CN116178509A - Preparation method and application of bifidobacterium surface protein - Google Patents
Preparation method and application of bifidobacterium surface protein Download PDFInfo
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- CN116178509A CN116178509A CN202210953264.3A CN202210953264A CN116178509A CN 116178509 A CN116178509 A CN 116178509A CN 202210953264 A CN202210953264 A CN 202210953264A CN 116178509 A CN116178509 A CN 116178509A
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Abstract
The invention discloses a preparation method and application of bifidobacterium surface protein, and belongs to the technical field of biology. In order to provide a bifidobacterium surface protein. The invention provides a preparation method of bifidobacterium surface protein, which is characterized in that bifidobacterium bifidum is cultured by taking 2' -FL as a unique carbon source, then thalli are collected, liCl solution is added for incubation, supernatant is centrifugally collected, then the supernatant is filtered by a hydrophilic microporous filter membrane to obtain filtrate, the filtrate is put into a dialysis bag for dialysis, and liquid after dialysis is collected and freeze-dried to obtain the bifidobacterium surface protein. The bifidobacterium surface protein helps to inhibit the growth and adhesion of pathogenic bacteria, inhibit the activation of inflammation-related pathways and reduce inflammatory reactions.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a preparation method and application of bifidobacterium surface protein.
Background
In recent years, researches of world health organization find that the incidence rate of intestinal inflammation such as infantile diarrhea is increased year by year due to the fact that the intestinal tract is infected by bacteria, viruses or parasites due to insufficient breast feeding, the related risk factors such as antibiotics used by infants and antibiotics used by mothers during breast feeding are commonly existed. Currently, more than about 50 tens of thousands of children die annually from diarrhea, and have developed into the second leading cause of death in children under 5 years of age, with recurrent or persistent diarrhea having a severe long-term impact on children's growth, nutrition and cognition, in addition to causing high mortality. The study by Bhutta et al found that early pure breast feeding was the most important and cost-effective intervention to reduce mortality in newborns and infants. Since the intestinal barrier is severely damaged due to the continuous deterioration of the environment in the intestine, research on improving the integrity and firmness of the intestinal barrier is of great importance in relieving diarrhea. As with lactobacillus, bifidobacteria surfaces have been found to help inhibit pathogenic bacteria growth and adhesion, inhibit activation of inflammation-related pathways, and reduce inflammatory responses.
Disclosure of Invention
The invention aims to provide a bifidobacterium surface protein.
The invention provides a preparation method of bifidobacterium surface protein, which is characterized in that bifidobacterium bifidum is cultured by taking 2' -FL as a unique carbon source, then thalli are collected, liCl solution is added for incubation, supernatant is centrifugally collected, then the supernatant is filtered by a hydrophilic microporous filter membrane to obtain filtrate, the filtrate is put into a dialysis bag for dialysis, and liquid after dialysis is collected and freeze-dried to obtain the bifidobacterium surface protein.
Further defined, the 2' -FL is added in an amount of 2% (wt/vol); the inoculum size of bifidobacterium bifidum was 3% (v/v).
Further defined, the culture conditions are 37 ℃ to a log phase of 18 hours; the condition for collecting the supernatant by centrifugation is 8000r/min,4 ℃ and 10min.
Further defined, the concentration of LiCl solution is 5mol/L and the addition amount is 0.05% (v/v); the conditions for incubation with LiCl solution were 200r/min,37℃for 30min.
Further defined, the pore size of the hydrophilic microporous filter membrane is 0.22 μm; the size of the dialysis bag is 12000kD.
The invention provides the surface protein of the bifidobacterium obtained by the preparation method.
The invention provides application of the bifidobacterium surface protein in preparing a medicament for treating or assisting in treating intestinal barrier inflammation.
Further defined, the treatment of intestinal barrier inflammation is to promote proliferation of intestinal epithelial cells, prevent expression and localized destruction of TJ proteins, inhibit secretion of pro-inflammatory cytokines, increase secretion of anti-inflammatory cytokines, and increase expression of the zonula compacta ZO-1, claudin-1, and Occludin.
The invention provides application of the bifidobacterium surface protein in preparing health care products or foods for relieving intestinal barrier inflammation.
Further defined, the alleviating intestinal barrier inflammation is promoting proliferation of intestinal epithelial cells, preventing expression and localized destruction of TJ proteins, inhibiting secretion of pro-inflammatory cytokines, and increasing secretion of anti-inflammatory cytokines.
The beneficial effects are that: by constructing a LPS-induced Caco-2 cell monolayer barrier injury model, the in vitro repair of intestinal barrier inflammation by bifidobacterium bifidum DNG6 surface proteins cultured and extracted by Lac, GOS and 2' -FL as single carbon sources is explored.
(1) Compared with Glu, lac, GOS and FOS, the bifidobacterium bifidum DNG6 has slower growth by using 2' -FL as a carbon source, but has stronger acid production capacity; 2' -FL is used as a carbon source for culture, so that the adhesion of thalli to Caco-2 cells is obviously promoted; chemical and enzymatic treatment results indicate that the surface protein is the most dominant adhesion medium in the adhesion process of bifidobacterium bifidum DNG 6.
(2) Compared with Lac and GOS, the surface protein of bifidobacterium bifidum DNG6 grown and extracted by using 2' -FL as a carbon source can remarkably enhance the activity of intestinal epithelial cells, reduce the LDH activity, and reduce intestinal inflammation by inhibiting the secretion of pro-inflammatory cytokines (TNF-alpha, IL-6 and IL-1 beta), and increasing the secretion of anti-inflammatory cytokines IL-10.
(3) The bifidobacterium bifidum DNG6 surface protein enhances the expression of the tight junction proteins (ZO-1, claudin-1 and Occludin) at the gene and protein level caused by LPS, and exhibits dose dependency, while significantly improving the continuity and integrity of the distribution of the tight junction proteins between cells.
Drawings
FIG. 1 shows the OD values of bifidobacterium bifidum DNG6 grown in different carbon sources, notes: data are expressed as mean ± standard deviation.
Fig. 2 shows the pH values of bifidobacterium bifidum DNG6 grown under different carbon sources, notes: data are expressed as mean ± standard deviation.
FIG. 3 is the effect of different treatments on the adhesion of bifidobacterium bifidum DNG6, notes: the different letters above the bar graph represent significance between the different treatments (P < 0.05).
Fig. 4 shows the surface structure of bifidobacterium bifidum DNG6 (x 6000) observed by transmission electron microscopy, and is annotated: a is the surface structure of bifidobacterium bifidum DNG6 before LiCl treatment; b shows the surface structure of bifidobacterium bifidum DNG6 after LiCl treatment.
FIG. 5 is a SDS-PAGE diagram of bifidobacterium bifidum DNG6 surface proteins, A being unpurified surface proteins; b is surface protein purified by a 1mol/L LiCl salting-out method.
FIG. 6 hydrophobicity and self-aggregation of Bifidobacterium bifidum DNG6 at different carbon sources. FIG. 6A shows the self-aggregation of B.bifidum DNG6 grown in different carbon sources; FIG. 6B shows the hydrophobicity of B.bifidum DNG6 at different carbon sources.
Fig. 7 shows the adhesion rate of bifidobacterium bifidum DNG6 at different carbon sources.
FIG. 8 shows TEER values of Caco-2 cell monolayer models over time.
FIG. 9 is the effect of LPS on TEER at various concentrations, notes: * The significance (P < 0.05) between different concentrations of LPS at the same time and control group is indicated.
FIG. 10 is the effect of LPS concentration on cell activity, notes: * Represents the significance between 100 μg/mL of LPS and control group (P < 0.05); * Represents the significance between 200 μg/mL,500 μg/mL LPS and control group (P < 0.001).
FIG. 11 is the effect of surface proteins on LPS-induced LDH activity of Caco-2 cell barrier, notes: panels A, B, C were assayed for Caco-2 cell monolayer supernatant LDH activity by adding 200, 400, 600, 800 and 1000. Mu.g/mL of Sp-L, sp-G and Sp-F, respectively, and incubating with 100. Mu.g/mL of LPS for 24 h.
FIG. 12 is the effect of surface proteins on LPS-induced Caco-2 cell viability, notes: panels A, B, C show cell activity of Sp-L, sp-G and Sp-F added at 200, 400, 600, 800 and 1000. Mu.g/mL incubated with 100. Mu.g/mL LPS for 24h, respectively.
FIG. 13 shows the effect of surface proteins on cytokine levels in a monolayer barrier, (A) TNF- α levels; (B) is the content of IL-6; (C) is the IL-1 beta content; (D) is the IL-10 content; and (3) injection: ELISA method measures the level of cytokines in supernatants of different treatment groups. Wherein the LPS concentration is 100 mug/mL, the Sp-L concentration, the Sp-G concentration and the Sp-F concentration are 800 mug/mL, and the incubation is carried out for 24 hours. The different letters above the bar graph represent significance between the different treatments (P < 0.05).
FIG. 14 shows mRNA expression of ZO-1, claudin-1 and Occludin.
FIG. 15 shows the expression of ZO-1, claudin-1 and Occludin proteins, A shows the expression of 3 tight junctions proteins; b, C, D are shown by the gray scale ratio of 3 kinds of tight junction proteins to internal reference beta-actin.
FIG. 16 shows immunofluorescence observation of tight junction proteins, annotated: occludin protein and Claudin-1 protein are labeled with green fluorescence, and ZO-1 is labeled with red fluorescence. FITC staining excitation showed green light and CY3 staining excitation red light.
Detailed Description
Bifidobacterium bifidum DNG6 is described in G Zhang, J Zhao, R Wen, et al 2' -Fucosyllactose promotes Bifidobacterium bifidum DNG addition to Caco-2cells [ J ]. Journal of Dairy Science,2020,103 (11).
Caco-2cells are human colon cancer epithelial cells purchased from Shanghai cell banks.
TABLE 1 reagents
| Reagent name | Manufacturing factories |
| MRS culture medium | BEIJING AOBOXING BIOLOGICAL TECHNOLOGY Co.,Ltd. |
| Cysteine hydrochloride | Beijing Soilebao Biotech Co.Ltd |
| Glucose (Glu) | Tianjin chemical reagent Co.Ltd |
| Lactose (Lac) | Tianjin chemical reagent Co.Ltd |
| Fructooligosaccharides (FOS) | SHANDONG LONGLIVE BIO-TECHNOLOGY Co.,Ltd. |
| Galacto-oligosaccharide (GOS) | SHANDONG LONGLIVE BIO-TECHNOLOGY Co.,Ltd. |
| 2'-fucosyllactose (2' -FL) | Shanghai auspicious tillage technology development Co.Ltd |
| DMEM culture solution | Gibco Co Ltd |
| Fetal bovine serum | Canadian Wisent Co Ltd |
| pancreatin-EDTA | Gibco Co Ltd |
| Double antibody | Gibco Co Ltd |
| LPS | Sigma Co., USA |
| Human ELISA kit | Shenzhen Xinbo biosciences and technologies company |
| LDH kit | Nanjing built institute of bioengineering |
| CCK-8 kit | Beijing Solarbio Biotechnology Co., ltd |
| Alkaline phosphatase kit | Nanjing built institute of |
| 4% paraformaldehyde universal tissue fixing solution | Shanghai Biosharp Biotechnology Co.Ltd |
| Fluorescein isothiocyanate | Wohan Sieve Co Ltd |
| CY3 sheep anti-rabbit staining agent | Wohan Sieve Co Ltd |
| RIPA lysate | Beijing Biyun biotechnology Co Ltd |
| BCA kit | Beijing Biyun biotechnology Co Ltd |
| ECL luminous kit | Millipore Co., USA |
| Rabbit anti-human ZO-1, claudin-1 antibodies | Affinity Co Ltd |
| Rabbit anti-human Occludin-1 antibodies | Abclonal Co., USA |
| Goat anti-rabbit secondary antibody (HRP) | Utibody Co., ltd |
| β-actin | Abcam Co |
| Low molecular weight protein Maker (14.4-97.4 kDa) | BEIJING SOLARBIO TECHNOLOGY Co.,Ltd. |
| 12% concentrated glue | BEIJING SOLARBIO TECHNOLOGY Co.,Ltd. |
| 5% separating gel | BEIJING SOLARBIO TECHNOLOGY Co.,Ltd. |
| 4 Xprotein loading buffer | BEIJING SOLARBIO TECHNOLOGY Co.,Ltd. |
| Coomassie brilliant blue | Dalian Mei Lun Biotechnology Co., ltd |
| Total RNA extraction kit | Japanese Takara Co |
| cDNA synthesis kit | Japanese Takara Co |
| Taq SYBRGreen qPCR Premix | Dalianbao bioengineering Co.Ltd |
| Lithium chloride | Dalian Mei Lun Biotechnology Co., ltd |
| Other chemical reagents | Domestic analytical grade |
Example 1.
Caco-2 cell activation and culture: fast slave liquid N 2 Taking out the cell freezing tube from the pot, rapidly thawing in a water bath at 37deg.C, transferring to cell space, sucking cell suspension in the freezing tube from light to centrifuge tube with DMEM, centrifuging (1000 r/min,5 min) to obtain cell precipitate, adding 4mL of DMEM culture solution containing 10% bovine serum, blowing uniformly, sucking into 25T cell culture bottle, and placing in cell culture box (5% CO) 2 Culturing at 37 ℃ overnight, discarding the culture solution after the cells adhere to 80-90%, washing 3 times by using preheated PBS, adding 1mL of trypsin for digestion for 3min, immediately adding 1mL of DMEM complete culture solution for stopping digestion, sucking the cell suspension into a 15mL centrifuge tube for centrifugation (1000 r/min,5 min) to obtain a precipitate, and continuously adding the cell culture solution into a cell culture solution transfer bottle or a seed plate for culturing according to test requirements. Activating and culturing bifidobacterium bifidum: thawing-20deg.C bifidobacterium bifidum DNG6 glycerol, inoculating 3% into sterilized and ultraviolet-irradiated TPY culture medium for activation (MRS culture medium + -0.05% cysteine hydrochloride), culturing in 37 deg.C constant temperature incubator to logarithmic phase, streaking plate, picking single colony, and continuously passaging for 3 times to obtain engineering bacteria for subsequent test.
2. Bifidobacterium bifidum growth characteristics
(1) Growth curve determination
Bifidobacterium bifidum DNG6 was inoculated to a medium of glucose (Glu), lactose (Lac), galactooligosaccharide (GOS), fructooligosaccharide (FOS) and 2-fucosyllactose (2' -FL) as sole carbon sources, respectively, and then placed in an anaerobic incubator for culturing at 37 ℃ for 36 hours. OD values at 600nm were measured, sampled every 2 hours for detection, and growth curves were prepared.
Results: the growth rate of bifidobacterium bifidum DNG6 was different from each other in terms of the availability of different carbon sources (Glu, lac, GOS, FOS and 2' -FL), and thus the growth of bifidobacterium bifidum DNG6 in TPY medium was first measured in this experiment. As can be seen from the growth curve of fig. 1, there was a significant difference in the growth of the strain in the medium of 5 carbon sources as a whole.
Firstly, the strain grows faster in a culture medium taking Lac and Glu as carbon sources, the hysteresis period is only 4 hours, and the strain reaches the stationary phase rapidly with higher bacterial count, wherein in the culture medium taking Lac as the carbon sources, the strain enters the stationary phase after 12 hours, the OD value is maintained at 1.88, in the culture medium taking Glu as the carbon sources, the strain enters the stationary phase after 14 hours, and the OD value is maintained at 1.72; secondly, compared with Lac and Glu, the strain grows at a slightly slower rate in a culture medium with GOS as a carbon source, the lag phase is 6h, the strain enters a stationary phase after 20h, and the OD value is maintained at 1.58; thirdly, the strain grows at the slowest rate in a culture medium taking FOS as a carbon source, the lag phase is 20h, the strain enters a stabilization phase after 24h, the OD value is maintained at 0.41, and the strain shows lower utilization rate; finally, the strain grew relatively slowly in the medium with 2' -FL as carbon source compared to Lac, glu and GOS, but significantly higher than the FOS utilization, with a lag phase of 12h, and after 26h entered the stationary phase, with an OD value maintained at 1.43.
(2) Determination of pH value
Bifidobacterium bifidum DNG6 was inoculated to a medium of glucose, lactose oligosaccharide, fructo-oligosaccharide and 2' -FL as sole carbon sources, respectively, and then placed in an anaerobic incubator for cultivation at 37 ℃ for 36 hours. The acidity value was measured using a pH meter, sampled and detected every 2 hours, and a pH curve was prepared.
Results: the growth pH of bifidobacterium bifidum DNG6 in TPY medium was measured in this test, as there was a difference in the acid production capacity of bifidobacterium bifidum DNG6 using different carbon sources (Glu, lac, GOS, FOS and 2' -FL).
As can be seen from fig. 2, bifidobacterium bifidum DNG6 has a significant difference in growth pH in the medium of 5 carbon sources as a whole. Wherein, the strain can ferment rapidly to produce acid by utilizing Lac and Glu as 2 carbon sources, and the pH value is maintained at 4.30 and 4.42 respectively when the strain reaches the stable period; in addition, when GOS is used as a carbon source, the strain slowly produces acid in a slow period, the acid production capacity rises along with the prolonged culture time, and the pH value reaches a stable period and is maintained at 4.77; the strain uses FOS as a carbon source, the pH value is maintained at 6.01 when the strain reaches a stable period, and the acid production degree is low. Finally, the strain was maintained at pH 4.56 at the time of reaching stationary phase using 2' -FL as a carbon source.
By combining with growth curve analysis, bifidobacterium bifidum DNG6 can rapidly proliferate to reach higher biomass and produce a large amount of acid by using Glu and Lac which are 2 carbon sources; the proliferation and acid production are slower by using GOS as a carbon source; it was also found that strain utilization of FOS was low in both growth and acidogenesis. In addition, the strain grows more slowly by using 2' -FL as a carbon source, and the acid production capacity is slowly improved with time, probably because bifidobacterium bifidum DNG6 uses a special oligosaccharide structure of 2' -FL, and more polysaccharide glycoside hydrolase and oligosaccharide transporter are needed, but the strain can decompose the trisaccharide of 2' -FL to produce a large amount of acid with the prolonged culture time. Therefore, since the research on the adhesion characteristics of the strain is required to be established on the basis that the thalli utilizes a carbon source and reaches a certain biomass, the subsequent experiment selects 4 carbon sources of Glu, lac, GOS and 2' -FL to research the adhesion characteristics of bifidobacterium bifidum DNG 6.
3. Determination of bifidobacterium bifidum adhesion under different treatments
(1) LiCl treatment
The LiCl treatment can remove surface proteins bound to the surface of the bacterial cells by non-covalent bonds. After the bifidobacterium bifidum DNG6 strain to be tested is cultured to the logarithmic phase, the bifidobacterium bifidum DNG6 strain to be tested is washed 3 times by PBS and centrifuged (8000 r,10 min) at 4 ℃ to obtain bacterial sludge, 0.05% (v/v) of 5mol/L LiCl solution is added, and the bacterial sludge is placed on a shaking table at 37 ℃ and is subjected to shaking treatment at 200r/min for 30min.
(2)NaIO 4 Treatment of
NaIO 4 Can destroy carbohydrate components on the surface of the bacteria. Culturing the bifidobacterium bifidum DNG6 strain to be tested to a logarithmic phase, and adding 0.05mol/L NaIO of 0.05% (v/v) 4 The solution was placed on a shaking table at 37℃and 200r/min and shaken for 30min.
(3) Pepsin and trypsin treatment
Pepsin and trypsin are both capable of destroying surface proteins of bacteria. Pepsin was dissolved in 0.05mol/L Gly-HCl buffer at ph=2.2, trypsin was dissolved in 0.2mol/mL PBS at pH 8.0, both of which were formulated as solutions with a concentration of 400U/mL, and bifidobacterium bifidum DNG6 puree was suspended on a shaking table at 37 ℃, and shaken at 200 r/min.
(4) Phenol treatment
Phenol treatment was used to destroy cell surface teichoic acid and non-fibrous proteins. After preparing 0.8mol/L phenol solution, the bifidobacterium bifidum DNG6 bacterial sludge is suspended in 0.05% (v/v) phenol solution, and is subjected to shaking treatment for 30min by a 65 table.
The bacterial sludge treated differently is centrifuged and resuspended in PBS solution, and the concentration of the bacterial liquid is adjusted to 10 8 CFU/mL, carrying out adhesion test after fluorescent marking, and obtaining the relative adhesion rate.
Results: since the surface properties of the bifidobacterium bifidum DNG6 under different carbon sources have no obvious difference, and the adhesion rate of the bifidobacterium bifidum DNG6 cultured by taking 2' -FL as the carbon source has obvious difference compared with other carbon sources, in order to explore the components of the bifidobacterium bifidum DNG6 playing a main role in adhesion, the experiment adopts different chemistry and enzyme treatment to analyze the contribution of the components on the surface of bacteria to the adhesion process of strains and Caco-2 cells.
As shown in FIG. 3, the bifidobacterium bifidum DNG6 has different sensitivities to lithium chloride, protease, sodium periodate and phenol, and different treatments have obvious inhibition effect on the adhesion capacity of the strain (P<0.05 Wherein, after LiCl treatment, the strain adhesion rate is reduced by 63.31+/-4.22%; after pepsin and trypsin treatment, the adhesion rate of the strain is reduced by 53.36 +/-2.83% and 54.93+/-3.23%, respectively, and the adhesion rate of the strain are not significantly different (P)>0.05 A) is provided; after phenol treatment, the adhesion rate of the strain is reduced by 44.58 +/-2.31%; through NaIO 4 The treatment is reduced by 26.42+/-1.83 percent. The results show that the carbohydrate, teichoic acid and other components on the surface of the bifidobacterium bifidum DNG6 are all involved in the adhesion, wherein the surface protein is stripped by LiCl treatment, so that the cell adhesion rate is reduced most remarkably, and the surface protein is the most main adhesion component in the bifidobacterium bifidum DNG6 adhesion process.
4. And (3) observing the surface structure of bifidobacterium bifidum by a transmission electron microscope: and (3) centrifugally collecting thalli, respectively re-suspending the thalli in a PBS solution (control group) and a 5mol/L LiCl solution (treatment group), after shaking table incubation for 30min, re-suspending the thalli in a 3% glutaraldehyde solution for 2h, washing the thalli for 3 times by the PBS solution, dyeing the thalli for 1h by using 1% phosphotungstic acid, and performing dehydration treatment by using ethanol and observing the thalli under a transmission electron microscope.
Results: to verify the surface protein composition of b.bifidum DNG6, the test used 5mol/L LiCl to treat the strain and observe the change in surface structure before and after treatment. As a result, as shown in FIG. 4, it was observed under a transmission electron microscope that the cell wall of the strain which had not been treated with LiCl was wrapped with a thicker surface villus structure, whereas the villus structure disappeared after LiCl peeling treatment, suggesting that the surface of the B.bifidum DNG6 strain contained abundant surface proteins.
Example 2 extraction and determination of Bifidobacterium bifidum surface proteins
1. (1) Extraction of bifidobacterium surface proteins
3% (v/v) bifidobacterium bifidum DNG6 was inoculated in TPY medium, 2% (wt/vol) filter sterilized lactose, 2% filter sterilized GOS and 2% filter sterilized 2' -FL were added as the only carbon sources, after incubation at 37℃for 18 hours in log phase, bacterial sludge was collected by centrifugation (8000 r/min,4℃for 10 min), washed 3 times with PBS, added with 0.05% (v/v) 5mol/L LiCl solution, and incubated on a shaker (200 r/min,37℃for 30 min). Centrifuging and collecting supernatant (8000 r/min,4 ℃ C., 10 min) to obtain crude extract of surface protein. The crude extract is firstly passed through a sterile hydrophilic microporous filter membrane with the diameter of 0.22 mu m, the filtrate is put into a 12000kD dialysis bag, the crude extract is placed into deionized water for dialysis at the temperature of 4 ℃ and the deionized water is replaced for a plurality of times, and 10g/L AgNO is adopted 3 The solution was checked for white precipitate. Collecting the dialyzed liquid, pre-freezing at-20deg.C for 2 hr, lyophilizing to obtain crude extract of surface protein, and storing in refrigerator at-80deg.C. Marking surface proteins of bifidobacterium bifidum DNG6 extracted by taking lactose as a carbon source by using Sp-L, marking the bifidobacterium bifidum DNG6 extracted by taking GOS as the carbon source by using Sp-G, and marking the surface proteins of the bifidobacterium bifidum DNG6 extracted by taking 2' -FL as the carbon source by using Sp-F.
(2) Purification of surface proteins
Purifying surface protein by salting-out method, taking appropriate amount of the above Sp-L, sp-G and Sp-F crude extracts in a centrifuge tube, adding LiCl (1 mol/L,10 mL), placing the centrifuge tube in ice water bath, stirring for 15min, and centrifuging (12000 r/min,4 deg.C, 20 min). Washing the precipitate with sterilized PBS for 2 times, centrifuging again, collecting the precipitate to obtain purified surface protein, and storing the purified surface protein in an ultralow temperature refrigerator at-80deg.C for use.
(3) SDS-PAGE identification of surface protein molecular weight
SDS-PAGE is adopted to determine the surface protein and the relative molecular mass, and 12% of separation gel and 5% of concentration gel are filled. Dissolving the crude extract of surface protein in 4 x protein loading buffer solution, boiling in boiling water bath for 3min, adjusting the loading amount, adding protein into a Maker (14.4-97.4 kDa), regulating the electrophoresis voltage of the concentrated gel to 80V, and regulating the electrophoresis voltage of the separating gel to 120V. After electrophoresis, the bands were stained with coomassie brilliant blue and decolorized with acetic acid, and the relative molecular masses of the bands were observed and calculated by photographing.
(4) BCA method for measuring surface protein concentration
The protein content in the test is determined by the Coomassie Brilliant blue G-250 method, the bovine serum albumin standard is determined according to the BCA kit, a standard curve is drawn, 200 mug/mL of surface protein is weighed and added into a sample hole, the absorbance of each sample is determined at 595nm, and the protein content of each surface is determined according to the standard curve.
Results: a5 mol/L LiCl solution is typically used to extract proteins that are non-covalently bound to the bacterial surface. FIG. 5 shows SDS-PAGE analysis of the bands of crude surface proteins, and it can be seen that the bands of surface proteins were reduced relative to those before purification after purification by 1mol/L LiCl salting-out, and that proteins were largely enriched at a molecular weight of about 41kDa, the major proteins extracted in this experiment. This is similar to the reported molecular weight of the Lactobacillus acidophilus surface protein of 41-49 kDa. Similarly, the test result is similar to the result of Zhao et al, namely the molecular weight of the surface protein of the bifidobacterium H3-R2 is 43kDa, which shows that the LiCl extraction method adopted in the study can effectively extract the surface protein of the B.bifidum DNG 6.
2. Surface Properties of bifidobacterium bifidum
(1) Self-aggregation
Centrifuging the suspension of Bifidobacterium bifidum DNG6 to obtain precipitate (4deg.C, 5000r,10 min), washing with sterilized PBS for 2 times, re-suspending in PBS solution to make the absorbance range of all the suspension at 600nm of 0.5+ -0.02, and recording absorbance as A 0 . Vortex 2mL of the cell suspension for 10s, stand at 37 ℃ for 2h, and take the supernatantThe absorbance value of the liquid detected at 600nm is A 1 The self-agglutination ability formula is as follows:
self-aggregation rate (%) = [ (a) 0 -A 1 )/A 0 ]×100
(2) Hydrophobicity of
Adopting PBS to adjust the absorbance of the bacteria liquid to be detected to be OD 600 =0.5±0.02 and the absorbance was recorded as a 0 . Taking bacterial liquid to be detected in a test tube, adding 1/3 of the volume of the solution into the test tube, carrying out vortex oscillation for 3min, standing for 20min at room temperature, sucking the lower water phase, and measuring the absorbance at 600nm to be A t The hydrophobicity formula is as follows:
hydrophobicity (%) = [ (a) 0 -A t )/A 0 ]×100
Results: the surface properties of the bacterial strain can indirectly reflect the adhesion characteristics of the bacterial strain, the hydrophobicity and self-agglomeration are key indexes for measuring the nonspecific adhesion of the bacterial strain, and the values of the hydrophobic and self-agglomeration are greatly related to factors such as the surface charge of the bacterial strain, the content of hydrophobic amino acid and the like. Self-aggregation is considered to be the first step in the cell adhesion process, enabling it to form a physical barrier, enhancing resistance to the environment and preventing the attachment of undesirable bacteria. Surface hydrophobicity is another property that is considered to be important for cell adhesion ability. De Souza et al found that cells with higher hydrophobicity could better bind to epithelial cells, affecting adhesion to some extent. Falah et al found that the hydrophobicity assay could be considered an important pre-test for the ability of probiotics to adhere to epithelial cells, one of the important properties to improve the initial contact of the bacteria with the host cell. The hydrophobicity and self-clotting properties of b.bifidum DNG6 were determined in this test.
FIG. 6A shows the self-aggregation of B.bifidum DNG6 grown in different carbon sources. The self-aggregation rate of the strain in 2' -FL is 33.18+ -0.73%, which is higher than that in GOS, lac and Glu, and is 31.85 + -1.50%, 29.92 + -1.87% and 30.49 + -2.37%, respectively, and the self-aggregation rates of B.bifidum DNG6 in 4 carbon source cultures are not significantly different (P > 0.05) similar to the hydrophobic results.
FIG. 6B shows the hydrophobicity of B.bifidum DNG6 at different carbon sources. The hydrophobicity of the strain in 2' -FL culture was 87.30+ -1.90%, slightly higher than that in GOS, lac and Glu, 83.47 + -1.40%, 82.59 + -3.83% and 86.12 + -1.73%, respectively, but there was no significant difference in hydrophobicity between groups (P > 0.05).
3. Determination of bifidobacterium bifidum adhesion under different carbon sources
(1) Preparation of Caco-2 cells
Method for adhesion test Wang Liqun of bifidobacterium bifidum DNG6 to Caco-2 cells. Caco-2 cells were cultured at 10 5 The concentration of each/well was inoculated in a 12-well Transwell, with medium changes every other day for the first 7 days and every other day for the next 14 days. After the cell attachment is complete, the test can be performed after culturing for 21 days to the polarized state.
(2) Preparation of Bifidobacterium bifidum suspension
After the bifidobacterium bifidum DNG6 is washed for 2 times by sterile PBS, the collected thalli are centrifuged, resuspended in complete DMEM culture solution and the concentration of the bacterial suspension is adjusted to 10 8 CFU/mL。
(3) Fluorescent labeling of bifidobacterium bifidum:
5.0mmg of carboxyfluorescein succinimidyl ester was weighed and dissolved in 8.969ml of DMSO to prepare a fluorescent labeling solution, which was stored at-20℃for further use. 1mmol/L fluorescence labeling solution cFDA-SE stock solution is added into the bifidobacterium bifidum DNG6 suspension to prepare 20 mu mol/L concentration, the mixture is subjected to light-shielding standing treatment at 37 ℃ for 20min and then centrifuged (4 ℃ for 5000r and 10 min), and the mixture is washed for 3 times by PBS to remove redundant fluorescent dye and resuspended in PBS solution. The fluorescence spectrophotometer detects the marking rate of the bifidobacterium bifidum DNG6 as the fluorescence intensity A before adhesion 1 Wavelength: EX is 492nm, EM is 517nm, and slit width is 2.5nm.
(4) Bifidobacterium bifidum adherent cells
Adhesion (%) = [ At/A1] ×100
The size of the cell adhesion rate directly influences the colonization degree in the intestinal tract. The adhesion rate of the whole B.bifidum DNG6 was measured in this test.
As shown in FIG. 7, the adhesion rate of B.bifidum DNG6 grown in different carbon sources to Caco-2 cells was between 7.43% and 9.15%. The adhesion rates of bifidum DNG6 in Glu, lac and GOS were 7.43±0.56%,7.58±0.39% and 7.89±0.27%, respectively, with no significant difference (P > 0.05). The adhesion rate in 2' -FL was 9.15±0.64%, significantly higher than that in other carbon sources (P < 0.05). The results indicate that 2' -FL can effectively promote the adhesion of B.bifidum DNG6 to Caco-2 cells.
As a result, b.bifidum DNG6 has no significant difference in hydrophobicity and self-coagulation property in different carbon sources, but the 2' -FL as a carbon source increases its adhesion rate. This is probably due to the fact that 2' -FL increases expression of cell surface adhesion component, thereby promoting adhesion of B.bifidum DNG6 to Caco-2 cells, according to Zhang et al.
EXAMPLE 3 construction of Caco-2 cell monolayer Barrier
1. (1) Construction of a Single layer Barrier model
Caco-2 cells differentiate into polarized monolayers that are morphologically and physiologically similar to colonic epithelium. The cell line is in the classical intestinal epithelial barrier model. The test uses a 12-well Transwell (bottom area 1.12 cm) 2 ) Suspension type culture dish is cultivated Caco-2 cell and is built a model, and the cell inoculation density is adjusted to be 2 multiplied by 10 5 mu.L of each well was inoculated into the bottom of a top side (AP) cell of the cell, and 1.5mL of complete medium DMEM was added to the bottom side (BL). The culture medium was changed every day for 7 days, and every other day for 14 days thereafter, for a total of 21 days. To evenly distribute the cells on the polycarbonate membrane using the splay shaking method, and observe under an inverted microscope.
(2) Determination of transepithelial resistance
TEER (Transepithelial Electrical Resistance) values were measured using a Millicell ERS ohmmeter to reflect the integrity and permeability of the intestinal epithelial barrier. Pre-preparationAfter ultraviolet irradiation and balancing electrodes in PBS, electrodes of a resistor are vertically placed in holes containing cross-hole inserts at 90 ℃, long ends are inserted into BL sides, short ends are inserted into AP sides, and films are prevented from contacting the electrodes until the resistance value in the meter is stable in reading. 3 wells around the cells were repeatedly measured, and stable results were recorded 3 times in succession, the average value of 3 wells was used as the resistance value measured in the cell, and finally the measured cell resistance value was recorded to draw a line graph. The measurements were taken every two days until the resistance reached a maximum and plateau, indicating that a tight and complete permeation barrier was formed. And when the resistance value of the intestinal epithelium tends to be stabilized at 350-400 omega cm 2 About, the cell connection under the light microscope is complete and compact, which means that the modeling is successful, and the monolayer barrier can be used for subsequent experiments.
The TEER calculation formula is:
teer= (TEER-blank TEER measured) ×1.12cm 2
Results: EER values are formed by ions crossing cell monolayer barriers, reflecting the integrity and permeability of the monolayer barrier. The experiment uses a voltmeter to measure Caco-2 cell monolayer resistance for 21 consecutive days. As shown in FIG. 8, TEER increases continuously with increasing culture time, and the TEER value increases slowly in the first 5 days, increases sharply in days 5 to 19, and reaches the maximum average value of 425 Ω.cm at day 21 2 When TEER value is significantly increased and is higher than 350Ω·cm 2 And in the process, the in-vitro model of the intestinal canal monolayer barrier is successfully constructed.
(3) Determination of alkaline phosphatase
Alkaline phosphatase (Alkaline Phosphatase Assay, AKP) is an enzyme secreted by differentiated mature intestinal cells, and the culture solutions on the AP side and the BL side were collected and assayed for AKP activity at 7 days, 14 days and 21 days, respectively, in a monolayer barrier culture, and absorbance at 405nm was read. And drawing an AKP standard curve according to an AKP kit specification, drawing an AKP activity ratio curve in the AP side/BL side culture solution, testing 3 compound wells for each sample, and repeating the test for 3 times. When the AKP ratio of the AP side to the BL side reaches 4.0-6.0, the marker cell monolayer is completely differentiated, and the barrier model is successfully established.
Results: alkaline phosphatase (AKP) expression and secretion are considered to be a good marker for crypt cell differentiation. The test respectively determines the AKP activity ratio of the AP side and the BL side in the Transwell cell, and when the AKP ratio of the AP side/BL side reaches 3.5-5.0, the marker cell monolayer is completely differentiated, and the barrier model is successfully established.
The results are shown in table 2, with increasing incubation time (7 d,14d and 21 d), significantly increasing (P < 0.05), indicating a sharp increase in polarity of the monolayer barrier. The AP/BP ratio reaches 4.51+/-0.47 on the 21 st day, and is obviously increased by 1.29 times compared with the 7 th day, and the AP/BP ratio shows obvious polarity, which is consistent with the rising trend of TEER value, and proves that the establishment of the Caco-2 cell monolayer barrier model is successful.
TABLE 2 AKP Activity in Caco-2 cell monolayer model
Note that: different letters represent the significance of each column (P < 0.05).
Lipopolysaccharide induced monolayer barrier inflammatory injury
2. The TEER value and CCK-8 method are adopted to screen the proper LPS concentration for establishing the intestinal barrier dysfunction model.
(1) Measurement of Single layer Barrier resistance values after LPS treatment at different concentrations
Caco-2 cells were cultured in a 12-well Transwell until differentiation and maturation. Experiments were performed by observing the effect of different concentrations of LPS (1. Mu.g/mL, 10. Mu.g/mL, 100. Mu.g/mL, 200. Mu.g/mL and 500. Mu.g/mL) on TEER values of in vitro intestinal barrier models at various time points of 0h,6h,12h,24h and 48 h.
(2) Determination of cell Activity after different concentrations of LPS treatment
The effect of different concentrations of LPS (100. Mu.g/mL, 200. Mu.g/mL and 500. Mu.g/mL) on cell viability was determined using the CCK-8 method. Will be 3X 10 4 Inoculating cell suspension with density of individual/mL into 96-well plate at volume of 100 μl/well, culturing for 12 hr until the cell suspension adheres to the wall, discarding old culture solution, adding LPS with different concentrations for 24 hr, adding 10 μl of CCK-8 solution, incubating for 2 hr, shaking for 1min under dark condition to make color reaction uniform, and treating at 450nmThe absorbance was measured.
The cell viability was calculated as follows:
(3) Test group
Caco-2 tight junction injury was induced with LPS at a concentration of 200 μg/mL. Caco-2 cells (1X 10) 5 Per mL) was plated in a 12-well Transwell cell culture chamber at 37℃with 5% CO 2 Culturing under conditions to establish a Caco-2 monolayer cell barrier model. The test was divided into 5 groups, each group being treated as follows:
1) A control group; only adding cell culture solution for treatment; bacterial Surface proteins Sp-L (Surface proteins-Lactobacillus), sp-G (Surface proteins-Galactooligosaccharide) and Sp-F (Surface proteins-2 '-Fucosyl) were cultured and extracted with Lac, GOS and 2' -FL together with 3 carbon sources, respectively.
2) LPS treatment group: adding 200 mug/mL LPS for independent treatment;
3) LPS+Sp-L group: 200. Mu.g/mL LPS was added to co-treat with different concentrations of Sp (Lac);
4) LPS+Sp-G group: 200. Mu.g/mL LPS was added to co-treat with different concentrations of Sp (GOS);
5) lps+sp-F group: 200. Mu.g/mL LPS was added to co-treat with different concentrations of Sp (2' -FL).
Sp-L, the concentrations of Sp-G and Sp-F were 200. Mu.g/mL, 400. Mu.g/mL, 600. Mu.g/mL, 800. Mu.g/mL, 1000. Mu.g/mL, and the surface proteins were added after being prepared into a cell culture solution and then being coated (0.22 μm).
Results: to screen the appropriate concentration of LPS for establishing a model of intestinal barrier inflammatory injury, 5 concentrations of LPS were used to stimulate Caco-2 cells at 1. Mu.g/mL, 10. Mu.g/mL, 100. Mu.g/mL, 200. Mu.g/mL and 500. Mu.g/mL, respectively, and TEER values for the in vitro intestinal barrier models were determined at 5 time points over 0h,6h,12h,24h and 48 h.
As shown in fig. 9, the monolayer barrier resistance value showed a significant decrease trend with increasing LPS concentration and treatment time. Wherein, compared with the control group, 1 mug/mL and 10 mug/mL of LPS do not significantly reduce TEER value (P > 0.05) within 0-48 h. In LPS treated groups at concentrations of 100. Mu.g/mL, 200. Mu.g/mL and 500. Mu.g/mL, LPS stimulation was significantly reduced and exhibited a significant time dependence (P < 0.05) for 6 to 48 hours compared to the control group, and the TEER was most significantly reduced by 14.92%,26.76% and 44.12% (P < 0.05) at 24 hours with LPS treatments of 100. Mu.g/mL, 200. Mu.g/mL and 500. Mu.g/mL, respectively.
3. The effect of 100. Mu.g/mL, 200. Mu.g/mL and 500. Mu.g/mL LPS on Caco-2 cell activity was determined using the CCK-8 method. As shown in fig. 10, 100 μg/mL of LPS significantly reduced the cell activity by 28.33% (P < 0.05) compared to the control group; cell activity was significantly reduced by 41.59% and 57.67% (P < 0.001) in the LPS treated groups of 200. Mu.g/mL and 500. Mu.g/mL, respectively. It can be seen that the 3 concentrations of LPS all caused significant disruption of cell activity.
In combination with the measurement result of the single-layer barrier resistance value, 100 mug/mL of LPS not only obviously reduces the single-layer barrier resistance value of Caco-2 cells, but also destroys the cell activity within the allowable addition range of the test, and meets the requirement of LPS for inducing intestinal barrier dysfunction, so the test selects 100 mug/mL of LPS for the establishment of a subsequent inflammation model.
4. Measurement of lactate dehydrogenase Activity: the LDH content in the culture solution is detected by adopting a lactate dehydrogenase (Lactic Dehydrogenase, LDH) detection kit. The differently treated Caco-2 monolayer barrier supernatants were collected and centrifuged (1000 r,4 ℃,5 min) to obtain a centrifuge supernatant. Adding a sample and a standard substance according to the instruction of a kit, sequentially adding a matrix buffer solution, coenzyme I, 2, 4-dinitrophenylhydrazine and 0.4mol/mL NaOH, carrying out water bath treatment, and measuring an OD value at a wavelength of 450 nm.
Calculation formula of LDH activity in cell culture solution:
LDH is a cytoplasmic enzyme that is released into the supernatant when the cell membrane is damaged as an indicator of the extent of damage to the cell. FIG. 11 shows the effect of adding different concentrations of Sp-L, sp-G, sp-F together with 3 surface proteins (200, 400, 600, 800 and 1000. Mu.g/mL) on LDH activity in LPS-induced cell monolayer barrier supernatants.
As shown in fig. 11: the LDH activity in the control group was 50.78.+ -. 5.30 (U/L), and the LDH activity in the LPS group was 189.13.+ -. 4.29 (U/L). Wherein, as shown in fig. 11A: after addition of different concentrations of Sp-L, the LDH activities were 176.06+ -5.83 (U/L), 164.4+ -4.45 (U/L), 149.47 + -9.92 (U/L), 144.35 + -6.61 (U/L) and 128.83 + -4.41 (U/L), respectively. The results show that the inhibition of the level of LDH by Sp-L is dose dependent, wherein 1000 mug/mL of Sp-L has the most remarkable inhibition effect (P < 0.05).
As shown in fig. 11B: LDH activities were 186.07.+ -. 4.27 (U/L), 175.8.+ -. 6.39 (U/L), 157.51.+ -. 7.06 (U/L), 113.52.+ -. 4.51 (U/L) and 108.94.+ -. 6.72, respectively, in sequence after addition of different concentrations of Sp-G. The results show that the inhibition of Sp-G on LDH content has dose dependency, wherein the Sp-G inhibition effect of 800 mug/mL and 1000 mug/mL is optimal, and the difference between the two is not obvious (P > 0.05).
As shown in FIG. 11C, LDH activities were 174.02.+ -. 8.75 (U/L), 169.77.+ -. 6.52 (U/L), 134.76.+ -. 6.74 (U/L), 103.51.+ -. 3.96 (U/L) and 91.32.+ -. 5.43, respectively, in the different concentrations of Sp-F. The results show that Sp-F has dose dependency on the inhibition of LDH content, wherein the Sp-F inhibition effect of 800 mug/mL and 1000 mug/mL is optimal, and the difference between the Sp-F inhibition effect and the Sp-F inhibition effect is not obvious (P is more than 0.05).
Overall, the addition of LPS significantly increased LDH release compared to the control (P < 0.05), whereas the addition of 3 surface proteins Sp-L, sp-G, sp-F was dose dependent for the decrease of LDH activity. And compared with Sp-L and Sp-G, the reduction degree of the release amount of the LDH by adding 800 mug/mL and 1000 mug/mL of Sp-F is most obvious, which shows that 2' -FL can better promote bifidobacterium bifidum DNG6 surface protein to relieve inflammatory barrier injury.
5. Measurement of cell Activity: the effect of surface proteins on cell viability was assessed using the CCK-8 method. Will be 3X 10 4 After 100. Mu.L of cells with a density of one cell/mL were inoculated into a 96-well plate and cultured for 12 hours until the cells were attached, the old culture solution was discarded, and after 24 hours treatment according to 2.2.3 test group treatment, 10. Mu.L of CCK-8 solution was addedIncubation was continued for 2h, shaking table 1min was kept away from light, mixing was performed and absorbance at 450nm was measured.
The cell viability was calculated as follows:
to assess the effect of surface proteins on viability of injured cells, the experiment used different doses (200. Mu.g/mL, 400. Mu.g/mL, 600. Mu.g/mL, 800. Mu.g/mL and 1000. Mu.g/mL) of Sp-L, sp-G and Sp-F to treat Caco-2 cells. As shown in fig. 12: compared with the control group, LPS significantly reduced the cell viability by 47.36% (P < 0.05)
As shown in fig. 12A: the activity of Sp-L added with different concentrations is 52.54% -75.87%, and the dose dependence trend is shown, wherein 1000 mug/mL of Sp-L most remarkably improves the activity of the cells, and the activity is 75.87% (P < 0.05);
as shown in fig. 12B: the activity of Sp-G added with different concentrations is between 53.60 and 78.10 percent, and the dose-dependent trend is presented, wherein the improvement degree of the Sp-G of 800 mug/mL and 1000 mug/mL on the cell activity is most obvious, and the Sp-G added with different concentrations has no obvious difference (P is more than 0.05) and is 71.50 percent and 78.10 percent respectively;
as shown in fig. 12C: sp-F cell activities at different concentrations are 53.73% -88.39%, and the cell activities show a dose-dependent trend, wherein the improvement degree of the Sp-F cell activities of 800 mug/mL and 1000 mug/mL is most obvious, and the Sp-F cell activities are not significantly different (P is more than 0.05), namely 86.59% and 88.39%, respectively.
Overall, the addition of LPS significantly reduced cellular activity (P < 0.05) compared to the control group, whereas the addition of 3 surface proteins Sp-L, sp-G, sp-F was dose dependent for the increase in cellular activity. And compared with Sp-L and Sp-G, the added Sp-F of 800 mug/mL and 1000 mug/mL has the most obvious improvement degree on the cell activity, which shows that 2' -FL can better promote bifidobacterium bifidum DNG6 surface protein to relieve the damage of LPS on the cell activity. Therefore, in combination with the results of the determination of LDH activity and cell viability, the test selects 3 surface proteins of 800. Mu.g/mL for subsequent study.
6. Determination of cytokine content in damaged barrier: cells were inoculated into 12-well Transwell, and subjected to test grouping according to 2.2.3, and 100. Mu.L of cell supernatants of different treatment groups were collected and centrifuged to obtain a supernatant (1000 r/min,4 ℃,15 min) for centrifugation, and cytokine release was detected strictly according to ELISA kit instructions. Firstly, adding an antigen to be detected at room temperature, combining the antigen in a pore plate, sealing a gel plate, incubating for 90min in a constant temperature box at 37 ℃, washing the plate for 5 times, adding a biotinylated antibody, incubating for 60min, adding an enzyme conjugate, incubating for 30min, washing the plate for 5 times, adding a chromogenic substrate TMB, incubating for 15min in a dark place, adding a reaction stopping solution, uniformly mixing, measuring an OD value at 450nm in 3min, and calculating the content of the corresponding cytokines according to a standard curve.
RT-qPCR detection of expression of claudin
(1) Extraction of total RNA from cells
(1) Cells were prepared according to 2.2.3, surface proteins were added and incubated with LPS for 24h, the supernatant was discarded and washed 3 times with PBS. Adding 0.5mL of Trizol lysate into each hole, repeatedly blowing and sucking until no obvious precipitate is generated in the lysate, adding 100 mu L of chloroform, mixing until the solution is emulsified to be milky, standing at room temperature for 5min, centrifuging (4 ℃,12000r/min,15 min), sucking colorless supernatant containing RNA, transferring to another new centrifuge tube, adding 0.5mL of isopropanol into the supernatant, fully mixing uniformly, centrifuging (4 ℃,12000r/min,10 min), obtaining RNA precipitate at the bottom of a test tube, adding 500 mu L of 75% ethanol, mixing uniformly, centrifuging (4 ℃,7500r/min,5 min) to obtain precipitate, drying at room temperature, adding a proper amount of RNase-free water to dissolve the precipitate, determining the purity and the integrity of the RNA, and guaranteeing the purity and the integrity of the A 260/280 The ratio of (2) is 1.8-2.0.
(2) Reverse transcription cDNA reaction
The PrimeScript kit was thawed at room temperature and quickly placed on ice. The following operations are sequentially carried out: reverse transcription is carried out at 42 ℃ for 15min, reverse transcriptase is inactivated at 85 ℃ for 5s, cDNA is obtained after the reaction is finished, and the cDNA is preserved at-20 ℃ for standby. Reverse transcription system formulation information is shown in table 3:
TABLE 3 reverse transcription reaction system
(3) Fluorescent quantitative PCR amplification reaction
Using TB
Premix Ex Taq TM The II kit was reacted on a fluorescent quantitative PCR instrument. Pre-denaturation at 95 ℃ for 30s; PCR reaction was performed at 95℃for 5s and 30s at 60℃for 45 cycles. The reaction system formulation information is shown in table 4:
table 4 real-time fluorescent quantitative PCR amplification reaction System
The primer sequence is designed by Jilin England Biotechnology Inc. and is compared with the academic verification of high-frequency reference literature, and beta-actin is adopted as an internal reference by Oligo synthesized by Shanghai biological engineering Co., ltd, 2 △△Ct The difference in gene expression was calculated by the method. The primer information for the target gene and the reference gene are shown in Table 5:
TABLE 5 real-time fluorescent quantitative PCR primer sequences
Note that: f (F) 1 ：Forwad；R 1 ：Reverse
8. To investigate the effect of 3 surface proteins, sp-L, sp-G, sp-F, on cytokines in the LPS-induced inflammatory barrier, ELISA was used for this assay. As shown in fig. 13:
(1) FIG. 13 (A) TNF- α: the level of LPS group was 12.12 times that of control group, and the addition of Sp-L, sp-G and Sp-F significantly reduced the TNF- α content by 24.92%,38.58% and 53.46%, respectively (P < 0.05), compared to LPS group, wherein the addition of Sp-F most significantly reduced the TNF- α content (P < 0.05).
(2) FIG. 13 (B) IL-6: the level of LPS group was 2.23 times that of control group, and the addition of Sp-L, sp-G and Sp-F reduced the IL-6 content by 19.20%,31.34% and 35.91%, respectively (P < 0.05), compared with LPS group, wherein there was no significant difference in the addition of Sp-G and Sp-F (P > 0.05).
(3) FIG. 13 (C) IL-1β: the level of LPS group was 2.03 times that of control group, and the addition of Sp-L, sp-G and Sp-F reduced the IL-1β content by 27.30%,29.49% and 37.05%, respectively (P < 0.05), compared to LPS group, wherein there was no significant difference in the addition of Sp-G and Sp-F (P > 0.05).
(4) FIG. 13 (D) IL-10: the control group level was 1.86 times that of the LPS group, and the addition of Sp-L, sp-G and Sp-F significantly increased the IL-10 anti-inflammatory cytokine content by 30.69%,32.30% and 40.13%, respectively (P < 0.05), compared to the LPS group.
Overall, the addition of LPS significantly increased secretion of TNF- α, IL-6 and IL-1β compared to the control group, decreasing secretion of IL-10 (P < 0.05). In addition, the addition of 3 surface proteins, namely Sp-L, sp-G and Sp-F, obviously reduces the secretion of proinflammatory cytokines and obviously increases the secretion of anti-inflammatory factors (P < 0.05), wherein the surface protein of bifidobacterium bifidum DNG6 cultured by taking 2' -FL as a carbon source can better relieve the single-layer barrier damage induced by LPS.
Western-Blot detection of expression of Tight-junction proteins
(1) Total protein extraction
The Caco-2 monolayer cell barrier model was prepared as described above, surface proteins at different concentrations were added and treated with LPS for 24h, and the supernatant was discarded and washed 3 times with PBS. Lysates were added to each well and shaking was performed for 15min with shaking, cells were collected with a cell scraper, and centrifuged (4 ℃,14000r,15 min) to obtain protein-rich supernatants.
(2) BCA assay for determining protein concentration in a sample
According to the BCA kit, protein standard and treatment fluid are sequentially added into an ELISA plate, BCA working fluid is prepared and added, incubation is carried out for 30min at a constant temperature of 37 ℃, and after cooling, the absorbance is measured at 562 nm. A standard curve was drawn and protein sample concentrations were calculated.
(3) SDS-PAGE electrophoresis
Pouring 12% of separating gel and 5% of concentrated gel in the middle of the mounted glass plate, removing the comb after polymerization, adding electrophoresis buffer solution, selecting a pore canal, and adding a Marker and a sample to be tested. The electrophoresis voltage of the separation gel is regulated to be 60V, and the electrophoresis voltage of the concentration gel is regulated to be 90V.
(4) Transfer film
Cutting off the glue containing the separated target strips, spreading the glue in pre-cooled transfer film liquid, clamping the glue into a transfer printing clamp according to the sequence of a fiber pad, filter paper, gel, NC/PVDF film, filter paper and the fiber pad, mounting the glue into a transfer tank filled with the transfer film liquid, regulating and controlling current to 200mA, and treating for 60-120 min.
(5) Antibody incubation and image analysis
NC/PVDF membrane was placed in a 5% nonfat milk powder blocking solution for shaking table treatment for 2h, and then washed 3 times with TBST for 5min each. Primary antibodies were formulated and added and incubated overnight with shaking at 4 ℃. The primary antibody was removed by pipetting, washing the membrane 3 times with TBST for 5min each time, adding the secondary antibody and shaking the reaction at room temperature for 2h. The membrane was washed 3 times with TBST for 5min each. Working solution is prepared according to ECL chemiluminescence kit instructions, the working solution is dripped on an NC/PVDF film of a asphalt liquid, a gel imager is used for imaging, and gray analysis is performed on the result by using Image J software, wherein the relative expression quantity of target protein=target protein gray value/beta-actin gray value.
Results: in order to explore the effect of 3 surface proteins, namely Sp-L, sp-G and Sp-F, on tight junctions in inflammatory barriers, the gene expression amounts of the tight junctions proteins ZO-1, claudin-1 and Occludin in a monolayer barrier were analyzed by an RT-qPCR method.
As shown in fig. 14: compared with the control group, LPS resulted in a significant decrease in gene expression levels of the 3 tightly-linked proteins of 79.51%, 79.87% and 61.49% (P < 0.05), respectively; the addition of surface proteins significantly increased the amount of expression of the tight junction protein compared to the LPS group (P < 0.05); wherein, the addition of Sp-L and Sp-G obviously improves the expression of Occludin, and on the basis of LPS group, 77.28 percent and 110.62 percent (P < 0.05) are obviously increased respectively; compared with Sp-L and Sp-G, the addition of Sp-F maximally improves the gene expression of ZO-1, claudin-1 and Occludin, and 146.21%, 268.53% and 137.22% (P < 0.05) are respectively increased on the basis of LPS group.
The results show that the surface protein treatment extracted under the culture of 3 carbon sources all relieves the damage of LPS to the tight junction protein and increases the gene expression of ZO-1, claudin-1 and Occludin in a single-layer barrier. And the expression promoting effect of the added Sp-F on the 3 kinds of the tight junction protein mRNA is obviously higher than that of Sp-L and Sp-G (P is less than 0.05).
10. The expression of the claudin in the Caco-2 cell monolayer barrier model was detected by Western Blot, and FIG. 15A shows the expression of 3 claudin, the gray scale ratio of which to the internal reference beta-actin is shown in FIGS. 15B, C, D:
as can be seen from the ratio of ZO-1/beta-actin in FIG. 15B, the addition of LPS reduced ZO-1 expression by 87.38% (P < 0.05) compared to the control group; compared with LPS group, the addition of Sp-L, sp-G and Sp-F significantly increased ZO-1 expression by 56.92%,81.25% and 255.37% (P < 0.05), respectively.
As can be seen from the ratio of Claudin-1/beta-actin in FIG. 15C, the addition of LPS reduced Claudin-1 expression by 82.14% (P < 0.05) compared to the control group; sp-L, sp-G and Sp-F increased Claudin-1 expression by 22.98%,69.27% and 273.41%, respectively (P < 0.05) compared to LPS group.
As can be seen from the ratio of Occludin/β -actin in fig. 15D, the addition of LPS reduced Occludin expression by 87.06% (P < 0.05) compared to the control group; the addition of Sp-L, sp-G and Sp-F increased Occludin expression by 71.28%,242.83% and 462.19%, respectively (P < 0.05) compared to LPS group.
Overall, the expression levels of the 3 kinds of tightly-linked proteins at the protein level were consistent with the trend of the variation in the gene expression levels. These results appear to indicate that the 3 surface proteins Sp-L, sp-G and Sp-F increase the expression level of the tight junction protein; compared with Lac and GOS which are 2 carbon sources, the surface protein of bifidobacterium bifidum DNG6 extracted by taking 2' -FL as the carbon source can better enhance physical barrier of intestinal canal by increasing the expression of the tight junction protein and relieve the inflammatory injury of the intestinal canal induced by LPS.
11. Immunofluorescence observation tight junctions
The Caco-2 monolayer cell barrier model was prepared as described above, and surface proteins and LPS were added for 24h. The Transwell was removed and the supernatant discarded, washed 3 times with 5min each with PBS. After fixing 4% paraformaldehyde for 30min, washing with PBS for 3 times, each time for 5min; cells were covered with 1% TrizonX-100 and permeabilized at room temperature for 5min, then washed 3 times with PBS for 5min each. Adding blocking solution containing 5% skimmed milk, blocking at 37deg.C for 2 hr, and washing with PBS for 3 times each for 5min. Rabbit anti-human Occludin antibody (1:200), claudin-1 antibody (1:200), ZO-1 antibody (1:200) were added, covered cells were added dropwise evenly, and treated overnight at 4 ℃. The next day, the liquid was discarded and washed 3 times with 5min each with PBS. Goat anti-rabbit secondary antibody (1:300) was added, incubated at 37℃for 1h, the liquid was discarded, and washed 3 times with PBS for 5min each. Dripping and uniformly mixing the anti-fluorescence quenching liquid, and rapidly placing the mixture under an inverted fluorescence microscope for observation and image acquisition.
Data analysis and processing data were analyzed using SPSS 19.0 software and data are expressed as mean ± standard deviation. Data from different groups were analyzed by one-way variance using Duncan multiple comparisons, and differences were considered significant when P <0.05, plotted using Origin 2018.
After 24h of Sp-F acting inflammation model at 800. Mu.g/mL, ZO-1, occidin and Claudin-1 expression and localization were observed by immunofluorescence assay. As shown in FIG. 16, the green fluorescence labeled the distribution of Occidin and Claudin-1 proteins, and the red fluorescence labeled the distribution of ZO-1 proteins.
In the control group, ZO-1, occidin and Claudin-1 are distributed along the cell gap, and are in a net-shaped complete structure, clear in boundary and obvious in fluorescence intensity; the continuity and the integrity of the intercellular tight junction proteins in the LPS group are obviously damaged, the structure of the tight junction proteins is loose, obvious zigzag distribution, even notch and crack-like appearance can be seen, and the fluorescence intensity is weakened; compared with the LPS group, the continuous and integral tight junction proteins in the LPS+Sp-F group are partially recovered, so that the distribution among cells is clearer, and the fluorescence intensity is obviously enhanced.
Overall, the bifidobacterium bifidum DNG6 surface protein extracted by culturing with 2' -FL as a carbon source can alleviate intestinal barrier inflammatory injury by improving the localization of the tight junction protein.
Claims (10)
1. A preparation method of bifidobacterium surface protein is characterized in that bifidobacterium bifidum is cultured by taking 2' -FL as a unique carbon source, then bacterial cells are collected, liCl solution is added for incubation, supernatant is centrifugally collected, then the supernatant is filtered by a hydrophilic microporous filter membrane to obtain filtrate, the filtrate is put into a dialysis bag for dialysis, and liquid after dialysis is collected and freeze-dried to obtain the bifidobacterium surface protein.
2. The method according to claim 1, wherein the 2' -FL is added in an amount of 2% (wt/vol); the inoculum size of bifidobacterium bifidum was 3% (v/v).
3. The method according to claim 1, wherein the culturing conditions are 37℃for 18 hours in log phase; the condition for collecting the supernatant by centrifugation is 8000r/min,4 ℃ and 10min.
4. The preparation method according to claim 1, wherein the concentration of the LiCl solution is 5mol/L and the addition amount is 0.05% (v/v); the conditions for incubation with LiCl solution were 200r/min,37℃for 30min.
5. The method according to claim 1, wherein the pore size of the hydrophilic microporous filter membrane is 0.22 μm; the size of the dialysis bag is 12000kD.
6. A surface protein of bifidobacterium obtainable by the process as claimed in any one of claims 1 to 4.
7. Use of a bifidobacterium surface protein as claimed in claim 6 in the manufacture of a medicament for the treatment or co-treatment of intestinal barrier inflammation.
8. The use according to claim 7, wherein the treatment of intestinal barrier inflammation is to promote proliferation of intestinal epithelial cells, prevent expression and localized destruction of TJ proteins, inhibit secretion of pro-inflammatory cytokines, increase secretion of anti-inflammatory cytokines and increase expression of zon-1, claudin-1 and Occludin.
9. Use of a bifidobacterium surface protein as claimed in claim 6 in the manufacture of a health product or food for alleviating inflammation of the intestinal barrier.
10. The use according to claim 9, wherein said alleviation of intestinal barrier inflammation is promotion of proliferation of intestinal epithelial cells, prevention of expression and localized destruction of TJ proteins, inhibition of pro-inflammatory cytokine secretion and increase of anti-inflammatory cytokine secretion.
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