Recombinant ARH77 cell line of overexpressed DC-SIGN gene and construction method and application of recombinant ARH77 cell line

Abstract

According to the invention, lentivirus is taken as a carrier, a virus receptor DC-SIGN gene can be integrated into an ARH77 cell genome, and an ARH77 cell line for long-time stable overexpression of the DC-SIGN gene is constructed. Compared with RPMI8266 cells, the growth condition of the cells transfected by the lentivirus has the advantages that the ARH77 cells can normally grow, the RPMI8266 cells are increased, cracked and broken, the growth speed is obviously reduced, and the cell state is not improved along with the prolonging of the culture time. More importantly, the SFTSV infects ARH77 cells expressing DC-SIGN protein, the virus titer is obviously increased, and cell transcriptome level gene expression is obviously changed. Therefore, the ARH77 cell model derived from the SFTSV infected B cells is successfully constructed.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and in particular to a recombinant ARH77 cell line overexpressing a DC-SIGN gene, and a construction method and application thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance understanding of the overall background of the invention and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to those skilled in the art.

[0003] Severe fever with thrombocytopenia syndrome (SFTS) is an emerging viral hemorrhagic fever with a mortality rate ranging from 12% to 50%. The causative agent is severe fever with thrombocytopenia syndrome virus (SFTSV), a member of the genus Bandavirus in the family Phenuiviridae. SFTS is primarily transmitted by ticks, but human-to-human transmission through close contact with infected secretions has also been reported. Due to its high mortality rate, complex pathogenic mechanism, and widespread distribution of transmission vectors, SFTS has garnered significant international attention. The World Health Organization has designated SFTSV as one of the ten infectious diseases requiring urgent research priorities. At present, the clinical treatment plan for patients with fever and thrombocytopenia syndrome mainly focuses on symptomatic supportive treatment. There are no approved specific antiviral drugs or protective vaccines. Therefore, it is urgent to strengthen basic research on SFTSV and analyze its pathogenic mechanism, which is of great significance to the development of drugs and vaccines and the formulation of prevention strategies.

[0004] Numerous previous studies have shown that SFTSV can cause humoral immune response defects, which is related to the virus's direct infection of B cells. However, due to the lack of an in vitro B cell infection model, the impact of SFTSV infection on the cells themselves during replication and release is still unclear. Therefore, an in vitro SFTSV infection B cell model is urgently needed. However, in order to improve the proliferation of SFTSV in B cell models, it is usually necessary to introduce viral receptors. However, during the model preparation process, it was found that some B cells could not be transfected with plasmids that overexpress the viral receptor gene, making it impossible to achieve high SFTSV proliferation in the B cell model. Summary of the Invention

[0005] In order to overcome the above problems, the present invention provides a recombinant ARH77 cell line overexpressing the DC-SIGN gene, and a construction method and application thereof.

[0006] In order to achieve the above technical objectives, the present invention adopts the following technical solutions: The first aspect of the present invention provides a recombinant ARH77 cell line overexpressing the DC-SIGN gene, wherein the recombinant ARH77 cell line overexpresses the DC-SIGN protein.

[0007] In one or more embodiments, the amino acid sequence of the DC-SIGN protein is shown as SEQ ID No. 1.

[0008] The second aspect of the present invention provides a method for constructing the recombinant ARH77 cell line overexpressing the DC-SIGN gene as described in the first aspect, comprising the following steps: (1) Construction of a recombinant plasmid overexpressing the DC-SIGN gene; (2) co-transfecting cells with the recombinant plasmid overexpressing the DC-SIGN gene and the auxiliary plasmid in step (1) to rescue the recombinant lentivirus overexpressing the DC-SIGN gene; (3) Infecting ARH77 cells with the recombinant lentivirus that overexpresses the DC-SIGN gene, and screening to obtain a recombinant ARH77 cell line that overexpresses the DC-SIGN gene.

[0009] In one or more embodiments, the method for constructing a recombinant plasmid overexpressing the DC-SIGN gene in step (1) comprises: A DC-SIGN gene fragment containing EcoR I and Not I restriction enzyme sites was synthesized, and the fragment was double-digested with EcoR I and Not I, respectively, and the restriction enzyme fragment and the linearized vector were ligated and identified to obtain a recombinant plasmid overexpressing the DC-SIGN gene.

[0010] Preferably, the plasmid is pLVX-IRES-ZsGreen1 plasmid.

[0011] In one or more embodiments, in step (2), the helper plasmids are psPAX2 and pMD2.G.

[0012] Preferably, the mass ratio of the recombinant plasmid overexpressing the DC-SIGN gene to psPAX2 and pMD2.G is 3:2:1.

[0013] In one or more embodiments, in step (2), the recombinant plasmid overexpressing the DC-SIGN gene in step (1) and the helper plasmid are co-transfected into HEK293T cells.

[0014] In one or more embodiments, in step (3), the screening method is flow cytometry sorting.

[0015] The third aspect of the present invention provides use of the recombinant ARH77 cell line overexpressing the DC-SIGN gene according to the first aspect in enhancing the replication of Severe fever with thrombocytopenia syndrome virus (SFTSV).

[0016] The fourth aspect of the present invention provides the use of the recombinant ARH77 cell line overexpressing the DC-SIGN gene described in the first aspect in basic research and clinical diagnosis and treatment of fever with thrombocytopenia syndrome.

[0017] The beneficial effects of the present invention are: (1) In the present invention, lentivirus is used as a vector to integrate the viral receptor DC-SIGN gene into the ARH77 cell genome, thereby constructing an ARH77 cell line that stably overexpresses the DC-SIGN gene for a long time.

[0018] By comparing the cell growth after lentiviral transfection, ARH77 cells were able to grow normally compared with RPMI8266 cells, while RPMI8266 cells showed enlargement, lysis, and fragmentation, and the growth rate decreased significantly. As the culture time prolonged, the cell state showed no signs of improvement; therefore, ARH77 cells are more suitable for constructing an SFTSV-infected B cell model.

[0019] Comparison of the viral titers in ARH77 before and after the introduction of the DC-SIGN receptor revealed that normal ARH77 had the ability to infect SFTSV, but after the introduction of the DC-SIGN receptor, the viral adsorption and proliferation abilities were significantly improved. Therefore, in the present invention, the viral adsorption and proliferation abilities were improved by introducing the viral vector.

[0020] The present invention adopts a defined lentiviral transfection method, which is more efficient and causes no cell loss compared with chemical transfection and electrofection, and is conducive to constructing an SFTSV-infected B cell model.

[0021] (2) In the present invention, after overexpression of the DC-SIGN gene in ARH77 cells, the adsorption and proliferation abilities of the SFTSV virus were significantly improved, and there was no typical cytopathic effect (CPE) phenomenon after SFTSV virus infection. Therefore, a SFTSV-infected plasmablast model was successfully constructed. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The accompanying drawings, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0023] Figure 1is a map of the recombinant plasmid pLVX DC-SIGN; Figure 2 The results of enzyme digestion identification are shown in Table 1. Column 1 represents the intact plasmid, column 2 represents the fragments after endonuclease digestion, and column M is the molecular weight standard marker. Figure 3 ARH77 and RPMI8266 cells were infected with recombinant lentiviral vectors; A is a bright field image of infected ARH77 cells; B is the fluorescence of infected ARH77 cells; C is an overlapping image of A and B; D is a bright field image of infected RPMI8266 cells; E is the fluorescence of infected RPMI8266 cells; F is an overlapping image of D and E; Figure 4 Figure 3 is ARH77-DC-SIGN after flow cytometry sorting; A is the gating strategy diagram for sorting positive cells by flow cytometry; B is the bright field image of ARH77-DC-SIGN cells after flow cytometry sorting; C is the fluorescence of ARH77-DC-SIGN cells; D is the overlapping image of Figures B and C; Figure 5 ARH77 cells were physically transfected; A is a bright field image of electroporated ARH77 cells 48 hours after transfection; B is a fluorescence image of electroporated ARH77 cells 48 hours after transfection; C is an overlapping image of A and B. Figure 6 ARH77 cells were chemically transfected; A is a bright field image of ARH77 cells transfected with liposomes 120 hours after transfection; B is a fluorescence image of ARH77 cells transfected with liposomes 120 hours after transfection; C is an overlapped image of A and B. Figure 7 Figure 3 is the SFTSV infection of ARH77-DC-SIGN; A is the growth curve of SFTSV in ARH77 and ARH77-DC-SIGN cells; B and C are the growth status of ARH77 and ARH77-DC-SIGN cells on the 7th day after SFTSV infection; Figure 8 Figure 3. SFTSV-induced transcriptional changes in ARH77-DC-SIGN cells. Figure A is a bar graph showing the number of differentially expressed genes 48 and 96 h after SFTSV infection. Figure B is a cluster heat map showing the enrichment of differentially expressed genes. Figure C is a volcano plot showing typical differentially expressed genes. Figure D is a bar graph showing typical pathways enriched in differentially expressed genes in the GO database. DETAILED DESCRIPTION

[0024] It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0025] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof.

[0026] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0027] Sources of biological materials: SFTSV (SDTA-1, GenBank: KX641909): propagated and maintained in our laboratory; ARH77: purchased from China Center for Type Culture Collection.

[0028] pLVX-IRES-ZsGreen1 plasmid was purchased from Youbao Bio (catalog number: VT1460); The brand of chemical transfection Lipofectamine 2000 is Thermo Fisher, and the product number is 11668019.

[0029] Example 1 Construction of recombinant ARH77 cell line overexpressing DC-SIGN gene: (1) The viral receptor DC-SIGN gene (SEQ ID No. 1) (nucleotide sequence shown in SEQ ID No. 2) was introduced into the pLVX-IRES-ZsGreen1 plasmid to obtain the recombinant plasmid pLVX DC-SIGN. The pLVX-IRES-ZsGreen1 plasmid carries the ZsGreen gene that expresses green fluorescence and can be used to subsequently observe cell transfection efficiency directly under a fluorescence microscope; the DC-SIGN gene was codon-optimized for human cells.

[0030] Specifically, a DC-SIGN gene fragment containing EcoR I and Not I restriction sites was synthesized, and the fragment was double-digested with EcoR I and Not I by plasmid pLVX-IRES-ZsGreen1. The restriction fragment and the linearized vector were ligated and identified to obtain the recombinant plasmid pLVX DC-SIGN that overexpressed the DC-SIGN gene.

[0031] The map of the recombinant plasmid pLVX DC-SIGN is as follows Figure 1 As shown, the enzyme digestion identification results are as follows Figure 2 shown.

[0032] (2) Lentivirus construction: HEK293T cells were digested with trypsin and plated into 6-well plates. Transfection was performed after the cell density reached 80%. First, the cells were transferred to fresh DMEM medium containing 10% fetal bovine serum, 2 mL per well. The recombinant plasmid pLVX DC-SIGN and the auxiliary plasmids psPAX2 and pMD2.G (mass ratio of 3:2:1) were added to serum-free DMEM and mixed thoroughly. The transfection complex was prepared using X-tremeGENE HP DNA transfection reagent (according to the instructions: the ratio of plasmid to transfection reagent was 1 μg:1 μL). After standing at room temperature for 20 minutes, the transfection complex was added dropwise to the cell culture supernatant for labeling. After culturing at 37°C for 48 hours, the cell supernatant was harvested, centrifuged at 5000 g to remove cell debris, filtered through a 0.45 μM filter membrane, and stored in a -80°C refrigerator.

[0033] ARH77 cells were placed in a 1.5 mL centrifuge tube and centrifuged at 1200 rpm for 5 min. The supernatant was discarded and 1 mL of 1640 medium was added for resuspending. The supernatant was discarded again and 100 μL of lentivirus was added for resuspending. The cells were mixed thoroughly and incubated at 37 °C for 3 h. The liquid was then transferred to a cell culture flask and supplemented with an appropriate amount of RPMI-1640 medium containing 15% fetal bovine serum. The cells were then cultured at 37 °C and observed under a fluorescence microscope for 48 h. Figure 3 As shown in A, B, and C, the results showed that ARH77 had green fluorescence, and the cells were observed to be round and densely arranged in the bright field, indicating that the lentiviral infection was successful, DC-SIGN was expressed in B cells with high density, and was in good condition.

[0034] (3) ARH77-DC-SIGN cell purification Since the positive infection efficiency of lentiviral infection of ARH77-DC-SIGN is only about 10%, flow cytometry was used to sort the positive cells in order to increase the positive cell rate. A: ARH77 cells overexpressing DC-SIGN were cultured to a volume of approximately 100 mL and then collected into a centrifuge tube. After centrifugation at 500 × g for 5 min, the supernatant was discarded and the cells were resuspended in culture medium. B: ZsGreen1-fluorescent cells were isolated using an ultrafast flow cytometry system. Working concentrations of penicillin and streptomycin (100 U / mL for penicillin and 100 μg / mL for streptomycin) and amphotericin B (5 μg / mL) were added and cultured at 37°C. Good cell morphology and growth were observed, and the cells were expanded for subsequent experiments to obtain a recombinant ARH77 cell line overexpressing the DC-SIGN gene, referred to as ARH77-DC-SIGN.

[0035] The flow cytometry results are as follows Figure 4 shown.

[0036] Comparative Example 1 Compared with Example 1, ARH77 cells were replaced with RPMI8266 cells, and the same lentivirus was transferred into RPMI8266 cells in the same manner.

[0037] Specifically: Take RPMI8266 cells in a 1.5 mL centrifuge tube, centrifuge at 1200 rpm for 5 min, discard the supernatant and add 1 mL 1640 medium to resuspend, centrifuge again and discard the supernatant, add 100 μL lentivirus to resuspend, mix thoroughly, incubate at 37 ° C incubator for 3 h, transfer the liquid to a cell culture flask, add an appropriate amount of RPMI-1640 medium containing 15% fetal bovine serum and place in a 37 ° C incubator for culture, observe under a fluorescence microscope for 48 h, if Figure 3 As shown in Figures D, E, and F, the results showed that compared with ARH77-DC-SIGN cells, there was no significant difference in the proportion of green fluorescence in RPMI8266 cells transfected with lentivirus, but the cells showed enlargement, lysis, and fragmentation, and the growth rate decreased significantly. As the culture time prolonged, the cell state showed no signs of improvement; therefore, ARH77 cells are more suitable for constructing an SFTSV-infected B cell model.

[0038] Comparative Example 2 Comparison of transfection methods: Physical transfection: First, clean the electroporation cuvette, soak it in 75% alcohol for 2 hours, and then irradiate it under UV light before use. Place the cell suspension in a 15 mL centrifuge tube and centrifuge at 1200 rpm for 5 minutes. Discard the supernatant and resuspend the cells in 1 mL of RPMI-1640 medium. Centrifuge again at 1200 rpm for 5 minutes to wash the cells. Discard the supernatant and resuspend the cells in 1 mL of RPMI-1640 medium. Count the cells at this time and calculate the number of cells required (1-3 × 10 6 ) Pipette the corresponding volume into a 1.5 mL centrifuge tube, add culture medium to 500 μL, add 10 μg of recombinant plasmid pLVX DC-SIGN, and mix evenly. Transfer the mixed solution to an electroporation cup, set the parameters according to the predetermined conditions, and perform the electric shock operation: 150V, 500 μF, ∞Ω, 4 mm. After the electric shock is completed, transfer the cell suspension from the electroporation cup to a culture flask, add an appropriate amount of prepared culture medium, and culture in a 37°C 5% CO2 incubator. Observe cell growth after 24 hours and observe cell fluorescence under a fluorescence microscope after 48 hours. The transfection results are shown in the figure. Figure 5As described above, the transfection efficiency is less than 1 / 100, the cell edges appear burr-like, many cells die, the background is blurred, and the cell state is poor.

[0039] Chemical transfection (Lipofectamine 2000): First, determine the cell status and activity before transfection. On the day of transfection, count the cell density and viability and adjust the cell density to 0.25~1×10 6 cells / mL; use Opti-MEM medium to dilute Lipofectamine reagent and recombinant plasmid pLVX DC-SIGN, add the diluted recombinant plasmid pLVX DC-SIGN to the diluted Lipofectamine 2000 reagent (volume ratio of 1:1), incubate at room temperature for 5 minutes, add the complex to the cells, shake well and return to the constant temperature incubator; observe the cell density, viability and fluorescence 4-6 days after transfection. Figure 6 , ARH77 cells did not emit green fluorescence, indicating that transfection failed and the plasmid did not successfully enter the cells.

[0040] The final experimental results proved that the lentiviral vector had a high transfection efficiency and no cell loss. Physical transfection and chemical transfection methods had low transfection rates or were unable to perform transfection. Therefore, the present invention used a lentiviral vector transfection method to introduce the pLVXDC-SIGN plasmid and successfully constructed ARH77-DC-SIGN.

[0041] Example 2 SFTSV proliferates in ARH77-DC-SIGN The constructed ARH77-DC-SIGN is based on 1×10 5 The cells were passaged at a density of 100 μg / mL and cultured until stable growth. Then, SFTSV was inoculated at a multiplicity of infection (MOI) of 10. After incubation at 37°C for 3 h, the cells were washed twice with PBS and resuspended in 1640 medium containing 2% FBS. The cells were cultured at 37°C in a 5% CO2 incubator. 400 μL aliquots were collected at 0, 24, 48, 72, 96, 120, 144, and 168 h and stored at -80°C. Fluorescence quantitative analysis was used to determine viral load, and viral growth curves were plotted.

[0042] RNA was extracted using a reagent kit machine. 3.2 μL of RNA was pipetted into an 8-well strip and denatured at 65°C for 5 min. The reaction system was prepared according to the TOYOBO kit instructions, mixed evenly, and then added to the denatured RNA for reverse transcription to obtain cDNA. The SFTSV gene was detected using a qPCR kit and a Quant Studio 5 qPCR instrument. The primers are shown in Table 1, and the qPCR reaction procedure was as follows: 95°C for 30 s; 95°C for 5 s, 60°C for 30 s, 40 cycles.

[0043] Table 1 SFTSV primer and probe sequences

[0044] Comparison of the viral titers of SFTSV in ARH77 before and after the introduction of DC-SIGN receptors revealed that normal ARH77 had the ability to infect SFTSV, but after the introduction of DC-SIGN receptors, the viral adsorption and proliferation abilities were significantly improved ( Figure 7 Although SFTSV can continuously propagate in ARH77-DC-SIGN cells, similar to Vero cells, no typical CPE phenomenon occurs after virus infection ( Figure 7 The above results indicate that the plasmablast model infected with SFTSV was successfully established.

[0045] Example 3: SFTSV infection causes significant cellular changes in ARH77-DC-SIGN To explore the pathogenic mechanism of SFTSV virus infection in ARH77-DC-SIGN, Figure 6 Based on the growth curve characteristics in Figure A, samples were taken at three points (adaptation phase, logarithmic phase, and stationary phase) after infection to detect the transcriptional gene content and compare the dynamic changes in gene expression after viral infection. The data showed that 48 hours after viral infection, 155 genes were upregulated and 496 genes were downregulated; 96 hours after infection, 176 genes were upregulated and 267 genes were downregulated. It can be seen that the number of downregulated genes is higher than that of upregulated genes, indicating that the cellular transcription level is significantly inhibited, and this is more obvious in the logarithmic phase of the virus ( Figure 8 The clustering of differentially expressed genes 48 h after infection revealed that differentially expressed genes between 0 h and 48 h could be clustered according to their expression levels ( Figure 8 Middle B). After SFTSV infection, the expression levels of genes such as TSC22D3, GPR157, ATF3, KLF4, NR4A1, IGHA1, JUN, and TNFRSF9 were significantly reduced, suggesting that the antiviral humoral immunity and inflammatory response processes were inhibited; on the contrary, the expression of genes such as BEST1, which regulates calcium ion channels, was upregulated ( Figure 8GO enrichment analysis of differentially expressed genes revealed that processes such as protein binding, DNA-binding transcription factor activity, and RNA ploymerase II were significantly upregulated, indicating that the SFTSV virus hijacked the cell's transcription and translation functions and rapidly expressed viral proteins ( Figure 8 These data strongly confirm that SFTSV infection of B cell-derived ARH77 can cause significant changes in cellular transcriptional levels, and it can be used as a model to study the pathogenic mechanism of SFTSV.

[0046] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A recombinant ARH77 cell line overexpressing the DC-SIGN gene, characterized in that: The recombinant ARH77 cell line overexpresses DC-SIGN protein.

2. The recombinant ARH77 cell line overexpressing the DC-SIGN gene according to claim 1, wherein The amino acid sequence of the DC-SIGN protein is shown in SEQ ID No.

1.

3. The method for constructing the recombinant ARH77 cell line overexpressing the DC-SIGN gene according to claim 1 or 2, characterized in that: The steps include: (1) Construction of a recombinant plasmid overexpressing the DC-SIGN gene; (2) co-transfecting cells with the recombinant plasmid overexpressing the DC-SIGN gene and the auxiliary plasmid in step (1) to rescue the recombinant lentivirus overexpressing the DC-SIGN gene; (3) Infecting ARH77 cells with the recombinant lentivirus that overexpresses the DC-SIGN gene, and screening to obtain a recombinant ARH77 cell line that overexpresses the DC-SIGN gene.

4. The construction method according to claim 3, wherein: The method for constructing a recombinant plasmid overexpressing the DC-SIGN gene in step (1) includes: A DC-SIGN gene fragment containing EcoR I and Not I restriction enzyme sites was synthesized, and the fragment was double-digested with EcoR I and Not I, respectively, and the restriction enzyme fragment and the linearized vector were ligated and identified to obtain a recombinant plasmid overexpressing the DC-SIGN gene.

5. The construction method according to claim 4, wherein: The plasmid is pLVX-IRES-ZsGreen1 plasmid.

6. The construction method according to claim 3, wherein: In step (2), the auxiliary plasmids are psPAX2 and pMD2.G; Preferably, the mass ratio of the recombinant plasmid overexpressing the DC-SIGN gene to psPAX2 and pMD2.G is 3:2:

1.

7. The construction method according to claim 3, wherein: In step (2), the recombinant plasmid overexpressing the DC-SIGN gene in step (1) and the auxiliary plasmid are co-transfected into HEK293T cells.

8. The construction method according to claim 3, wherein: In step (3), the screening method is flow cytometry sorting.

9. Use of the recombinant ARH77 cell line overexpressing the DC-SIGN gene according to claim 1 or 2, or the recombinant ARH77 cell line overexpressing the DC-SIGN gene constructed by the construction method according to any one of claims 3 to 8, in enhancing the replication of fever with thrombocytopenia syndrome virus.

10. Use of the recombinant ARH77 cell line overexpressing the DC-SIGN gene according to claim 1 or 2, or the recombinant ARH77 cell line overexpressing the DC-SIGN gene constructed by the construction method according to any one of claims 3 to 8, in basic research and clinical diagnosis and treatment of fever with thrombocytopenia syndrome.

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