CN1624145A - Process for structuring recombined adenovirus by high-flux direct directional clone - Google Patents
Process for structuring recombined adenovirus by high-flux direct directional clone Download PDFInfo
- Publication number
- CN1624145A CN1624145A CN 200410060972 CN200410060972A CN1624145A CN 1624145 A CN1624145 A CN 1624145A CN 200410060972 CN200410060972 CN 200410060972 CN 200410060972 A CN200410060972 A CN 200410060972A CN 1624145 A CN1624145 A CN 1624145A
- Authority
- CN
- China
- Prior art keywords
- carrier
- adenovirus
- asci
- clai
- recombinant adenovirus
- 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.)
- Pending
Links
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
A process for configuring the recombinant adenovirus includes such steps as directionally and directly linking the PCR product amplified by the specifically designed primer, screening resistivity to obtain the recombinant adenovirus plasmid, linearizing by restrictive endonuclearase Pacl, and transforming the packing cell line of mamal. Its advantages are high correctness, short period and low cost.
Description
Technical field
What the present invention relates to is biological gene clonal expression technology, and the direct directed cloning of particularly a kind of high-throughput makes up the method for recombinant adenovirus.Be particularly suitable for expressing and derive from mammiferous gene and make up expression library based on Mammals cDNA.
Background information
Genome times afterwards comprehensively, development can high-throughput, and the expression system of loyal as much as possible expressing human genoid receives people's urgent concern.The gland virus expression system becomes one of first-selected expression system with the superiority of himself.At present existing several companies have released the clonal expression system of adenovirus, Adeno-Quest system as Q-biogene, the Adeasy system of Strategene, the Adeno-X system of Clontech, the technological line that these several systems adopt has nothing in common with each other, but represents 3 kinds of main methods of current structure recombinant adenovirus.First kind is to be representative with the Adeno-Quest system, by at first being cloned into goal gene on the shuttle vectors, the shuttle vectors that has foreign gene is recombinated in the Mammals package cell line with the adenovirus skeleton carrier through after the linearizing, obtains recombinant adenovirus through screening.Second kind is representative with Adeasy, goal gene is cloned on the shuttle vectors, recombinate in intestinal bacteria with the adenovirus skeleton carrier after having the shuttle vectors linearizing of foreign gene, the recombinant adenovirus plasmid that obtains obtains recombinant adenovirus in Mammals packing cell internal packing after linearizing.The third is representative with Adeno-X, be cloned into goal gene on the intermediate carrier earlier, be allowed to condition at that rare enzyme I-CeuI and PI-SceI cut the site on the band of two ends, downcut this gene again with rare enzyme then, be directly connected on the adenovirus carrier, obtain recombinant adenovirus in Mammals packing cell internal packing after the recombinant adenovirus plasmid linearizing that obtains.These methods have improved conventional efficient to a certain extent, have reduced experimental cost, but all there is deficiency in these methods, mainly show following two aspects:
One, experimental period long, the efficient that obtains recombinant adenovirus is low.More than three kinds of methods all need realize the segmental clone of purpose by a kind of intermediate carrier, then by in mammalian cell or intestinal bacteria, recombinating, or connect by ligase enzyme goal gene imported on the adenoviral gene group.Therefore whole building process needs secondary to connect at least and transforms.First method need be recombinated in mammalian cell, the efficient of this reorganization is very low, therefore need identify virus in follow-up phase, to have the recombinant virus of goal gene separates with the adenovirus with goal gene not, need be through too much wheel choose the plaque purifying, this process wastes time and energy, and has the potential safety problem.Second method need be recombinated in intestinal bacteria.The third method needs rare enzyme, and needs other a kind of rare enzyme to reduce from connecting background.
Two, be not suitable for making up Mammals cDNA expression library.Preceding two kinds of methods all need earlier goal gene to be passed through restriction enzyme, perhaps Cre-loxP recombination system, or gene recombination technology such as Gateway technology clones on shuttle vectors, the recombinant plasmid of gained will with adenovirus skeleton carrier homologous recombination and obtain recombinant adenovirus in Mammals package cell line or intestinal bacteria.Through twice transformation, cause storage capacity very limited, be difficult to satisfy of the basic demand of structure library to storage capacity.Though the third method can be built the storehouse, building process does not need the homologous recombination step, and the clone connects and eliminates context process all needs rare enzyme, and experimental cost is higher, and is also uneconomical concerning large-scale research.
Three, be difficult to realization and express a plurality of genes simultaneously, preceding two kinds of systems need cotransformation, and step is loaded down with trivial details and qualification process is complicated time-consuming; A kind of method in back can not be expressed a plurality of genes at all.
(referring to publication " Hum.Gene Ther.1998 the 9th volume 2577-2583 page or leaf "; " Proc.Natl.Acad.Sci.1998 the 95th volume 2509-2514 page or leaf ")
Summary of the invention
The purpose of this invention is to provide a kind of technology of new structure recombinant adenovirus, really can accomplish high-throughput, directly, directed cloning is expressed, and need not rely on transport vehicle in making up the recombinant adenovirus process; Need not any homologous recombination process; Do not need many wheels plaque purifying of wasting time and energy yet, just can make things convenient for promptly to make up recombinant adenovirus, can amalgamation and expression, also can non-fusion expression.
The present invention realizes like this.Mainly comprise an existing adenovirus expression carrier system and through the goal gene of PCR primer amplification, by existing adenovirus system being carried out the site-directed mutagenesis of success and the modification of homologous recombination, two unique particular restriction endonuclease digestion site sequences and a resistant gene expression unit in this carrier, have been introduced; The amplification target gene one couple of PCR primers 5 ' end introduce 3～5 specific bases respectively, under dTTP or dATP or dGTP or dCTP existence condition, amplified production is through T
4The sticky end coupling that sticky end that archaeal dna polymerase digestion back produces and carrier double digestion obtain, thereby can directed cloning on the carrier of ClaI, AscI double digestion, transformed into escherichia coli is identified or Function Identification obtains recombinant plasmid by resistance screening and PCR.Recombinant plasmid is after linearizing, but direct transfection adenovirus packaging cell system obtains the recombinant adenovirus particle.
Specific practice is, an existing cover adenovirus system (comprising skeleton carrier and transport vehicle) is analyzed, select two proper restriction site ClaI and AscI, the condition that should satisfy is: 1. can obtain 5 after enzyme is cut ' and the base kind of outstanding sticky end is no more than 3 kinds; 2. the number of this restriction enzyme site of carrier existence itself is the least possible.Owing to still have two AscI sites on the existing adenovirus skeleton carrier, therefore at first this skeleton carrier is carried out site-directed mutagenesis, eliminate two AscI sites.Express the joint of polynucleotide of unitary two ends design then at a resistant gene, make its two ends introduce a ClaI, AscI restriction enzyme site respectively; By this joint resistant gene being cloned on the transport vehicle and with ClaI, AscI restriction enzyme site introduces.Have after the transport vehicle linearizing of ClaI and AscI restriction enzyme site adenovirus skeleton carrier with modified and carry out homologous recombination obtain the ultimate aim carrier in intestinal bacteria, this carrier produces such two sticky ends behind ClaI and AscI double digestion:
3′TA——CCGCGC 5′
5′CGAT——?GG 3′
5 ' end of amplification target gene one couple of PCR primers is introduced 5 ' CGA3 ' and such 3～5 Nucleotide of 5 ' CGCGA3 ' respectively, and the product that pcr amplification obtains adds dTTP and T
4The archaeal dna polymerase effect produces such sticky end:
5′CGA——T 3′
3′T——AGCGC 5′
The sticky end coupling that this sticky end just in time can produce with ultimate aim carrier double digestion, thus make the direct directed cloning of PCR product to carrier, and transformed into escherichia coli is identified or Function Identification obtains recombinant plasmid by resistance screening and PCR.Recombinant plasmid is after linearizing, but direct transfection adenovirus packaging cell system as the HEK293 cell, obtains the recombinant adenovirus particle.
The restriction enzyme at polynucleotide joint two ends can be any two kinds or AscI, Bsu36I, ClaI, CpoI, EarI, SapI, among the StyI any among AscI, Bsu36I, ClaI, CpoI, EarI, NotI, SapI, the StyI.That any enzyme is cut the back is getable 5 ' and restriction enzyme that the base kind of outstanding sticky end is no more than 3 kinds (containing 3 kinds) all can use.
The present invention can modify existing adenovirus carrier, eliminates the restriction enzyme digestion sites that is used to clone goal gene that carrier itself has, and then introduces specific restriction enzyme site, as ClaI and AscI, is used to clone goal gene.
The present invention can to build the method in storehouse compatible with all PCR-based.
The present invention can be used for making up the multiple mammalian cell and the recombinant adenovirus of tissue as the host that comprises the people.
The present invention compared with prior art has remarkable advantages:
One, through the PCR product T of the primer amplification gained of particular design
4Archaeal dna polymerase digestion, then with through special digestion with restriction enzyme by the adenovirus skeleton carrier of modified directly and orientation be connected, thereby a step makes up and obtains recombinant adenovirus plasmid.Whole building process is without any need for intermediate carrier, need be in the Mammals package cell line or carry out the step of homologous recombination in the intestinal bacteria yet, more do not need to use expensive rare enzyme.Easy and simple to handle, need not loaded down with trivial details plaque purifying, need not secondary connection and conversion, shortened experimental period, simplified the step that makes up recombinant adenovirus.Utilize this method to improve conventional efficient greatly, the work that makes up a recombinant adenovirus can be finished in two weeks.
Two, to build the method in storehouse compatible with present all PCR-based of report, can be used for the Mammals cDNA expression library of high flux construction based on adenovirus, introduce two or more specific restriction endonuclease sites if can express Mammals cDNA as far as possible really, can realize polygenic while clonal expression.
Embodiment
The present invention is further described with embodiment below:
Embodiment 1:
Utilize this method in the HEK293 cell, to express intestinal bacteria one galactoside enzyme.
Select a cover adenovirus carrier (comprising skeleton carrier pAdeasy and transport vehicle pShuttle-CMV) to analyze, select two proper restriction site ClaI and AscI, owing to contain two AscI sites on the pAdeasy, therefore at first pAdeasy is carried out site-directed mutagenesis, eliminate two AscI sites, obtain pmAdeasy.Design a polynucleotide joint then, its two ends are introduced a ClaI and AscI restriction enzyme site respectively, an ammonia benzyl of indirect resistant gene is expressed the unit, this joint is incorporated on the pShuttle-CMV, in intestinal bacteria BJ5183, carries out homologous recombination with pmAdeasy after the product linearizing and obtain ultimate aim carrier pAd-Ace.Design a pair of primer at target gene one galactoside enzyme gene order, its 5 ' end is introduced 5 ' CGA3 ' and these several extra Nucleotide of 5 ' CGCGA3 ' respectively, with intestinal bacteria BJ5183 genomic dna is template, (Pfu) carries out pcr amplification with high-fidelity DNA polymerase, the product that obtains is under the situation that dTTP exists, through T
4After archaeal dna polymerase is handled, its PCR product two ends respectively have a 5 ' distal process to go out the sticky end of 2 or 4 bases, this product energy directed cloning is on the pAd-Ace carrier of ClaI and AscI double digestion, transformed into escherichia coli again, the transformant that obtains is the recon of our expection through resistance screening and functional screening more than 90%.Select a recon and increase, direct transfection HEK293 cell after the recombinant plasmid linearizing detects enzyme and lives after 24 hours, collect the recombinant adenovirus particle after 7 days and infect the HEK293 cell again, detects one galactoside enzyme high level gene expression.
Embodiment 2:
DsRed is expressed in the HEK293 cell
Press the PCR primer of the method design amplification DsRed gene DsRed of embodiment 1, with Pfu polymeric enzymatic amplification DsRed gene, amplified production is used T equally under the condition that dTTP exists
4After archaeal dna polymerase was handled, directed cloning was on the pAd-Ace carrier of ClaI and AscI double digestion, and transformed into escherichia coli, the transformant that obtains pass through identifies more than 90% to be recon.Select a recon amplification, transfection HEK293 cell after the recombinant plasmid linearizing is collected the recombinant adenovirus particle and is infected the HEK293 cell again after 7 days, use fluorescence microscope after 24 hours, excite through green fluorescence, the cell that can see infection sends out red fluorescence tangible.
Claims (6)
1, the direct directed cloning of a kind of high-throughput makes up the method for recombinant adenovirus, mainly comprise an existing adenovirus expression carrier and through the goal gene of PCR primer amplification, it is characterized in that by existing adenovirus system being carried out the site-directed mutagenesis of success and the modification of homologous recombination, two unique particular restriction endonuclease digestion site sequences and a resistant gene expression unit in this carrier, have been introduced, 5 ' end in the one couple of PCR primers of amplification target gene is introduced 3～5 specific bases respectively, at dTTP, perhaps dATP, perhaps dGTP, perhaps under the dCTP existence condition, amplified production is through T
4The sticky end coupling that sticky end that archaeal dna polymerase digestion back produces and carrier double digestion obtain, thereby can directed cloning on the carrier of ClaI, AscI double digestion, transformed into escherichia coli, identify or Function Identification obtains recombinant plasmid by resistance screening and PCR, recombinant plasmid is after linearizing, but direct transfection adenovirus packaging cell system obtains the recombinant adenovirus particle.
2, make up the method for recombinant adenovirus according to the direct directed cloning of the described high-throughput of claim l, it is characterized in that the adenovirus skeleton carrier that designs by experiment in the existing adenovirus system carries out site-directed mutagenesis, eliminated its original two AscI restriction enzyme site; Express the joint of polynucleotide of unitary two ends design then at a resistant gene, make its two ends introduce a ClaI, AscI restriction enzyme site respectively; By this joint resistant gene being cloned on the transport vehicle and with ClaI, AscI restriction enzyme site introduces, product carries out homologous recombination with the adenovirus skeleton carrier of having eliminated the AscI restriction enzyme site after linearizing, obtain the final purpose carrier, the final purpose carrier produces such two sticky ends through ClaI and AscI double digestion:
3′ TA —————CCGCGC 5′
5′ CGAT ————— GG 3′
5 of goal gene one couple of PCR primers ' end is introduced 5 ' CGA3 ' and such 3～5 Nucleotide of 5 ' CGCGA3 ' respectively, and the product that pcr amplification obtains adds dTTP and T
4The archaeal dna polymerase effect produces such sticky end:
5′ CGA ————— T 3′
3′ T ————— AGCGC 5′
This sticky end just in time can mate with the sticky end that the carrier double digestion produces, thereby makes the direct directed cloning of PCR product to carrier.
3, high-throughout direct directed cloning according to claim 1 and 2 makes up the method for recombinant adenovirus, it is characterized in that two special restriction enzymes, can be enzyme cut the back getable 5 ' restriction enzyme that the base kind of outstanding sticky end is no more than 3 kinds (containing 3 kinds), two special restriction enzymes specifically can be ClaI or AscI or Bsu36I or CpoI or EarI or NotI or SapI or StyI and AscI or Bsu36I, ClaI, CpoI, EarI, NotI, SapI, StyI.
4, the direct directed cloning of high-throughput according to claim 1 makes up the method for recombinant adenovirus, it is characterized in that and to modify existing adenovirus carrier, eliminate the restriction enzyme digestion sites that will be selected for clone's goal gene that carrier itself has, and then introduce specific restriction enzyme site ClaI and AscI, be used to clone goal gene.
5, the direct directed cloning of high-throughput according to claim 1 makes up the method for recombinant adenovirus, and it is characterized in that can to build the method in storehouse compatible with all PCR-based.
6, the direct directed cloning of high-throughput according to claim 1 makes up the method for recombinant adenovirus, it is characterized in that can be used for making up the multiple mammalian cell and the recombinant adenovirus of tissue as the host that comprises the people.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410060972 CN1624145A (en) | 2004-10-19 | 2004-10-19 | Process for structuring recombined adenovirus by high-flux direct directional clone |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410060972 CN1624145A (en) | 2004-10-19 | 2004-10-19 | Process for structuring recombined adenovirus by high-flux direct directional clone |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1624145A true CN1624145A (en) | 2005-06-08 |
Family
ID=34764389
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 200410060972 Pending CN1624145A (en) | 2004-10-19 | 2004-10-19 | Process for structuring recombined adenovirus by high-flux direct directional clone |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1624145A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101139586B (en) * | 2007-08-06 | 2011-05-11 | 湖北大学 | Fast high-flux gene site-directed mutagenesis method |
| CN103146753A (en) * | 2013-03-18 | 2013-06-12 | 山东维真生物科技有限公司 | Novel adenovirus vector and production method for same |
| CN106868047A (en) * | 2017-03-07 | 2017-06-20 | 南方医科大学 | A kind of recombinant adenoviral vector and its construction method and purposes |
| CN110055278A (en) * | 2019-04-23 | 2019-07-26 | 中科广聚(北京)生物医学技术中心有限公司 | A kind of application of New-type adenovirus packing method in CRISPR/Cas9 gene editing method |
-
2004
- 2004-10-19 CN CN 200410060972 patent/CN1624145A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101139586B (en) * | 2007-08-06 | 2011-05-11 | 湖北大学 | Fast high-flux gene site-directed mutagenesis method |
| CN103146753A (en) * | 2013-03-18 | 2013-06-12 | 山东维真生物科技有限公司 | Novel adenovirus vector and production method for same |
| CN103146753B (en) * | 2013-03-18 | 2013-12-18 | 山东维真生物科技有限公司 | Adenovirus vector and production method for same |
| CN106868047A (en) * | 2017-03-07 | 2017-06-20 | 南方医科大学 | A kind of recombinant adenoviral vector and its construction method and purposes |
| CN106868047B (en) * | 2017-03-07 | 2020-04-28 | 南方医科大学 | Recombinant adenovirus vector and construction method and application thereof |
| CN110055278A (en) * | 2019-04-23 | 2019-07-26 | 中科广聚(北京)生物医学技术中心有限公司 | A kind of application of New-type adenovirus packing method in CRISPR/Cas9 gene editing method |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP2001500740A (en) | Method for stably cloning large repetitive DNA sequences | |
| EP3604524A1 (en) | New technique for genomic large fragment direct cloning and dna multi-molecular assembly | |
| Xu et al. | The complete mitochondrial DNA sequence of the white rhinoceros, Ceratotherium simum, and comparison with the mtDNA sequence of the Indian rhinoceros, Rhinoceros unicornis | |
| CA2064092A1 (en) | Efficient directional genetic cloning system | |
| US20030017552A1 (en) | Modular vector systems | |
| JP2010193896A (en) | Method for producing recombinant virus using site-specific recombination | |
| Westergren Jakobsson et al. | The human adenovirus 2 transcriptome: an amazing complexity of alternatively spliced mRNAs | |
| CN111094575A (en) | DNA assembly | |
| CN1624145A (en) | Process for structuring recombined adenovirus by high-flux direct directional clone | |
| Zhang et al. | Getting genetic access to natural adenovirus genomes to explore vector diversity | |
| CN1844401A (en) | Method for rapid construction of carrier for gene targeting recombination | |
| Yao et al. | High-frequency genetic recombination and reactivation of orthopoxviruses from DNA fragments transfected into leporipoxvirus-infected cells | |
| CN105950612B (en) | A kind of efficient DNA connector connecting method | |
| Ruzsics et al. | Transposon-Assisted Cloning and Traceless Mutagenesis of Adenoviruses: Development of a Novel Vector Based on SpeciesD | |
| Arbiol et al. | Mobile regulatory cassettes mediate modular shuffling in T4-type phage genomes | |
| CN1253561C (en) | Process for structuring combined rhabdovirus by high-flux clone | |
| CN113174373B (en) | Method for knocking out baculovirus essential gene | |
| CN113584064A (en) | Rapid TALE expression vector construction method based on codon degeneracy | |
| CN103088055B (en) | Construction method of plant dual-gene co-expression vector | |
| WO1996001320A3 (en) | Complete genomic sequence of autographa californica nuclear polyhedrosis virus C6 | |
| CN1165622C (en) | Zero-background high-flux directed cloning expression method | |
| Shaw | Quick and easy CRISPR engineering in Saccharomyces cerevisiae | |
| CN104357438A (en) | DNA assembly and cloning method | |
| Antolovic et al. | Short description of an alternative simplified method for screening recombinant clones within the" AdEasy-System" by Duplex-PCR | |
| ES2125842A1 (en) | INFECTIOUS VECTORS AND CLONES OF PLANTS DERIVED FROM THE TURNIP MOSAIC VIRUS (TuMV) |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C12 | Rejection of a patent application after its publication | ||
| RJ01 | Rejection of invention patent application after publication |