CN107012159A - A kind of Streptococcus mutans biomembrane pH indicating gages and its application based on gene recombination plasmid - Google Patents

Abstract

The invention discloses a kind of Streptococcus mutans biomembrane pH indicating gages based on gene recombination plasmid and its application, the indicating gage is expressed by the way that plasmid pDLpH is transformed into host's Streptococcus mutans UA159；The nucleotide sequence of the plasmid pDLpH such as SEQ ID NO:Shown in 1, its molecular weight is 66183.62Da；The indicating gage cost of the present invention is low, applied widely, on the growing environment of microorganism without influence, and build conveniently, culture is simple, propagation is fast.

Description

A kind of Streptococcus mutans biomembrane pH indicating gages based on gene recombination plasmid and its Using

Technical field

The present invention relates to biomembrane pH indicating gages, and in particular to a kind of Streptococcus mutans based on gene recombination plasmid are biological Film indicating gage, preparation and its application.

Background technology

Dental caries are one of most common mouth diseases, the aggregation of facing Dental plaque biofilm be dental caries formation it is primary because Element；Dental plaque biofilm forms rapid and more difficult to remove, and biomembrane inner structure is special；Microorganism therein is in special environment Grow, the acidic materials such as lactic acid that metabolism is produced are difficult outflow and are also difficult to be neutralized by saliva, therefore bacterial plaque bottom can be caused to adhere to The local long-term low ph conditions of facing；Dental caries start in the tooth body demineralization that local low ph conditions are caused；As can be seen here, phase For bacterium itself, the low ph conditions for the bacterial plaque attaching surface that its production acid is caused are only the pathogenetic direct factor of dental caries；Such as What intuitively and accurately reflects that bacterial plaque pH is also particularly important.

At present, bacterial plaque pH measuring method substantially has following several：1st, contact electrode mensuration：Microelectrode insertion is treated Position is surveyed to be detected；2nd, detection method is sampled：Detected part bacterial plaque is collected, then detected by pH meter；Both Method is simple, cheap, flexibly, but can upset that detected part biomembrane, testing time be discontinuous, the cushioning effect of saliva during measurement Influence is big, ex situ detection；3rd, inherent electrode measurement method：Microelectrode is placed in advance and does not form the body surface of biomembrane (such as Basal seat area, culture dish bottom etc.), be subsequently placed in patient it is intraoral or access bacterium, you can it is continuously long-term by radiotechnics Detect pH in ground；This method does not upset the formation of biomembrane and in situ detection, but its expensive, complex operation；4th, pH contaminates Material method：The pH fluorescent dyes of commodity in use are dyed and detected to biomembrane；The operation of pH dyeings is easy in the method Influenceed by factors such as biofilm thickness, biofilm surface degree of roughness, cause experimental error；On the whole, current biomembrane PH measuring method is not fully up to expectations.

The content of the invention

The present invention provides a kind of convenience, cheap, continuous in-situ measurement and the less 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：

Gene recombination plasmid pDLpH, the plasmid pDLpH a kind of nucleotide sequence such as SEQ ID NO:Shown in 1, its point Son amount is 66183.62Da.

A kind of Streptococcus mutans biomembrane pH indicating gages 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 used for the factor detection related to biomembrane pH changes.

Further, indicating gage is used for the screening that biomembrane produces acid inhibitor.

Further, indicating gage is detected for Streptococcus mutans biomembrane pH.

A kind of gene recombination plasmid pDLpH preparation method, comprises the following steps：

(1) streptococcus salivarius 57.I pureI genes and the encoding gene of green fluorescent protein are obtained by PCR method gfp；

(2) genetic fragment obtained using restriction endonuclease BamHI digestions step (1), is connected by DNA ligase Obtain to obtain recombination fragment pureI-gfp；

(3) the recombination fragment pureI-gfp obtained in step (2) and plasmid PDL278 are used into restricted core respectively Sour restriction endonuclease SacI and SalI double digestions；

(4) product obtained in step (3) is connected using DNA ligase, and connection product is transformed into Escherichia coli In DH5 α competent cells；

(5) it is coated with the fluid nutrient medium agar plate containing 100 μ g/mL spectinomycins, aerobic conditions and cultivates 24h, chooses Positive colony is selected to carry out Zengjing Granule 24h in fluid nutrient medium；

(6) extract plasmid and enter performing PCR and sequence verification, it is target product to verify correct recombinant plasmid.

A kind of preparation method of indicating gage, comprises the following steps：

(1) Streptococcus mutans UA159 is inoculated under BHI medium agar flat boards, 37 DEG C of anaerobic conditions and cultivates 24h, chosen Single bacterium colony is inoculated in passage in BHI culture mediums and stayed overnight；

(2) bacterium solution of incubated overnight is diluted after 20 times with BHI culture mediums, in being cultivated under anaerobic condition to OD_600nmIt is worth and is 0.2；

(3) the 500 above-mentioned bacterium solutions of μ L are taken in sterile EP pipes, final concentration of 1 μ g/mL Streptococcus mutans impression is added thereto State stimulator polypeptide CSP, is placed in being incubated in 37 DEG C of incubators；

(4) added after 10min into step (3) solution under the above-mentioned carrier pDLpH of 5 μ L, anaerobic condition and cultivate 2h；

(5) bacterium solution in 100 μ L steps (4) is taken to be coated with the BHI agar plates containing 1mg/mL spectinomycins, anaerobic condition Lower culture 48h；

(6) anti-spectinomycin positive colony is selected in the BHI fluid nutrient mediums containing 1mg/mL spectinomycins, and 37 DEG C are detested Zengjing Granule is carried out under the conditions of oxygen to stay overnight, that is, obtains the indicating gage.

The beneficial effects of the invention are as follows：

(1) indicating gage of the invention is one of constituent of biomembrane in itself, without operations such as dyeing elutions Detect the pH of its local environment；

(2) present invention uses the resident bacteria Streptococcus mutans of oral biological film as plasmid pDLpH host so that indicate Meter is applied widely；

(3) indicating gage is in condition of living organism in the present invention, without operation bidirectional can Continuous Observation change in fluorescence to detect life Thing film pH situations of change；

(4) indicating gage is built conveniently in the present invention, and culture is simple, propagation is fast；

(5) present invention in indicating gage on the growth of microorganism without influence, can more really reflect microorganism phenotype and ring Relation between the pH height of border；

(6) indicating gage cost of the present invention it is low, applied widely, can continuous in-situ detect its residing pH and related to pH changes Factor.

Brief description of the drawings

Fig. 1 is gene recombination plasmid pDLpH construction method schematic diagrames.

Fig. 2 is different pH, different disposal time-variance streptococcus pH indicating gage fluorograms.

Fig. 3 is different pH, different disposal time-variance streptococcus pH indicating gage unit area fluorescence intensity statistical charts.

Fig. 4 is that control group Δ codY/pDLpH indicates fluoroscopic image after different pH.

Fig. 5 is that control group UA159/pDL278-pldh-gfp indicates fluoroscopic image after different pH.

Embodiment

The present invention will be further described with specific embodiment below in conjunction with the accompanying drawings.

Gene recombination plasmid pDLpH, the plasmid pDLpH a kind of nucleotide sequence such as SEQ ID NO:Shown in 1, its point Son amount is 66183.62Da.

A kind of Streptococcus mutans biomembrane pH indicating gages 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 used for the factor detection related to biomembrane pH changes.

Further, indicating gage is used for the screening that biomembrane produces acid inhibitor.

Further, indicating gage is detected for Streptococcus mutans biomembrane pH.

A kind of gene recombination plasmid pDLpH preparation method, as shown in figure 1, comprising the following steps：

(1) streptococcus salivarius 57.I pureI genes and the encoding gene of green fluorescent protein are obtained by PCR method gfp；

Streptococcus mutans UA159 (Sichuan Universitys with streptococcus salivarius 57.I and containing green fluorescent protein encoding gene Mouth disease research National Key Laboratory preserve) genomic DNA be template, with pureI-SacI:5’- TTTGAGCTCATTCCTGGGAGACTAGCTG-3’；pureI-BamHI:5’- TTTGGATCCCTCCTAAGTTTTTTATGTTAATATC-3’.And gfp-BamHI:5’- AGAAGGATCCATGAGTAAAGGAGAAGAACTTTTC-3’； gfp-SalI:5’- TTTTGTCGACCTATTTGTATAGTTCATCCATGCCATG-3 ' is that primer distinguishes the pureI that PCR expands about 450bp sizes The gfp genes of gene and about 700bp sizes；

PCR processes are as follows：

Pcr amplification product is detected by 2% agarose gel electrophoresis, DNA gel QIAquick Gel Extraction Kit is reclaimed in PCR primer Target gene fragment；

(2) genetic fragment obtained using restriction endonuclease BamHI digestions step (1), is connected by DNA ligase Obtain to obtain recombination fragment pureI-gfp；

Obtained target gene fragment restriction endonuclease BamHI digestions will be expanded, then using 2% agarose Gel electrophoresis and DNA gel QIAquick Gel Extraction Kit reclaim the target gene fragment after digestion, and use the 16 DEG C of connections of T4DNA ligases 16 hours；Connection product is analyzed using 1.5% agarose gel electrophoresis, and cuts the band of 1200bp sizes and is carried out DNA is reclaimed, PCR enrichment restructuring connector fragments；

PCR processes are as follows：

(3) the recombination fragment 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 cells；

(5) it is coated with the LB fluid nutrient medium agar plates containing 100 μ g/mL spectinomycins, aerobic conditions and cultivates 24h, Select positive colony and Zengjing Granule 24h is carried out in LB fluid nutrient mediums；

(6) extract plasmid and enter performing PCR and sequence verification, it is target product to verify correct recombinant plasmid.

A kind of preparation method of indicating gage, comprises the following steps：

(1) Streptococcus mutans UA159 (preservation of mouth disease research National Key Laboratory of Sichuan University) is inoculated in BHI Cultivated under medium agar flat board, 37 DEG C of anaerobic conditions after 24h, smear for microscopic examination and biochemical identification, choose single bacterium colony and be inoculated in BHI Passage is stayed overnight in culture medium；

(2) bacterium solution of incubated overnight is diluted after 20 times with BHI culture mediums, in being cultivated under anaerobic condition to OD_600nmIt is worth and is 0.2；

(3) the 500 above-mentioned bacterium solutions of μ L are taken in sterile EP pipes, final concentration of 1 μ g/mL Streptococcus mutans impression is added thereto State stimulator polypeptide CSP, is placed in being incubated in 37 DEG C of incubators；

(4) added after 10min into step (3) solution under the above-mentioned carrier pDLpH of 5 μ L, anaerobic condition and cultivate 2h；

(5) bacterium solution in 100 μ L steps (4) is taken to be coated with the BHI agar plates containing 1mg/mL spectinomycins, anaerobic condition Lower culture 48h；

(6) anti-spectinomycin positive colony is selected in the BHI fluid nutrient mediums containing 1mg/mL spectinomycins, and 37 DEG C are detested Zengjing Granule is carried out under the conditions of oxygen to stay overnight；

Enter performing PCR using bacterium solution to verify, verification process is as follows：

Verify that correct mutants streptococcus strain is named as UA159/pDLpH, as described indicating gage.

Embodiment 1

In order to verify the effect of the indicating gage, tested as follows.

(1) restructuring mutants streptococcus strain UA159 is inoculated in containing final concentration of 15mg/mL acid-base buffer agents Pipes (Sigma, the U.S.) and the BHI+H of 1mg/mL spectinomycins₂O (volume ratios 1:1) in culture medium, cultivated in 37 DEG C of anaerobic culture boxes Overnight, bacterium solution is standby；

(2) the glass sequin after 9 are sterilized is respectively placed in 9 holes of 24 orifice plates, and it is above-mentioned to add 2mL thereto Mixed-culture medium and the 20 above-mentioned bacterium solutions of μ L；

After being cultivated 14 hours in (3) 37 DEG C of anaerobic culture boxes, pH 7.5, pH 5.5 and pH 3.5 BHI trainings are respectively placed in Support and 1min, 30min, 60min are handled in base；

(4) each sequin is taken out and be placed on slide, being added dropwise one to sequin center drips unstressed configuration mirror oil, carefully covers Cover glass；

(5) the above-mentioned sample of lower observation is excited in just putting fluorescence microscope blue violet light, each sample randomly selects five visuals field And its unit area fluorescence intensity is calculated using ImagePro-Plus, and use SNK check analysis group differences.

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 it is 7.5 and 5.5 to handle pH, with the growth of processing time, unit area fluorescence intensity It is significantly raised；When it is 3.5 to handle pH, processing 1min and 30min unit area fluorescence intensity no significant differences, but relatively handle It is low during 60min；When handling same time using different pH, unit area fluorescence intensity is raised with the reduction of pH value；Processing During 1min, with pH reduction, 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 and strengthened with the time.

Embodiment 2

Specificity for checking indicating gage is tested as follows.

Control group Δ codY/pDLpH and UA159/pDL278-pldh-gfp are built first.

1st, control group Δ codY/pDLpH structure

(1) Streptococcus mutans Δ codY is inoculated under improvement BHI medium agar flat boards, 37 DEG C of anaerobic conditions and cultivated 24h；

(2) choose single bacterium colony and be inoculated in respectively in improvement BHI culture mediums and pass on and stay overnight；

(3) bacterium solution of incubated overnight is diluted after 20 times using improvement BHI culture mediums, in cultivated under anaerobic condition to OD_600nmIt is worth for 0.2；

(4) take the 500 above-mentioned bacterium solutions of μ L in sterile EP pipes, final concentration of 1 μ g/mL Streptococcus mutans CSP added thereto, It is placed in being incubated in 37 DEG C of incubators；

(5) carrier pDLpH is added after 10min thereto.2.5h is cultivated under anaerobic condition；

(6) take 100 μ L bacterium solutions to be coated with the improvement BHI agar plates containing 1mg/mL spectinomycins, anaerobic condition to cultivate 48h；

(7) spectinomycin positive colony is selected in improvement BHI culture mediums, and Zengjing Granule is carried out under 37 DEG C of anaerobic conditions；

(8) using bacterium solution as template after 24h, gfp-BamHI and gfp-SalI are primer, enter performing PCR amplification checking.It will test Demonstrate,prove correct bacterial strain and be respectively designated as Δ codY/pDLpH, conservation freezes stand-by.

2nd, control group UA159/pDL278-pldh-gfp structure

(1) Streptococcus mutans UA159 is inoculated under improvement BHI medium agar flat boards, 37 DEG C of anaerobic conditions and cultivated 24h；

(2) choose single bacterium colony and be inoculated in respectively in improvement BHI culture mediums and pass on and stay overnight；

(3) bacterium solution of incubated overnight is diluted after 20 times using improvement BHI culture mediums, in cultivated under anaerobic condition to OD_600nmIt is worth for 0.2；

(4) take the 500 above-mentioned bacterium solutions of μ L in sterile EP pipes, final concentration of 1 μ g/mL Streptococcus mutans CSP added thereto, It is placed in being incubated in 37 DEG C of incubators；

(5) 5 μ L plasmids 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) take 100 μ L bacterium solutions to be coated with the improvement BHI agar plates containing 1mg/mL spectinomycins, anaerobic condition to cultivate 48h；

(7) spectinomycin positive colony is selected in improvement BHI culture mediums, and Zengjing Granule is carried out under 37 DEG C of anaerobic conditions；

(8) using bacterium solution as template after 24h, gfp-BamHI and gfp-SalI are primer, enter performing PCR amplification checking.It will test Demonstrate,prove correct bacterial strain and be respectively designated as UA159/pDL278-pldh-gfp, conservation freezes stand-by.

Control group carries out the test of fluorescence intensity

(1) Δ codY/pDLpH and UA159/pDL278-pldh-gfp are inoculated in containing final concentration of 15mg/mL respectively Acid-base buffer agent Pipes (Sigma, the U.S.) and 1mg/mL spectinomycins BHI+H₂O (volume ratios 1:1) in culture medium, 37 DEG C Overnight incubation in anaerobic culture box, bacterium solution is standby；

(2) the glass sequin after 18 are sterilized is respectively placed in 18 holes of two 24 orifice plates (each orifice plate 9 Hole), and the above-mentioned mixed-culture mediums of 2mL are added thereto；

(3) 20 μ L Δs codY/pDLpH bacterium solution is added in 9 holes into orifice plate A；Add in 9 holes into orifice plate B Enter 20 μ L UA159/pDL278-pldh-gfp bacterium solution；

After being cultivated 14 hours in (4) 37 DEG C of anaerobic culture boxes, pH 7.5, pH 5.5 and pH 3.5 BHI trainings are respectively placed in Support and 1min, 30min, 60min are handled in base；

(5) each sequin is taken out and be placed on slide, being added dropwise one to sequin center drips unstressed configuration mirror oil, carefully covers Cover glass；

(6) the above-mentioned sample of lower observation is excited in just putting fluorescence microscope blue violet light, each sample randomly selects five visuals field And its unit area fluorescence intensity is calculated using ImagePro-Plus, and use SNK check analysis group differences.

As shown in Figure 4 and Figure 5, Δ codY/pDLpH and UA159/pDL278-pldh-gfp unit area is glimmering for experimental result Luminous intensity respectively may be about 0.39IOD/ μm²And 0.42IOD/ μm², and do not become with processing pH height and the length of processing time Change；Compared with the indicating gage UA159/pDLPH in the present invention, Δ codY/pDLpH missing CodY albumen and UA159/pDL278- The sour evoked promoter pureI of pldh-gfp missings；Both in different pH and under the different disposal time, unit area fluorescence intensity without Significant change, illustrates it without the ability for indicating biomembrane pH；Meanwhile, also demonstrate the spy that this patent constructing system indicates pH The opposite sex.

The present invention is built pDLpH and be transferred in Streptococcus mutans using pureI sour induced expression characteristic makes biomembrane PH indicating gages in situ；Indicating gage is one of constituent of biomembrane in itself, without operations such as dyeing elutions, you can detection The pH of its local environment；Indicating gage is in condition of living organism all the time, and without operation bidirectional, can be examined with Continuous Observation change in fluorescence Survey biomembrane pH situations of change；Vector construction is convenient and swift, and instruction is calculated as thalline in itself, and the simple propagation of culture is fast；Make a variation hammer Bacterium is the resident bacteria of oral biological film, and the sour acid resistance of production is strong, as carrier pDLpH host so that indicating gage is applicable model Enclose wide；Detected available for biomembrane pH and the factor detection related to pH changes, such as biomembrane produces the screening of acid inhibitor.

Biomembrane in the present invention mutually sticks the complicated, metastable micro- of aggregation formation between referring to microorganism individual Biocenose；Bacterial plaque refers to Dental plaque biofilm, is a kind of Bacterial biofilms, be matrix parcel adhere to each other or adhere to tooth The soft and bacillary colony of non-mineralising between face, tooth or dummy surface, it is impossible to washed away by water or gargle.

SEQ ID NO:1

<110>Sichuan University

<120>A kind of Streptococcus mutans biomembrane pH indicating gages and its application based on plasmid pDLpH

<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 (9)

1. a kind of gene recombination plasmid pDLpH, it is characterised in that：The nucleotide sequence of the plasmid pDLpH such as SEQ ID NO:1 Shown, its molecular weight is 66183.62Da.

2. a kind of Streptococcus mutans biomembrane pH indicating gages based on plasmid described in claim 1, it is characterised in that：It is described to indicate Meter is expressed by the way that plasmid pDLpH is transformed into host's Streptococcus mutans UA159.

3. a kind of Streptococcus mutans biomembrane pH indicating gages based on gene recombination plasmid pDLpH according to claim 2, It is characterized in that：The plasmid pDLpH is expressed as green fluorescent protein in host's Streptococcus mutans UA159.

4. a kind of application of indicating gage as claimed in claim 2, it is characterised in that：Detection for biomembrane pH.

5. a kind of application of indicating gage as claimed in claim 2, it is characterised in that：For the factor related to biomembrane pH changes Detection.

6. a kind of application of indicating gage as claimed in claim 2, it is characterised in that：The screening of acid inhibitor is produced for biomembrane.

7. a kind of application of indicating gage as claimed in claim 2, it is characterised in that：For Streptococcus mutans biomembrane pH detections.

8. a kind of gene recombination plasmid pDLpH as claimed in claim 1 preparation method, it is characterised in that comprise the following steps：

(1) streptococcus salivarius 57.I pureI genes and the encoding gene gfp of green fluorescent protein are obtained by PCR method；

(2) genetic fragment obtained using restriction endonuclease BamHI digestions step (1), is obtained by DNA ligase connection Obtain recombination fragment pureI-gfp；

(3) by the recombination fragment pureI-gfp obtained in step (2) and plasmid PDL278 respectively using in restriction nuclease Enzyme cutting SacI and SalI double digestion；

(4) product obtained in step (3) is connected using DNA ligase, and connection product is transformed into bacillus coli DH 5 alpha In competent cell；

(5) it is coated with the fluid nutrient medium agar plate containing 100 μ g/mL spectinomycins, aerobic conditions and cultivates 24h, selects sun Property be cloned in fluid nutrient medium and carry out Zengjing Granule 24h；

(6) extract plasmid and enter performing PCR and sequence verification, it is target product to verify correct recombinant plasmid.

9. a kind of preparation method of indicating gage as claimed in claim 1, it is characterised in that comprise the following steps：

(1) Streptococcus mutans UA159 is inoculated under BHI medium agar flat boards, 37 DEG C of anaerobic conditions and cultivates 24h, choose single bacterium Fall to be inoculated in passage in BHI culture mediums to stay overnight；

(2) bacterium solution of incubated overnight is diluted after 20 times with BHI culture mediums, in being cultivated under anaerobic condition to OD_600nmIt is worth for 0.2；

(3) the 500 above-mentioned bacterium solutions of μ L are taken in sterile EP pipes, final concentration of 1 μ g/mL Streptococcus mutans competence thorn is added thereto Kassinin kinin CSP, is placed in being incubated in 37 DEG C of incubators；

(4) added after 10min into step (3) solution under the above-mentioned carrier pDLpH of 5 μ L, anaerobic condition and cultivate 2h；

(5) take the bacterium solution in 100 μ L steps (4) to be coated with the BHI agar plates containing 1mg/mL spectinomycins, anaerobic condition to train Support 48h；

(6) anti-spectinomycin positive colony is selected in the BHI fluid nutrient mediums containing 1mg/mL spectinomycins, 37 DEG C of anaerobism bars Zengjing Granule is carried out under part to stay overnight, that is, obtains the indicating gage.