CN108837152B - Application of flagella main regulatory gene flhDC or expression product thereof as target in antibacterial drug development - Google Patents

Abstract

The invention discloses application of flagella main regulatory gene flhDC or expression product thereof as a target in antibacterial drug development. The research of the invention finds that the flagella main control gene flhDC of salmonella or escherichia coli is over-expressed in the escherichia coli to kill the escherichia coli, namely, the product FlhD expressed by the flagella main control gene flhDC₄C₂The Escherichia coli can be killed due to the over-high total activity of the protein, so that the gene or an expression product thereof can be used as a target for developing antibacterial drugs, and a direction is provided for solving the problem of bacterial drug resistance.

Description

Application of flagella main regulatory gene flhDC or expression product thereof as target in antibacterial drug development

Technical Field

The invention relates to the technical field of biology, in particular to application of a flagella main regulatory gene flhDC or an expression product thereof as a target point in antibacterial drug development.

Background

The flagella are important moving organs of bacteria, promote the bacteria to tend to a nutrient-rich environment, escape from a hostile environment containing harmful substances, and benefit and avoid harm. Many bacteria contain flagella, including E.coli, Salmonella, Proteus, and the like.

The complete bacterial flagella (flagellum) structure comprises three parts, namely a basal body, a flagella hook and a flagella filament, spanning from the inner membrane to the cell wall and the outer membrane and extending to the outside of the cell (Evans L.D.B, Hughes C and Fraser G.M. building a flagella outer side the bacterial cell. trends in microbiology.2014,22(10): 566) 572.). Wherein, the matrix consists of a central flagpole (rod) and a plurality of annular structures embedded in cell membranes and cell walls. A part of the structure of the C-loop protrudes inward from the cytoplasm, and within this enclosed cavity is a flagellar type III secretion system (fT 3SS) which is responsible for flagellin secretion and self-assembly.

The gene cluster encoding the flagella is located at 4 different positions in the bacterial genome, contains more than 50 genes, and consists of at least 14 operons (Kalir S, McClure J, Pabbaraju K et al. organic genes in a flagellate pathway by analysis of expression kinetics from bacteria. science 2001,292(5524): 2080-. Expression of the bacterial flagella gene cluster exhibits a three-level ladder (see FIG. 1). The flhD and flhC genes of the first-stage ladder flhDC operon are main regulatory genes for flagella expression, the expression of other downstream flagella genes is controlled, the expression intensity is regulated, and the length, the number and the motility of flagella on the surface of bacteria are finally determined. The flhDC operon is regulated by a number of environmental factors, such as: ambient temperature, pH, osmotic pressure, oxygen, and other environmental factors (Fahrner K.A, Berg H.C. metals which is stuck flexible DC expression in Escherichia coli K-12.J.bacteriol.2015,197(19): 3087-96.). FlhDC gene expression product composition FlhD₄C₂Flagella major regulatory protein, FlhD₄C₂Binding to the gene promoter of the second step, activating the level II gene, opening expression of the matrix-flagellate sheath (HBB) gene group and assembly of HBB, and at the same time, FlhD₄C₂Also turn on expression of sigma required for class III promoter expression²⁸Factor (FliA) and antagonist factor (FlgM) thereof, before HBB assembly is completed, FliA cannot be combined with promoter of third ladder gene due to the presence of FlgM, III-level gene is temporarily not expressed, when HBB assembly is completed, antagonist factor FlgM is secreted to the outside of cells and antagonism is eliminated, III-level gene is subsequently expressed, flagella begin to assemble and finally complete, and bacteria obtain motility and chemotaxis capability.

Most salmonella have flagella and are motile, with the exception of pullorum and salmonella gallinarum. However, Chaubal and Holt (1.Holt P.S and Chaubal L.H.detection of motion and reactive synthesis of flagellar proteins in Salmonella pulverum culture.J.Clin.Micro.1997,35:1016-1020.2.Chaubal L.H and Holt P.S.characteristics of swammingmotion and identification of flagellar proteins in Salmonella pulverum isolates.am.J.vet.Res.1999,60:1322 1327.) have reported that Salmonella pullorum has flagellar activity in certain situations.

Killing or inhibiting bacterial growth is a major strategy for human control of bacterial diseases, however, with the widespread use of antibacterial drugs, particularly extensive abuse, the problem of bacterial resistance is becoming more serious, and new drugs are urgently sought for controlling bacterial diseases.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides a flagella main regulatory gene flhDC or an expression product FlhD thereof₄C₂The protein (flagella major regulatory protein) is used as a target point to be applied to the development of antibacterial drugs.

Experimental research shows that the flagella main control gene flhDC of salmonella or escherichia coli is overexpressed in escherichia coli to kill escherichia coli, namely, a product FlhD expressed by the flagella main control gene flhDC₄C₂The over-high total activity of the protein can kill Escherichia coli, which shows that the gene or its expression product can be used as target for developing antibacterial drug, and find a gene which can make flagella main control gene flhDC in bacteria over-express or enhance its expression product FlhD₄C₂Protein Activity, when the FlhDC Gene is overexpressed, the product FlhD₄C₂The amount of protein is increased, and the total activity is overhigh; while if FlhD can be enhanced₄C₂The activity of the protein is increased under the condition that the total amount of the protein is not changed, so that the aim of inhibiting the growth of bacteria or killing the bacteria can be fulfilled.

An action target of an antibacterial drug, wherein the target is flagella main regulatory gene flhDC or expression product FlhD thereof₄C₂A protein.

The invention also provides a flagella major regulatory gene flhDC or an expression product FlhD thereof₄C₂The protein is used as a target point in the development of antibacterial drugs.

The antibacterial drug can up-regulate the expression of flagella major regulatory gene flhDC or enhance FlhD₄C₂The activity of the protein.

The antibacterial agent is used for killing or inhibiting bacteria having flagellar activity. Preferably, the antibacterial agent is used for killing or inhibiting salmonella or escherichia coli.

The invention also provides an antibacterial drug which can up-regulate the expression of flagella major regulatory gene flhDC in bacteria or enhance FlhD₄C₂The activity of the protein. The bacteria have flagellar activity. Preferably, the bacteria are salmonella or escherichia coli.

According to the invention, through researches, the host bacteria can be killed by the overexpression of the flagella main control gene flhDC, a new target point is provided for the research and development of novel antibacterial drugs, and a direction is provided for solving the problem of drug resistance of bacteria.

Drawings

FIG. 1 is a schematic diagram of the tertiary regulation of flagella genes.

FIG. 2 is a diagram showing the result of electrophoretic detection of a PCR-amplified flhDC gene fragment, in which lane M is a standard molecular weight Marker DL2000 (the same applies below), and lane 1 is flhDC^STMLane 2 is flhDC^EcoliLane 3 is flhC^STMLane 4 is flhC^EcoliLane 5 is flhD^STMLane 6 is flhD^Ecoli。

FIG. 3 shows PCR amplificationThe electrophoresis detection result of the other flagella gene segments is shown in the figure, wherein the numbers of the lanes 1-9 are fliA respectively^Ecoli、fliA^STMflgK, fliI, flgM, fliT, fliZ, fliC, motA, and the fragment sizes are 795bp, 747bp, 1702bp, 1401bp, 353bp, 443bp, 610bp, 1223bp, and 848bp, respectively.

FIG. 4 shows pN15E6-flhDC^STMFIG. A shows the results of experiments on lethal E.coli DH31 soprocI, where A is a Kan + Chl plate and B is a Kan + Chl + IPTG plate.

FIG. 5 shows pN15E6-flhD^STMFIG. A is a graph showing the results of experiments on non-lethal E.coli DH31 soprocI, wherein A is a Kan + Chl plate and B is a Kan + Chl + IPTG plate.

FIG. 6 shows pN15E6-flhDC^STMFIG. of the results of growth curve measurement of E.coli DH31 soprocI strain.

FIG. 7 shows pN15E6-flhDC^EcoliThe result of the experiment for inhibiting Escherichia coli DH31 soprocI is shown in the figure, wherein A is Kan + Chl plate, and B is Kan + Chl + IPTG plate.

FIG. 8 shows pN15E6-flhD^EcoliFIG. A is a graph showing the results of experiments on non-lethal E.coli DH31 soprocI, wherein A is a Kan + Chl plate and B is a Kan + Chl + IPTG plate.

FIG. 9 is fliA^STMExperimental results of overexpression lethal E.coli DH31 soprocI, wherein A is Kan + Chl plate, and B is Kan + Chl + IPTG plate.

FIG. 10 is fliA^EcoliExperimental results of overexpression lethal E.coli DH31 soprocI, wherein A is Kan + Chl plate, and B is Kan + Chl + IPTG plate.

Detailed Description

The main reagents are as follows: NcoI enzyme, BglII enzyme, T4DNA ligase were purchased from New England Biolabs; DL2000marker, DL5000marker, Taq DNA polymerase, ExTaq DNA polymerase were purchased from TaKaRa, Inc., Boehringer Biotech (Dalian Co., Ltd.); the bacterial plasmid rapid extraction kit and the PCR cleaning kit are purchased from Axygen company; IPTG was purchased from Shanghai Biotechnology, Inc.

Plasmid and strain: the s.typhimurium STM542 strain was purchased from the chinese veterinary drug institute; the DH31 soprocI strain, pN15E6 plasmid, was given by the 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 vector based on the reproduction of the plasmid of temperature phase N15[ J ]. Gene 395(2007) 15-21.); JM109 strain was stored in this laboratory.

The pKDb plasmid is a plasmid which is self-ligated to the backbone of pKD46 plasmid (Datsenko KA & Wanner BL. one-step inactivation of chromosomal genes in Escherichia coli K-12using PCR products. PNAS (2000)97(12):6640-45.) and has 2 XcmI cleavage sites added thereto, and the plasmid is constructed and stored in the laboratory. The pKDb plasmid is cut by XcmI to form a linear T vector with two heads protruding 1T, and a PCR product with the end protruding 1A can be directly cloned.

Example 1

Amplification of various flagella gene fragments and homology comparison of flhD and flhC.

Using JM109 and STM542 genome DNA as templates, carrying out PCR amplification on flhDC, flhD, flhC and fliA gene fragments of escherichia coli and salmonella, and using STM542 genome DNA as templates, carrying out PCR amplification on fliT, fliZ, flgM, flgK, fliI, fliC and motA gene fragments. The primers and characteristics used for amplification are shown in Table 1, and primer synthesis and sequencing are available from Huada Biotech, Inc.

TABLE 1 amplification of Gene fragments of interest

Note: the underlined bases are recognition sites for NcoI or BglII, and the bases preceding the recognition sites are protecting bases.

Using the primers shown in table 1, PCR-amplifying the target gene fragment: FlhDC^STM、flhDC^Ecoli、flhC^STM、flhC^Ecoli、flhD^STM、flhD^Ecoli、fliA^Ecoli、fliA^STMflgK, fliI, flgM, fliT, fliZ, fliC, motA. Wherein, flhDC^STM、flhDC^Ecoli、flhC^STM、flhC^Ecoli、flhD^STM、flhD^EcoliThe sizes of the gene fragments are 973bp, 579bp, 351bp and 351bp respectively, which are shown in figure 2; fliA^Ecoli、fliA^STMThe sizes of the flgK, fliI, flgM, fliT, fliZ, fliC and motA gene fragments are 795bp, 747bp, 1702bp, 1401bp, 353bp, 443bp, 610bp, 1223bp and 848bp respectively, as shown in figure 3.

Reference to genomic sequence of 5 Salmonella enteritidis strains (Salmonella enterica) in GenBank: s.typhimurium LT2, s.galingarum 287/91, s.typhi CT18, s.cholereas SC-B67, s.enteritidis P125109; and 3 Escherichia coli (Escherichia coli) genomic sequences: MG1655, W3110 and DH10B were aligned with the deduced amino acids of the sequence of 5 Salmonella strains, 3 Escherichia coli flhD and flhC, respectively, and the homology was calculated.

The result shows that the deduced amino acids of 5 salmonella enteritidis flhD are completely the same, and the deduced amino acid homology of flhC is 98.96-99.83%. The deduced amino acid homology of the 3 strains of escherichia coli flhD and flhC is more than 97%. The deduced amino acid difference of flhD and flhC between salmonella and colibacillus is large, and the homology is 74.34-75.78% and 76.51-81.69% respectively. The result shows that the flhDC gene is different between salmonella and escherichia coli, and the regulation and control functions of the salmonella flhDC gene in escherichia coli host bacteria need to be further verified.

Example 2

Flagella main regulation gene flhDC^STMEffect of overexpression on the survival of E.coli DH31 soprocI strains.

(1) Construction and identification of pN15E6 expression vector:

the PCR target gene fragment and pN15E6 plasmid were digested simultaneously with NcoI and BglII, and ligated to construct a recombinant expression plasmid. The recombinant expression plasmids were identified by PCR using pN15-insChKUp3 and pN15-insChKDn2 as primers (Table 1).

(2)flhDC^STMOverexpression of lethal E.coli DH31 soplacI:

transforming pN15E6 recombinant plasmids of different flagellum genes into DH31 soprocI strains, selecting monoclone for overnight culture, and diluting an overnight culture 10 by taking sterile LB liquid⁶Double, spread on kanamycin chloride (Kan + Chl) double resistant LB agar plate and Kan + Chl double resistant LB agar plate containing 0.4mM IPTG, 37 ℃ culture for 24h, observe the results.

The recombinant plasmid pN15E6-flhDC^STMTransformation of DH31 soprocI strain followed by plate culture revealed that it contained flhDC^STMThe kanamycin-chloramphenicol plates had a large amount of bacterial growth, while the corresponding inducer-containing plates had no bacterial growth. Indicating flhDC^STMThe gene overexpression can kill Escherichia coli DH31 soprocI. The specific test results are shown in FIG. 4.

(3)flhD^STM、flhC^STMOverexpression of non-lethal E.coli DH31 soprocI alone:

the recombinant plasmid pN15E6-flhD was introduced^STMAnd pN15E6-flhC^STMAfter the DH31 soprocI strain is respectively transformed, the plate coating culture is carried out, and the result shows that flhD^STMAnd flhC^STMOverexpression alone did not kill E.coli DH31 soplacI. Thus indicating flhDC^STMGene overexpression lethal Escherichia coli DH31 soprocI is prepared from flhD^STMAnd flhC^STMTwo genes are expressed simultaneously (combined into FlhD)₄C₂Flagellin major regulatory protein) rather than either alone. The specific test results are shown in FIG. 5, and FIG. 5 shows pN15E6-flhD^STMResults of experiments with non-lethal E.coli DH31 soprocI due to flhC^STMAnd flhD^STMThe experimental results are similar and the figure is omitted.

(4)flhD^STMAnd flhC^STMCo-expression of lethal e.coli DH31 soplacI:

pKDb-flhD^STMconstruction and identification of recombinant expression plasmids: using pN15E6-flhD^STMPlasmid as template, Trrn _ up1 and T0_ dn1 as primers (Table 1), and flhD was PCR-amplified^STMThe expression cassette is connected with a T-vector prepared by XcmI enzyme digestion pKDb plasmid to construct a recombinant expression vector. And identifying the recombinant expression plasmid by PCR by taking pKDb _ SeqR and pKDb _ SeqF as primers.

Recombinant plasmid pKDb-flhD^STM(carrying the amp + resistance gene, ampicillin resistance in the transformant) and pN15E6-flhC^STM(with kan + resistance gene, kanamycin resistance of the transformant) simultaneous transformation of DH31soplacI strain followed by plating, and results showed flhD^STMAnd flhC^STMSimultaneous overexpression with flhDC^STMThe results of the two-gene over-expression lethal host bacteria are the same. Further indicating flhD^STMAnd flhC^STMAt the same time, the expression is performed to assemble FlhD₄C₂Flagella major regulatory protein, FlhD₄C₂An excess of flagellar major regulatory protein leads to death of the host bacteria.

(5) pN15E6-flhDC^STMDetermination of growth curves of E.coli DH31soplacI Strain:

for the vector containing pN15E6-flhDC^STMColi DH31soplacI strain of plasmid was subjected to the determination of growth curve. Culturing pN15E6-flhDC^STMAn overnight culture of plasmid E.coli DH31soplacI strain was inoculated in LB liquid medium at 0.5% and cultured at 37 ℃ to OD₆₀₀About.0.5, different concentrations of IPTG (final IPTG concentrations 0, 0.0004mM, 0.0015625mM, 0.00625mM, 0.025mM, 0.1mM, 0.4mM, respectively) were added and samples were taken each half hour of induction for OD determination₆₀₀And drawing a curve chart.

The results of the growth curve measurements are shown in FIG. 6. The results show that bacterial growth is similar to that of the control induced without IPTG when the IPTG concentration is 0.0004mM and 0.0015625 mM; when the IPTG concentration is 0.00625mM and 0.025mM, the bacteria begin to generate a certain degree of growth inhibition after being induced for 4 hours; whereas, at IPTG concentrations of 0.1mM and 0.4mM, growth inhibition occurred after 1.5h of induction of the bacteria, followed by OD₆₀₀The value begins to drop, indicating that the bacteria died and the amount of bacteria decreased.

(6) pN15E6-flhDC^STMDetermination of the E.coli DH31soplacI Strain motility test: for the vector containing pN15E6-flhDC^STMColi DH31soplacI strain of plasmid was subjected to the kinetic test assay. The method comprises the following steps: respectively taking 2 mu L of pN15E6-flhDC^STMAn overnight culture of the E.coli DH31 soprocI strain of the plasmid was spotted at different IPTG concentrations (0, 0.0004mM, 0.0015625mM, 0.00625mM, 0.025 mM)mM, 0.1mM) of a sports agar plate (without corresponding antibiotics), culturing for 6-7 h at 37 ℃, and evaluating the influence of the salmonella flhDC gene expression on the growth and the power of escherichia coli host bacteria by measuring the diameter of a colony on the semisolid plate.

The results show that at IPTG concentrations of 0.0004mM, 0.0015625mM and 0.00625mM, the bacterial motility (i.e.the size of the colony grown by the bacteria on the kinetic plate) is higher than that of the control without IPTG, and increases in sequence with increasing inducer concentration. When the IPTG concentration is 0.0004mM and 0.0015625mM, the growth curve of the induced strain is basically consistent with that of a control, the bacterial power is enhanced, and the flhDC gene is induced to express but the expression quantity does not reach the dose for inhibiting the growth of the host bacteria; when the concentration of IPTG is 0.00625mM, the growth curve of the induced strain shows a certain degree of inhibition, but the bacterial colony of the bacterial growth is still larger than that of a control, even the bacterial colony size of the IPTG induced strain is higher than that of the IPTG induced strain with the concentrations of 0.0004mM and 0.0015625mM, which indicates that the inhibition effect of the flhDC gene product on the bacterial power can be counteracted by the enhancement effect of the flhDC gene product on the bacterial power, and the comprehensive result still shows that the bacterial growth has larger bacterial colony, and the bacteria have stronger power; when the IPTG concentration is increased to 0.025mM, the inhibition effect of the flhDC product on the growth of host bacteria cannot be counteracted probably due to the enhancement effect of the flhDC product induced to express on the bacterial power, and the colony of the bacterial growth is reduced; when the IPTG concentration was increased to 0.1mM, bacterial growth was completely inhibited and no colony growth was observed. The results of the bacterial motility test are shown in Table 2.

TABLE 2 pN15E6-flhDC^STMResults of the kinetic test of Escherichia coli DH31soplacI Strain

Note: the kinetic test results are expressed in terms of colony size on the kinetic plate, and the test data is obtained from three reproducible test results.

Example 3

Flagella main regulation gene flhDC^EcoliOverexpression of the P.coliEffect of survival of bacillus DH31soplac strain.

(1)flhDC^EcoliOverexpression inhibits growth of E.coli DH31 soplacI:

the procedure is as in example 2, plasmid pN15E6-flhDC^EcoliTransformation of DH31soplacI strain followed by plating. The results showed that the contained flhDC^EcoliThe kanamycin-chloramphenicol plates had a large amount of bacteria growth, while the bacterial colonies growth was smaller on the corresponding inducer-containing plates. Indicating flhDC^EcoliOverexpression can inhibit the growth of E.coli DH31 soprocI. The specific test results are shown in FIG. 7.

(2)flhD^Ecoli、flhC^EcoliOverexpression of non-lethal E.coli DH31soplac alone:

the procedure is as in example 2, plasmid pN15E6-flhD^EcoliAnd pN15E6-flhC^EcoliThe DH31soplacI strain was transformed separately and plated for culture. The results showed flhD^EcoliAnd flhC^EcoliOverexpression alone did not kill E.coli DH31 soplacI. Thus indicating flhDC^EcoliThe gene over-expression inhibits the growth of Escherichia coli DH31 soprocI^EcoliAnd flhC^EcoliTwo genes are expressed simultaneously (combined into FlhD)₄C₂Flagellin major regulatory protein) rather than either alone. The specific test results are shown in FIG. 8, in which FIG. 8 shows pN15E6-flhD^EcoliResults of experiments with non-lethal E.coli DH31soplacI, flhC^EcoliAnd flhD^EcoliThe experimental results are similar and the figure is omitted.

Example 4

Effect of overexpression of other flagellar genes on the survival of E.coli DH31soplacI strains.

(1)fliA^STMOverexpression of lethal E.coli DH31 soplacI:

to verify whether other flagella genes involved also had a similar phenomenon of overexpression of the flhDC gene, lethal E.coli DH31soplacI, pN15E6-fliA was used in this experiment^STMpN15E6-fliT, pN15E6-fliZ, pN15E6-flgM, pN15E6-flgK, pN15E6-fliI, pN15E6-fliC and pN15E6-motA plasmids into DH31soplac strains, and then plating and culturing (the concrete method is the same as the embodiment for implementing the method for transforming the DH31soplac strains into the pN15E6-fliI strainsExample 2). The results showed fliA^STMThe gene over-expression can kill Escherichia coli DH31soplac I, while other flagella genes such as fliT, fliZ, flgM, fliI and the like do not have the effect of over-expressing the lethal Escherichia coli DH31soplac I, and a plate picture of a specific test result is shown in FIG. 9.

(2)fliA^EcoliOverexpression of lethal E.coli DH31 soplacI:

mixing pN15E6-fliA^EcoliThe DH31soplacI strain was transformed with the plasmid and plated (as described in example 2). The results showed fliA^EcoliOverexpression of the gene likewise leads to lethality in E.coli DH31 soplacI. The image of the plate for the specific test results is shown in FIG. 10.

Sequence listing

<110> Zhejiang university

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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 (1)

1. Flagella main regulation gene flhDC or its expression product FlhD₄C₂Application of protein as target spot in screening antibacterial drugs, and antibacterial drugsThe composition is used for killing or inhibiting bacteria with flagellar activity,

the antibacterial drug can up-regulate the expression of flagella major regulatory gene flhDC or enhance FlhD₄C₂The activity of the protein is determined by the activity of the protein,

the antibacterial agent is used for killing or inhibiting salmonella or escherichia coli.

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