CN112410364A

CN112410364A - Green fluorescent protein gene labeling method of acinetobacter baumannii

Info

Publication number: CN112410364A
Application number: CN202011313708.4A
Authority: CN
Inventors: 余道军; 潘平
Original assignee: Hangzhou First Peoples Hospital
Current assignee: Hangzhou First Peoples Hospital
Priority date: 2020-11-20
Filing date: 2020-11-20
Publication date: 2021-02-26

Abstract

The invention provides a method for marking acinetobacter baumannii by a green fluorescent protein gene, which constructs pET-RA-Y escherichia coli-acinetobacter baumannii shuttle plasmid (comprising a promoter sequence, a GFP sequence, an acinetobacter baumannii gene replication starting point sequence and a rifampicin resistance screening marker) and introduces the pET-RA-Y escherichia coli-acinetobacter baumannii shuttle plasmid into an acinetobacter baumannii standard strain ATCC 17978. The cells showed green fluorescence when observed under a fluorescence microscope. The invention also establishes a fluorescence labeling acinetobacter baumannii infected cell model, is used for the stability and reliability of the fluorescence labeling acinetobacter baumannii constructed by the method, provides technical support for researching the biological action of bacterial pathogenic factors and the interaction condition of bacteria and hosts, and provides a simple and visual method for researching the mutual relationship between the acinetobacter baumannii and the hosts by introducing the green fluorescent protein gene.

Description

Green fluorescent protein gene labeling method of acinetobacter baumannii

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a green fluorescent protein gene labeling method of acinetobacter baumannii, wherein a shuttle plasmid is used to obtain an acinetobacter baumannii fluorescent labeled strain capable of stably expressing green fluorescent protein, and a construction method and application thereof.

Background

Acinetobacter baumannii (Ab) is a gram-negative bacterium, and is the leading position in the pathogenic Acinetobacter (Acinetobacter pp.). As one of important pathogenic bacteria of domestic hospital infection, acinetobacter baumannii mainly causes upper respiratory tract infection, urethritis, catheter-related bacteremia, wound infection after burn, meningitis and the like in intensive care units and people with low immunity, and is also easy to fix and adhere to the surfaces of medical instruments and other environments, thereby seriously threatening the lives of patients. Especially in recent years, with the increasing number of multi-drug resistant and pan-drug resistant acinetobacter baumannii strains year by year and the continuous emergence of full-drug resistant bacteria, the clinical treatment is more challenging. Researches show that the pathogenicity of acinetobacter baumannii can be improved by influencing bacterial virulence, reducing the sensitivity of bacteria to antibiotics, increasing the invasion and infection of the bacteria to hosts and the like through biofilm formation, a protein secretion system, a quorum sensing system and the like. Therefore, the current research on acinetobacter baumannii mainly focuses on the expression regulation network of pathogenic factors, the preparation of specific antibodies of virulence factors, the search of novel drug targets and the like.

In these studies, if the physiological and pathological functions of the pathogenic factor need to be clarified, the relationship between acinetobacter baumannii and the host must be involved, so that the control mechanism of the pathogenic factor expression network in the interaction process of acinetobacter baumannii and the host needs to be researched in an effective way to observe the behavior state of the bacteria in the host in real time. The fluorescent protein labeling system can be used as a molecular labeling technology to visually observe the interaction process of pathogenic bacteria and a host, so that the fluorescent protein labeling system is widely applied to cell biology.

Disclosure of Invention

The invention aims to overcome the defects of the prior art, namely weak fluorescence signals, unstable expression, easy attenuation of fluorescence, more complicated construction method and the like of an Acinetobacter baumannii (Ab) fluorescence labeled strain, and provides a construction method of the Acinetobacter baumannii strain for stably expressing green fluorescent protein.

In order to achieve the purpose, the technical scheme provided by the invention is as follows: a green fluorescent protein gene marking method of Acinetobacter baumannii is characterized in that a gene synthesis method is adopted to construct a plasmid pET-RA-Y used in the research. pET-RA-Y comprises a promoter sequence, a GFP (Green fluorescent protein) sequence, an A.baumannii gene replication origin, a rifampicin resistance selection marker, and the like. In addition, the invention also establishes a fluorescence-labeled acinetobacter baumannii infected cell model to verify the influence of the acinetobacter baumannii introduced with the fluorescent plasmid on the cell infecting capacity (adhesion/invasion cells), is used for testing the stability and reliability of the fluorescence-labeled acinetobacter baumannii constructed by the method, and provides technical support for researching the biological action of bacterial pathogenic factors and the interaction condition of bacteria and hosts. The method comprises the following steps:

1. pET-RA-Y Escherichia coli-Acinetobacter baumannii shuttle plasmid is constructed by taking pET-RA sequence on Genebank (NO. HM219006.1) as basic sequence template (attached figure 1-A) and adopting gene synthesis method to construct plasmid pET-RA-Y (attached figure 1-B) used in the research. The pET-RA-Y sequence (SEQ ID NO.1) comprises a promoter sequence (SEQ ID NO.2), a GFP sequence (SEQ ID NO.3), a rifampin resistance selection marker (SEQ ID NO.4), an A.baumannii gene replication origin (SEQ ID NO.5) and the like.

2. Preparing Acinetobacter baumannii competent cells, introducing pET-RA-Y escherichia coli-Acinetobacter baumannii shuttle plasmid into Acinetobacter baumannii standard strain ATCC17978, and identifying

Adding 5 mu LpET-RA-Y into competent cells containing 100 mu LE. coliDH5 alpha, constructing pET-RA-Y escherichia coli, preparing competent cells of the baumannii standard strain ATCC17978, co-culturing the pET-RA-Y escherichia coli and the competent cells, carrying out PCR by using pET-RA-Y-F/pET-RA-Y-R primers, simultaneously using the strains for direct sequencing, checking whether the pET-RA-Y is successfully introduced into the baumannii standard strain ATCC17978, and marking the successfully introduced strain as an Ab-GFP strain.

3. Fluorescent strain observation:

and (3) performing LB broth amplification culture on the Ab-GFP strain identified by PCR and sequencing until the strain is obviously turbid to the naked eye, and observing whether the strain carries fluorescence or not by using a fluorescence microscope.

4. Detecting the stability and reliability of the fluorescence-labeled acinetobacter baumannii;

(1) human alveolar epithelial cells are selected as host cells,

(2) the fluorescence labeling strain and the human alveolar epithelial cells are cultured together,

(3) a cell fluorescence staining method is adopted, a confocal fluorescence microscope is utilized to observe a model of bacteria infected cells, the model comprises fluorescence intensity and fluorescence retention time of fluorescence labeling acinetobacter baumannii inside and outside the cells, and the influence of introduced fluorescence plasmids on the infected cell force (adhesion/invasion cells) is verified.

The fluorescence-labeled acinetobacter baumannii constructed by the invention is used for more intuitively researching the dynamic process of the interaction (including the processes of adhesion/invasion cells and the like) of bacteria and host cells, and provides a technical basis for the research of a molecular mechanism of bacteria infected cells. The method has the advantages that the infection process of the fluorescence-labeled acinetobacter baumannii infected host cells can be visually detected by adopting a self-built bacterial infection cell model and a common fluorescence microscope, the stability, reliability and fluorescence intensity of the fluorescence expression of the established fluorescence-labeled acinetobacter baumannii are verified at a cell level, and a tool strain is provided for the research of related bacteria-cell interaction.

The invention realizes the non-destructive marking of bacteria by constructing the Acinetobacter baumannii strain stably expressing the green fluorescent protein marker, provides technical support for researching important biological mechanisms such as the regulation and control mechanism of an Acinetobacter baumannii pathogenic factor network system and biological functions exerted in the interaction process of bacteria and hosts by combining a microscopic imaging technology, thereby realizing a model for observing the host cell infected by the Acinetobacter baumannii in real time and laying a research foundation for further clarifying the pathogenic mechanism of the Acinetobacter baumannii.

Drawings

FIG. 1 is a pET-RA sequence map, wherein A is a base sequence template; b: gene Synthesis method plasmid pET-RA-Y used in this study was constructed.

FIG. 2 is a PCR electrophoresis image of 24 single colonies transformed with pET-RA plasmid.

FIG. 3 shows Ab-GFP strain observed by fluorescence microscopy.

FIG. 4 is confocal microscopy of bacterial adhesion/invasion cells. In the figure, DAPI: DAPI staining of cellular nucleic acids (blue fluorescent nuclei within white boxes); GFP: acinetobacter baumannii fluorescent marker (Ab-GFP showing green fluorescence in white circle): phalloidin: red is staining of cell actin by phalloidin; merge: triple fluorescence was combined with color development (white dots in the triangle are white dot Ab-GFP invading cells as a schematic representation of bacteria invading cells). 400X.

Detailed Description

The invention is further described with reference to the accompanying drawings and examples, the following examples are illustrative only, and the invention is not limited to these examples.

Example 1Ab-GFP Strain construction

1. Experimental Material

Human alveolar epithelial cell (HPAEpic) cell line was purchased from CELLBIO (accession No.: CBR130583), RPMI 1640 (Gino, GNM31800), RPMI 1640 double antibody (Gino, GNM-31800-S), PBS buffer 1X (Gino, GNM20012), 0.25% pancreatin + EDTA (Gino, GNM-25260), fetal bovine serum (CAPRICORN SCIENTIFIC, FBS-11A), agar powder (Baisi, BS1068), peptone (Baisi, BS7006), yeast powder (Baisi, BS1069), sodium chloride (national drug group chemical Co., Ltd.), Triton X-100 (polyethylene glycol octylphenyl ether, Dalian American Biotechnology Co., Ltd., MB2486), iFluor^TM647 Phallocidin (Coprin-iFluor 647 conjugate, Dalian Melam Biotechnology Ltd., MB5939), DAPI (4', 6-diamidino-2-phenylindole, Thermo, 62247), paraformaldehyde (Meline, P804536-500g), bovine serum albumin (Shanghai-derived leaf Biotechnology Ltd., S25762), 10 Xpolylysine (Dalian Melamine Biotechnology Ltd., MA 0174). The strains, plasmids and primers of the invention are shown in table 1.

TABLE 1 strains, plasmids and primers

The promoter sequence, the GFP sequence, the rifampicin resistance coding gene sequence, the acinetobacter baumannii gene replication initiation point sequence and the pET-RA-Y sequence are shown in a sequence table.

2. Reagent preparation method

0.1% Triton X-100: mu.L of Triton X-100 to 100mL of PBS was dissolved and stored at 4 ℃ until use.

4% paraformaldehyde: 4g of paraformaldehyde is weighed into 100mL of PBS, and the mixture is fully dissolved and stored at 4 ℃ for later use.

0.01% polylysine: 10 mu.L of 10 Xpolylysine stock solution is taken to 100mL of sterilized water to prepare 0.01 percent polylysine solution, and the solution is stored at 4 ℃ for standby.

DAPI: adding 1mL of sterile water into a reagent bottle containing 10mgDAPI powder, completely dissolving to obtain 10mg/mL of storage solution, and storing at-20 ℃ in a dark place; 1mL of 10mg/mL DAPI stock solution is added into 10mL of sterile distilled water to prepare 1 mu g/mL working solution, and the working solution is subpackaged and stored at minus 20 ℃ in dark for later use.

iFluor^TM647 phaseolin: adding 30 mu LDMSO to dissolve the dye powder to prepare 1000 Xphallodin storage solution, and storing at-20 ℃ in the dark; 1 u L1000 x alpha lloidin stock solution in 1mL containing 1% BSA PBS fully dissolved, after subpackage-20 degrees C light protection for standby.

3. Experimental methods

3.1pET-RA-Y plasmid preparation

The plasmid pET-RA-Y used in this study (FIG. 1-B) was constructed by gene synthesis using the pET-RA sequence on Genebank (NO. HM219006.1) as the base sequence template (FIG. 1-A). pET-RA-Y comprises a promoter sequence, a GFP sequence, an acinetobacter baumannii gene replication origin, a rifampicin resistance selection marker and the like.

3.2E. coli DH5 alpha (pET-RA-Y) construction, the procedure is as follows:

(1)5 mu L pET-RA-Y plasmid is added into competent cells containing 100 mu LE. coliDH5 alpha; ice-cooling for 20min, heat-shocking for 60s at 42 deg.C, and freezing for 10 min; adding into 1mLLB liquid culture medium, and heating at 37 deg.C and 180rpm/min for 60 min; taking 100 mu L of supernatant transformed bacterium liquid to be coated on an LB plate containing riff with 100 mu g/mL (an LB plate containing rifampicin), and carrying out inverted culture at 37 ℃ for overnight;

(2) selecting a single colony to perform amplification culture in RifLB broth of 100 mu g/mL until obvious turbidity is seen by naked eyes, and performing PCR by using a pET-RA-Y-F/pET-RA-Y-R primer to verify whether the vector is successfully introduced (the reaction system is shown in tables 2 and 3);

TABLE 2E. coli DH5 alpha (pET-RA-Y) Positive clone screening PCR System (50. mu.L)

Composition of	Volume (μ L)
		2xPhanata Max Master Mix	25
pET-RA-Y-F	2
		pET-RA-Y-R	2
Suspected E.coliDH5 alpha (pET-RA-Y) positive clone bacterial liquid	2
		ddH₂O	19

TABLE 3E. coli DH5 alpha (pET-RA-Y) Positive clone screening PCR amplification program

(3) Performing electrophoresis gel verification on the PCR product, and performing sequencing verification on a suspected positive product; the positive strain is marked as E.coli DH5 alpha (pET-RA-Y).

3.3 preparation of Acinetobacter baumannii (Ab) competent cells

(1) The frozen Acinetobacter baumannii standard strain ATCC17978 strain is taken out by an aseptic inoculating loop, streaked on an LB plate, and cultured overnight at 37 ℃.

(2) Selecting well-grown single colony in 5mLLB liquid culture medium, performing shake culture at 37 deg.C overnight, inoculating 10% of the colony in 100mLLB liquid culture medium, and performing amplification culture for 2-4 hr to OD₆₀₀The value is about 0.8.

(3) The culture was dispensed into two 50mL centrifuge tubes and placed in an ice water mixture for 10 min.

(4) After ice-bath, the mixture was centrifuged at 4,000rpm for 5min at 4 ℃.

(5) The supernatant was discarded, and 10mL of ice-pre-cooled 0.1mol/L CaCl was added to each tube₂Resuspend the bacterial pellet and re-ice for 30 min.

(6) After ice-bath, centrifuging at 4,000rpm for 5min at 4 deg.C, sucking out the residue with a sterilized gun head, adding ice to each tube to pre-cool 0.1mol/L CaCl ₂2 mL of suspension was resuspended.

(7) After each tube is divided into 10 small tubes, the acinetobacter baumannii standard strain ATCC17978 competent cells are placed for 12 hours at 4 ℃ and then placed at-70 ℃ for storage.

3.4Ab-GFP Strain construction

(1) Coli DH5 alpha (pET-RA-Y) (100. mu.g/mL Rif) as donor, Acinetobacter baumannii standard strain ATCC17978 competent cells (100. mu.g/mL Tic and 100. mu.g/mL Amp) as recipient, and E.coli HB101(pRK2013) (50. mu.g/mL kan) helper were cultured to respective resistant plates at 37 ℃ with 5% CO₂Culturing overnight; single clones were picked up in LB broth containing the corresponding antibody, and cultured overnight at 37 ℃ at 180 rpm/min.

(2) 1ml of a. coliDH5 alpha (pET-RA-Y), an Acinetobacter baumannii standard strain ATCC17978 competent cell and an E. coliHB101(pRK2013) culture were respectively taken, washed 3 times by using nonreactive LB broth, mixed according to a ratio of 2:1:1 (donor bacteria: acceptor bacteria: auxiliary bacteria), 50. mu.L of nonreactive LB was taken, mixed and spread on nonreactive LB at 28 ℃ overnight.

(3) Scraping appropriate amount of bacteria on LB culture plate, mixing with 1mL of non-resistant LB, spreading appropriate amount of bacteria on corresponding resistant plate ATCC17978 in Rif (10 μ g/mL), 37 deg.C, 5% CO₂The culture was carried out overnight.

(4) And (3) selecting a single colony to perform amplification culture on a corresponding antibody LB broth, and when the naked eye shows obvious turbidity, performing PCR (polymerase chain reaction) by using a pET-RA-Y-F/pET-RA-Y-R primer, and checking whether the pET-RA-Y is successfully introduced into the Acinetobacter baumannii ATCC17978 strain, wherein the reaction conditions and the system are shown in tables 1 and 2.

(5) The PCR products were subjected to electrophoretic gel verification (FIG. 2), and those suspected positive products were subjected to sequencing verification, and the positive bacterial marker was ATCC17978-GFP (Ab-GFP).

3.5 fluorescent-labeled Acinetobacter baumannii Strain (Ab-GFP) Observation

The bacterium liquid which is confirmed by PCR and sequencing identification and successfully introduced into the pET-RA-Y plasmid is placed under a fluorescence microscope, and bacteria with visible green fluorescence marks are observed (figure 3), which shows that the Acinetobacter baumannii strain marked by green fluorescence protein is successfully constructed.

Example 2

1. Construction of fluorescence-labeled acinetobacter baumannii infected cell model

Acinetobacter baumannii mainly infects the respiratory tract to cause the occurrence and development of related respiratory infectious diseases, so in the present invention, Human alveolar epithelial cells (Human alveolar epithelial cells, HPAEpic, cell strain purchased from CELLBIO, No.: CBR130583) were used as a cell model for studying the bacteria-host interaction. As shown in FIG. 4, the fluorescence microscopy shows that a model of the host cell infected by the Acinetobacter baumannii GFP marker strain can be successfully constructed, and the relative position, adhesion or invasion of the bacteria and the host cell can be accurately positioned. Provides a basic experimental technology for dynamically observing the interaction research of bacteria and cells in real time and providing the biological functions of related pathogenic factors of acinetobacter baumannii.

The relevant experimental procedures were as follows:

(1) cover glass preparation: cleaning the cover glass, soaking in acid, washing again (washing with tap water for 2 times and then with distilled water for 3 times), autoclaving, oven drying, soaking in 75% ethanol for 10min for sterilization, air drying, soaking in 0.1mg/mL polylysine solution for 5min, taking out, and drying at room temperature for use.

(2) Cell preparation: in order to ensure the physiological characteristics of the cells, the human alveolar epithelial cells (HPAEpic) used in the experiment are within 10 generations. Cells were incubated at 37 ℃ with 5% CO₂Culturing to 60-80% cell confluence, digesting the cells with 0.25% pancreatin-EDTA, diluting the cells with fresh non-resistant RPMI 1640 cell culture solution to corresponding density, inoculating to a cell culture plate with a cover slip, and culturing with non-resistant and non-serum RPMI 1640 at 37 deg.C and 5% CO₂Incubate for 24h to about 60% cell confluence.

(3) Preparing bacteria: picking single colony of fluorescence labeling acinetobacter baumannii (Ab-GFP) constructed in example 2 to 5mL of corresponding LB broth culture solution, and culturing at 37 ℃ and 180r/min overnight; the bacterial suspension was adjusted to 1.0 turbidity (1.0 MCF 2 × 10)⁸CFU/mL), washed 3 times with non-resistant serum-free RPMI 1640 at 16,000r/min for 5min, and resuspended in non-resistant serum-free RPMI 1640 for further use.

(4) Bacteria-cell co-culture:

1) the plated cells were gently washed 3 times with PBS for 5min each;

2) adding 1mL of non-resistant serum-free RPMI 1640 cell culture solution; inoculating suspension Ab-GFP bacterial liquid according to the Infection Multiplicity (MOI) of 100:1, co-culturing for 5h, washing cells for 5min each time for 3 times with PBS, and washing off free bacteria;

3) fixing: washing with PBS for 3 times (pre-warming, washing with gentle shaking), discarding PBS, adding 4% paraformaldehyde immediately (storing at 4 deg.C in dark place), and fixing at room temperature for 20 min;

4) washed three times with PBS, 5min each time.

(5) Dyeing with fluorescent dyes

1) Permeabilizing the fixed cells with 0.1% Triton X-100 for 5 min;

2) washing cells with PBS for 5min for 3 times;

3) taking enough fresh and prepared1×iFluor^TM647 Phallodin to cover the cells, and staining for 60min at room temperature in dark place;

4) washing cells with PBS for 5min for 3 times;

5) adding enough 1 mug/mLDAPI solution to carry out counterstaining on cell nucleuses, and carrying out shading staining for 10min at room temperature;

6) washing cells with PBS for 5min for 3 times; washing with sterile distilled water for 1 time and 5 min;

7) by Fluorshield^TMThe resulting encapsulated tablets were subjected to encapsulation, dried and observed by a confocal microscope (FIG. 4).

Sequence listing

<110> first-person hospital in Hangzhou city

<120> green fluorescent protein gene marking method of acinetobacter baumannii

<160> 7

<170> SIPOSequenceListing 1.0

<210> 1

<211> 6818

<212> DNA

<213> Acinetobacter baumannii plasmid pET-RA-Y (Artificial sequence Unknown)

<400> 1

aaaaggatcc ggatccaaaa gatcctctag atttaagaag gagatataca tatgagtaaa 60

ggagaagaac ttttcactgg agttgtccca attcttgttg aattagatgg tgatgttaat 120

gggcacaaat tttctgtcag tggagagggt gaaggtgatg caacatacgg aaaacttacc 180

cttaaattta tttgcactac tggaaaacta cctgttccat ggccaacact tgtcactact 240

ttcgcgtatg gtcttcaatg ctttgcgaga tacccagatc atatgaaaca gcatgacttt 300

ttcaagagtg ccatgcccga aggttatgta caggaaagaa ctatattttt caaagatgac 360

gggaactaca agacacgtgc tgaagtcaag tttgaaggtg atacccttgt taatagaatc 420

gagttaaaag gtattgattt taaagaagat ggaaacattc ttggacacaa attggaatac 480

aactataact cacacaatgt atacatcatg gcagacaaac aaaagaatgg aatcaaagtt 540

aacttcaaaa ttagacacaa cattgaagat ggaagcgttc aactagcaga ccattatcaa 600

caaaatactc caattggcga tggccctgtc cttttaccag acaaccatta cctgtccaca 660

caatctgccc tttcgaaaga tcccaacgaa aagagagacc acatggtcct tcttgagttt 720

gtaacagctg ctgggattac acatggcatg gatgaactat acaaagaatc tctgaaaatc 780

tctcaggctg ttcacgctgc tcacgctgaa atcaacgaag ctggtcgtga agtagtaggt 840

taactgcagc caagcttccg atcgtagaaa tatctatgat tatcttgaag aacgcaaccc 900

tatagcagct attgaaattg atgatttaat tgaagaaaag acagatttag ttgttgataa 960

tcgactgatg gggcgcacag gcagacagaa agatactagg gagttagtga tacatccgca 1020

ttatgtggtt gtatatgaca tcactgatat aatacggata ctcagagtgc tacacacatc 1080

gcaggagtgg tcatgactta ctcatgtact ttggattatt tagtgttata aaatcctgat 1140

ttataaattt tttttgttaa aaaagataaa agccccttgc aattgcttgg ggctttaccg 1200

taatttatgg ggtacagatc ttcgatactg acatatcggc aatcgaaagc attaaggttt 1260

gacgaccgct aatgatttca ccacaggggc ttaatgtacc tgtcttaaat tctaaggttt 1320

taactcgctt tgtcaagcat agaccccaaa aatttagcca atgtctgtaa ctcaatctgt 1380

ccatgtgtgg gtgatgaggt acagtgacgc tagcacacat cggaaaaacg ctattactag 1440

gggaactgaa cagagtagcg gacgcaatga gtagtcattt aattggcggt tatgagcgtg 1500

ttcaggcggt gctatcaatc gtaatcataa cagtggcagc ttgatacagt gatgtcatcc 1560

ctgatgcgaa agcgaccgac cgacggtaca tcgaatggga atactttagg gtgattttta 1620

agaatcgctc tagggtgagt atttcccatt cagctctgct ccctccctct ggtactttaa 1680

tcaaaagcac tactaaacat atgtttttaa ataaaaaata ttgatataga gataatatta 1740

gtaagaataa ttaaacaatt gaatatagat aaatcattgt taaataaaga ttaattatta 1800

aaatgaatgt atacttatat ataaatcaat gatttaaaat atttgataaa gaaaactttt 1860

caaaaaaaat ataattgaga ttgtgtcatt tcggtcaatt cttaatatgt tccacgcaag 1920

ttttagctat ggtgctaaac agaaatttgc tgaaaaagaa cttttcactg aactggttaa 1980

aatgtaagca gcctgagagc cgccaaaaat tttaaaaaca aaccgcctta atcatcttca 2040

aaaaatacct ctaaaacctc accatttgcg ttttaagacc catatttcat cctgccctta 2100

tgttcccatg ctgatagcta taaagtgtct gtaatcgctt cctatgacgt tctaggctgt 2160

tgataacttt tggaacaacg caaaatgtta aaatccggaa gcttctgttt tggcggatga 2220

gagaagattt tcagcctgat acagattaaa tcagaacgca gaagcggtct gataaaacag 2280

aatttgcctg gcggcagtag cgcggtggtc ccacctgacc ccatgccgaa ctcagaagtg 2340

aaacgccgta gcgccgatgg tagtgtgggg tctccccatg cgagagtagg gaactgccag 2400

gcatcaaata aaacgaaagg ctcagtcgaa agactgggcc tttcgtttta tctgttgttt 2460

gtcggtgaac gctctcctga gtaggacaaa tccgccggga gcggatttga acgttgcgaa 2520

gcaacggccc ggagggtggc gggcaggacg cccgccataa actgccaggc atcaaattaa 2580

gcagaaggcc atcctgacgg atggcctttt tgcgtttcta caaactcttt tgtttatttt 2640

tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat 2700

aatattgaaa aaggaagagt aattcaggtg cgcggaaccc ctatttgttt atttttctaa 2760

atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat 2820

tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg 2880

gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa 2940

gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt 3000

gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt 3060

ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat 3120

tctcagaatg acttggtgtt atgcagccaa atcccaacaa ttaagggtct taaaatggta 3180

aaagattgga ttcccatctc tcatgataat tacaagcagg tgcaaggacc gttctatcat 3240

ggaaccaaag ccaatttggc gattggtgac ttgctaacca cagggttcat ctctcatttc 3300

gaggacggtc gtattcttaa gcacatctac ttttcagcct tgatggagcc agcagtttgg 3360

ggagctgaac ttgctatgtc actgtctggc ctcgagggtc gcggctacat atacatagtt 3420

gagccaacag gaccgttcga agacgatccg aatcttacga acaaaaaatt tcccggtaat 3480

ccaacacagt cctatagaac ctgcgaaccc ttgagaattg ttggcgttgt tgaagactgg 3540

gaggggcatc ctgttgaatt aataagggga atgttggatt cgttagagga cttaaagcgc 3600

cgtggtttac acgtcattga agactagcct ggacagatcg ctgagatagg tgcctcactg 3660

attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa 3720

cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa 3780

atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga 3840

tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg 3900

ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact 3960

ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac 4020

cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg 4080

gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg 4140

gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga 4200

acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc 4260

gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg 4320

agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc 4380

tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc 4440

agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt 4500

cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc 4560

gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc 4620

ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat atggtgcact 4680

ctcagtacaa tctgctctga tgccgcatag ttaagccagt atacactccg ctatcgctac 4740

gtgactgggt catggctgcg ccccgacacc cgccaacacc cgctgacgcg ccctgacggg 4800

cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt 4860

gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag 4920

cgtggtcgtg aagcgattca cagatgtctg cctgttcatc cgcgtccagc tcgttgagtt 4980

tctccagaag cgttaatgtc tggcttctga taaagcgggc catgttaagg gcggtttttt 5040

cctgtttggt cacttgatgc ctccgtgtaa gggggaattt ctgttcatgg gggtaatgat 5100

accgatgaaa cgagagagga tgctcacgat acgggttact gatgatgaac atgcccggtt 5160

actggaacgt tgtgagggta aacaactggc ggtatggatg cggcgggacc agagaaaaat 5220

cactcagggt caatgccagc gcttcgttaa tacagatgta ggtgttccac agggtagcca 5280

gcagcatcct gcgatgcaga tccggaacat aatggtgcag ggcgctgact tccgcgtttc 5340

cagactttac gaaacacgga aaccgaagac cattcatgtt gttgctcagg tcgcagacgt 5400

tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt gattcattct gctaaccagt 5460

aaggcaaccc cgccagccta gccgggtcct caacgacagg agcacgatca tgcgcacccg 5520

tggccaggac ccaacgctgc ctcagagtgc tacacacatc gcaggagtgg tcatgactta 5580

ctcatgtact ttggattatt tagtggtata aaatcctgat ttataaattt ttttttgtta 5640

aaaaagataa aagccccttg caattgcttg gggctttacc gtaatttatg gggtacagat 5700

cttcgatact gacatatcgg caatcgaaag cattaaggtt tgacgaccgc taatgatttc 5760

accacaggcc gagatgcgcc gcgtgcggct gctggagatg gcggacgcga tggatatgtt 5820

ctgccaaggg ttggtttgcg cattcacagt tctccgcaag aattgattgg ctccaattct 5880

tggagtggtg aatccgttag cgaggtgccg ccggcttcca ttcaggtcga ggtggcccgg 5940

ctccatgcac cgcgacgcaa cgcggggagg cagacaaggt atagggcggc gcctacaatc 6000

catgccaacc cgttccatgt gctcgccgag gcggcataaa tcgccgtgac gatcagcggt 6060

ccagtgatcg aagttaggct ggtaagagcc gcgagcgatc cttgaagctg tccctgatgg 6120

tcgtcatcta cctgcctgga cagcatggcc tgcaacgcgg gcatcccgat gccgccggaa 6180

gcgagaagaa tcataatggg gaaggccatc cagcctcgcg tcgcgaacgc cagcaagacg 6240

tagcccagcg cgtcggccgc catgccggcg ataatggcct gcttctcgcc gaaacgtttg 6300

gtggcgggac cagtgacgaa ggcttgagcg agggcgtgca agattccgaa taccgcaagc 6360

gacaggccga tcatcgtcgc gctccagcga aagcggtcct cgccgaaaat gacccagagc 6420

gctgccggca cctgtcctac gagttgcatg ataaagaaga cagtcataag tgcggcgacg 6480

atagtcatgc cccgcgccca ccggaaggag ctgactgggt tgaaggctct caagggcatc 6540

ggtcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg 6600

ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc 6660

ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg 6720

cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg 6780

gcgccggtga tgccggccac gatgcgtccg gcgtagag 6818

<210> 2

<211> 589

<212> DNA

<213> promoter (Artificial sequence Unknown) in Acinetobacter baumannii plasmid pET-RA-Y

<400> 2

ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc 60

agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt 120

cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt 180

caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc 240

tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa 300

ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac 360

ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg 420

gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga 480

gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact 540

tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaa 589

<210> 3

<211> 714

<212> DNA

<213> GFP (Artificial sequence Unknown) in Acinetobacter baumannii plasmid pET-RA-Y plasmid

<400> 3

atgagtaaag gagaagaact tttcactgga gttgtcccaa ttcttgttga attagatggt 60

gatgttaatg ggcacaaatt ttctgtcagt ggagagggtg aaggtgatgc aacatacgga 120

aaacttaccc ttaaatttat ttgcactact ggaaaactac ctgttccatg gccaacactt 180

gtcactactt tcgcgtatgg tcttcaatgc tttgcgagat acccagatca tatgaaacag 240

catgactttt tcaagagtgc catgcccgaa ggttatgtac aggaaagaac tatatttttc 300

aaagatgacg ggaactacaa gacacgtgct gaagtcaagt ttgaaggtga tacccttgtt 360

aatagaatcg agttaaaagg tattgatttt aaagaagatg gaaacattct tggacacaaa 420

ttggaataca actataactc acacaatgta tacatcatgg cagacaaaca aaagaatgga 480

atcaaagtta acttcaaaat tagacacaac attgaagatg gaagcgttca actagcagac 540

cattatcaac aaaatactcc aattggcgat ggccctgtcc ttttaccaga caaccattac 600

ctgtccacac aatctgccct ttcgaaagat cccaacgaaa agagagacca catggtcctt 660

cttgagtttg taacagctgc tgggattaca catggcatgg atgaactata caaa 714

<210> 4

<211> 492

<212> DNA

<213> Rifampicin resistance coding Gene sequence (Artificial sequence Unknown) in Acinetobacter baumannii plasmid pET-RA-Y plasmid

<400> 4

gtgttatgca gccaaatccc aacaattaag ggtcttaaaa tggtaaaaga ttggattccc 60

atctctcatg ataattacaa gcaggtgcaa ggaccgttct atcatggaac caaagccaat 120

ttggcgattg gtgacttgct aaccacaggg ttcatctctc atttcgagga cggtcgtatt 180

cttaagcaca tctacttttc agccttgatg gagccagcag tttggggagc tgaacttgct 240

atgtcactgt ctggcctcga gggtcgcggc tacatataca tagttgagcc aacaggaccg 300

ttcgaagacg atccgaatct tacgaacaaa aaatttcccg gtaatccaac acagtcctat 360

agaacctgcg aacccttgag aattgttggc gttgttgaag actgggaggg gcatcctgtt 420

gaattaataa ggggaatgtt ggattcgtta gaggacttaa agcgccgtgg tttacacgtc 480

attgaagact ag 492

<210> 5

<211> 1337

<212> DNA

<213> Acinetobacter baumannii Gene replication initiation sequence (Artificial sequence Unknown)

<400> 5

gatcgtagaa atatctatga ttatcttgaa gaacgcaacc ctatagcagc tattgaaatt 60

gatgatttaa ttgaagaaaa gacagattta gttgttgata atcgactgat ggggcgcaca 120

ggcagacaga aagatactag ggagttagtg atacatccgc attatgtggt tgtatatgac 180

atcactgata taatacggat actcagagtg ctacacacat cgcaggagtg gtcatgactt 240

actcatgtac tttggattat ttagtgttat aaaatcctga tttataaatt ttttttgtta 300

aaaaagataa aagccccttg caattgcttg gggctttacc gtaatttatg gggtacagat 360

cttcgatact gacatatcgg caatcgaaag cattaaggtt tgacgaccgc taatgatttc 420

accacagggg cttaatgtac ctgtcttaaa ttctaaggtt ttaactcgct ttgtcaagca 480

tagaccccaa aaatttagcc aatgtctgta actcaatctg tccatgtgtg ggtgatgagg 540

tacagtgacg ctagcacaca tcggaaaaac gctattacta ggggaactga acagagtagc 600

ggacgcaatg agtagtcatt taattggcgg ttatgagcgt gttcaggcgg tgctatcaat 660

cgtaatcata acagtggcag cttgatacag tgatgtcatc cctgatgcga aagcgaccga 720

ccgacggtac atcgaatggg aatactttag ggtgattttt aagaatcgct ctagggtgag 780

tatttcccat tcagctctgc tccctccctc tggtacttta atcaaaagca ctactaaaca 840

tatgttttta aataaaaaat attgatatag agataatatt agtaagaata attaaacaat 900

tgaatataga taaatcattg ttaaataaag attaattatt aaaatgaatg tatacttata 960

tataaatcaa tgatttaaaa tatttgataa agaaaacttt tcaaaaaaaa tataattgag 1020

attgtgtcat ttcggtcaat tcttaatatg ttccacgcaa gttttagcta tggtgctaaa 1080

cagaaatttg ctgaaaaaga acttttcact gaactggtta aaatgtaagc agcctgagag 1140

ccgccaaaaa ttttaaaaac aaaccgcctt aatcatcttc aaaaaatacc tctaaaacct 1200

caccatttgc gttttaagac ccatatttca tcctgccctt atgttcccat gctgatagct 1260

ataaagtgtc tgtaatcgct tcctatgacg ttctaggctg ttgataactt ttggaacaac 1320

gcaaaatgtt aaaatcc 1337

<210> 6

<211> 24

<212> DNA

<213> Artificial sequence (Unknown)

<400> 6

gaattagatg gtgatgttaa tggg 24

<210> 7

<211> 24

<212> DNA

<213> Artificial sequence (Unknown)

<400> 7

atcactaact ccctagtatc tttc 24

Claims

1. A green fluorescent protein gene labeling method of acinetobacter baumannii is characterized by comprising the following steps:

(1) constructing pET-RA-Y escherichia coli-acinetobacter baumannii shuttle plasmid: a plasmid pET-RA-Y is constructed by adopting a gene synthesis method by taking a pET-RA sequence of NO. HM219006.1 on Genebank as a basic sequence template.

(2) Preparing Acinetobacter baumannii competent cells: introducing pET-RA-Y escherichia coli-acinetobacter baumannii shuttle plasmid into a standard strain of the ambulania baumannii with the accession number of ATCC17978, identifying, and marking the successfully introduced strain as an Ab-GFP strain;

(3) fluorescent strain observation: performing LB broth amplification culture on the Ab-GFP strain until obvious turbidity can be seen by naked eyes, and observing whether the strain carries fluorescence or not by using a fluorescence microscope;

(4) detecting the stability and reliability of the fluorescence-labeled acinetobacter baumannii:

(a) human alveolar epithelial cells were selected as host cells.

(b) The fluorescence labeling strain and the human alveolar epithelial cells are cultured together.

(c) A cell fluorescent staining method is adopted, a confocal fluorescent microscope is utilized to observe a model of bacteria infected cells, the model comprises fluorescence intensity and fluorescence retention time of fluorescence labeling acinetobacter baumannii inside and outside the cells, and the influence of introduced fluorescent plasmids on the cell infection force is verified.

2. The method of claim 1, wherein the pET-RA-Y sequence of step (1) is represented by SEQ ID No.1, and the pET-RA-Y sequence comprises a promoter sequence of SEQ ID No.2, a GFP sequence of SEQ ID No.3, a rifampicin resistance selection marker sequence of SEQ ID No.4, and an A.baumannii gene replication start sequence of SEQ ID No. 5.

3. The method of claim 1, wherein the step (2) comprises adding pET-RA-Y to E.coli DH5 α -containing competent cells to construct pET-RA-Y E.coli, preparing competent cells of a stock strain of Acinetobacter baumannii, co-culturing the E.coli pET-RA-Y with the competent cells, performing PCR using pET-RA-Y-F/pET-RA-Y-R primers, and performing direct sequencing of the strain to determine whether pET-RA-Y is successfully introduced into the stock strain of Acinetobacter baumannii.