Immortalized chicken embryonic epithelial-like cells, methods of making and using the same

Abstract

The application discloses immortalized chicken embryo epithelioid cells and a preparation method and application thereof, and the preparation method of the immortalized chicken embryo epithelioid cells comprises the following steps: extracting primary chicken embryo epithelial cells from a chicken embryo; and culturing the primary chicken embryo epithelioid cells in a conditional medium containing chicken Noggin protein for subculture, so as to obtain the immortalized chicken embryo epithelioid cells. The immortalized chicken embryo epithelioid cells prepared by the preparation method can be stably subcultured for a long time, can maintain stable proliferation ability after multiple subcultures, reduces individual differences, and improves the reliability and repeatability of experimental results. In addition, the preparation method does not introduce exogenous genes or change the genotype of the cells, ensures the stability at the gene level, and avoids the interference of the exogenous genes on the stability of the cell phenotype.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of cell engineering, and in particular to an immortalized chicken embryo epithelial-like cell and a preparation method and application thereof. BACKGROUND

[0002] The importance and research challenges of Infectious Bronchitis Virus (IBV) in poultry farming. IBV, a highly contagious respiratory disease, mainly affects the respiratory, digestive and urogenital systems of chickens. The virus can spread rapidly among chicken populations, leading to high morbidity. IBV can cause stunted growth in chicks, reduced egg production in adult chickens, and even death in severe cases, causing significant economic losses to chicken farms. IBV has a significant impact on poultry farming, and effective prevention and control of IBV transmission is of great significance to the healthy development of the chicken farming industry, improvement of economic efficiency and maintenance of public health. By strengthening biosecurity measures, regular monitoring and timely treatment, the impact of IBV can be effectively reduced.

[0003] IBV can grow on a variety of cells, but only on Chicken Embryonic Kidney (CEK) or Chicken Kidney (CK) cells will it exhibit a clear Cytopathic Effect (CPE). IBV has a clear epithelial cell tropism, especially in kidney tubular epithelial cells, where it exhibits high replication characteristics. Currently, research on IBV at the cellular level relies mainly on chicken embryo kidney cells or chicken embryos. However, there is currently no stable passaged chicken embryo kidney cell line, which means that primary CEK cells need to be extracted for each experiment. Primary cells have the following disadvantages: individual differences between different chicken embryos, the health status of the chicken embryo itself and whether it carries pathogens will affect subsequent research, slow growth, limited proliferation capacity, and inability to perform long-term, large-scale experiments. In order to solve these problems, it is urgent to prepare immortalized chicken embryo epithelial-like cell lines to better support IBV research and related applications.

[0004] Therefore, the present application is proposed. SUMMARY

[0005] The application aims to provide immortalized chicken embryo kidney epithelioid cells, a preparation method and application thereof.

[0006] In order to achieve the above-mentioned purpose of the application, the following technical scheme is adopted:

[0007] The application provides a preparation method of immortalized chicken embryo kidney epithelioid cells, which comprises the following steps:

[0008] (a) extracting primary chicken embryo kidney epithelial cells from a chicken embryo;

[0009] (b) culturing the primary chicken embryo kidney epithelial cells in a culture medium containing chicken Noggin protein for subculture, to obtain the immortalized chicken embryo kidney epithelioid cells.

[0010] Preferably, the content of chicken Noggin protein in the culture medium is 80-120 ng / ml.

[0011] Preferably, 100 ml of the culture medium comprises the following components:

[0012] FBS 1.8-2.2 mL, non-essential amino acids 0.8-1.2 mL, penicillin-streptomycin-amphotericin B solution 0.8-1.2 mL, Y27632 80-120 μL, L-WRN conditioned medium 35-45 ml, and the rest is DMEM / F12 culture medium.

[0013] The penicillin-streptomycin-amphotericin B solution has a penicillin concentration of 9000-11000 U / mL, a streptomycin concentration of 8-12 mg / mL, and an amphotericin B concentration of 20-30 μg / mL.

[0014] The final concentration of Y27632 is 8-12 μM.

[0015] Preferably, the chicken embryo is a 16-18-day-old SPF chicken embryo.

[0016] Preferably, the subculture is carried out at a temperature of 39℃ and a carbon dioxide concentration of 5%.

[0017] Preferably, the culture medium is replaced once every 2-4 days during the subculture.

[0018] Preferably, the number of passages of the subculture is not less than 21 times.

[0019] The second aspect of the present application provides an immortalized chicken embryo epithelial-like cell prepared by the preparation method of the immortalized chicken embryo epithelial-like cell.

[0020] The third aspect of the present application provides an application of the immortalized chicken embryo epithelial-like cell prepared by the preparation method of the immortalized chicken embryo epithelial-like cell in constructing an in vitro infection model of infectious bronchitis virus.

[0021] The fourth aspect of the present application provides a construction method of an in vitro infection model of infectious bronchitis virus, characterized in that the construction method comprises using the immortalized chicken embryo epithelial-like cell prepared by the preparation method of the immortalized chicken embryo epithelial-like cell to infect the infectious bronchitis virus.

[0022] Compared with the prior art, the present application has at least the following beneficial effects:

[0023] Compared with the traditional primary cell stable construction method, the immortalized chicken embryo epithelial-like cell constructed by the present application does not have an exogenous gene in the form of insertion or recombination to change the gene of the cell itself, ensuring the stability at the gene level and avoiding the potential risk of genetic mutation. In addition, the immortalized chicken embryo epithelial-like cell can maintain stable proliferation ability after multiple passages, significantly improving the efficiency and repeatability of virus isolation culture and pathogenic mechanism research of IBV wild-type strains, solving the bottleneck of limited passage number of primary cells in the research of pathogenic mechanisms of avian coronaviruses and other pathogens, and improving the reliability and repeatability of experimental results. In addition, the immortalized chicken embryo epithelial-like cell can meet the demand of large-scale experiments, reduce the experimental cost and workload, reduce the dependence on fresh chicken embryos, and meet the requirements of animal welfare.

[0024] The immortalized chicken embryo epithelial-like cell of the present application can be stably passed for a long time, improves the biological safety, can be cultured for a long time under sterile conditions, reduces the risk of introducing pollution in the extraction and culture process, and ensures the safety of the laboratory environment. Moreover, the immortalized chicken embryo epithelial-like cell also has a wide application prospect, can provide more stable and reliable cell models for virology research, vaccine development, drug screening and other fields, and promote the progress of related research. BRIEF DESCRIPTION OF DRAWINGS

[0025] In order to more clearly illustrate the specific embodiments of the present application or the technical solutions in the prior art, the drawings needed in the specific embodiments or prior art description will be briefly introduced below. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, the elements or parts are not necessarily drawn according to the actual proportions.

[0026] Figure 1 Figure 2 is the vector map and agarose gel electrophoresis results of GL178 in Example 1 of the present application, wherein A is the vector map of GL178, and B is the agarose gel electrophoresis results;

[0027] Figure 2 Figure 3 is the blasticidin killing curve in Example 1 of the present application;

[0028] Figure 3 Figure 4 is the morphology of L-WRN-chNOG cells and L-WRN-Ctrl cells in Example 1 of the present application, wherein A is the morphology of L-WRN-Ctrl cells, and B is the morphology of L-WRN-chNOG cells;

[0029] Figure 4 Figure 5 is the Western Blot and RT-qPCR verification of chicken-derived Noggin expression effect in Example 1 of the present application, wherein A is the Western Blot results, and B is the RT-qPCR results;

[0030] Figure 5 Figure 6 is the morphology and population doubling level of primary CEKE cells at different culture days in Example 3 of the present application, wherein A is the morphology comparison of CEKE-Control and CEKE-Treated, with a magnification of 100x; B is the CCK-8 determination of CEKE-Control and CEKE-Treated cell viability; and C is the comparison of cell population doubling level of CEKE-Control and CEKE-Treated;

[0031] Figure 6 Figure 7 is the IFA detection of immortalized CEKE cell surface marker expression in Example 4 of the present application, wherein A is the CK-5 expression, B is the E-Cadherin expression, C is the Vimentin expression, and D is the α-SMA expression;

[0032] Figure 7 Figure 8 is the RT-qPCR detection of IBV-M41 post-infection intracellular virus 3'UTR mRNA expression amount in Example 5 of the present application. DETAILED DESCRIPTION

[0033] The embodiments of the technical solutions of the present application will be described in detail below in conjunction with the examples. The following examples are only used to more clearly illustrate the technical solutions of the present application, and therefore only serve as examples, but cannot limit the protection scope of the present application.

[0034] It should be noted that, unless otherwise specified, the technical terms or scientific terms used in the present application should be understood as the usual meanings understood by the skilled person in the field to which the present application belongs.

[0035] Currently, traditional primary CEK cells encounter a series of problems in virus research and cell culture: 1. Passage stability: Primary CEK cells are prone to lose proliferative capacity and functional characteristics during the passage process, making it difficult to maintain for a long time. 2. Individual differences: Individual differences between different chicken embryos can lead to inconsistent cell characteristics, affecting the repeatability of experimental results. 3. Health status: The health status of primary CEK cells is affected by the chicken embryo itself, which may carry the risk of carrying pathogens. 4. Growth rate and proliferative capacity: Primary CEK cells grow slowly and have limited proliferative capacity, making it difficult to meet the needs of large-scale experiments. 5. Sustainability of cell supply: Primary CEK cells need to be extracted from chicken embryos frequently, increasing experimental costs and workload. 6. Biological safety: There is a biological safety risk in the extraction and culture process of primary CEK cells, which may introduce contamination.

[0036] To solve the above problems, the present application provides a method for preparing immortalized chicken embryo epithelial-like cells, which comprises the following steps:

[0037] (a) Extracting primary chicken embryo epithelial cells from chicken embryos;

[0038] (b) Subculturing the primary chicken embryo epithelial cells in a culture medium containing chicken Noggin protein, thereby obtaining the immortalized chicken embryo epithelial-like cells.

[0039] Compared with traditional primary cell stable construction methods, the immortalized chicken embryo epithelial-like cells constructed by the present application do not have exogenous genes inserted or recombined to change the cell's own genes, ensuring stability at the genetic level and avoiding potential genetic mutation risks. In addition, the immortalized chicken embryo epithelial-like cells can maintain stable proliferative capacity after multiple passages, significantly improving the efficiency and repeatability of virus isolation and culture of IBV wild-type strains and the study of pathogenic mechanisms, solving the bottleneck of limited passage number of primary cells in the study of avian coronavirus and other pathogenic mechanisms, and improving the reliability and repeatability of experimental results. In addition, the immortalized chicken embryo epithelial-like cells can meet the needs of large-scale experiments, reducing experimental costs and workload, reducing dependence on fresh chicken embryos, and meeting the requirements of animal welfare.

[0040] In an embodiment, the content of chicken Noggin protein in the culture medium is 80-120 ng / ml.

[0041] In an embodiment, 100 ml of the culture medium comprises the following components:

[0042] FBS 1.8~2.2 mL, non-essential amino acids 0.8~1.2 mL, penicillin-streptomycin-amphotericin B solution 0.8~1.2 mL, Y27632 80~120 μL, L-WRN conditioned medium 35~45 mL, and the rest is DMEM / F12 medium;

[0043] The penicillin-streptomycin-amphotericin B solution has a penicillin concentration of 9000~11000 U / mL, a streptomycin concentration of 8~12 mg / mL, and an amphotericin B concentration of 20~30 μg / mL.

[0044] The final concentration of Y27632 is 8~12 μM.

[0045] In an embodiment, the chicken embryo is a 16~18-day-old SPF chicken embryo.

[0046] In an embodiment, the temperature of the subculture is 39℃, and the carbon dioxide concentration is 5%.

[0047] In an embodiment, the medium is replaced every 2~4 days during the subculture.

[0048] In an embodiment, the number of subcultures is not less than 21.

[0049] Another embodiment of the present application provides an immortalized chicken embryo epithelioid cell prepared by the above-mentioned method for preparing immortalized chicken embryo epithelioid cells.

[0050] Still another embodiment of the present application provides an application of an immortalized chicken embryo epithelioid cell prepared by the above-mentioned method for preparing immortalized chicken embryo epithelioid cells in constructing an in vitro infection model of chicken infectious bronchitis virus.

[0051] Yet another embodiment of the present application provides a method for constructing an in vitro infection model of chicken infectious bronchitis virus, which comprises infecting the immortalized chicken embryo epithelioid cell prepared by the above-mentioned method for preparing immortalized chicken embryo epithelioid cells with chicken infectious bronchitis virus.

[0052] The technical solutions of the present application are further described in detail through specific embodiments.

[0053] Preparation of L-WRN conditioned medium:

[0054] In a 150 mm cell culture flask, 5×10 6L-WRN cells, add 30 mL DMEM / F12 medium (containing 10% FBS, non-essential amino acids 1x, penicillin-streptomycin-amphotericin B 1x) culture. 72 h after the first batch of conditioned medium collection, 2000 g centrifugation for 10 min, temporary storage at 4℃, continue to add the above 30 mL DMEM / F12 medium culture for 48 h to collect the second batch of conditioned medium, 2000 g centrifugation for 10 min, temporary storage at 4℃, and then collect the third batch according to the same procedure. Mix the three batches of conditioned medium and mix well, filter through a 0.22 μm filter and store at -80℃.

[0055] Example 1

[0056] This example is a preparation method of chicken Noggin protein, which comprises the following steps:

[0057] (1) Based on the chicken-derived Noggin gene CDS sequence (GenBank ID: NM_204123.2), primers chNoggin-F (5'-ATGGATCATTCCCAGTGCCTT-3' SEQ ID NO: 1) and chNoggin-R (5'-CTAGCAGGAGCACTTGCACT-3' SEQ ID NO: 2) were designed. PCR amplification was performed using high-fidelity DNA polymerase (Takara, R010A). The amplification product was verified by agarose gel electrophoresis and gel recovery. Linearized vector (vector map as shown in Figure 1 A) was prepared using EcoR I restriction endonuclease (Takara, 1040S) and Xba I restriction endonuclease (Takara, 1093S). The target gene fragment was connected with the linearized vector at a molar ratio of 3:1 using T4 DNA ligase (Takara, 2011A). 10 μL of the ligation product was used to transform competent cells, and single colonies were picked and identified by PCR and sequencing to obtain the recombinant plasmid pcSLenti-EF1-BSR-CMV-chNOG-3xFlag-WPRE. After screening positive clones and extracting plasmids, the plasmids were identified by enzyme digestion Figure 1 B), a target gene band was observed at 672 bp; Figure 1 A: vector map; B: agarose gel electrophoresis M: DNA Marker; 1: uncut plasmid; 2: EcoR I / Xba I double enzyme digestion product;

[0058] (2) L-WRN cells were cultured at 5x10 4The density of 1 x 10 cells / well was seeded in 24-well plates. After the cells adhered, the fresh culture medium was replaced, and the culture medium containing 1 μg / mL, 2 μg / mL, 3 μg / mL, 4 μg / mL, 5 μg / mL blasticidin (Solarbio, B9300) was added. The culture medium containing the corresponding concentration of BSR was replaced every 48 h, and the cell survival rate was observed (the results are shown in FIG. 2). Figure 2 The lowest BSR concentration that caused 95%-99% cell death within 5-7 days was used as the screening concentration.

[0059] (3) 5 x 10 4 cells / well were seeded in 24-well plates and cultured for 24 h. When the cell density reached 30%-40%, the recombinant lentivirus and empty vector lentivirus were infected at an MOI of 40. The virus volume (μL / well) = MOI x cell number / dilution (TU / mL) x 1000. Virus solution containing 5 μg / mL Polybrene (Biyun Tian, C0351) was added. After 12 h of infection, the virus solution was discarded, and the culture medium containing 2% FBS was replaced for continuous culture. At 96 h after infection, 3 μg / mL blasticidin was added, and two cell strains, L-WRN-chNOG and L-WRN-Ctrl, were screened (the morphologies of the two cells are shown in FIG. 3). Figure 3 Figure 3 In FIG. 3, A is the morphology of L-WRN-Ctrl cells; B is the morphology of L-WRN-chNOG cells.

[0060] (4) L-WRN-chNOG and L-WRN-Ctrl cells were cultured, centrifuged, and the cells and supernatant were collected.

[0061] Supernatant (SUP) sample processing: centrifuge the supernatant to remove cell debris; weigh 10 g of TCA, add ultrapure water to make up to 10 ml to prepare a 100% TCA solution; add 100% TCA solution to the supernatant at a volume ratio of 4:1, and stand at 4°C for 10 min; then centrifuge at 4°C, 14000 rpm for 10 min, discard the supernatant, resuspend the protein precipitate with pre-cooled acetone, centrifuge at 4°C, 14000 rpm for 10 min, wash once with pre-cooled acetone, centrifuge at 4°C, 14000 rpm for 10 min, and then evaporate acetone at room temperature for 20 min; finally, add RIPA lysis buffer to the obtained protein precipitate and resuspend, and add 5x protein Loading Buffer at a volume ratio of 4:1, and denature at 100°C metal bath for 10 min.

[0062] ​Whole Cell Lysate (WCL) sample processing: The collected cells were washed three times with PBS (Savillex, G4202), and lysate was prepared: 1 mL RIPA lysis buffer (Bi Yun Tian, P0013C) was added to 10 μL PMSF (Bi Yun Tian, ST505) and lysed on ice for 30 min. Centrifuged at 4°C, 12000 r / min for 20 min, and the supernatant was added to 5x SDS loading buffer (Bi Yun Tian, P0015) and denatured at 100°C for 10 min.

[0063] The two samples were separated by 10% precast gel (Bio-Rad, AR1203) at 120 V constant voltage and 300 mA constant current to PVDF membrane. After blotting, 5% skim milk was used to block at room temperature for 1 h at 60 rpm; 4°C overnight incubation with Anti-Flag primary antibody (Huaan Biotech, M1403-2, 1:1000); the primary antibody was recovered, and the membrane was washed 6 times with TBST at 90 rpm for 5 min; then Anti-Rabbit secondary antibody (Savillex, GB23303, 1:10000) was incubated at room temperature for 1 h at 90 rpm. ECL luminous liquid was prepared and added to the PVDF membrane, and the target band was exposed using a full-automatic digital gel imaging analysis system.

[0064] Total RNA was extracted from L-WRN-chNOG cells and L-WRN-Ctrl control cells using a total RNA extraction kit (Meibio, R4132), and then cDNA was synthesized by reverse transcription kit (Tolobio, 22106). Real-time fluorescent quantitative PCR reaction was performed using TB Green Premix Ex Taq (Tolobio, 22201). The mouse GAPDH gene was selected as the internal reference gene for quantitative analysis, and the primer sequences are shown in Table 1. Finally, the relative expression of the gene was calculated by 2 -ΔΔCt Method for calculating the relative expression of the gene.

[0065] Table 1

[0066]

[0067] The Western Blot results showed that the protein size was consistent with the expected Figure 4 RT-qPCR Figure 4 Western Blot (Fig. 1A) and RT-qPCR (Fig. 1B) confirmed that Noggin protein was stably expressed in L-WRN cells and effectively secreted into the extracellular matrix; Figure 4In the middle, A: Western Blot results 1: L-WRN-chNOG cell lysate, 2: L-WRN-Ctrl lysate, 3: L-WRN-chNOG supernatant protein mixture, 4: L-WRN-Ctrl supernatant protein mixture; B: RT-qPCR results, **: P < 0.01;

[0068] (5) Extraction and purification of chicken-derived Noggin protein

[0069] The chicken-derived Noggin protein was purified using a FLAG tag (DYKDDDDK) fusion protein purification kit (Elabscience, item number: EA-TP-K001). First, reagents were prepared. 10×PBS was diluted with deionized water at a ratio of 9:1 to prepare 1×PBS (freshly prepared), 10 mg of 3×Flag peptide was centrifuged and then dissolved in 0.2 mL of 10×PBS, and 1.8 mL of deionized water was added to prepare a 5 mg / mL storage solution (stored at -20°C), which was diluted with 1×PBS to 0.2-0.5 mg / mL competitive eluent when used, and glycerol and 1×PBS were mixed at a ratio of 1:1 to prepare a gel storage solution (freshly prepared) with a preservative. Then, the sample was processed. The L-WRN-chNOG cell culture supernatant was collected and centrifuged at 12000 rpm at 4°C for 10 min to remove debris. Then, the column was loaded and incubated. The Anti-Flag affinity gel was gently resuspended, 2 mL of gel suspension (containing 1 mL of affinity gel) was added to the purification column, and after the liquid flowed out, 10 mL of 1×PBS was added for balancing, and then the pretreated supernatant was added. After sealing the purification column, it was incubated overnight at 4°C on a shaker. After incubation, the flow-through supernatant was collected (stored at 4°C temporarily), and the affinity gel was washed with 5 mL of 1×PBS for 3 times. Then, competitive elution was performed. First, 5 mL of pre-cooled acidic pre-washing solution was added for washing, and after the liquid flowed out, 2 mL of pre-cooled competitive eluent was added, sealed and incubated at 4°C on a shaker for 2 h (which can be extended), and the eluent was collected. If necessary, the elution was repeated once and combined. After elution, the affinity gel was immediately regenerated. 10 mL of 1×PBS was added for washing, 3 mL of acidic eluent was added for washing, and then 3 mL of 1×PBS was added for washing 3 times until the flow-through liquid was neutral. Finally, 1×PBS containing 50% glycerol and 0.2% sodium azide was added and mixed, and then stored at -20°C. A small amount of eluent was taken for SDS-PAGE to identify the purity of the purified protein. The eluent was sterilized through a 0.22 μm filter, and then stored at 4°C for short-term or at -80°C for long-term. Before use, the concentration was adjusted as needed.

[0070] Example 2

[0071] The present embodiment is a method for extracting primary chicken embryo kidney epithelial cells, which comprises the following steps:

[0072] Take 18-day-old SPF chicken embryos and sterilize them with 75% ethanol. Open the shell and shell membrane in a clean bench, and take out the chicken embryos aseptically. Cut open the abdominal cavity, peel off the internal organs and intestinal tract to expose the kidneys. Remove the kidneys and wash them twice with PBS to remove fat and blood clots. Cut the tissue into small pieces and add 0.25% trypsin (Dr. Scopes, PYG0107) for 10 minutes of digestion at 37°C in a water bath. When the tissue is loose and transparent, stop the digestion by adding complete culture medium and mix well; filter through a 100 μm cell strainer and centrifuge at 1200 r / min for 6 minutes; resuspend the cell pellet with DMEM / F12 culture medium containing 2% FBS (Pronova, 164250) and culture at 39°C, 5% CO2, and saturated humidity. Extract the chicken embryo kidney epithelial cells using the differential adhesion method: after 50 minutes of culture, the fibroblasts will preferentially adhere, while the epithelial cells will mostly remain in suspension. Carefully collect the supernatant (containing unattached epithelial cells) from the culture bottle and transfer it to a new sterile culture bottle. Supplement the new culture bottle with an appropriate amount of DMEM / F12 culture medium containing 2% FBS and continue to culture in a 39°C, 5% CO2 incubator to obtain primary chicken embryo kidney epithelial cells.

[0073] Example 3

[0074] The present embodiment is a comparison of the morphology and population doubling level of CEKE cells at different culture days.

[0075] Culture CEKE cells using different culture media. The treatment group (CEKE-Treated) uses the medium formulated in Table 2, and the control group uses a medium that removes the chicken-derived nogign protein and L-WRN cell culture medium based on the medium formulation in Table 2. Perform subculture at a temperature of 39°C and a carbon dioxide concentration of 5%. Replace the medium every 3 days. The CEKE cells cultured in the ordinary medium exhibit signs of aging at the 12th day: deterioration of cell morphology, increased mortality, and a significant decrease in proliferation rate. Figure 5 Figure 2A. By the 21st day, the cells are completely shriveled and dead. In contrast, the CEKE-Treated cells maintain stable proliferation ability and can be subcultured stably without signs of aging after 21 days of culture. Figure 5 Figure 2A. By the 21st day, the cells are completely shriveled and dead. In contrast, the CEKE-Treated cells maintain stable proliferation ability and can be subcultured stably without signs of aging after 21 days of culture. Figure 5 Figure 2B, C

[0076] Table 2

[0077]

[0078] Figure 5 (A) CEKE-Control vs. CEKE-Treated morphology, magnification 100x; (B) CCK-8 assay of CEKE-Control vs. CEKE-Treated cell viability (C) Comparison of CEKE-Control vs. CEKE-Treated cell population doubling level (PDL).

[0079] Population Doubling Level (PDL) refers to the number of times the cell number doubles from the initial inoculation to a certain cell density during the culture process. PDL is an important indicator for evaluating cell proliferation capacity and passage stability, especially in the preparation and identification of immortalized cell lines. PDL is calculated according to the following formula: PDL = [log(N t / N0) / log(2)] + PDL0 (N0: initial inoculation cell number; N t : cell number at harvest; PDL0: initial PDL value).

[0080] The cell viability of CEKE was detected by CCK-8 method: CEKE was digested, cell counting was performed and plating was carried out. In 96-well plates, 5000 cells were plated in each well, and incubated in a 39°C cell incubator for 12-24 hours. 100 μL of fresh culture medium was added, and the culture medium without cells was added to the control group as a blank control. 10 μL of CCK-8 solution was added to each well, and incubated in a 39°C cell incubator. The absorbance at 450 nm was measured at 1, 2, 4 hours, respectively, and the cell viability was calculated. Compared with the control group, the proliferation capacity and cell viability of CEKE-Treated cells were significantly higher. In summary, combined with cell morphology and stable passage culture, it is preliminarily judged that immortalized chicken embryo kidney epithelial-like cells are successfully obtained.

[0081] Example 4

[0082] This example is for IFA detection of the expression of surface markers of immortalized CEKE cells:

[0083] Different treated CEKE was taken, 2x10 4Cells were collected and 300 μL of culture medium was added. The cells were cultured at 39 °C and 5% CO2 for 24 hours. The culture medium was then aspirated, and 500 μL of PBS was added to each well. The cells were shaken at 150 rpm for 5 minutes at room temperature, and washed three times (the following washing steps are the same as this step). Add 300 μL of acetone to each well and fix at -20 °C for 10 min; wash 3 times; add 500 μL of blocking buffer (10% goat serum / 0.1% Tween 20 / PBS) to each well and block at 4 °C for 8 h; wash 3 times; dilute the primary antibodies CK-5 (Saiwell, GB111246), E-Cadherin (Proteintech, 60335-1-Ig), Vimentin (Huaan Bio, ET1610-39), and α-SMA (Huaan Bio, ET1607-53) with antibody dilution buffer (0.1% goat serum / 0.1% Tween 20 / PBS), add 200 μL to each well, and block at 4 °C for 8 h; Incubate overnight at ℃; wash 3 times; add 200 μL of fluorescent secondary antibody (FITC-labeled goat anti-rabbit IgG (H+L), Beyotime, A0562; FITC-labeled goat anti-mouse IgG (H+L), Beyotime, A0568) to each well in the dark, and incubate at room temperature for 1 h. Wash 3 times. Add a drop of mounting medium containing DAPI (Saiwell, G1012) to a glass slide, gently cover with a coverslip, remove air bubbles and excess mounting medium, and seal the edges of the coverslip with nail polish. Observe fluorescence using a BioTek Cytation 1 multi-functional cell imaging system. IFA results are as follows: Figure 6 As shown;

[0084] Figure 6 In the diagram, (A) CK-5; (B) E-Cadherin; (C) Vimentin; (D) α-SMA; DAPI, cell nucleus. Scale bar, 50 μm;

[0085] Depend on Figure 6 It can be seen that epithelial-like markers CK-5 and E-Cadherin are both expressed in CEKE, while mesenchymal cell marker Vimentin and endothelial cell marker α-SMA are not expressed. This indicates that immortalized CEKE has a stable and consistent phenotype with primary CEKE, reflecting that the cells maintain good genetic stability during passage.

[0086] Example 5

[0087] This example demonstrates the use of RT-qPCR to detect the expression level of viral 3'UTR mRNA in cells after IBV-M41 challenge.

[0088] After CEKE-Control and CEKE-Treated cells were plated and cultured at 39°C in a 5% CO2 incubator for 24 h, the supernatant was discarded, and the cells were washed three times with pre-cooled PBS. The medium was then replaced with serum-free medium (to eliminate the potential influence of serum on virus adsorption efficiency). IBV-M41 virus allantoic fluid was diluted 1:10 in serum-free medium and added to culture plates as the virus infection group (IBV group); an equal volume of virus-free allantoic fluid was added to the control group (MOCK group). The culture plates were gently shaken several times to mix, and then incubated at 39°C in a 5% CO2 incubator for 2 h. After adsorption, the virus-containing liquid was discarded, and the cells were washed three times with pre-cooled PBS to remove unadsorbed virus. The medium was then replaced with maintenance medium containing 2% fetal bovine serum (FBS) for continued culture. Cell morphology changes were observed and recorded every 12 hours post-infection. 72 h post-infection, total RNA was extracted from the cells, and cDNA was synthesized using a reverse transcription kit. The expression level of IBV 3'UTR was detected by RT-qPCR using cDNA as a template, with chicken GAPDH as an internal reference gene. Primer sequences are shown in Table 3.

[0089] Table 3 Primer information for real-time quantitative PCR

[0090]

[0091] The expression level of IBV 3'UTR can reflect viral replication activity and therefore can be used as a reliable indicator for assessing IBV infection status. Quantitative PCR results (…) Figure 7 The results showed that IBV 3'UTR expression was detected in both CEKE-Control and CEKE-Treated cells after challenge. Although the expression level of viral 3'UTR mRNA in the CEKE-Treated group was lower than that in the CEKE-Control group, it was significantly different from that in the unchallenged control group (MOCK group) (P < 0.0001). These results indicate that the immortalized CEKE cells constructed in this study can serve as an effective model for IBV challenge experiments, and that RT-qPCR can sensitively and accurately detect viral replication levels.

[0092] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention, and they should all be covered within the scope of the claims and specification of the present invention.

Claims

1. A method for preparing immortalized chicken embryo kidney epithelial-like cells, characterized in that, The method for preparing immortalized chicken embryo kidney epithelial-like cells includes the following steps: (a) Extraction of primary chicken embryo renal epithelial cells from chicken embryos; (b) Primary chicken embryo kidney epithelial cells were passaged in a culture medium containing chicken Noggin protein to obtain the immortalized chicken embryo kidney epithelial-like cells. The content of chicken Noggin protein in the culture medium is 80~120 ng / ml; 100 ml of the culture medium comprises the following components: 1.8-2.2 mL FBS, 0.8-1.2 mL non-essential amino acids, 0.8-1.2 mL penicillin-streptomycin-amphotericidal B solution, 80-120 μL Y27632, 35-45 mL L-WRN conditioned medium, with the remainder being DMEM / F12 medium; The penicillin-streptomycin-amphomycin B solution contains penicillin at a concentration of 9000-11000 U / mL, streptomycin at a concentration of 8-12 mg / mL, and aamphomycin B at a concentration of 20-30 μg / mL. The final concentration of Y27632 is 8~12 μM.

2. The method for preparing immortalized chicken embryo kidney epithelial-like cells according to claim 1, characterized in that, The chicken embryos are SPF chicken embryos aged 16-18 days.

3. The method for preparing immortalized chicken embryo kidney epithelial-like cells according to claim 1, characterized in that, The subculture temperature was 39°C and the carbon dioxide concentration was 5%.

4. The method for preparing immortalized chicken embryo kidney epithelial-like cells according to claim 1, characterized in that, The culture medium is changed every 2 to 4 days during the subculture process.

5. The method for preparing immortalized chicken embryo kidney epithelial-like cells according to claim 1, characterized in that, The number of subcultures shall not be less than 21.

6. The immortalized chicken embryo kidney epithelial-like cells prepared by the method described in any one of claims 1 to 5.

7. The application of the immortalized chicken embryo kidney epithelial-like cells prepared by the method described in any one of claims 1 to 5 in constructing an in vitro infection model of chicken infectious bronchitis virus.

8. A method for constructing an in vitro infection model of infectious bronchitis virus in chickens, characterized in that, The construction method includes the preparation of immortalized chicken embryo kidney epithelial-like cells by infecting them with chicken infectious bronchitis virus according to any one of the methods described in claims 1 to 5.

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