Fluorescent RNA (Ribonucleic Acid) aptamer-carrying influenza report virus as well as construction method and application thereof

Abstract

The invention discloses an influenza reporter virus carrying a fluorescent RNA aptamer and a construction method and application thereof, and the influenza reporter virus carrying the fluorescent RNA aptamer is obtained by introducing a termination codon TAA into the 328th nucleotide of an ORF region of an NS1 gene on the basis of a genome of an influenza A virus and terminating in advance, then introducing a terminating codon TAA into the 328th nucleotide of the ORF region of the NS1 gene of the influenza A virus; original nucleotides at the 328-480 sites are replaced by forward or reverse RNA aptamers, and the RNA aptamer is obtained. The influenza reporter virus carrying the fluorescent RNA aptamer constructed by the invention also has the characteristics of good virus replication ability, high RNA imaging sensitivity and good specificity on the basis of stable inheritance, can be used for real-time visual observation of virus RNA, and is used for research on virus replication cycle and molecular mechanism related to the virus RNA. Besides, the construction method of the influenza reporter virus carrying the fluorescent RNA aptamer can also be applied to construction of RNA fluorescent reporter viruses of other subtype influenza viruses such as H3N2 and H5N1, and an important tool is provided for real-time tracing of corresponding virus RNA.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an influenza reporter virus carrying a fluorescent RNA aptamer, its construction method, and its application. Background Technology

[0002] Influenza A virus (IAV) belongs to the Orthomyxoviridae family. Its genome consists of eight segments of single-stranded negative-sense RNA. These RNA segments (vRNA, mRNA, and cRNA) are encapsulated by head-to-tail NP oligomers and assembled with polymerase proteins to form ribonucleoprotein complexes (vRNP, mRNP, and cRNP). Based on their surface structure and genetic characteristics (HA and NA), IAV can be classified into 18 different HA subtypes (H1-H18) and 11 different NA subtypes (N1-N11). Currently, the most prevalent IAV subtypes among humans are H1N1 and H3N2. The IAV genome encodes at least 17 viral proteins, including two outer membrane proteins, hemagglutinin (HA) and neuraminidase (NA). The internal proteins of IAV mainly include nucleoprotein NP, three polymerases (PB1, PB2, and PA), matrix protein M, and the non-structural protein NS1.

[0003] Infectious influenza viruses (IAVs) have a wide host range, including humans, poultry, wild birds, dogs, and mink, and are important pathogens causing global influenza pandemics and seasonal influenza outbreaks. Due to the segmented genome structure of IAVs and the lack of calibration ability of their RNA-dependent RNA polymerases, they are highly susceptible to antigenic drift and transformation. During evolution, gene mutations or rearrangements can occur, leading to the emergence of novel IAVs, rendering existing vaccines and anti-influenza drugs ineffective, and even triggering influenza pandemics. Understanding the basis of viral pathogenicity and its genetic evolution is inseparable from basic research related to influenza virus RNA, such as exploring the interaction between viral RNA and host molecules and revealing the processes of viral genome RNA replication and translation in host cells. High-precision research on viral RNA requires the application of RNA imaging technology to accurately image the real-time translation, replication, and dynamic localization of viral RNA. Several RNA imaging techniques have been established, such as fluorescence in situ hybridization (FISH) and single-molecule fluorescence in situ hybridization (smFISH). However, due to the need for sample fixation, these methods are not suitable for visualizing RNA in live cells, cannot study RNA dynamics, and suffer from low sensitivity and weak labeling specificity. MS2-MCP and SunTag-based RNA imaging systems are also available, but these methods struggle to label and image endogenous, non-genetically modified natural RNA. Labeling imaging techniques based on CRISPR / dCas systems offer programmability, high efficiency, and versatility, and can directly target unmodified viral RNA, but require specific labeling methods. Transfecting multiple sgRNAs to recruit multiple fluorescent protein-Cas protein complexes on the target sequence can also cause background fluorescence and nonspecific nucleolar signals if the fluorescent protein is not bound. Viral RNA imaging based on quantum dot fluorescent labeling of nanomaterials has excellent photostability and high fluorescence intensity, but there are still problems with the preparation and intracellular delivery of quantum dot probes, and the aggregation of quantum dots in cells may lead to nonspecific background signals. RNA aptamer labeling systems such as Broccoli, Mango, Pepper and Peach can easily label multiple target RNA molecules with high sensitivity and can be used to achieve intracellular RNA imaging at the single molecule level. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide an influenza reporter virus carrying a fluorescent RNA aptamer, which has the characteristics of good genetic stability, good replication ability, and high specificity and sensitivity of fluorescence imaging, and can be used to conduct research on the visualization of viral RNA in living cells at the single-molecule level.

[0005] To achieve its purpose, the present invention adopts the following technical solution: The influenza reporter virus carrying fluorescent RNA aptamers provided by this invention is based on the influenza A virus genome. A stop codon is introduced at the 328th nucleotide position of the ORF region of the NS1 gene to terminate the virus early. Then, the original nucleotides 328-480 are replaced with positive or negative fluorescent RNA aptamers.

[0006] As a further preferred embodiment of the technical solution of the present invention, the RNA aptamer is F30-2×Broccoli, wherein the nucleotide sequence of the forward F30-2×Broccoli is shown in SEQ ID NO.1, and the nucleotide sequence of the reverse F30-2×Broccoli is shown in SEQ ID NO.2.

[0007] Furthermore, the nucleotide sequence of the recombinant influenza virus NS1 gene carrying forward F30-2×Broccoli is shown in SEQ ID NO.3, and the nucleotide sequence of the recombinant influenza virus NS1 gene carrying reverse F30-2×Broccoli is shown in SEQ ID NO.4.

[0008] Furthermore, the nucleotide sequence of the influenza A virus NP gene is shown in SEQ ID NO.5; The nucleotide sequence of the polymerase PB1 gene is shown in SEQ ID NO. 6; The nucleotide sequence of the polymerase PB2 gene is shown in SEQ ID NO.7; The nucleotide sequence of the polymerase PA gene is shown in SEQ ID NO. 8; The nucleotide sequence of the matrix protein M gene is shown in SEQ ID NO.9; The nucleotide sequence of the hemagglutinin HA gene is shown in SEQ ID NO.10; The nucleotide sequence of the neuraminidase NA gene is shown in SEQ ID NO.11.

[0009] Furthermore, the influenza A virus is the H1N1 influenza virus.

[0010] The above method for constructing an influenza reporter virus carrying a fluorescent RNA aptamer includes the following steps: (1) Prepare the forward RNA aptamer NS1∆-Broccoli(+) gene and the reverse RNA aptamer NS1∆-Broccoli(-) gene; (2) The NS1∆-Broccoli(+ / -) gene and the NP, PB1, PB2, PA, M, HA and NA genes of influenza A virus protein were respectively constructed into expression vectors to obtain recombinant expression vectors. (3) The recombinant expression vector was co-transfected into mammalian cells to rescue the virus and obtain an influenza reporter virus carrying a fluorescent RNA aptamer.

[0011] Further, in step (3), virus propagation is carried out after the virus is rescued; the method of virus propagation is to inoculate the rescued virus strain into chicken embryos for culture and collect the virus.

[0012] Furthermore, in step (3), virus rescue refers to culturing for 48 to 96 hours after co-transfection.

[0013] Furthermore, in step (3), the mammalian cell is a 293T cell.

[0014] The aforementioned influenza reporter virus carrying fluorescent RNA aptamers can be used for real-time tracking and imaging of live cell RNA. Specifically, this means using the influenza reporter virus to image and track the spatial localization, transcription and replication processes of viral RNA, as well as the interaction processes between other molecules and viral RNA.

[0015] The influenza reporter virus was used to infect the corresponding cells. Before fluorescence imaging, the old cell culture medium was discarded, and the cells were washed three times with PBS. A storage solution prepared with DMSO was added to the cells, and a working concentration of 20 μM of a membrane-permeable RNA aptamer-activated fluorescent probe DFHBI-1T (ex / em=472 nm / 507 nm) was prepared with serum-free culture medium. The cells were incubated with the dye in a 37°C, 5% CO2 humidified incubator for 10 minutes to fluorescently label the positive or negative strand RNA of the virus.

[0016] The beneficial effects of this invention are as follows: This invention provides two influenza reporter viruses carrying fluorescent RNA aptamers. The method involves introducing a stop codon (TAA) at nucleotide 328 of the ORF region of the NS1 gene of influenza A virus to prematurely terminate the genome, and then replacing the original nucleotides 328-480 with the F30-2×Broccoli nucleotide sequence. Experiments show that the influenza reporter viruses carrying fluorescent RNA aptamers constructed in this invention exhibit good viral replication ability, high RNA imaging sensitivity, and good specificity, in addition to stable genetic inheritance.

[0017] The method for constructing influenza reporter viruses carrying fluorescent RNA aptamers provided by the present invention can also be applied to the construction of RNA fluorescent reporter viruses for other subtypes of influenza viruses, such as H3N2 and H5N1, providing an important tool for real-time tracking of the corresponding viral RNA. Attached Figure Description

[0018] Figure 1This is a schematic diagram illustrating the construction of an influenza reporter virus carrying a fluorescent RNA aptamer according to the present invention. Figure 1 In diagram A, the original nucleotides 328-480 of the NS1 gene ORF region were replaced with forward or reverse Broccoli after the TAA stop codon was introduced at position 328 of the ORF region of the present invention to terminate the ORF prematurely. Figure 1 Figure B is a schematic diagram of the construction of influenza virus with positive or negative Broccoli markers on RNA according to the present invention. Figure 2 This invention demonstrates the fluorescence detection performance of the parent virus and two influenza reporter viruses carrying fluorescent RNA aptamers. Figure 2 In Figure A, fluorescence imaging results are shown for the parent virus and two influenza reporter viruses carrying fluorescent RNA aptamers. Figure 2 B stands for MOCK, representing the flow cytometry results of the parent virus and two influenza reporter viruses carrying fluorescent RNA aptamers. Figure 3 Results on the genetic stability of two influenza reporter viruses carrying fluorescent RNA aptamers. Figure 3 Figure A shows the evaluation of continuous passage of RNA fluorescently labeled influenza virus in chicken embryos according to the present invention. Figure 3 Figure B is a fluorescence imaging stability evaluation diagram of the influenza virus labeled by this invention after passage. Figure 4 The results of viral RNA fluorescence imaging after parental virus and two influenza reporter viruses carrying fluorescent RNA aptamers infected cells from different species; Figure 5 The results show a comparison of replication kinetics curves of parental virus and two influenza reporter viruses carrying fluorescent RNA aptamers after infecting MDCK cells with different doses; Figure 6 Results of mouse infection with parental virus and two influenza reporter viruses carrying fluorescent RNA aptamers; Figure 6 In the middle, A represents the parent virus and two influenza reporter viruses carrying fluorescent RNA aptamers, each at 10... 3 EID 50 / 100 μL, 10 4 EID 50 / 100 μL, 10 5 EID 50 / 100 μL and 10 6 EID 50 The curve of body weight change of BALB / c mice over 14 consecutive days after infection with a dose of 100 μL. Figure 6 In the middle, B was the parent virus and two influenza reporter viruses carrying fluorescent RNA aptamers, each at 10 3 EID 50 / 100 μL, 10 4 EID 50 / 100 μL, 10 5 EID 50 / 100 μL and 10 6 EID 50 Statistics on the survival of BALB / c mice and calculation of the median lethal dose for mice within 14 consecutive days after infection with a dose of 100 μL. Figure 7 RNA fluorescence imaging results at different time points after MDCK cells were infected with the parent virus and two influenza reporter viruses carrying fluorescent RNA aptamers. Detailed Implementation

[0019] This invention provides two influenza reporter viruses carrying fluorescent RNA aptamers, which involve, based on the influenza virus genome, introducing a stop codon TAA at nucleotide position 328 of the ORF region of the NS1 gene for early termination, and then replacing the original nucleotides 328-480 with the forward or reverse fluorescent RNA aptamer F30-2×Broccoli.

[0020] In this invention, the F30-2×Broccoli replaces nucleotides 328-480 between the NS1 gene effect domains. The Broccoli-labeled virus was passaged for five generations and its growth characteristics were studied. It was found that the virus can be stably inherited without affecting its replication and infectivity.

[0021] In this invention, the influenza virus is type A influenza virus. In this embodiment of the invention, the PR8 / H1N1 (A / Puerto Rico / 8 / 1934, PR8 / H1N1) influenza virus strain is used as the test strain for modification.

[0022] This invention provides a method for constructing an influenza reporter virus carrying the Broccoli tag, comprising the following steps: Preparation of NS1-Broccoli (+ / -) chimeric genes; The NS1-Broccoli(+) chimeric gene, NS1-Broccoli(-) chimeric gene, NP gene, PB1 gene, PB2 gene, PA gene, M gene, HA gene, and NA gene were respectively constructed into expression vectors to obtain recombinant expression vectors. The recombinant expression vector was co-transfected into mammalian cells to rescue the virus, resulting in influenza virus carrying a Broccoli label in positive or negative strand RNA.

[0023] The NS1-Broccoli(+ / -) chimeric gene described in this invention is obtained through artificial synthesis.

[0024] This invention does not impose any special restrictions on the type of expression vector used; any expression vector well-known in the art can be used, such as the pBD vector. The vector used in this invention is the pBD vector, constructed and donated by Academician Chen Hualan of the Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences (see prior art Li, Z., Chen, H., Jiao, P., Deng, G., Tian, ​​G., Li, Y., Hoffmann, E., Webster, RG, Matsuoka, Y., Yu, K., 2005. Molecularbasis of replication of duck H5N1 influenza viruses in a mammalian mousemodel. J Virol 79, 12058-12064,10.1128 / JVI.79.18.12058-12064.2005.). This invention does not impose any special restrictions on the construction method; any method well-known in the art for constructing recombinant expression vectors can be used, such as homologous recombination methods, artificial synthesis methods, etc. After construction, eight recombinant expression vectors were validated. The preferred verification method involves introducing the recombinant expression vector into competent E. coli cells, culturing them, extracting a large number of plasmids, and performing bacterial PCR and sequencing based on specific gene fragments.

[0025] After obtaining a positive recombinant expression vector, the present invention co-transfects the recombinant expression vector into mammalian cells for virus rescue, thereby obtaining an influenza reporter virus carrying an RNA fluorescent aptamer.

[0026] This invention does not impose any particular limitation on the co-transfection method; any co-transfection method well-known in the art can be used. In this embodiment, the co-transfection is preferably performed using the X-treme GENE™ HP DNATransfection Reagent (Roche). The mammalian cells are 293T cells. Virus rescue is preferably performed by culturing for 48 hours after the co-transfection.

[0027] In this invention, virus propagation is preferably performed after virus rescue; the method of virus propagation is to inoculate the rescued virus strain into chicken embryos for culture and collect the virus. The chicken embryos are preferably SPF chicken embryos. The age of the SPF chicken embryos is preferably 9-11 days.

[0028] In this invention, the recombinant influenza virus particles prepared using the above method achieve chimeric expression of the RNA aptamer and the NS gene. Because they carry the RNA fluorescent aptamer Broccoli, they achieve high signal-to-noise ratio, high sensitivity, good specificity, and fluorescence that is not easily quenched after specific binding to the dye DFHBI-1T, resulting in viral RNA imaging results. Stability evaluation experiments show that the constructed labeled virus replicates stably for five consecutive generations, and its fluorescence imaging results demonstrate excellent stability. Furthermore, in vitro and in vivo multi-cycle replication analysis results show that the infectivity of the labeled virus is similar to that of the wild-type virus. This indicates that the insertion of Broccoli does not significantly affect the replication and infectivity of the influenza virus, and the labeled virus achieves viral RNA fluorescence imaging in cells from various species.

[0029] In this invention, taking the migration process of viral RNA during the viral life cycle as an example, an experiment was conducted to detect changes in the intracellular RNA localization of influenza reporter virus carrying the RNA aptamer Broccoli label. The results showed that the RNA aptamer Broccoli label of influenza virus of this invention can be used as a tracking tool for the sublocalization of viral RNA in cells.

[0030] The following detailed description, in conjunction with embodiments, illustrates two influenza reporter viruses carrying the RNA aptamer Broccoli marker, their construction methods, and applications provided by the present invention. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0031] Example 1 Construction methods of two PR8 influenza RNA fluorescent reporter viruses The influenza virus strain PR8 / H1N1 (A / Puerto Rico / 8 / 1934, PR8 / H1N1) was selected. A stop codon TAA was introduced at nucleotide 328 of the ORF region of its NS1 gene to prematurely terminate the expression. Nucleotides 328-480 were then replaced with either a forward or reverse F30-2×Broccoli sequence to obtain the chimeric gene NS1△-Broccoli(+ / -). Gene fragments (PB2, PB1, PA, NP, M, NS-Broccoli, HA, NA) were recombined into the pBD vector to prepare the following nine recombinant expression plasmids: pBD-PB2, pBD-PB1, pBD-PA, pBD-NP, pBD-M, pBD-NS-Broccoli(+), pBD-NS-Broccoli(-), pBD-HA, and pBD-NA. 0.5 μg of each of the above nine recombinant expression plasmids was used. Add 200 μL of fresh Opti-MEM; transfect using X-treme GENE™ HP DNA Transfection Reagent (Roche); co-transfect into 293T cells, collect the virus from the supernatant after 48 hours, and multiply the virus on 9-11 day old SPF chicken embryos. Construct influenza RNA fluorescent reporter virus.

[0032] Figure 1 This diagram illustrates the construction of an influenza virus carrying a Broccoli marker on RNA, as described in this invention. The RNA aptamer Broccoli marker is introduced into the NS gene. This RNA aptamer specifically binds to DFHBI-1T, causing the viral RNA to exhibit green fluorescence upon binding.

[0033] Example 2 RNA fluorescence detection performance of influenza RNA reporter virus The fluorescence imaging performance of the influenza fluorescent RNA reporter virus constructed in Example 1 was evaluated: Figure 2 The specific operation of A in the middle is as follows: (1) Inoculate MDCK cells in confocal dishes and infect them with a specified virus (influenza fluorescent RNA reporter virus or parental virus); (2) Twelve hours after infection, the RNA aptamer-activated fluorescent probe DFHBI-1T with a working concentration of 20 μM was incubated with the infected target cells at 37°C and 5% CO2 for 10 minutes to fluorescently label the viral RNA. (3) Wash the cells three times with PBS to remove unbound DFHBI-1T, and add an appropriate volume of 4% paraformaldehyde to the dish for fixation. (4) Wash the cells three times with PBS, add an appropriate volume of DAPI dye to the dish to stain the cell nuclei for 10 minutes; use super-resolution laser confocal microscope to collect fluorescence imaging results; like Figure 2 As shown in Figure A, both positive-strand and negative-strand influenza virus RNA labeled with Broccoli can obtain clear viral RNA imaging results with low background values. From the perspective of subcellular localization, both positive-strand and negative-strand viral RNA are mainly located in the cell nucleus and diffusely distributed in the cytoplasm, while the parent virus cannot be collected.

[0034] Figure 2 The specific operation of B in the middle is as follows: (1) MDCK cells were seeded in 6-well plates and infected with a specified virus (Broccoli-labeled virus or parental virus); (2) Twelve hours after viral infection, the working concentration of 20 μM RNA aptamer activated fluorescent probe DFHBI-1T was incubated with infected target cells at 37°C and 5% CO2 for 10 minutes to fluorescently label the viral RNA. (3) After digesting the cells with 0.25% trypsin, add an appropriate amount of culture medium to neutralize the cells, then centrifuge to collect the cell pellet, add an appropriate amount of PBS to resuspend the cells, and then use flow cytometry to perform statistical analysis on the total number of cells and the number and percentage of green fluorescent positive cells. like Figure 2 As shown in B, the Broccoli marker is present in the viral particles and can be used to indicate viral infection in positive cell populations, while the parent virus cannot be collected.

[0035] Example 3 Stability evaluation of influenza RNA fluorescent reporter virus The specific steps are as follows: (1) The virus was labeled and passaged for 5 generations in SPF chicken embryos; (2) The hemagglutination titer of the collected viruses was determined by hemagglutination test. (3) The collected viruses were used to infect MDCK cells. 12 hours after infection, RNA fluorescent probe DFHBI-1T was added and co-incubated with the infected target cells for RNA fluorescence-specific staining. The cell nuclei were stained with DAPI dye and the fluorescent RNA was imaged using a super-resolution laser confocal microscope. (3) Comparative findings: The labeled virus replicated stably for five consecutive generations, and its fluorescence imaging results showed excellent stability and specificity.

[0036] like Figure 3As shown, the labeled virus was continuously passaged in chicken embryos, and the hemagglutination titer and RNA fluorescence imaging of the harvested virus (generations 1 to 5) were measured. The results showed that the labeled virus replicated stably for five consecutive generations, and its RNA fluorescence imaging results showed excellent stability and specificity.

[0037] Example 4 Detection of influenza RNA fluorescent reporter virus RNA imaging results in multi-species cells (1) Inoculate hamster kidney cells BHK21, chicken embryo fibroblast cells DF1 and porcine kidney cells PK15 in confocal dishes and infect them with the specified virus (Broccoli-labeled virus or parental virus); (2) Twelve hours after infection, the RNA aptamer-activated fluorescent probe DFHBI-1T with a working concentration of 20 μM was incubated with the infected target cells at 37°C and 5% CO2 for 10 minutes to fluorescently label the viral RNA. (3) Wash the cells three times with PBS to remove unbound DFHBI-1T, and add an appropriate volume of 4% paraformaldehyde to the dish for fixation. (4) Wash the cells three times with PBS, add an appropriate volume of DAPI dye to the dish to stain the cell nuclei for 10 minutes; use super-resolution laser confocal microscope to collect fluorescence imaging results; like Figure 4 As shown, both positive and negative strand RNA Broccoli-labeled viruses can successfully infect cells from the three species mentioned above and obtain clear viral RNA imaging with low background values, while parental viruses cannot be collected.

[0038] Example 5 In vitro multi-cycle replication analysis of influenza RNA fluorescent reporter virus 1. In vitro multi-cycle replication analysis of influenza RNA fluorescent reporter virus, the specific procedures are as follows: (1) Inoculate MDCK cells into 12-well plates and infect them with a specified virus (influenza RNA fluorescent reporter virus or parent virus); (2) Incubate at 37°C for 1 hour, then gently wash the cells 3 times with PBS and add fresh Opti-MEM containing 0.5 μg / mL TPCK-trypsin; (3) Collect the supernatant at the designated time point after the fluid change; (4) Chicken embryos were inoculated with wild-type virus at sequential 10-fold dilutions to determine EID. 50The value was calculated using the Reed-Muench method (see prior art Reed LJ, Muench H. A simple method of estimating fifty percent endpoints. Am J Hyg. 1938; 27(3): 493–497. DOI:10.1093 / oxfordjournals.aje.a118408.).

[0039] like Figure 5 As shown, the replication kinetics curves of the influenza RNA fluorescent reporter virus and the parent virus after infecting MDCK cells with different doses are compared. The results show that the influenza RNA fluorescent reporter virus and the parent virus have little difference in replication kinetics, which indicates that the substitution of Broccoli has little effect on influenza virus replication.

[0040] Example 6 In vivo multi-cycle replication analysis of influenza RNA fluorescent reporter virus The specific steps are as follows: (1) The parent virus and two influenza RNA fluorescent reporter viruses were respectively 10 3 EID 50 / 100μL, 10 4 EID 50 / 100μL, 10 5 EID 50 / 100μL, 10 6 EID 50 Infect BALB / c mice with a dose of 100 μL; (2) Observe and record the mortality and average weight change of surviving mice for 14 consecutive days after infection. Mice were considered dead when their weight dropped to 75% of their initial weight before inoculation. Plot the weight change curve and survival curve, and calculate the MLD. 50 .

[0041] like Figure 6 As shown, the median lethal dose (LD50) of influenza virus labeled with positive Broccoli, influenza virus labeled with reverse RNA Broccoli, and parental virus in BALB / c mice was 4.375 Log. 10 EID 50 4.833 Log 10 EID 50 and 3.833Log 10 EID 50 Broccoli markers reduced the pathogenicity of influenza virus in BALB / c mice to some extent.

[0042] Example 7 Application of the constructed influenza RNA fluorescent reporter virus in tracing viral invasion of host cells (1) Infect MDCK cells with Broccoli-labeled virus at a dose of 1 MOI; (2) After infection at 4℃ for 1 h, the supernatant was discarded, and the cells were gently washed 3 times with ice-cold PBS. Then, fresh Opti-MEM containing 0.5 μg / mL TPCK-trypsin was added. (3) Cells were collected at 0 h, 1 h, 2 h, 4 h, 6 h and 8 h after infection for incubation with fluorescent probes and staining of cell nuclei; (4) RNA imaging was acquired using a super-resolution laser confocal microscope; like Figure 7 As shown, RNA fluorescence imaging of influenza virus can basically indicate the viral invasion process, including viral adsorption, internalization and uncoating, as well as the transcription and replication of the genome in the cell nucleus.

Claims

1. An influenza reporter virus carrying a fluorescent RNA aptamer, characterized in that, The recombinant influenza virus is based on the genome of influenza A virus. A stop codon is introduced at nucleotide position 328 of the ORF region of the NS1 gene to terminate the virus early. Then, the original nucleotides 328-480 are replaced with fluorescent RNA aptamers in either forward or reverse orientation.

2. The influenza reporter virus carrying a fluorescent RNA aptamer as described in claim 1, characterized in that, The RNA aptamer is F30-2×Broccoli, wherein the nucleotide sequence of the forward F30-2×Broccoli is shown in SEQ ID NO.1, and the nucleotide sequence of the reverse F30-2×Broccoli is shown in SEQ ID NO.

2.

3. The influenza reporter virus carrying a fluorescent RNA aptamer as described in claim 2, characterized in that, The nucleotide sequence of the recombinant influenza virus NS1 gene carrying forward F30-2×Broccoli is shown in SEQ ID NO.3, and the nucleotide sequence of the recombinant influenza virus NS1 gene carrying reverse F30-2×Broccoli is shown in SEQ ID NO.

4.

4. The influenza reporter virus carrying a fluorescent RNA aptamer as described in claim 1, characterized in that, The nucleotide sequence of the influenza A virus NP gene is shown in SEQ ID NO. 5; The nucleotide sequence of the polymerase PB1 gene is shown in SEQ ID NO. 6; The nucleotide sequence of the polymerase PB2 gene is shown in SEQ ID NO.7; The nucleotide sequence of the polymerase PA gene is shown in SEQ ID NO. 8; The nucleotide sequence of the matrix protein M gene is shown in SEQ ID NO.9; The nucleotide sequence of the hemagglutinin HA gene is shown in SEQ ID NO.10; The nucleotide sequence of the neuraminidase NA gene is shown in SEQ ID NO.

11.

5. The influenza reporter virus carrying a fluorescent RNA aptamer as described in claim 4, characterized in that, The influenza A virus in question is the H1N1 influenza virus.

6. The method for constructing an influenza reporter virus carrying a fluorescent RNA aptamer according to any one of claims 1 to 5, characterized in that, Includes the following steps: (1) Prepare the forward RNA aptamer NS1∆-Broccoli(+) gene and the reverse RNA aptamer NS1∆-Broccoli(-) gene; (2) The NS1∆-Broccoli(+ / -) gene and the NP, PB1, PB2, PA, M, HA and NA genes of influenza A virus protein were respectively constructed into expression vectors to obtain recombinant expression vectors. (3) The recombinant expression vector was co-transfected into mammalian cells to rescue the virus and obtain an influenza reporter virus carrying a fluorescent RNA aptamer.

7. The method for constructing an influenza reporter virus carrying a fluorescent RNA aptamer as described in claim 6, characterized in that, In step (3), virus propagation is carried out after the virus is rescued; the method of virus propagation is to inoculate the rescued virus strain into chicken embryos for culture and collect the virus.

8. The method for constructing an influenza reporter virus carrying a fluorescent RNA aptamer as described in claim 7, characterized in that, In step (3), virus rescue refers to culturing for 48 to 96 hours after co-transfection.

9. The method for constructing an influenza reporter virus carrying a fluorescent RNA aptamer as described in claim 8, characterized in that, In step (3), the mammalian cell is a 293T cell.

10. The application of influenza reporter virus carrying fluorescent RNA aptamers as described in any one of claims 1 to 5 in real-time RNA tracking imaging in live cells, characterized in that, The application refers to using the influenza reporter virus to image and track the spatial localization, transcription and replication processes of viral RNA, as well as the interaction processes between other molecules and viral RNA.

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