CN107937437A - A kind of light-operated expression vector of insect cell and application - Google Patents
A kind of light-operated expression vector of insect cell and application Download PDFInfo
- Publication number
- CN107937437A CN107937437A CN201711177112.4A CN201711177112A CN107937437A CN 107937437 A CN107937437 A CN 107937437A CN 201711177112 A CN201711177112 A CN 201711177112A CN 107937437 A CN107937437 A CN 107937437A
- Authority
- CN
- China
- Prior art keywords
- light
- pmib
- operated
- expression
- insect cell
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/65—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression using markers
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
- C12N2015/8518—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic expressing industrially exogenous proteins, e.g. for pharmaceutical use, human insulin, blood factors, immunoglobulins, pseudoparticles
Landscapes
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Wood Science & Technology (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Veterinary Medicine (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a kind of light-operated expression vector of insect cell and application, applicant is based on the light-operated transcriptional activation systems of VP EL222, and positive plasmid pMIB C120 mCherry GFP EL222 and control plasmid pMIB C120 mCherry GFP are successfully built on insect cell expression vector pMIB/V5 HisA；Transfect Sf9 cells after, egfp expression and red fluorescent protein is not expressed.After blue light illumination stimulates, red fluorescent protein expression；Control plasmid pMIB C120 mCherry GFP transfection Sf9 cells after, egfp expression and red fluorescent protein is not expressed.Stimulated through blue light illumination, due to not having EL222 transcription factors, red fluorescent protein is not still expressed.Successful utilization on insect cell, has expanded the application range of the technology to the light-operated inducible expression first.Verification method plasmid constructed by the present invention using light-operated induced expression technology also to provide flexible and easy-to-use method.
Description
Technical field
The invention belongs to bioengineering field, and in particular to a kind of light-operated expression vector of insect cell and application.
Background technology
With the development of transgenic technology, controllable Gene expression regulation system becomes biomedical research and biotechnology
In indispensable instrument.Past decades, the gene expression system of chemical regulation are widely used in from time up regulation base
The expression of cause.But since these small molecule inducers can freely be spread, it is difficult to eliminate and potential to cell function
Undershooting-effect, so it is difficult to accurately controlling the expression of gene on room and time.However, a kind of just preferably gene table
It is controllable strong up to inducer, without toxicity, and it can realize the dual accurate control of the time to destination gene expression, space.Cause
This, light-operated gene expression system becomes the instrument had a bright future of accurate control gene expression.It is nevertheless, current main
Light-operated expression system still has the application that many weakness limit it, such as：The toxicity of albumen in itself, narrow modification scope and
Sluggish activation and deexcitation.A new light-operated expression system will be shown here, it does not have foregoing
Shortcoming, and it has been proved to wide applicability in mammalian cell and model animal zebra fish.The system can be not only
Innovative instrument is provided the basic research of the life science such as cell biology, Developmental Biology, Neurobiology, can also be extensive
The application field such as diagnosis and treatment for bioengineering, human diseases.
The light control system is that the light controlling gene based on C120DNA binding sequences and EL222 transcription factors expresses system
System (Motta-Mena et al., 2014；Rivera-Cancel,G et al.,2012).EL222 transcription factors, initially come from
Light-oxygen-pressure albumen of one bacterium, when illuminated by blue light will dimerization be attached on C120DNA.The light of this system relies on
Transcriptional activation only needs a small amount of element, a photosensitive structure domain LOV, and helix turn helix (H-T-H) DNA combines knot
Structure domain.LOV domains combine HTH domains when dark, cover for being attached to as dimer necessary to DNA
HTH4 α sites.Blue light illumination can trigger photochemical reaction：Flavoprotein compound in LOV domains has interrupted LOV and HTH
Interaction, make EL222 dimerizations, thus play DNA binding protein activity.These reaction in the dark again can in turn into
OK, its back reaction will occur quickly after blue light illumination is stopped, this is the basis of quick deexcitation.This system is to control
The very big adjustable expression scope of the target protein of expression, quick activation and deexcitation, and have with luminous intensity sufficiently high
Linear dependence.In short, every research show C120-EL222 systems broadness versatility and it as light science of heredity work
The advantage of tool.
At present, the light control system based on C120-EL222 is widely used in mammalian cell,
Shown in terms of the manipulation in time and space the obvious advantage of light control system (Nash et al., 2011；Motta-Mena et
al.,2014).Further, scientist attempts to demonstrate the feasibility of the light control system in zebra fish, although light can be realized
Control expression, but due to showing certain toxicity, limit application of the system in zebra fish.Next, pass through gene
Engineered, scientist's optimization is more applicable for the light control system of zebra fish, i.e. TAEL (TA4-EL222) system
(Reade,A.et al.,2017).Leap of the C120-EL222 systems from mammalian cell to zebra fish, it is light-operated to extend this
Systematic difference scope, while also a strong instrument is provided for the research of fish.
Based on this, applicant establishes the light-operated gene expression based on VP-EL222 in Sf9 insect cells
System, an insect cell expression is dexterously incorporated into by the element of C120-EL222 light control systems and fluorescent protein report gene
On carrier, so that the test of light control system efficiency is more quick and stablizes, make the regulation and control of exogenous protein expression in insect cell
It is more flexible.We are successfully realized the light-operated expression of C120-EL222 systems in insect cell first, and it is light-operated to extend this
The system scope of application.
The content of the invention
Object of the present invention is to provide a kind of light-operated expression vector pMIB-C120-MCS- applied to insect cell
GFP-EL222, the nucleotides sequence of the carrier are classified as shown in SEQ ID NO.1, which dexterously incorporates C120-EL222
The controlling element and fluorescent protein report gene of light control system, utilize the regulation and control GFP reporter gene series connection of opIE1 composition promoters
The expression of EL222 transcription factors；Using the expression of light-operated adjusting promoter C120DNA sequences control foreign genes, this is improved
The flexibility of kind light control system and ease for operation.Meanwhile the carrier also retains blasticidin S resistant gene, available for screening
The stable cell lines of light-operated expression, newly introduce GFP reporter genes, easy to monitor the transfection efficiency of carrier in real time, and pass through
Flow cytometer based on green fluorescent label rapidly and efficiently sorts the stable cell lines of light-operated expression.
It is another object of the present invention to provide a kind of application of the light-operated expression vector of insect cell, by external source egg
After in white insertion pMIB-C120-MCS-GFP-EL222, the light-operated expression, it can be achieved that functional protein is transfected to Insect cells Sf9.
To achieve the above object, the technical solution adopted by the present invention is：
A kind of preparation method of the light-operated expression vector of the insect cell with foreign protein, including：
1) foreign gene replaces the Luc genes in pC120-Luc plasmids by EcoR1 and Xba1 restriction enzyme sites, obtains
PC120- foreign gene plasmids；
2) using pC120- foreign genes plasmid as template, C120- exogenous genetic fragments are expanded, insertion pMIB/V5-his is carried
In BspH1 the and Age1 restriction enzyme sites of body, pMIB-C120- foreign gene plasmids are obtained；
3) nucleotide fragments shown in artificial synthesized SEQ ID NO.2, clone are placed in psimple-T carriers, obtain
Psimple-T/PFT2AN carriers, introduce fluorescin by the Nhe1 restriction enzyme sites in psimple-T/PFT2AN and report base
Cause, then EL222 genes are introduced by the BsiwI restriction enzyme sites in psimple-T/PFT2AN, obtain psimple-T/ fluorescence eggs
- EL222 plasmids in vain；
4) by the fluorescin-EL222 fragments in psimple-T/ fluorescin-EL222 plasmids, by PvuII and
BglII restriction enzyme sites are inserted into pMIB-C120- foreign gene plasmids, obtain pMIB-C120- foreign gene-fluorescins-
EL222 plasmid vectors；
The pMIB/V5-his carriers are pMIB/V5-his A or pMIB/V5-his B or pMIB/V5-his C.
Those skilled in the art, according to above-mentioned method, can prepare the elder brother for expressing different foreign proteins according to actual conditions
The light-operated expression vector of worm cell；Or foreign protein is directly inserted into light-operated expression vector pMIB-C120-MCS-GFP-EL222
In in (MCS after C120 carries a variety of restriction enzyme sites, for the insertion of foreign gene), come prepare carry foreign protein insect it is thin
The light-operated expression vector of born of the same parents.
The application of the light-operated expression vector of a kind of insect cell, using the light-operated expression vector establishment insect cell
Light-operated expression platform, or the expression using carrier control albumen.
In schemes described above, specifically, being by pMIB-C120- foreign gene-GFP-EL222 plamid vector transfections
Into insect cell, blue light illumination or dark processing are then carried out as required, to control the expression of foreign protein.
Compared with prior art, the present invention has the following advantages：
1. the element of C120-EL222 light control systems is dexterously incorporated into an insect cell expression vector, there is provided one
The normal form of kind plasmid construction.
2 realize the light-operated Expression and Application of the C120-EL222 light control systems in insect cell first, extend the light
The new scope of application of control system.
Carry, greatly improve 3. the element of former C120-EL222 light control systems is carried by more plasmids and is reduced to simple substance grain
The positive rate of aftereffect cell is transfected, also so that the test of the light control system is more quick and stablizes.This light control system works
Test index be：, can be by detecting red fluorescence in the Sf9 cells for thering is green fluorescent protein GFP to express after Induced by Blue Light
Albumen mCherry's expresses to reflect the task performance of light-dependent control element.
4. constitutive expression GFP reporter genes are introduced in light-operated plasmid, easy to thin by the streaming of green fluorescence label
Born of the same parents' instrument carries out positive cell screening, substantially increases the efficiency that light-operated cell line is stablized in screening.
Brief description of the drawings
Fig. 1 insect cell expression vector pMIB/V5-His collection of illustrative plates.
Fig. 2 is the light-operated expression system schematic diagram based on C120-EL222.
Fig. 3 builds basic flow chart for the light-operated expression vector pMIB-C120-mCherry-GFP-EL222 of insect cell.
The digestion identification electrophoretic image of Fig. 4 carrier constructions.
Functional verification schematic diagram of the light-operated expression vector of Fig. 5 insect cells in sf9 insect cells.
Embodiment
In order to make the purpose , technical scheme and advantage of the present invention be clearer, with reference to the accompanying drawings and embodiments, it is right
The present invention is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, and
It is not used in the restriction present invention.
Embodiment 1：
The structure of insect cell expression vector pMIB-C120-mCherry-GFP-EL222
1. experiment material
| PMIB/v5-hisA carriers | Invitrogen companies |
| PC120-Luc plasmids | Kevin professors HGardner present (Motta-Mena et al., 2014) |
| PVP-EL222 plasmids | Kevin professors HGardner present (Motta-Mena et al., 2014) |
| Gel Extraction kit plastic recovery kits | OMEGA bio-tek companies |
| Plasmid mini kit plasmid QIAquick Gel Extraction Kits | OMEGA bio-tek companies |
| E.coli DH-5 α competence | ATCC |
| Restriction enzyme | NEB companies |
| CIP | NEB companies |
| T4DNA ligase | NEB companies |
| Gel imager | BioRad companies |
The list of primers that plasmid cloning uses：
2. experimental method
1) carrier (is bought in Addgene) as template using pLV-mCherry, is expanded using primer E-mch-F and X-mch-R
Mcherry genetic fragments, replace the Luc genes in pC120-Luc plasmids by EcoR1 and Xba1 restriction enzyme sites, obtain
PC120-mcherry plasmids.
2) using pC120-mcherry plasmids as template, C120- is expanded using primer BspH-C120-F and Age-Mch-R
Mcherry fragments, are inserted into BspH1 the and Age1 restriction enzyme sites of pMIB/V5-hisA carriers, obtain pMIB-C120-mcherry
Plasmid.
3) for the ease of the structure of multicomponent integration vector plasmid, artificial synthesized section of DNA fragment PFT2AN, the fragment by
(shown in SEQ ID NO.2), clone is placed in psimple-T carriers 457 base-pair compositions, obtains plasmid psimple-T/
PFT2AN.Using primer Nhe-GFP-F and GFP-Nhe-R, carrier (is bought in Clontech) as template using pEGFP-N1, is expanded
GFP genes, introduce GFP reporter genes by the Nhe1 restriction enzyme sites in psimple-T/PFT2AN, utilize primer Bsiw-EL-F
EL222 genes are expanded with EL-Bsiw-R, EL222 genes are introduced by the BsiwI restriction enzyme sites in psimple-T/PFT2AN,
Obtain psimple-T/GFP-EL222 plasmids.
4) by the GFP-EL222 fragments in psimple-T/GFP-EL222 plasmids, PvuII and BglII restriction enzyme sites are passed through
It is inserted into pMIB-C120-mcherry plasmids, obtains pMIB-C120-mCherry-GFP-EL222 plasmid vectors
5) single endonuclease digestion directly is carried out to carrier pMIB-C120-mCherry-GFP-EL222 using BsiwI enzymes.Passed through after purifying
Connection conversion obtains pMIB-C120-mCherry-GFP plasmids, the negative control plasmids as EL222 afunction；
According to the vector construction strategy of Fig. 3, build positive plasmid pMIB-C120-mCherry-GFP-EL222 and feminine gender is right
It is completely correct (such as Fig. 4) through digestion verification according to plasmid pMIB-C120-mCherry-GFP.pMIB-C120-mCherry-GFP-
It is mCherry bands that 750bp or so band is released in the EcoRI/XbaI digestions of EL222 function carriers, and the digestion of BsiwI is released
900bp or so EL222 bands,；The EcoRI/XbaI of pMIB-C120-mCherry-GFP control vectors releases 750bp or so bar
Band is mCherry bands, and the digestion of BsiwI is without band.Qualification result meets expection, all cloned plasmids all sequence verifications, sequence
Correctly.
Each step of above vector construction is using conventional molecular cloning method：
1) design special primer and the fragment for containing specific restriction enzyme site in end is obtained by PCR amplification；At the same time to carrier and
Fragment distinguishes digestion, and purifies digestion products；Using T4 ligases, 16 DEG C of connection carrier segments are stayed overnight；
2) convert, shake bacterium, apply tablet (what is used in experiment is all ammonia benzyl resistant panel), tablet mistake in 37 DEG C of baking ovens
Night；Choose spot and shake bacterium, identified after cell concentration increase with corresponding primer bacterium solution PCR, expand culture identification as positive bacterium；Preserve bacterium
Kind of (fungi preservation in 10%-15% glycerites, -20 DEG C).
3) plasmid, and measured concentration are extracted with the small extraction reagent kit of plasmid；Institute's upgrading grain is carried out using rational restriction enzyme
Digestion is identified；Picking positive plasmid is sequenced, and is used to use in next step by plasmid after sequencing is correct.Plasmid need to be stored in -20 DEG C.
The present embodiment be using reporter gene mCherry as foreign gene exemplified by, to it is provided by the invention it is light-operated expression carry
Body illustrates, and those skilled in the art, according to above-mentioned method, can prepare according to actual conditions and express different foreign proteins
The light-operated expression vector of insect cell；Or foreign protein is directly inserted into light-operated expression vector pMIB-C120-MCS-GFP-
In EL222 (MCS after C120 carries a variety of restriction enzyme sites, for the insertion of foreign gene), to prepare the elder brother for carrying foreign protein
The light-operated expression vector of worm cell.
Embodiment 2：
Functions of the light-operated expression vector pMIB-C120-mCherry-GFP-EL222 of insect cell in Sf9 insect cells
Verification：1. experiment material
2. experimental method and result
The secondary culture of 2.1Sf9 cells：Use Grace ' s Insect Cell Medium increase serums and dual anti-culture
Base, the adhere-wall culture Sf9 cells in 28 DEG C of cell incubators, when cell growth to 90% or so, directly gently blow afloat cell
Passed on.
The light-operated expression vector transfection Sf 9 insect cell of 2.2 insect cells.The plasmid that this experiment needs to transfect has two：
PMIB-C120-mCherry-GFP-EL222 (function plasmid) and pMIB-C120-mCherry-GFP (negative control plasmids).
1) shift to an earlier date 24 it is small when by Sf9 cells by 8 × 105A cells/well is inoculated in 6 orifice plates, when cell confluency degree reaches
When 70~80%, transfected.Prepare 10ml plating mediums before transfection：1.5ml Grace cell culture fluids (contain 10%FBS)
+ 8.5ml Grace cell culture fluids (are free of FBS).There can not be antibiotic in plating medium.
2) Sf9 cells in 6 orifice plates to be transfected are placed and, on super-clean bench, absorb the culture medium in 6 orifice plate cells, are added
The above-mentioned fresh plating mediums of 2ml.
3) dilute 2~6 μ l transfection reagents (II Reagent) in 100 μ l Grace ' s culture mediums.Dilution 1
~3 μ g target plasmids are in 100 μ l Grace ' s culture mediums.
4) DNA after dilution and transfection reagent are uniformly mixed, are incubated at room temperature 15-30min.
5) plus the mixed liquor of 210 μ l plasmids and transfection reagent is in cell to be transfected, 28 DEG C of 4~6h of incubation.
6) absorption transfection reagent mixed liquor, addition 2ml Sf9 cells complete culture solution (containing dual anti-and 10%FBS), 28 DEG C
Incubator continues to cultivate, and cell state is tracked after 24h.According to different demands, give culture cell different illumination conditions.
This devises altogether four groups of transfection experiments, transfection positive plasmid pMIB-C120-mCherry-GFP-EL222's
Illumination is with two groups dark, two groups of illumination and the dark of transfection negative control plasmids pMIB-C120-mCherry-GFP.Blue light illumination
Condition is：20s, dark 60s are irradiated for a circulation.
2.3 plasmid pMIB-C120-mCherry-GFP-EL222 and pMIB-C120-mCherry-GFP transfect Sf9 cells
Afterwards, cultivated in 28 DEG C of incubators, 24h imposes blue light illumination and dark two kinds of conditions again after transfection, is stimulated respectively in blue light illumination
In fluorescence microscopy Microscopic observation red fluorescence expression (Fig. 5) after 24h and 48h.
1) after function plasmid pMIB-C120-mCherry-GFP-EL222 transfects sf9, blue light illumination and dark condition are set
Cultivate two groups, when transfection 24 is small after grant illumination (blue light illumination 20s stops 60s as a circulation) and dark two kinds of conditions, illumination
24 it is small when after fluorescence microscopy Microscopic observation, obtain result (A in Fig. 5).Green fluorescent protein GFP is held after the results show transfection
Continuous property expression, there is the expression of stronger red fluorescent protein mCherry after blue light illumination in GFP positive cells.The above results
Prove that the C120-EL222 light control systems there can be specific efficient functional activity in Sf9 cells.
2) it is same that blue light illumination and dark bar are set after negative control plasmids pMIB-C120-mCherry-GFP transfects Sf9
Two groups of part culture, when transfection 24 is small after give illumination (blue light illumination 20s stops 60s as a circulation) and dark two kinds of conditions, light
According to 24 it is small when after fluorescence microscopy Microscopic observation, obtain result (B in Fig. 5).The results show：Green fluorescent protein GFP after transfection
There is persistent expression, but in the presence of the light-operated transcription factors of no EL222, all there is no red fluorescence under blue light illumination and dark condition
The expression of albumen mCherry.The above results prove that C120-EL222 light control systems transcription with height in Sf9 cells swashs
Specificity living and preciseness, basic No leakage expression.
3) after plasmid pMIB-C120-mCherry-GFP-EL222 transfects Sf9, blue light illumination and dark condition culture are set
Two groups, when transfection 24 is small after to grant illumination (blue light illumination 20s stops 60s as a circulation) small with dark two kinds of conditions, illumination 48
When after fluorescence microscopy Microscopic observation, obtain result (C in Fig. 5).Blue light additional radiation 24h can be slightly increased red fluorescent cell
Quantity, but increase be not apparent.The above results prove that in Sf9 cells C120-EL222 light control systems are in blue light illumination
After 24h, the expression of red fluorescent protein mCherry has just reached higher level, continues red fluorescent protein after light induction
MCherry expression increased, and can maintain higher level.
In conclusion the C120-EL222 light control systems can have specific efficient functional activity in Sf9 cells
, being capable of sustained activation expression external source destination protein with the preciseness of height.
As it will be easily appreciated by one skilled in the art that the foregoing is merely illustrative of the preferred embodiments of the present invention, not to
The limitation present invention, all any modification, equivalent and improvement made within the spirit and principles of the invention etc., should all include
Within protection scope of the present invention.
Sequence table
<110>Wuhan Inst. of Physics and Mathematics, Chinese Academy of Sciences
<120>A kind of light-operated expression vector of insect cell and application
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 5000
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 1
catgatcgct agcctcgagt aggtagcctt tagtccatgc gttataggta gcctttagtc 60
catgcgttat aggtagcctt tagtccatgc gttataggta gcctttagtc catgcgttat 120
aggtagcctt tagtccatga agcttagaca ctagagggta tataatggaa gctcgacttc 180
cagcttggca atccggtact gttggtaaag gatccactag tccagtgtgg tggaattctg 240
cagatatcca gcacagtggc ggccgctcga gtctagaggg cccgcggttc gaaggtaagc 300
ctatccctaa ccctctcctc ggtctcgatt ctacgcgtac cggtcatcat caccatcacc 360
attgagttta tctgactaaa tcttagtttg tattgtcatg ttttaataca atatgttatg 420
tttaaatatg tttttaataa attttataaa ataatttcaa cttttattgt aacaacattg 480
tccatttaca cactcctttc aagcgcgtgg gatcgatgct cactcaaagg cggtaatacg 540
gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa 600
ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga 660
cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag 720
ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct 780
taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg 840
ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc 900
ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt 960
aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta 1020
tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac 1080
agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc 1140
ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat 1200
tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc 1260
tcagtggaac gaaaactcac gttaagggat tttggtcatg cgaaacacgc acggcgcgcg 1320
cacgcagctt agcacaaacg cgtcgttgca cgcgcccacc gctaaccgca ggccaatcgg 1380
tcggccggcc tcatatccgc tcaccagccg cgtcctatcg ggcgcggctt ccgcgcccat 1440
tttgaataaa taaacgataa cgccgttggt ggcgtgaggc atgtaaaagg ttacatcatt 1500
atcttgttcg ccatccggtt ggtataaata gacgttcatg ttggtttttg tttcagttgc 1560
aagttggctg cggcgcgcgc agcacctttg ccgggatctg ccgggctgca gcacgtgttg 1620
acaattaatc atcggcatag tatatcggca tagtataata cgacaaggtg aggaactaaa 1680
ccatggccaa gcctttgtct caagaagaat ccaccctcat tgaaagagca acggctacaa 1740
tcaacagcat ccccatctct gaagactaca gcgtcgccag cgcagctctc tctagcgacg 1800
gccgcatctt cactggtgtc aatgtatatc attttactgg gggaccttgc gcagaactcg 1860
tggtgctggg cactgctgct gctgcggcag ctggcaacct gacttgtatc gtcgcgatcg 1920
gaaatgagaa caggggcatc ttgagcccct gcggacggtg ccgacaggtt cttctcgatc 1980
tgcatcctgg gatcaaagcc atagtgaagg acagtgatgg acagccgacg gcagttggga 2040
ttcgtgaatt gctgccctct ggttatgtgt gggagggcca gactttgaat tttgaccttc 2100
tcaagttggc gggagacgtg gagtccaacc cagggcccgc tagcatggtg agcaagggcg 2160
aggagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc 2220
acaagttcag cgtgtccggc gagggcgagg gcgatgccac ctacggcaag ctgaccctga 2280
agttcatctg caccaccggc aagctgcccg tgccctggcc caccctcgtg accaccctga 2340
cctacggcgt gcagtgcttc agccgctacc ccgaccacat gaagcagcac gacttcttca 2400
agtccgccat gcccgaaggc tacgtccagg agcgcaccat cttcttcaag gacgacggca 2460
actacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac cgcatcgagc 2520
tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg gagtacaact 2580
acaacagcca caacgtctat atcatggccg acaagcagaa gaacggcatc aaggtgaact 2640
tcaagatccg ccacaacatc gaggacggca gcgtgcagct cgccgaccac taccagcaga 2700
acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg agcacccagt 2760
ccgccctgag caaagacccc aacgagaagc gcgatcacat ggtcctgctg gagttcgtga 2820
ccgccgccgg gatcactctc ggcatggacg agctgtacaa ggctagcgag ggcagaggaa 2880
gtctgctaac atgcggtgac gtcgaggaga atcctggccc acgtacgatg ggccctaaaa 2940
agaagcgtaa agtcgccccc ccgaccgatg tcagcctggg ggacgagctc cacttagacg 3000
gcgaggacgt ggcgatggcg catgccgacg cgctagacga tttcgatctg gacatgttgg 3060
gggacgggga ttccccgggg ccgggattta ccccccacga ctccgccccc tacggcgctc 3120
tggatatggc cgacttcgag tttgagcaga tgtttaccga tgcccttgga attgacgagt 3180
acggtgggga attcggggca gacgacacac gcgttgaggt gcaaccgccg gcgcagtggg 3240
tcctcgacct gatcgaggcc agcccgatcg catcggtcgt gtccgatccg cgtctcgccg 3300
acaatccgct gatcgccatc aaccaggcct tcaccgacct gaccggctat tccgaagaag 3360
aatgcgtcgg ccgcaattgc cgattcctgg caggttccgg caccgagccg tggctgaccg 3420
acaagatccg ccaaggcgtg cgcgagcaca agccggtgct ggtcgagatc ctgaactaca 3480
agaaggacgg cacgccgttc cgcaatgccg tgctcgttgc accgatctac gatgacgacg 3540
acgagcttct ctatttcctc ggcagccagg tcgaagtcga cgacgaccag cccaacatgg 3600
gcatggcgcg ccgcgaacgc gccgcggaaa tgctcaggac gctgtcgccg cgccagctcg 3660
aggttacgac gctggtggca tcgggcttgc gcaacaagga agtggcggcc cggctcggcc 3720
tgtcggagaa aaccgtcaag atgcaccgcg ggctggtgat ggaaaagctc aacctgaaga 3780
ccagtgccga tctggtgcgc attgccgtcg aagccggaat ctaacgtacg cgaaacttgt 3840
ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag 3900
catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg 3960
tctgagatct gcatgtctac taaactcaca aattagagct tcaatttaat tatatcagtt 4020
attacccatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc 4080
ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa 4140
gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt 4200
aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt 4260
ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc 4320
atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg 4380
gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg 4440
gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac 4500
atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca 4560
aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta 4620
actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat 4680
aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa 4740
tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag 4800
ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat 4860
agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt 4920
tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg 4980
aagatccttt ttgataatct 5000
<210> 2
<211> 457
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
cagctggcaa cctgacttgt atcgtcgcga tcggaaatga gaacaggggc atcttgagcc 60
cctgcggacg gtgccgacag gttcttctcg atctgcatcc tgggatcaaa gccatagtga 120
aggacagtga tggacagccg acggcagttg ggattcgtga attgctgccc tctggttatg 180
tgtgggaggg ccagactttg aattttgacc ttctcaagtt ggcgggagac gtggagtcca 240
acccagggcc cgctagcgag ggcagaggaa gtctgctaac atgcggtgac gtcgaggaga 300
atcctggccc acgtacgcga aacttgttta ttgcagctta taatggttac aaataaagca 360
atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt 420
ccaaactcat caatgtatct tatcatgtct gacatct 457
Claims (4)
1. a kind of light-operated expression vector applied to insect cell, the nucleotides sequence of the carrier is classified as shown in SEQ ID NO.1.
2. a kind of preparation method of the light-operated expression vector of the insect cell with foreign protein, including：
1）Foreign gene replaces the Luc genes in pC120-Luc plasmids by EcoR1 and Xba1 restriction enzyme sites, obtains
PC120- foreign gene plasmids；
2）Using pC120- foreign genes plasmid as template, C120- exogenous genetic fragments are expanded, are inserted into pMIB/V5-his carriers
BspH1 and Age1 restriction enzyme sites in, obtain pMIB-C120- foreign gene plasmids；
3）Nucleotide fragments shown in artificial synthesized SEQ ID NO.2, clone are placed in psimple-T carriers, obtain psimple-
T/PFT2AN carriers, fluorescent protein report gene is introduced by the Nhe1 restriction enzyme sites in psimple-T/PFT2AN, then is passed through
BsiwI restriction enzyme sites in psimple-T/PFT2AN introduce EL222 genes, obtain psimple-T/ fluorescins-EL222
Plasmid；
4）By the fluorescin-EL222 fragments in psimple-T/ fluorescin-EL222 plasmids, by PvuII and
BglII restriction enzyme sites are inserted into pMIB-C120- foreign gene plasmids, to obtain the final product.
3. carrier prepared by the carrier or claim 2 the method described in claim 1 is in the light-operated table of structure insect cell
Up to the application in platform.
4. the application in the expression of carrier control albumen prepared by the carrier or claim 2 the method described in claim 1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711177112.4A CN107937437B (en) | 2017-11-22 | 2017-11-22 | Light-operated expression vector of insect cell and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711177112.4A CN107937437B (en) | 2017-11-22 | 2017-11-22 | Light-operated expression vector of insect cell and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107937437A true CN107937437A (en) | 2018-04-20 |
| CN107937437B CN107937437B (en) | 2020-10-27 |
Family
ID=61929914
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201711177112.4A Active CN107937437B (en) | 2017-11-22 | 2017-11-22 | Light-operated expression vector of insect cell and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107937437B (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110241019A (en) * | 2019-05-17 | 2019-09-17 | 华中农业大学 | A kind of light-operated steady gene oscillatory system |
| CN110438057A (en) * | 2019-08-21 | 2019-11-12 | 江南大学 | A kind of the dynamic regulation system and its application of blue light control |
| CN111534531A (en) * | 2020-04-28 | 2020-08-14 | 天津大学 | Design method for blue light induced cell scorching |
| CN113186190A (en) * | 2021-05-25 | 2021-07-30 | 华中科技大学 | Blue light mediated regulation plasmid and construction method and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101270366A (en) * | 2008-04-30 | 2008-09-24 | 苏州大学 | Method for improving transgenic insect cell expression exogenous gene level |
| CN101688246A (en) * | 2007-04-26 | 2010-03-31 | 夏威夷生物技术公司 | Synthetic expression vectors for insect cells |
| WO2013074911A1 (en) * | 2011-11-18 | 2013-05-23 | Board Of Regents, The University Of Texas System | Blue-light inducible system for gene expression |
| CN104745632A (en) * | 2013-12-31 | 2015-07-01 | 苏州杰诺曼博生物科技有限公司 | Method for expressing hABCG2 in insect cell sf9 |
-
2017
- 2017-11-22 CN CN201711177112.4A patent/CN107937437B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101688246A (en) * | 2007-04-26 | 2010-03-31 | 夏威夷生物技术公司 | Synthetic expression vectors for insect cells |
| CN101270366A (en) * | 2008-04-30 | 2008-09-24 | 苏州大学 | Method for improving transgenic insect cell expression exogenous gene level |
| WO2013074911A1 (en) * | 2011-11-18 | 2013-05-23 | Board Of Regents, The University Of Texas System | Blue-light inducible system for gene expression |
| CN104745632A (en) * | 2013-12-31 | 2015-07-01 | 苏州杰诺曼博生物科技有限公司 | Method for expressing hABCG2 in insect cell sf9 |
Non-Patent Citations (5)
| Title |
|---|
| AKIRA TAKAKADO ET AL.: "Photoinduced dimerization of a photosensory DNA-binding protein EL222 and its LOV domain", 《PHYS. CHEM. CHEM. PHYS.》 * |
| FEDOR V SUBACH ET AL.: "Photoactivatable mCherry for high-resolution two-color fluorescence microscopy", 《NAT METHODS》 * |
| MOTTA-MENA ET AL.: "An optogenetic gene expression system with rapid activation and deactivation kinetics", 《NATURE CHEMICAL BIOLOGY》 * |
| RIVERA-CANCEL ET AL.: "Identification of Natural and Artificial DNA Substrates for Light-Activated LOV-HTH Transcription Factor EL222", 《BIOCHEMISTRY》 * |
| 张佑红等: "昆虫细胞培养及其应用进展", 《武汉化工学院学报》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110241019A (en) * | 2019-05-17 | 2019-09-17 | 华中农业大学 | A kind of light-operated steady gene oscillatory system |
| CN110241019B (en) * | 2019-05-17 | 2020-08-04 | 华中农业大学 | Light-operated steady gene oscillation system |
| CN110438057A (en) * | 2019-08-21 | 2019-11-12 | 江南大学 | A kind of the dynamic regulation system and its application of blue light control |
| CN110438057B (en) * | 2019-08-21 | 2021-10-29 | 江南大学 | Dynamic regulation and control system for blue light control and application thereof |
| CN111534531A (en) * | 2020-04-28 | 2020-08-14 | 天津大学 | Design method for blue light induced cell scorching |
| CN113186190A (en) * | 2021-05-25 | 2021-07-30 | 华中科技大学 | Blue light mediated regulation plasmid and construction method and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN107937437B (en) | 2020-10-27 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107937437B (en) | Light-operated expression vector of insect cell and application | |
| US20220315892A1 (en) | Engineered Artificial Antigen Presenting Cells for Tumor Infiltrating Lymphocyte Expansion | |
| CN109136193B (en) | Application of NW _006884764-1 stable expression protein in CHO cell genome | |
| CN111004814A (en) | Construction method of sensitive arsenic ion whole-cell biosensor and arsenic ion concentration detection method | |
| JP2024073654A (en) | Methods and compositions for inducible expression of neurotrophic factors | |
| CN109295092B (en) | Application of NW _003613638-1 stable expression protein in CHO cell genome | |
| US20220127637A1 (en) | Transcriptional regulatory element and its use in enhancing the expression of heterologous protein | |
| CN109321604B (en) | Application of NW _006882077-1 stable expression protein in CHO cell genome | |
| JP2016540518A (en) | Methods for recombinant expression of novel eukaryotic cells and products of interest | |
| JP2024096300A (en) | Enhanced expression system and methods of use thereof | |
| CN110684738A (en) | Method for obtaining immortalized cell strain by lentivirus vector mediated SV40T antigen transfection | |
| CN109207432B (en) | Application of NW _006883358-1 stable expression protein in CHO cell genome | |
| CN109295093B (en) | Application of NW _006882456-1 stable expression protein in CHO cell genome | |
| CN109337927B (en) | Application of NW _006880285-1 stable expression protein in CHO cell genome | |
| WO2019129175A1 (en) | Chimeric promoter with high transcriptional activity in t-cells | |
| EA037273B1 (en) | Expression cassette | |
| CN101402970B (en) | Gene amplification method | |
| CN112725373B (en) | Construction method for amplifying cadmium ion whole-cell biosensor circuit | |
| WO2018150345A1 (en) | An expression vector | |
| AU2018356538B2 (en) | Eukaryotic cell line | |
| RU2715314C1 (en) | VTvaf17-Act1-Cas9 GENE-THERAPEUTIC DNA VECTOR BASED ON VTvaf17 GENE-THERAPEUTIC DNA VECTOR CARRYING TARGET Cas9 GENE FOR HETEROLOGOUS EXPRESSION OF THIS TARGET GENE IN PLANT CELLS DURING GENOME EDITING OF PLANTS, METHOD OF PRODUCING AND USING GENE-THERAPEUTIC DNA VECTOR, STRAIN OF ESCHERICHIA COLI SCS110-AF/VTvaf17-Act1-Cas9 CARRYING GENE-THERAPEUTIC DNA VECTOR, METHOD FOR PREPARING THEREOF, METHOD FOR INDUSTRIAL PRODUCTION OF GENE-THERAPEUTIC DNA VECTOR | |
| RU2733429C1 (en) | Gene-therapeutic dna-vector based on gene-therapeutic dna-vector gdtt1_8nas12, carrying target hfe gene to increase expression level of this target gene, method for production and use thereof, escherichia coli strain jm110-nas/gdtt1_8nas12-hfe carrying gene-therapeutic dna vector, method for production thereof, method for industrial production of gene-therapeutic dna vector | |
| RU2714763C1 (en) | Gene-therapeutic dna vector for targeted gene therapy, method for production thereof (embodiments), strain for production thereof, method for production thereof | |
| RU2754930C1 (en) | Recombinant plasmid pvnv-gl-rbdind, providing synthesis and secretion of recombinant receptor-binding domain (rbd) of sars-cov-2 coronavirus line b.1.617 in mammalian cells | |
| KR102335242B1 (en) | Cho cell comprising site-specific integrated rmce landing pad in fer1l4 gene |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20211124 Address after: 518107 floor 17, building 4, Weiguang Life Science Park, Zhenmei community, Xinhu street, Guangming District, Shenzhen, Guangdong Patentee after: Brincase (Shenzhen) Biotechnology Co.,Ltd. Address before: 430071 Xiao Hong, Wuchang District, Wuhan District, Hubei, Shanxi, 30 Patentee before: Institute of precision measurement science and technology innovation, Chinese Academy of Sciences Effective date of registration: 20211124 Address after: 430071 Xiao Hong, Wuchang District, Wuhan District, Hubei, Shanxi, 30 Patentee after: Institute of precision measurement science and technology innovation, Chinese Academy of Sciences Address before: 430071 Xiao Hong, Wuchang District, Wuhan District, Hubei, Shanxi, 30 Patentee before: WUHAN INSTITUTE OF PHYSICS AND MATHEMATICS, CHINESE ACADEMY OF SCIENCES |
|
| TR01 | Transfer of patent right |