CN101884782A - Application of bifidobacterium longum adherence inhibition protein Aip for preventing and treating diarrhea - Google Patents
Application of bifidobacterium longum adherence inhibition protein Aip for preventing and treating diarrhea Download PDFInfo
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Abstract
The invention relates to application of bifidobacterium longum adherence inhibition protein Aip for preventing and treating diarrhea. The application is characterized in that: the adherence inhibition protein Aip inhibitory pathogenic bacteria is adhered and planted to the intestinal tract without influencing the adherence of probiotics, thereby preventing and treating the diarrhea caused by the pathogenic bacteria and maintaining the microecological balance of the intestinal tract. The invention has the advantages that: the large side effects and the drug tolerance can be overcome; and the adherence inhibition protein Aip is prepared into a functional product for preventing and treating the diarrhea without killing the pathogenic bacteria per se, but inhibiting the pathogenic bacteria from adhering to the gastrointestinal tract so that the pathogenic bacteria can lose planting copying conditions and is discharged from the body, therefore, the adherence inhibition protein Aip is safe and reliable, has no problems of drug tolerance transfer, and the like and is a new thought for treating diarrhea.
Description
Technical field
The present invention relates to a kind of application of bifidobacterium longum, relate in particular to the application of a kind of bifidobacterium longum adhesion inhibition protein A ip in control diarrhoea.
Background technology
Human gi-tract live away from home 400 surplus kind of intestinal flora, these microorganism species keep relative populations by quorum sensing, its physiological function is brought into play in vie each other nutrition and space.In recent years, the illness outbreak that causes of food-borne pathogens (listeria monocytogenes, Escherichia coli O 157 etc.) shows a rising trend.These pathogenic bacterium enter human gastrointestinal tract by food chain, amplification is bred after adhering to intestinal mucosa, the invasion enterocyte, by the monokaryon macrophage phagocytic, and be diffused into regional nodes thereupon, arrive internal organs at last, thereby cause the generation of disease, as listeria monocytogenes is a kind of strong pathogenic bacterium that nature exists, and 100 CFU just can cause death.
Adhesion is that pathogenic entero becteria causes the diarrheal prerequisite, suppresses the adhesion of pathogenic bacterium, makes it to become the bacterium that passes by on one's way and is excreted, and allows its forfeiture in the condition of intestinal breeding, thereby reaches control diarrheal purpose.Bacillus bifidus is in certain advantage in external sticking with the pathogenic bacterium competition in the epithelial cell.Discoveries such as Moroni are being sticked in the process with the bacillus bifidus competition, the aggressivity that the listeria monocytogenes of type is invaded in weak invasion type, medium invasion type and the trunk offering 38-90% that descended does not wait, and the preincubate of bacillus bifidus and cell monolayer can significantly suppress the adhesion of listeria monocytogenes pair cell.The 30min preincubate of report bacillus bifidus secreting type adhesins (10-30 μ g/ml) such as Zhong Shishun can obviously suppress to produce malicious escherichia coli or enterorrhagia escherichia coli sticking the Lovo cell.
The site is sticked in probiotic bacteria and pathogenic bacterium competition jointly, causes space occupy-place effect, is that probiotic bacteria suppresses the adherent important way of pathogenic bacterium.Asialo-GM1 is the important glucide that pathogenic bacterium and non-pathogenic bacteria stick the mankind and mucous membrane of animal surface, and Lactobacillus johnsonii La1 promptly comes from the site of sticking that it has occupied pathogenic bacterium to the inhibitory action of enteropathogenic E.Coli, enterohemorrhagic Escherichia coli.Mukai etc. find lactobacillus reuteri and helicobacter pylori bacterium competing phase with the glycosyl site, occupy the sticking of lactobacillus reuteri and sticking of enterocyte cause helicobacter pylori to lose behind the site sticking the site and be excreted.Therefore, intestinal cell surface carbohydrates material plays an important role in antifungal mechanism as the common directed binding site of probiotic bacteria and pathogenic bacterium.On the other hand, probiotic bacteria by with the different parts of pathogenic bacterium competitions glycosyl, play the effect of size exclusion, reach the purpose that suppresses pathogenic bacterium.Escherichia coli I type preference is sticked the mannose α 1-3 mannose site in the epithelial cell, this site is not the site of sticking of some Lactobacillus johnsonii bacterial strain, Lactobacillus johnsonii is possible be because it occupies near the glycosyl site in above-mentioned site for the inhibition of escherichia coli I type, cause the size exclusion effect of thalline, and influence the combination of pathogenic bacterium.
What treatment diarrhoea at present generally adopted is antibiotherapy, and antibiosis have certain side effect, may cause the generation of persister simultaneously, thereby further increases the treatment difficulty.In recent years, people especially prevent and treat the microbial infection of causing a disease, especially the focus of studying at present having done a large amount of work aspect the alternative antibiotic therapy diarrhoea of research by probiotic bacteria.Probiotic bacteria by stick, competition, repulsion, occupy-place and produce mechanism such as antibiotic substance, suppress the pathogenic bacterium in the gastrointestinal tract, keep the advantage of ancestral home flora, thereby promote the health of human body and animal body.The present invention be exactly adhesion inhibition protein A ip competition pathogenic bacterium by the source bifidobacterium longum at the binding site of intestinal cell, prevent the pathogen adhesion intestinal, reach control diarrheal purpose.
Summary of the invention
The object of the present invention is to provide the application of a kind of bifidobacterium longum adhesion inhibition protein A ip in control diarrhoea, Aip has the ability of stronger inhibition pathogen adhesion enterocyte, and do not influence the adhesion function of probiotic bacteria, safe in utilization, noresidue, do not develop immunity to drugs.
The present invention is achieved like this, and it is characterized in that: adhesion inhibition protein A ip prevents pathogen adhesion to be colonizated in intestinal, and does not influence the adhesion of probiotic bacteria, thereby the diarrhoea that the control pathogenic bacterium cause is kept the intestinal microecology balance.
The preparation method of described adhesion inhibition protein A ip is:
(1) bifidobacterium longum with fresh cultured inserts 300mL MRS fluid medium by 1%, 37 ℃, anaerobism cultivation 16-18h, and the centrifugal 3min of 8000rpm collects fermented liquid supernatant;
(2) respectively according to 15% and 75% saturation ammonium sulfate precipitation supernatant, precipitation is resuspended with 20mLPBS at last, desalination, vacuum lyophilization;
(3) crude protein of getting 1ml 0.5mg/ml carries out ion-exchange chromatography with prepackage ion column Hitrap Q FF, at first use initial buffer balance chromatographic column, described initial buffer is the mixture of 0.025mol/L kaliumphosphate buffer and 0.001mol/L EDTA and to regulate pH be 8.0, carry out the linear concentration gradient eluting with elution buffer, flow velocity is 1ml/min, collect each eluting peak respectively, electrophoresis detection, determine place, purpose peak, described elution buffer is the 0.025mol/L kaliumphosphate buffer, the mixture of 1mol/L potassium chloride and 0.001mol/L EDTA and to regulate pH be 8.0;
(4) the purpose peak reuse molecular sieve column HitrapSuperdex75 of Shou Jiing separates, and mobile phase is the Millipore ultra-pure water, and flow velocity is 0.5ml/min, collects each eluting peak, and electrophoresis detection is determined place, purpose peak.
Advantage of the present invention is: can overcome the big and drug resistance of side effect, adhesion inhibition protein A ip is prepared into control diarrheal functional product, kill pathogenic organisms itself not, but prevent it to adhere to gastrointestinal tract, make it to lose the condition that field planting is duplicated, be excreted, so safe and reliable, also impure in problems such as drug resistance transfers, be treatment diarrheal new approaches.
Description of drawings
Fig. 1 is the electrophoresis detection collection of illustrative plates of the present invention separation and purification Aip from the bifidobacterium longum fermentation liquid
Fig. 2 is nucleotide sequence and the aminoacid sequence of Aip of the present invention.
Fig. 3 makes up the enzyme action proof diagram of Aip expression vector for the present invention.
Fig. 4 is the electrophoresis detection collection of illustrative plates of gene engineering expression of the present invention and purification Aip.
Fig. 5 adheres to the lab diagram of enterocyte for the present invention verifies Aip.
Fig. 6 adheres to enterocyte figure for microscopic examination Aip of the present invention suppresses listeria monocytogenes.
Fig. 7 adheres to enterocyte figure for microscopic examination Aip of the present invention suppresses Escherichia coli O 157: H7.
Fig. 8 measures Aip for count plate of the present invention and suppresses listeria monocytogenes adhesion enterocyte figure.
Fig. 9 measures Aip for count plate of the present invention and suppresses Escherichia coli O 157: H7 adhesion enterocyte figure.
Figure 10 measures Aip adheres to enterocyte to bifidobacterium longum the figure that influences for count plate of the present invention.
Figure 11 measures Aip adheres to enterocyte to Lactobacillus plantarum the figure that influences for count plate of the present invention.
The specific embodiment
Embodiment 1:
1. the bifidobacterium longum with fresh cultured inserts 300mL MRS fluid medium by 1%, 37 ℃, anaerobism cultivation 16-18h, and the centrifugal 3min of 8000rpm collects fermented liquid supernatant.
2. respectively according to 15% and 75% saturation ammonium sulfate precipitation supernatant, precipitation is resuspended with 20mLPBS at last, desalination, vacuum lyophilization.
3. the crude protein of getting 1ml 0.5mg/ml carries out ion-exchange chromatography with prepackage ion column Hitrap Q FF.At first use initial buffer (0.025mol/L kaliumphosphate buffer, 0.001mol/L EDTA, pH8.0) balance chromatographic column is with elution buffer (0.025mol/L kaliumphosphate buffer, 1mol/L potassium chloride, 0.001mol/L EDTA, pH8.0) carry out the linear concentration gradient eluting, flow velocity is 1ml/min, collects each eluting peak respectively, electrophoresis detection is determined place, purpose peak.
4. the purpose peak reuse molecular sieve column HitrapSuperdex75 of Shou Jiing separates, and mobile phase is the Millipore ultra-pure water, and flow velocity is 0.5ml/min, collects each eluting peak, and electrophoresis detection is determined place, purpose peak.The SDS-PAGE collection of illustrative plates of Aip is the albumen about 45kD as shown in Figure 1 in each purification step, and 1 is albumen Marker (14-94kDa); 2 was the protein sample behind the intact molecular sieve column; 3 was the protein sample behind the intact ion column; 4 is the protein sample of ammonium sulfate precipitation.
Embodiment 2:
1. the method for pressing among the embodiment 1 is cultivated bifidobacterium longum, extracts genomic DNA.According to sequential design primer Aip-up (5`-GCGAATTCATGACAGTTAAGATTGGTATCAACGG-3`) in the GenBank and Aip-down (5`-CCGCGGCCGCTCACTCGGAGATCTCAGCGA-3`), amplification Aip gene, gene order and protein sequence are as shown in Figure 2.
2. after the gene of amplification being handled with restricted enzyme EcoRI and NotI double digestion, be connected with the expression vector pGEX-4T-1 of same double digestion, make up recombinant expression carrier pAip-G4, transformed into escherichia coli DH5 α, the picking positive colony, checking and order-checking, as shown in Figure 3,1 is pGEX-BIF (EcoR Iand Not I digested); 2 is DL2000DNA Marker.
3. get the correct positive colony bacterium of order-checking of fresh cultured, connect the bacterium amount by 1% and insert in the 300ml LB culture medium, be cultured to 0D
600=0.4-0.6 adds 0.5mM IPTG, 25 ℃ of abduction deliverings.After the centrifugal 5min of 4500rpm collects thalline, PBS washing thalline three times.The resuspended thalline of 30ml lysis buffer (PBS+2mM EDTA), behind the ultrasonic disruption, the centrifugal 10min of 13000rpm gets supernatant.Operate in contrast with the bacillus coli DH 5 alpha of band pGEX-4T-1 simultaneously.
4.20ml A buffer (PBS+2mM EDTA) carry out the balance of glutathion resin prepacked column GSTrapFF, flow velocity 3ml/min.After treating that curve steadily, get sample on the supernatant after the fragmentation, flow velocity 0.5-1ml/min.
7. after treating the curve smooth decreasing, with the destination protein eluting, flow velocity 1ml/min collects eluting peak with B buffer (PBS+2mM EDTA+10mM glutathion).
8. ultrafiltration pipe concentrate eluant is crossed desalting column Hitrap desalting, the pure water eluting, and flow velocity 3ml/min gets eluting peak, after the vacuum lyophilization, gets to be partly dissolved and carries out the evaluation of SDS-PAGE electrophoresis, sees Fig. 4, and 1 is the pGEX-BIF expressing protein; 2 is pGEX-4T-1 expressing protein (contrast); 3, the Aip expressing protein of purification; 4 is Blue Plus albumen Marker (12kDa-94kDa)
Embodiment 3:
1. the Aip that is purified to by the method among the embodiment 2, adopt active ester method, get 2 μ M quantum dot (CdSe/ZnS Core/Shell, Emission=620nm) with sodium dihydrogen phosphate-HCl buffer (0.2M, pH 5.0) resuspended, (carbodiimide, EDAC) and 0.86mgN-hydroxysulfosuccinimide (Suflo-NHS), 25 ℃ are reacted 40min to add the 1.52mg carbodiimide.
2. transfer pH to 7.8 with NaOH, add Aip fusion rotein 1mg, behind 25 ℃ of reaction 3h, 4 ℃ of placements are spent the night.After the centrifugal 10min of 10000rpm got supernatant, (=100,000g) for30min got precipitation to 40000rpm, and with the dissolving of 500 μ LPBS solution, 4 ℃ of preservations are stand-by again.With quadrat method labelling GST albumen in contrast.
3. get 95 μ L enterocyte HT-29 (10
4Individual/mL) with 5 μ L and quantum dot-labeled Aip albumen mixing after, 37 ℃ hatch 2h after, the centrifugal 5min of 3000rpm gets precipitation, wash three times after, 100 μ LPBS are resuspended.
4. get 5 μ L mixed liquor points on microscope slide, 450nm excites to observe and finds that Aip albumen can adhere to enterocyte under the fluorescence microscope, and as shown in Figure 5, A is the GST of labelling quantum dot; B is the Aip of labelling quantum dot.Quantum dot-labeled GST albumen in contrast.
Embodiment 4:
1. the Aip that is purified to by the method among the embodiment 2 gets 50 μ L enterocyte HT-29 (10
4Individual/mL) with 50 μ L30 μ g/mL Aip fusion rotein, behind the mixing, hatch 2h for 37 ℃.
2.3000rpm centrifugal 5min gets precipitation, wash three times after, 30 μ LPBS are resuspended, the coating slide surface after drying in the shade, adds heat fixation, adds 3%BSA solution sealing 1h.
3. after the washes clean, the listeria monocytogenes drop of getting fresh cultured is added on fixed cell surface, 37 ℃ hatch 1h after, the unconjugated thalline of flush away carries out Gram, again microexamination.GST albumen is similarly handled in contrast.Discovery is behind the enterocyte that Aip handles, and the bonded quantity of listeria monocytogenes is obviously compared according to few, and as shown in Figure 6, A is Aip; B is GST.
Embodiment 5:
1. the Aip that is purified to by the method among the embodiment 2 gets 50 μ L enterocyte HT-29 (10
4Individual/mL) with 50 μ L30 μ g/mL Aip fusion rotein, behind the mixing, hatch 2h for 37 ℃.
2.3000rpm centrifugal 5min gets precipitation, wash three times after, 30 μ LPBS are resuspended, the coating slide surface after drying in the shade, adds heat fixation, adds 3%BSA solution sealing 1h.
3. after the washes clean, get the Escherichia coli O 157 of fresh cultured: H7 bacterium drop is added on fixed cell surface, 37 ℃ hatch 1h after, the unconjugated thalline of flush away carries out Gram, again microexamination.GST albumen is similarly handled in contrast.Discovery is behind the enterocyte that Aip handles, and the bonded quantity of Escherichia coli O 157: H7 is obviously compared according to few, and as shown in Figure 7, A is Aip; B is GST.
Embodiment 6:
1. the Aip that is purified to by the method among the embodiment 2, get 50 μ L enterocyte HT-29 (104/mL) with Aip fusion rotein (5,30, the 50 μ g/mL) mixing of 50 μ L variable concentrations after, hatch 2h for 37 ℃.
2.3000rpm centrifugal 5min gets precipitation, wash three times after, 30 μ LPBS are resuspended, coat (1 * 1.5cm on the activatory pvdf membrane of methanol
2), after drying in the shade, adding in the 3%BSA solution and seal 1h, PBST washes film three times.
3. the listeria monocytogenes liquid of getting fresh cultured simultaneously is diluted to 10
4Behind individual/mL, get 500 μ L and hatch 30min for 37 ℃ with the film of handling, take out film, remaining bacterium liquid carries out count plate.GST albumen is similarly handled in contrast.
4. calculate the adhesion inhibition rate according to following formula:
Adhesion inhibition rate (%)=(total thalline-residue thalline) * 100/ total thalline
The result makes Fig. 8, shows along with the Aip protein concentration increases, and suppresses the effect of listeria monocytogenes and strengthens.
Embodiment 7:
1. the Aip that is purified to by the method among the embodiment 2, get 50 μ L enterocyte HT-29 (104/mL) with Aip fusion rotein (5,30, the 50 μ g/mL) mixing of 50 μ L variable concentrations after, hatch 2h for 37 ℃.
2.3000rpm centrifugal 5min gets precipitation, wash three times after, 30 μ LPBS are resuspended, coat (1 * 1.5cm on the activatory pvdf membrane of methanol
2), after drying in the shade, adding in the 3%BSA solution and seal 1h, PBST washes film three times.
3. get the colon bacillus 0157 of fresh cultured simultaneously: H7 bacterium liquid is diluted to 10
4Behind individual/mL, get 500 μ L and hatch 30min for 37 ℃ with the film of handling, take out film, remaining bacterium liquid carries out count plate.GST albumen is similarly handled in contrast.
4. calculate the adhesion inhibition rate according to following formula:
Adhesion inhibition rate (%)=(total thalline-residue thalline) * 100/ total thalline
The result makes Fig. 9, shows along with the Aip protein concentration increases, and suppresses the effect of colon bacillus 0157: H7 and strengthens.
Embodiment 8:
1. the Aip that is purified to by the method among the embodiment 2, get 50 μ L enterocyte HT-29 (104/mL) with Aip fusion rotein (5,30, the 50 μ g/mL) mixing of 50 μ L variable concentrations after, hatch 2h for 37 ℃.
2.3000rpm centrifugal 5min gets precipitation, wash three times after, 30 μ LPBS are resuspended, coat (1 * 1.5cm on the activatory pvdf membrane of methanol
2), after drying in the shade, adding in the 3%BSA solution and seal 1h, PBST washes film three times.
3. the bifidobacterium longum liquid of getting fresh cultured simultaneously is diluted to 10
4Behind individual/mL, get 500 μ L and hatch 30min for 37 ℃ with the film of handling, take out film, remaining bacterium liquid carries out count plate.GST albumen is similarly handled in contrast.
4. calculate the adhesion inhibition rate according to following formula:
Adhesion inhibition rate (%)=(total thalline-residue thalline) * 100/ total thalline
The result makes Figure 10, shows that Aip albumen does not influence the adhesion of bifidobacterium longum to enterocyte.
Embodiment 9:
1. the Aip that is purified to by the method among the embodiment 2, get 50 μ L enterocyte HT-29 (104/mL) with Aip fusion rotein (5,30, the 50 μ g/mL) mixing of 50 μ L variable concentrations after, hatch 2h for 37 ℃.
2.3000rpm centrifugal 5min gets precipitation, wash three times after, 30 μ L PBS are resuspended, coat (1 * 1.5cm on the activatory pvdf membrane of methanol
2), after drying in the shade, adding in the 3%BSA solution and seal 1h, PBST washes film three times.
3. the Lactobacillus plantarum liquid of getting fresh cultured simultaneously is diluted to 10
4Behind individual/mL, get 500 μ L and hatch 30min for 37 ℃ with the film of handling, take out film, remaining bacterium liquid carries out count plate.GST albumen is similarly handled in contrast.
4. calculate the adhesion inhibition rate according to following formula:
Adhesion inhibition rate (%)=(total thalline-residue thalline) * 100/ total thalline
The result makes Figure 11, shows that Aip albumen does not influence the adhesion of Lactobacillus plantarum to enterocyte.
Claims (2)
1. the application of bifidobacterium longum adhesion inhibition protein A ip in control diarrhoea, it is characterized in that: adhesion inhibition protein A ip prevents pathogen adhesion to be colonizated in intestinal, and do not influence the adhesion of probiotic bacteria, thereby the diarrhoea that the control pathogenic bacterium cause is kept the intestinal microecology balance.
2. the application of bifidobacterium longum adhesion inhibition protein A ip according to claim 1 in control diarrhoea is characterized in that the preparation method of described adhesion inhibition protein A ip is:
(1) bifidobacterium longum with fresh cultured inserts 300mL MRS fluid medium by 1%, 37 ℃, anaerobism cultivation 16-18h, and the centrifugal 3min of 8000rpm collects fermented liquid supernatant;
(2) respectively according to 15% and 75% saturation ammonium sulfate precipitation supernatant, precipitation is resuspended with 20mLPBS at last, desalination, vacuum lyophilization;
(3) crude protein of getting 1ml 0.5mg/ml carries out ion-exchange chromatography with prepackage ion column Hitrap Q FF, at first use initial buffer balance chromatographic column, described initial buffer is the mixture of 0.025mol/L kaliumphosphate buffer and 0.001mol/L EDTA and to regulate pH be 8.0, carry out the linear concentration gradient eluting with elution buffer, flow velocity is 1ml/min, collect each eluting peak respectively, electrophoresis detection, determine place, purpose peak, described elution buffer is the 0.025mol/L kaliumphosphate buffer, the mixture of 1mol/L potassium chloride and 0.001mol/L EDTA and to regulate pH be 8.0;
(4) the purpose peak reuse molecular sieve column Hi trapSuperdex75 of Shou Jiing separates, and mobile phase is the Millipore ultra-pure water, and flow velocity is 0.5ml/min, collects each eluting peak, and electrophoresis detection is determined place, purpose peak.
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| CN108834900B (en) * | 2018-07-31 | 2022-03-11 | 安徽农业大学 | Culture method for inhibiting explant oxidation browning and endophyte pollution in tea tree tissue culture |
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