CN103865861A - Streptococcus mutans AMC metabolic recovery strain based on LuxS defect - Google Patents

Abstract

The invention discloses a streptococcus mutans AMC metabolic recovery strain based on LuxS defect, wherein AMC metabolic recovery strain is StreptococcusmutansKO-SUA159:: delta luxS, pIB-sahH and has a preservation number of CCTCCM2013699. The LuxS-mediated metabolism is separated from density induction by heterologous expression of SahH in a Streptococcus mutans stain with LuxS defect, the obstacles to research on the LuxS mechanism, caused by dual-defect effect of the deletion of LuxS, is effectively overcome, and an effective molecular biology tool and experimental control are provided for the discuss about the regulation mechanism of Streptococcus mutans LuxS.

Description

Streptococcus mutans AMC metabolism based on LuxS defect recovers strain

Technical field

The present invention relates to microbe density induction mechanism research field, more particularly, the Streptococcus mutans AMC metabolism relating to based on LuxS defect recovers strain and structure thereof.

Background technology

Microbial film is the existence form of occurring in nature most of microbe, its formation can greatly strengthen the viability of microorganism and resist the ability from environment, host and antibacterials, and therefore biofilm formation becomes the Main Etiological Factors that many chronic and refractories catch.Density induction plays a significant role in biomembranous formation, development and maturation.Density induction (quorum sensing, QS) is a kind of groups exchange mechanism for bacterium, and bacterium is by the signaling molecule in synthetic, secretion perception environment, to regulate the expression of self some gene.In known multi-signal molecule, self-induction molecule-2(autoinducer-2, AI-2) because it produces, gene luxS is conservative is present in that in different bacterium, it has been considered to participate in the signal transmission between bacterial classification.By knocking out luxS gene, much research shows that the density induction defect of LuxS/ mediation can cause the many physiological performance damages of bacterium, comprises biofilm formation obstacle.But in fact, AI-2 is a kind of by product (accompanying drawing 1 is shown in methyl working cycle) in bacterium methyl circulation (AMC) process, from its production process, the generation of LuxS while catalysis AI-2 and HCY, therefore, LuxS disappearance not only can cause the density induction defect of AI-2 mediation, can cause AMC path obstacle simultaneously.Therefore, lacking by LuxS the dysfunction causing is also likely caused by AMC obstacle.Obviously, density induction and the metabolism double defect effect of LuxS disappearance, make the research of LuxS regulatory mechanism become difficulty, and thus, in LuxS control mechanisms, density induction becomes most important with separating of AMC metabolic pathway.

Streptococcus mutans is one of known main cariogenic bacteria, and microbial film is that plaque formation and acid resistance are its two large important cariogenic factors.Research shows that LuxS disappearance can cause many physiological barriers of Streptococcus mutans, comprises that the relevant pathogenic property of the sick generation of many dental caries changes.And in the research of obstacle mechanism, there is equally the dispute of density induction and metabolic regulation.The quorum sensing that generally believes in the past LuxS/AI-2 mediation is brought into play Main Function in these physiological change.But in recent years,, much research, by adding the method for external source AI-2, finds that LuxS lacks the many genetic transcription variations or the physiological property change that cause and is not restored.It is weakened gradually that the density of LuxS/AI-2 mediation is responded to impact in LuxS adjusting function thus.Obviously, as another contribution member of LuxS adjusting function, the regulating and controlling effect significant of metabolic pathway in Streptococcus mutans LuxS Related Physiological Characteristics.

The bacterium that lacks luxS genes at some as Pseudomonas aeruginosa in, the main dependence SahH that completes of AMC completes.The direct catalysis SAH of SahH generates HCY and can not produce by product DPD, therefore for density induction provides tool with separating of metabolism.SahH recovers studied proof (the Sylvio Redanz of ability of metabolism; Kerstin Standar; Andreas Podbielski, and Bernd Kreikemeyer. Heterologous Expression of Heterologous Expression of sahH Reveals That Biofilm Formation Is Autoinducer-2-independent in Streptococcus sanguinis but Is Associated with an Intact Activated Methionine Cycle [J]. J Biol Chem. 2012 October 19; 287 (43): 36111 – 36122.), based on this, we are take the strain of Streptococcus mutans LuxS defect as model, utilize intestinal bacteria-Streptococcus mutans shuttle expression carrier to introduce external source SahH, the metabolism having built under Streptococcus mutans density induction defect background recovers strain, has realized metabolism and separating that density is responded to.For LuxS from now on provides research tool to the further investigation of mutans streptococcus function controlling mechanism.

Summary of the invention

First object of the present invention is to provide the Streptococcus mutans AMC metabolism based on LuxS defect to recover strain, thereby realizes the pathways metabolism of LuxS mediation and separating of density induction approach.

Second object of the present invention is to provide the Streptococcus mutans AMC metabolism based on LuxS defect to recover the construction process of strain.

For realizing above first object, the present invention discloses following technical scheme: a kind of Streptococcus mutans AMC metabolism based on LuxS defect recovers strain, it is characterized in that, it is Streptococcus mutans KO-S UA159: that described AMC metabolism recovers strain: △ luxS, pIB-sahH, deposit number is CCTCC M 2013699.

For realizing above second object, a kind of Streptococcus mutans AMC metabolism based on LuxS defect recovers the construction process of strain, it is characterized in that, comprises the following steps:

(1) utilize intestinal bacteria-Streptococcus mutans shuttle expression plasmid pIB169, cut, connect by enzyme, the sahH that external pcr amplification is gone out and luxS gene fragment are connected on carrier, construct the shuttle expression plasmid of intestinal bacteria-Streptococcus mutans SahH albumen and the LuxS protein expressing plasmid as positive expression contrast;

(2) first plasmid is transformed into intestinal bacteria after Sequence Identification, detects SahH and LuxS protein expression, the ability to express of checking carrier and goal gene by western blot;

(3) utilize CSP induce variation suis competence to form, plasmid is transformed into Streptococcus mutans, transform successfully by plasmid extraction double digestion checking plasmid;

(4) transcribe by the success of RNA extracting, reverse transcription and PCR checking goal gene;

(5) utilize AI-2 reporting bacterial strain Vibrio harveyi BB170 to detect the secretion of AI-2, secrete the positive as evidence take the AI-2 of positive expression contrast, the successful expression of checking SahH albumen in Streptococcus mutans.

The Streptococcus mutans AMC metabolism that the present invention is based on LuxS defect recovers strain Classification And Nomenclature and is: Streptococcus mutans KO-S UA159:: △ luxS, pIB-sahH(calls KO-S in the following text), deposit number is: CCTCC M 2013699, depositary institution is: Chinese Typical Representative culture collection center (preservation address: No. 16, Luo Jia Shan road, Wuchang District, Wuhan City, Hubei Province Wuhan University's Chinese Typical Representative culture collection center postcode 430072), preservation date is: on December 24th, 2013.

The invention has the advantages that: the present invention is by heterogenous expression SahH in the strain of Streptococcus mutans LuxS defect, the metabolism of LuxS mediation is responded to and separated with density, effectively overcome the LuxS Mechanism Study obstacle that the double defect effect that lacked by LuxS causes, for the discussion of Streptococcus mutans LuxS regulatory mechanism provides effective biology tool and experiment contrast.

Accompanying drawing explanation

Fig. 1 is the generation of methyl circulation (AMC) process and AI-2.Sulfuradenosin methionine (SAM) provides methyl for DNA of bacteria, RNA and albumen are synthetic etc. as methyl donor, generate virose sulphur adenosyl homocysteine (SAH), in most bacteriums, the SAH of toxicity generates homocysteine (HCY) by Pfs and LuxS two-step catalysis approach, produce the precursor DPD of signaling molecule AI-2 simultaneously, and in part in the bacterium without luxS gene, toxicity SAH relies on a step catalysis of SahH and does not produce AI-2 to the conversion of HCY.Then, HCY finally generates SAM through methionine(Met) (MET), completes methyl circulation.

Fig. 2 is that plasmid pIB169 collection of illustrative plates and enzyme are cut evaluation figure.(A) be pIB169 schematic diagram, plasmid pIB169 size is about 4.6kb, the multiple clone site (MCS) of inserting for foreign gene with external source strong promoter Pveg, chloramphenicol resistance gene (Cmr) and the His label that can represent with object amalgamation and expression.Wherein, site EcoRI and BamHI are goal gene insertion point.(B) cut evaluation figure for enzyme, object stripe size is about 4.6kb.(C) be the multiple clone site sequence of plasmid.

Fig. 3 is that plasmid pIB-luxS and pIB-sahH build schematic diagram.Goal gene luxS is about 500bp, and sahH is about 1400bp.LuxS, sahH and plasmid pIB169 are all with restriction enzyme EcoRI and BamHI digestion, postdigestive luxS is connected with the pIB169 of digestion, generates the about 5.1kb of plasmid pIB-luxS(size); The sahH of digestion is connected with the pIB169 of digestion, generates the about 6.0kb of plasmid pIB-sahH(size).

Fig. 4 is that goal gene pcr amplification and plasmid double digestion are identified.(A) be luxS and sahH pcr amplification figure, wherein luxS size is about 500bp, and sahH size is about 1400bp.(B) being the double digestion evaluation figure of plasmid pIB-luxS and pIB-sahH, is plasmid fragment compared with big band, and size is about 4600bp, and less band is goal gene fragment, and luxS size is about 500bp, and sahH size is about 1400bp.

Fig. 5 is LuxS and the expression of SahH in intestinal bacteria.Plasmid pIB-luxS and pIB-sahH are transformed in e. coli bl21, and Western blot detects the His-LuxS albumen of fusion and the successful expression of His-SahH albumen.His-LuxS size is about 18KD, and His-SahH size is about 52KD.The negative contrast of NC, BL21 is the bacterium background contrast of not Pignus pignoris grain, and L represents LuxS albumen, and S represents SahH albumen.

Fig. 6 is LuxS and the expression checking of SahH in Streptococcus mutans.The detection of luxS and sahH transcript mRNA and albumen catalysis secretion AI-2 in Streptococcus mutans.(A) mRNA that (B) is respectively luxS and sahH detects, KO-L, and KO-S, KO-P is the PCR product take the cDNA of each representative strain as template, object stripe size meets, and shows that goal gene is successfully transcribed into mRNA in Streptococcus mutans.(C) be the detection of the each experimental strain of Streptococcus mutans and control strain secretion AI-2.The KO-L secretion AI-2 positive, KO-S secretion AI-2 feminine gender, shows that AMC metabolism recovers strain KO-S and successfully constructs.

Fig. 7 is that real-time fluorescence quantitative PCR detection metabolism recovers the impact on gene expression dose.Choose and Streptococcus mutans biofilm formation and acidproof 8 relevant gene (smu44, smu46, ciaH, aguA, smu238, gtfD, gbpA, gbpC), detect its transcriptional level in experimental strain and control strain by real-time quantity PCR, relatively metabolism recovers the impact on gene transcription level.Result shows that metabolism recovers to recover the change of transcribing of most of detected gene, shows that metabolism brings into play important regulative in the transcriptional expression of these genes.

Fig. 8 is that microbial film is quantitative.Utilize crystal violet staining assay to detect the amount of each bacterial strain biofilm formation, by the quantity of comparison test bacterial strain and control strain biofilm formation, research metabolism recovers the impact on biofilm formation.In the strain of diagram Streptococcus mutans LuxS defect, the recovery of AMC pathways metabolism can make its biofilm formation quantity reach wild strain level, illustrates that metabolism brings into play underlying condition effect in biofilm formation.

Fig. 9 is confocal microscopy biofilm structure.The dead bacterium test kit dyeing of living for microbial film, viable bacteria is dyed to green, uses confocal microscopy biofilm structure after film-making mounting.Diagram Streptococcus mutans wild strain structure all has densification, and the strain of LuxS defect and KO-P, KO-L contrast strain structure are discontinuous, show ditch and return structure and occur that many long and narrow ditches split.AMC metabolism recovers strain microbial film ditch and splits minimizing, and structural table reveals the trend of recovering fine and close, and the agglomerate of bacterium, also by becoming greatly intensive small-particle, shows that the recovery of metabolism can improve the biomembranous erratic composition of defect strain to a certain extent.

Figure 10 is that microbial film acid-fast ability detects.After forming, is exposed at the microbial film initial stage in the sour environment of different pH, take neutral culture environment as contrast.Continue to be cultured to the purple staining of 24h post crystallization quantitative, the amount reducing with the microbial film with respect to neutral control group represents that acid kills effect, and acid-fast ability and acid are killed and acted as inverse ratio.Be shown in pH and be 4.3 and at 2.8 o'clock, bacterial strain is separated into two groups by acid-fast ability, show as stronger group of the acid-fast ability that metabolism recovers strain and wild strain composition, and the weak groups of the acid-fast ability of three contrast strain compositions, show that AMC metabolism recovery strain has improved the acid-fast ability of defect strain.

Embodiment

Below in conjunction with specific embodiment, further set forth the present invention.The experimental technique using in following embodiment if no special instructions, is ordinary method.Material, reagent etc. used in following embodiment, if no special instructions, all can obtain from commercial channels.Should be understood that these embodiment are only not used in and limit the scope of the invention for the present invention is described.The experimental technique of unreceipted actual conditions in the following example, conventionally according to normal condition, the people such as such as Sambrook, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.

1. bacterial strain, plasmid and main agents

Bacterial strain: Streptococcus mutans (Streptococcus mutans) UA159(Shanghai City stomatology institute provides);

Streptococcus mutans luxS defect strain (Shanghai City stomatology institute provides);

Pseudomonas aeruginosa (Pseudomonas aeruginosa) PAO1 (Shanghai City stomatology institute provides);

Intestinal bacteria (Escherichia coli) BL21(Shanghai City stomatology institute provides);

Competence intestinal bacteria TOP10(Shanghai City stomatology institute provides);

Vibrio harveyi BB170, BB152 (Korea Spro of the Chinese Academy of Agricultural Sciences is dry present first);

Intestinal bacteria-Streptococcus mutans shuttle expression carrier pIB169(Shanghai City stomatology institute provides).

Substratum: LB, BHI, THB, AB, BMGS;

Enzyme: restriction endonuclease: BamHI, EcoRI;

Ligase enzyme: T4 DNA Ligase;

Main agents box: 1. bacterial genomes DNA extraction test kit;

2. the little extraction reagent kit of plasmid;

3. DNA purification kit;

4. DNA gel reclaims test kit;

5. PrimerScript gDNA eraser RT reagent kit；

6. THUNDERBIRD SYBR qPCR Mix；

7.LIVE/DEAD BacLightTM Bacterial Viability Kit；

1,2,3 is Beijing Tian Gen biochemical technology company limited product; 4 is vast Tyke, Beijing biological gene technology company product; 5 is Takara company product; 6 is TOYOBO company product; 7 is Molecular Probes company product.

Hot deactivation horse serum;

Competence stimulator polypeptide (CSP);

Antibody: the anti-His monoclonal antibody of mouse;

Anti-with green fluorescence group mark two.

2. the preparation of cloning by expression

Utilize molecular cloning method, first pcr amplification obtains goal gene sahH, luxS complete sequence, utilize two kinds of restriction enzyme EcoRI and BamHI to digest object fragment and plasmid simultaneously, goal gene is connected with plasmid with T4 DNA ligase, obtain sahH, vivoexpression cloning vector pIB-sahH and the pIB-luxS of luxS.

The amplification of 2.1 luxS genes and sahH gene

Utilize bacterial genomes extraction agent box from Streptococcus mutans UA159 wild strain and Pseudomonas aeruginosa PAO1 wild strain, to extract genomic dna respectively, take Streptococcus mutans UA159 wild strain and Pseudomonas aeruginosa PAO1 wild strain genomic dna as template, utilize the archaeal dna polymerase Primestar of high-fidelity to carry out pcr amplification to goal gene luxS and sahH, its upstream and downstream primer is respectively: luxS:LF (5 ' CCGGAATTCATGACAAAAGAAGTTACTG-3 ') (SEQ ID NO.1), LR (5 ' CGCGGATCCTTACACTAGATGACGCTCAA-3 ') (SEQ ID NO.2), sahH:SF (5 ' CCGGAATTCATGAGCGCTGTCATGACG-3 ') (SEQ ID NO.3), SR (5 ' CGCGGATCCTTAGTAGCGATAGGTGTCCGG-3 ') (SEQ ID NO.4).Whole pcr amplification products are carried out utilizing DNA product glue to reclaim test kit recovery object fragment after 1% agarose gel electrophoresis.

2.2 luxS genes and sahH gene connect into intestinal bacteria-Streptococcus mutans shuttle expression carrier pIB169

Digest respectively object fragment luxS by restriction enzyme EcoR I and BamH I, sahH gene and plasmid pIB169, after 37 ℃ of reaction 3h, utilize DNA product purification test kit to cut product to the enzyme of goal gene and carry out purifying recovery, simultaneously, the enzyme of plasmid pIB169 is cut to product and carry out 1% agarose gel electrophoresis, and utilize glue to reclaim the endonuclease bamhi of test kit recovery plasmid.Utilizing T4 ligase enzyme that the enzyme after purifying recovery is cut to object fragment is connected and spends the night under 4 ℃ of conditions with plasmid.By CaCl2 conversion method, connection product is transformed into competence intestinal bacteria TOP10, after 45min is cultivated in 37 ℃ of low speed recoveries, is applied to the LB flat board containing paraxin (20 μ g/ml) resistance, cultivate 24h for 37 ℃.

The extracting of 2.3 cloning vectors and Molecular Identification

From transforming flat board, 5 of picking mono-clonals carry out enlarged culturing in containing the LB liquid nutrient medium of chlorampenicol resistant, utilize the little test kit of taking out of bacterial plasmid to carry out plasmid extracting in a small amount by its specification sheets, products therefrom utilizes restriction enzyme EcoR I and BamH I to carry out 1% agarose gel electrophoresis evaluation after 37 ℃ of digestion 2h, electrophoresis result is carried out to imaging analysis by gel imaging analysis system.Choose the positive group of double digestion plasmid and serve Hai Boshang biotech company and carry out DNA sequencing, sequencing result and standard sequence are compared, by called after pIB-luxS and the pIB-sahH preservation respectively of correct recombinant cloning vector.

3. cloning vector ability to express is identified

By pIB169, pIB-luxS and pIB-sahH are transformed into respectively through calcium chloride method and prepare in also frozen competence e. coli bl21, and coated plate is cultivated rear picking mono-clonal overnight incubation.Get 4 ℃ of centrifugal supernatants of abandoning of 1ml bacterium liquid, sterilizing PBS adds 200ul PBS resuspended after washing bacterium 2 times, adds latter 100 ℃ of equivalent 2 × albumen sample-loading buffer (SDS, Tris, glycerine, water, bromjophenol blue, mercaptoethanol) to boil sample 15min and collect protein sample.The e. coli bl21 1ml bacterium liquid of unconverted plasmid is collected after sample as a control group in the same way.

Preparation 12%SDS-PAGE glue, get appropriate sample loading after constant voltage 80V electrophoresis to sample reach separation gel bottom.Albumen on PAGE glue is gone on nitrocellulose membrane, 5% skimmed milk room temperature sealing treatment 1h, cut off unnecessary film district, target film district add successively mouse-anti His monoclonal antibody (1:3000 dilution) and with fluorescently-labeled anti-mouse two anti-(1:5000 dilution) also respectively after incubated at room 1h TBST wash film 10min × 3 time.Finally carry out scanning imagery by Odyssey imaging system.

4. strain construction and evaluation

4.1 plasmid pIB169, pIB-luxS and pIB-sahH are transformed into Streptococcus mutans and transform and identify

Streptococcus mutans LuxS gene-deleted strain (KO) overnight incubation in THBYS substratum, dilute with 1:20 with the THBYS of fresh pre-temperature, after continue to be cultured to OD600=0.2 ~ 0.3, add competence stimulator polypeptide (the compeptence stimulating peptide of synthetic, and plasmid CSP), its concentration adding is respectively: CSP(500-1000ng/ml), and plasmid (1ug/ml).37 ℃ are continued to be coated with the flat board containing erythromycin (10ug/ml) and the dual anti-property of paraxin (20ug/ml) after cultivation 2h, and cultivate 48h in 37 ℃ of anaerobism, picking mono-clonal enlarged culturing is also collected bacterial sediment, sterilizing deionized water bacterium 2 times, thalline is with utilizing the little test kit of taking out of plasmid to extract plasmid after 37 ℃ of broken wall treatment 2h of N,O-Diacetylmuramidase, the plasmid of taking out is transformed into competence intestinal bacteria TOP10 and increases and extract plasmid (method is with 2), utilize restriction enzyme EcoR I and BamH I in 37 ℃ of digested plasmid 2h, enzyme is cut product and is carried out 1% agarose gel electrophoresis, gel imaging system carries out imaging analysis to electrophoresis result.The plasmid of the double digestion positive is served to Hai Boshang biotech company and check order, sequencing result and standard sequence are compared.After plasmid conversion evaluation is errorless, bacterial strain after conversion respectively called after KO-P(Streptococcus mutans LuxS gene-deleted strain proceeds to empty plasmid pIB169), KO-L(Streptococcus mutans LuxS gene-deleted strain proceeds to plasmid pIB-luxS), KO-S(Streptococcus mutans LuxS gene-deleted strain proceeds to plasmid pIB-sahH).

The checking of 4.2 luxS and sahH genetic transcription mRNA

Bacterial strain is overnight incubation in BHI substratum, after 1:10 dilution, in 10cm plate, 37 ℃ of cultivation 16-24h form microbial film, abandoning supernatant also washes 3 times with aseptic PBS, microbial film is washed down and is proceeded in 1.5ml EP pipe with 1ml PBS, the centrifugal supernatant of abandoning, 37 ℃ of water bath processing 10min of N,O-Diacetylmuramidase for precipitation, the centrifugal supernatant of abandoning, gained precipitation is resuspended with 1ml TRIZOL reagent, extracts the total RNA of each bacterial strain by the operation of TRIZOL Reagent specification sheets.Gained RNA is after concentration determination, get 1ug, utilize reverse transcription test kit by specification operation reverse transcription to obtain the cDNA of strand, by 10 times of DEPC-water dilutions for the cDNA of reverse transcription gained, get 1ul diluent as template, LF/LR and SF/SR are respectively primer, carry out PCR, gained PCR product carries out 1% agarose gel electrophoresis, utilizes gel imaging system to carry out imaging analysis to electrophoresis result, and the success of checking goal gene is transcribed.

The detection of expression of 4.3 goal gene in Streptococcus mutans

With the positive contrast of Streptococcus mutans UA159 wild strain (WT), the negative contrast of luxS defect strain (KO), utilize AI-2 reporting bacterial strain Vibrio harveyi BB170 experimental strain to be carried out to the detection of AI-2 secretion, and KO-L is successfully secreted to the foundation of AI-2 as protein positive expression.Low-speed centrifugal after Streptococcus mutans is cultured to stationary phase in BHI substratum, abandons supernatant.Precipitation is washed 1 time with AB substratum, and resuspended with AB substratum, regulates the OD to 0.4 of resuspended liquid, and in 37 ℃ cultivate 4h after high speed centrifugation, collect supernatant and also use the filtration sterilization of 0.22um filter.The aseptic supernatant of gained detects or is stored in-80 ℃ for AI-2 immediately.Vibrio harveyi BB170 and BB152 be 28 ℃ of overnight incubation in AB substratum, BB152 supernatant is collected the same, BB170 bacterium liquid dilutes with 1:5000 with fresh AB substratum, aseptic supernatant and BB170 diluent add in 96 orifice plates by 20/180 volume ratio, each sample establish three parallel, 96 orifice plates leave standstill and cultivate in 28 ℃, survey a luminous intensity every one hour noclilucence pattern by multi-functional microplate reader, until difference occurs.

5. application example

5.1 metabolism recover the impact on gene expression dose

The total RNA extraction of bacterium and reverse transcription see above states (4.2).Choosing with Streptococcus mutans biofilm formation and acidproof 8 relevant genes is detected object, utilizes qPCR test kit to carry out real-time fluorescence quantitative PCR detection.Result shows that the metabolism of Streptococcus mutans LuxS defect recovers strain and can recover most of change of transcribing that is detected gene.

5.2 metabolism recover the impact on biofilm formation

Streptococcus mutans is overnight incubation in BHI substratum, regulates OD600 after same level, with BMGS substratum 1:10 dilution.Getting dilution bacterium liquid adds in 96 orifice plates with the amount in 200ul/ hole, leave standstill and cultivate after 16-24 hour in 37 ℃ of aerobics, suck supernatant, after microbial film is washed 3 times with sterilizing deionization, every hole adds 50ul 0.1% Viola crystallina (crystal violent, CV) fixing dyeing 15 minutes under room temperature, suck excess dyestuff, wash 3 times with aseptic deionized water after drying at room temperature.Every hole adds 100ul dehydrated alcohol, detects the absorbancy at wavelength 570nm place after shaking table concussion 5-10min by multi-functional microplate reader.

Streptococcus mutans is cultivated in 24 orifice plates based on preset 1.8cm × 1.8cm cover glass and is cultivated microbial film with BMGS, and microbial film cultural method is as aforementioned.Microbial film washes with aseptic deionized water and filter paper blots after excessive moisture, with the dead bacterium staining kit lucifuge dyeing 15min that lives, removing excess dyestuff filter paper blots and cover glass is fixed on slide glass with mounting oil afterwards, use nail varnish mounting, after 4 ℃ of placement 2-3h, observe biofilm structure with under Laser Scanning Confocal Microscope.

5.3 metabolism recover the impact on acid-fast ability

Microbial film cultural method is as aforementioned.Microbial film sucks supernatant after cultivating 6-8h, and aseptic deionized water is washed after 3 times, adds the fresh BMGS substratum of following pH: 5.8,4.3,2.8, take the not sour standard BMGS substratum of adjusting as contrast, continue to be cultured to 16-4h.Suck supernatant, wash after 3 times, carry out violet staining by the method in microbial film quantitative test, and detect the absorbancy at wavelength 570nm place by multi-functional microplate reader.The amount that microbial film reduces with respect to standard pH under lower pH has represented that acid kills effect, and bacterium acid-fast ability is inversely proportional to this reduction.

6. experimental result example

The luxS gene of the method amplification Streptococcus mutans UA159 wild strain by PCR and the sahH gene of Pseudomonas aeruginosa, and insert in expression vector pIB169, double digestion is identified and is confirmed that recombinant clone successfully constructs that (accompanying drawing 2 is shown in plasmid map and evaluation, plasmid construction flow process is shown in accompanying drawing 3, and goal gene amplification figure and plasmid double digestion are identified and seen accompanying drawing 4).

Carrier carries His label, with target protein amalgamation and expression, utilizes the antibody of anti-His label, by protein blot hybridizing method, has verified the expression (result is referring to accompanying drawing 5) in intestinal bacteria of His-LuxS fusion rotein and His-SahH fusion rotein.

With the CSP induce variation suis competence formation of synthetic, and plasmid is transformed into Streptococcus mutans, after total RNA extracts, reverse transcription becomes cDNA, take cDNA as template, it is that primer carries out pcr amplification and electrophoresis that clone builds the primer, has verified that the success of goal gene is transcribed.(result is referring to accompanying drawing 6A-B); Utilize the resuspended method of AB substratum to obtain the supernatant of test strain, after filtration sterilization, aseptic supernatant is added in the Vibrio harveyi BB170 with AB culture medium culturing dilution, after cultivation, utilize multi-functional microplate reader to detect luminous intensity.The positive contrast of Vibrio harveyi BB152, AB substratum adds luminous contrast as a setting in BB170 diluent.In the supernatant of KO-L, AI-2 detected and do not detect in KO-S supernatant, having verified the successful expression (result is referring to accompanying drawing 6C) of goal gene.

The Streptococcus mutans metabolism isolated strains that utilization successfully constructs, the impact that detects metabolism and recover the transcriptional level on Streptococcus mutans biofilm formation and acidproof genes involved by quantifying PCR method, detected result shows, the AMC metabolism that SahH realizes recovers to make the LuxS disappearance property of most of detected gene to transcribe to change to occur recovering to express.Show that pathways metabolism brings into play important regulative (result is referring to accompanying drawing 7) in genetic transcription.

The Streptococcus mutans metabolism isolated strains that utilization successfully constructs, observes metabolism by violet staining method and recovers the impact on biofilm formation.Result shows, metabolism recovers to recover LuxS completely and lack the minimizing (result is referring to accompanying drawing 8) of the microbial film quantity causing.On the quantitative basis of microbial film, utilize Laser Scanning Confocal Microscope further to observe the impact of metabolism on biofilm structure, result shows, although fail to recover completely textural defect, there is recovery trend (result is referring to accompanying drawing 9) to a certain degree in the textural defect that metabolism recovers that LuxS is lacked and causes.Further verify the regulating effect of pathways metabolism in biofilm formation.

The Streptococcus mutans metabolism isolated strains that utilization successfully constructs, the impact of research pathways metabolism on microbial film acid-fast ability.Result shows, after metabolism recovers, the acid-fast ability defect that LuxS defect causes is resumed.Pathways metabolism important regulating effect (result is referring to accompanying drawing 10) of same performance in Streptococcus mutans acid-fast ability has been described.

The present invention's plasmid used does not need inductor induction in the time expressing target protein, thereby has effectively avoided inductive condition and the impact of inductor toxicity on bacterium, has improved the stability of protein expression and the reliability of experimental result.Secondly, metabolism isolated strains and positive expression bacterial strain that the present invention is constructed are object results strain, and all stable aspect growth and physiological property performance, can be directly used in Related Experimental Study or contrast.Again, the strategy that makes separately its metabolic pathway obstacle up by heterogenous expression SahH in Streptococcus mutans LuxS gene-deleted strain of the present invention was not yet reported, thereby there is enough novelties, simultaneously also for the similar research of other bacteriums provides Research Thinking.

The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.

SEQUENCE LISTING

<110> Shanghai Ninth People's Hospital Affiliated to Shanghai Jiao Tong University Sch

The Streptococcus mutans AMC metabolism of <120> based on LuxS defect recovers strain

<130> 。

<160> 4

<170> PatentIn version 3.5

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Claims (2)

1. the Streptococcus mutans AMC metabolism based on LuxS defect recovers a strain, it is characterized in that, it is Streptococcus mutans KO-S UA159: that described AMC metabolism recovers strain: △ luxS, and pIB-sahH, deposit number is CCTCC M 2013699.

2. described in claim 1, a kind of Streptococcus mutans AMC metabolism based on LuxS defect recovers the construction process of strain, it is characterized in that, comprises the following steps:

(1) utilize intestinal bacteria-Streptococcus mutans shuttle expression plasmid pIB169, cut, connect by enzyme, the sahH that external pcr amplification is gone out and luxS gene fragment are connected on carrier, construct the shuttle expression plasmid of intestinal bacteria-Streptococcus mutans SahH albumen and the LuxS protein expressing plasmid as positive expression contrast;

(2) first plasmid is transformed into intestinal bacteria after Sequence Identification, detects SahH and LuxS protein expression, the ability to express of checking carrier and goal gene by western blot;

(3) utilize CSP induce variation suis competence to form, plasmid is transformed into Streptococcus mutans, transform successfully by plasmid extraction double digestion checking plasmid;

(4) transcribe by the success of RNA extracting, reverse transcription and PCR checking goal gene;

(5) utilize AI-2 reporting bacterial strain Vibrio harveyi BB170 to detect the secretion of AI-2, secrete the positive as evidence take the AI-2 of positive expression contrast, the successful expression of checking SahH albumen in Streptococcus mutans.

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