CN105641682A - Application of Bifidobacterium longum protein in improvement of antibiotics sensitivity of Salmonella typhimurium - Google Patents
Application of Bifidobacterium longum protein in improvement of antibiotics sensitivity of Salmonella typhimurium Download PDFInfo
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- CN105641682A CN105641682A CN201610107536.2A CN201610107536A CN105641682A CN 105641682 A CN105641682 A CN 105641682A CN 201610107536 A CN201610107536 A CN 201610107536A CN 105641682 A CN105641682 A CN 105641682A
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- protein
- bifidobacterium longum
- salmonella typhimurium
- antibiotic
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
- A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
- A61K31/425—Thiazoles
- A61K31/429—Thiazoles condensed with heterocyclic ring systems
- A61K31/43—Compounds containing 4-thia-1-azabicyclo [3.2.0] heptane ring systems, i.e. compounds containing a ring system of the formula, e.g. penicillins, penems
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
- A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
- A61K31/7036—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin having at least one amino group directly attached to the carbocyclic ring, e.g. streptomycin, gentamycin, amikacin, validamycin, fortimicins
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/164—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
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- Proteomics, Peptides & Aminoacids (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention provides an adhesive protein sourced from Bifidobacterium longum. An amino acid sequence of the protein is determined, on the basis, experiments find that the adhesive protein not only can adhere to an enterocyte, but also has a function of improving antibiotics sensibility of microorganisms, and the antibiotics synergy on Salmonella typhimurium is particularly prominent. On the basis of the beneficial discovery, an application of the protein as an anti-bacterial synergist for Salmonella typhimurium is determined, an application range of the protein is extended, and a new path for improving the drug sensitivity of the microorganisms is provided. Prominent technological effects are obtained on the basis of strict experimental measures, and broad application prospect is provided.
Description
Technical field
The present invention relates to microbial technology field, be specifically related to a kind of bifidobacterium longum protein for promoting the application of Salmonella typhimurium Antibiotic Sensitivity.
Background technology
Bacillus bifidus is widely known by the people as beneficial bacteria of intestinal tract, finds for the research of its intestinal prebiotic effect in recent years, and some metabolite of bacillus bifidus can suppress other microorganisms in the field planting of intestinal inwall, and then reduces the infringement risk of pathogenic microorganism. This feature has become as screening bifidobacterium species at present, researches and develops the important evidence of functional probiotic bacteria microbial inoculum.
Prior art research finds, other Microorganism colonizations are reduced gut pH in the suppression mechanism of intestinal inwall mainly by secretion organic acid by bacillus bifidus, regulating intestinal canal microecological environment, secretion exoenzyme is thus affecting pathogenic bacterium or bacteriotoxic sticking site and specific receptor thereof simultaneously. Additionally, there is scholar to think in recent years, bacillus bifidus themselves the occupation time process caused in intestinal and space steric effect are probably the key factor hindering pathogenic bacterium to stick and invade. Adhesion protein sticks as bacillus bifidus and is colonizated in the important factor of GI epithelium, and its material base, metabolic characteristic there is no clear and definite conclusion in the prior art,
Although once report part has an adhering bacillus bifidus expressing protein in prior art, but the functional materials of its whether be exactly thalline be colonizated in intestinal inwall is still not clear; Additionally, clearly obtaining on the basis of a kind of Adhesion of Bifidobacteria albumen, for realizing further application, its expression, purification efficiency should be optimized; Again, for Adhesion of Bifidobacteria albumen, it should study its biological characteristics further, thus other purposes for this attachment proteins lay the foundation.
Bifidobacterium longum (Bifidobacteriumlongum) is the one of Bifidobacterium, belong to Gram-positive, without spore, do not move, anaerobic bacillus, it is widely present in the intestinal of human body especially baby, belongs to one of conventional strain of medicine, microecological microbial agent preparation field.
Summary of the invention
It is contemplated that for the technological deficiency of prior art, there is provided a kind of bifidobacterium longum protein for promoting the application of Salmonella typhimurium Antibiotic Sensitivity, there is circumscribed technical problem solving bifidobacterium longum protein range of application in prior art.
Another that the invention solves the problems that technical problem is that antibiotic sensitivity is had to be hoisted by Salmonella typhimurium.
For realizing above technical purpose, the present invention by the following technical solutions:
A kind of bifidobacterium longum protein, its aminoacid sequence is such as shown in SEQIDNO:1.
The preparation method of a kind of above-mentioned bifidobacterium longum protein, comprises the following steps:
1) the expression of recombinant e. coli bacterial strain of bifidobacterium longum protein is prepared;
2) step 1 is taken) the expression of recombinant e. coli bacterial strain of described bifidobacterium longum protein cultivates, add derivant IPTG when being 0.6��0.9 to culture fluid OD600nm to final concentration of 0.8��1.2mM, then in 35��39 DEG C, concussion when inducing culture 4��5h;
3) thalline, broken, collection supernatant are collected;
4) step 3 is taken) supernatant, utilize GST affinitive layer purification, be concentrated by ultrafiltration after collecting eluent, desalination, dry, namely obtain described bifidobacterium longum protein.
As preferably, step 3) described in broken be after thalline is resuspended in PBS, ultrasonication 20��30 times under condition of ice bath, the every time broken persistent period is 2��4s, every 2 times broken between interval be 4��6s.
As preferably, step 4) specifically include following operation:
A) take buffer A, balance glutathione Sepharose resin post with the flow velocity of 2.5��3.5mL/min;
B) step 2 is taken) supernatant, with the flow velocity loading of 1��2mL/min;
C) take buffer B, with the flow velocity eluting of 0.8��1.2mL/min, collect eluting peak place solution and be eluent;
D) taking eluent to be concentrated by ultrafiltration, then cross desalting column, recycling pure water, with the flow velocity eluting of 2.5��3.5mL/min, is collected eluting peak place solution and is the second eluent, and postlyophilization, namely obtain described bifidobacterium longum protein;
Wherein said buffer A is the PBS containing 1.5��2.5mMEDTA;
Described buffer B is the PBS containing 1.5��2.5mMEDTA, 15��25mM glutathion; It is further preferred that the buffer A consumption being used for balancing glutathione Sepharose resin post in step a) is 25��35mL, more optimizedly 30mL.
As preferably, step 1) described in concussion be that the shaking table of 150��200r/min rotating speed is cultivated, more optimizedly the shaking table of 180r/min rotating speed is cultivated.
The aminoacid sequence such as protein shown in SEQIDNO:1 is for promoting the application of Salmonella typhimurium Antibiotic Sensitivity; It is further preferred that be the application as the antibacterial synergist of antibiotic of the above-mentioned bifidobacterium longum protein.
As preferably, described antibiotic is beta-lactam antibiotic; It is further preferred that described beta-lactam antibiotic can be ampicillin, benzylpenicillin or cephamycin.
As preferably, described antibiotic is aminoglycoside antibiotics; It is further preferred that described aminoglycoside antibiotics is kanamycin.
As preferably, described antibiotic is amphenicols antibiotic; It is further preferred that described amphenicols antibiotic is chloromycetin.
As preferably, described application is to utilize the protein solution be 45��55 �� g/mL, aminoacid sequence being SEQIDNO:1 containing concentration, processes Salmonella typhimurium with the way of contact; It is further preferred that processed Salmonella typhimurium concentration in described protein solution is 106CFU/mL; It is further preferred that the described process time is 1.5��2.5h.
In above technical scheme, described sensitivity refers to the degree that microorganism is subject to antibiotic to suppress, described lifting microorganism Antibiotic Sensitivity, after referring to that the medium utilized containing bifidobacterium longum protein of the present invention hatches microorganism, processed microorganism fungistatic effect under antibiotic effect is more notable. Described bifidobacterium longum protein, is illustrate that under natural environment, this dietary protein origin is in bifidobacterium longum, and is not intended that the restriction effect to this DNA techniques feature; In the present invention, the technical characteristic of this protein is only limited by its sequence. The expression of recombinant e. coli bacterial strain of described bifidobacterium longum protein, refers to the recombination bacillus coli being integrated with the natural expressing gene of bifidobacterium longum of the present invention; Its preparation method generally includes the extraction of bifidobacterium longum STb gene, the amplification of genes of interest, the purification of amplified production, genes of interest and the enzyme action of carrier, connection, and recombinant vector is to steps such as the importing in escherichia coli, the screenings of recon; Actual preparation method can carry out adaptability design according to the general technology general knowledge of this area.
As it was noted above, the present invention can be used for promoting microorganism to antibiotic sensitivity. But the material after utilizing bifidobacterium longum protein of the present invention to process not necessarily gives antibiotic treatment, and when giving antibiotic treatment, its processing method can adopt below scheme: ampicillin activity is 1��50 �� g/mL; Benzylpenicillin activity is 1��50 �� g/mL; Cephamycin activity is 1��50 �� g/mL; Kanamycin activity is 1��25 �� g/mL; Chloromycetin activity is 1��25 �� g/mL.
The invention provides a kind of attachment proteins deriving from bifidobacterium longum, and specify that its aminoacid sequence; The expression strain of this albumen of strain is obtained on this basis based on gene engineering, and the characteristic for bacterial strain and attachment proteins carries out innovative design from aspects such as abduction delivering, chromatography purification, ultrafiltration concentrations, and then obtain the high efficiency preparation method of above-mentioned attachment proteins. Further, experiment finds that this attachment proteins can not only adhere to enterocyte, and there is the effect promoting microbial antibiotic sensitivity, based on this useful discovery, determine this albumen purposes as the antibacterial synergist of antibiotic, thus extending its purposes scope, provide a kind of new way promoting microorganism medicine sensitive simultaneously. The present invention obtains prominent technique effect based on rigorous laboratory facilities, has broad application prospects.
Accompanying drawing explanation
Fig. 1 is the expression and purification electrophoresis detection figure of bifidobacterium longum specific proteins AIP2 in the embodiment of the present invention 1; In figure, swimming lane M is ProteinMarker (12-80kDa); Swimming lane 1 is E.colipGEX-4T-1 expression product (comparison); Swimming lane 2 is the AIP2 albumen after purification; Swimming lane 3 is E.colipGEX-4T-AIP2 fusion protein expression products.
Fig. 2 is that in the embodiment of the present invention 5, bifidobacterium longum specific proteins AIP2 promotes 3 kinds of common food-borne pathogens experimental result picture to ampicillin-sensitive; In figure, part A is the bifidobacterium longum specific proteins AIP2 exercising result to listeria monocytogenes; Part B be bifidobacterium longum specific proteins AIP2 to Escherichia coli O 157: the exercising result of H7; C portion is the bifidobacterium longum specific proteins AIP2 exercising result to Salmonella typhimurium.
Fig. 3 be in the embodiment of the present invention 6 bifidobacterium longum specific proteins AIP2 promote 3 kinds of common food-borne pathogens to benzylpenicillin, cephamycin, kanamycin, chloramphenicol sensitivity experimental result picture.
Detailed description of the invention
Hereinafter the specific embodiment of the present invention will be described in detail. In order to avoid too much unnecessary details, in the examples below to belonging to known structure or function will not be described in detail.
The approximating language used in following example can be used for quantitative expression, it was shown that quantity can be allowed to have certain variation when not changing basic function. Therefore, the numerical value revised with the language such as " about ", " left and right " is not limited to this exact value itself. In certain embodiments, " about " represents that the numerical value allowing its correction changes in the positive and negative scope of 10 (10%), and such as, what " about 100 " represented can be any numerical value between 90 to 110. Additionally, in the statement of " about first numerical value to second value ", at about revise two numerical value of the first and second numerical value. In some cases, approximating language is likely to relevant with the precision measuring instrument.
Except having definition, technology used in following example and scientific terminology have the identical meanings being commonly understood by with those skilled in the art of the invention.
Test reagent consumptive material used in following example, if no special instructions, is routine biochemistry reagent; Described experimental technique, if no special instructions, is conventional method; Quantitative test in following example, is respectively provided with three times and repeats experiment, results averaged; % in following example, if no special instructions, is weight/mass percentage composition.
Embodiment 1 (preparing the expression of recombinant e. coli bacterial strain of bifidobacterium longum protein)
1.1 experiment materials
1.1.1 bacterial strain and carrier
Bifidobacterium longum, bacillus coli DH 5 alpha, vector pGEX-4T-1 all buys from market.
1.1.2 common agents
(1) TE buffer (pH8.0): 10mmol/LTris-HCl (pH8.0), 1mmol/LEDTANa2(pH8.0). Autoclaving after configuration, room temperature storage.
(2) 10mg/mL lysozyme: 100mg lysozyme is dissolved in 10mLddH2In O, 20 DEG C save backup.
(3) 10%SDS:5gSDS adds ddH2O dissolves and is settled to 50mL.
(4) 3MKAc:14.72gKAc is dissolved in ddH2In O and be settled to 50mL.
(5) 3MNaAc:12.35gNaAc is dissolved in ddH2In O and be settled to 50mL.
(6)0.1MCaCl2: 11.1gCaCl2It is dissolved in 1000mLddH2In O, 121 DEG C of autoclaving 15min, in 4 DEG C of preservations.
(7) 50 �� TAE electrophoresis buffer:12.2gTris, 2.85mL glacial acetic acid, 10mL0.25mol/LEDTA (pH8.0), add ddH2O dissolves and is settled to 50mL.
(8) 6 �� loadingbuffer (sample-loading buffer): 0.25% dimethylbenzene green grass or young crops FF, 0.25% bromophenol blue, 30% glycerol.
1.1.3 conventional culture medium
(1) MRS culture medium: Carnis Bovis seu Bubali cream 3g, yeast powder 5g, Tween 80 1mL, MgSO4��7H2O0.6406g, K2HPO45g, glacial acetic acid 4.3mL, MnSO4��H2O0.13g, peptone 7g, glucose 20g, sodium acetate 5g, peptone 7g, ammonium citrate 2g, Cys 0.5g, add ddH2O to 1000mL, adjusting pH is 6.5, then 115 DEG C of autoclaving 15min.
(2) LB fluid medium: yeast extract 5g, peptone 10g, NaCl10g, add ddH2O to 1000mL, adjusts pH to 7.0,121 DEG C of autoclaving 15min.
(3) preparation of LB flat board: add agar powder by 1.5% (w/v) at LB fluid medium, is down flat plate after 121 DEG C of autoclavings.
(4) LB/Amp (Ampicillin, ampicillin) preparation of flat board: add agar powder at LB fluid medium by 1.5% (w/v), then non-scald on hand (about 50 DEG C) it is cooled to after 121 DEG C of autoclavings, add the Amp to final concentration of 50 �� g/mL of 100mg/mL, it is down flat plate after mixing, is placed in superclean bench and dries standby.
1.2 experimental techniques
1.2.1 the extraction of bifidobacterium longum genomic DNA and detection
1.2.1.1 the extraction of bacillus bifidus genomic DNA
Extracting the genomic DNA of bifidobacterium longum, this experiment adopts " lysozyme-SDS-potassium acetate " method, and concrete grammar is as follows:
(1) bacillus bifidus is inoculated in the test tube filling MRS fluid medium, under 37 DEG C of conditions, stands Anaerobic culturel 24h.
(2) pipette the bacillus bifidus bottom middle pipe with 1mL suction pipe, be placed in 2mL centrifuge tube.
(3) centrifugal (4500rpm, 5min), abandon supernatant, at the bottom of residue pipe, be about 0.1g wet thallus.
(4) 1mLTE (pH8.0) resuspended washing is added once, centrifugal (4500rpm, 5min), abandon supernatant.
(5) add the lysozyme of 0.7mLTE (pH8.0) and 0.1mL10mg/mL, blow and beat with pipettor, make thalline even suspension in solution.
(6) putting 40 DEG C of water-bath effect 3h, period reverse mixing is for several times.
(7) it is rapidly added 0.1mL10%SDS, up and down even rearmounted 40 DEG C of water-bath effect 1h.
(8) centrifugal (10000rpm, 5min, 4 DEG C), pipette 0.5mL supernatant in new 2mL centrifuge tube, abandon precipitation.
(9) add the KAc (3M) of 0.1mL (about 1/6 volume) ice pre-cooling, put ice-water bath 15min.
(10) centrifugal (12000rpm, 10min, 4 DEG C), pipette 400 �� L of supernatant liquid in new 2mL centrifuge tube, abandon precipitation.
(11) add the chloroform of equal-volume (400 �� L): isoamyl alcohol (24:1) is stripped, mixing of gently turning upside down, stand 3min.
(12) centrifugal (12000rpm, 5min, 4 DEG C), draw supernatant in new 2mL centrifuge tube.
(13) add the dehydrated alcohol of the pre-cooling of the NaAc (3M) and 880 �� L (2 times of volumes) of 40 �� L (1/10 volume) ice pre-cooling, even be up and down placed on-20 DEG C of freezing 30min.
(14) centrifugal (12000rpm, 10min, 4 DEG C), remove supernatant.
(15) add 400 �� L pre-coolings 75% washing with alcohol.
(16) centrifugal (12000rpm, 10min, 4 DEG C), remove supernatant, and make ethanol volatilization clean.
(17) 40 �� LTE dissolution precipitations are added.
(18) RNaseA (final concentration of 50 �� g/mL), 37 DEG C of water-bath 1h are added.
(19)-20 DEG C save backup.
1.2.1.2 agarose gel electrophoresis
(1) glue:
Weigh 0.25g agarose and put in 150mL triangular flask, add 25mL1 �� TAE moderate heat heating for dissolving in microwave oven and become gel solution. Install electrophoresis mould and sample comb, take a small amount of gel solution and electrophoresis both mold ends is shut, add 2 �� LDNA coloring matter GoldView dyestuffs, pour mould after mixing into, note producing bubble.
(2) loading electrophoresis:
Electrophoresis tank holds 1 �� TAE electrophoresis buffer. After gelling is solid, electrophoresis mould is put in electrophoresis tank, then carefully take out sample comb. Separately sampled product add loading after 6 �� loadingbuffer mixes, correctly connecting electrode, the loading wells of agarose gel, near negative pole, carries out electrophoresis under 5V/cm constant-pressure conditions, stop electrophoresis after 20min, gel is put into gel imaging system and carries out imaging, analysis.
1.2.2AIP2 the amplification of gene
Forward primer: TTCGAATTCGTGAAAACTTTCACTCCGAAGCCEcoRI63.4 DEG C
Downstream primer: CGCGCGGCCGCTTGGCCTGCTGCGAGACNotI63.3 DEG C
Table 1PCR reaction system
Reactant liquor is mixed with liquid-transfering gun, slightly centrifugal, cover tightly lid, after pipe covers and carries out labelling with marking pen, program shown in table 2 expands in PCR instrument.
Table 2PCR response procedures
PCR reaction adopts 1.0% agarose gel electrophoresis detection PCR primer after terminating.Take 5 �� LPCR products and 1 �� L6 �� Loadingbuffer mixing, click and enter in agarose gel loading hole, with DNAMarker (DL2000) for comparison, 5V/cm constant voltage electrophoresis 30min in DNA level electrophoresis tank. Gel is put into gel imaging system after terminating and is carried out imaging, analysis by electrophoresis.
1.2.3PCR the purification of product
Adopt the PCR primer purification kit of Beijing SBS Genetech gene technology company limited, PCR primer is purified, specifically comprises the following steps that
(1) by the electrophoresis on the plain agar sugar gel of 1% of the PCR product without paraffin oil, cut required DNA band and load in 2mL centrifuge tube. Cut away the agarose gel without DNA as far as possible.
Adding 0.4mL Purification Resin (before use fully mixing) in (2) 200��400mg agarose gel, 70 DEG C of insulations 5��10min, every 2min overturn mixing 1 time, make agarose gel melt completely.
(3) mixed liquor being transferred to centrifugal purification post, 13000rpm is centrifuged 30s, outwells the waste liquid in collecting pipe.
(4) adding 500 �� L80% ethanol, 13000rpm is centrifuged 30s, outwells the waste liquid in collecting pipe.
(5) repeating previous step, this step 13000rpm is centrifuged 2min, is eliminated by ethanol.
(6) being entered in clean 1.5mL or 2mL centrifuge tube by centrifugal purification column sleeve, uncap placement 2��3min, and ethanol must be made fully to volatilize totally. Add 30 �� LddH2O is on Purification Resin. After placing 2min, 13000rpm is centrifuged 30s.
(7) namely the liquid in centrifuge tube be the DNA fragmentation of purification, takes 1 �� L electrophoresis detection.-20 DEG C save backup. 1.2.4 the fragment of order and the enzyme action of carrier process
Purpose fragment carries out the enzyme action of purpose fragment and carrier, prepares enzyme action system with reference to table 3 after reclaiming, and two enzyme action systems are placed in 37 DEG C of insulation 1.5h.
Table 3 enzyme action system
1.2.5 the connection of genes of interest and vector pGEX-4T-1
Reference method 2.2.3, by the fragment after enzyme action and carrier recovery, reclaims product and prepares coupled reaction system according to by table 4, and 22 DEG C connect 30min.
Table 4 coupled reaction system
1.2.6 convert
1.2.6.1 the preparation of competent cell
Taking bacillus coli DH 5 alpha and prepare competent cell, concrete grammar is as follows:
(1) bacillus coli DH 5 alpha streak inoculation-70 DEG C frozen is taken in non-resistant LB flat board, in 37 DEG C of overnight incubation.
(2) the well-grown single colony inoculation of picking is in 5mLLB fluid medium, is placed in 37 DEG C of shaking table 180rpm shaken cultivation overnight.
(3) by the inoculum concentration of 1% by activation culture bacterium solution overnight, being forwarded in the triangular flask (500mL) containing 100mLLB fluid medium, 37 DEG C of shaking table 180rpm shaken cultivation are to OD600It is 0.4��0.6.
(4) triangular flask is placed in frozen water and stands after 10min, centrifugal (5000rpm, 10min, 4 DEG C), abandon supernatant.
(5) 0.1MCaCl of 50mL ice pre-cooling is added2, with pipettor, thalline is dispelled gently, then ice bath 10min.
(6) centrifugal (5000rpm, 10min, 4 DEG C), abandon supernatant, add the 0.1MCaCl of 25mL ice pre-cooling2, with pipettor, thalline is dispelled gently, ice-water bath 10min.
(7) centrifugal (5000rpm, 10min, 4 DEG C), abandon supernatant, add the 0.1MCaCl of 2mL ice pre-cooling2, add sterilized glycerol to final concentration of 10%, mix gently with pipettor, often pipe 200 �� L subpackage.
(8)-70 DEG C frozen standby.
1.2.6.2 connection product is transformed into competent cell
Connection product is transformed in DH5 �� Bacillus coli cells. Specifically comprise the following steps that
(1) LB/Amp plate is prepared.
(2) take 200 �� L competent cells and be placed in frozen water to bathe cold.
(3) adding 5 �� L in competent cell suspension and connect product, rotating centrifugal pipe is to mix content gently, stands 30min in ice bath.
(4) centrifuge tube being placed in 42 DEG C of water-bath heat shock 90s, then transfers in ice bath by centrifuge tube rapidly, make cell cooling 2min, this process does not shake centrifuge tube.
(5) adding the sterile LB medium (without antibiotic) of 1mL37 DEG C of preheating in each centrifuge tube, mixing is placed on 37 DEG C of shaking table 150rpm shaken cultivation 40min.
(6) centrifuge tube content is mixed, draw the 500 �� L competent cell converted, be added on LB/Amp Agar Plating, with the coating of aseptic spreading rod uniformly. Flat board is placed in room temperature until liquid is absorbed, 37 DEG C of incubators are inverted cultivation 12��16h.
1.2.7 the checking of positive colony
1.2.7.1 bacterium colony PCR checking
Bacterium colony PCR checking (bacterium colony PCR system and PCR program are respectively in Table 5 and table 6) is carried out from picking individual colonies plating medium with toothpick, and toothpick is put in the LB fluid medium containing final concentration of 100 �� g/mLAmp to 5mL, 37 DEG C of shaking table 180rpm shaken cultivation 12h.
Table 5 bacterium colony PCR reaction system
Table 6 bacterium colony PCR response procedures
1.2.7.2 the extraction of recombiant plasmid
Take bacterium colony PCR and be verified as the corresponding bacterium solution of the positive, adopt EasyPurePlasmidMiniPrepKit to extract recombinant plasmid dna. Concrete grammar is as follows:
(1) taking the incubated overnight bacterium solution of 1��4mL, 10000g is centrifuged 1min, exhausts supernatant as far as possible.
(2) add 250 �� L colourless solution RB (containing RNaseA), shake suspension bacterial sediment, should not leave little truffle.
(3) adding 250 �� L blue solution LB, leniently spin upside down mixing 4��6 times, make thalline fully crack, form blue bright solution, color bright is become bright blueness from half, and instruction is cracking (no more than 5min) completely.
(4) adding 350 �� L yellow solution NB, be gently mixed 5��6 times (color, by the complete yellowing of blueness, indicates mix homogeneously, neutralizes completely), until forming the yellow coagulation block of consolidation, room temperature stands 2min.
(5) the centrifugal 5min of 15000g, careful supernatant of drawing adds in adsorption column.
(6) the centrifugal 1min of 15000g, abandons effluent.
(7) add the centrifugal 1min of 650 �� L solution W B, 15000g, abandon effluent.
(8) the centrifugal 1��2min of 15000g, thoroughly removes the WB of residual.
(9) being placed in by adsorption column in a clean centrifuge tube, the central authorities at post add 30 �� LEB or ddH2After O, room temperature stands 1min.
(10) the centrifugal 1min of 1000g, eluted dna, the DNA eluted is in-20 DEG C of preservations.
1.2.7.3 double digestion recombiant plasmid identifies Insert Fragment size
Plasmid is carried out double digestion by restriction enzyme site EcoRI and the NotI selecting recombiant plasmid multiple clone site both sides, verifies purpose clip size. Endonuclease reaction system is prepared by table 7. The condition of endonuclease reaction: 37 DEG C, 4h.
Table 7 endonuclease reaction system
Adopting the effect of 1% agarose gel electrophoresis detection plasmid enzyme restriction, gel is put into gel imaging system imaging, analysis by electrophoresis after terminating.
1.2.7.4 sequence verification and analysis
By the bacterial strain order-checking that Preliminary screening is positive, after checking is correct, naming this recombiant plasmid is pGEX-4T-AIP2, and naming this recombination bacillus coli is E.coliDH5 �� pGEX-4T-AIP2.
Embodiment 2 (expression and purity of bifidobacterium longum adhesion protein)
The first step: the abduction delivering of bifidobacterium longum adhesion protein
The bifidobacterium longum adhesion protein expression strain E.coliDH5 �� pGEX-4T-AIP2 IPTG of Example 1 preparation carries out abduction delivering, specifically comprises the following steps that
(1) the bacterial strain list bacterium colony of picking plate loop method, is seeded in 3mlLB fluid medium, and adds Amp to final concentration of 100 �� g/ml in test tube, cultivates 8h in 37 DEG C of constant temperature culture oscillator 180r/m.
(2) it is forwarded to 20ml less salt LB fluid medium by the inoculum concentration of 1%, adds Amp to final concentration of 100 �� g/ml, cultivate in 37 DEG C of constant temperature culture oscillator 180r/m.
(3) when cell density OD600nm=0.6-0.9, derivant IPTG to final concentration of 1mM is added, in 37 DEG C of constant temperature culture oscillator 180r/m inducing culture 4-5h.
(4) bacterium solution equivalent being transferred to two 50ml centrifuge tubes after inducing culture, 4 DEG C, 5000r/m is centrifuged 8min, abandons supernatant. Being separately added into the resuspended washing bacterial sediment of 10mlPBS buffer, 5000r/m is centrifuged 8min, abandons supernatant, the centrifugal gained thalline of 10ml bacterium solution adds 3mlPBS buffer resuspended, and ice-bath ultrasonic (180W, work 3s, gap 5s) broken 20-30 time, to bacterium solution clear.
(5) gained solution presses 1mL/ pipe subpackage, and-20 DEG C save backup.
Second step: SDS-PAGE detects expressing protein
Adopt the abduction delivering situation of PAGE gel electrophoresis detection adhesion protein, specifically comprise the following steps that
(1) taking each 100 �� l of gained sample in the first step respectively, 4 DEG C, 13000r/m is centrifuged 5min, takes 5 �� l supernatant respectively and adds 2 �� sds gel sample-loading buffer of equivalent, mixes standby.
(2) sample is boiled in 100 DEG C of boiling water baths loading after 6min, by 80V, 25-35min+140V, 1-1.5h electrophoresis.
(3), after electrophoresis, take gel and be placed in sds gel dyeing liquor, room temperature horizontal shaker shake dyeing 4-6h.
(4) reclaiming dyeing liquor, after distilled water flushing gel, add appropriate destaining solution, room temperature shaker rocks decolouring 6-8h, changes destaining solution 2-3 time therebetween. Observation analysis electrophoresis result.
3rd step: the affinity purification of bacillus bifidus adhesion protein
Adopting GST affinity chromatograph column purification destination protein, step is as follows:
(1) balance of glutathione resin prepacked column: using the buffer A (PBS+2mMEDTA) of 30ml to carry out chromatographic column balance, coutroi velocity is about 3mL/min.
(2) combination of fusion protein and chromatographic column: the cell crude extract loading that will clarify, coutroi velocity is about 1-2mL/min.
(3) eluting of fusion protein: by buffer B (PBS+2mMEDTA+20mM glutathion) by destination protein eluting after curve smooth decreasing, coutroi velocity is 1ml/min, collects eluting peak.
(4) protein concentrate: use super filter tube concentrate eluant, crosses desalting column, pure water eluting, and coutroi velocity is 3mL/min, takes eluting peak solution, after vacuum lyophilization, takes to be partly dissolved and carries out SDS-PAGE electroresis appraisal. Qualification result is as shown in Figure 1.
Embodiment 2 (a kind of aminoacid sequence is the method for producing protein of SEQIDNO:1)
1) take recombination bacillus coli E.coliDH5 �� pGEX-4T-AIP2 to cultivate, add derivant IPTG when being 0.6 to culture fluid OD600nm to final concentration of 0.8mM, then in 35 DEG C, concussion when inducing culture 4h;
2) collecting thalline, after thalline is resuspended in PBS, under condition of ice bath, ultrasonication 20 times (the every time broken persistent period is 2s, and the interval time between crushing for every 2 times is 4s), then collects supernatant;
3) take buffer A, balance glutathione Sepharose resin post with the flow velocity of 2.5mL/min; Take step 2) supernatant, with the flow velocity loading of 1mL/min; Take buffer B, with the flow velocity eluting of 0.8mL/min, collect eluting peak place solution and be eluent; Taking eluent to be concentrated by ultrafiltration, then cross desalting column, recycling pure water, with the flow velocity eluting of 2.5mL/min, is collected eluting peak place solution and is the second eluent, and postlyophilization, namely obtain described protein;
Wherein said buffer A is the PBS containing 1.5mMEDTA;
Described buffer B is the PBS containing 1.5mMEDTA, 15mM glutathion.
Embodiment 3 (a kind of aminoacid sequence is the method for producing protein of SEQIDNO:1)
1) take recombination bacillus coli E.coliDH5 �� pGEX-4T-AIP2 to cultivate, add derivant IPTG when being 0.9 to culture fluid OD600nm to final concentration of 1.2mM, then in 39 DEG C, concussion when inducing culture 5h;
2) thalline, broken, collection supernatant are collected;
3) take buffer A, balance glutathione Sepharose resin post with the flow velocity of 3.5mL/min; Take step 2) supernatant, with the flow velocity loading of 2mL/min; Take buffer B, with the flow velocity eluting of 1.2mL/min, collect eluting peak place solution and be eluent; Taking eluent to be concentrated by ultrafiltration, then cross desalting column, recycling pure water, with the flow velocity eluting of 3.5mL/min, is collected eluting peak place solution and is the second eluent, and postlyophilization, namely obtain described protein;
Wherein said buffer A is the PBS containing 2.5mMEDTA;
Described buffer B is the PBS containing 2.5mMEDTA, 25mM glutathion.
Embodiment 4 (a kind of aminoacid sequence is the method for producing protein of SEQIDNO:1)
1) take recombination bacillus coli E.coliDH5 �� pGEX-4T-AIP2 to cultivate, add derivant IPTG when being 0.7 to culture fluid OD600nm to final concentration of 0.9mM, then in 37 DEG C, concussion when inducing culture 4.5h;
2) collecting thalline, after thalline is resuspended in PBS, under condition of ice bath, ultrasonication 30 times (the every time broken persistent period is 4s, and the interval time between crushing for every 2 times is 6s), then collects supernatant;
3) step 2 is taken) supernatant, utilize GST affinitive layer purification, be concentrated by ultrafiltration after collecting eluent, desalination, dry, namely obtain described protein.
Embodiment 5 (albumen prepared by embodiment 2 is respectively on listeria monocytogenes, Escherichia coli O 157: H7, Salmonella typhimurium ampicillin-sensitive impact experiment)
Listeria monocytogenes, Escherichia coli O 157: H7 and Salmonella typhimurium all co-culture 2h with bifidobacterium longum specific proteins AIP2, respectively three kinds of pathogenic bacterium equivalent are transferred to after carrying out count plate the equal-volume LB fluid medium adding variable concentrations ammonia benzyl, adhesion protein process group and GST process group are carried out count plate contrast. Concrete grammar is as follows:
(1) by aseptic PBS solution, adhesion protein AIP2 being diluted to concentration respectively to be 45 �� g/mL, GST and be diluted to same concentrations as comparison, 4 DEG C save backup.
(2) listeria monocytogenes is cultured to mid-log phase, 8000r/min, under 4 DEG C of conditions after centrifugal 5min, collects thalline, and aseptic PBS washs 3 times, respectively with GST diluent, the resuspended thalline of adhesion protein AIP2 diluent, is diluted to bacterial concentration and is 106CFU/mL, processes 2h in 37 DEG C of antibacterial culturing oscillators.
(3) listeria monocytogenes, Escherichia coli O 157: H7 and the Salmonella typhimurium that equivalent inoculation adhesion protein processed and GST processes respectively adds the ammonia benzyl LB fluid medium of variable concentrations in 2mL, ammonia benzyl Concentraton gradient is 50,25,10,1,0.1 �� g/mL, all cultivates 6h in 37 DEG C of isothermal vibration incubators, carries out count plate. Experimental result is shown in Fig. 2.
As shown in Figure 2, compared with matched group, it is attached the microorganism fungistatic effect under antibiotic effect after albumen processes to become apparent from, owing to 0, antibiotic place matched group is suitable with the biomass level of attachment proteins process group, it can be considered that attachment proteins itself does not have direct fungistatic effect, and only play the antibacterial potentiation of antibiotic.
Embodiment 6 (albumen prepared by embodiment 2 is respectively on listeria monocytogenes, Escherichia coli O 157: the impact experiment of H7, the benzylpenicillin sensitivity of Salmonella typhimurium, cephamycin sensitivity, kanamycin sensitivity, chloramphenicol sensitivity)
Listeria monocytogenes, Escherichia coli O 157: H7 and Salmonella typhimurium all co-culture 2h with bifidobacterium longum specific proteins AIP2, respectively three kinds of pathogenic bacterium equivalent are transferred to after carrying out count plate the equal-volume LB fluid medium adding variable concentrations ammonia benzyl, adhesion protein process group and GST process group are carried out count plate contrast. Concrete grammar is as follows:
(1) by aseptic PBS solution, adhesion protein AIP2 being diluted to concentration respectively to be 55 �� g/mL, GST and be diluted to same concentrations as comparison, 4 DEG C save backup.
(2) listeria monocytogenes is cultured to mid-log phase, 8000r/min, under 4 DEG C of conditions after centrifugal 5min, collects thalline, and aseptic PBS washs 3 times, respectively with GST diluent, the resuspended thalline of adhesion protein AIP2 diluent, is diluted to bacterial concentration and is 106CFU/mL, processes 2h in 37 DEG C of antibacterial culturing oscillators.
(3) listeria monocytogenes, Escherichia coli O 157: H7 and the Salmonella typhimurium that equivalent inoculation adhesion protein processed and GST processes respectively adds the LB fluid medium of concentration respectively 1 �� g/mL benzylpenicillin, 1 �� g/mL cephamycin, 2 �� g/mL kanamycin or 2 �� g/mL chloromycetin in 2mL, in 37 DEG C of isothermal vibration incubators, all cultivate 6h, carry out count plate. Result is shown in Fig. 3.
As it is shown on figure 3, the microorganism after adhesion protein process receives more prominent suppression under antibiotic effect, cell density is commonly less than the 1/10 of matched group, it can be seen that, bifidobacterium longum protein of the present invention has antibacterial potentiation definite, significant.
Above embodiments of the invention are described in detail, but described content has been only presently preferred embodiments of the present invention, not in order to limit the present invention. All any amendment, equivalent replacement and improvement etc. made in the application range of the present invention, should be included within protection scope of the present invention.
Claims (5)
1. the aminoacid sequence such as protein shown in SEQIDNO:1 is for promoting the application of Salmonella typhimurium Antibiotic Sensitivity.
2. application according to claim 1, it is characterised in that described antibiotic is beta-lactam antibiotic.
3. application according to claim 1, it is characterised in that described antibiotic is aminoglycoside antibiotics.
4. application according to claim 1, it is characterised in that described antibiotic is amphenicols antibiotic.
5. application according to claim 1, its feature is utilize the protein solution be 45��55 �� g/mL, aminoacid sequence being SEQIDNO:1 containing concentration in described application, processes Salmonella typhimurium with the way of contact.
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| CN102206599A (en) * | 2011-04-11 | 2011-10-05 | 天津科技大学 | Oxygen-resistant acid-resistant Bifidobacterium longum |
| CN103911337A (en) * | 2013-01-09 | 2014-07-09 | 中国海洋大学 | High adhesion clostridium butyricum and its preparation method |
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| CN102206599A (en) * | 2011-04-11 | 2011-10-05 | 天津科技大学 | Oxygen-resistant acid-resistant Bifidobacterium longum |
| CN103911337A (en) * | 2013-01-09 | 2014-07-09 | 中国海洋大学 | High adhesion clostridium butyricum and its preparation method |
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| CN109628343A (en) * | 2018-12-26 | 2019-04-16 | 嘉兴益诺康生物科技有限公司 | Bifidobacterium breve YH68 and the application in reduction Salmonella Typhimurium Infection risk product |
| CN109628343B (en) * | 2018-12-26 | 2022-02-18 | 嘉兴益诺康生物科技有限公司 | Bifidobacterium breve YH68 and application thereof in product for reducing infection risk of salmonella typhimurium |
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