Immortalized muscovy duck embryo fibroblast line as well as establishment method and application thereof
By constructing the immortalized Muscovy duck embryo fibroblast cell line iMDEF/SV40-LT, the problems of limited lifespan and poor passage ability of Muscovy duck embryo fibroblasts have been solved, providing a stable platform for virus replication and tropism research, and supporting vaccine development and safety applications.
Patent Information
- Application Number
- CN202511426492.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-30
- Publication Date
- 2026-01-23
AI Technical Summary
The limited lifespan, poor passage capacity, and large batch-to-batch variability of existing Muscovy duck embryo fibroblasts restrict the stability and high-throughput requirements of waterfowl virology research.
Immortalized Muscovy duck embryo fibroblast cell line iMDEF/SV40-LT was constructed through lentiviral transfection and gene verification, stably expressing the SV40 LT gene. Immortalized cells were obtained by screening with puromycin to ensure cell stability and safety.
It achieves unlimited cell proliferation and functional stability, overcomes the limitations of primary cells, provides a stable platform for viral replication and tropism research, supports vaccine development and viral tropism analysis, and ensures safety and applicability.
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Figure CN121379971A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of animal cell engineering, specifically to an immortalized Muscovy duck embryo fibroblast cell line, its establishment method, and its application. Background Technology
[0002] Avian viral diseases, including waterfowl diseases, pose a significant challenge to global poultry health and sustainable agricultural development. Effective in vitro models are indispensable for understanding virus-host interactions, vaccine development, and pathogenicity studies. Primary avian cells, such as Muscovy duck embryonic fibroblasts (MDEF), have been widely used in related research; however, their limited lifespan, poor passage capacity, and large batch-to-batch variability hinder long-term development and high-throughput research. Immortalized cell lines offer a stable alternative, capable of unlimited proliferation while maintaining cell function, overcoming the limitations of primary cell culture.
[0003] Current waterfowl cell models (such as the DEF cell line derived from Cherry Valley ducks) suffer from limitations including high culture costs and dependence on exogenous cytokines. Muscovy duck cells exhibit unique susceptibility to specific waterfowl viruses (such as Muscovy duck adenovirus, Muscovy duck parvovirus, and Muscovy duck reovirus), making them particularly important in virus tropism studies. However, the limitations of their primary culture necessitate immortalization.
[0004] This invention aims to establish immortalized MDEF cells to address the aforementioned problems. Through lentiviral transfection and gene validation, this invention focuses on creating stable, phenotypically consistent cell lines to support viral replication and tropism studies. Understanding viral tropism, i.e., the specificity of viral infection of host cells, is crucial for elucidating pathogenic mechanisms. Immortalized cell lines, such as immortalized MDEF cells, provide a unified platform for analyzing viral entry, replication dynamics, and host responses. Recent advances in avian cell immortalization, such as peacock fibroblasts for avian virus production and a novel avian intestinal epithelial cell line as a better in vitro model for studying the chicken intestine, highlight the practicality of such models in vaccine development. Therefore, this invention provides a powerful and economical tool for advancing waterfowl virology research. Summary of the Invention
[0005] The purpose of this invention is to provide an immortalized Muscovy duck embryo fibroblast cell line and a method for establishing it, so as to solve the technical problems of limited lifespan, poor passage ability and large batch-to-batch variation of Muscovy duck embryo fibroblasts (MDEF).
[0006] The objective of this invention is achieved through the following technical solution:
[0007] This invention provides an immortalized Muscovy duck embryo fibroblast cell line, namely immortalized Muscovy duck embryo fibroblast iMDEF / SV40-LT, Latin name immortalized MDEF cell line, which was deposited at the China Center for Type Culture Collection on August 13, 2025, with accession number CCTCC NO: C2025243; deposit address: Wuhan University, Wuhan, China, postcode: 430072.
[0008] The method for establishing the immortalized Muscovy duck embryo fibroblast cell line involves inserting the cloned SV40 LT gene into a lentiviral plasmid via homologous recombination, transfecting Muscovy duck embryo fibroblasts with lentivirus to achieve stable expression of SV40 LT, and then selecting with puromycin to obtain immortalized Muscovy duck embryo fibroblasts (iMDEF / SV40-LT).
[0009] The aforementioned establishment method specifically includes the following steps:
[0010] (1) Isolation and culture of primary Muscovy duck embryo fibroblasts: Muscovy duck embryos that have been incubated for 15 days were taken out, and after removing the head, limbs and internal organs, they were digested with trypsin and isolated to obtain primary Muscovy duck embryo fibroblasts. A complete culture medium containing 10% fetal bovine serum and 1% double antibiotics (penicillin and streptomycin) DMEM was prepared to culture the primary Muscovy duck embryo fibroblasts.
[0011] (2) Cell immortalization: The target gene SV40 LT was cloned from pBABE-Puro SV40 LT by double enzyme digestion, and ligated into the shuttle plasmid plex-mcs by homologous recombination to obtain plex-SV40 LT; plex-SV40 LT was co-transfected into 293T cells with the backbone plasmids pMD2.G and psPAX2 according to the instructions; 10 μg of total DNA (i.e., the total DNA of plex-SV40 LT, backbone plasmid pMD2.G and psPAX2) was diluted to 500 μL. Mix thoroughly by vortexing in the buffer solution; before use... Vortex the reagent for 5 seconds and centrifuge, then add 20 μL. Vortex for 1 second, then briefly centrifuge; incubate at room temperature for 10 minutes, add 500 μL of transfection mixture (a mixture of the three plasmids) to each dish, and drop it onto the cells in serum-containing medium, distributing it evenly; gently shake the culture dish back and forth and side to side, incubate at 37°C, and after 4 hours replace with complete medium and return to the incubator; after incubation for 24-72 hours, obtain the virus expressing the SV40 LT gene, and perform virus purification and titer determination;
[0012] When second-generation Muscovy duck embryonic fibroblasts (MDEF) grew to a monolayer, a viral solution expressing the SV40 LT gene was added to the culture dish. The control group was not given a viral solution and was treated in the same way. After 12 hours of incubation, the culture medium was discarded and replaced with complete culture medium. After 24 hours, the complete culture medium was discarded and replaced with complete culture medium containing 1.0 μg / mL puromycin. After culturing for 96 hours, immortalized Muscovy duck embryonic fibroblasts iMDEF / SV40-LT were obtained, abbreviated as iMDEF / SV40-LT. These cells can be passaged for more than 50 generations, are free of endogenous viruses, have good safety, and are non-teratogenic and non-tumorigenic.
[0013] The specific operation method of step (1) is as follows: Take out the Muscovy duck embryo that has been incubated for 15 days on a sterile operating table, disinfect the air cell of the duck embryo with iodine, knock open the air cell, take out the embryo and place it in a petri dish, remove the head, limbs, internal organs and bones, wash with Hank's solution, digest with trypsin, pour the supernatant into another digestion bottle, shake the precipitate and add Hank's solution, shake well and pour out the supernatant, repeat the shaking and washing several times, collect the supernatant and filter; collect the filtrate, centrifuge, discard the supernatant, resuspend the cell pellet and count for later use. Seed the cells in T25 culture flasks, prepare complete culture medium in advance, and use complete culture medium to culture primary Muscovy duck embryo fibroblasts.
[0014] The complete culture medium is DMEM medium containing 10% fetal bovine serum and 1% penicillin + streptomycin.
[0015] The complete culture medium containing 1.0 μg / mL puromycin is DMEM medium containing 1.0 μg / mL puromycin, 10% fetal bovine serum, and 1% penicillin + streptomycin.
[0016] This invention also provides the use of the immortalized Muscovy duck embryo fibroblast cell line in any of the following:
[0017] 1) Cultivate the virus;
[0018] 2) Preparation of products containing cultured viruses;
[0019] 3) Isolate the virus;
[0020] 4) Prepare products for virus isolation;
[0021] 5) Virus testing not for diagnostic or treatment purposes;
[0022] 6) Prepare products for virus detection;
[0023] 7) Prepare viral vaccines;
[0024] 8) Drug screening;
[0025] The drug is used to prevent or treat diseases caused by viral infections.
[0026] The virus described is adapted to replicate with iMDEF / SV40-LT. The virus is at least one of the following: goose parvovirus (GPV), duck plague virus (DEV), duck adenovirus B2 (DAdVB2), Muscovy duck parvovirus (MDPV), Muscovy duck gosling plague virus (MDGPV), novel duck reovirus (NDRV), duck tembusu virus (DTMUV), Muscovy duck reovirus (MDRV), duck paramyxovirus (DPMV), and SBDSV.
[0027] The method for culturing the virus involves inoculating the virus into the immortalized Muscovy duck embryo fibroblast cell line and culturing it.
[0028] The method for preparing the viral vaccine involves inoculating the virus into the immortalized Muscovy duck embryo fibroblast cell line, culturing it, inactivating it, and obtaining the corresponding viral vaccine antigen.
[0029] Compared with the prior art, the advantages of the present invention are as follows:
[0030] (1) In this study, an immortalized Muscovy duck embryo fibroblast cell line (iMDEF / SV40-LT) was successfully constructed using the SV40 large T antigen (SV40 LT) gene. The iMDEF / SV40-LT cell line has grown stably for more than 50 generations, maintaining the fibroblast morphology and showing no signs of senescence or tumorigenesis.
[0031] (2) A key advantage of iMDEF / SV40-LT is its strong support for waterfowl virus replication. Similar to primary cells, iMDEF / SV40-LT exhibits comparable cytopathic effects and viral titers against NDRV, DAdVB2, MDDPV, and DPMV, confirming its suitability as a substitute for primary MDEF in virus tropism studies. This is particularly important because existing duck cell lines such as DEF require exogenous cytokines and have limited applicability. Notably, the iMDEF / SV40-LT cell line is free of endogenous viruses and maintains normal karyotype stability, resolving safety concerns in vaccine production and mechanism studies.
[0032] (3) Functional characterization showed that iMDEF / SV40-LT retained key characteristics of primary cells, including normal apoptosis rate and metabolic activity, which was confirmed by CCK-8 and flow cytometry. This stability is crucial for longitudinal studies, as primary cells typically lose viability after short passage times. Furthermore, the lack of tumorigenicity in nude mice distinguishes iMDEF from transformed cell lines, ensuring its suitability for preclinical applications.
[0033] (4) In the future, iMDEF / SV40-LT can serve as a safe and stable in vitro cell model, providing a platform for promoting high-throughput screening of antiviral compounds and vaccine development. Future research can explore the transcriptomic response of iMDEF / SV40-LT to emerging waterfowl viruses, or optimize its application in large-scale virus production based on the successful experience of avian cell vaccine platforms.
[0034] In summary, the iMDEF / SV40-LT cell line addresses a key gap in waterfowl virology research by providing a stable and economical model that reproduces primary cell function. Its broad viral tropism and safety profile make it a valuable tool for elucidating viral pathogenic mechanisms and advancing avian vaccine technology. Attached Figure Description
[0035] Figure 1 This is a photograph of a 15-day-old duck embryo of the present invention.
[0036] Figure 2 This is a morphological diagram of fibroblasts in a primary Muscovy duck embryo.
[0037] Figure 3 These are images showing the cell status of second-generation MDEF cells after 48 hours of culture in selection media with different concentrations of puromycin; among them, Figure 3 -A was used at a concentration of 0.5 μg / mL of puromycin. Figure 3 -B was used at a concentration of 1 μg / mL for puromycin. Figure 3 -C The concentration of puromycin used was 2 μg / mL. Figure 3 The concentration of puromycin used for -D was 3 μg / mL. Figure 3 -E was used at a puromycin concentration of 4 μg / mL. Figure 3 -F uses a puromycin concentration of 5 μg / mL.
[0038] Figure 4 This is a diagram of plex-SV40 double enzyme digestion identification.
[0039] Figure 5 These are morphological images of primary MDEF and iMDEF / SV40-LT cells from different passages; among them, Figure 5 -A is a cell morphology diagram of primary MDEF. Figure 5 -B is a cell morphology diagram of the 10th generation iMDEF / SV40-LT. Figure 5 -C represents the cell morphology of the 20th generation iMDEF / SV40-LT. Figure 5 -D is a cell morphology diagram of the 30th generation iMDEF / SV40-LT. Figure 5 -E is a cell morphology diagram of iMDEF / SV40-LT from the 40th generation. Figure 5-F is a cell morphology diagram of the 50th generation iMDEF / SV40-LT.
[0040] Figure 6 This is a graph showing the transcriptional level of SV40 LT mRNA.
[0041] Figure 7 This is a graph showing the proliferation activity of iMDEF / SV40-LT cells at different passages.
[0042] Figure 8 This is a diagram of cell apoptosis.
[0043] Figure 9 This is a graph showing the apoptosis rate.
[0044] Figure 10 This is a graph showing the detection results of endogenous viruses. 1-9 and 10-18 represent the detection results of the 10th and 40th generation iMDEF / SV40-LT, respectively. M: Maker; 1: GPV; 2: DEV; 3: DAdV B2; 4: MDPV; 5: NDRV; 6: DTMUV; 7: MDRV; 8: DPMV; 9: ddH2O; 10: GPV; 11: DEV; 12: DAdV B2; 13: MDPV; 14: NDRV; 15: DTMUV; 16: MDRV; 17: DPMV; 18: ddH2O.
[0045] Figure 11 This is a diagram showing the results of a tumorigenesis experiment in nude mice.
[0046] Figure 12 It is a tumor growth curve.
[0047] Figure 13 These are cell morphology images of iMDEF / SV40-LT cells infected with different waterfowl viruses after 72 hours of continuous culture; among them, Figure 13 -A represents the control group. Figure 13 -B shows the cell morphology of iMDEF / SV40-LT cells infected with NDRV and cultured for 72 hours. Figure 13 -C shows the cell morphology of iMDEF / SV40-LT cells infected with DAdVB2 and cultured for 72 hours. Figure 13 -D shows the cell morphology of iMDEF / SV40-LT cells infected with MDGPV and cultured for 72 hours. Figure 13 -E is a cell morphology diagram of iMDEF / SV40-LT cells infected with SBDSV and cultured for 72 hours.
[0048] Figure 14 These are cell morphology images of primary MDEF cells infected with different waterfowl viruses and cultured continuously for 72 hours; among them, Figure 14 -A represents the control group. Figure 14 -B shows the cell morphology of primary MDEF cells infected with NDRV and cultured for 72 hours. Figure 14 -C shows the cell morphology of primary MDEF cells infected with DAdV B2 and cultured for 72 hours. Figure 14 -D is a cell morphology diagram of primary MDEF cells infected with MDGPV and cultured for 72 hours. Figure 14 -E is a cell morphology diagram of primary MDEF cells infected with SBDSV and cultured for 72 hours.
[0049] Figure 15 This is a graph showing the results of IFA detection of different viruses infecting iMDEF / SV40-LT cells; among them, Figure 15 -A represents the control group. Figure 15 -B indicates NDRV infection. Figure 15 -C indicates DAdV B2 infection. Figure 15 -D indicates MDGPV infection. Figure 15 -E indicates SBDSV infection.
[0050] Figure 16 This is a graph showing the results of IFA detection of primary MDEF cells infected with different viruses; among them, Figure 16 -A represents the control group. Figure 16 -B indicates NDRV infection. Figure 16 -C indicates DAdV B2 infection. Figure 16 -D indicates MDGPV infection. Figure 16 -E indicates SBDSV infection.
[0051] Figure 17 This is a graph showing the results of detecting NDRV virus mRNA expression levels.
[0052] Figure 18 This is a graph showing the results of detecting the expression level of DAdV B2 virus mRNA.
[0053] Figure 19 This is a graph showing the results of detecting the mRNA expression level of MDGPV virus.
[0054] Figure 20 This is a graph showing the results of detecting the expression level of SBDSV viral mRNA.
[0055] The iMDEF shown in the attached diagram is iMDEF / SV40-LT. Detailed Implementation
[0056] The present invention will now be described in detail with reference to the accompanying drawings and embodiments:
[0057] 1. Materials and Methods
[0058] 1.1 Isolation and Culture of Primary Muscovy Duck Embryo Fibroblasts
[0059] Remove 15-day-old Muscovy duck embryos from a sterile operating table. Sterilize the air cells with iodine, open the air cells, remove the embryos, and place them in a petri dish. Remove the head, limbs, and internal organs. Wash with Hank's solution, digest with trypsin (Gibco), and transfer the supernatant to another digestion flask. Shake the precipitate well, add Hank's solution, shake well, and discard the supernatant. Repeat the shaking and washing process several times, collecting the supernatant and filtering. Centrifuge the filtrate, discard the supernatant, resuspend the cell pellet, and count the cells for later use. Seed the cells in T25 culture flasks, prepare complete culture medium in advance, and use the complete culture medium to culture primary Muscovy duck embryo fibroblasts.
[0060] 1.2 Cell Immortality
[0061] The target gene SV40 LT was cloned from pBABE-Puro SV40 LT via double enzyme digestion, and then ligated into the shuttle plasmid plex-mcs (OPEN BIOSYSTEMS) via homologous recombination to obtain plex-SV40 LT. Primers are shown in Table 1. plex-SV40 LT, along with the backbone plasmids pMD2.G and psPAX2 (OPEN BIOSYSTEMS), were co-transfected into 293T cells according to the manufacturer's instructions. The total DNA amount (i.e., the total DNA amount of plex-SV40 LT, backbone plasmid pMD2.G, and psPAX2) was diluted from 10 μg to 500 μL. Mix thoroughly by vortexing in the buffer solution. Before use... Vortex the reagent for 5 seconds and centrifuge, then add 20 μL. Vortex for 1 second, then briefly centrifuge. Incubate at room temperature for 10 minutes. Add 500 μL of transfection mixture (i.e., a mixture of the three plasmids) to each dish and drop it onto the cells in serum-containing medium, distributing it evenly. Gently shake the culture dish back and forth and side to side. Incubate at 37°C for 4 hours, then replace with complete medium and return to the incubator. After incubation for 24–72 hours, obtain the viral fluid expressing the SV40 LT gene, and perform virus purification and titer determination.
[0062] When MDEF grew to a monolayer, a viral solution expressing the SV40 LT gene was added to the culture dish. The control group was not given a viral solution and was treated in the same way. After 12 hours of incubation, the culture medium was discarded and replaced with complete culture medium. After 24 hours, the complete culture medium was discarded and replaced with complete culture medium containing 1.0 μg / mL puromycin. After culturing for 96 hours, immortalized Muscovy duck embryo fibroblasts iMDEF / SV40-LT were obtained, abbreviated as iMDEF / SV40-LT.
[0063] Subsequently, the immortalized Muscovy duck embryo fibroblasts iMDEF / SV40-LT were further passaged and cultured. Biological characteristics such as proliferation activity, apoptosis, endogenous virus, teratogenicity and tumorigenicity, and viral replication level in cells at different passages were measured. It was found that there was no significant difference between them and the primary Muscovy duck embryo fibroblasts, indicating that it is an immortalized cell line.
[0064] The complete culture medium is DMEM medium containing 10% fetal bovine serum and 1% penicillin + streptomycin.
[0065] The complete culture medium containing 1.0 μg / mL puromycin is DMEM medium containing 1.0 μg / mL puromycin, 10% fetal bovine serum, and 1% penicillin + streptomycin.
[0066] 1.3 CCK-8 cell proliferation assay
[0067] Using second-generation MDEF as a control group, the proliferation activity of 10th, 20th, 30th, 40th, and 50th generation iMDEF / SV40-LT was measured. In a 96-well plate, 100 μL of a solution containing 1×10⁻⁶ mcg of MDEF / SV40-LT was added to each well. 4 Cell suspensions of 10 cells were prepared, with 8 replicates per cell generation. After 24 hours, 10 μL of CCK-8 solution (TRANS) was added to each well, and the cells were incubated at 37°C for 4 hours. The OD450nm value was measured using a microplate reader to determine the cell viability of iMDEF / SV40-LT cells at different passages.
[0068] 1.4 Apoptosis Detection
[0069] Following the instructions of the Annexin V-FITC apoptosis kit (Beyotime), apoptosis was detected in passage 27 and passage 47 iMDEF / SV40-LT cells, with passage 2 MDEF cells as a control. 5 μL of Annexin V-FITC was added to each of the three groups, and after gentle mixing, 5 μL of propidium iodide was added, and the mixture was gently mixed again. The cells were incubated at room temperature in the dark for 30 minutes, and the cells were then analyzed by flow cytometry.
[0070] 1.5 Detection of endogenous viruses in cells
[0071] Nucleic acid was extracted from iMDEF / SV40-LT at passages 10 and 40 according to the Virus DNA / RNA Extraction Kit 2.0 (Vazyme) instructions. Common duck-derived viruses were detected by PCR, including goose parvovirus (GPV), duck plague virus (DEV), duck adenovirus B2 (DAdV B2), Muscovy duck parvovirus (MDPV), novel duck reovirus (NDRV), duck Tembusu virus (DTMUV), Muscovy duck reovirus (MDRV), and duck paramyxovirus (DPMV). The primers for each virus are shown in Table 1.
[0072] 1.6 Cell teratogenicity and tumorigenicity tests
[0073] Four-week-old male Balb / c nude mice were selected, and a concentration of 1.0 × 10⁻⁶ was injected subcutaneously into the left axilla. 7 100 μL of 47th generation iMDEF / SV40-LT cell suspension was used as a positive control, with the same concentration and volume of human cervical cancer cell line (HeLa) as the positive control, and PBS as a negative control. Tumor size was measured every 2-3 days, with the longest and shortest diameters of the tumor measured using calipers, and calculated using the formula V = 1 / 2(a × b). 2 (a is the major axis, b is the minor axis) Calculate the volume and observe for 1 month.
[0074] 1.7 Indirect Immunofluorescence Assay (IFA) for Viral Proliferation
[0075] Primary MDEF and iMDEF / SV40-LT cells were cultured in 6-well plates and washed once with PBS after forming monolayers. Both cell lines were inoculated with NDRV (1:10,000), DAdv B2 (1:10), MDGPV (1:10), and SBDSV (1:10). All viruses were stored in the laboratory. After incubation at 37°C for 1 hour, the virus solution was discarded, and the cells were replaced with fresh medium containing 1% FBS. The cells were then incubated at 37°C with 5% CO2 to observe lesions. Once typical lesions appeared, the medium was discarded, and cells were fixed with 4% paraformaldehyde. Cells were incubated with laboratory-preserved primary antibodies and FITC-labeled goat anti-mouse IgG was used as a secondary antibody. Normal cell controls were included for IFA (infectious viral adhesion) identification. 1.8 Real-time quantitative PCR (qPCR) for viral replication.
[0076] Total RNA was extracted from iMDEF / SV40-LT at passages 27 and 47 using FastPure Cell / Tissue Total RNA Isolation Kit V2 (Vazyme). Reverse transcription was performed using HiScript III RT SuperMix for qPCR (+gDNAwiper) (Vazyme). Finally, real-time PCR was performed using the CFX Connect real-time quantitative PCR system and PerfectStart GreenqPCR SuperMix (TRANS), repeated twice.
[0077] To assess the transcriptional level of SV40 LT in passaged cells, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control and detected by qPCR. The calculation formula is referenced: Relative transcriptional level = 2. -ΔΔCt ΔΔCt = (Ct value of target gene in experimental group - Ct value of internal reference gene) - (Ct value of target gene in control group - Ct value of internal reference gene). All primers are shown in Table 1.
[0078] Table 1. Gene and Primer Sequences
[0079]
[0080] 1.9 Data Statistics
[0081] Results are expressed as mean ± SD standard deviation. Plotting was performed using GraphPad Prism 8.0, and data were analyzed using SPSS (Version 24).
[0082] 2 Results and Analysis
[0083] 2.1 Isolation and Culture of Primary Muscovy Duck Embryo Fibroblasts
[0084] To prepare the Muscovy duck embryo fibroblast cell line (MDEF), Muscovy duck embryos that had been incubated for 15 days were aseptically removed. Figure 1 Then decapitate, remove limbs, internal organs, and bones, and cut the remaining tissue into 1cm pieces. 3 Size. After trypsin digestion, the cells were seeded into T25 culture flasks, and the seeded fibroblasts exhibited a typical fibrous morphology. Figure 2 Following this, puromycin selection was performed. Second-generation MDEF cells were seeded into 6-well plates, with each well containing different concentrations of puromycin (0.5 μg / mL, 1 μg / mL, 2 μg / mL, 3 μg / mL, 4 μg / mL, and 5 μg / mL). Cell status was observed after 48 hours. Figure 3 AF).
[0085] 2.2 Construction of an immortalized Muscovy duck embryo fibroblast cell line
[0086] The target gene SV40 LT, obtained by double enzyme digestion, was ligated into the shuttle plasmid plex-mcs via homologous recombination to obtain plex-SV40 LT. A target band of approximately 2127 bp was observed on 1% agarose gel electrophoresis, consistent with the expected size. Figure 4 This proves that the plasmid is intact and can be used for subsequent experiments.
[0087] Immortalized Muscovy duck embryo fibroblasts (iMDEF / SV40-LT) obtained through puromycin screening, compared with primary MDEF, showed that immortalized MDEF cells (iMDEF / SV40-LT) from different passages were more fibrous and spindle-shaped, with intact cell morphology. Passaged at a 1:2 density, they grew into a monolayer within 2 days. Figure 5 AF). SV40 LT gene expression was detected in iMDEF / SV40-LT cells at passages 27 and 47, but not in primary MDEF cells, indicating that this cell line can stably express the SV40 LT gene. Figure 6 The proliferation capacity of iMDEF / SV40-LT cells at different passages was measured. The results showed that the cell viability of iMDEF / SV40-LT cells at passages 10, 20, 30, 40, and 50 was higher than that of primary MDEF cells, with significantly enhanced viability at passages 20, 30, and 40 (P<0.05). Figure 7 Flow cytometry analysis revealed that the percentages of live, dead, and apoptotic cells were predominantly live cells in primary MDEF, and in passages 27 and 47 of iMDEF / SV40-LT. Figure 8 However, the percentage of apoptotic cells in primary MDEF was higher than the latter two (P<0.05). Figure 9 The results indicate that primary MDEF cells undergo senescence, while there is no significant difference in apoptosis rates between the 27th and 47th generations of iMDEF / SV40-LT (P>0.05), suggesting that iMDEF / SV40-LT cells have stable viability.
[0088] 2.3 Safety Study of Immortalized Muscovy Duck Embryo Fibroblast Cell Line
[0089] The PCR results of experiment 1.5 showed that the cells themselves did not carry waterfowl virus ( Figure 10 This ruled out viral infection during the passage process.
[0090] No subcutaneous swelling was observed in either the experimental group (injected with 47th generation iMDEF) or the control group (injected with PBS) in Experiment 1.6. Figure 11Nude mice injected with HeLa cells initially developed subcutaneous induration at the injection site, which gradually increased in size. The long axis (a) and short axis (b) of the tumor were measured weekly, and the tumor volume was calculated using a formula. A tumor growth curve was plotted over 5 weeks. Figure 12 During the observation period, no skin ulceration or death occurred in any of the nude mice, thus confirming that the cell line is non-tumorigenic in vivo.
[0091] 2.4 Viral tropism studies
[0092] In Experiment 1.7, primary MDEF cells and immortalized MDEF cells (iMDEF / SV40-LT) were infected with laboratory-preserved waterfowl virus, respectively. After continuous culture for 72 hours, both showed obvious cytopathic effects. Figure 13 AE, Figure 14 AE). IFA detects viral infection ( Figure 15 AE, Figure 16 AE), qPCR detection of viral amplification ( Figure 17-20 The results showed that iMDEF / SV40-LT developed typical lesions after inoculation with the above-mentioned waterfowl viruses, and the IFA results were basically consistent. The virus expression was not significantly different from that of MDEF (P>0.05), indicating that there was no difference in the proliferation of different waterfowl viruses in immortalized MDEF cells and primary MDEF cells.
[0093] 3 Discussion
[0094] The establishment of immortalized cell lines is crucial for advancing avian virology research, as primary cells typically have limited lifespans and inconsistent performance. In this study, we successfully constructed an immortalized Muscovy duck embryonic fibroblast cell line (iMDEF / SV40-LT) using the SV40 large T antigen (SV40 LT) gene. The iMDEF / SV40-LT cell line has grown stably for over 50 generations, maintaining fibroblast morphology without signs of senescence or tumorigenesis.
[0095] A key advantage of iMDEF / SV40-LT is its ability to be passaged multiple times while maintaining similar cellular bioactivity to primary cells, providing strong support for waterfowl virus replication. Similar to primary cells, iMDEF / SV40-LT exhibits comparable cytopathic effects and viral titers against NDRV, DAdV B2, MDDPV, and DPMV, confirming its suitability as a substitute for primary MDEF in virus tropism studies. This is particularly important because existing duck cell lines, such as DEF, require exogenous cytokines and have limited applicability. Notably, the iMDEF / SV40-LT cell line is free of endogenous viruses and maintains normal karyotype stability, resolving safety concerns in vaccine production and mechanism studies.
[0096] Functional characterization showed that iMDEF / SV40-LT retained key characteristics of primary cells, including normal apoptosis rate and metabolic activity, as confirmed by CCK-8 and flow cytometry. This stability is crucial for longitudinal studies, as primary cells typically lose viability after short passage times. Furthermore, the lack of tumorigenicity in nude mice distinguishes iMDEF / SV40-LT from transformed cell lines, ensuring its suitability for preclinical applications.
[0097] In the future, iMDEF / SV40-LT can serve as a safe and stable in vitro cell model, providing a platform for high-throughput screening of antiviral compounds and vaccine development. For example, its practicality in studying virus-host interactions is similar to that of immortalized bovine intestinal epithelial cells (BIECs-21) in parasitic infection research. Future research could explore the transcriptomic response of iMDEF / SV40-LT to emerging waterfowl viruses, or optimize its application in large-scale virus production based on the successful experience of avian cell vaccine platforms.
[0098] In summary, the iMDEF / SV40-LT cell line addresses a key gap in waterfowl virology research by providing a stable and economical model that reproduces primary cell function. Its broad viral tropism and safety profile make it a valuable tool for elucidating viral pathogenic mechanisms and advancing avian vaccine technology.
Claims
1. An immortalized Muscovy duck embryo fibroblast cell line, characterized in that: It is an immortalized Muscovy duck embryo fibroblast iMDEF / SV40-LT, which was deposited at the China Center for Type Culture Collection on August 13, 2025, with accession number CCTCCNO:C2025243, deposited at Wuhan University, Wuhan, China. This cell can be passaged for more than 50 generations, is free of endogenous viruses, has good safety, and is non-teratogenic and non-tumorigenic.
2. The method for establishing an immortalized Muscovy duck embryo fibroblast cell line as described in claim 1, characterized in that: The cloned SV40 LT gene was inserted into a lentiviral plasmid via homologous recombination. Muscovy duck embryonic fibroblasts were transfected with the lentivirus to stably express SV40 LT. Immortalized Muscovy duck embryonic fibroblasts iMDEF / SV40-LT were then obtained by selection with puromycin.
3. The method for establishing according to claim 2, characterized in that: Includes the following steps: (1) Preparation of lentiviral culture medium expressing the SV40 LT gene: The target gene SV40 LT was cloned from pBABE-Puro SV40 LT by double enzyme digestion, and ligated into the shuttle plasmid plex-mcs by homologous recombination to obtain plex-SV40 LT; plex-SV40 LT was co-transfected into 293T cells with backbone plasmids pMD2.G and psPAX2 according to the instructions; the total DNA was diluted from 10 μg to 500 μL. Mix thoroughly by vortexing in the buffer solution; before use... Vortex the reagent for 5 seconds and centrifuge, then add 20 μL. Vortex for 1 second, then briefly centrifuge; incubate at room temperature for 10 minutes, add 500 μL of transfection mixture to each dish, drop it onto cells in serum-containing medium, and distribute evenly; gently shake the culture dish back and forth and side to side, incubate at 37°C, replace with complete medium after 4 hours, and return to the incubator; after incubation for 24-72 hours, obtain the virus solution expressing the SV40 LT gene, and perform virus purification and titer determination; (2) Immortality of Muscovy duck embryo fibroblasts: Muscovy duck embryo fibroblasts were prepared using conventional methods. When the Muscovy duck embryo fibroblasts grew to a monolayer, lentivirus culture medium expressing the SV40 LT gene was added to the culture dish. The control group was not given lentivirus culture medium. The same treatment was performed. After incubation for 12 hours, the culture medium was discarded and replaced with complete culture medium. After 24 hours, the complete culture medium was discarded and replaced with complete culture medium containing 1.0 μg / mL puromycin. After culturing for 96 hours, immortalized Muscovy duck embryo fibroblasts iMDEF / SV40-LT were obtained, abbreviated as iMDEF / SV40-LT.
4. The method for establishing according to claim 3, characterized in that: The complete culture medium containing 1.0 μg / mL puromycin is DMEM medium containing 1.0 μg / mL puromycin, 10% fetal bovine serum, and 1% penicillin + streptomycin.
5. The use of the immortalized Muscovy duck embryo fibroblast cell line as described in claim 1 in any of the following: 1) Cultivate the virus; 2) Preparation of products containing cultured viruses; 3) Isolate the virus; 4) Prepare products for virus isolation; 5) Virus testing not for diagnostic or treatment purposes; 6) Prepare products for virus detection; 7) Prepare viral vaccines; 8) Drug screening; The drug is used to prevent or treat diseases caused by viral infections.
6. The application according to claim 5, characterized in that: The virus is adapted to replicate in iMDEF / SV40-LT.
7. The application according to claim 6, characterized in that: The virus mentioned is at least one of the following: goose parvovirus (GPV), duck plague virus (DEV), duck adenovirus B2 (DAdVB2), Muscovy duck parvovirus (MDPV), Muscovy duck gosling plague virus (MDGPV), novel duck reovirus (NDRV), duck tembusu virus (DTMUV), Muscovy duck reovirus (MDRV), duck paramyxovirus (DPMV), and SBDSV.
8. The application according to claim 5, characterized in that: The method for culturing the virus involves inoculating the virus into the immortalized Muscovy duck embryo fibroblast cell line as described in claim 1 and culturing it.
9. The application according to claim 5, characterized in that: The method for preparing the viral vaccine involves inoculating the virus into the immortalized Muscovy duck embryo fibroblast cell line described in claim 1, culturing it, inactivating it, and obtaining the corresponding viral vaccine antigen.