CN105755042B - The fluorescence report system and its construction method of screening and identification circular rna translated protein - Google Patents
The fluorescence report system and its construction method of screening and identification circular rna translated protein Download PDFInfo
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Abstract
The present invention provides a kind of fluorescence report system of screening and identification circular rna translated protein, the nucleotide sequence of the expression frame of the fluorescence report system is as shown in SEQ ID NO:1.The present invention also provides the construction methods of the fluorescence report system.The present invention being capable of Effective selection identification circular rna translated protein.
Description
Technical field
The invention belongs to molecular biology fields, and in particular to a kind of fluorescence of screening and identification circular rna translated protein
Reporting system and its construction method.
Background technique
Circular rna (Circular RNA, circRNA) is that the one kind for being different from conventional linear RNA has specific function
RNA molecule has closed hoop structure, is that objective big circRNA is formed by precursor RNA by shearing and then reverse splicing.Greatly
Partial circular rna is made of exon sequence, has conservative in different species, exists simultaneously tissue and different hairs
Educate the expression specificity in stage.As depth RNA sequencing and the development of scale biology information technology, researcher just really have found
Largely there is the RNA molecule of cyclisation in vivo, ring-type of the cyclisation RNA due to forming closure is very steady in vivo
It is fixed.Since circular rna is insensitive to nuclease, so more more stable than linear rna, this faces circular rna as novel
It has a clear superiority in the development and application of bed diagnostic marker.
For the concrete function of circular rna, there has been no clear, the sayings of only several hypothesis at present: (1) circular rna can be with
MiRNA is inhaled as " sponge " (sponge), inhibits its function；(2) circular rna by base pair complementarity direct regulation and control other
Rna level；(3) circular rna can be in conjunction with protein, the component or regulation that inhibit protein active, raise protein complex
The activity of protein；Also some researchers have reported that circular rna also can be used as the synthesis of the template guided protein of translation, for virus
And the report of artificial synthesized circular rna translated protein is more；For circular rna translated protein in the higher organisms such as the mankind
Discovery it is less, reason may be due to before to circular rna research understanding it is natively few, in addition lack effective scale
Change the tool and method of screening circular rna translation albumen.
Recently research begins to focus on and may play the role of in terms of disease pathology in circRNA.For example, ring-type ANRIL
(cANRIL) be long-chain non-coding RNA ANRIL cyclic annular splicing form, expression in human cell with it is several on the site
A possible SNP for influencing ANRIL splicing is related, is adjustable the level of INK4/ARF and increases the risk of atherosclerosis.This
Item research sufficiently proves that circRNA exists with the generation of disease and is associated with, and can be work perfectly well as disease new biomarker.
In addition, more evidences show that circular rna plays very important effect in the fine tuning of miRNA level, pass through
Competitive binding miRNA carrys out the expression of controlling gene.And then illustrate that circular rna can join with the interaction of disease association miRNA
It is adjusted with disease.
Gene is the nucleotide sequence with hereditary effect, is studied in the function of specific molecular through clonal expression gene pairs
It is of great significance for researcher.In order to make clone gene in host cell great expression it is necessary to according to different realities
It tests purpose and selects different expression systems, construct different expression frames.
Detailed description of the invention
Fig. 1 is the fluorescence report system expression frame of screening and identification circular rna translated protein of the invention.
Fig. 2 is SEQ ID NO:4 of the invention.
Fig. 3 is SEQ ID NO:5 of the invention.
Fig. 4 is the result using fluorescence report screening system identification circular rna translated protein of the invention.
Summary of the invention
It being capable of Effective selection identification ring-type the purpose of the present invention is aiming at the above technical problems to be solved, providing one kind
The fluorescence report system of RNA translated protein.
It is another object of the present invention to provide the construction methods of above-mentioned fluorescence report system.
The present invention also provides the construction methods of above-mentioned fluorescence report system.
In order to achieve the goal above, the present invention provides a kind of fluorescence for screening and identification circular rna translated protein
Reporting system, the nucleotide sequence of the expression frame of the fluorescence report system is as shown in SEQ ID NO:1.
The present invention also provides a kind of construction method of the fluorescence report system of screening and identification circular rna translated protein,
Method includes the following steps:
(1) by full genome chemical synthesis, the Target Nucleotide Sequence as shown in SEQ ID NO:1 is obtained；
(2) by NheI and XhoI, the Target Nucleotide Sequence that step (1) obtains is connected to expression vector
In pcDNA3.1 (+).
As a preferred embodiment, in the step (2), using PCR amplification method design primer when
It waits and introduces NheI and XhoI restriction enzyme site sequence.
It is highly preferred that the sequence of the primer are as follows:
Primer-F0:5 '-GCGCTAGCGAGTTCTAAAATTAAACTA-3 '
Primer-R0:5 '-CGCTCGAGCTCTAAAATTATTCGTTCATGGCTT-3 '.
As a kind of preferred embodiment, the reaction system of the PCR amplification is as follows: PCR is 30 μ l total systems, tool
Body is each 1.5 μ l of 2 × PCR MIX, 15 μ l, 10mM upstream and downstream primer, chemically synthesized 1 μ of gene frame SEQ ID NO:1 template
L supplies 30 μ l systems with sterile deionized water；Reaction condition are as follows: 95 DEG C of 5min initial denaturations, the interior 95 DEG C of 30s denaturation of circulation, 62
DEG C 30s annealing, 72 DEG C of extensions 2min, totally 30 circulations continue to extend 7min for 72 DEG C after PCR reaction cycle, then save for 4 DEG C.
As a kind of preferred embodiment, by the product recovery purifying of the PCR amplification, then to its with NheI,
After XhoI digestion, while pcDNA3.1 (+) carrier is also cut with same restriction endonuclease, then constructs the PCR product after digestion
In eukaryotic expression vector pcDNA3.1 (+) after to digestion.
Method provided by the invention is by using a kind of reading that can make linear rna that target sequence after being cyclized and being cyclized occur
Frame sequence and the construction of strategy of green fluorescent protein (GFP albumen) fusion have gone out can Effective selection identification circular rna translation albumen
The fluorescence report system of matter efficiently solves the problems, such as the screening of organism inner annular RNA translated protein, for find it is new by
The research of the functional protein of circular rna translation provides a kind of efficient, accurate reporting system tool and method.
Specific embodiment
Technical solution of the present invention is described in further detail in the following with reference to the drawings and specific embodiments, but guarantor of the invention
Shield range is not limited to this.As unspecified, agents useful for same, instrument can be obtained by the prior art in the embodiment of the present invention
?.
One, the design of the fluorescence report system expression frame of screening and identification circular rna translated protein
As shown in Fig. 1 and SEQ ID NO:1, the present invention designs the fluorescence report of screening and identification circular rna translated protein
System expression frame includes upstream Frame sequence, circular rna insertion restriction enzyme site, downstream Frame sequence.The base sequence of the frame
Column are as shown in SEQ ID NO:1.Wherein, in the base sequence of the frame, 1~515bp is upstream Frame sequence (bases longs
Total 515bp), 516~539bp be circular rna be inserted into restriction enzyme site sequence (grey mark), be successively kpnI, BamHI,
3 common restriction enzyme sites of EcoRI, for being inserted into the site of test circular rna；540~1260bp is that Frame sequence is swum in downstream
(the total 721bp of bases longs), entire frame is made of 1260 nucleotide.Wherein 253~515bp (263bp) is that green is glimmering
Photoprotein GFP encodes C-terminal nucleotide sequence (two-wire mark)；Wherein 540~990bp (451bp) is green fluorescent protein GFP
It encodes N-terminal nucleotide sequence (single line mark).
Two, the building of the fluorescence report system expression frame of circular rna translated protein
It is obtained according to the fluorescence report system expression frame of above-mentioned circular rna translated protein by full genome chemical synthesis
Target Nucleotide Sequence is obtained, then Frame sequence is connected in expression vector pcDNA3.1 (+) by NheI and XhoI.
Specific embodiment is as follows:
After target framework nucleotide sequence SEQ ID NO:1 is synthesized (Shanghai JaRa biotech firm), using PCR amplification
Method NheI and XhoI restriction enzyme site sequence, the sequence of the primer of design are introduced when design primer are as follows:
Primer-F0:5 '-GCGCTAGCGAGTTCTAAAATTAAACTA-3 ' (SEQ ID NO:2)
Primer-R0:5 '-CGCTCGAGCTCTAAAATTATTCGTTCATGGCTT-3 ' (SEQ ID NO:3)
The reaction system of PCR amplification is as follows: PCR is 30 μ l total systems, specifically 2 × PCR MIX 15 μ l, 10mM or more
Each 1.5 μ l of primer is swum, chemically synthesized 1 μ l of gene frame SEQ ID NO.1 template supplies 30 μ l bodies with sterile deionized water
System；Reaction condition are as follows: 95 DEG C of 5min initial denaturations, the interior 95 DEG C of 30s denaturation of circulation, 62 DEG C of 30s annealing, 72 DEG C of extension 2min, altogether
30 recycle, and continue to extend 7min for 72 DEG C after PCR reaction cycle, then 4 DEG C of preservations.
PCR product is by using plastic recovery kit recovery purifying, then after PCR product NheI, XhoI digestion, simultaneously
PcDNA3.1 (+) carrier is also cut with same restriction endonuclease, then by this framework establishment to eukaryotic expression vector pcDNA3.1 (+)
In, the carrier built names PcDNA-cirGFP-Report.
Three, the use of the fluorescence report system of screening and identification circular rna translated protein
It is described as follows using circular rna hsa_circ_0041407 as case:
1. circBase database (http://circrna.org/cgi-bin/listsearch.cgi) find ring-type
The sequence (referring to SEQ ID NO:4) of RNA hsa_circ_0041407, total sequence length are 1059bp；
2. by target sequence in ORF Finder (http://www.ncbi.nlm.nih.gov/gorf/orfig.cgi)
The reading frame sequence of circular rna translated protein, grey mark are predicted in online software；Underlining " ATG " and " TAA " are respectively
For translation initiation codon and terminator codon (as shown in Fig. 2 and SEQ ID NO:4)；
3. the sequence (front grey underlining in Fig. 2 sequence) before terminator codon is transferred to the end of sequence,
The sequence of generation is as shown in SEQ ID NO:5 and Fig. 3；
4. the sequence SEQ ID NO:5 after transformation is obtained target polynucleotide piece by the chemically synthesized method of full genome
Section；
5. the sequence SEQ ID NO:5 after being changed circular rna by BamHI and EcoRI restriction enzyme site is connected to ring-type
In the fluorescence report carrier PcDNA-cirGFP-Report of RNA translated protein.
The specific embodiment of step 5 is as follows:
After target framework nucleotide sequence SEQ ID NO:5 is synthesized (Shanghai JaRa biotech firm), using PCR amplification
Method BamHI and EcoRI restriction enzyme site sequence, the sequence of the primer of design are introduced when design primer are as follows:
Primer-F1:5 '-CCGGATCCTAAATTTGCAATTTTATATTT-3 ' (SEQ ID NO:6)
Primer-R1:5 '-GCGAATTCAAAAAATGGGATGCAAAAAAAAAAAAAAGGCGG-3 ' (SEQ ID NO:
7)
The reaction system of PCR amplification is as follows: PCR is 30 μ l total systems: being specifically 2 × PCR MIX 15 μ l, 10mM or more
Each 1.5 μ l of primer is swum, chemically synthesized 1 μ l of genetic fragment SEQ ID NO:5 supplies 30 μ l systems with sterile deionized water；Instead
Answer condition are as follows: 95 DEG C of 5min initial denaturations, 95 DEG C of 30s denaturation in circulation, 56 DEG C of 30s annealing, 72 DEG C of extension 1min, totally 30
It recycles, continues to extend 7min for 72 DEG C after PCR reaction cycle, then 4 DEG C of preservations.
PCR product is by using plastic recovery kit recovery purifying, then after PCR product BamHI and EcoRI digestion,
Report carrier PcDNA-cirGFP-Report is also cut with same restriction endonuclease simultaneously, then by this SEQ ID NO:5 sequence structure
It is built in the fluorescence report carrier PcDNA-cirGFP-Report of circular rna translated protein of the invention, the carrier built
It is named as PcDNA-cirGFP-Report-1059.
6. cell transfecting
Above-mentioned PcDNA-cirGFP-Report-1059 carrier is transfected into HEK293 cell with lipo2000 transfection reagent
In (with PcDNA-cirGFP-Report empty carrier be control), 1 μ g/ml of carrier transfection concentrations, then cultivate 48 hours after use
Fluorescence microscope is taken pictures, and the expression of green fluorescence GFP albumen is observed.Fluorescence takes pictures result referring to fig. 4.Fluorescence photo result
Display PcDNA-cirGFP-Report-1059 carrier transfection groups of cells has apparent green fluorescence to occur, PcDNA-cirGFP-
Report empty vector control group does not have green fluorescence appearance, illustrates the fluorescence report system of circular rna translated protein of the invention
System PcDNA-cirGFP-Report carrier can effectively make effectively circular rna translated protein after being connected into circular rna
Visualize selective mechanisms.
Claims (6)
1. a kind of fluorescence report system of screening and identification circular rna translated protein, which is characterized in that the fluorescence report system
Expression frame nucleotide sequence as shown in SEQ ID NO:1.
2. the construction method of fluorescence report system described in claim 1, the described method comprises the following steps:
(1) by full genome chemical synthesis, the Target Nucleotide Sequence as shown in SEQ ID NO:1 is obtained；
(2) by NheI and XhoI, the Target Nucleotide Sequence that step (1) obtains is connected to expression vector pcDNA3.1
In (+).
3. according to the method described in claim 2, it is characterized in that, the method using PCR amplification is being set in the step (2)
NheI and XhoI restriction enzyme site sequence is introduced when counting primer.
4. according to the method described in claim 3, it is characterized in that, the sequence of the primer are as follows:
Primer-F0:5 '-GCGCTAGCGAGTTCTAAAATTAAACTA-3 '
Primer-R0:5 '-CGCTCGAGCTCTAAAATTATTCGTTCATGGCTT-3 '.
5. according to the method described in claim 3, it is characterized in that, the reaction system of the PCR amplification is as follows: PCR is 30 μ l
Total system, 15 μ l, 10mM upstream and downstream primer of specifically 2 × PCR MIX each 1.5 μ l, chemically synthesized gene frame SEQ ID
1 μ l of NO:1 template supplies 30 μ l systems with sterile deionized water；Reaction condition are as follows: 95 DEG C of 5min initial denaturations, circulation are 95 DEG C interior
30s denaturation, 62 DEG C of 30s annealing, 72 DEG C of extension 2min, totally 30 recycle, and continue to extend 7min for 72 DEG C after PCR reaction cycle, so
4 DEG C of preservations afterwards.
6. the method according to claim 3 or 5, which is characterized in that by the product recovery purifying of the PCR amplification, then
After using NheI, XhoI digestion to it, while pcDNA3.1 (+) carrier is also cut with same restriction endonuclease, then will be after digestion
PCR product is building up in the eukaryotic expression vector pcDNA3.1 (+) after digestion.
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