CN109553663A

CN109553663A - A kind of local modulin and its regulation method of lactobacillus plantarum metabolism FOS

Info

Publication number: CN109553663A
Application number: CN201811553737.0A
Authority: CN
Inventors: 陈臣; 王琳琳; 田怀香; 于海燕; 卢艳青; 周文雅; 何晓葳; 陈小燕
Original assignee: Shanghai Institute of Technology
Current assignee: Shanghai Institute of Technology
Priority date: 2018-12-19
Filing date: 2018-12-19
Publication date: 2019-04-02

Abstract

The present invention provides the local modulin SacR1 of lactobacillus plantarum metabolism FOS a kind of and its regulation methods.The protein, which is characterized in that its amino acid sequence is as shown in SEQ ID NO:1 in sequence table；Or for by the amino acid sequence fusion protein that protein shown in SEQ ID NO:1 and protein purification label form such as in sequence table.The transcriptional regulation protein SacR1 provided by the invention for playing local regulating and controlling effect to metabolism FOS is a kind of modulin of GalR-LacI family, this is to find that the albuminoid participates in the regulation of lactobacillus plantarum metabolism FOS for the first time.

Description

A kind of local modulin and its regulation method of lactobacillus plantarum metabolism FOS

Technical field

The invention belongs to genetic engineering fields, and in particular to a kind of couple of lactobacillus plantarum metabolism FOS plays local regulating and controlling effect Transcription factor SacR1 (local modulin).

Background technique

Oligofructose (Fructooligosaccharides, FOS) is used as a kind of common prebiotics, cannot be by alimentary canal Gastric acid and enzymic digestion, the population of microorganism in enteron aisle can be improved by selective uses such as Bifidobacterium, lactobacillus in enteron aisle Ratio.In addition, the short chain fatty acids that FOS is generated after being metabolized, for maintaining the normal physiological function of enteron aisle to play an important role.Closely Nian Lai, with perfect, transcription group technology and the table of the application especially systems biology technology of various high throughput sequencing technologies Variation during being respectively intended to parsing lactic acid bacteria glycometabolism up to spectrum chip technology.Researcher has found that lactic acid bacteria utilizes the base of FOS Because being located in a gene cluster, mainly it is made of the gene of coding transport protein, hydrolysis enzyme system and corresponding modulin.

Studies have shown that the control that the glycometabolism process of lactic acid bacteria is regulated and controled by host metabolism, the relevant base of certain glycometabolisms Because cluster is that corotation is recorded, but glucose can inhibit their transcript and expression under specific sugar existence condition.These phenomenon tables Bright, the metabolic regulation of lactic acid bacteria plays an important role in above process.When external environment is varied, bacterium can experience It is conducted in real time to environmental change and by signal to intracellular, the transcriptional expression of related gene is adjusted by transcriptional control system, into And cause metabolic activity in cells.The controlling elements such as the binding site in transcription factor, target gene and promoter region constitute transcription Regulator control system, transcription factor activate or inhibit the transcriptional expression of downstream target gene by the specific binding with binding site. In recent years, scientists have done a large amount of research work to the transcription regulation mechanism during lactic acid bacteria glycometabolism, it is believed that lactic acid The double effect that the glycometabolism process of bacterium usually will receive global regulation and locally regulate and control.Global regulation [28] is usually by a kind of easy Induced by the sugar (such as glucose) that host is utilized Metabolism control albumin A (Catabolite control protein A, CcpA) completed with the combination of metabolic response element (Catabolite response element, cre), CcpA with positioned at opening Sub-area or the site cre downstream combine and prevent the transcription of the structural gene.Part regulation is the localized transcriptional factor (also at aporepressor) in the presence of no inducer, prevent structural gene is from normal transcription in conjunction with corresponding binding site； In the presence of inducer, transcription factor is in combination, and transcription factor is detached from from binding site, RNA desmoenzyme startup structure gene Normal transcription.

The ability using FOS is all had in view of many enteric microorganism, in selective proliferative specific bacterial strain, in addition to selection Outside different polymerization degree and the FOS of connection type, another point it is important explore the transcription regulation mechanism that lactic acid bacteria utilizes FOS, To understand in depth its in different environments to growth element selection and rationally utilize.In order to explore different lactic acid bacteria metabolism The approach of FOS, in early-stage study with lactobacillus plantarum (Lactobacillus plantarum) be research object, using turn Record omics technology and gene knockout means have studied the metabolic pathway that lactobacillus plantarum utilizes FOS.Research finds that a size is The gene cluster of 7.5kb (sacPTS1 gene cluster) participates in lactobacillus plantarum to the metabolic process of FOS.FOS is to pass through SacPTS1 PTS system transport into after intracellular, monosaccharide is hydrolyzed by fructosidase (SacA) intracellular.One is found in this gene cluster Gene sacR1 encodes the transcription factor of GalR-LacI family type, but sacR1 is sent out during lactobacillus plantarum is metabolized FOS The effect and its regulatory mechanism waved are still unclear.

Summary of the invention

The purpose of the present invention is to provide the transcriptional regulation proteins that a kind of couple of lactic acid bacteria metabolism FOS plays local regulating and controlling effect (i.e. SacR1) and its coli expression carrier (pTolo-EX5-SacR1), while the construction method of the carrier being provided.

Technical solution provided by the invention first is that:

A kind of isolated nucleic acid, which is characterized in that its encoding amino acid sequence protein as shown in SEQ ID NO:1.

Wherein the preparation method of the nucleic acid is the preparation method of this field routine, and the preparation method is preferably comprised:

From extracting naturally occurring encoding amino acid sequence albumen shown in SEQ ID NO:1 such as in sequence table in nature The nucleic acid molecules of matter, or encoding amino acid sequence is obtained as shown in SEQ ID NO:1 in sequence table by gene clone technology Protein nucleic acid molecules, or obtained in encoding amino acid sequence such as sequence table by artificial complete sequence synthetic method The nucleic acid molecules of protein shown in SEQ ID NO:1.

In the present invention, the nucleic acid preferably has the nucleotide sequence as shown in SEQ ID NO:2 in sequence table, more Preferably, the nucleotide sequence of the nucleic acid is as shown in SEQ ID NO:2 in sequence table.

It is as is known to the person skilled in the art: encoding amino acid sequence protein as shown in SEQ ID NO:1 in sequence table Nucleotide sequence can be suitably introduced into replacement, missing, change, insertion or increase the mode of one or more bases to provide one The homologue of a polynucleotide, as long as the protein of homologue coding is still able to maintain amino acid sequence composition such as sequence The function of protein shown in SEQ ID NO:1 in table.

Technical solution provided by the invention second is that: a kind of recombinant expression carrier comprising above-mentioned nucleic acid.

Wherein the recombinant expression carrier can be obtained by conventional method in that art, it may be assumed that be connected to above-mentioned nucleic acid molecules It is built-up on various expression vectors.The expression vector is the various carriers of this field routine.The carrier preferably wraps Include: various plasmids, clay, bacteriophage or viral vectors etc., preferred plasmid, carrier of the present invention are more preferably plasmid pTolo- EX5, i.e., recombinant expression carrier of the present invention more preferably above-mentioned nucleic acid is connected on plasmid pTolo-EX5 construct and At.

Preferably, the recombinant expression carrier includes the promoter region connecting with above-mentioned nucleic-acid manipulation.

Preferably, the recombinant expression carrier contains one section of His₆Sequence.

Technical solution provided by the invention third is that: a kind of recombinant expression transformants comprising above-mentioned recombinant expression carrier.

Wherein the preparation method of the recombinant expression transformants is that this field is conventional, such as can be by by above-mentioned recombinant expression Carrier, which is converted into host microorganism, to be made.The host microorganism is the various host microorganisms of this field routine, as long as Being able to satisfy replicates above-mentioned recombinant expression carrier steadily voluntarily, and entrained encoding amino acid sequence is made to form such as sequence table The gene of protein shown in middle SEQ ID NO:1 can be by effective expression.Wherein the host microorganism is preferably: large intestine Bacillus is more preferably e. coli bl21 (DE3) or bacillus coli DH 5 alpha.Aforementioned recombinant expression carrier is converted to Escherichia coli In BL21 (DE3), currently preferred engineering strain can be obtained.Wherein the method for transformation is the conversion of this field routine Method, preferably chemical transformation, heat shock method or electric robin.

Technical solution provided by the invention fourth is that:

A kind of recombinant bacterium, which is characterized in that contain SEQ in such as sequence table of energy express amino acid sequence composition in its genome The foreign gene of protein shown in ID NO:1.

Preferably, the recombinant bacterium is recombinant lactic acid bacteria.

It is highly preferred that the lactic acid bacteria is lactobacillus plantarum strain.

Technical solution provided by the invention fifth is that:

A kind of isolated protein, which is characterized in that its amino acid sequence is as shown in SEQ ID NO:1；Or to include ammonia The fusion protein of base acid sequence protein and protein purification label as shown in SEQ ID NO:1.

Protein of the present invention be it is a kind of participation lactic acid bacteria metabolism FOS local modulin, entitled SacR1, From lactobacillus plantarum, can be obtained by the method for this field routine, such as by the channel genes of code for said proteins Suitable host cell obtains the transformant for capableing of protein described in normal expression, the transformant is then cultivated, from culture The protein is separated in object.The protein can also be synthesized by artificial complete sequence.

As is known to the person skilled in the art: the amino acid sequence of the protein can be suitably introduced into replacement, lack, change Become, insertion or increase one or more amino acid and the protein homologue, as long as the homologue of the protein is still It is able to maintain the function of the protein.

In the present invention, the protein purification label is preferably His6 purification tag described in the routine of this field, i.e., this The invention fusion protein is preferably protein and His6 as shown in amino acid sequence such as SEQ ID NO:1 in sequence table The fusion protein of purification tag composition.

Technical solution provided by the present invention fifth is that: protein immunoblot (Western blot), a kind of identification recombination The method of protein expressioning product.

Wherein the method for identifying recombinant protein expression product is conventional method in that art, that is, uses polyacrylamide Gel electrophoresis separating sample proteins, using on solid phase carrier protein or polypeptide as antigen, risen with corresponding antibody immune Reaction, then react with the secondary antibody of enzyme or isotope labelling, by substrate colour developing or autoradiograph to detect electrophoresis point From specific destination gene expression protein ingredient.Prove that albumen has been expressed if forming apparent band on gel.

Technical solution provided by the present invention sixth is that:

Application of the above-mentioned protein in regulation lactic acid bacteria metabolism FOS, which is characterized in that the protein and FOS The related gene promoter region site cre combines, and is regulated and controled with realizing to the part that FOS is metabolized.

Technical solution provided by the present invention seventh is that: protein immunoblot (Western blot), a kind of identification recombination The method of protein expressioning product.

Technical solution provided by the present invention eighth is that: a kind of verifying transcription factor and related gene promoter region are special Property combine method, i.e., gel shift experiment (electrophoresis mobility shift assay, EMSA).

It is combined using the DNA probe of specific marker with the albumen of purifying, on non denatured polyacrylamide gel Hysteresis band is formed, to verify the specific binding of transcription factor Yu related gene promoter region.

Wherein method of the verifying transcription factor in conjunction with related gene promoter regiospecificity is that this field is conventional Method, that is, the DNA Specific binding proteins (SacR1) purified and labeled DNA probe are incubated for together, in non denatured polypropylene Protein isolate matter-DNA compound and free probe, prove to transcribe if forming hysteresis band on gel on acrylamide gel electrophoresis The factor has been combined with promoter region.

On the basis of common knowledge of the art, above-mentioned each optimum condition, can any combination to get each preferable reality of the present invention Example.

Compared with prior art, the beneficial effects of the present invention are:

The present invention is by EMSA the experiment proves that the knot of part modulin SacR1 and FOS related gene promoter region It closes, provides theoretical foundation for the whole regulated and control network that building lactobacillus plantarum is metabolized FOS and experiment is supported.It is provided by the invention It is a kind of modulin of GalR-LacI family to the metabolism FOS transcriptional regulation protein SacR1 for playing local regulating and controlling effect, this is Find that the albuminoid participates in the regulation of lactobacillus plantarum metabolism FOS for the first time.

Detailed description of the invention

The swimming lane that " 1 " is numbered in Fig. 1 is PCR product；The swimming lane for numbering " 2 " is pTolo-EX5-SacR1 through XhoI digestion Electrophoretogram afterwards；M1 is DL 2000Marker；M2 is DL 10000Marker.

Electrophoresis result figure of the Fig. 2 for recombinant protein expression identification and after purification, wherein M is albumen Marker；It numbers " 1 " Swimming lane be that E.coli BL21-SacR1 induces precellular broken supernatant；The swimming lane for numbering " 2 " is E.coli BL21- Clasmatosis supernatant after SacR1 induction；The swimming lane for numbering " 3 " is purpose albumen through Ni²⁺Electrophoresis result after column purification.

Fig. 3 is the building schematic diagram of expression vector pTolo-EX5-SacR1.

Fig. 4 is the experimental result picture of Western blot, wherein M is albumen Marker；For the purpose of the swimming lane for numbering " 1 " The electrophoresis result of Protein S acR1 after purification.

Fig. 5 is the EMSA experimental result picture verifying 2 promoter region SacR1-DNA and combining, wherein M is albumen Marker；The swimming lane for numbering " 1-4 " is His₆After-SacR1 albumen is mixed with the promoter Ppts1-sacA marked by FAM, coagulate Gel electrophoresis shows the band of lag, that is, forms SacR1-DNA compound, and with His₆- SacR1 protein concentration by Cumulative to add, the band color of lag is deeper and deeper, illustrates that SacR1-DNA compound bond strength is more and more stronger.Number the swimming of " 5 " When road is that label mixes the experiment of being at war with property with unlabelled DNA probe, the formation of hysteresis band is not found.

Specific embodiment

The present invention is further illustrated below by the mode of embodiment, but does not therefore limit the present invention to the reality It applies among a range.In the following examples, the experimental methods for specific conditions are not specified, according to conventional methods and conditions, or according to quotient The selection of product specification.

Agents useful for same and raw material of the present invention are commercially available in addition to special instruction.

The building of 1 recombinant expression plasmid of embodiment

With SEQ ID NO:3 (upstream primer-SacR1F): 5 '-AAGGCCTACGTCGACGAGGGCCAATGAAACCAAA ATTAAATG-3 ' and SEQ ID NO:4 (downstream primer SacR1R): 5 '-GCGGCCGCACTAGTTGAGGTTACGCGGTAAGG CCCTTG-3 ' is primer, and upstream 5 ' is held added with one section of sequence complementary with expression vector terminal homologous, and the end of the sequence is to contain XhoI digestion mouth；3 ' end of downstream primer is added with one section of sequence complementary with expression vector terminal homologous, the end of the carrier The mouth of digestion containing XhoI.Using lactobacillus plantarum genome as template, PCR reaction is carried out.PCR reaction system is 50 μ L, each reactant Amount are as follows: 2 μ L DNA profilings (9.652ng/ μ L), 2 μ L SacR1F (0.2 μM), 2 μ L SacR1R (0.2 μM), 25 μ L 2 × Gold Star Best Master Mix, uses ddH₂O complements to 50 μ L.PCR response parameter are as follows: 95 DEG C of initial denaturation 10min, 94 DEG C denaturation 30s, 54 DEG C of annealing 30s, 72 DEG C of extension 1min, 30 recycle；72 DEG C of extension 7min.1% agarose of amplified production Detected through gel electrophoresis, and amplified production is purified.Expression plasmid pTolo-EX5 (is revealed into port biotechnology purchased from Shanghai Co., Ltd) single endonuclease digestion (XhoI) is carried out, digestion system is referring to restriction enzyme specification, recovery purifying digestion products.It will Amplified production and digestion products after purification (is had using Ezmax one-step method quick clone kit purchased from port biotechnology is revealed Limit company) it is attached, linked system: 2 μ L amplified productions (143.217ng/ μ L), 5 μ L digestion products (carrier) (194.584ng/ μ L), 1 μ L Ezmax recombinase, 2 μ 5 × Buffer of L, reaction condition: 16 DEG C, 30min.Turned using thermostimulation Change method, which will connect reaction solution and be transformed into, to be added in e. coli bl21 competent cell, is vibrated through 37 DEG C, 180rpm, 60min shaking table After culture, room temperature 4000rpm is centrifuged 5min, is coated on the LB culture medium containing 150 μ g/mL sulfuric acid card that resistance, 37 DEG C of trainings It supports for 24 hours.Screening positive clone simultaneously carries out sequencing identification, obtains correct target recombinant expression carrier, is named as pTolo- EX5-SacR1, the recombinant expression carrier include a kind of nucleic acid, and base sequence is as shown in SEQ ID NO:2 in sequence table, the core A kind of isolated protein of acid encoding, the isolated protein be comprising amino acid sequence as shown in SEQ ID NO:1 The fusion protein of protein and protein purification label H is6, corresponding recombinant bacterium are named as BL21-SacR1.

Interpretation of result

Using lactobacillus plantarum genome as template, the sequence shown in SEQ ID NO:3 and SEQ ID NO:4 be primer into Row pcr amplification reaction obtains the genetic fragment of 1kb or so, and size and expection are consistent, as a result as shown in Fig. 1 number " 1 " swimming lane, Target gene sacR1 is contained in target fragment.By the PCR product segment of recovery purifying after amplification and corresponding single endonuclease digestion carrier PTolo-EX5 is attached, construction of expression vector pTolo-EX5-SacR1, and expression vector establishment schematic diagram is as shown in Figure 3.It will Recombinant plasmid attachment is converted to E.coli BL21 (DE3), and 37 DEG C of plate cultures for 24 hours, select positive transformant and are seeded to LB liquid Overnight incubation in body culture medium, thalline were collected by centrifugation, extract recombinant plasmid, with corresponding restriction enzyme to recombinant plasmid into Row identification.PTolo-EX5-SacR1 obtains the pTolo-EX5 linear fragment of 5kb or so and the mesh of 1kb or so through XhoI digestion Genetic fragment, as Fig. 1 number " 2 " swimming lane shown in, it was demonstrated that expression vector pTolo-EX5-SacR1 is successfully transferred to Escherichia coli In BL21 (DE3) host strain.Positive expression host is named as BL21-SacR1.

The inducing expression of 2 destination protein of embodiment

Picking e. coli bl21-SacR1 single bacterium falls within the liquid LB training of kanamycin sulfate of the 20mL containing 150 μ g/mL It supports in base, 37 DEG C, 200rpm shaking flask overnight incubation.It will be incubated overnight bacterium solution, 200mL is seeded to and contains 150 μ g/mL kanamycins LB liquid medium in, inoculum concentration 2%, 37 DEG C of cultivation temperature, shaking table oscillation rate be 200rpm, in OD_600nmReach When 0.4-0.6 or so, inducer IPTG to final concentration of 0.1mmol/L is added, respectively in 37 DEG C of 3h, 25 DEG C of 6h and 16 DEG C of mistakes Night shaken cultivation selects optimal inductive condition according to SDS-PAGE protein adhesive electrophoresis result, the results showed that in the training of 25 DEG C of 6h Inducing effect is best under the conditions of supporting.After induction, in 4 DEG C, 10000g is centrifuged 10min, collects somatic cells.Every gram of wet thallus Cell is added 5mL combination buffer (0.2M PB, 5M NaCl, 1M imidazoles, pH7.4) and somatic cells is resuspended, and lysozyme is added extremely Final concentration 1mg/mL and phenylmethylsulfonyl fluoride (PMSF) after ice bath 30min, carry out ultrasonication to final concentration 1mM.Breaker bars Part is as follows: ultrasonic 200W, ultrasonic 2s, interval 2s, acts on 10min, and whole process carries out in ice bath.Then plus RNase is to end 10 μ g/mL and DNase of concentration to 5 μ g/mL of final concentration is mixed, and for clasmatosis liquid in 4 DEG C, 12000rpm/min is centrifuged 10min, is received Collect supernatant, i.e. protein sample.

10 μ L protein samples are mixed with 6 × sample-loading buffer of 6 μ L, electric heater 100 even in shaking in vortex oscillator After DEG C heating 3min, race glue is carried out using 5% concentration glue and 12% separation gel, electrode buffer is the sweet ammonia of Tris- of pH8.3 Acid buffer.Voltage when sample is in concentration glue is set as 80V, and changing voltage after bromophenol blue indicator enters separation gel is 120V；After electrophoresis is complete, with coomassie brilliant blue staining liquid after room temperature dyes 2h, it is clear to protein band to be decolourized with destainer.Dye Color with completed on decolorization swinging table in decolorization.By gel imager gray scale scanning electrophoresis offset plate, albumen distribution is analyzed With concentration.

Interpretation of result

After IPTG is induced, biomass increased significantly e. coli bl21-SacR1, and supernatant intracellular has one at about 39kDa The apparent recombinant protein band of expression of item, the electrophoresis result of inducible protein are as shown in Figure 2.

3 protein purification of embodiment

5mL cell lysis buffer solution (0.2M PB, 5M NaCl, 1M miaow is added in every gram of resulting wet thallus of embodiment 2 Azoles, pH7.4) somatic cells, addition lysozyme to 1mg/mL and phenylmethylsulfonyl fluoride (PMSF) is resuspended to final concentration 1mM, ice bath After 30min, ultrasonication is carried out.Broken condition is as follows: ultrasonic 200W, ultrasonic 2s, interval 2s, act on 10min, whole process in It is carried out in ice bath.Then plus RNase to 10 μ g/mL and DNase to 5 μ g/mL is mixed, and clasmatosis liquid is in 4 DEG C, 12000rpm/ Min is centrifuged 10min, collects supernatant.By supernatant be packed into after affinity column with the combination buffers of 10 times of volumes (0.2M PB, 5MNaCl, 1M imidazoles, pH7.4) wash away foreign protein after, with elution buffer (0.2M PB, 5M NaCl and the concentration of various concentration The respectively imidazoles of 100mmol/L, 200mmol/L, 300mmol/L, 400mmol/L, 500mmol/L, 600mmol/L) gradient Elute target protein.10 μ L elution protein liquid is taken to mix with 6 × sample-loading buffer of 6 μ L, to carry out protein electrophoresis analysis.

Interpretation of result

Recombinant protein is because having His₆Label can be specifically adsorbed to affinity media (Ni²⁺) on to foreign protein it is fast Speed separation.The imidazoles of low concentration can play the role of removing foreign protein and purifying protein, and the recombination egg strong with medium affinity It is white to be eluted under the effect of the imidazoles of high concentration.Passed through by the recombinant protein SacR1 that e. coli bl21-SacR1 is expressed Ni²⁺Affinity chromatography is purified, and final concentration 1M imidazoles is added in combination buffer, it can most foreign protein is removed, So that recombinant protein is adsorbed on filler to the maximum extent；Target protein is eluted using gradient concentration later, the results showed that The imidazoles of 300mmol/L concentration can elute target protein (the i.e. His on affine filler completely₆- SacR1 albumen).

The Western blot of 4 recombinant protein expression product of embodiment is identified

The separation gel of configuration 12% adds isopropanol to stand 20min, and gelling to be separated is solid, absorbs isopropanol, then be rinsed with water Remaining isopropanol, blotting paper suck raffinate.Configured 5% concentration glue is added, is inserted into comb, stands 20min, it is to be concentrated Comb is extracted after gelling is solid；Install electrophoretic apparatus, loading, 120V, 90min；Transferring film, it is wet to turn 120V pressure stabilizing and turn 100min；Envelope It closes, cellulose acetate film is put into 5% skim milk, close 30min；PBS washes film 3 times, each 5min；It is (raw to be incubated for primary antibody Work bioengineering (Shanghai) limited liability company, D191001-0100), 4 DEG C of overnight incubations；PBS washes film 3 times, each 10min； It is incubated for secondary antibody (Shanghai Yi Sheng Biotechnology Co., Ltd, 33208ES60), is incubated at room temperature 2h；PBS washes film 3 times, each 10min； Two kinds of hairs in kit (Sangon Biotech (Shanghai) Co., Ltd., D601017-0010) are added in the ratio of 1:1 Photo etching is incubated for 1min；It is packaged, is put into exposure box with preservative film, film is put into exposure box in darkroom and is exposed, exposed Light length of time voluntarily regulates and controls according to result；Development, fixing, scanning result.

Interpretation of result

Western blot experimental result (such as Fig. 4) shows that sacR1 gene has an apparent protein print at 39kDa Thus mark band illustrates that SacR1 successfully carries out heterogenous expression in Escherichia coli.

5 EMSA of embodiment experiment

Contain the starting of 2 potential sites cre (SEQ ID NO:5-SEQ ID NO:6) in being located at gene cluster sacPTS1 Sub-district (P_pts1-sacA) design primer (SEQ ID NO:7-SEQ ID NO:8), PCR amplification is carried out, obtaining size is 200bp's PCR fragment；It constructs cloning vector respectively according to the method for embodiment 1, extracts plasmid.

(1) configuration of 2.0%TBE acrylamide gel is according to kit (the raw limited public affairs of work bioengineering (Shanghai) share Department, C631100-0200) specification progress.

(2) label of probe: contain the promoter region design primer in 2 potential sites cre in being located at gene cluster sacPTS1 (SEQ ID NO:7-SEQ ID NO:8) carries out PCR reaction using lactobacillus plantarum genome as template.PCR reaction system is 50 μ L, the amount of each reactant are as follows: 2 μ L DNA profilings (9.652ng/ μ L), 2 μ L primers Fs (0.2 μM), 2 μ L primer R (0.2 μM), 25 μ L 2XGold Star Best Master Mix, use ddH₂O complements to 50 μ L.PCR response parameter are as follows: 95 DEG C of initial denaturations 10min, 94 DEG C of denaturation 30s, 51 DEG C of annealing 30s, 72 DEG C of extension 20s, 30 recycle；72 DEG C of extension 5min are saved at 4 DEG C, are obtained The PCR fragment for being 200bp to size, by after purification amplified production and carrier T pMD19 (have purchased from precious bioengineering (Dalian) Limit company) it is attached, linked system: 3 μ L amplified productions (143.217ng/ μ L), 2 μ L carrier T pMD19 (50ng/ μ L), 5 μ L DNA Ligation Kit<Mighty Mix>, reaction condition: 16 DEG C, 30min.Connection is reacted using thermostimulation conversion method Liquid, which is transformed into, to be added in e. coli bl21 competent cell, after 37 DEG C, 180rpm, 60min shaking table shaken cultivation, room temperature 4000rpm is centrifuged 5min, is coated on the LB culture medium containing 150 μ g/mL sulfuric acid card that resistance, and 37 DEG C of cultures are for 24 hours.Screening sun Property clone and carry out sequencing identification, extract plasmid；Using obtained plasmid as template, using high-fidelity DpxDNAploymerase (purchased from port Biotechnology Co., Ltd is revealed), with M13F-47 (5 ' end contain FAM probe modification, SEQ ID NO:9) and M13R-48 (SEQ ID NO:10) is that primer carries out PCR amplification.PCR reaction system is 50 μ L, the amount of each reactant are as follows: 2 μ L DNA profiling (7.169ng/ μ L), 2 μ L M13F-47 (FAM) (0.2 μM), 2 μ L M13R-48 (0.2 μM), 25 μ L Dpx DNAploymerase uses ddH₂O complements to 50 μ L.PCR response parameter are as follows: 95 DEG C of initial denaturation 10min, 94 DEG C of denaturation 30s, 67 DEG C annealing 30s, 72 DEG C of extensions 1min, 30 recycle；72 DEG C of extension 5min save at 4 DEG C, obtain the probe marked containing FAM Ppts1-sacA；Using obtained plasmid as template, construct unlabelled probe Ppts1-sacA: with M13F-47 (SEQ ID NO: 9) and M13R-48 (SEQ ID NO:10) is that primer carries out PCR amplification.PCR reaction system is 50 μ L, the amount of each reactant are as follows: 2 μ L DNA profiling (7.169ng/ μ L), 2 μ L M13F-47 (0.2 μM), 2 μ L M13R-48 (0.2 μM), 25 μ L DpxDNAploymerase uses ddH₂O complements to 50 μ L.PCR response parameter are as follows: 95 DEG C of initial denaturations 10min, 94 DEG C of denaturation 30s, 67 DEG C of annealing 30s, 72 DEG C of extension 1min, 30 recycle；72 DEG C of extension 5min save at 4 DEG C, obtain unlabelled probe.It will Obtained PCR product, with PCR Clean-Up System kit, step is purified to specifications, and uses NanoDrop 2000C carries out quantitative analysis；

(3) reaction system: by the embodiment of the labeled DNA probe of 40ng, various concentration (0,0.4,0.8,1.2 μ g) 3 resulting have purified His₆- SacR1 albumen and 10 μ L combination buffers (50mMTris-HCl (pH 8.0), 100mM KCl, 2.5mM MgCl₂, 0.2mM DTT, 2 μ g polydIdC and10%glycerol) and mixing, with Nuclease-free water Complement to 20 μ L；30min is incubated at 30 DEG C；

(4) electrophoretic development；Use the 2.0%TBE acrylamide gel of pre-cooling as electrophoretic buffer, 120V constant pressure prerunning 30min；By protein-dna combination product loading, continue low temperature electrophoresis, 150V, 30min；Glue after electrophoresis is exposed development.

Interpretation of result

Whether for verifying SacR1 His can be used in vitro in conjunction with 2 potential sites cre₆The SacR1 albumen of label Heterogenous expression is carried out in Escherichia coli, uses His₆- SacR1 albumen carries out EMSA experiment.As a result as shown in Fig. 5 (swimming lane 1-4), His₆- SacR1 albumen and the promoter P marked by FAM_pts1-sacAAfter mixing, gel electrophoresis shows the band of lag, shape At SacR1-DNA compound, and with His₆- SacR1 protein concentration gradually increases, and the band color of lag is increasingly It is deep, illustrate that SacR1-DNA compound bond strength is more and more stronger.On the contrary, when label and unlabelled DNA probe mix When being at war with property is tested (swimming lane 5), the formation of hysteresis band is not found, the combination effect of SacR1 albumen and DNA probe is competing Striving property inhibits, it was demonstrated that the specific binding of SacR1 and these promoter regions.

SEQUENCE LISTING

<110>Shanghai application technology university

<120>a kind of local modulin and its regulation method of lactobacillus plantarum metabolism FOS

<160> 10

<170> PatentIn version 3.5

<210> 1

<211> 326

<212> PRT

<213> Lactobacillus plantarum

<400> 1

Met Lys Pro Lys Leu Asn Asp Val Ala Lys Leu Ala Gly Val Ser Ala

1 5 10 15

Thr Thr Val Ser Arg Val Ile Asn Asn His Gly Tyr Leu Ser Ser Gln

20 25 30

Thr Lys Glu Lys Val Phe Ala Ala Met Arg Glu Leu His Tyr Gln Pro

35 40 45

Asn Asn Met Ala Arg Ser Leu Gln Gly Lys Asn Thr Arg Leu Ile Gly

50 55 60

Val Ile Phe Ser Asp Ile Ser Asn Pro Phe Phe Gly Glu Leu Val Ser

65 70 75 80

Arg Ile Glu Lys Ile Leu Phe Ala Lys Asn Tyr Lys Val Ile Leu Cys

85 90 95

Asn Ser Ala Asp Asp Pro Gln Lys Glu Arg Asp Tyr Leu Gln Met Leu

100 105 110

Met Ala Asn Gln Val Asp Gly Ile Ile Ala Gly Ala His Asn Leu Gly

115 120 125

Ile Glu Glu Tyr Gln Gln Tyr Gly Leu Pro Ile Ile Ser Phe Asp Arg

130 135 140

Tyr Leu Ser Asp Asn Ile Pro Ile Val Ser Ser Asp Asn Tyr Gln Gly

145 150 155 160

Gly Trp Leu Ala Thr Gln Thr Leu His Gln Ala Gly Ala Thr Asn Val

165 170 175

Ala Ile Phe Thr Gly Lys Ser His Ala Gly Ser Pro Thr Asn Gly Arg

180 185 190

Arg Glu Gly Tyr Glu Ala Tyr Leu Thr Ala Gln Gln Leu Thr Pro His

195 200 205

Val His Glu Leu Pro Phe Glu Leu Thr Pro Ala Leu Lys Met Met Glu

210 215 220

Ile Lys Thr Ile Met Thr Gln His Gln Tyr Asp Gly Ile Phe Cys Ser

225 230 235 240

Asp Asp Leu Thr Ala Leu Leu Val Leu Asn Val Ala Arg Gln Leu Ser

245 250 255

Leu Thr Val Pro Glu Gln Leu Arg Val Val Gly Tyr Asp Gly Thr Ala

260 265 270

Leu Ile Arg Asp Tyr His Ser Glu Leu Thr Thr Val Glu Gln Pro Leu

275 280 285

Ala Asp Ile Ser Thr Leu Leu Val Ser Leu Leu Leu Gln Arg Ile Glu

290 295 300

Asp Ala Asn Cys Thr Leu Glu Ser Lys Tyr Thr Leu Pro Val Lys Leu

305 310 315 320

Ile Lys Gly Leu Thr Ala

325

<210> 2

<211> 981

<212> DNA

<213> Lactobacillus plantarum

<400> 2

atgaaaccaa aattaaatga tgttgcgaag ctagcggggg tctcagcgac aaccgtttct 60

cgggttatta ataaccatgg ctacctgagt agtcagacta aagaaaaagt tttcgcggcg 120

atgcgagaac tgcattatca gccgaataat atggcacgct cactgcaagg aaagaatacg 180

cgcttaattg gcgtcatctt ttcagatatt agtaatccgt tctttggtga actggtgtcc 240

cggattgaaa aaatcttatt tgctaaaaat tataaggtga ttttatgtaa tagtgcggat 300

gatccgcaaa aagaacgcga ctacctacaa atgttgatgg ctaatcaggt cgatgggatt 360

attgctggtg cccacaattt aggtattgaa gaatatcaac aatacggtct tccgattatt 420

tcatttgacc ggtacttatc cgacaatatt cccatcgtta gttcggataa ttaccagggg 480

ggctggctag caacccagac attgcatcag gcgggggcga ctaacgtggc tatttttact 540

ggtaagtcac acgctggttc gccaacgaat ggtcgccgtg aaggatatga agcctacttg 600

actgcccaac agctaacacc ccatgtccat gaattaccgt ttgaattgac ccccgcgctg 660

aagatgatgg aaattaagac gattatgaca caacatcagt atgatggtat tttttgtagt 720

gatgatttga ccgctttact agtgctcaac gtcgcgcgac aactgtcatt aacggttcct 780

gagcaattac gcgttgttgg ttatgatggc acggcgctca tacgtgatta tcattctgaa 840

ttaacaacag tagaacaacc gttagcggat attagcacgt tactggtttc tctactattg 900

cagcggattg aggatgcgaa ttgtacactg gaatcaaaat atacgttacc agttaagctc 960

atcaagggcc ttaccgcgta a 981

<210> 3

<211> 42

<212> DNA

<213> Artificial sequence

<400> 3

aaggcctacg tcgacgaggg ccaatgaaac caaaattaaa tg 42

<210> 4

<211> 38

<212> DNA

<213> Artificial sequence

<400> 4

gcggccgcac tagttgaggt tacgcggtaa ggcccttg 38

<210> 5

<211> 16

<212> DNA

<213> Lactobacillus plantarum

<400> 5

tgtaaagcgc ttgcat 16

<210> 6

<211> 16

<212> DNA

<213> Lactobacillus plantarum

<400> 6

tgtcaaacga ttgaca 16

<210> 7

<211> 20

<212> DNA

<213> Artificial sequence

<400> 7

taagtacgcg gtcggcaact 20

<210> 8

<211> 23

<212> DNA

<213> Artificial sequence

<400> 8

aggggtataa cgggttttac gat 23

<210> 9

<211> 24

<212> DNA

<213> Artificial sequence

<400> 9

cgccagggtt ttcccagtca cgac 24

<210> 10

<211> 24

<212> DNA

<213> Artificial sequence

<400> 10

agcggataac aatttcacac agga 24

Claims

1. a kind of isolated protein, which is characterized in that its amino acid sequence is as shown in SEQ ID NO:1 in sequence table；Or it is Fusion protein comprising amino acid sequence protein and protein purification label shown in SEQ ID NO:1 such as in sequence table.

2. a kind of isolated nucleic acid, which is characterized in that its encoding amino acid sequence egg as shown in SEQ ID NO:1 in sequence table White matter.

3. nucleic acid as claimed in claim 2, which is characterized in that its nucleotide sequence is as shown in SEQ ID NO:2 in sequence table.

4. a kind of recombinant expression carrier comprising nucleic acid as claimed in claim 2 or claim 3.

5. recombinant expression carrier as claimed in claim 4, which is characterized in that the sequence containing one section of His6.

6. recombinant expression carrier as claimed in claim 4, which is characterized in that the recombinant expression carrier passes through will be such as right It is required that nucleic acid described in 2 or 3 be connected to it is built-up on plasmid pTolo-EX5.

7. a kind of recombinant expression transformants comprising recombinant expression carrier as claimed in claim 4.

8. recombinant expression transformants as claimed in claim 7, which is characterized in that the recombinant expression transformants pass through will be such as power Benefit requires 4 recombinant expression carriers to convert into e. coli bl21 (DE3) to be prepared.

9. a kind of recombinant bacterium, which is characterized in that contain SEQ in such as sequence table of energy express amino acid sequence composition in its genome The foreign gene of protein shown in ID NO:1.

10. protein described in claim 1 is in the application in regulation lactic acid bacteria metabolism FOS, which is characterized in that the egg White matter regulates and controls the part that FOS is metabolized with realizing in conjunction with the metabolism FOS related gene promoter region site cre.