Multi-channel virus reporting system based on TAT trans-activation effect

Abstract

The invention relates to the technical field of molecular biology, in particular to a multichannel virus reporting system based on TAT trans-activation. The invention relates to a multichannel virus reporting system based on TAT trans-activation. The multichannel virus reporting system comprises an activated virus and a reporting cell, the activated virus is a human enterovirus A71 for expressing TAT protein; the reporter cells are cells transfected with reporter plasmids; the reporter plasmid comprises an LTR sequence and a plurality of reporter genes which are started to express by the LTR sequence. When the virus is activated to infect the reporter cell, the TAT protein carried by the virus enters the reporter cell to activate the expression of the downstream reporter gene, and the expression level of the reporter gene can reflect the virus replication condition, so that the screening or mechanism research of related antiviral drugs can be realized. According to the technical scheme, the technical problem that the flexibility, stability and high-throughput screening adaptability of a virus reporting system in the prior art are not ideal can be solved, and the virus reporting system has ideal popularization and application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biology, and in particular to a multi-channel virus reporting system based on TAT transactivation. Background Art

[0002] In the field of virology, in-depth exploration of the immune and anti-immune interactions between viruses and hosts is crucial for understanding infection mechanisms, disease progression, and the development of effective antiviral strategies. Progress in this field relies not only on a deep understanding of viral biology but also greatly benefits from innovations in modern biotechnology, particularly the introduction of high-throughput screening (HTS) technologies, such as the CRISPR / Cas9 gene editing system, which have become core tools for unraveling the complexity of virus-host interaction networks.

[0003] However, despite the revolutionary changes high-throughput screening (HTS) has brought to virology research, existing viral reporting systems have presented a technical bottleneck. Traditional viral reporting systems often utilize cis-reporting systems. In these systems, a reporter gene (such as a fluorescent protein or luciferase gene) is directly inserted into a target vector (e.g., a viral genome) and colocalized with the target regulatory elements, effectively coexisting within the same DNA molecule. By integrating a reporter gene (such as luciferase or fluorescent protein) into the viral genome, the cis-reporting system provides a visual "window" into viral replication and infection. While intuitive, this approach is limited by the compact and short size of viral genomes (typically only a few thousand base pairs), resulting in extremely limited viral vector capacity. Consequently, these systems often only carry a single reporter gene, which not only limits the diversity of visualization methods but also hinders the flexibility and comprehensiveness of information obtained during high-throughput screening.

[0004] Furthermore, some commonly used cis-acting reporter genes, such as Gaussia luciferase (Gluc) and Renilla luciferase (Rluc), are easily lost during serial viral passage, directly linked to the instability of the reporter system. This instability not only reduces the reliability of screening results but also limits the potential application of these systems in long-term, large-scale screening experiments. Reporter gene stability is particularly important when multiple rounds of screening are required to identify key interactors or antiviral targets.

[0005] Therefore, the existing traditional virus reporter system has shown deficiencies in multiple key dimensions, including but not limited to flexibility, stability, and adaptability to high-throughput screening. In order to overcome these limitations and promote virology research to a deeper level, it is particularly urgent to develop a new, efficient, stable and flexible virus reporter system. The ideal new reporter system should have the following characteristics: First, it can overcome the limitations of vector capacity and achieve the simultaneous expression of multiple reporter genes, thereby increasing the diversity of visualization pathways and the richness of screening information; second, it has excellent genetic stability to ensure that the reporter gene is not easily lost during the virus passage process, ensuring the accuracy and consistency of the screening results; third, it is compatible with high-throughput screening technology, improves screening efficiency, broadens the scope of application, and provides strong technical support for the development of antiviral drugs, analysis of viral pathogenic mechanisms and other fields. Summary of the Invention

[0006] The purpose of the present invention is to provide a multi-channel virus reporter system based on TAT transactivation to solve the technical problems of the prior art virus reporter system in terms of flexibility, stability and high-throughput screening adaptability.

[0007] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0008] A multi-channel viral reporter system based on TAT transactivation, comprising an activated virus and reporter cells;

[0009] The activated virus is human enterovirus A71 expressing TAT protein;

[0010] The reporter cells are cells transfected with a reporter plasmid; the reporter plasmid includes an LTR sequence and a plurality of reporter genes whose expression is promoted by the LTR sequence.

[0011] Furthermore, the activated virus is prepared by the following method:

[0012] S1: construct a viral plasmid with the sequence shown in SEQ ID NO.15;

[0013] S2: The viral plasmid is linearized by enzyme digestion and then transcribed in vitro to obtain viral mRNA;

[0014] S3: The viral mRNA is transferred into Vero cells, the Vero cells are cultured, and the activated virus is obtained after cell lysis.

[0015] Furthermore, in S1, the viral plasmid having a sequence as shown in SEQ ID NO.15 was constructed by the following method:

[0016] Using a first primer having a sequence as shown in SEQ ID NO.1 and a second primer having a sequence as shown in SEQ ID NO.2, the first fragment is PCR amplified to obtain a first PCR product; using a third primer having a sequence as shown in SEQ ID NO.3 and a fourth primer having a sequence as shown in SEQ ID NO.4, the second fragment is PCR amplified to obtain a second PCR product; and using the first primer and the fourth primer, the first PCR product and the second PCR product are amplified to obtain a first spliced fragment;

[0017] The third fragment is amplified by PCR using the fifth primer having a sequence as shown in SEQ ID NO. 5 and the sixth primer having a sequence as shown in SEQ ID NO. 6 to obtain a third PCR product; the fourth fragment is amplified by PCR using the seventh primer having a sequence as shown in SEQ ID NO. 7 and the eighth primer having a sequence as shown in SEQ ID NO. 8 to obtain a fourth PCR product; the third PCR product and the fourth PCR product are amplified using the fifth primer and the eighth primer to obtain a second spliced fragment;

[0018] The first splicing fragment and the second splicing fragment are double-enzyme digested and then connected to obtain a circular viral plasmid; the sequences of the first fragment, the second fragment, the third fragment and the fourth fragment can be spliced to form a circular viral plasmid.

[0019] The above-mentioned viral plasmid construction method is a conventional method for synthesizing expression plasmids in molecular biology and virology research, and ultimately produces the viral plasmid shown in SEQ ID NO. 15. In addition to the above-mentioned method, other methods can also be used to ultimately produce the viral plasmid with the sequence shown in SEQ ID NO. 15.

[0020] Furthermore, in S2, the viral plasmid was linearized by digestion with Stu1 restriction endonuclease, and then the linearized plasmid was transcribed into mRNA in vitro.

[0021] Furthermore, in S3, mRNA formed by in vitro transcription of the linearized plasmid was added to the Vero cell suspension, and electroporation was performed under the parameter settings of voltage 250V and time 20ms; after the electroporation was completed, the cells were continued to be cultured, and the activated virus was obtained after cell lysis.

[0022] Furthermore, the reporter genes contained in the reporter plasmid include thymidine kinase gene, green fluorescent protein gene, and Gaussia luciferase gene.

[0023] Furthermore, the gene sequence of the reporter plasmid is shown as SEQ ID NO.16.

[0024] Furthermore, the reporter plasmid was constructed by the following method: using a ninth primer having a sequence as shown in SEQ ID NO.9 and a tenth primer having a sequence as shown in SEQ ID NO.10, a fragment consisting of the T2A gene and the Gluc gene was amplified to obtain a T2A-Gluc amplification product; the fragment formed after enzyme digestion of the plasmid pEGFP-C1 and the T2A-Gluc amplification product were synthesized into a circular plasmid pEGFP-T2A-Gluc;

[0025] Using the eleventh primer with the sequence shown in SEQ ID NO.11 and the twelfth primer with the sequence shown in SEQ ID NO.12, the fragment consisting of the LTR gene and the tk gene was amplified to obtain an LTR-tk amplification product; the original CMV promoter region in pEGFP-T2A-Gluc was replaced with the LTR-tk amplification product to obtain a reporter plasmid.

[0026] The above reporter plasmid construction method is a conventional method for synthesizing expression plasmids in molecular biology research, and ultimately produces the reporter plasmid shown in SEQ ID NO. 16. In addition to the above method, other methods can also be used to ultimately produce the reporter plasmid with the sequence shown in SEQ ID NO. 16.

[0027] Reporter cells can be obtained by transfecting cells with a reporter plasmid using conventional methods and then screening. A549 cells can be used as transfected cells. A549 cells are seeded at a seeding density of approximately 40% and cultured in conventional culture medium for 20 hours. The reporter plasmid and transfection reagent are then added, mixed, and the cells are cultured again. After 72 hours, conventional G418 screening is performed. Surviving cells are considered cells successfully transfected with the reporter plasmid, i.e., reporter cells.

[0028] This technical solution also provides an application of a multi-channel virus reporting system based on TAT transactivation in virus detection for purposes other than disease treatment or diagnosis:

[0029] First, reporter cells are infected with activating viruses;

[0030] Then, the infected cells are cultured in an environment containing GCV to observe the activation of the virus to induce the death of the reporter cells; or the GFP fluorescence signal in the infected reporter cells is observed; or the Gluc content in the culture supernatant of the infected reporter cells is detected.

[0031] Furthermore, the reporter cells are infected with the activation virus at an MOI of 0.1-10; preferably, the MOI is 1;

[0032] GCV was added to the reporter cells infected with the activated virus, and after culture, the cell survival was observed;

[0033] Use the excitation light channel of a fluorescence confocal microscope to examine the reporter cells after activation of virus infection and observe the GFP fluorescence signal;

[0034] Collect the culture supernatant of reporter cells 6-72 hours after activation of virus infection; add Gaussia luciferase detection substrate to the supernatant, perform chemiluminescence detection after the reaction luminescence signal tends to be stable, and finally convert it into Gluc content information.

[0035] In summary, the technical principle of this technical solution is:

[0036] The present invention proposes a multi-channel virus reporter system based on TAT transactivation, which aims to solve the technical bottlenecks such as poor stability, easy loss of reporter genes, inability to realize multi-channel screening and insufficient adaptability of high-throughput screening in existing cis-reporter systems in virological research. This solution utilizes the transactivation of TAT (transcription activator). TAT is a small molecule protein that can specifically bind to the transactivation response region element in the LTR (long terminal repeat) promoter, thereby regulating the transcriptional expression of downstream genes. In the present invention, first construct a kind of activation virus expressing TAT protein, which is based on human enterovirus A71, and the TAT protein encoding gene is integrated into the viral genome by genetic engineering technology. At the same time, a reporter plasmid comprising LTR promoter and multiple reporter genes (such as thymidine kinase gene TK, green fluorescent protein gene GFP, Gaussia luciferase gene Gluc) is designed and transfected into specific reporter cells to form a stably expressed reporter cell line. When the activation virus infects the reporter cells, the TAT protein carried by the virus enters the cells and binds to the LTR promoter to activate the transcriptional expression of downstream reporter genes. Since the expression level of TAT protein is consistent with the replication of the viral genome, the expression level of the reporter gene can indirectly reflect the replication of the virus in the reporter cells. This design not only overcomes the problem of easy loss of reporter genes in traditional cis-reporter systems, but also realizes the construction of a multi-channel reporter system, making it possible for high-throughput screening. This reporter system can be flexibly applied to high-throughput screening. When the knockout reporter cells are in a pro-viral environment, the expression level of green fluorescent protein GFP is regulated to increase, and conversely, the expression level is low. Cells with different levels of green fluorescence expression are sorted by flow cytometry, and finally deep sequencing analysis can find potential antiviral targets. Similarly, relying on the expression strength of the TK suicide system and the secreted luciferase Gluc in the reporter cells can achieve high-throughput rapid screening of antiviral drugs.

[0037] More specifically, the innovation of this technical solution compared to the prior art lies in:

[0038] The present invention cleverly utilizes the transactivation characteristics of TAT protein, and TAT protein is designed to be expressed in activation virus, and when viral infection reporter cell, TAT protein then enters cell, and plays its transcriptional activation effect.Traditional cis reporter system usually integrates reporter gene directly into viral genome, and this not only limits the diversity of reporter gene, is also susceptible to the constraint of viral genome compactness (being difficult to realize multi-reporter gene while integration in virus).And the present invention, by the means that TAT gene is integrated in viral genome and reporter gene is integrated in virus-infected cell, realizes the separation of reporter gene and viral genome, thus overcomes the above-mentioned limitation.Therefore, using the technical means of this solution, it is possible to overcome the defect that cis reporter system cannot realize multichannel screening (cannot simultaneously integrate multiple reporter genes in same viral genome).

[0039] In this technical solution, since only the smaller molecular weight TAT gene (about 258bp) is integrated into the activated virus, the larger molecular weight reporter genes (for example, EGFP reporter gene 717bp, Gluc reporter gene 555bp) are not integrated into the viral genome, thus overcoming the defect of poor stability of the cis-reporter system. The cis-reporter system of the prior art usually integrates a single reporter gene directly into the viral genome, and the reporter gene is easily lost during viral passage, resulting in a lack of stability of the reporter system.

[0040] One of the reporter genes used in this technical solution is the Gaussia luciferase gene Gluc, which has secretory properties. If Gluc is integrated into the viral genome to construct a cis-reporting system, there are the following problems: since Gluc is integrated into the viral genome, the Gluc reporter gene contains background expression in the viral stock solution. After the viral stock solution is inoculated into the cells, background expression will cover up the newly generated Gluc signal, thus making it impossible to use for high-throughput screening. However, in this technical solution, through clever design, the regulatory factor TAT and the reporter gene Gluc are located in different spaces, and there will be no phenomenon that background expression affects the newly generated Gluc signal. Using this technical solution, it is expected that rapid high-throughput screening can be achieved by directly measuring the cell culture supernatant.

[0041] The beneficial effects of this technical solution are:

[0042] (1) Significantly improve the stability of the reporter system: Due to the small molecular weight of the TAT protein and the rational design of the viral plasmid, the probability of loss during viral replication is significantly reduced. At the same time, the reporter gene is on a reporter plasmid that is stably propagated in cells, further enhancing the stability of the system. This solves the problem of easy loss of the reporter gene in traditional cis-reporter systems and provides reliable technical support for long-term, large-scale screening experiments.

[0043] (2) Achieve multi-channel screening and improve information richness: By constructing a reporter plasmid containing multiple reporter genes, placing them in the same coding frame and regulated by the LTR promoter, the present invention achieves the construction of a multi-channel reporter system. This not only allows for simultaneous observation of multiple biological processes related to viral replication, but also provides a rich source of information for high-throughput screening. This design not only improves screening flexibility but also provides a powerful tool for in-depth understanding of the interaction between viruses and hosts.

[0044] (3) Solve the background interference problem in high-throughput screening: In the traditional cis-reporter system, due to the background expression of some reporter genes in the viral stock solution, the newly generated reporter gene signal is often obscured, resulting in inaccurate high-throughput screening results. The present invention avoids this problem by introducing the TAT transactivation system. When the virus-infected reporter cells are activated, the TAT protein activates the reporter gene in the reporter cells, rather than the background expression in the viral stock solution. This achieves rapid high-throughput screening and improves screening efficiency and accuracy.

[0045] (4) Broaden the scope of application and promote the progress of virology research: The multi-channel virus reporter system of the present invention can not only be used in the fields of antiviral drug development, virus pathogenic mechanism analysis, etc., but can also be flexibly applied to high-throughput screening experiments. For example, in antiviral drug screening, the effect of candidate antiviral drugs can be characterized by observing the level of Gaussia luciferase secreted into the culture supernatant of cells, thereby screening potential antiviral drugs; the antiviral effect of candidate drugs can also be judged by using the thymidine kinase suicide system and detecting the number of surviving cells through the MTT kit, thereby achieving drug screening. In addition, it should be further explained that: although this scheme integrates the TAT sequence into the EV-A71 virus genome (EV-A71 virus has the effect of lysing cells) to form an activated virus, this is used as an example and for explanation. In actual operation, the TAT sequence can be integrated into other types of viruses (for example: some viruses that do not have the effect of lysing cells) to form an effective activated virus. Using such a reporter system, drug screening for specific viruses can be achieved. Furthermore, in the study of viral pathogenic mechanisms, the interaction mechanism between the virus and the host can be revealed by analyzing the expression patterns of different reporter genes. This has brought new breakthroughs and broad development space to the field of virology research. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 Schematic diagram of the construction principle of a multi-channel viral reporter system based on TAT transactivation.

[0047] Figure 2 Schematic diagram of the structure of the EV-A71-TAT-FL plasmid in Example 1.

[0048] Figure 3 This is a gel electrophoresis diagram of the EV-A71-TAT-FL plasmid linearization and in vitro transcribed mRNA in Example 1 (in the figure, from left to right: EV-A71-TAT-FL plasmid before linearization, EV-A71-TAT-FL plasmid after linearization, and in vitro transcribed mRNA).

[0049] Figure 4 This is a diagram of the cytopathic effect (CPE) of Vero cells after electroporation of EV-A71-TAT-FL (EV-A71-TAT) mRNA in Example 1.

[0050] Figure 5 These are the results of viral RNA level detection at different times after the mRNA in Example 1 was electroporated into Vero cells (EV-A71-TAT refers to transfection using mRNA transcribed from EV-A71-TAT-FL; 3D-Mut refers to transfection using mRNA transcribed from a viral replication-defective mutant, where the viral replication-defective mutant has a mutation from T to A at amino acid position 313 of the 3D protein responsible for viral replication).

[0051] Figure 6 Schematic diagram of the structure of the reporter plasmid in Example 2.

[0052] Figure 7 Fluorescence microscopic image of the reporter cells expressing three reporter genes constructed in Example 2 (scale bar: 100 μm).

[0053] Figure 8 The tk / GCV system verification results of Example 3 (scale bar 100 μm).

[0054] Figure 9 The GFP expression verification results of Example 3 (scale bar: 100 μm).

[0055] Figure 10 This is the Gluc expression level verification result of Example 3 (Mock: control group; EV-A71-TAT refers to transfection using mRNA transcribed from EV-A71-TAT-FL; 3D-Mut refers to transfection using mRNA transcribed from a viral replication-deficient virus, and a viral replication-deficient virus refers to a mutation of amino acid T at position 313 of the 3D protein responsible for viral replication to A).

[0056] Figure 11 This is the stability test result of the EV-A71-TAT virus in Example 4.

[0057] Figure 12 These are the results of studying the Gluc expression levels in reporter cells after infection with different viruses in Example 5. DETAILED DESCRIPTION

[0058] The present invention will be further described in detail below with reference to the examples, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the technical means used in the following examples and experimental examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used are all commercially available.

[0059] A multi-channel viral reporter system based on TAT transactivation consists of two parts: activation virus and reporter cells. The construction flow chart of activation virus and reporter cells can be found in Figure 1 .

[0060] Example 1: Construction of activation virus (human enterovirus A71 expressing TAT protein)

[0061] (1) Construction of EV-A71-TAT-FL plasmid

[0062] To facilitate in vitro amplification, the EV-A71-TAT-FL plasmid sequence was fragmented into multiple segments that could be spliced together to form the final EV-A71-TAT-FL plasmid. A biotechnology company was commissioned to synthesize the plasmid fragments (divided into four segments), including the EV-A71-TAT-FL-F1 segment (the first segment), the EV-A71-TAT-FL-F2 segment (the second segment), and the EV-A71-TAT-FL-F3 segment (the third segment); and to obtain the base vector fragment (backbone, the fourth segment). The EV-A71-TAT-FL plasmid was obtained through multiple rounds of PCR amplification of these segments.

[0063] First, the EV-A71-TAT-FL-F1 fragment was PCR amplified using a first primer (SEQ ID NO. 1) and a second primer (SEQ ID NO. 2) using conventional PCR methods in the prior art to obtain an EV-A71-TAT-FL-F1 fragment amplification product (first PCR product). The EV-A71-TAT-FL-F2 fragment was PCR amplified using a third primer (SEQ ID NO. 3) and a fourth primer (SEQ ID NO. 4) using conventional PCR methods in the prior art to obtain an EV-A71-TAT-FL-F2 fragment amplification product (second PCR product). Next, the EV-A71-TAT-FL-F1 fragment amplification product (first PCR product) and the EV-A71-TAT-FL-F2 fragment amplification product (second PCR product) were subjected to conventional overlap extension PCR using the first and fourth primers. By extending the overlapping strands, the two amplified fragments were overlapped and spliced together (first spliced fragment, EV-A71-TAT-FL-F12).

[0064] Then, using the fifth primer (SEQ ID NO.5) and the sixth primer (SEQ ID NO.6) and adopting the conventional PCR means in the prior art, the EV-A71-TAT-FL-F3 fragment was PCR amplified to obtain the EV-A71-TAT-FL-F3 fragment amplified product (the third PCR product). Using the seventh primer (SEQ ID NO.7) and the eighth primer (SEQ ID NO.8) and adopting the conventional PCR means in the prior art, the basic vector fragment was PCR amplified to obtain the basic vector fragment amplified product (the fourth PCR product). Next, using the fifth primer and the eighth primer, the EV-A71-TAT-FL-F3 fragment amplified product (the third PCR product) and the basic vector fragment (the fourth PCR product) amplified product were subjected to conventional overlap extension PCR in the prior art. By extending the overlapping chains, the two amplified fragments were overlapped and spliced together (the second spliced fragment, EV-A71-TAT-FL-F3B).

[0065] Finally, the first spliced fragment EV-A71-TAT-FL-F12 and the second spliced fragment EV-A71-TAT-FL-F3B were double-digested using BamH1 (recognition sequence: GGATCC) and Not1 (recognition sequence: GCGGCCGC), and the two digested fragments were ligated with T4 DNA ligase to obtain a circular EV-A71-TAT-FL plasmid.

[0066] The nucleotide sequence of EV-A71-TAT-FL plasmid is shown in SEQ ID NO.15 (the wavy line indicates the TAT gene sequence; the dotted line indicates the T2A gene sequence, Indicates the cleavage site; the double underline indicates the gene sequence of the structural protein VP4 of the EV-A71 genome; the TAT gene sequence is inserted into the gene sequence of the structural protein VP4. For details, see the structural diagram Figure 2 ):

[0067]

[0068]

[0069] (Only 1-2000 bp sequence is shown, subsequent sequence is omitted, the complete sequence of SEQ ID NO. 15 is shown in the sequence listing).

[0070] (3) Viral mRNA synthesis

[0071] The EV-A71-TAT-FL plasmid was linearized using Stu1 (recognition sequence: AGGCCT) by conventional restriction endonuclease digestion, and then the linearized plasmid was transcribed into mRNA using a T7 in vitro transcription kit. For details on the gel electrophoresis of the EV-A71-TAT-FL plasmid linearization and in vitro transcribed mRNA, see Figure 3 .

[0072] (4) Preparation of EV-A71-TAT virus

[0073] Vero cells were seeded into a 10 cm culture dish and subsequent operations were performed after the Vero cells were fully grown (confluence: 90%). 1 mL of trypsin was used to digest the cells, and 2 mL of MeM medium was used to terminate the digestion. The digested cells were transferred to a centrifuge tube, filled to 10 mL with PBS, centrifuged at 800 rpm for 5 minutes, and the supernatant was discarded. The cells were set aside. The cells were resuspended and washed with 10 mL of PBS, centrifuged and the supernatant was discarded, and the cells were resuspended with 0.5 mL of PBS. The resuspended cells were transferred to a sterile enzyme-free EP tube, 5 μg of INT (in vitro transcribed) EV71 mRNA was added, pipetted 3-5 times, and transferred to a 4 mm electroporation cuvette. The electroporation parameters were set to voltage: 250 V, time: 20 ms, and the electroporation operation was performed. After the electroporation was completed, 1 mL of MeM medium was immediately added to the cells in a clean bench, and the cells were transferred to a 15 mL centrifuge tube. The culture medium was added to 10 mL, centrifuged, and the supernatant and dead cells were discarded. The cells were resuspended and transferred to a 10 cm dish. The viral mRNA in the cell is translated and packaged to form EV-A71-TAT virus, which is finally released by cell lysis and the infectivity of the produced virus is verified. Figure 4 The results of viral RNA level detection at different times after transfection are detailed in Figure 5 .

[0074] The schematic diagram of the process of EV-A71-TAT-FL plasmid construction, viral mRNA synthesis, and EV-A71-TAT virus preparation is shown in Figure 1. The EV-A71-TAT-FL plasmid adopted in this technical solution is constructed on the basis of the EV-A71 viral genome. The EV-A71 virus is a single-stranded positive-strand RNA virus with a genome of approximately 7.4kb, including a 5' non-coding region (5'UTR), an open reading frame (ORF) and a 3' non-coding region (3'UTR). ORF encodes a polymeric precursor protein, which is cleaved by viral proteases to form structural proteins (P1, including VP4, VP3, VP2, VP1 in sequence) and non-structural proteins (P2 and P3). This technical solution inserts a TAT sequence into the EV-A71 viral genome, and the TAT sequence is inserted in the middle of the VP4 sequence. Specifically, the insertion position is after the "atgggc" sequence of VP4, and the TAT sequence is connected to the T2A sequence (cleavage peptide sequence), and the T2A sequence is followed by the VP4 sequence. The use of the above sequence design and the insertion site of the TAT sequence in this solution plays a key role in ensuring the expression activity of TAT that activates the virus. This approach inserts the nucleotide sequence of the TAT transactivator protein into the viral genome, which differs from the conventional method of inserting reporter genes into the viral genome in the prior art and also involves differences in the design of the viral plasmid. For example, Chinese patent CN106399263B reports a recombinant human enterovirus EV71 virus expressing the green fluorescent protein (EGFP) gene and its application. This technical solution inserts the GFP reporter gene between the 3D region and the 3'UTR region. Unlike the prior art, the insertion position of TAT in this technical solution ensures its effective expression and genetic stability, allowing the multi-channel viral reporter system of this solution to operate normally.

[0075] Example 2: Construction of reporter cell lines

[0076] (1) Construction of reporter plasmid

[0077] The reporter plasmid was constructed using the existing plasmid pEGFP-C1. The ninth primer (SEQ ID NO. 9) and the tenth primer (SEQ ID NO. 10) were used to amplify the T2A-Gluc fragment (sequences of both the T2A and Gluc genes) to obtain a T2A-Gluc PCR product. These two fragments (the T2A-Gluc PCR product and the pEGFP-C1 digest) were then synthesized into pEGFP-T2A-Gluc using seamless cloning.

[0078] The LTR-tk fragment (sequences of both the LTR and tk genes) was amplified using the eleventh primer (SEQ ID NO. 11) and the twelfth primer (SEQ ID NO. 12) to obtain a PCR product of the LTR-tk fragment. Circular PCR was then used to replace the CMV fragment in pEGFP-T2A-Gluc with the LTR-tk fragment to obtain a reporter plasmid.

[0079] The nucleotide sequence of the reporter plasmid is shown in SEQ ID NO. 16 (double underline indicates LTR gene sequence; wavy line indicates tk gene sequence; single underline indicates GFP gene sequence; dotted line indicates T2A gene sequence, Indicates the cleavage site; the dotted line indicates the Gluc gene sequence; the structural diagram of the reporter plasmid is shown in Figure 6 ):

[0080]

[0081]

[0082] (Only 1-3515 bp sequence is shown, the subsequent sequence is omitted, the complete sequence of SEQ ID NO. 16 is shown in the sequence listing).

[0083] (2) Generation of reporter cell lines

[0084] The constructed reporter plasmid was transfected into A549 cells (human lung adenocarcinoma epithelial cell line) and screened with G418 (1000 μg / mL) to obtain a stable reporter cell line with low background expression level (fluorescence microscopy images are shown in Figure 7 , taken under conditions without viral infection, indicating that the background GFP expression level of the reporter cells is very low, and the fluorescence expression is activated only after the virus infection is activated). Transfection of cells with the reporter plasmid is a conventional operation method in the prior art. Specifically, the operation process is as follows:

[0085] 25cm 2 A549 cells (<90%) were plated evenly into six-well plates (cultured in antibiotic-free, normal serum-concentrated medium) at a 1:12 ratio, reaching a cell density of approximately 40%. The cells were cultured overnight for 20 hours. The cells in the six-well plates were observed (density: 40%-50%). Next, 200 μL of opti-MEM was added to a sterile, enzyme-free EP tube, followed by the reporter plasmid (total DNA: 2 μg). The tube was gently stirred to mix thoroughly. Six (2 μg plasmid x 3) μL of Fugene (a conventional transfection reagent) was added and gently stirred to mix thoroughly. After incubation at room temperature for 15 minutes, the mixture was evenly added to the cell culture medium in the six-well plates. After gentle mixing, the plates were returned to the incubator for overnight culture. 72 hours after transfection, G418 was added for selection. Surviving cells were considered to have been successfully transfected with the reporter plasmid. The first batch of surviving reporter cells were digested and added to 48 wells. Each well was examined under a microscope, and the wells with low green fluorescence expression were selected and collected as the final reporter cells (the purpose was to select the cell population with low background expression level).

[0086] The construction of reporter plasmids and the process of generating reporter cell lines can be found in Figure 1 . This technical solution is transformed on the basis of the prior art plasmid pEGFP-C1, and pEGFP-C1 is integrated with a CMV promoter, a green fluorescent protein sequence (GFP), and a multiple cloning site (MCS) is set after the green fluorescent protein sequence. This technical solution first integrates T2A-Gluc at the MCS, and then replaces the original CMV with LTR-tk to finally form a reporter plasmid. The reporter plasmid includes three reporter genes: TK (thymidine kinase), GFP (green fluorescent protein, Green Fluorescent Protein) and Gluc (Gaussia Luciferase, Gaussia Luciferase), and the expression of the above-mentioned reporter genes is regulated by LTR (long terminal repeats). LTR includes a promoter and an enhancer region, which can realize the transcriptional regulation of TK, GFP and Gluc. TAT protein plays its function by combining with a specific sequence (called TAR, Trans-Activation Response element) in LTR to realize its function of transactivating gene expression. The constructed reporter plasmid is transferred into the recipient cells, and reporter cells containing three reporter genes are obtained through screening.

[0087] Example 3: Reporting system experimental results

[0088] A multi-channel viral reporter system based on TAT transactivation consists of two parts: an activation virus and reporter cells. The activation virus is used to infect reporter cells, and by adding GCV, it is converted into a toxic product by TK, thereby achieving inhibitory screening. By using flow cytometry and fluorescence imaging, GFP signals are observed, thereby achieving single-cell monitoring. By detecting Gluc fluorescence, the overall status of the cell population can be monitored. For details, see Figure 1 This study exploits the transactivation effect of TAT, which specifically binds to the transactivation response region element in the LTR promoter to regulate gene transcriptional expression. Linking multiple reporter genes to the LTR promoter, placing them in the same coding frame and under LTR regulation, overcomes the limitations of traditional single-channel screening with reporter viruses.

[0089] The specific process of activating virus-infected reporter cells is as follows:

[0090] The reporter cells were cultured in DMEM medium without double antibody in a cell culture incubator with 5% (v / v) CO2 and 37°C. The reporter cells were infected with activated virus at an MOI of 1.

[0091] The specific process of tk / GCV system verification is as follows: 200 μM GCV is added 48 hours after activation of virus-infected reporter cells, and photos are taken 48 hours after the addition of GCV.

[0092] The specific process of GFP expression verification is as follows: GFP fluorescence characterization is performed using a LSM980 laser confocal microscope with a 488 nm excitation light channel.

[0093] The specific process of Gluc expression level verification is as follows:

[0094] (1) Cell preparation: Use a 96-well plate suitable for chemiluminescence detection, inoculate 100 μl of reporter cells per well, and activate the virus to infect the reporter cells at an MOI of 1.

[0095] (2) Preparation of detection reagents:

[0096] Thaw Gaussia Luciferase Assay Buffer and equilibrate to room temperature. Place Gaussia Luciferase Assay Substrate (100×) in an ice bath or on ice until ready to use. Calculate the amount of Gaussia Luciferase Assay Working Solution required based on the 50 μl working solution required for each sample. Mix the appropriate amount of Gaussia Luciferase Assay Substrate (100×) and Gaussia Luciferase Assay Buffer in a 1:100 ratio to prepare the Gaussia Luciferase Assay Working Solution.

[0097] (3) Preparation of cell samples: 50 μl of cell culture supernatant was collected at different time points into a 96-well plate and equilibrated to room temperature.

[0098] (4) Luciferase assay:

[0099] Take 5μl of cell culture supernatant sample and add it to a 96-well white plate. Add 50μl of Gaussia luciferase assay working solution to each well and mix well. Incubate at room temperature (about 25°C) for 5-10 minutes to allow the luminescent signal to stabilize. Use a multifunctional microplate reader with chemiluminescence detection function to perform chemiluminescence detection (detection wavelength: 480nm) for 1 second. Then, based on the test results, calculate the Gluc content information through conventional means of existing technology.

[0100] This example verifies the operability of the multi-channel reporter system. The reporter cells were infected with the EV-A71 virus encoding TAT (activation virus) to verify whether the multi-channel reporter cells can function normally. This includes: ① Whether the TK+GCV suicide system can successfully induce cell death under EV-A71-TAT virus infection; ② Whether the reporter cells can successfully express GFP when infected with EV-A71-TAT virus; ③ Whether the reporter cells can successfully express and secrete Gluc when infected with EV-A71-TAT virus. TK / GCV system verification results are available at Figure 8 GFP expression verification results can be found in Figure 9 The results of Gluc expression level verification can be found in Figure 10 .

[0101] Example 4: Stability of EV-A71-TAT virus

[0102] Stability test is carried out for the EV-A71-TAT virus constructed in Example 1.EV-A71-TAT virus is passaged in a conventional manner, and after passage 10 generations, after RT-PCR amplification and gel electrophoresis operation, DNA band situation is observed on gel, and it is found that TAT gene sequence is stably present on EV-A71-TAT virus. EV-A71-TAT virus is constructed on EV-A71 genome using TAT integration, relative to the mode by which reporter gene (for example: EGFP, Gluc) is integrated on EV-A71 genome, the probability of inserting gene lost in viral replication process is significantly reduced.The EV-A71-TAT virus constructed in this scheme has good genetic stability, and then improves the stability and application value of the overall multi-channel virus reporter system based on TAT transactivation.

[0103] The electrophoresis results are detailed in Figure 11 , after EV-A71-TAT virus passage 10 generations, the electrophoresis result of the amplified product of RT-PCR amplification is carried out to viral genome using the 13th primer (SEQ ID NO.13) and the 14th primer (SEQ ID NO.14). As can be seen from the experimental result, in all samples tested, there are TAT amplification bands, indicating that in these EV-A71-TAT viruses, TAT genes are stably present, and there is no situation of insertion gene loss. If the Gluc gene is inserted into the EV-A71 genome, after the virus is passaged 10 generations, there will be a situation of Gluc gene loss in some viruses, and in the viruses lost in these target genes, it is difficult to amplify the band corresponding to the target gene. It can be seen that the EV-A71-TAT virus set up in this program has obvious advantages in the genetic stability of integrated genes relative to the virus of the conventional integrated reporter gene of the prior art.

[0104] Example 5: Study on the background interference problem of the reporting system

[0105] The existing technology usually constructs a cis-reporter system for related research. Specifically, a virus expressing a reporter gene is constructed, and the virus is used to infect blank cells (the plasmid expressing the reporter gene is not transferred into the cell, and the reporter gene is on the viral genome). The expression level of the reporter gene in the blank cells after virus infection is observed, and the replication activity of the virus is then determined. The replication activity of the virus can reflect the effect of the applied drugs or other means on the activity of the virus, thereby achieving the purpose of drug screening (research or screening of factors affecting viral replication) and so on.

[0106] The inventors also constructed an EV-A71-Gluc virus expressing the Gaussia luciferase gene Gluc in the early stage (the corresponding EV-A71-Gluc plasmid is formed by integrating the Gluc gene as a reporter gene into the EV-A71 virus, which is a cis-reporter system; the TAT gene of SEQ ID NO.15 is replaced with the Gluc gene. The specific sequence of the Gluc gene can be shown in the dotted line of SEQ ID NO.16). Referring to the method of Example 3, the EV-A71-Gluc virus and the EV-A71-TAT virus were infected with the corresponding reporter cells, and the Gluc expression level was detected at different time points after infection. The experimental results are detailed in Figure 12 , it can be seen that the reporter system of EV-A71-Gluc virus+blank cell is used, and very strong Gluc activity signal is just arranged 4h after infection.Because infection time is shorter (4h after infection), the replication level of virus in cell is limited, therefore, the strength of Gluc activity signal now can not effectively reflect the replication activity situation of virus.Why there is such strong Gluc activity signal in EV-A71-Gluc virus 4h after infection, this is because EV-A71-Gluc virus itself has the background expression of Gluc gene, has caused higher background interference, can cover the reporter gene signal of new generation, finally causes high throughput screening result inaccurate.And adopt the system of EV-A71-TAT virus+reporter cell of this program, 4h after infection, Gluc activity signal is close to control group, avoids the background expression of Gluc gene (obvious background expression does not occur in the Gluc gene in the reporter cell; And do not contain Gluc gene in EV-A71-TAT virus), eliminates the background interference problem. The experimental results showed that over time, the Gluc activity signal of the EV-A71-Gluc virus + blank cell reporter system remained significantly higher than that of the EV-A71-TAT virus + reporter cell system, indicating that background expression of the Gluc gene seriously affects the detection accuracy and effectiveness. The EV-A71-TAT virus + reporter cell system using this protocol can more accurately reflect viral replication activity by detecting Gluc activity signals, thereby enabling more precise drug screening research.

[0107] The above is only an embodiment of the present invention, and the common knowledge such as the specific structure and characteristics of the scheme is not described in detail here. It should be pointed out that for those skilled in the art, several variations and improvements can be made without departing from the structure of the present invention, and these should also be regarded as the scope of protection of the present invention. These will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.

Claims

1. A multi-channel viral reporter system based on TAT transactivation, characterized in that: It involves activating the virus and reporter cells; The activated virus is human enterovirus A71 expressing TAT protein; The reporter cells are cells transfected with a reporter plasmid; the reporter plasmid includes an LTR sequence and a plurality of reporter genes whose expression is promoted by the LTR sequence.

2. The multi-channel virus reporting system based on TAT transactivation according to claim 1, characterized in that: The activated virus is prepared by the following method: S1: construct a viral plasmid with the sequence shown in SEQ ID NO.15; S2: The viral plasmid is linearized by enzyme digestion and then transcribed in vitro to obtain viral mRNA; S3: The viral mRNA is transferred into Vero cells, the Vero cells are cultured, and the activated virus is obtained after cell lysis.

3. The multi-channel virus reporting system based on TAT transactivation according to claim 2, characterized in that: In S1, the viral plasmid with the sequence shown in SEQ ID NO. 15 was constructed by the following method: Using a first primer having a sequence as shown in SEQ ID NO.1 and a second primer having a sequence as shown in SEQ ID NO.2, PCR amplification is performed on the first fragment to obtain a first PCR product; Using a third primer having a sequence as shown in SEQ ID NO. 3 and a fourth primer having a sequence as shown in SEQ ID NO. 4, PCR amplification is performed on the second fragment to obtain a second PCR product; Amplifying the first PCR product and the second PCR product using the first primer and the fourth primer to obtain a first spliced fragment; The third fragment was amplified by PCR using the fifth primer having a sequence as shown in SEQ ID NO. 5 and the sixth primer having a sequence as shown in SEQ ID NO. 6 to obtain a third PCR product; the fourth fragment was amplified by PCR using the seventh primer having a sequence as shown in SEQ ID NO. 7 and the eighth primer having a sequence as shown in SEQ ID NO. 8 to obtain a fourth PCR product; amplifying the third PCR product and the fourth PCR product using the fifth primer and the eighth primer to obtain a second spliced fragment; The first splicing fragment and the second splicing fragment are double-enzyme digested and then connected to obtain a circular viral plasmid; the sequences of the first fragment, the second fragment, the third fragment and the fourth fragment can be spliced to form a circular viral plasmid.

4. The multi-channel virus reporting system based on TAT transactivation according to claim 3, characterized in that: In S2, the viral plasmid is linearized using the Stu1 restriction endonuclease, and the linearized plasmid is then transcribed into mRNA in vitro.

5. The multi-channel virus reporting system based on TAT transactivation according to claim 4, characterized in that: In S3, the mRNA formed by in vitro transcription of the linearized plasmid was added to the Vero cell suspension, and electroporation was performed under the parameter settings of voltage 250V and time 20ms; after electroporation, the cells were continued to be cultured, and the activated virus was obtained after cell lysis.

6. The multi-channel virus reporting system based on TAT transactivation according to claim 1, characterized in that: The reporter genes contained in the reporter plasmid include thymidine kinase gene, green fluorescent protein gene and Gaussia luciferase gene.

7. A multi-channel viral reporter system based on TAT transactivation according to claim 6, characterized in that: The gene sequence of the reporter plasmid is shown in SEQ ID NO.

16.

8. The multi-channel viral reporter system based on TAT transactivation according to claim 7, characterized in that: The reporter plasmid was constructed by the following method: A fragment consisting of the T2A gene and the Gluc gene was amplified using the ninth primer having a sequence as shown in SEQ ID NO.9 and the tenth primer having a sequence as shown in SEQ ID NO.10 to obtain a T2A-Gluc amplification product; the fragment formed after enzyme digestion of the plasmid pEGFP-C1 and the T2A-Gluc amplification product were synthesized into a circular plasmid pEGFP-T2A-Gluc; Using the eleventh primer with the sequence shown in SEQ ID NO.11 and the twelfth primer with the sequence shown in SEQ ID NO.12, the fragment consisting of the LTR gene and the tk gene was amplified to obtain an LTR-tk amplification product; the original CMV promoter region in pEGFP-T2A-Gluc was replaced with the LTR-tk amplification product to obtain a reporter plasmid.

9. Use of a multi-channel virus reporter system based on TAT transactivation according to any one of claims 1 to 8 in virus detection for purposes other than treatment or diagnosis of a disease, characterized in that: First, reporter cells are infected with activating viruses; Then, the infected cells are cultured in an environment containing GCV to observe the activation of the virus to induce the death of the reporter cells; or the GFP fluorescence signal in the infected reporter cells is observed; or the Gluc content in the culture supernatant of the infected reporter cells is detected.

10. Use of a multi-channel virus reporter system based on TAT transactivation according to claim 9 in virus detection for purposes other than disease treatment or diagnosis, characterized in that: The reporter cells are infected with the activation virus at an MOI of 0.1-10; preferably, the MOI is 1; GCV was added to the reporter cells infected with the activated virus, and after culture, the cell survival was observed; Use the excitation light channel of a fluorescence confocal microscope to examine the reporter cells after activation of virus infection and observe the GFP fluorescence signal; Collect the culture supernatant of reporter cells 6-72 hours after activation of virus infection; add Gaussia luciferase detection substrate to the supernatant, perform chemiluminescence detection after the reaction luminescence signal tends to be stable, and finally convert it into Gluc content information.

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