A RNA interference virus vector

Abstract

The present invention relates to the field of RNA interference technology, and in particular to an RNA interference viral vector, wherein the RNA interference viral vector expresses shRNA carried by a miR-AB framework based on human miR-30pri-miRNA mir to interfere with the expression of target genes; the miR‑AB sequence is regulated by a variety of eukaryotic promoters; the RNA interference virus vector traces cells that have RNA interference through fluorescent protein reporters, or screens cells that have RNA interference through antibiotics. Among them, the miR‑AB sequence in the RNA interference virus vector is regulated by a variety of eukaryotic promoters, which is conducive to its application in a variety of eukaryotic cells, and the viral vector can rely on its Puromycin resistance to screen cells that have RNA interference through antibiotics, and can also rely on its fluorescent protein reporter molecules to trace cells that have RNA interference. Therefore, it can simultaneously perform single-gene interference and multi-gene arbitrary combination interference on multiple target genes in eukaryotic cells through a variety of viral co-infection methods, thereby simultaneously analyzing the functions of multiple target genes in eukaryotic cells.

Description

Technical Field

[0001] The present invention relates to the technical field of RNA interference, and in particular to an RNA interference virus vector. Background Art

[0002] Target gene deletion research has always been a commonly used method in life sciences to study target gene function.

[0003] RNA interference is a means of target gene deletion, which refers to the phenomenon that small double-stranded RNA can specifically degrade or inhibit the expression of homologous mRNA, thereby inhibiting or shutting down the expression of specific genes. For example, as long as people know the pathogenic gene of a certain disease, they can design small interfering RNA (siRNA) targeting the mRNA of the gene to inhibit or block the expression of the pathogenic gene, thereby achieving the purpose of treating the disease. Specifically, siRNA can specifically bind to the target gene mRNA, and then degrade this mRNA through the RISC complex, thereby interfering with the expression of the target gene mRNA.

[0004] However, since life activities rely on the joint function of multiple genes, it is of great practical significance to interfere with the expression of multiple genes at the same time, that is, multiple RNA interference. However, traditional multiple RNA interference can only interfere with multiple genes at the same time, but cannot simultaneously observe the interference effects of multiple genes alone, nor can it simultaneously observe the interference of any combination of multiple genes.

[0005] Therefore, how to effectively and reliably achieve the simultaneous individual interference and arbitrary combined interference of multiple target genes is a technical problem that technical personnel in this field urgently need to solve. Summary of the invention

[0006] The purpose of the present invention is to provide an RNA interference viral vector, which can simultaneously perform single gene interference and arbitrary combination of multi-gene interference on multiple target genes in eukaryotic cells through multiple viral co-infection methods, thereby simultaneously analyzing the functions of multiple target genes in eukaryotic cells.

[0007] The present invention provides an RNA interference virus vector, wherein the RNA interference virus vector expresses shRNA carried by a miR-AB framework based on human miR-30pri-miRNA. mir To interfere with the expression of target genes;

[0008] The miR-AB sequence is regulated by a variety of eukaryotic promoters;

[0009] The RNA interference virus vector tracks cells that have RNA interference through fluorescent protein reporter molecules, or screens cells that have RNA interference through antibiotics.

[0010] The RNA interference virus vector of the present invention expresses shRNA carried by the miR-AB framework based on human miR-30pri-miRNA mir To interfere with the expression of target genes, wherein the miR-AB sequence in the viral vector is regulated by a variety of eukaryotic promoters, thereby facilitating its application in a variety of eukaryotic cells, and the viral vector can rely on its Puromycin resistance to screen cells with RNA interference through antibiotics, and can also rely on its fluorescent protein reporter molecules to trace cells with RNA interference. The RNA interference viral vector of the present invention has a variety of fluorescent protein reporter molecules as options, so it can simultaneously perform single gene interference and multi-gene arbitrary combination interference on multiple target genes in eukaryotic cells through a variety of viral co-infection methods, thereby simultaneously analyzing the functions of multiple target genes in eukaryotic cells.

[0011] As a preferred embodiment of the present invention, the RNA interference virus vector includes a lentiviral vector and a retroviral vector.

[0012] The lentiviral vector includes 5'LTR, RRE, cPPT, eukaryotic promoter, miR-AB framework, PGK promoter, fluorescent protein or Puromycin resistance, WPRE and 3'LTR in order from 5' to 3';

[0013] The retroviral vector comprises 5'LTR, miR-AB framework, PGK promoter, fluorescent protein or Puromycin resistance, WPRE and 3'LTR in order from 5' end to 3' end.

[0014] As a preferred embodiment of the present invention, the 5' end of the miR-AB sequence includes a 5'-GGATCC-3' sequence, and the 5' end can be recognized and cut by a BamHI restriction endonuclease;

[0015] The 3' end of the miR-AB sequence contains a 5'-GGGCCC-3' sequence, and the 3' end can be recognized and cut by the Apal restriction endonuclease.

[0016] The nucleotide sequence of the miR-AB sequence is specifically shown below:

[0017]

[0018] Among them, bold black fonts represent the introduced BamHI and ApaI restriction sites, and the boxes represent the Guide and Passenger sequences of the gene-specific shRNA.

[0019] Specifically, the 5' end includes a 5'-GGATCC-3' sequence, and the 5' end can be recognized and cut by the BamHI restriction endonuclease; the 3' end includes a 5'-GGGCCC-3' sequence, and the 3' end can be recognized and cut by the Apal restriction endonuclease; and the sequence derived from miR-30 is included between the 5' end and the 3' end.

[0020] The miR-AB sequence is based on the pri-miRNA sequence of miR-30, with artificial restriction sites added to both ends of the gene-specific shRNA sequence, thereby ensuring that only very short gene-specific shRNA sequences are cloned during the cloning process. The latter can be synthesized using DNA oligonucleotide chemical synthesis technology, thereby ensuring the efficiency and accuracy of cloning. Therefore, the miR-AB sequence maintains the shRNA mir The original secondary structure of shRNA can still maximize its function mir The mediated RNA interference effect greatly simplifies the cloning process of gene-specific shRNA.

[0021] As a preferred embodiment of the present invention, the lentiviral vector expresses shRNA via a eukaryotic promoter. mir .

[0022] As a preferred embodiment of the present invention, the eukaryotic promoter includes hCMV, mCMV, CBH, hEF1a, EH and AH;

[0023] Wherein, the sequence of the mCMV is shown as SEQ ID NO.2;

[0024] The sequence of the CBH is shown in SEQ ID NO.3;

[0025] The sequence of hEF1a is shown in SEQ ID NO.4;

[0026] The sequence of the EH is shown in SEQ ID NO.5;

[0027] The sequence of AH is shown in SEQ ID NO.6.

[0028] As a preferred embodiment of the present technical solution, the fluorescent proteins include mTagBFP2, Azurite, Ametrine, EGFP, Venus, mOrange, mCherry2 and E2-Crimson;

[0029] Wherein, the sequence of Azurite is shown as SEQ ID NO.7;

[0030] The sequence of mTagBFP2 is shown in SEQ ID NO.8;

[0031] The sequence of EGFP is shown in SEQ ID NO.9;

[0032] The sequence of Ametrine is shown in SEQ ID NO.10;

[0033] The sequence of mOrange is shown in SEQ ID NO.11;

[0034] The sequence of mCherry2 is shown in SEQ ID NO.12;

[0035] The sequence of E2-Crimson is shown in SEQ ID NO.13.

[0036] As a preferred embodiment of the present technical solution, the RNA interference virus vector expressing Puromycin resistance is used to screen cells in which target gene interference occurs;

[0037] Preferably, the gene sequence of Puromycin resistance is shown as SEQ ID NO.14.

[0038] As a preferred embodiment of the present technical solution, RNA interference virus vectors expressing fluorescent protein are used to trace or sort cells in which target gene interference occurs.

[0039] As a preferred embodiment of the present technical solution, the RNA interference viral vector interferes with the expression of the target gene in eukaryotic cells.

[0040] As a preferred embodiment of the present technical solution, the RNA interference viral vectors perform multiple RNA interference in eukaryotic cells in any combination.

[0041] The present invention also discloses a method for preparing the RNA interference virus vector, and an analysis method for the single interference and any combined interference of multiple target genes, the specific steps of which are as follows:

[0042] 1. Design shRNA for multiple target genes mir The design method can use online tools such as shERWOOD and SplashRNA to select one or several shRNAs with the highest scores. mir You can also design one or several shRNAs according to your own experimental purpose. mir ;

[0043] 2. According to the experimental purpose and experimental conditions (such as the configuration of multicolor fluorescence microscope or flow cytometer), for each gene specific shRNA mir Choose a fluorescent protein as a reporter molecule to ensure that these gene-specific shRNAs mirThe corresponding fluorescent protein molecules can be distinguished and identified on a multi-color fluorescence microscope or flow cytometer;

[0044] 3. The present invention provides a viral vector capable of expressing 8 different fluorescent proteins, among which the combination of Azurite and mTagBFP2 or the combination of EGFP and Venus cannot be used, while any combination of other fluorescent proteins can be achieved;

[0045] 4. Add shRNA to each target gene mir They were cloned into these miR-AB viral vectors expressing different fluorescent proteins;

[0046] 5. Use 293T cells or other packaging cells to package the virus. If necessary, the virus can be concentrated, and the concentration method should ensure that there is no significant impact on the activity of the virus;

[0047] 6. Carry gene-specific shRNA with different fluorescent proteins as reporter molecules mir If there is a special purpose, such as expecting to obtain a certain gene-specific shRNA mir The high infection efficiency of the virus can appropriately increase the proportion of the virus. Before and after virus infection, the cells can be subjected to conventional cell biology treatments;

[0048] 72 hours or longer after virus infection, directly observe cells expressing a single fluorescent protein or various combinations of fluorescent proteins by multicolor fluorescence microscopy, or analyze or sort these cells by multicolor flow cytometry.

[0049] Among them, the shRNA of each target gene mir The specific steps for cloning into these miR-AB viral vectors expressing different fluorescent proteins are as follows;

[0050] 1. Design shRNA using online tools such as shERWOOD or SplashRNA mir Sequence, select the shRNA with the highest score mir ;

[0051] 2. Use miR-AB Oligo tool to transfer shRNA mir The sequence was converted into two miR-AB framework-based sequences;

[0052] 3. Synthesize the above two sequences using DNA oligonucleotide chemical synthesis technology;

[0053] 4. Annealing the two synthesized DNA oligonucleotides to form a double-stranded DNA sequence;

[0054] 5. Connect this double-stranded DNA sequence to the chronic viral vector or retroviral vector of miR-AB after BamHI and ApaI cutting and purification;

[0055] 6. Use physical and chemical methods to transform competent cells so that they absorb DNA molecules in the surrounding environment. The transformed competent cells are cultured in culture medium and positive clones carrying exogenous DNA molecules are screened.

[0056] Compared with the prior art, the RNA interference virus vector of the present invention has the following advantages:

[0057] 1. The RNA interference virus vector of the present invention expresses shRNA carried by the miR-AB framework based on human miR-30pri-miRNA mir To interfere with the expression of target genes, wherein the miR-AB sequence in the viral vector is regulated by a variety of eukaryotic promoters, thereby facilitating its application in a variety of eukaryotic cells, and the viral vector can rely on its Puromycin resistance to screen cells with RNA interference through antibiotics, and can also rely on its fluorescent protein reporter molecules to trace cells with RNA interference. The RNA interference viral vector of the present invention has a variety of fluorescent protein reporter molecules as options, so it can simultaneously perform single gene interference and multi-gene arbitrary combination interference on multiple target genes in eukaryotic cells through a variety of viral co-infection methods, thereby simultaneously analyzing the functions of multiple target genes in eukaryotic cells.

[0058] 2. The present invention adopts the latest shRNA based on miR-AB framework mir The RNA interference method mediated by the RNA interference virus vector is different from the traditional siRNA method or shRNA method. The siRNA is produced by simulating the processing of endogenous miRNA in eukaryotic cells. The biggest advantage of the present invention is that it overcomes the cytotoxicity of the traditional siRNA or shRNA method, that is, because the production of a large amount of shRNA or the transfection of a large amount of siRNA leads to an overload of the RISC complex, the endogenous miRNA processing site, thereby interfering with the processing of endogenous miRNA and ultimately causing cytotoxicity. shRNA mir This advantage is particularly important for multiplex RNA interference, which requires the expression of multiple shRNAs in cells. mir , and these "excess" shRNA mir The interference of multiple genes can be completed under the premise of ensuring the normal life activities of cells to the maximum extent. Therefore, the RNA interference virus vector of the present invention can efficiently and reliably complete multiple RNA interference.

[0059] 3. The present invention constructs an RNA interference virus vector with multiple fluorescent proteins as reporter molecules (such as Figure 1 As shown in Figure 2, these fluorescent protein molecules have their own unique spectral characteristics, so they can be identified using fluorescence microscopy or flow cytometry (as shown in Figure 2). Figure 2 Therefore, the combined use of RNA interference virus vectors of the present invention, i.e., multiple RNA interference based on multiple fluorescent proteins as reporter molecules, can identify cells labeled with various fluorescent protein combinations, thereby performing a detailed analysis of the combined functions of multiple target genes at the single cell level. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0061] Figure 1 This is a schematic diagram of the RNA interference virus vector based on miR-AB of the present invention;

[0062] Figure 2 For the present invention Figure 1 The excitation spectrum characteristics of the fluorescent protein molecules used in the RNA interference virus vector shown;

[0063] Figure 3 For the present invention Figure 1 The emission spectrum characteristics of the fluorescent protein molecules used in the RNA interference virus vector shown;

[0064] Figure 4 is the name of the RNA interference virus vector based on miR-AB of the present invention;

[0065] Figure 5 The flow cytometry of the present invention shows the discrimination of any combination of six fluorescent proteins in CD8+T cells;

[0066] Figure 6 The present invention is based on the single interference and any combination of interference of multiple target genes marked with multi-color;

[0067] Figure 7 This is a cytotoxicity analysis of the combined interference of multiple target genes of the present invention. DETAILED DESCRIPTION

[0068] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0069] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular form also includes the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this description, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0070] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0071] Example 1

[0072] Prepare a lentiviral vector (such as Figure 4 shown).

[0073] S11, using the PLVX-Puro lentiviral vector as a backbone, inserting an artificially synthesized sequence (SEQ ID NO.1), which includes a miR-AB sequence, a Venus fluorescent protein gene sequence controlled by a PGK promoter, and a WPRE sequence, into the vector through the AfeI and KpnI restriction sites, thereby preparing a LCAPVW vector;

[0074] S12, using LCAPVW vector as the backbone, using ClaI and AfeI restriction sites, replacing the hCMV promoter with mCMV (SEQ ID NO.2), CBH (SEQ ID NO.3), hEF1a (SEQ ID NO.4), EH (SEQ ID NO.5) or AH (SEQ ID NO.6) promoter, thereby preparing LMAPVW, LBAPVW, LEAPVW, LHAPVW and LAAPVW. These promoters have different transcriptional activities in eukaryotic cells, thereby ensuring that the RNA interference process based on miR-AB can be carried out smoothly in a variety of eukaryotic cells;

[0075] Since these promoters have some endogenous restriction sites that may interfere with subsequent cloning work, the present invention optimizes these sites to remove these restriction sites.

[0076] S13, using LCAPVW, LMAPVW, LBAPVW, LEAPVW, LHAPVW and LAAPVW vectors as the backbone, using the PacI and PmeI restriction sites at both ends of the Venus gene sequence, the Venus sequence was replaced with Azurite (SEQ ID NO.7), mTagBFP2 (SEQ ID NO.8), EGFP (SEQ ID NO.9), Ametrine (SEQ ID NO.10), mOrange (SEQ ID NO.11), mCherry2 (SEQ ID NO.12) or E2-Crimson (SEQ ID NO.13). NO.13) sequence to prepare LCAPZW, LMAPZW, LBAPZW, LEAPZW, LHAPZW, LAAPZW, LCAPTW, LMAPTW, LBAPTW, LEAPTW, LHAPTW, LAAPTW, LCAPGW, LMAPGW, LBAPGW, LEAPGW, LHAPGW, LAAPGW, LCAPAW, LMAPAW, LBAPAW, LEAPAW, LHAPAW, LAAPAW, LCAPOW, LMAPOW, LBAPOW, LEAPOW, LHAPOW, LAAPOW, LCAPCW, LMAPCW, LBAPCW, LEAPCW, LHAPCW, LAAPCW, LCAPEW, LMAPEW, LBAPEW, LEAPEW, LHAPEW, LAAPEW;

[0077] Since the wild-type sequences of these fluorescent proteins have some endogenous restriction sites that may interfere with subsequent cloning work, the present invention optimizes these sites to remove these restriction sites.

[0078] S14. Using LCAPVW, LMAPVW, LBAPVW, LEAPVW, LHAPVW and LAAPVW vectors as the backbone, the PacI and PmeI restriction sites at both ends of the Venus gene sequence were used to replace the Venus sequence with the antibiotic Puromycin resistance gene sequence (SEQ ID NO.14), thereby preparing LCAPPW, LMAPPW, LBAPPW, LEAPPW, LHAPPW, LAAPPW, which are convenient for use in antibiotic screening experiments.

[0079] Example 2

[0080] Prepare a retroviral vector (such as Figure 4 shown).

[0081] S21, using the MIGR1 retroviral vector as a backbone, inserting an artificially synthesized sequence (SEQ ID NO.1), which includes a miR-AB sequence, a Venus fluorescent protein gene sequence controlled by a PGK promoter, and a WPRE sequence, into the vector through the BglII and ClaI restriction sites, thereby preparing a MAPVW vector;

[0082] Using the MAPVW vector as the backbone, the PacI and PmeI restriction sites at both ends of the Venus gene sequence were used to replace the Venus sequence with Azurite (SEQ ID NO.7), mTagBFP2 (SEQ ID NO.8), EGFP (SEQ ID NO.9), Ametrine (SEQ ID NO.10), mOrange (SEQ ID NO.11), mCherry2 (SEQ ID NO.12) or E2-Crimson (SEQID NO.13) sequences, thereby preparing MAPZW, MAPTW, MAPGW, MAPAW, MAPOW, MAPCW, and MAPEW;

[0083] The present invention optimizes the coding sequence of the fluorescent protein and removes the endogenous restriction sites that may interfere with subsequent cloning work.

[0084] S23. Using the MAPVW vector as the backbone, utilizing the PacI and PmeI restriction sites at both ends of the Venus gene sequence, the Venus sequence was replaced with the antibiotic Puromycin resistance gene sequence (SEQ ID NO.14), thereby preparing MAPPW.

[0085] Figure 1 Schematic diagram of RNA interference virus vector based on miR-AB. LV represents lentiviral vector and RV represents retroviral vector. Lentiviral vector controls the expression of miR-AB sequence by various eukaryotic promoters (hCMV, mCMV, CBH, hEF1a, EH and AH), while retroviral vector controls the expression of miR-AB sequence by its own endogenous promoter. Both lentiviral vector and retroviral vector control the expression of various fluorescent proteins through PGK promoter, and WPRE sequence provides stability for the expression of this vector.

[0086] For research Figure 1The optical properties of the fluorescent protein molecules used in the RNA interference virus vector shown in the figure, the present invention provides the excitation spectrum (Wavelength of excitation) ( Figure 2 ) and emission spectrum (Wavelength of emission) ( Figure 3 ). The five excitation lights (355nm, 405nm, 488nm, 561nm and 633nm) currently widely used in flow cytometers and fluorescence microscopes have been marked in the excitation spectrum diagram.

[0087] To further verify the feasibility of the multi-color analysis of RNA interference virus vectors based on multiple fluorescent proteins of the present invention, the following experiments were conducted:

[0088] 1. Flow cytometry identification of cells labeled with different fluorescent proteins

[0089] The present invention demonstrates the feasibility of multi-color analysis by expressing any combination of six fluorescent proteins in CD8+ T cells of mice.

[0090] First, the CD4 molecule-specific shRNA designed by SplashRNA mir They were cloned into retroviral vectors expressing one of six fluorescent proteins, Azurite, EGFP, Ametrine, mOrange, mCherry2, or E2-Crimson as reporters;

[0091] Then, after packaging the virus using 293T cells, the viruses carrying the six fluorescent proteins were mixed and used to co-infect activated CD8+T cells;

[0092] After 72 hours, flow cytometry was used to distinguish the distribution of the six fluorescent protein-positive cells in the cell population.

[0093] like Figure 5 As shown, CD8+T cells co-infected with viruses carrying these six fluorescent proteins can be distinguished into four cell groups on a flow cytometer by any combination of two fluorescent proteins, namely, one double negative, two single positives, and one double positive. The boundaries between these four cell groups are clear, and the edges of each cell group are smooth, which is convenient for gate analysis. This shows that the multi-color analysis based on multiple fluorescent proteins of the present invention is feasible.

[0094] 2. Multiplex RNA interference based on multiple fluorescent proteins as reporter molecules

[0095] The present invention demonstrates multi-color multiple RNA interference effects by interfering with multiple cell surface molecules (CD127, CD90, CD44 and CD8a) of CD8+T cells of mice.

[0096] First, retroviruses expressing these molecular specific shRNAmir were prepared, namely, mouse CD127, CD90, CD44 and CD8a specific shRNA designed by SplashRNA mir The cells were cloned into retroviral vectors carrying Azurite, EGFP, Ametrine and mCherry2. At the same time, the mouse CD19-specific shRNA designed by SplashRNA was mir cloned into a retroviral vector carrying Azurite, EGFP, Ametrine, and mCherry2 as a negative control (because CD8+ T cells do not express CD19);

[0097] After the above vectors were packaged into viruses, the activated CD8+T cells were co-infected with these viruses, and the cells were stained with CD127-PE, CD90-PE / Cy7, CD44-APC, and CD8a-PerCP / Cy5.5 antibodies 96 hours later;

[0098] Finally, flow cytometry was used to analyze the expression levels of CD127, CD90, CD44 and CD8a in single-positive or diverse cells of these four fluorescent protein molecules.

[0099] like Figure 6 As shown, the flow cytometer can clearly distinguish the cell populations expressing these four fluorescent proteins. According to whether these four fluorescent proteins are expressed or not, these cell populations are divided into 16 types (left panel), including 1 negative (None in the figure), 4 single positive (Single+ in the figure), 6 double positive (Double+ in the figure), 4 triple positive (Triple+ in the figure) and 1 quadruple positive (Quadruple+ in the figure). By analyzing the expression levels of CD127, CD90, CD44 and CD8a in each cell population, it is possible to observe whether these cells have undergone RNA interference of one or several molecules. As shown in the panel on the right, each cell population has unique RNA interference characteristics, that is, the cells that undergo RNA interference of one or several genes are precisely their specific shRNA. mir Corresponding fluorescent protein reporter molecule positive cells. This shows that the multiple RNA interference method based on multiple fluorescent proteins as reporter molecules of the present invention is feasible and reliable. ( Figure 6The left histogram in the middle shows the expression level of fluorescent protein, in which the background of the fluorescent protein positive histogram is highlighted; the right histogram in the figure shows the expression level of each targeted molecule).

[0100] 3. Low cytotoxicity of multiple RNAi interference

[0101] Since this multiple RNA interference method requires the expression of multiple shRNAs in cells, whether these "excess" shRNAs will produce cytotoxicity needs further verification.

[0102] First, the CD4 or CD19 specific shRNA designed by SplashRNA mir Each shRNA was cloned into a lentiviral vector expressing one of four fluorescent proteins. mir Azurite, GFP, Ametrine, and mOrange were used as reporter molecules, respectively;

[0103] These vectors were then packaged into viruses and mixed to co-infect 293T cells;

[0104] The cells were cultured for a long time (22 days) and passaged at the time points shown in the figure. Cell counting and flow cytometry were performed at each time point to analyze the proportion of the four fluorescent protein-positive cells in the cell population;

[0105] Finally, the total number of cells expressing single-positive and full-positive cells of the four fluorescent proteins at each time point was calculated to express the cell proliferation of various fluorescent protein combinations.

[0106] like Figure 7 As shown, at each time point, there was no significant difference in the proliferation process of cells that did not express fluorescent protein, expressed one fluorescent protein, or expressed four fluorescent proteins. This shows that this multiple RNAi interference method has no significant toxicity to cells. ( Figure 7 The cell numbers shown in the figure are normalized based on the number of cells without fluorescent protein expression on day 4. The statistical differences between the numbers of cells positive for various fluorescent proteins were analyzed by independent sample Student's T test. N / A indicates no statistical difference (P>0.05).

[0107] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention. Sequence Listing <110> Binzhou Medical College <120> A RNA interference virus vector <160> 14 <170> SIPOSequenceListing 1.0 <210> 1 <211> 2920 <212> RNA <213> Artificial Sequence <400> 1 tcgactaggg ataacagggt aattgtttga atgaggcttc agtactttac agaatcgttg 60 cctgcacatc ttggaaacac ttgctgggat tacttcgact tcttaaccca acagaaggct 120 cgagaaggta tattgctgtt gacaggatcc acattaacaa ctttaagtcc agcaatttga 180 gttaagggtg ttgctctcaa tgatttcact aatggttcaa tttttaattt cttttcttct 240 ggttcaaaat tcaagtttca agtgaaagtg taatatgcac ccatttcttt caataaatct 300 tctaaatagt ccactaatgt tttattttgt tttttacaaa atcaagcagc ctctgctatc 360 aatatagaac cttgtattcc atctttatct ctagctgagt catcaattac atatccataa 420 ctttcttcat aagcaaaaac aaaatttaat ccgttatctt cttctttagc aatttctcta 480 cccattcatt tcaatccagt taaagttttt acaatcttaa ctccatattt ttcctgagcg 540 attctatcac ccaaatcact tgttacaaaa cttgaatata gagccggatt ttttggaatg 600 ctatttaagc gttttagatt tgataatttt caatcaatta aaattggtcc tgtttgattt 660 ccatctaatc ttacaaaatg accatcatgt tttattgcca ttccaaatct gtcagcatct 720 gggtcattca tataataat atctgcatca tgtttaatac catattcaag cggtattttt 780 catgcaggat caaattctgg atttggattt acaacatttt tcaatgtttc atcttcaaat 840 gcacgatctt caccctcaat aacgttatat cctgattcac gtaatctttt tggggtaaat 900 ttagttcctg ttccattaac tgcgctaaaa atagggccct gcctcggact tcaaggggct 960 agaattcgag caattatctt gttactaaa actgaatacc ttgctatctc tttgatacat 1020 ttttacaaag ctgaattaaa atggtataaa ttaaatcact ttttcaatt ctaccgggta 1080 ggggaggcgc ttttcccaag gcagtctgga gcatgcgctt tagcagcccc gctgggcact 1140 tggcgctaca caagtggcct ctggcctcgc acacattcca catccaccgg taggcgccaa 1200 ccggctccgt tctttggtgg ccccttcgcg ccaccttcta ctcctcccct agtcaggaag 1260 ttcccccccg ccccgcagct cgcgtcgtgc aggacgtgac aaatggaagt agcacgtctc 1320 actagtctcg tgcagatgga cagcaccgct gagcaatgga agcgggtagg cctttggggc 1380 agcggccaat agcagctttg ctccttcgct ttctgggctc agaggctggg aaggggtggg 1440 tccgggggcg ggctcagggg cgggctcagg ggcggggcgg gcgcccgaag gtcctccgga 1500 ggcccggcat tctgcacgct tcaaaagcgc acgtctgccg cgctgttctc ctcttcctca 1560 tctccgggcc tttcgacctt taattaacca ccatggtgag caagggcgag gagctgttca 1620 ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg 1680 tgtccggcga gggcgagggc gatgccacct acggcaagct gaccctgaag ctgatctgca 1740 ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccctgggc tacggcctgc 1800 agtgcttcgc ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc 1860 ccgaaggcta cgtccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc 1920 gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg 1980 acttcaagga ggacggcaac atcctggggc acaagctgga gtacaactac aacagccaca 2040 acgtctatat caccgccgac aagcagaaga acggcatcaa ggccaacttc aagatccgcc 2100 acaacatcga ggacggcggc gtgcagctcg ccgaccacta ccagcagaac acccccatcg 2160 gcgacggccc cgtgctgctg cccgacaacc actacctgag ctaccagtcc gccctgagca 2220 aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc gccgccggga 2280 tcactctcgg catggacgag ctgtacaagt aagtttaaac atcaacctct ggattacaaa 2340 atttgtgaaa gattgactgg tattcttaac tatgttgctc cttttacgct atgtggatac 2400 gctgctttaa tgcctttgta tcatgctatt gcttcccgta tggctttcat tttctctccc 2460 ttgtataaat cctggttgct gtctctttat gaggagttgt ggcccgttgt caggcaacgt 2520 ggcgtggtgt gcactgtgtt tgctgacgca accccactg gttggggcat tgccaccacc 2580 tgtcagctcc tttccgggac tttcgctttc cccctcccta ttgccacggc ggaactcatc 2640 gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt tgggcactga caattccgtg 2700 gtgttgtcgg ggaagctgac gtcctttcca tggctgctcg cctgtgttgc cacctggatt 2760 ctgcgcggga cgtccttctg ctacgtccct tcggccctca atccagcgga ccttccttcc 2820 cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt 2880 cggatctccc tttgggccgc ctccccgcct ggcctgcagg 2920 <210> 2 <211> 536 <212> mCMV <213> Artificial Sequence <400> 2 tggcacgtat actgagtcat tagggacttt ccaatgggtt ttgcccagta cataaggtca 60 ataggggtga atcaacagga aagtcccatt ggagccaagt acactgagtc aatagggact 120 ttccattggg ttttgcccag tacaaaaggt caataggggg tgagtcaatg ggtttttccc 180 attattggca cgtacataag gtcaataggg gtgagtcatt gggtttttcc agccaattta 240 attataacgc catgtacttt cccaccattg acgtcaatgg gctattgaaa ctaatgcaac 300 gtgaccttta aacggtactt tcccatagct gattaatggg aaagtaccgt tgtcgagcca 360 atacacgtca atgggaagtg aaagggcagc caaaacgtaa caccgccccg gttttcccct 420 ggaaattcca tattggcacg cattctattg gctgagctgc gttctacgtg ggtataagag 480 gcgcgaccag cgtccgtacc gtcgcagtct tcggtctgac caccgtagaa cgcaga 536 <210> 3 <211> 802 <212> CBH <213> Artificial Sequence <400> 3 agtcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc 60 attgacgtca atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt 120 acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat 180 tgacgtcaat gacggtaaat ggcccgcctg gcattgtgcc cagtacatga ccttatggga 240 ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgg tcgaggtgag 300 ccccacgttc tgcttcactc tccccatctc ccccccctcc ccacccccaa ttttgtattt 360 atttattttt taattatttt gtgcagcgat gggggcgggg gggggggggg ggcgcgcgcc 420 aggcggggcg gggcggggcg aggggcgggg cggggcgagg cggagaggtg cggcggcagc 480 caatcagagc ggcgcgctcc gaaagtttcc ttttatggcg aggcggcggc ggcggcggcc 540 ctaaaaag cgaagcgcgc ggcggggcggg agtcgctgcg acgctgcctt cgccccgtgc 600 cccgctccgc cgccgcctcg cccggcccgc cccggctctg actgaccgcg ttactcccac 660 aggtgagcgg gcgggacggc ccttctcctc cgggctgtaa ttagctgagc aagaggtaag 720 ggtttaaggg atggttggtt ggtggggtat taatgtttaa ttacctgttt tacaggcctg 780 aaatcacttg gttttaggtt gg 802 <210> 4 <211> 1164 <212> hEF1a <213> Artificial Sequence <400> 4 gggcagagcg cacatcgccc acagtccccg agaagttggg gggaggggtc ggcaattgaa ccggagccta gagaaggtgg cgcggggtaa actgggaaag tgatgtcgtg tactggctcc gccttttttcc cgagggtggg ggagaaccgt fathergc gtgacgttc gtgaacgttc 180 ttttttcgcaa cgggtttgcc gccagaacac aggtaagtgc cgtgtgtggt tcccgcgggc 240 ctggcctctt tacgggttat ggcccttgcg tgccttgaat tacttccacg cccctggctg 300 cagtacgtga ttcttgatcc cgagcttcgg gttggaagtg ggtgggagag ttcgaggcct 360 tgcgcttaag gagccccttc gcctcgtgct tgagttgagg cctggcttgg gcgctggggc 420 cgccgcgtgc gaatctggtg gcaccttcgc gcctgtctcg ctgctttcga taagtctcta 480 gccatttaaa atttttgatg acctgctgcg acgctttttt tctggcaaga tagtcttgta 540 aatgcgggcc aagatcggca cactggtatt tcggtttttg gggccgcggg cggcgacggg 600 gccggtgcgt cccagcgcac atgttcggcg aggcggggcc tgcgagcgcg gccaccgaga 660 atcggacggg ggtagtctca agctggccgg cctgctctgg tgcctggcct cgcgccgccg 720 tgtatcgccc cgccctgggc ggcaaggctg gcccggtcgg caccagttgc gtgagcggaa 780 agatggccgc ttcccggccc tgctgcaggg agctcaaaat ggaggacgcg gcgctcggga 840 gagcgggcgg gtgagtcacc cacacaaagg aaaagggcct ttccgtcctc agccgtcgct 900 tcatgtgact ccacggagta ccgggcgccg tccaggcacc tcgattagtt ctcgcgcttt 960 tggagtacgt cgtctttagg ttggggggag gggttttatg cgatggagtt tccccacact 1020 gagtgggtgg agactgaagt taggccagct tggcacttga tgtaattctc cttggaatt gccctttttg agtttggatc ttggttcatt ctcaagcctc agacagtggt tcaaagtttt 1140 tttcttccat ttcaggtgtc gtga 1164 <210> 5 <211> 479 <212> EH <213> Artificial Sequence <400> 5 gggcagagcg cacatcgccc acagtccccg agaagttggg gggaggggtc ggcaattgaa ccggagccta gagaaggtgg cgcggggtaa actgggaaag tgatgtcgtg tactggctcc gccttttttcc cgagggtggg ggagaaccgt fathergc gtgacgttc gtgaacgttc 180 240. tttttcgcaa cgggtttgcc gccagaacac agggctcgca tctctccttc acgcgcccgc cgccttacct gaggccgcca tccacgccgg ttgagtcgcg ttctgccgcc tccccgcctgt 300 ggtgcctcct gaactacgtc cgccgtctag gtaagtttag agctcaggtc gagaccgggc 360 ctttgtccgg cgctcccttg gagcctacct agactcagcc ggctctccac gctttgcctg 420 accctgcttg ctcaactcta cgtctttgtt tcgttttctg ttctgcgccg ttacagatc 479 <210> 6 <211> 1775 <212> AH <213> Artificial Sequence <400> 6 gttccatgtc cttatatgga ctcatctttg cctattgcga cacacactca atgaacacct 60 actacgcgct gcaaagagcc ccgcaggcct gaggtgcccc cacctcacca ctcttcctat 120 ttttgtgtaa aaatccagct tcttgtcacc acctccaagg agggggagga ggaggaaggc 180 aggttcctct aggctgagcc gaatgcccct ctgtggtccc acgccactga tcgctgcatg 240 cccaccacct gggtacacac agtctgtgat tcccggagca gaacggaccc tgcccacccg 300 gtcttgtgtg ctactcagtg gacagaccca aggcaagaaa gggtgacaag gacagggtct 360 tcccaggctg gctttgagtt cctagcaccg ccccgccccc aatcctctgt ggcacatgga 420 gtcttggtcc ccagagtccc ccagcggcct ccagatggtc tgggagggca gttcagctgt 480 ggctgcgcat agcagacata caacggacgg tgggcctaga cccaggctgt gtagacccag 540 cccccccgcc ccgcagtgac taggtcaccc actaacgccc caggcctggt cttggctggg 600 cgtgactgtt accctcaaaa gcaggcagct ccagggtaaa aggtgccctg ccctgtagag 660 cccaccttcc ttcccagggc tgcggctggg taggtttgta gccttcatca cgggccacct 720 ccagccactg gaccgctggc ccctgccctg tcctggggag tgtggtcctg cgacttctaa 780 gtggccgcaa gccacctgac tcccccaaca ccacactcta cctctcaagc ccaggtctct 840 ccctagtgac ccacccagca catttagcta gctgagcccc acagccagag gtcctcaggc 900 cctgctttca gggcagttgc tctgaagtcg gcaaggggga gtgactgcct ggccactcca 960 tgccctccaa gagctccttc tgcaggagcg tacagaaccc agggccttgg cacccgtgca 1020 gaccctggcc caccccacct gggcgctcag tgcccaagag atgtccacaa ctaggatgtc 1080 ccgcggtggg tggggggcca gagagacggg caggccgggg gcaggcctgg ccatgcgggg 1140 ccgaaccggg cactgcccag cgtggggcgc gggggccacg gcgcgcgccc ccagcccccc 1200 ggcccagcac cccaaggcgg ccaacgccaa aactctccct cctcctcttc ctcaatctcg 1260 ctctcgctct tttttttttt cgcaaaagga ggggagaggg ggtaaaaaaa tgctgcactg 1320 tgcggcgaag ccggtgagtg agcggcgcgg ggccaatcag cgtgcgccgt tccgaaagtt 1380 tgcggcgaag ccggtgagtg agcggcgcgg ggccaatcag cgtgcgccgt tccgaaagtt 1380 gccttttatg gctcgaacgg ccgcggcggc gccctataaa acccagcggc gcgacgcgcc 1440 gccttttatg gctcgaacgg ccgcggcggc gccctataaa acccagcggc gcgacgcgcc 1440 accaccgccg agaccgcgtc cgccccgcga gcacagagcc tcgcctttgc cgatccgccg 1500 accaccgccg agaccgcgtc cgccccgcga gcacagagcc tcgcctttgc cgatccgccg 1500 cccgtccagg ctcgcatctc tccttcacgc gcccgccgcc ttacctgagg ccgccatcca 1560 cccgtccagg ctcgcatctc tccttcacgc gcccgccgcc ttacctgagg ccgccatcca 1560 cgccggttga gtcgcgttct gccgcctccc gcctgtggtg cctcctgaac tacgtccgcc 1620 cgccggttga gtcgcgttct gccgcctccc gcctgtggtg cctcctgaac tacgtccgcc 1620 gtctaggtaa gtttagagct caggtcgaga ccgggccttt gtccggcgct cccttggagc 1680 gtctaggtaa gtttagagct caggtcgaga ccgggccttt gtccggcgct cccttggagc 1680 ctacctagac tcagccggct ctccacgctt tgcctgaccc tgcttgctca actctacgtc 1740 ctacctagac tcagccggct ctccacgctt tgcctgaccc tgcttgctca actctacgtc 1740 tttgtttcgt tttctgttct gcgccgttac agatc 1775 tttgtttcgt tttctgttct gcgccgttac agatc 1775 <210> 7<210> 7 <211> 717<211> 717 <212> Azurite <212> Azurite <213> 人工序列(Artificial Sequence) <213> Artificial Sequence <400> 7 <400> 7 atgagcaaag gcgaagagct gttcaccggc gtggtgccca tcctggtgga gctggacggc 60 atgagcaaag gcgaagagct gttcaccggc gtggtgccca tcctggtgga gctggacggc 60 gacgtgaacg gccacaagtt cagcgtgagc ggcgagggcg agggcgacgc cacctacggc 120 gacgtgaacg gccacaagtt cagcgtgagc ggcgagggcg agggcgacgc cacctacggc 120 aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctg 180 aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctg 180 gtgaccaccc tgagccacgg cgtgcagtgc ttcagccggt accccgacca catgaagcag 240 cacgacttct tcaagagcgc catgcccgag ggctacgtgc aggagcggac catcttcttc 300 aaggacgacg gcaactacaa gacccgggcc gaggtgaagt tcgagggcga caccctggtg 360 aaccggatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct gggccacaag 420 ctggagtaca acttcaacag ccacaacatc tacatcatgg ccgacaagca gaagaacggc 480 atcaaggtga acttcaagat ccggcacaac atcgaggacg gcagcgtgca gctggccgac 540 cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac 600 ctgagcaccc agagcgccct gagcaaggac cccaacgaga agcgggacca catggtgctg 660 ctggagttcc ggaccgccgc cggcatcacc cacggcatgg acgagctcta taagtaa 717 <210> 8 <211> 714 <212> mTagBFP2 <213> Artificial Sequence <400> 8 atggtgtcta agggcgaaga gctgattaag gagaacatgc acatgaagct gtacatggag 60 ggcaccgtgg acaaccatca cttcaagtgc acatccgagg gcgaaggcaa gccctacgag 120 ggcacccaga ccatgagaat caaggtggtc gagggcggcc ctctcccctt cgccttcgac 180 atcctggcta ctagcttcct ctacggcagc aagaccttca tcaaccacac ccagggcatc 240 cccgacttct tcaagcagtc cttccctgag ggcttcacat gggagagagt caccacatac 300 gaagacgggg gcgtgctgac cgctacccag gacaccagcc tccaggacgg ctgcctcatc 360 tacaacgtca agatcagagg ggtgaacttc acatccaacg gccctgtgat gcagaagaaa 420 acactcggct gggaggcgtt caccgagacg ctgtaccccg ctgacggcgg cctggaaggc 480 agaaacgaca tggccctgaa gctcgtgggc gggagccatc tgatcgcaaa cgccaagacc 540 acatatagat ccaagaaacc cgctaagaac ctcaagatgc ctggcgtgta ctatgtggac 600 tacagactgg aaagaatcaa ggaggccaac aacgagacct acgtcgagca gcacgaggtg 660 gcagtggcca gatactgcga cctccctagc aaactggggc acaagctaaa ttag 714 <210> 9 <211> 720 <212> EGFP <213> Artificial Sequence <400> 9 atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60 ggcgacgtaa acggccacaa gttcagcgtg tctggcgagg gcgagggcga tgccacctac 120 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 180 ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 240 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420 aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac 480 ggcatcaagg cgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 540 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600 tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660 ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa 720 <210> 10 <211> 720 <212> Ametrine <213> Artificial Sequence <400> 10 atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60 ggcgacgtaa acggccacag gttcagcgtg cgcggcgagg gcgagggcga tgccaccaac 120 ggcaagctga ccctgaagtt catctgcacc tccggcgagc tgcccgtgcc ctggcccacc 180 ctcgtgacca ccctgtctta cggcgtgcag tgcttcgccc gctaccccga ccacatgaag 240 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatctcc 300 ttcaaggacg acggcagcta caggacccgc gccgaggtga agttcgaggg cgacaccctg 360 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420 aagctggagt acaacatgaa cgcttgggac gcttatatca cggccgacaa gcagaagaac 480 ggcatcaaag cgaacttcaa gatcgagcac aacgtcaagg acggcggcgt gcagctcgcc 540 gacgcttacc agcagaacac ccccatcggc gacggctccg tgctgctgcc tgacaaccac 600 tacctgagct tccagagcaa gctgttcaaa gaccccaacg agcagcgcga tcacatggtc 660 ctgctggagt tcgttaccgc cgccgggata actcccggca tggacgagct gtacaagtaa 720 <210> 11 <211> 711 <212> mOrange <213> Artificial Sequence <400> 11 atggtgagca agggcgaaga gaataacatg gcaatcatca aggagttcat gcgcttcaag 60 gttcgcatgg agggctccgt gaacggccac gagttcgaga tcgagggcga gggcgagggc 120 cgcccctacg agggctttca gaccgctaag ctgaaggtga ccaagggtgg ccccctgccc 180 ttcgcctggg acatcctgtc ccctcagttc acctacggct ccaaggcata cgtgaagcac 240 cccgccgaca tccccgacta cttcaagctg tccttccccg agggcttcaa gtgggagcgc 300 gtgatgaact tcgaggacgg cggcgtggtg accgtgaccc aggactcgtc cctgcaagac 360 ggcgagttca tctacaaggt gaagctgcgc ggcaccaact tcccctccga cggccccgta 420 atgcagaaaa agactatggg ctgggaggca tcctccgagc ggatgtaccc cgaggacggc 480 gccctgaagg gcgagatcaa gatgaggctg aagctgaagg acggcggcca ctacacctcc 540 gaggtcaaga ccacctacaa ggccaagaag cccgtgcaac tgcccggcgc ctacatcgtc 600 ggcatcaagt tggacatcac ctcccacaac gaggactaca ccatcgtgga acagtacgaa 660 cgcgccgagg gccgccactc caccggcggc atggacgagc tatacaagta g 711 <210> 12 <211> 711 <212> mCherry2 <213> Artificial Sequence <400> 12 atggtgagca agggcgagga ggataacatg gccatcatca aggagttcat gcgcttcaag 60 gtgcacatgg agggctccgt gaacggccac gagttcgaga tcgagggcga gggcgagggc 120 cgcccctacg agggcaccca gaccgccaag ctgaaggtga ccaagggtgg ccccctgccc 180 ttcgcctggg acatcctgtc ccctcagttc atgtacggct ccaaggcata cgtgaagcac 240 cccgccgaca tccccgacta cttgaagctg tccttccccg agggcttcaa ctgggagcgc 300 gtgatgaact tcgaggacgg cggcgtggtg accgtgaccc aggactcctc cctacaggac 360 ggcgagttca tctacaaggt gaagctgcgc ggcaccaact tcccctccga cggccccgta 420 atgcagtgtc gtacaatggg ctgggaggct tccactgagc ggatgtaccc cgaggacggt 480 gccctgaagg gcgagatcaa gcagaggctg aagctgaagg acggcggcca ctacgacgct 540 gaagtcaaga ccacctacaa ggccaagaag cccgtgcagc tgcccggagc ctacaacgtg 600 gacatcaagt tggacatcct ttcccacaac gaggactaca ccatcgtgga acagtacgaa 660 cgcgccgagg gccgccactc caccggcggc atggacgagc tgtacaagta g 711 <210> 13 <211> 678 <212> E2-Crimson <213> Artificial Sequence <400> 13 atggatagca ctgagaacgt catcaagccc ttcatgcgct tcaaggtgca catggagggc 60 tccgtgaacg gccacgagtt cgagatcgag ggcgtgggcg agggcaagcc ctacgagggc 120 acccagaccg ccaagctgca agtgaccaag ggcggccccc tgcccttcgc ctgggacatc 180 ctgtcccccc agttcttcta cggctccaag gcgtacatca agcaccccgc cgacatcccc 240 gactacctca agcagtcctt ccccgagggc ttcaagtggg agcgcgtgat gaacttcgag 300 gacggcggcg tggtgaccgt gacccaggac tcatccctgc aagacggcac cctcatctac 360 cacgtcaagt tcatcggcgt gaacttcccc tccgacggcc ccgtaatgca gaagaaaact 420 cttggctggg agccctccac tgagcgcaac tacccccgcg acggcgtgct gaagggcgag 480 aaccacatgg cgctgaagct gaagggcggc ggccactacc tgtgtgagtt caagtccatc 540 tacatggcta agaagcccgt gaagctgccc ggctaccact acgtggacta caagctcgac 600 atcacctccc acaacgagga ctacaccgtg gtggagcagt acgaacgcgc cgaggcccgc 660 caccacctgt tccagtag 678 <210> 14 <211> 600 <212> Puromycin <213> Artificial Sequence <400> 14 atgaccgagt acaagcccac ggtgcgcctc gccacccgcg acgacgtccc cagggccgta 60 cgcaccctcg ccgccgcgtt cgccgactac cccgccacgc gccacaccgt cgatccggac 120 cgccacatcg agcgggtcac cgagctgcaa gaactcttcc tcacgcgcgt cgggctcgac 180 atcggcaagg tgtgggtcgc ggacgacggc gccgcggtgg cggtctggac cacgccggag 240 agcgtcgaag cgggggcggt gttcgccgag atcggcccgc gcatggccga gttgagcggt 300 tcccggctgg ccgcgcagca acagatggaa ggcctcctgg cgccgcaccg gcccaaggag 360 cccgcgtggt tcctggccac cgtcggcgtc tcgcccgacc accagggcaa gggtctgggc 420 agcgccgtcg tgctccccgg agtggaggcg gccgagcgcg ccggggtgcc cgccttcctg 480 gagacctccg cgccccgcaa cctccccttc tacgagcggc tcggcttcac cgtcaccgcc 540 gacgtcgagg tgcccgaagg accgcgcacc tggtgcatga cccgcaagcc cggtgcctga 600

Claims

1. An RNA interference virus vector, characterized in that: The preparation method of the RNA interference virus vector comprises the following steps: Design shRNA using shERWOOD or SplashRNA online tool mir Sequence, select the shRNA with the highest score mir ; shRNA mir The sequence was converted into two miR-AB framework-based sequences; The two sequences mentioned above were synthesized using DNA oligonucleotide chemical synthesis technology; Annealing the two synthesized DNA oligonucleotides to form a double-stranded DNA sequence; This double-stranded DNA sequence is connected to the chronic viral vector or retroviral vector of miR-AB after BamHI and ApaI cutting and purification; Use physical and chemical methods to transform competent cells so that they absorb DNA molecules in the surrounding environment. The transformed competent cells are cultured in a culture medium and positive clones carrying exogenous DNA molecules are screened out; The RNA interference virus vector expresses shRNA carried by the miR-AB framework based on human miR-30 pri-miRNA mir To interfere with the expression of target genes; The miR-AB sequence is regulated by a eukaryotic promoter; The RNA interference virus vector tracks cells that have RNA interference by using fluorescent protein reporter molecules, or screens cells that have RNA interference by using antibiotics; The nucleotide sequence of the miR-AB sequence is as follows: Among them, bold black fonts represent the introduced BamHI and ApaI restriction sites, and the boxes represent the Guide and Passenger sequences of the gene-specific shRNA; The 5' end of the miR-AB sequence includes a 5'-GGATCC-3' sequence, and the 5' end can be recognized and cut by the BamHI restriction endonuclease; The 3' end of the miR-AB sequence includes a 5'-GGGCCC-3' sequence, and the 3' end can be recognized and cut by the Apal restriction endonuclease.

2. The RNA interference virus vector according to claim 1, characterized in that The RNA interference viral vector is a lentiviral vector or a retroviral vector. The lentiviral vector includes 5' LTR, RRE, cPPT, eukaryotic promoter, miR-AB framework, PGK promoter, fluorescent protein or Puromycin resistance, WPRE and 3' LTR in order from 5' end to 3' end; The retroviral vector includes 5' LTR, miR-AB framework, PGK promoter, fluorescent protein or Puromycin resistance, WPRE and 3' LTR in order from 5' end to 3' end.

3. The RNA interference virus vector according to claim 2, characterized in that: The lentiviral vector expresses shRNA via a eukaryotic promoter mir .

4. The RNA interference virus vector according to claim 2, characterized in that The eukaryotic promoter is hCMV, mCMV, CBH, hEF1a, EH or AH; Wherein, the sequence of mCMV is shown as SEQ ID NO.2; The sequence of the CBH is shown in SEQ ID NO.3; The sequence of hEF1a is shown in SEQ ID NO.4; The sequence of the EH is shown in SEQ ID NO.5; The sequence of AH is shown in SEQ ID NO.

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5. The RNA interference virus vector according to claim 2, characterized in that: The fluorescent protein is Venus, Azurite, mTagBFP2, EGFP, Ametrine, mOrange, mCherry2 or E2-Crimson; Wherein, the sequence of Azurite is shown as SEQ ID NO.7; The sequence of mTagBFP2 is shown in SEQ ID NO.8; The sequence of EGFP is shown in SEQ ID NO.9; The sequence of Ametrine is shown in SEQ ID NO.10; The sequence of mOrange is shown in SEQ ID NO.11; The sequence of mCherry2 is shown in SEQ ID NO.12; The sequence of E2-Crimson is shown in SEQ ID NO.

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6. The RNA interference virus vector according to claim 2, characterized in that: The RNA interference virus vector expressing Puromycin resistance is used to screen cells where target gene interference occurs; The gene sequence of puromycin resistance is shown in SEQ ID NO.

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7. The RNA interference virus vector according to claim 2, characterized in that: RNA interference virus vectors expressing fluorescent proteins are used to track or sort cells where target gene interference occurs.

8. The RNA interference virus vector according to claim 1, characterized in that: The RNA interference virus vector interferes with the expression of the target gene in eukaryotic cells.

9. The RNA interference virus vector according to claim 1, characterized in that: The RNA interference viral vectors can perform multiple RNA interference in eukaryotic cells in any combination.

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