CN1563053A - Nucleotide peculiar to 0-antigen of 0120 type bacillus coli - Google Patents
Nucleotide peculiar to 0-antigen of 0120 type bacillus coli Download PDFInfo
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Abstract
The invention provides a nucleotide which is specific to O-antigen of Escherichia coli type O120, it is a nucleotide complete sequence of gene cluster for controlling synthesis of O-antigen in Escherichia coli type, as the separated nucleotide shown by SEQ ID No:1, its total length has 12485 bases; or the nucleotide of SEQ ID No:1, which has one or several inserted, deleted or substituted bases, and retains the function of the described separated nucleotide at the same time; it also includes the oligonucleotide of the glycosyltransferase gene and oligosaccharide unit treatment gene originated from O-antigen gene cluster of Escherichia coli type O120. Said invention utilizes PCR to verify that the oligonucleotide has high specificity to O-antigen of Escherichia coli type O120, and said invention also discloses the method for detecting and identifying escherichia coli type O120 in human body.
Description
Technical field
The present invention relates to the complete nucleotide sequence of control O-antigen synthetic gene cluster in the intestinal bacteria O120 type (Escherichia coli O120), particularly relate in the intestinal bacteria O120 type oligonucleotide in the control O-antigen synthetic gene cluster, can utilize these the oligonucleotide of the O-antigen-specific intestinal bacteria O120 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 " Trends in Microbiology, 4:495-503].In Shigellae, intestinal bacteria and Salmonellas, O-antigen gene [Lei Wang.et al (2001) " Sequence analysis of four Shigella boydii O-antigenloci:implication for Escherichia coli and Shigella relationships " .Infection andImmunity, 11:6923-6930 bunch between galF and gnd gene; Lei Wang and Peter Reeves (2000) " The Escherichia coliO111 and Salmonella enterica O35 gene clusters:gene clusters encoding the samecolitose-containing O antigen are highly conserved " .Journal ofBacteriology.182:5256-5261].The O-antigen gene bunch contains three genoids: sugared synthesis path gene, glycosyltransferase gene, oligosaccharide unit treatment gene, the required nucleoside diphosphate monose of enzymic synthesis O-antigen of wherein sugared synthesis path genes encoding; Thereby the enzyme of glycosyltransferase gene coding forwards nucleoside diphosphate monose and other molecule to and makes monose aggregate into oligosaccharide unit on the monose; The oligosaccharide unit treatment gene comprises transhipment enzyme gene and pol gene, and they transfer to the bacterium inner membrance outside with oligosaccharide unit, and repolymerization becomes polysaccharide.Glycosyltransferase gene and oligosaccharide unit treatment gene only are present in the gene cluster of carrying these genes.The difference of monose in the O-antigen, between monose between the difference of link button and the oligosaccharide unit difference of link button constituted the antigenic diversity of O-, and the composition of monose, the link button between monose and the link button between the oligosaccharide unit are by the Gene Handling in the O-antigen gene bunch, so the O-antigen gene bunch has determined O-antigenic synthetic, has also determined the antigenic diversity of O-.
Because O-antigen is extremely strong antigen, be one of important paathogenic factor of 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.andReeves of a supposition, P.R. (1995) Sequence and analysis of the O antigen gene (rfb) cluster ofEscherichia coli O111.Gene 164:17-23], and have this sugar in the antigenic structure of the O-of other bacterium, so sugared synthesis path gene is not a high special for O-antigen yet
Shigellae has 46 kinds of serotypes, 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 specificselection and bacterial populations " .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 and Ewing ' 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.clinMicrobiol, 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 O120 type.It is the Nucleotide in the O-antigen gene bunch of intestinal bacteria O120 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 O120 type.
Another object of the present invention has provided the gene of the O-antigen gene bunch that constitutes intestinal bacteria O120 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 orf6, orf9, orf10 gene.
Another purpose of the present invention has provided oligonucleotide, and the gene that they come from encoding glycosyl transferring enzyme in the O-antigen gene bunch of intestinal bacteria O120 type respectively comprises orf6, orf9, orf10 gene; The gene that coming from coding transhipment enzyme 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 of identity function is arranged; They are the oligonucleotide in the said gene, and length is at 10-20nt; They are special to the O-antigen of intestinal bacteria O120 type; Especially the oligonucleotide of listing in the table 1, they are high specials to the O-antigen of intestinal bacteria O120 type, and these oligonucleotide are also reconfigurable, the oligonucleotide after the combination also is a high special to the O-antigen of intestinal bacteria O120 type.
The above-mentioned oligonucleotide that a further object of the present invention provides can be used as primer and is used for nucleic acid amplification reaction, perhaps be used for hybridization as probe, perhaps be used to make gene chip or microarray, thereby pass through O-antigen and the detection and the identification of escherichia coli O120 type of these methods detections and identification of escherichia coli O120 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 O120 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 to the Nucleotide of the O-antigen-specific of intestinal bacteria O120 type, and it is the isolating Nucleotide shown in SEQ ID NO:1,12485 bases of total length; The base that perhaps has one or more insertions, disappearance or replacement keeps the Nucleotide of the SEQ ID NO:1 of described isolating functional nucleotide simultaneously.
The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O120 type, comprising called after rmlB, rmlD, rmlA, rmlC, wzx, orf6, wzy, orf8, orf9,10 genomic constitutions of orf10 are all between galF gene and gnd gene.
The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O120 type, the gene that has high degree of specificity in the wherein said gene is: the gene of transhipment enzyme comprises the wzx gene or with wzx the gene of identity function is arranged; Pol gene wzy gene or the gene of identity function is arranged with wzy; Glycosyltransferase gene comprises orf6, orf9, orf10 gene; Wherein said gene: wzx is the Nucleotide of 4636 to 6117 bases among the SEQ ID NO:1; Wzy is the Nucleotide of 6943 to 8202 bases among the SEQ ID NO:1; Orf6 is the Nucleotide of 6114 to 6959 bases among the SEQ ID NO:1; Orf9 is the Nucleotide of 8970 to 9890 bases among the SEQ ID NO:1; Orf10 is the Nucleotide of 9902 to 11005 bases among the SEQ ID NO:1.
The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O120 type, wherein it also comprises and comes from described wzx gene, wzy gene, glf gene or glycosyltransferase gene orf6, orf9, orf10 gene; And their mixing or their reorganization.
The Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O120 type, the oligonucleotide of the wherein said wzx of coming from gene is to being: the Nucleotide of 4729 to 4744 bases among the SEQ ID NO:1 and the Nucleotide of 5119 to 5134 bases; The Nucleotide of 5575 to 5590 bases among the SEQ ID NO:1 and the Nucleotide of 5939 to 5955 bases.The oligonucleotide that comes from the wzy gene is to being: the Nucleotide of 7123 to 7139 bases among the SEQ ID NO:1 and the Nucleotide of 7582 to 7598 bases; The Nucleotide of 7064 to 7079 bases among the SEQ ID NO:1 and the Nucleotide of 7770 to 7787 bases.
The application of the Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O120 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 O120 type is providing the O-antigen of expressing intestinal bacteria O120 type by inserting to express, and the application in the preparation bacterial vaccine.
The application of the Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O120 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 O120 type is characterized in that it comprises the steps:
(1) genomic extraction: in substratum, cultivate intestinal bacteria O120 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 O120 type bunch: with the genome of intestinal bacteria O120 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 O120 type;
(6) screening of specific gene: at wzx, the wzy gene design primer in the O-antigen gene of intestinal bacteria O120 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 O120 type;
(7) detection of primer sensitivity: cultivate intestinal bacteria O120, 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 O120.
The separation method of the Nucleotide of aforesaid O-antigen-specific to intestinal bacteria O120 type is characterized in that it comprises the steps:
(1) genomic extraction: 37 ℃ of incubated overnight intestinal bacteria O120 types in the LB of 5mL substratum, centrifugal collecting cell.With 500ul 50mM Tris-HCl (pH8.0) and 10ul 0.4M EDTA re-suspended cell, 37 ℃ of incubations 20 minutes, the N,O-Diacetylmuramidase that adds 10ul 10mg/ml then continues insulation 20 minutes.The Proteinase K, the 15ul 10%SDS that add 3ul 20mg/ml afterwards, 50 ℃ of incubations 2 hours, the RNase that adds 3ul 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 30ul TE; Genomic dna detects by 0.4% agarose gel electrophoresis;
(2) by the O-antigen gene in the pcr amplification intestinal bacteria O120 type bunch: with the genome of intestinal bacteria O120 type is that template is passed through its O-antigen gene of Long pcr amplification bunch, at first according to the galF sequences Design upstream primer (#1523-ATT GTG GCTGCA GGG ATC AAA GAA AT) that often is found in O-antigen gene bunch promoter region, again according to the gnd gene design downstream primer (#1524-TAG TCG CGT GNG CCT GGA TTA AGT TCG C) in O-antigen gene bunch downstream; With the Expand Long Template PCR method of Boehringer Mannheim company amplification O-antigen gene bunch, the PCR response procedures was as follows: 94 ℃ of pre-sex change 2 minutes; 94 ℃ of sex change are 10 seconds then, annealed 15 seconds for 60 ℃, 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 PCR Preps purification kit purified pcr product of Promega company;
(3) make up O-antigen gene bunch library: make up O-antigen gene bunch library with the Novagen DNaseI shot gun method that is modified, reaction system is a 300ng PCR purified product, 0.9ul 0.1M MnCl
2, the DNaseI of the 1mg/ml of 1ul dilution in 1: 2000, reaction is carried out at room temperature, and enzyme is cut the dna fragmentation size is concentrated between the 1.5kb-3kb, then adds 2ul 0.1M EDTA termination reaction.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 18ul water, in this mixture, add 2.5ul dNTP (1mMdCTP, 1mMdGTP, 1mMdTTP subsequently, 10mMdATP), 1.25ul the T4DNA polysaccharase of 100mMDTT 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 80ul, 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 90ul.10 * the buffer of 9ul and the T4DNA ligase enzyme of 25 units are wherein arranged, use the dehydrated alcohol precipitation of the 3M NaAc (pH5.2) of 1/10 volume and 2 times of volumes to be connected mixture at last, wash precipitation with 70% ethanol again, be dissolved in after the drying and obtain connecting product in the 30ul water; Preparation method with the electric transformed competence colibacillus cell of Bio-Rad company prepares the competence e.colidh5, get after 2-3ul connects product and 50ul competence bacillus coli DH 5 alpha mixes, forward in the electric shock cup of 0.2cm of Bio-Rad company and shock by electricity, voltage is 2.5 kilovolts, time is 5.0 milliseconds 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 O120 type;
(4) to the cloning and sequencing in the library: from the library, select 96 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 O120 type; The quality of sequence is mainly guaranteed by two aspects: 1) genome of intestinal bacteria O120 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 O120 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 10 openings, determine also that with the function of finding the reading frame that these are open what gene they are with the genetic comparison among the blast groupware and the GenBank, finish gene annotation with the Artemis software at Britain sanger center again, do accurate comparison between DNA and protein sequence with Clustral W software, obtain the structure of the O-antigen gene bunch of intestinal bacteria O120 type at last;
(6) screening of specific gene: at wzx, the wzy gene design primer in the O-antigen gene of intestinal bacteria O120 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 the 13rd group of intestinal bacteria O120, 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 O120 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 O120 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 4729 to 4744 bases among the SEQ ID NO:1 and the Nucleotide of 5119 to 5134 bases; The Nucleotide of 5575 to 5590 bases among the SEQ ID NO:1 and the Nucleotide of 5939 to 5955 bases; The Nucleotide of 7123 to 7139 bases among the SEQ ID NO:1 and the Nucleotide of 7582 to 7598 bases; The Nucleotide of 7064 to 7079 bases among the SEQ ID NO:1 and the Nucleotide of 7770 to 7787 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 O120 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 O120 type, its complete sequence shown in SEQ ID NO:1,12485 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 O120 type by method of the present invention, as shown in table 3, it comprises called after rmlB, rmlD, rmlA, rmlC, wzx, orf6, wzy, orf8, orf9,10 genomic constitutions of orf10 are all between galF gene and gnd gene.
Second aspect of the present invention provides the gene in the O-antigen gene bunch of intestinal bacteria O120 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 (orf6, orf9, orf10 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 O120 type.
The 3rd aspect of the present invention, wzx gene in the O-antigen gene bunch that comes from intestinal bacteria O120 type is provided or the gene, wzy gene of identity function is arranged or the gene and the glycosyltransferase gene of identity function are arranged with wzy with wzx, the oligonucleotide that comprises orf6, orf9, orf10 gene, they are any one section oligonucleotide in these genes.But, be that the oligonucleotide of listing in the table 1 is right preferentially by usefulness, in table 1, also listed these oligonucleotide to the position in O-antigen gene bunch and with these oligonucleotide to being the size of the product of the PCR reaction done of primer, the annealing temperature in these PCR reaction free lists is carried out.These primers are except that a band that has obtained the expection size in the 13rd group, and any product that all do not increase in other groups is so the O-antigen of wzx, wzy gene pairs intestinal bacteria O120 type all is high special.
The separation method of the Nucleotide of described O-antigen-specific to intestinal bacteria O120 type comprises the steps: 1) genomic extraction; 2) the O-antigen gene in the pcr amplification intestinal bacteria O120 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 refers to derive from one section nucleic acid molecule in the gene of the gene of gene, coding transhipment enzyme of the encoding glycosyl transferring enzyme in the O-antigen gene bunch and coding polysaccharase, 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 4636 to 6117 bases from SEQ ID NO:1); Wzy gene (nucleotide position is the Nucleotide of 6943 to 8202 bases from SEQ ID NO:1).Coming from above intragenic oligonucleotide is high special to intestinal bacteria O120 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 glycosyltransferase gene; Come from transhipment enzyme and pol gene, comprise the wzx gene or the gene, wzy gene of identity function arranged or the gene of identity function is arranged with wzy with wzx.The mixture of these genes is special to a special bacterial polysaccharides antigen, is special thereby make this cover oligonucleotide to the polysaccharide antigen of this bacterium.More particularly, the mixture of these oligonucleotide is to come from glycosyltransferase gene, wzx gene or the gene, wzy gene of identity function arranged or with wzy 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 O120 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 O120 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 O120 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 O120 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 glycosyltransferase gene, wzx gene or with wzx the gene, wzy gene of identity function arranged or have on the sequence of gene of identity function with wzy that one of them oligonucleotide molecules can hybridize to one.In addition, when two oligonucleotide can both be hybridized, they may be hybridized in same gene and also may hybridize on the different genes.Also promptly, when cross reaction goes wrong, can select the mixture of oligonucleotide to detect the blended gene so that the specificity of detection to be provided.
The present inventor believes that the present invention is not necessarily limited to the above nucleotide sequence coded specific O-antigen of carrying, and is widely used in detecting all expression O-antigens and identifies the antigenic bacterium of O-.And because O-antigen is synthetic and the similarity of other polysaccharide antigens (as bacterium born of the same parents exoantigen) between synthesizing, 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 O120 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 O120 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 O120 types in the LB of 5mL substratum, centrifugal collecting cell.With 500ul 50mM Tris-HCl (pH8.0) and 10ul 0.4M EDTA re-suspended cell, 37 ℃ of incubations 20 minutes, the N,O-Diacetylmuramidase that adds 10ul 10mg/ml then continues insulation 20 minutes.The Proteinase K, the 15ul 10%SDS that add 3ul 20mg/ml afterwards, 50 ℃ of incubations 2 hours add the RNase of 3ul 10mg/ml again, 65 ℃ 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 30ul 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 O120 type bunch:
With the genome of intestinal bacteria O120 type is that template is passed through its O-antigen gene of Long pcr amplification bunch.At first according to the galF sequences Design upstream primer (#1523-ATT GTG GCT GCA GGG ATC AAA GAA AT) that often is found in O-antigen gene bunch promoter region, again according to the gnd gene design downstream primer (#1524-TAG TCG CGT GNG CCT GGATTA AGT TCG C) in O-antigen gene bunch downstream; With the Expand Long Template PCR method of Boehringer Mannheim company amplification O-antigen gene bunch, the PCR response procedures was as follows: 94 ℃ of pre-sex change 2 minutes; 94 ℃ of sex change are 10 seconds then, 60 ℃ of annealing 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, 0.9ul 0.1M MnCl
2, 1ul1: 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 2ul 0.1M EDTA termination reaction.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 18ul water.In this mixture, add 2.5ul dNTP (1mMdCTP subsequently, 1mMdGTP, 1mMdTTP, 10mMdATP), the T4DNA polysaccharase of 1.25ul 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 80ul, 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 90ul.10 * the buffer of 9ul and the T4DNA ligase enzyme of 25 units are wherein arranged.Use the dehydrated alcohol precipitation of the 3M NaAc (pH5.2) of 1/10 volume and 2 times of volumes to be connected mixture at last, wash precipitation with 70% ethanol again, be dissolved in after the drying and obtain connecting product in the 30ul water.
Next is the preparation of competent cell: the method that provides with reference to Bio-Rad company prepares the competent cell bacillus coli DH 5 alpha.Get a ring bacillus coli DH 5 alpha list bacterium colony in the LB of 5ml substratum, 180rpm cultivated after 10 hours, get in the LB substratum that the 2ml culture is transferred to 200ml, 37 ℃ of 250rpm thermal agitations are cultivated OD600 about 0.5, ice bath cooling was 20 minutes then, in centrifugal 15 minutes of 4 ℃ of 4000rpm.Confide all supernatant, dispel thalline, in centrifugal 15 minutes of 4 ℃ of 4000rpm with the deionization aqua sterilisa 200ml of cold ice precooling.Deionization aqua sterilisa 100ml with cold ice precooling dispelled thalline again, in centrifugal 15 minutes of 4 ℃ of 4000rpm.With 10% glycerine suspension cell of cold ice precooling, centrifugal 10 minutes of 4 ℃ of 6000rpm abandon supernatant, precipitate 10% glycerine suspension cell with the precooling of 1ml ice at last, are competent cell.The competent cell that makes is packed as 50ul one pipe ,-70 ℃ of preservations.
Be electric transformed competence colibacillus cell at last: get after 2-3ul connects product and 50ul competence bacillus coli DH 5 alpha mixes, forward in the electric shock cup of 0.2cm of Bio-Rad company and shock by electricity, voltage is 2.5 kilovolts, and the time is 5.0 milliseconds-6.0 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 O120 type with the EcoRI enzyme.
Embodiment 4: to the cloning and sequencing in the library:
From the library, select insert 96 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 O120 type obtained.The quality of sequence is mainly guaranteed by two aspects: 1) genome of intestinal bacteria O120 type is done 5 Long PCR reactions, mix these products then to produce the library.2), guarantee high-quality fraction of coverage more than 3 to each base.After obtaining the nucleotide sequence of intestinal bacteria O120 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 10 openings, determine also that with the function of finding the reading frame that these are open what gene they are with the genetic comparison among the blast groupware and the GenBank, finish gene annotation with the Artemis software at Britain sanger center again, do accurate comparison between DNA and protein sequence with ClustralW software, obtain the structure of the O-antigen gene bunch of intestinal bacteria O120 type at last, as shown in table 3.
By retrieving and comparing, the albumen of finding rmlB genes encoding in orf1 encoded protein and the Shigella boydii O-antigen gene bunch has 98% consensus amino acid sequence, by search, find the very high (E value=1.6 * e of homology desired value of the consensus sequence of orf1 encoded protein and known RmlB to Pfam protein-based order sequenced data storehouse
-213).The albumen of rmlD genes encoding has 97% consensus amino acid sequence in the albumen of Orf2 genes encoding and the Shigella boydii O-antigen gene bunch, by search, find the very high (E value=2.6 * e of homology desired value of the consensus sequence of orf2 encoded protein and known RmlD to Pfam protein-based order sequenced data storehouse
-74).The albumen of rmlA genes encoding has 98% consensus amino acid sequence in the albumen of orf3 genes encoding and the Shigellaboydii O-antigen gene bunch, by search, find the very high (E value=1.8 * e of homology desired value of the consensus sequence of orf3 encoded protein and known RmlA to Pfam protein-based order sequenced data storehouse
-118).The albumen of rmlC genes encoding has 84% consensus amino acid sequence in the albumen of orf4 genes encoding and the Shigella boydii O-antigen gene bunch, by search, find the very high (E value=1.2 * e of homology desired value of the consensus sequence of orf4 encoded protein and known RmlC to Pfam protein-based order sequenced data storehouse
-125).RmlBDAC is responsible for a kind of rare monose in the synthetic rhamnosyl O-antigen.Therefore we are rmlB with these four unnamed genes, D, A, and C.
Orf5 and orf7 are the proteic genes that there is transmembrane segment in only two codings of intestinal bacteria O120 kind.The O-antigen transferring enzyme of orf5 encoded protein and Shigella boydii (AAL27344) has 43% sequence identity, it contains 13 uniform transmembrane segments by the proteic topology discovery of HMMTOP2.0 programanalysis, and this is the proteic characteristic feature of Wzx.So name orf5 is wzx.The O-antigen polysaccharase of orf7 encoded protein and intestinal bacteria (AAK64372) has 23% consistence, 43% similarity, it contains 10 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.
Orf6, the albumen of 9 and 10 3 genes encodings and other known glycosyltransferases have the sequence identity of 35-44% and the sequence similarity of 53-62%.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 is very high, therefore we infer this three genes encoding glycosyltransferases, and because each glycosyltransferase specificity catalysis forms a kind of disaccharide bond, so we infer that the antigenic oligosaccharide unit of O-of intestinal bacteria O120 may be made up of four monose.Because the definite function of these three genes can't be determined, so we are with these two genes temporary called after orf6, orf9 and orf10.
The Eps11P of coding has 29% consensus amino acid sequence in the albumen of orf8 genes encoding and the Streptococcus thermophilus O-antigen gene bunch, because the definite function of this gene can't be determined, so we are with the temporary called after orf8 of this gene.
Embodiment 6: the screening of specific gene:
Wzx, wzy gene design primer in the O-antigen gene of intestinal bacteria O120 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 the 13rd group of intestinal bacteria O120, 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 O120 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 O120 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 4729 to 4744 bases among the SEQ ID NO:l and the Nucleotide of 5119 to 5134 bases; The Nucleotide of 5575 to 5590 bases among the SEQ ID NO:1 and the Nucleotide of 5939 to 5955 bases.The Nucleotide of 7123 to 7139 bases among the SEQ ID NO:1 and the Nucleotide of 7582 to 7598 bases; The Nucleotide of 7064 to 7079 bases among the SEQ ID NO:1 and the Nucleotide of 7770 to 7787 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 O120 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 Microbiology 1993,7:239-252).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 O120 of the present invention can be applied to set up recombiant vaccine.
Nucleotide sequence (shown in the SEQ IDNO:1) according to the O-antigen-specific to intestinal bacteria O120 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 glycosyltransferase gene and oligosaccharide unit treatment gene and intragenic primer and PCR data in the O antigen gene bunch of intestinal bacteria O120 type.Glycosyltransferase gene, transhipment enzyme gene and pol gene and their function corresponding and the size of the O antigen gene bunch of intestinal bacteria O120 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 IDNO:1.Is that template carry out PCR with listed corresponding annealing temperature in the table with the genomes of all bacterium in the table 2 with every pair of primer, has obtained corresponding PCR product, and its size is also listed in the table.
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 O120 type in the 13rd 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 25ul.Template is dilution in 1: 20, gets 1 μ l.After reaction finishes, get the 10ulPCR product 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 13rd 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 O120 type and O-antigen thereof are high specials.
At last, from intestinal bacteria O120 type, screen gene by PCR: wzx, wzy to the O-antigen high special of intestinal bacteria O120 type.And the oligonucleotide of these intragenic any one section 10-20nt is special to the O-antigen of intestinal bacteria O120 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 O120 type.These all oligonucleotide all can be used for the intestinal bacteria O120 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 O120 type, in table, listed the structure of the O-antigen gene bunch of intestinal bacteria O120 type, altogether by 10 genomic constitutions, each gene box indicating, and in square frame, write the title of gene, numeral be the order of the open reading frame (orf) in the O-antigen gene bunch.Two ends at O-antigen gene bunch are galF gene and gnd gene, and 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 O120 type, listed the accurate position of all open reading frame in complete sequence in the O-antigen gene bunch of intestinal bacteria O120 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)
＜110〉Tianjin Biochip Technology Co., Ltd
＜120〉to the Nucleotide of the O antigen-specific of intestinal bacteria O120 type
＜130〉to the Nucleotide of the O antigen-specific of intestinal bacteria O120 type
<160>1
<170>PatentIn?version?3.2
<210>1
<211>12485
<212>DNA
<213>Escherichia?coli
<400>1
attgtggctg?cagggatcaa?agaaatcctc?ctggtgactc?atgcgtccaa?gaacgcggtc??????60
gaaaaccact?tcgacacctc?ttatgaatta?gaatctctcc?ttgaactgcg?cgtgaagcgt?????120
caactgctgg?cggaagtaca?gtccatctgt?ccgccgggcg?tgaccattat?gaacgtgcgt?????180
cagggcgaac?ctttaggttt?aggccactct?attttgtgtg?cacgacccgc?cattggtgac?????240
aatccatttg?tcgtggtact?gccggacgtt?gttatcgatg?atgccagcgc?cgacccgctg?????300
cgttacaacc?ttgccgctat?gattgcgcgc?ttcaacgaaa?cgggacgcag?ccaggtgctg?????360
gcaaaacgta?tgccgggtga?cctctccgaa?tactccgtca?tccagaccaa?agagccgctg?????420
gaccgtgagg?gtaaagtcag?ccgcattgtt?gaatttatcg?aaaaaccgga?tcagccgcag?????480
acgctggact?cagacatcat?ggccgtaggt?cgctatgtgc?tttctgccga?tatttggccg?????540
gaactggaac?gtactcagcc?tggtgcatgg?ggacgtattc?agctgactga?tgctattgcc?????600
gagctggcga?aaaaacaatc?cgttgatgca?acgctgatga?ccggcgacag?ttacgactgc?????660
ggcaaaaaaa?tgggctatat?gcaggcgttt?gtgaagtatg?gcctacgcaa?cctgaaagaa?????720
gggacgaagt?tccgtaaagg?tattgagaag?ctgttaagcg?aataatgaaa?atctgaccgg?????780
atgtaacggt?tgataagaaa?attataacgg?cagtgaagat?ttgcagcaaa?agtaatttgt?????840
tgcgaatatt?cctgccgttg?ttttatataa?accatcagaa?taacaacgag?ttagcagtag?????900
ggttttagtc?aaagttttcc?aggattttcc?ttgtttccag?agcggattgg?taagacaatt?????960
agcgtttgaa?tttttcgggt?ttggcgcgag?tgggtaaagc?tcgtcacatc?gtagacatgc????1020
atgcagtgct?ctggtagctg?taaagccagg?ggcggtagcg?tgcattaata?cctctattaa????1080
tcaaactgag?agccgcttat?ttcacagcat?gctctgaagt?aatatggaat?aaattaagtg????1140
aaaatacttg?ttactggtgg?cgcaggattt?attggttctg?ctgtagttcg?tcacattata????1200
aataatacgc?aggatagtgt?tgttaatgtc?gataaattaa?cgtacgccgg?aaacctggaa????1260
tcacttgctg?atgtttctga?ttccgaacgc?tatgtttttg?aacatgcgga?tatttgcgat????1320
gcagctgtaa?tggcacggat?ttttgctcag?catcagccgg?atgcagtgat?gcacctggct????1380
gctgaaagcc?atgttgaccg?ttcaattaca?ggtcctgcgg?catttattga?aaccaatatt????1440
gttggtactt?atgtcctttt?ggaagccgct?cgcaattact?ggtctgctct?tgatggcgac????1500
aagaaaaata?gcttccgttt?tcatcatatt?tctactgacg?aagtctatgg?tgatttgcct????1560
catcctgacg?aggtaaataa?tacagaagaa?ttacccttat?ttactgagac?aacagcttac????1620
gcgccaagca?gcccttattc?cgcatccaaa?gcatccagcg?atcatttagt?ccgcgcgtgg????1680
aaacgtacct?atggtttacc?gaccattgtg?actaattgct?ctaacaatta?tggtccttat????1740
catttcccgg?aaaaattgat?tccattggtt?attctcaatg?ctctggaagg?taaagcatta????1800
cctatttatg?gtaaagggga?tcaaattcgc?gactggttgt?atgttgaaga?tcatgcgcgt????1860
gcgttatata?ccgtcgtaac?cgaaggtaaa?gcggatgaaa?cttataacat?tggtgggcac????1920
aacgaaaaga?aaaacattga?tgtagtgctc?actatttgtg?atttgttgga?tgagattgtc????1980
ccgaaagaga?aatcttatcg?tgagcaaatc?acttatgttg?ccgatcgtcc?gggacacgat????2040
cgccgttatg?cgattgatgc?tgagaagatt?ggtcgcgaat?tgggatggaa?accacaggaa????2100
acgtttgaga?gtgggattcg?taaaacggtg?gaatggtacc?tgtccaatac?aaaatgggtt????2160
gataatgtga?aaagtggtgc?ctatcaatcg?tggattgaac?agaactatga?gggccgccag????2220
taatgaatat?cctccttttt?ggcaaaacag?ggcaggtagg?ttgggaacta?cagcgtgctc????2280
tggcacctct?gggtaatttg?attgctcttg?atgttcactc?cactgattac?tgtggtgatt????2340
ttagtaatcc?tgaaggtgtc?gctgaaaccg?taagaagcat?tcggcctgat?attattgtca????2400
acgcagccgc?tcacaccgca?gtagacaaag?cagaatcaga?accagagttt?gcacaattac????2460
ttaacgcgac?gggtgtcgaa?gcgatcgcga?aagcagccaa?tgaagtcggt?gcttgggtta????2520
ttcactactc?tactgactac?gtatttccgg?ggactggtga?aataccatgg?caggaggcgg????2580
atgcaaccgc?accgctaaat?gtttacggtg?aaactaagtt?agctggagaa?aaagcattac????2640
aagagcattg?tgcgaagcac?cttattttcc?ggaccagctg?ggtctatgca?ggtaaaggaa????2700
ataacttcgc?caaaacaatg?ttgcgtctgg?caaaagagcg?tgaagaatta?gccgttatta????2760
atgatcagtt?tggtgcgcca?actggtgcag?agttactggc?tgattgtacg?gcacatgcca????2820
ttcgtgtggc?actgattaaa?ccggaagtcg?caggcttgta?ccatctggta?gctagtggta????2880
ccacaacgtg?gcacgattat?gctgcgctgg?tttttgaaga?ggcacgcaaa?gcaggcattc????2940
cccttgcact?caacaagctc?aacgcagtac?caacaacagc?ctatcctaca?ccagctcgtc????3000
gtccacataa?ctctcgcctt?aatacagaaa?aatttcagca?gaactttgcg?cttgtcttgc????3060
ctgactggca?ggttggtgtg?aaacgaatgc?ttaacgaatt?atttacgaca?acagcaattt????3120
aatagttttt?gcatcttgtt?cgtgatggtg?gagcaagatg?tattaaaagg?aatgatgaaa????3180
tgaaaacgcg?taaaggtatt?attttggcgg?gtggttctgg?tactcgtctt?tatcctgtga????3240
ctatggcagt?cagtaaacag?ctgttaccga?tttatgataa?accgatgatc?tattatccgc????3300
tctctacact?gatgttggcg?ggtattcgcg?atattttgat?tattagtacg?ccacaggata????3360
ctcctcgttt?tcaacaactg?ctgggtgacg?gtagccagtg?ggggctaaat?cttcagtaca????3420
aagtgcaacc?gagtccggat?ggtcttgcgc?aggcgtttat?tatcggtgaa?gagtttattg????3480
gtggtgatga?ttgtgctttg?gtacttggtg?ataatatctt?ctacggtcac?gacctgccta????3540
agttaatgga?tgccgctgtt?aacaaagaaa?gtggtgcaac?ggtatttgcc?tatcacgtta????3600
atgatcctga?acgctacggt?gtcgttgagt?ttgataaaaa?cggtacggca?ataagcctgg????3660
aagaaaaacc?gttacaacca?aaaagtaatt?atgcggtaac?tgggctttat?ttctatgata????3720
acgacgttgt?cgaaatggcg?aaaaacctta?agccttctgc?ccgtggtgaa?ctagaaatta????3780
ccgatattaa?ccgtatttat?atggaacagg?ggcgtttatc?tgttgccatg?atgggacgtg????3840
gttatgcatg?gttggacacg?gggacacatc?aaagtcttat?tgaggcaagc?aatttcatcg????3900
cgacaataga?agaacgacag?gggctgaaag?tttcctgccc?ggaagaaatt?gcttaccgta????3960
aagggtttat?cgatgctgag?caggtgaaag?tattagctga?accgttgaag?aaaaatgctt????4020
atggccagta?cctgctaaaa?atgattaaag?gttattaata?aaatgaacgt?aattaaaaca????4080
gagattccag?acgtattaat?tttcgaaccg?aaagtttttg?gtgatgagcg?tggtttcttt????4140
atggaaagct?ttaatcagaa?agttttcgaa?gaagctgtag?gacgtaaggt?tgaatttgtt????4200
caggataacc?attcgaagtc?tagtaaaggt?gttttacgcg?ggctgcatta?tcagttagaa????4260
ccgtatgcgc?aagggaaatt?ggtacgttgc?gctgttggtg?aggtttttga?tgtagctgtt????4320
gatattcgta?aatcgtcgcc?tacctttggt?aaatgggttg?gggtgaattt?atctgctgag????4380
aataaacggc?aattgtggat?ccctgaggga?tttgcacatg?gttttttagt?gctgagtgag????4440
actgcggaat?ttttatataa?aacgacgaat?tattatcatc?ctgagagtga?tagagggatt????4500
atttggaatg?atcccaatat?aggtattgtt?tggcctgaaa?gagaaaatat?agttttatcg????4560
aaaaaggata?taaatcaacc?taatttatca?tatagcttct?ctctcaaaca?gttgttgtcg????4620
taagagaaaa?gatctatgtc?ccttctaagc?aatgcaaaat?gggtaagctt?atctcaattt????4680
ggacgtatta?ttatacaatt?gattagcttg?actgttttaa?caagatttat?accaccagca????4740
gattatggat?taatggcttt?aggcacagtt?gttatgaatt?ttgtcttcat?aatacgtgat????4800
cttggaactg?catcggccat?aatacaaaga?aaagaagtag?atgacgatat?tttaaattca????4860
gttttttggt?tgaatgtaac?tatgggagtg?gggttatgca?tactaattat?actatgtact????4920
ccactaattg?agctgttatt?taagaatgag?aagttagata?atattctgta?tttgttatgc????4980
ttttgctttc?cattagctag?tattggttct?gctcatcagg?cattaatgga?aagaaactct????5040
cattttaaaa?aaatagcaat?catagaattg?ctctcatctc?tttctggttt?tgttgttgca????5100
ttgctagtag?cgatcaataa?tggaggtgtt?tatgcattag?tataccaatt?aatttgttca????5160
tcatttattt?ctagtatatt?attcttttat?ctatcctctt?ggacgccatc?cttgcatttt????5220
aaatttgagg?gggttaaaaa?aattataggt?tttagtagca?ggttaactgt?ttttaattta????5280
gttaactatt?tctcacgtaa?tgcagatgct?atgataataa?gtagaaagta?tgatgctaat????5340
attctcggcg?cttactctct?agcttataga?attatgttgt?ttccgctcca?gagtttaaca????5400
tttattgcgt?caagatcact?atatccaata?ttaagtagaa?accaagatga?tttgaaactt????5460
attagggaga?tatatttaaa?aacaatattt?ataatttcaa?tgattaccat?accattaatg????5520
ttaggattag?tatttttaag?gacgccgttc?attacaatag?ctttagggcc?taactgggca????5580
ataacttcta?atattttact?ttggttggca?ccaactggaa?ttattcagtc?aattcttagt????5640
tgctccggta?gcatattaat?gtcaactaat?aacacgaaaa?cattgatgtt?attaggtata????5700
tttggggccg?cgatacaagt?attatcattc?ttcattggtt?ctttatttag?tataaatatt????5760
tttgtttttg?cttactttat?agcaaatgta?atcaatgcgc?tagttgtttt?atattttacg????5820
tttaaaagta?taaagcttaa?tatttattca?ttctgtaaga?tcctactgat?cccaacctta????5880
tcttgttcgg?tgatgataat?agcgcttcat?ttcgttaaaa?aaatcattta?cattaataat????5940
gtggtaaatg?atgtgagcta?tttaataatt?caggtaatca?tcggttgtat?tgtatacttt????6000
attgtttttt?atatcttcgt?aaatatatgt?cttcgattta?aaatcatgtc?agaaaatatg????6060
atcccagatt?ttctgttgag?aataaagttc?tttagccata?agagattttt?aaattgaata????6120
aacaattatt?tttgattgtt?gtttataata?agaaaatagc?tgagagttgt?acttttaata????6180
aggttgttaa?ctctaagttt?agtgatgatt?cattatgtgt?tatatggaac?aatggaccca????6240
caagttgtga?ggaggagttt?ttgaatgtat?atgagtataa?taaaaacatt?cgctatcatg????6300
aaacattgca?taatatctca?ttggccaaga?tttataacga?ggtaattaat?aaatataacg????6360
ctcagattta?tatcatccta?gatgatgatt?cttcattaac?tgatgcatat?tttgaagctg????6420
ttaaacaaat?atcacctaat?gaagtaggta?tgcctgtcat?atattatgat?agaaaaataa????6480
ttaacccatg?cataaatggt?cagatttata?gtaaaggaat?taatattgat?gctaatgata????6540
ccataacaac?tatcgggagt?ggattggttg?tgggtaggga?tgtagcatct?caattattca????6600
aagcatataa?ttctgtattt?gatgaacgtt?tttatctata?tggagtagat?actacgtttt????6660
gtttccgact?taataaattg?aaaataaatg?ataaaattaa?gattatagat?ggctttgagc????6720
acggcctttc?aaggcttgaa?gggaaaaatg?ataaattaac?tctttttaga?aaaatagaga????6780
gaagtaatga?tctcgcatta?cagatccgat?attataaaga?aaaaaaacat?tggtttttaa????6840
tgatggtgct?gttactcgcg?agttcaatta?agaagtttat?tacttttcag?ccacaacaat????6900
ataaatattc?tactattatt?aaaacatttg?ttaagggaaa?gcatgaacgt?cagagataat????6960
gataacgcaa?tcaagttatt?ggagtttgtt?ttaattttct?ctttttttat?tattgcatta????7020
tttttcttat?attccccatc?ttatcttatt?tacattgtag?ttagtgcgtt?tgttttgatt????7080
aagttcaata?ataactatag?ttttggtttg?tttatattat?tgataatggt?atggctgatt????7140
gtcaattcta?ctaatgtcat?ttcataccaa?aattttgtgc?tcgcgagtga?tgatttctct????7200
tcttattata?ataactatct?tggatttctt?cattacggga?taaatgaaga?aaatcttttt????7260
gaatttggct?attcagaaat?aatgttgcct?ttgttgaact?atattgcaag?tttaattatt????7320
gggcaaccag?aaccatataa?gttaaagttt?ctgtattctg?tattgcaaag?cactggtgta????7380
ttatattgct?gcgtaaaaat?atctcgtcac?tataaacttg?atttgagtaa?tagtatatta????7440
ttaacagctg?ccgtattggt?gttttataag?cctttagtag?gtattcaact?atctagacaa????7500
atgtttagct?ctatttttat?tatagctttt?attttcacga?ataataaatt?tgagcgaatt????7560
atcttttcat?taatggcctt?ttgttttcat?tcatcagcga?taatattatt?accaataacg????7620
caatatcttc?tgacaaaaag?gaaaagaaag?gacttctatt?atagtttaat?tatcgttctg????7680
cttgcatatg?ctgcatttat?tgttgtgttt?acatcgttta?aaaatgaatt?aataaatatt????7740
cctattttaa?ataaattaga?ctacactttt?cgtatgttta?atgaaccaga?aaaaataaag????7800
caatctgtat?ttacatcttt?tctcttgtta?atatatttaa?tccctgcgct?gattttttcc????7860
atcttaaata?aaaaatctga?tggatttaaa?tataatttag?ctatcatttg?tgtattttta????7920
atatcttttt?gtattgtacc?tggtttcaat?aacagattat?tctttgctga?gttatatgtc????7980
tttcttggtt?ttttttatta?tatggtgttt?ttttacaata?gagtgaatgc?aagagagcat????8040
gtatcgagat?taatcatctt?tattcttttt?actattctat?tttttcaatc?tcaattttca????8100
tcagaaaata?ggcagtttga?tagctttgaa?ctagaacctt?tctattatct?taaatattta????8160
gaatatagag?agacagaaat?aaacagagga?ctattagagt?aacatgccta?atttagttat????8220
ttatactgct?gttactgggc?attatgatga?actggggaat?gtttatgagg?aaccttcagt????8280
tgattatatc?tgctttacag?actacaactt?taccggtacg?gtacctaaac?cgtggaagca????8340
aattaaattg?ccgccttcaa?atttctctga?taaagactta?gcaagatatt?gtaagcttaa????8400
tcctcatgtc?ctattgcctc?agtatcagca?ttcgttgtgg?attgatggca?atattcgaat????8460
aaaaggtaaa?atacgaaatt?ttattattga?cattttgagc?aagcatcgaa?ttgcagcata????8520
tgaacattgg?tggcgagaca?aaacagaaca?ggaatttcat?gaatgtgcta?gaagtggttt????8580
cgatcctgct?tggaaattat?ataaacaaat?tgagcgctat?aagcatgaag?gatacacctc????8640
ctcagatttt?tttgagaata?atatactcat?gcgtaaccat?atggaaagtg?acataatcaa????8700
aatgcatgaa?atatggtggg?gggagtatat?ttcaggaggt?aagcgtgatc?aatattcttt????8760
tacttatgct?gcttataaaa?gtgatgtaaa?tatatattca?ttaggggttc?atgatgctag????8820
agttataaaa?caatattttg?actatgtgac?acactccaag?aaaaggccgt?taaaacaata????8880
tatctggatt?ataataaata?gaatctatat?ttctctaacg?agatggagcg?taaatgaacc????8940
atctcggaca?aataaattag?tgctaaataa?tgaaagagat?tcaaatgaaa?aataaagttg????9000
ctggagtcat?tgttacatat?aatccagaaa?ttaaaatact?tctcagtgca?ttggagtcat????9060
cgacaaaaca?gttagactat?atttatttga?tagataattg?ctcattaaat?aaaaatgagg????9120
tggctgattt?agctggaaga?tttgggaatg?taaaatttca?ggcgctctct?aaaaatattg????9180
gattagccag?cgcccaaaac?atgggcatca?atactgctaa?aaacaataac?ctaatagaat????9240
atgtagtttt?attcgatcag?gatagcataa?tagagaacaa?ttttattgct?tcattacttg????9300
atgattataa?aaaagtttta?gcaaaggatt?ctaagatagg?agctataggc?ccggtttttt????9360
atgatccatc?tactaaaatt?aattatcctg?caacagtata?tataggtcct?tttataaaaa????9420
aaatatctat?aaaggatgag?ccacaattcg?caacgtattt?aattgcctct?ggctgtttaa????9480
taaaaataga?tactttgtct?tcggttggat?taatgcagga?tgatttgttc?atcgattaca????9540
tcgatgttga?atggagttta?agagcaaaga?gtaaaggtta?taatttatat?atatcaccaa????9600
acgcaaaaat?ggcccataca?attggagacg?cgaggcgtaa?atttttgggg?cggacaatat????9660
ctattcactc?cccttttaga?cgttacttct?tggttagaaa?tagtttttac?atgataaggt????9720
taaattatat?tcctttaggg?tacaagacta?gagagtgtat?ttttaatatt?gccaggatat????9780
taacatctat?catatattcc?gatcaaaaaa?ttaaaacaat?aaaatcggct?ttttatggag????9840
tcattgatgg?aattgcaggt?cgatttggcc?ctctaaaacg?taaaatataa?agagaaattt????9900
catgaaaatt?ttatatataa?tcacaaaagc?tgatgaaatc?ggtggggcac?aaattcatgt????9960
gagagacctt?gctcgtcgtt?tacacaatga?tggtaataaa?gttacagtaa?tcgtaggaga???10020
ggatggcgct?ttagttagac?aacttaaaga?gcttcacatt?tcggtgcgaa?tagtagaaaa????10080
tttagttagg?caaatttcac?cactaaatga?tataaaagcc?atttttaaca?tcagaaagat????10140
aattaaagat?aattcaccgg?atattattgg?tcttcactcc?tctaaggctg?gaattgttgg????10200
tcgactagca?gcttgtcgat?taaatattcc?agtagtattt?accgctcacg?gttgggcatt????10260
tgcagagggt?atcagtgaca?aaaaaaggaa?attatttatt?ttcattgaga?aaatactatc????10320
tccttttttg?gataagataa?ttactgtttc?ggagcaagat?aaaaagttgg?cgctagattt????10380
aggcgttgca?tcctctgaaa?aacaagtcgt?tattcataac?ggaatgcctg?atattactat????10440
taagcgtaag?gattattcgg?attcaactaa?agttcgatta?atcagtgtag?ctcgtttttc????10500
agagcaaaaa?gatcatgaaa?ctttatttca?cgcactcaag?ttaatttcta?cagataactg????10560
gacactaact?ctggttggga?aaggtcctaa?gttagaaaat?attaaaaaac?ttgcatctga????10620
tttgaatatt?gcaaataata?ttctctttct?tggggaaaga?gatgatgtcg?ataatttatt????10680
aagtgaaagt?gatgttttct?gtttaataac?aaactgggag?ggattgccat?taagtattat????10740
cgaggctatg?cgtgcgtcct?tgccagtggt?agctacagat?gttggaggtg?tctctgagtt????10800
agtagagcat?aaaaaaaccg?gcttgcttca?ttctcctaaa?aacatacaag?aacttgctaa????10860
tatcctggat?actattatca?atgatagtga?cttgagatat?aaattgggaa?atgctggaag????10920
agttaaatac?caaaataatt?tcacatttga?acatatgtat?tatgcaacaa?tagatgaata????10980
taataaattg?atacatacta?aatagttaat?tatatttttc?ttgtgttcgc?tcttttctaa????11040
cagttttaat?ttctttaatt?ttaagagata?aatatattag?acaagttatt?ccaatatgag????11100
taaattggca?taacttgtaa?ctttagaccg?caacccgcag?taacccctga?caggagtaaa????11160
taatgtcaaa?gcaacagatc?ggcgtcgtcg?gtatggcagt?gatggggcgc?aaccttgcgc????11220
tcaatatcga?aagccgtggt?tataccgtct?ctattttcaa?ccgttcccgt?gaaaagaccg????11280
aagaagtgat?tgccgaaaat?ccaggcaaga?aactggttcc?ttactatacg?gtgaaagagt????11340
ttgttgaatc?tctggaaacg?cctcgtcgca?tcctgttaat?ggtgaaagca?ggtgcaggca????11400
cggatgctgc?tattgattcc?ctcaagccat?acctcgataa?aggtgacatc?atcattgatg????11460
gtggtaatac?cttcttccag?gacaccattc?gtcgtaaccg?tgagctttct?gccgaaggct????11520
ttaacttcat?tggtaccggt?gtctccggtg?gtgaagaagg?cgcgctgaaa?ggtccttcta????11580
ttatgcctgg?tgggcagaaa?gaagcctatg?aactggttgc?gccgatcctg?accaaaatcg????11640
ccgcagtggc?tgaagacggt?gagccatgtg?ttacctatat?tggtgccgat?ggcgcaggtc????11700
actatgtgaa?gatggttcat?aacggtattg?aatacggcga?tatgcagctg?attgctgaag????11760
cctattctct?acttaaaggt?ggtctgaacc?tcaccaacga?agaactggcg?cagaccttta????11820
ccgagtggaa?taacggtgaa?ctgagcagct?acctgatcga?cattaccaaa?gacatcttca????11880
ccaaaaaaga?tgaagacggt?aactacctgg?ttgatgtgat?cctggatgaa?gcggctaaca????11940
aaggtaccgg?taaatggacc?agccagagcg?cgctggatct?cggcgaaccg?ctgtcgctga????12000
ttaccgagtc?tgtgtttgca?cgttatatct?cgtctctgaa?agatcagcgc?gttgccgcgt????12060
ctaaagttct?ctctggcccg?caagcgcagc?cagtaggcga?caaagctgag?tttatcgaga????12120
aagttcgtcg?tgcgctgtat?ctgggcaaaa?tcgtttctta?cgctcagggc?ttctctcagc????12180
tgcgtgcggc?gtctgaagag?tacaactggg?atctgaacta?cggcgaaata?gcgaagattt????12240
tccgtgctgg?ctgcatcatt?cgtgcgcagt?tcctgcagaa?aatcaccgat?gcttatgccg????12300
aaaatccgca?gatcgctaac?ctgctgctgg?ctccttactt?caagcaaatt?gccgatgact????12360
accagcaggc?gctgcgcgat?gtcgtcgctt?atgcggtaca?gaacggtatc?ccggttccga????12420
ccttcgccgc?tgcggttgcc?tattatgaca?gctaccgtgc?cgctgttctg?cctgcgaacc????12480
taatc????????????????????????????????????????????????????????????????12485
Glycosyltransferase gene in the O antigen gene of table 1 intestinal bacteria O120 type bunch and oligosaccharide unit treatment gene and wherein
Primer and PCR data
| 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 | O-antigen transhipment enzyme | 4636-6117 | ????4729-4744 | ????5119-5134 | ????406bp | ??0 * | ??56 |
| ????5575-5590 | ????5939-5955 | ????381bp | ??0 * | ??56 | |||
| ??Wzy | O-antigen polysaccharase | 6943-8202 | ????7123-7139 | ????7582-7598 | ????376bp | ??0 * | ??56 |
| ????7064-7079 | ????7770-7787 | ????724bp | ??0 * | ??56 |
*Only in intestinal bacteria O120 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，O123，O125，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 6th group of bacterial strain adds intestinal bacteria reference culture O120 IMVS
a
For the convenience that detects, every 12-19 bacterium is divided into one group, and 12 groups altogether, the 13rd group as positive control
a.Institute?of?Medical?and?Veterinary?Science(IMVS)，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 O120 type O antigen gene structure iron
E.coli?O120?O-antigen?gene?cluster
orf#??galF??????rmlB????rmlD????rmlA??rmlC????wzx??????wzy?????orf7??orf8???orf9????orf10?????gnd
G+C％?content???43??????47??????43????38??????30???????29??????27????34?????32??????34
Table 4 intestinal bacteria O120 type O antigen gene cluster gene position
ATTGTGGCTG?CAGGGATCAA?AGAAATCCTC?CTGGTGACTC?ATGCGTCCAA?GAACGCGGTC??????60
GAAAACCACT?TCGACACCTC?TTATGAATTA?GAATCTCTCC?TTGAACTGCG?CGTGAAGCGT?????120
CAACTGCTGG?CGGAAGTACA?GTCCATCTGT?CCGCCGGGCG?TGACCATTAT?GAACGTGCGT?????180
CAGGGCGAAC?CTTTAGGTTT?AGGCCACTCT?ATTTTGTGTG?CACGACCCGC?CATTGGTGAC?????240
AATCCATTTG?TCGTGGTACT?GCCGGACGTT?GTTATCGATG?ATGCCAGCGC?CGACCCGCTG?????300
CGTTACAACC?TTGCCGCTAT?GATTGCGCGC?TTCAACGAAA?CGGGACGCAG?CCAGGTGCTG?????360
GCAAAACGTA?TGCCGGGTGA?CCTCTCCGAA?TACTCCGTCA?TCCAGACCAA?AGAGCCGCTG?????420
GACCGTGAGG?GTAAAGTCAG?CCGCATTGTT?GAATTTATCG?AAAAACCGGA?TCAGCCGCAG?????480
ACGCTGGACT?CAGACATCAT?GGCCGTAGGT?CGCTATGTGC?TTTCTGCCGA?TATTTGGCCG?????540
GAACTGGAAC?GTACTCAGCC?TGGTGCATGG?GGACGTATTC?AGCTGACTGA?TGCTATTGCC?????600
GAGCTGGCGA?AAAAACAATC?CGTTGATGCA?ACGCTGATGA?CCGGCGACAG?TTACGACTGC?????660
GGCAAAAAAA?TGGGCTATAT?GCAGGCGTTT?GTGAAGTATG?GCCTACGCAA?CCTGAAAGAA?????720
GGGACGAAGT?TCCGTAAAGG?TATTGAGAAG?CTGTTAAGCG?AATAATGAAA?ATCTGACCGG?????780
ATGTAACGGT?TGATAAGAAA?ATTATAACGG?CAGTGAAGAT?TTGCAGCAAA?AGTAATTTGT?????840
TGCGAATATT?CCTGCCGTTG?TTTTATATAA?ACCATCAGAA?TAACAACGAG?TTAGCAGTAG?????900
GGTTTTAGTC?AAAGTTTTCC?AGGATTTTCC?TTGTTTCCAG?AGCGGATTGG?TAAGACAATT?????960
AGCGTTTGAA?TTTTTCGGGT?TTGGCGCGAG?TGGGTAAAGC?TCGTCACATC?GTAGACATGC????1020
ATGCAGTGCT?CTGGTAGCTG?TAAAGCCAGG?GGCGGTAGCG?TGCATTAATA?CCTCTATTAA????1080
Orf1's is initial
TCAAACTGAG?AGCCGCTTAT?TTCACAGCAT?GCTCTGAAGT?AATATGGAAT?AAATTAA
GTG??1140
AAAATACTTG?TTACTGGTGG?CGCAGGATTT?ATTGGTTCTG?CTGTAGTTCG?TCACATTATA????1200
AATAATACGC?AGGATAGTGT?TGTTAATGTC?GATAAATTAA?CGTACGCCGG?AAACCTGGAA????1260
TCACTTGCTG?ATGTTTCTGA?TTCCGAACGC?TATGTTTTTG?AACATGCGGA?TATTTGCGAT????1320
GCAGCTGTAA?TGGCACGGAT?TTTTGCTCAG?CATCAGCCGG?ATGCAGTGAT?GCACCTGGCT????1380
GCTGAAAGCC?ATGTTGACCG?TTCAATTACA?GGTCCTGCGG?CATTTATTGA?AACCAATATT????1440
GTTGGTACTT?ATGTCCTTTT?GGAAGCCGCT?CGCAATTACT?GGTCTGCTCT?TGATGGCGAC????1500
AAGAAAAATA?GCTTCCGTTT?TCATCATATT?TCTACTGACG?AAGTCTATGG?TGATTTGCCT????1560
CATCCTGACG?AGGTAAATAA?TACAGAAGAA?TTACCCTTAT?TTACTGAGAC?AACAGCTTAC????1620
GCGCCAAGCA?GCCCTTATTC?CGCATCCAAA?GCATCCAGCG?ATCATTTAGT?CCGCGCGTGG????1680
AAACGTACCT?ATGGTTTACC?GACCATTGTG?ACTAATTGCT?CTAACAATTA?TGGTCCTTAT????1740
CATTTCCCGG?AAAAATTGAT?TCCATTGGTT?ATTCTCAATG?CTCTGGAAGG?TAAAGCATTA????1800
CCTATTTATG?GTAAAGGGGA?TCAAATTCGC?GACTGGTTGT?ATGTTGAAGA?TCATGCGCGT????1860
GCGTTATATA?CCGTCGTAAC?CGAAGGTAAA?GCGGATGAAA?CTTATAACAT?TGGTGGGCAC????1920
AACGAAAAGA?AAAACATTGA?TGTAGTGCTC?ACTATTTGTG?ATTTGTTGGA?TGAGATTGTC????1980
CCGAAAGAGA?AATCTTATCG?TGAGCAAATC?ACTTATGTTG?CCGATCGTCC?GGGACACGAT????2040
CGCCGTTATG?CGATTGATGC?TGAGAAGATT?GGTCGCGAAT?TGGGATGGAA?ACCACAGGAA????2100
ACGTTTGAGA?GTGGGATTCG?TAAAACGGTG?GAATGGTACC?TGTCCAATAC?AAAATGGGTT????2160
GATAATGTGA?AAAGTGGTGC?CTATCAATCG?TGGATTGAAC?AGAACTATGA?GGGCCGCCAG????2220
The termination orf2's of orf1 is initial
TAATGAATAT?CCTCCTTTTT?GGCAAAACAG?GGCAGGTAGG?TTGGGAACTA?CAGCGTGCTC???2280
TGGCACCTCT?GGGTAATTTG?ATTGCTCTTG?ATGTTCACTC?CACTGATTAC?TGTGGTGATT????2340
TTAGTAATCC?TGAAGGTGTC?GCTGAAACCG?TAAGAAGCAT?TCGGCCTGAT?ATTATTGTCA????2400
ACGCAGCCGC?TCACACCGCA?GTAGACAAAG?CAGAATCAGA?ACCAGAGTTT?GCACAATTAC????2460
TTAACGCGAC?GGGTGTCGAA?GCGATCGCGA?AAGCAGCCAA?TGAAGTCGGT?GCTTGGGTTA????2520
TTCACTACTC?TACTGACTAC?GTATTTCCGG?GGACTGGTGA?AATACCATGG?CAGGAGGCGG????2580
ATGCAACCGC?ACCGCTAAAT?GTTTACGGTG?AAACTAAGTT?AGCTGGAGAA?AAAGCATTAC????2640
AAGAGCATTG?TGCGAAGCAC?CTTATTTTCC?GGACCAGCTG?GGTCTATGCA?GGTAAAGGAA????2700
ATAACTTCGC?CAAAACAATG?TTGCGTCTGG?CAAAAGAGCG?TGAAGAATTA?GCCGTTATTA????2760
ATGATCAGTT?TGGTGCGCCA?ACTGGTGCAG?AGTTACTGGC?TGATTGTACG?GCACATGCCA????2820
TTCGTGTGGC?ACTGATTAAA?CCGGAAGTCG?CAGGCTTGTA?CCATCTGGTA?GCTAGTGGTA????2880
CCACAACGTG?GCACGATTAT?GCTGCGCTGG?TTTTTGAAGA?GGCACGCAAA?GCAGGCATTC????2940
CCCTTGCACT?CAACAAGCTC?AACGCAGTAC?CAACAACAGC?CTATCCTACA?CCAGCTCGTC????3000
GTCCACATAA?CTCTCGCCTT?AATACAGAAA?AATTTCAGCA?GAACTTTGCG?CTTGTCTTGC????3060
CTGACTGGCA?GGTTGGTGTG?AAACGAATGC?TTAACGAATT?ATTTACGACA?ACAGCAATT
T??3120
The termination of orf2
AATAGTTTTT?GCATCTTGTT?CGTGATGGTG?GAGCAAGATG?TATTAAAAGG?AATGATGAA
A?3180
Orf3's is initial
TGAAAACGCG?TAAAGGTATT?ATTTTGGCGG?GTGGTTCTGG?TACTCGTCTT?TATCCTGTGA???3240
CTATGGCAGT?CAGTAAACAG?CTGTTACCGA?TTTATGATAA?ACCGATGATC?TATTATCCGC????3300
TCTCTACACT?GATGTTGGCG?GGTATTCGCG?ATATTTTGAT?TATTAGTACG?CCACAGGATA????3360
CTCCTCGTTT?TCAACAACTG?CTGGGTGACG?GTAGCCAGTG?GGGGCTAAAT?CTTCAGTACA????3420
AAGTGCAACC?GAGTCCGGAT?GGTCTTGCGC?AGGCGTTTAT?TATCGGTGAA?GAGTTTATTG????3480
GTGGTGATGA?TTGTGCTTTG?GTACTTGGTG?ATAATATCTT?CTACGGTCAC?GACCTGCCTA????3540
AGTTAATGGA?TGCCGCTGTT?AACAAAGAAA?GTGGTGCAAC?GGTATTTGCC?TATCACGTTA????3600
ATGATCCTGA?ACGCTACGGT?GTCGTTGAGT?TTGATAAAAA?CGGTACGGCA?ATAAGCCTGG????3660
AAGAAAAACC?GTTACAACCA?AAAAGTAATT?ATGCGGTAAC?TGGGCTTTAT?TTCTATGATA????3720
ACGACGTTGT?CGAAATGGCG?AAAAACCTTA?AGCCTTCTGC?CCGTGGTGAA?CTAGAAATTA????3780
CCGATATTAA?CCGTATTTAT?ATGGAACAGG?GGCGTTTATC?TGTTGCCATG?ATGGGACGTG????3840
GTTATGCATG?GTTGGACACG?GGGACACATC?AAAGTCTTAT?TGAGGCAAGC?AATTTCATCG????3900
CGACAATAGA?AGAACGACAG?GGGCTGAAAG?TTTCCTGCCC?GGAAGAAATT?GCTTACCGTA????3960
AAGGGTTTAT?CGATGCTGAG?CAGGTGAAAG?TATTAGCTGA?ACCGTTGAAG?AAAAATGCTT????4020
The termination orf4's of orf3 is initial
ATGGCCAGTA?CCTGCTAAAA?ATGATTAAAG?GTTAT
TAATA?AA
ATGAACGT?AATTAAAACA4080
GAGATTCCAG?ACGTATTAAT?TTTCGAACCG?AAAGTTTTTG?GTGATGAGCG?TGGTTTCTTT????4140
ATGGAAAGCT?TTAATCAGAA?AGTTTTCGAA?GAAGCTGTAG?GACGTAAGGT?TGAATTTGTT????4200
CAGGATAACC?ATTCGAAGTC?TAGTAAAGGT?GTTTTACGCG?GGCTGCATTA?TCAGTTAGAA????4260
CCGTATGCGC?AAGGGAAATT?GGTACGTTGC?GCTGTTGGTG?AGGTTTTTGA?TGTAGCTGTT????4320
GATATTCGTA?AATCGTCGCC?TACCTTTGGT?AAATGGGTTG?GGGTGAATTT?ATCTGCTGAG????4380
AATAAACGGC?AATTGTGGAT?CCCTGAGGGA?TTTGCACATG?GTTTTTTAGT?GCTGAGTGAG????4440
ACTGCGGAAT?TTTTATATAA?AACGACGAAT?TATTATCATC?CTGAGAGTGA?TAGAGGGATT????4500
ATTTGGAATG?ATCCCAATAT?AGGTATTGTT?TGGCCTGAAA?GAGAAAATAT?AGTTTTATCG????4560
AAAAAGGATA?TAAATCAACC?TAATTTATCA?TATAGCTTCT?CTCTCAAACA?GTTGTTGTCG????4620
The termination orf5's of orf4 is initial
TAAGAGAAAA?GATCT
ATGTC?CCTTCTAAGC?AATGCAAAAT?GGGTAAGCTT?ATCTCAATTT?4680
GGACGTATTA?TTATACAATT?GATTAGCTTG?ACTGTTTTAA?CAAGATTTAT?ACCACCAGCA????4740
GATTATGGAT?TAATGGCTTT?AGGCACAGTT?GTTATGAATT?TTGTCTTCAT?AATACGTGAT????4800
CTTGGAACTG?CATCGGCCAT?AATACAAAGA?AAAGAAGTAG?ATGACGATAT?TTTAAATTCA????4860
GTTTTTTGGT?TGAATGTAAC?TATGGGAGTG?GGGTTATGCA?TACTAATTAT?ACTATGTACT????4920
CCACTAATTG?AGCTGTTATT?TAAGAATGAG?AAGTTAGATA?ATATTCTGTA?TTTGTTATGC????4980
TTTTGCTTTC?CATTAGCTAG?TATTGGTTCT?GCTCATCAGG?CATTAATGGA?AAGAAACTCT????5040
CATTTTAAAA?AAATAGCAAT?CATAGAATTG?CTCTCATCTC?TTTCTGGTTT?TGTTGTTGCA????5100
TTGCTAGTAG?CGATCAATAA?TGGAGGTGTT?TATGCATTAG?TATACCAATT?AATTTGTTCA????5160
TCATTTATTT?CTAGTATATT?ATTCTTTTAT?CTATCCTCTT?GGACGCCATC?CTTGCATTTT????5220
AAATTTGAGG?GGGTTAAAAA?AATTATAGGT?TTTAGTAGCA?GGTTAACTGT?TTTTAATTTA????5280
GTTAACTATT?TCTCACGTAA?TGCAGATGCT?ATGATAATAA?GTAGAAAGTA?TGATGCTAAT????5340
ATTCTCGGCG?CTTACTCTCT?AGCTTATAGA?ATTATGTTGT?TTCCGCTCCA?GAGTTTAACA????5400
TTTATTGCGT?CAAGATCACT?ATATCCAATA?TTAAGTAGAA?ACCAAGATGA?TTTGAAACTT????5460
ATTAGGGAGA?TATATTTAAA?AACAATATTT?ATAATTTCAA?TGATTACCAT?ACCATTAATG????5520
TTAGGATTAG?TATTTTTAAG?GACGCCGTTC?ATTACAATAG?CTTTAGGGCC?TAACTGGGCA????5580
ATAACTTCTA?ATATTTTACT?TTGGTTGGCA?CCAACTGGAA?TTATTCAGTC?AATTCTTAGT????5640
TGCTCCGGTA?GCATATTAAT?GTCAACTAAT?AACACGAAAA?CATTGATGTT?ATTAGGTATA????5700
TTTGGGGCCG?CGATACAAGT?ATTATCATTC?TTCATTGGTT?CTTTATTTAG?TATAAATATT????5760
TTTGTTTTTG?CTTACTTTAT?AGCAAATGTA?ATCAATGCGC?TAGTTGTTTT?ATATTTTACG????5820
TTTAAAAGTA?TAAAGCTTAA?TATTTATTCA?TTCTGTAAGA?TCCTACTGAT?CCCAACCTTA????5880
TCTTGTTCGG?TGATGATAAT?AGCGCTTCAT?TTCGTTAAAA?AAATCATTTA?CATTAATAAT????5940
GTGGTAAATG?ATGTGAGCTA?TTTAATAATT?CAGGTAATCA?TCGGTTGTAT?TGTATACTTT????6000
ATTGTTTTTT?ATATCTTCGT?AAATATATGT?CTTCGATTTA?AAATCATGTC?AGAAAATATG????6060
The termination of the initial orf5 of orf6
ATCCCAGATT?TTCTGTTGAG?AATAAAGTTC?TTTAGCCATA?AGAGATTTTT?AAA
TTGAATA??6120
AACAATTATT?TTTGATTGTT?GTTTATAATA?AGAAAATAGC?TGAGAGTTGT?ACTTTTAATA????6180
AGGTTGTTAA?CTCTAAGTTT?AGTGATGATT?CATTATGTGT?TATATGGAAC?AATGGACCCA????6240
CAAGTTGTGA?GGAGGAGTTT?TTGAATGTAT?ATGAGTATAA?TAAAAACATT?CGCTATCATG????6300
AAACATTGCA?TAATATCTCA?TTGGCCAAGA?TTTATAACGA?GGTAATTAAT?AAATATAACG????6360
CTCAGATTTA?TATCATCCTA?GATGATGATT?CTTCATTAAC?TGATGCATAT?TTTGAAGCTG????6420
TTAAACAAAT?ATCACCTAAT?GAAGTAGGTA?TGCCTGTCAT?ATATTATGAT?AGAAAAATAA????6480
TTAACCCATG?CATAAATGGT?CAGATTTATA?GTAAAGGAAT?TAATATTGAT?GCTAATGATA????6540
CCATAACAAC?TATCGGGAGT?GGATTGGTTG?TGGGTAGGGA?TGTAGCATCT?CAATTATTCA????6600
AAGCATATAA?TTCTGTATTT?GATGAACGTT?TTTATCTATA?TGGAGTAGAT?ACTACGTTTT????6660
GTTTCCGACT?TAATAAATTG?AAAATAAATG?ATAAAATTAA?GATTATAGAT?GGCTTTGAGC????6720
ACGGCCTTTC?AAGGCTTGAA?GGGAAAAATG?ATAAATTAAC?TCTTTTTAGA?AAAATAGAGA????6780
GAAGTAATGA?TCTCGCATTA?CAGATCCGAT?ATTATAAAGA?AAAAAAACAT?TGGTTTTTAA????6840
TGATGGTGCT?GTTACTCGCG?AGTTCAATTA?AGAAGTTTAT?TACTTTTCAG?CCACAACAAT????6900
The termination of the initial orf6 of orf7
ATAAATATTC?TACTATTATT?AAAACATTTG?TTAAGGGAAA?GC
ATGAACGT?CAGAGA
TAAT6960
GATAACGCAA?TCAAGTTATT?GGAGTTTGTT?TTAATTTTCT?CTTTTTTTAT?TATTGCATTA????7020
TTTTTCTTAT?ATTCCCCATC?TTATCTTATT?TACATTGTAG?TTAGTGCGTT?TGTTTTGATT????7080
AAGTTCAATA?ATAACTATAG?TTTTGGTTTG?TTTATATTAT?TGATAATGGT?ATGGCTGATT????7140
GTCAATTCTA?CTAATGTCAT?TTCATACCAA?AATTTTGTGC?TCGCGAGTGA?TGATTTCTCT????7200
TCTTATTATA?ATAACTATCT?TGGATTTCTT?CATTACGGGA?TAAATGAAGA?AAATCTTTTT????7260
GAATTTGGCT?ATTCAGAAAT?AATGTTGCCT?TTGTTGAACT?ATATTGCAAG?TTTAATTATT????7320
GGGCAACCAG?AACCATATAA?GTTAAAGTTT?CTGTATTCTG?TATTGCAAAG?CACTGGTGTA????7380
TTATATTGCT?GCGTAAAAAT?ATCTCGTCAC?TATAAACTTG?ATTTGAGTAA?TAGTATATTA????7440
TTAACAGCTG?CCGTATTGGT?GTTTTATAAG?CCTTTAGTAG?GTATTCAACT?ATCTAGACAA????7500
ATGTTTAGCT?CTATTTTTAT?TATAGCTTTT?ATTTTCACGA?ATAATAAATT?TGAGCGAATT????7560
ATCTTTTCAT?TAATGGCCTT?TTGTTTTCAT?TCATCAGCGA?TAATATTATT?ACCAATAACG????7620
CAATATCTTC?TGACAAAAAG?GAAAAGAAAG?GACTTCTATT?ATAGTTTAAT?TATCGTTCTG????7680
CTTGCATATG?CTGCATTTAT?TGTTGTGTTT?ACATCGTTTA?AAAATGAATT?AATAAATATT????7740
CCTATTTTAA?ATAAATTAGA?CTACACTTTT?CGTATGTTTA?ATGAACCAGA?AAAAATAAAG????7800
CAATCTGTAT?TTACATCTTT?TCTCTTGTTA?ATATATTTAA?TCCCTGCGCT?GATTTTTTCC????7860
ATCTTAAATA?AAAAATCTGA?TGGATTTAAA?TATAATTTAG?CTATCATTTG?TGTATTTTTA????7920
ATATCTTTTT?GTATTGTACC?TGGTTTCAAT?AACAGATTAT?TCTTTGCTGA?GTTATATGTC????7980
TTTCTTGGTT?TTTTTTATTA?TATGGTGTTT?TTTTACAATA?GAGTGAATGC?AAGAGAGCAT????8040
GTATCGAGAT?TAATCATCTT?TATTCTTTTT?ACTATTCTAT?TTTTTCAATC?TCAATTTTCA????8100
TCAGAAAATA?GGCAGTTTGA?TAGCTTTGAA?CTAGAACCTT?TCTATTATCT?TAAATATTTA????8160
The termination orf8's of orf7 is initial
GAATATAGAG?AGACAGAAAT?AAACAGAGGA?CTATTAGAG
T?AAC
ATGCCTA?ATTTAGTTAT8220
TTATACTGCT?GTTACTGGGC?ATTATGATGA?ACTGGGGAAT?GTTTATGAGG?AACCTTCAGT????8280
TGATTATATC?TGCTTTACAG?ACTACAACTT?TACCGGTACG?GTACCTAAAC?CGTGGAAGCA????8340
AATTAAATTG?CCGCCTTCAA?ATTTCTCTGA?TAAAGACTTA?GCAAGATATT?GTAAGCTTAA????8400
TCCTCATGTC?CTATTGCCTC?AGTATCAGCA?TTCGTTGTGG?ATTGATGGCA?ATATTCGAAT????8460
AAAAGGTAAA?ATACGAAATT?TTATTATTGA?CATTTTGAGC?AAGCATCGAA?TTGCAGCATA????8520
TGAACATTGG?TGGCGAGACA?AAACAGAACA?GGAATTTCAT?GAATGTGCTA?GAAGTGGTTT????8580
CGATCCTGCT?TGGAAATTAT?ATAAACAAAT?TGAGCGCTAT?AAGCATGAAG?GATACACCTC????8640
CTCAGATTTT?TTTGAGAATA?ATATACTCAT?GCGTAACCAT?ATGGAAAGTG?ACATAATCAA????8700
AATGCATGAA?ATATGGTGGG?GGGAGTATAT?TTCAGGAGGT?AAGCGTGATC?AATATTCTTT????8760
TACTTATGCT?GCTTATAAAA?GTGATGTAAA?TATATATTCA?TTAGGGGTTC?ATGATGCTAG????8820
AGTTATAAAA?CAATATTTTG?ACTATGTGAC?ACACTCCAAG?AAAAGGCCGT?TAAAACAATA????8880
TATCTGGATT?ATAATAAATA?GAATCTATAT?TTCTCTAACG?AGATGGAGCG?TAAATGAACC????8940
The termination of the initial orf8 of orf9
ATCTCGGACA?AATAAATTAG?TGCTAAATA
A?TGAAAGAGAT?TCAAATGAAA?AA
TAAAGTTG9000
CTGGAGTCAT?TGTTACATAT?AATCCAGAAA?TTAAAATACT?TCTCAGTGCA?TTGGAGTCAT????9060
CGACAAAACA?GTTAGACTAT?ATTTATTTGA?TAGATAATTG?CTCATTAAAT?AAAAATGAGG????9120
TGGCTGATTT?AGCTGGAAGA?TTTGGGAATG?TAAAATTTCA?GGCGCTCTCT?AAAAATATTG????9180
GATTAGCCAG?CGCCCAAAAC?ATGGGCATCA?ATACTGCTAA?AAACAATAAC?CTAATAGAAT????9240
ATGTAGTTTT?ATTCGATCAG?GATAGCATAA?TAGAGAACAA?TTTTATTGCT?TCATTACTTG????9300
ATGATTATAA?AAAAGTTTTA?GCAAAGGATT?CTAAGATAGG?AGCTATAGGC?CCGGTTTTTT????9360
ATGATCCATC?TACTAAAATT?AATTATCCTG?CAACAGTATA?TATAGGTCCT?TTTATAAAAA????9420
AAATATCTAT?AAAGGATGAG?CCACAATTCG?CAACGTATTT?AATTGCCTCT?GGCTGTTTAA????9480
TAAAAATAGA?TACTTTGTCT?TCGGTTGGAT?TAATGCAGGA?TGATTTGTTC?ATCGATTACA????9540
TCGATGTTGA?ATGGAGTTTA?AGAGCAAAGA?GTAAAGGTTA?TAATTTATAT?ATATCACCAA?????9600
ACGCAAAAAT?GGCCCATACA?ATTGGAGACG?CGAGGCGTAA?ATTTTTGGGG?CGGACAATAT?????9660
CTATTCACTC?CCCTTTTAGA?CGTTACTTCT?TGGTTAGAAA?TAGTTTTTAC?ATGATAAGGT?????9720
TAAATTATAT?TCCTTTAGGG?TACAAGACTA?GAGAGTGTAT?TTTTAATATT?GCCAGGATAT?????9780
TAACATCTAT?CATATATTCC?GATCAAAAAA?TTAAAACAAT?AAAATCGGCT?TTTTATGGAG?????9840
The termination of orf9
TCATTGATGG?AATTGCAGGT?CGATTTGGCC?CTCTAAAACG?TAAAATA
TAA?AGAGAAATTT???9900
Orf10's is initial
C
ATGAAAATT?TTATATATAA?TCACAAAAGC?TGATGAAATC?GGTGGGGCAC?AAATTCATGT???9960
GAGAGACCTT?GCTCGTCGTT?TACACAATGA?TGGTAATAAA?GTTACAGTAA?TCGTAGGAGA????10020
GGATGGCGCT?TTAGTTAGAC?AACTTAAAGA?GCTTCACATT?TCGGTGCGAA?TAGTAGAAAA????10080
TTTAGTTAGG?CAAATTTCAC?CACTAAATGA?TATAAAAGCC?ATTTTTAACA?TCAGAAAGAT????10140
AATTAAAGAT?AATTCACCGG?ATATTATTGG?TCTTCACTCC?TCTAAGGCTG?GAATTGTTGG????10200
TCGACTAGCA?GCTTGTCGAT?TAAATATTCC?AGTAGTATTT?ACCGCTCACG?GTTGGGCATT????10260
TGCAGAGGGT?ATCAGTGACA?AAAAAAGGAA?ATTATTTATT?TTCATTGAGA?AAATACTATC????10320
TCCTTTTTTG?GATAAGATAA?TTACTGTTTC?GGAGCAAGAT?AAAAAGTTGG?CGCTAGATTT????10380
AGGCGTTGCA?TCCTCTGAAA?AACAAGTCGT?TATTCATAAC?GGAATGCCTG?ATATTACTAT????10440
TAAGCGTAAG?GATTATTCGG?ATTCAACTAA?AGTTCGATTA?ATCAGTGTAG?CTCGTTTTTC????10500
AGAGCAAAAA?GATCATGAAA?CTTTATTTCA?CGCACTCAAG?TTAATTTCTA?CAGATAACTG????10560
GACACTAACT?CTGGTTGGGA?AAGGTCCTAA?GTTAGAAAAT?ATTAAAAAAC?TTGCATCTGA????10620
TTTGAATATT?GCAAATAATA?TTCTCTTTCT?TGGGGAAAGA?GATGATGTCG?ATAATTTATT????10680
AAGTGAAAGT?GATGTTTTCT?GTTTAATAAC?AAACTGGGAG?GGATTGCCAT?TAAGTATTAT????10740
CGAGGCTATG?CGTGCGTCCT?TGCCAGTGGT?AGCTACAGAT?GTTGGAGGTG?TCTCTGAGTT????10800
AGTAGAGCAT?AAAAAAACCG?GCTTGCTTCA?TTCTCCTAAA?AACATACAAG?AACTTGCTAA????10860
TATCCTGGAT?ACTATTATCA?ATGATAGTGA?CTTGAGATAT?AAATTGGGAA?ATGCTGGAAG????10920
AGTTAAATAC?CAAAATAATT?TCACATTTGA?ACATATGTAT?TATGCAACAA?TAGATGAATA????10980
The termination of orf10
TAATAAATTG?ATACATACTA?AA
TAGTTAAT?TATATTTTTC?TTGTGTTCGC?TCTTTTCTAA??11040
CAGTTTTAAT?TTCTTTAATT?TTAAGAGATA?AATATATTAG?ACAAGTTATT?CCAATATGAG????11100
TAAATTGGCA?TAACTTGTAA?CTTTAGACCG?CAACCCGCAG?TAACCCCTGA?CAGGAGTAAA????11160
TAATGTCAAA?GCAACAGATC?GGCGTCGTCG?GTATGGCAGT?GATGGGGCGC?AACCTTGCGC????11220
TCAATATCGA?AAGCCGTGGT?TATACCGTCT?CTATTTTCAA?CCGTTCCCGT?GAAAAGACCG????11280
AAGAAGTGAT?TGCCGAAAAT?CCAGGCAAGA?AACTGGTTCC?TTACTATACG?GTGAAAGAGT????11340
TTGTTGAATC?TCTGGAAACG?CCTCGTCGCA?TCCTGTTAAT?GGTGAAAGCA?GGTGCAGGCA????11400
CGGATGCTGC?TATTGATTCC?CTCAAGCCAT?ACCTCGATAA?AGGTGACATC?ATCATTGATG????11460
GTGGTAATAC?CTTCTTCCAG?GACACCATTC?GTCGTAACCG?TGAGCTTTCT?GCCGAAGGCT????11520
TTAACTTCAT?TGGTACCGGT?GTCTCCGGTG?GTGAAGAAGG?CGCGCTGAAA?GGTCCTTCTA????11580
TTATGCCTGG?TGGGCAGAAA?GAAGCCTATG?AACTGGTTGC?GCCGATCCTG?ACCAAAATCG????11640
CCGCAGTGGC?TGAAGACGGT?GAGCCATGTG?TTACCTATAT?TGGTGCCGAT?GGCGCAGGTC????11700
ACTATGTGAA?GATGGTTCAT?AACGGTATTG?AATACGGCGA?TATGCAGCTG?ATTGCTGAAG????11760
CCTATTCTCT?ACTTAAAGGT?GGTCTGAACC?TCACCAACGA?AGAACTGGCG?CAGACCTTTA????11820
CCGAGTGGAA?TAACGGTGAA?CTGAGCAGCT?ACCTGATCGA?CATTACCAAA?GACATCTTCA????11880
CCAAAAAAGA?TGAAGACGGT?AACTACCTGG?TTGATGTGAT?CCTGGATGAA?GCGGCTAACA????11940
AAGGTACCGG?TAAATGGACC?AGCCAGAGCG?CGCTGGATCT?CGGCGAACCG?CTGTCGCTGA????12000
TTACCGAGTC?TGTGTTTGCA?CGTTATATCT?CGTCTCTGAA?AGATCAGCGC?GTTGCCGCGT????12060
CTAAAGTTCT?CTCTGGCCCG?CAAGCGCAGC?CAGTAGGCGA?CAAAGCTGAG?TTTATCGAGA????12120
AAGTTCGTCG?TGCGCTGTAT?CTGGGCAAAA?TCGTTTCTTA?CGCTCAGGGC?TTCTCTCAGC????12180
TGCGTGCGGC?GTCTGAAGAG?TACAACTGGG?ATCTGAACTA?CGGCGAAATA?GCGAAGATTT????12240
TCCGTGCTGG?CTGCATCATT?CGTGCGCAGT?TCCTGCAGAA?AATCACCGAT?GCTTATGCCG????12300
AAAATCCGCA?GATCGCTAAC?CTGCTGCTGG?CTCCTTACTT?CAAGCAAATT?GCCGATGACT????12360
ACCAGCAGGC?GCTGCGCGAT?GTCGTCGCTT?ATGCGGTACA?GAACGGTATC?CCGGTTCCGA????12420
CCTTCGCCGC?TGCGGTTGCC?TATTATGACA?GCTACCGTGC?CGCTGTTCTG?CCTGCGAACC????12480
TAATC????????????????????????????????????????????????????????????????12485
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 O120 type, it is characterized in that: it is the isolating Nucleotide shown in SEQ ID NO:1,12485 bases of total length; The base that perhaps has 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 O120 type of claim 1, it is characterized in that: it comprises called after rmlB, rmlD, rmlA, rmlC, wzx, orf6, wzy, orf8, orf9,10 genomic constitutions of orf10 are all between galF gene and gnd gene.
3, according to the Nucleotide of the described O-antigen-specific to intestinal bacteria O120 type of claim 2, it is characterized in that the gene that has high degree of specificity in the described gene is: the gene of transhipment enzyme comprises the wzx gene or with wzx the gene of identity function is arranged; Pol gene comprises the wzy gene or with wzy the gene of identity function is arranged; Glycosyltransferase gene comprises orf6, orf9, orf10 gene; Wherein said gene: wzx is the Nucleotide of 4636 to 6117 bases among the SEQ ID NO:1; Wzy is the Nucleotide of 6943 to 8202 bases among the SEQ ID NO:1; Orf6 is the Nucleotide of 6114 to 6959 bases among the SEQ ID NO:1; Orf9 is the Nucleotide of 8970 to 9890 bases among the SEQ ID NO:1; Orf10 is the Nucleotide of 9902 to 11005 bases among the SEQ ID NO:1.
4, according to the Nucleotide of claim 1 or 2 described O-antigen-specifics to intestinal bacteria O120 type, it is characterized in that: it also comprises and comes from described wzx gene, wzy gene, glf gene or glycosyltransferase gene orf6, orf9, orf10 gene; And their mixing or their reorganization.
5, according to the Nucleotide of the described O-antigen-specific to intestinal bacteria O120 type of claim 4, it is characterized in that the oligonucleotide of the described wzx of coming from gene is to being: the Nucleotide of 4729 to 4744 bases among the SEQ ID NO:1 and the Nucleotide of 5119 to 5134 bases; The Nucleotide of 5575 to 5590 bases among the SEQ ID NO:1 and the Nucleotide of 5939 to 5955 bases; The oligonucleotide that comes from the wzy gene is to being: the Nucleotide of 7123 to 7139 bases among the SEQ ID NO:1 and the Nucleotide of 7582 to 7598 bases; The Nucleotide of 7064 to 7079 bases among the SEQ ID NO:1 and the Nucleotide of 7770 to 7787 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 O120 type of claim 1.
7, the recombinant molecule of the Nucleotide of the described O-antigen-specific to intestinal bacteria O120 type of claim 1 is providing the O-antigen of expressing intestinal bacteria O120 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 O120 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 bacterial detection.
9, the separation method of the Nucleotide of the described O-antigen-specific to intestinal bacteria O120 type of claim 1 is characterized in that it comprises the steps:
(1) genomic extraction: in substratum, cultivate intestinal bacteria O120 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 O120 type bunch: with the genome of intestinal bacteria O120 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 O120 type;
(6) screening of specific gene: at wzx, the wzy gene design primer in the O-antigen gene of intestinal bacteria O120 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 O120 type;
(7) detection of primer sensitivity: cultivate intestinal bacteria O120, 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 O120.
10, the separation method of the Nucleotide of the described O-antigen-specific to intestinal bacteria O120 type of claim 9 is characterized in that it comprises the steps:
(1) genomic extraction: 37 ℃ of incubated overnight intestinal bacteria O120 types in the LB of 5mL substratum, centrifugal collecting cell.With 500ul 50mM Tris-HCl (pH8.0) and 10ul 0.4M EDTA re-suspended cell, 37 ℃ of incubations 20 minutes, the N,O-Diacetylmuramidase that adds 10ul 10mg/ml then continues insulation 20 minutes, the Proteinase K, the 15ul 10%SDS that add 3ul 20mg/ml afterwards, 50 ℃ of incubations 2 hours, the RNase that adds 3ul 10mg/ml again, 65 ℃ of incubations 30 minutes.Add equal-volume phenol extracting mixture, get supernatant and use isopyknic phenol again: chloroform: twice of primary isoamyl alcohol (25: 24: 1) mixing solutions extracting, 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, DNA is resuspended among the 30ul TE with 70% ethanol; Genomic dna detects by 0.4% agarose gel electrophoresis;
(2) by the O-antigen gene in the pcr amplification intestinal bacteria O120 type bunch: with the genome of intestinal bacteria O120 type is that template is passed through its O-antigen gene of Long pcr amplification bunch, at first according to the galF sequences Design upstream primer #1523-ATT GTG GCTGCA GGG ATC AAA GAA AT that often is found in O-antigen gene bunch promoter region, again according to the gnd gene design downstream primer #1524-TAG TCG CGT GNG CCT GGA TTA AGT TCG C in O-antigen gene bunch downstream; With the Expand Long Template PCR method of BoehringerMannheim company amplification O-antigen gene bunch, the PCR response procedures was as follows: 94 ℃ of pre-sex change 2 minutes; 94 ℃ of sex change are 10 seconds then, annealed 15 seconds for 60 ℃, 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 PCR Preps purification kit purified pcr product of Promega company;
(3) make up O-antigen gene bunch library: make up O-antigen gene bunch library with the Novagen DNaseI shot gun method that is modified, reaction system is a 300ng PCR purified product, 0.9ul 0.1M MnCl
2The DNaseI of the 1mg/ml of 1ul dilution in 1: 2000, reaction is carried out at room temperature, enzyme is cut the dna fragmentation size is concentrated between the 1.5kb-3kb, then add 2ul 0.1M EDTA termination reaction, 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 18ul water, in this mixture, add 2.5ul dNTP (1mMdCTP subsequently, 1mMdGTP, 1mMdTTP, 10mMdATP), 1.25ul the T4DNA polysaccharase of 100mMDTT 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 80ul, 70 ℃ of reactions 20 minutes make 3 of DNA ' end add the dA tail, and 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 90ul, and the 10 * buffer of 9ul and the T4DNA ligase enzyme of 25 units are wherein arranged.Use the dehydrated alcohol precipitation of the 3M NaAc (pH5.2) of 1/10 volume and 2 times of volumes to be connected mixture at last, wash precipitation with 70% ethanol again, be dissolved in after the drying and obtain connecting product in the 30ul water; Preparation method with the electric transformed competence colibacillus cell of Bi0-Rad company prepares the competence e.colidh5, get after 2-3ul connects product and 50ul competence bacillus coli DH 5 alpha mixes, forward in the electric shock cup of 0.2cm of Bi0-Rad company and shock by electricity, voltage is 2.5 kilovolts, time is 5.0 milliseconds to 6.0 milliseconds, the SOC substratum that adds 1ml after the electric shock immediately in cup makes the bacterium recovery, then bacterium is coated in and contains penbritin, on the LB solid medium of X-Gal and IPTG, 37 ℃ of incubated overnight, obtain blue white bacterium colony next day, with the white colony that obtains promptly the white clone forward on the LB solid medium that contains acillin and cultivate, from each clone, extract plasmid simultaneously, and cutting the segmental size of evaluation insertion wherein with the EcoRI enzyme, the white that obtains clone group has constituted the O-antigen gene bunch library of intestinal bacteria O120 type;
(4) to the cloning and sequencing in the library: from the library, select 96 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 O120 type; The quality of sequence is mainly guaranteed by two aspects: 1) genome of intestinal bacteria O120 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 O120 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 10 openings, determine also that with the function of finding the reading frame that these are open what gene they are with the genetic comparison among the blast groupware and the GenBank, finish gene annotation with the Artemis software at Britain sanger center again, do accurate comparison between DNA and protein sequence with Clustral W software, obtain the structure of the O-antigen gene bunch of intestinal bacteria O120 type at last;
(6) screening of specific gene: at wzx, the wzy gene design primer in the O-antigen gene of intestinal bacteria O120 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 the 13rd group of intestinal bacteria O120, 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 O120 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 O120 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 4729 to 4744 bases among the SEQ ID NO:1 and the Nucleotide of 5119 to 5134 bases; The Nucleotide of 5575 to 5590 bases among the SEQ ID NO:1 and the Nucleotide of 5939 to 5955 bases.The Nucleotide of 7123 to 7139 bases among the SEQ ID NO:1 and the Nucleotide of 7582 to 7598 bases; The Nucleotide of 7064 to 7079 bases among the SEQ ID NO:1 and the Nucleotide of 7770 to 7787 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 O120 in the pork filling when using aforesaid method.
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