HEK293 cell capable of stably expressing pigeon adenovirus I type Fiber2 protein as well as construction method and application of HEK293 cell
By introducing a recombinant expression vector into HEK293 cells and acclimating them into a suspension cell line, the problems of low expression level and poor stability of Fiber2 protein were solved, achieving efficient and stable expression and purification. The prepared subunit vaccine has good protective effect.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- 乾元浩生物股份有限公司
- Filing Date
- 2026-01-21
- Publication Date
- 2026-05-12
AI Technical Summary
In the existing technology, the expression level of pigeon adenovirus type I Fiber2 protein is low and the stability is poor, making it difficult to achieve efficient and sustained expression. Furthermore, there is a lack of stable expression cell lines for the preparation of subunit vaccines.
HEK293 cells were used as the host cells. The recombinant expression vector pCDNA-PAdV-Fiber2 was introduced into the cells via liposome transfection. Cells that stably expressed Fiber2 protein, namely QYH-PAF2-A1, were screened and bred into a suspension cell line. The protein was purified using the His tag and a subunit vaccine was prepared.
The efficient and stable expression and purification of Fiber2 protein were achieved, significantly improving protein expression efficiency. The prepared subunit vaccine achieved a protection rate of 80% in the immune challenge experiment, providing biological materials for subunit vaccines and diagnostic reagents.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of biotechnology, specifically relating to HEK293 cells that stably express pigeon adenovirus type I Fiber2 protein, their construction methods, and applications. Background Technology
[0002] Pigeon adenovirus type I is a major pathogen causing clinical symptoms in pigeons such as vomiting, weakness, slow digestion, green or watery droppings, and weight loss. The disease spreads rapidly and has a morbidity rate as high as 100%. In pigeons with weak immune systems and those experiencing mixed infections, symptoms often persist for several months, including decreased physical strength, emaciation, lethargy, and green, watery droppings, ultimately leading to death. Pigeon adenovirus type I's Fiber2 protein is one of the virus's main structural proteins, located on the surface of the virus particle. It is a key antigen that induces the production of neutralizing antibodies and has core value in vaccine preparation.
[0003] Currently, Fiber2 protein is mostly prepared through transient transfection or prokaryotic expression systems, but these methods suffer from low expression levels, poor stability, and incomplete protein modification. The HEK293 cell line possesses advantages such as strong suspension growth ability, high protein expression efficiency, and ease of large-scale culture. Constructing a stable HEK293 suspension cell line expressing Fiber2 protein can achieve efficient and sustained expression of the target protein, providing crucial materials for research on pigeon adenovirus type I subunit vaccines. Currently, there are no reports on such cell lines; therefore, there is an urgent need to develop an efficient and stable cell preparation technology. Summary of the Invention
[0004] The purpose of this invention is to provide HEK293 cells that stably express pigeon adenovirus type I Fiber2 protein, their construction method, and their applications.
[0005] To achieve the objectives of this invention, in a first aspect, this invention provides HEK293 cells QYH-PAF2-A1 that stably express pigeon adenovirus type I Fiber2 protein. This cell strain is currently deposited at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China, accession number CGMCC No. 46766, deposited on December 9, 2025.
[0006] Secondly, the present invention provides a method for constructing the cell, comprising the following steps: (1) The recombinant expression vector containing the pigeon adenovirus type I fiber2 gene was introduced into HEK293 adherent cells; (2) Screening to obtain positive clones that can stably express Fiber2 protein; (3) The positive clone cells are subjected to suspension acclimatization culture.
[0007] Furthermore, the recombinant expression vector described in step (1) is constructed by cloning the pigeon adenovirus type I fiber2 gene into the pCDNA3.1(+) vector.
[0008] Further, in step (1), the recombinant expression vector is introduced into HEK293 adherent cells using liposome transfection.
[0009] Furthermore, the screening in step (2) includes drug screening using G418 and monoclonalization using limiting dilution.
[0010] Furthermore, the suspension acclimatization culture in step (3) involves gradually reducing the serum concentration in the culture medium until serum-free culture is achieved.
[0011] Thirdly, the present invention provides the use of the cells in the preparation of pigeon adenovirus type I Fiber2 protein.
[0012] Fourthly, the present invention provides the use of the cells in the preparation of a subunit vaccine for preventing pigeon adenovirus type I infection.
[0013] In this invention, pigeon adenovirus ( Aviadenovirus columbae Type I includes, but is not limited to, strain QYH-HB-1, which is currently deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China, with accession number CGMCC No. 47084 and deposit date November 26, 2025.
[0014] By employing the above technical solution, the present invention has at least the following advantages and beneficial effects: This invention uses mammalian HEK293 cells as the host cell line. Cells stably expressing the Fiber2 protein (QYH-PAF2-A1) were obtained via liposome transfection, and these adherent cells were domesticated into a suspension cell line (QYH-PAF2-A1). By adding a His tag to the C-terminus of the fiber2 gene and purifying the protein using a nickel column, a pigeon adenovirus type I Fiber2 protein subunit vaccine was prepared, and its protection against immune challenge was demonstrated. This cell line stably and continuously expresses the protective antigen Fiber2 protein of pigeon adenovirus type I, significantly improving protein expression efficiency. The protein produced through large-scale culture using this cell line can provide biomaterials for subunit vaccines, diagnostic reagents, and basic research. Attached Figure Description
[0015] Figure 1This invention provides a preferred embodiment of the Western blot identification of four HEK293 cell clones stably expressing pigeon adenovirus type I Fiber2 protein. In this embodiment, M represents the standard molecular weight of the protein, lanes 1-4 represent the four HEK293 cell clones stably expressing pigeon adenovirus type I Fiber2 protein, and lane 5 represents HEK293 cells.
[0016] Figure 2 This is a Western blot analysis of QYH-PAF2-A1 suspension cells in a preferred embodiment of the present invention. Wherein, M is the standard molecular weight of the protein, and lanes 1-3 represent QYH-PAF2-A1 suspension cells, QYH-PAF2-A1 adherent cells, and HEK293 cells, respectively.
[0017] Figure 3 Western blot identification of generation 1, 5, and 10 QYH-PAF2-A1 suspension cells in a preferred embodiment of the present invention. Wherein, M is the standard molecular weight of the protein, and lanes 1-5 represent generation 1 QYH-PAF2-A1 suspension cells, generation 5 QYH-PAF2-A1 suspension cells, generation 10 QYH-PAF2-A1 suspension cells, QYH-PAF2-A1 adherent cells, and HEK293 cells, respectively.
[0018] Figure 4 This invention provides an SDS-PAGE assay to determine the purification efficiency of pigeon adenovirus type I Fiber2 protein in a preferred embodiment. M represents the standard molecular weight of the protein. Lanes 1-5 are respectively: QYH-PAF2-A1 suspension cell culture, supernatant of QYH-PAF2-A1 suspension cell culture after one freeze-thaw cycle at -80°C, cell debris of QYH-PAF2-A1 suspension cell culture after one freeze-thaw cycle at -80°C, Fiber2 protein purification flow buffer, and Fiber2 protein purification elution buffer. Detailed Implementation
[0019] This invention provides the construction and application of a HEK293 suspension cell line that stably expresses pigeon adenovirus type I Fiber2 protein.
[0020] The present invention adopts the following technical solution: This invention introduces the pigeon adenovirus type I Fiber2 gene into HEK293 adherent cells via liposome transfection. After drug screening and molecular identification, cells stably expressing the Fiber2 protein, QYH-PAF2-A1, are obtained. These adherent cells are then domesticated into the suspension cell line QYH-PAF2-A1. The construction method of the suspension cell line includes the following steps: 1. Construction of recombinant expression vector pCDNA-PAdV-Fiber2 First, pigeon adenovirus (preserved in the laboratory) was used separately. Aviadenovirus columbae Using type I QYH-HB-1 strain (CGMCC No. 47084) DNA and pCDNA3.1(+) plasmid as templates, the fiber2 target gene and pCDNA3.1(+) linearized vector were obtained by PCR. Then, the fiber2 target gene was cloned into the mammalian cell expression vector pCDNA3.1(+) using homologous recombinase, transformed into TOP10 E. coli competent cells, screened for positive clones and sequenced to verify, and obtained the recombinant expression vector pCDNA-PAdV-Fiber2.
[0021] 2. Transfection of recombinant expression vector pCDNA-PAdV-Fiber2 HEK293 cells in good growth condition were seeded into 6-well plates and cultured at 37°C and 5% CO2 until the cell density reached 80%. Following the instructions of the Lipofectamine 3000 liposome transfection reagent, 2 μg of the recombinant expression vector pCDNA-PAdV-Fiber2 was transfected into HEK293 adherent cells, and the medium was replaced with DMEM complete medium after 6 hours.
[0022] 3. Screening and identification of cell lines expressing pigeon adenovirus type I Fiber2 protein Forty-eight hours after transfection, the culture medium was replaced with DMEM complete medium containing G418 (800 μg / ml) for drug screening, which continued for 14 days. Cells from 6-well plates were then passaged into 96-well plates using a limiting dilution method for further culture, and healthy cell clones were selected and expanded. Four healthy cell clones were selected, and total cellular protein was collected using cell lysis buffer for Western blotting. His-tagged monoclonal antibody (1:1000 dilution) was used as the primary antibody, and HRP-labeled goat anti-mouse IgG antibody (1:5000 dilution) was used as the secondary antibody. The results showed a specific band at approximately 110 kDa, indicating successful expression of Fiber2 protein, and the HEK293 cell line QYH-PAF2-A1, stably expressing pigeon adenovirus type I Fiber2 protein, was obtained.
[0023] 4. Domestication of QYH-PAF2-A1 suspension cells Adherent QYH-PAF2-A1 cells were continuously cultured using a slow serum reduction method (10% → 8% → 4% → 2% → 0% bovine serum by volume) until the cells were fully adapted to the serum-free HEK293 cell medium. QYH-PAF2-A1 cells were then suspended in HEK293 serum-free medium, and the cell density was adjusted to 2 × 10⁶ cells / year. 6Cells / ml were dispensed into Erlenmeyer flasks and cultured in suspension at 37°C, 5% CO2, and 100 rpm on a shaker. After five consecutive passages, QYH-PAF2-A1 cells were fully adapted to suspension culture. Cells were then harvested, and total cellular protein was collected using cell lysis buffer for Western blotting. The results showed a specific band at approximately 110 kDa, indicating that QYH-PAF2-A1 adherent cells, after being adapted to suspension culture, can still express pigeon adenovirus type I Fiber2 protein.
[0024] 5. Identification of the stability of QYH-PAF2-A1 suspension cells Suspension cells QYH-PAF2-A1 were cultured for 10 generations. Cells from generations 1, 5, and 10 were harvested, treated with cell lysis buffer, and analyzed by Western blotting. The results showed that pigeon adenovirus type I Fiber2 protein could be stably expressed in the suspension cell line QYH-PAF2-A1.
[0025] This invention also provides the application of the suspension QYH-PAF2-A1 cell line in the preparation of pigeon adenovirus type I Fiber2 protein subunit vaccine.
[0026] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.
[0027] Example 1: Construction of the recombinant expression vector pCDNA-PAdV-Fiber2 Using the DNA of pigeon adenovirus type 1 strain QYH-HB-1 (CGMCC No. 47084) preserved in the laboratory as a template, the fiber2 target gene was obtained by PCR using primers Fiber2-F and Fiber2-R (Table 1). Simultaneously, using pCDNA3.1(+) plasmid as a template, the vector was linearized by PCR using primers pCDNA3.1-F and pCDNA3.1-R (Table 1). A 50 μL reaction system was established with the following components: template, 1 μL; Primer Star Mix (2×), 25 μL; upstream primer, 1 μL; downstream primer, 1 μL; ddH2O, 22 μL. Then, 1% agarose gel electrophoresis was performed, and the gel was recovered according to the instructions of the gel recovery kit. The fiber2 target gene was cloned into the mammalian cell expression vector pCDNA3.1(+) using homologous recombinase, transformed into TOP10 E. coli competent cells, positive clones were screened and sequenced to verify, and the recombinant expression vector pCDNA-Fiber2 was obtained.
[0028] Table 1 PCR amplification primers
[0029] Example 2: Transfection of recombinant expression vector pCDNA-PAdV-Fiber2 HEK293 cells in good growth condition were seeded into 6-well plates and cultured at 37°C in a 5% CO2 incubator until the cell density reached 80%. Following the instructions of the Lipofectamine 3000 liposome transfection reagent, 4 μl of liposomes were added to 200 μl of Opti-MEM and incubated at room temperature for 5 minutes. Then, 2 μg of the recombinant expression vector pCDNA-PAdV-Fiber2 was added, gently mixed, and incubated at room temperature for 15 minutes. The mixture was then added dropwise to the HEK293 cell culture medium, and after 6 hours, it was replaced with DMEM complete medium.
[0030] Example 3: Screening and Identification of Cell Lines Expressing Pigeon Adenovirus Type I Fiber2 Protein Forty-eight hours after transfection, the culture medium was replaced with DMEM complete medium containing G418 (800 μg / ml) for drug screening. The medium was changed every 72-84 hours for 14 days. When the remaining cells in the wells proliferated to 50%, the cells were digested from the six-well plates and passaged into 96-well plates using limiting dilution. Cell clones with good growth were selected and cultured on a larger scale. Subsequently, four cell clones with good growth were selected and seeded into six-well plates. After the cells reached confluence, total cellular protein was collected using cell lysis buffer and Western blot analysis was performed. His-tagged monoclonal antibody (1:1000 dilution) was used as the primary antibody, and HRP-labeled goat anti-mouse IgG antibody (1:5000 dilution) was used as the secondary antibody. The results showed a specific band at approximately 110 kDa. Figure 1 This indicates that the Fiber2 protein was successfully expressed in HEK293 cells, and the HEK293 cell line QYH-PAF2-A1, which can express pigeon adenovirus type I Fiber2 protein, was obtained.
[0031] Example 4: Domestication of QYH-PAF2-A1 suspension cells HEK293-PAdV-Fiber2 cells were continuously cultured using a slow serum reduction method with bovine serum (volume percentage) of 10% → 8% → 4% → 2% → 0% until the cells were fully adapted to the serum-free HEK293 cell culture medium. Three flasks of (T75) QYH-PAF2-A1 cells were cultured in HEK293 serum-free medium, and cells were collected for cell counting and viability testing. The cell density was then adjusted to 2 × 10⁶ cells / year. 6Cells / ml were aliquoted into Erlenmeyer flasks and placed in a 37°C, 5% CO2 incubator at 100 rpm for 72 hours of suspension culture. Cells were sampled daily for counting and viability analysis. The culture was continued until the cell density reached 6 × 10⁶ cells / ml. 6 When the cell density reaches a level higher than 100 cells / ml, the cells are passaged. After five consecutive passages, the QYH-PAF2-A1 cells are fully adapted to suspension culture. Then, when the cell density reaches 6 × 10⁶ cells / ml... 6 At a cell / ml ratio, centrifugation was performed at 1000 rpm. Cells were collected, and total cellular protein was collected using cell lysis buffer for Western blotting. His-tagged monoclonal antibody (1:1000 dilution) was used as the primary antibody, and HRP-labeled goat anti-mouse IgG antibody (1:5000 dilution) was used as the secondary antibody. The results showed a specific band at approximately 110 kDa. Figure 2 This indicates that QYH-PAF2-A1 adherent cells can still express pigeon adenovirus type I Fiber2 protein after being domesticated into suspension cells.
[0032] Example 5: Identification of the stability of QYH-PAF2-A1 suspension cells Suspension cells QYH-PAF2-A1 were cultured for 10 generations. Cells from generations 1, 5, and 10 were harvested, treated with cell lysis buffer, and identified by Western blotting. His-tagged monoclonal antibody (1:1000 dilution) was used as the primary antibody (purchased from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., catalog number TA-02), and HRP-labeled goat anti-mouse IgG antibody (1:5000 dilution) was used as the secondary antibody. The results showed a specific band at approximately 110 kDa. Figure 3 This indicates that pigeon adenovirus type I Fiber2 protein can be stably expressed in the suspension cell line QYH-PAF2-A1.
[0033] Example 6: Preparation of Pigeon Adenovirus Type I Fiber2 Protein Subunit Vaccine and Immunological Challenge Protection Experiment First, the QYH-PAF2-A1 suspension cell culture was freeze-thawed once at -80℃ and centrifuged at 8000 rpm for 30 minutes at 4℃. Next, the supernatant was added to a pre-equilibrated nickel column and incubated on a shaker at 4℃ for 1 hour. The flow-through was repeated twice. Impurities were washed with 6 column volumes of washing buffer (25 mM imidazole, 20 mM Tris-HCl, 500 mM NaCl, pH=8.0). Third, the target protein was eluted with 1 / 5 volume of elution buffer (500 mM imidazole, 20 mM Tris-HCl, 500 mM NaCl, pH=8.0). Fourth, the target protein was concentrated using the saturated ammonium sulfate method and redissolved in PBS containing 10% glycerol for later use. Fifth, the protein purification effect was verified by SDS-PAGE. Figure 4 Finally, 0.1% formaldehyde was added to the protein solution and inactivated at 37°C for 24 hours. Two portions of the oil phase were added to the emulsification tank, and the motor was turned on to stir at low speed. At the same time, one portion of the aqueous phase was slowly added. After the addition was complete, the mixture was stirred at 10,000 r / min for 5 minutes to ensure that the oil phase and the aqueous phase were fully mixed and emulsified, thus preparing the pigeon adenovirus type I Fiber2 protein oil emulsion subunit vaccine (the effective content of Fiber2 protein is 100 μg / ml).
[0034] Thirty healthy 30-day-old pigeons (fecal samples tested negative for pigeon adenovirus infection via PCR) were randomly divided into three groups of 10 each. Group 1 was the immunization group, immunized with pigeon adenovirus type I Fiber 2 protein subunit vaccine via intramuscular injection, 0.2 ml / pigeon. Fourteen days after the first immunization, a second immunization was administered via intramuscular injection of pigeon adenovirus type I Fiber 2 protein subunit vaccine, 0.2 ml / pigeon. Fourteen days after the second immunization, pigeon adenovirus QYH-HB-1 strain was challenged, 0.1 ml / pigeon. Group 2 was the challenge control group, challenged with pigeon adenovirus QYH-HB-1 strain 28 days after immunization, 0.1 ml / pigeon. Group 3 was the blank control group. Groups 1-3 were observed for 14 consecutive days after challenge. After 14 days, all pigeons were necropsies to observe for any lesions in their internal organs. The results showed that in the group vaccinated with pigeon adenovirus type I Fiber2 protein subunit vaccine, 10 pigeons had normal mental state, feed intake, and water intake, while 2 pigeons showed organ lesions upon necropsy. In the challenge control group, pigeons showed clinical symptoms such as lethargy and green diarrhea, and 1 pigeon died. Four other pigeons showed organ lesions upon necropsy. In the blank control group, pigeons had normal mental state, feed intake, and water intake, and no pigeons died. The specific results are shown in Table 2.
[0035] Table 2. Protective Experiments of Pigeon Adenovirus Type I Fiber2 Protein Subunit Vaccine Against Virus Challenge
[0036] In summary, this invention provides a method for constructing a HEK293 suspension cell line stably expressing pigeon adenovirus type I Fiber2 protein. By constructing a recombinant expression vector for pigeon adenovirus type I Fiber2 protein, the recombinant expression vector was introduced into HEK293 adherent cells using liposome transfection to obtain the cell line QYH-PAF2-A1 capable of expressing pigeon adenovirus type I Fiber2 protein, which was then acclimated to suspension cell culture. This cell line exhibited good suspension growth and stable passage, maintaining stable expression of Fiber2 protein even after 10 consecutive passages. Subsequently, the Fiber2 protein expressed by this cell line was used to prepare a subunit vaccine, which, after immune challenge experiments, demonstrated a protection rate of 80%, effectively preventing pigeon adenovirus type I infection. This invention's construction of the QYH-PAF2-A1 suspension cell line stably expressing pigeon adenovirus type I Fiber2 protein has broad application prospects in the development of pigeon adenovirus type I subunit vaccines, the preparation of diagnostic reagents, and basic research on viral invasion mechanisms.
[0037] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.
Claims
1. HEK293 cells QYH-PAF2-A1 stably expressing pigeon adenovirus type I Fiber2 protein, preservation number CGMCCNo.46766.
2. The method for constructing cells according to claim 1, characterized in that, Includes the following steps: (1) The recombinant expression vector containing the pigeon adenovirus type I fiber2 gene was introduced into HEK293 adherent cells; (2) Screening to obtain positive clones that can stably express Fiber2 protein; (3) The positive clone cells are subjected to suspension acclimatization culture.
3. The method according to claim 2, characterized in that, The recombinant expression vector mentioned in step (1) is constructed by cloning the pigeon adenovirus type I fiber2 gene into the pCDNA3.1(+) vector.
4. The method according to claim 2, characterized in that, In step (1), the recombinant expression vector was introduced into HEK293 adherent cells using liposome transfection.
5. The method according to claim 2, characterized in that, The screening in step (2) includes drug screening using G418 and monoclonalization using limiting dilution.
6. The method according to any one of claims 2-5, characterized in that, The suspension acclimatization culture in step (3) involves gradually reducing the serum concentration in the culture medium until serum-free culture is achieved.
7. The use of the cell described in claim 1 in the preparation of pigeon adenovirus type I Fiber2 protein.
8. The use of the cells of claim 1 in the preparation of a subunit vaccine for the prevention of pigeon adenovirus type I infection.
9. The application according to claim 8, characterized in that, The pigeon adenovirus type I strain is QYH-HB-1, with the accession number CGMCC No. 47084.