CN110563820A

CN110563820A - Local regulation protein for lactobacillus plantarum metabolism FOS and regulation method thereof

Info

Publication number: CN110563820A
Application number: CN201910821753.1A
Authority: CN
Inventors: 陈臣; 王琳琳; 田怀香; 于海燕; 黄轲; 陈小燕; 石其璇; 袁佳杰; 陈彬
Original assignee: Shanghai Institute of Technology
Current assignee: Shanghai Institute of Technology
Priority date: 2019-09-02
Filing date: 2019-09-02
Publication date: 2019-12-13

Abstract

the invention discloses a local regulation protein for lactobacillus plantarum metabolism FOS and a regulation method thereof. The amino acid sequence of the protein is shown as SEQ ID NO 1 in a sequence table; or a fusion protein consisting of a protein with an amino acid sequence shown as SEQ ID NO. 1 in the sequence table and a protein purification label. The transcription regulation protein SacR2 which has a local regulation effect on the metabolism FOS provided by the invention is a regulation protein of a GalR-LacI family, and the protein participates in the regulation of the metabolism FOS of lactobacillus plantarum for the first time.

Description

Local regulation protein for lactobacillus plantarum metabolism FOS and regulation method thereof

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to a transcription protein SacR2 (local regulatory protein) which has a local regulatory effect on the metabolism FOS of lactobacillus plantarum.

Background

Fructo-oligosaccharides (FOS) refers to carbohydrates in which 2-10 fructosyl groups are linked together by β (2-1) or β (2-6) glycosidic bonds. Besides the physicochemical properties of common oligosaccharides, FOS is a commonly used prebiotic, and the most remarkable physiological characteristic of FOS is that the population proportion of microorganisms in the intestinal tract can be obviously improved. Thus, FOS has been widely used in the food industry as an excellent ingredient capable of achieving the microecological balance of the human body. In recent years, with the improvement of various high-throughput sequencing technologies, particularly system biology technologies, transcriptomics technology and expression profiling chip technology are respectively used for analyzing changes in the sugar metabolism process of lactobacillus.

The transcription regulation system is composed of a transcription factor, a target gene and a regulation element such as a binding site in a promoter region thereof, and the transcription factor can activate or inhibit the transcription expression of the downstream target gene by the specific binding with the binding site. In recent years, scientists have made a lot of research work on transcriptional regulation and control in the sugar metabolism process of lactic acid bacteria, and it is considered that the sugar metabolism process of lactic acid bacteria is often subjected to dual effects of global regulation and local regulation. Global regulation is usually accomplished by the association of a metabolic control protein a (CcpA), which is easily induced by sugars (e.g., glucose) available to the host, with metabolic response elements (cre), and CcpA, which binds to cre sites located in or downstream of the promoter region to prevent transcription of the structural gene. Local regulation is the binding of a local transcription factor (also known as a repressor) to the corresponding binding site in the absence of an inducer, rendering the structural gene incapable of normal transcription; in the presence of an inducer, transcription factors bind to it, transcription factors dissociate from the binding sites, and RNA binding enzymes initiate normal transcription of the structural gene.

In order to explore the pathway of FOS metabolism by different lactic acid bacteria, in the previous studies, with Lactobacillus plantarum (Lactobacilli) as the subject, it was discovered that a gene cluster of size 4.5kb (sacPTS2 gene cluster) participates in FOS metabolism by Lactobacillus plantarum (ref: Chen Chen Chen, Guozhong Zhao, Wei Chen, Benheng Guo.metabolism of structural genes in Lactobacillus ST-III vitamin differential gene transcription and transformation of cell metabolism, 2015, 81(22): 7697-7707). FOS is transported into cells by the PTS system of SacPTS2, and then hydrolyzed into monosaccharides by fructosidase (SacA) in the cells. In this gene cluster, a gene sacR2 was found to encode a GalR-LacI family type transcription factor, but the role that sacR2 plays in the metabolism of FOS by Lactobacillus plantarum and its regulation mechanism are still unclear.

Disclosure of Invention

The technical problem to be solved by the invention is as follows: provides a transcription regulatory protein (SacR2) which has local regulation effect on FOS metabolism of lactic acid bacteria and an escherichia coli expression vector (pET28a-SacR2) thereof, and also provides a construction method of the vector.

In order to solve the technical problems, one of the technical solutions provided by the present invention is:

An isolated nucleic acid encoding a protein having the amino acid sequence set forth in SEQ ID NO. 1.

Wherein the nucleic acid is prepared by a method conventional in the art, preferably comprising:

The nucleic acid molecule of the protein with the amino acid sequence shown as SEQ ID NO. 1 in the sequence table is extracted from the nature, or the nucleic acid molecule of the protein with the amino acid sequence shown as SEQ ID NO. 1 in the sequence table is obtained by gene cloning technology, or the nucleic acid molecule of the protein with the amino acid sequence shown as SEQ ID NO. 1 in the sequence table is obtained by artificial complete sequence synthesis method.

In the present invention, the nucleic acid preferably has a nucleotide sequence shown as SEQ ID NO. 2 of the sequence Listing, and more preferably, the nucleotide sequence of the nucleic acid is shown as SEQ ID NO. 2 of the sequence Listing.

Optimizing an isolated nucleotide sequence for heterologous expression in an E.coli host bacterium, wherein the isolated nucleotide comprises a nucleotide sequence encoding a protein;

ligating the optimized nucleotide sequence into an expression vector;

wherein the optimization of the nucleotides comprises the steps of:

Identifying and modifying rare codons that are not commonly used in E.coli host bacteria from the nucleotide sequence by replacing the rare codons with commonly occurring codons;

Identifying and modifying a putative internal ribosome binding site sequence from the nucleotide sequence;

Identifying and modifying extended G or C nucleotide repeats by eliminating five or more G nucleotide flanking nucleic acid sequences;

identifying and minimizing the RBS of the nucleotide sequence and the mRNA secondary structure in the coding region of the gene;

Undesirable enzyme restriction sites are identified and modified from the nucleotide sequence to form an optimized nucleotide sequence.

As known to those skilled in the art: the nucleotide sequence encoding the protein having an amino acid sequence represented by SEQ ID NO. 1 of the sequence Listing may be appropriately introduced with substitution, deletion, alteration, insertion or addition of one or more bases to provide a homologue of a polynucleotide, as long as the protein encoded by the homologue can still retain the function of the protein having an amino acid sequence represented by SEQ ID NO. 1 of the sequence Listing.

The second technical scheme provided by the invention is as follows: a recombinant expression vector comprising the nucleic acid.

Wherein said recombinant expression vector is obtainable by methods conventional in the art, i.e.: the nucleic acid molecules are ligated to various expression vectors to construct the vector. The expression vector is any vector conventionally used in the art. The carrier preferably comprises: the vector of the present invention is preferably a plasmid, and more preferably a plasmid pET28a, that is, the recombinant expression vector of the present invention is preferably constructed by ligating the above-mentioned nucleic acid to plasmid pET28 a.

preferably, the recombinant expression vector includes a promoter region operably linked to the nucleic acid.

Preferably, the recombinant expression vector contains a His6 sequence.

The third technical scheme provided by the invention is as follows: a recombinant expression transformant comprising the above recombinant expression vector.

Wherein the recombinant expression transformant is prepared by a method conventional in the art, such as being prepared by transforming the above recombinant expression vector into a host microorganism. The host microorganism is various host microorganisms which are conventional in the field, and the host microorganism only needs to be capable of ensuring that the recombinant expression vector can stably replicate by itself and ensure that the gene of the protein which is carried by the recombinant expression vector and has an amino acid sequence and is shown as SEQ ID NO. 1 in a sequence table can be effectively expressed. Wherein the host microorganism is preferably: escherichia coli, more preferably Escherichia coli BL21(DE3) or Escherichia coli DH5 α. The recombinant expression vector is transformed into Escherichia coli BL21(DE3) to obtain the preferred genetic engineering strain of the present invention. Wherein the transformation method is a transformation method conventional in the art, preferably a chemical transformation method, a thermal shock method or an electric transformation method.

The fourth technical scheme provided by the invention is as follows:

A recombinant bacterium is characterized in that the genome of the recombinant bacterium contains an exogenous gene capable of expressing a protein with an amino acid sequence shown as SEQ ID NO. 1 in a sequence table.

Preferably, the recombinant bacterium is a recombinant lactic acid bacterium.

more preferably, the lactic acid bacteria are lactobacillus plantarum strains.

The fifth technical scheme provided by the invention is as follows:

an isolated protein, characterized in that its amino acid sequence is shown in SEQ ID NO 1; or fusion protein comprising protein with amino acid sequence shown in SEQ ID NO. 1 and protein purification label.

the protein of the present invention is a local regulatory protein involved in the metabolism of FOS by lactic acid bacteria, named SacR2, derived from lactobacillus plantarum, and can be obtained by a method that is conventional in the art, such as introducing a gene encoding the protein into a suitable host cell to obtain a transformant that can normally express the protein, culturing the transformant, and isolating the protein from the culture. The protein can also be obtained by artificial complete sequence synthesis.

as known to those skilled in the art: the amino acid sequence of the protein may be suitably modified by the introduction of substitutions, deletions, alterations, insertions or additions of one or more amino acids to the protein homologue, as long as the protein homologue retains the function of the protein.

in the present invention, the protein purification tag is a conventional tag in the art, preferably a His6 purification tag, i.e. the fusion protein of the present invention is preferably a fusion protein composed of a protein with an amino acid sequence shown as SEQ ID No. 1 in the sequence table and a His6 purification tag.

The fifth technical scheme provided by the invention is as follows: western blot (Western blot), a method for identifying recombinant protein expression products.

the method for identifying the recombinant protein expression product is a conventional method in the field, namely, a protein sample is separated by polyacrylamide gel electrophoresis, protein or polypeptide on a solid phase carrier is taken as an antigen, immunoreaction is carried out with a corresponding antibody, then the antibody reacts with a second antibody marked by enzyme or isotope, and the protein component expressed by the specific target gene separated by electrophoresis is detected by substrate chromogenic or autoradiography. Protein expression is demonstrated if a distinct band forms on the gel.

The sixth technical scheme provided by the invention is as follows:

the application of the protein in regulating and controlling FOS metabolism of lactic acid bacteria is characterized in that the protein is combined with cre sites of a promoter region cre of an FOS related gene to realize local regulation and control of FOS metabolism.

The seventh technical scheme provided by the invention is as follows: western blot (Western blot), a method for identifying recombinant protein expression products.

the eighth technical scheme provided by the invention is as follows: one method for verifying the specific binding of transcription factors to promoter regions of related genes is the gel migration assay (EMSA). The invention verifies the specific binding of the regulatory protein SacR2 and the promoter region binding site of the FOS metabolism related gene through EMSA experiments.

Specifically labeled DNA probes are combined with purified proteins to form a hysteresis band on a non-denatured polyacrylamide gel, so that the specific combination of transcription factors and related gene promoter regions is verified.

The method for verifying the specific binding of the transcription factor and the related gene promoter region is a conventional method in the field, namely, a purified DNA specific binding protein (SacR2) and a labeled DNA probe are incubated together, a protein-DNA complex and a free probe are separated on non-denaturing polyacrylamide gel electrophoresis, and if a hysteresis band is formed on the gel, the binding of the transcription factor and the promoter region is proved.

Compared with the prior art, the invention has the beneficial effects that:

The invention proves the combination of the local regulatory protein SacR2 and the FOS related gene promoter region through EMSA experiments, and provides theoretical basis and experimental support for constructing an overall regulatory network of lactobacillus plantarum metabolism FOS. The transcription regulation protein SacR2 which has a local regulation effect on the metabolism FOS provided by the invention is a regulation protein of a GalR-LacI family, and the first discovery shows that the protein participates in the regulation of the metabolism FOS of lactobacillus plantarum.

Drawings

FIG. 1 shows lanes of "1" as the gel recovery product; lane No. 2 is an electrophoretogram of pUC57-SacR2 digested with NheI and HindIII; lane number "3" is an electrophoretogram of pET28a-SacR2 digested with NheI and HindIII; m1 is DL 2000 Marker; m2 is DL 10000 Marker;

FIG. 2 is a diagram showing the result of electrophoresis after recombinant protein expression identification and purification, wherein M is a protein Marker; lane No. 1 is broken supernatant of E.coli BL21-SacR2 pre-induced cells; lane No. 2 is cell disruption supernatant after e.coli BL21-SacR2 induction; the lane numbered "3" is the electrophoresis result of the target protein purified by Ni2+ column;

FIG. 3 is a schematic diagram of the construction of expression vector pET28a-SacR 2;

FIG. 4 is a diagram showing the experimental results of Western blot, wherein M is a protein Marker; the lane numbered "1" shows the result of electrophoresis after purification of the target protein SacR 2;

FIG. 5 is a graph showing the results of EMSA experiments to verify the binding of 2 promoter regions SacR2-DNA, wherein M is a protein Marker; after the His6-SacR2 protein and the FAM-labeled promoter Pagl4-SacR2 in the lanes of numbers "1-4" are mixed, gel electrophoresis shows a delayed band, namely a SacR2-DNA complex is formed, and the delayed band becomes darker and darker along with the gradual increase of the concentration of the His6-SacR2 protein, which indicates that the SacR2-DNA complex is stronger and stronger; no formation of a hysteresis band was observed in the competitive experiment in which the labeled and unlabeled DNA probes were mixed in lane 5.

Detailed Description

In order to make the invention more comprehensible, preferred embodiments are described in detail below with reference to the accompanying drawings.

Example 1: codon optimization of nucleic acid sequences of interest

the sequence to be synthesized is analyzed to check whether a particularly complex secondary structure and a repetitive sequence linkage exist inside the gene. According to the result of gene sequence analysis, the design and synthesis of primer single-stranded oligos are performed, respectively. The synthetic oligos were spliced into the complete gene sequence using PCR. The synthesized sequence was loaded into pUC57 vector and transformed into competent cell DH5 α (this was partially done by Shanghai Bailegg Biotechnology Co., Ltd.).

Example 2: construction of recombinant expression plasmids

The pUC57-SacR2 plasmid and expression plasmid pET28a provided by Shanghai Bailey Biotechnology Limited company are respectively subjected to double digestion (NheI and HindIII), the digestion system refers to restriction enzyme instruction, and the digestion products are recovered and purified. Connecting the purified enzyme digestion products, wherein a connecting system comprises: mu.L of amplification product (201.168 ng/. mu.L), 5. mu.L of digestion product (vector) (216.451 ng/. mu.L), 1. mu.L of Ezmax recombinase, 2. mu.L of 5 XBuffer, reaction conditions: at 16 deg.C for 30 min. The ligation reaction solution is transformed into competent cells added with escherichia coli BL21 by adopting a thermal stimulation transformation method, and after shaking culture of a shaking table at 37 ℃ and 180rpm for 60min, the cells are centrifuged at 4000rpm at room temperature for 5min, and then the cells are coated on LB culture medium containing 150 mu g/mL kanamycin sulfate resistance and cultured for 24h at 37 ℃. Screening positive clones, sequencing and identifying to obtain a correct target recombinant expression vector, and naming the correct target recombinant expression vector as pET28a-SacR2, wherein the recombinant expression vector comprises a nucleic acid, the base sequence of the nucleic acid is shown as SEQ ID NO. 2 in a sequence table, the nucleic acid encodes a separated protein, the separated protein is a fusion protein of the protein with the amino acid sequence shown as SEQ ID NO. 1 and a protein purification tag His6, and a corresponding recombinant bacterium is named as BL21-SacR 2.

And (4) analyzing results:

The Puc57-SacR2 plasmid provided by Shanghai Bailey biology, Inc. and the vector pET28a are subjected to double enzyme digestion (NheI and HindIII), the enzyme digestion products are recovered and purified and are connected to construct an expression vector pET28a-SacR2, and the construction schematic diagram of the expression vector is shown in FIG. 3. Transforming the recombinant plasmid connection substance into E.coli BL21(DE3), carrying out plate culture at 37 ℃ for 24h, selecting positive transformants, inoculating the positive transformants into LB liquid culture medium for overnight culture, carrying out centrifugation to collect thalli, extracting recombinant plasmids, and identifying the recombinant plasmids by using corresponding restriction enzymes. pET28a-SacR2 was digested with NheI and HindIII to obtain pET28a linear fragment of about 5kb and target gene fragment of about 1kb, as shown in the lane numbered "3" in FIG. 1, which proved that the expression vector pET28a-SacR2 was successfully transferred into the host bacterium of Escherichia coli BL21(DE 3). The positive expression host is named as BL21-SacR 2.

Example 3: inducible expression of a protein of interest

Single colonies of E.coli BL21-SacR2 were picked up and cultured overnight in 20mL of liquid LB medium containing 150. mu.g/mL kanamycin sulfate in shaking flasks at 37 ℃ and 200 rpm. Inoculating overnight culture liquid into 200mL liquid LB culture medium containing 150 ug/mL kanamycin, wherein the inoculum size is 2%, the culture temperature is 37 ℃, the shaking table oscillation speed is 200rpm, and the OD is_600nmWhen the concentration reaches about 0.4-0.6, adding inducer IPTG to the final concentration of 0.1mmol/L, respectively carrying out shaking culture at 37 ℃ for 3h, 25 ℃ for 6h and 16 ℃ overnight, and selecting the optimal induction condition according to the electrophoresis result of SDS-PAGE protein gel, wherein the result shows that the induction effect is best under the culture condition of 25 ℃ for 6 h. After the induction, the cells were centrifuged at 10000g for 10min at 4 ℃ to collect the cells. Adding 5mL of binding buffer (0.2M PB, 5M NaCl, 1M imidazole, pH7.4) into each gram of wet bacterial cells, re-suspending the bacterial cells, adding lysozyme to a final concentration of 1mg/mL and phenylmethylsulfonyl fluoride (PMSF) to a final concentration of 1mM, carrying out ice bath for 30min, and carrying out ultrasonic disruption. The crushing conditions were as follows: performing ultrasonic treatment at 200W for 2s, and performing intermittent treatment for 2s for 10min in an ice bath. Then RNase was added to a final concentration of 10. mu.g/mL and DNase was added to a final concentration of 5. mu.g/mL, and the mixture was mixed well, and the cell disruption solution was centrifuged at 12000rpm for 10min at 4 ℃ to collect the supernatant, i.e., the protein sample.

Mixing 10 μ L protein sample with 6 μ L6 × loading buffer solution, vibrating on vortex oscillator, heating with electric heater at 100 deg.C for 3min, running gel with 5% concentrated gel and 12% separation gel, and electrode buffer solution is Tris-glycine buffer solution with pH of 8.3. When the sample is in the concentrated gel, the voltage is set to 80V, and the voltage is changed to 120V after the bromophenol blue indicator enters the separation gel; after electrophoresis, the gel is dyed by Coomassie brilliant blue staining solution for 2 hours at room temperature, and then is decolored by decoloration solution until protein bands are clear. The dyeing and the decoloring processes are finished on a decoloring shaking table. And (3) analyzing the protein distribution and concentration by a gel imager gray scanning electrophoresis gel plate.

And (4) analyzing results:

After the escherichia coli BL21-SacR2 is induced by IPTG, the bacterial quantity is obviously increased, an obvious recombinant protein expression band is arranged at the position of about 42kDa of an intracellular supernatant, and the electrophoresis result of the induced protein is shown in figure 2.

example 4: protein purification

The wet cells obtained in example 2 were added to 5mL of cell lysis buffer (0.2M PB, 5M NaCl, 1M imidazole, pH7.4) per gram of the wet cells, and the cells were resuspended, lysozyme was added to 1mg/mL, and phenylmethylsulfonyl fluoride (PMSF) was added to a final concentration of 1mM, and after ice-bath for 30min, ultrasonication was performed. The crushing conditions were as follows: performing ultrasonic treatment at 200W for 2s, and performing intermittent treatment for 2s for 10min in an ice bath. Then RNase was added to 10. mu.g/mL and DNase was added to 5. mu.g/mL, and the mixture was mixed well, and the cell disruption solution was centrifuged at 12000rpm for 10min at 4 ℃ to collect the supernatant. After loading the supernatant into an affinity chromatography column, the contaminating proteins were washed off with 10 volumes of binding buffer (0.2M PB, 5M NaCl, 1M imidazole, pH7.4), and the target proteins were eluted in a gradient with elution buffers of different concentrations (0.2M PB, 5M NaCl and imidazole at concentrations of 100mmol/L, 200mmol/L, 300mmol/L, 400mmol/L, 500mmol/L, 600mmol/L, respectively). mu.L of the eluted protein solution was mixed with 6. mu.L of 6 Xloading buffer for electrophoretic analysis of the protein.

And (4) analyzing results:

the recombinant protein has His₆the label can be specifically adsorbed to the affinity media (Ni)²⁺) Thereby rapidly separating from the hetero-protein. The imidazole with low concentration can remove the hybrid protein and purify the protein, and the recombinant protein with strong affinity with the medium can be eluted under the action of the imidazole with high concentration. The recombinant protein SacR2 expressed by Escherichia coli BL21-SacR2 is passed through Ni²⁺Affinity chromatography, adding 1M imidazole to the binding buffer to remove most of the impurity proteins, and allowing the recombinant protein to be absorbed to the maximumAttaching to the packing; then eluting the target protein by adopting gradient concentration, and the result shows that the imidazole with the concentration of 300mmol/L can completely elute the target protein (namely His) on the affinity filler₆-SacR2 protein).

Example 5: western blot identification of recombinant protein expression product

Preparing 12 wt% of separation gel, adding isopropanol, standing for 20min, absorbing and removing the isopropanol after the separation gel is solidified, washing the residual isopropanol with water, and absorbing residual liquid with absorbent paper. Adding prepared 5% concentrated glue, inserting into a comb, standing for 20min, and pulling out the comb after the concentrated glue is solidified; installing an electrophoresis device, loading, and carrying out 120V for 90 min; rotating the membrane, and wet-rotating at 120V for 100 min; sealing, namely putting the cellulose acetate membrane into 5% of skimmed milk, and sealing for 30 min; washing the membrane with PBS for 3 times, 5min each time; incubating the primary antibody (Biotechnology engineering (Shanghai) Co., Ltd., D191001-0100) overnight at 4 ℃; washing the membrane with PBS for 3 times, each time for 10 min; incubating a secondary antibody (Shanghai assist in san Francisco Biotech Co., Ltd., 33208ES60) at room temperature for 2 h; washing the membrane with PBS for 3 times, each time for 10 min; according to the following steps: 1, adding two luminescent agents in a kit (biological engineering (Shanghai) Co., Ltd., D601017-0010) in proportion, and incubating for 1 min; wrapping with preservative film, placing into an exposure box, placing the film into the exposure box in a darkroom for exposure, and automatically regulating and controlling the exposure time according to the result; developing, fixing and scanning the result.

Analysis of results

The result of Western blot (as shown in FIG. 4) shows that the sacR2 gene has a distinct Western blot band at 42kDa, thereby indicating that SacR2 is successfully expressed heterologously in Escherichia coli.

Example 6: EMSA experiment

Promoter region (P) containing 2 potential cre sites (SEQ ID NO: 3-SEQ ID NO: 4) in sacPTS2 gene cluster_agl4-sacR2) Design of primers (SEQ ID NO: 5-SEQ ID NO: 6) carrying out PCR amplification to obtain a PCR fragment with the size of 200 bp; cloning expression vectors were constructed and plasmids were extracted, respectively, according to the method of example 2.

(1) The preparation of 2.0% TBE acrylamide gel was carried out according to the instructions of the kit (Biotechnology engineering (Shanghai) Co., Ltd., C631100-0200).

(2) Labeling of the probe: primers (SEQ ID NO: 5-SEQ ID NO: 6) were designed in a promoter region containing 2 potential cre sites in the gene cluster sacPTS2, and PCR was performed using Lactobacillus plantarum genome as a template. The PCR reaction system is 50 μ L, and the amount of each reactant is: mu.L of DNA template (13.174 ng/. mu.L), 2. mu.L of primer F (0.2. mu.M), 2. mu.L of primer R (0.2. mu.M), 25. mu.L of 2 Xpolarized Star Best Master Mix with ddH₂O make up to 50. mu.L. The PCR reaction parameters are as follows: pre-denaturation at 95 ℃ for 10min, denaturation at 94 ℃ for 30s, annealing at 56 ℃ for 30s, extension at 72 ℃ for 20s, and 30 cycles; extending for 5min at 72 ℃, storing at 4 ℃ to obtain a PCR fragment with the size of 200bp, connecting the purified amplification product with a T vector pMD19 (purchased from Takara Bio-engineering Co., Ltd.) in a connection system: mu.L of the amplification product (127.951 ng/. mu.L), 2. mu. L T vector pMD19(50 ng/. mu.L), and 5. mu.L of DNA Ligation Kit<Mighty Mix>and the reaction conditions are as follows: at 16 deg.C for 30 min. The ligation reaction solution is transformed into competent cells added with escherichia coli BL21 by adopting a thermal stimulation transformation method, and after shaking culture of a shaking table at 37 ℃ and 180rpm for 60min, the cells are centrifuged at 4000rpm at room temperature for 5min, and then the cells are coated on LB culture medium containing 150 mu g/mL kanamycin sulfate resistance and cultured for 24h at 37 ℃. Screening positive clones, sequencing and identifying, and extracting plasmids; the obtained plasmid was used as a template, and PCR amplification was carried out using high fidelity Dpx DNAPloymease (purchased from Tou Luo Biotech Co., Ltd.) and using M13F-47 (5' -terminal containing FAM probe modification, SEQ ID NO: 7) and M13R-48(SEQ ID NO: 8) as primers. The PCR reaction system is 50 μ L, and the amount of each reactant is: mu.L of DNA template (13.174 ng/. mu.L), 2. mu. L M13F-47(FAM) (0.2. mu.M), 2. mu. L M13R-48 (0.2. mu.M), 25. mu.L of LDpx DNA ploymerase, and the same DNA fragment as described above was purified by ddH₂O make up to 50. mu.L. The PCR reaction parameters are as follows: pre-denaturation at 95 ℃ for 10min, denaturation at 94 ℃ for 30s, annealing at 67 ℃ for 30s, extension at 72 ℃ for 1min, and 30 cycles; extending for 5min at 72 ℃, and storing at 4 ℃ to obtain a probe P containing FAM markers_agl4-sacR(ii) a Constructing an unlabeled probe P by using the obtained plasmid as a template_agl4-sacR: PCR amplification was performed using M13F-47(SEQ ID NO: 7) and M13R-48(SEQ ID NO: 8) as primers. The PCR reaction system is 50 μ L, and the amount of each reactant is: 2 μ L DNA template (13.174 ng/. mu.L), 2 μ L M13F47 (0.2. mu.M), 2. mu. L M13R-48 (0.2. mu.M), 25. mu.L of Dpx DNAPloymease using ddH₂O make up to 50. mu.L. The PCR reaction parameters are as follows: pre-denaturation at 95 ℃ for 10min, denaturation at 94 ℃ for 30s, annealing at 67 ℃ for 30s, extension at 72 ℃ for 1min, and 30 cycles; extension was carried out at 72 ℃ for 5min, and the resulting product was stored at 4 ℃ to obtain an unlabeled probe. Purifying the obtained PCR product by using a PCR Clean-Up System kit according to the instruction steps, and carrying out quantitative analysis by using NanoDrop 2000C;

(3) Reaction system: 50ng of the labeled DNA probe, and different concentrations (0, 2, 5, 10. mu.g) of the purified His obtained in example 3₆SacR2 protein and 10. mu.L of binding buffer (50mM Tris-HCl (pH 8.0),100mM KCl,2.5mM MgCl₂0.2mM DTT, 2. mu.g polydIdC and 10% glycerol) and made up to 20. mu.L with nucleic-free water; incubating at 30 deg.C for 30 min;

(4) Carrying out electrophoretic development; pre-cooling 2.0% TBE acrylamide gel as electrophoresis buffer, and performing 120V constant-pressure pre-electrophoresis for 30 min; loading the protein-DNA combined product, and continuing low-temperature electrophoresis at 150V for 30 min; and exposing and developing the gel after electrophoresis.

And (4) analyzing results:

to verify whether SacR2 could bind to 2 potential cre sites in vitro, His was used₆Heterologous expression of the tagged SacR2 protein in E.coli with His₆the SacR2 protein was subjected to EMSA experiments. The results are shown in FIG. 5 (lanes 1-4), His₆SacR2 protein with FAM-tagged promoter P_agl4-sacRAfter mixing, gel electrophoresis showed a band with hysteresis, forming a SacR2-DNA complex with His₆The gradual increase of the concentration of SacR2 protein and the darker and darker color of the lagging band indicate that the SacR2-DNA complex has stronger and stronger binding strength. In contrast, when labeled and unlabeled DNA probes were mixed together for competitive experiments (lane 5), no formation of a hysteresis band was observed, and the binding effect of SacR2 protein to the DNA probes was competitively inhibited, demonstrating the specific binding of SacR2 to these promoter regions.

SEQUENCE LISTING

<110> Shanghai applied technology university

<120> local regulation protein for lactobacillus plantarum metabolism FOS and regulation mode thereof

<130> 8

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 333

<212> PRT

<213> Lactobacillus plantarum

<400> 1

Met Thr Thr Ile Ser Glu Ile Ala Ala Glu Ala Gly Val Gly Val Gly

1 5 10 15

Thr Val Ser Arg Tyr Leu Asn His Arg Pro Ser Val Ser Val Ala Lys

20 25 30

Lys Lys Gln Ile Gln Ala Ala Ile Glu Lys Leu Asp Tyr Thr Pro Asn

35 40 45

Ala Ile Ala Ser Gln Leu Arg Ala Gln Asn Thr Asn Thr Ile Gly Val

50 55 60

Leu Val Ser Arg Ile Ser Asn Pro Phe Phe Ala Gln Leu Phe Asp Ala

65 70 75 80

Leu Glu Arg Glu Leu Asn Gly Tyr Gly Phe Gln Val Met Val Met Gln

85 90 95

Thr His Asp Asp Ala Leu Ala Glu Gln His Phe Leu Asp Lys Leu Lys

100 105 110

Gln Gln Gln Val Asp Gly Val Ile Leu Ala Ser Ile Glu Asn Gln Gln

115 120 125

Leu Val Ala Gln Leu Ser Ala Thr Tyr Ala Asn Gln Met Val Leu Leu

130 135 140

Asn Glu Glu Ala Thr Asp Ile Gly Ile Pro Met Ile Ser Leu Asn His

145 150 155 160

Tyr Gln Ala Thr Lys Asp Ala Leu Ala Tyr Leu Tyr His Gln Gly His

165 170 175

Arg Arg Ile Ala Tyr Ala Thr Gly Gly Asp Phe Pro Ser Thr His His

180 185 190

Gly Arg Ser Arg Thr Gln Ala Tyr Leu Asp Phe Cys Glu Glu Gln Gln

195 200 205

Leu Val Val Asn Asn Asn Trp Val Phe Ala Gln Gln His Thr Ile Ala

210 215 220

Asp Gly Gln Ala Leu Gly Lys Gln Leu Ala Ser Leu Asn Pro Ser Asp

225 230 235 240

Arg Pro Thr Ala Val Phe Thr Asn Ser Asp Glu Val Ala Val Gly Val

245 250 255

Ile Asp Glu Leu Gln Gln Gln His Phe Arg Val Pro Asp Asp Met Ala

260 265 270

Val Met Gly Tyr Asp Asp Gln Pro Phe Ala Ala Val Ala Gln Val Pro

275 280 285

Leu Thr Thr Ile Arg Gln Pro Val Ala Ala Met Ala Gln Leu Ala Val

290 295 300

Asp Gln Leu Leu His His Leu Gly Arg Leu Gln Arg Pro Glu Leu Ala

305 310 315 320

Ile Ala Leu Ser Leu Asp Leu Ile Lys Arg Lys Ser Ala

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<210> 2

<211> 1002

<212> DNA

<213> Lactobacillus plantarum

<400> 2

atgaccacca tctctgaaat cgctgctgaa gctggtgttg gtgttggtac cgtttctcgt 60

tacctgaacc accgtccgtc tgtttctgtt gctaaaaaaa aacagatcca ggctgctatc 120

gaaaaactgg actacacccc gaacgctatc gcttctcagc tgcgtgctca gaacaccaac 180

accatcggtg ttctggtttc tcgtatctct aacccgttct tcgctcagct gttcgacgct 240

ctggaacgtg aactgaacgg ttacggtttc caggttatgg ttatgcagac ccacgacgac 300

gctctggctg aacagcactt cctggacaaa ctgaaacagc agcaggttga cggtgttatc 360

ctggcttcta tcgaaaacca gcagctggtt gctcagctgt ctgctaccta cgctaaccag 420

atggttctgc tgaacgaaga agctaccgac atcggtatcc cgatgatctc tctgaaccac 480

taccaggcta ccaaagacgc tctggcttac ctgtaccacc agggtcaccg tcgtatcgct 540

tacgctaccg gtggtgactt cccgtctacc caccacggtc gttctcgtac ccaggcttac 600

ctggacttct gcgaagaaca gcagctggtt gttaacaaca actgggtttt cgctcagcag 660

cacaccatcg ctgacggtca ggctctgggt aaacagctgg cttctctgaa cccgtctgac 720

cgtccgaccg ctgttttcac caactctgac gaagttgctg ttggtgttat cgacgaactg 780

cagcagcagc acttccgtgt tccggacgac atggctgtta tgggttacga cgaccagccg 840

ttcgctgctg ttgctcaggt tccgctgacc accatccgtc agccggttgc tgctatggct 900

cagctggctg ttgaccagct gctgcaccac ctgggtcgtc tgcagcgtcc ggaactggct 960

atcgctctgt ctctggacct gatcaaacgt aaatctgctt aa 1002

<210> 3

<211> 19

<212> DNA

<213> Lactobacillus plantarum

<400> 3

aaaccttagc aaaggtatt 19

<210> 4

<211> 20

<212> DNA

<213> Lactobacillus plantarum

<400> 4

taaaccttag ctaaggtgaa 20

<210> 5

<211> 21

<212> DNA

<213> Artificial sequence

<400> 5

caaccggggt aacatttgga t 21

<210> 6

<211> 22

<212> DNA

<213> Artificial sequence

<400> 6

Ggcggtcgat cagctattac at 22

<210> 7

<211> 24

<212> DNA

<213> Artificial sequence

<400> 7

cgccagggtt ttcccagtca cgac 24

<210> 8

<211> 24

<212> DNA

<213> Artificial sequence

<400> 8

agcggataac aatttcacac agga 24

Claims

1. The separated protein is characterized in that the amino acid sequence of the protein is shown as SEQ ID NO. 1 in a sequence table, or the protein is a fusion protein comprising the protein and a protein purification label, wherein the amino acid sequence of the protein is shown as SEQ ID NO. 1 in the sequence table.

2. An isolated nucleic acid encoding a protein having an amino acid sequence set forth in SEQ ID NO. 1 of the sequence Listing.

3. the nucleic acid of claim 2, wherein the nucleotide sequence is as set forth in SEQ ID NO 2 of the sequence Listing.

4. A recombinant expression vector comprising the nucleic acid of claim 2 or 3.

5. The recombinant expression vector of claim 4, comprising a His₆The sequence of (a).

6. the recombinant expression vector of claim 4, constructed by ligating the nucleic acid of claim 2 or 3 to plasmid pET28 a.

7. A recombinant expression transformant comprising the recombinant expression vector of claim 4.

8. The recombinant expression transformant according to claim 7, which is prepared by transforming the recombinant expression vector according to claim 4 into E.coli BL21(DE 3).

9. A recombinant bacterium is characterized in that the genome of the recombinant bacterium contains an exogenous gene capable of expressing a protein with an amino acid sequence shown as SEQ ID NO. 1 in a sequence table.

10. The use of the protein of claim 1 for regulating the metabolism of FOS in a lactic acid bacterium, wherein the protein is associated with the cre site of the promoter region of a gene associated with the metabolism of FOS, to achieve local regulation of FOS metabolism.