Method for obtaining and purifying chicken oviduct epithelial cells and application thereof

Abstract

The application belongs to the field of biotechnology and cell engineering, and particularly relates to a method for obtaining and purifying chicken oviduct epithelial cells and application thereof. The method comprises the following steps: selecting chicken oviduct funnel mucosa tissue, obtaining a mixed cell suspension after enzyme digestion, and adopting a combination purification strategy of culture for 2 hours and short-time differential digestion for 1 minute to efficiently remove fibroblasts and obtain high-purity chicken oviduct epithelial cells. The cells are transfected with an expression vector carrying hTERT and / or c-Myc genes, and a stable passable immortalized chicken oviduct epithelial cell line is established through screening. The cell line can maintain an epithelial-like morphology and protein secretion function under the action of estrogen, can be used as an in vitro model for screening and expressing exogenous recombinant proteins, and constitutes an in vitro screening and verification platform of a chicken egg bioreactor. The application solves the problems of low purity, easy aging and functional decline of primary cells, significantly shortens the development cycle of recombinant protein drugs, and reduces the research and development cost.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and cell engineering, specifically relating to a method for obtaining and purifying high-purity chicken oviduct epithelial cells, an immortalized chicken oviduct epithelial cell line constructed by the method, and the application of the cell line as an in vitro model for screening and expressing exogenous recombinant proteins, and for constructing an in vitro screening and verification platform for egg bioreactors. Background Technology

[0002] With the rapid development of the biopharmaceutical industry, there is an increasingly urgent need for the large-scale, low-cost production of recombinant protein drugs, vaccines, antibodies, etc. Although traditional mammalian cell culture systems (such as CHO cells) are widely used, they face challenges such as high production costs, complex culture conditions, and limited production capacity.

[0003] The "egg bioreactor" technology, which uses genetically modified poultry, especially laying hens, as a platform, has attracted much attention due to its unique advantages. This technology utilizes the natural ability of laying hens to efficiently synthesize and secrete proteins in the oviduct, enriching the target recombinant protein in the egg white or yolk. It has significant advantages such as low production cost, high yield of target protein, simple downstream separation and purification, good biosafety, and a product glycosylation modification pattern that is highly similar to that of humans. It is regarded as a strategic platform for next-generation biopharmaceutical production.

[0004] However, the industrial application of this technology faces a key bottleneck: the breeding cycle from constructing transgenic chicken lines to obtaining homozygous laying hens that stably express the target recombinant protein and efficiently is extremely long (usually exceeding 18 months). During this long period, researchers cannot conduct timely and effective assessments of the target protein's expression level, modification correctness, and biological function, resulting in high uncertainty and significant time and economic costs in candidate protein screening, expression system optimization, and product pipeline advancement. Therefore, there is an urgent need to develop a cell model that can efficiently and reliably simulate the function of an egg bioreactor in vitro for high-throughput screening and preliminary validation before in vivo animal experiments.

[0005] Chicken oviduct epithelial cells (cOECs) are ideal cells for constructing the aforementioned in vitro models. However, there are two major technical challenges in the culture of primary cOECs in the current technology: (1) Low cell purity and serious interference from impurity cells: Primary cOECs are often mixed with a large number of impurities such as fibroblasts, blood cells, and immune cells. In particular, fibroblasts proliferate rapidly and are very likely to gain a growth advantage during the culture process, leading to rapid degeneration or even collapse of the epithelial cell culture. Although some studies have attempted to sample from different parts of the oviduct, none of them have effectively solved the problem of efficiently obtaining high-purity cOECs. (2) Limited in vitro proliferation capacity and easy functional decline: Primary cOECs have a very limited number of passages in vitro (usually only 3-5 passages), and the cell morphology is prone to change in the later stages of passage (from epithelial-like cobblestone to fibrous, undergoing epithelial-mesenchymal transition), accompanied by a significant decrease or loss of specific secretory functions (such as secretion of ovalbumin OVA), resulting in poor experimental reproducibility and making it unsuitable as a long-term, stable in vitro research tool.

[0006] Therefore, establishing a method that can efficiently obtain high-purity cOECs and immortalize them while maintaining their key biological functions is of great practical significance and application value for overcoming the technological bottleneck of egg bioreactors and accelerating the development of related drugs. Summary of the Invention

[0007] The first objective of this invention is to overcome the above-mentioned deficiencies of the prior art and provide a method for efficiently obtaining high-purity chicken oviduct epithelial cells (cOECs).

[0008] The second objective of this invention is to provide an immortalized chicken oviduct epithelial cell line that can be stably passaged for a long period of time and maintain its secretory function, which is further constructed from cOECs obtained by the above method.

[0009] A third objective of this invention is to provide the application of the immortalized chicken oviduct epithelial cell line as an in vitro model for screening and expressing exogenous recombinant proteins, as well as an in vitro screening and validation platform for an egg bioreactor containing the cell line.

[0010] To achieve the above objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides a method for obtaining and purifying chicken oviduct epithelial cells.

[0011] The overall concept of the method of this invention is as follows: First, by selecting the tissue source site, the proportion of target cells is increased from the source and the introduction of impurities is reduced; second, by combining the differential adhesion method and the differential digestion method, a "two-step combined purification strategy" is adopted to efficiently remove the main impurity cells in the culture - fibroblasts, thereby obtaining high-purity cOECs.

[0012] Specifically, the method of the present invention includes the following steps: (1) Tissue acquisition: Select healthy laying hens, euthanize them, and aseptically separate the oviduct infundibulum tissue.

[0013] This invention, through extensive experimental comparison, has found that selecting the infundibulum of the chicken oviduct as the tissue source has significant advantages compared to the enlarged portion, which is also used in traditional studies. While the enlarged portion is the main site of egg white protein secretion, its thick mucosal layer easily introduces a large number of impurities such as fibroblasts after digestion, and the isolated epithelial cells have relatively weak proliferative capacity. In contrast, the infundibulum, as the starting segment of the oviduct, has mucosal epithelial cells with stronger proliferative potential, and the tissue itself introduces relatively less fibroblast contamination. Therefore, prioritizing the infundibulum as the tissue source is a crucial first step in improving the efficiency and purity of cOECs from the source.

[0014] Preferably, the laying hens in step (1) are 25-35 weeks old. Hens in this age range have mature and functional oviducts, making them more suitable for obtaining high-quality cOECs.

[0015] (2) Pre-treatment of tissue: Remove the serosal layer and mesentery of the infundibulum tissue, and separate and obtain the mucosal fold tissue.

[0016] This step aims to further precisely obtain target tissue rich in epithelial cells. The fallopian tube wall consists of the mucosa, muscle layer, and serosa from the inside out. The mucosal folds are where the epithelial cells are located. By carefully peeling away the outer serosa and mesentery, and precisely cutting the mucosal folds, contamination from fibroblasts and other cells in non-target tissues such as the muscle layer and serosa can be minimized.

[0017] (3) Digestion treatment: The mucosal layer folds are cut into pieces and then digested with enzymes to obtain a mixed cell suspension.

[0018] The mucosal tissue obtained in step (2) is cut into small fragments to increase the contact area with the digestive enzymes. Preferably, the enzyme digestion is pancreatic digestion, and the digestion time is 10-60 minutes, more preferably 30 minutes. Intermittent vortexing can be used during digestion to promote tissue dissociation. After digestion, the enzyme reaction is terminated and the mixture is filtered. The cells are collected by centrifugation to obtain a mixed cell suspension.

[0019] (4) Differential adhesion purification: The mixed cell suspension was seeded in a culture plate and cultured for 2 hours. Then, the non-adherent cell suspension was collected and transferred to a new culture plate.

[0020] This step utilizes the significant difference in adhesion speed between fibroblasts and cOECs. This invention is the first to discover and verify that chicken fibroblasts (such as the DF-1 cell line) have extremely strong adhesion ability, with over 90% adhering firmly within 2 hours; while the adhesion process of cOECs is much slower, with only a very small number adhering within 2 hours. Therefore, by transferring the culture supernatant containing a large number of unadhered cOECs to a new culture plate during the critical 2-hour post-inoculation culture window, while leaving the adhered fibroblasts in the original plate, efficient and simple preliminary purification can be achieved, removing the vast majority of fibroblasts.

[0021] (5) Differential digestion and purification: After the cells adhere to the wall, use trypsin to digest for 1 minute, remove the digested fibroblasts, and retain the adhered chicken oviduct epithelial cells.

[0022] This step serves as the second purification step, further removing any fibroblasts that may remain from step (4) or proliferate subsequently. This invention has found that fibroblasts are significantly more sensitive to trypsin than cOECs. Upon addition of trypsin, fibroblasts rapidly (within approximately 1 minute) undergo morphological changes, shrinking and rounding, and detach from the culture plate wall, while cOECs remain adherent. Therefore, in passage or purification operations, the trypsin digestion time is precisely controlled to 1 minute, and the digestion solution containing detached fibroblasts is immediately removed. Trypsin is then added again for digestion, and cOECs are collected. This method can be repeated with each passage to continuously purify the cell population.

[0023] By employing the combined purification strategy of "2-hour differential adhesion + 1-minute differential digestion" in steps (4) and (5) above, the method of this invention can significantly improve the purity of cOECs in the culture. This provides high-quality cell material for subsequent immortalization line establishment and functional studies.

[0024] In a second aspect, the present invention provides an immortalized chicken oviduct epithelial cell line, which is constructed by co-transfecting an expression vector carrying the hTERT gene and / or the c-Myc gene into chicken oviduct epithelial cells obtained and purified by the method described in the first aspect above.

[0025] The hTERT (human telomerase reverse transcriptase) gene encodes the catalytic subunit of telomerase, which can extend telomere length and thus delay cellular replicative senescence. c-Myc (mouse proto-oncogene) is a key transcription factor that can activate the expression of various cell cycle proteins, accelerate cell cycle progression, and promote cell proliferation. Introducing one or both of these genes into high-purity cOECs can work synergistically to overcome the bottleneck of limited in vitro proliferation capacity of primary cells, endowing them with unlimited proliferative capacity, i.e., achieving immortalization.

[0026] Preferably, the immortalized chicken oviduct epithelial cell line is cultured in a medium supplemented with estrogen to maintain or enhance the epithelial-like paving stone morphology and OVA gene expression ability of the cell line.

[0027] Estrogen is a key hormone for maintaining the differentiation state and specific secretory function of fallopian tube epithelial cells. In in vitro culture systems, cOECs deprived of the in vivo hormonal environment rapidly undergo functional decline. This invention discovers that adding exogenous estrogen to the culture medium can significantly delay or even reverse this process, enabling immortalized cell lines to maintain typical epithelial-like cobblestone morphology and high levels of OVA (ovalbumin) gene and protein expression during long-term culture.

[0028] More preferably, the concentration of the estrogen in the culture medium is 10-500 μM.

[0029] More preferably, the cell line is cultured in a culture medium containing estrogen for at least 72 hours.

[0030] Experiments have shown that estrogen has a concentration- and time-cumulative effect on the regulation of cOECs function, and the best function maintenance effect can be obtained under the above conditions.

[0031] Thirdly, the present invention provides the application of the above-mentioned immortalized chicken oviduct epithelial cell line as an in vitro model for screening and / or expressing exogenous recombinant proteins.

[0032] The immortalized cOECs line established by the method of this invention retains the core functions of fallopian tube epithelial cells in vivo, namely the ability to synthesize and secrete proteins, and can be infinitely expanded and manipulated in vitro. Therefore, it can serve as an ideal in vitro model for applications in the following areas: High-throughput screening of exogenous recombinant protein expression vectors: Exogenous gene expression vectors carrying different promoters, enhancers or signal peptide sequences are introduced into the cell line, and the optimal combination of expression regulatory elements is screened by rapidly detecting the expression level of reporter genes or target proteins.

[0033] Expression and preliminary functional validation of exogenous recombinant proteins: Before investing significant resources in live chicken transgenic experiments, the target recombinant protein was pre-expressed using this cell model, and its yield, molecular weight, glycosylation modification, and biological activity were preliminarily evaluated.

[0034] As a preferred application method, the application includes: using gene editing technology (such as CRISPR / Cas9 technology) to knock in the gene encoding the target exogenous recombinant protein into the genome of the immortalized chicken oviduct epithelial cell line, and culturing the cell line to express and / or secrete the target exogenous recombinant protein.

[0035] By knocking exogenous genes into endogenous high-expression gene sites such as OVA, the powerful transcriptional regulatory environment of these sites can be utilized to drive the efficient and stable expression of exogenous proteins. In one specific embodiment, the present invention successfully knocked the human lysosomal lipase (hLAL) gene into the OVA gene site of immortalized cOECs and successfully detected the expression of the OVA-hLAL fusion protein, verifying the feasibility of this application method.

[0036] Fourthly, the present invention provides an in vitro screening and verification platform for egg bioreactors, which includes the immortalized chicken oviduct epithelial cell line described in the second aspect above.

[0037] This platform is a comprehensive in vitro system integrating high-purity cell acquisition technology, cell immortalization technology, and function maintenance technology. It can simulate key steps of protein expression in egg bioreactors at the cellular level, providing reliable preliminary validation data for the efficient production of target proteins in transgenic eggs.

[0038] Fifthly, the present invention provides the application of the above-mentioned in vitro screening and validation platform for predicting the expression performance of exogenous recombinant proteins in an egg bioreactor.

[0039] The core value of this application lies in prediction and bridging. Specifically, the application includes: using gene editing technology to knock in the gene encoding a target exogenous recombinant protein into the genome of the immortalized chicken oviduct epithelial cell line, culturing the cell line and analyzing the expression level of the target exogenous recombinant protein, thereby predicting the expression performance of the exogenous recombinant protein in an egg bioreactor.

[0040] This platform allows researchers to evaluate one or more candidate protein expression constructs within weeks and predict their expression effects in the final transgenic chicken based on in vitro data. This significantly shortens the early-stage development cycle of egg bioreactors from the traditional 18 months or more to just a few months or even weeks, greatly reducing the trial-and-error costs and time risks in research and development. It represents a milestone in promoting the industrialization of egg bioreactor technology.

[0041] Beneficial effects of the present invention Compared with the prior art, the present invention has the following significant advantages: High cell purity provides reliable material for subsequent research: A combined purification strategy of "optimal sampling from the funnel + 2-hour differential adhesion + 1-minute differential digestion" achieved, for the first time, highly efficient and stable purification of primary cOECs, significantly improving cell purity. Single-cell transcriptome sequencing data (50.84% ​​of cells expressing the OVA gene) provides strong molecular-level evidence for this purity, solving a long-standing technical challenge in this field.

[0042] A functional cell line capable of long-term stable passage was established: an immortalized chicken oviduct epithelial cell line was successfully constructed using the hTERT and c-Myc genes. This cell line not only broke through the limitations of primary cell proliferation capacity, but more importantly, by adding estrogen during culture, it was able to maintain its epithelial-like cobblestone morphology and secrete specific proteins such as ovalbumin (OVA), making it a truly valuable in vitro model.

[0043] This invention provides a highly efficient tool for screening and validating recombinant proteins in vitro: the cell line of this invention can serve as the recipient cell for gene editing and successfully express exogenous recombinant proteins (such as hLAL). This demonstrates its feasibility and effectiveness as an in vitro alternative model for egg bioreactors. Using this platform, exogenous gene constructs can be rapidly screened and functionally validated at the cellular level, thereby predicting their expression performance in live egg bioreactors.

[0044] Significantly shortening the R&D cycle and reducing R&D costs: The platform of this invention drastically shortens the early evaluation cycle of exogenous recombinant proteins in the chicken oviduct system from over 18 months using traditional transgenic methods to just a few weeks. This allows researchers to eliminate inefficient constructs and optimize expression strategies before investing in costly transgenic animal experiments. This not only greatly improves R&D efficiency but also significantly reduces economic costs and research risks, providing crucial technical support for the development of novel biopharmaceuticals based on egg bioreactors, and possesses extremely high industrial application value and broad commercial prospects. Attached Figure Description

[0045] Figure 1 The anatomical structure of the chicken oviduct is shown. (A) Schematic diagram of the chicken oviduct anatomy. (B) Schematic diagram of the endothelial mucosa layer of the chicken oviduct.

[0046] Figure 2 The image shows the morphology of cOECs cultured to the second day.

[0047] Figure 3The results of RT-PCR detection of cOECs marker genes are shown. M. Marker; 1. OVA; 2. OVM; 3. AVD; 4. LYZ; 5. NC; 6. β-actin.

[0048] Figure 4 The expression of the cOECs marker protein OVA was shown.

[0049] Figure 5 The cell morphology before and after screening is shown. (A) before c-Myc-puro screening; (B) before hTERT-puro screening; (C) after c-Myc-puro screening; (D) after hTERT-puro screening.

[0050] Figure 6 The cell morphology of immortalized cell lines is shown. (A) cMyc-cOECs cell line; (B) hTERT-cOECs cell line.

[0051] Figure 7 The expression of c-Myc and hTERT in cOECs immortalized cells is shown. Left panel: M. Marker; 1, 2, 3. c-Myc; 4. c-Myc positive plasmid; 5. NC; Right panel: M. Marker; 1, 2, 3. hTERT; 4. hTERT positive plasmid; 5. NC.

[0052] Figure 8 The growth morphology of immortalized and wild-type cells is shown. (A) hTERT-mediated immortalized cell line; (B) wild-type cells.

[0053] Figure 9 This demonstrates the cell's growth vitality.

[0054] Figure 10 The effects of estrogen on OVA expression in cOECs were shown. (A) Effect of different concentrations of estrogen on OVA expression. (B) Effect of estrogen treatment time on OVA expression.

[0055] Figure 11 Different morphologies of cOECs are shown. (A) Untreated cOECs; (B) Estrogen-treated cOECs.

[0056] Figure 12 The image shows a fluorescence pattern indicating that the LAL plasmid has been transfected into the cell.

[0057] Figure 13 The expression of the OVA-His-LAL fusion protein was shown.

[0058] Figure 14 The image shows fluorescence after LTF plasmid transfection into cells.

[0059] Figure 15 The expression of the OVA-His-LTF fusion protein was shown. Detailed Implementation

[0060] In selecting the site for obtaining cOECs in this invention, a comprehensive comparison was made between the functionally distinct ampulla and the proliferative infundibulum. The infundibulum, located adjacent to the ovary, is the site of sperm-egg fusion, while the ampulla, the longest segment of the fallopian tube, is glandular tissue containing numerous tubular glands and is the main site for secreting proteins from egg white. Previous studies have included sampling from both the infundibulum and the ampulla. This invention samples from both the infundibulum and the ampulla separately for digestion and culture. The results show that the mucosal layer of the ampulla is thicker, requiring a longer digestion time, and the cultured material contains a large number of fibroblasts, which dominate and severely affect cOEC growth, hindering subsequent operations. These results are consistent with previous findings, further supporting the rationale and necessity of using the fallopian tube infundibulum as an ideal tissue source for establishing immortalized cOEC cell lines.

[0061] After tissue is obtained from the infundibulum and digested, only about half of the cells are fallopian tube epithelial cells; the other cells include fibroblasts, blood cells, and immune cells. Among these, fibroblasts are generally considered contaminants in epithelial cell cultures. Rapidly growing fibroblasts often lead to epithelial culture collapse after a relatively long period, making it difficult to maintain the epithelial characteristics of primary cell cultures in their presence.

[0062] Therefore, this invention removes a portion of fibroblasts using differential adhesion 2 hours after digesting the cultured tissue cells. During the culture process, differential digestion is employed with varying digestion times. When selecting the differential adhesion time, we analyzed fibroblast adhesion separately and found that over 90% of DF-1 cells adhered within 2 hours, while only a small portion of cOECs adhered within the same timeframe. To maximize DF-1 removal and preserve cOECs, we ultimately chose 2 hours as the differential adhesion time. Fibroblasts are more sensitive to trypsin; we precisely controlled the digestion time to 1 minute. After 1 minute of digestion, most fibroblasts floated, while cOECs remained attached to the cell plate. After removing the fibroblast trypsin suspension, trypsin was added again for further digestion until the cOECs completely floated, at which point digestion was terminated. Combining these two methods significantly improves the purity of our cultured cOECs.

[0063] The present invention will be further illustrated below with reference to specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field.

[0064] Example 1: Obtaining and purifying chicken oviduct epithelial cells (cOECs) This embodiment describes in detail the process of obtaining and purifying cOECs using the method of the present invention.

[0065] 1. Tissue Acquisition and Pre-processing Healthy laying hens aged 25-35 weeks were selected and euthanized after anesthesia. The abdominal cavity was aseptically opened, the oviduct was located, and it was cut at the infundibulum near the ovary and near the swollen portion, and the intact infundibulum tissue was removed. The tissue was longitudinally dissected and rinsed with DPBS. The serosal layer and mesentery were carefully removed with ophthalmic scissors and forceps, leaving only the mucosal folds on the inner surface.

[0066] 2. Digestion Cut the mucosal tissue into pieces approximately 2 mm in size. 3 Cells were sized and digested at 37°C for 30 minutes with intermittent vortexing. Digestion was terminated by adding FBS. Cells were then passed through a 70 μm cell sieve. After centrifugation, cells were resuspended in complete culture medium (DMEM / F12 basal medium supplemented with the following components: 10% fetal bovine serum, 2% chicken serum, 1× antibiotic-antifungal agent, non-essential amino acids, β-mercaptoethanol, epidermal growth factor, insulin-transferrin-selenium, L-glutamine, and hydrocortisone) and seeded into culture plates.

[0067] 3. Differential adhesion purification (a critical step lasting 2 hours) Incubate statically at 37°C and 5% CO2 for 2 hours. After 2 hours, gently aspirate the unattached cell suspension from the top layer and transfer it to a new culture plate for further culture (this is the preliminarily purified cOECs).

[0068] 4. Differential digestion and purification (a key step in 1 minute) When the cells reach 80%-90% confluence and require passage, discard the culture medium and wash with DPBS. Add 0.25% trypsin solution and place in an incubator. After precisely timing for 1 minute, observe under a microscope; most spindle-shaped fibroblasts will have shrunk, rounded, and detached from the cell wall. Immediately discard the trypsin digest containing fibroblasts. Then, add fresh 0.25% trypsin solution and continue digestion for 2-3 minutes until the cobblestone-like cOECs detach. Add complete culture medium to stop digestion, centrifuge to collect the cells, and passage.

[0069] 5. Purity Verification Single-cell sequencing of fallopian tube infundibulum-derived tissue: The infundibulum mucosal tissue prepared in step 1 was prepared into a single-cell suspension, and a comprehensive analysis was performed using 10x Genomics single-cell transcriptome sequencing (scRNA-seq). Based on the single-cell transcriptome sequencing data, the expression profile of cOECs-specific markers OVA (encoding genes SERPINB14 and SERPINB14B) was systematically analyzed. The results showed that in the cell samples from the fallopian tube infundibulum-derived tissue, 50.84% ​​of the cells expressed SERPINB14, and 30.42% of the cells expressed SERPINB14B, indicating that the proportion of cells expressing the OVA gene in this sample was close to half.

[0070] Morphological observation: After 48 hours of culture, under a microscope, the vast majority of cells in the field of view showed a typical epithelial-like cobblestone morphology with tight intercellular connections and very few spindle-shaped fibroblasts. Figure 2 ).

[0071] RT-PCR detection of cOECs marker genes: To identify whether isolated cells possessed cOECs molecular characteristics, total RNA was extracted from cells and reverse transcribed. PCR amplification was then performed targeting the fallopian tube epithelial characteristic genes OVA, AVD, OVM, LYZ, and the internal control β-actin. Results showed that all target genes exhibited clear bands of the expected size. Figure 3 This indicates that the isolated cells express cOECs marker genes.

[0072] Expression of cOECs marker proteins: Ovalbumin (OVA) expression was detected by Western blot. Results showed an OVA-specific band at approximately 45 kDa. Figure 4 This study confirmed that isolated cells retain key functional characteristics of synthesizing and secreting OVA, and can serve as a reliable cell model for immortalization.

[0073] Example 2: Establishment of an immortalized chicken oviduct epithelial cell line Screening for optimal puromycin concentration: Wild-type cOECs were seeded in 96-well plates, and culture medium containing 0, 1.0, 1.5, 2.0, 2.5, and 3.0 μg / mL puromycin were added, respectively. The cells were cultured continuously for 5 days, with daily observations. The results showed that a puromycin concentration of 1.5 μg / mL completely killed all wild-type cells within 5 days; therefore, this concentration was determined to be the optimal screening concentration.

[0074] Viral infection and screening: Second-generation cOECs purified in Example 1 were collected and, when they reached 60%-70% confluence, retroviral solutions carrying the hTERT and c-Myc genes were added at an MOI of 10. The infection was repeated 48 hours later to enhance efficiency. After infection, the medium was replaced with complete medium containing 1.5 μg / mL puromycin for screening, with the medium changed every two days. When all uninfected negative control cells died, the surviving cells in the experimental group were considered to have stably integrated the target gene. Figure 5 ).

[0075] Monoclonalization: After digestion of the selected polyclonal cells, the density was adjusted to approximately 6 cells / mL using limiting dilution, and seeded into 96-well plates at 100 μL per well. After 7-10 days of culture, wells containing only a single cell cluster were labeled. Once the cells reached 80% confluence, the culture was gradually expanded to culture flasks to obtain immortalized cell lines derived from monoclonal cells, named cMyc-cOECs and hTERT-cOECs, respectively. Monoclonal cell lines are as follows... Figure 6 As shown, the monoclonal cell line has clear cell boundaries and is in good growth condition. Genomic PCR and Sanger sequencing verified the successful integration of the hTERT and c-Myc genes. Figure 7 ).

[0076] Example 3: Characterization of Immortalized Cell Lines Proliferation capacity assay: The growth morphology and proliferative activity of immortalized cell lines (hTERT-cOECs) and wild-type cOECs were compared using the CCK-8 assay. Results ( Figure 8 and Figure 9 The results showed that throughout the culture period, the immortalized cell line transduced by c-Myc / hTERT consistently exhibited more vigorous proliferation and better growth status, with higher cell density, tighter intercellular connections, and significantly higher absorbance values ​​than wild-type cells (P<0.05), indicating that it achieved enhanced proliferation capacity and successfully achieved immortalization.

[0077] Functional Maintenance Validation: Estrogen is the core hormone regulating the function of cOECs. In the in vivo environment, it directly promotes the synthesis and secretion of various egg proteins, including OVA, by activating specific receptor signaling pathways. However, in the in vitro culture system, cells are removed from the in vivo hormonal environment, leading to a rapid decline in their functional integrity. Specifically, in the later stages of culture, the typical epithelial morphology of cOECs is difficult to maintain, gradually transforming from a tightly connected cobblestone-like structure to a mesenchymal-like fibrous structure; simultaneously, the expression levels of their specific marker genes (such as OVA and OVM) and protein secretion function are also significantly weakened. Therefore, supplementing the in vitro culture system with exogenous estrogen aims to reconstruct and maintain the functional phenotype of cOECs. This invention sets up treatment groups with different concentrations of estrogen and treatment groups with estrogen stimulation for different durations. The expression level of the OVA gene was detected by RT-PCR technology, and the effect of estrogen concentration on marker gene expression was analyzed. The results are as follows: Figure 10 As shown in the figure. The results showed that high-concentration estrogen treatment had a certain promoting effect on OVA expression; after continuous estrogen treatment for 72 h, the expression level of OVA was significantly enhanced compared with the 24 h and 48 h groups, indicating that estrogen has a concentration- and time-cumulative effect on the regulation of the OVA gene. After estrogen treatment, the morphology of cOECs changed significantly, gradually transforming from the original elongated spindle shape to an irregular polygon, and the cell morphology was closer to the classic cobblestone structure of epithelial cells, as shown in the figure. Figure 11 As shown.

[0078] Example 4: Immortalized cOECs for the expression of exogenous proteins Construction of CRISPR-Cas9 gene editing vectors: Using the CHOPCHOP platform, three highly efficient sgRNAs with low off-target effects were screened by targeting the end of exon 8 of the chicken OVA gene (Table 1). The corresponding single-stranded DNA was synthesized and annealed to double strands, then ligated into the linearized pX459 vector. Sanger sequencing confirmed that all three sgRNAs were accurately inserted into the gRNA scaffold region, and the pX459-sgRNA expression plasmid was successfully constructed.

[0079] Table 1: Results of sgRNA design Construction of exogenous gene donor plasmids: Lactoferrin (LTF) and lysosomal acid lipase (LAL) were selected as model exogenous proteins to evaluate the potential of immortalized cOECs to express complex pharmaceutical proteins. Using the pGL3 vector as a backbone, a 5' homologous arm, a chicken codon-optimized LTF or LAL expression cassette, a puromycin resistance gene, and a 3' homologous arm were sequentially inserted to construct a donor plasmid for site-directed integration via homologous recombination.

[0080] Validation of lysosomal acid lipase (LAL) expression: CRISPR-Cas9 editing plasmid and LAL donor plasmid were co-transfected into cOECs. Extensive GFP signal was observed under a fluorescence microscope 48 hours later, indicating that the plasmid was successfully introduced and the cells were in good condition. Figure 12 Western blot analysis showed an endogenous OVA band at approximately 43 kDa, while an OVA-His-LAL fusion protein-specific band was detected at approximately 90 kDa. Figure 13 This confirms that the expression system can successfully express exogenous LAL protein.

[0081] Validation of lactoferrin (LTF) expression: GFP signal was also observed after co-transfection with LTF plasmid, but the fluorescence intensity was weaker than that of the LAL group. Figure 14 Western blot analysis showed that endogenous OVA bands were detected in cMyc-cOECs, but the expected 120 kDa OVA-His-LTF fusion protein was not detected; neither was detected in hTERT-cOECs. Figure 15 This indicates that LTF expression efficiency is low in this system.

[0082] It should be noted that while the preferred embodiments of the present invention are given in the specification and accompanying drawings, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. These embodiments are not intended to impose additional limitations on the content of the present invention; their purpose is to provide a more thorough and comprehensive understanding of the disclosure of the present invention. Furthermore, the above-described technical features can be combined with each other to form various embodiments not listed above, all of which are considered to be within the scope of the present invention specification. Moreover, those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. A method for obtaining and purifying chicken oviduct epithelial cells, characterized in that, Includes the following steps: (1) Tissue acquisition: Select healthy laying hens, euthanize them, and aseptically separate the oviduct infundibulum tissue; (2) Pre-treatment of tissue: Remove the serosal layer and mesentery of the infundibulum tissue, and separate and obtain the mucosal fold tissue; (3) Digestion treatment: The mucosal layer folds are cut into small pieces and then digested with enzymes to obtain a mixed cell suspension; (4) Differential adhesion purification: The mixed cell suspension was seeded in a culture plate and cultured for 2 hours. Then, the non-adherent cell suspension was collected and transferred to a new culture plate. (5) Differential digestion and purification: After the cells adhere to the wall, use trypsin to digest for 1 minute, remove the digested fibroblasts, and retain the adhered chicken oviduct epithelial cells.

2. The method according to claim 1, characterized in that, The laying hens mentioned in step (1) are 25-35 weeks old.

3. The method according to claim 1, characterized in that, The enzyme digestion described in step (3) is pancreatic enzyme digestion, and the digestion time is 10-60 minutes.

4. An immortalized chicken oviduct epithelial cell line, characterized in that, The cell line was constructed by co-transfecting expression vectors carrying the hTERT gene and / or the c-Myc gene into chicken oviduct epithelial cells obtained and purified by any one of claims 1-3.

5. The immortalized chicken oviduct epithelial cell line according to claim 4, characterized in that, The cell lines were cultured in a medium supplemented with estrogen to maintain or enhance the epithelial-like paving stone morphology and OVA gene expression in the cell lines.

6. The immortalized chicken oviduct epithelial cell line according to claim 5, characterized in that, The concentration of the estrogen in the culture medium is 10-500 μM; Furthermore, the cell line is cultured in a medium containing estrogen for at least 72 hours.

7. The immortalized chicken oviduct epithelial cell line according to any one of claims 4-6 is used as an in vitro model for screening and / or expressing exogenous recombinant proteins.

8. The application according to claim 7, characterized in that, The application includes: using gene editing technology to knock in the gene encoding the target exogenous recombinant protein into the genome of the immortalized chicken oviduct epithelial cell line, and culturing the cell line to express and / or secrete the target exogenous recombinant protein.

9. An in vitro screening and validation platform for egg bioreactors, characterized in that, Including the immortalized chicken oviduct epithelial cell line according to any one of claims 4-6.

10. The application of the in vitro screening and validation platform according to claim 9 for predicting the expression performance of exogenous recombinant proteins in an egg bioreactor; Furthermore, the application includes: Gene editing technology was used to knock the gene encoding the target exogenous recombinant protein into the genome of the immortalized chicken oviduct epithelial cell line. The cell line was cultured and the expression level of the target exogenous recombinant protein was analyzed, thereby predicting the expression performance of the exogenous recombinant protein in an egg bioreactor.

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