Recombinant plasmid marked by double fluorescent proteins, recombinant influenza virus, and construction method and application of recombinant plasmid and recombinant influenza virus
By constructing a recombinant influenza virus with dual fluorescent protein labeling, using mNeonGreen and mCherry fluorescent proteins to label NS1 and NS2 proteins, the problem of difficult to visualize and quantify IAV in real-time detection of NS1 and NS2 proteins in host cells in the prior art is solved, and efficient and accurate detection of viral markers and protein expression is achieved.
Patent Information
- Application Number
- CN202411678092.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-22
- Publication Date
- 2025-03-28
AI Technical Summary
The prior art is difficult to visualize and quantify the expression of NS1 and NS2 proteins in host cells in real time, and the traditional methods are costly, cumbersome and have low accuracy.
By constructing a double fluorescent protein-tagged recombinant influenza virus, using mNeonGreen fluorescent protein to label NS1 protein and mCherry fluorescent protein to label NS2 protein, combined with P2A and T2A self-splitting polypeptide technology, real-time labeling and tracking of NS1 and NS2 proteins is achieved.
Real-time visual quantitative detection of NS1 and NS2 proteins of IAV in host cells is realized, reducing detection cost and operational complexity, and improving detection accuracy and repeatability.
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Figure CN119709801A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of genetic engineering, and specifically relates to a recombinant influenza virus labeled with double fluorescent proteins, and a construction method and application thereof. Background Art
[0002] Influenza A virus (IAV) is a single-stranded negative-strand segmented RNA virus of the family Orthomyxoviridae. Its genome consists of 8 segments with a total length of about 13.6 kb, encoding PB1, PB2, PA, HA, NP, NA, MP and NS (NS1 and NS2 (NEP)), of which NP is connected with viral RNA and PB1, PB2, PA to form RdRp, which participates in the assembly of the virus and the synthesis of RNA. The typing of IAV is based on the combination of viral surface proteins HA and NA to form HnNn, such as highly pathogenic influenza H5N1 and H7N7.
[0003] The nonstructural protein NS (NS) protein is encoded by the 8th gene segment of IAV and can be encoded by all influenza strains with good conservation. The NS1 protein is translated from the unspliced primary mRNA transcript, while the second protein NS2 / NEP encoded by this segment is translated from the spliced mRNA. The NS1 protein is involved in a variety of other activities, such as the regulation of vRNA synthesis and mRNA translation, viral particle morphogenesis, pathogenesis and splicing, and can also act as an inhibitor of host immune response; the NS2 (NEP) protein has been shown to play a role in the nuclear export of viral ribonucleoproteins (RNPs) and the regulation of transcription and replication during the viral life cycle. Therefore, NS1 and NS2 play an important role in host-virus interaction. However, a method for real-time observation of the expression of NS1 and NS2 proteins in virus-infected host cells has not yet been found. Therefore, a system that does not affect the NS splicing process in vivo and can be used to trace NS1 and NS2 proteins during virus-host interaction is needed.
[0004] The quantitative detection methods of IAV include qRT-PCR, plaque assay, immunofluorescence, TCID50 and other methods. These methods are costly, cumbersome to operate, low in accuracy and repeatability, and cannot quantify and locate live viruses in infected cells in real time. Therefore, it is necessary to develop a visual virus system. At present, the visual quantification of IAV is to integrate fluorescent proteins into viral proteins through gene editing to achieve real-time labeling of viral proteins. The genetic material of IAV is composed of 8 segmented RNAs, which are PB1, PB2, PA, HA, NP, NA, MP and NS (NS1 and NS2 (NEP)) in descending order. The larger the RNA encoding the protein, the more limited its ability to accommodate exogenous genes, and the more it will affect the vitality and replication ability of the virus. Summary of the invention
[0005] In view of the above technical problems to be solved, the object of the present invention is to provide a recombinant influenza virus that can achieve effective real-time virus labeling and NS protein tracking.
[0006] Another object of the present invention is to provide a method for constructing the recombinant influenza virus.
[0007] Another object of the present invention is to provide the use of the recombinant influenza virus.
[0008] In order to achieve the above invention objectives, the present invention provides a dual fluorescent protein-labeled recombinant plasmid, which includes the following DNA fragments: a DNA fragment expressing an NS1 protein mutant, a DNA fragment expressing a P2A short peptide, a DNA fragment expressing an mNeonGreen fluorescent protein, a DNA fragment expressing an NS2 protein, a DNA fragment expressing a T2A short peptide, and a DNA fragment expressing an mCherry protein.
[0009] Preferably, the DNA fragment expressing the NS1 protein mutant has the following synonymous mutations in the CDs region of the NS protein: 139D GCC>GAT, 164P CCT>CCA, 165S TCT>TCA, 166L CTT>CTG, 167P CCA>CCC.
[0010] Preferably, the nucleotide sequence of the DNA fragment expressing the NS1 protein mutant is as shown in SEQ ID NO.1.
[0011] Preferably, the recombinant plasmid comprises the nucleotide sequence shown as SEQ ID NO.2.
[0012] Preferably, the nucleotide sequence of the recombinant plasmid is as shown in SEQ ID NO.5.
[0013] On the other hand, the present invention also provides the use of the recombinant plasmid according to the present invention in constructing a recombinant influenza virus.
[0014] On the other hand, the present invention further provides a recombinant influenza virus labeled with dual fluorescent proteins, which comprises the recombinant plasmid according to the present invention.
[0015] Preferably, the influenza virus is influenza A virus, in particular H1N1 influenza virus.
[0016] On the other hand, the present invention also provides a method for constructing the recombinant influenza virus labeled with dual fluorescent proteins, the method comprising the following steps:
[0017] (1) constructing a recombinant plasmid labeled with dual fluorescent proteins according to the present invention;
[0018] (2) The recombinant plasmid and an 8-plasmid system expressing PB1, PB2, PA, HA, NP, NA and MP proteins and RNA were co-transfected into 293T cells, and the cells were cultured continuously. The supernatant was collected and the virus was amplified using MDCK cells to obtain the recombinant influenza virus labeled with the dual fluorescent proteins.
[0019] On the other hand, the present invention also provides the use of the recombinant influenza virus labeled with dual fluorescent proteins in the visualization and quantification of influenza viruses.
[0020] On the other hand, the present invention also provides the use of the recombinant influenza virus labeled with dual fluorescent proteins in the study of influenza virus infection mechanism.
[0021] On the other hand, the present invention also provides the use of the recombinant influenza virus labeled with dual fluorescent proteins in the dynamic expression, positioning and tracing research of NS1 and NS2.
[0022] On the other hand, the present invention also provides the use of the recombinant influenza virus labeled with dual fluorescent proteins in the dynamic expression, positioning and tracing research of the NS shearing process.
[0023] NS protein is a non-structural protein of the virus and does not participate in the construction of the virus structure. RNA is also the shortest. Combining the above two advantages, the present invention uses NS RNA as the integration target vector and retains the original splicing function to construct a dual fluorescent protein labeled virus that can simultaneously display NS1 and NS2. The NS1 protein is labeled with the brightest mNeoGreen green fluorescent protein, and the NS2 protein produced by the splicing body is labeled with mCherry red fluorescent protein. This system can be successfully rescued and a recombinant influenza virus labeled with dual fluorescent proteins can be obtained. This virus can be used for visual detection and NS transcript splicing tracing, positioning and quantification.
[0024] Fluorescent protein mNeonGreen is the brightest green fluorescent protein, and its excitation spectrum and emission spectrum are 506nm and 517nm respectively. mCherry is a red fluorescent protein, and its excitation spectrum and emission spectrum are 527nm and 610nm respectively. The emission spectra of the two do not overlap, and different proteins can be well labeled in the same system. The present invention uses the brightest fluorescent protein mNeonGreen to label NS1, and adopts the method of introducing self-cleaving polypeptide P2A in the middle. This method is different from the method of directly fusing proteins, which will increase the burden of the target protein and change its structure, affecting its function. P2A is located between NS1 and mNeonGreen, and will form the same open reading frame during translation. Initially, it will become a fusion protein of NS1-P2A-mNeonGreen. Next, P2A will cut NS1 and mNeonGreen proteins into two proteins at a ratio of 1:1. Therefore, the fluorescent protein mNeonGreen can be used to label viruses on the one hand, and its fluorescence intensity can also quantify the expression level of NS1 in cells on the other hand. Similarly, the red fluorescent protein mCherry was used to label NS2, and another cleavage polypeptide T2A was introduced in the middle, which could cut NS2 protein and mCherry protein to produce a 1:1 ratio for labeling and quantification of NS2 protein.
[0025] The present invention integrates DNA expressing P2A short peptide and mNeonGreen into the back of the NS1 gene with mutated SA splicing site, integrates DNA fragments expressing T2A short peptide and mCherry into the back of the gene of the part of NS2 carrying the SA splicing site, and then constructs a recombinant influenza virus labeled with dual fluorescent proteins after virus rescue using a reverse genetic system. The system can produce live viruses and can infect cells and be used for virus labeling, NS1 and NS2 protein labeling. The present invention does not directly integrate DNA fragments expressing two proteins of NS1 and NS2 into a vector in a 1:1 manner, but retains the splicing function of the original NS gene in the cell. The NS gene of the present invention can use the endogenous splicing system of the host cell to complete the production of its own NS2 protein, realize the labeling of NS1 and NS2 proteins in the host cell, and more realistically restore the transcription and splicing process of NS in the host cell. The present invention can be used for virus labeling, studying the splicing process of NS gene in host cells, and other basic research and development of antiviral drugs.
[0026] Compared with the prior art, the present invention can directly construct live viruses by reverse genetic rescue of viruses, and can be used for real-time visualization and quantification of influenza viruses; the present invention selects NS as the target protein for virus labeling, retains the splicing function of NS transcripts, and restores the function of normal NS in cells, which can be used to study the dynamic expression, positioning and tracing of the NS splicing process; the present invention marks the two transcripts of NS with two different colors respectively, so the system can be directly used for the dynamic expression, positioning and tracing research of NS1 and NS2 splicing copies. BRIEF DESCRIPTION OF THE DRAWINGS
[0027] Figure 1 It is a schematic diagram of the structure of the dual fluorescent protein labeling system of the recombinant influenza virus of the present invention.
[0028] Figure 2 This is the plasmid map of pM-PR8-NS.
[0029] Figure 3 This is the plasmid map of pM-PR8-NS-mNeonGreen-mCherry.
[0030] Figure 4 This is the plasmid map of pM-PoI-NS-mNeonGreen-mCherry.
[0031] Figure 5 The results of hemagglutination assays of IAV viruses rescued by reverse genetics methods, including wild-type viruses (IAV) and recombinant viruses (IAV-Dual-Fluor).
[0032] Figure 6 PCR gel running was used to verify that the dual-fluorescence-labeled recombinant influenza virus of the present invention has integrated a fluorescent label into the RNA of the NS. IAV-WT: wild type; IAV-dual fluor.: recombinant virus.
[0033] Figure 7 The results of the second-generation genotype sequencing showed that the target fragment was successfully integrated into the virus.
[0034] Figure 8 Schematic diagram of the design for expressing NS-mNeonGreen-mCherry vRNA.
[0035] Fig. 9 In order to express the transcriptase and all viral proteins of IAV using the plasmid system, NS integrated vRNA can be reverse transcribed into mRNA and express fluorescent protein.
[0036] Fig.10 This is the fluorescence detection result of MDCK cells infected with dual-fluorescence-labeled recombinant influenza virus (IAV-dual fluor).
[0037] Fig.11 Flow cytometry was used to detect the ratio of green fluorescence to red fluorescence of the NS-mNeonGreen-mCherry plasmid vector (A), as well as the ratio of NS2 expression over time (B).
[0038] Fig.12 The dual fluorescence reporter system for unidirectionally expressing NS vRNA was constructed: (a) Schematic diagram of the plasmid design for expressing the dual fluorescence virus IAV-dual vRNA; (b) The IAV-dual vRNA of the NS dual fluorescence reporter system can be rescued by RdRp to produce green-labeled NS1 and red-labeled NS2 proteins; (c) Flow cytometry was used to detect the ratio of green fluorescence and red fluorescence of the NS-mNeonGreen-mCherry plasmid vector; (d) The ratio of NS2 expression level changed over time; (e) The results of the hemagglutination test of the IAV virus rescued by the reverse genetic method. DETAILED DESCRIPTION
[0039] In order to better understand the technical solution of the present invention, the present invention is further described below in conjunction with specific examples. It should be understood that the following examples are only used to illustrate the present invention, rather than to limit the scope of the present invention.
[0040] It should be understood that, unless otherwise specified, the reagents and experimental instruments used in the following examples are known to those skilled in the art and are commercially available.
[0041] The virus strain used in the embodiment of the present invention is influenza virus type A strain A / Puerto Rico / 8 / 34 (H1N1) (abbreviated as PR8).
[0042] The dual fluorescent protein labeling system of the recombinant influenza virus of the present invention comprises: integrating DNA expressing a P2A short peptide and mNeonGreen behind the gene of an NS1 mutant to form an NS1-mNeonGreen fragment; integrating a DNA fragment expressing a T2A short peptide and mCherry behind the gene of a portion of NS2 carrying an SA cleavage site to form an NS2-mCherry fragment.
[0043] Specifically, the dual fluorescent protein-labeled recombinant plasmid provided by the present invention comprises the following DNA fragments: a DNA fragment expressing a mutant of NS1 protein, a DNA fragment expressing a short P2A peptide, a DNA fragment expressing mNeonGreen fluorescent protein, a DNA fragment expressing NS2 protein, a DNA fragment expressing a short T2A peptide, and a DNA fragment expressing mCherry protein. Preferably, the recombinant plasmid comprises the nucleotide sequence shown in SEQ ID NO.2.
[0044] The NS1 protein of the present invention is derived from the H1N1 strain, and the splice acceptor site (SA) is mutated by genetic engineering. Since SA is located in the CDs region of NS1, a synonymous mutation is performed. Specifically, the DNA fragment expressing the NS1 protein mutant has the following synonymous mutations in the CDs region of the NS protein: 139D GCC>GAT, 164PCCT>CCA, 165S TCT>TCA, 166L CTT>CTG, 167P CCA>CCC. The nucleotide sequence of the DNA fragment expressing the NS1 protein mutant is shown in SEQ ID NO.1.
[0045] The fluorescent protein mNeonGreen is the brightest fluorescent protein and is used to label the NS1 protein. The nucleotide sequence encoding the fluorescent protein mNeonGreen is shown in SEQ ID NO.3.
[0046] The P2A short peptide is located between the NS1 protein and the mNeonGreen fluorescent protein. NS1, P2A and mNeonGreen form the same open reading frame, which is translated into NS1-P2A-mNeonGreen fusion protein. In theory, P2A can cut NS1 and mNeonGreen proteins into two proteins at a 1:1 ratio. Therefore, the mNeonGreen fluorescent protein can be used to mark viruses on the one hand, and on the other hand, the intensity of the mNeonGreen fluorescent protein can also be used to reflect the expression level of the NS1 protein.
[0047] The NS2 protein of the present invention is derived from the H1N1 strain and is obtained from the spliced transcript of NS. The expressed DNA fragment includes two parts, one of which is the 1-30nt fragment before the splice donor site (SD) on the NS1 mRNA; the other is the 503-835nt of NS2 containing SA. The expression of NS2 depends on the host cell splicing system.
[0048] The fluorescent protein mCherry is used to label the NS2 protein, and its encoding nucleotide sequence is shown in SEQ ID NO.4.
[0049] The T2A short peptide is located between the NS2 protein and the mNeonGreen fluorescent protein. NS2, T2A and mCherry form the same open reading frame, and T2A can theoretically cut NS2 and mCherry proteins into two proteins at a 1:1 ratio. Therefore, the mCherry fluorescent protein can be used to label viruses and reflect the expression level of NS2.
[0050] Figure 1The schematic diagram of the structure of the dual fluorescent protein labeling system of the recombinant influenza virus of the present invention is shown.
[0051] Example 1. Construction of pM-PR8-NS-mNeonGreen-mCherry plasmid
[0052] Design and synthesize the NS-Dual-fluor-Template template plasmid (including the following elements in sequence: NS1, GSG-P2A, mNeonGreen, NS2-2, GST-T2A and mCherry, such as Figure 1 The nucleotide sequence of the plasmid is shown in SEQ ID NO.5.
[0053] The pM-PR8-NS plasmid was cut with SalI+FseI restriction endonucleases (pM-PR8-NS plasmid map is shown in Figure 2 As shown, the NS-Dual-fluor-Template template plasmid synthesized by Jinweizhi Company was used to analyze the enzyme product and PCR product by 0.8% (mass volume fraction) agarose gel electrophoresis. The 3035bp and 2763bp bands were recovered by gel cutting, and the concentration of the recovered fragments was determined by NanoDrop. The linearized NS-Dual-fluor-Template template plasmid and the pM-PR8-NS plasmid were connected by T4 DNA ligase purchased from NEB Company, and then the connection product was transformed into DH5α Escherichia coli, spread on LB solid plates containing 100μg / mL ampicillin, and cultured at 37°C overnight. Then a single clone was picked, and the plasmid was extracted after shaking at 37°C and 250rpm for DNA sequencing. The constructed plasmid vector was screened and named pM-PR8-NS-mNeonGreen-mCherry. The pM-PR8-NS-mNeonGreen-mCherry plasmid map is shown in Figure 3 shown.
[0054] The enzyme digestion system is 50 μL, including the following components:
[0055] SalI-HF restriction endonuclease 0.5μL FseI restriction endonuclease 0.5μL <![CDATA[10ⅹCutSmart TM Buffer]]> 5μL Plasmids 3μg ddH2O Make up to 50 μL
[0056] Reaction conditions: 37°C, 4 hours.
[0057] T4 ligation system 10 μL, including the following components:
[0058] T4 Ligase 1μL 10x T4 Ligase Buffer 1μL Linearize NS-Dual-fluor-Template plasmid 2μL Linearize pM-PR8-NS plasmid 2μL ddH2O 4μL
[0059] Reaction conditions: 25°C water bath for 1 hour.
[0060] Example 2. Construction of pM-PoI-NS-mNeonGreen-mCherry plasmid
[0061] Design and synthesize the following partially complementary sequences:
[0062] OLIGO-F:5'-CGCGTAGCTTTCTAGAAGGTTTAAACGAT-3'(SEQ ID NO.6)
[0063] OLIGO-R:5'-ATCGTTTAAACCTTCTAGAAAGCTA-3'(SEQ ID NO.7)
[0064] The two partially complementary single-stranded DNA fragments OLIGO-F and OLIGO-R are annealed to synthesize a double-stranded DNA fragment, namely Oligo-dsDNA. The specific steps are as follows:
[0065] (1) 10ul 100uM OLIGO-F and 10ul 100uM OLIGO-R were premixed in a 1.5ml EP tube;
[0066] (2) Boil 800 ml of distilled water in a beaker;
[0067] (3) Place the 1.5 ml EP tube in boiling water for 5 minutes;
[0068] (4) Take out the 1.5 ml EP tube and place it at room temperature overnight.
[0069] The pM-PR8-NS-mNeonGreen-mCherry plasmid was cut with MluI+EcoRV restriction endonuclease ( Figure 3 ), the enzyme product and PCR product were analyzed by 0.8% (mass volume fraction) agarose gel electrophoresis, the 5341bp band was recovered by gel excision, and the concentration of the recovered fragment was determined by NanoDrop, the linearized pM-PR8-NS-mNeonGreen-mCherry and the Oligo-dsDNA were connected by T4 DNA ligase purchased from NEB, and then the connection product was transformed into DH5α Escherichia coli, coated on LB solid plates containing 100μg / mL ampicillin, cultured at 37°C overnight, and then a single clone was picked, and the plasmid was extracted after shaking at 37°C and 250rpm for DNA sequencing, thereby screening the constructed vector, named pM-PoI-NS-mNeonGreen-mCherry. The pM-PoI-NS-mNeonGreen-mCherry plasmid map is shown in Figure 4 shown.
[0070] The enzyme digestion system is 50 μL, including the following components:
[0071] MluI-HF restriction endonuclease 0.5μL EcoRV-HF restriction endonuclease 0.5μL <![CDATA[10ⅹCutSmart TM Buffer]]> 5μL Plasmids 3μg ddH2O Make up to 50 μL
[0072] Reaction conditions: 37°C, 4 hours.
[0073] T4 ligation system 10 μL, including the following components:
[0074] T4 Ligase 1μL 10x T4 Ligase Buffer 1μL Linearize pM-PR8-NS-mNeonGreen-mCherry plasmid 2μL Oligo-dsDNA 1μL ddH2O 4μL
[0075] Reaction conditions: 25°C water bath for 1 hour.
[0076] Example 3. Rescue of IAV-dual-Fluor virus using 8-plasmid reverse genetics method:
[0077] Human renal epithelial cells 293T were cultured in DMEM complete medium (Gibco-DMEM basal medium-GgiC11995500BT containing 10% (mass volume fraction) FBS) and 1.5×10 5 / mL of cells were placed in a culture dish at 37°C and 5% CO 2 Incubate for 18-28 hours until the cell density reaches 70-80% for later use. Premix the plasmid to be transfected (see Table 1 for details) in 30 μL Opti-MEM TM The cells were placed in reduced serum medium (Thermo Fisher Scientific) at room temperature for 5 min, mixed with polyethyleneimine (PEI), and allowed to stand at room temperature for 15 min before being placed at 37°C and 5% CO 2 The cells were cultured in an incubator, and fresh virus culture medium (DMEM basal culture medium with 1 ug / mL TPCK enzyme added) was replaced after 8 hours. The supernatant virus solution was collected after 72 hours.
[0078] Canine kidney cells (MDCK) were cultured in DMEM complete medium (the composition is the same as that of the above DMEM complete medium) at a density of 1.5*10 5 / mL of cells were pre-plated in a T75 cell culture flask, washed once with PBS after 24 hours, and the virus solution obtained above was diluted with virus culture medium (DMEM basal culture medium with 1ug / mL of TPCK enzyme) at a volume ratio of 1:5 (virus solution: virus culture medium), and then transferred into T75 cells for expansion. The virus was collected after 72 hours.
[0079] The transfection concentration of liposome transfection reagent polyethyleneimine (PEI) was 1 mg / mL and the pH was 7.0.
[0080] Transfection conditions: PEI: total DNA = 1.5: 1 (g / g).
[0081] The transfection system is shown in Table 1 below.
[0082] Table 1
[0083]
[0084] The virus solution was collected, RNA was extracted, and influenza virus hemagglutination (HA) test was performed. The wild-type virus Influenza A virus (IAV H1N1-VR-95) (A / PR / 8 / 34) purchased from ATCC was used as a control. The experimental results are shown in Figure 5 As shown, the hemagglutination titer of the dual fluorescent protein-labeled virus (IAV-dual-Fluor) is comparable to that of the wild-type virus (IAV).
[0085] Viral RNA was extracted using a viral RNA extraction kit (Magen HiPure Viral RNA Kit-R4171-03) and reversed to obtain cDNA. Primers were designed and synthesized for genotype identification and second-generation sequencing. The specific results are as follows Figure 6 and Figure 7 The results showed that the IAV-Dual-Fluor virus with a dual fluorescent protein labeling system was successfully obtained.
[0086] The genotype identification primer sequences are as follows:
[0087]
[0088]
[0089] Example 4. Successful transcription of the NS-mNeonGreen-mCherry system using IAV transcriptase and production of fluorescent protein for labeling of NS protein
[0090] The constructed IAV-dual-Fluor is a virus rescued based on the reverse genetics system. Therefore, the plasmid system constructed by the present invention is first tested to see whether it affects the effect of IAV transcriptase on the transcription of vRNA into mRNA. The RNA polymerase of IAV is composed of PB1, PB2, and PA, and is assisted by the structural protein NP to participate in the assembly of the virus and the synthesis of RNA. Thus, a plasmid vector pM-PoI-NS-mNeonGreen-mCherry (schematic diagram of the structure is shown in FIG. 1 ) expressing only NS-mNeonGreen-mCherry vRNA was constructed. Figure 8 The plasmid map is shown in Figure 4), and co-transfected with 4 plasmids or 7 plasmids system (transfection method see Example 3, transfection conditions see Table 2), and co-transfected into 293T cells, wherein 4 plasmids include pM-PR8-PA, pM-PR8-PB1, pM-PR8-PB2 and pM-PR8-NP, and 7 plasmids include pM-PR8-PA, pM-PR8-PB1, pM-PR8-PB2, pM-PR8-HA, pM-PR8-NA, pM-PR8-M and pM-PR8-NP. After 72 hours, the cells were observed to express green and red fluorescence, and the specific results are as follows. Fig. 9 As shown, only the plasmid vector expressing NS-mNeonGreen-mCherry vRNA is transfected, and due to the lack of IAV RNA polymerase, it will not be reversed into mRNA and translated into fluorescent protein, while the 4-plasmid and 7-plasmid systems can produce RNA polymerase composed of PB1, PB2 and PA, and can produce green and red fluorescent proteins. The results show that the plasmid vector constructed by the present invention will not affect viral transcription, and can successfully transcribe the NS-mNeonGreen-mCherry system and produce fluorescent protein.
[0091] The transfection conditions are shown in Table 2 below.
[0092] Table 2
[0093]
[0094] Example 5. IAV-Dual-Fluor Virus Imaging
[0095] The dual fluorescent protein labeled virus IAV-Dual-Fluor obtained in Example 3 was diluted with virus culture medium (DMEM basal culture medium plus 1ug / mL TPCK enzyme) at a volume ratio of 1:5 (virus liquid: virus culture medium), and added to MDCK cells. After 48 hours, the virus infection was observed under a fluorescence microscope. The results are shown in Fig.10 The results showed that cells expressing green and red fluorescence were successfully observed, indicating that the constructed IAV-Dual-Fluor virus can be imaged and is suitable for visual detection.
[0096] Example 6. Using the NS-mNeonGreen-mCherry system to demonstrate the proportion of each transcript of NS protein in the host cell and the time of splicing
[0097] NS-mNeonGreen-mCherry and the 4-plasmid system expressing IAV RNA polymerase were transferred into 293T cells using PEI. Flow cytometry was used to detect the ratio of green fluorescence to red fluorescence at different times. The specific results are as follows: Fig.11The results showed that green fluorescence appeared first, followed by red fluorescence, and the red fluorescence accounted for 20%, which was consistent with the reported data.
[0098] In addition, a dual fluorescence reporter system NS-mNeonGreen-mCherry that unidirectionally expresses NS vRNA was constructed as above. This system can be used to rescue the IAV virus by reverse genetics. This system, together with the 4-plasmid and 7-plasmid systems expressing IAV RNA polymerase, was transferred into 293T cells using PEI. Flow cytometry was used to detect the proportion of green fluorescence and red fluorescence at different times. The specific results are shown in the figure. Fig.12 As shown in the figure, the IAV-dualvRNA of the NS dual fluorescence reporter system can be rescued by RdRp to produce green-labeled NS1 and red-labeled NS2 proteins (b), and the flow cytometry test results show that the NS2 expression accounts for about 20% (c, d), and the hemagglutination test results show that the IAV virus rescued by the reverse genetic method (Pol NS-GR(-)) has a hemagglutination titer equivalent to that of the wild-type virus (IAV) (e).
[0099] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.
[0100] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.
[0101] In addition, various embodiments of the present invention may be arbitrarily combined, and as long as they do not violate the concept of the present invention, they should also be regarded as the contents disclosed by the present invention.
Claims
1. A recombinant plasmid labeled with dual fluorescent proteins, characterized in that The following DNA fragments are included: a DNA fragment expressing a NS1 protein mutant, a DNA fragment expressing a P2A short peptide, a DNA fragment expressing an mNeonGreen fluorescent protein, a DNA fragment expressing an NS2 protein, a DNA fragment expressing a T2A short peptide, and a DNA fragment expressing an mCherry protein.
2. The dual fluorescent protein-labeled recombinant plasmid according to claim 1, characterized in that: The DNA fragment expressing the NS1 protein mutant has the following synonymous mutations in the CDs region of the NS protein: 139D GCC>GAT, 164P CCT>CCA, 165STCT>TCA, 166L CTT>CTG, 167P CCA>CCC.
3. The dual fluorescent protein-labeled recombinant plasmid according to claim 1, characterized in that: The nucleotide sequence of the DNA fragment expressing the NS1 protein mutant is shown in SEQ ID NO.
1.
4. The dual fluorescent protein-labeled recombinant plasmid according to claim 1, characterized in that: The recombinant plasmid includes the nucleotide sequence shown as SEQ ID NO.
2.
5. The dual fluorescent protein-labeled recombinant plasmid according to claim 1, characterized in that: The nucleotide sequence of the recombinant plasmid is shown in SEQ ID NO.
5.
6. Use of the dual fluorescent protein-labeled recombinant plasmid according to any one of claims 1 to 5 in constructing a recombinant influenza virus.
7. A recombinant influenza virus labeled with dual fluorescent proteins, comprising the recombinant plasmid labeled with dual fluorescent proteins according to any one of claims 1 to 5.
8. The recombinant influenza virus according to claim 1, characterized in that The influenza virus is an influenza A virus.
9. The method for constructing the recombinant influenza virus according to claim 7 or 8, comprising the following steps: (1) constructing a recombinant plasmid labeled with dual fluorescent proteins according to the present invention; (2) The recombinant plasmid and an 8-plasmid system expressing PB1, PB2, PA, HA, NP, NA and MP proteins and RNA were co-transfected into 293T cells, and the cells were cultured continuously. The supernatant was collected and the virus was amplified using MDCK cells to obtain the recombinant influenza virus labeled with the dual fluorescent proteins.
10. Use of the recombinant influenza virus according to claim 7 or 8 in any of the following aspects: visualization and quantification of influenza virus; study of influenza virus infection mechanism; dynamic expression, localization and tracing study of NS1 and NS2; dynamic expression, localization and tracing study of NS shearing process.