CN109517772A - The building of Lactococcus lactis MG1363 a kind of and its application in treatment puerpera's cracked nipple - Google Patents
The building of Lactococcus lactis MG1363 a kind of and its application in treatment puerpera's cracked nipple Download PDFInfo
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- CN109517772A CN109517772A CN201811273048.4A CN201811273048A CN109517772A CN 109517772 A CN109517772 A CN 109517772A CN 201811273048 A CN201811273048 A CN 201811273048A CN 109517772 A CN109517772 A CN 109517772A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/52—Cytokines; Lymphokines; Interferons
- C07K14/521—Chemokines
- C07K14/522—Alpha-chemokines, e.g. NAP-2, ENA-78, GRO-alpha/MGSA/NAP-3, GRO-beta/MIP-2alpha, GRO-gamma/MIP-2beta, IP-10, GCP-2, MIG, PBSF, PF-4, KC
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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
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/19—Cytokines; Lymphokines; Interferons
- A61K38/195—Chemokines, e.g. RANTES
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P15/00—Drugs for genital or sexual disorders; Contraceptives
- A61P15/14—Drugs for genital or sexual disorders; Contraceptives for lactation disorders, e.g. galactorrhoea
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- 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
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
Abstract
The building of Lactococcus lactis MG1363 a kind of and its application in treatment puerpera's cracked nipple.First by chemical synthesis source of people and source of mouse Chemokine CXCL12, also known as stromal cell derived factor-1 (SDF-1), belong to the small molecular protein of CXC class chemotactic factor (CF) family.The distinctive secreting, expressing cell-penetrating peptide SPusp45 of Lactococcus lactis is added, is built into pMG36e-CXCL12 recombinant plasmid, electricity rotates into the secreting, expressing for realizing Chemokine CXCL12 into Lactococcus lactis MG1363 later.It is administered by external application nipple wound surface smearing mode, the direct secreting, expressing Chemokine CXCL12 of one side bacterium solution can realize that the nipple surface of a wound is quickly repaired；On the other hand, baby or newborn can get probiotics by the lactation mode directly sucked and feed, and baby or newborn's balance intestinal flora is helped to improve autoimmunity.
Description
Technical field
The present invention relates to the preparation method of the Lactococcus lactis MG1363 of secreting, expressing Chemokine CXCL12 a kind of and its
Application in puerpera's cracked nipple, belongs to field of biological pharmacy.
Background technique
Breast milk is the optimal natural food of baby, and breast-feeding on infants and puerpera are in the recent period and at a specified future date helpful.But
First 6 months Pure breast feeding rates only 37% after whole world birth at present, there are many factor for influencing early stage Pure breast feeding rate,
In the most common main suit be exactly sore nipples, chap.Cracked nipple pain is the main shadow that puerpera abandons Pure breast feeding early stage
One of the main reason for ringing one of factor, and leading to early weaning.
Cracked nipple is one of nursing period common disease, and high incidence period is 24-72 hours after childbirth, less serious case only nipple surface
There is breach, notably burn wound exudates oozing of blood, is not with the passing of time cured recurrent exerbation and easily forms aphtha, deal with improperly and easily cause cream
Carbuncle.It is explored by studying for a long period of time, the method for a variety of prevention and treatment cracked nipples clinically occurs, what wherein effect was more significant is sheep
Hair rouge and breast milk.Cracked nipple is prevented and treated using breast milk, although the method facilitates economy, and not all puerpera point
Puerperium can lactation immediately；High-purity anhydrous lanolin is nonirritant, hypoallergenic, odorless tasteless and more more effective than breast milk,
But every 10 grams about 100-200 yuans of commercially available suet cream differs, this be difficult to meet the lower puerpera of income level there is an urgent need to.
The damage and reparation of tissue and organ are the processes of a DYNAMIC COMPLEX, are in cytokine profiles and repair cell
Between interact under process of tissue reparation.Chemotactic factor (CF) is the important component of cell factor, they are on a class formation
Cell factor that is similar, having orientation chemotactic recruitment to leucocyte and stem cell, the status in wound healing is very
It is important, they can not only chemotactic leucocyte and repair cell raise to the surface of a wound, and these cells can also be activated, and enhance it
Function.Chemokine CXCL12, also known as stromal cell derived factor-1 (stromal cell-derived factor-1,
It SDF-1 is) to mankind CD34+Progenitor cells have the CXC class chemotactic factor (CF) of significant chemotaxis, can be by mobilizing simultaneously chemotactic inflammation
Cell and all kinds of bone marrow-derived cells are migrated to damage location, participate in local inflammation reaction and tissue repair.
Based on Chemokine CXCL12 and its receptor CXCR 4 wide expression in Various Tissues cell, and to some organizers
Reparative regeneration plays the function of important regulative after official's development and damage, and becoming CXCL12 treatment puerpera's cracked nipple wound can
Energy.But CXCL12 is easily degraded by dipeptide aminopeptidase IV (DPP IV) in blood of human body, and when severe tissue damage is repaired
The endogenous CXCLl2 level of generation is not able to satisfy the needs of tissue repair, it is therefore necessary to introduce exogenous CXCLl2.
The present invention constructs pMG36e-CXCL12 recombinant expression carrier, using Lactococcus lactis MG1363 as expression bacterium
Strain.Lactococcus lactis belongs to generally acknowledged probiotics, has good prebiotic effect to human body, breast milk is visible to be obtained baby by sucking
Probiotics supply is obtained, help constructs its intestinal flora stable state and improves immunity；Lactic acid bacteria has good acidproof, production acid spy simultaneously
Property, its surface can be maintained to keep acidic environment after surface of a wound administered recombinant bacterial strain, this is to reduction bacteria breed and raising
CXCL12 bioavilability (environment can inhibit the bioactivity of DPP IV in acidity) is of great advantage.The surface of a wound is applied in summary
CXCL12 engineered strain can be expressed perhaps and can reach treatment puerpera's cracked nipple effect.
Summary of the invention
It is an object of the invention in view of the shortcomings of the prior art, providing a kind of lactic acid of secreting, expressing Chemokine CXCL12
The preparation method of galactococcus MG1363 and its application in treatment puerpera's cracked nipple, establish a kind of CXCL12 Lactococcus lactis
Bacterium expression system expresses CXCL12 in wound surface continuous release by Lactococcus lactis, participates in local inflammation reaction and tissue
It repairs, to realize the therapeutic effect to puerpera's cracked nipple；
The present invention is realized by following technical step:
A kind of building of the Lactococcus lactis MG1363 of secreting, expressing Chemokine CXCL12, comprises the steps of:
(1) pMG36e-CXCL12 construction of recombinant plasmid: PstI-SPusp45-CXCL12-HindIII complete sequence chemistry closes
At obtaining
Obtain pUC57-CXCL12 recombinant plasmid；
(2) pMG36e-CXCL12 recombinant plasmid is constructed:
A: using the small extraction reagent kit of OMEGA plasmid, extracts pMG36e, pUC57-CXCL12 plasmid；
B: digestion: pMG36e, pUC57-CXCL12 digestion system are as follows, and total system is 25 μ L, 37 DEG C of digestion 4h；
C: 2% agarose gel electrophoresis, 110V electrophoresis 45min are prepared；In 276bp and 3600bp or so gel extraction；
D: enzyme connects: enzyme disjunctor system is as follows, and total system 10 μ L, Fragment:Vector=5:1 and 10:1,10 DEG C overnight；
E: conversion comprises the steps of:
A: 200 μ LMC1061 competent cell suspensions are taken from -80 DEG C of refrigerators, are thawed on ice；
B: being added 2 μ L plasmid solutions, and plasmid concentration is not less than 200ng/ μ L, gently shakes up, after placing 30min on ice；
Thermal shock 90s in c:42 DEG C of water-bath is immediately placed in cooled on ice 3-5min after thermal shock；
D: 800 μ L LB liquid mediums being added into pipe, and 37 DEG C of shaken cultivation 1h after mixing make bacterium restore normal raw
Long status.It is centrifuged 4000rpm, 1min.800 μ L of supernatant is abandoned, 200 μ L bacterium solutions are stayed, is mixed；
E: it takes 200 μ L to be coated in the screening flat board of the L erythromycin of μ containing 300ng/ after above-mentioned bacterium solution is shaken up, faces up
Half an hour is placed, is cultured after base absorbs completely after bacterium solution and is inverted culture dish, 37 DEG C of culture 16-24h, picking monoclonal；
F: sequencing；
G: after being sequenced successfully, recombinant plasmid pMG36e-CXCL12 is extracted using the small extraction reagent kit of OMEGA plasmid, is saved standby
With.
It is Lactococcus lactis MG1363 that prepared pMG36e-CXCL12, which is passed through electrotransformation, and conversion process is by following step
Rapid composition:
A: the preparation of lactic acid bacterium competence:
A: -80 DEG C of Lactococcus lactis MG1363 frozen are taken to be inoculated in the M17 fluid nutrient medium that 5mL contains 5% glucose
In, 30 DEG C are incubated overnight；
B: will obtain bacterium solution and be inoculated in the M17 fluid nutrient medium containing 2.5%Gly and 5% glucose according to 1%, and 30
DEG C stationary culture to thallus OD600 value is 0.3-0.4, is collected spare；
C: thalline culture the ice bath 10min, 4 DEG C of centrifugations 5000rpm/min, 5min that will be collected above；Collect thallus；
D: precipitating is cleaned twice with 10% ice-cold sucrose and 10% glycerol mixed solution, 1/10 volume, 4 DEG C of centrifugations
8000rpm/min, 5min collect precipitating；
E: finally precipitating is resuspended in 1/100 volume, 10% sucrose and 10% glycerol mixed solution, after ice bath 10min
It uses；
B: Lactococcus lactis MG1363 electrotransformation:
A: taking 2 μ L recombinant plasmids, and concentration is not less than 150ng/ μ L, the ice-cold lactic acid bacteria sense with 50 μ L of above method preparation
After being mixed gently by state cell suspension, it is added in the spacing 0.1cm electric shock cup of pre-cooling, is placed in and stands 5min on ice, condition is set
For the μ F of 1800V, 200 Ω, 25；
Liquid in the cup that shocks by electricity: after electric shock, being drawn into centrifuge tube rapidly by b, at the same be included in 800 μ L containing 5%
In the M17 fluid nutrient medium of glucose, 30 DEG C of culture 2h, product is coated on containing containing Erythromycinresistant after taking 100 μ L to convert
On the M17 plate of 5% glucose, 30 DEG C of stationary culture 24-72h, screening positive clone；
Prepared pMG36e-CXCL12 detects its secreting, expressing in Lactococcus lactis using Western, specifically by with
Lower step composition:
1.ELISA detects Chemokine CXCL12 secreting, expressing
A: sample treatment: by the Lactococcus lactis without plasmid and contain the Lactococcus lactis of pMG36e-CXCL12
MG1363 is incubated overnight respectively, is centrifugated supernatant of bacteria solution and precipitating；
B: the secreting, expressing situation of mouse ELISA detection kit detection CXCL12 is used.
2.Westernblot detects Chemokine CXCL12 secreting, expressing
A: electrophoresis, specific steps are as follows:
A: 12%SDS-PAGE gel is prepared；
B: sample treatment
Lactococcus lactis MG1363 containing pMG36e-CXCL12 is cultivated into 36h-48h, collects supernatant precipitating；It will be upper
It states and 4 × SDS-PAGE of concentration albumen sample-loading buffer is added in the protein sample of collection, final concentration of 1 × protein sample, 100 DEG C
Or boiling water bath heats 10min, with abundant albuminate；
C: loading and electrophoresis
After being cooled to room temperature, protein sample is directly loaded in SDS-PAGE glue well；Electrophoresis liquid uses when electrophoresis
SDS-PAGE electrophoresis liquid-the P0014A/P0014B of green skies production；Electrophoretic parameters are as follows: 40V electrophoresis 60min, 80V electrophoresis
90min；
B:Western detection, specific steps are as follows:
A: pvdf membrane transferring film is used；Condition is 100V, 45min；
B: after transferring film, being placed into protein film in preprepared Western cleaning solution immediately, rinses 1-
2min, to wash away the transferring film liquid on film；
C: being added Western confining liquid, i.e. 5% skim milk slowly shakes on shaking table, and room temperature closes 90min；
D: rabbit source CXCL12 polyclonal antibody is diluted according to 1:1000 using 5% skim milk；Protein film is transferred to and is contained
Overnight incubation is slowly shaken on the side shaker for 4 DEG C in 5% skim milk of primary antibody, TBST is washed three times, each 10min；
E: horseradish peroxidase-labeled goat anti-rabbit igg is diluted according to 1:1000 using 1% skim milk；By protein film
It is transferred in 1% skim milk containing secondary antibody, slowly shakes be incubated for 60min on the side shaker, TBST is washed three times, every time
10min；
F: developing solution A liquid and each 250 μ L 1:1 of B liquid are protected from light mixing, ready-to-use, exposure.
In such a way that external application wound surface smearing is administered, so that Lactococcus lactis MG1363 continuous expression chemotactic factor (CF)
CXCL12, to realize alleviation and therapeutic effect to puerpera's cracked nipple.
Beneficial effects of the present invention:
(1) present invention is by building pMG36e-CXCL12 constitutive expression carrier, using the method that electricity turns by recombinant plasmid
It is transferred to the secreting, expressing that Lactococcus lactis MG1363 realizes CXCL12；
(2) Lactococcus lactis CXCL12 may be implemented in skin wound is continuously expressed, and lactic acid bacteria is acidproof, produces
The characteristic of acid can keep wound surface acidic environment, this has positive effect to the bioactivity for inhibiting DPP IV, to improve
The bioavilability of CXCL12, therefore enough CXCL12 may participate in local inflammation reaction and tissue repair to reach treatment puerpera
The effect of cracked nipple.
Detailed description of the invention
Fig. 1 modeling after 1 day, 3 days, 5 days, 7 days each experimental groups and control group appearance chap degree score trend chart.
Specific embodiment
Embodiment detailed description: the drug effect is assessed by the experiment of mouse cracked nipple model wound healing
This research is quasi- by cracked nipple model experiment, in conjunction with appearance chap degree, Histological Study, immunohistochemistry and
The methods of inflammatory protein expression, determine engineered strain to the appearance of cracked nipple model, histology, immunology role,
Solves the problems, such as that cracked nipple provides new theoretical foundation and practices thinking for clinic, thus nipple chap when reaching reduction breast-feeding
The purpose of generation is split, the Pure breast feeding rate in baby due 6 months is improved.
Embodiment 1
One, the foundation of mouse cracked nipple model
(1) experimental group is negative control group (C group), model group (M group), Lactococcus lactis engineered strain low dose therapy
Group (LT group), Lactococcus lactis engineered strain high-dose therapy group (HT group), every group 8；
(2) Balb/c mouse 32 are taken, at random marked as No. 1-32.In operation consent 12h fasting, water can't help.Use fixed frame
Mouse is fixed, is close to skin with automatic shaving machine and cuts off back two sides experiment position coat roughly, then slough fine, soft fur with depilatory cream.It is de-
It is thoroughly cleaned with warm water immediately after hair, keeps skin exposed with spare.The operation same day is with 3.6% abdominal cavity chloraldurate 10mL/kg note
Penetrate anesthesia.Metal disk is immersed in -196 DEG C of liquid nitrogen, is close to back of mice after taking-up immediately and lost hair or feathers position, continue 5
Second, form circle II degree frostbite skin.
Two, the treatment of mouse cracked nipple model
(1) coating (1 × 10 the surface of a wound of every back of mice models the same day (after modeling 5 hours) since animal7Or 1 ×
109The mixed bacteria liquid of CFU/mL), it is 3-5 times daily, coating 7 days, every mouse sub-cage rearing.
(2) the 1st after modeling, successively put to death some animals within 3,5,7 days, it is each every time to put to death 4, it is de- for cervical vertebra to put to death method
Mortar.The back of mice surface of a wound and its a little perienchyma are cut, as deep as muscle layer, the part each group wound tissue sample cut is used
4% paraformaldehyde solution is fixed, and hematoxylin-eosin (HE) dyeing, Masson dyeing and Ki67, VEGF immuning tissue are respectively used to
Chemical staining.- 80 DEG C of refrigerators preservations are transferred to after being partially disposed in liquid nitrogen flash freezer, to carry out Western-blot, ELISA, q-PCR
Deng detection.Coating to experiment terminates after remaining mouse self-modeling, the 1st after coating, visually observes its chapped skin journey within 3,5,7 days
It spends and records its scoring.
Embodiment 2
One, ocular estimate after the treatment of zoopery cracked nipple model
1, appearance chap degree scoring
The standards of grading are evaluated based on wound depth and degree of tissue damage, and scoring is by 1 to this experiment
Unwitting laboratory technician assesses.The scoring is higher, shows that chap degree is more serious；0-1 points represent normal or recovery from illness.Chap
Degree standards of grading see the table below.
Chap degree standards of grading
Embodiment 3
One, histopathology after the treatment of zoopery cracked nipple model
It is assessed in terms of the regeneration situation of epidermis and corium, granulation tissue thickness and angiogenesis situation etc. three.
HE dyeing is used to observe surface of a wound pathological change.The reparation situation of cicatrix of skin after Masson dyeing observation is caused injury.
1, histological score-wound tissue HE dyeing
(1) it is dehydrated.Already fixed wound tissue is taken out, is sufficiently rinsed with PBS, then ethanol dehydration is used respectively
70% ethyl alcohol, 80% ethyl alcohol, 90% ethyl alcohol, 95% ethyl alcohol, 95% ethanol dehydration 150min；Then with dehydrated alcohol dehydration two
It is secondary, respectively 60min；
(2) it embeds.The dewatered tissue 30min of dimethylbenzene processing, paraffin are embedded；
(3) slice and mounting.Rotary press cuts out flat surface, and setting section thickness is 55 μm, carefully provokes the drawing cut
Piece is placed in the exhibition piece machine sink that water temperature is 45 DEG C, will have the wax disk(-sc) of fold to evaluate by exhibit；Wax disk(-sc) is placed on glass slide, is placed
On 60 DEG C of roasting piece machines, 4h is toasted；
(4) it dewaxes.Respectively with dimethylbenzene (I), (II) dewaxing 15min, dimethylbenzene is then used: at dehydrated alcohol 1:1 solution
5min is managed, uses the ethanol solution of descending concentrations, 100% ethyl alcohol, 95% ethyl alcohol, 80% ethyl alcohol, 70% alcohol treatment 5min respectively.
(5) it dyes.Harris haematoxylin dyeing 5min uses the ethanol solution of descending concentrations, 70% ethyl alcohol, 80% second respectively
Alcohol, 95% ethyl alcohol, 100% alcohol treatment 5min；0.5% eosin stains 1min；Successively with 100% ethyl alcohol (I), 100% ethyl alcohol
(II) 5min is handled；It is separately added into dimethylbenzene (I), transparent 10min in dimethylbenzene (II)；Neutral gum mounting；
(6) it observes.Mounting is placed under optical microscopy, respectively in 200 times and observation of taking pictures respectively under 400 times of mirrors, and
The infiltration degree of analyzing skin edge of wound and surrounding inflammatory cell, whether there is or not abscess, bacterial aggregate and wound repair situation and granulations
Tissue generation etc..
2, histological score-wound tissue Masson dyeing
(1) it dewaxes, dimethylbenzene I 15min, the dimethylbenzene II 15min more renewed.
(2) aquation, 100% ethyl alcohol I 5min, 100% ethyl alcohol II 5min, 95% ethyl alcohol I 5min, 80% ethyl alcohol 3min,
Tissue, which is placed in distilled water, impregnates 30min.
(3) core is contaminated, haematoxylin is added dropwise, places 5min, tap water is slightly washed.
(4) it is put into PBS and impregnates 10min, oil blackeite.
(5) 0.2% acetums are slightly washed, and contaminate 7min in Ponceaux acid fuchsin liquid.
(6) 0.2% acetums are slightly washed, and the differentiation of 1% phosphomolybdic acid, when differentiation, which asks, to be not fixed, and the specific time is under microscope
It grasps.
(7) 0.2% acetums are slightly washed, and toluidine blue dye liquor is added, and dyeing time is not fixed, and the specific time is in most micro mirror
Lower grasp.
(8) gradient alcohol dehydration: 80% ethyl alcohol 10-15sec, 95% ethyl alcohol II 10-15sec, 100% ethyl alcohol, III 5min,
100% ethyl alcohol IV 5min.
(9) III 10min of dimethylbenzene, dimethylbenzene IV 10min.
(10) resinene mounting dries, under the microscope in optical microphotograph, the reparation situation of photographic analysis cicatrix of skin.
3, judge cell-proliferation activity-wound tissue immunohistochemistry
Ki67 is mainly used for the proliferation activity that observation judges cell between granulation tissue, is expressed in all movable cell cycles
G1, S, G2 and mitosis are interim, and do not express in the G0 phase.Ki67 positive staining is brown yellow granule, is primarily targeted for cell
In core.
VEGF is vascular endothelial growth factor, can stimulating endothelial cell proliferation, be able to reflect in surface of a wound expression contents
New vessels and lymphatic vessel ability, wound healing.
(1) conventional dewaxing, dehydration are dyed with HE；
(2) for the 3% fresh configuration of inactivating endogenous enzyme aqueous hydrogen peroxide solution room temperature processing 5-10min, spend from
Son washing 3 times；
(3) slice is immersed concentration is to stop after being heated to boiling in 0.01mol/LpH=6.0 citrate buffer
It heats, after natural cooling 5-10min, is repeating this operation 1-2 times, the PBS of pH=7.2-7.6, washing 1-2 times are used after cooling；
(4) it is incubated for slice 10min under room temperature with antigen retrieval buffers 1, antigen retrieval buffers 1 are added dropwise upper in slice and use pH=7.2-
7.6 PBS is washed 1-2 times；
(5) slice 20min is closed with 5% calf serum under room temperature, eliminates extra moisture；
(6) Ki67, VEGF primary antibody are added dropwise respectively, 37 DEG C of incubation 30min, 4 DEG C overnight, and the phosphate of pH=7.2-7.6 is slow
Fliud flushing is washed 2min, is washed 3 times；
(7) biotin secondary antibody, 37 DEG C of incubation 30min is added dropwise, the phosphate buffer of pH=7.2-7.6 is washed 3 times；
(8) 1:150SP compound, 37 DEG C of incubation 30min, the phosphate buffer of pH=7.2-7.6, washing is added dropwise
5min is washed 3 times；
(9) micropipette rifle draws 1mL deionized water, adds each 1 drop of DAB color developing agent A, B, C, mixes；
(10) 50 μ L color developing agents are drawn under room temperature, and the control reaction time is 5-30min under mirror, is spent after colour developing
Ionized water is sufficiently washed to terminate reaction；
(11) haematoxylin redyes 1min, according to the dehydration of HE dyeing conventional steps, de- wrong, transparent, mounting, under the microscope, point
Not in 200 times and observation of taking pictures respectively under 400 times of mirrors, and make Pathological Physiology analysis.
Embodiment 4
One, tissue immunology detects after the treatment of zoopery cracked nipple model
1, the expression of engineered strain regulation inflammatory signals pathway associated protein
Detection bacterium cracked nipple mouse model inflammation associated signal paths in wound tissue after engineered strain is treated
The protein expression situation of TLR4-NF- κ B, MAPK, PI3K/Akt, to analyze surface of a wound local immunity situation and clinical therapeutic efficacy.
This evaluation is shown by Western-blot.
After putting to death mouse, wound tissue albumen is extracted, carries out Western-blot.
(1) wounds in mice histone extracts
1. tissue is rinsed for several times with the sterile ice deionized water containing PMSF (1mM)；
2. tissue is placed in homogenizer, appropriate lysate buffer RIPA and protease inhibitors, ice water mixing is added
It is homogenized in bath；
3. the protein solution for cracking generation is fully transferred in 1.5mL EP pipe；
4. 1.5mL pipe is placed on ice, whirlpool shakes 10s, places 5min, repeats 3-4 times；
5. 4 DEG C, 13000rpm is centrifuged 10min；
6. drawing supernatant into completely new 1.5mL EP pipe, -80 DEG C of refrigerators are saved, and supernatant is holoprotein.
(2)Western-blot
1. suitable 4x albumen sample-loading buffer is added in the protein sample of collection, 100 DEG C of boiling water baths heat 10min,
With abundant albuminate；
2. preparing 5% concentration glue, 10% or 12% separation gel immerses glue in 1x electrophoretic buffer after being gelled admittedly,
Loading electrophoresis；
3. after the completion of electrophoresis, glue is removed glass plate, transferring film is stand-by.By pvdf membrane 1x transferring film buffer complete wetting
Afterwards, glue and film are clipped with clamping plate, carry out transferring film；
4. after the completion of transferring film, taking out pvdf membrane, after 5% skimmed milk or 5%BSA closing, the primary antibody of target protein is added
4 DEG C of overnight incubations afterwards, then the secondary antibody incubation 2h of HRP label is added, carry out colour developing exposure；
5. taking pictures to colour developing content, gray scale, mapping analysis are analyzed using software.
2, the expression of engineered strain regulation inflammatory factor
Mouse carries out excision eyeball and takes blood before putting to death, be centrifugated serum, passes through ELISA and detects cracked nipple mouse model
After engineered strain is treated TNF-α in serum, IL-1 β, IL-6 and IL-10 expression quantity, to analyze surface of a wound local immunity situation
It is imitated with clinical treatment.
After mouse is put to death, wound tissue RNA is extracted, reverse transcription detects inflammation in wound tissue at q-PCR is carried out after cDNA
The expression of factor TNF-α, IL-1 β, IL-6 and IL-10, TGF-β transcriptional level.
(1) ELISA experimental procedure
1. saying that kit balances half an hour at room temperature before use；
2. blank well is not loaded, only add A, B and terminate liquid for returning to zero；
3. standard sample wells: every hole adds the good 50 μ L of standard items diluted, and 50 μ L of standard items/sample diluting liquid is added in zero hole,
Then 50 μ L of biotin antigen working solution is added；
4. sample well: the 50 μ L of sample of 3 times of dilution is added, 50 μ L of biotin antigen working solution is then added；
5. jiggling, sealer plate, 37 DEG C of culture 60min are covered；
6. by spare after 25 times of concentrated cleaning solutions, 25 times of distilled water dilutions；
7. washing for the first time: carefully opening sealer plate, discard liquid, dry, 200 μ L are added in every hole, abandon after standing 30s
It goes, pats dry, be so repeated 3 times；
8. 50 μ L Avidin-HRP are added into standard sample wells and sample well, jiggle, covers sealer plate, 37 DEG C of cultures
60min；
9. second is washed: carefully opening sealer plate, discard liquid, dry, 200 μ L are added in every hole, abandon after standing 30s
It goes, pats dry, be so repeated 3 times；
10. colour developing: color developing agent A50 μ L is added in every hole, adds 50 μ L of color developing agent B, and gently concussion mixes, 37 DEG C be protected from light it is aobvious
Color 10min；
Terminate: 50 μ L of terminate liquid is added in every hole, terminates reaction (blue is vertical at this time switchs to yellow).
Measurement: microplate reader, 450nm wavelength survey OD value.
(2) extraction of wounds in mice tissue RNA
1. taking the wounds in mice tissue of phase homogenous quantities, 1mLTrizol is added, is shredded with scissors, and even with tissue refiner
Slurry, 5000rpm 10min shift supernatant into new EP pipe；
2. 160 μ L of chloroform is added, sufficiently oscillation is mixed, and 4 DEG C of 13000rpm 15min, centrifugation finishes, and is drawn supernatant, is paid attention to
Albumin layer is not drawn onto；
3. isometric isopropanol is added, mix well, 4 DEG C of 13000rpm 10min remove supernatant；
4. 75% ethanol washing of 1mL is added, oscillation, after completely dissolution, 4 DEG C of 13000rpm 10min abandon supernatant；
5. the washing of 1mL dehydrated alcohol is added, oscillation, after completely dissolution, 4 DEG C of 13000rpm 10min abandon supernatant；
6. room temperature dries 30min；
7. plus 160 μ L RNase Free Water, 55 DEG C of dissolutions；
8. measuring RNA concentration.
(3)RT-PCR
It is operated referring to TAKARA kit specification.
The new method being used in combination using probiotics provided by the invention with micromolecule polypeptide, formation it is novel
ATCC55221-pMG36e-CXCL12 engineered strain treats cracked nipple mouse model wound healing, to by introducing external source
Property CXCL12 accelerate nipple wound healing, solve the problems, such as that cracked nipple provides new theoretical foundation and practices thinking for clinic, from
And cracked nipple occurs when achieving the purpose that reduce breast-feeding, and the Pure breast feeding rate in baby due 6 months is continuously improved.
Preliminary result is shown by ocular estimate, will do it a series of realities such as histopathology and tissue immunology later
It tests, overall merit expresses therapeutic effect of the engineered strain to cracked nipple mouse model of Chemokine CXCL12.It can push away simultaneously
It is proved into clinical ethics, will do it clinical trial in conditions permit.Specific test result is shown in Figure of description 1.
Claims (4)
1. a kind of building of the Lactococcus lactis MG1363 of secreting, expressing Chemokine CXCL12, which is characterized in that by following step
Rapid composition:
(1) pMG36e-CXCL12 construction of recombinant plasmid: PstI-SPusp45-CXCL12-HindIII complete sequence chemical synthesis obtains
Obtain pUC57-CXCL12 recombinant plasmid；
(2) pMG36e-CXCL12 recombinant plasmid is constructed:
A: using the small extraction reagent kit of OMEGA plasmid, extracts pMG36e, pUC57-CXCL12 plasmid；
B: digestion: pMG36e, pUC57-CXCL12 digestion system are as follows, and total system is 25 μ L, 37 DEG C of digestion 4h；
C: 2% agarose gel electrophoresis, 110V electrophoresis 45min are prepared；In 276bp and 3600bp or so gel extraction；
D: enzyme connects: enzyme disjunctor system is as follows, and total system 10 μ L, Fragment:Vector=5:1 and 10:1,10 DEG C overnight；
E: conversion comprises the steps of:
A: 200 μ L MC1061 competent cell suspensions are taken from -80 DEG C of refrigerators, are thawed on ice；
B: being added 2 μ L plasmid solutions, and plasmid concentration is not less than 200ng/ μ L, gently shakes up, after placing 30min on ice；
Thermal shock 90s in c:42 DEG C of water-bath is immediately placed in cooled on ice 3-5min after thermal shock；
D: being added 800 μ L LB liquid mediums into pipe, 37 DEG C of shaken cultivation 1h after mixing, and bacterium is made to restore normal growth shape
State.It is centrifuged 4000rpm, 1min.800 μ L of supernatant is abandoned, 200 μ L bacterium solutions are stayed, is mixed；
E: taking 200 μ L to be coated in the screening flat board of the L erythromycin of μ containing 300ng/ after above-mentioned bacterium solution is shaken up, face up placement
Half an hour is cultured after base absorbs completely after bacterium solution and is inverted culture dish, 37 DEG C of culture 16-24h, picking monoclonal；
F: sequencing；
G: after being sequenced successfully, recombinant plasmid pMG36e-CXCL12 is extracted using the small extraction reagent kit of OMEGA plasmid, saves backup, walks
It is rapid to be same as above.
2. the building of the Lactococcus lactis MG1363 of secreting, expressing Chemokine CXCL12 according to claim 1 a kind of,
It is characterized by: it is Lactococcus lactis MG1363 that prepared pMG36e-CXCL12, which is passed through electrotransformation, conversion process is by following
Step composition:
A: the preparation of lactic acid bacterium competence:
A: taking -80 DEG C of Lactococcus lactis MG1363 frozen to be inoculated in 5mL and contain in the M17 fluid nutrient medium of 5% glucose, and 30
It DEG C is incubated overnight；
B: bacterium solution will be obtained and be inoculated in the M17 fluid nutrient medium containing 2.5%Gly and 5% glucose according to 1%, 30 DEG C quiet
Culture is set to thallus OD600Value is 0.3-0.4, is collected spare；
C: thalline culture the ice bath 10min, 4 DEG C of centrifugations 5000rpm/min, 5min that will be collected above；Collect thallus；
D: precipitating is cleaned twice with 10% ice-cold sucrose and 10% glycerol mixed solution, 1/10 volume, 4 DEG C of centrifugation 8000rpm/
Min, 5min collect precipitating；
E: finally precipitating is resuspended in 1/100 volume, 10% sucrose and 10% glycerol mixed solution, can be made after ice bath 10min
With；
B: Lactococcus lactis MG1363 electrotransformation:
A: taking 2 μ L recombinant plasmids, and concentration is not less than 150ng/ μ L, the ice-cold lactic acid bacterium competence with 50 μ L of above method preparation
After cell suspension mixes gently, it is added in the spacing 0.1cm electric shock cup of pre-cooling, is placed in and stands 5min on ice, setting condition is
1800V, 200 Ω, 25 μ F；
Liquid in the cup that shocks by electricity: after electric shock, being drawn into centrifuge tube rapidly by b, at the same be included in 800 μ L containing 5% grape
In the M17 fluid nutrient medium of sugar, 30 DEG C of culture 2h, product, which is coated on, after taking 100 μ L to convert contains 5% Portugal containing Erythromycinresistant
On the M17 plate of grape sugar, 30 DEG C of stationary culture 24-72h, screening positive clone.
3. the building of the Lactococcus lactis MG1363 of secreting, expressing Chemokine CXCL12 according to claim 1 a kind of,
It is characterized by: prepared pMG36e-CXCL12 detects its secretion table in Lactococcus lactis using ELISA and Western
It reaches, specifically comprises the steps of:
(1) ELISA detects Chemokine CXCL12 secreting, expressing
A: sample treatment: by the Lactococcus lactis without plasmid and the Lactococcus lactis MG1363 containing pMG36e-CXCL12 points
It is not incubated overnight, is centrifugated supernatant of bacteria solution and precipitating；
B: the secreting, expressing situation of mouse ELISA detection kit detection CXCL12 is used；
(2) Western blot detects Chemokine CXCL12 secreting, expressing
A: electrophoresis, specific steps are as follows:
A: 12%SDS-PAGE gel is prepared；
B: sample treatment
Lactococcus lactis MG1363 containing pMG36e-CXCL12 is cultivated into 36h-48h, collects supernatant precipitating；By above-mentioned receipts
4 × SDS-PAGE of concentration albumen sample-loading buffer, final concentration of 1 × protein sample, 100 DEG C or boiling are added in the protein sample of collection
Heating water bath 10min, with abundant albuminate；
C: loading and electrophoresis
After being cooled to room temperature, protein sample is directly loaded in SDS-PAGE glue well；Electrophoresis liquid uses green cloud when electrophoresis
SDS-PAGE electrophoresis liquid-the P0014A/P0014B of its production；Electrophoretic parameters are as follows: 40V electrophoresis 60min, 80V electrophoresis 90min；
B:Western detection, specific steps are as follows:
A: pvdf membrane transferring film is used；Condition is 100V, 45min；
B: after transferring film, being placed into protein film in preprepared Western cleaning solution immediately, rinses 1-2min, with
Wash away the transferring film liquid on film；
C: being added Western confining liquid, i.e. 5% skim milk slowly shakes on shaking table, and room temperature closes 90min；
D: rabbit source CXCL12 polyclonal antibody is diluted according to 1:1000 using 5% skim milk；Protein film is transferred to containing primary antibody
5% skim milk in 4 DEG C slowly shake overnight incubation on the side shaker, TBST is washed three times, each 10min；
E: horseradish peroxidase-labeled goat anti-rabbit igg is diluted according to 1:1000 using 1% skim milk；By albumen film transfer
It is incubated for 60min to being slowly shaken in 1% skim milk containing secondary antibody on the side shaker, TBST is washed three times, each 10min；
F: developing solution A liquid and each 250 μ L 1:1 of B liquid are protected from light mixing, ready-to-use, exposure.
4. the building of Lactococcus lactis MG1363 of secreting, expressing Chemokine CXCL12 a kind of and its treatment puerpera's nipple chap
Application in splitting, which is characterized in that application are as follows: by external application nipple wound surface smearing administration mode, bacterium solution can be achieved at the same time and suffering from
Place's secreting, expressing Chemokine CXCL12 helps the nipple surface of a wound quickly to repair；And baby or newborn pass through the food in one's mouth directly sucked
Newborn mode obtains probiotics supply indirectly, and baby or newborn's balance intestinal flora is helped to improve autoimmunity.
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