CN1554759A - O-antigen specific nucleotide of E.coli 081 type - Google Patents
O-antigen specific nucleotide of E.coli 081 type Download PDFInfo
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Abstract
The present invention provides a kind of nucleotide specific to O-antigen of Escherichia coli O81, and it is the total nucleotide sequence of gene cluster of Escherichia coli O81 to control the synthesis of O-antigen, such as separated nucleotide shown in SEQ ID No. 1 with whole length of 12200 bases; nucleotide similar to that in SEQ ID No. 1 with one or several inserted, deleted or substituted bases; and oligonucleotide originated glycosyltransferase gene and oligose unit processing gene of Escherichia coli O81 antigen gene cluster. PCR proves the high specificity of the oligonucleotide on the Escherichia coli O81 antigen. The present invention also discloses the method of detecting and identifying the Escherichia coli O81 in human body and in environment with 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 O81 type (Escherichia coli O81), particularly relate in the intestinal bacteria O81 type oligonucleotide in the control O-antigen synthetic gene cluster, can utilize these the oligonucleotide of the O-antigen-specific intestinal bacteria O81 type in human body and the environment and identify O-antigen in these pathogenic bacterium quickly and accurately.
Background technology
O-antigen is the O specific polysaccharide composition in the gram negative bacterium lipopolysaccharides, and it is made up of many multiple oligosaccharide unit.The antigenic building-up process of O-is studied clearlyer: by glycosyltransferase nucleoside diphosphate monose is transferred on the fat molecule that is fixed on the cell inner membrance earlier, then in the inboard synthesis of oligose unit of inner membrance, the antigenic oligosaccharide unit of O-is transferred to the inner membrance outside by the transhipment enzyme again, then aggregate into polysaccharide by polysaccharase, be connected to again and form lipopolysaccharide molecule [Whitfield, C. (1995) " Biosynthesis of lipopolysaccharide O antigens " .Trends inMicrobiology.3:178-185 on the glycolipid molecule; Schnaitman, C.A.and J.D.Klena. (1993) " Genetics oflipopolysaccharide biosynthesis in entericbacteria " .MicrobiologicalReviews, 57 (3): 655-682].Coding is responsible for the generally adjacent arrangement on karyomit(e) of gene of all enzyme molecules of O-antigen synthetic, form a gene cluster [Reeves, P.R., et al. (1981) " Bacterialpolysaccharide synthesis and gene nomenclature " Trends in Microbiology, 4:495-503].In Shigellae, intestinal bacteria and Salmonellas, O-antigen gene [Lei Wang.et al (2001) " Sequence analysis of four Shigella boydii O-antigenloci:implication for Escherichia coli and Shigella relationships " .Infection andImmunity, 11:6923-6930 bunch between galF and gnd gene; Lei Wang and Peter Reeves (2000) " The Escherichia coliO111 and Salmonella enterica O35 gene clusters:gene clusters encoding the samecolitose-containing O antigen are highly conserved " .Journal ofBacteriology.182:5256-5261].The O-antigen gene bunch contains three genoids: sugared synthesis path gene, glycosyltransferase gene, oligosaccharide unit treatment gene, the required nucleoside diphosphate monose of enzymic synthesis O-antigen of wherein sugared synthesis path genes encoding; Thereby the enzyme of glycosyltransferase gene coding forwards nucleoside diphosphate monose and other molecule to and makes monose aggregate into oligosaccharide unit on the monose; The oligosaccharide unit treatment gene comprises transhipment enzyme gene and pol gene, and they transfer to the bacterium inner membrance outside with oligosaccharide unit, and repolymerization becomes polysaccharide.Glycosyltransferase gene and oligosaccharide unit treatment gene only are present in the gene cluster of carrying these genes.The difference of monose in the O-antigen, between monose between the difference of link button and the oligosaccharide unit difference of link button constituted the antigenic diversity of O-, and the composition of monose, the link button between monose and the link button between the oligosaccharide unit are by the Gene Handling in the O-antigen gene bunch, so the O-antigen gene bunch has determined O-antigenic synthetic, has also determined the antigenic diversity of O-.
Because O-antigen is extremely strong antigen, be one of important paathogenic factor of intestinal bacteria, it has extremely strong diversity again simultaneously, and this enlightens us can study a kind of intestinal bacteria and good, highly sensitive method of the antigenic specificity of O-thereof of detecting quickly and accurately.With surperficial polysaccharide is that the serology immune response of target has been used to somatotype and the evaluation to bacterium always since the thirties in last century, is unique means of identifying pathogenic bacterium.This diagnostic method needs a large amount of antiserum(antisera)s, and the antiserum(antisera) general classes is incomplete, quantity not sufficient, and also there are some difficulties in a large amount of antiserum(antisera)s in preparation with in storing.On the other hand this method length consuming time, sensitivity is low, loss is high, poor accuracy, so, generally believe that now this traditional serology detection method will be that the modern molecular biology method replaces.1993, Luk, J.M.C et.al has identified the O-antigen [Luk of Salmonellas with the specific nucleotide sequence of Salmonellas (S.enterica) O-antigen gene bunch by PCR method, J.M.C.et.al. (1993) " Selective amplification of abequose andparatose synthase genes (rfb) by polymerase chain reaction for identification ofS.enterica major serogroups (A; B; C2; andD) ", J.Clin.Microbiol.31:2118-2123].Luk, the method for et.al is with corresponding to Salmonellas serotype E 1, D1 obtains the oligonucleotide special to the Salmonellas of different serotypes after the nucleotide sequence of the CDP-abequose in the A, the O-antigen of B and C2 and the synthetic gene of CDP-tyvelose is arranged.1981, Paton, the A.W et.al serotype [" Molecular microbiological investigation of an outbreak of Hemolytic-Uremic Syndrome caused by dry fermented sausage contaminated with Shiga-liketoxin producing Escherichia coli " .J.Clin.Microbiol.34:1622-1627] 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 the Enterobacteriaceae " .Elsevier SciencePublishers, Amsterdam, The Netherlands; T.cheasty, et al. (1983) " Antigenicrelationships between the enteroinvasive 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 O81 type.It is the Nucleotide in the O-antigen gene bunch of intestinal bacteria O81 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 O81 type.
Another object of the present invention has provided the gene of the O-antigen gene bunch that constitutes intestinal bacteria O81 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; Mutase gene is the Gne gene or with Gne the gene of identity function is arranged; Monose conjugate synthetic gene orf2, orf4; Glycosyltransferase gene comprises orf5, orf7, orf8, orf9 gene.
Another purpose of the present invention has provided oligonucleotide, and they come from gene that intestinal bacteria O81 type comes from coding transhipment enzyme respectively is the wzx gene or with wzx the gene of identity function is arranged; The gene that comes from the coding polysaccharase is the wzy gene or with wzy the gene of identity function is arranged; They are the oligonucleotide in the said gene, and length is at 10-20nt; They are special to the O-antigen of intestinal bacteria O81 type; Especially the oligonucleotide of listing in the table 1, they are high specials to the O-antigen of intestinal bacteria O81 type, and these oligonucleotide are also reconfigurable, the oligonucleotide after the combination also is a high special to the O-antigen of intestinal bacteria O81 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 O81 type of these methods detections and identification of escherichia coli O81 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 O81 type.Can obtain the complete sequence of the O-antigen gene bunch of other bacteriums according to present method operation, the complete sequence of the gene cluster of the bacterium of other polysaccharide antigens that also can obtain to encode.
The objective of the invention is to realize by following technical scheme.
The present invention is to the Nucleotide of the O-antigen-specific of intestinal bacteria O81 type, and it is the isolating Nucleotide shown in SEQ ID NO:1,12200 bases of total length; The base that perhaps has one or more insertions, disappearance or replacement keeps the Nucleotide of the SEQ ID NO:1 of described isolating functional nucleotide simultaneously.
The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O81 type, it is by 9 genomic constitutions, all between galF gene and gnd gene.
The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O81 type, wherein said gene is: the gene of transhipment enzyme comprises the wzx gene or with wzx the gene of identity function is arranged; Pol gene comprises the wzy gene or with wzy the gene of identity function is arranged; Mutase gene is the Gne gene or with Gne the gene of identity function is arranged; Monose conjugate synthetic gene orf2, orf4; Glycosyltransferase gene comprises orf5, orf7, orf8, orf9 gene.Wherein said gene: wzx is the Nucleotide of 3407 to 4567 bases among the SEQ ID NO:1; Wzy is the Nucleotide of 6516 to 7745 bases among the SEQ ID NO:1; Orf1 is the Nucleotide of 1415 to 2434 bases among the SEQ ID NO:1; Orf2 is the Nucleotide of 2800 to 3171 bases among the SEQ ID NO:1; Orf4 is the Nucleotide of 4551 to 5723 bases among the SEQ ID NO:1; Orf5 is the Nucleotide of 5720 to 6526 bases among the SEQ ID NO:1; Orf7 is the Nucleotide of 8021 to 8755 bases among the SEQ ID NO:1; Orf8 is the Nucleotide of 8800 to 9846 bases among the SEQ ID NO:1; Orf9 is the Nucleotide of 9843 to 10631 bases among the SEQ ID NO:1.
The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O81 type, wherein it is to come from described wzx gene, wzy gene or glycosyltransferase gene orf5, orf7, orf8, orf9 gene and their mixing or their reorganization.
The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O81 type, the oligonucleotide of the described wzx of coming from gene is to being: the Nucleotide of 3441 to 3458 bases among the SEQ ID NO:1 and the Nucleotide of 4084 to 4101 bases; The Nucleotide of 4054 to 4071 bases among the SEQ ID NO:1 and the Nucleotide of 4281 to 4298 bases.The oligonucleotide that comes from the wzy gene is to being: the Nucleotide of 6791 to 6808 bases among the SEQ ID NO:1 and the Nucleotide of 7517 to 7534 bases; The Nucleotide of 6823 to 6840 bases among the SEQ IDNO:1 and the Nucleotide of 7136 to 7153 bases.
The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O81 type is detecting the application of expressing the antigenic bacterium of O-, identify other polysaccharide antigen of the O-antigen of bacterium and bacterium in diagnosis.
The recombinant molecule of the Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O81 type, and can provide the O-antigen of expressing intestinal bacteria O81 type by inserting to express, and become bacterial vaccine.
The application of the Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O81 type, wherein it is used for PCR, is used for hybridization and fluoroscopic examination or is used to make gene chip or microarray, the bacterium in human body and the environment as probe as primer.
The separation method of the Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O81 type is characterized in that it comprises the steps:
(1) genomic extraction: 37 ℃ of incubated overnight intestinal bacteria O81 types in the LB of 5mL substratum, centrifugal collecting cell.With 500ul 50mM Tris-HCl (pH8.0) and 10ul 0.4M EDTA re-suspended cell, 37 ℃ of incubations 20 minutes, the N,O-Diacetylmuramidase that adds 10ul 10mg/ml then continues insulation 20 minutes.The Proteinase K, the 15ul 10%SDS that add 3ul 20mg/ml afterwards, 50 ℃ of incubations 2 hours, the RNase that adds 3ul 10mg/ml again, 65 ℃ of incubations 30 minutes, add equal-volume phenol extracting mixture, get supernatant liquor and use isopyknic phenol again: chloroform: primary isoamyl alcohol (25: 24: 1) mixing solutions extracting twice, get supernatant liquor again with isopyknic ether extracting to remove remaining phenol.Supernatant liquor rolls out DNA and washes DNA with 70% ethanol with glass yarn with 2 times of volume ethanol deposit D NA, and DNA is resuspended among the 30ul TE; Genomic dna detects by 0.4% agarose gel electrophoresis;
(2) by the O-antigen gene in the pcr amplification intestinal bacteria O81 type bunch: with the genome of intestinal bacteria O81 type is that template is passed through its O-antigen gene of Long pcr amplification bunch, at first according to the JumpStart sequences Design upstream primer (#1523-ATT GTGGCT GCA GGG ATC AAA GAA AT) that often is found in O-antigen gene bunch promoter region, again according to the gnd gene design downstream primer (#1524-TAG TCG CGT GNG CCT GGA TTA AGT TCG C) in O-antigen gene bunch downstream; With the Expand Long Template PCR method of Boehringer Mannheim company amplification O-antigen gene bunch, the PCR response procedures was as follows: 94 ℃ of pre-sex change 2 minutes; 94 ℃ of sex change are 10 seconds then, annealed 15 seconds for 60 ℃, 68 ℃ were extended 15 minutes, carry out 30 circulations like this, last, continue to extend 7 minutes at 68 ℃, obtain the PCR product, detect the size and the specificity thereof of PCR product with 0.8% agarose gel electrophoresis, merge 5 pipe longPCR 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 Bi0-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 Bi0-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 O81 type;
(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 O81 type; The quality of sequence is mainly guaranteed by two aspects: 1) genome of intestinal bacteria O81 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 O81 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 9 openings, determine also that with the function of finding the reading frame that these are open what gene they are with the genetic comparison among the blast groupware and the GenBank, finish gene annotation with the Artemis software at Britain sanger center again, do accurate comparison between DNA and protein sequence with Clustral W software, obtain the structure of the O-antigen gene bunch of intestinal bacteria O81 type at last;
(6) specific gene screening: at wzx, the wzy gene design primer in the O-antigen gene of dysentery intestinal bacteria O81 type bunch; Respectively designed two pairs of primers in each gene, every pair of primer is distributed in the different places in the corresponding gene, to guarantee its specificity; Is that template is carried out PCR with these primers with the genomes of 166 strain intestinal bacteria and 43 strain Shigellaes, except that a band that in the 13rd group, has obtained the expection size, any product that all do not increase in other groups is so the O-antigen of wzx, wzy gene pairs intestinal bacteria O81 type all is high special.
Just, first aspect of the present invention provides the full length nucleotide sequence of the O-antigen gene bunch of intestinal bacteria O81 type, its complete sequence shown in SEQ ID NO:1,12200 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 O81 type by method of the present invention, as shown in table 3, it is altogether by 9 genomic constitutions, all between galF gene and gnd gene.
Second aspect of the present invention provides the gene in the O-antigen gene bunch of intestinal bacteria O81 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); Special sugared synthesis path gene in glycosyltransferase gene (orf5, orf7, orf8, orf9) and the bacterial polysaccharides antigen comprises the gne 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 transhipment enzyme gene that the present invention relates to and pol gene are high specials to the O-antigen of intestinal bacteria O81 type.
The 3rd aspect of the present invention, wzx gene in the O-antigen gene bunch that comes from intestinal bacteria O81 type is provided or the gene, wzy gene of identity function is arranged or the gene and the glycosyltransferase gene of identity function are arranged, comprised orf5, orf7, orf8, orf9 gene with wzy with wzx.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 O81 type all is high special.
The separation method of the Nucleotide of described O-antigen-specific to intestinal bacteria O81 type comprises the steps: 1) genomic extraction; 2) the O-antigen gene in the pcr amplification intestinal bacteria O81 type bunch; 3) make up O-antigen gene bunch library; 4) to the cloning and sequencing in the library; 5) splicing of nucleotide sequence and analysis; 6) screening of specific gene.
Other aspects of the present invention are because disclosing of the technology of this paper is conspicuous to those skilled in the art.
As used herein, " oligonucleotide " mainly refers to derive from gene, the gene of coding transhipment enzyme and intragenic one section nucleic acid molecule of coding polysaccharase of the encoding glycosyl transferring enzyme in the O-antigen gene bunch, they can change on length, generally change in 10 to 20 Nucleotide scopes; More definite these oligonucleotide of saying are to come from wzx gene (nucleotide position is the Nucleotide of 3407 to 4567 bases from SEQ ID NO:1); Wzy gene (nucleotide position is the Nucleotide of 6516 to 7745 bases from SEQ ID NO:I); Coming from above intragenic oligonucleotide is high special to intestinal bacteria O81 type.
In addition, the antigenic gene cluster of the different O-of the coding of two genetic resemblances produces new O-antigen by gene recombination or sudden change sometimes, thereby produces new bacteria types, new mutant strain.In this environment, need filter out many specificitys that oligonucleotide is detected with raising with recombination hybridization.Therefore, the invention provides a whole set of many mixtures to oligonucleotide, they come from glycosyltransferase gene; Come from transhipment enzyme and pol gene, comprise the wzx gene or the gene, wzy gene of identity function arranged or the gene of identity function is arranged with wzy with wzx.The mixture of these genes is special to a special bacterial polysaccharides antigen, is special thereby make this cover oligonucleotide to the polysaccharide antigen of this bacterium.More particularly, the mixture of these oligonucleotide is to come from glycosyltransferase gene, wzx gene or the gene, wzy gene of identity function arranged or with wzy the combination of the oligonucleotide in the gene of identity function is arranged with wzx.
On the other hand, the present invention relates to the evaluation of oligonucleotide, they can be used for detecting the O-antigen of expressing the antigenic bacterium of O-and identifying bacterium in diagnosis.
The present invention relates to a kind of antigenic method of one or more bacterial polysaccharideses that detects in the food, these antigens can make sample can with the oligonucleotide specific hybrid of following at least one gene, these genes are: (i) gene of the encoding glycosyl transferring enzyme gene of transhipment enzyme and polysaccharase of (ii) encoding comprises the wzx gene or the gene, wzy gene of identity function is arranged or with wzy the gene of identity function is arranged with wzx.At least one oligonucleotide can be hybridized with at least one more than one such gene specific of expressing the special antigenic bacterium of O-under the situation of condition permission, and these bacteriums are intestinal bacteria O81 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 O81 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 O81 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 O81 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 O81 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 O81 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 O81 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 liquor and use isopyknic phenol again: chloroform: primary isoamyl alcohol is taken out (25: 24: 1) mixing solutions and is carried twice, get supernatant liquor again with isopyknic ether extracting to remove remaining phenol, supernatant liquor is with 2 times of volume ethanol deposit D NA, roll out DNA and wash DNA with glass yarn, at last DNA is resuspended among the 30ul TE with 70% ethanol.Genomic dna detects by 0.4% agarose gel electrophoresis.
Embodiment 2: by the O-antigen gene in the pcr amplification intestinal bacteria O81 type bunch:
With the genome of intestinal bacteria O81 type is template by increase its O-antigen gene bunch of LongPCR.At first according to the JumpStart sequences Design upstream primer (#1523-ATT GTG GCT GCA GGG ATC AAA GAA AT) that often is found in O-antigen gene bunch promoter region, again according to the gnd gene design downstream primer (#1524-TAG TCG CGT GNG CCT GGATTA 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, 60 ℃ of annealing 15 seconds, and 68 ℃ were extended 15 minutes, and carried out 30 circulations like this.At last, continue to extend 7 minutes at 68 ℃, obtain the PCR product, the agarose gel electrophoresis with 0.8% detects the size and the specificity thereof of PCR product.Merge 5 pipe long PCR products, and with the Wizard PCR Preps purification kit purified pcr product of Promega company.
Embodiment 3: make up O-antigen gene bunch library:
At first be the acquisition that connects product: make up O-antigen gene bunch library with the Novagen DNaseI shot gun method that is modified.Reaction system is a 300ng PCR purified product, 0.9ul 0.1M MnCl
2, 1ul1: the DNaseI of the 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 100mM DTT and 5 units, 11 ℃ 30 minutes, enzyme is cut product mend into flush end, after 75 ℃ of termination reactions, add the Tth archaeal dna polymerase of 5 units and corresponding damping fluid thereof and system is expanded as 80ul, 70 ℃ were reacted 20 minutes, made 3 of DNA ' end add the dA tail.This mixture is through the equal-volume chloroform: after primary isoamyl alcohol (24: 1) mixing solutions extracting and the extracting of equal-volume ether with 3 * 10 of Promega company
-3The pGEM-T-Easy carrier connect 10 hours in 16 ℃, cumulative volume is 90ul.10 * the buffer of 9ul and the T4DNA ligase enzyme of 25 units are wherein arranged.Use the dehydrated alcohol precipitation of the 3M NaAc (pH5.2) of 1/10 volume and 2 times of volumes to be connected mixture at last, wash precipitation with 70% ethanol again, be dissolved in after the drying and obtain connecting product in the 30ul water.
Next is the preparation of competent cell: the method that provides with reference to Bio-Rad company prepares competent cell bacillus coli DH 5.Get the single bacterium colony of a ring bacillus coli DH 5 in the LB of 5ml substratum, 180rpm cultivated after 10 hours, got in the LB substratum that the 2ml culture is transferred to 200ml, and 37 ℃ of 250rpm thermal agitations are cultivated OD600 about 0.5, ice bath cooling was 20 minutes then, in centrifugal 15 minutes of 4 ℃ of 4000rpm.Confide all supernatant liquor, dispel thalline, in centrifugal 15 minutes of 4 ℃ of 4000rpm with the deionization aqua sterilisa 200ml of cold ice precooling.Deionization aqua sterilisa 100ml with cold ice precooling dispelled thalline again, in centrifugal 15 minutes of 4 ℃ of 4000rpm.With 10% glycerine suspension cell of cold ice precooling, centrifugal 10 minutes of 4 ℃ of 6000rpm abandon supernatant liquor, precipitate 10% glycerine suspension cell with the precooling of 1ml ice at last, are competent cell.The competent cell that makes is packed as 50ul one pipe ,-70 ℃ of preservations.
Be electric transformed competence colibacillus cell at last: get after 2-3ul connects product and 50ul competence bacillus coli DH 5 mixes, forward in the electric shock cup of 0.2cm of Bio-Rad company and shock by electricity, voltage is 2.5 kilovolts, and the time is 5.0 milliseconds-6.0 milliseconds.The SOC substratum that adds 1ml after the electric shock immediately in cup makes the bacterium recovery.Immediately bacterium is coated in 37 ℃ of inversion incubated overnight on the LB solid medium that contains penbritin, X-Gal and IPTG then, obtains blue white bacterium colony next day.With the white colony that obtains promptly the white clone forward on the LB solid medium that contains penbritin and cultivate, from each clone, extract plasmid and cut the segmental size of evaluation insertion wherein simultaneously, obtain the O-antigen gene bunch library that white clone group has constituted intestinal bacteria O81 type 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 O81 type obtained.The quality of sequence is mainly guaranteed by two aspects: 1) genome of intestinal bacteria O81 type is done 5 LongPCR 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 O81 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 10 openings, determine also that with the function of finding the reading frame that these are open what gene they are with the genetic comparison among the blast groupware and the GenBank, finish gene annotation with the Artemis software at Britain sanger center again, do accurate comparison between DNA and protein sequence with ClustralW software, obtain the structure of the O-antigen gene bunch of intestinal bacteria O81 type at last, as shown in table 3.
By retrieving and comparing, the albumen of finding Gne genes encoding in orf1 encoded protein and the Yersinia O-antigen gene bunch has very high consensus amino acid sequence (56%), by search, find the very high (2.8 * e of homology desired value of the consensus sequence of orf1 encoded protein and known Gne to Pfam protein-based order sequenced data storehouse
-148).Gne is responsible for UDP-GlcNac is transformed into UDP-GalNac, and UDP-GalNac is a kind of rare monose in the O-antigen.Its precursor UDP-GlcNac is the common monose in the O-antigen, is responsible for the genes encoding of its synthetic enzyme by other positions of genome, and therefore these genes do not repeat in O-antigen gene bunch.The UDP-GalNac mutase then only comes across in the surface antigen gene bunch of bacterium of UDP-GalNac.We name orf1 is Gne, and the existence of UDP-GalNac is arranged in the O-antigen of supposition intestinal bacteria O81.
Orf3 and orf6 are the proteic genes that there is transmembrane segment in only two codings of intestinal bacteria O81 kind.The O-antigen transferring enzyme of Orf3 encoded protein and rersinia pestis CO92 has 29% sequence identity, and it contains 12 uniform transmembrane segments by the proteic topology discovery of HMMTOP2.0 programanalysis, and this is the proteic characteristic feature of Wzx.So name orf3 is wzx.The O-antigen polysaccharase of Orf6 encoded protein and intestinal bacteria O6 has 37% consistence, 48% similarity, it contains 9 transmembrane segments by the proteic topology discovery of HMMTOP2.0 programanalysis, and hydrophilic loop (loop) in big (61 an amino acid) kytoplasm is arranged, and this is the proteic characteristic feature of Wzy.So name orf6 is wzy.
Orf5, the albumen of 7,8,9 four genes encodings and other known glycosyltransferases have the sequence identity of 24-36% and the sequence similarity of 44-53%.By the 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 1 * e
-12To 5 * e
-17, so 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 O81 may be made up of five monose.Because the definite function of these four genes can't be determined, so we are with these four genes temporary called after orf5, orf7, orf8 and orf9
Embodiment 6: the screening of specific gene:
At wzx, wzy gene design primer in the O-antigen gene of intestinal bacteria O81 type bunch, the position of these genes in nucleotide sequence sees Table 1.
Table 1 has been listed glycosyltransferase gene and oligosaccharide unit treatment gene and intragenic primer and PCR data in the O antigen gene bunch of intestinal bacteria O81 type.Glycosyltransferase gene, transhipment enzyme gene and pol gene and their function corresponding and the size of the O antigen gene bunch of intestinal bacteria O81 type in table, have been listed.In each gene, we have respectively designed two pairs of primers, and the difference that every pair of primer is distributed in the corresponding gene is local to guarantee its specificity.In table, also listed position and the size of each primer in SEQ IDNO:1.Is that template carry out PCR with listed corresponding annealing temperature in the table with the genomes of all bacterium in the table 2 with every pair of primer, has obtained corresponding PCR product, and its size is also listed in the table.
Table 2 is 166 strain intestinal bacteria and 43 strain Shigellaes and their sources that are used to screen specific gene, and for the convenience that detects, we are divided into one group with their every 8-10 bacterium, and 13 groups altogether, all list in the table in their source.
The genomic dna that contains intestinal bacteria O81 type 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 13rd 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 O81 type and O-antigen thereof are high specials.
At last, from intestinal bacteria O81 type, screen gene by PCR: wzx, wzy and three glycosyltransferase genes to the O-antigen high special of intestinal bacteria O81 type.And the oligonucleotide of these intragenic any one section 10-20nt is special to the O-antigen of intestinal bacteria O81 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 O81 type.These all oligonucleotide all can be used for the intestinal bacteria O81 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 O81 type, in table, listed the structure of the O-antigen gene bunch of intestinal bacteria O81 type, altogether by 9 genomic constitutions, each gene box indicating, and in square frame, write the title of gene, numeral be the order of the open reading frame (orf) in the O-antigen gene bunch.Two ends at O-antigen gene bunch are galF gene and 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 O81 type, listed the accurate position of all open reading frame in complete sequence in the O-antigen gene bunch of intestinal bacteria O81 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.
SEQUENCE?LISTING
＜110〉Nankai University
＜120〉to the Nucleotide of the O antigen-specific of intestinal bacteria O81 type
＜130〉to the Nucleotide of the O antigen-specific of intestinal bacteria O81 type
<160>1
<170>PatentIn?version?3.2
<210>1
<211>12220
<212>DNA
<213>Escherichia?coli
<400>1
attgtggctg?cagggatcaa?agaaatcctc?ctggtaactc?atgcgtccaa?gaacgcggtc?????60
gaaaaccact?tcgacacctc?ttatgaatta?gaatctctcc?ttgaacagcg?cgtgaagcgt????120
caactgctgg?cggaagtaca?gtctatctgt?ccgccgggcg?tgaccattat?gaacgtgcgt????180
cagggcgaac?ctttaggttt?gggccactcc?attttgtgtg?cacgacccgc?cattggtgac????240
aacccatttg?tcgtggtact?gccggacgtt?gtgatcgacg?acgccagtgc?cgacccgctg????300
cgctacaacc?ttgctgccat?gattgcgcgt?ttcaatgaaa?cgggccgtag?ccaggtgctg????360
gcaaaacgta?tgccgggtga?cctttctgaa?tactccgtta?ttcagaccaa?agagccgctg????420
gatcgtgaag?gcaaagtcag?ccgcattgtt?gaatttatcg?aaaaaccgga?tcagccgcag????480
acgctggact?cagacatcat?ggccgttggt?cgctatgtgc?tttctgccga?tatttggccg????540
gaacttgaac?gcactcaacc?tggtgcatgg?ggacgtatcc?aactgactga?tgccattgct????600
gaactggcga?agaaacagtc?cgttgatgcc?atgctgatga?ctggtgacag?ctacgactgc????660
ggtaaaaaaa?tgggctatat?gcaggcgttt?gtgaagtatg?gactgcgcaa?cctgaaagaa????720
ggggcgaagt?tccgtaaagg?gattgagaag?ctgttaagcg?aataatgaaa?atctgaccgg????780
atgtaacggt?tgataagaaa?attataacgg?cagtgaagat?tcgtggtgaa?agtaatttgt????840
tgcgaatatt?cctgccgttg?ttttatataa?acaatcagaa?taacaacgag?ttagcaatag????900
gattttagtc?aaagttttcc?aggattttcc?ttgtttccag?agcggattgg?taagacaatt????960
agcgtttgaa?tttttcgagt?ttagcgcgag?taggtaacgc?tagtcacatc?gtagacatgt???1020
aagcagtgct?ctggtagctg?taaagccagg?ggcggtagca?ttgttcttga?tatttttaag???1080
tataaaaatg?acacataaaa?ataaaataat?tagttatcgg?tatgttttta?attgtgttat???1140
tattgaaaaa?tatcaccatt?aattatttaa?tgcgataaag?gccaaataaa?tcactcattt???1200
atttctacat?aggatagggg?actatcagat?gggaattaaa?taaaaatcca?agtgctaaat???1260
atttcaatct?taaattccat?tgtaaaaagt?ttaagggatt?ttattctaaa?aaagaattgg???1320
atgaattgaa?agttattttg?agcttaattt?attaaacttg?aagttttttt?actgagtttt???1380
tctggaaaat?aatttattgt?taatggagcc?atatatgtca?attttagtta?ccggaggttg???1440
tggttatata?ggtgtccaca?caatttatac?gttaatttct?cagggctacg?atgttattgt???1500
actggataac?ctgactaact?catcaataga?gtcattagag?agagtcaagt?taatgacaaa???1560
tcataacata?cccttttata?aaggagatgt?ggggaataaa?aaaatattgg?tagacatatt???1620
caatgagcat?caaataaaag?cggttatgca?ttttgcaggc?ttgaaatcag?taagtgaatc???1680
tataaaaaaa?ccgattgaat?attattcaag?taacgtaatg?caaacgttat?cactaatatc???1740
tacaatgata?gagtataacg?taaaaagctt?tatctttagt?tcatctgcta?ctgtatatgg???1800
taaaccaaag?ttatgcccta?taaatgaaac?tcatcctatt?ggcaaaacta?caaatcctta???1860
tggaacatca?aaattaatga?tggagttaat?gttgtctgat?ttgtgtaatg?catatgatga???1920
ttttaatgtg?acaattttac?gatattttaa?tccaattggt?gctcatgaat?caggtaatat???1980
tggtgaacat?ccaaacggta?taccaaataa?cctatttcct?tatcttacaa?aagttgctat???2040
tggagagtat?ccttatttaa?aagtgtatgg?ttgtgattac?ccaacgtctg?atggttctgg???2100
tgttcgagat?tatattcatg?tagttgatct?tgctgaagca?catgtaaaag?ctttatcaaa???2160
aaattcagag?tcgactgggc?taaaaattta?taatttagga?accggaaaag?gatattctgt???2220
actcgaagtt?atagaagctt?ttgaaaagat?aactggaatc?aaaataccat?ataaaattat???2280
gcctcgaagg?gatggtgatg?ttgcggagtg?ttggtctgat?agcacgaagg?caaatgataa???2340
attagaatgg?aaagcaaaca?gagaattata?tgacatgatt?agagacgctt?ggaattggca???2400
atcaaaaaat?cctcatggtt?atgataatac?atgatgttat?agtatattca?atgaatataa???2460
tttatgcttt?ggagtaaaag?ttgtcacagc?gttttcttgc?agaactaaga?tgtttttagc???2520
ataattagac?acagaattaa?aaatattatc?aggccgtatc?agtacatatc?ttcagtatgc???2580
actatcatat?attaatatga?gaaaatattt?gtatttaaaa?tatgatgttc?taataagatt???2640
aaattaactg?cctaggatat?taaaagctaa?tttggactag?ctttattaga?taagtaaatc???2700
taacaaaata?ttactatgtc?cgatatgtta?taccactatt?aatgacatca?tgtaatatta???2760
ttttaataaa?ctattttttg?atcgcagagg?taattataaa?tgagaagagt?tatcaccttt???2820
ggaacattcg?acgtgtttca?tattggacat?gttaatatat?taaaaagggc?gaaggagttt???2880
ggtgatttgc?tgattgtggg?agtttcatcc?gattcattaa?atttttctaa?aaaacaacgt???2940
tatccaatct?atagccagga?ggagcgcatt?gagattatat?cctcacttaa?gtttgtagat???3000
catgtcttta?tagaagaatc?tttagatctt?aagttagaat?atattagaaa?gtatgaggca???3060
aatttacttg?taatgggaga?tgattggaag?ggcagatttg?actgggtaaa?gatgagtgcg???3120
aagttattta?tttgcccaaa?acaccatcga?tatcaactac?cgagattata?gaaattgtcc???3180
ggagaataaa?atgataactc?atttgttgat?agtgatttat?attgtataac?taatactagg???3240
taaatgatat?atatgcaaat?acaaatgacg?tcatgtaata?actttaaaag?tataaaacat???3300
tatctttata?tccgttggtt?tatcctctat?tcgtgtcttt?ttggggccaa?taacattact???3360
tgtactagcc?aaaaaccttt?tccactgagg?aactaggatt?tttattatgc?tttttttttc???3420
gttaacagca?atggcacagt?tgctggaagt?tgggatgtct?agcgtattga?aacagtatta???3480
tagccattct?tatgctattc?atgatatcag?tgaaaaaaat?ataaaaatat?cgaactattt???3540
taacttttca?atctattggt?atttaatatt?atcaatactt?ttttttatat?tgggaggggg???3600
ggtgtcatat?gttatgttta?atgattataa?cggaatagtt?aactggaaat?taccatggtt???3660
ttttttgctt?tttgtttcat?gtactaattt?attagtattg?cccttaaatg?caatgcttga???3720
tggaacacaa?aggcaacaat?tattgatgaa?agctaatatt?gttagtcagt?taatgatagc???3780
gatatcatta?tggctgacaa?tttatttagg?cttggggcta?tattctcttg?gtctatcaca???3840
attggtagga?atattatttt?ttttactgtc?agtttttata?tttaataaat?gtagattcca???3900
gttcatagaa?atacataaaa?aaaacttttc?atttaaaaca?gtatttaatg?agttgtggcc???3960
gttattgaaa?aaaactagta?tcgtatggtt?tcttggttat?ttctactgga?acggttttaa???4020
tataatatca?tttaaatatc?ttggtgcaga?gatcgcaggc?ttcgttggta?tgtcgatgtc???4080
aattatgcga?gcgggacaga?atatagcaat?atctattttg?aattctcaga?tgacattgta???4140
tgcaagcaat?attgcgatgg?gaaagattga?tgaagcaaga?agagtattta?agaaatattt???4200
tatcatttca?tttttgttat?taatttctgg?ttactcaata?ttttatattt?tctattatgt???4260
taagcctgat?tttttcttgt?ttaagaaggt?cctgccctta?gaacaaatga?tttatgtgtc???4320
aatatttttt?gcctttacat?ttataatatc?aggaactgaa?acttttattc?gttgttttaa???4380
agtggagaag?ttcgttatca?ctcagacaat?caattcaatt?ttaactccat?tcttatttgt???4440
tttggggctt?aacttgcatt?atcaatattt?ttgtttacca?ataatatctg?tactgatagt???4500
gtcaatctta?acgttatata?taagtcgacg?tttttacacg?gggtattcga?atgcaaaaaa???4560
tacttaagta?tataatgaaa?ttaatgatgt?ggagtattta?tcctatcggc?tttttaatta???4620
aaagggataa?aagcatttgg?cttttcggta?gttatagcgg?ttttactgat?aatagtcgat???4680
atttatttga?atctaccact?aaaaagaatg?atgtaagatg?catttggata?tctgatgata???4740
taaagctgat?aaaaaaaatt?agatgtaaag?gttatgaggc?atattacaaa?tatagtgtta???4800
aaggtatata?tttttgttat?attgcaaagg?tgtatatata?ttcaaattat?gtatcaacga???4860
taaactttta?tacatctgca?ggcgcggtat?tggtaaattt?atggcatggg?acgccactta???4920
aaaaaatcga?atatgatata?tctaaatctc?ctttatttaa?ttattttaaa?ggagcctcat???4980
ttataattaa?aatattgatg?cctgagaaac?ataaaaagtg?taatttcatt?cttgctccga???5040
gtcaatttgt?ttatgattat?tcatttaaaa?gtgcatttag?ggtgtatgat?atgcaatatg???5100
ccattattgc?tgatccacct?aggatatcta?atcccttatc?aagagcctat?cacaaagtga???5160
ataatgataa?gaattcaatt?aaaaaaacaa?agaaaacatt?tttatatgca?ccaacatgga???5220
gagatgatcg?ctcggatttt?ataagtattt?caggtttaaa?tttcaacgaa?attaataatt???5280
ttctattaaa?aaataacgcg?tatttacaaa?tcaggttgca?tcctaataca?actttaaata???5340
ttgatatgag?tgaaatggaa?aatattcaca?ttgttgataa?ttatgaacca?gctgaagata???5400
gtatgctaaa?ttctgatttt?ctcataactg?attactcttc?aatatatttt?gactacctct???5460
atcttaatag?acctataatc?ttcttcccat?ttgatcttga?aaaatatacc?gcatcgcgtg???5520
atttttattt?gagttataac?gaagcaactc?caggggttaa?agtatataat?caaggtgaat???5580
taatccagga?gatggataac?ctattgaagg?gcattgacaa?ttggatgaac?tatcgaaagg???5640
agcttgctga?aaaattctgt?tcgctcgaaa?gacgtaacga?tgattatatc?ataaaaaaaa???5700
ttaaaaaagg?tgtatctgaa?tgaatcttgg?taaaattata?aattatatag?agtctaaggt???5760
tattagtata?aattttgatt?tcaatcattt?agctggctat?ttaagtcgtg?atgagcgaga???5820
taaaataacc?ttaaaacatc?taagcagtat?tattgataga?agaaaaaaac?atccatctgt???5880
ttctgctata?tacagagtga?aaaatggagc?tcaatttatt?gaactatcta?ttctttcaat???5940
agctgcattt?tgtcgtgaaa?taattattgt?agacaataat?tcaacagatg?atactttact???6000
aatagtagaa?cgtcttaaaa?gcgaacttca?ggaaatatgt?gaaattaaaa?tctatcagta???6060
tactagtcag?gtcgctttgg?cgggtaagaa?ttataatgat?gaaatcagaa?aatatcctga???6120
gcggtcattg?gcaaaatatt?ataactatgc?tttttcattg?gcaacatcgg?aattcttaat???6180
gaagtgtgat?gctcattgta?tattcacacc?tgcgggtata?gatagtatac?aacgaaaatt???6240
aatttcaaat?cataagtcag?ttgatgttat?atgttatcgt?ggcaccgaag?tatttggagc???6300
catgttagat?tgtgaaccct?atctaatacg?gaaaaatagt?tttaaatatt?acgacactca???6360
atattttgaa?cgtttgacat?taaacaagaa?gagaagtttt?attcaaaaaa?ttaaagggag???6420
aatttcatca?ccttgcttta?tacatgttaa?acgtttgagt?tatgttaagt?cagttcatct???6480
atgtgaaata?aaccctgtgg?agtatatgta?taaaaatgat?aattaaatta?cataatattt???6540
gcgtaactgg?gaagaatgat?aatcatgacg?ggtcttttcg?cctacaaata?tactatttcc???6600
tatgtatttt?aatatctatt?ctatttttca?gtgcataccc?tttggtatcg?ttacctctta???6660
tagctctttc?aagtccattt?gttaataact?atgcaagatg?gactatgtca?ctattaatga???6720
ttttttattt?tattttatta?tactcatcta?ttacgccttt?ttcagacata?gcagagtata???6780
tgcttatcta?tagaggagta?accgatgttg?atatatttag?ttttggtagg?tttggtggtg???6840
gaatcgagat?cttcatatta?gctctcatgc?gcttggtgta?ttttatatcc?gatggcaatg???6900
aatatctttt?tttatttttt?tgctttacta?ttattcaaat?atttcttttt?gtattttgtt???6960
atcgtgttaa?tcgtcgttta?tcatgtttat?attttttagt?atttaatctt?tcatatgctt???7020
attattcttt?taatgcatat?tttttacgaa?gcatgttatc?cgcggttttt?ttaattaatg???7080
cctttttgtt?tatttctaaa?aaaaagtatg?tgtattattt?ttttgcaatt?atatcgcatt???7140
tgtcatccgt?cctatatata?ggagtatggg?aatatttaag?ggcaagaaat?aaaaaattaa???7200
aaatatgggt?tgctagtatg?gcaactgtaa?ctattatatg?ttgttttatt?tttttgcgag???7260
ataggattga?attttattta?tttaaaagct?ctggaaattc?gatggggtat?gtacaagttg???7320
taaccattgc?ttttaattat?attttgtgtt?tgttgttatt?attcttttca?aatgcaagaa???7380
aagaattgtc?aactcaaaga?ataatattgt?ttttgacttt?tttctttatc?ttggccttgg???7440
taatttataa?aataccacaa?aattttggga?taagaatctc?tattttgctt?tacgcactat???7500
caccgttttt?cttgtatcca?cttcttacct?cctgcaatgt?aacaatatta?aataagatca???7560
aatcgttgtc?tctttatatg?attgttaact?gttgctttct?ctttgttgtg?ttatataagg???7620
gcgatgatat?ttcaaatcac?atgcgttcaa?atccattaga?tactagtttt?attgatttat???7680
taataaagga?catagattat?tttgttaaag?gatattatcc?agtaatatat?tctagagacg???7740
aataagggac?attgtttaga?tgtgcaaggt?ggaaatgtat?caaaggaaat?tgtagttact???7800
acagtaacag?ttactttgta?aagtatattt?cggtaaaacc?tgcaataacc?aagggcgcta???7860
aactccctaa?caactccgct?aatggaatta?gaattagaga?gtgacgagtt?taaatatgag???7920
agtttagggt?aatagaacaa?atttttaagt?ttatagagta?agaacagatt?aagttaagtt???7980
tgcttatttt?ataataagct?ctttcttaaa?ccatatatag?tattatctac?aattgagtga???8040
tatcaaattg?aatattgaga?tcggtttatc?agcgctgaat?tcaggaattt?ataaaattaa???8100
atttcgtaat?gattataatt?atcttattat?tcatcagatt?actgatgaat?gtgattactc?????8160
tgattatgtg?gcatctttcc?ctgaaaatgt?tagatacata?tcttcatatg?agtctgggtt?????8220
gtccaaaagt?cgaaatatgg?ccattgaaaa?tgcaaaatac?gattatcttt?ggttaatgga?????8280
tgatgatatg?gaaatagatg?atgcggcttg?ggattatctg?acgaaattta?ttagtgatta?????8340
taaaaatagt?cctttgctgg?ttgtttctca?ttcattggca?aaaaagtata?ataatgaaaa?????8400
agagaggatt?aaaagattga?atcctataac?tgctgcggga?atttgttctg?tagatatgat?????8460
attaaaaatt?tcagcactgg?acggtattag?atttaactca?gattttgggt?taggagctaa?????8520
atatccttcc?ggtgaggaat?atatttttgc?atgtgatttg?atgagagctg?gaaaaaaaat?????8580
atataaatct?aataaaattt?gctcgaatca?tcctgacacg?acttctagtg?ataaaacata?????8640
tccaaataaa?gacgctttta?gtacgaagaa?gaaaatgttt?aaagtcgcaa?atgggaaata?????8700
cttagggagc?attttgtata?ttgcttttgt?tgtcaaaaaa?ttactatata?aataataatt?????8760
tacataattt?tatttttata?taatctattt?attgtctaga?tgcctatgaa?aaaagtatat?????8820
atttttattg?atgatatatc?ccgttccggt?ggcacagaac?gagttgcgac?atttctctct?????8880
aatcaaatgt?ttttatccgg?attttcagtc?acactaatat?cattagcatt?agaaggaggg?????8940
aatacatact?atccgctgca?acccggagtc?aagttagagg?ttttaagtga?atcaacatta?????9000
tacgaattgt?ttcttttttt?aagaaaaaat?tcatgtgatg?tgttgatttc?tgtttccatg?????9060
ggtagtttat?cgtttaagct?tgcatgttta?cattttctat?tacgattaaa?atctcgtctt?????9120
attttaagtg?agcatgtggc?ttttgaatcc?tcatcaaagt?tcattcgttc?tctaaaatgg?????9180
ttaagctatc?aattcgctga?tgatcttgta?ttattgacaa?agcatgatta?taaattgctt?????9240
aagtcaaatg?ttagggctca?tactcacgtt?atcaataatg?ctagtggttt?cgaacaatta?????9300
tctgaatctg?ttcttattca?aaagcaaaag?atagtattag?cagtgggacg?tttaacttat?????9360
cagaaggggt?ttgataggtt?aattcgtatt?tgggcttcca?tatcggacaa?tcacggttgg?????9420
gttttacgga?tcgtagggga?tggagaattg?cgttcagaac?tacaaaaaat?gattgttgac?????9480
ctgaatgtta?cacataatgt?ccaacttata?ccagcaacaa?aatgtataga?acaagaatat?????9540
actaatgcag?ctatattagc?tatgacttct?cgctatgaag?gactaccatt?ggttcttata?????9600
gaggctaaat?cttttggatt?agcagcagtc?gcttttgatt?gtaaaacagg?tcctgctgaa?????9660
attattcatc?acaaaaaaga?tgggttcttg?attactgaag?ataatgaacc?tgaattttca?????9720
tatcgtcttc?agtctttaat?gatcgatgat?gtttatcgaa?acaaaataca?aagtatggcc?????9780
ttaagaaatg?ctaatgatta?ttctgttgaa?aaaataagtc?ggcagtggtt?ggagttaata?????9840
tcatgagtag?ttatgaaaca?gtatctataa?tcatgcctac?gtttaatgct?gcatcttata?????9900
tttcgtatag?catcatttct?gtaattgaac?aatcatataa?tgattggcat?ttatatataa?????9960
tagatgatgg?ttcaactgac?aataccgcac?atgaaatatc?taattttacc?gatctgcgaa????10020
taacatatgt?aatgcttcct?atcaatagtg?gagttgcaaa?tgctagaaat?ataggtatta????10080
aaatggcaaa?agggaaattt?attgcgtttc?ttgatagtga?tgacatttgg?gaaaaagaaa????10140
aattatcctt?gcaaatccct?ttactcgaaa?aaggatatga?tgtagtttgt?tcaaattatg????10200
ccatttttaa?aaatgaacca?aaaaaaataa?tcggttctcg?aactgcacca?caacagatca????10260
cctattcgcg?tttgttacgc?ggtaattgta?tcggcaattt?aacggggatt?tataaccgag????10320
aacgactagg?taaatgctta?caggaagaat?gtggacatga?agactatcta?atgtggttac????10380
agttggtaaa?gctttctggt?agcgcttatt?gcatacagaa?aaatttagcc?cggtatcgag????10440
ttacggaaca?ttcagtatca?tcaaataaat?taaaagcagc?attttggcaa?tggaatatat????10500
atcgtaaaaa?actcaagctt?aatattttga?gcagtatata?ttactggtgt?aattattctt????10560
tttacgcatt?tatgcacatg?ttaaaaatga?agtttcatgt?gaataaaagt?aatctaatat????10620
ataagagata?acaagatata?tagtgttaat?tgtcaccttc?attatataaa?gtagattaat????10680
ttgcgatggt?gacttttaca?ttaacatcga?agtaaatatt?gagataaata?ttatacaata????10740
ttaataatat?gttatgaatt?taaattttat?tttgaattgc?ccaataatat?ttttttaaat????10800
tattacttta?agtagtcatt?ttaggttaat?atattaccgt?taagcaaacc?atgcagtata????10860
ttactgacag?gagtaaacaa?tgtcaaagca?acagattggc?gtcgtcggta?tggcagtgat????10920
ggggcgcaac?cttgcgctca?acatcgaaag?ccgtggttat?accgtctcta?ttttcaaccg????10980
ttcccgtgag?aagacggaag?aagtgattgc?cgaaaatcca?ggcaagaaat?tggttcctta????11040
ctttacggta?aaagagtttg?ttgaatctct?ggaaacgcct?cgtcgcatcc?tgttaatggt????11100
gaaagcaggt?gcaggcacgg?atgctgctat?tgattctctc?aagccatacc?tcgataaagg????11160
tgacatcatc?attgatggtg?gtaacacctt?cttccaggac?accattcgtc?gtaaccgtga????11220
gctgtctgca?gaaggtttta?actttatcgg?tactggtgtt?tccggtggtg?aagaaggtgc????11280
actgaaagga?ccttccatca?tgcctggtgg?ccagaaagaa?gcctatgaac?tggttgcacc????11340
gatcctgacc?aaaatcgccg?cagtggctga?agatggggag?ccatgtgtta?cctatattgg????11400
tgccgatggt?gctggtcact?atgtgaagat?ggttcacaac?ggtattgaat?acggagatat????11460
gcaactgatt?gctgaagcct?attctctgct?aaaaggtggc?ctgaacctca?ccaacgaaga????11520
actggcgcag?acctttaccg?agtggaataa?cggtgaactg?agcagctacc?tgatcgacat????11580
caccaaagat?atcttcacca?aaaaagatga?agacggtaac?tacctggttg?atgtgattct????11640
ggatgaagct?gcaaacaaag?gtacgggtaa?atggaccagc?cagagtgcgc?tggatctcgg????11700
cgaaccgctg?tcgctgatta?ccgagtctgt?gtttgcacgt?tatatctctt?ctctgaaaga????11760
tcagcgcgtt?gccgcatcta?aagttctctc?tggcccgcaa?gcgcagccag?caggcgacaa????11820
aggtgagttc?atcgaaaaag?ttcgccgtgc?gttgtatctg?ggcaaaatcg?tttcttacgc????11880
ccagggcttc?tctcagctgc?gtgcggcgtc?tgaagagtac?aactgggatc?tgaactacgg????11940
cgaaatcgcg?aagattttcc?gtgctggctg?catcatccgt?gcgcagttcc?tgcagaaaat????12000
caccgatgct?tataccgaaa?atccgcagat?cgctaacttg?ctgttggctc?catacttcaa????12060
gcaaattgcc?gatgactatc?agcaggcgct?gcgtgatgtt?gttgcttatg?cagtacagaa????12120
cggtatcccg?gttcccacct?tcgccgctgc?ggttgcctat?tatgacagct?accgtgccgc????12180
tgttctgcct?gcgaacctga?ccaggcacag?cgcgactaag??????????????????????????12220
Glycosyltransferase gene in the O antigen gene of table 1 intestinal bacteria O81 type bunch and oligosaccharide unit treatment gene and wherein
Primer and PCR data
| Gene | Function | The base position of gene | Forward primer | Reverse primer | The length of PCR product | Produce the group number of correct big or small electrophoresis band | The annealing temperature of PCR (℃) |
| Wzx | O-antigen transhipment enzyme | 3407-4567 | ????3441-3424 | ????4094-4101 | ????661bp | ????0 * | ????56.5 |
| ????4054-4071 | ????4281-4298 | ????245bp | ????0 * | ????58 | |||
| Wzy | O-antigen polysaccharase | 6516-7745 | ????6791-6808 | ????7517-7534 | ????744bp | ????0 * | ????56 |
| ????6823-6840 | ????7136-7153 | ????331bp | ????0 * | ????58 |
*Only in intestinal bacteria O81 type, obtain a correct band
Table 2166 strain intestinal bacteria and 43 strain Shigellaes and their source
The bacterium source that contains in this group of group number
1, wild-type e. coli O1, O2, O5, O7, O12, O13, O14, O15, O16, O17, O19ab, O20, IMVS
a
O21，O22，O23，O24，O59
2, wild-type e. coli O25, O26, O27, O28, O29, O30, O32, O33, O35, O36, O37, IMVS
a
O38，O40，O41，O42，O43
3, wild-type e. coli O44, O45, O46, O48, O49, O50, O51, O52, O54, O55, O56, IMVS
a
O57，O58，O60，O61，O62
4, wild-type e. coli O63, O65, O66, O69, O70, O71, O72, O74, O75, O76, O77, O78, IMVS
a
O79，O80，O82，O83
5, wild-type e. coli O84, O85, O86, O87, O88, O89, O91, O92, O98, O99, O101, IMVS
a
O102，O103，O104，O106
6, wild-type e. coli O107, O108, O109, O110, O111, O112ab, O112ac, O113, IMVS
a
O115，O116，O118，O120，O123，O125，O126，O128
7, wild-type e. coli O129, O130, O131, O132, O133, O134, O135, O136, O137, IMVS
a
O138，O139，O140，O141，O142，O143，O144，O145
8, wild-type e. coli O146, O147, O148, O150, O152, O154, O156, O157, O158, IMVS
a
O159，O160，O161，O163，O164，O165，O166?????????????????????????????????b
9, wild-type e. coli O168, O169, O170, O171, O172, O173, c
D1，D2，D3，D4，D5，D6，D7，D8，D9，D10，D11，D12????????????????????????d
10, wild-type e. coli B1, B2, B3, B4, B6, B7, B8, B9, B10, B11, B12, B13, B14, B15, d
B16，B17，B18
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, wild-type e. coli O3, O11, O39, O59, O64, O73, O96, O95, O100, O114, O151, IMVS
a
O155，O124
13, the 4th group of bacterial strain adds intestinal bacteria reference culture O81 IMVS
*For the convenience that detects, we are divided into one group with every 13-17 bacterium, 13 groups altogether
a.Institude?of?Medical?and?Veterinary?Science，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 O81 type O antigen gene structure iron
Escherichia?coli?type?81?O-antigen?gene?cluster
galF????orf1????orf2????wzx????orf4????orf5????wzy????orf7????orf8????orf9????gnd
G+C％???32.8????34.7????29.3???29.0????29.7????27.6???30.2????34.1????31.8
Table 4 intestinal bacteria O81 type O antigen gene cluster gene position
ATTGTGGCTG?CAGGGATCAA?AGAAATCCTC?CTGGTAACTC?ATGCGTCCAA?GAACGCGGTC?????60
GAAAACCACT?TCGACACCTC?TTATGAATTA?GAATCTCTCC?TTGAACAGCG?CGTGAAGCGT????120
CAACTGCTGG?CGGAAGTACA?GTCTATCTGT?CCGCCGGGCG?TGACCATTAT?GAACGTGCGT????180
CAGGGCGAAC?CTTTAGGTTT?GGGCCACTCC?ATTTTGTGTG?CACGACCCGC?CATTGGTGAC????240
AACCCATTTG?TCGTGGTACT?GCCGGACGTT?GTGATCGACG?ACGCCAGTGC?CGACCCGCTG????300
CGCTACAACC?TTGCTGCCAT?GATTGCGCGT?TTCAATGAAA?CGGGCCGTAG?CCAGGTGCTG????360
GCAAAACGTA?TGCCGGGTGA?CCTTTCTGAA?TACTCCGTTA?TTCAGACCAA?AGAGCCGCTG????420
GATCGTGAAG?GCAAAGTCAG?CCGCATTGTT?GAATTTATCG?AAAAACCGGA?TCAGCCGCAG????480
ACGCTGGACT?CAGACATCAT?GGCCGTTGGT?CGCTATGTGC?TTTCTGCCGA?TATTTGGCCG????540
GAACTTGAAC?GCACTCAACC?TGGTGCATGG?GGACGTATCC?AACTGACTGA?TGCCATTGCT????600
GAACTGGCGA?AGAAACAGTC?CGTTGATGCC?ATGCTGATGA?CTGGTGACAG?CTACGACTGC????660
GGTAAAAAAA?TGGGCTATAT?GCAGGCGTTT?GTGAAGTATG?GACTGCGCAA?CCTGAAAGAA?????720
GGGGCGAAGT?TCCGTAAAGG?GATTGAGAAG?CTGTTAAGCG?AATAATGAAA?ATCTGACCGG?????780
ATGTAACGGT?TGATAAGAAA?ATTATAACGG?CAGTGAAGAT?TCGTGGTGAA?AGTAATTTGT?????840
TGCGAATATT?CCTGCCGTTG?TTTTATATAA?ACAATCAGAA?TAACAACGAG?TTAGCAATAG?????900
GATTTTAGTC?AAAGTTTTCC?AGGATTTTCC?TTGTTTCCAG?AGCGGATTGG?TAAGACAATT?????960
AGCGTTTGAA?TTTTTCGAGT?TTAGCGCGAG?TAGGTAACGC?TAGTCACATC?GTAGACATGT????1020
AAGCAGTGCT?CTGGTAGCTG?TAAAGCCAGG?GGCGGTAGCA?TTGTTCTTGA?TATTTTTAAG????1080
TATAAAAATG?ACACATAAAA?ATAAAATAAT?TAGTTATCGG?TATGTTTTTA?ATTGTGTTAT????1140
TATTGAAAAA?TATCACCATT?AATTATTTAA?TGCGATAAAG?GCCAAATAAA?TCACTCATTT????1200
ATTTCTACAT?AGGATAGGGG?ACTATCAGAT?GGGAATTAAA?TAAAAATCCA?AGTGCTAAAT????1260
ATTTCAATCT?TAAATTCCAT?TGTAAAAAGT?TTAAGGGATT?TTATTCTAAA?AAAGAATTGG????1320
ATGAATTGAA?AGTTATTTTG?AGCTTAATTT?ATTAAACTTG?AAGTTTTTTT?ACTGAGTTTT????1380
Orf1 is initial
TCTGGAAAAT?AATTTATTGT?TAATGGAGCC?ATAT
ATGTCA?ATTTTAGTTA?CCGGAGGTTG?1440
TGGTTATATA?GGTGTCCACA?CAATTTATAC?GTTAATTTCT?CAGGGCTACG?ATGTTATTGT????1500
ACTGGATAAC?CTGACTAACT?CATCAATAGA?GTCATTAGAG?AGAGTCAAGT?TAATGACAAA????1560
TCATAACATA?CCCTTTTATA?AAGGAGATGT?GGGGAATAAA?AAAATATTGG?TAGACATATT????1620
CAATGAGCAT?CAAATAAAAG?CGGTTATGCATTTTGCAGGC?TTGAAATCAG?TAAGTGAATC?????1680
TATAAAAAAA?CCGATTGAAT?ATTATTCAAG?TAACGTAATG?CAAACGTTAT?CACTAATATC????1740
TACAATGATA?GAGTATAACG?TAAAAAGCTT?TATCTTTAGT?TCATCTGCTA?CTGTATATGG????1800
TAAACCAAAG?TTATGCCCTA?TAAATGAAAC?TCATCCTATT?GGCAAAACTA?CAAATCCTTA????1860
TGGAACATCA?AAATTAATGA?TGGAGTTAAT?GTTGTCTGAT?TTGTGTAATG?CATATGATGA????1920
TTTTAATGTG?ACAATTTTAC?GATATTTTAA?TCCAATTGGT?GCTCATGAAT?CAGGTAATAT????1980
TGGTGAACAT?CCAAACGGTA?TACCAAATAA?CCTATTTCCT?TATCTTACAA?AAGTTGCTAT????2040
TGGAGAGTAT?CCTTATTTAA?AAGTGTATGG?TTGTGATTAC?CCAACGTCTG?ATGGTTCTGG????2100
TGTTCGAGAT?TATATTCATG?TAGTTGATCT?TGCTGAAGCA?CATGTAAAAG?CTTTATCAAA????2160
AAATTCAGAG?TCGACTGGGC?TAAAAATTTA?TAATTTAGGA?ACCGGAAAAG?GATATTCTGT????2220
ACTCGAAGTT?ATAGAAGCTT?TTGAAAAGAT?AACTGGAATC?AAAATACCAT?ATAAAATTAT????2280
GCCTCGAAGG?GATGGTGATG?TTGCGGAGTG?TTGGTCTGAT?AGCACGAAGG?CAAATGATAA????2340
ATTAGAATGG?AAAGCAAACA?GAGAATTATA?TGACATGATT?AGAGACGCTT?GGAATTGGCA????2400
Orf1 stops
ATCAAAAAAT?CCTCATGGTT?ATGATAATAC?A
TGATGTTAT?AGTATATTCA?ATGAATATAA??2460
TTTATGCTTT?GGAGTAAAAG?TTGTCACAGC?GTTTTCTTGC?AGAACTAAGA?TGTTTTTAGC????2520
ATAATTAGAC?ACAGAATTAA?AAATATTATC?AGGCCGTATC?AGTACATATC?TTCAGTATGC????2580
ACTATCATAT?ATTAATATGA?GAAAATATTT?GTATTTAAAA?TATGATGTTC?TAATAAGATT????2640
AAATTAACTG?CCTAGGATAT?TAAAAGCTAA?TTTGGACTAG?CTTTATTAGA?TAAGTAAATC????2700
TAACAAAATA?TTACTATGTC?CGATATGTTA?TACCACTATT?AATGACATCA?TGTAATATTA????2760
Orf2 is initial
TTTTAATAAA?CTATTTTTTG?ATCGCAGAGG?TAATTATAA
A?
TGAGAAGAGT?TATCACCTTT2820
GGAACATTCG?ACGTGTTTCA?TATTGGACAT?GTTAATATAT?TAAAAAGGGC?GAAGGAGTTT????2880
GGTGATTTGC?TGATTGTGGG?AGTTTCATCC?GATTCATTAA?ATTTTTCTAA?AAAACAACGT????2940
TATCCAATCT?ATAGCCAGGA?GGAGCGCATT?GAGATTATAT?CCTCACTTAA?GTTTGTAGAT????3000
CATGTCTTTA?TAGAAGAATC?TTTAGATCTT?AAGTTAGAAT?ATATTAGAAA?GTATGAGGCA????3060
AATTTACTTG?TAATGGGAGA?TGATTGGAAG?GGCAGATTTG?ACTGGGTAAA?GATGAGTGCG????3120
Orf2 stops
AAGTTATTTA?TTTGCCCAAA?ACACCATCGA?TATCAACTAC?CGAGATTA
TA?
GAAATTGTCC3180
GGAGAATAAA?ATGATAACTC?ATTTGTTGAT?AGTGATTTAT?ATTGTATAAC?TAATACTAGG????3240
TAAATGATAT?ATATGCAAAT?ACAAATGACG?TCATGTAATA?ACTTTAAAAG?TATAAAACAT????3300
TATCTTTATA?TCCGTTGGTT?TATCCTCTAT?TCGTGTCTTT?TTGGGGCCAA?TAACATTACT????3360
Orf3 is initial
TGTACTAGCC?AAAAACCTTT?TCCACTGAGG?AACTAGGATT?TTTATT
ATGC?TTTTTTTTTC??3420
GTTAACAGCA?ATGGCACAGT?TGCTGGAAGT?TGGGATGTCT?AGCGTATTGA?AACAGTATTA????3480
TAGCCATTCT?TATGCTATTC?ATGATATCAG?TGAAAAAAAT?ATAAAAATAT?CGAACTATTT????3540
TAACTTTTCA?ATCTATTGGT?ATTTAATATT?ATCAATACTT?TTTTTTATAT?TGGGAGGGGG????3600
GGTGTCATAT?GTTATGTTTA?ATGATTATAA?CGGAATAGTT?AACTGGAAAT?TACCATGGTT????3660
TTTTTTGCTT?TTTGTTTCAT?GTACTAATTT?ATTAGTATTG?CCCTTAAATG?CAATGCTTGA????3720
TGGAACACAA?AGGCAACAAT?TATTGATGAA?AGCTAATATT?GTTAGTCAGT?TAATGATAGC????3780
GATATCATTA?TGGCTGACAA?TTTATTTAGG?CTTGGGGCTA?TATTCTCTTG?GTCTATCACA????3840
ATTGGTAGGA?ATATTATTTT?TTTTACTGTC?AGTTTTTATA?TTTAATAAAT?GTAGATTCCA????3900
GTTCATAGAA?ATACATAAAA?AAAACTTTTC?ATTTAAAACA?GTATTTAATG?AGTTGTGGCC????3960
GTTATTGAAA?AAAACTAGTA?TCGTATGGTT?TCTTGGTTAT?TTCTACTGGA?ACGGTTTTAA????4020
TATAATATCA?TTTAAATATC?TTGGTGCAGA?GATCGCAGGC?TTCGTTGGTA?TGTCGATGTC????4080
AATTATGCGA?GCGGGACAGA?ATATAGCAAT?ATCTATTTTG?AATTCTCAGA?TGACATTGTA????4140
TGCAAGCAAT?ATTGCGATGG?GAAAGATTGA?TGAAGCAAGA?AGAGTATTTA?AGAAATATTT????4200
TATCATTTCA?TTTTTGTTAT?TAATTTCTGG?TTACTCAATA?TTTTATATTT?TCTATTATGT????4260
TAAGCCTGAT?TTTTTCTTGT?TTAAGAAGGT?CCTGCCCTTA?GAACAAATGA?TTTATGTGTC????4320
AATATTTTTT?GCCTTTACAT?TTATAATATC?AGGAACTGAA?ACTTTTATTC?GTTGTTTTAA????4380
AGTGGAGAAG?TTCGTTATCA?CTCAGACAAT?CAATTCAATT?TTAACTCCAT?TCTTATTTGT????4440
TTTGGGGCTT?AACTTGCATT?ATCAATATTT?TTGTTTACCA?ATAATATCTG?TACTGATAGT????4500
Orf4 is initial
GTCAATCTTA?ACGTTATATA?TAAGTCGACG?TTTTTACACG?GGGTATTCGA?
ATGCAAAAAA??4560
Orf3 stops
TACT
TAAGTA?TATAATGAAA?TTAATGATGT?GGAGTATTTA?TCCTATCGGC?TTTTTAATTA??4620
AAAGGGATAA?AAGCATTTGG?CTTTTCGGTA?GTTATAGCGG?TTTTACTGAT?AATAGTCGAT????4680
ATTTATTTGA?ATCTACCACT?AAAAAGAATG?ATGTAAGATG?CATTTGGATA?TCTGATGATA????4740
TAAAGCTGAT?AAAAAAAATT?AGATGTAAAG?GTTATGAGGC?ATATTACAAA?TATAGTGTTA????4800
AAGGTATATA?TTTTTGTTAT?ATTGCAAAGG?TGTATATATA?TTCAAATTAT?GTATCAACGA????4860
TAAACTTTTA?TACATCTGCA?GGCGCGGTAT?TGGTAAATTT?ATGGCATGGG?ACGCCACTTA????4920
AAAAAATCGA?ATATGATATA?TCTAAATCTC?CTTTATTTAA?TTATTTTAAA?GGAGCCTCAT????4980
TTATAATTAA?AATATTGATG?CCTGAGAAAC?ATAAAAAGTG?TAATTTCATT?CTTGCTCCGA????5040
GTCAATTTGT?TTATGATTAT?TCATTTAAAA?GTGCATTTAG?GGTGTATGAT?ATGCAATATG????5100
CCATTATTGC?TGATCCACCT?AGGATATCTA?ATCCCTTATC?AAGAGCCTAT?CACAAAGTGA????5160
ATAATGATAA?GAATTCAATT?AAAAAAACAA?AGAAAACATT?TTTATATGCA?CCAACATGGA????5220
GAGATGATCG?CTCGGATTTT?ATAAGTATTT?CAGGTTTAAA?TTTCAACGAA?ATTAATAATT????5280
TTCTATTAAA?AAATAACGCG?TATTTACAAA?TCAGGTTGCA?TCCTAATACA?ACTTTAAATA????5340
TTGATATGAG?TGAAATGGAA?AATATTCACA?TTGTTGATAA?TTATGAACCA?GCTGAAGATA????5400
GTATGCTAAA?TTCTGATTTT?CTCATAACTG?ATTACTCTTC?AATATATTTT?GACTACCTCT????5460
ATCTTAATAG?ACCTATAATC?TTCTTCCCAT?TTGATCTTGA?AAAATATACC?GCATCGCGTG????5520
ATTTTTATTT?GAGTTATAAC?GAAGCAACTC?CAGGGGTTAA?AGTATATAAT?CAAGGTGAAT????5580
TAATCCAGGA?GATGGATAAC?CTATTGAAGG?GCATTGACAA?TTGGATGAAC?TATCGAAAGG????5640
AGCTTGCTGA?AAAATTCTGT?TCGCTCGAAA?GACGTAACGA?TGATTATATC?ATAAAAAAAA????5700
The initial orf4 of orf5 stops
TTAAAAAAGG?TGTATCTGA
A?
TGAATCTTGG?TAAAATTATA?AATTATATAG?AGTCTAAGGT?5760
TATTAGTATA?AATTTTGATT?TCAATCATTT?AGCTGGCTAT?TTAAGTCGTG?ATGAGCGAGA?????5820
TAAAATAACC?TTAAAACATC?TAAGCAGTAT?TATTGATAGA?AGAAAAAAAC?ATCCATCTGT?????5880
TTCTGCTATA?TACAGAGTGA?AAAATGGAGC?TCAATTTATT?GAACTATCTA?TTCTTTCAAT?????5940
AGCTGCATTT?TGTCGTGAAA?TAATTATTGT?AGACAATAAT?TCAACAGATG?ATACTTTACT?????6000
AATAGTAGAA?CGTCTTAAAA?GCGAACTTCA?GGAAATATGT?GAAATTAAAA?TCTATCAGTA?????6060
TACTAGTCAG?GTCGCTTTGG?CGGGTAAGAA?TTATAATGAT?GAAATCAGAA?AATATCCTGA?????6120
GCGGTCATTG?GCAAAATATT?ATAACTATGC?TTTTTCATTG?GCAACATCGG?AATTCTTAAT?????6180
GAAGTGTGAT?GCTCATTGTA?TATTCACACC?TGCGGGTATA?GATAGTATAC?AACGAAAATT?????6240
AATTTCAAAT?CATAAGTCAG?TTGATGTTAT?ATGTTATCGT?GGCACCGAAG?TATTTGGAGC?????6300
CATGTTAGAT?TGTGAACCCT?ATCTAATACG?GAAAAATAGT?TTTAAATATT?ACGACACTCA?????6360
ATATTTTGAA?CGTTTGACAT?TAAACAAGAA?GAGAAGTTTT?ATTCAAAAAA?TTAAAGGGAG?????6420
AATTTCATCA?CCTTGCTTTA?TACATGTTAA?ACGTTTGAGT?TATGTTAAGT?CAGTTCATCT?????6480
The initial orf5 of orf6 stops
ATGTGAAATA?AACCCTGTGG?AGTATATGTA?TAAAA
ATGAT?AAT
TAAATTA?CATAATATTT?6540
GCGTAACTGG?GAAGAATGAT?AATCATGACG?GGTCTTTTCG?CCTACAAATA?TACTATTTCC?????6600
TATGTATTTT?AATATCTATT?CTATTTTTCA?GTGCATACCC?TTTGGTATCG?TTACCTCTTA?????6660
TAGCTCTTTC?AAGTCCATTT?GTTAATAACT?ATGCAAGATG?GACTATGTCA?CTATTAATGA?????6720
TTTTTTATTT?TATTTTATTA?TACTCATCTA?TTACGCCTTT?TTCAGACATA?GCAGAGTATA?????6780
TGCTTATCTA?TAGAGGAGTA?ACCGATGTTG?ATATATTTAG?TTTTGGTAGG?TTTGGTGGTG?????6840
GAATCGAGAT?CTTCATATTA?GCTCTCATGC?GCTTGGTGTA?TTTTATATCC?GATGGCAATG?????6900
AATATCTTTT?TTTATTTTTT?TGCTTTACTA?TTATTCAAAT?ATTTCTTTTT?GTATTTTGTT?????6960
ATCGTGTTAA?TCGTCGTTTA?TCATGTTTAT?ATTTTTTAGT?ATTTAATCTT?TCATATGCTT?????7020
ATTATTCTTT?TAATGCATAT?TTTTTACGAA?GCATGTTATC?CGCGGTTTTT?TTAATTAATG?????7080
CCTTTTTGTT?TATTTCTAAA?AAAAAGTATG?TGTATTATTT?TTTTGCAATT?ATATCGCATT?????7140
TGTCATCCGT?CCTATATATA?GGAGTATGGG?AATATTTAAG?GGCAAGAAAT?AAAAAATTAA?????7200
AAATATGGGT?TGCTAGTATG?GCAACTGTAA?CTATTATATG?TTGTTTTATT?TTTTTGCGAG?????7260
ATAGGATTGA?ATTTTATTTA?TTTAAAAGCT?CTGGAAATTC?GATGGGGTAT?GTACAAGTTG?????7320
TAACCATTGC?TTTTAATTAT?ATTTTGTGTT?TGTTGTTATT?ATTCTTTTCA?AATGCAAGAA?????7380
AAGAATTGTC?AACTCAAAGA?ATAATATTGT?TTTTGACTTT?TTTCTTTATC?TTGGCCTTGG?????7440
TAATTTATAA?AATACCACAA?AATTTTGGGA?TAAGAATCTC?TATTTTGCTT?TACGCACTAT?????7500
CACCGTTTTT?CTTGTATCCA?CTTCTTACCT?CCTGCAATGT?AACAATATTA?AATAAGATCA?????7560
AATCGTTGT℃TCTTTATATG?ATTGTTAACT?GTTGCTTTCT?CTTTGTTGTG?TTATATAAGG?????7620
GCGATGATAT?TTCAAATCAC?ATGCGTTCAA?ATCCATTAGA?TACTAGTTTT?ATTGATTTAT?????7680
TAATAAAGGA?CATAGATTAT?TTTGTTAAAG?GATATTATCC?AGTAATATAT?TCTAGAGACG?????7740
Orf6 stops
AA
TAAGGGAC?ATTGTTTAGA?TGTGCAAGGT?GGAAATGTAT?CAAAGGAAAT?TGTAGTTACT???7800
ACAGTAACAG?TTACTTTGTA?AAGTATATTT?CGGTAAAACC?TGCAATAACC?AAGGGCGCTA?????7860
AACTCCCTAA?CAACTCCGCT?AATGGAATTA?GAATTAGAGA?GTGACGAGTT?TAAATATGAG?????7920
AGTTTAGGGT?AATAGAACAA?ATTTTTAAGT?TTATAGAGTA?AGAACAGATT?AAGTTAAGTT?????7980
Orf7 is initial
TGCTTATTTT?ATAATAAGCT?CTTTCTTAAA?CCATATATAG?
TATTATCTAC?AATTGAGTGA???8040
TATCAAATTG?AATATTGAGA?TCGGTTTATC?AGCGCTGAAT?TCAGGAATTT?ATAAAATTAA?????8100
ATTTCGTAAT?GATTATAATT?ATCTTATTAT?TCATCAGATT?ACTGATGAAT?GTGATTACTC?????8160
TGATTATGTG?GCATCTTTCC?CTGAAAATGT?TAGATACATA?TCTTCATATG?AGTCTGGGTT?????8220
GTCCAAAAGT?CGAAATATGG?CCATTGAAAA?TGCAAAATAC?GATTATCTTT?GGTTAATGGA?????8280
TGATGATATG?GAAATAGATG?ATGCGGCTTG?GGATTATCTG?ACGAAATTTA?TTAGTGATTA????8340
TAAAAATAGT?CCTTTGCTGG?TTGTTTCTCA?TTCATTGGCA?AAAAAGTATA?ATAATGAAAA????8400
AGAGAGGATT?AAAAGATTGA?ATCCTATAAC?TGCTGCGGGA?ATTTGTTCTG?TAGATATGAT????8460
ATTAAAAATT?TCAGCACTGG?ACGGTATTAG?ATTTAACTCA?GATTTTGGGT?TAGGAGCTAA????8520
ATATCCTTCC?GGTGAGGAAT?ATATTTTTGC?ATGTGATTTG?ATGAGAGCTG?GAAAAAAAAT????8580
ATATAAATCT?AATAAAATTT?GCTCGAATCA?TCCTGACACG?ACTTCTAGTG?ATAAAACATA????8640
TCCAAATAAA?GACGCTTTTA?GTACGAAGAA?GAAAATGTTT?AAAGTCGCAA?ATGGGAAATA????8700
Orf7 stops
CTTAGGGAGC?ATTTTGTATA?TTGCTTTTGT?TGTCAAAAAA?TTACTATATA?AA
TAATAATT??8760
Orf8 is initial
TACATAATTT?TATTTTTATA?TAATCTATTT?ATTGTCTAG
A?
TGCCTATGAA?AAAAGTATAT8820
ATTTTTATTG?ATGATATATC?CCGTTCCGGT?GGCACAGAAC?GAGTTGCGAC?ATTTCTCTCT????8880
AATCAAATGT?TTTTATCCGG?ATTTTCAGTC?ACACTAATAT?CATTAGCATT?AGAAGGAGGG????8940
AATACATACT?ATCCGCTGCA?ACCCGGAGTC?AAGTTAGAGG?TTTTAAGTGA?ATCAACATTA????9000
TACGAATTGT?TTCTTTTTTT?AAGAAAAAAT?TCATGTGATG?TGTTGATTTC?TGTTTCCATG????9060
GGTAGTTTAT?CGTTTAAGCT?TGCATGTTTA?CATTTTCTAT?TACGATTAAA?ATCTCGTCTT????9120
ATTTTAAGTG?AGCATGTGGC?TTTTGAATCC?TCATCAAAGT?TCATTCGTTC?TCTAAAATGG????9180
TTAAGCTATC?AATTCGCTGA?TGATCTTGTA?TTATTGACAA?AGCATGATTA?TAAATTGCTT????9240
AAGTCAAATG?TTAGGGCTCA?TACTCACGTT?ATCAATAATG?CTAGTGGTTT?CGAACAATTA????9300
TCTGAATCTG?TTCTTATTCA?AAAGCAAAAG?ATAGTATTAG?CAGTGGGACG?TTTAACTTAT????9360
CAGAAGGGGT?TTGATAGGTT?AATTCGTATT?TGGGCTTCCA?TATCGGACAA?TCACGGTTGG????9420
GTTTTACGGA?TCGTAGGGGA?TGGAGAATTG?CGTTCAGAAC?TACAAAAAAT?GATTGTTGAC????9480
CTGAATGTTA?CACATAATGT?CCAACTTATA?CCAGCAACAA?AATGTATAGA?ACAAGAATAT????9540
ACTAATGCAG?CTATATTAGC?TATGACTTCT?CGCTATGAAG?GACTACCATT?GGTTCTTATA????9600
GAGGCTAAAT?CTTTTGGATT?AGCAGCAGTC?GCTTTTGATT?GTAAAACAGG?TCCTGCTGAA????9660
ATTATTCATC?ACAAAAAAGA?TGGGTTCTTG?ATTACTGAAG?ATAATGAACC?TGAATTTTCA????9720
TATCGTCTTC?AGTCTTTAAT?GATCGATGAT?GTTTATCGAA?ACAAAATACA?AAGTATGGCC????9780
TTAAGAAATG?CTAATGATTA?TTCTGTTGAA?AAAATAAGTC?GGCAGTGGTT?GGAGTTAATA????9840
The initial orf8 of orf9 stops
TC
ATGAGTAG?TTATGAAACA?GTATCTATAA?TCATGCCTAC?GTTTAATGCT?GCATCTTATA??9900
TTTCGTATAG?CATCATTTCT?GTAATTGAAC?AATCATATAA?TGATTGGCAT?TTATATATAA????9960
TAGATGATGG?TTCAACTGAC?AATACCGCAC?ATGAAATATC?TAATTTTACC?GATCTGCGAA????10020
TAACATATGT?AATGCTTCCT?ATCAATAGTG?GAGTTGCAAA?TGCTAGAAAT?ATAGGTATTA????10080
AAATGGCAAA?AGGGAAATTT?ATTGCGTTTC?TTGATAGTGA?TGACATTTGG?GAAAAAGAAA????10140
AATTATCCTT?GCAAATCCCT?TTACTCGAAA?AAGGATATGA?TGTAGTTTGT?TCAAATTATG????10200
CCATTTTTAA?AAATGAACCA?AAAAAAATAA?TCGGTTCTCG?AACTGCACCA?CAACAGATCA????10260
CCTATTCGCG?TTTGTTACGC?GGTAATTGTA?TCGGCAATTT?AACGGGGATT?TATAACCGAG????10320
AACGACTAGG?TAAATGCTTA?CAGGAAGAAT?GTGGACATGA?AGACTATCTA?ATGTGGTTAC????10380
AGTTGGTAAA?GCTTTCTGGT?AGCGCTTATT?GCATACAGAA?AAATTTAGCC?CGGTATCGAG????10440
TTACGGAACA?TTCAGTATCA?TCAAATAAAT?TAAAAGCAGC?ATTTTGGCAA?TGGAATATAT????10500
ATCGTAAAAA?ACTCAAGCTT?AATATTTTGA?GCAGTATATA?TTACTGGTGT?AATTATTCTT????10560
TTTACGCATT?TATGCACATG?TTAAAAATGA?AGTTTCATGT?GAATAAAAGT?AATCTAATAT????10620
Orf9 stops
ATAAGAGA
TA?
ACAAGATATA?TAGTGTTAAT?TGTCACCTTC?ATTATATAAA?GTAGATTAAT10680
TTGCGATGGT?GACTTTTACA?TTAACATCGA?AGTAAATATT?GAGATAAATA?TTATACAATA????10740
TTAATAATAT?GTTATGAATT?TAAATTTTAT?TTTGAATTGC?CCAATAATAT?TTTTTTAAAT????10800
TATTACTTTA?AGTAGTCATT?TTAGGTTAAT?ATATTACCGT?TAAGCAAACC?ATGCAGTATA????10860
TTACTGACAG?GAGTAAACAA?TGTCAAAGCA?ACAGATTGGC?GTCGTCGGTA?TGGCAGTGAT????10920
GGGGCGCAAC?CTTGCGCTCA?ACATCGAAAG?CCGTGGTTAT?ACCGTCTCTA?TTTTCAACCG????10980
TTCCCGTGAG?AAGACGGAAG?AAGTGATTGC?CGAAAATCCA?GGCAAGAAAT?TGGTTCCTTA????11040
CTTTACGGTA?AAAGAGTTTG?TTGAATCTCT?GGAAACGCCT?CGTCGCATCC?TGTTAATGGT????11100
GAAAGCAGGT?GCAGGCACGG?ATGCTGCTAT?TGATTCTCTC?AAGCCATACC?TCGATAAAGG????11160
TGACATCATC?ATTGATGGTG?GTAACACCTT?CTTCCAGGAC?ACCATTCGTC?GTAACCGTGA????11220
GCTGTCTGCA?GAAGGTTTTA?ACTTTATCGG?TACTGGTGTT?TCCGGTGGTG?AAGAAGGTGC????11280
ACTGAAAGGA?CCTTCCATCA?TGCCTGGTGG?CCAGAAAGAA?GCCTATGAAC?TGGTTGCACC????11340
GATCCTGACC?AAAATCGCCG?CAGTGGCTGA?AGATGGGGAG?CCATGTGTTA?CCTATATTGG????11400
TGCCGATGGT?GCTGGTCACT?ATGTGAAGAT?GGTTCACAAC?GGTATTGAAT?ACGGAGATAT????11460
GCAACTGATT?GCTGAAGCCT?ATTCTCTGCT?AAAAGGTGGC?CTGAACCTCA?CCAACGAAGA????11520
ACTGGCGCAG?ACCTTTACCG?AGTGGAATAA?CGGTGAACTG?AGCAGCTACC?TGATCGACAT????11580
CACCAAAGAT?ATCTTCACCA?AAAAAGATGA?AGACGGTAAC?TACCTGGTTG?ATGTGATTCT????11640
GGATGAAGCT?GCAAACAAAG?GTACGGGTAA?ATGGACCAGC?CAGAGTGCGC?TGGATCTCGG????11700
CGAACCGCTG?TCGCTGATTA?CCGAGTCTGT?GTTTGCACGT?TATATCTCTT?CTCTGAAAGA????11760
TCAGCGCGTT?GCCGCATCTA?AAGTTCTCTC?TGGCCCGCAA?GCGCAGCCAG?CAGGCGACAA????11820
AGGTGAGTTC?ATCGAAAAAG?TTCGCCGTGC?GTTGTATCTG?GGCAAAATCG?TTTCTTACGC????11880
CCAGGGCTTC?TCTCAGCTGC?GTGCGGCGTC?TGAAGAGTAC?AACTGGGATC?TGAACTACGG????11940
CGAAATCGCG?AAGATTTTCC?GTGCTGGCTG?CATCATCCGT?GCGCAGTTCC?TGCAGAAAAT????12000
CACCGATGCT?TATACCGAAA?ATCCGCAGAT?CGCTAACTTG?CTGTTGGCTC?CATACTTCAA????12060
GCAAATTGCC?GATGACTATC?AGCAGGCGCT?GCGTGATGTT?GTTGCTTATG?CAGTACAGAA????12120
CGGTATCCCG?GTTCCCACCT?TCGCCGCTGC?GGTTGCCTAT?TATGACAGCT?ACCGTGCCGC????12180
TGTTCTGCCT?GCGAACCTGA?CCAGGCACAG?CGCGACTAAG??????????????????????????12220
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 (9)
1, a kind of Nucleotide of the O-antigen-specific to intestinal bacteria O81 type, it is characterized in that: it is the isolating Nucleotide shown in SEQ ID NO:1,12200 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.
2, according to the Nucleotide of the described O-antigen-specific to intestinal bacteria O81 type of claim 1, it is characterized in that: it is by 9 genomic constitutions, all between galF gene and gnd gene.
3, according to the Nucleotide of the described O-antigen-specific to intestinal bacteria O81 type of claim 2, it is characterized in that described gene is: the gene of transhipment enzyme comprises the wzx gene or with wzx the gene of identity function is arranged; Pol gene comprises the wzy gene or with wzy the gene of identity function is arranged; Glycosyltransferase gene comprises orf5, orf7, orf8, orf9 gene; Isomerase orf1 and orf1 have the gene of identity function; It is the Nucleotide of 3407 to 4567 bases among the SEQ ID NO:1 that monose conjugate synthetic gene orf2, orf4 and orf2, orf4 have gene, wherein said gene: the wzx of identity function; Wzy is the Nucleotide of 6516 to 7745 bases among the SEQ ID NO:1; Orf1 is the Nucleotide of 1415 to 2434 bases among the SEQ ID NO:1; Orf2 is the Nucleotide of 2800 to 3171 bases among the SEQ ID NO:1; Orf4 is the Nucleotide of 4551 to 5723 bases among the SEQ ID NO:1; Orf5 is the Nucleotide of 5720 to 6526 bases among the SEQ IDNO:1; Orf7 is the Nucleotide of 8021 to 8755 bases among the SEQ ID NO:1; Orf8 is the Nucleotide of 8800 to 9846 bases among the SEQ ID NO:1; Orf9 is the Nucleotide of 9843 to 10631 bases among the SEQ ID NO:1.
4, according to the Nucleotide of claim 1 or 2 described O-antigen-specifics to intestinal bacteria O81 type, it is characterized in that: it is to come from described wzx gene, wzy gene; The orf5 that glycosyltransferase gene comprises, orf7, orf8, orf9 gene; And their mixing or their reorganization.
5, according to the Nucleotide of the described O-antigen high special to intestinal bacteria O81 type of claim 4, it is characterized in that the oligonucleotide of the described wzx of coming from gene is to being: the Nucleotide of 3441 to 3458 bases among the SEQ ID NO:1 and the Nucleotide of 4084 to 4101 bases; The Nucleotide of 4054 to 4071 bases among the SEQ ID NO:1 and the Nucleotide of 4281 to 4298 bases; The oligonucleotide that comes from the wzy gene is to being: the Nucleotide of 6791 to 6808 bases among the SEQ ID NO:1 and the Nucleotide of 7517 to 7534 bases; The Nucleotide of 6823 to 6840 bases among the SEQ ID NO:1 and the Nucleotide of 7136 to 7153 bases.
6, the Nucleotide of the described O-antigen-specific to intestinal bacteria O81 type of claim 1 is detecting the application of expressing the antigenic bacterium of O-, identify other polysaccharide antigen of the O-antigen of bacterium and bacterium in diagnosis.
7, the recombinant molecule of the Nucleotide of the described O-antigen-specific to intestinal bacteria O81 type of claim 1, and can provide the O-antigen of expressing intestinal bacteria O81 type by inserting to express, and become bacterial vaccine.
8, according to the application of the Nucleotide of the described O-antigen-specific to intestinal bacteria O81 type of claim 1, 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, the bacterium in human body and the environment as probe as primer.
9, the separation method of the Nucleotide of the described O-antigen-specific to intestinal bacteria O81 type of claim 1 is characterized in that it comprises the steps:
(1) genomic extraction: 37 ℃ of incubated overnight intestinal bacteria O81 types in the LB of 5mL substratum, centrifugal collecting cell.With 500ul 50mM Tris-HCl (pH8.0) and 10ul 0.4M EDTA re-suspended cell, 37 ℃ of incubations 20 minutes, the N,O-Diacetylmuramidase that adds 10ul 10mg/ml then continues insulation 20 minutes, the Proteinase K, the 15ul 10%SDS that add 3ul 20mg/ml afterwards, 50 ℃ of incubations 2 hours, the RNase that adds 3ul 10mg/ml again, 65 ℃ of incubations 30 minutes; Add equal-volume phenol extracting mixture, get supernatant liquor and use isopyknic phenol again: chloroform: twice of primary isoamyl alcohol (25: 24: 1) mixing solutions extracting, get supernatant liquor again with isopyknic ether extracting to remove remaining phenol, supernatant liquor is with 2 times of volume ethanol deposit D NA, roll out DNA and wash DNA with glass yarn, DNA is resuspended among the 30ul TE with 70% ethanol; Genomic dna detects by 0.4% agarose gel electrophoresis;
(2) by the O-antigen gene in the pcr amplification intestinal bacteria O81 type bunch: with the genome of intestinal bacteria O81 type is that template is passed through its O-antigen gene of Long pcr amplification bunch, at first according to the JumpStart sequences Design upstream primer (5 '-ATT GTG GCTGCA GGG ATC AAA GAA AT-3 ') that often is found in O-antigen gene bunch promoter region, again according to the gnd gene design downstream primer (5 '-TAG TCG CGT GNG CCT GGA TTA AGT TCG C-3 ') in O-antigen gene bunch downstream; With the Expand Long Template PCR method of 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 the 300ngPCR purified product, 0.9ul 0.1M MnCl
2The DNaseI of the 1mg/ml of 1ul dilution in 1: 2000, reaction is carried out at room temperature, enzyme is cut the dna fragmentation size is concentrated between the 1.5kb-3kb, then add 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), 1.25ul the T4DNA polysaccharase of 100mM DTT and 5 units, 11 ℃ 30 minutes, enzyme is cut product mends into flush end, after 75 ℃ of termination reactions, add the Tth archaeal dna polymerase of 5 units and corresponding damping fluid thereof and system is expanded as 80ul, 70 ℃ of reactions 20 minutes make 3 of DNA ' end add the dA tail, and 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, and the 10 * 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 Bi0-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 Bi0-Rad company and shock by electricity, voltage is 2.5 kilovolts, time is 5.0 milliseconds to 6.0 milliseconds, the SOC substratum that adds 1ml after the electric shock immediately in cup makes the bacterium recovery, then bacterium is coated in and contains penbritin, on the LB solid medium of X-Gal and IPTG, 37 ℃ of incubated overnight, obtain blue white bacterium colony next day, with the white colony that obtains promptly the white clone forward on the LB solid medium that contains acillin and cultivate, from each clone, extract plasmid simultaneously, and cutting the segmental size of evaluation insertion wherein with the EcoRI enzyme, the white that obtains clone group has constituted the O-antigen gene bunch library of intestinal bacteria O81 type;
(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 O81 type; The quality of sequence is mainly guaranteed by two aspects: 1) genome of intestinal bacteria O81 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 O81 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 9 openings, determine also that with the function of finding the reading frame that these are open what gene they are with the genetic comparison among the blast groupware and the GenBank, finish gene annotation with the Artemis software at Britain sanger center again, do accurate comparison between DNA and protein sequence with Clustral W software, obtain the structure of the O-antigen gene bunch of intestinal bacteria O81 type at last;
(6) screening of specific gene: at wzx, the wzy gene design primer in the O-antigen gene of intestinal bacteria O81 type bunch; Respectively designed two pairs of primers in each gene, every pair of primer is distributed in the different places in the corresponding gene, to guarantee its specificity; Is that template is carried out PCR with these primers with the genomes of 166 strain intestinal bacteria and 43 strain Shigellaes, except that a band that in the 13rd group, has obtained the expection size, any product that all do not increase in other groups is so the O-antigen of wzx, wzy gene pairs intestinal bacteria O81 type all is high special.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2003101178639A CN1324134C (en) | 2003-12-22 | 2003-12-22 | O-antigen specific nucleotide of E.coli 081 type |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2003101178639A CN1324134C (en) | 2003-12-22 | 2003-12-22 | O-antigen specific nucleotide of E.coli 081 type |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1554759A true CN1554759A (en) | 2004-12-15 |
| CN1324134C CN1324134C (en) | 2007-07-04 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB2003101178639A Expired - Fee Related CN1324134C (en) | 2003-12-22 | 2003-12-22 | O-antigen specific nucleotide of E.coli 081 type |
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| Country | Link |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113950524A (en) * | 2019-03-18 | 2022-01-18 | 杨森制药公司 | Methods of producing bioconjugates of E.coli O-antigen polysaccharides, compositions thereof, and methods of use thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113950524A (en) * | 2019-03-18 | 2022-01-18 | 杨森制药公司 | Methods of producing bioconjugates of E.coli O-antigen polysaccharides, compositions thereof, and methods of use thereof |
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