Bovine kidney cell line and its use

Abstract

This invention relates to the NBLS (Niobium Bovine Kidney Cell Line), with accession number CCTCC NO: C2022197. This invention also provides its application in culturing BVDV virus. This invention successfully introduced the SV40-LT gene into primary Niobium Bovine Kidney Cells, activating their telomerase activity and extending the cell lifespan in vitro. Experiments showed that the NBLS Niobium Bovine Kidney Cell Line of this invention can be passaged for more than 50 generations without changing its growth and proliferation characteristics, thus obtaining an immortalized cell line. This F50 generation Niobium Bovine Kidney Cell Line NBLS is highly sensitive to BVDV virus and can efficiently proliferate the virus. The virus titer proliferated using this cell line shows increased sensitivity compared to primary Niobium Bovine Kidney Cells, reducing production costs. Therefore, this invention provides a new technical means for the large-scale production of BVDV vaccines.

Description

Technical Field

[0001] This invention belongs to the field of cell engineering technology, and relates to the yak kidney cell line, specifically the immortalized yak kidney epithelial cell line NBLS and its applications. Background Technology

[0002] The Bos Cattle-yak is an interspecific F1 hybrid of yak and cattle (Bos Taurus). Both male and female Bos Cattle-yaks exhibit significant hybrid vigor. They possess marked advantages in physique, meat production, milk production, adaptability, productivity, disease resistance, and reproduction, and are well-suited to harsh weather conditions. Locally, they are primarily produced by crossing male cattle and female yaks. The male offspring are mainly used for fattening and meat production, while the female offspring are primarily used for milk production; however, the economic benefits from their milk and meat production are not ideal.

[0003] Currently, a variety of passaged cell lines have been discovered and established, such as MDCK cells and VERO cells, and these cell lines have been used in vaccine production, but they originated from foreign research institutions.

[0004] Cell culture is a method of enabling cells to survive, grow, divide, and maintain their original structure and function in vitro, mimicking the in vivo environment. Through this technology, a single cell can be cultured into a single cell or a multi-cell cluster; therefore, cell culture is also known as cell cloning technology or cell culture technique. This technology can not only culture large numbers of cells but also be applied to research in signal transduction, anabolic metabolism, and growth and proliferation. Furthermore, cell culture technology has made significant contributions to biomedicine and related fields. For example, recent advancements in basic medicine have largely relied on cell culture technology. In addition, practical technologies based on cell culture have been developed in fields such as biomedicine, involving vaccine production, toxicity assessment, and assisted reproduction. Therefore, cell culture is not only an important technology in cell biology but also plays a vital role in the entire field of bioengineering and biomedicine.

[0005] The key to establishing a stable cell line is maintaining the normal function of primary cells while maximizing cell proliferation. Cell immortalization refers to the process by which animal cells, after failing to differentiate and being exposed to viral infection, exogenous gene transfection, radiation, etc., lose their cell division limit mechanisms and DNA-based apoptosis detection mechanisms, resulting in cells with unlimited division and growth capacity. Immortalized cells are largely the same as passaged cell lines, possessing not only unlimited proliferative capacity but also the same biological characteristics as primary cells. According to the standards for immortalized cell lines in the *Pharmacopoeia of the People's Republic of China*, cells that can be stably passaged more than 50 times are called immortalized cells. To date, several methods have been discovered to achieve cell immortalization, including transfection with simian vacuolating virus 40 (SV40) large T antigen (LT), mediation by human papillomavirus (HPV) E6 / E7 proteins, overexpression of human telomerase reverse transcriptase (hTERT), as well as spontaneous, irradiated, and external stimuli.

[0006] SV40, short for simian virus 40, is composed of VP1, VP2, VP3, LT, and small T antigen (ST). The LT antigen activates E2F-mediated transcription by binding to the Rb-E2F signaling pathway pocket site complex, and can also bind to Rb proteins in an ATP-dependent manner via the LxCxE motif and J domain. The ATPase domain can further bind to p53 protein, promoting cell transformation. Therefore, LT can achieve cell immortalization through the co-regulation of pRB and p53. Studies have shown that LT, after binding to p300 / CBP histone acetyltransferase, can act on p53 and lead to cell immortalization. ST possesses various oncogenic properties and can synergistically work with LT to induce complete cell transformation, playing a stabilizing role. Because SV40 expression can cause genomic instability due to p53 deletion, this problem can be avoided by using the piggyBac transposon to excise the selective marker gene, which can restore cells to their primary state. Alternatively, the tsA58 temperature-sensitive mutant of SV40 can be used to eliminate SV40-LT expression through temperature regulation. Recent studies have shown that mouse podocytes undergo conditional immortalization via interferon-gamma, requiring approximately 14 days at temperatures above 37.5°C to completely lose the activity of the SV40-LT antigen.

[0007] Bovine viral diarrhea virus (BVDV) is one of the major and common viral threats in the cattle industry. Researchers have conducted extensive studies on BVDV from the perspectives of molecular biology, pathogenesis, immune response, and prevention and control measures. Vaccination helps control and eliminate BVDV infection and is an effective way to prevent clinical disease and fetal infection. However, the continuous emergence of new BVDV variants and strains can hinder diagnostic analysis and vaccine potency assessment. Therefore, the use of high-potency novel cell substrates is crucial for BVDV vaccine production and technological upgrades. Currently, the cultured cells used in BVDV vaccines are MDBK cells, but the viral titer is low.

[0008] In China, research on hybrid cattle is limited, focusing primarily on feeding and fur applications, with little broader practical value. Research on hybrid cattle kidney cells is also scarce, with no studies on their immortalization. The preparation of BVDV vaccines is complex, resulting in low viral titers, hindering large-scale production and limiting profits for vaccine manufacturers. Furthermore, MDBK cells were introduced from foreign research institutions. Therefore, developing a suitable passaged cell line for BVDV vaccine production is of significant practical importance to vaccine companies.

[0009] To further explore the utilization value of yak calves, and considering that the cell substrates currently used in vaccine production (such as MDCK cells and VERO cells) are all kidney cells, this study used lentivirus transfection to construct and evaluate immortalized cell lines. Summary of the Invention

[0010] To address the problems existing in current technologies, and considering the very limited in vitro culture time of primary yak kidney cells, which hinders research on cell growth, differentiation, and metabolic mechanisms, a cell model suitable for long-term in vitro culture and various experimental research purposes is needed. The applicant will attempt to establish an independently passaged cell line in my country, contributing to the field of basic research.

[0011] The inventors of this application used the SV40-LT gene introduction method to introduce a viral fluid encoding SV40-LT into limited-passage broiler kidney cells to obtain the broiler kidney cell line NBLS. Then, the broiler kidney cell line was immortalized. It was found that at passage 50, the F50 generation NBLS cell line was largely identical to the passaged cell line, possessing not only unlimited proliferative capacity but also the same biological characteristics as the primary cells, thus obtaining an immortalized broiler kidney cell line. Experiments also showed that the F50 generation broiler kidney cell line NBLS could efficiently proliferate BVDV virus. Therefore, the broiler kidney cell line NBLS, passaged to the F50 generation, was deposited on June 20, 2022, at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, China, with accession number CCTCCNO: C2022197.

[0012] This invention also provides the application of the NBLS (bicycle kidney cell line) in the culture of BVDV virus.

[0013] The present invention also provides a method for culturing BVDV virus, wherein culture medium is added to BVDV virus solution, and then the solution is added to a container containing cells for culture; wherein the cells are the above-mentioned yak kidney cell line NBLSCCTCCNO:C2022197.

[0014] Preferably, the culture medium is formulated as DMEM medium containing 2% fetal bovine serum by volume.

[0015] Preferably, the culture conditions are: cultured at 36±1℃ for 24-48h.

[0016] The beneficial effects of this invention are: the invention successfully introduced the SV40-LT gene into primary yak kidney cells, activated their telomerase activity, and extended the lifespan of the cells in vitro.

[0017] This invention, through experiments, has shown that the NBLS (Niobium Bovine Kidney Cell Line) can be passaged for more than 50 generations without changing its growth and proliferation characteristics, thus obtaining an immortalized NBLS cell line. This F50 generation NBLS cell line is highly sensitive to BVDV virus and can efficiently proliferate the virus. The viral titer proliferated using this cell line shows increased sensitivity compared to primary Niobium Bovine Kidney Cells, reducing production costs. Therefore, this invention provides a new technical means for the large-scale production of BVDV vaccines. Attached Figure Description

[0018] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used in conjunction with embodiments of the invention to explain the invention and do not constitute a limitation thereof. In the drawings:

[0019] Figure 1 Morphological observation of yak kidney cells. A: Bright field × 40; B: Bright field × 100.

[0020] Figure 2 The minimum screening concentration of puromycin for yak kidney cells was determined. A: 7 days, 0 μg / ml; B: 7 days, 1 μg / ml; C: 7 days, 2 μg / ml.

[0021] Figure 3 MOI values ​​were used to screen lentiviruses from yak kidney cells. The AF plots represent the following values ​​in order: A: MOI = 30; B: MOI = 50; C: MOI = 80; D: MOI = 100; E: MOI = 120; F: MOI = 150.

[0022] Figure 4 Images of SV40-LT transfected fluorescence. Image A is a 100x NBLS bright-field image, and image B is a 100x NBLS-green fluorescence image.

[0023] Figure 5 For NBLS cell morphology analysis.

[0024] Figure 6 This study analyzed gene stability in NBLS cells. Figure A shows the SV40-LT gene level in cells; Figure B shows the WB pattern comparison of SV40-LT; Figure C shows the protein level in cells; and Figure D shows the immunofluorescence of SV40-LT in NBLS cells.

[0025] Figure 7 This is for the analysis of cell growth characteristics. Figure A: Cell growth curve; Figure B: Cell cycle detection; Figure C: Apoptosis detection.

[0026] Figure 8 This section describes the analysis of cellular genetic characteristics. Figure A shows karyotype analysis; Figure B shows vimentin analysis; and Figure C shows cytokeratin-18 analysis. Detailed Implementation

[0027] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as a result. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions of the present invention without departing from the spirit and scope of the present invention, but all such modifications and substitutions fall within the protection scope of the present invention.

[0028] Unless otherwise specified, the culture medium used in this invention is a complete culture medium containing 10% fetal bovine serum (10% fetal bovine serum + DMEM).

[0029] Example 1

[0030] 1. Establishment of the NBLS (Niu Bovine Kidney Cell Line)

[0031] 1.1 Resuscitation and passage culture of yak kidney cells

[0032] To avoid overuse and waste of primary cells, this invention selects F2 generation yak kidney cells from the germplasm resource bank and resuscitates, passages, expands, and freezes them to ensure the smooth progress of subsequent experiments.

[0033] Three vials of F2 generation cytoplasmic kidney cells were taken from liquid nitrogen and quickly thawed in a 37°C water bath. After thawing, the cell suspension was aspirated and seeded into T-25 cell culture flasks. Complete culture medium containing 10% fetal bovine serum (FBS + DMEM) was added to a culture volume of 5 ml. The cells were cultured in a 37°C, 5% CO2 incubator for 6 hours, then the medium was replaced with fresh complete culture medium to reduce the damage to the cells caused by dimethyl sulfoxide (DMSO) in the cryopreservation solution. The cells were cultured for an extended period, and their growth status was recorded by photograph.

[0034] The growth status of F2 generation broiler kidney cells was observed. When the cells reached a confluence of over 85%, they were passaged at a ratio of 1:3. The culture medium was discarded, the cells were washed three times with PBS, 2 ml of 0.25% trypsin was added, and the cells were incubated in a cell culture incubator for 1 min. Observation was then performed under a microscope. When the cells dispersed and shrank into round shapes, the trypsin was discarded, and 3 ml of complete culture medium was added to resuspend the cells. The cells were then transferred to T-25 cell culture flasks at different ratios, with a culture volume of 5 ml, and continued to be cultured in an incubator. When the F3 generation broiler kidney cell density reached over 85%, the cells were washed and digested, and observed under a microscope. Afterwards, they were cryopreserved for later use: the resuspended cell solution was aliquoted into cryovials at 1 ml / vial, and frozen sequentially in the order of 4℃ for 0.5 h, -20℃ for 1 h, and -80℃ overnight, and finally stored in liquid nitrogen tubes.

[0035] After digestion, the cells adhered to the flask and gradually covered the bottom, growing in sheets with tight intercellular connections, forming a spindle-shaped monolayer arrangement (see...). Figure 1 ).

[0036] Figure 1 Morphological observation of yak kidney cells (A: bright field × 40; B: bright field × 100).

[0037] 1.2 Screening of yak kidney cells for puromycin

[0038] For subsequent positive cell screening, it is necessary to determine the minimum lethal concentration of puromycin in yak kidney cells for reference in later experiments. The specific experimental procedure is as follows:

[0039] Resuscitate the yak-cattle kidney cells of F3 generation and inoculate them in a cell culture plate at a density of 1×10

[0048] cells / well. Conduct experiments when the cells grow to 50%-60%. Configure puromycin into different screening concentrations with the culture medium in the range of 0 μg / ml - 10 μg / ml, with a gradient of 1 μg / ml for each. Replace the screening culture medium every 2 days. The concentration at which all cells die after 4 - 7 days is the minimum lethal concentration of puromycin.

[0040] The screening results of puromycin show that all cells in the 2 μg / ml concentration group and higher concentration groups died on the 7th day. That is, 2 μg / ml can be used as the minimum screening concentration of puromycin.

[0041] Figure 2 The minimum screening concentration of puromycin for yak-cattle kidney cells (A: 7 days, 0 μg / ml; B: 7 days, 1 μg / ml; C: 7 days, 2 μg / ml).

[0042] 1.3 Determination of the MOI value of lentivirus for yak-cattle kidney cells

[0043] Due to the limited amount of virus solution, it is necessary to determine the MOI value of the lentivirus for yak-cattle kidney cells for reference in transfection.

[0044] The experiment is divided into three groups: 1) Group M: lentiviral vector GV367; 2) Group P: lentiviral vector GV367 + HitransG P enhancer; 3) Control group: blank control group. The experimental groups are divided into 6 gradients: MOI = 30, MOI = 50, MOI = 80, MOI = 100, MOI = 120, MOI = 150.

[0045] Among them, the lentiviral vector GV367 is purchased from Shanghai GeneChem Co., Ltd., which contains the puro screening gene and the EGFP green fluorescent gene and does not contain the SV40-LT gene.

[0046] HitransGP enhancer is a supporting reagent for the lentiviral vector GV367. The specific experimental process is as follows:

[0047] Resuscitate the yak-cattle kidney cells of F3 generation and inoculate them into a 96-well plate at a density of 3×10 3 cells / well, and continue to culture for about 24 h. When the cell growth density reaches 80%-90%, add each gradient of the experimental groups respectively for the pre-experiment of lentivirus infection. After about 72 hours of infection, when the fluorescence expression abundance is observed to be relatively high, the infection efficiency is about 50%, and the infection conditions and MOI value corresponding to the group with good cell growth can be used as the basis for the subsequent formal infection experiment.

[0048] After lentiviral empty vector infection of F3 generation yak kidney cells for 72 h, the fluorescence efficiency of group P cells was observed to first increase and then decrease with increasing MOI value. High fluorescence abundance was observed at MOI = 100. When MOI exceeded 100, the fluorescence rate began to decrease. Since the high fluorescence abundance at MOI = 100 met the criteria for subsequent functional testing, lentiviral cells with MOI = 100 were selected for subsequent experiments (see details in [link to results]). Figure 3 (and Table 1).

[0049] Figure 3 MOI values ​​were used to screen lentiviruses from yak kidney cells. The AF plot represents the experimental data for group P, and is shown in the following order: A: MOI = 30; B: MOI = 50; C: MOI = 80; D: MOI = 100; E: MOI = 120; F: MOI = 150.

[0050] Table 1. Screening of MOI values ​​for lentiviral vectors from yak kidney cells.

[0051]

[0052] 1.4SV40-LT virus transfection

[0053] The SV40-LT gene was transfected into yak kidney cells using a lentiviral transfection method. Specifically, the SV40-LT gene was constructed into the lentiviral vector GV367 to obtain SV40-LT-lentivirus, which was then transfected using SV40-LT-lentivirus packaging solution. The optimal MOI for infection, determined in step 1.3 of the lentiviral infection pre-experiment, was used for the formal infection experiment. Transfection with SV40-LT-lentivirus packaging solution: F3 generation yak kidney cells were resuscitated and transfected with 1×10⁻⁶ lentiviral packaging solution. 4 Cells were seeded per well in cell culture plates. Lentiviral transfection was performed when cells reached 60% confluence. SV40-LT-lentivirus and HitransGP enhancement medium were added based on cell MOI and viral titer. The calculation formula was: Virus volume = (MOI × cell number) / viral titer. Cells were incubated at 37°C for 12 hours, then replaced with complete culture medium and cultured further.

[0054] The target gene SV40-LT gene sequence is as follows:

[0055]

[0056]

[0057] To determine whether NBLS cells were successfully transfected after 72 hours, images were taken under a fluorescence microscope and analyzed using ImageJ to observe whether fluorescence was observed in the cells. The results are shown below. Figure 4 And Table 2.

[0058] Figure 4 Images show fluorescence images of SV40-LT-lentivirus transfection. Image A is a 100× NBLS bright-field image, and image B is a 100× NBLS-green fluorescence image.

[0059] Table 2. Fluorescence analysis of SV40-LT72h after lentiviral transfection. (ImageJ analysis)

[0060]

[0061] Cells transfected with SV40-LT-lentivirus were observed at 24h, 48h, and 72h. Results showed that the fluorescence intensity and fluorescence rate were higher at 72h after transfection, indicating suitability for further evaluation experiments. For ease of description, cells transfected with SV40-LT-lentivirus are collectively referred to as NBLS.

[0062] 1.5 Screening for positive cells

[0063] To screen for NBLS cells with high fluorescence intensity and high transfection efficiency, NBLS cells were screened 72 hours after transfection.

[0064] NBLS cells were screened using medium containing 2 μg / ml puromycin. The screening medium was changed every 2 days, and cell death was observed. When the control cells were completely dead, NBLS cells were screened again using medium containing 2 μg / ml puromycin. When the cells reached a confluence of more than 90%, the cells were transferred to culture flasks for further culture.

[0065] SV40-LT lentivirus-transfected yak kidney cells were screened with 2 μg / ml puromycin to obtain puromycin-resistant positive cell clones. RNA was extracted from the puromycin-resistant positive cell clones, and qPCR detection showed that all puromycin-resistant positive cells expressed the SV40-LT gene. This indicates that the SV40-LT gene was successfully transformed into primary yak kidney cells and effectively expressed. The selected puromycin-resistant positive cells were named the yak kidney cell line NBLS. After passage, the performance of cells at different passages was analyzed. It was found that at passage 50, the F50 passage NBLS yak kidney cell line could efficiently proliferate BVDV virus. Therefore, the NBLS yak kidney cell line passaged to F50 was deposited at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, China, with accession number CCTCCNO: C2022197, on June 20, 2022.

[0066] Analysis of the NBLS (Niu Bovine Kidney Cell Line)

[0067] 2.1 Morphological Analysis

[0068] NBLS cells were passaged, and cell fluorescence was observed and cryopreserved every 5 generations. Biological characteristics were tested in 5 aspects every 10 generations.

[0069] To compare the morphological differences of cells before and after transfection, F3 generation yak kidney cells, and F3, F10, F20, F30, F40, and F50 generation NBLS cells were seeded into six-well plates. When the cell density reached approximately 70%–80%, cell morphology was observed under an inverted microscope. Results are shown below. Figure 5 .

[0070] Figure 5 For NBLS cell morphology analysis.

[0071] Observations of F3 generation NBLS cells from hybrid cattle kidneys, and F3, F10, F20, F30, F40, and F50 generations, revealed that the cells were spindle-shaped, morphologically intact, and adhered to the culture vessel after passage, gradually covering the bottom of the vessel. With increasing passage number, the F3, F10, F20, F30, F40, and F50 generations of NBLS cells gradually elongated, decreased in size, and exhibited a multi-layered arrangement.

[0072] 2.2 Gene stability analysis

[0073] Using F3 generation broiler kidney cells as a control, the gene and protein levels in F3, F10, F20, F30, F40, and F50 generation NBLS cells were detected by qPCR, Western Blot, and immunofluorescence.

[0074] 2.2.1 qPCR analysis and identification

[0075] (1) RNA extraction

[0076] Total RNA was extracted from cells using TRIzol Reagent reagent. F3 generation yak kidney cells, and F3, F10, F20, F30, F40, and F50 generation NBLS cells frozen in liquid nitrogen were revived and seeded in T25 cell flasks. cDNA was extracted from the cells when they reached 80%-90% cell growth and exhibited good morphology and growth status.

[0077] (2) Primer design

[0078] Based on the hTERT gene, SV40-LT gene, and internal reference actin-β (ACTB) sequence from yak kidney cells in GenBank, primers suitable for qPCR were designed and analyzed using Primer5 and Oligo6 within the conserved regions of the nucleic acid sequence, and primer specificity was detected using NCBIBlast.

[0079] (3) Removal of genomic DNA and reverse transcription

[0080] DNA removal and cDNA synthesis were performed according to the reverse transcription kit instructions.

[0081] (4) Reverse transcription reaction

[0082] Dilute the primers to 10 nmol using DEPC water, set up control wells, prepare according to the ratio, and perform qPCR experiments.

[0083] The gene level of SV40-LT in NBLS cells was identified using qPCR. The results showed that the relative level of SV40-LT mRNA in NBLS cells increased with each generation, while the SV40-LT mRNA level in F3 generation yak kidney cells was almost zero. Figure 6 -A).

[0084] 2.2.2 Western Blot Identification

[0085] F3 generation yak kidney cells, F3, F10, F20, F30, F40 and F50 generation NBLS cells were revived and seeded in T25 cell flasks. When the cell growth density was about 80%-90%, the following steps were taken to perform the SV40-LT Western Blot experiment: (1) Protein sample preparation (2) Protein content determination (3) SDS-PAGE electrophoresis (4) Imaging detection.

[0086] Western blotting was used to identify and quantify SV40-LT protein in NBLS cells. The results showed that SV40-LT protein was expressed in NBLS cells at a high level, with a significant difference in expression compared to yak kidney cells (P<=0.05). However, there was no significant difference in SV40-LT expression among different generations from F3 to F50 (P>0.05) (Figure 6-B). Figure 6 -C).

[0087] 2.2.3 Immunofluorescence identification

[0088] Cell smears were placed in 12-well plates, and F3 generation HYK cells, F3, F10, F20, F30, F40, and F50 generation NBLS cells were resuscitated at 5 × 10⁻⁶ cells / well. 3 Seed cells per well. When the cell growth density is about 80%-90%, perform immunofluorescence detection of SV40-LT according to the following steps: 1) Add 4% paraformaldehyde and fix at room temperature for 30 min; 2) Remove the fixative, add 1 ml of 0.1% Triton-100 and permeate for 15 min; 3) Add healthy fetal sheep serum and block at 37℃ for 30 min.

[0089] 4) Discard the blocking solution, add SV40-LT primary antibody, and incubate overnight at 4°C; 5) Add FITC-labeled goat anti-rabbit IgG secondary antibody, and incubate at 37°C in the dark for 2 hours; 6) Add 2 ml of DAPI stock solution and stain in the dark for 4 minutes; 7) Add anti-fluorescence quenching agent, mount with glycerol, seal the slide on a glass slide and store in the dark, and observe and photograph under a laser confocal microscope.

[0090] Immunofluorescence was used to identify the localization of SV40-LT in NBLS cells (exogenous genes expressed in the cytoplasm are shown as red fluorescence; exogenous genes not expressed in the nucleus are shown as blue fluorescence). The results showed that the cytoplasm was stained red due to SV40-LT expression, while the nucleus, which was not expressed, was stained blue. Figure 6 -D).

[0091] Figure 6 This study analyzed gene stability in NBLS cells. Figure A shows the SV40-LT gene level in cells; Figure B shows the WB pattern comparison of SV40-LT; Figure C shows the protein level in cells; and Figure D shows the immunofluorescence of SV40-LT in NBLS cells.

[0092] 2.3 Growth Characteristics Analysis

[0093] 2.3.1 Cell viability determination

[0094] To determine the viability and quantity of live cells, a cell counter was used to measure the cell viability of resuscitated F3 generation yak kidney cells and NBLS cells from F3, F10, F20, F30, F40, and F50 generations. Cell suspension was aspirated and added to fill the counting chamber, which was then placed in the counter for measurement.

[0095] The results showed that the average viability of F3 generation cytoplasmic kidney cells was above 90%, with a doubling time of approximately 27 hours. The average and minimum viability of NBLS cells from F3, F10, F20, F30, F40, and F50 generations were all above 90%. See Table 2 for details.

[0096] Table 2. Cell viability and doubling time determination

[0097]

[0098] 2.3.2 Cell growth curve determination and doubling time determination

[0099] Resuscitate F3 generation cytoplasmic kidney cells, and F3, F10, F20, F30, F40, and F50 generation NBLS cells at a concentration of 1×10⁻⁶. 4Seed cells per well. Collect digested cells daily, centrifuge at 1000 rpm for 5 min, discard the supernatant, and prepare a single-cell suspension. After thorough mixing, count the cells, perform three replicates, and calculate the average cell count across the three wells. Repeat the above method for 12 consecutive days, calculating the average. Plot a cell growth curve with culture time on the x-axis and cell count on the y-axis. The cell population doubling time is calculated using the following formula: PDT = t × lg2lgNt - lgN0. Where t represents the culture time, N0 represents the initial cell count, and Nt represents the cell count after culture time t.

[0100] Growth curve analysis revealed that F3 generation broiler kidney cells proliferated slowly from day 1 to day 3, entered the logarithmic growth phase from day 3, and reached a plateau phase by day 5, but the cell number did not decrease. NBLS cells from F3, F10, F20, F30, F40, and F50 generations all exhibited slow proliferation from day 1 to day 3, entered the logarithmic growth phase from day 3, reached their peak on day 8, then entered a plateau phase, and decreased on day 9. Figure 7 -A).

[0101] 2.3.3 Cell cycle assay

[0102] F3 generation yak kidney cells, and F3, F10, F20, F30, F40 and F50 generation NBLS cells were revived until the growth density reached about 70% to 80%, and then cell cycle measurements were performed.

[0103] Flow cytometry analysis of cell cycle time revealed that F3 generation broiler kidney cells had approximately 40% completion in both the S and G1 phases, and 11.78% in the G2 phase. From F3 to F50 generation NBLS cells, both G1 and S phases exceeded 40%, similar to the results for broiler kidney cells, with no significant difference (P>0.05). Figure 7 -B).

[0104] 2.3.4 Apoptosis assay

[0105] F3 generation yak kidney cells, and F3, F10, F20, F30, F40, and F50 generation NBLS cells were revived. Once the growth density reached approximately 70%–80%, apoptosis was measured.

[0106] The percentage of live, dead, and apoptotic cells was detected by flow cytometry. The results showed that the apoptosis rate of F3 generation broiler kidney cells was less than 2%, while the live cell rate was higher than 95%. The apoptosis rate of NBLS cells from F3 to F50 generation was less than 2%, and the live cell rate was higher than 95%, which was similar to the results of broiler kidney cells and there was no significant difference (P>0.05) (Figure 7-C).

[0107] Figure 7 This is for the analysis of cell growth characteristics. Figure A: Cell growth curve; Figure B: Cell cycle detection; Figure C: Apoptosis detection.

[0108] 2.4 Genetic Characteristics Analysis

[0109] 2.4.1 Karyotype analysis

[0110] F3 generation NBLS cells were resuscitated, and F3, F10, F20, F30, F40, and F50 generation cells were seeded into 6-well culture plates until the growth density reached approximately 70%-80%. The following steps were performed: 1) Add 0.1 μg / ml colchicine to each well for 6 h, digest, and resuspend in preheated 0.075 mol / L potassium chloride solution, then incubate at 37°C for 30 min; 2) Discard the potassium chloride solution, add 400 μL of fixative, and centrifuge at 1500 rpm for 5 min; 3) Retain 1 ml of supernatant and centrifuge again, repeating the above process; 4) After resuspending the cells, remove the slide from the water bath, aspirate the resuspended solution, and drip it from a height of 50 cm, then air dry for 30 min; 5) After the slide is dry, stain with 10% Giemsa stain for 20 min, rinse with distilled water, mount, and observe under a microscope.

[0111] Karyotype analysis was used to identify mutations in chromosome number and morphology in cells. The results showed that most NBLS cells from generations F3, F10, F20, F30, F40, and F50 had 2n = 60 chromosomes, which is roughly the same as the chromosome number in yak kidney cells. Figure 8 -A).

[0112] 2.4.2 Immunofluorescence assay

[0113] Cell slides were placed in 12-well plates, and F3 generation NBLS cells were resuscitated. F3, F10, F20, F30, F40, and F50 generation NBLS cells were cultured at 5 × 10⁻⁶ cells / well. 3 Inoculate cells per well and wait until the growth density reaches about 70%-80% before conducting experiments. The primary antibody is cytokeratin-18 and vimentin, which are blocked using fetal sheep serum. The secondary antibody is FITC-labeled goat anti-rabbit IgG.

[0114] Immunofluorescence was used to identify the expression of cytokeratin-18 and vimentin in cells. The results showed that cytokeratin-18 was expressed in F3-F50 generation NBLS cells and was expressed in the cytoplasm, appearing as red fluorescence. Vimentin, however, was not expressed and appeared as blue fluorescence. The results for NBLS cells were consistent with those for yak kidney cells. Figure 8 -B、 Figure 8 -C).

[0115] Figure 8 This section describes the analysis of cellular genetic characteristics. Figure A shows karyotype analysis; Figure B shows vimentin analysis; and Figure C shows cytokeratin-18 analysis.

[0116] 2.5 Functional Verification

[0117] 2.5.1 BVDV Virus Amplification

[0118] F3 generation cyba kidney cells were resuscitated and seeded into T25 cell culture flasks. When the confluence of cyba kidney cells reached 80%, the cell culture medium was discarded under aseptic conditions. The cells were washed three times with 2 ml of sterile PBS, and 4.5 ml of DMEM medium containing 2% fetal bovine serum was added, followed by 0.5 ml of BVDV virus stock solution. The cell culture flasks were then incubated at 36°C in a CO2 incubator. The cell status was observed daily. After all cells had died, on the 5th day after virus inoculation, the inoculated cell culture flasks were placed at -80°C and subjected to three freeze-thaw cycles. The frozen-thawed virus culture medium was collected, centrifuged at 12,000 rpm for 10 min, and the supernatant was collected. The supernatant was then filtered through a 0.22 μm filter, aliquoted into centrifuge tubes, and stored at -80°C.

[0119] 2.5.2 TCID50 Experiment

[0120] F3 generation cybotomyces kidney cells and F50 generation cybotomyces kidney cell line NBLSCCTCCNO:C2022197 were seeded in 96-well plates and cultured at 37°C for 24 h until a monolayer formed. Once the cells reached confluence, the BVDV virus suspension collected in step 2.5.1 was serially diluted 10-fold using DMEM medium containing 2% fetal bovine serum (FBS). The diluted solution was dispensed into 10 test tubes (0.9 ml per tube). 0.1 ml of the virus suspension was added to the first tube and mixed thoroughly to obtain 10 separate tubes. -1 Change to a different straw, in 10 -1 Aspirate and dispense the virus solution 3-4 times, take 0.1 ml and transfer it to a second tube, then shake well. Dilute to 10⁻⁶ in this way. -10 Remove the original culture medium and inoculate the diluted virus solution into the cells: add 100 μL of virus solution to each well, and use F3 generation yak kidney cells without virus solution as a virus control. Incubate at 37°C, and observe the results after 24 h, 48 h, 72 h, and 96 h. Observe the normal cell control first, and then observe the experimental group cells. Calculate and plot the results according to the Reed-Muench formula: TCID50 = logarithm of the reciprocal of the high critical dilution for 50% infection + distance ratio × logarithm of the dilution factor. Where: distance ratio = (50% critical infection rate - 50%) / (50% critical infection rate - 50% low critical infection rate);

[0121] The TCID50 assay was used to determine the half-maximal infection dose and infection efficiency of the cells. The results showed that: lgTCID50 (F3 bovine kidney epithelial cells) = 10 -5.62 / 0.1ml, lgTCID50 (F50NBLS cells) = 10 -6.59 / 0.1ml, see Table 3 for details.

[0122] Table 3 Statistical analysis of TCID50 measurement results

[0123]

[0124]

[0125] In summary, this invention successfully introduced the SV40-LT gene into the cell genome. SV40-LT activates telomerase in cells, preventing telomere shortening and maintaining a stable telomere length, ultimately achieving cell immortalization and successfully constructing the yak kidney cell line NBLSCCTCCNO:C2022197. The yak kidney cell line NBLSCCTCCNO:C2022197 retains the normal biological characteristics of its parent cells (primary yak kidney cells) and can efficiently proliferate BVDV virus.

[0126] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The NBLS (Nephrodisiac Bacterialis Kidney Cell Line) has the accession number CCTCC NO: C2022197.

2. The bovine kidney cell line NBLS according to claim 1, characterized in that: The NBLS (Niu kidney cell line) is an immortalized cell line.

3. The bovine kidney cell line NBLS according to claim 1, characterized in that: It was obtained by introducing the SV40-LT gene into the kidney epithelial cells of yak.

4. The use of the NBLS (Niobium Bifidobacterium L.) cell line as described in any one of claims 1-3 in the culture of BVDV virus.

5. A method of culturing a BVDV virus, characterized by: Culture medium is added to the BVDV virus solution, and then it is added to a container containing cells for culture; the cells are the yak kidney cell line as described in any one of claims 1-3.

6. The method of claim 5, wherein: The culture medium formulation is: DMEM medium containing 2% fetal bovine serum by volume.

7. The method of claim 5, wherein: The culture conditions were: cultured at 36±1℃ for 24-48 hours.

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