CN106434732A - Expression vector applicable to corynebacterium glutamicum and application thereof - Google Patents
Expression vector applicable to corynebacterium glutamicum and application thereof Download PDFInfo
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- CN106434732A CN106434732A CN201610619141.0A CN201610619141A CN106434732A CN 106434732 A CN106434732 A CN 106434732A CN 201610619141 A CN201610619141 A CN 201610619141A CN 106434732 A CN106434732 A CN 106434732A
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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
- C12N15/77—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Corynebacterium; for Brevibacterium
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/34—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Corynebacterium (G)
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- C12P21/00—Preparation of peptides or proteins
- C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
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Abstract
The invention provides an expression vector applicable to corynebacterium glutamicum, aims at solving the technical problem that corynebacterium glutamicum is low in foreign protein expression level, and further provides application of the expression vector. The expression vector applicable to the corynebacterium glutamicum comprises an initiator. The expression vector is characterized in that the nucleotide sequence of the initiator is as shown in SEQ ID NO:1, or a nucleotide sequence complementary to the nucleotide sequence.
Description
Technical field
The present invention relates to genetic engineering and metabolic engineering technical field are and in particular to a kind of be applied to Corynebacterium glutamicum
Expression vector and its application.
Background technology
Corynebacterium glutamicum is a kind of gram-positive bacteria of non-pathogenic, is widely used in industrial production amino acid, organic
The industrial products such as acid, higher alcohol；Meanwhile, Corynebacterium glutamicum endotoxin-free and extracellular no hydrolytic enzyme activities, it can be secreted
Correctly fold has bioactive functions protein, so Corynebacterium glutamicum is also a kind of the extremely advantageous of expression foreign protein
Host.
Promoter is the important DNA sequence dna that one-stage control gene initiates transcription, positioned at transcription initiation site upstream.And gene
MRNA transcriptional level be one of major influence factors of final protein expression level, have research existed by substantial amounts of analytical proof
Control in the various region of sequence of protein expression level, the influence power of promoter region, more than 45%, is most important sequence area
Domain.Therefore, a potent promoter passes through to make to carry the high transcription of mRNA of gene order, thus realizing high-level albumen table
Reach.
For Corynebacterium glutamicum, people are still confined to apply with cspB, trc or tac as promoter at present
Expression vector, the exogenous protein expression level of the wherein expression vector of application tac promoter is higher, but still cannot meet people
The demand growing to foreign protein.
Content of the invention
For the problems referred to above, the invention provides a kind of expression vector being applied to Corynebacterium glutamicum, it can solve paddy
The low technical problem of propylhomoserin bar bacterium exogenous protein expression level, present invention also offers the application of this expression vector.
A kind of expression vector being applied to Corynebacterium glutamicum, its comprise promoter it is characterised in that：Described promoter
Nucleotide sequence such as SEQ ID NO:Shown in 1, or the nucleotide sequence being complementary to.
A kind of exogenous protein expression system, it includes Corynebacterium glutamicum and described expression vector.
The application in exogenous protein expression of above-mentioned expression vector or exogenous protein expression system.
The above-mentioned expression vector of the present invention, comprises SEQ ID NO:The tufA promoter of the bacillus subtilis shown in 1, warp
It is experimentally confirmed that the expression effect of this carrier is more than tac promoter, and the classics in pXMJ19 combine with conservative SD sequence, Neng Gouyou
Effect improves the expression of foreign protein.
Brief description
Fig. 1 is the plasmid map of carrier p10-A0
Fig. 2 is the SDS-PAGE analysis chart of the different expression vector protein expressions of promoter of the present invention and tac promoter structure.
Specific embodiment
Used bacterial strain in the present invention：Bacillus subtilis 168, Corynebacterium glutamicum ATCC 13032 and Escherichia coli
DH5 α is purchased from Chinese Universities ' microbial resources data platform.
Restriction enzymeHind III、BamH I、EcoR V, T4 DNA ligase etc. is purchased from Thermo company；BsaI
Two type restriction endonucleases, the super proofreading polymerase of Q5 are purchased from NEB company；Plasmid extraction kit, PCR primer purification kit, glue reclaim
Kit is purchased from Axygen company；Genome extracts kit is purchased from Tiangeng company；Infusion recombination kit is purchased from
Clontech company；The antibiotic such as chloramphenicol, kanamycins are purchased from Shanghai bioengineering Co., Ltd；Remaining reagent is domestic
Or Import Analysis are pure.High speed freezing centrifuge and PCR thermal cycler, ThermoFisher company of the U.S.；Synergy H4 enzyme
Mark instrument, BioTek company of the U.S.；Constant-temperature metal bath, Beijing Tiangeng company；Dark Reader, it is biotech firm that Beijing reaches section；Core
Acid and protein electrophoresis instrument, Bio-Rad company of the U.S..
The nucleotide sequence of used carrier p19-A0 of the present invention such as SEQ ID NO:Shown in 3, its plasmid map as shown in figure 1,
Its construction method referring to：Liu Xiuxia, Zhao Zihao, Sun Yang, etc. the screening [J] of Corynebacterium glutamicum endogenous expression elements.
Microbiology is circulated a notice of, 2016-05-24 16:42, carrier p19-A0 containsegfpReporter gene.
1st, the clone of promoter.
Comprise the following steps：
A, the acquisition of Bacillus subtilis genes group DNA：Extract the base of bacillus subtilis 168 by genome extracts kit
Because organizing DNA.
B, PCR expand：
PCR amplification condition is：98 °C 、3 min；98 °C, 15 s, 72 °C, 3.25 min, 30 circulations；72 °C 、2
min.Reaction system is：10 × Buffer 5 μ l, dNTPs 4 μ l (each 2.5 mmol/L), each 1 μ l of upstream and downstream primer
(0.2 μm of ol/L), Q5 archaeal dna polymerase 0.5 μ l (2 U/ μ l), template 1 μ l, add water to 50 μ l；
The tufA promoter of the present invention（Hereinafter referred to as PtufA）Primer：
tufA-F：5‘-TTTTGAAGCTTCTGGTTACGAAGAAGTGCCGAAGAG -3’
tufA-R：5‘-CGCCCTTGCTCACCATTCTAAAATCCTCCTTAAGAGCTTT -3’；
Above-mentioned primer 5 ' end introduce homology arm sequence " TTTTGAAGCTTCTGGT " and " CGCCCTTGCTCACCA " respectively so that
Two sequences after amplification withBsaCarrier p19-A0 two terminal sequence after I linearization for enzyme restriction is consistent, homologous recombination enzyme in vitro
With the help of, homologous recombination can occur, so that promoter and reporter geneegfpIt is seamlessly connected, construct containing promoter
Obtain EGFP expression plasmid.
2nd, the structure of expression vector.
The structure of the plasmid containing PtufA, comprises the following steps：
A, pass through II type restriction endonucleaseBsaI cut vector p19-A0 obtains linear plasmid；
B, promoter and linear plasmid are occurred in reactant liquor by homologous recombination by Infusion recombination kit；
C, by the reactant liquor obtaining after homologous recombination convert bacillus coli DH 5 alpha, you can construct the expression plasmid containing NtufA,
Referred to as ptufA.
The restriction enzyme site that conventional method uses is such asEcoR I、BamH I etc. can disturb the correct mensure of promoter, and this
Bright used carrier p19-A0 avoids presence and the interference to result of these additional sequences.
Containing tac promoter（Referred to as Ptac）Positive control plasmid structure, comprise the following steps：
A, clone such as SEQ ID NO:Corynebacterium glutamicum shown in 2 guard SD sequence andegfpGenetic fragment, required primer
For：
SD-EGFP-F: 5‘- CCCAAGCTTAAAGGAGGACAACTAATGGTGAGCAAGGGCG -3’
SD-EGFP-R: 5‘-CCCGGATCCTTACTTGTACAGCTCGTCCATG-3’
PCR amplification condition is identical with PCR amplification condition in the clone of promoter " 1, "；
Article two, " AAGCTT " " GGATCC " of primer point represents the restriction enzyme site building the required introducing of this positive control plasmid, point
It is notHinD III andBamH I.
b、HinD III andBamH I double digestion,
C, T4 connect.
Digestion is all carried out according to the instructions direct of commodity enzymatic reagent with being connected.
Corynebacterium glutamicum guard SD sequence andegfpGenetic fragment is combined with the Ptac on plasmid pXMJ19, builds composition
The positive control plasmid of type expression（Referred to as pTAC）, the efficient table of Corynebacterium glutamicum that this classical positive control represents
Reach the level of recombinant protein.
3rd, convert.
Comprise the following steps：
A, ptufA, pTAC are extracted respectively by plasmid extraction kit；
B, conversion Corynebacterium glutamicum ATCC 13032.
Concrete grammar is：
（1）The preparation of Corynebacterium glutamicum competent cell
Corynebacterium glutamicum picking after cremating on culture medium flat plate is seeded in BHI culture medium, in 30 °C, 230 rpm
Under the conditions of incubated overnight, bacterium solution is forwarded to the LB Liquid Culture containing 3% glycine, 0.1%Tween 80 and 0.5% glucose
In 250 ml triangular flasks of base, control its initial OD600For 0.3, cultivate 4-6 h under the conditions of 30 °C, 230 rpm, to OD600Reach
To 0.6-0.9.Again bacterium solution is transferred in sterile centrifugation tube, places 15 min on ice.Hereinafter operate all in low temperature, aseptic condition
Under carry out.
4 °C, 4000 rpm centrifugation 5min, remove supernatant, collects thalline, with the resuspended thalline of 10% glycerine of precooling, floated use
The careful piping and druming of liquid-transfering gun.Repeat above-mentioned centrifugation, go supernatant step 3 time, with 1.5 ml 10% glycerine re-suspended cell, dispense to
In 1.5ml centrifuge tube, often pipe fills 100 μ l, be stored in -80 °C standby.
（2）Corynebacterium glutamicum is competent electroporated
Competent cell is removed from -80 °C of refrigerators, as thawed on ice, gently mixes after adding 10 μ l recombinant plasmids, turn
Move in 0.1 cm electric shock cup of precooling, shock by electricity under the conditions of 1.8 kv, 200 Ω, 25 μ F cell.Add immediately after electric shock and contain
LB fluid nutrient medium 1 ml of 9.2% sorbierite of 1.85% brain heart oxoid, mixes, proceeds in 1.5 ml centrifuge tubes, in 46 °C
Water-bath 6 min, 30 °C, 150 rpm shaken cultivation 2 h.Then coat after bacterium solution being concentrated containing 1.85% brain heart oxoid
On the LB solid medium of 9.2% sorbierite, cultivate 48h in 30 °C.
4th, the determination of activity of promoter.
Comprise the following steps：
A, culture, the Corynebacterium glutamicum containing ptufA or pTAC building in the structure of expression vector " 2, " is inoculated respectively
Contain the brain heart oxoid of 10 mg/L chloramphenicol to 2 ml（BHI）In culture medium, 30 °C, 230 r/min overnight incubation, so
Afterwards according to 1:100 dilution proportion is transferred in 2 new ml BHI culture mediums, using high flux 24 hole depth well culture plate, in phase
Cultivate 24 hours under conditions of same；
B, microorganism collection, cultured bacterium solution are immediately placed in precooling on ice, thalline are collected by centrifugation, clean two with PBS
Secondary, resuspended；
C, EGFP expression measures：The EGFP expression of unit thalline is defined as the OD of fluorescence measurement value F and sample600Ratio,
Measure OD using multi-function microplate reader600And fluorescence intensity, its result is as shown in the table.
5th, application PtufA promoter expresses amylase in Corynebacterium glutamicum.
A, the structure of amylase expression vector
By the same way promoter PtufA is inserted in amylase carrier detection p19-B0, amylase carrier detection p19-B0
It is replaced by amylase gene with the reporter gene that differs only in of green fluorescent protein carrier detection p19-A0.By the matter building
Grain is transformed in Corynebacterium glutamicum.
B, enzyme activity determination method：Take 1%, pH6.0 soluble starch solution 50 μ l, water-bath at 60 DEG C preheats 5 min
Afterwards, after adding crude enzyme liquid 250 μ l accurately to react 5 min, 450 μ l dinitrosalicylic acids are added（DNS）, 5 points of boiling water bath reaction
Clock, is diluted with water 20 times and measures light absorption value at 540 nm with spectrophotometer afterwards, calculate the reduction sugar amount producing.Calibration curve
Drafting referring to：Shi Yongchang, Jiang Yongming. the comparative studies [J] of five kinds of AMS measuring method for activity. microbiology is circulated a notice of,
1996-06-14 6:23 enzyme activity definition：One amylase unit is equivalent under the conditions of 60 DEG C, pH6.0, and catalysis per minute produces
1 μm of ol reduced sugar（With glucose meter）Required enzyme amount.Enzyme activity computing formula is：（5.1159╳OD540-0.0353）Dilution
Multiple ÷ 5
Through the detection of amylase activity, the amylase gene expression vector with promoter PtufA sequence is in glutamic acid bar
Vigor in bacterium is 33.6U/ml, and is not detected by amylase activity vigor without the control vector of promoter sequence.
Above-mentioned the results show：
PtufA and albumen coded sequence can start protein expression after being joined directly together, and can lure without any chemistry
Lead constitutive expression recombinant protein in the case of agent or physics inducement condition；
Under the evaluation and test of Green fluorescent protein fusion vector, the protein expression level of the carrier containing PtufA of the present invention is obvious
More than the Ptac protein expression level that the classics in pXMJ19 combine with conservative SD sequence.
Analyzed by SDS-PAGE, the EGFP protein expression test result of the Corynebacterium glutamicum containing different carriers is as schemed
Shown in 2.Wherein, the EGFP protein band color corresponding to the Corynebacterium glutamicum containing ptufA is deeper than the paddy ammonia containing pTAC
EGFP protein band color corresponding to sour bar bacterium, the protein expression level also having confirmed PtufA is higher than the albumen table of Ptac
Reach level.
Additionally, the expression through amylase gene is verified it was demonstrated that PtufA is also applied for other genes in Corynebacterium glutamicum
Expression.
Claims (3)
1. a kind of expression vector being applied to Corynebacterium glutamicum, its comprise promoter it is characterised in that：The core of described promoter
Acid sequence such as SEQ ID NO:Shown in 1 or the nucleotide sequence that is complementary to.
2. a kind of exogenous protein expression system, it includes Corynebacterium glutamicum and expression vector as claimed in claim 1.
3. expression vector as claimed in claim 1 or exogenous protein expression system as claimed in claim 2 are in foreign protein table
Application in reaching.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107164371A (en) * | 2017-03-21 | 2017-09-15 | 武汉远大弘元股份有限公司 | It is a kind of that the corynebacteria constitutive expression carrier promoter built is sequenced based on transcript profile |
| CN107164370A (en) * | 2017-03-21 | 2017-09-15 | 武汉远大弘元股份有限公司 | A kind of corynebacteria constitutive expression carrier promoter, the expression vector containing the promoter and gnd genes |
| CN107164369A (en) * | 2017-03-21 | 2017-09-15 | 武汉远大弘元股份有限公司 | A kind of corynebacteria constitutive expression carrier promoter, the expression vector containing the promoter and lrp genes |
| US11639512B2 (en) | 2017-03-21 | 2023-05-02 | Wuhan Grand Hoyo Co., Ltd. | Corynebacterium constitutive expression vector promoter screened on the basis of transcriptome sequencing, screening method thereof, and applications thereof |
| CN116355940A (en) * | 2023-03-24 | 2023-06-30 | 江南大学 | Expression vector of structural protein VP1 of porcine foot-and-mouth disease virus O-type foot-and-mouth disease virus, construction method and application thereof |
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| CN101698844A (en) * | 2009-06-29 | 2010-04-28 | 中国科学院微生物研究所 | Promoter from corynebacterium glutamicum and application thereof |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107164371A (en) * | 2017-03-21 | 2017-09-15 | 武汉远大弘元股份有限公司 | It is a kind of that the corynebacteria constitutive expression carrier promoter built is sequenced based on transcript profile |
| CN107164370A (en) * | 2017-03-21 | 2017-09-15 | 武汉远大弘元股份有限公司 | A kind of corynebacteria constitutive expression carrier promoter, the expression vector containing the promoter and gnd genes |
| CN107164369A (en) * | 2017-03-21 | 2017-09-15 | 武汉远大弘元股份有限公司 | A kind of corynebacteria constitutive expression carrier promoter, the expression vector containing the promoter and lrp genes |
| CN107164371B (en) * | 2017-03-21 | 2019-09-20 | 武汉远大弘元股份有限公司 | A kind of corynebacteria constitutive expression carrier promoter based on transcript profile sequencing building |
| CN107164370B (en) * | 2017-03-21 | 2019-09-20 | 武汉远大弘元股份有限公司 | A kind of corynebacteria constitutive expression carrier promoter, the expression vector containing the promoter and gnd gene |
| CN107164369B (en) * | 2017-03-21 | 2019-09-20 | 武汉远大弘元股份有限公司 | A kind of corynebacteria constitutive expression carrier promoter, the expression vector containing the promoter and lrp gene |
| US11639512B2 (en) | 2017-03-21 | 2023-05-02 | Wuhan Grand Hoyo Co., Ltd. | Corynebacterium constitutive expression vector promoter screened on the basis of transcriptome sequencing, screening method thereof, and applications thereof |
| CN116355940A (en) * | 2023-03-24 | 2023-06-30 | 江南大学 | Expression vector of structural protein VP1 of porcine foot-and-mouth disease virus O-type foot-and-mouth disease virus, construction method and application thereof |
| CN116355940B (en) * | 2023-03-24 | 2024-03-22 | 江南大学 | Expression vector of structural protein VP1 of porcine foot-and-mouth disease virus O-type foot-and-mouth disease virus, construction method and application thereof |
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