CN115976080B - Carrier for identifying protein interacted with human brain lncRNA and application thereof - Google Patents
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- 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
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Abstract
The invention belongs to the field of biotechnology, and particularly relates to a carrier for identifying proteins interacted with human brain lncRNA and application thereof; the SBP-Csy4 fusion protein combines with gRNA sequence in cells to form SBP-Csy4_gRNA-lncRNA structure, and simultaneously combines with human brain lncRNA interacting protein to form SBP-Csy4_gRNA-lncRNA interacting protein complex, and the protein interacting with human brain lncRNA is qualitatively and quantitatively analyzed by means of mass spectrum and other technologies, so that the method has the advantages of small cell consumption, no restriction by antibodies and high flux.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a vector for identifying proteins interacted with human brain lncRNA and application thereof.
Background
The human brain is the most complex organ, and many studies have found that the gene copy number variation (Copy number variation, CNV) region and the differentially expressed gene (DIFFERENTIALLY-expressed genes, DEGs) in brain tissue of brain disease patients contain a large number of long non-coding RNA (lncRNA) genes. These lncRNA may play an important role in brain development and their expression or dysfunction may be involved in the development and progression of complex brain diseases. Currently, GENCODE databases record the number of human lncRNA genes next to the number of protein-encoding genes, but the function of most lncRNA genes is unknown. Protein participation is accompanied in the whole life cycle of lncRNA transcription, processing, functioning and degradation; identification of proteins that interact with lncRNA is an important method to study lncRNA function. Therefore, an experimental method capable of identifying proteins interacting with abnormal lncRNA in human brain diseases on a large scale is established, the process of constructing an lncRNA-protein interaction network is quickened, and the lncRNA function and the molecular mechanism thereof in the pathogenesis of the brain diseases are favorably researched.
Currently, the methods for studying lncRNA-protein interactions are: (1) in vitro transcription method: firstly, constructing the lncRNA gene on a vector with a T7 promoter; in vitro transcribing lncRNA genes, and inserting biotin-marked ribonucleotides into the transcribed lncRNA sequences; purifying the transcription product; incubating the purified transcript with a cell lysate to allow interactions of the lncRNA with the protein to mimic intracellular lncRNA-protein interactions; carrying out a pull-down experiment (pulldown) on the biotin-labeled transcripts by using a magnetic shelf and streptavidin magnetic beads, wherein proteins interacted with the lncRNA are pulled down together; the pulled-down proteins were qualitatively analyzed by Western Blot (WB) or mass spectrometry (Mass Spectrometry, MS). (2) immunoprecipitation method: immunoprecipitation of a protein of interest using an antibody (Immunoprecipitation, IP), the lncRNA interacting with the protein also precipitates; the lncRNA was analyzed qualitatively and quantitatively by reverse transcription quantitative PCR (Quantitative reverse transcription PCR, RT-qPCR). (3) Probe method: designing a biotin-marked probe aiming at the lncRNA sequence, wherein the probe sequence is reversely complementary with the lncRNA sequence; incubating the probe with the cell lysate, and pulling down the biotin-labeled probe using a magnetic rack and streptavidin magnetic beads; WB or MS identified 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 lncRNA-protein interactions (one-to-many) studies, without extensive, systematic, many-to-many lncRNA-protein interaction network studies. Some calculation and prediction methods are used for researching lncRNA-protein interaction, but the prediction results are quite different from experimental results, and the accuracy of the prediction results is to be studied.
Although there are a number of methods available to study lncRNA-protein interactions, these methods have certain limitations. In vitro transcription methods use in vitro methods to mimic lncRNA-protein interactions within cells, which have the problem that in vitro methods may not completely mimic intracellular interaction events, as the two-dimensional and three-dimensional spatial structures formed by lncRNA in the in vitro and in the in-cell environment may differ, and the interactions between lncRNA and protein are often spatially dependent, and thus the results of interactions between lncRNA and protein obtained by in vitro transcription methods may differ from those that actually occur within cells.
The immunoprecipitation method requires the use of antibodies specific for the protein of interest, which has the disadvantages that antibodies are expensive, only one protein can be identified for each experiment to interact with one lncRNA, and the method is limited in use without antibodies specific for the protein of interest, so that the immunoprecipitation method is a protein-centered study of lncRNA sequences interacting with the protein of interest, is not suitable for lncRNA-centered study protocols, and the use of immunoprecipitation method is limited to specific antibodies.
The probe method relies on endogenous lncRNA sequences, and experimental results can reflect the actually occurring lncRNA-protein(s) interaction, but the method faces the realistic challenge that the expression level of the endogenous lncRNA is very low, so that the probe method needs 20-200 megacells, and more manpower and financial support is needed in large-scale experiments. These shortcomings make the above three approaches unsuitable for large scale, intracellular-realistic lncRNA-protein interaction studies.
The invention mainly solves the problem of providing a preparation method of a pulldown system suitable for the systematic and large-scale research of human brain lncRNA-protein interaction.
Disclosure of Invention
Aiming at the problems existing in the prior art, the invention aims to provide a carrier for identifying proteins interacted 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 proteins that interact with human brain lncRNA, the vector comprising a vector p-gRNA and SBP-Csy4 fusion protein vector for loading human brain lncRNA sequences; the p-gRNA is used for connecting target human brain lncRNA, so that the lncRNA sequence is provided with a gRNA label.
The invention adopts the steps that human brain lncRNA is connected to the p-gRNA carrier, so that the lncRNA sequence is provided with a gRNA label and is transcribed into a gRNA-lncRNA sequence in cells; the SBP-Csy4 fusion protein is combined with the gRNA sequence in cells to form an SBP-Csy4_gRNA-lncRNA structure, and simultaneously, a protein with interaction with human brain lncRNA is combined with the SBP-Csy4_gRNA-lncRNA-interaction protein complex, and the protein with interaction with human brain lncRNA is qualitatively and quantitatively analyzed through the existing mass spectrometry technology and the like.
Preferably, the nucleotide sequence of the gRNA is 5'-ACTGCCGTATAGGCAG-3'.
Preferably, the nucleotide sequence of the gRNA is transcribed with a sequence 5'-ACUGCCGUAUAGGCAG-3'.
Preferably, in the vector p-gRNA, the sequence (5'-ACUGCCGUAUAGGCAG-3') obtained after transcription of the gRNA sequence forms a hairpin structure that is specifically recognized and bound by the Csy4 protein of Pseudomonas aeruginosa.
Preferably, the nucleotide sequence from the 5 'end to the 3' end of the vector p-gRNA is shown in SEQ ID NO. 1.
The gRNA in the p-gRNA vector of the invention is located 100bp downstream of the CMV promoter. The sequence obtained after transcription of the gRNA sequence can form a hairpin structure, and can be specifically identified and combined by Csy4 protein of pseudomonas aeruginosa. The gateway cloning sites and ccdB resistance genes of attR1 and attR2 are reserved on the p-gRNA vector, and a large-scale vector construction can be carried out by using a gateway cloning method. The gRNA sequence of the p-gRNA vector is followed by EcoRV and HindIII restriction sites, and a gateway cloning site and ccdB resistance genes of attR1 and attR2 are arranged between the two restriction sites.
The lncRNA sequence was successfully tagged with a gRNA tag by ligating the target lncRNA sequence to the gRNA sequence of the p-gRNA vector of the invention, which would be transcribed into a gRNA-lncRNA sequence when expressed in the cell.
Preferably, the construction method of 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 the gRNA sequence; connecting the obtained double-stranded gRNA sequence fragment to a vector framework, and obtaining a p-gRNA vector through transformation, cloning and sequencing;
the Grna-F has the sequence:
5’-CGgctagcggtggcggtggctctGTTCACTGCCGTATAGGCAGgatatc-3’；
The Grna-R has the sequence:
5’-CGgatatcCTGCCTATACGGCAGTGAACagagccaccgccaccgctagc-3’。
Preferably, the SBP-Csy4 fusion protein vector comprises a CMV promoter, an SBP peptide fragment, 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 fragment and the Csy4 protein, so that the SBP peptide fragment and the Csy4 protein respectively keep the space conformation and the function;
The Csy4 protein sequence is obtained by introducing an amino acid mutation based on a wild type Pseudomonas aeruginosa Csy4 protein sequence, namely, the 29 th amino acid 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 the 5 'end to the 3' end of the SBP-Csy4 fusion protein vector is shown as SEQ ID NO. 5.
The sequence characteristics of the SBP-Csy4 fusion protein vector of the invention are as follows: the CMV promoter is followed by A139 bp random sequence followed by A38 amino acid SBP peptide fragment of the translation product, followed by A20 amino acid linker sequence (amino acid sequence ggggsggggsggsggggs) followed by A csy4 protein sequence, A T2A sequence, and A Blasticidin (BSD) resistance gene.
The SBP-Csy4 fusion protein carrier of the invention also has an ampicillin resistance gene and a kana resistance gene. SBP peptide fragments can bind to streptavidins 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 a space for forming a space conformation of the SBP peptide segment and the Csy4 protein respectively so as to exert respective functions; multiple resistance genes facilitate screening of clones. The Csy4 protein sequence is a functional protein molecule which introduces an amino acid mutation based on a wild type Pseudomonas aeruginosa Csy4 protein sequence, namely, histidine (H) at position 29 is changed into alanine (A) -H29A mutation, and the introduced H29A mutation changes Csy4 protein from a restriction enzyme activity which can be combined with a gRNA sequence and is cut at the downstream of the gRNA sequence to a restriction enzyme activity which only keeps the binding activity with the gRNA sequence but loses the restriction enzyme activity. The SBP-Csy4 fusion protein can bind to a gRNA sequence on the gRNA-lncRNA in a cell, and the SBP peptide can bind to a strepavidin magnetic bead when the cell breaks to release the intracellular protein. The invention can form covalent connection between the SBP-Csy4 protein and the gRNA in cells simultaneously expressing the SBP-Csy4 protein and the gRNA-lncRNA by utilizing ultraviolet crosslinking or formaldehyde crosslinking, and can pull down the complex of SBP-Csy4_gRNA-lncRNA_interaction protein by using pulluown, and can perform qualitative and quantitative analysis on the protein interacted with the lncRNA by using a mass spectrometry technology.
Preferably, the construction method of the SBP-Csy4 fusion protein vector comprises the following steps:
1) Synthesizing Csy4 gene sequence containing H29A mutation;
2) Obtaining SBP-Csy4 fragment and T2A-BSD fragment based on the corresponding primers, respectively;
3) And (3) carrying out enzyme digestion and recovery on the carrier skeleton, the SBP-Csy4 fragment and the T2A-BSD fragment, sequentially connecting the adhesive tail ends of the carrier skeleton, the SBP-Csy4 fragment and the T2A-BSD fragment, and obtaining the SBP-Csy4 fusion protein carrier through sequencing identification.
In a second aspect, the invention provides a method for identifying proteins interacting with human brain lncRNA based on the vector, comprising the steps of:
Ligating human brain lncRNA to the p-gRNA vector to form p-gRNA-lncRNA; the p-gRNA-lncRNA is transcribed in the cell to a gRNA-lncRNA sequence;
The SBP-Csy4 fusion protein expressed by the SBP-Csy4 fusion protein carrier is combined with the gRNA sequence in cells to form an SBP-Csy4_gRNA-lncRNA structure, and simultaneously, a protein with interaction with human brain lncRNA is combined with the SBP-Csy4_gRNA-lncRNA-interacting protein complex, and the protein interacted with human brain lncRNA is identified based on the complex.
The method for identifying the protein interacted with the human brain lncRNA does not need an antibody, only the lncRNA is required to be constructed 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 SBP peptide can be combined with a strepavidin magnetic bead when the cell is broken to release the intracellular protein. The invention utilizes ultraviolet crosslinking or formaldehyde crosslinking to form covalent connection between the SBP-Csy4 protein and the gRNA in cells simultaneously expressing the SBP-Csy4 protein and the gRNA-lncRNA, pulls down the complex of SBP-Csy4_gRNA-lncRNA_interacting protein by pulldown, and utilizes mass spectrometry technology to perform qualitative and quantitative analysis on the protein interacted with the lncRNA.
The method of the invention requires less cells than the traditional probe method, which is about 1/20 of the probe method; the method of the invention does not require the use of antibodies, and has the advantages of being free of antibody limitations and higher throughput than immunoprecipitation.
Compared with the prior art, the invention has the following beneficial effects:
The invention connects human brain lncRNA to the p-gRNA carrier, so that the lncRNA sequence carries a gRNA label and is transcribed into a gRNA-lncRNA sequence in cells; the SBP-Csy4 fusion protein is combined with the gRNA sequence in cells to form an SBP-Csy4_gRNA-lncRNA structure, and simultaneously, a protein with interaction with human brain lncRNA is combined with the SBP-Csy4_gRNA-lncRNA-interaction protein complex, and the protein with interaction with human brain lncRNA is qualitatively and quantitatively analyzed through the existing mass spectrometry technology and the like.
Compared with the probe method, the method for identifying the protein interacted with the human brain lncRNA requires fewer cells, is only 1/20 of that of the probe method, does not need to use an antibody, and has the advantages of being free from the limitation of the antibody and higher in flux compared with the immunoprecipitation method.
Detailed Description
For a better description of the objects, technical solutions and advantages of the present invention, the present invention will be further described with reference to the following specific examples. It will be appreciated by persons skilled in the art that the specific embodiments described herein are for purposes of illustration only and are not intended to be limiting.
The test methods used in the examples are conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are all commercially available.
Example 1
The embodiment provides a carrier p-gRNA of a pulldown system loaded with lncRNA sequences, wherein the nucleotide sequence from the 5 'end to the 3' end of the p-gRNA is shown as SEQ ID NO. 1.
Wherein the gRNA sequence in the p-gRNA vector is 5'-ACTGCCGTATAGGCAG-3' (SEQ ID NO. 2) located 100bp downstream of the CMV promoter. The sequence obtained after transcription of the gRNA sequence can form a hairpin structure, and can be specifically identified and combined by Csy4 protein of pseudomonas aeruginosa. The gateway cloning sites and ccdB resistance genes of attR1 and attR2 are reserved on the p-gRNA vector, and a large-scale vector construction can be carried out by using a gateway cloning method.
The gRNA sequence of the p-gRNA vector is followed by EcoRV and HindIII restriction sites, between which are the gateway cloning sites for attR1 and attR2 and the ccdB resistance gene. The lncRNA sequence was successfully tagged with a gRNA tag by ligating the target lncRNA sequence to the gRNA sequence of the p-gRNA vector of the invention, which would be transcribed into a gRNA-lncRNA sequence when expressed in the cell.
The construction method of the carrier 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 this laboratory and the sequence of the original vector (Original vector sequence) is shown in SEQ ID NO. 14. The double digestion was performed by incubating at 37℃for 2 hours according to the system described in Table 1 below, then fragment analysis was performed by 1% agarose gel electrophoresis, and fragments of 6000 to 7000bp in size were recovered by gel digestion, and the concentration of the backbone fragments was determined by ultraviolet spectrophotometry.
TABLE 1
| Component (A) | Usage amount |
| Nhe Ⅰ(FastDigest,Thermo Scientific) | 2μl |
| EcoR Ⅴ(FastDigest,Thermo Scientific) | 2μl |
| FastDigest buffer | 5μl |
| Original plasmid | 12μg |
| ddH2O | Make up to 50. Mu.l |
(2) Primers carrying gRNA sequences were synthesized in Huada genes:
Grna-F:5’-CGgctagcggtggcggtggctctGTTCACTGCCGTATAGGCAGgatatc-3’(SEQ ID NO.3)；
Grna-R:5’-CGgatatcCTGCCTATACGGCAGTGAACagagccaccgccaccgctagc-3’(SEQ ID NO.4)；
(3) Obtaining a double-stranded gRNA sequence fragment: and (3) diluting Grna-F and Grna-R primers into 100 mu M, preparing an annealing reaction system by taking 1 mu L of each PCR tube in 200 mu L according to a system shown in the following table 2, annealing to form double chains, placing 20 mu L of the PCR tube of the annealing reaction system into water when a water bath is heated to 95 ℃, closing a power supply of the water bath after 5min, carrying out enzyme digestion on 20 mu L of the annealing reaction product by the same method in (1) when the water temperature in the water bath is reduced to room temperature, recovering the enzyme digestion product by a nucleic acid purification column, and measuring the concentration of double-chain gRNA sequence fragments by an ultraviolet spectrophotometry.
TABLE 2
| Component (A) | Usage amount |
| Grna-F(100μM) | 1μl |
| Grna-R(100μM) | 1μl |
| Ex taq buffer | 2μl |
| ddH2O | Up to 20. Mu.l |
(4) Obtaining p-gRNA vector: and (3) connecting the vector skeleton obtained in the step (1) and the double-stranded gRNA sequence fragment obtained in the step (3) by using T4 ligase, transforming DH5 alpha competent cells by using the connection product, plating on LB plates carrying ampicillin antibiotics, selecting a monoclonal after overnight culture for Sanger sequencing identification, and returning clones with the sequencing result consistent with the expected result to obtain the p-gRNA vector.
A nucleotide sequence from 5 'end to 3' end of the pulldown system SBP-Csy4 fusion protein vector is shown as SEQ ID NO.5, and the sequence of the SBP-Csy4 fusion protein vector is characterized in that: the CMV promoter is followed by a 139bp random sequence followed by an SBP peptide fragment of 38 amino acids as translation product, followed by a 20 amino acid linker sequence of amino acid: GGGGSGGGGSGGGGSGGGGS (SEQ ID NO. 6) is followed by the Csy4 protein sequence, the T2A sequence and the Blasticidin (BSD) resistance gene. The vector also has an ampicillin resistance gene and a kana resistance gene.
The amino acid sequence of the SBP peptide fragment is as follows:
MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP(SEQ ID
No. 7), SBP peptide fragment can bind to streptavidine 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 a space for forming a space conformation of the SBP peptide segment and the Csy4 protein respectively so as to exert respective functions; multiple resistance genes facilitate screening of clones.
The Csy4 protein sequence is a functional protein molecule which introduces an amino acid mutation based on a wild type Pseudomonas aeruginosa Csy4 protein sequence, namely 29 th histidine (H) is changed into alanine (A) -H29A mutation, and the introduced H29A mutation changes Csy4 protein from a restriction enzyme activity which can be combined with gRNA sequence and is cut downstream of gRNA sequence to a functional protein molecule which only retains the binding activity with gRNA sequence but loses the restriction enzyme activity.
The SBP-Csy4 fusion protein can bind intracellular to the gRNA sequence on the gRNA-lncRNA, and the SBP peptide fragment can bind to the strepavidin magnetic beads when the cells are disrupted to release the intracellular protein. The invention can form covalent connection between the SBP-Csy4 protein and the gRNA in cells simultaneously expressing the SBP-Csy4 protein and the gRNA-lncRNA by utilizing ultraviolet crosslinking or formaldehyde crosslinking, and can pull down the complex of SBP-Csy4_gRNA-lncRNA_interaction protein by using pulluown, and can perform qualitative and quantitative analysis on the protein interacted with the lncRNA by using a mass spectrometry technology.
The construction method of the SBP-Csy4 fusion protein carrier of the pulldown system comprises the following steps:
(1) Synthesis of Csy4 (already containing H29A mutation) gene sequence: linker-Csy4 sequences were synthesized at the 11/v (Shanghai) and subcloned into pcDNA3.1 (+) vector;
(2) Acquisition of SBP-Csy4 sequence and T2A-BSD sequence: the following primers were synthesized in the 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, 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) are obtained by PCR through a 2X Phanta Max Master Mix kit (the PCR system is carried out according to the specification), and 1% agarose gel analysis and gel recovery of target sequences (41 mu l ddH2O elution) are carried out on PCR products; SBP sequence was then added to the 5' end (primer) of linker-Csy4 sequence by overlap extension PCR (Splicing by overlap extension PCR, SOE-PCR) to obtain SBP-Csy4 sequence (41. Mu.l ddH2O eluted) by agarose gel analysis and gel recovery, SOE-PCR system as shown in Table 3 below, SOE-PCR conditions were set according to the 2X Phanta Max Master Mix kit instructions.
TABLE 3 Table 3
| Component (A) | Usage amount |
| 2×Phanta mix | 25μl |
| sbp-f(10μM) | 1.2μl |
| sbp-r(10μM) | 0.3μl |
| CSY4-R(10μM) | 1.2μl |
| Linker-Csy4 template | 50ng |
| ddH2O | Up to 20. Mu.l |
(3) Double cleavage of vector backbone and SBP-Csy4 fragment, T2A-BSD fragment: the double cleavage systems of SBP-Csy4 fragment and T2A-BSD fragment are shown in the following Table 4, the cleavage conditions are that the cleavage is carried out at 37 ℃ for 2 hours, the cleavage products are recovered by a nucleic acid purification column and the concentration of the recovered fragments is determined by ultraviolet spectrophotometry; mu.g of pcDNA3.1 (+) vector was taken, the digested product was recovered by a nucleic acid purification column (44. Mu.l ddH 2 O eluted) after single digestion with EcoRI for 2h, then single digestion was performed with NotI for 4h, the digested product was recovered by a nucleic acid purification column and the concentration of pcDNA3.1 (+) -EcoRI_NotI backbone fragment was determined by UV spectrophotometry.
TABLE 4 Table 4
(4) SBP-Csy4 fusion protein vector acquisition: bglII and BamHI are isoceric enzymes, so that the cohesive ends of the pcDNA3.1 (+) -EcoRI_NotI backbone, SBP-Csy4 fragment and T2A-BSD fragment recovered after double digestion can be ligated in sequence using T4 ligase, the system is shown in Table 5 below, 8. Mu.l ligation product is used to transform DH 5. Alpha. Competent cells after ligation and plated on LB plates carrying ampicillin antibiotics, and after overnight incubation, the single clones are selected for Sanger sequencing identification, and clones whose sequencing results match those expected are returned to obtain the SBP-Csy4 fusion protein vector.
TABLE 5
| Component (A) | Usage amount |
| T4 DNA Ligase(Thermo Scientific) | 1μl |
| T4 10x T4 buffer(Thermo Scientific) | 2μl |
| PcDNA3.1 (+) -EcoRI-NotI backbone | 100ng |
| SBP-Csy4 fragment | 200ng |
| T2A-BSD fragment | 200ng |
| ddH2O | Up to 20. Mu.l |
The existing methods for studying the interaction of lncRNA-protein(s) comprise an in vitro transcription method, an immunoprecipitation method, a probe method and the like. The results of interactions between lncRNA and protein obtained by in vitro transcription may differ from the results of interactions actually occurring within the cell; immunoprecipitation methods, which focus on protein-based studies of lncRNA sequences interacting with target proteins, are not applicable to lncRNA-centered research protocols, and are limited to use with specific antibodies; the probe method relies on endogenous expression of lncRNA, and experimental results can reflect the actually occurring lncRNA-protein(s) interaction, but the method faces the realistic challenge that the endogenous expression level of lncRNA is very low, so that 20-200 megacells are required to be used in the probe method, and more manpower and financial support is required in large-scale experiments.
Compared with the existing probe method, the pulludown system has the advantages that the required cell number is less, and the required cell number is about 1/20 of that of the probe method; the pulldown system provided by the invention does not need an antibody, and can complete the construction of the p-gRNA-lncRNA vector by only placing the lncRNA sequence and the gRNA sequence on the same downstream of the promoter on the target vector, so that the qualitative and quantitative analysis of the proteins interacted with the lncRNA can be realized by combining with the SBP-Csy4 fusion protein vector.
Finally, it should be noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present invention may be modified or substituted equally without departing from the spirit and scope of the technical solution of the present invention.
Claims (4)
1. A vector for identifying a protein that interacts with human brain lncRNA, wherein the vector comprises a vector p-gRNA and SBP-Csy4 fusion protein vector for loading human brain lncRNA sequences; the p-gRNA is used for connecting target human brain lncRNA, so that the lncRNA sequence is provided with a gRNA label; the nucleotide sequence of the gRNA is 5'-ACTGCCGTATAGGCAG-3'; in the vector p-gRNA, 5'-ACUGCCGUAUAGGCAG-3' sequences obtained after transcription of the gRNA sequences form a hairpin structure, and can be specifically identified and combined by Csy4 protein of pseudomonas aeruginosa; the nucleotide sequence from the 5 'end to the 3' end of the vector p-gRNA is shown as SEQ ID NO. 1; the nucleotide sequence from the 5 'end to the 3' end of the SBP-Csy4 fusion protein vector is shown as SEQ ID NO. 5.
2. The method for constructing the vector p-gRNA for loading the human brain lncRNA sequence according to claim 1, wherein the method comprises the following steps:
Synthesizing a double-stranded gRNA sequence fragment based on primers Grna-F and Grna-R carrying the gRNA sequence; connecting the obtained double-stranded gRNA sequence fragment to a vector framework, and obtaining a p-gRNA vector through transformation, cloning and sequencing;
the Grna-F has the sequence:
5’-CGgctagcggtggcggtggctctGTTCACTGCCGTATAGGCAGgatatc-3’；
The Grna-R has the sequence:
5’-CGgatatcCTGCCTATACGGCAGTGAACagagccaccgccaccgctagc-3’。
3. The method for constructing a vector for identifying a human brain lncRNA interacting protein according to claim 1, wherein the method for constructing the SBP-Csy4 fusion protein vector comprises the steps of:
1) Synthesizing Csy4 gene sequence containing H29A mutation;
2) Obtaining SBP-Csy4 fragment and T2A-BSD fragment based on the corresponding primers, respectively;
3) And (3) carrying out enzyme digestion and recovery on the carrier skeleton, the SBP-Csy4 fragment and the T2A-BSD fragment, sequentially connecting the adhesive tail ends of the carrier skeleton, the SBP-Csy4 fragment and the T2A-BSD fragment, and obtaining the SBP-Csy4 fusion protein carrier through sequencing identification.
4. A method for identifying proteins interacting with human brain lncRNA based on the vector of any one of claims 1-3, comprising the steps of:
Ligating human brain lncRNA to the p-gRNA vector to form p-gRNA-lncRNA; the p-gRNA-lncRNA is transcribed in the cell to a gRNA-lncRNA sequence;
The SBP-Csy4 fusion protein expressed by the SBP-Csy4 fusion protein carrier is combined with the gRNA sequence in cells to form an SBP-Csy4_gRNA-lncRNA structure, and simultaneously, a protein with interaction with human brain lncRNA is combined with the SBP-Csy4_gRNA-lncRNA-interacting protein complex, and the protein interacted with human brain lncRNA is identified based on the complex.
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