CN105779497A - Dual-fluorescence protein report system for verifying activity of Internal Ribosome Entry Site (IRES) - Google Patents
Dual-fluorescence protein report system for verifying activity of Internal Ribosome Entry Site (IRES) Download PDFInfo
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- CN105779497A CN105779497A CN201610178729.7A CN201610178729A CN105779497A CN 105779497 A CN105779497 A CN 105779497A CN 201610178729 A CN201610178729 A CN 201610178729A CN 105779497 A CN105779497 A CN 105779497A
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Abstract
The invention provides a dual-fluorescence protein report system for verifying activity of an Internal Ribosome Entry Site (IRES). A nucleotide sequence of an expression cassette of the dual-fluorescence protein report system is shown as SEQ ID NO: 1. The invention further provides a construction method of the dual-fluorescence protein report system. The dual-fluorescence protein report system for verifying the activity of the IRES can effectively detect the activity of the IRES.
Description
Technical field
The invention belongs to biology field, be specifically related to double; two fluorescin reporting systems and the construction method thereof of a kind of validation of kernel sugar body insertion point IRES activity.
Background technology
Internal ribosome entry site (InternalRibosomeEntrySite, IRES) is one section of nucleotide sequence, it is possible to makes protein translation initiate and does not rely on messenger RNA 5 ' cap sequence, it is possible to initial RNA starts translated protein from centre.Generally, eukaryotic translation is from the 5 ' ends of mRNA, because translation initiation need to rely on the 5 ' cap sequences held.Internal ribosome entry site is usually located at the 5 ' untranslated regions (UTR) of rna virus cdna group, and the translation of such virus protein just can not rely on 5 ' cap sequences.Along with deepening continuously of research have also discovered many genes in higher organism and there is natural IRES bioactive sequence, the genome of the mankind finds successively verify there is IRES sequence inside a lot of gene RNAs, and there is important biologic activity, the IRES bioactive sequence such as existed at common proto-oncogene C-MYC gene internal, there is also IRES bioactive sequence inside important tumor suppressor gene TP53.So would be likely to occur the IRES bioactive sequence not much being found in eukaryote body, the qualification of IRES and activity research are contributed to the basic law of more deep announcement bioscience.
Accompanying drawing explanation
Double; two fluorescin reporting systems that Fig. 1 is the validation of kernel sugar body insertion point IRES sequence active of the present invention express framework.
Fig. 2 is the result of the double; two fluorescin reporting systems detection IRES sequence active utilizing the present invention.
Summary of the invention
It is an object of the invention to for solve the technical problem that above, it is provided that a kind of can effectively validation of kernel sugar body insertion point IRES activity double; two fluorescin reporting systems.
Further object is that the construction method that above-mentioned pair of fluorescin reporting system is provided.
In order to realize object above, the invention provides double; two fluorescin reporting systems of a kind of validation of kernel sugar body insertion point IRES activity, the nucleotide sequence expressing framework of described pair of fluorescin reporting system is such as shown in SEQIDNO:1.
Present invention also offers the construction method of a kind of double; two fluorescin reporting systems for validation of kernel sugar body insertion point IRES activity, the method comprises the following steps:
(1) by full genome chemosynthesis, it is thus achieved that the Target Nucleotide Sequence as shown in SEQIDNO:1；
(2) by NheI and XhoI, the described Target Nucleotide Sequence that step (1) obtains is connected to expression vector pcDNA3.1 (+) in.
As one preferred embodiment, in described step (2), the method for pcr amplification is adopted to introduce NheI and XhoI restriction enzyme site sequence when design primer.
It is highly preferred that the sequence of described primer is:
Primer-F0:5 ' GCGCTAGCATGAAGGGCGAGGAGGATAAC-3 ' (SEQIDNO:3)
Primer-R0:5 ' CGCTCGAGTTACTTGTACAGCTCGTCCATG-3 ' (SEQIDNO:4)
As a kind of preferred embodiment, the reaction system of described pcr amplification is as follows: PCR is the 50 total systems of μ l: specifically 2 × PCRMIX15 μ l, the each 2 μ l of 10mM upstream and downstream primer, the gene framework SEQIDNO.1 template 1 μ l of chemosynthesis, supply 50 μ l systems with sterilizing deionized water；Reaction condition is: 95 DEG C of 5min denaturations, circulates interior 95 DEG C of 30s degeneration, 60 DEG C of 30s annealing, and 72 DEG C extend 1min30s, totally 35 circulations, and after PCR reaction cycle, 72 DEG C are continued to extend 7min, then 4 DEG C of preservations.
As a kind of preferred embodiment, the product of described pcr amplification is reclaimed purification, then to it with after NheI, XhoI enzyme action, simultaneously pcDNA3.1 (+) carrier also cuts with same restriction endonuclease, then the PCR primer after enzyme action is building up to the eukaryotic expression vector pcDNA3.1 after enzyme action (+) in.
The present invention devises a kind of expression framework connected by mCherry red fluorescent protein and GFP green fluorescent protein, and red fluorescent protein leans on 5 ' cap sequence initiation of translation.If the nucleotide sequence of test has IRES activity, then green fluorescence will send fluorescence in vain as reporter fluorescence；If target sequence does not have IRES activity, green fluorescence will do not had to occur.Finally can carry out effective Visual retrieval checking according to the fluorescence of green fluorescent protein internal ribosomal entry site IRES activity.
Detailed description of the invention
Below in conjunction with the drawings and specific embodiments, technical scheme is described in further detail, but protection scope of the present invention is not limited to this.As unspecified, in the embodiment of the present invention, agents useful for same, instrument all can be obtained by prior art.
One, double; two fluorescin reporting systems of validation of kernel sugar body insertion point IRES activity express the design of framework
As shown in Fig. 1 and SEQIDNO:1, double; two fluorescin reporting systems of the validation of kernel sugar body insertion point IRES activity of present invention design are expressed framework and are included MCHERRY red fluorescent protein sequence, restriction enzyme site sequence, GFP green fluorescent protein sequence.The base sequence of this framework is such as shown in SEQIDNO:1.Wherein, in the base sequence of this framework, 1～708bp is that (single line underscore marks mCherry red fluorescent protein sequence, bases longs is 708bp altogether), 709～732bp is restriction enzyme site sequence (the Lycoperdon polymorphum Vitt mark inserted, bases longs is 24bp altogether), it is KPNI, BAMHI, ECORI3 conventional restriction enzyme site successively, for inserting the site of test IRES；733～1452bp is GFP green fluorescent protein sequence (two-wire underscore marks, and bases longs is 720bp altogether), and whole framework is made up of 1452 nucleotide.
Two, double; two fluorescin reporting systems of validation of kernel sugar body insertion point IRES activity express the structure of framework
Double; two fluorescin reporting systems according to above-mentioned validation of kernel sugar body insertion point IRES activity express framework, by full genome chemosynthesis, obtain Target Nucleotide Sequence, then pass through NheI and XhoI Frame sequence is connected to expression vector pcDNA3.1 (+) in.
Specific embodiment is as follows:
After target framework nucleotide sequence SEQIDNO:1 is synthesized (Shanghai JaRa biotech firm), adopting the method for pcr amplification to introduce NheI and XhoI restriction enzyme site sequence when design primer, the sequence of the primer of design is:
Primer-F0:5 ' GCGCTAGCATGAAGGGCGAGGAGGATAAC-3 ' (SEQIDNO:3)
Primer-R0:5 ' CGCTCGAGTTACTTGTACAGCTCGTCCATG-3 ' (SEQIDNO:4)
The reaction system of pcr amplification is as follows: PCR is the 50 total systems of μ l: the specifically each 2 μ l of 2 × PCRMIX15 μ l, 10mM upstream and downstream primer, the gene framework SEQIDNO.1 template 1 μ l of chemosynthesis, supplies 50 μ l systems with sterilizing deionized water；Reaction condition is: 95 DEG C of 5min denaturations, circulates interior 95 DEG C of 30s degeneration, 60 DEG C of 30s annealing, and 72 DEG C extend 1min30s, totally 35 circulations, and after PCR reaction cycle, 72 DEG C are continued to extend 7min, then 4 DEG C of preservations.
PCR primer reclaims purification by using glue to reclaim test kit, then PCR primer is with after NheI, XhoI enzyme action, simultaneously pcDNA3.1 (+) carrier also cuts with same restriction endonuclease, then by this framework establishment to eukaryotic expression vector pcDNA3.1 (+) in, the carrier that builds name P-RGFP-IRES-Report.
Three, the use of double; two fluorescin reporting systems of validation of kernel sugar body insertion point IRES activity
Arranging with the nucleotides sequence with IRES activity having verified that describes as follows into case:
1. with psin-EF2-IRES-puro carrier for pcr template, adopting the method amplification of PCR to obtain IRES positive control nucleotide sequence SEQIDNO:2, introduce BamHI and EcoRI restriction enzyme site sequence when design primer, the sequence of the primer of design is:
Primer-F1:5 ' CCGGATCCGCCCCTCTCCCTCCCCCCCCCCTAACGT-3 ' (SEQIDNO:5)
Primer-R1:5 ' GCGAATTCTTTTTCAAAGGAAAACCACGTC-3 ' (SEQIDNO:6)
The reaction system of pcr amplification is as follows: PCR is the 30 total systems of μ l: the specifically each 1.5 μ l of 2 × PCRMIX15 μ l, 10mM upstream and downstream primer, the genetic fragment SEQIDNO:21 μ l of chemosynthesis, supplies 30 μ l systems with sterilizing deionized water；Reaction condition is: 95 DEG C of 5min denaturations, circulates interior 95 DEG C of 30s degeneration, 58 DEG C of 30s annealing, and 72 DEG C extend 40s, totally 30 circulations, and after PCR reaction cycle, 72 DEG C are continued to extend 7min, then 4 DEG C of preservations.
PCR primer reclaims purification by using glue to reclaim test kit, then after PCR primer BamHI and EcoRI enzyme action, report carrier P-RGFP-IRES-Report is also with same restriction endonuclease cutting simultaneously, then by double; two fluorescin reporting system carrier P-RGFP-IRES-Report of this SEQIDNO:2 sequence construct to internal ribosome insertion point IRES activity, the carrier built names P-RGFP-IRES-Report-564.
2. cell transfecting
nullAbove-mentioned P-RGFP-IRES-Report-564 carrier lipo2000 transfection reagent is transfected in HEK293 cell (with P-RGFP-IRES-Report empty carrier for comparison)，Carrier transfection concentrations 1 μ g/ml，Turn and after then cultivating 48 hours, adopt fluorescence microscope to take pictures，Observe the expression of green fluorescence GFP albumen，Fluorescence takes pictures result referring to Fig. 2，Fluorescence photo result display P-RGFP-IRES-Report-564 vector-transfected cell group has obvious green fluorescence to occur，P-RGFP-IRES-Report empty vector control group does not have green fluorescence to occur，This illustrates that double; two fluorescin reporting system P-RGFP-IRES-Report carriers active for internal ribosome insertion point IRES of the present invention are after being connected into activity IRES sequence，The activity of IRES can be made effective Visual retrieval.
Claims (6)
1. double; two fluorescin reporting systems of a validation of kernel sugar body insertion point IRES activity, it is characterised in that the nucleotide sequence expressing framework of described pair of fluorescin reporting system is such as shown in SEQIDNO:1.
2. the construction method of double; two fluorescin reporting systems described in claim 1, said method comprising the steps of:
(1) by full genome chemosynthesis, it is thus achieved that the Target Nucleotide Sequence as shown in SEQIDNO:1；
(2) by NheI and XhoI, the described Target Nucleotide Sequence that step (1) obtains is connected to expression vector pcDNA3.1 (+) in.
3. method according to claim 2, it is characterised in that in described step (2), adopts the method for pcr amplification to introduce NheI and XhoI restriction enzyme site sequence when design primer.
4. method according to claim 3, it is characterised in that the sequence of described primer is:
Primer-F0:5 ' GCGCTAGCATGAAGGGCGAGGAGGATAAC-3 '
Primer-R0:5 ' CGCTCGAGTTACTTGTACAGCTCGTCCATG-3 '.
5. method according to claim 3, it is characterized in that, the reaction system of described pcr amplification is as follows: PCR is the 50 total systems of μ l: specifically 2 × PCRMIX15 μ l, the each 2 μ l of 10mM upstream and downstream primer, the gene framework SEQIDNO.1 template 1 μ l of chemosynthesis, supplies 50 μ l systems with sterilizing deionized water；Reaction condition is: 95 DEG C of 5min denaturations, circulates interior 95 DEG C of 30s degeneration, 60 DEG C of 30s annealing, and 72 DEG C extend 1min30s, totally 35 circulations, and after PCR reaction cycle, 72 DEG C are continued to extend 7min, then 4 DEG C of preservations.
6. the method according to any one of claim 3 to 5, it is characterized in that, the product of described pcr amplification is reclaimed purification, then to it with after NheI, XhoI enzyme action, simultaneously pcDNA3.1 (+) carrier also cuts with same restriction endonuclease, then the PCR primer after enzyme action is building up to the eukaryotic expression vector pcDNA3.1 after enzyme action (+) in.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102634538A (en) * | 2012-05-07 | 2012-08-15 | 扬州大学 | Red fluorescence protein yeast cell expression vector and construction method thereof |
| CN104593512A (en) * | 2015-02-04 | 2015-05-06 | 中国人民解放军第三军医大学第一附属医院 | Application of bifluorescence reporting system in tumor stem cell targeted drug screening and method thereof |
| CN104593417A (en) * | 2015-02-04 | 2015-05-06 | 江苏省农业科学院 | Xylanase intestinal directional expression vector and cell line thereof |
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2016
- 2016-03-25 CN CN201610178729.7A patent/CN105779497A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102634538A (en) * | 2012-05-07 | 2012-08-15 | 扬州大学 | Red fluorescence protein yeast cell expression vector and construction method thereof |
| CN104593512A (en) * | 2015-02-04 | 2015-05-06 | 中国人民解放军第三军医大学第一附属医院 | Application of bifluorescence reporting system in tumor stem cell targeted drug screening and method thereof |
| CN104593417A (en) * | 2015-02-04 | 2015-05-06 | 江苏省农业科学院 | Xylanase intestinal directional expression vector and cell line thereof |
Non-Patent Citations (4)
| Title |
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| ARUN VENKATESAN ET AL.: "Novel Fluorescence-Based Screen To Identify Small Synthetic Internal Ribosome Entry Site Elements", 《MOLECULAR AND CELLULAR BIOLOGY》 * |
| GAGOSKI D ET AL.: "Cell-free gateway cloning vector C-term mcherry myc-tag pCellFree_G08, complete sequence，GenBank: KJ541672.1", 《GENBANK》 * |
| MICHEL U ET AL.: "Cloning vector pAAV-9(5)-CMV-EGFP-CytbAS, complete sequence，GenBank: KT343252.1", 《GENBANK》 * |
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