Method for visually screening agonist for activating cell RIG-I signal channel in vitro
By constructing a cell screening platform based on FRET and CRISPR-Cas9, the interaction intensity of RIG-I and MAVS was detected, and the problem of difficulty in detecting RIG-I signaling pathway activation in real time at the level of living cells was solved, and the rapid and convenient live cell detection.
Patent Information
- Application Number
- CN202411485471.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-23
- Publication Date
- 2025-02-14
AI Technical Summary
The prior art is difficult to detect the activation status of the RIG-I signaling pathway in real time at the level of living cells, and traditional methods cause damage to cells and cannot dynamically study protein interactions in cells.
Using fluorescence resonance energy transfer (FRET) technology and CRISPR-Cas9 gene editing technology, a cell screening platform of EGFP-RIG-I and Tag-RFP-MAVS was constructed, and the interaction intensity between RIG-I and MAVS was detected through a high-connotation imaging system. , evaluating the activation of RIG-I signaling pathway.
It realizes rapid and convenient detection of the activation state of the RIG-I signaling pathway at the level of living cells, avoids cell damage, and can dynamically study protein interactions in cells.
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Figure CN119432912A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of RIG-I signaling pathway activation detection, and particularly relates to a method for visually screening agonists for activating the cell RIG-I signaling pathway in vitro. Background Art
[0002] One of the key pattern recognition receptors in the innate immune system is the retinoic acid-induced gene I (RIG-I)-like receptor. These receptors are found in a variety of cell and tissue types. Type I interferon (IFN-I) and inflammatory cytokines are key sensors of viral infection and are induced by RIG-I-like receptors (RLRs). The RLR family has three members: RIG-I, MDA5, and LGP2, encoded by the DDX58, IFIH1, and DHX58 genes, respectively.
[0003] RIG-I primarily recognizes single-stranded RNA (ssRNA) or short double-stranded RNA (dsRNA) with a triphosphate group (PPP) at its 5' end. Under normal circumstances, RIG-I exists in an inactive and inhibited state in the cytoplasm, with its CARDs domain bound to the insertion region of the helicase domain. Upon viral invasion, the CTD domain senses and binds to the 5' triphosphate double-stranded RNA, causing the RIG-I molecule to transition to an open conformation and release the CARDs domain. The released CARDs domain then forms a tetramer and, through CARD-CARD interactions, recruits the MAVS protein to the outer mitochondrial membrane. The RIG-I-MAVS signaling cascade ultimately leads to the phosphorylation and activation of IRF3, IRF7, NF-κB, and AP-1, which together induce the expression of interferons and proinflammatory cytokines, thereby inhibiting viral replication.
[0004] However, recent studies have found that in addition to recognizing exogenous viral RNA, RIG-I is also activated by binding to various endogenous RNAs, such as microRNAs, snRNAs, and endogenous retroviruses (ERVs). Interestingly, quercetin and actin cytoskeleton remodeling can also activate RIG-I.
[0005] In addition to its role in host antiviral defense, RIG-I is also associated with cancer. Existing studies have shown that the RIG-I signaling pathway can mediate cancer cell death through multiple pathways, including apoptosis, pyroptosis, and autophagy, and is also associated with drug resistance in certain types of cancer cells.
[0006] Fluorescence resonance energy transfer (FRET) is a new, recently emerging technique for detecting protein-protein interactions, enabling real-time detection in living cells. While traditional techniques for studying protein-protein interactions facilitate this research, they also suffer from limitations. Methods such as yeast two-hybrid, immunoprecipitation, immunofluorescence, and radiolabeling require cell disruption or damage, making it impossible to dynamically study protein-protein interactions in real time under physiological conditions within living cells. FRET addresses this limitation.
[0007] Therefore, it is of positive significance to construct a drug screening platform based on fluorescence resonance energy transfer technology to detect the activation of the RIG-I signaling pathway. Summary of the Invention
[0008] The present invention aims to provide a method for visualizing in vitro screening of agonists for activating the RIG-I signaling pathway in cells, so as to conveniently and quickly detect the interaction strength between RIG-I and MAVS at the living cell level and evaluate whether the RIG-I signaling pathway is activated.
[0009] This protocol provides a method for visually screening agonists for activating the RIG-I signaling pathway in cells in vitro, comprising the following steps:
[0010] Step 1: Construct EGFP-RIG-I and Tag-RFP-MAVS subcloning fluorescent protein plasmids;
[0011] Step 2: Construct CRISPR-Cas9 sgRNA plasmids corresponding to RIG-I and MAVS;
[0012] Step 3: Build a cell screening platform for EGFP-RIG-I / Tag-RFP-MAVS;
[0013] Step 4: RIG-I agonist drug screening efficiency test.
[0014] Furthermore, the construction of EGFP-RIG-I and Tag-RFP-MAVS subcloning fluorescent protein plasmids includes the following steps:
[0015] (1) PCR was used in vitro to amplify the 500 bp fragments before and after the start codons of RIG-I and MAVS, respectively, and named them R1, R2, M1, and M2;
[0016] (2) Using plasmids containing EGFP and Tag-RFP fluorescent tags as templates, PCR amplification or synthesis was performed to obtain EGFP and Tag-RFP;
[0017] (3) Through polymerase chain reaction, EGFP was connected between R1 and R2, and it was named RER; similarly, Tag-RFP was connected between M1 and M2, and it was named MRM; and the two fragments of RER and MRM were connected to the T vector to obtain EGFP-RIG-I and Tag-RFP-MAVS subcloned fluorescent protein plasmids.
[0018] Furthermore, when constructing RER and MRM, a linker protein sequence is inserted between the fluorescent tag protein and the target protein. The insertion of the linker protein sequence ensures the correct folding of the target protein and the fluorescent tag protein and reduces the impact of the fluorescent tag on the structure and function of the target protein.
[0019] Furthermore, when constructing CRISPR-Cas9 sgRNA plasmids corresponding to RIG-I and MAVS, the following steps were performed: sgRNAs corresponding to RIG-I and MAVS were designed and synthesized; the CRISPR-Cas9 vector was digested with restriction enzymes and then ligated with the synthesized sgRNA under the action of DNA ligase; the ligation products were transformed and cultured; single colonies were selected and positive clones were screened by colony PCR using U6 upstream primers and sgRNA downstream primers; after sequencing verification and expanded culture, the endotoxin-free plasmids were extracted to obtain CRISPR-Cas9 sgRNA plasmids corresponding to RIG-I and MAVS.
[0020] Furthermore, the following steps were included when establishing the EGFP-RIG-I / Tag-RFP-MAVS cell screening platform:
[0021] (1) Cell transfection: After passage, untransfected normal cells were plated into 6-well plates. When the cell density reached 70%-80%, donor plasmid and sgRNA plasmid were added for transfection. The cells were cultured and passaged into T25 culture flasks for expansion and observed under a fluorescence microscope.
[0022] (2) Cell sorting: First, use untransfected cells to set up a fluorescence-negative control area, based on which the fluorescence signal-positive area is determined. Positive cells are collected and cultured. When the cell density reaches 80%-95%, they are subcultured;
[0023] (3) Monoclonal cell verification: Determine the protein expression of monoclonal cells and identify the accuracy of the fluorescent tag insertion site by PCR.
[0024] Furthermore, in step 4, when testing the efficiency of RIG-I agonist drug screening, the monoclonal cells screened in step 3 are treated with a RIG-I agonist, and images are collected using a high-content imaging system. The images are analyzed using image analysis software, and the FRET intensity is calculated to determine whether the RIG-I signaling pathway is activated.
[0025] Compared with the prior art, the present invention has the following advantages:
[0026] This study, based on fluorescence resonance energy transfer (FRET) and CRISPR-Cas9 gene editing technology, has constructed a drug screening platform for RIG-I signaling pathway activation. This platform can conveniently and quickly assess RIG-I signaling pathway activation by measuring the interaction strength between RIG-I and MAVS in living cells. BRIEF DESCRIPTION OF THE DRAWINGS
[0027] Figure 1 This is the agarose gel electrophoresis diagram of the six fragments R1, R2, EGFP, M1, M2 and Tag-RFP amplified by PCR technology;
[0028] Figure 2 Obtain agarose gel electrophoresis patterns of RER and MRM fragments for overlap-PCR;
[0029] Figure 3 Screening diagram of positive clones of RER and MRM linked to pEASY-blunt (A: RER; B: MRM);
[0030] Figure 4 Electrophoresis after digestion with BsmBI restriction enzyme for lentiCRISPR V2 vector (A) and verification after ligation (B);
[0031] Figure 5 Fluorescence images of 293 cells 48 hours after co-transfection of RER and RIG-I-sgRNA, MRM and MAVS-sgRNA, respectively. DETAILED DESCRIPTION
[0032] The following is further described in detail through specific implementation methods:
[0033] Drugs and Reagents: The primers used in the experiment were synthesized by Hunan Aikerui Biotechnology. The DNA extraction kit was purchased from Hunan Aikerui Biotechnology, DNA Polymerase Dye (No. RR371A) was purchased from TaKaRa, the gel recovery kit (B518131-0100) was purchased from Shanghai Sangon Biotechnology, BsmBI restriction enzyme was purchased from Thermo Scientific, T4 DNA ligase was purchased from Shanghai Beyotime Biotechnology, and the pEASY-blunt kit was purchased from Beijing Qingke Biotechnology.
[0034] A method for visually screening agonists for activating the RIG-I signaling pathway in cells in vitro, comprising the following steps:
[0035] Step 1: Digest and collect the cells in the culture flask, extract gDNA using a DNA extraction kit, and use it as a template to perform PCR amplification using DNA Polymerase Dye to obtain four fragments: R1, R2, M1, and M2. That is, 500bp fragments before and after the start codons of RIG-I and MAVS were amplified in vitro by PCR technology and named R1, R2, M1, and M2, respectively. As for EGFP and Tag-RFP, PCR amplification was performed using plasmids containing the corresponding fluorescent tags as templates. The six fragments (R1, R2, M1, M2, EGFP, and Tag-RFP) were excised, recovered, purified, and set aside.
[0036] Step 2: 50 ng of each fragment recovered from gel excision of R1, EGFP, and R2 was first subjected to the first step reaction in a PCR instrument. After the reaction was completed, the fragments were removed and amplified with primers R1-F and R2-R for 20 cycles. The reaction solution was subjected to agarose gel electrophoresis, and the band containing the correct product was cut and purified to obtain RER for later use. The same treatment was performed as above to obtain MRM for M1, Tag-RFP, and M2. 50 ng of each fragment recovered from gel excision of M1, Tag-RFP, and M2 was first subjected to the first step reaction in a PCR instrument. After the reaction was completed, the fragments were removed and amplified with primers M1-F and M2-R for 20 cycles. The reaction solution was subjected to agarose gel electrophoresis, and the band containing the correct product was cut and purified to obtain MRM.
[0037] Step 3: Connect RER and MRM to the pEASY-blunt vector according to the pEASY-blunt vector kit instructions. Spread the transformed plate and incubate at 37°C. After 12-16 hours, observe the colony growth and select a single colony for colony PCR verification.
[0038] Step 4: Digest 1 μg of lentiCRISPR V2 plasmid using the BsmBI restriction enzyme instructions. Ligate the plasmid to the sgRNA using the T4 DNA ligase instructions. Transform the ligated plasmid into DH-5α cells and plate culture. After 12-16 hours, select a single colony and expand it in LB liquid medium. Extract the plasmid and verify the results by agarose gel electrophoresis and sequencing.
[0039] Step 5: Transfect 293 cells with the EGFP-RIG-I and Tag-RFP-MAVS subcloned fluorescent protein plasmids and the CRISPR-Cas9 sgRNA plasmid at a 1:1 mass ratio according to the transfection reagent instructions. Replace the medium with fresh medium after 6 hours. Observe fluorescence and capture images after 24 hours.
[0040] RIG-I agonist drug screening efficiency test
[0041] After treating the selected monoclonal cells with the RIG-I agonist poly(I:C), images were collected using a high-content imaging system, analyzed using image analysis software, and FRET intensity was calculated to detect the interaction between RIG-I and MAVS after poly(I:C) treatment.
[0042] High alcohol detection: average values, I(DA1)=65332; I(DA0)=64311; I(D0)=42243; I(D1)=40463;
[0043]
[0044] Note: I(DA1) is the light intensity after RFP test, I(DA0) is the initial light intensity of RFP, I(D0) is the light intensity of GFP test, and I(D1) is the light intensity after GFP test.
[0045] Results: After the addition of the positive Poly(I:C) agonist, the activation efficiency of the RIG-I signaling pathway was 69.6%; the FRET efficiency was 31.4%.
[0046] sequence:
[0047] R1:5`-ttcacaccgaccaagatggaagttaaaattaaaaaaaaaaaagaaaggctgaccaatgtgtctatctactggagctctcctactgcgggtgggaatgtaaactggatgtaatcagtcattttggacaacaggtggcggtttcctataaagctaaacatagacttaccctggcacccagccgacgtgggagaactgggtacacaggtgtacactttacaaaactcatcgaattgcacactcagattgtgtgcattttactgtgtctaaacgttagcgctataaaagcaaaacaaaatataagatcataaaaataaaagcaattcctaggggtcctctccgttcccgggcttcctcggtgcggagggaaacgaaactagcccgaggcaaaacagcctccgcgaaccccgcccgccgctagttgcactttcgattttccctttagttattaaagttcctatgcagctccgcctcgcgtccggcctcatttcctcggaaaatcc-3`
[0048] R2:5`-agggtgagtgtctccagcggcgccttctcggcggaaaacaattgaggacttgtttttctcttgcttcgttcccttcttttaatcaatgcagagagcctgccagactccccaggtttgtggtaagatctccgcagcattttaggctagaggttgccagagtttgaggagccctcggaggcctaggtggggaaactgagttccagcgaggtgaagtgacttgcttaagggtgctcatctatttaggttcaaagctggggaagaaattttgtttcatttattttcactaatattaaagtctactgtgtgcttggctgggtactgacagagattgagaaataagataagggctcttccttagtatacattacagtccaagggatgggacacaaagggcagataattacagtactgagcacaaagtgcggtctgagcttaaaggaagttgatgcttactctgccgaatcttatttgcacgcctaaagaagtggaagctggagagg-3`
[0049] M1:5`-gttaatcccccaaattggcctcccaaagtgctgggattataggcgtgaaccactgtgcccagcccgagaccccatctcttaaaaacaaaataaaacaaaacaaaaacggccaggtgtggtggctcacacctgtaatccccaaacttgggaggccgaggcgggtggaccacttgaggtcaggagtctgtgaccagcttgccaacatggtgaaaccccatctctactaaaaatacaaaaattagctgggcatggtggtgcgcacctgtaatcccagctactcagaagggaggctgaggcaagagactcaattgaacccaggaggcggaggttgcagtgagccgagattgccccactgcactccagcctgggtgacaaagtgagactcgctctcaaaaaaaaaaaaaagaagaaattaaagatgaaagaaaacaaacattccaaaaagttgagaaagaattgccttttgtccagccccactcccaaccccccaaccctgttgt-3`
[0050] M2:5`-gagaccaggtgagcaagggaagtgacagcccgacactggcctgggggcagggctgtggaattcaaagctcagccccatcctagttcctcacccaagcctgggctggctccttccttcttcctcttgctgtgtcttgctccttgtccttgctgcttttcttttttttttttttttttgagattgagtctcgttctgtcgccaggctggagtgcagtggcacgatcttggctcattgcaacctccgcctcctgggttcaagtgattctcctgcctcagcctcctgagtagctgggattacaggtgcgtgccaccacgcccagctaatttttttgtttttaatagagacggggtttcaccatgttggccaggatggtcttgatctcttgaccttgtgatccgcctgcctcggcctcccaaagtgctgggattacaggcgtgagccaccgcacccttgctgcttttctaacttttggatggagtgtggctcagggtggcgttgc-3`
[0051] EGFP:5`-gtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaag-3`
[0052] Tag-RFP:5`-atggtgtctaagggcgaagagctgattaaggagaacatgcacatgaagctgtacatggagggcaccgtgaacaaccaccacttcaagtgcacatccgagggcgaaggcaagccctacgagggcacccagaccatgagaatcaaggtggtcgagggcggccctctccccttcgccttcgacatcctggctaccagcttcatgtacggcagcagaaccttcatcaaccacacccagggcatccccgacttctttaagcagtccttccctgagggcttcacatgggagagagtcaccacatacgaagacgggggcgtgctgaccgctacccaggacaccagcctccaggacggctgcctcatctacaacgtcaagatcagaggggtgaacttcccatccaacggccctgtgatgcagaagaaaacactcggctgggaggccaacaccgagatgctgtaccccgctgacggcggcctggaaggcagaagcgacatggccctgaagctcgtgggcgggggccacctgatctgcaacttcaagaccacatacagatccaagaaacccgctaagaacctcaagatgcccggcgtctactatgtggaccacagactggaaagaatcaaggaggccgacaaagagacctacgtcgagcagcacgaggtggctgtggccagatactgcgacctccctagcaaactggggcacaaacttaat-3`
[0053] RER:5`-gagaccaggtgagcaagggaagtgacagcccgacactggcctgggggcagggctgtggaattcaaagctcagccccatcctagttcctcacccaagcctgggctggctccttccttcttcctcttgctgtgtcttgctccttgtccttgctgcttttcttttttttttttttttttgagattgagtctcgttctgtcgccaggctggagtgcagtggcacgatcttggctcattgcaacctccgcctcctgggttcaagtgattctcctgcctcagcctcctgagtagctgggattacaggtgcgtgccaccacgcccagctaatttttttgtttttaatagagacggggtttcaccatgttggccaggatggtcttgatctcttgaccttgtgatccgcctgcctcggcctcccaaagtgctgggattacaggcgtgagccaccgcacccttgctgcttttctaacttttggatggagtgtggctcagggtggcgttgc-3`
[0054]
[0055] sgRNA:5`-gtgagcaagggcgaggagctgtt-3`
[0056] R1-F:ttcacaccgaccaagatggaag
[0057] R2-R:cctctccagcttccacttctttag
[0058] M1-F:gttaatcccccaaattggcctc
[0059] M2-R:gcaacgccaccctgagc
[0060] The above is only an embodiment of the present invention, and the common knowledge such as the specific structure and characteristics of the scheme is not described in detail here. It should be pointed out that for those skilled in the art, without departing from the structure of the present invention, several variations and improvements can be made, which should also be regarded as the scope of protection of the present invention, and these will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.
Claims
1. A method for visualizing in vitro screening of agonists for activating the RIG-I signaling pathway in cells, characterized in that: The following steps are involved: Step 1: construct EGFP-RIG-I and Tag-RFP-MAVS subcloning fluorescent protein plasmids; Step 2: Construct CRISPR-Cas9 sgRNA plasmids corresponding to RIG-I and MAVS; Step 3: Build a cell screening platform for EGFP-RIG-I / Tag-RFP-MAVS; Step 4: RIG-I agonist drug screening efficiency test.
2. The method for visualizing in vitro screening of agonists for activating the cell RIG-I signaling pathway according to claim 1, characterized in that: The construction of EGFP-RIG-I and Tag-RFP-MAVS subcloning fluorescent protein plasmids includes the following steps: (1) The 500 bp fragments before and after the start codons of RIG-I and MAVS were amplified in vitro by PCR and named R1, R2, M1, and M2, respectively; (2) using plasmids containing EGFP and Tag-RFP fluorescent tags as templates to perform PCR amplification or synthesis to obtain EGFP and Tag-RFP; (3) Through polymerase chain reaction, EGFP was connected between R1 and R2, which was named RER; similarly, Tag-RFP was connected between M1 and M2, which was named MRM; and the two fragments of RER and MRM were connected to the T vector to obtain EGFP-RIG-I and Tag-RFP-MAVS subcloned fluorescent protein plasmids.
3. The method for visualizing in vitro screening of agonists for activating the cell RIG-I signaling pathway according to claim 2, characterized in that: When constructing RER and MRM, a linker protein sequence was inserted between the fluorescent tag protein and the target protein.
4. The method for visualizing in vitro screening of agonists for activating the cell RIG-I signaling pathway according to claim 3, characterized in that: When constructing the CRISPR-Cas9 sgRNA plasmids corresponding to RIG-I and MAVS, the specific steps are as follows: sgRNA corresponding to RIG-I and MAVS are designed and synthesized, the CRISPR-Cas9 vector is digested with restriction enzymes, and then connected with the synthesized sgRNA under the action of DNA ligase, the ligation product is transformed and cultured, and a single colony is selected to screen out positive clones by colony PCR using U6 upstream primers and sgRNA downstream primers, and after sequencing verification, the endotoxin-free plasmid is extracted after expanded culture to obtain the CRISPR-Cas9 sgRNA plasmids corresponding to RIG-I and MAVS.
5. The method for visualizing in vitro screening of agonists for activating the cell RIG-I signaling pathway according to claim 4, characterized in that: The following steps are included when setting up the EGFP-RIG-I / Tag-RFP-MAVS cell screening platform: (1) Cell transfection: After subculturing, untransfected normal cells were plated in 6-well plates. When the cell density reached 70%-80%, donor plasmid and sgRNA plasmid were added for transfection. The cells were cultured and subcultured in T25 culture flasks for expansion and observed under a fluorescence microscope. (2) Cell sorting: First, use untransfected cells to set up a fluorescent negative control area, based on which the fluorescent signal positive area is determined, and the positive cells are collected and cultured. When the cell density reaches 80%-95%, they are subcultured; (3) Monoclonal cell verification: Determine the protein expression of monoclonal cells and identify the accuracy of the fluorescent tag insertion site by PCR.
6. The method for visualizing in vitro screening of agonists for activating the cell RIG-I signaling pathway according to claim 5, characterized in that: In step 4, when testing the efficiency of RIG-I agonist drug screening, the monoclonal cells screened in step 3 are treated with RIG-I agonists, images are collected using a high-content imaging system, and image analysis software is used to analyze and calculate the FRET intensity to determine whether the RIG-I signaling pathway is activated.