CN106497953A - A kind of green fluorescence protein expression carrier of improvement and its construction method - Google Patents
A kind of green fluorescence protein expression carrier of improvement and its construction method Download PDFInfo
- Publication number
- CN106497953A CN106497953A CN201610942279.4A CN201610942279A CN106497953A CN 106497953 A CN106497953 A CN 106497953A CN 201610942279 A CN201610942279 A CN 201610942279A CN 106497953 A CN106497953 A CN 106497953A
- Authority
- CN
- China
- Prior art keywords
- pbe
- gfpst
- carrier
- sal1
- hind3
- 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
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
- C12N15/65—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
- C12N15/66—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
Landscapes
- Genetics & Genomics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a kind of construction method of the green fluorescence protein expression carrier of improvement.The present invention is according to carrier pBE GFP sequences (from pBin19, which is that GFP genes are inserted between Age1 the and Sac1 restriction enzyme sites of pBin19 carriers), select a pair of upstream primer pBE GFPST Hind3 F of sequences Design and downstream primer pBE GFPST Sal1 R of wherein one section of about 1000bp, new unique restriction enzyme site Spe1 is inserted at its two in cohesive end the enzyme Hind3 and Sal1 at its two, Xho1, Fse1, so that the enzyme sum of cohesive end reaches 8, and pulled open the distance between several conventional Cheap highly effective enzymes, ensure that enough protection base quantity.Additionally, also above connecting S Tag label A AAGAAACCGCTGCTGCTAAATTCGAACGCCAGCACATGGACAGC.After PCR, digestion connection obtains new expression vector.
Description
Technical field
The invention belongs to gene engineering technology field, relates in particular to a kind of green fluorescence protein expression carrier of improvement
And its construction method.
Background technology
Egfp expression system with 35S promoter is usually used in the transient expression in eucaryote body, can use
In the application such as Subcellular Localization or in vivo functionality identification.But the MCS of the GFP carriers of a lot of business is limited, and is delivering
During paper, the enzyme activity for identifying target protein after responsible reader requires to remove GFP again is had, while will also demonstrate,prove by Western blot
The bright albumen is expressed in vivo, is at this moment accomplished by introducing other antibody labels.By taking the pBE-GFP expressed in tobacco as an example,
Its MCS only has Xba1, Sal1, Kpn1, Sma1, BamH1 and Age1, not only selectable negligible amounts, and contains
The relatively low flat end restriction enzyme site of specific joint efficiency.Some restriction enzymes are less common, and a lot of laboratories need separately
Purchase, and the price corresponding to unit enzyme activity is also costly.Additionally, these restriction enzyme site intervals are too near, shortage is effectively protected
Base ensures the efficiency of double digestion, even if having selected two enzymes therein, but affects cloning efficiency because of too near apart.
Content of the invention
The purpose of the present invention be for MCS quantity in existing green fluorescence protein expression carrier pBE-GFP few,
The deficiency such as many, the used enzyme poor universality of flat end site, price be high and restriction enzyme site is spaced closely together, there is provided a kind of many grams
The enzyme versatility that grand site is more, flat end site is few, used is high, and the egfp expression of the high improvement of joint efficiency is carried
Body and its construction method.
The green fluorescence protein expression carrier of the improvement of the present invention, which builds by the following method, including following step
Suddenly：With carrier pBE-GFP as template, with upstream primer pBE-GFPST-Hind3-F and downstream primer pBE-GFPST-Sal1-R
Enter performing PCR amplification as primer, amplified production Hind3 and Sal1 double digestions obtain the product after double digestion, are named as insertion
Son；By carrier pBE-GFP Hind3 and Sal1 double digestions, the product after double digestion is obtained, be named as digestion carrier, will insertion
Son and digestion carrier connect the green fluorescence protein expression carrier for obtaining improvement；
The nucleotides sequence of described upstream primer pBE-GFPST-Hind3-F is classified as：
5’-CGCCAAGCTTGCATGCCTGCAGGTCC-3’；(as shown in SEQ ID NO.1).
The nucleotides sequence of described downstream primer pBE-GFPST-Sal1-R is classified as:5’-
AATGTCGACGGCCGGCCTCGAGATGGACTAGTAGCTCTAGAGCTGTCCATGTGCTGGCGTTCGAATTTAGCAGCAGC
GGTTTCTTTCATGTCCCCCGTGTTC-3 ' (as shown in SEQ ID NO.2).
According to carrier pBE-GFP sequences, (from pBin19, which is that GFP genes are inserted into pBin19 carriers to the present invention
Between Age1 and Sac1 restriction enzyme sites), select a pair of upstream primer pBE-GFPST- of sequences Design of wherein one section of about 1000bp
Hind3-F and downstream primer pBE-GFPST-Sal1-R, at its two in cohesive end the enzyme Hind3 and Sal1 at its two
New unique restriction enzyme site Spe1, Xho1, Fse1 is inserted, so that the enzyme sum of cohesive end reaches 8, and is pulled open
The distance between several conventional Cheap highly effective enzymes, it is ensured that enough protection base quantity.Additionally, also above connecting S-Tag
Label A AAGAAACCGCTGCTGCTAAATTCGAACGCCAGCACATGGACAGC.After PCR, digestion connection obtains new table
Reach carrier.
GFP sequences (GenBank accession number：HF675000.1)：
ATGAGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAAT
GGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTTAAATTTATTTGCAC
TACTGGAAAACTACCTGTTCCTTGGCCAACACTTGTCACTACTTTCTCTTATGGTGTTCAATGCTTTTCAAGATACC
CAGATCATATGAAGCGGCACGACTTCTTCAAGAGCGCCATGCCTGAGGGATACGTGCAGGAGAGGACCATCTTCTTC
AAGGACGACGGGAACTACAAGACACGTGCTGAAGTCAAGTTTGAGGGAGACACCCTCGTCAACAGGATCGAGCTTAA
GGGAATCGATTTCAAGGAGGACGGAAACATCCTCGGCCACAAGTTGGAATACAACTACAACTCCCACAACGTATACA
TCATGGCAGACAAACAAAAGAATGGAATCAAAGTTAATTTCAAAATTAGACACAACATTGAAGATGGAAGCGTTCAA
CTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCCAC
ACAATCTGCCCTTTCGAAAGATCCCAACGAAAAGAGAGACCACATGGTCCTTCTTGAGTTTGTAACAGCTGCTGGGA
TTACACATGGCATGGATGAACTATACAAATAA
Description of the drawings：
Fig. 1 is single endonuclease digestion inspection figure, and wherein 1 is pBE-GFP (Xba1 digestions)；2 is new support pBE-GFPST (Xho1 enzymes
Cut)；
Fig. 2 is new support pBE-GFPST sequencing results (reverse complementary sequence)；
Fig. 3 is the MCS schematic diagram of former pBE-GFP；
Fig. 4 is the MCS schematic diagram of new support pBE-GFPST, the ATG bases after 5814bp start to
It is restriction enzyme site and the S-TAG labels (green background) for newly increasing between Sal1；
Fig. 5 is pBE-GFPST::The tobacco of CsTPS conversions, by gene constructed for CsTPS on new support pBE-GFPST,
Fluorescence is given expression in tobacco；
Fig. 6 is to check expression in tobacco in Western blot with the S-TAG labels in new support pBE-GFPST
CsTPS albumen.
Specific embodiment：
Following examples are that the present invention is further illustrated, rather than limitation of the present invention.
Embodiment 1：
A) PCR of Insert Fragment：
With pBE-GFP as template, upstream primer pBE-GFPST-Hind3-F and downstream primer pBE-GFPST-Sal1-R be
Primer.PrimeSTAR Max Premix (2 ×) 20 μ l, upstream primer pBE-GFPST-Hind3-F of the PCR amplifications with Takara
Each with downstream primer pBE-GFPST-Sal1-R 10 μM, 0.1 μ l of pBE-GFP templates, plus distilled water supplies 40 μ l.PCR expands journey
Sequence is 98 DEG C of 30sec, and 1 circulates；98 DEG C of 15sec, 45 DEG C of 15sec, 72 DEG C of 15sec, 1 circulation；98 DEG C of 15sec, 72 DEG C
15sec, 32 circulations；72 DEG C of 10min, 1 circulation, thus obtain the PCR primer of Insert Fragment
The nucleotides sequence of described upstream primer pBE-GFPST-Hind3-F is classified as：
5’-CGCCAAGCTTGCATGCCTGCAGGTCC-3’；
The nucleotides sequence of described downstream primer pBE-GFPST-Sal1-R is classified as：
5’-AATGTCGACGGCCGGCCTCGAGATGGACTAGTAGCTCTAGAGCTGTCCATGTGCTGGCGTTCGAAT
TTAGCAGCAGCGGTTTCTTTCATGTCCCCCGTGTTC-3’.
B) double digestion of Insert Fragment：
The PCR primer of Insert Fragment is reclaimed after target stripe, with the restriction enzyme of NEB through agarose gel electrophoresis
Do digestion.40 μ l of reaction system, wherein 4 μ l of Cutsmart buffer, 15 μ l of PCR primer, each 0.3 μ l of Hind3 and Sal1, enzyme
It is recovered by filtration after cutting two hours, obtains the intron after double digestion.
C) double digestion of carrier：
PBE-GFP is double digestion, wherein 40 μ l of reaction system, 4 μ l of Cutsmart buffer, 15 μ of pBE-GFP plasmids
The each 0.3 μ l of l, Hind3 and Sal1, digestion tap rubber after two hours and reclaim, and obtain the carrier after double digestion.
D) connection conversion：
The carrier after intron and double digestion after by double digestion is connected with the T4 ligases of Promega, in 10 μ l systems
Containing 4 μ l of intron, the 1 μ l of carrier after double digestion after double digestion.Ice bath half an hour after DH5a Escherichia coli is transformed into after connection,
Add LB culture mediums 230rpm cultures 1h in 37 DEG C of shaking tables after 42 DEG C of heat shocks, take the culture plate that 200 μ l apply kalamycin resistance.12
Positive monoclonal is chosen after hour, survey identification is sent with universal primer M13R and PBEGFPN3 ', is thus obtained new support pBE-GFPST
(its be by double digestion after intron and the carrier after double digestion be formed by connecting).
E) result verification：
The result that new support pBE-GFPST carries out single endonuclease digestion inspection is normal (Fig. 1), and sequence verification result is correct (Fig. 2).
Original vector (Fig. 3) shows new introducing with the comparison in the MCS region of new support pBE-GFPST (Fig. 4)
Between MCS and new mutual alignment relation of the MCS on new support.
F) application example
Terpene synthase gene CsTPS9 (the NCBI accession number of tealeaves is screened from transcript profile database：
GEFQ01060445.1), when the function of expressing in tobacco body is verified, on old carrier pBE-GFP, limited MCS exists
Exist in CsTPS9, at this moment just used the Xho1 restriction enzyme sites of new insertion in new support pBE-GFPST.
Using tealeaves transcript profile RNA as template, terpene synthase gene CsTPS9 is obtained by PCR amplifications, two before and after which
End is respectively provided with restriction enzyme site Xho1 and BamH1, PCR primer after Xho1 and BamH1 double digestions with also pass through Xho1 and
The new support pBE-GFPST connections of BamH1 double digestions, connection product (pBE-GFPST::CsTPS9) conversion enters tobacco, experiment
As a result show conversion after tobacco there is good fluorescence (Fig. 5), after CsTPS9 albumen is through expression and purification, CsTPS followed by
Enter in the case that terminator removes GFP, check to have arrived by the S-TAG labels inserted in new support in Western blot and weigh
The successful expression (Fig. 6) of histone.
Sequence table
<110>South China Botanical Garden Chinese Academy of Sciences
<120>A kind of green fluorescence protein expression carrier of improvement and its construction method
<160>2
<210>1
<211>26
<212>DNA
<213>Artificial sequence
<400>1
CGCCAAGCTT GCATGCCTGC AGGTCC 26
<210>2
<211>102
<212>DNA
<213>Artificial sequence
<400>2
AATGTCGACG GCCGGCCTCG AGATGGACTA GTAGCTCTAG AGCTGTCCAT GTGCTGGCGT 60
TCGAATTTAG CAGCAGCGGT TTCTTTCATG TCCCCCGTGT TC 102
Claims (2)
1. a kind of construction method of the green fluorescence protein expression carrier of improvement, it is characterised in that comprise the following steps：With carrier
PBE-GFP is template, is entered using upstream primer pBE-GFPST-Hind3-F and downstream primer pBE-GFPST-Sal1-R as primer
Performing PCR is expanded, and amplified production Hind3 and Sal1 double digestions obtain the product after double digestion, are named as intron；By carrier
PBE-GFP Hind3 and Sal1 double digestions, obtain the product after double digestion, are named as digestion carrier, and intron and digestion are carried
Body connection obtains the green fluorescence protein expression carrier of improvement；
The nucleotides sequence of described upstream primer pBE-GFPST-Hind3-F is classified as：
5’-CGCCAAGCTTGCATGCCTGCAGGTCC-3’；
The nucleotides sequence of described downstream primer pBE-GFPST-Sal1-R is classified as:
5’-AATGTCGACGGCCGGCCTCGAGATGGACTAGTAGCTCTAGAGCTGTCCATGTGCTGGCGTTCGAATTTAG
CAGCAGCGGTTTCTTTCATGTCCCCCGTGTTC-3’.
2. a kind of construction method according to described in claim 1 builds the green fluorescence protein expression carrier of the improvement for obtaining.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610942279.4A CN106497953A (en) | 2016-11-01 | 2016-11-01 | A kind of green fluorescence protein expression carrier of improvement and its construction method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610942279.4A CN106497953A (en) | 2016-11-01 | 2016-11-01 | A kind of green fluorescence protein expression carrier of improvement and its construction method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106497953A true CN106497953A (en) | 2017-03-15 |
Family
ID=58321071
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610942279.4A Pending CN106497953A (en) | 2016-11-01 | 2016-11-01 | A kind of green fluorescence protein expression carrier of improvement and its construction method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106497953A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109679994A (en) * | 2018-12-13 | 2019-04-26 | 湖北汇智铭传生物科技股份有限公司 | Pass through the episomal vector and its construction method of tetracycline inducing expression foreign gene |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101240290A (en) * | 2008-02-29 | 2008-08-13 | 杭州师范大学 | Plant transgene expression vector containing green fluorescence protein gene and its construction method and application |
| CN101899470A (en) * | 2010-02-25 | 2010-12-01 | 杭州师范大学 | Method for breeding root-knot nematodes by using transgenic adventitious roots |
| CN102703496A (en) * | 2012-06-05 | 2012-10-03 | 江南大学 | Construction method and use method of pichia pastoris expression vector |
| WO2013071295A2 (en) * | 2011-11-10 | 2013-05-16 | Rutgers, The State University Of New Jersey | Transcript optimized expression enhancement for high-level production of proteins and protein domains |
-
2016
- 2016-11-01 CN CN201610942279.4A patent/CN106497953A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101240290A (en) * | 2008-02-29 | 2008-08-13 | 杭州师范大学 | Plant transgene expression vector containing green fluorescence protein gene and its construction method and application |
| CN101899470A (en) * | 2010-02-25 | 2010-12-01 | 杭州师范大学 | Method for breeding root-knot nematodes by using transgenic adventitious roots |
| WO2013071295A2 (en) * | 2011-11-10 | 2013-05-16 | Rutgers, The State University Of New Jersey | Transcript optimized expression enhancement for high-level production of proteins and protein domains |
| CN102703496A (en) * | 2012-06-05 | 2012-10-03 | 江南大学 | Construction method and use method of pichia pastoris expression vector |
Non-Patent Citations (2)
| Title |
|---|
| HADI KHALIL等: "The role of endogenous and exogenous RasGAP-derived fragment N in protecting cardiomyocytes from peroxynitrite-induced apoptosis", 《FREE RADICAL BIOLOGY AND MEDICINE》 * |
| 朱宽鹏 等: "芪合酶基因Fm-STS 在何首乌毛状根中的过量表达分析", 《现代生物医学进展》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109679994A (en) * | 2018-12-13 | 2019-04-26 | 湖北汇智铭传生物科技股份有限公司 | Pass through the episomal vector and its construction method of tetracycline inducing expression foreign gene |
| CN109679994B (en) * | 2018-12-13 | 2021-02-02 | 湖北汇智铭传生物科技股份有限公司 | Free vector for inducing expression of exogenous gene by tetracycline and construction method thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP6814141B2 (en) | Fungal Genome Modification System and Usage | |
| JP6937740B2 (en) | Genome editing system and usage | |
| US10202619B2 (en) | Compositions and methods directed to CRISPR/Cas genomic engineering systems | |
| CN107223157B (en) | Compositions and methods for helper-strain-mediated modification of fungal genomes | |
| JP2019528797A5 (en) | ||
| US20190292537A1 (en) | Prokaryotic Argonaute Proteins and Uses Thereof | |
| MX2021004455A (en) | Compositions and methods for delivering transgenes. | |
| CN104911199B (en) | Cloned dna molecule method | |
| CN106497953A (en) | A kind of green fluorescence protein expression carrier of improvement and its construction method | |
| CN101899467A (en) | Method for inserting target DNA fragment into vector | |
| CN106086060A (en) | A kind of system and method screening Pichia sp. promoter | |
| Klahre et al. | Extreme heterogeneity of polyadenylation sites in mRNAs encoding chloroplast RNA-binding proteins in Nicotiana plumbaginifolia | |
| JP4665190B2 (en) | Gene transcription regulation method | |
| CN106566840A (en) | Improved pichia pastoris protein expression vector and construction method thereof | |
| CN102268434A (en) | Quick construction method for arabidopsis thaliana artificial miRNA (micro Ribonucleic Acid) gene interference vector | |
| CN104789564A (en) | Promoter and recombinant expression vector as well as application thereof and method for expressing heterologous proteins | |
| FI3690052T3 (en) | Chimeric promoter for use in metabolic engineering | |
| CN109234273A (en) | Primer, kit and cloning process for limit dextrinase gene cloning | |
| CN116286904B (en) | Bovine-derived CRISPR/boCas13a gene editing system, method and application | |
| CN107012146A (en) | The tetrahymena expression vector recombinated based on locus specificity and its structure and application | |
| CN107022570B (en) | A kind of regulated gene expression regulator control system | |
| CN108220314A (en) | DNA segment quickly connect new method and its application of conversion with carrier | |
| CN104059932A (en) | Construction method of single enzyme digestion vector | |
| CN103805624A (en) | Simplified method for constructing TAL effector and TALENs and plasmid | |
| CN118139979A (en) | Enzymes with HEPN domains |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20170315 |
|
| RJ01 | Rejection of invention patent application after publication |