CN108753998A - The application of flagellum two level controlling gene fliA or its expression product as target spot in antibacterials exploitation - Google Patents

The application of flagellum two level controlling gene fliA or its expression product as target spot in antibacterials exploitation Download PDF

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CN108753998A
CN108753998A CN201810501616.5A CN201810501616A CN108753998A CN 108753998 A CN108753998 A CN 108753998A CN 201810501616 A CN201810501616 A CN 201810501616A CN 108753998 A CN108753998 A CN 108753998A
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flia
flagellum
escherichia coli
stm
antibacterials
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CN108753998B (en
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余旭平
王明晓
余子豪
刘金泽
李统战
张远星
李倩文
刘蕾
刘云惠
相国
李永霞
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Zhejiang University ZJU
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G01N33/56916—Enterobacteria, e.g. shigella, salmonella, klebsiella, serratia

Abstract

Application the invention discloses flagellum two level controlling gene fliA or its expression product as target spot in antibacterials exploitation.Present invention research is found, the flagellum two level controlling gene fliA of salmonella or Escherichia coli is overexpressed in Escherichia coli, it being capable of lethal Escherichia coli, namely the product FliA albumen of flagellum two level controlling gene fliA expression general activity it is excessively high being capable of lethal Escherichia coli, illustrate the target spot that the gene or its expression product can be developed as antibacterials, for solve bacterial resistance sex chromosome mosaicism provider to.

Description

Flagellum two level controlling gene fliA or its expression product are opened as target spot in antibacterials Application in hair
Technical field
The present invention relates to biotechnologies, more particularly to flagellum two level controlling gene fliA or its expression product conduct Application of the target spot in antibacterials exploitation.
Background technology
Flagellum is the important locomotive organ of bacterium, it pushes bacterium to tend to environment full of nutrition, and is fled from containing harmful The evil of substance omits environment, goes after profits and advoids disadvantages.Many bacteriums are containing amphitrichous, including Escherichia coli, salmonella, proteus etc..
Complete bacterial flagellum (flagellum) structure includes matrix (basal body), flagellum hook (hook) and flagellum Silk (helical filament) three parts, from inner membrance cross over cell wall and outer membrane, extend to it is extracellular (Evans L.D.B, Hughes C and Fraser G.M.Building a flagellum outside the bacterial cell.Trends in Microbiology.2014,22(10):566-572.).Wherein, matrix by center flagellum bar (rod) and it is multiple be embedded in cell membrane, cell wall cyclic structure composition.A part of structure of C rings is protruded inwardly from endochylema, This enclose cave in there are one flagellum type III secernent (flagellar type III secretion system, FT3SS), it is responsible for the secretion and self assembly of flagellin.
The gene cluster of coding flagellum is located at 4 different locations of bacterial genomes, including a gene more than 50, at least by 14 Operon forms (Kalir S, McClure J, Pabbaraju K et al.Ordering genes in a flagella pathway by analysis of expression kinetics from living bacteria.Science 2001, 292(5524):2080–2083.).Three-level ladder (three classes are presented in the expression of bacterial flagellum gene cluster Hierarchical order) (see Fig. 1).Wherein flhD the and flhC genes of first order ladder flhDC operons are flagellum table The master regulation gene reached controls the expression of the other flagellin genes in downstream, regulating and expressing intensity, the final flagellum for determining bacterium surface Length, quantity and motoricity.FlhDC operons are regulated and controled by many environmental factors, such as：Environment temperature, pH value, osmotic pressure, oxygen The various environmental factors such as gas (Fahrner K.A, Berg H.C.Mutations that stimulate flhDC expression in Escherichia coli K-12.J.Bacteriol.2015,197(19):3087-96.)。flhDC Gene expression product forms FlhD4C2Flagellum master regulation albumen, FlhD4C2It is combined, is activated with the gene promoter of second level ladder II grade of gene opens the expression of matrix-flagellum sheath (hook-basal body, HBB) gene group and the assembling of HBB, same with this When, FlhD4C2Also open the σ expressed needed for III grade of promoter expression28The factor (FliA) and its antagonism factor (FlgM), Before HBB is completed, due to the presence of FlgM, FliA can not be combined with the promoter of third ladder gene, and III grade of gene is temporary It does not express, when HBB is completed, antagonism factor FlgM is secreted into extracellular, and antagonism is eliminated, III grade of later on expression of gene, Flagellum starts to assemble and be finally completed, and bacterium obtains movement and chemotactic ability.
Most of salmonella amphitrichous have motoricity, except white diarrhea and avian infectious bronchitis nephritis virus.But Chaubal and Holt (1.Holt P.S and Chaubal L.H.Detection of motility and putative synthesis of flagellar protein in Salmonella pullorum culture.J.Clin.Micro.1997,35:1016-1020.2.Chaubal L.H and Holt P.S.Characterization of swimming motility and identification of flagellar proteins in Salmonella pullorum isolates.Am.J.Vet.Res.1999,60:1322-1327.) once report There is flagellum activity under specific circumstances in road, S. pullonum.
It is the main policies that the mankind control bacteriosis to kill or inhibit bacterial growth, however as the wide of antibacterials General use, especially a large amount of abuses, bacterial resistance sex chromosome mosaicism getting worse, therefore it is bacillary to control to be badly in need of searching newtype drug Disease.
Invention content
The present invention is directed to the deficiencies in the prior art, provides flagellum two level controlling gene fliA or its expression product Application of the FliA albumen as target spot in antibacterials exploitation.
The present invention pass through experimental studies have found that, by the flagellum two level controlling gene fliA of salmonella or Escherichia coli big It is overexpressed in enterobacteria, is capable of the product FliA eggs of lethal Escherichia coli namely whip two level controlling gene fliA expression White general activity it is excessively high can lethal Escherichia coli, illustrate what the gene or its expression product can be developed as antibacterials Target spot finds a kind of flagellum two level controlling gene fliA that can make in bacterium and is overexpressed or enhances its expression product FliA Protein active, when fliA gene overexpressions, the amount of product FliA albumen increases, and general activity is excessively high；And if can enhance FliA protein actives, then in the case where the Tot Prot is constant, general activity also increases, and inhibits bacterium to just reach Growth or the purpose for killing bacterium.
A kind of action target spot of antibacterials, the target spot are flagellum two level controlling gene fliA or its expression product FliA Albumen.
Invention further provides flagellum two level controlling gene fliA or its expression product FliA albumen as target spot in antibacterial Application in drug development.
The antibacterials can raise the expression of flagellum two level controlling gene fliA or enhance the activity of FliA albumen.
The antibacterials are for killing or inhibiting with the active bacterium of flagellum.Preferably, the antibacterials are used for Kill or inhibit salmonella or Escherichia coli.
The present invention also provides a kind of antibacterials, can raise tables of the flagellum two level controlling gene fliA in bacterium It reaches, or enhances the activity of FliA albumen.The bacterium has flagellum activity.Preferably, the bacterium is salmonella or big Enterobacteria.
The present invention is it has been investigated that flagellum two level controlling gene fliA is overexpressed the lethal host strain of meeting, for novel antibacterial drug Research and development provide new target spot, for solve bacterial resistance sex chromosome mosaicism provider to.
Description of the drawings
Fig. 1 is that the three-level of flagellin gene regulates and controls schematic diagram.
Fig. 2 is the flhDC genetic fragment electrophoresis detection result figures of PCR amplification, wherein swimming lane M is standard molecular weight Marker DL2000 (similarly hereinafter), swimming lane 1 are flhDCSTM, swimming lane 2 is flhDCEcoli, swimming lane 3 is flhCSTM, swimming lane 4 is flhCEcoli, swimming lane 5 is flhDSTM, swimming lane 6 is flhDEcoli。
Fig. 3 is other flagellin gene fragment electrophoretic testing result figures of PCR amplification, wherein swimming lane 1~9 is respectively fliAEcoli、fliASTM, flgK, fliI, flgM, fliT, fliZ, fliC, motA, clip size be respectively 795bp, 747bp, 1702bp, 1401bp, 353bp, 443bp, 610bp, 1223bp and 848bp.
Fig. 4 is pN15E6-flhDCSTMThe experimental result picture of lethal Escherichia coli DH31soplacI, wherein A are Kan+Chl Tablet, B are Kan+Chl+IPTG tablets.
Fig. 5 is pN15E6-flhDSTMThe experimental result picture of not lethal Escherichia coli DH31soplacI, wherein A are Kan+ Chl tablets, B are Kan+Chl+IPTG tablets.
Fig. 6 is containing pN15E6-flhDCSTMEscherichia coli DH31soplacI strain growth curve determination result figures.
Fig. 7 is pN15E6-flhDCEcoliIt is Kan+ to inhibit the experimental result picture of Escherichia coli DH31soplacI, wherein A Chl tablets, B are Kan+Chl+IPTG tablets.
Fig. 8 is pN15E6-flhDEcoliThe experimental result picture of not lethal Escherichia coli DH31soplacI, wherein A are Kan+ Chl tablets, B are Kan+Chl+IPTG tablets.
Fig. 9 is fliASTMIt is overexpressed the experimental result picture of lethal Escherichia coli DH31soplacI, wherein A is flat for Kan+Chl Plate, B are Kan+Chl+IPTG tablets.
Figure 10 is fliAEcoliIt is overexpressed the experimental result picture of lethal Escherichia coli DH31soplacI, wherein A is Kan+Chl Tablet, B are Kan+Chl+IPTG tablets.
Specific implementation mode
Main agents：NcoI enzymes, II enzymes of Bgl, T4DNA ligases are purchased from New England Biolabs companies； DL2000marker, DL5000marker, Taq archaeal dna polymerase, ExTaq archaeal dna polymerases, which are purchased from precious bioengineering (Dalian), to be had Limit company (TaKaRa companies)；Bacterial plasmid rapid extraction kit, PCR cleaning agents boxes are purchased from purchased from Axygen companies； IPTG is purchased from Shanghai Sheng Gong bioengineering limited liability company.
Plasmid and bacterial strain：542 bacterial strains of S.Typhimurium STM are purchased from China Veterinary Drugs Supervisory Inst.；DH31soplacI bacterium Strain, pN15E6 plasmids by Russian scientist Ravin NV (Andrey V.Mardanov, Taisia S.Strakhova, Vladimir A.Smagin,Nikolai V.Ravin.Tightly regulated,high-level expression from controlled copy number vectors based on the replicon of temperate phage N15 [J] .Gene 395 (2007) 15-21.) team's present；JM109 bacterial strains are preserved by this laboratory.
PKDb plasmids are by pKD46 plasmids (Datsenko KA&Wanner BL.One-step inactivation of chromosomal genes in Escherichia coli K-12using PCR products.PNAS(2000)97 (12):6640-45.) skeleton is built by laboratory and is preserved from the plasmid for connecting and increasing by 2 XcmI restriction enzyme sites, plasmid. PKDb plasmids form the carrier T that linear and two head process go out 1 T after XcmI digestions, can be with Direct Cloning end 1 A's of protrusion PCR product.
Embodiment 1
The amplification of a variety of flagellin gene segments and flhD, flhC tetraploid rice.
Using JM109, STM542 genomic DNA as template, flhDC, flhD of PCR amplification Escherichia coli and salmonella, FlhC, fliA genetic fragment, using STM542 genomic DNAs template, PCR amplification fliT, fliZ, flgM, flgK, fliI, FliC, motA genetic fragment.Amplification the primer and characteristic are shown in Table 1, and primer synthesis and sequencing commission Hua Da biotechnology are limited Company completes.
The amplification of 1 target gene fragment of table
Note：The base for marking underscore is the recognition site of NcoI or Bgl II, and the base before recognition site is protection alkali Base.
Utilize primer shown in table 1, PCR amplification target gene fragment：flhDCSTM、flhDCEcoli、flhCSTM、flhCEcoli、 flhDSTM、flhDEcoli、fliAEcoli、fliASTM,flgK,fliI,flgM,fliT,fliZ,fliC,motA.Wherein, flhDCSTM、flhDCEcoli、flhCSTM、flhCEcoli、flhDSTM、flhDEcoliGenetic fragment size be respectively 973bp, 973bp, 579bp, 579bp, 351bp, 351bp are shown in Fig. 2；fliAEcoli、fliASTM、flgK、fliI、flgM、fliT、fliZ、fliC、 MotA genetic fragment sizes be respectively 795bp, 747bp, 1702bp, 1401bp, 353bp, 443bp, 610bp, 1223bp, 848bp is shown in Fig. 3.
With reference to 5 plants of Bacterium enteritidis (Salmonella enterica) genome sequences in GenBank： S.Typhimurium LT2、S.Gallinarum 287/91、S.Typhi CT18、S.Choleraesuis SC-B67、 S.Enteritidis P125109；And 3 plants of Escherichia coli (Escherichia coli) genome sequences：MG1655, W3110, DH10B, compare respectively 5 plants of salmonellas, 3 plants of Escherichia coli flhD, flhC sequences derivation amino acid, it is same to calculate it Source property.
The results show that the derivation amino acid of 5 plants of Bacterium enteritidis flhD is identical, the derivation amino acid homology of flhC Property be 98.96%~99.83%.The derivation amino acid identity of 3 plants of Escherichia coli flhD, flhC are 97% or more.And it is husky The derivation amino acid of differences of flhD, flhC between door Salmonella and Escherichia coli are larger, and homology is respectively 74.34%~ 75.78%, 76.51%~81.69%.It, need to be into the result shows that flhDC genes are variant between salmonella and Escherichia coli One step demonstrate,proves adjusting function of the salmonella flhDC genes in e. coli host bacteria.
Embodiment 2
Flagellum master regulation gene flhDCSTMIt is overexpressed the influence to Escherichia coli DH31soplacI Strain survivals.
(1) structure of pN15E6 expression vectors and identification：
With NcoI and II double digestion PCR target gene fragments of Bgl and pN15E6 plasmids, the two is connected into structure recombinant expression Plasmid.Using pN15-insChKUp3 and pN15-insChKDn2 as primer (table 1), PCR identifies recombinant expression plasmid.
(2)flhDCSTMIt is overexpressed lethal Escherichia coli DH31soplacI：
By the pN15E6 recombinant plasmid transformed DH31soplacI bacterial strains of different flagellin genes, chooses monoclonal and is incubated overnight, Take sterile LB liquid dilutings overnight culture 106Times, it is coated on that Double LB agar plate of chloramphenicol (Kan+Chl) of card and contains The Double LB agar plates of Kan+Chl of 0.4mM IPTG, 37 DEG C of cultures for 24 hours, observe result.
By recombinant plasmid pN15E6-flhDCSTMCoated plate culture after conversion DH31soplacI bacterial strains, as a result display contain flhDCSTMCard that chloramphenicol tablet on have a large amount of bacterial growths, and without bacterial growth on the corresponding tablet containing derivant.Show flhDCSTMGene overexpression being capable of lethal Escherichia coli DH31soplacI.Specific test result is shown in Fig. 4.
(3)flhDSTM、flhCSTMIndividually it is overexpressed not lethal Escherichia coli DH31soplacI：
By recombinant plasmid pN15E6-flhDSTMAnd pN15E6-flhCSTMCoated plate is trained after converting DH31soplacI bacterial strains respectively It supports, as a result shows flhDSTMAnd flhCSTMIndividually it is overexpressed not lethal Escherichia coli DH31soplacI.It is indicated above flhDCSTMThe lethal Escherichia coli DH31soplacI of gene overexpression is by flhDSTMAnd flhCSTMTwo genes express (group simultaneously Synthesize FlhD4C2Flagellum master regulation albumen) caused by, rather than the result that one of any of which is individually overexpressed.Specific experiment knot Fruit sees Fig. 5, Fig. 5 pN15E6-flhDSTMThe experimental result of not lethal Escherichia coli DH31soplacI, due to flhCSTMWith flhDSTMExperimental result it is similar, figure omit.
(4)flhDSTMAnd flhCSTMCo-express lethal Escherichia coli DH31soplacI：
pKDb-flhDSTMThe structure of recombinant expression plasmid and identification：With pN15E6-flhDSTMPlasmid is template, with Trrn_ Up1 and T0_dn1 is primer (table 1), PCR amplification flhDSTMExpression cassette is connected to the T- prepared through XcmI digestion pKDb plasmids and carries Body builds recombinant expression carrier.Using pKDb_SeqR and pKDb_SeqF as primer, PCR identifies recombinant expression plasmid.
By recombinant plasmid pKDb-flhDSTM(carrying amp+ resistant genes, transformed bacteria is in ammonia benzyl resistance) and pN15E6- flhCSTM(carrying kan+ resistant genes, transformed bacteria is in that resistance of card) while coated plate culture after DH31soplacI bacterial strains is converted, tied Fruit shows flhDSTMAnd flhCSTMOverexpression and flhDC simultaneouslySTMThe dual-gene result for being overexpressed lethal host strain is the same.Into one Step shows flhDSTMAnd flhCSTMIt is assembled into FlhD after being overexpressed simultaneously4C2Flagellum master regulation albumen, FlhD4C2Flagellum master regulation The excess of albumen causes host strain dead.
(5) contain pN15E6-flhDCSTMEscherichia coli DH31soplacI strain growth curves measurement：
To containing pN15E6-flhDCSTMThe Escherichia coli DH31soplacI bacterial strains of plasmid carry out the measurement of growth curve. Culture contains pN15E6-flhDCSTMThe Escherichia coli DH31soplacI bacterial strains of plasmid are incubated overnight bacterium, by 0.5% switching in LB Fluid nutrient medium, 37 DEG C of cultures to OD600≈ 0.5, addition various concentration IPTG induction (IPTG final concentrations are respectively 0, 0.0004mM, 0.0015625mM, 0.00625mM, 0.025mM, 0.1mM, 0.4mM), often induce half an hour, sampling to survey it OD600, make curve graph.
Growth curve measurement result is shown in Fig. 6.The results show that as a concentration of 0.0004mM and 0.0015625mM of IPTG, carefully Bacterium grow be not added with IPTG induce compare it is similar；As a concentration of 0.00625mM and 0.025mM of IPTG, bacterium is through inducing 4h Afterwards, start a degree of growth inhibition occur；And as a concentration of 0.1mM and 0.4mM of IPTG, bacterium after inducing 1.5h just There is growth inhibition phenomenon, subsequent OD600Value is begun to decline, and it is dead to illustrate that bacterium occurs, amount of bacteria is reduced.
(6) contain pN15E6-flhDCSTMEscherichia coli DH31soplacI bacterial strain dynamic tests measurement：To containing pN15E6-flhDCSTMThe Escherichia coli DH31soplacI bacterial strains of plasmid carry out dynamic test measurement.Method and step：2 μ are taken respectively L contains pN15E6-flhDCSTMThe overnight culture of the Escherichia coli DH31soplacI bacterial strains of plasmid, dibbling is in dense containing different IPTG The movement agar plate of (0,0.0004mM, 0.0015625mM, 0.00625mM, 0.025mM, 0.1mM) is spent (without corresponding anti- Raw element), after 37 DEG C are cultivated 6h~7h, pass through the diameter for measuring bacterium colony on semisolid tablet, assesses salmonella flhDC gene tables Up to e. coli host bacteria is grown, the influence of power.
When as a result showing IPTG a concentration of 0.0004mM, 0.0015625mM and 0.00625mM, (i.e. bacterium exists bacterium power The bacterium colony size of power flat bed upper growth) higher than the control for being not added with IPTG, and improve with inducer concentrations and enhance successively.In conjunction with Growth curve (Fig. 6) interpretation of result is found, as a concentration of 0.0004mM and 0.0015625mM of IPTG, the growth of inducible strain Curve with compare almost the same, bacterium power enhancing, display flhDC genes are induced to express, but inhibition has not yet been reached in expression quantity The dosage of host germ grew；As a concentration of 0.00625mM of IPTG, a degree of suppression has been displayed in the growth curve of inducible strain It makes, but the bacterium colony of bacterial growth is still bigger than compareing, even higher than 0.0004mM and 0.0015625mM concentration IPTG inducible strains Bacterium colony size, prompt the flhDC gene outcomes of induced expression that can offset it to growth to the humidification of bacterium power Inhibiting effect, synthesis result still show that bacterial growth has the bacterium colony of bigger, bacterium to have stronger power；When IPTG concentration increases When greatly to 0.025mM, may it can not be offset to host to the humidification of bacterium power due to the flhDC products of induced expression The inhibiting effect of bacterium growth, as a result the bacterium colony reduction of bacterial growth；And when IPTG concentration increases to 0.1mM, bacterial growth is complete It is complete to be suppressed, have no that bacterium colony is grown.The results are shown in Table 2 for bacterium dynamic test.
Table 2 contains pN15E6-flhDCSTMEscherichia coli DH31soplacI bacterial strain dynamic tests measurement result
Note：Dynamic test with power flat bed upper bacterium colony size the result is that indicated, test data is by the experiment that repeats three times As a result it obtains.
Embodiment 3
Flagellum master regulation gene flhDCEcoliIt is overexpressed the influence to Escherichia coli DH31soplacI Strain survivals.
(1)flhDCEcoliIt is overexpressed the growth for inhibiting Escherichia coli DH31soplacI：
Method is with embodiment 2, by plasmid pN15E6-flhDCEcoliCoated plate culture after conversion DH31soplacI bacterial strains.As a result Display contains flhDCEcoliCard that chloramphenicol tablet on have a large amount of bacterial growths, and bacterial clump on the corresponding tablet containing derivant It grows less than normal.Show flhDCEcoliThe growth of Escherichia coli DH31soplacI can be inhibited by being overexpressed.Specific test result is shown in figure 7。
(2)flhDEcoli、flhCEcoliIndividually it is overexpressed not lethal Escherichia coli DH31soplac：
Method is with embodiment 2, by plasmid pN15E6-flhDEcoliAnd pN15E6-flhCEcoliIt converts respectively Coated plate culture after DH31soplacI bacterial strains.As a result flhD is shownEcoliAnd flhCEcoliIndividually it is overexpressed not lethal Escherichia coli DH31soplacI.It is indicated above flhDCEcoliGene overexpression inhibit the growth of Escherichia coli DH31soplacI be by flhDEcoliAnd flhCEcoliTwo genes are expressed simultaneously (is combined into FlhD4C2Flagellum master regulation albumen) caused by, rather than its In one of any result being individually overexpressed.Specific test result is shown in Fig. 8, Fig. 8 pN15E6-flhDEcoliNot lethal large intestine bar The experimental result of bacterium DH31soplacI, flhCEcoliWith flhDEcoliExperimental result it is similar, figure omit.
Embodiment 4
Other flagellin genes are overexpressed the influence to Escherichia coli DH31soplacI Strain survivals.
(1)fliASTMIt is overexpressed lethal Escherichia coli DH31soplacI：
In order to verify it is other in relation to flagellin gene whether also with the similar lethal Escherichia coli of overexpression of flhDC genes The phenomenon that DH31soplacI, this experiment is by pN15E6-fliASTM、pN15E6-fliT、pN15E6-fliZ、pN15E6-flgM、 After pN15E6-flgK, pN15E6-fliI and pN15E6-fliC and pN15E6-motA plasmids convert DH31soplacI bacterial strains Coated plate culture (specific method is with embodiment 2).As a result fliA is shownSTMGene overexpression can lethal Escherichia coli DH31soplacI, and other flagellin genes such as fliT, fliZ, flgM, fliI do not have the lethal Escherichia coli of overexpression The tablet picture of the effect of DH31soplacI, specific test result is shown in Fig. 9.
(2)fliAEcoliIt is overexpressed lethal Escherichia coli DH31soplacI：
By pN15E6-fliAEcoliPlasmid converts coated plate culture after DH31soplacI bacterial strains (specific method is with embodiment 2). As a result fliA is shownEcoliGene overexpression equally can lethal Escherichia coli DH31soplacI.The tablet picture of specific test result See Figure 10.
Sequence table
<110>Zhejiang University
<120>The application of flagellum two level controlling gene fliA or its expression product as target spot in antibacterials exploitation
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tggcagaaac caatcaactg gtttgtcact tccgttttga cagccaccag acgattactc 240
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<400> 2
ccatgggaat aatgcatacc tccgagttgc tgaaacacat ttatgacatc aacttgtcat 60
atttactact tgcacagcgt ttgattgttc aggacaaagc gtccgctatg tttcgtctcg 120
gcataaatga agaaatggcg acaacgttag cggcactgac tcttccgcaa atggttaagc 180
tggcagaaac caatcaactg gtttgtcact tccgttttga cagccaccag acgattactc 240
agttgacgca agattcccgc gttgacgatc tccagcaaat tcataccggc atcatgctct 300
caacacgctt gctgaatgat gttaatcagc ctgaagaagc gctgcgcaag aaaagggcct 360
gatcatgagt gagatct 377
<210> 3
<211> 613
<212> DNA
<213>Escherichia coli (Escherichia coli)
<400> 3
ccatgggtga aaaaagcatt gttcaggaag cgcgggatat tcagctggca atggaattga 60
tcaccctggg cgctcgtttg cagatgctgg aaagcgaaac acagttaagt cgcggacgcc 120
tgataaaact ttataaagaa ctgcgcggaa gcccaccgcc gaaaggcatg ctgccattct 180
caaccgactg gtttatgacc tgggaacaaa acgttcatgc ttcgatgttc tgtaatgcat 240
ggcagttttt actgaaaacc ggtttgtgta atggcgtcga tgcggtgatc aaagcctacc 300
gtttatacct tgaacagtgc ccacaagcag aagaaggacc actgctggca ttaacccgtg 360
cctggacatt ggtgcggttt gttgaaagtg gattactgca actttccagc tgcaactgct 420
gcggcggcaa ttttattacc cacgctcacc agcctgttgg cagctttgcc tgcagcttat 480
gtcaaccgcc atcccgggca gtaaaaagac gtaaactttc ccagaatcct gccgatatta 540
tcccacaact gctggatgaa cagagagtac aggctgttta actgatacgg tgaggcgcaa 600
cattccaaga tct 613
<210> 4
<211> 789
<212> DNA
<213>Escherichia coli (Escherichia coli)
<400> 4
ccatgggaaa ttcactctat accgctgaag gtgtaatgga taaacactcg ctgtggcagc 60
gttatgtccc gctggtgcgt cacgaagcat tgcgcctgca ggttcgactg cccgcgagcg 120
tggaacttga cgatctgcta caggcgggcg gcattgggtt acttaatgcc gtcgaacgct 180
atgacgccct acaaggaacg gcatttacaa cttacgcagt gcagcgtatc cgtggcgcta 240
tgctggatga acttcgcagc cgtgactggg tgccgcgcag cgtgcgacgc aacgcgcgtg 300
aagtggcaca ggcaataggg caactggagc aggaacttgg ccgcaacgcc acggaaactg 360
aggtagcgga acgtttaggg atcgatattg ccgattatcg ccaaatgttg ctcgacacca 420
ataacagcca gctcttctcc tacgatgagt ggcgcgaaga gcacggcgat agcatcgaac 480
tggttactga tgatcatcag cgagaaaacc cgctacaaca actactggac agtaatctgc 540
gccagcgggt gatggaagcc atcgaaacgt tgccggagcg cgaaaaactg gtattaaccc 600
tctattacca ggaagagctg aatctcaaag agattggcgc ggtgctggag gtcggggaat 660
cgcgggtcag tcagttacac agccaggcta ttaaacggtt acgcactaaa ctgggtaagt 720
tataacgtca gtaaatgccg cactttaact ttgactacca ggagttctta atgatggtgc 780
agcagatct 789
<210> 5
<211> 966
<212> DNA
<213>Salmonella typhimurium (S.Typhimurium)
<400> 5
ccatgggaac aatgcataca tccgagttgc taaaacacat ttatgacatc aatttgtcat 60
atttactcct tgcacagcgt ttgatcgtcc aggacaaagc atctgcgatg ttccgcctcg 120
gtatcaacga agagatggca aacacactgg gcgcgttgac cctgccgcag atggtcaaac 180
tggcggagac gaaccagtta gtttgtcatt tccggtttga cgatcatcag acgatcaccc 240
gtttgactca ggattcgcgc gtcgatgact tacagcagat tcacacaggt atcatgcttt 300
caacgcgtct gctcaatgaa gtggacgata cggcgcgtaa gaaaagggca tgataatgag 360
tgaaaaaagc attgttcagg aagctcgcga tatccagttg gcgatggagt tgattaatct 420
tggcgctcgt ctacaaatgc tggaaagcga aacacagctc agccgtggtc gcctcatcag 480
gctgtacaaa gaattacgcg gtagcccgcc gcctaaaggg atgctgccat tttcgacaga 540
ctggtttatg acctgggagc aaaatattca tgcctccatg ttctgcaacg cctggcaatt 600
tttactgaag accggcttat gcagcggtgt ggatgcggtg attaaagctt atcggcttta 660
tcttgagcag tgtccgcaac cgcctgaagg gccgttgttg gcgctgactc gcgcatggac 720
gctggtgcgt tttgttgaaa gtgggttgct tgaattgtcg agctgtaact gctgcggtgg 780
gaactttatt acccatgcgc atcagcccgt aggcagcttt gcgtgtagtt tatgccagcc 840
gccatcccgc gcagtaaaaa gacgtaaact ttcccgagat gctgccgata ttattccaca 900
actgctggat gaacagatcg aacaggctgt ttaaccgaaa cggtgtggac aaacactcca 960
agatct 966
<210> 6
<211> 368
<212> DNA
<213>Salmonella typhimurium (S.Typhimurium)
<400> 6
ccatgggaac aatgcataca tccgagttgc taaaacacat ttatgacatc aatttgtcat 60
atttactcct tgcacagcgt ttgatcgtcc aggacaaagc atctgcgatg ttccgcctcg 120
gtatcaacga agagatggca aacacactgg gcgcgttgac cctgccgcag atggtcaaac 180
tggcggagac gaaccagtta gtttgtcatt tccggtttga cgatcatcag acgatcaccc 240
gtttgactca ggattcgcgc gtcgatgact tacagcagat tcacacaggt atcatgcttt 300
caacgcgtct gctcaatgaa gtggacgata cggcgcgtaa gaaaagggca tgataatgag 360
tgagatct 368
<210> 7
<211> 613
<212> DNA
<213>Salmonella typhimurium (S.Typhimurium)
<400> 7
ccatgggtga aaaaagcatt gttcaggaag ctcgcgatat ccagttggcg atggagttga 60
ttaatcttgg cgctcgtcta caaatgctgg aaagcgaaac acagctcagc cgtggtcgcc 120
tcatcaggct gtacaaagaa ttacgcggta gcccgccgcc taaagggatg ctgccatttt 180
cgacagactg gtttatgacc tgggagcaaa atattcatgc ctccatgttc tgcaacgcct 240
ggcaattttt actgaagacc ggcttatgca gcggtgtgga tgcggtgatt aaagcttatc 300
ggctttatct tgagcagtgt ccgcaaccgc ctgaagggcc gttgttggcg ctgactcgcg 360
catggacgct ggtgcgtttt gttgaaagtg ggttgcttga attgtcgagc tgtaactgct 420
gcggtgggaa ctttattacc catgcgcatc agcccgtagg cagctttgcg tgtagtttat 480
gccagccgcc atcccgcgca gtaaaaagac gtaaactttc ccgagatgct gccgatatta 540
ttccacaact gctggatgaa cagatcgaac aggctgttta accgaaacgg tgtggacaaa 600
cactccaaga tct 613
<210> 8
<211> 741
<212> DNA
<213>Salmonella typhimurium (S.Typhimurium)
<400> 8
ccatggctaa ttcactgtat accgctgaag gtgtaatgga taaacactcg ctgtggcagc 60
gttatgtacc gctggtgcgt cacgaagcat tgcgcctgca ggtgcgattg ccggcgagcg 120
tggaactgga cgatctgcta caagcgggcg gcatcgggtt attaaatgcg gtcgaccgat 180
atgacgcttt gcaaggaacg gcatttacca cttacgcagt gcagcgtatt cgtggggcga 240
tgctggatga attacgcagc cgcgattggg tgccgcgtag cgtccggcgt aatgcccgcg 300
aagtggcgca ggcgatggga caactggagc aggaactggg gcgtaatgcg acggaaaccg 360
aagtggcgga acgtcttggc atccctgttg cggagtatcg tcagatgttg ctcgatacca 420
acaacagcca acttttctct tacgatgagt ggcgggaaga gcatggcgat agcatcgaac 480
tggtgactga agaacatcaa caggaaaacc cgttacatca actgctggag ggcgacctgc 540
gacagcgggt aatggatgcg attgaatcgc tgccggaacg cgagcaactg gtgttaacgc 600
tgtattacca ggaagagctc aatctcaaag agattggcgc ggtactggaa gtcggcgaat 660
cgcgggtcag ccagttgcat agtcaggcca tcaaacgatt acgcaccaaa ctgggtaagt 720
tataggtcgc gcatgagatc t 741
<210> 9
<211> 437
<212> DNA
<213>Salmonella typhimurium (S.Typhimurium)
<400> 9
ccatgggtac ctcaaccgtg gagtttatca accgttggca gcgtattgcg ctgctcagtc 60
aatcgctgct tgaacttgcg cagcgaggtg aatgggatct cttactgcaa caagaggtct 120
cctatctgca aagtattgaa acggtgatgg aaaagcaaac tccaccgggc attacgcgaa 180
gtattcagga tatggtcgcc ggatacatca aacaaacgct ggacaatgag cagctcctga 240
aagggctgct gcaacagcga ctggatgaac tgagtagttt gatcggacaa tccacccgcc 300
aaaaatcact caacaacgcg tatggccgtc tttccggtat gttactcgtg ccagatgcgc 360
ctggcgcctc ataatatttt cccgtctcgt atgaaaattc ttccatactc cagaggtcgg 420
ctaaacgact tagatct 437
<210> 10
<211> 603
<212> DNA
<213>Salmonella typhimurium (S.Typhimurium)
<400> 10
ccatgggtac ggtgcagcaa cctaaaaggc ggcctttgag ccgctatctt aaagacttta 60
aacacagcca gacgcattgc gcgcattgtc acaaactgct cgaccgcatt acgctggttc 120
gccgtggcaa gatcgttaat aaaatcgcta tttcacagct ggatatgcta cttgacgacg 180
ctgcctggca gcgggagcag aaggagtggg tggcgctgtg tcgcttttgc ggcgatttgc 240
actgcaaaaa gcagagtgat tttttcgata ttatcggttt caagcagtat ttgtttgaac 300
aaaccgagat gagccatggc acggtgcggg aatatgtcgt gcgtttacga cgccttggca 360
attacctcag cgagcaaaac atttcccacg atctgctgca ggacggtttt ctcgatgaaa 420
gcctggcgcc atggttgccg gaaaccagca ccaataatta ccgtatcgca ctgcgtaaat 480
accagcaata taaagcgcat cagcagattg cgcccagaca gaaatccccc tttaccgcca 540
gttctgatat atattaaaaa agcataagat gtagcagagt cgtgttggtg aaacgtgaga 600
tct 603
<210> 11
<211> 347
<212> DNA
<213>Salmonella typhimurium (S.Typhimurium)
<400> 11
ccatgggtag cattgaccgt acctcacctt tgaaacccgt tagcactgtc cagacgcgcg 60
aaaccagcga cacgccggta caaaaaacgc gtcaggaaaa aacgtccgcc gcgacgagcg 120
ccagcgtaac gttaagcgac gcgcaagcga agctcatgca gccaggcgtc agcgacatta 180
atatggaacg cgtcgaagca ttaaaaacgg ctatccgtaa cggtgagtta aaaatggata 240
cgggaaaaat agcagactcg ctcattcgcg aggcgcagag ctacttacag agtaaataag 300
cgtatgactc gtttgtcaga aatacttgac cagatgacca cagatct 347
<210> 12
<211> 1696
<212> DNA
<213>Salmonella typhimurium (S.Typhimurium)
<400> 12
ccatgggttc cagcttgatt aatcacgcca tgagcggact taacgccgcg caggccgcgt 60
taaatacggt cagtaataac atcaacaatt ataacgttgc gggttatacc cggcagacaa 120
ctattctggc gcaggcaaac agtacgttag gggctggcgg ctggataggt aatggcgttt 180
acgtttcagg cgtacagcgc gaatatgatg cgtttatcac taatcagcta cgcggcgcgc 240
aaaaccagag cagcggctta accacgcgct atgaacaaat gtcgaaaatc gacaacctgc 300
tggccgataa atccagctca ctgtctggtt cgttgcagag tttttttacc agcctgcaaa 360
cgttagtcag taacgcggaa gatcctgcgg cgcgtcaggc gctgattggt aaagcggaag 420
ggctggtaaa ccagttcaaa accaccgatc agtatctgcg cgatcaggat aaacaggtca 480
atatcgcgat tggctccagc gtggcgcaaa tcaacaatta cgcgaagcag atagctaacc 540
tgaacgatca aatctcccgt atgacgggcg taggcgcggg cgcatcgccg aacgacctgc 600
tcgatcagcg tgatcagttg gtcagcgagc ttaacaagat cgttggcgtc gaggtgagtg 660
tacaggacgg cggcacctat aacctgacga tggccaatgg ctatacgctg gtgcaggggt 720
cgacggcgcg tcagttggcg gcggttccct ccagcgccga cccgacgcga acgactgtcg 780
cttatgtcga tgaggccgcc ggtaacatcg aaattccgga aaagttgctg aacaccggtt 840
cgctcggcgg gctactgacg ttccgttctc aggatctgga tcagactcgt aatacgctgg 900
gccagttggc gttggcgttt gccgatgcgt ttaacgcgca gcataccaaa ggttatgacg 960
ccgacggcaa taaagggaaa gacttcttta gcattggctc gccggtggta tatagcaaca 1020
gtaataatgc cgataaaacg gtatcgctaa ccgctaaggt ggtcgacagc acgaaggttc 1080
aggcgacgga ttataagatt gtttttgacg gtacagactg gcaggttact cgcactgcgg 1140
ataacaccac cttcacggcg acaaaagatg ctgacggaaa actggagatt gacggtctga 1200
aagtgacggt agggaccggc gcacagaaaa atgacagttt tcttctcaag ccggtcagca 1260
atgctatcgt cgacatgaac gttaaagtga caaatgaagc cgagattgcg atggcgtctg 1320
agtcaaaact cgatcctgac gtggataccg gcgacagcga taaccgcaat ggtcaggcat 1380
tgctggactt acaaaacagc aatgtagtgg gcggcaacaa aacttttaac gatgcttacg 1440
ccacgttggt cagcgatgtg ggtaacaaaa cgtcaacgct gaaaaccagc agcaccacgc 1500
aggcgaatgt ggttaaacag ctttataaac agcaacagtc ggtttccggc gttaacctcg 1560
acgaagagta cggcaatttg cagcgttatc agcagtatta tctggcgaat gcgcaagtat 1620
tgcagaccgc gaatgcgctg tttgatgcgt tattgaatat tcgctaaagg agaaggatga 1680
catgcgtatc agatct 1696
<210> 13
<211> 1398
<212> DNA
<213>Salmonella typhimurium (S.Typhimurium)
<400> 13
ccatgggtat gactactcgt ctgacccgct ggcttaccgc gctcgacaac tttgaagcca 60
aaatggcgtt attgccggcg gtgcgtcgtt atggacgttt aacccgcgcc actggcctgg 120
tactggaggc caccggtctc cagcttccgc tgggcgccac ctgcattatt gagcgccagg 180
acggccctga aaccaaagag gtggaatcag aagtcgtcgg ttttaacggc cagcgtctgt 240
ttctaatgcc gctggaagag gtcgaaggca ttctgcccgg tgcccgcgtt tacgcccgta 300
acgggcatgg cgacggtctg caaagcggca aacagttacc gctcggcccg gccctgcttg 360
gtcgggtgct ggatggcggc ggtaaaccgc tcgacggact gcctgcgccg gatacgctgg 420
aaaccggcgc gttaatcacg ccgccgttta acccgctaca gcgaacgcca atcgaacatg 480
tgctggatac cggcgtacgc gctatcaacg cgttgttaac cgtagggcgc ggtcagcgta 540
tgggactctt tgccggttcc ggcgttggta aatcggttct gcttggcatg atggcgcgct 600
acacgcgggc ggacgtgatt gtcgtgggac ttatcggcga acgtggccgc gaagttaaag 660
attttatcga aaatattctc ggccccgacg gtcgcgcgcg ttcggtggtg atcgccgccc 720
cggcggatgt ctcgccgctg ctgcgaatgc agggcgccgc ctatgccacc cgcatcgccg 780
aagactttcg cgatcgcgga cagcatgtgt tgctgattat ggattcgctg acgcgctatg 840
caatggcgca gcgtgagatt gcgctggcaa tcggcgaacc gccagccacc aaaggttatc 900
cgccctcggt gttcgcaaag ctgccggcgc tggtcgagcg tgccggtaat ggcatccacg 960
gcggtggctc tatcaccgcg ttttataccg tgttgaccga gggggacgac caacaagatc 1020
ccattgccga ctcggcacgc gcaatcctcg acgggcacat tgtcttgtcc cgccgtctgg 1080
cggaggccgg gcactatccg gccattgata tcgaggcgtc aatcagccgg gcgatgaccg 1140
cgctcattac cgagcagcac tatgcgcggg tacggctatt taaacagttg ctttccagtt 1200
tccagcgtaa ccgcgatctg gtcagcgtcg gcgcctatgc caaaggcagc gatccgatgc 1260
tcgataaagc cattacctta tggccgcaac tggaagcgtt tttacagcaa ggcatttttg 1320
aacgggccga ctgggaggac tctctgcagg cgctcgattt aattttcccg acggtgtgat 1380
aaagcaggag ggagatct 1398
<210> 14
<211> 1529
<212> DNA
<213>Salmonella typhimurium (S.Typhimurium)
<400> 14
ccatggcaca agtcattaat acaaacagcc tgtcgctgtt gacccagaat aacctgaaca 60
aatctcagtc ctcactgagt tccgctattg agcgtctgtc ctctggtctg cgtatcaaca 120
gcgcgaaaga cgatgcggca ggccaggcga ttgctaaccg cttcacttct aatatcaaag 180
gtctgactca ggcttcccgt aacgctaacg acggcatttc tattgcgcag accactgaag 240
gtgcgctgaa tgaaatcaac aacaacctgc agcgtgtgcg tgagttgtct gttcaggcca 300
ctaacgggac taactctgat tccgatctga aatctatcca ggatgaaatt cagcaacgtc 360
tggaagaaat cgatcgcgtt tctaatcaga ctcaatttaa cggtgttaaa gtcctgtctc 420
aggacaacca gatgaaaatc caggttggtg ctaacgatgg tgaaaccatt accatcgatc 480
tgcaaaaaat tgatgtgaaa agccttggcc ttgatgggtt caatgttaat gggccaaaag 540
aagcgacagt gggtgatctg aaatccagct tcaagaatgt tacgggttac gacacctatg 600
cagcgggtgc cgataaatat cgtgtagata ttaattccgg tgctgtagtg actgatgcag 660
cagcaccgga taaagtatat gtaaatgcag caaacggtca gttaacaact gacgatgcgg 720
aaaataacac tgcggttgat ctctttaaga ccactaaatc tactgctggt accgctgaag 780
ccaaagcgat agctggtgcc attaaaggtg gtaaggaagg agataccttt gattataaag 840
gcgtgacttt tactattgat acaaaaactg gtgatgacgg taatggtaag gtttctacta 900
ccatcaatgg tgaaaaagtt acgttaactg tcgctgatat tgccactggc gcgacggatg 960
ttaatgctgc taccttacaa tcaagcaaaa atgtttatac atctgtagtg aacggtcagt 1020
ttacttttga tgataaaacc aaaaacgaga gtgcgaaact ttctgatttg gaagcaaaca 1080
atgctgttaa gggcgaaagt aaaattacag taaatggggc tgaatatact gctaacgcca 1140
cgggtgataa gatcacctta gctggcaaaa ccatgtttat tgataaaaca gcttctggcg 1200
taagtacatt aatcaatgaa gacgctgccg cagccaagaa aagtaccgct aacccactgg 1260
cttcaattga ttctgcattg tcaaaagtgg acgcagttcg ttcttctctg ggggcaattc 1320
aaaaccgttt tgattcagcc attaccaacc ttggcaatac ggtaaccaat ctgaactccg 1380
cgcgtagccg tatcgaagat gctgactatg caacggaagt ttctaatatg tctaaagcgc 1440
agattctgca gcaggctggt acttccgttc tggcgcaggc taaccaggtt ccgcaaaacg 1500
tcctctcttt actgcgttaa tccagatct 1529
<210> 15
<211> 842
<212> DNA
<213>Salmonella typhimurium (S.Typhimurium)
<400> 15
ccatgggtac cggcggacac cttggggcac tctatcaacc tgctgaactg gtcatcattg 60
gcggcgcggg gataggggcg ttcattgtcg gcaacaacgg gaaggccatc aaaggcacga 120
tgaaagctat cccgttgtta tttcgtcgtt cgaaatacac aaaatctatg tacatggatt 180
tgctggcgtt gctctatcgc ctgatggcca aatcacgcca gcaggggatg ttctcccttg 240
aacgcgatat tgaaaatcca aaagagagtg aaatcttcgc cagttatccg cgtattctgg 300
ccgatgcggt aatgcttgat tttattgtcg attatctgcg cctgatcatc agcggcaaca 360
tgaatacgtt cgaaattgaa gcgttgatgg atgaagagat tgaaacccat gaaagcgagg 420
cggaagtccc ggccaacagt ctggcgatgg tgggggattc gctgcctgcc tttggtatcg 480
tcgcggcggt aatgggggtg gttcacgctc tggcttcagc cgatcgtccg gcagcggagt 540
tgggggcgct gattgcccat gccatggtag gtacgttcct cggtatttta ctggcttatg 600
gattcatttc accgttagcg accgttttgc gccagaagag cgccgaaacc accaagatga 660
tgcagtgcgt aaaaatcaca ctgctgtcta atctgaacgg ctatgcgccg ccgattgccg 720
tggaatttgg tcgtaaaacg ctttattcca gtgagcgtcc atcgtttatt gagttggaag 780
aacacgttcg cgcagtgaga aacccaaacc agcagcagac gactgaggaa gcatgaagat 840
ct 842
<210> 16
<211> 29
<212> DNA
<213>Artificial sequence (Artificial)
<400> 16
catgccatgg gaataatgca tacctccga 29
<210> 17
<211> 32
<212> DNA
<213>Artificial sequence (Artificial)
<400> 17
gaagatcttg gaatgttgcg cctcaccgta tc 32
<210> 18
<211> 32
<212> DNA
<213>Artificial sequence (Artificial)
<400> 18
gaagatcttg gagtgtttgt ccacaccgtt tc 32
<210> 19
<211> 29
<212> DNA
<213>Artificial sequence (Artificial)
<400> 19
catgccatgg gaacaatgca tacatccga 29
<210> 20
<211> 33
<212> DNA
<213>Artificial sequence (Artificial)
<400> 20
gtagatctca ctcatgatca ggcccttttc ttg 33
<210> 21
<211> 33
<212> DNA
<213>Artificial sequence (Artificial)
<400> 21
gtagatctca ctcatgatca ggcccttttc ttg 33
<210> 22
<211> 35
<212> DNA
<213>Artificial sequence (Artificial)
<400> 22
ctagccatgg gtgaaaaaag cattgttcag gaagc 35
<210> 23
<211> 29
<212> DNA
<213>Artificial sequence (Artificial)
<400> 23
gaagatctta ccgctgctgg agtgtttgt 29
<210> 24
<211> 34
<212> DNA
<213>Artificial sequence (Artificial)
<400> 24
catgccatgg gtgaaaaaag cattgttcag gaag 34
<210> 25
<211> 32
<212> DNA
<213>Artificial sequence (Artificial)
<400> 25
catgccatgg gaaattcact ctataccgct ga 32
<210> 26
<211> 32
<212> DNA
<213>Artificial sequence (Artificial)
<400> 26
gtagatctgc tgcaccatca ttaagaactc ct 32
<210> 27
<211> 42
<212> DNA
<213>Artificial sequence (Artificial)
<400> 27
catgccatgg ctaattcact gtataccgct gaaggtgtaa tg 42
<210> 28
<211> 32
<212> DNA
<213>Artificial sequence (Artificial)
<400> 28
gtagatctca tgcgcgacct ataacttacc ca 32
<210> 29
<211> 33
<212> DNA
<213>Artificial sequence (Artificial)
<400> 29
catgccatgg gtacctcaac cgtggagttt atc 33
<210> 30
<211> 31
<212> DNA
<213>Artificial sequence (Artificial)
<400> 30
gtagatctaa gtcgtttagc cgacctctgg a 31
<210> 31
<211> 32
<212> DNA
<213>Artificial sequence (Artificial)
<400> 31
catgccatgg gtacggtgca gcaacctaaa ag 32
<210> 32
<211> 31
<212> DNA
<213>Artificial sequence (Artificial)
<400> 32
gtagatctca cgtttcacca acacgactct g 31
<210> 33
<211> 36
<212> DNA
<213>Artificial sequence (Artificial)
<400> 33
catgccatgg gtagcattga ccgtacctca cctttg 36
<210> 34
<211> 33
<212> DNA
<213>Artificial sequence (Artificial)
<400> 34
gtagatctgt ggtcatctgg tcaagtattt ctg 33
<210> 35
<211> 34
<212> DNA
<213>Artificial sequence (Artificial)
<400> 35
catgccatgg gttccagctt gattaatcac gcca 34
<210> 36
<211> 31
<212> DNA
<213>Artificial sequence (Artificial)
<400> 36
gtagatctga tacgcatgtc atccttctcc t 31
<210> 37
<211> 38
<212> DNA
<213>Artificial sequence (Artificial)
<400> 37
cagtccatgg gtactactcg tctgacccgc tggcttac 38
<210> 38
<211> 30
<212> DNA
<213>Artificial sequence (Artificial)
<400> 38
gtagatctcc ctcctgcttt atcacaccgt 30
<210> 39
<211> 32
<212> DNA
<213>Artificial sequence (Artificial)
<400> 39
catgccatgg cacaagtcat taatacaaac ag 32
<210> 40
<211> 31
<212> DNA
<213>Artificial sequence (Artificial)
<400> 40
gtagatctgg attaacgcag taaagagagg a 31
<210> 41
<211> 28
<212> DNA
<213>Artificial sequence (Artificial)
<400> 41
catgccatgg gtaccggcgg acaccttg 28
<210> 42
<211> 31
<212> DNA
<213>Artificial sequence (Artificial)
<400> 42
gtagatcttc atgcttcctc agtcgtctgc t 31
<210> 43
<211> 21
<212> DNA
<213>Artificial sequence (Artificial)
<400> 43
cctggctgat acgttggtcc t 21
<210> 44
<211> 24
<212> DNA
<213>Artificial sequence (Artificial)
<400> 44
gcaaccgagc gttctgaaca aatc 24
<210> 45
<211> 25
<212> DNA
<213>Artificial sequence (Artificial)
<400> 45
gtcaagccgt caattcctgg tatga 25
<210> 46
<211> 23
<212> DNA
<213>Artificial sequence (Artificial)
<400> 46
gtcacgttgc ggccgcattc tca 23
<210> 47
<211> 24
<212> DNA
<213>Artificial sequence (Artificial)
<400> 47
acggaaggat ctgaggttct tatg 24
<210> 48
<211> 24
<212> DNA
<213>Artificial sequence (Artificial)
<400> 48
ggttattgtc tcatgagcgg atac 24

Claims (8)

1. a kind of action target spot of antibacterials, which is characterized in that the target spot is flagellum two level controlling gene fliA or its table Up to product FliA albumen.
2. the application of flagellum two level controlling gene fliA or its expression product FliA albumen as target spot in antibacterials exploitation.
3. application as claimed in claim 2, which is characterized in that the antibacterials can raise flagellum two level controlling gene The expression of fliA or the activity for enhancing FliA albumen.
4. application as claimed in claim 2, which is characterized in that the antibacterials are for killing or inhibiting with flagellum activity Bacterium.
5. application as claimed in claim 4, which is characterized in that the antibacterials are used to kill or inhibit salmonella or big Enterobacteria.
6. a kind of antibacterials, which is characterized in that expression of the flagellum two level controlling gene fliA in bacterium can be raised, or Enhance the activity of FliA albumen.
7. antibacterials as claimed in claim 6, which is characterized in that the bacterium has flagellum activity.
8. antibacterials as claimed in claim 7, which is characterized in that the bacterium is salmonella or Escherichia coli.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109706105A (en) * 2019-01-07 2019-05-03 集美大学 One plant of deformation pseudomonad fliA gene silencing bacterial strain

Citations (2)

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WO2009012589A1 (en) * 2007-07-23 2009-01-29 University Of Saskatchewan Vaccines and methods for treatment or prevention of gram negative bacterial infection in a vertebrate subject
US20120027786A1 (en) * 2010-02-23 2012-02-02 Massachusetts Institute Of Technology Genetically programmable pathogen sense and destroy

Patent Citations (2)

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WO2009012589A1 (en) * 2007-07-23 2009-01-29 University Of Saskatchewan Vaccines and methods for treatment or prevention of gram negative bacterial infection in a vertebrate subject
US20120027786A1 (en) * 2010-02-23 2012-02-02 Massachusetts Institute Of Technology Genetically programmable pathogen sense and destroy

Non-Patent Citations (1)

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Title
KAZUHIRO KUTSUKAKE等: "Role of the FliA-FlgM Regulatory System on the Transcriptional Control of the Flagellar Regulon and Flagellar Formation in Salmonella typhimurium", 《JOURNAL OF BACTERIOLOGY》 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109706105A (en) * 2019-01-07 2019-05-03 集美大学 One plant of deformation pseudomonad fliA gene silencing bacterial strain
CN109706105B (en) * 2019-01-07 2020-11-06 集美大学 Pseudomonas proteorum fliA gene silencing strain

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