CN107012159B - A kind of streptococcus mutans biomembrane pH indicating gage and its application based on gene recombination plasmid - Google Patents
A kind of streptococcus mutans biomembrane pH indicating gage and its application based on gene recombination plasmid Download PDFInfo
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- CN107012159B CN107012159B CN201710333236.0A CN201710333236A CN107012159B CN 107012159 B CN107012159 B CN 107012159B CN 201710333236 A CN201710333236 A CN 201710333236A CN 107012159 B CN107012159 B CN 107012159B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
- C12N15/746—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for lactic acid bacteria (Streptococcus; Lactococcus; Lactobacillus; Pediococcus; Enterococcus; Leuconostoc; Propionibacterium; Bifidobacterium; Sporolactobacillus)
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/315—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/65—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression using markers
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
Abstract
The streptococcus mutans biomembrane pH indicating gage and its application, the indicating gage that the invention discloses a kind of based on gene recombination plasmid are expressed by the way that plasmid pDLpH to be transformed into host's Streptococcus mutans UA159；The nucleotide sequence of the plasmid pDLpH is as shown in SEQ ID NO:1, molecular weight 66183.62Da；Indicating gage of the invention is at low cost, it is applied widely, on the growing environment of microorganism without influence, and construct conveniently, culture is simple, proliferation is fast.
Description
Technical field
The present invention relates to biomembrane pH indicating gages, and in particular to a kind of Streptococcus mutans biology based on gene recombination plasmid
Film indicating gage, preparation and its application.
Background technique
Dental caries are one of most common mouth diseases, the aggregation of facing Dental plaque biofilm be dental caries formed it is primary because
Element；Dental plaque biofilm forms rapid and more difficult to remove, and structure is special in biomembrane；Microorganism therein is in special environment
Growth, be metabolized the acidic materials such as the lactic acid of generation be not easy outflow be also not easy to be neutralized by saliva, therefore can cause plaque bottom adhere to
The long-term low ph conditions in the part of facing；Dental caries start in tooth body demineralization caused by local low ph conditions；It can be seen that phase
Itself for bacterium, the low ph conditions for producing bacterial plaque attaching surface caused by acid are only the pathogenetic direct factor of dental caries；Such as
Intuitively and accurately reflection bacterial plaque pH is also particularly important for what.
Currently, the measurement method of bacterial plaque pH substantially have it is following several: 1, contact electrode mensuration: by microelectrode insertion to
Position is surveyed to be detected；2, sample detection method: detected part bacterial plaque is collected, then is detected by pH meter；Both
Method is simple, cheap, flexibly, but while measuring can be upset that detected part biomembrane, testing time be discontinuous, buffer function of saliva
Influence big, ex situ detection；3, microelectrode inherent electrode measurement method: is placed in the body surface of not formed biomembrane (such as in advance
Basal seat area, culture dish bottom etc.), it is subsequently placed in patient mouthful or accesses bacterium, it can be continuously long-term by radio technology
Detect pH in ground；This method does not upset the formation of biomembrane and is in situ detection, but it is expensive, complicated for operation；4, pH contaminates
Material method: the pH fluorescent dye of commodity in use is dyed and is detected to biomembrane；The operation of pH dyeing is easy in the method
It is influenced by factors such as biofilm thickness, biofilm surface degree of roughness, causes experimental error；On the whole, biomembrane at present
The measurement method of pH is not fully up to expectations.
Summary of the invention
The present invention provides a kind of convenience, cheap, continuous in-situ measurement and the lesser change based on gene recombination plasmid of error
Different streptococcus organism film indicating gage, preparation and its application.
The technical solution adopted by the present invention is that:
A kind of nucleotide sequence of gene recombination plasmid pDLpH, the plasmid pDLpH as shown in SEQ ID NO:1, point
Son amount is 66183.62Da.
A kind of streptococcus mutans biomembrane pH indicating gage based on plasmid pDLpH, the indicating gage is by by plasmid pDLpH
It is transformed into host's Streptococcus mutans UA159 and is expressed.
Further, the plasmid pDLpH is expressed as green fluorescent protein in host's Streptococcus mutans UA159.
Further, indicating gage is detected for biomembrane pH.
Further, indicating gage is detected for factor relevant to biomembrane pH variation.
Further, indicating gage produces the screening of acid inhibitor for biomembrane.
Further, indicating gage is detected for streptococcus mutans biomembrane pH.
A kind of preparation method of gene recombination plasmid pDLpH, comprising the following steps:
(1) the pureI gene of streptococcus salivarius 57.I and the encoding gene of green fluorescent protein are obtained by PCR method
gfp；
(2) genetic fragment obtained using restriction endonuclease BamHI digestion step (1), is connected by DNA ligase
Obtain to obtain recombination segment pureI-gfp；
(3) recombination segment pureI-gfp obtained in step (2) and plasmid PDL278 are used into restricted core respectively
Sour restriction endonuclease SacI and SalI double digestion；
(4) product obtained in step (3) is connected using DNA ligase, and connection product is transformed into Escherichia coli
In DH5 α competent cell；
(5) it is coated with the fluid nutrient medium agar plate containing 100 μ g/mL spectinomycins, cultivates for 24 hours, chooses under aerobic conditions
Positive colony is selected to carry out Zengjing Granule for 24 hours in fluid nutrient medium；
(6) it extracts plasmid and carries out PCR and sequence verification, verifying correct recombinant plasmid is target product.
A kind of preparation method of indicating gage, comprising the following steps:
(1) Streptococcus mutans UA159 is inoculated in BHI medium agar plate, cultivates for 24 hours, chooses under 37 DEG C of anaerobic conditions
Single colonie is inoculated in BHI culture medium and passes on overnight；
(2) it after the bacterium solution being incubated overnight being diluted 20 times with BHI culture medium, cultivates under anaerobic condition to OD600nmValue is
0.2；
(3) it takes the 500 above-mentioned bacterium solutions of μ L in sterile EP tube, final concentration of 1 μ g/mL Streptococcus mutans impression is added thereto
State stimulator polypeptide CSP is placed in 37 DEG C of incubators and is incubated for；
(4) the above-mentioned carrier pDLpH of 5 μ L is added after 10min into step (3) solution, cultivates 2h under anaerobic condition；
(5) bacterium solution in 100 μ L steps (4) is taken to be coated with the BHI agar plate containing 1mg/mL spectinomycin, anaerobic condition
Lower culture 48h；
(6) anti-spectinomycin positive colony is selected in the BHI fluid nutrient medium containing 1mg/mL spectinomycin, and 37 DEG C are detested
Zengjing Granule is carried out under the conditions of oxygen to stay overnight to get the indicating gage is arrived.
The beneficial effects of the present invention are:
(1) indicating gage of the invention itself is one of the constituent of biomembrane, does not need to be dyed the operation such as elution
Detect the pH of its local environment；
(2) host of the present invention using the resident bacteria Streptococcus mutans of oral biological film as plasmid pDLpH, so that instruction
It counts applied widely；
(3) indicating gage is in condition of living organism in the present invention, and change in fluorescence can be observed continuously to detect life without operation bidirectional
Object film pH situation of change；
(4) indicating gage building is convenient in the present invention, and culture is simple, proliferation is fast；
(5) present invention in indicating gage on the growth of microorganism without influence, can more really reflect microorganism phenotype and ring
Relationship between the pH height of border；
(6) indicating gage of the present invention it is at low cost, it is applied widely, can continuous in-situ detect its locating pH and related to pH variation
Factor.
Detailed description of the invention
Fig. 1 is gene recombination plasmid pDLpH schematic diagram of construction method.
Fig. 2 is different pH, different disposal time-variance streptococcus pH indicating gage fluorogram.
Fig. 3 is different pH, different disposal time-variance streptococcus pH indicating gage unit area fluorescence intensity statistical chart.
Fig. 4 is that control group Δ codY/pDLpH indicates fluorescent image after different pH.
Fig. 5 is that control group UA159/pDL278-pldh-gfp indicates fluorescent image after different pH.
Specific embodiment
The present invention will be further described in the following with reference to the drawings and specific embodiments.
A kind of nucleotide sequence of gene recombination plasmid pDLpH, the plasmid pDLpH as shown in SEQ ID NO:1, point
Son amount is 66183.62Da.
A kind of streptococcus mutans biomembrane pH indicating gage based on plasmid pDLpH, the indicating gage is by by plasmid pDLpH
It is transformed into host's Streptococcus mutans UA159 and is expressed.
Further, the plasmid pDLpH is expressed as green fluorescent protein in host's Streptococcus mutans UA159.
Further, indicating gage is detected for biomembrane pH.
Further, indicating gage is detected for factor relevant to biomembrane pH variation.
Further, indicating gage produces the screening of acid inhibitor for biomembrane.
Further, indicating gage is detected for streptococcus mutans biomembrane pH.
A kind of preparation method of gene recombination plasmid pDLpH, as shown in Figure 1, comprising the following steps:
(1) the pureI gene of streptococcus salivarius 57.I and the encoding gene of green fluorescent protein are obtained by PCR method
gfp；
Streptococcus mutans UA159 (Sichuan University with streptococcus salivarius 57.I and containing green fluorescent protein encoding gene
Mouth disease research National Key Laboratory save) genomic DNA be template, with pureI-SacI:5 '-
TTTGAGCTCATTCCTGGGAGACTAGCTG-3'；pureI-BamHI:5'-TTTGGATCCCTCCTAAGTTTTTTATGTTA
ATATC-3'.And gfp-BamHI:5 '-AGAAGGATCCATGAGTAAAGGAGAAGAACTTTTC-3 '； gfp-SalI:5'-
TTTTGTCGACCTATTTGTATAGTTCATCCATGCCATG-3 ' is the pureI that primer distinguishes PCR amplification about 450bp size
The gfp gene of gene and about 700bp size；
PCR process is as follows:
Pcr amplification product is detected by 2% agarose gel electrophoresis, DNA gel QIAquick Gel Extraction Kit recycles in PCR product
Target gene fragment；
(2) genetic fragment obtained using restriction endonuclease BamHI digestion step (1), is connected by DNA ligase
Obtain to obtain recombination segment pureI-gfp；
Then the target gene fragment restriction endonuclease BamHI digestion that amplification is obtained uses 2% agarose
Target gene fragment after gel electrophoresis and DNA gel QIAquick Gel Extraction Kit recycling digestion, and use 16 DEG C of T4DNA ligase connections
16 hours；Connection product is analyzed using 1.5% agarose gel electrophoresis, and the band for cutting 1200bp size carries out
DNA recycling, PCR enrichment recombination connector segment；
PCR process is as follows:
(3) recombination segment pureI-gfp obtained in step (2) and plasmid pDL278 (are preserved in Sichuan University
Mouth disease research National Key Laboratory) restriction endonuclease SacI and SalI double digestion is used respectively；
(4) product obtained in step (3) is connected using DNA ligase, and connection product is transformed into Escherichia coli
In DH5 α competent cell；
(5) it is coated with the LB liquid medium agar plate containing 100 μ g/mL spectinomycins, is cultivated for 24 hours under aerobic conditions,
It selects positive colony and carries out Zengjing Granule for 24 hours in LB liquid medium；
(6) it extracts plasmid and carries out PCR and sequence verification, verifying correct recombinant plasmid is target product.
A kind of preparation method of indicating gage, comprising the following steps:
(1) Streptococcus mutans UA159 (preservation of mouth disease research National Key Laboratory, Sichuan University) is inoculated in BHI
Medium agar plate is cultivated for 24 hours under 37 DEG C of anaerobic conditions, after smear for microscopic examination and biochemical identification, chooses single colonie and be inoculated in BHI
It is passed on overnight in culture medium；
(2) it after the bacterium solution being incubated overnight being diluted 20 times with BHI culture medium, cultivates under anaerobic condition to OD600nmValue is
0.2；
(3) it takes the 500 above-mentioned bacterium solutions of μ L in sterile EP tube, final concentration of 1 μ g/mL Streptococcus mutans impression is added thereto
State stimulator polypeptide CSP is placed in 37 DEG C of incubators and is incubated for；
(4) the above-mentioned carrier pDLpH of 5 μ L is added after 10min into step (3) solution, cultivates 2h under anaerobic condition；
(5) bacterium solution in 100 μ L steps (4) is taken to be coated with the BHI agar plate containing 1mg/mL spectinomycin, anaerobic condition
Lower culture 48h；
(6) anti-spectinomycin positive colony is selected in the BHI fluid nutrient medium containing 1mg/mL spectinomycin, and 37 DEG C are detested
Zengjing Granule is carried out under the conditions of oxygen to stay overnight；
PCR verifying is carried out using bacterium solution, verification process is as follows:
It verifies correct mutants streptococcus strain and is named as UA159/pDLpH, the as described indicating gage.
Embodiment 1
In order to verify the effect of the indicating gage, tested as follows.
(1) recombination mutants streptococcus strain UA159 is inoculated in containing final concentration of 15mg/mL acid-base buffer agent Pipes
The BHI+H in (Sigma, the U.S.) and 1mg/mL spectinomycin2In O (volume ratio 1:1) culture medium, culture in 37 DEG C of anaerobic culture boxes
Overnight, bacterium solution is spare；
(2) the glass sequin after 9 sterilizings is respectively placed in 9 holes of 24 orifice plates, and it is above-mentioned that 2mL is added thereto
Mixed-culture medium and the 20 above-mentioned bacterium solutions of μ L；
In (3) 37 DEG C of anaerobic culture boxes after culture 14 hours, it is respectively placed in the BHI training of pH 7.5, pH 5.5 and pH 3.5
It supports and handles 1min, 30min, 60min in base；
(4) taking-up of each sequin is placed on glass slide, a drop unstressed configuration mirror oil is added dropwise to sequin center, carefully covers
Coverslip；
(5) in just setting the above-mentioned sample of the lower observation of fluorescence microscope blue violet light excitation, each sample randomly selects five visuals field
And its unit area fluorescence intensity is calculated using ImagePro-Plus, and use SNK check analysis group difference.
Its fluorogram is as shown in Fig. 2, unit area fluorescence intensity statistical chart is as shown in Figure 3；
As shown in Figures 2 and 3, when handling pH is 7.5 and 5.5, with the growth of processing time, unit area fluorescence intensity
It is significantly raised；When handling pH is 3.5, processing 1min and 30min unit area fluorescence intensity no significant difference, but relatively handle
It is low when 60min；When handling same time using different pH, unit area fluorescence intensity is increased with the reduction of pH value；Processing
When 1min, with the reduction of pH, indicating gage fluorescence intensity is remarkably reinforced；Illustrate that the indicating gage can rapidly reflect detected part
PH height；With the growth of processing time, Streptococcus mutans growth and breeding produces acid simultaneously, reduces the pH of local environment, therefore
Indicating gage fluorescence intensity increases at any time and enhances.
Embodiment 2
Specificity for verifying indicating gage is tested as follows.
Control group Δ codY/pDLpH and UA159/pDL278-pldh-gfp is constructed first.
1, the building of control group Δ codY/pDLpH
(1) Streptococcus mutans Δ codY is inoculated in improvement BHI medium agar plate, is cultivated under 37 DEG C of anaerobic conditions
24h；
(2) choose single colonie be inoculated in respectively improvement BHI culture medium in passage overnight；
(3) using improvement BHI culture medium will the bacterium solution that be incubated overnight dilute 20 times after, cultivated under anaerobic condition to
OD600nmValue is 0.2；
(4) it takes the 500 above-mentioned bacterium solutions of μ L in sterile EP tube, final concentration of 1 μ g/mL Streptococcus mutans CSP is added thereto,
It is placed in 37 DEG C of incubators and is incubated for；
(5) carrier pDLpH is added after 10min thereto.2.5h is cultivated under anaerobic condition；
(6) it takes 100 μ L bacterium solutions to be coated with the improvement BHI agar plate containing 1mg/mL spectinomycin, is cultivated under anaerobic condition
48h；
(7) spectinomycin positive colony is selected in improvement BHI culture medium, carries out Zengjing Granule under 37 DEG C of anaerobic conditions；
(8) for 24 hours after using bacterium solution as template, gfp-BamHI and gfp-SalI are primer, carry out PCR amplification verifying.It will test
It demonstrate,proves correct bacterial strain and is respectively designated as Δ codY/pDLpH, conservation freezes for use.
2, the building of control group UA159/pDL278-pldh-gfp
(1) Streptococcus mutans UA159 is inoculated in improvement BHI medium agar plate, is cultivated under 37 DEG C of anaerobic conditions
24h；
(2) choose single colonie be inoculated in respectively improvement BHI culture medium in passage overnight；
(3) using improvement BHI culture medium will the bacterium solution that be incubated overnight dilute 20 times after, cultivated under anaerobic condition to
OD600nmValue is 0.2；
(4) it takes the 500 above-mentioned bacterium solutions of μ L in sterile EP tube, final concentration of 1 μ g/mL Streptococcus mutans CSP is added thereto,
It is placed in 37 DEG C of incubators and is incubated for；
(5) 5 μ L plasmid pDL278-pldh-gfp are added after 10min thereto and (are preserved in Sichuan University's mouth disease research
National Key Laboratory), 2.5h is cultivated under anaerobic condition；
(6) it takes 100 μ L bacterium solutions to be coated with the improvement BHI agar plate containing 1mg/mL spectinomycin, is cultivated under anaerobic condition
48h；
(7) spectinomycin positive colony is selected in improvement BHI culture medium, carries out Zengjing Granule under 37 DEG C of anaerobic conditions；
(8) for 24 hours after using bacterium solution as template, gfp-BamHI and gfp-SalI are primer, carry out PCR amplification verifying.It will test
It demonstrate,proves correct bacterial strain and is respectively designated as UA159/pDL278-pldh-gfp, conservation freezes for use.
The test of control group progress fluorescence intensity
(1) Δ codY/pDLpH and UA159/pDL278-pldh-gfp are inoculated in respectively containing final concentration of 15mg/mL
The BHI+H in acid-base buffer agent Pipes (Sigma, the U.S.) and 1mg/mL spectinomycin2In O (volume ratio 1:1) culture medium, 37 DEG C
Overnight incubation in anaerobic culture box, bacterium solution are spare；
(2) the glass sequin after 18 sterilizings is respectively placed in 18 holes of two 24 orifice plates (each orifice plate 9
Hole), and the above-mentioned mixed-culture medium of 2mL is added thereto；
(3) bacterium solution of 20 μ L Δ codY/pDLpH is added in 9 holes into orifice plate A；Add in 9 holes into orifice plate B
Enter the bacterium solution of 20 μ L UA159/pDL278-pldh-gfp；
In (4) 37 DEG C of anaerobic culture boxes after culture 14 hours, it is respectively placed in the BHI training of pH 7.5, pH 5.5 and pH 3.5
It supports and handles 1min, 30min, 60min in base；
(5) taking-up of each sequin is placed on glass slide, a drop unstressed configuration mirror oil is added dropwise to sequin center, carefully covers
Coverslip；
(6) in just setting the above-mentioned sample of the lower observation of fluorescence microscope blue violet light excitation, each sample randomly selects five visuals field
And its unit area fluorescence intensity is calculated using ImagePro-Plus, and use SNK check analysis group difference.
Experimental result is as shown in Figure 4 and Figure 5, and Δ codY/pDLpH and UA159/pDL278-pldh-gfp unit area is glimmering
Luminous intensity respectively may be about 0.39IOD/ μm2And 0.42IOD/ μm2, and not with the height of processing pH and handle the length of time and become
Change；Compared with the indicating gage UA159/pDLPH in the present invention, Δ codY/pDLpH lacks CodY albumen and UA159/pDL278-
Pldh-gfp lacks sour evoked promoter pureI；The two in different pH and under the different disposal time, unit area fluorescence intensity without
Significant change illustrates its ability for not having instruction biomembrane pH；Meanwhile also demonstrating the spy of this patent building system instruction pH
It is anisotropic.
The present invention constructs pDLpH and be transferred in Streptococcus mutans using the sour inducing expression characteristic of pureI makes biomembrane
PH indicating gage in situ；Indicating gage itself is one of the constituent of biomembrane, does not need to be dyed the operation such as elution, can be detected
The pH of its local environment；Indicating gage is in condition of living organism always, and is not necessarily to operation bidirectional, and change in fluorescence can be observed continuously to examine
Survey biomembrane pH situation of change；Vector construction is convenient and efficient, and instruction is calculated as thallus itself, and it is fast to cultivate simple proliferation；Make a variation hammer
Bacterium is the resident bacteria of oral biological film, and the sour acid resistance of production is strong, as the host of carrier pDLpH, so that indicating gage is applicable in model
It encloses wide；It can be used for biomembrane pH detection and factor relevant to pH variation detection, such as biomembrane produces the screening of acid inhibitor.
Biomembrane in the present invention refer to microorganism individual between mutually stick aggregation formed structure is complicated, metastable micro-
Biocenose；Bacterial plaque refers to Dental plaque biofilm, is a kind of Bacterial biofilms, adheres to each other or be adhered to tooth for matrix package
The bacillary group of the soft and non-mineralising between face, tooth or dummy surface, cannot be washed away by water or gargle.
。
SEQ ID NO:1
<110>Sichuan University
<120>a kind of based on the streptococcus mutans biomembrane pH indicating gage of plasmid pDLpH and its application
<130>
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 7885
<212> DNA
<213>artificial sequence
<400> 1
ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc 60
gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct 120
cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg 180
tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc 240
cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga 300
aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct 360
cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg 420
gcgctttctc aatgctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag 480
ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat 540
cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac 600
aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac 660
tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc 720
ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt 780
tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc 840
ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg 900
agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca 960
atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca 1020
cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag 1080
ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac 1140
ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc 1200
agaagtggtc cgataaaccc agcgaaccat ttgaggtgat aggtaagatt ataccgaggt 1260
atgaaaacga gaattggacc tttacagaat tactctatga agcgccatat ttaaaaagct 1320
accaagacga agaggatgaa gaggatgagg aggcagattg ccttgaatat attgacaata 1380
ctgataagat aatatatctt ttatatagaa gatcgatttt cgttcgtgaa tacatgttat 1440
aataactata actaataacg taacgtgact ggcaagagat atttttaaaa caatgaatag 1500
gtttacactt actttagttt tatggaaatg aaagatcata tcatatataa tctagaataa 1560
aattaactaa aataattatt atctagataa aaaatttaga agccaatgaa atctataaat 1620
aaactaaatt aagtttattt aattaacaac tatggatata aaataggtac taatcaaaat 1680
agtgaggagg atatatttga atacatacga acaaattaat aaagtgaaaa aaatacttcg 1740
gaaacattta aaaaataacc ttattggtac ttacatgttt ggatcaggag ttgagagtgg 1800
actaaaacca aatagtgatc ttgacttttt agtcgtcgta tctgaaccat tgacagatca 1860
aagtaaagaa atacttatac aaaaaattag acctatttca aaaaaaatag gagataaaag 1920
caacttacga tatattgaat taacaattat tattcagcaa gaaatggtac cgtggaatca 1980
tcctcccaaa caagaattta tttatggaga atggttacaa gagctttatg aacaaggata 2040
cattcctcag aaggaattaa attcagattt aaccataatg ctttaccaag caaaacgaaa 2100
aaataaaaga atatacggaa attatgactt agaggaatta ctacctgata ttccattttc 2160
tgatgtgaga agagccatta tggattcgtc agaggaatta atagataatt atcaggatga 2220
tgaaaccaac tctatattaa ctttatgccg tatgatttta actatggaca cgggtaaaat 2280
cataccaaaa gatattgcgg gaaatgcagt ggctgaatct tctccattag aacataggga 2340
gagaattttg ttagcagttc gtagttatct tggagagaat attgaatgga ctaatgaaaa 2400
tgtaaattta actataaact atttaaataa cagattaaaa aaattataaa aaaattgaaa 2460
aaatggtgga aacacttttt tcaatttttt tgttttatta tttaatattt gggaaatatt 2520
cattctaatt ggtaatcaga ttttagaaaa caataaaccc ttgcataact ttctcgtcca 2580
tatcggaaac tactttttta ttcccttttt ttcgaccgag attattttgc gataaaatcc 2640
gattaagata ctgcctacta actcccaatt cttgagctaa ttctgatact gttttactca 2700
tcagctacct cacaaaataa ttctttctat cccaattcca aaaggcaaca atttcacgac 2760
cagtactttc tgcagattct tctgctccgg tctgaacaag attcccttct tcgacatcat 2820
caagagctaa ctccgccttg attttcttga acaactttcc aaaaccaatc tgcctttttc 2880
ggtgcaaacc attcgttaaa tcttctgtta tccgttcttt gaccatttca gagaataatg 2940
tcacatcttc tgtatctaca tcaaaaggct taacaggata tttagctgtt tcgataatag 3000
ctgacttcaa gtttttacgt ttatgagctt tgacggttcg aatatcaact acaggtgtat 3060
aatccaattt catcgcctta gcccatagtt tcgtccattc ttcttggcta atgtaatttg 3120
tattatttcc agtaaaatag ctagatttca caaataacaa tacatgcaaa tgagggtgat 3180
aattctcatg ctcagttgaa taagttacct cagtggctcg caaatagcca ataacatttt 3240
tatcgacttt cttgtatttc ataagacgat taaaagctcg ccctatttct gaaattgatt 3300
tatttaattt ttcaccacta atgttcttaa tcgtcaaggt caaaaagaga aagcgacctt 3360
ttggatagcg tttcattgct tcatttacca ctaattcagc ttgataagca tacttcattg 3420
accgtctcca gttacaaatt gggcagagct tatttttgca aaaataagac tgatagagtt 3480
ttttcgtgcc gtcttgttgt tcgataaact tcaacacttc ggcacattga tagacccgtt 3540
caaacgaacg atatcctaaa cgctctaaac gaccagcaag ctcaatattt tttaacttgc 3600
gttctcgcca ctttctatct ttgccattct tagaatagtc cttaaagact tgattttcag 3660
tacctgtcat gttataattt tcttgcaaaa aaattttttt gagtatataa aaaagttcct 3720
gcaattctcc taagtttatt attttaaatt ggacacttaa attataaact ttattttatt 3780
aaattgcaag ggcttttttg ttacctaaaa acccagcaat atcaagggtt tgaggtgctt 3840
taaaacaaga aaataaaaaa actcattatt tctatatgta tcaagataag aaaaaaccct 3900
tgctacgcaa ttgggttttt aagggctttt cagccctctt tttcgtttca cgaaaaattc 3960
acccctaaaa cctcaaattt tgactttcta aatccaaacg tggtataata attttaacgc 4020
tagaagttct aggaaacgaa tagcacaacg caaaaaaaac agtagctgga actactgtct 4080
ttcttttttg tcctcaaaaa gaactcaggt taatttttgt cgcgtttgtt tcgaacatac 4140
cgaatcaacc aatcagcact tacctctgca gcaacgccaa ccacaaaagg tatcaaaaca 4200
agttctagga aagtcttcac agcgctcacc tccaatctaa gttcgtaact tagcgaaaga 4260
acgttgcgac ctagctatta taccatgtcc gcttacttta ttccatggtc taattgacaa 4320
caagtaacca agggcggacg tcctttgtcc gtgtcggctc gaaacgctaa agcctttcgg 4380
ctcgtcacgt cctagcgtac tttgcccttg cttattgtca attagctttc acggcataaa 4440
tcgctcaaag gcctagccct ttttcaatca ctcgtttaac tacccttacg attggctgaa 4500
tagctcttaa ctctgatata aaatttttta aggctttatc tggcataaac tctttcagtt 4560
ttcctagcat tttgtcattt tgctgtctca aaactgagtt ttcacttcta agcgctctat 4620
tttcgttcct aagacgctct atgctctctc ccttactttt ttctttagct cgttccaact 4680
cgcttcaaac cgtttagagc gtctcagagg cactatccgc cttaattgct cattctctag 4740
ctctttttga tgaacaaggt ttctgcttcg gtatatcagc cctttaaatt cttcaaactg 4800
ctctttagtc aattccacat attctttacc gaacacgcct tttttagggg taatagccgc 4860
tagttctttc tcagacatga tttttaggtc agctatttgt gtttttagtt tttctaattt 4920
cagcccctcg cgctctaaac gctctatttt cgcttctaag ctcttcgaat aattttctaa 4980
ggtatttata tatccctcag ctttagatag ctctgagagc ctctcagagg tctcagaatc 5040
gccaggaccc aacgctgccc gactgctttc ctgatgcaaa aacgaggcta gtttaccgta 5100
tctgtggggg gatggcttgt agatatgacg acaggaagag tttgtagaaa cgcaaaaagg 5160
ccatccgtca ggatggcctt ctgcttaatt tgatgcctgg cagtttatgg cgggcgtcct 5220
gcccgccacc ctccgggccg ttgcttcgca acgttcaaat ccgctcccgg cggatttgtc 5280
ctactcagga gagcgttcac cgacaaacaa cagataaaac gaaaggccca gtctttcgac 5340
tgagcctttc gttttatttg atgcctggca gttccctact ctcgcatggg gagaccccac 5400
actaccatcg gcgctacggc gtttcacttc tgagttcggc atggggtcag gtgggaccac 5460
cgcgctactg ccgccaggca aattctgttt tatcagaccg cttctgcgtt ctgatttaat 5520
ctgtatcagg ctgaaaatct tctctcatcc gccaaaacag ctgctttcct gatgcaaaaa 5580
cgaggctagt ttaccgtatc tgtgggggga tggcttgtag atatgacgac aggaagagtt 5640
tgtagaaacg caaaaaggcc atccgtcagg atggccttct gcttaatttg atgcctggca 5700
gtttatggcg ggcgtcctgc ccgccaccct ccgggccgtt gcttcgcaac gttcaaatcc 5760
gctcccggcg gatttgtcct actcaggaga gcgttcaccg acaaacaaca gataaaacga 5820
aaggcccagt ctttcgactg agcctttcgt tttatttgat gcctggcagt tccctactct 5880
cgcatgggga gaccccacac taccatcggc gctacggcgt ttcacttctg agttcggcat 5940
ggggtcaggt gggaccaccg cgctactgcc gccaggcaaa ttctgtttta tcagaccgct 6000
tctgcgttct gatttaatct gtatcaggct gaaaatcttc tctcatccgc caaaacagcc 6060
gagatgcgcc gcgtgcggct gctggagatg gcggacgcga tggatatgtt ctgccaaggg 6120
ttggtttgcg cattcacagt tctccgcaag aattgattgg ctccaattct tggagtggtg 6180
aataattccc ggcattcgcc attcaggctg cgcaactgtt gggaagggcg atcggtgcgg 6240
gcctcttcgc tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg 6300
gtaacgccag ggttttccca gtcacgacgt tgtaaaacga cggccagtga attcgagctc 6360
attcctggga gactagctgt aaatcagtag tgtagttatt tgtttccatc ataattctcc 6420
ttttggattt ttgtccaaaa aaagacttta tcataaaaaa cgtttgactt tgttacccaa 6480
ctgtaaaatt actaaaaata ggtctatgga cttaggtctg gagaatgagt tggaaaaata 6540
ggcgagaaaa aaatataatg ctcacattgg atgatagatt gtacggacta tattgtcaga 6600
aacagtcaaa tactaaagga agctttttat agattaactg tttatttatc tgggattaag 6660
caaaggactc ctattgagaa ttaccaaatg taatgtcatt ttttgacacc acatgttagc 6720
ttgactaata tgtaaatgtt gcaaaatttc tgaaaattcg ttgacatgtg ttgtcaaagt 6780
agtatgatat taacataaaa aacttaggag ggatccatga gtaaaggaga agaacttttc 6840
actggagttg tcccaattct tgttgaatta gatggtgatg ttaatgggca caaattttct 6900
gtcagtggag agggtgaagg tgatgcaaca tacggaaaac ttacccttaa atttatttgc 6960
actactggaa aactacctgt tccatggcca acacttgtca ctactttctc ttatggtgtt 7020
caatgctttt caagataccc agatcatatg aaacggcatg actttttcaa gagtgccatg 7080
cccgaaggtt atgtacagga aagaactata tttttcaaag atgacgggaa ctacaagaca 7140
cgtgctgaag tcaagtttga aggtgatacc cttgttaata gaatcgagtt aaaaggtatt 7200
gattttaaag aagatggaaa cattcttgga cacaaattgg aatacaacta taactcacac 7260
aatgtataca tcatggcaga caaacaaaag aatggaatca aagttaactt caaaattaga 7320
cacaacattg aagatggaag cgttcaacta gcagaccatt atcaacaaaa tactccaatt 7380
ggcgatggcc ctgtcctttt accagacaac cattacctgt ccacacaatc tgccctttcg 7440
aaagatccca acgaaaagag agaccacatg gtccttcttg agtttgtaac agctgctggg 7500
attacacatg gcatggatga actatacaaa taggtcgacc tgcaggcatg caagcttggc 7560
gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa 7620
catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac 7680
attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca 7740
ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attggagctt gttgtaactg 7800
aaaaaggaaa attattgtgc caggcagttg aaagtcagca ccttttaacg agtgctgaaa 7860
tgacggctaa atgggaaacg tattt 7885
Claims (7)
1. a kind of streptococcus mutans biomembrane pH indicating gage based on gene recombination plasmid, it is characterised in that: the genetic recombination
The nucleotide sequence of plasmid pDLpH is as shown in SEQ ID NO:1, molecular weight 66183.62Da；The indicating gage pass through by
Plasmid pDLpH is transformed into host's Streptococcus mutans UA159 and is expressed.
2. a kind of streptococcus mutans biomembrane pH indicating gage based on gene recombination plasmid pDLpH according to claim 1,
It is characterized by: the plasmid pDLpH is expressed as green fluorescent protein in host's Streptococcus mutans UA159.
3. a kind of application of indicating gage as described in claim 1, it is characterised in that: the detection for biomembrane pH.
4. a kind of application of indicating gage as described in claim 1, it is characterised in that: be used for factor relevant to biomembrane pH variation
Detection.
5. a kind of application of indicating gage as described in claim 1, it is characterised in that: produce the screening of acid inhibitor for biomembrane.
6. a kind of application of indicating gage as described in claim 1, it is characterised in that: detected for streptococcus mutans biomembrane pH.
7. a kind of preparation method of indicating gage as described in claim 1, which comprises the following steps:
(1) Streptococcus mutans UA159 is inoculated in BHI medium agar plate, is cultivated for 24 hours under 37 DEG C of anaerobic conditions, chooses single bacterium
It falls to be inoculated in BHI culture medium and pass on overnight；
(2) it after the bacterium solution being incubated overnight being diluted 20 times with BHI culture medium, cultivates under anaerobic condition to OD600nmValue is 0.2；
(3) it takes the 500 above-mentioned bacterium solutions of μ L in sterile EP tube, final concentration of 1 μ g/mL Streptococcus mutans competence thorn is added thereto
Kassinin kinin CSP is placed in 37 DEG C of incubators and is incubated for；
(4) the above-mentioned carrier pDLpH of 5 μ L is added after 10min into step (3) solution, cultivates 2h under anaerobic condition；
(5) it takes the bacterium solution in 100 μ L steps (4) to be coated with the BHI agar plate containing 1mg/mL spectinomycin, is trained under anaerobic condition
Support 48h；
(6) anti-spectinomycin positive colony is selected in the BHI fluid nutrient medium containing 1mg/mL spectinomycin, 37 DEG C of anaerobism items
Zengjing Granule is carried out under part to stay overnight to get the indicating gage is arrived.
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