Method for preparing suspension-adapted chicken embryo fibroblasts, AQP1 mutant DF-1 cell line and use thereof
Patent Information
- Application Number
- PCT/CN2025/083119
- Authority / Receiving Office
- WO · WO
- Patent Type
- Applications
- Current Assignee / Owner
- Filing Date
- 2025-03-18
- Publication Date
- 2026-09-24
Smart Images
Figure CN2025083119_24092026_PF_FP_ABST
Abstract
Description
Methods for preparing suspension chicken embryo fibroblasts, AQP1 mutant DF-1 cell line and its applications Technical Field
[0001] This invention belongs to the field of cell preparation, virus proliferation and viral vaccine technology, and particularly relates to a method for preparing suspension chicken embryo fibroblasts, an AQP1 mutant DF-1 cell line and its application. Background Technology
[0002] The preparation of avian propolis vaccines often requires high-titer viral antigens. Increasing the viral concentration in the antigen solution and enhancing viral proliferation capacity are key focuses in the development of inactivated avian vaccines. Currently, for large-scale industrial vaccine production, suspension culture is superior to microcarrier culture and roller bottle culture due to its higher cell density, higher viral titer, lower production cost, and easier process control. Therefore, suspension culture will be the trend in cell culture methods for producing antiviral vaccines.
[0003] There are many strategies for adapting cells to suspension culture, but traditional acclimatization methods are only effective for a subset of cells with suspension growth potential, remaining ineffective for most. Currently, the types of suspension cells used in industrial vaccine production are still very scarce, and viral vaccine production still relies heavily on cells with low production efficiency, such as primary cells like chicken embryo fibroblasts or passaged cell lines like the chicken embryo fibroblast (DF-1). To improve the efficiency of poultry vaccine production, there is an urgent need to find new suspension acclimatization methods and technologies to change the adherent growth characteristics of poultry cell lines such as the DF-1 cell line, enabling large-scale production.
[0004] Genetic engineering to prepare suspension cell lines can overcome the uncertainties and randomness inherent in traditional suspension culture methods regarding cell yield, genetic background, and culture time. Some studies have shown that altering certain adhesion-related genes can adapt cells to suspension culture. However, the loss of cell adhesion does not equate to cell growth in suspension; the deletion of many adhesion genes does not necessarily result in suspension-capable cells. Furthermore, many adhesion genes are closely linked to cancer, and inhibiting or overexpressing these genes may lead to cell carcinogenesis. Therefore, in developing suspension cell lines, improving cell suspension performance by altering the function of relevant proteins without inducing tumorigenesis is a pressing issue.
[0005] Aquaporins (AQPs) are a series of specific pores on the cell membrane that efficiently and selectively transport water molecules. These include AQP0 to AQP12, which function in transporting water, urine, glycerol, and other substances, and participate in various physiological mechanisms. In poultry cells, AQP1 is expressed in tissues and cells such as the kidney, stomach, intestine, ovary, and oviduct, and is also significantly expressed in the ectodermal and endoderm epithelial cells of the chicken embryo's chorioallantoic membrane. Currently, no studies have reported the relationship between the structure of the AQP1 protein and the adherent growth state of poultry cells. Summary of the Invention
[0006] The purpose of this invention is to provide a method for preparing suspended chicken embryo fibroblasts, an AQP1 mutant DF-1 cell line, and its applications.
[0007] This invention serendipitously discovered that disrupting the AQP1 protein structure can significantly alter the adherent growth state of poultry cells, without producing tumorigenic effects in continuous passages. Therefore, to improve the suspension capacity of the chicken embryo fibroblast cell line DF-1, a portion of the transmembrane region of the AQP1 gene in DF-1 cells was knocked out through gene editing, altering the function of the AQP1 protein and thereby enhancing cell suspension growth ability, accelerating suspension acclimatization, and providing an excellent cell line for the large-scale production of avian viral disease vaccines. Thus, this invention improves cell suspension acclimatization efficiency without inducing tumorigenic risk by knocking out or mutating the AQP1 gene in the chicken embryo fibroblast cell line DF-1, thereby preparing a high-density cultured poultry cell line.
[0008] To achieve the above-mentioned objectives, the present invention provides the following technical solution:
[0009] This invention provides a method for preparing suspended chicken embryo fibroblasts, including the step of knocking down AQP1 in chicken embryo fibroblasts.
[0010] In this invention, the method for knocking down AQP1 includes knocking down with sgRNA, the nucleotide sequence of which is preferably as shown in SEQ ID NO: 3; the chicken embryo fibroblasts include the DF-1 cell line.
[0011] This invention provides an AQP1 mutant gene, prepared by editing chicken embryo fibroblasts using sgRNA gene editing as shown in SEQ ID NO: 3. Preferably, the AQP1 mutant gene has nucleotides 92-214 deleted from the wild-type AQP1 gene, and the preferred nucleotide sequence of the AQP1 mutant gene is shown in SEQ ID NO: 1.
[0012] This invention provides a mutant protein encoding the aforementioned AQP1 mutant gene. The preferred amino acid sequence of the mutant protein is shown in SEQ ID NO: 2: MASEFKKKMFWRAVVAEFLAMILFIFISIGFCGAHLNPAVTLGLLLSCQISIFKALMYILAQCLGAVVATAILSGVTSSLPYNSLGLNALAKGINAGQGLGIEIIATLQLVLCVLATTDRRRNDVSGSAPLAIGLSVALGHLLAIDYTGCGINPARSFGSALIANNFENHWIFWVGPIIGGAGAALIYDFILAPRSSDLTDRVKVWTSGQVEEYDLEGDDMNSRVEMKPK.
[0013] This invention provides an expression cassette, expression vector, cell line, or recombinant bacterial strain containing the aforementioned AQP1 mutant gene. The host cell of the cell line preferably includes the DF-1 cell line.
[0014] This invention provides a method for preparing an AQP1 mutant DF-1 cell line, comprising the following steps: knocking out the AQP1 gene in the DF-1 cell line using a CRISPR / Cas9 system. The CRISPR / Cas9 system preferably includes sgRNA with nucleotide sequences as shown in SEQ ID NO: 3 and Cas9 protein. As a specific embodiment, the method includes the following steps: designing sgRNA targeting the AQP1 gene sequence of DF-1 cells; applying the CRISPR / Cas9 system to knock out nucleotides 92-214 of the first and second transmembrane regions of the AQP1 gene in DF-1 cells; and purifying the resulting sgRNA through cloning and screening.
[0015] The present invention provides an AQP1 mutant DF-1 cell line prepared by the above preparation method.
[0016] This invention provides the application of the above-mentioned AQP1 mutant gene, mutant protein, expression cassette, expression vector, cell line or recombinant strain or AQP1 mutant DF-1 cell line in cell suspension culture, preparation of products that promote viral proliferation or preparation of viral vaccines.
[0017] In this invention, the virus includes infectious bursal virus, such as infectious bursal virus strain BJQ902.
[0018] This invention provides a method for preparing a propolis inactivated vaccine, comprising the following steps:
[0019] The virus was proliferated in the AQP1 mutant chicken embryo fibroblast cell line, and then the antigen solution was collected. The antigen solution was mixed with propolis immune enhancer to obtain the final product.
[0020] Preferably, the preparation method of the propolis immune enhancer includes freezing propolis at -20 to -10°C for more than 24 hours, pulverizing it, passing it through a 100 to 140 mesh sieve to obtain propolis powder, then dissolving it in 3 to 5 times the amount of 95% ethanol solution, sealing it, and extracting it at 20 to 25°C for 70 to 74 hours, stirring it once every 8.5 to 9.5 hours during extraction, filtering the settled propolis solution, and collecting the filtrate to obtain the product.
[0021] In this invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art.
[0022] Compared with the prior art, the present invention has the following beneficial effects:
[0023] This invention alters the function of the AQP1 protein by knocking out the first and second transmembrane spacer sequences in the AQP1 gene of DF-1 cells through gene editing. The resulting AQP1 mutant cells have better suspension properties and stable genetic traits, increasing virus yield. They can be applied to the production of poultry virus vaccines or propolis inactivated vaccines, replacing traditional cell vaccine production processes and significantly improving production efficiency. Attached Figure Description
[0024] Figure 1 shows the PCR identification results of DF-1 cells after transfection. M is the DNA molecular weight standard, 1 is the transfected cell culture, and 2-4 are the untransfected cell control, blank control, and culture medium control, respectively. The 437bp band in each lane is the unedited amplification product, the 315bp band is the amplification product of the edited mutant gene, and the others are non-specific amplification bands.
[0025] Figure 2 shows DF-1 / AQP1 － Predicted transmembrane sites for AQP1 protein expression in DF-1 cells, 2A representing DF-1 / AQP1 — The predicted transmembrane site of the AQP1 encoding gene in cell line 2B is shown in Figure 2B, where 33-51aa is the intramembrane region. Figure 2B is shown in Figure 2B, where 36-54aa is the extramembrane region.
[0026] Figure 3 shows DF-1 / AQP1 － The images show the observation of cells during passage. 3A shows cells that have grown to a confluence just before passage; 3B shows cells that have been simply pipetted without trypsin digestion; 3C shows cells that have been dispersed by pipetting and inoculated into a new bottle and have grown for 24 hours; and 3D shows cells that have grown for 48 hours after inoculation.
[0027] Figure 4 shows DF-1 / AQP1－ Growth curves of DF-1 cells cultured in shake flasks.
[0028] Figure 5 shows DF-1 / AQP1 － In a nude mouse tumorigenesis assay with DF-1 cells, 5A represents DF-1 / AQP1. — Nude mice in the cell group, 5B is the CEF cell group, 5C is the positive control group, and the arrows point to the injection site or tumor site. Detailed Implementation
[0029] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.
[0030] Example 1 DF-1 / AQP1 － Cell construction
[0031] 1.1 sgRNA Design and Preparation
[0032] Based on the chicken AQP1 gene sequence (ENSGALG00000005209) registered in the Ensembl database, a sequence from -20 to 437 nt near the start codon in exon 2 was selected. A guide RNA sequence was designed online using a design tool (genscript.com) and named AQP1_g1 (Table 1). Off-target effects were analyzed by comparing the sequence with human, monkey, bovine, and ovine genomes. AQP1_g1 in vitro transcription primers were designed and synthesized. PCR amplification and sgRNA transcription were performed according to the instructions of a commercially available one-step sgRNA in vitro transcription kit (Inovogen Technology), and the sgRNA concentration was adjusted to 400 ng / μL.
[0033] Table 1. sgRNA and in vitro transcription primers used in the experiment.
[0034] 1.2 Transfection
[0035] DF-1 cells were obtained from the ATCC cell bank (ATCC CRL-12203). Cells were cultured to the logarithmic growth phase and passaged into 24-well plates to prepare a monolayer with 90% confluence at the bottom of each well. 2.5 μL of the transcribed sgRNA from step 1.1 was added sequentially to the first 1.5 mL tube along with 2.5 μL of commercially available Cas9 protein (Genescript, 5 μM). After incubation at room temperature for 10 minutes, 20 μL of CRISPR buffer was added to obtain the Cas9 / gRNA complex. Following the instructions of the commercially available CRISPR RNP transfection kit (Viromer, Origene), 25 μL of transfection reagent (0.4 μL Viromer + 24.6 μL CRISPR buffer) was prepared in the second 1.5 mL tube and rapidly mixed with an equal volume of the Cas9 / gRNA complex from the first tube. After incubation at room temperature for 15 minutes, the mixture was added to the 24-well plate and cultured for 72 hours.
[0036] 1.3 Cloning screening of gene-deleted cell lines
[0037] 72 hours after transfection, the transfected cells in the 24-well plates were digested and resuspended into single cells, and subcloning was performed using the limiting dilution method in 96-well plates. Wells that produced single-cell clones were selected, and AQP1 gene-deleted cell clones were screened using the established PCR method (primers are shown in Table 2).
[0038] Table 2. Deletion identification primers and sequencing primers used in the experiment.
[0039] The selected DF-1 / AQP1 － Cells were washed three times with PBS, resuspended in PBS at a density of 10,000 cells / mL, and 200 μL of supernatant was collected for nucleic acid extraction using a DNA / RNA extraction kit. Using the extracted cellular RNA as a template, RT-PCR amplification was performed using the designed primers AQP1F and AQP1R. Cells showing deleted bands were selected for further culture.
[0040] After the first round of cell subcloning culture and PCR identification, a clone with a deletion band of approximately 120 bp was obtained (see Figure 1). This clone was selected for further subcloning and purification to obtain a well-growing, PCR-purified gene-deleted cell clone. This clone was then passaged and preserved, and named DF-1 / AQP1. － .
[0041] 1.4 DF-1 / AQP1 － Strain gene sequencing identification
[0042] Primers were designed based on the chicken AQP1 gene sequence (ENSGALG00000005209) registered in the Ensembl database, and primers were used to target DF-1 / AQP1 as described in section 1.3. － The full-length AQP1 protein-coding gene of the strain was amplified and sequenced (primers AQP1wF and AQP1wR are shown in Table 3). The sequencing results were compared with the relevant sequences in the reference gene.
[0043] Table 3. Deletion identification primers and sequencing primers used in the experiment.
[0044] Sequencing comparisons revealed that DF-1 / AQP1 － The full-length AQP1 gene coding region of the cells is 693 bp. Compared with the parental strain's 813 bp, the CRISPR system caused a double-strand break near the PAM site in the AQP1_g1 target sequence, resulting in a deletion of more than 120 bp at the cleavage site. This deletion was subsequently repaired and rejoined, forming a mutant gene. Compared with the parental cells, the mutant cell line lacked a 123 bp sequence from 92 to 214 nt, and also had 3 bp of inserted bases due to random repair (see SEQ ID NO: 1 in the sequence listing). Deducing its amino acid sequence and comparing it with the parental cells, the mutant cell's AQP1 gene encodes a 230 aa protein, with a mutation of 42 aa from 31 to 72 aa (including a deletion of 40 aa) compared to the parental cells, covering the first and second transmembrane spacer sequences (see Figure 2). DF-1 / AQP1 － The coding sequence of the AQP1 gene and its amino acid sequence of the strain are shown in SEQ ID NO: 2.
[0045] In summary, this embodiment involved knocking out a portion of the AQP1 gene near the N-terminus in DF-1 cells, resulting in a cell clone. The molecular characteristic of this cell is the absence of the first and second transmembrane spacer sequences in its AQP1 protein; hereinafter referred to as DF-1 / AQP1. － cell.
[0046] Example 2 DF-1 / AQP1 － Cell growth characteristics
[0047] 2.1 DF-1 / AQP1 － Cell adherent culture and morphological observation
[0048] Prepare DMEM medium (GIBCO) containing 5% newborn calf serum as the cell growth medium, and disperse DF-1 / AQP1 by pipetting. － Cells, at a rate of 1×10⁶ cells per T25 culture flask 6 The cells were passaged at a specific concentration, and the parental line was passaged in the same manner to observe cell morphology and growth status. Mutant clone DF-1 / AQP1－ The cell morphology of the mutant cells was not significantly different from that of the parent cells. However, the mutant cells were easily mechanically dispersed after adhering to the culture vessel, especially during the late logarithmic growth phase and plateau phase. They could be easily detached from the culture vessel wall by pipetting, dispersing into single cells or clusters of 4–16 cells. After subculturing, the cells spread well and were still able to adhere to the culture vessel wall, exhibiting a relatively uniform polygonal or spindle-shaped structure (see Figure 3).
[0049] 2.2 DF-1 / AQP1 － Cell suspension culture
[0050] Cells in logarithmic growth phase are processed at 5 × 10⁻⁶. 5 Cells were seeded at a concentration of 5 × 10⁶ cells / mL into 125 mL shake flasks, and 30 mL of DMEM medium containing 5% serum was added. The cells were incubated at 37°C and 150 rpm for 72 hours using a 5% CO₂ shaker. Cells were collected by centrifugation at 1200 rpm for 5 min, resuspended in 30 mL of medium, and aliquoted into separate flasks at an initial cell concentration of 5 × 10⁶ cells / mL. 5 Subculture at 100 cells / mL. If the cell concentration exceeds 1×10⁻⁶ after 72 hours... 6 If the number of cells / mL is 1, then the initial seeding density needs to be reduced to 1×10⁻⁶. 5 Cells / mL to 3×10 5 Cells / mL. If the cell concentration is below 1×10⁻⁶ cells / mL after 72 hours of culture. 6 If the cell concentration was found to be 1 × 10⁶ cells / mL, the culture medium was replaced with fresh medium and cultured again. Unmutated parental DF-1 cells were used as a control group, also cultured in shake flasks for comparison. Cell concentration was continuously passaged and recorded every 72 hours. It was observed that parental DF-1 cells could not consistently reach 1 × 10⁶ cells / mL even after 5 generations of continuous suspension culture. 6 cells / mL, while DF-1 / AQP1 － Cells reached 1×10⁶ cells / day after 48 hours of suspension culture in the second generation. 6 Cells / mL, and the cells were cultured for 5 generations and the growth was basically stable (see Figure 4).
[0051] Example 3 DF-1 / AQP1 － Cell tumorigenicity test
[0052] Select DF-1 / AQP1 buds that are in good growth condition and in the logarithmic growth phase. － Cells were collected when they reached 80-90% confluence. The cells were resuspended in PBS or serum-free medium to a concentration of 5 × 10⁶ cells / mL. 7 Cells / mL. Simultaneously set 5×10⁻⁶ cells / mL. 7 CEF of 10 cells / mL was used as the negative control group, 1×10 7BHK-21 cells / mL were used as the positive control group. Nude mice aged 5-8 weeks, weighing 18-20g, were subcutaneously inoculated with cells on the back of their necks, with 5 mice per group and 0.2mL per mouse. Before inoculation, the cell suspension was thoroughly dispersed to prevent clumping. Mice were observed for 35 to 12 weeks post-inoculation, checking for nodules or tumors. In the positive control group, mice developed a noticeable mass at the inoculation site, up to 10mm in diameter, and exhibited lethargy and decreased appetite. No nodules or suspected tumor tissue were observed in either the negative control group or the experimental group, and mice showed no abnormalities in mental state or appetite (see Figure 5). Necropsy and histological examination at 35 days and 12 weeks revealed no tumor cells.
[0053] Example 4 DF-1 / AQP1 － Passage stability of AQP1 mutations in cells
[0054] DF-1 / AQP1 － Cells were passaged for more than 20 generations. Samples from the 20th generation were used to amplify a 693bp target fragment of the AQP1 coding region using the primers and steps described in Example 1. The amplified fragment was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequencing results were compared with the AQP1 coding sequence of earlier passages in the mutant cell line; the sequence homology was 100%, and no reversion mutations were observed. DF-1 / AQP1 － The genetics of the strain are relatively stable.
[0055] Example 5 DF-1 / AQP1 － Cell culture of infectious bursal virus vaccine antigen
[0056] DF-1 / AQP1 in the logarithmic growth phase － Cells are arranged at 5 × 10 5 Cells were seeded at a concentration of [number] cells / mL into 125 mL shake flasks, and 30 mL of DMEM medium was added. The flasks were incubated at 150 rpm with 5% CO2 for 48 hours. Infectious bursal virus strain BJQ902 (vaccine strain from Shandong Lvdou Biotechnology Co., Ltd.) was then inoculated at a MOI of 0.005. The flasks were then continued in suspension culture for 72 hours, during which glucose consumption was monitored periodically. When the glucose concentration fell below 1 g / L, it was replenished to 4 g / L. The culture was then administered with TCID45. 50 An assay was used to detect the content of infectious bursal disease virus. Suspension culture of DF-1 / AQP1 was applied. － Cellular proliferative infectious bursal virus (IPV) can achieve a viral titer of 10 within 36-48 hours after inoculation. 9.1 TCID 50 / mL, compared with the same number of DF-1 parental cells cultured by the traditional adherent culture method, as shown in Table 4, its unit yield can be increased by 3.98-19.95 times.
[0057] Table 4 Comparison of viral replication after inoculation with infectious bursal disease virus (TCID) 50 / mL)
[0058] Example 6 DF-1 / AQP1 － Efficacy of Propolis Inactivated Vaccine for Infectious Bursal Disease (IBD)
[0059] DF-1 / AQP1 cultured in suspension － Infectious bursal disease virus strain BJQ902, proliferating in cells, was cultured and subjected to a freeze-thaw cycle 48 hours after inoculation. The culture was aseptically harvested and used as the antigen solution. After passing sterility and mycoplasma-free tests, the titer was determined to be greater than or equal to 10. 8.9 TCID 50 / mL, which can be inactivated with formaldehyde solution (0.1% V / V) for 16 hours. The inactivated antigen that passes the test is then mixed with propolis immune enhancer to synthesize the vaccine, resulting in a vaccine with an IBDV antigen content of 10. 8.5 The dry matter content of propolis is 1 mg / mL or more.
[0060] The preparation method of propolis immune enhancer is as follows: Select pure, high-quality propolis, remove impurities such as beeswax, grass clippings, and sawdust, freeze at -15℃ for 24 hours, then pulverize the propolis into fine powder using a cryogenic grinder, pass it through a 120-mesh sieve to obtain propolis powder, then dissolve it in 4 times its volume of 95% ethanol solution (w / v), seal it, and extract it at 25℃ for 72 hours, stirring once every 9 hours at a stirring speed of 200 rpm. After standing for 24 hours, filter the propolis solution under positive pressure and collect the filtrate to obtain the product, whose dry matter percentage is not less than 45%.
[0061] Ten susceptible chicks under 28 days of age were vaccinated, with each chick receiving a single subcutaneous injection of 1 mL in the neck. After 14 days of observation, no adverse systemic reactions were observed, and there was no swelling or pain upon pressure at the injection site.
[0062] Ten 21-day-old SPF chickens were vaccinated and administered a single subcutaneous injection of one dose of vaccine as the immunization group. Twenty-eight days after vaccination, all ten chickens were challenged with live BJQ902 strain virus, with each chicken receiving 0.2 mL via eye drops or nasal drops. 5.0 ID 50 A total of one challenge was conducted, with lesions in the bursa of Fabricius examined 4 days later. Simultaneously, five 21-day-old SPF chickens were used as a control group, receiving the same volume of physiological saline as the vaccine group, but challenged with 0.2 mL (10) of the virus on day 28. 5.0 ID 50The BJQ902 strain virus was administered once. Five 21-day-old SPF chickens were used as a healthy control group and were inoculated with the same volume of physiological saline as the vaccine group. On day 28, 0.2 mL of physiological saline was injected once. Bursal disease lesions were examined by necropsy on the same day as the experimental group.
[0063] Compared with the healthy control group, 5 out of 5 animals in the challenged control group showed lesions such as bursal swelling, while 9 out of 10 animals in the immunized group had bursae of normal size. The antigen prepared using the virus culture method of this invention can be used in vaccine production.
[0064] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.
Claims
1. A method for preparing suspended chicken embryo fibroblasts, characterized in that, This includes the step of knocking down AQP1 in chicken embryo fibroblasts.
2. The method according to claim 1, characterized in that, Methods for knocking down AQP1 include using sgRNA.
3. The method according to claim 2, characterized in that, The nucleotide sequence of the sgRNA is shown in SEQ ID NO:
3.
4. An AQP1 mutant gene, characterized in that, The AQP1 mutant gene was prepared by editing chicken embryo fibroblasts using sgRNA gene with the nucleotide sequence shown in SEQ ID NO:
3.
5. The AQP1 mutant gene according to claim 4, characterized in that, The AQP1 mutant gene has nucleotides 92-214 missing from the AQP1 wild-type gene.
6. The AQP1 mutant gene according to claim 5, characterized in that, The nucleotide sequence of the AQP1 mutant gene is shown in SEQ ID NO:
1.
7. A mutant protein encoding the AQP1 mutant gene as described in any one of claims 4 to 6.
8. The mutant protein according to claim 7, characterized in that, The amino acid sequence of the mutant protein is shown in SEQ ID NO:
2.
9. An expression cassette, expression vector, cell line, or recombinant bacterial strain, characterized in that, It contains the AQP1 mutant gene as described in any one of claims 4 to 6.
10. The expression cassette, expression vector, cell line, or recombinant strain according to claim 9, characterized in that, The host cells of the cell line include chicken embryo fibroblast cell lines.
11. A method for preparing an AQP1 mutant DF-1 cell line, characterized in that, Includes the following steps: The AQP1 gene was knocked out in the DF-1 cell line using the CRISPR / Cas9 system.
12. The preparation method according to claim 11, characterized in that, The CRISPR / Cas9 system includes sgRNA and Cas9 protein with nucleotide sequences as shown in SEQ ID NO:
3.
13. The preparation method according to claim 11, characterized in that, The nucleotide sequence of the AQP1 mutant is shown in SEQ ID NO:
1.
14. The AQP1 mutant DF-1 cell line prepared by the preparation method according to any one of claims 11 to 13.
15. The use of the AQP1 mutant gene according to any one of claims 4 to 6, the mutant protein according to claim 7 or 8, the expression cassette, expression vector, cell line or recombinant strain according to claim 9, or the AQP1 mutant DF-1 cell line according to claim 14 in cell suspension culture, preparation of products with increased viral yield, or preparation of viral vaccines.
16. The application according to claim 15, characterized in that, The virus mentioned includes infectious bursal virus.
17. A method for preparing a propolis inactivated vaccine, characterized in that, Includes the following steps: The virus is proliferated in the AQP1 mutant chicken embryo fibroblast cell line of claim 14, and then the antigen solution is collected. The antigen solution is mixed with propolis immune enhancer to obtain the final product.
18. The preparation method according to claim 17, characterized in that, The virus mentioned includes infectious bursal virus.
19. The preparation method according to claim 17, characterized in that, The preparation method of the propolis immune enhancer includes freezing propolis at -20 to -10°C for more than 24 hours, pulverizing it, passing it through a 100 to 140 mesh sieve to obtain propolis powder, then dissolving it in 3 to 5 times the amount of 95% ethanol solution, sealing it, and extracting it at 20 to 25°C for 70 to 74 hours, stirring it once every 8.5 to 9.5 hours during extraction, filtering the settled propolis solution, and collecting the filtrate to obtain the product.