Virus protein dynamic monitoring system and method based on fluorescent gene editing technology

Abstract

The invention discloses a virus protein dynamic monitoring system and method based on a fluorescent gene editing technology, and relates to the technical field of fluorescent tracer, the system comprises a gene editing module, a fluorescent gene and a Tax response promoter are used for constructing a co-transcription unit by using CRISPR / Cas9, the co-transcription unit is inserted into a virus genome non-functional region through homologous recombination repair, and the gene editing module is used for editing the gene. Subculturing and screening a stable cell line with the fluorescence positive rate reaching the standard; the signal response module is used for realizing synchronous expression of fluorescent protein and Tax through a co-transcription mechanism; the cell carrier module simulates infection through co-culture; the detection and analysis module quantifies the fluorescence intensity and the number of fluorescent cells through a fluorescence microscope and a flow cytometer, the detection period is shortened, the detection efficiency is improved, the system does not need antibiotic treatment, and Tax dynamics can be monitored in real time.

Description

Technical Field

[0001] The present invention relates to the field of fluorescent tracing technology, and in particular to a viral protein dynamic monitoring system and method based on fluorescent gene editing technology. Background Art

[0002] Dynamic monitoring of viral proteins is a core component of virology research and antiviral drug development. Traditional viral model protein detection processes are cumbersome, requiring multiple steps including monoclonal cloning, flow cytometry, and antibiotic treatment to identify target cells for testing. This approach precludes real-time monitoring, and results in long detection cycles and low sensitivity. Therefore, the development of a system and method for dynamic viral protein monitoring based on fluorescent gene editing technology is crucial.

[0003] The existing technology also has the following defects, which are specifically reflected in: 1. In the existing technology, the detection cycle is long and the operation is cumbersome. It relies on complex processes such as multiple rounds of cell lysis and antibody incubation, and requires multiple operations such as monoclonal screening, flow cytometry, and antibiotic treatment. The experimental cost is high and the efficiency is low. It is easy to introduce errors due to manual operation, resulting in the inability to quickly obtain experimental data in virological research, especially in the screening of antiviral drugs, which greatly delays the drug development cycle.

[0004] 2. The existing technology can only provide static detection results of the viral protein Tax, and cannot track its dynamic changes such as nucleocytoplasmic shuttling in real time. It is also difficult to capture the low-expression Tax protein in the early stage of infection, resulting in the inability to fully reflect the entire infection process of the HTLV-1 virus, which limits the research on the mechanism of action and rapid evaluation of antiviral drugs. Summary of the Invention

[0005] The purpose of the present invention is to provide a viral protein dynamic monitoring system and method based on fluorescent gene editing technology, which solves the problems existing in the background technology.

[0006] To solve the above technical problems, the present invention adopts the following technical solution: The present invention provides a viral protein dynamic monitoring system based on fluorescent gene editing technology, including: a gene editing module: used to insert a fluorescent gene sequence into the non-functional region of the HTLV-1 viral genome through the CRISPR / Cas9 system, the fluorescent gene sequence is connected to the viral promoter activated by the Tax protein to form a co-transcription unit, which is transferred into the recipient cells through the viral genome, and cells that stably express the fluorescent protein are screened by subculture.

[0007] It should be noted that the CRISPR / Cas9 system is a powerful gene editing technology derived from the adaptive immune system of bacteria and archaea, and is used to resist the invasion of foreign viruses or plasmids. CRISPR, or clustered regularly interspaced short palindromic repeats, is a special DNA sequence composed of numerous short repeat sequences and spacer sequences. The spacer sequences usually come from DNA fragments of viruses or plasmids that have invaded bacteria. Cas9 is a nuclease, the full name of which is CRISPR-associated protein 9. Under the guidance of guide RNA (gRNA), it can recognize and bind to a specific DNA sequence, and then cut the DNA sequence to cause double-strand breaks.

[0008] It should also be noted that the fluorescent gene sequence is inserted into the non-functional region of the HTLV-1 viral genome by the CRISPR / Cas9 system. The specific implementation method is as follows: first, the non-functional region in the HTLV-1 viral genome needs to be determined as the insertion site. According to the selected insertion site, a gRNA sequence complementary to it is designed. The gene encoding the Cas9 protein is cloned into a suitable expression vector. The designed gRNA sequence is cloned into another expression vector. A promoter is also required to drive the expression of the gRNA. A donor vector containing the fluorescent gene sequence is constructed, which also needs to contain a promoter that is complementary to the insertion site. Homologous DNA sequences on both sides of the dot are inserted. A cell line that can be infected by the HTLV-1 virus is selected as the recipient cell. Using an appropriate transfection method, such as lipofection or electroporation, the Cas9 expression vector, gRNA expression vector, and fluorescent gene donor vector are introduced into the recipient cell. Once inside the cell, the Cas9 protein binds to the gRNA to form a complex. The gRNA guides this complex to recognize and bind to the target insertion site of the HTLV-1 viral genome. The Cas9 protein cuts the DNA at this site, causing a double-strand break. When a DNA double-strand break occurs, the cell activates its own DNA repair mechanism. At this point, the fluorescent gene sequence on the fluorescent gene donor vector is integrated into the broken DNA site through homologous recombination, thereby achieving site-specific insertion of the fluorescent gene sequence.

[0009] Signal response module: used for the co-transcription unit to drive the co-transcription of the fluorescent gene and the viral gene to express the self-luminous fluorescent protein when the Tax protein binds to the promoter, thereby realizing real-time tracking of the Tax protein expression by the fluorescent signal.

[0010] Cell vector module: comprising a recipient cell integrated with the co-transcription unit, and used for constructing an HTLV-1 virus infection model.

[0011] Detection and analysis module: used to detect fluorescence intensity through an inverted fluorescence microscope and the number of fluorescent cells through a flow cytometer, and to quantify Tax protein expression and virus infection efficiency respectively.

[0012] Preferably, the gene editing module achieves fluorescent gene integration through homologous recombination repair strategy, and the insertion site does not affect the nucleocytoplasmic shuttling function of the Tax protein.

[0013] Preferably, the fluorescent gene is a self-luminous fluorescent protein gene, and the promoter is the HTLV-1 virus pX promoter, which drives the co-transcription of the fluorescent gene and the viral gene after activation.

[0014] Preferably, the cells stably expressing fluorescent protein are screened out by subculture, and the specific implementation method is: after using CRISPR / Cas9 to transfer the co-transcription unit into the recipient cells, the cells are diluted to a single cell density and plated, and observed under an inverted fluorescence microscope, monoclonal cells are selected for expansion culture, and after continuous subculture, the consistency of fluorescence expression of each generation of cells is detected, and cell lines with a fluorescence positivity rate higher than a preset threshold and a stable signal intensity are retained, and stable clones can be obtained without antibiotic treatment.

[0015] Preferably, the fluorescence intensity is linearly positively correlated with the Tax protein concentration, and the number of fluorescent cells is positively correlated with the virus infection efficiency.

[0016] The second aspect of the present invention includes a method for a viral protein dynamic monitoring system based on fluorescent gene editing technology, comprising: step 1, gene editing: using the CRISPR / Cas9 system to insert a fluorescent gene sequence into a non-functional region of the HTLV-1 viral genome, the fluorescent gene sequence is connected to a viral promoter activated by the Tax protein to form a co-transcription unit, which is transferred into recipient cells through the viral genome, and cells that stably express fluorescent protein are screened by subculture.

[0017] Step 2: Signal response: When the Tax protein binds to the promoter, the co-transcription unit drives the fluorescent gene and the viral gene to co-transcribe and express the self-luminous fluorescent protein, thereby achieving real-time tracking of the Tax protein expression by the fluorescent signal.

[0018] Step 3: Cell vector: comprising a recipient cell integrated with the co-transcription unit, for constructing an HTLV-1 virus infection model.

[0019] Step 4. Detection and analysis: Fluorescence intensity was detected by inverted fluorescence microscopy and the number of fluorescent cells was detected by flow cytometry to quantify Tax protein expression and virus infection efficiency, respectively.

[0020] The beneficial effects of the present invention are: 1. In the present invention, the CRISPR / Cas9 system is combined with the homologous recombination repair strategy to site-specifically insert the fluorescent gene into the non-functional region of the viral genome, thereby improving the integration success rate. No multiple rounds of monoclonal screening and antibiotic treatment are required, and a stable cell line can be obtained directly through subculture; abandoning the traditional cumbersome process and using direct detection of fluorescent signals, the detection cycle is shortened, the detection efficiency is improved, the reagent consumption and manual errors are reduced, and the antiviral drug screening process is accelerated.

[0021] 2. In the present invention, through the design of a "co-transcription-autoluminescence" system, the fluorescent gene and the viral gene are synchronously transcribed when the Tax protein activates the promoter, and the nucleocytoplasmic shuttling dynamics of the Tax protein and its low-abundance expression in the early stage of infection are captured in real time; the fluorescence intensity and the proportion of fluorescent cells are directly quantified through an inverted fluorescence microscope and flow cytometer, realizing dynamic tracking of the entire process from the early stage of viral infection to the replication stage, providing real-time multi-dimensional data support for the study of drug action mechanisms. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0023] Figure 1 This is a schematic diagram of the system structure connection of the present invention.

[0024] Figure 2 The figure is a flow chart of the steps for implementing the method of the present invention.

[0025] Figure 3 Schematic diagram of an embodiment of the present invention. DETAILED DESCRIPTION

[0026] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0027] Reference Figure 1As shown, the present invention provides a viral protein dynamic monitoring system based on fluorescent gene editing technology, including: a gene editing module: used to insert a fluorescent gene sequence into a non-functional region of the HTLV-1 viral genome through the CRISPR / Cas9 system, the fluorescent gene sequence is connected to a viral promoter activated by the Tax protein to form a co-transcription unit, which is transferred into recipient cells through the viral genome, and cells that stably express fluorescent protein are screened by subculture.

[0028] In the present invention, the CRISPR / Cas9 system is combined with the homologous recombination repair strategy to site-specifically insert the fluorescent gene into the non-functional region of the viral genome, thereby improving the integration success rate. There is no need for multiple rounds of monoclonal screening and antibiotic treatment, and stable cell lines can be obtained directly through subculture. Abandoning the traditional cumbersome process, direct detection using fluorescent signals shortens the detection cycle, improves detection efficiency, reduces reagent consumption and human errors, and accelerates the antiviral drug screening process.

[0029] It should be noted that the non-functional region of the viral genome, in the embodiment, specifically includes: the spacer region between the env and gag genes; the co-transcription unit, which activates RNA polymerase II only when the Tax protein binds, driving the co-transcription of the fluorescent gene and the viral gene.

[0030] In a specific embodiment, the gene editing module achieves fluorescent gene integration through a homologous recombination repair strategy, and the insertion site does not affect the nucleocytoplasmic shuttling function of the Tax protein.

[0031] In a specific embodiment, the fluorescent gene is a self-luminous fluorescent protein gene, and the promoter is the HTLV-1 virus pX promoter, which drives the co-transcription of the fluorescent gene and the viral gene after activation.

[0032] In the present invention, through the design of a "co-transcription-autoluminescence" system, the fluorescent gene and the viral gene are synchronously transcribed when the Tax protein activates the promoter, and the nucleocytoplasmic shuttling dynamics of the Tax protein and the low-abundance expression in the early stage of infection are captured in real time; the fluorescence intensity and the proportion of fluorescent cells are directly quantified through an inverted fluorescence microscope and a flow cytometer, realizing dynamic tracking of the entire process from the early stage of viral infection to the replication stage, providing real-time multi-dimensional data support for the study of drug action mechanisms.

[0033] It should be noted that the fluorescent protein spontaneously generates fluorescence through an enzymatic reaction, specifically in the embodiment: NanoLuciferase catalyzes the substrate to produce bioluminescence; the fluorescent gene mRNA and the Tax protein mRNA share the same transcription start site;

[0034] In a specific embodiment, the cells stably expressing fluorescent protein are screened out by subculture, and the specific implementation method is: after using CRISPR / Cas9 to transfer the co-transcription unit into the recipient cells, the cells are diluted to a single cell density and plated, and observed under an inverted fluorescence microscope, monoclonal cells are selected for expansion and culture, and after continuous subculture, the consistency of fluorescence expression of each generation of cells is detected, and cell lines with a fluorescence positivity rate higher than a preset threshold and a stable signal intensity are retained, and stable clones can be obtained without antibiotic treatment.

[0035] It should be noted that the recipient cells in the embodiment are specifically: JETWT35 cells (HTLV-1 susceptible cells) or ATL-T cells (HTLV-1 immortalized cells); the continuous passage is generally 5-10 generations in the embodiment; and screening is performed by the puromycin resistance gene in the embodiment.

[0036] Signal response module: used for the co-transcription unit to drive the co-transcription of the fluorescent gene and the viral gene to express the self-luminous fluorescent protein when the Tax protein binds to the promoter, thereby realizing real-time tracking of the Tax protein expression by the fluorescent signal.

[0037] Cell vector module: comprising a recipient cell integrated with the co-transcription unit, and used for constructing an HTLV-1 virus infection model.

[0038] It should be noted that the HTLV-1 virus infection model is constructed. In the embodiment, the intercellular transmission process of the virus is simulated by a co-culture system. The co-culture is a 1:3 mixture of ATL-T and JETWT35 cells. The drugs to be screened are added at 0h (before infection) or 12h (after infection) of the co-culture, and the drugs with the inhibition rate reaching the standard are screened out by detecting the change in fluorescence intensity. The drugs include FDA-approved drugs or natural product compounds. According to the correlation between the drug treatment time and the fluorescence inhibition effect, the drug mechanism of action is judged. If it is effective when added before infection, the drug inhibits the viral infection process. If it is effective when added after infection, the drug inhibits Tax protein expression or cell proliferation.

[0039] Detection and analysis module: used to detect fluorescence intensity through an inverted fluorescence microscope and the number of fluorescent cells through a flow cytometer, and to quantify Tax protein expression and virus infection efficiency respectively.

[0040] In a specific embodiment, the fluorescence intensity is linearly positively correlated with the Tax protein concentration, and the number of fluorescent cells is positively correlated with the virus infection efficiency.

[0041] Reference Figure 2As shown, a method for implementing the viral protein dynamic monitoring system of the gene editing technology described in the present invention includes: Step 1, gene editing: using the CRISPR / Cas9 system to insert a fluorescent gene sequence into the non-functional region of the HTLV-1 viral genome, the fluorescent gene sequence is connected to the viral promoter activated by the Tax protein to form a co-transcription unit, which is transferred into the recipient cells through the viral genome, and cells stably expressing the fluorescent protein are screened by subculture.

[0042] Step 2: Signal response: When the Tax protein binds to the promoter, the co-transcription unit drives the fluorescent gene and the viral gene to co-transcribe and express the self-luminous fluorescent protein, thereby achieving real-time tracking of the Tax protein expression by the fluorescent signal.

[0043] Step 3: Cell vector: comprising a recipient cell integrated with the co-transcription unit, for constructing an HTLV-1 virus infection model.

[0044] Step 4. Detection and analysis: Fluorescence intensity was detected by inverted fluorescence microscopy and the number of fluorescent cells was detected by flow cytometry to quantify Tax protein expression and virus infection efficiency, respectively.

[0045] In the embodiment, five effective drugs against HTLV-1 virus were screened through this model, and the effects of nearly 100 drugs in the two drug libraries of FDA-ApprovedDrug and NaturaProduct on HTLV-1 virus infection were detected. First, ATL-T and JETWT35 were co-cultured at a ratio of 1:3, and the cells were treated for 48 hours in two ways: "adding drugs after co-culture 0 hours (i.e., adding drugs immediately after co-culture)" and "adding drugs after co-culture 12 hours (i.e., after HTLV-1 infection is basically completed)". Figure 3 , then used an inverted fluorescence microscope to observe the expression of fluorescent protein in JETWT35 cells. Using this method, five compounds (Ritonavir, MK2048, Lopinavir, GSK1349572, and GSK1349572A) that can significantly inhibit the HTLV-1 virus were screened. Adding Ritonavir, MK2048, Lopinavir, GSK1349572, or GSK1349572A after 0 hours of co-culture all inhibited the expression of red fluorescent protein in JETWT35 cells to varying degrees. However, adding the drugs after 12 hours of co-culture had no significant effect on the expression of red fluorescent protein, except for the Lopinavir group. This suggests that Ritonavir, MK2048, GSK1349572, and GSK1349572A inhibit cell-to-cell contact-mediated HTLV-1 infection, while Lopinavir may have a certain inhibitory effect on the expression of the viral protein Tax or cell proliferation.

[0046] The above contents are merely examples and explanations of the concept of the present invention. Those skilled in the art may make various modifications or additions to the described specific embodiments or replace them in a similar manner. As long as they do not deviate from the concept of the invention or exceed the scope defined by the present invention, they should all fall within the scope of protection of the present invention.

Claims

1. A viral protein dynamic monitoring system based on fluorescent gene editing technology, characterized by: include: Gene editing module: used to insert a fluorescent gene sequence into the non-functional region of the HTLV-1 viral genome using the CRISPR / Cas9 system. The fluorescent gene sequence is connected to the viral promoter activated by the Tax protein to form a co-transcription unit. The viral genome is then transferred into recipient cells, and cells that stably express the fluorescent protein are screened through subculture. Signal response module: used for the co-transcription unit to drive the co-transcription of the fluorescent gene and the viral gene to express the self-luminous fluorescent protein when the Tax protein binds to the promoter, thereby achieving real-time tracking of the Tax protein expression by the fluorescent signal; Cell vector module: comprising a recipient cell integrated with the co-transcription unit, used for constructing an HTLV-1 virus infection model; Detection and analysis module: used to detect fluorescence intensity through an inverted fluorescence microscope and the number of fluorescent cells through a flow cytometer, and to quantify Tax protein expression and virus infection efficiency respectively.

2. The viral protein dynamic monitoring system based on fluorescence gene editing technology according to claim 1 is characterized in that: The gene editing module achieves fluorescent gene integration through a homologous recombination repair strategy, and the insertion site does not affect the nucleocytoplasmic shuttling function of the Tax protein.

3. The viral protein dynamic monitoring system based on fluorescence gene editing technology according to claim 1 is characterized in that: The fluorescent gene is a self-luminous fluorescent protein gene, and the promoter is the HTLV-1 virus pX promoter, which drives the co-transcription of the fluorescent gene and the viral gene after activation.

4. The viral protein dynamic monitoring system based on fluorescence gene editing technology according to claim 1 is characterized in that: The cells stably expressing the fluorescent protein are screened out through subculture, and the specific implementation method is as follows: After using CRISPR / Cas9 to transfer the co-transcription unit into the recipient cells, the cells are diluted to a single cell density and plated. Under inverted fluorescence microscopy, single clones are selected for expansion and culture. After continuous passaging, the consistency of fluorescence expression of each generation of cells is tested. Cell lines with a fluorescence positivity rate higher than the preset threshold and stable signal intensity are retained. Stable clones can be obtained without antibiotic treatment.

5. The viral protein dynamic monitoring system based on fluorescence gene editing technology according to claim 1 is characterized in that: The fluorescence intensity is linearly positively correlated with the Tax protein concentration, and the number of fluorescent cells is positively correlated with the virus infection efficiency.

6. A method for implementing the viral protein dynamic monitoring system based on fluorescence gene editing technology according to any one of claims 1 to 5, characterized in that: include: Step 1: Gene editing: The fluorescent gene sequence is inserted into the non-functional region of the HTLV-1 viral genome using the CRISPR / Cas9 system. The fluorescent gene sequence is connected to the viral promoter activated by the Tax protein to form a co-transcription unit. The viral genome is then transferred into the recipient cells, and cells that stably express the fluorescent protein are screened by subculture. Step 2: Signal response: When the Tax protein binds to the promoter, the co-transcription unit drives the fluorescent gene and the viral gene to co-transcribe and express the self-luminescent fluorescent protein, realizing real-time tracking of Tax protein expression by the fluorescent signal; Step 3: Cell vector: comprising a recipient cell integrated with the co-transcription unit, for constructing an HTLV-1 virus infection model; Step 4. Detection and analysis: Fluorescence intensity was detected by inverted fluorescence microscopy and the number of fluorescent cells was detected by flow cytometry to quantify Tax protein expression and virus infection efficiency, respectively.

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