CN115976080A - Vector for identifying protein interacting with human brain lncRNA and application thereof - Google Patents
Vector for identifying protein interacting with human brain lncRNA and application thereof Download PDFInfo
- Publication number
- CN115976080A CN115976080A CN202211516312.9A CN202211516312A CN115976080A CN 115976080 A CN115976080 A CN 115976080A CN 202211516312 A CN202211516312 A CN 202211516312A CN 115976080 A CN115976080 A CN 115976080A
- Authority
- CN
- China
- Prior art keywords
- lncrna
- grna
- sequence
- protein
- csy4
- 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.)
- Granted
Links
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 125
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 99
- 108020005198 Long Noncoding RNA Proteins 0.000 title claims abstract description 87
- 210000004556 brain Anatomy 0.000 title claims abstract description 42
- 108020005004 Guide RNA Proteins 0.000 claims abstract description 50
- 108020001507 fusion proteins Proteins 0.000 claims abstract description 30
- 102000037865 fusion proteins Human genes 0.000 claims abstract description 30
- 230000003993 interaction Effects 0.000 claims abstract description 26
- 150000001875 compounds Chemical class 0.000 claims abstract description 10
- 235000018102 proteins Nutrition 0.000 claims description 89
- 238000000034 method Methods 0.000 claims description 53
- 239000012634 fragment Substances 0.000 claims description 22
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 16
- 230000035772 mutation Effects 0.000 claims description 11
- 238000013518 transcription Methods 0.000 claims description 11
- 230000035897 transcription Effects 0.000 claims description 11
- 235000001014 amino acid Nutrition 0.000 claims description 10
- 150000001413 amino acids Chemical class 0.000 claims description 10
- 239000002773 nucleotide Substances 0.000 claims description 9
- 125000003729 nucleotide group Chemical group 0.000 claims description 9
- 241000589517 Pseudomonas aeruginosa Species 0.000 claims description 8
- 238000010367 cloning Methods 0.000 claims description 8
- 238000001976 enzyme digestion Methods 0.000 claims description 8
- 238000010276 construction Methods 0.000 claims description 7
- 238000012163 sequencing technique Methods 0.000 claims description 6
- 238000011084 recovery Methods 0.000 claims description 5
- 230000002194 synthesizing effect Effects 0.000 claims description 5
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 claims description 4
- 108010033276 Peptide Fragments Proteins 0.000 claims description 4
- 102000007079 Peptide Fragments Human genes 0.000 claims description 4
- 235000004279 alanine Nutrition 0.000 claims description 4
- 238000006243 chemical reaction Methods 0.000 claims description 4
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 claims description 4
- 238000011068 loading method Methods 0.000 claims description 4
- 102220555352 Caspase-4_H29A_mutation Human genes 0.000 claims 1
- 238000005516 engineering process Methods 0.000 abstract description 5
- 230000004907 flux Effects 0.000 abstract description 3
- 238000001819 mass spectrum Methods 0.000 abstract 1
- 239000000523 sample Substances 0.000 description 16
- 238000001114 immunoprecipitation Methods 0.000 description 12
- 239000000047 product Substances 0.000 description 10
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 9
- 238000000338 in vitro Methods 0.000 description 9
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 8
- 108010090804 Streptavidin Proteins 0.000 description 7
- 239000011324 bead Substances 0.000 description 7
- 230000000694 effects Effects 0.000 description 7
- 230000003834 intracellular effect Effects 0.000 description 7
- 238000004949 mass spectrometry Methods 0.000 description 7
- 238000004132 cross linking Methods 0.000 description 6
- 108091008146 restriction endonucleases Proteins 0.000 description 6
- 125000003275 alpha amino acid group Chemical group 0.000 description 5
- 238000000137 annealing Methods 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- 208000014644 Brain disease Diseases 0.000 description 4
- 229960000723 ampicillin Drugs 0.000 description 4
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 4
- 229960002685 biotin Drugs 0.000 description 4
- 235000020958 biotin Nutrition 0.000 description 4
- 239000011616 biotin Substances 0.000 description 4
- 101150102092 ccdB gene Proteins 0.000 description 4
- 238000001821 nucleic acid purification Methods 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 3
- 239000012154 double-distilled water Substances 0.000 description 3
- 238000010828 elution Methods 0.000 description 3
- 239000000499 gel Substances 0.000 description 3
- 238000004451 qualitative analysis Methods 0.000 description 3
- 238000004445 quantitative analysis Methods 0.000 description 3
- 238000001262 western blot Methods 0.000 description 3
- 102000003960 Ligases Human genes 0.000 description 2
- 108090000364 Ligases Proteins 0.000 description 2
- 239000011543 agarose gel Substances 0.000 description 2
- 239000013592 cell lysate Substances 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 230000008606 intracellular interaction Effects 0.000 description 2
- 229930027917 kanamycin Natural products 0.000 description 2
- 229960000318 kanamycin Drugs 0.000 description 2
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 2
- 229930182823 kanamycin A Natural products 0.000 description 2
- 238000011173 large scale experimental method Methods 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000007480 sanger sequencing Methods 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 238000002798 spectrophotometry method Methods 0.000 description 2
- 238000000870 ultraviolet spectroscopy Methods 0.000 description 2
- 102000012410 DNA Ligases Human genes 0.000 description 1
- 108010061982 DNA Ligases Proteins 0.000 description 1
- 101150036477 Gen gene Proteins 0.000 description 1
- 101000702488 Rattus norvegicus High affinity cationic amino acid transporter 1 Proteins 0.000 description 1
- 108091028664 Ribonucleotide Proteins 0.000 description 1
- 108091046869 Telomeric non-coding RNA Proteins 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 230000005856 abnormality Effects 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000004641 brain development Effects 0.000 description 1
- 210000005013 brain tissue Anatomy 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000003278 mimic effect Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000009456 molecular mechanism Effects 0.000 description 1
- 238000007857 nested PCR Methods 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000003753 real-time PCR Methods 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 239000002336 ribonucleotide Substances 0.000 description 1
- 125000002652 ribonucleotide group Chemical group 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000009897 systematic effect Effects 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Peptides Or Proteins (AREA)
Abstract
The invention belongs to the technical field of biology, and particularly relates to a vector for identifying protein interacting with human brain lncRNA and application thereof, wherein the human brain lncRNA is connected to a p-gRNA vector, so that an lncRNA sequence is provided with a gRNA label and is transcribed into a gRNA-lncRNA sequence in a cell; the SBP-Csy4 fusion protein is combined with a gRNA sequence in a cell to form an SBP-Csy4_ gRNA-lncRNA structure, and simultaneously, a protein which has interaction with human brain lncRNA is combined with the gRNA to form an SBP-Csy4_ gRNA-lncRNA-interacting protein compound, and the protein which interacts with the human brain lncRNA is qualitatively and quantitatively analyzed by means of technologies such as mass spectrum and the like, so that the SBP-Csy4_ gRNA-lncRNA-interacting protein compound has the advantages of small cell consumption, no antibody limitation and high flux.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a carrier for identifying a protein interacting with human brain lncRNA and application thereof.
Background
The human brain is the most complex organ, and many studies have found that large amounts of Long non-coding RNA (incrna) genes are contained in the Copy Number Variation (CNV) regions and Differentially Expressed Genes (DEGs) in brain tissues of patients with brain diseases. These lncRNAs may play an important role in brain development, and the expression or function abnormality thereof may be involved in the occurrence and development of complex brain diseases. At present, the number of human lncRNA genes recorded in the gengene database is second only to the number of protein-encoding genes, but the functions of most lncRNA genes are unknown. The whole life cycle of lncRNA transcription, processing, function and degradation is accompanied by protein participation; the identification of proteins that interact with lncRNA is an important method for studying lncRNA function. Therefore, an experimental method capable of identifying the protein interacting with abnormal lncRNA in human brain diseases in a large scale is established, the process of constructing the lncRNA-protein interaction network is accelerated, and the research on the function of lncRNA and the molecular mechanism of lncRNA in the disease process of brain diseases is facilitated.
Currently, methods for studying lncRNA-protein interactions are: (1) in vitro transcription method: firstly, constructing lncRNA genes on a vector with a T7 promoter; in vitro transcribing the lncRNA gene, wherein biotin-labeled ribonucleotides are inserted into a transcribed lncRNA sequence; purifying the transcription product; incubating the purified transcript with a cell lysate to allow lncRNA to interact with the protein to mimic intracellular lncRNA-protein interaction; a pulling-down experiment (pulldown) is carried out on the transcript marked by the biotin by using a magnetic frame and streptavidin magnetic beads, and the protein interacted with the lncRNA is pulled down together; the pulled-down protein was qualitatively analyzed by Western Blot (WB) or Mass Spectrometry (MS). (2) immunoprecipitation: immunoprecipitation (IP) of the protein of interest using antibodies, lncRNA that interacts with the protein will also precipitate; the lncRNA was qualitatively and quantitatively analyzed by reverse transcription Quantitative PCR (RT-qPCR). (3) a probe method: designing a biotin-labeled probe aiming at the lncRNA sequence, wherein the probe sequence is reversely complementary with the lncRNA sequence; incubating the cell lysate with a probe, and pulling down the biotin-labeled probe by using a magnetic frame and streptavidin magnetic beads; WB or MS identifies proteins that bind to lncRNA sequences. However, these methods have limitations and are not suitable for large-scale lncRNA research experiments.
Currently, most experimental studies are single incrna-protein interaction (one-to-many) studies, without large-scale, systematic, many-to-many incrna-protein interaction network studies. Some calculation and prediction methods study lncRNA-protein interaction, but the results obtained by prediction are greatly different from those obtained by experiments, and the accuracy of the prediction results needs to be studied.
Although a variety of methods have been available to study lncRNA-protein interactions, these methods all have certain limitations. The in vitro transcription method simulates the intracellular lncRNA-protein interaction by using an in vitro method, and has the problems that the in vitro method cannot completely simulate the intracellular interaction events, and the interaction result of the lncRNA and the protein obtained by the in vitro transcription method can be different from the actual intracellular interaction result because the two-dimensional and three-dimensional space structures formed by the lncRNA in the in vitro and intracellular environments are different, and the interaction of the lncRNA and the protein is often space structure dependent.
The immunoprecipitation method requires the use of an antibody specific to the target protein, and has disadvantages in that the antibody is expensive, only one protein can be identified with each experiment whether or not it interacts with one incrna, and the method is limited in use when there is no antibody specific to the target protein, so that the immunoprecipitation method is to study the incrna sequence interacting with the target protein centering on the protein, is not suitable for the study scheme centering on incrna, and the use of the immunoprecipitation method is limited to a specific antibody.
The probe method depends on endogenously expressed lncRNA sequences, and experimental results can reflect the real occurrence of lncRNA-protein(s) interaction, but the practical challenge of the method is that the expression level of endogenous lncRNA is very low, so that the probe method needs 20-200 million cells and needs more manpower and financial support in large-scale experiments. These disadvantages make the above three methods unsuitable for large-scale, intracellular real lncRNA-protein interaction studies.
The invention mainly solves the problem of providing a preparation method of a pulldown system which is suitable for systematically and massively researching the interaction of lncRNA-protein in human brain.
Disclosure of Invention
Aiming at the problems in the prior art, the invention aims to provide a vector for identifying protein interacting with human brain lncRNA and application thereof.
Based on the above purpose, the technical scheme adopted by the invention is as follows:
in a first aspect, the invention provides a vector for identifying a human brain lncRNA interacting protein, wherein the vector comprises a vector p-gRNA and an SBP-Csy4 fusion protein vector for loading a human brain lncRNA sequence; the p-gRNA is used for connecting lncRNA of a target human brain, so that an lncRNA sequence is provided with a gRNA label.
The method comprises the steps that a human brain lncRNA is connected to a p-gRNA vector, so that an lncRNA sequence is provided with a gRNA label and is transcribed into a gRNA-lncRNA sequence in a cell; the SBP-Csy4 fusion protein is combined with a gRNA sequence in a cell to form an SBP-Csy4_ gRNA-lncRNA structure, and simultaneously combined with a protein with interaction with human brain lncRNA to form an SBP-Csy4_ gRNA-lncRNA-interacting protein compound, and the protein with interaction with the human brain lncRNA is qualitatively and quantitatively analyzed by the prior mass spectrometry and other technologies.
Preferably, the nucleotide sequence of gRNA is 5-.
Preferably, the sequence transcribed by the nucleotide sequence of gRNA is 5-.
Preferably, in the vector p-gRNA, the sequence (5 'ACUGCCGUAUAGGCAG-3') obtained after transcription of the gRNA sequence forms a hairpin structure which can be specifically recognized and bound by the Csy4 protein of Pseudomonas aeruginosa.
Preferably, the nucleotide sequence of the vector p-gRNA from 5 'end to 3' is shown as SEQ ID NO. 1.
The gRNA in the p-gRNA vector is positioned at the downstream 100bp of the CMV promoter. The sequence obtained after transcription of the gRNA sequence of the invention can form a hairpin structure and can be specifically identified and combined by the Csy4 protein of pseudomonas aeruginosa. Gateway cloning sites and ccdB resistance genes of attR1 and attR2 are reserved on the p-gRNA vector, and a gateway cloning method can be used for large-scale vector construction. The gRNA sequence of the p-gRNA vector is followed by EcoRV and Hind III restriction enzyme sites, and gateway cloning sites and ccdB resistance genes of attR1 and attR2 are arranged between the two restriction enzyme sites.
After the target lncRNA sequence is connected to the gRNA sequence of the p-gRNA vector, the p-gRNA-lncRNA expresses a sequence which is transcribed into gRNA-lncRNA in a cell, and the lncRNA sequence is successfully provided with a gRNA label.
Preferably, the method for constructing the vector p-gRNA for loading the human brain lncRNA sequence comprises the following steps:
synthesizing a double-stranded gRNA sequence fragment based on primers Grna-F and Grna-R carrying a gRNA sequence; connecting the obtained double-chain gRNA sequence fragment to a vector framework, and obtaining a p-gRNA vector through conversion, cloning and sequencing;
the sequence of the Grna-F is as follows:
5’-CGgctagcggtggcggtggctctGTTCACTGCCGTATAGGCAGgatatc-3’；
the sequence of the Grna-R is as follows:
5’-CGgatatcCTGCCTATACGGCAGTGAACagagccaccgccaccgctagc-3’。
preferably, the SBP-Csy4 fusion protein vector sequentially comprises a CMV promoter, an SBP peptide segment, a linker sequence, a Csy4 protein sequence, a T2A sequence and a BSD resistance gene;
the linker sequence is used for connecting the SBP peptide segment and the Csy4 protein, so that the SBP peptide segment and the Csy4 protein respectively keep the spatial conformation and the function;
the Csy4 protein sequence is characterized in that an amino acid mutation is introduced on the basis of the wild Pseudomonas aeruginosa Csy4 protein sequence, namely the amino acid at the 29 th position is changed from histidine to alanine (H29A).
Preferably, the amino acid sequence of the SBP peptide fragment is:
MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP。
the amino acid sequence of the linker is as follows: GGGGSGGGGSGGGGSGGGGS.
Preferably, the nucleotide sequence from 5 'end to 3' end of the SBP-Csy4 fusion protein vector is shown in SEQ ID NO. 5.
The sequence characteristics of the SBP-Csy4 fusion protein carrier are as follows: the CMV promoter was followed by a 139bp random sequence, which was followed by a 38 amino acid SBP peptide translated product, followed by a 20 amino acid linker sequence (whose amino acid sequence was GGGGSGGGGSGGGGSGGGGS), followed by a Csy4 protein sequence, a T2A sequence, and a Blistic (BSD) resistance gene.
The SBP-Csy4 fusion protein carrier vector of the invention also has an ampicillin resistance gene and a kanamycin resistance gene. SBP peptide fragments can be bound to streptavidin magnetic beads (KD =2.5 × 10) -9 M); the linker sequence plays a role in connecting the SBP peptide segment and the Csy4 protein, and enables the fusion protein to have space for the SBP peptide segment and the Csy4 protein to respectively form own space conformation so as to play respective functions; multiple resistance genes provide convenience for screening clones. The Csy4 protein sequence is a functional protein molecule which is introduced with an amino acid mutation on the basis of a wild Pseudomonas aeruginosa Csy4 protein sequence, namely, the 29 th histidine (H) is changed into alanine (A) -H29A mutation, and the introduced H29A mutation enables the Csy4 protein to be changed from 'a restriction endonuclease activity capable of being combined with the gRNA sequence and cutting downstream of the gRNA sequence' into 'a protein molecule which only retains the combination activity with the gRNA sequence but loses the restriction endonuclease activity'. The SBP-Csy4 fusion protein can be combined with a gRNA sequence on gRNA-lncRNA in a cell, and an SBP peptide segment can be combined with streptavidin magnetic beads when the cell is broken to release intracellular proteins. The invention utilizes the ultraviolet crosslinking or formaldehyde crosslinking effect to simultaneously express SBP-Csy4 protein and gRNA-lncRNA in cellsCovalent linkage is formed between Csy4 protein and gRNA, the SBP-Csy4_ gRNA-lncRNA _ interacting protein complex can be pulled down by pulldown, and the protein interacting with lncRNA can be qualitatively and quantitatively analyzed by mass spectrometry.
Preferably, the construction method of the SBP-Csy4 fusion protein carrier comprises the following steps:
1) Synthesizing a Csy4 gene sequence containing the H29A mutation;
2) Respectively obtaining an SBP-Csy4 fragment and a T2A-BSD fragment based on corresponding primers;
3) And carrying out enzyme digestion and recovery on the vector framework, the SBP-Csy4 fragment and the T2A-BSD fragment, sequentially connecting the viscous ends of the vector framework, the SBP-Csy4 fragment and the T2A-BSD fragment, and carrying out sequencing identification to obtain the SBP-Csy4 fusion protein vector.
In a second aspect, the invention provides a method for identifying a protein interacting with lncRNA in human brain based on the vector, which comprises the following steps:
connecting human brain lncRNA to the p-gRNA vector to form p-gRNA-lncRNA; the p-gRNA-lncRNA is transcribed into a gRNA-lncRNA sequence in the cell;
SBP-Csy4 fusion protein obtained by SBP-Csy4 fusion protein vector expression is combined with a gRNA sequence in a cell to form an SBP-Csy4_ gRNA-lncRNA structure, and simultaneously combined with a protein with interaction with human brain lncRNA to form an SBP-Csy4_ gRNA-lncRNA-interaction protein compound, and the protein with interaction with the human brain lncRNA is identified based on the compound.
The method for identifying the protein interacting with the lncRNA of the human brain does not need an antibody, only needs to construct the lncRNA into a p-gRNA-lncRNA vector containing a gRNA label, SBP-Csy4 fusion protein can be combined with a gRNA sequence on the gRNA-lncRNA in a cell, and an SBP peptide segment can be combined with streptavidin magnetic beads when the cell is broken to release intracellular protein. According to the invention, by utilizing ultraviolet crosslinking or formaldehyde crosslinking, covalent connection can be formed between Csy4 protein and gRNA in a cell simultaneously expressing SBP-Csy4 protein and gRNA-lncRNA, the compound of SBP-Csy4_ gRNA-lncRNA _ interacting protein is pulled down by pulldown, and qualitative and quantitative analysis is carried out on the protein interacting with lncRNA by utilizing a mass spectrometry technology.
Compared with the traditional probe method, the method of the invention needs less cells, which is about 1/20 of the traditional probe method; the method does not need to use antibodies, and has the advantages of no antibody limitation and higher throughput compared with an immunoprecipitation method.
Compared with the prior art, the invention has the following beneficial effects:
the human brain lncRNA is connected to the p-gRNA vector, so that an lncRNA sequence is provided with a gRNA label and is transcribed into a gRNA-lncRNA sequence in a cell; the SBP-Csy4 fusion protein is combined with a gRNA sequence in a cell to form an SBP-Csy4_ gRNA-lncRNA structure, and simultaneously combined with a protein with interaction with human brain lncRNA to form an SBP-Csy4_ gRNA-lncRNA-interacting protein compound, and the protein with interaction with the human brain lncRNA is qualitatively and quantitatively analyzed by the prior mass spectrometry and other technologies.
Compared with a probe method, the method for identifying the lncRNA interacting protein with the human brain requires less cells which are only 1/20 of the number of the cells required by the probe method, does not need to use an antibody, and has the advantages of no limitation of the antibody and higher flux compared with an immunoprecipitation method.
Detailed Description
To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to specific examples. It will be understood by those skilled in the art that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
The test methods used in the examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are commercially available unless otherwise specified.
Example 1
This example provides a vector p-gRNA loaded with lncRNA sequence by pulldown system, the nucleotide sequence of the p-gRNA from 5 'end to 3' is shown in SEQ ID No. 1.
Wherein the gRNA sequence in the p-gRNA vector is 5 'ACTGCCGTATAGGCAG-3' (SEQ ID NO. 2) and is positioned at 100bp downstream of the CMV promoter. The sequence obtained after transcription of the gRNA sequence can form a hairpin structure and can be specifically recognized and combined by the Csy4 protein of pseudomonas aeruginosa. Gateway cloning sites and ccdB resistance genes of attR1 and attR2 are reserved on the p-gRNA vector, and a gateway cloning method can be used for large-scale vector construction.
The gRNA sequence of the p-gRNA vector is followed by EcoRV and HindIII restriction sites, between which are gateway cloning sites for attR1 and attR2 and the ccdB resistance gene. After the target lncRNA sequence is connected to the gRNA sequence of the p-gRNA vector, the p-gRNA-lncRNA expresses a sequence which is transcribed into gRNA-lncRNA in cells, and the lncRNA sequence is successfully provided with a gRNA label.
The construction method of the vector p-gRNA loaded with the lncRNA sequence by the pulldown system comprises the following steps:
(1) Obtaining a carrier framework: the Original vector was obtained from the laboratory and the sequence of the Original vector (Original vector sequence) is shown in SEQ ID NO. 14. The double digestion was carried out by incubation at 37 ℃ for 2h according to the system described in table 1 below, followed by fragment analysis by 1% agarose gel electrophoresis, and the fragments of 6000 to 7000bp size were recovered by gel cutting and the concentration of the backbone fragments was determined by uv spectrophotometry.
TABLE 1
| Components | Amount of the composition used |
| Nhe Ⅰ(FastDigest,Thermo Scientific) | 2μl |
| EcoR Ⅴ(FastDigest,Thermo Scientific) | 2μl |
| FastDigest buffer | 5μl |
| Original plasmid | 12μg |
| ddH 2 O | Make up to 50 μ l |
(2) Synthesizing a primer carrying a gRNA sequence in Huada gene:
Grna-F:5’-CGgctagcggtggcggtggctctGTTCACTGCCGTATAGGCAGgatatc-3’(SEQ ID NO.3)；
Grna-R:5’-CGgatatcCTGCCTATACGGCAGTGAACagagccaccgccaccgctagc-3’(SEQ ID NO.4)；
(3) Obtaining double-stranded gRNA sequence fragments: diluting the Grna-F and Grna-R primers to 100 mu M, taking 1 mu l of each PCR tube, configuring an annealing reaction system according to the system shown in the following table 2 in 200 mu l of each PCR tube, annealing to form double chains, placing 20 mu l of the annealing reaction system PCR tube in water under the annealing condition that the temperature of a water bath is raised to 95 ℃, closing the power supply of the water bath after 5min, carrying out enzyme digestion on 20 mu l of annealing reaction products by the same method in the step (1) when the temperature of the water in the water bath is lowered to room temperature, recovering the enzyme digestion products by a nucleic acid purification column, and determining the concentration of double-chain gRNA sequence fragments by an ultraviolet spectrophotometry.
TABLE 2
| Components | Amount of the use |
| Grna-F(100μM) | 1μl |
| Grna-R(100μM) | 1μl |
| Ex taq buffer | 2μl |
| ddH 2 O | Make up to 20 μ l |
(4) Obtaining of p-gRNA vector: and (3) connecting the vector framework obtained in the step (1) and the double-stranded gRNA sequence fragment obtained in the step (3) by using T4 ligase, converting a ligation product into a DH5 alpha competent cell, coating a plate on an LB plate carrying ampicillin, selecting a single clone after overnight culture for Sanger sequencing identification, and returning the clone with the sequencing result conforming to the expected sequence to obtain the p-gRNA vector.
A pulldown system SBP-Csy4 fusion protein carrier, the nucleotide sequence from 5 'end to 3' end is shown in SEQ ID NO.5, the sequence characteristics of the SBP-Csy4 fusion protein carrier are: the CMV promoter is followed by a 139bp random sequence, which is followed by an SBP peptide segment with a translation product of 38 amino acids, followed by a 20 amino acid linker sequence, the amino acid sequence of which is: GGGGSGGGGSGGSGGGGS (SEQ ID NO. 6) followed by the Csy4 protein sequence, the T2A sequence and the Blastidin (BSD) resistance gene. The vector also contains ampicillin resistance gene and kanamycin resistance gene.
The amino acid sequence of the SBP peptide segment is as follows:
MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP(SEQ ID
no. 7), SBP peptide fragments can be bound to streptavidin beads (KD =2.5 × 10) -9 M); the linker sequence plays a role in connecting the SBP peptide segment and the Csy4 protein, and enables the fusion protein to have space for the SBP peptide segment and the Csy4 protein to respectively form own space conformation so as to play respective functions; multiple resistance genes provide convenience for screening clones.
The Csy4 protein sequence is characterized in that on the basis of the wild Pseudomonas aeruginosa Csy4 protein sequence, an amino acid mutation is introduced, namely, the 29 th histidine (H) is changed into alanine (A) -H29A mutation, and the introduced H29A mutation enables the Csy4 protein to be changed from 'restriction endonuclease activity capable of being combined with the gRNA sequence and cutting at the downstream of the gRNA sequence' into 'functional protein molecule only retaining the combination activity with the gRNA sequence but losing the restriction endonuclease activity'.
The SBP-Csy4 fusion protein can be combined with a gRNA sequence on a gRNA-lncRNA in a cell, and an SBP peptide segment can be combined with streptavidin magnetic beads when the cell is broken to release intracellular proteins. According to the invention, by utilizing ultraviolet crosslinking or formaldehyde crosslinking, covalent connection can be formed between Csy4 protein and gRNA in a cell simultaneously expressing SBP-Csy4 protein and gRNA-lncRNA, the compound of SBP-Csy4_ gRNA-lncRNA _ interacting protein can be pulled down by pulldown, and qualitative and quantitative analysis can be carried out on the protein interacting with lncRNA by a mass spectrometry technology.
The construction method of the pulldown system SBP-Csy4 fusion protein vector comprises the following steps:
(1) Synthesis of Csy4 (already containing the H29A mutation) gene sequence: the linker-Csy4 sequence was synthesized in Biotechnology engineering (Shanghai) GmbH and subcloned into pcDNA3.1 (+) vector;
(2) Obtaining the SBP-Csy4 sequence and the T2A-BSD sequence: the following primers were synthesized in Huada gene:
sbp-f:5’-GGTGGAATTCATGGACGAGAAGACCACCGGCTGGCGGGGCGGCCACGTGGTGGAGGGCCTGGCCGGCGAGCTGGAGCAGCTGCGGGCCAGGCTGGA-3’(SEQ ID NO.8)；
sbp-r:5’-CAGATCCTccTCCTCCGGATCCACCTCCTCCAGAGCCACCGCCACCGGGCTCCCGCTGGCCCTGAGGGTGGTGCTCCAGCCTGGCCCGCAGCTGCT-3’(SEQ ID NO.9)；
CSY4-F:5’-TGGATCCGGAGGAGGAGGATCTGGAGGTGGAGGAAGTATGGACCACTACCTCGACATTC-3’(SEQ ID NO.10)；
CSY4-R:5’-GGAAGATCTGAACCAGGGAACGAAACCTCCTTTGC-3’(SEQ ID NO.11)；
T2A-BSD-F:5’-CGGGATCCggcggcgggtccggaggagagggcag-3’(SEQ ID NO.12)；
T2A-BSD-R:5’-gttgagcggccgctcagccctcccacacataaccaga-3’(SEQ ID NO.13)；
firstly, carrying out PCR by using a 2 x Phanta Max Master Mix kit (a PCR system is carried out according to the instruction) to obtain a linker-Csy4 sequence (primers are CSY4-F and CSY 4-R) and a T2A-BSD sequence (primers are T2A-BSD-F and T2A-BSD-R), and carrying out 1% agarose gel analysis on a PCR product and gel recovery of a target sequence (41 mu l of ddH2O elution); SBP-Csy4 sequences (41. Mu.l ddH2O elution) were then obtained by agarose gel analysis and gel recovery by adding SBP sequences to the 5' end of the linker-Csy4 sequences using split by overlap extension PCR (SOE-PCR) set up according to the 2 × Phanta Max Master Mix kit instructions, as shown in Table 3 below.
TABLE 3
| Components | Amount of the composition used |
| 2×Phanta mix | 25μl |
| sbp-f(10μM) | 1.2μl |
| sbp-r(10μM) | 0.3μl |
| CSY4-R(10μM) | 1.2μl |
| Linker-Csy4 template | 50ng |
| ddH 2 O | Make up to 20 μ l |
(3) Carrying out double enzyme digestion on the vector framework, the SBP-Csy4 fragment and the T2A-BSD fragment: the SBP-Csy4 fragment and T2A-BSD fragment double enzyme digestion system is shown in the following table 4, the enzyme digestion conditions are all incubation for 2 hours at 37 ℃, enzyme digestion products are recovered by a nucleic acid purification column, and the concentration of the recovered fragments is measured by an ultraviolet spectrophotometry; mu.g of pcDNA3.1 (+) vector was digested with EcoRI for 2h and the digested product (44. Mu.l ddH) was recovered on a nucleic acid purification column 2 O elution), followed by 4h single cleavage with Not I, recovery of the cleaved product using a nucleic acid purification column and determination of the concentration of the recovered pcDNA3.1 (+) _ EcoRI _ Not I backbone fragment using UV spectrophotometry.
TABLE 4
(4) Acquisition of SBP-Csy4 fusion protein vector: bgl II and BamH I are isocaudarner, so the cohesive ends of pcDNA3.1 (+) _ EcoRI _ Not I skeleton, SBP-Csy4 fragment and T2A-BSD fragment recovered after double digestion can be connected in turn, and connected by T4 ligase, the system is as shown in Table 5 below, 8 mul of the ligation product is taken to transform DH5 alpha competent cell and coated on LB plate carrying ampicillin, after overnight culture, single clone is selected for Sanger sequencing identification, and clone with sequencing result consistent with expectation is returned to obtain SBP-Csy4 fusion protein vector.
TABLE 5
| Components | Amount of the use |
| T4 DNA Ligase(Thermo Scientific) | 1μl |
| T4 10x T4 buffer(Thermo Scientific) | 2μl |
| pcDNA3.1 (+) _ EcoRI _ Not I backbone | 100ng |
| SBP-Csy4 fragment | 200ng |
| T2A-BSD fragments | 200ng |
| ddH2O | Make up to 20 μ l |
The existing methods for researching the lncRNA-protein(s) interaction comprise an in vitro transcription method, an immunoprecipitation method, a probe method and the like. The interaction result of the lncRNA and the protein obtained by the in vitro transcription method is possibly different from the interaction result actually generated in the cell; the immunoprecipitation method studies lncRNA sequence interacting with target protein centering on protein, is not suitable for lncRNA centering study scheme, and the use of the immunoprecipitation method is limited to specific antibody; the probe method depends on endogenously expressed lncRNA, and the experimental result can reflect the real occurrence of lncRNA-protein(s) interaction, but the method faces the practical challenge that the endogenous expression level of lncRNA is very low, so that the probe method needs 20-200 million cells and needs more manpower and financial support during large-scale experiments.
Compared with the existing probe method, the pulldown system of the invention needs less cells, which is about 1/20 of the probe method; the pulldown system disclosed by the invention does not need to use an antibody, can complete the construction of a p-gRNA-lncRNA vector by only placing an lncRNA sequence and a gRNA sequence on a standard vector at the downstream of the same promoter, and further realizes the qualitative and quantitative analysis of the protein interacted with lncRNA by combining an SBP-Csy4 fusion protein vector, so that the pulldown system has the advantages of wide application range, no limitation of the antibody and higher flux compared with an immunoprecipitation method.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
Claims (10)
1. A carrier for identifying a protein interacting with human brain lncRNA is characterized by comprising a carrier p-gRNA and an SBP-Csy4 fusion protein carrier for loading a human brain lncRNA sequence; the p-gRNA is used for connecting lncRNA of a target human brain, so that an lncRNA sequence is provided with a gRNA label.
2. The vector for identifying a protein interacting with human brain lncRNA of claim 1, wherein the nucleotide sequence of gRNA is 5.
3. The vector for identifying the protein interacting with human brain lncRNA as claimed in claim 1, wherein in the vector p-gRNA, 5 'ACUGCCGUAUAGGCAG-3' sequence obtained after transcription of gRNA sequence forms hairpin structure which can be specifically recognized and combined by Csy4 protein of Pseudomonas aeruginosa.
4. The vector for identifying the lncRNA interacting protein in the human brain according to claim 1, wherein the nucleotide sequence from 5 'end to 3' end of the vector p-gRNA is shown as SEQ ID NO. 1.
5. The vector for identifying the protein interacting with the human brain lncRNA according to claim 1, wherein the method for constructing the vector p-gRNA for loading the human brain lncRNA sequence comprises the following steps:
synthesizing a double-chain gRNA sequence fragment based on primers Grna-F and Grna-R carrying gRNA sequences; connecting the obtained double-chain gRNA sequence fragment to a vector framework, and obtaining a p-gRNA vector through conversion, cloning and sequencing;
the sequence of the Grna-F is as follows:
5’-CGgctagcggtggcggtggctctGTTCACTGCCGTATAGGCAGgatatc-3’；
the sequence of the Grna-R is as follows:
5’-CGgatatcCTGCCTATACGGCAGTGAACagagccaccgccaccgctagc-3’。
6. the vector for identifying the lncRNA interacting protein with the human brain as claimed in claim 1, wherein the SBP-Csy4 fusion protein vector comprises a CMV promoter, an SBP peptide segment, a linker sequence, a Csy4 protein sequence, a T2A sequence and a BSD resistance gene in sequence;
the linker sequence is used for connecting the SBP peptide segment and the Csy4 protein, so that the SBP peptide segment and the Csy4 protein respectively keep the spatial conformation and the function;
the Csy4 protein sequence is characterized in that an amino acid mutation is introduced on the basis of the wild Pseudomonas aeruginosa Csy4 protein sequence, namely the amino acid at the 29 th position is changed from histidine to alanine.
7. The vector for identifying a protein interacting with lncRNA of human brain according to claim 6, wherein the amino acid sequence of the SBP peptide fragment is:
MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP。
the amino acid sequence of the linker is as follows: GGGGSGGGGSGGGGSGGGGS.
8. The vector for identifying the lncRNA interacting protein of human brain according to claim 1, wherein the nucleotide sequence from 5 'end to 3' end of the SBP-Csy4 fusion protein vector is shown as SEQ ID No. 5.
9. The vector for identifying the protein interacting with the human brain lncRNA as claimed in claim 1, wherein the construction method of the SBP-Csy4 fusion protein vector comprises the following steps:
1) Synthesizing a Csy4 gene sequence containing H29A mutation;
2) Respectively obtaining an SBP-Csy4 fragment and a T2A-BSD fragment based on corresponding primers;
3) And carrying out enzyme digestion and recovery on the vector framework, the SBP-Csy4 fragment and the T2A-BSD fragment, sequentially connecting the viscous ends of the vector framework, the SBP-Csy4 fragment and the T2A-BSD fragment, and carrying out sequencing identification to obtain the SBP-Csy4 fusion protein vector.
10. Method for identifying proteins interacting with human brain lncRNA based on the vector according to any one of claims 1 to 9, comprising the following steps:
connecting human brain lncRNA to the p-gRNA vector to form p-gRNA-lncRNA; the p-gRNA-lncRNA is transcribed into a gRNA-lncRNA sequence in the cell;
SBP-Csy4 fusion protein obtained by SBP-Csy4 fusion protein vector expression is combined with a gRNA sequence in a cell to form an SBP-Csy4_ gRNA-lncRNA structure, and simultaneously combined with a protein with interaction with human brain lncRNA to form an SBP-Csy4_ gRNA-lncRNA-interaction protein compound, and the protein with interaction with the human brain lncRNA is identified based on the compound.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211516312.9A CN115976080B (en) | 2022-11-28 | 2022-11-28 | Carrier for identifying protein interacted with human brain lncRNA and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211516312.9A CN115976080B (en) | 2022-11-28 | 2022-11-28 | Carrier for identifying protein interacted with human brain lncRNA and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN115976080A true CN115976080A (en) | 2023-04-18 |
| CN115976080B CN115976080B (en) | 2024-04-19 |
Family
ID=85963714
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202211516312.9A Active CN115976080B (en) | 2022-11-28 | 2022-11-28 | Carrier for identifying protein interacted with human brain lncRNA and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115976080B (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100999760A (en) * | 2006-01-10 | 2007-07-18 | 中国科学院生物物理研究所 | Reversible fixture for aglucon of streptavidin envelope chip surface |
| US20130130248A1 (en) * | 2010-05-10 | 2013-05-23 | The Regents Of The University Of California | Endoribonuclease compositions and methods of use thereof |
| US20210340592A1 (en) * | 2020-04-30 | 2021-11-04 | City University Of Hong Kong | Method for Determining Long Non-Coding Ribonucleic Acid Interaction Proteins |
-
2022
- 2022-11-28 CN CN202211516312.9A patent/CN115976080B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100999760A (en) * | 2006-01-10 | 2007-07-18 | 中国科学院生物物理研究所 | Reversible fixture for aglucon of streptavidin envelope chip surface |
| US20130130248A1 (en) * | 2010-05-10 | 2013-05-23 | The Regents Of The University Of California | Endoribonuclease compositions and methods of use thereof |
| US20210340592A1 (en) * | 2020-04-30 | 2021-11-04 | City University Of Hong Kong | Method for Determining Long Non-Coding Ribonucleic Acid Interaction Proteins |
Also Published As
| Publication number | Publication date |
|---|---|
| CN115976080B (en) | 2024-04-19 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| AU724651B2 (en) | Phage-display of immunoglobulin heavy chain libraries for identification of inhibitors of intracellular constituents | |
| TWI626315B (en) | DNA 5-methylcytosine and 5-hydroxymethylcytosine gene mapping method | |
| CN108368503A (en) | Method for controlled dn A fragmentations | |
| CA2709994A1 (en) | Non-antibody scaffold protein fusions phage display via fusion to pix of m13 phage | |
| CA2400058A1 (en) | Protein scaffolds for antibody mimics and other binding proteins | |
| Li et al. | Single-cell multi-omics in the medicinal plant Catharanthus roseus | |
| Waldo | Improving protein folding efficiency by directed evolution using the GFP folding reporter | |
| CN109593757B (en) | Probe and method for enriching target region by using same and applicable to high-throughput sequencing | |
| CN110567788B (en) | Enrichment and identification method of RNA-protein complex | |
| WO2023221842A1 (en) | High-throughput single-cell transcriptome sequencing method and use thereof | |
| Barber et al. | CRISPR-based peptide library display and programmable microarray self-assembly for rapid quantitative protein binding assays | |
| CN114507711A (en) | Single cell transcriptome sequencing method and application thereof | |
| JP5733784B2 (en) | Efficient synthesis of cDNA / mRNA-protein conjugates | |
| CN115976080A (en) | Vector for identifying protein interacting with human brain lncRNA and application thereof | |
| CN111549034A (en) | Aptamer specifically binding with chemokine ligand-5 and application thereof | |
| CN113584135B (en) | Method for mixed sample detection of RNA modification and realization of accurate quantification | |
| Haynes et al. | Rapid single-pot assembly of modular chromatin proteins for epigenetic engineering | |
| CN115807068A (en) | Quality detection method of adapter primer for high-throughput sequencing and high-throughput sequencing method | |
| WO2015176339A1 (en) | Primer-free gene synthesis method | |
| Liu et al. | Genome-wide identification of protein binding sites on RNAs in mammalian cells | |
| JP2016123343A (en) | Nucleic acid linker | |
| He et al. | Production of protein arrays by cell-free systems | |
| US20240093185A1 (en) | Circular rnas and preparation methods thereof | |
| Morselli et al. | Design and Construction of a Designed Ankyrin Repeat Protein (DARPin) Display Library | |
| CN114606227B (en) | High-precision adenine base editor and application thereof |
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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |