CHO-S cell strain capable of stably expressing H5N1 hemagglutinin protein and construction method of CHO-S cell strain

Abstract

The invention relates to a CHO-S cell strain capable of stably expressing H5N1 hemagglutinin protein and a construction method of the CHO-S cell strain, and belongs to the field of bioengineering.H5N1 hemagglutinin protein expression plasmids are obtained in an in-vitro synthesis and seamless cloning mode, 293T cells are transfected through the H5N1 hemagglutinin protein expression plasmids, and it is proved that the H5N1 hemagglutinin protein is expressed; the CHO-S cell strain capable of stably expressing the H5N1 hemagglutinin protein is obtained by transfecting CHO-S cells by using the H5N1 hemagglutinin protein expression plasmids and carrying out multiple rounds of cloning and screening, so that a basis is provided for obtaining H5N1 recombinant protein influenza vaccines.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of bioengineering, and particularly relates to a CHO-S cell strain stably expressing H5N1 hemagglutinin protein and a construction method thereof. BACKGROUND

[0002] Since 1996, highly pathogenic avian influenza (HPAI) H5 viruses have been isolated from wild birds and poultry.

[0003] Influenza A (H5N1) virus belongs to the Orthomyxoviridae family, and is an influenza A virus in the influenza A virus, with two antigens on the surface of glycoprotein, hemagglutinin (HA) and neuraminidase (NA), which are crucial for the virus to enter host cells and its pathogenicity. However, the influenza A H5N1 virus currently spreading in cattle has undergone several mutations, including mutations in HA, which has been confirmed to be an immunodominant target for vaccine-induced immunity, and therefore, HA is considered a key target for the development of H5N1 influenza vaccines.

[0004] In mammalian cells, CHO cell lines are the main cell types for the industrial production of recombinant proteins and monoclonal antibodies (mAbs), and compared with prokaryotic expression systems, CHO cell expression systems show less immunogenicity. They also have the ability to perform post-translational modifications. CHO cell lines are the most popular expression system for the production of pharmaceutical proteins. The advantages of using CHO cells include high productivity, consistent growth phenotype, ease of industrial scale-up, and ease of adaptation to serum-free media, and can be inoculated at high cell density to facilitate large-scale production processes. CHO-S cells, as a highly efficient and stable CHO cell expression system, are widely used in the production of recombinant proteins.

[0005] Vaccination is the most effective strategy for preventing infectious diseases, and developing an effective vaccine against highly pathogenic avian influenza virus is crucial for preventing pandemics. Although inactivated vaccines are safe, their immunogenicity is low. Recombinant protein vaccines have the advantages of high safety, low cost, and good stability, and obtaining stable expression cells that stably express target antigens is a necessary condition for obtaining recombinant protein vaccines. SUMMARY

[0006] In order to solve the problems in the background art, the present application provides a CHO-S cell strain stably expressing H5N1 hemagglutinin protein and a construction method thereof, the present application first constructs an H5N1 hemagglutinin protein expression plasmid, then transfects the H5N1 hemagglutinin protein expression plasmid into CHO-S cells, and obtains a cell strain that is stably expressing H5N1 hemagglutinin protein by combination screening of puromycin and methotrexate.

[0007] To achieve the above-mentioned purpose, the present application is realized by the following technical scheme: The H5N1 hemagglutinin protein expression plasmid is obtained by cloning the optimized base sequence of the H5N1 hemagglutinin protein into the AvrII site of pCHO1.0.

[0008] The optimized base sequence of the H5N1 hemagglutinin protein comprises: a signal peptide base sequence as shown in SEQ ID NO. 1; an H5N1 hemagglutinin protein base sequence as shown in SEQ ID NO. 2; an enterokinase cleavage site base sequence as shown in SEQ ID NO. 3; and a histidine base sequence as shown in SEQ ID NO. 4.

[0009] The stable H5N1 hemagglutinin protein-expressing CHO-S cell strain is obtained after transfecting CHO-S cells with the H5N1 hemagglutinin protein expression plasmid.

[0010] The construction method of the stable H5N1 hemagglutinin protein-expressing CHO-S cell strain comprises the following steps: (1) adding the H5N1 hemagglutinin protein expression plasmid into OptiPRO™ SFM and mixing; (2) adding FreeStyle™ MAX reagent into OptiPRO™ SFM and mixing; (3) adding the mixed solution of step (2) into the mixed solution of step (1) to obtain a DNA-lipid complex; (4) adding the DNA-lipid complex of step (3) into a CHO-S cell culture flask for incubation; (5) screening the stable H5N1 hemagglutinin protein-expressing CHO-S cells; (6) expanding and screening to obtain the stable H5N1 hemagglutinin protein-expressing CHO-S cell strain.

[0011] Further, the screening of step (5) is drug screening, which is performed by culturing in a culture medium containing puromycin and methotrexate.

[0012] Further, the screening of step (5) specifically comprises the following steps: (1) 10P / 100M selection medium is used to plate two 96-well plates, 2.1 ml of cell suspension of the transfected cells at 48 h is taken, 40 ul of puromycin, 4 ul of methotrexate, and 37.9 ml of CDForti CHO complete medium are added, and the mixture is mixed and plated in a 96-well plate at 200 ul per well; (2) 10P selection medium is used to plate two 96-well plates, 2.1 ml of cell suspension of the transfected cells at 48 h is taken, 40 ul of puromycin and 37.9 ml of CDForti CHO complete medium are added, and the mixture is mixed and plated in a 96-well plate at 200 ul per well.

[0013] A recombinant protein vaccine comprising a CHO-S cell line stably expressing H5N1 hemagglutinin protein.

[0014] The beneficial effects of this invention are: This invention obtains an H5N1 hemagglutinin protein expression plasmid through in vitro synthesis and seamless cloning. This H5N1 hemagglutinin protein expression plasmid has two resistance selection conditions: a puromycin resistance gene and a dihydrofolate reductase selective marker. It can be successfully transfected into CHO-S cells. By adding puromycin and methotrexate for combined screening, a CHO-S cell line that stably expresses H5N1 hemagglutinin protein is obtained.

[0015] By transfecting CHO-S cells with the H5N1 hemagglutinin protein expression plasmid, a CHO-S cell line stably expressing H5N1 hemagglutinin protein was obtained. This stable CHO-S cell line expressing H5N1 hemagglutinin protein can be used as a raw material for the preparation of H5N1 recombinant protein vaccine, providing a direction and foundation for the subsequent acquisition of H5N1 recombinant protein vaccine. Attached Figure Description

[0016] Figure 1 This is the Western blot result of 293T cells transfected with the H5N1 hemagglutinin protein expression plasmid in Example 1 of this invention; Figure 2 This is the Western blot result of CHO-S cells transfected with the H5N1 hemagglutinin protein expression plasmid in Example 2 of this invention for 48 hours; Figure 3 These are the WB results of the cell culture supernatant and cell lysis supernatant from the 2-well plate of the present invention. Figure 4 This is the result of two Western blot experiments conducted on a 6-well plate of the H5N1 hemagglutinin protein expression plasmid in Example 2 of this invention. Figure 5 This is the D6 WB result of the monoclonal 6-well plate in Example 2 of the present invention (2#); Figure 6 These are the D6 WB results (14#, 15#) of a monoclonal 6-well plate in Example 2 of this invention. Figure 7 This is the D6 WB result (12#) of a monoclonal 6-well plate in Example 2 of this invention. Detailed Implementation

[0017] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the technical solutions of this invention will be described in detail below. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. Other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are all within the scope of protection of this invention.

[0018] To illustrate the present invention more clearly, the following embodiments will be described in detail.

[0019] Example 1

[0020] Construction of H5N1 hemagglutinin protein expression plasmid Sequence design and optimization: To facilitate the extracellular secretion of synthesized proteins, we introduced an optimized signal peptide sequence of influenza virus (SEQ ID NO.1), followed by the H5N1 hemagglutinin protein sequence (SEQ ID NO.2), the enterokinase cleavage site sequence (SEQ ID NO.3), and a histidine sequence consisting of 10 histidines for protein purification (SEQ ID NO.4). We further optimized the base codon bias according to the species characteristics of CHO cells. The optimized H5N1 hemagglutinin protein base sequence combination is shown in Table 1.

[0021] Sequence synthesis and plasmid construction: The optimized base sequence was synthesized in vitro, and then the H5N1 hemagglutinin protein sequence was cloned into the AvrII site of pCHO1.0 using a seamless cloning method to obtain the pCHO1.0-H5N1-HA plasmid (H5N1 hemagglutinin protein expression plasmid).

[0022] Table 1. Optimized base sequence combinations of H5N1 hemagglutinin protein

[0023] The constructed H5N1 hemagglutinin protein expression plasmid (pCHO1.0-H5N1-HA plasmid) was transfected into 293T cells. After 48 hours, the protein was extracted and separated by SDS-PAGE electrophoresis. The separated protein was then transferred to a membrane, blocked with blocking buffer, incubated with primary antibody, and incubated with secondary antibody. Finally, imaging was performed using a gel imaging system (e.g.,...). Figure 1 As shown in the figure, the results indicate that H5N1 hemagglutinin protein was expressed.

[0024] Example 2

[0025] The construction of a CHO-S cell line stably expressing H5N1 hemagglutinin protein includes the following steps: (1) Cell preparation before transfection: Twenty-four hours before transfection, CHO-S™ cells were passaged in CD FortiCHO™ complete medium at a density of 5×10^5 to 6×10^5 viable cells / mL. The culture flasks were placed on a ring shaker at 130 rpm (37°C, 8% CO2) and cultured for 24 hours. Cell counting was performed (all cell counts were performed using a cell counter), and the cell counting results are shown in Table 2.

[0026] Table 2 Cell preparation before transfection

[0027] (2) Transfected cells 2.1 Add 50 μg of H5N1 hemagglutinin protein expression plasmid (pCHO1.0-H5N1-HA plasmid) to OptiPRO™ SFM to make a final volume of 1.5 mL, and mix gently. 2.2 Add 50 μL of FreeStyle™ MAX reagent to 1.45 mL of OptiPRO™ SFM, bringing the final volume to 1.5 mL, and mix gently.

[0028] 2.3 Immediately add the solution mixed in 2.2 to the solution mixed in 2.1 using a pipette. First, immerse the pipette tip in the pipette tip, then slowly add the solution while shaking the pipette tip.

[0029] 2.4 Shake the test tube and incubate the mixture at room temperature for 10 min. Gently mix the DNA-FreeStyle™ MAX mixture to form a DNA-lipid complex.

[0030] 2.5 Add 3 mL of DNA-lipid complex (DNA-FreeStyle™ MAX mixture) dropwise to a 125 mL culture flask containing CHO-S cells and gently shake the flask.

[0031] 2.6 The transfected cell cultures were incubated on a ring shaker at 130 rpm (37°C, 8% CO2). The transfection data are shown in Table 3.

[0032] Table 3 Cell transfection data

[0033] On the day of transfection, live cell counts were performed, and the cell viability was approximately 98.53%.

[0034] (3) Continue to screen cell lines that stably express proteins for protein expression. 3.1 After incubation for 48 hours, collect 1 ml of transfected cell suspension, centrifuge to lyse the cells, and collect the cell lysis supernatant for Western blot analysis.

[0035] Cell lysate supernatant proteins were separated using SDS-PAGE electrophoresis. The separated proteins were then transferred to a membrane, blocked with blocking buffer, incubated with primary antibody, and incubated with secondary antibody. Imaging was then performed using a gel imaging system. The results are shown below. Figure 2 As shown in the figure, the results indicate that H5N1 hemagglutinin protein was expressed.

[0036] 3.2 Cell counting and seeding at 2×10^4 viable cells / well / 200µl in 96-well plates, followed by drug screening: (1) Two 96-well plates were prepared using 10P / 100M selective medium: 2.1 ml of cell suspension after 48 h of transfection was taken, 40 μl of puromycin (P), 4 μl of methotrexate (M), and 37.9 ml of CDForti CHO complete medium were added, and after mixing, 200 μl / well was prepared for 96-well plate preparation; (2) Two 96-well plates were prepared using 10P selective medium: 2.1 ml of cell suspension after 48 h of transfection was taken, 40 μl of puromycin (P) and 37.9 ml of CDForti CHO complete medium were added, and after mixing, 200 μl / well was prepared for 96-well plate preparation. See Table 4.

[0037] Table 4. 96-well plate pressure screening

[0038] The dosage of puromycin (10 mg / ml) is: 10P (1 μl / ml); 30P (3 μl / ml).

[0039] The dosage of methotrexate (MTX) (1 mmol) added is: 100M (0.1 μl / ml); 300M (0.3 μl / ml).

[0040] CDFortiCHO™ Complete Medium (CDFortiCHO™ Medium with 4% added glutamine).

[0041] The pCHO 1.0 vector contains a puromycin resistance gene and a dihydrofolate reductase-selectively labeled resistance. This invention uses both puromycin and methotrexate for screening. Only cells stably transfected with H5N1 hemagglutinin protein particles can grow in CD FortiCHO™ medium containing both puromycin and methotrexate. Therefore, to obtain stable cell lines that generate high levels of H5N1 hemagglutinin protein, we use a two-stage (10P & 10P / 100M → 30P / 300M) selection scheme to generate multiple groups of stable cell lines expressing H5N1 hemagglutinin protein particles. The H5N1 hemagglutinin protein particles in these cells have been integrated into the host cell genome; therefore, only these stable cell lines can grow in CD FortiCHO™ complete medium containing both puromycin and methotrexate.

[0042] (4) 24-well plate propagation 4.1 In 10P-CC1-96-well plates, the cell confluence under a microscope was approximately 40% at D10. 150 μL of 30P selective medium was added to each well (see Table 4), and the cells were cultured until the cell confluence under a microscope was >50% at D13. 48 wells were randomly selected and expanded into 24-well plates, with 0.5 ml of 30P / 300M selective medium per well. 4.2 Cell confluence > 50% under a microscope in 10P / 100M-CC1-96-well plates. 70 wells were randomly selected and cultured into 24-well plates, with 0.5 ml of 30P / 300M selective medium per well.

[0043] (5) First expansion culture of 6-well plate On day 7 of a 24-well plate, 18 wells were randomly selected and transferred to 6-well plates, using 2 ml of 30P / 300M per well. On day 7, the cell culture supernatant and cell lysis supernatant were collected for Western blotting (WB) analysis. The results are as follows: Figure 3 As shown in Table 5, samples 2, 12, and 15 were each cultured in 15 ml of water, and samples 1, 5, 10, 13, 14, and 16 were cultured in 6-well plates. The cell count results are shown in Table 5.

[0044] Table 5. Cell count results after cell expansion culture

[0045] (6) Second expansion culture of 6-well plate Western blot analysis was performed on the cell culture supernatant collected from the 6-well plate on day 7. The results are as follows: Figure 4 As shown. Based on the test results, wells #2, #12, #14, and #15 were selected for monoclonal plate formation, with 0.8 live cells / well and 120 μL / well. Four wells were formed from wells #2, #12, and #14, and seven wells were formed from well #15. On day 7 of plate formation, 100 μL of monoclonal complete culture medium was added to each well.

[0046] (7) Expand monoclonal cells to 24-well plates Once the confluence of monoclonal cells is observed under a microscope (>10%), they can be expanded into 24-well plates with 0.5 ml of 30P / 300M selective medium per well. Based on observation, 31, 60, 50, and 65 mini pools of monoclonal cells #2, #12, #14, and #15 were selected and expanded into 24-well plates, respectively.

[0047] (8) Expand monoclonal cells to 6-well plates On day 5 of a 24-well plate, 11, 24, 6, and 19 cells were randomly selected from wells 2#, 12#, 14#, and 15#, respectively, and then transferred to 6-well plates. 2 ml of 30P / 300M selective medium was added per well. On day 6, the supernatant was collected for Western blotting (WB) analysis. The results are shown below. Figures 5 to 7 As shown ( Figure 5 It is #2. Figure 6 For #14 and #15, Figure 7 (12#). Based on the WB test results, 17 monoclonal cells stably expressing hemagglutinin protein were selected for cell counting and cultured to 125°C in shake flasks. After culture, the cells were cryopreserved.

[0048] Through the above rounds of cloning and screening, we finally obtained 17 monoclonal cell lines that stably express H5N1 hemagglutinin protein.

[0049] We can select cell lines with high H5N1 hemagglutinin protein expression levels from these 17 stable cell lines to establish a primary seed cell bank, a master seed cell bank, and a working seed cell bank, providing conditions for the later preparation of H5N1 influenza vaccines.

[0050] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made to it in form and detail without departing from the scope defined by the claims of the present invention.

Claims

1. An H5N1 hemagglutinin protein expression plasmid, characterized in that, The optimized base sequence of the H5N1 hemagglutinin protein was cloned into the AvrII site of pCHO1.

0. The optimized base sequence combination of the H5N1 hemagglutinin protein is as follows: signal peptide base sequence as shown in SEQ ID NO.1; H5N1 hemagglutinin protein base sequence as shown in SEQ ID NO.2; enterokinase cleavage site base sequence as shown in SEQ ID NO.3; and histidine base sequence as shown in SEQ ID NO.

4.

2. A CHO-S cell line stably expressing H5N1 hemagglutinin protein obtained by transfecting CHO-S cells with the H5N1 hemagglutinin protein expression plasmid as described in claim 1.

3. The method for constructing a CHO-S cell line stably expressing H5N1 hemagglutinin protein as described in claim 2, characterized in that, Includes the following steps: (1) Add the H5N1 hemagglutinin protein expression plasmid to OptiPRO™ SFM and mix well; (2) Add FreeStyle™ MAX reagent to OptiPRO™ SFM and mix well; (3) Add the solution mixed in step (2) to the solution mixed in step (1) to obtain the DNA-lipid complex; (4) Add the DNA-lipid complex from step (3) into a CHO-S cell culture flask and incubate; (5) Screening for CHO-S cells that stably express H5N1 hemagglutinin protein; (6) Expand culture and screen to obtain CHO-S cell lines that stably express H5N1 hemagglutinin protein.

4. The construction method according to claim 3, characterized in that, The screening described in step (5) is a drug-assisted screening, specifically a culture medium containing puromycin and methotrexate for screening.

5. The construction method according to claim 4, characterized in that, The screening described in step (5) is as follows: (1) Two 96-well plates are plated with 10P / 100M selective medium. Take 2.1ml of cell suspension after 48h transfection, add 40ul of puromycin, 4ul of methotrexate, and 37.9ml of CDForti CHO complete medium. After mixing, plate the 96-well plates with 200ul / well. (2) Two 96-well plates are plated with 10P selective medium. Take 2.1ml of cell suspension after 48h transfection, add 40ul of puromycin and 37.9ml of CDForti CHO complete medium. After mixing, plate the 96-well plates with 200ul / well.

6. A recombinant protein vaccine comprising a CHO-S cell line stably expressing H5N1 hemagglutinin protein as described in claim 2.

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