SiRNA screening method and application thereof

Abstract

The invention discloses a siRNA screening method, a fluorescence reporting system and application thereof. Specifically, the siRNA screening method disclosed by the invention comprises the following steps: constructing a stable cell strain with a fluorescence reporter gene and a target gene, transfecting siRNA to be screened into a stable cell strain cell, and detecting the siRNA silencing efficiency through fluorescence intensity. The siRNA silencing efficiency can be detected through fluorescence intensity only by transfecting siRNA into the stable cell strain cell independently by using the stable cell strain provided by the invention. The siRNA screening method is rapid, simple to operate and low in detection cost, and silence efficiency of siRNA rapid and large-scale batch screening can be achieved.

Description

Technical Field

[0001] The present invention relates to the field of gene medicine, and in particular to a siRNA screening method, a fluorescent reporter system and applications thereof. Background Art

[0002] siRNA drugs refer to a gene silencing phenomenon induced by double-stranded siRNA. They mainly play a role in regulating gene expression at the gene level and are an important category in the field of gene therapy. Because they do not need to identify the complex spatial conformation of proteins, they avoid the process of identifying target molecules with high activity, affinity and specificity, breaking through the dilemma of innovative drugs that many diseases cannot be treated with small molecules and antibody drugs. Globally, many siRNA drugs have entered clinical trials or are on the market, and have become the fast track for the development of innovative drugs. The difficulties in the development of siRNA drugs are concentrated in three aspects: (1) siRNA design; (2) siRNA screening; and (3) siRNA delivery system.

[0003] There are currently three main methods for siRNA screening:

[0004] (1) qPCR detection: This method involves transfecting siRNA into cells that highly express the target gene. 48 or 72 hours after transfection, the cells are harvested, RNA is extracted, and cDNA is reverse transcribed. qPCR detection is then performed using primers. This method has a long experimental cycle, low detection efficiency, and poor consistency in qPCR results using different primers.

[0005] (2) WB detection: This method also involves transfecting siRNA into cells that highly express the target gene. 48 or 72 hours after transfection, the cells are harvested, total protein is extracted, and WB detection is performed. This method also has a long experimental cycle, low detection efficiency, and the detection effect is limited by the specificity of the antibody.

[0006] (3) The psiCHECK-2 plasmid screening system with dual fluorescent reporter genes contains two luciferase reporter genes, of which the main reporter gene is the Renilla reporter gene, the target gene is connected in series downstream of the Renilla reporter gene, and is used to detect the silencing efficiency of siRNA. The auxiliary reporter gene is the firefly reporter gene, which is used to detect the cell state. This method is to co-incubate siRNA and psiCHECK-2 plasmids, transfect 293T cells, add two reporter gene substrates, and calculate the silencing efficiency of siRNA by detecting the chemiluminescence value on an ELISA instrument. Although this method is efficient, its disadvantage is that siRNA and psiCHECK-2 plasmids need to be transfected simultaneously during transfection, and two luminescent substrates need to be added during detection, which increases the experimental operation and detection costs.

[0007] Therefore, developing a faster and simpler siRNA screening method is of great value to the development of siRNA drugs. Summary of the invention

[0008] In view of the above problems, the present invention aims to provide a method which is faster, simpler to operate and has lower detection cost than the existing siRNA screening methods, and can achieve rapid and large-scale batch screening of the silencing efficiency of siRNA.

[0009] The first aspect of the present invention provides a siRNA screening method, which comprises: constructing a stable cell line with a fluorescent reporter gene and a target gene, transfecting the siRNA to be screened into the stable cell line cells, and detecting the silencing efficiency of the siRNA by fluorescence intensity, that is, detecting the silencing efficiency of the siRNA on the target gene.

[0010] In a specific embodiment of the present invention, the stable cell line with a fluorescent reporter gene and a target gene can be constructed by inserting the cDNA of the target gene in series into the downstream of the fluorescent reporter gene on a lentiviral vector (pLV), and then constructing the stable cell line through the mediation of the lentiviral vector.

[0011] The second aspect of the present invention provides a fluorescent reporter system suitable for the above siRNA screening method, which includes a lentiviral vector and an auxiliary packaging system for lentiviral packaging. The cDNA of the target gene in the above lentiviral vector is inserted in series downstream of the fluorescent reporter gene.

[0012] In a specific embodiment of the present invention, the above-mentioned fluorescent reporter system also includes positive / negative control siRNA.

[0013] The third aspect of the present invention provides a siRNA screening kit, which includes a lentiviral vector, an auxiliary packaging system for lentiviral packaging, and positive / negative control siRNA. Specifically, the above-mentioned lentiviral vector includes at least one of a pLV-Rluc plasmid or a pLV-Fluc plasmid. The pLV-Rluc plasmid is used to express Renilla luciferase, and the structure is: promoter + T7 Promoter + 5′UTR + Renilla luciferase + CDS cloning site + 3′UTR sequence + PolyA. The pLV-Fluc plasmid is used to express firefly luciferase, and the structure is: promoter + T7 Promoter + 5′UTR + firefly luciferase + CDS cloning site + 3′UTR sequence + PolyA. The above-mentioned CDS cloning site is used to insert the cDNA of the target gene X. The positive control siRNA in the kit includes PC Rluc-siRNA or PC Fluc-siRNA, wherein the nucleotide sequence of PC Rluc-siRNA is shown in SEQ ID NO.17 and SEQ ID NO.18, and the nucleotide sequence of PC Fluc-siRNA is shown in SEQ ID NO.19 and SEQ ID NO.20. The nucleotide sequence of the negative control siRNA is shown in SEQ ID NO.21 and SEQ ID NO.22.

[0014] Preferably, the promoter in the above kit includes any one of SV40 promoter, CMV promoter, EF1α promoter, CBA promoter, EFS promoter, SFFV promoter, MSCV promoter or PGK promoter. The 5′UTR sequence in the pLV-Rluc plasmid or pLV-Fluc plasmid includes any one of 5′UTR1, 5′UTR2, 5′UTR3, 5′UTR4, 5′UTR5, 5′UTR6, 5′UTR7 or 5′UTR8. The 3′UTR sequence in the pLV-Rluc plasmid or pLV-Fluc plasmid includes any one of 3′UTR1, 3′UTR2, 3′UTR3, 3′UTR4, 3′UTR5, 3′UTR6, 3′UTR7 or 3′UTR8.

[0015] In a specific embodiment of the present invention, a siRNA screening kit for NOX1 gene is provided, wherein the gene X in the pLV-Rluc plasmid or the pLV-Fluc plasmid in the kit is the NOX1 gene, the 5′UTR sequence is 5′UTR1, and its nucleotide sequence is shown in SEQ ID NO.1, and the 3′UTR sequence is 3′UTR1, and its nucleotide sequence is shown in SEQ ID NO.9.

[0016] The present invention utilizes a stable cell line with a fluorescent reporter gene and a target gene, and only needs to transfect siRNA into the stable cell line cells separately, and the siRNA silencing efficiency can be detected by fluorescence intensity. Compared with the qPCR and WB detection methods, the present invention has high detection efficiency and short time consumption; compared with the psiCHECK-2 dual fluorescence reporter system detection method, the present invention has simpler experimental operation, only needs to transfect siRNA, no need to transfect plasmid, reducing the operation steps, and can reduce the uncertainty caused by each plasmid transfection efficiency. When detecting, only one substrate needs to be added, saving time and experimental costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The pLV-Rluc plasmid map in Example 1 of the present invention;

[0018] Figure 2 The pLV-Fluc plasmid map in Example 1 of the present invention;

[0019] Figure 3 The plasmid map of pLV-Rluc-NOX1 in Example 1 of the present invention;

[0020] Figure 4 The plasmid map of pLV-Fluc-NOX1 in Example 1 of the present invention;

[0021] Figure 5 The identification results of 293T-FLuc-NOX1 cell line in Example 4 of the present invention;

[0022] Figure 6 The identification results of 293T-RLuc-NOX1 cell line in Example 4 of the present invention;

[0023] Figure 7 Statistical graph of the silencing results of NOX1 siRNA on 293T-RLuc-NOX1 cell line in Example 6 of the present invention;

[0024] Figure 8 Statistical graph of the silencing results of NOX1 siRNA on 293T-FLuc-NOX1 cell line in Example 6 of the present invention;

[0025] Fig. 9 A statistical diagram of the silencing results of different concentration gradients of NOX1 siRNA on 293T-RLuc-NOX1 cell lines in Example 7 of the present invention;

[0026] Fig.10 A statistical diagram of the silencing results of different concentration gradients of NOX1 siRNA on 293T-FLuc-NOX1 cell line in Example 7 of the present invention. DETAILED DESCRIPTION

[0027] The purpose of the present invention is to provide a method that is faster, simpler to operate, and has lower detection costs than existing siRNA screening methods. The inventive concept of the present invention is to construct a stable cell line with a fluorescent reporter gene and a target gene, then transfect the siRNA to be screened into the above stable cell line cells, and detect the silencing efficiency of the siRNA to be tested by fluorescence intensity.

[0028] In a specific embodiment of the present invention, a lentiviral vector can be used to achieve the construction of a stable cell line. Specifically, the cDNA of the target gene can be inserted in series downstream of the fluorescent reporter gene on the lentiviral vector, and then the stable cell line is constructed through the lentiviral vector.

[0029] The host cells suitable for constructing stable cell lines of the present invention include, but are not limited to, human embryonic kidney cell 293 (HEK293 cells), CHO-K1, NIH / 3T3, Hela, COS-7 and HepG2 cell lines. In a specific embodiment of the present invention, 293T cells are used as host cells for the construction of stable cell lines. 293T cells are easily infected and easily transfected, and have good experimental operability and stability. After repeated studies, the present invention makes full use of the characteristics of 293T cells that are easily infected and easily transfected, and adopts the downstream of the luciferase Luc gene on the lentiviral vector pLV-siScreen by inserting the cDNA of the target gene X in series, and constructing a 293T-Luc-X stable cell line by a lentiviral LV, and the stable cell line constructed has good fluorescence stability.

[0030] Fluorescent reporter genes applicable to the present invention include, but are not limited to, luciferase genes. At the same time, luciferases applicable to the present invention include, but are not limited to, firefly luciferase (Firefly Luciferase, Fluc) and Renilla Luciferase (Rluc). In an exemplary embodiment of the present invention, at least one of pLV-Rluc plasmid or pLV-Fluc plasmid is specifically used, wherein:

[0031] 1) pLV-Rluc plasmid, used to express Renilla luciferase, has the following structure: promoter + T7 Promoter + 5′UTR + Renilla luciferase + CDS cloning site + 3′UTR sequence + PolyA. The CDS cloning site is used to insert the cDNA of the target gene.

[0032] 2) pLV-Fluc plasmid, used to express firefly luciferase, has the following structure: promoter + T7Promoter + 5′UTR + firefly luciferase + CDS cloning site + 3′UTR sequence + PolyA. The CDS cloning site is used to insert the cDNA of the target gene.

[0033] In a specific embodiment of the present invention, the promoter in the above-mentioned plasmid includes but is not limited to: SV40 promoter, CMV promoter, EF1α promoter, CBA promoter, EFS promoter, SFFV promoter, MSCV promoter, PGK promoter and the like.

[0034] In a specific embodiment of the present invention, the 5′UTR sequence in the above-mentioned plasmid is preferably the following specific sequence as recorded in Table 1: 5′UTR1, 5′UTR2, 5′UTR3, 5′UTR4, 5′UTR5, 5′UTR6, 5′UTR7, 5′UTR8, etc.

[0035] Table 1 Related nucleotide sequences of 5′UTR sequences

[0036]

[0037]

[0038] In a specific embodiment of the present invention, the 3′UTR sequence in the above-mentioned plasmid is preferably the following specific sequence as recorded in Table 2: 3′UTR1, 3′UTR2, 3′UTR3, 3′UTR4, 3′UTR5, 3′UTR6, 3′UTR7, 3′UTR8, etc.

[0039] Table 2 Related nucleotide sequences of 3′UTR sequences

[0040]

[0041] It should be noted that the present invention obtained the above UTR sequence by screening different genes through repeated experiments, and those skilled in the art can use it more specifically according to the experiments.

[0042] In a specific embodiment of the present invention, when siRNA screening is performed, one of the two expression plasmids can be selected to construct pLV-Rluc-X or pLV-Fluc-X (X refers to the target gene), both of which can achieve the purpose of efficient siRNA screening. Those skilled in the art can select appropriate elements to construct plasmids based on the above scheme disclosed in the present invention.

[0043] After the construction of the lentiviral vector is completed, the auxiliary packaging system of the lentiviral packaging can be used to achieve transfection and construct a stable cell line. In a specific embodiment of the present invention, the auxiliary packaging system of the lentiviral packaging used contains three auxiliary plasmids required for lentiviral packaging: pLp1, pLp2, and pLp-VSVG, which express the envelope protein required for virus packaging. pLV-Rluc-X or pLV-Fluc-X is transfected into 293T cells together with the three auxiliary plasmids, and the lentiviral particles are harvested, and then the lentiviral particles are infected into 293T host cells to obtain a 293T-Luc-X stable cell line.

[0044] After obtaining a stable cell line, the siRNA to be tested can be transfected into the obtained stable cell line cells. After a certain period of culture, a substrate detection reagent corresponding to the fluorescent reporter gene is added to the cell culture to be tested, for example, the substrate for FLuc is luciferin, and the substrate for RLuc is coelenterazine. After standing in the dark, chemiluminescence detection is performed on a fluorescence detection instrument such as an ELISA instrument, and the silencing efficiency of the siRNA to be tested is evaluated by the fluorescence intensity (RLU). The higher the fluorescence intensity, the lower the silencing efficiency, and conversely, the lower the fluorescence intensity, the higher the silencing efficiency. After screening for siRNA with high silencing efficiency, the stable cell line cells can be further used for gradient screening of different concentrations to determine the relationship between the silencing effect and the siRNA dosage, so as to explore suitable conditions for the actual usage of siRNA.

[0045] The above-mentioned diffuse viral vector and the auxiliary packaging system for lentiviral packaging can constitute a fluorescent reporter system for efficient screening of siRNA. The system can further include negative control NC siRNA and positive control PC siRNA. Among them, the positive control PC siRNA includes PC Rluc-siRNA or PC Fluc-siRNA. As a preferred example, the specific sequences of the relevant control siRNAs such as PC Rluc-siRNA, PC Fluc-siRNA, and negative control NC siRNA used in the embodiments of the present invention are shown in Table 3. Among them, PC Rluc-siRNA and PC Fluc-siRNA are respectively suitable for their corresponding luciferases Rluc and Fluc.

[0046] Table 3 Sequence information of the comparison

[0047]

[0048] By constructing the 293T-Luc-X stable cell line with the above fluorescent reporter system, the siRNA, PC siRNA and NC siRNA of the target gene are transfected separately as controls, and the silencing efficiency of siRNA can be screened quickly and in large quantities. Therefore, the above siRNA screening method and fluorescent reporter system provided by the present invention can further prepare the above lentiviral vector, the auxiliary packaging system for lentiviral packaging and the positive / negative control siRNA into a kit, which is widely used for siRNA target gene verification.

[0049] The present invention will be further described in detail below through specific implementation modes in conjunction with the accompanying drawings.

[0050] The fluorescent reporter system for siRNA screening of the present invention is to insert the target gene cDNA (X) in series into the downstream of the luciferase Luc gene on the lentiviral vector pLV-Rluc or pLV-Fluc, and construct a 293T-Luc-X stable cell line through the lentiviral LV. Only a single siRNA transfection into the 293T-Luc-X cells is required, and the siRNA silencing efficiency can be detected by the fluorescence intensity. The system has good stability.

[0051] In order to verify the feasibility of the present invention, the following is an illustration using siRNA screening targeting NOX1 (nicotinamide adenine dinucleotide phosphate oxidase 1) as a specific example.

[0052] Example 1: Construction of pLV-Rluc-NOX1 and pLV-Fluc-NOX1 vectors

[0053] Please refer to Figure 1 , the NOX1 gene was constructed into the pLV-Rluc plasmid ( Figure 1 ) and pLV-Fluc plasmid ( Figure 2 ), wherein the 5′UTR sequence uses the 5′UTR1 in Table 1, and the 3′UTR sequence uses the 3′UTR1 in Table 2. First, the NOX1 gene sequence is amplified and recovered by PCR using specific primers. Secondly, the pLV-Rluc and pLV-Fluc plasmids are double-digested with XhoI and NotI, the linearized vector is recovered, and seamless cloning is performed with the corresponding linearized plasmid and PCR recovered product. Take 5μL of seamless cloning connection product and add it to 50μL TOP10 competent cells, ice bath for 30 minutes, heat shock for 45 seconds and immediately put it on ice for 2 minutes, add 500μL LB liquid culture medium and shake at 37°C, 200rpm for 30 minutes, collect the bacterial liquid by centrifugation and evenly apply it to the plate of LB culture medium containing Amp antibiotics, culture at 37°C overnight, pick 3 single clones and shake them for sequencing, and the successfully constructed plasmid is pLV-Rluc-NOX1( Figure 3) and pLV-Fluc-NOX1( Figure 4 ).

[0054] Example 2: Packaging and harvesting of lentivirus

[0055] Rluc-NOX1 and Fluc-NOX1 viruses were packaged respectively. 293T cells in the logarithmic growth phase were taken and the cell density was adjusted to 1×10 7 Inoculate 10cm cell culture dish with 10 cells, and use it for transfection when the cell confluence reaches 90%. Take a 15ml centrifuge tube, add 3 auxiliary plasmids and pLV-Rluc-NOX1 or pLV-Fluc-NOX1 to opti-MEM culture medium (1ugDNA / 100μL Opti MEM) according to Table 4, and mix well. Take 22.5μL Lip2000 reagent (1ugDNA / 100μL Opti MEM) and add it to the above mixture, mix gently, and let it stand at room temperature for 5-10min. Add the plasmid-Lip2000 complex dropwise to the culture dish and mix gently. Harvest the virus supernatant once 48h and 72h after transfection, filter it with a 0.22um filter, add 4× virus concentrate at a ratio of 3:1, mix well, mix every half an hour, at least 3 times, and let it stand overnight at 4℃. 1500g, 4℃, centrifuge for 45min. After centrifugation, remove the supernatant, add 500 μL serum-free medium or PBS to resuspend the virus, and store it in aliquots at -80°C for later use.

[0056] Table 4 Plasmid dosage table

[0057]

[0058]

[0059] Example 3 Construction of 293T-FLuc-NOX1 and 293T-RLuc-NOX1 stable cell lines

[0060] Count the 293T cells in good condition, plate them, and inoculate them into 24-well plates. The number of cells is 1x10 5 / well, add 500μL culture medium to each well and culture overnight. Before virus infection, replace fresh culture medium, then add Rluc-NOX1 and Fluc-NOX1 viruses respectively and mix well, the corresponding virus amount MOI = 2, and add Polybrene at the same time to make its concentration 5ug / mL. After 24 hours of virus infection, observe the cell state and replace fresh culture medium. After 72 hours of virus infection of cells, replace with screening medium containing the corresponding optimal concentration of puromycin for pressure screening, the concentration of puromycin is 2ug / mL, maintain this condition to continue culture or passage, observe the cell state every day, and after 7 days, almost all surviving cells can stably express RLuc-NOX1 gene and Fluc-NOX1 gene.

[0061] Example 4 Identification of stable cell lines

[0062] The cells of the 293T-FLuc-NOX1 and 293T-RLuc-NOX1 stable cell lines in good condition were counted and inoculated into a 96-well white plate. The inoculation volume of the first well was 1×10 5 The subsequent wells were diluted to 0.5×10 5 pcs / hole, 0.25×10 5 pcs / hole, 0.125×10 5 pcs / hole, 0.0625×10 5 pcs / hole, 0.03125×10 5 Each well of the 96-well plate was placed in an incubator for 1 hour. After the plate was taken out and equilibrated to room temperature, 100 μL / well of the substrate luciferin was added to each well of the 293T-FLuc-NOX1 cells and 100 μL / well of the substrate coelenterazine was added to each well of the 293T-RLuc-NOX1 cells. The plate was placed in a dark place and chemiluminescence detection was performed on an ELISA reader within 5-10 minutes. From the analysis of the detection results, that is, Figure 5-6 The constructed stable cell line has high fluorescence intensity, strong stability of luminescence signal, and linear relationship R 2 ≥0.99, among which the fluorescence intensity of 293T-RLuc-NOX1 cell line was higher than that of 293T-FLuc-NOX1 cell line.

[0063] Example 5 Transfection of NOX1 siRNA

[0064] 293T-Fluc-NOX1 cells and 293T-Rluc-NOX1 cells were cultured separately and counted and plated on 96-well white plates. The cell plating volume was 2×10 4 / well, volume 100μL. After overnight culture, the cell culture plate culture medium was replaced with 90μL of opti-MEM medium, and 15 siRNA sequences designed for NOX1mRNA were transfected with Lip3000 reagent. The final transfection concentration of each siRNA was 1nM. For each siRNA: Prepare solution A: Take 4μL of 100nM siRNA and add it to 16μL of opti-MEM medium and mix well. Prepare solution B: Take 0.8μL of Lip3000 and add it to 19.2μL of opti-MEM medium and mix well. Add solution A to solution B, mix well and let stand at room temperature for 15 minutes. Take 10μL of each siRNA-Lip3000 complex and add it to a culture plate containing 90μL of culture medium (3 replicates), shake it slightly, and place it in a cell culture incubator for 48 hours.

[0065] Example 6 Fluorescence reporter system detection

[0066] Take out the 96-well white plate transfected with siRNA from the incubator and place it at room temperature for 30 minutes to allow the temperature of the culture plate to equilibrate to room temperature. Add a substrate detection reagent of the same volume as the cell culture to be tested. If 293T-FLuc-NOX1 cells are selected, add 100 μL / well of substrate luciferin to each well. If 293T-RLuc-NOX1 cells are selected, add 100 μL / well of substrate coelenterazine to each well. Keep it away from light and perform chemiluminescence detection on an enzyme reader within 5-10 minutes. Evaluate the silencing efficiency of siRNA by relative fluorescence intensity (RLU). From the analysis of the test results, that is, Figure 7-8 Whether it is the test results of transfected 293T-RLuc-NOX1 cells or transfected 293T-FLuc-NOX1 cells, the silencing efficiency of 15 NOX1-siRNAs on the two types of cells is consistent in trend, with a small error. Among them, the silencing effects of NOX1-siRNA7 and NOX1-siRNA9 are better and can be used for further concentration gradient screening experiments.

[0067] Example 7 Concentration gradient screening of siRNA sequences with high silencing efficiency

[0068] 293T-Fluc-NOX1 cells and 293T-Rluc-NOX1 cells were cultured separately and counted and plated on 96-well white plates. The cell plating volume was 2×10 4 / well, volume 100μL. After overnight culture, the cell culture plate culture medium was replaced with 90μL of opti-MEM medium. The experimental groups were: Blank group, PC siRNA group (1nM), NC siRNA group (1nM), NOX1-siRNA7 group (1nM / 0.1nM / 0.01nM), NOX1-siRNA9 group (1nM / 0.1nM / 0.01nM). The PC siRNA group and NC siRNA group are shown in Table 3. Lip3000 reagent was used for transfection. After 48 hours, the substrate was added for chemiluminescence detection as in step 6 to evaluate the siRNA silencing efficiency. From the analysis of the test results, that is, Figure 9-10 The silencing effects of NOX1-siRNA7 and NOX1-siRNA9 sequences showed a dose-dependent relationship with the siRNA concentration. At a concentration of 0.01 nM, the silencing efficiency of siRNA was poor.

[0069] For comparison, in this example, the 15 siRNA sequences designed for NOX1 mRNA in Example 5 were subjected to control experiments using qPCR, WB, and psiCHECK-2 plasmid screening systems. The results are as follows:

[0070] The qPCR test results showed that among the above 15 siRNAs, NOX1-siRNA7 and NOX1-siRNA9 also had better silencing efficiency.

[0071] WB test results showed that the above 15 siRNAs showed the same trend, and NOX1-siRNA7 and NOX1-siRNA9 had higher silencing efficiency.

[0072] The psiCHECK-2 plasmid screening system was used for further verification, and the test results showed that among the above 15 siRNAs, NOX1-siRNA7 and NOX1-siRNA9 also performed better.

[0073] The results of the control experiment are basically consistent with the results of the present invention, but from the perspective of the time period, qPCR needs to extract RNA and reverse transcribe cDNA after harvesting cells before qPCR detection, and the experimental cycle is obviously too long. The WB test also has the same problem of long extraction cycle, time-consuming and unstable. The psiCHECK-2 plasmid screening system must add two plasmids for transfection at the same time during each screening, while the present invention only needs to transfect siRNA after constructing cells to achieve detection, which greatly reduces the experimental operation steps and detection costs when conducting a large number of siRNA censuses to be tested. In summary, the siRNA screening technology of this patent is fast, simple and low-cost, and can efficiently perform rapid screening and large-scale batch processing of siRNA silencing effects.

[0074] The above specific examples are used to illustrate the present invention, which is only used to help understand the present invention and is not intended to limit the present invention. For those skilled in the art, according to the concept of the present invention, some simple deductions, modifications or substitutions can be made.

Claims

1. A siRNA screening method, It is characterized in that include: A stable cell line with a fluorescent reporter gene and a target gene is constructed, the siRNA to be screened is transfected into the stable cell line cells, and the silencing efficiency of the siRNA is detected by fluorescence intensity.

2. The screening method according to claim 1, It is characterized in that The method of constructing a stable cell line with a fluorescent reporter gene and a target gene comprises: inserting the cDNA of the target gene in series into the downstream of the fluorescent reporter gene on a lentiviral vector, and then constructing a stable cell line through the mediation of the lentiviral vector.

3. The screening method according to claim 2, It is characterized in that The fluorescent reporter gene includes a luciferase gene.

4. The screening method according to claim 2, It is characterized in that The stable cell line uses human embryonic kidney cell 293 as host cell; Preferably, the human embryonic kidney cells 293 are 293T cells.

5. The screening method according to claim 3, It is characterized in that The lentiviral vector comprises at least one of a pLV-Rluc plasmid or a pLV-Fluc plasmid, wherein: The pLV-Rluc plasmid is used to express Renilla luciferase, and has a structure of: promoter+T7Promoter+5′UTR+Renilla luciferase+CDS cloning site+3′UTR sequence+PolyA, wherein the CDS cloning site is used to insert the cDNA of the target gene; The pLV-Fluc plasmid is used to express firefly luciferase, and its structure is: promoter + T7Promoter + 5′UTR + firefly luciferase + CDS cloning site + 3′UTR sequence + PolyA, and the CDS cloning site is used to insert the cDNA of the target gene; Preferably, the promoter includes any one of SV40 promoter, CMV promoter, EF1α promoter, CBA promoter, EFS promoter, SFFV promoter, MSCV promoter or PGK promoter; Preferably, the 5′UTR sequence includes any one of 5′UTR1, 5′UTR2, 5′UTR3, 5′UTR4, 5′UTR5, 5′UTR6, 5′UTR7 or 5′UTR8; Preferably, the 3'UTR sequence includes any one of 3'UTR1, 3'UTR2, 3'UTR3, 3'UTR4, 3'UTR5, 3'UTR6, 3'UTR7 or 3'UTR8.

6. A fluorescent reporter system for use in the screening method according to claim 1, It is characterized in that The fluorescent reporter system comprises a lentiviral vector and an auxiliary packaging system for lentiviral packaging, wherein the cDNA of the target gene in the lentiviral vector is inserted in series into the downstream of the fluorescent reporter gene.

7. The fluorescent reporter system as claimed in claim 6, It is characterized in that The system also includes positive / negative control siRNA; Preferably, the positive control siRNA includes PC Rluc-siRNA or PC Fluc-siRNA, the nucleotide sequence of the PC Rluc-siRNA is shown in SEQ ID NO.17 and SEQ ID NO.18; the nucleotide sequence of the PC Fluc-siRNA is shown in SEQ ID NO.19 and SEQ ID NO.20; Preferably, the nucleotide sequence of the negative control siRNA is shown as SEQ ID NO.21 and SEQ ID NO.

22.

8. The fluorescent reporter system as claimed in claim 6, It is characterized in that The lentiviral vector comprises at least one of a pLV-Rluc plasmid or a pLV-Fluc plasmid; The pLV-Rluc plasmid is used to express Renilla luciferase, and has a structure of: promoter+T7Promoter+5′UTR+Renilla luciferase+CDS cloning site+3′UTR sequence+PolyA, wherein the CDS cloning site is used to insert the cDNA of the target gene X; The pLV-Fluc plasmid is used to express firefly luciferase, and has a structure of: promoter + T7Promoter + 5′UTR + firefly luciferase + CDS cloning site + 3′UTR sequence + PolyA, and the CDS cloning site is used to insert the cDNA of the target gene X; Preferably, the promoter includes any one of SV40 promoter, CMV promoter, EF1α promoter, CBA promoter, EFS promoter, SFFV promoter, MSCV promoter or PGK promoter; Preferably, the 5′UTR sequence includes any one of 5′UTR1, 5′UTR2, 5′UTR3, 5′UTR4, 5′UTR5, 5′UTR6, 5′UTR7 or 5′UTR8; Preferably, the 3'UTR sequence includes any one of 3'UTR1, 3'UTR2, 3'UTR3, 3'UTR4, 3'UTR5, 3'UTR6, 3'UTR7 or 3'UTR8.

9. Use of the screening method according to any one of claims 1 to 5 or the fluorescent reporter system according to any one of claims 6 to 9 in the field of siRNA screening.

10. A siRNA screening kit, It is characterized in that The kit comprises a lentiviral vector, an auxiliary packaging system for lentiviral packaging, and a positive / negative control siRNA; the lentiviral vector comprises at least one of a pLV-Rluc plasmid or a pLV-Fluc plasmid; The pLV-Rluc plasmid is used to express Renilla luciferase, and has a structure of: promoter+T7Promoter+5′UTR+Renilla luciferase+CDS cloning site+3′UTR sequence+PolyA, wherein the CDS cloning site is used to insert the cDNA of the target gene X; The pLV-Fluc plasmid is used to express firefly luciferase, and has a structure of: promoter + T7Promoter + 5′UTR + firefly luciferase + CDS cloning site + 3′UTR sequence + PolyA, and the CDS cloning site is used to insert the cDNA of the target gene X; The positive control siRNA includes PC Rluc-siRNA or PC Fluc-siRNA, the nucleotide sequence of the PC Rluc-siRNA is shown in SEQ ID NO.17 and SEQ ID NO.18; the nucleotide sequence of the PC Fluc-siRNA is shown in SEQ ID NO.19 and SEQ ID NO.20; The nucleotide sequences of the negative control siRNA are shown in SEQ ID NO.21 and SEQ ID NO.22; Preferably, the promoter includes any one of SV40 promoter, CMV promoter, EF1α promoter, CBA promoter, EFS promoter, SFFV promoter, MSCV promoter or PGK promoter; Preferably, the 5′UTR sequence includes any one of 5′UTR1, 5′UTR2, 5′UTR3, 5′UTR4, 5′UTR5, 5′UTR6, 5′UTR7 or 5′UTR8; Preferably, the 3′UTR sequence includes any one of 3′UTR1, 3′UTR2, 3′UTR3, 3′UTR4, 3′UTR5, 3′UTR6, 3′UTR7 or 3′UTR8; Preferably, the gene X is NOX1 gene, the 5′UTR sequence is 5′UTR1, the 3′UTR sequence is 3′UTR1, the nucleotide sequence of the 5′UTR1 is shown in SEQ ID NO.1, and the nucleotide sequence of the 3′UTR1 is shown in SEQ ID NO.9.

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