A recombinant BHK-21 cell line overexpressing HS3ST5 gene and its application in enhancing virus replication

Abstract

The application provides a recombinant BHK-21 cell line overexpressing HS3ST5 gene and application thereof in enhancing virus replication, and belongs to the technical field of genetic engineering. The application provides that a recombinant lentivirus plasmid is constructed by cloning a HS3ST5 fragment into a lentivirus vector, and then the lentivirus is packaged by virus rescue, and BHK-21 cells are infected to obtain the recombinant BHK-21 cell line overexpressing HS3ST5. The recombinant BHK-21 cell line overexpressing HS3ST5 can promote adsorption and replication of FMDV, which provides an effective tool for preparation of virus vaccine antigen, and lays a foundation for subsequent research on virus infection and prevention.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of genetic engineering, and particularly relates to a recombinant BHK-21 cell line overexpressing HS3ST5 gene and application thereof in enhancing virus replication. BACKGROUND

[0002] In 1961, British scholars first isolated and established a baby hamster kidney cell line (BHK-21) from 1-day-old Syrian hamster kidney cells. BHK-21 cells are fibrous and can be continuously passaged, and are one of the most common animal cell types. BHK-21 cells have the advantages of being able to be cultured in suspension, fast growth, and wide virus sensitivity spectrum, and are widely used in large-scale preparation of viral vaccine antigens. At present, a variety of viruses have been propagated using BHK-21 cells, including Food-and-mouth disease virus (FMDV), Japanese encephalitis virus (JEV), Rabies virus (RV), Newcastle disease virus (NDV), and Sindbis virus (SV). Since BHK-21 cells have been domesticated to grow in suspension, they have been most widely used in the field of FMD vaccine production and are one of the ideal cell lines for FMDV proliferation. Today, there are also reports of using BHK-21 suspension cells to express recombinant proteins to prepare subunit vaccines. Therefore, BHK-21 cells are an important engineering cell in the biological product industry and show great application potential. In order to improve the production performance of the cells, the modification of BHK-21 cells using different strategies is continuously updated.

[0003] Heparan sulfate (HS) is a class of linearly sulfated biomolecules. Functional HS carries a large negative charge under physiological conditions, recognizing and binding to positively charged viral particles through weak interactions, thereby enhancing the virus's ability to infect host cells. Therefore, HS can act as a receptor in cell-virus interactions, improving viral replication. HS has been shown to assist various viruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), herpes simplex virus type 1 (HSV-1), and FMDV, in invading cells, significantly enhancing viral proliferation on host cells. The sulfatyltransferase HS3ST5 modifies HS through sulfation, forming 3-O-sulfated HS, which plays a role in regulating the biological functions of HS. Currently, there are no reports of BHK-21 cell lines stably expressing HS3ST5, and it remains unclear whether recombinant BHK-21 cells stably expressing HS3ST5 can enhance the replication performance of viruses that use HS as a receptor.

[0004] FMDV is a classic small RNA virus capable of infecting host cells using the HS receptor. FMDV attaches to host cells by recognizing and binding to the HS receptor on the cell membrane surface, and then enters the host cell via HS-mediated endocytosis, initiating viral replication. Studies on the interaction between FMDV and HS have largely focused on the utilization efficiency and capacity of key amino acid residues on different FMDV strains and within the same strain for HS. The effect of the HS-modifying enzyme HS3ST5 on FMDV replication remains unclear. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a recombinant BHK-21 cell line that overexpresses the HS3ST5 gene. The cell line that overexpresses the HS3ST5 gene can enhance viral replication and can be used to prepare viral vaccine antigens on a large scale.

[0006] The present invention provides a recombinant BHK-21 cell line that overexpresses the HS3ST5 gene, wherein the recombinant BHK-21 cell line overexpresses the HS3ST5 protein.

[0007] Preferably, the amino acid sequence of the HS3ST5 protein is shown in SEQ ID NO: 2.

[0008] This invention provides a method for constructing the recombinant BHK-21 cell line, comprising the following steps:

[0009] 1) Construct a recombinant lentiviral plasmid overexpressing the HS3ST5 gene;

[0010] 2) The recombinant lentiviral plasmid overexpressing the HS3ST5 gene described in step 1) and the helper plasmid were co-transfected into cells to rescue the recombinant lentivirus overexpressing the HS3ST5 gene.

[0011] 3) Infect BHK-21 cells with the recombinant lentivirus overexpressing the HS3ST5 gene, and obtain a recombinant BHK-21 cell line that stably expresses HS3ST5 after screening.

[0012] Preferably, the construction method described in step 1) involves amplifying the HS3ST5 gene using primers containing Xba I and Not I restriction sites. The amplified fragment and the lentiviral vector pLOV-CMV-EGFP are then digested with Xba I and Not I, respectively. The digested fragments are ligated with the linearized vector and identified to obtain a recombinant lentiviral plasmid overexpressing the HS3ST5 gene.

[0013] Preferably, primers containing Xba I and Not I restriction sites include a forward primer with a nucleotide sequence as shown in SEQ ID NO:3 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO:4.

[0014] Preferably, the screening method in step 3) is to use puromycin for screening;

[0015] The concentration of the puromycin was 4 μg / mL.

[0016] This invention provides the application of the recombinant BHK-21 cell line or the recombinant BHK-21 cell line constructed by the construction method in enhancing viral replication.

[0017] This invention provides the application of the recombinant BHK-21 cell line or the recombinant BHK-21 cell line constructed by the construction method in the production of viral vaccines.

[0018] Preferably, the virus includes viruses that invade cells using the HS receptor.

[0019] Preferably, the virus that invades cells using the HS receptor includes one or more of foot-and-mouth disease virus, severe acute respiratory syndrome coronavirus, and herpes simplex virus.

[0020] This invention provides a recombinant BHK-21 cell line overexpressing the HS3ST5 gene, wherein the recombinant BHK-21 cell line overexpresses the HS3ST5 protein. Using FMDV as a viral model, this invention measured the adsorption and replication levels of FMDV on the recombinant BHK-21 cell line. The results showed that recombinant BHK-21 cells stably expressing HS3ST5 promoted FMDV adsorption and gene replication. It is evident that overexpression of the HS3ST5 gene in BHK-21 cells can affect FMDV adsorption to cells and promote FMDV infection. Furthermore, given that BHK-21 cells are frequently used for large-scale preparation of viral vaccine antigens, the recombinant BHK-21 cell line overexpressing the HS3ST5 gene provided by this invention offers an effective tool for the preparation of viral vaccine antigens, laying the foundation for subsequent research on viral infection and prevention. Attached Figure Description

[0021] Figure 1 The results of HS3ST5 gene sequence alignment in CHO-K1 and BHK-21 cells;

[0022] Figure 2 Results of HS3ST5 gene amplification;

[0023] Figure 3 The results show the expression of recombinant lentiviral plasmids with the EGFP tag in HEK-293T cells;

[0024] Figure 4 The results show the expression of EGFP in the monoclonal recombinant BHK-21 cell line;

[0025] Figure 5 Results of HS3ST5 protein expression analysis in the BHK-HS3ST5-OE-1 recombinant cell line;

[0026] Figure 6 Results of HS3ST5 mRNA level detection in BHK-HS3ST5-OE-1 recombinant cell line;

[0027] Figure 7 The results of the effect of recombinant BHK-21 cells stably expressing HS3ST5 on FMDV adsorption;

[0028] Figure 8 The effect of recombinant BHK-21 cells stably expressing HS3ST5 on FMDV replication. Detailed Implementation

[0029] The present invention provides a recombinant BHK-21 cell line that overexpresses the HS3ST5 gene, wherein the recombinant BHK-21 cell line overexpresses the HS3ST5 protein.

[0030] In this invention, the amino acid sequence of the HS3ST5 protein is preferably as shown in SEQ ID NO: 2.

[0031] This invention provides a method for constructing the recombinant BHK-21 cell line, comprising the following steps:

[0032] 1) Construct a recombinant lentiviral plasmid overexpressing the HS3ST5 gene;

[0033] 2) The recombinant lentiviral plasmid overexpressing the HS3ST5 gene described in step 1) and the helper plasmid were co-transfected into cells to rescue the recombinant lentivirus overexpressing the HS3ST5 gene.

[0034] 3) Infect BHK-21 cells with the recombinant lentivirus overexpressing the HS3ST5 gene, and obtain a recombinant BHK-21 cell line that stably expresses HS3ST5 after screening.

[0035] This invention constructs a recombinant lentiviral plasmid that overexpresses the HS3ST5 gene.

[0036] In this invention, the construction method preferably uses primers containing Xba I and NotI restriction sites to amplify the HS3ST5 gene. The amplified fragment and the lentiviral vector pLOV-CMV-EGFP are then digested with Xba I and Not I, respectively. The digested fragments are ligated with the linearized vector, and after identification, a recombinant lentiviral plasmid overexpressing the HS3ST5 gene is obtained.

[0037] In this invention, the nucleotide sequence of the HS3ST5 gene is shown in SEQ ID NO:1.

[0038] In this invention, the primers containing Xba I and Not I restriction sites preferably include a forward primer with a nucleotide sequence as shown in SEQ ID NO:3 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO:4. The preferred amplification reaction system is as follows: reverse transcription at 50°C for 30 min; pre-denaturation at 94°C for 5 min; denaturation at 94°C for 1 min, annealing at 57°C for 1 min, extension at 72°C for 2.5 min, repeated 35 times; and a final extension at 72°C for 10 min.

[0039] This invention does not impose any particular limitation on the method of double digestion and ligation with Xba I and Not I; any method well-known in the art for double digestion and ligation with Xba I and Not I can be used. The preferred method for identification is vector sequencing, and the nucleotide sequence of the primers used for sequencing is shown in SEQ ID NO:5.

[0040] After obtaining the recombinant lentiviral plasmid overexpressing the HS3ST5 gene, the present invention co-transfects cells with the recombinant lentiviral plasmid overexpressing the HS3ST5 gene and the helper plasmid to rescue the recombinant lentivirus overexpressing the HS3ST5 gene.

[0041] In this invention, the co-transfected cells preferably comprise the following components by weight: 10 parts recombinant lentiviral plasmid, 7.5 parts psPAX2 helper plasmid, and 2.5 parts pMD2.G helper plasmid. The preferred transfection reagent for co-transfecting the cells is Lipofectamine 2000. This invention does not limit the type of cell; any cell well-known in the art can be used. In this embodiment, HEK-293T cells are used as the infecting cells.

[0042] After obtaining the recombinant lentivirus, the present invention infects BHK-21 cells with the recombinant lentivirus and selects a recombinant BHK-21 cell line that stably expresses HS3ST5.

[0043] In this invention, the preferred method for infecting BHK-21 cells with recombinant lentivirus is to mix lentivirus solution and complete cell culture medium at a volume ratio of 1:1 to prepare a mixed culture medium, add it to BHK-21 cells in the logarithmic growth phase, and replace the mixed culture medium sequentially every 24 hours.

[0044] In this invention, the screening method preferably uses puromycin. The concentration of puromycin is 4 μg / mL.

[0045] In this invention, the recombinant BHK-21 cell line preferably includes verification. The verification method preferably includes RT-qPCR detection and Western Blot analysis. In this embodiment, RT-qPCR detection showed a significant increase in HS3ST5 mRNA levels in the selected cell lines, indicating successful construction of a recombinant cell line overexpressing HS3ST5. Western Blot analysis showed no significant difference in HS3ST5 protein expression levels between the 5th and 20th generations of the recombinant BHK-21 cell line, indicating that the recombinant BHK-21 cell line can stably express HS3ST5 protein.

[0046] In this invention, recombinant BHK-21 cells that stably express HS3ST5 promote the adsorption and replication of FMDV.

[0047] This invention provides the application of the recombinant BHK-21 cell line or the recombinant BHK-21 cell line constructed by the construction method in enhancing viral replication.

[0048] This invention provides the application of the recombinant BHK-21 cell line or the recombinant BHK-21 cell line constructed by the construction method in the production of viral vaccines.

[0049] In this invention, the virus preferably includes a virus that invades cells using the HS receptor. The virus that invades cells using the HS receptor includes one or more of foot-and-mouth disease virus, severe acute respiratory syndrome coronavirus, and herpes simplex virus. In this embodiment of the invention, the application of the recombinant BHK-21 cell line in enhancing viral replication is illustrated using foot-and-mouth disease virus as an example.

[0050] The following examples illustrate a recombinant BHK-21 cell line overexpressing the HS3ST5 gene and its application in enhancing viral replication, but these examples should not be construed as limiting the scope of protection of this invention.

[0051] Description of the source of experimental materials and reagents

[0052] 1. Cells, plasmids, and viruses

[0053] BHK-21 cells were obtained from the China Center for Type Culture Collection, HEK-293T cells were obtained from the China Center for Type Culture Collection, JM109 competent cells were purchased from TaKaRa, pMD2.G, psPAX2 and pLOV-CMV-EGFP plasmids were purchased from Invitrogen, and the classic O-type foot-and-mouth disease vaccine strain O / HN / CHA / 93 (Cathay) was provided by the National Foot-and-Mouth Disease Reference Laboratory of China.

[0054] 2. Main reagents and instruments

[0055] Rabbit HS3ST5 polyclonal antibody was purchased from Novus, mouse β-actin monoclonal antibody from Kangwei Century, RNeasy Mini Kit from Qiagen, phosphate-buffered saline (PBS) from BI Biosciences, astragalus gum from MPBiomedicals, high-glucose DMEM, MEM, Lipofectamine 2000, Opti-MEM and trypsin from Invitrogen, fetal bovine serum (FBS) and puromycin from Gibco, plasmid mini-extraction kit and plasmid mass extraction kit from Tiangen Biotech (Beijing) Co., Ltd., lentivirus rapid detection card from Beijing Bio-Long Immunotherapy Co., Ltd., and ChamQ SYBR qPCR MasterMix from Novizan.

[0056] 3. Primer design and synthesis

[0057] Primers were designed based on the HS3ST5 gene sequence from NCBI. The full-length HS3ST5 gene in BHK-21 cells was amplified by RT-PCR. The designed upstream primer was 5'-ATGAAAAGCGTAGTAGTGATGAGT-3' (SEQ ID NO:3), and the downstream primer was 5'-TTAGGGCCAGTTCAATGTCCTCCCAGTGAT-3' (SEQ ID NO:4). The upstream primer sequence for RT-qPCR amplification of the HS3ST5 gene fragment was 5'-CCATTTGCCCTGTTGAAAGCC-3' (SEQ ID NO:6), and the downstream primer sequence was 5'-CCGGAATTCATGCAGCAGAC-3' (SEQ ID NO:7). The upstream primer sequence for the internal reference gene GAPDH was 5'-CAAGAAGGTGGTGAAGCA-3' (SEQ ID NO:8), and the downstream primer sequence was 5'-AAGTGGAAGAGTGAGTGTC-3' (SEQ ID NO:9). The upstream primer sequence for RT-qPCR amplification of the FMDV 3D gene was 5'-ACTGGGTTTTACAAACCTGTGA-3' (SEQ ID NO:10), and the downstream primer sequence was 5'-GCGAGTCCTGCCACGACGGA-3' (SEQ ID NO:11). All primers were synthesized by Genewiz Biotechnology Co., Ltd.

[0058] Example 1

[0059] Amplification of the HS3ST5 gene in BHK-21 cells

[0060] The HS3ST5 gene in BHK-21 cells was amplified using the primers designed and synthesized above. The PCR reaction system is shown in Table 1, and the reaction conditions are as follows: reverse transcription at 50℃ for 30 min; pre-denaturation at 94℃ for 5 min; denaturation at 94℃ for 1 min, annealing at 57℃ for 1 min, extension at 72℃ for 2.5 min, for 35 cycles; and a final extension at 72℃ for 10 min. The PCR products were subjected to 1% agarose gel electrophoresis, followed by DNA agarose gel recovery, and the recovered products were sent to Genewiz Biotechnology Co., Ltd. for sequencing.

[0061] Table 1 RT-PCR system

[0062]

[0063] Results of HS3ST5 gene amplification in BHK-21 cells:

[0064] Using extracted BHK-21 cell genome as a template, the full-length HS3ST5 gene was amplified using designed primers. Agarose gel electrophoresis results showed successful amplification of the HS3ST5 gene in BHK-21 cells. (Electrophoresis image shown below.) Figure 1 The amplified band was approximately 1000 bp in size. After the target band was recovered from the gel, it was sent to the company for sequencing. The sequencing results showed that the HS3ST5 gene sequence length in BHK-21 was 1038 nts, while the previously sequenced HS3ST5 gene sequence length in CHO-K1 was 981 nts.

[0065] The HS3ST5 sequence of BHK-21 and CHO-K1 cells was further aligned using Megalign software, and significant differences in the HS3ST5 gene sequence were found between the two cell types.

[0066] The results are as follows Figure 2 As shown, compared with the HS3ST5 gene in BHK-21 cells, the HS3ST5 gene in CHO-K1 cells has a continuous deletion of 46 nts at the N-terminus, suggesting that the function of the HS3ST5 gene may be different between the two cell types.

[0067] Example 2

[0068] Construction of recombinant lentiviral plasmid overexpressing BHK-21 cell-derived HS3ST5

[0069] The lentiviral vector pLOV-CMV-EGFP was digested with Xba I and NotI enzymes, and an 8000 bp fragment was recovered. The recovered fragment was ligated with the HS3ST5 amplified fragment to construct a recombinant lentiviral plasmid, named pLOV-EGFP-BHK-HS3ST5. The pLOV-EGFP-BHK-HS3ST5 plasmid was identified by agarose gel electrophoresis. Positive plasmids were sent to Genewiz Biotechnology Co., Ltd. for sequencing using the primer 5'-CGGTGAATGCTGGTGGCATC-3' (SEQ ID NO: 5). Numerous positive recombinant plasmids, as well as the helper plasmids pMD2.G and psPAX2, were extracted and identified.

[0070] Construction and identification results of HS3ST5 recombinant lentiviral plasmid:

[0071] The pLOV-CMV-EGFP vector was double-digested with Xba I and Not I. Two fragments were observed by agarose gel electrophoresis. The 8000bp fragment was recovered and ligated with an amplified fragment from HS3ST5 derived from BHK-21 cells. After preliminary identification by agarose gel electrophoresis, positive clones were sent for further analysis, confirming the successful construction of the recombinant lentiviral plasmid pLOV-EGFP-BHK-HS3ST5. The correctly sequenced positive plasmid was extracted and stored for later use.

[0072] Example 3

[0073] 1. Rescue of recombinant lentiviruses overexpressing HS3ST5

[0074] Normal HEK-293T cells were seeded in 10cm cell culture dishes. When the cells were in good condition and the density reached 70%, plasmid transfection was performed using Lipofectamine 2000 transfection reagent (10μg recombinant lentiviral plasmid + 7.5μg psPAX2 helper plasmid + 2.5μg pMD2.G helper plasmid). After 6 hours, 4ml of high-glucose DMEM complete medium was gently added to the cell culture dish, and the cells were incubated at 37°C. During incubation, the green fluorescence intensity of the transfected cells was observed multiple times using a fluorescence microscope. After 48 hours, the cell supernatant was collected and filtered through a 0.45μm filter. The lentivirus titer in the supernatant was determined using a lentivirus rapid detection card. The filtered lentivirus solution was stored at -40°C for later use.

[0075] 2. Determination of the screening concentration of puromycin

[0076] BHK-21 cells were seeded into six-well plates. Once the cell density reached 90%, puromycin was added at concentrations of 1, 2, 3, 4, 5, and 6 μg / mL, respectively, with treatment repeated every 24 hours. Cell viability was observed after 7 days, and the lowest drug concentration at which no cells survived was the optimal concentration for puromycin screening.

[0077] 3. Construction of a recombinant BHK-21 cell line stably expressing HS3ST5

[0078] Lentiviral solution and complete cell culture medium were mixed at a 1:1 volume ratio to prepare a mixed culture medium. Normal BHK-21 cells were seeded in six-well plates and cultured in the mixed culture medium. After the cells reached confluence, they were transferred to cell culture flasks, and the mixed culture medium was added and changed every 24 hours. After 7 days, the degree of green fluorescence of the cells after lentivirus infection was observed using a fluorescence microscope. Lentivirally infected cells were treated with the optimal concentration of puromycin, and the complete culture medium containing puromycin was changed every 24 hours. After 7 days, almost all surviving BHK-21 cells were recombinant cells carrying the recombinant lentiviral plasmid. Recombinant BHK-21 cells overexpressing HS3ST5 were counted, diluted to single cells, and added to 96-well plates. After the cells grew into clusters, they were observed under a microscope to see if they were single clones. After the cells reached confluence, the single-clone recombinant cells were expanded and cryopreserved, and the expression level of HS3ST5 in the single-clone recombinant cells was detected by Western blotting and real-time quantitative PCR (RT-qPCR). The primary antibodies used in Western blotting experiments were a rabbit-derived polyclonal antibody against HS3ST5 and a mouse-derived monoclonal antibody against β-actin. The secondary antibodies used were HRP-labeled goat anti-mouse IgG and HRP-labeled goat anti-rabbit IgG. The reverse transcription system for RT-qPCR is shown in Table 2, with a program of 37℃ for 15 min and 85℃ for 5 s. The amplification system for RT-qPCR is shown in Table 3, with a program of 95℃ for 30 s pre-denaturation; 95℃ for 5 s, 60℃ for 30 s, 72℃ for 30 s, for 40 cycles; then 95℃ for 15 s, 60℃ for 1 min, 95℃ for 30 s, and 60℃ for 15 s. The relative mRNA copy number of HS3ST5 was calculated using the ΔΔCT method.

[0079] Table 2. RT-qPCR reverse transcription system for HS3ST5

[0080]

[0081] Table 3. RT-qPCR amplification system for HS3ST5

[0082]

[0083] Establishment and identification of recombinant BHK-21 cell lines stably expressing HS3ST5:

[0084] HEK-293T cells were transfected with the positive recombinant lentiviral plasmid pLOV-EGFP-BHK-HS3ST5. After 48 hours, the cells were observed under a fluorescence microscope to determine if they emitted green fluorescence. The results are as follows: Figure 3As shown, strong green fluorescence was observed in the transfected cells under a fluorescence microscope. Since the recombinant lentiviral plasmid carries an EGFP protein tag, the successful expression of EGFP protein in HEK-293T cells indicates successful transfection of the recombinant lentiviral plasmid into HEK-293T cells, demonstrating successful rescue of the recombinant lentiviral overexpression. Treatment of BHK-21 cells with gradient concentrations of puromycin revealed that all BHK-21 cells died after one week of selection with a minimum concentration of 4 μg / mL of puromycin. Therefore, the optimal puromycin concentration for screening the BHK-21 recombinant cell line was determined to be 4 μg / mL.

[0085] After successful infection with recombinant lentivirus and selection with optimal concentrations of puromycin, BHK-21 cells were observed under a fluorescence microscope to determine whether they emitted green fluorescence. Results are as follows: Figure 4 As shown, fluorescence microscopy revealed strong green fluorescence in BHK-21 cells infected with the recombinant lentivirus, indicating successful infection and the initial successful construction of the recombinant BHK-21 cell line overexpressing HS3ST5. The constructed recombinant cell line was named BHK-HS3ST5-OE, and three monoclonal cell lines with strong fluorescence were selected for cryopreservation.

[0086] BHK-HS3ST5-OE-1 was selected to validate the expression levels of HS3ST5 protein and mRNA. The results are as follows: Figure 5 As shown, Western blotting results indicated that the HS3ST5 protein level was significantly increased in the recombinant cell line BHK-HS3ST5-OE-1 compared to normal BHK-21 cells.

[0087] RT-qPCR results are as follows Figure 6 As shown, the HS3ST5 mRNA level in BHK-HS3ST5-OE-1 cells was significantly increased compared to normal BHK-21 cells. These results indicate that a recombinant BHK-21 cell line stably expressing HS3ST5 was successfully established.

[0088] Example 4

[0089] Effect of recombinant BHK-21 cells stably expressing HS3ST5 on FMDV adsorption

[0090] The effect of recombinant BHK-21 cells on FMDV adsorption was evaluated by detecting FMDV 3D mRNA expression levels. Cells were first counted, then FMDV pre-chilled at 4°C with an infection dose (MOI) of 1 was added, and the cells were incubated at 4°C for 2 h to allow for sufficient FMDV adsorption. Unadsorbed FMDV was then washed away with pre-chilled PBS buffer at 4°C. RNA lysis buffer was then added, and RNA was extracted from the samples. The 3D mRNA level of FMDV adsorbed to the cell surface was detected using RT-qPCR. The reverse transcription system for RT-qPCR is shown in Table 4, with a reverse transcription program of 37°C for 15 min, 85°C for 5 s. The amplification system for RT-qPCR is shown in Table 5, with an amplification program of 95°C for 30 s pre-denaturation; 95°C for 5 s, 60°C for 30 s, 72°C for 30 s, 40 cycles; 95°C for 15 s, 60°C for 1 min, 95°C for 30 s, 60°C for 15 s. The relative mRNA copy number of FMDV 3D was calculated using the ΔΔCT method.

[0091] Table 4. RT-qPCR reverse transcription system for FMDV 3D

[0092] Ingredients Volume RNA (1000 / concentration) μL 5x RF Mix 4 μL Water to 20 μL

[0093] Table 5. Amplification system of FMDV 3D RT-qPCR

[0094] Ingredients Volume 2x TB Green Premix EX Taq 10 μL cDNA 2 μL Upstream primer 0.4 μL Downstream primer 0.4 μL ROX Dye 0.4 μL Water 6.8 μL

[0095] result:

[0096] The effect of BHK-HS3ST5-OE-1 recombinant cells on FMDV adsorption was detected using RT-qPCR. Results are as follows: Figure 7 As shown, compared with normal BHK-21 cells, the FMDV mRNA level in recombinant BHK-21 cells stably expressing HS3ST5 was significantly increased. The viral mRNA level in BHK-HS3ST5-OE-1 cells was 3.5 times that in control cells, indicating that recombinant BHK-21 cells stably expressing HS3ST5 are conducive to FMDV adsorption.

[0097] Example 5

[0098] Effect of recombinant BHK-21 cells stably expressing HS3ST5 on FMDV replication

[0099] Recombinant BHK-21 cells were inoculated with the virus at an MOI of 1 and incubated at 37°C. RNA lysis buffer was added and samples were collected at 4, 6, 8, 10, 12, and 16 hours. RNA was extracted, and the replication level of FMDV was detected using RT-qPCR.

[0100] result:

[0101] The effect of BHK-HS3ST5-OE-1 recombinant cells on FMDV gene replication was detected using RT-qPCR. Results are as follows: Figure 8 As shown, for all time points, the FMDV mRNA level in recombinant BHK-21 cells stably expressing HS3ST5 was significantly increased compared to normal BHK-21 cells. The FMDV mRNA levels in both BHK-HS3ST5-OE-1 recombinant cells and normal BHK-21 cells peaked at 12 h, at which point the FMDV mRNA level in BHK-HS3ST5-OE-1 cells was 1.8 times that in control cells. These results indicate that recombinant BHK-21 cells stably expressing HS3ST5 increased FMDV replication levels.

[0102] As shown in the above examples, the HS3ST5 gene in BHK-21 cells was amplified by RT-PCR and cloned into the pLOV-CMV-EGFP vector to construct a recombinant lentiviral plasmid. Then, the recombinant lentiviral plasmid pLOV-EGFP-BHK-HS3ST5, along with the helper plasmids pMD2.G and psPAX2, was transfected into HEK-293T cells using liposome transfection technology. The lentivirus packaged from HEK-293T cells was harvested and then used to infect BHK-21 cells. After 10 days of continuous selection with puromycin, a recombinant BHK-21 cell line expressing HS3ST5 was obtained. The monoclonal recombinant BHK-21 cell line stably expressing HS3ST5 was screened by limiting dilution and observation of green fluorescence intensity.

[0103] Using recombinant BHK-21 cells stably expressing HS3ST5 as a viral model, the effect of FMDV adsorption and replication on recombinant BHK-21 cells was determined. The results showed that recombinant BHK-21 cells stably expressing HS3ST5 promoted FMDV adsorption and replication. Therefore, recombinant BHK-21 cells stably expressing HS3ST5 significantly enhanced FMDV replication capacity.

[0104] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A recombinant BHK-21 cell line overexpressing a HS3ST5 gene derived from a BHK-21 cell line, characterized in that, The recombinant BHK-21 cell line overexpresses a HS3ST5 protein, and the amino acid sequence of the HS3ST5 protein is shown as SEQ ID NO:

2.

2. A method of constructing the recombinant BHK-21 cell line of claim 1, wherein, The method comprises the following steps: 1) constructing a recombinant lentivirus plasmid overexpressing a HS3ST5 gene from a BHK-21 cell line; 2) co-transfecting the recombinant lentivirus plasmid overexpressing the HS3ST5 gene and a helper plasmid in step 1) into cells to rescue a recombinant lentivirus overexpressing the HS3ST5 gene; 3) infecting BHK-21 cells with the recombinant lentivirus overexpressing the HS3ST5 gene, and screening to obtain a recombinant BHK-21 cell line stably expressing HS3ST5.

3. The method of claim 2, wherein, The construction method in step 1) uses a primer containing Xba I and Not I restriction sites to amplify the HS3ST5 gene, and the obtained amplification fragment and the lentiviral vector pLOV-CMV-EGFP are respectively digested by Xba I and Not I, the digested fragments and the linearized vector are connected, identified, and the recombinant lentiviral plasmid overexpressing the HS3ST5 gene is obtained.

4. The method of claim 3, wherein, containing Xba I and Not Primers including a restriction enzyme site include a forward primer having a nucleotide sequence as set forth in SEQ ID NO: 3 and a reverse primer having a nucleotide sequence as set forth in SEQ ID NO:

4.

5. The construction method according to any one of claims 2 to 4, characterized in that, The screening method in step 3) is screening by using puromycin. The concentration of the puromycin is 4 μg / mL.

6. The recombinant BHK-21 cell line of claim 1 or the recombinant BHK-21 cell line constructed by the method of any one of claims 2-5 is applied to enhancing virus replication; and the virus is foot-and-mouth disease virus.

7. The recombinant BHK-21 cell line of claim 1 or the recombinant BHK-21 cell line constructed by the method of any one of claims 2-5 is applied to producing a virus vaccine; and the virus is foot-and-mouth disease virus.

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