CN114645063B - Melioidosis bacteria expression plasmid, fluorescence labeling expression plasmid, construction method and application thereof - Google Patents

Abstract

The invention provides a group of expression plasmids for fluorescence labeling of Boeck hollandis melioidis living bodies, and further provides an expression plasmid containing red, green or blue fluorescent protein. The invention also discloses a constituent element, a restriction enzyme site and a gene sequence of the fluorescence expression plasmid, a method for carrying out living fluorescence labeling on melioidosis bacteria, and further discloses application of the red fluorescence labeling melioidosis bacteria in infection pathogenicity research. The invention provides a group of live fluorescence labeling melioidosis bacteria plasmid, which is helpful for dynamic research on melioidosis bacteria infection pathogenic process.

Description

Melioidosis bacteria expression plasmid, fluorescence labeling expression plasmid, construction method and application thereof

Technical Field

The invention belongs to the technical field of bacterial genetic engineering, and particularly relates to three expression vectors, reagents, preparation methods and applications for living body fluorescent tracing of burkholderia melioides.

Background

Burkholderia melioidis (B. Pseudomelioidis) is short for farinosus melioidis, is a gram-negative short bacillus capable of causing farinosis diseases of people and livestock, has the characteristics of B-class biological threat factors and I-class biological terrorist agents, seriously threatens the health and public health safety of human beings, and is mainly distributed in tropical and subtropical regions, including cities such as Hainan, guangzhou, hongkong and the like in China and military strategic regions such as south sea, taihai and the like. In view of the potential threat of melioidosis to public health safety, the strengthening of the research of diagnosis and treatment of melioidosis has important significance, and the strengthening depends on the elucidation of the molecular mechanism of infection and pathogenesis of melioidosis. The development of a convenient and simple live bacteria marking tool is helpful for dynamically observing the infection and pathogenic process of the bacteria in cells, tissues or animal models, and is an important tool for researching the infection and pathogenic molecular mechanism of melioidosis.

At present, some conventional marking methods are established, such as DAPI and other related organic fluorescent dyes direct staining, melioidosis bacteria specific antibody immunofluorescence staining and other traditional means. The organic dye needs to enter living cells to be combined with DNA, inevitably has toxic action on organisms, influences the normal physiological activity of the marked objects, and can cause environmental pollution and harm to experimenters in the using process. For immunofluorescence staining, an antibody of melioidosis bacteria needs to be obtained in advance, the preparation process of the antibody is long in time consumption and high in cost, whether other bacteria of the same species can be distinguished or not needs to be considered in the prepared antibody, such as Burkholderia Thailand and Burkholderia cepacia, and the purpose that cells need to be fixed and cannot be observed in real time needs to be achieved by the method. In addition, the existing labeling technology mainly depends on fluorescent dye, and the fluorescent dye also has the problem that a fluorescent signal is easy to quench, so that the existing melioidosis tracing technology is inconvenient to dynamically observe the interaction between melioidosis and a host in real time. The fluorescent protein marking system is a molecular marking technology different from a fluorescent dye method, and can achieve the aim of visually and dynamically observing the interaction between pathogenic bacteria and a host by expressing fluorescent protein in melioidosis bacteria without influencing the normal activity of the bacteria so as to carry out living body marking, and has the advantages of simple operation, economy and the like. At present, the method is not applied to the research of melioidosis bacteria.

Disclosure of Invention

The invention aims to: aiming at the defects or improvement requirements of the prior research technology, three different fluorescent labeled melioidosis bacteria tracing vectors, preparation methods and applications are provided, and the purpose is to meet the requirement of direct real-time observation of melioidosis bacteria in an infection model.

The technical scheme provided by the invention is as follows:

in order to achieve the above objects, according to one aspect of the present invention, there is provided a melioidosis bacterium expression plasmid, which is prepared by replacing a lactose operon of a backbone plasmid of pUCP28T by a 16s ribosomal RNA endogenous Promoter (16 s-Promoter).

In one embodiment according to the present invention, the nucleotide sequence of the melioidosis bacteria expression plasmid is SEQ ID NO. 1.

The invention provides a fluorescence labeling expression plasmid for melioidosis bacteria on the other hand, which is prepared by inserting the coding gene of fluorescent protein into the reading frame of 16s-Promoter of the melioidosis bacteria expression plasmid; the fluorescent protein is selected from any one of GFP, CFP, BFP, RFP, EGFP, ECFP, EBFP, YFP, mHoneyde, mBanana, mOrange, tdTomato, mTangerine, mStrawberry, mCherry, mGrape1, mRaspberry, mGrape2 and mPlum; preferably, the fluorescent protein is selected from one of GFP, BFP and RFP.

In one embodiment according to the invention, the nucleotide sequence of the 16s-Promoter is SEQ ID NO 18.

In one embodiment according to the present invention, the 16s-Promoter is obtained by amplifying a primer pair having the nucleotide sequences of SEQ ID NO. 7 and SEQ ID NO. 8 from a gene vector containing the 16s ribosomal RNA endogenous Promoter of the genus melioidosis; preferably, the gene vector is melioidosis bacteria BPC006 genome DNA.

In one embodiment according to the present invention, the nucleotide sequence of BFP is SEQ ID NO 2, the nucleotide sequence of GFP is SEQ ID NO 3 and the nucleotide sequence of RFP is SEQ ID NO 4.

In one embodiment according to the present invention, the nucleotide sequence of the fluorescently labeled expression plasmid of melioidosis bacteria is SEQ ID NO. 15, SEQ ID NO. 16 or SEQ ID NO. 17.

In another aspect of the present invention, a method for preparing the above fluorescence labeling expression plasmid is provided, which comprises:

1) Amplifying by taking a fluorescent protein coding gene vector as a template to obtain a coding gene of the fluorescent protein; preferably, the fluorescent protein is selected from any one of GFP, CFP, BFP, RFP, EGFP, ECFP, EBFP, YFP, mHoneydew, mbana, mororange, tdTomato, mTangerine, mStrawberry, mCherry, mgrap 1, mrasperry, mgrap 2 and mPlum; preferably, the fluorescent protein is selected from one of GFP, BFP and RFP;

2) Inserting the fluorescent protein coding gene into the 16s-Promoter reading frame of the rhinoceroids expression plasmid to obtain the recombinant rhinoceroids.

The expression plasmid is characterized in that on the basis of pUCP28T, a lactose operon sequence is replaced by a melioidosis bacterium 16s ribosomal RNA endogenous Promoter 16s-Promoter sequence; the expression region is a fluorescent protein gene sequence of which the 5 'end is added with an XbaI enzyme cutting site and the 3' end is added with a HindIII enzyme cutting site; the fluorescent protein gene sequence is positioned in the reading frame of 16s-Promoter of 16s ribosomal RNA endogenous Promoter of melioidosis bacteria.

In an embodiment according to the present invention, the nucleotide sequences of the melioidosis bacteria expression plasmid pUCP28TL and the three fluorescent protein genes are SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, and SEQ ID NO. 4, respectively.

In an embodiment according to the present invention, the nucleotide sequences of the upstream and downstream primers of the pUCP28T plasmid amplified fragment are SEQ ID NO. 5 and SEQ ID NO. 6, respectively; the nucleotide sequences of the upstream and downstream primers of the 16s-Promoter are respectively SEQ ID NO. 7 and SEQ ID NO. 8; the nucleotide sequences of the upstream and downstream gfp primers are respectively SEQ ID NO 9 and SEQ ID NO 10; the nucleotide sequences of the rfp upstream and downstream primers are respectively SEQ ID NO. 11 and SEQ ID NO. 12; the nucleotide sequences of the upstream and downstream bfp primers are respectively SEQ ID NO. 13 and SEQ ID NO. 14.

In one embodiment according to the invention, the nucleotide sequence of the pUCP28TL-GFP plasmid is SEQ ID NO. 15, the nucleotide sequence of the pUCp28TL-BFP plasmid is SEQ ID NO. 16, the nucleotide sequence of the pUCP28TL-RFP plasmid is SEQ ID NO. 17, and the resistance screening gene is a trimethoprim resistance gene; the expression regions each individually comprise a fluorescent protein; preferably, the expression region of the plasmid is cut by using restriction endonuclease, and other target proteins can be expressed alternatively; more preferably, the promoter region of the plasmid is cleaved with a restriction endonuclease, which can replace other prokaryotic promoters.

In one embodiment according to the present invention, the fluorescent protein is selected from any one of a BFP-encoding gene primer pair consisting of SEQ ID NO. 9 and SEQ ID NO. 10, a GFP-encoding gene primer pair consisting of SEQ ID NO. 11 and SEQ ID NO. 12, and an RFP-encoding gene primer pair consisting of SEQ ID NO. 13 and SEQ ID NO. 14; preferably, the melioidosis bacteria expression plasmid and the fluorescent protein coding gene are respectively subjected to enzyme digestion by using restriction enzymes, and then the enzyme digestion products are connected, so that the fluorescent protein coding gene is inserted into the melioidosis bacteria expression plasmid.

The invention further provides a fluorescence-labeled melioidosis bacterium, which comprises the fluorescence-labeled expression plasmid.

The invention also provides a preparation method and an electrotransformation method of the melioidosis bacterium electrotransformation competence.

The invention further provides detection of the fluorescence expression stability of the recombinant plasmid.

The invention further provides application of the recombinant plasmid in immunofluorescence detection and dynamic observation of melioidosis bacteria and autophagy marker molecules.

The invention has the beneficial effects that:

the red/green/blue three fluorescence labeling expression plasmids provided by the invention can safely and stably label melioidosis bacteria. After the melioidosis bacterium electrotransformation competence is obtained according to the embodiment 3, the fluorescence labeling expression plasmid required by the experiment is electrically transformed into the melioidosis bacterium according to the embodiment 4, and the melioidosis bacterium required to be labeled by fluorescence can be obtained on a trimethoprim LB plate, so that the traditional method for the transformation of melioidosis bacterium through double-parent hybridization or triple-parent hybridization is simplified. Meanwhile, the plasmid can also be used for tentatively marking other bacteria including Escherichia coli. The invention provides a group of tools for marking melioidosis bacteria by living fluorescence, which can systematically and accurately observe the behavior state change of the living melioidosis bacteria after infecting a host, the interaction with the host and the like, does not influence the proliferation and the living toxicity of the bacteria, provides a necessary practical tool for the distribution, colonization and survival conditions of the melioidosis bacteria in organisms and the research of pathogenic mechanisms thereof, and has good application prospect and development space.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

FIG. 1 shows the result of amplification of the endogenous Promoter 16s-Promoter of melioidosis bacterium in example 1 of the present invention; wherein M is DL-5000Marker;1 is pUCP28T vector amplification product; 2 is 16s-Promoter gene amplification product; 3 XbaI/KpnI double enzyme digestion identification of pUCP28TL;

FIG. 2 shows the result of PCR amplification of gfp/bfp/rfp genes in example 2 of the present invention; wherein M is DL-2000Marker;1 is gfp amplification product; 2 is bfp amplification product; 3 is rfp amplification product; 4 is negative control;

FIG. 3 shows the restriction enzyme analysis result of the recombinant plasmid in example 2 of the present invention; wherein M is DL-2000Marker;1 is pUCP28TL;2 is pUCP28TL-GFP;3 is pUCP28TL-RFP;4 is pUCP28TL-BFP;5-8, respectively identifying pUCP28TL, pUCP28TL-GFP, pUCP28TL-RFP and pUCP28TL-BFP vectors by double digestion of XbaI/HindIII;

FIG. 4 is a plasmid map of fluorescence labeling expression plasmids containing BFP, GFP or RFP melioidis respectively prepared by the present invention;

FIG. 5 is a fluorescence micrograph of an electrically transformed recombinant melioidosis smear in example 4 of the present invention;

FIG. 6 is a fluorescent colony of recombinant fluorescent melioidosis bacterium of example 5 of the present invention after passage of 30 generations;

FIG. 7 is an immunofluorescence map of RAW264.7 cells infected with recombinant red fluorescent gangrene bacteria in example 6 of the present invention;

FIG. 8 is the dynamic process of LC 3-encapsulated pathogenic bacteria after infection with red fluorescence-labeled melioidosis in example 6 of the present invention.

Detailed Description

The following detailed description of the preferred embodiments of the present invention is provided to enable those skilled in the art to more readily understand the advantages and features of the present invention and to clearly define the scope of the invention.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The following examples are merely illustrative of the present invention and should not be construed as limiting the scope of the invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

Example 1 construction of melioidosis bacterium expression vector pUCP28TL

The nucleotide sequence of 16s-Promoter gene obtained by amplifying the Promoter 16s-Promoter gene using farg Y, huang Y, li Q, chen H, yao Z, pan J, gu J, tang B, wang HG, yu B, tong YG, zou QM, mao XH.first genome sequence of a Burkholderia pseudolari in China, strain BPC006, and organism free from a melioidosis source in Hainan.J.Bacteriol.2012Dec 194 (23): 6604-5.) as template and primers SEQ ID NO:7 and SEQ ID NO:8 synthesized by HuamaoGeneSci Limited, is SEQ ID NO:18. Similarly, pUCP28T vector gene fragments were amplified using plasmid pUCP28T (purchased from Biovector NTCC) as a template and primers SEQ ID NO:5 and SEQ ID NO:6, which were synthesized by Huada Gene science, inc., and the sequences of the primers are shown in Table 1.

PCR reaction (50. Mu.L): 2 x Prime

GC Buffer 25. Mu.L, dNTP mix 4. Mu.L, upstream and downstream primers (10. Mu. Mol/L) each 1. Mu.L, template DNA 1. Mu.L, prime ^ 4>

HS DNA Polymerase (2.5U/. Mu.L) 0.5. Mu.L, DMSO 2. Mu.L, ddH2O 16.5mL, where Prime @>

HS DNA Polymerase, dNTP mix and 2 XPrime

GC Buffer (shanghai basheng organism); and (3) PCR reaction conditions: pre-denaturation at 95 ℃ for 5min; denaturation at 98 ℃ for 10s, annealing at 58 ℃ for 30s, and extension at 72 ℃ for 1min for 35 cycles; extending for 1.5min at 72 ℃; keeping at 4 ℃.

Preparing 1% agarose gel, fully and uniformly mixing 6 × Loading Buffer and PCR reaction products, loading, confirming the position of a strip by a gel imager, and cutting a target gene fragment by an ultraviolet instrument as shown in figure 1; recovering the target fragment by using a gel recovery kit (Tiangen Biochemical technology Co., ltd.); the purified fragment is subjected to double enzyme digestion by XbaI and KpnI, then is subjected to overnight ligation at 16 ℃ by using T4 DNA ligase (Baori doctor technology), a ligation product is subjected to heat shock to transform escherichia coli DH5 alpha competent cells, then is coated on an LB plate containing 250 mu g/mL trimethoprim for screening, a positive monoclonal colony is picked and subjected to shaking culture in an LB liquid culture medium containing 250 mu g/mL trimethoprim, a small amount of bacterial liquid is taken and sent to Huada science and technology Limited for sequencing verification, the rest bacterial liquid is centrifuged to obtain an upgraded particle, and the double enzyme digestion verification is carried out by using XbaI and KpnI. The size of the constructed carrier enzyme cutting electrophoresis strip is correct, the sequencing result is consistent with the theoretical sequence, the melioidosis bacteria expression plasmid pUCP28TL is successfully constructed, and the base sequence is SEQ ID NO:1.

Example 2 construction of recombinant expression plasmid pUCP28TL-GFP/BFP/RFP

Designing primers GFP-F/R and RFP-F/R according to GFP and RFP gene sequences of a GFP-RFP-LC3 plasmid (purchased from Biovector NTCC), inserting XbaI and HindIII enzyme sites into the 5 'end of the GFP-F/R primer respectively, and inserting XbaI and SpeI enzyme sites into the 5' end of the RFP-F/R primer respectively; similarly, primers BFP-F/R were designed based on the BFP gene of pCMV-N-BFP plasmid (purchased from Shanghai Binshi Biotechnology Co., ltd.), and XbaI and HindIII enzyme sites were inserted into the 5' ends of the upstream and downstream primers. The primers were synthesized by Huada Gene science and technology, inc. The gene sequences of the three fluorescent proteins are shown in a sequence table at the end of the text, and the primer sequences are shown in a table 1 in detail. The amplification results are shown in FIG. 2.

The gfp/bfp gene recovered from the gel and melioidosis bacteria expression plasmid pUCP28TL are subjected to double enzyme digestion by XbaI and HindIII, T4 DNA ligase (Baoriri doctor technology) is used for overnight connection at 16 ℃, a connection product is subjected to heat shock to transform escherichia coli DH5 alpha competent cells, then the competent cells are coated on an LB plate containing 250 mu g/mL trimethoprim for screening, a positive monoclonal colony is selected and is subjected to shaking culture in an LB liquid culture medium containing 250 mu g/mL trimethoprim, a small amount of bacterial liquid is taken and sent to Huada technology Limited for sequencing verification, the rest bacterial liquid is centrifuged, and then quality-improving grains are obtained and are subjected to double enzyme digestion verification by XbaI and HindIII. Because two HindIII enzyme cutting sites exist on the RFP gene, the constructed pUCP28TL-BFP plasmid and the RFP gene need to be connected after XbaI and SpeI double enzyme cutting, the pUCP28TL-RFP plasmid is obtained in the same way, and XbaI and SpeI double enzyme cutting is used for verification. Electrophoresis results show that corresponding bands appear at the corresponding molecular weight positions of gfp/bfp/rfp genes after double enzyme digestion of the three recombinant plasmids (figure 3), and sequencing results of the corresponding plasmids are correct. The map of the resulting expression plasmid is shown in FIG. 4.

TABLE 1 construction and validation of fluorescent plasmids ^a Restriction sites are underlined.

Example 3 preparation of melioidosis bacteria BPC006 electrotransformation competence

Selecting BPC006 single colony from LB solid plate, adding 5mL SOB culture medium (containing tryptone 20g, yeast extract 5g, naCl 0.5g,250mM KCl 10mL, sterilizing at 121 deg.C, and adding sterile 2M MgCl 5mL ₂ ) Shaking the flask overnight, inoculating 250. Mu.L of overnight culture solution into 25mL of SOB medium according to the proportion of 100 is 0.6-0.8 (detected by a microplate reader, bio-Rad, USA). Placing the bacterial liquid on ice for 20min, then centrifuging for 10min at 4 ℃ and 2000g, washing 50mL of precooled sterile ddH2O once, then centrifuging, washing the bacterial precipitate for 2 times by 25mL of precooled 10% sterile glycerol, centrifuging, retaining the bacterial precipitate, adding 500 mu L of 10% glycerol for resuspension, and packaging each tube of 100 mu L of the bacterial precipitate at-80 ℃ for temporary storage.

EXAMPLE 4 electroporation of recombinant plasmid BPC006 competent cells and selection of fluorescent Strain

50 μ L of the Vibrio paranasal ulcer sensitive cells BPC006 and 5 μ L of the recombinant plasmid (500-1000 ng) are mixed on ice, added into a 0.2cm electric cup precooled on ice, and placed on ice for 10min. Micropulser (Bio-Rad, USA) has a reaction condition of 3kV,200 omega, after 25 muF electric shock (time constant is 4.5-5 mS), 1mL SOC culture medium preheated at 37 ℃ (2 mL sterile 1M glucose solution is added to each 100mL SOB culture medium), the SOC culture medium is transferred to 1.5mL centrifuge tube, the cell is incubated at 37 ℃ with oscillation for 2 hours, 200 muL bacterial liquid is taken to coat LB plate containing 250 mug/mL trimethoprim, the cell is incubated at 37 ℃ for 36 hours, single colony is picked up and suspended in appropriate 4% paraformaldehyde for fixation, 10 muL is taken to drop on a glass slide, observation is carried out under a fluorescence microscope, and melioidia melioidis successfully screened to express three fluorescent proteins (figure 5).

Example 5 detection of expression stability of fluorescent vector in BPC006

Inoculating fluorescent melioidosis-like strain BPC006 into a nonresistant LB culture medium, culturing at 37 ℃, transferring into a fresh nonresistant LB culture medium at an interval of 16-18 h according to a proportion of 0.1%, continuously carrying out passage for 30 times, sampling, diluting in a proper amount, coating on nonresistant LB plates (3 times), placing in an incubator at 37 ℃ for culturing for 36 hours, and carrying out iBright imaging in a multifunctional intelligent imaging system ^TM And (3) photographing and observing the fluorescent colonies under CL1500 (Thermo company in America), and calculating the number of the fluorescent colonies/the total number of the colonies, namely the stable expression rate of the fluorescent vector. The results show that after 30 times of serial dilution and passage, colonies with fluorescence can still be cultured on a non-anti LB plate (figure 6), and the percentage of the fluorescent colonies to the total colonies is 92 plus or minus 5 percent (green fluorescent bacteria) and 95 plus or minus 13 percent (red fluorescent bacteria), which indicates that the recombinant fluorescent melioidosis bacteria can still express fluorescent protein when the passage is 30,is labeled with fluorescence.

Example 6 immunofluorescence detection and kinetic visualization of Fluorogenic Fangmei bacteria and LC3

Autophagy is a means by which host cells immunoreact with a pathogen via the autophago-lysosomal pathway by encapsidating the intracellular pathogen with autophagosomes. Wherein LC3 is an autophagy-related protein light chain 3, is a marker protein of autophagy process and is mainly involved in the formation of autophagosomes.

Mouse macrophage RAW264.7 (Henan bioscience) stably expressing GFP-LC3 at 1.5X 10 ⁵ Inoculated into a laser confocal dish (. PHI.20 mm), and cultured overnight in a cell culture chamber (5% CO2, 37 ℃). Selecting red fluorescent gangrene bacteria to infect RAW264.7 cells with the multiplicity of infection MOI of 10, after 1.5 hours of infection, washing a confocal dish for 3 times by PBS, then adding kanamycin to 250 mu g/mL, after 0.5 hour of killing extracellular bacteria, washing for 3 times by PBS, replacing a fresh culture medium containing 25 mu g/mL of kanamycin, and carrying out immunofluorescence detection on a fluorescence microscope. Under a fluorescence microscope, the red fluorescence labeled melioidosis can be well distinguished from green fluorescence of GFP-LC3 (see figure 7), fluorescent antibody labeling of melioidosis is not needed, and the dynamic development process of melioidosis and host autophagy can be directly observed.

RAW264.7 cells stably expressing GFP-LC3 are cultured on a high-resolution live cell imaging system (DeltaVision, GE company, USA), and dynamic processes of pathogenic bacteria wrapped by LC3 after the cells are infected by red fluorescence-labeled melioidosis bacteria are observed. After the melioidosis bacteria is infected, macrophages can form tentacles to actively capture the melioidosis bacteria, the bacteria are taken into cells through phagocytosis, and the melioidosis bacteria enter host cells to trigger LC3 aggregation to quickly form autophagosome wrapping the melioidosis bacteria (see figure 8).

In conclusion, the three fluorescent expression plasmids provided by the invention comprise the components, the enzyme cutting sites and the gene sequences, and the method for marking the living fluorescence in Burkholderia melioidea, and the plasmids can be stably expressed after being transformed into the Burkholderia melioidea. The application result of the red fluorescence labeling Burkholderia meliloti in the infection pathogenesis research proves that the method carries out fluorescence tracing on the Burkholderia meliloti, and has the characteristics of high safety coefficient and convenient and simple operation. The above summary and the detailed description are intended to demonstrate the practical application of the technical solutions provided by the present invention, and should not be construed as limiting the scope of the present invention. Various modifications, equivalent substitutions, or improvements may be made by those skilled in the art within the spirit and principles of the invention. The scope of the invention is defined by the appended claims.

Sequence listing

<110> China people liberation army, military and medical university

<120> melioidosis bacterium expression plasmid, fluorescence labeling expression plasmid, construction method and application thereof

<141> 2022-04-26

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ttccataaca tcaaacatcg acccacggcg taacgcgctt agctagcttg gatgcccgag 2640

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gacatagcga cgatggcggg tgtccctatc gaatcttcct acaaccagct caaagaagcg 3420

gccctgcgcc tgaaacggcg ggaagtccgg ttaacccaag agcccaatgg caaggggaaa 3480

agaccgagtg tgatgattac cggctgggtg caaacaatca tctaccggga gggtgagggc 3540

cgtgtagaac tcaggttcac caaagacatg ctgccgtacc tgacggaact caccaaacag 3600

ttcaccaaat acgccttggc tgacgtggcc aagatggaca gcacccacgc gatcaggctt 3660

tacgagctgc tcatgcaatg ggacagcatc ggccagcgcg aaatagaaat tgaccagctg 3720

cgaaagtggt ttcaactgga aggccggtat ccctcgatca aggacttcaa gttgcgagtg 3780

cttgatccag ccgtgacgca gatcaacgag cacagcccgc tacaggtgga gtgggcgcag 3840

cgaaagaccg ggcgcaaggt cacacatctg ttgttcagtt ttggaccgaa gaagcccgcc 3900

aaggcggtgg gtaaggcccc agcgaagcgc aaggccggga agatttcaga tgctgagatc 3960

gcgaaacagg ctcgccctgg tgagacatgg gaagcggccc gcgctcgact aacccagatg 4020

ccgctggatc tggcctagag gccgtggcca ccacggcccg gcctgccttt caggctgcgc 4080

aactgttggg aagggcgatc ggtgcgggcc tcttcgctat tacgccagct ggcgaaaggg 4140

ggatgtgctg caaggcgatt aagttgggta acgccagggt tttcccagtc acgacgttgt 4200

aaaacgacgg ccagtgccaa gcttgcatgc ctgcaggtcg actctagacc 4250

<210> 2

<211> 693

<212> DNA

<213> Boeck Hold's vibrio paranasal (B. Pseudofollallalei)

<400> 2

atgagcgagc tgattaagga gaacatgcac atgaagctgt acatggaggg caccgtggac 60

aaccatcact tcaagtgcac atccgagggc gaaggcaagc cctacgaggg cacccagacc 120

atgagaatca aggtggtcga gggcggccct ctccccttcg ccttcgacat cctggctact 180

agcttcctct acggcagcaa gaccttcatc aaccacaccc agggcatccc cgacttcttc 240

aagcagtcct tccctgaggg cttcacatgg gagagagtca ccacatacga agacgggggc 300

gtgctgaccg ctacccagga caccagcctc caggacggct gcctcatcta caacgtcaag 360

atcagagggg tgaacttcac atccaacggc cctgtgatgc agaagaaaac actcggctgg 420

gaggccttca ccgagacgct gtaccccgct gacggcggcc tggaaggcag aaacgacatg 480

gccctgaagc tcgtgggcgg gagccatctg atcgcaaaca tcaagaccac atatagatcc 540

aagaaacccg ctaagaacct caagatgcct ggcgtctact atgtggacta cagactggaa 600

agaatcaagg aggccaacaa cgagacctac gtcgagcagc acgaggtggc agtggccaga 660

tactgcgacc tccctagcaa actggggcac taa 693

<210> 3

<211> 717

<212> DNA

<213> Boeck Hold's bacterium melioideus (B. Pseudomalalei)

<400> 3

atgattaaag gagaagaact tttcactgga gttgtcccaa ttcttgttga attagatggt 60

gatgttaatg ggcacaaatt ttctgtcagt ggagagggtg aaggtgatgc aacatacgga 120

aaacttaccc ttaaatttat ttgcactact ggaaaactac ctgttccatg gccaacactt 180

gtcactactt tcacttatgg tgttcaatgc ttttcccgtt atccggatca tatgaaacgg 240

catgactttt tcaagagtgc catgcccgaa ggttatgtac aggaacgcac tatatctttc 300

aaagatgacg ggaactacaa gacgcgtgct gaagtcaagt ttgaaggtga tacccttgtt 360

aatcgtatcg agtttaaagg tattgatttt aaagaagatg gaaacattct cggacacaaa 420

ctcgagtaca accataactc acacaatgta tacatcacgg cagacaaaca aaagaatgga 480

atcaaagcta acttcaaaat tcgccacaac attgaagatg gatccgttca actagcagac 540

cattatcaac aaaatactcc aattggcgat ggccctgtcc ttttaccaga caaccattac 600

ctgtcgacac aatctgccct ttcgaaagat cccaacgaaa agcgtgacca catggtcctt 660

cttgagtttg taactgctgc tgggattaca catggcatgg atgagctcta caaataa 717

<210> 4

<211> 678

<212> DNA

<213> Boeck Hold's bacterium melioideus (B. Pseudomalalei)

<400> 4

atggcgagta gcgaagacgt tatcaaagag ttcatgcgtt tcaaagttcg tatggaaggt 60

tccgttaacg gtcacgagtt cgaaatcgaa ggtgaaggtg aaggtcgtcc gtacgaaggt 120

acccagaccg ctaaactgaa agttaccaaa ggtggtccgc tgccgttcgc ttgggacatc 180

ctgtccccgc agttccagta cggttccaaa gcttacgtta aacacccggc tgacatcccg 240

gactacctga aactgtcctt cccggaaggt ttcaaatggg aacgtgttat gaacttcgaa 300

gacggtggtg ttgttaccgt tacccaggac tcctccctgc aagacggtga gttcatctac 360

aaagttaaac tgcgtggtac caacttcccg tccgacggtc cggttatgca gaaaaaaacc 420

atgggttggg aagcttccac cgaacgtatg tacccggaag acggtgctct gaaaggtgaa 480

atcaaaatgc gtctgaaact gaaagacggt ggtcactacg acgctgaagt taaaaccacc 540

tacatggcta aaaaaccggt tcagctgccg ggtgcttaca aaaccgacat caaactggac 600

atcacctccc acaacgaaga ctacaccatc gttgaacagt acgaacgtgc tgaaggtcgt 660

cactccaccg gtgcttaa 678

<210> 5

<211> 41

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

atggtaccca tatggaattc aattgcgttg cgctcactgc c 41

<210> 6

<211> 34

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

cctctagagt cgacctgcag gcatgcaagc ttgg 34

<210> 7

<211> 24

<212> DNA

<213> Boeck Hold's bacterium melioideus (B. Pseudomalalei)

<400> 7

tcggtaccgc agtaaaaaag ttgt 24

<210> 8

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

attctagaaa ttcctgttac tgttttcggt tgtttcc 37

<210> 9

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

gctctagaat gagcgagctg attaaggaga acatg 35

<210> 10

<211> 38

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

ccaagcttac tagtttagtg ccccagtttg ctagggag 38

<210> 11

<211> 34

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

tttctagaat gattaaagga gaagaacttt tcac 34

<210> 12

<211> 32

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

ccaagctttt atttgtagag ctcatccatg cc 32

<210> 13

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

gctctagaat ggcgagtagc gaagacg 27

<210> 14

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

ggactagtta agcaccggtg gagtgacgac 30

<210> 15

<211> 4947

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa 60

atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga 120

ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt 180

gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg 240

agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga 300

gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga 360

agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg 420

catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc 480

aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc 540

gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca 600

taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtttcttag 660

gccacacgtt caagtgcagc cacaggataa atttgcactg agcctgggtg ggattcggac 720

tcgaccgcat agccttcagg agtgagtttt gtgcaatacc aaccgacgac ttgaccctgc 780

caagcggcac cagatttctt gcgtacgcga tcccctaagc caaaggtggc actcagggga 840

agcgcaaact gccctgcaac gggagcgttg gcttcatcgc tactttgacc catgtcgaat 900

ccttcttgtg aatctattat ggcgacaagc aaatcgaact ctgactgcct accccacaac 960

aactactaga aagcaccagc acaacggctg cctaactttg ttttagggcg actaccctgc 1020

tgcgtaacat cgttgctgtt ccataacatc aaacatcgac ccacggcgta acgcgcttag 1080

ctagcttgga tgcccgaggc tagactgtac aaaaaaacag tcataacaag ccatgaaaac 1140

cgccactgcg ccgttaccac cgctgcgttc ggtcaaggtt ctggaccagt tgcgtgagcg 1200

catacgctac ttgcattaca gtttacgaac cgaacaggct tatgtcaact gggttcgtga 1260

acgccgctgg tgccgctggt tggacgccaa gggtgaatcc gcctcgatac cctgattact 1320

cgcttcctgc gccctctcag gcggcgatag gggactggta aaacggggat tgcccagacg 1380

cctcccccgc cccttcaggg gcacaaatgc ggccccaacg gggccacgta gtggtgcgtt 1440

ttttgcgttt ccaccctttt cttccttttc ccttttaaac cttttaggac gtctacaggc 1500

cacgtaatcc gtggcctgta gagtttaaaa agggacggat ttgttgccat taagggacgg 1560

atttgttgtt aagaagggac ggatttgttg ttgtaaaggg acggatttgt tgtattgtgg 1620

gacgcagata cagtgtcccc ttatacacaa ggaatgtcga acgtggcctc acccccaatg 1680

gtttacaaaa gcaatgccct ggtcgaggcc gcgtatcgcc tcagtgttca ggaacagcgg 1740

atcgttctgg cctgtattag ccaggtgaag aggagcgagc ctgtcaccga tgaagtgatg 1800

tattcagtga cggcggagga catagcgacg atggcgggtg tccctatcga atcttcctac 1860

aaccagctca aagaagcggc cctgcgcctg aaacggcggg aagtccggtt aacccaagag 1920

cccaatggca aggggaaaag accgagtgtg atgattaccg gctgggtgca aacaatcatc 1980

taccgggagg gtgagggccg tgtagaactc aggttcacca aagacatgct gccgtacctg 2040

acggaactca ccaaacagtt caccaaatac gccttggctg acgtggccaa gatggacagc 2100

acccacgcga tcaggcttta cgagctgctc atgcaatggg acagcatcgg ccagcgcgaa 2160

atagaaattg accagctgcg aaagtggttt caactggaag gccggtatcc ctcgatcaag 2220

gacttcaagt tgcgagtgct tgatccagcc gtgacgcaga tcaacgagca cagcccgcta 2280

caggtggagt gggcgcagcg aaagaccggg cgcaaggtca cacatctgtt gttcagtttt 2340

ggaccgaaga agcccgccaa ggcggtgggt aaggccccag cgaagcgcaa ggccgggaag 2400

atttcagatg ctgagatcgc gaaacaggct cgccctggtg agacatggga agcggcccgc 2460

gctcgactaa cccagatgcc gctggatctg gcctagaggc cgtggccacc acggcccggc 2520

ctgcctttca ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta 2580

cgccagctgg cgaaaggggg atgtgctgca aggcgattaa gttgggtaac gccagggttt 2640

tcccagtcac gacgttgtaa aacgacggcc agtgccaagc ttttatttgt agagctcatc 2700

catgccatgt gtaatcccag cagcagttac aaactcaaga aggaccatgt ggtcacgctt 2760

ttcgttggga tctttcgaaa gggcagattg tgtcgacagg taatggttgt ctggtaaaag 2820

gacagggcca tcgccaattg gagtattttg ttgataatgg tctgctagtt gaacggatcc 2880

atcttcaatg ttgtggcgaa ttttgaagtt agctttgatt ccattctttt gtttgtctgc 2940

cgtgatgtat acattgtgtg agttatggtt gtactcgagt ttgtgtccga gaatgtttcc 3000

atcttcttta aaatcaatac ctttaaactc gatacgatta acaagggtat caccttcaaa 3060

cttgacttca gcacgcgtct tgtagttccc gtcatctttg aaagatatag tgcgttcctg 3120

tacataacct tcgggcatgg cactcttgaa aaagtcatgc cgtttcatat gatccggata 3180

acgggaaaag cattgaacac cataagtgaa agtagtgaca agtgttggcc atggaacagg 3240

tagttttcca gtagtgcaaa taaatttaag ggtaagtttt ccgtatgttg catcaccttc 3300

accctctcca ctgacagaaa atttgtgccc attaacatca ccatctaatt caacaagaat 3360

tgggacaact ccagtgaaaa gttcttctcc tttaatcatt ctagaaattc ctgttactgt 3420

tttcggttgt ttccaaccgg tcgctcactc aaagctgaca ggtatatgaa tctcttcata 3480

aacctgactt actttagtgt gagactcttg atactttcgc ttgcgatccg aaaatcgccc 3540

gcttccatca agcgcccaca cttatcggct gttcattgtt aaagagcata tccgcgagaa 3600

gattcgacta cttaccgcac ccttctcagc agcgctgcgt tgtcagcagc agagaaatga 3660

gattatgatc acttattcgc agcgcgtcaa caactttttt actgcggtac ccatatggaa 3720

ttcaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca 3780

ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc 3840

ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc 3900

aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatggggat 3960

tccttaaggt atactttccg ctgcataacc ctgcttcggg gtcattatag cgattttttc 4020

ggtatatcca tcctttttcg cacgatatac aggattttgc caaagggttc gtgtagactt 4080

tccttggtgt atccaacggc gtcagccggg caggataggt gaagtaggcc cacccgcgag 4140

cgggtgttcc ttcttcactg tcccttattc gcacctggcg gtgctcaacg ggaatcctgc 4200

tctgcgaggc tggccgataa gctagcttat gtgagcaaaa ggccagcaaa aggccaggaa 4260

ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca 4320

caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc 4380

gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata 4440

cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta 4500

tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca 4560

gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga 4620

cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg 4680

tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg 4740

tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg 4800

caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag 4860

aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa 4920

cgaaaactca cgttaaggga ttttggt 4947

<210> 16

<211> 4931

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa 60

atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga 120

ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt 180

gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg 240

agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga 300

gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga 360

agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg 420

catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc 480

aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc 540

gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca 600

taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtttcttag 660

gccacacgtt caagtgcagc cacaggataa atttgcactg agcctgggtg ggattcggac 720

tcgaccgcat agccttcagg agtgagtttt gtgcaatacc aaccgacgac ttgaccctgc 780

caagcggcac cagatttctt gcgtacgcga tcccctaagc caaaggtggc actcagggga 840

agcgcaaact gccctgcaac gggagcgttg gcttcatcgc tactttgacc catgtcgaat 900

ccttcttgtg aatctattat ggcgacaagc aaatcgaact ctgactgcct accccacaac 960

aactactaga aagcaccagc acaacggctg cctaactttg ttttagggcg actaccctgc 1020

tgcgtaacat cgttgctgtt ccataacatc aaacatcgac ccacggcgta acgcgcttag 1080

ctagcttgga tgcccgaggc tagactgtac aaaaaaacag tcataacaag ccatgaaaac 1140

cgccactgcg ccgttaccac cgctgcgttc ggtcaaggtt ctggaccagt tgcgtgagcg 1200

catacgctac ttgcattaca gtttacgaac cgaacaggct tatgtcaact gggttcgtga 1260

acgccgctgg tgccgctggt tggacgccaa gggtgaatcc gcctcgatac cctgattact 1320

cgcttcctgc gccctctcag gcggcgatag gggactggta aaacggggat tgcccagacg 1380

cctcccccgc cccttcaggg gcacaaatgc ggccccaacg gggccacgta gtggtgcgtt 1440

ttttgcgttt ccaccctttt cttccttttc ccttttaaac cttttaggac gtctacaggc 1500

cacgtaatcc gtggcctgta gagtttaaaa agggacggat ttgttgccat taagggacgg 1560

atttgttgtt aagaagggac ggatttgttg ttgtaaaggg acggatttgt tgtattgtgg 1620

gacgcagata cagtgtcccc ttatacacaa ggaatgtcga acgtggcctc acccccaatg 1680

gtttacaaaa gcaatgccct ggtcgaggcc gcgtatcgcc tcagtgttca ggaacagcgg 1740

atcgttctgg cctgtattag ccaggtgaag aggagcgagc ctgtcaccga tgaagtgatg 1800

tattcagtga cggcggagga catagcgacg atggcgggtg tccctatcga atcttcctac 1860

aaccagctca aagaagcggc cctgcgcctg aaacggcggg aagtccggtt aacccaagag 1920

cccaatggca aggggaaaag accgagtgtg atgattaccg gctgggtgca aacaatcatc 1980

taccgggagg gtgagggccg tgtagaactc aggttcacca aagacatgct gccgtacctg 2040

acggaactca ccaaacagtt caccaaatac gccttggctg acgtggccaa gatggacagc 2100

acccacgcga tcaggcttta cgagctgctc atgcaatggg acagcatcgg ccagcgcgaa 2160

atagaaattg accagctgcg aaagtggttt caactggaag gccggtatcc ctcgatcaag 2220

gacttcaagt tgcgagtgct tgatccagcc gtgacgcaga tcaacgagca cagcccgcta 2280

caggtggagt gggcgcagcg aaagaccggg cgcaaggtca cacatctgtt gttcagtttt 2340

ggaccgaaga agcccgccaa ggcggtgggt aaggccccag cgaagcgcaa ggccgggaag 2400

atttcagatg ctgagatcgc gaaacaggct cgccctggtg agacatggga agcggcccgc 2460

gctcgactaa cccagatgcc gctggatctg gcctagaggc cgtggccacc acggcccggc 2520

ctgcctttca ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta 2580

cgccagctgg cgaaaggggg atgtgctgca aggcgattaa gttgggtaac gccagggttt 2640

tcccagtcac gacgttgtaa aacgacggcc agtgccccaa gcttactagt ttagtgcccc 2700

agtttgctag ggaggtcgca gtatctggcc actgccacct cgtgctgctc gacgtaggtc 2760

tcgttgttgg cctccttgat tctttccagt ctgtagtcca catagtagac gccaggcatc 2820

ttgaggttct tagcgggttt cttggatcta tatgtggtct tgatgtttgc gatcagatgg 2880

ctcccgccca cgagcttcag ggccatgtcg tttctgcctt ccaggccgcc gtcagcgggg 2940

tacagcgtct cggtgaaggc ctcccagccg agtgttttct tctgcatcac agggccgttg 3000

gatgtgaagt tcacccctct gatcttgacg ttgtagatga ggcagccgtc ctggaggctg 3060

gtgtcctggg tagcggtcag cacgcccccg tcttcgtatg tggtgactct ctcccatgtg 3120

aagccctcag ggaaggactg cttgaagaag tcggggatgc cctgggtgtg gttgatgaag 3180

gtcttgctgc cgtagaggaa gctagtagcc aggatgtcga aggcgaaggg gagagggccg 3240

ccctcgacca ccttgattct catggtctgg gtgccctcgt agggcttgcc ttcgccctcg 3300

gatgtgcact tgaagtgatg gttgtccacg gtgccctcca tgtacagctt catgtgcatg 3360

ttctccttaa tcagctcgct cattctagaa attcctgtta ctgttttcgg ttgtttccaa 3420

ccggtcgctc actcaaagct gacaggtata tgaatctctt cataaacctg acttacttta 3480

gtgtgagact cttgatactt tcgcttgcga tccgaaaatc gcccgcttcc atcaagcgcc 3540

cacacttatc ggctgttcat tgttaaagag catatccgcg agaagattcg actacttacc 3600

gcacccttct cagcagcgct gcgttgtcag cagcagagaa atgagattat gatcacttat 3660

tcgcagcgcg tcaacaactt ttttactgcg gtacccatat ggaattcaat tgcgttgcgc 3720

tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa 3780

cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg 3840

ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg 3900

ttatccacag aatcagggga taacgcagga aagaacatgg ggattcctta aggtatactt 3960

tccgctgcat aaccctgctt cggggtcatt atagcgattt tttcggtata tccatccttt 4020

ttcgcacgat atacaggatt ttgccaaagg gttcgtgtag actttccttg gtgtatccaa 4080

cggcgtcagc cgggcaggat aggtgaagta ggcccacccg cgagcgggtg ttccttcttc 4140

actgtccctt attcgcacct ggcggtgctc aacgggaatc ctgctctgcg aggctggccg 4200

ataagctagc ttatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg 4260

ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca 4320

agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc 4380

tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc 4440

ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag 4500

gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc 4560

ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca 4620

gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg 4680

aagtggtggc ctaactacgg ctacactaga aggacagtat ttggtatctg cgctctgctg 4740

aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct 4800

ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa 4860

gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa 4920

gggattttgg t 4931

<210> 17

<211> 4915

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa 60

atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga 120

ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt 180

gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg 240

agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga 300

gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga 360

agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg 420

catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc 480

aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc 540

gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca 600

taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtttcttag 660

gccacacgtt caagtgcagc cacaggataa atttgcactg agcctgggtg ggattcggac 720

tcgaccgcat agccttcagg agtgagtttt gtgcaatacc aaccgacgac ttgaccctgc 780

caagcggcac cagatttctt gcgtacgcga tcccctaagc caaaggtggc actcagggga 840

agcgcaaact gccctgcaac gggagcgttg gcttcatcgc tactttgacc catgtcgaat 900

ccttcttgtg aatctattat ggcgacaagc aaatcgaact ctgactgcct accccacaac 960

aactactaga aagcaccagc acaacggctg cctaactttg ttttagggcg actaccctgc 1020

tgcgtaacat cgttgctgtt ccataacatc aaacatcgac ccacggcgta acgcgcttag 1080

ctagcttgga tgcccgaggc tagactgtac aaaaaaacag tcataacaag ccatgaaaac 1140

cgccactgcg ccgttaccac cgctgcgttc ggtcaaggtt ctggaccagt tgcgtgagcg 1200

catacgctac ttgcattaca gtttacgaac cgaacaggct tatgtcaact gggttcgtga 1260

acgccgctgg tgccgctggt tggacgccaa gggtgaatcc gcctcgatac cctgattact 1320

cgcttcctgc gccctctcag gcggcgatag gggactggta aaacggggat tgcccagacg 1380

cctcccccgc cccttcaggg gcacaaatgc ggccccaacg gggccacgta gtggtgcgtt 1440

ttttgcgttt ccaccctttt cttccttttc ccttttaaac cttttaggac gtctacaggc 1500

cacgtaatcc gtggcctgta gagtttaaaa agggacggat ttgttgccat taagggacgg 1560

atttgttgtt aagaagggac ggatttgttg ttgtaaaggg acggatttgt tgtattgtgg 1620

gacgcagata cagtgtcccc ttatacacaa ggaatgtcga acgtggcctc acccccaatg 1680

gtttacaaaa gcaatgccct ggtcgaggcc gcgtatcgcc tcagtgttca ggaacagcgg 1740

atcgttctgg cctgtattag ccaggtgaag aggagcgagc ctgtcaccga tgaagtgatg 1800

tattcagtga cggcggagga catagcgacg atggcgggtg tccctatcga atcttcctac 1860

aaccagctca aagaagcggc cctgcgcctg aaacggcggg aagtccggtt aacccaagag 1920

cccaatggca aggggaaaag accgagtgtg atgattaccg gctgggtgca aacaatcatc 1980

taccgggagg gtgagggccg tgtagaactc aggttcacca aagacatgct gccgtacctg 2040

acggaactca ccaaacagtt caccaaatac gccttggctg acgtggccaa gatggacagc 2100

acccacgcga tcaggcttta cgagctgctc atgcaatggg acagcatcgg ccagcgcgaa 2160

atagaaattg accagctgcg aaagtggttt caactggaag gccggtatcc ctcgatcaag 2220

gacttcaagt tgcgagtgct tgatccagcc gtgacgcaga tcaacgagca cagcccgcta 2280

caggtggagt gggcgcagcg aaagaccggg cgcaaggtca cacatctgtt gttcagtttt 2340

ggaccgaaga agcccgccaa ggcggtgggt aaggccccag cgaagcgcaa ggccgggaag 2400

atttcagatg ctgagatcgc gaaacaggct cgccctggtg agacatggga agcggcccgc 2460

gctcgactaa cccagatgcc gctggatctg gcctagaggc cgtggccacc acggcccggc 2520

ctgcctttca ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta 2580

cgccagctgg cgaaaggggg atgtgctgca aggcgattaa gttgggtaac gccagggttt 2640

tcccagtcac gacgttgtaa aacgacggcc agtgccccaa gcttactagt taagcaccgg 2700

tggagtgacg accttcagca cgttcgtact gttcaacgat ggtgtagtct tcgttgtggg 2760

aggtgatgtc cagtttgatg tcggttttgt aagcacccgg cagctgaacc ggttttttag 2820

ccatgtaggt ggttttaact tcagcgtcgt agtgaccacc gtctttcagt ttcagacgca 2880

ttttgatttc acctttcaga gcaccgtctt ccgggtacat acgttcggtg gaagcttccc 2940

aacccatggt ttttttctgc ataaccggac cgtcggacgg gaagttggta ccacgcagtt 3000

taactttgta gatgaactca ccgtcttgca gggaggagtc ctgggtaacg gtaacaacac 3060

caccgtcttc gaagttcata acacgttccc atttgaaacc ttccgggaag gacagtttca 3120

ggtagtccgg gatgtcagcc gggtgtttaa cgtaagcttt ggaaccgtac tggaactgcg 3180

gggacaggat gtcccaagcg aacggcagcg gaccaccttt ggtaactttc agtttagcgg 3240

tctgggtacc ttcgtacgga cgaccttcac cttcaccttc gatttcgaac tcgtgaccgt 3300

taacggaacc ttccatacga actttgaaac gcatgaactc tttgataacg tcttcgctac 3360

tcgccattct agaaattcct gttactgttt tcggttgttt ccaaccggtc gctcactcaa 3420

agctgacagg tatatgaatc tcttcataaa cctgacttac tttagtgtga gactcttgat 3480

actttcgctt gcgatccgaa aatcgcccgc ttccatcaag cgcccacact tatcggctgt 3540

tcattgttaa agagcatatc cgcgagaaga ttcgactact taccgcaccc ttctcagcag 3600

cgctgcgttg tcagcagcag agaaatgaga ttatgatcac ttattcgcag cgcgtcaaca 3660

acttttttac tgcggtaccc atatggaatt caattgcgtt gcgctcactg cccgctttcc 3720

agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg 3780

gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc 3840

ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag 3900

gggataacgc aggaaagaac atggggattc cttaaggtat actttccgct gcataaccct 3960

gcttcggggt cattatagcg attttttcgg tatatccatc ctttttcgca cgatatacag 4020

gattttgcca aagggttcgt gtagactttc cttggtgtat ccaacggcgt cagccgggca 4080

ggataggtga agtaggccca cccgcgagcg ggtgttcctt cttcactgtc ccttattcgc 4140

acctggcggt gctcaacggg aatcctgctc tgcgaggctg gccgataagc tagcttatgt 4200

gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc 4260

ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa 4320

acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc 4380

ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg 4440

cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc 4500

tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc 4560

gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca 4620

ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact 4680

acggctacac tagaaggaca gtatttggta tctgcgctct gctgaagcca gttaccttcg 4740

gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt 4800

ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct 4860

tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggt 4915

<210> 18

<211> 300

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

aattcctgtt actgttttcg gttgtttcca accggtcgct cactcaaagc tgacaggtat 60

atgaatctct tcataaacct gacttacttt agtgtgagac tcttgatact ttcgcttgcg 120

atccgaaaat cgcccgcttc catcaagcgc ccacacttat cggctgttca ttgttaaaga 180

gcatatccgc gagaagattc gactacttac cgcacccttc tcagcagcgc tgcgttgtca 240

gcagcagaga aatgagatta tgatcactta ttcgcagcgc gtcaacaact tttttactgc 300

Claims (14)

1. An melioidosis-like bacteria expression plasmid is characterized in that pUCP28T is used as a skeleton plasmid, and the melioidosis-like bacteria expression plasmid is prepared by replacing 16s-Promoter of a melioidosis-like bacteria 16s ribosomal RNA endogenous Promoter with a lactose operon of the skeleton plasmid; the nucleotide sequence is SEQ ID NO. 1.

2. A fluorescence-labeled expression plasmid for melioidosis bacteria, which is prepared by inserting a gene encoding a fluorescent protein into the reading frame of 16s-Promoter of the melioidosis bacteria expression plasmid according to claim 1; the fluorescent protein is selected from any one of GFP, CFP, BFP, RFP, EGFP, ECFP, EBFP, YFP, mHoneyde, mBanana, mOrange, tdTomato, mTangerine, mStrawberry, mCherry, mGrape1, mRaspberry, mGrape2 and mPlum.

3. The fluorescently labeled expression plasmid according to claim 2, wherein: the fluorescent protein is selected from one of GFP, BFP and RFP.

4. The fluorescently labeled expression plasmid of claim 2, wherein the nucleotide sequence of said 16s-Promoter is SEQ ID No. 18.

5. The fluorescent marker expression plasmid of claim 2, wherein the 16s-Promoter is obtained by amplification from a gene vector containing the endogenous Promoter of 16s ribosomal RNA of melioidosis bacteria with a primer pair having the nucleotide sequences SEQ ID No. 7 and SEQ ID No. 8.

6. The fluorescent marker expression plasmid of claim 5 wherein the gene vector is melioidosis BPC006 genomic DNA.

7. The fluorescent marker expression plasmid of claim 2, wherein the nucleotide sequence of BFP is SEQ ID NO. 2, the nucleotide sequence of GFP is SEQ ID NO. 3, and the nucleotide sequence of RFP is SEQ ID NO. 4.

8. The fluorescent marker expression plasmid of claim 2 wherein the nucleotide sequence is SEQ ID No. 15, SEQ ID No. 16 or SEQ ID No. 17.

9. The method of producing a fluorescent-labeled expression plasmid according to any one of claims 2 to 8, comprising:

1) Amplifying by taking a fluorescent protein coding gene vector as a template to obtain a coding gene of the fluorescent protein;

2) Inserting the fluorescent protein coding gene into the 16s-Promoter reading frame of the rhinoceroid expression plasmid of claim 1 or 2.

10. The method of claim 9, wherein: the fluorescent protein is selected from any one of GFP, CFP, BFP, RFP, EGFP, ECFP, EBFP, YFP, mHoneyde, mBanana, mOrange, tdTomato, mTangerine, mStrawberry, mCherry, mGrape1, mRaspberry, mGrape2 and mPlum.

11. The method of claim 9, wherein: the fluorescent protein is selected from one of GFP, BFP and RFP.

12. The method of claim 9, wherein the fluorescent protein is amplified from any one of a BFP-encoding gene primer pair consisting of SEQ ID NO. 9 and SEQ ID NO. 10, a GFP-encoding gene primer pair consisting of SEQ ID NO. 11 and SEQ ID NO. 12, and an RFP-encoding gene primer pair consisting of SEQ ID NO. 13 and SEQ ID NO. 14.

13. The method of manufacturing according to claim 12, wherein: the vibrio paranasal bacteria expression plasmid and the fluorescent protein coding gene are respectively subjected to enzyme digestion by using restriction enzyme, then enzyme digestion products are connected, and the fluorescent protein coding gene is inserted into the melioidosis paranasal bacteria expression plasmid.

14. A fluorescence-labeled melioidosis bacterium, comprising the fluorescence-labeled expression plasmid according to any one of claims 2 to 8.

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