Stable passage chick embryo kidney cell as well as preparation method and application thereof

Abstract

The invention discloses stable passage chick embryo kidney cells as well as a preparation method and application thereof. The preparation method comprises the following steps: (a) extracting primary generation chick embryo kidney cells from chick embryos; (b) performing in-vitro subculture on the primary chicken embryo kidney cells in a specific culture medium to obtain stable subculture chicken embryo kidney cells; the stable passage chick embryo kidney cell is successfully constructed by activating a classical Wnt / beta-catenin signal channel in the chick embryo kidney cell, compared with a traditional primary cell stable construction method, the stable passage cell does not have an exogenous gene in an insertion or recombination form, the gene of the cell is changed, the stability on the gene level is ensured, and the stable passage of the chick embryo kidney cell is realized. The potential gene mutation risk is avoided; in addition, the stable passage CEK cells can still keep stable multiplication capacity after multiple passages, the bottleneck that the research of pathogenic mechanisms such as avian coronavirus is limited by the limited passage number of primary cells is solved, and the reliability and repeatability of experimental results are improved.

Description

Technical Field

[0001] The invention relates to the technical field of cell engineering, and in particular to a stable passage chicken embryo kidney cell and a preparation method and application thereof. Background Art

[0002] Importance and research challenges of infectious bronchitis virus (IBV) in poultry farming. IBV belongs to the gammacoronavirus, a highly contagious respiratory disease that mainly affects the respiratory, digestive and urogenital systems of chickens. The virus can spread rapidly among chickens, leading to high morbidity. IBV can cause growth retardation in chicks, decreased egg production in adult chickens, and even death in severe cases, causing significant economic losses to chicken farms. IBV has an extremely important impact in the poultry farming industry. Effective prevention and control of the spread of IBV is of great significance to ensuring the healthy development of the chicken farming industry, improving economic benefits and maintaining public health. The impact of IBV can be effectively reduced by strengthening biosecurity measures, regular monitoring and timely treatment.

[0003] Research status and challenges of IBV. IBV can grow on a variety of cells, but it will only show obvious cytopathic effects (CPE) on chicken embryonic kidney cells (CEK) or chicken kidney cells (CK). At present, the study of IBV at the cellular level mainly relies on chicken embryonic kidney cells or chicken embryos. However, there is currently no stable chicken embryonic kidney cell line, which means that primary CEK needs to be extracted for each experiment. Primary cells have the following disadvantages: there are individual differences between different chicken embryos, the health status of the chicken embryos themselves and whether they carry pathogens will affect subsequent research, they grow slowly and have limited proliferation capacity, and long-term, large-scale experiments cannot be carried out. In order to solve these problems, it is urgent to prepare a stable CEK cell line to better support the research and related applications of IBV.

[0004] In view of this, the present invention is proposed. Summary of the invention

[0005] The purpose of the present invention is to provide a stable passage chicken embryo kidney cell and a preparation method and application thereof. The stable passage chicken embryo kidney cell prepared by the preparation method of the present invention can be stably passaged for a long time, can still maintain a stable proliferation ability after multiple passages, reduces individual differences, and improves the reliability and repeatability of experimental results. In addition, the preparation method of the present invention does not introduce exogenous genes or change the genotype of the cell itself, ensures stability at the gene level, and avoids potential gene mutation risks.

[0006] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0007] The first aspect of the present invention provides a method for preparing stable passage chicken embryo kidney cells, the method comprising the following steps: (a) Primary chicken embryo kidney cells were extracted from chicken embryos; (b) placing the primary chicken embryo kidney cells in a culture medium for subculture, thereby obtaining the stable subculture chicken embryo kidney cells;

[0008] 100 mL of the culture medium consists of the following components:

[0009] FBS 1.8~2.2 mL, NAAS non-essential amino acids 0.8~1.2 mL, penicillin-streptomycin solution 0.8~1.2 mL, GlutaMAX 0.8~1.2 mL, CHIR99021 40~45 μL, Y27632 95~105 μL, valproic acid 12~13 μL, DMH-1 12~14 μL, FOXO1-inhibitor 1.8~2.2 μL, and the rest is DMEM / F12 medium.

[0010] The Wnt signaling pathway is an ancient and highly conserved signaling pathway, and its function is particularly prominent in embryonic development and cancer. The pathway shows a high degree of genetic conservation among different animal species, and its structure and function are extremely similar. The Wnt pathway is mainly divided into two categories: the classical signaling pathway that depends on β-catenin (β-catenin) and the non-classical signaling pathway that does not depend on β-catenin. The classical Wnt / β-catenin signaling pathway plays a key role in maintaining cell stemness and regulating cell differentiation, and is mainly involved in processes such as cell proliferation, cell differentiation, cell migration and angiogenesis. CHIR99021 and valproic acid are GSK-3β inhibitors, which mainly promote cell proliferation and self-renewal by activating the Wnt / β-catenin signaling pathway. GSK-3β is a key negative regulator in the Wnt signaling pathway. Inhibiting GSK-3β can stabilize β-catenin, causing it to accumulate in the nucleus and activate the transcription of downstream genes. DMH-1 is a BMP (bone morphogenetic protein) signaling pathway inhibitor that blocks the conduction of BMP signals by inhibiting the activity of BMP receptors. The BMP signaling pathway plays an important role in cell differentiation and organ development. Y27632 is a ROCK (Rho-associated protein kinase) inhibitor that reduces apoptosis and improves cell survival by inhibiting ROCK kinase activity. ROCK plays an important role in cytoskeleton reorganization, cell migration and apoptosis. FOXO1 is a transcription factor that is involved in regulating apoptosis, metabolism and stress response. FOXO1-Inhibitor inhibits the activity of FOXO1 and reduces its transcriptional regulation of downstream genes, thereby promoting cell proliferation and survival. In the present invention, the canonical Wnt / β-catenin signaling pathway in embryonic renal cells is continuously activated by adding compounds such as CHIR99021, valproic acid, DMH-1, Y27632, and FOXO1-inhibitor to the culture medium, thereby maintaining the stemness of the cells and achieving stable cell passage.

[0011] Preferably, the chicken embryo is a 16-18 day old SPF chicken embryo.

[0012] Preferably, the temperature of the subculture is 38-39.5° C. and the carbon dioxide concentration is 5%.

[0013] Preferably, the culture medium is replaced every 2 to 4 days during the subculture process.

[0014] Preferably, the number of subcultures is no less than 40.

[0015] The second aspect of the present invention provides stable passage chicken embryonic kidney cells prepared by the preparation method of stable passage chicken embryonic kidney cells.

[0016] The third aspect of the present invention provides an application of the stable passage chicken embryo kidney cells prepared by the above-mentioned preparation method of the stable passage chicken embryo kidney cells in constructing an in vitro infection model of chicken infectious bronchitis virus.

[0017] The fourth aspect of the present invention provides a method for constructing an in vitro infection model of chicken infectious bronchitis virus, the construction method comprising using chicken infectious bronchitis virus to infect the stable passage chicken embryo kidney cells prepared by the preparation method of the stable passage chicken embryo kidney cells.

[0018] Compared with the prior art, the beneficial effects of the present invention include at least:

[0019] The present invention successfully constructs a stable passage chicken embryo kidney cell by activating the classical Wnt / β-catenin signaling pathway in chicken embryo kidney cells. Compared with the traditional primary cell stable construction method, the stable passage cells constructed by the present invention do not have exogenous genes inserted or recombined to change the cell's own genes, thereby ensuring stability at the gene level and avoiding potential gene mutation risks; in addition, the stable passage CEK cells can still maintain a stable proliferation ability after multiple passages, solving the bottleneck of the limited number of primary cell passages in the study of pathogenic mechanisms such as avian coronavirus, while improving the reliability and repeatability of experimental results; in addition, the stable passage CEK cells can meet the needs of large-scale experiments, reduce experimental costs and workload, reduce dependence on fresh chicken embryos, and meet animal welfare requirements.

[0020] The stably propagated CEK cells of the present invention can be stably propagated for a long time, thereby improving biosafety, being able to be cultured for a long time under sterile conditions, reducing the risk of introducing contamination during extraction and culture, and ensuring the safety of the laboratory environment; moreover, the stably propagated CEK cells also have broad application prospects, and can provide a more stable and reliable cell model for virology research, vaccine development, drug screening and other fields, thereby promoting the progress of related research. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following is a brief introduction to the drawings required for the specific embodiments or the description of the prior art. 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 scale.

[0022] Figure 1 This is a process diagram for extracting primary CEK in Example 1 of the present invention;

[0023] Figure 2 The growth condition of the cells after 40 generations in Example 1 of the present invention;

[0024] Figure 3 The results of the identification of the proliferation ability and cell viability of CEK-F40 in Example 2 of the present invention;

[0025] Figure 4 The results of IFA detection of cell surface marker expression of CEK cells of different generations in Example 3 of the present invention;

[0026] Figure 5 The expression of different CEK-related genes after IBV challenge detected by RT-qPCR in Example 4 of the present invention;

[0027] Figure 6 The distribution of virus particles in CEK after virus attack observed by transmission electron microscopy in Example 5 of the present invention;

[0028] Figure 7 TCID in Example 6 of the present invention 50 The experiment detected the virus titer in the CEK supernatant after virus attack. DETAILED DESCRIPTION

[0029] The following embodiments of the technical solution of the present invention are described in detail in conjunction with the embodiments. The following embodiments are only used to more clearly illustrate the technical solution of the present invention, and are therefore only used as examples, and cannot be used to limit the protection scope of the present invention.

[0030] It should be noted that, unless otherwise specified, the technical terms or scientific terms used in this application should have the common meanings understood by those skilled in the art to which the present invention belongs.

[0031] At present, 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 proliferation ability and functional characteristics during the passaging process and are difficult to maintain for a long time. 2. Individual differences: Individual differences between different chicken embryos may 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 embryos themselves, and there may be a risk of carrying pathogens. 4. Growth rate and proliferation ability: primary CEK cells grow slowly and have limited proliferation ability, making it difficult to meet the needs of large-scale experiments. 5. Sustainability of cell supply: primary CEK cells need to be frequently extracted from chicken embryos, which increases experimental costs and workload. 6. Biosafety: There are biosafety risks in the extraction and culture of primary CEK cells, which may introduce contamination.

[0032] In order to solve the above problems, an embodiment of the present invention provides a method for preparing stable passage chicken embryo kidney cells, and the method for preparing stable passage chicken embryo kidney cells comprises the following steps: (a) Primary chicken embryo kidney cells were extracted from chicken embryos; (b) placing the primary chicken embryo kidney cells in a culture medium for subculture, and after cell clusters appear, separating the cell cluster cells to obtain the stable subculture chicken embryo kidney cells;

[0033] 100 mL of the culture medium consists of the following components:

[0034] FBS 1.8~2.2 mL, NAAS non-essential amino acids (NAAS non-essential amino acids (100×): Solarbio, catalog number: N1250) 0.8~1.2 mL, penicillin-streptomycin solution (Penicillin-streptomycin solution (100X penicillin-streptomycin solution): Biosharp, catalog number: BL505A) 0.8~1.2 mL, GlutaMAX 0.8~1.2 mL, CHIR99021 40~45 μL, Y27632 95~105 μL, valproic acid 12~13 μL, DMH-1 12~14 μL, FOXO1-inhibitor 1.8~2.2 μL, and the remainder is DMEM / F12 medium.

[0035] The present invention successfully constructs a stable passage chicken embryo kidney cell by activating the classical Wnt / β-catenin signaling pathway in chicken embryo kidney cells. Compared with the traditional cell stable passage method, the present invention does not introduce exogenous genes or change the genotype of the cell itself, thereby ensuring stability at the gene level and avoiding potential gene mutation risks; in addition, the stable passage CEK cells can still maintain a stable proliferation ability after multiple passages, reducing individual differences and improving the reliability and repeatability of experimental results; in addition, the stable passage CEK cells can meet the needs of large-scale experiments, reduce experimental costs and workload, reduce dependence on fresh chicken embryos, and meet the requirements of animal welfare.

[0036] In some embodiments, the chicken embryo is a 16-18 day old SPF chicken embryo.

[0037] In some embodiments, the temperature of the subculture is 38-39.5° C. and the carbon dioxide concentration is 5%.

[0038] In some embodiments, the culture medium is replaced every 2 to 4 days during the subculture process.

[0039] In some embodiments, the subculture is performed for no less than 40 times.

[0040] Another embodiment of the present invention provides stable passage chicken embryonic kidney cells prepared by the preparation method of stable passage chicken embryonic kidney cells.

[0041] Yet another embodiment of the present invention provides an application of the stable passage chicken embryo kidney cells prepared by the above-mentioned preparation method of the stable passage chicken embryo kidney cells in constructing an in vitro infection model of chicken infectious bronchitis virus.

[0042] Another embodiment of the present invention provides a method for constructing an in vitro infection model of chicken infectious bronchitis virus, the construction method comprising using chicken infectious bronchitis virus to infect the stable passage chicken embryo kidney cells prepared by the preparation method of the stable passage chicken embryo kidney cells.

[0043] The technical solution of the present invention is further described in detail below through specific embodiments.

[0044] Example 1

[0045] This embodiment is a method for preparing stable passage chicken embryo kidney cells, which comprises the following steps: (a) Extraction method of primary chicken embryo kidney cells, extraction process such as Figure 1 As shown: Disinfect 16-18 day old chicken embryos with alcohol, and move them into the clean bench with the air chamber facing upwards; gently knock open the chicken embryo shell with the blunt end of tweezers, remove the shell membrane, take out the chicken embryo, and transfer it to a dissecting dish; use sterile scissors and tweezers to cut open the chicken embryo, peel off the internal organs and intestines, expose the kidneys, take out the kidneys embedded in both sides of the spine, transfer them to a plate containing PBS, carefully remove impurities such as blood vessels and fat, and wash them twice in PBS; transfer the kidneys to 2 mL of PBS, chop them up and aspirate the liquid with a pipette, leaving the tissue blocks, add 3 mL of 0.25% trypsin, blow and mix well, and digest in a 37 ℃ water bath for 10 min, shaking the centrifuge tube every 3 min to ensure complete digestion; when the tissue blocks increase in size, become loose, transparent, and flocculent, add 4 mL of complete culture medium to terminate digestion, carefully blow and mix with a pipette; filter with a 100 μm cell sieve to obtain a single cell suspension, transfer it to a 15 mL centrifuge tube, and centrifuge at 1200 r / min for 6 min; discard the supernatant, resuspend the cells in culture medium, and adjust the cell density to 1×10 7 The cells were cultured statically in a 39°C cell culture incubator.

[0046] (b) Primary chicken embryo kidney cells were placed in culture medium at 39°C and 5% CO2 for subculture. The culture medium was replaced every 3 days. After 40 generations, the cell growth was as follows: Figure 2As shown, cell cluster cells were digested separately using cell cloning rings for culture, thus obtaining stable passage chicken embryo kidney cells;

[0047] 100 mL of the culture medium consists of the following components: FBS 2 mL, NAAS non-essential amino acids (NAAS non-essential amino acids (100×): Solarbio, catalog number: N1250) 1 mL, penicillin-streptomycin solution (Penicillin-streptomycin solution (100X penicillin-streptomycin solution): Biosharp, catalog number: BL505A) 1 mL, GlutaMAX 1 mL, CHIR99021 43 μL, Y27632 100 μL, valproic acid 12.5 μL, DMH-1 13 μL, FOXO1-inhibitor 2 μL, and the remainder is DMEM / F12 medium.

[0048] Example 2

[0049] This example is to identify the proliferation ability and cell viability of stable passage chicken embryo kidney cells: Population Doubling Level (PDL) refers to the number of times the number of cells doubles during the culture process, from the initial inoculation to reaching a certain cell density. PDL is an important indicator for evaluating cell proliferation ability and cell stability, especially in the preparation and identification of stable cell lines.

[0050] The calculation method of PDL is: PDL=log2 （N 2 / N 0 ） , where N2 is the number of cells after doubling and N0 is the initial number of cells.

[0051] The cell viability of CEK-F40 (stable passage chicken embryo kidney cells of CEK passage 40) and CEK-F0 (primary chicken embryo kidney cells) was detected using the CCK-8 method: CEK-F0 and CEK-F40 were digested, the cells were counted and plated. In a 96-well plate, 5000 cells were plated in each well and incubated in a 39°C cell culture incubator for 18 hours. 100 μL of fresh culture medium (DMEM / F12) was added, and a cell-free culture medium (DMEM / F12) 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 culture incubator. The absorbance at 450 nm was measured at 1, 2, and 4 hours, respectively, to calculate the cell viability.

[0052] Specific test results such as Figure 3 As shown, Figure 3Middle, (A) CEK-F0 and (B) CEK-F40 morphology comparison, magnification 100×; (C) CEK-F0 and CEK-F40 cell population doubling level comparison (PDL); (D) CCK-8 assay for cell viability;

[0053] Depend on Figure 3 It can be seen that compared with CEK-F0, the cell proliferation ability and cell viability of CEK-F40 are significantly higher. Therefore, combined with the cell morphology and stable subculture, it was preliminarily judged that stable subculture chicken embryo kidney cells were successfully obtained.

[0054] Example 3

[0055] This example is an IFA test for the expression of cell surface markers of different generations of CEK: CEK-F15 (CEK chicken embryo kidney cells at the 15th generation), CEK-F45 (CEK chicken embryo kidney cells at the 45th generation), and CEK-F45-Nor (cells at the 45th generation obtained by culturing in ordinary medium after culturing to the 44th generation) with different passage times were inoculated with 2×10 cells per well of an 8-chamber cell culture slide. 4 cells and add 300 μL of culture medium (DMEM / F12). Incubate at 39 ℃, 5 % CO2 for 24 hours. Aspirate the culture medium, add 500 μL of PBS to each well, shake at 150 rpm for 5 minutes at room temperature, and wash 3 times (the following washing steps are the same as this step). Add 300 μL of acetone to each well and fix at -20 ℃ for 10 minutes. Wash 3 times. Add 500 μL of blocking buffer (10% goat serum / 0.1% Tween 20 / PBS) to each well, block for 8 h at 4°C, wash 3 times, dilute the primary antibody (Vimentin, α-SMA, CD31, CK-5, E-Cadherin, etc.) with antibody diluent (0.1% goat serum / 0.1% Tween 20 / PBS), add 200 μL to each well, incubate overnight at 4°C, wash 3 times, add 200 μL of fluorescent secondary antibody to each well, incubate at room temperature in the dark for 1 h, wash 3 times; add a drop of sealing agent containing DAPI to the slide, gently cover the coverslip, remove bubbles and excess sealing agent, and seal the edges of the coverslip with nail polish; use the BioTek Cytation1 cell imaging multifunctional detection system to observe fluorescence; IFA results are as follows Figure 4 As shown, Figure 4 Immunofluorescence detection (IFA) of markers in CEK of different generations: (A) CK-5; (B) CD31; (C) CE-Cadherin; (D) Vimentin; (E) α-SMA; DAPI, cell nucleus; scale bar, 100 μm.

[0056] Depend on Figure 4 It can be seen that CK-5 and E-Cadherin are highly expressed in CEK of different generations, while the expression levels of CD31 and Vimentin are low, and α-SMA is not expressed in CEK; this shows that CEK of different generations have stable and consistent phenotypes, reflecting that the cells maintain good genetic stability during the passaging process; there is no significant difference in phenotype between CEK-F45 and CEK-F45-Nor, which shows that after removing the organoid culture medium, CEK cells can also maintain a stable growth state in ordinary culture medium and maintain the consistency of their phenotype.

[0057] Example 4

[0058] This example is RT-qPCR detection of the expression levels of different CEK-related genes after IBV challenge: Example 1 After stably passaged chicken embryo kidney cells are plated, they are cultured for 24 hours, the culture medium is discarded, the cells are washed 3 times with PBS, and then replaced with a serum-free culture medium (DMEM / F12) (serum will affect the efficiency of virus adsorption to cells). IBV-M41 virus allantoic fluid is taken at a final concentration of 1:10 and added to the culture dish as the virus attack group (IBV group), and a control group (MOCK group) is set up. After gently shaking several times to mix, the cells are placed in an incubator at 39°C and 5% CO2 and incubated for 3 hours. The liquid in the well is aspirated, the cells are washed 3 times again with PBS, and the culture medium (DMEM / F12) containing 2% serum is replaced and cultured continuously, and the cell morphology is observed every 12 hours. After 72 hours, the cells are collected to extract RNA, and further reverse transcribed to obtain cDNA, which is detected by RT-qPCR; the test results are as follows Figure 5 As shown; Statistical analysis was performed using GraphPad Prism 8.0.2 software, and one-way ANOVA analysis of variance was used. If P > 0.05, it means there is no significant difference, which is marked with "ns"; if P < 0.05, it means there is a significant difference, which is marked with "ns". If P < 0.01, If P < 0.001, If P < 0.0001, mark.

[0059] Figure 5 In the figure, (AC) expression of epithelial markers in CEK-F0; (D) expression level of 3'UTR of IBV-S1 gene in CEK-F0; (EG) expression of epithelial markers in CEK-F40; (H) expression level of 3'UTR of IBV-S1 gene in CEK-F40.

[0060] Depend on Figure 5 It can be seen that:

[0061] The results of fluorescence quantification showed that CEK-F40 could be infected by IBV, but the level of viral infection was lower than that of primary CEK-F0. The expression of CEK-F40 epithelial marker was downregulated after infection, which may be the reason for the decrease in IBV infection ability.

[0062] Example 5

[0063] This example is a transmission electron microscope (TEM) observation of the distribution of virus particles in CEK after virus attack:

[0064] The virus was diluted according to different amounts: 1:10 (High-Dose) for the high-dose group and 1:100 (Low-Dose) for the low-dose group. Following the cell infection procedure described above, 72 hours after infection, the cells were digested and centrifuged at 1500 rpm for 10 minutes to collect the cells. Electron microscopy fixative precooled at 4°C was added along the tube wall and allowed to stand at 4°C overnight. The samples were sent to the Scientific Dog Instrument Testing Platform for ultrathin sectioning, and the virus particles were observed using a transmission electron microscope; the observation results are shown in Figure 2. Figure 6 As shown;

[0065] Figure 6 Middle, (A) CEK-F0 control group, IBV-M41 low-dose group, and IBV-M41 high-dose group. (B) CEK-F30 control group, IBV-M41 low-dose group, and IBV-M41 high-dose group. (C) CEK-F40(Nor) control group, IBV-M41 low-dose group, and IBV-M41 high-dose group;

[0066] Depend on Figure 6 It can be seen that all cells in the challenge group contained viral particles, indicating that CEK cells can be infected by IBV and have the ability to be used as a tool for studying IBV.

[0067] Example 6

[0068] This example is TCID 50 Experimental detection of virus titer in CEK supernatant after virus attack:

[0069] According to the above-mentioned CEK infection steps, CEK-F15 and CEK-F45 cells were taken. After 72 hours of infection, the cell culture supernatant (F15-IBV-72 h, F45-IBV-72 h) was collected, and the virus titer in the culture medium was determined using the TCID50 method. The specific steps are as follows: CEK-F0 cells were inoculated into a 96-well plate at a density of 5000 cells per well and cultured for 24 hours. The collected cell culture supernatant and IBV-M41 were diluted at a dilution ratio of 10-1-10-8, and the virus samples with different dilutions were inoculated into the cells respectively, with 8 replicate wells inoculated for each dilution. After incubation for 5-7 days, the cytopathic effect (CPE) was observed and the corresponding TCID50 value was calculated using the Karber method based on the number of positive wells (results are shown in Tables 1 and Figure 7 ).lgTCID 50 =LD(S-0.5). (L: logarithm of the highest dilution, D: difference between logarithms of dilutions, S: sum of positive well ratios).

[0070] Table 1 Virus titer in CEK supernatant after challenge

[0071] From Table 1 and Figure 7 It can be seen that: The virus was detected in the supernatant of CEK cells 72 h after infection, indicating that stably cultured CEK cells have the ability to be infected by IBV.

[0072] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents. These modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention, and they should all be included in the scope of the claims and specification of the present invention.

Claims

1. A method for preparing stable passage chicken embryo kidney cells, characterized in that: The preparation method of the stable passage chicken embryo kidney cells comprises the following steps: (a) Primary chicken embryo kidney cells were extracted from chicken embryos; (b) placing the primary chicken embryo kidney cells in a culture medium for subculture, thereby obtaining the stable subculture chicken embryo kidney cells; 100 mL of the culture medium consists of the following components: FBS 1.8~2.2 mL, NAAS non-essential amino acids 0.8~1.2 mL, penicillin-streptomycin solution 0.8~1.2 mL, GlutaMAX 0.8~1.2 mL, CHIR99021 40~45 μL, Y27632 95~105 μL, valproic acid 12~13 μL, DMH-1 12~14 μL, FOXO1-inhibitor 1.8~2.2 μL, and the rest is DMEM / F12 medium.

2. The method for preparing stable passage chicken embryo kidney cells according to claim 1, characterized in that: The chicken embryos are 16-18 day old SPF chicken embryos.

3. The method for preparing stable passage chicken embryo kidney cells according to claim 1, characterized in that: The temperature of the subculture is 38-39.5° C., and the carbon dioxide concentration is 5%.

4. The method for preparing stable passage chicken embryo kidney cells according to claim 1, characterized in that: During the subculture process, the culture medium is replaced every 2 to 4 days.

5. The method for preparing stable passage chicken embryo kidney cells according to claim 1, characterized in that: The number of subcultures is no less than 40.

6. Stable passage chicken embryonic kidney cells prepared by the method for preparing stable passage chicken embryonic kidney cells according to any one of claims 1 to 5.

7. Use of the stable passage chicken embryo kidney cells prepared by the method for preparing stable passage chicken embryo kidney cells according to 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 avian infectious bronchitis virus, characterized in that: The construction method comprises using chicken infectious bronchitis virus to infect the stable passage chicken embryo kidney cells prepared by the preparation method of any one of claims 1 to 5.

Images