CN1429832A - Nucleotide specific to O-antigen of shigella boydii 7 - Google Patents

Nucleotide specific to O-antigen of shigella boydii 7 Download PDF

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CN1429832A
CN1429832A CN 03100536 CN03100536A CN1429832A CN 1429832 A CN1429832 A CN 1429832A CN 03100536 CN03100536 CN 03100536 CN 03100536 A CN03100536 A CN 03100536A CN 1429832 A CN1429832 A CN 1429832A
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gene
nucleotide
bases
antigen
seq
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CN1168827C (en
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王磊
陶江
冯露
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Tianjin Biochip Corp
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Nankai University
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Abstract

A nucleotide specific to the O-antigen of Shigella boydii 7, the structure of the said O-antigen genom, the oligonucleotide of the glycosyltransferase gene and oligose unit treating gene in the said O-antigen genom, the process for obtaining bacterial O-antigen genom, and the method for detecting shigella boydii 7 in human body and environment are disclosed.

Description

The Nucleotide of the O-antigen-specific of shigella boydii 7
Technical field
The present invention relates to the complete nucleotide sequence of control O-antigen synthetic gene cluster in the shigella boydii 7 (Shigella boydii7), particularly relate in the shigella boydii 7 oligonucleotide in the control O-antigen synthetic gene cluster, can utilize these the oligonucleotide of the O-antigen-specific shigella boydii 7 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 boydii 7 just has identical O-antigen [Ewing, W.H. (1986) " Edwards and Ewing ' sidentification of the Enterobacteriaceae " .Elsevier SciencePublishers, 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 of shigella boydii 7, it is the Nucleotide in the O-antigen gene bunch of shigella boydii 7, 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 boydii 7.
Another object of the present invention has provided the gene of the O-antigen gene bunch that constitutes shigella boydii 7: 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, orf10, orf11, orf12 gene; Sugar synthesis path gene comprises flmA, flmB, neuA, flmD, flmH, nue, glf.
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 boydii 7 respectively comprises orf8, orf10, orf11, orf12 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 boydii 7; Especially the oligonucleotide of listing in the table one, they are high specials to the O-antigen of shigella boydii 7, and these oligonucleotide are also reconfigurable, the oligonucleotide after the combination also is a high special to the O-antigen of shigella boydii 7.
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 the detection and the evaluation shigella boydii 7 of shigella boydii 7 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 boydii 7.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 boydii 7 it is the isolating Nucleotide shown in SEQ ID NO:1,15796 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 boydii 7 is characterized in that it is by 11 genomic constitutions, and they are all between galF gene and gnd gene.
The Nucleotide of aforesaid O-antigen-specific to shigella boydii 7 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, orf10, orf11, orf12 gene; Wherein said wzx gene is the Nucleotide of 6714 to 7928 bases among the SEQ ID NO:1; The orf8 gene is the Nucleotide of 7936 to 8877 bases among the SEQ ID NO:1; The wzy gene is the Nucleotide of 8889 to 10184 bases among the SEQ ID NO:1; The orf10 gene is the Nucleotide of 10186 to 11148 bases among the SEQ ID NO:1; The orf11 gene is the Nucleotide of 11355 to 12272 bases among the SEQ IDNO:1; The orf12 gene is the Nucleotide of 12247 to 13176 bases among the SEQ ID NO:1;
The Nucleotide of aforesaid O-antigen-specific to shigella boydii 7 is characterized in that it is orf8, orf10, orf11, the orf12 gene that comes from described wzx gene, wzy gene or glycosyltransferase 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 boydii 7 is characterized in that the oligonucleotide of the described wzx of coming from is to being:
The Nucleotide of 6782 to 6799 bases among the SEQ ID NO:1 and the Nucleotide of 7449 to 7466 bases,
The Nucleotide of 6971 to 6989 bases among the SEQ ID NO:1 and the Nucleotide of 7481 to 7498 bases,
The Nucleotide of 7035 to 7053 bases among the SEQ ID NO:1 and the Nucleotide of 7588 to 7607 bases;
The oligonucleotide that comes from orf8 is to being:
The Nucleotide of 8065 to 8082 bases among the SEQ ID NO:1 and the Nucleotide of 8789 to 8806 bases,
The Nucleotide of 8134 to 8152 bases among the SEQ ID NO:1 and the Nucleotide of 8685 to 8704 bases,
The Nucleotide of 8198 to 8215 bases among the SEQ ID NO:1 and the Nucleotide of 8459 to 8476 bases;
The oligonucleotide that comes from wzy is to being:
The Nucleotide of 9091 to 9108 bases among the SEQ ID NO:1 and the Nucleotide of 9457 to 9474 bases,
The Nucleotide of 9357 to 9376 bases among the SEQ ID NO:1 and the Nucleotide of 9704 to 9721 bases,
The Nucleotide of 9120 to 9137 bases among the SEQ ID NO:1 and the Nucleotide of 9945 to 9962 bases;
The oligonucleotide that comes from orf10 is to being:
The Nucleotide of 10271 to 10288 bases among the SEQ ID NO:1 and the Nucleotide of 11060 to 11077 bases,
The Nucleotide of 10375 to 10393 bases among the SEQ ID NO:1 and the Nucleotide of 10790 to 10808 bases,
The Nucleotide of 10510 to 10528 bases among the SEQ ID NO:1 and the Nucleotide of 11090 to 11108 bases;
The oligonucleotide that comes from orf11 is to being:
The Nucleotide of 11737 to 11754 bases among the SEQ ID NO:1 and the Nucleotide of 12013 to 12030 bases,
The Nucleotide of 11367 to 11384 bases among the SEQ ID NO:1 and the Nucleotide of 12154 to 12171 bases,
The Nucleotide of 11482 to 11598 bases among the SEQ ID NO:1 and the Nucleotide of 11839 to 11856 bases;
The oligonucleotide that comes from orf12 is to being:
The Nucleotide of 12274 to 12291 bases among the SEQ ID NO:1 and the Nucleotide of 13016 to 13033 bases,
The Nucleotide of 12489 to 12506 bases among the SEQ ID NO:1 and the Nucleotide of 13101 to 13120 bases,
The Nucleotide of 12384 to 12401 bases among the SEQ ID NO:1 and the Nucleotide of 12959 to 12976 bases.
The Nucleotide of aforesaid O-antigen-specific to shigella boydii 7 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 boydii 7, and can provide the O-antigen of expressing shigella boydii 7 by inserting to express, and become bacterial vaccine.
The application of the Nucleotide of aforesaid O-antigen-specific to shigella boydii 7, 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 boydii 7 is characterized in that it comprises the steps:
(1) extracts genome.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 primary isoamyl alcohol (25: 24: 1) 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 the Long pcr amplification shigella boydii 7 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 15796 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, 0.9ul0.1M MnCl 2, 1ul1: the DNaseI of the 1mg/ml of 2000 dilutions, 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 2ul0.1M 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, 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 1.25ul100mM 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 (24: 1) 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 boydii 7;
(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.The sequence of residue 20% is again according to the sequences Design primer that has obtained, two pairs of primers, as follows: 5 '-GCATGGGTTACTGTACTAGC-3 ' and 3 '-AATGGCATCAATACCCGC-5 ' and 5 '-TGGCGGTATTGAGAGAGT-3 ' and 3 '-TTACAGGCTACTTCTCTTC-5 ', direct PCR and from the genomic dna of shigella boydii 7 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 the Nucleotide full length sequence (seeing sequence list) of the O-antigen gene bunch of shigella boydii 7 obtained.The quality of sequence is mainly guaranteed by two aspects: 1) genome of shigella boydii 7 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 boydii 7 O-antigen gene bunch, with American National biotechnology information science center (The NationalCenter for Biotechnology Information, NCBI) orffinder finds gene, find the reading frame of 13 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 boydii 7 at last
(6) screening of specific gene.At wzx, wzy, orf8, orf10, orf11, the orf12 gene design primer in the O-antigen gene of shigella boydii 7 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, the correct band of any size does not all increase in each group, 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, orf10, orf11, orf12 gene pairs shigella boydii 7 all is high special.
Just, of the present invention aspect first, provide the full length nucleotide sequence of the O-antigen gene bunch of shigella boydii 7, its complete sequence shown in SEQ ID NO:1,15796 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 boydii 7 by method of the present invention, as shown in table 3, it is altogether by 11 genomic constitutions, all between galF gene and gnd gene.
Aspect second of the present invention, the gene in the O-antigen gene bunch of shigella boydii 7 is provided, promptly transport 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, orf10, orf11, orf12 gene; Special sugared synthesis path gene in the bacterial polysaccharides antigen comprises flmA, flmB, and neuA, flmD, flmH, nue, the glf gene, their zero positions in O-antigen gene bunch and final position and nucleotide sequence all are listed in the table 4.The invention particularly relates to glycosyltransferase gene, transhipment enzyme gene and pol gene, because sugared synthesis path gene is that the gene of synthetic nucleosides bisphosphate monose is common, common by indication to more exocellular polysaccharide now, O-antigen to bacterium is not very special, and the glycosyltransferase gene that the present invention relates to, transhipment enzyme gene and pol gene are high specials to the O-antigen of shigella boydii 7.
Aspect the 3rd of the present invention, wzx gene in the O-antigen gene bunch that comes from shigella boydii 7 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, orf10, orf11, orf12 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 being to obtain expecting the product of size in the pcr amplification that carries out of template with the shigella boydii 7 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 two 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 one be high special to shigella boydii 7 and their O-antigen.
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 6714 to 7928 bases from SEQ ID NO:1), orf8 gene (nucleotide position is 7936 to 8877 bases from SEQ ID NO:1), wzy gene (nucleotide position is 8889 to 10184 bases from SEQ ID NO:1), orf10 gene (nucleotide position is 10186 to 11148 bases from SEQ ID NO:1), orf11 gene (nucleotide position is 11355 to 12272 bases from SEQID NO:1), orf12 gene (nucleotide position is 12247 to 13176 bases from SEQ ID NO:1).Coming from above intragenic oligonucleotide all is high special to shigella boydii 7 (SEQID NO:1).
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 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 be shigella boydii 7 or.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 boydii 7 or 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 be shigella boydii 7 or.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 be shigella boydii 7 or.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 boydii 7 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 boydii 7 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 the pcr amplification shigella boydii 7 bunch.
The O-antigen gene of shigella boydii 7 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.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: 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 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 boydii 7 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 boydii 7 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 boydii 7, 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 boydii 7 obtained.The quality of sequence is mainly guaranteed by two aspects: 1) genome of shigella boydii 7 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 boydii 7 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 13 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 boydii 7 at last, as shown in table 3.
By retrieving and relatively, finding that the synthetic enzyme flmA gene of orf1 and Aeromonas punctata all has 53% homogeny in 197 amino acid whose sequences, show the homology that height is arranged between them.So can determine orf1 is the flmA gene.The synthetic enzyme flmB gene of orf2 and Aeromonas punctata all has 61% homogeny in 378 amino acid whose sequences, show the homology that height is arranged between them.So can determine orf2 is the flmB gene.The nueA gene of orf3 and Aeromonas punctata has 56% homogeny in 227 amino acid whose sequences, show the homology that height is arranged between them.So, can determine that orf3 is the neuA gene.The flmD gene of orf4 and Aeromonas punctata has 33% homogeny in 361 amino acid whose sequences, 53% similarity shows the homology that height is also arranged between them.So, can determine that orf4 is the flmD gene.The flmH gene of orf5 and Aeromonaspunctata has 35% homogeny in 227 amino acid whose sequences, 59% similarity shows the homology that height is arranged between them.So, can determine that orf5 is the flmH gene.The neuB gene of Orf6 and Aeromonas punctata has 73% homogeny in 361 amino acid whose sequences, 84% similarity shows the homology that height is also arranged between them.So, can determine that orf6 is the neuB gene.Orf7 and colibacillary wzx gene have 23% homogeny in 307 aminoacid sequences, and algorithm [Eisenberg by people such as Eisenberg, D, Schwarz, E.etal (1984) .Analysis ofmembrane and surface protein sequences with the hydrophobic momentplot.J.Mol.Biol.179:125-142] find that orf7 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 orf7 is the wzx gene, called after wzx.The glycosyltansferase gene of Orf8 and Salmonella enterica has 29% homogeny in 286 amino acid, be a glycosyltransferase gene, called after orf8.Orf9 and colibacillary wzy gene have 20% homogeny in 306 aminoacid sequences, learn that by the Eisenberg algorithm orf9 has 10 potential transmembrane domains in addition, to other O-antigen polysaccharase similar secondary structure is arranged, so determine that orf9 is exactly the wzy gene, called after wzy.The glycosyltransferase gene of Orf11 and S.boydii has 32% homogeny in 239 aminoacid sequences, 51% similarity is so determine that orf11 is an acetyltransferase gene, called after orf11.The glycosyltransferase gene of Orf12 and Methanothermobacter thermautotrophicas has 36% homogeny in 286 aminoacid sequences, 51% similarity is so determine that orf12 is a glycosyltransferase gene, called after orf12.The synthetic enzyme wbpS gene of Orf12 and Pseudomonas aeruginosa has 56% homogeny in 628 aminoacid sequences, 72% similarity, so determine that orf12 is a synthetic enzyme enzyme gene, called after orf12, the glf gene of Orf13 and Pseudomonastyphimurium LT2 has 58% homogeny in 381 aminoacid sequences, 73% similarity is the glf gene so can determine orf13, called after glf.O-antigenic structure according to shigella boydii 7 also needs 1 glycosyltransferase gene as can be known, and orf10 and known array do not have obvious homology, but have the feature of glycosyltransferase, is 1 glycosyltransferase, called after orf10.Embodiment 6: the screening of specific gene.At wzx, wzy, orf8, orf10, orf11, orf12 gene design primer in the O-antigen gene of shigella boydii 7 bunch, the position of these genes in nucleotide sequence is shown in 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 boydii 7.Glycosyltransferase gene, transhipment enzyme gene and pol gene and their function corresponding and the size of the O antigen gene bunch of shigella boydii 7 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.
In the 23rd group, contain the genomic dna of shigella boydii 7 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, orf8, orf10, orf11, orf12 gene, each gene all has 1 to 3 pair of primer detected, 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 23rd group after.After being template PCR with the genomic dna of each bacterium in the 18th group, only find in obtained positive findings.In more detail, more than each gene each to primer all in obtain expecting the correct PCR product band of size.It is reported that the O-antigenic structure of shigella boydii 7 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, orf10, orf11, orf12 gene pairs shigella boydii 7 and O-antigen thereof all are high specials.
At last, from shigella boydii 7, screen gene by PCR: wzx, wzy and four glycosyltransferase genes to the O-antigen high special of shigella boydii 7.And the oligonucleotide of these intragenic any one section 10-20nt is special to the O-antigen of shigella boydii 7, and the primer in especially above-mentioned each gene is that oligonucleotide is high special to detecting the back confirmation through PCR to shigella boydii 7.These all oligonucleotide all can be used for the shigella boydii 7 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 boydii 7, in table, listed the structure of the O-antigen gene bunch of shigella boydii 7, altogether by 13 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 boydii 7, in table, listed the accurate position of all open reading frame in complete sequence in the O-antigen gene bunch of shigella boydii 7, 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 boydii 7
＜130〉to the Nucleotide of the O-antigen-specific of shigella boydii 7
<160>1
<170>PatentIn?version?3.1
<210>1
<211>15796
<212>DNA
<213>Shigella?boydii
<400>1
ctcctggtga?ctcatgcgtc?caagaacgcg?gtcgaaaacc?acttcgacac?ctcatatgaa?????60
ttagaatcgc?tccttgaaca?gcgtgtaaaa?cgccagttgc?tggcggaagt?tcagtccatc????120
tgcccaccgg?gcgtcaccat?tatgaatgtg?cgtcagagcg?aacctttagg?tttgggccac????180
tccattttgt?gcgccagacc?cgccattggc?gacaatccat?tcgttgtggt?gctgcctgat????240
gtcgttattg?atgatgccag?cgccgatccg?ctgcgttaca?accttgccgc?tatgattgcg????300
cgattcaacg?aaacgggtcg?cagtcaggtt?ctggcaaaac?gtatgccggg?tgacctctct????360
gaatactctg?tcatccagac?taaagaaccg?ctggatcgtg?aaggcaaagt?cagtcgcatt????420
gttgaattta?tcgaaaaacc?cgatcagccg?cagacactgg?attcagacat?tatggcggtt????480
ggtcgctatg?tgctttctgc?agacatttgg?cctgaactgg?agcggactca?acccggtgca????540
tggggacgta?ttcagctaac?tgacgcaatt?gccgaactgg?cgaaaaaaca?gtccgttgat????600
gcaatgttaa?tgacgggtga?cagctacgac?tgcggtaaaa?aaatgggcta?catgcaggca????660
tttgtgaagt?acggactacg?caacctgaaa?gaaggagtga?agttccgcaa?agggattgag????720
cagctgttaa?gcgaataaaa?atcttaccgg?atgtaacggt?taataagaaa?atcataacgg????780
cagtgaagat?tcgtggtgaa?agtaaattgc?aacgaatgtt?cctgccgtta?ttgctttata????840
aaacactata?ataacaatga?gttagcaata?agattttagt?caaatttttc?caggattttc????900cttgtttcca?gagcggattg?gtaagacaat?tagcgtttga?atttttcggg?tttagcgcga????960gtgggtaatg?ctcgtcacat?cgtaggtatg?catgcagtgc?tctggtagct?gttaagccag???1020gggcggtagc?gtgtgttaaa?cttaaggggg?ttgttgcgag?taatcaagat?cgcggcaatt???1080tcggattgtg?tcttattttg?ataataaaac?tatcctgata?actggtggta?ctggctcatt???1140cggcaataag?tttgtgcgca?tgacattaga?taaatataac?cctaaaaaga?ttattattta???1200ttctcgtgat?gaaatgaaac?agtgggaaat?ggcaaaacat?ttcaaggacg?aaagccgaat???1260tcgattcttt?attggggatg?ttcgtgataa?agatcgtttg?tatcgtgcgt?tggatggtgt???1320tgactttgtt?gttcacgccg?cagcaacaaa?aatcgtacct?acagcagagt?ataatccctt???1380cgaatgtgta?aaaacaaata?tcaatggtgc?gatgaatgtg?attgatgctt?gtatcgataa???1440aggtatcgaa?cgtgttgttg?cactttctac?tgataaagca?agcagccctg?cgaatctgta???1500tggtgcaaca?aaattagcat?ccgataaact?tttcgttgct?ggtaactctt?attctggtgc???1560gacaaaaact?cgttttgctg?ttgttcgcta?cggaaatgta?atgggatcac?gtggttcggt???1620aataccgttc?ttcttatcta?tcaaagaaaa?aggagaattg?cctattactg?atgatcgtat???1680gactcgcttt?atgattactc?tggagcaggg?ggttgagtta?gtttggcatg?cgtttgaaga???1740tatggtcggt?ggtgaaattt?atgtaaaaaa?aataccttcg?atgaaagtaa?ctgatatagc???1800aacagctgtt?gcgcctaatg?ctacacaaaa?aatggtcggt?attcgaccgg?gcgagaaact???1860acacgagcag?atgatcagtg?ctgaagatgc?ttattacact?tatgaatatc?cagagcattt???1920taagattctt?ccagccattc?acaactggtg?taactcacct?gaaaggatta?aagatggtaa???1980aaaagtgcca?gaaggcttcg?tctatgaaag?tgatagtaac?gcagaatgga?tgagtattga???2040agaacttcgt?caatggatcg?aggataatag?agaaaaagta?ggtaacattt?gatgaaattt???2100attccctatg?gacggcagga?tgtttctgat?gaagatatcc?atgctgtggt?ggatgtactg???2160aagtccgatt?ttttgacgca?aggaccatgt?gttccccaat?ttgagaaggc?aattgctgat???2220tatgtgaatg?ctaagtatgc?agttgcagta?aatagtgcta?cttcagcact?tcacattgct???2280tgtctcgcgt?tggggatgaa?gaaaggcgat?tggctttgga?catcgccaaa?tacatttgtt???2340gcttctgcga?actgtgctct?ttattgcggt?gctaatgtca?gttttgtcga?tattgatgcg???2400cggacttata?atatgagtgt?tagcgcgtta?gaagcgaaac?ttatgtctgc?aaggaacaat???2460ggaacattgc?ctaaggttgt?tgttcctgtc?gcatttgcag?gacagtcatg?tgaaatggag???2520gaaatatata?aactctcaaa?agagtatgga?ttttcaatta?tcgaagatgc?ttcgcatgct???2580attggaggaa?attaccaggg?aaagaaaata?ggtaattcgc?gctttgcaga?tattactata???2640ttgagcttcc?atcctgttaa?aattattaca?actgcagaag?gtggtatggc?tctaaccaat???2700aatgatgatt?tggcagaaaa?aatgcagtta?ttccgtagtc?atgggatcac?tcgtgatagt???2760aatcatatga?cgaaggtaag?tgaaggagga?tggtattatc?aacaagttga?tcttggtctg???2820aattaccgaa?tgactgaact?gcaggctgca?ttaggcgtga?gtcagctgaa?gagtattgat???2880gattttgtaa?cacgccgcca?tgaactggtt?gaacgttaca?aaaaagctct?cgaagatatt???2940cctatttccc?taccatacca?ggcggaaagt?ggtcacagtg?cctttcacct?ttatcctgta???3000accgttaaaa?attctgcaca?gcggaaaact?ttgtttgatt?atctgcggaa?taaaaatatc???3060ggggttaatg?tccattatat?tccagttcat?actcaaccgt?attatgaaaa?gttagggcat???3120aaaatcggag?attatcctgt?cgctgaggat?tattactctc?gaaccatcag?cataccgatg???3180tattctgctt?tgacaaatga?agaccaggat?tatgtaatcc?aatgcattcg?ggagtttttt???3240taaatgaatg?tggcaatcat?tccagctcgt?ggtggtagta?aacgtatccc?tcgaaaaaat???3300attaaaatgt?tttgcgggaa?accaatgatt?gcctggtcga?ttaatgccgc?tcgaaagagc???3360ggcgtgtttg?acagaatcat?tgtttcgacg?gatgatatgg?agattgcagc?tgtagcaaga???3420aaattcggcg?ccgaagttcc?gtttatgcgg?ccagaagaac?tttcgaatga?ttttactgga???3480acaatccctg?ttatacggca?tgcggctgaa?tggcttattt?ctcagggaaa?taaaataaaa???3540tatatttgct?gcatttatgc?aacggccccc?tttattagag?atgaagattt?aaaactaggt???3600ttgaacctta?tcaaagcaaa?ggggggggat?tattgttttt?ctgttactac?ttattcgtac???3660cctattcaaa?gagcattaaa?aattaatgag?aataacgaaa?taaccatgtt?tgatccgact???3720cagtttgaaa?tgcgatctca?agatcttgtg?gacgcttggc?atgatgccgg?gcaattttat???3780tgggggaata?ttgatgcatg?gataaaagaa?aaaccggtat?ttaagtcaca?ggctatacca???3840ttacctttgc?ctagagaacg?agtgcaagat?attgatacgc?cggaagattg?gcatttcgct???3900gaggtgttat?ataggataaa?tacaataaaa?aatgaagatt?gtatttaggg?ttgatgcttc???3960gctttcaatt?ggttcaggac?atgtttttcg?ttgtctaaat?cttgctggtg?cattgagaaa???4020gaatggtttt?gaatgcattt?ttcttacaaa?ggaacatgca?ggaaacttaa?ttcaatatat???4080aaagacatat?ttgttccaag?tatatgttat?ttcaaaggaa?gataatgttg?atgataaata???4140tattgaaaat?gaaaaagagt?ggttaggagg?gagtcaaaaa?gaagacgctg?aaaaaacttg???4200tgaaattatt?tacagaaata?attttgcacc?aggcatattt?attattgatc?attattcatt???4260agattctgaa?tgggaaagtt?taattaaatg?gaaatttcca?agtacaaaaa?ttgttgttat???4320tgacgacttg?tgtaatagaa?aacattattg?tgatatcctg?attgattcaa?catttgagcg???4380tacttctgag?gaatatagga?gattagttcc?tgaatattgt?actattttgg?ccggtactga???4440ctatgcttta?ctgaaagatg?aatttttgca?attgcgcaaa?gaagccatat?taaaacgtac???4500atctatatta?agccctcaga?aaattttaat?tactatggga?ggggttgata?tacataacgt???4560gactgggaaa?gttttaaaag?aattaaataa?tagagctgat?cttcattata?aaaagatcac???4620tgttgtatta?ggggtgaatt?gccctcataa?aaaaaacatt?gaaaatattg?cacaatcgat???4680aaaatatgat?gtggatataa?aagtgaatac?caacaatatg?ccacaactta?tgttagaaca???4740tgatttatct?attggagctt?taggaagtac?tacttgggag?agagctgtgc?ttggattacc???4800cacagtaaat?attgctattg?ctgacaatca?actggttatt?gtagagaaac?taaggcgaaa???4860tggttttatt?gtctttgata?gcattaattt?tactggggtg?gaattagctc?gttctttaat???4920aaaggtaact?gatttctatg?atgaaatggt?gtctcgctcg?ctctctatat?gtgatggcag???4980gggattacaa?aggacactaa?aatttataat?atctttagct?acaaataaat?aatgagacat???5040gcttatgact?ggcaaaaata?taaaaaaatt?aaatttgctt?aacattgttc?attcttcgga???5100ggatattcaa?gaaaaagttc?gatgtataag?aaatgaacct?caaattagga?agtggatgta???5160tacagatcat?cttatatcaa?aagatgagca?taaaaattgg?attgaaagat?taagaaatga???5220atcaagaaat?tttgtgtatg?tcgtaatgaa?tgactacgat?gagcctattg?gtgtcgttag???5280tgtaaattcg?attgatttga?aaaacaaaaa?aggagactgg?gcatattatt?tgacagcaaa???5340tacacgtact?ggtattggtg?ctgtattaga?attttttgtg?attgattatg?tttttaacta???5400tttgcatctt?gaaaaattaa?attgtgaggt?aattgaacat?aatgataccg?tcgttcgatt???5460acatgagaaa?tttgggttta?agcaggaagg?atttagagag?tctaatatta?taaaggatga???5520taagcgtttt?ggagtttatt?attttggttt?aacgaaagaa?aaatggatgc?taacacgaga???5580atgtttaaag?aataaatatt?ataagctttt?ttctcaatac?gcggttgatt?ttcattaata???5640ggtttatttt?tggggtgata?aatgaaagca?attactattg?caggacgtaa?aattggtaaa???5700gattttcctc?catatatcat?tgctgaatta?tcggctaatc?ataatggaga?tataaatcgt???5760gcatttaaga?taatggaaga?ggcaaaagca?gctggagctg?atgctgtaaa?attacagaca???5820tatagagcag?atactataac?gctggattgt?gattctgaag?agtttcaaat?acatggtggt???5880ttatggggtg?gtcagacatt?gtatagctta?tatcaaagtg?ctcaaatgcc?ttgggaatgg???5940cataagccat?tatttgataa?ggcaaaagag?ttaggcatta?ctatttttag?cagtcctttt???6000gattttactg?ctgttgattt?attagaagaa?ttaggtgctc?cggcttataa?aatagcgtca???6060ttcgaagcta?ttgatttacc?gctaattcga?tatgtagctc?aaactgggaa?accgatgatt???6120atctcgacag?ggatggctga?tgaacaggag?attcaggagg?cgatccatgt?agctaaagaa???6180ggcggttgta?aggagctcgt?tgtcttgcat?tgcgtaagtg?gatatccggc?tccaccagag???6240gattacaatc?ttgccacaat?ccctgacatg?gctaaaaaat?ttaacgttat?aacaggtctg???6300tcagaccata?caatagataa?tactacggcg?atagcatccg?ttgcattagg?agcgagtgtg???6360atcgaaaagc?atgtaacact?tgatcgtaat?ggaggagggc?cagacgatag?tttctcactt???6420gaaccaagtg?agcttacagt?tctttgccgt?gatgcaaaga?tcgcttggca?ggctttaggt???6480agaataaact?ataccagaaa?gccaagtgag?atgggaaacg?ttcaatttag?acgttcattg???6540tattttgtta?aagatatgaa?agctggtgat?attattgatg?aaacatgtgt?gcgcagtgtg???6600cggccaggct?atggattacc?acctaaatat?tttgatgatt?taattggtaa?gcgagttatt???6660agtgatgtta?tggccaatac?ccgaacatca?ttggaattaa?ttgcagattt?aaaatgaaag???6720ttattaaaga?tagtgttctg?tatgtaatag?gtgagttgat?atctaaaagt?gttccatttc???6780tattgcttcc?ctatcttaca?aggcggttgg?gaattgatgg?ttttggagaa?atgtcatttt???6840atcagacaat?acttgcttta?ttggttatcg?ttataagctt?agggcaagat?ggggctgttg???6900ctagatattt?ttatttctac?ggagcccgtt?ctataaattt?gattgtgact?gtaggtgctt???6960tatattcact?tgtgacgaca?tccctattgt?ttttatcgtt?ttcatttttt?aaatccacga???7020tattatcaat?tattgtgttg?actgctggat?ttcaatcact?tttcaatacg?cagctaagtc???7080ttaggcagtg?ccaaaaagag?tcagttcagt?atattgtcat?gcaaatatta?gttgcgttga???7140cttctggtgg?tataaccttt?tgtttactgg?aaatatatag?tcatgattta?gttgaaaaga???7200gatttattgc?attgcttttg?agtaatatag?tttgtgtttt?tattgcttat?cttatttata???7260tgaggagacg?gaaatataat?cacgcatttt?tatttcgacg?atattatagt?gcattgctat???7320acttgattgg?ttttggaggc?ccattgattt?tgcatcaaat?aagtggcttt?cttaaaggac???7380aattagatcg?agttttgata?tatagccgtt?tttctgcttc?agatttaggg?ggctattctg???7440ctgcatttca?aatagcctca?gttctcttga?tactgataat?ggcgttgaat?aaagcactag???7500ttccatatta?ttatagtgag?ctcaaaaaaa?agcatatagt?ctttgatgat?ataaaaaaaa???7560cttgcgtttt?tttatttcta?ttgattccac?ttccatcaat?catatcgttg?attattcctg???7620ctgatttttg?gtgttttatt?ttaggaaatg?aatttgttga?tgttaaatat?tacatcgcta???7680tattcctatt?aggtataggt?tttacaatac?cttatctttt?actagtgaat?tatctttttt???7740attatggtaa?aaataaaagt?ataactatct?gttcagtcat?ttcaacatct?atttattgca???7800ttgttcttta?ttttttatca?tgtttaggta?tttcttatgt?tccatattct?atgttgatag???7860caaatatgct?aatcgttcct?ttactttata?tggctgccaa?aaaaacatca?atgcttgaac???7920gccagtgaga?ttaatatgcc?ctcgttattt?atatgtgtta?cgccactgca?aatgttgatt???7980gctgaaaaaa?taattgataa?aaccaggcca?gtaaacatag?aaattatagt?gcttgcatat???8040cagaaaaatg?ataaatatat?gcattacata?aagcggttgg?agaaaaaatg?tactaatttt???8100acagttttgg?ctgtaacgcc?aaaaaataaa?tttgtaaccg?tgatcgcctt?tgcaaaatta???8160cataccatct?taaataaaaa?tatgagtaaa?acttactctg?aggtttatct?gtctagtatt???8220gataacaagt?atgtacaatt?aattgtttct?aagctcaatt?atgcaaggtt?atatacattt???8280gacgatggaa?cagcgaatat?aattaaatct?agtgcatatt?atcaagagga?aaaaaaaact???8340ctcaagacta?acattttgag?atggattttt?ggaattaata?aaggattgca?agaaattaag???8400agtgaaatat?gcaagcacta?tacaatttat?ccaagtgtta?gtaatattgt?ctctaataca???8460gaattgatag?agatgtttac?acaatgcaaa?aaaaggaagc?aagataaaaa?agttgtgaga???8520gtctttatag?gacaaccttt?tgatgaatta?ggaataccat?tatcactaat?agaagagttt???8580ttctttaaat?acaaaatgga?ctattattat?ccacacccaa?gagagaaaat?aattaataat???8640aagttcactt?atatacactc?acatttgatt?tttgaagaat?atataattga?aggcttacaa???8700catcaagata?tcgtatacaa?aatatatggt?gctgtgtgta?caagtatatt?aaatttagcg???8760tcctcgaaaa?atgatatcga?gatatgttcc?atttatacgg?atgagctgag?aactaaatat???8820agtgattatt?atgcactggc?tgaaaaaatg?aatattacct?tgttaaaact?tacttgatat???8880gataaaatat?gactaatata?acgttcagaa?ctctgagaat?tccaggatat?ttactctcat???8940tatttgttct?tttaatcgga?tttataacat?atttcatctt?taatgaaact?cttgcagcga???9000atttattagc?agtgggggct?attggcatta?tcattcttga?tagtttacat?gataagcgga???9060ttttttattt?aacacttttt?cccgtgtatg?tcctcgtagg?tcaactcatt?tcattattat???9120tagttgagca?cggatggatt?ctaatcgaac?ttggaggtat?taaaagctat?cccattggtt???9180caattctttt?tatggcgtta?acaatagttt?tattccattt?tatcatattt?ttaactgtaa???9240aaatttcaat?attcaaattt?gatcgaacat?gtattgataa?gttaagtggt?aattctttta???9300tttattactt?gccgattttt?tatctatgct?tagtatatat?tcctgttttt?atttatggct???9360ctgcattatc?tgtaacaaat?ggtaatcgag?ttgtatacaa?tcaaataata?tcacctgttt???9420ttttatattt?atttcaaatt?aaacaattta?tacttcctgt?tgcaggattg?tatttgctta???9480aaaataaaaa?aatatttact?atatatctgt?ttgcaatttt?actatggaat?atattgattg???9540gagagaaggc?aacgggcatt?tggcagtcat?tgtatccaat?gctgttacct?tatgttttga???9600taaattatga?taaaataaaa?acaaaaaata?tattaatagt?attaggtttt?tgtattgtat???9660ttataaccag?tagcatagta?attaattata?tatttattga?aaagtcaggt?gctacgttta???9720tatttgatag?aatatcaatg?caaggacaat?tgtggtggta?ttattttaat?gagcacgttt???9780tgttagctaa?gacccctcat?gaattatctg?aagagttctc?gtcagaatat?agtgggcttc???9840ttaatctgat?gtatcattca?atgcctgccc?atcttttcaa?tagctatatc?gagcgcggtg???9900tagtcttgac?gagtggtttc?cccgcaatat?ttctttttta?ctttggacaa?tactggctcc???9960ttcctacgtt?attatcacct?tttctttttg?gtttagttgt?ctattttttt?tcaagaagtt??10020tatatagcgg?tagtatattg?agtttgctta?tatcaagtaa?actatttttt?gcttttactg??10080ttttttttgc?ccgtggagat?atcgcaactt?ttttagatta?taaattgctt?atttatctat??10140tgattataat?aattttacaa?tatttgccaa?gggttaaggt?ttaatatggc?gtatacttct??10200cactttatat?ataaagtagc?taataagatt?agcattttta?taagaaaaat?catcaactta??10260tttagattat?ttctttacgg?tgatgttgtt?tcatatgatt?ttgaaatgga?ggagaataga??10320agaattatag?tgtcaggaag?tctaacaaag?agagataaca?tacctaaaat?aatttggatt??10380tattggcatg?atgagcaaat?tccattatta?atagaggctt?gtataaaacg?ccttaaagat??10440ttaaatccta?attatcaatt?aaatgtgcta?aataaattta?caattcatga?atacttacct??10500gagagtctaa?tgtggagaca?ggatctttct?gtccaaataa?gatctgatct?gattcgcttg??10560ggattattat?ataaatttgg?tggagtatgg?atagatgcta?gtgtaatgtt?ttttgaggat??10620ctttcatggt?tagaaagact?ttcagcagag?aataaatatg?atttaatcgg?tttttatcga??10680ggcagactat?ctatcgactg?ctataatcca?atagtagaaa?gttggatgct?gggggcacca??10740aaatacaata?agtttataaa?gcgatggtat?gacaactttt?atccagtatc?ttatttaggg??10800ttagatgaat?atttcaacac?aattaagaaa?agaagtgatt?ataaaattat?caaacagaat??10860ataaatattc?ccaaatattt?atctgtttat?ctggcacaac?aaatttcgtc?gcgagaaatg??10920agagattatt?gcgcttattt?aagaaaggta?gaggatagtg?catatttata?tcaagagtca??10980tatccacagc?gtgattgtgt?tctctcggag?gtttggtgta?aaagaagaaa?accacagaaa??11040attcctccac?taatcaaatt?aacaagccag?aacagaaata?caattttagc?ctgtagtaaa??11100ataaatccac?gtagtatcgt?aggtgagttt?ttagataata?agaattagga?atagagaaag??11160tgcgagataa?aattgtattt?gtagtcgctg?atatcacggc?tactggcggg?attgaacgag??11220ttattactta?tcttgccagt?tatttatgta?ataatgggta?tagtgtagag?atattatcta??11280ttcatagaag?taatcgtaat?ttaccttatc?ctgttcatgc?agacgttcat?atctcttttg??11340ttgataaaac?tataatgaag?gcaagaaagc?caggatctgt?cagcaaatta?ctttcacatt??11400ttaaatcctc?atttcattta?aatataaaat?tatttaagag?aagaaaatgt?attattgtag??11460caaatagttt?tcctgttgct?ttattttcct?gcttctcggc?cttattttca?tcaaaaatgc??11520tcgttgtcga?gcatgttcat?tatcactatt?acagtaagtg?cttagttaat?gtaaggcgtt??11580tcatttataa?gtttttttat?ggggtagttg?cgttaactga?gcgtgacagt?gagctttata??11640ttaatgataa?actaaattct?ctaacaatac?ctaacgctct?tagttctttt?cctgaaaaaa??11700tagcgaatcc?atctgtggaa?aagaaaaaaa?taattgccgc?tggaagatta?gaacatcaaa??11760aagggtttga?tattctaata?aaatcctttg?ctgaaataga?ctattctgtg?aggaaagaat??11820ggatcatgga?tatttatggg?gatggaaatg?aaagagctgc?cttacagaaa?ttaattcatg??11880atttaaatat?tgaggattgt?gttaatttat?tgggtaacag?tcgagacctt?atgaacactt??11940atgctgaata?tgattttttt?gtattatctt?caagatttga?aggtttcggg?atggttttac??12000ttgaagccat?gagttgcggt?cttccttgta?ttgccatcga?ttgtcctaca?gggcctagag??12060aaatacttga?tggaggcaag?tatgggatac?tttgtgacaa?ccaatcaaat?ctgggggaga??12120gtattgcatc?tctaattgtt?aataaggatc?taagaactat?gctatccggc?aaaagcattt??12180tacgtagtca?tgattataat?attcagatta?taagtgaaaa?atggcagggt?ttatttgaaa??12240gcttaaatga?aaaaaatata?tatgaaaaat?aatatcgcac?ttgttcttgt?tacgtataat??12300agagagtctc?ttttaaaaga?agtcctttca?tcgataaagc?atttaaaaga?taaaccgaaa??12360catgtttata?taatcgataa?taatagctca?gatggaacct?gtaatataat?tatagatttt??12420attcaacaga?attcatctat?tctaagtatg?agttatcata?atacaggaga?taatctcggt??12480ggggctggcg?gatttgctta?cggtagccgg?cttgcttatg?ccgatggatt?ccaatggatt??12540tggttagctg?atgatgatgt?tgtttttgag?caaacatgtc?tgactcaatt?gatgctttat??12600tcttcagatg?ccgatatatt?acagccaatg?agaatcaata?ctgacggtag?ctgtgcagaa??12660atttcaggaa?tagattacga?aataaacaat?atcttcaggt?taaaccctaa?aaaacttaaa??12720attactgata?tatatgaaga?aaagtgggat?attcaagaaa?tcaaaacgat?accttttgaa??12780ggaccattaa?tacatcgacg?tgtatttgag?cagattggtt?ttcccaaccc?cgattttttt??12840attttttatg?atgatctaga?ttttgcattg?agggctcaaa?gagcaggctt?caagattaaa??12900tgtgtaaaat?ctgctcatct?gattcgtaag?attagatttg?ttcaaagcgt?cgctttgacg??12960tcttggaaag?gttattttat?gtatcgtaac?ttttttaagg?ttcaacttac?gtatgcgaga??13020ataccgatag?gattatttag?agttattgct?atatttttta?ttgtaataat?ttattctcta??13080ctatttggag?gtgcaaaaaa?catttcgact?ttgataagtg?cattgcgtga?tgcattaagg??13140aaaaattttc?cacttgatca?acgttataaa?ccctaattta?aaagttaaaa?tcatgaaaaa??13200taaaatagct?attgtcggtg?caggcttaag?tggggcagta?atagctcgtg?aacttgctga??13260tgctggatat?ttagttgatg?tttttgataa?acgtcaacat?attggaggta?actgctatac??13320cgctagggat?cctcaaacaa?atattatggt?tcatacatat?ggcccacata?tatttcatac??13380taacagtaaa?tttgtttggg?cttatattaa?taagctgggg?gtatttaaac?cgtttactaa??13440ccgcgttaaa?gctgttacta?gaggacaggt?tttttctttg?cctattaacc?tattaactat??13500aaatcaattc?ttcaataaat?tatttacacc?agcagaagca?aaaaaatata?ttgaatctat??13560atctgataaa?tatattaaag?atcctaatac?gtttgaagaa?caagctttat?cctttattgg??13620acgtgaatta?tatgaggctt?ttttcaagtc?ttatacaatt?aaacagtggg?ggattttacc??13680taccgatata?ccagccagta?ttctcaaacg?actgcctgtc?cgttttaatt?atgatgataa??13740ttatttttcg?catcaatatc?agggaatgcc?ggaggacggc?tatacacctc?tattcgaaaa??13800tcttctgaat?cactccaata?tcaatgttat?tctttctacg?gaattctcgg?caattgattg??13860tgataaatat?attcatactt?tttatagtgg?cacaatcgat?gggttctttg?attatgattt??13920aggtcgtttg?ctatatagaa?ctctagactt?taaaacggaa?gtgttttggg?gagattatca??13980gggatgtgca?gtaatgaatt?attgtgataa?ttctaaacca?tatacacgta?ttactgaaca??14040taaatatttt?tctccttggg?aacaacatga?caaaacaata?atttacaaag?aatatagtag??14100agaatgtaat?gatagtgata?ttccctatta?ccccgtacga?ctggtcagtg?ataaagaaat??14160attaaaaaaa?tatgtggaac?gtgccagtaa?acttgaaaat?gttacttttg?ttggccgatt??14220gggtacgtat?cgatatttag?atatggatgt?aactatcact?gaggccttaa?acaccgccga??14280acagttttta?gaacttcacc?aaaataatca?aagaatacca?gctttttttg?ttgatgtttt??14340atagcgagtc?aacatgatta?tattgtagta?cataatatta?cgttcatcct?gataaattta??14400atctatattt?tgctcatggc?tttaaattta?aagctattct?tcatataata?gcactaattg??14460cggtaacccc?tgacaggagt?aaacaatgtc?aaagcaacag?atcggcgtcg?tcggtatggc??14520ggtgatgggg?cgcaaccttg?cgctcaatat?cgaaagccgt?ggttataccg?tctctatttt??14580caaccgttcc?cgtgaaaaga?cggaagaagt?gattgccgaa?aattctggca?aaaaactggt??14640tccttactat?acggtgaaag?agttcgttga?atctcttgaa?acgcctcgtc?gcatcctgtt??14700aatggttaaa?gcaggtgcag?gcacagatgc?tgctattgat?tcccttaagc?cataccttga??14760aaaaggcgac?atcatcattg?atggcggtaa?caccttcttc?ctggacacca?ttcgtcgtaa??14820ccgtgagctt?tctgcggaag?gttttaactt?cattggtacg?ggggtttccg?gtggtgaaga??14880aggtgcgctg?aaaggtcctt?ctatcatgcc?tggcggtcag?aaagaagcct?atgaattggt??14940tgcgccgatc?ctgaccaaaa?tcgctgccgt?agctgaagat?ggcgaaccgt?gcgttaccta??15000tattggtgca?gacggcgcag?gtcattatgt?gaagatggtt?cacaacggta?ttgaatacgg??15060tgatatgcag?ttgattgccg?aagcttattc?actgcttaaa?ggtggcctga?atctctccaa??15120cgaagaacta?gcgcagacct?ttaccgagtg?gaataacggt?gaactgagca?gttacctgat??15180
cgacataacc?aaagacatct?tcaccaaaaa?agatgaagac?ggtaactacc?tggttgatgt????15240
gattctggat?gaagcggcta?acaaaggtac?cggtaaatgg?accagccaga?gtgcgctgga????15300
tctcggcgaa?ccgctgtcgc?tgattaccga?gtctgtgttt?gcacgttata?tctcgtctct????15360
gaaagatcag?cgtgttgccg?cgtctaaagt?tctctctggt?ccgcaggcac?agccagcagg????15420
cgacaaagcg?gagttcatcg?agaaagttcg?ccgtgcgctg?tatcttggca?aaatcgtttc????15480
ttatgctcag?ggcttctctc?aattgcgtgc?cgcgtctgaa?gagtatcact?gggatctgaa????15540
ctacggtgaa?atcgcgaaga?ttttccgtgc?tggctgcatc?atccgtgcgc?agttcctgca????15600
gaaaatcacc?gatgcctatg?ccgaaaacct?aaaaatcgct?aacctgctgt?tggctccgta????15660
ttttaagaaa?atcgctgacg?actaccaaca?agcactgcgt?gatgtcgtcg?cttatgcagt????15720
gcaaaacggt?attccggttc?caaccttctc?tgcggcggtt?gcttattacg?acagctatcg????15780
Glycosyltransferase gene and oligosaccharide unit treatment gene in the tgccgctgtcctgcct 15796 table one shigella boydii 7 O antigen genes bunch
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 6714 to 7928 #103 (6782 to 6799) #104 (7449 to 7466) ??685bp ????0 ????52
#147 (6971 to 6989) #148 (7481 to 7498) ??528bp ????0 ????59
#173 (7035 to 7053) #174 (7588 to 7607) ??573bp ????0 ????45
?Orf8 Glycosyltransferase 7936 to 8877 #105 (8065 to 8082) #106 (8789 to 8806) ??742bp ????0 ????58
#149 (8134 to 8152) #150 (8685 to 8704) ??571bp ????0 ????59
#175 (8198 to 8215) #176 (8459 to 8476) ??279bp ????0 ????50
??Wzy Polysaccharase 8889 to 10184 #107 (9091 to 9108) #108 (9457 to 9474) ??384bp ????0 ????55
#155 (9357 to 9376) #156 (9704 to 9721) 365bp ????0 ????59
#177 (9120 to 9137) #178 (9945 to 9962) 843bp ????0* ????50
?Orf10 Glycosyltransferase 10186 to 11148 #109 (10271 to 10288) #110 (11060 to 11077) 807bp ????0** ????50
#125 (10375 to 10393) #126 (10790 to 10808) 444bp ????0*** ????48
#411 (10510 to 10528) #412 (11090 to 11108) 599bp ????0*** ????45
?Orf11 Glycosyltransferase 11355 to 12272 #111 (11737 to 11754) #112 (12013 to 12030) 294bp ????0 ????58
#151 (11367 to 11384) #152 (12154 to 12171) 805bp ????0 ????59
#179 (11482 to 11598) #180 (11839 to 11856) 375bp ????0*** ????50
?Or f12 Glycosyltransferase 12247 to 13176 #113 (12274 to 12291) #114 (13016 to 13033) 767bp ????0 ????50
#153 (12489 to 12506) #154 (13101 to 13120) 632bp ????0 ????59
#181 (12384 to 12401) #182 (12959 to 12976) 593bp ????0 ????50
* in all groups, obtain the band of 2 wrong sizes, * obtains the band of 1 wrong size in 6 groups, * * obtains the bacterium source 1 wild-type e. coli O1 that contains in the band table two 166 strain intestinal bacteria of 1 wrong size and 43 strain Shigellaes and their this group of source group number in all groups, O2, O3, O10, O16, O18, O39 IMVS a2 wild-type e. coli O40, O41, O48, O49, O71, O73, O88, O100 IMVS3 wild-type e. coli O102, O109, O119, O120, O121, O125, O126, O137 IMVS4 wild-type e. coli O138, O139, O149, O7, O5, O6, O11, O12 IMVS5 wild-type e. coli O13, O14, O15, O17, O19ab, O20, O21, O22 IMVS6 wild-type e. coli O23, O24, O25, O26, O27, O28, O29, O30 IMVS7 wild-type e. coli O32, O33, O34, O35, O36, O37, O38, O42 IMVS8 wild-type e. coli O43, O44, O45, O46, O50, O51, O52, O53 IMVS9 wild-type e. coli O54, O55, O56, O57, O58, O59, O60, O61 IMVS10 wild-type e. coli O62, O63, O64, O65, O66, O68, O69, O70 IMVS11 wild-type e. coli O74, O75, O76, O77, O78, O79, O80, O81 IMVS12 wild-type e. coli O82, O83, O84, O85, O86, O87, O89, O90 IMVS13 wild-type e. coli O91, O92, O95, O96, O97, O98, O99, O101 IMVS14 wild-type e. coli O112, O162, O113, O114, O115, O116, O117, O118 IMVS15 wild-type e. coli O123, O165, O166, O167, O168, O169, O170, O171 See b16 wild-type e. coli O172, O173, O127, O128, O129, O130, O131, O132, See c17 wild-type e. coli O133, O134, O135, O136, O140, O141, O142, O143 IMVS18 wild-type e. coli O144, O145, O146, O147, O148, O150, O151, O152 IMVS19 wild-type e. coli O153, O154, O155, O156, O157, O158, O159, O164 IMVS20 wild-type e. coli O160, O161, O163, O8, O9, O124, O111 IMVS21 wild-type e. coli O103, O104, O105, O106, O107, O108, O110 IMVS22 Shigella bogdii serotypes B 4, B5, B6, B8, B9, B11, B12, B14 Seed23 Shigella bogdii serotypes B 1, B3, B7, B8, B10, B13, B15, B16, B17, B18 Seed24 shigella dysenteriae serotype D1, D2, D3, D4, D5, D6, D7, D8 Seed25 shigella dysenteriae serum D9, D10, D11, D12, D13 Seed26 shigella flexneri F6a, F1a, F1b, F2a, F2b, F3, F4a, F4b, F5 (v:7) F5 (v:4) Song Seed27 Nei Shi will Hayes 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, epidemiological study institute of the rest from IMVS d. China Preventive Medicial Science Institute table 3 are structural tables of the O-antigen gene bunch of shigella boydii 7
1kb47O-ctcctggtga ctcatgcgtc caagaacgcg gtcgaaaacc acttcgacac ctcatatgaa 60ttagaatcgc tccttgaaca gcgtgtaaaa cgccagttgc tggcggaagt tcagtccatc 120tgcccaccgg gcgtcaccat tatgaatgtg cgtcagagcg aacctttagg tttgggccac 180tccattttgt gcgccagacc cgccattggc gacaatccat tcgttgtggt gctgcctgat 240gtcgttattg atgatgccag cgccgatccg ctgcgttaca accttgccgc tatgattgcg 300cgattcaacg aaacgggtcg cagtcaggtt ctggcaaaac gtatgccggg tgacctctct 360gaatactctg tcatccagac taaagaaccg ctggatcgtg aaggcaaagt cagtcgcatt 420gttgaattta tcgaaaaacc cgatcagccg cagacactgg attcagacat tatggcggtt 480ggtcgctatg tgctttctgc agacatttgg cctgaactgg agcggactca acccggtgca 540tggggacgta ttcagctaac tgacgcaatt gccgaactgg cgaaaaaaca gtccgttgat 600gcaatgttaa tgacgggtga cagctacgac tgcggtaaaa aaatgggcta catgcaggca 660tttgtgaagt acggactacg caacctgaaa gaaggagtga agttccgcaa agggattgag 720cagctgttaa gcgaataaaa atcttaccgg atgtaacggt taataagaaa atcataacgg 780cagtgaagat tcgtggtgaa agtaaattgc aacgaatgtt cctgccgtta ttgctttata 840aaacactata ataacaatga gttagcaata agattttagt caaatttttc caggattttc 900cttgtttcca gagcggattg gtaagacaat tagcgtttga atttttcggg tttagcgcga 960gtgggtaatg ctcgtcacat cgtaggtatg catgcagtgc tctggtagct gttaagccag 1020gggcggtagc gtgtgttaaa cttaaggggg ttgttgcgag taatcaagat cgcggcaatt 1080tcggattgtg tcttattttg ataataaaac tatcctgata actggtggta ctggctcatt 1140
The initial cggcaataag tttgtgcgca of orf1 Tgacattaga taaatataac cctaaaaaga ttattattta 1200ttctcgtgat gaaatgaaac agtgggaaat ggcaaaacat ttcaaggacg aaagccgaat 1260tcgattcttt attggggatg ttcgtgataa agatcgtttg tatcgtgcgt tggatggtgt 1320tgactttgtt gttcacgccg cagcaacaaa aatcgtacct acagcagagt ataatccctt 1380cgaatgtgta aaaacaaata tcaatggtgc gatgaatgtg attgatgctt gtatcgataa 1440aggtatcgaa cgtgttgttg cactttctac tgataaagca agcagccctg cgaatctgta 1500tggtgcaaca aaattagcat ccgataaact tttcgttgct ggtaactctt attctggtgc 1560gacaaaaact cgttttgctg ttgttcgcta cggaaatgta atgggatcac gtggttcggt 1620aataccgttc ttcttatcta tcaaagaaaa aggagaattg cctattactg atgatcgtat 1680gactcgcttt atgattactc tggagcaggg ggttgagtta gtttggcatg cgtttgaaga 1740tatggtcggt ggtgaaattt atgtaaaaaa aataccttcg atgaaagtaa ctgatatagc 1800aacagctgtt gcgcctaatg ctacacaaaa aatggtcggt attcgaccgg gcgagaaact 1860acacgagcag atgatcagtg ctgaagatgc ttattacact tatgaatatc cagagcattt 1920taagattctt ccagccattc acaactggtg taactcacct gaaaggatta aagatggtaa 1980
The termination aaaagtgcca gaaggcttcg tct of the initial orf1 of orf2 Atgaaag tgatagtaac gcagaatgga tgagtattga 2040agaacttcgt caatggatcg aggataatag agaaaaagta ggtaacattt gatgaaattt 2100attccctatg gacggcagga tgtttctgat gaagatatcc atgctgtggt ggatgtactg 2160aagtccgatt ttttgacgca aggaccatgt gttccccaat ttgagaaggc aattgctgat 2220tatgtgaatg ctaagtatgc agttgcagta aatagtgcta cttcagcact tcacattgct 2280tgtctcgcgt tggggatgaa gaaaggcgat tggctttgga catcgccaaa tacatttgtt 2340gcttctgcga actgtgctct ttattgcggt gctaatgtca gttttgtcga tattgatgcg 2400cggacttata atatgagtgt tagcgcgtta gaagcgaaac ttatgtctgc aaggaacaat 2460ggaacattgc ctaaggttgt tgttcctgtc gcatttgcag gacagtcatg tgaaatggag 2520gaaatatata aactctcaaa agagtatgga ttttcaatta tcgaagatgc ttcgcatgct 2580attggaggaa attaccaggg aaagaaaata ggtaattcgc gctttgcaga tattactata 2640ttgagcttcc atcctgttaa aattattaca actgcagaag gtggtatggc tctaaccaat 2700aatgatgatt tggcagaaaa aatgcagtta ttccgtagtc atgggatcac tcgtgatagt 2760aatcatatga cgaaggtaag tgaaggagga tggtattatc aacaagttga tcttggtctg 2820aattaccgaa tgactgaact gcaggctgca ttaggcgtga gtcagctgaa gagtattgat 2880gattttgtaa cacgccgcca tgaactggtt gaacgttaca aaaaagctct cgaagatatt 2940cctatttccc taccatacca ggcggaaagt ggtcacagtg cctttcacct ttatcctgta 3000accgttaaaa attctgcaca gcggaaaact ttgtttgatt atctgcggaa taaaaatatc 3060ggggttaatg tccattatat tccagttcat actcaaccgt attatgaaaa gttagggcat 3120aaaatcggag attatcctgt cgctgaggat tattactctc gaaccatcag cataccgatg 3180tattctgctt tgacaaatga agaccaggat tatgtaatcc aatgcattcg ggagtttttt 3240orf2 orf3Taaatgaatg tggcaatcat tccagctcgt ggtggtagta aacgtatccc tcgaaaaaat 3300attaaaatgt tttgcgggaa accaatgatt gcctggtcga ttaatgccgc tcgaaagagc 3360ggcgtgtttg acagaatcat tgtttcgacg gatgatatgg agattgcagc tgtagcaaga 3420aaattcggcg ccgaagttcc gtttatgcgg ccagaagaac tttcgaatga ttttactgga 3480acaatccctg ttatacggca tgcggctgaa tggcttattt ctcagggaaa taaaataaaa 3540tatatttgct gcatttatgc aacggccccc tttattagag atgaagattt aaaactaggt 3600ttgaacctta tcaaagcaaa ggggggggat tattgttttt ctgttactac ttattcgtac 3660cctattcaaa gagcattaaa aattaatgag aataacgaaa taaccatgtt tgatccgact 3720cagtttgaaa tgcgatctca agatcttgtg gacgcttggc atgatgccgg gcaattttat 3780tgggggaata ttgatgcatg gataaaagaa aaaccggtat ttaagtcaca ggctatacca 3840ttacctttgc ctagagaacg agtgcaagat attgatacgc cggaagattg gcatttcgct 3900
The termination gaggtgttat ataggataaa tacaataaaa a of the initial orf3 of orf4 AtgAagatt gtatt Taggg ttgatgcttc 3960gctttcaatt ggttcaggac atgtttttcg ttgtctaaat cttgctggtg cattgagaaa 4020gaatggtttt gaatgcattt ttcttacaaa ggaacatgca ggaaacttaa ttcaatatat 4080aaagacatat ttgttccaag tatatgttat ttcaaaggaa gataatgttg atgataaata 4140tattgaaaat gaaaaagagt ggttaggagg gagtcaaaaa gaagacgctg aaaaaacttg 4200tgaaattatt tacagaaata attttgcacc aggcatattt attattgatc attattcatt 4260agattctgaa tgggaaagtt taattaaatg gaaatttcca agtacaaaaa ttgttgttat 4320tgacgacttg tgtaatagaa aacattattg tgatatcctg attgattcaa catttgagcg 4380tacttctgag gaatatagga gattagttcc tgaatattgt actattttgg ccggtactga 4440ctatgcttta ctgaaagatg aatttttgca attgcgcaaa gaagccatat taaaacgtac 4500atctatatta agccctcaga aaattttaat tactatggga ggggttgata tacataacgt 4560gactgggaaa gttttaaaag aattaaataa tagagctgat cttcattata aaaagatcac 4620tgttgtatta ggggtgaatt gccctcataa aaaaaacatt gaaaatattg cacaatcgat 4680aaaatatgat gtggatataa aagtgaatac caacaatatg ccacaactta tgttagaaca 4740tgatttatct attggagctt taggaagtac tacttgggag agagctgtgc ttggattacc 4800cacagtaaat attgctattg ctgacaatca actggttatt gtagagaaac taaggcgaaa 4860tggttttatt gtctttgata gcattaattt tactggggtg gaattagctc gttctttaat 4920aaaggtaact gatttctatg atgaaatggt gtctcgctcg ctctctatat gtgatggcag 4980
The initial gggattacaa aggacactaa aatttataat atctttagct acaaataaa of the termination orf5 of orf4 T aaTgagac At5040gcttatgact ggcaaaaata taaaaaaatt aaatttgctt aacattgttc attcttcgga 5100ggatattcaa gaaaaagttc gatgtataag aaatgaacct caaattagga agtggatgta 5160tacagatcat cttatatcaa aagatgagca taaaaattgg attgaaagat taagaaatga 5220atcaagaaat tttgtgtatg tcgtaatgaa tgactacgat gagcctattg gtgtcgttag 5280tgtaaattcg attgatttga aaaacaaaaa aggagactgg gcatattatt tgacagcaaa 5340tacacgtact ggtattggtg ctgtattaga attttttgtg attgattatg tttttaacta 5400tttgcatctt gaaaaattaa attgtgaggt aattgaacat aatgataccg tcgttcgatt 5460acatgagaaa tttgggttta agcaggaagg atttagagag tctaatatta taaaggatga 5520taagcgtttt ggagtttatt attttggttt aacgaaagaa aaatggatgc taacacgaga 5580
The termination atgtttaaag aataaatatt ataagctttt ttctcaatac gcggttgatt ttcat of orf5 TaaTa 5640
The initial ggtttatttt tggggtgata a of orf6 Atgaaagca attactattg caggacgtaa aattggtaaa 5700gattttcctc catatatcat tgctgaatta tcggctaatc ataatggaga tataaatcgt 5760gcatttaaga taatggaaga ggcaaaagca gctggagctg atgctgtaaa attacagaca 5820tatagagcag atactataac gctggattgt gattctgaag agtttcaaat acatggtggt 5880ttatggggtg gtcagacatt gtatagctta tatcaaagtg ctcaaatgcc ttgggaatgg 5940cataagccat tatttgataa ggcaaaagag ttaggcatta ctatttttag cagtcctttt 6000gattttactg ctgttgattt attagaagaa ttaggtgctc cggcttataa aatagcgtca 6060ttcgaagcta ttgatttacc gctaattcga tatgtagctc aaactgggaa accgatgatt 6120atctcgacag ggatggctga tgaacaggag attcaggagg cgatccatgt agctaaagaa 6180ggcggttgta aggagctcgt tgtcttgcat tgcgtaagtg gatatccggc tccaccagag 6240gattacaatc ttgccacaat ccctgacatg gctaaaaaat ttaacgttat aacaggtctg 6300tcagaccata caatagataa tactacggcg atagcatccg ttgcattagg agcgagtgtg 6360atcgaaaagc atgtaacact tgatcgtaat ggaggagggc cagacgatag tttctcactt 6420gaaccaagtg agcttacagt tctttgccgt gatgcaaaga tcgcttggca ggctttaggt 6480agaataaact ataccagaaa gccaagtgag atgggaaacg ttcaatttag acgttcattg 6540tattttgtta aagatatgaa agctggtgat attattgatg aaacatgtgt gcgcagtgtg 6600cggccaggct atggattacc acctaaatat tttgatgatt taattggtaa gcgagttatt 6660
The termination agtgatgtta tggccaatac ccgaacatca ttggaattaa ttgcagattt aaa of the initial orf6 of orf7 Atgaaag 6720ttattaaaga tagtgttctg tatgtaatag gtgagttgat atctaaaagt gttccatttc 6780tattgcttcc ctatcttaca aggcggttgg gaattgatgg ttttggagaa atgtcatttt 6840atcagacaat acttgcttta ttggttatcg ttataagctt agggcaagat ggggctgttg 6900ctagatattt ttatttctac ggagcccgtt ctataaattt gattgtgact gtaggtgctt 6960tatattcact tgtgacgaca tccctattgt ttttatcgtt ttcatttttt aaatccacga 7020tattatcaat tattgtgttg actgctggat ttcaatcact tttcaatacg cagctaagtc 7080ttaggcagtg ccaaaaagag tcagttcagt atattgtcat gcaaatatta gttgcgttga 7140cttctggtgg tataaccttt tgtttactgg aaatatatag tcatgattta gttgaaaaga 7200gatttattgc attgcttttg agtaatatag tttgtgtttt tattgcttat cttatttata 7260tgaggagacg gaaatataat cacgcatttt tatttcgacg atattatagt gcattgctat 7320acttgattgg ttttggaggc ccattgattt tgcatcaaat aagtggcttt cttaaaggac 7380aattagatcg agttttgata tatagccgtt tttctgcttc agatttaggg ggctattctg 7440ctgcatttca aatagcctca gttctcttga tactgataat ggcgttgaat aaagcactag 7500ttccatatta ttatagtgag ctcaaaaaaa agcatatagt ctttgatgat ataaaaaaaa 7560cttgcgtttt tttatttcta ttgattccac ttccatcaat catatcgttg attattcctg 7620ctgatttttg gtgttttatt ttaggaaatg aatttgttga tgttaaatat tacatcgcta 7680tattcctatt aggtataggt tttacaatac cttatctttt actagtgaat tatctttttt 7740attatggtaa aaataaaagt ataactatct gttcagtcat ttcaacatct atttattgca 7800ttgttcttta ttttttatca tgtttaggta tttcttatgt tccatattct atgttgatag 7860caaatatgct aatcgttcct ttactttata tggctgccaa aaaaacatca atgcttgaac 7920 orf7 orf8gccagTgaGa ttaat Atgcc ctcgttattt atatgtgtta cgccactgca aatgttgatt 7980gctgaaaaaa taattgataa aaccaggcca gtaaacatag aaattatagt gcttgcatat 8040cagaaaaatg ataaatatat gcattacata aagcggttgg agaaaaaatg tactaatttt 8100acagttttgg ctgtaacgcc aaaaaataaa tttgtaaccg tgatcgcctt tgcaaaatta 8160cataccatct taaataaaaa tatgagtaaa acttactctg aggtttatct gtctagtatt 8220gataacaagt atgtacaatt aattgtttct aagctcaatt atgcaaggtt atatacattt 8280gacgatggaa cagcgaatat aattaaatct agtgcatatt atcaagagga aaaaaaaact 8340ctcaagacta acattttgag atggattttt ggaattaata aaggattgca agaaattaag 8400agtgaaatat gcaagcacta tacaatttat ccaagtgtta gtaatattgt ctctaataca 8460gaattgatag agatgtttac acaatgcaaa aaaaggaagc aagataaaaa agttgtgaga 8520gtctttatag gacaaccttt tgatgaatta ggaataccat tatcactaat agaagagttt 8580ttctttaaat acaaaatgga ctattattat ccacacccaa gagagaaaat aattaataat 8640aagttcactt atatacactc acatttgatt tttgaagaat atataattga aggcttacaa 8700catcaagata tcgtatacaa aatatatggt gctgtgtgta caagtatatt aaatttagcg 8760tcctcgaaaa atgatatcga gatatgttcc atttatacgg atgagctgag aactaaatat 8820
The initial agtgattatt atgcactggc tgaaaaaatg aatattacct tgttaaaact tact of the termination orf9 of orf8 Tgat At8880gataaaatat gactaatata acgttcagaa ctctgagaat tccaggatat ttactctcat 8940tatttgttct tttaatcgga tttataacat atttcatctt taatgaaact cttgcagcga 9000atttattagc agtgggggct attggcatta tcattcttga tagtttacat gataagcgga 9060ttttttattt aacacttttt cccgtgtatg tcctcgtagg tcaactcatt tcattattat 9120tagttgagca cggatggatt ctaatcgaac ttggaggtat taaaagctat cccattggtt 9180caattctttt tatggcgtta acaatagttt tattccattt tatcatattt ttaactgtaa 9240aaatttcaat attcaaattt gatcgaacat gtattgataa gttaagtggt aattctttta 9300tttattactt gccgattttt tatctatgct tagtatatat tcctgttttt atttatggct 9360ctgcattatc tgtaacaaat ggtaatcgag ttgtatacaa tcaaataata tcacctgttt 9420ttttatattt atttcaaatt aaacaattta tacttcctgt tgcaggattg tatttgctta 9480aaaataaaaa aatatttact atatatctgt ttgcaatttt actatggaat atattgattg 9540gagagaaggc aacgggcatt tggcagtcat tgtatccaat gctgttacct tatgttttga 9600taaattatga taaaataaaa acaaaaaata tattaatagt attaggtttt tgtattgtat 9660ttataaccag tagcatagta attaattata tatttattga aaagtcaggt gctacgttta 9720tatttgatag aatatcaatg caaggacaat tgtggtggta ttattttaat gagcacgttt 9780tgttagctaa gacccctcat gaattatctg aagagttctc gtcagaatat agtgggcttc 9840ttaatctgat gtatcattca atgcctgccc atcttttcaa tagctatatc gagcgcggtg 9900tagtcttgac gagtggtttc cccgcaatat ttctttttta ctttggacaa tactggctcc 9960ttcctacgtt attatcacct tttctttttg gtttagttgt ctattttttt tcaagaagtt 10020tatatagcgg tagtatattg agtttgctta tatcaagtaa actatttttt gcttttactg 10080ttttttttgc ccgtggagat atcgcaactt ttttagatta taaattgctt atttatctat 10140
The initial tgattataat aattttacaa tatttgccaa gggttaaggt t of the termination orf10 of orf9 Taat Atggc gtatacttct 10200cactttatat ataaagtagc taataagatt agcattttta taagaaaaat catcaactta 10260tttagattat ttctttacgg tgatgttgtt tcatatgatt ttgaaatgga ggagaataga 10320agaattatag tgtcaggaag tctaacaaag agagataaca tacctaaaat aatttggatt 10380tattggcatg atgagcaaat tccattatta atagaggctt gtataaaacg ccttaaagat 10440ttaaatccta attatcaatt aaatgtgcta aataaattta caattcatga atacttacct 10500gagagtctaa tgtggagaca ggatctttct gtccaaataa gatctgatct gattcgcttg 10560ggattattat ataaatttgg tggagtatgg atagatgcta gtgtaatgtt ttttgaggat 10620ctttcatggt tagaaagact ttcagcagag aataaatatg atttaatcgg tttttatcga 10680ggcagactat ctatcgactg ctataatcca atagtagaaa gttggatgct gggggcacca 10740aaatacaata agtttataaa gcgatggtat gacaactttt atccagtatc ttatttaggg 10800ttagatgaat atttcaacac aattaagaaa agaagtgatt ataaaattat caaacagaat 10860ataaatattc ccaaatattt atctgtttat ctggcacaac aaatttcgtc gcgagaaatg 10920agagattatt gcgcttattt aagaaaggta gaggatagtg catatttata tcaagagtca 10980tatccacagc gtgattgtgt tctctcggag gtttggtgta aaagaagaaa accacagaaa 11040attcctccac taatcaaatt aacaagccag aacagaaata caattttagc ctgtagtaaa 11100
The termination ataaatccac gtagtatcgt aggtgagttt ttagataata agaat of orf10 TagGa atagagaaag 11160tgcgagataa aattgtattt gtagtcgctg atatcacggc tactggcggg attgaacgag 11220ttattactta tcttgccagt tatttatgta ataatgggta tagtgtagag atattatcta 11280ttcatagaag taatcgtaat ttaccttatc ctgttcatgc agacgttcat atctcttttg 11340
The initial ttgataaaac tata of orf11 Atgaag gcaagaaagc caggatctgt cagcaaatta ctttcacatt 11400ttaaatcctc atttcattta aatataaaat tatttaagag aagaaaatgt attattgtag 11460caaatagttt tcctgttgct ttattttcct gcttctcggc cttattttca tcaaaaatgc 11520tcgttgtcga gcatgttcat tatcactatt acagtaagtg cttagttaat gtaaggcgtt 11580tcatttataa gtttttttat ggggtagttg cgttaactga gcgtgacagt gagctttata 11640ttaatgataa actaaattct ctaacaatac ctaacgctct tagttctttt cctgaaaaaa 11700tagcgaatcc atctgtggaa aagaaaaaaa taattgccgc tggaagatta gaacatcaaa 11760aagggtttga tattctaata aaatcctttg ctgaaataga ctattctgtg aggaaagaat 11820ggatcatgga tatttatggg gatggaaatg aaagagctgc cttacagaaa ttaattcatg 11880atttaaatat tgaggattgt gttaatttat tgggtaacag tcgagacctt atgaacactt 11940atgctgaata tgattttttt gtattatctt caagatttga aggtttcggg atggttttac 12000ttgaagccat gagttgcggt cttccttgta ttgccatcga ttgtcctaca gggcctagag 12060aaatacttga tggaggcaag tatgggatac tttgtgacaa ccaatcaaat ctgggggaga 12120gtattgcatc tctaattgtt aataaggatc taagaactat gctatccggc aaaagcattt 12180tacgtagtca tgattataat attcagatta taagtgaaaa atggcagggt ttatttgaaa 12240 orf12 orf11gcttaaAtgA aaaaaatata tatgaaaaa t Aatatcgcac ttgttcttgt tacgtataat 12300agagagtctc ttttaaaaga agtcctttca tcgataaagc atttaaaaga taaaccgaaa 12360catgtttata taatcgataa taatagctca gatggaacct gtaatataat tatagatttt 12420attcaacaga attcatctat tctaagtatg agttatcata atacaggaga taatctcggt 12480ggggctggcg gatttgctta cggtagccgg cttgcttatg ccgatggatt ccaatggatt 12540tggttagctg atgatgatgt tgtttttgag caaacatgtc tgactcaatt gatgctttat 12600tcttcagatg ccgatatatt acagccaatg agaatcaata ctgacggtag ctgtgcagaa 12660atttcaggaa tagattacga aataaacaat atcttcaggt taaaccctaa aaaacttaaa 12720attactgata tatatgaaga aaagtgggat attcaagaaa tcaaaacgat accttttgaa 12780ggaccattaa tacatcgacg tgtatttgag cagattggtt ttcccaaccc cgattttttt 12840attttttatg atgatctaga ttttgcattg agggctcaaa gagcaggctt caagattaaa 12900tgtgtaaaat ctgctcatct gattcgtaag attagatttg ttcaaagcgt cgctttgacg 12960tcttggaaag gttattttat gtatcgtaac ttttttaagg ttcaacttac gtatgcgaga 13020ataccgatag gattatttag agttattgct atatttttta ttgtaataat ttattctcta 13080ctatttggag gtgcaaaaaa catttcgact ttgataagtg cattgcgtga tgcattaagg 13140
The initial aaaaattttc cacttgatca acgttataaa ccc of the termination orf13 of orf12 TaaTtta aaagttaaaa tc Atgaaaaa 13200taaaatagct attgtcggtg caggcttaag tggggcagta atagctcgtg aacttgctga 13260tgctggatat ttagttgatg tttttgataa acgtcaacat attggaggta actgctatac 13320cgctagggat cctcaaacaa atattatggt tcatacatat ggcccacata tatttcatac 13380taacagtaaa tttgtttggg cttatattaa taagctgggg gtatttaaac cgtttactaa 13440ccgcgttaaa gctgttacta gaggacaggt tttttctttg cctattaacc tattaactat 13500aaatcaattc ttcaataaat tatttacacc agcagaagca aaaaaatata ttgaatctat 13560atctgataaa tatattaaag atcctaatac gtttgaagaa caagctttat cctttattgg 13620acgtgaatta tatgaggctt ttttcaagtc ttatacaatt aaacagtggg ggattttacc 13680taccgatata ccagccagta ttctcaaacg actgcctgtc cgttttaatt atgatgataa 13740ttatttttcg catcaatatc agggaatgcc ggaggacggc tatacacctc tattcgaaaa 13800tcttctgaat cactccaata tcaatgttat tctttctacg gaattctcgg caattgattg 13860tgataaatat attcatactt tttatagtgg cacaatcgat gggttctttg attatgattt 13920aggtcgtttg ctatatagaa ctctagactt taaaacggaa gtgttttggg gagattatca 13980gggatgtgca gtaatgaatt attgtgataa ttctaaacca tatacacgta ttactgaaca 14040taaatatttt tctccttggg aacaacatga caaaacaata atttacaaag aatatagtag 14100agaatgtaat gatagtgata ttccctatta ccccgtacga ctggtcagtg ataaagaaat 14160attaaaaaaa tatgtggaac gtgccagtaa acttgaaaat gttacttttg ttggccgatt 14220gggtacgtat cgatatttag atatggatgt aactatcact gaggccttaa acaccgccga 14280acagttttta gaacttcacc aaaataatca aagaatacca gctttttttg ttgatgtttt 14340orf13aTagcgagtc aacatgatta tattgtagta cataatatta cgttcatcct gataaattta 14400atctatattt tgctcatggc tttaaattta aagctattct tcatataata gcactaattg 14460cggtaacccc tgacaggagt aaacaatgtc aaagcaacag atcggcgtcg tcggtatggc 14520ggtgatgggg cgcaaccttg cgctcaatat cgaaagccgt ggttataccg tctctatttt 14580caaccgttcc cgtgaaaaga cggaagaagt gattgccgaa aattctggca aaaaactggt 14640tccttactat acggtgaaag agttcgttga atctcttgaa acgcctcgtc gcatcctgtt 14700aatggttaaa gcaggtgcag gcacagatgc tgctattgat tcccttaagc cataccttga 14760aaaaggcgac atcatcattg atggcggtaa caccttcttc ctggacacca ttcgtcgtaa 14820ccgtgagctt tctgcggaag gttttaactt cattggtacg ggggtttccg gtggtgaaga 14880aggtgcgctg aaaggtcctt ctatcatgcc tggcggtcag aaagaagcct atgaattggt 14940tgcgccgatc ctgaccaaaa tcgctgccgt agctgaagat ggcgaaccgt gcgttaccta 15000tattggtgca gacggcgcag gtcattatgt gaagatggtt cacaacggta ttgaatacgg 15060tgatatgcag ttgattgccg aagcttattc actgcttaaa ggtggcctga atctctccaa 15120cgaagaacta gcgcagacct ttaccgagtg gaataacggt gaactgagca gttacctgat 15180cgacataacc aaagacatct tcaccaaaaa agatgaagac ggtaactacc tggttgatgt 15240gattctggat gaagcggcta acaaaggtac cggtaaatgg accagccaga gtgcgctgga 15300tctcggcgaa ccgctgtcgc tgattaccga gtctgtgttt gcacgttata tctcgtctct 15360gaaagatcag cgtgttgccg cgtctaaagt tctctctggt ccgcaggcac agccagcagg 15420cgacaaagcg gagttcatcg agaaagttcg ccgtgcgctg tatcttggca aaatcgtttc 15480ttatgctcag ggcttctctc aattgcgtgc cgcgtctgaa gagtatcact gggatctgaa 15540ctacggtgaa atcgcgaaga ttttccgtgc tggctgcatc atccgtgcgc agttcctgca 15600gaaaatcacc gatgcctatg ccgaaaacct aaaaatcgct aacctgctgt tggctccgta 15660ttttaagaaa atcgctgacg actaccaaca agcactgcgt gatgtcgtcg cttatgcagt 15720gcaaaacggt attccggttc caaccttctc tgcggcggtt gcttattacg acagctatcg 15780tgccgctgtcctgcct 15796
The above, it only is preferred embodiment of the present invention, be not that the present invention is done any pro forma restriction, every foundation technical spirit of the present invention all still belongs in the scope of technical solution of the present invention any simple modification, equivalent variations and modification that above embodiment did.

Claims (9)

1, a kind of Nucleotide of the O-antigen-specific to shigella boydii 7 is characterized in that it is the isolating Nucleotide shown in SEQ ID NO:1,15796 bases of total length; The base that perhaps has one or more insertions, disappearance or replacement keeps the Nucleotide of the SEQ ID NO:1 of described isolating functional nucleotide simultaneously.
2, according to the Nucleotide of the described O-antigen-specific to shigella boydii 7 of claim 1, it is characterized in that it is by 11 genomic constitutions, they are all between galF gene and gnd gene.
3, according to the Nucleotide of the described O-antigen-specific to shigella boydii 7 of claim 2, it 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, orf10, orf11, orf12 gene; Wherein said wzx gene is the Nucleotide of 6714 to 7928 bases among the SEQ ID NO:1; The orf8 gene is the Nucleotide of 7936 to 8877 bases among the SEQ ID NO:1; The wzy gene is the Nucleotide of 8889 to 10184 bases among the SEQ ID NO:1; The orf10 gene is the Nucleotide of 10186 to 11148 bases among the SEQ ID NO:1; The orf11 gene is the Nucleotide of 11355 to 12272 bases among the SEQ ID NO:1; The orf12 gene is the Nucleotide of 12247 to 13176 bases among the SEQ ID NO:1;
4, according to the Nucleotide of claim 1 or 2 described O-antigen-specifics to shigella boydii 7, it is characterized in that it is orf8, orf10, orf11, the orf12 gene that comes from described wzx gene, wzy gene or glycosyltransferase gene; Or the oligonucleotide in the sugared synthesis path gene; And their mixing or their reorganization.
5, according to the Nucleotide of the described O-antigen-specific to shigella boydii 7 of claim 4, it is characterized in that the oligonucleotide of the described wzx of coming from gene is to being: the Nucleotide of 6782 to 6799 bases among the SEQ ID NO:1 and the Nucleotide of 7449 to 7466 bases; The Nucleotide of 6971 to 6989 bases among the SEQ ID NO:1 and the Nucleotide of 7481 to 7498 bases; The Nucleotide of 7035 to 7053 bases among the SEQ ID NO:1 and the Nucleotide of 7588 to 7607 bases; The oligonucleotide that comes from the orf8 gene is to being: the Nucleotide of 8065 to 8082 bases among the SEQ ID NO:1 and the Nucleotide of 8789 to 8806 bases; The Nucleotide of 8134 to 8152 bases among the SEQ ID NO:1 and the Nucleotide of 8685 to 8704 bases; The Nucleotide of 8198 to 8215 bases among the SEQ ID NO:1 and the Nucleotide of 8459 to 8476 bases; The oligonucleotide that comes from the wzy gene is to being: the Nucleotide of 9091 to 9108 bases among the SEQ ID NO:1 and the Nucleotide of 9457 to 9474 bases; The Nucleotide of 9357 to 9376 bases among the SEQ ID NO:1 and the Nucleotide of 9704 to 9721 bases; The Nucleotide of 9120 to 9137 bases among the SEQ ID NO:1 and the Nucleotide of 9945 to 9962 bases; The oligonucleotide that comes from the orf10 gene is to being: the Nucleotide of 10271 to 10288 bases among the SEQ ID NO:1 and the Nucleotide of 11060 to 11077 bases; The Nucleotide of 10375 to 10393 bases among the SEQ ID NO:1 and the Nucleotide of 10790 to 10808 bases; The Nucleotide of 10510 to 10528 bases among the SEQ ID NO:1 and the Nucleotide of 11090 to 11108 bases; The oligonucleotide that comes from the orf11 gene is to being: the Nucleotide of 11737 to 11754 bases among the SEQ ID NO:1 and the Nucleotide of 12013 to 12030 bases; The Nucleotide of 11367 to 11384 bases among the SEQ ID NO:1 and the Nucleotide of 12154 to 12171 bases; The Nucleotide of 11482 to 11598 bases among the SEQ ID NO:1 and the Nucleotide of 11839 to 11856 bases; The oligonucleotide that comes from the orf12 gene is to being: the Nucleotide of 12274 to 12291 bases among the SEQ ID NO:1 and the Nucleotide of 13016 to 13033 bases; The Nucleotide of 12489 to 12506 bases among the SEQ ID NO:1 and the Nucleotide of 13101 to 13120 bases; The Nucleotide of 12384 to 12401 bases among the SEQ ID NO:1 and the Nucleotide of 12959 to 12976 bases.
6, the Nucleotide of the described O-antigen-specific to shigella boydii 7 of claim 1 is detecting the application of expressing the antigenic bacterium of O-, identify other polysaccharide antigen of bacterium O-antigen and bacterium in diagnosis.
7, the recombinant molecule of the Nucleotide of the described O-antigen-specific to shigella boydii 7 of claim 1, and can provide the O-antigen of expressing shigella boydii 7 by inserting to express, and become bacterial vaccine.
8, according to the application of the Nucleotide of the described O-antigen-specific to shigella boydii 7 of claim 1, it is characterized in that, it is used for PCR, is used for hybridization and fluoroscopic examination or is used to make gene chip or microarray, the bacterium in human body and the environment as probe as primer.
9, according to the separation method of the Nucleotide of the described O-antigen-specific to shigella boydii 7 of claim 1, it is characterized in that it comprises the steps:
(1) extracts genome.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 primary isoamyl alcohol (25: 24: 1) 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 the Long pcr amplification shigella boydii 7 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 15796 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: primary isoamyl alcohol (25: 24: 1) 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 1.25ul100mM 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 (24: 1) 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, and the 10 * buffer of 9ul and the T4DNA ligase enzyme of 25 units are wherein arranged, and connects mixture with the dehydrated alcohol precipitation at last, is dissolved in after 70% ethanol is washed obtaining 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 37 ℃ of incubated overnight on the LB solid medium that contains penbritin, X-Gal and IPTG, 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 has constituted the O-antigen gene bunch library of shigella boydii 7;
(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, 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 boydii 7 again to the order-checking of PCR product, thus obtain all sequences of O-antigen gene bunch.
(5) splicing of nucleotide sequence and analysis.The Pregap4 and the splicing of Gap4 software of the Staden package software package of publishing with Britain Camb MRC (Medical ResearchCouncil) Molecular Biology Lab and edit all sequences, thus the Nucleotide full length sequence (seeing sequence list) of the O-antigen gene bunch of shigella boydii 7 obtained.The quality of sequence is mainly guaranteed by two aspects: 1) genome of shigella boydii 7 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 boydii 7 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 13 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 boydii 7 at last
(6) screening of specific gene.At wzx, wzy, orf8, orf10, orf11, the orf12 gene design primer in the O-antigen gene of shigella boydii 7 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, 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, orf10, orf11, orf12 gene pairs shigella boydii 7 all is high special.
CNB031005365A 2003-01-20 2003-01-20 Nucleotide specific to O-antigen of shigella boydii 7 Expired - Fee Related CN1168827C (en)

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Publication number Priority date Publication date Assignee Title
CN103323597A (en) * 2012-11-21 2013-09-25 北京农学院 Colloidal gold rapid detecting card for shigella detection and preparation method thereof

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103323597A (en) * 2012-11-21 2013-09-25 北京农学院 Colloidal gold rapid detecting card for shigella detection and preparation method thereof

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