WO2018223417A1

WO2018223417A1 - Bacillus subtilis with cephalosporin resistance and high expression of sir2 protein and application thereof

Info

Publication number: WO2018223417A1
Application number: PCT/CN2017/089077
Authority: WO
Inventors: 孙晗笑; 郭晴; 谢亚杰; 利时雨; 刘梦鸽; 陈露平; 黄金群
Original assignee: 广州弘宝元生物科技有限公司
Priority date: 2017-06-10
Filing date: 2017-06-19
Publication date: 2018-12-13
Also published as: CN107254419B; CN107254419A

Abstract

Provided are a Bacillus subtilis BS16 with cephalosporin resistance and high expression of Sir2 protein and an application thereof. The Bacillus subtilis BS16 with cephalosporin resistance and high expression of Sir2 protein can enhance the aerobic oxidation of glucose in cells, increase mitochondrial respiration, and regulate an ATP energy metabolism pathway. The Bacillus subtilis BS16 can be used for treatment of a number of metabolism-associated diseases such as cell aging, mitochondrial dysfunction, and metabolic syndrome.

Description

Bacillus subtilis with cephalosporin resistance and high expression of Sir2 protein and application thereof

Technical field

The present invention belongs to the field of screening and application mechanisms in the new army, and more particularly, to a Bacillus subtilis BS16 having cephalosporin resistance and high expression of Sir2 protein and application thereof.

Background technique

The Sir2 protein family (Sirtuin) is a conserved protein that acts primarily as a NAD+-dependent protein deacetylase, which is found in a variety of organisms, from archaea to mammals. A number of studies have now shown that the mammalian family of Sirtuin proteins is involved in regulating cellular metabolism and helps delay or even prevent metabolic related diseases. In addition, more and more studies have shown that probiotics can improve metabolic diseases and the like. Probiotics also have Sir2 homologous proteins, however, little is known about the biological function of the sir2 protein in probiotics.

Studies have shown that sirtuins are widely involved in regulating the regulation of energy metabolism in the body and maintaining the homeostasis of glucose and lipid metabolism. Among them, SIRT1, SIRT3 and SIRT6 are the most widely studied. SIRT1 is the most studied member of this family. Current research has shown that it not only regulates the deacetylation of histones, but also regulates non-histones. In vivo and in vitro, SIRT1 regulates gluconeogenesis and glycolysis through PGC-1α transcription factors, resulting in an increase in the number and function of mitochondria. In addition, studies have shown that SIRT1 can improve mitochondrial dysfunction and improve high glucose-induced insulin resistance in skeletal muscle cells when overexpressing SIRT1. Further studies have found that this is achieved by regulating SIRT1-SIRT3-mitochondria. In muscle, SIRT1 is able to enhance fatty acid oxidation in mitochondria by deacetylating PGC1-α. In addition, SIRT3, SIRT4 and SIRT5 belong to the mitochondrial Sirtuin protein, which are located in the mitochondria. Currently, SIRT3 is the most important mitochondrial deacetylase, and many of its target proteins have been identified, many of which are involved in the regulation of important metabolic balances. For example, SIRT3 deacetylates to activate isocitrate dehydrogenase 2 (IDH2; it participates in the tricarboxylic acid cycle (TCA cycle)). SIRT3 also activates the TCA circulating glutamate dehydrogenase (GDH), although the physiological significance of GDH deacetylation is unclear. In addition, SIRT3 deacetylated complex I, complex II and complex III, which are involved in oxidative phosphorylation (OXPHOS), the final stage of mitochondrial aerobic respiration. Overexpression of mitochondrial sirtuin protein in HEK293T cells alters glycolysis and mitochondrial function. SIRT6 is a more intensive Sirtuin protein compared to SIRT1 and SIRT3. SIRT6 participates in the stabilization and repair, metabolism and senescence of pre-genomic DNA. Studies have shown that SIRT6-deficient mice develop symptoms of hypoglycemia, while in SIRT6-deficient cells, Hif1α activity increases, glucose uptake increases, glycolysis increases, and mitochondrial respiratory function diminishes. Summarizing the above studies, it is not difficult to find that Sirtuin proteins are closely related to cellular energy metabolism, and to some extent they act as switches for glycolysis to oxidative metabolism.

Probiotics, which act as intestinal microbes, show good effects in improving host metabolic diseases, but the specific molecular mechanisms are not clear. The Sir2 gene family is a conserved NAD+-dependent histone/non-histone deacetylase from archaea to mammals. Many studies have shown that the mammalian Sirtuin protein family can regulate cell metabolism, including cellular respiration, glycolysis, gluconeogenesis, and fatty acid metabolism. Maintenance of normal activity of sirtuins helps to delay or even prevent the development of metabolic related diseases. At present, there are few reports on the biological functions of the probiotic sir2 protein. Whether the probiotic Sir2 protein is involved in the regulation of host metabolism and as a target for probiotics to regulate metabolic diseases is worth studying.

At present, the study of the probiotic Sir2 homologous protein is still in its infancy, and only the B. subtilis Sir2 homologous protein is identified and encoded by the yhdz gene. There are no related studies on the sir2 homologous proteins of other probiotics. Previous studies on the mammalian Sirtuin protein family have shown that it is widely involved in the regulation of glycolipid metabolism and maintains energy metabolism balance, and is expected to be a target for metabolic diseases. So whether the probiotic Sir2 protein is involved in cell metabolism and serves as a molecular target for probiotics to regulate host metabolism is currently not relevant, and these are worthy of further study.

Summary of the invention

The present invention screens Bacillus subtilis BS16 with cephalosporin resistance and high expression of Sir2 protein and Study its mechanism of action to make it play a role in the treatment of disease.

The invention provides a Bacillus subtilis BS16 with cephalosporin resistance and high expression of Sir2 protein, which is preserved in the Guangdong Provincial Collection of Microorganisms and Cultures, the deposit number is GDMCC No. 60193, and the deposit address is No. 100, Xianlie Middle Road, Guangzhou. The 5th floor of Building 59, the date of the deposit is June 2, 2017.

The Bacillus subtilis BS16 of the present invention having cephalosporin resistance and high expression of the Sir2 protein can be used for the preparation of a medicament for treating anti-aging, mitochondrial dysfunction or metabolic syndrome.

The present invention also provides an anti-aging food comprising an effective amount of a Bacillus subtilis BS16 having a cephalosporin resistance and a high expression of a Sir2 protein as an active ingredient and/or a bacterial protein component thereof.

The present invention also provides an anti-aging medicine comprising an effective amount of a cephalosporin-resistant and highly expressed Sir2 protein-producing Bacillus subtilis BS16 and/or a bacterial protein component thereof as an active ingredient, and a pharmaceutically acceptable compound Carrier.

The present invention also provides a food for preventing and treating mitochondrial dysfunction of a cell comprising an effective amount of a Bacillus subtilis BS16 having a cephalosporin resistance and a high expression of a Sir2 protein as an active ingredient and/or a bacterial protein component thereof.

The present invention also provides a medicament for preventing and treating mitochondrial dysfunction of a cell comprising an effective amount of a cephalosporin-resistant and highly expressed Sir2 protein-producing Bacillus subtilis BS16 and/or a bacterial protein component thereof as an active ingredient. And a pharmaceutically acceptable carrier.

The present invention also provides a food for preventing and treating metabolic syndrome comprising an effective amount of a Bacillus subtilis BS16 having a cephalosporin resistance and a high expression of a Sir2 protein as an active ingredient and/or a bacterial protein component thereof.

The present invention also provides a medicament for preventing and treating metabolic syndrome comprising an effective amount of a cephalosporin-resistant and highly expressed Sir2 protein-producing Bacillus subtilis BS16 and/or a bacterial protein component thereof as an active ingredient, and A pharmaceutically acceptable carrier.

Compared with the prior art, the present inventors have found that Bacillus subtilis BS16 with cephalosporin resistance and high expression of Sir2 protein can enhance the aerobic oxidation of cells, increase mitochondrial respiration, and regulate the ATP energy metabolism pathway. Bacillus subtilis BS16 can treat and prevent various metabolic related diseases such as cell senescence, mitochondrial dysfunction and metabolic syndrome.

DRAWINGS

Figure 1 shows the results of identification of the sir2 gene of Bacillus subtilis of Example 1.

Figure 2 is a diagram showing the electrophoresis pattern of the Bacillus subtilis protein of Example 1.

Figure 3 is a result of Western blotting of the B. subtilis Sir2 protein of Example 1.

Figure 4 is a PCR fragment size identification of the B. subtilis sir2 gene of Example 2.

Figure 5 is a result of double enzyme digestion identification of Example 2 pEGFP-N1-BS-sir2.

Figure 6 is a fluorescence microscopy of the transfection of HEK293T cells transfected with pEGFP-N1-BS-sir2 in Example 2, wherein the control plasmid, empty plasmid transfection vector, and recombinant plasmid pEGFP-N1-BS-sir2 were transfected from left to right. Group Sir2.

Figure 7 shows the results of Western Blot detection of the fusion protein expressed by HEK293T cells transfected with the recombinant plasmid of Example 2, wherein 1 is a blank control group, 2 is a pEGFP-N1 empty plasmid group, and 3 is a pEGFP-N1-BS-sir2 group. .

Figure 8 is the ATP level of the recombinant plasmid of Example 2 after transfection of HEK293T cells.

Figure 9 is a graph showing the effect of the BS-sir2 gene on the glucose metabolism enzyme activity in the co-immunoprecipitation of Example 2.

Figure 10 is a graph showing the effect of the lactic acid level of the BS-sir2 gene of Example 2.

Figure 11 is a graph showing the effect of BS-sir2 on mitochondrial respiration in Example 2.

detailed description

The present invention will be further described in detail below with reference to the embodiments.

Example 1

1. Experimental method materials

1.1 Probiotics screening

Collect 1.0g of stool samples from 3 young patients from Nanyang People's Hospital, amplify and culture with LB medium, remove the plasmid by ethidium bromide, dilute to 10 ^{-8 in} distilled water, take 1 dilution, take 1mL, coat On LB solid medium, placed in an incubator and incubated at 37 ° C for 48 h. After the completion of the culture, the plate was removed and the surface was rough and opaque, stained with white or yellowish colonies, and transferred to LB liquid medium for 48 hours at 37 ° C, and stored in a refrigerator at 4 ° C for use.

1.2 Morphological observation of the strain.

The pure culture of the isolated and purified strain was inoculated into the enrichment liquid medium, incubated at 37 ° C for 24 h, and after Gram staining, the cell morphology was observed under a light microscope, and a microscope with a digital imaging system was used. Select a suitable field of view for photographic recording.

1.3 Physiological and biochemical characteristics

Isolated strains were identified according to the Berger's Bacterial Identification Manual and the Common Bacterial System Identification Manual.

1.4 Bacillus subtilis molecular biology - 16S rDNA identification.

16S rDNA full length universal primer:

Primer 1:27F--SEQ ID NO:1;

Primer 2: 1492R--SEQ ID NO: 2;

PCR reaction system (25 μL): primer 1/1.0 μL; primer 2/1.0 μL; 10×PCR Buffer/2.5 μL; dNTP mix/2.0 μL; Taq enzyme/0.3 μL; DNA template/1.0 μL; ultrapure water/25 μL .

The PCR reaction conditions were: first 94 ° C - 4 min; then 94 ° C - 30 s, 55 ° C - 40 s, 72 ° C - 90 s cycle 30 times, and finally 72 ° C - 10 min.

The template was replaced with sterile deionized water as a negative control. After the completion of the amplification, 3.0 μL of the amplified product was subjected to agarose gel electrophoresis. After the electrophoresis is finished, the tape to be tested is sent to Shanghai Biotech for sequence determination. The sequence results of the assay strains were compared with the known 16S rDNA sequences in NCBI by Blast to obtain the closest strain to determine the species of the bacteria isolated from the experiment.

1.5 Determination of antibacterial activity

The test bacteria were Bacillus subtilis BS12, Bacillus subtilis BS14, and Bacillus subtilis BS16 obtained above, and 100 μL of each strain was subjected to activation and resuscitation in 100 mL of LB liquid medium. The indicator bacteria were Escherichia coli ATCC11105, Clostridium difficile NY-5, Staphylococcus aureus NT-12.

1.6 drug sensitivity test

Select 10 antibacterial drug sensitive papers: cefepime (FEP, 20μg), cefotaxime (CTX, 20μg), rifampicin (Rf, 10μg), ampicillin (AM, 10μg), tetracycline (TE, 20μg ), chloramphenicol (CHL, 20 μg), ciprofloxacin (CIP, 10 μg), amikacin (AK, 20 μg), gentamicin (GM, 10 μg), trimethoprim (TMP, 10 μg) ), all purchased from Guangzhou Yikang Biotechnology Co., Ltd. (Oxoid).

Susceptibility testing was performed according to the KB method promulgated by the American Association of Clinical Laboratory Standards. First, use a sterile tweezers to separately take the drug-sensitive papers containing different antibiotics, and paste them on each plate that has been inoculated with the test bacteria (200 μL of the suspension of the bacteria to be tested is 3.0×108 CFU/mL, respectively, and added to the temperature. In an LB agar medium of about 50 ° C, it was quickly mixed and poured, and the plate was inverted. Approximately 5 sheets of susceptibility paper are attached to each plate, and the distance between the sheets is approximately equal and marked. Incubate overnight at 37 ° C, observe the inhibition of the plate, measure the size of the inhibition zone by vernier caliper and record

Identification of the 1.7Sir2 gene.

Using the genomic DNA of the test bacteria as a template;

Bacillus subtilis sir2 primer:

BS-sir2-F: SEQ ID NO: 3;

BS-sir2-R: SEQ ID NO: 4.

PCR reaction system: template - 1 μL; primer BS-sir2-F - 0.5 μL; primer BS-sir2-R - 0.5 μL; dNTP (10 mM) - 0.5 μL; LA Taq (5 U / μL) - 0.5 μL; 10 × PCR buffer - 2.5 μL; sterile water - 19.5 μL.

1.8 (1) Configure the protein gel. The separation gel and concentrate formula are as follows:

Table 1

(2) Glue: After mixing, use a pipette to carefully inject the gel solution along the glass rod into the slit between the long and short glass plates (the glue height is about 1 cm from the lower edge of the sample template comb);

(3) Liquid seal: gently add a layer of water along the edge of the short glass plate to isolate the air on the surface of the gel to make the rubber surface smooth. After standing for about 30 minutes, observe the change of the rubber surface. When the water and the solidified rubber surface have different refractive index limits, it indicates that the glue has completely solidified, the upper layer water is poured off, and the residual water liquid is absorbed by the filter paper;

(4) Insert the rubber comb: After mixing, inject the gel solution into the slit between the long and short glass plates (above the separation gel), gently add the sample template comb, and carefully avoid the appearance of bubbles. About 30 minutes, the polymerization is complete;

(5) Install the electrophoresis tank: Remove the prepared gel plate and carefully remove the comb. Two 10% gel plates are inserted into the concave grooves on both sides of the U-shaped rubber frame, and can be lifted up to make the gel plate close to the rubber. Place the plastic mold frame with the glass plate flat on the sump frame on the bottom, and align the lower edge with the lower edge of the sump frame and place it in the electrophoresis tank. Pour 1X tris-gly running buffer;

(6) Sample treatment: For the protein sample, directly take 80 μl of the sample, add 20 μl of 5× buffer (with B-mercaptoethanol), and mix. For solid samples such as cells or tissues, take a small amount of sample and add 100ul 2x buffer (with B-mercaptoethanol) to boil for 10 minutes;

(7) Loading: 10 μl of the treated sample solution was pipetted, carefully added to each gel concave sample tank in turn, and the marker was added to one of the grooves. To distinguish the two plates, the marker can be added. In different holes;

(8) Stripping glue: take out the electrophoresis tank, take off the two plates, use a doctor blade to lift from the middle of the long and short slides, then scrape off the concentrated glue and remove it;

(9) Dyeing: placed in a dyeing dish with R250 staining solution, the dyeing solution can be passed through the gel, placed on a shaker at a speed of about 45r/min, and the time is about 1 hour. After the completion, the dye solution is drained and used. Wash off the dye solution;

(10) The gel imaging system takes a picture of the protein electrophoresis gel and saves the picture.

1.9Western blotting

(1) Glue: Dispose 12% of the separation gel and inject it into the gap of the glass plate to 1.5cm from the upper edge, add the appropriate amount of 75% ethanol; to solidify, pour off the upper liquid, inject 5% concentrated glue, insert the comb, naturally Dry.

(2) Loading: The protein sample was bathed at 100 ° C for 3 min, and the newly configured electrophoresis solution was poured into the electrophoresis tank. 8 μl and 5 μl of protein maker were added to the two lanes, and the volume was made up to 10 μl with 1× protein loading buffer. Add 10 μl samples to each of the remaining lanes and run at 50 V constant pressure.

(3) Transfer film: After electrophoresis, cut 11cm×8cm filter paper and 0.22μm PVDF film of appropriate size and activate with methanol for 5min, according to “sponge-6 layer filter paper-gel-PVDF film-6 layer filter paper-sponge "Assemble the transfer interlayer, the splint was assembled and transferred to the transfer tank, and transferred at a constant current of 200 mA for 60 min.

(4) Incubation of antibody: After the membrane is transferred, the membrane is washed for 5 min in TBS, 5% skim milk powder is blocked for 1 h, and TBST solution is washed 3 times for 5 min each time. Add anti-human anti-human SIRT3 polyclonal antibody dilution 4 °C Overnight incubation; TBST liquid wash membrane 3 After 5 min, add the secondary anti-diluted anti-dilution solution for 1 h at room temperature.

(5) Luminescence detection: TBST liquid washing membrane, incubated with luminescent liquid for 3 min, blotting the luminescent liquid with filter paper, placing the film and PVDF film into the tableting box for 1 min, taking out the film fixing for 30 s, washing with water, drying, scanning.

2. Experimental results

2.1 Physiological and biochemical results of Bacillus subtilis are shown in Table 2.

Table 2 physiological and biochemical tests

2.2 16S rDNA identification results.

Enter the NCBI Nucleic Acid Database http://blast.ncbi.nlm.nih.gov/Blast.cgi, enter the 16S rDNA sequence of the strain to be tested, and click Blast to enter the alignment.

The results of the comparison showed that the 16S rDNA sequence of the three strains was 99% similar to the other 16S rDNA sequences of Bacillus subtilis, and was initially identified as Bacillus subtilis, which was named Bacillus subtilis BS12, Bacillus. Subtilis BS14, Bacillus subtilis BS16.

2.3 Determination of antibacterial activity

The test results of the antibacterial activity are shown in Table 3. Among them, Bacillus subtilis BS12 and Bacillus subtilis BS16 have certain antibacterial ability, but Bacillus subtilis BS14 has weak antibacterial ability.

Table 3 Test results of bacteriostatic activity of Bacillus subtilis

2.4 drug sensitivity test, the results are shown in Table 4. The two Bacillus subtilis BS12 and Bacillus subtilis BS16 we screened had strong cephalosporin resistance.

Table 4 Results of drug susceptibility test of Bacillus subtilis

Note: S-sensitive; R-resistant; I-moderate drug resistance.

2.5Sir2 gene PCR identification results

As shown in Fig. 1, two Bacillus subtilis sir2 gene bands were amplified at about 744 bp.

2.6SDS-PAGE results

As shown in Figure 2, the two strains of Bacillus subtilis Sir2 protein had a bright band at 27 KD.

2.7Western blotting

Western blot analysis showed that the expression of Sir2 protein in Bacillus subtilis BS16 was the highest. Therefore, a strain of Bacillus subtilis BS16 with cephalosporin resistance and high expression of Sir2 protein was successfully screened.

Example 2 Transfection of Bacillus subtilis BS16sir2 (BS-sir2) Gene and Its Intracellular Function

Experimental material

1.1 Bacteria, plasmids and cells

1.2 main medium and reagents

(1) Buffer A: From 150 mM sodium chloride, 500 mM Tris-HCl, 0.1% TritoX-100, pH 8.0.

(2) Buffer B: consisted of 50 mM Tris-HCl, 0.1% Triton X-100, pH 8.0.

(3) Antibody dilution: 10 g of BSA powder was weighed and dissolved in 200 mL of TBST buffer, which was a 5% BSA solution, and stored at 4 ° C until use.

(4) Cell growth solution: Fetal bovine serum (final concentration: 10%) and penicillin streptomycin solution (final concentration: 100 units/ml) were added to the culture solution, and stored at 4 ° C until use.

(5) Cell maintenance solution: fetal bovine serum and penicillin streptomycin solution (final concentration: 100 units/ml) (final concentration: 2%) were added to the culture solution, and stored at 4 ° C until use.

(6) Trypsin solution: Weigh 8.0g of sodium chloride, 0.2g of potassium chloride, 0.2g of disodium hydrogen phosphate, 0.2g of potassium dihydrogen phosphate, 2.5g of trypsin, and make up to 1L with ultrapure water. Filter and sterilize, dispense and seal, and store at -20 °C for use.

(7) 10% SDS (sodium lauryl sulfate): 10 g of sodium lauryl sulfate dissolved in 90 mL of deionized water, after Adjust the pH of the solution to 7. 4% SDS: SDS 4g was dissolved in 96 mL of deionized water and the pH of the solution was adjusted to 7.0.

(8) PBS phosphate buffer: Weigh 1.6g of sodium chloride, 0.04g of potassium chloride, 0.698g of NaHPO4-12H2O, 0.04g of potassium dihydrogen phosphate, add ultrapure water to 200mL, mix evenly, adjust PH to 7.2 The prepared PBS solution was autoclaved for 30 minutes, and then the bottle was dispensed and sealed, and stored at -20 ° C for use.

(9) Primary reagent

1.4 Experimental methods

1.4.1 Construction and identification of eukaryotic expression vector

1.4.1.1BS-sir2 gene amplification

(1) Specific primers were designed using Primier 5.0 software, and EcoR I and BamH I restriction sites were added to both ends of BS-sir2 gene.

BS-sir2-FF: SEQ ID NO: 5, wherein GAATTC is an EcoR I cleavage site

BS-sir2-RR: SEQ ID NO: 6, wherein GGATCC is a BamH I restriction site)

(2) PCR reaction system:

A total of 48 μL.

(3) PCR reaction conditions

The PCR product was stored at 4 ° C and identified by 1% agarose gel electrophoresis.

1.4.1.2BS-sir2 gene is ligated into plasmid pEGFP-N1

(1) Double digestion of the inserted fragment

16μl in total

(2) Double digestion of pEGFP-N1

16μl in total

(3) Endonuclease inactivation: after the end of the enzyme digestion, it is placed in a water bath at 60 ° C for 25 min.

(4) Connection reaction

A total of 18μl

After the ligation, the PCR instrument was thermostatically reacted at 37 ° C for 20 h, and the target plasmid pEGFP-N1-BS-sir2 was detected and recovered on a 1% agarose gel.

1.4.1.3 Identification of recombinant plasmid pEGFP-N1-BS-sir2

The ligation system pEGFP-N1-BS-sir2 was transformed into E. coli competent cells by CaCl2 method, and then plated on Kan-resistant LB solid plate for screening, and placed in a 37 ° C biochemical constant temperature incubator for inversion culture overnight. The transformants were randomly selected for colony PCR verification, and positive colonies were screened. The plasmid was extracted and identified by double digestion with EcoR I and BamH I. The positive clones were selected for sequencing analysis.

1.4.2 liposome transfection

According to the Lipofectamine 2000 liposome transfection kit, the specific operation is as follows:

(1) Inoculate 500 μl of DMEM medium containing no antibiotics and serum in a 24-well plate (1-2×10 ⁵ cells per well) until the cells can be as long as 90-95% confluent when transfected;

(2) Plasmid DNA and Lipofectamine 2000 were separately diluted with 50 μl of Opti-ME I serum-free medium. Mix well and incubate for 5 minutes at room temperature;

(3) The above diluted DNA and Lipofectamine 2000 were mixed (total volume of 100 μl), and allowed to stand at room temperature for 20 minutes;

(4) Add 100 μl of transfection solution to each well of the cells and shake gently;

(5) The medium can be changed after incubation for 4-6 hours at 37 ° C, 5% CO 2 , and the cells are lysed 24 h later for detection of gene expression.

1.4.3. Detection of green fluorescent protein (GFP) expression

The plasmid pEGFP-N1-BS-sir2 was transfected into 293T cells by Lipofectamine 2000, and cultured in a 37 °C CO2 incubator for 4 hours. The anti-free serum-free medium was replaced with normal medium to continue the culture. The green fluorescence was observed by fluorescence microscope at around 48 h and photographed.

1.4.4 Detection of the expression of BS-sir2 by Western blot

(1) The screened stable expression cell line was collected in a 1.5 ml EP tube, 100 ul of pre-cooled cell lysate and 1 ul of protease inhibitor were added, and vortexed twice, each time 5 min, 5 s each time.

(2) After standing on ice for 20 min, centrifuge at 7 ° C and 12000 g for 7 min, and collect the supernatant as the total protein of the cells. Quantitation was performed using the BCA Protein Assay kit. Quantitation was performed using the BCA Protein Assay kit.

(3) Take 20ug protein into the loading buffer, boil in boiling water for 5min, then perform SDS-PAGE separation (separation gel concentration is 10%), transfer 100V constant pressure for 1h50min onto PVDF membrane, buffer in TBST containing 5% skim milk powder. The solution was blocked for 1 h, and the mouse anti-Anti-EGFP mAb (1:500) was diluted with blocking solution overnight at 4 °C.

(4) The next day, wash with three times of 0.1% PBST for 10 min, add goat anti-mouse IgG-HRP secondary antibody diluted with PBS (1:1000), incubate for 1 h at room temperature in a light shaker, wash three times with 0.1% PBST, wash Same as above, the membrane was washed after washing with 0.1 M PBS for 5 min. Take β-actin as an internal reference.

1.4.5 Determination of ATP content

According to the ATP content determination kit instructions:

(1) Dissolving the reagent to be used on ice, and diluting the ATP standard solution to a concentration of 0.1, 1, 10 μM/L by ATP assay lysate to prepare a standard curve.

(2) Add 200 μl of lysate to the cell culture dish, repeatedly blow until fully lysed, centrifuge at 12000 rpm for 10 min at 4 ° C, and take the supernatant.

(3) Dilute the ATP detection working solution, repeat each sample 3 times, add 100μl ATP detection working solution to each monitoring hole, place it at room temperature for 5min, consume local ATP, add 100μl sample or standard sample to the detection hole. , mix thoroughly, interval 2s, immediately use bioluminescent instrument to detect CMP value. The ATP concentration in the sample to be tested is calculated using the measured standard curve according to the calculation formula.

1.4.6 pyruvate kinase activity assay

According to the instructions of the pyruvate kinase kit:

(1) 400 μl of cell extract was taken, and the cells were disrupted by ultrasonic wave (power 20%, ultrasonic 3 s, interval 10 s, repeated 30 times), 8000 g, centrifuged at 4 ° C for 10 min, and the supernatant was stored for use.

(2) Mix the reagent four in the kit with the reagent five, and then bathe at 37 ° C for 5 min, add 30 μl of the sample, start timing, and record the initial absorbance A1 at 20 s at a wavelength of 340 nm.

(3) After quickly, the cuvette water bath was accurately reacted in a 37 ° C water bath for 2 min, and the cuvette was quickly taken out and then dried with a mirror paper. The colorimetric color at 340 nm was recorded, and the absorbance A2 at 2 min 20 s was recorded to calculate ΔA=A1-A2. .

(4) PK enzyme activity calculation formula: PK (U/10 ⁴ cell) = total reaction volume ÷ sample volume ÷ reaction time ÷ NADPH extinction coefficient × ΔA ÷ live cell density.

1.4.7 fructose-6-phosphate kinase activity assay

The instructions for the kit are as follows:

(1) The cell treatment method is the same as step 1 in 3.3.7.

(2) PFK working solution 800μl, sample 30μl, reagent six 5μl, reagent seven 5μl sequentially added to 1mL cuvette In the case, the absorbance at a wavelength of 340 nm recorded at 20 s is A1.

(3) Then, the mixture was washed in a water bath at 37 ° C for 10 min, and then dried by a mirror paper, and then colorimetrically measured at 340 nm, and the absorbance at 10 min and 20 s was measured as A2, and ΔA = A1 - A2 was calculated.

(4) Calculation formula of PFK enzyme activity: Calculated in the same formula as in 4.3.3.

1.4.8 Lactate dehydrogenase activity assay

According to the instructions of the lactate dehydrogenase kit, the operation is as follows:

(1) Cell treatment is the same as step 1 in 3.3.3;

(2) The sample was 50 liters, the reagent was L 250, the reagent was 50 L (experimental group only), and the distilled water was 50 liters (only the control group was added), except that it was thoroughly stirred, and then a 37 ° C water bath for 15 minutes. Finally, add reagent three and continue to bath for 15 minutes. Then add reagent four, mix thoroughly, let stand for 3 min at room temperature, measure absorbance at 450 nm and record;

(3) Calculation formula of LDH enzyme activity:

LDH (U/10 ⁴ cell) = 1379 × (measurement tube absorbance - control tube absorbance) / cell density (10 ⁴ cell / mL).

1.4.9 Cellular oxygen consumption test

(1) The cells were inoculated into a 24-well microplate of hippocampus test, in which 200 μl of ordinary high-sugar DMEM was added to each well, and then placed in a constant-temperature cell incubator of 5% CO ₂ and cultured overnight at 37 ° C;

(2) After culture for 24 hours, the cells were washed twice with high-sugar hippocampus XF test medium (25 mmol/L glucose concentration), and then the culture system was supplemented. Finally, the volume of hippocampus XF test medium per well was 525 μl, and finally 24 holes. The microplate was incubated for 1 h at 37 ° C in a constant temperature incubator without CO ₂ .

(3) The test plate is then added to the hippocampus bioenergy meter XF24 to complete the calibration procedure and then placed in the cell culture plate test.

(4) The hippocampus bioenergy tester XF24 needs to be preheated 24 hours before the experiment. The hippocampal test plate was added to the hippocampal activation solution 24 hours in advance and placed in a 37 ° C, CO ₂ free incubator for activation of the detection probe.

(5) The degree of oxidative phosphorylation in cells is measured by the bioenergy analyzer XF24 by measuring the oxygen consumption rate per well (oxygen consumption, rate, OCR).

1.4.10 lactic acid content detection

According to the instructions of the lactic acid content determination kit:

(1) Add 200 μl of lysate to the cell culture dish, repeatedly blow until thoroughly lysed, centrifuge at 12000 rpm for 10 min at 4 ° C, and take the supernatant.

(2) Take 5 mL of the sample to be tested, add 40 μl of LD coloring solution, mix, and boil at 37 ° C for 5 min, then immediately add 240 μl of stop solution to terminate the reaction.

(3) The absorbance at 340 nm was measured with a microplate reader.

1.4.11 Statistical processing

Data were analyzed by SPSS18.0 software. The two groups were compared for t-test. One-way ANOVA was used to compare the multiple groups. The significant difference was expressed by p<0.05 or p<0.01.

2. Experimental results

2.1 Construction and identification of recombinant eukaryotic expression plasmid

The BS-sir2 gene was amplified by PCR (the EcoRI restriction site was introduced at the 5' end and the BamH I restriction site was introduced at the 3' end), and the specificity bar at the level of about 750 bp was observed by agarose gel electrophoresis analysis. The band size corresponds to the theoretical value of the target gene of 744 bp, as shown in Figure 4.

After cloning of the BS-sir2 gene, it was digested with EcoR I and BamH I and ligated into the pEGFP-N1 plasmid to obtain the recombinant plasmid plasmid pEGFP-N1-BS-sir2 expressing BS-sir2, and the recombinant plasmid pEGFP-N1-sir2 was recombined by CaCl2 method. The plasmid was transformed into E. coli competent cells and plated on a Kan-resistant LB solid plate for screening, and placed in a 37 ° C biochemical constant temperature incubator for inversion culture overnight. The transformants were randomly selected for colony PCR verification, and positive colonies were screened. The plasmid was extracted and identified by double digestion with EcoR I and BamH I. The positive clones were selected for sequencing analysis. EcoR I, BamH I enzyme production The agarose gel electrophoresis identification shown in Figure 5 showed a target band around 4700 and 750 bp, which was consistent with expectations. Positive plasmids were sequenced and sequenced for NCBI. The results showed that the sequence was identical to the template gene, the amino acid sequence was 100% correct, and pEGFP-N1-BS-sir2 was successfully constructed.

2.2BS-sir2 gene transfection into HEK293T cells

2.2.1 Fluorescence microscopy

Recombinant plasmid pEGFP-N1-BS-sir2 and empty plasmid pEGFP-N1 were transfected into HEK293 cells by Lipofectamine2000 liposome. After 48 hours, the expression of GFP was observed by inverted fluorescence microscope. The recombinant plasmid pEGFP-N1-BS was found. GFP expression was observed in the -sir2 group; GFP expression was observed in the empty plasmid pEGFP-N1 group; GFP expression was not observed in the untransfected HEK293 cell group. See Figure 6 for the 48-hour three-group fluorescence microscope observation from left to right.

2.2.2 Western blot to verify fusion protein expression

The molecular weight of the BS-sir2 protein is about 27 KD, the molecular weight of the enhanced green fluorescent protein (EGFP) is about 27 KD, and the molecular weight of the EGFP-BS-sir2 fusion protein is about 54 KD. Lanes 1-3 were blank control group (blank), empty vector transfection group (Vetor), and pEGFP-N1-BS-sir2 transfection group (Sir2). In the third lane, a 54 kDa band was observed at the GFP detection level, and the recombinant plasmid was transfected into the fusion protein expressed by HEK293T cells (Fig. 7).

Effect of 2.3BS-Sir2 on cellular aerobic oxidation pathway

Glycolysis and the TCA cycle are central to the carbon metabolism of mammalian cells. Through these two pathways, glucose is oxidized, producing energy in the form of NADH and ATP, or into precursors of amino acids, lipids, and nucleotides.

2.3.1 Effect of BS-sir2 transfection on ATP production by cells

After transfection of HEK293T cells with BS-sir2, intracellular ATP levels were significantly increased. The BS-sir2 transfected cells had approximately a 2-fold higher level of ATP than the WT and empty vector-transfected cells after 48 h of transfection, with a slight increase after 24 h (Fig. 8).

2.3.2 Effect of BS-sir2 transfection on cell glucose metabolism enzyme activity

The key rate-limiting enzymes regulated by the glycolytic pathway are fructose-6-phosphate kinase (PFK-1) and pyruvate kinase (PK). BS-sir2 significantly increased PFK-1 and PK activity after 48 h of transfection (Figure 9). At the same time, lactate dehydrogenase (LDH) activity was also analyzed. In contrast, BS-sir2 significantly reduced LDH activity after transfection for 48 h compared to empty vector and normal cell groups.

2.3.3 Effect of BS-sir2 transfection on cell lactic acid production

The above results demonstrate that BS-sir2 enhances glucose metabolism, and in order to further demonstrate an increase in glucose aerobic oxidation, the lactate level is detected by a kit. The results showed that BS-sir2 transfected cells showed significantly lower levels of lactate than empty vector transfected and wild-type cells (Fig. 10).

2.3.4 Effect of BS-sir2 transfection on mitochondrial respiration in cells

Cellular energy is measured using a cellular energy meter. Among them, oligomycin, uncoupler (FCCP), rotenone (Rot.). The results showed that BS-sir2 transfection also resulted in increased cellular oxygen consumption and increased mitochondrial respiration. These results indicate that BS-sir2 transfection enhances glucose aerobic oxidation, showing enhanced mitochondrial respiration and inhibition of glycolysis (anaerobic respiration) (Figure 11).

The eukaryotic expression vector pEGFP-N1-sir2 was successfully constructed and transfected into HEK293T cells. Fluorescence microscopy and Western blot showed that the B. subtilis Sir2 protein was stably expressed in HEK293T cells. Then, the enzyme activities of ATP, PFK-1, PK and LDH in the aerobic oxidation of glucose were detected by using the relevant kits. The facts show that BS-sir2 increases the glucose metabolism and ATP content of cells; in addition, BS-sir2 The oxygen consumption and lactate levels of the transfected cells were detected, and it was found that the oxygen consumption of the cells increased and the level of lactate decreased. These results indicate that BS-sir2 transfection of HEK293T cells increases cellular aerobic oxidation and attenuates cellular anaerobic respiration.

Claims

Bacillus subtilis BS16 with cephalosporin resistance and high expression of Sir2 protein, which is preserved in the Guangdong Provincial Collection of Microorganisms and Cultures, with the deposit number GDMCC No. 60193, and the deposit address is No. 100, Xianlie Middle Road, Guangzhou The 5th floor of Building 59, the date of the deposit is June 2, 2017.
The use of the cephalosporin-resistant and high-expressing Sir2 protein-producing Bacillus subtilis BS16 according to claim 1 for the preparation of a medicament for treating anti-aging, mitochondrial dysfunction or metabolic syndrome.
An anti-aging food comprising an effective amount of the cephalosporin-resistant Bulillus subtilis BS16 having a cephalosporin resistance and high expression of a Sir2 protein and/or a bacterial protein component thereof as an active ingredient.
An anti-aging medicine comprising: an effective amount of the cephalosporin-resistant and high-expressing Sir2 protein-producing Bacillus subtilis BS16 and/or its bacterial protein component, and pharmaceutics, as an active ingredient An acceptable carrier.
A food for preventing and treating mitochondrial dysfunction of a cell, comprising an effective amount of the Bacillus subtilis BS16 and/or its bacterium having cephalosporin resistance and high expression of Sir2 protein according to claim 1 as an active ingredient Body protein component.
A medicament for preventing and treating mitochondrial dysfunction of a cell, comprising an effective amount of the cephalosporin-resistant Bulillus subtilis BS16 having a cephalosporin resistance and high expression of Sir2 protein as claimed in claim 1, and/or a bacterium thereof Body protein component, and a pharmaceutically acceptable carrier.
A food for preventing and treating metabolic syndrome, comprising an effective amount of the Bacillus subtilis BS16 having a cephalosporin resistance and high expression of a Sir2 protein according to claim 1 and/or a bacterial cell thereof as an active ingredient Protein composition.
A medicament for preventing and treating metabolic syndrome, comprising an effective amount of the cephalosporin-resistant Bulillus subtilis BS16 having a cephalosporin resistance and high expression of a Sir2 protein as claimed in claim 1, and/or a bacterial cell thereof a protein component, and a pharmaceutically acceptable carrier.