaaacaac 6600
ctgcttattg cattaaaaat aaactagccg cataccgaat ttcaaataaa tctctttctt 6660
ctaataaaat gaaagtggta atttggcaat ggaaaatata tcgtaaagca ttaggtatga 6720
atattatcaa gtctttatat tacttcttga attatatatg ttttgcgata aaaaaaagaa 6780
The termination of orf6
ac
taatattc aataagtttt aatttttttt tcattcaatt tcagtaataa ttgtagttat 6840
ttgcgtatag ttatacccta accgaacata cccgcagaca acaccccctg acaggagtaa 6900
acaatgtcaa agcaacagat cggcgtcgtc ggtatggcag tgatggggcg caaccttgcg 6960
ctcaatatcg aaagccgtgg ttataccgtc tctattttca accgttcccg tgaaaagacg 7020
gaagaagtaa ttgctgaaaa tccaggcaag aaactggttc cttactatac ggtgaaagag 7080
tttgttgaat ctctggaaac gcctcgtcgc atcctgttaa tggtgaaagc aggtgcaggc 7140
acggatgctg ctattgattc cctcaagcca tacctcgata aaggtgacat catcattgat 7200
ggtggtaata ccttcttcca ggacaccatt cgtcgtaatc gtgagctttc tgcagaaggc 7260
tttaacttca ttggtaccgg tgtttccggc ggtgaagaag gtgcgctgaa aggtccttcc 7320
attatgcctg gtgggcagaa agaagcctat gaactggttg caccgatcct gaccaaaatc 7380
gccgcagtgg ctgaagacgg cgaaccgtgc gttacctata ttggtgccga tggcgcaggt 7440
cactatgtga agatggttca caacggtatt gaatacggtg atatgcagct gattgctgaa 7500
gcctattctc tgctaaaagg tggcctgaac 7530
The invention has the beneficial effects as follows: the special molecular marker (molecular probe) that the objective of the invention is to seek and use this bacterium, it is specific nucleotide sequence, therefore technical scheme of the present invention has uniqueness, the difference of itself and conventional study method is, search out and have specific nucleotide sequence, and guarantee the specificity of molecule marker by reliable experimental, can utilize and well known to a person skilled in the art mature technology (as PCR or gene chip etc.), use the technology of this marker bacterial detection, make all those skilled in the art can be, realize purpose of the present invention easily and obtain the result of use of expection according to technology contents provided by the invention.Already provided experimental data among the present invention, fully proved the specificity of this nucleotide sequence, and can be applied to the detection of this bacterium, use modern molecular biology method for determining bacteria of the present invention with respect to traditional serology immune response, have fast, accurately, advantage cheaply.The present invention order-checking is also inferred the function of each gene with information biology software, be in order to seek specificity Nucleotide, the gene that some specific functions are arranged in the bacterium is a high special, gene that utilizes above method to infer these specific functions and their position, utilize experiment to prove then, the function of these genes that will be inferred, remove to search out better faster specificity Nucleotide as a kind of road sign, like this, can reduce the blindness of research experiment, accelerate the progress of experiment, reduce experiment fees.
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 (3)
1, a kind of oligonucleotide of the O-antigen-specific to intestinal bacteria O134 type is characterized in that it is the Nucleotide that is selected from 2146 to 2164 bases among the SEQ ID NO:1; The Nucleotide of 2907 to 2924 bases among the SEQID NO:1; The Nucleotide of 2709 to 2726 bases among the SEQ ID NO:1; The Nucleotide of 3169 to 3190 bases among the SEQ ID NO:1; The Nucleotide of 4558 to 4577 bases among the SEQ ID NO:1; The Nucleotide of 5003 to 5023 bases among the SEQ ID NO:1; The Nucleotide of 4712 to 4732 bases among the SEQ ID NO:1; The Nucleotide of 5120 to 5137 bases among the SEQ ID NO:1.
2, a kind of oligonucleotide of the O-antigen-specific to intestinal bacteria O134 type is right, it is characterized in that it is to be selected from the Nucleotide of 2146 to 2164 bases among the SEQ ID NO:1 and the Nucleotide of 2907 to 2924 bases; The Nucleotide of 2709 to 2726 bases among the SEQ ID NO:1 and the Nucleotide of 3 169 to 3190 bases; The Nucleotide of 4558 to 4577 bases among the SEQ ID NO:1 and the Nucleotide of 5003 to 5023 bases; The Nucleotide of 4712 to 4732 bases among the SEQ ID NO:1 and the Nucleotide of 5120 to 5137 bases.
3, the right application of the oligonucleotide of the oligonucleotide of claim 1 or claim 2, it is characterized in that, it is used for PCR as primer, perhaps is used for hybridization and fluoroscopic examination, manufacturing gene chip or microarray as probe, for detecting intestinal bacteria O134 type.
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| CN1546509A (en) * | 2003-12-03 | 2004-11-17 | 南开大学 | Nucleotide specific for escherichia coli 054 O-antigen |
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