CN1687419A - Nucleotide specific to O antigen of 078 type bacillus coli - Google Patents

Nucleotide specific to O antigen of 078 type bacillus coli Download PDF

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CN1687419A
CN1687419A CNA2004100941162A CN200410094116A CN1687419A CN 1687419 A CN1687419 A CN 1687419A CN A2004100941162 A CNA2004100941162 A CN A2004100941162A CN 200410094116 A CN200410094116 A CN 200410094116A CN 1687419 A CN1687419 A CN 1687419A
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gene
nucleotide
antigen
intestinal bacteria
type
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CN100355890C (en
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王磊
韩巍青
冯露
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Tianjin Biochip Corp
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Tianjin Biochip Corp
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Abstract

This invention provides a specific nucleotide O-antigen for Escherichia coli 078, a complete sequence of nucleotide which controls the synthesis of O-antigen, e.g. as show in the SEQ ID NO: 1 the nucleic acid for separation has 12655 alkali bases, or compromising one or several insert, lost, or substitutes alkali bases, while keeping the function of the nucleic acid; It also compromises oligonucleotide which processing gene in the single-surge unit of O-antigen gene group form Escherichia coli; this invention proves that the oligonucleotide has a high specific function on Escherichia coli 078 by PCR, and discloses a method of identifying and detecting Escherichia coli by the oligonucleotide.

Description

Nucleotide to the O-antigen-specific of intestinal bacteria O78 type
Technical field
The present invention relates to the complete nucleotide sequence of control O-antigen synthetic gene cluster in the intestinal bacteria O78 type (Escherichia coli O78), particularly relate in the intestinal bacteria O78 type oligonucleotide in the control O-antigen synthetic gene cluster, can utilize these the oligonucleotide of the O-antigen-specific intestinal bacteria O78 type in human body and the environment and identify O-antigen in these pathogenic bacterium quickly and accurately.
Background technology
O-antigen is the O specific polysaccharide composition in the gram negative bacterium lipopolysaccharides, and it is made up of many multiple oligosaccharide unit.The antigenic building-up process of O-is studied clearlyer: by glycosyltransferase nucleoside diphosphate monose is transferred on the fat molecule that is fixed on the cell inner membrance earlier, then in the inboard synthesis of oligose unit of inner membrance, the antigenic oligosaccharide unit of O-is transferred to the inner membrance outside by the transhipment enzyme again, then aggregate into polysaccharide by polysaccharase, be connected to again and form lipopolysaccharide molecule [Whitfield, C. (1995) " Biosynthesis of lipopolysaccharide O antigens " .Trends inMicrobiology.3:178-185 on the glycolipid molecule; Schnaitman, C.A.and J.D.Klena. (1993) " Genetics oflipopolysaccharide biosynthesis in entericbacteria " .MicrobiologicalReviews, 57 (3): 655-682].Coding is responsible for the generally adjacent arrangement on karyomit(e) of gene of all enzyme molecules of O-antigen synthetic, form a gene cluster [Reeves, P.R., et al. (1996) " Bacterialpolysaccharide synthesis and gene nomenclature " Trendsin Microbiology, 4:495-503].In Shigellae, intestinal bacteria and Salmonellas, O-antigen gene [Lei Wang.et al (2001) " Sequence analysis of fourShigella boydii O-antigen loci:implication for Escherichia coli and Shigellarelationships " .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 geneclusters:gene clusters 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 intestinal bacteria, it has extremely strong diversity again simultaneously, and this enlightens us can study a kind of intestinal bacteria and good, highly sensitive method of the antigenic specificity of O-thereof of detecting quickly and accurately.With surperficial polysaccharide is that the serology immune response of target has been used to somatotype and the evaluation to bacterium always since the thirties in last century, is unique means of identifying pathogenic bacterium.This diagnostic method needs a large amount of antiserum(antisera)s, and the antiserum(antisera) general classes is incomplete, quantity not sufficient, and also there are some difficulties in a large amount of antiserum(antisera)s in preparation with in storing.On the other hand this method length consuming time, sensitivity is low, loss is high, poor accuracy, so, generally believe that now this traditional serology detection method will be that the modern molecular biology method replaces.1993, Luk, J.M.C et.al has identified the O-antigen [Luk of Salmonellas with the specific nucleotide sequence of Salmonellas (S.enterica) O-antigen gene bunch by PCR method, J.M.C.et.al. (1993) " Selective amplification of abequose andparatose synthase genes (rfb) by polymerase chain reaction for identification ofS.enterica major serogroups (A; B; C2; andD) ", J.Clin.Microbiol.31:2118-2123].Luk, the method for et.al is with corresponding to Salmonellas serotype E 1, D1 obtains the oligonucleotide special to the Salmonellas of different serotypes after the nucleotide sequence of the CDP-abequose in the A, the O-antigen of B and C2 and the synthetic gene of CDP-tyvelose is arranged.1996, Paton, the A.W et.al serotype [" Molecular microbiological investigation of an outbreak of Hemolytic-Uremic Syndrome caused by dry fermented sausage contaminated with Shiga-liketoxin producing Escherichia coli " .J.Clin.Microbiol.34:1622-1627] of the oligonucleotide that comes from the wbdI gene of the O-antigen-specific of E.coli O111 having been identified the toxogenic E.coli O111 of a strain, but afterwards studies show that Paton, the usefulness of A.W et.al comes from the oligonucleotide of wbdI gene and identifies that the method for the serotype of E.coli O111 has false positive results to occur.Bastin D.A.and Reeves, P.R. think, this is because the wbdI gene is sugared synthesis path gene [the Bastin D.A.and Reeves of a supposition, P.R. (1995) Sequence and analysis of the O antigen gene (rfb) cluster of Escherichiacoli O111.Gene 164:17-23], and in the antigenic structure of the O-of other bacterium, also has this sugar, so sugared synthesis path gene is not that the Shigellae of high special has 46 kinds of serotypes for O-antigen, 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 there are 12 kinds to be intestinal bacteria and the total [Ewing of Shigellae, W.H. (1986) " Edwards andEwing ' s identification of the Enterobacteriaceae " .Elsevier SciencePublishers, Amsterdam, The Netherlands; T.cheasty, et al. (1983) " Antigenicrelationships between the enteroinvasive Escherichia coli antigensO28ac; O112ac; O124; O136, O143, O144; O152 and and Shigella O antigens " J.clin Microbiol, 17 (4): 681-684].
Summary of the invention
The Nucleotide that the purpose of this invention is to provide a kind of O-antigen-specific to intestinal bacteria O78 type.It is the Nucleotide in the O-antigen gene bunch of intestinal bacteria O78 type, is the special Nucleotide that comes from glycosyltransferase gene and transhipment enzyme gene and pol gene.
A time purpose of the present invention has provided the full length nucleotide sequence of the O-antigen gene bunch of intestinal bacteria O78 type.
Another object of the present invention provides the gene of the O-antigen gene bunch that constitutes intestinal bacteria O78 type: the gene of transhipment enzyme is the wzx gene or with wzx the gene of identity function is arranged; Pol gene is the wzy gene or with wzy the gene of identity function is arranged; Glycosyltransferase gene comprises orf1, orf2, orf6 gene.
Another purpose of the present invention has provided oligonucleotide, and the gene that they come from coding transhipment enzyme in the O-antigen gene bunch of intestinal bacteria O78 type respectively is the wzx gene or with wzx the gene of identity function is arranged; The gene that comes from the coding polysaccharase is the wzy gene or with wzy the gene (table 1) of identity function is arranged; They are the oligonucleotide in the said gene, and length is at 10-25nt; They are high specials to the O-antigen of intestinal bacteria O78 type; And these oligonucleotide are also reconfigurable, and the oligonucleotide after the combination also is a high special to the O-antigen of intestinal bacteria O78 type.
A further object of the present invention provides above-mentioned oligonucleotide and can be used as primer and be used for nucleic acid amplification reaction, perhaps be used for hybridization as probe, perhaps be used to make gene chip or microarray, thereby pass through O-antigen and the detection and the identification of escherichia coli O78 type of these methods detections and identification of escherichia coli O78 type.
An also purpose of the present invention has provided the method for complete sequence of the O-antigen gene bunch of separating Escherichia coli O78 type.Can obtain the complete sequence of the O-antigen gene bunch of other bacteriums according to present method operation, the complete sequence of the gene cluster of the bacterium of other polysaccharide antigens that also can obtain to encode.
The objective of the invention is to realize by following technical scheme.
The present invention is characterized in that the Nucleotide of the O-antigen-specific of intestinal bacteria O78 type: it is the isolating Nucleotide shown in SEQ ID NO:1,12655 bases of total length; Perhaps described base with one or more insertions, disappearance or replacement keeps the Nucleotide of the SEQ IDNO:1 of described isolating functional nucleotide simultaneously.
The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O78 type, comprising called after orf1, orf2, manC, manB, orf5, orf6, wzy, orf8, orf9, orf10,11 genomic constitutions of wzx are all between JUMPStart sequence and gnd gene.
The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O78 type, the gene that has high degree of specificity in the wherein said gene is: transhipment enzyme gene, it comprises the wzx gene; Pol gene, it comprises the wzy gene; Glycosyltransferase gene, it comprises orf1, orf2, orf6 gene; Wherein said gene: wzx is the Nucleotide of 11139 to 12554 bases among the SEQ ID NO:1; Wzy is the Nucleotide of 7291 to 8412 bases among the SEQ ID NO:1; Orf1 is the Nucleotide of 74 to 1216 bases among the SEQ ID NO:1; Orf2 is the Nucleotide of 1237 to 2346 bases among the SEQ ID NO:1; Orf6 is the Nucleotide of 6168 to 7289 bases among the SEQID NO:1.The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O78 type wherein also comprises coming from described wzx gene, wzy gene and their mixing or their reorganization.
The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O78 type is characterized in that the oligonucleotide that wherein comes from the wzx gene is to being: the Nucleotide of 11344 to 11363 bases among the SEQ ID NO:1 and the Nucleotide of 11937 to 11955 bases; The Nucleotide of 11935 to 11953 bases among the SEQ ID NO:1 and the Nucleotide of 12503 to 12520 bases.The oligonucleotide that comes from the wzy gene is to being: the Nucleotide of 7568 to 7586 bases among the SEQ ID NO:1 and the Nucleotide of 8175 to 8193 bases; The Nucleotide of 7728 to 7744 bases among the SEQ ID NO:1 and the Nucleotide of 8204 to 8224 bases.
The application of the Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O78 type in detecting other polysaccharide antigen of expressing the antigenic bacterium of O-, the O-antigen of identifying bacterium and bacterium.
The recombinant molecule of the Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O78 type is providing the O-antigen of expressing intestinal bacteria O78 type and the application in the preparation bacterial vaccine by inserting to express.
The application of the Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O78 type 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 as probe as primer, for the application of bacterial detection.
The separation method of the Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O78 type is characterized in that it comprises the steps:
(1) genomic extraction: in substratum, cultivate intestinal bacteria O78 type, centrifugal collecting cell; The genomic dna that obtains detects by agarose gel electrophoresis;
(2) by the O-antigen gene in the pcr amplification intestinal bacteria O78 type bunch: with the genome of intestinal bacteria O78 type is template by increase its O-antigen gene bunch of LongPCR, with the PCR product that obtains, detect the size and the specificity thereof of PCR product with agarose gel electrophoresis, merge this long PCR product, and with DNA purification kit purified pcr product;
(3) make up O-antigen gene bunch library: Long PCR purified product is used shotgun make up O-antigen gene bunch library;
(4) to the cloning and sequencing in the library: from the library, select the clone of insertion fragment more than 1kb and the insertion fragment among the clone is checked order with laboratory automatic dna sequencer commonly used, sequence reaches 100% fraction of coverage, thereby obtains all sequences of O-antigen gene bunch;
(5) splicing of nucleotide sequence and analysis: applying biological information science software splicing and edit all sequences, thus obtain the Nucleotide full length sequence of the O-antigen gene bunch of intestinal bacteria O78 type;
(6) screening of specific gene: at wzx, the wzy gene design primer in the O-antigen gene of intestinal bacteria O78 type bunch; Respectively designed two pairs of primers in each gene, every pair of primer is distributed in the different places in the corresponding gene, to guarantee its specificity; Is that template is carried out PCR with these primers with the genomes of 166 strain intestinal bacteria and 43 strain Shigellaes, determines the antigenic high degree of specificity of O-of wzx, wzy gene pairs intestinal bacteria O78 type;
(7) detection of primer sensitivity: cultivate intestinal bacteria O78, after the bacterial count respectively with 5 * 10 3, 5 * 10 2, 5 * 10 15 and 0 viable bacteria join in a certain amount of certain thing to be detected, sneak into the thing to be detected of bacterium and use sample as detecting, sample is added the LB substratum, getting the LB substratum that some and sample mix cross filters, filtered liquid is cultivated, carried out the PCR reaction as pcr template with oligonucleotide after the peek milliliter is handled from cultured bacterium liquid, detect its sensitivity intestinal bacteria O78.
The separation method of the Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O78 type is characterized in that it comprises the steps:
(1) genomic extraction: 37 ℃ of incubated overnight intestinal bacteria O78 types in the LB of 5mL substratum, centrifugal collecting cell.With 500 μ l 50mM Tris-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, the RNase that adds 3 μ l 10mg/mL again, 65 ℃ of incubations 30 minutes, add equal-volume phenol extracting mixture, get supernatant and use isopyknic phenol again: chloroform: primary isoamyl alcohol (25: 24: 1) mixing solutions 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, and DNA is resuspended among the 30 μ l TE; Genomic dna detects by 0.4% agarose gel electrophoresis;
(2) by the O-antigen gene in the pcr amplification intestinal bacteria O78 type bunch: with the genome of intestinal bacteria O78 type is that template is passed through its O-antigen gene of Long pcr amplification bunch, at first according to the JUMPStart sequences Design upstream primer (#wl-1098-ATTGGT AGC TGT AAG CCA AGG GCG GTA GCG T) 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 AGTTCG 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, 55-annealing 15 seconds, 68 ℃ were extended 15 minutes, carry out 30 circulations like this, last, continue to extend 7 minutes at 68 ℃, obtain the PCR product, detect the size and the specificity thereof of PCR product with 0.8% agarose gel electrophoresis, merge 5 pipe long PCR products, and with the Wizard PCRPreps 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, 0.9 μ l 0.1MMnCl 2, the DNaseI of the 1mg/mL of 1 μ l dilution in 1: 2000, reaction is carried out at room temperature, and enzyme is cut the dna fragmentation size is concentrated between the 1.5kb-3kb, then adds 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) mixing solutions extracting once, after using isopyknic ether extracting once again, dehydrated alcohol deposit D NA with 2.5 times of volumes, and wash precipitation with 70% ethanol, be resuspended at last in the 18 μ l water, in this mixture, add 2.5 μ l dNTP (1mMdCTP, 1mMdGTP, 1mMdTTP subsequently, 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 corresponding damping fluid thereof and system is expanded as 80 μ l, 70 ℃ of reactions 20 minutes make 3 of DNA ' end add the dA tail.This mixture is through the equal-volume chloroform: after primary isoamyl alcohol (24: 1) mixing solutions extracting and the extracting of equal-volume ether with 3 * 10 of Promega company -3The pGEM-T-Easy carrier connect 10 hours in 16 ℃, cumulative volume is 90 μ l.10 * the buffer of 9 μ l and the T4DNA ligase enzyme of 25 units are wherein arranged, use the 3M NaAc (pH5.2) of 1/10 volume and the dehydrated alcohol precipitation of 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; 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 to 6.0 milliseconds, the SOC substratum that adds 1mL after the electric shock immediately in cup makes the bacterium recovery, then bacterium is coated in and contains penbritin, on the LB solid medium of X-Gal and IPTG, 37 ℃ of incubated overnight, obtain blue white bacterium colony next day, with the white colony that obtains promptly the white clone forward on the LB solid medium that contains penbritin and cultivate, from each clone, extract plasmid simultaneously, and cutting the segmental size of evaluation insertion wherein with the EcoRI enzyme, the white that obtains clone group has constituted the O-antigen gene bunch library of intestinal bacteria O78 type;
(4) to the cloning and sequencing in the library: from the library, select 97 clones of insertion fragment more than 1kb and the insertion fragment among the clone is checked order with this lab A BI3730 type automatic dna sequencer, sequence reaches 100% fraction of coverage, thereby obtains all sequences of O-antigen gene bunch;
(5) splicing of nucleotide sequence and analysis: the Pregap4 and the splicing of Gap4 software of the Staden package software package of publishing with Britain Camb MRC (Medical Research Council) Molecular Biology Lab and edit all sequences, thus obtain the Nucleotide full length sequence of the O-antigen gene bunch of intestinal bacteria O78 type; The quality of sequence is mainly guaranteed by two aspects: 1) genome of intestinal bacteria O78 type is done 5 Long PCR reactions, mix these products then to produce the library, 2) to each base, guarantee high-quality fraction of coverage more than 3, after obtaining the nucleotide sequence of intestinal bacteria O78 type O-antigen gene bunch, with American National biotechnology information science center (The National Center forBiotechnology Information, NCBI) Orffinder finds gene, find the reading frame of 11 openings, determine also that with the function of finding the reading frame that these are open what gene they are with the genetic comparison among the Blast groupware and the GenBank, finish gene annotation with the Artemis software at Britain Sanger center again, do accurate comparison between DNA and protein sequence with Clustral W software, obtain the structure of the O-antigen gene bunch of intestinal bacteria O78 type at last;
(6) specific gene screening: at wzx, the wzy gene design primer in the O-antigen gene of dysentery intestinal bacteria O78 type bunch; Respectively designed two pairs of primers in each gene, every pair of primer is distributed in the different places in the corresponding gene, to guarantee its specificity; Is that template is carried out PCR with these primers with the genomes of 166 strain intestinal bacteria and 43 strain Shigellaes, except that a band that in containing intestinal bacteria O78 group, has obtained the expection size, the correct product of the expection clip size that all do not increase in other groups is so the O-antigen of wzx, wzy gene pairs intestinal bacteria O78 type all is high special.
(7) detection of primer sensitivity: buy the live pig meat stuffing on the market, stir, be divided into the 20g portion, exist in-40 ℃ of refrigerators standby.The frozen bacterium liquid of 10 μ l intestinal bacteria O78 is inoculated in the triangular flask of 20mL LB substratum, in 37 ℃, 200 rev/mins, cultivate 12 hours to saturated, the cultured bacterium liquid that takes a morsel does 10 6With 10 7Dilution doubly, remaining bacterium liquid are put in 4 ℃ the refrigerator standby, get 50 μ l dilution bacterium liquid coating LB agar plate, and 37 degree are cultivated 12h, to being coated with plate count, calculate viable bacteria concentration in the stoste.In 5 portions of live pig meat stuffings, mix 5 * 10 respectively 3, 5 * 10 2, 5 * 10 1, 5 and 0 viable bacteria stir, and add 200mL LB substratum, and through 6 layers of filtered through gauze, filtered liquid 200 rev/mins, is cultivated 12h in 37 ℃.Get 3mL bacterium liquid in 6 from cultured bacterium liquid, centrifugal 5 minutes of 000g removes supernatant, adds 100 μ l MQ ultrapure waters and blows precipitation and mixing open, puts into 100 degree boiling water and boils 15 minutes, and lysate is in 12, and centrifugal 8 minutes of 000g gets 1 μ supernatant as pcr template.Right with 4 pairs of oligonucleotide, the Nucleotide of 11344 to 11363 bases among the SEQ ID NO:1 and the Nucleotide of 11937 to 11955 bases; The Nucleotide of 11935 to 11953 bases among the SEQ ID NO:1 and the Nucleotide of 12503 to 12520 bases; The Nucleotide of 7568 to 7586 bases among the SEQ ID NO:1 and the Nucleotide of 8175 to 8193 bases; The Nucleotide of 7728 to 7744 bases among the SEQ ID NO:1 and the Nucleotide of 8204 to 8224 bases carry out the PCR reaction, and the PCR reaction system is as follows: MQ:15.7 μ l, Mg 2+: 2.5 μ l, Buffer:2.5 μ l, dNTP:1 μ l, Taq enzyme: 0.3 μ l, P1:1 μ l, P2:1 μ l, template DNA: 1 μ l.The PCR reaction conditions is: 95 ℃: 5 ', 95 ℃: 30 ", 56 ℃: 45 ", 72 ℃: 1 ', 72 ℃: 5 ', totally 30 circulations.Reaction is got 10 μ l reaction product electrophoresis after finishing, if the amplified band that conforms to the expection size is arranged, then the result is positive, if do not have, then the result is negative.Participated in 5 * 10 3, 5 * 10 2, 5 * 10 1And every part of pork filling of 5 viable bacterias all obtains positive findings in the PCR of 4 pairs of primers reaction.The pork filling that participates in 0 viable bacteria obtains negative findings in the PCR of 4 pairs of primers reaction.Illustrate that these 4 pairs of primers are 0.25 bacterium/g to the detection sensitivity of the intestinal bacteria O78 in the pork filling when using aforesaid method.
Just, first aspect of the present invention provides the full length nucleotide sequence of the O-antigen gene bunch of intestinal bacteria O78 type, its complete sequence shown in SEQ ID NO:1,12655 bases of total length; The base that perhaps has one or more insertions, disappearance or replacement keeps the Nucleotide of the SEQ ID NO:1 of described isolating functional nucleotide simultaneously.Obtained the structure of the O-antigen gene bunch of intestinal bacteria O78 type by method of the present invention, as shown in table 3, it comprises called after orf1, orf2, manC, manB, orf5, orf6, wzy, orf8, orf9, of10,11 genomic constitutions of wzx are all between JUMPStart sequence and gnd gene.
Second aspect of the present invention provides the gene in the O-antigen gene bunch of intestinal bacteria O78 type, promptly transports enzyme gene (wzx gene or the gene of identity function arranged with wzx): pol gene (wzy gene or the gene of identity function arranged with wzy); Glycosyltransferase gene (orf1, orf2, orf6 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 intestinal bacteria O78 type.
The 3rd aspect of the present invention, wzx gene in the O-antigen gene bunch that comes from intestinal bacteria O78 type is provided or the gene of identity function and wzy gene is arranged or with wzy the oligonucleotide (table 1) of the gene of identity function is arranged with wzx, they are any one section oligonucleotide in these genes.In table 1, also listed these oligonucleotide to the position in O-antigen gene bunch and with these oligonucleotide to being the size of the product of the PCR reaction done of primer, the annealing temperature in these PCR reaction free lists is carried out.These primers are except that obtain in the 4th group expecting that the band of size, any product that all do not increase is so the O-antigen of wzx, wzy gene pairs intestinal bacteria O78 type all is high special in other groups.
The separation method of the Nucleotide of described O-antigen-specific to intestinal bacteria O78 type comprises the steps: 1) genomic extraction; 2) the O-antigen gene in the pcr amplification intestinal bacteria O78 type bunch; 3) make up O-antigen gene bunch library; 4) to the cloning and sequencing in the library; 5) splicing of nucleotide sequence and analysis; 6) screening of specific gene; 7) detection of primer sensitivity.
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 refer to derive from the gene of the coding transhipment enzyme in the O-antigen gene bunch and intragenic one section nucleic acid molecule of coding polysaccharase, and they can change on length, generally change in 10 to 20 Nucleotide scopes; More definite these oligonucleotide of saying are to come from wzx gene (nucleotide position is the Nucleotide of 11139 to 12554 bases from SEQ ID NO:1); Wzy gene (nucleotide position is the Nucleotide of 7291 to 8412 bases from SEQ ID NO:1); Coming from above intragenic oligonucleotide is high special to intestinal bacteria O78 type.
In addition, the antigenic gene cluster of the different O-of the coding of two genetic resemblances produces new O-antigen by gene recombination or sudden change sometimes, thereby produces new bacteria types, new mutant strain.In this environment, need filter out many specificitys that oligonucleotide is detected with raising with recombination hybridization.Therefore, the invention provides a whole set of many mixtures to oligonucleotide, they come from transhipment enzyme and pol gene, comprise the wzx gene or the gene, wzy gene of identity function are arranged or with wzy the gene of identity function is arranged with wzx.The mixture of these genes is special to a special bacterial polysaccharides antigen, is special thereby make this cover oligonucleotide to the polysaccharide antigen of this bacterium.More particularly, the mixture of these oligonucleotide is to come from the wzx gene or the gene, wzy gene of identity function arranged or with wzy the combination of the oligonucleotide in the gene of identity function is arranged with wzx.
On the other hand, the present invention relates to the evaluation of oligonucleotide, they can be used for detecting the O-antigen of expressing the antigenic bacterium of O-and identifying bacterium in diagnosis.
The present invention relates to a kind of antigenic method of one or more bacterial polysaccharideses that detects in the food, these antigens can make sample can with the oligonucleotide specific hybrid of following at least one gene, these genes are: (i) gene of the encoding glycosyl transferring enzyme gene of transhipment enzyme and polysaccharase of (ii) encoding comprises the wzx gene or the gene, wzy gene of identity function is arranged or with wzy the gene of identity function is arranged with wzx.At least one oligonucleotide can be hybridized with at least one more than one such gene specific of expressing the special antigenic bacterium of O-under the situation of condition permission, and these bacteriums are intestinal bacteria O78 types.Available PCR method detects, more can with behind the Nucleotide mark in the inventive method as probe by hybridization such as Southern-blot or fluoroscopic examination, perhaps by antigen and bacterium in gene chip or the microarray assay sample.
Planner of the present invention considers following situation: when one special oligonucleotide detects when invalid, the mixture of oligonucleotide can with the target region specific hybrid with test sample.Therefore the invention provides a cover oligonucleotide and be used for detection method of the present invention.Here said oligonucleotide is meant the gene that comes from the encoding glycosyl transferring enzyme, the gene of coding transhipment enzyme and the gene of polysaccharase, comprises the wzx gene or the gene, wzy gene of identity function is arranged or with wzy the oligonucleotide of the gene of identity function is arranged with wzx.This cover oligonucleotide is special to the O-antigen of a special bacterium, and this special bacterium O-antigen is expressed by intestinal bacteria O78 type.
On the other hand, the present invention relates to a kind of antigenic method of one or more bacterial polysaccharideses that detects in the movement, these antigens can make sample can with the oligonucleotide specific hybrid of following at least one gene, these genes are: (i) gene of the encoding glycosyl transferring enzyme gene of transhipment enzyme and polysaccharase of (ii) encoding comprises the wzx gene or the gene, wzy gene of identity function is arranged or with wzy the gene of identity function is arranged with wzx.At least one oligonucleotide can be expressed more than one such gene specific hybridization of the special antigenic bacterium of O-with at least one under the situation of condition permission.These bacteriums are intestinal bacteria O78 types.Oligonucleotide among available the present invention is made the method test sample of primer by PCR, also can with behind the oligonucleotide molecules mark among the present invention as probe by hybridization such as Southern-blot or fluoroscopic examination, perhaps by antigen and bacterium in gene chip or the microarray assay sample.
General a pair of oligonucleotide may with same gene recombination also can with different gene recombinations, but must have in them an oligonucleotide can specific hybrid to the distinguished sequence of special antigenic type, another oligonucleotide can be hybridized in non-specific zone.Therefore, when the oligonucleotide in the special polysaccharide antigen gene cluster is reconfigured, can select specific gene mixture hybridization in a pair of oligonucleotide and the polysaccharide antigen gene cluster at least, perhaps select many mixture hybridization oligonucleotide and specific gene.Even even when all genes were all unique in the specific genes bunch, this method also can be applied to discern the nucleic acid molecule of the gene mixture in this gene cluster.Therefore the invention provides a whole set of is used to detect the many to oligonucleotide of the inventive method, many here is that the gene of the gene that comes from the encoding glycosyl transferring enzyme, coding transhipment enzyme and polysaccharase comprises the wzx gene or the gene, wzy gene of identity function arranged or with wzy the gene of identity function is arranged with wzx to oligonucleotide, this cover oligonucleotide is special to a special bacterial polysaccharides, and this cover oligonucleotide may be the Nucleotide of necessary gene during sugar synthesizes.
On the other hand, the present invention also relates to the antigenic method of one or more bacterial polysaccharideses in the sample that a kind of detection comes from patient.One or more bacterial polysaccharides antigens in the sample can make sample can with a specific hybrid in a pair of oligonucleotide in following at least one gene, these genes are: (i) gene of the encoding glycosyl transferring enzyme gene of transhipment enzyme and polysaccharase of (ii) encoding comprises the wzx gene or the gene, wzy gene of identity function is arranged or with wzy the gene of identity function is arranged with wzx.Under the situation of condition permission at least one oligonucleotide can with sample at least one express more than one such gene specific hybridization of the special antigenic bacterium of O-, these bacteriums are intestinal bacteria O78 types.Oligonucleotide among available the present invention is made the method test sample of primer by PCR, also can will pass through hybridization as probe behind the oligonucleotide mark among the present invention, perhaps by antigen and bacterium in gene chip or the microarray assay sample.
In more detail, method described above can be understood as when oligonucleotide when being used, it is not to derive from the wzx gene or with wzx the gene, wzy gene of identity function arranged or have on the sequence of gene of identity function with wzy that one of them oligonucleotide molecules can hybridize to one.In addition, when two oligonucleotide can both be hybridized, they may be hybridized in same gene and also may hybridize on the different genes.Also promptly, when cross reaction goes wrong, can select the mixture of oligonucleotide to detect the blended gene so that the specificity of detection to be provided.
The present inventor believes that the present invention is not necessarily limited to the above nucleotide sequence coded specific O-antigen of carrying, and is widely used in detecting all expression O-antigens and identifies the antigenic bacterium of O-.And because O-antigen is synthetic and the similarity of other polysaccharide antigens (as bacterium born of the same parents exoantigen) between synthesizing, method of the present invention and molecule also are applied to these other polysaccharide antigen.
The present invention discloses the full length sequence of the O-antigen gene bunch of intestinal bacteria O78 type first, and can from the sequence of this total length gene cluster of not cloned, produce recombinant molecule, can produce the O-antigen of expressing intestinal bacteria O78 type by inserting to express, and become useful vaccine.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should understand these embodiment only is used to the present invention is described and is not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, molecular cloning: the condition described in the laboratory manual (NewYork:Cold Spring Harbor Laboratory Press, 1989).
Embodiment 1: genomic extraction:
37 ℃ of incubated overnight intestinal bacteria O78 types in the LB of 5mL substratum, centrifugal collecting cell.With 500 μ l 50mM Tris-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 ℃ of incubations 30 minutes.Add equal-volume phenol extracting mixture, get supernatant and use isopyknic phenol again: chloroform: primary isoamyl alcohol is taken out (25: 24: 1) mixing solutions and is carried twice, get supernatant again with isopyknic ether extracting to remove remaining phenol, supernatant is with 2 times of volume ethanol deposit D NA, roll out DNA and wash DNA with glass yarn, at last DNA is resuspended among the 30 μ l TE with 70% ethanol.Genomic dna detects by 0.4% agarose gel electrophoresis.
Embodiment 2: by the O-antigen gene in the pcr amplification intestinal bacteria O78 type bunch:
With the genome of intestinal bacteria O78 type is that template is passed through its O-antigen gene of Long pcr amplification bunch.At first according to the JUMPStart sequences Design upstream primer (#wl-1098-ATT GGT AGC TGT AAG CCA AGG GCG GTA GCG T) that often is found in O-antigen gene bunch promoter region, again according to the gnd gene design downstream primer (#1524-TAG TCG CGTGNG CCT GGA TTA AGT TCG C) in O-antigen gene bunch downstream; With the ExpandLong Template PCR method of Boehringer Mannheim company amplification O-antigen gene bunch, the PCR response procedures was as follows: 94 ℃ of pre-sex change 2 minutes; 94 ℃ of sex change are 10 seconds then, 55 annealing 15 seconds, and 68 ℃ were extended 15 minutes, and carried out 30 circulations like this.At last, continue to extend 7 minutes at 68 ℃, obtain the PCR product, the agarose gel electrophoresis with 0.8% detects the size and the specificity thereof of PCR product.Merge 5 pipe long PCR products, and with the Wizard PCR Preps purification kit purified pcr product of Promega company.
Embodiment 3: make up O-antigen gene bunch library:
At first be the acquisition that connects product: make up O-antigen gene bunch library with the Novagen DNaseI shot gun method that is modified.Reaction system is a 300ng PCR purified product, 0.9 μ l 0.1M MnCl 2, 1 μ l1: the DNaseI of the 1mg/mL of 2000 dilutions, reaction is carried out at room temperature.Enzyme is cut the dna fragmentation size is concentrated between the 1.5kb-3kb, then adds 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) mixing solutions extracting once, after using isopyknic ether extracting once again, dehydrated alcohol deposit D NA with 2.5 times of volumes, and wash precipitation with 70% ethanol, be resuspended at last in the 18 μ l water.In this mixture, add 2.5 μ l dNTP (1mMdCTP subsequently, 1mMdGTP, 1mMdTTP, 10mMdATP), the T4DNA polysaccharase of 1.25 μ l 100mMDTT 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 80 μ l, 70 ℃ were reacted 20 minutes, made 3 of DNA ' end add the dA tail.This mixture is through the equal-volume chloroform: after primary isoamyl alcohol (24: 1) mixing solutions extracting and the extracting of equal-volume ether with 3 * 10 of Promega company -3The pGEM-T-Easy carrier connect 10 hours in 16 ℃, cumulative volume is 90 μ l.10 * the buffer 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.
Next is the preparation of competent cell: the method that provides with reference to Bio-Rad company prepares the competent cell bacillus coli DH 5 alpha.Get a ring bacillus coli DH 5 alpha list bacterium colony in the LB of 5mL substratum, 180rpm cultivated after 10 hours, got in the LB substratum that the 2mL culture is transferred to 200mL, and 37 ℃ of 250rpm thermal agitations are cultivated OD600 about 0.5, ice bath cooling was 20 minutes then, in centrifugal 15 minutes of 4 ℃ of 4000rpm.Confide all supernatant, dispel thalline, in centrifugal 15 minutes of 4 ℃ of 4000rpm with the deionization aqua sterilisa 200mL of cold ice precooling.Deionization aqua sterilisa 100mL with cold ice precooling dispelled thalline again, in centrifugal 15 minutes of 4 ℃ of 4000rpm.With 10% glycerine suspension cell of cold ice precooling, centrifugal 10 minutes of 4 ℃ of 6000rpm abandon supernatant, precipitate 10% glycerine suspension cell with the precooling of 1mL ice at last, are competent cell.The competent cell that makes is packed as 50 μ l, one pipe ,-70 ℃ of preservations.
Be electric transformed competence colibacillus cell at last: 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, from each clone, extract plasmid and cut the segmental size of evaluation insertion wherein simultaneously, obtain the O-antigen gene bunch library that white clone group has constituted intestinal bacteria O78 type with the EcoRI enzyme.
Embodiment 4: to the cloning and sequencing in the library:
From the library, select insert 97 clones of fragment more than 1kb with this lab A BI3730 type automatic dna sequencer to unidirectional order-checking of insertion fragment among the clone, make sequence reach 100% fraction of coverage, thus all sequences of acquisition O-antigen gene bunch.
Embodiment 5: the splicing of nucleotide sequence and analysis:
The Pregap4 and the splicing of Gap4 software of the Staden package software package of publishing with Britain Camb MRC (Medical Research Council) Molecular Biology Lab and edit all sequences, thus the Nucleotide full length sequence (seeing sequence list) of the O-antigen gene bunch of intestinal bacteria O78 type obtained.The quality of sequence is mainly guaranteed by two aspects: 1) genome of intestinal bacteria O78 type is done 5 LongPCR reactions, mix these products then to produce the library.2), guarantee high-quality fraction of coverage more than 3 to each base.After obtaining the nucleotide sequence of intestinal bacteria O78 type O-antigen gene bunch, with American National biotechnology information science center (The National Center for BiotechnologyInformation, NCBI) Orffinder finds gene, find the reading frame of 11 openings, determine also that with the function of finding the reading frame that these are open what gene they are with the genetic comparison among the Blast groupware and the GenBank, finish gene annotation with the Artemis software at Britain Sanger center again, do accurate comparison between DNA and protein sequence with ClustralW software, obtain the structure of the O-antigen gene bunch of intestinal bacteria O78 type at last, as shown in table 3.
By retrieving and comparing, the albumen of finding the manC genes encoding in orf3 encoded protein and Escherichia coli K12 (AAC75110) the O-antigen has 90% consistence and 96% similarity in 472 amino acid whose sequences, show the homology that height is arranged between them, therefore, be manC with the temporary transient called after of orf3.The albumen of the manB genes encoding in orf4 encoded protein and Escherichia coli K12 (AAC75109) the O-antigen has 96% consistence and 98% similarity in 454 amino acid whose sequences, show the homology that height is arranged between them, therefore, be manB with the temporary transient called after of Drf4.
Orf5, orf8, orf9, the orf10 encoded protein respectively with Vibrio cholerae O1 biovareltor str.N16961 (AAF93404), Vibrio cholerae O1 biovar eltor str.N16961 (AAF93408), Vibrio cholerae O1 biovar eltor str.N16961 (AAF93407), the albumen of supposition has the similarity of sequence identity and the 50%-76% of 29%-52% among the Vibrio cholerae O1 biovar eltor str.N16961 (AAF93406), but the function of these four genes be can not determine, temporarily with its called after orf5, orf8, orf9, orf10.
Orf7 and orf11 are the proteic genes that there is transmembrane segment in only two codings in the O-antigen gene bunch of intestinal bacteria O78.The O-antigen polysaccharase of orf7 encoded protein and Shigella boydii (AAL27339) has 24% consistence and 47% similarity, it contains 9 transmembrane segments by the proteic topology discovery of HMMTOP2.0 programanalysis, and hydrophilic loop (loop) in the big kytoplasm is arranged, and this is the proteic characteristic feature of Wzy.So name orf7 is wzy.The O-antigen transhipment enzyme of orf11 encoded protein and Escherichia coli (AAK64374) has 22% sequence identity and 46% similarity, it contains 12 uniform transmembrane segments by the proteic topology discovery of HMMTOP2.0 programanalysis, and this is the proteic characteristic feature of Wzx.So name orf11 is wxz.
The albumen of orf1, orf2, three genes encodings of orf6 and other known glycosyltransferases have the sequence identity of 28-31% and the sequence similarity of 47-52%.By search to glycosyltransferase motif database among the Pfam, the homology desired value of the consensus sequence of the albumen of these three genes encodings and known glycosyltransferase family 1 and 2 is very high, therefore we infer this three genes encoding glycosyltransferases, and because each glycosyltransferase specificity catalysis forms a kind of disaccharide bond, this is consistent with the O-antigenic structure of intestinal bacteria O78.Because the definite function of these three genes can't be determined, so we are with the temporary called after orf1 of these three genes, orf2, orf6.
Embodiment 6: the screening of specific gene.
Wzx, wzy gene design primer in the O-antigen gene of intestinal bacteria O78 type bunch have respectively designed two pairs of primers in each gene, every pair of primer is distributed in different local in the corresponding gene, to guarantee its specificity; Is that template is carried out PCR with these primers with the genomes of 166 strain intestinal bacteria and 43 strain Shigellaes, except that a band that in containing intestinal bacteria O78 group, has obtained the expection size, the correct product of the expection clip size that all do not increase in other groups is so the O-antigen of wzx, wzy gene pairs intestinal bacteria O78 type all is high special; The position of these genes in nucleotide sequence sees Table 1.
Embodiment 7: the detection of primer sensitivity.
Buy the live pig meat stuffing on the market, stir, be divided into the 20g portion, exist in-40 ℃ of refrigerators standby.The frozen bacterium liquid of 10 μ l intestinal bacteria O78 is inoculated in the triangular flask of 20mL LB substratum, in 37 ℃, 200 rev/mins, cultivate 12 hours to saturated, the cultured bacterium liquid that takes a morsel is done 106 and 107 times dilution, remaining bacterium liquid is put in 4 ℃ the refrigerator standby, get 50 μ l dilution bacterium liquid coating LB agar plate, 37 degree are cultivated 12h, to being coated with plate count, calculate viable bacteria concentration in the stoste.In 5 portions of live pig meat stuffings, mix 5 * 10 respectively 3, 5 * 10 2, 5 * 10 1, 5 and 0 viable bacteria stir, and add 200mL LB substratum, and through 6 layers of filtered through gauze, filtered liquid 200 rev/mins, is cultivated 12h in 37 ℃.Get 3mL bacterium liquid in 6 from cultured bacterium liquid, centrifugal 5 minutes of 000g removes supernatant, adds 100 μ l MQ ultrapure waters and blows precipitation and mixing open, puts into 100 degree boiling water and boils 15 minutes, and lysate is in 12, and centrifugal 8 minutes of 000g gets 1 μ supernatant as pcr template.Right with 4 pairs of oligonucleotide, the Nucleotide of 11344 to 11363 bases among the SEQ ID NO:1 and the Nucleotide of 11937 to 11955 bases; The Nucleotide of 11935 to 11953 bases among the SEQ ID NO:1 and the Nucleotide of 12503 to 12520 bases; The Nucleotide of 7568 to 7586 bases among the SEQ IDNO:1 and the Nucleotide of 8175 to 8193 bases; The Nucleotide of 7728 to 7744 bases among the SEQ IDNO:1 and the Nucleotide of 8204 to 8224 bases carry out the PCR reaction, and the PCR reaction system is as follows: MQ:15.7 μ l, Mg 2+: 2.5 μ l, Buffer:2.5 μ l, dNTP:1 μ l, Taq enzyme: 0.3 μ l, P1:1 μ l, P2:1 μ l, template DNA: 1 μ l.The PCR reaction conditions is: 95 ℃: 5 ', 95 ℃: 30 ", 55 ℃: 45 ", 72 ℃: 1 ', 72 ℃: 5 ', totally 30 circulations.Reaction is got 10 μ l reaction product electrophoresis after finishing, if the amplified band that conforms to the expection size is arranged, then the result is positive, if do not have, then the result is negative.Participated in 5 * 10 3, 5 * 10 2, 5 * 10 1And every part of pork filling of 5 viable bacterias all obtains positive findings in the PCR of 4 pairs of primers reaction.The pork filling that participates in 0 viable bacteria obtains negative findings in the PCR of 4 pairs of primers reaction.Illustrate that these 4 pairs of primers are 0.25 bacterium/g to the detection sensitivity of intestinal bacteria O78 in the pork filling when using aforesaid method.
By clone and the expression in the vaccine strains of attenuation, can set up recombiant vaccine to O-antigen gene bunch.O-antigen is the surface antigen of topmost Gram-negative bacteria, can cause the intensive immune response, is one of best target molecule of making recombiant vaccine.Viret laboratory success in 1993 the O-antigen gene of Shigellae Sonnei bunch is expressed in a strain Salmonellas Tyziai vaccine bacterium, experimentation on animals proof can cause rabbit immune response (Molecular Microbiology1993,7:239-252).The group of China Military Medical Science Institute also similarly works being engaged in the Viret laboratory.Bunch express the O-antigen gene of intestinal bacteria O111 success in 1999 in the Wang Lei laboratory in salmonella vaccine STM-1, and the bacterial strain set up of proof can cause the blood of mouse and humoral response (Microbial Pathogenesis 1999,27:55-59).So the O-antigen-specific gene order of intestinal bacteria O78 of the present invention can be applied to set up recombiant vaccine.
When molecular probe nucleotide sequence and target DNA sequence homology greater than 85% the time, can accurately aim sequence be hybridized out.The homology that requires both in the Southern of low preciseness hybridization is greater than 65% (" molecular cloning experiment guide " third edition, the 509th page, low preciseness hybridization).The homology search of specific nucleotide sequence among the present invention in Genebank shows does not have homology to exist greater than other genes of 65%.So in hybrid experiment, the specific nucleotide sequence among the present invention can only draw positive findings to the purpose bacterium as molecular probe.The Southern hybrid method is not strict with for the length of molecular probe, can use hybridization to whole sequence from 20bp or above oligonucleotide in the specific nucleotide sequence among the present invention.In a Southern experiment, utilize the relevant specific gene (more than the 1000bp) of Salmonellas to do molecular probe, success tell this bacterium (LiuD, VermaNK, Romana LK, Reeves PR., 1991 Relationships among the rfb regions ofSalmonella serovars A, B, and D.J Bacteriol.173 (15): 4814-4819.), the experiment of a lot of this areas shows that all the gene order about 1000-2000bp can be used as molecular probe.Gene chip is the same with Southern hybridization ratio juris, also similar in the requirement of selecting molecular probe for use, so specific nucleotide sequence among the present invention and oligonucleotide fragment wherein can detect this purpose bacterium as molecular probe in hybridization, comprise the ordinary method of multiple hybridization such as Southern, gene chip.
Nucleotide sequence (shown in the SEQ IDNO:1) according to the O-antigen-specific to intestinal bacteria O78 type of the present invention, structure specific nucleic acid probe, be fixed on the carrier of chip and make biochip, after the sample that will detect is suitably handled, carry out hybridization with biochip, utilize the biochip signal analysis equipment just can obtain corresponding bacteria situation in the sample then.The DNA chip that this intestinal bacteria O-antigen is identified can be directly used in clinical and other check place (as food-processing and production industry, the Micro biological Tests of animal and veterinary industry customs quarantine control etc.).This chip only need enlarge output, just can industrialization under identical condition.
Table 1 has been listed transhipment enzyme gene and pol gene and intragenic primer and PCR data in the O-antigen gene bunch of intestinal bacteria O78 type.Transhipment enzyme gene and pol gene and their function corresponding and the size of the O-antigen gene bunch of intestinal bacteria O78 type in table, have been listed.In each gene, we have respectively designed two pairs of primers, and the difference that every pair of primer is distributed in the corresponding gene is local to guarantee its specificity.In table, also listed position and the size of each primer in SEQ 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.
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 12-19 bacterium, and 12 groups altogether, all list in the table in their source.
The genomic dna that contains intestinal bacteria O78 type in the 4th group is as positive control.Do template with every group of bacterium, be PCR by following condition with every pair in the table 1 primer: 95 ℃ of pre-sex change after 5 minutes, 95 ℃ of sex change 30 seconds, annealing time is 30 seconds, temperature sees Table 1,72 ℃ extended 2 minutes, carried out 25 circulations like this.Continue to extend 5 minutes at 72 ℃ at last, reaction system is 25 μ l.Template is dilution in 1: 20, gets 1 μ l.After reaction finishes, get 10 μ lPCR products and detect the fragment that amplifies by 0.8% agarose gel electrophoresis.
For wzx, wzy gene, each gene all has two pairs of primers detected, every pair of primer has obtained except be PCR in the 4th group after the correct band of expection size, the correct band of any size does not all increase in other groups, that is to say, not obtaining any PCR product band in the array mostly, so wzx, wzy gene pairs intestinal bacteria O78 type and O-antigen thereof are high specials.And the oligonucleotide of these intragenic any one section 10-25nt is special to the O-antigen of intestinal bacteria O78 type, and the primer in especially above-mentioned each gene is that oligonucleotide is high special to detecting the back confirmation through PCR to intestinal bacteria O78 type.These all oligonucleotide all can be used for the intestinal bacteria O78 type in the human body and environment rapidly and accurately, and can identify their O-antigen.
Table 3 is structural tables of the O-antigen gene bunch of intestinal bacteria O78 type, in table, listed the structure of the O-antigen gene bunch of intestinal bacteria O78 type, altogether by 11 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 JUMPStart sequence and gnd gene, and they do not belong to O-antigen gene bunch, and we are just with the increase full length sequence of O-antigen gene bunch of their one section sequences Design primer.
Table 4 is site plan of the gene in the O-antigen gene bunch of intestinal bacteria O78 type, listed the accurate position of all open reading frame in complete sequence in the O-antigen gene bunch of intestinal bacteria O78 type in the drawings, at the underscoring of the initiator codon and the terminator codon of each open reading frame.The initiator codon of open reading frame has two in intestinal bacteria: ATG and GTG.
SEQ ID NO:1 sequence (SEQUENCE LISTING)
SEQUENCE?LISTING
＜110〉Tianjin Biochip Technology Co., Ltd
＜120〉to the Nucleotide of the O-antigen-specific of intestinal bacteria O78 type
＜130〉to the Nucleotide of the O-antigen-specific of intestinal bacteria O78 type
<160>1
<170>PatentIn?version?3.2
<210>1
<211>12655
<212>DNA
<213>Escherichia?coli
<400>1
attggtagct?gtaagccaag?ggcggtagcg?tggattttta?ttgcgtaaaa?acgtcagtaa?????60
acatagaggt?ttaatgaaaa?aaatactatt?aatatccatg?agtttgggaa?agggcgatta????120
tggtggtggt?atagtatcaa?atacaaactt?ttttgcttta?aaggaattag?agcattatga????180
gttattttca?gtaggtattg?taaaaaacac?caacgatgca?ccaaacttta?ttaacatggt?????240
tttgcctggc?aacgcgagta?aattctcaac?tgcaattaat?aatatattag?ggtttgccgg?????300
gcagttaaat?aataaaacta?cgaaaaacat?aaaatacata?attgacgagt?ttgagcctga?????360
tattgtatac?cttgattcaa?gccttctggg?gtgtatagct?tcttattgca?agaaaaaaca?????420
taaaactatt?caaataataa?cgttctttca?taatattgag?tttgattttg?aaattgcacg?????480
tattatgtca?gggttattgc?atttttttcc?ctcgttaata?tccacaactc?tcgcagaata?????540
tgcggctgta?aggtatagtg?acaagattat?cgcattacat?aaaaaagatt?cttttagact?????600
agaggaaaaa?tatggaagga?aggctgatta?tatcgtaccc?gtttgcatta?aagatacaca?????660
aagagaaaaa?agttttaaac?tcgtcaacga?aaagaaaaaa?gaaggcaaaa?agttaaaagt?????720
tggctttata?ggtacagctt?tttttgcaaa?tgtagagtct?gctaagatta?tatctcaata?????780
cattgcgcca?aaggtggagg?ggattgctaa?tttttatata?tgtggcaatg?gatttgaaaa?????840
atataaagca?ttgaattcaa?ctaatgtaaa?tgtttcaggc?tatattgatt?ctttggatga?????900
tttctataat?gaaatggatg?tgatgatatt?tcctattttt?agcggcgcag?gaatgaaggt?????960
gaaaatagca?gagtcattaa?tgtataataa?accaattcta?gcttctgcat?ttgcattagt????1020
tggttacgaa?aaaataatag?atggaactaa?tgtgattagc?tgtgagtctc?acgaatcttt????1080
tgtttatcat?ataagacaat?ttagaaggga?taataacaca?ttatataata?ggaagtctta????1140
ttataaatat?ttctctgata?aagcatgtct?gcattatttt?agaaatatat?tacgagaaat????1200
tgttaataac?gaatagatag?atggactaat?aacgtcatga?atataataca?attatcaaaa????1260
ttctaccctc?cgatatttgg?tggaatagag?caagttgcac?aggacataac?cgaagggatg????1320
caaaatttgt?gcaatgttga?tgttttaagc?gtaaataaca?gttctaaaac?aatatattgt????1380
aaaaatataa?ttcgagcttc?attactattt?acactattat?ctaccccagt?atcgattagc????1440
tatatactaa?tctggagtag?aatcagaaat?aattatgata?taattcatgt?tcatttgcca????1500
aatcctcttg?ctattattgc?actgttgctg?ttcccgccca?aagctcccgt?cgtagtgcat????1560
tggcatagcg?atatagtaaa?acagaaaata?gcgttaaagt?tttttcagcc?tttacagaat????1620
ttatttttga?ataaagtaga?aaaaattatt?gtgacatctg?aaatttatgg?gagttcttct????1680
cctcaattac?aaagatttca?agataaaata?atttgtatac?cgatcggtat?aaaatcagag????1740
cgattaccta?agaatgaaac?gctattaaag?catctaaaag?aaaaatataa?aaataaaaag????1800
atagttttct?ctcttgggag?gctggtttat?tacaaagggt?ttgaaaatct?agttaatgca????1860
gctaactttc?ttcctgaaga?tacaataatc?cttattggtg?ggggcggtga?gttatatgac????1920
gagttggctg?atagcatttt?atccaataaa?ctagaaggga?aagtggtttt?gctgggggag????1980
ataaaatatg?aacagctttc?tgcttattat?caggtttgtg?acgtattttg?tcttccttcc????2040
attcatgaat?cagaagcttt?tggcgtggtt?caaattgagg?caatgagcta?tggaaaacct????2100
gttgtatcca?caaatattaa?aaatagtggg?gtcccatggg?taaatgaaaa?tggtatttca????2160
ggtgttgtag?ttgaacctaa?tgagcctcat?gaactagcca?aagctatctt?gacaatttta????2220
aataatccag?ctggtttttc?cttaggggca?ttagagcgat?atcgcaaatt?attcactaga????2280
gataaaatga?tctcaagttt?gattggactt?tatcagaata?ttaaaatagg?aaatgaaaaa????2340
gaatgatctt?acctgttatt?ctggccggtg?gtgcaggtag?tcgcctttgg?ccactttccc????2400
gcttacttta?ccccaaacag?tttttatgtc?tgaaaggcga?gctttctatg?ctgcaaacaa????2460
ctatctgccg?cctgaatggt?ttggggtgcg?aaaatccggt?ggtgatttgt?aatgagcagc????2520
accgctttat?tgttgcggaa?cagttgtatc?aactgaacaa?actcaccgag?aacattattc????2580
ttgaaccggc?aggacgtaat?actgcacctg?ctattgcgct?ggcggcgcta?gcaacacaat????2640
ggcataaacc?agatagtgat?ccgttactgt?tggtgctggc?agcggatcat?gtgattgctg????2700
atgaagacgc?attccgcgcc?gctgtgagta?atgctatgcc?gtatgccgaa?gcgggcaagt????2760
tggtgacttt?tggcattgtg?ccaaggcatc?cagaaacagg?ttatgggtat?attcgtcgtg????2820
gtgcagtgtc?tgcgggcgag?caggagggag?tagcctttga?agtggcacag?tttgtcgaaa????2880
aaccgaatct?ggaaaccgcg?caggcctatg?tggcaagcgg?cgaatactac?tggaacagcg????2940
gtatgttcct?gttccgcgcc?ggacgctatc?tcgaagaact?gaaaaagtat?cgtccggata????3000
ttctcgacgc?ctgtaaaaaa?gcgatgagcg?ccgttgagcc?agatcttaat?tttattcgtg????3060
tagatgaaga?agcgtttctc?gcctgtccgg?aagagtcggt?tgattacgcg?gtaatggaac????3120
gaacagcgga?tgctgttgta?gtaccgatgg?atgcgggctg?gagtgatgtg?ggctcttggg????3180
cttcgttatg?ggagagctgc?actcacacag?ctgaaggcaa?cgtttgccgc?ggtgacgtta????3240
ttaattataa?atccgaaaac?agttatgtgt?atgccgaatc?gggcctggtc?accaccgtcg????3300
gggtgaaaga?tttggtggta?gtgcagacca?aagatgcggt?gttaatagcc?gatcgcagct????3360
cggttcaaga?tgtgaaacat?gtggttgagc?agctcaaagc?cgacggtcga?cacgagtatc????3420
ggatgcatcg?cgaagtatac?cgtccgtggg?ggcaatatga?ctctatcgat?gcaggcgctc????3480
gctaccaggt?gaaacgcata?actgtgaaac?cgggcgaggg?cttgtcggta?cagatgcatc????3540
atcaccgcgc?agaacactgg?gtggtggttg?cgggaacggc?aaaagtcact?attaatggtg????3600
acatcaaact?gcttggtgaa?aatgagtcca?tctatattcc?actgggggcg?acacattgtt????3660
tggaaaatcc?aggaaaaata?cctttagaaa?taattgaggt?tcgctcaggt?tcttatcttg????3720
aagaagatga?tatagttaag?ttttatgatc?gttatgggcg?agattaattt?cattactgaa????3780
gttgctaatt?gtttaacaaa?atacttctgt?gtattttttc?atataagttt?gtaaatatga????3840
ctagattaag?ctgctttaaa?gcctatgata?ttcgcgggaa?attaggcgaa?gaactgaatg????3900
aagatatcgc?ctggcgcatt?ggtcgcgcct?atggcgaatt?tcttaaaccg?aaaaccattg????3960
ttttaggcgg?tgatgtccgc?ctcaccagcg?aaaccttaaa?acgggcgctg?gcgaaaggtt????4020
tacaggatgc?gggcgtcgac?gtgctggata?tcggtatgtc?cggcacagaa?gagatctatt????4080
tcgccacgtt?ccatctcggc?gtggatggcg?gcattgaagt?taccgccagc?cataacccga????4140
tggattataa?cggcatgaag?ctggtgcgtg?agggggcccg?cccgatcagc?ggcgataccg????4200
gactgcgcga?tgtccagcgt?ctggcagaag?ccaacgactt?tccccccgtc?gatgaaacca????4260
aacgcggtcg?ctatcagcaa?atcaacctgc?gtgatgcata?cgttgatcac?ctgttcggtt????4320
atatcaatgt?caaaaacctt?actccgctga?aacttgtgat?caactccgga?aacggcgcag????4380
caggtccggt?ggtggacgcc?atcgaagccc?gctttaaagc?cctcggcgca?ccggtggaat????4440
taatcaaagt?gcacaacacg?ccggacggca?atttccccaa?cggtattcct?aacccgctgc????4500
tgccggaatg?ccgcgacgac?acccgcaatg?cggtcatcaa?acacggcgcg?gatatgggca????4560
ttgcctttga?tggcgatttt?gaccgctgtt?ttctgtttga?cgaaaaaggg?cagtttatcg????4620
agggctacta?cattgtcggc?ctgctggcag?aagcgttcct?cgaaaaaaat?cccggcgcga????4680
agatcatcca?cgatccacgt?ctctcctgga?acaccgttga?tgtggtgact?gccgcaggcg????4740
gcaccccggt?aatgtcgaaa?accggacacg?cctttattaa?agaacgtatg?cgcaaggaag????4800
acgccatcta?cggtggcgaa?atgagcgccc?accattactt?ccgtgatttc?gcttactgcg????4860
acagcggcat?gatcccgtgg?ctgctggtcg?ccgaactggt?gtgcctgaaa?ggaaaaacgc????4920
tgggcgaact?ggtgcgcgac?cggatggcgg?tgttcccggc?aagcggtgag?atcaacagca????4980
aactggcgca?acccgttgag?gcgattaacc?gtgtggaaca?gcattttagc?cgtgaggcgc????5040
tggcggtgga?tcgcaccgat?ggcatcagca?tgacctttgc?caactggcgc?tttaacctgc????5100
gctcctccaa?caccgaaccg?gtggtgcggt?tgaatgtaga?atctcgtggt?gatttaggac????5160
ttgttgaaaa?gcagacaaca?tatatcatta?acttattaac?aacccccccc?aatgatgaaa????5220
agtgaaaata?ttacattcgt?agtgcaaggt?ccagtcagaa?atgaaacaaa?agatacttta????5280
gaaagtatta?ggctaaattt?taaagaagct?aaaataattt?tgtccacatg?ggaggggagt????5340
aacgttaaag?gtctttcatt?tgacgatatt?gtttttagta?acgatcctgg?ccctttaaat????5400
ataaaaagag?gtactaaaat?agttgctcag?gaaaatacaa?atcgtcaaat?aatttcaaca????5460
taccaaggtt?taattaaggt?tcgaacacca?tatgcggtta?agttgagaac?agatacgcca????5520
ttgcgaaatg?ataatattat?aaaaaactat?ataaaagctc?aagcatatgg?gagagattat????5580
aatttttcat?acttagagga?aaggattctt?gtatcatcta?taaatactat?agatccaaaa????5640
agctatattc?aattcccata?ccatatttct?gattggatct?actttggtaa?aactaacgat????5700
ttattaaaaa?tttgggatgg?ggaattaata?gatgataatg?attattttac?tgagcaggat????5760
ataaaagaag?agattagttg?caaaagagga?tttgagtttg?gaagatacac?agctgaacag????5820
cttgttctat?actcattttt?aaaaaaaaat?aaaattagcg?aatataaaca?ttattgtgat????5880
acaaataatg?acagggttga?aaaggtatta?aaagtaatcc?tttctaattt?ctattgcgta????5940
agtcctaaac?aaatgggact?ctgttttgat?aaatactcag?acttaataac?ccctagaatg????6000
aattacagga?gtctccgtag?tttcttaggg?ttttattttg?ttacaattaa?tgagagagtt????6060
tggcgaaatt?attacagcca?attctataaa?cttgacacta?caatatcaga?aaaaatagaa????6120
atatgtgcac?gcaggatatt?tataaagatc?atgaagaaga?ggaaaaaatg?agtaaaagca????6180
tgttatatat?ctacacacat?aatggttaca?atgatgcatt?tttaaaaaaa?gatgtaggga????6240
tggtaccatc?tgttatgtta?aatggtggga?aagttgattc?gctaatttat?tgtaaagaaa????6300
agaaaggaaa?ggctgttttt?aataatttag?aaatatattt?tactggatgt?ggatggttta????6360
ttagtaactt?caaggcgata?ctgtttcttt?gtcgaaatgc?aatatttaat?agacatttag????6420
atttattgat?atttcatctc?agccttaaaa?atatcgtgcc?tgtgttaaca?tataaacttt????6480
tatttaaagg?ggaattaata?tgtaagatgg?atttaaatac?tgaaagtgcc?atcgaatatt????6540
ctgataaaag?ccataaaata?aatgggatca?aaaaaaaact?aataagaatg?attatttccc????6600
gcacagatca?attttatgtt?gagactataa?gaaattataa?cataataaat?aaaggcattt????6660
ttgatgtgcc?gatttcagga?aagttaaaat?taatgcccaa?cggagttgat?gctaaattag????6720
ttagggaggt?tattaaaact?gaaggtcctg?atgtatttaa?agaaaataca?ataacaataa????6780
tctctcgcca?tgcttgtgaa?gcaaaggctc?caaacagaat?attcgatgta?attgacgtaa?????6840
tgtctgaatt?gaaacttaat?aactggacat?tgcaaatatt?gggctatttc?ccaacaatat?????6900
ttacggatgc?tctaagaata?agagctcttg?aatgtggagt?gaatatcata?tgtgacggtt?????6960
ataatttaga?gctctatgat?gtgtataaaa?gattatttaa?aagtaagatg?tttatatgtc?????7020
tttcgctgca?agagtcatat?tgtatatcac?tagttgaagc?tgctgtattt?aataatcata?????7080
taatatctac?aaatgttgga?gtagcagaag?atctttcatt?aaaatacggt?aatattaaaa?????7140
ttatcgaaaa?ttattcgaaa?aatttactat?ccaaaactat?tgaaaagaca?attaaaaatg?????7200
atattaaaat?aaaaaaagca?agcatttctg?atagaatgtt?agactcatat?tcttgggata?????7260
atatcataac?aggggtcatg?agcgaataat?atgggagtat?atttcatttt?aataacagtg?????7320
gcactattga?tctctattat?cgtattattc?cacaatgatg?ttaaatttac?gaatgtatat?????7380
atatatactt?atattttact?gctctcatta?cttgctggtt?taagatttca?taccggctgg?????7440
gattgggaag?cttatgacta?ttttttttat?gagctgacaa?attctaattt?atttgaaata?????7500
agcaaagcaa?atatttttaa?ttatgaacca?gggttcgttt?tactatctta?tatatctgtt?????7560
ttattgcaca?taccaccatt?tctctttttc?tctttaataa?ccgttacatt?aattataaag?????7620
tcggcttata?agtatttagg?gaactttgtt?tacatttttt?tattaatata?tttatattac?????7680
ggttattttc?ataatttttc?tattgtacga?caaggtgttg?ctgctgcgtt?attcgcttat?????7740
tccattcgtt?atttgatagc?gaaaagctat?aaatattata?tattaatatt?tgtcgctgca?????7800
ttattccata?tgtctgctat?tatgttatta?attgtcccat?ttctttataa?aatcgctaat?????7860
cgtataccat?tttccgccct?attgctattt?tcattgttaa?tggtttatac?ctctgtaagt?????7920
gagctcatag?gtatgaaatc?attattgtca?ggaataaatg?cactaaatat?atatatcaat?????7980
aatagttctt?taagctataa?agttggtttt?tcgtttaaat?atttggagtt?attaatcatt?????8040
ctagttctat?tttataataa?gagattttca?gaactaattg?ctagcaaatt?tagtaaaaag?????8100
caatattatg?tatttagatg?tttgattgta?attgaagtga?tgatatattc?gcttttcaat?????8160
gactttagca?ttatttatga?aaggctaact?gtttacttcg?aattttctca?tgctatatgt?????8220
atagcaatga?ttatatcagc?ctttaagtat?aaaagagttc?agttgttttt?actgttaatt?????8280
ttattgtctc?ttatttttgt?aagatattat?caattattca?attctcctgt?tcgtgtcgat?????8340
ggagaaatga?cacattacga?acgatttgaa?aactattgtt?ctgtttttaa?cacaaaagat?????8400
tgtcaaaggt?aacaatgaag?cgaattatta?tagacttgga?taatactatc?acaatccata?????8460
acgatgaacc?atacggtaaa?aaaccagtaa?atcaagatgt?tgtaaataaa?atgaagatgt?????8520
ataaagagct?gggttataca?atcacaatat?ttaccgcccg?taatatgaat?actttttcag?????8580
gtaacattgg?gaagataaat?gttcatacaa?tgcctgttat?aattgactgg?ttaaatcgta?????8640
ataatattcc?atatgatgaa?attattgtgg?gtaaaccatg?gtgtgggaca?aatggatttt?????8700
atgttgatga?taaggcgatt?aggccttcag?agtttataag?cttgtcagaa?acggagattg?????8760
ctgaactact?ggataaggag?gtaaaaaaat?gattctaata?aactctgctg?cttacgttaa?????8820
tgacgaccta?agagctgaat?tcggtttgct?accaccgtgc?tttttacccc?ttggtaatag?????8880
aagactatat?catcatcaga?ttgagtcgtt?aaggaaagct?tttcctgaag?aaaaaatata?????8940
tttatctctg?ccggaagagt?atcacattgg?taggtatgat?caacaatact?ttaatgaaaa?????9000
tgatattaca?atactaaaaa?cttcttatac?tttatcacaa?ggacatagtc?tgtatcggtt?????9060
gttaaatgat?tttaacttaa?tcgaatgtga?taaactaatt?attaattatg?gtgatacttt?????9120
ttatcatacc?tttgaagcta?cgagtgatga?ttatttttat?gtatctaata?acatcggcta?????9180
ctataaccga?gcatctgttg?taattaatgg?tgataagctt?attataaata?aagataatcc?????9240
agctgttgat?aatggtgagt?tggttatttc?tggtttcttt?tgtttcagta?atattgggag?????9300
gttatgcgaa?tgcttgaaaa?agatgaacta?tgattttctg?tcgtcttctg?aattgtatta?????9360
ctcgtcggtt?gcaacaaaag?ttttaaaaaa?tgatcattgg?tatgattttg?gtcatttaaa?????9420
cagctttttt?acatcaagaa?cgagtatcac?tactgagcgt?gagtctaata?gtcttcaaat?????9480
aaataatttt?tgtgtgataa?aaaaaagtaa?gaagaaagca?aaaatgctag?gcgaggcgaa?????9540
ttggtttttg?agtttgccgc?ctgcattgag?tatcttcact?ccgagggttt?ttgatgttag?????9600
agaagataat?gattatgcag?agtatacaat?agaatatctt?tataatcttc?ctttgtctga?????9660
tatggcagtg?ttttgtgagt?tacctaagtc?atgttgggtg?aatgtgttta?actcgtgtcg?????9720
agatttccta?aatagatcaa?aagaatactc?taatagtaaa?aaaacgctag?atatgtctga?????9780
actggcatca?tatgataaat?tgtactatga?taagacccta?aaaaggttac?atgattttga?????9840
attgacaaca?aacatacaat?tgactaaaga?tgttatattt?aatggtaaaa?gattcccatc?????9900
gataaaaaaa?atagcagaaa?tatcatcacg?ctttattaac?ccaactaaga?gtgatgatgt?????9960
tataattaca?catggcgatt?tttgctttag?taatatatta?tacgatttta?gaagtcagag????10020
aattaaatta?atcgatccca?gaggtattaa?ctataacaat?gagttaagca?tatggggaga????10080
tataaggtat?gacttggcta?agttatgtca?ctcggtaatt?ggatgttatg?atcttattat????10140
cgctcagaaa?tttttgctgg?aatacgatta?tgtagataac?agaattgaat?ttgaactata????10200
tgatgataac?tacagcgaca?tagtgaattc?atttttaggc?gtgtttaatt?cttttgaaga????10260
atttgggtat?actattgaag?agatacaatg?tataaccata?catttatttt?tatcaatgtt????10320
gccgcttcat?tctgattcta?aacttagaca?agaagcattt?atagcaaatg?cttatagact????10380
gttcagtttg?ttaaatttgg?agtatttatg?acaacgattg?taattcctat?ggcaggacaa????10440
tcatcacgct?tttataaagc?aggttatgat?gttcctaagt?ataagttaaa?aattaataac????10500
aaaagcgtat?tttttcattc?actacgaagt?tttctaaaat?ttaaggacca?tttttttcta????10560
attataggta?ttaaaggcat?actagatgaa?aagtttgttc?aatatgaaat?ggccgaatta????10620
ggtataaatg?attttgaaat?tgttttgctc?gaacagcaaa?caaatggtca?agcagagacg????10680
gttgtgaaag?gagtaaaaaa?ttgcaagaga?gatattagtg?acattttaat?ttttaatata????10740
gatacttttc?gcagagagat?taatttgcct?gataatttta?gtttgaaaga?ttgctctggg????10800
tatttggaaa?catttttagg?gtcaggtgaa?aattggagta?atattatgcc?tgaatcagat????10860
gtccctaatc?aagtaaaaat?gactgcggaa?aaacaaaata?tatctgaata?ttgttgcact????10920
ggtttatatt?attttggtga?ttataagttg?ttgaatatag?cttataataa?ctggctagaa????10980
aatgaagata?ctactaaaga?ggtgtatatc?gctcctctat?ataatcattt?gattacatgt????11040
gggcacaaaa?ttttttatac?cgttataaac?agatgtgatg?tggtgttctg?tggtgtgcct????11100
aatgaatatg?aaaaaataaa?gaatgggttt?gactggaaat?gagacagcac?agaaaattag????11160
tcagttatgc?tgtgattatt?acttttttgt?ttatacctat?acaagttatt?ctgtcgttaa????11220
tttttaacta?ttatttagct?aaaaacgttg?ctctggatgt?tttaaatgct?tggtatatgt????11280
tttttgcact?catttccctt?ttttctttgt?tggaattttc?ctatcccata?ttagcaatta????11340
atttttttaa?cagctatcag?ggaaattgta?aaggagctct?gtctttcttc?ataaagaaat????11400
cattaggggt?agtaataccc?cttcagatca?tatttgttgt?aatttatagt?ttatttgtta????11460
acccgctttt?tatttatttt?ggtatgggtt?tggtggtacg?tagtatatcg?aatattatta????11520
atgcatgtgt?atatgcaaaa?caaaatatta?ttatcgataa?agtttatcga?tttctgtacg????11580
ccataattat?gccaactgct?ttccttttct?tgttttatca?ttctgataaa?aacattgatt????11640
tgtatgattt?aactcttgta?tggtttttga?gctcactatt?ctgcttaata?tattctttgt????11700
catttttact?ccttaagcat?aagaagtctg?tcaacaggag?cgaaaaatca?ttccatctac????11760
cattcaagta?taaccttaag?ctttttttta?ctgtttttcc?tgcaattttc?atctatacat????11820
tgtctatata?ttatttgaaa?atatttggtt?cagaaagcag?ttctagttct?actattatat????11880
atggtttctt?tttgcaaata?tttaatgttt?ataatttgat?aattattttg?gttgcaagtt????11940
atttgatgcc?tacgctctca?aagcgtttcc?atgatggtgt?tgatgttatc?cctctcacac????12000
ttaaactatt?agatatcagt?gtgcttatat?ctgttatttc?tacttctttt?gtggcattgt????12060
taggtattcc?tgttgcggag?tatattctta?aggggaaaat?agatgtaata?tcctactatt????12120
atatagtagc?aataattatt?atattctata?ttgaaacatg?tcaggttatt?ctaacctcaa????12180
ttggcactgc?cattggtatt?tacgattttc?ataggcaaag?tattatatca?gcagttatgg????12240
ttttaattct?gtcatgtttt?ttaatacctc?attacaatgc?tgagggtttg?atgtatagta????12300
tcattatatc?tcaattcttt?acatgcttta?tttataaccc?taaacgtgtt?ataaataaaa????12360
ttggattaga?taaagttaat?tatattaaaa?gattaatggt?gcactttttg?tttatgttga????12420
ctcttatttt?tatggggtta?tattttcaaa?atgatgatat?attgccgaga?gttgtgattc????12480
tttcttttat?cacattgatt?ggtgtttgtt?ttctctaccc?aaatgtgaaa?aaaataattg????12540
ttgatttctt?gtagtttttt?aattttattt?gcattttaaa?tggaaaatct?tgttctatct????12600
tagaaaggag?ttaacaatgc?tgaagcaaca?gatcggcgtc?gtcggtatgg?cagtg?????????12655
Oligosaccharide unit treatment gene in the O-antigen gene of table 1 intestinal bacteria O78 type bunch and primer and PCR data wherein
Gene Function The base position of gene Forward primer Reverse primer The length of PCR product Produce the group number of correct big or small electrophoresis band The annealing temperature of PCR (℃)
????wzx The transhipment enzyme ????11139-12554 ??11134-11363 ??11937-11955 ????822 ????0 * ????50
??11935-11953 ??12503-12520 ????586 ????0 * ????55
????wzy Polysaccharase ????7291-8412 ??7568-7586 ??8175-8193 ????626 ????0 * ????55
??7728-7744 ??8204-8224 ????497 ????0 * ????50
*Only in intestinal bacteria O78 type, obtain a correct band
Table 2 166 strain intestinal bacteria and 43 strain Shigellaes and their source
The bacterium source that contains in this group of group number
1, wild-type e. coli O1, O2, O5, O7, O8, O9, O12, O13, O14, O15, O16, O17, O18, IMVS a
O19ab，O20，O21，O22，O23，O24
2, wild-type e. coli O4, O10, O25, O26, O27, O28, O29, O30, O32, O33, O34, O35, IMVS a
O36，O37，O38，O40，O41，O42，O43
3, wild-type e. coli O6, O44, O45, O46, O48, O49, O50, O51, O52, O54, O55, O56, IMVS a
O57，O58，O60，O61，O62，O53
4, wild-type e. coli O63, O65, O66, O69, O70, O71, O74, O75, O76, O77, O78, IMVS a
O79，O80，O81，O82，O83，O68
5, wild-type e. coli O84, O85, O86, O87, O88, O89, O90, O91, O92, O98, O99, IMVS a
O101，O102，O103，O104，O105，O106，O97
6, wild-type e. coli O107, O108, O109, O110, O111, O112ab, O112ac, O113, IMVS a
O115，O116，O118，O120，O123，O126，O128，O117
7, wild-type e. coli O129, O130, O131, O132, O133, O134, O135, O136, O137, IMVS a
O138，O139，O141，O142，O143，O144，O145，O140
8, wild-type e. coli O146, O147, O148, O150, O152, O154, O156, O157, O158, IMVS a
O159，O160，O161，O163，O164，O165，O166，O153???????????????????b
9, wild-type e. coli O168, O169, O170, O171, O172, O173, c
Shigella dysenteriae D1, D2, D3, D4, D5, D6, D7, D8, D9, D10, D11, D12, D13 d
10, Shigella bogdii B1, B2, B3, B4, B6, B7, B8, B9, B10, B11, B12, B13, B14, B15, d
B16，B17，B18
11, shigella flexneri F1a, F1b, F2a, F2b, F3, F4a, F4b, F5 (v:4), F5 (v:7), F6, d
DS，DR
12, wild-type e. coli O3, O11, O39, O59, O64, O73, O96, O95, O100, O114, O151, O155, IMVS a
O124，O167，O162，O121，O127，O149，O119
13, the 4th group of bacterial strain deducts intestinal bacteria reference culture O78 IMVS a
For the convenience that detects, every 12-19 bacterium is divided into one group, and 12 groups altogether, the 13rd group as negative control
a.?Institude?of?Medical?and?Veterinary?Science，Anelaide，Australia
b.?Statens?Serum?Institut，Copenhagen，Denmark
C. O172 and O173 come from Statens Serum Institut, Copenhagen, and Denmark, all the other come from IMVS
D. China Preventive Medicial Science Institute's epidemiological study institute
Table 3 intestinal bacteria O78 type O antigen gene structure iron
orf#
orf1?????orf2????manC????manB????orf5????orf6????wzy?????orf8????orf9????orf10???wzx
G+C％????40.0????33.4????49.5????54.1????30.2????28.9????27.1????32.8????31.2????30.5???28.7
content
Table 4 intestinal bacteria O78 type O-antigen gene cluster gene position
attggtagct?gtaagccaag?ggcggtagcg?tggattttta?ttgcgtaaaa?acgtcagtaa?????60
Orf1's is initial
acatagaggt?ttaatgaaaa?aaatactatt?aatatccatg?agtttgggaa?agggcgatta????120
tggtggtggt?atagtatcaa?atacaaactt?ttttgcttta?aaggaattag?agcattatga????180
gttattttca?gtaggtattg?taaaaaacac?caacgatgca?ccaaacttta?ttaacatggt?????240
tttgcctggc?aacgcgagta?aattctcaac?tgcaattaat?aatatattag?ggtttgccgg?????300
gcagttaaat?aataaaacta?cgaaaaacat?aaaatacata?attgacgagt?ttgagcctga?????360
tattgtatac?cttgattcaa?gccttctggg?gtgtatagct?tcttattgca?agaaaaaaca?????420
taaaactatt?caaataataa?cgttctttca?taatattgag?tttgattttg?aaattgcacg?????480
tattatgtca?gggttattgc?atttttttcc?ctcgttaata?tccacaactc?tcgcagaata?????540
tgcggctgta?aggtatagtg?acaagattat?cgcattacat?aaaaaagatt?cttttagact?????600
agaggaaaaa?tatggaagga?aggctgatta?tatcgtaccc?gtttgcatta?aagatacaca?????660
aagagaaaaa?agttttaaac?tcgtcaacga?aaagaaaaaa?gaaggcaaaa?agttaaaagt?????720
tggctttata?ggtacagctt?tttttgcaaa?tgtagagtct?gctaagatta?tatctcaata?????780
cattgcgcca?aaggtggagg?ggattgctaa?tttttatata?tgtggcaatg?gatttgaaaa?????840
atataaagca?ttgaattcaa?ctaatgtaaa?tgtttcaggc?tatattgatt?ctttggatga?????900
tttctataat?gaaatggatg?tgatgatatt?tcctattttt?agcggcgcag?gaatgaaggt?????960
gaaaatagca?gagtcattaa?tgtataataa?accaattcta?gcttctgcat?ttgcattagt????1020
tggttacgaa?aaaataatag?atggaactaa?tgtgattagc?tgtgagtctc?acgaatcttt????1080
tgtttatcat?ataagacaat?ttagaaggga?taataacaca?ttatataata?ggaagtctta????1140
ttataaatat?ttctctgata?aagcatgtct?gcattatttt?agaaatatat?tacgagaaat????1200
The termination orf2's of orf1 is initial
tgttaataac?gaa tagatag?atggactaat?aacgtc atga?atataataca?attatcaaaa1260
ttctaccctc?cgatatttgg?tggaatagag?caagttgcac?aggacataac?cgaagggatg????1320
caaaatttgt?gcaatgttga?tgttttaagc?gtaaataaca?gttctaaaac?aatatattgt????1380
aaaaatataa?ttcgagcttc?attactattt?acactattat?ctaccccagt?atcgattagc????1440
tatatactaa?tctggagtag?aatcagaaat?aattatgata?taattcatgt?tcatttgcca????1500
aatcctcttg?ctattattgc?actgttgctg?ttcccgccca?aagctcccgt?cgtagtgcat????1560
tggcatagcg?atatagtaaa?acagaaaata?gcgttaaagt?tttttcagcc?tttacagaat????1620
ttatttttga?ataaagtaga?aaaaattatt?gtgacatctg?aaatttatgg?gagttcttct????1680
cctcaattac?aaagatttca?agataaaata?atttgtatac?cgatcggtat?aaaatcagag????1740
cgattaccta?agaatgaaac?gctattaaag?catctaaaag?aaaaatataa?aaataaaaag????1800
atagttttct?ctcttgggag?gctggtttat?tacaaagggt?ttgaaaatct?agttaatgca????1860
gctaactttc?ttcctgaaga?tacaataatc?cttattggtg?ggggcggtga?gttatatgac????1920
gagttggctg?atagcatttt?atccaataaa?ctagaaggga?aagtggtttt?gctgggggag????1980
ataaaatatg?aacagctttc?tgcttattat?caggtttgtg?acgtattttg?tcttccttcc????2040
attcatgaat?cagaagcttt?tggcgtggtt?caaattgagg?caatgagcta?tggaaaacct????2100
gttgtatcca?caaatattaa?aaatagtggg?gtcccatggg?taaatgaaaa?tggtatttca????2160
ggtgttgtag?ttgaacctaa?tgagcctcat?gaactagcca?aagctatctt?gacaatttta????2220
aataatccag?ctggtttttc?cttaggggca?ttagagcgat?atcgcaaatt?attcactaga????2280
gataaaatga?tctcaagttt?gattggactt?tatcagaata?ttaaaatagg?aaatgaaaaa????2340
The termination of the initial orr2 of orf3
ga atgatctt?acctgttatt?ctggccggtg?gtgcaggtag?tcgcctttgg?ccactttccc??2400
gcttacttta?ccccaaacag?tttttatgtc?tgaaaggcga?gctttctatg?ctgcaaacaa????2460
ctatctgccg?cctgaatggt?ttggggtgcg?aaaatccggt?ggtgatttgt?aatgagcagc????2520
accgctttat?tgttgcggaa?cagttgtatc?aactgaacaa?actcaccgag?aacattattc????2580
ttgaaccggc?aggacgtaat?actgcacctg?ctattgcgct?ggcggcgcta?gcaacacaat????2640
ggcataaacc?agatagtgat?ccgttactgt?tggtgctggc?agcggatcat?gtgattgctg????2700
atgaagacgc?attccgcgcc?gctgtgagta?atgctatgcc?gtatgccgaa?gcgggcaagt????2760
tggtgacttt?tggcattgtg?ccaaggcatc?cagaaacagg?ttatgggtat?attcgtcgtg????2820
gtgcagtgtc?tgcgggcgag?caggagggag?tagcctttga?agtggcacag?tttgtcgaaa????2880
aaccgaatct?ggaaaccgcg?caggcctatg?tggcaagcgg?cgaatactac?tggaacagcg????2940
gtatgttcct?gttccgcgcc?ggacgctatc?tcgaagaact?gaaaaagtat?cgtccggata????3000
ttctcgacgc?ctgtaaaaaa?gcgatgagcg?ccgttgagcc?agatcttaat?tttattcgtg????3060
tagatgaaga?agcgtttctc?gcctgtccgg?aagagtcggt?tgattacgcg?gtaatggaac????3120
gaacagcgga?tgctgttgta?gtaccgatgg?atgcgggctg?gagtgatgtg?ggctcttggg????3180
cttcgttatg?ggagagctgc?actcacacag?ctgaaggcaa?cgtttgccgc?ggtgacgtta????3240
ttaattataa?atccgaaaac?agttatgtgt?atgccgaatc?gggcctggtc?accaccgtcg????3300
gggtgaaaga?tttggtggta?gtgcagacca?aagatgcggt?gttaatagcc?gatcgcagct????3360
cggttcaaga?tgtgaaacat?gtggttgagc?agctcaaagc?cgacggtcga?cacgagtatc????3420
ggatgcatcg?cgaagtatac?cgtccgtggg?ggcaatatga?ctctatcgat?gcaggcgctc????3480
gctaccaggt?gaaacgcata?actgtgaaac?cgggcgaggg?cttgtcggta?cagatgcatc????3540
atcaccgcgc?agaacactgg?gtggtggttg?cgggaacggc?aaaagtcact?attaatggtg????3600
acatcaaact?gcttggtgaa?aatgagtcca?tctatattcc?actgggggcg?acacattgtt????3660
tggaaaatcc?aggaaaaata?cctttagaaa?taattgaggt?tcgctcaggt?tcttatcttg????3720
The termination of orf3
aagaagatga?tatagttaag?ttttatgatc?gttatgggcg?agat taattt?cattactgaa??3780
Orf4's is initial
gttgctaatt?gtttaacaaa?atacttctgt?gtattttttc?atataagttt?gtaaat atga??3840
ctagattaag?ctgctttaaa?gcctatgata?ttcgcgggaa?attaggcgaa?gaactgaatg????3900
aagatatcgc?ctggcgcatt?ggtcgcgcct?atggcgaatt?tcttaaaccg?aaaaccattg????3960
ttttaggcgg?tgatgtccgc?ctcaccagcg?aaaccttaaa?acgggcgctg?gcgaaaggtt????4020
tacaggatgc?gggcgtcgac?gtgctggata?tcggtatgtc?cggcacagaa?gagatctatt????4080
tcgccacgtt?ccatctcggc?gtggatggcg?gcattgaagt?taccgccagc?cataacccga????4140
tggattataa?cggcatgaag?ctggtgcgtg?agggggcccg?cccgatcagc?ggcgataccg????4200
gactgcgcga?tgtccagcgt?ctggcagaag?ccaacgactt?tccccccgtc?gatgaaacca????4260
aacgcggtcg?ctatcagcaa?atcaacctgc?gtgatgcata?cgttgatcac?ctgttcggtt????4320
atatcaatgt?caaaaacctt?actccgctga?aacttgtgat?caactccgga?aacggcgcag????4380
caggtccggt?ggtggacgcc?atcgaagccc?gctttaaagc?cctcggcgca?ccggtggaat????4440
taatcaaagt?gcacaacacg?ccggacggca?atttccccaa?cggtattcct?aacccgctgc????4500
tgccggaatg?ccgcgacgac?acccgcaatg?cggtcatcaa?acacggcgcg?gatatgggca????4560
ttgcctttga?tggcgatttt?gaccgctgtt?ttctgtttga?cgaaaaaggg?cagtttatcg????4620
agggctacta?cattgtcggc?ctgctggcag?aagcgttcct?cgaaaaaaat?cccggcgcga????4680
agatcatcca?cgatccacgt?ctctcctgga?acaccgttga?tgtggtgact?gccgcaggcg????4740
gcaccccggt?aatgtcgaaa?accggacacg?cctttattaa?agaacgtatg?cgcaaggaag????4800
acgccatcta?cggtggcgaa?atgagcgccc?accattactt?ccgtgatttc?gcttactgcg????4860
acagcggcat?gatcccgtgg?ctgctggtcg?ccgaactggt?gtgcctgaaa?ggaaaaacgc????4920
tgggcgaact?ggtgcgcgac?cggatggcgg?tgttcccggc?aagcggtgag?atcaacagca????4980
aactggcgca?acccgttgag?gcgattaacc?gtgtggaaca?gcattttagc?cgtgaggcgc????5040
tggcggtgga?tcgcaccgat?ggcatcagca?tgacctttgc?caactggcgc?tttaacctgc????5100
gctcctccaa?caccgaaccg?gtggtgcggt?tgaatgtaga?atctcgtggt?gatttaggac????5160
ttgttgaaaa?gcagacaaca?tatatcatta?acttattaac?aacccccccc?aatgatgaaa????5220
The termination orf5's of orf4 is initial
ag tgaaaata?ttacattcgt?a gtgcaaggt?ccagtcagaa?atgaaacaaa?agatacttta5280
gaaagtatta?ggctaaattt?taaagaagct?aaaataattt?tgtccacatg?ggaggggagt????5340
aacgttaaag?gtctttcatt?tgacgatatt?gtttttagta?acgatcctgg?ccctttaaat????5400
ataaaaagag?gtactaaaat?agttgctcag?gaaaatacaa?atcgtcaaat?aatttcaaca????5460
taccaaggtt?taattaaggt?tcgaacacca?tatgcggtta?agttgagaac?agatacgcca????5520
ttgcgaaatg?ataatattat?aaaaaactat?ataaaagctc?aagcatatgg?gagagattat????5580
aatttttcat?acttagagga?aaggattctt?gtatcatcta?taaatactat?agatccaaaa????5640
agctatattc?aattcccata?ccatatttct?gattggatct?actttggtaa?aactaacgat????5700
ttattaaaaa?tttgggatgg?ggaattaata?gatgataatg?attattttac?tgagcaggat????5760
ataaaagaag?agattagttg?caaaagagga?tttgagtttg?gaagatacac?agctgaacag????5820
cttgttctat?actcattttt?aaaaaaaaat?aaaattagcg?aatataaaca?ttattgtgat????5880
acaaataatg?acagggttga?aaaggtatta?aaagtaatcc?tttctaattt?ctattgcgta????5940
agtcctaaac?aaatgggact?ctgttttgat?aaatactcag?acttaataac?ccctagaatg????6000
aattacagga?gtctccgtag?tttcttaggg?ttttattttg?ttacaattaa?tgagagagtt????6060
tggcgaaatt?attacagcca?attctataaa?cttgacacta?caatatcaga?aaaaatagaa????6120
The termination of the initial orf5 of orf6
atatgtgcac?gcaggatatt?tataaagatc?atgaagaaga?ggaaaaa atg?agtaaaagca??6180
tgttatatat?ctacacacat?aatggttaca?atgatgcatt?tttaaaaaaa?gatgtaggga????6240
tggtaccatc?tgttatgtta?aatggtggga?aagttgattc?gctaatttat?tgtaaagaaa????6300
agaaaggaaa?ggctgttttt?aataatttag?aaatatattt?tactggatgt?ggatggttta????6360
ttagtaactt?caaggcgata?ctgtttcttt?gtcgaaatgc?aatatttaat?agacatttag????6420
atttattgat?atttcatctc?agccttaaaa?atatcgtgcc?tgtgttaaca?tataaacttt????6480
tatttaaagg?ggaattaata?tgtaagatgg?atttaaatac?tgaaagtgcc?atcgaatatt????6540
ctgataaaag?ccataaaata?aatgggatca?aaaaaaaact?aataagaatg?attatttccc????6600
gcacagatca?attttatgtt?gagactataa?gaaattataa?cataataaat?aaaggcattt????6660
ttgatgtgcc?gatttcagga?aagttaaaat?taatgcccaa?cggagttgat?gctaaattag????6720
ttagggaggt?tattaaaact?gaaggtcctg?atgtatttaa?agaaaataca?ataacaataa????6780
tctctcgcca?tgcttgtgaa?gcaaaggctc?caaacagaat?attcgatgta?attgacgtaa????6840
tgtctgaatt?gaaacttaat?aactggacat?tgcaaatatt?gggctatttc?ccaacaatat????6900
ttacggatgc?tctaagaata?agagctcttg?aatgtggagt?gaatatcata?tgtgacggtt????6960
ataatttaga?gctctatgat?gtgtataaaa?gattatttaa?aagtaagatg?tttatatgtc????7020
tttcgctgca?agagtcatat?tgtatatcac?tagttgaagc?tgctgtattt?aataatcata????7080
taatatctac?aaatgttgga?gtagcagaag?atctttcatt?aaaatacggt?aatattaaaa????7140
ttatcgaaaa?ttattcgaaa?aatttactat?ccaaaactat?tgaaaagaca?attaaaaatg????7200
atattaaaat?aaaaaaagca?agcatttctg?atagaatgtt?agactcatat?tcttgggata????7260
The termination orf7's of orf6 is initial
atatcataac?aggggtcatg?agcgaa taat? atgggagtat?atttcatttt?aataacagtg7320
gcactattga?tctctattat?cgtattattc?cacaatgatg?ttaaatttac?gaatgtatat????7380
atatatactt?atattttact?gctctcatta?cttgctggtt?taagatttca?taccggctgg????7440
gattgggaag?cttatgacta?ttttttttat?gagctgacaa?attctaattt?atttgaaata????7500
agcaaagcaa?atatttttaa?ttatgaacca?gggttcgttt?tactatctta?tatatctgtt????7560
ttattgcaca?taccaccatt?tctctttttc?tctttaataa?ccgttacatt?aattataaag????7620
tcggcttata?agtatttagg?gaactttgtt?tacatttttt?tattaatata?tttatattac????7680
ggttattttc?ataatttttc?tattgtacga?caaggtgttg?ctgctgcgtt?attcgcttat????7740
tccattcgtt?atttgatagc?gaaaagctat?aaatattata?tattaatatt?tgtcgctgca????7800
ttattccata?tgtctgctat?tatgttatta?attgtcccat?ttctttataa?aatcgctaat????7860
cgtataccat?tttccgccct?attgctattt?tcattgttaa?tggtttatac?ctctgtaagt????7920
gagctcatag?gtatgaaatc?attattgtca?ggaataaatg?cactaaatat?atatatcaat????7980
aatagttctt?taagctataa?agttggtttt?tcgtttaaat?atttggagtt?attaatcatt????8040
ctagttctat?tttataataa?gagattttca?gaactaattg?ctagcaaatt?tagtaaaaag????8100
caatattatg?tatttagatg?tttgattgta?attgaagtga?tgatatattc?gcttttcaat????8160
gactttagca?ttatttatga?aaggctaact?gtttacttcg?aattttctca?tgctatatgt????8220
atagcaatga?ttatatcagc?ctttaagtat?aaaagagttc?agttgttttt?actgttaatt????8280
ttattgtctc?ttatttttgt?aagatattat?caattattca?attctcctgt?tcgtgtcgat????8340
ggagaaatga?cacattacga?acgatttgaa?aactattgtt?ctgtttttaa?cacaaaagat????8400
The termination orf8's of orf7 is initial
tgtcaaagg t?aaca atgaag?cgaattatta?tagacttgga?taatactatc?acaatccata8460
acgatgaacc?atacggtaaa?aaaccagtaa?atcaagatgt?tgtaaataaa?atgaagatgt????8520
ataaagagct?gggttataca?atcacaatat?ttaccgcccg?taatatgaat?actttttcag????8580
gtaacattgg?gaagataaat?gttcatacaa?tgcctgttat?aattgactgg?ttaaatcgta????8640
ataatattcc?atatgatgaa?attattgtgg?gtaaaccatg?gtgtgggaca?aatggatttt????8700
atgttgatga?taaggcgatt?aggccttcag?agtttataag?cttgtcagaa?acggagattg????8760
The termination of the initial orf8 of orf9
ctgaactact?ggataaggag?gtaaaaaa at?gattctaata?aactctgctg?cttacgttaa??8820
tgacgaccta?agagctgaat?tcggtttgct?accaccgtgc?tttttacccc?ttggtaatag????8880
aagactatat?catcatcaga?ttgagtcgtt?aaggaaagct?tttcctgaag?aaaaaatata????8940
tttatctctg?ccggaagagt?atcacattgg?taggtatgat?caacaatact?ttaatgaaaa????9000
tgatattaca?atactaaaaa?cttcttatac?tttatcacaa?ggacatagtc?tgtatcggtt????9060
gttaaatgat?tttaacttaa?tcgaatgtga?taaactaatt?attaattatg?gtgatacttt????9120
ttatcatacc?tttgaagcta?cgagtgatga?ttatttttat?gtatctaata?acatcggcta????9180
ctataaccga?gcatctgttg?taattaatgg?tgataagctt?attataaata?aagataatcc????9240
agctgttgat?aatggtgagt?tggttatttc?tggtttcttt?tgtttcagta?atattgggag????9300
gttatgcgaa?tgcttgaaaa?agatgaacta?tgattttctg?tcgtcttctg?aattgtatta????9360
ctcgtcggtt?gcaacaaaag?ttttaaaaaa?tgatcattgg?tatgattttg?gtcatttaaa????9420
cagctttttt?acatcaagaa?cgagtatcac?tactgagcgt?gagtctaata?gtcttcaaat????9480
aaataatttt?tgtgtgataa?aaaaaagtaa?gaagaaagca?aaaatgctag?gcgaggcgaa????9540
ttggtttttg?agtttgccgc?ctgcattgag?tatcttcact?ccgagggttt?ttgatgttag?????9600
agaagataat?gattatgcag?agtatacaat?agaatatctt?tataatcttc?ctttgtctga?????9660
tatggcagtg?ttttgtgagt?tacctaagtc?atgttgggtg?aatgtgttta?actcgtgtcg?????9720
agatttccta?aatagatcaa?aagaatactc?taatagtaaa?aaaacgctag?atatgtctga?????9780
actggcatca?tatgataaat?tgtactatga?taagacccta?aaaaggttac?atgattttga?????9840
attgacaaca?aacatacaat?tgactaaaga?tgttatattt?aatggtaaaa?gattcccatc?????9900
gataaaaaaa?atagcagaaa?tatcatcacg?ctttattaac?ccaactaaga?gtgatgatgt?????9960
tataattaca?catggcgatt?tttgctttag?taatatatta?tacgatttta?gaagtcagag????10020
aattaaatta?atcgatccca?gaggtattaa?ctataacaat?gagttaagca?tatggggaga????10080
tataaggtat?gacttggcta?agttatgtca?ctcggtaatt?ggatgttatg?atcttattat????10140
cgctcagaaa?tttttgctgg?aatacgatta?tgtagataac?agaattgaat?ttgaactata????10200
tgatgataac?tacagcgaca?tagtgaattc?atttttaggc?gtgtttaatt?cttttgaaga????10260
atttgggtat?actattgaag?agatacaatg?tataaccata?catttatttt?tatcaatgtt????10320
gccgcttcat?tctgattcta?aacttagaca?agaagcattt?atagcaaatg?cttatagact????10380
The termination of the initial orf9 of orf10
gttcagtttg?ttaaatttgg?agtattt atg?acaacgattg?taattcctat?ggcaggacaa??10440
tcatcacgct?tttataaagc?aggttatgat?gttcctaagt?ataagttaaa?aattaataac????10500
aaaagcgtat?tttttcattc?actacgaagt?tttctaaaat?ttaaggacca?tttttttcta????10560
attataggta?ttaaaggcat?actagatgaa?aagtttgttc?aatatgaaat?ggccgaatta????10620
ggtataaatg?attttgaaat?tgttttgctc?gaacagcaaa?caaatggtca?agcagagacg????10680
gttgtgaaag?gagtaaaaaa?ttgcaagaga?gatattagtg?acattttaat?ttttaatata????10740
gatacttttc?gcagagagat?taatttgcct?gataatttta?gtttgaaaga?ttgctctggg????10800
tatttggaaa?catttttagg?gtcaggtgaa?aattggagta?atattatgcc?tgaatcagat????10860
gtccctaatc?aagtaaaaat?gactgcggaa?aaacaaaata?tatctgaata?ttgttgcact????10920
ggtttatatt?attttggtga?ttataagttg?ttgaatatag?cttataataa?ctggctagaa????10980
aatgaagata?ctactaaaga?ggtgtatatc?gctcctctat?ataatcattt?gattacatgt????11040
gggcacaaaa?ttttttatac?cgttataaac?agatgtgatg?tggtgttctg?tggtgtgcct????11100
The termination of the initial orf10 of orf11
aatgaatatg?aaaaaataaa?gaatgggttt?gactggaa at?gagacagcac?agaaaattag??11160
tcagttatgc?tgtgattatt?acttttttgt?ttatacctat?acaagttatt?ctgtcgttaa????11220
tttttaacta?ttatttagct?aaaaacgttg?ctctggatgt?tttaaatgct?tggtatatgt????11280
tttttgcact?catttccctt?ttttctttgt?tggaattttc?ctatcccata?ttagcaatta????11340
atttttttaa?cagctatcag?ggaaattgta?aaggagctct?gtctttcttc?ataaagaaat????11400
cattaggggt?agtaataccc?cttcagatca?tatttgttgt?aatttatagt?ttatttgtta????11460
acccgctttt?tatttatttt?ggtatgggtt?tggtggtacg?tagtatatcg?aatattatta????11520
atgcatgtgt?atatgcaaaa?caaaatatta?ttatcgataa?agtttatcga?tttctgtacg????11580
ccataattat?gccaactgct?ttccttttct?tgttttatca?ttctgataaa?aacattgatt????11640
tgtatgattt?aactcttgta?tggtttttga?gctcactatt?ctgcttaata?tattctttgt????11700
catttttact?ccttaagcat?aagaagtctg?tcaacaggag?cgaaaaatca?ttccatctac????11760
cattcaagta?taaccttaag?ctttttttta?ctgtttttcc?tgcaattttc?atctatacat????11820
tgtctatata?ttatttgaaa?atatttggtt?cagaaagcag?ttctagttct?actattatat????11880
atggtttctt?tttgcaaata?tttaatgttt?ataatttgat?aattattttg?gttgcaagtt????11940
atttgatgcc?tacgctctca?aagcgtttcc?atgatggtgt?tgatgttatc?cctctcacac????12000
ttaaactatt?agatatcagt?gtgcttatat?ctgttatttc?tacttctttt?gtggcattgt????12060
taggtattcc?tgttgcggag?tatattctta?aggggaaaat?agatgtaata?tcctactatt????12120
atatagtagc?aataattatt?atattctata?ttgaaacatg?tcaggttatt?ctaacctcaa????12180
ttggcactgc?cattggtatt?tacgattttc?ataggcaaag?tattatatca?gcagttatgg????12240
ttttaattct?gtcatgtttt?ttaatacctc?attacaatgc?tgagggtttg?atgtatagta????12300
tcattatatc?tcaattcttt?acatgcttta?tttataaccc?taaacgtgtt?ataaataaaa????12360
ttggattaga?taaagttaat?tatattaaaa?gattaatggt?gcactttttg?tttatgttga????12420
ctcttatttt?tatggggtta?tattttcaaa?atgatgatat?attgccgaga?gttgtgattc????12480
tttcttttat?cacattgatt?ggtgtttgtt?ttctctaccc?aaatgtgaaa?aaaataattg????12540
The termination of orf11
ttgatttctt?g tagtttttt?aattttattt?gcattttaaa?tggaaaatct?tgttctatct??12600
tagaaaggag?ttaacaatgc?tgaagcaaca?gatcggcgtc?gtcggtatgg?cagtg?????????12655
The invention has the beneficial effects as follows: the special molecular marker (molecular probe) that the objective of the invention is to seek and use this bacterium, it is specific nucleotide sequence, therefore technical scheme of the present invention has uniqueness, the difference of itself and conventional study method is, search out and have specific nucleotide sequence, and guarantee the specificity of molecule marker by reliable experimental, can utilize and well known to a person skilled in the art mature technology (as PCR or gene chip etc.), use the technology of this marker bacterial detection, make all those skilled in the art can be, realize purpose of the present invention easily and obtain the result of use of expection according to technology contents provided by the invention.Already provided experimental data among the present invention, fully proved the specificity of this nucleotide sequence, and can be applied to the detection of this bacterium, use modern molecular biology method for determining bacteria of the present invention with respect to traditional serology immune response, have fast, accurately, advantage cheaply.The present invention order-checking is also inferred the function of each gene with information biology software, be in order to seek specificity Nucleotide, the gene that some specific functions are arranged in the bacterium is a high special, gene that utilizes above method to infer these specific functions and their position, utilize experiment to prove then, the function of these genes that will be inferred, remove to search out better faster specificity Nucleotide as a kind of road sign, like this, can reduce the blindness of research experiment, accelerate the progress of experiment, reduce experiment fees.
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 (10)

1, a kind of Nucleotide of the O-antigen-specific to intestinal bacteria O78 type, it is characterized in that: it is the isolating Nucleotide shown in SEQ ID NO:1,12655 bases of total length; Perhaps described base with one or more insertions, disappearance or replacement keeps the Nucleotide of the SEQ IDNO:1 of described isolating functional nucleotide simultaneously.
2, according to the Nucleotide of the described O-antigen-specific to intestinal bacteria O78 type of claim 1, it is characterized in that: it comprises called after orf1, orf2, manC, manB, orf5, orf6, wzy, orf8, orf9, orf10,11 genomic constitutions of wzx are all between JUMPStart sequence and gnd gene.
3, according to the Nucleotide of the described O-antigen-specific to intestinal bacteria O78 type of claim 2, it is characterized in that the gene that has high degree of specificity in the described gene is: transhipment enzyme gene, it comprises the wzx gene; Pol gene, it comprises the wzy gene; Glycosyltransferase gene, it comprises orf1, orf2, orf6 gene; Wherein said gene: wzx is the Nucleotide of 11139 to 12554 bases among the SEQ ID NO:1; Wzy is the Nucleotide of 7291 to 8412 bases among the SEQ ID NO:1; Orf1 is the Nucleotide of 74 to 1216 bases among the SEQ IDNO:1; Orf2 is the Nucleotide of 1237 to 2346 bases among the SEQ ID NO:1; Orf6 is the Nucleotide of 6168 to 7289 bases among the SEQ ID NO:1.
4, according to the Nucleotide of claim 1 or 2 described O-antigen-specifics to intestinal bacteria O78 type, it is characterized in that: it also comprises and comes from described wzx gene, wzy gene and their mixing or their reorganization.
5, according to the Nucleotide of the described O-antigen-specific to intestinal bacteria O78 type of claim 4, it is characterized in that, the oligonucleotide that wherein comes from the wzx gene is to being: the Nucleotide of 11344 to 11363 bases among the SEQ ID NO:1 and the Nucleotide of 11937 to 11955 bases, the Nucleotide of 11935 to 11953 bases among the SEQ ID NO:1 and the Nucleotide of 12503 to 12520 bases; The oligonucleotide that comes from the wzy gene is to being: the Nucleotide of 7568 to 7586 bases among the SEQ ID NO:1 and the Nucleotide of 8175 to 8193 bases, the Nucleotide of 7728 to 7744 bases among the SEQ ID NO:1 and the Nucleotide of 8204 to 8224 bases.
6, the application of Nucleotide in detecting other polysaccharide antigen of expressing the antigenic bacterium of O-, the O-antigen of identifying bacterium and bacterium of the described O-antigen-specific to intestinal bacteria O78 type of claim 1.
7, the recombinant molecule of the Nucleotide of the described O-antigen-specific to intestinal bacteria O78 type of claim 1 is providing the O-antigen of expressing intestinal bacteria O78 type by inserting to express, and the application in the preparation bacterial vaccine.
8, according to the application of the Nucleotide of the described O-antigen-specific to intestinal bacteria O78 type of claim 1, it is characterized in that, it is used for PCR, is used for hybridization and fluoroscopic examination or is used to make gene chip or microarray as probe as primer, for the application of bacterial detection.
9, the separation method of the Nucleotide of the described O-antigen-specific to intestinal bacteria O78 type of claim 1 is characterized in that it comprises the steps:
(1) genomic extraction: in substratum, cultivate intestinal bacteria O78 type, centrifugal collecting cell; The genomic dna that obtains detects by agarose gel electrophoresis;
(2) by the O-antigen gene in the pcr amplification intestinal bacteria O78 type bunch: with the genome of intestinal bacteria O78 type is that template is passed through its O-antigen gene of Long pcr amplification bunch, with the PCR product that obtains, detect the size and the specificity thereof of PCR product with agarose gel electrophoresis, merge this long PCR product, and with DNA purification kit purified pcr product;
(3) make up O-antigen gene bunch library: Long PCR purified product is used shotgun make up O-antigen gene bunch library;
(4) to the cloning and sequencing in the library: from the library, select the clone of insertion fragment more than 1kb and the insertion fragment among the clone is checked order with laboratory automatic dna sequencer commonly used, sequence reaches 100% fraction of coverage, thereby obtains all sequences of O-antigen gene bunch;
(5) splicing of nucleotide sequence and analysis: applying biological information science software splicing and edit all sequences, thus obtain the Nucleotide full length sequence of the O-antigen gene bunch of intestinal bacteria O78 type;
(6) screening of specific gene: at wzx, the wzy gene design primer in the O-antigen gene of intestinal bacteria O78 type bunch; Respectively designed two pairs of primers in each gene, every pair of primer is distributed in the different places in the corresponding gene, to guarantee its specificity; Is that template is carried out PCR with these primers with the genomes of 166 strain intestinal bacteria and 43 strain Shigellaes, determines the antigenic high degree of specificity of O-of wzx, wzy gene pairs intestinal bacteria O78 type;
(7) detection of primer sensitivity: cultivate intestinal bacteria O78, after the bacterial count respectively with 5 * 10 3, 5 * 10 2, 5 * 10 15 and 0 viable bacteria join in a certain amount of certain thing to be detected, sneak into the thing to be detected of bacterium and use sample as detecting, sample is added the LB substratum, getting the LB substratum that some and sample mix cross filters, filtered liquid is cultivated, carried out the PCR reaction as pcr template with oligonucleotide after the peek milliliter is handled from cultured bacterium liquid, detect its sensitivity intestinal bacteria O78.
10, the separation method of the Nucleotide of the O-antigen-specific to intestinal bacteria O78 type according to claim 9 is characterized in that it comprises the steps:
(1) genomic extraction: 37 ℃ of incubated overnight intestinal bacteria O78 types in the LB of 5mL substratum, centrifugal collecting cell; With the pH value is 8.0 500 μ l 50mM Tris-HCl and 10 μ l 0.4M EDTA re-suspended cells, 37 ℃ of incubations 20 minutes, and 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, the RNase that adds 3 μ l 10mg/mL again, 65 ℃ of incubations 30 minutes, add equal-volume phenol extracting mixture, get supernatant and use isopyknic phenol again: chloroform: primary isoamyl alcohol mixing solutions extracting twice, get supernatant again with isopyknic ether extracting to remove remaining phenol, phenol: chloroform: the mixed volume ratio of primary isoamyl alcohol is 25: 24: 1; Supernatant rolls out DNA and washes DNA with 70% ethanol with glass yarn with 2 times of volume ethanol deposit D NA, and DNA is resuspended among the 30 μ l TE; Genomic dna detects by 0.4% agarose gel electrophoresis;
(2) by the O-antigen gene in the pcr amplification intestinal bacteria O78 type bunch: with the genome of intestinal bacteria O78 type is that template is passed through its O-antigen gene of Long pcr amplification bunch, be #wl-1098-ATTGGT AGC TGT AAG CCA AGG GCG GTA GCG T according to the JUMPStart sequences Design upstream primer that often is found in O-antigen gene bunch promoter region at first, the gnd gene design downstream primer according to O-antigen gene bunch downstream is #1524-TAG TCG CGT GNG CCT GGA TTA AGTTCG C again; 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, 55 annealing 15 seconds, 68 ℃ were extended 15 minutes, carry out 30 circulations like this, last, continue to extend 7 minutes at 68 ℃, obtain the PCR product, detect the size and the specificity thereof of PCR product with 0.8% agarose gel electrophoresis, merge 5 pipe long PCR products, and with the Wizard PCRPreps 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, 0.9 μ l 0.1MMnCl 2, the DNaseI of the 1mg/mL of 1 μ l dilution in 1: 2000, reaction is carried out at room temperature, and enzyme is cut the dna fragmentation size is concentrated between the 1.5kb-3kb, then adds 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 mixing solutions extracting of primary isoamyl alcohol once, phenol: chloroform: the mixed volume ratio of primary isoamyl alcohol is after using isopyknic ether extracting once again at 25: 24: 1, dehydrated alcohol deposit D NA with 2.5 times of volumes, and wash precipitation with 70% ethanol, be resuspended at last in the 18 μ l water, in this mixture, add 2.5 μ ldNTP (1mMdCTP, 1mMdGTP, 1mMdTTP subsequently, 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 corresponding damping fluid thereof and system is expanded as 80 μ l, 70 ℃ of reactions 20 minutes make 3 of DNA ' end add the dA tail; This mixture is through the equal-volume chloroform: after the mixing solutions extracting of primary isoamyl alcohol and the extracting of equal-volume ether with 3 * 10 of Promega company -3The pGEM-T-Easy carrier connect 10 hours in 16 ℃, cumulative volume is 90 μ l, chloroform: the mixed volume ratio of primary isoamyl alcohol is 24: 1; 10 * the buffer of 9 μ l and the T4DNA ligase enzyme of 25 units are wherein arranged, be that 5.2 the 3M NaAc and the dehydrated alcohol precipitation of 2 times of volumes are connected mixture with the pH value of 1/10 volume at last, wash precipitation with 70% ethanol again, be dissolved in after the drying 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 to 6.0 milliseconds, the SOC substratum that adds 1mL after the electric shock immediately in cup makes the bacterium recovery, then bacterium is coated in and contains penbritin, on the LB solid medium of X-Gal and IPTG, 37 ℃ of incubated overnight, obtain blue white bacterium colony next day, with the white colony that obtains promptly the white clone forward on the LB solid medium that contains penbritin and cultivate, from each clone, extract plasmid simultaneously, and cutting the segmental size of evaluation insertion wherein with the EcoRI enzyme, the white that obtains clone group has constituted the O-antigen gene bunch library of intestinal bacteria O78 type;
(4) to the cloning and sequencing in the library: from the library, select 97 clones of insertion fragment more than 1kb and the insertion fragment among the clone is checked order with this lab A BI3730 type automatic dna sequencer, sequence reaches 100% fraction of coverage, thereby obtains all sequences of O-antigen gene bunch;
(5) splicing of nucleotide sequence and analysis: the Pregap4 and the splicing of Gap4 software of the Staden package software package of publishing with Britain Camb MRC Molecular Biology Lab and edit all sequences, thus obtain the Nucleotide full length sequence of the O-antigen gene bunch of intestinal bacteria O78 type; The quality of sequence is mainly guaranteed by two aspects: 1) genome of intestinal bacteria O78 type is done 5 Long PCR reactions, mix these products then to produce the library, 2) to each base, guarantee high-quality fraction of coverage more than 3, after obtaining the nucleotide sequence of intestinal bacteria O78 type O-antigen gene bunch, Orffinder with American National biotechnology information science center finds gene, find the reading frame of 11 openings, determine also that with the function of finding the reading frame that these are open what gene they are with the genetic comparison among the Blast groupware and the GenBank, finish gene annotation with the Artemis software at Britain Sanger center again, do accurate comparison between DNA and protein sequence with Clustral W software, obtain the structure of the O-antigen gene bunch of intestinal bacteria O78 type at last;
(6) specific gene screening: at wzx, the wzy gene design primer in the O-antigen gene of intestinal bacteria O78 type bunch; Respectively designed two pairs of primers in each gene, every pair of primer is distributed in the different places in the corresponding gene, to guarantee its specificity; Is that template is carried out PCR with these primers with the genomes of 166 strain intestinal bacteria and 43 strain Shigellaes, except that a band that in containing intestinal bacteria O78 group, has obtained the expection size, the correct product of the expection clip size that all do not increase in other groups is so the O-antigen of wzx, wzy gene pairs intestinal bacteria O78 type all is high special.
(7) detection of primer sensitivity: buy the live pig meat stuffing on the market, stir, be divided into the 20g portion, exist in-40 ℃ of refrigerators standby; The frozen bacterium liquid of 10 μ l intestinal bacteria O78 is inoculated in the triangular flask of 20mL LB substratum, in 37 ℃, 200 rev/mins, cultivate 12 hours to saturated, the cultured bacterium liquid that takes a morsel does 10 6With 10 7Dilution doubly, remaining bacterium liquid are put in 4 ℃ the refrigerator standby, get 50 μ l dilution bacterium liquid coating LB agar plate, and 37 degree are cultivated 12h, to being coated with plate count, calculate viable bacteria concentration in the stoste.In 5 portions of live pig meat stuffings, mix 5 * 10 respectively 3, 5 * 10 2, 5 * 10 1, 5 and 0 viable bacteria stir, and add 200mL LB substratum, and through 6 layers of filtered through gauze, filtered liquid 200 rev/mins, is cultivated 12h in 37 ℃.Get 3mL bacterium liquid in 6 from cultured bacterium liquid, centrifugal 5 minutes of 000g removes supernatant, adds 100 μ l MQ ultrapure waters and blows precipitation and mixing open, puts into 100 degree boiling water and boils 15 minutes, and lysate is in 12, and centrifugal 8 minutes of 000g gets 1 μ supernatant as pcr template; Right with 4 pairs of oligonucleotide, the Nucleotide of 11344 to 11363 bases among the SEQ ID NO:1 and the Nucleotide of 11937 to 11955 bases; The Nucleotide of 11935 to 11953 bases among the SEQ ID NO:1 and the Nucleotide of 12503 to 12520 bases; The Nucleotide of 7568 to 7586 bases among the SEQ ID NO:1 and the Nucleotide of 8175 to 8193 bases; The Nucleotide of 7728 to 7744 bases among the SEQ ID NO:1 and the Nucleotide of 8204 to 8224 bases carry out the PCR reaction, and the PCR reaction system is as follows: MQ:15.7 μ l, Mg 2+: 2.5 μ l, Buffer:2.5 μ l, dNTP:1 μ l, Taq enzyme: 0.3 μ l, P1:1 μ l, P2:1 μ l, template DNA: 1 μ l.The PCR reaction conditions is: 95 ℃: 5 ', 95 ℃: 30 ", 55 ℃: 45 ", 72 ℃: 1 ', 72 ℃: 5 ', totally 30 circulations; Reaction is got 10 μ l reaction product electrophoresis after finishing, if the amplified band that conforms to the expection size is arranged, then the result is positive, if do not have, then the result is negative; Participated in 5 * 10 3, 5 * 10 2, 5 * 10 1And every part of pork filling of 5 viable bacterias all obtains positive findings in the PCR of 4 pairs of primers reaction; The pork filling that participates in 0 viable bacteria obtains negative findings in the PCR of 4 pairs of primers reaction; Illustrate that these 4 pairs of primers are 0.25 bacterium/g to the detection sensitivity of the intestinal bacteria O78 in the pork filling when using aforesaid method.
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