CN106591367B - A method of it obtains in vivo and purifies a large amount of purpose LncRNA - Google Patents
A method of it obtains in vivo and purifies a large amount of purpose LncRNA Download PDFInfo
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- CN106591367B CN106591367B CN201710040358.0A CN201710040358A CN106591367B CN 106591367 B CN106591367 B CN 106591367B CN 201710040358 A CN201710040358 A CN 201710040358A CN 106591367 B CN106591367 B CN 106591367B
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Abstract
The present invention relates to the methods that one kind obtains in vivo and purifies a large amount of purpose LncRNA.The present invention identifies base in 3 ' tip designs PPR protein-specific of LncRNA gene order, PPR albumen and LncRNA are overexpressed plasmid co-transfection cell, make PPR and LncRNA great expression in the cell, then PPR albumen is enriched with by Anti-Flag antibody, and then indirect enrichment LncRNA, it realizes and successfully obtains and purify a large amount of purpose LncRNA in vivo.The present invention can obviously simplify is transcribed in vitro the step of obtaining purpose LncRNA in the prior art, and economical and convenient, and feasibility is strong, convenient for the research of LncRNA related experiment, provides brand-new thinking in the study on mechanism field of LncRNA.
Description
Technical field
The present invention relates to field of biotechnology, obtain in vivo specifically, being related to one kind and purify a large amount of purposes
The method of LncRNA.
Background technique
Currently, the research of long-chain non-coding RNA (LncRNA) is very extensive.LncRNA is proved in the occurrence and development of tumour
In play a key effect, and have tissue specificity and Space-time speciality.When studying the functional mechanism of LncRNA, Duo Shuoyan
Study carefully personnel to need to obtain a large amount of LncRNA in vitro, then be proved between LncRNA and nucleic acid and albumen by experiment in vitro
Interaction, thus study its cell and tissue in functional characteristics.Pass through corresponding DNA profiling, RNA transcriptase, NTP
And referred to as " method is transcribed in vitro " in other corresponding reaction conditions, the method for largely obtaining LncRNA in vitro.By turning in vitro
The method that record method obtains LncRNA is the method for the acquisition LncRNA of current mainstream, but this method has its defect: one, obtaining in vitro
The LncRNA obtained is stringent to experimental condition research: obtaining LncRNA by transcription in vitro, transcription is usually imperfect, and due to body
It is difficult to outside and thoroughly removes RNA enzyme, if the LncRNA length transcribed out is longer, can usually degrade in transcription, thus shadow
Ring subsequent experiment；Two, the experimental cost being transcribed in vitro is larger: the related reagent of in-vitro transcription is usually expensive；Three, external
Transcription subsequent experimental difficulty is big: the LncRNA of acquisition is transcribed in vitro, the experiment of follow-up study is generally also completed in vitro, is such as studied
LncRNA and albumen dependent interaction need to interact with LncRNA again after protein cleavage, bright by Mass Spectrometer Method or WB
The albumen really to interact, the condition of experiment requires strictly, costly.Four, relevant subsequent due to being transcribed in vitro in LncRNA
Research is also completed in vitro, and the LncRNA of acquisition can not be restored well due to carrying out nature processing and synthesis not in cell
True intracellular environment frequently can lead to a large amount of false positive results.
In conclusion a kind of economical and convenient is needed, the strong acquisition of feasibility and the method for purifying a large amount of purpose LncRNA.
PPR (pentatricopeptide repeat) albumen has sequence-specific recognition mode to single stranded RNA, specifically
Mode are as follows: by number, equal repetitive unit is not connected in series PPR albumen, and each classics PPR repetitive unit contains 35 amino acid,
It can specifically identify a RNA base, and the 5th and the 35th amino acids play during RNA specific recognition
Important function, referred to as password amino acid.
The method about application PPR albumen efficiently concentrating purpose LncRNA in vivo has not been reported at present.
Summary of the invention
The purpose of the present invention is aiming at the shortcomings in the prior art, provide one kind to obtain a large amount of purpose LncRNA's in vivo
Method.
To achieve the above object, the technical solution adopted by the present invention is that:
A method of it obtains in vivo and purifies a large amount of purpose LncRNA, comprising the following steps:
A) over-express vector of the PPR albumen with label protein and LncRNA over-express vector cotransfection tool is thin
Born of the same parents, the LncRNA gene order downstream of the LncRNA over-express vector have the specific recognition base sequence of PPR albumen；
B) vehicles cells are cultivated；
C) vehicles cells, enrichment and purifying PPR albumen are cracked, and then is enriched with indirectly and purifying obtains LncRNA.
The amino acid sequence of the PPR albumen is as shown in SEQ ID NO.1.
The gene order of the PPR albumen is as shown in SEQ ID NO.3.
The construction method of the PPR protein overexpression carrier are as follows: the gene order of PPR albumen is inserted into pcDNA3.1
Between the site HindIII and EcoRI of carrier, and in the coded sequence of the gene sequence upstream of PPR albumen insertion label protein.
The specific recognition base sequence of the PPR albumen is as shown in SEQ ID NO.2.
The LncRNA over-express vector is selected from one of the following:
A) gene order of H19 shown in SEQ ID NO.4 pcDNA3.1-H19: is inserted into pcDNA3.1 carrier
Between the site HindIII and EcoRI；
B) pcDNA3.1-HOTAIR: the gene order of HOTAIR shown in SEQ ID NO.5 is inserted into pcDNA3.1 and is carried
Between the site HindIII and EcoRI of body；
C) gene order of BANCR shown in SEQ ID NO.6 pcDNA3.1-BANCR: is inserted into pcDNA3.1 carrier
The site HindIII and EcoRI between.
The vehicles cells are HEK-293T cells.
The label protein is Flag label.
Step c) is to be enriched with PPR albumen by the antibody of label protein.
Each concrete operations in step c) use RNase inhibitor.
The invention has the advantages that:
1, successful design of the present invention is in the method for obtaining in vivo and purifying a large amount of purpose LncRNA.By in LncRNA base
Because 3 ' tip designs PPR protein-specific of sequence identifies base, by PPR albumen and LncRNA cotransfection cells, make PPR and
LncRNA great expression in the cell.Then PPR albumen is enriched with by Anti-Flag antibody, since PPR albumen can specificity
It identifies LncRNA, can successfully obtain in vivo and purify a large amount of mesh to realize with indirect enrichment LncRNA by this method
LncRNA.The present invention can obviously simplify the step of in-vitro transcription obtains purpose LncRNA, and economical and convenient, and feasibility is strong,
The research method that LncRNA related experiment can largely be simplified provides fine in the study on mechanism field of LncRNA
New Research Thinking；
2, the high expression of PPR albumen may be implemented in the PPR over-express vector that the present invention designs, and then can be enriched with more
LncRNA；
3, the PPR protein-specific identification base sequence for the LncRNA that the present invention designs can be identified by PPR albumen height
And combination, it ensure that the high-recovery of LncRNA；
4, in a specific embodiment of the present invention, the over-express vector of the LncRNA of design can realize the high table of LncRNA
It reaches, is further ensured that and can get a large amount of LncRNA.
Detailed description of the invention
The expression of Fig. 1 .PPR albumen.
The expression of Fig. 2 .H19, HOTAIR, BANCR.***P<0.01.
Specific embodiment
It elaborates with reference to the accompanying drawing to specific embodiment provided by the invention.
Embodiment 1
One, material and method
1. cell line culture
Using HEK-293T vehicles cells (purchase is in ACTT company) in the test, with DMEM culture medium, (purchase is in GIBCO
Company), 10% fetal calf serum (purchase is in HyClone company), 1% dual anti-mixed-culture medium cultivates (37 in cell incubator
DEG C, 5%CO2)。
2.qRT-RCR experiment
The reagent TransStart Green qPCRSuperMix that qRT-RCR is used in the test is bought in the full formula in Beijing
King Company, detecting instrument 7900HT Fast Real-Time PCR System are purchased from Life company.Reverse transcription makes in the test
Kit PrimeScriptTMRT reagent Kit is bought in Takara company.Three LncRNA points in the test
It Wei not H19, HOTAIR and BANCR.The PCR detection primer design of three kinds of LncRNA is as follows: H19:S-
AGGAATCGGCTCTGGAAGG (SEQ ID NO.7), A-GCGTAATGGAATGCTTGAAGG (SEQ ID NO.8)；HOTAIR:
S-AAGAATTACAAGGAAGCGAAGG (SEQ ID NO.9), A-TAATAGCAGGAGGAAGTTCAGG (SEQ ID NO.10)；
BANCR:S-TTCTTGTAGGGTCTGGATTGG (SEQ ID NO.11), A-TGAACTGGGAAACTGTTGAAAC (SEQ ID
NO.12)。
3. plasmid construction
Constructed in the test with Flag label PPR albumen (SEQ ID NO.1,26.7kilodaltons, wherein
LRVAPT and ELTYRRVVEY plays a protective role to triangular shape pentapeptide structure) overexpression plasmid (pcDNA3.1-PPR, in
Beijing Jin Weizhi company building).The overexpression plasmid (carrier pcDNA3.1) of H19, HOTAIR and BANCR, and set behind
Count the base sequence (SEQ ID NO.2) of PPR protein-specific identification.In laboratory by PCR, digestion is connected, and converts, and is applied
Screen choosing verifies three kinds of LncRNA after being sequenced and is overexpressed plasmid construction success.
Each overexpression specific construction method of plasmid is as follows:
(1) pcDNA3.1-PPR:pcDNA3.1 is purchased from Beijing Jin Weizhi company, then by the gene order of PPR albumen
(SEQ ID NO.3) is inserted between the site HindIII-EcoRI of pcDNA3.1, and in the gene sequence upstream of PPR albumen
It is inserted into the coded sequence (SEQ ID NO.13) of Flag label, the amino acid sequence of Flag label is as shown in SEQ ID NO.14.
(2) pcDNA3.1-H19:pcDNA3.1 is purchased from Beijing Jin Weizhi company, then by the gene order of H19 (SEQ ID
NO.4 it) is inserted between the site HindIII-EcoRI of pcDNA3.1.
(3) pcDNA3.1-HOTAIR:pcDNA3.1 is purchased from Beijing Jin Weizhi company, then by the gene order of HOTAIR
(SEQ ID NO.5) is inserted between the site HindIII-EcoRI of pcDNA3.1.
(4) pcDNA3.1-BANCR:pcDNA3.1 is purchased from Beijing Jin Weizhi company, then by the gene order of BANCR
(SEQ ID NO.6) is inserted between the site HindIII-EcoRI of pcDNA3.1.
4. cell transfecting
Over-express vector PPR, H19, HOTAIR and BANCR are used into DNA transfection reagent (Biotool, B35101) wink respectively
It rotates into HEK-293T cell, cultivates 48 hours.Experimental group: PPR+H19, PPR+HOTAIR, PPR+BANCR；Control group: PPR.
Transfection reagent (purchase is in Biotool company) uses step and dosage to specifications.
5. cell cracking and antibody incubation
With 1%NP40 by HEK-293T cell cracking, be added protease inhibitors (purchase in Biotool company,
B14001), inhibitors of phosphatases (Biotool company, B15001), and RNA enzyme inhibition is added in each step later
Agent (Shanghai Sangon Biotech Company, B54004) is to prevent the degradation of RNA.Using Anti-Flag Affinity Gel, (purchase is in Biotool
Company) enrichment PPR albumen, before albumen sample-loading buffer is added, every group stay 3 μ l precipitated liquids for LncRNA reverse transcription with
PCR。
6.SDS-PAGE electrophoresis and Coomassie brilliant blue dyeing
Albumen sample-loading buffer will be added in above-mentioned precipitating, boil 10min, 10 μ l of supernatant is taken to carry out SDS-PAGE after centrifugation
Electrophoresis (80V, 120min).Then SDS-PAGE glue is subjected to Coomassie brilliant blue dyeing with the expression of clear PPR albumen.
Two, experimental result
1, the expression of PPR albumen
See that Fig. 1, PPR albumen have apparent high expression in experimental group and control group.Should the result shows that, PPR albumen mistake
Expression vector Successful transfection HEK-293T cell, and great expression wherein.
2, LncRNA:H19, HOTAIR, BANCR expression
See Fig. 2, pass through the method for qRT-PCR, it was demonstrated that the expression quantity of three kinds of LncRNA is apparently higher than control in experimental group
Group.The test result shows intracellular in HEK-293T, and the PPR albumen of overexpression can be held with specific recognition 3 ' with identification
The LncRNA of base, LncRNA are by efficiently concentrating and purifying, in obtaining a large amount of LncRNA in vitro.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
Member, under the premise of not departing from the method for the present invention, can also make several improvement and supplement, these are improved and supplement also should be regarded as
Protection scope of the present invention.
SEQUENCE LISTING
<110>Second Military Medical University, PLA
<120>a kind of method for obtaining in vivo and purifying a large amount of purpose LncRNA
<130> /
<160> 14
<170> PatentIn version 3.3
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gcaggcagtg gggaactctg actcgcctgt gctctggagc ttgatccgaa agcttccaca 180
gtgaggactg ctccgtgggg gtaagagagc accaggcact gaggcctggg agttccacag 240
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tccatgagga aaagggaaaa tctagggaac gggagtacag agagaataat ccgggtccta 840
gctcgccaca tgaacgccca gagaacgctg gaaaaacctg agcgggtgcc ggggcagcac 900
ccggctcggg tcagccactg ccccacaccg ggcccaccaa gccccgcccc tcgcggccac 960
cggggcttcc ttgctcttct tatcatctcc atctttatga tgaggcttgt taacaagacc 1020
agagagctgg ccaagcacct ctatctcagc cgcgcccgct cagccgagca gcggtcggtg 1080
gggggactgg gaggcgctaa ttaattgatt cctttggact gtaaaatatg gcggcgtcta 1140
cacggaaccc atggactcat aaacaatata tctgttgggc gtgagtgcac tgtctctcaa 1200
ataatttttc cataggcaaa tgtcagaggg ttctggattt ttagttgcta aggaaagatc 1260
caaatgggac caattttagg aggcccaaac agagtccgtt cagtgtcaga aaatgcttcc 1320
ccaaaggggt tgggagtgtg ttttgttgga aaaaagcttg ggttatagga aagcctttcc 1380
ctgctacttg tgtagaccca gcccaattta agaattacaa ggaagcgaag gggttgtgta 1440
ggccggaagc ctctctgtcc cggctggatg caggggactt gagctgctcc ggaatttgag 1500
aggaacatag aagcaaaggt ccagcctttg cttcgtgctg attcctagac ttaagattca 1560
aaaacaaatt tttaaaagtg aaaccagccc tagcctttgg aagctcttga aggttcagca 1620
cccacccagg aatccacctg cctgttacac gcctctccaa gacacagtgg caccgctttt 1680
ctaactggca gcacagagca actctataat atgcttatat taggtctaga agaatgcatc 1740
ttgagacaca tgggtaacct aattatataa tgcttgttcc atacaggagt gattatgcag 1800
tgggaccctg ctgcaaacgg gactttgcac tctaaatata gaccccagct tgggacaaaa 1860
gttgcagtag aaaaatagac ataggagaac acttaaataa gtgatgcatg tagacacaga 1920
aggggtattt aaaagacaga aataatagaa gtacagaaga acagaaaaaa aatcagcaga 1980
tggagattac cattcccaat gcctgaactt cctcctgcta ttaagattgc tagagaattg 2040
tgtcttaaac agttcatgaa cccagaagaa tgcaatttca atgtatttag tacacacaca 2100
gtatgtatat aaacacaact cacagaatat attttccata cattgggtag gtatgcactt 2160
tgtgtatata taataatgta ttttccatgc agttttaaaa tgtagatata ttaatatctg 2220
gatgcatttt ctgtgcactg gttttatatg ccttatggag tatatactca catgtagcta 2280
aatagactca ggactgcaca ttccttgtgt aggttgtgtg tgtgtggtgg ttttatgcat 2340
aaataaagtt ttacatgtgg tgaatataaa ttttaatttt aattttaatt tt 2392
<210> 6
<211> 710
<212> DNA
<213>artificial sequence
<400> 6
attcccttac tttcttaata aactcgcttt cactttatgg attcaactgt aattctttct 60
tgtgtgagat ccaagaacct tcttgtaggg tctggattgg gacccttttc tggtaacatc 120
ttcctggtga ccatgaaggg acaatactga agagacccct gaccctaagg aaatagactg 180
cagcaccaat gggccaactt tggggcgatc atcttgccca gaaacatcat gttgaaactc 240
ttggtcagag gttggatgaa agctgacagg gtccatccag gagcaagttt gagccttgcc 300
agttccattt tgggtgctga gtggagtggc gactatagca aacctgtgat ctctggctgc 360
tgctcagaag aaacaagagg gagggatgaa taatgtaaaa ctctggatca atattctaat 420
tctgagcctc tattggaatc agctagcaac cacatatcag cttggtttca acagtttccc 480
agttcatgct gctgagaagt tcagagtcaa acctgaatct cacctctgca aagagcacag 540
gactccatgg caaacgttgt atatacgcaa gtcatccctg gcaccacatt gatttactgc 600
accaggcttt cttcattgtg atgatgttct ctctcttttc taaaaaaaaa ataaaaataa 660
aatttaaaaa atcctaaaaa aaaaaaaatt ttaattttaa ttttaatttt 710
<210> 7
<211> 19
<212> DNA
<213>artificial sequence
<400> 7
aggaatcggc tctggaagg 19
<210> 8
<211> 21
<212> DNA
<213>artificial sequence
<400> 8
gcgtaatgga atgcttgaag g 21
<210> 9
<211> 22
<212> DNA
<213>artificial sequence
<400> 9
aagaattaca aggaagcgaa gg 22
<210> 10
<211> 22
<212> DNA
<213>artificial sequence
<400> 10
taatagcagg aggaagttca gg 22
<210> 11
<211> 21
<212> DNA
<213>artificial sequence
<400> 11
ttcttgtagg gtctggattg g 21
<210> 12
<211> 22
<212> DNA
<213>artificial sequence
<400> 12
tgaactggga aactgttgaa ac 22
<210> 13
<211> 24
<212> DNA
<213>artificial sequence
<400> 13
gattacaagg atgacgacga taag 24
<210> 14
<211> 8
<212> PRT
<213>artificial sequence
<400> 14
Asp Tyr Lys Asp Asp Asp Asp Lys
1 5
Claims (10)
1. the method that one kind obtains in vivo and purifies purpose LncRNA, which comprises the following steps:
A) by the over-express vector and LncRNA over-express vector cotransfection vehicles cells of the PPR albumen with label protein, institute
The LncRNA gene order downstream for the LncRNA over-express vector stated has the specific recognition base sequence of PPR albumen；
B) vehicles cells are cultivated；
C) vehicles cells, enrichment and purifying PPR albumen are cracked, and then is enriched with indirectly and purifying obtains LncRNA.
2. the method according to claim 1, wherein the amino acid sequence of the PPR albumen such as SEQ ID
Shown in NO.1.
3. according to the method described in claim 2, it is characterized in that, the gene order of the PPR albumen such as SEQ ID NO.3
It is shown.
4. according to the method described in claim 3, it is characterized in that, the construction method of the PPR protein overexpression carrier are as follows:
The gene order of PPR albumen is inserted between the site HindIII and EcoRI of pcDNA3.1 carrier, and in the base of PPR albumen
Because of the coded sequence of Sequences upstream insertion label protein.
5. the method according to claim 1, wherein the specific recognition base sequence of the PPR albumen is such as
Shown in SEQ ID NO.2.
6. the method according to claim 1, wherein the LncRNA over-express vector in following one
Kind:
A) gene order of H19 shown in SEQ ID NO.4 pcDNA3.1-H19: is inserted into pcDNA3.1 carrier
Between the site HindIII and EcoRI；
B) gene order of HOTAIR shown in SEQ ID NO.5 pcDNA3.1-HOTAIR: is inserted into pcDNA3.1 carrier
Between the site HindIII and EcoRI；
C) gene order of BANCR shown in SEQ ID NO.6 pcDNA3.1-BANCR: is inserted into pcDNA3.1 carrier
Between the site HindIII and EcoRI.
7. the method according to claim 1, wherein the vehicles cells are HEK-293T cells.
8. the method according to claim 1, wherein the label protein is Flag label.
9. according to the method described in claim 8, it is characterized in that, step c) is to be enriched with PPR egg by the antibody of label protein
It is white.
10. the method according to claim 1, wherein each concrete operations in step c) are pressed down using RNA enzyme
Preparation.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101921766A (en) * | 2010-06-29 | 2010-12-22 | 清华大学 | miRNA-1425 in response to H2O2 and application thereof |
| WO2013155555A1 (en) * | 2012-04-16 | 2013-10-24 | The University Of Western Australia | Peptides for the binding of nucleotide targets |
| CN105392796A (en) * | 2013-04-22 | 2016-03-09 | 国立大学法人九州大学 | Dna binding protein using ppr motif, and use thereof |
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| JP2013128413A (en) * | 2010-03-11 | 2013-07-04 | Kyushu Univ | Method for modifying rna-binding protein using ppr motif |
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2017
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101921766A (en) * | 2010-06-29 | 2010-12-22 | 清华大学 | miRNA-1425 in response to H2O2 and application thereof |
| WO2013155555A1 (en) * | 2012-04-16 | 2013-10-24 | The University Of Western Australia | Peptides for the binding of nucleotide targets |
| EP2838912A1 (en) * | 2012-04-16 | 2015-02-25 | The University Of Western Australia | Peptides for the binding of nucleotide targets |
| CN105392796A (en) * | 2013-04-22 | 2016-03-09 | 国立大学法人九州大学 | Dna binding protein using ppr motif, and use thereof |
Non-Patent Citations (2)
| Title |
|---|
| Long noncoding RNAs are generated from the mitochondrial genome and regulated bu nuclear-encoded proteins;Oliver Rackham等;《RNA》;20111231;第17卷(第12期);第2042-2052页 * |
| Site-specific binding of a PPR protein defines and stabilizes 5" and 3" mRNA termini in chloroplasts;Jeannette Pfalz等;《the EMBO JOURNAL》;20090507;第28卷(第14期);第2085-2093页 * |
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