CN106497840B - A kind of method of directional separation immune regulative enteron aisle lactobacillus - Google Patents

A kind of method of directional separation immune regulative enteron aisle lactobacillus Download PDF

Info

Publication number
CN106497840B
CN106497840B CN201610975479.XA CN201610975479A CN106497840B CN 106497840 B CN106497840 B CN 106497840B CN 201610975479 A CN201610975479 A CN 201610975479A CN 106497840 B CN106497840 B CN 106497840B
Authority
CN
China
Prior art keywords
lactobacillus
thallus
added
buffer
magnetic bead
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN201610975479.XA
Other languages
Chinese (zh)
Other versions
CN106497840A (en
Inventor
孙进
朱华玲
齐策
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Jiangnan University
Original Assignee
Jiangnan University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Jiangnan University filed Critical Jiangnan University
Priority to CN201610975479.XA priority Critical patent/CN106497840B/en
Publication of CN106497840A publication Critical patent/CN106497840A/en
Application granted granted Critical
Publication of CN106497840B publication Critical patent/CN106497840B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/02—Separating microorganisms from their culture media

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Biotechnology (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Microbiology (AREA)
  • Biomedical Technology (AREA)
  • Virology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

The invention discloses a kind of methods of directional separation immune regulative enteron aisle lactobacillus, belong to biologic product technology field.The present invention biotinylated anti-IgA antibody processing animal or people's saliva, intestinal contents or fecal bacteria suspension, then it is sorted using the nanometer magnetic bead of streptavidin, the enteron aisle lactobacillus with immunoloregulation function is obtained after enrichment culture and Selective Separation.Be compared with the traditional method, this method can quickly, directional separation immune regulative beneficial bacterium, the bacterial strain of acquisition can be used for feed, ordinary food or functional food after further verifying acid resistance, resisting biliar and adhesiveness.

Description

A kind of method of directional separation immune regulative enteron aisle lactobacillus
Technical field
The present invention relates to a kind of methods of directional separation immune regulative enteron aisle lactobacillus, belong to biologic product technology neck Domain.
Background technique
Lactobacillus is the important member of probiotics, is widely present in human body and animal gastrointestinal tract.Home and abroad is existing a large amount of Test confirms that host is adjusted to function of immune system in intracorporal a variety of lactobacillus, this is their infection prevention diseases, improves Food hypersenstivity, anticancer, firm gut barrier, the basis for improving immunologic adjuvant performance gentle the effects of solving intestinal inflammatory.It is made With mechanism, directly immune cell activated is related with secretory immune instrumentality.
The research of a large amount of separation intestine immunity modulability lactobacillus is carried out both at home and abroad.Separation screening intestine immunity tune Section property lactobacillus is had forefathers and is screened using conventional traditional method, is carried out spread plate with MRS solid medium and is obtained To a large amount of bacterial strains, Gram's staining is then carried out, microscopy discards gram negative strain, tests later by catalase With C.perfringens stormy fermentation test, discard catalase positive and C.perfringens, finally by Gram-positive, Negative catalase and the PCR of lactobacillus specificity is finally for lactobacillus finally determines and to sift out without rod-shaped fix tentatively of gemma Bacterium is lactobacillus.This method has certain limitation, sieves the bacterium not necessarily intestine immunity modulability that the bacterium period is long and sifts out The uncertainty of lactobacillus, conventional method leads to the uncertainty of screening operation.In addition, this cultivation method detection limit is 10cfu/g。
To sum up, intestine immunity modulability lactobacillus is to be screened to obtain using the traditional separation method of experience directiveness 's.A large amount of enteron aisle lactobacillus strains are obtained first with selective medium separation, it is then investigated respectively and adheres to enteric bacteria Ability, acid resistance and external evoked cytokine activity.This separation method workload is very big, needs a large amount of human and material resources, It is also very long that active bacterial strain screens the period.But the activity in vivo difference that many Physiological in vitro show similar bacterial strain is very big [Ibnouzekri et al., 2003], more increases the complexity of screening operation.
Summary of the invention
To solve the above-mentioned problems, the present invention establishes a kind of side of targeting separation high-immunity regulation activity lactobacillus strain Method.Quick, orientation the method for separating high immune regulative lactobacillus from human or animal's intestinal contents or excrement of the invention, The advantages of based on immunomagnetic bead technique, is easy to the characteristics of combining using biotin and Streptavidin, fast and effeciently from excrement Middle separation IgA wraps up lactobacillus, then recycles special media isolated strains, and carried out fastly with lactobacillus specific PCR Fast Preliminary Identification.
Lactobacillus can enter small intestine peyer's patches (PP) by M cell.PP is small intestine most important antigen acquisition position, interior There are a large amount of macrophages, dendritic cells, T cell and B cells in portion.Dendritic cells absorb lactobacillus, are stimulated maturation, will induce B cell, which becomes, produces IgA thick liquid cell.B cell can produce two kinds of IgA, and one kind is with the most of enteric bacteria of weak force combination Natural IgA, another is with the IgA of high-affinity combination immune induction bacterium.The immunoregulation effect of lactobacillus has obvious Bacterial strain dependence, high activity bacterial strain can enter PP induction high-affinity IgA generate, therefore, the stronger intestines of immunostimulatory activity Lactobacillus its cell surface in road is all wrapped up by high-affinity IgA.It also can promote lactobacillus after IgA package and enter PP successive induction Immune response, also can be enhanced its anti-inflammatory activity.
The method of directional separation immune regulative enteron aisle lactobacillus of the invention successively includes: that (1) prepares thallus suspension Liquid；(2) lowlenthal serum or bovine serum albumin(BSA) are added in suspension, ice bath is for a period of time；Then biotin labeling is added Rabbit-anti people IgA ice bath for a period of time；Streptavidin MagneSphere is added, ice bath is for a period of time；Last magnetic absorption is combined with The Streptavidin MagneSphere of thallus, washing obtain thallus；(3) by thallus enrichment culture obtained in the previous step；(4) identification enrichment The bacterial strain cultivated, the bacterial strain for being accredited as lactobacillus is the lactobacillus strain of high-immunity regulation activity.
In one embodiment of the invention, the thallus suspension liquid is saliva, intestinal contents or excrement preparation Bacterial suspension.
In one embodiment of the invention, the thallus suspension liquid is by sample when sample is solid-state or semisolid Product are added in the buffer of sterilizing, and whirlpool mixes, and low-speed centrifugal takes supernatant and handles through cell filtering net, the liquid being obtained by filtration Then body is resuspended in buffer through multiple high speed centrifugation, washing, obtain the thallus suspension liquid of mixed cell；When sample is liquid When body, directly sample is added in the buffer of sterilizing, whirlpool mixes, and high speed centrifugation takes precipitating, repeatedly washed, is centrifuged, so After be resuspended in buffer and obtain thallus suspension liquid.
In one embodiment of the invention, the low-speed centrifugal is in 800~1200rpm；The high speed centrifugation is In 8000~12000rpm.
In one embodiment of the invention, the buffer is peptone buffer agent.
In one embodiment of the invention, the effect of the lowlenthal serum or bovine serum albumin(BSA) is that closing is non-specific Property combine.
In one embodiment of the invention, the additive amount of the lowlenthal serum or bovine serum albumin(BSA) is in bacteria suspension Final concentration be respectively 10%, 0.05~0.5%.
In one embodiment of the invention, the additive amount of the Streptavidin MagneSphere is 0.1-1.0mg/mL.
In one embodiment of the invention, the preparation of the Streptavidin MagneSphere is first to prepare amination nano magnetic Then pearl connect with glutaraldehyde, the amination nanometer magnetic bead for connecting upper glutaraldehyde is reacted in buffer with Streptavidin, After reaction using the nanometer magnetic bead of externally-applied magnetic field absorption streptavidin, clean to get Streptavidin MagneSphere is arrived.
In one embodiment of the invention, the preparation of the Streptavidin MagneSphere is specifically:
1. amination nanometer magnetic bead: experimental ware used is impregnated overnight in chloroazotic acid, cleans drying, Xiang Fangyou rotor 60mL ethylene glycol, 13g hexamethylene diamine, 4g anhydrous sodium acetate, 2g FeCl are added in round-bottomed flask3·6H2O, in 50 DEG C of oil baths to molten Liquid is uniform, and liquid is transferred in the autoclave with tetrafluoroethene liner, in 198 DEG C of reaction 6h, after being cooled to room temperature It is transferred in beaker, after dehydrated alcohol and the multiple black solid of deionized water repeated flushing, by black solid in 50 DEG C of items 10h is dried under part, then dry black solid arrives amination nanometer magnetic bead using agate mortar grind into powder.
2. glutaraldehyde connects: weighing 2mg amination magnetic bead in centrifuge tube, glutaraldehyde/PBS buffer solution (volume is added Than reacting 2h in 37 DEG C of shaking tables, magnetic bead being sucked using the magnetic force of magnet, cleans 5 repeatedly with PBS buffer solution for 5%) 2mL More than secondary, unbonded glutaraldehyde is removed.
3. Streptavidin MagneSphere: adding 2mL PBS buffer solution in the amination magnetic bead for connecting upper glutaraldehyde, then again The Streptavidin 250uL of 1mg/mL is added, reacts 12h in 37 DEG C of shaking tables, is cleaned repeatedly using externally-applied magnetic field with PBS buffer solution 5 times or more, unbonded Streptavidin is removed, collects precipitating to get the nanometer magnetic bead of streptavidin is arrived.Magnetic bead cleaning After be placed in 4 DEG C of environment and save backup.
In one embodiment of the invention, the time of the ice bath is 10-30min.
In one embodiment of the invention, the step (3) is specifically:
1. 5% lowlenthal serum, ice is added in the thallus suspension liquid that mixed cell is suspended in that peptone buffer agent obtains Bathe 15min；Rabbit-anti the people IgA, ice bath 20min of biotin labeling is added；Then Streptavidin MagneSphere, ice bath 20min is added.
2. adsorbing magnetic bead combination bacterium with magnet or magnetic bead sorting device, supernatant is outwelled；Peptone buffer agent is used later Clean 3 thallus.
3. finally the thallus that Streptavidin MagneSphere adsorbs is resuspended in peptone buffer agent.
In one embodiment of the invention, the enrichment culture is the first enrichment culture 6-8h on enriched medium, Then it repeatedly crosses, purify on lactobacillus specific isolation culture medium (LAMVAB), obtain candidate strain.
In one embodiment of the invention, the formula of the enriched medium: arabinose 20mM；Ribose 20mM； MgSO41mM；MnSO40.1mM；FeSO45uM。
In one embodiment of the invention, the formula of the lactobacillus specific isolation culture medium: solution A: MRS training Base 250mL, cysteine hydrochloride 0.0125g, bromocresol green 0.0012g are supported, pH to 5.0 ± 0.1 is adjusted；Solution B: 10g agar is molten In 250mL deionized water；Solution C: 5mL vancomycin hydrochloride, 2mg/mL, sterile water are prepared；Equivalent A, 121 DEG C of B solution go out Bacterium 15min, B are cooled to 50 DEG C, and A is cold to be gone to room temperature to add solution C, mix, then A, B solution are mixed, inverted plate.
In one embodiment of the invention, the identification is to carry out PCR identification using lactobacillus specific primer.
In one embodiment of the invention, the identification is specifically: extracting candidate strain genomic DNA, utilizes cream Bacillus Genus-specific primers carry out PCR, and the primer is (sequence is respectively as shown in SEQ ID NO:1 and SEQ ID NO:2):
LbLMA1-rev (5 '-CTCAAAACTAAACAAAGTTTC-3 ') and R16-1 (5 '- CTTGTACACACCGCCCGTCA-3')；The obtained bacterial strain containing 200~500bp distinctive products is that high immunological regulation is living The lactobacillus strain of property.
Advantages of the present invention:
The method of the present invention, preparation, intestinal contents or the preparation of fecal bacteria suspension, magnetic bead including immunomagnetic beads combine Reaction and magnetic bead sorting, Enrichment of bacteria and selectivity culture, the identification of PCR method lactobacillus.The method of the present invention is based on high activity bacterial strain The IgA of secretion inducing, which enters enteric cavity, may specifically bind the bacterial strain, affine magnetic bead and fecal bacteria using targeting in conjunction with IgA Suspension is acted on and is cleaned.During the experiment, the natural IgA in conjunction with bacterium and magnetic bead in combination be in the process of cleaning It can be washed off, only retain magnetic bead with high-affinity IgA in conjunction with, by magnetic bead sorting, and enrichment culture, thus quick separating work Property bacterial strain.Meanwhile by separation method, the additive amount of lowlenthal serum or bovine serum albumin(BSA), Streptavidin MagneSphere The optimization such as additive amount, ice bath time further improves screening efficiency, shortens the screening period.
Compared with conventional lactobacillus screening, there are situation separation, screening by cell surface high-affinity IgA by the present invention Lactobacillus greatly improves immunological regulation bacterium separation selectivity, is also obviously shortened the bacterial strain screening period.
Detailed description of the invention
Fig. 1 is the flow chart of directional separation high-affinity IgA combination lactobacillus；
Fig. 2 is the lactobacillus specific PCR electrophoresis result figure of the IgA package lactobacillus screened from excrement；
Fig. 3 is the lactobacillus specific PCR electrophoresis result figure of the IgA package lactobacillus screened from saliva；
Fig. 4 is the immune regulative lactobacillus inducing mouse peritoneal macrophage Raw264.7 cell factor of separate sources The generation of TNF-α (a), IL-6 (b), IL-12 (c) and IL-10 (d)；Wherein, LPS represents lipopolysaccharides；Magnetic bead in F1 generation table excrement The lactobacillus of absorption；FS1 represents the lactobacillus after magnetic bead adsorbs in supernatant；T1 represents the lactobacillus that magnetic bead adsorbs in saliva； TS1 represents the lactobacillus after magnetic bead adsorbs in supernatant；Wherein * indicates that there were significant differences compared to the blank group, P < 0.05；* table Showing compared to the blank group has extremely significant difference, P < 0.01；# indicates that there were significant differences compared with LPS negative control group, P < 0.05；## expression has extremely significant difference, P < 0.01 compared with LPS negative control group；
Fig. 5 is TNF-α/IL-10 (e), IL-6/IL-10 (f) and IL-12/IL-10 (g)；Wherein, LPS represents lipopolysaccharides； The bacterium that magnetic bead adsorbs in F1 generation table excrement；FS1 represents the bacterium after magnetic bead adsorbs in supernatant；T1 represents what magnetic bead in saliva adsorbed Bacterium；TS1: the bacterium after magnetic bead absorption in supernatant；Wherein * indicates that there were significant differences compared to the blank group, P < 0.05；* indicate with Blank group, which is compared, extremely significant difference, P < 0.01；# indicates that there were significant differences compared with LPS negative control group, P < 0.05；## table Show has extremely significant difference, P < 0.01 compared with LPS negative control group.
Specific embodiment
Below in conjunction with case study on implementation, the present invention is further described；Following embodiments are illustrative, are not limited , it cannot be limited the scope of protection of the present invention with following embodiments.
Embodiment 1: the preparation of Streptavidin MagneSphere
Streptavidin MagneSphere is prepared in accordance with the following methods
(1) amidized Fe3O4The preparation of nanometer magnetic bead
1. round-bottomed flask, rotor and reaction kettle liner are all impregnated in chloroazotic acid overnight, then cleaned up with dish washing liquid, It is put into baking oven and dries after being cleaned again with deionized water.
2. 30mL ethylene glycol is added into the round-bottomed flask for be placed with rotor, 6.5g 1 is added, 6- hexamethylene diamine adds 2.0g Anhydrous sodium acetate, finally plus 1.0g FeCl3·6H2O。
3. oil bath adds reflux unit, and the general 0.5h-1h of magnetic agitation in 50 DEG C by round-bottomed flask.It reacts to flask Interior solution is uniform.
4. the solution in round-bottomed flask is carefully transferred in the autoclave liner of tetrafluoroethene liner, and will be anti- Answer kettle to tighten, be placed under 198 DEG C of high temperature and react 6h, after after cooled to room temperature (cooling overnight).
5. liquid in reaction kettle is stirred evenly with glass bar, carefully pours into 250mL beaker, repeatedly rushed with dehydrated alcohol Reaction kettle is washed, cleaning solution also pours into beaker together.
6. the black magnetic particle in beaker is dispersed (about 5min) using ultrasound, lifted down from ultrasonic pond by beaker It being placed on magnet, is collected using Magneto separate, black particle will be attracted on bottom of a cup, the solution in beaker poured out with magnet, Remaining liquid is sucked out with liquid-transfering gun.
7. 40mL deionized water and washes of absolute alcohol is used alternatingly, the black magnetic particle in beaker is divided using ultrasound It dissipates (about 5min), beaker is lifted down from ultrasonic pond and is placed on magnet, is collected using Magneto separate, black particle will be inhaled In bottom of a cup, the solution in beaker is poured out with magnet, remaining liquid is sucked out with liquid-transfering gun.
8. repeating step 7 twice.
Dry 10h under the conditions of obtained black solid is placed on 50 DEG C.
9. grind into powder is in dry black solid particle agate mortar to get arriving amidized Fe3O4Nano magnetic Pearl is placed in 4 DEG C and stores for future use.
(2) glutaraldehyde is connected in amination magnetic bead
The above-mentioned amination magnetic bead 2mg made is weighed in centrifuge tube, glutaraldehyde/PBS buffer solution is added, and (volume ratio is 5%) 2mL reacts 2h in 37 DEG C of shaking tables, magnetic bead is sucked using the magnetic force of magnet, with PBS buffer solution clean repeatedly 5 times with On, remove unbonded glutaraldehyde.
(3) Streptavidin MagneSphere
Add 2mLPBS buffer solution in connection in the amination magnetic bead of glutaraldehyde, the strepto- for then adding 1mg/mL is affine Plain 250uL is reacted 12h in 37 DEG C of shaking tables, is cleaned 5 times or more, removed unbonded repeatedly with PBS buffer solution using externally-applied magnetic field Streptavidin collects precipitating to get the nanometer magnetic bead of streptavidin is arrived.It is placed in 4 DEG C of environment and protects after magnetic bead cleaning It deposits spare.
Embodiment 2: Streptavidin MagneSphere technology separation screening immunological regulation lactobacillus from human faecal mass is used
The embodiment be using Streptavidin MagneSphere technology from human faecal mass separation screening immunological regulation lactobacillus, Fig. 1 For the flow chart of directional separation high-affinity IgA combination lactobacillus.
Specifically includes the following steps:
Using Streptavidin MagneSphere method, separation screening IgA wraps up lactobacillus from excrement:
1. weighing the excrement of 1g Healthy People, the peptone buffer agent of 10mL sterilizing is added, whirlpool mixes 5min.
2. being centrifuged 5min at 1000rpm, supernatant, big fragment and supernatant is taken to pass through 200 sterile aim cells In filter screen filtration to a new pipe.
3. the obtained centrifuge tube of filtering is centrifuged 5min at 10000rpm, supernatant is outwelled.The peptone of 5mL is added Buffer, piping and druming mix, and are centrifuged 5min under 10000rpm, outwell supernatant, repeat primary.
4. precipitating is resuspended in 5mL peptone buffer agent again, 5% lowlenthal serum, ice bath 15min is added.It is added Rabbit-anti the people IgA, ice bath 20min of biotin labeling.Then entering the nano immune magnetic bead of marked by streptavidin, (i.e. strepto- is affine Biscuit porcelain pearl), ice bath 20min.
5. being adsorbed with magnet in tube bottom, supernatant is poured in a new sterilizing pipe.Later with sterilizing Peptone buffer agent cleans 3 thallus.
6. finally the thallus that immunomagnetic beads adsorb is resuspended in peptone buffer agent, the experiment of next step is carried out.
The IgA package bacterium enrichment culture 6-8h on enriched medium obtained in the previous step, then in LAMVAB spy Scribing line culture is carried out on anisotropic culture medium flat plate, 37 DEG C of culture 48h are then selected on each plate with specificity, bacterium 4 plants of different bacterium of form are fallen, then this 4 plants of bacterium are inoculated into respectively in MRS fluid nutrient medium, 37 DEG C are cultivated for 24 hours, then Scribing line purifying is carried out on LAMVAB special media plate, chooses single colonie culture, so purifying 3 times, obtains tens plants of bacterium.
The basis of enriched medium are as follows: arabinose 20mM；Ribose 20mM；MgSO41mM；MnSO40.1mM； FeSO45uM。
After MRS fluid nutrient medium expands culture, extracts bacterial strain bacterium group DNA and extracts:
Lactobacillus specific PCR:
To prove whether above-mentioned isolated bacterial strain is that sIgA wraps up lactobacillus, the DNA of purifying bacterial strain obtained above is used Carry out lactobacillus specific PCR.
PCR reaction system 25uL:10buffer-MgCl2 2.5uL；MgCl2 2uL；dNTP 0.5uL；Upstream primer 0.5uL；Downstream primer 0.5uL；Template DNA 3uL；Taq enzyme 0.5uL；DEPC water 13.5uL.
PCR amplification program are as follows: 94 DEG C of initial denaturation 5min；94 DEG C of denaturation 30s, 64 DEG C of annealing 30s, 72 DEG C of extension 60s, 30 Circulation；72 DEG C of extension 15min, are cooled to 12 DEG C.
After PCR, in 1% agarose gel electrophoresis, the bacterial strain for not running out of band is rejected, to obtain from excrement The sIgA of screening wraps up lactobacillus.
Embodiment 3: Streptavidin MagneSphere technology separation screening immunological regulation lactobacillus from saliva is used
The embodiment is that separation screening IgA wraps up lactobacillus from saliva using Streptavidin MagneSphere technology.
It is a kind of based on Streptavidin MagneSphere technology separation IgA package lactobacillus method specifically includes the following steps:
The preparation of Streptavidin MagneSphere: in the same manner as in Example 1.
Using Streptavidin MagneSphere method, separation screening IgA wraps up lactobacillus from saliva:
1. drawing the saliva of 1mL Healthy People, the peptone buffer agent of 9mL sterilizing is added, whirlpool mixes 5min.
2. being centrifuged 5min at 10000rpm after mixing, supernatant is outwelled.The peptone buffer agent of 5mL sterilizing is added, blows It beats and mixes, be centrifuged 5min under 10000rpm, outwell supernatant, repeat primary.
4. precipitating is resuspended in 5mL peptone buffer agent again, 5% lowlenthal serum, ice bath 15min is added.It is added Rabbit-anti the people IgA, ice bath 20min of biotin labeling.Then enter the nano immune magnetic bead of marked by streptavidin, ice bath 20min.
5. being adsorbed with magnet in tube bottom, supernatant is poured in a new sterilizing pipe.Later with sterilizing Peptone buffer agent cleans 3 thallus.
6. finally the thallus that immunomagnetic beads adsorb is resuspended in peptone buffer agent, the experiment of next step is carried out.
The IgA package bacterium enrichment culture 6-8h in enriched medium obtained in the previous step, then in LAMVAB spy Scribing line culture is carried out on anisotropic culture medium flat plate, 37 DEG C of culture 48h are then selected on each plate with specificity, bacterium 4 plants of different bacterium of form are fallen, then this 4 plants of bacterium are inoculated into respectively in MRS fluid nutrient medium, 37 DEG C are cultivated for 24 hours, then Scribing line purifying is carried out on LAMVAB special media plate, chooses single colonie culture, so purifying 3 times, obtains tens plants of bacterium.
After MRS fluid nutrient medium expands culture, a part saves thallus in 40% Freezing Glycerine, and a part is for extracting Bacterial genomes DNA.
Bacterial strain bacterium group DNA is purified to extract:
Use Ezup pillar bacterial genomes DNA extraction agent box extraction purification strain gene group DNA.
Lactobacillus specific PCR:
To prove whether above-mentioned isolated bacterial strain is that IgA wraps up lactobacillus, using purifying bacterial strain obtained above DNA into Row lactobacillus specific PCR.
PCR reaction system 25uL:10buffer-MgCl2 2.5uL；MgCl2 2uL；dNTP 0.5uL；Upstream primer 0.5uL；Downstream primer 0.5uL；Template DNA 3uL；Taq enzyme 0.5uL；DEPC water 13.5uL.
PCR amplification program are as follows: 94 DEG C of initial denaturation 5min；94 DEG C of denaturation 30s, 64 DEG C of annealing 30s, 72 DEG C of extension 60s, 30 Circulation；72 DEG C of extension 15min, are cooled to 12 DEG C.
After PCR, in 1% agarose gel electrophoresis, the bacterial strain for not running out of band is rejected, to obtain from saliva The sIgA of separation screening wraps up lactobacillus.
Fig. 2-3 is from the electrophoretogram after the lactobacillus specific PCR that the IgA screened in excrement and saliva wraps up lactobacillus. F6, F7, F21 as shown in Figure 2 do not have band, remaining just to wrap up lactobacillus for IgA；T2 as shown in Figure 3 does not run out of band, remaining Just for IgA wrap up lactobacillus.
Embodiment 4: cell experiment
Lactobacillus obtained in the obtained slave excrement of above-described embodiment 2-3 and saliva is subjected to cell experiment, verifying obtains The ability that generates of the external evoked cell factor of lactobacillus.
Supernatant bacterial strain after Turnover of Mouse Peritoneal Macrophages Raw264.7 and the above-mentioned bacterium filtered out and magnetic bead absorption It is co-cultured, experiment is divided into following four groups:
(1) 100uL PBS blank group: is added；
(2) experimental group: it is 1 × 10 that 100uL concentration, which is added,5(CFU)/mL F1 (lactobacillus that magnetic bead adsorbs in excrement), F1 supernatant (lactobacillus after magnetic bead absorption in supernatant), T1 (lactobacillus that magnetic bead adsorbs in saliva), T1 supernatant (magnetic bead Lactobacillus after absorption in supernatant)；
(3) positive controls: it is 1 × 10 that 100uL concentration, which is added,5(CFU)/mL L.rhamnosus GG (LGG) is (positive Control)；
(4) negative control group: 1 μ g/mL LPS；The concentration of Turnover of Mouse Peritoneal Macrophages Raw264.7 is adjusted to 5 × 105 (CFU)/mL。
37 DEG C, 5%CO2Cell incubator culture 12h, is collected by centrifugation supernatant, wants in strict accordance with ELISA kit specification It asks, measures IL-6, TNF-α, the level of IL-12 and IL-10 in Turnover of Mouse Peritoneal Macrophages Raw264.7.
By can be seen that LPS can significantly raise the expression of the inflammatory cytokines such as IL-6, TNF-α and IL-12 in Fig. 4 and Fig. 5 Amount, LPS group reaches extremely significant difference (P < 0.01) to the secretory volume of IL-6, TNF-α and IL-12 compared to the blank group；But from Fig. 4 In that F1 and T1 can be obtained is low compared with the expression quantity of the inflammatory cytokines such as the IL-6 of FS1 and TS1, TNF-α and IL-12, but to IL-10 Expression do not have an impact, illustrate preliminary identification immune regulative lactobacillus immunological regulation performance in the present invention；Equally, exist The raising of TNF-α/IL-10 ratio represents the uncoordinated of cytokine secretion in Fig. 5, will lead to inflammatory reaction, LPS, FS1 and TNF-α/IL-10 of TS1 is significantly higher than blank group, and LPS, FS1 and TS1 group reach aobvious to TNF-α/IL-10 compared to the blank group It writes difference (P < 0.05).Based on above-mentioned conclusion, the method for the present invention can obtain intestine immunity modulability lactobacillus with separation screening.
Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not intended to limit the invention, any to be familiar with this skill The people of art can do various change and modification, therefore protection model of the invention without departing from the spirit and scope of the present invention Enclosing subject to the definition of the claims.
<110>Southern Yangtze University
<120>a kind of method of directional separation immune regulative enteron aisle lactobacillus
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 21
<212> DNA
<213>artificial sequence
<400> 1
ctcaaaacta aacaaagttt c 21
<210> 2
<211> 20
<212> DNA
<213>artificial sequence
<400> 2
cttgtacaca ccgcccgtca 20

Claims (8)

1. a kind of method of directional separation immune regulative enteron aisle lactobacillus, which is characterized in that the method successively includes: (1) Prepare thallus suspension liquid；(2) bovine serum albumin(BSA) or lowlenthal serum ice bath is added for a period of time in suspension；Then it is added The rabbit-anti people IgA ice bath of biotin labeling is for a period of time；Streptavidin MagneSphere is added, ice bath is for a period of time；It is last magnetic Absorption is combined with the Streptavidin MagneSphere of thallus, and washing obtains thallus；(3) by thallus enrichment culture obtained in the previous step； (4) bacterial strain that identification enrichment culture arrives, the bacterial strain for being accredited as lactobacillus is the lactobacillus strain of high-immunity regulation activity.
2. the method according to claim 1, wherein the thallus suspension liquid is saliva, intestinal contents or excrement Just the bacterial suspension prepared.
3. the method according to claim 1, wherein the bovine serum albumin(BSA) or lowlenthal serum additive amount are It is respectively 10%, 0.05~0.5% in the final concentration of bacteria suspension.
4. the method according to claim 1, wherein the additive amount 0.1-1.0mg/ of the Streptavidin MagneSphere mL。
5. the method according to claim 1, wherein the time of the ice bath is 10-30min.
6. the method according to claim 1, wherein the thallus suspension liquid, when sample is solid-state or semisolid When, it is sample to be added in the buffer of sterilizing, whirlpool mixes, and low-speed centrifugal takes supernatant and handles through cell filtering net, mistake Obtained liquid is filtered through multiple high speed centrifugation, washing, is then resuspended in buffer, obtains the thallus suspension liquid of mixed cell； When sample is liquid, directly sample is added in the buffer of sterilizing, whirlpool mixes, and high speed centrifugation takes precipitating, repeatedly washed It washs, be centrifuged, be then resuspended in buffer and obtain thallus suspension liquid.
7. according to the method described in claim 6, it is characterized in that, the buffer is peptone buffer agent.
8. any the method for claim 1~7 is in the application of feed, ordinary food or functional food field.
CN201610975479.XA 2016-11-07 2016-11-07 A kind of method of directional separation immune regulative enteron aisle lactobacillus Active CN106497840B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201610975479.XA CN106497840B (en) 2016-11-07 2016-11-07 A kind of method of directional separation immune regulative enteron aisle lactobacillus

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201610975479.XA CN106497840B (en) 2016-11-07 2016-11-07 A kind of method of directional separation immune regulative enteron aisle lactobacillus

Publications (2)

Publication Number Publication Date
CN106497840A CN106497840A (en) 2017-03-15
CN106497840B true CN106497840B (en) 2019-06-21

Family

ID=58323834

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201610975479.XA Active CN106497840B (en) 2016-11-07 2016-11-07 A kind of method of directional separation immune regulative enteron aisle lactobacillus

Country Status (1)

Country Link
CN (1) CN106497840B (en)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108456644A (en) * 2018-06-08 2018-08-28 保睿集团有限公司 High vigor high-purity human body intestines bacterium capsule preparation method thereof

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101575582A (en) * 2008-05-08 2009-11-11 景岳生物科技股份有限公司 Lactobacillus separation strains with anti-inflammatory activity and application thereof
CN102115721A (en) * 2008-05-08 2011-07-06 景岳生物科技股份有限公司 Lactobacillus isolated strains with anti-inflammatory activity and use thereof
CN102488717A (en) * 2006-06-05 2012-06-13 生命大地女神有限公司 Use of selected lactic acid bacteria for reducing infantile colic

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102488717A (en) * 2006-06-05 2012-06-13 生命大地女神有限公司 Use of selected lactic acid bacteria for reducing infantile colic
CN101575582A (en) * 2008-05-08 2009-11-11 景岳生物科技股份有限公司 Lactobacillus separation strains with anti-inflammatory activity and application thereof
CN102115721A (en) * 2008-05-08 2011-07-06 景岳生物科技股份有限公司 Lactobacillus isolated strains with anti-inflammatory activity and use thereof

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
Lactobacilli Differentially Modulate Expression of Cytokines and Maturation Surface Markers in Murine Dendritic Cells;Hanne R. Christensen等;《The Journal of Immunology》;20021231;第168卷;第171-178页

Also Published As

Publication number Publication date
CN106497840A (en) 2017-03-15

Similar Documents

Publication Publication Date Title
Majewski et al. Restriction endonuclease analysis of the genome of Campylobacter pylori with a rapid extraction method: evidence for considerable genomic variation
Sashihara et al. An analysis of the effectiveness of heat-killed lactic acid bacteria in alleviating allergic diseases
Young et al. Bifidobacterial species differentially affect expression of cell surface markers and cytokines of dendritic cells harvested from cord blood
Guglielmetti et al. Implication of an outer surface lipoprotein in adhesion of Bifidobacterium bifidum to Caco-2 cells
CN103097513B (en) Lactic acid bacteria for chylous diarrhea
JP4942831B2 (en) Antiallergic composition
RU2768030C2 (en) Bifidobacterium longum, able to modulate the immune response to a respiratory viral infection
CN101160134A (en) Selection and use of lactic acid bacteria for reducing inflammation in mammals
Wang et al. Isolation of adhesive strains and evaluation of the colonization and immune response by Lactobacillus plantarum L2 in the rat gastrointestinal tract
CN111286550A (en) Specific primer for amplifying lactobacillus paracasei and application thereof
CN102399718A (en) Strain of Lactobacillus paracasei GMNL-133 and composition for improving atopic dermatitis or other allergic diseases and purpose thereof
CN106497840B (en) A kind of method of directional separation immune regulative enteron aisle lactobacillus
CN109439778A (en) Derived from mark primer pair, identification method and its application of the Lactobacillus rhamnosus hsryfm 1301 of bar horse Changshou village
CN102309001B (en) Food composition and medicinal composition of lactobacillus strain for treating allergy
JP5927730B2 (en) Screening method for lactic acid bacteria having immunomodulating action
Wang et al. Protection against Trichinella spiralis in BALB/c mice via oral administration of recombinant Lactobacillus plantarum expressing murine interleukin-4
JP4847038B2 (en) Novel microbial strain GM-080 of Lactobacillus paracasei and its use for treating allergy related diseases
KR0170374B1 (en) Bifidobacterium strains having high iga induction potential
CN106497854A (en) Lactobacilluss D8 and its application
CN106520817A (en) Preparation method and applications of porcine circovirus type II recombinant lactobacillus plantarum oral vaccine
Abdel-Haleem et al. Potential anti‐hypercholesterolemic activity and acidogenic ability of probiotic lactic acid bacteria isolated from camel milk
CN111206069B (en) Method for rapidly capturing three pathogenic bacteria in cosmetics by utilizing nano immunomagnetic beads
CN107746893A (en) The IC LAMP detection primers group of salmonella and its application
JP6219728B2 (en) Oral immune tolerance screening method and oral immune tolerance enhancing composition using Foxp3 positive regulatory T cells and IFN-γ producing IL-10 producing T cells as indices
TW201231057A (en) Lactic acid bacteria isolated and their use

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant