CN1249234C - Nucleotide specific to O-antigen of shigella boydii I and Bacillus coli 0149 - Google Patents
Nucleotide specific to O-antigen of shigella boydii I and Bacillus coli 0149 Download PDFInfo
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Abstract
The present invention provides a specific nucleotide for the O-antigens of Shigella boydii 1 and Escherichia coli 0149. The specific nucleotide is the nucleotide complete sequence of gene clusters in Shigella boydii 1 for controlling the synthesis of O-antigens, such as a separated nucleotide with the total length of 10005 basic groups shown as SEQ ID NO: 1 or the nucleotide of SEQ ID NO: 1 which has one or a plurality of inserted, deleted or substituted basic groups and can simultaneously keep the functions of the separated nucleotide. The present invention also comprises the oligonucleotide of glycosyltransferase genes and unit processing genes stemmed from the O-antigen gene clusters of Shigella boydii 1. The present invention verifies that the specific nucleotide and the oligonucleotide have high specificity to all of the O-antigen genes Shigella boydii 1 and Escherichia coli 0149 through PCR. The present invention also discloses a method for using the oligonucleotide of the present invention to detect and identify Shigella boydii 1 and Escherichia coli 0149 in human bodies and environment.
Description
Technical field
The present invention relates to the complete nucleotide sequence of control O-antigen synthetic gene cluster in Shigella bogdii 1 type (Shigella boydii 1), particularly relate in Shigella bogdii 1 type oligonucleotide in the control O-antigen synthetic gene cluster, can utilize these oligonucleotide of O-antigen-specific Shigella bogdii 1 type and intestinal bacteria 0149 (Escherichia coliO149) in human body and the environment and identify O-antigen in these pathogenic bacterium quickly and accurately.
Background technology
Shigellae is the pathogenic bacterium that grow up along with the human evolution, can attack colon film epithelial cell, causes self limiting pyogenic infection focus, causes human bacillary dysentery.Human have higher susceptibility to Shigellae, only need be less than the infection that ten bacterium just can cause the people, children and adult easy infection, particularly children, easily cause acute poisoning dysentery, and the O-antigen of Shigellae is the one of the main reasons that Shigellae causes disease.
O-antigen is the O specific polysaccharide composition in the gram negative bacterium lipopolysaccharides, and it is made up of many multiple oligosaccharide unit.The antigenic building-up process of O-is studied clearlyer: by glycosyltransferase nucleoside diphosphate monose is transferred on the fat molecule that is fixed on the cell inner membrance earlier, then in the inboard synthesis of oligose unit of inner membrance, the antigenic oligosaccharide unit of O-is transferred to the inner membrance outside by the transhipment enzyme again, then aggregate into polysaccharide by polysaccharase, be connected to again and form lipopolysaccharide molecule [Whitfield, C. (1995) " Biosynthesis of lipopolysaccharide Oantigens " .Trends in Microbiology.3:178-185 on the glycolipid molecule; Schnaitman, C.A.andJ.D.Klena. (1993) " Genetics of lipopolysaccharide biosynthesis inentericbacteria " .Microbiological Reviews, 57 (3): 655-682].Coding is responsible for the generally adjacent arrangement on karyomit(e) of gene of all enzyme molecules of O-antigen synthetic, form a gene cluster [Reeves, P.R., et al. (1996) " Bacterial polysaccharide synthesis and genenomenclature " Trends in Microbiology, 4:495-503].In Shigellae, intestinal bacteria and Salmonellas, O-antigen gene [Lei Wang.et al (2001) " Sequence analysis of four Shigella boydii O-antigen loci:implicationfor Escherichia coli and Shigella relationships " .Infection andImmunity, 11:6923-6930 bunch between galF and gnd gene; Lei Wang and Peter Reeves (2000) " TheEscherichia coli O111 and Salmonella enterica O35 gene clusters:geneclusters encoding the same colitose-containing O antigen are highlyconserved " .Journal of Bacteriology.182:5256-5261].The O-antigen gene bunch contains three genoids: sugared synthesis path gene, glycosyltransferase gene, oligosaccharide unit treatment gene.The required nucleoside diphosphate monose of enzymic synthesis O-antigen of wherein sugared synthesis path genes encoding; Thereby the enzyme of glycosyltransferase gene coding forwards nucleoside diphosphate monose and other molecule to and makes monose aggregate into oligosaccharide unit on the monose; The oligosaccharide unit treatment gene comprises transhipment enzyme gene and pol gene, and they transfer to the bacterium inner membrance outside with oligosaccharide unit, and repolymerization becomes polysaccharide.Glycosyltransferase gene and oligosaccharide unit treatment gene only are present in the gene cluster of carrying these genes.The difference of monose in the O-antigen, between monose between the difference of link button and the oligosaccharide unit difference of link button constituted the antigenic diversity of O-, and 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 Shigellae, it has extremely strong diversity again simultaneously, and this enlightens us can study a kind of Shigellae and good, highly sensitive method of the antigenic specificity of O-thereof of detecting quickly and accurately.With surperficial polysaccharide is that the serology immune response of target has been used to somatotype and the evaluation to bacterium always since the thirties in last century, is unique means of identifying pathogenic bacterium.This diagnostic method needs a large amount of antiserum(antisera)s, and the antiserum(antisera) general classes is incomplete, quantity not sufficient, and also there are some difficulties in a large amount of antiserum(antisera)s in preparation with in storing.On the other hand this method length consuming time, sensitivity is low, loss is high, poor accuracy, so, generally believe that now this traditional serology detection method will be that the modern molecular biology method replaces.1993, Luk, J.M.C et.al has identified the O-antigen [Luk of Salmonellas with the specific nucleotide sequence of Salmonellas (S.enterica) O-antigen gene bunch by PCR method, J.M.C.et.al. (1993) " Selective amplification ofabequose and paratose synthase genes (rfb) by polymerase chain reactionfor identification of S.enterica major serogroups (A; B; C2; andD) ", J.Clin.Microbiol.31:2118-2123].Luk, the method for et.al is with corresponding to Salmonellas serotype E 1, D1 obtains the oligonucleotide special to the Salmonellas of different serotypes after the nucleotide sequence of the CDP-abequose in the A, the O-antigen of B and C2 and the synthetic gene of CDP-tyvelose is arranged.1996, Paton, the A.W et.al serotype [" Molecularmicrobiological investigation of an outbreak of Hemolytic-UremicSyndrome caused by dry fermented sausage contaminated with Shiga-liketoxin producing Escherichiacoli " .J.Clin.Microbiol.34:1622-1627] of having identified the toxogenic E.coli O111 of a strain at the oligonucleotide that comes from the wbdI gene of the O-antigen-specific of E.coli O111, but afterwards studies show that Paton, the usefulness of A.W et.al comes from the oligonucleotide of wbdI gene and identifies that the method for the serotype of E.coli O111 has false positive results to occur.Bastin D.A.and Reeves, P.R. think, this is because the wbdI gene is sugared synthesis path gene [the Bastin D.A.andReeves of a supposition, P.R. (1995) Sequence and analysis of the O antigen gene (rfb) cluster of Escherichia coli O111.Gene 164:17-23], and have this sugar in the antigenic structure of the O-of other bacterium, so sugared synthesis path gene is not a high special for O-antigen yet.
Shigellae has 46 kinds of serotypes, intestinal bacteria have 166 kinds of different O-antigens, the two sibship is very near, and it is that intestinal bacteria and Shigellae are total that 12 kinds of O-antigens are arranged, wherein Shigella bogdii 1 type just has identical O-antigen [Ewing with intestinal bacteria O149, W.H. (1986) " Edwardsand Ewing ' s identification of the Enterobacteriaceae " .ElsevierScience Publishers, Amsterdam, The Netherlands; T.cheasty, et al. (1983) " Antigenic relationships between the enteroinvasive Escherichiacoli antigens O28ac; O112ac; O124, O136, O143; O144; O152 and O164 andShigella O antigens " J.clin Microbiol, 17 (4): 681-684], traditional serotype method can not be distinguished them.
Summary of the invention
The Nucleotide that the purpose of this invention is to provide a kind of O-antigen-specific to Shigella bogdii 1 type and intestinal bacteria O149.It is the Nucleotide in the O-antigen gene bunch of Shigella bogdii 1 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 Shigella bogdii 1 type.
Another object of the present invention has provided the gene of the O-antigen gene bunch that constitutes Shigella bogdii 1 type: the gene of transhipment enzyme is the wzx gene or with wzx the gene of identity function is arranged; Pol gene is the wzy gene or with wzy the gene of identity function is arranged; Glycosyltransferase gene comprises orf8, orf9 gene; Sugar synthesis path gene comprises rmlB, rmlD, rmlA, rmlC, GumL.
Another purpose of the present invention has provided oligonucleotide, and the gene that they come from encoding glycosyl transferring enzyme in the O-antigen gene bunch of Shigella bogdii 1 type respectively comprises orf8, orf9 gene; The gene that coming from coding transhipment enzyme be the wzx gene or with wzx the gene of identity function, the gene that comes from the coding polysaccharase are arranged is the wzy gene or with wzy the gene of identity function is arranged; They are the oligonucleotide in the said gene, and length is at 10-20nt; They are special to the O-antigen of Shigella bogdii 1 type and intestinal bacteria O149; Especially the oligonucleotide of listing in the table 1, they are high specials to the O-antigen of Shigella bogdii 1 type and intestinal bacteria O149, and these oligonucleotide are also reconfigurable, and the oligonucleotide after the combination also is a high special to the O-antigen of Shigella bogdii 1 type and intestinal bacteria O149.
The above-mentioned oligonucleotide that a further object of the present invention provides can be used as primer and is used for nucleic acid amplification reaction, perhaps be used for hybridization as probe, perhaps be used to make gene chip or microarray, thereby detect and identify O-antigen and detection and evaluation Shigella bogdii 1 type and the intestinal bacteria O149 of Shigella bogdii 1 type and intestinal bacteria O149 by these methods.
An also purpose of the present invention has provided the method for the complete sequence of the O-antigen gene bunch that separates Shigella bogdii 1 type.Can obtain the complete sequence of the O-antigen gene bunch of other bacteriums according to present method operation, the complete sequence of the gene cluster of the bacterium of other polysaccharide antigens that also can obtain to encode.
The objective of the invention is to realize by following technical scheme.
The present invention is characterized in that to the Nucleotide of the O-antigen-specific of Shigella bogdii 1 type and intestinal bacteria O149 it is the isolating Nucleotide shown in SEQ ID NO:1,10005 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 Shigella bogdii 1 type and intestinal bacteria O149 is characterized in that it is by 10 genomic constitutions, and they are all between galF gene and gnd gene.
The Nucleotide of aforesaid O-antigen-specific to Shigella bogdii 1 type and intestinal bacteria O149 is characterized in that described gene comprises: transhipment enzyme gene 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 orf8, orf9 gene; Wherein, described wzx gene is the Nucleotide of 4570 to 5754 bases among the SEQ ID NO:1; The wzy gene is the Nucleotide of 6605 to 7669 bases among the SEQ ID NO:1; The orf8 gene is the Nucleotide of 7666 to 8487 bases among the SEQ ID NO:1; The orf9 gene is the Nucleotide of 8529 to 9611 bases among the SEQ ID NO:1.
The Nucleotide of aforesaid O-antigen-specific to Shigella bogdii 1 type and intestinal bacteria O149 is characterized in that it is to come from described wzx gene, wzy gene or glycosyltransferase gene orf8, orf9 gene; Or the oligonucleotide in the sugared synthesis path gene; And their mixing or their reorganization.
The Nucleotide of aforesaid O-antigen-specific to Shigella bogdii 1 type and intestinal bacteria O149, it is characterized in that the oligonucleotide of the described wzx of coming from gene is to being: the Nucleotide of 5051 to 5069 bases among the SEQ ID NO:1 and the Nucleotide of 5724 to 5742 bases; The Nucleotide of 4654 to 4672 bases among the SEQ ID NO:1 and the Nucleotide of 5276 to 5294 bases; The Nucleotide of 5164 to 5172 bases among the SEQ ID NO:1 and the Nucleotide of 5535 to 5553 bases; The oligonucleotide that comes from the wzy gene is to being: the Nucleotide of 6607 to 6625 bases among the SEQ ID NO:1 and the Nucleotide of 7545 to 7563 bases; The Nucleotide of 6667 to 6685 bases among the SEQ ID NO:1 and the Nucleotide of 7424 to 7441 bases; The Nucleotide of 6755 to 6773 bases among the SEQ ID NO:1 and the Nucleotide of 7235 to 7253 bases; The oligonucleotide that comes from the orf8 gene is to being: the Nucleotide of 7800 to 7818 bases among the SEQ ID NO:1 and the Nucleotide of 8400 to 8417 bases; The Nucleotide of 7676 to 7694 bases among the SEQ ID NO:1 and the Nucleotide of 8280 to 8298 bases; The Nucleotide of 7896 to 8014 bases among the SEQ ID NO:1 and the Nucleotide of 8293 to 8311 bases; The oligonucleotide that comes from the orf9 gene is to being: the Nucleotide of 8697 to 8715 bases among the SEQID NO:1 and the Nucleotide of 9307 to 9325 bases; The Nucleotide of 9002 to 9020 bases among the SEQ IDNO:1 and the Nucleotide of 9483 to 9502 bases; The Nucleotide of 8965 to 8983 bases among the SEQ IDNO:1 and the Nucleotide of 9425 to 9443 bases.
The Nucleotide of aforesaid O-antigen-specific to Shigellae 1 type and intestinal bacteria O149 is detecting the application of expressing the antigenic bacterium of O-, identify other polysaccharide antigen of the O-antigen of bacterium and bacterium in diagnosis.
The recombinant molecule of the Nucleotide of aforesaid O-antigen-specific to Shigella bogdii 1 type and intestinal bacteria O149, and can provide the O-antigen of expressing Shigella bogdii 1 type and intestinal bacteria O149 by inserting to express, and become bacterial vaccine.
The application of the Nucleotide of aforesaid O-antigen-specific to Shigella bogdii 1 type and intestinal bacteria O149, 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.
The separation method of the Nucleotide of aforesaid O-antigen-specific to Shigella bogdii 1 type and intestinal bacteria O149 is characterized in that it comprises the steps:
(1) genomic extraction: 37 ℃ of incubated overnight Shigellaes in the LB of 5mL substratum, centrifugal collecting cell.With 500ul 50mM Tris-HCl (pH8.0) and 10ul 0.4M EDTA re-suspended cell, 37 ℃ of incubations 20 minutes, the N,O-Diacetylmuramidase that adds 10ul 10mg/ml then continues insulation 20 minutes.The Proteinase K, the 15ul 10%SDS that add 3ul20mg/ml afterwards, 50 ℃ of incubations 2 hours add the RNase of 3ul 10mg/ml again, 65 ℃ 30 minutes.Add equal-volume phenol extracting mixture, get supernatant liquor liquid and use isopyknic phenol again: chloroform: primary isoamyl alcohol (25: 24: 1) extracting twice, get supernatant liquor liquid again with isopyknic ether extracting to remove remaining phenol.Supernatant liquor liquid rolls out DNA and washes DNA with 70% ethanol with glass yarn with 2 times of volume ethanol deposit D NA, at last DNA is resuspended among the 30ul TE.Genomic dna detects by 0.4% agarose gel electrophoresis;
(2) by the O-antigen gene in pcr amplification Shigella bogdii 1 type bunch: the O-antigen gene of Shigella bogdii 1 type is bunch by the Long pcr amplification.At first according to the JumpStart sequences Design upstream primer (#1523-ATT GTG GCT GCA GGG ATCAAA GAA AT) that often is found in O-antigen gene bunch promoter region, again according to the gnd gene design downstream primer (#1524-TAGTCG CGT GNG CCT GGA TTA AGT TCG C) in O-antigen gene bunch downstream.With the ExpandLong Template PCR method of Boehringer Mannheim company amplification O-antigen gene bunch, the PCR response procedures was as follows: 94 ℃ of pre-sex change 2 minutes; 94 ℃ of sex change are 10 seconds then, 60 ℃ of annealing 30 seconds, and 68 ℃ were extended 15 fens, and carried out 30 circulations like this.At last, continue to extend 7 fens at 68 ℃, obtain the PCR product, the agarose gel electrophoresis with 0.8% detects the size and the specificity thereof of PCR product.Merge 6 pipe long PCR products, and with the Wizard PCR Preps purification kit purified pcr product of Promega company;
(3) make up O-antigen gene bunch library: make up O-antigen gene bunch library with the Novagen DNaseI shot gun method that is modified.Reaction system is a 300ng PCR purified product, 0.9ul 0.1M MnCl
2, the DNaseI of the 1mg/ml of 1ul dilution in 1: 2000, reaction is carried out at room temperature.Enzyme is cut the dna fragmentation size is concentrated between the 1kb-3kb, then adds 2ul 0.1M EDTA termination reaction.Merge the same reaction system of 4 pipes, use isopyknic phenol and phenol respectively: chloroform: primary isoamyl alcohol (25: 24: 1) extracting once, use twice of isopyknic ether extracting again after, with the dehydrated alcohol deposit D NA of 2.5 times of volumes, and wash precipitation with 70% ethanol, be resuspended at last in the 18ul water.In this mixture, add 2.5uldNTP (1mMdCTP subsequently, 1mMdGTP, 1mMdTTP, 10mMdATP), the T4DNA polysaccharase of 1.25ul 100mM DTT and 5 units, 11 ℃ 30 minutes, enzyme is cut product mend into flush end, after 75 ℃ of termination reactions, add the Tth archaeal dna polymerase of 5 units and corresponding damping fluid thereof and system is expanded as 80ul, 70 ℃ were reacted 20 fens, made the 3 ' end of DNA add the dA tail.This mixture is through the equal-volume chloroform: after primary isoamyl alcohol (24: 1) extracting and the extracting of equal-volume ether with 3 * 10 of Promega company
-3The pGEM-T-Easy carrier connect 24 hours in 16 ℃, cumulative volume is 90ul.10 * the buffer of 9ul and the T4DNA ligase enzyme of 25 units are wherein arranged.Use the dehydrated alcohol precipitation of the 3M NaAc (pH5.2) of 1/10 volume and 2 times of volumes to be connected mixture at last, wash precipitation with 70% ethanol again, be dissolved in after the drying and obtain connecting product in the 30ul water; Preparation method with the electric transformed competence colibacillus cell of BiO-Rad company prepares competence bacillus coli DH 5 a cell, get after 2-3ul connects product and 50ul competence bacillus coli DH 5 a mixes, forward in the electric shock cup of 0.2cm of BiO-Rad company and shock by electricity, voltage is 2.5 kilovolts, time is 5.0 milliseconds to 6.0 milliseconds, the SOC substratum that adds 1ml after the electric shock immediately in cup makes the bacterium recovery, then bacterium is coated in and contains penbritin, on the LB solid medium of X-Gal and IPTG, 37 ℃ of incubated overnight, obtain blue white bacterium colony next day, with the white colony that obtains promptly the white clone forward on the LB solid medium that contains acillin and cultivate, from each clone, extract plasmid simultaneously, and cutting the segmental size of evaluation insertion wherein with the EcoRI enzyme, the white that obtains clone group has constituted the O-antigen gene bunch library of Bao Shi will Hayes 1 type;
(4) to the cloning and sequencing in the library: from the library, select insert 120 clones of fragment more than 700bp by Shanghai biotechnology company limited with ABI377 type automatic dna sequencer to unidirectional order-checking of insertion fragment in cloning, make sequence reach 80% fraction of coverage.Residue 20% sequence is again according to the sequences Design primer that has obtained, these two pairs of primers, and as follows: 5 '-GCATGGGTTACTGTACTAGC-3 ' and 3 '-AATGGCATCAATACCCGC-5 ' reaches
5 '-TGGCGGTATTGAGAGAGT-3 ' and 3 '-TTACAGGCTACTTCTCTTC-5 '.Direct PCR and from the genomic dna of Shigella bogdii 1 type again to the order-checking of PCR product, thus all sequences of O-antigen gene bunch obtained;
(7) 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 ResearchCouncil) Molecular Biology Lab and edit all sequences, thus obtain the Nucleotide full length sequence (seeing sequence list) of the O-antigen gene bunch of Shigella bogdii 1 type.The quality of sequence is mainly guaranteed by two aspects: 1) genome of Shigella bogdii 1 type is done 6 Long PCR reactions, mix these products then to produce the library.2), guarantee high-quality fraction of coverage more than 3 to each base.After obtaining the nucleotide sequence of Shigella bogdii 1 type O-antigen gene bunch, with American National biotechnology information science center (TheNational Center for Biotechnology 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 Shigella bogdii 1 type at last;
(8) screening of specific gene: at wzx, wzy, orf8, the orf9 gene design primer in the O-antigen gene of Shigella bogdii 1 type bunch; Respectively designed three pairs of primers in each gene, every pair of primer is distributed in the different places in the corresponding gene, to guarantee its specificity; Is that template is carried out PCR with these primers with the genomes of 166 strain intestinal bacteria and 43 strain Shigellaes, all primers all obtain positive findings in intestinal bacteria O149, the correct band of any size does not all increase in other groups, promptly do not obtaining any PCR product band in the array mostly, though in the minority group, obtain PDR product band, but its size does not meet the expection size, so the O-antigen of wzx, wzy, orf8, orf9, gene pairs Shigella bogdii 8 types all is high special.
First aspect just of the present invention provides the full length nucleotide sequence of the O-antigen gene bunch of Shigella bogdii 1 type, its complete sequence shown in SEQ ID NO:1,11005 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 (listing) of the O-antigen gene bunch of Shigella bogdii 1 type by method of the present invention in table 3, it is altogether by 10 genomic constitutions, all between galF gene and gnd gene.
Second aspect of the present invention provides the gene in the O-antigen gene bunch of Shigella bogdii 1 type, promptly transports enzyme gene (wzx gene or the gene of identity function arranged with wzx); Pol gene (wzy gene or the gene of identity function arranged with wzy); Glycosyltransferase gene comprises orf8, orf9 gene; Special sugared synthesis path gene in the bacterial polysaccharides antigen comprises rmlB, rmlD, rmlA, rmlC gene, and their zero positions in O-antigen gene bunch and final position and nucleotide sequence all are listed in the table 4.The invention particularly relates to glycosyltransferase gene, transhipment enzyme gene and pol gene, because sugared synthesis path gene is that the gene of synthetic nucleosides bisphosphate monose is common, common by indication to more exocellular polysaccharide now, O-antigen to bacterium is not very special, and the glycosyltransferase gene that the present invention relates to, transhipment enzyme gene and pol gene are high specials to the O-antigen of Shigella bogdii 1 type and intestinal bacteria O149.
The 3rd aspect of the present invention, wzx gene in the O-antigen gene bunch that comes from Shigella bogdii 1 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 with wzy with wzx, the oligonucleotide that comprises orf8, orf9 gene, they are any one section oligonucleotide in these genes.But, be that the oligonucleotide of listing in the table 1 is right preferentially by usefulness, in table 1, also listed these oligonucleotide to the position in O-antigen gene bunch and with these oligonucleotide to being the size of the product of the PCR reaction done of primer, the annealing temperature in these PCR reaction free lists is carried out.These primers are being to obtain expecting the product of size in the pcr amplification that carries out of template with Shigella bogdii 1 type and intestinal bacteria O149 only, and are all not obtain expecting the product of size in the pcr amplification that carries out of template the 166 strain intestinal bacteria listed with table 2 and 43 strain Shigellaes.In more detail, with these oligonucleotide to being that PCR that primer is done is reflected at and does not all obtain spawn in most of bacteriums, though in some bacterium, obtained PCR product band, but its size does not meet the expection size, this is that this problem can be avoided by being PCR with intragenic other primer because primer is attached to genomic other position and causes.So, can determine these primers promptly the listed oligonucleotide of table 1 be high special to Shigella bogdii 1 type and intestinal bacteria O149 and their O-antigen.
The separation method of the Nucleotide of the O-antigen-specific to Shigella bogdii 1 type and intestinal bacteria O149 provided by the invention comprises the steps: (1) genomic extraction; (2) the O-antigen gene in pcr amplification Shigella bogdii 1 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 " is meant the gene that derives from the encoding glycosyl transferring enzyme in the O-antigen gene bunch, the gene of coding transhipment enzyme and intragenic one section nucleic acid molecule of coding polysaccharase, they can change on length, generally change in 10 to 20 Nucleotide scopes.More precisely these oligonucleotide are to come from wzx gene (nucleotide position is 4570 to 5754 bases from SEQ ID NO:1), wzy gene (nucleotide position is 6605 to 7669 bases from SEQ ID NO:1), orf8 gene (nucleotide position is 7666 to 8487 bases from SEQ ID NO:1), orf9 gene (nucleotide position is 8529 to 9611 bases from SEQ ID NO:1), coming from above intragenic oligonucleotide all is high special to Shigella bogdii 1 type (SEQ ID NO:1) and intestinal bacteria O149.
In addition, the antigenic gene cluster of the different O-of the coding of two genetic resemblances produces new O-antigen by gene recombination or sudden change sometimes, thereby produces new bacteria types, new mutant strain.In this environment, need filter out many specificitys that oligonucleotide is detected with raising with recombination hybridization.Therefore, the invention provides a whole set of many mixtures to oligonucleotide, they come from glycosyltransferase gene; Come from transhipment enzyme and pol gene, comprise the wzx gene or the gene, wzy gene of identity function arranged or the gene of identity function is arranged with wzy with wzx; Also come from sugared synthesis path gene.The mixture of these genes is special to a special bacterial polysaccharides antigen, is special thereby make this cover oligonucleotide to the polysaccharide antigen of this bacterium.More particularly, the mixture of these oligonucleotide is to come from glycosyltransferase gene, wzx gene or the gene, wzy of identity function arranged or with wzy oligonucleotide and the combination that comes from the oligonucleotide in the sugared synthesis path gene in the gene of identity function are arranged with wzx.
On the other hand, the present invention relates to the evaluation of oligonucleotide, they can be used for detecting the O-antigen of expressing the antigenic bacterium of O-and identifying bacterium in diagnosis.
The present invention relates to a kind of antigenic method of one or more bacterial polysaccharideses that detects in the food, these antigens can make sample can with the oligonucleotide specific hybrid of following at least one gene, these genes are: (i) gene of the encoding glycosyl transferring enzyme gene of transhipment enzyme and polysaccharase of (ii) encoding comprises the wzx gene or the gene, wzy gene of identity function is arranged or with wzy the gene of identity function is arranged with wzx.At least one oligonucleotide can be hybridized with at least one more than one such gene specific of expressing the special antigenic bacterium of O-under the situation of condition permission, and these bacteriums are Shigella bogdii 1 type or intestinal bacteria O149.Available PCR method detects, more can with behind the Nucleotide mark in the inventive method as probe by hybridization such as southern-blot or fluoroscopic examination, perhaps by antigen and bacterium in gene chip or the microarray assay sample.
The present inventor considers following situation: when one special oligonucleotide detects when invalid, the mixture of oligonucleotide can with the target region specific hybrid with test sample.Therefore the invention provides a cover oligonucleotide and be used for detection method of the present invention.Here said oligonucleotide is meant the gene that comes from the encoding glycosyl transferring enzyme, the gene of coding transhipment enzyme and the gene of polysaccharase, comprises the wzx gene or the gene, wzy gene of identity function is arranged or with wzy the oligonucleotide of the gene of identity function is arranged with wzx.This cover oligonucleotide is special to the O-antigen of a special bacterium, and this special bacterium O-antigen is expressed by Shigella bogdii 1 type or intestinal bacteria O149.
On the other hand, the present invention relates to a kind of antigenic method of one or more bacterial polysaccharideses that detects in the movement, these antigens can make sample can with the oligonucleotide specific hybrid of following at least one gene, these genes are: (i) gene of the encoding glycosyl transferring enzyme gene of transhipment enzyme and polysaccharase of (ii) encoding comprises the wzx gene or the gene, wzy gene of identity function is arranged or with wzy the gene of identity function is arranged with wzx.At least one oligonucleotide can be expressed more than one such gene specific hybridization of the special antigenic bacterium of O-with at least one under the situation of condition permission.These bacteriums are Shigella bogdii 1 type or intestinal bacteria O149.Oligonucleotide among available the present invention is made the method test sample of primer by PCR, also can with behind the oligonucleotide molecules mark among the present invention as probe by hybridization such as southern-blot or fluoroscopic examination, perhaps by antigen and bacterium in gene chip or the microarray assay sample.
Generally, a pair of oligonucleotide may with same gene recombination also can with different gene recombinations, but must have in them an oligonucleotide can specific hybrid to the distinguished sequence of special antigenic type, another oligonucleotide can be hybridized in non-specific zone.Therefore, when the oligonucleotide in the special polysaccharide antigen gene cluster is reconfigured, can select specific gene mixture hybridization in a pair of oligonucleotide and the polysaccharide antigen gene cluster at least, perhaps select many mixture hybridization oligonucleotide and specific gene.Even even when all genes were all unique in the specific genes bunch, this method also can be applied to discern the nucleic acid molecule of the gene mixture in this gene cluster.Therefore the invention provides a whole set of is used to detect the many to oligonucleotide of the inventive method, many here is that the gene of the gene that comes from the encoding glycosyl transferring enzyme, coding transhipment enzyme and polysaccharase comprises the wzx gene or the gene, wzy gene of identity function arranged or with wzy the gene of identity function is arranged with wzx to oligonucleotide, this cover oligonucleotide is special to a special bacterial polysaccharides, and this cover oligonucleotide may be the Nucleotide of necessary gene during sugar synthesizes.
On the other hand, the present invention also relates to the antigenic method of one or more bacterial polysaccharideses in the sample that a kind of detection comes from patient.One or more bacterial polysaccharides antigens in the sample can make sample can with a specific hybrid in a pair of oligonucleotide in following at least one gene, these genes are: (i) gene of the encoding glycosyl transferring enzyme gene of transhipment enzyme and polysaccharase of (ii) encoding comprises the wzx gene or the gene, wzy gene of identity function is arranged or with wzy the gene of identity function is arranged with wzx.Under the situation of condition permission at least one oligonucleotide can with sample at least one express more than one such gene specific hybridization of the special antigenic bacterium of O-, these bacteriums are Shigella bogdii 1 type or intestinal bacteria O149.Oligonucleotide among available the present invention is made the method test sample of primer by PCR, also can will pass through hybridization as probe behind the oligonucleotide mark among the present invention, or by antigen and bacterium in gene chip or the microarray assay sample.
In more detail, method described above can be understood as when oligonucleotide when being used, it is not to derive from glycosyltransferase gene, wzx gene or with wzx the gene, wzy gene of identity function arranged or have on the sequence of gene of identity function with wzy that one of them oligonucleotide molecules can hybridize to one.In addition, when two oligonucleotide can both be hybridized, they may be hybridized in same gene and also may hybridize on the different genes.Also promptly, when cross reaction goes wrong, can select the mixture of oligonucleotide to detect the blended gene so that the specificity of detection to be provided.
The present inventor believes that the present invention is not necessarily limited to the above nucleotide sequence coded specific O-antigen of carrying, and is widely used in detecting all expression O-antigens and identifies the antigenic bacterium of O-.And because O-antigen is synthetic and the similarity of other polysaccharide antigens (as bacterium born of the same parents exoantigen) between synthesizing, the inventor believes that method of the present invention and molecule also are applied to these other polysaccharide antigen.
The present invention discloses the full length sequence of the O-antigen gene bunch of Shigella bogdii 1 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 Shigella bogdii 1 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 Shigellaes in the LB of 5mL substratum, centrifugal collecting cell.With 500ul50mM Tris-HCl (pH8.0) and 10ul 0.4M EDTA re-suspended cell, 37 ℃ of incubations 20 minutes, the N,O-Diacetylmuramidase that adds 10ul 10mg/ml then continues insulation 20 minutes.The Proteinase K, the 15ul 10%SDS that add 3ul 20mg/ml afterwards, 50 ℃ of incubations 2 hours add the RNase of 3ul 10mg/ml again, 65 ℃ 30 minutes.Add equal-volume phenol extracting mixture, get supernatant liquor liquid and use isopyknic phenol again: chloroform: primary isoamyl alcohol (25: 24: 1) extracting twice, get supernatant liquor liquid again with isopyknic ether extracting to remove remaining phenol.Supernatant liquor liquid rolls out DNA and washes DNA with 70% ethanol with glass yarn with 2 times of volume ethanol deposit D NA, at last DNA is resuspended among the 30ul TE.Genomic dna detects by 0.4% agarose gel electrophoresis.
Embodiment 2: by the O-antigen gene in pcr amplification Shigella bogdii 1 type bunch
The O-antigen gene of Shigella bogdii 1 type bunch is by the Long pcr amplification.At first according to the JumpStart sequences Design upstream primer (#1523-ATTGTG GCT GCA GGG ATC AAA GAA AT) that often is found in O-antigen gene bunch promoter region, again according to the gnd gene design downstream primer (#1524-TAG TCG CGT GNG CCT GGA TTA AGT TCG C) in O-antigen gene bunch downstream.With the Expand Long Template PCR method of BoehringerMannheim company amplification O-antigen gene bunch, the PCR response procedures was as follows: 94 ℃ of pre-sex change 2 minutes; 94 ℃ of sex change are 10 seconds then, 60 ℃ of annealing 30 seconds, and 68 ℃ were extended 15 fens, and carried out 30 circulations like this.At last, continue to extend 7 fens at 68 ℃, obtain the PCR product, the agarose gel electrophoresis with 0.8% detects the size and the specificity thereof of PCR product.Merge 6 pipe long PCR products, and with the Wizard PCR Preps purification kit purified pcr product of Promega company.
The structure in embodiment 3:O-antigen gene bunch library
At first formula connects the acquisition of product: make up O-antigen gene bunch library with the Novagen DNaseI shot gun method that is modified.Reaction system is a 300ng PCR purified product, 0.9ul 0.1M MnCl
2, the DNaseI of the 1mg/ml of lul dilution in 1: 2000, reaction is carried out at room temperature.Enzyme is cut the dna fragmentation size is concentrated between the 1kb-3kb, then adds 2ul 0.1M EDTA termination reaction.Merge the same reaction system of 4 pipes, use isopyknic phenol and phenol respectively: chloroform: primary isoamyl alcohol (25: 24: 1) extracting once, use twice of isopyknic ether extracting again after, with the dehydrated alcohol deposit D NA of 2.5 times of volumes, and wash precipitation with 70% ethanol, be resuspended at last in the 18ul water.In this mixture, add 2.5uldNTP (1mMdCTP subsequently, 1mMdGTP, 1mMdTTP, 10mMdATP), the T4DNA polysaccharase of 1.25ul 100mM DTT and 5 units, 11 ℃ 30 minutes, enzyme is cut product mend into flush end, after 75 ℃ of termination reactions, add the Tth archaeal dna polymerase of 5 units and corresponding damping fluid thereof and system is expanded as 80ul, 70 ℃ were reacted 20 fens, made the 3 ' end of DNA add the dA tail.This mixture is through the equal-volume chloroform: after primary isoamyl alcohol (24: 1) extracting and the extracting of equal-volume ether with 3 * 10 of Promega company
-3The pGEM-T-Easy carrier connect 24 hours in 16 ℃, cumulative volume is 90ul.10 * the buffer of 9ul and the T4DNA ligase enzyme of 25 units are wherein arranged.Use the dehydrated alcohol precipitation of the 3M NaAc (pH5.2) of 1/10 volume and 2 times of volumes to be connected mixture at last, wash precipitation with 70% ethanol again, be dissolved in after the drying and obtain connecting product in the 30ul water.
Next is the preparation of competent cell: the method that provides with reference to Bio-Rad company prepares the competent cell bacillus coli DH 5 alpha.Get a ring bacillus coli DH 5 alpha list bacterium colony in the LB of 5ml substratum, 180rpm cultivated after 10 hours, got in the LB substratum that the 2ml culture is transferred to 200ml, and 37 ℃ of 250rpm thermal agitations are cultivated OD600 about 0.5, ice bath cooling was 20 minutes then, in centrifugal 15 minutes of 4 ℃ of 4000rpm.Confide all supernatant liquor liquid, 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 liquid, precipitate 10% glycerine suspension cell with the precooling of 1ml ice at last, are competent cell.The competent cell that makes is packed as 50ul one pipe ,-70 ℃ of preservations.
Be electric transformed competence colibacillus cell at last: get after 2-3ul connects product and 50ul competence bacillus coli DH 5 alpha mixes, forward in the electric shock cup of 0.2cm of Bio-Rad company and shock by electricity, voltage is 2.5 kilovolts, and the time is 5.0 milliseconds-6.0ms millisecond.The SOC substratum that adds 1ml after the electric shock immediately in cup makes the bacterium recovery.Immediately bacterium is coated in 37 ℃ of inversion incubated overnight on the LB solid medium that contains penbritin, X-Gal and IPTG then, obtains blue white bacterium colony next day.With the white colony that obtains promptly the white clone forward on the LB solid medium that contains penbritin and cultivate, from each clone, extract plasmid and cut the segmental size of evaluation insertion wherein simultaneously, obtain the O-antigen gene bunch library that white clone group has constituted Shigella bogdii 1 type with the EcoRI enzyme.
Embodiment 4: to the cloning and sequencing in the library
From the library, select insert 120 clones of fragment more than 700bp by Shanghai biotechnology company limited with ABI377 type automatic dna sequencer to unidirectional order-checking of insertion fragment in cloning, make sequence reach 80% fraction of coverage.Residue 20% sequence is again according to the sequences Design primer that has obtained, direct PCR and from the genomic dna of Shigella bogdii 1 type again to the order-checking of PCR product, thus obtain all sequences of O-antigen gene bunch.We have designed two pairs of primers in Shigella bogdii 1 type, and are as follows: 5 '-GCATGGGTTACTGTACTAGC-3 ' and 3 '-AATGGCATCAATACCCGC-5 ' 5 '-TGGCGGTATTGAGAGAGT-3 ' and 3 '-TTACAGGCTACTTCTCTTC-5 '
Embodiment 5: the splicing of nucleotide sequence and analysis
The Pregap4 and the splicing of Gap4 software of the Staden package software package of publishing with Britain Camb MRC (Medical Research Council) Molecular Biology Lab and edit all sequences, thus the Nucleotide full length sequence (seeing sequence list) of the O-antigen gene bunch of Shigella bogdii 1 type obtained.The quality of sequence is mainly guaranteed by two aspects: 1) genome of Shigella bogdii 1 type is done 6 Long PCR reactions, mix these products then to produce the library.2), guarantee high-quality fraction of coverage more than 3 to each base.After obtaining the nucleotide sequence of Shigella bogdii 1 type O-antigen gene bunch, with American National biotechnology information science center (The National Center for BiotechnologyInformation, NCBI) orffinder finds gene, find the reading frame of 9 openings, determine also that with the function of finding the reading frame that these are open what gene they are with the genetic comparison among the blast groupware and the GenBank, finish gene annotation with the Artemis software at Britain sanger center again, do accurate comparison between DNA and protein sequence with Clustral W software, obtain the structure of the O-antigen gene bunch of Shigella bogdii 1 type at last, as shown in table 3.
By retrieving and relatively, finding that orf 1 and the rmlB gene of Escherichia coli K12 all have 95% homogeny in 297 amino acid whose sequences, show the homology that height is arranged between them.So can determine orf 1 is the rmlB gene.Orf 2 has 97% homogeny with the rmlD gene of Shigella boydii in 299 amino acid whose sequences, show the homology that height is arranged between them.So, can determine that orf 2 is rmlD genes.Orf 3 has 98% homogeny with the rmlA gene of Escherichia coli K12 in 293 amino acid whose sequences, show the homology that height is arranged between them.So, can determine that orf 3 is rmlA genes.Orf 4 has 90% homogeny with the rmlC gene of Shigella boydii in 171 amino acid whose sequences, show the homology that height is also arranged between them.So, can determine that orf 4 is rmlC genes.In addition, in Shigellae and intestinal bacteria, rmlB, rmlD, rmlA, rmlC are four genes that synthesize rhamnosyl specially, in the antigenic structure of the O-of Shigella bogdii 1 type rhamnosyl are arranged also.Orf 5 has 24% homogeny with the wzx gene of salmonella enterica in 361 amino acid whose sequences, 48% similarity, and algorithm [Eisenberg by people such as Eisenberg, D, Schwarz, E.etal (1984) .Analysis of membrane and surfaceprotein sequences with the hydrophobic moment plot.J.Mol.Biol.179:125-142] find that orf6 has 12 potential transmembrane domains, it and many wzx protein similars, and about 50 amino acid whose conservative motifs are arranged at the proteic aminoterminal of wzx, so can determine orf5 is the wzx gene, called after wzx.The gumL gene of orf6 and Xanthomonas oryzae has 37% homogeny in 199 amino acid, can determine that orf6 is the gumL gene, called after gumL.The wzy gene of orf7 and S.boydii has 24% homogeny in 343 aminoacid sequences, 44% similarity, learn that by the Eisenberg algorithm orf7 has 10 potential transmembrane domains in addition, to other O-antigen polysaccharase similar secondary structure is arranged, so determine that orf7 is exactly the wzy gene, called after orf7.Orf8 and colibacillary glycosyltransferase gene have 29% homogeny in 227 aminoacid sequences, 49% similarity is so determine that orf8 is a glycosyltransferase gene.The glycosyltransferase gene of orf9 and S.boydii has 51% homogeny in 348 aminoacid sequences, 68% similarity is so determine that orf9 is a glycosyltransferase gene, called after orf9.
Embodiment 6: the screening of specific gene.At wzx, wzy, orf8, orf9 gene design primer in the O-antigen gene of Shigella bogdii 1 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 Shigella bogdii 1 type.Glycosyltransferase gene, transhipment enzyme gene and pol gene and their function corresponding and the size of the O antigen gene bunch of Shigella bogdii 1 type in table, have been listed.In each gene, we have respectively designed three pairs of primers, and the difference that every pair of primer is distributed in the corresponding gene is local to guarantee its specificity.In table, also listed position and the size of each primer in SEQ IDNO:1.Is that template carry out PCR with listed corresponding annealing temperature in the table with the genomes of all bacterium in the table 2 with every pair of primer, has obtained corresponding PCR product, and its size is also listed in the table.
Mdh (malate dehydrogenase) gene is to be present in all colibacillary genomes and a gene of high conservative, so we according to the mdh gene design primer #101 (TTC ATC CTAAAC TCC TTA TT) and #102 (TAA TCG CAG GGG AAA GCA GG), extract genome then from 166 strain intestinal bacteria, method as previously mentioned.With this to primer from the colibacillary genome of 166 strains PCR with identification of escherichia coli and detect its genomic quality.
Table 2 is 166 strain intestinal bacteria and 43 strain Shigellaes and their sources that are used to screen specific gene, and for the convenience that detects, we are divided into one group with their every 8-10 bacterium, and 27 groups altogether, all list in the table in their source.
The genomic dna that contains Shigella bogdii 1 type in the 25th group is as positive control.Do template with every group of bacterium, be PCR by following condition with every pair in the table 1 primer: 94 ℃ of pre-sex change after 2 minutes, 94 ℃ of sex change 15 seconds, annealing temperature is because of the difference different (with reference to table 1) of primer, annealing time is 50 seconds, and 72 ℃ were extended 2 fens, and carried out 30 circulations like this.Continue to extend 10 fens at 72 ℃ at last, reaction system is 25ul.After reaction finishes, get 10ul PCR product and detect the fragment that amplifies by 0.8% agarose gel electrophoresis.
For wzx, wzy, orf8, orf9 gene, each gene all has three pairs of primers detected, and every pair of primer has obtained also all obtaining onesize specificity band the correct band of expection size in the 18th group except be PCR in the 25th group after.After being template PCR with the genomic dna of each bacterium in the 18th group, find only in intestinal bacteria O149, to have obtained positive findings.In more detail, each of each gene all obtains expecting the correct PCR product band of size to primer more than in intestinal bacteria O149.It is reported that the O-antigenic structure of Shigella bogdii 1 type and intestinal bacteria O149 is the same, our result has confirmed this point from another side.In addition, all primers are the correct band of any size that all do not increase in other groups, that is to say, do not obtaining any PCR product band in the array mostly, though in the minority group, obtain PCR product band, but its size does not meet the expection size, so wzx, wzy, orf8, orf9 gene pairs Shigella bogdii 1 type and intestinal bacteria O149 and O-antigen thereof all are high specials.
At last, from Shigella bogdii 1 type, screen gene by PCR: wzx, wzy and four glycosyltransferase genes to the O-antigen high special of Shigella bogdii 1 type and intestinal bacteria O149.And the oligonucleotide of these intragenic any one section 10-20nt is special to the O-antigen of Shigella bogdii 1 type and intestinal bacteria O149, and the primer in especially above-mentioned each gene is that oligonucleotide is high special to detecting the back confirmation through PCR to Shigella bogdii 1 type and intestinal bacteria O149.These all oligonucleotide all can be used for Shigella bogdii 1 type and the intestinal bacteria O149 in the human body and environment rapidly and accurately, and can identify their O-antigen.
Table 3 is structural tables of the O-antigen gene bunch of Shigella bogdii 1 type, in table, listed the structure of the O-antigen gene bunch of Shigella bogdii 1 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 these two not responsible O-of gene are antigenic synthetic, and we are just with the increase full length sequence of O-antigen gene bunch of their one section sequences Design primer.
Table 4 is location tables of the gene in the O-antigen gene bunch of Shigella bogdii 1 type, in table, listed the accurate position of all open reading frame in complete sequence in the O-antigen gene bunch of Shigella bogdii 1 type, at the underscoring of the initiator codon and the terminator codon of each open reading frame.
Sequence list
SEQUENCE LISTING
＜110〉Nankai University
＜120〉to the Nucleotide of the O-antigen-specific of Shigella bogdii 1 type and intestinal bacteria O149
＜130〉to the Nucleotide of the O-antigen-specific of Shigella bogdii 1 type and intestinal bacteria O149
<160>1
<170>PatentIn version 3.1
<210>1
<211>11005
<212>DNA
<213>Shigella boydii
<400>1
ctcctggtaa ctcacgcgtc caaaaacgcg gtcgaaaacc acttcgacac ctcttatgaa 60
ttagaatctc tccttgaact gcgcgtgaag cgtcagctgc tggcggaagt acagtccatc 120
tgtccgccgg gcgtgaccat tatgaacgtg cgtcagggcg aacctttagg tttaggccac 180
tccattttgt gtgcacgacc cgccattggt gacaacccat ttgttgtggt attgccggac 240
gttgtgatcg acgacgccag tgccgacccg ctgcgctaca accttgctgc catgattgcg 300
cgcttcaatg aaacgggccg cagccaggtg ctggcaaaac gtatgccggg tgacctctct 360
gaatactccg tcattcagac caaagaacca ctggatcgtg aaggtaaagt cagccgcatc 420
gtcgaattta tcgaaaaacc ggatcagccg cagacgctgg actcagacat catggccgtt 480
ggtcgctatg tgctttctgc cgatatttgg ccggaactgg aacgtactca gcctggtgca 540
tggggacgta ttcagctgac tgatgctatt gccgagctgg cgaaaaaaca gtccgttgat 600
gccatgctga tgactgggga cagttacgac tgcggtaaaa aaatgggtta tatgcaggca 660
tttgtgaagt atggactacg caacctcaaa gaaggggcga agttccgtaa agggattgag 720
aagctgttaa gcgaataatg aaaatctgac cggatgtaac ggttgataag aaaattataa 780
cggcagtgaa gattcgtggc gaaagtaatt tgttgcgaat attcctgccg ttgttttata 840
taaacaatca gaataacaac gagttagcaa taggatttta gtcaaagttt tccaggattt 900
tccttgtttc cagagcggat tggtaagaca attagcgttt gaatttttcg ggtttagcgc 960
gagtgggtaa cgctcgtcac atcgtagaca tgcatgtagt gctctggtag ctgtaaagcc 1020
aggggcggta gcgtgcatta acacctctat taatcaacct aagagccgct tatttcaccg 1080
catgctctga agttatatgg aataaataag tgaagatact tgttactggt ggcgcaggat 1140
ttattggttc tgctgtagtt cgtcacatta taaataatac gcaggatagt gttgttaatg 1200
tcgataaatt aacgtacgcc ggaaacctgg aatcacttgc tgatgtttct gattctgaac 1260
gctatgtttt tgaacatgcg gatatttgcg atgctgctgc aatggcgcga attttttctc 1320
agcatcagcc ggatgcagtg atgcacctgg ctgctgaaag ccatgttgac cgttcaatta 1380
caggccctgc ggcatttatt gaaaccaata ttgttggtac ttatgtcctt ttggacgctg 1440
ctcgcaatta ctggtctgct cttgatagcg acaagaaaaa tagcttccgt tttcatcata 1500
tttctactga cgaagtctat ggtgatttgc ctcatcctga cgaagtaaat aataaagaaa 1560
aattaccctt atttattgag acaacagctt acgcaccaag cagcccttat tccgcatcca 1620
aagcatccag cgatcattta gtccgcgcgt ggaaacgtac ctatggttta ccgaccattg 1680
tgactaattg ctctaacaat tatggccctt atcatttccc ggaaaaattg attccattgg 1740
ttattctgaa tgctctggaa ggtaagatgt tgcctattta tggcaaaggg gatcaaattc 1800
gtgattggct gtatgtagag gatcatgctc gagcgttata tacagttgtt actgaaggta 1860
aagcaggtga aacctataat attggtggac acaacgaaaa gaaaaacatc gatgtagtgc 1920
tcactatttg tgatttgttg gatgagattg tcccgaaaga gaaatcttac cgcgagcaaa 1980
ttacttatgt tgccgatcgt ccgggacacg atcgccgtta tgcgattaat gctgagaaga 2040
ttggtcgcga attgggctgg aaaccacagg aaacgtttga gagcgggatt cgtaaaacag 2100
tggaatggta ccttgctaat gcaaaatggg tcgaaaatat gaaaagtggt gcctatcaat 2160
cgtggattga acagaactat gagggccgcc agtaatgaat attctccttt tcggcaaaac 2220
agggcaggta ggttgggaac tacagcgtgc tctggcacct ctgggtaatt tgattgctct 2280
tgatgttcac tccactgatt attgtggtga ttttagtaat cctgaaggtg tagctgaaac 2340
cgtaagaagc attcggcctg atattattgt caacgcagcc gctcacaccg cagtagacaa 2400
agcagaatca gaaccggagt ttgcacaatt acttaacgcg acaagtgtcg aagcgattgc 2460
gaaagcagcc aatgaagtcg gcgcctgggt tattcactac tctactgact acgtatttcc 2520
ggggaccggt gaaataccat ggcaggagac ggatgcaacc gcaccgctaa atgtgtacgg 2580
tgaaaccaag ttagctggag aaaaagcatt acaagagcat tgtgcgaagc accttatttt 2640
ccgtaccagc tgggtatatg caggtaaagg aaacaatttt gccaagacaa tgttacgtct 2700
ggcaaaagag cgcgaagaat tagctgtcat taacgatcag tttggtgcac caacaggtgc 2760
tgagttgctg gctgattgta cggcacatgc cattcgtgtt gcactgaata aaccagaagt 2820
cgcaggctta taccatctgg tagccagtgg taccacaacc tggtacgatt atgctgcgct 2880
ggtttttgaa gaggcgcgcg aagcaggcat tccccttgca ctcaacaagc tcaacgcagt 2940
accaacaaca gcctatccta cactagctcg tcgtccgcat aactctcgcc ttgatacaga 3000
aaaatttcag cagaactttg cgcttgtctt gcctgactgg cagattggcg tgaaacgcat 3060
gctcaacgaa ttatttacga ctacagctat ttaatagttt ttgcatcttg tttgtgatgg 3120
tggagcaaga tgaattaaaa ggaatgatga aatgaaaacg cgtaaaggta ttattttagc 3180
gggtggttct ggtactcgtc tttatcctgt gactatggca gtcagtaaac agctgctacc 3240
gatttatgat aaaccgatga tctattatcc gctctctaca ctgatgttgg cgggtattcg 3300
cgatattttg attatcagta cgccacagga tactcctcgt tttcaacaac tgttgggtga 3360
cggtagccag tgggggctga atcttcagta caaagtgcaa gcgagtccgg atggtcttgc 3420
gcaggcgttt attatcggtg aagagtttat tggtggtgat gattgtgctt tgattcttgg 3480
tgataatatc ttttacggtc acgatctgcc gaagttaatg gatgccgctg ttaacaaaga 3540
aagtggtgca acggtatttg catatcacgt aaatgatcct gaacgctatg gtgtcgtgga 3600
gtttgataat aacggaacag ccattagcct tgaagaaaaa ccgttagagc caaaaagcaa 3660
ttatgctgta accggacttt atttctatga caatgatgtt gtagaaatgg cgaaaaacct 3720
taagccttct gcccgtggcg aactggaaat taccgatatt aaccgtattt atatggagca 3780
gggacgttta tctgttgcca tgatgggacg tggatatgca tggttggaca cggggacaca 3840
tcaaagtctt attgaggcaa gcaattttat cgcaacaata gaagaacgtc aggggctgaa 3900
agtttcctgc ccggaagaaa ttgcttaccg taaagggttt atcgatgctg agcaggtgaa 3960
agtattagct gaaccgttga agaaaaatgc ttatggtcag tatctgctga aaatgattaa 4020
aggttattaa taaaatgaac gtaattaaaa ctgaaattcc tgatgtgtta attttcgagc 4080
cgaaagtttt tggtgatgag cgtggtttct ttatggaaag ctttaatcag aaagttttcg 4140
aagaggctgt agggcgtaag gttgaatttg ttcaggataa tcattcgaag tctagtaaag 4200
gcgttttacg tgggctgcac tatcagttag aaccttatgc gcaaggaaaa ctggtacgct 4260
gcgttgttgg tgaagttttt gacgtagcgg ttgatattcg taaatcgtca ccaacttttg 4320
gcaaatgggt tggggtgaat ttatctgctg agaataagcg ccagttgtgg atccctgaag 4380
gatttgcaca tggttttttg gtgctgagtg aaacggcgga gtttttgtat aagacgacta 4440
attattatca tccgataagt gaaagagggg ttaaatggga tgatccctgt attgatatta 4500
agtggcctct agtatcgtct cccctgttat cagaaaaaga taagtgttat tcatggctaa 4560
cgaaacaaaa tgaacagtaa taagagacaa actttattct tttcgcttga tcaaataata 4620
gattctggac ttgtttttat ttttattgct attggcggta atttacttga caaatcagat 4680
atggctaatg ttgtgtcatt ccaatctgtt gcattagtat ctgtattgtt ttgttcctgt 4740
tttacaacgc aatatttact gttgcgctat aaaaaacaat cttcactttt ttggctaaag 4800
ttatttttat ttttttgtat tattactatg ttgattgtat cattatggtt tcaatcggca 4860
gaaatattat tcttgttttt tgttggaata tcttcagaat acataaaacg atactgcttt 4920
tatattaata attcttgtct ctctttcttt tcaacgataa taacggcatt tatttttttt 4980
gtttttatat tgatgtgttg gctgaatttc atgcgcatta attcgactat atatatatat 5040
atatatagcg gtgcaaagtt aattccgcta acgataatat gttgttttct ttttattaaa 5100
acaaagttca atagtgatgg tgaatatgaa atgccatctt tatggtatgc aattactgat 5160
tcattaagat ttggaggcgt gttttctgtt attaccatta tttattggat tacaaatcaa 5220
ggtttcttta tattctttca tgacagaatg cctgcagcag agttggtaga aattagagtt 5280
acgcagaatg tgtttggtgt agttacaatg ctaattgcat tgtacgactc tatgttttta 5340
aagaaaaaca tagagaataa aaataaaata tttgattggc gatcttattt taaattcctt 5400
gtaatttcaa ttgttttgat tacatttaat tttattttat tatatatatt atcaataaca 5460
atatataaac atttggatgt ttttaaatat tcgatatgtc ttgcactggc ccaatttttt 5520
tatctttccg caaggatgcc tgtacttatt ttgaaattgc gctatagttt aagtattatt 5580
ctgtggttat acgtgtgttc gctttttttg tcctcgtgtt acttattctt gaaacataat 5640
gattatgatt ttcaatatat agtgcaatct ataacattat ctaacttctt tatttttcta 5700
ttttcattat taatagtgtt ttacaaagag aggatttatg ggaagaatta ttgaagctct 5760
gtatgggata tcaacggaac tgaaagagcg aatcttgaat atgggagaga aaacagttcc 5820
ggtgaattac tatacaagac ataataactg gggtgatcaa ttaaataaat atttaattga 5880
aaaaattacc aataaaaagg tagtaaagaa caacttcaaa aagctacctc atatattagc 5940
tataggaagc gttctttcaa gtgcatcaaa taaaagtatc gtatggggga gtggttttat 6000
atctaaagat gcgccattac gagaaacagc ccctgatatt agggctgttc gaggtgtttt 6060
gactagaaat aaacttcaga aagatttttc aattgactgc cctgatgtgt tgggagaccc 6120
ggccgttctt ttgcctttat tttataaagc aaaacataga agtaaaaaat acaaagtcgg 6180
tattattcca cactataaag ataagcaacg agctgttgta caggattttt tacataaagg 6240
atgtgttctt gttgatattc aagatgacat tgaagttttt attgataaaa ttaacgaatg 6300
tgaggttgta atttcatcgt cacttcatgg attaattgct gcagatactt atggtatacc 6360
taatttatgg gtttcatttg gtgaccaagt attaggtggt gattttaagt ttttggacta 6420
ttatagtact actaataata taaatcctaa aatgatacga ctagataatg atcataaata 6480
ttctattgat gatttggtta gaatttctag atgtaataaa tttattagaa gttctgaaga 6540
gttacttgaa gcgtttccta gggagtacat atgaattact attatataat tgtgtaggtg 6600
aaaaatgcta gttacaatcc tggctttatt tctgtttata caattactct atttaatatc 6660
gtataaacgt aatttcaata caaaaagtat aaatgacatt ttgtcattta taatttttct 6720
atttttaatt gtattgtttt tctttaggaa tgaagatttt ggtgcagata ctcagaacta 6780
tttaaatgaa tttgatgaat attgttctaa tcctagtgga tatataggag tggattacac 6840
atataaatat atatttgatt taattaattt tctaatgttg gatagttgcc atattagttg 6900
gttaatttgg atatggccaa tatttgttat tggttgcatc tttttagcaa taacaatatt 6960
tagagttgat aaaatttata tcatggcttt gttttcttca tttataggca ttgagttgtt 7020
aactaatgct ctgaggcagg gattctcaat agcagtactg ttattatcgt tctgtttgtt 7080
tataaagagg cgatattttt tattcttgat agctgtaacc atttcttttc tttttcatca 7140
agcttcgatt ctgatcgttt ctatttttat tatttctaga tgtagcatta gaattgtagt 7200
tcctagtatt gttgtaggtg tgtttttgtt gtttgcgaca aatattctag atgttgtgcc 7260
ttgggttatt gagtttaaaa agttaatata taaatatttg ccgtatgcag atgaggactt 7320
tattattcga atcataagta taattaatac tttatgtctt tgtgtattat atttaattgt 7380
catgattaaa tcacattgta aggataaaat aataaacaat attgttatta atgctacctt 7440
tgtttgttgt atcgcatcta ttgttccgta tcttggtttt cgaattatat atggcgtata 7500
tccattatta ttacttttaa cctactgtag cataagaaat tatatcggca gagtgtcttc 7560
atataattat ttatcgcttt cgatatgtat taatatcttt atctcaatta tgtggttgtg 7620
tggttcctcc catatgaaca aaataccatt tgtgagcatt attatatgaa aaaaaggagt 7680
ttcggtattt ctgttgttgt acctgtgtat aacaggaaag aaaagttaat cagggctatt 7740
gactcagttg atactttatc cccacatctt gtggagatta ttgtaattga tgattgttca 7800
gatttggctc ctgacacatt tttagagaag agtaataagt atggggttaa tgttcacatt 7860
tatcgcaata aatataataa ggggccgcaa atttgtagga acattgggat taggagagct 7920
aaatttaatt atattgcctt tttagattca gatgattatt tttgcatggg aaaaatagat 7980
tggctattga atattctgaa ggatgaggat atagattttc tctaccatgc agtaaatgga 8040
tgtgaaaaat ataatcgaat ttcatcatat tggtttaata cagtaggtaa gtttattcat 8100
tttagatggt ttttgatttt tttgaatcct tgtgttacac caagtgtagt gattaaaagg 8160
aaagtttgtt tatttaatcc atgtttgaga tattcagagg attatgcata ttttttatct 8220
tatattaatt caaaaactaa agttaaatat tttgaaagca tatatacaac agttcctcga 8280
gaaataggca caagaggtgg tatttcgcat aatattataa aaatgagaaa gggagaattt 8340
tatggaaaaa aaaatatatt aaggaagagg cgaaatatat cgaattatat tcggtttctg 8400
atatctttag gcatggcttg tcttagagtt ttctttgata tagtgagaaa acggtataaa 8460
gtgaataatt tatttaaaga tatatagtta aatttctaat attagcaaga taaattaagg 8520
tcatcagtat gacagaacaa ttttcagaga aaaaaataga tgttgttgga attgttggat 8580
tgccagcatg ttatggtgga tttgaatcat tagttcaaaa tttagttgat tatcaaagtc 8640
aaaatataaa atacaatgta tattgttcac gaaaaaaata taaaaacacc ccaaaaaaat 8700
acaagcgtgc tgacttgaag tatataccat ttgacgccaa tggtagttct agtatcttgt 8760
atgatatcta ttcgttattt ttgtcattgt ttaacaaggt agatgttgtc ctaattctcg 8820
gggtttcagg gtgtgttttt ctaccaattt acagattttt ttcgtcatca aaggttattg 8880
taaatattga tggattagaa tggaaaagag caaaatggaa gggtattgct aagtggtatt 8940
taaaaaattc agagaaaata gctgttaaat attcagatgt tgttgtggct gataatgaag 9000
ctattgctaa atatgtctta aaaaaatacg gtttagaagc taaaattatt gcttatggtg 9060
gtgatcattc attggttaag aagccaatat cagttataaa agaggactat ttttttactg 9120
tctgcaggat tgaaccagaa aataacataa gaatgatatt agaagcattt aaaaatacaa 9180
cacacagtct aaaaattgtt ggtaattggg attccagttt gtatgggcga cgactaaaag 9240
aagaatttgg gaattataat aatattgaaa ttatagaccc tatatatgat tctgatatcc 9300
tttttaactt taggtcttta tgccgtggct atattcatgg gcattcagct ggtgggacaa 9360
atccttcttt agtcgaggct atgcattttc aaattcctat tattgccttt gattgtgatt 9420
tcaatcggtt tacaactgac aactatgcgt tttattttaa aaataaaaat gagctatctt 9480
ttatcgtcaa tgatatatta aatggcaatc agaatgaaca ggcagaaatc tgcgcaaaaa 9540
aaatgaagga aattgctaca aaaaaataca cgtgggatac catagccaaa atgtatgaag 9600
aactttattg atatttaata aatgaacgat tatttaaaga ttggcatata atttagttca 9660
gaagcaagcc ataaccccct gacaggagta aacaatgtca aagcaacaga tcggcgtcgt 9720
cggtatggca gtgatggggc gcaaccttgc gctcaacatc gaaagccgtg gttataccgt 9780
ctctattttc aaccgttccc gtgaaaaaac ggaagaagtg attgccgaaa atccaggcaa 9840
aaaactggtt ccttactata cggtgaaaga gtttgttgaa tctctggaaa cgcctcgtcg 9900
catcctgtta atggtgaaag caggtgcagg cacggatgct gctattgatt ccctcaagcc 9960
atacctcgat aaaggtgaca tcatcattga tggtggtaac accttcttcc aggacaccat 10020
tcgtcgtaac cgtgagcttt ctgccgaagg ttttaacttt atcggtaccg gtgtttccgg 10080
aggcgaagaa ggcgcgttga aaggtccttc cattatgcct ggtgggcaga aagaagccta 10140
cgaactggtt gcgccgatcc tgaccaaaat cgccgccgtt gctgaagacg gcgagccatg 10200
cgttacctat attggtaccg atggtgcagg tcactatgtg aagatggttc acaacggtat 10260
tgaatacggc gatatgcaac tgattgctga agcctattct ctcctgaaag gcggcctgaa 10320
tctctctaac gaagaactgg cacagacctt taccgagtgg aataacggtg aactgagtag 10380
ctacctgatc gacatcacca aagacatctt caccaaaaaa gatgaagacg gtaactacct 10440
ggttgatgtg atcctggatg aagcagcaaa caaaggtacg ggcaaatgga ccagccagag 10500
cgcgctggat ctcggcgaac cgctgtcgct gattaccgag tctgtgtttg cacgttatat 10560
ctcgtctctg aaagatcagc gtgttgccgc gtctaaagtt ctctctggtc cgcaagcgca 10620
gccagcaggc gacaaggctg agtttatcga gaaagttcgt cgtgcgctgt atctgggcaa 10680
aatcgtttct tatgctcagg gcttctctca actgcgtgct gcgtctgaag agtacaactg 10740
ggatctgaac tacggcgaga tcgcgaagat tttccgtgct ggttgcatca tccgtgcgca 10800
gttcctgcag aaaatcaccg atgcttatgc cgaaaatccg caaatcgcta acctgctgct 10860
ggctccgtac ttcaagcaaa ttgcagatga ctaccagcag gctctgcgtg atgtcgttgc 10920
ttatgcagta cagaacggta tcccggttcc gaccttcgcc gctgcggttg cctattacga 10980
tagctaccgt gccgctgttc tgcct 11005
Glycosyltransferase gene in the table 1 Shigella bogdii 1 type O antigen gene bunch and oligosaccharide unit treatment gene and wherein primer and PCR data
| Gene | Function | The base position of gene | Forward primer | Reverse primer | PCR product length | Produce the group number of correct big or small electrophoresis band | The annealing temperature of PCR (℃) |
| Wzx | The transhipment enzyme | 4570 to 5754 | #13 (5051 to 5069) | #14 (5724 to 5742) | 692bp | 0 | 55 |
| #127 (4654 to 4672) | #128 (5276 to 5294) | 641bp | 0 | 52 | |||
| #129 (5164 to 5172) | #130 (5535 to 5553) | 390bp | 0 | 50 | |||
| wzy | Polysaccharase | 6605 to 7669 | #21 (6607 to 6625) | #22 (7545 to 7563) | 957bp | 0 * | 55 |
| #23 (6667 to 6685) | #24 (7424 to 7441) | 775bp | 0 *** | 50 | |||
| #27 (6755 to 6773) | #28 (7235 to 7253) | 499bp | 0 *** | 45 | |||
| Orf8 | Glycosyltransferase | 7666 to 8487 | #25 (7800 to 7818) | #26 (8400 to 8417) | 618bp | 0 | 55 |
| #131 (7676 to 7694) | #132 (8280 to 8298) | 623bp | 0 | 55 | |||
| #133 (7896 to 8014) | #134 (8293 to 8311) | 416bp | 0 | 55 | |||
| orf9 | Glycosyltransferase | 8529 to 9611 | #29 (8697 to 8715) | #30 (9307 to 9325) | 629bp | 0 ** | 55 |
| #31 (9002 to 9020) | #32 (9483 to 9502) | 501bp | 0 *** | 45 | |||
| #33 (8965 to 8983) | #34 (9425 to 9443) | 479bp | 0 *** | 45 |
*In 4 groups, obtain the band of 1 wrong size, in 3 groups, obtain the band of 2 wrong sizes
*In 3 groups, obtain the band of 1 wrong size, in 6 groups, obtain the band of 2 wrong sizes
* *In all groups, all obtain the band of 1 wrong size
Table 2 166 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, O3, O10, O16, O18, O39 IMVS
a
2 wild-type e. coli O40, O41, O48, O49, O71, O73, O88, O100 IMVS
3 wild-type e. coli O102, O109, O119, O120, O121, O125, O126, O137 IMVS
4 wild-type e. coli O138, O139, O7, O5, O6, O11, O12 IMVS
5 wild-type e. coli O13, O14, O15, O17, O19ab, O20, O21, O22 IMVS
6 wild-type e. coli O23, O24, O25, O26, O27, O28, O29, O30 IMVS
7 wild-type e. coli O32, O33, O34, O35, O36, O37, O38, O42 IMVS
8 wild-type e. coli O43, O44, O45, O46, O50, O51, O52, O53 IMVS
9 wild-type e. coli O54, O55, O56, O57, O58, O59, O60, O61 IMVS
10 wild-type e. coli O62, O63, O64, O65, O66, O68, O69, O70 IMVS
11 wild-type e. coli O74, O75, O76, O77, O78, O79, O80, O81 IMVS
12 wild-type e. coli O82, O83, O84, O85, O86, O87, O89, O90 IMVS
13 wild-type e. coli O91, O92, O95, O96, O97, O98, O99, O101 IMVS
14 wild-type e. coli O112, O162, O113, O114, O115, O116, O117, O118 IMVS
15 wild-type e. coli O123, O165, O166, O167, O168, O169, O170, O171 See b
16 wild-type e. coli O172, O173, O127, O128, O129, O130, O131, O132, See c
17 wild-type e. coli O133, O134, O135, O136, O140, O141, O142, O143 IMVS
18 wild-type e. coli O144, O145, O146, O147, O148, O150, O151, O152 IMVS
19 wild-type e. coli O153,0154, O155, O156, O157, O158, O159, O164 IMVS
20 wild-type e. coli O160, O161, O163, O8, O9, O124, O111 IMVS
21 wild-type e. coli O103, O104, O105, O106, O107, O108, O110 IMVS
22 Shigella bogdii serotypes B 4, B5, B6, B8, B9, B11, B12, B14 See d
23 Shigella bogdii serotypes B 3, B7, B8, B10, B13, B15, B16, B17, B18 See d
24 shigella dysenteriae serotype D1, D2, D3, D4, D5, D6, D7, D8 See d
25 shigella dysenteriae serum D9, D10, D11, D12, D13 See d
26 shigella flexneri F6a, F1a, F1b, F2a, F2b, F3, F4a, F4b, F5 (v:7) F5 (v:4) See d
27 bacillus ceylonensis A D5, DR Seed
a. Institude of Medical and Veterinary Science，Anelaide，Australia
b. O123 from IMVS；the rest from Statens Serum Institut，Copenhagen，Denmark
c. 172 and 173 from Statens Serum Institut，Copenhagen，Denmark，the rest from IMVS
D. China Preventive Medicial Science Institute's epidemiological study institute
Table 3 is the structural table of the O-antigen gene bunch of Shigella bogdii 1 type
1Kb
Table 4 is the location tables of the gene in the O-antigen gene bunch of Shigella bogdii 1 type
ctcctggtaa ctcacgcgtc caaaaacgcg gtcgaaaacc acttcgacac ctcttatgaa 60
ttagaatctc tccttgaact gcgcgtgaag cgtcagctgc tggcggaagt acagtccatc 120
tgtccgccgg gcgtgaccat tatgaacgtg cgtcagggcg aacctttagg tttaggccac 180
tccattttgt gtgcacgacc cgccattggt gacaacccat ttgttgtggt attgccggac 240
gttgtgatcg acgacgccag tgccgacccg ctgcgctaca accttgctgc catgattgcg 300
cgcttcaatg aaacgggccg cagccaggtg ctggcaaaac gtatgccggg tgacctctct 360
gaatactccg tcattcagac caaagaacca ctggatcgtg aaggtaaagt cagccgcatc 420
gtcgaattta tcgaaaaacc ggatcagccg cagacgctgg actcagacat catggccgtt 480
ggtcgctatg tgctttctgc cgatatttgg ccggaactgg aacgtactca gcctggtgca 540
tggggacgta ttcagctgac tgatgctatt gccgagctgg cgaaaaaaca gtccgttgat 600
gccatgctga tgactgggga cagttacgac tgcggtaaaa aaatgggtta tatgcaggca 660
tttgtgaagt atggactacg caacctcaaa gaaggggcga agttccgtaa agggattgag 720
aagctgttaa gcgaataatg aaaatctgac cggatgtaac ggttgataag aaaattataa 780
cggcagtgaa gattcgtggc gaaagtaatt tgttgcgaat attcctgccg ttgttttata 840
taaacaatca gaataacaac gagttagcaa taggatttta gtcaaagttt tccaggattt 900
tccttgtttc cagagcggat tggtaagaca attagcgttt gaatttttcg ggtttagcgc 960
gagtgggtaa cgctcgtcac atcgtagaca tgcatgtagt gctctggtag ctgtaaagcc 1020
aggggcggta gcgtgcatta acacctctat taatcaacct aagagccgct tatttcaccg 1080
catgctctga agttatatgg aataaataag tgaagatact tgttactggt ggcgcaggat 1140
ttattggttc tgctgtagtt cgtcacatta taaataatac gcaggatagt gttgttaatg 1200
tcgataaatt aacgtacgcc ggaaacctgg aatcacttgc tgatgtttct gattctgaac 1260
Orf1's is initial
gctatgtttt tgaacatgcg gatatttgcg atgctgctgc a
atggcgcga attttttctc 1320
agcatcagcc ggatgcagtg atgcacctgg ctgctgaaag ccatgttgac cgttcaatta 1380
caggccctgc ggcatttatt gaaaccaata ttgttggtac ttatgtcctt ttggacgctg 1440
ctcgcaatta ctggtctgct cttgatagcg acaagaaaaa tagcttccgt tttcatcata 1500
tttctactga cgaagtctat ggtgatttgc ctcatcctga cgaagtaaat aataaagaaa 1560
aattaccctt atttattgag acaacagctt acgcaccaag cagcccttat tccgcatcca 1620
aagcatccag cgatcattta gtccgcgcgt ggaaacgtac ctatggttta ccgaccattg 1680
tgactaattg ctctaacaat tatggccctt atcatttccc ggaaaaattg attccattgg 1740
ttattctgaa tgctctggaa ggtaagatgt tgcctattta tggcaaaggg gatcaaattc 1800
gtgattggct gtatgtagag gatcatgctc gagcgttata tacagttgtt actgaaggta 1860
aagcaggtga aacctataat attggtggac acaacgaaaa gaaaaacatc gatgtagtgc 1920
tcactatttg tgatttgttg gatgagattg tcccgaaaga gaaatcttac cgcgagcaaa 1980
ttacttatgt tgccgatcgt ccgggacacg atcgccgtta tgcgattaat gctgagaaga 2040
ttggtcgcga attgggctgg aaaccacagg aaacgtttga gagcgggatt cgtaaaacag 2100
tggaatggta ccttgctaat gcaaaatggg tcgaaaatat gaaaagtggt gcctatcaat 2160
The termination orf2's of orf1 is initial
cgtggattga acagaactat gagggccgcc ag
taatgaat attctccttt tcggcaaaac 2220
agggcaggta ggttgggaac tacagcgtgc tctggcacct ctgggtaatt tgattgctct 2280
tgatgttcac tccactgatt attgtggtga ttttagtaat cctgaaggtg tagctgaaac 2340
cgtaagaagc attcggcctg atattattgt caacgcagcc gctcacaccg cagtagacaa 2400
agcagaatca gaaccggagt ttgcacaatt acttaacgcg acaagtgtcg aagcgattgc 2460
gaaagcagcc aatgaagtcg gcgcctgggt tattcactac tctactgact acgtatttcc 2520
ggggaccggt gaaataccat ggcaggagac ggatgcaacc gcaccgctaa atgtgtacgg 2580
tgaaaccaag ttagctggag aaaaagcatt acaagagcat tgtgcgaagc accttatttt 2640
ccgtaccagc tgggtatatg caggtaaagg aaacaatttt gccaagacaa tgttacgtct 2700
ggcaaaagag cgcgaagaat tagctgtcat taacgatcag tttggtgcac caacaggtgc 2760
tgagttgctg gctgattgta cggcacatgc cattcgtgtt gcactgaata aaccagaagt 2820
cgcaggctta taccatctgg tagccagtgg taccacaacc tggtacgatt atgctgcgct 2880
ggtttttgaa gaggcgcgcg aagcaggcat tccccttgca ctcaacaagc tcaacgcagt 2940
accaacaaca gcctatccta cactagctcg tcgtccgcat aactctcgcc ttgatacaga 3000
aaaatttcag cagaactttg cgcttgtctt gcctgactgg cagattggcg tgaaacgcat 3060
The termination of orf2
gctcaacgaa ttatttacga ctacagctat t
taatagttt ttgcatcttg tttgtgatgg 3120
Orf3's is initial
tggagcaaga tgaattaaaa ggaatgatga a
atgaaaacg cgtaaaggta ttattttagc 3180
gggtggttct ggtactcgtc tttatcctgt gactatggca gtcagtaaac agctgctacc 3240
gatttatgat aaaccgatga tctattatcc gctctctaca ctgatgttgg cgggtattcg 3300
cgatattttg attatcagta cgccacagga tactcctcgt tttcaacaac tgttgggtga 3360
cggtagccag tgggggctga atcttcagta caaagtgcaa gcgagtccgg atggtcttgc 3420
gcaggcgttt attatcggtg aagagtttat tggtggtgat gattgtgctt tgattcttgg 3480
tgataatatc ttttacggtc acgatctgcc gaagttaatg gatgccgctg ttaacaaaga 3540
aagtggtgca acggtatttg catatcacgt aaatgatcct gaacgctatg gtgtcgtgga 3600
gtttgataat aacggaacag ccattagcct tgaagaaaaa ccgttagagc caaaaagcaa 3660
ttatgctgta accggacttt atttctatga caatgatgtt gtagaaatgg cgaaaaacct 3720
taagccttct gcccgtggcg aactggaaat taccgatatt aaccgtattt atatggagca 3780
gggacgttta tctgttgcca tgatgggacg tggatatgca tggttggaca cggggacaca 3840
tcaaagtctt attgaggcaa gcaattttat cgcaacaata gaagaacgtc aggggctgaa 3900
agtttcctgc ccggaagaaa ttgcttaccg taaagggttt atcgatgctg agcaggtgaa 3960
agtattagct gaaccgttga agaaaaatgc ttatggtcag tatctgctga aaatgattaa 4020
The termination orf4's of orf3 is initial
aggttattaa
taaa
atgaac gtaattaaaa ctgaaattcc tgatgtgtta attttcgagc 4080
cgaaagtttt tggtgatgag cgtggtttct ttatggaaag ctttaatcag aaagttttcg 4140
aagaggctgt agggcgtaag gttgaatttg ttcaggataa tcattcgaag tctagtaaag 4200
gcgttttacg tgggctgcac tatcagttag aaccttatgc gcaaggaaaa ctggtacgct 4260
gcgttgttgg tgaagttttt gacgtagcgg ttgatattcg taaatcgtca ccaacttttg 4320
gcaaatgggt tggggtgaat ttatctgctg agaataagcg ccagttgtgg atccctgaag 4380
gatttgcaca tggttttttg gtgctgagtg aaacggcgga gtttttgtat aagacgacta 4440
attattatca tccgataagt gaaagagggg ttaaatggga tgatccctgt attgatatta 4500
agtggcctct agtatcgtct cccctgttat cagaaaaaga taagtgttat tcatggctaa 4560
The termination of the initial orf4 of orf5
cgaaacaaa
a tgaacag
taa taagagacaa actttattct tttcgcttga tcaaataata 4620
gattctggac ttgtttttat ttttattgct attggcggta atttacttga caaatcagat 4680
atggctaatg ttgtgtcatt ccaatctgtt gcattagtat ctgtattgtt ttgttcctgt 4740
tttacaacgc aatatttact gttgcgctat aaaaaacaat cttcactttt ttggctaaag 4800
ttatttttat ttttttgtat tattactatg ttgattgtat cattatggtt tcaatcggca 4860
gaaatattat tcttgttttt tgttggaata tcttcagaat acataaaacg atactgcttt 4920
tatattaata attcttgtct ctctttcttt tcaacgataa taacggcatt tatttttttt 4980
gtttttatat tgatgtgttg gctgaatttc atgcgcatta attcgactat atatatatat 5040
atatatagcg gtgcaaagtt aattccgcta acgataatat gttgttttct ttttattaaa 5100
acaaagttca atagtgatgg tgaatatgaa atgccatctt tatggtatgc aattactgat 5160
tcattaagat ttggaggcgt gttttctgtt attaccatta tttattggat tacaaatcaa 5220
ggtttcttta tattctttca tgacagaatg cctgcagcag agttggtaga aattagagtt 5280
acgcagaatg tgtttggtgt agttacaatg ctaattgcat tgtacgactc tatgttttta 5340
aagaaaaaca tagagaataa aaataaaata tttgattggc gatcttattt taaattcctt 5400
gtaatttcaa ttgttttgat tacatttaat tttattttat tatatatatt atcaataaca 5460
atatataaac atttggatgt ttttaaatat tcgatatgtc ttgcactggc ccaatttttt 5520
tatctttccg caaggatgcc tgtacttatt ttgaaattgc gctatagttt aagtattatt 5580
ctgtggttat acgtgtgttc gctttttttg tcctcgtgtt acttattctt gaaacataat 5640
gattatgatt ttcaatatat agtgcaatct ataacattat ctaacttctt tatttttcta 5700
The termination of the initial orf5 of orf6
ttttcattat taatagtgtt ttacaaagag aggattt
atg ggaagaa
tta ttgaagctct 5760
gtatgggata tcaacggaac tgaaagagcg aatcttgaat atgggagaga aaacagttcc 5820
ggtgaattac tatacaagac ataataactg gggtgatcaa ttaaataaat atttaattga 5880
aaaaattacc aataaaaagg tagtaaagaa caacttcaaa aagctacctc atatattagc 5940
tataggaagc gttctttcaa gtgcatcaaa taaaagtatc gtatggggga gtggttttat 6000
atctaaagat gcgccattac gagaaacagc ccctgatatt agggctgttc gaggtgtttt 6060
gactagaaat aaacttcaga aagatttttc aattgactgc cctgatgtgt tgggagaccc 6120
ggccgttctt ttgcctttat tttataaagc aaaacataga agtaaaaaat acaaagtcgg 6180
tattattcca cactataaag ataagcaacg agctgttgta caggattttt tacataaagg 6240
atgtgttctt gttgatattc aagatgacat tgaagttttt attgataaaat taacgaatg 6300
tgaggttgta atttcatcgt cacttcatgg attaattgct gcagatactt atggtatacc 6360
taatttatgg gtttcatttg gtgaccaagt attaggtggt gattttaagt ttttggacta 6420
ttatagtact actaataata taaatcctaa aatgatacga ctagataatg atcataaata 6480
ttctattgat gatttggtta gaatttctag atgtaataaa tttattagaa gttctgaaga 6540
The termination orf7's of orf6 is initial
gttacttgaa gcgtttccta gggagtacat a
tgaattact attatataat tgtgtagg
tg 6600
aaaaatgcta gttacaatcc tggctttatt tctgtttata caattactct atttaatatc 6660
gtataaacgt aatttcaata caaaaagtat aaatgacatt ttgtcattta taatttttct 6720
atttttaatt gtattgtttt tctttaggaa tgaagatttt ggtgcagata ctcagaacta 6780
tttaaatgaa tttgatgaat attgttctaa tcctagtgga tatataggag tggattacac 6840
atataaatat atatttgatt taattaattt tctaatgttg gatagttgcc atattagttg 6900
gttaatttgg atatggccaa tatttgttat tggttgcatc tttttagcaa taacaatatt 6960
tagagttgat aaaatttata tcatggcttt gttttcttca tttataggca ttgagttgtt 7020
aactaatgct ctgaggcagg gattctcaat agcagtactg ttattatcgt tctgtttgtt 7080
tataaagagg cgatattttt tattcttgat agctgtaacc atttcttttc tttttcatca 7140
agcttcgatt ctgatcgttt ctatttttat tatttctaga tgtagcatta gaattgtagt 7200
tcctagtatt gttgtaggtg tgtttttgtt gtttgcgaca aatattctag atgttgtgcc 7260
ttgggttatt gagtttaaaa agttaatata taaatatttg ccgtatgcag atgaggactt 7320
tattattcga atcataagta taattaatac tttatgtctt tgtgtattat atttaattgt 7380
catgattaaa tcacattgta aggataaaat aataaacaat attgttatta atgctacctt 7440
tgtttgttgt atcgcatcta ttgttccgta tcttggtttt cgaattatat atggcgtata 7500
tccattatta ttacttttaa cctactgtag cataagaaat tatatcggca gagtgtcttc 7560
atataattat ttatcgcttt cgatatgtat taatatcttt atctcaatta tgtggttgtg 7620
The termination of the initial orf7 of orf8
tggttcctcc catatgaaca aaataccatt tgtgagcatt attat
atgaa aaaaaggagt 7680
ttcggtattt ctgttgttgt acctgtgtat aacaggaaag aaaagttaat cagggctatt 7740
gactcagttg atactttatc cccacatctt gtggagatta ttgtaattga tgattgttca 7800
gatttggctc ctgacacatt tttagagaag agtaataagt atggggttaa tgttcacatt 7860
tatcgcaata aatataataa ggggccgcaa atttgtagga acattgggat taggagagct 7920
aaatttaatt atattgcctt tttagattca gatgattatt tttgcatggg aaaaatagat 7980
tggctattga atattctgaa ggatgaggat atagattttc tctaccatgc agtaaatgga 8040
tgtgaaaaat ataatcgaat ttcatcatat tggtttaata cagtaggtaa gtttattcat 8100
tttagatggt ttttgatttt tttgaatcct tgtgttacac caagtgtagt gattaaaagg 8160
aaagtttgtt tatttaatcc atgtttgaga tattcagagg attatgcata ttttttatct 8220
tatattaatt caaaaactaa agttaaatat tttgaaagca tatatacaac agttcctcga 8280
gaaataggca caagaggtgg tatttcgcat aatattataa aaatgagaaa gggagaattt 8340
tatggaaaaa aaaatatatt aaggaagagg cgaaatatat cgaattatat tcggtttctg 8400
atatctttag gcatggcttg tcttagagtt ttctttgata tagtgagaaa acggtataaa 8460
The termination of orf8
gtgaataatt tatttaaaga tata
tagtta aatttctaat attagcaaga taaattaagg 8520
Orf9's is initial
tcatcagt
at qacagaacaa ttttcagaga aaaaaataga tgttgttgga attgttggat 8580
tgccagcatg ttatggtgga tttgaatcat tagttcaaaa tttagttgat tatcaaagtc 8640
aaaatataaa atacaatgta tattgttcac gaaaaaaata taaaaacacc ccaaaaaaat 8700
acaagcgtgc tgacttgaag tatataccat ttgacgccaa tggtagttct agtatcttgt 8760
atgatatcta ttcgttattt ttgtcattgt ttaacaaggt agatgttgtc ctaattctcg 8820
gggtttcagg gtgtgttttt ctaccaattt acagattttt ttcgtcatca aaggttattg 8880
taaatattga tggattagaa tggaaaagag caaaatggaa gggtattgct aagtggtatt 8940
taaaaaattc agagaaaata gctgttaaat attcagatgt tgttgtggct gataatgaag 9000
ctattgctaa atatgtctta aaaaaatacg gtttagaagc taaaattatt gcttatggtg 9060
gtgatcattc attggttaag aagccaatat cagttataaa agaggactat ttttttactg 9120
tctgcaggat tgaaccagaa aataacataa gaatgatatt agaagcattt aaaaatacaa 9180
cacacagtct aaaaattgtt ggtaattggg attccagttt gtatgggcga cgactaaaag 9240
aagaatttgg gaattataat aatattgaaa ttatagaccc tatatatgat tctgatatcc 9300
tttttaactt taggtcttta tgccgtggct atattcatgg gcattcagct ggtgggacaa 9360
atccttcttt agtcgaggct atgcattttc aaattcctat tattgccttt gattgtgatt 9420
tcaatcggtt tacaactgac aactatgcgt tttattttaa aaataaaaat gagctatctt 9480
ttatcgtcaa tgatatatta aatggcaatc agaatgaaca ggcagaaatc tgcgcaaaaa 9540
aaatgaagga aattgctaca aaaaaataca cgtgggatac catagccaaa atgtatgaag 9600
The termination of orf9
aactttat
tg atatttaata aatgaacgat tatttaaaga ttggcatata atttagttca 9660
gaagcaagcc ataaccccct gacaggagta aacaatgtca aagcaacaga tcggcgtcgt 9720
cggtatggca gtgatggggc gcaaccttgc gctcaacatc gaaagccgtg gttataccgt 9780
ctctattttc aaccgttccc gtgaaaaaac ggaagaagtg attgccgaaa atccaggcaa 9840
aaaactggtt ccttactata cggtgaaaga gtttgttgaa tctctggaaa cgcctcgtcg 9900
catcctgtta atggtgaaag caggtgcagg cacggatgct gctattgatt ccctcaagcc 9960
atacctcgat aaaggtgaca tcatcattga tggtggtaac accttcttcc aggacaccat 10020
tcgtcgtaac cgtgagcttt ctgccgaagg ttttaacttt atcggtaccg gtgtttccgg 10080
aggcgaagaa ggcgcgttga aaggtccttc cattatgcct ggtgggcaga aagaagccta 10140
cgaactggtt gcgccgatcc tgaccaaaat cgccgccgtt gctgaagacg gcgagccatg 10200
cgttacctat attggtaccg atggtgcagg tcactatgtg aagatggttc acaacggtat 10260
tgaatacggc gatatgcaac tgattgctga agcctattct ctcctgaaag gcggcctgaa 10320
tctctctaac gaagaactgg cacagacctt taccgagtgg aataacggtg aactgagtag 10380
ctacctgatc gacatcacca aagacatctt caccaaaaaa gatgaagacg gtaactacct 10440
ggttgatgtg atcctggatg aagcagcaaa caaaggtacg ggcaaatgga ccagccagag 10500
cgcgctggat ctcggcgaac cgctgtcgct gattaccgag tctgtgtttg cacgttatat 10560
ctcgtctctg aaagatcagc gtgttgccgc gtctaaagtt ctctctggtc cgcaagcgca 10620
gccagcaggc gacaaggctg agtttatcga gaaagttcgt cgtgcgctgt atctgggcaa 10680
aatcgtttct tatgctcagg gcttctctca actgcgtgct gcgtctgaag agtacaactg 10740
ggatctgaac tacggcgaga tcgcgaagat tttccgtgct ggttgcatca tccgtgcgca 10800
gttcctgcag aaaatcaccg atgcttatgc cgaaaatccg caaatcgcta acctgctgct 10860
ggctccgtac ttcaagcaaa ttgcagatga ctaccagcag gctctgcgtg atgtcgttgc 10920
ttatgcagta cagaacggta tcccggttcc gaccttcgcc gctgcggttg cctattacga 10980
tagctaccgt gccgctgttc tgcct 11005
The above, it only is preferred embodiment of the present invention, be not that the present invention is done any pro forma restriction, every foundation technical spirit of the present invention all still belongs in the scope of technical solution of the present invention any simple modification, equivalent variations and modification that above embodiment did.
Claims (2)
1, a kind of oligonucleotide of the O-antigen-specific to Shigella bogdii 1 type and intestinal bacteria O149 is characterized in that described oligonucleotide is to being: the Nucleotide of 5051 to 5069 bases among the SEQ ID NO:1 and the Nucleotide of 5724 to 5742 bases; The Nucleotide of 4654 to 4672 bases among the SEQ ID NO:1 and the Nucleotide of 5276 to 5294 bases; The Nucleotide of 5164 to 5172 bases among the SEQ ID NO:1 and the Nucleotide of 5535 to 5553 bases; The Nucleotide of 6607 to 6625 bases among the SEQ ID NO:1 and the Nucleotide of 7545 to 7563 bases; The Nucleotide of 6667 to 6685 bases among the SEQ ID NO:1 and the Nucleotide of 7424 to 7441 bases; The Nucleotide of 6755 to 6773 bases among the SEQ ID NO:1 and the Nucleotide of 7235 to 7253 bases; The Nucleotide of 7800 to 7818 bases among the SEQ ID NO:1 and the Nucleotide of 8400 to 8417 bases; The Nucleotide of 7676 to 7694 bases among the SEQ ID NO:1 and the Nucleotide of 8280 to 8298 bases; The Nucleotide of 7896 to 8014 bases among the SEQ ID NO:1 and the Nucleotide of 8293 to 8311 bases; The Nucleotide of 8697 to 8715 bases among the SEQ ID NO:1 and the Nucleotide of 9307 to 9325 bases; The Nucleotide of 9002 to 9020 bases among the SEQ ID NO:1 and the Nucleotide of 9483 to 9502 bases; The Nucleotide of 8965 to 8983 bases among the SEQ ID NO:1 and the Nucleotide of 9425 to 9443 bases.
2, the application of the described Nucleotide of claim 1 is characterized in that, it is used for PCR as primer, perhaps is used for hybridization and fluoroscopic examination, makes gene chip or microarray confession detection Shigella bogdii 1 type and intestinal bacteria O149 type as probe.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 03100357 CN1249234C (en) | 2003-01-20 | 2003-01-20 | Nucleotide specific to O-antigen of shigella boydii I and Bacillus coli 0149 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 03100357 CN1249234C (en) | 2003-01-20 | 2003-01-20 | Nucleotide specific to O-antigen of shigella boydii I and Bacillus coli 0149 |
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| CN1451662A CN1451662A (en) | 2003-10-29 |
| CN1249234C true CN1249234C (en) | 2006-04-05 |
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| CN 03100357 Expired - Fee Related CN1249234C (en) | 2003-01-20 | 2003-01-20 | Nucleotide specific to O-antigen of shigella boydii I and Bacillus coli 0149 |
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| CN1307307C (en) * | 2004-12-30 | 2007-03-28 | 天津生物芯片技术有限责任公司 | Nucleotide specific for Shigella bogdii B14-type O-antigen |
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