CN1234719C - Nucleotide with specificity to o-antigen of type-12 shigella shigae and colibacillus 0152 - Google Patents
Nucleotide with specificity to o-antigen of type-12 shigella shigae and colibacillus 0152 Download PDFInfo
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Abstract
The present invention relates to a specific nucleotide for the O-antigens of Shigella dysenteriae 12 and Escherichia coli O152. The specific nucleotide is the nucleotide complete sequence of gene clusters in Shigella dysenteriae 12 for controlling the synthesis of O-antigens. The present invention also comprises the structure of O-antigen gene clusters and the oligonucleotide of glycosyltransferase genes and unit processing genes (comprising wzx genes or genes with the similar functions of wzx, wzy genes or genes with the similar functions of wzy) stemmed from the O-antigen gene clusters of Shigella dysenteriae 12, and a method for obtaining bacteria O-antigen gene clusters. The present invention verifies that the oligonucleotide has high specificity to all of the O-antigen genes of Shigella dysenteriae 12 and Escherichia coli O152 through PCR. The present invention also discloses a method for using the oligonucleotide of the present invention to detect and identify Shigella dysenteriae 12 and Escherichia coli O152 in human bodies and environment.
Description
Technical field
The present invention relates to the complete nucleotide sequence of control O-antigen synthetic gene cluster in shigella dysenteriae 12 types (Shigella dysenteriae 12), particularly relate in shigella dysenteriae 12 types oligonucleotide in the control O-antigen synthetic gene cluster, can utilize these oligonucleotide of O-antigen-specific shigella dysenteriae 12 types and the intestinal bacteria O152 (Escherichia coli O152) 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 O antigens " .Trends inMicrobiology.3:178-185 on the glycolipid molecule; Schnaitman, C.A.and J.D.Klena. (1993) " Genetics of lipopolysaccharidebiosynthesis in entericbacteria " .Microbiological Reviews, 57 (3): 655-682].Coding is responsible for the generally adjacent arrangement on karyomit(e) of gene of all enzyme molecules of O-antigen synthetic, form a gene cluster [Reeves, P.R., etal. (1996) " Bacterial polysaccharide synthesis and gene nomenclature " Trends in Microbiology, 4:495-503].In Shigellae, intestinal bacteria and Salmonellas, O-antigen gene [Lei Wang.etal (2001) " Sequence analysis of four Shigella boydii O-antigen loci:implication for Escherichiacoli and Shigella relationships " .Infection and Immunity, 11:6923-6930 bunch between galF and gnd gene; Lei Wang and PeterReeves (2000) " The Escherichia coli O111 and Salmonella enterica O35 gene clusters:geneclusters encoding the same colitose-containing O antigen are highly conserved " .Journal ofBacteriology.182:5256-5261].The O-antigen gene bunch contains three genoids: sugared synthesis path gene, glycosyltransferase gene, oligosaccharide unit treatment gene, the required nucleoside diphosphate monose of enzymic synthesis O-antigen of wherein sugared synthesis path genes encoding; Thereby the enzyme of glycosyltransferase gene coding forwards nucleoside diphosphate monose and other molecule to and makes monose aggregate into oligosaccharide unit on the monose; The oligosaccharide unit treatment gene comprises transhipment enzyme gene and pol gene, and they transfer to the bacterium inner membrance outside with oligosaccharide unit, and repolymerization becomes polysaccharide.Glycosyltransferase gene and oligosaccharide unit treatment gene only are present in the gene cluster of carrying these genes.The difference of monose in the O-antigen, between monose between the difference of link button and the oligosaccharide unit difference of link button constituted the antigenic diversity of O-, and the composition of monose, the link button between monose and the link button between the oligosaccharide unit are by the Gene Handling in the O-antigen gene bunch, so the O-antigen gene bunch has determined O-antigenic synthetic, has also determined the antigenic diversity of O-.
Because O-antigen is extremely strong antigen, be one of important paathogenic factor of 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 of abequose and paratose synthase genes (rfb) by polymerase chain reaction for identification of S.enterica major serogroups (A; B; C2; and D) ", 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 [" Molecular microbiological investigation of an outbreak of Hemolytic-Uremic Syndromecaused by dry fermented sausage contaminated with Shiga-like toxin producing Escherichiacoli " .J.Clin.Microbiol.34:1622-1627] of the oligonucleotide that comes from the wbdI gene of the O-antigen-specific of R.coli O111 having been identified the toxogenic E.coli O111 of a strain, but afterwards studies show that Paton, the usefulness of A.W et.al comes from the oligonucleotide of wbdI gene and identifies that the method for the serotype of E.coli O111 has false positive results to occur.Bastin D.A.andReeves, 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 coliO111.Gene 164:17-23], and have this sugar in the antigenic structure of the O-of other bacterium, so sugared synthesis path gene is not a high special for O-antigen yet.
Shigellae has 46 kinds of serotypes, but have only 33 kinds of different O-antigens, intestinal bacteria have 166 kinds of different O-antigen [Reeves, P.R (1992) " Variation in O antigens; niche specific selection and bacterialpopulations " .FEMS Microbiol.Lett, 100:509-516], 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 dysenteriae 12 types just have identical O-antigen [Ewing with intestinal bacteria O152, W.H. (1986) " Edwards and Ewing ' s identification of theEnterobacteriaceae " .Elsevier Science Publishers, Amsterdam, The Netherlands; T.cheasty, etal. (1983) " Antigenic relationships between the enteroinvasive Escherichia coli antigensO28ac; O112ac; O124, O136, O143; O144; O152 and O164 and Shigella O antigens " J.clinMicrobiol, 17 (4): 681-684], traditional serotype method can not be distinguished them.
Summary of the invention
The Nucleotide that the purpose of this invention is to provide a kind of O-antigen-specific to shigella dysenteriae 12 types and intestinal bacteria O152.It is the Nucleotide in the O-antigen gene bunch of shigella dysenteriae 12 types, is the special Nucleotide that comes from glycosyltransferase gene and transhipment enzyme gene and pol gene.
An object of the present invention is to provide the full length nucleotide sequence of the O-antigen gene bunch of shigella dysenteriae 12 types.
Another object of the present invention has provided the gene of the O-antigen gene bunch that constitutes shigella dysenteriae 12 types: the gene of transhipment enzyme is the wzx gene or with wzx the gene of identity function is arranged; Pol gene is the wzy gene or with wzy the gene of identity function is arranged; Glycosyltransferase gene comprises orf5, orf7, orf9, orf10 gene; Sugar synthesis path gene comprises rmlB, rmlD, rmlA, rmlC.
Another object 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 dysenteriae 12 types respectively comprises orf5, orf7, orf9, orfl0 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 dysenteriae 12 types and intestinal bacteria O152; Especially the oligonucleotide of listing in the table 1, they are high specials to the O-antigen of shigella dysenteriae 12 types and intestinal bacteria O152, and these oligonucleotide are also reconfigurable, and the oligonucleotide after the combination also is a high special to the O-antigen of shigella dysenteriae 12 types and intestinal bacteria O152.
The above-mentioned oligonucleotide that another object of the present invention provides can be used as primer and is used for nucleic acid amplification reaction, perhaps be used for hybridization as probe, perhaps be used to make gene chip or microarray, thereby detect and identify O-antigen and detection and evaluation shigella dysenteriae 12 types and the intestinal bacteria O152 of shigella dysenteriae 12 types and intestinal bacteria O152 by these methods.
Another object of the present invention has provided the method for the complete sequence of the O-antigen gene bunch that separates shigella dysenteriae 12 types.Can obtain the complete sequence of the O-antigen gene bunch of other bacteriums according to present method operation, the complete sequence of the gene cluster of the bacterium of other polysaccharide antigens that also can obtain to encode.
Of the present invention aspect first, the full length nucleotide sequence of the O-antigen gene bunch of shigella dysenteriae 12 types is provided, its complete sequence shown in SEQ ID NO:1,12235 bases of total length; The base that perhaps has one or more insertions, disappearance or replacement keeps the Nucleotide of the SEQ ID NO:1 of described isolating functional nucleotide simultaneously.Obtained the structure (listing) of the O-antigen gene bunch of shigella dysenteriae 12 types by method of the present invention in Fig. 1, it is altogether by 10 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 dysenteriae 12 types 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 orf5, orf7, orf9, orfl0 gene; Special sugared synthesis path gene in the bacterial polysaccharides antigen comprises rmlB, rmlD, rmlA, rmlC gene, and their zero positions in O-antigen gene bunch and final position and nucleotide sequence all are listed among Fig. 2.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 dysenteriae 12 types and intestinal bacteria O152.
Aspect the 3rd of the present invention, wzx gene in the O-antigen gene bunch that comes from shigella dysenteriae 12 types is provided or the gene, wzy gene of identity function is arranged or the gene and the glycosyltransferase gene of identity function are arranged with wzy with wzx, the oligonucleotide that comprises orf5, orf7, orf9, orf10 gene, they are any one section oligonucleotide in these genes.But, be that the oligonucleotide of listing in the table 1 is right preferentially by usefulness, in table 1, also listed these oligonucleotide to the position in O-antigen gene bunch and with these oligonucleotide to being the size of the product of the PCR reaction done of primer, the annealing temperature in these PCR reaction free lists is carried out.These primers are being to obtain expecting the product of size in the pcr amplification that carries out of template with shigella dysenteriae 12 types and intestinal bacteria O152 only, and are all not obtain expecting the product of size in the pcr amplification that carries out of template the 166 strain intestinal bacteria listed with table 2 and 43 strain Shigellaes.In more detail, with these oligonucleotide to being that PCR that primer is done is reflected at and does not all obtain spawn in most of bacteriums, though in some bacterium, obtained PCR product band, but its size does not meet the expection size, this is that this problem can be avoided by being PCR with intragenic other primer because primer is attached to genomic other position and causes.So, can determine these primers promptly the listed oligonucleotide of table 1 be high special to shigella dysenteriae 12 types and intestinal bacteria O152 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 " mainly 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 orf5 gene (nucleotide position is 4626 to 5663 bases from SEQ ID NO:1), wzx gene (nucleotide position is 5663 to 6874 bases from SEQ ID NO:1), orf7 gene (nucleotide position is 6877 to 7869 bases from SEQ ID NO:1), wzy gene (nucleotide position is 7882 to 8955 bases from SEQ ID NO:1), orf9 gene (nucleotide position is 8960 to 9721 bases from SEQ ID NO:1), orf10 gene (nucleotide position is 9718 to 10788 bases from SEQ ID NO:1).Coming from above intragenic oligonucleotide all is high special to shigella dysenteriae 12 types (SEQ ID NO:1) and intestinal bacteria O152.
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 gene of identity function arranged or with wzy oligonucleotide and the combination that comes from the oligonucleotide in the sugared synthesis path gene in the gene of identity function are arranged with wzx.
On the other hand, the present invention relates to the evaluation of oligonucleotide, they can be used for detecting the O-antigen of expressing the antigenic bacterium of O-and identifying bacterium in diagnosis.
The present invention relates to a kind of antigenic method of one or more bacterial polysaccharideses that detects in the food, these antigens can make sample can with the oligonucleotide specific hybrid of following at least one gene, these genes are: (i) gene of the encoding glycosyl transferring enzyme gene of transhipment enzyme and polysaccharase of (ii) encoding comprises the wzx gene or the gene, wzy gene of identity function is arranged or with wzy the gene of identity function is arranged with wzx.At least one oligonucleotide can be hybridized with at least one more than one such gene specific of expressing the special antigenic bacterium of O-under the situation of condition permission, and these bacteriums are shigella dysenteriae 12 types or intestinal bacteria O152.Available PCR method detects, more can with behind the Nucleotide mark in the inventive method as probe by hybridization such as southern-blot or fluoroscopic examination, perhaps by antigen and bacterium in gene chip or the microarray assay sample.
The present inventor considers following situation: when one special oligonucleotide detects when invalid, the mixture of oligonucleotide can with the target region specific hybrid with test sample.Therefore the invention provides a cover oligonucleotide and be used for detection method of the present invention.Here said oligonucleotide is meant the gene that comes from the encoding glycosyl transferring enzyme, the gene of coding transhipment enzyme and the gene of polysaccharase, comprises the wzx gene or the gene, wzy gene of identity function is arranged or with wzy the oligonucleotide of the gene of identity function is arranged with wzx.This cover oligonucleotide is special to the O-antigen of a special bacterium, and this special bacterium O-antigen is expressed by shigella dysenteriae 12 types or intestinal bacteria O152.
On the other hand, the present invention relates to a kind of antigenic method of one or more bacterial polysaccharideses that detects in the movement, these antigens can make sample can with the oligonucleotide specific hybrid of following at least one gene, these genes are: (i) gene of the encoding glycosyl transferring enzyme gene of transhipment enzyme and polysaccharase of (ii) encoding comprises the wzx gene or the gene, wzy gene of identity function is arranged or with wzy the gene of identity function is arranged with wzx.At least one oligonucleotide can be expressed more than one such gene specific hybridization of the special antigenic bacterium of O-with at least one under the situation of condition permission.These bacteriums are shigella dysenteriae 12 types or intestinal bacteria O152.Oligonucleotide among available the present invention is made the method test sample of primer by PCR, also can with behind the oligonucleotide molecules mark among the present invention as probe by hybridization such as southern-blot or fluoroscopic examination, perhaps by antigen and bacterium in gene chip or the microarray assay sample.
Generally, a pair of oligonucleotide may with same gene recombination also can with different gene recombinations, but must have in them an oligonucleotide can specific hybrid to the distinguished sequence of special antigenic type, another oligonucleotide can be hybridized in non-specific zone.Therefore, when the oligonucleotide in the special polysaccharide antigen gene cluster is reconfigured, can select specific gene mixture hybridization in a pair of oligonucleotide and the polysaccharide antigen gene cluster at least, perhaps select many mixture hybridization oligonucleotide and specific gene.Even even when all genes were all unique in the specific genes bunch, this method also can be applied to discern the nucleic acid molecule of the gene mixture in this gene cluster.Therefore the invention provides a whole set of is used to detect the many to oligonucleotide of the inventive method, many here is that the gene of the gene that comes from the encoding glycosyl transferring enzyme, coding transhipment enzyme and polysaccharase comprises the wzx gene or the gene, wzy gene of identity function arranged or with wzy the gene of identity function is arranged with wzx to oligonucleotide, this cover oligonucleotide is special to a special bacterial polysaccharides, and this cover oligonucleotide may be the Nucleotide of necessary gene during sugar synthesizes.
On the other hand, the present invention also relates to the antigenic method of one or more bacterial polysaccharideses in the sample that a kind of detection comes from patient.One or more bacterial polysaccharides antigens in the sample can make sample can with a specific hybrid in a pair of oligonucleotide in following at least one gene, these genes are: (i) gene of the encoding glycosyl transferring enzyme gene of transhipment enzyme and polysaccharase of (ii) encoding comprises the wzx gene or the gene, wzy gene of identity function is arranged or with wzy the gene of identity function is arranged with wzx.Under the situation of condition permission at least one oligonucleotide can with sample at least one express more than one such gene specific hybridization of the special antigenic bacterium of O-, these bacteriums are shigella dysenteriae 12 types or intestinal bacteria O152.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 dysenteriae 12 types first, and can from the sequence of this total length gene cluster of not cloned, produce recombinant molecule, can produce the O-antigen of expressing shigella dysenteriae 12 types by inserting to express, and become useful vaccine.
Description of drawings
Fig. 1 is the structure iron of the O-antigen gene bunch of shigella dysenteriae 12 types, listed the structure of the O-antigen gene bunch of shigella dysenteriae 12 types in the drawings, altogether by 10 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.
Fig. 2 is the site plan of the gene in the O-antigen gene bunch of shigella dysenteriae 12 types, listed the accurate position of all open reading frame in complete sequence in the O-antigen gene bunch of shigella dysenteriae 12 types in the drawings, at the underscoring of the initiator codon and the terminator codon of each open reading frame.
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 (New York:Cold Spring Harbor LaboratoryPress, 1989).
Embodiment 1: genomic extraction
37 ℃ of incubated overnight Shigellaes in the LB of 5mL substratum, centrifugal collecting cell.With 500 μ l 50mMTris-HCl (pH8.0) and 10 μ l 0.4M EDTA re-suspended cells, 37 ℃ of incubations 20 minutes, the N,O-Diacetylmuramidase that adds 10 μ l 10mg/ml then continues insulation 20 minutes.The Proteinase K, the 15 μ l 10%SDS that add 3 μ l 20mg/ml afterwards, 50 ℃ of incubations 2 hours add the RNase of 3 μ l 10mg/ml again, 65 ℃ 30 minutes.Add equal-volume phenol extracting mixture, get supernatant and use isopyknic phenol again: chloroform: primary isoamyl alcohol extracting twice, get supernatant again with isopyknic ether extracting to remove remaining phenol.Supernatant rolls out DNA and washes DNA with 70% ethanol with glass yarn with 2 times of volume ethanol deposit D NA, at last DNA is resuspended among the 30 μ l TE.Genomic dna detects by 0.4% agarose gel electrophoresis.
Embodiment 2: by the O-antigen gene in pcr amplification shigella dysenteriae 12 types bunch
The O-antigen gene of shigella dysenteriae 12 types bunch is by the Long pcr amplification.At first according to the JumpStart sequences Design upstream primer (#1523-ATT GTG GCT GCA GGG ATCAAA GAA AT) that often is found in O-antigen gene bunch promoter region, again according to the gnd gene design downstream primer (#1524-TAG TCG CGT GNGCCT GGA TTA AGT TCG C) in O-antigen gene bunch downstream.With the Expand Long Template PCR method of Boehringer Mannheim company amplification O-antigen gene bunch, the PCR response procedures was as follows: 94 ℃ of pre-sex change 2 minutes; 94 ℃ of sex change are 10 seconds then, 60 ℃ of annealing 30 seconds, and 68 ℃ were extended 15 minutes, and carried out 30 circulations like this.At last, continue to extend 7 minutes at 68 ℃, obtain the PCR product, the agarose gel electrophoresis with 0.8% detects the size and the specificity thereof of PCR product.Merge 6 pipe longPCR products, and with the Wizard PCR Preps purification kit purified pcr product of Promega company.
The structure in embodiment 3:O-antigen gene bunch library
Make up O-antigen gene bunch library with the Novagen DNaseI shot gun method that is modified.Reaction system is a 300ng PCR purified product, 0.9 μ l 0.1M MnCl
2, the DnaseI of the 1mg/ml of 1 μ l 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 2 μ l 0.1M EDTA termination reactions.Merge the same reaction system of 4 pipes, with isopyknic phenol extracting once, use isopyknic phenol: chloroform: primary isoamyl alcohol (25: 24: 1) extracting once, after using isopyknic ether extracting once again, dehydrated alcohol deposit D NA with 2.5 times of volumes, and with 70% ethanol, wash precipitation, be resuspended at last in the 18 μ l water.In this mixture, add 2.5 μ l dNTP (1mMdCTP subsequently, 1mMdGTP, 1mMdTTP, 10mMdATP), 1.25 the T4DNA polysaccharase of μ l 100mM DTT and 5 units, 11 ℃ 30 minutes, enzyme is cut product mends into flush end, after 75 ℃ of termination reactions, add the Tth archaeal dna polymerase of 5 units and the damping fluid of Tth archaeal dna polymerase thereof, and system expanded as 80 μ l, 70 ℃ of reactions 20 minutes make the 3 ' end of DNA add the dA tail.This mixture is through the equal-volume chloroform: after primary isoamyl alcohol (24: 1) extracting and the extracting of equal-volume ether with 3 * 10 of Promega company
-3The pGEM-T-Easy carrier of (μ g) was in 16 ℃ of connections 24 hours, and cumulative volume is 90 μ l.The damping fluid of 10 times of T4DNA ligase enzymes of 9 μ l and the T4DNA ligase enzyme of 25 units are wherein arranged.Use the dehydrated alcohol precipitation of the 3M NaAc (pH5.2) of 1/10 volume and 2 times of volumes to be connected mixture at last, wash precipitation with 70% ethanol again, be dissolved in after the drying in the 30 μ l water and obtain connecting product.
Embodiment 4: the preparation of competent cell
The method that provides with reference to Bio-Rad company prepares the competent cell bacillus coli DH 5 alpha.Get a ring bacillus coli DH 5 alpha list bacterium colony in the LB of 5ml substratum, 180rpm cultivated after 10 hours, got in the LB substratum that the 2ml culture is transferred to 200ml, and 37 ℃ of 250rpm thermal agitations are cultivated OD600 about 0.5, ice bath cooling was 20 minutes then, in centrifugal 15 minutes of 4 ℃ of 4000rpm.Confide all supernatant, dispel thalline, in centrifugal 15 minutes of 4 ℃ of 4000rpm with the deionization aqua sterilisa 200ml of cold ice precooling.Deionization aqua sterilisa 100ml with cold ice precooling dispelled thalline again, in centrifugal 15 minutes of 4 ℃ of 4000rpm.With 10% glycerine suspension cell of cold ice precooling, centrifugal 10 minutes of 4 ℃ of 6000rpm abandon supernatant, precipitate 10% glycerine suspension cell with the precooling of 1ml ice at last, are competent cell.The competent cell that makes is packed as 50 μ l, one pipe ,-70 ℃ of preservations.
Embodiment 5: electric transformed competence colibacillus cell
Get after 2-3 μ l connects product and 50 μ l competence bacillus coli DH 5 alphas mix, forward in the electric shock cup of 0.2cm of Bio-Rad company and shock by electricity, voltage is 2.5 kilovolts, and the time is 5.0 milliseconds-6.0 milliseconds.The SOC substratum that adds 1ml after the electric shock immediately in cup makes the bacterium recovery.Immediately bacterium is coated in 37 ℃ of inversion incubated overnight on the LB solid medium that contains penbritin, X-Gal and IPTG then, obtains blue white bacterium colony next day.With the white colony that obtains promptly the white clone forward on the LB solid medium that contains penbritin and cultivate, extract plasmid and cut the segmental size of evaluation insertion wherein with the EcoRI enzyme from each clone simultaneously, the white that obtains clone group has constituted the O-antigen gene bunch library of shigella dysenteriae 12 types.
Embodiment 6: 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 dysenteriae 12 types again to the order-checking of PCR product, thus obtain all sequences of O-antigen gene bunch.In shigella dysenteriae 12 types we according to the sequences Design that has obtained two pairs of primers, as follows:
5 '-GCATGGGTTACTGTACTAGC-3 ' and 3 '-AATGGCATCAATACCCGC-5 '
5 '-TGGCGGTATTGAGAGAGT-3 ' and 3 '-TTACAGGCTACTTCTCTTC-5 '
Embodiment 7: 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 dysenteriae 12 types obtained.The quality of sequence is mainly guaranteed by two aspects: 1) genome of shigella dysenteriae 12 types is done 6 Long PCR reactions, mix these products then to produce the library.2), guarantee high-quality fraction of coverage more than 3 to each base.After obtaining the nucleotide sequence of shigella dysenteriae 12 type O-antigen genes bunch, with American National biotechnology information science center (The National Center for BiotechnologyInformation, NCBI) orffinder finds gene, find the reading frame of 10 openings, determine also that with the function of finding the reading frame that these are open what gene they are with the genetic comparison among the blast groupware and the GenBank, finish gene annotation with the Artemis software at Britain sanger center again, do accurate comparison between DNA and protein sequence with Clustral W software, obtain the structure (see figure 1) of the O-antigen gene bunch of shigella dysenteriae 12 types at last.
By retrieving and relatively, finding that the rmlB gene of orf1 and Shigella boydii and Escherichia coli K12 all has 97% homogeny in 297 amino acid whose sequences, show the homology that height is arranged between them.So can determine orf1 is the rmlB gene.The rmlD gene of orf2 and Shigella boydii has 98% homogeny in 299 amino acid whose sequences, aminoacid sequence with the rmlD gene of Escherichia coli K12 also has 95% homogeny in addition, shows the homology that height is arranged between them.So, can determine that orf2 is the rmlD gene.The rmlA gene of orf3 and Shigella boydii and Escherichia coli K12 has 97% homogeny in 292 amino acid whose sequences, show the homology that height is arranged between them.So, can determine that orf3 is the rmlA gene.The rmlC gene of orf4 and Shigella boydii and Escherichia coli K12 all has 86% homogeny in 179 amino acid whose sequences, show the homology that height is also arranged between them.So, can determine that orf4 is the rmlC gene.In addition, in Shigellae and intestinal bacteria, rmlB, rmlD, rmlA, rmlC are four genes that synthesize rhamnosyl specially, so can know by inference in the antigenic structure of the O-of shigella dysenteriae 12 types rhamnosyl are arranged also.The eps4k gene of orf5 and Streptococcus thernophilus has 33% homogeny in 384 amino acid whose sequences, 51% similarity, and the eps4k gene is a glycosyltransferase gene, so orf5 also is a glycosyltransferase gene, and called after orf5.The wzx gene of orf6 and Pseudomonasaeruginosa has 30% homogeny in 410 amino acid, in 410 amino acid, 35% homogeny is arranged with the wzx gene of Shigella bodyii, in 402 amino acid, 30% homogeny is arranged with the wzx gene of Edwardsiella ictaluri.And algorithm [Eisenberg by people such as Eisenberg, D, Schwarz, E.etal (1984) .Analysis ofmembrane and surface protein sequences with the hydrophobic moment plot.J.Mol.Biol.179:125-142] find that orf6 has 12 potential transmembrane domains, it and many wzx protein similars, and about 50 amino acid whose conservative motifs are arranged at the proteic aminoterminal of wzx, so can determine orf6 is the wzx gene, called after wzx.The Eps10H gene of orf7 and Thermophilus has 31% homogeny in 347 aminoacid sequences, in addition also with Streptococcus salivariusyou in the cpsJ gene in 190 aminoacid sequences, 41% homogeny is arranged, and the Eps10H gene is a galactosyltransferase gene, the cpsJ gene also is a glycosyltransferase gene, so determine that orf7 is the gene of an encoding glycosyl transferring enzyme, called after orf7.The wzy gene of orf8 and intestinal bacteria O104 has 27% homogeny in 340 aminoacid sequences, in 320 aminoacid sequences, 23% homogeny is arranged with the O-antigen polysaccharase of Shigella boydii, learn that by the Eisenberg algorithm orf8 has 10 potential transmembrane domains in addition, to other O-antigen polysaccharase similar secondary structure is arranged, so determine that orf8 is exactly the wzy gene, called after wzy.The wbdN gene of orf9 and colibacillary O157:H7 has 33% homogeny in 256 aminoacid sequences, 52% similarity; OB2925 gene with Oceanobacillus iheyensis has 41% homogeny in 240 aminoacid sequences in addition, 60% similarity, and these two genes all are glycosyltransferase genes, feature with glycosyltransferase, so determine that orf9 is a glycosyltransferase gene, called after orf9.The capM gene of orf10 and Campylobacter jejuni has 27% homogeny in 365 aminoacid sequences, 48% similarity, amsD gene with Erwinia amylovom has 30% homogeny in 379 aminoacid sequences in addition, 48% similarity, and these two genes all are glycosyltransferase genes, so determine that orf10 also is a glycosyltransferase gene, called after orf10.
Embodiment 8: the screening of specific gene
At wzx, wzy, orf5, orf7, orf9, orf10 gene design primer in the O-antigen gene of shigella dysenteriae 12 types bunch, the position of these genes in nucleotide sequence sees Table 1.
Table 1 has been listed glycosyltransferase gene and oligosaccharide unit treatment gene and intragenic primer and PCR data in the O antigen gene bunch of shigella dysenteriae 12 types.Glycosyltransferase gene, transhipment enzyme gene and pol gene and their function corresponding and the size of the O antigen gene bunch of shigella dysenteriae 12 types in table, have been listed.In each gene, we have respectively designed three pairs of primers, and the difference that every pair of primer is distributed in the corresponding gene is local to guarantee its specificity.In table, also listed position and the size of each primer in SEQ ID NO:1.Is that template carry out PCR with listed corresponding annealing temperature in the table with the genomes of all bacterium in the table 2 with every pair of primer, has obtained corresponding PCR product, and its size is also listed in the table.
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 CTA AAC TCC TTA TT) and #102 (TAA TCG CAG GGG AAA GCA GG), extract genome then from 166 strain intestinal bacteria, method as previously mentioned.With this to primer from the colibacillary genome of 166 strains PCR with identification of escherichia coli and detect its genomic quality.
Table 2 is 166 strain intestinal bacteria and 43 strain Shigellaes and their sources that are used to screen specific gene, and for the convenience that detects, we are divided into one group with their every 8-10 bacterium, and 27 groups altogether, all list in the table in their source.
The genomic dna that contains shigella dysenteriae 12 types in the 25th group is as positive control.Do template with every group of bacterium, be PCR by following condition with every pair in the table 1 primer: 94 ℃ of pre-sex change after 2 minutes, 94 ℃ of sex change 15 seconds, annealing temperature is because of the difference different (with reference to table 1) of primer, annealing time is 50 seconds, and 72 ℃ were extended 2 minutes, and carried out 30 circulations like this.Continue to extend 10 minutes 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, orf5, orf7, orf9, orf10 gene, each gene all has three pairs of primers detected, every pair of primer has obtained also all obtaining onesize specificity band the correct band of expection size in the 18th group except be PCR in the 25th group after.After being template PCR with the genomic dna of each bacterium in the 18th group, find only in intestinal bacteria O152, to have obtained positive findings.In more detail, each of each gene all obtains expecting the correct PCR product band of size to primer more than in intestinal bacteria O152.It is reported that the O-antigenic structure of shigella dysenteriae 12 types and intestinal bacteria O152 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, orf5, orf7, orf9, orf10 gene pairs shigella dysenteriae 12 types and intestinal bacteria O152 and O-antigen thereof all are high specials.
At last, from shigella dysenteriae 12 types, screen gene by PCR: wzx, wzy and four glycosyltransferase genes to the O-antigen high special of shigella dysenteriae 12 types and intestinal bacteria O152.And the oligonucleotide of these intragenic any one section 10-20nt is special to the O-antigen of shigella dysenteriae 12 types and intestinal bacteria O152, and the primer in especially above-mentioned each gene is that oligonucleotide is high special to detecting the back confirmation through PCR to shigella dysenteriae 12 types and intestinal bacteria O152.These all oligonucleotide all can be used for shigella dysenteriae 12 types and the intestinal bacteria O152 in the human body and environment rapidly and accurately, and can identify their O-antigen.
Sequence list
SEQUENCE?LISTING
＜110〉Nankai University
＜120〉to the Nucleotide of the O-antigen-specific of shigella dysenteriae 12 types and intestinal bacteria O152
＜130〉to the Nucleotide of the O-antigen-specific of shigella dysenteriae 12 types and intestinal bacteria O152
<160>1
<170>PatentIn?version?3.1
<210>1
<211>12235
<212>DNA
<213>Shigella?dysenteriae
<400>1
attgtggctg?cagggatcaa?agaaatcctc?ctggtaactc?acgcgtccaa?gaacgcggtc 60
gaaaaccact?tcgacacctc?ttatgaatta?gaatctctcc?ttgagcagcg?cgtgaagcgt 120
caactgttag?cggaagttca?gtccatctgc?ccgccgggcg?tgactattat?gaacgtgcgt 180
cagggcgaac?ctttaggttt?gggccactcc?attttatgtg?cacgacctgc?cattggtgac 240
aacccatttg?tcgtggtact?gccagacgtt?gtgatcgatg?acgccagcgc?cgacccgctg 300
cgctacaacc?ttgcagccat?gattgcacga?ttcaacgaaa?cgggtcgtag?ccaggtgctg 360
gcaaaacgca?tgccggggga?cctctctgaa?tactctgtca?tccagaccaa?agagccgctg 420
gatcgtgaag?gcaaagtcag?ccgcattgtt?gaatttatcg?aaaaaccgga?tcagccgcag 480
acgttggagt?cagacatcat?ggccgttggt?cgctatgtgc?tttctgccga?tatttggccg 540
gaacttgaac?gcacgcagcc?aggtgcatgg?gggcgtattc?agctgactga?tgccattgcc 600
gaactggcga?aaaaacagtc?cgttgatgcc?atgctgatga?caggtgacag?ctacgactgc 660
ggtaaaaaaa?tgggttatat?gcaggcattt?gtgaagtatg?gactacgcaa?cctgaaagaa 720
ggggcgaagt?tccgtaaagg?cattgagaag?ctgttaagcg?aataatgaaa?atctgaccgg 780
atgtaacggt?tgataagaaa?attataacgg?cagtgaagat?tcgtggcgaa?agtaatttgt 840
tacgaatttt?cctgccgttg?ttttatataa?acaatcagaa?taacaacgag?ttagcaatag 900
gattttagtc?aaagttttcc?aggattttcc?ttgtttccag?agcggattgg?taagacaatt 960
agcgtttgaa?tttttcgggt?ttagcgcgag?tgggtaacgc?tcgtcacatc?gtagacatga 1020
atgcagtgct?ctggtagctg?taaagccagg?ggcggtagcg?tgcattaata?cctctattaa 1080
tcaaactaag?agccgcttat?ttcacagcat?gctctgaagt?aatatggaat?aaaagagtga 1140
agatacttgt?tactggtggc?gcaggattta?ttggttctgc?tgtagttcgt?cacattataa 1200
ataatacgca?ggatagtgtt?gttaatgtcg?ataaattaac?gtacgccgga?aacctggaat 1260
cacttgctga?tgtttctgat?cctgaacgct?atatttttga?acatgcggat?atttgcgatg 1320
ctgctgcaat?ggcacggatt?tttgctcagc?atcagccgca?tgcagtgatg?cacttggctg 1380
ctgaaagcca?tgttgaccgt?tcaattacag?gtcctgcggc?atttattgaa?accaatattg 1440
ttggtactta?tgtccttttg?gaagccgctc?gcaattactg?gtctgctctt?gatagcgaca 1500
agaaaaatag?cttccgtttt?catcatattt?ctactgacga?agtctatggt?gatttgcctc 1560
atcctgacga?ggtaaataat?aaagaacaat?tacccctctt?tactgagacg?acagcttacg 1620
cgccaagtag?cccatattct?gcttcgaaag?catcaagcga?tcatttagtc?cgcgcttgga 1680
aacgtaccta?tggtttaccg?accattgtga?ctaattgttc?gaataactac?ggtccttatc 1740
actttccgga?aaaattgatt?ccactagtaa?ttcttaatgc?tctggaaggt?aaggcattac 1800
ctatttatgg?caaaggggat?caaattcgtg?actggctgta?tgttgaagat?catgcgcgtg 1860
cgttatatac?cgtcgtaacc?gaaggtaaag?cgggtgaaac?ttataacatt?ggtggacaca 1920
acgaaaagaa?aaacatcgat?gtagtgctca?ctatttgtga?tttgttggat?gagattgtac 1980
cgaaagagaa?atcttatcgt?gagcaaatta?cttatgttgc?cgaccgcccg?ggacatgatc 2040
gccgttatgc?gattgatgct?gagaagatta?gccgcgaatt?gggttggaaa?ccacaggaaa 2100
cgtttgagag?cgggattcgt?aaaacggtgg?aatggtacct?ggctaatgca?aaatgggtcg 2160
agaatgtgaa?aagtggtgcc?tatcaatcgt?ggattgaaca?gaactatgag?ggccgccagt 2220
aatgaatatt?ctccttttcg?gcaaaacagg?gcaggtaggt?tgggaactac?agcgtgctct 2280
ggcacctttg?ggtaatttga?ttgctcttga?tgttcactcc?actgattatt?gtggtgattt 2340
tagtaatcct?gaaggtgtgg?ctgaaaccgt?caaaaaaatt?cgccctgatg?taattgttaa 2400
tgctgcggct?cataccgcag?tagataaggc?tgagtcagaa?cccgaatttg?cacaattact 2460
caatgcgacc?agcgttgaat?caattgccaa?agcggctaat?gaagttgggg?cctgggtaat 2520
tcattactca?actgactacg?tatttccggg?aaccggtgaa?ataccatggc?tggagacgga 2580
tgcaaccgca?ccgctaaatg?tttacggtga?aaccaagtta?gccggagaaa?aagcgttaca 2640
ggaacattgc?gcgaagcatc?ttattttccg?taccagctgg?gtatacgcag?gtaaaggaaa 2700
taacttcgcc?aaaacgatgt?tgcgtctggc?aaaagagcgc?gaagaactgg?ctgtgattaa 2760
tgatcaattt?ggtgcgccaa?caggtgctga?gctgctggca?gattgtacgg?cacatgctat 2820
tcgtgtggca?ctgaataaac?cagaagtcgc?aggcatgtac?catctggtag?ccagtggtac 2880
cacaacctgg?cacgattatg?ctgcgctggt?ttttgaagag?gcgcgcaaag?caggtattcc 2940
ccttgcactc?aacaagctca?acgcagtacc?agcaacagcc?tatcctacac?cagctcgtcg 3000
tccacataac?tctcgcctta?atacagaaaa?atttcagcag?aactttgcgc?ttgtcttgcc 3060
tgactggcag?gttggcgtga?aacgaatgct?taacgaatta?tttacgacta?cagcaattta 3120
atagtttttg?catcttgttc?gtgatggtgg?aacaagatga?attaaaagga?atgatgaaat 3180
gaaaacgcgt?aaaggtatta?ttttagcggg?gggggctggt?actcgtcttt?atcctgtgac 3240
tatggctgta?agcaaacagc?tgttaccgat?ttatgataaa?ccgatgatct?attacccgct 3300
ttctacactg?atgttagcgg?gtattcgcga?tattctgatt?atcagtacgt?cacaggatac 3360
tcctcgtttt?caacaactgc?tgggtgatgg?gagccagtgg?gggctaaatc?ttcagtacaa 3420
agtgcaaccg?agtccagatg?gtcttgcgca?ggcatttatc?atcggtgaag?agtttatcgg 3480
tggtgatgat?tgtgctctgg?ttctcggtga?taatatcttt?tacggtcacg?atctgccgaa 3540
gttaatggat?gtcgctgtca?acaaagaaag?tggtgcaacg?gtatttgcct?atcacgttaa 3600
tgatcctgaa?cgctacggtg?ttgttgagtt?tgataaaaac?ggtacggcaa?tcagcctgga 3660
agaaaaaccg?ctacaaccaa?aaagtaatta?tgcggtaacc?gggctttatt?tctatgataa 3720
cgacgttgtc?gaaatggcga?aaaaccttaa?gccttctgcc?cgtggtgaac?tagaaattac 3780
cgatattaac?cgtatttata?tggaacaggg?gcgtttatcc?gttgccatga?tggggcgtgg 3840
ttatgcatgg?ctggatacgg?ggacacatca?gagtcttatt?gaagcaagca?acttcattgc 3900
caccattgaa?gagcgccagg?gactaaaggt?ttcctgccca?gaagaaattg?cttaccgtaa 3960
agggtttatt?gatgctgagc?aggtgaaagc?attagctgaa?cctctgaaaa?aaaatgctta 4020
tggtcagtac?ctgataaaaa?tgattaaagg?ttattaataa?aatgaatgtt?attaaaacag 4080
aaatcccaga?tgtattgatt?tttgaaccga?aagtttttgg?tgatgagcgt?ggtttctttt 4140
ttgagagctt?taaccagaag?gtttttgagg?aagctgtagg?ccgcaaagtt?gaatttgttc 4200
aggataacca?ttcgaagtct?agtaaaggcg?ttttacgcgg?actgcattat?cagttagaac 4260
cttatgcgca?aggaaaattg?gtacgctgtg?ttgttggtga?ggtttttgat?gttgcggttg 4320
atattcgtaa?atcgtcccct?acttttggca?aatgggttgg?ggtgaatttg?tctgctgaga 4380
ataagcgcca?gttgtggata?cctgaaggat?ttgcgcatgg?atttttggtg?ctgagtgaga 4440
ctgcggagtt?tgtttataag?acaactaatt?attatcatcc?ggaaagtgac?agaggtatta 4500
gatgggatga?cccctatata?ggaattaaat?gggagaatga?gtacaatgta?agattgtcag 4560
ataaagataa?aaaaaatcca?acaatgaaag?acgttttttt?gtttatgtaa?gtaataggct 4620
tttatatgaa?aaaaaatatt?atcgcatatt?atcttcctca?atatcatgag?gttaaagaaa 4680
ataatgagtg?gtgggggaaa?ggatttactg?aatggacagc?tttaaaaaaa?gcaaagaaat 4740
atttcccttc?tcaaaaaata?agattgccta?ctgattttct?tggatattat?gatttgacag 4800
aatcacgtat?tatcgagaag?cagtttgaac?tagctgaaga?aaatggtgta?agtgggtttt 4860
gtctatggac?atattggttc?ggtaatggag?aaaaaatttt?agaaaaacct?cttactctta 4920
ttcttgaaaa?caaacttaat?gttcgatact?gtgtggcatg?ggcaaaccac?tcttggtaca 4980
ataaatcaaa?agggctactt?ttaaaagagc?aaaaatacct?tggagaaaaa?gattatactg 5040
atttcttcta?ttacttactt?cctcatttta?agtcagataa?ttatctaaaa?aaagataata 5100
agccaattgt?aacaatcttt?gatcctaatt?ccataccaga?tttgtttttg?tttatatccc 5160
tttggaataa?tcttgcaaaa?gagaacggat?acgagggtat?ctattttatc?ggggatttca 5220
catcgtataa?ttcaaattat?gtaggagtat?ttgatggtta?tttggattct?agagtaatgt 5280
acaggaagag?aacctttatt?caaaaggtga?gggagaaatt?agttcgaaag?catagagtga 5340
aatttttggg?gcctataagg?tataattatg?aaaaaatgat?aagctcatta?tggcacaatc 5400
agacaaagga?tataaaagag?attccaatta?tattttctgg?atgggataca?acgatacgac 5460
atggaaaaca?gggggttttt?tatagtgatt?tctctgagca?ttcgtttgag?gttaatgtta 5520
aaaatgctat?aaattataac?ccacaacaag?atattgtttt?cttaaaatca?tggaatgaat 5580
gggctgaggg?aaatacagtt?gaaccagaca?ctatattctc?agataaatta?cttagaataa 5640
tttcaaaata?caataaatac?tgatgagaaa?aaatatattt?tatttactgt?tggtgcagtt 5700
atctaactat?attttcccac?tgattacatt?accatatctt?gtcagaactt?taggagtaga 5760
taaatttgga?tatcttatgc?ttggtcaagc?attgattcaa?tactttattt?tgatgactga 5820
ttacggtttt?aatttcagtg?caacaaaaaa?aatagctaca?tcaacaagta?ttgatgaaag 5880
aaatgaagtc?tacacagaga?cattaaatgc?caaaatgata?ttggtgacat?tatgtttatt 5940
cttgatgtta?atcattatta?atatcatacc?aatgtttcac?tctatatcta?tagtttgcat 6000
aatacttttt?attggggttc?taggtaatat?tttttttcca?gtctatctgt?ttcaaggtat 6060
ggagaaaatg?aaaagtattg?catgggttac?tgtactagca?aggaccttga?tgttgttatc 6120
tgtctttctg?tttgtaaaag?ataataatga?tatcaatgct?gctgcactag?ctttttctat 6180
tgcgcttatc?gttccaggga?tgatgtcaat?ttatctaata?cgaaaaaacc?acattgttgc 6240
ttataaaggg?ttttcttttt?caaaagcctt?tatcgctatt?aaagcaagta?cacctttgtt 6300
tatttctcaa?atcgctataa?cattttatac?tacttttaat?tcgatactta?tagggcatgt 6360
atttaatgca?aggcaagtag?gtatttattc?tgcagcagac?aagttaagag?gcgcggttca 6420
atctttattt?attcctatac?aacaggttgt?gtttccgaga?ataaatgttg?agcgtggaaa 6480
tataaaggaa?aaactaaagt?tttacggtag?tctttttatt?ttattttcat?taattatatg 6540
tgtgtcgata?tttttaattg?gagataagat?tgttatttta?tacttcggaa?ctcagtatgc 6600
tgagtcagct?aatttattta?aatggatgtc?agtccttatt?tttattgtct?ctgtcgctat 6660
tgtcttttca?caatggggaa?tgataacttt?aggtagagaa?aaaatgttaa?caaaaattta 6720
tattgtcggt?gccttattgc?attgcacgta?tgctcctttc?ttcactaaaa?atttcgggct 6780
ttttggtacg?ttaacggcag?taatattgac?agagttaaca?attaccgtag?ttatgggcgt 6840
gtttttgtac?aagataatta?aaaggcataa?ataaaaatga?aaagcaacac?aattgacatt 6900
gtaattccat?gggtagatag?ttctgatcct?aaatggcaag?aggattatat?gaagtttgca 6960
tgtgaacaaa?gaaatgttga?tgcaaatatt?catcgttata?gagataatgg?gttattaaaa 7020
ttctggtttc?gtggcattga?aaaaaatgcc?ccttggatta?ggaaagttca?ttttattaca 7080
tatggacatg?ttccggcgtg?gcttaagcaa?gatcacccta?aattacatat?tgtaaagcgc 7140
tccgactata?taccccaaaa?atacctacca?acatttaata?gccatgttat?tgaattaaat 7200
ttgcataaaa?tagatggatt?gagtgataag?ttcatctatt?ataatgacga?tacatttttg 7260
attaatccag?tagatgaaag?tttttacttc?aaaaatggaa?agcctcgaga?ttgtgcaatc 7320
ctgaacgcag?ttcaacctga?tgggctttca?catgttattt?taaataatct?ttcaattatc 7380
aataagcatt?tttgtaaaaa?acaggtgatc?agaaaaaata?ttttaaaatg?gataaatgtt 7440
aaatatggtg?cggcgttaag?tagaacgata?tgtctaatgc?cttggccgaa?gtttgtaggt 7500
atcaaagata?ctcatcttcc?aattccttat?cttaagtcaa?catttgataa?attatgggag 7560
ctcgagccag?aaatattaaa?tgctacaaat?cggtcaaaat?ttagacagaa?ttcagatatt 7620
aatcaatatc?tctttagata?ttggcagtta?gtttcagggg?agtttgaatg?ctctaataca 7680
atggcatctt?cagcctattt?agatataggt?aaggatagta?ttataaaaat?tcgaaaggaa 7740
ctattttgtt?caagaaataa?acttgttgtt?ataaatgatg?ctgatacttt?ggaattcgat 7800
tccaaaacaa?aaaaaataca?agaagaattt?gcaaagatat?ttccttttaa?atcaacgttt 7860
gaaatataat?acggtagaaa?aatgttattt?acctacgcct?catatttatg?tatattggtg 7920
ttttctacat?cggttgcatt?ttgttacgaa?cattcaaaaa?gtaattttaa?agtattgcta 7980
tggtcagtat?tgtttttgtc?tctttggata?cctgcagcaa?ttcgttttgg?agtaggcgct 8040
gattacttcc?gatatttaag?tatggttgaa?tcagttaggt?tgggatatat?ttctactgaa 8100
gttggatttt?attttataaa?ttattttgtt?gtttggtttg?atttaaatcc?acaatggtct 8160
attgcaatat?caagtttcat?tgtaatcttt?ttaagtttga?aggcgattcc?acgaaactat 8220
ttttctgttt?cagtttttgt?tttagtatgt?accttttatc?tgccttcgtt?ttctcttgtt 8280
agacaagcta?ttgctatagc?tttcatagcg?tatgcagtga?aaaattatat?cgatggtaat 8340
aaaaaaaact?atattatttt?tattttgata?ggctctctat?ttcatctctc?ggcattatta 8400
cttgtaccca?tttattttat?tgcgcgattt?agagtccgta?gttcaattct?ctttttgggt 8460
attttaataa?taagtttatt?gctattcctg?ggtagtggat?taaatgcact?tttgaatagt 8520
gagttttttt?tacaaagcaa?atacggttat?tacgcaacaa?gtgatttcat?acaggatgct 8580
aaaatcggaa?gtggggtagg?tgtatttatc?aaaatgctat?tacctttaat?ttttataaca 8640
aacgcaaaaa?aaatactaaa?aacaaatcag?aatcttaata?ttctattgtg?gatgtctgtt 8700
ggatgtgtgc?ttgctaatgt?atgttcaatg?aagattcata?tttttaatcg?tctagcagat 8760
atattccttt?ttgttaattt?catggtaacc?tcatatatta?tcaattcatt?agaaaaaaat 8820
gttataaaac?aattatcttc?aatggcaatt?attattgttt?ttattgtgtt?ttattgcaga 8880
acaattcagg?ccaattataa?tgatgattta?ggtggcattg?gtatttctcc?ctacaaaaca 8940
atactttcta?agtgatatta?tggatgatta?tttagtatca?attattatgc?cttcatataa 9000
cgcagagcat?acgatttctg?catcaattag?ttccgtgtta?aagcaaacat?atgctaactg 9060
ggaattgctc?gtttgcgatg?atgattcaag?tgataataca?agatttaaag?ttttggagtt 9120
ttctgattca?agaattaaac?ttttaacaaa?tgaatacgca?aaaggtgcag?caggtgctag 9180
aaataccgct?ctaaaatatg?caagcggccg?atttatagct?ttcttagata?gtgatgatat 9240
atggatcgct?aacaaattag?agatgcaaat?atctatgatg?ctaaagaata?atatttcttt 9300
tatgtatggt?aattatgaaa?ttataaataa?taattcaata?gttggtaagt?ttgtagctcc 9360
ccaaaaaatc?acgtacaaca?aattgctaaa?aaattgtggt?attggttgtt?tgactgtcgt 9420
tttagacagg?actttactaa?atcctttcag?ctttcctttc?gtgcataaag?aagattatta 9480
tctatggctg?agtattctaa?aagataacaa?tattagtgca?attaattgcg?gctttatttg 9540
ttctaaatat?agactttcac?aaagttcaat?ttcatctaat?aaatttaaag?agttgaaaag 9600
gcaatgggat?gttttggggg?attttgttga?aaacccattg?gctcggatat?attatttgct 9660
aaattatatt?gttatcggta?taaagaaaca?tgcttttgac?tataaaaacg?gtaaaaaatg 9720
aaaaaaatag?cttttgtaat?tgctgatatt?acatttgttg?gcggtattga?gagagtaaat 9780
acaatacttg?ccaataaatt?tatcagcgaa?ggatactctg?tagaaataat?atctctctat 9840
aaaactaatc?taaaaattaa?ttacgagctt?gacaatcaca?ttaaaattag?ttttgttaat 9900
gatggctctt?atttaggtga?gcctggaagt?tttggacggc?tgaagaaaca?catagggtca 9960
cagtttaaat?tatttaaagc?cattaaaaag?tcaaatgctg?atttttatat?cattaataca 10020
ttccctatgg?cttttttgtc?atttttcagt?ctcttttcaa?aaaaaaagta?tgttgttatt 10080
gaacatgtac?attatgacta?ttatagtgca?attgttagag?cattaagaaa?tttattgtat 10140
agattctttt?ataaagtaat?ctgtattaat?aaaaatgact?tgtataagtt?ttctaaacat 10200
ttagataatg?ttgttaaaat?tagtaacccg?ctgagttttt?cttgttcaaa?agtagctgat 10260
ttaagcgcaa?aaaaaattat?ggcggttggc?aggctagagc?atcaaaaggg?ttttgattta 10320
ttaatagata?tttttgcgaa?agttaacaaa?tcaaatcctg?gttgggaact?ccatatttat 10380
ggtgtcggaa?cgtgcgaaaa?atttctcaca?gataaaataa?ataagcatgg?attaaataat 10440
gtaaaactga?tgggaagtgt?cgatcatata?caacaatatt?atcctaaata?ttccattttt 10500
gcattctcat?caaggtttga?agggttcggg?atggtgttat?tggaagcaat?ggaatgtggt 10560
ttgccatgta?tttcatttga?ttgccctacc?ggcccatctg?aaatattagg?ggatggggag 10620
tatggaatct?tagttgagaa?tgggaatctt?attaaattta?gcgcggaatt?agcgaattta 10680
atgtccgatg?aagagaagaa?aattaaattt?tcaattttat?caaagagtcg?tgcacaagag 10740
tttaatatag?acaagatttt?tgacgaatgg?agaaagttaa?taagttagta?cttatagtaa 10800
atgactgaaa?acgttgtaca?aataatttat?atcaaaaggt?tgcttattcg?attaatataa 10860
ttaaataagt?tacctagccc?tgacaggagt?aaacaatgtc?aaagcaacag?atcggcgtcg 10920
tcggtatggc?agtgatgggg?cgcaaccttg?cgctcaacat?cgaaagccgt?ggttataccg 10980
tctctatttt?caaccgttcc?cgtgaaaaga?cggaagaagt?gattgccgaa?aatctaggca 11040
agaaactggt?tccttactat?acggtgaaag?agtttgttga?atctctggaa?acgcctcgtc 11100
gcatcctgtt?aatggtgaaa?gcaggtgcag?gcacggatgc?tgctattgat?tccctcaagc 11160
catacctcga?taaaggtgac?atcatcattg?atggtggtaa?caccttcttc?caggacacca 11220
ttcgtcgtaa?ccgtgagctt?tctgcagaag?gctttaactt?tatcggtacc?ggtgtttccg 11280
gtggtgaaga?aggcgcgctg?aaaggtcctt?ccattatgcc?tggtgggcag?aaagaagcct 11340
atgaactggt?tgcgccgatc?ctgaccaaaa?tcgccgcagt?ggctgaagac?ggtgagccat 11400
gcgttaccta?tattggtgcc?gatggtgcag?gtcactatgt?gaagatggtt?cacaacggta 11460
ttgaatacgg?tgatatgcaa?ctgattgctg?aagcctattc?tctgcttaaa?ggtggtctga 11520
acctctccaa?cgaagaactg?gcgcagacct?ttaccgagtg?gaataacggt?gaactgagca 11580
gctacctgat?tgacatcact?aaagacatct?tcactaaaaa?agatgaagac?ggtaactacc 11640
tggttgatgt?gattctggat?gaagcagcta?acaaaggtac?cggtaaatgg?accagccaga 11700
gcgcgctgga?tctcggtgaa?ccgctgtcgc?tgattaccga?gtctgtgttt?gcacgttata 11760
tctcttctct?gaaagatcag?cgtgttgccg?catctaaagt?tctctctggc?ccgcaagcac 11820
agccagcagg?cgacaaggct?gagttcatcg?aaaaagttcg?ccgtgcgctg?tatctgggca 11880
aaatcgtttc?ttacgctcag?ggcttctctc?agctacgcgc?cgcgtctgaa?gagtacaact 11940
gggatctgaa?ctacggtgaa?atcgcgaaga?ttttccgtgc?tggctgcatc?atccgtgcgc 12000
agttcctgca?gaaaatcacc?gatgcatatg?ccgaaaatcc?gcagatcgct?aacctgatgc 12060
tggctccgta?cttcaagcaa?atcgeegatg?actaceagca?ggegctgcgc?gatgtcgtcg 12120
cttacgcggt?acagaacggt?atcccggttc?cgaccttcgc?cgctgcggtt?gcctattatg 12180
acagctaccg?cgccgctgtt?ctgcctgcga?acctaatcca?ggcacagcgc?gacta 12235
Glycosyltransferase gene in the table 1 shigella dysenteriae 12 type O antigen gene clusters and oligosaccharide unit treatment gene and wherein primer and PCR data
| Gene | Function | The base position of gene | Forward primer | Reverse primer | PCR product length | Produce the group number of correct big or small electrophoresis band | The annealing temperature of PCR (℃) |
| Orf5 | Glycosyl transferase | 4626-5663 | #221(4866-4886) | #222(5573-5591) | 762bp | 0 | 52 |
| #223(5156-5176) | #224(5587-5595) | 440bp | 0 | 52 | |||
| #241(4641-4660) | #242(5302-5320) | 680bp | 0 | 50 | |||
| wzx | The transhipment enzyme | 5663-6874 | #225(6189-6207) | #226(6731-6750) | 562bp | 0 * | 54 |
| #227(5713-5733) | #228(6724-6741) | 1029bp | 0 | 57 | |||
| #237(6463-6481) | #238(6821-6839) | 377bp | 0 | 57 | |||
| orf7 | Glycosyl transferase | 6877-7869 | #229(7024-7041) | #230(7779-7800) | 777bp | 0 | 57 |
| #243(6972-6992) | #244(7578-7596) | 625bp | 0 | 50 | |||
| #245(7131-7148) | #246(7693-7674) | 513bp | 0 | 55 | |||
| wzy | Polymerase | 7882-8955 | #235(7883-7903) | #236(8903-8921) | 1039bp | 0 | 52 |
| #253(8049-8069) | #254(8876-8894) | 846bp | 0 | 59 | |||
| #255(8197-8214) | #256(8689-8709) | 513bp | 0 | 59 | |||
| orf9 | Glycosyl transferase | 8960-9721 | #231(9020-9040) | #232(9630-9647) | 628bp | 0 ** | 52 |
| #233(8982-9000) | #234(9443-9462) | 463bp | 0 | 57 | |||
| #247(9165-9183) | #248(9525-9543) | 378bp | 0 | 58 | |||
| orf10 | Glycosyl transferase | 9718-10788 | #239(9752-9770) | #240(10567-10586) | 863bp | 0 *** | 55 |
| #247(9165-9183) | #248(9525-9543) | 378bp | 0 | 59 | |||
| #249(9934-9952) | #250(10513-10530) | 597bp | 0 | 55 |
*In one group, obtain the band of a wrong size
**In three groups, all obtain the band of a wrong size
***In one group, obtain the band of three wrong sizes
Table 2166 strain Escherichia coli and 43 strain Shigellas and their source
The bacterium source that contains in this group of group number
1 wild-type e. coli O1, O2, O3, O4, O10, O16, O18, O39 IMVSa
2 wild-type e. coli O40, O41, O48, O49, O71, O73, O88, O100 IMVS
3 wild-type e. coli O102, O109, O119, O120, O121, O125, O126, O137 IMVS
4 wild-type e. coli O138, O139, O149, O7, O5, O6, O11, O12 IMVS
5 wild-type e. coli O13, O14, O15, O17, O19ab, O20, O21, O22 IMVS
6 wild-type e. coli O23, O24, O25, O26, O27, O28, O29, O30 IMVS
7 wild-type e. coli O32, O33, O34, O35, O36, O37, O38, O42 IMVS
8 wild-type e. coli O43, O44, O45, O46, O50, O51, O52, O53 IMVS
9 wild-type e. coli O54, O55, O56, O57, O58, O59, O60, O61 IMVS
10 wild-type e. coli O62, O63, O64, O65, O66, O68, O69, O70 IMVS
11 wild-type e. coli O74, O75, O76, O77, O78, O79, O80, O81 IMVS
12 wild-type e. coli O82, O83, O84, O85, O86, O87, O89, O90 IMVS
13 wild-type e. coli O91, O92, O95, O96, O97, O98, O99, O101 IMVS
14 wild-type e. coli O112, O162, O113, O114, O115, O116, O117, O118 IMVS
15 wild-type e. coli O123, O165, O166, O167, O168, O169, O170, O171 See b
16 wild-type e. coli O172, O173, O127, O128, O129, O130, O131, O132, See c
17 wild-type e. coli O133, O134, O135, O136, O140, O141, O142, O143 IMVS
18 wild-type e. coli O144, O145, O146, O147, O148, O150, O151, O152 IMVS
19 wild-type e. coli O153,0154, O155, O156, O157, O158, O159, O164 IMVS
20 wild-type e. coli O160, O161, O163, O8, O9, O124, O111 IMVS
21 wild-type e. coli O1O3, O104, O105, O106, O107, O108, O110 IMVS
22 Shigella bogdii serotypes B 4, B5, B6, B8, B9, B11, B12, B14 See d
23 Shigella bogdii serotypes B 1, B3, B7, B8, B10, B13, B15, B16, B17, B18 See d
24 shigella dysenteriae serotype D1, D2, D3, D4, D5, D6, D7, D8 See d
25 shigella dysenteriae serum D9, D10, D11, D12, D13 See d
26 shigella flexneri F6a, F1a, F1b, F2a, F2b, F3, F4a, F4b, F5 (v:7) F5 (v:4) See d
27 bacillus ceylonensis A D5, DR See d
a.Institude?of?Medical?and?Veterinary?Science，Anelaide，Australia
b.O123?from?IMVS；the?rest?from?Statens?Serum?Institut，Copenhagen，Denmark
c.172?and?173?from?Statens?Serum?Institut，Copenhagen，Denmark，the?rest?from?IMVS
D. China Preventive Medicial Science Institute's epidemiological study institute
Claims (8)
1, a kind of Nucleotide of the O-antigen-specific to shigella dysenteriae 12 types and intestinal bacteria O152 is characterized in that it is an isolating Nucleotide shown in SEQ ID NO:1,12235 bases of total length.
2, according to the Nucleotide of the described O-antigen-specific to shigella dysenteriae 12 types and intestinal bacteria O152 of claim 1, it is characterized in that it is by 10 genomic constitutions, they are all between galF gene and gnd gene.
3, according to the Nucleotide of the described O-antigen-specific to will dysentery Hayes bacterium 12 types and intestinal bacteria O152 of claim 2, it is characterized in that described gene is:
About the gene of transhipment and processing, comprise the wzx gene or the gene of identity function is arranged, wzy gene or the gene of identity function is arranged with wzy with wzx;
Glycosyltransferase gene comprises orf5, orf7, orf9, orf10 gene; Wherein said gene:
Orf5 is the Nucleotide of 4626 to 5663 bases among the SEQ ID NO:1;
Wzx is the Nucleotide of 5663 to 6874 bases among the SEQ ID NO:1;
Orf7 is the Nucleotide of 6877 to 7869 bases among the SEQ ID NO:1;
Wzy is the Nucleotide of 7882 to 8955 bases among the SEQ ID NO:1;
Orf9 is the Nucleotide of 8960 to 9721 bases among the SEQ ID NO:1;
Orf10 is the Nucleotide of 9718 to 10788 bases among the SEQ ID NO:1.
4, according to the Nucleotide of the described O-antigen-specific to shigella dysenteriae 12 types and intestinal bacteria O152 of claim 1-2, it is characterized in that it is to come from described wzx gene, wzy gene or glycosyltransferase gene orf5, orf7, orf9, orf10 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 dysenteriae 12 types and intestinal bacteria O152 of claim 4, it is characterized in that described oligonucleotide is:
The oligonucleotide that comes from orf5 is right:
The Nucleotide of 4866 to 4886 bases among the SEQ ID NO:1 and the Nucleotide of 5573 to 5591 bases,
The Nucleotide of 5156 to 5176 bases among the SEQ ID NO:1 and the Nucleotide of 5587 to 5595 bases,
The Nucleotide of 4641 to 4660 bases among the SEQ ID NO:1 and the Nucleotide of 5302 to 5320 bases;
The oligonucleotide that comes from wzx is right:
The Nucleotide of 6189 to 6207 bases among the SEQ ID NO:1 and the Nucleotide of 6731 to 6750 bases,
The Nucleotide of 5713 to 5733 bases among the SEQ ID NO:1 and the Nucleotide of 6724 to 6741 bases,
The Nucleotide of 6463 to 6481 bases among the SEQ ID NO:1 and the Nucleotide of 6821 to 6839 bases;
The oligonucleotide that comes from orf7 is right:
The Nucleotide of 7024 to 7041 bases among the SEQ ID NO:1 and the Nucleotide of 7779 to 7800 bases,
The Nucleotide of 6972 to 6992 bases among the SEQ ID NO:1 and the Nucleotide of 7578 to 7596 bases,
The Nucleotide of 7131 to 7148 bases among the SEQ ID NO:1 and the Nucleotide of 7693 to 7674 bases;
The oligonucleotide that comes from wzy is right:
The Nucleotide of 7883 to 7903 bases among the SEQ ID NO:1 and the Nucleotide of 8903 to 8921 bases,
The Nucleotide of 8049 to 8069 bases among the SEQ ID NO:1 and the Nucleotide of 8876 to 8894 bases,
The Nucleotide of 8197 to 8214 bases among the SEQ ID NO:1 and the Nucleotide of 8197 to 8214 bases;
The oligonucleotide that comes from orf9 is right:
The Nucleotide of 9020 to 9040 bases among the SEQ ID NO:1 and the Nucleotide of 9630 to 9647 bases,
The Nucleotide of 8982 to 9000 bases among the SEQ ID NO:1 and the Nucleotide of 9443 to 9462 bases,
The Nucleotide of 9165 to 9183 bases among the SEQ ID NO:1 and the Nucleotide of 9525 to 9543 bases;
The oligonucleotide that comes from orf10 is right:
The Nucleotide of 9752 to 9770 bases among the SEQ ID NO:1 and the Nucleotide of 10567 to 10586 bases,
The Nucleotide of 9165 to 9183 bases among the SEQ ID NO:1 and the Nucleotide of 9525 to 9543 bases,
The Nucleotide of 9934 to 9952 bases among the SEQ ID NO:1 and the Nucleotide of 10513 to 10530 bases.
6, the Nucleotide of the described O-antigen-specific to shigella dysenteriae 12 types and intestinal bacteria O152 of claim 1 is expressed the application of the antigenic bacterium of O-in vitro detection.
7,, it is characterized in that it is used for PCR, is used for hybridization and fluoroscopic examination, makes gene chip or microarray is used for vitro detection shigella dysenteriae 12 types or intestinal bacteria O152 as probe as primer according to the application of the Nucleotide of the described O-antigen-specific to shigella dysenteriae 12 types and intestinal bacteria O152 of claim 1.
8, the separation method of the Nucleotide of the described O-antigen-specific to shigella dysenteriae 12 types and intestinal bacteria O152 of claim 1 is characterized in that it comprises the steps:
1) genomic extraction
37 ℃ of incubated overnight Shigellaes in the LB substratum, centrifugal collecting cell is 8.0 Tris-HCl and EDTA re-suspended cell with the pH value, and 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; Add equal-volume phenol extracting mixture, get supernatant and use isopyknic phenol again: chloroform: enzyme and albumen that the primary isoamyl alcohol extracting removes wherein for twice, remove 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 30 μ lTE, genomic dna detects by 0.4% agarose gel electrophoresis;
2) by the O-antigen gene in Long pcr amplification shigella dysenteriae 12 types bunch
At first according to the JumpStart sequences Design upstream primer-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-TAG TCG CGT GNG CCT GGA TTA AGT TCG C in O-antigen gene bunch downstream; With the Expand LongTemplate PCR method of Boehringer Mannheim company amplification O-antigen gene bunch, the PCR response procedures was as follows: 94 ℃ of pre-sex change 2 minutes; 94 ℃ of sex change are 10 seconds then, 60 ℃ of annealing 30 seconds, and 68 ℃ were extended 15 minutes, and carried out 30 circulations like this; At last, continue to extend 7 minutes, obtain the PCR product at 68 ℃; Merge 6 pipe long PCR products, and with the WizardPCR Preps purification kit purified pcr product of Promega company;
3) structure in O-antigen gene bunch library
Make up O-antigen gene bunch library with the Novagen DNaseI shot gun method that is modified, reaction system is the 300ngPCR purified product, 0.9 μ l 0.1M MnCl
2, the DNaseI of the 1mg/ml of 1 μ l dilution in 1: 2000, reaction is carried out at room temperature; The dna fragmentation size that enzyme is cut after enzyme being cut in 10 minutes concentrates between the 1kb-3kb, then add 2 μ l 0.1M EDTA termination reactions, merge the same reaction system of 4 pipes, with isopyknic phenol extracting once, use isopyknic phenol: chloroform: the extracting of different one-tenth mixed alkoxide solution once, phenol: chloroform: primary isoamyl alcohol mixed volume ratio is 25: 24: 1; After using isopyknic ether extracting once again,, and wash precipitation, be resuspended at last in the 18 μ l water with 70% ethanol with the dehydrated alcohol deposit D NA of 2.5 times of volumes; In this mixture, add 2.5 μ l dNTP subsequently, wherein comprise 1mMdCTP, 1mMdGTP, 1mMdTTP and 10mMdATP, 1.25 the T4DNA polysaccharase of μ l 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 the damping fluid of Tth archaeal dna polymerase thereof, and system expanded as 80 μ l, make the 3 ' end of DNA add the dA tail, this mixture is 24: 1 chloroform through the mixed volume ratio: extracting of primary isoamyl alcohol mixed solution and ether extracting, be connected 24 hours with the pGEM-T-Easy carrier in 16 ℃ then, cumulative volume is 90 μ l, the damping fluid that 10 times of T4DNA ligase enzymes of the T4DNA ligase enzyme of 25 units and 9 μ l are wherein arranged, be connected mixture with the 3M NaAc of 1/10 volume pH=5.2 and the dehydrated alcohol precipitation of 2 times of volumes at last, be dissolved in after 70% ethanol is washed in the 30 μ l water and obtain connecting product, preparation method with the electric transformed competence colibacillus cell of Bio-Rad company prepares the competence e.colidh5, get after 2-3 μ l connects product and 50 μ l competence bacillus coli DH 5 alphas mix, forward in the electric shock cup of 0.2cm of Bio-Rad company and shock by electricity, voltage is 2.5 kilovolts, time is 5.0 milliseconds-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 group has constituted the O-antigen gene bunch library of shigella dysenteriae 12 types;
4) acquisition of O-antigen gene bunch complete sequence
From O-antigen gene bunch 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, direct PCR and the PCR product checked order from the genomic dna of shigella dysenteriae 12 types again, thereby obtain all sequences of O-antigen gene bunch, the Pregap4 and the splicing of Gap4 software of the Staden package software package of publishing with Britain Camb Medical Research Council Molecular Biology Lab and edit all sequences, thereby obtain the Nucleotide full length sequence of the O-antigen gene bunch of shigella dysenteriae 12 types, the quality of sequence is mainly guaranteed by two aspects: (1) does 6 Long PCR reactions to each genome, mixes these products then to produce the library; (2), guarantee high-quality fraction of coverage more than 3 to each base;
5) acquisition of O-antigen gene clustering architecture
Find the gene in the nucleotide sequence of shigella dysenteriae 12 type O-antigen genes bunch with the orffinder of The National Center for Biotechnology Information, find the reading frame of 10 openings, determine also that with the function of finding the reading frame that these are open what gene they are with the genetic comparison among the blast groupware and the GenBank, finish gene annotation with the Artemis software at Britain sanger center again, do accurate comparison between DNA and protein sequence with Clustral W software, obtain the structure of the O-antigen gene bunch of shigella dysenteriae 12 types at last.
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| CN1171999C (en) | Nucletide specific to O-antigen of shigella dysenteriae 8 | |
| CN1252267C (en) | Nucleotide | |
| CN1257279C (en) | Nucleotide peculiar to 0-antigen of 0163 type bacillus coli | |
| CN1257178C (en) | Nucleotide peculiar to 0-antigen of 021 type bacillus coli | |
| CN1256432C (en) | Nucleotide peculiar to 0-antigen of 033 type bacillus coli | |
| CN1285728C (en) | Nucleotide to 0-antigen specificity of escherichia coli 0154 type | |
| CN1234857C (en) | Nucleotide peculiar to 0-antigen of 051 type bacillus coli | |
| CN1274831C (en) | Nucleotide specific to O-antigen of shigella dysenteriae I and Bacillus coli 0121 | |
| CN1256435C (en) | Nucleotide peculiar to 0-antigen of 110 type bacillus coli | |
| CN1253571C (en) | Nucleotide peculiar to 0-antigen of 0114 type bacillus coil | |
| CN1252263C (en) | O-antigen specific nucleotide of E.coli 0138 type | |
| CN1285727C (en) | O-antigen specific nucleotide of E.coli 0139 type | |
| CN1234866C (en) | Nucleotide peculiar to 0-antigen of 17 type Baoshi Sh. dysenterae | |
| CN1234863C (en) | Nucleotide peculiar to 0-antigen of 0130 type bacillus coli | |
| CN1252266C (en) | nucleotide and its use | |
| CN1324135C (en) | O-antigen specific nucleotide of E.coli 0109 type | |
| CN1234861C (en) | Nucleotide peculiar to 0-antigen of 095 type bacillus coli |
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