CN109097380A - Application of the bacillus subtilis swrC gene as lactic acid bacteria selection markers - Google Patents
Application of the bacillus subtilis swrC gene as lactic acid bacteria selection markers Download PDFInfo
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Abstract
The present invention relates to lactic acid bacteria genetic engineering field, specifically application of the bacillus subtilis swrC gene as lactic acid bacteria selection markers.The present invention expresses SwrC albumen by building swrC expression casette in lactic acid bacteria, and positive transformant can be suppressed in the plate normal growth for being added with surfactin, control transformant in the growth of surfactin plate.Surfactin has excellent fungistatic effect to most of gram-positive bacteria, and it is safer compared with antibiotic, therefore having exploitation with surfactin and swrC gene constructed lactic acid bacteria selection markers system is the potentiality of wide host's security filtering system of substitute antibiotics resistance screening.
Description
Technical field
The present invention relates to microbiological genetic engineering fields, and in particular to bacillus subtilis swrC gene is sieved as lactic acid bacteria
Select the application of label.
Background technique
Lactic acid bacteria is widely used in each field, sends out with technique for gene engineering as a kind of generally recognized as safe bacterial strain (GRAS)
Exhibition, lactic acid bacteria can be modified and assign new good characteristic.But in genetic engineering operation, often mostly use antibiotic anti-
Property screening, and these resistance screenings labels the case where may having genetic drift phenomenon, endurance strain is caused to spread unchecked, destroy
The characteristics of lactobacillus food grade bacterial strain.Therefore developing the new selection markers to replace antibiotic resistance screening has great meaning
Justice.
Antibacterial lipopeptid is gradually obtained the approval of people, is slowly opened with its excellent antibacterial activity and preferable safety
Hair is to replace traditional antibiotic.Surfactin from bacillus subtilis is then that one type research is more early, more thoroughly
Thorough antibacterial lipopeptid, surfactin have inhibiting effect to a variety of gram-positive bacterias.Safety for surfactin is asked
The research of topic, the concentration that discovery surfactin only reaches 50 μm of ol/L (about 52mg/mL) all can just show hemolytic, and
The discovery of mouse administering transgenic, the feeding of long-term 10mg not will cause apparent toxicity, these results illustrate that surfactin is opposite and pacify
Entirely.Surfactin has the potentiality of the additive as safety, is expected to be developed as a kind of new screening pressure.
Immunologic mechanism to bacillus subtilis surfactin is the study found that swrC gene encodes a kind of memebrane protein, root
It is reported that more drug resistance efflux pump (multidrug resistances of this protein exhibits similar to Escherichia coli AcrB family
Effluxpumps function).There has been no using swrC gene as selection markers, carry out positive son in lactic acid bacteria to turn at present
Change the paper or report of screening.
Summary of the invention
Technical problem
The present invention is directed to develop antibacterial lipopeptid surfactin correlation swrC gene to be immunized as selection markers,
Lactic acid bacteria wide host screening system of the surfactin as screening pressure.
Application of the bacillus subtilis swrC gene of the present invention as lactic acid bacteria selection markers.
Specific step is as follows:
1) vector construction
Lactic acid bacteria constitutive promoter sequence is connect with swrC gene order by fusion DNA vaccine, recycles one-step cloning reagent
Box, the antibiotic-resistance marker on lactic acid bacteria vector that fusion segment replacement is screened based on antibiotic resistance, constructs swrC
Target gene is passed through the method for PCR amplification, digestion connection or one-step cloning, is cloned into swrC screening vector by screening vector
Multiple cloning sites, complete exogenous protein expression carrier building；
2) carrier converts
According to lactic acid bacteria Electroporation-competent cells preparation condition, NZ3900, LP163 or other related lactic acid bacteria impressions are prepared
State cell takes 100 μ L competent cells, and the exogenous protein expression carrier that 5 μ g are built is added thereto, according to electrotransformation condition
It carries out electroporated, the electroporated recovery medium of pre-cooling is added, after being sufficiently incubated for culture, take 100-150 μ L to be coated on and contain
Having surfactin concentration is the GM17 plate of 100-250 μ g/mL, to carry out positive transformant screening；
3) transformant screening
Above-mentioned conversion plate is placed in 30 DEG C of incubators, and after culture for 24 hours, the lactic acid bacteria containing swrC selection markers grows on plate
The mellow and full bacterium colony of white, the lactic acid bacteria without containing swrC gene can not be 100-250 μ g/mL's containing surfactin concentration
It is grown on GM17 plate, picking positive transformant, further uses swrC gene primer and carry out PCR verifying, verify positive transformants
Son.
The lactic acid bacteria constitutive promoter refers to P32, PpepN, PlacA or P170.The swrC gene refers to withered grass
The swrC gene in 168 source of bacillus or the codon optimised sequence of swrC gene-correlation and related high homology sequence.Institute
It states lactic acid bacteria and refers to that Lactococcus lactis, lactobacillus plantarum, lactobacillus fermenti or streptococcus thermophilus etc. have suppression to surfactin
Make active lactic acid bacteria.
The utility model has the advantages that
1. positive rate.SwrC gene is used as lactic acid bacteria selection markers, and for 24 hours, picking transformant is verified for culture, and positive rate is
100%, use surfactin and swrC gene as selection markers, positive rate is high.With ampicillin, chloramphenicol or
The antibiotic such as erythromycin are compared, and swrC gene is as selection markers, and screening has phase with antibiotic resistance screening in lactic acid bacteria
Same positive rate.
2. stability.Under 100 μ g/mL surfactin screening pressures, the plasmid culture 18h based on swrC genescreen has
95% mitotic stability, the gene and surfactin have good stability as selection markers.It screens and marks with antibiotic resistance
The stability of the lactic acid bacteria plasmid of note is compared, and swrC gene is as selection markers, and screening has with antibiotic resistance in lactic acid bacteria
There is similar mitotic stability.
3. safety.SwrC gene will not impact the expression of foreign protein, not influence acetaldehyde-dehydrogenase as selection markers
Enzyme activity.Additionally as the albumen from withered grass, antibiotic resistance and antibiotic resistance will not be caused to drift about and asked
Topic.Therefore, swrC gene screens safer as lactic acid bacteria selection markers compared with antibiotic resistance.
Detailed description of the invention
Fig. 1 bacillus subtilis antibacterial lipopeptid surfactin structural schematic diagram
The analysis of Fig. 2 swrC gene expression product TMHMM protein transmembrane is as a result, TMHMM analyzes network address: http: //
www.cbs.dtu.dk/services/TMHMM/
Fig. 3 swrC gene PCR amplification, stripe size 3198bp
Fig. 4 swrC gene is verified in Lactococcus lactis NZ3900 screening effect, agar diffusion test results
Fig. 5 swrC gene is verified in Lactococcus lactis NZ3900 screening effect, and different surfactin concentration flat-plate spottings are real
It tests
Fig. 6 swrC gene is verified in lactobacillus plantarum LP163 screening effect, agar diffusion test results
Fig. 7 swrC gene is verified in Lactococcus lactis LP163 screening effect, and different surfactin concentration flat-plate spottings are real
It tests
Specific embodiment
Following embodiment does not limit the present invention convenient for better understanding the present invention.Involved in the embodiment
Experimental method is unless otherwise specified general chemistry and molecular biology conventional method；It is related to experimental material, if special
Illustrate, is domestic chemical analysis reagent；It is related to primer synthesis and examining order is completed by Nanjing Jin Sirui company.
Embodiment 1surfactin preparation
Take -80 DEG C of Bacillus.amyloliquefaciens fmb50 frozen (bacillus amyloliquefaciens mode bacterium,
It is public, deposit number CGMCC N0.943) in BPY solid plate (beef extract 5g, peptone 10g, yeast extract 5g, glucose
10g, sodium chloride 5g, deionized water are settled to 1000mL, pH 7.0) scribing line, 37 DEG C of constant temperature stationary culture 18h；Picking single colonie
It is inoculated in BPY liquid, 37 DEG C, shaken cultivation 18h in 180rpm constant-temperature table；It is cultivated according to 3% inoculum concentration switching in Landy
Base (glucose 42.0g, sodium glutamate 14.0g, MgSO4·7H2O 0.5g, KCl 0.5g, KH2PO41.0g, FeSO4·7H2O
0.15mg, MnSO4·H2O 5mg, CuSO4·5H2O 0.16mg, distilled water 1000mL, pH7.0), 37 DEG C, 180rpm constant temperature shakes
Shaken cultivation 18h in bed.
Collection takes culture, and 0.5g/L chitosan is added, and 0.3g/L sodium alginate, adjusting pH is 5.0, and flocculate 30min, takes out
Filter.Filter cake is recycled, dehydrated alcohol is added and disperses filter cake, adjusts pH to 7.5, carries out second step suction filtration.Filtrate is collected, is freezed
It is dry, prepare surfactin.According to experiment, surfactin is dissolved in ethyl alcohol, prepares related concentrations reservoir.
Embodiment 2swrC gene cloning
Take -80 DEG C of Bacillus.subtilus frozen 168 (bacillus subtilis mode bacterium, public, deposit number
ATCC23857 it) is activated in BPY plate streaking, activated strains switching BPY liquid, 33 DEG C of shaken cultivation 16h draw 1mL culture
Object, 8000rpm are centrifuged 5min, wash and collect thallus.Referring to a small amount of extraction agent box steps of Shanghai Sangon Biotech Company's genomic DNA
Suddenly, 168 genomic DNA of B.subtilus is extracted.
According to swrC gene in ncbi database (Gene ID:938055), amplimer is designed to P1:5 '-GAG CTCATG ACC AGT CAG TCA ATA-3'；P2:5 '-GCA TGC TTA CTC TTC TTC CGT TCC-3 ', underscore
Part is restriction enzyme site Sac I and Sph I and using 168 genomic DNA of the B.subtilus of above-mentioned acquisition as template, is carried out
PCR amplification.Amplification system are as follows: 10 × Pfu Buffer, 5.0 μ L；2.5mmol/L dNTP, 4.0 μ L；P1,2.0 μ L；P2,2.0 μ
L；168,1.0 μ L of genome DNA fromB.subtilus；Pfu DNApolymerase, 2.0 μ L；ddH2O, 34.0 μ L.Instead
Answer condition are as follows: 94 DEG C of initial denaturation, 5min；94 DEG C of denaturation, 30sec；55 DEG C of annealing temperature, 30sec；72 DEG C of elongating temperature, 3min；
After 30 reaction cycles, extend 10min.
Embodiment 3swrC screens resistant vector pMG36e-swrC and pFMB4191-swrC building
PMG36e-swrC building: directly using the means of digestion connection, Sac I and Sph I restriction enzyme are used respectively
It to the PCR product digestion in carrier pMG36e (purchased from excellent precious biology, product number VT1092) and embodiment 2, and connects, obtains
The carrier obtained is named as pMG36e.
PFMB4191-swrC building: synthesis PpepNPromoter sequence:
1 AGATCTGTCG ACCTGCAGTA GACAGTTTTT TTAATAAGTT AAAGAAAAGA TGTAATTTTT
61 CTTTGTACTC GAAATTTTCT ATTCAATTTG ATATAATTAT ATTAATACTG AATATTTAGG
121 AGAAGGCATG
Design primer P3:5 '-TTA AAT ATG GGT CGA TCG AAT TCA GAT CTG TCG ACC TGC AGTA-
3 ', P4:5 '-TAG AGG ATC GAT CCC CGG GCG AGC TCC TTC TCC TAA ATA TTC AGT A-3 ', make
With overlap extension pcr, the P above pMG36e carrier is replaced32The new support of promoter sequence, acquisition is named as
pFMB4191.Respectively with Sac I and Sph I restriction enzyme to the PCR product enzyme in carrier pFMB4191 and embodiment 2
It cuts, and connects, the carrier of acquisition is named as pFMB4191-swrC.
Embodiment 4swrC gene is verified in lactic acid bacteria screening function
The preparation of 4.1 Lactococcus lactis NZ3900 competent cells
NZ3900 (being purchased from MoBiTec company of Germany, number VS-ELS03900-01) is taken out from -80 DEG C, in GM17
(0.5% glucose, M17 meat soup, Qingdao Hai Bo) plate streaking, 30 DEG C of inversion cultures.Picking single colonie is inoculated in 5mL
GSGM17 culture medium (GM17 adds 0.5mol/L sucrose, 25g/L glycine) after 30 DEG C of stationary culture 12h, connects according to 1%
Kind amount access 50mL GSGM17 culture medium transfers 50mL culture into fresh 400mL GSGM17 after 30 DEG C of cultures for 24 hours
Culture medium, 30 DEG C of stationary cultures to OD600For 0.2-0.3.Bacterium solution is taken out, 10min is stood on ice, is centrifuged in 4 DEG C with 6000xg
20min collects thallus.The buffer washing thalline containing 0.5mol/L sucrose and 10% glycerol being pre-chilled on ice with 400mL, in
4 DEG C are centrifuged 20min with 6000xg, collect thallus.With 200mL be pre-chilled on ice containing 0.5mol/L sucrose, 10% glycerol and
Thallus is resuspended in the buffer of 50mmol/LEDTA, stands 20min on ice.20min is centrifuged with 6000xg in 4 DEG C, collects thallus, is used
The buffer containing 0.5mol/L sucrose and 10% glycerol that 100mL is pre-chilled on ice washing thalline one time again, 6000xg centrifugation.Most
Thallus is resuspended containing the buffer of 0.5mol/L sucrose and 10% glycerol with what 4mL was pre-chilled on ice afterwards, after standing 2min on ice, often
100uL competent cell is distributed into the EP pipe of 1.5mL, and -80 DEG C freeze.
4.2 carrier pMG36e-swrC and pMG36e convert NZ3900
It takes out -80 DEG C and freezes the pipe of NZ3900 competent cell two, melt in standing on ice, each concentration that is added is 5 μ g's
PMG36e-swrC and pMG36e Plasmid DNA, gently rotation mixes, and stands 20min on ice.By mixed liquor it is careful be transferred to ice
In the internal diameter 2mm electrotransformation cup of upper pre-cooling, shock parameters are set are as follows: 2000V, 200 Ω, 25 μ F are accurate to place electrotransformation cup, into
Row is electroporated, and (general discharge time is in 4.5ms to 5ms).After electroporated, it is rapidly added GM17MC electrotransformation renewal cultivation
Base, resuspended bacterium solution are transferred in 1.5mL sterile EP tube, in 30 DEG C of incubator stationary incubation culture 1.5h.It draws and is incubated for culture solution
100-150uL is coated in containing the GM17 plate that erythromycin concentration is 5 μ g/mL, is inverted in 30 DEG C of incubator cultures.Two is positive
Conversion is respectively designated as NZ3900/swrC and NZ3900/pMG36e.
4.3swrC gene is in NZ3900 screening verification
It takes two parts of GM17 solid mediums to melt, is cooled to 50 DEG C, portion is added 1mL and is incubated overnight NZ3900/
PMG36e pours into 9cm culture dish after mixing, uniformly punched with 6mm punch after cooling, and 50 μ L concentration of injection are 250 μ g/
ML, 500 μ g/mL, 1000 μ g/mL, the surfactin solution of 2500 μ g/mL, 50 μ L are injected containing 500 μ g/mL concentration in another hole
Nisin solution.The NZ3900/swrC that 1mL is incubated overnight is added in another, is similarly operated.30 DEG C are just set overnight incubation,
It observes and measures surfactin diffusion pore size.Inject the experimental group of the surfactin solution of 250 μ g/mL, NZ3900/
SwrC plate has the inhibition zone that diameter is 7.13mm and NZ3900/pMG36e plate then has the inhibition zone of diameter 8.94mm；
Inject the experimental group of the surfactin solution of 500 μ g/mL, NZ3900/swrC plate there is the inhibition zone that diameter is 7.21mm and
NZ3900/pMG36e plate then has the inhibition zone of diameter 9.38mm；Inject the experiment of the surfactin solution of 1000 μ g/mL
Group, NZ3900/swrC plate has the inhibition zone that diameter is 7.97mm and NZ3900/pMG36e plate then has diameter
10.02mm inhibition zone.Under identical surfactin concentration, surfactin is to the antibacterial circle diameter of NZ3900/swrC with right
The antibacterial circle diameter of NZ3900/pMG36e is compared to small by 20% or more.
Preparation contains the GElliker plate that surfactin concentration is respectively 0,50,100 μ g/mL, by NZ3900/
PMG36e and NZ3900/swrC is cultivated respectively to OD600Value is 0.6, draws 2 μ L bacterium solutions drop in containing different surfactin's
GElliker plate, observation bacterium colony growth, determines the surfactin concentration to screen.It is 0 in surfactin concentration
Gelliker plate, NZ3900/swrC and NZ3900/pMG36e and bacterium colony having the same are formed；It is in surfactin concentration
The Gelliker plate of 50 μ g/mL, NZ3900/swrC has apparent bacterium colony, but NZ3900/pMG36e bacterium colony is not apparent；
In the Gelliker plate that surfactin concentration is 100 μ g/mL, NZ3900/swrC has apparent bacterium colony, but NZ3900/
PMG36e is formed without obvious bacterium colony.
4.4swrC gene is verified in lactobacillus plantarum screening function
Lactobacillus plantarum LP163 competent cell is made, swrC gene is expressed to simultaneously authentication function in LP163.Specifically
Competence production method and LP163 method for transformation obtain swrC genetic transformation bacterial strain referring to described in embodiment 4.1 and 4.2
The LP163/pFMB4191-swrC and unloaded conversion bacterial strain LP163/pFMB4191 of control.Screening verification is referring to 4.3 institute of embodiment
It states.It takes two parts of GM17 solid mediums to melt, is cooled to 50 DEG C, portion is added 1mL and is incubated overnight LP163/pFMB4191, mixes
9cm culture dish is poured into after even, is uniformly punched with 6mm punch after cooling, and 50 μ L concentration of injection are 250 μ g/mL, 500 μ g/
The nisin solution that 50 μ L contain 500 μ g/mL concentration is injected in the surfactin solution of mL and 1000 μ g/mL, another hole.Another adds
Enter the LP163/pFMB4191-swrC that 1mL is incubated overnight, is similarly operated.30 DEG C are just set overnight incubation, are observed and are measured
Surfactin spreads pore size.Inject the experimental group of the surfactin solution of 250 μ g/mL, LP163/pFMB4191-swrC
Plate has the inhibition zone that diameter is 8.07mm and LP163/pFMB4191 plate then has the inhibition zone of diameter 8.47mm；Injection
The experimental group of the surfactin solution of 500 μ g/mL, it is the antibacterial of 8.63mm that LP163/pFMB4191-swrC plate, which has diameter,
Circle and LP163/pFMB4191 plate then has the inhibition zone of diameter 9.38mm.Under identical surfactin addition concentration,
Surfactin is to the antibacterial circle diameter of LP163/pFMB4191-swrC compared with the antibacterial circle diameter to LP163/pFMB4191
It is small by 4% or more.
Embodiment 5surfactin screening acetaldehyde dehydrogenase expression vector establishment simultaneously verifies mitotic stability and fermentative activity
Acetaldehyde dehydrogenase expression vector pFMB4197 building
Use primer 4197P1:5 '-AAG GCG GCG GTC AAG GTA CCA GAT CTG TCG ACC TGC AGT A-
3'；4197P2:5 '-GTG GTG GTG GTG GTG GTG CAT CAT GCC TTC TCC TAA ATAT-3 ', with synthesis
PpepN sequence is template, carries out PCR to PpepN promoter；Use primer 4197P3:5 '-ATG CAC CAC CAC CAC
CAC CAC CTT AGA ACT GCA ACT AGA A-3'；4197P4:5'-AAT ATC GTA GCG CCG GGG TAC
CTT ATT GTG GGC CAT CGT TAA TGG-3 ', with the acetaldehyde dehydrogenase istALDH gene of research department's preservation (GI:
257782115) it is template, PCR amplification is carried out to acetaldehyde dehydrogenase istALDH gene.
The method for using SOE PCR again, by PpepNPromoter connects istALDH gene, and is inserted into carrier pFMB4191-swrC
On, acquisition can be named with the acetaldehyde dehydrogenase expression vector of Erythromycinresistant screening and surfactin resistance screening simultaneously
For pFMB4197.
Plasmid is transferred into containing 5 μ g/ simultaneously in surfactin screening pressure stability inferior verifying NZ3900/pFMB4197
The GM17 liquid of mL erythromycin after being incubated overnight, is transferred respectively into the GM17 culture for containing 0,25,100 μ g/mL surfactin
Base (not adding erythromycin) stationary culture 18h, every 6h draw 1mL, and dilution spread is in GM17 plate, 100 single bacteriums of random picking
Photocopy is fallen to the GM17 plate containing 5 μ g/mL erythromycin, stationary culture records the bacterium colony number scale C of photocopy plate, plasmid stability
=C/100 × 100%.
Under 100 μ g/mL surfactin screening pressures, plasmid pFMB4197 culture 18h has 95% stability.
The method of acetaldehyde-dehydrogenase expression of enzymes verifying acetaldehyde dehydrogenase enzyme activity determination, reaction under surfactin screening pressure
System is as follows: system total volume 3mL, includes 2mmol/L acetaldehyde, 0.5mmol/L NAD+, 0.1mol/L Tris-HCl
(pH8.0), 10mmol/L 2 mercapto ethanol and 0.1mol/L KCl are eventually adding 0.1mL enzyme solution to be measured to start enzyme activity inspection
Reaction is surveyed, at 25 DEG C, the variation of absorbance is measured under the Detection wavelength of 340nm.Definition generates 1 μm of ol NADH institute per minute
Needing enzyme amount is an enzyme-activity unit (U).Finally calculate fermentation liquid vigor=absorbance change/0.00622*10/10 per minute.?
Under 100 μ g/mL surfactin screening pressures, measuring NZ3900/pFMB4197 acetaldehyde-dehydrogenase enzyme activity after culture 18h is
2.6U/mL.Show to use swrC gene as selection markers, the expression of foreign protein will not be impacted, external source will not be caused
Protein active loss.
The present invention is by the way that the gene constructed lactic acid bacteria expression cassette of correlation swrC is immunized using bacillus subtilis surfactin, i.e., newborn
Sour bacterium promoter lactic acid bacterias constitutive promoters such as (including but) being not only limited to P32, PpepN, PlacA, P170 plus swrC base
Because (including but be not only limited to the codon optimised sequence of 168 source swrC gene of bacillus subtilis or swrC gene-correlation
And related high homology sequence), as new selection markers, cooperate the surfactin of suitable concentration, replacement lactic acid bacteria (including
But it is not limited to Lactococcus lactis, lactobacillus plantarum, lactobacillus fermenti, a variety of surfactin such as streptococcus thermophilus, which have, to be inhibited
Active lactic acid bacteria) in antibiotic resistance screening.
Sequence table
<110>Agricultural University Of Nanjing
<120>application of the bacillus subtilis swrC gene as lactic acid bacteria selection markers
<141> 2018-07-24
<160> 9
<170> SIPOSequenceListing 1.0
<210> 2
<211> 125
<212> DNA
<213> Lactococcus lactis
<400> 2
gatctgtcga cctgcagtag acagtttttt taataagtta aagaaaagat gtaatttttc 60
tttgtactcg aaattttcta ttcaatttga tataattata ttaatactga atatttagga 120
gaagg 125
<210> 2
<211> 24
<212> DNA
<213>bacillus subtilis (Bacillus subtilis)
<400> 2
gagctcatga ccagtcagtc aata 24
<210> 3
<211> 24
<212> DNA
<213>bacillus subtilis (Bacillus subtilis)
<400> 3
gcatgcttac tcttcttccg ttcc 24
<210> 4
<211> 43
<212> DNA
<213> Lactococcus lactis
<400> 4
ttaaatatgg gtcgatcgaa ttcagatctg tcgacctgca gta 43
<210> 5
<211> 46
<212> DNA
<213> Lactococcus lactis
<400> 5
tagaggatcg atccccgggc gagctccttc tcctaaatat tcagta 46
<210> 6
<211> 40
<212> DNA
<213> Lactococcus lactis
<400> 6
aaggcggcgg tcaaggtacc agatctgtcg acctgcagta 40
<210> 7
<211> 40
<212> DNA
<213> Lactococcus lactis
<400> 7
gtggtggtgg tggtggtgca tcatgccttc tcctaaatat 40
<210> 8
<211> 40
<212> DNA
<213> Lactococcus lactis
<400> 8
atgcaccacc accaccacca ccttagaact gcaactagaa 40
<210> 9
<211> 45
<212> DNA
<213> Lactococcus lactis
<400> 9
aatatcgtag cgccggggta ccttattgtg ggccatcgtt aatgg 45
Claims (9)
1. application of the bacillus subtilis swrC gene as lactic acid bacteria selection markers.
2. application according to claim 1, the specific steps are as follows:
1) vector construction
Lactic acid bacteria constitutive promoter sequence is connect with swrC gene order by fusion DNA vaccine, recycles one-step cloning reagent
Box, the antibiotic-resistance marker on lactic acid bacteria vector that fusion segment replacement is screened based on antibiotic resistance, constructs swrC
Target gene is passed through the method for PCR amplification, digestion connection or one-step cloning, is cloned into swrC screening vector by screening vector
Multiple cloning sites, complete exogenous protein expression carrier building；
2) carrier converts
According to lactic acid bacteria Electroporation-competent cells preparation condition, NZ3900, LP163 or other related lactic acid bacteria impressions are prepared
State cell takes 100 μ L competent cells, and the exogenous protein expression carrier that 5 μ g are built is added thereto, according to electrotransformation condition
It carries out electroporated, the electroporated recovery medium of pre-cooling is added, after being sufficiently incubated for culture, take 100-150 μ L to be coated on and contain
Having surfactin concentration is the GM17 plate of 100-250 μ g/mL, to carry out positive transformant screening；
3) transformant screening
Above-mentioned conversion plate is placed in 30 DEG C of incubators, and after culture for 24 hours, the lactic acid bacteria containing swrC selection markers grows on plate
The mellow and full bacterium colony of white, the lactic acid bacteria without containing swrC gene can not be 100-250 μ g/mL's containing surfactin concentration
It is grown on GM17 plate, picking positive transformant, further uses swrC gene primer and carry out PCR verifying, verify positive transformants
Son.
3. application according to claim 1 or 2, which is characterized in that lactic acid bacteria constitutive promoter refer to P32,
PpepN, PlacA or P170.
4. application according to claim 1 or 2, which is characterized in that swrC gene refers to 168 source of bacillus subtilis
SwrC gene or swrC gene-correlation codon optimised sequence and related high homology sequence.
5. application according to claim 3, which is characterized in that swrC gene refers to 168 source of bacillus subtilis
The codon optimised sequence and related high homology sequence of swrC gene or swrC gene-correlation.
6. application according to claim 1 or 2, which is characterized in that lactic acid bacteria refers to Lactococcus lactis, plant cream bar
Bacterium, lactobacillus fermenti or streptococcus thermophilus etc. have the lactic acid bacteria of inhibitory activity to surfactin.
7. application according to claim 3, which is characterized in that lactic acid bacteria refers to Lactococcus lactis, lactobacillus plantarum, hair
Kefir milk bacillus or streptococcus thermophilus etc. have the lactic acid bacteria of inhibitory activity to surfactin.
8. application according to claim 4, which is characterized in that lactic acid bacteria refers to Lactococcus lactis, lactobacillus plantarum, hair
Kefir milk bacillus or streptococcus thermophilus etc. have the lactic acid bacteria of inhibitory activity to surfactin.
9. application according to claim 5, which is characterized in that lactic acid bacteria refers to Lactococcus lactis, lactobacillus plantarum, hair
Kefir milk bacillus or streptococcus thermophilus etc. have the lactic acid bacteria of inhibitory activity to surfactin.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102220367A (en) * | 2011-04-27 | 2011-10-19 | 南京农业大学 | Method for increasing yield of antibacterial peptide of bacillus subtilis through overexpression of yerP gene |
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| CN102220367A (en) * | 2011-04-27 | 2011-10-19 | 南京农业大学 | Method for increasing yield of antibacterial peptide of bacillus subtilis through overexpression of yerP gene |
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| DANIEL B. KEARNS等: "Genes governing swarming in Bacillus subtilis and evidence for a phase variation mechanism controlling surface motility", 《MOLECULAR MICROBIOLOGY》 * |
| KENJI TSUGE等: "Gene yerP, Involved in Surfactin Self-Resistance in Bacillus subtilis", 《ANTIMICROBIAL AGENTS AND CHEMOTHERAPY》 * |
| 伯沄等主编: "《病理学技术》", 30 June 2000, 北京：人民卫生出版社 * |
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