CN100383247C - Constitutive-expression vector and its construction method and application - Google Patents
Constitutive-expression vector and its construction method and application Download PDFInfo
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- CN100383247C CN100383247C CNB2004100838496A CN200410083849A CN100383247C CN 100383247 C CN100383247 C CN 100383247C CN B2004100838496 A CNB2004100838496 A CN B2004100838496A CN 200410083849 A CN200410083849 A CN 200410083849A CN 100383247 C CN100383247 C CN 100383247C
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Abstract
The present invention discloses a constitutive expression vector, a construction method and the application thereof. The aim of the present invention is to provide a constitutive expression vector, a construction method and the application thereof in GL-7-ACA acylase expression. The provided constitutive expression vector is an induced plasmid expansion vector used for regulating a gene to be knocked out or deleted. The double-copper composition uses plasmid expression vectors containing a tac promoter, containing pMAL-p2x, pGEX, pMBP, pKK223, and the like as templates, both ends of a gene lac I<q> are provided with primers, whole plasmids except for the gene lac I<q> are enlarged by PCR, and the optimized constitutive expression vector pMALC is obtained. The dry weight of thallus obtained by the method is approximately 1 g/L, the content of total protein is approximately 200 mg/L, enzyme accounts for 40 to 50% of the total protein, the amount of the expressed GL-7-ACA acylase does not have significant difference from that of inducing expression, and the enzyme activity is 220 U/L. The constitutive expression vector performs the important action in foreign protein production, particularly the GL-7-ACA acylase.
Description
Technical field
The present invention relates to a kind of constitutive expression carrier and construction process thereof and application, the application in expressing the GL-7-ACA acylase of particularly a kind of constitutive expression carrier and construction process thereof and this expression vector.
Background technology
7-amino-cephalosporanic acid (7-aminocephalosporanic acid, 7-ACA) be the important source material of present semi-synthetic cynnematin, mainly (Cephalosporin C CPC) obtains by the D-alpha-amino group hexanedioyl side chain that chemical method or enzymatic cleavage are sloughed the 7-position by cephalosporin.
Use enzyme process to substitute traditional chemical method cracking cephalosporin and produce 7-ACA, have advantages such as simple, efficient, safe, the pollution-free and constant product quality of technology, bigger competitive edge is all arranged in economy and environment protection.In recent years, people have dropped into a large amount of man power and materials to the research and development of Production by Enzymes semisynthetic antibiotics.Produce 7-ACA with enzymatic cleavage, be divided into a step enzyme method and two step enzyme methods.Wherein a step enzyme method utilizes directly catalysis cephalosporin of cephalosporin C acrylase (Cephalosporin C acylase), sloughs the D-alpha-amino group hexanedioyl side chain of 7-position, generates 7-ACA.Though this method step is simple, the CPC acylase vigor of having found at present is all very low, far can not satisfy the needs of Industrial Catalysis.Two step enzyme methods are to utilize two different enzymes of source to carry out two-step catalysis, and at first, cephalosporin is at D-amino-acid oxidase (D-Amino Acid Oxidase, DAAO) under the effect, produce the intermediate with ketone group, this intermediate is unstable, is easy to the H that is generated simultaneously
2O
2The chemical oxidation decarboxylation, be transformed into glutaryl--7-amino-cephalosporanic acid (Glutaryl-7-amidocephalosporanicAcid, GL-7-ACA); Under the effect of GL-7-ACA acylase, slough its side chain then, generate 7-ACA.
Most of bacterial strain that produces the GL-7-ACA acylase is pseudomonas (Pseudomonas sp.); can fall into 5 types having the active enzyme of catalysis GL-7-ACA generation 7-ACA; same zymoid gene all has the homology more than 95%, and zymologic property is almost completely identical.Along with development of biology, people more and more pay attention to producing enzyme and transforming enzyme with engineered method.(Luo Hui etc. such as Luo Hui; produce the structure and the expression of GL-7-ACA acylase recombination bacillus coli; the microbiology circular; 2004; 31 (4), 38-42) used a kind of method of obtaining gene fast, obtained the GL-7-ACA acylase gene through the method that microbial selective is cultivated and specific PCR increases fast from occurring in nature; and made up genetic engineering bacterium BL21 (DE3)/pET-Acy, realized the cloning and expression of GL-7-ACA acylase.
The expression of gene in intestinal bacteria can be divided into inducible expression and constitutive expression.A lot of foreign genes all are to express under the effect of the evoked promoter that can control, this gene is not expressed or minute quantity is expressed inductive the time, then can change condition (add certain chemical substance, change temperature, increase radiation etc.) when needing and induce efficiently expressing of this gene, avoided its disadvantageous effect that host is grown like this, but the specific protein of this genetic expression of high efficiency extraction again, but the use of inductor has not only improved production cost, also has pollution problem.It is stably to remain on a level that some expression of gene are also arranged, and the difference because of organ or cell category is not different, and this genoid is called the constitutive expression gene; The sudden change of some generegulation factor (comprise and regulate albumen or adjusting order) and gene is not expressed not modulatedly, this genoid also claims the constitutive expression gene.And this class sudden change is called constitutive mutation.
Summary of the invention
The purpose of this invention is to provide a kind of constitutive expression carrier.
Constitutive expression carrier provided by the present invention is that the induction type plasmid that regulatory gene is knocked out or lacks reaches carrier.
Wherein, to can be the genetically engineered field existing by laq I for the carrier that sets out that is used for making up described constitutive expression carrier
qThe inducible expression vector of aporepressor regulation and control contains the plasmid expression vector of tac promotor as pMAL-p2x, pGEX, pMBP, pKK223 etc.; Be preferably pMAL-p2x.
Described constitutive expression carrier is preferably pMALC (its physical map as shown in Figure 1), and it is a double-stranded cyclic DNA, and length is 5.6kb.The lacI of this carrier
qGenetically deficient contains ampicillin resistance gene (Amp
r), the tac promotor, the tac promotor is positioned at before the multiple clone site.
The present invention also provides a kind of method that makes up above-mentioned constitutive expression carrier, and this method is by pcr amplification lacI
qWhole plasmid outside the gene is with lac I
qGenetically deficient.
Construction process provided by the present invention may further comprise the steps:
1) be template with the plasmid expression vector that contains the tac promotor, use the primer of being made up of following forward primer and reverse primer to carrying out pcr amplification: described forward primer is the sequence 1 in the sequence table, and described reverse primer is the sequence 2 in the sequence table; Or 5 ' end of described forward primer and reverse primer all contains a restriction endonuclease of the same race or isocaudarner recognition sequence on the same group, and described forward primer is 3 ' terminal oligonucleotide sequence from the 25-40nt of 5 ' end 9-25 bit base for sequence 1, and described reverse primer is 3 ' terminal oligonucleotide sequence from the 25-40nt of 5 ' end 9-26 bit base for sequence 2;
2) pcr amplification product that obtains is carried out enzyme to the restriction endonuclease of the recognition sequence that has and cuts with discerning described primer, use T then
4Dna ligase connects and obtains constitutive expression carrier.
The plasmid expression vector of the described tac of containing promotor can be pMAL-p2x, pGEX, pMBP, pKK223 etc.
Described template is preferably pMAL-p2x, and described constitutive expression carrier is preferably pMALC.
Expression vector of the present invention can be used for expressing the foreign protein that the host is grown and do not have significant adverse to influence, as the GL-7-ACA acylase.
The present invention also provides and has utilized this constitutive expression carrier to express the method for GL-7-ACA acylase.
The method of expression GL-7-ACA acylase provided by the present invention is the GL-7-ACA acylase gene to be inserted above-mentioned constitutive expression carrier obtain GL-7-ACA acylase gene expression vector, imports host cell, expresses obtaining the GL-7-ACA acylase.
Described GL-7-ACA acylase gene has the nucleotide sequence of sequence 3 in the sequence table.
Described constitutive expression carrier is preferably pMALC.
Described GL-7-ACA acylase gene expression vector is preferably pMALC-Acy, and it is a double-stranded cyclic DNA, and length is 7.8kb.
Described host is intestinal bacteria, yeast, Bacillus subtilus; Be preferably intestinal bacteria; E.coliTB1 more preferably.
The fermention medium that is imported into the host cell that is loaded with described GL-7-ACA acylase gene constitutive expression carrier is identical with its corresponding host cell of fermentation condition.
Experimental results show that the dry cell weight that utilizes method of the present invention to obtain is about 1g/L, total protein content is about 200mg/L, and enzyme accounts for 40～50% of total protein, and the amount and the abduction delivering of the GL-7-ACA acylase of expression do not have marked difference, and enzyme is lived and is 220U/L.Adopt constitutive expression carrier of the present invention to express the process that foreign protein can be simplified fermentation greatly and express, and avoid the cost that use caused and the pollution problem of inductor.Constitutive expression carrier of the present invention will play a significant role in the production of foreign protein (particularly GL-7-ACA acylase).
Description of drawings
Fig. 1 is the physical map of composing type plasmid vector pMALC
Fig. 2 is the physical map of recombinant plasmid pMAILC-Acy
Fig. 3 is the full bacterium SDS-PAGE electrophorogram behind the genetic engineering bacterium abduction delivering
Embodiment
Method therefor is ordinary method if no special instructions among the following embodiment, and used enzyme is TaKaRa company product if no special instructions.
The structure of embodiment 1, constitutive expression plasmid pMALC
With expression plasmid pMAL-p2x (Amp
r, the tac promotor; Available from NEB company) be template, be that primer carries out pcr amplification with primer 1 and primer 2.Wherein, primer 1:5 '-TA
GGGCCCGCGCAACGCAATTAATG-3 ' (base of band underscore is an Apa I recognition site) (sequence 1 in the sequence table); Primer 2: 5 '-TA
GGGCCCATTCACCACCCTGAATTG-3 ' (base of band underscore is an Apa I recognition site) (sequence 2 in the sequence table).In the 0.2mL PCR thin-walled tube of a sterilization, add 10 * PCR damping fluid of 20 μ L sterilized waters, 5 μ L, the dNTP (10mM of 1 μ L successively, plug Parkson company), the primer 1 (final concentration 50pmol) of 1 μ L, the primer 2 (final concentration 50pmol) of 1 μ L, the plasmid pMAL-p2x solution (concentration is 50ng/ μ L) of 0.5 μ L, LA-Taq archaeal dna polymerase (the 5U/ μ L of 0.5 μ L, treasured biotechnology (Dalian) company limited), complement to cumulative volume 50 μ L with sterilized water, centrifuge tube is placed temperature varying system.In the PCR reaction, the temperature changing process of PCR reaction is: be warming up to 94 ℃ earlier, kept 5 minutes, then by following temperature variation program loop 28 times: be warming up to 94 ℃, kept 30 seconds, be cooled to 56 ℃, kept 1 minute and 30 seconds, and be warming up to 72 ℃, kept 5 minutes and 30 seconds; Kept the end amplified reaction 10 minutes in 72 ℃ at last.Through pcr amplification, obtain the linear plasmid that two ends have Apa I restriction enzyme enzyme recognition site respectively, with the linear plasmid that obtains Apa I digestion with restriction enzyme, and use T
4Dna ligase is connected to become cyclic plasmid.To connect product CaCl
2Method transformed into escherichia coli TOP-10F ' (F-mcrA Δ (mrr-hsdRMS-mcrBC) φ 80lacZ Δ M15 Δ lacX74deoRrecAlaraD139 (ara-leu) 7697galU galK rpsL (Str
R) endA1 nupG, available from Invitrogen company) competent cell, after cultivating 16hr on the LB solid medium that contains 100 μ g/mL penbritins, several bacterium colonies of picking are overnight incubation in liquid LB substratum, collect thalline and extract plasmid, after Apa I enzyme is cut, detect with 0.7% agarose gel electrophoresis, obtain the 5.6kbp fragment, prove that the gained plasmid is disappearance lac I
qThe plasmid vector pMALC of gene (its physical map as shown in Figure 1).
The clone of embodiment 2, GL-7-ACA acylase gene
According to document (Luo Hui etc., the structure and the expression of producing GL-7-ACA acylase recombination bacillus coli, the microbiology circular, 2004,31 (4), method 38-42) makes up the recombinant vectors pET-Acy that contains the GL-7-ACA acylase gene.Being about to the GL-7-ACA acylase gene of pcr amplification and pET-28a plasmid (available from Novagen company) carries out enzyme with Nde I and HindIII respectively and cuts; enzyme is cut product and is reclaimed with the agarose gel electrophoresis purifying; 4 ℃ of following spending the night with the T4 dna ligase connect goal gene and plasmid fragment; connect product transformed into escherichia coli TOP-10F ' (available from Invitrogen company); picking colony carries out the PCR screening, and the pET-Acy plasmid obtains recombinating.
With the recombinant plasmid pET-Acy that has the GL-7-ACA acylase gene is template, is that primer carries out pcr amplification with primer 3 and primer 4.Wherein, primer 3:5 '-GAC
GAATTCATGGAGCCGACCTCGA-3 ' (base of band underscore is an EcoR I recognition site); Primer 4:5 '-TAG
AAGCTTGTCATGGCTTGAAGTTG-3 ' (base of band underscore is a Hind III recognition site).In the 0.2mL PCR thin-walled tube of a sterilization, add 10 * PCR damping fluid of 5 μ L sterilized waters, 10 μ L, the dNTP (10mM of 0.3 μ L successively, plug Parkson company), the primer 3 (final concentration 50pmol) of 1 μ L, the primer 4 (final concentration 50pmol) of 1 μ L, the plasmid pET-Acy solution (concentration is 50ng/ μ L) of 0.5 μ L, pfu archaeal dna polymerase (the 5U/ μ L of 0.2 μ L, Qi Huasheng biotech development center, Beijing), complement to cumulative volume 20 μ L with sterilized water, centrifuge tube is placed temperature varying system.In the PCR reaction, the temperature changing process of PCR reaction is: be warming up to 94 ℃ earlier, kept 1 minute, then by following temperature variation program loop 30 times: be warming up to 94 ℃, kept 30 seconds, be cooled to 56 ℃, kept 1 minute and 30 seconds, and be warming up to 72 ℃, kept 2 minutes and 30 seconds; Kept the end amplified reaction 10 minutes in 72 ℃ at last.Through pcr amplification; obtain having the GL-7-ACA acylase gene of EcoR I and Hind III restriction enzyme site; and the GL-7-ACA acylase gene that obtains is carried out enzyme with EcoR I and Hind III cuts, and with the same intestinal bacteria constitutive expression plasmid pMALC T that cuts with EcoR I and Hind III enzyme
4Dna ligase connects, and will connect product CaCl
2Method transformed into escherichia coli TOP-10F ' competent cell (F-mcrA Δ (mrr-hsdRMS-mcrBC) φ 80lacZ Δ M15 Δ lacX74deoRrecAlaraD139 (ara-leu) 7697galU galK rpsL (Str
R) endA1 nupG; available from Invitrogen company); after cultivating 16hr on the LB solid medium that contains 100 μ g/mL penbritins; several bacterium colonies of picking are overnight incubation in liquid LB substratum; collect thalline and extract plasmid, behind EcoR I and Hind III double digestion, detect with 0.7% agarose gel electrophoresis; obtain the band of 2.1kbp and 5.6kbp, show the recombinant plasmid pMALC-Acy (its physical map as shown in Figure 2) that obtains having the GL-7-ACA acylase gene.
The expression of embodiment 3, GL-7-ACA acylase
PMALC-Acy transformed host cell E.coli TB1 (M15 Tn10 (ter will recombinate
r) HsdS gal (λ cIts857 indl Sam7 nin5 lacUV5-T7 Genel), available from NEB company), obtain genetic engineering bacterium E.coli TB1/pMALC-Acy.Genetic engineering bacterium is inoculated in the LB liquid nutrient medium (the LB substratum consists of: yeast powder 0.5%, Tryptones 1%, NaCl 1%, pH7.0), 37 ℃ of shaking table overnight incubation; Be transferred to the 300mL that 50mL LB substratum is housed with 5% inoculum size again and shake in the bottle, continue 28 ℃ cultivate 24hr after, centrifugal collection thalline.Measure the expression amount and the activity thereof of GL-7-ACA acylase in the genetic engineering bacterium; the result shows that the dry cell weight of acquisition is about 1g/L; total protein content is about 200mg/L; enzyme accounts for 40～50% of total protein; the amount and the abduction delivering of the GL-7-ACA acylase of expressing do not have marked difference, and enzyme is lived and is 220U/L.
The thalline of collecting is carried out full bacterium SDS-PAGE electrophoresis, and the result shows that genetic engineering bacterium has given expression to fusion rotein as shown in Figure 3, and swimming lane M is standard protein molecular weight (is respectively 97,66,45,30,20.1,14.4kD) among the figure.Two tangible protein bands about 66kD and 58kD, have been given expression to; wherein the α subunit of the corresponding GL-7-ACA acylase of 66kD protein band and the fusion rotein of MBP; the β subunit of the corresponding GL-7-ACA acylase of the protein band about 58kD; the α subunit and the β subunit that show the GL-7-ACA acylase are correctly processed, and enzyme is successful expression.
Sequence table
<160>3
<210>1
<211>25
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>1
tagggcccgc?gcaacgcaat?taatg 25
<210>2
<211>26
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>2
tagggcccat?tcaccaccct?gaattg 26
<210>3
<211>2163
<212>DNA
＜213〉pseudomonas (Pseudomonas sp.)
<400>3
atgctgagag?ttctgcaccg?ggcgacgtcc?gccctggtta?tggcgactgt?gatcggectt 60
gcgcccggcg?tcgcctttgc?gctggccgag?ccgacctcga?cgccgcaggc?gccgattgcg 120
gcctataaac?cgagaagcaa?tgagatcctg?tgggacggct?acggcgtccc?gcacatctac 180
ggggtcgacg?cgccccccgc?cttctacggc?tacggctggg?cccaggcgcg?aagccacggc 240
gacaatatcc?tgcgcctgta?tggagaagcg?cggggcaagg?gggccgaata?ctggggcccg 300
gattacgaac?agacgaccgt?ctggctgctg?accaacggcg?tgccggagcg?cgcccagcag 360
tggtatgcgc?agcagtcgct?ggatttccgc?gccaacctcg?acgccttcgc?ggcgggcatc 420
aacgcctatg?ccgaacagaa?cccagacgac?atctcgcccg?aggtgcggca?ggtgctgccg 480
gtttccggcg?ccgacgtggt?ggcgcacgcc?catcgcctga?tgaacttcct?ctatgtcgcg 540
tcgcccggcc?gcaccctggg?cgagggcgat?ccgccggacc?tggccgatca?ggggtccaac 600
tcctgggccg?tggcgccggg?caagacggcg?aacgggaacg?ccctgctgct?gcggaacccg 660
cacctgtcct?ggacgacgga?ctacttcacc?tactacgagg?cgcatctcgt?cacgccggac 720
ttcgaagtct?atggcgcgac?ccagatcggc?ctgccggtca?tccgcttcgc?cttcaatcag 780
cggatgggca?tcaccaatac?cgtcaacggc?atggtggggg?ccaccaacta?tcggctgacg 840
cttcaggacg?gcggctatct?gtacgacggt?caggtgcggc?cgttcgagcg?gcgtcaggcc 900
tcgtatcgcc?tgcgtcaggc?ggacgggtcg?acggtcgaca?agccgttgga?gattcgctcc 960
agcgtccatg?gcccggtctt?cgagcgcgcg?gacggcacgg?ccgtcgccgt?tcgggtcgcc 1020
ggtttggatc?ggccgggcat?gctcgagcag?tatttcgaca?tgatcacggc?cgacagcttc 1080
gacgactacg?aagccgccat?ggcgcggatg?caggtgccga?ccttcaacat?cgtctgcgcc 1140
gaccgcgaag?ggaccatcaa?ctacagcttc?aacggcgtgg?cgccccaacg?ggccgagggc 1200
gacatcgcct?tctggcaggg?gctcgtgcct?ggcgattcct?cgcgttacct?gtggaccgag 1260
acccacccgc?tggacgatct?gccgcgcgtc?accaatccgc?cgggcggctt?cgtgcaggac 1320
tccaatgatc?cgccgtggac?gccgacctgg?cccgtcacct?acacgcccaa?ggacttcccc 1380
tcctatctgg?cgccccagac?gccgcattcc?ctgcgcgcgc?aacaaagcgt?gcgtctgatg 1440
tccgagaacg?acgacctgac?gctggagcgc?ttcatggcgc?tgcagttgag?ccaccgcgcc 1500
gtcatggccg?accgcacgtt?gccggatctg?attccggccg?ccctgatcga?ccccgatccc 1560
gaggtccagg?cggcggcgcg?cctgctggcg?gcgtgggatc?gcgagttcgc?cagcgacagc 1620
cgggccgccc?tgctgttcga?ggaatgggcg?cgtctgttcg?ccggtcagaa?tttcgccggc 1680
caggcgggtt?tcgccacgcc?ctggtcgctg?gataagccgg?tcagcacgcc?ctacggcgtc 1740
tgcgacacca?aggccgccgt?cgatcaactg?cggaccgcca?tcgccaacac?caagcgcaag 1800
tacggcgcga?tcgaccggcc?gttcggcgac?gcctcgcgca?tgatcctgaa?cgatgtgaat 1860
gttccgggcg?ccgccggcta?cggcaacctg?ggttccttcc?gggtcttcac?ctggtccgat 1920
cctgacgaaa?acggggttcg?cacgcccgtc?cacggcgaga?cgtgggtggc?gatgatcgag 1980
ttctccaccc?cggtgcgggc?ctatggcctg?atgagctacg?gcaattctcg?ccagccgggc 2040
acgacgcact?acagcgatca?gatcgaacgc?gtgtcgcgcg?ccgacttccg?cgagctgttg 2100
ttgcggcgag?agcaggtcga?ggccgccgtc?caggaacgca?cgcccttcaa?cttcaagcca 2160
tga 2163
Claims (7)
1. a host is colibacillary constitutive expression carrier, is to contain the induction type plasmid expression vector that the regulatory gene lacIq of tac promotor is knocked out or lacks.
2. constitutive expression carrier according to claim 1 is characterized in that: the carrier that sets out that is used to make up described constitutive expression carrier is pMAL-p2x.
3. constitutive expression carrier according to claim 2 is characterized in that: described constitutive expression carrier is pMALC; This plasmid is to be template with expression plasmid pMAL-p2x, and the primer of forming with forward primer shown in the SEQ ID NO:1 and the reverse primer shown in the SEQID NO:2 is to carrying out pcr amplification, the linear plasmid that obtains ApaI digestion with restriction enzyme, and use T
4Dna ligase connects and obtains.
4. method that makes up the described constitutive expression carrier of claim 2 may further comprise the steps:
1) be template with the induction type plasmid expression vector pMAL-p2x that contains the tac promotor, use the primer of forming by the reverse primer shown in forward primer shown in the SEQ ID NO:1 and the SEQ ID NO:2 carrying out pcr amplification:
2) pcr amplification product that obtains is carried out enzyme to the restriction endonuclease of the recognition sequence that has and cuts with discerning described primer, use T then
4Dna ligase connects and obtains constitutive expression carrier.
5. method according to claim 4 is characterized in that: described constitutive expression carrier is pMALC as shown in Figure 1; This plasmid is to be template with expression plasmid pMAL-p2x, and the primer of forming with the reverse primer shown in forward primer shown in the SEQ ID NO:1 and the SEQ ID NO:2 is to carrying out pcr amplification, and the linear plasmid that obtains is with Apa I digestion with restriction enzyme, and uses T
4Dna ligase connects and obtains.
6. one kind is utilized the described constitutive expression carrier of claim 1 to express the method for GL-7-ACA acylase, be that the GL-7-ACA acylase gene is inserted the described constitutive expression carrier of claim 1, obtain GL-7-ACA acylase gene expression vector, import e. coli host cell, express obtaining the GL-7-ACA acylase; Described GL-7-ACA acylase gene expression vector is pMALC-Acy as shown in Figure 2; this plasmid is to be template with plasmid pET-Acy; the primer of forming with the primer shown in SEQ ID NO:3 and the SEQID NO:4 is to carrying out pcr amplification; obtain having the GL-7-ACA acylase gene of EcoR I and Hind III restriction enzyme site; and the GL-7-ACA acylase gene that obtains is carried out enzyme with EcoR I and Hind III cut, with the same intestinal bacteria constitutive expression plasmid pMALC T that cuts with EcoR I and Hind III enzyme
4Dna ligase connects and obtains.
7. method according to claim 6 is characterized in that: described host is E.coli TB1.
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| CN101240285B (en) * | 2008-03-19 | 2010-06-02 | 清华大学 | Cephalosporin C acrylase and its vector and application |
| CN101560516B (en) * | 2009-01-19 | 2010-12-08 | 广西大学 | Constitutive expression promoter in escherichia coli and applications in escherichia coli |
| CN106086082B (en) * | 2016-06-01 | 2019-11-15 | 苏州华赛生物工程技术有限公司 | A method of improvement recombination bacillus coli produces 9- decenol |
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Non-Patent Citations (4)
| Title |
|---|
| 2002 Feb,184(4): Links Regulation and adaptive evolutionof lactose operon expression in Lactobacillus delbrueckii.. Lapierre L, Mollet B, Germond JE.J Bacteriol.,Vol.184 No.4. 2002 * |
| Analysis of mouse keratin 6a regulatory sequencesintransgenic mice reveals constitutive,tissue-specificexpression by a keratin 6a minigene.. Mahony D, Karunaratne S, Cam G, Rothnagel JA.J Invest Dermatol.,Vol.115 No.5. 2000 * |
| 产GL-7-ACA酰化酶重组大肠杆菌的构建和表达. 罗晖等.微生物学通报，第31卷第4期. 2004 * |
| 玉米基因枪转化Bt基因的研究. 孙红炜等.山东农业科学，第3卷. 2004 * |
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