CN121006317A

CN121006317A - An immortalized chicken embryo kidney epithelial-like cell, its preparation method and application

Info

Publication number: CN121006317A
Application number: CN202511508771.6A
Authority: CN
Inventors: 覃尧; 宗永泰; 丁仁博; 符敏; 黄文强; 陈云
Original assignee: Sanya Nanfan Research Institute Of Hainan University
Current assignee: Sanya Nanfan Research Institute Of Hainan University
Priority date: 2025-10-22
Filing date: 2025-10-22
Publication date: 2025-11-25
Anticipated expiration: 2045-10-22
Also published as: CN121006317B

Abstract

The invention discloses an immortalized chick embryo kidney epithelial-like cell and a preparation method and application thereof, wherein the preparation method of the immortalized chick embryo kidney epithelial-like cell comprises the steps of extracting primary chick embryo kidney epithelial cells from chick embryos; the immortalized chicken embryo kidney epithelial-like cells prepared by the preparation method can be stably passaged for a long time, can still maintain stable proliferation capacity after multiple passaging, reduce individual difference, improve reliability and repeatability of experimental results, and in addition, the preparation method does not introduce exogenous genes or change genotype of cells per se, ensures stability on gene level and avoids interference of exogenous genes on phenotypic stability of the cells.

Description

Immortalized chick embryo kidney epithelial-like cell and preparation method and application thereof

Technical Field

The invention relates to the technical field of cell engineering, in particular to an immortalized chick embryo kidney epithelial-like cell, a preparation method and application thereof.

Background

The importance of avian infectious bronchitis virus (Infectious Bronchitis Virus, IBV) in the avian farming industry and research challenges. IBV belongs to gamma coronavirus, is a highly infectious respiratory disease, and mainly affects the respiratory system, digestive system and genitourinary system of chickens. The virus can spread rapidly in chicken flocks, leading to high morbidity. IBV can cause growth retardation of chickens, reduction of egg yield of adult chickens, even death in severe cases, and brings remarkable economic loss to chicken farms. IBV has extremely important influence in the poultry farming industry, effectively prevents and controls the transmission of IBV, and has important significance for guaranteeing the healthy development of the chicken farming industry, improving the economic benefit and maintaining public health. By enhancing biosafety, periodic monitoring and timely treatment, the effects of IBV can be effectively reduced.

IBV is capable of growing on a variety of cells, but a significant cytopathic effect (Cytopathic Effect, CPE) occurs on chicken embryo kidney cells (Chicken Embryonic Kidney, CEK) or chicken kidney cells (CHICKEN KIDNEY, CK). Avian Infectious Bronchitis Virus (IBV) has defined epithelial cell tropism and exhibits highly efficient replication characteristics, particularly in tubular epithelial cells. Currently, studies on IBV at the cellular level are mainly dependent on chick embryo kidney cells or chick embryos. However, there is no chick embryo kidney cell line that is stable for passage, meaning that extraction of primary CEKs is required for each experiment. The primary cells have the following defects that individual differences exist among different chick embryos, the health condition of the chick embryos and whether the chick embryos carry pathogens influence the subsequent research, the chick embryos grow slowly and have limited reproductive capacity, and long-term and large-batch experiments cannot be carried out. To address these issues, there is an urgent need to prepare immortalized chicken embryo epithelial-like cell lines in order to better support the research and related applications of IBV.

In view of this, the present invention has been made.

Disclosure of Invention

The invention aims to provide an immortalized chick embryo kidney epithelial-like cell, a preparation method and application thereof, wherein the chick embryo kidney epithelial-like Cell (CEKE) prepared by the preparation method can be stably passaged for a long time, can still maintain stable proliferation capacity after multiple passaging, reduces individual difference, improves reliability and repeatability of experimental results, and in addition, the preparation method does not introduce exogenous genes or change the genotype of the cells, ensures the stability on the gene level and avoids potential gene mutation risks.

In order to achieve the above object of the present invention, the following technical solutions are specifically adopted:

the first aspect of the invention provides a method for preparing immortalized chick embryo kidney epithelial-like cells, comprising the following steps:

(a) Extracting primary chick embryo kidney epithelial cells from chick embryos;

(b) Placing the primary chick embryo kidney epithelial cells in a culture medium containing chick Noggin protein for subculturing, and obtaining the immortalized chick embryo kidney epithelial-like cells.

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

Preferably, 100ml of said medium comprises the following components:

1.8-2.2 mL of FBS, 0.8-1.2 mL of nonessential amino acid, 0.8-1.2 mL of penicillin-streptomycin-amphotericin B solution, 35-45 mL of Y27632 80-120 mu L, L-WRN conditioned medium and the balance of DMEM/F12 medium;

Penicillin concentration in the penicillin-streptomycin-amphotericin B solution is 9000-11000U/mL, streptomycin concentration is 8-12 mg/mL, and amphotericin B concentration is 20-30 mug/mL;

the final concentration of Y27632 is 8-12 mu M.

Preferably, the chick embryo is an SPF chick embryo of 16-18 days old.

Preferably, the subculture temperature is 39 ℃ and the carbon dioxide concentration is 5%.

Preferably, the culture medium is replaced at intervals of 2-4 days in the subculture process.

Preferably, the number of passages of the subculture is not less than 21.

The invention also provides the immortalized chick embryo kidney epithelial-like cell prepared by the preparation method of the immortalized chick embryo kidney epithelial-like cell.

The third aspect of the invention provides an application of the immortalized chick embryo kidney epithelial-like cells prepared by the preparation method of the immortalized chick embryo kidney epithelial-like cells in constructing an in-vitro infection model of infectious bronchitis viruses.

The invention provides a construction method of an in-vitro infection model of infectious bronchitis viruses, which is characterized by comprising the step of transfecting the immortalized chick embryo kidney epithelial-like cells prepared by the preparation method of the immortalized chick embryo kidney epithelial-like cells by using the infectious bronchitis viruses.

Compared with the prior art, the invention has the beneficial effects that at least:

Compared with the traditional primary cell stable construction method, the constructed immortalized chicken embryo kidney epithelial-like cells have no foreign genes in an insertion or recombination mode, change the genes of the cells, ensure the stability on the gene level, avoid potential gene mutation risks, further, the immortalized chicken embryo kidney epithelial-like cells can still keep stable proliferation capacity after multiple passages, the efficiency and the repeatability of virus isolation culture and pathogenic mechanism research of IBV wild-type strains are obviously improved, the bottleneck of limited passage times of the primary cells in pathogenic mechanism research of avian coronaviruses and the like is solved, meanwhile, the reliability and the repeatability of experimental results are improved, in addition, the immortalized chicken embryo kidney epithelial-like cells can meet the requirement of large-scale experiments, reduce the experimental cost and the workload, reduce the dependence on fresh chicken embryos and meet the requirements of animal welfare.

The immortalized chick embryo kidney epithelial-like cells can be stably passaged for a long time, the biological safety is improved, the long-term culture can be performed under the sterile condition, the risk of introducing pollution in the extraction and culture process is reduced, the safety of laboratory environment is ensured, the immortalized chick embryo kidney epithelial-like cells also have wide application prospects, more stable and reliable cell models can be provided for the fields of virology research, vaccine development, drug screening and the like, and the development of related researches is promoted.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. Like elements or portions are generally identified by like reference numerals throughout the several figures. In the drawings, elements or portions thereof are not necessarily drawn to scale.

FIG. 1 shows the GL178 carrier map and agarose gel electrophoresis result in example 1 of the present invention, wherein A is the GL178 carrier map and B is the agarose gel electrophoresis result;

FIG. 2 shows the blasticidin killing profile of example 1 of the invention;

FIG. 3 shows the morphology of L-WRN-chNOG cells and L-WRN-Ctrl cells in example 1 of the present invention, wherein A is the morphology of L-WRN-Ctrl cells, and B is the morphology of L-WRN-chNOG cells;

FIG. 4 shows the effect of Western Blot and RT-qPCR in the embodiment 1 of the invention on verifying the expression of chicken-origin Noggin, wherein A is the result of Western Blot and B is the result of RT-qPCR;

FIG. 5 shows the morphology and population doubling levels of primary CEKE cells of example 3 of the present invention on different days of culture, wherein A is CEKE-Control versus CEKE-Treated morphology, magnification is 100×, B is CCK-8 assay CEKE-Control versus CEKE-Treated cell viability, and C is CEKE-Control versus CEKE-Treated cell population doubling levels;

FIG. 6 shows the expression of the immortalized CEKE cell surface markers detected by IFA in example 4 of the present invention, wherein A is the CK-5 expression, B is the E-Cadherin expression, C is the Vimentin expression, and D is the alpha-SMA expression;

FIG. 7 shows the RT-qPCR assay of the expression level of intracellular virus 3' UTR mRNA after IBV-M41 challenge in example 5 of the present invention.

Detailed Description

Embodiments of the technical scheme of the present invention will be described in detail below with reference to the embodiments. The following examples are only for more clearly illustrating the technical aspects of the present invention, and thus are merely examples, and are not intended to limit the scope of the present invention.

It is noted that unless otherwise indicated, technical or scientific terms used herein should be given the ordinary meaning as understood by one of ordinary skill in the art to which this application belongs.

At present, a series of problems of 1 and passage stability of the traditional primary CEK cells in virus research and cell culture are that the primary CEK cells easily lose proliferation capacity and functional characteristics in the passage process, and are difficult to maintain for a long time. 2. Individual differences-individual differences between different chick embryos may lead to inconsistent cell characteristics, affecting the reproducibility of experimental results. 3. Health status the health status of primary CEK cells is affected by the chick embryo itself, and there may be a risk of carrying pathogens. 4. The primary CEK cells grow slowly and have limited proliferation capacity, so that the requirements of large-scale experiments are difficult to meet. 5. Sustainability of cell supply primary CEK cells need to be extracted from chick embryos frequently, increasing experimental cost and effort. 6. Biosafety-biosafety risks exist in the process of extracting and culturing primary CEK cells, and pollution can be introduced.

In order to solve the above problems, the embodiment of the invention provides a method for preparing immortalized chick embryo kidney epithelial-like cells, which comprises the following steps:

(a) Extracting primary chick embryo kidney epithelial cells from chick embryos;

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

In one embodiment, 100ml of the medium comprises the following components:

the final concentration of Y27632 is 8-12 mu M.

In one embodiment, the chick embryo is a 16-18 day old SPF chick embryo.

In one embodiment, the subculture is at a temperature of 39 ℃ and the carbon dioxide concentration is 5%.

In one embodiment, the medium is replaced at intervals of 2-4 days during the subculture.

In one embodiment, the number of passages of the subculture is not less than 21.

The invention also provides an application of the immortalized chick embryo kidney epithelial-like cells prepared by the preparation method of the immortalized chick embryo kidney epithelial-like cells in constructing an in-vitro infection model of the infectious bronchitis virus.

The invention also provides a construction method of the in-vitro infection model of the avian infectious bronchitis virus, which comprises the step of adopting the avian infectious bronchitis virus to infect the immortalized avian embryo kidney epithelial-like cells prepared by the preparation method of the immortalized avian embryo kidney epithelial-like cells.

The technical scheme of the invention is further described in detail through specific examples.

Preparation of L-WRN conditioned Medium:

5X 10 ⁶ L-WRN cells were inoculated into a 150 mm cell culture flask, and then cultured in 30 mL DMEM/F12 medium (10% FBS, 1X, the nonessential amino acids, and 1X, penicillin-streptomycin-amphotericin B). 72 After h, the first batch of conditioned medium was collected, centrifuged at 2000 g for 10min at 4℃and the second batch was collected by further adding the 30 mL DMEM/F12 medium culture 48 h, centrifuged at 2000 g for 10min at 4℃and the third batch was collected again according to the same procedure. The conditioned medium collected three times was mixed and homogenized, filtered through a 0.22 μm filter and sub-packaged for freezing at-80 ℃.

Example 1

The embodiment is a preparation method of chicken Noggin protein, which comprises the following steps:

(1) Primers chNoggin-F (5'-ATGGATCATTCCCAGTGCCTT-3' SEQ ID NO: 1) and chNoggin-R (5 '-CTAGCAGGAGCACTTGCACT-3' SEQ ID NO: 2) were designed based on chicken-derived Noggin gene CDS sequence (GenBank ID: NM-204123.2). PCR amplification was performed using high fidelity DNA polymerase (Takara, R010A). The amplified products were verified by agarose gel electrophoresis and gel recovery was performed. Linearized vectors were prepared with EcoRI restriction enzyme (Takara, 1040S) and XbaI restriction enzyme (Takara, 1093S) (vector map shown in FIG. 1A). The gene fragment of interest was ligated to the linearized vector using T4 DNA ligase (Takara, 2011A) at a 3:1 molar ratio. And (3) transforming 10 mu L of the connection product into competent cells, and selecting single colony for PCR and sequencing identification to obtain recombinant plasmid pcSLenti-EF1-BSR-CMV-chNOG-3 xFlag-WPRE. Screening positive clones, extracting plasmids, and performing enzyme digestion identification (B in FIG. 1), wherein a target gene band is observed at 672 bp, in FIG. 1, A is a vector map, B is agarose gel electrophoresis M is DNA MARKER, 1 is an uncleaved plasmid, and 2 is EcoRI/XbaI double enzyme digestion products;

(2) L-WRN cells were seeded at a density of 5X 10 ⁴ cells/well in 24-well plates. After cell attachment, fresh medium was changed and medium containing 1. Mu.g/mL, 2. Mu.g/mL, 3. Mu.g/mL, 4. Mu.g/mL, 5. Mu.g/mL blasticidin (Solarbio, B9300) was added. Cell viability was observed (results are shown in FIG. 2) by changing the medium containing BSR at corresponding concentrations every 48h days, taking as screening concentration the minimum BSR concentration that resulted in 95% -99% cell death within 5-7 days;

(3) 5X 10 ⁴ cells/well were seeded in 24 well plates and cultured 24: 24 h. When the cell density reached 30% -40%, the recombinant lentivirus, empty vector lentivirus was infected at moi=40. Viral volume (μl/well) =moi×cell number/titer (TU/mL) ×1000. Virus solution containing 5. Mu.g/mL Polybrene (Biyun Tian, C0351) was added. After 12 h is infected, the virus liquid is discarded, the culture medium containing 2% FBS is replaced for continuous culture, 96 mug/mL blasticidin is added after the infection, and two cells of an over-expression L-WRN-chNOG cell strain and an empty vector control L-WRN-Ctrl cell strain are obtained through screening (the two cell forms are shown in figure 3, A in figure 3 is the L-WRN-Ctrl cell form; B is the L-WRN-chNOG cell form);

(4) Culturing the L-WRN-chNOG and L-WRN-Ctrl cells, centrifuging, and collecting cells and supernatant;

Sample treatment of cell supernatant (Supernatant, SUP) comprises centrifuging supernatant to remove cell fragments, weighing 10gTCA, adding ultrapure water to a volume of 10ml to prepare 100% TCA solution, adding 100% TCA solution into supernatant according to a volume ratio of 4:1, standing for 10min at 4 ℃, centrifuging at 4 ℃ and 14000rpm for 10min, discarding supernatant, re-suspending protein precipitate with pre-cooled acetone, centrifuging at 4 ℃ and 14000rpm for 10min, washing with pre-cooled acetone once, centrifuging at 4 ℃ and 14000rpm for 10min, volatilizing acetone at room temperature for 20min, finally adding RIPA lysate to re-suspend the obtained protein precipitate, adding 5x protein LoadingBuffer according to a volume ratio of 4:1, and denaturing in a metal bath at 100 ℃ for 10min;

Whole cell lysate (white CELL LYSATE, WCL) samples were treated by washing the collected cells three times with PBS (seville, G4202), and 1 mL RIPA lysate (bi-cloudy days, P0013C) was prepared and 10 μl PMSF (bi-cloudy days, ST 505) was added for on-ice lysis of 30 min. Centrifugation at 20min at 12000 r/min at 4℃and denaturation at 100℃of 10 min was performed by adding 5 XSDS loading buffer (Biyun Tian, P0015).

The two samples were separated by constant pressure electrophoresis at 120V with 10% pre-gel (doctor, AR 1203), and constant flow at 300 mA was transferred to PVDF membrane. After the transfer, the membrane was blocked with 5% nonfat milk powder at room temperature at 60rpm for 1h, the Anti-Flag primary antibody (Hua' an organism, M1403-2, 1:1000) was incubated overnight at 4℃and recovered, washed 6 times with TBST at 90 rpm for 5min, followed by incubation of the Anti-Rabbit secondary antibody (Saiwel, GB23303, 1:10000) at room temperature of 90 rpm for 1 h. Preparing ECL luminous solution, dripping the ECL luminous solution onto a PVDF film, and exposing a target strip by using a full-automatic digital gel imaging analysis system.

Total RNA from the L-WRN-chNOG cells and L-WRN-Ctrl control cells was extracted using a total RNA extraction kit (Meitz. RTM., R4132) followed by cDNA synthesis by reverse transcription kit (Tolobio, 22106). Real-time fluorescent quantitative PCR reactions were performed using TB Green Premix Ex Taq (Tolobio, 22201). The murine GAPDH gene was selected as an internal reference gene for quantitative analysis, the primer sequences are shown in Table 1, and the relative expression levels of the genes were finally calculated by the 2 ^-ΔΔCt method.

TABLE 1

Western Blot results show that the overall protein size accords with expectations (A in FIG. 4) and RT-qPCR (B in FIG. 4) and that Noggin protein is stably expressed and effectively secreted outside cells in L-WRN cells, wherein in FIG. 4, A is 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, and B is RT-qPCR results, with the formula P < 0.01;

(5) Chicken-source Noggin protein extraction and purification

Purifying chicken-origin Noggin protein by using FLAG tag (DYKDDDDK) fusion protein purification kit (Elabscience, cat# EA-TP-K001), namely firstly preparing reagent, diluting 10 XPBS with deionized water according to the proportion of 9:1 to prepare 1 XPBS (on-the-spot preparation), centrifuging 10 mg of 3X FLAG PEPTIDE, adding 0.2 mL of 10 XPBS to dissolve, adding 1.8 mL of deionized water to prepare 5 mg/mL of stock solution (-20 ℃ for preservation), diluting 1 XPBS to 0.2-0.5 mg/mL of competitive eluent when in use, and simultaneously mixing glycerol and 1 XPBS according to the proportion of 1:1 and adding preservative to prepare gel preservation solution (on-the-spot preparation); then processing the sample, collecting L-WRN-chNOG cell culture supernatant, centrifuging at 4 ℃ and 12000rpm for 10min to remove fragments, then loading and incubating, gently suspending Anti-FLAG affinity gel, taking 2mL of gel suspension (containing 1mL of affinity gel), adding into a purification column, balancing by adding 10mL of 1 XPBS after the liquid is completely discharged, adding the pretreated supernatant, sealing the purification column, incubating overnight at 4 ℃ by shaking bed, collecting the flow-through supernatant (temporary storage at 4 ℃) after incubation, washing the affinity gel 3 times by 5mL of 1 XPBS, then performing competitive elution, washing by adding 5mL of precooled acidic pre-washing liquid, adding 2mL of precooled competitive eluent after the liquid is completely discharged, incubating for 2h (prolonged) by shaking bed at 4 ℃ after sealing, collecting the eluent, repeating the elution 1 time and merging when necessary, immediately regenerating the affinity gel after the elution is completed, washing by 10mL of 1 XPBS, adding 3mL of acidic eluent, washing 3mL of 1 XPBS until the flow-through gel is neutral, finally, adding 1 XPBS containing 50% glycerol and 0.2% sodium azide, uniformly mixing, preserving at-20 ℃, finally, processing purified protein, taking a small amount of eluent to carry out SDS-PAGE to identify purity, sterilizing by a 0.22 mu m filter membrane, preserving at 4 ℃ for a short period or at-80 ℃ for a long period, and adjusting concentration as required before use.

Example 2

The embodiment is a method for extracting primary chick embryo kidney epithelial cells, comprising the following steps:

SPF chick embryo of 18 days old is sterilized with 75% ethanol. And opening the outer shell and the shell membrane in the super clean bench, and taking out the chick embryo aseptically. The abdominal cavity is incised, and the viscera and intestinal tract are stripped to expose the kidneys. The kidneys were removed and washed twice with PBS to remove fat and blood clots. The tissue was minced, 0.25% trypsin (bosch, PYG 0107) was added and digested in a 37 ℃ water bath at 10 min. The digestion was stopped by adding complete medium until the tissue was loose and clear, and mixed well, 100 μm cell sieve filtered, centrifuged at 1200 r/min for 6min, and the cell pellet was resuspended in DMEM/F12 (Punuoxepin, PM 153312) medium containing 2% FBS (Punuoxepin, 164250) and incubated at 39℃under 5% CO ₂ at saturated humidity. The differential adherence method is used for extracting chick embryo kidney epithelial cells, namely, after 50 min of chick embryo kidney epithelial cells are cultured, the fibroblast cells are preferentially adhered, most of the epithelial cells are still in a suspension state, the supernatant (containing non-adhered epithelial cells) in the culture flask is carefully collected, and the chick embryo kidney epithelial cells are transferred into a new sterile culture flask. And supplementing a proper amount of DMEM/F12 culture medium containing 2% FBS into the new culture flask, and continuously culturing in a constant temperature incubator with 39 ℃ and 5% CO ₂ to obtain primary chick embryo kidney epithelial cells.

Example 3

This example shows the morphology of CEKE cells on different days of culture compared to population doubling levels;

CEKE were cultured using different media, the treatment group (CEKE-Treated) was a medium of the formulation of Table 2, the control group was a medium for removing the nogign protein of chicken and the L-WRN cells based on the formulation of the medium of Table 2, subculturing was performed separately at 39℃and carbon dioxide concentration of 5%, medium was replaced once every 3 days, and CEKE cells cultured in the ordinary medium exhibited aging characteristics at day 12, such as deterioration in cell morphology, increase in mortality, and significant decrease in proliferation rate (FIG. 5A). By day 21, the cells were completely collapsed and dead. In contrast, CEKE-Treated cells maintained stable proliferative capacity, and were stably passaged after 21 days of culture without signs of aging (a in fig. 5). The CCK-8 cell viability assay and cell Population Doubling Level (PDL) analysis further prove that CEKE-Treated cells have significantly improved cell viability and proliferation capacity and stable passage capacity (B, C in FIG. 5)

TABLE 2

In FIG. 5, (A) CEKE-Control is compared with CEKE-Treated morphology at 100X magnification, and (B) CCK-8 determines the cell population doubling level comparison (PDL) of CEKE-Control and CEKE-Treated cell viability (C) CEKE-Control and CEKE-Treated.

Cell population doubling (Population Doubling Level, PDL) refers to the number of times the number of cells doubles from initial inoculation to a certain cell density during the culture process. PDL is an important index for assessing cell proliferation capacity and passaging stability, especially in the preparation and identification of immortalized cell lines. PDL is calculated as pdl= [ log (N _t / N₀) / log(2)] + PDL₀（N₀: initial number of inoculated cells; N _t: number of cells at harvest; PDL ₀: initial PDL value).

And cell viability was measured CEKE using CCK-8 by digestion of CEKE, cell counting and plating. 5000 cells were plated in each well in 96-well plates and incubated in a 39 ℃ cell incubator for 12-24 hours. 100. Mu.L of fresh medium was added and the control group was added with cell-free medium as a blank. mu.L of CCK-8 solution was added to each well and incubated in a 39℃cell incubator. The absorbance at 450 nm was measured at 1,2, and 4 hours, respectively, to calculate cell viability. Compared with the control group, CEKE-Treated cells have obviously higher proliferation capacity and cell activity, and in conclusion, the immortalized chick embryo kidney epithelial-like cells are successfully obtained by combining cell morphology and preliminary judgment of stable subculture.

Example 4

This example shows the expression of the cell surface markers of immortalized CEKE for IFA detection:

The different treatments CEKE were taken, 2X 10 ⁴ cells were seeded per well using 8-well cell culture slides, and 300. Mu.L of medium was added. The culture was carried out at 39℃under 5% CO ₂ for 24 hours. The culture broth was aspirated, 500. Mu.L of PBS was added to each well, and the mixture was shaken for 5 minutes by shaking table 150 and rpm at room temperature, and washed 3 times (the washing step was the same as the following). 300. Mu.L of acetone was added to each well and the wells were fixed at-20℃for 10 minutes, washed 3 times, 500. Mu.L of blocking buffer (10% goat serum/0.1% Tween 20/PBS) was added to each well, 4℃blocked for 8H, washed 3 times, anti-CK-5 (Sivel, GB 111246), E-Cadherin (Proteintech, 60335-1-Ig), vimentin (Hua's organism, ET 1610-39), α -SMA (Hua's organism, ET 1607-53) was diluted with antibody dilution (FITC-labeled goat anti-rabbit IgG (H+L), bicloud days, A0562, FITC-labeled anti-mouse IgG (H+L), bidays, A0568) and cloud-incubated at room temperature for 1H in a dark environment, and washed 3 times. Washing 3 times. A drop of a caplet containing DAPI (seville, G1012) was added to the slide, the coverslip was gently covered, the air bubbles and excess caplet removed, and the edges of the slide were sealed with nail polish. Fluorescence was observed using BioTek Cytation cell imaging multifunctional detection system. The IFA results are shown in fig. 6;

In FIG. 6, (A) CK-5, (B) E-Cadherin, (C) Vimentin, (D) alpha-SMA, DAPI, nuclei. Scale bar, 50 μm;

as can be seen from FIG. 6, both the epithelial-like markers CK-5 and E-Cadherin were expressed in CEKE, while both the mesenchymal cells MARKER VIMENTIN and the endothelial cells Marker alpha-SMA were not expressed, indicating that immortalized CEKE had a stable and consistent phenotype with primary CEKE, reflecting that the cells maintained good genetic stability during passage.

Example 5

This example shows the RT-qPCR detection of the expression level of the intracellular virus 3' UTR mRNA after IBV-M41 challenge.

After CEKE-Control and CEKE-Treated cells were plated, 24h were cultured in a 39℃5% CO ₂ incubator. The supernatant was discarded, and the cells were washed 3 times with pre-chilled PBS and replaced with serum-free medium (to eliminate the potential effect of serum on virus adsorption efficiency). IBV-M41 virus allantoic fluid was respectively taken and diluted 1:10 in serum-free medium, and the culture plate was added as virus-infected group (IBV group) and control group (MOCK group) was added with equal volume of allantoic fluid containing no virus. After gently shaking the plates several times, they were incubated in a 39 ℃ 5% CO ₂ incubator for 2 h. After adsorption, the virus-containing liquid was discarded, and the cells were washed 3 times with pre-chilled PBS to remove unadsorbed virus. The culture was continued by changing to a maintenance medium containing 2% Fetal Bovine Serum (FBS). Cell morphology changes were observed and recorded every 12 hours after infection. 72 h after infection, total RNA was extracted from the collected cells and cDNA was synthesized using a reverse transcription kit. And using cDNA as a template, adopting RT-qPCR to detect the expression level of IBV 3' UTR, using GAPDH of chicken as an internal reference gene, and the primer sequence is shown in Table 3.

TABLE 3 real-time fluorescent quantitative PCR primer information

The expression level of the IBV 3' UTR can reflect the replication activity of the virus and thus can be used as a reliable indicator for evaluating the infection status of IBV. The results of the fluorescent quantitative measurements (FIG. 7) show that both CEKE-Control and CEKE-Treated cells detected IBV 3' UTR expression after challenge. Although the expression level of viral 3' UTR mRNA was lower in CEKE-Treated groups than in CEKE-Control groups, CEKE-Treated groups exhibited significant differences (P < 0.0001) compared to the non-challenged Control group (MOCK group). The result shows that the immortalized CEKE cells constructed by the research can be used as an effective model of an IBV virus attack experiment, and the virus replication level can be sensitively and accurately detected by an RT-qPCR method.

It should be noted that the above embodiments are only used to illustrate the technical solution of the present invention, but not to limit the technical solution of the present invention, and although the detailed description of the present invention is given with reference to the above embodiments, it should be understood by those skilled in the art that the technical solution described in the above embodiments may be modified or some or all technical features may be equivalently replaced, and these modifications or substitutions do not make the essence of the corresponding technical solution deviate from the scope of the technical solution of the embodiments of the present invention, and all the modifications or substitutions are included in the scope of the claims and the 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, wherein the chicken embryo is an SPF chicken embryo aged 16-18 days.

3. The method for preparing immortalized chicken embryo kidney epithelial-like cells according to claim 1, characterized in that the passage culture temperature is 39°C and the carbon dioxide concentration is 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 passage culture process.

5. The method for preparing immortalized chicken embryo kidney epithelial-like cells according to claim 1, characterized in that the number of passages in the subculture is not 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 chicken infectious bronchitis virus, characterized in that the method comprises preparing immortalized chicken embryo kidney epithelial-like cells by infecting them with chicken infectious bronchitis virus using any one of the methods for preparing immortalized chicken embryo kidney epithelial-like cells according to claims 1 to 5.