Cell lines based on spacer gene-directed insertion of hibit tags, recombinant viruses and methods and uses thereof

Abstract

The application provides a cell line based on Spacer gene directional insertion of HiBit tags, a recombinant virus and a preparation method and application thereof, linearized fragments are obtained by double enzyme digestion of a vector pCDN3.1-CMV-G418 through SacI / PmeI, fragments 1 and 2 are obtained by amplifying a D-type HBV 1.3-fold plasmid through primers shown as SEQ ID NO. 4-SEQ ID NO. 5 and primers shown as SEQ ID NO. 6-SEQ ID NO. 7, and the linearized fragments and the recombinant plasmid are obtained through seamless cloning, the recombinant plasmid is transfected into hepatoma cells after single enzyme digestion through ScaI, and a cell line with replication ability based on Spacer gene directional insertion of HiBit tags is obtained through resistance screening. A novel recombinant virus with replication and infection ability can be obtained from the cell model, the whole life cycle of the virus can be completed, simple and rapid detection and high-throughput screening of antiviral drugs can be realized.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to a cell line, recombinant virus, preparation method and application based on the directional insertion of the Spacer gene into the HiBit tag. Background Technology

[0002] Hepatitis B virus (HBV) belongs to the Hepatotropic DNA Virus family and primarily infects human liver cells, causing acute or chronic hepatitis. Chronic HBV infection significantly increases the incidence of liver fibrosis, cirrhosis, and even liver cancer. Although preventative vaccines against hepatitis B have been available for decades, approximately 257 million people worldwide are still suffering from HBV infection, which remains a serious public health concern globally (WHO. Hepatitis B Fact Sheet. Available at: https: / / www.who.int / news-room / fact-sheets / detail / hepatitis-b (Accessed: March 2020)). Currently, the U.S. Food and Drug Administration (FDA) has approved two main classes of therapeutic drugs for chronic HBV infection: type I interferon (IFNA) and nucleoside analogues (NAs). Both of these drug treatments inhibit HBV replication to some extent, but they cannot completely eliminate the hepatitis B virus covalently closed circular DNA (cccDNA) in hepatocytes, thus failing to achieve a functional cure for HBV (Kwon H, LokAS. Hepatitis B therapy. Nat Rev Gastroenterol Hepatol 2011.8:275-284. Nassal M. HBV cccDNA: viral persistence reservoir and key obstacle for a cure of chronic hepatitis B. Gut 2015.64:1972-1984.). Therefore, exploring the mechanism of chronic HBV infection and developing and screening novel drugs that can cure hepatitis B remain important scientific problems that need to be solved.

[0003] To address these issues, the ideal approach is to construct recombinant viruses capable of rapidly and efficiently monitoring the HBV replication and infection process and stably expressing reporter genes or fluorescent elements. Detecting reporter genes or markers is often much easier than detecting HBV indicators (HBV-DNA, HBsAg, HBeAg, HBV RNA, and HBV cccDNA). Constructing a recombinant viral reporter system by directionally inserting exogenous genes into the viral genome, while generating recombinant viruses to a certain extent without affecting their life cycle, is extremely difficult. For HBV, viral labeling itself has certain limitations. First, the HBV genome is only 3.2 kb, tightly packed, containing four overlapping open reading frames (preC / C, P, preS1 / preS2 / S, and X), as well as multiple cis-acting control elements, such as replication control regions for pregenomic RNA (pgRNA) synthesis, packaging, and reverse transcription. Therefore, finding suitable insertion sites in the tightly packed HBV genome is difficult. Second, the size of the genome that can be effectively assembled into the HBV capsid is strictly limited; exogenous gene insertion severely affects viral packaging efficiency. Furthermore, even if the complete HBV protein is replenished through complementation, it is difficult to restore the functional loss caused by the insertion of foreign genes. At present, although various marker strategies have been reported to prepare recombinant HBV, attempts to produce recombinant hepatitis B virus with replication and infection capabilities have not been successful (Bai WY, Cui XX, Xie YH, Liu J. Engineering Hepadnaviruses as Reporter-Expressing Vectors: Recent Progress and Future Perspectives. Viruses. 2016 May 10; 8(5). E125.).

[0004] The reason for long-term chronic HBV infection is the stable presence of HBV cccDNA in host cells. Currently, the main drugs used clinically for chronic hepatitis B are interferon and nucleoside analogs. While these drugs can inhibit HBV replication to some extent, they have not achieved a functional cure. Therefore, the development of novel anti-HBV drugs is urgently needed. There is an urgent need to establish an efficient HBV cell culture system to simulate the entire HBV life cycle and to monitor HBV replication and infection levels simply, rapidly, and efficiently. Therefore, it is necessary to develop a novel recombinant virus reporter system with replication and infection capabilities for rapid, efficient, and high-throughput in vitro screening. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a cell line, recombinant virus, and its preparation method and application based on the Spacer gene-directed insertion of the HiBit tag. Using a NanoBiT fluorescence complementary reporter system, the HiBit coding sequence is directionally inserted into the genome of type D HBV1.3, specifically binding to the LgBiT large subunit and emitting a stable fluorescent signal under the action of the substrate, successfully constructing a recombinant viral reporter system. This construction strategy can obtain a novel recombinant virus (HBV-HiBit) without significantly affecting the virus's replication and infectivity.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] In a first aspect of the present invention, a method for preparing a cell line based on the directional insertion of a Spacer gene into a HiBit tag is provided, the method comprising:

[0008] The vector pCDN3.1-CMV-G418 was digested with SacI / PmeI to obtain the linearized vector backbone fragment.

[0009] Primers shown in SEQ ID NO.4-SEQ ID NO.5 and primers shown in SEQ ID NO.6-SEQ ID NO.7 were used to amplify the D-type HBV 1.3 ploid plasmid, obtaining fragment 1 and fragment 2, respectively;

[0010] The linearized vector backbone fragment, fragment 1, and fragment 2 were subjected to homologous recombination and transformation to obtain the recombinant plasmid pCDN3.1-CMV-HBV1.3-HiBit-G418;

[0011] The recombinant plasmid pCDN3.1-HBV1.3-HiBit-G418 was digested with ScaI to obtain a linearized fragment, which was then transfected into HepG2 cells and selected for G418 resistance to obtain a polyclonal cell line. The polyclonal cell line was then selected for G418 resistance to obtain a cell line based on the directional insertion of the Spacer gene into the HiBit tag.

[0012] The sequence of the vector pCDN3.1-CMV-G418 plasmid is shown in SEQ ID NO.2;

[0013] The nucleotide sequence of HBV1.3 is shown in SEQ ID NO.3;

[0014] The sequence of the recombinant plasmid pCDN3.1-HBV1.3-HiBit-G418 is shown in SEQ ID NO.1.

[0015] In a second aspect of the invention, a cell line based on the Spacer gene-directed insertion of a HiBit tag is provided, prepared using the method described above.

[0016] In a third aspect of the invention, a method for preparing a recombinant virus based on the directional insertion of a Spacer gene into a HiBit tag is provided, the method comprising:

[0017] When the cell line based on the Spacer gene directionally inserted into the HiBit tag is expanded and cultured to a cell density of 70%-90%, it is replaced with a medium containing DMSO for induction. The cell culture supernatant is collected and replaced with a fresh toxin-producing medium.

[0018] The cell culture supernatant was centrifuged and filtered to obtain concentrated viral fluid, which is a recombinant virus based on the Spacer gene with a HiBit tag inserted in a directional manner.

[0019] Furthermore, the volume of DMSO added to the DMSO-containing culture medium is 2% ± 0.5% of the culture medium volume.

[0020] In a fourth aspect of the invention, a recombinant virus based on the Spacer gene-directed insertion of a HiBit tag is provided, prepared using the method described above.

[0021] In a fifth aspect of the invention, the application of the aforementioned cell line based on the Spacer gene-directed insertion of the HiBit tag is provided in the screening and evaluation of novel anti-hepatitis B virus drugs.

[0022] In a sixth aspect of the invention, the application of the recombinant virus based on the Spacer gene-directed insertion of the HiBit tag is provided in the screening and evaluation of novel anti-hepatitis B virus drugs.

[0023] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0024] This invention provides a cell line based on the targeted insertion of the Spacer gene into the HiBit tag, its preparation method, and its application. Based on NanoBiT technology, the small subunit HiBit can specifically bind to the large subunit LgBit, emitting a stable fluorescent signal under the action of the substrate. This allows for rapid and efficient detection using a microplate reader. Specifically:

[0025] (1) This invention obtains a cell model with stable integration of HBV1.3-HiBit DNA by directionally inserting the novel tag HiBit as a reporter gene into the genome of type D HBV1.3 and screening stable cell lines. This model is used to prepare novel recombinant viruses with replication and infection capabilities. Directly inserting exogenous sequences into the HBV genome to construct recombinant viruses has certain limitations. One of the innovations of this invention lies in the insertion site of the exogenous sequence: First, the HBV genome is only 3.2kb and tightly packed, containing four overlapping open reading frames (preC / C, P, preS1 / preS2 / S, and X). Its genome contains multiple cis-acting control elements, making it difficult to find a suitable insertion site. Second, the size of the genome for effectively packaging the HBV capsid is strictly limited, and the insertion of exogenous genes will seriously affect packaging efficiency.

[0026] (2) The second innovation of this invention lies in the ability of the recombinant virus HBV1.3-HiBit, which fused with an exogenous tag, to achieve viral infection and replication. Based on the correct selection of the genomic insertion site and the size of the exogenous HiBit fragment, a recombinant virus reporter system was successfully constructed. This reporter system only requires the detection of the intensity of chemical signals in the cell culture supernatant using chemiluminescence to replace the detection of relevant HBV indicators, making it simple, rapid, and efficient. The novel recombinant virus reporter system constructed in this invention can be used for basic research on virus-host interactions and for the development and evaluation of novel antiviral drugs. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0028] Figure 1 This is a schematic diagram of the directional insertion of exogenous tags into the viral genome;

[0029] Figure 2 The pCDN3.1-CMV-HBV1.3-HiBit-G418 recombinant plasmid was constructed using seamless cloning technology.

[0030] Figure 3 It is for phenotypic validation of recombinant plasmids expressing exogenous tags; among which Figure 3 A is a flowchart of plasmid transfection time; Figure 3 B and 3C represent the detection of HBeAg and HBsAg secretion levels in cell culture supernatants 2 and 4 days after transfection with wild-type and recombinant plasmids, respectively. Figure 3D represents the expression levels of intracellular proteins after transfection with wild-type and recombinant plasmids;

[0031] Figure 4 The project focuses on the construction and screening of HBV1.3-HiBit DNA stable integration cell models; among which, Figure 4 A represents the construction process of the HBV1.3-HiBit DNA stable integration cell model; Figure 4 B represents the detection results of HBV-related replication indicators during the screening process. The horizontal axis represents the cell lines to be tested, with HepAD38 serving as the positive control.

[0032] Figure 5 It involves the preparation of novel recombinant viruses; among which, Figure 5 A represents the preparation process of the novel recombinant virus; Figure 5 B represents the detection of recombinant virus titer;

[0033] Figure 6 It is the detection of HBV replication markers related to infection with novel recombinant viruses; Figure 6 A is a flowchart of the recombinant virus infection process; Figure 6 B represents the detection of HBeAg levels in the supernatant after recombinant virus infection; Figure 6 C represents the protein expression status after infection with the recombinant virus. Detailed Implementation

[0034] The present invention will be described in detail below with reference to specific embodiments and examples, thereby making the advantages and various effects of the present invention more clearly apparent. Those skilled in the art should understand that these specific embodiments and examples are for illustrative purposes only and are not intended to limit the present invention.

[0035] Throughout this specification, unless otherwise specified, the terminology used herein should be understood as having the meaning commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In the event of any conflict, this specification shall prevail.

[0036] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be purchased from the market or prepared by existing methods.

[0037] The effects of this application will be described in detail below with reference to embodiments and experimental data. Unless otherwise specified, the molecular cloning methods, protein expression and purification methods, cell culture methods, and various detection methods mentioned in the following schemes are all traditional experimental methods, which can be obtained by consulting the literature; the relevant reagents used can be purchased from the corresponding reagent suppliers. The room temperature mentioned in the following embodiments of the present invention refers to the natural room temperature conditions in all four seasons, without additional cooling or heating treatment, and is generally controlled at 10-30°C, preferably 15-25°C.

[0038] Example 1: Cell line based on Spacer gene-directed insertion of HiBit tag and its preparation method

[0039] I. Preparation of Linearized Vector Fragments

[0040] The plasmid template used to obtain the linearized vector backbone fragment was pCDN3.1-CMV-G418 (this is a commonly used vector backbone sequence). The restriction enzyme sites used to obtain the linearized vector backbone fragment were SacI (#R3156S) and PmeI (#R0560S), and the size of the pCDN3.1-G418 linear fragment was 5.6 kb. Double digestion was performed using NEB's restriction endonucleases SacI and PmeI. The total reaction volume was: 1 μl SacI, 1 μl PmeI, 2 μl template, 5 μl CutSmart buffer, and sterile distilled water to a final volume of 50 μl. The reaction conditions were 37°C for 1 h.

[0041] II. Obtaining the target segment for insertion

[0042] The target fragment was amplified using PCR technology. The template used for amplifying the HBV target fragment was the D-type HBV1.3 ploid plasmid (genotype D, sequence shown in SEQ ID NO.3). Primers were designed and synthesized as shown in the table below. Vazyme high-fidelity DNA polymerase (catalog number P510-01) was used. The reaction system consisted of 25 μl of polymerase, 12.5 μl of PrimeSTAR Max Premix (2×), 0.5 μl each of primers F and R, 1 μl (1 ng) of template, and 10.5 μl of sterile distilled water. The reaction conditions were 98℃, 10 s, 56℃, 5 s or 15 s, 72℃, 5 s / kb, for 32 cycles. The template used for amplifying HBV1.3-HiBit was the HBV1.3 ploid, and the amplification primers used were HBV1.3-F1 / HBV1.3-HiBit-R1 and HBV1.3-HiBit-F2 / HBV1.3-R2. In this embodiment, the novel HiBit sequence was added to the primers for amplifying the HBV1.3 sequence, and the target fragment was obtained by PCR amplification in two segments. The primer sequences involved in this embodiment are shown in Table 1:

[0043] Table 1

[0044]

[0045] III. Target Fragment for Gel Recovery and Purification

[0046] The purified target fragment was obtained using a DNA gel extraction kit (Vazyme, catalog number DC301). The specific procedures are as follows:

[0047] 1. Perform DNA electrophoresis on the linear fragment obtained by double enzyme digestion and the target fragment obtained by PCR, respectively. Rapidly recover the gel containing the target DNA fragment under UV irradiation. Weigh the gel (excluding the weight of empty tubes); 100 mg of gel is equivalent to 100 μl, which is taken as one gel volume.

[0048] 2. Add an equal volume of Buffer GDP. Incubate in a 50-55°C water bath for 7-10 minutes to ensure the gel is completely dissolved. Invert the container twice during the water bath to accelerate the dissolution process.

[0049] 3. Briefly centrifuge to collect droplets on the tube wall. Place the Fastpure DNA Mini Columns-G adsorption column in a 2ml Collection Tube, transfer ≤700μl of sol to the adsorption column, and centrifuge at 12000×g for 30-60s. If the sol volume is greater than 700μl, return the adsorption column to the collection tube, transfer the remaining sol to the adsorption column, and centrifuge at 12000×g for 30-60s.

[0050] 4. Discard the filtrate and place the adsorption column in the collection tube. Add 300 μl of Buffer GDP to the adsorption column. Let stand for 1 min. Centrifuge at 12000×g for 30-60 s.

[0051] 5. Discard the filtrate and place the adsorption column in the collection tube. Add 600 μl of Buffer GW (with anhydrous ethanol added) to the adsorption column. Centrifuge at 12000×g for 30–60 s.

[0052] 6. Repeat step 5.

[0053] 7. Discard the filtrate and return the adsorption column to the collection tube. Centrifuge at 12000×g for 2 min.

[0054] 8. Place the adsorption column in a 1.5 ml sterile centrifuge tube, add 7-30 μl of Elution Buffer to the center of the adsorption column, and incubate for 2 min. Centrifuge at 12000 × g for 1 min. Discard the adsorption column and store the DNA at -20℃.

[0055] IV. Homologous Recombination of Linearized Vectors and Inserted Fragments

[0056] The three purified fragments were subjected to homologous recombination using a seamless cloning method, following the instructions of the ABclonalMultiF Seamless Assembly Mix (catalog number RK21020) kit. The reaction volume was 10 μl.

[0057] Table 2

[0058]

[0059] Reaction conditions: 50℃ for 30 min. Insert 1-2 fragments into the vector, with a recommended total DNA amount of 0.02-0.5 pmols (including the total DNA of all fragments and the vector). The pmols of each fragment can be calculated based on its length and mass using the following formula: pmols = (mass ng) × 1000 / (number of base pairs × 650 Daltons).

[0060] V. Transformation of Recombinant Products

[0061] 1. Take 100 μl of DH5α competent cells that have been frozen at -80℃ and place them on ice to thaw;

[0062] 2. Quickly add the above homologous recombination ligation product into competent cells, gently tap the tube wall to mix (do not shake to mix), and let stand on ice for 30 min;

[0063] 3. After heat shock in a 42℃ water bath for 45 seconds, immediately place it on ice to cool for 2-3 minutes;

[0064] 4. Add 900 μl of SOC or LB medium (without antibiotics) and shake at 37°C for 1 hour;

[0065] 5. Preheat the LB agar plates with the corresponding resistance in a 37°C incubator;

[0066] 6. Centrifuge the culture at 5000 rpm for 5 min, discard 900 μl of supernatant with a pipette, resuspend the bacterial culture in the remaining culture medium, and gently spread it evenly on a plate containing the correct antibiotic using a sterile spreader. Incubate in an inverted incubator at 37°C overnight.

[0067] VI. Bacterial culture PCR identification

[0068] After overnight incubation, hundreds of single colonies formed on the transformation plates. Several colonies were selected from the transformation plates for colony PCR identification. The PCR reaction system consisted of: 1 μl of the test bacterial culture, 0.5 μl each of forward and reverse primers, 12.5 μl of 2× DNA polymerase mix, and 10.5 μl of deionized water. The PCR program was as follows: pre-denaturation: 95℃, 3 min; denaturation: 95℃, 30 s; annealing: 56℃, 15 s; extension: 72℃, 1 min, 32 amplification cycles. The amplified PCR products were subjected to 1% agarose gel electrophoresis and imaged.

[0069] VII. Plasmid Sequencing

[0070] Select the correctly identified single colonies from step six and inoculate them into 50ml centrifuge tubes at a ratio of 1:1000. Incubate at 37℃ on a shaker at 200 rpm for 14-16 hours. Centrifuge the bacterial culture, discard the supernatant, and obtain the bacterial cells. Extract plasmids according to the instructions of the plasmid extraction kit. Simultaneously, perform plasmid sequencing.

[0071] VIII. Recombinant plasmid transfection and phenotypic verification

[0072] 1. Plasmid transfection: Huh7 cells (102) were seeded into 24-well cell culture plates. 5 (cell / well) When the cell density is approximately 60%, PEI transfection of the plasmid is performed. The PEI transfection method is as follows: Dilute 500 ng of plasmid (referred to as solution A) and 1.5 μl of PEI (referred to as solution B) with Opti-MEM in a 1.5 ml sterile EP tube. Then add solution B to solution A, vortex to mix, let stand at room temperature for 20 min, and then evenly add the mixture to the well plate.

[0073] 2. Indicator Detection: After transfection of Huh7 cells with the recombinant plasmid, cell culture supernatant and cells were collected for the detection of relevant HBV replication indicators. Results are as follows: Figure 3 As shown, this indicates that the recombinant plasmid was successfully constructed.

[0074] IX. Construction and Screening of Novel Cell Models for Stable Integration of HBV1.3-HiBit DNA

[0075] (a) Construction and screening of polyclonal cell lines

[0076] The recombinant plasmid pCDN3.1-HBV1.3-HiBit-G418 was linearized using a single enzyme digestion method and transfected into HepG2 cells. Polyclonal cell lines were obtained through G418 resistance selection. The normal functioning of the polyclonal cell lines was determined by detecting HBeAg and HBsAg levels in the cell culture supernatant. The specific methods are as follows:

[0077] (1) Obtaining the linearized vector: The recombinant plasmid pCDN3.1-HBV1.3-HiBit-G418 was digested with ScaI to obtain the linearized vector. The digestion system was: 2 μl ScaI, 5 μl CutSmart Buffer, 2 μl (2 μg) of recombinant plasmid, and sterile water to a final volume of 50 μl. The mixture was incubated at 37°C for 1 h. The digested product was then purified using a DNA purification kit to obtain the purified linearized fragment.

[0078] (2) Transfection: Taking a 6-well plate as an example, transfection is performed when the cell density reaches 80-90%. Take 100 μl of the transfection solvent Opti-MEM, and the ratio of the linearized plasmid fragment to the transfection reagent FuGENE-HD (promega) is 1:4. Mix well using a pipette or vortex. Let the mixture stand at room temperature for 15-20 min, then slowly add it to the wells and mix well. Incubate at 37℃. Change the medium 8 h after transfection with fresh medium.

[0079] (3) Screening: 48 h after transfection, the medium was replaced with one containing G418 resistance for pressure screening. The resistance screening was repeated 2-3 times to obtain polyclonal cell lines.

[0080] (4) HBV-related index detection: Collect the cell culture supernatant of the cell lines obtained by the above screening, and detect the expression levels of HBeAg and HBsAg by ELISA. If both are expressed, the polyclonal cell line construction screening is successful.

[0081] (b) Screening of monoclonal cell lines

[0082] To improve the toxin-producing capacity of cell lines, monoclonal cell lines were constructed and screened. The specific methods are as follows:

[0083] (1) Cell seeding: The polyclonal cell lines HepG2-1.3HBV-HiBit obtained by the above screening were seeded into 96-well plates at a ratio of 1:5 with HepG2;

[0084] (2) Resistance screening: Observe the above-mentioned seeded cells under a microscope. When cell clusters are generated in the wells, add screening medium containing G418 and perform resistance screening 2-3 times. Cell lines with only one cell cluster in the well after resistance screening can be used as the cell lines obtained from the initial screening.

[0085] (3) HBV marker detection: The culture supernatant of the cell lines obtained from the preliminary screening was collected for HBV marker detection (hepatitis B surface antigen / e antigen diagnostic kit and hepatitis B virus nucleic acid detection kit). The results are as follows: Figure 4 As shown, this indicates that a single-clonal cell line was successfully obtained through screening.

[0086] Example 2: Recombinant virus based on Spacer gene-directed insertion into HiBit tag and its preparation method

[0087] A novel cell model for the stable integration of HBV1.3-HiBit DNA was used for the preparation of recombinant viruses. The specific method is as follows:

[0088] (1) Cell seeding: The monoclonal cell line was expanded and seeded into T175 cell flasks;

[0089] (2) Obtaining cell culture supernatant: When the cell density reaches 90%, the medium is replaced with a medium containing 2% DMSO for induction. The cell culture supernatant is collected every two days and replaced with fresh toxin-producing medium.

[0090] (3) Recombinant virus filtration and concentration: The cell culture supernatant collected above was centrifuged at 2000 rpm / min for 10 min to remove cell debris, and then filtered through a 0.22 μm filter before use. The virus was concentrated using a Plus-70 Millipore column and centrifuged at 5000 rpm / min at 4°C for 60 min to obtain concentrated virus solution. The concentrated virus solution was then aliquoted and frozen at -80°C.

[0091] (4) Recombinant Virus Titer Detection: The recombinant virus titer was detected using a hepatitis B virus nucleic acid detection kit. Detailed procedures are described in the kit's instruction manual. Results are as follows: Figure 5 As shown, this model can yield recombinant viruses with titers greater than 10. 9 copies / ml.

[0092] Example 3: In vitro infection with novel recombinant hepatitis B virus

[0093] The specific method for using cell lines based on Spacer gene-directed insertion of HiBit tags in screening and evaluating novel anti-hepatitis B virus drugs includes:

[0094] (1) Cell seeding: Taking HepG2-NTCP cells as an example, after counting, cells were seeded at a density of 3×10⁶ cells per cell line. 5 Cells / wells were seeded in collagen-coated 24-well culture plates.

[0095] (2) When the HepG2-NTCP cells reach 100% confluence, replace the medium with DMEM containing 2% DMSO, 10% fetal bovine serum and 1% penicillin-streptomycin.

[0096] (3) After incubating HepG2-NTCP cells in DMEM medium containing 2% DMSO for 48 h, HepG2-NTCP cells were infected with hepatitis B virus at MOI 100 for 24 h, with MyrB as the control group. After 24 h, the virus solution was discarded, and the cells were washed three times with PBS. Subsequently, DMEM medium containing 2% DMSO, 10% fetal bovine serum, and 1% penicillin-streptomycin was added, and the cells were cultured for another 7 days. Subsequent indicators were detected 7 days after infection.

[0097] The results are as follows Figure 6 As shown, this indicates that the novel recombinant virus has the ability to replicate and infect, and can support the entire life cycle of the virus.

[0098] Finally, it should be noted that the terms “comprising,” “including,” or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0099] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of the invention.

[0100] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A method for preparing a cell line based on the directional insertion of a Spacer gene into a HiBit tag, characterized in that, The method includes: The vector pCDN3.1-CMV-G418 was digested with SacI / PmeI to obtain the linearized vector backbone fragment; Primers shown in SEQ ID NO.4-SEQ ID NO.5 and primers shown in SEQ ID NO.6-SEQ ID NO.7 were used to amplify the D-type HBV 1.3 ploid plasmid, obtaining fragment 1 and fragment 2, respectively; The linearized vector backbone fragment, fragment 1, and fragment 2 were subjected to homologous recombination and transformation to obtain the recombinant plasmid pCDN3.1-CMV-HBV1.3-HiBit-G418; The recombinant plasmid pCDN3.1-HBV1.3-HiBit-G418 was digested with ScaI to obtain a linearized fragment, which was then transfected into HepG2 cells and selected for G418 resistance to obtain a polyclonal cell line. The polyclonal cell line was then selected for G418 resistance to obtain a cell line based on the directional insertion of the Spacer gene into the HiBit tag.

2. A cell line based on the Spacer gene-directed insertion of a HiBit tag, prepared using the method of claim 1.

3. A method for preparing a recombinant virus based on the directional insertion of a Spacer gene into a HiBit tag, characterized in that, The method includes: When the cell line based on the Spacer gene directionally inserted into the HiBit tag as described in claim 2 is expanded and cultured to a cell density of 70%-90%, it is replaced with a medium containing DMSO for induction. The cell culture supernatant is collected and replaced with a fresh toxin-producing medium. The cell culture supernatant was centrifuged and filtered to obtain concentrated viral fluid, which is a recombinant virus based on the Spacer gene with a HiBit tag inserted in a directional manner.

4. The method according to claim 3, characterized in that, The volume of DMSO added to the DMSO-containing culture medium is 2% ± 0.5% of the culture medium volume.

5. A recombinant virus based on the Spacer gene-directed insertion of a HiBit tag, prepared by the method of claim 3 or 4.

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