CN108441516A - A kind of slow virus CMV-CBh double-promoters transformation vector construction and application - Google Patents

A kind of slow virus CMV-CBh double-promoters transformation vector construction and application Download PDF

Info

Publication number
CN108441516A
CN108441516A CN201810257388.1A CN201810257388A CN108441516A CN 108441516 A CN108441516 A CN 108441516A CN 201810257388 A CN201810257388 A CN 201810257388A CN 108441516 A CN108441516 A CN 108441516A
Authority
CN
China
Prior art keywords
tcbh
cmv
mcherry
slow virus
carrier
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.)
Withdrawn
Application number
CN201810257388.1A
Other languages
Chinese (zh)
Inventor
杨蕊菊
孙子豪
杨兴林
潘讴东
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Yuan Biotechnology (shanghai) Ltd By Share Ltd
Original Assignee
Yuan Biotechnology (shanghai) Ltd By Share Ltd
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Yuan Biotechnology (shanghai) Ltd By Share Ltd filed Critical Yuan Biotechnology (shanghai) Ltd By Share Ltd
Priority to CN201810257388.1A priority Critical patent/CN108441516A/en
Publication of CN108441516A publication Critical patent/CN108441516A/en
Withdrawn legal-status Critical Current

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
    • C12N15/86—Viral vectors
    • 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
    • C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
    • 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
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Abstract

The present invention relates to a kind of slow virus CMV CBh double-promoters transformation vector construction and applications.The slow virus carrier pLenti CMV 3FLAG EGFP tCBh mCherry T2A Puro obtain PGK in tCBh segments replacement pLenti CMV 3FLAG EGFP PGK mCherry T2A Puro carriers by PCR amplification and obtain.TCBh promoters driving expression mCherry genes and Puromycin resistant genes can use Puromycin to filter out the cell stablized and be overexpressed target gene；MCherry genes help to judge viral packaging efficiency and efficiency of infection, while also contributing to stablize the screening of cell.By transfecting Fluirescence observation, it was demonstrated that slow virus dual-promoter vector expression system construction success provides carrier is carrier further to study interested target gene.

Description

A kind of slow virus CMV-CBh double-promoters transformation vector construction and application
Technical field
The invention belongs to genetic engineering fields, are transformed and carry more specifically to a kind of slow virus CMV-CBh double-promoters Body is built and application.
Background technology
Bidirectional promoter：Section of DNA sequence between two adjacent and gene that transcriptional orientation is opposite.
Bidirectional promoter is widely distributed in eukaryotic gene group, and most of bidirectional promoter lacks TATA boxes, and With higher G/C content and the two-way starting transcription of the abundant islands CpG bidirectional promoters, and can exist to the gene of its bidirectional transcription Certain effect is played in coexpression and stability expression regulation.
During carrying out transgene improvement to biology, it may be necessary to be transferred to the foreign gene of more than one specific Organism in, these foreign genes needs carried out under the regulation and control of respective promoter sequence expression due to being used to lose The promoter limited amount of improvement is passed, so often repeatedly specific using one when multiple genes are transferred in organism Although the hope for being transferred to multiple genes simultaneously may be implemented in this way in the promoter of promoter or Sequences similar, this carrier Time-consuming and laborious is built on the other hand, because the promoter sequence introduced is homologous, even if there was only the sequence of 90bp between two promoters Arrange it is homologous, import organism in will cause gene expression " co-suppression " phenomenon, lead to the silence of genetic transcription, this is to turn base Because of the difficulty and challenge to be faced in technology
Since bidirectional promoter can instruct the generation of mRNA in two directions, carried out in this way using bidirectional promoter When transgenosis, can its both ends merge two genes of certain physiology or biochemical process or multiple genes and combinations thereof this not only Tedious steps when different genes introduce are avoided, the promoter number being introduced into receptor biological is reduced, more there is no concern that drawing Enter between the promoter in organism transgene silencing caused by sequence homology and show example and few, and son carries out transgenosis The emphasis of research, which is still placed on bidirectional promoter, to carry out the emphasis of transgenic research and is still placed on the application of bidirectional promoter and is It solves transgenic technology problem and provides possibility, possibility is provided to solve transgenic technology problem in the application of genetic engineering, There is immeasurable effect in genetic engineering research and application field, just become the frontier of transgenic technology research.
Invention content
The present invention is described in further detail with specific implementation mode below in conjunction with the accompanying drawings：
1. present disclosure, which is structure, shares the reversed double-promoter Lentiviral of enhancer
2. the technical solution adopted by the present invention is：
A kind of slow virus CMV-CBh double-promoters transformation vector construction and application.The slow virus carrier pLenti-CMV- 3FLAG-EGFP-tCBh-mCherry-T2A-Puro obtains tCBh segments by PCR amplification and replaces pLenti-CMV-3FLAG- PGK is obtained in EGFP-PGK-mCherry-T2A-Puro carriers.
3. the invention further relates to the method for the structure slow virus carrier, the method includes：
(1) amplification of segment tCBh：Using plasmid pX330A-1x2 as template, design primer amplified obtains.
The primer sequence of the amplified fragments tCBh is as follows：
tCBh-F:5'-ACGAGCTGTACAAGTCTAGATAATCGAGGTGAGCCCCACGT-3'
tCBh-R:5'-TGCTCACCATGGTAAGCTTGGGCTGCAGGTCCAACCTGAAAAAAAGTGATTTC-3'
(2) structure of slow virus carrier pLenti-CMV-3FLAG-EGFP-tCBh-mCherry-T2A-Puro：Use XbaI With BspMI digestions pLenti-CMV-3FLAG-EGFP-PGK-mCherry-T2A-Puro carrier recoveries large fragment and tCBh segments Seamless clone is carried out to obtain.
4. the beneficial effects are mainly as follows：The slow virus carrier that the present invention is built has double-promoter-CMV And CBh can be detected using green fluorescent protein EGFP and mcherry as reporter gene with existing fluorescence microscope arrangement, Efficiency of infection further to study slow virus carrier infection cell provides experiment basis.
Description of the drawings
Attached drawing 1:Slow virus carrier pLenti-CMV-3FLAG-EGFP-tCBh-mCherry-T2A-Puro plasmid maps；
Attached drawing 2:In the Multiplex CRISPR/Cas9Assembly System Kitprotocol of Addgene buyings PX330A-1x2 plasmid maps；
Attached drawing 3:Slow virus carrier pLenti-CMV-3FLAG-EGFP-PGK-mCherry-T2A-Puro plasmid maps；
Attached drawing 4:Segment tCBh amplification figures；M:DNA Marker：DL2000, Lane1:PCR amplificationg of tCBh；
Attached drawing 5:Plasmid pLenti-CMV-3FLAG-EGFP-PGK-mCherry-T2A-Puro XbaI/BspMI double digestions Gel recycles result；M:DNA Marker 1kb,Lane1:Digestion of plasmid pLenti-CMV-3FLAG- EGFP-PGK-mCherry-T2A-Puro。
Specific implementation mode
The chemical reagent and biological products being related to below are all commercially produced product if not otherwise specified.In addition, other are not noted Bright experimental implementation is carried out according to conventional molecular biological operating method.With reference to specific embodiment to the present invention into advance one Step description, but protection scope of the present invention is not limited to that：
Embodiment
1. building pLenti-EGFP-2A-Puro-CMV-tCBh slow virus expression plasmids
1.1tCBh fragment amplification：
Using carrier pX330A-1x2 as template, with tCBh-F and tCBh-R primers (No.1~2 SEQ ID) to carrying out PCR Obtain the tCBh segments of 565bp, gel extraction；
PCR system is as follows：
Plasmid template (100ng/ μ l) 2μl
Primer Star polymerase(3.5U/μl) 0.5μl
dNTPs(10mM；2.5mM each) 4μl
2X GC Buffer 25μl
tCBh-F(10pmol/μl) 1μl
tCBh-R(10pmol/μl) 1μl
ddH2O 16.5μl
PCR reaction conditions：
1.2 use XbaI and BspMI digestion pLenti-CMV-3FLAG-EGFP-PGK-mCherry-T2A-Puro plasmids, 2h, 37 DEG C：
Digestion system is as follows：
2μg(2μl) pLenti-CMV-3FLAG-EGFP-PGK-mCherry-T2A-Puro
1μl XbaI(NEB)
1μl BspMI(NEB)
5μl NEBuffer3.1
41μl ddH2O
50μl It is total
The segment that 525bp and 9244bp are obtained after digestion purifies the matter of the 9244bp after digestion using plastic recovery kit Grain large fragment；
The segment tCBh that 1.3 amplifications obtain is with the seamless clone of carrier large fragment after digestion at purpose carrier pLenti- CMV-3FLAG-EGFP-tCBh-mCherry-T2A-Puro。
System is as follows：
11.3ng Insert Fragment (tCBh)
92.4ng Linearized vector (pLenti-CMV-3FLAG-EGFP-PGK-mCherry-T2A-Puro)
1μl Seamless clone enzyme
4μl 5X reacts Buffer
Up to 20μl ddH2O
30 points of kinds are incubated after mixing at 37 DEG C, is then transferred into and places 5 minutes on ice.
Plasmid after connection is converted into competent cell DH5 α, is uniformly applied in LB solid medium tablets, is placed in It is cultivated 12-16 hours in 37 DEG C of incubators, single bacterium colony may occur in which.
The several single bacterium colonies expansions of 1.4 pickings, which are cultivated, and plasmid is small carries.
1.5 identification plasmid construction successes.The purpose plasmid that correct plasmid is constructed is sequenced, is named as pLenti- CMV-3FLAG-EGFP-tCBh-mCherry-T2A-Puro。
2. transfection efficiency is verified
It is based on 5%CO with the DMEM in high glucose culture containing 10% fetal calf serum2, 37 DEG C of constant temperature incubation 293T cells (are purchased from U.S. State's ATCC cell banks).Take logarithmic phase cell with 1 × 105/ hole is inoculated into 24 orifice plate cultures.Wait for that cell fusion degree reaches 70%~8 Opti-MEM culture mediums are replaced with when 0%, by pLenti-CMV-3FLAG-EGFP-tCBh-mCherry-T2A- after 1 hour 0.8 μ g of Puro plasmids are transfected into through Lipo2000 reagents in 293T cells, and as a child, fluorescence microscopy turns under the microscope for transfection 48 Contaminate effect.
Sequence table
<110>With first biotechnology (Shanghai) limited liability company
<120>A kind of slow virus CMV-CBh double-promoters transformation vector construction and application
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 41
<212> DNA
<213>Artificial sequence (Artifical synthesis)
<400> 1
<210> 2
<211> 53
<212> DNA
<213>Artificial sequence (Artifical synthesis)
<400> 2
<210> 3
<211> 512
<212> DNA
<213>Artificial sequence (Artifical synthesis)
<400> 3
<210> 4
<211> 9769
<212> DNA
<213>Artificial sequence (Artifical synthesis)
<400> 4
<210> 5
<211> 9774
<212> DNA
<213>Artificial sequence (Artifical synthesis)
<400> 5

Claims (5)

1. a kind of slow virus CMV-CBh double-promoters transformation vector construction and application, the slow virus carrier pLenti-CMV- 3FLAG-EGFP-tCBh-mCherry-T2A-Puro obtains tCBh segments by PCR amplification (sequence is as shown in SEQ ID No. 3) It replaces in pLenti-CMV-3FLAG-EGFP-PGK-mCherry-T2A-Puro (shown in sequence such as SEQ ID No. 4) carrier PGK is obtained.
2. building the slow virus carrier pLenti-CMV-3FLAG-EGFP-tCBh-mCherry-T2A- as described in claim 1 Puro, which is characterized in that the base sequence of the carrier is as shown in SEQ ID No.5.
3. building the slow virus carrier pLenti-CMV-3FLAG-EGFP-tCBh-mCherry-T2A- as described in claim 1 Puro, which is characterized in that the carrier structure is as shown in Figure 1.
4. the method for building the slow virus CMV-CBh double-promoters transformation carrier as described in claim 1, the method includes：
(1) amplification of segment tCBh：The Multiplex CRISPR/Cas9 Assembly System purchased with Addgene PX330A-1x2 plasmids (carrier structure is as shown in Figure 2) in Kitprotocol is template, designs specific primer Amplification obtains；The primer sequence of the amplified fragments tCBh is as follows：
tCBh-F: 5'- ACGAGCTGTACAAGTCTAGATAATCGAGGTGAGCCCCACGT-3'
tCBh-R: 5'-TGCTCACCATGGTAAGCTTGGGCTGCAGGTCCAACCTGAAAAAAAGTGATTTC-3'
(2) structure of slow virus carrier pLenti-CMV-3FLAG-EGFP-tCBh-mCherry-T2A-Puro：With XbaI and The BspMI digestion carriers pLenti-CMV-3FLAG-EGFP-PGK-mCherry-T2A-Puro (carrier structure such as Fig. 3 institutes Show), it recycles after large fragment and tCBh segments carries out seamless clone.
5. CMV-CBh double-promoters Lentiviral the answering in slow virus carrier structure as described in claim 1 With.
CN201810257388.1A 2018-03-27 2018-03-27 A kind of slow virus CMV-CBh double-promoters transformation vector construction and application Withdrawn CN108441516A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201810257388.1A CN108441516A (en) 2018-03-27 2018-03-27 A kind of slow virus CMV-CBh double-promoters transformation vector construction and application

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201810257388.1A CN108441516A (en) 2018-03-27 2018-03-27 A kind of slow virus CMV-CBh double-promoters transformation vector construction and application

Publications (1)

Publication Number Publication Date
CN108441516A true CN108441516A (en) 2018-08-24

Family

ID=63196780

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201810257388.1A Withdrawn CN108441516A (en) 2018-03-27 2018-03-27 A kind of slow virus CMV-CBh double-promoters transformation vector construction and application

Country Status (1)

Country Link
CN (1) CN108441516A (en)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109679977A (en) * 2019-01-21 2019-04-26 贵州大学 It is a kind of carry cytochrome C gene slow virus expression plasmid building and application method
CN110055281A (en) * 2019-04-25 2019-07-26 山东大学第二医院 A kind of slow virus carrier being used to prepare CAR-T and its construction method and application
CN110305902A (en) * 2019-07-12 2019-10-08 和元生物技术（上海）股份有限公司 A kind of method and its application activating hSyn promoter in vehicles cells
CN113106091A (en) * 2021-03-04 2021-07-13 杭州师范大学 Plant leaf high-expression bidirectional promoter and application thereof
CN116218844A (en) * 2022-09-07 2023-06-06 吉满生物科技(上海)有限公司 Bidirectional promoter, over-expression vector thereof, lentiviral expression plasmid and application

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090210952A1 (en) * 2005-12-16 2009-08-20 Xiaoyun Wu Compositions and Methods Related to Controlled Gene Expression Using Viral Vectors
CN102268448A (en) * 2011-06-28 2011-12-07 华东理工大学 Expression equipment for expressing heterologous protein in Trichoderma reesei cell, and gene engineering bacteria
CN102311960A (en) * 2011-07-13 2012-01-11 深圳大学 Marine penicillium Swollenin gene, protein coded by Swollenin gene and application of Swollenin gene
CN103290056A (en) * 2012-02-27 2013-09-11 上海诺百生物科技有限公司 Lentivirus vector, preparation and application of and virus particle thereof
CN107475298A (en) * 2017-09-11 2017-12-15 扬州大学 CdtB gene overexpressions slow virus carrier and its construction method and the slow virus comprising cdtB genes and its application

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090210952A1 (en) * 2005-12-16 2009-08-20 Xiaoyun Wu Compositions and Methods Related to Controlled Gene Expression Using Viral Vectors
CN102268448A (en) * 2011-06-28 2011-12-07 华东理工大学 Expression equipment for expressing heterologous protein in Trichoderma reesei cell, and gene engineering bacteria
CN102311960A (en) * 2011-07-13 2012-01-11 深圳大学 Marine penicillium Swollenin gene, protein coded by Swollenin gene and application of Swollenin gene
CN103290056A (en) * 2012-02-27 2013-09-11 上海诺百生物科技有限公司 Lentivirus vector, preparation and application of and virus particle thereof
CN107475298A (en) * 2017-09-11 2017-12-15 扬州大学 CdtB gene overexpressions slow virus carrier and its construction method and the slow virus comprising cdtB genes and its application

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
MANLI NA 等: "Design of Ad5F35 vectors for coordinated dual gene expression in candidate human hematopoietic stem cells", 《EXPERIMENTAL HEMATOLOGY》 *
REGINE HEILBRONN 等: "Viral Vectors for Gene Transfer: Current Status of Gene Therapeutics", 《HANDBOOK OF EXPERIMENTAL PHARMACOLOGY》 *
张玲 等: "重组慢病毒载体介导不同启动子驱动的绿色荧光蛋白在多种（不同）细胞中的表达影响", 《分子诊断与治疗杂志》 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109679977A (en) * 2019-01-21 2019-04-26 贵州大学 It is a kind of carry cytochrome C gene slow virus expression plasmid building and application method
CN110055281A (en) * 2019-04-25 2019-07-26 山东大学第二医院 A kind of slow virus carrier being used to prepare CAR-T and its construction method and application
CN110305902A (en) * 2019-07-12 2019-10-08 和元生物技术（上海）股份有限公司 A kind of method and its application activating hSyn promoter in vehicles cells
CN110305902B (en) * 2019-07-12 2020-08-21 和元生物技术（上海）股份有限公司 Method for activating hSyn promoter in tool cell and application thereof
CN113106091A (en) * 2021-03-04 2021-07-13 杭州师范大学 Plant leaf high-expression bidirectional promoter and application thereof
CN116218844A (en) * 2022-09-07 2023-06-06 吉满生物科技(上海)有限公司 Bidirectional promoter, over-expression vector thereof, lentiviral expression plasmid and application

Similar Documents

Publication Publication Date Title
CN108441516A (en) A kind of slow virus CMV-CBh double-promoters transformation vector construction and application
US20220033858A1 (en) Crispr oligoncleotides and gene editing
CN104087610B (en) Shuttle vector and construction process thereof and application
JP2022548062A (en) Modified bacterial retroelements with enhanced DNA production
CN101368188B (en) Quick efficient plant manpower fine RNA expression vector construction method
CN109750035B (en) sgRNA for targeting and guiding Cas9 protein to efficiently cleave TCR and B2M gene locus
CN107338266A (en) A kind of VIGS silencing systems for identifying mulberry tree MmPDS genes and its construction method and application
CN113278635A (en) Sequence combination for promoting cyclic RNA cyclization and application thereof
CN108559731A (en) A kind of human embryonic stem cell line of tetracycline-regulated gene expression and its application
CN116004681B (en) Method and kit for improving carrier connection efficiency in TOPO cloning
CN102628057A (en) Vector for non-background directed cloning of PCR products, preparation method thereof and application thereof
Li et al. Strategy for efficient cloning of biosynthetic gene clusters from fungi
KR20220061078A (en) Method for modifying a target nucleic acid in the genome of a cell
CN112359092B (en) Construction method of genome short fragment library
CN111662932B (en) Method for improving homologous recombination repair efficiency in CRISPR-Cas9 gene editing
CN110791503B (en) Low-phosphorus inducible promoter and application thereof
CN112877332A (en) Method for detecting activity of chicken RIPK2 promoter by using dual-luciferase reporter gene
CN116240209B (en) Enhancer strongly induced by powdery mildew in Chinese spring wheat and application thereof
CN114891791B (en) sgRNA of specific targeting canine Rosa26 gene and application thereof
CN112662668B (en) Primer group for CKO vector construction
CN113025621B (en) Application of CIPK14 gene in improving drought resistance of pigeon pea
CN108823210B (en) Promoter for specific and efficient expression of pig muscle tissue and application
US20230203479A1 (en) UnGE promoter sequence and its uses
Su et al. An efficient gene disruption method using a positive–negative split-selection marker and Agrobacterium tumefaciens-mediated transformation for Nomuraea rileyi
CN110804626B (en) Method for constructing high-efficiency expression vector by combining high CG segment and low CG promoter

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
WW01 Invention patent application withdrawn after publication

Application publication date: 20180824

WW01 Invention patent application withdrawn after publication