A multi-channel viral reporter system based on tat transactivation

Abstract

The application relates to the technical field of molecular biology, in particular to a multi-channel virus reporting system based on TAT transactivation. The multi-channel virus reporting system based on TAT transactivation comprises an activation virus and a reporting cell; the activation virus is human enterovirus A71 expressing a TAT protein; the reporting cell is a cell transfected with a reporting plasmid; the reporting plasmid comprises an LTR sequence and a plurality of reporter genes expressed by the LTR sequence. When the activation virus infects the reporting cell, the TAT protein carried by the virus enters the reporting cell, activates the expression of downstream reporter genes, and the expression level of the reporter genes can reflect the virus replication situation, so that related antiviral drug screening or mechanism research can be realized. The technical scheme can solve the technical problems of the flexibility, stability and high-throughput screening adaptability of the virus reporting system in the prior art, and has an ideal popularization and application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of molecular biology, and particularly relates to a multi-channel virus reporting system based on TAT transactivation. BACKGROUND

[0002] In the field of virology research, in-depth exploration of the immune and anti-immune interaction between viruses and hosts is the key to understanding infection mechanisms, disease progression, and developing effective antiviral strategies. The progress in this field not only depends on a deep understanding of the biological characteristics of viruses, but also greatly benefits from the innovation of modern biotechnology, especially the introduction of high-throughput screening technology (HTS), such as the CRISPR / Cas9 gene editing system, which has become a core tool for revealing the complexity of virus-host interaction networks.

[0003] However, despite the revolutionary changes brought about by high-throughput screening technology in virology research, the existing virus reporting system has to some extent constituted a technical bottleneck. The traditional virus reporting system often uses a cis-reporting system. In the cis-reporting system, the reporter gene (such as the fluorescent protein gene, luciferase gene, etc.) is directly inserted into the target vector (for example: viral genome), and is located in the same molecule with the target regulatory element, that is, the reporter gene and the regulatory sequence are "coexist" in the same DNA molecule. By integrating the reporter gene (such as luciferase or fluorescent protein) into the viral genome, the cis-reporting system provides a visual "window" for the process of viral replication and infection. Although this method is intuitive, it is limited by the compact and short characteristics of the viral genome (usually only a few thousand base pairs), resulting in extremely limited capacity of the viral vector. Therefore, these systems can usually only carry one reporter gene, which not only limits the diversity of visualization methods, but also affects the flexibility of the high-throughput screening process and the comprehensiveness of information acquisition.

[0004] In addition, some commonly used cis-reporter genes, such as Gaussia Luciferase (Gluc) and Renilla Luciferase (Rluc), are prone to be lost during the continuous passage of viruses, which is directly related to the instability of the reporter system. The instability of the reporter gene not only reduces the reliability of the screening results, but also limits the application potential of these systems in long-term, large-scale screening experiments, especially in the case of multiple rounds of screening to identify key interaction factors or antiviral targets, the stability of the reporter gene is particularly important.

[0005] Therefore, the existing conventional virus reporter system shows deficiencies in multiple key dimensions, including but not limited to flexibility, stability, and high-throughput screening adaptability. 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 vector capacity limit and realize the simultaneous expression of multiple reporter genes, thereby increasing the diversity of visualization paths and the richness of screening information; second, it has excellent genetic stability, ensuring that the reporter gene is not easily lost during virus passage, and ensuring the accuracy and consistency of the screening results; third, it is compatible with high-throughput screening technology, improves screening efficiency, and broadens the application range, providing strong technical support for the fields of antiviral drug research, virus pathogenesis analysis, etc. SUMMARY

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

[0007] To achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows:

[0008] A multi-channel virus reporter system based on TAT transactivation, comprising an activating virus and a reporter cell;

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

[0010] The reporter cell is a cell transfected with a reporter plasmid; the reporter plasmid comprises an LTR sequence and a plurality of reporter genes expressed by the LTR sequence.

[0011] Further, the activating virus is prepared by the following method:

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

[0013] S2: linearize the virus plasmid by enzyme digestion, and then obtain virus mRNA by in vitro transcription;

[0014] S3: transfer the virus mRNA into Vero cells, culture the Vero cells, lyse the cells, and obtain the activated virus.

[0015] Further, in S1, the virus plasmid with the sequence shown in SEQ ID NO. 15 is constructed by the following method:

[0016] PCR-amplify the first fragment using the first primer with the sequence shown in SEQ ID NO. 1 and the second primer with the sequence shown in SEQ ID NO. 2 to obtain a first PCR product; PCR-amplify the second fragment using the third primer with the sequence shown in SEQ ID NO. 3 and the fourth primer with the sequence shown in SEQ ID NO. 4 to obtain a second PCR product; and amplify the first PCR product and the second PCR product using the first primer and the fourth primer to obtain a first splicing fragment.

[0017] PCR-amplify the third fragment using the fifth primer with the sequence shown in SEQ ID NO. 5 and the sixth primer with the sequence shown in SEQ ID NO. 6 to obtain a third PCR product; PCR-amplify the fourth fragment using the seventh primer with the sequence shown in SEQ ID NO. 7 and the eighth primer with the sequence shown in SEQ ID NO. 8 to obtain a fourth PCR product; and amplify the third PCR product and the fourth PCR product using the fifth primer and the eighth primer to obtain a second splicing fragment.

[0018] double-digest the first splicing fragment and the second splicing fragment, and then connect them to obtain a circular virus plasmid; the sequences of the first fragment, the second fragment, the third fragment, and the fourth fragment can be spliced to form the circular virus plasmid.

[0019] The virus plasmid construction method described above is a conventional method for synthesizing and expressing plasmids in molecular biology and virology research, and ultimately the virus plasmid with the sequence shown in SEQ ID NO. 15 can be obtained. In addition to the above method, other methods can also be used to ultimately obtain the virus plasmid with the sequence shown in SEQ ID NO. 15.

[0020] Further, in S2, the virus plasmid is linearized by using Stu1 restriction enzyme, and then the linearized plasmid is transcribed into mRNA in vitro.

[0021] Further, in S3, the mRNA formed by in vitro transcription of the linearized plasmid is added to the Vero cell suspension, and the electric transformation operation is performed under the parameter settings of voltage 250V and time 20ms; after the electric transformation is completed, the cells are continuously cultured, and the activated virus is obtained after the cells are lysed.

[0022] Further, the reporter gene contained in the reporter plasmid includes a thymidine kinase gene, a green fluorescent protein gene, and a Gaussia luciferase gene.

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

[0024] Further, the reporter plasmid is constructed by the following method: using a ninth primer with a sequence shown in SEQ ID NO. 9 and a tenth primer with a sequence shown in SEQ ID NO. 10 to amplify a fragment composed of a T2A gene and a Gluc gene, to obtain a T2A-Gluc amplification product; synthesizing a fragment formed after the plasmid pEGFP-C1 is cut and the T2A-Gluc amplification product into a circular plasmid pEGFP-T2A-Gluc.

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

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

[0027] Using conventional means of the prior art, the reporter plasmid is used to transfect cells, and after transfection, the reporter cells can be obtained by screening. Among them, the cells to be transfected can be A549 cells, which are seeded at a seeding density of about 40%, and cultured in a conventional culture medium for 20 hours. Then, the reporter plasmid and transfection reagent are added, mixed, and the cell culture is continued. After 72 hours, conventional G418 screening is performed, and the surviving cells are the cells successfully transfected with the reporter plasmid, i.e., the reporter cells.

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

[0029] First, the reporter cells are infected with an activated virus;

[0030] Then, the infected cells are cultured in an environment containing GCV, and the activation virus-induced reporter cell death is observed, or the GFP fluorescence signal in the infected reporter cells is observed, or the Gluc content in the supernatant of the infected reporter cells is detected.

[0031] Further, the MOI of the activation virus for infecting the reporter cells is 0.1-10; preferably, the MOI is 1;

[0032] GCV is added to the reporter cells after being infected by the activation virus, and after culture, the cell survival is observed.

[0033] The reporter cells infected by the activation virus are observed by using a fluorescence confocal microscope to excite the light channel, and the GFP fluorescence signal is observed.

[0034] The culture medium supernatant of the reporter cells infected by the activation virus for 6-72 hours is collected; the luciferase detection substrate is added to the supernatant, and after the reaction luminescence signal tends to be stable, chemiluminescence detection is performed, and finally the Gluc content information is converted.

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

[0036] The application provides a multi-channel virus reporting system based on TAT transactivation, aiming at solving the technical bottlenecks of poor stability, easy loss of reporter genes, inability to realize multi-channel screening and insufficient adaptability of high-throughput screening of existing cis-reporting systems in virology research. The present scheme utilizes the transactivation of TAT (transcription activator). TAT is a small molecular protein, which can specifically bind to the transactivation response element in the LTR (long terminal repeat) promoter, thereby regulating the transcription and expression of downstream genes. In the present application, an activated virus expressing TAT protein is first constructed, which is based on human enterovirus A71, and the TAT protein coding gene is integrated into the viral genome through genetic engineering technology. At the same time, a reporter plasmid containing an LTR promoter and multiple reporter genes (such as thymidine kinase gene TK, green fluorescent protein gene GFP and Gaussian luciferase gene Gluc) is designed, and is transfected into specific reporter cells to form a stable reporter cell line. When the activated virus infects the reporter cells, the TAT protein carried by the virus enters the cells and binds to the LTR promoter, activating the transcription and expression of the downstream reporter genes. Since the expression amount of TAT protein is consistent with the replication of viral genome, the expression level of reporter genes can indirectly reflect the replication of virus in reporter cells. This design not only overcomes the problem of easy loss of reporter genes in traditional cis-reporting systems, but also realizes the construction of multi-channel reporting system, and provides the possibility for high-throughput screening. This reporting system can be flexibly applied to high-throughput screening. When the reporter cells are knocked out in a pro-virus environment, the expression level of green fluorescent protein GFP is regulated to increase, and vice versa. By flow sorting, cells with different expression levels of green fluorescent protein are obtained, and finally deep sequencing analysis can find potential antiviral target points. Similarly, the expression strength of the secreted luciferase Gluc and the TK suicide system in the reporter cells can realize the high-throughput and rapid screening of antiviral drugs.

[0037] More specifically, the technical scheme is innovative compared with the prior art in that:

[0038] The present application ingeniously utilizes the transactivation property of TAT protein, which is designed to be expressed in the activating virus, and when the virus infects the reporter cell, the TAT protein enters the cell and exerts its transcription activation effect. The traditional cis-reporter system usually directly integrates the reporter gene into the viral genome, which not only limits the diversity of the reporter gene, but also is prone to be restricted by the compactness of the viral genome (it is difficult to simultaneously integrate multiple reporter genes in the virus). However, the present application realizes the separation of the reporter gene from the viral genome by integrating the TAT gene in the viral genome and the reporter gene in the virus-infected cell, thereby overcoming the above limitations. Therefore, by using the technical means of the present application, the defect of the cis-reporter system that cannot realize multi-channel screening (cannot simultaneously integrate multiple reporter genes in the same viral genome) can be overcome.

[0039] In the present technical solution, only the TAT gene with a small molecular weight (about 258 bp) is integrated in the activating virus, and the reporter gene with a large molecular weight (for example, the EGFP reporter gene 717 bp, the Gluc reporter gene 555 bp) is not integrated into the viral genome, thereby overcoming the defect of poor stability of the cis-reporter system. The existing cis-reporter system usually directly integrates a single reporter gene into the viral genome, and the reporter gene is prone to be lost during virus passage, resulting in poor stability of the reporter system.

[0040] One of the reporter genes used in the present technical solution is the Gaussian luciferase gene Gluc, which has a secretion property. If Gluc is integrated into the viral genome to construct a cis-reporter system, the following problems exist: since Gluc is integrated into the viral genome, the Gluc reporter gene contains background expression in the virus stock solution, and after the virus stock solution is inoculated into cells, the background expression will mask the newly generated Gluc signal, thereby cannot be used for high-throughput screening. However, in the present technical solution, the regulatory factor TAT and the reporter gene Gluc are located in different spaces through ingenious design, and the phenomenon that the background expression affects the newly generated Gluc signal does not occur. By using the present technical solution, it is expected to realize rapid high-throughput screening by directly determining the cell culture supernatant.

[0041] The present technical solution has the following beneficial effects:

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

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

[0044] (3) Solve the problem of background interference in high-throughput screening: In traditional cis-reporter systems, the background expression of reporter genes in virus stock often masks the newly generated reporter gene signal, resulting in inaccurate high-throughput screening results. The present application avoids this problem by introducing a TAT transactivation system. When activating virus infection of reporter cells, the TAT protein activates the reporter genes in the reporter cells, not the background expression in the virus stock. This realizes rapid high-throughput screening and improves screening efficiency and accuracy.

[0045] (4) Broaden the application range and promote the progress of virology research: The multi-channel virus reporter system of the present application can be used not only in the fields of antiviral drug development and virus pathogenesis analysis, but also flexibly applied in high-throughput screening experiments. For example, in antiviral drug screening, the effect of candidate antiviral drugs can be characterized by observing the level of secreted Gaussia luciferase in the cell supernatant, and then screening potential antiviral drugs; the antiviral effect of candidate drugs can also be determined by using the thymidine kinase suicide system and detecting cell survival number by MTT kit, thereby realizing drug screening. In addition, it needs to be further explained that: Although the TAT sequence is integrated into the EV-A71 virus genome (EV-A71 virus has the effect of lysing cells) to form an activated virus, which is used as an example and described. 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 realized. Further, in the study of virus pathogenesis, the expression patterns of different reporter genes can be analyzed to reveal the interaction mechanism between viruses and hosts. This brings new breakthroughs and broad development space to the field of virology research. BRIEF DESCRIPTION OF DRAWINGS

[0046] Figure 1 It is a schematic diagram of the construction principle of a multi-channel virus reporter system based on TAT transactivation.

[0047] Figure 2 It is a schematic diagram of the structure of EV-A71-TAT-FL plasmid of Example 1.

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

[0049] Figure 4 Cellular pathogenic effect (CPE) diagram of EV-A71-TAT-FL (EV-A71-TAT) mRNA electroporated Vero cells of Example 1.

[0050] Figure 5 Virus RNA level detection results of mRNA electroporated Vero cells at different times of Example 1 (EV-A71-TAT refers to mRNA transfection using EV-A71-TAT-FL transcription; 3D-Mut refers to mRNA transfection using virus replication-defective transcription, and the virus replication-defective refers to the mutation of T to A at the 313th amino acid of the 3D protein responsible for replication).

[0051] Figure 6 Structural schematic diagram of the reporter plasmid of Example 2.

[0052] Figure 7 Fluorescence microscopic image of the reporter cell of the constructed three reporter genes of Example 2 (scale 100 μm).

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

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

[0055] Figure 10 Gluc expression level verification results of Example 3 (Mock: control group; EV-A71-TAT refers to mRNA transfection using EV-A71-TAT-FL transcription; 3D-Mut refers to mRNA transfection using virus replication-defective transcription, and the virus replication-defective refers to the mutation of T to A at the 313th amino acid of the 3D protein responsible for replication).

[0056] Figure 11 Stability detection results of EV-A71-TAT virus of Example 4.

[0057] Figure 12 Gluc expression level research results of the reporter cells infected with different viruses of Example 5. DETAILED DESCRIPTION

[0058] The application will be further described in connection with the following examples, which do not limit the scope of the application. If not specified, the technical means used in the following examples and experimental examples are conventional means known to those skilled in the art, and the materials, reagents, etc. used can be obtained from commercial channels.

[0059] A multi-channel virus reporter system based on TAT transactivation includes two parts of an activating virus and a reporter cell. The flow chart for constructing the activating virus and the reporter cell can be found in Figure 1 .

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

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

[0062] In order to facilitate in vitro amplification, the sequence of the EV-A71-TAT-FL plasmid is divided into multiple sequences, which can be spliced into the final EV-A71-TAT-FL plasmid. The plasmid fragments (divided into four fragments) are synthesized by a biotechnology company, including EV-A71-TAT-FL-F1 fragment (first fragment), EV-A71-TAT-FL-F2 fragment (second fragment), EV-A71-TAT-FL-F3 fragment (third fragment); and a basic vector fragment (Backbone, fourth fragment) is obtained. The EV-A71-TAT-FL plasmid is obtained by multiple rounds of PCR amplification of the above-mentioned fragments.

[0063] First, the EV-A71-TAT-FL-F1 fragment is subjected to PCR amplification using the first primer (SEQ ID NO. 1) and the second primer (SEQ ID NO. 2) and conventional PCR means of the prior art, to obtain the EV-A71-TAT-FL-F1 fragment amplification product (first PCR product). The EV-A71-TAT-FL-F2 fragment is subjected to PCR amplification using the third primer (SEQ ID NO. 3) and the fourth primer (SEQ ID NO. 4) and conventional PCR means of the prior art, to obtain the 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) are subjected to conventional overlap extension PCR of the prior art using the first primer and the fourth primer, and the two amplification fragments are spliced together by extension of the overlapping strands (first spliced fragment, EV-A71-TAT-FL-F12).

[0064] Then, the EV-A71-TAT-FL-F3 fragment was amplified by PCR using the fifth primer (SEQ ID NO. 5) and the sixth primer (SEQ ID NO. 6) and by means of the conventional PCR method of the prior art, to obtain an EV-A71-TAT-FL-F3 fragment amplification product (third PCR product). The basic vector fragment was amplified by PCR using the seventh primer (SEQ ID NO. 7) and the eighth primer (SEQ ID NO. 8) and by means of the conventional PCR method of the prior art, to obtain a basic vector fragment amplification product (fourth PCR product). Next, the EV-A71-TAT-FL-F3 fragment amplification product (third PCR product) and the basic vector fragment (fourth PCR product) were subjected to the conventional overlap extension PCR of the prior art using the fifth primer and the eighth primer, and the two amplification fragments were spliced together by overlapping chain extension (second splicing fragment, EV-A71-TAT-FL-F3B).

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

[0066] The nucleotide sequence of the EV-A71-TAT-FL plasmid is shown in SEQ ID NO. 15 (the wavy line represents the TAT gene sequence; the dotted line represents the T2A gene sequence, the cleavage site; the double underlined part represents 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, and the structural schematic diagram is shown in Figure 2 ):

[0067]

[0068]

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

[0070] (3) Synthesis of viral mRNA

[0071] EV-A71-TAT-FL plasmid was linearized by restriction enzyme digestion using Stul (recognition sequence: AGGCCT) according to the conventional method of the prior art, and then the linearized plasmid was transcribed into mRNA using a T7 in vitro transcription kit. The gel electrophoresis diagram of EV-A71-TAT-FL plasmid linearization and in vitro transcribed mRNA is shown in Figure 3 .

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

[0073] Vero cells were seeded in a 10 cm culture dish, and after the Vero cells were full (confluence: 90%), the subsequent operation was performed. The cells were digested using 1 mL trypsin, and the digestion was terminated using 2 mL MeM medium. The digested cells were transferred to a centrifuge tube, supplemented with PBS to 10 mL, centrifuged at 800 rpm for 5 min, and the supernatant was discarded, and the cells were reserved. The cells were resuspended and washed with 10 mL PBS, the supernatant was discarded by centrifugation, and the cells were resuspended with 0.5 mL PBS. The resuspended cells were transferred to a sterile enzyme-free EP tube, 5 μg of INT (in vitro transcribed) EV71 mRNA was added, and the cells were blown and sucked for 3-5 times, and then transferred to a 4 mm electroporation cup. The electroporation parameters were set as voltage: 250 V, time: 20 ms, and the electroporation operation was performed. After the electroporation was completed, 1 mL MeM medium was immediately added to the cells in the clean bench, transferred to a 15 mL centrifuge tube, supplemented with medium 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 cells was translated and packaged to form EV-A71-TAT virus, and finally the virus particles were released by cell lysis, and the infectivity of the produced virus was verified. The cytopathic effect (CPE) diagram of EV-A71-TAT-FL mRNA electroporated Vero cells is shown in Figure 4 . The results of viral RNA level detection at different times after transfection are shown in Figure 5 .

[0074] The process diagram of the construction of EV-A71-TAT-FL plasmid, the synthesis of viral mRNA, and the preparation of EV-A71-TAT virus is shown in Figure 1The EV-A71-TAT-FL plasmid used in the technical solution is constructed on the basis of the EV-A71 virus genome. The EV-A71 virus is a single-stranded positive RNA virus, and the genome is about 7.4 kb, including a 5' non-coding region (5'UTR), an open reading frame (ORF), and a 3' non-coding region (3'UTR). The ORF encodes a polyprotein precursor, which is cleaved by viral proteases to form structural proteins (P1, including VP4, VP3, VP2, and VP1 in turn) and non-structural proteins (P2 and P3). In the technical solution, the TAT sequence is inserted into the EV-A71 virus 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, the TAT sequence is followed by the T2A sequence (a cleavage peptide sequence), and the T2A sequence is immediately followed by the VP4 sequence. The sequence design and insertion site of the TAT sequence used in the present solution play a key role in ensuring the expression activity of the activated virus TAT. The nucleotide sequence of the TAT transactivation protein inserted in the virus genome is different from the conventional method of inserting a reporter gene in the virus genome, and there are differences in the design of the virus plasmid. For example, Chinese Patent CN106399263B reports a human enterovirus EV71 type recombinant virus expressing a green fluorescent protein EGFP gene and its application. The technical solution inserts the GFP reporter gene between the 3D region and the 3'UTR region. In the present technical solution, the insertion position of TAT ensures the effective expression and genetic stability of TAT, so that the multi-channel virus reporter system of the present solution can function normally.

[0075] Example 2: Construction of a reporter cell line

[0076] (1) Construction of a reporter plasmid

[0077] The existing technology plasmid pEGFP-C1 was used to construct the reporter plasmid. The ninth primer (SEQ ID NO. 9) and the tenth primer (SEQ ID NO. 10) were used to amplify the T2A-Gluc (the sequence of two genes T2A and Gluc) fragment, and the PCR product of T2A-Gluc was obtained. Then the two fragments (the PCR product of T2A-Gluc and the pEGFP-C1 enzyme digestion fragment) were ligated into pEGFP-T2A-Gluc by seamless cloning.

[0078] The eleventh primer (SEQ ID NO. 11) and the twelfth primer (SEQ ID NO. 12) were used to amplify the LTR-tk (the sequence of two genes LTR and tk) fragment, and the PCR product of LTR-tk was obtained. Then the CMV in pEGFP-T2A-Gluc was replaced with LTR-tk by circular PCR to obtain the reporter plasmid.

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

[0080]

[0081]

[0082] (only the sequence of 1-3515 bp 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 G418 (1000 μg / mL) was used for screening to obtain a stable reporter cell line with low background expression level (fluorescence microscopic image is shown in Figure 7 , which was taken under non-viral infection conditions, indicating that the background expression level of the reporter cell is very low, and the fluorescence expression is activated only after viral infection). Transfection of the reporter plasmid into cells is a conventional means in the prior art, and the operation process is as follows:

[0085] A 25 cm 2 culture flask of A549 cells (<90%) was evenly distributed into six-well plates at a ratio of 1:12 (without antibiotics, normal serum concentration medium culture), with a cell density of about 40%, and was incubated overnight for 20 hours. The cell state in the six-well plate (density: 40%-50%) was observed. Then, a sterile enzyme-free EP tube was used, 200 uL of opti-MEM was added first, and then 6 (2 ug of plasmid x 3) uL of Fugene (a conventional transfection reagent in the prior art) was added, and the EP tube was gently mixed by hand. After incubation at room temperature for 15 min, the mixed solution was evenly added to the cell culture solution in the six-well plate, and the six-well plate was gently mixed and then placed back into the incubator for overnight culture. G418 was added for screening after 72 hours of transfection, and the surviving cells were the cells successfully transfected with the reporter plasmid. The first batch of surviving reporter cells were digested and added to 48-well plates, and each well was examined by microscopy, and wells with low green fluorescence expression were selected and collected as the final reporter cells (the purpose is to select a cell population with low background expression level).

[0086] The construction of the reporter plasmid and the process of generating the reporter cell line can be seen in​Figure 1 The technical solution is based on the modification of the existing plasmid pEGFP-C1. The pEGFP-C1 integrates a CMV promoter, a green fluorescent protein sequence (GFP), and a multiple cloning site (MCS) after the green fluorescent protein sequence. The technical solution first integrates T2A-Gluc at the MCS, then replaces the original CMV with LTR-tk, and finally forms a reporter plasmid. The reporter plasmid includes three reporter genes: TK (Thymidine Kinase), GFP (Green Fluorescent Protein), and Gluc (Gaussia Luciferase), and the expression of the above reporter genes is regulated by LTR (Long Terminal Repeat). LTR contains a promoter and an enhancer region, which can achieve transcriptional regulation of TK, GFP, and Gluc. TAT protein exerts its function by binding to a specific sequence (called TAR, Trans-Activation Response element) in LTR to achieve its function of transactivating gene expression. The constructed reporter plasmid is transferred into recipient cells, and through screening, reporter cells containing three reporter genes are obtained.

[0087] Example 3: Experimental effect of the reporter system

[0088] A multi-channel virus reporter system based on TAT transactivation includes two parts: activating virus and reporter cells. The activating virus is used to infect reporter cells, and by adding GCV, it is converted into a toxic product by TK, thereby achieving inhibition screening; by using flow cytometry and fluorescence imaging, the GFP signal is observed, thereby achieving single-cell monitoring; by detecting Gluc fluorescence, the overall situation of the cell population can be monitored, as described in detail in Figure 1 In this study, the transactivation of TAT is used, which can specifically bind to the transactivation response element in the LTR promoter to regulate gene transcription and expression. By linking several reporter genes after the LTR promoter, they are in the same coding frame and are regulated by LTR, which can overcome the shortcomings of traditional single-channel reporter viruses.

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

[0090] The reporter cells are cultured in DMEM medium without double antibodies in a cell culture incubator with 5% (v / v) CO2 at 37°C. The activating virus is used to infect the reporter cells at a MOI of 1.

[0091] The specific process of tk / GCV system verification is as follows: 48 hours after the activating virus infects the reporter cells, 200 μM of GCV is added, and 48 hours after the addition of GCV, the cells are photographed.

[0092] The specific process for GFP expression verification is: using LSM980 laser confocal microscope 488nm excitation light channel microscopy, GFP fluorescence characterization is carried out.

[0093] The specific process for Gluc expression level verification is:

[0094] (1) Preparation of cells: using a 96-well plate suitable for chemiluminescence detection, 100 μl of reporter cells were inoculated in each well, and the reporter cells were infected with activated virus at MOI = 1.

[0095] (2) Preparation of detection reagents:

[0096] The luciferase detection buffer was thawed and equilibrated to room temperature. The luciferase detection substrate (100x) was placed in an ice bath or ice box for standby. According to the amount of 50 μl detection working solution required for detecting each sample, the amount of luciferase detection working solution required was calculated. The appropriate amount of luciferase detection substrate (100x) and luciferase detection buffer were mixed at a ratio of 1:100 to prepare the luciferase detection 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 detection:

[0099] 5 μl of cell culture supernatant sample was taken and added to a 96-well white plate. 50 μl of luciferase detection working solution was added to each well and mixed well. Incubate at room temperature (about 25°C) for 5-10 minutes to allow the luminescence signal to stabilize. Use a multifunctional enzyme label instrument with chemiluminescence detection function to detect chemiluminescence (detection wavelength: 480nm) for 1 second. Then according to the detection results, through the conventional means of the prior art, the Gluc content information is calculated.

[0100] This example verifies the working nature of the multi-channel reporter system. The EV-A71 virus encoding TAT (activated virus) infects the reporter cells to verify whether the multi-channel of the reporter cells can work normally, which 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 under EV-A71-TAT virus infection; ③ Whether the reporter cells can successfully express and secrete Gluc under EV-A71-TAT virus infection. The results of tk / GCV system verification are shown in Figure 8 . The results of GFP expression verification are shown in Figure 9 . The results of Gluc expression level verification are shown in Figure 10 .

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

[0102] Stability test was performed on the EV-A71-TAT virus constructed in Example 1. The EV-A71-TAT virus was passaged in a conventional manner, and after 10 passages, the DNA band on the gel was observed after RT-PCR amplification and gel electrophoresis operation, and it was found that the TAT gene sequence was stably present on the EV-A71-TAT virus. The EV-A71-TAT virus was constructed by using TAT integrated on the EV-A71 genome, and compared with the method of integrating a reporter gene (such as EGFP, Gluc) on the EV-A71 genome, the probability of the inserted gene being lost during virus replication was significantly reduced. The EV-A71-TAT virus constructed by this scheme has good genetic stability, thereby improving the stability and application value of the overall multi-channel virus reporter system based on the TAT transactivation effect.

[0103] The electrophoresis results are shown in detail in Figure 11 After the EV-A71-TAT virus was passaged for 10 generations, the thirteenth primer (SEQ ID NO. 13) and the fourteenth primer (SEQ ID NO. 14) were used to perform RT-PCR amplification on the virus genome. From the experimental results, it can be seen that in all the samples tested, the TAT amplification band appeared, indicating that the TAT gene was stably present in these EV-A71-TAT viruses, and there was no loss of the inserted gene. If the Gluc gene is inserted into the EV-A71 genome, after the virus is passaged for 10 generations, there will be a situation that the Gluc gene is lost in some viruses, and in these viruses where the target gene is lost, it is difficult to amplify the band corresponding to the target gene. Therefore, the EV-A71-TAT virus established by this scheme has obvious advantages in the genetic stability of the integrated gene compared with the conventional virus integrating a reporter gene in the prior art.

[0104] Example 5: Research on the background interference problem of the reporter system

[0105] The prior art 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 (a plasmid expressing a reporter gene is not transferred into the cells, and the reporter gene is on the virus genome), and the reporter gene expression level of the blank cells after virus infection is observed, and then the replication activity of the virus is judged. Through the replication activity of the virus, the influence of the applied drug or other means on the activity of the virus can be reflected, and then the purpose of drug screening (research or screening of factors affecting virus replication) and the like can be achieved.

[0106] The inventors have also constructed EV-A71-Gluc virus expressing the Gaussia luciferase gene Gluc (the corresponding EV-A71-Gluc plasmid is integrated with Gluc gene as a reporter gene in EV-A71 virus to form a cis reporter system; the TAT gene of SEQ ID NO. 15 is replaced with Gluc gene, and the specific sequence of Gluc gene can be seen in SEQ ID NO. 16 underlined). Referring to the manner of Example 3, EV-A71-Gluc virus and EV-A71-TAT virus are used to infect the corresponding reporter cells, and the Gluc expression level is detected at different time points after infection. The experimental results are shown in Figure 12 It can be seen that the reporter system using EV-A71-Gluc virus + blank cells has very strong Gluc activity signal at 4h after infection. Because the infection time is short (4h after infection), the replication level of the virus in the cells is limited, and therefore the strength of the Gluc activity signal at this time cannot effectively reflect the replication activity of the virus. The reason why EV-A71-Gluc virus has such strong Gluc activity signal at 4h after infection is that EV-A71-Gluc virus itself has background expression of Gluc gene, which causes high background interference and masks the newly generated reporter gene signal, ultimately leading to inaccurate high-throughput screening results. The system of EV-A71-TAT virus + reporter cells using the present scheme has Gluc activity signal similar to the control group at 4h after infection, avoiding the background expression of Gluc gene (the Gluc gene in the reporter cells does not have obvious background expression; and EV-A71-TAT virus does not contain Gluc gene), eliminating the background interference problem. The experimental results show that with the passage of time, the Gluc activity signal of the reporter system of EV-A71-Gluc virus + blank cells is always significantly higher than that of the system of EV-A71-TAT virus + reporter cells, indicating that the background expression of Gluc gene seriously affects the detection accuracy and detection effect. The system of EV-A71-TAT virus + reporter cells using the present scheme can more accurately reflect the viral replication activity by detecting the Gluc activity signal, and thus realize more accurate drug screening research.

[0107] The above is only an embodiment of the present application, and the specific structure and characteristics of the scheme are not described in detail. It should be noted that for those skilled in the art, without departing from the structure of the present application, a number of modifications and improvements can be made, which should also be considered as the protection scope of the present application, which will not affect the effect and practicality of the patent. The protection scope claimed in this application should be subject to the content of its claims, and the specific implementation mode and the like recorded in the specification can be used to explain the content of the claims.

Claims

1. A multi-channel viral reporter system based on TAT transactivation, characterized by: It comprises an activating virus and a reporter cell; The activating virus is human enterovirus A71 expressing TAT protein; The reporter cell is a cell transfected with a reporter plasmid; the reporter plasmid comprises an LTR sequence and a plurality of reporter genes expressed by the LTR sequence; The activating virus is prepared by the following method: S1: constructing a viral plasmid with a sequence as shown in SEQ ID NO. 15; S2: linearizing the viral plasmid after enzyme digestion, and then obtaining viral mRNA by in vitro transcription; S3: transferring the viral mRNA into Vero cells, culturing the Vero cells, and obtaining the activating virus after cell lysis; The gene sequence of the reporter plasmid is as shown in SEQ ID NO.

16.

2. The multi-channel viral reporter system based on TAT transactivation according to claim 1, characterized in that: In S2, the viral plasmid is linearized by using Stu1 restriction endonuclease digestion, and then the linearized plasmid is in vitro transcribed into mRNA.

3. The multi-channel viral reporter system based on TAT transactivation according to claim 2, characterized in that: In S3, the mRNA formed by in vitro transcription of the linearized plasmid is added to the Vero cell suspension, and the electric transformation operation is performed under the parameter setting of voltage 250V and time 20ms; after the electric transformation is completed, the cells are further cultured, and the activating virus is obtained after cell lysis.

4. Use of the multi-channel virus reporter system based on TAT transactivation in virus detection for purposes other than treatment or diagnosis of diseases according to any one of claims 1-3, characterized in that: First, the reporter cell is infected with the activating virus; Then, the infected cells are cultured in an environment containing GCV, and the death of the reporter cells induced by the activating virus is observed; or the GFP fluorescence signal in the infected reporter cells is observed; or the Gluc content in the supernatant of the culture medium of the infected reporter cells is detected.

5. Use of a multi-channel viral reporter system based on TAT transactivation according to claim 4 for the detection of viruses not for the purpose of treatment or diagnosis of a disease, characterized in that: The MOI of the activating virus used to infect the reporter cell is 0.1-10; GCV is added to the reporter cell infected with the activating virus, and after culture, the cell survival condition is observed; The reporter cell infected with the activating virus is observed by using a fluorescence confocal microscope to excite the light channel, and the GFP fluorescence signal is observed; The culture medium supernatant of the reporter cell infected with the activating virus for 6-72h is collected; the Gaussia luciferase detection substrate is added to the supernatant, and after the luminescence signal tends to be stable, chemiluminescence detection is performed, and finally the Gluc content information is converted.

6. Use of a multi-channel viral reporter system based on TAT transactivation according to claim 5 for the detection of viruses not for the purpose of treatment or diagnosis of a disease, characterized in that: The MOI is 1.

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