CN1252267C - Nucleotide - Google Patents
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- CN1252267C CN1252267C CNB031005381A CN03100538A CN1252267C CN 1252267 C CN1252267 C CN 1252267C CN B031005381 A CNB031005381 A CN B031005381A CN 03100538 A CN03100538 A CN 03100538A CN 1252267 C CN1252267 C CN 1252267C
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Abstract
The present invention provides a specific nucleotide for the O-antigens of Shigella boydii 15, Shigella dysenteriae 2 and Escherichia coli 0112, which is a nucleotide complete sequence of gene clusters for controlling O-antigen synthesis in Shigella boydii 15. The present invention also comprises the structure of O-antigenic gene clusters, the oligonucleotide of glycosyl transferase genes and oligosaccharide unit processing genes (comprising wzx genes or genes with the similar functions with wzx, wzy genes or genes with the similar functions with wzy) in the O-antigenic gene clusters stemmed from Shigella boydii 15, and a method for obtaining bacteria O-antigenic gene clusters. The present invention verifies that the specific nucleotide, the structure of O-antigenic gene clusters and the oligonucleotide have high specificity to all of O-antigens of Shigella boydii 15, Shigella dysenteriae 2 and Escherichia coli 0112 through PCR. The present invention also discloses a method for using the oligonucleotide of the present invention to detect and identify Shigella boydii 15, Shigella dysenteriae 2 and Escherichia coli 0112 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 15 types (Shigella boydii 15), particularly relate to the oligonucleotide in the control O-antigen synthetic gene cluster in Shigella bogdii 15 types, can utilize these to the oligonucleotide of O-antigen-specific Shigella bogdii 15 types in human body and the environment quickly and accurately, shigella dysenteriae 2 types (Shigelladysenteriae 2) and intestinal bacteria O112 (Escherichia coli O112) also identify O-antigen in these pathogenic bacterium.
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 link button between synthetic, the monose of 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, Shigella bogdii 15 types wherein, shigella dysenteriae 2 types just have identical O-antigen [Ewing with intestinal bacteria O112, W.H. (1986) " Edwards and Ewing ' s identification of theEnterobacteriaceae " .Elsevier Science Publishers, Amsterdam, TheNetherlands; T.cheasty, et al. (1983) " Antigenic relationships betweenthe enteroinvasive Escherichia coli antigensO28ac; O112ac; O124, O136, O143; O144; O152 and O164 and Shigella Oantigens " J.clin Microbiol, 17 (4): 681-684], traditional serotype method can not be distinguished them.
Summary of the invention
The purpose of this invention is to provide a kind of to Shigella bogdii 15 types, the Nucleotide of the O-antigen-specific of shigella dysenteriae 2 types and intestinal bacteria O112.It is the Nucleotide in the O-antigen gene bunch of Shigella bogdii 15 types, is the special Nucleotide that comes from glycosyltransferase gene and transhipment enzyme gene and pol gene.
A time purpose of the present invention has provided the full length nucleotide sequence of the O-antigen gene bunch of Shigella bogdii 15 types.
Another object of the present invention has provided the gene of the O-antigen gene bunch that constitutes Shigella bogdii 15 types: the gene of transhipment enzyme is the wzx gene or with wzx the gene of identity function is arranged; Pol gene is the wzy gene or with wzy the gene of identity function is arranged; Glycosyltransferase gene comprises orf3, orf4, orf6, orf7 gene.
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 15 types respectively comprises orf3, orf4, orf6, orf7 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 to Shigella bogdii 15 types, and the O-antigen of shigella dysenteriae 2 types and intestinal bacteria O112 is special; Especially the oligonucleotide of listing in the table one, they are to Shigella bogdii 15 types, the O-antigen of shigella dysenteriae 2 types and intestinal bacteria O112 is high special, and these oligonucleotide are also reconfigurable, oligonucleotide after the combination is to Shigella bogdii 15 types, and the O-antigen of shigella dysenteriae 2 types and intestinal bacteria O112 also is high special.
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 Shigella bogdii 15 types by these methods, the O-antigen of shigella dysenteriae 2 types and intestinal bacteria O112 and detection and evaluation Shigella bogdii 15 types, shigella dysenteriae 2 types and intestinal bacteria O112.
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 15 types.Can obtain the complete sequence of the O-antigen gene bunch of other bacteriums according to present method operation, the complete sequence of the gene cluster of the bacterium of other polysaccharide antigens that also can obtain to encode.
The technical scheme that the present invention is adopted for achieving the above object is:
The present invention is to the Nucleotide of the O-antigen-specific of Shigella bogdii 15 types, shigella dysenteriae 2 types and intestinal bacteria O112, and it is the isolating Nucleotide shown in SEQ ID NO:1,10812 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 15 types, shigella dysenteriae 2 types and intestinal bacteria O112, it all is positioned between galF gene and gnd gene by 8 genomic constitutions.
The Nucleotide of aforesaid O-antigen-specific to Shigella bogdii 15 types, shigella dysenteriae 2 types and intestinal bacteria O112, wherein said gene is: 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 orf3, orf4, orf6, orf7 gene; Wherein said gene: wzx is the Nucleotide of 1041 to 2432 bases among the SEQ ID NO:1; Wzy is the Nucleotide of 2452 to 3630 bases among the SEQ ID NO:1; Orf3 is the Nucleotide of 3630 to 4610 bases among the SEQ ID NO:1; Orf4 is the Nucleotide of 4617 to 5798 bases among the SEQ ID NO:1; Orf6 is the Nucleotide of 6766 to 7647 bases among the SEQID NO:1; Orf7 is the Nucleotide of 7653 to 8393 bases among the SEQ ID NO:1.
The Nucleotide of aforesaid O-antigen-specific to Shigella bogdii 15 types, shigella dysenteriae 2 types and intestinal bacteria O112 wherein comes from described wzx gene, wzy gene or glycosyltransferase gene orf3, orf4, orf6, orf7 gene; And their mixing or their reorganization.
The Nucleotide of aforesaid O-antigen-specific to Shigella bogdii 15 types, shigella dysenteriae 2 types and intestinal bacteria O112, the oligonucleotide that wherein comes from the wzx gene is to being: the Nucleotide of 1148 to 1165 bases among the SEQ ID NO:1 and the Nucleotide of 1856 to 1873 bases; The Nucleotide of 1233 to 1250 bases among the SEQ ID NO:1 and the Nucleotide of 1859 to 1866 bases; The Nucleotide of 1460 to 1477 bases among the SEQ ID NO:1 and the Nucleotide of 1861 to 1878 bases; The oligonucleotide that comes from the wzy gene is to being: the Nucleotide of 2515 to 2533 bases among the SEQ ID NO:1 and the Nucleotide of 3087 to 3105 bases; The Nucleotide of 2935 to 2952 bases among the SEQ ID NO:1 and the Nucleotide of 3392 to 3411 bases; The Nucleotide of 2718 to 2734 bases among the SEQ ID NO:1 and the Nucleotide of 3536 to 3553 bases; The oligonucleotide that comes from the orf3 gene is to being: the Nucleotide of 3723 to 3740 bases among the SEQ ID NO:1 and the Nucleotide of 4121 to 4138 bases; The Nucleotide of 4103 to 4121 bases among the SEQ ID NO:1 and the Nucleotide of 4506 to 4525 bases; The Nucleotide of 3856 to 3874 bases among the SEQ ID NO:1 and the Nucleotide of 4548 to 4564 bases; The oligonucleotide that comes from the orf4 gene is to being: the Nucleotide of 4644 to 4661 bases among the SEQ ID NO:1 and the Nucleotide of 5246 to 5263 bases; The Nucleotide of 4649 to 4667 bases among the SEQ ID NO:1 and the Nucleotide of 5452 to 5471 bases; The Nucleotide of 5255 to 5272 bases among the SEQ ID NO:1 and the Nucleotide of 5661 to 5680 bases; The oligonucleotide that comes from the orf6 gene is to being: the Nucleotide of 6861 to 6880 bases among the SEQ ID NO:1 and the Nucleotide of 7309 to 7328 bases; The Nucleotide of 6995 to 7013 bases among the SEQ ID NO:1 and the Nucleotide of 7413 to 7430 bases; The Nucleotide of 7141 to 7159 bases among the SEQ ID NO:1 and the Nucleotide of 7613 to 7630 bases; The oligonucleotide that comes from the orf7 gene is to being: the Nucleotide of 7731 to 7747 bases among the SEQ ID NO:1 and the Nucleotide of 8096 to 8113 bases; The Nucleotide of 7855 to 7872 bases among the SEQ ID NO:1 and the Nucleotide of 8222 to 8240 bases; The Nucleotide of 7919 to 7937 bases among the SEQ ID NO:1 and the Nucleotide of 8341 to 8360 bases.
The Nucleotide of aforesaid O-antigen-specific to Shigella bogdii 15 types, shigella dysenteriae 2 types and intestinal bacteria O112 is detecting the application of expressing the antigenic bacterium of O-, identify other polysaccharide antigen of bacterium O-antigen and bacterium in diagnosis.
The recombinant molecule of the Nucleotide of aforesaid O-antigen-specific to Shigella bogdii 15 types, shigella dysenteriae 2 types and intestinal bacteria O112, and can provide Shigella bogdii 15 types of expressing by insert expressing, the O-antigen of shigella dysenteriae 2 types and intestinal bacteria O112, and become bacterial vaccine.
The application of the Nucleotide of aforesaid O-antigen-specific to Shigella bogdii 15 types, shigella dysenteriae 2 types and intestinal bacteria O112, wherein be used for PCR, be used for hybridization and fluoroscopic examination or be 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 15 types, shigella dysenteriae 2 types and intestinal bacteria O112, comprising following step:
(1) genomic extraction: 37 ℃ of incubated overnight Shigellaes in the LB substratum, centrifugal collecting cell, with Tris-HCl (pH8.0) and EDTA re-suspended cell, 37 ℃ of incubations add N,O-Diacetylmuramidase cracking bacterium after 20 minutes, add Proteinase K and SDS degrade proteins, add RNase again and remove RNA; Use equal-volume phenol and isopyknic phenol then: chloroform: enzyme and albumen is wherein removed in the primary isoamyl alcohol extracting, removes remaining phenol with isopyknic ether extracting again; With 2 times of volume ethanol deposit D NA, wash DNA with 70% ethanol after rolling out DNA with glass yarn, at last DNA is resuspended among the 30ulTE, genomic dna detects by 0.4% agarose gel electrophoresis;
(2) by the O-antigen gene in Long pcr amplification Shigella bogdii 15 types bunch: at first according to the JumpStart sequences Design upstream primer (#1523-ATT GTG GCT GCA GGG ATC AAA GAA AT) that often is found in O-antigen gene bunch promoter region, again according to the gnd gene design downstream primer (#1524-TAG TCG CGT GNG 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 30 seconds for 60 ℃, 68 ℃ were extended 15 fens, and carried out 30 circulations like this; At last, continue to extend 7 fens at 68 ℃, obtaining length is the PCR product of 12235 bases; 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; Select the dna fragmentation size after the suitable reaction times is cut enzyme to concentrate between the 1kb-3kb, then add 2ul 0.1M EDTA termination reaction, merge the same reaction system of 4 pipes, use isopyknic phenol and phenol respectively: chloroform: the primary isoamyl alcohol extracting once, after using twice of isopyknic ether extracting again, with the dehydrated alcohol deposit D NA of 2.5 times of volumes, and wash precipitation, be resuspended at last in the 18ul water with 70% ethanol; 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, make the 3 ' end of DNA add the dA tail, this mixture is through chloroform: after primary isoamyl alcohol extracting and the ether extracting 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, connect mixture with the dehydrated alcohol precipitation at last, be dissolved in after 70% ethanol is washed and obtain connecting product in the 30ul water, preparation method with the electric transformed competence colibacillus cell of Bio-Rad company prepares the competence e.colidh5, get after 2-3ul connects product and 50ul competence bacillus coli DH 5 alpha mixes, forward in the electric shock cup of 0.2cm of Bio-Rad company and shock by electricity, voltage is 2.5 kilovolts, time is 5.0 milliseconds-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, 37 ℃ of incubated overnight on the LB solid medium of X-Gal and IPTG 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, extract plasmid and cut the segmental size of evaluation insertion wherein with the EcoRI enzyme from each clone simultaneously, the white that obtains clone has constituted the O-antigen gene bunch library of Shigella bogdii 15 types;
(4) to the cloning and sequencing in the library: from the library, select insert 100 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 90% fraction of coverage.Residue 10% sequence is again according to the sequences Design primer that has obtained, and this primer is as follows: 5 '-GGAGCGATCGTCCGGTCAC-3 ' and 5 '-CAGCGCAGCTATGTGTCC-3 '; Direct PCR and from the genomic dna of Shigella bogdii 15 types again to the order-checking of PCR product, thus all sequences of O-antigen gene bunch obtained.
(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 (seeing sequence list) of the O-antigen gene bunch of Shigella bogdii 15 types.The quality of sequence is mainly guaranteed by two aspects: 1) genome of Shigella bogdii 15 types 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 15 type O-antigen genes bunch, with American National biotechnology information science center (TheNational Center for Biotechnology Information, NCBI) orffinder finds gene, find the reading frame of 8 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 15 types at last.
(6) screening of specific gene: at wzx, wzy, orf3, orf4, orf6, the orf7 gene design primer in the O-antigen gene of Shigella bogdii 15 types 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 shigella dysenteriae 2 types and intestinal bacteria O112, 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, orf3, orf4, orf6, orf7 gene pairs Shigella bogdii 15 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 15 types, its complete sequence shown in SEQ ID NO:1,10812 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 Shigella bogdii 15 types by method of the present invention, as shown in table 3, it is altogether by 8 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 15 types, promptly transports enzyme gene (wzx gene or the gene of identity function arranged with wzx); Pol gene (wzy gene or the gene of identity function arranged with wzy); Glycosyltransferase gene comprises orf3, orf4, orf6, orf7 gene, and their zero positions in O-antigen gene bunch and final position and nucleotide sequence all are listed among the figure two.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 be to Shigella bogdii 15 types, and the O-antigen of shigella dysenteriae 2 types and intestinal bacteria O112 is high special.
The 3rd aspect of the present invention, wzx gene in the O-antigen gene bunch that comes from Shigella bogdii 15 types 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 orf3, orf4, orf6, orf7 gene, they are any one section oligonucleotide in these genes.But, be that the oligonucleotide of listing in the table one is right preferentially by usefulness, also listed in Table 1 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 only with Shigella bogdii 15 types, shigella dysenteriae 2 types and intestinal bacteria O112 obtain expecting the product of size in the pcr amplification that carries out of template, and are all not obtain expecting the product of size in the pcr amplification that carries out of template all the other 165 strain intestinal bacteria listed with table two and 41 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.So promptly the listed oligonucleotide of table one is to Shigella bogdii 15 types can to determine these primers, shigella dysenteriae 2 types and intestinal bacteria O112 and their O-antigen are high specials.
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 1041 to 2432 bases from SEQ ID NO:1), wzy gene (nucleotide position is 2452 to 3630 bases from SEQ ID NO:1), orf3 gene (nucleotide position is 3630 to 4610 bases from SEQ ID NO:1), orf4 gene (nucleotide position is 4617 to 5798 bases from SEQ ID NO:1), orf6 gene (nucleotide position is 6766 to 7647 bases from SEQ ID NO:1), orf7 gene (nucleotide position is 7653 to 8393 bases from SEQ ID NO:1).Come from above intragenic oligonucleotide to Shigella bogdii 15 types (SEQ ID NO:1), shigella dysenteriae 2 types and intestinal bacteria O112 are high specials.
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 15 types, shigella dysenteriae 2 types or intestinal bacteria O112.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 by Shigella bogdii 15 types or shigella dysenteriae 2 types or intestinal bacteria O112 expression.
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 15 types or shigella dysenteriae 2 types or intestinal bacteria O112.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 Shigella bogdii 15 types or shigella dysenteriae 2 types or intestinal bacteria O112.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, 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 15 types first, and can from the sequence of this total length gene cluster of not cloned, produce recombinant molecule, can produce the O-antigen of expressing Shigella bogdii 15 types by inserting to express, and become useful vaccine.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should understand these embodiment only is used to the present invention is described and is not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, molecular cloning: the condition described in the laboratory manual (NewYork:Cold Spring Harbor Laboratory Press, 1989).Embodiment 1: genomic extraction: 37 ℃ of incubated overnight 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 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 and use isopyknic phenol again: chloroform: primary isoamyl alcohol extracting twice, get supernatant again with isopyknic ether extracting to remove remaining phenol.Supernatant rolls out DNA and washes DNA with 70% ethanol with glass yarn with 2 times of volume ethanol deposit D NA, 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 15 types bunch: the O-antigen gene of Shigella bogdii 15 types is bunch by the Long pcr amplification.At first according to the JumpStart sequences Design upstream primer (#1523-ATT GTG GCT GCA GGGATC 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, 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.
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, 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: the primary isoamyl alcohol 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 2.5ul 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 TthDNA polysaccharase 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 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, dispel thalline, in centrifugal 15 minutes of 4 ℃ of 4000rpm with the deionization aqua sterilisa 200ml of cold ice precooling.Deionization aqua sterilisa 100ml with cold ice precooling dispelled thalline again, in centrifugal 15 minutes of 4 ℃ of 4000rpm.With 10% glycerine suspension cell of cold ice precooling, centrifugal 10 minutes of 4 ℃ of 6000rpm abandon supernatant, precipitate 10% glycerine suspension cell with the precooling of 1ml ice at last, are competent cell.The competent cell that makes is packed as 50ul one pipe ,-70 ℃ of preservations.
Be electric transformed competence colibacillus cell at last: get after 2-3ul connects product and 50ul competence bacillus coli DH 5 alpha mixes, forward in the electric shock cup of 0.2cm of Bio-Rad company and shock by electricity, voltage is 2.5 kilovolts, and the time is 5.0 milliseconds-6.0ms 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, extract plasmid and cut the segmental size of evaluation insertion wherein with the EcoRI enzyme from each clone simultaneously, the white that obtains clone group has constituted the O-antigen gene bunch library of Shigella bogdii 15 types.
Embodiment 4: to the cloning and sequencing in the library:
From the library, select insert 100 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 90% fraction of coverage.Residue 10% sequence is again according to the sequences Design primer that has obtained, direct PCR and from the genomic dna of Shigella bogdii 15 types again to the order-checking of PCR product, thus obtain all sequences of O-antigen gene bunch.We have designed a pair of primer in Shigella bogdii 15 types, and are as follows: 5 '-GGAGCGATCGTCCGGTCAC-3 ' and 5 '-CAGCGCAGCTATGTGTCC-3 '.
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 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 15 types.The quality of sequence is mainly guaranteed by two aspects: 1) genome of Shigella bogdii 15 types 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 15 type O-antigen genes bunch, with American National biotechnology information science center (TheNational Center for Biotechnology Information, NCBI) orffinder finds gene, find the reading frame of 8 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 15 types at last, as shown in table 3.
By retrieving and comparing, the wzx gene of finding orf1 and Streptococcus agalactiae has 21% homogeny in 466 amino acid whose sequences, 45% similarity, also in 473 amino acid whose sequences, 19% homogeny is arranged in addition with the wzx gene of Streptococcus thermophilus, 43% similarity, show very high homology is arranged between them, and algorithm [Eisenberg by people such as Eisenberg, D, Schwarz, E.etal (1984) .Analysis of membrane andsurface protein sequences with the hydrophobic momentplot.J.Mol.Biol.179:125-142] find that orf1 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 orf1 is the wzx gene, called after wzx.The wzy gene of orf2 and Escherichia coli has 20% homogeny in 395 amino acid whose sequences, 42% similarity, show very high homology is arranged between them, learn that by the Eisenberg algorithm orf2 has 10 potential transmembrane domains in addition, to other O-antigen polysaccharase similar secondary structure is arranged, so determine that orf2 is the wzy gene, called after wzy.The N-acetylglucosamine transferase gene of orf3 and Streptococcus agalactiae homogeny of 26% in 333 amino acid whose sequences, 48% similarity, in addition also with the Eps7G gene of Streptococcus thermophilus 28% homogeny in 328 amino acid whose sequences, 47% similarity, Eps7G is a glycosyltransferase gene, so infer that orf3 is the gene of an encoding glycosyl transferring enzyme, called after orf3.The glycosyltransferase gene of orf4 and Streptococcus pneumoniae has 28% homogeny in 367 amino acid whose sequences, 47% similarity, also in 365 amino acid whose sequences, 26% homogeny is arranged in addition with the wlaE gene of Campylobacter jejuni, 43% similarity shows the homology that height is arranged between them.The wlaE gene is a glycosyltransferase gene, so, can infer that orf4 is a glycosyltransferase gene.The D-glucuronyl C5 epimerase gene of Orf5 and Bos taurus has 32% homogeny in 617 amino acid, 46% similarity, showing the homology that height is arranged between them, is a D-glucuronyl C5 dystopy enzyme gene so can infer orf5, called after orf5.The WbgQ gene of Orf6 and Shigella boydii has 44% homogeny in 287 aminoacid sequences, 60% similarity, in addition also with Bacteroides fragilis in the WcgB gene in 315 aminoacid sequences, 35% homogeny is arranged, 53% similarity, and WbgQ and WcgB gene all are glycosyltransferase genes, so infer that orf6 is the gene of an encoding glycosyl transferring enzyme, called after orf6.The glycosyltransferase gene of Orf7 and Escherichia coli O157:H7 has 39% homogeny in 260 aminoacid sequences, 62% similarity, in addition also with Oceanobacillus iheyensis in glycosyltransferase gene in 258 aminoacid sequences, 44% homogeny is arranged, 64% similarity, so infer that orf7 is the gene of an encoding glycosyl transferring enzyme, called after orf7.The gne gene of orf8 and Yersinia enterocolitica has 57% homogeny in 336 aminoacid sequences, 74% similarity, and showing has high homology between them, so determine that orf8 is the gne gene, called after wzy.
Embodiment 6: the screening of specific gene: at wzx, wzy, orf3, orf4, orf6, orf7 gene design primer in the O-antigen gene of Shigella bogdii 15 types 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 15 types.Glycosyltransferase gene, transhipment enzyme gene and pol gene and their function corresponding and the size of the O antigen gene bunch of Shigella bogdii 15 types 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 two 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 15 types in the 23rd group is as positive control.Do template with every group of bacterium, be PCR by following condition with every pair in the table one 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 one) 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 the 10ulPCR product and detect the fragment that amplifies by 0.8% agarose gel electrophoresis.
For wzx, wzy, orf3, orf4, orf6, orf7 gene, each gene all has three pairs of primers detected, every pair of primer has obtained also all obtaining onesize specificity band the correct band of expection size in the 14th group and the 24th group except be PCR in the 23rd group after.After being template PCR with the genomic dna of each bacterium in the 14th group, find only in intestinal bacteria O112, to have obtained positive findings.After being template PCR with the genomic dna of each bacterium in the 24th group, find only in shigella dysenteriae 2 types, 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 shigella dysenteriae 2 types and intestinal bacteria O112.It is reported, Shigella bogdii 15 types, the O-antigenic structure of shigella dysenteriae 2 types and intestinal bacteria O112 is the same, our result has confirmed this point from another side.In addition, all primers are any band that all do not increase in other groups, so wzx, wzy, orf3, orf4, orf6, orf7 gene pairs Shigella bogdii 15 types, shigella dysenteriae 2 types and intestinal bacteria O112 and O-antigen thereof all are high specials.
At last, from Shigella bogdii 15 types, screen to Shigella bogdii 15 types the gene of the O-antigen high special of shigella dysenteriae 2 types and intestinal bacteria O112 by PCR: wzx, wzy and four glycosyltransferase genes.And the oligonucleotide of these intragenic any one section 10-20nt is to Shigella bogdii 15 types, the O-antigen of shigella dysenteriae 2 types and intestinal bacteria O112 is special, especially the primer in above-mentioned each gene is that oligonucleotide confirms that to after detecting through PCR shigella dysenteriae 2 types and intestinal bacteria O112 are high specials to Shigella bogdii 15 types.These all oligonucleotide all can be used for Shigella bogdii 15 types in the human body and environment rapidly and accurately, shigella dysenteriae 2 types and intestinal bacteria O112, and can identify their O-antigen.
Table 3 is structural tables of the O-antigen gene bunch of Shigella bogdii 15 types, in table, listed the structure of the O-antigen gene bunch of Shigella bogdii 15 types, altogether by 8 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 15 types, in table, listed the accurate position of all open reading frame in complete sequence in the O-antigen gene bunch of Shigella bogdii 15 types, 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 Shigella bogdii 15 types, the Nucleotide of the O-antigen-specific of shigella dysenteriae 2 types and intestinal bacteria O112
＜130〉to Shigella bogdii 15 types, the Nucleotide of the O-antigen-specific of shigella dysenteriae 2 types and intestinal bacteria O112
<160>1
<170>PatentIn version 3.1
<210>1
<211>10812
<212>DNA
<213>Shigella boydii
<400>1
CTCCTGGTAA CTCATGCGTC CAAGAACGCG GTCGAAAACC ACTTCGACAC CTCTTATGAA 60
TTAGAATCTC TCCTTGAACA GCGCGTGAAG CGTCAACTGC TGGCGGAAGT ACAGTCTATC 120
TGTCCGCCGG GCGTGACCAT TATGAACGTG CGTCAGGGCG AACCTTTAGG TTTGGGCCAC 180
TCCATTTTGT GTGCACGACC CGCCATTGGT GACAACCCAT TTGTCGTGGT ACTGCCGGAC 240
GTTGTGATCG ACGACGCCAG TGCCGACCCG CTGCGCTACA ACCTTGCTGC CATGATTGCG 300
CGTTTCAATG AAACGGGCCG TAGCCAGGTG CTGGCAAAAC GTATGCCGGG TGACCTTTCT 360
GAATACTCCG TTATTCAGAC CAAAGAGCCG CTGGATCGTG AAGGCAAAGT CAGCCGCATT 420
GTTGAATTTA TCGAAAAACC GGATCAGCCG CAGACGCTGG ACTCAGACAT CATGGCCGTT 480
GGTCGCTATG TGCTTTCTGC CGATATTTGG CCGGAACTTG AACGCACTCA ACCTGGTGCA 540
TGGGGGCGTA TTCAGCTGAC TGATGCCATT GCTGAACTGG CGAAAAAACA GTCCGTTGAT 600
GCAATGCTGA TGACTGGTGA CAGCTACGAC TGCGGTAAAA AAATGGGCTA TATGCAGGCG 660
TTTGTGAAGT ATGGCTTACG CAACCTGAAA GAAGGGGCGA AGTTCCGTAA AGGTATTGAG 720
AAACTGTTAA GCGAATAATG AAAATCTGAC CGGATGTAAC GGTTGATAAG AAAATTATAA 780
CGGCGGTGAA GATTCCTGTG GAGAGTAATT TGTTGCGATT ACCACTGTCG TTATTCAACT 840
ATAAGCATAA AAAAGTTAAA TATAATTTGG GGATTTTAAG TATATGGAAA CGCCTTTGTC 900
TACTGAATAA GTAAGGTATT AACTGTAAGA AAAGCGTGAA TGGTGTTCCC TACATAATTT 960
AAAGATGTTC AGTCTACTGG TAGCTGTTAA GCCAGGGGCG GTAGCGTGGA TAATTTTGAA 1020
AAAAAAGATT AGAGCATAAA ATGAATTTAC TTAAAGGGAC AATAATATAT ACATTGTCAA 1080
ATTTGACAAT TAAATTTGGT GCAGTTTTAC TGCTTCCTAT ATTAACTCAT TTACTTAATC 1140
CAGAAGAGTA TGGGATGGTA GGATTGTATG TTACACTTAC TTCCTTTCTT ACAATAATTT 1200
TAGGGCTTGG TTTTTATACA CCCTTAATGA AAACTCAGTC GGAGCAGAAA AGCAATATTG 1260
CAACTTCTAG CCATGTGAAT TTTTGTGCAA TAATATTTCT TATCGCAGTT TATTTATTAT 1320
TGCTTTTATT TCTTTACTTT ATATTGAAGT CGTCTTTAGT TATTGATTTG TTGGGAGAGG 1380
TGAAACTAGA TAGCAAATTA ATTTATTTGG CAGTTATTGT ATCAATGTTT TCTGCAATTA 1440
ATATTATTAT GAACACGTCG TTTAGAATGG ACGAGAATTA TATATTAGTT GCAATATTGT 1500
CAATTTTATC TTTTGTGTTA TTTTATTCCT CTGCTATAAT ATTCATTAAA CAGTTCAATT 1560
ATGGAGGCCT TGGCTATATA TATGGCAATT TATTAAGTTC AATAATCATT TTTTTTATAA 1620
GTTTCACTTG TTACTATAGA AAATTATCTA TTGCATTTTC ATGGCACAAC GTGAAATTTT 1680
TGTGTGGGAA TGGAATTCCA ATGGTTTTTG TAGAGTTATC GGATAAAATA ATTGAAGCCA 1740
GCGATAGATT TATTTTAGTC AGATATATTT CACTATCCTC TTTAGGCGTA TATACACTAG 1800
CACTAACAGG ATGTAAAGTA TTAAACGTAG TTTTCAATTC TTATATTAGC GCTATCCTTC 1860
CCTCTATCTA TAAAAGCGTT GAAAGCCGTG AAGATTCAAA TTATATAAAA GTTAAGCTTG 1920
AGAATGCATA CGTGCTTGTA GTAATGATGG TTTTTTTGGG ACAGTTGATA TCAAAAGAAA 1980
TAATTGACAT AATTTTTCCT GCATCATATT CAAATTTATT TTTTGTATTT ATATTAGCTT 2040
TGCCTGCAAT ATCTCTACAA TATTATTATT TTTTAGATTT CTATTTTCAT AGAAGTGAAG 2100
ATAGCCGATT TATTTTATGT TTTACAATAG CAACCTCGAT AATAAATATT ATTCTAAACT 2160
TAATTTTTGT TCCCAAATAT GGAGTTTATG CATCTCTTTT ATCAACGTAT ATATCCTATT 2220
TAATCAGAAC GCTAGTAGAA ATAAAATTTA TAGCGAGACG GTATAAATTG AAGTTTAGTG 2280
TGCTTAAAGT CCTTATGGGA AGCTTTGTTC TGTATACTGT TCCTTTATTA TATCATTATG 2340
GTTATTATAC AGTGTTGTTG AGAGTAACTT TGTCAATTGT TTTATTTATA GGTATTATTA 2400
TGTTTTATAA AAAAAGTAAG ATATTCAATT AAAAGTGAGT GCTAAGTGAT CATGGTTAAT 2460
GCTAAAAATT TTGTATTAAT TATTTTTGAT GTAATATGTC TTTTTCTTAA TGTTTTTGTT 2520
TATGTCCCAC GTCTAATTAT TGGTGTTTTA ACAGTAGATA AAACATTGAT TATTACATTT 2580
CCCTTGATGT TATCAGTGTT GGCATATAAT TTTATTCCAC CAGAAACATA TGATCTTTAC 2640
AGACATTATG AAAACTATCA AAATTTTTTG AATAATAATG AAGTAAGTTT TATCAGGGAT 2700
TTTTTTCTTT ATTTTTTATT TACCGTAGGG AGGCTTTTTG ATTTAAATAA TGGATTTATA 2760
GCATTTGTAT CTTGTTATAT CCTCTATTTT TATTGGTTAA AAATATTATA CATTAACTTT 2820
GATTATAAAG CCAACAAGAA AAATTTCCCG TTATACTTTT TTATATTTTT TATTACTCCA 2880
CCTCTTGTTA TATATACAGG CGTTCGATTT TCCACAGGGC TTATATTAAC ACTATATGGG 2940
ATTGTAAACT GGATTCAATT ACGGAATGTA AAACTTGGTA TAGTATATAT TATTTGCGGG 3000
TTAACAGCCC ATTTTTCAAT GGCATTGATT GCTTTAATAT TTATTTTCAC ATCATATTTC 3060
AATAATATCT TTTCAAATAA ATATTTAAGG GTTGGGCTCG TATTTTCTGC ATTACTTGTA 3120
ATCTCATTTA ATGAACAGTT AGTAAGGTTT TTGACAACAG TAATTGAGTT CATTAATCAG 3180
TTATTTGATT GGAACTTTAT TAGTATCGAT ACATATATTA CTGGTAAATG GGGGCTGGAG 3240
AGGGCGCAGG AATTGAATTC AACCGGACAA TTAGTATTAG GTATACGTAA TTATGGTATG 3300
CTCTTTCTAA TATTGCTATT TTGCCCACTA ATATCTTTTA ATAAAAATAA ATATACGAAC 3360
TTTTATTATG TAATGTGTGC TGTTACAATA TTAGTTTTCC CTTTTACAGC AGTATTTGAT 3420
AGATATGGAA CATTTTTAAT TTTTTTTATT GTATACTATC TTAGTACTCT TCAGAAAAAA 3480
ACTAACATTG AAATAATATA TTTTTTAATG TTGATTGTAT ATTTGATTTT CTTTAGAACA 3540
ACAGACATCA AAGATAATAT TTCCGTATAC ACTGAAAGCT ATAGTAATAT ATTGAATCTT 3600
TCTTTAATTA AAGTACTGTT AGGAATATAA TGCGTAAATA TGAAAATAAC ATTGTTATAA 3660
GTGTGATTAT TCCAACATAT AACAATGATA AAACAATCAT AGAGGCTATT CAATCTATAG 3720
TAAATGTTGA GATGCCTGTG AAATATGAGG TACTAGTAAT AGACGATGGT TCGGTAGATC 3780
AGACATCAAA TTTAATTGCA GAATATATAA GTCAAAGAAA AGATAATACT ATTAAATATT 3840
TTTATCAAAA AAACAGTGGT GTAAGCGCTG CGAGAAACGT GGGAATAAAA AATGCATTAG 3900
GGAAATATGT TCTATTTTTA GACGCAGATG ATACATATAG TGAACATGTG TTAAAAGAAA 3960
TAGATGAAAT ATTAAGTACT AACAATGATA TATATGTGTT TTCATATGAA AAACTGAATA 4020
ATAAAAAGGC TATAATATCA AATAAAAAAA ATATTATAAC TCATTATAAT AATAAATATG 4080
ATTTTTTAAA CGCCTATATC TCAGGTGAAC TTTTCGGGTG CGTTAGTGTT TGCTCTTGCA 4140
TTTTTAGTAA AGAATCATTA ATTAGCAATA ATATTTTTTA CGATAGTAAA ATTGCTTATG 4200
GGGAAGATCA ATGGTTTTTG ATAAATGCTC TCAAGGTTTG CAATGATGTA GAGTTCAGAT 4260
TAGAACTAAA TTTACTGAAT TATACTTATA ATACGGAGTC GGCGACTAAT CGTTTCAACC 4320
TAAGACGTTT TGACGCGATT ACAATGCTAG AAAGAATCAA TCAAGAAGCA TTTAAGGTTA 4380
ATCAAGATAT AATTAATGCA AGAATTAATA AAGAATTGGT AAATATAGCA ACAATATATA 4440
CGAAATTCAA TTCGTTACAG AAATCAATAA TGTTTGTTAA TAACGTTATA CTCCCAAAAA 4500
TAAAAACGGA TAGTGACAAA AGACTATATA TGTTCAAAGA TATGGTTTTC ACTAGATTAC 4560
CATGTTTATA TATAATACTT TATAAACTCT ATATTCAGCT ATTTAAGTAA ACTAATATGA 4620
ATATTCTTAT AATTATTCCT AAGTTATGTA ACGGTGGGAT TGAAAAGATT GCATCAAACT 4680
TTTCTCAATA CTTACCAGAA AGATATAATC AATTTGTATA TTCAATCATG TCACAAGACT 4740
CTTCTTATGA TTTTGCGGTA AAGCCCATTA TATTAAATAA AAGTTTAGGT AAAACCTTTA 4800
TTGGAAAATT GATTACATTT TTCTACAGGC TTCATTTGGC ACGTAAAATT ATTAGAGAAA 4860
ATAACATAGA TATATGCATT AGTTTTGGGG AGAGATGTAA TATTATAAAT ATTCTATCTA 4920
TGGGGAAAAC AAAAAAAATC ATTACTATAC ATAGTCAGCT ATCTATTGAA AATAAAACGA 4980
AAGGATTATA TGGAAAAGTA ACAACACTAT TTTCAAAATT GCTTTATAAA AATGCGGATG 5040
CTACTGTAGC CGTATCAGAA ATAGTGAAGA AGGATGCATG CGGTTTATTA AATTTAGATG 5100
CGAATAATGT AGAGATAATT TATAATGGAC ACGATATTGG TTATATCAAA GATAAGTCTA 5160
CTGATTATAA AGAATTTGAT ACACCTGTGA TCGACTTCGT GTCTGTAGGG CGAATTACAT 5220
ATGCTAAAGG GCATTATCAT CTATTGAGAA GTCCTGCCAT AGTAAAGGAG ACGTATCCTA 5280
ATGTAATACT ATATATTGTT GGCACGTATG AAAAAGATAA TTTAAAAAGC ATAATTGATC 5340
ATTTAATTGA GAAATACGAT TTATATGATA ATGTGATTTT TACCGGCTTT AGTGACAATC 5400
CATATCCTTA CATAAAATCA GCAAAGGCTC TTATACTTAG TTCAATTTTT GAAGGTTTTC 5460
CAGGCGTTGT TATCGAGAGT ATTGCGCTTG GAACTCCTGT CATAGCGACA GATTGTGGCG 5520
GTGCTTCAGA AGTTTTAAGA AGTCCAGATG CCAAAATAAA GAATAATACG GGAGATGTGG 5580
AAATTACTAA ATTAGGAGTA CTGTTACCAA AATTCGATGG GATTTATGAT ATTAATTTGG 5640
ATTTTTCGCC CACAGAAAAA AAGATGGCGC AAGTTATGAA TGATATTATA TCAGGGAAGC 5700
TGAAATTTAA TGAAGACTTT TTAATTACAA AAGCAAACGA ATATGATATA GATTCTATGA 5760
TGAAAAAATA TACGGACTTA ATAGAGAAAA TAAAATAATG AAAGAAGCTA CTACCTCATA 5820
TAAATCAATT CATACTATAT TAGAATATTT TGGTTGGTCT CATGAAGATT ATTGGCATAT 5880
CAACTATAAA GTAAATTATG ATTTTTCAAA ATTTAATTAT TATTGGGATT TGACAAAAAA 5940
GTATAATTGG TTTAGTGGAC CTTTTGATCA AAATAATATT CCTTTATATC TTGGCACTGA 6000
TTCAAAACTG CATTACTCAC CAATTTTTTT AGGGCATTAT GCTCTTGGAG CATATCAAGA 6060
ATATTTATTA TCAAATGATA AAAAAGCATA TGACGATTTT ATAAGAATAG CGGATTGGCT 6120
GGTTGAAAAT ACAACGACAT ATAAAAAATG TTCTGGAATA TGGATAAATA CATACCCAAT 6180
GTCAACTTTT AACTTAAAAG GTAAATGGCA ATCGTGTTTA GCCCAGGCAA AAGGTATATC 6240
TGTCCTATGT AGAGCTTATT ATCTTACGCA AAATTTAAAA TATTTGAAGG TAGCTCTTGA 6300
CGCATTTGAA TCTTTCAAAA TATATAAAGA AGAAGGTGGA GTTAAGGTAA GGCATAAAAA 6360
CGTATTCTTT TGGGAGGAAT ATCCAACTAT AACCAATTCT ATCGTATTGA ATGGACATAT 6420
TTTTGCAGTT TGGGCATTGT ACGATTTATC TTTTGTATTA AAAGATAAGA ACGAGTTTAA 6480
GCAACAATGG GCGGATATTT ACAGTTATTA TAAAACATCA CTGATTATAT TAAAAGAGAA 6540
TTATAAGTTA TGGGATACGC ACTATTGGAC TAGATATGAT ATATGGGATA AACATTTTAA 6600
TGTTTCAAGT TTGTTTTATC ATAATCTCCA TATTAAACAA TTCTATATAT TGGCTGTTTT 6660
GGGAGATGAT GATGATTTCA AGAAAATTGC TAATAAATGG GCTGGTAAAT TAAAAAATCC 6720
GCTTTTCAGA ATGTATGCGC TTATAAAGAA AGTAGTTTTT AGGTTATGAT TATGAATGTT 6780
ATTGATAAAA TTATTATGGG TTTGAAAAGT ATATATTTAA GCTTTCTTTA TACAATATAT 6840
AATTTATTTC TTAATGAAAA TAGATTCGAA GAGACAAATG AAGTACATGT TGTTGTAAGT 6900
TTGACGTGTT TTCCTGGACG TATAAAAAAA GTATATCTTA CTTTAGAGTC AATTATGGCT 6960
CAACAATATA ATAACTTTAA AGTGGTATTG TACTTGTCTC ATGAAGAGTT TCCAAAGGGT 7020
TTAGAGGATT TACCTCGTAG TTTAATACGA TTACATAAAA GAGGTGTCGA TATTAATTTT 7080
ACGTGTGAAA ATATTCGATC TTATAAGAAG TTACATTATG CGCTGAGTGA TTTCCCAGAA 7140
CTACCTGTAA TAACAGCTGA TGATGATGTC TTATATCCCT CGCGATGGGT AAATGATTTT 7200
ATGGAGTCAC ATAAGCTTTT TCATGATGAT ATATTATTTG CGAGGGGGCA TCAGATAACA 7260
TTTGACCGAA ATGGAAATGT TAAAAAATAT ATCTCTTTCG GAAAACCAGC TGGATATTCA 7320
GCATCAAGTC TATATATTCC TACAGGAGTA AGTGGAATTC TATATCCGCC AGGATGTTTT 7380
TTTCAAGATG TGCAGAATAA AGATATTTTT ATGAAGTTGG CACCTAACGC AGATGATATA 7440
TGGTATAAAG TAATGACTTT GTTAAACGGT CGAAAATCCA GATTGATCTA TAAAAAGCCA 7500
ATTCATTATC CACCTATCCT TGGCACGCAG AAGATCTCCT TGCGTAAACA AAACTTATCG 7560
AAACAACATT TAAATAATGA TACACAATTA TTAAATTTAA TTGATTATTA TTCTATCGAA 7620
TTAGCTGATT TTAAGAAAAA GGAATAGCTC CTATGGAATT AGTTTCAATT ATAATGCCTT 7680
GTTTTAATAA TGAAGCAACT ATAAAAGAAA GTATTAATAG CGTACTATCT CAAAGTTATA 7740
AATACTGGGA GTTAATTATT ATCAATGACA ACTCAACAGA TGATTCACAA AATATTATTA 7800
ACGACTATGT GAATAGTGAT GAAAGAATAA AACTGATTAC TAATGAAAAA AATATCGGAG 7860
CTGGAGGTAG TAGAAATAAA GGAATTGAAA TTGCAAATGG AAGATTCATT GCTTTTTTAG 7920
ATGCAGATGA TGTTTGGTTG CCAGAAAAAC TTGCAAAACA AATAAAGTTT ATGAGAGATG 7980
GTAATTATGC ACTGACATAT AGTTATTATC AAAAGTTTGA TTCAACTGGA TTGGGAGCGA 8040
TCGTCCGGTC ACCTGCGACA ACAACTTATA GGAAATTGTT ATACAGTAAT GTTATTGGAT 8100
GTCTAACAGG AATGTATGAT ACTGATTTTT TGGGTAAATG CTATATGCCT TTAATTCGTA 8160
AACGACAAGA TATGGGGCTT TGGTTGTCAA TATTGAAAAA GTGTAATAAA GCATATTGCT 8220
TACCTGAAGT TTTGGCATAT TATAGAATTG ACTCAGGAAT GACAAAAAAT AAACTTGATG 8280
CGGCAAAATA TCAGTGGCGA TTTTATAGGG ATGTAACTAA ACTTAATTTG ATTCAATCAA 8340
GTTGGTATTT TGTTTGGTAT TCAGTTTTAG GTTTGGTAAA GTACAAAAAA TAATAAGGAG 8400
GAGTGATGAC AATTTTAGTC ACTGGTGGTG CTGGATATAT TGGCAGTCAC ACTGTTATTG 8460
AATTACAGAA AAATGGTTTT GACGTGGTAA TAGTAGATAA TTTTTGTAAT TCATCGCCAT 8520
CGGTTTTAAA AAGAATTGAA TTAATTACAG GAAAGGAAAT TAAATGTTAC GATATTGATC 8580
TTCTGGATTT TAATTCTTTG CAGAAGGTTT TTGTAACTAA TAATATTAAC TCTGTGATTC 8640
ATTTTGCATC TTTAAAATCA GTTTCTGAAT CACTAATCAA ACCAACTGAA TATTATCATA 8700
ATAATTTGAG TGGTTGCCTC AATGTCTTGA AAATAATGAA AGAACATAAC GTGAATAATT 8760
TCATTTTCAG TTCTTCGGCA ACTGTATATG GCAGTAATAA CAACAATCCT GTTTCAGAAT 8820
CTTCATCAGT CGGCACCGCA ACAAATCCTT ATGGAAAATC CAAGATTTTT TTAGAGCAAA 8880
TATTGTCTGA TTGCTGTAAA TCAAATCCTA ATTTAAATGT AACTTGCCTG AGGTATTTCA 8940
ATCCGGTTGG TGCACATTCT TCAGGTTTGA TAGGAGAATC TCCGAATGGT GTTCCTGCCA 9000
ATTTAGTTCC ATATTTAACT CAGGTTGCTT TAGGTAAATT GCCCGTATTA CATATATATG 9060
GTAATGACTA TGATACCAAG GATGGGACGG GAGTAAGAGA TTTTATTCAT GTTACGGATT 9120
TAGCTAATGG ACACATAGCT GCGCTGAAAA AAATAAATGG TCATAATGGT TTTAAAGTTT 9180
ATAATCTTGG TACTGGGCGA GGTTATAGTG TGTTAGAAGT CGTTAGATGT TTTGAGGCTA 9240
TTACAGGAAA AAGTATTCCA ATCGAATTTT CGCCTCGCAG AGAAGGTGAT ATCGCTGAAA 9300
GTTGGGCAAA TGTTTCCGCA GCAAATTATG AGTTAGAGTG GTTCGCTAAA AAGACATTAA 9360
CTGATATGTT ACGAGATGCA TGGAAATGGC AAACGTTAAA TCCAAATGGA TTATAAAGAT 9420
ATTTAATTTA TATTAAACCT TGCTTATTTA AGATAATGTA AACCGCACCT TTGCGGTAAC 9480
CACCCCTGAC AGGAGTAAAC AATGTCAAAG CAACAGATCG GCGTCGTCGG TATGGCAGTG 9540
ATGGGGCGCA ACCTTGCGCT CAACATCGAA AGCCGTGGTT ATACCGTCTC TATTTTCAAC 9600
CGTTCCCGTG AAAAGACCGA AGAAGTGATT GCCGAAAATC CAGGCAAGAA ACTGGTTCCT 9660
TACTATACGG TGAAAGAGTT TGTTGAATCT TTGGAAACGC CTCGTCGCAT CCTGTTAATG 9720
GTGAAAGCAG GTGCAGGCAC GGATGCTGCT ATTGATTCCC TTAAGCCATA TCTCGATAAA 9780
GGCGACATCA TCATTGATGG CGGTAACACC TTCTTCCAGG ACACCATTCG TCGTAACCGT 9840
GAGCTTTCTG CCGAAGGCTT TAACTTCATT GGTACCGGTG TCTCCGGTGG TGAAGAAGGC 9900
GCGCTGAAAG GTCCTTCCAT TATGCCTGGT GGGCAGAAAG AAGCCTATGA ACTGGTTGCT 9960
CCGATCCTGA CCAAAATCGC TGCAGTGGCT GAAGACGGCG AGCCATGCGT TACCTATATT 10020
GGTGCCGATG GTGCAGGTCA CTATGTGAAG ATGGTTCACA ACGGTATTGA ATACGGCGAT 10080
ATGCAGCTGA TTGCCGAAGC CTATTCTCTG CTAAAAGGTG GCCTGAACCT TACCAACGAA 10140
GAATTGGCAC AGACCTTTAC CGAATGGAAT AACGGTGAAC TGAGCAGCTA CCTGATCGAC 10200
ATTACCAAAG ACATCTTCAC TAAAAAAGAT GAAGACGGTA ACTACCTGGT TGATGTGATC 10260
CTGGATGAAG CGGCAAACAA AGGTACGGGC AAATGGACCA GCCAGAGCGC ACTGGATCTC 10320
GGCGAACCGC TGTCGCTGAT TACCGAGTCT GTGTTTGCAC GTTATATCTC TTCTCTGAAA 10380
GATCAGCGCG TTGCCGCTTC TAAAGTTCTC TCTGGCCCGC AAGCGCAGCC AGCAGGCGAC 10440
AAGGCTGAGT TCATCGAAAA AGTTCGTCGT GCGCTGTATC TGGGCAAAAT CGTTTCTTAC 10500
GCTCAGGGCT TCTCTCAGTT GCGTGCTGCG TCTGAAGAAT ACAACTGGGA TCTGAACTAC 10560
GGCGAAATCG CGAAGATTTT CCGTGCTGGC TGCATTATCC GTGCGCAGTT CCTGCAGAAA 10620
ATCACCGATG CCTATGCCGA AAATCCGCAG ATCGCTAACC TGCTGCTGGC TCCGTACTTC 10680
AAGCAAATTG CCGATGACTA TCAGCAGGCG CTGCGTGATG TCGTTGCTTA TGCAGTACAG 10740
AACGGTATCC CGGTTCCGAC CTTCGCCGCT GCGGTTGCCT ATTATGACAG CTACCGTGCC 10800
GCTGTTCTGC CT 10812
Glycosyltransferase gene in the table one Shigella bogdii 15 type O antigen genes 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 | 1041-2432 | #401(1148-1165) | #402(1856-1873) | 726bp | 0 * | 50 |
| #403(1233-1250) | #404(1859-1866) | 634bp | 0 * | 50 | |||
| #405(1460-1477) | #406(1861-1878) | 418bp | 0 * | 50 | |||
| wzy | Polysaccharase | 2452-3630 | #407(2515-2533) | #408(3087-3105) | 591bp | 0 * | 50 |
| #409(2935-2952) | #410(3392-3411) | 477bp | 0 * | 53 | |||
| #411(2718-2734) | #412(3536-3553) | 836bp | 0 * | 53 | |||
| Orf3 | Glycosyltransferase | 3630-4610 | #413(3723-3740) | #414(4121-4138) | 416bp | 0 * | 50 |
| #415(4103-4121) | #416(4506-4525) | 423bp | 0 * | 54 | |||
| #417(3856-3874) | #418(4548-4564) | 709bp | 0 * | 53 | |||
| Orf4 | Glycosyltransferase | 4617-5798 | #419(4644-4661) | #420(5246-5263) | 620bp | 0 * | 50 |
| #421(4649-4667) | #422(5452-5471) | 823bp | 0 * | 54 | |||
| #423(5255-5272) | #424(5661-5680) | 426bp | 0 * | 50 | |||
| Orf6 | Glycosyltransferase | 6766-7647 | #425(6861-6880) | #426(7309-7328) | 468bp | 0 * | 52 |
| #427(6995-7013) | #428(7413-7430) | 436bp | 0 * | 54 | |||
| #429(7141-7159) | #430(7613-7630) | 490bp | 0 * | 54 | |||
| Orf7 | Glycosyltransferase | 7653-8393 | #431(7731-7747) | #432(8096-8113) | 383bp | 0 * | 55 |
| #433(7855-7872) | #434(8222-8240) | 386bp | 0 * | 59 | |||
| #435(7919-7937) | #436(8341-8360) | 442bp | 0 * | 55 |
0*: the band that only in Shigella bogdii 15 types, shigella dysenteriae 2 types and intestinal bacteria O112, obtains correct size
Table two 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, O4, 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, O149, 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 1, B3, 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 See d
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 structural tables of the O-antigen gene bunch of Shigella bogdii 15 types
Table 4 is location tables of the gene in the O-antigen gene bunch of Shigella bogdii 15 types
CTCCTGGTAA CTCATGCGTC CAAGAACGCG GTCGAAAACC ACTTCGACAC CTCTTATGAA 60
TTAGAATCTC TCCTTGAACA GCGCGTGAAG CGTCAACTGC TGGCGGAAGT ACAGTCTATC 120
TGTCCGCCGG GCGTGACCAT TATGAACGTG CGTCAGGGCG AACCTTTAGG TTTGGGCCAC 180
TCCATTTTGT GTGCACGACC CGCCATTGGT GACAACCCAT TTGTCGTGGT ACTGCCGGAC 240
GTTGTGATCG ACGACGCCAG TGCCGACCCG CTGCGCTACA ACCTTGCTGC CATGATTGCG 300
CGTTTCAATG AAACGGGCCG TAGCCAGGTG CTGGCAAAAC GTATGCCGGG TGACCTTTCT 360
GAATACTCCG TTATTCAGAC CAAAGAGCCG CTGGATCGTG AAGGCAAAGT CAGCCGCATT 420
GTTGAATTTA TCGAAAAACC GGATCAGCCG CAGACGCTGG ACTCAGACAT CATGGCCGTT 480
GGTCGCTATG TGCTTTCTGC CGATATTTGG CCGGAACTTG AACGCACTCA ACCTGGTGCA 540
TGGGGGCGTA TTCAGCTGAC TGATGCCATT GCTGAACTGG CGAAAAAACA GTCCGTTGAT 600
GCAATGCTGA TGACTGGTGA CAGCTACGAC TGCGGTAAAA AAATGGGCTA TATGCAGGCG 660
TTTGTGAAGT ATGGCTTACG CAACCTGAAA GAAGGGGCGA AGTTCCGTAA AGGTATTGAG 720
AAACTGTTAA GCGAATAATG AAAATCTGAC CGGATGTAAC GGTTGATAAG AAAATTATAA 780
CGGCGGTGAA GATTCCTGTG GAGAGTAATT TGTTGCGATT ACCACTGTCG TTATTCAACT 840
ATAAGCATAA AAAAGTTAAA TATAATTTGG GGATTTTAAG TATATGGAAA CGCCTTTGTC 900
TACTGAATAA GTAAGGTATT AACTGTAAGA AAAGCGTGAA TGGTGTTCCC TACATAATTT 960
AAAGATGTTC AGTCTACTGG TAGCTGTTAA GCCAGGGGCG GTAGCGTGGA TAATTTTGAA 1020
Orf1's is initial
AAAAAAGATT AGAGCATAAA
ATGAATTTAC TTAAAGGGAC AATAATATAT ACATTGTCAA 1080
ATTTGACAAT TAAATTTGGT GCAGTTTTAC TGCTTCCTAT ATTAACTCAT TTACTTAATC 1140
CAGAAGAGTA TGGGATGGTA GGATTGTATG TTACACTTAC TTCCTTTCTT ACAATAATTT 1200
TAGGGCTTGG TTTTTATACA CCCTTAATGA AAACTCAGTC GGAGCAGAAA AGCAATATTG 1260
CAACTTCTAG CCATGTGAAT TTTTGTGCAA TAATATTTCT TATCGCAGTT TATTTATTAT 1320
TGCTTTTATT TCTTTACTTT ATATTGAAGT CGTCTTTAGT TATTGATTTG TTGGGAGAGG 1380
TGAAACTAGA TAGCAAATTA ATTTATTTGG CAGTTATTGT ATCAATGTTT TCTGCAATTA 1440
ATATTATTAT GAACACGTCG TTTAGAATGG ACGAGAATTA TATATTAGTT GCAATATTGT 1500
CAATTTTATC TTTTGTGTTA TTTTATTCCT CTGCTATAAT ATTCATTAAA CAGTTCAATT 1560
ATGGAGGCCT TGGCTATATA TATGGCAATT TATTAAGTTC AATAATCATT TTTTTTATAA 1620
GTTTCACTTG TTACTATAGA AAATTATCTA TTGCATTTTC ATGGCACAAC GTGAAATTTT 1680
TGTGTGGGAA TGGAATTCCA ATGGTTTTTG TAGAGTTATC GGATAAAATA ATTGAAGCCA 1740
GCGATAGATT TATTTTAGTC AGATATATTT CACTATCCTC TTTAGGCGTA TATACACTAG 1800
CACTAACAGG ATGTAAAGTA TTAAACGTAG TTTTCAATTC TTATATTAGC GCTATCCTTC 1860
CCTCTATCTA TAAAAGCGTT GAAAGCCGTG AAGATTCAAA TTATATAAAA GTTAAGCTTG 1920
AGAATGCATA CGTGCTTGTA GTAATGATGG TTTTTTTGGG ACAGTTGATA TCAAAAGAAA 1980
TAATTGACAT AATTTTTCCT GCATCATATT CAAATTTATT TTTTGTATTT ATATTAGCTT 2040
TGCCTGCAAT ATCTCTACAA TATTATTATT TTTTAGATTT CTATTTTCAT AGAAGTGAAG 2100
ATAGCCGATT TATTTTATGT TTTACAATAG CAACCTCGAT AATAAATATT ATTCTAAACT 2160
TAATTTTTGT TCCCAAATAT GGAGTTTATG CATCTCTTTT ATCAACGTAT ATATCCTATT 2220
TAATCAGAAC GCTAGTAGAA ATAAAATTTA TAGCGAGACG GTATAAATTG AAGTTTAGTG 2280
TGCTTAAAGT CCTTATGGGA AGCTTTGTTC TGTATACTGT TCCTTTATTA TATCATTATG 2340
GTTATTATAC AGTGTTGTTG AGAGTAACTT TGTCAATTGT TTTATTTATA GGTATTATTA 2400
The termination of Orf1
TGTTTTATAA AAAAAGTAAG ATATTCAAT
T AAAAGTGAGT GCTAAGTGAT CATGGTTAAT 2460
Orf2's is initial
GCTAAAAATT TTGTATTAAT TATTTTTGAT GTAATATGTC TTTTTCTTA
A TGTTTTTGTT 2520
TATGTCCCAC GTCTAATTAT TGGTGTTTTA ACAGTAGATA AAACATTGAT TATTACATTT 2580
CCCTTGATGT TATCAGTGTT GGCATATAAT TTTATTCCAC CAGAAACATA TGATCTTTAC 2640
AGACATTATG AAAACTATCA AAATTTTTTG AATAATAATG AAGTAAGTTT TATCAGGGAT 2700
TTTTTTCTTT ATTTTTTATT TACCGTAGGG AGGCTTTTTG ATTTAAATAA TGGATTTATA 2760
GCATTTGTAT CTTGTTATAT CCTCTATTTT TATTGGTTAA AAATATTATA CATTAACTTT 2820
GATTATAAAG CCAACAAGAA AAATTTCCCG TTATACTTTT TTATATTTTT TATTACTCCA 2880
CCTCTTGTTA TATATACAGG CGTTCGATTT TCCACAGGGC TTATATTAAC ACTATATGGG 2940
ATTGTAAACT GGATTCAATT ACGGAATGTA AAACTTGGTA TAGTATATAT TATTTGCGGG 3000
TTAACAGCCC ATTTTTCAAT GGCATTGATT GCTTTAATAT TTATTTTCAC ATCATATTTC 3060
AATAATATCT TTTCAAATAA ATATTTAAGG GTTGGGCTCG TATTTTCTGC ATTACTTGTA 3120
ATCTCATTTA ATGAACAGTT AGTAAGGTTT TTGACAACAG TAATTGAGTT CATTAATCAG 3180
TTATTTGATT GGAACTTTAT TAGTATCGAT ACATATATTA CTGGTAAATG GGGGCTGGAG 3240
AGGGCGCAGG AATTGAATTC AACCGGACAA TTAGTATTAG GTATACGTAA TTATGGTATG 3300
CTCTTTCTAA TATTGCTATT TTGCCCACTA ATATCTTTTA ATAAAAATAA ATATACGAAC 3360
TTTTATTATG TAATGTGTGC TGTTACAATA TTAGTTTTCC CTTTTACAGC AGTATTTGAT 3420
AGATATGGAA CATTTTTAAT TTTTTTTATT GTATACTATC TTAGTACTCT TCAGAAAAAA 3480
ACTAACATTG AAATAATATA TTTTTTAATG TTGATTGTAT ATTTGATTTT CTTTAGAACA 3540
ACAGACATCA AAGATAATAT TTCCGTATAC ACTGAAAGCT ATAGTAATAT ATTGAATCTT 3600
The termination of Orf2, Orf3's is initial
TCTTTAATTA AAGTACTGTT AGGAATA
TAA TGCGTAAATA TGAAAATAAC ATTGTTATAA 3660
GTGTGATTAT TCCAACATAT AACAATGATA AAACAATCAT AGAGGCTATT CAATCTATAG 3720
TAAATGTTGA GATGCCTGTG AAATATGAGG TACTAGTAAT AGACGATGGT TCGGTAGATC 3780
AGACATCAAA TTTAATTGCA GAATATATAA GTCAAAGAAA AGATAATACT ATTAAATATT 3840
TTTATCAAAA AAACAGTGGT GTAAGCGCTG CGAGAAACGT GGGAATAAAA AATGCATTAG 3900
GGAAATATGT TCTATTTTTA GACGCAGATG ATACATATAG TGAACATGTG TTAAAAGAAA 3960
TAGATGAAAT ATTAAGTACT AACAATGATA TATATGTGTT TTCATATGAA AAACTGAATA 4020
ATAAAAAGGC TATAATATCA AATAAAAAAA ATATTATAAC TCATTATAAT AATAAATATG 4080
ATTTTTTAAA CGCCTATATC TCAGGTGAAC TTTTCGGGTG CGTTAGTGTT TGCTCTTGCA 4140
TTTTTAGTAA AGAATCATTA ATTAGCAATA ATATTTTTTA CGATAGTAAA ATTGCTTATG 4200
GGGAAGATCA ATGGTTTTTG ATAAATGCTC TCAAGGTTTG CAATGATGTA GAGTTCAGAT 4260
TAGAACTAAA TTTACTGAAT TATACTTATA ATACGGAGTC GGCGACTAAT CGTTTCAACC 4320
TAAGACGTTT TGACGCGATT ACAATGCTAG AAAGAATCAA TCAAGAAGCA TTTAAGGTTA 4380
ATCAAGATAT AATTAATGCA AGAATTAATA AAGAATTGGT AAATATAGCA ACAATATATA 4440
CGAAATTCAA TTCGTTACAG AAATCAATAA TGTTTGTTAA TAACGTTATA CTCCCAAAAA 4500
TAAAAACGGA TAGTGACAAA AGACTATATA TGTTCAAAGA TATGGTTTTC ACTAGATTAC 4560
The termination Orf4's of Orf3 is initial
CATGTTTATA TATAATACTT TATAAACTCT ATATTCAGCT ATTTAAG
TAA ACTAAT
ATGA 4620
ATATTCTTAT AATTATTCCT AAGTTATGTA ACGGTGGGAT TGAAAAGATT GCATCAAACT 4680
TTTCTCAATA CTTACCAGAA AGATATAATC AATTTGTATA TTCAATCATG TCACAAGACT 4740
CTTCTTATGA TTTTGCGGTA AAGCCCATTA TATTAAATAA AAGTTTAGGT AAAACCTTTA 4800
TTGGAAAATT GATTACATTT TTCTACAGGC TTCATTTGGC ACGTAAAATT ATTAGAGAAA 4860
ATAACATAGA TATATGCATT AGTTTTGGGG AGAGATGTAA TATTATAAAT ATTCTATCTA 4920
TGGGGAAAAC AAAAAAAATC ATTACTATAC ATAGTCAGCT ATCTATTGAA AATAAAACGA 4980
AAGGATTATA TGGAAAAGTA ACAACACTAT TTTCAAAATT GCTTTATAAA AATGCGGATG 5040
CTACTGTAGC CGTATCAGAA ATAGTGAAGA AGGATGCATG CGGTTTATTA AATTTAGATG 5100
CGAATAATGT AGAGATAATT TATAATGGAC ACGATATTGG TTATATCAAA GATAAGTCTA 5160
CTGATTATAA AGAATTTGAT ACACCTGTGA TCGACTTCGT GTCTGTAGGG CGAATTACAT 5220
ATGCTAAAGG GCATTATCAT CTATTGAGAA GTCCTGCCAT AGTAAAGGAG ACGTATCCTA 5280
ATGTAATACT ATATATTGTT GGCACGTATG AAAAAGATAA TTTAAAAAGC ATAATTGATC 5340
ATTTAATTGA GAAATACGAT TTATATGATA ATGTGATTTT TACCGGCTTT AGTGACAATC 5400
CATATCCTTA CATAAAATCA GCAAAGGCTC TTATACTTAG TTCAATTTTT GAAGGTTTTC 5460
CAGGCGTTGT TATCGAGAGT ATTGCGCTTG GAACTCCTGT CATAGCGACA GATTGTGGCG 5520
GTGCTTCAGA AGTTTTAAGA AGTCCAGATG CCAAAATAAA GAATAATACG GGAGATGTGG 5580
AAATTACTAA ATTAGGAGTA CTGTTACCAA AATTCGATGG GATTTATGAT ATTAATTTGG 5640
ATTTTTCGCC CACAGAAAAA AAGATGGCGC AAGTTATGAA TGATATTATA TCAGGGAAGC 5700
TGAAATTTAA TGAAGACTTT TTAATTACAA AAGCAAACGA ATATGATATA GATTCTATGA 5760
The termination of Orf4, Orf5's is initial
TGAAAAAATA TACGGACTTA ATAGAGAAAA TAAAA
TAATG AAAGAAGCTA CTACCTCATA 5820
TAAATCAATT CATACTATAT TAGAATATTT TGGTTGGTCT CATGAAGATT ATTGGCATAT 5880
CAACTATAAA GTAAATTATG ATTTTTCAAA ATTTAATTAT TATTGGGATT TGACAAAAAA 5940
GTATAATTGG TTTAGTGGAC CTTTTGATCA AAATAATATT CCTTTATATC TTGGCACTGA 6000
TTCAAAACTG CATTACTCAC CAATTTTTTT AGGGCATTAT GCTCTTGGAG CATATCAAGA 6060
ATATTTATTA TCAAATGATA AAAAAGCATA TGACGATTTT ATAAGAATAG CGGATTGGCT 6120
GGTTGAAAAT ACAACGACAT ATAAAAAATG TTCTGGAATA TGGATAAATA CATACCCAAT 6180
GTCAACTTTT AACTTAAAAG GTAAATGGCA ATCGTGTTTA GCCCAGGCAA AAGGTATATC 6240
TGTCCTATGT AGAGCTTATT ATCTTACGCA AAATTTAAAA TATTTGAAGG TAGCTCTTGA 6300
CGCATTTGAA TCTTTCAAAA TATATAAAGA AGAAGGTGGA GTTAAGGTAA GGCATAAAAA 6360
CGTATTCTTT TGGGAGGAAT ATCCAACTAT AACCAATTCT ATCGTATTGA ATGGACATAT 6420
TTTTGCAGTT TGGGCATTGT ACGATTTATC TTTTGTATTA AAAGATAAGA ACGAGTTTAA 6480
GCAACAATGG GCGGATATTT ACAGTTATTA TAAAACATCA CTGATTATAT TAAAAGAGAA 6540
TTATAAGTTA TGGGATACGC ACTATTGGAC TAGATATGAT ATATGGGATA AACATTTTAA 6600
TGTTTCAAGT TTGTTTTATC ATAATCTCCA TATTAAACAA TTCTATATAT TGGCTGTTTT 6660
GGGAGATGAT GATGATTTCA AGAAAATTGC TAATAAATGG GCTGGTAAAT TAAAAAATCC 6720
Orf6's is initial, the termination of Orf5
GCTTTTCAGA ATGTATGCGC TTATAAAGAA AGTAGTTTTT AGGTT
ATGAT TATGAATGTT 6780
ATTGATAAAA TTATTATGGG TTTGAAAAGT ATATATTTAA GCTTTCTTTA TACAATATAT 6840
AATTTATTTC TTAATGAAAA TAGATTCGAA GAGACAAATG AAGTACATGT TGTTGTAAGT 6900
TTGACGTGTT TTCCTGGACG TATAAAAAAA GTATATCTTA CTTTAGAGTC AATTATGGCT 6960
CAACAATATA ATAACTTTAA AGTGGTATTG TACTTGTCTC ATGAAGAGTT TCCAAAGGGT 7020
TTAGAGGATT TACCTCGTAG TTTAATACGA TTACATAAAA GAGGTGTCGA TATTAATTTT 7080
ACGTGTGAAA ATATTCGATC TTATAAGAAG TTACATTATG CGCTGAGTGA TTTCCCAGAA 7140
CTACCTGTAA TAACAGCTGA TGATGATGTC TTATATCCCT CGCGATGGGT AAATGATTTT 7200
ATGGAGTCAC ATAAGCTTTT TCATGATGAT ATATTATTTG CGAGGGGGCA TCAGATAACA 7260
TTTGACCGAA ATGGAAATGT TAAAAAATAT ATCTCTTTCG GAAAACCAGC TGGATATTCA 7320
GCATCAAGTC TATATATTCC TACAGGAGTA AGTGGAATTC TATATCCGCC AGGATGTTTT 7380
TTTCAAGATG TGCAGAATAA AGATATTTTT ATGAAGTTGG CACCTAACGC AGATGATATA 7440
TGGTATAAAG TAATGACTTT GTTAAACGGT CGAAAATCCA GATTGATCTA TAAAAAGCCA 7500
ATTCATTATC CACCTATCCT TGGCACGCAG AAGATCTCCT TGCGTAAACA AAACTTATCG 7560
AAACAACATT TAAATAATGA TACACAATTA TTAAATTTAA TTGATTATTA TTCTATCGAA 7620
The termination Orf7's of Orf6 is initial
TTAGCTGATT TTAAGAAAAA GGAA
TAGCTC CT
ATGGAATT AGTTTCAATT ATAATGCCTT 7680
GTTTTAATAA TGAAGCAACT ATAAAAGAAA GTATTAATAG CGTACTATCT CAAAGTTATA 7740
AATACTGGGA GTTAATTATT ATCAATGACA ACTCAACAGA TGATTCACAA AATATTATTA 7800
ACGACTATGT GAATAGTGAT GAAAGAATAA AACTGATTAC TAATGAAAAA AATATCGGAG 7860
CTGGAGGTAG TAGAAATAAA GGAATTGAAA TTGCAAATGG AAGATTCATT GCTTTTTTAG 7920
ATGCAGATGA TGTTTGGTTG CCAGAAAAAC TTGCAAAACA AATAAAGTTT ATGAGAGATG 7980
GTAATTATGC ACTGACATAT AGTTATTATC AAAAGTTTGA TTCAACTGGA TTGGGAGCGA 8040
TCGTCCGGTC ACCTGCGACA ACAACTTATA GGAAATTGTT ATACAGTAAT GTTATTGGAT 8100
GTCTAACAGG AATGTATGAT ACTGATTTTT TGGGTAAATG CTATATGCCT TTAATTCGTA 8160
AACGACAAGA TATGGGGCTT TGGTTGTCAA TATTGAAAAA GTGTAATAAA GCATATTGCT 8220
TACCTGAAGT TTTGGCATAT TATAGAATTG ACTCAGGAAT GACAAAAAAT AAACTTGATG 8280
CGGCAAAATA TCAGTGGCGA TTTTATAGGG ATGTAACTAA ACTTAATTTG ATTCAATCAA 8340
The termination of Orf7
GTTGGTATTT TGTTTGGTAT TCAGTTTTAG GTTTGGTAAA GTACAAAAAA
TAATAAGGAG 8400
Orf8's is initial
GAGTG
ATGAC AATTTTAGTC ACTGGTGGTG CTGGATATAT TGGCAGTCAC ACTGTTATTG 8460
AATTACAGAA AAATGGTTTT GACGTGGTAA TAGTAGATAA TTTTTGTAAT TCATCGCCAT 8520
CGGTTTTAAA AAGAATTGAA TTAATTACAG GAAAGGAAAT TAAATGTTAC GATATTGATC 8580
TTCTGGATTT TAATTCTTTG CAGAAGGTTT TTGTAACTAA TAATATTAAC TCTGTGATTC 8640
ATTTTGCATC TTTAAAATCA GTTTCTGAAT CACTAATCAA ACCAACTGAA TATTATCATA 8700
ATAATTTGAG TGGTTGCCTC AATGTCTTGA AAATAATGAA AGAACATAAC GTGAATAATT 8760
TCATTTTCAG TTCTTCGGCA ACTGTATATG GCAGTAATAA CAACAATCCT GTTTCAGAAT 8820
CTTCATCAGT CGGCACCGCA ACAAATCCTT ATGGAAAATC CAAGATTTTT TTAGAGCAAA 8880
TATTGTCTGA TTGCTGTAAA TCAAATCCTA ATTTAAATGT AACTTGCCTG AGGTATTTCA 8940
ATCCGGTTGG TGCACATTCT TCAGGTTTGA TAGGAGAATC TCCGAATGGT GTTCCTGCCA 9000
ATTTAGTTCC ATATTTAACT CAGGTTGCTT TAGGTAAATT GCCCGTATTA CATATATATG 9060
GTAATGACTA TGATACCAAG GATGGGACGG GAGTAAGAGA TTTTATTCAT GTTACGGATT 9120
TAGCTAATGG ACACATAGCT GCGCTGAAAA AAATAAATGG TCATAATGGT TTTAAAGTTT 9180
ATAATCTTGG TACTGGGCGA GGTTATAGTG TGTTAGAAGT CGTTAGATGT TTTGAGGCTA 9240
TTACAGGAAA AAGTATTCCA ATCGAATTTT CGCCTCGCAG AGAAGGTGAT ATCGCTGAAA 9300
GTTGGGCAAA TGTTTCCGCA GCAAATTATG AGTTAGAGTG GTTCGCTAAA AAGACATTAA 9360
The termination of Orf8
CTGATATGTT ACGAGATGCA TGGAAATGGC AAACGTTAAA TCCAAATGGA TTA
TAAAGAT 9420
ATTTAATTTA TATTAAACCT TGCTTATTTA AGATAATGTA AACCGCACCT TTGCGGTAAC 9480
CACCCCTGAC AGGAGTAAAC AATGTCAAAG CAACAGATCG GCGTCGTCGG TATGGCAGTG 9540
ATGGGGCGCA ACCTTGCGCT CAACATCGAA AGCCGTGGTT ATACCGTCTC TATTTTCAAC 9600
CGTTCCCGTG AAAAGACCGA AGAAGTGATT GCCGAAAATC CAGGCAAGAA ACTGGTTCCT 9660
TACTATACGG TGAAAGAGTT TGTTGAATCT TTGGAAACGC CTCGTCGCAT CCTGTTAATG 9720
GTGAAAGCAG GTGCAGGCAC GGATGCTGCT ATTGATTCCC TTAAGCCATA TCTCGATAAA 9780
GGCGACATCA TCATTGATGG CGGTAACACC TTCTTCCAGG ACACCATTCG TCGTAACCGT 9840
GAGCTTTCTG CCGAAGGCTT TAACTTCATT GGTACCGGTG TCTCCGGTGG TGAAGAAGGC 9900
GCGCTGAAAG GTCCTTCCAT TATGCCTGGT GGGCAGAAAG AAGCCTATGA ACTGGTTGCT 9960
CCGATCCTGA CCAAAATCGC TGCAGTGGCT GAAGACGGCG AGCCATGCGT TACCTATATT 10020
GGTGCCGATG GTGCAGGTCA CTATGTGAAG ATGGTTCACA ACGGTATTGA ATACGGCGAT 10080
ATGCAGCTGA TTGCCGAAGC CTATTCTCTG CTAAAAGGTG GCCTGAACCT TACCAACGAA 10140
GAATTGGCAC AGACCTTTAC CGAATGGAAT AACGGTGAAC TGAGCAGCTA CCTGATCGAC 10200
ATTACCAAAG ACATCTTCAC TAAAAAAGAT GAAGACGGTA ACTACCTGGT TGATGTGATC 10260
CTGGATGAAG CGGCAAACAA AGGTACGGGC AAATGGACCA GCCAGAGCGC ACTGGATCTC 10320
GGCGAACCGC TGTCGCTGAT TACCGAGTCT GTGTTTGCAC GTTATATCTC TTCTCTGAAA 10380
GATCAGCGCG TTGCCGCTTC TAAAGTTCTC TCTGGCCCGC AAGCGCAGCC AGCAGGCGAC 10440
AAGGCTGAGT TCATCGAAAA AGTTCGTCGT GCGCTGTATC TGGGCAAAAT CGTTTCTTAC 10500
GCTCAGGGCT TCTCTCAGTT GCGTGCTGCG TCTGAAGAAT ACAACTGGGA TCTGAACTAC 10560
GGCGAAATCG CGAAGATTTT CCGTGCTGGC TGCATTATCC GTGCGCAGTT CCTGCAGAAA 10620
ATCACCGATG CCTATGCCGA AAATCCGCAG ATCGCTAACC TGCTGCTGGC TCCGTACTTC 10680
AAGCAAATTG CCGATGACTA TCAGCAGGCG CTGCGTGATG TCGTTGCTTA TGCAGTACAG 10740
AACGGTATCC CGGTTCCGAC CTTCGCCGCT GCGGTTGCCT ATTATGACAG CTACCGTGCC 10800
GCTGTTCTGC CT 10812
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 15 types, shigella dysenteriae 2 types and intestinal bacteria O112, it is characterized in that described oligonucleotide is to being: the Nucleotide of 1148 to 1165 bases among the SEQ ID NO:1 and the Nucleotide of 1856 to 1873 bases; The Nucleotide of 1233 to 1250 bases among the SEQ ID NO:1 and the Nucleotide of 1859 to 1866 bases; The Nucleotide of 1460 to 1477 bases among the SEQ ID NO:1 and the Nucleotide of 1861 to 1878 bases; The Nucleotide of 2515 to 2533 bases among the SEQ ID NO:1 and the Nucleotide of 3087 to 3105 bases; The Nucleotide of 2935 to 2952 bases among the SEQ ID NO:1 and the Nucleotide of 3392 to 3411 bases; The Nucleotide of 2718 to 2734 bases among the SEQ ID NO:1 and the Nucleotide of 3536 to 3553 bases; The Nucleotide of 3723 to 3740 bases among the SEQ ID NO:1 and the Nucleotide of 4121 to 4138 bases; The Nucleotide of 4103 to 4121 bases among the SEQ ID NO:1 and the Nucleotide of 4506 to 4525 bases; The Nucleotide of 3856 to 3874 bases among the SEQ ID NO:1 and the Nucleotide of 4548 to 4564 bases; The Nucleotide of 4644 to 4661 bases among the SEQ ID NO:1 and the Nucleotide of 5246 to 5263 bases; The Nucleotide of 4649 to 4667 bases among the SEQ ID NO:1 and the Nucleotide of 5452 to 5471 bases; The Nucleotide of 5255 to 5272 bases among the SEQ ID NO:1 and the Nucleotide of 5661 to 5680 bases; The Nucleotide of 6861 to 6880 bases among the SEQ ID NO:1 and the Nucleotide of 7309 to 7328 bases; The Nucleotide of 6995 to 7013 bases among the SEQ ID NO:1 and the Nucleotide of 7413 to 7430 bases; The Nucleotide of 7141 to 7159 bases among the SEQ ID NO:1 and the Nucleotide of 7613 to 7630 bases; The Nucleotide of 7731 to 7747 bases among the SEQ ID NO:1 and the Nucleotide of 8096 to 8113 bases; The Nucleotide of 7855 to 7872 bases among the SEQ ID NO:1 and the Nucleotide of 8222 to 8240 bases; The Nucleotide of 7919 to 7937 bases among the SEQ ID NO:1 and the Nucleotide of 8341 to 8360 bases.
2, the application of the described Nucleotide of claim 1, it 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 15 types, shigella dysenteriae 2 types and intestinal bacteria O112 type as probe.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB031005381A CN1252267C (en) | 2003-01-20 | 2003-01-20 | Nucleotide |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB031005381A CN1252267C (en) | 2003-01-20 | 2003-01-20 | Nucleotide |
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| CN1438230A CN1438230A (en) | 2003-08-27 |
| CN1252267C true CN1252267C (en) | 2006-04-19 |
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| CNB031005381A Expired - Fee Related CN1252267C (en) | 2003-01-20 | 2003-01-20 | Nucleotide |
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| CN (1) | CN1252267C (en) |
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| CN100372945C (en) * | 2004-12-29 | 2008-03-05 | 深圳市疾病预防控制中心 | Method of multiplex fluorescence PCR - improved molecule beacon for detecting pathogenesis bacterium stemmed from eating source |
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