Method for preparing pseudovirus of monkey B virus by lentiviral vector system and application of pseudovirus

Abstract

The invention discloses a method for preparing a simian B virus pseudovirus through a lentiviral vector system and application, and relates to the technical field of biology.The method comprises the following specific steps that a modified lentiviral vector is constructed, and a gag gene specific base sequence in psPAX2 plasmids is modified through a site-specific mutagenesis technology; constructing a lentiviral vector for optimizing infection characteristics and expression efficiency; the lentivirus packaging plasmids are precisely modified, so that interspecific obstacles of a traditional human immunodeficiency virus (HIV) lentivirus system are broken through, the prepared pseudovirus has double infection capacity on monkey cells and human cells, the production efficiency and safety of the pseudovirus are improved, and the pseudovirus has good application prospects. The technical scheme is combined with pseudoviruses packaged after replacing VSV-G genes in pCMV-VSV-G plasmids with monkey B virus glycoprotein, the natural infection process of the monkey B virus can be highly simulated, and a safe and reliable technical platform is provided for virus pathogenic mechanism research, neutralizing antibody evaluation, vaccine research and development and antiviral drug screening.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method and application of preparing monkey B virus pseudoviruses using a lentiviral vector system. Background Art

[0002] Macaque herpesvirus type 1, commonly known as simian B virus (BV), is a pathogen that is widely spread in macaque populations. Its natural hosts are mainly rhesus macaques and crab-eating macaques. Macaque BV infection usually manifests as asymptomatic or mild herpes. BV is neurotoxic. Although B virus infection of the central nervous system is extremely rare in natural hosts and is usually associated with immunosuppression or other complications, as a zoonotic pathogen, after infecting humans, if not treated in time, the B virus will replicate in cells at the infected site and spread to the central nervous system through sensory ganglia, causing neurological symptoms or fatal progressive encephalomyelitis, with a mortality rate of up to 80%. Therefore, BV has become an important pathogen that macaque breeders and experimentalists need to pay attention to.

[0003] In the field of virology research, pseudovirus systems serve as an important research tool, effectively simulating the viral infection process while greatly reducing the biosafety risks associated with the use of live viruses. Currently, commonly used pseudovirus systems include HIV-1-based lentivirus systems, vesicular stomatitis virus systems, murine leukemia virus systems, and systems modified from adenoviruses and herpes simplex viruses. However, each of these systems has significant limitations. For example, lentivirus systems have species-restricted infection issues, making it difficult to simultaneously meet the research needs of monkey and human cells. VSV systems may exhibit nonspecific infection, affecting the accuracy of experimental results. MLV systems can only infect dividing cells, limiting their scope of application. Adenovirus systems are prone to triggering immune responses, interfering with studies of viral infection mechanisms. HSV systems are relatively complex to operate and may produce cytotoxicity, increasing the difficulty and risk of experiments. These shortcomings make it difficult for traditional pseudovirus systems to play an ideal role in simian B virus research and cannot meet the needs of in-depth research on this virus. It is urgent to develop a more efficient, safe, and widely applicable pseudovirus preparation method. Summary of the Invention

[0004] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a method and application for preparing monkey B virus using a lentiviral vector system. The method can precisely modify the gag gene sequence in the psPAX2 plasmid by implementing site-directed modification based on a lentiviral packaging plasmid routinely used in the laboratory, thereby constructing a lentiviral vector with optimized infection characteristics and higher pseudovirus expression efficiency. At the same time, suitable transfer plasmids, packaging plasmids, and envelope plasmids are selected, and the CDS region sequence of the monkey B virus glycoprotein is obtained by artificial synthesis and recombined into the envelope plasmid to construct multiple recombinant plasmids. During the pseudovirus packaging process, co-transfection operations are carried out strictly according to specific experimental groupings and plasmid dosages. The culture supernatant is collected, concentrated, and stored, and the titer of the pseudovirus is accurately determined by real-time fluorescence quantitative PCR reaction. This breaks the interspecies barrier existing in traditional HIV lentiviral systems, enables the prepared pseudovirus to have the ability to dually infect monkey and human cells, and provides strong technical support for the research of monkey B virus and its application in related fields.

[0005] To solve the above technical problems, the present invention provides the following technical solutions: On the one hand, a method for preparing simian B virus pseudoviruses through a lentiviral vector system, the method comprising the following specific steps:

[0006] Construction of modified lentiviral vectors: Using site-directed mutagenesis to modify the specific base sequence of the gag gene in the psPAX2 plasmid, a lentiviral vector with optimized infection characteristics and expression efficiency was constructed;

[0007] Prepare relevant plasmids: Select specific transfer, packaging, and envelope plasmids, synthesize the simian B virus glycoprotein gene and recombinant it into the envelope plasmid. After transformation into competent cells, use the corresponding kit to extract and test the concentration and purity;

[0008] Pseudovirus packaging: After replacing the culture medium of cells in a specific growth state, the relevant plasmids were co-transfected according to the calcium phosphate method according to different groups. After 18 hours, the culture medium was replaced with serum and the supernatant was collected 24 and 48 hours later.

[0009] Pseudovirus concentration and storage: The collected culture supernatant was centrifuged and filtered, concentrated with PEG 6000 and NaCl, and allowed to stand overnight at 4°C. After high-speed centrifugation, the precipitate was resuspended in DPBS and stored in aliquots at -70°C.

[0010] Pseudovirus titer determination: Take concentrated pseudovirus samples to extract nucleic acid, digest, elute, reverse transcribe to cDNA, and react with specific PCR mixture. The titer is determined using a Bio-Rad CFX96 PCR instrument.

[0011] Furthermore, in the step of preparing relevant plasmids, pLVX-mCMV-ZsGreen1-Puro vector and pLVX-Luc2-P2A-MCS-puro were selected as transfer plasmids; the packaging plasmids included the original pSPAX2 and the modified psPAX2-Mutant; and the envelope plasmid was selected as pCMV-VSV-G.

[0012] Furthermore, in the step of preparing the relevant plasmids, the corresponding sequences were obtained by artificial synthesis based on the CDS region sequences of the simian B virus glycoproteins gB, gD, gH and gL in Genbank, and recombined into the envelope plasmid pCMV-VSV-G vector to construct four recombinant plasmids, pCMV-VSV-ΔG-BVgB, pCMV-VSV-ΔG-BVgD, pCMV-VSV-ΔG-BVgH and pCMV-VSV-ΔG-BVgL, respectively. Each plasmid was transformed into competent Escherichia coli DH5α cells, and the extraction operation was performed using the TIANpure Mini plasmid kit, ExdoFree Midi plasmid kit or EndoFreeMaxi plasmid kit according to the subsequent use of the plasmid.

[0013] Furthermore, in the pseudovirus packaging step, HEK293T cells were seeded into a 15 cm culture dish one day before virus packaging. When the cell confluence reached 70% the next day, the culture medium was replaced with serum-free high-glucose DMEM medium. According to different experimental groups, the envelope plasmids, packaging plasmids and reporter plasmids before and after transformation were co-transfected according to the calcium phosphate method. After 18 hours of co-transfection, the culture medium was replaced with high-glucose DMEM medium containing 10% fetal bovine serum, and the culture supernatant was collected 24 hours and 48 hours later.

[0014] Furthermore, in the pseudovirus packaging step, according to different experimental groups, the envelope plasmids, packaging plasmids and reporter plasmids before and after transformation were co-transfected according to the calcium phosphate method. The specific groups are:

[0015] LV-Mutant pseudovirus group: 17 μg pCMV-VSV-G, 40 μg pLVX-mCMV-ZsGreen1-Puro or pLVX-Luc2-P2A-MCS-puro, and 34 μg psPAX2-Mutant were used per dish;

[0016] LV-Wild-Type pseudovirus group: 17 μg pCMV-VSV-G, 40 μg pLVX-mCMV-ZsGreen1-Puro or pLVX-Luc2-P2A-MCS-puro, and 34 μg psPAX2 were used per dish;

[0017] PV-M-BV-BDHL pseudovirus group: 40 μg pLVX-mCMV-ZsGreen1-Puro, 34 μg psPAX2-Mutant, 20 μg pCMV-VSV-ΔG-BVgB, 16 μg pCMV-VSV-ΔG-BVgD, 15 μg pCMV-VSV-ΔG-BVgH, and 15 μg pCMV-VSV-ΔG-BVgL were used per dish;

[0018] PV-WT-BV-BDHL pseudovirus group: 40 μg pLVX-mCMV-ZsGreen1-Puro, 34 μg psPAX2, 20 μg pCMV-VSV-ΔG-BVgB, 16 μg pCMV-VSV-ΔG-BVgD, 15 μg pCMV-VSV-ΔG-BVgH and 15 μg pCMV-VSV-ΔG-BVgL were used per dish.

[0019] Furthermore, in the pseudovirus concentration and preservation step, the collected culture supernatant was first centrifuged at 500g for 5 minutes to remove cell debris, filtered through a 0.45μm filter, and the lentivirus was concentrated using a PEG 6000 solution to achieve final concentrations of PEG 6000 and NaCl of 8.5% and 0.3M, respectively. The mixed solution was allowed to stand overnight at 4°C.

[0020] Furthermore, in the pseudovirus concentration and storage step, after high-speed centrifugation, the precipitate is resuspended in DPBS and stored in aliquots at -70°C. Specifically, a high-speed centrifuge is used to centrifuge at 7000g for 10 minutes at 4°C. After the centrifugation, the supernatant is discarded, the precipitate is resuspended in DPBS, the vector suspension is aliquoted, and stored in a -70°C environment for future use.

[0021] Furthermore, in the pseudovirus titer determination step, 5 μL of the concentrated pseudovirus sample was taken, and the nucleic acid extraction operation was performed according to the steps specified in the RNA extraction kit instructions. DNase I was added for digestion, and RNA was eluted with 100 μL of RNase-free water. 10 μL of RNA sample was taken and reverse transcribed into cDNA using random primers and reverse transcriptase M-MLV.

[0022] Furthermore, in the pseudovirus titer determination step, random primers and reverse transcriptase M-MLV are used to reverse transcribe it into cDNA. Specifically, 5 μL of cDNA is thoroughly mixed with 15 μL of PCR mixture, wherein the PCR mixture contains 10 μL of 2×Probe Premix Ex Taq, 0.4 μL of a forward primer with a concentration of 20 μM, 0.4 μL of a reverse primer with a concentration of 20 μM, 0.4 μL of a probe with a concentration of 20 μM, and 3.8 μL of DNase / RNase-free distilled water. The reaction program is set as follows: pre-denaturation at 95°C for 30 seconds, 40 cycles, each cycle including denaturation at 95°C for 5 seconds and annealing and extension at 60°C for 30 seconds, and real-time fluorescence quantitative PCR reaction is performed using a Bio-Rad CFX96 PCR instrument to accurately determine the titer of the pseudovirus.

[0023] On the other hand, a lentiviral vector system is used to prepare a monkey B virus pseudovirus, and the pseudovirus is used in the study of viral infection mechanisms, antiviral drug development, vaccine development and gene therapy research.

[0024] Compared with the existing technology, the method and application of preparing simian B virus pseudovirus by a lentiviral vector system has the following beneficial effects:

[0025] 1. The present invention overcomes the interspecies barriers of traditional HIV lentiviral systems by precisely modifying the lentiviral packaging plasmid, enabling the prepared pseudovirus to have the dual infection ability of monkey cells and human cells. At the same time, it improves the efficiency and safety of pseudovirus production. In the field of virology research, it can effectively simulate the monkey B virus infection process, reduce biosafety risks, and provide strong technical support for in-depth research on the monkey B virus infection mechanism and the development of antiviral drugs.

[0026] 2. The present invention uses a method for preparing monkey B virus glycoprotein pseudovirus to accurately test the inhibitory effect of drugs on the pseudovirus infection process of cells, greatly reducing biosafety risks. It is more convenient to operate and has lower costs, which helps to accelerate the development of effective antiviral drugs for monkey B virus and provides an efficient and safe solution to the threat posed by the virus to human health.

[0027] Other advantages, objects and features of the present invention will be described in part in the following description and, in part, will be apparent to those skilled in the art based on an examination of the following or may be learned from the practice of the invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] 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. Those skilled in the art can also derive other drawings based on these drawings without inventive effort.

[0029] Figure 1 A method for preparing simian B virus pseudovirus in a lentiviral vector system and an amino acid sequence alignment diagram of the packaging plasmid before and after modification in its application;

[0030] Figure 2 A diagram showing a method for preparing simian B virus pseudoviruses using a lentiviral vector system and the expression of a tagged fluorescent protein during pseudovirus packaging;

[0031] Figure 3 A diagram showing a method for preparing simian B virus pseudoviruses using a lentiviral vector system and the expression of firefly luciferase after cells are infected with the pseudoviruses used in the application;

[0032] Figure 4 A method for preparing monkey B virus pseudoviruses using a lentiviral vector system and a protein immunoblot of the pseudovirus PV-BV-BDHL packaged in the application;

[0033] Figure 5 A method for preparing simian B virus pseudovirus using a lentiviral vector system and a diagram showing the expression of firefly luciferase after cells are infected with the packaged pseudovirus PV-BV-BDHL;

[0034] Figure 6 The present invention is a flowchart of a method for preparing simian B virus pseudovirus using a lentiviral vector system and its application. DETAILED DESCRIPTION

[0035] In order to further illustrate the technical means and effects adopted by the present invention to achieve the predetermined purpose of the invention, the specific implementation methods, structures, features and effects of the present invention are described in detail below in conjunction with the accompanying drawings and preferred embodiments.

[0036] Example 1

[0037] HEK 293T cells were cultured in high-glucose DMEM supplemented with 10% fetal bovine serum (FBS); African green monkey kidney cells (Vero), rhesus monkey kidney cells (LLC-MK2), human glioblastoma cells (U-251MG, U-87MG, and SNB19), and human neuroblastoma cells (SK-N-SH) were cultured in DMEM supplemented with 10% FBS, human glioma cells (LN229) were cultured in DMEM supplemented with 5% FBS, and CHO-K1 cells were cultured in Ham's F12 medium supplemented with 10% FBS. All cells were cultured in a 37°C, 5% CO2 incubator and passaged every 2 to 3 days.

[0038] The transfer plasmids used in the experiment included pLVX-mCMV-ZsGreen1-Puro vector and pLVX-Luc2-P2A-MCS-puro vector, the packaging plasmid was pSPAX2, the envelope plasmid was pCMV-VSV-G, and the gag modified sequence in the psPAX2 Mutant plasmid was inserted into the original psPAX2 plasmid by homologous recombination technology. The amino acid sequences of psPAX2 Gag before and after modification are shown in the figure. Figure 1 shown.

[0039] According to the BV sequence in Genbank, the CDS regions of the synthesized BV glycoproteins gB, gD, gH and gL were recombined into the envelope plasmid pCMV-VSV-G vector. By replacing the VSV-G gene sequence in the vector, four recombinant plasmids, pCMV-VSV-ΔG-BVgB, pCMV-VSV-ΔG-BVgD, pCMV-VSV-ΔG-BVgH and pCMV-VSV-ΔG-BVgL, were constructed respectively.

[0040] The plasmid was transformed into competent Escherichia coli DH5α cells. According to the kit instructions and the subsequent use of the plasmid, the plasmid was extracted using the TIANpure Mini Plasmid Kit, ExdoFree Midi Plasmid Kit, or EndoFree Maxi Plasmid Kit. The concentration and purity of the extracted plasmid were measured using a NanoVue microspectrophotometer to ensure that the quality of the plasmid met the requirements of subsequent experiments.

[0041] HEK 293T cells were seeded into 15 cm culture dishes one day before virus packaging. The next day, the cell confluence was ensured to reach approximately 70%. The culture medium was then replaced with serum-free high-glucose DMEM. Based on the experimental group, the envelope plasmids before and after transformation, the packaging plasmids, and the reporter plasmids were co-transfected according to the instructions of the calcium phosphate cell transfection kit. The specific plasmid dosages are as follows:

[0042] LV-Mutant pseudovirus: 17 μg pCMV-VSV-G, 40 μg pLVX-mCMV-ZsGreen1-Puro or pLVX-Luc2-P2A-MCS-puro, and 34 μg psPAX2Mutant were used per dish;

[0043] LV-Wild-Type pseudovirus: 17 μg pCMV-VSV-G, 40 μg pLVX-mCMV-ZsGreen1-Puro or pLVX-Luc2-P2A-MCS-puro, and 34 μg psPAX2 were used per dish;

[0044] PV-M-BV-BDHL pseudovirus: 40 μg pLVX-Luc2-P2A-MCS-puro, 34 μg psPAX2Mutant, 20 μg pCMV-VSV-ΔG-BVgB, 16 μg pCMV-VSV-ΔG-BVgD, 15 μg pCMV-VSV-ΔG-BVgH, and 15 μg pCMV-VSV-ΔG-BVgL were used per dish.

[0045] PV-WT-BV-BDHL pseudovirus: 40 μg pLVX-Luc2-P2A-MCS-puro, 34 μg psPAX2, 20 μg pCMV-VSV-ΔG-BVgB, 16 μg pCMV-VSV-ΔG-BVgD, 15 μg pCMV-VSV-ΔG-BVgH, and 15 μg pCMV-VSV-ΔG-BVgL were used per dish.

[0046] 18 hours after plasmid co-transfection, the culture medium was replaced with high-glucose DMEM medium containing 10% FBS, and the culture supernatant was collected 24 hours and 48 hours later for subsequent pseudovirus purification and experimental analysis.

[0047] The collected culture supernatant was centrifuged at 500 g for 5 minutes and then filtered through a 0.45 μm filter to remove cell debris. The lentivirus was concentrated using a PEG 6000 solution to achieve final concentrations of PEG 6000 and NaCl of 8.5% and 0.3 M, respectively. The mixture was stored at 4°C overnight and then centrifuged at 7000 g for 10 minutes at 4°C using a Thermo Fisher Sorvall LYNX 4000 high-speed centrifuge. The supernatant was discarded, the pellet was resuspended in DPBS, and the vector suspension was aliquoted and stored at -70°C for subsequent experimental use.

[0048] 5 μL of the concentrated pseudovirus sample was extracted and nucleic acid was extracted according to the instructions of the RNA Easy Fast Animal Tissue / Cell Total RNA Extraction Kit. DNase I was added for digestion to ensure RNA purity, and RNA was finally eluted with 100 μL of RNase-free water. According to the kit instructions, 10 μL of RNA was reverse transcribed into cDNA using random primers and reverse transcriptase M-MLV (RNase H-). 5 μL of cDNA was mixed with 15 μL of PCR mixture, where the PCR mixture consisted of 10 μL of 2× Probe Premix ExTaq, 0.4 μL of forward primer (20 μM), 0.4 μL of reverse primer (20 μM), 0.4 μL of probe (20 μM), and 3.8 μL of DNase- / RNase-free distilled water. The reaction program was set as follows: an initial step of 95°C for 30 seconds, followed by 40 cycles of 95°C for 5 seconds and 60°C for 30 seconds. Real-time fluorescence quantitative PCR reactions were performed using a Bio-Rad CFX96 PCR instrument to determine the titer of the pseudovirus.

[0049] CHO-K1, Vero, LLC-MK2, HEK 293T, U-87MG, U-251MG, SNB19, LN229, and SK-N-SH cells were seeded into 48-well plates and cultured overnight at 37°C, 5% CO2. Before pseudovirus infection, the culture medium in each well was replaced with 0.25 ml of the corresponding serum-free medium. Subsequently, LV-Mutant and LV-Wild-type pseudoviruses were inoculated into the cells at a multiplicity of infection (MOI) of 10. After 24 hours of infection, the culture medium was replaced with complete medium and cultured for another 48 hours. The fluorescence expression in the cells was observed using a fluorescence microscope to assess the infection efficiency of the pseudovirus.

[0050] CHO-K1, U-87MG, SK-N-SH, HEK 293T, Vero, LLC-MK2, U-251MG, SNB19, and LN229 cells were seeded into 48-well plates, with equal numbers of cells per well. The cells were cultured overnight at 37°C in 5% CO2. Prior to pseudovirus infection, the medium in each well was replaced with 0.05 ml of fresh serum-free medium. Subsequently, 0.05 ml of a 1:10 dilution of PV-BV-BDHL and LV-Mutant pseudoviruses was inoculated into each well. After infection for 24 hours, the medium was replaced with complete medium and cultured for an additional 48 hours. The remaining medium was then aspirated, and 0.1 ml of Firefly Luciferase Reporter Gene Assay Cell Lysis Buffer was added to each well to lyse the cells. The Firefly Luciferase Reporter Gene Assay Kit and the corresponding substrate were used, and luciferase activity was measured on a Spark multi-microplate reader to assess pseudovirus infection efficiency.

[0051] During the pseudovirus packaging process, the fluorescence expression of the reporter gene was observed under a fluorescence microscope, and no significant difference was found between the LV-Mutant experimental group and the LV-Wild-type control group. Figure 2 As shown, the viral gene copy number was further detected by qPCR. The results showed that there was no significant difference in the viral gene copy number between the pseudovirus LV-Mutant packaged with the modified lentiviral packaging plasmid and the pseudovirus LV-Wild-type packaged with the original packaging plasmid. Figure 3 shown.

[0052] After the pseudovirus was infected with different cells, the fluorescence tag protein was observed and it was found that in the hamster cell CHO-K1 and the human cell HEK 293T, U-87MG, U-251MG, SNB19, LN229 and SK-N-SH, there was no significant difference in the fluorescence expression between the LV-Mutant group and the LV-Wild-type group. However, in the monkey cell Vero and LLC-MK2, the number of fluorescent cells in the LV-Mutant group was significantly more than that in the LV-Wild-type group. Figure 4 As shown, the modified packaging plasmid can enable the pseudovirus to break through species restrictions and successfully infect monkey-derived cells, providing an important tool for related research.

[0053] To further quantify the advantages of the modified lentiviral system, chemiluminescence detection was performed by packaging pseudoviruses carrying the firefly luciferase gene. The results showed that both LV-Wild-type and LV-Mutant could successfully infect human and hamster cells. In monkey Vero and LLC-MK2 cells, the luminescence intensity of the LV-Mutant group was significantly better than that of the control group (P<0.05). The luminescence intensity of LLC-MK2 cells was about 6 times that of the LV-Wild-type group, and even higher than that of human LN229 and Hela cells. Figure 5 As shown, in addition, the study also found that there were significant differences in the infection efficiency of lentivirus in different cells: U251 cells were the most sensitive to lentivirus, followed by HEK293T and U87-MG cells, while Vero cells were the least susceptible.

[0054] For further verification, the pseudoviruses PV-M-BV-BDHL and PV-WT-BV-BDHL packaged with BV glycoprotein were used to infect Vero, LLC-MK2 and U-251MG cells, respectively. The results showed that PV-WT-BV-BDHL could infect U-251MG and cells, but could hardly infect Vero and LLC-MK2 cells, while PV-M-BV-BDHL could successfully infect the above three cell types, indicating that the monkey B virus pseudovirus packaged with the modified psPAX2 Mutant plasmid successfully overcame the species restriction and achieved efficient infection in monkey cells, laying an important foundation for the subsequent study of the cell entry mechanism of B virus at the monkey and human cell levels.

[0055] Example 2

[0056] Human embryonic kidney cells (HEK293T) were used for pseudovirus packaging, while human glioblastoma cells (U-251MG) and rhesus monkey kidney cells (LLC-MK2) were used for subsequent pseudovirus infection experiments. The cells were cultured in DMEM medium containing 10% FBS (for HEK293T cells) and DMEM medium containing 10% FBS (for U251-MG and LLC-MK2 cells) at 37°C in a 5% CO2 incubator. When the cell confluency reached 80%-90%, they were subcultured. The day before the experiment, both cell types were digested and counted, and seeded into 96-well plates at an appropriate cell density. 100 μL of culture medium was added to each well to keep the cells in the logarithmic growth phase during the experiment.

[0057] Based on the lentiviral packaging plasmid commonly used in the laboratory, the specific base sequence of the gag gene of the psPAX2 plasmid was modified using site-directed mutagenesis technology. Primers carrying the mutation site were designed based on the gag gene sequence. The mutant fragment was obtained by PCR amplification and introduced into the original psPAX2 plasmid through enzyme digestion and ligation to construct the psPAX2 Mutant lentiviral vector. After construction, the recombinant plasmid was sequenced to ensure the accuracy of the modified base sequence.

[0058] Prepare pLVX-Luc2-P2A-MCS-puro as a transfer plasmid. This plasmid carries the firefly luciferase reporter gene for subsequent detection. Also prepare the psPAX2 Mutant packaging plasmid, the pCMV-VSV-G envelope plasmid, and the recombinant plasmids pCMV-VSV-ΔG-BVgB, pCMV-VSV-ΔG-BVgD, pCMV-VSV-ΔG-BVgH, and pCMV-VSV-ΔG-BVgL, obtained by recombining the simian B virus glycoprotein gB, gD, gH, and gL genes into the envelope plasmid. Transform each plasmid into competent Escherichia coli DH5α cells and extract the plasmid using an appropriate plasmid extraction kit (e.g., the TIANpure Mini Plasmid Kit or the EndoFree Maxi Plasmid Kit, depending on the desired plasmid yield). Determine the concentration and purity of the extracted plasmid using a Nano Vue microspectrophotometer to ensure that the quality meets the requirements of subsequent experiments.

[0059] One day before virus packaging, HEK293T cells in the logarithmic growth phase were seeded into 15 cm culture dishes. The next day, the cell confluence was observed under an inverted microscope. When it reached about 70%, the medium was replaced with serum-free high-glucose DMEM medium. According to the grouping set in the experiment, plasmid co-transfection was performed using a calcium phosphate cell transfection kit. Taking the preparation of pseudovirus for antiviral drug screening as an example, 40 μg pLVX-Luc2-P2A-MCS-puro, 34 μg psPAX2-Mutant, 20 μg pCMV-VSV-ΔG-BVgB, 16 μg pCMV-VSV-ΔG-BVgD, 15 μg pCMV-VSV-ΔG-BVgH and 15 μg pCMV-VSV-ΔG-BVgL, the transfection process was carried out strictly according to the kit instructions to ensure transfection efficiency. After 18 hours of plasmid transfection, the high-glucose DMEM medium containing 10% FBS was replaced, and the culture supernatant was collected 24 hours and 48 hours later.

[0060] The collected culture supernatant was first centrifuged at 500g for 5 minutes to remove larger cell debris, and then the supernatant was filtered through a 0.45μm filter to further remove tiny impurities. The filtered supernatant was transferred to a new centrifuge tube, and PEG 6000 solution was added to make the final concentrations of PEG 6000 and NaCl reach 8.5% and 0.3M, respectively. The solution was gently mixed and allowed to stand overnight at 4°C. The next day, the solution was centrifuged at 7000g for 10 minutes at 4°C using a Thermo Fisher Sorvall LYNX4000 high-speed centrifuge. After centrifugation, the supernatant was carefully discarded and the precipitate was resuspended with an appropriate amount of DPBS. After sufficient dissolution, the pseudovirus suspension was divided into sterile cryovials and stored in a -70°C refrigerator for later use.

[0061] An appropriate amount of concentrated pseudovirus sample was taken out from the -70°C freezer and slowly thawed on ice. 5 μL of sample was taken and nucleic acid was extracted according to the instructions of the RNA Easy Fast Animal Tissue / Cell Total RNA Extraction Kit. DNase I was added for digestion during the extraction process to remove possible residual genomic DNA. RNA was eluted with 100 μL of RNase-free water. 10 μL of RNA was taken and reverse transcribed into cDNA using random primers and reverse transcriptase M-MLV (RNaseH-). 5 μL of cDNA was mixed with 15 μL of PCR mixture, which contained 10 μL of 2×Probe Premix Ex Taq, 0.4 μL of forward primer (20 μM), 0.4 μL of reverse primer (20 μM), 0.4 μL of probe (20 μM) and 3.8 μL of DNase / RNase-free distilled water. The extension reaction was carried out in a PCR instrument. The reaction program was: pre-denaturation at 95°C for 30 seconds, followed by 40 cycles, each cycle including denaturation at 95°C for 5 seconds and annealing and extension at 60°C for 30 seconds. After the reaction was completed, the titer of the pseudovirus was calculated based on the standard curve.

[0062] The U-251MG cells and LLC-MK2 cells seeded in the 96-well plate were removed from the incubator, the original culture medium was aspirated, and 100 μL of serum-free culture medium was added to each well. The antiviral drug to be screened was gradiently diluted with serum-free culture medium, and different drug concentration gradients were set (such as 100 μM, 50 μM, 25 μM, 12.5 μM, etc.). The diluted drug solution was added to each well, 100 μL per well, and the drug was added to a new 96-well plate. Then, according to the measured pseudovirus titer, an appropriate amount of PV-M-BV-BDHL pseudovirus was added to each well to keep the MOI consistent. The culture plate was gently shaken to evenly distribute the pseudovirus and drug. 3-5 replicates were set for each group. At the same time, a control group was set, including a normal cell control group that was not infected with the pseudovirus and was not drug-added, and a virus-infected control group that was infected with the pseudovirus but not drug-added. Different drug-virus action times can be set. Then, the corresponding drug and pseudovirus mixture was added to the cell wells and incubated at 37 ℃, 5% CO2 incubator for 2 hours, then aspirate the culture medium containing pseudovirus and drug, add 200 μL complete culture medium (DMEM culture medium containing 10% FBS) to each well, and continue to culture for 24 hours. After the end of the culture, aspirate the culture medium in the wells, add 100 μL firefly luciferase reporter gene detection cell lysis buffer to each well, incubate at room temperature for 15-30 minutes to fully lyse the cells, use a firefly luciferase reporter gene detection kit, add the corresponding substrate, and detect the luciferase activity of each well on a Spark multifunctional microplate reader. The inhibitory effect of the drug on pseudovirus-infected cells is evaluated based on the level of luciferase activity. The lower the luciferase activity, the stronger the inhibitory effect of the drug on pseudovirus infection. Drugs with potential anti-simian B virus activity and their effective concentration ranges are screened.

[0063] The above description is merely a preferred embodiment of the present invention and does not constitute any form of limitation to the present invention. Although the present invention has been disclosed as above in terms of a preferred embodiment, it is not intended to limit the present invention. Any person skilled in the art can, without departing from the scope of the technical solution of the present invention, make some changes or modifications to equivalent embodiments using the technical contents disclosed above. However, any brief modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A method for preparing simian B virus pseudovirus using a lentiviral vector system, characterized in that: The method comprises the following specific steps: Construction of modified lentiviral vectors: Using site-directed mutagenesis to modify the specific base sequence of the gag gene in the psPAX2 plasmid, a lentiviral vector with optimized infection characteristics and expression efficiency was constructed; Prepare relevant plasmids: Select packaging and envelope plasmids before and after specific transfer and transformation, synthesize the simian B virus glycoprotein gene and recombinant it into the envelope plasmid. After transformation into competent cells, use the corresponding kit to extract and test the concentration and purity; Pseudovirus packaging: After replacing the culture medium of cells in a specific growth state, the relevant plasmids were co-transfected according to the calcium phosphate method according to different groups. After 18 hours, the culture medium was replaced with serum and the supernatant was collected at 24 and 48 hours; Pseudovirus concentration and storage: The collected culture supernatant was centrifuged and filtered, concentrated with PEG 6000 and NaCl, and allowed to stand overnight at 4°C. After high-speed centrifugation, the precipitate was resuspended in DPBS and stored in aliquots at -70°C. Pseudovirus titer determination: Take concentrated pseudovirus samples to extract nucleic acid, digest, elute, reverse transcribe to cDNA, and react with specific PCR mixture. The titer is determined using a Bio-Rad CFX96 PCR instrument.

2. The method for preparing simian B virus pseudovirus using a lentiviral vector system according to claim 1, characterized in that: In the step of preparing the relevant plasmids, pLVX-mCMV-ZsGreen1-Puro vector and pLVX-Luc2-P2A-MCS-puro are selected as transfer plasmids; the packaging plasmids include the original pSPAX2 and the modified psPAX2-Mutant, and the envelope plasmids are selected as pCMV-VSV-G and the modified pCMV-VSV-ΔG-BVgB, pCMV-VSV-ΔG-BVgD, pCMV-VSV-ΔG-BVgH and pCMV-VSV-ΔG-BVgL.

3. The method for preparing simian B virus pseudovirus using a lentiviral vector system according to claim 1, characterized in that: In the step of preparing the relevant plasmids, the corresponding sequences are obtained by artificial synthesis based on the coding sequence (CDS) region sequences of the simian B virus glycoproteins gB, gD, gH and gL in the gene library (GenBank), and are recombined into the envelope plasmid pCMV-VSV-G vector to construct four recombinant plasmids, pCMV-VSV-ΔG-BVgB, pCMV-VSV-ΔG-BVgD, pCMV-VSV-ΔG-BVgH and pCMV-VSV-ΔG-BVgL, respectively. Each plasmid is transformed into competent Escherichia coli cells, and the extraction operation is performed using a TIANpure Mini plasmid kit, an ExdoFree Midi plasmid kit or an EndoFree Maxi plasmid kit.

4. The method for preparing simian B virus pseudovirus using a lentiviral vector system according to claim 1, characterized in that: In the pseudovirus packaging step, human embryonic kidney cells (HEK293T) are inoculated into a 15 cm culture dish one day before virus packaging. When the cell confluence reaches 70% the next day, the culture medium is replaced with serum-free high-glucose DMEM medium. According to different experimental groups, the envelope plasmids, packaging plasmids and reporter plasmids before and after transformation are co-transfected according to the calcium phosphate method. After 18 hours of co-transfection, the culture medium is replaced with high-glucose DMEM medium containing 10% fetal bovine serum, and the culture supernatant is collected 24 hours and 48 hours later.

5. The method for preparing simian B virus pseudovirus using a lentiviral vector system according to claim 1, characterized in that: In the pseudovirus packaging step, according to different experimental groups, the envelope plasmids, packaging plasmids and reporter plasmids before and after transformation were co-transfected according to the calcium phosphate method. The specific groups are: LV-Mutant pseudovirus group: 17 μg pCMV-VSV-G, 40 μg pLVX-mCMV-ZsGreen1-Puro or pLVX-Luc2-P2A-MCS-puro, and 34 μg psPAX2-Mutant were used per dish; LV-Wild-Type pseudovirus group: 17 μg pCMV-VSV-G, 40 μg pLVX-mCMV-ZsGreen1-Puro or pLVX-Luc2-P2A-MCS-puro, and 34 μg psPAX2 were used per dish; PV-M-BV-BDHL pseudovirus group: 40 μg pLVX-Luc2-P2A-MCS-puro, 34 μg psPAX2-Mutant, 20 μg pCMV-VSV-ΔG-BVgB, 16 μg pCMV-VSV-ΔG-BVgD, 15 μg pCMV-VSV-ΔG-BVgH, and 15 μg pCMV-VSV-ΔG-BVgL were used per dish; PV-WT-BV-BDHL pseudovirus group: 40 μg pLVX-Luc2-P2A-MCS-puro, 34 μg psPAX2, 20 μg pCMV-VSV-ΔG-BVgB, 16 μg pCMV-VSV-ΔG-BVgD, 15 μg pCMV-VSV-ΔG-BVgH and 15 μg pCMV-VSV-ΔG-BVgL were used per dish.

6. The method for preparing simian B virus pseudovirus using a lentiviral vector system according to claim 1, characterized in that: In the pseudovirus concentration and preservation step, the collected culture supernatant was first centrifuged at 500g for 5 minutes to remove cell debris, filtered through a 0.45μm filter, and the lentivirus was concentrated using a PEG 6000 solution to achieve final concentrations of PEG 6000 and NaCl of 8.5% and 0.3M, respectively. The mixed solution was allowed to stand overnight at 4°C.

7. The method for preparing simian B virus pseudovirus using a lentiviral vector system according to claim 1, characterized in that: In the pseudovirus concentration and storage step, after high-speed centrifugation, the precipitate is resuspended in DPBS and stored in aliquots at -70°C. Specifically, a high-speed centrifuge is used to centrifuge at 7000g for 10 minutes at 4°C. After the centrifugation is completed, the supernatant is discarded, the precipitate is resuspended in DPBS, the vector suspension is aliquoted, and stored in a -70°C environment for future use.

8. The method for preparing simian B virus pseudovirus using a lentiviral vector system according to claim 1, characterized in that: In the pseudovirus titer determination step, 5 μL of the concentrated pseudovirus sample was taken, and nucleic acid extraction was performed according to the steps specified in the RNA extraction kit instructions. DNase I was added for digestion, and RNA was eluted with 100 μL of RNase-free water. 10 μL of the RNA sample was taken and reverse transcribed into cDNA using random primers and reverse transcriptase M-MLV.

9. The method for preparing simian B virus pseudovirus using a lentiviral vector system according to claim 1, characterized in that: In the pseudovirus titer determination step, random primers and reverse transcriptase M-MLV are used to reverse transcribe it into cDNA. Specifically, 5 μL of cDNA is thoroughly mixed with 15 μL of PCR mixture, wherein the PCR mixture contains 10 μL of 2×Probe Premix Ex Taq, 0.4 μL of a forward primer with a concentration of 20 μM, 0.4 μL of a reverse primer with a concentration of 20 μM, 0.4 μL of a probe with a concentration of 20 μM, and 3.8 μL of DNase / RNase-free distilled water. The reaction program is set to: pre-denaturation at 95°C for 30 seconds, 40 cycles, each cycle including denaturation at 95°C for 5 seconds and annealing and extension at 60°C for 30 seconds, and real-time fluorescence quantitative PCR reaction is performed using a Bio-Rad CFX96 PCR instrument to accurately determine the titer of the pseudovirus.

10. A use of a lentiviral vector system to prepare a simian B virus pseudovirus, wherein the simian B virus pseudovirus prepared using a lentiviral vector system is characterized in that: The application areas of the virus are: research on viral infection mechanisms, development of antiviral drugs, vaccine development and gene therapy research.

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