Lactic acid bacteria signal molecule AI-2 transport protein as well as coding gene and application thereof

Abstract

The invention discloses a lactic acid bacteria signal molecule AI-2 transport protein as well as a coding gene and application thereof. The amino acid sequence of the AI-2 transport protein is shown as SEQ ID NO. 1. The nucleotide sequence of the coding gene of the AI-2 transport protein is as shown in SEQ ID NO. 2. The coding gene controls the target protein to regulate and control the synthesis of the transporter of the fermentation mucus lactobacillus quorum sensing signal molecule AI-2, so that the intracellular signal molecule AI-2 is transported to the outside of the cell, and a LuxS / AI-2 quorum sensing system is activated, thereby directionally regulating and controlling the synthesis of lactobacillus metabolites such as exopolysaccharides, and further improving the metabolism of the lactobacillus. The exopolysaccharide has better physiological activity and physical properties, and has huge application potential in food and medicine industries.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a lactic acid bacteria signal molecule AI-2 transporter protein, a coding gene and an application thereof. Background Art

[0002] The quorum sensing (QS) system of lactic acid bacteria is a population density-dependent gene expression regulation system in which lactic acid bacteria rely on population density, and chemically communicate through the production, release, accumulation and induction of signal molecules, ultimately showing that multiple bacteria cooperate to complete specific physiological functions. It plays an important role in the physiological regulation of lactic acid bacteria. The LuxS / AI-2-QS system is an important system in lactic acid bacteria, carrying the signal transmission and communication of various behaviors in lactic acid bacteria. This system plays an important role not only in the formation of regulatory functional factors, but also in regulating the stress resistance of strains.

[0003] AI-2 (Autoinducer-2) is a universal signal molecule that mediates the LuxS / AI-2 quorum sensing system, carrying the signal transmission and communication of various behaviors in lactic acid bacteria. First, lactic acid bacteria themselves encode the luxS gene and LuxS protein that synthesize the signal molecule AI-2. The synthesized signal molecule AI-2 is transported to the extracellular space through transport proteins. When the extracellular AI-2 reaches a certain concentration, AI-2 binds to the receptor protein on the cell membrane and is internalized into the cell, thereby regulating the expression of downstream genes and proteins, and ultimately regulating the synthesis of functional factors such as lactic acid bacteria biofilm, exopolysaccharides, and bacteriocins. Studies have shown that the LuxS / AI-2 quorum sensing system regulates the synthesis of exopolysaccharides of fermentative mucus Lactobacillus A51 by mediating the Wzx / Wzy pathway, but the key signal molecule AI-2 transporter is still unclear.

[0004] Signal molecule AI-2 transporter is a type of transmembrane protein that binds to signal molecule AI-2 on the cell membrane. Studies have found that signal molecule AI-2 transporter plays an extremely important role in the physiological regulation and functional factor synthesis of lactic acid bacteria. Signal molecule AI-2 must bind to the corresponding transporter to be transported to the extracellular space, thereby activating the LuxS / AI-2 quorum sensing system and mediating the synthesis of lactic acid bacteria functional factors. However, there are no reports on the research of lactic acid bacteria signal molecule AI-2 transporter. Therefore, it is necessary to further study the lactic acid bacteria signal molecule AI-2 transporter. Summary of the invention

[0005] In view of the above problems, the present invention aims to provide a lactic acid bacteria signal molecule AI-2 transporter and the use of its encoding gene A0A1L7GXS4 in synthesizing the signal molecule AI-2 transporter.

[0006] The present invention provides a lactic acid bacteria signal molecule AI-2 transporter, the amino acid sequence of which is shown in SEQ ID NO. 1. The lactic acid bacteria signal molecule AI-2 transporter provided by the present invention is cloned in fermented mucus lactobacillus A51, the deposit number of which is CCTCC NO: M2023861, and has been publicly deposited in the existing patent CN116769673A.

[0007] The present invention also provides a gene encoding the lactic acid bacteria signal molecule AI-2 transporter protein, and the nucleotide sequence of the gene encoding is shown in SEQ ID NO.2.

[0008] The invention also provides a recombinant plasmid containing the encoding gene.

[0009] Preferably, the recombinant plasmid is obtained by homologous recombination of the gene encoding the signal molecule AI-2 transporter and the pET-22b vector, and is named pET-22b-XS4-His.

[0010] The present invention also provides a genetically engineered bacterium, wherein the genetically engineered bacterium contains the recombinant plasmid, or the coding gene is integrated into the genome of the genetically engineered bacterium.

[0011] As a further description of the above scheme: the genetically modified engineered bacteria is the Escherichia coli BL21 (DE3) strain.

[0012] The present invention also provides the use of the transport protein in promoting the transport of the signal molecule AI-2 from the intracellular to the extracellular or promoting the synthesis of the signal molecule AI-2; the transport protein can be applied to the quorum sensing system of lactic acid bacteria to realize the transport of the signal molecule AI-2 from the intracellular to the extracellular, activate the LuxS / AI-2 quorum sensing system, and directionally regulate the synthesis of functional factors such as lactic acid bacteria extracellular polysaccharides.

[0013] The present invention also provides a method for synthesizing a lactic acid bacteria signal molecule AI-2 transporter, the method comprising the following steps: Step (1) preparation of target gene cDNA template; Step (2) using the cDNA as a template and using a DNA polymerase to amplify and recover the target gene; Step (3) Construction and identification of gene recombination vector; Step (4) recombinant protein expression and purification; Step (5) determination of the ability of the recombinant protein to bind AI-2; The construction and identification of the gene recombination vector in step (3) includes vector linearization and gene recombination: the vector linearization is to use BamHI and NdeI double enzymes to digest the pET-22b vector to obtain a linearized vector pET-22b, and use a kit to purify and recover it; the gene recombination is to connect the target gene amplified by the kit with the linearized pET-22b vector, transform the strain into E.coli BL21 (DE3) competent cells, and obtain a bacterial solution containing the transport protein.

[0014] As a further description of the above scheme: the preparation operation of the cDNA template in step (1) is as follows: the fermented mucus lactobacillus suspension with the deposit number of CCTCC NO: M2023861 is centrifuged, the centrifuged bacterial sludge is quickly frozen in liquid nitrogen and then RNA is extracted, and the RNA is reverse transcribed into cDNA using a TAKARA reverse transcription kit.

[0015] As a further description of the above scheme: the protein expression and purification steps in step (4) are as follows: the bacterial liquid is taken for recovery culture, and inoculated into fresh LB liquid culture medium for expansion culture, isopropyl-β-D-thiogalactoside (IPTG) is added to the culture medium to induce protein expression under low temperature conditions; after the induction expression is completed, the bacteria are collected by centrifugation, the bacteria are resuspended in buffer, ultrasonically broken, and centrifuged at high speed, and the supernatant is loaded onto a His affinity chromatography column, eluted with different concentrations of imidazole and the filtrate is collected, and the eluate and the precipitate are subjected to SDS-PAGE protein electrophoresis to detect the results.

[0016] The determination of the ability of the recombinant protein to bind to AI-2 in step (5) comprises the following steps: determining the ability of the recombinant protein to bind to and release the signal molecule AI-2, and determining the binding energy of the recombinant protein to AI-2 by surface plasmon resonance technology (SPR).

[0017] Compared with the prior art, the present invention has the following beneficial effects: (1) The present invention discovered for the first time a signal molecule AI-2 transporter A0A1L7GXS4; the present invention can achieve rapid and efficient expression of the AI-2 transporter A0A1L7GXS4 through recombinant plasmids, in vitro cloning, expression and purification, and can be used to promote the synthesis of AI-2.

[0018] (2) The study of the gene of the AI-2 transporter, a signaling molecule of lactic acid bacteria, and its in vitro synthesis will lay the foundation for the study of the synthesis of functional factors of lactic acid bacteria, physiological regulation, and the function of AI-2 transporters. It will also provide a new direction for the directional regulation of the synthesis of functional factors of lactic acid bacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1This is the vector map of pET-22b-XS4. Note: XS4 in the figure is the transporter protein A0A1L7GXS4; Figure 2 This is the electrophoresis diagram of the recombinant plasmid pET22b-XS4 after PCR amplification; Figure 3 This is the SDS-PAGE test result of recombinant protein A0A1L7GXS4; Figure 4 This is the elution test result of the recombinant protein A0A1L7GXS4; Figure 5 This is the result of gel analysis of the recombinant protein A0A1L7GXS4 purification; Figure 6 To determine the ability of the recombinant protein A0A1L7GXS4 to release AI-2; Figure 7 It is the determination of the ability of the recombinant protein A0A1L7GXS4 to bind to the exogenous AI-2 molecule (the fluorescence intensity in the figure refers to the luminescence intensity of Vibrio harveyi BB170, and its fluorescence intensity is proportional to the activity of the signal molecule AI-2); Figure 8 It is the determination of the ability of the recombinant protein A0A1L7GXS4 to bind to the exogenous AI-2 molecule (DPD in the figure is the signal molecule AI-2; Cycle DPD 62.5 μM refers to the binding energy between the signal molecule AI-2 at a concentration of 62.5 μM and the recombinant protein A0A1L7GXS4, and Cycle DPD 125 μM, Cycle DPD 250 μM, Cycle DPD 500 μM and Cycle DPD 1000 μM and so on); Fig. 9 To analyze the hydrophilicity and hydrophobicity of the recombinant protein A0A1L7GXS4; Fig.10 For signal peptide analysis of recombinant protein A0A1L7GXS4; Fig.11 For the transmembrane structure analysis of the recombinant protein A0A1L7GXS4; Fig.12 This is the secondary structure analysis of the recombinant protein A0A1L7GXS4. Note: The purple line is a random coil, the blue line is an α-helix, the red line is an extended chain, and the green line is a β-turn; Fig.13 For the tertiary structure analysis of the recombinant protein A0A1L7GXS4; Fig.14 Schematic diagram of the synthesis of extracellular polysaccharides in lactic acid bacteria mediated by the signal molecule AI-2 transporter. DETAILED DESCRIPTION

[0020] In order to enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention is further described in detail below in conjunction with specific embodiments. The description in this section is only exemplary and explanatory and should not have any limiting effect on the scope of protection of the present invention.

[0021] Example 1 Through bioinformatics analysis, target gene analysis was performed on the whole genome and proteome data of fermentative mucus Lactobacillus A51, and a coding gene with a length of 1,041bp was obtained. The nucleotide sequence is shown in SEQ ID NO.2. The encoded protein is named A0A1L7GXS4, and the amino acid sequence is shown in SEQ ID NO.1. It can be used to promote the transport of signal molecule AI-2 from intracellular to extracellular.

[0022] Example 2 Construction of recombinant plasmid and its application in AI-2 transporter The construction of recombinant plasmid and its application in AI-2 transporter mainly include the following steps: preparation of cDNA template, amplification and recovery of target gene, construction and identification of gene recombination vector, protein expression and purification of target gene (coding gene of A0A1L7GXS4), and determination of the ability of recombinant protein A0A1L7GXS4 to bind to AI-2.

[0023] (1) Preparation of cDNA template The centrifuged bacterial sludge of fermented Lactobacillus mucilaginosus A51 was taken and quickly frozen in liquid nitrogen for RNA extraction. The total RNA was extracted using the TSINGKE Plant DNA Extraction Kit (Universal), and RNA was extracted according to the operating steps in the kit manual. After passing the test, the RNA was reverse transcribed into cDNA using the TAKARA reverse transcription kit and stored at -80°C for later use.

[0024] (2) Gene amplification and recovery SnapGene software was used to design specific primers for the full-length sequence of the gene coding region. The cDNA obtained by reverse transcription was used as a template, and DNA polymerase was used to amplify the target gene. The amplification system was as follows: 2 μL of cDNA, 25 μL of 2 × KODPCR Master mix, 1 μL of upstream and downstream primers (10 μM), 22 μL of ddH2O, and the amplification program was as follows: 94°C, 2 min, 98°C, 10 s, 58°C, 10 s, 68°C, 1 S, 30 cycles; 68°C, 5 min. After the PCR amplification program was completed, 1.2% agarose gel electrophoresis was used to detect whether the length of the amplified gene band was consistent with that of the target gene band. If the length was similar, the target gene was recovered using a kit, the recovery concentration was determined, and finally stored in a -80°C refrigerator.

[0025] (3) Construction and identification of gene recombination vectors ① Vector linearization: Use BamHI and NdeI to digest the PCR product of the target gene and the pET-22b plasmid extract to obtain the linearized vector pET-22b-XS4 (map as shown in Figure 1 As shown), the enzyme digestion system is: BamHI 1μL, NdeI 1μL, 2× buffer 2μL, ddH2O to 20μL; use the kit to purify and recover, determine its concentration and store in a -80℃ refrigerator for later use; ②Gene recombination: The target gene amplified by the kit and the pET-22b prokaryotic expression vector were double-digested with BamHI / NdeI, and then connected with T4 enzyme to construct the recombinant expression vector pET-22b-XS4. The vector was directly heat-shocked into the competent cells of E. coli BL21 (DE3), and a single colony was picked for PCR verification with universal primers. The positive recombinant expression vector was sent for sequencing. The sequencing results were as follows: Figure 2 As shown, the inserted target fragment (A0A1L7GXS4 encoding gene) is exactly the same as the sequence on the genome, indicating that the recombinant vector pET-22b-XS4 was successfully constructed.

[0026] (4) Protein expression and purification of target gene Recombinant protein A0A1L7GXS4 expression: ① Transform the expression plasmid into BL21(DE3) competent cells and culture them inverted overnight at 37℃; ②Pick a single colony and place it in 5 mL of LB medium. Culture it overnight at 37°C and 250 rpm for 16 to 18 hours as seed solution. ③ Transfer the seed solution to fresh LB medium at a ratio of 1:100, culture at 37°C and 250rpm. When the bacterial solution OD600 = 0.6, add isopropyl-β-D-thiogalactoside (IPTG) inducer to a final concentration of 0.5mM, and culture at 18°C ​​for 16-18h. ④ Collect the bacteria by low-temperature centrifugation; ⑤ Resuspend the cells in lysis buffer and then disrupt them by ultrasonication; ⑥ After ultrasonication, centrifuge the sample at low temperature, take the supernatant and precipitate respectively and perform protein analysis using SDS-PAGE gel.

[0027] Purification of recombinant protein A0A1L7GXS4 ① Take the cultured bacteria, add lysis buffer (50 mM Tris, 0.5 M NaCl), resuspend and then ultrasonically disrupt. The ultrasonic conditions are: work 3s, off 2s, time 15min, repeat once; ② Centrifuge the bacterial suspension after ultrasonic disruption at low temperature (12,000 rpm, 30 min, 4°C) and keep the supernatant; ③ Filter the supernatant with a 0.45 μm filter membrane and purify the protein through a His affinity chromatography column. The steps are as follows: Wash with 5 column volumes of deionized water to remove air and 20% ethanol; Equilibrate the column with 5-10 column volumes of Buffer A, Buffer A: 50 mM Tris, 150 mM NaCl, pH 7.4; The sample was flowed through the His column at a rate of 0.25 mL / min; The column was equilibrated with Buffer A and eluted with 50 mM, 200 mM, and 50 mM imidazole; The eluted samples were run on SDS-PAGE to analyze whether there was the target protein, and the concentration of the purified protein was detected.

[0028] After the recombinant protein A0A1L7GXS4 expression strain was induced by IPTG, the expression, elution and purification of the protein were detected by SDS-PAGE. The results are as follows Figure 3 , Figure 4 and Figure 5 As shown. The size of the recombinant protein A0A1L7GXS4 is roughly similar to the predicted result, and the molecular weight of the protein is about 40kDa, indicating that the recombinant protein A0A1L7GXS4 is successfully expressed in BL21. And from the results of protein solubility analysis, the recombinant protein A0A1L7GXS4 is expressed in a soluble manner in the supernatant in an appropriate amount, and sufficient soluble protein can be obtained by increasing the culture volume of the strain. The results of protein concentration determination show that the concentration of the recombinant protein A0A1L7GXS4 is 0.172mg / mL.

[0029] Example 3 Determination of the ability of recombinant protein A0A1L7GXS4 to bind to AI-2 (1) Detection of the ability of recombinant protein A0A1L7GXS4 to release exogenous AI-2 molecules Purify the recombinant protein A0A1L7GXS4 expressed by BL21 (DE3) according to the above method, and determine the protein concentration. Transfer the protein to a 10kDa protein ultrafiltration tube, centrifuge at 7,500rpm at 4°C, observe every 30 minutes, and discard the filtrate until the liquid on the filter membrane is basically filtered out and the protein is precipitated. Resuspend the protein in PBS filtered with a 0.22μm filter membrane, and centrifuge again to remove the liquid. Resuspend the precipitated protein with an appropriate amount of sterile PBS to a concentration of 5mg / mL. The obtained protein is stored at 4°C and used within 24h.

[0030] ① Release group: Take 200 μL of recombinant protein A0A1L7GXS4 into EP tubes, set up 3 tubes. Place in 40, 50, and 60℃ water bath for 10 min respectively, centrifuge and ultrafilter at 7,500rpm at 4℃ for 10kDa protein ultrafiltration tube. Take the filtrate, filter and sterilize with 0.22μm filter membrane, use V. harveyi The activity of AI-2 was determined by BB170 luminescence assay.

[0031] ② Negative control group: Take 200 μL of recombinant protein A0A1L7GXS4 into an EP tube and directly use a 10kDa protein ultrafiltration tube to centrifuge and ultrafilter at 4°C and 7,500 rpm without temperature treatment. Take the filtrate and filter it with a 0.22 μm filter membrane for sterilization. V. harveyi The activity of AI-2 was determined by BB170 luminescence assay.

[0032] ③ AI-2 positive control: Take 200 μL of sterile PBS into an EP tube, add exogenous AI-2 with a final concentration of 20 μM, and use a 10 kDa protein ultrafiltration tube to centrifuge and ultrafilter at 4°C and 7,500 rpm. Take the filtrate, filter it with a 0.22 μm filter membrane to sterilize it, and use V. harveyi The activity of AI-2 was determined by BB170 luminescence assay.

[0033] The results of AI-2 release by recombinant protein A0A1L7GXS4 are as follows Figure 6 As shown. The activity of AI-2 was very low under 40℃ treatment because the recombinant protein A0A1L7GXS4 was tightly bound to the signal molecule AI-2, resulting in a low fluorescence value. After the recombinant protein A0A1L7GXS4 was heat-treated at 50℃ and 60℃, the recombinant protein A0A1L7GXS4 released the bound AI-2, resulting in an increase in the free AI-2 content and fluorescence intensity. The above results show that the recombinant protein A0A1L7GXS4 can bind to AI-2 produced by BL21 (DE3) and release AI-2 under heat inactivation conditions. The amount of AI-2 released is about 12μM, indicating that the recombinant protein A0A1L7GXS4 is a transporter of the signal molecule AI-2, which can transport the intracellular signal molecule AI-2 to the extracellular space, activate and upregulate the strain quorum sensing system, and thus promote the synthesis of functional factors. The untreated recombinant protein A0A1L7GXS4 (negative control group) cannot release AI-2.

[0034] (2) Detection of the ability of recombinant protein A0A1L7GXS4 to bind to exogenous AI-2 molecules The recombinant protein A0A1L7GXS4 was purified according to the above method and resuspended in PBS to obtain 5 mg / mL BL21 (DE3) expression. The obtained protein was stored at 4°C and used within 24 hours.

[0035] Take 200 μL of recombinant protein A0A1L7GXS4 into an EP tube, add exogenous AI-2 at final concentrations of 50 μM and 20 μM, and repeat 3 groups at each concentration: ① Binding group: Take 200 μL of recombinant protein A0A1L7GXS4 into an EP tube, add exogenous AI-2 at final concentrations of 50 μM and 20 μM respectively. Mix well and incubate at 37°C for 1 hour, centrifuge and ultrafilter at 7,500 rpm in a 10 kDa protein ultrafiltration tube at 4°C. Take the filtrate, filter and sterilize with a 0.22 μm filter membrane, and use V. harveyi The activity of AI-2 was determined by BB170 luminescence assay.

[0036] ② Release group: Take 200 μL of recombinant protein A0A1L7GXS4 into an EP tube, add exogenous AI-2 at final concentrations of 50 μM and 20 μM respectively. After mixing, incubate at 37°C for 1 hour, and then place in a 60°C water bath for 10 minutes to release the bound AI-2. Centrifuge ultrafiltration at 7500 rpm at 4°C in a 10 kDa protein ultrafiltration tube. Take the filtrate, filter and sterilize with a 0.22 μm filter membrane, and use V. harveyi The activity of AI-2 was determined by BB170 luminescence assay.

[0037] ③ Denaturation group: After treating the recombinant protein A0A1L7GXS4 in a 100℃ water bath for 10 min, repeat the steps of the binding group.

[0038] ④ Positive control group: Take 200 μL of sterile PBS into an EP tube, add exogenous AI-2 with a final concentration of 50 μM and 20 μM, and repeat the steps of ① binding group.

[0039] To detect the binding ability of recombinant protein A0A1L7GXS4 to AI-2, the recombinant protein A0A1L7GXS4 (3 mg / mL) expressed by BL21 (DE3) was combined with 50μM and 20μM AI-2, respectively. At the same time, a binding group, a release group, a denaturation group and an AI-2 positive control group were set up. After 20μM signal molecule AI-2 and recombinant protein A0A1L7GXS4 were reacted at 37℃ for 1h, the activity of AI-2 in the reaction solution (binding group) was detected. The results showed that the activity of AI-2 was 12.8% of that in the AI-2 positive control group, indicating that the recombinant protein A0A1L7GXS4 binds to AI-2, thereby reducing the free AI-2 in the reaction solution. High temperature can lead to the destruction of the spatial conformation of the protein and cause the loss of its biological properties. After 20μM AI-2 and recombinant protein A0A1L7GXS4 were reacted at 37℃ for 1h, they were placed in a 60℃ water bath for 10min to heat denature the recombinant protein A0A1L7GXS4, causing its binding to AI-2 molecular domain to be destroyed, thereby releasing the bound AI-2 (release group). The results of the AI-2 activity detection in the release group showed that the AI-2 activity in the release group was 163.3% of that in the binding group, indicating that the recombinant protein A0A1L7GXS4 released the signal molecule AI-2 after denaturation, resulting in an increase in the free AI-2 in the reaction solution. After 20μM AI-2 and recombinant protein A0A1L7GXS4 denatured in a 100℃ water bath for 10min were reacted at 37℃ for 1h, the results of the AI-2 activity detection in the reaction solution (denatured group) showed that the AI-2 activity was slightly lower than that in the positive control group, indicating that the denatured recombinant protein A0A1L7GXS4 could not bind to AI-2. When the concentration of exogenous AI-2 was increased to 50 μM, the results obtained were also similar to the inhibition trend of 20 μM results (see Figure 7 ), indicating that the recombinant protein A0A1L7GXS4 can bind to AI-2 and is a potential transporter of AI-2. The recombinant protein A0A1L7GXS4 directly acts as a transporter to bind to AI-2 and transport it to the extracellular space, activating and upregulating the quorum sensing system of the strain, thereby promoting the synthesis of functional factors.

[0040] (3) Surface plasmon resonance (SPR) technique to measure the binding energy between recombinant protein A0A1L7GXS4 and AI-2 ①Chip preparation The ligand recombinant protein A0A1L7GXS4 was diluted 10 times with the immobilization reagent (10 mM sodium acetate, pH 4.0). First, the surface of the CM5 chip was activated with 400 mM N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and 100 mM N-hydroxysuccinimide at a flow rate of 10 μL / min for 900 s. Secondly, the diluted ligand recombinant protein A0A1L7GXS4 was injected into the experimental channels (Fc2, FC4) at a flow rate of 10 μL / min, and the immobilization amount was about 14,600 RU and 14,400 RU respectively. Finally, the chip was blocked with 1M ethanolamine at 10 μL / min for 900 s. The reference channels (Fc1, FC3) were only activated and blocked, and no ligand was coupled.

[0041] ② Preliminary test of affinity determination The analyte (S)-4,5-Dihydroxy-2,3-pentandione (DPD / AI-2) was diluted to 200 μM and 1000 μM using running buffer 1. The diluted analyte was injected into the experimental channel (Fc2, FC4) and the reference channel (Fc1, FC3) at a flow rate of 30 μL / min, with a binding time of 60 s, and the signal value change was recorded.

[0042] The analyte (S)-4,5-Dihydroxy-2,3-pentandione (DPD / AI-2) was diluted to 1,000 μM using running buffer 2. The diluted analyte was injected into the experimental channel (Fc2, FC4) and the reference channel (Fc1, FC3) at a flow rate of 30 μL / min, with a binding time of 120 s, and the signal value changes were recorded.

[0043] ③ Affinity determination formal test The analyte (S)-4,5-Dihydroxy-2,3-pentandione (DPD / AI-2) was diluted in running buffer 2 (1,000, 500, 250, 125, 62.5 and 0 μM). The diluted analyte was injected into the experimental channel (Fc2, FC4) and the reference channel (Fc1, FC3) at a flow rate of 30 μL / min, with a binding time of 120 s and a dissociation time of 300 s. The temperature of the chip chamber and the sample chamber was selected to be 25°C, and the affinity between the analyte and the ligand was determined.

[0044] Using Biacore T200, the ligand (PET-22b-XS4) was covalently coupled to the CM5 chip surface by amino coupling method as a ligand. HBS-EP+ was used as the running buffer. No obvious binding signal was detected when DPD / AI-2 was 200μM and 1,000μM. Using PBS as the running buffer, the analyte AI-2 had a certain affinity with the A0A1L7GXS4 protein at 1,000μM, which was a specific binding ( Figure 8 ), indicating that the recombinant protein A0A1L7GXS4 can bind to AI-2 and is a potential transporter of AI-2, defining the recombinant protein A0A1L7GXS4 as the transporter A0A1L7GXS4.

[0045] Example 4 Structural properties of the transporter A0A1L7GXS4 (1) Analysis of the physicochemical properties of the transporter protein A0A1L7GXS4 The physicochemical parameters of the transporter protein A0A1L7GXS4 were predicted by the ProtParam tool, and the results are shown in Table 1. The transporter protein A0A1L7GXS4 is a protein encoded by 366 amino acids with a molecular formula of C 1888 H 3022 N 450 O 467 S 17 The relative molecular mass is 40042.52 Da (40.0 kDa), the total number of atoms is 5,844; the theoretical isoelectric point pI is 9.89, the number of positively charged residues (Arg + Lys) is 10, the number of negatively charged residues (Asp + Glu) is 29, and it is an acidic protein; the half-life is > 10h (Escherichia coli), the instability coefficient is 29.04, which is less than 40, indicating that the XS4 protein has good stability, and the aliphatic coefficient is 135.22. Analysis by ProtScale software showed that the hydrophobic region of the transport protein A0A1L7GXS4 is more than the hydrophilic region, among which the 297th and 298th positions have the strongest hydrophobicity, which is 3.4560, and the 196th position has the strongest hydrophilicity, which is -1.711. The total average hydrophilicity is 1.006, indicating that the protein is a hydrophobic protein ( Fig. 9 ).

[0046] Table 1 Physicochemical properties of transporter A0A1L7GXS4

[0047] (2) Structural properties analysis of transporter protein A0A1L7GXS4 ① Analysis of signal peptide structure and transmembrane structure of transporter A0A1L7GXS4 SignalP 5.0 prediction results showed that the probability of the transporter A0A1L7GXS4 having a signal peptide was 0.0345%, and it did not contain a signal peptide sequence or a signal peptide cleavage site, suggesting that the XS4 protein was a non-secretory membrane protein ( Fig.10 ). According to the prediction of transmembrane structure, XS4 protein is a transmembrane protein with 8 transmembrane regions, 4 of which are located from outside the membrane to inside the membrane (at amino acids 7-26, 36-58, 191-251 and 312-356); 4 of which are located from inside the membrane to outside the membrane (at amino acids 79-101, 171-191, 252-274 and 279-301) ( Fig.11 ).

[0048] ② Subcellular localization of transporter A0A1L7GXS4 The results of PSORT Ⅱ online prediction showed that the transporter A0A1L7GXS4 was mainly located in the plasma membrane outside the cell (52.2%), followed by the endoplasmic reticulum and mitochondria, with proportions of 43.5% and 4.3%, respectively. Therefore, it is speculated that the transporter A0A1L7GXS4 is a non-secretory membrane protein.

[0049] ③ Secondary structure and spatial structure of transporter protein A0A1L7GXS4 The online prediction analysis of SOPMA and SWISS-MODEL showed that the secondary structure of the transporter A0A1L7GXS4 consisted of 77.32% α-helix, 9.84% extended chain, 1.64% β-turn and 11.02% random coil, and α-helix was the main secondary structure of the protein ( Fig.12 ). The functional role of protein is closely related to its spatial structure. The a-helix in the secondary structure can stabilize the protein skeleton and play a role in transmembrane transmission, and proteins with a high proportion of a-helix are mostly conservative proteins. Random coils are important functional regions of proteins, which can maximize the free energy in the spatial structure and promote the stability of protein structure. The a-helix accounts for a high proportion in the secondary structure of the transport protein A0A1L7GXS4, indicating that it plays an important role in transmembrane transport and signal transmission, such as the transmembrane transport of the signal molecule AI-2; in addition, a high content of random coils promotes the stability of the structure of the transport protein A0A1L7GXS4. The SWISS-MODEL prediction results show that the three-dimensional structure of the transport protein A0A1L7GXS4 is mallet-shaped, with a GMQE of 0.81 and a QMEAN of 0.53 in the tertiary structure, and a sequence similarity of 99.18% ( Fig.13The three-dimensional structure of XS4 protein was tested by PROCHECK. The pull-type conformation results showed that the amino acid residues of XS4 occupied 96% of the favorable region, 3% of the extra allowed region, 0.3% of the general allowed region and 0.6% of the unallowed region. The total proportion of amino acid residues occupying the favorable region and the extra allowed region was higher than 90%, which was in line with the rules of stereochemistry.

[0050] Example 5 Signal molecule AI-2 transporter mediates exopolysaccharide synthesis In order to further clarify the regulatory relationship between the signal molecule AI-2 transporter and the synthesis of extracellular polysaccharides, the expression level of the AI-2 transporter encoding gene (AI-2E), AI-2 activity and extracellular polysaccharide production in the normal group and the AI-2 quorum sensing system inhibition group (addition of 200 μmo1 / L furanone) were compared. First, RNA was isolated from the normal group and the inhibition group strains using an RNA extraction kit. The A260 / 280 ratio of the extracted RNA was approximately 2.1. The total RNA was reverse transcribed into cDNA using the Goldenstar RT6cDNA Synthesis Kit Ver 2. Real-time PCR analysis of the target gene was performed using an ABI, QuantStudioTM 1 Plus real-time fluorescence quantitative PCR instrument. Table 2 lists the gene-specific primers used in the experiment. Gene expression was measured using 2 -ΔΔCt Method for analysis.

[0051] Table 2 Primer sequences used for real-time fluorescence quantitative PCR

[0052] The activity of signal molecule AI-2 of normal group and inhibition group strains was determined by Vibrio harveyi luminescence method. Vibrio harveyi BB170 was cultured in AB medium overnight until the OD600nm value reached 0.9-1.1. Fresh AB medium was used to dilute the volume ratio of 1:100. V. harveyi BB170 solution. Filter with a 0.22 μm microbial filter L. fermentum A51 cell-free supernatant. Positive control is V. harveyi The cell-free supernatant of BB170, the negative control is sterile AB medium, and the medium control is sterile MRS medium. V. harveyi The BB170 bacterial solution was mixed at a volume ratio of 1:50 and placed under shaking culture at 30°C. The fluorescence intensity was detected by an ELISA instrument every 2 hours. When the fluorescence intensity of the negative control was the lowest, the AI-2 activity of each sample group was calculated. The AI-2 activity was the ratio of the fluorescence value of each sample group to the fluorescence value of the negative control.

[0053] The phenol-sulfuric acid method was used to determine the extracellular polysaccharide production of the normal group and the inhibition group strains. The third-generation activated strain was inoculated into fresh sterile MRS liquid medium at a 5% inoculation rate and cultured at 37°C for 30 hours to obtain the fermentation broth; the fermentation broth was inactivated in boiling water for 10 minutes, cooled and centrifuged at 4°C for 20 minutes at 8,000 rpm, the bacteria were removed to obtain the supernatant, filtered with gauze, 80% trichloroacetic acid was added to the supernatant to a final concentration of 4% (w / v), stirred for 5 minutes, and allowed to stand at 4°C for 10 hours, and centrifuged to remove the protein. Then it was concentrated to 1 / 3 of the original volume using a rotary evaporator, and 3 times the volume of pre-cooled anhydrous ethanol was added, allowed to stand at 4°C overnight, centrifuged, and the precipitate was resuspended in deionized water and dialyzed for 48 hours. Take 20 mL of the dialyzed EPS solution, add 6% phenol and 100 mL of concentrated sulfuric acid, and measure its absorbance at 490 nm after 30 minutes of reaction. The extracellular polysaccharide content was determined using a standard curve.

[0054] The effects of adding AI-2 inhibitor on gene expression, activity and extracellular polysaccharide production of the strain were determined, and the results are shown in Table 3.

[0055] Table 3 Effects of adding AI-2 inhibitor on gene expression, activity and extracellular polysaccharide production

[0056] Note: Different superscript letters in the same column indicate significant differences (P < 0.05).

[0057] As shown in Table 3, the expression levels of AI-2E gene and exopolysaccharide synthesis gene in the normal group ( gtf, Wzx, Wy ), AI-2 activity and extracellular polysaccharide production were significantly higher than those in the inhibition group, and the expression of AI-2E gene and extracellular polysaccharide synthesis gene was consistent with the activity of AI-2, and was positively correlated with the extracellular polysaccharide production, indicating that when the AI-2 quorum sensing system was inhibited, the production of signal molecule AI-2 synthesized by the strain decreased, resulting in a decrease in the amount of AI-2 transported by the AI-2 transporter, which ultimately affected the synthesis of extracellular polysaccharides in the strain. The gene expression level proved that the signal molecule AI-2 transporter mediated the synthesis of extracellular polysaccharides ( Fig.14 ).

[0058] The above-mentioned implementation cases are preferred implementations of the present invention, but the implementations of the present invention are not limited to the above-mentioned implementation cases. Any other changes made without departing from the spirit and principle of the present invention shall be equivalent replacement modes and shall be included in the protection scope of the present invention.

Claims

1. A lactic acid bacteria signal molecule AI-2 transporter, characterized in that: The amino acid sequence of the transporter is shown in SEQ ID NO.

1.

2. A gene encoding the lactic acid bacteria signal molecule AI-2 transporter as claimed in claim 1, characterized in that: The nucleotide sequence of the coding gene is shown in SEQ ID NO.

2.

3. A recombinant plasmid containing the coding gene according to claim 2.

4. The recombinant plasmid according to claim 3, characterized in that The recombinant plasmid is obtained by homologous recombination of the coding gene of the signal molecule AI-2 transporter and the pET-22b vector, and is named pET-22b-XS4-His.

5. A genetically modified bacteria, characterized in that: The genetically engineered bacteria contains the recombinant plasmid described in claim 3 or 4, or the coding gene described in claim 2 is integrated into the genome of the genetically engineered bacteria.

6. The genetically modified bacteria according to claim 5, characterized in that: The genetically modified bacteria is the Escherichia coli BL21 strain.

7. Use of the transporter protein according to claim 1 in promoting the transport of signal molecule AI-2 from intracellular to extracellular or promoting the synthesis of signal molecule AI-2.

8. A method for synthesizing the lactic acid bacteria signal molecule AI-2 transporter as claimed in claim 1, characterized in that: The method comprises the following steps: Step (1) preparation of target gene cDNA template; Step (2) using the cDNA as a template and using a DNA polymerase to amplify and recover the target gene; Step (3) Construction and identification of gene recombination vector; Step (4) recombinant protein expression and purification; Step (5) determination of the ability of the recombinant protein to bind AI-2; The construction and identification of the gene recombination vector in step (3) includes vector linearization and gene recombination: the vector linearization is to use BamHI and NdeI double enzymes to digest the pET-22b vector to obtain a linearized vector pET-22b, and use a kit to purify and recover it; the gene recombination is to connect the target gene amplified by the kit with the linearized pET-22b vector, and transform the strain into E. coli BL21 competent cells are used to obtain a bacterial solution containing the transporter protein.

9. The method for synthesizing the signal molecule AI-2 transporter according to claim 8, characterized in that: The preparation operation of the cDNA template in step (1) is as follows: centrifuge the suspension of fermented mucus lactobacillus with a preservation number of CCTCC NO: M2023861, quickly freeze the centrifuged bacterial sludge with liquid nitrogen, extract RNA, and use a TAKARA reverse transcription kit to reverse transcribe the RNA into cDNA.

10. The method for synthesizing the signal molecule AI-2 transporter according to claim 8, characterized in that: The protein expression and purification steps in step (4) are as follows: the bacterial solution is revived and cultured, and inoculated into a fresh LB liquid culture medium for expansion culture, isopropyl-β-D-thiogalactoside is added to the culture medium to induce protein expression under low temperature conditions; after the induction expression is completed, the bacteria are collected by centrifugation, the bacteria are resuspended in a buffer, ultrasonically disrupted, and centrifuged at high speed, the supernatant is loaded onto a His affinity chromatography column, and different concentrations of imidazole are used to elute the column and collect the filtrate, and the eluate and the precipitate are subjected to SDS-PAGE protein electrophoresis to detect the results.

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