Construction method of herpes simplex virus
Patent Information
- Application Number
- CN202510524506.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-24
- Publication Date
- 2025-08-22
Smart Images
Figure CN120519404A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of biotechnology, and in particular relates to a method for constructing herpes simplex virus. Background Art
[0002] Herpes simplex virus (HSV), belonging to the α-subfamily of the Herpesviridae family, is an enveloped, double-stranded DNA virus. It is divided into two serotypes, HSV-1 and HSV-2, based on antigenic differences. HSV-1 is a double-stranded DNA virus with a 152kb genome consisting of a unique long segment (UL) and a short segment (US), flanked by terminal inverted repeats (TRL) and TRS, and joined by an internal inverted repeat (IR) region. HSV-1 encodes approximately 90 proteins, most of which have well-defined functions. Approximately half are essential for viral replication, while others are non-essential. Deletion and replacement of these non-essential genes with exogenous genes does not affect viral replication, making HSV-1 amenable to viral vector development.
[0003] The HSV virus particle is about 150-200nm in diameter and consists of four parts: core, capsid, cortex and envelope. Its genome is a linear double-stranded DNA, about 152kb in length, containing at least 74 open reading frames (ORFs), encoding more than 80 proteins. A significant biological characteristic of HSV is the ability to establish a lifelong latent infection after infection. The viral genome exists in the form of a circular episome in the nucleus of neurons in the sensory ganglia. When the host's immunity declines (such as fever, fatigue, stress or immunosuppression), the latent virus can be reactivated and migrate along the nerve axons to the skin or mucosal surface, causing recurrent lesions.
[0004] In clinical applications, HSV possesses dual value due to its unique biological properties: as a pathogen requiring control and treatment, it is also being developed as a gene therapy vector and oncolytic virus platform. As a gene therapy vector, HSV offers the following advantages: a large genome that can accommodate multiple exogenous genes; its natural neurotropism, particularly suitable for gene therapy of neurological diseases; the ability to infect both dividing and quiescent cells; and its latent infection properties, enabling long-term gene expression. As an oncolytic virus, genetically modified HSV can selectively replicate and lyse tumor cells, stimulating an anti-tumor immune response while minimizing the effects on normal cells.
[0005] However, the clinical application of HSV faces many challenges, including the control of viral virulence, insufficient targeting, rapid immune clearance, and unpredictable reactivation during the latent period.
[0006] Currently, common technologies for constructing herpes simplex virus (HSV) include traditional homologous recombination, bacterial artificial chromosome systems, and bacteria-yeast shuttle vector systems. However, traditional homologous recombination suffers from extremely low efficiency, while bacterial artificial chromosome systems pose potential toxicity issues when HSV genes are expressed in bacteria, making large genomes difficult to manipulate, and the resulting rescued virus may contain unwanted bacterial sequences. Furthermore, bacteria-yeast shuttle vector systems suffer from lengthy operational procedures, high technical barriers, and the risk of genome instability. Therefore, finding an efficient HSV construction technology is crucial. Summary of the Invention
[0007] The purpose of the present invention is to provide a method for constructing herpes simplex virus, which is applicable to the construction and modification of various types of HSV viruses and improves the efficiency of herpes simplex virus construction.
[0008] The purpose of the present invention can be achieved through the following technical solutions:
[0009] A method for constructing a herpes simplex virus comprises the following steps:
[0010] S1. HSV virus plaque purification: Inoculate and culture Vero cells; infect Vero cells with HSV-1 virus; culture until clear monoclonal plaques are visible; select and mark independent plaques, inoculate Vero cells for expansion culture, and obtain virus A;
[0011] S2. CRISPR-CAS9-mediated TK gene editing: Design TK-sgRNA1 target site and TK-sgRNA2 target site, construct the two target sites into the vector, and construct the VP103 plasmid; construct the Phdr-TK (fLUC) homology repair plasmid; co-transfect the VP103 plasmid and the Phdr-TK (fLUC) homology repair plasmid into 293T cells; inoculate virus A after transfection, collect virus B, and freeze it; dilute virus B and infect Vero cells, pick single clones for culture, and extract the genome for PCR amplification after culture; run gel to screen positive clones; purify and amplify the positive clones to obtain HSV recombinant virus.
[0012] Plaque purification is a technique for isolating and purifying phages or viruses by obtaining a single plaque to ensure genetic consistency. The core steps include: diluting the phage suspension in a gradient manner (e.g., 10 -6 ~10 -8), mixed with the host bacteria, soft agar is added and spread on the surface of the solid culture medium. After cultivation, the phage lyses the host bacteria to form transparent "plaques" (1-2 mm in diameter), each of which represents a phage clone. A single plaque is then picked up with a sterile pipette tip, eluted into a buffer solution, and infected with a new host bacteria for amplification. This is repeated 2-3 times to eliminate contamination by other bacteria. This ensures that phages with a single genetic background are obtained, which can be used to study gene function or prepare vectors, and is crucial in gene editing (such as CRISPR) or phage display technology.
[0013] Furthermore, the specific steps of HSV virus plaque purification include: one day before infection, Vero cells were plated in a 6-well plate at a density of about 2-5x10^ 6 / well, the next day, the HSV-1 virus strain was diluted 10 times, and the dilution 10^ 2 -10^ 7 Vero cells were infected with the virus in a volume of 1 mL. After infection at 37°C for 6 h, the virus solution was discarded and the cells were rinsed twice with 1x PBS buffer. A 0.4% agar-medium mixture was slowly poured along the side wall of the culture plate, and 2 mL was added to each well.
[0014] Plaque purification: Place the cell culture plate in an incubator at 37°C and 5% CO2 for 3-6 days. Plaque formation can be observed. Single clones of plaques are selected and marked with a marker. Single plaques are selected and inoculated into Vero cells for expansion.
[0015] After repeating 2-3 rounds of plaque purification, the final plaque was expanded and purified to obtain virus A.
[0016] Furthermore, the specific steps of the CRISPR-CAS9-mediated TK gene editing include:
[0017] Two sgRNA targets were designed, namely TK-sgRNA1 and TK-sgRNA2; the two targets were respectively constructed into vectors to construct the VP103 plasmid; and the homology repair plasmid Phdr-TK (fLUC) was constructed;
[0018] The two constructed plasmids VP103 and Phdr-TK (fLUC) were co-transfected into 293T cells. 24 hours after transfection, virus A was infected at an MOI of 5-10. After about 24 hours of infection, the cells showed complete pathological changes, and virus B was obtained after collection. The cells were stored at -80°C.
[0019] Virus B was diluted 10-fold, and the dilution 10^ 2 -10^ 6The diluted virus was used to infect Vero cells plated in 6-well plates. After infection, 2 mL of a 0.4% agar-medium mixture was added to each well. After culturing for 3-6 days, single clones were picked and plated in 48-well plates for 3-6 days. After culturing, a portion was removed for genomic extraction and PCR amplification. The amplified fragment was 364 bp in size and spanned the TK and fLUC genes.
[0020] A single clone of HSV-1 that can amplify the exogenous fragment is selected and cultured in Vero cells. After 2-3 rounds of plaque purification, the final plaque is expanded and purified to obtain the constructed HSV recombinant virus.
[0021] Furthermore, the gel running is performed by agarose gel electrophoresis.
[0022] Furthermore, the HSV-1 virus is numbered as NCBI Reference Sequence: NC001806.2.
[0023] Furthermore, the two sgRNA targets are TK-sgRNA1 and TK-sgRNA2; the sequence of TK-sgRNA1 is shown in Seq ID No.1; the sequence of TK-sgRNA2 is shown in Seq ID No.2.
[0024] Furthermore, the vector is VP103-U6-sgRNA-U6-sgRNA-EF1-CAS9-P2A-puro; the sequence is shown in Seq ID No.3.
[0025] Furthermore, the template sequence of the Phdr-TK (fLUC) homologous repair plasmid is shown in Seq ID No.4.
[0026] Furthermore, the amplification primers are QC-FP and QC-RP; the sequence of the QC-FP is shown in Seq ID No. 5; the sequence of the QC-RP is shown in Seq ID No. 6.
[0027] Beneficial effects of the present invention:
[0028] (1) The present invention significantly improves gene editing efficiency through the synergistic effect of dual sgRNAs. Two sgRNAs (TK-sgRNA1 and TK-sgRNA2) designed for the HSV-1 thymidine kinase (TK) gene target the upstream and downstream regions of the TK gene, respectively, and introduce double-strand breaks (DSBs) at specific sites in the genome through the CRISPR-CAS9 system, greatly enhancing the probability of homology-directed repair. In addition, the long homology arms designed in the homology repair plasmid Phdr-TK (fLUC) further ensure the precise insertion of the luciferase gene (fLUC) to avoid off-target effects.
[0029] (2) The present invention ensures a high degree of consistency in the genetic background of the recombinant virus through plaque purification technology. Each round of purification starts from a monoclonal plaque to amplify the virus, effectively eliminating contamination by wild-type virus or non-recombinant virus. After the last round of purification, the sustained expression of luciferase protein is verified by Western Blot, and the stability of the TK-fLUC gene structure is confirmed in combination with sequencing. The method of the present invention is convenient and fast, and can construct a herpes simplex virus (HSV) quickly and at a low cost. This method is suitable for the construction and modification of various types of HSV viruses. BRIEF DESCRIPTION OF THE DRAWINGS
[0030] The present invention will be further described below with reference to the accompanying drawings.
[0031] Figure 1 This is a schematic diagram of the VP103-U6-sgRNA-U6-sgRNA-EF1-CAS9-P2A-puro vector map of the present invention;
[0032] Figure 2 is a schematic diagram of the Phdr-TK (fLUC) homology repair plasmid of the present invention;
[0033] Figure 3 It is a schematic diagram of the results of agarose gel electrophoresis of the present invention. DETAILED DESCRIPTION
[0034] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described 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 any creative efforts shall fall within the scope of protection of the present invention.
[0035] Obviously, the following descriptions are only some examples or embodiments of the present application. For ordinary technicians in this field, the present application can also be applied to other similar scenarios without expending creative work. In addition, it can also be understood that although the efforts made in this development process may be complex and lengthy, for ordinary technicians in the field related to the content disclosed in this application, some changes in design, manufacturing or production based on the technical content disclosed in this application are just conventional technical means and should not be understood as the content disclosed in this application is insufficient. However, there will be cases where unnecessary detailed descriptions are omitted. For example, there are cases where detailed descriptions of well-known matters and repeated descriptions of actually the same structures are omitted. This is to avoid the following description from becoming unnecessarily lengthy and to facilitate the understanding of those skilled in the art. In addition, the following description is provided for those skilled in the art to fully understand the present application and is not intended to limit the subject matter recorded in the claims.
[0036] Unless otherwise specified, all embodiments and optional embodiments of the present application can be combined with each other to form a new technical solution, and all technical features and optional technical features of the present application can be combined with each other to form a new technical solution.
[0037] Example
[0038] This embodiment provides a method for constructing herpes simplex virus, comprising the following steps:
[0039] S1. HSV virus plaque purification:
[0040] Vero cells were plated in 6-well plates at a density of 5 x 10^ 6 / well, culture for 18 hours, and make 10-fold gradient dilution of the above HSV-1 strain. 5 Vero cells were infected with the virus in an infection volume of 1 mL. After infection at 37°C for 6 h, the virus solution was discarded and the cells were rinsed twice with 1x PBS buffer. An agar-medium mixture with a final agar concentration of 0.4% was slowly poured along the side of the culture plate, and 2 mL was added to each well.
[0041] Plaque purification: Place the cell culture plate in an incubator at 37°C and 5% CO2 for 5 days. Plaque formation can be observed. Monoclonal plaques are selected and marked with a marker. Single plaques are selected and inoculated into Vero cells for expansion.
[0042] After repeating three rounds of plaque purification, the final plaque was expanded and purified to obtain the virus;
[0043] S2. Using CRISPR-CAS9 technology to modify the HSV-1 genome, a HSV-1 luciferase virus was constructed;
[0044] Two sgRNA targets were designed, namely TK-sgRNA1 and TK-sgRNA2, and the two targets were constructed into the vector VP103-U6-sgRNA-U6-sgRNA-EF1-CAS9-P2A-puro. The vector map is shown in Figure 1 As shown; VP103 plasmid was constructed; homologous repair plasmid Phdr-TK (fLUC) was constructed, and the plasmid map is shown Figure 2 As shown: the two plasmids were subjected to large-scale extraction of plasmid DNA respectively;
[0045] After culturing 293T cells for 18 hours, the density was 60%, and the two constructed plasmids VP103 and Phdr-TK (fLUC) were co-transfected into the 293T cells. 24 hours after transfection, the 293T cells were infected with the purified HSV-1 at an MOI of 10. After about 24 hours of infection, the cells showed complete pathological changes, and virus B was collected and stored at -80°C.
[0046] Virus B was diluted 10-fold, and the dilution 10^ 5 The diluted virus was used to infect Vero cells plated in 6-well plates. After infection, 2 mL of a 0.4% agar-medium mixture was added to each well. After 5 days of culture, monoclonal plaques were picked and cultured in 48-well plates.
[0047] After culture expansion, a portion was removed and separated for genomic extraction and PCR amplification. The amplified fragment spanned the TK and fLUC genes. The amplification primers were QC-FP and QC-RP. The amplified fragment size was 364 bp.
[0048] Based on efficient verification technology for specific PCR primers, recombinant virus identification can be completed within 24 hours. The designed primers QC-FP and QC-RP span the junction region of the TK gene and the fLUC gene. Only when fLUC is successfully inserted, PCR amplification can produce a specific band of 364bp. The amplified product is directly detected by agarose electrophoresis, and the success of the recombination can be preliminarily determined without sequencing. For example, in the embodiment, only one of the five monoclonal plaques showed the target band after PCR screening, with a false positive rate of less than 20%, significantly shortening the screening cycle.
[0049] After PCR amplification, the PCR product was subjected to agarose gel electrophoresis experiment, and the agarose gel electrophoresis result was obtained as shown in the following figure: Figure 3 As shown;
[0050] Agarose gel electrophoresis is a separation technique based on differences in nucleic acid molecular weight. It is particularly suitable for rapid verification of luciferase gene (fLUC) insertion in recombinant HSV-1 viruses following CRISPR-CAS9 editing. The method first requires preparation of a 1.5% agarose gel. Agarose powder is dissolved in 1× TAE buffer by microwave heating and then a nucleic acid dye is added after cooling to 50-60°C. This solution is poured into a gel casting tank and a comb is inserted to form sample wells. The wells are then allowed to solidify at room temperature for 20-30 minutes. During sample preparation, the PCR product is mixed with 6× DNA loading buffer and, if necessary, a DNA molecular weight standard is added as a quantitative reference. The total sample volume per well should not exceed 10 μL to avoid cross-contamination. Electrophoresis is performed at a constant voltage of 120 V for 30-40 minutes, until the bromophenol blue indicator migrates to the two-thirds mark of the gel. Electrophoresis is stopped when the target band (364 bp) is clearly separated. Results were analyzed using a UV gel imager, with the gel viewed at an excitation wavelength of 300 nm and an emission wavelength of 590 nm. If a chromogenic dye such as EB was used, observation should be performed under a UV transilluminator. A positive result is indicated by the presence of a single bright band at the 364 bp position, while a negative result indicates the absence of a band or the presence of nonspecific bands.
[0051] Of the five monoclonal clones in this round of screening, only one was able to amplify the target fragment, as shown in the gel electrophoresis diagram, indicating that this monoclonal plaque was a successfully recombinant HSV virus. This monoclonal plaque was expanded and cultured in Vero cells; after three rounds of plaque purification, the final plaque was expanded and purified to obtain the constructed HSV recombinant virus.
[0052] The obtained labeled viruses play an important role and significance in the study of HSV-1 infection and latent sites.
[0053] It should be noted that, in this document, relational terms such as first and second, etc., are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the terms "comprises," "comprising," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a list of elements includes not only those elements but also other elements not explicitly listed, or elements inherent to such process, method, article, or apparatus.
[0054] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.
Claims
1. A method for constructing a herpes simplex virus, characterized in that: The specific steps include: S1. HSV virus plaque purification: HSV-1 virus was infected into Vero cells; independent plaques were selected and labeled, and inoculated into Vero cells for expansion culture to obtain virus A; S2. CRISPR-CAS9-mediated TK gene editing: construct VP103 plasmid; construct Phdr-TK (fLUC) homologous repair plasmid; co-transfect VP103 plasmid and Phdr-TK (fLUC) homologous repair plasmid into 293T cells; inoculate virus A after transfection and collect virus B; dilute virus B and infect Vero cells, pick single clones for culture, and extract the genome for amplification after culture; screen positive clones; purify and amplify positive clones to obtain HSV recombinant virus.
2. The method for constructing a herpes simplex virus according to claim 1, wherein: The HSV virus plaque purification specifically comprises the following steps: The specific steps of HSV virus plaque purification include: one day before infection, Vero cells were plated in a 6-well plate at a density of about 2-5×10 ^6 / well, the next day, the HSV-1 virus strain was diluted 10 times, and the dilution 10 ^2 -10 ^7 Vero cells were infected with the virus in a volume of 1 mL. After 6 h of infection at 37°C, the virus solution was discarded and the cells were rinsed twice with 1x PBS buffer. A 0.4% agar-medium mixture was slowly poured along the side of the culture plate, and 2 mL was added to each well. Plaque purification: Place the cell culture plate in an incubator at 37°C and 5% CO2 for 3-6 days. Plaque formation can be observed. Single clones of plaques are selected and marked with a marker. Single plaques are selected and inoculated into Vero cells for expansion. After repeating 2-3 rounds of plaque purification, the final plaque was expanded and purified to obtain virus A.
3. The method for constructing a herpes simplex virus according to claim 1, wherein: The construction steps of the VP103 plasmid include: Design the TK-sgRNA1 target site and the TK-sgRNA2 target site, and construct the two target sites into the vector.
4. The method for constructing a herpes simplex virus according to claim 1, wherein: The HSV-1 virus is numbered as NCBI Reference Sequence: NC001806.
2.
5. The method for constructing a herpes simplex virus according to claim 1, wherein: The specific steps of CRISPR-CAS9-mediated TK gene editing include: Two sgRNA targets were designed, namely TK-sgRNA1 and TK-sgRNA2; the two targets were respectively constructed into vectors to construct the VP103 plasmid; and the homology repair plasmid Phdr-TK (fLUC) was constructed; The two constructed plasmids VP103 and Phdr-TK (fLUC) were co-transfected into 293T cells. 24 hours after transfection, virus A was infected at an MOI of 5-10. After about 24 hours of infection, the cells showed complete pathological changes, and virus B was obtained after collection. The cells were stored at -80°C. Virus B was diluted 10-fold. ^2 -10 ^6 The diluted virus was used to infect Vero cells plated in 6-well plates. After infection, 2 mL of a 0.4% agar-medium mixture was added to each well. After culturing for 3-6 days, single clones were picked and plated in 48-well plates for 3-6 days. After culturing, a portion was removed for genomic extraction and PCR amplification. The amplified fragment was 364 bp in size and spanned the TK and fLUC genes. Screen positive clones; select a single clone that can amplify the exogenous fragment to construct HSV-1, and expand and culture it in Vero cells; and after 2-3 rounds of plaque purification, expand and purify the final plaque to obtain the constructed HSV recombinant virus.
6. The method for constructing a herpes simplex virus according to claim 5, wherein: The sequence of the TK-sgRNA1 is shown in Seq ID No. 1; the sequence of the TK-sgRNA2 is shown in Seq ID No.
2.
7. The method for constructing a herpes simplex virus according to claim 5, wherein: The vector is VP103-U6-sgRNA-U6-sgRNA-EF1-CAS9-P2A-puro; the sequence is shown in Seq ID No.
3.
8. The method for constructing a herpes simplex virus according to claim 1, wherein: The template sequence of the Phdr-TK (fLUC) homologous repair plasmid is shown in Seq ID No.
4.
9. The method for constructing a herpes simplex virus according to claim 1, wherein: The primers for the amplification are QC-FP and QC-RP; The sequence of the QC-FP is shown in Seq ID No. 5; the sequence of the QC-RP is shown in Seq ID No.
6.
10. The method for constructing a herpes simplex virus according to claim 1, wherein: The method for screening positive clones is agarose gel electrophoresis.