CN108220219A - A set of lactobacillus plantarum food-grade expression system and its application in heterologous protein expression - Google Patents
A set of lactobacillus plantarum food-grade expression system and its application in heterologous protein expression Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- 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
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- C—CHEMISTRY; METALLURGY
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- 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
- C12N15/65—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression using markers
Abstract
The invention discloses a set of lactobacillus plantarum food-grade expression system and its applications in heterologous protein expression.The lactobacillus plantarum food-grade expression system includes food-grade host plant lactobacillus（Lactobacillus plantarum）NZ5333, food grade expression vector pSIP497, culture medium MRS Y and culture medium MRS+N；The food-grade host plant lactobacillus（Lactobacillus plantarum）NZ5333 is knocked out using Cre loxP technologies on lactobacillus plantarum WCFS1 genomesglmSGene and obtain, lose the ability of 6 phosphate synthase of synthesis of glucose amine.The expression system is applied in heterologous protein expression, and expression effect is good.The expression system is food-grade expression system, and DNA element comes from the microorganism of generally recognized as safe, without antibiotic resistance selection markers.
Description
Technical field
The present invention relates to biology fields, and in particular to a set of lactobacillus plantarum food-grade expression system and its different
Application in source protein expression.
Background technology
With the development of science and technology, it is more and more research shows that, lactic acid bacteria in immunological regulation, maintain intestinal flora
Balance the absorption of nutrient ingredients that promotes, alleviates lactose intolerance, inhibits pathogenic bacteria and reduces serum cholesterol content and prevention painstaking effort
Pipe disease etc. has a significant impact.Since lactic acid bacteria has generally acknowledged biological safety (generally recognized as
Safe, GRAS), it is developed and used as engineering carrier bacterial strain then in food development, nutrient health and medicine and hygiene fields
In have sizable potentiality and value.At present, had using lactic acid bacteria engineering carrier production functional protein, developed
The report of the researchs such as oral vaccine and developing new drug.It is worth noting that, in lactic acid bacteria to the research of Bacillus acidi lactici especially
The hot spot of research, the report on the one hand with the lactobacillus of notable prebiotic function emerge in an endless stream, such as lactobacillus acidophilus, rhamnose breast
Bacillus and lactobacillus reuteri etc., on the other hand since lactobacillus derives from a wealth of sources, fermenting property is good and industrially especially eats
The industrial important strain of product, such as Lactobacillus delbrueckii.
Traditional lactic acid bacteria expression vectors are all anti-with one or more coding certain antibiotics (such as erythromycin, chloramphenicol)
The gene of property, although resistance marker ensure that effective progress of transformant screening, the transferability of resistance marker and potential prestige
The side of body significantly reduces practical value of the lactic acid bacteria in food and medical domain of molecular genetic modification, thus, there is an urgent need to develop
Food-grade selection markers establish the food-grade expression system expressed in heterologous protein.
It is divided into two major class about food-grade selection markers in the prior art, i.e., dominant selection markers and complementation label.
Dominant selection markers are typically to reach sieve using characteristic such as bacteriocin resistance, thermal sensitivity, carbohydrate utilization rate etc. of host strain
Select purpose, such as develop it is more mature be most widely used at present from Lactococcus lactis nisalpin Nisin selection markers；
Complementation marks, and the missing of the certain genes of bacterium such as housekeeping gene is generally utilized or mutation causes bacterial growth variation to reach
To screening purpose, such as Lactococcus lactis thymidylate synthase gene thyA genescreens label, lactobacillus plantarum pyruvic acid racemization
Enzyme alr selection markers and Lactobacillus casei Q-5/pQJ5 screening systems.The lactobacillus food grade screening mark of current development and application
Note is seldom, it is impossible to meet the needs of many destination protein food-grade expression, more new selection markers wait to develop.It is of the invention public
The expression system using lactobacillus plantarum glmS genes as selection markers has been opened, has been the new screening mark in lactic acid bacteria heterogenous expression
Note.Glucosamine-6-phosphate synzyme (Glucosamine-6-phosphate synthase, GlmS) can promote 6- phosphoric acid
Fructose and L-Glutamine are converted to 6- phosphorylated amino glucoses, are glucose metabolic pathways Hexosamine biosynthesis pathway and Cell wall synthesis
The rate-limiting enzyme of reaction.Glucosamine-6-phosphate synzyme is N-Acetyl-D-glucosamine (GlcNAc) and ammonia in the HBP products being catalyzed
Base glucose (GlcN), both gucosamines are of great significance to organism vital movement, and the polysaccharide of many biological cells is such as
Chitin and glutinous polysaccharide are all finally synthesized by N-Acetyl-D-glucosamine (GlcNAc), N-Acetyl-D-glucosamine (GlcNAc)
It is also the important as precursors of the double qi factors of synthesis.GlmS is the encoding gene of glucosamine-6-phosphate synzyme, research shows that not having
GlmS encoding genes, bacterial strain in the culture medium without aminoglucose or N-Acetyl-D-glucosamine can not normal growth, cell
It can rapid cleavage death.Further, since glucosamine-6-phosphate synzyme is food-grade molecule, therefore glmS genes is selected to make
For selection markers, the food-grade expression system that it is lactobacillus plantarum is developed, is application of the lactic acid bacteria in food and medical domain
Research provides a kind of means of effective and safe.
Invention content
For prior art problem, the purpose of the present invention is to provide a set of lactobacillus plantarum food-grade expression system and its
Application in heterologous protein expression, the set lactobacillus plantarum food-grade expression system expression effect is good, and all DNA are former in system
Part comes from the microorganism of generally recognized as safe, without antibiotic resistance selection markers.
A set of lactobacillus plantarum food-grade expression system includes food-grade host plant lactobacillus (Lactobacillus
Plantarum) NZ5333, food grade expression vector pSIP497, culture medium MRS-Y and culture medium MRS+N；The food-grade place
Main lactobacillus plantarum (Lactobacillus plantarum) NZ5333 is to knock out lactobacillus plantarum using Cre-loxP technologies
GlmS genes on WCFS1 genomes and obtain, lose the ability of synthesis of glucose amine -6- phosphate synthases.
Food-grade host plant lactobacillus (Lactobacillus plantarum) NZ5333, in December, 2017
08, it is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center CGMCC, preservation address：Court of Beijing
The positive institute 3 of area's North Star West Road 1, deposit number are CGMCC No.15039.
The construction method of the food grade expression vector pSIP497, on the basis of lactic acid bacteria expression vectors pSIP409,
The base sequence of coding erythromycin resistance gene is knocked out, is inserted into the glmS genes from lactobacillus plantarum WCFS1, is inserted into source
In the P of lactobacillus plantarum WCFS1 bacterial strainsldhLPromoter.
The glmS genes of the lactobacillus plantarum WCFS1 are food-grade host plant lactobacillus (Lactobacillus
Plantarum) NZ5333 and food grade expression vector pSIP497 completes the food-grade label of complementation.
Application of the above-mentioned lactobacillus plantarum food-grade expression system in heterologous protein expression.
Application of the above-mentioned lactobacillus plantarum food-grade expression system in heterologous protein expression, includes the following steps：Using
PCR amplification reporter gene red fluorescent protein gene mCherry is inserted into food grade expression vector pSIP497 multiple cloning sites, adopts
The carrier built is transferred in lactobacillus plantarum NZ5333 with electrotransformation, using SppIP induction peptide induced expressions, detection is red
Expression of the color fluorescin in lactobacillus plantarum NZ5333 cells, expression process do not add antibiotic.
The method for detecting the expression of red fluorescent protein：It measures its fluorescence intensity level with microplate reader or utilizes and be inverted fluorescence
The red fluorescence of microscopic.
Advantageous effect：
The a set of lactobacillus plantarum food-grade expression system of the present invention, the system have food-grade host plant lactobacillus
NZ5333 and food grade expression vector pSIP497, and host and carrier have the food-grade label that can complete complementation,
Antibiotic need not be added during the heterogenous expression of reporter gene, is avoided because of the biological safety that resistance factor transition zone comes
Potential hazard.
Description of the drawings
Fig. 1 is the verification result for knocking out plasmid pNZ5326, wherein, M-DNA molecular weight standards, 1-5 ' homologous recombination arms, length
Spend 1064bp, 2-3 ' homologous recombination arms, length 1001bp；
Fig. 2 is glmS gene knockout PCR verification results, wherein, M-DNA molecular weight standards, 3-glmS genes, length
1818bp, 4- upstream outer amplified production, length 1194bp；Amplified production on the outside of 5- downstreams, length 1283bp；
Fig. 3 is PCR verification results after chloromycetin gene removal on WCFS1 genomes, wherein, M-DNA molecular weight standards, 6-
Outside amplified production, length 2183bp；
Fig. 4 for ldhL promoters and glmS gene magnifications as a result, wherein, M-DNA molecular weight standards, 7-PldhLAmplification production
Object, length 500bp, 8-glmS gene amplification product, length 1818bp；
Fig. 5 removes verification result for food grade carrier pSIP497 erythromycin, wherein, M-DNA molecular weight standards, 9-PldhL-
GlmS genes, length 2318bp；10- erythromycin genes, length 602bp；
Fig. 6 is the structure flow chart of the food grade expression vector pSIP497 of the present invention；
Fig. 7 is mCherry channel genes host strain PCR verification results, wherein, M-DNA molecular weight standards, 11-mCherry
Gene, length 711bp；
Fig. 8 expresses bacterial strain fluorescence intensity and growth curve chart for mCherry；
Fig. 9 is the design sketch using the food-grade expression system expression red fluorescent protein of the present invention.
Specific embodiment
The present invention is further described in detail below by specific embodiment.
Embodiment 1
Bacterium bacterial strain, plasmid, main material and the reagent for using and being related in present invention experiment：
Lactobacillus plantarum WCFS1 is purchased from Huankai Microbes Tech Co., Ltd., Guangdong；
Food-grade host plant lactobacillus NZ5333；
Bacillus coli DH 5 alpha competent cell is purchased from Beijing Quanshijin Biotechnology Co., Ltd；
Plasmid pNZ5319, pNZ5348 are given by Dutch dairy products research institute Jolanda M.Lambert；
Plasmid pSIP409 is purchased from BioVector NTCC plasmid vector bacterium cell protein antibodies gene collections；
The E.coli K12 Δ glmS competent cells of glmS gene knockouts are preserved by laboratory structure early period；
Bacterial genomes extracts kit；
The a small amount of extraction agent boxes of SanPrep pillar Plasmid DNA；
Ion-exchange type plasmid extraction kit；
2-acetylamino-2-deoxy-D-glucose (GlcNAc)；
Erythromycin, chloramphenicol, ampicillin, agarose, 4S nucleic acid dyes etc., which are purchased from Shanghai life work bioengineering, to be had
Limit company；The polymerases such as Ex taq and rtaq, Ex taq, PrimeSTAR Max Premix (2 ×), restriction enzyme, even
Connect enzyme, pMD19-T carriers, DNA Marker, DNA gel QIAquick Gel Extraction Kit carries by Beijing Bao Yi Bioisystech Co., Ltd
For；Other main agents are domestic analysis net product；
The special MRS culture mediums of lactic acid bacteria；Mutant strain MRS+N culture mediums：Peptone 10g, powdered beef 8g, dusty yeast
4g, glucose 20g, dipotassium hydrogen phosphate 2g, citric acid hydrogen diamine 2g, sodium acetate 5g, magnesium sulfate 0.2g, manganese sulfate 0.04g, tween
80 1g, 2-acetylamino-2-deoxy-D-glucose (GlcNAc) 22.121g；Escherichia coli LB culture mediums.
Recombinant lactic acid bacteria screening and culturing medium MRS-Y culture mediums in the present invention：Peptone 10g, powdered beef 8g, glucose 20g,
Dipotassium hydrogen phosphate 2g, citric acid hydrogen diamine 2g, sodium acetate 5g, magnesium sulfate 0.2g, manganese sulfate 0.04g, Tween 80 1g.
Recombination bacillus coli screening and culturing medium MT (M9 adds 1% peptone) culture medium in the present invention.
Embodiment 2
1st, the structure of food-grade host plant lactobacillus NZ5333.
(1) lactobacillus plantarum WCFS1 complete genome DNAs extract
It is extracted using bacterial genomes extracts kit with reference to specification.
(2) the structure I of the PCR amplification and suicide plasmid pNZ5326 of glmS gene 5 's end homology arm and 3 ' end homology arms
Design of primers and synthesis
With NCBI Lactobacillus plantarum WCFS1genome (GenBank:AL935263.2 it is) template,
5 ' the end homology arms and 3 ' end homology arms of glmS genes are separately designed,
GlmS gene 5 's end homology arm primer：
A:5’-GGGCTCGAGCAGGATACAGTCGTCACAACG-3’
B:5’-GGGATTTAAATCCACACATAAATTAATCTTCC-3’
GlmS genes 3 ' hold homology arm primer：
C:5’-CCCGAGCTCGTAAGTGTGTGGGCACCAAG-3’
D:5’-GGGAGATCTGATTGCCACAAGTAGCCAC-3’
Amplification, clone and the sequencing of II .glmS gene 5 's homology arm and 3 ' homology arms
(I) PCR reaction systems are as follows：
(II) PCR reaction conditions are as follows：
95 DEG C of pre-degeneration 5min；(95 DEG C of unwinding, 45s；60 DEG C of annealing, 45s；72 DEG C of extension, 1min) × 30 cycles；72
DEG C, 10min；4 DEG C of preservations
(III) TA cloning and sequencings
The PCR product of 5 ' homology arms and the PCR product of 3 ' homology arms respectively with pMD19-T simple carriers 16 DEG C connect
1h, rear Transformed E .coli DH5 α Competent cells, after be coated on LB/Amp (100 μ g/mL) tablet, 37 DEG C of cultures to list
Clone formation.Picking monoclonal is corresponded in 3mL in culture medium, 37 DEG C, 220rpm shaken cultivations.By the clone of acquisition through plasmid
Extraction, respectively with XhoI-SwaI and Ecl136II-BglII digestion verifications, will verify correct plasmid with M13Forward
Primer and M13Reverse Primer primers are sent to the sequencing of Shanghai life work biology Co., Ltd.
(3) structure for knocking out plasmid pNZ5326 of homologous recombination
Distinguish a large amount of digestions using XhoI-SwaI and Ecl136II-BglII and correct T clones upstream and downstream homology arm is sequenced
Plasmid is detached through 1% Ago-Gel, extracts DNA fragmentation under ultraviolet lamp, elution is isolated and purified with plastic recovery kit.
PNZ5319 is largely recycled through ion exchange column kit, about 5 μ g/ μ L of plasmid concentration, first carries out the bis- enzymes of Ecl136II and BglII
Large fragment carrier is cut back to close, is connect overnight for 16 DEG C with T4 ligases with 3 ' end homology arm recovery products.Connection product converts
E.coli DH5 α Competent cells, are coated on LB tablets (containing 10 μ g/mL chloramphenicol and 250 μ g/mL erythromycin), 37 DEG C
Culture.Clone through Liquid Culture is extracted into plasmid, digestion verification.It verifies correct clone large quantity extracting plasmid, passes through
The processing of XhoI-SwaI double digestions connect overnight (16 DEG C) with 5 ' end homology arm recovery products, converts Escherichia coli Competent
Cell is coated on the LB solid mediums containing chloramphenicol and erythromycin, plasmid is extracted after culture.With XhoI-SwaI and
Ecl136II-BglII difference double digestions verify 5 ' homology arms and 3 ' homology arms, and the result is shown in Figure 1 obtains striking for homologous recombination
Except plasmid pNZ5326.DNA molecular amount standard used from top to bottom molecular weight be followed successively by 2000,1000,750,500,250,
100bp。
(4) using the glmS genes of suicide plasmid pNZ5326 and pNZ5348 traceless knockout WCFS1
The preparation of I lactobacillus plantarum WCFS1 competent cells
It chooses from MRS tablets in WCFS1 monoclonals inoculation 50mL MRS culture mediums, after 30 DEG C of Anaerobic culturels are stayed overnight, will train
For nutrient solution under the conditions of 4 DEG C, 1000g centrifuges 10 minutes sedimentation cells；It is light (containing 10% glycerine) with the 0.5M sucrose being pre-chilled in equal volume
Soft suspension cell, sedimentation cell under the same terms, incline supernatant, then washed once.Contain the 0.5M sucrose of 10% glycerine with 100 μ L
Solution suspension cell, by 40 μ L volumes packing, two pipe.
II electricity turns to knock out plasmid in competent cell
PNZ5326 is largely extracted using ion exchange pillar plasmid extraction kit, in 40 μ L lactobacillus plantarum competence
5 μ L plasmid pNZ5326 are added in cell, are transferred in the 2mm electricity revolving cup (eppendorf) of precooling after mixing, in 1800V voltages
Lower electric shock (eppendorf AG electroporations).After electric shock, suspended immediately with 1mL MRS culture mediums (containing 10mMGlcNAc) thin
Bacterium is transferred to 2mL eppendorf pipes, and MRS+N tablets (containing 10 μ g/mL chloramphenicol), 30 DEG C of trainings are coated on after putting 30 DEG C of incubation 3h
It supports.The monoclonal grown extracts full-length genome after respective liquid medium culture, and glmS genes are carried out with glmS Outside primers
Identification, as shown in Figure 2.
(5) removal of chloramphenicol resistance marker
First step knockout eliminates glmS genes, using chloramphenicol as selection markers, obtains chloramphenicol resistant strain.By the bacterium
Strain is prepared into competent cell (preparation method with WCFS1 competence prepare consistent), 1800V it is electroporated enter 3 μ L ion exchanges
The pNZ5348 of column extraction.Immediately with 1mL MRS culture mediums (GlcNAc containing 10mM) suspension thalline, after 30 DEG C are incubated 3h, coating
MRS+N solid mediums, 37 DEG C are cultivated 2 days.Target clone verifies (such as Fig. 3) through PCR, confirms that chloromycetin gene is successfully moved
It removes, the mutant strain of acquisition is named as NZ5333.
The structure of the food grade expression vector pSIP497 of embodiment 3glmS labels
1.PldhLConnection with glmS genetic fragments is cloned
I expands ldhL promoter genes and glucosamine-6-phosphate synthetase-coding gene glmS respectively
Lactobacillus plantarum WCFS1 full-length genomes are extracted using bacterial genomes extracts kit, with reference to lactobacillus plantarum
Whole genome sequence (the GenBank that WCFS1 is announced:AL935263.2), retrieval obtains glmS gene orders, designs primer, leads to
It crosses PCR and expands glmS genes (1818bp) and P respectivelyldhLSegment (500bp) is shown in Fig. 4, primer sequence, reaction system and reaction
Condition is as follows：
(I) primer sequence：
Pldhl-F:5’-AGATCTAATCTTCTCACCGTCTTG-3’
Pldhl-R:5’-CACCAACAATTCCACACATTCCTACGAGAGGATGACTTATT-3’
glmSF:5’-AATAAGTCATCCTCTCGTAGGAATGTGTGGAATTGTTGGTG-3’
glmSR:5’-GGGCTCGAGTCAGTGGTGGTGGTGGTGGTGTTCAACGGTCACACTCTTG-3’
(II) reaction system：
(III) reaction condition：95 DEG C of pre-degeneration 5min；(95 DEG C of unwinding, 60s；60 DEG C of annealing, 90s；72 DEG C of extension, 3min)
× 30 cycles；72 DEG C, 10min；4 DEG C of preservations.
II .ldhL promoter genes connected with glucosamine-6-phosphate synthetase-coding gene glmS after overlap-
PCR amplification
The P amplifiedldhLPurified respectively with plastic recovery kit with glmS genetic fragments, using two segments of purifying as
Template uses primer pair Pldhl-F and glmSR ligation amplification PldhL- glmS segments, PCR system and reaction condition are as follows.
(I) reaction system：
(II) reaction condition：95 DEG C of pre-degeneration 5min；(95 DEG C of unwinding, 60s；60 DEG C of annealing, 90s；72 DEG C of extension, 3min)
× 30 cycles；72 DEG C, 10min；4 DEG C of preservations
Amplified production after purification, connect with carrier T (16 DEG C), converts DH5 α competent cells, is coated on containing 100 μ
On the LB solid mediums of g/mL Amp, 37 DEG C of cultures.Plasmid DNA is extracted after choosing Colony Culture, double digestion verification is carried out, tests
Card correct plasmid compares sieve through sequencing and takes positive colony.
II utilizes overlap PldhL- glmS segments removal pSIP409 carriers erythromycin label
BglII-XhoI digestions processing T cloning vector recycling PldhL- glmS segments, then with BamHI and SalI double digestions
Linear carrier segments are recycled in pSIP409 carriers, purifying.PldhL- glmS segments are attached processing with the carrier, and 16 DEG C overnight.
After mixing ice bath heat shock, 1h is incubated with 1mL MT culture mediums for next day Transformed E .coli K12 Δs glmS Competent cells, in
It is cultivated in nonreactive MT screening flat boards, 37 DEG C overnight.Picking Colony Culture extracts plasmid, utilizes primer pair Pldhl-F-glmSR
PCR verifications are carried out with EryintF-EryintR, EryintF-EryintR primer sequences, PCR reaction systems and reaction condition are such as
Under, as a result see Fig. 5, successfully construct food grade expression vector pSIP497.
2nd, using on the lactobacillus plantarum food-grade expression system heterogenous expression mCherry fluorescins constructed by the present invention
Application of the lactobacillus plantarum food-grade expression system in heterologous protein expression is stated, is included the following steps：It is reported using PCR amplification
Gene red fluorescent protein gene mCherry is inserted into food grade expression vector pSIP497 multiple cloning sites, using electrotransformation
The carrier built is transferred in lactobacillus plantarum NZ5333, using SppIP induction peptide induced expressions, detects red fluorescent protein
Expression in lactobacillus plantarum NZ5333 cells, expression process do not add antibiotic.The results are shown in Figure 7, red fluorescence egg
It is expressed in lactobacillus plantarum NZ5333 cells in vain good.
In addition, the present invention is not limited to the above embodiment, as long as in without departing from the scope of the present invention, can take various
Mode implements the present invention.
Sequence table
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Claims (6)
1. a set of lactobacillus plantarum food-grade expression system, which is characterized in that include food-grade host plant lactobacillus
（Lactobacillus plantarum）NZ5333, food grade expression vector pSIP497, culture medium MRS-Y and culture medium MRS
+N；The food-grade host plant lactobacillus（Lactobacillus plantarum）NZ5333 is using Cre-loxP technologies
It knocks out on lactobacillus plantarum WCFS1 genomesglmSGene and obtain, lose synthesis of glucose amine -6- phosphate synthases energy
Power.
2. a set of lactobacillus plantarum food-grade expression system according to claim 1, which is characterized in that the food-grade table
Up to the construction method of carrier pSIP497, on the basis of lactic acid bacteria expression vectors pSIP409, coding Erythromycinresistant base is knocked out
The base sequence of cause is inserted into from lactobacillus plantarum WCFS1'sglmSGene is inserted into from lactobacillus plantarum WCFS1 bacterium
The P of strain ldhL Promoter.
3. a set of lactobacillus plantarum food-grade expression system according to claim 1, which is characterized in that the plant breast bar
Bacterium WCFS1'sglmSGene is that food-grade host plant lactobacillus NZ5333 and food grade expression vector pSIP497 complete complementation
The food-grade label of screening.
4. the application based on a set of lactobacillus plantarum food-grade expression system described in claim 1 in heterologous protein expression.
5. application of a set of lactobacillus plantarum food-grade expression system according to claim 4 in heterologous protein expression,
It is characterized by comprising the following steps：Using PCR amplification reporter gene red fluorescent protein genemCherry, it is inserted into food-grade
The carrier built is transferred in lactobacillus plantarum NZ5333 by expression vector pSIP497 multiple cloning sites using electrotransformation, profit
With SppIP induction peptide induced expressions, process is expressed in expression of the detection red fluorescent protein in lactobacillus plantarum NZ5333 cells
Do not add antibiotic.
6. application of a set of lactobacillus plantarum food-grade expression system according to claim 4 in heterologous protein expression,
It is characterized in that, the method for the expression of detection red fluorescent protein：Its fluorescence intensity level is measured with microplate reader or utilizes inversion
The red fluorescence of fluorescence microscopy.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110643561A (en) * | 2019-10-30 | 2020-01-03 | 上海市农业科学院 | Application of glms gene in lactobacillus biosafety screening marker |
| CN110846268A (en) * | 2019-12-03 | 2020-02-28 | 齐鲁工业大学 | Food-grade lactobacillus plantarum expression system and application thereof in yoghourt preparation |
| CN112080451A (en) * | 2020-07-13 | 2020-12-15 | 宁波大学 | Food-grade gene expression system of lactobacillus acidophilus and preparation method and application thereof |
| CN110791522B (en) * | 2019-12-03 | 2021-04-02 | 齐鲁工业大学 | Double-plasmid food-grade lactobacillus plantarum expression system and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103757048A (en) * | 2013-05-22 | 2014-04-30 | 仙奕生物科技（南京）有限公司 | Construction and application of drug resistant gene-free yeast-bacterial shuttle vector |
| CN105734072A (en) * | 2016-03-08 | 2016-07-06 | 吉林农业大学 | alr (alanine racemase)-deficient lactobacillus plantarum NC8 |
-
2018
- 2018-02-08 CN CN201810127250.XA patent/CN108220219B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103757048A (en) * | 2013-05-22 | 2014-04-30 | 仙奕生物科技（南京）有限公司 | Construction and application of drug resistant gene-free yeast-bacterial shuttle vector |
| CN105734072A (en) * | 2016-03-08 | 2016-07-06 | 吉林农业大学 | alr (alanine racemase)-deficient lactobacillus plantarum NC8 |
Non-Patent Citations (3)
| Title |
|---|
| GUOGAN WU等: "Application of GFAT as a Novel Selection Marker to Mediate Gene Expression", 《PLOS ONE》 * |
| 乐易林等: "微生物遗传转化筛选标记及其生物安全性的研究进展", 《微生物学通报》 * |
| 李自刚等: "《食品微生物检验技术》", 31 January 2016, 中国轻工业出版社 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110643561A (en) * | 2019-10-30 | 2020-01-03 | 上海市农业科学院 | Application of glms gene in lactobacillus biosafety screening marker |
| CN110846268A (en) * | 2019-12-03 | 2020-02-28 | 齐鲁工业大学 | Food-grade lactobacillus plantarum expression system and application thereof in yoghourt preparation |
| CN110791522B (en) * | 2019-12-03 | 2021-04-02 | 齐鲁工业大学 | Double-plasmid food-grade lactobacillus plantarum expression system and application thereof |
| CN110846268B (en) * | 2019-12-03 | 2021-06-25 | 齐鲁工业大学 | Food-grade lactobacillus plantarum expression system and application thereof in yoghourt preparation |
| CN112080451A (en) * | 2020-07-13 | 2020-12-15 | 宁波大学 | Food-grade gene expression system of lactobacillus acidophilus and preparation method and application thereof |
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| CN108220219B (en) | 2022-02-22 |
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