CN117568369A - Protein system for rapidly screening binding with decoy RNA, method and application - Google Patents
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Abstract
The invention discloses a protein system for rapidly screening and combining with decoy RNA, a method and application thereof, wherein the system comprises an induction expression suicide gene-decoy vector and an RNAse-to-be-selected RNA combined protein fusion expression vector. The inducible expression suicide gene-decoy vector is an inducible expression hybrid RNA, and contains a suicide gene expression frame and decoy RNA; the fusion expression vector of the RNase-the RNA binding protein to be selected is a fusion protein formed by the RNase and the RNA binding protein to be selected through a genetic engineering method; when the RNA-binding protein of the fusion protein specifically recognizes the decoy RNA, the RNase activity of the fusion protein destroys the expression-inducing hybrid RNA, and reduces the expression-inducing suicide gene, thereby allowing the cell to survive, or else the cell dies. The invention realizes the specific, efficient and simple screening of the protein combined with RNA, can be used for developing any RNA targeting drug, and is simple and practical.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to a protein system for rapidly screening binding with decoy RNA, a method and application thereof.
Background
RNA Binding Proteins (RBPs) are generally considered as proteins that bind RNA and alter the fate or function of the bound RNA through one or more RNA Binding Domains (RBDs). These RBPs widely control each stage of the RNA lifecycle, including mRNA synthesis and maturation, activation, cleavage, degradation of non-coding RNAs (ncrnas), etc., and thus RNA-protein interactions are increasingly being considered as a key loop in RNA-protein interactions, transcriptome and proteome regulation between viruses/pathogens and host cells. Therefore, the dynamic change of the RBPs in cell functions and disease states is of great importance for researchers to know the physiological functions and disease progression in complex systems, and has great clinical significance for screening effective interfering polypeptide drugs.
In recent years, in order to fully identify these RNA binding proteins and elucidate the dynamic assembly behavior of RBPs, researchers have developed a number of technological means for capturing RNA binding proteins. The more commonly used methods for capturing RNA binding proteins can be mainly divided into a targeting method and an unbiased method, wherein the former method is used for capturing RNA to be studied, and the interaction proteins are pulled down by a series of methods based on AP-MS or APEX; the latter is based on the inherent properties of the nucleic acid-protein complex. Although a series of research techniques have been developed, there are more or less insufficient factors in either technique. Two important problems derived from this: first, how much does the in vitro identified RNA-protein interactions reflect in vivo? Second, do there interaction mean there is a function? In view of these problems, there is still a need to further develop new research techniques for RNA-protein interactions. In addition, false positive signals, high sample consumption, strong background interference and high cost are main defects in the prior art.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a protein system for rapidly screening and combining with decoy RNA, a method and application thereof, which can be applied to the field of virus gene therapy, can be used for researching physiological functions and disease progression, provides beneficial information for drug research and development, has the advantages of accuracy, rapidness, simple operation and the like, and is suitable for the fields of gene function research, virus nucleic acid removal and the like.
The invention is realized by the following technical scheme:
a protein system for rapid screening of binding to decoy RNA, comprising inducible expression of a suicide gene-decoy vector and an rnase-candidate RNA binding protein fusion expression vector;
the inducible expression suicide gene-decoy vector is an inducible expression hybrid RNA, and contains a suicide gene expression frame and decoy RNA; the suicide gene is any gene coded by the gene and can cause cytotoxicity to cause cell death;
the RNA enzyme-to-be-selected RNA binding protein fusion expression vector is SA-NCD-to-be-selected RNA binding protein fusion expression vector, wherein the RNA enzyme is RNA enzyme activity domain NCD of human MCPIP1, streptavidin SA and NCD are constructed together to form recombinant protein of SA-NCD through a genetic engineering means, and then the SA-NCD is connected with a to-be-selected RNA binding protein coding sequence to construct the SA-NCD-to-be-selected RNA binding protein fusion expression vector;
when the RNA to be selected in the RNase-RNA to be selected binding protein fusion expression vector specifically recognizes the decoy RNA, the RNase activity in the fusion expression vector damages the hybrid RNA of induced expression, so that the suicide gene of induced expression is reduced, and the cell is alive, otherwise, the cell dies, and the gene information combined with the decoy RNA can be known by detecting the information of the fusion expression vector of the living cell, so that the RBP gene sequence is obtained, and the rapid screening is realized.
Preferably, the suicide gene is a saporin protein sequence as shown in SEQ ID NO. 2; the sequence of the SA-NCD recombinant protein is shown as SEQ ID NO. 1.
Preferably, the candidate RNA binding protein is a defined RNA binding protein or a gene expression library.
A screening method based on the rapid screening of protein systems binding to decoy RNA described above, comprising the steps of:
step 1) construction of an inducible expression suicide gene-bait vector:
selecting any gene coded by the gene and capable of causing cytotoxicity to cause cell death as a suicide gene, and constructing a suicide gene-bait inducing parent vector; synthesizing a decoy RNA sequence to be selected by using RT-PCR or chemical synthesis technology; introducing a decoy RNA sequence into a multiple cloning site of the suicide gene-decoy inducing parent vector by a conventional genetic engineering technology to form a lentivirus expression vector, namely the suicide gene-decoy inducing vector;
step 2) construction of an RNase-candidate RNA binding protein fusion expression vector:
constructing a parent vector of an RNase-candidate RNA binding protein fusion expression vector; according to different purposes, a method for constructing or directly amplifying/synthesizing a coding sequence of the RNA binding protein to be selected by using an expression library; introducing the coding sequence of the RNA binding protein to be selected into the multiple cloning site of the parent vector of the RNase-RNA binding protein to be selected fusion expression vector through a conventional genetic engineering technology to form a lentiviral expression vector, namely the RNase-RNA binding protein to be selected fusion expression vector;
step 3) establishing a stable expression cell line for inducing suicide gene expression, and screening the concentration of the lowest lethal induction reagent:
infecting eukaryotic cells 293 with the induction expression suicide gene-bait vector obtained in the step 1) in a slow virus mode, selecting single cell clones, and amplifying and breeding to obtain steady-state expression cell strains; the steady-state expression cell strain induces suicide gene expression by adding exogenous compounds with different concentrations, and according to different used induction reagents, systematic concentration attempt is required until the lowest concentration capable of killing all cells is found out and is used as the concentration of the induction reagent;
step 4) infecting the steady-state expression cell strain obtained in step 3) by using the RNase-candidate RNA binding protein fusion expression vector obtained in step 2), adding an induction reagent to kill cells incapable of cutting the suicide gene in a targeted manner, wherein the surviving cells contain a vRBD sequence:
infecting the steady-state expression cell strain obtained in the step 3) in a slow virus form by using the RNase-candidate RNA binding protein fusion expression vector obtained in the step 2); screening by using the concentration of the induction reagent obtained in the step 3) until the cells are still viable under the screening pressure, wherein the obtained cells possibly contain proteins with target RNA cells;
step 5) obtaining a vRBP gene sequence by DNA recovery and sequencing, and finishing screening:
extracting the genome DNA of the cells obtained in the step 4), and designing primers by using the sequences at two sides of the multiple cloning sites on the RNase-candidate RNA binding protein fusion expression vector obtained in the step 2); and then taking the genomic DNA of the cells obtained in the step 4) as a template, carrying out PCR amplification by using a primer, then carrying out sequencing by using a high-throughput sequencing method, and analyzing the sequence information to obtain the protein information possibly combined with target RNA so as to finish screening.
Preferably, the parent vector of the suicide gene-inducing bait in the step 1) is PCDH-SA-NCD-MCS, and the construction method is as follows:
performing PCR amplification by using the SA-NCD sequence as a template through primers SA-NCD-F and SA-NCD-R, purifying a PCR product, and treating the purified product by using restriction enzymes XbaI and EcoR 1; simultaneously taking PCDH vector, also using XbaI and EcoR1 for enzyme digestion, then performing gel electrophoresis, cutting large fragments and recovering products; connecting the enzyme digestion products by adopting T4 DNA ligase; adding the connection product into competent cells for connection, and coating the connection product on an LB solid plate; performing enzyme digestion and sequencing identification on the monoclonal antibody to obtain the monoclonal antibody;
wherein, the SA-NCD coding sequence is shown as SEQ ID NO. 3; the sequence of the primer SA-NCD-F is shown as SEQ ID NO. 4; the sequence of the primer SA-NCD-R is shown as SEQ ID NO. 5.
Preferably, the master vector of the RNase-candidate RNA binding protein fusion expression vector in the step 2) is pLVX-TetOne-mCherry-2A-SAP2-MCS-Puro, and the construction method is as follows:
PCR amplification was performed by using the mCherry-2A-SAP2 sequence as a template through the primers mCherry-SAP2-F and mCherry-SAP2-R, and after purification of the PCR product, the purified product was treated with restriction enzymes BamH1 and EcoR 1; simultaneously taking pLVX-TetOne-Puro vector, also using BamH1 and EcoR1 for enzyme digestion, then performing gel electrophoresis, cutting large fragments and recovering products; connecting the enzyme digestion products by adopting T4 DNA ligase; adding the connection product into competent cells for connection, and coating the connection product on an LB solid plate; performing enzyme digestion and sequencing identification on the monoclonal antibody to obtain the monoclonal antibody;
wherein the mCherry-2A-SAP2 coding sequence is shown in SEQ ID NO. 6; the primer mCherry-SAP2-F has a sequence shown in SEQ ID NO. 7; the primer mCherry-SAP2-R has a sequence shown in SEQ ID NO. 8.
Preferably, the exogenous compound of step 3) is doxycycline or a derivative thereof.
The use of the above described rapid screening protein system that binds decoy RNA in the preparation of a drug for targeting RNA.
The application of the screening method in preparing medicaments for targeting RNA.
The beneficial effects of the invention are as follows:
(1) The invention provides a protein system for rapidly screening binding with decoy RNA, which mainly comprises two parts: inducing expression of suicide gene-decoy vector and RNAse-candidate RNA binding protein fusion expression vector. The inducible expression suicide gene-decoy vector is an inducible expression hybrid RNA, and contains a suicide gene expression frame and decoy RNA; the fusion expression vector of the RNase and the RNA binding protein to be selected is a fusion protein formed by the RNase and the RNA binding protein to be selected through a genetic engineering method; when the RNA-binding protein of the fusion protein specifically recognizes the decoy RNA, the RNase activity of the fusion protein destroys the expression-inducing hybrid RNA, and reduces the expression-inducing suicide gene, thereby allowing the cell to survive, or else the cell dies. The method of the invention realizes specific, efficient and simple screening of the protein combined with RNA, can be used for developing any RNA targeting drug, and is simple and practical.
(2) Compared with other RNA binding protein screening methods (such as bacterial or yeast three-hybrid systems), the method can fully modify the protein, and better reflect the state of the protein in the mammalian cells, so that more real results can be obtained; the method is carried out according to popular molecular biology methods, and the required reagents and instruments are all commonly used without special purchase.
Drawings
FIG. 1 is a schematic diagram of the major elements of the PCDH-SA-NCD-MCS master vector of example 1;
FIG. 2 is a schematic diagram of the pLVX-TetOne-Puro vector in example 2;
FIG. 3 is a schematic diagram of a rapid screening protein system for binding to decoy RNA in example 3;
FIG. 4 is a flow chart of the fluorescent reporting method used in example 4 to verify that SA-NCD-PP7cp cleaves PP7bs containing PP7cp protein target;
FIG. 5 shows the results of an experiment in example 4 in which SA-NCD-PP7cp cleavage contains mCherry-PP7 cp: a is the experimental procedure (left) and the fluorescence display result (right); b is the result of real-time quantitative PCR identification of mCherry mRNA levels.
Detailed Description
The invention will be described in further detail with reference to the drawings and the specific embodiments.
EXAMPLE 1PCDH-SA-NCD-MCS master vector construction
1. Chemical synthesis of SA-NCD and primer sequences for vector construction
The SA-NCD coding sequence is synthesized and is shown as SEQ ID NO. 3.
Synthesizing a primer SA-NCD-F sequence shown as SEQ ID NO. 4;
the sequence of the synthetic primer SA-NCD-R is shown as SEQ ID NO. 5.
2. 1nmol of synthesized SA-NCD coding sequence is taken as a template, and a PCR amplification system (NEB) is configured by primers SA-NCD-F and SA-NCD-R with final concentration of 0.25 mu MDNA polymerase, cat# M0254V) and PCR reagents were added according to the instructions, 100. Mu.L of PCR product was purified after amplification, and then restriction enzyme Xba was usedTreatment of the purified products with I (NEB) and EcoR1 (NEB); simultaneously, 10. Mu.g of PCDH vector (supplied from Nanjing PPL plasmid and protein sharing library) was taken, and the same was digested with XbaI and EcoR1 at 37℃for 2 hours, followed by 1% agarose gel electrophoresis, and the large fragment was excised with a blade under an ultraviolet lamp and the product was recovered.
3. The cleavage products obtained in the above steps were ligated using T4 DNA ligase (NEB), and the ligation was performed according to the instructions.
4. mu.L of the ligation product was added to 100. Mu.L of competent cells (Shanghai) and transformed into recombinant plasmid according to instructions and plated on LB solid plates containing ampicillin.
5. And (3) carrying out enzyme digestion and sequencing identification on the monoclonal to obtain a recombinant vector PCDH-SA-NCD-MCS (parent vector).
As shown in FIG. 1, the main element of the parent vector is SA-NCD, wherein MCS is a multiple cloning site, and the expressed gene of the RNA binding protein to be selected can be inserted into MCS by conventional enzyme digestion connection or by using the current more popular seamless cloning mode, and finally the SA-NCD-the RNA binding protein to be selected is expressed in eukaryotic cells such as common 293 cells.
EXAMPLE 2pLVX-TetOne-mCherry-2A-SAP2-MCS-Puro Master vector construction
1. Chemical synthesis of mCherry-2A-SAP2 and primer sequences for vector construction
The mCherry-2A-SAP2 coding sequence is synthesized and shown as SEQ ID NO. 6.
The sequence of a primer mCherry-SAP2-F is synthesized and is shown as SEQ ID NO. 7;
the sequence of the primer mCherry-SAP2-R is synthesized and is shown as SEQ ID NO. 8.
2. 1nmol of the synthesized mCherry-2A-SAP2 coding sequence was used as a template, and primers mCherry-SAP2-F and mCherry-SAP2-R were used to prepare PCR amplification systems (NEB) at a final concentration of 0.25. Mu.MDNA polymerase, cat# M0254V) and PCR reagents were added according to the instructions, 100. Mu.L of the PCR product was purified after amplification, and restriction enzymes BamH1 (NEB) and EcoR1 #NEB) treating the purified product; simultaneously, 10. Mu.g of pLVX-TetOne-Puro vector (supplied by Nanjing PPL plasmid and protein sharing library) was taken, and the same was digested with BamH1 and EcoR1 at 37℃for 2 hours, followed by 1% agarose gel electrophoresis, and the large fragment was excised with a blade under an ultraviolet lamp and the product was recovered.
The pLVX-TetOne-Puro vector is shown in FIG. 2, wherein the fluorescent protein mCherry gene and the suicide gene are both regulated and controlled by an induction expression system together for transcription of mCherry-SAP, the SAP gene is followed by a multiple cloning site MCS for insertion of a bait sequence, and the transcription level of the mCherry-SAP-bait has a positive correlation with the quantity of the induction drug.
3. The cleavage products obtained in the above steps were ligated using T4 DNA ligase (NEB), and the ligation was performed according to the instructions.
4. mu.L of the ligation product was added to 100. Mu.L of competent cells (Shanghai) and transformed into recombinant plasmid according to instructions and plated on LB solid plates containing ampicillin.
5. And (3) carrying out enzyme digestion and sequencing identification on the monoclonal to obtain a recombinant vector pLVX-TetOne-mCherry-2A-SAP2-MCS-Puro (parent vector).
Example 3
A rapid screening protein system for binding to decoy RNA, as shown in FIG. 3, comprises inducible expression of a suicide gene-decoy vector and an RNase-candidate RNA binding protein fusion expression vector.
The inducible expression suicide gene-decoy vector is an inducible expression hybrid RNA, and contains a suicide gene expression frame and decoy RNA; the suicide gene may be any gene encoded by a gene that causes cytotoxicity resulting in cell death, including but not limited to selecting the gene coding region of Saporin (Saporin) as the suicide gene.
The rnase in the rnase-candidate RNA binding protein fusion expression vector may be any protein or active domain having rnase activity, including but not limited to, the rnase active domain NCD of the selected human MCPIP1, and in order to form multimeric active protein from NCD, streptavidin SA and NCD are genetically engineered together to form a recombinant protein of SA-NCD, the SA-NCD may be followed by ligation to the coding sequence of the candidate RNA binding protein, and the sequence of the recombinant protein of SA-NCD according to this example is shown in SEQ ID No. 1.
The candidate RNA binding protein may be the exact RNA binding protein obtained by other means, or may be a gene expression library.
When the RNA to be selected in the RNase-RNA to be selected binding protein fusion expression vector specifically recognizes the decoy RNA, the RNase activity in the fusion expression vector damages the hybrid RNA of induced expression, so that the suicide gene of induced expression is reduced, and the cell is alive, otherwise, the cell dies, and the gene information combined with the decoy RNA can be known by detecting the information of the fusion expression vector of the living cell, so that the RBP gene sequence is obtained, and the rapid screening is realized.
Based on the above system, the embodiment provides a method for rapidly screening RNA binding proteins, which comprises the following specific steps:
(1) Construction of inducible expression suicide Gene-bait vector
Any gene which is coded by the gene and can cause cytotoxicity to cause cell death is selected as a suicide gene, the suicide gene related in the embodiment is a protein sequence of Saporin (Saporin) (shown as SEQ ID NO. 2), and therefore, the used parent vector for inducing suicide gene-bait is the vector pLVX-TetOne-mCherry-2A-SAP2-MCS-Puro obtained in the embodiment 2; synthesizing a decoy RNA sequence of interest by RT-PCR or chemical synthesis; the bait RNA sequence is led into the multiple cloning site of the mother vector pLVX-TetOne-mCherry-2A-SAP2-MCS-Puro by the conventional genetic engineering technology to form a slow virus expression vector, namely the induction expression suicide gene-bait vector.
The decoy RNA of interest is a fixed sequence, corresponding to a fixed subject, and the method involves screening for proteins or polypeptides that bind to the RNA.
(2) Construction of an RNase-alternative RNA binding protein fusion expression vector
The mother vector of the RNase-candidate RNA binding protein fusion expression vector in this example is the PCDH-SA-NCD-MCS obtained in example 1; constructing or directly amplifying/synthesizing the coding sequence of the RNA binding protein to be selected by using an expression library according to different purposes; the coding sequence of the RNA binding protein to be selected is led into the multiple cloning site of the parent vector PCDH-SA-NCD-MCS by the conventional genetic engineering technology to form a lentiviral expression vector, namely the RNAse-RNA binding protein to be selected fusion expression vector.
(3) The steady-state expression cell strain for inducing suicide gene expression is established, and the concentration of the lowest lethal induction reagent is screened, and the method is as follows:
and (3) mixing the inducible expression suicide gene-bait vector obtained in the step (1) with psPAX2 and pMD2.G in a molar ratio of 1:1 to 1, co-transfecting the mixture into 293T cells, packaging the cells into slow viruses, infecting the newly inoculated 293T cells with a plurality of infection numbers of 1, screening the cells infected with the slow viruses with 1 mug/mL puromycin for 2 days after 3-5 days, carrying out monoclonalization after screening, and culturing the selected positive clones to obtain steady-state expression cell strains.
Steady state expression cell lines induce suicide gene expression by adding exogenous compounds such as tetracycline (tetracycline) or its derivative doxycycline (doxycycline Dox) at different concentrations, depending on the inducing agent used, a systematic concentration attempt is required until the lowest concentration that kills all cells is found.
The establishment of the steady-state expression cell strain for inducing suicide gene expression is to infect eukaryotic cells 293 in a slow virus form through the suicide gene-bait vector, select single cell clone and expand and reproduce to obtain the steady-state expression cell strain; the concentration of the inducing agent is confirmed by adding a medium containing a series of concentrations of the inducing agent and then observing the lowest concentration that can cause all cell death, and it is necessary to determine the concentration of the inducing agent according to the specific vector since the inserted bait sequence may affect the protein expression level.
(4) Infecting the cells obtained in the step (3) by using an RNase-alternative RNA binding protein fusion expression vector, adding an induction reagent to kill the cells incapable of targeted cleavage of the suicide gene, wherein the surviving cells contain a vRBD sequence, and the method comprises the following steps of:
co-transfecting the RNase-candidate RNA binding protein fusion expression vector obtained in the step (2) with psPAX2 and pMD2.G in a molar ratio of 1:1:1 into 293T cells, packaging the cells into lentiviruses, and then infecting the steady-state expression cells obtained in the step (3) by a plurality of 1 s, wherein the amount of the infected cells is determined according to the reservoir capacity of the constructed RNase-candidate RNA binding protein fusion expression vector, and if the RNase-candidate RNA binding protein fusion expression vector contains n mixtures, the number of the cells to be infected is not less than 2 n. 3 days after infection with the virus, the cells obtained may contain proteins with the target RNA cells by screening with the lowest concentration that kills all cells obtained in step (3) until the cells remain viable under the screening pressure.
(5) The vRBP gene sequence is obtained through DNA recovery and sequencing, and the screening is completed, specifically as follows:
extracting the genomic DNA of the cells obtained in the step (4), and then designing the following pair of primers by using the sequences on two sides of the multiple cloning site on the RNase-candidate RNA binding protein fusion expression vector obtained in the step (2):
SEQ-F(SEQ ID NO.9)：CTGCGTAAGAAGCCACTCAC；
SEQ-R(SEQ ID NO.10)：AGCGATCGCAGATCCTTCGC；
the gene DNA is used as a template, SEQ-F and SEQ-R are used as primers, PCR amplification is carried out, then sequencing is carried out by a high-throughput sequencing method, and protein information possibly combined with target RNA can be obtained by analyzing sequence information.
Example 4
Identification of phage PP7 envelope protein PP7cp and target binding PP7bs using a rapid screening protein system that binds to decoy RNA as described in example 3 demonstrates that eukaryotic expression of SA-NCD-PP7cp can act on PP7bs. The principle of verifying whether SA-NCD-PP7cp protein degrades RNA containing target PP7bs is shown in FIG. 4, specifically by the following method:
1. the PP7bs primer sequence is synthesized, as shown in SEQ ID No.11-12, two primers are subjected to phosphorylation modification by using T4 DNA kinase (NEB) according to the specification, and the two primers are subjected to double digestion with BamH1 and EcoR1 to form a fluorescent report carrier mCherry-C by a carrier construction method under the action of T4 DNA ligase, so that the carrier mCherry-PP7bs is constructed.
2. Chemical synthesis of PP7cp and primer sequences for vector construction
The PP7cp coding sequence is synthesized as shown in SEQ ID NO. 13.
The sequence of the primer PP7cp-F is synthesized and is shown as SEQ ID NO. 14;
the sequence of the synthetic primer PP7cp-R is shown as SEQ ID NO. 15.
3. The recombinant vector PCDH-SA-NCD-PP7cp was obtained by constructing on the PCDH-SA-NCD-MCS vector of example 1 by the conventional vector construction method.
4. The constructed vector mCherry-PP7bs and PCDH-SA-NCD-PP7cp were co-expressed in 293T cells, and the intensity of red fluorescence mCherry was detected by fluorescence microscopy after 48 hours, as shown in fig. 5.
5. Experimental results
As shown in FIG. 5A, the co-transfected SA-NCD-PP7cp can reduce the expression of red light, namely mCherry, whereas the co-transfected empty vector shows stronger red fluorescence, which indicates that the SA-NCD-PP7cp can effectively degrade target RNA; the results of real-time quantitative PCR identification of mRNA levels of mCherry are shown in fig. 5B, consistent with the results (protein levels) of a in fig. 5.
The analysis results show that SA-NCD-PP7cp can effectively reduce fluorescence intensity, and the SA-NCD-PP7cp binding protein can degrade the binding sequence containing the corresponding RNA binding protein.
Example 5
The protein bound to the RRE sequence in HIV1 was screened based on the system and method described in example 3.
The method comprises the following specific steps:
(1) RRE sequence (SEQ ID NO. 16) is constructed on the pLVX-TetOne-mCherry-2A-SAP2-MCS-Puro master vector obtained in example 2 by a vector construction method to obtain an inducible expression suicide gene-RRE recombinant vector pLVX-TetOne-mCherry-2A-SAP2-RRE-Puro; meanwhile, 9 genes expressed by HIV1 are respectively constructed on the PCDH-SA-NCD-MCS master vector obtained in the example 1, so as to obtain the RNAse-candidate RNA binding protein fusion expression vector.
(2) Packaging the recombinant vector of the induced expression suicide gene-RRE constructed in the step (1) into slow virus, then infecting 293T cells, and screening a stable expression cell strain under the action of puromycin.
(3) And (3) inducing suicide gene expression of the stably expressed cell strain obtained in the step (2) under the action of doxycycline (doxycycline Dox) to obtain the lowest concentration of 5ng/mL capable of killing the stably expressed cell strain.
(4) Transferring the RNase-to-be-selected RNA binding protein fusion expression vector constructed in the step (1) into the stable expression cell strain in the step (2), then screening by using doxycycline Dox not lower than 5ng/mL, and obtaining the gene bound with RRE as Rev by DNA sequencing of the living cells, thereby completing the screening.
Example 6
Based on the system and method described in example 3, binding sequences of Tat protein in HIV1 were screened as follows:
(1) Dividing HIV1 full-length RNA into fragments of about 2000bp, wherein each fragment has an overlapping region of 300bp, and constructing the fragments on the pLVX-TetOne-mCherry-2A-SAP2-MCS-Puro master vector in example 2 respectively to obtain a recombinant vector for inducing expression of suicide genes; meanwhile, tat was constructed on the PCDH-SA-NCD-MCS master vector of example 1 to obtain an RNase-Tat fusion expression vector.
(2) Packaging the recombinant vector constructed in the step (1) for inducing expression of suicide genes into slow viruses, then infecting 293T cells, and screening stable expression cell lines under the action of puromycin.
(3) And (3) inducing suicide gene expression by the stable expression cell strain obtained in the step (2) under the action of doxycycline (doxycycline Dox) to obtain the minimum concentration of 3 ng/. Mu.L capable of killing the stable expression cell strain.
(4) Transferring the RNase-Tat fusion expression vector constructed in the step (1) into the stable expression cell strain in the step (2), then screening with doxycycline Dox not lower than 3 ng/. Mu.L, and obtaining the RNA sequence combined with Tat containing TAR sequence from the living cells through DNA sequencing to complete the screening.
(5) In order to obtain the binding sequence of Tat more finely, the sequence can be further shortened by the obtained preliminary Tat binding sequence, and the above operation can be repeated.
The foregoing has shown and described the basic principles, principal features and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the foregoing embodiments, which have been described in the foregoing description merely illustrates the principles of the invention, and that various changes and modifications may be made therein without departing from the spirit and scope of the invention, which is defined in the appended claims.
Claims (9)
1. A protein system for rapid screening of binding to decoy RNA comprising an inducible expression suicide gene-decoy vector and an rnase-candidate RNA binding protein fusion expression vector;
the inducible expression suicide gene-decoy vector is an inducible expression hybrid RNA, and contains a suicide gene expression frame and decoy RNA; the suicide gene is any gene coded by the gene and can cause cytotoxicity to cause cell death;
the RNA enzyme-to-be-selected RNA binding protein fusion expression vector is SA-NCD-to-be-selected RNA binding protein fusion expression vector, wherein the RNA enzyme is RNA enzyme activity domain NCD of human MCPIP1, streptavidin SA and NCD are constructed together to form recombinant protein of SA-NCD through a genetic engineering means, and then the SA-NCD is connected with a to-be-selected RNA binding protein coding sequence to construct the SA-NCD-to-be-selected RNA binding protein fusion expression vector;
when the RNA to be selected in the RNase-RNA to be selected binding protein fusion expression vector specifically recognizes the decoy RNA, the RNase activity in the fusion expression vector damages the hybrid RNA of induced expression, so that the suicide gene of induced expression is reduced, and the cell is alive, otherwise, the cell dies, and the gene information combined with the decoy RNA can be known by detecting the information of the fusion expression vector of the living cell, so that the RBP gene sequence is obtained, and the rapid screening is realized.
2. The rapid screening protein system for binding to decoy RNA of claim 1, wherein the suicide gene is saporin protein sequence as shown in SEQ ID No. 2; the sequence of the SA-NCD recombinant protein is shown as SEQ ID NO. 1.
3. The rapid screening protein system for binding to decoy RNA of claim 1, wherein the candidate RNA binding protein is a defined RNA binding protein or gene expression library.
4. A screening method based on the rapid screening of a protein system binding to decoy RNA according to any one of claims 1 to 3, comprising the steps of:
step 1) construction of an inducible expression suicide gene-bait vector:
selecting any gene coded by the gene and capable of causing cytotoxicity to cause cell death as a suicide gene, and constructing a suicide gene-bait inducing parent vector; synthesizing a decoy RNA sequence to be selected by using RT-PCR or chemical synthesis technology; introducing a decoy RNA sequence into a multiple cloning site of the suicide gene-decoy inducing parent vector by a conventional genetic engineering technology to form a lentivirus expression vector, namely the suicide gene-decoy inducing vector;
step 2) construction of an RNase-candidate RNA binding protein fusion expression vector:
constructing a parent vector of an RNase-candidate RNA binding protein fusion expression vector; according to different purposes, a method for constructing or directly amplifying/synthesizing a coding sequence of the RNA binding protein to be selected by using an expression library; introducing the coding sequence of the RNA binding protein to be selected into the multiple cloning site of the parent vector of the RNase-RNA binding protein to be selected fusion expression vector through a conventional genetic engineering technology to form a lentiviral expression vector, namely the RNase-RNA binding protein to be selected fusion expression vector;
step 3) establishing a stable expression cell line for inducing suicide gene expression, and screening the concentration of the lowest lethal induction reagent: infecting eukaryotic cells 293 with the induction expression suicide gene-bait vector obtained in the step 1) in a slow virus mode, selecting single cell clones, and amplifying and breeding to obtain steady-state expression cell strains; the steady-state expression cell strain induces suicide gene expression by adding exogenous compounds with different concentrations, and according to different used induction reagents, systematic concentration attempt is required until the lowest concentration capable of killing all cells is found out and is used as the concentration of the induction reagent;
step 4) infecting the steady-state expression cell strain obtained in step 3) by using the RNase-candidate RNA binding protein fusion expression vector obtained in step 2), adding an induction reagent to kill cells incapable of cutting the suicide gene in a targeted manner, wherein the surviving cells contain a vRBD sequence:
infecting the steady-state expression cell strain obtained in the step 3) in a slow virus form by using the RNase-candidate RNA binding protein fusion expression vector obtained in the step 2); screening by using the concentration of the induction reagent obtained in the step 3) until the cells are still viable under the screening pressure, wherein the obtained cells possibly contain proteins with target RNA cells;
step 5) obtaining a vRBP gene sequence by DNA recovery and sequencing, and finishing screening:
extracting the genome DNA of the cells obtained in the step 4), and designing primers by using the sequences at two sides of the multiple cloning sites on the RNase-candidate RNA binding protein fusion expression vector obtained in the step 2); and then taking the genomic DNA of the cells obtained in the step 4) as a template, carrying out PCR amplification by using a primer, then carrying out sequencing by using a high-throughput sequencing method, and analyzing the sequence information to obtain the protein information possibly combined with target RNA so as to finish screening.
5. The screening method according to claim 4, wherein the suicide gene-decoy-inducing parent vector of step 1) is PCDH-SA-NCD-MCS, and the construction method is as follows:
performing PCR amplification by using the SA-NCD sequence as a template through primers SA-NCD-F and SA-NCD-R, purifying a PCR product, and treating the purified product by using restriction enzymes XbaI and EcoR 1; simultaneously taking PCDH vector, also using XbaI and EcoR1 for enzyme digestion, then performing gel electrophoresis, cutting large fragments and recovering products; connecting the enzyme digestion products by adopting T4 DNA ligase; adding the connection product into competent cells for connection, and coating the connection product on an LB solid plate; performing enzyme digestion and sequencing identification on the monoclonal antibody to obtain the monoclonal antibody;
wherein, the SA-NCD coding sequence is shown as SEQ ID NO. 3; the sequence of the primer SA-NCD-F is shown as SEQ ID NO. 4; the sequence of the primer SA-NCD-R is shown as SEQ ID NO. 5.
6. The method according to claim 4, wherein the master vector of the RNase-candidate RNA binding protein fusion expression vector of step 2) is pLVX-TetOne-mCherry-2A-SAP2-MCS-Puro, and the construction method is as follows:
PCR amplification was performed by using the mCherry-2A-SAP2 sequence as a template through the primers mCherry-SAP2-F and mCherry-SAP2-R, and after purification of the PCR product, the purified product was treated with restriction enzymes BamH1 and EcoR 1; simultaneously taking pLVX-TetOne-Puro vector, also using BamH1 and EcoR1 for enzyme digestion, then performing gel electrophoresis, cutting large fragments and recovering products; connecting the enzyme digestion products by adopting T4 DNA ligase; adding the connection product into competent cells for connection, and coating the connection product on an LB solid plate; performing enzyme digestion and sequencing identification on the monoclonal antibody to obtain the monoclonal antibody;
wherein the mCherry-2A-SAP2 coding sequence is shown in SEQ ID NO. 6; the primer mCherry-SAP2-F has a sequence shown in SEQ ID NO. 7; the primer mCherry-SAP2-R has a sequence shown in SEQ ID NO. 8.
7. The method of screening according to claim 4, wherein the exogenous compound in step 3) is doxycycline or a derivative thereof.
8. Use of a rapid screening protein system binding to decoy RNA according to any of claims 1 to 3 for the preparation of a medicament for targeting RNA.
9. Use of the screening method of claim 5 for the preparation of a medicament for targeting RNA.
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