CN109022475A - The building of Lactobacillus crispatus BT1386 and its application in treatment bacterial vaginitis - Google Patents
The building of Lactobacillus crispatus BT1386 and its application in treatment bacterial vaginitis Download PDFInfo
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Abstract
The building of Lactobacillus crispatus BT1386 and its application in treatment bacterial vaginitis.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 Lactobacillus crispatus BT1386 later.The continuous expression Chemokine CXCL12 in such a way that vaginal plug thrusts medicine, to realize alleviation and therapeutic effect to bacterial vaginitis.
Description
Technical field
The present invention relates to the preparation method of the Lactobacillus crispatus BT1386 of secreting, expressing Chemokine CXCL12 a kind of and its
Application in bacterial vaginitis, belongs to field of biological pharmacy.
Background technique
Chemotactic factor (CF) is a kind of micromolecule polypeptide substance to leucocyte and stem cell with orientation chemotactic recruitment,
Their structure and function is similar, and the overwhelming majority has 4 conservative cysteines on amino acid sequence, and according to its N-terminal half
The quantity of cystine and the difference of spacing are divided into C-, CXC-, CC- and CX3C-4 class chemotactic factor (CF).Chemokine CXCL12 is also known as
Stromal cell derived factor-1 (SDF-1), there are two types of form, SDF-1 α/CXCL12a and SDF-1 β/CXCL12b for it.CXCR4 is
The specific receptor of Chemokines CC XCLl2, belongs to the transmembrane receptor family that G-protein couples, and has the affine of height with CXCLl2
Power, its wide expression is in blood, immune and central nervous system cell.Research shows that CXCL12/CXCR4 effect axis is primarily involved in
The reparation of hypoxic-ischemic and injury tissue, in human endometrial CXCL12 function research shows that: CXCL12 may be in inner membrance
Proliferation plays a significant role in embryo nidation, they can not only chemotactic leucocyte and repair cell raise to the surface of a wound, but also
These cells can be activated, and enhance their function.Therefore it is contemplated that could can to participate in local inflammation by CXCL12 anti-
Bacterial leaf steak should be treated with the property of tissue repair.But CXCL12 in blood of human body easily by dipeptide aminopeptidase
Enzyme IV (DPP IV) degradation, and the endogenous CXCLl2 level generated when severe tissue damage is repaired is not able to satisfy tissue repair
Needs, it is therefore necessary to introduce exogenous CXCLl2.
The present invention constructs pMG36e-CXCL12 recombinant expression carrier, using Lactobacillus crispatus BT1386 as expression bacterium
Strain.Lactococcus lactis belongs to generally acknowledged probiotics, has good prebiotic effect, while the good acidproof, production of lactic acid bacteria to human body
Sour characteristic plays a role in vagina for recombinant bacterial strain and provides primary condition, and Lactococcus lactis can in human vagina
To realize that CXCL12 is continuously expressed, lactic acid bacteria, which produces environment in the acidity of acid and vagina, can inhibit the biological living of DPP IV
Property, to improve the bioavilability of CXCL12 to reach treatment Bacterial leaf steak 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 curling of secreting, expressing Chemokine CXCL12
The preparation method of lactobacillus BT1386 and its application in treatment Bacterial leaf steak establish a kind of CXCL12 lactic acid cream
Coccus expression system expresses CXCL12 in intravaginal continuous release by Lactococcus lactis, participates in local inflammation reaction and tissue
It repairs, to realize the therapeutic effect to Bacterial leaf steak；
The present invention is realized by following technical step:
The building of Lactobacillus crispatus BT1386, comprises the steps of:
(1) pMG36e-CXCL12 construction of recombinant plasmid: PstI-SPusp45-CXCL12-HindIII complete sequence chemistry closes
At acquisition pUC57-CXCL12 recombinant plasmid；
(2) pMG36e-CXCL12 recombinant plasmid is constructed:
A uses 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 prepares 2% agarose gel electrophoresis, 110V electrophoresis 45min；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: 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 Lactobacillus crispatus BT1386 that prepared pMG36e-CXCL12, which is passed through electrotransformation, and conversion process is by following step
Rapid composition:
The preparation of A lactic acid bacterium competence:
A: -80 DEG C of Lactobacillus crispatus BT1386 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 is 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 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 Lactobacillus crispatus BT1386 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:
A electrophoresis, specific steps are as follows:
A: 12%SDS-PAGE gel is prepared；
B: sample treatment
Lactobacillus crispatus BT1386 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 vaginal plug thrusts medicine, so that Lactobacillus crispatus BT1386 continuous expression Chemokine CXCL12, with
Realize the alleviation and therapeutic effect to bacterial vaginitis.
Beneficial effects of the present invention:
(1) present invention is by building pMG36e-CXCL12 constitutive expression carrier, in addition Lactococcus lactis cell-penetrating peptide
After SPusp45, recombinant plasmid is transferred to by the secreting, expressing that Lactobacillus crispatus BT1386 realizes CXCL12 using the method that electricity turns；
(2) the good acidproof, characteristic of lactic acid production of lactic acid bacteria plays a role the item that provides the foundation for recombinant bacterial strain in vagina
Part；
(3) Lactococcus lactis CXCL12 may be implemented in human vagina is continuously expressed, and lactic acid bacteria produces acid
And environment can inhibit the bioactivity of DPP IV in the acidity of vagina, to improve the bioavilability of CXCL12, therefore foot
Enough CXCL12 may participate in local inflammation reaction and tissue repair to reach treatment Bacterial leaf steak effect.
Detailed description of the invention
Fig. 1 is rat uterus tissue HE colored graph under different pharmaceutical.
Specific embodiment
Embodiment 1
Embodiment detailed description: the drug effect is assessed by bacterial vaginitis rat model
One, the foundation of rat vagina inflammation model
(1) experimental group is negative control group, model group, metronidazole treatment, Lactococcus lactis engineered strain treatment group, volume
Bent lactobacillus engineered strain treatment group, every group 5；
(2) medical absorbent gelfoam is cut into 0.5cm × 0.5cm size by antibiotic treatment, is drawn 50 μ L concentration and is
The antibiotic of 100mg/mL；After gelatin fully absorbs antibiotic saturation, press from both sides antibiotic gelfoam, plug to vagina plug and
In uterine cavity；Antibiotic treatment 6 times, restore within continuous processing three days primary；
(3) after antibiotic treatment by beta hemolytic streptococcus, staphylococcus aureus, Escherichia coli in 1:1:2 ratio,
It is configured to the mixed cell solution that concentration is 3,000,000,000/mL；Medical absorbent gelfoam is cut into 0.5cm × 0.5cm size,
Drawing 50 μ L fully absorbs gelfoam；After gelatin fully absorbs antibiotic saturation, antibiotic gelfoam is pressed from both sides, is filled in
In to vagina and uterine cavity and keep mouse handstand 1-2min；Blank group is PBS processing；Modeling is handled 7 times, is discontinued one during modeling
It, stopping a day after modeling.
Two, the treatment of rat bacterial vaginitis model
(1) 7 drug treatments are carried out after modeling success.Metronidazole treatment group dosage, according to bought metronidazole medicine
Product illustrate and obtain after equivalent dose converts, rat dosage 16.2mg/200g.By medical absorbent gelfoam
It is cut into 0.5cm × 0.5cm size, 50 μ L drugs of absorption are put into rat vagina after so that gelfoam is fully absorbed drug；This hair
The bright novel B T1386-pMG36e-CXCL12 engineered strain prepares 1 × 109The mixed bacteria liquid of CFU/mL；By medical absorbent
Gelfoam is cut into 0.5cm × 0.5cm size, draws 50 μ L bacterium solutions, after so that gelfoam is fully absorbed drug, is put into rat yin
In road, every experimental rat 5 × 107CFU；
(2) after treatment restores 7 days, each group rat vagina secretion is taken within continuous three days, vaginal flora is extracted, utilizes high throughput
The type and quantity variation of rat vagina microorganism after the treatment of sequencing technologies analysis project bacterial strain；
(3) extracing eyeball takes cervical dislocation after blood to put to death experimental rat.Solution takes rat vagina tissue, is partially disposed in 4%
Paraformaldehyde solution saves, and to carry out HE dyeing and immunohistochemistry detection, is transferred to -80 DEG C of refrigerators after being partially disposed in liquid nitrogen flash freezer
It saves, to carry out the detection such as Western-blot, ELISA, q-PCR.
Three, indices detect after the treatment of zoopery vaginitis model
1, rat uterus tissue HE is dyed
(1) it is dehydrated.Already fixed uterus is taken out, is sufficiently rinsed with PBS, then ethanol dehydration, respectively with 70%
Ethyl alcohol, 80% ethyl alcohol, 90% ethyl alcohol, 95% ethyl alcohol, 95% ethanol dehydration 150min；Then twice with dehydrated alcohol dehydration, divide
It Wei not 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, respectively use descending concentrations ethanol solution, 70% ethyl alcohol 5,80%
Ethyl alcohol, 95% ethyl alcohol, 100% alcohol treatment 5min；0.5% eosin stains 1min；Successively with 100% ethyl alcohol (I), 100% second
Alcohol (II) handles 5min；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
Make Pathological Physiology analysis.
2, rat uterus tissue immunohistochemistry
(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/L pH=6.0 citrate buffer
It heats, after natural cooling 5-l0min, 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
The PBS of=7.2-7.6 is washed 1-2 times；
(5) slice 20min is closed with 5% calf serum under room temperature, eliminates extra moisture；
(6) 1:400 rabbit-anti Caspase-3, Caspase-8, FasL primary antibody, 37 DEG C of incubation 30min, 4 DEG C of mistakes are added dropwise respectively
Night, the phosphate buffer of pH=7.2-7.6 wash 2min, wash 3 times；
(7) 1:150 biotin secondary antibody (goat antirabbit Caspase-3, Caspase-8, FasL) is added dropwise, 37 DEG C of incubations
The phosphate buffer of 30min, pH=7.2-7.6 are 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.
3, the expression of engineered strain regulation inflammatory signals pathway associated protein
Detection bacterium vaginitis rat model inflammation associated signal paths in vagina tissue after engineered strain is treated
The protein expression situation of TLR4-NF- κ B, MAPK, PI3K/Akt, to analyze vagina local immunity situation and clinical treatment effect.This
Item evaluation is shown by Western-blot.
After rat is put to death, vagina tissue albumen is extracted, carries out Western-blot.
(1) rat vagina 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 or boiling water bath heating
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.
4, the expression of engineered strain regulation inflammatory factor
Detection bacterium vaginitis rat model after engineered strain is treated TNF-α in vagina tissue, IL-1 β, IL-6,
The expression quantity of TNF-α in IL-17, IL-4, IL-22, TGF-β and serum, IL-1 β, IL-6 and IL-4, is locally exempted from analyzing vagina
Epidemic disease situation and clinical treatment effect.The novel MG1363-pMG36e-CXCL12 engineered strain inhibits part to cause scorching in intravaginal
The release of the factor, further, the pro-inflammatory cytokine are TNF-α, IL-1 β, IL-6, IL-17；Promote to press down scorching factor IL-4, IL-
22, the expression of TGF-β.This inhibition and promotion are embodied in mRNA level in-site, influence proinflammatory and suppression inflammatory factor transcription, lower,
The expression quantity of inflammatory mediator mRNA is raised, this evaluation is shown by q-PCR.Be also embodied in protein level, influence proinflammatory and
The translation for pressing down the scorching factor, is lowered, the expression quantity of up-regulation inflammatory mediator albumen, this evaluation is shown by ELISA.
After rat is put to death, vagina tissue RNA is extracted, reverse transcription detects inflammation in vagina tissue at q-PCR is carried out after cDNA
Factor TNF-α, IL-1 β, IL-6, IL-17 and IL-4, IL-22, TGF-β transcriptional level expression.
(1) extraction of rat vagina tissue RNA
1. taking the rat endometrium tissue of phase homogenous quantities, 1mL Trizol is added, is shredded with scissors, and be homogenized with tissue
Machine homogenate, 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.
(2)RT-PCR
It is operated referring to TAKARA kit specification.
Rat, which carries out extracing eyeball before putting to death, takes blood, is centrifugated serum, ELISA detect inflammatory factor TNF-α in serum,
The expression of IL-1 β, IL-6 and IL-4 protein 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: 50 μ L of color developing agent A is added in every hole, adds 50 μ L of color developing agent B, and gently concussion mixes, and 37 DEG C are protected from light
Develop the 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.
5, rat vagina flora high-flux sequence is analyzed
Flora type and quantity become detection bacterium vaginitis rat model in vaginal fluid after engineered strain is treated
Change, to analyze vagina local immunity situation and clinical treatment effect.The vagina microorganism genomic DNA of extraction send promise standing grain in Beijing to cause
Source company carries out 16S rRNA sequencing.Using the genomic DNA of extraction as template, the primer pair 338F/ with encoding gene is used
The area V3-V4 of 806R amplification 16S rRNA gene.It constructs library and upper machine is sequenced, to obtain the OTUs of each sample.According to
Obtained OTUs number can carry out the analysis of the data such as carry out abundance, diversity indices, while right in each categorization levels
Species annotation carries out the statistical analysis of structure of community.On this basis, it can carry out gathering based on OTUs, a series of of species composition
The difference between sample sets with species in group is further excavated in alanysis.
The new method being used in combination using probiotics provided by the invention with micromolecule polypeptide, the novel B T1386- of formation
PMG36e-CXCL12 engineered strain treats bacterial vaginitis rat model, to by restoring vaginal flora balance, participating in
Local inflammation reaction and tissue repair improve BV therapeutic efficiency.
Claims (4)
1. the building of Lactobacillus crispatus BT1386, which is characterized in that comprise the steps of:
(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 uses 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 prepares 2% agarose gel electrophoresis, 110V electrophoresis 45min；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 is centrifuged 4000rpm, 1min, abandons 800 μ L of supernatant, stays 200 μ L bacterium solutions, mixes；
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, is saved backup.
2. the building of Lactobacillus crispatus BT1386 according to claim 1, it is characterised in that: by prepared pMG36e-
CXCL12 is Lactobacillus crispatus BT1386 by electrotransformation, and conversion process comprises the steps of:
The preparation of A lactic acid bacterium competence:
A: taking -80 DEG C of Lactobacillus crispatus BT1386 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 Lactobacillus crispatus BT1386 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 Lactobacillus crispatus BT1386 according to claim 1, it is characterised in that: prepared pMG36e-
CXCL12 detects its secreting, expressing in Lactococcus lactis using Western, specifically comprises the steps of:
A electrophoresis, specific steps are as follows:
A: 12%SDS-PAGE gel is prepared；
B: sample treatment
Lactobacillus crispatus BT1386 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 Lactobacillus crispatus BT1386 and its application in treatment bacterial vaginitis, the application are as follows: pass through yin
The mode of road embolism administration, so that Lactobacillus crispatus BT1386 continuous expression Chemokine CXCL12, to realize to bacillary yin
The alleviation and therapeutic effect of road inflammation.
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| CN109517772A (en) * | 2018-10-30 | 2019-03-26 | 南昌大学 | The building of Lactococcus lactis MG1363 a kind of and its application in treatment puerpera's cracked nipple |
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| CN103074270A (en) * | 2012-11-15 | 2013-05-01 | 上海交大昂立股份有限公司 | Lactobacillus crispatus and application |
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| CN103074270A (en) * | 2012-11-15 | 2013-05-01 | 上海交大昂立股份有限公司 | Lactobacillus crispatus and application |
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Cited By (1)
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| CN109517772A (en) * | 2018-10-30 | 2019-03-26 | 南昌大学 | The building of Lactococcus lactis MG1363 a kind of and its application in treatment puerpera's cracked nipple |
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