BHK-21 cell line stably expressing encephalitis virus NS1 protein and construction method and application thereof

Abstract

The application discloses a BHK-21 cell line stably expressing Japanese encephalitis virus NS1 protein and a construction method and application thereof, and the method comprises the following steps: (1) amplifying a Japanese encephalitis virus NS1 gene and constructing a recombinant lentivirus plasmid; (2) co-transfecting the recombinant lentivirus plasmid and an auxiliary plasmid into cells of 293T and a derivative cell line to obtain a recombinant lentivirus particle through packaging; (3) infecting BHK-21 cells with the recombinant lentivirus particle, and obtaining a mixed cell line stably expressing NS1 protein through antibiotic screening and screening a monoclonal cell strain; and (4) verifying the expression of the NS1 protein at a gene and protein level respectively. The construction method firstly utilizes a lentivirus expression system to construct a BHK-21 cell line stably expressing Japanese encephalitis virus (JEV) NS1 protein. The cell line can efficiently and stably express the JEV NS1 protein, and has important scientific research and clinical application values.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, specifically to a BHK-21 cell line that stably expresses the NS1 protein of Japanese encephalitis virus, its construction method, and its applications. Background Technology

[0002] Japanese encephalitis (JE) is an important zoonotic disease caused by Japanese encephalitis virus (JEV), primarily transmitted between hosts by Culex mosquitoes. Pigs are the main amplification host for this virus, while humans and horses are the definitive hosts. JEV belongs to the genus Flaviviridae in the family Flaviviridae. It is a single-stranded positive-sense RNA virus with a genome length of approximately 11 kb, encoding a polyprotein that, after cleavage, forms three structural proteins (C, prM / M, and E) and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5).

[0003] NS1 protein is a key glycoprotein that plays multiple roles in viral infection, replication, and pathogenicity. It is highly expressed in virus-infected cells, can be secreted extracellularly, and even exists in patient serum as a soluble hexamer. NS1 protein not only participates in viral RNA replication but also regulates the host's innate immune response, making it an important target for developing diagnostic reagents and subunit vaccines. Therefore, obtaining cell lines that stably and efficiently express JEV NS1 protein is of significant application value for in-depth research into the pathogenic mechanism of JEV, the biological function of NS1 protein, the screening of antiviral drugs, and the establishment of serological diagnostic methods.

[0004] Currently, while conventional transient transfection methods can rapidly express exogenous proteins, they suffer from drawbacks such as unstable expression levels, fluctuating transfection efficiency, and limitations in large-scale application. Constructing stable expression cell lines using lentiviral vector systems allows for the integration of exogenous genes into the host cell genome, achieving long-term, stable expression of the target protein. However, existing lentiviral vector systems exhibit the following technical limitations when applied to the stable expression of JEV NS1 protein:

[0005] First, in many lentiviral expression vectors, the resistance gene (such as the puromycin resistance gene) and the target gene are expressed by separate promoters. This design leads to some cells integrating only the resistance gene without integrating or expressing low levels of the target gene, allowing them to survive antibiotic selection and generating a large number of "false positive" clones. For proteins like JEV NS1, which may have some adaptive cost to the host cell or slight cytotoxicity, the proportion of false positive clones is even higher, severely interfering with the acquisition of stable expression cell lines.

[0006] Second, the insertion site of the exogenous gene and the restriction enzyme cleavage strategy directly affect the lentiviral packaging efficiency and the expression level of the target gene. Inappropriate restriction enzyme site selection may introduce additional nucleotide sequences, which may form secondary structures that interfere with the initiation of mRNA translation or destroy cis-acting elements necessary for lentiviral genome packaging, leading to a decrease in viral titer and integration efficiency, ultimately resulting in extremely low NS1 protein expression levels after screening and difficulty in maintaining long-term stability.

[0007] Third, the JEV NS1 protein itself has the function of regulating the host immune response and affecting cell signaling pathways. Its high expression in BHK-21 cells may trigger cell stress or growth inhibition. Conventional infection multiples, screening drug concentrations and screening cycles often fail to achieve a balance between "effectively killing unintegrated cells" and "protecting potentially high-expressing cells", which can easily lead to screening failure or rapid decay of the expression level of the obtained cell lines.

[0008] In summary, to achieve stable and efficient expression of JEV NS1 protein in BHK-21 cells, the following technical challenges must be overcome: (1) Selecting or designing lentiviral vectors that enable translational coupling between the resistance gene and the target gene, ensuring that resistance selection is strictly linked to the expression of the target protein, and eliminating false positive escapes; (2) Optimizing the enzyme digestion cloning strategy to complete the targeted insertion of the target gene without introducing redundant sequences or disrupting viral packaging signals; (3) Systematically exploring lentiviral infection conditions, antibiotic selection concentrations, and maintenance protocols to balance cell viability and expression stability. Currently, there are no reports of BHK-21 cell lines that have proposed effective solutions to the above technical challenges and obtained stable expression of JEV NS1 protein. Summary of the Invention

[0009] The purpose of this invention is to provide a BHK-21 cell line that can stably and efficiently express the NS1 protein of Japanese encephalitis virus and its construction method.

[0010] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0011] In a first aspect, the present invention provides a method for constructing a BHK-21 cell line stably expressing the NS1 protein of Japanese encephalitis virus, comprising the following steps:

[0012] (1) Construction of recombinant plasmid: The NS1 gene of Japanese encephalitis virus was amplified and cloned into a lentiviral expression vector to construct a recombinant lentiviral plasmid;

[0013] (2) Preparation of lentiviral particles: The recombinant lentiviral plasmid constructed in step (1) and the helper plasmid were co-transfected into 293T and its derived cell lines, and the recombinant lentiviral particles were packaged.

[0014] (3) Screening of stable expression cell lines: BHK-21 cells were infected with the recombinant lentivirus particles obtained in step (2), and mixed cell lines that stably expressed NS1 protein were obtained by antibiotic screening. Single clone cell lines were then screened by limiting dilution method.

[0015] (4) Detection of NS1 gene nucleic acid and protein levels: For the monoclonal cell lines screened in step (3), the expression of NS1 protein was verified at the gene and protein levels, respectively.

[0016] Preferably, in step (1), the nucleotide sequence of the Japanese encephalitis virus NS1 gene is as shown in SEQ ID NO.1, which is the complete NS1 sequence.

[0017] Preferably, the primer pair for amplifying the NS1 gene includes a forward primer NS1-F and a reverse primer NS1-R, the sequence of the forward primer NS1-F is shown in SEQ ID NO.2, and the sequence of the reverse primer NS1-R is shown in SEQ ID NO.3.

[0018] Preferably, in step (1), the lentiviral expression vector is pSin-EGFP-Ires-Puro. The inventors analyzed the limitations of using other lentiviral expression vectors as follows: Based on the basic principles of lentiviral vector design and numerous literature reports, vectors where the resistance gene and target gene are driven by independent promoters (such as pCDH-CMV-MCS-EF1-Puro, pLenti-CMV-Puro, etc.) are highly prone to false-positive clones due to resistance escape when expressing proteins with slight cytotoxicity or adaptive costs (such as JEV NS1). In contrast, the preferred pSin-EGFP-Ires-Puro vector of this invention utilizes the IRES element to place the puromycin resistance gene and the NS1 gene on the same mRNA, achieving strict coupling between resistance selection and target protein expression, thereby fundamentally avoiding the generation of false-positive clones. If an uncoupled vector is used, even if the infection and selection parameters of this invention are strictly followed, it is difficult to obtain a highly expressed and long-term stable monoclonal cell line. This is also an important innovation of this invention compared to conventional lentiviral construction methods.

[0019] Preferably, the lentiviral system used for transfection includes a recombinant lentiviral plasmid and an auxiliary packaging plasmid, wherein the auxiliary packaging plasmid contains psPAX2 and pMD2.G. Using psPAX2 and pMD2.G together is the standard method for lentiviral packaging. Its core advantages are: 1. High-titer production: Synergistic action is the basis for producing stable, high-titer viral particles. The two work together to maximize viral production efficiency and avoid overall yield reduction caused by the instability of single-function components. 2. Broad host range: Co-expression of the VSV-G protein encoded by pMD2.G endows lentivirus with broad cellular infectivity (pantropy), enabling the virus to efficiently infect various cell types, from nerve cells to immune cells. 3. psPAX2: It provides the viral structural proteins (Gag, Pol, Rev), assembles the lentiviral "capsid," and completes key steps such as reverse transcription of RNA into DNA. 4. pMD2.G: It provides the viral envelope protein (VSV-G), acting like a universal key that can recognize and open the "doors" of various cells. Using either one alone will not produce complete, infectious virus particles. Using psPAX2 or pMD2.G alone will significantly reduce the virus yield, or even fail to obtain a sufficient amount of virus.

[0020] Preferably, in step (1), the NS1 gene amplification product and the lentiviral expression vector are ligated using T4 DNA ligase after being double-digested with EcoRI and SpeI, and then transformed into DH5α competent bacteria.

[0021] In step (2) of the present invention, 293T and its derived cell lines (including 293FT, Lenti-X 293T, 293V) are the most commonly used lentivirus packaging tools due to their high transfection efficiency and high viral yield (i.e. viral production capacity), with 293T being the preferred choice.

[0022] In step (3) of the present invention, the antibiotic used for screening depends on the antibiotic resistance gene on the recombinant lentiviral plasmid in the recombinant lentiviral particle. Preferably, in step (3), the antibiotic is puromycin.

[0023] Preferably, in step (3), the recombinant lentivirus particles infect BHK-21 cells twice.

[0024] Preferably, in step (3), each infection lasts for 24 hours.

[0025] Preferably, in step (3), the screening concentration of puromycin is 6 μg / mL.

[0026] Preferably, in step (3), the screening time is 10 days.

[0027] Preferably, in step (3), the recombinant lentiviral particles infect BHK-21 cells twice, each time for 24 hours; the screening concentration of puromycin is 6 μg / mL, and the screening time is 10 days.

[0028] In step (4) of the present invention, the methods for verifying the expression of NS1 protein at the gene and protein levels include PCR, IFA and Western Blot. At the gene level, specific bands are amplified by PCR, at the protein level, fluorescence is verified by IFA, and at the protein level, protein bands of the appropriate size are verified by Western Blot (or wb).

[0029] Secondly, the present invention provides a BHK-21 cell line that stably expresses the NS1 protein of Japanese encephalitis virus, wherein the BHK-21 cell line is constructed by the method for constructing a BHK-21 cell line that stably expresses the NS1 protein of Japanese encephalitis virus according to any one of claims 1-7.

[0030] Thirdly, the present invention provides a primer set for amplifying the NS1 gene of Japanese encephalitis virus, including a forward primer NS1-CX-F and a reverse primer NS1-CX-R, wherein the sequence of the forward primer NS1-CX-F is shown in SEQ ID NO.4 and the sequence of the reverse primer NS1-CX-R is shown in SEQ ID NO.5.

[0031] Fourthly, the present invention provides a method for constructing a BHK-21 cell line stably expressing the NS1 protein of Japanese encephalitis virus as described above, the BHK-21 cell line stably expressing the NS1 protein of Japanese encephalitis virus as described above, and the application of the primer set described above for amplifying the NS1 gene of Japanese encephalitis virus in the preparation of diagnostic reagents for Japanese encephalitis virus, a screening model for anti-Japanese encephalitis virus drugs, or in the study of NS1 protein function.

[0032] By adopting the above technical solution, the beneficial effects of the present invention include at least the following:

[0033] This invention provides the first-ever construction of a BHK-21 cell line stably expressing the Japanese encephalitis virus (JEV) NS1 protein using a lentiviral expression system. This cell line can efficiently and stably express the JEV NS1 protein, providing an ideal cell model for in-depth research into the biological function of the NS1 protein, elucidating the pathogenic mechanism of JEV, developing NS1 protein-based diagnostic reagents, and establishing antiviral drug screening models. It has significant scientific research and clinical application value.

[0034] Other features and advantages of the present invention will be described in detail in the following detailed description section. Attached Figure Description

[0035] Figure 1 Figure 1 This is a schematic diagram illustrating the construction of the recombinant lentiviral plasmid pSin-NS1.

[0036] Figure 2 The image shows the results of double enzyme digestion identification of recombinant lentiviral plasmid pSin-NS1 (M: DNA Marker; 1: pSin-NS1 digested with EcoRI and SpeI).

[0037] Figure 3 PCR identification results of monoclonal cell lines stably expressing NS1 protein (M: DNA Marker; 1-5: different monoclonal cell lines; 6: negative control).

[0038] Figure 4 Western Blot identification results of monoclonal cell lines stably expressing NS1 protein (1: NS1 positive control; 2: parental BHK-21 cells; 3, 4: different monoclonal cell lines).

[0039] Figure 5 The image shows the results of indirect immunofluorescence (IFA) identification of a monoclonal cell line stably expressing NS1 protein. Detailed Implementation

[0040] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described in the following description are merely illustrative examples of specific implementations of this invention and are intended to explain the invention, but do not constitute a limitation thereof.

[0041] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein. In this invention, when a specific numerical value is mentioned, it means that the value can vary within ±5%. In this invention, unless otherwise stated, terms such as "multiple / a plurality of" mean two / a kind or more. The terms "containing," "comprising," or "including" can be open-ended, semi-closed, or closed.

[0042] The present invention will be described below with reference to specific embodiments. It should be noted that these embodiments are merely illustrative and should not be construed as limiting the invention. It should also be noted that if specific conditions are not specified in the embodiments, the techniques or conditions described in the literature in the art or the product instructions should be followed. If the manufacturers of the reagents or instruments used are not specified, they are all conventional products that can be purchased commercially.

[0043] Example 1 Construction of recombinant lentiviral plasmid pSin-NS1

[0044] 1. Amplification of the NS1 gene

[0045] Based on the NS1 gene sequence of Japanese encephalitis virus strain SD12-F120 (GenBank accession number: MN544779) published in GenBank (SEQ ID NO.1 sequence shown below), specific primers were designed. EcoRI and Spe I restriction sites and protective bases were introduced into the upstream and downstream primers, respectively. A Kozak sequence and an atg start codon were introduced at the N-terminus of the NS1 gene using the upstream primer. The primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd., and the primer sequences are shown in Table 1.

[0046]

[0047]

[0048] Using cDNA from JEV SD12-F120 strain as a template, PCR amplification was performed using the primers described above. The PCR reaction mixture consisted of: 25 μL of 2×Phanta PCR Master Mix, 2 μL each of forward and reverse primers (10 μM), 2 μL of cDNA template, and ddH2O to a final volume of 50 μL. The reaction program was as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 1 min, for a total of 35 cycles; and a final extension at 72℃ for 5 min.

[0049] The PCR product was identified by 1% agarose gel electrophoresis, and the target band of approximately 1056 bp was recovered.

[0050] 2. Enzyme digestion and ligation, including the following steps:

[0051] The PCR product recovered above and the lentiviral expression vector pSin-EGFP-Ires-Puro were double-digested using EcoRI and SpeI restriction endonucleases, respectively. The digestion system was as follows: 1 μg target fragment / vector, 1 μL EcoRI, 1 μL SpeI, 5 μL 10×CutSmart Buffer, and ddH2O to a final volume of 50 μL. The reaction was carried out at 37 °C for 3 h.

[0052] After gel recovery of the digested products, ligation was performed using T4 DNA ligase. The ligation system consisted of: 50 ng of digested pSin-EGFP-Ires-Puro vector, 100 ng of digested NS1 gene fragment, 1 μL of T4 DNA ligase, 1 μL of 10×T4 DNA ligase buffer, and ddH2O to a final volume of 10 μL. Ligation was carried out overnight at 16°C.

[0053] The ligation product was transformed into DH5α competent cells (purchased from Shanghai Weidi Biotechnology Co., Ltd.), plated on LB agar plates containing ampicillin (100 μg / mL), and incubated overnight at 37 ℃. Single colonies were picked for PCR identification, and plasmids were extracted for double enzyme digestion identification (results are shown in Figure 1). Figure 2 (As shown in the diagram) and sequencing verification. The correctly constructed recombinant plasmid was named pSin-NS1 (as shown in the diagram). Figure 1 (As shown).

[0054] Example 2 Preparation of Lentiviral Particles

[0055] 1. Cell transfection

[0056] 24 hours before transfection, healthy 293T cells were passaged into 10 cm culture dishes. Transfection was performed when the cell density reached 70%–80%. Using Lipofectamine™ 3000 transfection reagent (Thermo Fisher Scientific), recombinant lentiviral plasmid pSin-NS1 (15 μg) was co-transfected with helper packaging plasmids psPAX2 (10 μg) and pMD2.G (5 μg). The specific steps included:

[0057] (1) Prepare solution A in a sterile EP tube: Mix 15 μg pSin-NS1, 10 μg psPAX2 and 5 μg pMD2.G, add Opti-MEM medium to a total volume of 500 μL, add 30 μL P3000™ reagent, and gently pipette to mix.

[0058] (2) Prepare solution B in another sterile EP tube: Add 20 μL of Lipofectamine™ 3000 reagent to 500 μL of Opti-MEM medium and mix gently.

[0059] (3) Mix solution A and solution B in a 1:1 ratio and let stand at room temperature for 15 min to form a liposome-DNA complex.

[0060] (4) Discard the old culture medium in the 293T cell culture dish, add 5 mL of Opti-MEM culture medium, and add the above liposome-DNA complex drop by drop evenly into the culture dish, and gently shake to mix evenly.

[0061] (5) After incubating the cells in a 37°C, 5% CO2 incubator for 6 h, discard the transfection mixture and replace it with 10 mL of DMEM containing 2% fetal bovine serum and continue culturing.

[0062] 2. Virus collection and concentration

[0063] Cell supernatants were collected at 48 h and 72 h post-transfection and centrifuged at 3000 rpm for 10 min at 4 °C to remove cell debris. The supernatant was filtered through a 0.22 μm filter and then centrifuged in an ultracentrifuge tube at 25000 rpm for 2 h at 4 °C. The supernatant was discarded, and the virus pellet was resuspended in an appropriate amount of serum-free DMEM medium, aliquoted, and stored at -80 °C for later use.

[0064] Example 3: Screening of BHK-21 cell lines stably expressing NS1 protein

[0065] 1. Lentiviral infection

[0066] 24 hours before infection, BHK-21 cells were seeded into 6-well plates. When the cell density reached 50% to 60%, the culture medium was discarded, and 1 mL of DMEM medium containing 2% fetal bovine serum and 100 μL of concentrated lentivirus stock solution were added. After incubation at 37 °C for 24 hours, the infection was repeated for a second time.

[0067] 2. Screening of stable cell lines

[0068] Forty-eight hours after viral infection, puromycin was added to the culture medium to a final concentration of 6 μg / mL for selection. The culture medium containing puromycin was replaced every 2–3 days. After approximately 10 days of selection, all uninfected control cells died, while resistant cell clones appeared in the infected group. The resistant cells were cultured on a large scale to obtain a mixed cell line stably expressing NS1 protein.

[0069] 3. Screening of monoclonal cell lines

[0070] The above-mentioned mixed cell lines were seeded into 96-well plates using a limiting dilution method, ensuring that each well contained no more than one cell. The cells were then cultured in medium containing 6 μg / mL puromycin. Wells with single-clone growth were selected, and the culture was gradually expanded to obtain single-clone cell lines.

[0071] Example 4 Identification of cell lines stably expressing NS1 protein

[0072] 1. PCR identification

[0073] Single-clonal cell lines were collected, and total RNA was extracted using the Trizol method and reverse transcribed into cDNA. Using the cDNA as a template, PCR amplification was performed using the NS1 gene-specific primers (SEQ ID NO.4 and NO.5) listed in Table 2. Parental BHK-21 cells were used as a negative control. Results are as follows: Figure 3 As shown, the monoclonal cell lines in lanes 2 and 3 successfully amplified a specific band of approximately 500 bp (i.e., successfully expressed cells), while the negative control showed no band, indicating that the NS1 gene has been successfully integrated into the cell genome. Lanes 1, 4, and 5 represent cells that could not express the gene.

[0074]

[0075] 2. Western Blot Identification

[0076] Total protein was extracted from PCR-identified NS1-positive monoclonal cell lines and parental BHK-21 cells using RIPA lysis buffer. After SDS-PAGE electrophoresis, the extracted protein was transferred to an NC membrane. The membrane was blocked with 5% skim milk at room temperature for 1 h, then incubated overnight at 4°C with rabbit anti-JEV NS1 polyclonal antibody (1:1000 dilution). After washing with TBST, HRP-labeled goat anti-rabbit IgG secondary antibody (1:10,000 dilution) was added and incubated at room temperature for 1 h. The results were observed using a gel imaging system after ECL chemiluminescence staining. The results are as follows: Figure 4 As shown, a specific band was detected at approximately 46 kD in both monoclonal cell lines, consistent with the expected size of the NS1 protein, while the parental BHK-21 cells did not show this band, indicating that the NS1 protein was stably expressed.

[0077] 3. Indirect immunofluorescence (IFA) identification

[0078] Monoclonal cell lines were seeded into 24-well plates and cultured for 24 h. After fixation with 4% paraformaldehyde, permeabilization with 0.2% Triton X-100, and blocking with 5% BSA, rabbit anti-JEV NS1 monoclonal antibody (1:200 dilution) was added, and the plates were incubated at 37°C for 1 h. After washing with PBST, FITC-labeled goat anti-rabbit IgG secondary antibody (1:500 dilution) was added, and the plates were incubated at 37°C in the dark for 1 h. Finally, the nuclei were stained with DAPI and observed under a fluorescence microscope. Results are as follows: Figure 5 As shown, bright red fluorescence was visible in the cytoplasm, while the negative control group showed no fluorescence, further confirming the stable expression of NS1 protein in cells.

[0079] In summary, this invention successfully constructed a stable and efficient BHK-21 cell line expressing the NS1 protein of Japanese encephalitis virus, providing a powerful tool for subsequent research and applications.

[0080] To further demonstrate the key role of the technical solutions selected in this invention (especially the number of infections, screening concentration, and enzyme digestion cloning strategy) in achieving stable and efficient expression of JEV NS1 protein in BHK-21 cells, the inventors conducted the following comparative experiments.

[0081] Comparative Example 1: Effect of single infection combined with low-concentration puromycin screening on stable expression.

[0082] Experimental methods: The method of Example 3 was followed, except that: (1) BHK-21 cells were infected with lentivirus only once (24 h after infection), instead of twice; (2) 2 μg / mL of puromycin (instead of 6 μg / mL) was added to the culture medium 48 h after infection for screening, and the screening time was 5 days (instead of 10 days). The remaining steps (including cell culture, virus preparation, limiting dilution method, etc.) were consistent with the example.

[0083] Results: Five days after screening, most of the uninfected control cells died, but a large number of resistant cell clones (more than 100 per 6-well plate) appeared in the infected group. Expanding these resistant cells and detecting NS1 expression, Western blotting showed only a weak NS1-specific band, with the vast majority of clones showing no expression, indicating a very high proportion of false-positive clones.

[0084] The mixed cell lines were further passaged in 2 μg / mL puromycin. By the 5th generation, NS1 expression was almost undetectable by Western blotting. Even when single clones were selected using limiting dilution, NS1 expression was rapidly lost in the obtained clones after the 3rd generation.

[0085] Conclusion: Single infection leads to insufficient viral integration efficiency. Low concentration and short-term screening cannot effectively eliminate cells that have not integrated the target gene, nor can they maintain long-term screening pressure. Therefore, it is impossible to obtain a cell line that stably and efficiently expresses NS1 protein. This demonstrates the necessity of the two-infection, high concentration of 6 μg / mL, and long-term screening of 10 days used in this invention.

[0086] Comparative Example 2: Effects of Single-Enzyme Digestion Cloning Strategy on Viral Packaging and Stable Expression

[0087] Experimental Methods: Recombinant plasmids were constructed according to the method in Example 1, with the difference that a single enzyme digestion (EcoRI) strategy was used instead of EcoRI / SpeI double digestion. Specifically, EcoRI restriction sites were introduced into both the upstream and downstream primers for amplifying the NS1 gene. The PCR product was digested with EcoRI and then ligated into the pSin-EGFP-Ires-Puro vector, which had also been digested with EcoRI. Other steps, such as ligation, transformation, identification, lentivirus packaging, and cell screening, were the same as in the example.

[0088] Results: After transformation of the ligation product, 20 single colonies were selected for sequencing. 13 (65%) were found to be reverse insertions, 3 (15%) had frameshift mutations, and only 4 (20%) were correct forward insertions. The accuracy rate was significantly lower than that of double-enzyme digestion directional cloning (typically >95%).

[0089] The correctly sequenced recombinant plasmid (named pSin-NS1-EcoRⅠ) was selected for lentiviral packaging. BHK-21 cells were then infected with this virus, and the selection protocol of Example 3 (two infections, selection with 6 μg / mL puromycin for 10 days) was followed. The number of resistant clones obtained was significantly reduced. After expansion culture, NS1 expression was almost undetectable by Western blotting, and cell growth was slow, making long-term culture difficult.

[0090] Conclusion: The single-enzyme digestion cloning strategy not only leads to a low proportion of correct recombinants, but may also introduce additional restriction enzyme sites or disrupt the spatial structure of IRES elements due to non-directional insertion, severely affecting lentiviral packaging efficiency and the translation level of the target protein. The EcoRI / SpeI double-enzyme digestion directional cloning used in this invention is a key step to ensure subsequent success.

[0091] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and do not constitute a limitation on the content of the present invention. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, including combining various technical features in any other suitable manner. These simple modifications and combinations should also be regarded as the content disclosed in the present invention and all fall within the protection scope of the present invention.

Claims

1. A method for constructing a BHK-21 cell line stably expressing Japanese encephalitis virus NS1 protein, characterized in that, Includes the following steps: (1) Construction of recombinant plasmid: The NS1 gene of Japanese encephalitis virus was amplified and cloned into a lentiviral expression vector to construct a recombinant lentiviral plasmid; (2) Preparation of lentiviral particles: The recombinant lentiviral plasmid constructed in step (1) and the helper plasmid were co-transfected into 293T and its derived cell lines, and the recombinant lentiviral particles were packaged. (3) Screening of stable expression cell lines: BHK-21 cells were infected with the recombinant lentivirus particles obtained in step (2), and mixed cell lines that stably expressed NS1 protein were obtained by antibiotic screening. Single clone cell lines were then screened by limiting dilution method. (4) Detection of NS1 gene nucleic acid and protein levels: For the monoclonal cell lines screened in step (3), the expression of NS1 protein was verified at the gene and protein levels, respectively.

2. The construction method according to claim 1, characterized in that, In step (1), the nucleotide sequence of the Japanese encephalitis virus NS1 gene is shown in SEQ ID NO.

1.

3. The construction method according to claim 1, characterized in that, In step (1), the primer pair for amplifying the NS1 gene includes a forward primer NS1-F and a reverse primer NS1-R. The sequence of the forward primer NS1-F is shown in SEQ ID NO.2, and the sequence of the reverse primer NS1-R is shown in SEQ ID NO.

3.

4. The construction method according to claim 1, characterized in that, In step (1), the lentiviral expression vector is pSin-EGFP-Ires-Puro.

5. The construction method according to claim 2, characterized in that, In step (1), the NS1 gene amplification product and the lentiviral expression vector are ligated using T4 DNA ligase after being double-digested with EcoRI and SpeI.

6. The construction method according to claim 1, characterized in that, In step (3), the antibiotic is puromycin.

7. The construction method according to claim 1, characterized in that, In step (3), the recombinant lentiviral particles are used to infect BHK-21 cells twice, each time for 24 hours; the screening concentration of puromycin is 6 μg / mL, and the screening time is 10 days.

8. A BHK-21 cell line stably expressing Japanese encephalitis virus NS1 protein, characterized in that, The BHK-21 cell line is constructed according to the method for constructing a BHK-21 cell line stably expressing Japanese encephalitis virus NS1 protein according to any one of claims 1-7.

9. A primer set for amplifying the NS1 gene of Japanese encephalitis virus, characterized in that, It includes a forward primer NS1-CX-F and a reverse primer NS1-CX-R, the sequence of which is shown in SEQ ID NO.4 and the sequence of which is shown in SEQ ID NO.

5.

10. The method for constructing a BHK-21 cell line stably expressing the NS1 protein of Japanese encephalitis virus according to any one of claims 1-7, the BHK-21 cell line stably expressing the NS1 protein of Japanese encephalitis virus according to claim 8, and the application of the primer set for amplifying the NS1 gene of Japanese encephalitis virus according to claim 9 in the preparation of diagnostic reagents for Japanese encephalitis virus, screening models for anti-Japanese encephalitis virus drugs, or the study of NS1 protein function.

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